Microwave-assisted synthesis of triazolothiadiazole analogs as anticancer agents

Article abstract of DOI:10.1007/s00044-014-0944-x

A series of S-alkyl derivatives of 3-(substituted-(1,1′-biphenyl)-3- yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiol (4a-j) were synthesized by conventional and microwave irradiation methods. Microwave method proved to be a rapid and better yield compare to that of conventional method. The synthesized compounds were characterized by ¹H NMR, ¹³C NMR, ¹⁹F NMR, IR, LCMS, and elemental spectroscopic analysis. These compounds were screened for their in vitro anticancer activity by MTT assay. Among the tested compounds, the compound 4c was the most promising anticancer agent with IC₅₀ value 12 μM in HT29 cell line. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0944-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0944-x
ORIGINAL RESEARCH
Microwave-assisted synthesis of triazolothiadiazole analogs
as anticancer agents
•
G. C. Ramaprasad Balakrishna Kalluraya
•
B. Sunil Kumar
Received: 22 July 2013 / Accepted: 7 February 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of S-alkyl derivatives of 3-(substi-
tuted-(1,10-biphenyl)-3-yl)[1,2,4]triazolo[3,4-b][1,3,4]thia-
diazole-6-thiol (4a–j) were synthesized by conventional
and microwave irradiation methods. Microwave method
proved to be a rapid and better yield compare to that of
conventional method. The synthesized compounds were
characterized by 1H NMR, 13C NMR, 19F NMR, IR,
LCMS, and elemental spectroscopic analysis. These com-
pounds were screened for their in vitro anticancer activity
by MTT assay. Among the tested compounds, the com-
pound 4c was the most promising anticancer agent with
IC50 value 12 lM in HT29 cell line.
products such as taxol and vinca alkaloids are known for
effective antimitotic drugs. However, the complex synthesis,
difficult formulations, lack of oral availability make these
drugssuboptimumfor clinicaltreatmentofcancer(Kavallaris
et al., 2001). Therefore, the search for novel and selective
anticancer agents is urgently required due to problems asso-
ciated with currently available anticancer agents.
Designing new drug is based on the development of
hybrid molecules by combining different pharmacophore
fragments in a single structure, which may lead to com-
pounds with interesting biological profiles.
The chemistry of 1,2,4-triazoles and their fused hetero-
cyclic derivatives has received considerable attention owing
to their synthetic and effective biological importance. For
example, a triazolo-thiadiazole system may be viewed as a
cyclic analog of two important components: thiosemicar-
bazide and biguanide (Mathew et al., 2006), which often
display antibacterial (Palekar et al., 2009), antifungal (Isloor
et al., 2009), analgesic (Mathew et al., 2007), anti-inflam-
matory (Husain and Naseer, 2011), antitubercular (Kart-
hikeyan et al., 2007), and anticancer (Holla et al., 2002; Sunil
et al., 2009) activities. Fluorinated heterocycles have
attracted attention due to the ability of fluorine to act as
polar hydrogen or hydroxyl mimic (Filler and Kobayashi,
1982). Therefore, substitution of hydrogen by fluorine
has been a strategy in designing molecules for biological
activity studies (Holla et al., 2006). The microwave-induced
organic reaction received considerable attention due to
their simplicity and operational convenience. The use of
microwave irradiation as a source of heat in synthetic
chemistry offers a promising alternative (Jyothi et al., 2007;
Kalluraya et al., 2008). Microwave-assisted heating under
controlled conditions is an invaluable technology because it
often dramatically reduces reactions times, with increased
Keywords 1,2,4-Triazoles ꢀ Triazolothiadiazoles ꢀ
Microwave irradiation ꢀ Anticancer activity ꢀ MTT assay
Introduction
Cancer is a serious disease with complex pathogenesis, which
threats human life greatly. A number of chemotherapeutic
drugshave been developed to treatcancerthat includes DNA-
alkylating agents and antimitotic agents. Many natural
G. C. Ramaprasad ꢀ B. Kalluraya
Department of Studies in Chemistry, Mangalore University,
Mangalagangothri 574199, Karnataka, India
G. C. Ramaprasad (&)
Syngene Inernaional Ltd., Biocon Park, Plot No. 2&3,
Bommasandra IV Phase, Bangalore 99, India
e-mail: ramaprasadbhatt@yahoo.com
B. Sunil Kumar
Department of Pharmaceutical Chemistry, N.E.T Pharmacy
College, Raichur 584103, India
´
yield (Escalante and Dıaz-Coutin˜o, 2009). Nowadays,
123

Med Chem Res
H
N
N
N
N
N
N
S
SH
SH
H2N-NH2 /EtOH
N
NH
N
O
NH
2
2
R
R
1a-b
R
2a-b
CS2 / KOH
EtOH
N
N
N
N
N
N
R1-Br / K2CO3
DMF
N
N
S
N
S
S
N
N
S
S
N
R1
SH
H
MW /
R
R
R
4a-j
3a-b
R
R1
R
R1
4a
4b
4c
4d
4e
5'-F, 2'-OMe
-(CH2)3CO2Me
-(CH2)3CO2Me
-(CH2)4F
4f
4g
4h
4i
2'-F
-(CH2)3CN
-(CH2)3NH2
-(CH2)3NH2
-(CH2)2OH
-(CH2)2OH
2'-F
5'-F, 2'-OMe
2'-F
5'-F, 2'-OMe
2'-F
-(CH2)4F
5'-F, 2'-OMe
2'-F
5'-F, 2'-OMe
-(CH2)3CN
4j
Scheme 1 Synthesis of S-alkyl derivatives of 3-(substituted-(1,10-biphenyl)-3-yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiol
microwave-assisted organic synthesis has become a new era
in the field of synthetic chemistry (Algul et al., 2009).
Encouraged by the afore-mentioned findings and in a
continuation of an ongoing program aiming at finding new
structural leads with potential chemotherapeutic activities
(Ramaprasad et al., 2010, 2012, 2013), it was rationalized to
synthesize 3-(substituted-(1,10-biphenyl)-3-yl)[1,2,4]triaz-
olo[3,4-b][1,3,4]thiadiazole-6-thiol and its S-alkyl deriva-
tives in order to study their anticancer activity.
10-biphenyl)-3-yl)-1,3,4-oxadiazole-2-thiol (1a–b) with
hydrazine hydrate (Ramaprsad et al., 2012).
The compounds 3a–b were prepared by heating the
triazoles 2a–b with carbon disulfide, potassium hydroxide
in ethyl alcohol as solvent. Our initial effort toward the
regioselective synthesis of compounds 4a–j was heating
the compounds 3a–b with alkyl halides, potassium car-
bonate in N,N-dimethylformamide at 85 °C for 2 h, while
under microwave irradiation, it required shorter reaction
time (5 min) and higher product yields making it superior
method. The yield and physical data of synthesized com-
pounds are summarized in Table 1.
Results and discussion
In the IR spectra of compounds 2a–b, NH2 bands were
observed at 3,131–3,225 cm-1. The disappearance of NH2
bands and new stretching frequencies of N–N=C group
formed by thiadiazole ring closure are observed at
1,260 cm-1 in compound 3a and 1,259 cm-1 in compound
3b. Analysis of spectral data of compounds 3a–b leads to the
Chemistry
As described in Scheme 1, the 4-amino-5-(substituted-(1,
10-biphenyl)-3-yl)-4H-1,2,4-triazole-3-thiol (2a–b) was
prepared in good yield by the reaction of 5-(substituted-(1,
123

Med Chem Res
Table 1 Characterization data of compounds 4a–j
Compound no.
Mol. formula
Yield (%) (microwave)
Nature
M. Pt. (°C)
Analysis (%) found (calculated)
C
H
N
4a
4b
4c
4d
4e
4f
C21H19FN4O3S2
63
Colorless liquid
Colorless liquid
Pale yellow liquid
Colorless liquid
Colorless oil
–
54.96
4.15
12.18
(80)
60
(55.01)
56.11
(4.18)
4.03
(12.22)
13.05
C
C
C
C
C
C
C
C
C
20H17FN4O2S2
20H18F2N4OS2
19H16F2N4S2
20H16FN5OS2
19H14FN5S2
–
(79)
75
(56.06)
55.51
(4.00)
4.15
(13.08)
12.93
–
(84)
71
(55.54)
56.68
(4.19)
4.03
(12.95)
13.89
–
(83)
71
(56.70)
56.41
(4.01)
3.75
(13.92)
16.42
–
(85)
72
(56.45)
57.68
(3.79)
3.56
(16.46)
17.72
Colorless oil
–
(82)
61
(57.70)
54.89
(3.57)
4.34
(17.71)
16.83
4g
4h
4i
19H18FN5OS2
18H16FN5S2
White solid
176–178
166–167
156–158
166–168
(77)
63
(54.92)
56.05
(4.37)
4.15
(16.85)
18.18
White solid
(78)
64
(56.08)
53.69
(4.18)
3.72
(18.17)
13.89
18H15FN4O2S2
17H13FN4OS2
Off white solid
White solid
(81)
60
(53.72)
54.81
(3.76)
3.51
(13.92)
15.06
4j
(79)
(54.82)
(3.52)
(15.04)
conclusion that these compounds can exist in two tautomeric
forms, thione form in solution and thiol form in solid state.
The IR spectra recorded in KBr pellets show a band at
2,351 cm-1 (for 3a) and 2,355 cm-1 (for 3b) can be attrib-
uted to SH group. The predominant tautomer of 3 was
identified as thione form by means of NMR spectroscopy.
The signal at 3.16 ppm (for 3a) and 3.21 ppm (for 3b) in the
1H NMR can be attributed to the NH-thiadiazolic proton, and
(human kidney cancer), and MDA231 (human breast
cancer) cancer cell lines using MTT assay (Mosmann,
1983). Results were compared with reference drug doxo-
rubicin. From the anticancer results, it is clear that the
compounds having the fluoro and methoxy groups in the
biphenyl ring (3a, 4a, 4c, 4e, 4g, and 4i) showed better
activity than respective fluoro substitution (3b, 4b, 4d, 4f,
4h, and 4j) against all the three cancer cell lines. This can
be attributed due to the presence of electron-donating
methoxy group. From the structure–anticancer activity
relationship of the compounds 4a–j, it was observed that
the functional group in the aliphatic side chain of thiadi-
azole ring played an important role. Compounds 4a and
4b having ester functional group showed moderate activity
against all the three cancer cell lines. The replacement of
ester by fluoro (4c and 4d) improved the potency in all
cell lines especially against HT29 cell line (IC50 = 12 lM
for 4c and 15 lM for 4d). Nitrile-substituted com-
pounds (4e and 4f) could not significantly improve the
cytotoxic activity in comparison with fluoro substituent.
The amine- and hydroxyl-substituted compounds 4g–j
were inactive against K293 cell line. However, compounds
4g and 4i showed moderate activity against HT29
and MDA231 cell lines. The results are summarized in
Table 2.
the peak at 182.1 ppm (for 3a) and 181.9 (for 3b) in the 13
C
NMR spectra can be assigned to C=S group in thione form.
For compounds 4a–j, the formation of S-alkyl derivative was
indicated by the presence of a peak around 3.11–3.52 ppm
for the S–CH2 group in the 1H NMR spectra. Similarly, the
absence of C=S peak at 181.9–182.1 ppm in the 13C NMR of
4a–j is the strong evidence for S-alkyaltion.
In 19F NMR of compounds 3a–b and 4a–j, the signals
observed from -128.2 to -122.8 were due to fluorines at
the aromatic ring. In compounds 4c and 4d fluorine
attached to an aliphatic system the signal was seen at
-218.3 and -217.5 ppm, respectively.
Anticancer activity
The in vitro cytotoxic activity of the synthesized compounds
was evaluated against HT29 (human adenocarcinoma), K293
123

Med Chem Res
resulting mixture was heated under reflux for 6 h. The
mixture was cooled, diluted with water, and acidified with
3 N hydrochloric acid. The resulting solid was filtered,
washed with water, dried, and recrystallized from ethyl
alcohol to give compounds 2a–b.
Table 2 In vitro cytotoxicity data (IC50 lM) of compounds 3a–b and
4a–j against cancer cell lines by MTT assay
Compound no.
HT29
K293
MDA231
3a
33
37
20
24
12
15
24
28
19
26
24
31
08
53
57
50
55
33
36
53
57
67
71
69
75
25
41
44
35
38
23
25
38
40
31
39
34
45
16
3b
4a
4-amino-5-[50-fluoro-20-methoxy-(1,10-biphenyl)-3-yl]-4H-
4b
1,2,4-triazole-3-thiol (2a)
4c
4d
Yield: 86 %, White solid, M.p. 210–212 °C; IR (KBr,
cm-1): 3,225, 3,143 (NH2), 1,600 (C=N); 1H NMR
(DMSO-d6) d: 3.68 (bs, 2H, NH2), 3.78 (s, 3H, OCH3),
7.13–7.23 (m, 3H, Ar–H), 7.55–7.61 (m, 2H, Ar–H), 8.21
(d, 1H, J = 9.1 Hz, Ar–H), 8.35 (s, 1H, Ar–H), 13.6 (bs,
1H, NH); 13C NMR (DMSO-d6) d: 55.2 (OCH3), 109.9,
111.2, 112.3, 114.7, 117.1, 117.4, 125.2, 127.2, 128.6,
131.4 (aromatic ring carbons), 151.2 (C5-triazole ring),
155.8 (d, J = 237.2 Hz) (C50-Ar), 158.3 (C20-Ar), 167.3
(C=S); 19F NMR (CDCl3) d: -122.3 (m, 1F); LCMS M?1:
317; Anal. Cacld for C15H13FN4OS: C, 56.95; H, 4.14; N,
17.71. Found: C, 56.91; H, 4.11; N, 17.69.
4e
4f
4g
4h
4i
4j
Doxorubicin
Conclusion
In summary, we have synthesized a novel series of S-alkyl
derivatives of 3-(substituted-(1,10-biphenyl)-3-yl)[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole-6-thiol by both conven-
tional and microwave irradiation methods. Microwave
method proved to be a rapid and better yield compared to that
of conventional method. The synthesized compounds also
screened for their anticancer activity. Compound 4c was
found to be most promising anticancer agent.
4-amino-5-[20-fluoro-(1,10-biphenyl)-3-yl]-4H-1,2,4-
triazole-3-thiol (2b)
Yield: 81 %, White solid, M.p. 190–192 °C; IR (KBr,
cm-1): 3,215, 3,122 (NH2), 1,596 (C=N); 1H NMR
(DMSO-d6) d: 3.91 (bs, 2H, NH2), 7.03–7.09 (m, 2H,
Ar–H), 7.18–7.35 (m, 3H, Ar–H), 7.51–7.62 (m, 2H, Ar–
H), 7.72 (s, 1H, Ar–H), 13.2 (bs, 1H, NH); 13C NMR
(DMSO-d6) d: 113.2, 114.8, 115.5, 116.7, 117.3, 118.5,
121.3, 121.8, 122.5, 122.9, 131.3 (aromatic ring carbons),
150.2 (C5-triazole ring), 156.7 (d, J = 241.2 Hz) (C20-Ar),
168.1 (C=S); 19F NMR (CDCl3) d: -123.1 (m, 1F); LCMS
M?1: 287; Anal. Cacld for C14H11FN4S: C, 58.73; H, 3.87;
N, 19.57. Found: C, 58.69; H, 3.85; N, 19.51.
Experimental
Chemistry
Thin layer chromatography was used to analyze the reac-
tion progress and purity of the compounds synthesized.
Melting points were determined in open glass capillary
methods and were uncorrected. IR spectra were obtained in
KBr disks on a Shimadzu-8400 FTIR spectrophotometer;
1H NMR spectra were recorded on Brucker spectrometer
(400 MHz) in DMSO-d6/CDCl3 using TMS as an internal
standard; mass spectra were recorded on Agilent 6320 Ion
Trap method; and 13C NMR spectra were recorded on
General procedure for the synthesis of compounds 3a–b
To a solution of 2a–b (8.0 g, 25.31 mmol) in ethyl alcohol
(80 mL), potassium hydroxide (1.41 g, 25.31 mmol) and
carbon disulfide (3.85 g, 50.63 mmol) were added, and the
reaction mixture was refluxed for 4 h. The solvent was
removed under reduced pressure, and then the residue was
poured into an ice-water mixture with stirring. The solid
product was filtered, washed with water, dried, and re-
crystallized from ethyl alcohol to give compounds 3a–b.
Brucker spectrometer (100 MHz) in DMSO-d6/CDCl3. 19
F
NMR spectra were recorded on 376 MHz in CDCl3 as
solvent. Microwave reactions were carried out in Biotage
Initiator (400 W) microwave synthesizer.
3-[50-fluoro-20-methoxy-(1,10-biphenyl)-3-
General procedure for the synthesis of compounds 2a–b
yl][1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiol (3a)
To a solution of 1a–b (66.22 mmol) in ethyl alcohol
(200 mL), hydrazine hydrate (231.7 mmol) was added and
Yield: 89 %, white solid, M.p. 201–203 °C; IR (KBr,
cm-1): 3,073 (aromatic CH), 2,351 (SH), 1260 (N–N=C);
123

Med Chem Res
1H NMR (DMSO-d6) d: 3.16 (bs, 1H, NH), 3.78 (s, 3H,
–OCH3), 7.14–7.24 (m, 3H, Ar–H), 7.56–7.62 (m, 2H, Ar–H),
8.21 (d, 1H, J = 8.2 Hz, Ar–H), 8.36 (s, 1H, Ar–H); 13C
NMR (DMSO-d6) d: 56.7 (OCH3), 113.7, 113.8, 115.3,
117.1, 125.1, 126.7, 129.1, 130.6, 131.1, 137.9 (aromatic
ring carbons), 143.2 (C3-triazole ring), 153.1 (C5-triazole
ring), 157.1 (d, J = 237.1 Hz) (C50-Ar), 158.1 (C20-Ar),
182.1(C=S); 19F NMR (CDCl3) d: -128.2 (m, 1F); LCMS
M?1: 359; Anal. Cacld for C16H11FN4OS2: C, 53.62; H,
3.09; N, 15.63. Found: C, 53.59; H, 3.06; N, 15.61.
chromatography [30–35 % ethyl acetate in petroleum
ether] to afford compounds 4a–f.
Methyl-4-{[3-(50-fluoro-20-methoxy-(1,10-biphenyl)-3-
yl)[1,2,4]triazolo[3,4-b][1,3,4]-thiadiazol-6-
yl]sulfanyl}butanoate (4a)
IR (KBr, cm-1): 3,069 (aromatic CH), 1,734 (carbonyl of
ester), 1,252 (N–N=C); 1H NMR (CDCl3) d: 2.19–2.23 (m,
2H, CH2), 2.59 (t, 2H, J = 7.1 Hz, CH2), 3.42 (t, 2H,
J = 7.1 Hz, SCH2), 3.68 (s, 3H, ester CH3), 3.81 (s, 3H,
OCH3), 6.92–6.96 (m, 1H, Ar–H), 7.02–7.07 (m, 1H, Ar–H),
7.13 (d, 1H, J = 8.2, 4.2 Hz, Ar–H), 7.56–7.61 (m, 1H,
Ar–H), 7.68 (d, 1H, J = 8.1 Hz, Ar–H), 8.28 (d, 1H,
J = 8.1 Hz, Ar–H), 8.47 (s, 1H, Ar–H); 13C NMR (CDCl3)
d: 24.1, 32.2, 33.3 (aliphatic side chain carbons), 51.8
(OCH3), 56.3 (OCH3), 112.5, 114.7, 117.2, 117.4, 125.2,
125.5, 127.2, 128.7, 131.1, 131.4 (aromatic ring carbons),
138.1 (C3-triazole ring), 152.6 (C5-triazole ring), 155.9 (d,
J = 237.1 Hz) (C50-Ar), 158.3 (C20-Ar), 159.3 (C=N-
thiadiazole ring), 172.6 (C=O); 19F NMR (CDCl3) d:
-123.2 (m, 1F); LCMS M?1: 459.
3-[20-fluoro-(1,10-biphenyl)-3-yl][1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole-6-thiol (3b)
Yield: 81 %, white solid, M.p. 189–191 °C; IR (KBr,
cm-1): 3,069 (aromatic CH), 2,355 (SH), 1,259 (N–N=C);
1H NMR (DMSO-d6) d: 3.21 (bs, 1H, NH), 7.03–7.10 (m,
2H, Ar–H), 7.19–7.29 (m, 3H, Ar–H), 7.47–7.61 (m, 2H,
Ar–H), 7.69 (s, 1H, Ar–H); 13C NMR (DMSO-d6) d: 113.1,
114.5, 115.6, 116.5, 117.9, 119.1, 121.3, 122.1, 122.8,
123.2, 135.6 (aromatic ring carbons), 141.2 (C3-triazole
ring), 153.1 (C5-triazole ring), 155.2 (d, J = 260.2 Hz)
(C20-Ar), 181.9 (C=S); 19F NMR (CDCl3) d: -122.8 (m,
1F); LCMS M?1: 329; Anal. Cacld for C15H9FN4S2: C,
54.86; H, 2.76; N, 17.06. Found: C, 54.81; H, 2.75; N,
17.01.
Methyl-4-{[3-(20-fluoro-(1,10-biphenyl)-3-
yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
yl]sulfanyl}butanoate (4b)
IR (KBr, cm-1): 3,051 (aromatic CH), 1,737 (carbonyl of
ester), 1,259 (N–N=C); 1H NMR (CDCl3) d: 2.17–2.21 (m,
2H, CH2), 2.51 (t, 2H, J = 7.2 Hz, CH2), 3.42 (t, 2H,
J = 7.2 Hz, SCH2), 3.66 (s, 3H, ester CH3), 7.03–7.09 (m,
3H, Ar–H), 7.19–7.29 (m, 2H, Ar–H), 7.45–7.59 (m, 2H,
Ar–H), 7.67 (s, 1H, Ar–H); 13C NMR (CDCl3) d: 25.1,
32.2, 34.3 (aliphatic side chain carbons), 51.2 (OCH3),
112.8, 113.5, 115.2, 116.5, 117.9, 119.6, 121.3, 122.4,
122.8, 123.2, 134.6 (aromatic ring carbons), 139.1 (C3-
triazole ring), 151.7 (C5-triazole ring), 155.4 (d,
J = 238.1 Hz) (C20-Ar), 159.4 (C=N-thiadiazole ring),
172.8 (C=O); 19F NMR (CDCl3) d: -123.8 (m, 1F); LCMS
M?1: 429.
General procedure for synthesis of compounds 4a–f
Conventional method
To a solution of 3a–b (1.39 mmol) in dry N,N-dimethyl-
formamide (5 mL), dry potassium carbonate (1.67 mmol)
and alkyl halide (1.67 mmol) were added and the resulting
solution was heated to 85 °C for 1 h. The solvent was
removed by distillation in vacuum and the residue was
taken in ethyl acetate (50 mL), washed with water
(20 mL), and dried over sodium sulfate. The resulting
solution was concentrated and purified by column chro-
matography [30–35 % ethyl acetate in petroleum ether] to
afford compounds 4a–f.
6-[(4-fluorobutyl)sulfanyl]-3-[50-fluoro-20-methoxy-(1,
10-biphenyl)-3-yl][1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
(4c)
Microwave method
A mixture of compounds 3a–b (1.39 mmol), dry N,N-
dimethylformamide (5 mL), dry potassium carbonate
(1.67 mmol), and alkyl halide (1.67 mmol) were subjected
to microwave irradiation 70 W at 50 °C for 5 min. The
solvent was removed by distillation in vacuum and the
residue was taken in ethyl acetate (50 mL), washed with
water (20 mL), and dried over sodium sulfate. The result-
ing solution was concentrated and purified by column
IR (KBr, cm-1): 3,056 (aromatic CH), 1,253 (N–N=C); 1H
NMR (CDCl3) d: 1.82–1.91 (m, 2H, CH2), 2.02–2.06 (m,
2H, CH2), 3.41 (t, 2H, J = 7.1 Hz, SCH2), 3.81 (s, 3H,
OCH3), 4.41 (t, 1H, J = 5.7 Hz, F–CH), 4.52 (t, 1H,
J = 5.8 Hz, F–CH), 6.92–6.96 (m, 1H, Ar–H), 7.02–7.05
(m, 1H, Ar–H), 7.13 (dd, 1H, J = 8.2, 4.2 Hz, Ar–H),
7.56–7.61 (m, 1H, Ar–H), 7.69 (d, 1H, J = 8.1 Hz, Ar–H),
8.28 (d, 1H, J = 8.1 Hz, Ar–H), 8.46 (s, 1H, Ar–H); 13C
123

Med Chem Res
NMR (CDCl3) d: 25.2, 29.2, 33.7 (aliphatic side chain
carbons), 56.3 (OCH3), 83.3 (d, J = 165.1 Hz) (CF-ali-
phatic), 112.5, 114.7, 117.1, 117.4, 125.2, 125.5, 127.2,
128.6, 131.1, 131.4 (aromatic ring carbons), 138.1 (C3-
triazole ring), 151.9 (C5-triazole ring), 152.7 (C20-Ar),
155.2 (d, J = 235.5 Hz) (C50-Ar), 159.4 (C=N-thiadiazole
ring); 19F NMR (CDCl3) d: -124.2 (m, 1F), -218.3
(m,1F); LCMS M?1: 433.
SCH2), 7.01–7.09 (m, 3H, Ar–H), 7.18–7.29 (m, 2H, Ar–H),
7.47–7.59 (m, 2H, Ar–H), 7.66 (s, 1H, Ar–H); 13C NMR
(CDCl3) d: 16.3, 25.5, 32.9 (aliphatic side chain carbons),
112.5, 113.8, 115.2, 116.7, 117.3, 117.7 (aromatic ring
carbons), 119.8 (CN), 122.3, 122.8, 123.2, 124.2, 134.6
(aromatic ring carbons), 139.5 (C3-triazole ring), 151.8
(C5-triazole ring), 155.8 (d, J = 238.1 Hz) (C20-Ar), 158.9
(C=N-thiadiazole ring); 19F NMR (CDCl3) d: -125.1
(m, 1F); LCMS M?1: 396.
3-[20-fluoro-(1,10-biphenyl)-3-yl]-6-[(4-
fluorobutyl)sulfanyl][1,2,4]triazolo[3,4-b][1,3,4]-
thiadiazole (4d)
General procedure for the synthesis of compounds 4g–h
A
mixture of compounds 3a–b (1.39 mmol), dry
IR (KBr, cm-1): 3,066 (aromatic CH), 1,252 (N–N=C); 1H
NMR (CDCl3) d: 1.83–1.91 (m, 2H, CH2), 2.04–2.07 (m,
2H, CH2), 3.44 (t, 2H, J = 7.1 Hz, SCH2), 4.42 (t, 1H,
J = 5.8 Hz, F–CH), 4.53 (t, 1H, J = 5.8 Hz, F–CH),
7.02–7.10 (m, 3H, Ar–H), 7.18–7.29 (m, 2H, Ar–H),
7.47–7.59 (m, 2H, Ar–H), 7.65 (s, 1H, Ar–H); 13C NMR
(CDCl3) d: 25.2, 29.2, 33.7 (aliphatic side chain carbons),
83.3 (d, J = 165.1 Hz) (CF-aliphatic), 112.2, 113.8, 115.2,
116.7, 116.9, 119.8, 122.3, 122.8, 123.2, 124.2, 134.6
(aromatic ring carbons), 139.5 (C3-triazole ring), 151.8
(C5-triazole ring), 155.5 (d, J = 239.1 Hz) (C20-Ar), 159.9
(C=N-thiadiazole ring); 19F NMR (CDCl3) d: -123.5 (m,
1F), -217.5 (m, 1F); LCMS M?1: 403.
N,N-dimethylformamide (5 mL), dry potassium carbonate
(0.23 g, 1.67 mmol), and 2-(3-bromopropyl)-1H-isoindole-
1,3(2H)-dione (1.67 mmol) were subjected to Microwave
irradiation 70 W at 50 °C for 5 min. The mixture was
concentrated and taken in ethyl acetate (50 mL), washed
with water (20 mL), dried over sodium sulfate, concen-
trated, and crude was dissolved in ethyl alcohol (4 mL);
hydrazine hydrate (4 mL) was added; and the reaction
mixture was stirred at room temperature for 16 h. The solid
was filtered and washed with methanol, the solvents were
concentrated, and resulting solid was purified by column
chromatography [2–3 % methanol in chloroform] to afford
compounds 4g–h.
4-{[3-(50-fluoro-20-methoxy-(1,10-biphenyl)-3-
yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-
yl]sulfanyl}butanenitrile (4e)
3-{[3-(50-fluoro-20-methoxy-(1,10-biphenyl)-3-yl)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl]
sulfanyl}propan-1-amine (4g)
1
IR (KBr, cm-1): 3,066 (aromatic CH), 2,246 (CN), 1,251
IR (KBr): 3,375, 3,118 (NH), 1,253 (N–N=C) cm-1; H
1
(N–N=C); H NMR (CDCl3) d: 2.25–2.32 (m, 2H, CH2),
NMR (DMSO-d6) d: 1.28 (bs, 2H, NH2), 1.99 (m, 2H,
CH2), 3.11 (t, 2H, J = 7.2 Hz, SCH2), 3.31 (t, 2H,
J = 7.2 Hz, NCH2), 3.84 (s, 3H, OCH3), 6.96–6.99 (m,
1H, Ar–H), 7.04–7.08 (m, 1H, Ar–H), 7.12 (dd, 1H,
J = 8.1, 4.2 Hz, Ar–H), 7.59–7.62 (m, 1H, Ar–H), 7.67 (d,
1H, J = 8.1 Hz, Ar–H), 8.27 (d, 1H, J = 8.2 Hz, Ar–H),
8.44 (s, 1H, Ar–H); 13C NMR (DMSO-d6) d: 29.2, 33.7,
38.3 (aliphatic side chain carbons), 56.3 (OCH3), 112.2,
114.5, 116.5, 117.4, 125.3, 125.4, 127.2, 128.6, 131.1,
131.4 (aromatic ring carbons), 138.3 (C3-triazole ring),
151.6 (C5-triazole ring), 152.5 (C20-Ar), 155.2 (d,
J = 238.5 Hz) (C50-Ar), 159.2 (C=N-thiadiazole ring); 19F
NMR (CDCl3) d: -125.2 (m, 1F); LCMS M?1: 416.
2.55 (t, 2H, J = 7.1 Hz, CH2), 3.51 (t, 2H, J = 7.2 Hz,
SCH2), 3.81(s, 3H, OCH3), 6.94–6.98 (m, 1H, Ar–H),
7.03–7.08 (m, 1H, Ar–H), 7.12 (dd, 1H, J = 8.2, 4.2 Hz,
Ar–H), 7.58–7.62 (m, 1H, Ar–H), 7.69 (d, 1H, J = 8.1 Hz,
Ar–H), 8.27 (d, 1H, J = 8.2 Hz, Ar–H), 8.45 (s, 1H, Ar–H);
13C NMR (CDCl3) d: 16.2, 25.1, 32.9 (aliphatic side chain
carbons), 56.2 (OCH3), 111.5, 114.2, 116.9, 117.4 (aro-
matic ring carbons), 118.2 (CN), 125.2, 125.3, 127.1,
127.6, 131.1, 131.8 (aromatic ring carbons), 138.7 (C3-
triazole ring), 152.3 (C5-triazole ring), 152.5 (C20-Ar),
156.1 (d, J = 238.5 Hz) (C50-Ar), 159.7 (C=N-thiadiazole
ring); 19F NMR (CDCl3) d: -124.3 (m, 1F); LCMS M?1:
426.
3-{[3-(20-fluoro-(1,10-biphenyl)-3-yl)[1,2,4]triazolo[3,
4-{[3-(20-fluoro-(1,10-biphenyl)-3-yl)[1,2,4]triazolo[3,
4-b][1,3,4]thiadiazol-6-yl]sulfanyl}-propan-1-amine (4h)
4-b][1,3,4]thiadiazol-6-yl]sulfanyl}-butanenitrile (4f)
1
IR (KBr): 3,371, 3,120 (NH), 1,249 (N–N=C) cm-1; H
IR (KBr, cm-1): 3,068 (aromatic CH), 2,247 (CN), 1,257
NMR (DMSO-d6) d: 1.28 (bs, 2H, NH2), 1.98 (m, 2H,
CH2), 3.13 (t, 2H, J = 7.2 Hz, SCH2), 3.33 (t, 2H,
J = 7.2 Hz, NCH2), 6.99–7.00 (m, 3H, Ar–H), 7.16–7.28
1
(N–N=C); H NMR (CDCl3) d: 2.24–2.31 (m, 2H, CH2),
2.56 (t, 2H, J = 7.3 Hz, CH2), 3.52 (t, 2H, J = 7.3 Hz,
123

Med Chem Res
(m, 2H, Ar–H), 7.47–7.59 (m, 2H, Ar–H), 7.65 (s, 1H, Ar–H);
13C NMR (DMSO-d6) d: 29.2, 33.7, 38.3 (aliphatic side
chain carbons), 111.8, 113.6, 115.1, 116.5, 117.1, 119.8,
122.3, 122.8, 123.4, 124.5, 134.6 (aromatic ring carbons),
139.7 (C3-triazole ring), 151.8 (C5-triazole ring), 155.9
(d, J = 238.1 Hz) (C20-Ar), 159.7 (C=N-thiadiazole ring);
19F NMR (CDCl3) d: -124.1 (m, 1F); LCMS M?1: 386.
6.98–7.00 (m, 3H, Ar–H), 7.15–7.27 (m, 2H, Ar–H),
7.47–7.59 (m, 2H, Ar–H), 7.65 (s, 1H, Ar–H); 13C NMR
(DMSO-d6) d: 32.2, 56.3 (aliphatic side chain carbons),
112.2, 113.5, 115.4, 117.2, 117.9, 119.8, 122.3, 122.9,
123.4, 124.8, 134.6 (aromatic ring carbons), 139.9 (C3-
triazole ring), 151.8 (C5-triazole ring), 154.9 (d,
J = 238.6 Hz) (C-20), 158.9 (C=N-thiadiazole ring); 19F
NMR (CDCl3) d: -125.2 (m, 1F); LCMS M?1: 373.
General procedure for the synthesis of compounds 4i–j
Biological activity
A mixture of compounds 3a–b (1.39 mmol), dry N,N-
dimethylformamide (5 mL), dry potassium carbonate
(1.67 mmol), and 2-(2-bromoethoxy)tetrahydro-2H-pyran
(1.67 mmol) were subjected to microwave irradiation
70 W at 50 °C for 5 min. The mixture was concentrated
and taken in ethyl acetate (50 mL), washed with water
(20 mL), dried over sodium sulfate, concentrated, and
dissolved in methanol (5 mL); 1 N HCl (5 mL) was added
at 0 °C; and the resulting mixture was stirred at room
temperature for 2 h. Methanol was concentrated and the
reaction mixture was neutralized with sodium bicarbonate
solution, and taken in ethyl acetate (50 mL), washed with
water (20 mL), saturated sodium chloride solution
(20 mL), and dried over sodium sulfate. The resulting
solution was concentrated and purified by column chro-
matography [1–2 % methanol in chloroform] to afford
compounds 4i–j.
Anticancer activity
Cells (HT29, K293, and MDA231) were seeded in 96-well
plates at a density of 2 9 105 cells per well for 12 h. Cells
were incubated with different concentrations (10, 25, 50,
100, and 200 lg/mL) of the test compounds for period of
72 h. Then, 50 lL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] was added, and plates were
incubated at 37 °C for 4 h. 150 lL of DMSO was added to
dissolve the formazan crystals. Absorbance of the colored
solution was measured spectrophotometrically at 570 nm
using ELISA micro plate reader (BIOTECH, USA). Three
independent experiments in triplicate were performed and
inhibitory concentration (IC) values were calculated as
follows:
%
inhibition = [(Control absorbance - Test absor-
bance)/Control absorbance] 9 100.
2-{[3-(50-fluoro-20-methoxy-(1,10-biphenyl)-3-yl)
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl]
sulfanyl}ethanol (4i)
IC50 value, defined as the drug concentration at which
50 % survival of cells, is listed in Table 2.
Acknowledgments The authors thank the Head, Syngene Interna-
tional Ltd. for laboratory facilities. The authors are also thankful to
Dr. Ashis Baran Mandal, Dr. Sandipan Sarkar, Dr. Sateesh Rai, and
Dr. Prashanth Latte for their valuable support. The authors are
thankful to Dr. Chandrashekhar, Department of Pharmacology, HSK
College of Pharmacy, Belgam for anticancer activities.
1
IR (KBr): 3,600 (OH), 1,255 (N–N=C) cm-1; H NMR
(DMSO-d6) d: 3.21 (t, 2H, J = 7.2 Hz, SCH2), 3.22 (t, 1H,
J = 7.8 Hz, OH), 3.64 (t, 2H, J = 7.1 Hz, OCH2), 3.82 (s,
3H, OCH3), 6.99–7.02 (m, 1H, Ar–H), 7.04–7.07 (m, 1H,
Ar–H), 7.12 (dd, 1H, J = 8.1, 4.2 Hz, Ar–H), 7.58–7.63
(m, 1H, Ar–H), 7.67 (d, 1H, J = 8.1 Hz, Ar–H), 8.28 (d,
1H, J = 8.2 Hz, Ar–H), 8.41 (s, 1H, Ar–H); 13C NMR
(DMSO-d6) d: 32.2, 56.3 (aliphatic side chain carbons),
59.2 (OCH3), 111.2, 113.5, 116.5, 118.4, 125.3, 125.4,
127.2, 128.3, 130.1, 131.4 (aromatic ring carbons), 138.5
(C3-triazole ring), 151.6 (C5-triazole ring), 153.5 (C20-Ar),
156.2 (d, J = 232.5 Hz) (C50-Ar), 158.2 (C=N-thiadiazole
ring); 19F NMR (CDCl3) d: -127.2 (m, 1F), LCMS M?1:
403.
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