Synthesis and cytotoxicity of 3,4-disubstituted-5-(3,4,5-trimethoxyphenyl)- 4H-1,2,4-triazoles and novel 5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4] thiadiazole derivatives bearing 3,4,5-trimethoxyphenyl moiety

Article abstract of DOI:10.1016/j.bmcl.2012.03.023

A series of 3,4-disubstituted-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazoles and some novel 5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles bearing 3,4,5-trimethoxyphenyl moiety were synthesized and screened for their anticancer activity. The preliminary bioassay results indicated that compounds 14 and 16 showed much stronger cytotoxicity than Doxorubicin against HepG2 cell lines with IC₅₀ values of 0.58 and 3.17 μM, respectively. Meanwhile compound 16 also exhibited a broad spectrum of antitumor activity against MCF-7 and MKN45 with IC₅₀ values of 10.92 and 13.79 μM, respectively.

Full text of DOI:10.1016/j.bmcl.2012.03.023

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4471–4474
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and cytotoxicity of 3,4-disubstituted-5-(3,4,5-trimethoxyphenyl)-4H-
1,2,4-triazoles and novel 5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole
derivatives bearing 3,4,5-trimethoxyphenyl moiety
⇑
⇑
Pei-Liang Zhao, An-Na Duan, Min Zou, Hai-Kui Yang, Wen-Wei You , Shu-Guang Wu
Department of Chemistry, College of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3,4-disubstituted-5-(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazoles and some novel 5,6-dihydro-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles bearing 3,4,5-trimethoxyphenyl moiety were synthesized and
screened for their anticancer activity. The preliminary bioassay results indicated that compounds 14
and 16 showed much stronger cytotoxicity than Doxorubicin against HepG2 cell lines with IC50 values
Received 18 January 2012
Revised 22 February 2012
Accepted 3 March 2012
Available online 11 March 2012
of 0.58 and 3.17
lM, respectively. Meanwhile compound 16 also exhibited a broad spectrum of antitumor
activity against MCF-7 and MKN45 with IC50 values of 10.92 and 13.79
l
M, respectively.
Keywords:
Ó 2012 Published by Elsevier Ltd.
5,6-Dihydro-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazole
Synthesis
Antitumor activity
In recent years, increasing attentions have been focused on the
1,2,4-triazoles and their fused heterocyclic derivatives owing to
their promising biological activities as well as great utility in pre-
parative organic chemistry.1–5 Especially some sulfur-containing
1,2,4-triazole derivatives always possess broad-spectrum biologi-
cal activities, such as herbicidal,6 insecticidal,7 antifungal,8 antimi-
crobial,9 antiviral,10 and anti-inflammatory properties.11 Recently
substituted 3-thio-1,2,4-triazoles have emerged as potential anti-
tumor agents inhibiting epidermal growth factor (EGFR) tyrosine
kinase.12
Therefore, as a part of our research work on the development of
novel bioactive nitrogen-containing heterocycle,19–21 we devel-
oped an idea that introducing 3-thio-1,2,4-triazole ring or fused
1,2,4-triazole ring as the locked cis-type bridge might also result
in new compounds with high cytotoxic activity (Fig. 1). In this
Letter, we described the design, synthesis, and in vitro cytotoxic
activities of
a series of 3,4-disubstituted-5-(3,4,5-trimethoxy-
phenyl)-4H-1,2,4-triazole 5–13 and some novel 5,6-dihydro-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives bearing 3,4,
5-trimethoxyphenyl moiety 14–16.
As a cis-stilbene natural product, combretastatin A-4 (CA-4, 1,
Fig. 1) with simple structure, has been identified as one of the most
potent anti-mitotic activity, which make it an attractive lead com-
pound in the development of new antitumor agents. The extensive
structure–activity relationship analysis revealed that both the
3,4,5-trimethoxy substitution pattern on the A-ring and the cis-
olefin configuration at the bridge were essential for optimal activ-
ity, while the B-ring is greater tolerance of structural modifica-
tions.13 However, the cis-stilbenes tend to isomerize to the more
stable trans-forms which show dramatic reduction in cytotoxic
activities.14,15 By replacement of the double bond bridge of CA-4
with a rigid triazole ring, compounds 2, 3 and 4 (Fig. 1) have been
found to exhibit potent anticancer activities comparable with
CA-4.16–18
Our synthetic strategy for the target compounds is illustrated in
Scheme 1. Starting from 3,4,5-trimethoxybenzoic acid, according to
the reported method,22 the key intermediate in the present study
4-amino-3-(3,4,5-trimethoxy phenyl)-1H-1,2,4-triazole-5(4H)-thi-
one III was synthesized in four steps employing esterification,
hydrazidation, salt formation, and cyclization. Subsequently, the
compound III was converted into their corresponding Schiff base
IV by condensation reaction with commercially purchased substi-
tuted benzaldehyde. Finally, compound IV reacted with various
alkylation reagents to afford the target compounds 5–13 in yields
of 64–89%. The structures of title compounds 5–13 were elucidated
by comprehensive 1H NMR, EI-MS and elemental analysis, and all
the analytical data were documented in the Supplementary data.
In addition, the crystal structure of 13 was determined by X-ray dif-
fraction analyses (Fig. 2), which revealed that the carbon–nitron
double bond in the molecule was of E-configuration.23 Most inter-
estingly, using phenacyl bromide or ethyl 2-bromoacetate as
alkylation reagents, some novel 3,6,6-trisubstituted-5,6-dihydro
⇑
Corresponding authors. Tel./fax: +86 (0)20 61648196 (S.-G.W.).
E-mail addresses: youww@smu.edu.cn (W.-W. You), shuguang@smu.edu.cn
(S.-G. Wu).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.03.023

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P.-L. Zhao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4471–4474
N
N
O
O
O
O
N
N
N
N
N
O
O
A
O
O
O
N
N
B
O
O
OH
O
R
O
R
N
4
1 (CA-4)
2
3
N
N
N
N
R2
O
O
O
S
N
N
HN
S
+
O
N
R2
R1
O
R1
5~13
Figure 1. Design strategy of the target compounds 5–16.
O
14~16
O
O
O
O
O
O
O
O
O
O
O
O
a
b
S
c
NHNHCS-K+
O
NHNH2
OH
O
O
O
O
Ⅱ
Ⅰ
N
N
R2
S
O
f
N
N
O
N
NH
N
NH
O
R1
N
O
O
O
O
S
e
d
S
N
NH2
5~13
N
N
N
O
O
O
R1
N
HN
S
g
O
R2
Ⅳ
Ⅲ
R1
O
14~16
Scheme 1. General synthetic route for compounds 5–16. Reagents and conditions: (a) concd. H2SO4, ethanol, reflux; (b) 60%NH2H2ÁH2O, ethanol, reflux; (c) KOH, CS2, ethanol,
rt; (d) 60%NH2NH2ÁH2O, H2O, reflux, HAc; (e) R1CHO, HAc, reflux; (f) K2CO3, acetone, substituted benzyl bromides, ethyl 2-bromoacetate or methyl iodide, reflux; (g) K2CO3
acetone, phenacyl bromide or ethyl 2-bromoacetate, reflux.
derivatives of 1,2,4-triazolo[3,4-b]1,3,4-thiadiazole 14–16 were ob-
tained in good yields, and their structures were fully characterized
by 1H NMR, 13C NMR, EI-MS and elemental analysis.24 Further opti-
mization of reaction conditions and study of the reaction mecha-
nism are well under way.
The cytotoxicity of all the title compounds 5–16 against four
human cancer cell lines, including HepG2 (hepatocellular carci-
noma cell line), MKN45 (gastric carcinoma cell line), MCF-7 (breast
cancer cell line) and HT-29 (human colon carcinoma cell line),
were evaluated by MTT assay.25 Doxorubicin was used as the refer-
MKN45 with the IC50 values of 0.58 and 7.47 lM, respectively.
Interestingly, compound 16 exhibited a broad spectrum of antitu-
mor activity against HepG2, MCF-7 and MKN45 with IC50 values of
3.17, 10.92 and 13.79 lM, respectively.
Further analysis on the structure–activity relationship revealed
that, in most cases, 1,2,4-triazole derivatives 5–13 did not display
high activity against the four tested human cancer cell lines, while
most 5,6-dihydro-1,2,4-triazolo[3,4-b]1,3,4-thiadiazole derivatives
showed highly potent cytotoxic activity. These results indicated
that iminoacyl moiety, on the 4-position of 1,2,4-triazole ring, is
too flexible to keep in cis-orientation between the aryl ring and
3,4,5-trimethoxyphenyl ring, which can be verified by the crystal
structure of 13. Nevertheless, within the series of fused 1,2,4-tria-
zole compounds, anticancer activities were influenced by the C-6
substituents on the 5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadi-
azole ring and phenacyl substitution always gave higher activities
(14 and 16). Replacement of phenacyl group with ester group led
ence drug and the results expressed as IC50 (lM) were summarized
in Table 1. As the results indicated that some compounds showed
excellent antiproliferative activity against selected human tumor
cell lines. Among them, two representative compounds 14 and
16 displayed much higher antitumor activity against HepG2 cell
lines than positive control Doxorubicin. In particular, the com-
pound 14 showed the most active in inhibiting HepG2 and

P.-L. Zhao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4471–4474
4473
Figure 2. X-ray crystal structures of compound 13 (CCDC 820019).
Table 1
In vitro cytotoxicity (IC50
a
,
l
M) of synthesized compounds 5–13 and 14–16
N
N
N
N
R2
O
O
O
O
S
N
N
N
HN
S
R2
R1
O
O
R1
14~16
5~13
Compd
R1
R2
Cytotoxicity
HepG2
MCF-7
MKN45
HT-29
5
6
7
8
9
10
11
12
13
14
15
16
C6H5
C6H5
C6H5
C6H5
2,4-(MeO)2C6H3
2,4-(MeO)2C6H3
2,4-(MeO)2C6H3
2,4-(MeO)2C6H3
2,4-(MeO)2C6H3
C6H5
Me
C6H5CH2
4-FC6H4CH2
2-ClC6H4CH2
Me
4-FC6H4CH2
2-ClC6H4CH2
C6H5CH2
>200
>200
>200
31.72
>200
>200
>200
60.94
>200
0.58
>200
>200
>200
>200
>200
>200
63.72
152.59
>200
>200
>200
10.92
17.67
28.50
>200
>200
>200
>200
>200
>200
49.00
>200
7.47
>200
>200
>200
>200
143.87
126.40
>200
>200
>200
EtCO2CH2
4-MeC6H4COCH2
EtCO2CH2
150.79
>200
C6H5
2,4-(MeO)2C6H3
>200
3.17
5.57
>200
13.79
0.168
4-MeC6H4COCH2
66.81
b
Doxorubicin
—
a
IC50 values are presented as mean values of three independent experiments done in quadruplicates. Coefficients of variation were <10%.
Not tested.
b
to a dramatic decrease in antitumor activity (15). It can be con-
preliminary bioassay showed that compound 14 and 16 exhibited
much stronger cytotoxicity than Doxorubicin against HepG2 cell
lines. Meanwhile compound 16 also displayed potent and broad-
spectrum antitumor activity against MCF-7 and MKN45. These
results indicated that 5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thi-
adiazoles could be used as a promising lead for further developing
cluded that phenacyl group at the 6-position of the fused 1,2,4-tri-
azoles play a crucial role in modulating the antitumor activity.
In conclusion, we synthesized a series of 3,4-disubstituted-5-
(3,4,5-trimethoxyphenyl)-4H-1,2,4-triazoles and three novel 5,6-
dihydro-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives. The

4474
P.-L. Zhao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4471–4474
22. Sahoo, P. K.; Sharma, R.; Pattanayak, P. Med. Chem. Res. 2010, 19, 127.
23. Crystallographic data for the structural analysis have been deposited at the
Cambridge Crystallographic Data Centre, CCDC No. 820019. (E-mail:
deposit@ccdc.cam.ac.uk).
new antitumor agents. Further structural optimization and the
mechanism of reaction in detail will be studied in our laboratory.
24. 2-(6-Phenyl-3-(3,4,5-trimethoxyphenyl)-5,6-dihydro-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazol-6-yl)-1-p-tolylethanone (14): Following the general procedure
described in the Supplementary data, compound 14 was purified by
recrystallization from acetone. White solid, yield: 78%. Mp: 191–192 °C. 1H
NMR (400 MHz, CDCl3) d: 2.41 (s, 3H, SCH3), 3.80 (s, 6H, 2 Â OCH3), 3.86 (s, 3H,
OCH3), 5.14 (d, J = 4.4 Hz, 1H, SCH), 5.29 (d, J = 2.8 Hz, 1H, SCH), 6.54 (s, 1H,
NH), 7.24–7.31 (m, 5H, ArH), 7.36 (s, 2H, ArH), 7.45 (t, J = 4.0 Hz, 2H, ArH), 7.80
(d, J = 8.0 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3) d: 21.8, 41.6, 56.1, 59.1, 60.9,
105.0, 121.3, 127.2, 128.8, 128.9, 129.1, 129.9, 131.6, 136.0, 139.5, 143.0, 145.9,
152.2, 153.1, 194.5. ESI MS: m/z = 525.22 [M+Na]+, 503.14 [M+1]+. Anal. Calcd
for C27H26N4O4S: C, 64.52; H, 5.21; N, 11.15; S, 6.38. Found: C, 64.37; H, 5.43;
N, 10.94; S, 6.12.
Acknowledgments
This work was financially supported by the National Natural
Science Foundation of China (No. 21102069), Natural Science
Foundation of Guangdong Province (10451051501004725) and
the Specialized Research Fund for the Doctoral Program of Higher
Education (New Teachers) (No. 20104433120011).
Supplementary data
Ethyl
2-(6-phenyl-3-(3,4,5-trimethoxyphenyl)-5,6-dihydro-[1,2,4]triazolo[3,4-
b][1,3,4]thiadiazol-6-yl) acetate (15): Following the general procedure
described in the Supplementary data, compound 15 was purified by
recrystallation from acetone. White solid, yield: 67%. Mp: 145–146 °C. 1H
NMR (400 MHz, CDCl3) d: 1.27 (t, J = 7.0 Hz, 3H, OCH2CH3), 3.83 (s, 6H,
2 Â OCH3), 3.87 (s, 3H, OCH3), 4.21–4.29 (m, 2H, OCH2CH3), 4.38 (d, J = 4.0 Hz,
1H, SCH), 4.96 (t, J = 4.0 Hz, 1H, SCH), 6.38 (s, 1H, NH), 7.29 (d, J = 3.0 Hz,
J = 7.0 Hz, 3H, ArH), 7.36 (d, J = 5.6 Hz, 4H, ArH). 13C NMR (100 MHz, CDCl3) d:
13.8, 41.0, 56.1, 58.8, 60.9, 62.7, 104.9, 121.2, 126.9, 128.9, 129.0, 134.9, 139.4,
142.3, 152.0, 153.0, 170.4. ESI MS: m/z = 457.11 [M+1]+. Anal. Calcd for
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.
023.
References and notes
1. Wang, L. Y.; Tseng, W. C.; Wu, T. S.; Kaneko, K.; Takayama, H.; Kimura, M.; Yang,
W. C.; Wu, J. B.; Juang, S. H.; Wong, F. F. Bioorg. Med. Chem. Lett. 2011, 21, 5358.
2. Thottempudi, V.; Shreeve, J. M. J. Am. Chem. Soc. 2011, 133, 19982.
3. Papadopoulou, M. V.; Trunz, B. B.; Bloomer, W. D.; McKenzie, C.; Wilkinson, S.
R.; Prasittichai, C.; Brun, R.; Kaiser, M.; Torreele, E. J. Med. Chem. 2011, 54, 8214.
4. Moulin, A.; Demange, L.; Ryan, J.; Mousseaux, D.; Sanchez, P.; Bergé, G.; Gagne,
D.; Perrissoud, D.; Locatelli, V.; Torsello, A.; Galleyrand, J. C.; Fehrentz, J. A.;
Martinez, J. J. Med. Chem. 2008, 51, 689.
C
22H24N4O5S: C, 57.88; H, 5.30; N, 12.27; S, 7.02. Found: C, 57.61; H, 5.49; N,
11.98; S, 6.86.
2-(6-(2,4-Dimethoxyphenyl)-3-(3,4,5-trimethoxyphenyl)-5,6-dihydro-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-6-yl)-1-p-tolylethanone (16): Following the general
procedure described in the Supplementary data, compound 16 was purified
by recrystallation from acetone. White solid, yield: 72%. Mp: 184–185 °C. 1H
NMR (400 MHz, CDCl3) d: 2.35 (s, 3H, Ar –CH3), 3.70 (s, 3H, OCH3), 3.72 (s, 6H,
2 Â OCH3), 3.80 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 5.04 (dd, J = 6.4 Hz, J = 8.4 Hz,
1H, SCH), 5.36 (d, J = 6.4 Hz, 1H, SCH), 6.28–6.33 (m, 2H, ArH), 6.41 (d,
J = 2.0 Hz, 1H, NH), 7.08 (d, J = 8.4 Hz, 1H, ArH), 7.18 (d, J = 8.0 Hz, 2H, ArH),
7.43 (s, 2H, ArH), 7.71 (d, J = 8.4 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3) d:
21.6, 42.7, 55.3, 55.5, 55.9, 57.9, 60.7, 99.0, 104.5, 104.8, 117.0, 121.5, 128.6,
129.5, 129.7, 131.9, 139.2, 143.9, 145.3, 151.6, 152.9, 157.5, 161.0, 194.0. ESI
MS: m/z = 563.23 [M+1]+. Anal. Calcd for C29H30N4O6S: C, 61.91; H, 5.37; N,
9.96; S, 5.70. Found: C, 61.83; H, 5.14; N, 10.08; S, 5.54.
5. Nakamura, T.; Takegami, A.; Abe, M. J. Org. Chem. 2010, 75, 1956.
6. Wang, B. L.; Shi, Y. X.; Ma, Y.; Liu, X. H.; Li, Y. H.; Song, H. B.; Li, B. J.; Li, Z. M. J.
Agric. Food Chem. 2010, 58, 5515.
7. Hull, J. W., Jr.; Romer, D. R.; Adaway, T. J.; Podhorez, D. E. Org. Process Res. Dev.
2009, 13, 1125.
8. Ezabadi, I. R.; Camoutsis, C.; Zoumpoulakis, P.; Geronikaki, A.; Sokovic´, M.;
Glamocilija, J.; Ciric´, A. Bioorg. Med. Chem. 2008, 16, 1150.
9. Pardeshi, S.; Bobade, V. D. Bioorg. Med. Chem. Lett. 2011, 21, 6559.
10. Upmanyu, N.; Gupta, J. K.; Shah, K.; Mishra, P. J. Pharm. Bioallied Sci. 2011, 3,
259.
11. Navidpour, L.; Shafaroodi, H.; Abdi, K.; Amini, M.; Ghahremani, M. H.; Dehpour,
A. R.; Shafiee, A. Bioorg. Med. Chem. 2006, 14, 2507.
12. Abou-Seri, S. M. Eur. J. Med. Chem. 2010, 45, 4113.
13. Romagnoli, R.; Baraldi, P. G.; Cruz-Lopez, O.; Lopez-Cara, C.; Carrion, M. D.;
Brancale, A.; Hamel, E.; Chen, L.; Bortolozzi, R.; Basso, G.; Viola, G. J. Med. Chem.
2010, 53, 4248.
14. Odlo, K.; Hentzen, J.; Chabert, J. F.; Ducki, S.; Gani, A. B. S. M. O.; Sylte, I.; Skrede,
M.; Flenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16, 4829.
15. Liu, T.; Dong, X. W.; Xue, N.; Wu, R.; He, Q. J.; Yang, B.; Hua, Y. Z. Bioorg. Med.
Chem. 2009, 17, 6279.
16. Zhang, Q.; Peng, Y.; Wang, X. I.; Keenan, S. M.; Arora, S.; Welsh, W. J. J. Med.
Chem. 2007, 50, 749.
17. Wu, M. J.; Sun, Q. M.; Yang, C. H.; Chen, D. D.; Ding, J.; Chen, Y.; Lin, L. P.; Xie, Y.
Y. Bioorg. Med. Chem. Lett. 2007, 17, 869.
25. Evaluation of cytotoxicity (MTT): The antitumor activity of compounds 5–16
were evaluated with HepG2, MCF-7, MKN45 and HT-29 cell lines by the
standard MTT assay in vitro. The cancer cell lines were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS). Cells
were passaged at 70–80% confluence, about twice a week by trypsinization.
Exponentially growing cells were plated in 96-well plates (8 Â 104 cells/well
for MKN45, HT-29 and MCF-7 cells, 5 Â 104 cells/well for HepG2 cells) and
incubated at 37 °C for 24 h for attachment. Test compounds were prepared by
dissolving in dimethyl sulfoxide (DMSO) at 10 mM and diluted with the
medium. Then, culture medium was changed, and cells grew in medium with
the test compounds. DMSO (0.1%) was used as negative control. Cells were
incubated at 37 °C for 48 h. Then the medium was replaced with MTT solution
(5 mg/ml, 100
lL) followed by incubation for another 3 h. The medium was
then aspirated and formazan crystals were dissolved in DMSO (100
l
L) for
about 10 min. The absorbance at 570 nm (Abs) of the suspension was
measured by an enzyme-linked immunosorbent assay (ELISA) reader. The
18. Akselsen, O. W.; Odlo, K.; Cheng, J. J.; Maccari, G.; Botta, M.; Hansen, T. V.
Bioorg. Med. Chem. 2012, 20, 234.
19. Zhao, P. L.; Wang, L.; Zhu, X. L.; Huang, X.; Zhan, C. G.; Wu, J. W.; Yang, G. F. J.
Am. Chem. Soc. 2010, 132, 185.
20. Zhao, P. L.; Wang, F.; Zhang, M. Z.; Liu, Z. M.; Huang, W.; Yang, G. F. J. Agric. Food
Chem. 2008, 56, 10767.
inhibition percentage was calculated using the following formula:
%
inhibition = (Abscontrol À Abscompound)/Abscontrol  100%. The IC50 values of the
test compounds and Doxorubicin were measured by treating cells with drugs
of varying concentrations, and analyzing by use of the prism statistical package
(GraphPad Software, San Diego, CA, USA).
21. Zhao, P. L.; Wang, F.; Huang, W.; Chen, Q.; Liu, Z. M. Chin. J. Org. Chem. 2010, 30,
1567.