Derivatives of benzothiadiazole-7-carboxylates: Synthesis and biological activity

Article abstract of DOI:10.1007/s00706-008-0887-3

Salicylic acid (SA) and methyl jasmonate (MJ) are important plant signal molecules to cause systemic acquired resistance (SAR), while it's reported that they also have wide spectrum antitumor activities. Benzothiadiazole-7- carboxylates are plant activators which can cause SAR just like SA and MJ. To investigate whether the benzothiadiazole-7-carboxylate family is endowed with anticancer activities, several benzothiadiazole-7-carboxylate derivatives are synthesized and their inhibition to P388 murine leukemia cell and A549 human lung cancer cell compared with MJ are evaluated. The data indicated that benzo-1,2,3-thiadiazole-7-carboxylic acid 2-benzoyloxyethyl ester has a higher inhibition ability to the cancer cell P388 and A549, compared with MJ.

Full text of DOI:10.1007/s00706-008-0887-3

Monatsh Chem 139, 1067–1071 (2008)
DOI 10.1007/s00706-008-0887-3
Printed in The Netherlands
Derivatives of benzothiadiazole-7-carboxylates: synthesis
and biological activity
Weiping Zhu1;2, Zhenjiang Zhao1;2, Yufang Xu1;2
1
Shanghai Key Lab of Chemical Biology, Shanghai, China
2
School of Pharmacy, East China University of Science and Technology, Shanghai, China
Received 19 December 2007; Accepted 8 January 2008; Published online 18 February 2008
# Springer-Verlag 2008
Abstract Salicylic acid (SA) and methyl jasmonate
(MJ) are important plant signal molecules to cause
systemic acquired resistance (SAR), while it’s re-
ported that they also have wide spectrum antitumor
activities. Benzothiadiazole-7-carboxylates are plant
activators which can cause SAR just like SA and MJ.
To investigate whether the benzothiadiazole-7-car-
boxylate family is endowed with anticancer activities,
several benzothiadiazole-7-carboxylate derivatives
are synthesized and their inhibition to P388 murine
leukemia cell and A549 human lung cancer cell com-
pared with MJ are evaluated. The data indicated that
benzo-1,2,3-thiadiazole-7-carboxylic acid 2-benzo-
yloxyethyl ester has a higher inhibition ability to the
cancer cell P388 and A549, compared with MJ.
rial, and viral pathogens and provides a signal for
expression of pathogenesis-related proteins and oth-
er potentially protective factors induced following
pathogen challenge [1]. Jasmonic acid (JA) and
methyl jasmonate (MJ), also a kind of plant hormone
and produced by the octadecanoid pathway via
lipoxygenation of linolenic acid, serve as a signal
for expression of a number of proteins inducing
polyphenoloxidase and proteinase inhibitors [2–4].
SA and MJ are well known important plant signal
molecules which can cause systemic acquired resis-
tance (SAR) to pathogens and injury and they can
elicit secondary metabolism in Taxus cell cultures
[5–8]. S-Methyl benzo-1,2,3-thiadiazole-7-carboxyl-
ate (BTH), the potent synthetic salicylate mimic [9–
11], has been invented by Ciba-Geigy Co. As the
first commercial SAR activator for plants. It is no-
ticed that BTH could induce the same set of defense
response as salicylic acid in signal transduction.
Recently, we have reported that the synthetic benzo-
1,2,3-thiadiazole-7-carboxylates could be used for
eliciting taxoid biosynthesis and resulted in nearly
40% increase in taxuyunnanine C content and pro-
duction in comparison with methyl jasmonate, which
was previously reported as the most powerful chem-
ical elicitor for taxoid biosynthesis [12] Scheme 1.
SA has been reported to be able to induce intracel-
lular biochemical events typical of a stress response
in mammalian cells as well and induce apoptosis
(programmed cell death) in human myeloid leuke-
Keywords Jasmonate; Benzothiadiazole-7-carboxylates;
Hydrogen peroxide; Biological activity.
Introduction
Plant stress hormones are defined as a group of nat-
urally occurring organic substances, which influ-
ence physiological processes at low concentrations.
Salicylic acid (SA), a kind of plant hormone, is a key
compound that regulates resistance of fungal, bacte-
Correspondence: Yufang Xu, School of Pharmacy, East China
University of Science and Technology, Shanghai 200237,
China. E-mail: yfxu@ecust.edu.cn

1068
W. Zhu et al.
Scheme 1
atives may be effective to some mammalian cancer
cells. To verify this inference, several benzothia-
diazole-7-carboxylate derivatives (Scheme 2) were
synthesized and their cytotoxicity to P388 murine
leukemia cell and A549 human lung cancer cell
are evaluated.
Results and Discussion
Scheme 2
Benzothiadiazole-7-carboxylate derivatives are pre-
pared from 2-chlorobenzonic acid by nitration, sub-
stitution of Cl by benzyl mercaptan, then reduction
and diazotization and cyclization [20]. The series of
compounds are then prepared from benzothiadia-
zole-7-carboxylic acid chloride and the correspond-
ing alcohol or amine (Scheme 3) [10] and purified
by recrystallization and silica gel chromatography.
Their structures were confirmed with HNMR, HR-
MS, and elemental analyses.
Compound 1b was prepared by acylation of ben-
zothiadiazole. However, esterification of benzothia-
mia, FS-4 fibroblasts and pancreatic cancer [13].
Recently, Flescher et al. reported that jasmonates
induce suppression of cellular proliferation and
death in various human and mouse cancer lines, in-
cluding breast, prostate, melanoma, lymphoblastic
leukemia, and lymphoma cells [14–19]. As the mim-
ic of SA, BTH has some similar biological activities
in plant cells in comparison with MJ and there
are some similarities between jasmonate actions in
plants cells, we hypothesized that BTH and its deriv-
1
Scheme 3

Derivatives of benzothiadiazole-7-carboxylates
1069
diazole-7-carboxylic acid with trifluoroethanol did
not work in the preparation of 1c under the same
conditions because of the acidity of trifluoroethanol,
so condensation of benzothiadiazole-7-carboxylic
acid chloride and trifluoroethanol in the presence
of triethylamine was adopted. In the process of pre-
paring 1e, the protection of hydroxyl group with
acetic anhydride was not required. Because of steric
factors and intramolecular hydrogen bond, the free
phenolic hydroxyl group would not participate in
the reaction as long as no excess acid chloride was
added.
The in vitro antitumor activities of these com-
pounds were evaluated by MTT tetrazolium dye as-
say against P388 (murine leukemia cell) (Table 1)
and Sulforhodamine B (SRB) assay against A549
(human lung cancer cell) (Table 2).
As shown in Tables 1 and 2, compounds 1a–1e
show some ability for cancer cell inhibition, espe-
cially 1e has a higher inhibition ability than MJ.
Actually, 1e was found to induce plant defense
responses as effectively as MJ, and it could be used
for eliciting taxoid biosynthesis and resulted in near-
ly 40% increase in taxuyunnanine C content and
production in comparison with MJ [12].
It is well-known that jasmonates are plant lipid
derivatives, similar to mammalian eicosanoid, that
play a critical role in plant defenses against her-
bivores and pathogens through up-regulating the
expression of defense-related genes. Recently, jas-
monates were shown to induce cell cycle arrest or
apoptosis in human leukemia, prostate and breast can-
cer cells, but not in normal lymphocytes. Kim et al.
reported that MJ induces apoptosis through induction
of Bax=Bcl-XS and activation of caspase-3 via hy-
drogen peroxide (H2O2) generation in A549 human
lung adenocarcinoma cells [21]. Flescher et al. even
thought that jasmonates can induce similar metabolic,
signaling, and stress-associated modifications (that
could lead to decreased growth and viability) in plant
cells as well as in cancerous animal cells [19]. In
order to find some biological evidences for the above
results, MJ and 1e were selected to demonstrate as
typical examples the elicitor-induced plant defense
responses, while the cultures with non-elicitor addi-
tion was used as controls (Fig. 1).
Figure 1 shows that an increased level of H2O2
production was observed in MJ- and 1e-elicited T.
chinensis suspension cultures. It can be seen that the
time course of H2O2 production of 1e-elicited cells
was almost the same as that of MJ-treated cells and
in the first hour the H2O2 production of 1e-elicited
cells was higher than that of MJ-treated cells, which
is in accordance with the cancer cell inhibition abil-
ity shown in Tables 1 and 2.
To the best of our knowledge, benzothiadiazole-7-
carboxylates were never studied as antitumor agents.
The mechanism of plant activator is to activate the
Table 1 Inhibition ratio to P388 %
Compound 10ꢀ4M 10ꢀ5
M
10ꢀ6
M
10ꢀ7
M
10ꢀ8
M
MJ
1a
1b
1c
1d
1e
8.6
67.0
44.9
54.0
44.5
85.8
5.7
0
9.3
11.4
0
14.4
0
9.9
6.9
0
10.2
0
0
1.0
0
8.0
8.6
0
0
0
0
9.2
14.2
11.4
Table 2 Inhibition ratio to A549 %
Compound 10ꢀ4
M
10ꢀ5
M
10ꢀ6
M
10ꢀ7
M
10ꢀ8
M
Fig. 1 Methyl jasmonate (MJ) and 1e elicitor-induced H2O2
production in T. chinensis suspension cultures. MJ or 1e (at
100 mM) was added to the cultures in 1 mm3 ethanol per
1 cm3 culture medium on day 7 of cultivation and elicited
cell cultures were sampled at certain intervals for H2O2 anal-
ysis. Data are the means of three flasks and vertical bars
show standard deviations.
MJ
1a
1b
1c
1d
1e
23.9
8.9
37.8
28.3
34.9
41.0
11.9
0
0
0
0
13.6
28.1
29.9
23.7
22.2
0
28.8
20.9
19.1
17.5
0
19.0
7.0
6.3
6.9
0
15.5
29.9
22.2

1070
W. Zhu et al.
ppm; IR (KBr): ꢁꢀ¼ 3000, 1625, 1540, 1470, 1398, 1287,
1071, 908, 780, 730 cmꢀ1; HRMS (EI): m=z (%) ¼ 209.9920
[Mþ], C8H6N2OS2 requires 209.9922.
plants’ own immune system which is called SAR,
they do not have direct antimicrobial activity, and
work by inducing resistance to the same spectrum
of pathogens and lead to expression of the same
biochemical markers, such as the PR-proteins in
the plant [22]. They usually have lower cytotoxicity,
and the same function mechanism as SA and MJ to
some extent. So, as one kind of unnatural plant stress
hormones, benzothiadiazole-7-carboxylates may be
further evaluated as novel anticancer agents, and it
is much easier to synthesize these compounds than
to synthesize methyl jasmonate derivatives [23–27].
2,2,2-Trifluoroethyl benzo-1,2,3-thiadiazole-7-carboxylate
(1c, C9H5F3N2O2S)
Light yellow crystals; mp 119–121ꢂC; 1HNMR (500MHz,
DMSO-d6): ꢀ ¼ 5.16 (q, J ¼ 9.0 Hz, CH2CF3), 7.98 (dd, J ¼
8.35, 7.36 Hz, 5-Ar-H), 8.49 (d, J ¼ 7.37Hz, 4-Ar-H), 9.10 (d,
J ¼ 8.35 Hz, 6-Ar-H) ppm; IR (KBr): ꢁꢀ¼ 3074, 2978, 1718,
1558, 1410, 1305, 1180, 1135, 1050, 980, 820, 753 cmꢀ1
;
HRMS (EI): m=z (%) ¼ 262.0026 [Mþ], C9H5F3N2O2S re-
quires 262.0024.
N-(2-(Dimethylamino)ethyl)benzo-1,2,3-thiadiazole-7-
carboxamide (1d, C11H14N4OS)
Experimental
White solid; mp 172–174ꢂC; 1HNMR (500MHz, DMSO-d6):
ꢀ ¼ 2.18 (s, N(CH3)2), 2.46(t, J ¼ 6.80 Hz, CH2), 3.47 (dt,
J1 ¼ 6.79, J2 ¼ 5.37 Hz, CH2), 7.92(dd, J1 ¼ 8.21Hz, J2 ¼
7.36Hz, Ar-H-5), 8.47(d, J ¼ 7.35Hz, Ar-H-4), 8.89(d, J ¼
8.21Hz, Ar-H-6), 9.14(t, NH, J ¼ 5.37Hz) ppm; IR (KBr):
ꢁꢀ¼ 3563(NH), 3259, 2948, 1636, 1540, 1465, 1313, 1170,
1050, 940, 770cmꢀ1; HRMS (EI): m=z (%) ¼ 250.0884 [Mþ]
C11H14N4OS requires 250.0888.
Triethylamine was dried over KOH and distilled. Methyl jas-
monate was purchased from Tokyo Kasei Kogyo Co., Ltd
(Tokyo, Japan). Trifluoroethanol was purchased from Fluka
Chem. Co. (Switzerland). All other solvents and chemicals
were reagent grade and used without further purification.
Melting points were recorded by an electrothermal digital
apparatus. The 1HNMR spectra were recorded with a Brucker
AM-500 spectrometer. The MS spectra were measured with a
HR-MS Micromass GCT CA 055 spectrometer. Elemental
analyses (C, H, and N) were conducted using the Elemental
Vario EL III Analyzer, their results were found to be in good
agreement (ꢁ0.3%) with the calculated values. Compound 1a
(methyl benzo-1,2,3-thiadiazole-7-carboxylate), benzo-1,2,3-
thiadiazole-7-carboxylic acid, and benzo-1,2,3-thiadiazole-7-
carboxylic acid chloride were prepared according to Ref. [19].
H2O2 produced by the cells and released into the medium
was determined by the scopoletin fluorescence oxidative
quenching method (excitation wavelength: 350 nm, emission:
460 nm) according to Ref. [28]. To measure H2O2 accumula-
tion, samples were taken at various intervals over the 180-min
period following elicitation. Aliquots of 4 cm3 extracellular
medium were mixed with 40 mm3 5 mM stock solution of
scopoletin in DMSO and 40 mm3 1 mg=cm3 stock solution
of peroxidase (Sino-American Biotechnology Co., Shanghai).
The concentration of H2O2 in the medium was calculated from
the fluorescence decrease using a calibration curve established
in the presence of H2O2. A standard curve by adding scopo-
letin to the solutions at different H2O2 concentrations was
prepared by using cell-free medium. Effects of various jasmo-
nate elicitors on peroxidase-dependent assay for H2O2 de-
termination were tested. Various jasmonates were added to
cell-free medium to obtain final concentrations of 10, 50, or
100 mM. Under the assay conditions, an addition of jasmonate
elicitors had no obvious effect on the decrease of scopoletin
fluorescence because of H2O2 addition.
Benzo-1,2,3-thiadiazole-7-carboxylic acid 2-benzoyloxyethyl
ester (1e, C16H12N2O5S)
White crystals; mp 92–94ꢂC; 1HNMR (500MHz, DMSO-d6):
ꢀ ¼ 4.71–4.80 (m, OCH2-CH2O), 6.88–6.91 (m, Ar-H), 6.95–
6.97 (m, Ar-H), 7.48–7.51 (m, Ar-H), 7.77–7.80 (m, Ar-H),
7.94 (dd, J ¼ 7.34 and 8.34Hz, 5-Ar-H), 8.45 (d, J ¼ 7.33Hz,
4-Ar-H), 9.04 (d, J ¼ 8.34 Hz, 6-Ar-H), 10.38 (s, OH) ppm; IR
(KBr): ꢁꢀ¼ 3280 (OH), 3059, 2963, 1722, 1666, 1606, 1580,
1480, 1400, 1290, 1250, 1161, 1080, 860, 756, 700 cmꢀ1
;
HRMS (EI): m=z (%) ¼ 344.0451 [Mþ], C16H12N2O5S re-
quires 344.0467.
Acknowledgements
This work was under the auspices of The National Basic
Research Program of China (2003CB114400), National Natu-
ral Science Foundation of China, The Science and Technology
Foundation of Shanghai, Program of Shanghai Subject Chief
Scientist and The Shanghai Leading Academic Discipline
Project (Project Number: B507).
References
1. Alexander JE, Nasser Yalpanl (1992) Cell 70:879
2. Lidija K (1999) Blood 93:2386
3. Grundlach H, Mu¨ller MJ, Kutchan TM (1992) Proc Natl
Acad Sci 89:2389
4. Demole E, Lederer E, Mercier D (1962) Helv Chim Acta
45:675
5. Haider G, Schrader TV, Fußlein M, Blechert S, Kutchan
TM (2000) Biol Chem 381:741
6. Zhao ZJ, Xu YF, Qian ZG, Tian WH, Qian XH, Zhong JJ
(2004) Bioorg Med Chem Lett 14:4755
S-Methyl benzo-1,2,3-thiadiazole-7-carboxylate
(1b, C8H6N2OS2)
White solid; mp 134–135ꢂC; 1HNMR (500MHz, DMSO-d6):
ꢀ ¼ 2.59 (s, SCH3), 7.98 (dd, 1H, J ¼ 7.26, 7.89 Hz, 5-Ar-H),
8.60 (d, J ¼ 7.26 Hz, 4-Ar-H), 9.08 (d, J ¼ 7.88 Hz, 6-Ar-H)

Derivatives of benzothiadiazole-7-carboxylates
1071
7. Qian ZG, Zhao ZJ, Xu YF, Qian XH, Zhong JJ (2004)
Biotech Bioeng 86:809
8. Qian ZG, Zhao ZJ, Tian WH, Xu YF, Zhong JJ, Qian XH
(2004) Biotech Bioeng 85:595
9. Desiree LC (1999) Crop Protection 18:267
10. Binningen RS, Oberwil WK, Nyfeler R (1990) US Patent
4931581
20. Kunz W, Schurter R, Maetzke T (1997) Pestic Sci 50:275
21. Kim JH, Lee SY, Oh SY, Han SI, Park HG, Yoo MA,
Kang HS (2004) Oncol Rep 12:1233
22. Enyedi AJ, Yalpanl N, Silverman P, Raskin L (1992) Cell
70:879
23. Kiyota H, Saitoh M, Oritani T, Yoshihara T (1996)
Phytochemistry 42:1259
11. Thaler JS, Fidantsef AL (1999) J Chemical Ecology 25:7
12. Xu YF, Zhao ZJ, Qian XH, Qian ZG, Tian WH, Zhong JJ
(2006) J Agric Food Chem 54:8793
24. Blechert S, Bockelmann C, Bru¨mmer O, Fu¨blein M,
¨
Gundlach H, Haider G, Holder S, Kutchan TM, Weiler
EW, Zenk MH (1997) J Chem Soc Perkin Trans I 1:3549
13. Schwenger P, Bellosta P, Vietor I, Basilico C, Skolnik EY,
ꢁ
25. Krumm T, Bandemer K, Boland W (1995) FEBS Lett
377:523
Vilcek J (1997) Proc Natl Acad Sci 94:2869
14. Flescher E, Fingrut O (2002) Leukemia 16:608
15. Rotem R, Fingrut O, Moskovitz J, Flescher E (2003)
Leukemia 17:2230
26. Lauchli R, Schu¨ler G, Boland W (2002) Phytochemistry
61:807
¨
27. Schu¨ler G, Mithofer A, Baldwin IT, Berger S, Ebel J,
16. Heyfets A, Flescher E (2007) Cancer Lett 250:300
17. Flescher E (2005) Anticancer Drugs 16:911
18. Rotem R, Heyfets A, Fingrut O, Blickstein D, Shaklai M,
Flescher E (2005) Cancer Res 65:1984
Santos JG, Herrmann G, Holscher D, Kramell R, Kutchan
TM, Maucher H, Schneider B, Stenzel I, Wasternack C,
Boland W (2004) FEBS Lett 563:17
´
28. Cazale AC, Rouet-Mayer MA, Barbier-Brygoo H,
`
Mathieu Y, Lauriere C (1998) Plant Physiol 116:659
19. Flescher E (2007) Cancer Lett 245:1