Synthesis and anti-cancer activity of benzothiazole containing phthalimide on human carcinoma cell lines

Article abstract of DOI:10.1016/j.bmc.2008.02.005

Phthalic anhydride is a highly toxic substance, facing, however, the problem of hydrolysis. In fact, it is rapidly hydrolyzed in aqueous medium, generating phthalic acid as the final product, which is almost harmless to viable cells. Here we describe the 'one pot' condensation reaction for the synthesis of phthalic imide derivative (benzothiazole containing phthalimide), exhibiting in vitro cytotoxic potential on human cancer cell lines. We further demonstrated that both caspase-dependent and -independent pathways are involved in our novel benzothiazole containing phthalimide induced apoptosis on cancer cells.

Full text of DOI:10.1016/j.bmc.2008.02.005

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3626–3631
Synthesis and anti-cancer activity of benzothiazole containing
phthalimide on human carcinoma cell lines
Stanton Hon Lung Kok,a,b Roberto Gambari,c Chung Hin Chui,a,e
Marcus Chun Wah Yuen,b Eva Lin,a,d Raymond Siu Ming Wong,e Fung Yi Lau,e
Gregory Yin Ming Cheng,e Wing Sze Lam,a Sau Hing Chan,a Kim Hung Lam,a
Chor Hing Cheng,a Paul Bo Shan Lai,f Michael Wing Yiu Yu,a Filly Cheung,a,*
a,
Johnny Cheuk On Tanga,b, and Albert Sun Chi Chan
*
*
aLo Ka Chung Centre for Natural Anti-cancer Drug Development (Laboratory B) and State Key Laboratory of Chinese Medicine
and Molecular Pharmacology, Hong Kong Polytechnic University, Yuk Choi Road, Hung Hom, Kowloon, Hong Kong, China
bInstitute of Textiles and Clothing, Hong Kong Polytechnic University, Hong Kong, China
cER-Gentech, Department of Biochemistry and Molecular Biology, The University of Ferrara, Ferrara, Italy
dDepartment of Pharmaceutical Sciences, Danish University, Denmark
eDepartment of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China
fDepartment of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China
Received 5 December 2007; revised 3 February 2008; accepted 5 February 2008
Available online 8 February 2008
Abstract—Phthalic anhydride is a highly toxic substance, facing, however, the problem of hydrolysis. In fact, it is rapidly hydrolyzed
in aqueous medium, generating phthalic acid as the final product, which is almost harmless to viable cells. Here we describe the ‘one
pot’ condensation reaction for the synthesis of phthalic imide derivative (benzothiazole containing phthalimide), exhibiting in vitro
cytotoxic potential on human cancer cell lines. We further demonstrated that both caspase-dependent and -independent pathways
are involved in our novel benzothiazole containing phthalimide induced apoptosis on cancer cells.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
blocker,7–9 calmodulin (CaM) antagonists,10 neuropro-
tective agent11,12 and agents exhibiting other interesting
biological activities.13–19 Recently, structurally novel
benzothiazole derivatives have been shown to have im-
proved biological activity. Some of the 2-amino deriva-
tives have been reported to possess cytotoxicity on
tumour cells which are comparable to that of cisplatin.1
Cantharidin-containing benzothiazole represents our re-
cently explored series possessing antitumour properties
in hepatocellular carcinoma, breast cancer, acute mye-
logenous leukaemia and non-small cell lung carcinoma.
At the same time, the cytotoxicity towards non-malig-
nant haematological disorder bone marrow cells is sig-
nificantly low. Our results also demonstrated that
cantharimide analogues bearing 2-amino-benzothiazole
moiety were effective to minimize the original cytotoxic-
ity, whilst maintaining potency towards cancer cell lines
in vitro. As part of our on-going studies of cantharimide
analogues, further modifications were elucidated and
evaluated in vitro, in order to expand structure–activity
Cancer chemotherapy targeting tumour progression rep-
resents one of the most relevant challenges of chemists
and oncologist. In order to gain new insights into the
complexity of the disease, robust screening methods
for evaluating different natural or synthetic drugs have
been carried out from the science community.
In this respect, the benzothiazole constitutes an impor-
tant scaffold of drugs, possessing several pharmacologi-
cal functions, rendering this molecule and its derivatives
powerful antitumour agents,1–6 neurotransmission
Keywords: Antiproliferation; Benzothiazole; Neoplasm; Phthalimide.
*
Corresponding authors. Tel.: +852 3400 8702; fax: +852 2364 9932
(F.C.); tel.: +852 3400 8727; fax: +852 2364 9932 (J.C.O.T.); tel.:
+852 2766 5222; fax: +852 2364 9932 (A.S.C.C.); e-mail addresses:
bcfilly@inet.polyu.edu.hk; bccotang@inet.polyu.edu.hk; bcachan@
inet.polyu.edu.hk
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.005

S. H. L. Kok et al. / Bioorg. Med. Chem. 16 (2008) 3626–3631
3627
relationship studies for possibly finding novel active
templates. Replacement of the bicyclic counterpart is
however the most straightforward structural modifica-
tion (Fig. 1). The phthalimide substituted benzothiazole
was then synthesized and its biological activity screened
on cancer cell lines. Our results showed that it mediates
a significant cytotoxic response to cancer cell lines
according to striking differences in electronic property
and potency.
2. Results and discussion
2.1. Identification of our novel synthesized benzothiazole
containing phthalimide
Figure 2. Growth inhibitory activity of benzothiazole containing
phthalimide on CA46, K562 and SKHep1 human cancer cells after
48 h as investigated by quantitating the cellular ATP levels. Reported
results represent the mean + standard deviation from triplicate tests.
The figure shows a representative experiment taken from three
independent experiments giving similar results.
To confirm the structure of our novel benzothiazole
containing phthalimide, we have performed both 1H
spectrum and 13C spectrum NMR analysis.
2.2. Cytotoxic activity of benzothiazole containing
phthalimide on human cancer cell lines
The human hepatoma cell line SKHep1, the Burkitt’s
lymphoma cell line (B cell type) CA46 and the chronic
myelogenous leukaemia (CML) K562 were firstly se-
lected for the purpose of preliminary anti-cancer screen-
ing of novel synthesized compounds. As shown in
Figure 2, our novel synthesized benzothiazole contain-
ing phthalimide showed significant cytotoxicity towards
all the three human cancer cell lines tested. The mean
concentration of this benzothiazole containing phthali-
mide compound reducing by 50% the cellular adenosine
triphosphate (ATP) content in all the cancer cell lines
was found to be around 69 lM (25 lg/mL). Phthalic
anhydride, on the other hand, did not show significant
cytotoxicity in all the three cell lines tested. The results
are shown in Figure 3. Figure 4 demonstrates that incu-
bating the SKHep1 hepatoma cells with 110 lM (40 lg/
mL) of benzothiazole containing phthalimide after 48 h
showed morphological changes including cell rounding
and detachment from the substratum.
Figure 3. Effects of phthalic anhydride (16.9 lM = 25 lg/mL) on cell
proliferation of CA46, K-562 and SKHep1 human cancer cells after
48 h as investigated by quantitating the cellular ATP levels. Reported
results represent the mean + standard deviation from triplicate tests.
The figure shows a representative experiment taken from three
independent experiments giving similar results.
2.3. Caspase-dependent and -independent pathways are
involved in benzothiazole containing phthalimide induced
apoptosis on human cancer cells
Failure to activate apoptotic pathways in response to
drug treatment may lead to resistance of tumours cells
to anti-cancer therapies. Therefore, factors affecting
caspase activation and apoptosis might be important
determinants for the drug sensitivity. In addition to cas-
pase-dependent apoptosis, caspase-independent forms
of cell death may also play a crucial role for treatment
response. Among the different caspases, caspase 3 is con-
sidered to be the central machinery of apoptosis. In con-
clusion, insights into the mechanisms regulating
apoptosis as well as other forms of cell death pathways
provide information greatly valuable for the develop-
ment of novel strategies targeting resistance of tumour
Figure 1. Chemical structures of cantharidin, phthalic anhydride,
benzothiazole containing cantharimide and benzothiazole containing
phthalimide.

3628
S. H. L. Kok et al. / Bioorg. Med. Chem. 16 (2008) 3626–3631
Figure 5. Caspase 3 activity assay to study the possible apoptotic
potential of benzothiazole containing phthalimide (BP) at a concen-
tration of 110 lM (40 lg/mL) on CA46 and SKHep1 human cancer
cells after 48 h incubation. The zVADfmk (20 lM) is a caspase
inhibitor used to inhibit the activation of caspase 3. Reported results
represent the mean + standard deviation from triplicate tests. The
figure shows a representative experiment taken from three independent
experiments giving similar results.
Figure 4. Morphological analysis for the effects of benzothiazole
containing phthalimide on SKHep1 hepatoma cells after 48 h of
incubation and methylene blue staining. (A) Untreated control. (B)
Benzothiazole containing phthalimide at 110 lM (40 lg/mL).
cells. Since both CA46 and SKHep1 cancer cells were
found to be more susceptible to the action of our benzo-
thiazole containing phthalimide (Fig. 2), we further se-
lected them to study the possible involvement of
caspase 3 for the biological activity of benzothiazole
containing phthalimide. As anticipated, we found that
the benzothiazole containing phthalimide at a concen-
tration of 110 lM (40 lg/mL) for 48 h could signifi-
Figure 6. Analysis of the possible reversal effects of caspase inhibitor
zVADfmk (20 lM) on the cytotoxicity of benzothiazole containing
phthalimide (BP) at a concentration of 110 lM (40 lg/mL) on CA46
and SKHep1 human cancer cells after 48 h of incubation. Reported
results represent the mean + standard deviation from triplicate tests.
The figure shows a representative experiment taken from three
independent experiments giving similar results.
cantly activate caspase
3 as determined by the
luminescence kit purchased from Promega (Fig. 5) while
pre-incubating the cancer cells with 20 lM of the pan-
caspase inhibitor (zVADfmk, purchased from Promega)
for 2 h before the addition of the tested compound could
significantly inhibit the proteolytic activity of caspase 3.
However, pre-incubating the cancer cells with the
caspase inhibitor could only partially reverse the benzo-
thiazole containing phthalimide mediated cytotoxic
activity [increased from a mean of 30.2–37.1% for
CA46 and 37.8–42.6% for SKHep1 for their cellular
ATP content when compared with untreated control
(Fig. 6)]. Thus we speculated that both caspase-depen-
dent and -independent apoptotic pathways are impor-
tant for the actions our benzothiazole containing
phthalimide on these two cancer cell lines.
cells. We found that the trend of inhibition of our ben-
zothiazole containing phthalimide on bone marrow cells
showed similar toxicity as those on cancer cells [with
50% of cellular ATP content reduction around 69 lM
(25 lg/mL)].
In order to identify its physicochemical properties in
relation to its biological activity or binding, LogP and
p values were estimated using ChemDraw Ultra 11
(Fig. 7). In the presence of OCF3 substituting group,
the overall hydrophobicity (i.e., LogP) increases signifi-
cantly for both phthalimide and cantharidimide in rela-
tion to the corresponding H derivatives. The extra gain
of hydrophobicity changing from cantharidimide to
phthalimide (entries 2 and 4) suggests that aromatic
structure of phthalimide could show dominant effect on
hydrophobicity. However, the overall substituent hydro-
phobicity constant (i.e., pOCF3) remains unchanged. For
the case of using hepatoma cell line SKHep1, the struc-
tural modification on imide, however, did not appear
As mentioned before, the selectivity between normal and
malignant cells is one of the critical issues for the
research and development of chemotherapeutic re-
agents. Accordingly, we have also tested the tolerance
of non-malignant haematological disorder bone marrow

S. H. L. Kok et al. / Bioorg. Med. Chem. 16 (2008) 3626–3631
3629
Figure 7. Physicochemical properties in relation to each compound’s biological activity or binding. LogP and p values of our benzothiazole
containing phthalimide and related compounds. *LogP values are predicted using ChemDraw Ultra 11.
to significantly alter the hydrophobicity; on the contrary,
it induced a significant drop of 50% inhibitory concentra-
tion (more than a double), suggesting a further investiga-
tion on structure–activity relationship.
with moderate yield (Scheme 1). Based upon the similar-
ity in structure, one would predict that benzothiazole
containing cantharimide and phthalimide would be
metabolized and degraded in a similar manner, retaining
potential applications as active anti-cancer drugs.
3. Conclusions
Unless otherwise indicated, all reactions were carried
out under nitrogen atmosphere. NMR spectra were re-
corded on a Varian 500 MHz Fourier transform spec-
trometer. 1H and 13C{1H} NMR spectra were
recorded relative to residual protiated solvent; a positive
value of the chemical shift denotes a resonance down-
field from TMS. Mass analyses were performed on a
Finnigan model Mat 95 ST mass spectrometer. Phthalic
anhydride and 2-amino-6-trifluoromethoxy-benzothia-
zole were purchased from Sigma–Aldrich. All other
chemicals were purchased from commercial suppliers
and used without further purification. Toluene and tri-
ethylamine were freshly distilled from sodium under
nitrogen. All reactions were monitored by analytical
thin-layer chromatography (TLC) on Merck alumin-
ium-precoated plates of silica gel 60F254 with detection
by spraying with 5% (w/v) dodecamolybdophosphoric
acid in ethanol or 5% (w/v) ninhydrin in ethanol and
subsequent heating. E. Merck silica gel 60 (230–400
mesh) was used for flash chromatography. Chemical
In conclusion, we have here described a facile synthesis
of an anti-cancer compound, which showed significant
cytotoxic activity towards three human cancer cell lines
tested. Further experimental investigation for other po-
tential partners with benzothiazole is on-going to select
potential combinations not only with improved ability
to discriminate between non-malignant cells and carci-
noma cells but may also possess anti-microbial activity
including the inhibition of b-lactamase, which could be
versatile in textile industry.
4. Experimental
4.1. Preparation of phthalic imide
For the synthesis of phthalic imide derivative, a ‘one
pot’ condensation reaction was employed as a key step
Scheme 1. Condensation of phthalic anhydride with benzothiazole.

3630
S. H. L. Kok et al. / Bioorg. Med. Chem. 16 (2008) 3626–3631
structure was confirmed by the nuclear magnetic reso-
nance including 1H and 13C spectra, respectively.
ported by the post of ‘Senior Research Fellow’ kindly
offered by Dr. J.C.O. Tang (Niche Area Grant: BB8P
to Dr. J.C.O. Tang). Special thank is given to Professor
R. Gambari and Dr. C.H. Chui who gave us valuable
advice on the anti-cancer drug screening experiments
and their constructive criticism on this paper. Professor
R. Gambari is sponsored by AIRC (Italian Association
for Cancer Research). Lastly, we would like to thank the
assistance from Miss Monica Kwok, Dr. R.S.M. Wong
and Professor G.Y.M. Cheng for providing the cancer
cell lines and non-malignant haematological disorder
bone marrow samples for our study.
A mixture of phthalic anhydride (50 mg, 0.34 mmol),
2-amino-6-trifluoromethoxy-benzothiazole (410 mg,
1.7 mmol), dried toluene and dried triethylamine (6 mL;
2:1, v/v) in sealed tube was heated for 5 h at 180 °C. Con-
centration followed by flash chromatography gave the
product (80.5 mg) in 65% yield and Rf = 0.91 (DCM/
1
MeOH, 10:1). H NMR (CDCl3, 500 MHz): d 7.39 (d,
J = 8.5 Hz, 1H), 7.77 (s, 1H), 7.88–7.90 (m, 2H), 8.06–
8.08 (m, 2H), 8.14 (dd, J = 9 Hz and 1.5 Hz, 1H); 13C
NMR (dimethylsulfoxide, DMSO-d6): d 115.7, 120.9,
124.3, 124.9, 131.7, 134.4, 136.3, 145.9, 146.0, 148.6,
154.4, 165.1; MS (ESI) m/z (relative intensity) 387.3
[(M+Na]+, 100); HRMS (ESI) [M+Na]+ Calcd for
C16H7F3N2NaO3S 387.0027, found 387.0024.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2008.02.005.
4.2. Bioassay of phthalic imide
Human Burkitt’s lymphoma cell line (B cell type) CA46,
chronic myelogenous leukaemia (CML) K562 and hep-
atoma cell line SKHep1 were used for preliminary
anti-cancer screening of the novel synthesized com-
pound. Cancer cells seeded in the 96 wells microtitre
plates for 24 h were prepared for the benzothiazole con-
taining pthalimide screening. The benzothiazole con-
References and notes
1. Hutchinson, I.; Bradshaw, T. D.; Matthews, C. S.;
Stevens, M. F. G.; Westwell, A. D. Bioorg. Med. Chem.
Lett. 2003, 13, 471–474.
2. Hutchinson, I.; Jennings, S. A.; Vishnuvajjala, B. R.;
Westwell, A. D.; Stevens, M. F. G. J. Med. Chem. 2002,
45, 744–747.
taining pthalimide was prepared as
a
stock
concentration of 69 mM (25 mg/mL) in DMSO. Phtha-
lic anhydride purchased from Sigma chemical was also
prepared as a stock concentration of 16.9 mM (25 mg/
mL) in DMSO. Both compounds were added at a start-
ing concentration of 25 lg/mL and followed by a serial
of twofold dilutions and incubated for a further 48 h.
Untreated control received either total complete med-
ium or 0.1% of DMSO. Afterwards, the evaluation of
possible antiproliferative and cytotoxicity of our novel
synthesized compound was assayed by the One Step
ATP lite assay purchased from PerkinElmer (Nether-
lands) according to the technical manual provided.19
3. Lin, P. Y.; Shi, S. J.; Shu, H. L.; Chen, H. F.; Lin, C. C.;
Liu, P. C.; Wang, L. F. Bioorg. Chem. 2000, 28, 266–272.
4. Hall, I. Arch. Pharm. (Weinheim) 1999, 332, 115–123.
5. Beneteau, V.; Besson, T.; Guillard, J.; Leonce, S.; Pfeiffer,
B. Eur. J. Med. Chem. 1999, 34, 1053–1060.
6. Bradshaw, T. D.; Wrigley, S.; Shi, D. F.; Schultz, R. J.;
Paull, K. D.; Stevens, M. F. G. Br. J. Cancer 1998, 77,
745–752.
7. Jimonet, P.; Audiau, F.; Barreau, M.; Blanchard, J. C.;
´
Boireau, A.; Bour, Y.; Coleno, M. A.; Doble, A.;
Doerflinger, G.; Hu, C. D.; Donat, M. H.; Duchesne, J.
´ ´
´
M.; Ganil, P.; Gueremy, C.; Honore, E.; Just, B.;
Kerphirique, R.; Gontier, S.; Hubert, P.; Laduron, P.
M.; Le Blevec, J.; Meunier, M.; Miquet, J. M.; Nemecek,
C.; Pasquet, M.; Piot, O.; Pratt, J.; Rataud, J.; Reibaud,
M.; Stutzmann, J. M.; Mignani, S. J. Med. Chem. 1999,
42, 2828–2843.
4.3. Caspase 3 activity assay
CA46 and SK-Hep1 cancer cells were incubated with
110 lM (40 lg/mL) of benzothiazole containing phthal-
imide for 48 h. Twenty micrograms of total cellular pro-
tein was analysed for the caspase 3 activity using the
PerkinElmer Trupoint caspase 3 assay system according
to the manufacturer’s instruction.19
8. Benavides, J.; Camelin, J. C.; Mitrani, N.; Flamand, F.;
´ ´
Uzan, A.; Legrand, J. J.; Gueremy, C.; Le Fur, G.
Neuropharmacology 1985, 24, 1085–1092.
9. Mizoule, J.; Meldrum, B.; Mazadier, M.; Croucher, M.;
Ollat, C.; Uzan, A.; Legrand, J. J.; Gueremy, C.; Le Fur,
G. Neuropharmacology 1985, 24, 767–773.
10. Tanaka, T.; Umekawa, H.; Saitoh, M.; Ishikawa, T.; Shin,
T.; Ito, M.; Itoh, H.; Kawamatsu, Y.; Sugihara, H.;
Hidaka, H. Mol. Pharmacol. 1986, 29, 264–269.
11. Malgouris, C.; Bardot, F.; Daniel, M.; Pellis, J.; Rataud,
A.; Uzan, A.; Blanchard, J. C.; Laduron, P. M.
J. Neurosci. 1989, 9, 3720–3727.
12. Pratt, J.; Rataud, J.; Bardot, F.; Roux, M.; Blanchard, J.
C.; Laduron, P. M.; Stutzmann, J. M. Neurosci. Lett.
1992, 140, 225–230.
Acknowledgements
We acknowledge a donation fund from Mr. K.C. Lo to
Dr. J.C.O. Tang for the establishment of Lo Ka Chung
Centre for Natural Anti-cancer Drug Development
(Laboratory B) and a Niche area grant offered by the
Hong Kong Polytechnic University to Dr. F. Cheung
(BB8R). Drs. C.H. Chui and F.Y. Lau are the honou-
rary tutors kindly offered by Professor J. Sung and Pro-
fessor G.Y.M. Cheng from Department of Medicine
and Therapeutics, Prince of Wales Hospital, The Chi-
nese University of Hong Kong. Dr. C.H. Chui is sup-
´
13. Quiroga, J.; Hernandez, P.; Insuasty, B.; Abonıa, R.;
Cobo, J.; Sanchez, A.; Nogueras, M.; Low, J. N. J. Chem.
´
´
Soc., Perkin Trans. 1 2002, 555–559, and references cited
therein.
14. Kok, S. H. L.; Chui, C. H.; Lam, W. S.; Chen, J.; Lau, F.
Y.; Wong, R. S. M.; Cheng, G. Y. M.; Lai, P. B. S.;

S. H. L. Kok et al. / Bioorg. Med. Chem. 16 (2008) 3626–3631
3631
Leung, R. W. T.; Tang, J. C. O.; Chan, A. S. C. Bioorg.
Med. Chem. Lett. 2007, 17, 1155–1159.
15. Kok, S. H. L.; Gambari, R.; Chui, C. H.; Lam, W. S.;
Chen, J.; Lau, F. Y.; Wong, R. S. M.; Cheng, G. Y. M.;
Lai, P. B. S.; Leung, T. W. T.; Chan, A. S. C.; Tang, J. C.
O. Minerva Biotechnol. 2006, 18, 153–157.
16. Kok, S. H. L.; Chui, C. H.; Lam, W. S.; Chen, J.; Lau, F.
Y.; Wong, R. S. M.; Cheng, G. Y. M.; Tang, W. K.; Teo,
I. T. N.; Cheung, F.; Cheng, C. H.; Chan, A. S. C.; Tang,
J. C. O. Int. J. Mol. Med. 2006, 18, 1217–1221.
17. Kok, S. H. L.; Chui, C. H.; Lam, W. S.; Chen, J.; Lau, F.
Y.; Wong, R. S. M.; Cheng, G. Y. M.; Tang, W. K.;
Cheng, C. H.; Tang, J. C. O.; Chan, A. S. C. Int. J. Mol.
Med. 2006, 18, 375–379.
18. Kok, S. H. L.; Chui, C. H.; Lam, W. S.; Chen, J.; Tang, J.
C. O.; Lau, F. Y.; Cheng, G. Y. M.; Wong, R. S. M.;
Chan, A. S. C. Int. J. Mol. Med. 2006, 17, 945–949.
19. Kok, S. H. L.; Chui, C. H.; Lam, W. S.; Chen, J.; Tang, J.
C. O.; Lau, F. Y.; Cheng, G. Y. M.; Wong, R. S. M.;
Chan, A. S. C. Int. J. Mol. Med. 2006, 17, 151–157.