Synthesis, structure-activity relationships and preliminary antitumor evaluation of benzothiazole-2-thiol derivatives as novel apoptosis inducers

Article abstract of DOI:10.1016/j.bmcl.2010.12.124

A series of novel benzothiazole-2-thiol derivatives were synthesized, and their anti-proliferative activities on HepG2 and MCF-7 cells were investigated. Most compounds had inhibitory effects on cell growth, and some of them were more effective than cisplatin. Compounds 6m and 6t displayed good inhibitory activities against a panel of different types of human cancer cell lines, with IC₅₀ values in the low micromolar range. Further biological evaluation indicated that 6m induced apoptosis in HepG2 cancer cells. Structure-activity relationships were also proposed.

Full text of DOI:10.1016/j.bmcl.2010.12.124

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1097–1101
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, structure–activity relationships and preliminary antitumor
evaluation of benzothiazole-2-thiol derivatives as novel apoptosis inducers
Zhao Wang a, Xuan-Hong Shi a, Jia Wang b, Tian Zhou a, You-Zhi Xu a, Ting-Ting Huang a, Yan-Fang Li b,
a
Ying-Lan Zhao a, Li Yang a, Sheng-Yong Yang a, Luo-Ting Yu a, , Yu-Quan Wei
⇑
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Sichuan 610041, China
b Department of Pharmaceutical and Bioengineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, 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 novel benzothiazole-2-thiol derivatives were synthesized, and their anti-proliferative activi-
ties on HepG2 and MCF-7 cells were investigated. Most compounds had inhibitory effects on cell growth,
and some of them were more effective than cisplatin. Compounds 6m and 6t displayed good inhibitory
activities against a panel of different types of human cancer cell lines, with IC50 values in the low micro-
molar range. Further biological evaluation indicated that 6m induced apoptosis in HepG2 cancer cells.
Structure–activity relationships were also proposed.
Received 1 October 2010
Revised 16 December 2010
Accepted 28 December 2010
Available online 31 December 2010
Keywords:
Tumor
Ó 2010 Elsevier Ltd. All rights reserved.
Benzothiazole-2-thiol
Structure–activity relationships (SAR)
Apoptosis
Cancer is a notably complex, widespread and lethal disease that
can affect almost every tissue lineage in the human body,1 and
poses great challenges to medical science. Although tumor cells
are diverse and heterogeneous, they all share the property that
they proliferate rapidly.2 The discovery and development of novel
antitumor drugs that could effectively inhibit proliferative path-
ways is needed urgently. Apoptosis, a physiological mode of cell
death in higher eukaryotes, plays a crucial role in the prevention
of tumor cell proliferation.3 Apoptosis serves as a defense mecha-
nism to remove unwanted and potentially dangerous cells, such
as tumor cells. Its deregulation is widely believed to be involved
in the pathogenesis of cancer, and there are many antitumor com-
pounds that induce the apoptotic process in tumor cells.4 Thus,
development of drugs that can effectively trigger apoptosis in tu-
mor cells has been receiving considerable attention. Whether new-
ly developed antitumor agents can induce apoptosis is usually
studied first during their preliminary biological evaluation.
fonamide,13 imidazole14 and aryl,15 were shown to be important,
and the resulting compounds had an inhibitory effect against cer-
tain cancer cell lines. However, most investigators focused on
designing new benzothiazole compounds by substituting 2-aryl-
benzothiazoles or 2-aminobenzothiazoles; only a few employed
the benzothiazole-2-thiol as a functional group. Recent findings
have indicated that compounds derived from benzothiazole-2-
thiol could achieve good antitumor potency; but these compounds
were not explored further.16 Furthermore, as far as we are aware,
none of the reported benzothiazole derivatives were reported to
induce apoptosis in cancer cells.
In a previous cell-based screening study of anticancer activity of
benzothiazole compounds, we found a hit compound 1 (Fig. 1), a
benzothiazole-2-thiol derivative bearing amide linkages and phe-
nyl rings, could inhibit HepG2 cell growth and induce its apoptosis
in vitro. Compound 1 exhibited moderate inhibition against the
Benzothiazole derivatives are known for different biological
properties, including antitubercular,5 antimalarial,6 anticonvul-
sant,7 antihelmintic,8 analgesic,9 anti-inflammtory,10 antidiabetic11
and antitumor12 activities. In recent years, extensive research has
focused on developing novel benzothiazole derivatives to improve
antitumor activities. Among these attempts, modifying the benzo-
thiazole moiety with some functional groups, such as benzenesul-
⇑
Corresponding author. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail address: yuluot@scu.edu.cn (L.-T. Yu).
Figure 1. The structure of compound 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.124

1098
Z. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1097–1101
human hepatocellular carcinoma cell line, HepG2 (IC50 = 34.2
and the human mammary carcinoma cell line, MCF-7 (IC50
35.6 M), in the MTT assay. To develop novel tumor growth inhib-
itors and apoptosis inducers as potential anticancer agents, we
designed and synthesized a series of benzothiazole-2-thiol deriva-
tives, based on compound 1, employing a structure–activity rela-
tionship (SAR) study.
l
M),
=
with an electron-attracting nitro group resulted in complete loss
of activity (compounds 6i–l). However, replacing the hydrogen at
the 2-position of the phenyl ring with an electron-donating meth-
oxy group resulted in the activities increasing significantly; for
example, compounds 6m and 6n. Compound 6m in particular
was the most active analog, and, compared with compound 1, en-
hanced the potency by approximately 57-fold against HepG2 cells,
and 32-fold against MCF-7 cells.
In another variation, we replaced the substituted phenyl ring
with methyl and chloromethyl groups at R2 for Region 2, affording
6p–s and 6t–w. As shown in Table 1, 6p–r had considerably better
activity than compound 1, indicating methyl might be a good sub-
stitution for modification. However, the lost potency of compound
6s suggested that the phenyl rings on both sides of the compound
should not be removed simultaneously. In contrast, the potent
l
The two regions of compound 1 used for SAR evaluation are
shown in Figure 1. The route adapted for the synthesis of com-
pound 1 and other benzothiazole derivatives 6a–w is outlined in
Scheme 1, using a modified literature method.17,18 The starting
compounds 3a–c were prepared from commercially available ben-
zylamine derivatives 2a–c, and 2-chloroacetyl chloride. Compound
3d could be obtained through aqueous methylamine and 2-chloro-
acetyl chloride at room temperature. In the next step, 3a–d were
condensed with 6-aminobenzothiazole-2-thiol in acetone with
refluxing, affording the amines, 5a–d. Then, target compounds, 1
and 6a–w, were obtained, respectively, by the reaction between
5a–d and the corresponding acyl chloride in the presence of so-
dium bicarbonate, at room temperature, in dichloromethane. The
yield of all reactions was satisfactory. All the products were char-
acterized by 1H NMR, 13C NMR and MS data. The structures of
two representative compounds, 6m and 6t, were further confirmed
by IR data.19
Twenty-four benzothiazole-2-thiol derivatives were synthe-
sized to survey the SAR by evaluating the cell growth inhibitory
activities (IC50) in cultures of HepG2 and MCF-7 cells, using the
MTT assay.20 As shown in Table 1, most compounds were able to
increase the inhibitory activities against the two cancer cell lines,
and some were even better than cisplatin, while five compounds
had no activity.
activities of 6t–w, especially 6t (IC50 = 1.1 lM), suggested that
chloromethyl could improve greatly the potency of the parent
compound. Interestingly, when the phenyl ring in Region 2 was re-
placed by chloromethyl, the absence of another phenyl ring in Re-
gion 1 did not result in the loss of activity (6w). This observation
confirmed further that chloromethyl could play an important role
in the biological activities of benzothiazole derivatives.
Through the structure–activity relationship study, we found that
chloromethyl could improve significantly the antitumor activity of
this series of benzothiazole derivatives in vitro. Region 2 seemed
to play a more important role in the activities of these compounds
than Region 1; this observation provided us with important infor-
mation for further molecular structure modification.
To further study the cytotoxic profile, the most potent analogs
6m and 6t were selected for evaluation of their inhibitory activities
against a panel of different types of human cancer cell lines:
colon cancer cell lines HCT-116, SW480 and SW620, lung cancer
cell line A549, prostate cancer cell line PC-3, pancreatic cancer cell
line BxPC-3, epidermoid cancer cell line A431, cervical cancer cell
line HeLa, melanoma cancer cell line A375, ovarian cancer cell line
SKOV-3, and breast cancer cell lines MDA-MB-468 and SKRB-3. As
shown in Table 2, compounds 6m and 6t exhibited good antitumor
activities against the different types of human cancer cell lines in
For Region 1, the structure–activity relationships were not very
clear among analogs that had the same substituent in Region 2. The
activities of 6b, 6f, and 6q were higher than their parent com-
pounds, but the activities of 6a, 6u and 6v were closed to their par-
ent compounds, and the potency of 6r even decreased.
Replacement of 4-position on the left phenyl ring with chlorine
or methoxy groups showed no clear SAR trends.
For Region 2, replacement of the hydrogen at the 4-position of
the phenyl ring with chlorine altered slightly the activity against
the tumor cells tested (compound 6d vs 1, 6e vs 6a, 6f vs 6b, 6g
vs 6c). The increased potency could be attributed most probably
to a +
Replacement of the hydrogen at the 4-position of the phenyl ring
vitro, with IC50 values ranging from 1.1 lM to 8.8 lM. This
broad-spectrum anti-proliferative activity is encouraging for the
development of new anticancer drugs, and adds to the current
armamentarium that includes cisplatin, doxorubicin and paclitaxel.
As mentioned above, the antitumor efficacy of chemotherapeu-
tic agents is correlated with their apoptosis inducing ability.
p effect, a +r effect, or to a combination of +p
and +r21
Scheme 1. Reagents and conditions: (a) 3a–c: 2-chloroacetyl chloride, K2CO3, CH2Cl2, reflux, 4 h, 65–76%; 3d: 2-chloroacetyl chloride, H2O, 0–10 °C, 36%; (b) K2CO3, acetone,
reflux, 4 h, 92–98%; (c) benzoyl chloride/4-chlorobenzoyl chloride/4-nitrobenzoyl chloride/2-methoxybenzoyl chloride/acetyl chloride/2-chloroacetyl chloride, NaHCO3,
CH2Cl2, rt, 4–26 h, 80–96%.

Z. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1097–1101
1099
Table 1
SAR evaluation of benzothiazole derivatives, 6a–w
R1
R2
Yield (%)
IC50 (lM)
a
Compounds
HepG2
MCF-7
1
Phenyl
4-Chlorophenyl
4-Methoxyphenyl
H
Phenyl
4-Chlorophenyl
4-Methoxyphenyl
H
Phenyl
4-Chlorophenyl
4-Methoxyphenyl
H
Phenyl
4-Chlorophenyl
4-Methoxyphenyl
H
Phenyl
4-Chlorophenyl
4-Methoxyphenyl
H
Phenyl
4-Chlorophenyl
4-Methoxyphenyl
H
—
Phenyl
Phenyl
Phenyl
Phenyl
96
90
88
85
94
80
81
86
85
82
83
86
91
85
92
89
91
92
91
87
90
91
92
88
—
34.2
30.1
12.7
26.5
25.4
17.8
7.4
15.3
>100
>100
>100
>100
8.0
0.6
2.1
18.5
9.2
6.7
11.2
>100
1.1
1.8
2.0
35.6
33.7
21.2
34.4
25.9
29.3
19.2
29.4
>100
>100
>100
>100
17.2
1.1
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
4-Nitrophenyl
2-Methoxyphenyl
2-Methoxyphenyl
2-Methoxyphenyl
2-Methoxyphenyl
Methyl
Methyl
Methyl
Methyl
Chloromethyl
Chloromethyl
Chloromethyl
Chloromethyl
—
9.3
23.4
17.6
10.2
20.5
>100
4.2
4.9
5.1
5.0
8.4
6s
6t
6u
6v
6w
Cisplatin
1.9
16.3
a
Values are means of three experiments. See Ref. 20.
Table 2
The anti-proliferative activities for compounds 6m and 6t against various cancer cell lines
a
Compounds
IC50 (lM)
HCT116
SW480
SW620
A549
PC-3
BxPC-3
A431
HeLa
A375
SKOV-3
MDA-MB-468
SKRB-3
6m
6t
Cisplatin
1.5
1.3
17.9
5.4
5.2
20.2
4.2
1.2
17.0
2.0
4.0
27.2
4.6
6.1
23.1
4.4
6.6
17.6
1.1
4.4
15.0
4.2
4.9
25.9
2.2
1.8
9.7
6.0
4.8
12.6
2.3
4.1
14.4
1.2
8.8
18.1
a
Values are means of three independent experiments. See Ref. 20.
Figure 2. Induction of apoptosis in HepG2 cancer cells following treatment with 6m. Cells were treated with various concentrations of 6m for 24 h: 0
0.6 M; 1.2 M. The cells in the sub-G1 phase were considered as apoptotic cells (n = 5).
lM (control); 0.3 lM;
l
l
Therefore, we examined whether our compounds could induce the
apoptosis. As compound 6m exhibited the best potency against
HepG2 cancer cells in vitro, we took it as a lead for further study.
To quantitatively assess apoptosis, we used flow cytometry
analysis to identify sub-G1 cells/apoptotic cells, and to measure
the percentage of sub-G1 cells after PI-staining.22 The percentage

1100
Z. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1097–1101
Figure 3. Effect of 6m on cell morphology. Bright-field microscopy images (A), and fluorescence microscopic appearance of PI-stained nuclei (B) of HepG2 cancer cells, after
incubation with 6m for 24 h at varying concentrations: 0 M (control); 0.3 M; 0.6 M; 1.2
l
l
l
lM (original magnification 400Â). Apoptotic cells containing condensed and
fragmented fluorescent nuclei are visible in 6m-treated cells, but not in control cells.
of apoptotic cells were 19.2%, 35.9% and 55.4%, respectively, after
Acknowledgment
treatment with 0.3 lM, 0.6 lM and 1.2 lM 6m for 24 h (Fig. 2).
The results suggested that 6m inhibited the proliferation of HepG2
cells by inducing apoptosis in a concentration-dependent manner.
Furthermore, fluorescence microscopic examination of
PI-stained cell confirmed the apoptosis inducing effect of 6m.23
The apoptotic cells are often characterized by DNA fragmenta-
tion, chromatin condensation, cell shrinkage, and membrane
blebbing.24 When treated with 6m for 24 h, HepG2 cancer cells
shrank and rounded up. Moreover, these phenomena became
more significant as the 6m concentration was increased. In con-
trast, the untreated HepG2 cells retained their normal size and
shape, kept proliferating with time and became over-crowded
by 24 h (Fig. 3A). The morphological changes were also character-
istic of apoptosis after PI-staining: bright red fluorescent, con-
densed nuclei (intact or fragmented) were observed, and the
change was concentration-dependent. In contrast, the untreated
cells showed red, diffusely stained intact nuclei (Fig. 3B). These
findings also indicated that 6m induced apoptosis in HepG2 can-
cer cells.
In conclusion, a novel series of benzothiazole-2-thiol deriva-
tives were synthesized and their anti-proliferative activities were
evaluated in vitro. The data acquired by the MTT assay showed that
most of the compounds exhibited good inhibitory activities, and
some were even better than cisplatin. Through the SAR study, the
inhibition was found to depend on substitution pattern of the side
chains, especially in Region 2, and chloromethyl played a crucial
role in this series of benzothiazole-2-thiol derivatives. Compounds
6m and 6t displayed good inhibitory activities against a panel of
different types of human cancer cell lines with IC50 values in the
low micromolar range. The results of flow cytometry analysis
and morphological analysis indicated that compound 6m induced
apoptosis in HepG2 cancer cells in a concentration-dependent
manner, suggesting a new method for the evaluation of the antitu-
mor evaluation of benzothiazole derivatives. Our results will pro-
vide useful information for the design of novel benzothiazole
derivatives with better potency as antitumor agents. The exact bio-
logical mechanism of these compounds is currently under investi-
gation in our laboratory.
This work was supported by National Science and Technology
Major Project of China (2009ZX09103-132). The authors gratefully
thank Yong-Qiu Mao (State Key Laboratory of Biotherapy, Huaxi
hospital, Sichuan University) for helpful technical assistance and
discussion in the flow cytometry analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.12.124.
References and notes
1. Varmus, H. Science 2006, 312, 1162.
2. Evan, G. I.; Vousden, K. H. Nature 1992, 411, 342.
3. Thompson, C. B. Science 1995, 267, 1456.
4. Cummings, J.; Ward, T. H.; Ranson, M.; Dive, C. Biochim. Biophys. Acta 2004,
1705, 53.
5. Palmer, P. J.; Trigg, R. B.; Warrington, J. V. J. Med. Chem. 1971, 14, 248.
6. Burger, A.; Sawhney, S. N. J. Med. Chem. 1968, 11, 270.
7. Chakole, R. D.; Amnerkar, N. D.; Khedekar, P. B.; Bhusari, K. P. Indian J.
Heterocycl. Chem. 2005, 15, 27.
8. Jayachandran, E.; Bhatia, K.; Naragud, L. V. G.; Roy, A. Indian Drugs 2003, 40, 408.
9. Siddiqui, N.; Alam, M.; Siddiqui, A. A. Asian J. Chem. 2004, 16, 1005.
10. Gurupadayya, B. M.; Gopal, M.; Padmashali, B.; Vaidya, V. P. Indian J. Heterocycl.
Chem. 2005, 15, 169.
11. Diaz, H. M.; Molina, R. V.; Andrade, R. O.; Coutino, D. D.; Franco, L. M.; Webster,
S. P.; Binnie, M.; Soto, S. E.; Barajas, M. I.; Rivera, I. L.; Vazquez, G. N. Bioorg.
Med. Chem. Lett. 2008, 18, 2871.
12. (a) Rana, A.; Siddiqui, N.; Khan, S. A. Indian J. Pharm. Sci. 2007, 69, 10; (b) Shi, D.-
F.; Bradshaw, T. D.; Wrigley, S.; McCall, C. J.; Lelieveld, P.; Fichtner, I.; Stevens,
M. F. G. J. Med. Chem. 1996, 39, 3375; (c) Hutchinson, I.; Chua, M. S.; Browne, H.
L.; Trapani, V.; Bradshaw, T. D.; Westwell, A. D.; Stevens, M. F. G. J. Med. Chem.
2001, 44, 1446; (d) Kok, S. H. L.; Gambari, R.; Chui, C. H.; Yuen, M. C. W.; Lin, E.;
Wong, R. S. M.; Lau, F. Y.; Cheng, G. Y. M.; Lam, W. S.; Chan, S. H.; Lam, K. H.;
Cheng, C. H.; Lai, P. B. S.; Yu, M. W. Y.; Cheung, F.; Tanga, J. C. O.; Chan, A. S. C.
Bioorg. Med. Chem. 2008, 16, 3626.
13. Kamal, A.; Khanl, M. N. A.; Reddyl, K. S. R.; Srikanthl, Y. V. V.; Sridhar, B. Chem.
Biol. Drug Des. 2008, 71, 78.
14. (a) Trapani, G.; Franco, M.; Latrofa, A.; Reho, A.; Liso, G. Eur. J. Pharm. Sci. 2001,
14, 209; (b) Srimanth, K.; Rao, V. R.; Krishna, D. R. Arzneimittelforschung 2002,
52, 388.
15. Song, E. Y.; Kaur, N.; Park, M.-Y.; Jin, Y.; Lee, K.; Kim, G.; Lee, K.-Y.; Yang, J. S.;
Shin, J. H.; Nam, K. Y.; No, K. T.; Han, G. Eur. J. Med. Chem. 2008, 43, 1519.

Z. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1097–1101
1101
16. (a) Zhang, L.; Fan, J.; Vu, K.; Hong, K.; Le Brazidec, J.-Y.; Shi, J.; Biamonte, M.;
Busch, D. J.; Lough, R. E.; Grecko, R.; Ran, Y.; Sensintaffar, J. L.; Kamal, A.;
Lundgren, K.; Burrows, F. J.; Mansfield, R.; Timony, G. A.; Ulm, E. H.; Kasibhatla,
S. R.; Boehm, M. F. J. Med. Chem. 2006, 49, 5352; (b) Fréchette, S.; Leit, S.; Woo,
S. H.; Lapointe, G.; Jeannotte, G.; Moradei, O.; Paquin, I.; Bouchain, G.; Raeppel,
S.; Gaudette, F.; Zhou, N.; Vaisburg, A.; Fournel, M.; Yan, P. T.; Trachy-Bourget,
M.-C.; Kalita, A.; Robert, M.-F.; Lu, A.; Rahil, J.; MacLeod, A. R.; Besterman, J. M.;
Li, Z.; Delorme, D. Bioorg. Med. Chem. Lett. 2008, 18, 1502; (c) Zhang, J.-T.; Qi, J.;
Peng, H.; Dong, Z. WO 2010/048603 A1, 2010.
17. Baraldi, P. G.; Preti, D.; Tabrizi, M. A.; Fruttarolo, F.; Saponaro, G.; Baraldi, S.;
Romagnoli, R.; Moorman, A. R.; Gessi, S.; Varani, K.; Borea, P. A. Bioorg. Med.
Chem. 2007, 15, 2514.
18. Musso, D. L.; Cochran, F. R.; Kelley, J. L.; McLean, E. W.; Selph, J. L.; Rigdon, G. C.;
Orr, G. F.; Davis, R. G.; Cooper, B. R.; Styles, V. L.; Thompson, J. B.; Hall, W. R. J.
Med. Chem. 2003, 46, 399.
127.09 (2C), 128.19 (2C), 135.26, 135.46, 138.96, 149.01, 164.76, 164.84,
166.38; IR (KBr, cmÀ1): 3268, 3043, 1635, 1574, 1537, 1443, 1403, 1226, 1012,
750, 697; ESI-MS m/z: 404.0 (MÀHÀ).
20. Briefly, cells (3 Â 103/well) were seeded in 96-well plates and cultured for 24 h,
followed by compounds treatment for 48 h. A volume of 10 ll of 10 mg/ml
MTT was added per well and incubated for another 2 h at 37 °C, then the
supernatant fluid was removed and DMSO was added 150 l/well for 15–
l
20 min. The light absorptions (OD) were measured at 570 nm with
SpectraMAX M5 microplate spectrophotometer (Molecular Devices). The
effect of compounds on tumor cells viability was expressed by IC50 of each
cell line.
21. Topliss, J. G. J. Med. Chem. 1972, 15, 1006.
22. Briefly, cells were seeded in 6-well plates at the density of 1 Â 105/well and
cultured for 24 h, followed by 6m treatment for another 24 h. At the indicated
intervals, both attached cells and floating cells were harvested, sediments were
19. Compound 6m: White solid, yield 85%, mp 163 °C; 1H NMR (DMSO-d6) d: 3.91
(s, 3H), 4.19 (s, 2H), 4.31 (d, J = 6.0 Hz, 2H), 7.08 (t, J = 7.6 Hz, 1H), 7.20 (d,
J = 8.4 Hz, 1H), 7.27–7.34 (m, 4H), 7.50–7.54 (m, 1H), 7.65 (dd, J = 7.6, 1.6 Hz,
1H), 7.70 (dd, J = 8.8, 2.0 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 8.53 (d, J = 2.0 Hz, 1H),
8.85 (t, J = 6.0 Hz, 1H), 10.35 (s, 1H); 13C NMR (DMSO-d6) d: 36.49, 41.89, 55.87,
111.84, 111.97, 119.16, 120.48, 120.95, 124.76, 128.12, 128.95 (2C), 129.66
(2C), 131.33, 132.11, 135.40, 135.94, 138.09, 148.79, 156.48, 164.34, 164.58,
166.55; IR (KBr, cmÀ1): 3413, 3318, 3066, 1678, 1653, 1577, 1534, 1482, 1445,
1401, 1294, 1235, 1009, 800, 750; ESI-MS m/z: 496.1 (MÀHÀ). Compound 6t:
White solid, yield 90%, mp 168 °C; 1H NMR (DMSO-d6) d: 4.19 (s, 2H), 4.30 (s,
2H), 4.33 (d, J = 5.6 Hz, 2H), 7.21–7.29 (m, 5H), 7.56 (dd, J = 8.8, 2.0 Hz, 1H),
7.79 (d, J = 8.8 Hz, 1H), 8.37 (d, J = 2.0 Hz, 1H), 8.82 (t, J = 5.6 Hz, 1H), 10.55 (s,
1H); 13C NMR (DMSO-d6) d: 36.52, 42.52, 43.55, 111.76, 118.73, 121.10, 126.76,
resuspended in 1 ml hypotonic fluorochrome solution containing 50 lg/ml PI
in 0.1% sodium citrate plus 0.1% Triton X-100, and then analyzed by flow
cytometer (ESP Elite, Beckman-Coulter, Miami, FL). Apoptotic cells appeared in
the cell cycle distribution as cells with a DNA content of less than that of G1
cells and were estimated with Listmode software.
23. Briefly, cells (1 Â 105/well) were seeded in 6-well plates and cultured for 24 h,
followed by 6m treatment for another 24 h. After rinsing with PBS, the cells
were fixed using 75% of ethanol. The morphological change of cell was
examined by inverted microscope, then the cells were stained with PI (1 lg/ml,
in PBS) and analyzed under fluorescence microscope (Zeiss, Axiovert 200,
Germany) to identify the apoptotic cells.
24. Fan, C. D.; Zhao, B. X.; Wei, F.; Zhang, G. H.; Dong, W. L.; Miao, J. L. Bioorg. Med.
Chem. Lett. 2008, 18, 3860.