Synthesis and biological evaluation of novel 7-O-lipophilic substituted baicalein derivatives as potential anticancer agents

Article abstract of DOI:10.1039/c5md00163c

We synthesized derivatives of baicalein, wogonin, and chrysin through alkylation at the 7-O-position of the A ring with lipophilic terphenyl or long chain n-alkyl groups, and studied the in vitro anticancer activity of the derivatives through the growth inhibition MTT assay. We discovered that baicalein and two of its derivatives were good free radical scavengers. Among the 20 synthesized derivatives, 7-O-farnesylbaicalein (5d) and 7-O-dodecylbaicalein (5i) demonstrated stronger growth inhibition against human colon cancer SW480 cells compared with baicalein, with half maximal inhibitory concentration (IC₅₀) values of 1.15 and 1.57 μM, respectively. Furthermore, 5d and 5i dose- and time-dependently inhibited the growth of SW480 cells. Cell cycle distribution analysis showed that 5d and 5i induced SW480 cell arrest at the S phase through an apoptotic mechanism, which was associated with an increase in the generation of reactive oxygen species. In conclusion, the potent anticancer activity of the baicalein derivatives (5d and 5i) suggested that the derivatives are potential anticancer agents for human colon cancer.

Full text of DOI:10.1039/c5md00163c

View Article Online
View Journal
MedChemComm
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: S. Wang, C.-H.
Chen, C. Lo, J. Feng, H. Lin, P. Chang, L. Yang, L. Chen, Y. Liu, C. Kuo and J. Wu, Med. Chem. Commun.,
2015, DOI: 10.1039/C5MD00163C.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/medchemcomm

Page 1 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
1
2
Synthesis and Biological Evaluation of Novel 7-O-Lipophilic
Substituted Baicalein Derivatives as Potential Anticancer Agents
3
4
5
6
Shao-Hung Wang,a Ching-Hsein Chen,a Chih-Yu Lo,b Ji-Zhen Feng,a Hong-Jhih Lin,a
Po-Ya Chang,a Ling-Ling Yang,c Lih-Geeng Chen,a Yi-Wen Liu,a Cheng-Deng Kuod
and Jin-Yi Wu*a
7
8
9
a Department of Microbiology, Immunology and Biopharmaceuticals, College of Life
Sciences, National Chiayi University, Chiayi 60004, Taiwan
10
11
b Department of Food Science, College of Life Sciences, National Chiayi University,
Chiayi 60004, Taiwan
12
c College of LOHAS, Fo Guang University, Yilan County 26247, Taiwan
13
14
d Department of Medical Research and Education, Taipei Veterans General Hospital,
Taipei 11217, Taiwan
15
16
17
18
19
20
21
22
23
Running title: Anticancer activity of 7-O-substituted baicalein derivatives
24
25
26
27
*Corresponding author: Jin-Yi Wu, Ph.D., Department of Microbiology, Immunology
and Biopharmaceuticals, College of Life Sciences, National Chiayi University, 300
University Road, Chiayi 60004, Taiwan; Tel.: +886-5-2717925; Fax: +886-5-2717778;
E-mail: jywu@mail.ncyu.edu.tw
1

MedChemComm
Page 2 of 27
View Article Online
DOI: 10.1039/C5MD00163C
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Abstract
We synthesized derivatives of baicalein, wogonin, and chrysin through alkylation
at the 7-O-position of the A ring with lipophilic terpenyl or long chain n-alkyl groups,
and studied the in vitro anticancer activity of the derivatives through the growth
inhibition MTT assay. We discovered that baicalein and two of its derivatives were
good
free
radical
scavengers.
Among 20
synthesized
derivatives,
7-O-farnesylbaicalein (5d) and 7-O-dodecylbaicalein (5i) demonstrated stronger
growth inhibition against human colon cancer SW480 cells compared with baicalein,
with half maximal inhibitory concentration (IC50) values of 1.15 and 1.57 ꢀM,
respectively. Furthermore, 5d and 5i dose- and time-dependently inhibited the growth
of SW480 cells. A cell cycle distribution analysis showed that 5d and 5i induced
SW480 cell arrest at the S phase through an apoptotic mechanism, which was
associated with an increase in the generation of reactive oxygen species. In conclusion,
the potent anticancer activity of the baicalein derivatives (5d and 5i) suggested that
the derivatives are potential anticancer agents for human colon cancer.
Keywords: Baicalein, Lipophilic substituent, Colon cancer, Cytotoxic activity, Flow
cytometry
2

Page 3 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
61 1. Introduction
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
Colorectal cancer (CRC) is the most commonly diagnosed cancer and is a
leading cause of cancer mortality. It remains a considerable major heath concern both
globally1 and in Taiwan, where the number of patients diagnosed with CRC has
increased; such patients generally have poor prognosis. Therapeutic strategies to treat
CRC include surgery, chemotherapy, and radiation therapy;2 however, the side effects
associated with chemotherapy, and radiation therapy, the high mortality rates and local
tumour recurrence associated with surgical procedures3,4 necessitate alternative
therapeutic options for CRC patients.
Flavonoids are a group of compounds found in several plant sources, such as
citrus fruits, seeds, olive oil, and cocoa, as well as in tea and red wine.5 They are
low-molecular-weight compounds containing a three-ring (polyphenolic) structure
with various substituents.6 For decades, flavonoids have been utilized for their
significant pharmacological activities, including anticancer activity.7 Studies have
shown that flavonoids reduce the risk of cancer, inflammation, and heart diseases.8
Additional studies have demonstrated that flavonoids possess an antioxidant,
anti-inflammatory, anti-allergic, antiviral, and hepatoprotective activity.9 Both in vitro
and in vivo xenograft models have shown that flavonoids are cytotoxic to various
human cancer cell lines, suggesting their potential as anticancer agents.10–12
Flavonoids are present in abundant quantities in traditional Chinese medicinal
herbs, such as Huang-Qin (Scutellaria baicalensis Georgi). Four major flavonoids
present in Huang-Qin are baicalein, baicalin, oroxylin A, and wogonin (Fig. 1).
Baicalein is widely used as an antioxidant, anti-inflammatory, and anticancer
agent.13,14 A hepatic metabolic study demonstrated that the bioavailability and
effectiveness of baicalein decreased rapidly in the intestinal tract upon
glucuronidation or sulfation of the hydroxyl group at the 7-O-position. More than
90% of baicalein is converted to baicalein-7-O-glucuronide (baicalin) in the
intestines.15–17 Baicalein is a selective inhibitor of 12-lipooxygenase, which is
responsible for the production of reactive oxygen species (ROS) during arachidonic
acid metabolism.18 Shieh et al. demonstrated that baicalein acts as a strong scavenger
of the superoxide radicals in a cell-free system through rapid donation of hydrogen
ions.19 Thus, these polyphenols may be vital in preventing human oxidative stress by
scavenging hydroxyl, DPPH, and alkyl free radicals.20,21 Various methods, such as
3

MedChemComm
Page 4 of 27
View Article Online
DOI: 10.1039/C5MD00163C
94
95
DPPH and ABTS•+ radical scavenging, have been used to estimate the in vitro
antioxidant activity of baicalein derivatives. The other two flavonoid components
isolated from S. baicalensis Georgi, oroxylin A and wogonin, have also been reported
to possess anticancer activity.18–23
96
97
98
The current study was conducted to determine whether baicalein derivatives
with lipophilic moieties exhibit increased cell permeability and thus higher
intracellular oxidative stress and cytotoxicity. We synthesized and evaluated a series
of lipophilic substituted baicalein derivatives, with a focus on substitution at the
7-O-position of the A-ring with terpenyl or long chain n-alkyl groups. Moreover, we
examined the effects of the synthesized derivatives on cell proliferation, cell cycle
progress, and apoptosis against the three human colon cancer cell lines, one human
liver cancer cell line, and one mouse normal cell line.
99
100
101
102
103
104
105
106 2. Chemistry
107
108
109
110
111
112
Derivatives of baicalein (1), wogonin (3), and chrysin (4) were synthesized
through 7-O-alkylation with a long chain n-alkyl or terpenyl bromide. As shown in
Scheme 1 and Scheme 2, 5a-i, 6a-d, and 7a-d were synthesized according to a
procedure reported in the literature by reacting baicalein (1), wogonin (3), and chrysin
(4) with selected n-alkyl or terpenyl bromides in anhydrous acetone using anhydrous
potassium carbonate (K2CO3) as a base under N2 for 8‒24 h.14
113
114
115
116
117
118
119
120
121
122
In brief, the reaction mixture was refluxed for 8–24 h, and the progress of the
reaction was monitored using TLC. Subsequently, the reaction mixture was cooled to
room temperature and the solvent was removed under reduced pressure. The crude
product was chromatographed on a silica gel column and eluted with EtOAc/n-Hex
(1/3) to afford the desired baicalein, wogonin, and chrysin derivatives (5a-i, 6a-d, and
7a-d, respectively) in 40.0–72.3% yield (Scheme 1 and 2). The 7-O-substituted
derivatives were the major products and the 6,7-O-disubstituted derivatives were only
minor products in yields less than 10%. The structures of the flavonoid derivatives
1 13
were determined through H and C NMR spectra, and liquid chromatography–mass
spectrometry.
123 3. Results and discussion
4

Page 5 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
124
3.1. Analysis of antioxidant activity of baicalein derivatives
125
126
127
128
129
130
The hydroxyl group of the A ring of baicalein was first alkylated at the
7-O-position with terpenyl or long chain n-alkyl groups, and free-radical scavenging
activity examined using both the DPPH and ABTS•+ scavenging methods.20,21 For
comparison, the antioxidant activities of baicalein, oroxylin A, ascorbic acid and
quercetin were analyzed. As shown in Table 1, a minor difference in DPPH and
ABTS•+ free radical activities was observed among analyzed derivatives.
131
3.2. Analysis of in vitro anti-proliferative activity using MTT assay
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
The cytotoxic activity of the synthesized derivatives was evaluated using in vitro
growth inhibition assays using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT).22 The derivatives were rated according to their half maximal
inhibitory concentration values (IC50), a measure of the effectiveness of a compound in
inhibiting biological and biochemical function; the lower the IC50 value, the more
effective the compound at inhibiting cancer cell growth. The cytotoxic activity of 20
flavonoid derivatives, along with those of 5-fluorouracil (5-Fu), cisplatin, and
doxorubicin as positive controls, were examined against four human cancer cell lines,
including SW480 (colon carcinoma), HT29 (colon carcinoma), DLD-1 (colon
carcinoma), and HepG2 (liver carcinoma), and one normal murine embryonic liver
BNL CL.2 cell line. The cLogP and IC50 values of tested compounds are listed in
Table 2. The anticancer activity of the derivatives increased with the chain length, or
lipophilic characteristics, of the substitutes. The lipophilic activity was evident in the
two striking bioactive derivatives, namely 7-O-farnesylbaicalein (5d) and
7-O-dodecylbaicalein (5i). Both showed significant cell growth inhibition in all four
human cancer cell lines with IC50 values of 1.15
0.15 and 1.57
0.20 ꢀM,
respectively, against the SW480 cell line. These values were 16- to 11-fold more
active compared with baicalein (1), which exhibited an IC50 value of 18.18 0.89 ꢀM.
When treated with baicalein (1), 5d, or 5i, no significant cell death was detected in the
normal murine embryonic liver BNL CL.2 cell line. Furthermore, only a marked effect
on cell death (< 20%) was observed at the highest concentration tested (20 ꢀM) for 5d
and 5i after 48 h treatment. Baicalein (1) exhibited a slight cytotoxic effect after 48 h,
suggesting that both 5d and 5i were cytotoxic to human colon cancer cells with no
significant adverse effects on normal murine embryonic liver cells.
5

MedChemComm
Page 6 of 27
View Article Online
DOI: 10.1039/C5MD00163C
156
157
158
159
160
161
162
163
164
165
166
167
In the light of the cytotoxicity-related findings described previously, we examined
the cytotoxicity of 5d and 5i against SW480 cells as a function of time. As shown in
Table 3, the in vitro cytotoxicity of 5d and 5i demonstrated a 12- and 8-fold increase
on SW480 cells for 48 h treatment, respectively, compared with baicalein (1).
We hypothesized that the increased activity of synthesized derivatives
attributable to their enhanced bioavailability and cell membrane permeability that
resulted from the increased lipophilicity. To investigate this hypothesis, we calculated
cLogP values of the 7-O-substituted baicalein derivatives and compared their
anticancer activity. The cLogP values correlated satisfactorily with IC50 values (Table
3). Hence, these results support our hypothesis that lipophilicity, or the chain length,
of n-dodecyl and farnesyl moieties of 7-O-substituted baicalein derivatives played a
vital role in the anticancer activity of those derivatives.
168
3.3. Cell morphological assessment
169
170
171
172
173
174
175
176
177
178
179
180
Cell apoptosis was observed by examining Hoechst 33258 stained cell nuclei
through fluorescent microscopy. Apoptosis was determined as changes in cell
morphology, such as chromatin condensation, nuclear shrinking, and DNA
fragmentation.23 To further investigate the role of apoptosis in the cytotoxicity of 5d
and 5i, SW480 cells were incubated with 20 ꢀM of baicalein (1), 5d, or 5i, and in 0.1%
DMSO as control, for 48 h. The cells were stained with Hoechst 33258 and examined
using fluorescence microscopy for topical morphological changes. The nuclei of the
cells in the control sample were round and stained homogenously, whereas those
treated with baicalein (1), 5d, and 5i exhibited typical morphological features of
apoptosis such as nuclear shrinkage, chromatin condensation and DNA fragmentation
(Fig. 3).24 These results demonstrated that 5d and 5i induced apoptosis in SW480
cells.
181
3.4. Cell cycle distribution analysis through flow cytometry
182
183
184
185
186
To analyze the apoptotic effects of baicalein (1), 5d, and 5i on cell cycle
progression, SW480 cells were treated with the synthesized derivatives at their
different concentrations for 48 h. The cell cycle distribution and the subG1 phase were
analyzed through flow cytometry after propidium iodide (PI) staining.25 Untreated cells
were used as the control. The percentage of cells at the S phase increased by 33.33%
6

Page 7 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
187
188
189
190
191
192
193
194
195
196
197
198
199
and 50.00% when treated with 10 and 20 ꢀM of 5d, respectively, and by 32.12% and
51.02% when treated with 10 and 20 ꢀM of 5i, respectively, compared with a 26.19%
increase in the control (Fig. 4). The increased percentage of cells at the S phase and
the cytotoxic activity of 5d and 5i suggested that the synthesized derivatives induced
SW480 cells arrest in the S phase. In addition, baicalein was also found to increase the
percentage of cells at the S phase following the 24 h exposure at the concentration of
50 ꢀM for the test compounds. Analysis of the S-phase arrest, however, showed that
the concentration of all cell cycle regulatory molecules cyclin-dependent kinase 4
(CDK4), cyclin B1, and cyclin D1 showed a decreased level.26 Our results indicated
that baicalein did not affect cell cycle in the SW480 cells. A study26 showed that
baicalein dose-dependently inhibited the growth of human lung squamous carcinoma
CH27 cells. The results of the current study may suggest that the apoptotic effect of
baicalein depends on the type of the cancer cells under treatment.
200
3.5. Annexin V-FITC/PI staining
201
202
203
204
205
206
207
208
209
210
211
212
213
To study the bioactivity of baicalein (1), 5d, and 5i against SW480 cells, the
cancer cells were treated with vehicle alone as control, or with one of the three test
compounds at their different concentrations (5, 10, and 20 ꢀM). After 48 h, the samples
were double-stained with annexin V-FITC and PI.27 The percentages of cells at various
stages of apoptosis are shown in Fig. 5. The data indicated that apoptotic cell death
resulting from treatment with 5d or 5i was dose-dependent; however, this was not
observed in cells treated with baicalein (1). Starting from a dose of 10 ꢀM, both 5d and
5i induced a higher degree of apoptosis in SW480 cells compared with baicalein (1)
and cytotoxic effects at both the early and late stages, as determined through annexin
V-FITC/PI staining. For baicalein (1), the effect was observed only at a higher
concentration (20 ꢀM). The analysis confirmed that the superior efficiency of both 5d
and 5i, in inducing cytotoxicity and inhibiting the proliferation of human colorectal
cancer cells.
214
3.6. Measurement of intracellular ROS production
215
216
217
Several flavonoids induce apoptosis by generating reactive oxygen species
(ROS) in mitochondria.28 Baicalein and its derivatives are hypothesized to induce
apoptosis by increasing the concentration of intracellular ROS. Therefore, we
7

MedChemComm
Page 8 of 27
View Article Online
DOI: 10.1039/C5MD00163C
218
219
220
221
222
223
224
225
226
227
228
229
230
investigated whether baicalein (1), 5d, or 5i could stimulate generation of ROS in
SW480 cells. The fluorescence intensity of dihydroethidium (DHE) in the cells was
right-shifted after the cells were treated with all three compounds in a
concentration-dependent manner (Fig. 6), indicating that both 5d and 5i could
•‒
stimulate the release of intracellular O2 from SW480 cells. As expected, 5d and 5i,
at the concentrations of 5, 10, and 20 ꢀM, exhibited a more profound effect (mean
values = 102.13, 169.24, and 192.86, respectively, for 5d) and (mean values = 117.62,
165.78, and 215.12, respectively, for 5i), compared with baicalein (mean values =
•–
104.84, 99.49, and 98.79, respectively) on O2 generation in SW480 cells after 48 h
of treatment (P < 0.05, Fig. 6B). Thus, the results showed that 5d and 5i induced
apoptosis by increasing the intracellular oxidative stress of SW480 cells, and
exhibited a strong capacity to induce apoptosis in SW480 cells in a ROS-dependent
manner.
231 4. Conclusions
232
233
234
235
236
237
238
239
240
241
242
Among 20 analyzed derivatives, the baicalein derivatives 5d and 5i, which have a
farnesyl group and a dodecyl group at the 7-O-position, respectively, showed a
superior level of cytotoxicity in all human cancer cell lines studied. Furthermore, we
discovered that 5d and 5i were the most cytotoxic in vitro, against human colon
adenocarcinoma SW480 cells. In the cell cycle distribution and apoptotic analysis, 5d
and 5i induced SW480 cells arrest at the S phase. In the Hoechst 33258 staining
analysis, 5d and 5i markedly induced apoptosis, which was confirmed by the positive
rate of annexin V-FITC/PI double staining. Both derivatives induced apoptosis in
SW480 cells, by the inducing ROS generation. The results indicate that 5d and 5i
have an enhanced anticancer activity against human colon cancer cells compared with
standard treatments.
243 Acknowledgements
244
245
246
The authors thank Dr. Christopher Liu for his valuable editorial revision. This
study was supported by grants from Taipei Veterans General Hospital, Taiwan
(V103E2-002 and V104E2-002).
247 Notes and references
8

Page 9 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
1. WHO Report. http://www.who.int/mediacentre/factsheets/fs297/en/index.html.
2. A. Jemal, F. Bray, M. M. Center, J. Ferlay, E. Ward and D. Forman, CA Cancer J.
Clin., 2011, 61, 69‒90.
3. S. Carnesecchi, K. Langley, F. Exinger, F. Gosse and F. Raul, J. Pharmacol. Exp.
Ther., 2002, 301, 625–630.
4. M. Line-Edwige, F. Raymond, E. Francois and N. E. Edouard, Afr. J. Tradit.
Complement. Altern. Med., 2009, 6, 112–117.
5. K. A. Youdim, J. P. E. Spencer, H. Schroeter and C. Rice-Evans, Biol. Chem.,
2005, 383, 503–519.
6. A. Pick, H. Müller, R. Mayer, B. Haenisch, I. K. Pajeva, M. Weigt, H. Bönisch, C.
E. Müller and M. Wiese, Bioorg. Med. Chem., 2011, 19, 2090–2102.
7. M. López-Lázaro, Curr. Med. Chem. Anticancer Agents, 2002, 2, 691–714.
8. E. J. Middleton, C. Kandaswami and T. C. Theoharides, Pharmacol. Rev., 2000,
52, 673–751.
9. O. A. Aiyegoro and A. I. Okoh, Int. J. Mol. Sci., 2009, 10, 4990–5001.
10. R. J. Nijveldt, E. van Nood, D. E. van Hoorn, P. G. Boelens, K. van Norren and P.
A. van Leeuwen, Am. J. Clin. Nutr., 2001, 74, 418–425.
11. Y. Suh, F. Afaq, J. J. Johnson and H. Mukhtar, Carcinogenesis, 2009, 30, 300–307.
12. H. L. Liu, W. B. Jiang and M. X. Xie, Recent Pat. Anticancer Drug Discov., 2010,
5, 152–164.
13. Z. Gao, K. Huang, X. Yang and H. Xu, Biochim. Biophys. Acta, 1999, 1492, 643‒
650.
14. J. Y. Wu, K. T. Chung, Y. W. Liu, F. J. Lu, R. S. Tsai, C. H. Chen and C. H. Chen,
J. Agric. Food Chem., 2008, 56, 2838‒2845.
15. C. Li, L. Zhang, L. Zhou, S. K. Wo, G. Lin and Z. Zuo, AAPS J., 2012, 14, 23–34.
16. L. Zhang, G. Lin, B. Kovács, M. Jani, P. Krajcsi and Z. Zuo, Eur. J. Pharm. Sci.,
2007, 31, 221–231.
17. L. Zhang, G. Lin and Z. Zuo, Pharm. Res., 2007, 24, 81–89.
18. K. Sekiya and H. Okuda, Biochem. Biophys. Res. Commun., 1982, 105, 1090–
1095.
19. D. E. Shieh, L. T. Liu and C. C. Lin, Anticancer Res., 2000, 20, 2861–2865.
20. T. Yamaguchi, H. Takamura, T. Matoba and J. Terao, Biosci. Biotechnol. Biochem.,
1998, 62, 1201–1204.
9

MedChemComm
Page 10 of 27
View Article Online
DOI: 10.1039/C5MD00163C
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
21. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-Evans,
Free Radic. Biol. Med., 1999, 26, 1231–1237.
22. T. Mosmann, J. Immunol. Methods, 1983, 65, 55‒63.
23. C. Y. Lo, L. C. Hsu, M. S. Chen, Y. J. Lin, L. G. Chen, C. D. Kuo and J. Y. Wu,
Bioorg. Med. Chem. Lett., 2013, 23, 305‒309.
24. H. B. Zhang, P. Lu, Q. Y. Guo, Z. H. Zhang and X. Y. Meng, Oncol. Lett., 2013, 5,
722–728.
25. S. J. Kim, H. J. Kim, H. R. Kim, S. H. Lee, S. D. Cho, C. S. Choi, J. S. Nam and J.
Y. Jung, Mol. Med. Rep., 2012, 6, 1443–1449.
26. H. Z. Lee, H. W. Leung, M. Y. Lai and C. H. Wu, Anticancer Res., 2005, 5, 959–
964.
27. R. Luo, J. Wang, L. Zhao, N. Lu, Q. You, Q. Guo and Z. Li, Bioorg. Med. Chem.
Lett., 2014, 24, 1334–1338.
28. L. Burnaugh, K. Sabeur and B. A. Ball, Theriogenology 2007, 67, 580–589.
10

Page 11 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
Figures and Tables captions
Scheme 1 Synthesis of 7-O-substituted baicalein derivatives.
Scheme 2 Synthesis of 7-O-substituted wogonin and chrysin derivatives.
Fig. 1 Chemical structures of baicalein (1), baicalin (2), wogonin (3), and chrysin (4).
Fig. 2 In vitro cytotoxicity of (A) baicalein (1), (B) 5d, and (C) 5i in SW480 cells
after drug exposure over various time periods.
Fig. 3 Morphological changes in SW480 cells treated with baicalein (1), 5d, or 5i at
20 µM for 48 h (magnification, 200×). Upper panels show the cell morphology under
phase‒contrast microscopy, and the lower panels display the Hoechst 33258-stained
nuclear patterns detected though fluorescence microscopy (magnification, 200×). Red
arrows indicate the apoptotic cells.
Fig. 4 Effect of baicalein (1) and its derivatives on SW480 cell cycle distribution. (A)
Cell cycle distribution after treatment with baicalein (1), 5d, or 5i at 5‒20 ꢀM in
SW480 cells for 48 h. (B) Quantitative difference of cell cycle distribution changed
after treatment with baicalein (1), 5d, and 5i at 5‒20 ꢀM in SW480 for 48 h. Data are
shown as the mean SD of three independent experiments, and *P < 0.05, **P < 0.01,
***P < 0.001 compared with control.
Fig. 5 Effect of baicalein (1), 5d, or 5i on cell apoptosis and necrosis of SW480 cells
assessed through flow cytometry. (A) Analysis of cell death pathway after the
treatment with baicalein (1), 5d, or 5i at 5‒20 µM in SW480 cells for 48 h. (B)
Quantitative analysis of cell death pathway after the treatment with baicalein (1), 5d,
and 5i at 5‒20 µM in SW480 cells for 48 h. Plates were examined for apoptotic cells
using an Annexin V-FITC apoptosis detection kit. Annexin V‒positive/PI‒negative
cells are in the early stages of apoptosis and double positive cells are in late apoptosis,
whereas Annexin V–negative/PI–positive cells are necrotic. Each value represents the
mean SD of three independent experiments. *P < 0.05, compared with control.
11

MedChemComm
Page 12 of 27
View Article Online
DOI: 10.1039/C5MD00163C
•‒
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
Fig. 6 Effect of baicalein (1), 5d, or 5i on ROS (O2 ) generation in SW480 cells. (A)
Analysis after treatment with baicalein (1), 5d, or 5i at 5‒20 ꢀM in SW480 cells for
48 h to detect O2•‒ content. (B) Quantitative analysis after treatment with baicalein (1),
5d, or 5i at 5‒20 ꢀM in SW480 cells for 48 h to detect O2•– content. Data are shown as
the mean SD of three independent experiments, and *P < 0.05, compared with
control.
Table 1 Antioxidant activity of baicalein (1) and its derivatives (5a-i), trolox, ascorbic
acid, and quercetin
Table 2 cLogP values and cytotoxic activities (IC50, ꢀM) of baicalein, wogonin,
chrysin and their derivatives against four human cancer cell lines and one normal cell
line after drug exposure for 48 h
Table 3 In vitro cytotoxicity of baicalein (1), 5d, and 5i against a panel of human
colon cancer cell lines over various time periods
12

Page 13 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
381
382
Scheme 1 Synthesis of 7-O-substituted baicalein derivatives.
383
384
aReagents and conditions: (a) n-alkyl or terpenyl bromide, anhydrous K2CO3,
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
anhydrous acetone, reflux, 8–24 h.
13

MedChemComm
Page 14 of 27
View Article Online
DOI: 10.1039/C5MD00163C
403
404
Scheme 2 Synthesis of 7-O-substituted wogonin and chrysin derivatives
OH
O
O
OH
O
O
a
R
HO
O
R'
R'
Wogonin (3), R = OCH3
Chrysin (4), R = H
6a d and 7a d
6a (R' = OCH3); 7a (R' = H), R = -CH3
6b (R' = OCH3); 7b (R' = H), R =
6c (R' = OCH3); 7c (R' = H), R =
6d (R' = OCH3); 7d (R' = H), R =
405
406
aReagents and conditions: (a) methyl iodide or terpenyl bromide, anhydrous K2CO3,
407
408
409
410
411
412
413
414
415
416
anhydrous acetone, reflux, 8–24 h.
14

Page 15 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
Fig. 1 Chemical structures of baicalein (1), baicalin (2), wogonin (3) and chrysin (4).
15

MedChemComm
Page 16 of 27
View Article Online
DOI: 10.1039/C5MD00163C
Fig. 2 In vitro cytotoxicity of (A) baicalein (1), (B) 5d, and (C) 5i in SW480 cells after drug
exposure over various time periods.
16

Page 17 of 27
MedChemComm
Fig. 3 Morphological changes in SW480 cells treated with baicalein (1), 5d, or 5i at 20 ꢀM for 48 h (magnification, 200×). Upper panels show
the cell morphology under phase‒contrast microscopy, and the lower panels display the Hoechst 33258-stained nuclear patterns detected though
fluorescence microscopy (magnification, 200×). Red arrows indicate the apoptotic cells.
17

MedChemComm
Page 18 of 27
18

Page 19 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
Fig. 4 Effect of baicalein (1) and its derivatives on SW480 cell cycle distribution. (A)
Cell cycle distribution after treatment with baicalein (1), 5d, or 5i at 5‒20 ꢀM in
SW480 cells for 48 h. (B) Quantitative difference of cell cycle distribution changed
after treatment with baicalein (1), 5d, and 5i at 5‒20 ꢀM in SW480 for 48 h. Data are
shown as the mean SD of three independent experiments, and *P < 0.05, **P < 0.01,
***P < 0.001 compared with control.
19

MedChemComm
Page 20 of 27
20

Page 21 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
Fig. 5 Effect of baicalein (1), 5d, or 5i on cell apoptosis and necrosis of SW480 cells
assessed through flow cytometry. (A) Analysis of cell death pathway after the
treatment with baicalein (1), 5d, or 5i at 5‒20 ꢀM in SW480 cells for 48 h. (B)
Quantitative analysis of cell death pathway after the treatment with baicalein (1), 5d,
and 5i at 5‒20 ꢀM in SW480 cells for 48 h. Plates were examined for apoptotic cells
using an Annexin V-FITC apoptosis detection kit. Annexin V‒positive/PI‒negative
cells are in the early stages of apoptosis and double positive cells are in late apoptosis,
whereas Annexin V–negative/PI–positive cells are necrotic. Each value represents the
mean SD of three independent experiments, and *P < 0.05, compared with control.
21

MedChemComm
Page 22 of 27
22

Page 23 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
•‒
Fig. 6 Effect of baicalein (1), 5d, or 5i on ROS (O2 ) generation in SW480 cells. (A)
Analysis after treatment with baicalein (1), 5d, or 5i at 5‒20 ꢀM in SW480 cells for
48 h to detect O2•‒ content. (B) Quantitative analysis after treatment with baicalein (1),
5d, or 5i at 5‒20 ꢀM in SW480 cells for 48 h to detect O2•– content. Data are shown as
the mean SD of three independent experiments, and *P < 0.05, compared with
control.
23

MedChemComm
Page 24 of 27
View Article Online
DOI: 10.1039/C5MD00163C
Table 1 Antioxidant effect of baicalein (1) and its derivatives (5a-i), trolox, ascorbic
acid, and quercetin
IC50 (ꢀM)a (mean SD)
Compound
DPPH
ABTS•+
Baicalein (1)
17.18 1.26
28.40 2.06
23.97 1.13
24.34 1.56
24.59 2.30
12.91 0.57
23.59 0.30
21.53 0.29
28.58 0.52
23.51 0.60
22.70 0.03
27.30 0.05
8.41 0.64
15.54 1.26
20.22 1.36
18.83 0.47
20.43 1.66
23.99 1.64
13.98 0.45
20.37 0.79
20.27 1.50
23.40 0.59
26.27 1.85
16.80 0.02
18.30 0.55
7.26 0.01
5a
5b
5c
5d
5e
5f
5g
5h
5i
Trolox
Ascorbic acid
Quercetin
a Compound concentration required to eliminate rate by 50 %. Data are expressed as the mean SD
from the dose response curves of at 3‒5 independent experiment.
24

Page 25 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
Table 2 cLogP values and cytotoxic activities (IC50, ꢀM) of baicalein, wogonin, chrysin and their
derivatives against four human cancer cell lines and one normal cell line after drug exposure for 48 h
IC50 (ꢀM)b (mean SD)
Compound
Baicalein (1)
clogPa
SW480c
HT-29c
DLD-1c
HepG2d
BNL CL.2e
> 40
> 40
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 40
> 20
> 20
> 20
> 20
> 40
> 20
> 20
> 20
> 20
-
3.00
3.33
5.03
7.06
9.09
4.92
5.98
7.03
8.09
9.15
3.33
3.77
5.48
7.51
9.54
3.56
4.15
5.85
7.88
9.91
18.18 0.89 30.61 0.46 27.88 0.31 28.09 0.51
29.41 0.46
8.60 0.37
> 40
> 20
30.93 0.65
> 20
> 40
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
5a
5b
2.84 0.43 17.02 0.25
1.15 0.15 14.95 0.63
9.77 0.93
6.97 0.15
> 20
5c
5d
> 20
19.48 0.35
5e
9.48 0.47 17.65 0.16 19.01 0.52
3.03 0.46 16.25 0.62 15.41 0.76
5f
5g
1.99 0.38
1.57 0.20
> 20
> 20
15.52 0.47
9.26 0.10
5h
5i
Wogonin (3)
35.06 3.84 39.55 0.23 36.87 0.65 37.89 2.26
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
2.72 0.84
> 20
6a
6b
> 20
6c
> 20
6d
Chrysin (4)
7a
31.08 2.96 19.49 0.38 18.62 0.81 16.50 0.36
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
7b
> 20
7c
> 20
7d
f
Cisplatin
5-Fu
40.72 1.18 24.07 0.03
-
36.07 3.11
40.18 7.63
0.30 0.02
32.72 8.32
> 100
-
-
-
Doxorubicin
0.53 0.07
1.70 0.20
-
a Calculated log value of partition coefficient by ChemDraw Ultra 11.0.
b Compound concentration (ꢀM) required to inhibit tumor cell proliferation rate by 50 %. Data are expressed as the mean
SD from the dose response curves of at 3–5 independent experiments.
c Human colon adenocarcinoma cell lines
d Human hepatocarcinoma cell lines
e Normal murine embryonic liver cell lines
f Not tested
25

MedChemComm
Page 26 of 27
Table 3 In vitro cytotoxicity of baicalein (1), 5d, and 5i against a panel of human colon cancer cell lines over various time periods
IC50 (ꢀM)a (mean SD)
Compound
SW480b
48 h
HT-29b
48 h
DLD-1b
48 h
24 h
72 h
24 h
> 20
72 h
24 h
> 20
72 h
> 20
Baicalein (1) 16.95 0.47 14.05 1.09 11.55 2.63
18.05 1.68 18.36 2.98
> 20
> 20
> 20
1.21 0.03 1.26 0.13
1.84 0.08 1.64 0.58
> 20
> 20
16.80 0.68 15.40 0.46 16.15 1.02 4.88 0.58 6.86 2.59
5d
5i
> 20
> 20
> 20
7.23 1.42 6.19 1.71
a Compound concentration required to inhibit tumor cell proliferation rate by 50 %. Data are expressed as the mean SD from the dose response curves of at 3–5
independent experiment.
b Human colon adenocarcinoma cell lines
26

Page 27 of 27
MedChemComm
View Article Online
DOI: 10.1039/C5MD00163C
Synthesis and Biological Evaluation of Novel 7-O-Lipophilic
Substituted Baicalein Derivatives as Potential Anticancer Agents
Shao-Hung Wang, Ching-Hsein Chen, Chih-Yu Lo, Ji-Zhen Feng, Hong-Jhih Lin,
Po-Ya Chang, Ling-Ling Yang, Lih-Geeng Chen, Yi-Wen Liu, Cheng-Deng Kuo and
Jin-Yi Wu*
Graphical abstract
A series of lipophilic 7-O-substituted baicalein derivatives were synthesized and evaluate
for their anticancer activity against four human cancer cell lines.
OH
O
OH
O
O
HO
HO
HO
Extracted
Modified
R
O
7
7
1
)
Baicalein (
5d
: R =
Scutellaria baicalensis Georgi
5i: R =