Synthesis and biological evaluation of baicalein derivatives as potent antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2014.01.053

Baicalein (5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one), a major flavonoid extracted from the root of Scutellaria baicalensis Georgi (Chinese name: Huangqin), showed potent anti-proliferative activity against a broad panel of human cancer cell lines both in vitro and in vivo. A novel series of baicalein derivatives were synthesized by introducing a group to C6-OH and a nitrogen-containing hydrophilic heterocyclic ring to C7-OH via a length of 3 or 4-carbon chain in this study. The in vitro antiproliferative activities of the 30 derivatives against HepG2, A549, BCG-823 cancer cell lines were evaluated. Among them, 10 compounds exhibit more potent cytotoxicity than baicalein against the three cancer cell lines. The most potent compound 9b possesses highest anti-proliferative potency against HepG2, A549, and BCG-823 with an IC ₅₀ value of 2.0 μM, 0.8 μM and 3.2 μM, respectively. Preliminary mechanism studies with compound 9b using Annexin V/PI double-staining assay and DAPI staining assay indicated that 9b inhibits tumor cell proliferation potentially through inducing apoptosis.

Full text of DOI:10.1016/j.bmcl.2014.01.053

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1334–1338
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of baicalein derivatives as potent
antitumor agents
Rong Luo a, Jubo Wang a, Li Zhao b, Na Lu b, Qidong You a, Qinglong Guo b, Zhiyu Li a,
⇑
a Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China
b Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Baicalein (5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one), a major flavonoid extracted from the root of
Scutellaria baicalensis Georgi (Chinese name: Huangqin), showed potent anti-proliferative activity against
a broad panel of human cancer cell lines both in vitro and in vivo. A novel series of baicalein derivatives
were synthesized by introducing a group to C6-OH and a nitrogen-containing hydrophilic heterocyclic
ring to C7-OH via a length of 3 or 4-carbon chain in this study. The in vitro antiproliferative activities
of the 30 derivatives against HepG2, A549, BCG-823 cancer cell lines were evaluated. Among them, 10
compounds exhibit more potent cytotoxicity than baicalein against the three cancer cell lines. The most
potent compound 9b possesses highest anti-proliferative potency against HepG2, A549, and BCG-823
Received 1 November 2013
Revised 7 January 2014
Accepted 18 January 2014
Available online 27 January 2014
Keywords:
Flavanoid
Baicalein
Synthesis
Antitumor
Apoptosis
with an IC50 value of 2.0 lM, 0.8 lM and 3.2 lM, respectively. Preliminary mechanism studies with com-
pound 9b using Annexin V/PI double-staining assay and DAPI staining assay indicated that 9b inhibits
tumor cell proliferation potentially through inducing apoptosis.
Ó 2014 Elsevier Ltd. All rights reserved.
Flavonoids are a broad category of polyphenolic compounds
that present ubiquitously in fruits, vegetables and beverages de-
rived from plants such as tea and red wine. Flavonoids have been
demonstrated to play an important role in human health benefits,
including anti-atherosclerotic, anti-inflammatory, anticancer, anti-
thrombogenic, antiviral and anti-osteoporotic.1–6
ous study also revealed that 1 inhibits the adhesion, proliferation,
migration and invasive properties of human hepatoma cells.12 All
these results suggest that 1 possess potent antitumor activities both
in vitro and in vivo and has great therapeutic potentials.
Unfortunately, some perceived disadvantages of 1 limits its
clinical application. These include low aqueous solubility and poor
oral bioavailability. It has been reported that two metabolites of 1
in human plasma after oral administration had been identified as
baicalein 7-O-glucuronide (baicalin) and baicalein 6-O-sulfate,13
and the metabolism would less likely happened at 5-OH position
due to the intra-molecular hydrogen bond on this position. In view
of these disadvantages of 1, a nitrogen-containing hydrophilic het-
erocyclic ring was introduced to 6-OH via a certain length of car-
bon chain in order to block the metabolism of hydroxyl group of
A ring. Meanwhile, methylation or benzylation of 7-OH was con-
ducted to investigate the impact on the antitumor activity with dif-
ferent linking groups at 7-OH. 30 baicalein derivatives were
synthesized and divided into three categories according to the link-
ing groups at 7-OH.
The synthesis of baicalein derivatives was described in Scheme 1.
Benzylation of 1 was conducted by reaction with benzyl bromide in
the presence of KHCO3 to afford intermediate 2. A three or four-car-
bon linker was introduced to 6-OH to form intermediates 3 and 4,
which was subsequently reacted with several structurally diverse
amines to provide compounds 9a–9j. Debenzylation of compounds
9a–9j gave rise to compounds 11a–11h. Intermediates 3 and 4 were
Baicalein (1), 5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one, is a
major flavonoid from the root of a traditional Chinese herbal medi-
cine: Scutellaria baicalensis Georgi (Chinese name: Huangqin).7 It at-
tracted lots of attention due to its cytotoxicity to various human
cancer cell lines in vitro and inhibitory potency of tumor growth
in vivo. Great efforts have been carried put into exploring the
mechanism of the antitumor activity of 1 recent years. It has been re-
ported that 1 induces proliferation inhibition in B16F10 melanoma
cells by generating reactive oxygen species via 12-lipoxygenase.8
Another study demonstrated that the pro-apoptotic effect of 1 in
PaCa cells is mediated through reducing the expression of the pro-
survival protein Mcl-1, which is highly expressed in PaCa cell lines.9
It was shown that the anti-proliferative potency of 1 was due to cell
cycle inhibition at G0/G1, also associated with suppression of cyclin
D1 and D3 protein levels.10 Chan et al. identified 1 as an agent
preventing carcinogen–DNA adduct formation.11 In addition, previ-
⇑
Corresponding author. Tel.: +86 13951678592.
E-mail address: zhiyuli@cpu.edu.cn (Z. Li).
http://dx.doi.org/10.1016/j.bmcl.2014.01.053
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

R. Luo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1334–1338
1335
Scheme 1. Synthesis of baicalein derivatives.
first debenzylated and then methylated at 7-position with dimeth-
ylsulfate to give the intermediates 7 and 8, which then reacted with
various amines to give the final compounds 10a–10j. During the
nucleophilic substitution reaction between intermediates 7, 8 and
amines, two by-products (compounds 12, 13) were observed, and
the structures of which were identified by 1H NMR and HRMS to
be intramolecular substituted products (shown in Scheme 1). This
side reaction could be avoided through synthesizing the nitrogen-
containing side chain firstly.
The in vitro antiproliferative evaluation of all baicalein
derivatives were carried out on three human cancer cell lines
HepG2 (human liver carcinoma), A549 (adenocarcinomic human
alveolar basal epithelial) and BCG-823(gastric carcinoma) by MTT
assay with baicalein as a comparative control. All the compounds
substituent and baicalein scaffold but also the linking groups at
the 5-position, 6-position and 7-position have pronounced effects
on the biological activities of the molecules. Except for 9d and 9i,
the antiproliferative activities of compounds 9a–9j were particu-
larly potent against all the three cell lines. Compound 9b was
found to be the most potent, with IC50 values of 2.0
lM (HepG2),
0.8 M (A549) and 3.2 M (BCG-823), approximately 6–16-fold
l
l
more potent than baicalein. The substituent at 7-OH has great ef-
fect on its antitumor activity. The bulky benzyl group is the most
preferred. For example, compounds 9a–9j with a benzyl group at
7-OH were generally more active than compounds 10a–10j with
a methyl group. Introducing a hydrophilic hydroxyl group at 7-
OH resulted in a rapid decrease in activity, for instance, compounds
11a–11g were mostly inactive even at a relative high concentration
were treated over a range of concentrations from 0.1 to 100
lM
of 100 lM. This result suggest that the properties of the substitu-
for 48 h, and then the IC50 values were calculated, represents the
concentration of a drug that is required for 50% inhibition in vitro.
From the antiproliferative data presented in Table 1, not only
the length of carbon chain between the terminal heterocyclic
ents at 7-position is a crucial factor for the potency and that fol-
lows this trends: benzyloxy > methoxy ꢀ hydroxyl. It may be
attributed to the volume of the substituents. Among the deriva-
tives with the 7-benzyloxy structure (9a–9j), the molecules with

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R. Luo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1334–1338
Table 1
The structures and the in vitro antiproliferative activities of baicalein derivatives
Antitumor cell proliferation (IC50
A549
, lM)
Compound
n
R3
HepG2
BCG-823
19.1 2.0
4.2 0.5
Baicalein
0
4
H
H
28.3 2.4
2.2 0.3
13.0 1.6
1.8 0.1
9a
Bn–
9b
9c
9d
4
4
4
Bn–
Bn–
Bn–
2.0 0.2
2.4 0.2
>100
0.8 0.1
1.9 0.3
>100
3.2 0.4
3.0 0.3
83.6 4.4
9e
9f
4
3
Bn–
Bn–
2.5 0.3
2.2 0.4
4.4 0.6
2.6 0.1
7.2 0.7
5.5 0.5
9g
9h
9i
3
3
3
Bn–
Bn–
Bn–
4.2 0.4
2.2 0.3
75.8 2.8
1.4 0.2
3.10 0.3
>100
>100
4.6 0.5
51.4 4.1
9j
3
4
4
4
4
4
3
3
3
3
Bn–
2.4 0.3
7.1 0.3
4.9 0.6
21.0 3.3
19.8 2.8
36.7 2.7
3.7 0.2
4.3 0.3
22.3 2.8
9.2 0.8
1.3 0.2
4.1 0.5
10a
10b
10c
10d
10e
10f
10g
10h
10i
H3C–
H3C–
H3C–
H3C–
H3C–
H3C–
H3C–
H3C–
H3C–
35.0 1.7
13.0 2.3
34.0 3.9
25.0 3.5
32.0 2.0
4.9 0.8
17.4 2.3
10.6 1.7
21.9 2.9
27.2 2.1
53.5 6.7
5.7 0.9
6.0 0.9
6.5 0.8
17.0 2.5
14.0 2.1
25.3 3.9
43.1 7.0
10j
3
4
4
4
H3C–
H
15.1 1.6
>100
18.0 2.7
51.0 2.9
75.0 3.1
>100
31.3 4.1
86.4 3.7
52.7 2.8
58.7 8.0
11a
11b
11c
H
>100
H
67.2 8.5
11d
11e
11f
11g
4
3
3
3
H
H
H
H
93.7 6.3
>100
>100
>100
>100
>100
82.9 3.4
>100
70.6 8.8
60.9 2.1
86.2 5.7
88.3 6.4
12
13
3
4
H3C–
H3C–
—
—
69.0 9.1
>100
51.3 3.1
>100
>100
>100
The results are reported as mean value SEM for n = 3.
morpholinyl substitution at the terminal of 6-O-alkyl (9d and 9i)
exhibited much lower activities than that with N,N-diethylamino
(9a), pyrrolidinyl (9b and 9g), piperidyl (9c and 9h), and N-methyl
piperazinyl (9e and 9j). A marked decreasing in potency is
observed when morpholinyl is linking to 6-O-alkyl, which means
the morpholinyl is a poor linking group at this position. The com-
pounds containing a 4-carbon spacer between the terminal hetero-
cyclic substitutes and baicalein scaffold seemed slightly more

R. Luo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1334–1338
1337
potent than the others. This result probably indicates that the
length of the linker between the terminal heterocyclic substitutes
and baicalein scaffold could impact the activity as well and the
4-carbon length is optimal. Compounds (12 and 13) that formed
through the intramolecular nucleophilic substitution between 5-
OH and halogen displayed lower potency than baicalein. It re-
vealed that the 5-phenolic hydroxyl is also crucial to the activity.
In general, optimizing the structure of baicalein by introducing
benzyl at 7-OH, linking amine (N,N-diethylamino, pyrrolidinyl,
piperidyl or N-methyl piperazinyl) to 6-postion through a 4-carbon
alkyl and remaining 5-OH unsubstituted proved to be able to en-
hance the cytotoxic activities of baicalein.
Based on our previous research, the antitumor effect of
baicalein probably arose through apoptosis. In order to further
characterize the potential mechanism of anticancer activities of
baicalein derivatives, an apoptotic assay by flow cytometry with
Annexin V and PI staining was carried out on compound 9b, since
9b showed the highest in vitro efficiency against HepG2 cell. An-
nexin V can identify the externalization of phosphatidylserine
during the process of apoptosis, so it can be detected in both
the early and late stages of apoptosis. PI enters the cell in late
apoptosis or necrosis. Viable cells were negative for both annexin
V and PI (lower left quadrant); early apoptotic cells were positive
for annexin V and negative for PI (lower right quadrant); late
apoptotic or necrotic cells displayed both positive annexin V
and PI (upper right quadrant); non-viable cells which underwent
necrosis were positive for PI and negative for annexin V (upper
left quadrant).14 As shown in Figure 1,following the treatment
with 0.75, 1.5 and 3 lM of compound 9b for 72 h in HepG2 cells,
the percentage of early apoptotic cells (right low quadrant)
increased (from 1.4% to 30.0%), and the percentage of late apop-
totic and necrotic cells (right upper quadrant) were also increased
(from 1.4% to 50.6%).
We could characterize apoptosis under a fluorescent micro-
scope after staining the cell nucleus by DAPI because the apoptotic
cell changes in morphology such as chromatin condensation and
nuclear shrinking. In our experiments, HepG2 cells were incubated
with 0.75 lM, 1.5 lM, 3.0 lM of compound 9b and 0.2% DMSO as
control for 72 h. As shown in Figure 2, the control cells emitted
blue fluorescence with consistent intensity, indicating that the
Figure 1. Compound 9b-induced apoptosis in HepG2 cells. The cells were treated
with 9b for 72 h, and apoptosis was determined by Annexin V/PI double-staining
assay.
Figure 2. Fluorescent micrographs of DAPI staining (400Â). The arrows indicate the apoptotic cells.

1338
R. Luo et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1334–1338
9. Takahashi, H.; Chen, M. C.; Pham, H.; Angst, E.; King, J. C.; Park, J.; Brovman, E.
Y.; Ishiguro, H.; Harris, D. M.; Reber, H. A.; Hines, O. J.; Gukovskaya, A. S.; Go, V.
L.; Eibl, G. Biochim. Biophys. Acta 2011, 1813, 1465.
10. Pidgeon, G. P.; Kandouz, M.; Meram, A.; Honn, K. V. Cancer Res. 2002, 62, 2721.
11. Chan, H. Y.; Chen, Z. Y.; Tsang, D. S.; Leung, L. K. Biomed. Pharmacother. 2002, 56,
269.
12. Chiu, Y. W.; Lin, T. H.; Huang, W. S.; Teng, C. Y.; Liou, Y. S.; Kuo, W. H.; Lin, W. L.;
Huang, H. I.; Tung, J. N.; Huang, C. Y.; Liu, J. Y.; Wang, W. H.; Hwang, J. M.; Kuo,
H. C. Toxicol. Appl. Pharmacol. 2011, 255, 316.
13. Muto, R.; Motozuka, T.; Nakano, M.; Tatsumi, Y.; Sakamoto, F.; Kosaka, N.
Yakugaku Zasshi 1998, 118, 79.
chromatin was equivalently distributed in the nuclei. The test
group displayed chromatin congregated and karyopyknosis, which
emitting bright fluorescence. These results indicated that com-
pound 9b is capable of inducing apoptosis in HepG2 cells.
In conclusion, we have synthesized 30 novel baicalein deriva-
tives and evaluated their biological activities in cellular prolifera-
tion assay. Our work has led to the discovery of more potent
compounds than baicalein. 8 compounds with the moiety of 7-ben-
zyloxy group exhibited more potent anti-proliferation activities
against three tumor cell lines than baicalein. Especially, compound
9b was found to be 14-fold, 16-fold, and 6-fold more potent than
baicalein against HepG2, A549 and BCG-823 cancer cell lines,
respectively. The evidences of Annexin V/PI double-staining assay
and DAPI staining assay on 9b indicated that 9b is a potential apop-
tosis-inducing agent. Further investigation about the cellular
mechanism and other biological activities of the potent com-
pounds are in progress.
14. Wang, C. Z.; Li, X. L.; Wang, Q. F.; Mehendale, S. R.; Yuan, C. S. Phytomedicine
2010, 17, 63.
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This work was supported by the National Natural Science Foun-
dation of China (Nos. 21072232 and 21372260).
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Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
01.053. These data include MOL files and InChiKeys of the most
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References and notes
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269. http://www.sciencedirect.com/science/article/pii/S0753332202001920.
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Huang, H. I.; Tung, J. N.; Huang, C. Y.; Liu, J. Y.; Wang, W. H.; Hwang, J. M.; Kuo,
H. C. Toxicol. Appl. Pharmacol. 2011, 255, 316. http://www.sciencedirect.com/
science/article/pii/S0041008X11002687.
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Yakugaku zasshi. 1998, 118, 79. http://www.ncbi.nlm.nih.gov/pubmed/
9549431.
1. Lampila, P.; van Lieshout, M.; Gremmen, B.; Lähteenmäki, L. Food Res. Int. 2009,
42, 122.
2. Hodgson, J. M.; Croft, K. D. Mol. Aspects Med. 2010, 31, 495.
3. Fang, F.; Li, J.-M.; Pan, Q.-H.; Huang, W.-D. Food Chem. 2007, 101, 428.
4. Galati, G.; O’Brien, P. J. Free Radical Biol. Med. 2004, 37, 287.
5. Steinberg, F. M.; Bearden, M. M.; Keen, C. L. J. Am. Diet. Assoc. 2003, 103, 215.
6. Peng, S.; Zhang, G.; Zhang, B. T.; Guo, B.; He, Y.; Bakker, A. J.; Pan, X.; Zhen, W.;
Hung, L.; Qin, L.; Leung, W. N. Bone 2013, 55, 230.
7. Chou, D. S.; Hsiao, G.; Lai, Y. A.; Tsai, Y. J.; Sheu, J. R. Free Radical Biol. Med. 2009,
46, 1197.
8. Bednar, W.; Holzmann, K.; Marian, B. Food Chem. Toxicol. 2007, 45, 508.
14. Wang, C. Z.; Li, X. L.; Wang, Q. F.; Mehendale, S. R.; Yuan, C. S. Phytomedicine.
2010, 17, 63. http://www.sciencedirect.com/science/article/pii/S0944711309
001810.