Synthesis and biological evaluation of 7-O-modified oroxylin A derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.11.117

Oroxylin A (5,7-dihydroxy-6-methoxyflavone) is a naturally occurring monoflavonoid isolated from the root of Scutellaria baicalensis Georgi, and exhibits potent anticancer activities in vitro and in vivo. In this study, we synthesized three series of orox

Full text of DOI:10.1016/j.bmcl.2011.11.117

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1118–1121
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 7-O-modified oroxylin A derivatives
Wei Fu a, Jubo Wang a, Liqin Yu a, Li Zhao b, Na Lu b, Qidong You a, Qinglong Guo b, Zhiyu Li a,
⇑
a Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
b Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Oroxylin A (5,7-dihydroxy-6-methoxyflavone) is a naturally occurring monoflavonoid isolated from the
root of Scutellaria baicalensis Georgi, and exhibits potent anticancer activities in vitro and in vivo. In this
study, we synthesized three series of oroxylin derivatives by connecting a nitrogen-containing hydro-
philic, heterocyclic ring to the C7-OH via a varying length of carbon chain. All the derivatives were
screened for anti-proliferative activities against three tumor cell lines. Some of the derivatives displayed
higher activities compared to oroxylin A. The most potent antitumor compound, 5f, also induced apop-
tosis in HepG2 cell. The difference of 5f between the inhibiting rates of cell proliferation and the apoptotic
rates indicated that 5f was more likely to be a necrosis-inducing agent or both apoptosis/necrosis
inducer.
Received 6 August 2011
Revised 7 November 2011
Accepted 28 November 2011
Available online 6 December 2011
Keywords:
Flavanoid
7-O-Modified oroxylin A
Synthesis
Anticancer
Ó 2011 Elsevier Ltd. All rights reserved.
Apoptosis
Flavanoids belong to a big class of polyphenolic secondary
metabolites in plants and in various common foods such as apple,
onion, tea, and red wine. In addition to their biological roles in
nitrogen fixation and chemical defense, flavanoids possess a broad
range of pharmacological properties, including anti-atheroscle-
rotic, anti-inflammatory, anticancer, anti-thrombogenic, antiviral
and anti-osteoporotic effects,1–6 and hence demonstrate consider-
able therapeutic potentials. However, many flavanoids have poor
oral bioavailability and limited in vivo efficacy.7
bioavailability due to its extensive first-pass metabolism, mainly
glucuronidation in gut.13 Glucuronidation of oroxylin A would
most likely happen at the 7-OH position due to the intra-molec-
ular hydrogen bond on the 5-OH position. In order to block the 7-
OH site from glucuronidation, and hence improve the oral bio-
availability of oroxylin A, a nitrogen-containing hydrophilic, het-
erocyclic ring was introduced to the 7-OH via a certain length
of carbon chain, and the antitumor activities of the derivatives
were evaluated. The length of carbon chain and the amino substi-
tution at the end of the chain was explored to investigate their
impact on the biological activities.
Oroxylin
A (1), 5,7-dihydroxy-6-methoxyflavone, is an O-
methylated flavones found in the roots of traditional Chinese
medicine Scutellaria baicalensis.8 Recently, preliminary studies by
our groups revealed the antitumor mechanisms of oroxylin A.
Hu et al demonstrated that oroxylin A effectively induced pro-
grammed cell death and suggested that it could be a promising
antitumor drug.9 Yang et al reported that oroxylin A induced
G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated
expression of Cdc2/p34 in human gastric carcinoma BGC-823
cells.10 Sun et al showed that oroxylin A suppressed MDA-MB-
435 human breast cancer cell invasion through down-regulating
the expression of matrix metalloproteinase-2/9.11 It was recently
reported that oroxylin A had an antitumor effect on human cervi-
cal cancer HeLa cell line in vitro and in vivo.12 All these reports
suggest that oroxylin A has potent anticancer activities in vitro
and in vivo.
Alkylation of 114 was carried out by reaction with di-haloge-
nated hydrocarbon in the presence of K2CO3 to afford intermedi-
ates 2. Then primary or secondary amines were coupled with the
halogenated hydrocarbons to afford the final products 3–5
(Scheme 1). Compounds in series I (3d–3h) contained a two-car-
bon alkyl chain between oroxylin A skeleton and heterocyclic
moieties; compounds in series II (4d–4h) had a 3-carbon spacer;
and compounds in series III (5d–5h) contained a 4-carbon spacer
between oroxylin A and the substituent.15
The anti-proliferative activities of all the novel 7-O-alkylamino
derivatives of oroxylin A were evaluated by MTT assay against
three human tumor cell lines (HepG2, HCT-116 and BCG-823)
with oroxylin A as a comparative control. All the treatments for
MTT assay lasted for 48 h.
Although various aforementioned in vitro studies have demon-
strated therapeutic potentials of oroxylin A, it has very low oral
As shown in Table 1, the majority of the compounds displayed
comparable or higher (lower IC50 values) anti-proliferative activi-
ties than oroxylin A. Our results confirmed that the introduction
of a side chains at 7-OH does reduce their activities against the
three cancer cell lines.
⇑
Corresponding author.
E-mail addresses: zhiyuli@263.net, zhiyuli@cpu.edu.cn (Z. Li).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.117

W. Fu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1118–1121
1119
X
HO
O
O
O
O
O
O
O
K2CO3/Acetone,dibromoalkanes
reflux,7 hrs
n
OH
OH
1
2a-2c
2a, n=0, X=Br
2b, n=1, X=Cl
2c, n=2, X=Br
R,CH3CN
Reflux,4-5 hrs
R
O
O
(2a-2c)
n
O
OH
O
3d-3h, n=0
4d-4h, n=1
5d-5h, n=2
(3d, 4d, 5d),
(3e, 4e, 5e),
(3f, 4f, 5f),
R=N,N-Diethylamine
R=Pyrrolidine
R=Piperidine
R=Morpholine
(3g, 4g, 5g),
(3h, 4h, 5h), R=N-methyl piperizane
Scheme 1. Synthesis of 7-O-alkylamino derivatives of oroxylin A.
Table 1
Antitumor cell proliferation activities of 7-O-alkylamino derivatives of oroxylin A by MTT assay
Compound
IC50 valuea
HCT116
(lM)
HepG2
BCG-823
Oroxylin A
22.7
12.0
9.7
17.1
67.6
15.4
18.0
20.5
6.8
74.8
30.4
6.6
4.7
2.4
33.9
39.6
23.4
21.1
56.3
54.9
23.4
26.6
22.1
52.4
42.5
5.1
25.5
13.0
22.7
26.9
38.2
20.5
3.5
12.0
9.7
41.6
42.3
6.3
3d
3e
3f
3g
3h
4d
4e
4f
4g
4h
5d
5e
5f
1.4
3.0
9.5
6.6
5g
5h
50.6
27.2
32.4
12.5
26.8
35.8
a
The data shown were the mean from three parallel experiments (n = 3).
Compounds (5d–5f) containing a 4-carbon spacer were particu-
IC50 at 30.4–74.8 lM) also exhibited lower activities than substitu-
larly potent with respect to cellular inhibition of all the three cell
lines, with IC50 value ranging from 1.42 to 9.52 M, approximately
5- to 20-fold more potent than oroxylin A, except for 5g and 5h.
Compound 5e and 5f were found to be the most potent, with
IC50 of 1.42 lM (HCT116) and 2.98 lM (HepG2), respectively. The
results indicate that the length of chain between the terminal het-
erocyclic substitutes and oroxylin A is an important factor for their
potency. However, with a morpholinyl or N-methyl piperazinyl
group at the terminal of 7-O-alkyl oroxylin A, compound 5g and
tions with N,N-diethylamino (3d and 4d), pyrrolyl (3e and 4e) or a
piperidyl (3f and 4f) group, respectively, regardless to the length of
carbon chain. It may be attributed to the volume of the side chain.
Based on our previous research, the antitumor effect of oroxylin
A might be through apoptosis. In order to further explore the apop-
tosis induced by 7-O-alkylamino derivative of oroxylin A, Annexin
V/PI staining assays were performed with compound 5f, which was
demonstrated the highest in vitro efficacy against HepG2 cells
among the derivatives.
l
5h (with IC50 at 12.5–50.6
l
M) displayed moderate levels of inhi-
Apoptosis was determined by staining with Annexin V/PI label-
ing, as Annexin V can identify the externalization of phosphatidyl-
serine during the process of apoptosis.16 As shown in Figure 1,
bition, which was similar to that of oroxylin A but over 4–25-fold
(HepG2), 2–30-fold (HCT116), 3–6-fold (BCG823) less potent than
5d–5f. The morpholinyl or N-methyl piperazinyl substitution in
following the treatment with 1, 2 and 4
lM of compound 5f for
the compounds 3g–h (with IC50 at 15.4–67.6 lM) and 4g–h (with
72 h in HepG2 cells, the percentage of early apoptotic cells (right

1120
W. Fu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1118–1121
low section of fluorocytogram) increased (from 1.4% to 14.0%), and
the percentage of late apoptotic and necrotic cells (right upper sec-
tion of fluorocytogram) were also increased (from 1.4% to 17.9%).
It is well-known that apoptosis can be characterized by mor-
phological and biochemical changes in the cell nucleus, such as
chromatin condensation and nuclear shrinking. In our experi-
ments, the changes in nuclei in HepG2 cells by 5f were observed
under a fluorescent microscope. As shown in Figure 2, the control
cells emitted blue fluorescence with consistent intensity, indicat-
ing that the chromatin was equivalently distributed in the nuclei.
Following incubation with compound 5f for 72 h, the HepG2 cells
displayed chromatin congregated and karyopyknosis, which emit-
ting bright fluorescence.
These above results demonstrate that compound 5f is capable
of inducing apoptosis in HepG2 cells. However, the value of MTT
assay (represents cell viability) is much more efficient than that
of Annexin V (represents cell apoptosis). The percentage of necrosis
cells induced by 5f is far away larger than that of apoptotic cells.
Thus, we hypothesize that 5f is more likely to be a necrosis-induc-
ing agent or both apoptosis/necrosis inducer, which need for fur-
ther studies in the future.
In conclusion, we have synthesized the novel 7-O-alkylamino
derivatives of oroxylin A and assessed their biological activities
in cellular proliferation assay. Among the three structural series
of derivatives, the most active compounds (5d–5f) contained a 4-
carbon spacer with appropriate amino substitutions such as N,N-
diethylamino, pyrrolyl or piperidyl groups. A 4-carbon alkyl chain
yields higher in vitro antitumor activities than the 2- and 3-carbon
chains. Our results showed that compound 5f exhibited strong
antitumor effect in HepG2 cell line, involving with apoptosis
induction. The difference of 5f between the inhibition of cell prolif-
eration and the apoptotic induction indicated that 5f was more
likely to be a necrosis-inducing agent or both apoptosis/necrosis
inducer. Thus, the novel 7-O-alkylamino derivative of oroxylin A,
compound 5f, could be a promising antitumor candidate, and fur-
ther in vitro and in vivo biological characters evaluations are
warranted.
Figure 1. Compound5f-induced apoptosis in HepG2 cells. Thecells were treatedwith
5f for 72 h, and apoptosis was determined by Annexin V/PI double-staining assays.
Figure 2. Fluorescent micrographs of DAPI staining (400Â). The arrows indicate the apoptotic cells.

W. Fu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1118–1121
1121
11. Sun, Y.; Lu, N.; Ling, Y.; Gao, Y.; Chen, Y.; Wang, L.; Hu, R.; Qi, Q.; Liu, W.; Yang,
Y.; You, Q.; Guo, Q. Eur. J. Pharmacol. 2009, 603, 22.
Acknowledgments
12. Li, H. N.; Nie, F. F.; Liu, W.; Dai, Q. S.; Lu, N.; Qi, Q.; Li, Z. Y.; You, Q. D.; Guo, Q. L.
Toxicology 2009, 257, 80.
13. Tukey, R. H.; Strassburg, C. P. Annu. Rev. Pharmacol. Toxicol. 2000, 40, 581.
This work was supported by the National Natural Science Foun-
dation of China (No. 21072232).
14. Oroxylin
A was initially obtained from Baicalein according a modified
procedure as in the reference of Sarin, P. S.; Seshadri, T. R. J. Sci. Ind. Res.
India 1960, 19B, 117.
Supplementary data
15. General procedures for the preparation of 7-O-alkylamino derivatives of oroxylin
A: (i) Procedures for the preparation of 7-O-halogenalkyl derivatives of
oroxylin A (2a–2b): To a mixture of oroxylin A (1, 1.00 g, 3.52 mmol) and
anhydrous potassium carbonate (1.46 g, 10.6 mmol) in 20 ml acetone,
corresponding di-halogenated hydrocarbon (1,2-dibromo ethane for 2a, 1-
bromo-3-chloropropane for 2b, 1,4-dibromo butane for 2c) was added. The
mixture was refluxed under a nitrogen atmosphere for 7–8 h. After completion
of the reaction, potassium carbonate was filtered and washed with excess
acetone (2 Â 50 ml). The combined acetone layer was concentrated under
vacuum. The residue was purified by column chromatography on silica gel
(60–120 mesh) to yield 7-O-alkyl oroxylin A (2a, 2b and 2c) in pure form. (ii)
Procedures for the preparation of 7-O-alkylamino derivatives of oroxylin A: To
a solution of halogenalkyl oroxylin A (2a, 2b and 2c) in 20 ml acetonitrile, the
corresponding amine was added. The mixture was refluxed under a nitrogen
atmosphere for 3–4 h. The reaction mixture was then concentrated under
vacuum. The residue was purified by column chromatography on silica gel
(60–120 mesh) to give the corresponding 7-O-alkylamino derivatives of
oroxylin A (3d–3h, 4d–4h and 5d–5h) in yields of 60–85%.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.117.
References and notes
1. Arai, Y.; Watanabe, S.; Kimira, M.; Shimoi, K.; Mochizuki, R.; Kinae, N. J. Nutr.
2000, 130, 2243.
2. Lin, J. K.; Tsai, S. H.; Lin, S. Y. Drugs Future 2001, 26, 145.
3. Birt, D. F.; Hendrich, S.; Wang, W. Pharmacol. Ther. 2001, 90, 157.
4. Tzeng, S. H.; Ko, W. C.; Ko, F. N.; Teng, C. M. Thromb. Res. 1991, 64, 91.
5. Bae, E. A.; Han, M. J.; Lee, M.; Kim, D. H. Biol. Pharm. Bull. 2000, 12, 1122.
6. Hegarty, V. M.; May, H. M.; Khaw, K. T. Am J. Clin. Nutr. 2000, 71, 1003.
7. Yang, C. S.; Sang, S.; Lambert, J. D.; Lee, M. J. Mol. Nutr. Food Res. 2008, 52, S139.
8. Huen, M. S.; Leung, J. W.; Ng, W.; Lui, W. S.; Chan, M. N.; Wong, J. T.; Xue, H.
Biochem. Pharmacol. 2003, 66, 125.
9. Hu, Y.; Yang, Y.; You, Q. D.; Liu, W.; Gu, H. Y.; Zhao, L.; Zhang, K.; Wang, W.;
Wang, X. T.; Guo, Q. L. Biochem. Biophys. Res. Commun. 2006, 351, 521.
10. Yang, Y.; Hu, Y.; Gu, H. Y.; Lu, N.; Liu, W.; Qi, Q.; Zhao, L.; Wang, X. T.; You, Q. D.;
Guo, Q. L. J. Pharm. Pharmacol. 2008, 60, 1459.
16. Urech, K.; Scher, J. M.; Hostanska, K.; Becker, H. J. Pharm. Pharmacol. 2005, 57,
101.