Structural modification of luteolin from Flos Chrysanthemi leads to increased tumor cell growth inhibitory activity

Article abstract of DOI:10.1016/j.bmcl.2016.06.043

The luteolin from Flos Chrysanthemi was found to directly bind to the Bcl-2 protein and inhibit the tumor cell growth in our previous study. However, it has been shown to possess wide and week biological activities. In this study, a series of derivatives of luteolin were designed and synthesized, and their tumor cell growth inhibitory activities were evaluated against human leukemia cell line HL-60. The results showed that compounds 1B-2, 2A-3, and 2B-5, with hydrophobic substituted benzyl groups introduced to B ring and hydrogen or methyl introduced to 7-OH group of luteolin, exhibited the strongest inhibitory activity with the IC₅₀lower than 10?μM, which were significantly more potent than luteolin. The studies presented here offer a good example for modifications of flavones to improve their tumor cell growth inhibitory activities.

Full text of DOI:10.1016/j.bmcl.2016.06.043

Bioorganic & Medicinal Chemistry Letters 26 (2016) 3464–3467
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structural modification of luteolin from Flos Chrysanthemi leads
to increased tumor cell growth inhibitory activity
Chao Yang a,y, Hui Chen b,y, Shihai Lu a,y, Meng Zhang a, Wei Tian a, Mingping Wang a,c, Ling Zhang a,
Yunlong Song a, Aijun Shen d, Youjun Zhou a, , Ju Zhu a, , Canhui Zheng a,
⇑
⇑
⇑
a School of Pharmacy, Second Military Medical University, Shanghai 200433, China
b Changhai Hospital, Second Military Medical University, Shanghai 200433, China
c Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
d State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The luteolin from Flos Chrysanthemi was found to directly bind to the Bcl-2 protein and inhibit the tumor
cell growth in our previous study. However, it has been shown to possess wide and week biological activ-
ities. In this study, a series of derivatives of luteolin were designed and synthesized, and their tumor cell
growth inhibitory activities were evaluated against human leukemia cell line HL-60. The results showed
that compounds 1B-2, 2A-3, and 2B-5, with hydrophobic substituted benzyl groups introduced to B ring
and hydrogen or methyl introduced to 7-OH group of luteolin, exhibited the strongest inhibitory activity
with the IC50 lower than 10 lM, which were significantly more potent than luteolin. The studies
presented here offer a good example for modifications of flavones to improve their tumor cell growth
inhibitory activities.
Received 27 October 2015
Revised 18 January 2016
Accepted 16 June 2016
Available online 16 June 2016
Keywords:
Luteolin
Flos Chrysanthemi
Structural modification
Bcl-2 protein
Anti-tumor
Ó 2016 Elsevier Ltd. All rights reserved.
In China, the Flos Chrysanthemi is both a common health food as
chrysanthemum tea and a traditional Chinese medicine included in
the Chinese Pharmacopoeia.1,2 Luteolin (Fig. 1), a main substance
found in Flos Chrysanthemi, is a flavonoid contained in many plants,
and has been shown to possess a variety of pharmacological activ-
ities, including antioxidant, anti-inflammatory and anticancer
activities. Several possible mechanisms involved in the biological
activities of luteolin were reported.3–5 In our previous study, that
luteolin directly binds to and shows inhibitory activity against
the activity of Bcl-2 protein, and its anti-tumor activity is related
to the effect were reported.6
Flavones are a class of natural products widely distributed in
different plants with a wide range of biological activities. However,
many natural flavones including luteolin have been shown to pos-
sess week biological activities and low specificity.7–10 It is possible
to improve their specificity by rational designing their analogues
based on a particular mechanism. In our previous study, the
derivatives of luteolin with a benzyl group introduced to the B ring
(30-benzyl luteolin and 20-benzyl luteolin) (Fig. 1) were designed,
and showed increased inhibitory activity against the activity of
Bcl-2 protein and tumor cell growth inhibitory activity.6 In addi-
tion, the benzyl-substituted flavone compounds are rare in nat-
ure,11–14 which are a structural type worthy of further study.
The oxidative cyclization of 20-hydroxy-chalcone and the acidic
rearrangement of benzyl groups in aryl benzyl ethers were used to
totally synthesize benzyl-substituted flavone compounds based on
our previous study (Scheme 1A).6,15 In the re-synthesis process of
30-benzyl luteolin and 20-benzyl luteolin, two new byproducts of
the last demethylation reaction were found. Through exhaustive
isolation, purification and structural characterization, they were
confirmed to be the products of partial demethylation (30-benzyl-
7-methoxy luteolin and 20-benzyl-7-methoxy luteolin) (Fig. 1).
The bioactivities of these derivatives in inhibiting the growth of
human leukemia cell line HL-60 were evaluated by the MTT assay
(Table 1). In order to further improve its tumor cell growth inhibi-
tory activity, a series of derivatives of luteolin were designed and
synthesized with different substituted benzyl groups introduced
to B ring and hydrogen or methyl introduced to 7-OH group
(Fig. 1). The structures of the target compounds are listed in
Table 1.
⇑
Corresponding authors. Tel.: +86 021 81871231 (Y.Z.), +86 021 81871238 (J.Z.),
+86 021 81871240 (C.Z.).
E-mail addresses: zhouyoujun@smmu.edu.cn (Y. Zhou), zhuju@smmu.edu.cn (J.
Zhu), canhuizheng@smmu.edu.cn (C. Zheng).
y
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2016.06.043
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

C. Yang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3464–3467
3465
OH
OH
OH
OH
HO
O
O
HO
O
O
OH
OH
3'-benzyl luteolin
2'-benzyl luteolin
OH
OH
OH
OH
OH
O
O
O
OH
O
O
HO
O
O
OH
OH
O
2'-benzyl-7-methoxy
luteolin
3'-benzyl-7-methoxy luteolin
OH
luteolin
OH
OH
OH
OH
R2O
O
R2O
O
O
R1
R1
OH
O
OH
2
1
Figure 1. The structures of luteolin and its derivatives.
The target derivatives of luteolin were synthesized by the
modification of our previous procedure of benzyl-substituted fla-
vone compounds (Scheme 1B), which was reduced two steps to
improve the synthesis efficiency. Starting from key intermediate
11, a serial of flavone benzyl ether compounds 12 were synthe-
sized by the benzylation reaction in the presence of different sub-
stituted benzyl chlorides with yields higher than 80%. Using
microwave, the intermediate 12 underwent a rearrangement
reaction in the presence of methylsulfonic acid. This reaction
yielded rearrangement products 13 and 14 with the benzyl group
introduced to the 20 or 30 on the B ring with moderate yields
respectively, and debenzylated product 11. Finally, intermediates
13 and 14 were treated with boron tribromide to obtain the prod-
ucts of completely demethylation 1A and 2A, and the products of
partial demethylation 1B and 2B with moderate yields respec-
tively. Although the yield of the single reaction via this route
was not high, the intermediate 11 could be re-used to improve
the overall yield of the multiple reactions.
and 1A, respectively. It suggested that the methyl introduced to
7-OH group of luteolin were also good for improving its activities.
However, the more detailed SAR of them was not clear, which
should be with the help of more derivatives synthesized in the
future study.
To test whether the target compounds in this study would bind
to Bcl-2 protein and inhibit it activity, representative compound 20-
benzyl-7-methoxy was subjected to evaluation by fluorescence
polarization-based binding assay (FP assay) (Table 2).6,16,17 Lute-
olin, 20-benzyl luteolin and AT-101 were also evaluated by this
assay as controls. The IC50 of this compound was 1.3 lM, lower
than luteolin, 20-benzyl luteolin and slight higher than AT-101.
Considering that it was consistent with their tumor cell growth
inhibitory activities, it seems that Bcl-2 protein represents an
important potential target for these compounds. In addition, the
toxicity of these compounds was evaluated by testing the growth
inhibitory activities against the normal human HEK-293 cell line.
The results indicated their low toxicity (Table 1S).
The growth inhibitory activities of the target compounds on HL-
60 were tested (Table 1). AT-101 which also derivated from the
natural product and has been investigated in clinical research
was used as a positive control. Among them, compounds 1B-2,
2A-3, and 2B-5 showed the strongest inhibitory activity with the
In summary, the luteolin from Flos Chrysanthemi was found to
directly bind to the Bcl-2 protein and inhibit the tumor cell growth
in our previous study. However, it has been shown to possess wide
and week biological activities. In this study, a series of derivatives
of luteolin were designed and synthesized, and their tumor cell
growth inhibitory activities were evaluated on human leukemia
cell line HL-60. The results showed that compounds 1B-2, 2A-3,
and 2B-5, with hydrophobic substituted benzyl groups introduced
to B ring and hydrogen or methyl introduced to 7-OH group of lute-
olin, exhibited the strongest inhibitory activity with the IC50 lower
IC50 lower than 10 lM, which were significantly more potent than
luteolin and even equivalent to AT-101. The main objective of this
study was achieved. In addition, the preliminary structure–activity
relationships (SAR) of these compounds were further discussed.
Most of the compounds showed stronger inhibitory activity than
luteolin, and half of them showed stronger inhibitory activity than
30-benzyl luteolin and 20-benzyl luteolin. It indicated that the
hydrophobic substituted benzyl groups introduced to B ring of
luteolin were good for improving its activities. Moreover, most of
compounds 2B and 1B were more potent than compounds 2A
than 10 lM, which were significantly more potent than luteolin.
The studies presented here provide a new structural type for the
development of novel antitumor agents, and also offer a good
example for future modifications of flavones to improve their
tumor cell growth inhibitory activities.

3466
C. Yang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3464–3467
A
OH
3
O
O
H
OH
a
O
O
HO
OH
O
7
O
4
c
O
OH
O
d
O
O
O
O
O
b
O
H
OH
OH
Cl
HO
HO
O
O
5
6
OH
O
3'-benzyl luteolin
OH
f
OH
O
O
O
OH
O
O
O
O
9
OH
O
O
3'-benzyl-7-methoxy luteolin
O
OH
O
O
OH
OH
e
O
O
O
HO
O
O
O
O
f
8
O
OH
10
11
2'-benzyl luteolin
OH
OH
OH
O
O
O
Cl
O
O
O
O
b
OH
O
2'-benzyl-7-methoxy luteolin
B
OH
Cl
R1
O
O
O
O
b
O
11
OH
OH
O
O
O
R1
OH
O
O
R2O
O
O
O
O
O
f
e
R1
R1
O
O
OH
O
12
1A R2 = H
13
1B R2= Me
R1
-1 2-
-2 3-
-3 4-
-4 2-
-5 3-
-6 4-
Cl
OH
Cl
Cl
Me
Me
Me
O
OH
OH
R2O
O
f
O
O
O
R1
R1
OH
O
O
2A R2 = H
14
2B R2 = Me
Reagents and conditions:
a: DMS, K2CO3, Acetone, rt, 75%;
c: 50% KOH,CH3OH, rt, 70%;
b: K2CO3, DMF, reflux, 80-90%;
d: I2, Pyridine, reflux, 71%;
e: MSA, CHCl3, microwave, 20-40%; f: BBr3, DCM, rt., 20-40%.
Scheme 1. Synthesis of target compounds.

C. Yang et al. / Bioorg. Med. Chem. Lett. 26 (2016) 3464–3467
3467
Table 1
The structures and biological activity of luteolin and its analogues
Name
Structure
HL-60 (MTT)
IR (100M)
44.00
R1
/
R2
/
IC50
>100
44.0
>100
28.80
>100
>100
20.93
100.00
>100
13.93
2.92
(
l
M)
Luteolin
/
30-Benzyl luteolin
1A-1
1A-2
1A-3
1A-4
–H
2-Cl
3-Cl
4-Cl
2-Me
3-Me
4-Me
–H
2-Cl
3-Cl
–H
–H
–H
–H
–H
78
30.58
OH
OH
76.44
48.61
31.49
71.86
49.11
37.48
80.79
85.17
68.94
55.02
R2O
O
O
R1
1A-5
1A-6
–H
–H
OH
30-Benzyl-7-methoxy luteolin
–CH3
–CH3
–CH3
–CH3
–CH3
1B-1
1B-2
1B-4
1B-5
2-Me
3-Me
14.76
62.95
20-Benzyl luteolin
2A-1
–H
2-Cl
–H
–H
80.00
73.43
35.90
41.83
OH
OH
2A-2
2A-3
2A-6
3-Cl
4-Cl
4-Me
–H
2-Cl
3-Cl
4-Cl
2-Me
3-Me
4-Me
/
–H
–H
–H
65.22
100.00
19.90
81.95
85.83
85.55
92.11
90.17
86.13
96.40
92.00
64.37
7.05
R2O
O
O
>100
14.90
20.54
10.31
11.68
14.91
9.97
R1
20-Benzyl-7-methoxy luteolin
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
–CH3
/
OH
2B-1
2B-2
2B-3
2B-4
2B-5
2B-6
AT-101
10.91
6.60
/
Table 2
The inhibitory activity against the Bcl-2 and Bcl-xL proteins of luteolin and its analogues
Name
Bcl-2 (FP)
Bcl-xL (FP)
Inhibition rate (50
Inhibition rate (50
l
M) (%)
Ki (lM)
lM) (%)
Ki (lM)
Luteolin
46
96
100
100
6
3
/
49
70
100
100
5
4
/
20-Benzyl luteolin
20-Benzyl-7-methoxy luteolin
AT-101
4.6 1.0
1.3 0.6
0.44 0.18
8.4 1.3
2.5 0.8
0.31 0.14
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Supplementary data (materials and methods of chemistry and
biology experiments, the NMR spectrum of 20-benzyl-7-methoxy
luteolin, and the growth inhibitory activities against the normal
human HEK-293 cell line) associated with this article can be found,
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References and notes
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Beijing, China, 2010; p 292. vol. 1.