Luteolin from Flos Chrysanthemi and its derivatives: New small molecule Bcl-2 protein inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.08.034

Over-expression of the Bcl-2 anti-apoptotic proteins is closely related to tumorigenesis and associated with drug resistance. Here we report that luteolin, a main substance found in Flos Chrysanthemi, directly binds to and shows inhibitory activity against the Bcl-2 protein. We studied the binding mode of luteolin and its derivatives with target proteins, their structure-activity relationship, and their effect on the human leukemia cell line HL-60. The results suggest that luteolin and its derivatives with a benzyl group introduced to the B ring, are new small molecule Bcl-2 protein inhibitors, and their anti-tumor activity is likely related to their effect on the Bcl-2 protein.

Full text of DOI:10.1016/j.bmcl.2014.08.034

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4672–4677
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Luteolin from Flos Chrysanthemi and its derivatives: New small
molecule Bcl-2 protein inhibitors
Can-Hui Zheng a, , Meng Zhang a, , Hui Chen b, , Chong-Qing Wang a, Min-Min Zhang a, Jun-Hang Jiang a,
Wei Tian a, Jia-Guo Lv a, Tie-Jun Li a, , Ju Zhu a, , You-Jun Zhou a,
⇑
⇑
⇑
a School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
b Changhai Hospital, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Over-expression of the Bcl-2 anti-apoptotic proteins is closely related to tumorigenesis and associated
with drug resistance. Here we report that luteolin, a main substance found in Flos Chrysanthemi, directly
binds to and shows inhibitory activity against the Bcl-2 protein. We studied the binding mode of luteolin
and its derivatives with target proteins, their structure-activity relationship, and their effect on the
human leukemia cell line HL-60. The results suggest that luteolin and its derivatives with a benzyl group
introduced to the B ring, are new small molecule Bcl-2 protein inhibitors, and their anti-tumor activity is
likely related to their effect on the Bcl-2 protein.
Received 16 May 2014
Revised 1 August 2014
Accepted 13 August 2014
Available online 20 August 2014
Keywords:
Luteolin
Flos Chrysanthemi
Bcl-2 protein
Apoptosis
Ó 2014 Elsevier Ltd. All rights reserved.
Anti-tumor
Apoptosis is autonomous programmed cell death for the pur-
pose of maintaining homeostasis.1 The Bcl-2 protein family plays
a key regulatory role in this process via its effect on the interaction
between anti-apoptotic proteins (Bcl-2, Bcl-xL, etc.) and pro-
apoptotic proteins (Bak, Bid, etc.).2–4 Over-expression of the Bcl-2
anti-apoptotic proteins is closely related to tumorigenesis and
associated with drug resistance.2,3 This is why Bcl-2 anti-apoptotic
proteins have become attractive targets for anti-cancer drug
development.5
small-molecular inhibitors, ABT-263,21 ABT-199,19 AT-101,22 and
GX15-070,23 have been investigated in clinical research.
Though many anticancer natural products are known to indi-
rectly affect the activity of the Bcl-2 family proteins by modulating
the expression of either anti- or pro-apoptotic proteins, there are
not many that can bind directly to anti-apoptotic proteins and
inhibit their activity. There are mainly four types of natural prod-
ucts that can bind directly to anti-apoptotic proteins, and AT-101
(gossypol acetic acid) is one of these.3,24
It has been shown that Bcl-2 anti-apoptotic proteins exert their
anti-apoptotic effects by antagonizing the pro-apoptotic members.2
This antagonistic function is mediated by binding of a hydrophobic
groove on the surface of anti-apoptotic proteins with the BH3 region
in pro-apoptotic proteins.6 In recent years, a series of small-
molecule inhibitors with different structures have been
reported,3,7–20 which show anti-tumor effects by directly binding
to the hydrophobic groove of the anti-apoptotic proteins, inhibiting
the binding of these proteins to pro-apoptotic proteins, and thereby
inhibiting the activity of anti-apoptotic proteins. At present, four
In China, the Flos Chrysanthemi is both a common health food
(chrysanthemum tea) and a traditional Chinese medicine included
in the Chinese Pharmacopoeia.25 The Flos Chrysanthemi was found
to have a variety of effects including angiocardiopathy and cancer
prevention and treatment.26–28 Luteolin (Fig. 1A), 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.29–31 Several possible mechanisms involved in the biolog-
ical activities of luteolin such as modulation of ROS levels, inhibi-
tion of topoisomerases, inhibition of PI3 K, and indirectly effect
on the activity of the Bcl-2 family proteins by modulating of the
expression and degradation of the Bcl-2 family proteins were
reported.30–33 However, the mechanism of luteolin that binding
directly to Bcl-2 anti-apoptotic proteins and thereby inhibiting
their activity has not been reported previously to our knowledge.
⇑
Corresponding authors. Tel.: +86 021 81871272 203 (T.-J.L.), +86 021 81871238
(J.Z.), +86 021 81871240 (Y.-J.Z.).
E-mail addresses: ltj204@163.com (T.-J. Li), zhuju@smmu.edu.cn (J. Zhu),
zhouyoujun@smmu.edu.cn (Y.-J. Zhou).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmcl.2014.08.034
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4672–4677
4673
Figure 1. The structures (A) and the binding modes to Bcl-2 of luteolin and its derivatives 1 and 10 (B, C, D).
Table 1
The structures and biological activity of luteolin and its analoguesa
Name
Structure
Bcl-2 (FP)
M) Ki (
Bcl-xL (FP)
M) Ki (
HL-60 (MTT)
M) IC50 (lM)
IRb (50
46 6%
l
lM)
IRb (50
49 5%
l
lM)
IRb (100
l
Luteolin
/
/
44 7%
>100
Luteolin tetraacetate
N.A.c
/
N.A.c
/
/
/
Flavone
Chrysin
N.A.c
N.A.c
/
/
N.A.c
N.A.c
/
/
/
/
/
/
Compound 1
96 3%
4.6 1.0
70 4%
8.4 1.3
80 8%
35.9 3.4
(continued on next page)

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C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4672–4677
Table 1 (continued)
Name
Structure
Bcl-2 (FP)
M) Ki (
Bcl-xL (FP)
M) Ki (
HL-60 (MTT)
M) IC50 (lM)
IRb (50
l
lM)
IRb (50
l
lM)
IRb (100
l
Compound 10
93 4%
100%
3.6 0.9
87 5%
100%
5.4 1.1
78 5%
44.0 4.2
6.6 1.9
AT-101
/
0.44 0.18
0.31 0.14
92% 6%
a
The results of the biological assay are the mean of three independent determinations.
IR: inhibition ratio.
N.A. = no observed activity.
b
c
Scheme 1. Synthesis of target compounds. Reagents and conditions: (a) DMS, K2CO3, Acetone, rt; (b) K2CO3, DMF, reflux; (c) 50% KOH,CH3OH, rt; (d): I2, Pyridine, reflux;
(e) MSA, CHCl3, microwave; (f) BBr3, DCM, rt.
In this study, we report that luteolin directly binds to and shows
inhibitory activity against the Bcl-2 protein. We studied the bind-
ing mode of luteolin and its derivatives with target proteins, their
structure-activity relationship, and their effect on the human leu-
kemia cell line HL-60. The results suggest that luteolin and its
derivatives are new small molecule Bcl-2 protein inhibitors, and
their anti-tumor activity is likely related to their effect on the
Bcl-2 protein.
When studying the bioactivity of chrysanthemum’s main com-
ponents, it was found that luteolin directly binds to and shows
inhibitory activity against the Bcl-2 and Bcl-xL proteins by fluores-
cence polarization-based binding assay (FP) (Table 1). It may be a
new small molecule Bcl-2 inhibitor. Under the same experimental
conditions, a number of other natural polyphenols, including sev-
eral natural flavonoids with different substituent groups from lute-
olin (Table 1 and Table S1 in Supplementary material), did not
show significant inhibitory activity. These suggested that this
experiment method is specificity and the biological effect of luteo-
lin to binds to Bcl-2 protein is specificity.
To further explore the binding mode of luteolin to Bcl-2 protein,
the molecular docking between luteolin and Bcl-2 protein was sim-
ulated.34–36 The result showed that luteolin bound well to the pro-
tein binding groove (Fig. 1B). The A ring and C ring of luteolin were
found to bind to the L1 domain, and the B ring entered the P1
hydrophobic pocket to form hydrophobic interactions with the
protein binding groove. The four phenolic hydroxyl groups of lute-
olin formed hydrogen bonds with G145, Y108 and E136 residues in
the protein binding groove.
Based on the luteolin binding model, we predicted that the four
phenolic hydroxyl groups in luteolin are crucial to target binding.
To test this hypothesis, a luteolin analog with the four hydroxyls
acetylated (luteolin tetraacetate) was synthesized, and two flavo-
noids without the four free hydroxyls (flavone) or two of them
(chrysin) were purchased. These compounds had significantly

C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4672–4677
4675
Figure 2. The apoptosis analysis by flow cytometry and Western blot analysis of cytochrome C expression. HL-60 cells were treated with different concentrations of Luteolin
or compound 1 for 24 h and subjected to Annexin V and PI double staining, and apoptosis was detected by flow cytometry. Typical apoptosis in HL60 cells was induced by
Luteolin (A) or compound 1 (B) (P < 0.05 and P < 0.01 vs control group). Luteolin or compound 1 induced apoptosis in HL-60 cells was dose-dependent and compound 1 was
more potent in induction of apoptosis. (C) Translocation of Cyt C into the cytoplasm of HL-60 cells was induced by Luteolin in a dose-dependent manner (25 lM and 100 lM,
P < 0.05).
reduced inhibitory activity compared to luteolin consistent with
the prediction (Table 1).
Molecular docking revealed that the benzyl group in the com-
pounds 1 and 10 could bind to the P1 pocket well, thus improve
the binding capacity between the compounds and the target pro-
tein (Fig. 1C and D). This was confirmed experimentally (Table 1).
The compounds 1 and 10 showed higher inhibitory activity than
luteolin against the Bcl-2 and Bcl-xL proteins by FP assay. The
compounds 10 showed only 8 times less inhibitory activity
against the Bcl-2 protein than AT-101 which has been investi-
gated in clinical research. All of the above results verify our model
for the binding between luteolin and the target protein, suggest-
ing that luteolin and its derivatives are new small molecule Bcl-
2 protein inhibitors.
Next, the bioactivities of luteolin and its derivatives 1 and 10 in
inhibiting the growth of human tumor cell line HL-60 were evalu-
ated by the MTT assay (Table 1). This cell line was used because it
has high Bcl-2 protein expression. The results show that these sub-
stances exhibited inhibitory activity, but the compounds 1 and 10
were significantly stronger versus luteolin, consistent with their
binding capacity to the target protein. The compound 1 showed
only 5 times less inhibitory activity than AT-101.
Further use of this model suggested that if a hydrophobic group
introduced into the B ring of luteolin can deeply reach the P1
pocket of the binding groove of Bcl-2, the binding capacity
between luteolin and Bcl-2 should increase. To test this hypothesis,
we synthesized new compounds 1 and 10 with a benzyl group
introduced to 50 and 60 on the B ring (Scheme 1). For synthesis of
the flavonoid skeleton, 2’-hydroxyl chalcone oxidative cyclization
was used. Trihydroxyacetophenone (2) was treated with control-
ling ratio (1: 2) dimethyl sulfate and provided 3 by selective meth-
ylation. Vanillin (4) was benzylated to form 5. Then intermediate 3
and 5 was condensed to provide 6 by aldol reaction, and formed 40-
benzyloxy-30,5,7-trimethoxy-flavone (7) by iodine mediated
cyclization. The introduction of the benzyl group to the flavonoid
skeleton was achieved using the benzyl rearrangement reaction
of the aromatic ether. Intermediate 7 was treated with methylsulf-
onic acid under microwave to provide 8 and 9 by benzyl rearrange-
ment. Then 8 and 9 were treated with boron tribromide to form
target compounds 1 and 10 by demethylation.

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C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4672–4677
In order to investigate whether the decreased viability attrib-
Supplementary data
uted to the apoptosis induced by the tested compounds, HL-60
cells were treated with Luteolin or compound 1, and subjected
to flow cytometry analysis, respectively. As shown in Figure 2A
and B, HL-60 cells treatment with Luteolin or compound 1 for
24 h resulted in apoptosis in a dose dependent manner. Treat-
Supplementary data (materials and methods of chemistry,
molecular docking and biology experiments) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2014.08.034.
ment with 50 lM of Luteolin or compound 1 resulted in
27.19 3.34% or 54.08 5.50% PI/Annexin V double-labeled apop-
totic cells, suggesting that compound 1 is more potent in the
induction of apoptosis in HL-60 cells. Cytochrome C is widely
believed to be localized solely in the mitochondrial intermem-
brane space under normal physiological conditions. The release
of cytochrome C from mitochondria to the cytosol, where it acti-
vates the caspase family of proteases is believed to be primary
trigger leading to the onset of apoptosis.37 Cytochrome C release
from the HL-60 cells in the presence of luteolin was also exam-
ined (Fig. 2C), and it was found that the cytosolic cytochrome C
concentration increased progressively with increasing concentra-
tion of the compound, indicating that the mitochondrial apoptotic
pathway was activated. The results of these biological experi-
ments and structure-activity relationship study suggest that the
anti-tumor activity of luteolin and its derivatives are very likely
associated with their directly binding to and inhibiting the activ-
ity of the Bcl-2 protein.
However, it was reported previously that luteolin could indi-
rectly affects the activity of the Bcl-2 family proteins by modulat-
ing the expression and degradation of either anti- or pro-apoptotic
proteins.30–32 A decrease of Bcl-2 protein was observed upon lute-
olin treatment in various cancer cell lines.38,39 The cleavage of the
proapoptotic Bcl-2 proteins to produce their truncated forms, and
the cleavage of the antiapoptotic Bcl-2 proteins into their potent
pro-apoptotic fragments was observed upon luteolin treatment
in HL-60 cells.33 Therefore, the inhibitory activity against the Bcl-
2 protein of luteolin maybe the combination of its directly and
indirect effects on the protein.
In addition, many natural flavonoids including luteolin have
been shown to possess a variety of pharmacological activities
and low specificity.40,41 It is possible to improve their specificity
by identifying the molecular mechanisms of the bioactivity of
them, and rational designing their analogues based on a particular
mechanism. In this Letter, luteolin was found to directly bind to
and show inhibitory activity against the Bcl-2 protein. Two ana-
logues of luteolin were designed based on the binding mode of
luteolin to Bcl-2 protein, and they were confirmed experimentally
to show higher affinity than luteolin with the Bcl-2 protein. We are
currently conducting experiments to further modify the structure
of luteolin to improve its inhibitory activity against the target pro-
tein and the specificity of such an effect.
In summary, this study suggests luteolin and its derivatives
with a benzyl group introduced to the B ring, are new small mole-
cule Bcl-2 protein inhibitors, and their anti-tumor activity is likely
related to their effect on the Bcl-2 protein. This Letter also provides
a potential molecular mechanism underlying the anti-tumor activ-
ity of luteolin, and sheds light on the molecular mechanisms of the
bioactivity of Flos Chrysanthemi, a plant widely utilized as both
food and medicine in China.
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