Cytisine-flavonoid conjugates: Synthesis and antitumor structure-activity relationship research

Article abstract of DOI:10.1016/j.tetlet.2020.151803

In research of anti-triple negative breast cancer (TNBC) agents, a series of cytisine-flavonoid conjugates (A-1 ~ G-1) were designed and synthesized in high yields with (?)-cytisine and flavonoids via N,N-4-dimethyl-4-aminopyridine (DMAP)-catalyzed synthe

Full text of DOI:10.1016/j.tetlet.2020.151803

Journal Pre-proofs
Cytisine-flavonoid conjugates: synthesis and antitumor structure-activity rela‐
tionship research
Renhao Liu, Xiaoze Bao, Xuanrong Sun, Yue Cai, Tianwei Zhang, Xinyi Ye,
Xing-Nuo Li
PII:
S0040-4039(20)30238-0
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.151803
TETL 151803
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
27 January 2020
19 February 2020
3 March 2020
Please cite this article as: Liu, R., Bao, X., Sun, X., Cai, Y., Zhang, T., Ye, X., Li, X-N., Cytisine-flavonoid
conjugates: synthesis and antitumor structure-activity relationship research, Tetrahedron Letters (2020), doi:
https://doi.org/10.1016/j.tetlet.2020.151803
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1
Cytisine-flavonoid conjugates: synthesis and antitumor structure-activity
relationship research
Renhao Liu a, Xiaoze Bao a, Xuanrong Sun a, Yue Cai a, Tianwei Zhang a, Xinyi Ye a,
Xing-Nuo Li a,
aCollege of Pharmaceutical Science & Collaborative Innovation Center of Yangtze
River Delta Region Green Pharmaceuticals, Zhejiang University of Technology,
Hangzhou 310014, P. R. China
*Corresponding author: Tel: +86-571-88320613
E-mail: li_xingnuo@163.com

2
Abstract: In research of anti-triple negative breast cancer (TNBC) agents, a series of
cytisine-flavonoid conjugates (A-1~G-1) were designed and synthesized in high yields
with (-)-cytisine and flavonoids via N,N-4-dimethyl-4-aminopyridine (DMAP)-
catalyzed synthetic strategy. In addition, the in vitro cytotoxicity of the conjugates was
tested, and the results showed that some compounds had better cytotoxicity against
human breast cancer cells (MDA-MB-231) than the positive control drug Paclitaxel.
The studies of structure-activity relationship showed that the conjugates formed by
cytisine featuring 5-chloro and flavonoids had better anti-proliferative effect on MDA-
MB-231 cells.
Keywords: Cytisine, Flavonoid, Antitumor, Structure-activity relationship

3
1. Introduction
Natural products have afforded a rich source of compounds and made a huge
contribution to human health, especially in the anticancer area, as many anticancer
drugs are from natural products and their derivatives1-3. Nowadays, the treatment of
cancer remains a major challenge, encouraging the sustaining discovery of new lead
compounds. Among the research subjects of anticancer drug discovery, the treatment
of triple negative breast cancer (TNBC), characterized by tumors that do not express
estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor
receptor 2 (HER2)4, has been a tough task for breast oncologists for a long time.
(-)-Cytisine is a quinolone alkaloid in the family Leguminosae or Fabaceae, which
has been used as respiratory analgesic5 and smoking cessation drug in Europe for many
years6. Now, it has been used clinically to treat nicotine addiction7. Moreover, recent
studies clearly showed that chemically modified cystine showed interesting anti-
proliferative activity against human cancer cells8. Therefore, the development of
rational structural modification of (-)-cytisine9 for the lead compound discovering for
the treatment of TNBC is meaningful.
The combination of pharmacophores is a useful strategy for the rational design of
lead compounds. In this context, the natural product tonkinensine B isolated from the
roots of Sophora tonkinensis, featuring the biologically active (-)-maackiain and (-)-
cytisine pharmacophores, was found to having good cytotoxicity against human breast
cancer cells (MDA-MB-231) and low cytotoxic effects on the central nervous system
in our previous research10. Interestingly, tonkinensine B could be synthesized easily by

4
our biomimetic strategy from (-)-maackiain and (-)-cytisine. However, the small
amount of (-)-maackiain in plants greatly limited the synthesis of tonkinensine B in
large quantities11,12, and made the further biological research of tonkinensine B difficult.
As we all know, (-)-maackiain belongs to the flavonoid family, which is the part of
polyphenols that widely distributed in natural plants. The structures of flavonoids are
consisted of two benzene rings (A and B) and an oxygen-containing heterocyclic ring
(C) (Figure 1). The subclasses of flavonoids included flavones, isoflavones, flavanones,
and others13,14. In addition, flavonoids have a wide range of pharmacological properties
in terms of antioxidant activity, hepatoprotective activity, antibacterial activity and
anticancer activity due to their structure diversity15,16. For instance, epicatechin and
rutin are strong radical scavengers and inhibitors of lipid peroxidation in vitro17.
Silymarin can be used to treat cirrhosis, ischemic injury, and toxic hepatitis18. In recent
years, a large number of studies have been carried out on the structural modification of
flavonoids, as well as the structure-activity relationship, and the modes of action of the
corresponding compounds19-21. The results showed that flavonoids were known to be
good cytotoxic agents22-27, and certain anti-proliferative effects on human cancer cells
were also determined28-30
.
In this context, we hypothesized that the replacement of (-)-maackiain with readily
available natural flavonoids to form a new array of conjugate with (-)-cytisine could
circumvent the low yield of tonkinensine B. Herein, a new series of cytisine-flavonoid
conjugates and their structure-activity relationship against MDA-MB-231 cells were
reported.

5
2. Results and discussion
2.1. Synthesis
According to previous research, the methylene linker of the conjugates could be
installed via established aminomethyiation process31-35. Thus, with the good
nucleophilicity of flavonoids, the cytisine-flavonoid conjugates can be synthesized
readily with (-)-cytisine and flavonoids via N,N-4-dimethyl-4-aminopyridine (DMAP)-
catalyzed aminomethylation under mild conditions. Accordingly, several (-)-cytisine
derivatives (1 ~ 5) and natural flavonoids (A ~ E) were used as substrates (Figure 1).
To our delight, with the participation of formaldehyde (HCHO), the desired conjugates
(A-1 ~ E-5) were obtained in good yields (Scheme 1) by using N,N-4-dimethyl-4-
aminopyridine as the catalyst and 1,4-dioxane as the solvent. The result showed that
the aminomethylation of 7-hydroxyflavonoids occurred predominantly at 8-position of
the chromone core. In the aim of increasing the structure diversity of the conjugates,
we assumed that the occupation of 8-position will release the nucleophilicity at 6-
position. To prove this hypothesis, a one-pot iodination/Suzuki cross-coupling cascade
of flavonoid A and C was conducted (Scheme 2) to block the 8-position. The high
regioselectivity of the iodination with N-iodosuccinimide (NIS) was facilitated by the
good nucleophilicity at 8-position and the fluorinated alcohols36. Thus, the 8-phenyl
substituted flavonoid derivatives F and G were obtained in good yields.
With the 8-position blocked flavonoid derivatives F and G in hand, the
aminomethylation process was tested again (Scheme 3). Expectingly, the 6-
cytisinylmethyl derivatives F-1 and G-1 were formed smoothly under the same

6
conditions. Therefore, a conclusion can be drawn that aminomethylation of 7-
hydroxyflavonoids mainly occurred at 8-position of the chromone core. When 8-
position was occupied, the reaction occurred at 6-position. The yields of all this new
series of conjugates (A-1 ~ G-1) were listed in Table 1. It’s worth noting that all of the
synthesized compounds (except E-133) are first reported.
2.2. Biological Evaluation
It is known that natural flavonoids and (-)-cytisine have antitumor activities8, 28-30
.
Since the synthesis process completely retains the skeletons of these two natural
compounds, it is expected that the newly obtained conjugates will have meliorative
biological activities. Thus, the cytotoxicity efficacy of all synthesized compounds (A-
1 ~ G-1) were tested on MDA-MB-231 cells by using 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. The results obtained were summarized
in Figure 2 (A ~ D), with the drug Paclitaxel (PTX) as the positive control.
The experiment results showed that the newly prepared compounds had better anti-
proliferation effects on MDA-MB-231 cells than the starting materials. Some
compounds even had better cytotoxicity against MDA-MB-231 cells than PTX (IC50 =
25.2 ± 6.3 µM), such as A-2 (IC50 = 22.3 ± 1.6 µM), A-3 (IC50 = 21.9 ± 0.8 µM), A-4
(IC50 = 18.4 ± 0.3 µM), B-4 (IC50 = 24.6 ± 2.2 µM), and E-4 (IC50 = 20.1 ± 0.1 µM).
The same set of compounds (A-1 ~ A-5) exhibited good anti-proliferative effects on
MDA-MB-231 cells.
2.3. Structure-activity relationship
Structure-activity relationship (SAR) studies of the synthesized compounds revealed

7
the dependence of activity on the type of substitutions presenting both on the cytisine
and flavonoids pharmacophores. It should be noted that the cytisine-flavonoid
conjugates (A-4, B-4, E-4) formed from 5-chlorocytisine (4) and flavonoids (A, B, E)
had good performance of anti-proliferative effect on MDA-MB-231 cells. The better
potent compound A-4 (IC50 = 18.4 ± 0.3 µM), which exhibited about 1.4 times
improvement in activity compared to the positive control drug Paclitaxel (IC50 =ꢀ25.2 ±
6.3 µM). Therefore, 5-chlorocytisine is of great significance for improving the
antitumor activity among cytisine-flavonoid derived compounds. Conversely, the
introduction of electron withdrawing groups (such as chloro-, bromo-, and nitro-) at 3-
position of (-)-cytisine has no positive effect and may even reduce the activity.
At the same time, we found that the cytisine-flavonoid conjugates synthesized from
7-hydroxyflavonoid (A) had better antiproliferative effect than the ones from 7-
hydroxyisoflavone and 7-hydroxyflavanone. Interestingly, with the effects of the
hydroxyl or methoxy group at 4-position of 7-hydroxyisoflavones (D and E), the
potency of (-)-cytisine-7-hydroxyisoflavones conjugates against MDA-MB-231 cells
can be improved. However, not only the 8-cytisinylmethyl derivatives had a better
cytotoxicity against MDA-MB-231 cells, but also the 6-cytisinylmethyl derivatives (F-
1 and G-1) exhibited good performance of the anti-proliferative effect. (Figure 3)
3. Conclusion
In summary, a novel class of cytisine-flavonoid conjugates were designed and
synthesized from readily available flavonoids and (-)-cytisine in order to improve the

8
inhibitory activity against MDA-MB-231 cells. The results showed that most of the
compounds had good cytotoxic activity against MDA-MB-231 cells, and five analogs
A-2, A-3, A-4, B-4, and E-4 showed better inhibitory activity. Compound A-4
containing mono substituted electron-withdrawing chlorine group at 5-position of (-)-
cytisine was the better potent inhibitor (IC50 = 18.4 ± 0.3 µM), and it was found to have
about 1.4 times better activity compared to paclitaxel. Thus, further modification and
structure derivatization may lead to the development of selective inhibitors against
MDA-MB-231 cells with more potency for the treatment of TNBC patients.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
Acknowledegments
We thank National Natural Science Foundation of China (NO. 81502941) and
Zhejiang Provincial Natural Science Foundation of China (NO. LY16H300006) for
financial support of this work.
Appendix A. Supplementary data
Supplementary dataassociated with this article can be found, in the online version, at
http://

9
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12
Table 1. Appearance, Yields and IC50 values of Compounds
Entry
Compounds
Structure
Appearance
Yields (%)
IC50 (µM)
N
N
O
1
A-1
White solid
73%
35.0 ± 1.1
HO
O
O
Cl
Cl
N
N
O
2
3
4
A-2
A-3
A-4
Yellow solid
White solid
White solid
61%
64%
75%
22.3 ± 1.6
21.9 ± 0.8
18.4 ± 0.3
HO
O
O
Br
Br
N
N
O
HO
O
O
Cl
N
N
O
HO
O
O
O2N
N
N
O
5
6
A-5
B-1
Yellow solid
Yellow solid
85%
70%
58.6 ± 7.9
57.0 ± 1.2
HO
O
O
N
N
O
HO
O
O
Cl
Cl
N
N
O
7
8
9
B-2
B-3
B-4
Yellow solid
Yellow solid
White solid
44%
57%
44%
>200
>200
HO
O
O
Br
Br
N
N
O
HO
O
O
Cl
N
N
O
24.6 ± 2.2
HO
O
O

13
O2N
N
N
O
10
11
B-5
C-1
Yellow solid
White solid
70%
82%
>200
HO
O
O
N
N
O
HO
O
O
93.0 ± 6.4
Cl
Cl
N
N
O
12
13
14
C-2
C-3
C-4
White solid
White solid
White solid
82%
84%
78%
>200
HO
O
O
Br
Br
N
N
O
>200
HO
O
O
Cl
N
N
O
34.6 ± 6.3
HO
O
O
O2N
N
N
O
HO
O
15
16
C-5
D-1
Yellow solid
White solid
84%
82%
>200
O
N
N
O
HO
O
O
137.6 ± 16.2
OH
Cl
Cl
N
N
O
17
18
D-2
D-3
White solid
63%
63%
74.8 ± 0.2
HO
O
O
OH
Br
Br
N
N
O
White solid
104.3 ± 0.6
HO
O
O
OH

14
Cl
N
N
O
19
D-4
White solid
59%
32.0 ± 0.4
HO
O
O
OH
O2N
N
N
O
HO
O
O
20
21
D-5
E-1
Yellow solid
White solid
55%
83%
156.4 ± 16.8
OH
N
N
O
HO
O
O
58.1 ± 2.5
O
Cl
Cl
N
N
O
22
23
24
E-2
E-3
E-4
White solid
White solid
White solid
83%
76%
63%
＞200
＞200
HO
O
O
O
Br
Br
N
N
O
HO
O
O
O
Cl
N
N
O
20.1 ± 0.1
HO
O
O
O
O2N
N
N
O
HO
O
25
26
27
E-5
F-1
G-1
Yellow solid
White solid
White solid
82%
58%
62%
107.0 ± 6.9
83.1 ± 4.9
38.7 ± 2.2
O
O
HO
O
O
N
N
O
HO
O
N
N
O
O
28
29
Positive Control
1
Paclitaxel
White solid
—
—
25.2 ± 6.3
Yellow solid
105.1 ± 2.3
N
NH
O

15
Cl
Br
30
31
32
33
2
3
4
5
Yellow solid
Yellow solid
Yellow solid
Yellow solid
40%
79%
45%
61%
＞200
Cl
Br
N
NH
NH
O
O
＞200
＞200
＞200
N
Cl
N
NH
O
O2N
N
NH
O
HO
O
O
34
35
A
B
Yellow solid
Redsolid
—
—
＞200
＞200
HO
HO
O
O
O
36
37
38
C
D
E
Yellow solid
White solid
White solid
—
—
—
＞200
＞200
＞200
O
HO
O
O
OH
HO
O
O
O
HO
O
O
39
40
F
White solid
White solid
72%
51%
＞200
HO
O
O
G
109.26 ± 13.68

16
Figure Captions
Figure 1. The structures of flavonoids (A ~ E) and cytisine derivatives (1 ~ 5)
Figure 2. Comparing of the cytotoxicity efficacy of cytisine-flavonoid conjugates
(except IC50 > 200).
Figure 3. SAR study of cytisine-flavonoid conjugates as potent anti-breast cancer
agent.

17
3'
2'
1'
4'
5'
1
O
8
B
HO
O
HO
HO
O
7
6
2
6'
A
C
3
4
5
O
O
O
R1
1
A
B
C:
R = H
1
D
: R = OH
2
3
R3
1
1
: R = R = H;
E
: R = OCH3
2
3
2
3
4
5
: R = R = CI;
R2
2
3
: R = R = Br;
N
NH
2
3
: R = H, R = CI;
O
2
3
: R = NO2, R = H
Figure 1.

18
Figure 2.

19
Electron withdrawing group has no
positive effect on enhancing the
antibreast cancer activity and may
even reduce it.
Introduction of CI group on 5-position
of cytisine showed good activity against
MDA-MB-231cells.
R3
R2
The hydroxyl or methoxy group
can improve the potency against
MDA-MB-231 cells.
N
N
O
CH2
HO
R4
O
O
R1
s
an
activity
group
play
cytisine
substituted
The
linke
ids
flavono
with
d
ng
improvi
in
t role
importan
cells.
MDA-MB-231
nst
agai
The substituted aryl group linked with the
position 2 of chromone ring showed good
activity against MDA-MB-231 cells.
The cytisinylmethyl group linked with
flavonoids exhibits good performance
in the anti-proliferative effect.
The carbon-carbon double bond of
chromone moiety is essential to
enhance the antibreast cancer activity.
Figure 3.

20
Scheme Captions
Scheme 1. Synthesis of cytisine-flavonoid conjugates A-1 ~ E-5.
Scheme 2. Synthesis of flavonoid derived compounds F and G.
Scheme 3. Synthesis of cytisine-flavonoid conjugates F-1 and G-1.

21
R3
4'' 5''
7''
6''
13''
3''
R2
N12'' 14''
R3
O
2''
N
8''
1''
CH2
8
O
HCHO
DMAP
1
HO
O
NH
10''
9'' 11'
3'
2'
R1
2
O
1'
4' R1
HO
N
7
R2
6
3
4
6' 5'
O
5
O
A ~ E
1 ~ 5
A-1 ~ E-5
1
1
1
2
2
3
2
3
2
3
= OCH3; : R = R3 = H; : R = R = CI; : R = R = Br; : R = H, R = CI;
A, B, C:
D:
E:
1
2
1
3
4
R
= H;
R
= OH;
R
2
3
1
2
3
2
3
1
2
3
5
A-1, B-1, C-1:
D-1:
E-1:
: R = NO2, R = H;
R = R = R = H;
R = OH, R = R = H;
R = OCH3, R = R = H;
1
2
3
1
2
3
1
2
3
1
A-2, B-2, C-2:
D-2:
E-2:
A-3, B-3, C-3:
R
R = H, R = R = CI;
R = OH, R = R = CI;
R = OCH3, R = R = CI;
2
3
1
2
3
1
2
3
1
2
3
D-3:
E-3:
A-4, B-4, C-4:
R = R = H, R = CI;
= H, R = R = Br;
R = OH, R = R = Br;
R = OCH3, R = R = Br;
1
2
3
1
2
3
2
1
1 = R3 = H, R = NO2;
R
D-4:
E-4:
A-5, B-5, C-5:
D-5:
R
= OH, R = H, R = CI;
R
= OCH3, R = H, R = CI;
R
2
3
1
2
3
E-5:
= OH, R = NO2, R = H;
R = OCH3, R = NO2, R = H
Scheme 1. Synthesis of cytisine-flavonoid conjugates A-1 ~ E-5.
a Conditions: A ~ E (1.0 mmol), 1 ~ 5 (1.2 mmol), 1,4-dioxane (10 mL), DMAP (0.02
mmol, 2.5 mg), HCHO (2.0 mmol, 37 ~ 40%), 110 ◦C, 2 ~ 3h.

22
I
HO
or
O
O
HO
O
O
HO
or
O
O
NIS
ArB(OH)2, PdCI2(PPh3)2
Na2CO3, H2O
R
R
R
HFIP, rt
HFIP, 100 oC, 16h
A
C
: R=2-phenyl
: R=3-phenyl
F
: R=2-phenyl
: R=3-phenyl
no isolated
G
HO
O
O
HO
O
O
F
G
, 51%
, 72%
Scheme 2. Synthesis of flavonoid derived compounds F and G.
a Conditions: A or C (0.5 mmol) and NIS (0.5 mmol) in 2 mL HFIP for the first step.
Then ArB(OH)2 (0.75 mmol), Na2CO3 (1 mmol), PdCl2(PPh3)2 (1.5 mol %) and 2 mL
H2O were added for the second step.

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HO
O
O
HO
O
O
HCHO
DMAP
N
NH
N
N
O
O
F
G
F-1 G-1
or
or
1
Scheme 3. Synthesis of cytisine-flavonoid conjugates F-1 and G-1.
a Conditions: F or G (0.25 mmol), 1 (0.3 mmol), 1,4-dioxane (2.5 mL), DMAP (0.005
mmol, 0.6 mg), HCHO (0.5 mmol, 37 ~ 40%), 110◦C, 2 ~ 3h.
Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which
may be considered as potential competing interests:
A series of cytisine-flavonoid conjugates were synthesized.
Some of the synthesized compounds showed good antineoplastic activities.
Compound A-4 showed more potent antitumor activity against MDA-MB-231 cells.
Structure-activity relationship (SAR) of the synthesized compounds were studied.

24
Graphical Abstract
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Cytisine-flavonoid conjugates: synthesis and
antitumor structure-activity relationship
research
Renhao Liu a, Xiaoze Bao a, Xuanrong Sun a, Yue Cai a, Tianwei Zhang a, Xinyi Ye a, Xing-Nuo Li a
a
College of Pharmaceutical Science & Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of
Technology, Hangzhou 310014, P. R. China

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