Design, synthesis and biological evaluation of pyridine-chalcone derivatives as novel microtubule-destabilizing agents

Article abstract of DOI:10.1016/j.ejmech.2019.04.008

Further optimization of the trimethoxyphenyl scaffold of parent chalcone compound (2a) by introducing a pyridine ring afforded a series of novel pyridine-chalcone derivatives as potential anti-tubulin agents. All the target compounds were evaluated for their antiproliferative activities. Among them, representative compound 16f exhibited the most potent activity with the IC₅₀ values ranging from 0.023 to 0.045 μM against a panel of cancer cell lines. Further mechanism study results demonstrated that compound 16f effectively inhibited the microtubule polymerization by binding to the colchicine site of tubulin. Moreover, cellular mechanism studies disclosed that 16f caused G2/M phase arrest, induced cell apoptosis and disrupted the intracellular microtubule network. Also, 16f reduced the cell migration and disrupted the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs). Importantly, 16f significantly inhibited tumor growth in H22 xenograft models without apparent toxicity, which was stronger than the reference compound CA-4, indicating that it is worthy to investigate 16f as a potent microtubule-destabilizing agent for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2019.04.008

Accepted Manuscript
Design, synthesis and biological evaluation of pyridine-chalcone derivatives as novel
microtubule-destabilizing agents
Feijie Xu, Wenlong Li, Wen Shuai, Limei Yang, Yi Bi, Cong Ma, Hequan Yao,
Shengtao Xu, Zheying Zhu, Jinyi Xu
PII:
S0223-5234(19)30307-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.008
EJMECH 11243
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2019
Revised Date: 10 March 2019
Accepted Date: 2 April 2019
Please cite this article as: F. Xu, W. Li, W. Shuai, L. Yang, Y. Bi, C. Ma, H. Yao, S. Xu, Z. Zhu, J. Xu,
Design, synthesis and biological evaluation of pyridine-chalcone derivatives as novel microtubule-
destabilizing agents, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.04.008.
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ACCEPTED MANUSCRIPT
Graphic Abstract

ACCEPTED MANUSCRIPT
Design,
Pyridine-Chalcone
Microtubule-Destabilizing Agents
Synthesis
and
Biological
Evaluation
as
of
Derivatives
Novel
Feijie Xua,#, Wenlong Lia,#, Wen Shuaia, Limei Yanga, Yi Bib, Cong Mac, Hequan Yaoa,
Shengtao Xua,*, Zheying Zhud, Jinyi Xua,*
aState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China
bSchool of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation, Yantai
University, Yantai 264005. P. R. China
cState Key Laboratory of Chemical Biology and Drug Discovery, and Department of Applied
Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kon
g
dDivision of Molecular Therapeutics & Formulation, School of Pharmacy, The University of
Nottingham, University Park Campus, Nottingham NG7 2RD, U. K.
# These authors contributed equally to this work.
*Corresponding Author:
E-mail addresses: cpuxst@163.com (S. Xu); jinyixu@china.com (J. Xu)
Abstract
Further optimization of the trimethoxyphenyl scaffold of parent chalcone
compound (2a) by introducing a pyridine ring afforded a series of novel
pyridine-chalcone derivatives as potential anti-tubulin agents. All the target
compounds were evaluated for their antiproliferative activities. Among them,
representative compound 16f exhibited the most potent activity with the IC50 values
ranging from 0.023 to 0.045 µM against a panel of cancer cell lines. Further
mechanism study results demonstrated that compound 16f effectively inhibited the
microtubule polymerization by binding to the colchicine site of tubulin. Moreover,

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cellular mechanism studies disclosed that 16f caused G2/M phase arrest, induced cell
apoptosis and disrupted the intracellular microtubule network. Also, 16f reduced the
cell migration and disrupted the capillary-like tube formation of human umbilical vein
endothelial cells (HUVECs). Importantly, 16f significantly inhibited tumor growth in
H22 xenograft models without apparent toxicity, which was stronger than the
reference compound CA-4, indicating that it is worthy to investigate 16f as a potent
microtubule-destabilizing agent for cancer therapy.
Keywords: pyridine; chalcone; microtubule-destabilizing agent; colchicine site;
anti-vascular; antitumor
1. Introduction
Microtubules as the key components of the cytoskeleton, are long, filamentous,
tube-shaped protein polymers which are essential in all eukaryotic cells [1].
Microtubules, which are composed of α-tubulin and β-tubulin heterodimers, play vital
roles in cell proliferation, trafficking, signaling, migration, cell division and mitosis
[2]. For this reason, microtubules become an important target for anticancer drugs.
Microtubule-active drugs generally bind to one of three main classes of sites on
tubulin, the paclitaxel site, the vinca domain and the colchicine domain [3]. Those
inhibitors that bind to the taxane or vinca alkaloid binding sites have many
disadvantages such as extremely structural complexity, low aqueous solubility,
multidrug resistance and dose-limiting toxicity [4]. Hence, colchicine binding site
inhibitors (CBSIs) are undergoing intensive investigation in recent decades [5].
Combretastatin A-4 (1, CA-4), an excellent colchicine binding site inhibitor which
was isolated from the bark of the African willow tree Combretum caffrum, displayed
potent antimitotic and vascular disrupting profiles [6]. Other typical colchicine
binding site inhibitors were chalcone compounds 2a and 2b which were discovered by
Ducki et al. in 1998, both showing remarkable antiproliferative activities [7]. The
3,4,5-trimethoxyphenyl (TMP) moiety of CA-4 was vital for its activity since the
4-methoyl group formed a critical hydrogen bond with the residue Cys241 [8-10].

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Attempts to modify the TMP moiety usually led to reduced anticancer potency
[11-14]. However, in 2017, Yin et al. introduced a selenium atom to replace the
oxygen atom at the C-3 position of TMP to afford compound 3a, which showed
improved activities both in vitro and in vivo compared to its parent compound
isocombretastatin A-4 (IsoCA-4) [15]. Recently, Li et al. discovered a unique
3-methoxybenzo[4,5]-dioxene moiety 3b which exhibited improved antiproliferative
activity compared with the compound bearing TMP moiety [16]. Other efforts have
also been made to discover new moieties to replace the TMP moiety. In 2015, Alami
and colleagues reported the discovery of compound 3c which bears a quinazoline
nucleus [17], and it displayed nanomolar-level cytotoxicity against various human
cancer cell. In 2017, Alami’s group reported an isoCA-4 analog 3d with a quinoline
moiety [18]. In 2018, Millier et al. first reported the high-resolution X-ray crystal
structure of the heterocyclic-fused pyrimidine compound 3e complexed with tubulin,
and the results showed that the N-1 atom formed hydrogen bond with the residue
Cys241, demonstrating that quinoline or quinazoline moieties that have N-1 atom
might be the suitable surrogates of the traditional TMP moiety for binding to the
colchicine site [19]. Meanwhile, our group also discovered a quinoline-chalcone
compound 3f by replacing TMP with quinoline moiety, and its antiproliferative
activity was improved for 8-10 folds as well as the improvement of the solubility [20].
Nitrogenous heterocycles, such as pyridine, quinoline, pyrimidine and piperidine,
are privileged building blocks in drugs [21]. Pyridine plays important roles in drug
development, which can be found in a vast number of drugs like imatinib [22],
telithromycin [23], indinavir [24] and acalabrutinib [25]. Even though the number of
CA-4 analogs has been more than 28000 [26], compounds containing pyridine ring
replacing of TMP have rarely been reported.
Recently, our group has focused on discovering and developing novel anticancer
agents targeting tubulin-microtubule system, such as chalcone analogs bearing vinyl
sulfone skeleton [27], 4-arylisochromenes [28], quinazolines that occupy three zones
of colchicine domain [29] and quinoline-indole derivatives [30]. Inspired by Millier’s
work [19], we assumed that the N-1 atom of pyridine might also form a hydrogen

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bond with Cys241 in the colchicine, thus pyridine might be introduced into the
structures of CBSIs as an alternative ring of TMP moiety.
In this work, we continued our efforts in the modification works of the parent
compound 2a by replacing the TMP moiety with a pyridine ring. Thus, a series of
novel pyridine-chalcone derivatives were designed and synthesized (Figure 2). Herein,
we would like to report the synthesis and evaluation of their antitumor activities in
vitro and in vivo.
Figure 1. A) Structures of the representative colchicine binding site inhibitors (CBSIs); B) CBSIs
reported to modify TMP; C) CBSIs containing nitrogenous heterocycles in replace of TMP.

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Figure 2. Design strategy of novel pyridine-chalcone derivatives.
2. Results and discussion
2.1 Chemistry
The synthetic route for the synthesis of pyridine-chalcone derivatives 15a-f and
16a-f is outlined in Scheme 1. The intermediates 9a, 9b and 14 are synthesized by
two different routes. The starting material 2,6-dichloroisonicotinic acid (4) underwent
nucleophilic attack by ethylmagnesium bromide (EtMgBr) to give intermediate 5
which was then treated with 2-bromoethanol to give intermediate 6. Intermediates 7
and 8 were prepared by treating intermediate 6 with sodium methoxide in 60 and
100 ℃, respectively, followed by deprotection with hydrochloric acid to afford
intermediates 9a and 9b, respectively. As for intermediate 14, the starting material
2,6-dichloroisonicotinic acid (4) was first treated with N-methoxymethanamine to
obtain intermediate 10 then underwent nucleophilic attack by methylmagnesium
bromide (MeMgBr) to give intermediate 11. Then the procedures are the same as 9a
and 9b. Subsequently, ketone 9a, 9b and 14 were underwent Aldol reaction with a
variety of aldehydes to obtain target compounds 15a-r and 16a-f.
Scheme 1. The synthetic route for the preparation of compounds 15a-f and 16a-f a.

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O
O
O
e
d
O
O
N
O
O
OH
O
O
N
O
b
a
O
8
9b
Cl
N
Cl
Cl
N
Cl
O
Cl
N
Cl
O
c
O
e
6
4
5
Cl
N
O
Cl
N
O
9a
7
OMe
O
O
OH
O
N
O
Me
O
f
g
b
Cl
N
Cl
Cl
N
Cl
Cl
N
Cl
Cl
N
Cl
12
4
11
10
O
O
O
e
d
O
N
O
O
O
N
O
13
14
O
CHO
15a R1=R3=H, R2=OMe
15b R1=H, R2=R3=OMe
15c R1=R2=R3=OMe
15d R1=R3=H, R2=SMe
15e R1=H, R2=OMe, R3=OH
15f R1=H, R2=OMe, R3=F
MeO
R3
R2
h
+
N
R1
R3
O
N
O
R2
A1-A6
OMe
R1
14
15a-f
O
CHO
O
16a R1=R2=R4=OMe, R3=R5=H
16b R1=R2=R3=R4=OMe, R5=H
16c R1=R2=R3=R4=R5=OMe
16d R1=R2=Cl, R3=OH, R4=OMe, R5=H
16e R1=Cl, R2=R4=OMe, R3=OH, R5=H
16f R1=R2=R4=OMe, R3=OH, R5=H
R1
R5
R4
h
+
N
R3
R5
R1
N
R2
R4
R2
R3
5 or 9a or 9b
A1-A3, A5
16a-f
a
Reagents and conditions: (a) EtMgBr, HCOOEt, THF, N2, rt, 1 h; (b) BrCH2CH2OH, DBU, Tol,
80 , overnight; (c) MeONa, MeOH, 60 ℃, overnight; (d) MeONa, MeOH, 100 ℃, overnight; (e)
1M HCl, MeOH, 60 ℃, 1 h; (f) EDCI, HOBt, N-methoxymethanamine, Et3N, DCM, rt, 8 h; (g)
CH3MgBr, THF, N2, rt, 1 h; (h) NaOH, EtOH, rt, 3 h.
Pyridine-indole derivatives 18a-i with different substituted positions on the indole
ring were synthesized as shown in Scheme 2. Indolealdehyde derivatives were
prepared by Vilsmeier-Haack reaction with DMF and phosphoryl chloride. Target
compounds 18a-i were synthesized through Aldol reactions by treating
indolealdehyde with ketone 9 in the presence of piperidine.

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Scheme 2. The synthetic route for the preparation of compounds 18a-i a.
a Reagents and conditions: (a) POCl3, DMF, 0 , 10 min; (b) piperidine, EtOH, reflux, overnight.
Compounds 19a-g were prepared as shown in Scheme 3. The compounds 18a and
18h was treated with methyl iodine and ethyl iodine to give the target compounds 19a,
19b and 19h, respectively. And compound 18a was converted to 19c using
formaldehyde with sodium hydroxide under refluxing condition. Also, compound 18a
was reacted with acetyl chloride, 2-furoyl chloride and benzoyl chloride to provide
the corresponding compounds 19d-f.
Scheme 3. The synthetic route for the preparation of compounds 18a-g a.
a Reagents and conditions: (a) NaH, MeI, DMF, 0 , 1 h; (b) HCHO, EtOH, NaOH, reflux, 1 h; (c)
acyl chloride, NaOH, DCM, rt, 2 h.
2.2 In vitro antiproliferative activity
Target compounds 15a-f, 16a-f, 18a-i and 19a-g were evaluated for their in vitro
antiproliferative efficacy against K562 cells by the MTT assays with CA-4 as the
reference. As shown in Table 1, compounds with indole moieties as ring B showed
favorable activity except 18b, 18f, 18h and 19b. Compound 16f that bear

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3-hydroxy-4-methoylphenyl moiety displayed the most potent activity with the IC50
value of 0.023 µM, which was about 3-fold increase in activity when compared with
the parent compound 2a (IC50 = 0.068 µM), and was slightly less potent than the CA-4
(IC50 = 0.011 µM). The methyl substituted at the α-position of unsaturated carbonyl
group was favorable for the improvement of activity (15a vs 16a, 15b vs 16b, 15c vs
16c and 15e vs 16f), which was accordance with the results in the previous reports [7].
Besides, the exposed hydroxyl on ring B was essential for maintaining the activity
(16b vs 16f). Moreover, the substituents on the pyridine were also critical to the
activity (16d, 16e and 16f), indicating that the electron-donating groups (EDG) were
more favorable than the electron-withdraw groups (EWG). We proposed that the EDG
increased the electron density of the N-1 of pyridine, which was beneficial to form a
hydrogen bond with the critical residue Cys241. Also, the results indicated that the
3-substitued indoles were more favorable (18a vs 18h and 18i), and while substituents
at N-1 of indole ring were unfavorable to activity (18a vs 18b-g, 18a vs 19a-f ).
Table 1. Antiproliferative activities of compounds 15a-f, 16a-f, 18a-g and 19a-g against K562
cell line a.
IC50 values (µM)b
IC50 values (µM)b
Compd.
Compd.
K562
> 1
K562
15a
15b
15c
15d
15e
15f
18d
18e
18f
0.352 ± 0.004
0.191 ± 0.008
> 1
> 1
> 1
> 1
18g
18h
18i
0.106 ± 0.006
> 1
0.983 ± 0.013
> 1
0.142 ± 0.036
0.700 ± 0.023
> 1
16a
16b
16c
16d
16e
16f
0.554 ± 0.017
> 1
19a
19b
19c
19d
19e
19f
> 1
0.058 ± 0.006
0.086 ± 0.008
0.060 ± 0.003
0.070 ± 0.003
0.952 ± 0.017
0.068± 0.002
0.011 ± 0.001
0.156 ± 0.012
0.138 ± 0.011
0.023 ± 0.002
0.051 ± 0.003
> 1
18a
18b
18c
19g
2a
0.983 ± 0.018
CA-4
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was measured by the

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MTT assay as described in the Experimental Section.
b IC50 values are indicated as the mean ± SD (standard error) of at least three independent experiments.
Table 2. Antiproliferative activities of representative compounds against five cancer cell lines and
one normal cell line a
IC50 values (µM)b
Compd.
MDA-MB-23
1
HepG2
KB
HCT-8
H22
L-O2
15e
16d
16e
16f
0.991 ± 0.021
0.183 ± 0.008
0.132 ± 0.010
0.047 ± 0.002
0.085 ± 0.005
0.392 ± 0.009
0.364 ± 0.012
0.124 ± 0.012
0.157 ± 0.003
0.092 ± 0.003
0.068 ± 0.002
0.093 ± 0.001
0.102± 0.010
0.012± 0.001
1.018 ± 0.015
0.162 ± 0.003
0.147 ± 0.012
0.045 ± 0.001
0.090 ± 0.003
0.324 ± 0.013
0.421 ± 0.022
0.137 ± 0.013
0.143 ± 0.008
0.109 ± 0.006
0.072 ± 0.006
0.107 ± 0.001
0.108 ± 0.005
0.011 ± 0.002
0.997 ± 0.010
0.177 ± 0.005
0.139 ± 0.012
0.047 ± 0.003
0.095 ± 0.003
0.335 ± 0.011
0.379 ± 0.018
0.127 ± 0.010
0.152 ± 0.006
0.121 ± 0.009
0.091 ± 0.008
0.110 ± 0.003
0.104 ± 0.010
0.010 ± 0.001
1.128 ± 0.011
0.195 ± 0.009
0.162 ± 0.013
0.041 ± 0.004
0.079 ± 0.002
0.320 ± 0.016
0.364 ± 0.015
0.153 ± 0.014
0.148 ± 0.005
0.088 ± 0.007
0.082 ± 0.008
0.093 ± 0.007
0.102 ± 0.009
0.010 ± 0.001
0.928 ± 0.065
0.177 ± 0.011
0.142 ± 0.014
0.043 ± 0.002
0.088 ± 0.012
0.336 ± 0.025
0.428 ± 0.041
0.127 ± 0.013
0.138 ± 0.008
0.098 ± 0.007
0.075 ± 0.002
0.108 ± 0.011
0.132 ± 0.008
0.008 ± 0.001
4.226 ± 0.321
0.399 ± 0.031
0.768 ± 0.054
0.157 ± 0.013
0.408 ± 0.036
1.271 ± 0.121
1.102 ± 0.104
0.628 ± 0.057
0.728 ± 0.069
0.417± 0.032
0.338 ± 0.031
0.528 ± 0.042
0.421 ± 0.037
0.095 ± 0.011
18a
18d
18e
18g
18i
19c
19e
19f
2a
CA-4
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was measured by the
MTT assay as described in the Experimental Section.
b IC50 values are indicated as the mean ± SD (standard error) of at least three independent experiments.
In consideration of the prominent antiproliferative activities of the newly
pyridine-chalcone derivatives against K562 cells, we tend to further evaluate the
biological functions against more cancer cell lines. Human hepatocellular carcinoma
(HepG2), epidermoid carcinoma of the nasopharynx (KB), human ileocecal cancer
cells (HCT-8) human breast cancer cells (MDA-MB-231), and hepatoma 22 cells
(H22) were selected for the further evaluation of activity. In addition, one normal
non-cancerous cell line - human normal liver cells (L-O2) - was chosen to evaluate
the toxicity of these compounds. The cytotoxic data of representative compounds

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against these four cancer cell lines were shown in Table 2, which showed that all
these chosen compounds displayed potent activities against these four cancer cell
lines. Moreover, the structure activity relationships (SARs) were similar to their
performances in K562 assays. Also, the K562 cell was the most sensitive cell line
among five cancer cell lines tested, and the most active compound 16f exhibited
remarkable activity with IC50 values ranging from 0.023 to 0.047 µM, which was
about 3-fold improvement of activity when compared with the parent compound 2a.
The SARs of synthesized compounds were summarized in Figure 3.
Figure 3. Summarized SARs of synthesized compounds.
2.3 In vitro tubulin polymerization inhibitory assay
To verify whether the target molecules interacted with microtubule systems, 16f,
the most cytotoxic compounds, was selected for the experiment of effects on
microtubule dynamics. Also colchicine（a typical microtubule-destabilizing agent）
and taxol (microtubule-stabilizing agent) were chosen as the controls. As shown in
Figure 4, the fluorescence emission of 16f was less than the control group and similar
to the colchicine group, indicating that 16f was a microtubule-destabilizing agent.
Moreover, the calculated IC50 value of 16f in inhibiting tubulin polymerization was
2.08 µM, which was better than the CA-4 (IC50 = 2.17 µM) and the parent compound
2a (IC50 =3.05 µM) (Table 3). In addition, the test of competing with [3H]-colchicine
in binding to tubulin was performed, indicating that 16f bound to the colchicine
binding site with the potency about 87.9% at 5 µM, (Table 3). Hence, 16f was chosen

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for further mechanism studies due to its excellent activities both in the in vitro
antiproliferative assay and tubulin polymerization inhibition assay.
Figure 4. Effects of 16f on tubulin polymerization in vitro. Purified tubulin protein at 2 mg/mL in
a reaction buffer was incubated at 37 °C in the presence of 1% DMSO, test compounds (16f at 5
or 10 µM), Colchicine (10 µM) or Taxol (10 µM). Polymerizations were followed by an increase
in fluorescence emission at 350 nm over a 60 min period at 37 °C. The experiments were
performed three times.
Table 3. Inhibition of tubulin polymerizationa and colchicine binding to tubulinb
inhibition of colchicine binding
inhibition of tubulin polymerization
Compd.
(%) inhibition ± SD
IC50 (µM)
2.08 ± 0.12
2.17 ± 0.14
3.05 ± 0.15
1 µM
5 µM
16f
CA-4
2a
75.1 ± 1.63
81.2 ± 2.01
70.2 ± 1.71
87.9 ± 2.18
91.7 ± 4.40
78.3 ± 3.39
a The tubulin assembly assay measured the extent of assembly of 2 mg/mL tubulin after 60 min at
37 °C. Data are presented as mean from three independent experiments.
b Tubulin, 1 µM; [3H]-colchicine, 5 µM; and inhibitors, 1 or 5 µM.
2.4. Anti-microtubule effects in K562 cells
Immunofluorescent assay was performed to investigate the effects of compound
16f on microtubule networks. As shown in Figure 5, K562 cells and MAD-MB-231

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cells without drug treatment exhibited normal filamentous arrays. However, after
exposure to 16f (10 nM, 20 nM, 40 nM to K562, and 20 nM, 40 nM, 80 nM to
MAD-MB-231) for 24 h, the microtubule networks in cytosol were disrupted; these
results indicated that 16f can induce a dose-dependent collapse of the microtubule
networks.
Figure 5. A) Effects of 16f on the cellular microtubule network visualized by
immunofluorescence. K562 cells were treated with vehicle control 0.1% DMSO, 16f (10 nM), 16f
(20 nM), and 16f (40 nM). B) Effects of 16f on the cellular microtubule network visualized by
immunofluorescence. MAD-MB-231 cells were treated with vehicle control 0.1% DMSO, 16f (20
nM), 16f (40 nM), and 16f (80 nM).Then, the cells were fixed and stained with
anti-α-tubulin-FITC antibody (green), Alexa Fluor 488 dye and counterstained with DAPI (blue).
The detection of the fixed and stained cells was performed with an LSM 570 laser confocal
microscope (Carl Zeiss, Germany).
2.5 Cell cycle analysis
Since most microtubule polymerization inhibitors disrupt cell mitosis and exert cell
cycle arrest effects, the effect of 16f on cell cycle progression using propidiumiodide
(PI) staining in K562 cells was examined. As illustrated in Figure 6A and 6B,
incubation with 16f blocked the cell cycle at the G2/M phase. Compared to the

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control cells incubated with DMSO, the incubation of K562 cells with increasing
concentrations of 16f (15, 30, and 60 nM) increased the percentage of cells in the
G2/M phase from 7.10% to 33.22%.
Figure 6．A) Compound 16f induced G2/M arrest in K562 cells. K562 cells were incubated with
varying concentrations of 16f (0, 15, 30, and 60 nM) for 48 h. Cells were harvested and stained
with PI and then analyzed by flow cytometry. The percentages of cells in different phases of the
cell cycle were analyzed by ModFit 4.1; B) Histograms display the percentage of cell cycle
distribution after treatment with 16f.
2.6 Cell apoptosis analysis
Most microtubule polymerization inhibitors can induce cell apoptosis by
up-regulating the expression of pro-apoptotic proteins and down-regulating the
expression of antiapoptotic proteins. Aiming to assess whether compound 16f would
induce cell apoptosis, an annexin VFITC and propidium iodide (PI) assay was carried
out. As shown in Figure 7A and 7B, the percentage of apoptotic cells after the 48 h
treatment was only 3.42% in the control group, but the total numbers of early
(annexin-V+/PI−) and late (annexin-V+/PI+) apoptotic cells increased to 15.79%, 39.01%
and 56.07% after treatment with 16f at 10, 20, and 40 nM for 48 h, respectively. These
results confirmed that compound 16f effectively induced cell apoptosis in K562 cells

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in a dose-dependent manner.
Figure 7. A) Compound 16f induced apoptosis in K562 cells. K562 cells were incubated with
varying concentrations of 16f (0, 10, 20, and 40 nM). After 48 h of incubation, cells were collected
and stained with Annexin V/PI, followed by flow cytometric analysis. The percentages of cells in
each stage of cell apoptosis were quantified by flow cytometry: (upper left quadrant) necrosis cells;
(upper right quadrant) late-apoptotic cells; (bottom left quadrant) live cells; and (bottom right
quadrant) early apoptotic cells. B) Histograms display the percentage of cell distribution after
treatment with 16f.
2.7 Mitochondrial membrane potential analysis.
Decreased mitochondrial membrane potential (MMP) has been implicated as an
early event in apoptotic cells. Thus, the effect of compound 16f on MMP of K562
cells was determined by staining with lipophilic cationic JC-1 dye. When treated with
16f at concentrations of 0, 10, 20 and 40 nM for 24 h, the number of K562 cells with
collapsed MMP increased to 5.75%, 16.44%, 28.10% and 44.49%, respectively.
(Figure 8), suggesting that 16f caused mitochondrial depolarization of K562 cells in
the process of apoptosis.

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Figure 8. Effects of 16f on the mitochondrial membrane potential of K562 cells. A) Incubation
with different concentrations (0, 10, 20, and 40 nM) of 16f in K562 cells for 24 h prior to staining
with JC-1 dye, the number of cells with collapsed mitochondrial membrane potentials was
determined by flow cytometry analysis. B) Histograms display the percentage of green
fluorescence.
2.8 In vitro evaluation of anti-vascular activity
In view of such facts that most CBSIs possess vascular disrupting activity, we
further evaluated the anti-vascular activity of compound 16f. The HUVECs culture
assays were performed to assess the ability of 16f to inhibit HUVECs migration
which is the key step to generate new blood vessels. As shown in Figure 9A, the
untreated cells migrated to fill the area that was initially scraped after 24 h. In contrast,
16f significantly inhibited the HUVEC migration in a dose-dependent manner.
Then we further assessed the anti-vascular activity of 16f in a tube formation assay.
After being seeded on Matrigel, HUVECs formed the capillary-like tubules with
multicentric junctions. After exposure to 16f at 10, 20, and 40 nM for 6 h, the
capillary-like tubes were interrupted in different levels (Figure 9B). These results
showed that compound 16f effectively has an effect on tubule-like disruption of
HUVECs.
The antiproliferative activity of 16f against HUVECs was also determined by an
MTT assay to exclude the possibility that the anti-vascular activity of 16f was due to a

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cytotoxic action of 16f. The calculated IC50 value of 16f against HUVECs after a 24 h
treatment was 0.126 ± 0.007 µM, which is higher than the concentration of 40 nM
required for the obvious inhibition of cell migration and tube formation. These results
indicate that 16f exhibited anti-vascular activity.
Figure 9. Effects on the HUVECs migration and tube formation. A) Scratches were created with
sterile 200 µL pipette and images were captured using phase contrast microscopy at 0 h and 24 h
after treatments with 0, 10, 20 and 40 nM of 16f. B) Images depicting the formation of HUVEC
capillary-like tubular network by treatments with 0, 10, 20 and 40 nM of 16f for 6 h. C)
Histograms display the length of the scratches at 0 h and 24 h after treatments with 0, 10, 20 and
40 nM of 16f, ***, P < 0.001 vs control group.

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2.9 In vivo antitumor activity of 16f
To evaluate the in vivo antitumor activities of 16f, liver cancer allograft mouse
model was established by subcutaneous inoculation of H22 cells into the right flank of
mice. The tumor size and body weights of the mice were monitored and recorded
every 2 days. Paclitaxel (PTX), CA-4 and the disodium phosphate of CA-4 (CA-4P)
were selected as the positive controls. As shown in Figure 10A and Figure 10B, 16f at
the dose of 20 mg/kg per day significantly decreased the tumor volume. Obviously,
the reduction in tumor weight reached 65.8% at dose of 20 mg/kg per day (i.v.) of 16f,
which was slightly less potent than PTX (72.7% at a dose of 6 mg/kg per 2 days).
Particularly, 16f at the dose of 20 mg/kg displayed more potent antitumor activity than
CA-4 (inhibition rate: 50.9% at 20 mg/kg) or CA-4P (inhibition rate: 62.7% at 20
mg/kg) (Figure 10D). Moreover, 16f did not significantly affected body weight even
at the doses of 20 mg/kg, while treatment with PTX at a dose of 6 mg/kg per 2 days
led to a significant influence on body weight (Figure 10C). Thus, compound 16f is
worthy of further investigation for the treatment of cancers.

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Figure 10. 16f inhibited liver cancer allograft growth in vivo. After administered with vehicle,
PTX (6 mg/kg per 2 days, group 2), CA-4 (20 mg/kg per day), CA-4P (20 mg/kg per day), 16f (10
mg/kg per day), and 16f (20 mg/kg per day) for three weeks, the mice were sacrificed, and the
tumors were weighed. A) The images of tumors from mice at 21 days after initiation of treatment.
B) Tumor volume changes of mice during treatment. C) Body weight changes of mice during
treatment. D) The weight of the excised tumors of each group. **P < 0.05, ***P < 0.001 vs
control group.
2.10 Molecular modelling studies
To explain the binding modes of the most active compound 16f with tubulin, we
performed a docking study of 16f into the colchicine binding pocket of tubulin (PDB:
5lyj) by using the DOCK program in the Discovery Studio 3.0 software. As shown in
figure 11A, 16f adopted a very similar positioning to the colchicine at the colchicine
binding pocket. As displayed in figure 11B, the N-1 of pyridine moiety formed a
critical hydrogen bond with residue Cys241 resembling that of the 4-methoxy group
in compound 2a. Moreover, the hydroxy group of ring B formed two additional

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hydrogen bonds with Val181 and Asn258, which illustrated the vital role of the
exposed hydroxyl group.
Figure 11. Proposed binding model for 16f (violet) binding with tubulin (PDB code: 5lyj), and
overlapping with colchicine (green) and compound 2a (yellow).
3. Conclusion
In summary, we designed and synthesized a series of novel pyridine-chalcone
derivatives as microtubule destabilizing agents, which may have some potential
advantages over the reported chalcones.
All compounds were evaluated for their anti-proliferative activities, and their
detailed SARs were obtained. Compound 16f exhibited the most potent activities
against five cancer cell lines with IC50 values ranging from 23 to 45 nM, which was
3-fold more potent than parent compound 2a. Compared to chalcone derivatives
without nitrogenous heterocycles, such as 2a and 2b, the introduction of pyridine
moiety, which can be further prepared as the hydrochloride salts, provides an
alternative method to improve the solubility of CBSIs. On the other hand, compound
16f has a promising application in development for its simpler processing than that of
the quinoline-chalcone derivatives in our previous study [20].
Besides, by using colchicine competition inhibition assay and molecular
modelling studies, representative compound 16f was verified to bind to the colchicine
binding site as microtubule-destabilizing agent with the IC50 value of 2.08 µM.
Further mechanism studies demonstrated that 16f disrupted microtubule networks,
arrested cell cycle at G2/M phase, induced apoptosis and depolarized mitochondria of

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K562 cells in a dose-dependent manner. Furthermore, the wound healing and tube
formation assays also identified 16f as a novel vascular disrupting agent. The most
importantly, the in vivo antitumor experiments displayed that 16f effectively
suppressed the tumor volume and reduced tumor weight by 65.8% at the dose of 20
mg/kg per day (i.v.), which was more potent than control compound CA-4 (inhibition
rate: 50.9% at 20 mg/kg) or CA-4P (inhibition rate: 62.7% at 20 mg/kg). Collectively,
the present study demonstrates that pyridine-chalcone derivative 16f represents a
novel class of anti-tubulin agent with potent anti-vascular and antitumor activities and
deserves further investigation.
EXPERIMENTAL SECTION
4.1. Chemistry
4.1.1. General
Most chemicals and solvents were purchased from commercial sources. Further
1
purification and drying by standard methods were employed when necessary. H
NMR and 13C NMR spectra were recorded on Bruker-300 spectrometers in the
indicated solvents (TMS as internal standard). Data are reported as follows: chemical
shift in ppm (d), multiplicity (s =singlet, d =doublet, t =triplet, q =quartet, brs = broad
singlet, m =multiplet), coupling constant (Hz), and integration. High Resolution Mass
measurement was performed on Agilent QTOF 6520 mass spectrometer with electron
spray ionization (ESI) as the ion source. Flash column chromatography was carried
out using commercially available silica gel (200-300 mesh) under pressure.
4.1.2 Synthesis of intermediate 5
To a solution of 2,6-dichloroisonicotinic acid (10 g, 52 mmol) in 50 mL anhydrous
THF under nitrogen atmosphere at 0 °C was added C2H5MgBr in diethyl ether (2 M,
80 mL, 160 mmol). After stirring for 2 h, the reaction was quenched by NH4Cl
aqueous, and extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were
then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to

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provide the crude product, which was purified by column chromatography with
petroleum/ethyl acetate (4:1) to give intermediate 5 as white solid (9.0 g, 85.1 %).1H
NMR (300 MHz, CDCl3) δ 7.67 (s, 2H), 2.96 (q, J = 7.1 Hz, 2H), 1.22 (t, J = 7.1 Hz,
3H), 13C NMR (75 MHz, CDCl3) δ 197.15, 151.85, 147.70, 120.91, 32.64, 7.54;
ESI-MS m/z: 202.9 [M+Na]+ 225.9.
4.1.3 Synthesis of intermediate 6
To a solution of 5 (6 g, 29.4 mmol) in anhydrous toluene was added DBU (12.43
mL, 88.2 mmol), 2-bromoethanol (10.42 mL, 147 mmol). The mixture was stirred at
80 °C overnight. Then the solvent was removed in vacuo, and the residue was diluted
by H2O, and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were
then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to
provide the crude product, which was purified by column chromatography with
petroleum/ethyl acetate (40:1) to give intermediate 6 as colorless oil (6 g, 83.2 %).1H
NMR (300 MHz, CDCl3) δ 7.33 (s, 2H), 4.05 (t, J = 6.8 Hz, 2H), 3.78 (t, J = 6.9 Hz,
2H), 1.85 (q, J = 7.6 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H); ESI-MS m/z: 247.0 [M+Na]+
270.0.
4.1.4 Synthesis of intermediate 9a
To a solution of 6 (2.5 g, 10.1 mmol) in anhydrous MeOH was added sodium
methoxide solid (5.45 g, 101 mmol). The mixture was stirred at 60 °C in sealed tube
overnight. Then the reaction was quenched by H2O, the solvent was removed in vacuo,
and the residue was extracted with CH2Cl2 (3 × 20 mL). The combined organic layers
were then washed with brine, dried over anhydrous Na2SO4, and concentrated in
vacuo to provide the crude product, which was dissolved in MeOH, and concentrated
hydrochloric acid (10 mL) was added. Then the mixture was stirred at room
temperature for 2 h. Then, the solution was neutralized with 2 M NaOH aqueous to
pH 7, and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were then
washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to
provide the crude product, which was purified by column chromatography with
petroleum/ethyl acetate (4:1) to give intermediate 9a as yellow solid (1.5 g, 79.1%).1H
NMR (300 MHz, CDCl3) δ 7.33 (s, 1H), 7.09 (s, 1H), 3.98 (s, 3H), 2.93 (q, J = 7.1 Hz,

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2H), 1.25 - 1.17 (m, 3H); ESI-MS m/z: 199.0 [M+Na]+ 222.0.
4.1.5 Synthesis of intermediate 9b
To a solution of 6 (5 g, 20.2 mmol) in anhydrous MeOH was added sodium
methoxide solid (10.89 g, 202 mmol). The mixture was stirred at 100 °C in sealed
tube overnight. Then the reaction was quenched by H2O, the solvent was removed in
vacuo, and the residue was extracted with CH2Cl2 (3 × 20 mL). The combined organic
layers were then washed with brine, dried over anhydrous Na2SO4, and concentrated
in vacuo to provide the crude product, which was dissolved in MeOH, and
concentrated hydrochloric acid (10 mL) was added. Then the mixture was stirred at
room temperature for 2 h. Then, the solution was neutralized with 2 M NaOH aqueous
to pH 7, and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were
then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to
provide the crude product, which was purified by column chromatography with
petroleum/ethyl acetate (4:1) to give intermediate 9b as yellow solid (3.2 g,
81.1 %).1H NMR (300 MHz, CDCl3) δ 6.75 (s, 2H), 3.95 (s, 6H), 2.92 (q, J = 6.5 Hz,
2H), 1.20 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 200.01, 164.01, 148.70,
99.41, 53.89, 32.44, 7.85; ESI-MS m/z: 195.0 [M+Na]+ 218.0.
4.1.6 Synthesis of intermediate 10
To a solution of 6 (1.5 g, 4.1 mmo) in anhydrous DCM was added HOBt (1.3 g, 4.8
mmol), EDCI (1.8 g, 4.8 mmol), Et3N (0.62 g, 6.15 mmol) and
N-methoxymethanamine (1.2 g, 6.6 mmol). The mixture was stirred at room
temperature for 2 h. The precipitate was filtered and the filtrate was removed under
vacuum. The resulting mixture was then purified by flash column chromatography
with petroleum/ethyl acetate (4:1) to give intermediate 10 as white solid (1.6 g,
85.1 %). 1H NMR (300 MHz, CDCl3) δ 7.50 (s, 2H), 3.58 (s, 3H), 3.37 (s, 3H);
ESI-MS m/z: 233.9 [M+Na]+ 256.9.
4.1.7 Synthesis of intermediate 11
To a solution of 10 (1.1 g, 4.68 mmol) in 10 ml anhydrous THF under nitrogen
atmosphere at 0 °C was added CH3MgBr in diethyl ether (3 M, 2.34 mL, 4.80 mmol).
After stirring for 2 h, the reaction was quenched by NH4Cl aqueous, and extracted

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with CH2Cl2 (3 × 20 mL). The combined organic layers were then washed with brine,
dried over anhydrous Na2SO4, and concentrated in vacuo to provide the crude product,
which was purified by column chromatography with petroleum/ethyl acetate (4:1) to
give intermediate 11 as white solid (0.820 g, 92.1 %).1H NMR (300 MHz, CDCl3) δ
7.69 (s, 2H), 2.64 (s, 3H); ESI-MS m/z: 188.9 [M+Na]+ 211.9.
4.1.8 Synthesis of intermediate 14
To a solution of 11 (1.2 g，6.32 mmol)in anhydrous toluene was added DBU (2.67
mL , 19.0 mmol), 2-bromoethanol (2.24 mL, 31.6 mmol). The mixture was stirred at
80 °C overnight. Then the solvent was removed in vacuo, and the residue was diluted
by H2O, and extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were
then washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to
provide the crude product 12 as colorless oil (1.25 g, 83.1%). To a solution of 12
(1.25 g, 5.05 mmol) in anhydrous MeOH was added sodium methoxide (2.72 g, 50.5
mmol). The mixture was stirred at 100 °C in sealed tube overnight. Then the reaction
was quenched by H2O, the solvent was removed in vacuo, and the residue was
extracted with CH2Cl2 (3 × 20 mL). The combined organic layers were then washed
with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to provide the
crude product, which was dissolved in MeOH, and concentrated hydrochloric acid (10
ml) was added. Then the mixture was stirred at room temperature for 2 h. Then, the
solution was neutralized with 2 M NaOH aqueous to pH 7, and extracted with CH2Cl2
(3 × 20 mL). The combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude product, which
was purified by column chromatography with petroleum/ethyl acetate (4:1) to give
intermediate 14 as white solid (0.8 g, 81.1 %).1H NMR (300 MHz, CDCl3) δ 6.74 (s,
2H), 3.95 (s, 6H), 2.55 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 212.48, 164.06, 148.74,
99.70, 53.94, 27.01; ESI-MS m/z: 181.0 [M+Na]+ 204.
4.1.9 The procedure for the preparation of 15a-f and 16a-f.
To a stirred solution of ketone (0.28 mmol) and aldehyde (0.28 mmol) in anhydrous
EtOH, NaOH (55.1 mg, 1.38 mmol) was slowly added at room temperature. After
stirring for 3 h, the reaction mixture was concentrated and extracted with CH2Cl2 (3 ×

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20 mL). The combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude product, which
was purified by column chromatography with petroleum/ethyl acetate (4:1) to give the
corresponding pure compounds 15a-f and 16a-f.
4.1.9.1 (E)-1-(2,6-dimethoxypyridin-4-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (15a).
1
Yellow solid, yield 76.2%. H NMR (300 MHz, CDCl3) δ 7.63 (d, J = 15.7 Hz, 1H),
7.45 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 15.7 Hz, 1H), 6.81 (d, J = 8.6 Hz, 2H), 6.64 (s,
2H), 3.84 (s, 6H), 3.73 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 189.92, 163.88, 162.04,
150.74, 146.20, 130.47, 127.21, 119.61, 114.50, 99.85, 55.43, 53.89; HR-MS (ESI)
m/z: calcd for C17H18NO4 [M+H]+ 300.1230 found 300.1229.
4.1.9.2
(E)-3-(3,4-dimethoxyphenyl)-1-(2,6-dimethoxypyridin-4-yl)prop-2-en-1-one
(15b). Yellow solid, yield 72.3%. 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J = 15.7 Hz,
1H), 7.09 – 6.98 (m, 3H), 6.75 (d, J = 8.3 Hz, 1H), 6.63 (s, 2H), 3.83 (s, 6H), 3.81 (s,
3H), 3.79 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 189.87, 163.85, 151.84, 150.69,
149.31, 146.50, 127.43, 123.71, 119.76, 111.11, 109.96, 99.86, 56.00, 55.96, 53.89.
HR-MS (ESI) m/z: calcd for C18H20NO5 [M+H]+ 330.1336, found 330.1331.
4.1.9.3 (E)-1-(2,6-dimethoxypyridin-4-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(15c). Yellow solid, yield 83.7%. 1H NMR (300 MHz, CDCl3) δ 7.57 (d, J = 15.7 Hz,
1H), 7.10 (s, 1H), 6.72 (s, 2H), 6.65 (s, 2H), 3.86 (s, 6H), 3.80 (s, 6H), 3.78 (s, 3H),
13C NMR (75 MHz, CDCl3) δ 189.88, 163.89, 153.51, 150.47, 146.49, 140.88, 129.87,
121.20, 105.88, 99.92, 61.01, 56.25, 53.92; HR-MS (ESI) m/z: calcd for C19H22NO6
[M+H]+ 360.1442, found 360.1440.
4.1.9.4 (E)-1-(2,6-dimethoxypyridin-4-yl)-3-(4-(methylthio)phenyl)prop-2-en-1-one
(15d). Yellow solid, yield 77.6%. 1H NMR (300 MHz, CDCl3) δ 7.74 (d, J = 15.7 Hz,
1H), 7.53 (d, J = 8.1 Hz, 2H), 7.30 (s, 1H), 7.24 (d, J = 5.0 Hz, 2H), 6.77 (s, 2H), 3.97
(s, 6H), 2.52 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 205.60, 163.92, 156.40, 150.51,
145.81, 128.97, 125.92, 120.80, 99.86, 98.74, 53.93, 15.06. HR-MS (ESI) m/z: calcd
for C17H18NO3S [M+H]+ 316.1002, found 316.0998.
4.1.9.5
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one

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(15e). Yellow solid, yield 57.3%. 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J = 15.7 Hz,
1H), 7.25 (d, J = 3.0 Hz, 1H), 7.21 (s, 1H), 7.15 – 7.11 (m, 1H), 6.88 (d, J = 8.3 Hz,
1H), 6.76 (s, 2H), 5.68 (s, 1H), 3.97 (s, 6H), 3.95 (s, 3H), 13C NMR (75 MHz, CDCl3)
δ 163.60, 163.36, 161.07, 153.08, 152.69, 149.77, 136.54, 106.04, 105.03, 99.33,
98.74, 98.03, 56.15, 53.65. HR-MS (ESI) m/z: calcd for C17H18NO5 [M+H]+316.1179,
found 316.1174.
4.1.9.6
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(3-fluoro-4-methoxyphenyl)prop-2-en-1-one
1
(15f). White solid, yield 63.7%. H NMR (300 MHz, CDCl3) δ 7.56 (d, J = 15.7 Hz,
1H), 7.24 (t, J = 11.2 Hz, 2H), 7.05 (d, J = 15.7 Hz, 1H), 6.85 (t, J = 8.4 Hz, 1H), 6.63
(s, 2H), 3.85 (s, 6H), 3.81 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 189.61, 163.93,
154.05, 150.36, 150.04 (d, J = 24.0 Hz), 147.85, 146.01, 144.95, 127.69, 126.31,
123.80, 120.75 (d, J = 228.8 Hz), 115.15, 114.90 (d, J = 18.8 Hz), 113.21, 99.83,
56.27, 53.91; HR-MS (ESI) m/z: calcd for C17H17FNO4 [M+H]+ 318.1136, found
318.1140.
4.1.9.7
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(4-methoxyphenyl)-2-methylprop-2-en-1-one
(16a). Yellow solid, yield 78.7%. 1H NMR (300 MHz, CDCl3) δ 7.40 (d, J = 8.4 Hz,
2H), 7.23 (s, 1H), 6.96 – 6.89 (m, 2H), 6.46 (s, 2H), 3.96 (s, 6H), 3.85 (s, 3H), 2.23 (s,
3H), 13C NMR (75 MHz, CDCl3) δ 197.33, 162.70, 159.85, 151.75, 144.55, 133.53,
131.43, 127.49, 113.52, 100.07, 54.86, 53.29, 12.99. HR-MS (ESI) m/z: calcd for
C20H24NO6 [M+H]+ 374.1598, found 313.1596.
4.1.9.8
(E)-3-(3,4-dimethoxyphenyl)-1-(2,6-dimethoxypyridin-4-yl)-2-methylprop-2-en-1-one
(16b). Yellow solid, yield 86.9%. 1H NMR (300 MHz, CDCl3) δ 7.22 (s, 1H), 7.07 (d,
J = 8.0 Hz, 1H), 6.95 – 6.89 (m, 2H), 6.46 (s, 2H), 3.96 (s, 6H), 3.93 (s, 3H), 3.90 (s,
3H), 2.26 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 197.26, 162.72, 151.73, 149.49,
148.25, 144.67, 133.82, 127.73, 123.30, 112.58, 110.41, 100.07, 55.46, 53.30, 13.07.
HR-MS (ESI) m/z: calcd for C19H22NO5 [M+H]+ 344.1492, found 313.1493.
4.1.9.9

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(E)-1-(2,6-dimethoxypyridin-4-yl)-2-methyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-o
ne (16c). Yellow solid, yield 85.3%. 1H NMR (300 MHz, CDCl3) δ 7.19 (s, 1H), 6.64
(s, 2H), 6.47 (s, 2H), 3.97 (s, 6H), 3.89 (s, 3H), 3.88 (s, 6H), 2.25 (s, 3H), 13C NMR
(75 MHz, CDCl3) δ 197.17, 162.76, 152.59, 151.40, 144.43, 138.52, 134.97, 130.29,
106.93, 100.10, 60.47, 55.74, 53.31, 13.16. HR-MS (ESI) m/z: calcd for C18H20NO4
[M+H]+ 313.1387, found 313.1385.
4.1.9.10
(E)-1-(2,6-dichloropyridin-4-yl)-3-(3-hydroxy-4-methoxyphenyl)-2-methylprop-2-en-1
-one (16d). Yellow solid, yield 75.1%. 1H NMR (300 MHz, CDCl3) δ 7.13 (s, 1H),
7.09 (s, 1H), 7.04 (s, 1H), 6.96 (d, J = 9.5 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H), 6.77 (s,
1H), 3.95 (s, 3H), 2.24 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 198.78, 160.43, 141.55,
135.76, 131.55, 126.42, 123.28, 120.95, 118.52, 111.52, 107.06, 103.53, 55.57, 15.01.
HR-MS (ESI) m/z: calcd for C16H14Cl2NO3 [M+H]+ 338.0345, found 338.0352.
4.1.9.11
(E)-1-(2-chloro-6-methoxypyridin-4-yl)-3-(3-hydroxy-4-methoxyphenyl)-2-methylprop
-2-en-1-one (16e). Yellow solid, yield 65.2%. 1H NMR (300 MHz, DMSO-d6) δ 9.24
(s, 1H), 7.19 (s, 1H), 7.11 (s, 1H), 7.06 (s, 1H), 7.00 (s, 2H), 6.93 (s, 1H), 3.91 (s, 3H),
3.82 (s, 3H), 2.15 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 197.26, 162.70, 147.23,
144.57, 136.24, 133.68, 128.15, 127.51, 126.98, 126.69, 124.14, 115.28, 110.85,
100.06, 55.51, 53.30, 12.86. HR-MS (ESI) m/z: calcd for C17H17ClNO4 [M+H]+
334.0841, found 334.0834.
4.1.9.12
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(3-hydroxy-4-methoxyphenyl)-2-methylprop-2-en
1
-1-one (16f). Yellow solid, yield 75.3%. H NMR (300 MHz, CDCl3) δ 7.18 (s, 1H),
7.08 (d, J = 2.1 Hz, 1H), 6.97 – 6.92 (m, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.45 (s, 2H),
13
5.64 (s, 1H), 3.96 (s, 6H), 3.94 (s, 3H), 2.23 (s, 3H), C NMR (75 MHz, CDCl3) δ
162.72, 151.65, 146.91, 144.95, 144.51, 128.36, 127.81, 127.44, 122.83, 115.47,
109.87, 100.10, 55.49, 53.30, 13.01. HR-MS (ESI) m/z: calcd for C18H20NO5 [M+H]+
330.1336, found 330.1332.
4.1.10 The procedure for the preparation of 18a-i.

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To a stirred solution of ketone 9b (50 mg, 0.34 mmol) and aldehyde (0.34 mmol) in
anhydrous EtOH, piperidine (144.9 mg, 1.72 mmol) was slowly added. After stirring
for 10 h at 80 °C, the reaction mixture was concentrated and extracted with CH2Cl2 (3
× 20 mL). The combined organic layers were then washed with brine, dried over
anhydrous Na2SO4, and concentrated in vacuo to provide the crude product, which
was purified by column chromatography with petroleum/ethyl acetate (4:1) to give the
corresponding pure compounds 18a-i.
4.1.10.1
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(1H-indol-3-yl)-2-methylprop-2-en-1-one (18a).
Yellow solid, yield 85.9%. 1H NMR (300 MHz, CDCl3) δ 8.74 (s, 1H), 7.70 (s, 1H),
7.65 (d, J = 2.9 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.29 (d, J
13
= 7.5 Hz, 1H), 7.19 (d, J = 7.3 Hz, 1H), 6.51 (s, 2H), 3.98 (s, 6H), 2.28 (s, 3H), C
NMR (75 MHz, DMSO-d6) δ 195.46, 162.62, 153.39, 137.13, 135.76, 129.10, 128.89,
127.15, 122.59, 120.57, 117.59, 112.13, 111.21, 99.95, 53.52, 13.77. HR-MS (ESI)
m/z: calcd for C19H19N2O3[M+H]+ 323.1390, found 323.1388.
4.1.10.2
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(5-methoxy-1H-indol-3-yl)-2-methylprop-2-en-1-
1
one (18b). Yellow solid, yield 77.9%. H NMR (300 MHz, CDCl3) δ 8.66 (s, 1H),
7.63 (d, J = 10.0 Hz, 2H), 7.32 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 10.9 Hz, 2H), 6.52 (s,
13
2H), 3.97 (s, 6H), 3.84 (s, 3H), 2.26 (s, 3H), C NMR (75 MHz, CDCl3) δ 196.85,
163.22, 155.22, 136.87, 131.52, 130.81, 130.53, 128.26, 127.16, 113.21, 112.15,
110.40, 101.08, 100.70, 55.92, 53.78, 14.29. HR-MS (ESI) m/z: calcd for
C20H21N2O4[M+H]+ 353.1496, found 353.1497.
4.1.10.3
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(6-methoxy-1H-indol-3-yl)-2-methylprop-2-en-1-
1
one (18c). Yellow solid, yield 78.3%. H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H),
7.64 (s, 1H), 7.53 (d, J = 2.8 Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 6.89 (d, J = 2.1 Hz,
1H), 6.85 (dd, J = 8.7, 2.2 Hz, 1H), 6.49 (s, 2H), 3.97 (s, 6H), 3.85 (s, 3H), 2.25 (s,
3H), 13C NMR (75 MHz, CDCl3) δ 196.50, 162.71, 156.89, 152.44, 148.19, 136.68,
130.68, 125.14, 121.14, 118.81, 112.85, 110.56, 100.05, 94.23, 55.18, 53.32, 13.67.

ACCEPTED MANUSCRIPT
HR-MS (ESI) m/z: calcd for C20H21N2O4[M+H]+ 353.1496, found 353.1499.
4.1.10.4
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(5-fluoro-1H-indol-3-yl)-2-methylprop-2-en-1-on
1
e (18d). Yellow solid, yield 66.5%. H NMR (300 MHz, CDCl3) δ 8.76 (s, 1H), 7.68
(s, 1H), 7.56 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 7.17 (d, J = 9.2 Hz, 1H), 7.04 (d, J =
9.2 Hz, 1H), 6.49 (s, 2H), 3.99 (s, 6H), 2.26 (s, 3H), 13C NMR (75 MHz, DMSO-d6) δ
196.03, 163.11, 153.58, 137.14, 132.90, 131.22, 129.64, 113.88 (d, J = 186.8 Hz),
113.75, 111.94, 111.39, 111.03, 103.63, 103.31, 100.58, 53.97, 14.40. HR-MS (ESI)
m/z: calcd for C19H18FN2O3[M+H]+ 341.1296, found 341.1298.
4.1.10.5
(E)-1-(2,6-dimethoxypyridin-4-yl)-3-(6-fluoro-1H-indol-3-yl)-2-methylprop-2-en-1-on
e (18e). Yellow solid, yield 66.5%. 1H NMR (300 MHz, CDCl3) δ 8.73 (s, 1H), 7.62 (s,
2H), 7.47 – 7.41 (m, 1H), 7.11 (d, J = 8.9 Hz, 1H), 7.00 – 6.92 (m, 1H), 6.50 (s, 2H),
3.98 (s, 6H), 2.26 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 197.00, 163.24, 152.69,
136.41, 131.81, 126.82, 124.02, 119.67, 119.54, 113.30, 110.08 (d, J = 241.5 Hz),
109.76, 100.56, 98.05, 97.70, 53.84, 14.23. HR-MS (ESI) m/z: calcd for
C19H18FN2O3[M+H]+ 341.1296, found 341.1296.
4.1.10.6
(E)-3-(5-chloro-1H-indol-3-yl)-1-(2,6-dimethoxypyridin-4-yl)-2-methylprop-2-en-1-o
ne (18f). Yellow solid, yield 73.5%. 1H NMR (300 MHz, CDCl3) δ 8.68 (s, 1H), 7.66
(s, 1H), 7.55 (s, 1H), 7.49 (s, 1H), 7.35 (d, J = 8.8 Hz, 2H), 6.49 (s, 2H), 3.99 (s, 6H),
2.26 (s, 3H), 13C NMR (75 MHz, DMSO-d6) δ 196.10, 163.10, 153.50, 136.78,
134.79, 130.95, 130.13, 128.83, 125.63, 123.06, 117.75, 114.21, 111.51, 100.64, 54.00,
14.48. HR-MS (ESI) m/z: calcd for C19H18ClN2O3[M+H]+ 357.1000, found 357.1001.
4.1.10.7
(E)-3-(6-chloro-1H-indol-3-yl)-1-(2,6-dimethoxypyridin-4-yl)-2-methylprop-2-en-1-o
ne (18g). Yellow solid, yield 53.7%. 1H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 7.64
– 7.58 (m, 2H), 7.42 (s, 1H), 7.16 (dd, J = 8.4, 1.9 Hz, 1H), 6.74 (s, 1H), 6.50 (s, 2H),
3.98 (s, 6H), 2.26 (s, 3H), 13C NMR (75 MHz, CDCl3) δ 200.37, 163.25, 152.62,
136.18, 135.85, 131.91, 129.38, 127.01, 126.00, 121.85, 119.63, 113.33, 111.43,