Synthesis, biological evaluation and molecular modeling of imidazo[1,2-a]pyridine derivatives as potent antitubulin agents

Article abstract of DOI:10.1016/j.bmc.2017.05.057

Two series of novel 5,7-diarylimidazo[1,2-a]pyridine-8-carbonitrile derivatives (3a–3q and 7a–7n) were designed by modification of CA-4 pharmacophore to develop colchicine targeted antitubulin agents. All compounds were efficiently synthesized and evaluated for their cytotoxicity against five selected cancer cell lines (HT-29, H460, A549, MKN-45 and SMMC-7721) which got an insight in structure and activity relationships (SARs). Several molecules (7e, 7f, 7h–7j and 7m) were disclosed to exhibit promising antiproliferative activity with IC₅₀ values in double-digit nanomolar degree. Optimization toward these compounds led to the discovery of a promising lead 7e, which showed noteworthy potency with IC₅₀ value ranging from 0.01 to 3.2?μM superior to CA-4 and Crolibulin. Importantly, immunofluorescence staining and colchcine competitive binding assay revealed that microtubule dynamics was disrupted by 7e by binding at the colchicine site of tubulin. Moreover, molecular docking studies suggested the binding of this mimic at colchcine-binding site is similar to Crolibulin, as was in conformity with the observed SARs for these compounds.

Full text of DOI:10.1016/j.bmc.2017.05.057

Accepted Manuscript
Synthesis, biological evaluation and molecular modeling of imidazo[1,2-a]pyr-
idine derivatives as potent antitubulin agents
Jin Liu, Daiying Zuo, Tongfei Jing, Ming Guo, Lingyun Xing, Wenyu Zhang,
Jianwen Zhao, Jiwei Shen, Ping Gong, Dajun Zhang, Xin Zhai
PII:
S0968-0896(17)30821-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.057
BMC 13776
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 April 2017
15 May 2017
27 May 2017
Please cite this article as: Liu, J., Zuo, D., Jing, T., Guo, M., Xing, L., Zhang, W., Zhao, J., Shen, J., Gong, P., Zhang,
D., Zhai, X., Synthesis, biological evaluation and molecular modeling of imidazo[1,2-a]pyridine derivatives as
potent antitubulin agents, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2
017.05.057
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Synthesis, biological evaluation and molecular modeling of
imidazo[1,2-a]pyridine derivatives as potent antitubulin agents
1
,#
1,#
1
1
1
1
Jin Liu , Daiying Zuo , Tongfei Jing , Ming Guo , Lingyun Xing , Wenyu Zhang , Jianwen
2
1
1
2,*
1,∗
Zhao , Jiwei Shen , Ping Gong , Dajun Zhang , Xin Zhai
1
Key Laboratory of Structure-Based Drug Design and Discovery,Ministry of Education, Shenyang
2
Pharmaceutical University, Shenyang 110016, PR China; key lab. of behavioral and cognitive
neuroscience of Liaoning province, Shenyang Medical College, Shenyang 110034, PR China.
Abstract
Two series of novel 5,7-diarylimidazo[1,2-a]pyridine-8-carbonitrile derivatives (3a-3q and
7a-7n) were designed by modification of CA-4 pharmacophore to develop colchicine targeted
antitubulin agents. All compounds were efficiently synthesized and evaluated for their cytotoxicity
against five selected cancer cell lines (HT-29, H460, A549, MKN-45 and SMMC-7721) which got
an insight in structure and activity relationships (SARs). Several molecules (7e, 7f, 7h-7j and 7m)
were disclosed to exhibit promising antiproliferative activity with IC50 values in double-digit
nanomolar degree. Optimization toward these compounds led to the discovery of a promising lead
7e, which showed noteworthy potency with IC50 value ranging from 0.01 to 3.2 µM superior to
CA-4 and Crolibulin. Importantly, immunofluorescence staining and colchcine competitive
binding assay revealed that microtubule dynamics was disrupted by 7e by binding at the
colchicine site of tubulin. Moreover, molecular docking studies suggested the binding of this
mimic at colchcine-binding site is similar to Crolibulin, as was in conformity with the observed
SARs for these compounds.
Keywords: Imidazo[1,2-a]pyridines; Tubulin; Synthesis; Cytotoxicity; Molecular modeling
1. Introduction
Microtubules are a key cytoskeletal structure which exert their effects by disrupting microtubule
dynamics and inhibiting the noncovalent polymerization of tubulin into microtubules, resulting in
disruption of cell progression, signal transduction and, subsequently, induction of cell death [1-2].
The essential role of microtubules in mitotic spindle formation makes them one of the most
attractive targets for development of antitumor molecules [3-5]. There are four main binding sites
on tubulin which include colchicine site, vinca domain, taxane site and laulimalide domain [6-8]
and some of the well-known natural occurring tubulin binding ligands that inhibit microtubule
dynamics by binding to the distinct colchicine domains are colchicine [9], combretastatin
A-4(CA-4) [10], Crolibulin [11], and diverse hit-like synthetic antitubulin ligands [12-14] (Fig. 1).
CA-4, a natural antitubulin agent isolated from South Africa tree Combretumcaffrum, was
reported to inhibit microtubules dynamics strongly by binding to the colchicine site and showed
excellent cytotoxicity [15]. However, the poor solubility and acquired significant toxicity toward
*
Corresponding author. E-mail address: zhangdajun2008@126.com (D.J. Zhang); zhaixin_syphu@126.com (X. Zhai).
Authors contributed equally to this work.
#

normal cells impinged its clinical development and thus, stimulated a surge of research endeavors
in discovery of more effective CA-4 structurally analogs [16]. CA-4 is usually divided into three
pharmacological substructures, two hydrophobic aryl rings (A and B) and a cis-olefin linkage.
Since 3,4,5-trimethoxyaryl (A-ring) and cis-olefin linking bridge are generally believed to be
essential for biological activity, previous structural modification was mainly focused on B-ring
[
17]. When we explored the relevant CA-4 literature, it was found that dihalogenation with either
bromine or iodine was a tolerated modification when compared to the parent compound CA-4 and
had less effect than B-ring modification on potency [18]. Therefore, 2,3,4-trimethoxyphenyl,
2,4-diflourophenyl or 2-chloro-4-flourophenyl groups were introduced to A-ring in this paper to
extending the SARs. Besides, it is noteworthy that several attempts have been reported to modify
the cis-olefin to prevent its isomerization under storage and metabolism, which led to the
development of diverse CA-4 derivatives with a five to six member heteroaromatic rings, for
instance, indolyl and imidazolyl as cis-restricted CA-4 analogues (e.g. compounds a in Fig. 1)
[
12-14].
Figure 1.The colchicine-site-binding antitubulincandidates and compounds 3a-3q and 7a-7n.
Crolibulin, a 4-aryl-4H-chromene derivative in phase II clinical trials, is one of the best
characterized antimitotic agents with potent vascular disrupting activity and in vivo efficacy [11,
9-20] (Fig. 1). Crolibulin-based SARs studies suggested that the cyano group at C-3 position is
1
essential for its antitumor potency, and substituted phenyl are the preferred group at C-4 position
of 4H-chromenes [11, 21].
Imidazo[1,2-a]pyridines (IMPs) framework was recognized as a “drug prejudice” scaffold due
to its diverse biological activities and adopted in a variety of clinical drugs [22-25]. Herein, our
researches have been focused on optimization of the 4-aryl-4H-chromenes-3-carbonitrile scaffold
of Crolibulin by replacing with imidazo[1,2-a]pyridine-8-carbonitrile fragment.
In our effort to search for antitubulin agents targeting colchicines-binding site, we were inspired
to further optimize the 7-arylimidazo[1,2-a]pyridine-8-carbonitrile skeleton by introducing aryl
group at C-5 position as the B-ring based on bioisosterism and molecular hybridization principles.
In the continuation of SARs study of restricted CA-4 derivatives, the B-ring was replaced by the
pharmacologically relevant phenyl or indolyl scaffolds, and the cis configuration of olefin was
restricted by incorporation of imidazo[1,2-a]pyridine-8-carbonitrile framework which is envisaged
by the structural resemblance to CA-4. Moreover, substituted dimethoxyphenyl or halogenated
phenyl motifs were incorporated into the C-7 position to mimic CA-4. Additionally, with the

purpose of increasing water solubility, various hydrophilic N-aliphatic aminomethyl groups were
drawn into C-2 or C-3 positions on the IMPs skeleton. Accordingly, two series of
5,7-diarylimidazo[1,2-a]pyridine-8-carbonitrile derivatives (3a-3q and 7a-7n) were designed and
synthesized. Wherein, diverse substituents were introduced to aryl groups (A and B) and terminal
aminomethyl fragments in an attempt to investigate the influence on antitumor efficacy by
regulating the electronic and steric effects.
In this paper, all newly synthesized compounds were evaluated for their cytotoxic activities
against a panel of human cancer cell lines in vitro. Also, the antitubulin potency of candidate 7e
was identified by immunofluorescence staining and colchcine competitive binding assay, and the
results were further confirmed by molecular docking mode analysis.
2. Chemistry
The target compounds 3a-3q and 7a-7n were synthesized according to the pathways depicted in
Schemes 1-2. As shown in Scheme 1, 2-amino-4,6-diarylnicotinonitrile skeleton of 1a-1c was
concisely assembled in a one-pot 4-component reaction with commercially available aromatic
formaldehyde, substituted acetophenones and malononitrile in the presence of ammonium acetate
in refluxing toluene [26]. Subsequently, 1a-1c were cyclization with 40% acetaldehyde in
refluxing ethanol on exposure to sodium bicarbonate afforded intermediates 2a-2c in good yields
[
27]. Finally, 2a-2c were subjected to Mannich reaction at the existence of N-aliphatic amines and
formaldehyde in acetic acid at 30 ºC, and the resultant crude products were purified by
chromatography on silica gel using CH Cl /MeOH (20:1) to generate the desired compounds
a-3q.
2
2
3
Scheme 1. Reagents and conditions: (a) toluene, reflux; (b) 40% acetaldehyde aqueous solution, NaHCO
reflux; (c) HCHO, N-aliphatic amine, acetic acid, 30 ºC.
3
, ethanol,
Target compounds 7a-7n were prepared following the procedure outlined in Scheme 2.
Acylation reaction of 1H-indole with acetic anhydride in the presence of anhydrous stannic
chloride in nitromethane afforded 1-(1H-indol-3-yl)ethan-1-one 4 [28]. 4 was utilized in a
convenient 4-component reaction with aromatic formaldehyde, malononitrile and ammonium
acetate in refluxing toluene to give desired intermediates 5a-5b, as was similar to the previously
described protocol of 1a-1c in scheme 1. Then, The entire imidazo[1,2-a]pyridine framework was
packaged in one step with intermediates 5a-5b in the presence of 1,3-dichloropropanone in
ethanol giving rise to 6a-6b. Eventually, nucleophilic substitution of the resultant 6a-6b with
appropriate N-aliphatic amines in refluxing ethanol afforded target compounds 7a-7n in satisfied

yields.
4
Scheme 2. Reagents and conditions: (a) acetic anhydride, anhydrous SnCl , nitromethane, 0 ºC to r.t.; (b) toluene,
reflux; (c) 1,3-dichloropropan-2-one, ethanol,reflux; (d) N-aliphatic amine, ethanol, reflux.
3. Results and discussions
3.1 Antiproliferative activity and SARs
To determine whether the imidazo[1,2-a]pyridine-8-carbonitrile skeleton is a suitable mimic for
the cis-olefin presented in CA-4, two series of 5,7-diphenylimidazo[1,2-a]pyridine-8-carbonitrile
derivatives (3a-3q and 7a-7n) were examined for their cytotoxic activity against five cancer cells
lines, HT29 (human colon cancer), H460 (human lung cancer), A549 (human lung carcinoma),
MKN45 (human gastric cancer), and SMMC-7721 (human liver cancer) by a MTT assay. CA-4
and Crolibulin were used as the positive control. The results expressed as half-maximal inhibitory
concentration (IC50) values are depicted in Table 1-2, as mean values of experiments performed in
triplicate.
Table 1. Cytotoxicity of 3a-3q against HT29, H460, A549, MKN45 and SMMC-7721 cell lines in vitro.
a
IC50(µM) ±SD
Compd.
R1
R2
-NR3R4
HT29
H460
A549
MKN45
SMMC-7721
24.62±2.65
3
3
3
3
3
a
b
c
2,3,4-tri-OMe
2,3,4-tri-OMe
2,3,4-tri-OMe
2,3,4-tri-OMe
2,3,4-tri-OMe
4-F
4-F
4-F
4-F
4-F
2.04±0.20
3.89±0.46
12.40±1.94
5.48±0.70
0.91±0.09
1.95±0.22
1.03±0.22
0.80±0.11
4.54±0.54
15.62±1.86
9.75±1.13
9.33±1.05
3.24±0.71
1.33±0.41
3.56±0.48
0.67±0.01
7.88±0.92
5.84±0.78
10.48±1.15
6.99±0.90
6.02±0.79
5.12±0.77
4.03±0.48
3.02±0.42
d
e

3
3
3
f
2,3,4-tri-OMe
2,3,4-tri-OMe
2,3,4-tri-OMe
4-F
3.01±0.44
0.83±0.12
0.21±0.02
3.08±0.39
1.88±0.22
0.06±0.01
1.97±0.28
0.71±0.09
1.04±0.15
23.40±2.77
3.04±0.35
6.79±0.72
6.91±0.86
4.42±0.49
1.31±0.18
g
h
4-F
2,4-di-F
3
3
3
3
3
i
2,3,4-tri-OMe
2,3,4-tri-OMe
2,3,4-tri-OMe
2,3,4-tri-OMe
2,3,4-tri-OMe
2,4-di-F
2,4-di-F
2,4-di-F
2,4-di-F
2,4-di-F
1.34±0.13
0.07±0.01
0.23±0.03
0.09±0.01
0.14±0.03
0.06±0.01
0.02±0.007
0.20±0.02
0.03±0.008
0.06±0.01
0.80±0.13
0.13±0.02
1.98±0.34
0.17±0.03
1.46±0.25
7.26±0.83
2.7±0.31
2.38±0.32
1.17±0.21
6.48±0.74
1.32±0.23
3.48±0.45
j
k
l
9.08±1.12
3.52±0.62
1.68±0.21
m
3
3
3
3
n
o
p
q
2,3,4-tri-OMe
3,4,5-tri-OMe
3,4,5-tri-OMe
3,4,5-tri-OMe
2,4-di-F
2,4-di-F
2,4-di-F
2,4-di-F
0.47±0.08
3.51±0.45
0.36±0.04
0.08±0.01
0.76±0.09
0.67±0.11
1.15±0.22
>100
2.38±0.29
40.57±5.03
14.02±2.11
3.2±0.41
>100
31.0±3.39
62.5±5.32
0.70±0.08
0.05±0.01
0.13±0.03
0.08±0.01
1.2±0.23
11.6±1.46
0.11±0.02
2.93±0.44
1.92±0.11
CA-4
0.43±0.053
b
b
b
b
b
Crolibulin
0.52±0.02
0.03±0.01
ND
0.17±0.03
ND
a
Data presented is the mean ± SD value of three independent determinations.
b
22
reported IC50 value .
Table 2. Cytotoxicity of 7a-7q against HT29, H460, A549, MKN45 and SMMC-7721 cell lines in vitro.
a
IC50(µM)±SD
Compd.
R1
-NR2R3
HT29
H460
A549
MKN45
SMMC-7721
5.30±0.62
7
7
7
a
b
c
2,4-di-F
2,4-di-F
2,4-di-F
0.06±0.008
0.19±0.02
1.12±0.16
1.34±0.21
0.16±0.02
0.05±0.06
0.2±0.03
8.90±0.92
0.92±0.94
18.2±2.12
2.77±0.34
16.25±1.76
6.12±0.68
0.15±0.02
7
d
2,4-di-F
0.08±0.01
0.18±0.03
1.92±0.21
3.34±0.36
6.54±0.74

7
7
7
7
7
7
7
7
7
7
e
f
2,4-di-F
2,4-di-F
2,4-di-F
2,4-di-F
2,4-di-F
2,4-di-F
2,4-di-F
2-Cl, 4-F
2-Cl, 4-F
2-Cl, 4-F
0.01±0.002
0.04±0.006
0.11±0.01
0.01±0.002
0.04±0.009
0.02±0.003
2.7±0.27
0.04±0.006
0.05±0.003
0.17±0.02
0.20±0.03
0.28±0.04
0.11±0.01
0.19±0.02
0.22±0.03
0.08±0.01
0.54±0.06
1.03±0.19
0.92±0.09
3.64±0.47
9.66±1.02
0.08±0.01
3.33±0.42
1.55±0.18
2.53±0.26
2.4±0.28
5.6±0.56
0.48±0.06
2.71±0.32
2.62±0.32
3.68±0.45
3.22±0.36
7.86±0.85
3.62±0.38
3.68±0.42
5.04±0.62
3.82±0.48
3.55±0.38
6.8±0.72
g
h
i
j
1.92±0.21
12.84±1.34
7.12±0.76
4.88±0.53
k
l
0.14±0.02
0.02±0.004
m
n
0.12±0.01
0.05±0.01
0.25±0.03
0.08±0.01
1.04±0.15
2.44±0.26
0.11±0.02
5.66±0.87
1.92±0.11
CA-4
0.43±0.053
b
b
b
b
b
Crolibulin
0.52±0.02
0.03±0.01
ND
0.17±0.03
ND
a
Data presented is the mean ± SD value of three independent determinations.
b
22
reported IC50 value .
As illustrated in Table 1, an expected observation was that most compounds showed moderate
to excellent cytotoxic activity toward five tested cell lines, producing double-digit nanomolar IC50
values in some instances (e.g., analogues 3j, 3l, 7a, 7c, 7e, 7f, 7h-7j and 7m). Six compounds (7e,
7f, 7h-7j and 7m) were optimized for their prominent antiproliferative activity with IC50 values
ranging from 0.01 to 6.80 µM. Notably, the most promising compound 7e displayed remarkable
cytotoxicity whose IC50 values were 0.01 µM, 0.04 µM, 0.54 µM, 2.4 µM and 5.6 µM against
HT29, H460, A549, MKN45 and SMMC-7721 cell lines respectively, which was 5-fold more
potent than CA-4 and 52 times higher than Crolibulin selectively on HT29 cell, reflecting a good
selectivity for colon cancer. As a general trend, the title compounds, especially for 7a-7n, were
more potent on HT-29 and H460 cells than on other cells. By contrast, the molecules displayed
about 6- to 510-fold less sensitivity on SMMC-7721 cell as indicated by the IC50 values.
The pharmacological data disclosed a clear tendency related to the IMPs framework. As shown
in Table 1, aromatic substituents (A and B ring) attached on IMPs skeleton plays a vital role in
determining the cytotoxicity and selectivity of these inhibitors. To our surprise, introduction of
2,3,4-trimethoxyphenyl group at C-7 position of IMPs led to an obvious improvement in potency
as compared with the relative 3,4,5-trimethoxyphenyl group (3h vs. 3o, 3k vs. 3p,and 3i vs. 3q), as
extend the modify limitation of A-ring restricted to 3,4,5-trimethoxyphenyl motif in CA-4.
2
Moreover, replacement of 2,4-difluorophenyl group (R ) at C-5 position (3h-3n) with
4-fluorophenyl moiety (3a-3g) resulted in a dramatic decrease in activity, indicating 2,4-difluoro

group is a favorable moiety in this region. For example, 2,4-difluorophenyl derivatives 3h was
approximate 3.4- to 51.4-fold more active than the corresponding 4-fluorophenyl analog 3f.
Further structural optimization was conducted by introducing of diverse aliphatic aminomethyl
groups on C-3 position of IMPs scaffold. The SARs based on IC50 values showed that variations
of terminal aliphatic amino groups have a marked impact on activity. Interestingly, compound 3j
with the small methylamino motif exhibited the best activity against tested cells with IC50 values
ranging from 0.07 to 2.7 µM, which was comparable to that of CA-4 and Crolibulin. By contrast,
compounds 3m (4-methylpiperidinyl) and 3i (pyrrolidinyl) with bulky heterocyclic amino groups
demonstrated an approximate 2- to 19-fold diminished potency against tested cells, except for on
MKN-45. Accordingly, secondary amino derivatives, such as 3j (HT-29, IC50 = 0.07 µM) and 3l
(
HT-29, IC50 = 0.09 µM), exhibited better cytotoxicity than tertiary amino derivatives 3h (HT-29,
IC50 = 0.21 µM), 3i (HT-29, IC50 = 1.34 µM) and 3k (HT-29, IC50 = 0.23 µM). The loss in activity
might be due to the steric clash between alkylamino chain and aryl B-ring which prevent the test
molecule from reaching the colchicine binding site of tubulin. The same trend was observed
among compounds 3a-3g.
To further optimize structural skeleton, we prepared a series of
5-indolyl-7-arylimidazo[1,2-a]pyridine-8-carbonitrile derivatives 7a-7n by introducing diverse
N-aliphatic amino groups into the C-2 position on IMP skeleton. Inspired by the outstanding
cytotoxicity of 3j, the favorable 2,4-difluorophenyl group was introduced to C-7 position
producing analogs 7a-7k. Additionally, 7l-7n with 2-chloro-4-flourophenyl moiety were
synthesized to explore the extending SARs. It is worth noting that embedment of indolyl group
turned out to enhance the antiproliferative potency as compared with the relative phenyl
derivatives 3a-3q, in good agreement with the low expand ability and of the B ring pocket at the
colchicine site. Meanwhile, replacement of 2,4-diflourophenyl group (R
-chloro-4-flourophenyl (7l, 7m and 7n) altered cytotoxicity slightly, indicating that halogen at
phenyl R fragment contributed little to potency.
1
) with
2
1
The data presented in Table 2 indicated that the cytotoxicity of 7a-7n was generally higher than
the corresponding 3a-3q, suggesting that the substituted amino group at C-2 position is favor for
activity. Interestingly, an altered or even reverse trend was observed when the aliphatic amino
groups were shifted from C-3 to C-2 position on IMP skeleton. Differ from 3j, compound 7b with
tiny methylamino group displayed poor potency as compared to others. Contrary to 3a-3q,
compounds bearing secondary amino (e.g. methyl group, ethyl group, isopropyl group) indicated
1.2- to 16-fold decrease in potency than tertiary alicyclic amino analogs (e.g. cyclehexyl,
4-methylpiperidinyl and pyrrolidinyl) as comparison of 7a and 7b, 7d and 7f. To our delight,
2-((cyclohexylamino)methyl)-7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyridine-8-c
arbonitrile 7e exhibited prominent cytotoxicity whose IC50 value ranging from 0.01 to 6.80 µM,
superior to positive control and other analogs. However, a dramatic loss in cytotoxic potency was
observed for derivatives 7g (HT-29, IC50 = 0.11 µM) or 7k (HT-29, IC50 = 2.7 µM) with
4-methylpiperizino or morpholino groups respectively, indicating the heterocyclic amino was not
favorable for this region. Additional SARs and further optimization are in progress and will be
reported in due course.
3.2 7e disturbs microtubule polymerization
To further ascertain the promising cytotoxic potency of the target compounds are due to

interference with the mitotic machinery, we analyzed the antitubulin effect of the lead 7e on
cellular microtubule networks via tubulin immunofluorescence in HT29 cells [29]; CA-4 was
tested for comparison. As shown in Fig. 2, the immunofluorescence analysis using the specific
antibodies to α-tubulin revealed that the microtubule network of the cells in negative control group
was homogenous, intact arranged and well organized. However, cells exposed to either CA-4 (20
nM) or 7e (88 nM) for 24 h induced abnormal microtubule network arrangement and exhibited
cellular microtubule depolymerization with scattered short microtubule fragments in the
cytoplasm of HT-29 cells. The results confirm that tubulin polymerization was disturbed by 7e,
indicating the potential tubulin-targeted activity possessed by this lead compound.
Figure 2. Immunofluorescence assay on 7e in HT29 cells. HT29 cells were treated with CA-4 (20nM) or 7e (88
nM) for 24 h. After incubation, the cells were fixed, stained with monoclonalα-tubulin antibody (green) at 4 ℃
overnight and then reacted with FITCconjugatedsecondary antibody. DAPI (blue) was used for nuclear
counterstaining. The cellular microtubules were observed by fluorescence microscopy (scalebar = 20 µm).
3.3 7e binds to tubulin at the colchicine site.
These IMP derivatives showed similar inhibitory effects on tubulin assembly to that of CA-4
and which prompted us to investigate whether 7e bind to the colchicine site of the tubulin by
employing fluorescence based colchicine binding assay in parallel with CA-4 and Taxol as
reference [30]. It was observed that at low concentration the test compound 7e and CA-4 showed
significant affinity towards colchicine site, whereas at higher concentrations both of them showed
more binding affinity as shown in Fig. 3. The decrease in fluorescence of the colchicine-tubulin
complex in the presence of 7e was in a dose‑dependent manner site on tubulin. The taxol was
used as a negative control, which is known to binding at different site and shows no effect on
tubulin-colchicine complex [31]. Therefore, the results supported a notion that 7e binds to the
colchicine site on tubulin although inferior to CA-4.
Figure 3. Fluorescence based colchicine competitive binding assay on 7e. Tubulin was co-incubated with

indicated concentrations of Taxol, 7e or CA-4 for 1 h, and then 5 µmol/L colchicine was added. The fluorescence
was measured by Hitachi F-2500 spectrofluorometer.
3. 4 Molecular modeling analysis
A molecular docking study was conducted to elucidate the hypothetical binding mode of
,7-diarylimidazo[1,2-a]pyridine-8-carbonitrile derivative 7e that exhibited potent cytotoxic
5
activity in the colchicine-binding site cavity using Discovery Studio Program 3.5. The
surflex-Dock program in Sybyl-X 2.1.1 was used to dock 7e into the colchicine-binding site of
tubulin following the removal of the ligand from the 3D crystal structure of the
DAMA-colchicines-tubulin complex (PDB code: 4O2B) [32].
According to the docking model, 7e occupied the colchicine binding site of tubulin (Fig.4B/C).
Most of all, the cyano group on IMPs skeleton formed a strong hydrogen bond with the amino
group of Asn249 of β-tubulin and inferior Pi-donor hydrogen bond with protein residue Leu248.
Moreover, the IMP framework could also generate several alkyl interactions by linking up to
Ala180, Ala354, Lys352 and Leu248 (Fig. 4A), suggesting introduction of IMPs scaffold could
foster potent tubulin-binding affinity leading to significant antiproliferative activities. And also,
the phenyl and indolyl groups were involved in the alkyl, Pi-donor hydrogen bond and halogen
interactions (Fig. 4A). Concretely, the diflourophenyl moiety could form hydrophobic interactions
with the hydrophobic pocket made up by Ala250, Thr179, Asn101, Gly143 and Ser178.
Significantly, the fluorine at C-2 position forms halogen interaction well with Asn249, as may be
the reason that introducing halogen groups could enhance the antitubulin activity. Meanwhile, a
Pi-sulfur interaction between the indolyl group and Met 259 residue, and a few alkyl interactions
with Ala180 and Lys352 were formed successively. In addition, the cyclohexyl moiety was
responsible for the hydrophobic and alkyl interactions with Ala317, Ile378 and Ile 318. Notably,
the overlapped 3D model of candidate 7e and Crolibulin (Fig.4D) indicated that 7e was oriented in
the similar manner with matching occupancy at the colchicine-binding site of tubulin. Thus, the
results from the docking study were in accordance with the SARs analysis.
Figure 4.The binding mode between the active conformation of 7e and tubulin. (A) 2D diagram of the interaction
between 7e and colchicine-binding site.(B/C)3D diagram of the interaction between 7e and colchicine-binding site.

For clarity, only interacting residues are displayed. The H-bond (green arrows) is highlighted as dotted arrows in
B. (D)Predicted modes for 7e(green) and Crolibulin (pink) binding at the colchicine-binding site of tubulin (PDB
code: 4O2B), and overlapping with each other.
4
4. Conclusions
Imidazo[1,2-a]pyridine-8-carbonitriles serving as the “privileged medicinal scaffolds” are of
particular utility for generation of small-molecule ligands with pronounced antitumor efficacy.
Herein, with an aim to develop antitubulin agents targeting colchicine-binding site, we discovered
two series of 5,7-diarylimidazo[1,2-a]pyridine-8-carbonitrile derivatives (3a-3q and 7a-7n)
according to bioisosterism and hybridization principles. All the compounds were prepared via a
concise three-step process involving acylation reaction, one-pot coupling reaction and cyclization
transformation, and their antiproliferative activities were evaluation against a panel of human
cancer cell lines (HT29, H460, A549, MKN-45, and SMMC-7721) in MTT assay. The preliminary
investigation showed that several molecules (e.g. 3j, 3l, 7e, 7f, 7h-7j and 7m) displayed promising
cytotoxic activity with IC50 values in a double-digit nanomolar range. Optimization towards these
series of compounds resulted in the discovery of 7e, which displayed noteworthy antitumor
potency with IC50 value of 0.01 µM, 0.04 µM, 0.54 µM, 2.4 µM, and 5.6 µM against HT29, H460,
A549, MKN45 and SMMC-7721 cell lines respectively, superior to positive CA-4 and Crolibulin
on most of the tested cells. The preliminary SARs provided a useful insight that could be utilized
in developing improved leads based on IMPs skeleton. Notably, fluorescence based colchicine
competitive binding assay as well as immunofluorescence assay were undertook which
demonstrated that tubulin polymerization was disturbed by 7e by binding at colchicine site of
tubulin. Moreover, a docking mode revealed that 7e could foster potent tubulin-binding affinity by
forming critical hydrogen bonding and hydrophobic interactions, and orient in the similar manner
with Crolibulin occupied at the colchicine-binding site in overlap 3D model, in good agreement
with the pharmacological activity and the observed SARs for these series of compounds.
In summary, the SARs-guided discovery process and docking models led to identify a candidate
7e, which could provide helpful guidance in development of new chemical entities as effective
colchicines-binding-site targeted tubulin polymerization inhibitors.
5. Experimental protocols
5
.1 Chemistry
All melting points were obtained on a Büchi Melting Point B-540apparatus
BüchiLabortechnik, Flawil, Switzerland) and were uncorrected.Mass spectra (MS) were taken in
(
1
13
ESI mode on Agilent100 LC-MS (Agilent, Palo Alto, CA, USA). H NMR and C NMR
spectroscopy was performed using Bruker ARX-400 and ARX-600spectrometers (Bruker
Bioscience, Billerica, MA, SA) with TMS as an internal standard. Column chromatography was
run on silica gel (200-300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China).
Unless otherwise noted, all materials were obtained from commercially available sources and
were used without further purification.
5.1.1. General procedure for preparation of 2-amino-4,6-diarylnicotinonitrile (1a-1c)
To a solution of aromatic formaldehydes (0.1mol) in toluene (300 mL) was added substituted
acetophenones (0.1mol), malononitrile (6.6 g, 0.1 mol) and ammonium acetate (61.6 g, 0.8mol).
The reaction mixture was refluxed for 8 h. Upon cooling to room temperature, the solvent was
evaporated under vacuum and the residue was triturated with ethanol (150 mL). After stirring for

10 min, the resulting precipitant was filtered, and the filtrate was further stirred with ethanol (200
mL) overnight. Thus, the resultant solid were collected by filtration to afford compounds 1a-1c.
5
5
5
5
.1.1.1. 2-amino-6-(4-fluorophenyl)-4-(2,3,4-trimethoxyphenyl)nicotinonitrile (1a)
+
Pale yellow solid.Yield: 51.3%. MS (ESI) m/z (%): 380.2 [M+H] .
.1.1.2. 2-amino-6-(2,4-difluorophenyl)-4-(2,3,4-trimethoxyphenyl)nicotinonitrile (1b)
+
Pale yellow solid.Yield: 46.5%. MS (ESI) m/z (%):398.2 [M+H] .
.1.1.3. 2-amino-6-(2,4-difluorophenyl)-4-(3,4,5-trimethoxyphenyl)nicotinonitrile (1c)
+
Pale yellow solid.Yield: 41.9%. MS (ESI) m/z (%): 398.2 [M+H] .
.1.2. General procedure for preparation of 5,7-diarylimidazo[1,2-a]pyridine-8-carbonitrile
(2a-2c)
A mixture of 2-amino-4,6-diarylnicotinonitrile (1a-1c) (46.1 mmol), 40% acetaldehyde
aqueous solution (90 mL, 46.1 mmol), sodium bicarbonate (7.7 g, 92.2 mmol) in ethanol (180 mL)
was heated to reflux and stirred for 7 h, whereby a yellow suspension was formed. After
completion of the reaction indicated by TLC, the mixture was cooled to room temperature, and the
solvent was evaporated under reduced pressure. The residue was dissolved in the mixture solution
of H
CH Cl
over anhydrous Na
isopropyl ether (150 mL) for 1 h, and the solid were filtered and air-dried to furnish compounds
2
O/CH
twice. The combined organic layer was washed with water (200 mL×2) and brine, dried
SO and concentrated in vacuum. The residue was triturated with
2 2
Cl (1:1, 400 mL) and stirred for 10 min, and the aqueous layer was extracted with
2
2
2
4
2a-2c.
5
.1.2.1. 5-(4-fluorophenyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]pyridine-8-carbonitrile (2a)
+
Pale while solid. Yield: 74.4%. MS (ESI) m/z (%):404.2 [M+H] .
5.1.2.2. 5-(2,4-difluorophenyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]pyridine-8-carbonitrile
(2b)
+
Pale while solid. Yield: 82.7%. MS (ESI) m/z (%):422.2 [M+H] .
.1.2.3. 5-(2,4-difluorophenyl)-7-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]pyridine-8-carbonitrile
2c)
5
(
+
Pale while solid. Yield: 79.1%. MS (ESI) m/z (%):422.2 [M+H] .
.1.3. General procedure for preparation compounds (3a-3q)
To a mixture of 37% formalin (3.6 mmol) in acetic acid (15 mL) was added various
5
N-aliphatic amine (3.6 mmol). After stirring for 30 min, 2a-2c (1.2 mmol) was added at room
temperature. Until dissolved completely, the solution was heat to 30ºC and stirred. After
completion of the reaction monitored by TLC, the solution was cooled to room temperature, and
poured into water (50 mL).Insoluble residues were removed by filtration, and the filtrate was
basified with ammonium hydroxide (25%) to pH 8. The resulting precipitate was filtered, dried to
give crude products. The crude products was purified by chromatography on silica gel using
CH
.1.3.1. 5-(4-fluorophenyl)-3-((4-methylpiperazin-1-yl)methyl)-7-(2,3,4-trimethoxyphenyl)imidazo
1,2-a]pyridine-8-carbonitrile (3a)
White solid.Yield:95.8%.m.p.: 189.3-190.8 ºC. MS(ESI) m/z(%):516.2 [M+H] . H NM
R (400 MHz, CDCl ) δ: 7.59 (s, 1H), 7.47 (dd, J = 7.8, 5.5 Hz, 2H), 7.19–7.11 (m, 3H),
.76 (t, J = 4.2 Hz, 2H), 3.88 (d, J = 3.8 Hz, 6H), 3.81 (s, 3H), 3.22 (s, 2H), 1.95 (m,
H), 2.69 (m, 2H), 2.50 (m, 2H), 2.29 (m, 2H), 2.12 (s, 3H).Anal. calcd. for C29
(%):C, 67.56, H, 5.87, N, 13.58. Found (%):C, 67.51, H, 5.78, N, 13.52.
2 2
Cl /MeOH (20:1) to afford the desired compounds 3a-3q.
5
[
+
1
3
6
2
H30FN
5
O
3

5
.1.3.2. 3-((ethylamino)methyl)-5-(4-fluorophenyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]pyri
dine-8-carbonitrile (3b)
White solid.Yield:93.6%.m.p.: 195.2-197.0ºC. MS(ESI) m/z(%):461.1 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.59 (s, 1H), 7.47 (dd, J = 7.8, 5.5 Hz, 2H), 7.19 –7.11 (m, 3H),
.76 (t, J = 4.2 Hz, 2H), 3.88 (d, J = 3.8 Hz, 6H), 3.81 (s, 3H), 3.22 (s, 2H), 2.49 (m,
H), 1.84 (m, 1H), 1.35 (s, 1H), 1.19 (t, J = 5.1 Hz, 3H).Anal.calcd. for C26
%):C, 67.81, H, 5.47, N, 12.17. Found (%):C, 67.76, H, 5.39, N, 12.12.
+
1
(
3
6
1
4 3
H25FN O
(
5.1.3.3. 5-(4-fluorophenyl)-3-(pyrrolidin-1-ylmethyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]py
ridine-8-carbonitrile (3c)
White solid.Yield: 89.1%. m.p.: 176.5-179.0 ºC. MS(ESI) m/z(%):487.2 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.60 (s, 1H), 7.49 (dd, J = 7.8, 5.5 Hz, 2H), 7.22 –7.10 (m, 3H), 6.76 (t, J =
.2 Hz, 2H), 3.88 (d, J= 3.8 Hz, 6H), 3.82 (s, 3H), 3.23 (s, 2H), 2.00 (s, 4H), 1.56 (s, 4H).
+
1
(
3
4
Anal.calcd. forC28
N,11.46.
4 3
H27FN O (%):C, 69.12, H, 5.59, N, 11.52. Found (%): C, 69.07, H, 5.52,
5
.1.3.4. 5-(4-fluorophenyl)-3-(morpholinomethyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]pyrid
ine-8-carbonitrile (3d)
White solid.Yield:77.8%.m.p.: 170.0-171.6ºC. MS(ESI) m/z(%):503.0 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.61 (s, 1H), 7.51 (dd, J = 8.5, 5.2 Hz, 2H), 7.18 (dd, J= 17.2,
.6 Hz, 3H), 6.83 – 6.73 (m, 2H), 3.90 (d, J= 4.9 Hz, 6H), 3.85 (s, 3H), 3.43 (s, 4H), 3.
+
1
(
3
8
1
1
5
7 (s, 2H), 1.93 – 1.80 (m, 4H).Anal.calcd. forC28
5. Found (%):C, 66.84, H, 5.37, N, 11.07.
4 4
H27FN O (%):C, 66.92, H, 5.42, N, 11.
.1.3.5. 3-((dimethylamino)methyl)-5-(4-fluorophenyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]
pyridine-8-carbonitrile (3e)
White solid.Yield:91.9%.m.p.: 147.5-149.0ºC. MS(ESI) m/z(%):461.2 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.59 (s, 1H), 7.47 (dd, J = 7.8, 5.5 Hz, 2H), 7.19 –7.11 (m, 3H),
.76 (t, J= 4.2 Hz, 2H), 3.88 (d, J= 3.6 Hz, 6H), 3.81 (s, 3H), 3.22 (s, 2H), 2.29 (s, 6
H).Anal.calcd. for C26 (%):C, 67.81, H, 5.47, N, 12.17. Found (%):C, 67.74, H,
+
1
(
3
6
4 3
H25FN O
5.41, N, 12.11.
5.1.3.6. 3-((diethylamino)methyl)-5-(4-fluorophenyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]py
ridine-8-carbonitrile (3f)
White solid.Yield:83.2%. m.p.: 119.2-121.0 ºC. MS(ESI) m/z(%): 489.2 [M+H] . H N
MR (400 MHz, CDCl ) δ: 7.66 (s, 1H), 7.47 (dd, J = 7.8, 5.5 Hz, 2H), 7.21 – 7.12 (m,
H), 6.76 (d, J= 4.2 Hz, 2H), 3.88 (d, J = 3.8 Hz, 6H), 3.80 (s, 3H), 3.27 (s, 2H), 2.1
– 2.00 (m, 4H), 0.71 (t, J= 7.0 Hz, 6H).Anal.calcd. forC28 (%):C, 68.84, H, 5.9
, N, 11.47. Found (%):C, 68.78, H, 5.94, N, 11.43.
.1.3.7. 5-(4-fluorophenyl)-3-((methylamino)methyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]py
+
1
3
3
6
8
5
4 3
H29FN O
ridine-8-carbonitrile (3g)
White solid. Yield: 74.7%.m.p.: 152.4-153.9 ºC. MS(ESI) m/z(%):447.0 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.79 (s, 1H), 7.61 (dd, J = 8.3, 5.2 Hz, 2H), 7.27 – 7.18 (m, 3H), 6.84 (s,
H), 6.79 (d, J= 8.8 Hz, 1H), 3.91 (d, J= 4.9 Hz, 6H), 3.81 (s, 3H), 3.47 (s, 3H), 2.15 (s,
+
1
(
3
1
3
1
5
H).Anal.calcd. forC25
2.50.
4 3
H23FN O (%):C, 67.25, H, 5.19, N, 12.55. Found (%): C, 67.19, H, 5.14, N,
.1.3.8.3-((diethylamino)methyl)-5-(2,4-difluorophenyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]
pyridine-8-carbonitrile (3h)

+
1
White solid.Yield:77.8%.m.p.: 149.1-150.7ºC. MS(ESI) m/z(%):533.3 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.66 (s, 1H), 7.58 (dd,J= 14.3, 7.6 Hz, 1H), 7.24 (d, J= 8.7 Hz,
H), 7.02 (td, J = 8.1, 1.9 Hz, 1H), 6.95 (td, J = 9.1, 2.4 Hz, 1H), 6.85 (s, 1H), 6.79
d, J = 8.7 Hz, 1H), 3.92 (s, 3H), 3.88 (s, 6H), 3.08 (s, 2H), 2.55 (m, 4H), 1.05 (t, J =
(
3
1
(
5
6
.1 Hz, 6H).Anal.calcd. for C28
6.35, H, 5.55, N, 11.02.
28 2 4 3
H F N O (%):C, 66.39, H, 5.57, N, 11.06. Found (%):C,
5.1.3.9. 5-(2,4-difluorophenyl)-3-(pyrrolidin-1-ylmethyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-
a]pyridine-8-carbonitrile (3i)
White solid.Yield:83.9%.m.p.: 189.3-190.8 ºC. MS(ESI) m/z(%):531.1 [M+H] . H NM
R (400 MHz, CDCl ) δ: 7.66 (s, 1H), 7.48 (dd, J = 14.3, 7.6 Hz,1H), 7.24 (d, J = 8.7
Hz, 1H), 7.02 (td, J = 8.1, 1.9 Hz, 1H), 6.95 (td, J = 9.1, 2.4 Hz, 1H), 6.85 (s, 1H), 6.
+
1
3
7
9 (d, J = 8.7 Hz, 1H), 3.92 (s, 3H), 3.88 (s, 6H), 3.08 (s, 2H),3.02 (m, 2H), 2.36 (m,
H), 1.56 (s, 4H).Anal.calcd. for C28 (%):C, 66.66, H, 5.19, N, 11.10. Found
2
26 2 4 3
H F N O
(
%):C, 66.64, H, 5.11, N, 11.05.
5.1.3.10. 5-(2,4-difluorophenyl)-3-((methylamino)methyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-
a]pyridine-8-carbonitrile (3j)
White solid. Yield: 90.9%.m.p.: 132.4-134.2ºC. MS(ESI) m/z(%):491.4 [M+H] . H NM
R (400 MHz, CDCl )δ: 7.60 (s, 1H), 7.48 (d, J = 6.0 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H),
.03 – 6.97 (m, 1H), 6.95 (d, J = 8.7 Hz, 1H), 6.85 (s, 1H), 6.79 (d, J = 8.7 Hz, 1H),
.91 (d, J = 3.8 Hz, 6H), 3.82 (s, 3H), 3.13 (d, J = 4.4 Hz, 2H), 2.15 (s, 3H). Anal.cal
(%):C, 64.65, H, 4.77, N, 12.06. Found(%): C, 64.57, H, 4.68, N, 12.
+
1
3
7
3
22 2 4 3
cd.for C25H F N O
0
5
2
0.
.1.3.11. 5-(2,4-difluorophenyl)-3-((dimethylamino)methyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,
-a]pyridine-8-carbonitrile (3k)
+
1
White solid.Yield:87.4%.m.p.: 152.1-155.0 ºC. MS(ESI) m/z(%):479.3 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.63 (s, 1H), 7.48 (dd, J = 14.3, 7.6 Hz, 1H), 7.24 (d, J = 8.7 H
z, 1H), 7.02 (td, J = 8.1, 1.9 Hz, 1H), 6.95 (td, J = 9.1, 2.4 Hz, 1H), 6.85 (s, 1H), 6.79
d, J = 8.7 Hz, 1H), 3.91 (d, J = 3.8 Hz, 6H), 3.82 (s, 3H), 3.20-3.07 (m, 2H), 1.75 (s,
(
3
(
6
4
H).Anal.calcd. forC26
.99, N, 11.66.
24 2 4 3
H F N O (%):C, 65.26, H, 5.06, N, 11.71. Found (%):C, 65.23, H,
5
.1.3.12. 5-(2,4-difluorophenyl)-3-((ethylamino)methyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]
pyridine-8-carbonitrile (3l)
White solid.Yield: 97.4%.m.p.: 162.1-163.8 ºC. MS(ESI) m/z(%):505.2 [M+H] . H NM
R (400 MHz, CDCl ) δ: 7.59 (s, 1H), 7.44 (dd, J = 14.3, 7.6 Hz, 1H), 7.26 (d, J = 8.8
+
1
3
Hz, 1H), 7.04 – 6.91 (m, 2H), 6.85 (s, 1H), 6.78 (d, J = 8.8 Hz, 1H), 3.91 (d, J = 3.4
Hz, 6H), 3.82 (s, 3H), 3.16 (dd, J = 26.1, 13.2 Hz, 2H),2.69 (m, 1H), 2.19 (m, 1H), 1.4
8
(s, 1H), 1.20 (t, J = 5.0 Hz, 3H).Anal.calcd. for C26
N, 11.71. Found (%):C, 65.23, H, 4.99, N, 11.66.
.1.3.13. 5-(2,4-difluorophenyl)-3-((4-methylpiperidin-1-yl)methyl)-7-(2,3,4-trimethoxyphenyl)imi
dazo[1,2-a]pyridine-8-carbonitrile (3m)
White solid.Yield:92.1%.m.p.: 193.3-197.1 ºC. MS(ESI) m/z(%):533.1 [M+H] . H NM
R (400 MHz, CDCl ) δ: 7.59 (s, 1H), 7.44 (d, J = 5.9 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1
H), 7.03 – 6.97 (m, 1H), 6.94 (d, J=8.7 Hz, 1H), 6.85 (s, 1H), 6.78 (d, J = 8.6 Hz, 1H),
.91 (d, J = 3.4 Hz, 6H), 3.82 (s, 3H), 3.16 (dd, J = 26.8, 13.9 Hz, 2H), 2.01 (d,J=0.6
24 2 4 3
H F N O (%): C, 65.26, H, 5.06,
5
+
1
3
3

Hz, 2H), 1.59 (d, J = 9.6 Hz, 2H), 1.39 (d, J = 11.5 Hz, 2H), 1.23 (d, J = 12.8 Hz, 3
30 2 4 3
H), 0.83 (d, J = 6.3 Hz, 3H).Anal.calcd. forC30H F N O (%):C, 67.66, H, 5.68, N, 10.52.
Found (%):C, 67.61, H, 5.64, N, 10.47.
5
.1.3.14. 5-(2,4-difluorophenyl)-3-(morpholinomethyl)-7-(2,3,4-trimethoxyphenyl)imidazo[1,2-a]
pyridine-8-carbonitrile (3n)
White solid.Yield:69.9%.m.p.: 149.1-150.6 ºC. MS(ESI) m/z(%):521.0 [M-H] . H NMR
400 MHz, CDCl ) δ: 8.16 (td, J = 8.9, 6.7 Hz, 1H), 7.90 (d, J = 1.1 Hz, 1H), 7.12 (d,
J = 8.6 Hz, 2H), 7.03 (td, J = 8.2, 2.1 Hz, 1H), 6.95 (ddd, J = 11.3, 8.6, 2.4 Hz, 1H),
+
1
(
3
6
3
.81 (d, J = 8.7 Hz, 1H), 6.30 (t, J = 3.1 Hz, 1H), 6.27 (s, 1H), 3.94 (s, 6H), 3.93 (s,
H), 3.71 (s, 2H), 3.34 (s, 3H), 2.19 (s, 3H).Anal.calcd. forC28 (%):C, 64.61,
26 2 4 4
H F N O
H, 5.03, N, 10.76. Found (%):C, 64.54, H, 4.97, N, 10.67.
.1.3.15.3-((diethylamino)methyl)-5-(2,4-difluorophenyl)-7-(3,4,5-trimethoxyphenyl)imidazo[1,2-
a]pyridine-8-carbonitrile (3o)
White solid.Yield:66.0%.m.p.: 173.0-174.7 ºC. MS(ESI) m/z(%):507.0 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.66 (s, 1H), 7.58 (td, J = 8.5, 6.3 Hz, 1H), 7.07 – 6.96 (m, 2H),
.81 (d, J= 1.3 Hz, 1H), 6.75 (s, 2H), 3.92 (s, 3H), 3.88 (s, 6H), 3.10 (s, 2H),2.75 (m,
H), 2.54 (m, 2H), 1.01 (t, J = 5.1 Hz, 6H).Anal.calcd. for C28 (%):C, 66.39,
H, 5.57, N, 11.06.Found (%):C, 66.33, H, 5.55, N, 10.98.
.1.3.16.5-(2,4-difluorophenyl)-3-((dimethylamino)methyl)-7-(3,4,5-trimethoxyphenyl)imidazo[1,2
a]pyridine-8-carbonitrile (3p)
White solid.Yield:87.2%.m.p.: 143.7-145.8 ºC. MS(ESI) m/z(%):479.1 [M+H] . H NMR
400 MHz, CDCl ) δ: 7.66 (s, 1H), 7.58 (td, J = 8.5, 6.3 Hz, 1H), 7.07 – 6.96 (m, 2H),
.81 (d, J = 1.3 Hz, 1H), 6.75 (s, 2H), 3.92 (s, 3H), 3.88 (s, 6H), 3.10 (s, 2H), 1.86 (s,
H).Anal.calcd. for C26 (%):C, 65.26, H, 5.06, N, 11.71. Found (%):C, 65.23,
5
+
1
(
3
6
2
28 2 4 3
H F N O
5
-
+
1
(
3
6
6
24 2 4 3
H F N O
H, 4.99, N, 11.66.
.1.3.17.5-(2,4-difluorophenyl)-3-(pyrrolidin-1-ylmethyl)-7-(3,4,5-trimethoxyphenyl)imidazo[1,2-
a]pyridine-8-carbonitrile (3q)
White solid.Yield: 92.0%.m.p.: 189.3-190.8 ºC. MS(ESI) m/z(%):531.1 [M+H] . H NM
R (400 MHz, CDCl ) δ: 7.66 (s, 1H), 7.58 (td, J = 8.5, 6.3 Hz, 1H), 7.07 – 6.96 (m, 2
H), 6.81 (d, J = 1.3 Hz, 1H), 6.75 (s, 2H), 3.92 (s, 3H), 3.88 (s, 6H), 3.10 (s, 2H), 3.02
m, 2H), 2.33 (m, 2H), 1.70 (m, 4H).Anal.calcd. for C28 (%):C, 66.66, H, 5.19,
5
+
1
3
(
26 2 4 3
H F N O
N, 11.10. Found (%):C, 66.59, H, 5.12, N, 11.04.
5.1.4. Preparation of 1-(1H-indol-3-yl)ethan-1-one (4)
A 500mL oven-dried three-necked flask was dealed with septa and backfilled with nitrogen
gas (N
2
) three times before starting the reaction.To a stirring solution of indole (23.4 g, 0.2 mol) in
(200 mL), a suspension of anhydrous SnCl (62.4g, 0.24 mol) in dried CH Cl (150 mL)
CH Cl
2
2
4
2
2
was dropped, and the mixture was stirring at 0 ºC for 30 min. Then acetic anhydride (15.5 g, 0.2
mol) in CH Cl (100 mL) was added dropwise. Upon the completion of reaction, a brown
2
2
nitromethane (50 mL) solvent was added slowly, and the mixture was stirred at 0 ºC for 30 min.
Then the mixture was warmed to room temperature and further stirred for 2 h. After being
quenched with ice aqueous solution (30 mL), the suspension solution was filtered and the filtrate
was extracted with ethyl acetate twice (100 mL×2). The combined organic phase was washed with
2 4
water and brine, then dried overNa SO and concentrated at reduced pressure to give a dark brown
solid. The solid was triturated with ethanol (100 mL) for 1 h, and collected by filtration to

+
generate 4 as an off white solid in 79.3%yield. MS (ESI) m/z (%):160.1[M+H] .
5.1.5. General procedure for preparation of 2-amino-6-(1H-indol-3-yl)-4-arylnicotinonitrile
(5a-5b)
To a solution of substituted benzaldehyde (0.1mol) in toluene (250 mL) was added
1-(1H-indol-3-yl)ethan-2-one 4 (9.1 mL, 0.1 mol), malononitrile (6.6 g, 0.1 mol) and ammonium
acetate (61.6 g, 0.8 mol) at room temperature, then the solution was refluxed for 8 h. Upon cooling
to room temperature, the solvent was evaporated under vacuum and the residue was triturated with
ethanol (100 mL). After stirring for 10 min, the precipitant was filtered to give the corresponding
crude product. Afterward, the filter cake was transited to anhydrous ethanol (120 mL) and stirred
overnight at room temperature. Finally, the suspension liquid was filtered, off dried over MgSO
4
to afford 5a-5b.
5
5
5
.1.5.1.2-amino-4-(2,4-difluorophenyl)-6-(1H-indol-3-yl)nicotinonitrile (5a)
+
Pale yellow solid.Yield: 59.7%. MS (ESI) m/z (%):347.1[M+H] .
.1.5.2. 2-amino-4-(2-chloro-4-fluorophenyl)-6-(1H-indol-3-yl)nicotinonitrile (5b)
+
Pale yellow solid.Yield: 74.6%. MS (ESI) m/z (%):363.0 [M+H] .
.1.6. General procedure for preparation of 2-(chloromethyl)-5-(1H-indol-3-yl)-7-arylimidazo
[1,2-a]pyridine-8-carbonitrile (6a-6b)
To a stirred solution of 5a-5b (14.4 mmol) in ethanol (50 mL) was added
1,3-dichloropropan-2-one (18.2 g,144.1 mmol). Then the reaction mixture was refluxed and stirred
for 10 h. After the completion of the reaction, the solvent was concentrated in vacuum and the
residue was dissolved in diethyl ether (50 mL) and stirred for 2 h. The corresponding crude
products 6a-6b were obtained by filtration without further purification.
5.1.6.1. 2-(chloromethyl)-7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyridine-8-carb
onitrile (6a)
+
Yellow solid.Yield:77.4%. MS (ESI) m/z (%):419.1 [M+H] .
5.1.6.2. 7-(2-chloro-4-fluorophenyl)-2-(chloromethyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyridine-8
-
carbonitrile (6b)
+
Yellow solid. Yield:72.9%. MS (ESI) m/z (%):435.0 [M+H] .
5.1.7. General procedure for preparation compounds (7a-7n)
A stirring mixture of an appropriate 6a-6b (1.2 mmol) and an appropriateN-aliphatic amines
(
1.8 mmol) in ethanol(25 mL) was stirred at reflux for 5 h.After cooling to room temperature, the
solvent was evaporated in vacuum to afford yellow residue. The formed precipitate was dissolved
in DMF and purified on a silica gel column using petroleum ether/ ethyl acetate (3:1) to give pure
target product 7a-7n.
5
.1.7.1. 7-(2,4-difluorophenyl)-2-((dimethylamino)methyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyridi
ne-8-carbonitrile (7a)
Pale while solid. Yield: 66.6%.m.p.: 246.9-248.4 ºC. MS(ESI) m/z(%): 428.2 [M+H] . H
NMR (400 MHz, DMSO-d ) δ: 12.17 (s, 1H), 8.25 (d,J= 2.0 Hz, 1H), 8.00 (s, 1H), 7.84 (dd,J=
5.2, 8.6 Hz, 1H), 7.60 (d,J = 8.4 Hz, 2H), 7.57 – 7.51 (m, 1H), 7.34 (td,J= 8.5, 2.3 Hz, 1H), 7.28
+
1
6
1
(
(
19 2 5
t, J = 7.7 Hz, 1H), 7.21 – 7.16 (m, 2H), 3.64 (s, 2H), 2.23 (s, 6H).Anal.calcd. forC25H F N
%):C, 70.25, H, 4.48, N, 16.38. Found (%):C, 70.19, H, 4.39, N, 16.31.
5
.1.7.2. 7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)-2-((methylamino)methyl)imidazo[1,2-a]pyridine
8-carbonitrile (7b)
Pale while solid. Yield:69.8%.m.p.: 219.3-221.2ºC. MS(ESI) m/z(%): 414.1 [M+H] . H
-
+
1

NMR (400 MHz, DMSO-d ) δ:12.29 (s, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.19 (s, 1H), 7.
6
8
3 (dd, J = 15.2, 8.2 Hz, 1H), 7.60 – 7.53 (m, 3H), 7.33 (td, J = 8.5, 2.3 Hz, 1H), 7.2
(t, J = 7.6 Hz, 1H), 7.21 – 7.15 (m, 2H), 4.01 (s, 2H), 2.42 (s, 3H), 1.21 (s, 1H).Anal.
7
17 2 5
calcd. forC24H F N (%):C, 69.72, H, 4.14, N, 16.94. Found (%):C, 69.65, H, 4.08, N, 16.
8
5
8
9.
.1.7.3. 7-(2,4-difluorophenyl)-2-((ethylamino)methyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyridine-
-carbonitrile (7c)
+
1
Pale while solid. Yield: 79.3%. m.p.: 171.1-177.1 ºC.MS(ESI) m/z(%): 428.2 [M+H] . H
NMR (400 MHz, DMSO-d ) δ: 12.32 (s, 1H), 8.26 (s, 1H), 8.13 (s, 1H), 7.83 (dd, J= 15.2, 8.6 Hz,
H), 7.65 – 7.59 (m, 2H), 7.58 – 7.51 (m, 1H), 7.33 (td,J= 8.5, 2.2 Hz, 1H), 7.27 (t, J = 7.6 Hz,
H), 7.21 – 7.16 (m, 2H), 3.96 (s, 2H), 2.67 (q, J = 7.1 Hz, 2H), 1.21 (s, 1H), 1.06 (t, J = 7.1 Hz,
6
1
1
3
1
5
H).Anal.calcd. forC25
6.33.
19 2 5
H F N (%):C, 70.25, H, 4.48, N, 16.38. Found (%):C, 70.19, H, 4.42, N,
.1.7.4. 7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)-2-((isopropylamino)methyl)imidazo[1,2-a]pyrid
ine-8-carbonitrile (7d)
Pale while solid. Yield:79.8%.m.p.: 246.3-248.9 ºC. MS(ESI) m/z(%): 440.1 [M-H] . H
NMR (400 MHz, DMSO-d ) δ: 12.25 (d, J = 0.7 Hz, 1H), 8.26 (s, 1H), 8.20 (s, 1H), 7.85 (td, J =
.6, 6.6 Hz, 1H), 7.62 (t, J = 8.1 Hz, 2H), 7.55 (dd, J = 10.4, 2.4 Hz, 1H), 7.35 (td, J = 8.4, 2.4 Hz,
H), 7.32 – 7.27 (m, 1H), 7.20 (dd, J = 13.6, 5.7 Hz, 2H), 4.04 (s, 2H), 1.23 (s, 2H), 1.19 (d, J =
+
1
6
8
1
6
1
3
.3 Hz, 1H), 1.10 (d, J = 6.3 Hz, 5H). C NMR (101 MHz, DMSO) δ:160.97, 158.46, 148.90, 143.76,
37.97, 137.91, 137.00, 133.45, 133.35, 128.86, 125.11, 123.19, 121.37, 119.91, 115.95, 113.21,
(%):C,
1
1
7
5
12.89, 112.73, 111.30, 107.60, 105.28, 96.32, 48.09, 45.14, 23.02. Anal.calcd. forC26
H
21
F
2
N
5
0.74, H, 4.79, N, 15.86. Found (%):C, 70.69, H, 4.75, N, 15.80.
.1.7.5. 2-((cyclohexylamino)methyl)-7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyri
dine-8-carbonitrile (7e)
Pale while solid. Yield:78.4%.m.p.: 193.0-194.8 ºC. MS(ESI) m/z(%): 482.2 [M+H] .
H NMR (400 MHz, DMSO-d ) δ: 12.29 (s, 1H), 8.25 (s, 1H), 8.13 (s, 1H), 7.82 (td, J =
.7, 6.6 Hz, 1H), 7.60 (d, J = 8.6 Hz, 2H), 7.54 (td, J = 10.6, 2.5 Hz, 1H), 7.33 (td, J
+
1
6
8
=
8.4, 2.3 Hz, 1H), 7.30 – 7.24 (m, 1H), 7.22 – 7.09 (m, 2H), 3.99 (s, 2H), 2.00 – 1.7
8
(m, 2H), 1.66 (d, J = 12.5 Hz, 2H), 1.53 (d, J = 11.7 Hz, 1H), 1.29 – 1.01 (m, 6H).
Anal.calcd. for C29
N, 14.45.
25 2 5
H F N (%):C, 72.33, H, 5.23, N, 14.54. Found (%):C, 72.27, H, 5.19,
5
.1.7.6. 7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)-2-(pyrrolidin-1-ylmethyl)imidazo[1,2-a]pyridine
8-carbonitrile (7f)
Pale while solid. Yield:90.6%.m.p.: 200.5-203.2 ºC. MS(ESI) m/z(%): 454.2 [M+H] .
H NMR (400 MHz, DMSO-d ) δ: 12.19 (s, 1H), 8.25 (s, 1H), 8.03 (s, 1H), 7.82 (dd, J
15.2, 8.6 Hz, 1H), 7.60 (s, 1H), 7.58 (s, 1H), 7.57 – 7.50 (m, 1H), 7.33 (td, J = 8.5,
.2 Hz, 1H), 7.26 (t, J = 7.7 Hz, 1H), 7.20 – 7.14 (m, 2H), 3.86 (s, 2H), 2.61 (s, 4H),
-
+
1
6
=
2
1
4
5
.69 (s, 4H).Anal.calcd. forC27
4, H, 4.58, N, 15.39.
21 2 5
H F N (%):C, 71.51, H, 4.67, N, 15.44. Found (%): C, 71.
.1.7.7. 7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)-2-((4-methylpiperazin-1-yl)methyl)imidazo[1,2-
a]pyridine-8-carbonitrile (7g)
Pale while solid. Yield:91.6%.m.p.: 245.1-247.9 ºC. MS(ESI) m/z(%): 482.2 [M+H] .
H NMR (400 MHz, DMSO-d ) δ: 12.21 (s, 1H), 8.25 (d, J = 2.3 Hz, 1H), 8.00 (s, 1H),
+
1
6

7
.83 (td, J = 8.7, 6.6 Hz, 1H), 7.62 – 7.57 (m, 2H), 7.54 (dd, J = 10.0, 1.7 Hz, 1H),
7
.34 (td, J = 8.5, 2.4 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.21–7.15 (m, 2H), 3.67 (s, 2
1
3
H), 2.54–2.45 (m, 8H), 2.14 (s, 3H). C NMR (101 MHz, DMSO) δ:160.81, 158.45, 146.17,
43.77, 138.03, 137.03, 133.44, 133.31, 128.97, 125.03, 123.13, 121.27, 119.91, 115.93, 113.27,
12.94, 112.67, 112.57, 112.39, 107.52, 105.27, 96.43, 56.12, 55.03, 52.92, 45.98.Anal.calcd. f
or C28 (%):C, 69.70, H, 5.01, N, 17.42. Found (%): C, 69.65, H, 4.96, N, 17.35.
.1.7.8. 7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)-2-((4-methylpiperidin-1-yl)methyl)imidazo[1,2-
a]pyridine-8-carbonitrile (7h)
Pale while solid. Yield:87.4%.m.p.: 219.3-221.0ºC. MS(ESI) m/z(%): 482.1 [M+H] . H
NMR (400 MHz, DMSO-d ) δ: 12.16 (s, 1H), 8.23 (s, 1H), 7.97 (s, 1H), 7.81 (td, J =
.6, 6.6 Hz, 1H), 7.57 (t, J = 7.3 Hz, 2H), 7.52 (dd, J = 9.9, 1.8 Hz, 1H), 7.32 (td, J =
.4, 2.2 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.18 – 7.13 (m, 2H), 3.65 (s, 2H), 2.88 (d,
J = 11.1 Hz, 2H), 2.00 (t, J = 11.0 Hz, 2H), 1.52 (d, J = 11.6 Hz, 2H), 1.19 (s, 1H),
.13 – 1.04 (m, 2H), 0.83 (d, J = 6.4 Hz, 3H).Anal.calcd. forC29 (%):C, 72.33, H,
.23, N, 14.54. Found (%): C, 72.27, H, 5.18, N, 14.45.
.1.7.9. 2-((cyclopropylamino)methyl)-7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyr
1
1
24 2 6
H F N
5
+
1
6
8
8
1
25 2 5
H F N
5
5
idine-8-carbonitrile (7i)
Pale while solid. Yield:81.5%.m.p.: 205.0-207.6ºC. MS(ESI) m/z(%): 426.3 [M+H] . H
NMR (400 MHz, DMSO-d ) δ: 12.15 (s, 1H), 8.23 (d, J = 2.0 Hz, 1H), 7.98 (s, 1H), 7.
+
1
6
8
6
7
0
5
2 (dd, J = 15.2, 8.6 Hz, 1H), 7.60 – 7.50 (m, 3H), 7.33 (td, J = 8.5, 2.3 Hz, 1H), 7.2
(t, J = 7.7 Hz, 1H), 7.20 – 7.14 (m, 2H), 3.62 (s, 2H), 2.09 (m, 1H), 1.17 (s, 1H), 0.
9 (m, 4H). Anal.calcd. forC26
1, H, 4.32, N, 15.88.
19 2 5
H F N (%):C, 71.06, H, 4.36, N, 15.94. Found (%): C, 71.
.1.7.10.2-((diethylamino)methyl)-7-(2,4-difluorophenyl)-5-(1H-indol-3-yl)imidazo[1,2-a]pyridine
-
8-carbonitrile (7j)
+
1
Pale while solid. Yield:89.7%.m.p.: 200.3-202.0ºC. MS(ESI) m/z(%): 456.2 [M+H] . H
NMR (400 MHz, DMSO-d6) δ: 12.43 (s, 1H), 8.34 (s, 1H), 8.29 (d, J = 1.8 Hz, 1H),
.83 (td, J = 8.6, 6.6 Hz, 1H), 7.62 (dd, J = 7.9, 4.8 Hz, 2H), 7.53 (td, J = 10.7, 2.5 H
7
z, 1H), 7.33 (td, J = 8.4, 2.2 Hz, 1H), 7.26 (dd, J = 13.4, 4.9 Hz, 2H), 7.16 (t, J = 7.5
23 2 5
Hz, 1H), 4.17 (s, 2H), 2.88 (s, 4H), 1.18 (t, J = 6.9 Hz, 5H).Anal.calcd. for C27H F N
(
%):C, 71.19, H, 5.09, N, 15.37. Found (%): C, 71.16, H, 4.98, N, 15.33.
5.1.7.11. 7-(2-chloro-4-fluorophenyl)-5-(1H-indol-3-yl)-2-(morpholinomethyl)imidazo[1,2-a]pyri
dine-8-carbonitrile (7k)
Pale while solid. Yield: 91.2%. m.p.: 225.1-227.0ºC.MS(ESI) m/z(%): 486.2 [M+H] .
H NMR (400 MHz, DMSO-d ) δ:12.21 (s, 1H), 8.25 (d, J = 2.3 Hz, 1H), 8.00 (s, 1H),
+
1
6
7
.83 (td, J = 8.7, 6.6 Hz, 1H), 7.62 – 7.57 (m, 2H), 7.54 (dd, J = 10.0, 1.7 Hz, 1H), 7.
4 (td, J = 8.5, 2.4 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.21 – 7.15 (m, 2H), 3.66 (s, 2
3
H), 3.54 (s, 6H),1.21 (s, 2H).Anal.calcd. for C27
Found (%): C, 69.02, H, 4.46, N, 14.87.
21 2 5
H F N O (%):C, 69.07, H, 4.51, N, 14.92.
5
.1.7.12. 7-(2-chloro-4-fluorophenyl)-5-(1H-indol-3-yl)-2-((4-methylpiperazin-1-yl)methyl)imidaz
o[1,2-a]pyridine-8-carbonitrile (7l)
Pale while solid. Yield: 63.5%.m.p.: 185.7-187.9ºC. MS(ESI) m/z(%): 499.2 [M+H] .
H NMR (400 MHz, DMSO-d ) δ: 12.17 (s, 1H), 8.26 (d, J = 2.4 Hz, 1H), 8.04 (s, 1H),
.77 (dd, J = 8.7, 6.2 Hz, 1H), 7.73 (dd, J = 8.9, 2.6 Hz, 1H), 7.59 (d, J = 8.3 Hz, 2
+
1
6
7

H), 7.46 (td, J = 8.5, 2.6 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H),
.13 (s, 1H), 3.67 (s, 2H), 2.64 – 2.42 (m, 8H), 2.15 (s, 3H).Anal.calcd. forC28
%): C, 67.40, H, 4.85, N, 16.84. Found (%):C, 67.36, H, 4.79, N, 16.77.
7
H24ClFN
6
(
5.1.7.13.7-(2-chloro-4-fluorophenyl)-2-((cyclohexylamino)methyl)-5-(1H-indol-3-yl)imidazo[1,2-a]
pyridine-8-carbonitrile (7m)
Pale while solid. Yield: 79.3%.m.p.: 189.3-190.8 ºC. MS(ESI) m/z(%):498.0 [M+H] .
H NMR (400 MHz, DMSO-d ) δ: 12.21 (s, 1H), 8.24 (d, J = 2.4 Hz, 1H), 8.18 (s, 1H),
.78 – 7.70 (m, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.45 (td, J = 8.5, 2.6 Hz, 1H), 7.27 (t,
J = 7.5 Hz, 1H), 7.20 – 7.13 (m, 2H), 3.99 (s, 2H), 2.43 (m, 1H), 1.79 (m, 5H), 1.36
m, 5H), 1.17 (s, 1H).Anal.calcd. for C29 (%):C, 69.94, H, 5.06, N, 14.06. Found
%): C, 69.88, H, 5.00, N, 13.97.
.1.7.14. 7-(2-chloro-4-fluorophenyl)-5-(1H-indol-3-yl)-2-((methylamino)methyl)imidazo[1,2-a]p
yridine-8-carbonitrile (7n)
Pale while solid. Yield: 83.3%.m.p.: 219.5-223.5 ºC. MS(ESI) m/z(%): 430.1 [M+H] .
H NMR (400 MHz, DMSO-d ) δ: 12.23 (s, 1H), 8.26 (s, 1H), 8.20 (s, 1H), 7.78 (dd, J
+
1
6
7
(
H25ClFN
5
(
5
+
1
6
=
8.6, 6.3 Hz, 1H), 7.75 – 7.71 (m, 1H), 7.65 – 7.57 (m, 2H), 7.47 (td, J = 8.5, 2.6 Hz,
H), 7.29 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.17 (d, J = 4.1 Hz, 1H), 4.0
1
1
(s, 2H), 2.43 (s, 3H).Anal.calcd. forC24
%): C, 66.97, H, 3.93, N, 16.25.
5
H17ClFN (%):C, 67.06, H, 3.99, N, 16.29. Found
(
5
.2. Biological evaluation
.2.1. MTT assay
5
The cytotoxic activity of compounds (3a-3q and7a-7n) was evaluated against HT-29,H460,
A549, MKN-45 and SMMC-7721 cells by the MTT assay in vitro, with Colchicine and CA-4 as
references. The cancer cells were cultured in minimum essential medium (MEM) supplemented
3
with 10% fetal bovine serum (FBS). Approximately 4×10 cells, suspended in MEM medium,
2
were plated onto each well of a 96-well plate and incubated in 5% CO at 37°C for24 h. The tested
compounds were added to the culture medium at the indicated final concentrations and the cell
cultures were continued for 72 h. Fresh MTT was added to each well at a final concentration of
5.0µg/mL and incubated with cells at 37 °C for 4 h. The formazan crystals were dissolved in 100.0
µLDMSO per each well, and the absorbency at 492 nm (for the absorbance of MTT formazan)
and 630 nm (for the reference wavelength) was measured with the ELISA reader. All of the
compounds were tested three times in each of the cell lines. The results expressed as IC50
(
inhibitory concentration of 50%) were the mean ± SD and were calculated by using the Bacus
Laboratories Incorporated Slide Scanner (Bliss) software.
5.2.2. Immunofluorescence staining
Immunostaining was carried out to detect tubulin protein after exposure to CA-4 and 7e. The
4
HT-29 cell was seeded at 1 × 10 per well on a 24 well plate and grown for 24 h. Cells were treated
with CA-4 or 7e for 24 h. Cells in the control group were treated with culture medium. The control
and treated cells were fixed with 4% formaldehyde in PBS for 30 min at -20ºC, then washed three
times with PBS and permeabilized with 0.2% (v/v) Triton X-100 in PBS for 20 min. Then, the
cells were blocked with 5% bovine serum albumin (BSA) in PBS for 30 min. The primary
a-tubulin antibody was diluted (1:100) with 2% BSA in PBS and incubated overnight at 4 ºC. The

cells were washed with PBS to remove unbound primary antibody and then cells were incubated
with FITC-conjugated antimousesecondary antibody, diluted (1:100) with 2% BSA in PBS, for 2 h
at 37 ºC. The cells were washed with PBS to remove unboundsecondary antibody, nucleus was
stained with 4,6-diamino-2-phenolindoldihydrochloride (DAPI) and then, immunofluorescence
was detected using a fluorescence microscope (Olympus, Tokyo, Japan).
5.2.3. Competitive tubulin-binding assay
Tubulin was coin cubated with various concentrations of Taxol, 7e or CA-4 respectively at 37ºC
for 1 h. Then colchicine was added to a final concentration of 5 µM. Fluorescence was determined
using a Hitachi F-2500 spectrofluorometer (Tokyo, Japan) at excitation wavelengths of 365 nm
and emission wavelengths of 435 nm. Blank values (buffer alone) as background were subtracted
from all samples. Then the inhibition rate (IR) was calculated as follows: IR=(F÷F0)×100 %
where F0 is the fluorescence of the 5 µM colchicine-tubulin complex, and F is the fluorescence of
a given concentration of Taxol, 7e or CA-4 (0 µM, 1.6 µM, 5 µM and 15 µM) competition with the
5
µM colchicine-tubulin complex. Taxol, not binding in the colchicine-site of tubulin, was added
as a negative control.
5.2.4. Molecular Docking
The molecular modeling studies were performed with Accelrys Discovery Studio 3.5. The
crystal structure of tubulin complexed with DAMA-colchicine (PDB code: 4O2B) was retrieved
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/). In the docking process, the protein
protocol was prepared by several operations, including standardization of atom names and
insertion of missing atoms in residues. Then, the receptor model was typed with the CHARMm
force field and a binding sphere with radius of 15.0 Å was defined through the original ligand
(
DAMA-colchicine) as the binding site. The Crolibulin and 7e were drawn with Chemdraw and
fully minimized using the CHARMm force field. Finally, they were docked into the binding site
using the CDOCKER protocol with the default settings.
Acknowledgments
The work was supported by National Natural Science Foundation of China (No. 81273357),
Doctoral Research Foundation of Shenyang Medical College (No. 2014-3057) and Development
Project of Ministry of Education Innovation Team (No. IRT1073).
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Graphical Abstract

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Two series of novel IMP derivatives (3a-3q & 7a-7n) were designed and synthesized.
7e showed better cytotoxicity against tested cells than CA-4 and Crolibulin.
Immunofluorescence assay ascertain the mitotic machinery interfering potency of 7e.
Colchicine competitive binding assay showed that 7e binds at colchicine site.
Docking mode revealed 7e could overlap well with Crolibulinin the colchcine site.