Design, synthesis, and structure activity relationship (SAR) studies of novel imidazo[1,2-a] pyridine derivatives as Nek2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115775

Never in mitosis (NIMA) related kinase 2 (Nek2) is involved in multiple cellular processes such as cell cycle checkpoint regulation, cell division, DNA damage response and cell apoptosis. Nek2 has been reported to be overexpressed in various tumors and correlated with poor prognosis. Herein, a series of imidazo[1,2-a] pyridines Nek2 inhibitors were designed, synthesized, and their biological activities were investigated. Besides, structure activity relationship analysis of these compounds were performed in the MGC-803 cell. The screening results are promising, and compound 28e shows good proliferation inhibitory activity with an IC₅₀ of 38 nM. The results would be helpful to design and develop more effective Nek2 inhibitors for the treatment of gastric cancer.

Full text of DOI:10.1016/j.bmc.2020.115775

Bioorg. Med. Chem. 28 (2020) 115775
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and structure activity relationship (SAR) studies of novel
imidazo[1,2-a] pyridine derivatives as Nek2 inhibitors
,
,
Haili Wanga 1, Yunzhong Chena 1, Xiaofan Gua, Jianbei Xia, Ziwei Rena, Shuting Wanga,
Yanhong Duanb, Hongyu Lic, Tong Zhua, Yijie Dud *, Xiongwen Zhanga *, Mingliang Maa
,
e,
,
,b,*
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, College of Chemistry and Molecular Engineering, East China Normal
University, 3663 North Zhongshan Road, Shanghai 200062, China
b Key Laboratory of Brain Functional Genomics-Ministry of Education, School of Life Science, East China Normal University, 3663 North Zhongshan Road, Shanghai,
China
c Department of Pharmaceutical Science, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
d Department of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China
e Institutes of Integrative Medicine, Fudan University, Shanghai 200040, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Never in mitosis (NIMA) related kinase 2 (Nek2) is involved in multiple cellular processes such as cell cycle
checkpoint regulation, cell division, DNA damage response and cell apoptosis. Nek2 has been reported to be
overexpressed in various tumors and correlated with poor prognosis. Herein, a series of imidazo[1,2-a] pyridines
Nek2 inhibitors were designed, synthesized, and their biological activities were investigated. Besides, structure
activity relationship analysis of these compounds were performed in the MGC-803 cell. The screening results are
promising, and compound 28e shows good proliferation inhibitory activity with an IC50 of 38 nM. The results
would be helpful to design and develop more effective Nek2 inhibitors for the treatment of gastric cancer.
Gastric cancer
Nek2
Imidazo[1,2-a]pyridine
Antitumor activities
1. Introduction
target.19,21,22
To extensively explore the biological role of Nek2 and validate Nek2
Never in mitosis (NIMA) related kinase 2 (Nek2) is a key regulator for
cell cycle progression.1,2 Nek2 is enriched in the centrosome and triggers
centrosome separation by phosphorylating β-catenin, C-Nap1, rootletin,
and centrosomal proteins Nlp (ninein-like protein) during the G2/M
phase of the cell cycle.3–7 Furthermore, Nek2 plays a crucial role in
modulating chromosome alignment and the spindle assembly check-
point (SAC) signaling through the direct interaction with Mad1/28 and
the phosphorylation of Hec1 and Sgo1.9 Overexpression of Nek2 has
been reported induced chromosome instability (CIN),10 cell prolifera-
tion,11,12 drug resistance13,14 and poor prognosis.15–17 Overcoming drug
resistance and inhibiting cancer cell growth in vitro and in vivo have
been reported via targeting Nek2.18–20 Thus, Nek2 represents a novel
as a fruitful and novel anti-cancer target, Nek2 inhibitors have attracted
great attentions recently. Benzimidazole-based derivatives and
pyrazine-based derivatives were developed and showed mild potent
activities against Nek2 kinase with IC50 of micromole range (Fig. 1).23,24
Subsequently, based on the exploration of the hybrid of the benzimid-
azole core and the pyrazine core scaffold, a series of aminopyridine-
based compounds have been discovered, and they showed highly effi-
cient and reversible Nek2 inhibitory activity (Fig. 1).25 We previously
described a series of novel imidazo[1,2-a]pyridine Nek2 inhibitors,
exemplified by MBM-55 (Fig. 1), which obtained an IC50 of 1 nM,
showing high selectivity for Nek2 kinase and proliferation inhibition on
MGC-803 cells.26 Despite their sufficient potency on Nek2 kinase,
structural optimization based on imidazo[1,2-a]pyridine Nek2
biomarker of cancer cells and could be
a valuable anticancer
* Corresponding authors at: Department of Integrative Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China (Y. Du); Shanghai Engineering
Research Center of Molecular Therapeutics and New Drug Development, College of Chemistry and Molecular Engineering, East China Normal University, 3663 North
Zhongshan Road, Shanghai 200062, China (X. Zhang); Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, College of
Chemistry and Molecular Engineering, Key Laboratory of Brain Functional Genomics-Ministry of Education, School of Life Science, East China Normal University,
3663 North Zhongshan Road, Shanghai 200062, China (M. Ma).
E-mail addresses: duyijie@huashan.org.cn (Y. Du), xwzhang@sat.ecnu.edu.cn (X. Zhang), mlma@brain.ecnu.edu.cn (M. Ma).
1
These authors contribute to the paper equally.
https://doi.org/10.1016/j.bmc.2020.115775
Received 4 July 2020; Received in revised form 14 September 2020; Accepted 15 September 2020
Available online 21 September 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
into the 3 position of the imidazopyridine skeleton and extend the
structure ꢀ activity relationship (SAR) study. Herein, we report a novel
series of 3-(thiophen-2-yl)imidazo[1,2-a]pyridine-based compounds
and investigate their potential antitumor properties on MGC-803 cells.
2. Results and discussion
2.1. Synthesis
Compound 2 was synthesized from 2-amino-4-bromopyridine 1 and
2-chloroacetaldehyde by a cyclization reaction in high yield.26 Com-
pound 3 was synthesized by a Suzuki coupling reaction of compound 2
with 1-methyl-4-pyrazole boronic acid pinacol ester. And following NIS
iodination reaction, the left iodide fragment 4 could be obtained.
Cyclization of methyl thioglycolate 5 with ethyl propiolate provided
methyl 3-hydroxythiophene-2-carboxylate 6,27 which was treated with
various substituted 1-phenylethanol 7a-e upon Mitsunobu conditions to
achieve 8a-e, respectively. Boronic esters 9a-e were formed via Miyaura
Borylation reactions. 8a-e subsequently were coupled with 4 through
Suzuki reaction to generate relative ester 10a-e. Finally, esters 10a-e
were heated in methanol with ammonia to afford target primary amide
11a-e (Scheme 1).
Fig. 1. Structures of Nek2 inhibitors.
inhibitors is still necessary in order to achieve favorable cellular activity.
As part of our continued research, the initial design was based on our
published Nek2 inhibitor MBM-55, which feature the core of imidazo
[1,2-a]pyridine. In this work, we intend to introduce the thiazole ring
Pyrazole borate compound 12 were treated with 2-chloroethyldime-
thylamine to form compound 13 through nucleophilic reaction. By
Scheme 1. Synthesis of Compound 10a-e and 11a-ea. aReagents and conditions: (a) 2-chloroacetaldehyde, EtOH/H2O, NaHCO3, 80 ◦C; (b) 1-Methyl-4-pyrazole
boronic acid pinacol ester, Pd(dppf)Cl2⋅DCM, K2CO3 (1 M), DMF, 80 ◦C; (c) NIS, DCM, r.t; (d) ethyl propiolate, MeONa, MeOH, 40 ◦C; (e) PPh3, DIAD, DCM; (f)
LDA, B(OMe)3, THF, ꢀ 78 ◦C; (g) Pd(dppf)Cl2⋅DCM, Na2CO3, 70 ◦C; (h) NH4OH, NH3/MeOH, 78 ◦C.
2

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
Suzuki coupling reaction, compound 13 was treated with bromide 2 to
form coupling product 14 in high yield, which was treated with NIS
iodination to form iodide 15 as the left segment. Compounds 16a-e were
synthesized by Suzuki coupling reaction of iodide 15 with boric acid
compounds 9a-e, and then following ammonolysis reaction, target
compounds 17a-e were obtained (Scheme 2).
high potency in the inhibition of MGC-803 cell proliferation. A series of
novel imidazo[1,2-a] pyridine derivatives was designed and synthe-
sized. Their structure–activity relationship studies were focused on the
different substituents connecting to the 3 position of the thiophene ring
through an ether bond (Table 1), and their anti-cell proliferation ac-
tivities were carried out in MGC-803 cells. It has been found that o-
CF3Ph-substituted 17a and 11a had submicromolar cellular potency
against MGC-803 cells, slightly more potent than m-CF3Ph-substituted
Pyrazole borate 12 was treated with various substituted hydrochlo-
ride salts of chloroalkylamines to form corresponding borate 19a-
d through nucleophilic substitution reaction. Then, through Suzuki
cross-coupling reaction, pyrazole borate 19a-d were treated with bro-
mide 7 to form the coupling product 20a-d in high yield. And then, NIS
iodination was used to form the iodide 21a-d as the left segment.
Various substituted hydroxyl compounds 23a-c was treated with
methanesulfonyl chloride to form corresponding methanesulfonyl
compounds 24a-c through affinity substitution reaction. Bromide 2 and
1-Boc-pyrazole-4-boronic acid pinacol ester were coupled by Suzuki
reaction to form the key intermediate 22. It was unexpectedly found that
in the reaction process, with the leaving of Boc group, one reaction step
was omitted and the synthesis efficiency was improved. Then, NaH was
used as base and compound 25a-c were formed by nucleophilic substi-
tution reaction. The iodide 26a-c as the left segment of the target
compound were generated by NIS iodination reaction. Compounds 27a-
g were produced by a Suzuki coupling reaction using the previously
synthesized iodides 21a-d and 26a-c with the boric acid compound 9a.
Finally, the target compounds 28a-g were obtained by aminolysis re-
action (Scheme 3).
17b (MGC-803, IC50 = 6.81
μ
M), o-FPh-substituted 17c (MGC-803, IC50
M), or
M). Then we explored
= 2.91 M), m-FPh-substituted 11d (MGC-803, IC50 = 16.41
μ
μ
Phenyl-substituted 11e (MGC-803, IC50 = 2.98
μ
the effect of the 2-position substitution of the thiophene ring, and found
that the carboxamide substitution effect is significantly better than the
ester substitution. 11a has obvious MGC-803 inhibitory activity (MGC-
803, IC50 = 0.57 μM), which is better than 10a (MGC-803, IC50 = 3.92
μ
M). The cellular inhibitory activity was significantly improved by 6.8
times. In addition, we found that the change of the pyrazole ring sub-
stituent will greatly affect its inhibitory activity, such as methyl
substituted 11d (MGC-803, IC50 = 16.41 μM) is significantly inferior to
2-(N,N-dimethylamino)ethyl derivative 17d (MGC-803, IC50 = 6.02
M). Therefore, in order to obtain a more effective Nek2 inhibitor, we
μ
explored the connection of several different substituents to the 1-posi-
tion of the pyrazole ring to obtain compounds 27a-28 g (Table 2).
When pyrrolidine, piperidine, morpholine and piperazine rings were
used as solubilizing groups, the introduction of N-containing groups
usually showed good tumor cell inhibitory activity. Similarly, 27a-g
thiophene ring of 2-position ester group is replaced by an amide bond to
obtain compounds 28a-g. The cell efficacy of the carboxamide-
substituted compound is still superior to the ester-substituted com-
pound. Consistent with earlier findings, the addition of amide bonds
plays an important role in improving activity. Surprisingly, replacing the
ester group of 27c-e with an amide bond for 28c-e, the cell activity is
improved by 100–500 times. Gratifyingly, 28c (MGC-803, IC50 = 44
nM), 28d (MGC-803, IC50 = 54 nM), 28e (MGC-803, IC50 = 38 nM)
showed nanomolar cell inhibitory activity, which was more potent than
MBM-55. The cell research results also showed that when a nitrogen-
containing heterocycle containing a Boc group as a solubilizing group,
azetidine is more active than pyrrolidine and piperidine. In particular,
the tumor inhibitory activity of 28e reached 38 nM, which was 17 times
2.2. Results
A series of imidazo[1,2-a] pyridine compounds was designed and
synthesized, and their antitumor activities against gastric cancer cell
lines including MGC-803 cell were investigated (Tables 1–3). Compound
MDM-55 synthesized in our previous work was used as a control in
different cell lines.
2.3. Discussion
Herein we report the sophisticated process of structural optimization
toward a previously disclosed Nek2 inhibitor, MBM-55, which showed
Scheme 2. Synthesis of Compound 16a-e and 17a-ea. aReagents and conditions: (a) 2-chloroethyldimethylamine, Cs2CO3, CH3CN, 78 ◦C; (b) 2, Pd(dppf)Cl2⋅DCM,
K2CO3 (1 M), DMF, 78 ◦C; (c) NIS, DCM, r.t.; (d) 15, Pd(dppf)Cl2⋅DCM, Na2CO3, 70 ◦C; (e) NH4OH, NH3/MeOH, 78 ◦C.
3

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
Scheme 3. Synthesis of Target Derivatives 27a-g and 28a-ga. aReagents and conditions: (a) 18a-d, Cs2CO3, CH3CN, 78 ◦C; (b) Pd(dppf)Cl2⋅DCM, K2CO3 (1 M), DMF,
78 ◦C; (c) NIS, DCM, r.t.; (d) MsCl, TEA, DMAP, DCM, 0 ◦C; (e) 1-Boc-pyrazole-4-boronic acid pinacol ester, Pd(dppf)Cl2⋅DCM, K2CO3 (1 M), DMF, 78 ◦C; (f) NaH,
DMF, 80 ◦C; (g) NIS, DCM, r.t.; (h)21a-d, 26a-c, Pd(dppf)Cl2⋅DCM, Na2CO3, 70 ◦C; (i) NH4OH, NH3/MeOH, 78 ◦C.
more potent than that of MBM-55 (IC50 = 0.65
μ
M).
Most of the synthesized compounds showed moderate anti-tumor ac-
tivities to MGC-803 cells. Among them, 28e is a molecule which has the
best inhibitory activity against MGC-803 cells (IC50 = 38 nm). In addi-
tion, 28c-e exhibited excellent inhibitory activities against MGC-803
On the basis of their excellent in vitro efficacy in gastric cancer cell
line, 28c, 28d, 28e were selected for further anti-cell proliferation
analysis in other tumor cells. As shown in Table 3, these compounds
effectively inhibited cell proliferation in both hepatoma cell (Hep-3B,
BEL-7402) and colon cancer cell (HCT-116) (MBM-55 as the positive
control). Finally, we found that compounds 28c, 28d, 28e and 28f had
similar inhibitory activity as MBM-55 in these cell lines. Wherein,
compound 28e showed the best potential for inhibition of MGC-803
cells, supporting its further research as a lead compound for Nek2.
Docking analysis were carried out to examine the binding affinity
and binding modes of MBM-55 and 28e to Nek2 (Fig. 2). The docking
results show that 28e forms three conservative hydrogen bonds with
Nek2 kinase, similarity to MBM-55. Two of them were formed between
the nitrogen atom on the amide bond and ASP159, and the third one was
cells with less than 0.1 μM IC50. Therefore, our research indicates that
compound 28e is a potential and prospects candidate, which has high
research value.
4. Experimental section
4.1. General material and methods
Starting reagents, materials, and solvents were reagent grade and
used as purchased. Chromatography solvents were HPLC grade and were
used directly without further purification. Reaction mixtures were
monitored via thin-layer chromatography (TLC) on silica gel F-254 TLC
plates. Flash column chromatography was carried out using silica gel
(300–400 mesh). Chemical shifts (δ) are reported in parts per million
(ppm) and coupling constants (J) are reported in Hertz (Hz). NMR
spectra were recorded on a Brucker Ascend-400 MHz spectrometer with
CDCl3, CD3OD, and DMSO‑d6 as solvent. Data for 1H and 13C spectra are
recorded as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d
= doublet, t = triplet, m = multiplet, q = quartet, br = broad). HRMS
(ESI) Mass spectra were recorded on Bruker micro TOF-Q 10,198 mass
spectrometer. Compound purity is determined by high performance
liquid chromatography (HPLC), and all final test compounds display
purity higher than 95%.
formed with LYS37. Besides, the
π-π interaction between 28e and
PHE148 is retained. Particularly, one halogen bond was detected be-
tween the CF3 group of 28e and residue TYR19. The introduction of tert-
butyl 3-isobutylazetidine-1-carboxylate group makes the hydrogen bond
between 28e and TYP88 weaker, while the larger shape of 28e makes it
form a stronger VDW interaction with the protein (Table S1). The
binding affinity of 28e (11.210 kcal/mol) was stronger to that of com-
pound MBM-55 (10.693 kcal/mol). Therefore, the proposed binding
mode supports the inhibitory activity of 28e on Nek2 kinase.
3. Conclusions
In summary, a series of pyridazo [1,2-a] imidazole compounds were
designed and synthesized and their cell viabilities were investigated.
4

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
Table 1
Table 1 (continued)
Comp.
Structures of synthesized compounds, and their inhibitory activities in MGC-803
R1
R2
R3
MGC-803 cell
IC50 M)
(μ
cells.
16e
17a
1.29
0.17
Comp.
R1
R2
R3
MGC-803 cell
IC50 M)
(
μ
17b
17c
6.81
2.91
MBM-
55
–
–
–
0.65
10a
3.92
10b
10c
17.27
11.13
17d
17e
6.02
1.95
10d
10e
>30
4.2. Chemistry.
4.2.1. 7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (3)
9.18
To a solution of 2 (3.10 g, 15.7 mmol) in mixed solution of DMF (55
mL) and H2O (5 mL), 1-methyl-4-pyrazole boronic acid pinacol ester
(4.00 g, 1.2 eq.), K2CO3(7.70 g, 55.80 mmol) and Pd (diphenylphos-
phino) ferrocene] dichloropalladium (II)⋅DCM(1.00 g, 1.22 mmol) were
added, then the reaction mixture was stirred at 80 ◦C overnight under N2
atmosphere. The reaction mixture was filtered over a pad of Celite and
concentrated under vacuum. The residue was purified by flash chro-
matography, eluting with a gradient of 2%-6% MeOH in DCM, to give 3
(2.50 g, 78%) as a brownish yellow solid. 1H NMR (400 MHz, Chloro-
form-d) δ 7.97 (dd, J = 7.1, 4.5 Hz, 1H), 7.70 (d, J = 3.8 Hz, 1H), 7.57 (d,
J = 4.6 Hz, 1H), 7.56 (s, 1H), 7.50 (d, J = 3.0 Hz, 1H), 7.42 (d, J = 3.8
Hz, 1H), 6.79 (td, J = 5.6, 4.4, 1.8 Hz, 1H), 3.85 (s, 3H).13C NMR (100
MHz, Chloroform-d) δ 144.86, 135.74, 132.85, 128.27, 126.33, 124.72,
120.17, 111.02, 110.75, 110.38, 38.18. MS (ESI) m/z calcd. for
11a
0.57
11b
11c
3.48
3.52
11d
11e
16.41
2.98
C
11H10N4 (M+H)+ 199.09.
4.2.2. 3-iodo-7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (4)
To a solution of 3 (1.93 g, 3.68 mmol) in acetonitrile (30 mL), NIS
(2.32 g, 10.31 mmol) was added, then the reaction mixture was stirred
at room temperature for 2 h. Concentrated the organic solvent, the
residue was purified by flash chromatography, eluting with a gradient of
1%-5% MeOH in DCM, to give 4(2.50 g, 78%)as a brownish yellow solid.
1H NMR (400 MHz, Chloroform-d) δ 8.09 (d, J = 7.1 Hz, 1H), 7.85 (s,
1H), 7.73 (s, 1H), 7.68 (s, 1H), 7.66 (s, 1H), 7.06(dd, J = 7.1, 1.6 Hz)
3.99 (s, 3H).13C NMR (100 MHz, Chloroform-d) δ 148.18, 140.68,
136.97, 129.96, 127.54, 125.95, 120.89, 112.29, 111.85, 59.97, 39.32.
MS (ESI) m/z calcd. for C11H9IN4 (M+H)+ 324.99.
16a
0.32
16b
16c
4.71
2.47
4.2.3. Methyl 3-(1-(2-(trifluoromethyl) phenyl) ethoxy) thiophene-2-
carboxylate (8a)
16d
1.57
To a solution of 6 (2.10 g, 13.29 mmol) in DCM (50 mL), commercial
compound 7a (2.90 g, 15.26 mmol) and triphenylphosphine (4.66 g,
17.79 mmol) was added. After stirring at 0 ◦C for 20 min, diisopropyl
5

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
Table 2
Table 3
The IC50 values of selected compounds against other cancer cells
Structures of 27a-g and 28a-g, and their inhibitory activities in MGC-803
IC50
Cell
(
μ
M)
MBM-55
28c
28d
28e
1.25
Hep-3B
1.07
6.37
0.70
1.25
4.33
0.36
1.18
5.99
0.46
Hepatoma cells
BEL-7402
10.44
0.48
Hepatoma cells
HCT-116
cells.
Colon cancer cells
(d, J = 7.9 Hz, 1H), 7.55 (t, J = 7.3 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.23
(dd, J = 5.4, 2.0 Hz, 1H), 6.68 (dd, J = 5.6, 1.6 Hz, 1H), 5.78 (q, J = 6.3
Hz, 1H), 3.87 (s, 3H), 1.71 (d, J = 6.3 Hz, 3H). 13C NMR (101 MHz,
Chloroform-d) δ 161.00, 158.75, 142.16–140.00 (m), 131.81, 129.40,
126.54 (d, J = 35.8 Hz), 125.66 (d, J = 30.4 Hz), 125.06 (d, J = 30.4
Hz), 124.49 (q, J = 5.8 Hz), 123.36 (d, J = 273.8 Hz), 116.99, 110.13,
74.45, 50.51, 23.62. MS (ESI) m/z calcd. for C15H13F3O3S (M+H)+
331.05.
Comp.
R1
R2
MGC-803 cell IC50
2.05
(μM)
27a
27b
27c
27d
7.36
5.90
3.76
4.2.4. Methyl 3-(1-(3-(trifluoromethyl) phenyl) ethoxy) thiophene-2-
carboxylate (8b)
Following the procedure described for preparing 8a (Yields: 92%).1H
NMR (400 MHz, Chloroform-d) δ 7.74 (s, 1H), 7.64 (d, J = 7.7 Hz, 1H),
7.52 (d, J = 7.9 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 5.6 Hz,
1H), 6.69 (d, J = 5.5 Hz, 1H), 5.44 (q, J = 6.4 Hz, 1H), 3.86 (s, 3H), 1.68
(d, J = 6.5 Hz, 3H).13C NMR (100 MHz, Chloroform-d) δ 162.01, 159.62,
143.61, 130.87 (d, J = 32.3 Hz), 130.44, 129.18, 129.06 (d, J = 1.5 Hz),
124.55 (q, J = 3.8 Hz), 124.07(d, J = 272.3 Hz), 122.49 (q, J = 3.8 Hz),
118.53, 111.97, 79.37, 51.48, 23.90. MS (ESI) m/z calcd. for
27e
27f
19.33
20.27
C
15H13F3O3S (M+H)+ 331.05.
4.2.5. Methyl 3-(1-(2-fluorophenyl) ethoxy) thiophene-2-carboxylate (8c)
Following the procedure described for preparing 8a (Yields: 65%)1H
NMR (400 MHz, Chloroform-d) δ 7.61 (td, 1H), 7.26 (d, J = 5.4 Hz, 1H),
7.21 (d, J = 5.5 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 9.3 Hz, 1H),
6.70 (d, J = 5.6 Hz, 1H), 5.71 (q, J = 6.4 Hz, 1H), 3.86 (s, 3H), 1.70 (d, J
= 6.4 Hz, 3H).13C NMR (100 MHz, Chloroform-d) δ 162.06, 159.98,
159.27(d, J = 245.0 Hz), 130.38, 129.49 (d, J = 13.4 Hz), 129.24(d, J =
8.2 Hz), 127.21 (d, J = 3.9 Hz), 124.77 (d, J = 3.4 Hz), 117.91, 115.14
(d, J = 21.5 Hz), 110.89, 73.15 (d, J = 3.0 Hz), 51.54, 22.99. MS (ESI)
m/z calcd. for C14H13FO3S (M+H)+ 281.07.
27g
28a
28b
28c
28d
20.44
8.40
8.68
0.044
0.054
4.2.6. Methyl 3-(1-(3-fluorophenyl) ethoxy) thiophene-2-carboxylate (8d)
Following the procedure described for preparing 8a (Yields: 81%).1H
NMR (400 MHz, Chloroform-d) δ 7.33–7.27 (m, 1H), 7.26 (d, J = 5.6 Hz,
1H), 7.20–7.13 (m, 2H), 6.94 (tdd, J = 8.7, 2.6, 1.2 Hz, 1H), 6.65 (d, J =
5.5 Hz, 1H), 5.35 (q, J = 6.4 Hz, 1H), 3.86 (s, 3H), 1.67 (d, J = 6.5 Hz,
3H).13C NMR (100 MHz, Chloroform-d) δ 163.00 (d, J = 246.4 Hz),
162.04 , 159.94 , 145.21 (d, J = 6.9 Hz), 130.30 , 130.22 , 121.23 (d, J =
2.9 Hz), 118.49 , 114.66 (d, J = 21.2 Hz), 112.64 (d, J = 22.2 Hz),
111.38 , 79.31 (d, J = 1.8 Hz), 51.57 , 24.12 . MS (ESI) m/z calcd. for
28e
28f
0.038
0.55
C
14H13FO3S (M+H)+ 281.07.
4.2.7. Methyl 3-(1-phenylethoxy) thiophene-2-carboxylate (8e)
Following the procedure described for 8a (Yields: 71%).1H NMR
(400 MHz, Chloroform-d) δ 7.30 (d, J = 7.6 Hz, 2H), 7.22 (t, J = 7.5 Hz,
2H), 7.15 (t, J = 7.2 Hz, 1H), 7.09 (d, J = 5.5 Hz, 1H), 6.53 (d, J = 5.5
Hz, 1H), 5.24 (q, J = 6.5 Hz, 1H), 3.74 (s, 3H), 1.58 (d, J = 6.4 Hz,
3H).13C NMR (100 MHz, Chloroform-d) δ 161.05, 159.31, 141.45,
129.12, 127.58(2C), 126.69, 124.53(2C), 117.55, 109.83, 78.93, 50.46,
23.32. MS (ESI) m/z calcd. for C14H14O3S (M+H)+ 263.07.
28g
1.81
azodicarboxylate (3.60 g, 17.82 mmol) was added and stirred at room
temperature for 5 h. The reaction solution concentrated under vacuum
and the residue was purified by flash chromatography, eluting with a
gradient of 1%-5% EA in PE, to give 8a(4.03 g, 92%)as a light yellow
solid. 1H NMR (400 MHz, Chloroform-d) δ 7.91 (d, J = 7.8 Hz, 1H), 7.61
4.2.8. Methyl-5-(7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-
yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (10a)
Compounds 8a (2.80 g, 8.48 mmol) and tetrahydrofuran (40 mL)
6

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
Fig. 2. Binding mode of 28e (left) and MBM-55 (right) to Nek2 (PDB code for NEK2 is 4AFE).
were added to the triple-neck flask. The mixture was stirred evenly with
nitrogen replacing air three times. The reaction system was lowered to
ꢀ 78 ◦C with dry ice, and 2 M diisopropylaminolium lithium (8.4 mL,
2.0 eq.) was slowly injected into the syringe with a needle. At this
temperature, the reaction mixture was stirred for 45 min. Trimethyl
borate (2.8 mL, 3.0 eq.) was injected into the syringe and stirred for 30
min at ꢀ 78 ◦C. The reaction liquid was gradually raised to room tem-
perature and stirred for 30 min. 2 M HCl aqueous solution was added
slowly until the reaction solution is acidic, stirred for 10 min at room
temperature, extracted with ethyl acetate (40 mL) three times, washed
with saturated salt water, dried over Na2SO4 and concentrated under
vacuum to afford yellow solid 9a, which was not purified and directly
used for the next step.
527.1365, found 527.1378.
4.2.10. Methyl-3-(1-(2-fluorophenyl)ethoxy)-5-(7-(1-methyl-1H-pyrazol-
4-yl)imidazo[1,2-a]pyridine-3-yl)thiophene-2-carboxylate (10c)
Following the procedure described for preparing 10a (Yields: 65%,
two steps). 1H NMR (400 MHz, Chloroform-d) δ 8.13 (d, J = 7.2 Hz, 1H),
7.74 (s, 1H), 7.66 (s, 1H), 7.62 (s, 1H), 7.58 (s, 1H), 7.55 (d, J = 7.2 Hz,
1H), 7.25–7.14 (m, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 9.3 Hz, 1H),
6.90 (d, J = 5.5 Hz, 1H), 6.79 (s, 1H), 5.71 (q, J = 6.4 Hz, 1H), 3.88 (s,
3H), 3.82 (s, 3H), 1.67 (d, J = 6.3 Hz, 3H).13C NMR (100 MHz, Chlo-
roform-d) δ 161.75, 160.53, 159.77, 158.09, 147.63, 136.89, 134.92,
134.37, 130.18, 129.50 (d, J = 8.2 Hz), 129.20 (d, J = 13.2 Hz), 127.53,
127.36 (d, J = 3.7 Hz), 124.96 (d, J = 3.4 Hz), 123.94, 120.73, 118.92,
115.21 (d, J = 21.6 Hz), 114.25, 112.33 (d, J = 17.5 Hz), 109.29, 73.33
(d, J = 2.9 Hz), 51.72, 39.28, 22.98. HRMS (ESI) m/z calcd. for
To a solution of 4 (250 mg, 0.77 mmol) in mixed solution of THF (16
mL) and H2O (2 mL), 9a (434 mg, 1.16 mmol), Na2CO3 (268 mg, 2.70
mmol) and Pd(diphenylphosphino)ferrocene]dichloropalladium(II)⋅
DCM (126 mg, 0.15 mmol) were added, then the reaction mixture was
stirred at 80 ◦C overnight under N2 atmosphere. The reaction mixture
was filtered over a pad of Celite and concentrated under vacuum. The
residue was purified by flash chromatography, eluted with a gradient of
2%-6% MeOH in DCM to give 10a (310 mg, 76%, two steps) as a light
yellow solid. 1H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 7.2 Hz,
1H), 7.85 (d, J = 8.0 Hz, 1H), 7.74 (s, 1H), 7.63 (d, J = 1.9 Hz, 2H), 7.57
(d, J = 8.5 Hz, 2H), 7.51 (t, J = 7.7 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H), 6.90
(dd, 1H), 6.76 (s, 1H), 5.76 (q, J = 6.3 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 3H),
1.68 (d, J = 6.2 Hz, 3H).13C NMR (100 MHz, Chloroform-d) δ 160.71,
158.61, 146.64, 140.54, 135.88, 133.93, 133.59, 132.07, 129.19,
126.96, 126.64 (d, J = 178.5 Hz), 126.45 (d, J = 15.8 Hz), 125.31 (d, J
= 30.3 Hz), 124.60 (q, J = 5.8 Hz), 122.90, 122.09, 119.70, 117.92,
112.81, 111.37, 111.23, 108.07, 74.78, 50.70, 38.25, 23.74. HRMS
(ESI) m/z calcd. for C26H21F3N4O3S (M+H)+ 527.1365, found 527.1378.
C
25H21FN4O3S (M+H)+ 477.1397, found 477.1361.
4.2.11. Methyl-3-(1-(3-fluorophenyl)ethoxy)-5-(7-(1-methyl-1H-pyrazol-
4-yl)-imidazo [1,2-a]pyridin-3-yl)thiophene-2-carboxylate (10d)
Following the procedure described for preparing 10a (Yields: 71%,
two steps). 1H NMR (400 MHz, Chloroform-d) δ 8.09 (d, J = 7.1 Hz, 1H),
7.74 (s, 1H), 7.65 (s, 1H), 7.62 (s, 1H), 7.59 (s, 1H), 7.32–7.21 (m, 1H),
7.13 (t, J = 8.0 Hz, 2H), 6.96–6.86 (m, 2H), 6.72 (s, 1H), 5.35 (q, J = 6.4
Hz, 1H), 3.88 (s, 3H), 3.83 (s, 3H), 1.66 (d, J = 6.4 Hz, 3H).13C NMR
(100 MHz, Chloroform-d) δ 162.06 (d, J = 246.9 Hz), 160.73, 158.71,
146.60, 143.98 (d, J = 6.9 Hz), 135.87, 133.82, 133.17, 129.41 (d, J =
8.2 Hz), 129.23, 126.52, 122.90, 120.30 (d, J = 2.8 Hz), 119.69, 117.85,
114.11, 113.86 (d, J = 21.1 Hz), 111.72 (d, J = 22.2 Hz), 111.42,
111.20, 108.84, 78.65, 50.77, 38.27, 23.12. HRMS (ESI) m/z calcd. for
C
25H21FN4O3S (M+H)+ 477.1397, found 477.1361.
4.2.12. Methyl-5-(7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine-
3-yl)-3-(1-phenyl ethoxy)thiophene-2-carboxylate (10e)
4.2.9. Methyl-5-(7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-
yl)-3-(1-(3-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (10b)
Following the procedure described for preparing 10a (Yields: 66%,
two steps). 1H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 7.1 Hz, 1H),
7.70 (s, 1H), 7.68 (s, 1H), 7.63 (s, 1H), 7.59 (d, J = 6.6 Hz, 2H), 7.54 (s,
1H), 7.48 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 7.1
Hz, 1H), 6.73 (s, 1H), 5.43 (q, J = 6.5 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 3H),
1.66 (d, J = 6.3 Hz, 3H).13C NMR (100 MHz, Chloroform-d) δ 161.74,
159.77, 159.31 (d, J = 245.0 Hz), 147.63, 136.89, 134.92, 134.37,
130.18, 129.50 (d, J = 8.2 Hz), 129.20 (d, J = 13.2 Hz), 127.53, 127.36
(d, J = 3.7 Hz), 124.96 (d, J = 3.4 Hz), 123.94, 120.73, 118.92, 115.32,
115.11, 114.25, 112.33 (d, J = 17.5 Hz), 109.29, 73.33 (d, J = 2.9 Hz),
51.72, 39.28, 22.98. HRMS (ESI) m/z calcd. for C26H21F3N4O3S (M+H)+
Following the procedure described for 10a (Yields: 76%, two steps).
1H NMR (400 MHz, Chloroform-d) δ 7.91 (d, J = 7.1 Hz, 1H), 7.68 (s,
1H), 7.56 (d, J = 8.3 Hz, 2H), 7.50 (s, 1H), 7.36 (s, 1H), 7.34 (s, 1H),
7.27 (t, J = 7.4 Hz, 2H), 7.20 (d, J = 7.1 Hz, 1H), 6.79 (d, J = 7.1 Hz,
1H), 6.67 (s, 1H), 5.33 (q, J = 6.5 Hz, 1H), 3.82 (s, 3H), 3.79 (s, 3H),
1.64 (d, J = 6.4 Hz, 3H).13C NMR (100 MHz, Chloroform-d) δ 161.79,
160.09, 147.47, 142.35, 136.78, 134.66, 133.92, 130.09, 128.78(2C),
127.91, 127.53, 125.71(2C), 123.88, 120.60, 118.88, 115.21, 112.25,
112.00, 109.28, 80.35, 51.69, 39.21, 24.33. HRMS (ESI) m/z calcd. for
C
25H22N4O3S (M+H)+ 459.1491, found 459.1516.
7

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
4.2.13. 5-(7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine-3-yl)-3-
(1-(2-(trifluoromethyl) phenyl) ethoxy) thiophene-2-carboxamide (11a)
A mixed solution of 10a (200 mg, 0.38 mmol) in 7 M ammonia-
methanol solution (4 mL) and ammonia-water (2 mL) was stirred for
3 days at 78 ◦C. When the reaction mixture was clear, it was concen-
trated under vacuum. The residue was purified by flash chromatog-
raphy, eluted with a gradient of 2%-8% MeOH in DCM to give 11a(143
mg, 72%)as a light yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ 8.40 (d,
J = 9.7 Hz, 2H), 8.13 (s, 1H), 7.92 (d, J = 8.5 Hz, 1H), 7.90 (s, 1H), 7.80
(s, 2H), 7.77 (d, J = 7.7 Hz, 2H), 7.56 (t, J = 7.7 Hz, 1H), 7.33 (d, J = 7.3
Hz, 1H), 7.15 (s, 1H), 7.10 (s, 1H), 6.02 (q, J = 6.4 Hz, 1H), 3.91 (s, 3H),
1.77 (d, J = 6.1 Hz, 3H).13C NMR (100 MHz, DMSO‑d6) δ 161.90,
153.65, 147.02, 140.49, 136.76, 134.17, 133.54, 131.91, 130.26,
129.00, 128.70, 127.23, 125.87 (d, J = 5.5 Hz), 125.59 (d, J = 7.5 Hz),
124.95, 124.39, 124.08 (d, J = 236.4 Hz), 119.69, 118.41, 115.91,
113.69, 112.15, 110.76, 75.65, 54.86, 23.88. HRMS (ESI) m/z calcd. for
7.48–7.38 (m, 3H), 7.34 (d, J = 7.2 Hz, 2H), 7.12 (s, 1H), 5.86 (q, J =
6.6 Hz, 1H), 3.91 (s, 3H), 1.73 (d, J = 6.3 Hz, 3H).13C NMR (100 MHz,
DMSO‑d6) δ 162.11, 154.52, 146.97, 141.71, 136.74, 134.30, 131.69,
130.10, 128.94, 128.66(2C), 128.09, 126.10(2C), 124.80, 119.75,
118.60, 115.29, 114.89, 112.08, 110.69, 79.27, 54.88, 23.27. HRMS
(ESI) m/z calcd. for C24H21N5O2S (M+H)+ 444.1494, found 444.1460.
4.2.18. 2-(4-(imidazo[1,2-a]pyridin-6-yl)-1H-pyrazol-1-yl)-N,N-
dimethylethan-1-amine (14)
To a solution of pinacol 4-pyrazole borate 12 (1.0 equiv) in aceto-
nitrile, cesium carbonate (3.5 equiv) and 2-chloro-N,N-dimethylethane-
1-amine hydrochloride (1.5 equiv) were added. The mixture was
refluxed for 24 h, then cooled to room temperature and poured onto ice-
water, and extracted with EA. The combined organics were washed with
saturated aqueous NaHCO3, dried over anhydrous Na2SO4, and
concentrated in vacuo to afford esters 13, which was used in the next
step without further purification.
C
25H20F3N5O2S (M+H)+ 512.1368, found 512.1382.
To a solution of the crude 13 (1.3 equiv) in DMF, 2 (1.0 equiv), 1 N
K2CO3 aqueous and Pd(diphenylphosphino)ferrocene]dichloropalla-
dium(II)⋅DCM (0.1 equiv) were added and then the reaction mixture was
stirred at 80 ◦C overnight under N2 atmosphere. The reaction mixture
was filtered over a pad of Celite and concentrated under vacuum. The
residue was purified by flash chromatography, eluted with a gradient of
2%-5% MeOH in DCM to give 14 in 61% yields (two steps). 1H NMR
(400 MHz, Chloroform-d) δ 8.23 (s, 1H), 7.73 (d, J = 5.4 Hz, 2H), 7.61 (s,
1H), 7.57 (d, J = 5.6 Hz, 2H), 7.25 (dd, J = 9.5, 1.8 Hz, 1H), 4.26 (t, J =
6.5 Hz, 2H), 2.80 (t, J = 6.5 Hz, 2H), 2.29 (s, 6H).13C NMR (100 MHz,
Chloroform-d) δ 136.25(2C), 133.66, 126.64, 124.36, 121.05, 118.80,
118.45, 117.55, 112.62, 58.92, 50.40, 45.45(2C). MS (ESI) m/z calcd.
for C14H17N5 (M+H)+ 256.17.
4.2.14. 5-(7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridine-3-yl)-3-
(1-(3-(trifluoromethyl) phenyl) ethoxy) thiophene-2-carboxamide (11b)
Following the procedure described for preparing 11a (Yields:
63%).1H NMR (400 MHz, DMSO‑d6) δ 8.49 (d, J = 7.2 Hz, 1H), 8.40 (s,
1H), 8.12 (s, 1H), 8.00 (s, 1H), 7.91 (d, J = 7.0 Hz, 2H), 7.88 (s, 1H),
7.72 (s, 1H), 7.70 (s, 1H), 7.68 (t, J = 8.4 Hz, 1H), 7.52 (s, 1H), 7.31 (dd,
J = 7.3, 1.8 Hz, 1H), 7.16 (s, 1H), 5.98 (q, J = 6.4 Hz, 1H), 3.92 (s, 3H),
1.77 (d, J = 6.3 Hz, 3H).13C NMR (100 MHz, DMSO‑d6) δ 161.99,
154.16, 147.01, 143.12, 136.70, 134.40, 131.95, 130.29, 130.10,
129.80, 129.30 (d, J = 31.6 Hz), 128.90, 128.15, 124.87(q, J = 3.7 Hz),
124.09 (d, J = 272.5 Hz), 122.99 (q, J = 4.0 Hz), 119.75, 118.60,
115.70, 114.69, 112.05, 110.75, 78.59, 38.77, 22.93. HRMS (ESI) m/z
calcd. for C25H20F3N5O2S (M+H)+ 512.1368, found 512.1382.
4.2.19. 2-(4-(3-iodoimidazo[1,2-a]pyridin-6-yl)-1H-pyrazol-1-yl)-N,N-
dimethylethan-1-amine (15)
4.2.15. 3-(1-(2-fluorophenyl)ethoxy)-5-(7-(1-methyl-1H-pyrazol-4-yl)
imidazo[1,2-a] pyridine-3-yl) thiophene-2-carboxamide (11c)
To a solution of 14 (1.00 g, 3.92 mmol) in acetonitrile, NIS (0.88 g,
3.92 mmol) was added and then the reaction mixture was stirred at room
temperature for 2 h. Concentrated the organic solvent, the residue was
purified by flash chromatography, eluted with a gradient of 1%-20%
MeOH in DCM to give 15 (0.80 g, 54%) as a brown solid.
Following the procedure described for preparing 11a (Yields:
70%).1H NMR (400 MHz, DMSO‑d6) δ 8.50 (d, J = 7.2 Hz, 1H), 8.40 (s,
1H), 8.13 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.73 (s, 1H), 7.66 (t, J = 7.6
Hz, 1H), 7.46 (s, 1H), 7.40 (t, J = 6.8 Hz, 1H), 7.36 (d, J = 7.2 Hz, 1H),
7.29 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 6.05 (q, J = 6.4 Hz, 1H), 3.91 (s,
3H), 1.77 (d, J = 6.3 Hz, 3H).13C NMR (100 MHz, DMSO‑d6) δ 161.99,
159.55 (d, J = 245.7 Hz), 154.06, 147.04, 136.74, 134.40, 131.98,
130.34 (d, J = 8.3 Hz), 130.14, 128.93, 128.19 (d, J = 4.1 Hz), 128.05
(d, J = 12.8 Hz), 124.90 (d, J = 3.2 Hz), 124.75, 119.77, 118.57, 115.85
(d, J = 21.4 Hz), 115.69, 114.26, 112.14, 110.74, 74.44, 54.86, 21.63.
HRMS (ESI) m/z calcd. for C24H20FN5O2S (M+H)+ 462.1400, found
462.1368.
4.2.20. Methyl-5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)
imidazo[1,2-a]pyridin-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)
thiophene-2-carboxylate (16a)
To a solution of 15 (350 mg, 0.92 mmol) in mixed solution of THF
(20 mL) and H2O (2 mL), 9a (516 mg, 1.38 mmol), Na2CO3 (341 mg,
3.22 mmol) and Pd(diphenylphosphino)ferrocene]dichloropalladium
(II)⋅DCM (147 mg, 0.18 mmol) were added, and then the reaction
mixture was stirred at 80 ◦C overnight under N2 atmosphere. The re-
action mixture was filtered over a pad of Celite and concentrated under
vacuum. The residue was purified by flash chromatography, eluted with
a gradient of 2%-6% MeOH in DCM to give 16a (284 mg, 53%) as a light
yellow solid. 1H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 7.2 Hz,
1H), 7.94 (d, J = 7.9 Hz, 1H), 7.86 (s, 2H), 7.73 (s, 1H), 7.70 (s, 1H),
7.67 (d, J = 7.9 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H),
7.02 (d, J = 7.2 Hz, 1H), 6.85 (s, 1H), 5.86 (q, J = 5.8, 5.4 Hz, 1H), 4.28
(t, J = 6.3 Hz, 2H), 3.93 (s, 3H), 2.82 (t, J = 6.3 Hz, 2H), 2.31 (s, 6H),
1.77 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 161.75,
159.64, 147.70, 141.54, 136.84, 134.94, 134.66, 133.09, 130.38,
127.99, 127.38, 127.23, 126.77, 126.32 (d, J = 30.3 Hz), 125.63 (q, J =
5.6 Hz), 124.47 (d, J = 273.8 Hz), 123.88, 120.43, 118.93, 113.80,
112.37 (d, J = 23.1 Hz), 109.04, 75.80, 58.99, 51.74, 50.65, 45.60(2C),
24.77. HRMS (ESI) m/z calcd. for C29H28F3N5O3S (M+H)+ 584.1943,
found 584.1902.
4.2.16. 3-(1-(3-fluorophenyl)ethoxy)-5-(7-(1-methyl-1H-pyrazol-4-yl)
imidazo[1,2-a]pyridin-3-yl)thiophene-2-carboxamide (11d)
Following the procedure described for 11a (Yields: 57%).1H NMR
(400 MHz, DMSO‑d6) δ 8.48 (d, J = 7.2 Hz, 1H), 8.40 (s, 1H), 8.12 (s,
1H), 7.91 (s, 1H), 7.88 (s, 1H), 7.70 (s, 1H), 7.48 (t, J = 5.1 Hz, 2H), 7.41
(t, J = 8.0 Hz, 2H), 7.33 (d, J = 7.2 Hz, 1H), 7.17 (dd, J = 8.9, 2.4 Hz,
1H), 7.13 (d, J = 8.4 Hz, 1H), 5.87 (q, J = 6.4 Hz, 1H), 3.91 (s, 3H), 1.73
(d, J = 6.2 Hz, 3H).13C NMR (100 MHz, DMSO‑d6) δ 162.28 (d, J =
244.1 Hz), 162.02, 154.27, 147.00, 144.62 (d, J = 7.0 Hz), 136.72,
134.36, 131.86, 130.68 (d, J = 8.3 Hz), 130.13, 128.93, 124.86, 122.31
(d, J = 2.7 Hz), 119.76, 115.56, 114.93 (d, J = 21.0 Hz), 114.73, 112.98
(d, J = 21.9 Hz), 112.10, 110.72, 78.59, 54.87, 22.98. HRMS (ESI) m/z
calcd. for C24H20FN5O2S (M+H)+ 462.1400, found 462.1368.
4.2.17. 5-(7-(1-methyl-1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)-3-
(1-phenylethoxy) thiophene-2-carboxamide (11e)
Following the procedure described for preparing 11a (Yields:
69%).1H NMR (400 MHz, DMSO‑d6) δ 8.43 (d, J = 9.2 Hz, 2H), 8.14 (s,
1H), 7.92 (s, 1H), 7.87 (s, 1H), 7.74 (s, 1H), 7.57 (d, J = 7.6 Hz, 2H),
8

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Bioorganic & Medicinal Chemistry 28 (2020) 115775
4.2.21. Methyl-5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)
imidazo[1,2-a]pyridin-3-yl)-3-(1-(3-(trifluoromethyl)phenyl)ethoxy)
thiophene-2-carboxylate (16b)
4.2.25. 5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)imidazo[1,2-
a]pyridin-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-
carboxamide (17a)
Following the procedure described for preparing 16a (Yields:
43%).1H NMR (400 MHz, Chloroform-d) δ 8.18 (d, J = 7.2 Hz, 1H), 7.85
(s, 2H), 7.76 (s, 2H), 7.69 (d, J = 11.7 Hz, 2H), 7.59 (d, J = 7.9 Hz, 1H),
7.52 (t, J = 7.8 Hz, 1H), 7.01 (dd, 1H), 6.82 (s, 1H), 5.51 (q, J = 6.5 Hz,
1H), 4.28 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 2.82 (t, J = 6.5 Hz, 2H), 2.31
(s, 6H), 1.77 (d, J = 6.4 Hz, 3H).13C NMR (100 MHz, Chloroform-d) δ
161.79, 159.49, 147.72, 143.37, 136.86, 134.93, 134.50, 131.07 (d, J =
32.3 Hz), 131.07 (d, J = 97.0 Hz), 130.47, 129.47, 129.11, 127.24,
124.85 (q, J = 3.7 Hz), 124.00 (d, J = 272.5 Hz), 123.82, 122.57 (q, J =
3.8 Hz), 120.42, 118.79, 114.99, 112.59, 112.27, 110.29, 79.78, 58.99,
51.86, 50.65, 45.62(2C), 24.14. HRMS (ESI) m/z calcd. for
A mixed solution of 16a (200 mg, 0.34 mmol) in 7 M ammonia-
methanol solution (4 mL) and ammonia-water (2 mL) was stirred for
3 days at 78 ◦C. When the reaction mixture was clear liquid, it was
concentrated under vacuum. The residue was purified by flash chro-
matography, eluted with a gradient of 2%-10% MeOH in DCM to give
17a(123 mg, 62%)as a light yellow solid. 1H NMR (400 MHz, Chloro-
form-d) δ 8.24 (d, J = 7.1 Hz, 1H), 7.86 (d, J = 1.4 Hz, 2H), 7.72 (d, J =
7.7 Hz, 2H), 7.69 (d, J = 5.7 Hz, 2H), 7.61 (t, J = 7.7 Hz, 1H), 7.45 (t, J
= 7.6 Hz, 1H), 7.25 (s, 1H), 7.00 (dd, J = 7.1, 1.9 Hz, 1H), 6.76 (s, 1H),
6.72 (s, 1H), 5.89 (q, J = 6.2 Hz, 1H), 4.29 (t, J = 6.3 Hz, 2H), 2.82 (t, J
= 6.3 Hz, 2H), 2.31 (s, 6H), 1.80 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz,
Chloroform-d) δ 163.66, 154.66, 147.62, 140.51, 136.85, 134.66,
133.89, 133.31, 130.29, 128.49, 127.28, 126.48 (d, J = 30.4 Hz),
126.47, 126.14 (q, J = 5.7 Hz), 124.37 (d, J = 273.7 Hz), 123.85,
120.45, 119.08, 115.33, 113.01, 112.44, 112.23, 76.28, 58.98, 50.62,
45.59(2C), 24.94. HRMS (ESI) m/z calcd. for C28H27F3N6O2S (M+H)+
569.1947, found 569.1978.
C
29H28F3N5O3S (M+H)+ 584.1943, found 584.1902.
4.2.22. Methyl-5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)
imidazo[1,2-a]pyridin-3-yl)-3-(1-(2-fluorophenyl)ethoxy)thiophene-2-
carboxylate (16c)
Following the procedure described for preparing 16a (Yields:
19%).1H NMR (400 MHz, Chloroform-d) δ 8.36 (s, 1H), 8.17 (d, J = 7.2
Hz, 1H), 7.87 (s, 1H), 7.73 (d, J = 7.5 Hz, 2H), 7.63 (td, J = 7.6, 1.8 Hz,
1H), 7.32 (ddd, J = 7.8, 5.6, 2.0 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.09 (t,
1H), 7.04 (d, J = 7.2 Hz, 1H), 6.89 (s, 1H), 5.80 (q, J = 6.3 Hz, 1H), 4.88
(t, J = 6.0 Hz, 2H), 3.91 (s, 3H), 3.75 (t, J = 6.0 Hz, 2H), 2.81 (s, 6H),
1.76 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 161.70,
159.73, 159.33 (d, J = 245.1 Hz), 137.94, 134.07, 133.73, 130.11,
129.63 (d, J = 8.2 Hz), 129.17, 129.15, 129.04, 127.37, 127.33, 124.99
(d, J = 3.6 Hz), 124.12, 121.10, 115.30 (d, J = 21.6 Hz), 114.81, 112.75,
112.14, 109.75, 73.41 (d, J = 2.9 Hz), 56.63, 53.31, 51.78, 44.00(2C),
22.97 .HRMS (ESI) m/z calcd. for C28H28FN5O3S (M+H)+ 534.1975,
found 534.1970.
4.2.26. 5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)imidazo[1,2-
a]pyridin-3-yl)-3-(1-(3-(trifluoromethyl)phenyl)ethoxy)thiophene-2-
carboxamide (17b)
Following the procedure described for preparing 17a (Yields:
13%).1H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 7.2 Hz, 1H), 7.76
(d, J = 3.2 Hz, 2H), 7.62 (s, 1H), 7.60 (d, J = 5.8 Hz, 2H), 7.55 (d, J =
7.9 Hz, 2H), 7.49 (d, J = 7.6 Hz, 1H), 7.08 (s, 1H), 6.90 (d, J = 7.2 Hz,
1H), 6.67 (s, 1H), 6.61 (s, 1H), 5.47 (q, J = 6.5 Hz, 1H), 4.20 (t, J = 6.5
Hz, 2H), 2.74 (t, J = 6.5 Hz, 2H), 2.23 (s, 6H), 1.72 (d, J = 6.4 Hz, 3H).
13C NMR (100 MHz, Chloroform-d) δ 162.47, 153.63, 146.59, 141.34,
135.76, 133.60, 132.64, 130.40 (q, J = 32.5 Hz), 129.33, 128.88,
127.75, 126.26, 124.36 (q, J = 3.8 Hz), 122.78 (d, J = 272.7 Hz),
122.76, 121.36 (d, J = 3.9 Hz), 119.38, 117.90, 114.94, 112.55, 111.47,
111.11, 78.90, 57.95, 49.58, 44.55(2C), 23.13. HRMS (ESI) m/z calcd.
for C28H27F3N6O2S (M + Na)+ 591.1766, found 591.1738.
4.2.23. Methyl-5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)
imidazo[1,2-a]pyridin-3-yl)-3-(1-(3-fluorophenyl)ethoxy)thiophene-2-
carboxylate (16d)
Following the procedure described for preparing 16a (Yields:
19%).1H NMR (400 MHz, Chloroform-d) δ 8.19 (d, J = 7.2 Hz, 1H), 7.85
(s, 2H), 7.74 (s, 1H), 7.70 (s, 1H), 7.36 (q, J = 7.5 Hz, 1H), 7.22 (t, J =
8.2 Hz, 2H), 7.01 (d, J = 7.2 Hz, 2H), 6.80 (s, 1H), 5.44 (q, J = 6.5 Hz,
1H), 4.28 (t, J = 6.5 Hz, 2H), 3.92 (s, 3H), 2.81 (t, J = 6.5 Hz, 2H), 2.31
(s, 6H), 1.75 (d, J = 6.4 Hz, 3H).13C NMR (100 MHz, Chloroform-d) δ
163.09 (d, J = 246.9 Hz), 161.79, 159.75, 147.66, 144.98 (d, J = 6.7
Hz), 136.85, 134.86, 134.26, 130.49, 130.40 (d, J = 1.2 Hz), 127.23,
123.88, 121.31 (d, J = 2.9 Hz), 120.43, 118.85, 115.07, 114.90 (d, J =
21.2 Hz), 112.73 (d, J = 22.2 Hz), 112.53, 112.25, 109.77, 79.66 (d, J =
1.8 Hz), 58.99, 51.82, 50.66, 45.62(2C), 24.18. HRMS (ESI) m/z calcd.
for C28H28FN5O3S (M+H)+ 534.1975, found 534.1970.
4.2.27. 5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)imidazo[1,2-
a]yridine-3-yl)-3-(1-(2-fluorophenyl)ethoxy)thiophene-2-carboxamide
(17c)
Following the procedure described for preparing 17a (Yields:
44%).1H NMR (400 MHz, Chloroform-d) δ 8.28 (dd, J = 7.2, 1.0 Hz, 1H),
7.86 (d, J = 2.2 Hz, 2H), 7.73 (d, J = 1.3 Hz, 1H), 7.71 (s, 1H), 7.44 (td,
J = 7.5, 1.7 Hz, 1H), 7.34 (dd, J = 14.1, 6.9 Hz, 1H), 7.25 (s, 1H), 7.20 (t,
J = 7.6 Hz, 1H), 7.18–7.07 (m, 1H), 7.01 (dt, J = 7.2, 1.5 Hz, 1H), 6.86
(d, J = 1.1 Hz, 1H), 6.65 (s, 1H), 5.81 (q, J = 6.4 Hz, 1H), 4.29 (t, J = 6.5
Hz, 2H), 2.83 (t, J = 6.5 Hz, 2H), 2.32 (s, 6H), 1.80 (d, J = 6.4 Hz, 3H).
13C NMR (100 MHz, Chloroform-d) δ 163.64, 159.59 (d, J = 245.9 Hz),
154.84, 147.57, 136.85, 134.59, 133.58, 130.26 (d, J = 7.0 Hz), 130.14,
128.05 (d, J = 13.1 Hz), 127.31, 126.95 (d, J = 3.7 Hz), 125.09 (d, J =
3.3 Hz), 123.92, 120.47, 119.07, 115.84 (d, J = 21.5 Hz), 115.65,
113.35, 112.48, 112.18, 74.27 (d, J = 2.9 Hz), 58.95, 50.58, 45.57(2C),
22.85. HRMS (ESI) m/z calcd. for C27H27FN6O2S (M+H)+ 519.1978,
found 519.1969.
4.2.24. Methyl-5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)
imidazo[1,2-a]pyridin-3-yl)-3-(1-phenylethoxy)thiophene-2-carboxylate
(16e)
Following the procedure described for preparing 16a (Yields:
45%).1H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J = 7.2 Hz, 1H), 7.77
(d, J = 2.4 Hz, 2H), 7.64 (s, 1H), 7.61 (s, 1H), 7.38 (d, J = 6.9 Hz, 2H),
7.32 (t, J = 7.5 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H), 6.89 (d, J = 5.4 Hz, 1H),
6.70 (s, 1H), 5.35 (q, J = 6.5 Hz, 1H), 4.21 (t, J = 6.5 Hz, 2H), 3.84 (s,
4.2.28. 5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)imidazo[1,2-
a]pyridin-3-yl)-3-(1-(3-fluorophenyl)ethoxy)thiophene-2-carboxamide
(17d)
3H), 2.75 (t, J = 6.5 Hz, 2H), 2.24 (s, 6H), 1.69 (d, J = 6.4 Hz, 3H).13
C
NMR (100 MHz, Chloroform-d) δ 160.84, 159.12, 146.57, 141.31,
135.83, 133.74, 132.95, 129.24, 127.81(2C), 126.92, 126.19, 124.67
(2C), 122.87, 119.44, 117.88, 114.33, 111.39, 111.21, 108.35, 79.40,
57.94, 50.74, 49.59, 44.58(2C), 23.38. HRMS (ESI) m/z calcd. for
Following the procedure described for 17a (Yields: 55%).1H NMR
(400 MHz, Methanol‑d4) δ 8.18 (s, 1H), 8.15 (d, J = 7.4 Hz, 1H), 7.90 (s,
1H), 7.63 (s, 1H), 7.54 (s, 1H), 7.43 (q, J = 7.4 Hz, 1H), 7.34 (d, J = 7.7
Hz, 1H), 7.28 (d, J = 9.9 Hz, 1H), 7.12 (d, J = 6.4 Hz, 2H), 7.06 (d, J =
2.3 Hz, 1H), 5.76 (q, J = 6.4 Hz, 1H), 4.39 (t, J = 6.5 Hz, 2H), 3.09 (t, J
= 6.5 Hz, 2H), 2.51 (s, 6H), 1.76 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz,
Methanol‑d4) δ 165.40, 164.52 (d, J = 245.7 Hz), 156.90, 148.39,
145.97 (d, J = 6.8 Hz), 138.34, 134.38, 134.10, 132.40, 132.00 (d, J =
C
28H29N5O3S (M+H)+ 516.2069, found 516.2075.
9

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Bioorganic & Medicinal Chemistry 28 (2020) 115775
8.1 Hz), 130.02, 125.67, 123.08 (d, J = 2.9 Hz), 121.69, 120.52, 116.36
(d, J = 11.4 Hz), 116.09, 116.04, 113.90 (d, J = 22.4 Hz), 113.87,
111.63, 81.12 (d, J = 1.9 Hz), 59.30, 49.95, 45.21(2C), 24.14. HRMS
(ESI) m/z calcd. for C27H27FN6O2S (M+H)+ 519.1978, found 519.1969.
4.2.33. 7-(1-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrazol-4-yl)imidazo
[1,2-a]pyridine (20d)
Following the procedure described for preparing 20a (Yields:
82%).1H NMR (400 MHz, Chloroform-d) δ 8.11 (d, J = 7.0 Hz, 1H), 7.82
(s, 1H), 7.76 (s, 1H), 7.67 (s, 1H), 7.60 (s, 1H), 7.55 (s, 1H), 6.93 (d, J =
7.1 Hz, 1H), 4.23 (t, J = 6.8 Hz, 2H), 2.75–2.50 (m, 8H), 2.39 (s, 5H),
2.09 (p, J = 7.0 Hz, 2H). 13C NMR (100 MHz, Chloroform-d) δ 145.78,
136.76, 133.65, 129.45, 126.90, 125.80, 120.65, 112.08, 111.58,
111.44, 54.49, 54.43(2C), 52.02(2C), 50.17, 45.18, 27.12. MS (ESI) m/z
calcd. for C17H22N6 (M+H)+ 311.19.
4.2.29. 5-(7-(1-(2-(dimethylamino)ethyl)-1H-pyrazol-4-yl)imidazo[1,2-
a]yridine-3-yl)-3-(1-phenylethoxy)thiophene-2-carboxamide (17e)
Following the procedure described for 17a (Yields: 42%).1H NMR
(400 MHz, Chloroform-d) δ 8.17 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 2.7 Hz,
2H), 7.69 (d, J = 3.3 Hz, 2H), 7.44–7.39 (m, 4H), 7.36 (dd, J = 6.1, 2.4
Hz, 1H), 7.25 (s, 1H), 6.97 (dd, J = 7.1, 1.8 Hz, 1H), 6.78 (s, 1H), 6.47 (s,
1H), 5.48 (q, J = 6.4 Hz, 1H), 4.29 (t, J = 6.4 Hz, 2H), 2.83 (t, J = 6.4 Hz,
2H), 2.32 (s, 6H), 1.78 (d, J = 6.2 Hz, 3H). 13C NMR (100 MHz, Chlo-
roform-d) δ 163.63, 155.25, 147.52, 141.27, 136.84, 134.49, 133.26,
130.24, 129.13(2C), 128.45, 127.29, 125.48(2C), 123.92, 120.48,
119.09, 115.59, 114.21, 112.41, 112.16, 80.68, 58.96, 50.59, 45.57
(2C), 24.31. HRMS (ESI) m/z calcd. for C27H28N6O2S (M+H)+ 501.2073,
found 501.2092.
4.2.34. 3-iodo-7-(1-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrazol-4-yl)imidazo
[1,2-a]pyridine (21a)
To a solution of 20a (1.0 equiv) in acetonitrile, NIS (1.0 equiv) was
added, and then the reaction mixture was stirred at room temperature
for 2 h. After concentrated the organic solvent, the residue was purified
by flash chromatography, eluted with a gradient of 1%-20% MeOH in
DCM to give 21a (Yields: 50%).1H NMR (400 MHz, DMSO‑d6) δ 8.55 (s,
1H), 8.32 (d, J = 7.1 Hz, 1H), 8.24 (s, 1H), 7.89 (s, 1H), 7.69 (s, 1H),
7.34 (d, J = 7.1 Hz, 1H), 4.55 (t, J = 6.3 Hz, 2H), 3.66 (t, J = 6.1 Hz, 2H),
3.25 (s, 4H), 1.93 (d, J = 6.8 Hz, 4H). 13C NMR (100 MHz, DMSO‑d6) δ
147.3, 140.0, 137.5, 129.3, 129.0, 126.5, 120.2, 111.9, 110.5, 64.9,
53.7(2C), 53.4, 48.0, 22.6(2C). MS (ESI) m/z calcd. for C16H18IN5
(M+H)+ 408.06.
4.2.30. 7-(1-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrazol-4-yl)imidazo[1,2-a]
pyridine (20a)
To a solution of pinacol 4-pyrazole borate 8 (1.0 equiv) in acetoni-
trile, cesium carbonate (3.5 equiv) and various substituted chlorides of
hydrochloric acid (1.5 equiv) were added. The mixture was refluxed for
24 h, then cooled to room temperature and poured onto ice-water, and
extracted with EA. The combined organics were washed with saturated
aqueous NaHCO3, dried over anhydrous Na2SO4, and concentrated in
vacuo to afford esters 19a, which was used in the next step without
further purification.
4.2.35. 3-iodo-7-(1-(2-(piperidin-1-yl)ethyl)-1H-pyrazol-4-yl)imidazo
[1,2-a]pyridine (21b)
Following the procedure described for preparing 21a (Yields: 50%).
1H NMR (400 MHz, DMSO‑d6) δ 8.59 (s, 1H), 8.33 (d, J = 7.1 Hz, 1H),
8.25 (s, 1H), 7.89 (s, 1H), 7.72 (s, 1H), 7.36 (d, J = 7.2 Hz, 1H), 4.64 (t, J
= 6.6 Hz, 2H), 3.66 (t, J = 6.5 Hz, 2H), 3.50–2.95 (m, 4H), 1.77 (s, 4H),
1.57 (s, 2H). 13C NMR (100 MHz, DMSO‑d6) δ 147.05, 139.62, 137.61,
129.45, 129.03, 126.62, 120.23, 112.03, 110.39, 65.47, 54.75, 52.50
(2C), 46.00, 22.53(2C), 21.10. MS (ESI) m/z calcd. for C17H20IN5
(M+H)+ 422.10.
To a solution of the crude 19a (1.3 equiv) in DMF, 2 (1.0 equiv), 1 N
K2CO3 aqueous and 1,1′-Bis(diphenylphosphino)ferrocene]dichlor-
opalladium(II) (0.1 equiv) in dichloromethane were added, and then the
reaction mixture was stirred at 80 ◦C overnight under N2 atmosphere.
The reaction mixture was filtered over a pad of Celite and concentrated
under vacuum. The residue was purified by flash chromatography,
eluted with a gradient of 2%-5% MeOH in DCM to give 20a (Yields:
79%).1H NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 7.1 Hz, 1H), 7.72
(d, J = 6.2 Hz, 2H), 7.56 (s, 1H), 7.49 (s, 1H), 7.41 (s, 1H), 6.80 (d, J =
7.1 Hz, 1H), 4.20 (t, J = 6.8 Hz, 2H), 2.88 (t, J = 6.8 Hz, 2H), 2.45 (t, J =
6.0 Hz, 4H), 1.68 (q, J = 3.3 Hz, 4H). 13C NMR (100 MHz, Chloroform-d)
δ 144.9, 135.6, 132.9, 128.3, 125.9, 124.7, 119.9, 111.0, 110.7, 110.4,
54.8, 53.2(2C), 50.8, 22.5(2C). MS (ESI) m/z calcd. for C16H19N5
(M+H)+ 282.16.
4.2.36. 4-(2-(4-(3-iodoimidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-yl)
ethyl)morpholine (21c)
Following the procedure described for preparing 21a (Yields: 80%).
1H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J = 7.1 Hz, 1H), 7.77 (s,
2H), 7.58 (s, 2H), 6.97 (d, J = 7.1 Hz, 1H), 4.22 (t, J = 6.5 Hz, 2H), 3.63
(t, J = 4.4 Hz, 4H), 2.79 (t, J = 6.5 Hz, 2H), 2.44 (t, J = 4.4 Hz, 4H). 13
C
NMR (100 MHz, Chloroform-d) δ 147.15, 139.66, 135.85(2C), 128.95,
126.16, 124.91, 119.59, 111.23, 110.79, 65.92(2C), 57.11, 52.65(2c),
48.92. MS (ESI) m/z calcd. for C16H18IN5O (M+H)+ 424.08.
4.2.31. 8-(1-(2-(piperidin-1-yl)ethyl)-1H-pyrazol-4-yl)-4H-pyrido[1,2-a]
pyrimidine (20b)
Following the procedure described for preparing 20a (Yields:
81%).1H NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 7.0 Hz, 1H), 7.74
(s, 1H), 7.71 (s, 1H), 7.57 (s, 1H), 7.50 (s, 1H), 7.42 (s, 1H), 6.81 (d, J =
6.6 Hz, 1H), 4.17 (t, J = 6.7 Hz, 2H), 2.69 (t, J = 6.7 Hz, 2H), 2.34 (t, J =
5.2 Hz, 4H), 1.48 (p, J = 5.6 Hz, 4H), 1.34 (p, J = 6.0 Hz, 2H).13C NMR
(100 MHz, Chloroform-d) δ 145.93, 136.48, 133.90, 129.36, 127.03,
125.67, 120.78, 111.98, 111.70, 111.36, 58.50, 54.60(2C), 50.18, 26.00
(2C), 24.13. MS (ESI) m/z calcd. for C17H21N5 (M+H)+ 296.18.
4.2.37. 3-iodo-7-(1-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrazol-4-yl)
imidazo[1,2-a] pyridine (21d)
Following the procedure described for preparing 21a (Yields:
65%).1H NMR (400 MHz, DMSO‑d6) δ 8.39 (s, 1H), 8.27 (d, J = 7.2 Hz,
1H), 8.09 (s, 1H), 7.84 (s, 1H), 7.66 (s, 1H), 7.31 (d, J = 7.1 Hz, 1H),
4.15 (t, J = 7.0 Hz, 2H), 2.51 (t, J = 2.1 Hz, 2H), 2.43–2.29 (m, 4H), 2.27
(t, J = 7.0 Hz, 4H), 2.16 (s, 3H), 1.96 (p, J = 7.0 Hz, 2H).13C NMR (100
MHz, DMSO‑d6) δ 147.48, 140.07, 136.61, 129.76, 128.15, 126.36,
119.52, 111.95, 110.28, 63.52, 54.64(2C), 54.54, 52.48(2C), 49.73,
45.61, 26.97. MS (ESI) m/z calcd. for C18H23IN6 (M+H)+ 451.11
[M+H]+
4.2.32. 4-(2-(4-(imidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-yl)ethyl)
morpholine (20c)
Following the procedure described for preparing 20a (Yields:
53%).1H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 7.0 Hz, 1H), 7.74
(d, J = 3.7 Hz, 2H), 7.59 (s, 1H), 7.54 (s, 1H), 7.46 (s, 1H), 6.84 (dd, J =
7.2, 1.6 Hz, 1H), 4.21 (t, J = 6.6 Hz, 2H), 3.63 (t, J = 4.6 Hz, 4H), 2.77 (t,
J = 6.6 Hz, 2H), 2.43 (t, J = 4.6 Hz, 4H). 13C NMR (100 MHz, Chloro-
form-d) δ 135.72(2C), 133.02, 128.22, 125.95, 124.72, 119.97, 111.06,
110.88, 110.37, 65.92(2C), 57.12, 52.64(2C), 48.88. MS (ESI) m/z
calcd. for C16H19N5O (M+H)+ 298.18.
4.2.38. tert-butyl 3-(((methylsulfonyl)oxy)methyl)azetidine-1-carboxylate
(24a)
Compound
3 ??- (hydroxymethyl) azacyclobutane-1-tert-butyl
carboxylate 23a (2.40 g, 12.83 mmol) was dissolved in DCM (60 mL)
and stirred in ice bath for 15 min to 0 ◦C. DMAP (156 mg, 1.28 mmol),
triethylamine (3.6 mL, 25.56 mmol) and methyl sulfonyl chloride (1.8
mL, 15.3 mmol) were added to the mixture respectively. The mixture
was stirred for 15 min in ice bath and then wormed to room temperature
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Bioorganic & Medicinal Chemistry 28 (2020) 115775
for 12 h. Stop the reaction, quench the saturated NaHCO3 aqueous so-
lution (25 mL), then diluted with EA (60 mL), washed with H2O (60 mL),
brine, dried over Na2SO4, and concentrated under vacuum. The crude
product was purified by flash chromatography on silica gel with a
gradient of 10%-20% EA in PE to give 24a (8.45 g, 92%) as a brown
solid. 1H NMR (400 MHz, Chloroform-d) δ 4.35 (d, J = 6.7 Hz, 2H), 4.04
(t, J = 8.6 Hz, 2H), 3.77–3.65 (m, 2H), 3.06 (s, 3H), 2.93 (p, 1H), 1.43 (s,
9H). 13C NMR (100 MHz, Chloroform-d) δ 156.08, 79.61, 77.95–76.44
(m), 70.34(2C), 37.35, 28.27(3C), 27.87. MS (ESI) m/z calcd. for
75%).1H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J = 7.0 Hz, 1H), 7.75
(s, 1H), 7.65 (s, 1H), 7.56 (s, 1H), 7.51 (s, 1H), 7.44 (s, 1H), 6.80 (d, J =
7.0 Hz, 1H), 4.82 (q, J = 6.3 Hz, 1H), 3.94–3.32 (m, 3H), 2.46–2.22 (m,
J = 6.9, 6.2 Hz, 2H), 1.39 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ
153.30, 144.84, 136.08, 133.03, 127.96, 124.79, 123.99, 120.26,
111.09, 111.01, 110.33, 78.80, 59.46, 50.03, 43.28, 30.93, 28.64, 27.45
(2C). MS (ESI) m/z calcd. for C19H23N5O2 (M+H)+ 354.19.
4.2.44. tert-butyl-4-(4-(imidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-yl)
piperidine-1-carboxylate (25c)
C
10H19NO5S (M+H)+ 266.11.
Following the procedure described for preparing 25a (Yields:67%).
1H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J = 7.0 Hz, 1H), 7.75 (s,
1H), 7.66 (s, 1H), 7.57 (s, 1H), 7.51 (s, 0H), 7.44 (s, 1H), 6.82 (dd, J =
7.3, 1.7 Hz, 1H), 4.21 (tt, J = 12.0, 4.1 Hz, 3H), 2.90–2.76 (m, 2H),
2.15–2.02 (m, 2H), 1.88 (qd, J = 12.3, 4.4 Hz, 2H), 1.40 (s, 9H). 13C
NMR (100 MHz, Chloroform-d) δ 153.50, 144.91, 135.54, 133.02,
128.19, 124.75, 123.09, 119.83, 111.03, 110.90, 110.35, 78.90(2C),
58.58(2C), 41.72, 31.34, 27.40(3C). MS (ESI) m/z calcd. for C20H25N5O2
(M+H)+ 368.20.
4.2.39. tert-butyl 3-((methylsulfonyl)oxy)pyrrolidine-1-carboxylate (24b)
Following the procedure described for preparing 24a (Yields: 99%).
1H NMR (400 MHz, Chloroform-d) δ 5.19 (dt, J = 4.9, 2.4 Hz, 1H),
3.66–3.48 (m, 2H), 3.52–3.31 (m, 2H), 2.99 (s, 3H), 2.30–2.03 (m, 2H),
1.39 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 153.15 (d, J = 13.3
Hz), 79.12, 78.74 (d, J = 13.3 Hz), 50.98 (d, J = 42.4 Hz), 42.44 (d, J =
34.0 Hz), 37.65, 31.13 (d, J = 92.7 Hz), 27.42(3C). MS (ESI) m/z calcd.
for C10H19NO5S (M+H)+ 266.11.
4.2.40. tert-butyl 4-((methylsulfonyl)oxy)piperidine-1-carboxylate (24c)
Following the procedure described for preparing 24a (Yields: 53%).
1H NMR (400 MHz, Chloroform-d) δ 4.88 (tt, J = 7.7, 3.7 Hz, 1H),
3.77–3.66 (m, 2H), 3.36–3.24 (m, 2H), 1.97 (ddt, J = 14.7, 7.6, 3.9 Hz,
2H), 1.82 (ddt, J = 13.1, 8.5, 4.0 Hz, 2H), 1.46 (s, 8H). 13C NMR (100
MHz, Chloroform-d) δ 154.54, 79.95(2C), 40.44(2C), 38.80(2C), 31.62,
28.36(3C). MS (ESI) m/z calcd. for C11H21NO5S (M+H)+ 280.12.
4.2.45. tert-butyl-3-((4-(3-iodoimidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-
yl)methyl) azetidine-1-carboxylate (26a)
To a solution of 25a (1.0 equiv) in acetonitrile, NIS (1.0 equiv) was
added, then the reaction mixture was stirred at room temperature for 2
h. Concentrated the organic solvent, the residue was purified by flash
chromatography, eluted with a gradient of 1%-20% MeOH in DCM to
give 26a (Yields: 77%). 1H NMR (400 MHz, Chloroform-d) δ 8.05 (d, J =
7.1 Hz, 1H), 7.85 (s, 1H), 7.75 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.02 (d,
J = 7.1 Hz, 1H), 4.38 (d, J = 7.6 Hz, 2H), 4.08 (t, J = 8.5 Hz, 2H), 3.78
(dd, J = 8.9, 5.1 Hz, 2H), 3.13 (qd, J = 8.6, 8.1, 4.1 Hz, 1H), 1.44 (s, 9H).
13C NMR (100 MHz, Chloroform-d) δ 156.24, 148.03, 140.67, 137.41,
129.59, 126.77, 125.94, 120.77, 112.17, 111.88, 79.65, 60.27, 55.17,
52.46(b, 2C), 29.12, 28.36(3C). MS (ESI) m/z calcd. for C19H22IN5O2
(M+H)+ 480.12.
4.2.41. 7-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine (22)
To a solution of 2 (12.20 g, 61.93 mmol) in mixed solution of DMF
(220 mL) and H2O(100 mL), 1-Boc-pyrazole-4-boronic acid pinacol ester
(20.00 g, 68.03 mmol), K2CO3 (31.80 g, 230.43 mmol) and Pd (diphe-
nylphosphino) ferrocene] dichloropalladium(II)⋅DCM(7.60 g, 9.30
mmol) were added, then the reaction mixture was stirred at 80 ◦C
overnight under N2 atmosphere. The reaction mixture was filtered over a
pad of Celite and concentrated under vacuum. The residue was purified
by flash chromatography, eluting with a gradient of 4%-8% MeOH in
DCM to give 22 (8.45 g, 92%) as a brown solid. 1H NMR (400 MHz,
DMSO‑d6) δ 13.07 (s, 1H), 8.87 (s, 1H), 8.12 (s, 2H), 7.90 (s, 1H), 7.61
(d, J = 9.6 Hz, 1H), 7.59 (s, 1H), 7.55 (dd, J = 9.3, 1.7 Hz, 1H). 13C NMR
(100 MHz, DMSO‑d6) δ 143.44, 133.12, 124.24(2C), 121.61, 118.10,
117.52, 116.83(2C), 113.09. MS (ESI) m/z calcd. for C10H8N4 (M+H)+
185.07.
4.2.46. tert-butyl 3-(4-(3-iodoimidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-
yl)pyrrolidine-1-carboxylate (26b)
Following the procedure described for preparing 26a (Yields:
70%).1H NMR (400 MHz, Chloroform-d) δ 7.88 (d, J = 7.1 Hz, 1H), 7.74
(s, 1H), 7.69 (s, 1H), 7.50 (d, J = 3.0 Hz, 2H), 6.87 (d, J = 7.1 Hz, 1H),
4.81 (q, J = 6.2 Hz, 1H), 3.86–3.39 (m, 4H), 2.32 (p, J = 6.6 Hz, 2H),
1.37 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 154.21, 147.86,
140.49, 137.11, 129.57, 125.83, 125.33, 120.67, 112.06, 111.64, 79.72,
60.47, 60.40, 51.02, 44.27, 31.44, 28.44(3C). MS (ESI) m/z calcd. for
4.2.42. tert-butyl-3-((4-(imidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-yl)
methyl)azetidine-1-carboxylate (25a)
C
19H22IN5O2 (M+H)+ 480.12.
Compound 22 (1.60 g, 9.00 mmol) was dissolved in DMF (90 mL).
The solution was stirred in ice bath for 15 mim. 60% NaH (500 mg,
11.70 mmol) was added in batches and stirred in ice bath for 1 h. Then,
compound 24a (3.10 g, 11.70 mmol) was added. After the solution was
raised to room temperature, it was placed in an oil bath at 100 ◦C and
stirred for 12 h. The reaction mixture was filtered over a pad of Celite
and concentrated under vacuum. The residue was purified by flash
chromatography, eluted with a gradient of 2%-10% MeOH in DCM to
give 25a(2.20 g, 81%)as a Canary yellow solid. 1H NMR (400 MHz,
Chloroform-d) δ 8.10 (d, J = 7.0 Hz, 1H), 7.83 (s, 1H), 7.73 (s, 1H), 7.66
(s, 1H), 7.61 (s, 1H), 7.54 (s, 1H), 6.90 (d, J = 7.1 Hz, 1H), 4.36 (d, J =
7.7 Hz, 2H), 4.06 (t, J = 8.6 Hz, 2H), 3.77 (dd, J = 8.8, 5.1 Hz, 2H), 3.10
(p, 1H), 1.44 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 156.2, 145.8,
137.2, 134.0, 128.9, 126.6, 125.8, 121.1, 112.1, 111.9, 111.3, 79.6,
55.1, 52.06(b, 2C) 29.1, 28.3(3C). MS (ESI) m/z calcd. for C19H23N5O2
(M+H)+ 354.19.
4.2.47. tert-butyl-4-(4-(3-iodoimidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-
yl)piperidine-1-carboxylate (26c)
Following the procedure described for 26a (Yields: 69%). 1H NMR
(400 MHz, Chloroform-d) δ 7.96 (d, J = 7.1 Hz, 1H), 7.77 (s, 1H), 7.69 (s,
1H), 7.55 (d, J = 3.4 Hz, 2H), 6.94 (dd, J = 7.2, 1.7 Hz, 1H), 4.23 (tt,
3H), 2.83 (t, J = 12.9 Hz, 2H), 2.09 (dd, 2H), 1.89 (qd, J = 12.3, 4.3 Hz,
2H), 1.40 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 153.47, 147.07,
139.62, 135.66, 128.87, 124.88, 123.31, 119.39, 111.17, 110.73, 78.91,
59.12, 58.63(2C), 41.68, 31.34(2C), 27.41(3C). MS (ESI) m/z calcd. for
C
20H24IN5O2 (M+H)+ 494.11.
4.2.48. Methyl-5-(7-(1-(2-(pyrrolidin-1-yl)ethyl)-1H-pyrazol-4-yl)
imidazo[1,2-a] pyridine-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)
thiophene-2-carboxylate (27a)
To a solution of 21a (1.0 eq.) in mixed solution of THF and H2O, 9a
(1.5 eq.), Na2CO3(3.5 eq.) and Pd(diphenylphosphino) ferrocene]
dichloropalladium (II)⋅DCM (0.2 eq.) were added, then the reaction
mixture was stirred at 80 ◦C overnight under N2 atmosphere. The re-
action mixture was filtered over a pad of Celite and concentrated under
vacuum. The residue was purified by flash chromatography, eluted with
4.2.43. tert-butyl-3-(4-(imidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-yl)
pyrrolidine-1-carboxylate (25b)
Following the procedure described for preparing 25a (Yields:
11

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
a gradient of 5%-10% MeOH in DCM, to give 27a (Yields: 40%). 1H NMR
(400 MHz, Chloroform-d) δ 8.21 (d, J = 7.2 Hz, 1H), 7.94 (d, J = 7.9 Hz,
1H), 7.86 (s, 2H), 7.73 (s, 1H), 7.69 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H),
7.60 (t, J = 7.8 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.02 (dd, J = 7.3, 1.8
Hz, 1H), 6.85 (s, 1H), 5.85 (q, J = 6.4 Hz, 1H), 4.32 (t, J = 6.8 Hz, 2H),
4.2.52. 3.3.3. Tert-butyl-3-((4-(3-(5-(methoxycarbonyl)-4-(1-(2-
(trifluoromethyl)-phenyl) ethoxy)thiophen-2-yl)imidazo[1,2-a]pyridin-7-
yl)-1H-pyrazol-1-yl)methyl) azetidine-1-carboxylate (27e)
Following the procedure described for preparing 27a (Yields:
18%).1H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 7.1 Hz, 1H), 7.94
(d, J = 7.9 Hz, 1H), 7.86 (s, 1H), 7.78 (s, 1H), 7.73 (d, J = 1.3 Hz, 1H),
7.67 (d, J = 10.5 Hz, 2H), 7.60 (t, J = 7.7 Hz, 1H), 7.40 (t, J = 7.6 Hz,
1H), 7.00 (dd, J = 7.2, 1.9 Hz, 1H), 6.86 (s, 1H), 5.86 (q, J = 6.3 Hz, 1H),
4.39 (d, J = 7.4 Hz, 2H), 4.09 (t, J = 8.4 Hz, 2H), 3.93 (s, 3H), 3.79 (dd,
J = 8.9, 5.0 Hz, 2H), 3.14 (qd, J = 7.4, 3.6 Hz, 1H), 1.77 (d, J = 6.2 Hz,
3H), 1.45 (s, 9H). 13C NMR (100 MHz, Chloroform-d) δ 161.71, 159.60,
156.26, 147.56, 141.50 (d, J = 1.6 Hz), 137.40, 134.95, 134.53, 133.09,
129.91, 128.53, 127.99, 127.36, 126.82, 126.28 (d, J = 30.4 Hz),
125.61 (q, J = 5.7 Hz), 124.46 (d, J = 273.8 Hz), 123.96, 120.66,
118.98, 113.83, 112.38, 112.35, 109.08, 79.67, 75.78, 55.19, 52.40(b,
2C), 51.73, 29.12, 28.34(3C), 24.75. HRMS (ESI) m/z calcd. for
3.00 (t, J = 6.8 Hz, 2H), 2.57 (t, J = 5.9 Hz, 4H), 1.84–1.73 (m, 7H). 13
C
NMR (100 MHz, Chloroform-d) δ 161.73 , 159.63 , 147.69 , 141.53 (d, J
= 1.8 Hz), 136.79 , 134.95 , 134.64 , 133.08 , 127.98 , 127.37 , 127.21 ,
126.31 (d, J = 30.2 Hz), 125.85, 125.61 (q, J = 5.7 Hz), 124.46 (d, J =
273.7 Hz), 123.88 , 120.40 , 118.92 , 113.76 , 112.44 , 112.22 , 109.02 ,
75.77 , 55.76 , 54.26(2C) , 51.84 , 51.72 , 24.76 , 23.55(2C). HRMS (ESI)
m/z calcd. for C31H30F3N5O3S (M+H)+ 610.2100, found: 610.2062.
4.2.49. Methyl-5-(7-(1-(2-(piperidin-1-yl)ethyl)-1H-pyrazol-4-yl)imidazo
[1,2-a]pyridin-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-
carboxylate (27b)
Following the procedure described for preparing 27a (Yields:
39%).1H NMR (400 MHz, Chloroform-d) δ 8.22 (d, J = 7.2 Hz, 1H), 7.93
(d, J = 8.0 Hz, 1H), 7.88 (s, 1H), 7.84 (s, 1H), 7.73 (s, 1H), 7.70 (s, 1H),
7.67 (d, J = 8.0 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H),
7.02 (dd, J = 7.2, 1.7 Hz, 1H), 6.85 (s, 1H), 5.85 (q, J = 6.4 Hz, 1H), 4.30
(t, J = 6.7 Hz, 2H), 3.93 (s, 3H), 2.81 (t, J = 6.7 Hz, 2H), 2.46 (t, J = 5.2
Hz, 4H), 1.77 (d, J = 6.2 Hz, 3H), 1.60 (p, J = 5.6 Hz, 4H), 1.46 (q, J =
6.2 Hz, 2H). 13C NMR (100 MHz, Chloroform-d) δ 161.77, 159.66,
147.71, 141.55, 136.70, 134.89, 134.64, 133.10, 130.47, 127.99,
127.39, 127.37, 126.34 (d, J = 30.2 Hz), 125.63 (q, J = 5.7 Hz), 124.47
(d, J = 273.8 Hz), 123.92, 120.34, 118.93, 113.85, 112.49, 112.18,
109.06, 75.81, 58.50, 54.68(2C), 51.75, 50.28, 26.01(2C), 24.77, 24.16.
HRMS (ESI) m/z calcd. for C32H32F3N5O3S (M+H)+ 624.2256, found:
624.2286.
C
35H36F3N5O5S (M+H)+ 682.2311, found: 682.2285.
4.2.53. Tert-butyl 3-(4-(3-(5-(methoxycarbonyl)-4-(1-(2-
(trifluoromethyl) phenyl)ethoxy) thiophen-2-yl)imidazo[1,2-a]pyridin-7-
yl)-1H-pyrazol-1-yl)pyrrolidine-1-carboxylate (27f)
Following the procedure described for preparing 27a (Yields: 68%).
1H NMR (400 MHz, Chloroform-d) δ 8.14 (d, J = 7.1 Hz, 1H), 7.85 (d, J
= 8.0 Hz, 1H), 7.80 (s, 1H), 7.70 (s, 1H), 7.65 (s, 1H), 7.60 (d, J = 9.0 Hz,
1H), 7.57 (s, 1H), 7.51 (t, J = 7.7 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 6.92
(d, J = 7.2 Hz, 1H), 6.77 (s, 1H), 5.77 (q, J = 6.3 Hz, 1H), 4.85 (q, J =
6.1 Hz, 1H), 3.84 (s, 3H), 3.82–3.64 (m, 2H), 3.64–3.44 (m, 2H), 2.35 (q,
J = 7.4, 6.9 Hz, 2H), 1.69 (d, J = 6.3 Hz, 3H), 1.41 (s, 9H). 13C NMR
(101 MHz, Chloroform-d) δ 160.74, 158.63, 153.38 (d, J = 4.7 Hz),
146.55, 140.52, 136.23, 133.88, 133.50, 132.09, 129.06, 129.02 (d, J =
1.4 Hz), 126.99, 126.38, 125.33 (d, J = 30.2 Hz), 124.63 (q, J = 4.9, 4.5
Hz), 124.17, 123.46 (d, J = 273.7 Hz), 122.97, 119.78, 118.00, 112.97,
111.41, 108.22, 78.96(2C), 74.83, 50.74(2C), 28.67, 27.46(3C), 23.76
(2C). HRMS(ESI) m/z calcd. for C34H34F3N5O5S (M+H)+ 682.2311,
found: 682.2285.
4.2.50. Methyl-5-(7-(1-(2-morpholinoethyl)-1H-pyrazol-4-yl)imidazo
[1,2-a]pyridin-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-
carboxylate (27c)
Following the procedure described for 27a (Yields: 52%). 1H NMR
(400 MHz, Chloroform-d) δ 8.23 (dd, J = 7.2, 0.9 Hz, 1H), 7.93 (d, J =
7.9 Hz, 1H), 7.85 (s, 2H), 7.73 (s, 1H), 7.70 (dd, J = 1.8, 0.9 Hz, 1H),
7.67 (dd, J = 7.9, 1.2 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.40 (t, J = 7.6
Hz, 1H), 7.02 (dd, J = 7.2, 1.8 Hz, 1H), 6.86 (s, 1H), 5.85 (q, J = 6.3 Hz,
1H), 4.30 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 3.71 (t, 4H), 2.87 (t, J = 6.5
Hz, 2H), 2.55–2.44 (m, 3H), 1.77 (d, J = 6.2 Hz, 3H). 13C NMR (100
MHz, Chloroform-d) δ 161.76, 159.65, 147.68, 141.54 (d, J = 1.8 Hz),
136.88, 134.98, 134.61, 133.12, 130.27, 128.00, 127.39, 127.22,
126.33 (d, J = 30.4 Hz), 125.64 (q, J = 5.7 Hz), 124.48 (d, J = 273.7
Hz), 123.96, 120.51, 118.98, 113.86, 112.43, 112.31, 109.10, 75.81,
66.9(2C), 58.1, 53.6(2C), 51.7, 49.9, 24.8. HRMS (ESI) m/z calcd. for
4.2.54. Tert-butyl 4-(4-(3-(5-(methoxycarbonyl)-4-(1-(2-
(trifluoromethyl) phenyl) ethoxy) thiophen-2-yl)imidazo[1,2-a]pyridin-7-
yl)-1H-pyrazol-1-yl)piperidine-1-carboxylate (27g)
Following the procedure described for preparing 27a (Yields:
73%).1H NMR (400 MHz, Chloroform-d) δ 8.12 (d, J = 7.2 Hz, 1H), 7.84
(d, J = 7.9 Hz, 1H), 7.77 (s, 1H), 7.70 (s, 1H), 7.64 (s, 1H), 7.60 (s, 1H),
7.57 (d, J = 8.0 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.30 (t, J = 7.7 Hz, 1H),
6.92 (dd, J = 7.2, 1.7 Hz, 1H), 6.76 (s, 1H), 5.76 (q, J = 6.4 Hz, 1H),
4.33–4.11 (m, 3H), 3.83 (s, 3H), 2.85 (t, J = 12.7 Hz, 2H), 2.10 (dd, 2H),
1.90 (qd, J = 12.3, 4.3 Hz, 2H), 1.68 (d, J = 6.2 Hz, 3H), 1.40 (s, 9H). 13
C
C
31H30F3N5O4S (M+H)+ 626.2049, found: 626.2017.
NMR (100 MHz, Chloroform-d) δ 161.70, 159.60, 154.50, 147.65,
141.54 (d, J = 1.6 Hz), 136.67, 134.96, 134.58, 133.08, 130.20, 127.98,
127.38, 126.31 (d, J = 30.4 Hz), 125.85, 125.61 (q, J = 5.8 Hz), 124.46
(d, J = 273.7 Hz), 124.33, 123.93, 120.79, 118.95, 113.83, 112.37,
112.28, 109.10, 79.94, 75.79 (d, J = 2.2 Hz), 59.71, 51.71(2C), 32.37,
28.40(3C), 24.75(2C). HRMS (ESI) m/z calcd. for C35H36F3N5O5S
(M+H)+ 696.2468, found: 696.2471.
4.2.51. Methyl-5-(7-(1-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrazol-4-
yl)imidazo[1,2-a] pyridin-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)
thiophene-2-carboxylate (27d)
Following the procedure described for 27a (Yields: 42%). 1H NMR
(400 MHz, Chloroform-d) δ 8.22 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 7.9 Hz,
1H), 7.85 (s, 2H), 7.71 (d, J = 6.7 Hz, 2H), 7.67 (d, J = 8.0 Hz, 1H), 7.60
(t, J = 7.8 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.86
(s, 1H), 5.85 (q, J = 6.4 Hz, 1H), 4.26 (t, J = 6.8 Hz, 2H), 3.93 (s, 3H),
2.81 (s, 4H), 2.71 (s, 4H), 2.50 (s, 3H), 2.45 (t, J = 7.0 Hz, 2H), 2.12 (p, J
= 6.9 Hz, 2H), 1.77 (d, J = 6.2 Hz, 3H). 13C NMR (100 MHz, Chloroform-
d) δ 161.75, 159.62, 147.66, 141.52, 136.96, 134.85, 134.54, 133.11,
130.33, 128.02, 127.34 (d, J = 7.7 Hz), 126.31 (d, J = 30.4 Hz), 125.85,
125.64 (q, J = 5.6 Hz), 124.47 (d, J = 273.8 Hz), 123.97, 120.28,
118.98, 113.91, 112.45, 112.20, 109.16, 75.81, 54.44(2C), 51.77, 51.53
(2C), 50.17, 46.26, 45.04, 27.07, 24.78 . HRMS (ESI) m/z calcd. for
4.2.55. 5-(7-(1-(2-(pyridine ꢀ 1-yl) ethyl)-1H-pyrazol-4-yl) imidazo[1,2-
a]pyridine-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-
carboxamide (28a)
A mixed solution of 27a (150 mg, 0.25 mmol) in 7 M ammonia-
methanol solution (3 mL) and ammonia-water (1.5 mL) was stirred for
3 days at 78 ◦C. When the reaction mixture was clear, it was concen-
trated under vacuum. The residue was purified by flash chromatog-
raphy, eluted with a gradient of 2%-5% MeOH in DCM to give 28a(72
mg, 48%)as a light yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ 8.58 (s,
1H), 8.46 (d, J = 7.2 Hz, 1H), 8.30 (s, 1H), 7.97 (s, 1H), 7.93 (d, J = 8.2
Hz, 1H), 7.82 (s, 1H), 7.79 (d, J = 8.2 Hz, 3H), 7.56 (t, J = 7.7 Hz, 1H),
7.36 (d, J = 7.2 Hz, 1H), 7.18 (s, 1H), 7.11 (s, 1H), 6.02 (q, J = 6.6 Hz,
C
33H35F3N6O3S (M+H)+ 653.2522, found: 653.2489.
12

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
1H), 4.55 (t, J = 6.4 Hz, 2H), 3.67 (s, 2H), 3.47–3.11 (m, 4H), 1.93 (s,
4H), 1.78 (d, J = 6.2 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 161.89,
153.64, 146.95, 140.49, 137.69, 134.31, 133.57, 131.82, 129.82,
129.18, 128.72, 127.24, 125.87 (d, J = 5.6 Hz), 125.57 (d, J = 12.2 Hz),
124.54, 124.06 (d, J = 231.9 Hz), 120.11, 118.52, 115.94, 113.76,
112.09, 111.08, 75.64, 53.65 (2C), 53.28, 47.84, 23.91, 22.56 (2C).
HRMS (ESI) m/z calcd. for C30H29F3N6O2S (M+H)+ 595.2103, found:
595.2140.
6.80 (s, 1H), 6.52 (d, J = 23.5 Hz, 1H), 5.92 (q, J = 6.0 Hz, 1H), 4.41 (d,
J = 7.6 Hz, 2H), 4.10 (t, J = 8.5 Hz, 2H), 3.80 (dd, J = 8.8, 5.1 Hz, 2H),
3.15 (dtd, J = 12.7, 7.6, 2.5 Hz, 1H), 1.83 (d, J = 6.2 Hz, 3H), 1.47 (s,
9H).13C NMR (126 MHz, Chloroform-d) δ 163.56, 156.33, 154.69,
147.55, 140.53, 137.47, 134.74, 133.86, 133.34, 129.89, 128.53,
126.84, 126.52(d, J = 30.4 Hz), 126.50, 126.18(q, J = 5.8 Hz), 124.40
(d, J = 273.8 Hz), 123.98, 120.76, 119.18, 115.47, 113.17, 112.46,
112.38, 79.76, 76.34, 55.27, 52.36(b, 2C), 29.17, 28.39(3C), 24.96.
HRMS (ESI) m/z calcd. for C33H33F3N6O4S (M+H)+ 667.2314, found
667.2335.
4.2.56. 5-(7-(1-(2-(piperidin-1-yl)ethyl)-1H-pyrazol-4-yl)imidazo[1,2-a]
pyridin-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-
carboxamide (28b)
4.2.60. Tert-butyl 3-(4-(3-(5-carbamoyl-4-(1-(2-(trifluoromethyl) phenyl)
ethoxy) thiophen-2-yl) imidazo[1,2-a] pyridin-7-yl)-1H-pyrazol-1-yl)
pyrrolidine-1-carboxylate (28f)
Following the procedure described for preparing 28a (Yields: 70%).
1H NMR (400 MHz, Chloroform-d) δ 8.24 (d, J = 7.2 Hz, 1H), 7.90 (s,
1H), 7.84 (s, 1H), 7.73 (s, 1H), 7.71 (s, 1H), 7.70 (s, 2H), 7.62 (t, J = 7.8
Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 7.7 Hz, 1H), 7.00 (d, J =
7.4 Hz, 1H), 6.96 (s, 1H), 6.77 (s, 1H), 5.89 (q, J = 6.4 Hz, 1H), 4.31 (t, J
= 6.7 Hz, 2H), 2.84 (t, J = 6.7 Hz, 2H), 2.48 (t, J = 5.4 Hz, 4H), 1.80 (d,
Following the procedure described for preparing 28a (Yields: 68%).
1H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 7.0 Hz, 1H), 7.80 (s,
1H), 7.70 (s, 1H), 7.65 (d, J = 4.0 Hz, 2H), 7.63 (s, 2H), 7.54 (t, J = 7.7
Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 9.1 Hz, 1H), 6.90 (d, J =
7.2 Hz, 1H), 6.69 (s, 1H), 6.47 (s, 1H), 5.81 (q, J = 6.4 Hz, 1H), 4.86 (p,
J = 5.9 Hz, 1H), 3.91–3.68 (m, 2H), 3.64–3.36 (m, 2H), 2.47–2.28 (m, J
= 8.5, 7.1 Hz, 2H), 1.72 (d, J = 6.1 Hz, 3H), 1.41 (s, 9H). 13C NMR (100
MHz, Chloroform-d) δ 162.52, 153.64, 153.34, 146.51, 139.49, 136.21,
133.71, 132.82, 132.30, 128.89, 127.49, 125.48 (d, J = 30.5 Hz),
125.46, 125.13 (q, J = 5.9 Hz), 124.17, 123.35 (d, J = 273.6 Hz),
122.94, 119.78, 118.15, 114.47, 112.13, 111.44, 111.34, 78.92(2C),
76.24, 75.29(2C), 28.67(3C), 27.46(2C). HRMS (ESI) m/z calcd. for
J = 6.2 Hz, 3H), 1.61 (p, J = 5.6 Hz, 4H), 1.46 (q, J = 5.9 Hz, 2H). 13
C
NMR (100 MHz, Chloroform-d) δ 163.75, 154.66, 147.64, 140.52,
136.70, 134.69, 133.86, 133.32, 130.31, 128.48, 127.40, 126.91,
126.49, 126.12 (d, J = 5.8 Hz), 124.36 (d, J = 273.9 Hz), 123.86,
120.37, 119.07, 115.34, 113.00, 112.40, 112.17, 76.26, 58.46, 54.66
(2C), 50.19, 25.94(2C), 24.93, 24.10. HRMS (ESI) m/z calcd. for
C
31H31F3N6O2S (M+H)+ 609.2260, found: 609.2273.
4.2.57. 5-(7-(1-(2-morpholinoethyl)-1H-pyrazol-4-yl)imidazo[1,2-a]
pyridine-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-
carboxamide (28c)
C
33H33F3N6O4S (M+H)+ 667.2314, found 667.2335.
4.2.61. Tert-butyl 4-(4-(3-(5-carbamoyl-4-(1-(2-(trifluoromethyl) phenyl)
ethoxy) thiophen-2-yl)imidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-yl)
piperidine-1-carboxylate (28g)
Following the procedure described for preparing 28a (Yields: 39%).
1H NMR (400 MHz, Chloroform-d) δ 8.26 (d, J = 7.1 Hz, 1H), 7.85 (s,
2H), 7.76–7.68 (m, 4H), 7.62 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H),
7.27 (d, J = 7.7 Hz, 1H), 7.00 (dd, J = 7.1, 1.9 Hz, 1H), 6.77 (s, 1H), 6.63
(s, 1H), 5.89 (q, J = 6.3 Hz, 1H), 4.31 (t, J = 6.4 Hz, 2H), 3.72 (t, 4H),
2.87 (t, J = 6.4 Hz, 2H), 2.52 (t, 4H), 1.80 (d, J = 6.1 Hz, 3H).13C NMR
(100 MHz, Chloroform-d) δ 163.62, 154.69, 147.59, 140.50, 136.87,
134.66, 133.87, 133.33, 130.21, 128.51, 127.25, 126.48 (d, J = 30.5
Hz), 126.47, 126.15 (q, J = 5.7 Hz), 124.37 (d, J = 273.7 Hz), 123.93,
120.51, 119.12, 115.36, 113.08, 112.39, 112.25, 76.30, 66.93(2C),
58.13, 53.67(2C), 49.95, 29.69. HRMS (ESI) m/z calcd. for
Following the procedure described for preparing 28a (Yields: 52%).
1H NMR (400 MHz, Chloroform-d) δ 8.18 (d, J = 7.2 Hz, 1H), 7.79 (s,
1H), 7.70 (s, 1H), 7.65 (d, J = 7.7 Hz, 2H), 7.62 (s, 2H), 7.54 (t, J = 7.7
Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.17 (s, 1H), 6.92 (d, J = 7.2 Hz, 1H),
6.69 (s, 1H), 6.07 (s, 1H), 5.82 (q, J = 6.4 Hz, 1H), 4.32–4.14 (m, 3H),
2.83 (t, 2H), 2.14–2.08 (m, 2H), 1.92 (td, J = 12.4, 11.9, 4.0 Hz, 2H),
1.73 (d, J = 6.2 Hz, 3H), 1.42 (s, 9H). 13C NMR (100 MHz, Chloroform-
d) δ 162.33, 153.64, 153.52, 146.58, 139.49, 135.68, 133.68, 132.92,
132.29, 129.16, 127.50, 125.50 (d, J = 30.4 Hz), 125.44, 125.15 (d, J =
5.9 Hz), 123.35 (d, J = 273.6 Hz), 123.30, 122.92, 119.35, 118.11,
114.40, 112.11, 111.36, 111.29, 78.99, 75.32, 58.73(2C), 31.39, 28.68,
27.41(3C), 23.92(2C). HRMS (ESI) m/z calcd. for C34H35F3N6O4S
(M+H)+ 680.2393, found: 680.2398.
C
30H29F3N6O3S (M+H)+ 611.2052, found: 611.2036.
4.2.58. 5-(7-(1-(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrazol-4-yl)
imidazo[1,2-a] pyridin-3-yl)-3-(1-(2-(trifluoromethyl)phenyl)ethoxy)
thiophene-2-carboxamide (28d)
Following the procedure described for preparing 28a (Yields:
24%).1H NMR (400 MHz, Chloroform-d) δ 8.23 (d, J = 7.2 Hz, 1H), 7.83
(d, J = 12.3 Hz, 2H), 7.71 (dd, J = 12.0, 5.7 Hz, 4H), 7.62 (t, J = 7.8 Hz,
1H), 7.45 (t, J = 7.7 Hz, 1H), 7.28 (d, J = 11.4 Hz, 1H), 7.00 (d, J = 7.2
Hz, 1H), 6.76 (s, 2H), 5.89 (q, J = 6.4 Hz, 1H), 4.25 (t, J = 7.0 Hz, 2H),
2.61 (d, J = 19.4 Hz, 8H), 2.40 (s, 5H), 2.10 (t, J = 6.9 Hz, 2H), 1.80 (d,
J = 6.2 Hz, 3H). 13C NMR (100 MHz, Chloroform-d) δ 163.62, 154.68,
147.60, 140.49, 136.89, 134.63, 133.83, 133.32, 130.27, 128.50,
127.25, 126.48, 126.44 (d, J = 30.5 Hz), 126.13 (q, J = 5.5 Hz), 124.36
(d, J = 273.8 Hz), 124.27, 120.20, 119.06, 115.33, 113.04, 112.37,
112.13, 76.3, 54.80(2C), 54.57, 52.34(2C), 50.27, 45.56, 27.17, 24.93.
HRMS (ESI) m/z calcd. for C32H34F3N7O2S (M+H)+ 638.2525, found:
638.2531.
4.3. Detection of the cell proliferation
The human gastric cancer cell line MGC-803, colorectal cancer cell
line HCT-116, liver carcinoma cell line Hep-3B and Bel-7402 obtained
from Cell Bank of China Science Academy (Shanghai, China) were
cultured in recommended growth medium with 10% FBS, Penicillin
(100 U/ml) and streptomycin (100 U/ml) at 37 ◦C in a 5% CO2 atmo-
sphere. The three cell lines were exposed to various concentrations of
compounds and further incubated for 72 h at 37 ◦C. Cell proliferation
was then determined by MTT assay.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4.2.59. Tert-butyl 3-((4-(3-(5-carbamoyl-4-(1-(2-(trifluoromethyl)
phenyl) ethoxy) thiophen-2-yl) imidazo[1,2-a] pyridine-7-yl)-1H-pyrazol-
1-yl)methyl)azetidine-1-carboxylate (28e)
Following the procedure described for preparing 28a (Yields: 27%).
1H NMR (501 MHz, Chloroform-d) δ 8.27 (d, J = 7.2 Hz, 1H), 7.88 (s,
1H), 7.78 (s, 1H), 7.77–7.73 (m, 2H), 7.72 (s, 2H), 7.64 (t, J = 7.6 Hz,
1H), 7.47 (t, J = 7.6 Hz, 1H), 7.28 (s, 1H), 7.00 (dd, J = 7.2, 1.6 Hz, 1H),
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (21572067); the Chinese medicine innovation project of
13

H. Wang et al.
Bioorganic & Medicinal Chemistry 28 (2020) 115775
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Shanghai Health Committee (ZYKC201601023); and the State Key
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115775.
16 Neal CP, Fry AM, Moreman C, et al. Overexpression of the Nek2 kinase in colorectal
cancer correlates with beta-catenin relocalization and shortened cancer-specific
survival. J Surg Oncol. 2014;110:828–838.
17 Takahashi Y, Iwaya T, Sawada G, et al. Up-regulation of NEK2 by MicroRNA-128
methylation is associated with poor prognosis in colorectal cancer. Ann Surg Oncol.
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