Discovery of 5-(3-Chlorophenylamino)benzo[ c][2,6]naphthyridine Derivatives as Highly Selective CK2 Inhibitors with Potent Cancer Cell Stemness Inhibition

Article abstract of DOI:10.1021/acs.jmedchem.1c00131

Multifunctional entities have recently been attractive for the development of anticancer chemotherapeutic drugs. However, such entities with concurrent CK2 along with cancer stem cell (CSC) inhibitory activities are rare in a single small molecule. Herein, a series of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine derivatives were synthesized using a known CK2 inhibitor, silmitasertib (CX-4945), as the lead compound. Among the resulting compounds, 1c exhibited stronger CK2 inhibitory activity with higher Clk2/CK2 selectivity than CX-4945. Significantly, 1c could modulate the Akt1(ser129)-GSK-3β(ser9)-Wnt/β-catenin signaling pathway and inhibit the expression of the stemness marker ALDH1A1, CSC surface antigens, and stem genes, showing potent CSC inhibitory activity. Moreover, 1c also displayed superior pharmacokinetics and antitumor activity compared with CX-4945 sodium salt, without obvious toxicity. The favorable antiproliferative and antitumor activity of 1c, its high inhibitory selectivity for CK2, and its potent inhibition of cancer cell stemness make this molecule a candidate for the treatment of cancer.

Full text of DOI:10.1021/acs.jmedchem.1c00131

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Article
Discovery of 5‑(3-Chlorophenylamino)benzo[c][2,6]naphthyridine
Derivatives as Highly Selective CK2 Inhibitors with Potent Cancer
Cell Stemness Inhibition
Yuanjiang Wang, Zhaodan Lv, Feihong Chen, Xing Wang, and Shaohua Gou*
Cite This: J. Med. Chem. 2021, 64, 5082−5098
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ABSTRACT: Multifunctional entities have recently been attractive for the development of anticancer chemotherapeutic drugs.
However, such entities with concurrent CK2 along with cancer stem cell (CSC) inhibitory activities are rare in a single small
molecule. Herein, a series of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine derivatives were synthesized using a known CK2
inhibitor, silmitasertib (CX-4945), as the lead compound. Among the resulting compounds, 1c exhibited stronger CK2 inhibitory
activity with higher Clk2/CK2 selectivity than CX-4945. Significantly, 1c could modulate the Akt1(ser129)-GSK-3β(ser9)-Wnt/β-
catenin signaling pathway and inhibit the expression of the stemness marker ALDH1A1, CSC surface antigens, and stem genes,
showing potent CSC inhibitory activity. Moreover, 1c also displayed superior pharmacokinetics and antitumor activity compared
with CX-4945 sodium salt, without obvious toxicity. The favorable antiproliferative and antitumor activity of 1c, its high inhibitory
selectivity for CK2, and its potent inhibition of cancer cell stemness make this molecule a candidate for the treatment of cancer.
probably affect multiple targets rather than a single target,
leading to an outcome that likely favors cancer treatment.9
Consisting of two kinds of subunits, catalytic α/α′ along
with modulatory β ones, CK2 has large amino acid residues,
which make the ATP binding site of CK2 relatively small.10
Therefore, the ATP competitive inhibitors of CK2 must satisfy
two requirements: high specificity and relatively small
molecular weight. Researchers have developed a number of
CK2 inhibitors including DRB, TBB, Emodin, and CX-5011
with sub-micromolar Ki values in the past 40 years (Figure
1).11−14 However, due to the limited selectivity and the weak
specificity and potency, apart from the possibly potential long-
term toxicity, none of them did fulfill the requirement for
successful clinical settings except silmitasertib (CX-4945,
INTRODUCTION
■
Casein kinase 2 (CK2), a pleiotropic, acidophilic, and highly
conserved serine/threonine protein kinase, plays a crucial role
in various essential and pathological biological processes.1,2
Unlike most other kinases, CK2 is constitutively active and
more than 300 substrates are phosphorylated by CK2, making
it possibly one of the most pleiotropic proteins in eukaryotic
systems.3,4 It has been confirmed that CK2 is involved in
circadian rhythms, gene expression, cell cycle progression,
apoptosis, cell growth and differentiation, embryogenesis, and
many other cellular processes in a variety of tumors.5,6 As
almost all cancers can be associated with CK2-dependent
phosphorylation and tumors rely on CK2 for their survival,
cancer cells are more sensitive to CK2 inhibition.7,8
Significantly, CK2 is constitutively active, but tumor trans-
formation caused by gain-of-function CK2 mutations has not
been reported so far. Thus, the widely accepted view is that the
increasing levels of CK2 provide a permissive cellular
environment that favors malignant transformation through a
mechanism referred to as “non-oncogene dependent”. Differ-
ent from classical protein kinase inhibitors, CK2 inhibitors will
Received: January 23, 2021
Published: April 9, 2021
https://doi.org/10.1021/acs.jmedchem.1c00131
© 2021 American Chemical Society
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Figure 1. Structures representing known ATP-competitive CK2 inhibitors.
Figure 1). To our knowledge, so far, only CX-4945 as a small
molecule inhibitor of CK2 has entered Phase I/II clinical
trials.6 CX-4945 has been found to exhibit a strong potency
against CK2 with an IC50 value of 1 nM; however,
unfortunately, it also represses 12 other kinases in nanomolar
affinity. Particularly, CX-4945 has strong inhibition against
Clk2, which is even stronger than its inhibition against
CK2.15−17 The potent inhibition of CX-4945 on Clk2 most
likely causes the altered serine/arginine-rich (SR) protein
phosphorylation in cells.18 Although the success of drug
development does not always require absolute specificity, cell
permeable inhibitors that are highly selective are invaluable
tools for studying the role of phosphorylation networks of
proteins in the modulation of cellular processes.19,20
donor to form two essential hydrogen bonds with the amino
acid residues near the ATP-binding site in CK2 protein, which
accounts for the potent activity of CX-4945.29 Since amide
groups can also act as hydrogen bond donors and/or receptors
like carboxylic acid, they were selected to modify the skeleton
of CX-4945. In addition, ester groups were introduced to the
framework of CX-4945 as well. Previous research indicated
that CK2 has large amino acid residues that make the ATP
binding site of CK2 relatively small.10 Upon considering the
pros and cons of various strategies, some aliphatic amines/
alcohols with different side chains were selected to get CX-
4945 derivatives (Figure 2). Herein, we report the synthesis
CK2 has more than 300 phosphorylated substrates that are
different signal transduction cascades including Notch, JAK/
STAT, PI3K/Akt, Wnt, NF-κB and Hedgehog (Hh).2 Among
them, Wnt/β-catenin, Notch, and Hedgehog/Gli1 are also the
main signaling transduction pathways in stem cell biology,
providing cancer stem cells (CSCs) with unlimited prolifer-
ative capability.21 CSCs, a small subpopulation of tumor cells,
can differentiate into different phenotypes with the ability of
continuous self-renewal and differentiation and are a highly
tumorigenic subpopulation.22,23 Unlike most of the tumor
cells, CSCs have similar characteristics to stem cells, which are
essentially involved in tumorigenesis, tumor recurrence, cancer
progression, invasion, metastasis, and therapeutic toler-
ance.24,25 Therefore, CSCs provide a new direction for modern
cancer research and a critical index for prevention, diagnosis, as
well as treatment of cancer.26 Generally, most of the clinical
chemotherapeutic drugs like cisplatin, gemcitabine, and
hydroxycamptothecin acting only on common cancer cells
have no effect on CSCs, and some even promote the growth of
CSCs.27,28 As a consequence, research and development of
targeting drugs to suppress CSCs have been a great challenge
and formidable task in cancer research and clinical application.
As stated above, we believe that it is worthwhile to make a
reasonable structural modification on the framework of CX-
4945 to obtain novel compounds with increasing anticancer
activity and CK2 selectivity and suitable physical and chemical
properties as novel CK2 inhibitors. It has been reported that
the carboxylic group in CX-4945 can act as an acceptor or
Figure 2. Structure modification strategy for the target compounds.
and biological evaluation of these compounds together with
their CK2 inhibition activities and cytotoxicity against a panel
of cancer cells. Among the resulting compounds, a potent
bifunctional inhibitor (1c) was obtained. The inhibition
activity of 1c toward CK2 and CSCs as well as its in vivo
tumor growth inhibition on an HCT-116 xenograft model was
investigated.
RESULTS AND DISCUSSION
■
Synthesis of Target Compounds. Compounds BTE and
CX-4945 were synthesized following the previously docu-
mented procedures (Scheme S1).29 Target compounds were
prepared according to the ways shown in Scheme 1. Treatment
of BTE with hydroxylamine hydrochloride in refluxing
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Scheme 1. Synthesis of Target Compounds
methanol afforded 1a that precipitated from the reaction
mixture upon cooling. Target compounds 1b−1o and 2a−2g
were obtained through the condensation reaction of CX-4945
with different aliphatic amines/alcohols. Compounds 1p and
1q were obtained by hydrolysis of 1i and 1o, respectively, in
the presence of lithium hydroxide.
hydrogen bonding donors and/or acceptors maintained or
even enhanced their inhibitory effects on CK2 and decreased
their inhibitory activity against ClK2. For instance, compounds
1c, 1d, 1i ,and 1l have lower IC50 values than CX-4945 against
CK2, and the inhibitory effect on Clk2 was also lower than
CX-4945. 1l containing a morpholinoethyl group showed the
most potent CK2 inhibitory activity (IC50 = 0.46 nM).
Significantly, its SF value for Clk2/CK2 reached 47.61, which
is 136 times over that of CX-4945. The inhibitory activity of
1c containing a 2-hydroxyethyl side chain against CK2 was
0.66 nM with a high selectivity for Clk2/CK2 (142 times
higher than that of CX-4945). However, compounds 1g, 1j,
and 1k displayed weaker CK2 inhibitory effects with IC50
values over 10 nM. Although these amide compounds contain
a tertiary amine nitrogen atom that can serve as a hydrogen
bond receptor, they are less capable of forming hydrogen
bonds than those containing an oxygen atom. Introduction of
−(CH2)2− chains with potential hydrogen bonding donors
and/or acceptors like compounds 1m, 1n, 1o, and 1q seemed
not to be beneficial to increase their CK2 inhibitory activity in
contrast to CX-4945. As for the ester compounds with a
−(CH2)2− chain containing hydrogen bonding acceptors like
2a, 2c, and 2e and containing a 2-iodoethyl side chain (2b),
they also exhibited stronger CK2 inhibitory activity than CX-
4945. Especially, the IC50 value of 2c was 6.67 times less than
Physicochemical Properties and Analysis of CK2 and
ClK2 Inhibitory Activity. As potential CK2 inhibitors, the
inhibitory effects of all the target compounds on CK2 were first
detected by a CK2 Kinase (Human) Assay/Inhibitor Screening
Assay Kit with CX-4945 as the positive control. Lipophilic
ligand efficiency (LLE) is considered to be an important
indicator for predicting absorption, distribution, metabolism,
and excretion properties.30 To find better CK2 inhibitors as
potential anticancer agents, we attempted to modulate the LLE
values of target compounds while maintaining their CK2
inhibitory effect. Since Clk2 is one of the main off-targets of
CX-4945, the Clk2 Kinase Assay/Inhibitor Screening Assay
Kit was also used to detect those compounds with IC50 < 2 nM
against CK2. The results are summarized in Table 1. CX-4945
exhibited a potent inhibitory effect on CK2 but with a small
selection factor (SF) for Clk2/CK2. As expected, the
introduction of amide with different functional chains could
remarkably adjust the CK2 activity of the resulting compounds.
Particularly, those containing −(CH2)2− chains with potential
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that of CX-4945. However, this kind of compounds did not
effectively improve their selectivity for CK2 compared with the
corresponding amide compounds. Based on the above
structure−activity relationship analysis, we concluded that
the amide compounds with a −(CH2)2− side chain containing
hydrogen bond acceptors and/or donors would be helpful to
promote the inhibitory activity and selectivity of the
compound against CK2.
Kinase Selectivity Profiling. To demonstrate the
inhibitor’s kinase selectivity, 1c was profiled for suppression
against a panel of 208 human kinases using the ADP-Glo and
HTRF kinase assay. As illustrated in Figure 3 and Table S1, 1c
exhibits the expected high selectivity for CK2α within the
human kinome. The activity of CK2α can be completely
inhibited by 1c at 1 μM. It is observed that the inhibition rate
of 1c on Clk2, ZAP70, EGFR(ErbB1), PLK1, and EPHA1
kinases is more than 50% but less than 78%. Among them, the
highest inhibition rate of Clk2 is only about 80% (Figure 3c).
Overall, compound 1c exhibits strong and selective inhibition
against CK2α.
Investigations on the Antiproliferative Activity In
Vitro. The antiproliferative influences of all the target
compounds against five CK2-overexpressed cancer cell lines
(prostate cancer, PC-3; colon cancer, HCT-116 and HT-29;
breast cancer, MCF-7; and bladder cancer, T24) together with
normal embryonic liver cells (LO2) were assessed by an MTT
assay in which CX-4945 was employed as a positive control.
The IC50 values derived from the dose−response curves are
indicated in Table 2. It was observed that the cell proliferation
inhibitory activities of the target compounds were related to
their effects on enzyme inhibition. Compounds 1c, 1d, 1i, and
1l with strong inhibitory potency against CK2 displayed potent
antiproliferative influence against the tested cancer cells. For
Table 1. Physicochemical Properties and CK2 and ClK2
Inhibitory Activities of the Target Compounds
IC50 SD (nM)
a
b
compound clogP LLE
CK2
ClK2
SF
c
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
2a
2b
2c
2d
2e
4.09
4.32
4.13
4.21
5.42
5.27
5.10
5.11
5.03
5.74
5.31
5.09
4.45
4.53
5.36
4.91
4.55
4.53
6.54
5.67
6.02
5.99
6.50
4.42
5.44
4.15
4.04
5.05
4.84
3.27
3.10
3.78
3.82
4.07
2.99
3.59
4.24
4.55
4.19
3.23
3.83
4.23
4.68
2.58
3.97
2.92
3.23
2.24
4.45
3.37
5.66 0.38
9.33 0.23
0.66 0.03
0.89 0.07
1.78 0.09
14.28 0.26
21.30 0.08
18.14 0.05
0.79 0.01
16.82 0.02
12.6 0.15
0.46 0.01
8.81 0.04
5.32 0.16
4.53 0.15
9.84 0.08
21.65 0.05
0.61 0.02
0.76 0.16
0.23 0.04
7.29 0.11
0.60 0.08
3.43 0.56
11.34 0.34
1.54 0.07
NT
NT
32.69 0.05
19.64 0.02
24.69 0.07
NT
49.53
22.07
13.87
NT
NT
9.7 0.04
NT
12.29
47.61
NT
21.9 0.08
NT
NT
NT
NT
NT
2.38 0.05
1.69 0.08
1.06 0.06
NT
1.54 0.11
NT
3.90
2.22
4.60
2.54
2f
2g
CX-4945
NT
0.55 0.02
0.35
a
b
LLE was calculated as follows: LLE = pIC50 (M) − cLogP. SF
c
(selection factor) is defined as IC50 (ClK2)/IC50 (CK2). NT = not
tested.
Figure 3. Compound 1c demonstrates high potency as well as selectivity for CK2α in vitro. (a) Graphical illustration of the kinase inhibition profile
of 1c with 208 kinases (a list of the tested kinases is shown in Table S1). The KinMap software tool provided by Cell Signaling Technology, Inc.
(https://www.cellsignal.com) was employed to map the TREEspot image. (b) Hierarchical clustering depicting the inhibition ratio of 1c on the top
50 kinases (the higher the level, the better the inhibitory activity). (c) Kinases’ activity less than 50% in the presence of compound 1c at 1 μM.
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a
Table 2. In Vitro Cytotoxicity Profiles of the Target Compounds against Several Human Cancer Cell Lines
IC50 (μM)
cpd.
PC-3
HCT-116
MCF-7
HT-29
T24
LO2
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
2a
2b
2c
2d
2e
2f
15.09 0.15
4.16 0.09
4.53 0.43
5.48 0.31
8.76 0.11
6.12 0.56
11.08 0.25
7.60 0.18
9.94 0.24
10.07 0.57
6.08 0.39
3.28 0.10
8.94 0.12
14.10 0.25
4.89 0.10
16.18 0.36
4.20 0.25
9.15 0.69
10.01 0.37
6.40 0.18
11.00 0.27
22.09 1.25
13.53 0.31
19.18 0.15
12.75 0.21
8.62 0.25
13.25 0.58
3.07 0.67
7.26 0.49
28.11 1.42
16.54 0.92
12.76 0.78
21.21 0.96
8.13 0.92
8.79 0.36
8.60 0.20
7.98 1.40
13.68 0.30
15.92 0.85
5.91 0.24
4.60 0.10
24.77 1.57
10.91 2.55
17.73 1.15
4.28 0.27
6.91 0.18
7.52 1.60
20.83 1.20
9.71 0.19
13.21 0.52
7.66 0.11
12.16 0.45
7.50 0.15
4.88 0.65
14.86 0.72
10.43 018
6.01 0.18
13.88 0.69
4.51 0.52
8.60 0.10
3.74 0.06
11.01 0.15
7.71 0.13
19.71 0.54
19.80 0.70
24.55 0.96
19.63 0.98
15.18 0.28
10.69 0.39
5.98 0.22
10.10 0.30
9.85 0.80
14.23 0.85
13.58 0.20
15.31 0.45
9.98 0.19
17.81 0.70
5.18 0.12
11.37 0.38
8.01 0.12
13.95 0.31
5.08 0.10
16.43 0.52
3.52 0.16
7.94 0.15
13.80 0.17
13.37 0.25
11.75 0.15
9.87 0.09
20.60 0.35
19.90 0.27
14.51 0.64
10.90 0.12
17.13 0.30
7.80 0.21
9.52 0.25
8.09 0.13
18.40 0.80
12.62 0.08
16.00 0.30
3.89 0.10
23.49 0.70
6.10 0.90
14.42 0.48
8.95 0.18
18.13 0.35
5.93 0.10
18.49 0.26
10.67 0.29
3.23 0.03
3.24 0.07
8.73 0.55
18.80 0.53
37.28 0.90
12.93 0.15
24.42 0.50
21.14 0.58
16.61 0.56
17.48 0.62
5.91 0.86
22.53 0.50
22.73 0.39
20.62 0.46
14.08 0.38
12.92 0.25
21.42 0.69
28.43 1.59
96.68 1.28
111.13 2.46
66.73 3.53
18.37 0.95
17.26 0.67
28.41 2.01
>200
16.04 0.93
14.31 0.70
37.81 1.74
74.16 2.40
84.97 1.23
89.12 6.26
72.84 4.53
32.24 0.82
45.13 3.20
43.46 1.60
57.76 3.16
24.92 1.26
83.91 1.18
21.98 1.05
30.70 1.39
22.98 2.52
2g
CX-4945
a
IC50 values were given as the means SD of three independent experiments.
Figure 4. Western blot analysis on the specific CK2 phosphorylation targets: (a) phosphorylation of the Akt1 serine 129 and (b) Cdc37 serine 13
in HCT-116 cells inoculated with varying levels of 1c (5, 10, and 20 μM) for 24 h.
example, 1c exhibited potent antiproliferative activities against
five different CK2-overexpressed cancer cells, especially PC-3
cells and HCT-116 cells with IC50 values of 4.53 and 3.07 μM,
respectively. 1i showed considerable potency against MCF-7
cells (4.51 μM) and HT-29 cells (3.52 μM); more importantly,
it was less toxic to LO2 with an IC50 value higher than 200 μM.
In addition, 1m showed a more potent activity against PC-3
cells, and the IC50 value is 3.28 μM. However, compounds 1m,
1p, 1q, 2f, and 2g with lower CK2 inhibitory activities than
CX-4945, probably because of their steric hindrances or long
methylene chains, had less antiproliferation activities against
cancer cells than CX-4945. It was also worth noting that
compounds 1j and 1k with moderate CK2 inhibitory activities
also exhibited strong anticancer activity. When ester bonding
served as the linker, the resulting compounds (2a−e)
demonstrated much better cytotoxicity than CX-4945. In
particular, 2c not only displayed potent activity against CK2
but also showed strong inhibitory influence toward five
different cancer cells. However, it was a little toxic to normal
liver cells LO2. The possible reason is that functional groups
varied from carboxylic acid to amide or ester in CX-4945
might change the pharmacokinetic properties and the
antitumor and CK2 inhibitory activity of the resulting
compounds. Based on the above study, 1c with high selectivity
for CK2, potent inhibitory activity against the tested cancer
cells, low toxicity to normal liver cells, and a suitable LLE value
was chosen for further investigation.
Investigation of the Representative Compound on
Its CK2α Targeting. To confirm that 1c can act directly on
endogenous CK2α as well, the phosphorylation level of Akt1
serine 129 (p-Akt1S129) and Cdc37 serine 13 (p-Cdc37S13), the
two specific phosphorylation targets of CK2, was also
investigated by western blot in HCT-116 cells. As shown in
Figure 4a,b, compound 1c can obviously suppress the
expression of p-Akt1S129 and p-Cdc37S13 in a dose-dependent
manner, suggesting that 1c can not only inhibit exogenous
CK2α but also inhibit endogenous CK2α in HCT-116 cells.
In addition, molecular docking simulations of 1c with CK2α
and Clk2 were also performed using AutoDock Vina. CK2α
(PDB ID: 6isj) and Clk2 (PDB ID: 6khe) co-structures
complexed with CX-4945 were used as a template. As shown
in Figure 5a, compound 1c is bound tightly to the surface of
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hydroxyl group generate hydrogen bonding with ASP-127
residues, respectively, while the N atom in ring A has hydrogen
bonds with GLU-224 residues. The results illustrate that 1c
binds CK2α more tightly than Clk2.
Investigation on the Inhibition of ALDH1A1. It has
been proven that CK2α positively modulates Notch1 tran-
scriptional activity and the population of CD44+/CD24−,
which participate in stem cell maintenance.31 With the
inherent CK2 inhibitory activity, we hypothesize that 1c may
also inhibit cancer cell stemness by regulating stem-cell-related
signaling pathways. As aldehyde dehydrogenase 1-A1
(ALDH1A1), one of the strongest CSC hallmarks, plays a
core role in sustaining the stemness of HCT-116 cells,32 it was
detected to quantify the proportion of ALDH+ cells. As shown
in Figure 6a (see also Supporting Information Figure S1), the
ratio of the ALDH+ cell subpopulation was up to 63% in HCT-
116 cells, indicating that 1c could strongly inhibit the
proportion of intracellular ALDH+ cell subsets and present a
concentration-dependent inhibitory manner. The inhibitory
ratio of 1c reached 96% at 20 μM and 77% even at 5 μM,
significantly higher than that of CX-4945 (61%) at the same
concentration of 5 μM. This was further validated by the
related western blotting analysis (Figure 6a). Moreover, the
impact of 1c on ALDH1A1 expression in HCT-116 cells was
investigated. As expected, the transcriptional level of
ALDH1A1 in HCT-116 cells decreased significantly after
being treated with 1c (20 μM) for 24 h, which was only half of
that in the CX-4945-treated group (Figure 6c). In addition, we
also evaluated the inhibitory activity of 1c against the
exogenous ALDH1A1 enzyme. As shown in Figure 6d, both
CX-4945 and 1c can inhibit exogenous ALDH1A1 enzyme
activity in a dose-dependent manner, especially 1c, with an
IC50 value of 0.10 μM that is 8.8 times higher than that of CX-
4945. To further elucidate the binding mechanism between 1c
Figure 5. Molecular docking. Docking of 1c into (a) CK2α (PDB ID:
6isj) and (b) Clk2 (PDB ID: 6khe) kinase X-ray crystal structure.
Potentially relevant residues to the docking are labeled. Interactions
are illustrated by dashed lines (H bonds, yellow).
the protein with good compatibility. The amide serving as both
a hydrogen bond donor and a hydrogen bond acceptor forms
two important hydrogen bonds with TRP-175 and THR-112
residues via its O and H atom, respectively. The N atom on the
ring A of naphthyridine produces a hydrogen bond with the
VAL-115 residue. Meanwhile, the hydroxyl group on the side
chain of 2-aminoethanol also forms hydrogen bonding with
ASP-174 and GLU-80 residues by its O and H atom,
respectively. The five hydrogen bonds enable the entire 1c
skeleton to be firmly anchored deep in the CK2α active cavity.
Compared with CK2α, 1c is not inserted deep into the activity
cavity of Clk2 (Figure 5b). There are only three hydrogen
bonds between 1c and the amino acid residues in the active
cavity of Clk2. The hydrogen atoms from the amide and the
Figure 6. Investigation on the inhibition of 1c on ALDH1A1. (a) A statistical analysis of the ALDH1A1 inhibitory ratio. (b) Western blotting
assessment on expression of ALDH1A1 protein in HCT-116 cells treated with CX-4945 (5 μM) and different quantities of 1c (5, 10, and 20 μM)
for 24 h, respectively. (c) mRNA transcription levels of ALDH1A1 after exposure to CX-4945 and 1c at 20 μM, respectively, in the HCT-116 cells
for 24 h. (d) Inhibition rate of 1c and CX-4945 at different concentrations on the enzyme activity of ALDH1A1. Data were subjected to statistical
analysis by using Student’s t test and given as the mean SD (n = 3). *P < 0.05 and **P < 0.01 vs CX-4945 group or the control group.
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Figure 7. Investigation on the inhibition of cancer cell stemness. (a) A statistical analysis of the CD44+/CD133+ inhibitory ratio. (b) qRT-PCR of
mRNA transcription levels of stemness genes after exposure to BBI608, CX-4945, and 1c, respectively, at 20 μM in the HCT-116ALDH+ cells for 24
h. (c) The number and (d) the morphological observation of the HCT-116AHDH+ cell tumorspheres treated with vehicle (DMSO), BBI608 (5
μM), CX-4945 (5 μM), and 1c (5 μM) for 24 h and then cultivated for 10 days. Comparison of gene expression level was performed using the
2
−ΔΔCt approach. Data were subjected to statistical analysis by using Student’s t test and given as the mean SD (n = 3). *P < 0.05 and **P < 0.01
vs BBI608 group or the control group.
and ALDH1A1, we performed a related molecular docking
study. As shown in Figure S2, the N atom in ring A, O atom in
amide, and OH group in 2-aminoethanol form hydrogen
bonding with GLY-458, CYS-303, and ASN-170 residues,
respectively. These hydrogen bonds hold 1c firmly in the
ALDH1A1 active cavity. These results demonstrate that 1c can
target ALDH1A1 protein and inhibit its activity, and regulate
certain signaling pathways to inhibit the transcription and
protein expression of ALDH1A1.
quantitatively analyzed. As shown in Figure 7b, noticeable
differences in mRNA expression were observed for the
transcription factor. CX-4945 had the weakest inhibitory
activity, whose transcription levels of OCT4, SOX2, and
Nanog were 2.27, 2.34, and 1.57 times as many as those of 1c,
respectively. It is noteworthy that the level of SOX2
transcription in HCT-116ALDH+ cells treated with BBI608
was 1.75 times higher than the 1c group despite the
transcription levels of OCT4 and Nanog being slightly lower
than the 1c group. It has been documented that the high
expression level of SOX2 enhances the tumorigenic potential
by regulating the expressions of genes that participate in
proliferation, apoptosis, and cell cycle modulation.35 In
addition, SOX2 overexpressed in human colon CSCs promotes
chemotherapy resistance and tumor growth. The capability of
tumorsphere formation was also tested. As shown in Figure
7c,d, obvious phenotypic alterations were observed in HCT-
116ALDH+-derived tumorspheres in all the groups. HCT-
116ALDH+ cells treated with 1c exhibited fewer sphere number
and smaller size than those treated with either CX-4945 or
BBI608. This also proved that 1c has a stronger inhibitory
effect on the cancer cell stemness than BBI608.
Investigation on the Signaling Pathway of Wnt/β-
Catenin. The Wnt/β-catenin signaling pathway plays a core
role in tumor invasion, especially in tumor metastasis and
differentiation.36,37 β-Catenin binds to the TCF/LEF tran-
scription factor family in the nucleus to activate the promoter
of β-catenin target genes, thus activating the target genes of the
Wnt signaling pathway like CD44, CD24, CD133, ABCG2,
ALDH1A1, EpCAM, MMP7, Vimentin, and Slug. It has been
confirmed that Wnt1 can enhance ALDH1A1 expression,
hence enhancing the expression of Akt as well as β-catenin.38
Phosphorylated Akt inactivates GSK-3β through phosphorylat-
ing GSK-3β at ser9, repressing its capacity to degrade β-
catenin.39,40 Therefore, it is suggested that 1c might regulate
the expression of ALDH1A1 through the Akt-GSK-3β(ser9)-
Wnt/β-catenin signaling pathway, thereby affecting tumor
Investigation on the Inhibition of Cancer Cell
Stemness. Based on the inhibitory effect of 1c on
ALDH1A1, we further explored the influence of 1c on the
stemness driven by ALDH in HCT-116 cells. ALDH+
population cells in HCT-116 cells were sorted by flow
cytometer and named HCT-116ALDH+ cells. It is known that
CSC surface antigens’ expression is cancer type-distinct or
cancer subtype-distinct. Since they play a vital role in
invasiveness and promoting cancer recurrence, it is pivotal to
comprehend how CSCs are modulated and maintained at the
molecular level.22 To examine the influence of 1c on the cancer
cell stemness, the expression of CD44+ and CD133+ in HCT-
116ALDH+ cells treated with 1c was determined by flow
cytometry using CX-4945 and napabucasin (BBI608) as the
positive controls. BBI608, currently in Phase II/III clinical
trials, is a known STAT3 inhibitor that can potently block
spherogenesis, kill stem cancer cells isolated from numerous
kinds of cancer, and repress stemness gene expression.33 As
shown in Figure 7a and Figure S3, compound 1c shows a
potent inhibitory effect on CD44+/CD133+, with an inhibitory
rate of about 84%, second only to BBI608 (89%), which is
1.45 times higher than that of CX-4945 (58%). SOX2, OCT4,
and Nanog, essential transcription factors to maintain the
characteristics of stem cells, are currently recognized as the
signature genes of CSCs, which have an important role in
maintaining self-renewal along with proliferation in malignant
tumors.34 The influence of 1c on the transcription levels of the
signature genes of CSCs in HCT-116ALDH+ cells was
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Figure 8. Compound 1c regulated the GSK-3β(ser9)-Wnt/β-catenin signaling pathway. Western blotting images of (a) p-GSK-3β(ser9), GSK-3β,
(c) DKK1, and β-catenin proteins induced by different concentrations of 1c for 24 h in HCT-116 cells. (b) Decreased mRNA levels of Wnt target
genes in HCT-116 cells upon exposure to 1c (20 μM) for 24 h. (d) Transwell invasion assays. The HCT-116 cells were inoculated with 1c and CX-
4945, respectively, at 5 μM for 24 h. Data were subjected to statistical analysis by using Student’s t test and given as the mean SD (n = 3). *P <
0.05 and **P < 0.01 vs the control group.
Figure 9. Cell apoptosis and cell cycle arrest analysis. (a) Cell apoptosis in HCT-116 cells treated with CX-4945 (5 μM) and varying levels of 1c
(5, 10, and 20 μM) was assessed using flow cytometry (stained with Annexin V-FITC and PI). (b) A statistical analysis of apoptotic ratio. (c) Cell
cycle arrest in HCT-116 cells inoculated with CX-4945 (20 μM) and 1c (20 μM), respectively, for 24 h; stained with PI; and explored with flow
cytometry. (d) A statistical analysis of cell cycle arrest. The results were given as the mean SD (n = 3). **P < 0.01 vs the control group.
invasion and metastasis. As illustrated in Figure 8a, compound
1c significantly inhibited GSK-3β(ser9) protein phosphoryla-
tion in HCT-116 cells dose-dependently. In addition, the
mRNA levels of Wnt target genes consisting of c-Myc, cyclin
D1, MMP7, and S100A4 were remarkably reduced upon
treatment of 1c (Figure 8b). Meanwhile, the protein level of
DKK1 was upregulated with the increasing concentrations of
1c, while that of β-catenin was significantly downregulated
(Figure 8c). In addition, 1c also exhibited a superior capacity
to repress the invasion of HCT-116 cells (Figure 8d). The
above results indicated that 1c decreased the activity of CK2
by specifically targeting CK2 and then reduced ALDH1A1
expression and inhibited the invasion of cancer cells by
suppressing the Akt1(ser129)-GSK-3β(ser9)-Wnt/β-catenin
signaling pathway.
Investigation on the Cell Apoptosis and Cell Cycle
Arrest. Cell apoptosis was measured via flow cytometric
assays. HCT-116 cells after being treated with CX-4945 (5
μM) and varying levels of 1c (5, 10, and 20 μM) for 24 h were
analyzed. As shown in Figure 9a,b, at the same concentration
(5 μM), the ratio of apoptosis triggered by 1c was much higher
in contrast with that of CX-4945. With the increasing
concentration, the proportion of apoptosis induced by 1c
also increased gradually. At the concentration of 20 μM, the
apoptotic ratio reached about 55%, exhibiting a dose-
dependent behavior. The effect of 1c on HCT-116 cell cycle
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Figure 10. Study on the mechanism of apoptotic pathway. (a) Western blotting assessment of Cyt c, Bcl-2, Bax, pro-caspase 9, cleaved caspase 9,
pro-caspase 3, cleaved caspase 3, total PARP, cleaved PARP as well as β-actin after 24 h treatment of HCT-116 cells with CX-4945 (20 μM) and 1c
(20 μM), respectively. (b) Expression levels of (a) acquired from western blotting and standardized with β-actin. The results are given as the mean
SD (n = 3). **P < 0.01 and *P < 0.05 vs the control group.
progression was examined via flow cytometry, with CX-4945
serving as the positive control. Due to the analogous structure,
both CX-4945 and 1c arrested the cell cycle at the G0−G1
phase in HCT-116 cells as illustrated in Figure 9c,d.
Table 3. Pharmacokinetic Parameters of Compound 1c in
SD Rats
a
parameter
Tmax (h)
Cmax (ng·mL−1
t1/2 (h)
CL (L·h−1·kg−1
AUC0−t (ng·h·mL−1
AUC0−∞ (ng·h·mL−1
Vd (L·kg−1
CX-4945·Na
1c
2.00 0.00
6786.32 223.88
5.82 0.27
2.33 0.58
7017.81 297.85
6.67 1.59
Investigation on the Mechanism of Apoptotic Path-
way. The mitochondrial linked apoptotic proteins including
cytochrome c (Cyt c), Bax, Bcl-2, pro-caspase 9, cleaved
caspase 9, pro-caspase 3, cleaved caspase 3, total PARP, and
cleaved PARP were assessed in HCT-116 cells after
inoculation with 1c by western blotting, using CX-4945 as
the positive control. The results are indicated in Figure 10a,b.
In contrast with the control, 1c induced an obvious decrease in
the level of Bcl-2 and increased the Bax expression to a higher
degree than CX-4945. Moreover, treatment of 1c in HCT-116
cells released more Cyt c than that of CX-4945, resulting in the
activation of the downstream caspase cascade. The activations
of downstream caspases and PARP were observed, which were
able to trigger the apoptosis of cancer cells. As expected,
exposure of HCT-116 cells to 1c caused more starkly
downregulation in the levels of pro-caspase 9, pro-caspase 3,
and total PARP and upregulation in the levels of cleaved
caspase 9, cleaved caspase 3, and cleaved PARP relative to CX-
4945. These results indicate that 1c can induce HCT-116 cell
apoptosis through a mitochondrial-mediated signaling pathway
and caspase cascade.
Pharmacokinetic Study. The pharmacokinetic profiles of
1c and CX-4945 were evaluated in Sprague−Dawley (SD) rats
following a single 25 mg/kg dose administration by the oral
route. To be consistent with previous reports,29 CX-4945 was
transformed into its sodium salt (CX-4945·Na). As summar-
ized in Table 3, the PK profile of 1c is similar to that of CX-
4945·Na with a slight improvement. The maximal drug
concentration (Cmax) reached 7017.8 ng/mL in plasma,
elimination half-life (t1/2) was approximately 6.67 h, and the
plasma exposure AUC0−t and AUC0−∞ were above 4000 ng·h/
mL, which were a little better than those of CX-4945·Na
except for the clearance. These results reveal that 1c presents a
favorable pharmacokinetic behavior.
)
)
0.63 0.01
0.60 0.03
)
38,919.58 90.48
39,994.05 74.29
2.39 0.50
40,460.61 2353.48
41,450.07 2378.31
2.77 0.25
)
)
MRT0−t (h)
MRT0−∞ (h)
6.45 0.55
6.52 0.61
6.21 0.26
6.26 0.29
a
T
max, time for peak concentration; Cmax, peak value; t1/2, elimination
half-life period; CL, plasma clearance; AUC, area under the time
concentration curve; Vd, apparent volume of distribution; and MRT,
mean residue time.
illustrated in Figure 11a−c, the inhibitory rate (59%) of 1c at
the low dose is 3.9 times higher than that (15%) of CX-4945·
Na. In addition, it is noticed that 1c obviously inhibits the
tumor growth dose-dependently with a maximum inhibitory
rate of 69% at a dose of 90 mg/kg. There is no conspicuous
change in body weight of all the mice treated with 1c.
Furthermore, the HE staining of slices from the heart, liver,
spleen, lung, and kidney of an animal randomly selected from
each tested group in HCT-116 models demonstrates that 1c at
the used dosages has hardly toxic effects on organ tissues
(Figure S4), revealing that 1c has low systemic toxicity and
good safety (Figure 11d). These results indicate that 1c
exhibits potent antitumor activity with low toxicity in the
HCT-116 xenograft mice model.
CONCLUSIONS
■
Herein, a series of 5-(3-chlorophenylamino)benzo[c][2,6]-
naphthyridine derivatives were synthesized with CX-4945 as
the lead compound. 1c stands out for its excellent selectivity
and inhibitory activity on CK2. Biological studies indicated
that 1c exhibited stronger CK2 inhibitory activity with much
higher ClK2/CK2 selectivity than CX-4945, which greatly
reduces the off-target toxicity of CX-4945. In addition, 1c also
exhibited a potent inhibitory effect on cancer cell stemness.
Further research on the mechanism of action reveals that 1c
can inhibit the Akt1(ser129)-GSK-3β(ser9)-Wnt/β-catenin
In Vivo Antitumor Activity. The antitumor activity of 1c
in HCT-116 xenograft models was tested, in which animals
were grouped randomly into four groups as vehicle, CX-4945·
Na (60 mg/kg), and 1c at two doses (60 and 90 mg/kg) and
administrated po twice a day for 4 weeks. The volume of
tumors and body weight were assessed every 3 days. As
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Figure 11. In vivo HCT-116 xenograft model study of 1c. Twenty nude mice inoculated with HCT-116 cells in the right armpit were divided into
four groups as vehicle (equivalent volume of CMC-Na), CX-4945·Na (60 mg/kg), and 1c (60 and 90 mg/kg). Animals were orally administrated
twice a day for 4 weeks. (a) Excised tumor images from each group. (b) The relative volume of the tumors. (c) The relative tumor weight resected
from all groups of sacrificed mice on the last day. (d) The body weights of each group mice in the period of 28 days. Data are given as the mean
SD. *P < 0.05 and **P < 0.01 vs the control group.
1H), 8.34 (t, J = 1.9 Hz, 1H), 8.17 (dd, J = 6.3, 1.4 Hz, 2H), 7.90 (dd,
J = 8.4, 1.7 Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.12 (dd, J = 7.9, 1.4 Hz,
1H), 3.92 (s, 3H) ppm; CX-4945: H NMR (600 MHz, DMSO-d6)
δ12.04 (s, 1H), 10.30 (s, 1H), 10.16(s, 1H), 9.08 (d, J = 5.8 Hz, 1H),
9.00−8.97 (m, 1H), 8.94 (d, J = 8.5 Hz, 1H), 8.31−8.28 (m, 2H),
8.12 (d, J = 8.2 Hz, 1H), 8.03 (dd, J = 8.4, 1.5 Hz, 1H), 7.45 (t, J =
8.1 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H) ppm.
signaling pathway and inhibit the expression of the stemness
markers ALDH1A1, CSCs surface antigens (CD44+ and
CD133+), and stem genes (SOX2, OCT4, and Nanog),
thereby inhibiting the CSCs. Furthermore, 1c displayed
superior pharmacokinetics and antitumor activity compared
with CX-4945·Na without obvious toxicity. The development
of novel antitumor drugs and therapeutic strategies targeting
the CK2 and CSCs in tumors is a still unmet need, but it may
represent a definite advantage in cancer treatment. The
identification of 1c is a step forward in this direction. The
favorable antiproliferative and antitumor activities of 1c make
it possible for 1c to act as a multifunctional drug candidate to
suppress CK2 protein and CSCs for the treatment of cancer.
1
Synthesis of 1a. KOH (1.12 g, 20 mmol) and NH2OH·HCl (0.695
g, 10 mmol) were dissolved in 5 and 8 mL MeOH, respectively, to get
solutions A and B. Then, solution A was added dropwise to solution B
at 0 °C. The temperature of the reaction system was slowly raised to
room temperature, and the reaction was continued for 30 min. At the
end of the reaction, the precipitated solid was removed by suction
filtration to obtain an alkaline hydroxylamine solution. BTE (0.36 g, 1
mmol) was dissolved in alkaline hydroxylamine solution (5 mL),
stirred 1 h at room temperature, and monitored by TLC. After the
substrate was completely consumed, the solvent was removed under
vacuum, and the residue was acidified with 2 M HCl to pH = 5−6,
filtering the precipitate; washed with H2O (3 × 15 mL); and dried to
obtain the crude product of 1b. The precipitate was recrystallized with
MeOH (10 mL) to get a bright yellow solid (0.332 g). Yield: 91.2%.
1H NMR (600 MHz, DMSO-d6) δ 11.52 (s, 1H), 10.16 (s, 1H), 9.65
(s, 1H), 9.19 (s, 1H), 8.97 (d, J = 5.4 Hz, 1H), 8.83 (d, J = 8.4 Hz,
1H), 8.57 (d, J = 5.2 Hz, 1H), 8.33 (s, 1H), 8.15 (d, J = 1.2 Hz, 1H),
8.07 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 1.2 Hz, 1H), 7.43 (t, J = 8.0 Hz,
1H), 7.13 (dd, J = 7.8, 1.2 Hz, 1H) ppm. 13C NMR (150 MHz,
DMSO-d6) δ 164.19, 150.52, 148.13, 147.73, 143.74, 142.35, 134.16,
133.27, 130.53, 127.49, 126.08, 124.19, 122.95, 122.90, 122.64,
121.54, 120.60, 119.60, 116.81 ppm. HR-MS (m/z) (ESI): calcd for
C19H13ClN4O2 [M + H]+: 365.0805; found: 365.0800.
General Procedure for Preparation of Compounds 1b−1o. 5-((3-
Chlorophenyl)amino)-N-(methylsulfonyl)benzo[c][2,6]-
naphthyridine-8-carboxamide (1b). To a solution of CX-4945
(0.210 g, 0.6 mmol) in N,N-dimethylformamide (DMF) (5 mL) was
added 2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hex-
afluorophosphate (HATU) (0.273 g, 0.72 mmol) and stirred at room
temperature for 10 min. Then, N,N-diisopropylethylamine (DIPEA)
(0.197 mL, 1.2 mmol) was added to the solution and stirred for 10
min at the same temperature, followed by addition of methanesulfo-
namide (0.048 g, 0.5 mmol), and the solution was stirred overnight in
EXPERIMENTAL SECTION
■
Materials and Instruments. All chemical reagents along with the
solvents were commercially acquired from Energy Chemical, Acros,
Alfa, Maybridge, or Adamas-beta and utilized without further
purification. Flash column chromatography employed a silica gel
(100−200 mesh). NMR spectra were obtained in DMSO-d6, unless
otherwise stated, on Bruker 600 (1H at 600 MHz, 13C at 150 MHz)
NMR spectrometers, and tetramethylsilane (TMS) was employed as
reference. An Agilent 6224 ESI/TOF MS instrument was employed
to measure the mass spectra. Purities of the tested compounds,
determined by RP-HPLC analysis using a Waters e2965 HPLC with
ODS column (5 μm, 250 × 4.6 mm column and methanol/water),
were ≥ 95%. All cell lines utilized herein were purchased from Biorn
Lifescience Co., Ltd. (Nanjiang, Jiangsu, China). p-Akt1S129, Akt, p-
Cdc37S13, Cdc37, p-GSK-3β(ser9), GSK-3β, DKK1, β-catenin, Cyt c,
Bax, Bcl-2, pro-caspase 9, cleaved caspase 9, pro-caspase 3, cleaved
caspase 3, total PARP, cleaved PARP, and β-actin antibodies were
supplied by Beyotime Biotechnology. The HRP-conjugated secondary
antibody (1: 2000) was provided by Santa Cruz Biotechnologies.
Synthesis and Characterization of the Compounds. Com-
pound BTE and CX-4945 were synthesized following the previously
documented procedures29 and verified by 1H NMR. Compound
BTE: 1H NMR (600 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.91 (s, 1H),
8.94 (d, J = 5.6 Hz, 1H), 8.79 (d, J = 8.5 Hz, 1H), 8.77 (d, J = 5.6 Hz,
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a nitrogen atmosphere. The progress of the reaction was monitored
with TLC. After the substrate was completely consumed, the solvent
was removed under vacuum, and the residue was treated with EtOAc
(50 mL). The organic layers were washed with saturated salt water (3
× 20 mL) and dried with anhydrous Na2SO4. The organic layers were
concentrated under vacuum to obtain the crude product and further
purified with silica gel chromatography with the eluent of MeOH and
DCM (1:100−1:50) to give a yellow solid 1b (0.093 g). Yield: 43.5%.
1H NMR (600 MHz, DMSO-d6) δ 13.26 (s, 1H), 10.19 (s, 1H), 9.69
(s, 1H), 9.01 (d, J = 5.5 Hz, 1H), 8.87 (d, J = 8.4 Hz, 1H), 8.60 (d, J =
5.6 Hz, 1H), 8.33 (s, 1H), 8.28 (s, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.99
(d, J = 8.1 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.16−7.13 (d, J = 7.8 Hz,
1H), 2.51 (s, 3H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 150.71,
150.60, 148.30, 148.09, 143.76, 142.33, 133.24, 130.62, 128.72,
127.42, 124.70, 124.47, 123.13, 123.08, 122.80, 122.67, 120.57,
119.61, 116.86, 63.26 ppm. HR-MS (m/z) (ESI): calcd for
C20H15ClN4O3S [M + H]+: 427.0631; found: 427.0643.
1H), 8.98 (d, J = 5.6 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H), 8.72 (t, J = 5.4
Hz, 1H), 8.59 (d, J = 5.6 Hz, 1H), 8.28 (t, J = 1.8 Hz, 1H), 8.23 (d, J
= 1.4 Hz, 1H), 8.11 (dd, J = 8.4, 1.0 Hz, 1H), 7.94 (dd, J = 8.4, 1.6
Hz, 1H), 7.45 (t, J = 8.2 Hz, 1H), 7.15 (dd, J = 7.8, 1.4 Hz, 1H), 3.45
(dd, J = 12.8, 6.6 Hz, 2H), 2.53−2.51 (m, 2H), 2.25 (s, 6H) ppm. 13C
NMR (150 MHz, DMSO-d6) δ 166.19, 150.54, 148.13, 147.70,
143.77, 142.39, 135.94, 133.27, 130.54, 127.53, 126.29, 124.20,
123.35, 122.76, 122.66, 121.50, 120.71, 119.70, 116.83, 58.59, 45.64,
37.92 ppm. HR-MS (m/z) (ESI): calcd for C23H22ClN5O [M + H]+:
420.1591; found: 420.1595.
5-((3-Chlorophenyl)amino)-N-(prop-2-yn-1-yl)benzo[c][2,6]-
1
naphthyridine-8-carboxamide (1h). Yellow solid. Yield: 76.4%. H
NMR (600 MHz, DMSO-d6) δ 10.17 (s, 1H), 9.96 (s, 1H), 9.36 (t, J
= 5.2 Hz, 1H), 8.96 (d, J = 5.4 Hz, 1H), 8.85 (d, J = 8.4 Hz, 1H), 8.82
(d, J = 5.5 Hz, 1H), 8.40 (s, 1H), 8.28 (s, 1H), 8.23 (d, J = 8.2 Hz,
1H), 8.00 (d, J = 8.3 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.15 (d, J = 7.8
Hz, 1H), 4.18 (d, J = 3.2 Hz, 2H), 3.21 (s, 1H) ppm. 13C NMR (1500
MHz, DMSO-d6) δ 166.06, 150.67, 147.69, 143.78, 142.50, 135.11,
133.17, 130.40, 128.23, 127.42, 126.52, 124.31, 123.26, 122.83,
122.54, 121.73, 120.71, 119.74, 117.23, 81.83, 73.37, 29.12 ppm. HR-
MS (m/z) (ESI): calcd for C22H15ClN4O [M + H]+: 387.1012;
found: 387.1011.
Methyl 3-(5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxamido)propanoate (1i). Yellow solid.
Yield: 65.4%. 1H NMR (600 MHz, DMSO-d6) δ 10.18 (s, 1H),
9.66 (s, 1H), 8.99 (d, J = 5.6 Hz, 1H), 8.88 (t, J = 5.4 Hz, 1H), 8.85
(d, J = 8.4 Hz, 1H), 8.58 (d, J = 5.6 Hz, 1H), 8.28 (t, J = 1.8 Hz, 1H),
8.22 (d, J = 1.4 Hz, 1H), 8.10 (dd, J = 8.2, 1.2 Hz, 1H), 7.93 (dd, J =
8.4, 1.6 Hz, 1H), 7.45 (t, J = 8.2 Hz, 1H), 7.15 (dd, J = 7.8, 1.2 Hz,
1H), 3.65 (s, 3H), 3.58 (dd, J = 12.6, 6.8 Hz, 2H), 2.68 (t, J = 7.0 Hz,
2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 172.28, 166.39, 150.55,
148.15, 147.73, 143.76, 142.37, 135.72, 133.27, 130.55, 127.51,
126.31, 124.21, 123.33, 122.81, 122.68, 121.58, 120.71, 119.71,
116.82, 51.89, 36.13, 34.02 ppm. HR-MS (m/z) (ESI): calcd for
C23H19ClN4O3 [M + H]+: 435.1224; found: 435.1220.
5-((3-Chlorophenyl)amino)-N-(2-hydroxyethyl)benzo[c][2,6]-
1
naphthyridine-8-carboxamide (1c). Yellow solid. Yield: 72.1%. H
NMR (600 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.89 (s, 1H), 8.97 (d, J
= 5.4 Hz, 1H), 8.84 (t, J = 6.0 Hz, 2H), 8.77 (d, J = 5.4 Hz, 1H), 8.35
(s, 1H), 8.27 (s, 1H), 8.21 (d, J = 8.2 Hz, 1H), 8.00 (d, J = 8.2 Hz,
1H), 7.45 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 7.6 Hz, 1H), 4.91 (t, J = 5.6
Hz, 1H), 3.61 (q, J = 5.8 Hz, 2H), 3.43 (dd, J = 11.2, 5.4 Hz, 2H)
ppm. 13C NMR (150 MHz, DMSO-d6) δ 166.40, 150.61, 148.05,
147.64, 143.77, 142.51, 135.92, 133.19, 130.46, 127.52, 126.40,
124.25, 123.38, 122.71, 122.56, 121.46, 120.70, 119.73, 117.16, 60.20,
42.88 ppm. HR-MS (m/z) (ESI): calcd for C21H17ClN4O2 [M + H]+:
393.1118; found: 393.1120.
N-(2-Aminoethyl)-5-((3-chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxamide (1d). Yellow solid. Yield: 81.6%.
Compound 1d may also be prepared by the method reported in
the literature.41 1H NMR (600 MHz, DMSO-d6) δ 10.12 (s, 1H),
9.60 (s, 1H), 8.95 (d, J = 5.4 Hz, 1H), 8.81−8.79 (m, 1H), 8.54 (s,
1H), 8.53 (d, J = 5.6 Hz, 1H), 8.24 (t, J = 1.8 Hz, 1H), 8.20 (d, J = 1.4
Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.93 (dd, J = 8.2, 1.4 Hz, 1H), 7.42
(t, J = 8.2 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H), 3.45 (dd, J = 12.6, 6.4
Hz, 2H), 3.35 (m, 2H), 1.94−1.88 (m, 2H) ppm. 13C NMR (150
MHz, DMSO-d6) δ 166.29, 150.46, 148.07, 147.66, 143.75, 142.37,
135.96, 133.26, 130.52, 127.48, 126.25, 124.13, 123.30, 122.74,
122.62, 121.45, 120.65, 119.65, 116.78, 37.98, 29.60 ppm. HR-MS
(m/z) (ESI): calcd for C21H18ClN5O [M + H]+: 393.1278; found:
393.1280.
5-((3-Chlorophenyl)amino)-N-(2-(pyrrolidin-1-yl)ethyl)benzo[c]-
[2,6]naphthyridine-8-carboxamide (1j). Yellow solid. Yield: 72.8%.
1H NMR (600 MHz, DMSO-d6) δ 10.19 (s, 1H), 9.69 (s, 1H), 9.00
(d, J = 5.6 Hz, 1H), 8.97 (d, J = 5.2 Hz, 1H), 8.88 (d, J = 8.4 Hz, 1H),
8.58 (d, J = 5.6 Hz, 1H), 8.26 (d, J = 4.2 Hz, 2H), 8.05 (d, J = 8.0 Hz,
1H), 7.96 (d, J = 8.2 Hz, 1H), 7.44 (t, J = 8.2 Hz, 1H), 7.16 (d, J = 7.8
Hz, 1H), 3.66 (dd, J = 10.8, 4.8 Hz, 2H), 3.37 (m, 6H), 1.96 (m, 4H)
ppm. 13C NMR (150 MHz, DMSO-d6) δ 166.99, 150.73, 148.20,
147.90, 143.79, 142.32, 135.29, 133.29, 130.59, 127.49, 126.43,
124.32, 123.43, 122.92, 122.83, 121.85, 120.90, 119.89, 116.88, 54.06,
53.91, 36.50, 23.00 ppm. HR-MS (m/z) (ESI): calcd for
C25H24ClN5O [M + H]+: 446.1747; found: 446.1750.
N-(2-Bromoethyl)-5-((3-chlorophenyl)amino)benzo[c][2,6]-
1
naphthyridine-8-carboxamide (1e). Yellow solid. Yield: 61.4%. H
NMR (600 MHz, DMSO-d6) δ 10.20 (s, 1H), 9.67 (s, 1H), 9.12 (t, J
= 5.0 Hz, 1H), 9.00 (d, J = 5.4 Hz, 1H), 8.82 (d, J = 3.8 Hz, 1H), 8.59
(d, J = 5.4 Hz, 1H), 8.29 (s, 1H), 8.28 (s, 1H), 8.13 (d, J = 8.0 Hz,
1H), 7.97 (d, J = 8.2 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.15 (d, J = 7.6
Hz, 1H), 4.85 (t, J = 5.2 Hz, 2H), 3.88−3.84 (m, 2H) ppm.13C NMR
(150 MHz, DMSO-d6) δ 166.69, 152.29, 150.61, 148.16, 147.77,
143.79, 142.37, 135.05, 133.28, 130.55, 127.50, 126.41, 124.25,
123.36, 122.87, 122.71, 120.76, 119.76, 116.84, 79.74, 38.61 ppm.
HR-MS (m/z) (ESI): calcd for C21H16BrClN4O [M + H]+: 455.0274;
found: 455.0272.
5-((3-Chlorophenyl)amino)-N-(2-(piperidin-1-yl)ethyl)benzo[c]-
[2,6]naphthyridine-8-carboxamide (1k). Yellow solid. Yield: 77.1%.
1H NMR (600 MHz, DMSO-d6) δ 10.19 (s, 1H), 9.69 (s, 1H), 9.00
(d, J = 5.4 Hz, 2H), 8.88 (d, J = 8.4 Hz, 1H), 8.59 (d, J = 5.6 Hz, 1H),
8.27 (s, 1H), 8.25 (d, J = 0.8 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.96
(dd, J = 8.4, 1.0 Hz, 1H), 7.45 (t, J = 8.2 Hz, 1H), 7.16 (dd, J = 7.8,
1.2 Hz, 1H), 3.70 (dd, J = 11.8, 5.8 Hz, 2H), 3.59 (t, J = 5.8 Hz, 2H),
3.30 (t, J = 6.2 Hz, 2H), 2.97 (t, J = 6.4 Hz, 2H), 1.91−1.61 (m, 6H)
ppm. 13C NMR (150 MHz, DMSO-d6) δ 167.00, 151.59, 148.20,
147.90, 143.79, 142.31, 135.24, 133.28, 130.59, 129.32, 126.43,
124.32, 123.39, 122.96, 122.83, 121.19, 120.89, 119.88, 116.88, 55.62,
52.81, 34.77, 22.99, 21.68 ppm. HR-MS (m/z) (ESI): calcd for
C26H26ClN5O [M + H]+: 460.1904; found: 460.1900.
5-((3-Chlorophenyl)amino)-N-(2-morpholinoethyl)benzo[c][2,6]-
naphthyridine-8-carboxamide (1l). Yellow solid. Yield: 68.7%.
Compound 1l may also be prepared by the method reported in the
literature.41 1H NMR (600 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.98 (s,
1H), 8.94 (d, J = 5.6 Hz, 1H), 8.84 (d, J = 5.2 Hz, 2H), 8.82 (s, 1H),
8.38 (s, 1H), 8.22 (s, 1H), 8.19 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 8.2
Hz, 1H), 7.42 (t, J = 8.1 Hz, 1H), 7.12 (dd, J = 7.8, 0.9 Hz, 1H), 3.62
(m, 4H), 3.49 (m, 2H), 3.38 (m, 4H), 2.61 (m, 2H) ppm. 13C NMR
(150 MHz, DMSO-d6) δ 166.30, 150.71, 148.04, 147.67, 143.80,
142.56, 135.89, 133.16, 130.42, 127.53, 126.36, 124.31, 123.32,
5-((3-Chlorophenyl)amino)-N-(2,2-difluoroethyl)benzo[c][2,6]-
1
naphthyridine-8-carboxamide (1f). Yellow solid. Yield: 61.8%. H
NMR (600 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.99 (s, 1H), 9.28 (s,
1H), 8.96 (d, J = 4.6 Hz, 1H), 8.86 (d, J = 8.2 Hz, 1H), 8.83 (d, J =
4.6 Hz, 1H), 8.37 (s, 1H), 8.29 (s, 1H), 8.22 (d, J = 7.4 Hz, 1H), 8.01
(d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 7.2 Hz, 1H),
6.23 (t, J = 13.2 Hz, 1H), 3.77 (t, J = 14.8 Hz, 2H) ppm. 13C NMR
(150 MHz, DMSO-d6) δ 166.98, 150.76, 148.05, 147.77, 143.80,
142.50, 134.89, 133.17, 130.42, 127.44, 126.57, 124.37, 123.31,
122.90, 122.59, 121.87, 120.80, 119.82, 117.29, 115.09 (t, J = 240.2
Hz), 42.25 (t, J = 26.6 Hz) ppm. HR-MS (m/z) (ESI): calcd for
C21H15ClF2N4O [M + H]+: 413.0980; found: 413.0967.
5-((3-Chlorophenyl)amino)-N-(2-(dimethylamino)ethyl)benzo-
[c][2,6]naphthyridine-8-carboxamide (1g). Yellow solid. Yield:
1
69.8%. H NMR (600 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.67 (s,
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122.79, 122.55, 121.53, 120.79, 119.81, 117.37, 66.36, 63.29, 57.60,
53.54 ppm. HR-MS (m/z) (ESI): calcd for C25H24ClN5O2 [M + H]+:
462.1697; found: 462.1695.
2.35 (t, J = 7.3 Hz, 2H), 1.87−1.81 (m, 2H) ppm. 13C NMR (150
MHz, DMSO-d6) δ 174.80, 166.28, 150.50, 148.13, 147.68, 143.76,
142.38, 135.96, 133.27, 130.55, 127.52, 126.30, 124.17, 123.37,
122.74, 122.64, 121.47, 120.67, 119.67, 116.81, 39.29, 31.72, 25.05
ppm. HR-MS (m/z) (ESI): calcd for C23H19ClN4O3 [M + H]+:
435.1224; found: 435.1221.
5-((3-Chlorophenyl)amino)-N-(3-hydroxypropyl)benzo[c][2,6]-
1
naphthyridine-8-carboxamide (1m). Yellow solid. Yield: 79.8%. H
NMR (600 MHz, DMSO-d6) δ 10.19 (s, 1H), 9.67 (s, 1H), 8.99 (d, J
= 5.6 Hz, 1H), 8.85 (d, J = 8.4 Hz, 1H), 8.77 (t, J = 5.6 Hz, 1H), 8.59
(d, J = 5.6 Hz, 1H), 8.28 (s, 1H), 8.23 (s, 1H), 8.12 (dd, J = 8.2, 1.0
Hz, 1H), 7.95 (dd, J = 8.4, 1.6 Hz, 1H), 7.45 (t, J = 8.2 Hz, 1H), 7.15
(dd, J = 7.8, 1.2 Hz, 1H), 4.54 (t, J = 5.2 Hz, 1H), 3.52 (q, J = 6.2 Hz,
2H), 3.40 (dd, J = 12.8, 7.0 Hz, 2H), 1.75 (p, J = 6.6 Hz, 2H) ppm.
13C NMR (150 MHz, DMSO-d6) δ 166.27, 150.53, 148.15, 147.70,
143.78, 142.39, 136.06, 133.27, 130.56, 127.54, 126.27, 124.19,
123.35, 122.77, 122.66, 121.46, 120.68, 119.69, 116.84, 59.13, 37.22,
32.92 ppm.HR-MS (m/z) (ESI): calcd for C22H19ClN4O2 [M + H]+:
407.1269; found: 407.1264.
General Procedure for Preparation of Compounds 2a−2g. 2-
Hydroxyethyl 5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxylate (2a). General procedure C: To a
solution of CX-4945 (0.210 g, 0.6 mmol) in DMF (5 mL) was added
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluorobo-
rate (TBTU) (0.231 g, 0.72 mmol) and stirred at 45 °C for 10
min. Then, triethylamine (TEA) (0.167 mL, 1.2 mmol) was added to
the solution and stirred for 10 min at the same temperature, followed
by the addition of ethylene glycol (0.062 g, 1 mmol), and the solution
was stirred for 24 h in a nitrogen atmosphere. The progress of the
reaction was monitored with TLC. After the substrate was completely
consumed, the solvent was removed under vacuum, and the residue
was treated with EtOAc (25 mL). The organic layers were washed
with saturated salt water (3 × 20 mL) and dried with anhydrous
Na2SO4. The organic layers were concentrated under vacuum to
obtain the crude product and further purified with silica gel
chromatography with the eluent of MeOH and DCM (1:100−1:50)
N-(3-Aminopropyl)-5-((3-chlorophenyl)amino)benzo[c][2,6]-
1
naphthyridine-8-carboxamide (1n). Yellow solid. Yield: 57.3%. H
NMR (600 MHz, DMSO-d6) δ 10.19 (s, 1H), 10.01 (s, 1H), 8.96 (d,
J = 5.6 Hz, 1H), 8.91 (t, J = 5.4 Hz, 1H), 8.87 (d, J = 3.8 Hz, 1H),
8.38 (s, 1H), 8.25−8.21 (m, 2H), 8.04 (s, 1H), 7.99 (d, J = 8.4 Hz,
1H), 7.44 (t, J = 8.2 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H), 3.36 (dd, J =
12.6, 6.4 Hz, 2H), 3.18 (dd, J = 12.8, 6.2 Hz, 2H), 1.76−1.70 (m,
2H), 1.22 (s, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 166.25,
161.58, 150.71, 148.07, 147.68, 143.82, 142.56, 135.91, 133.16,
130.46, 127.55, 126.34, 124.30, 123.21, 122.79, 122.56, 120.77,
119.82, 117.38, 37.53, 35.51, 29.61 ppm. HR-MS (m/z) (ESI): calcd
for C22H20ClN5O [M + H]+: 406.1434; found: 406.1431.
Methyl 4-(5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxamido)butanoate (1o). Orange solid.
Yield: 65.8%. 1H NMR (600 MHz, DMSO-d6) δ 10.17 (s, 1H),
9.94 (s, 1H), 8.95 (d, J = 6.0 Hz, 1H), 8.87−8.83 (m, 2H), 8.81 (d, J
= 5.0 Hz, 1H), 8.34 (s, 1H), 8.22 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H),
7.95 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.13 (d, J = 7.4 Hz,
1H), 3.59 (s, 3H), 3.35 (dd, J = 12.6, 6.8 Hz, 2H), 2.41 (t, J = 6.8 Hz,
2H), 1.87−1.81 (m, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ
173.68, 166.36, 150.69, 148.03, 147.66, 143.79, 142.52, 135.91,
133.17, 130.46, 127.54, 126.30, 124.29, 123.35, 122.76, 122.59,
121.49, 120.78, 119.81, 117.30, 51.75, 39.15, 31.36, 24.94 ppm. HR-
MS (m/z) (ESI): calcd for C24H21clN4O3 [M + H]+: 449.1380;
found: 449.1386.
General Procedure for Preparation of Compounds 1p−1q. 3-(5-
((3-Chlorophenyl)amino)benzo[c][2,6]naphthyridine-8-
carboxamido)propanoic Acid (1p). General procedure B: To a
solution of 1i (0.434 g, 1 mmol) in MeOH (10 mL) were added H2O
(5 mL) and LiOH·H2O (0.105 g, 2.5 mmol) and stirred at 60 °C
overnight. The progress of the reaction was monitored with TLC.
After the substrate was completely consumed, the solvent was
removed under vacuum, and the residue was acidified with 1 M HCl
to pH = 5, filtering the precipitate; washed with water (3 × 10 mL);
and dried to obtain the crude product of 1p. The crude product was
recrystallized with MeOH (15 mL) to get a yellow solid (0.370 g).
Yield: 88.1%. 1H NMR (600 MHz, DMSO-d6) δ 12.37 (s, 1H), 10.15
(s, 1H), 9.63 (s, 1H), 8.96 (s, 1H), 8.84 (s, 1H), 8.81 (d, J = 7.4 Hz,
1H), 8.55 (s, 1H), 8.24 (t, J = 1.8 Hz, 1H), 8.21 (d, J = 1.2 Hz, 1H),
8.09 (d, J = 6.8 Hz, 1H), 7.92 (dd, J = 7.2, 1.8 Hz, 1H), 7.42 (d, J =
6.8 Hz, 1H), 7.13 (d, J = 6.4 Hz, 1H), 3.54 (dd, J = 10.6, 5.8 Hz, 2H),
2.58 (t, J = 6.4 Hz, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ
173.63, 166.29, 150.52, 148.12, 147.69, 143.75, 142.37, 135.77,
133.27, 130.54, 127.50, 126.30, 124.18, 123.32, 122.76, 122.65,
121.53, 120.68, 119.68, 116.80, 36.31, 34.40 ppm. HR-MS (m/z)
(ESI): calcd for C22H17ClN4O3 [M + H]+: 421.1067; found:
421.1060.
1
to give a yellow solid (0.204 g). Yield: 86.5%. H NMR (600 MHz,
DMSO-d6)δ 10.17 (s, 1H), 9.95 (s, 1H), 8.99 (d, J = 5.6 Hz, 1H),
8.88 (d, J = 8.5 Hz, 1H), 8.79 (d, J = 5.6 Hz, 1H), 8.32 (t, J = 2.0 Hz,
1H), 8.27 (d, J = 1.6 Hz, 1H), 8.18 (dd, J = 8.2, 1.3 Hz, 1H), 8.01
(dd, J = 8.4, 1.7 Hz, 1H), 7.45 (t, J = 8.1 Hz, 1H), 7.15 (dd, J = 7.9,
1.4 Hz, 1H), 5.12 (t, J = 5.4 Hz, 1H), 4.39−4.35 (m, 2H), 3.78 (dd, J
= 9.4, 4.8 Hz, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) 13C NMR
(151 MHz, DMSO) δ 166.18, 150.85, 148.21, 148.16, 143.76, 142.34,
133.15, 131.11, 130.52, 128.59, 127.27, 124.62, 124.41, 123.23,
123.15, 122.71, 120.79, 119.85, 117.26, 67.36, 59.52 ppm. HR-MS
(m/z) (ESI): calcd for C21H16ClN3O3 [M + H]+: 394.0958; found:
394.0961.
2-Iodoethyl 5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxylate (2b). Yellow solid. Yield: 77.3%. 1H
NMR (600 MHz, DMSO-d6) δ 10.16 (s, 1H), 10.04 (s, 1H), 8.98 (d,
J = 5.6 Hz, 1H), 8.82 (d, J = 8.5 Hz, 1H), 8.52 (dd, J = 8.3, 1.0 Hz,
1H), 8.34 (s, 1H), 8.26 (d, J = 8.2 Hz, 1H), 8.02 (d, J = 1.4 Hz, 1H),
7.73 (dd, J = 8.4, 1.5 Hz, 1H), 7.48 (t, J = 8.1 Hz, 1H), 7.16 (dd, J =
7.9, 1.2 Hz, 1H), 5.11−5.08 (t, J = 8.2 Hz, 2H), 4.78 (t, J = 7.8 Hz,
2H). 13C NMR (150 MHz, DMSO-d6) δ 165.62, 152.34, 150.89,
148.19, 143.68, 142.39, 133.15, 130.50, 130.26, 128.49, 127.20,
124.66, 124.04, 123.26, 123.17, 122.71, 120.89, 119.95, 117.39, 79.06,
63.61. HR-MS (m/z) (ESI): calcd for C21H15ClF2N4O [M + H]+:
503.9975; found: 503.9980.
2-Fluoroethyl 5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxylate (2c). Yellow solid. Yield: 82.8%. 1H
NMR (600 MHz, DMSO-d6) δ 10.11 (s, 1H), 9.64 (s, 1H), 8.97 (d, J
= 5.4 Hz, 1H), 8.82 (d, J = 8.2 Hz, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.22
(s, 1H), 8.20 (s, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.2 Hz,
1H), 7.44 (t, J = 8.0 Hz, 1H), 7.14 (dd, J = 7.8, 1.2 Hz, 1H), 4.88−
4.86 (m, 1H), 4.80−4.77 (m, 1H), 4.65−4.62 (m, 1H), 4.59−4.57
(m, 1H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 165.79, 150.59,
148.15, 148.11, 143.62, 142.15, 133.19, 130.51, 130.46, 128.53,
127.08, 124.44, 124.18, 123.20, 123.18, 122.70, 120.64, 119.64,
116.72, 82.22 (d, J = 166.2 Hz), 64.72 (d, J = 18.2 Hz) ppm. HR-MS
(m/z) (ESI): calcd for C21H15ClFN3O2 [M + H]+: 396.0915; found:
396.0911.
2-(Pyridin-2-yl)ethyl 5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxylate (2d). Yellow solid. Yield: 77.2%. 1H
NMR (600 MHz, DMSO-d6) δ 10.15 (s, 1H), 10.02 (s, 1H), 8.97 (s,
1H), 8.87 (d, J = 7.0 Hz, 1H), 8.83 (s, 1H), 8.61 (s, 1H), 8.36 (s,
1H), 8.27 (s, 1H), 8.18 (d, J = 6.1 Hz, 1H), 8.00 (d, J = 6.7 Hz, 1H),
7.88 (s, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.43 (s, 1H), 7.39 (s, 1H), 7.13
(d, J = 5.9 Hz, 1H), 5.50 (s, 2H), 3.40 (s, 1H), 3.18 (s, 1H) ppm. 13C
NMR (150 MHz, DMSO-d6) δ 165.78, 156.03, 150.96, 149.74,
148.25, 143.82, 142.36, 137.58, 133.11, 130.64, 130.47, 128.69,
127.23, 124.71, 124.32, 123.63, 123.44, 122.68, 122.50, 122.10,
4-(5-((3-Chlorophenyl)amino)benzo[c][2,6]naphthyridine-8-
carboxamido)butanoic Acid (1q). Yellow solid. Yield: 83.6%. 1H
NMR (600 MHz, DMSO-d6) δ 12.43 (s, 1H), 10.18 (s, 1H), 9.66 (d,
J = 5.3 Hz, 1H), 8.99 (d, J = 5.4 Hz, 1H), 8.84 (d, J = 8.4 Hz, 1H),
8.81 (t, J = 6.4 Hz, 1H), 8.58 (d, J = 5.4 Hz, 1H), 8.27 (s, 1H), 8.24
(s, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.46 (t, J =
8.1 Hz, 1H), 7.15 (d, J = 7.5 Hz, 1H), 3.38 (dd, J = 12.6, 6.5 Hz, 2H),
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120.83, 119.88, 118.27, 117.41, 67.41, 49.03 ppm. HR-MS (m/z)
(ESI): calcd for C26H19ClN4O2 [M + H]+: 455.1275; found:
455.1269.
2,2-Difluoroethyl 5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxylate (2e). Earth-yellow solid. Yield: 65.7%.
1H NMR (600 MHz, DMSO-d6) δ 10.10 (s, 1H), 9.90 (s, 1H), 8.96
(d, J = 5.6 Hz, 1H), 8.83 (d, J = 8.5 Hz, 1H), 8.76 (d, J = 5.4 Hz, 1H),
8.31 (d, J = 1.7 Hz, 1H), 8.19 (d, J = 1.7 Hz, 1H), 8.16−8.14 (m,
1H), 7.93 (dd, J = 8.4, 1.8 Hz, 1H), 7.44 (t, J = 8.2 Hz, 1H), 7.14 (dd,
J = 7.8, 1.2 Hz, 1H), 6.51 (tt, J = 54.2, 3.2 Hz, 1H), 4.68 (td, J = 15.2,
3.2 Hz, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ 165.21, 150.86,
148.21, 148.12, 143.70, 142.25, 133.12, 130.41, 129.78, 128.69,
127.07, 124.63, 124.17, 123.51, 123.37, 122.69, 120.82, 119.82,
117.19, 114.12 (t, J = 238.9 Hz), 63.04 (t, J = 26.4 Hz).HR-MS (m/z)
(ESI): calcd for C21H14ClF2N3O2 [M + H]+: 414.0821; found:
414.0820.
Cell Line Culture. The CK2-overexpressed cell lines PC-3
(prostate cancer cell line), HCT-116 (colorectal cancer cell line),
MCF-7 ( breast cancer cell line), HT-29 ( colorectal cancer cell line),
and T24 (bladder Cancer Cell line) and the normal human liver cell
line LO2 were grown under 5% CO2 and 95% air at 37 °C conditions.
All these cells were grown in monolayer in DMEM or RPMI-1640
medium enriched with 10% FBS, 100 μg/mL streptomycin, and 100
μg/mL penicillin solutions.
Antiproliferative Assay. MTT assay was used to detect the
antiproliferative effect of all the target compounds against several
cancer cell lines. About 5 × 103 cells per well were planted in a 96-well
plate and incubated at 37 °C in 5% CO2 overnight. Compounds were
dissolved with DMSO and diluted with medium to various
concentrations, and added to these different cells culture medium,
respectively, for 72 h. After that, 10 μL of 5 mg/mL MTT was
introduced to all the wells, and incubation of the cells was continued
for 4 h. The medium was discarded, and 150 μL of DMSO was added
to dissolve the dark blue crystals on the bottom. The value of O.D.
was determined with an enzyme-labeling instrument at 490 nm. The
3,3-Dichloropropyl 5-((3-Chlorophenyl)amino)benzo[c][2,6]-
naphthyridine-8-carboxylate (2f). Yellow solid (0.141 g). Yield:
1
61.5%. H NMR (600 MHz, DMSO-d6) δ 10.14 (s, 1H), 10.02 (s,
IC50 value of each compound presented as the mean
obtained by three parallel experiments.
SD was
1H), 8.97 (d, J = 5.6 Hz, 1H), 8.87 (d, J = 3.6 Hz, 1H), 8.86 (s, 1H),
8.39 (t, J = 1.8 Hz, 1H), 8.24 (d, J = 1.4 Hz, 1H), 8.17 (dd, J = 8.2,
1.2 Hz, 1H), 7.98 (dd, J = 8.4, 1.6 Hz, 1H), 7.43 (t, J = 8.2 Hz, 1H),
7.14 (dd, J = 7.8, 1.2 Hz, 1H), 4.84−4.80 (m, 1H), 4.70 (dd, J = 11.8,
4.6 Hz, 1H), 4.65 (dd, J = 11.8, 6.2 Hz, 1H), 4.14 (t, J = 5.2 Hz, 2H)
ppm. 13C NMR (150 MHz, DMSO-d6) δ 165.47, 150.91, 148.21,
148.17, 143.75, 142.34, 133.12, 130.40, 130.23, 128.72, 127.17,
124.67, 124.27, 123.44, 123.36, 122.67, 120.86, 119.87, 117.38, 65.77,
58.78, 46.48 ppm. HR-MS (m/z) (ESI): calcd for C22H16Cl3N3O2 [M
+ H]+: 460.0386; found: 460.0380.
2-(2-(2-Hydroxyethoxy)ethoxy)ethyl 5-((3-Chlorophenyl)amino)-
benzo[c][2,6]naphthyridine-8-carboxylate (2g). Yellow solid. Yield:
72.6%. 1H NMR (600 MHz, DMSO-d6) δ 10.12 (s,1H), 9.63 (s, 1H),
8.98 (d, J = 6.0 Hz, 1H), 8.82 (d, J = 6.0 Hz, 1H), 8.54 (d, J = 6.0 Hz,
1H), 8.27 (t, J = 2.0 Hz, 1H), 8.20 (d, J = 1.6 Hz, 1H), 8.07 (dd, J =
8.2, 1.4 Hz, 1H), 7.94 (dd, J = 8.4, 1.8 Hz, 1H), 7.45 (t, J = 8.1 Hz,
1H), 7.15 (dd, J = 6.0, 1.4 Hz, 1H), 4.57 (t, J = 6.0 Hz, 1H), 4.47 (t, J
= 6.0 Hz, 2H), 3.83 (t, J = 6.0 Hz, 2H), 3.66 (dd, J = 5.8, 3.8 Hz, 2H),
3.58 (dd, J = 5.8, 3.8 Hz, 2H), 3.48 (dd, J = 10.4, 4.8 Hz,2H), 3.44
(dd, J = 7.8, 3.0 Hz, 2H) ppm. 13C NMR (150 MHz, DMSO-d6) δ
165.97, 150.65, 148.22, 148.16, 143.67, 142.19, 133.22, 130.88,
130.54, 128.53, 127.19, 124.49, 124.25, 123.21, 123.15, 122.73,
120.67, 119.67, 116.78, 72.86, 70.41, 70.27, 68.84, 64.79, 60.69 ppm.
HR-MS (m/z) (ESI): calcd for C25H24ClN3O5 [M + H]+: 482.1482;
found: 482.1483.
CK2, ClK2, and ALDH1A1 Assay. CK2, Clk2, and the ALDH1A1
Kinase (Human) Assay/Inhibitor Screening Assay Kit as well as CK2,
Clk2, and ALDH1A1 enzymes were supplied by Shanghai Fusheng
Industrial Co., Ltd. (Shanghai, China). The inhibitory activity of
tested compounds on CK2, ClK2, and ALDH1A1 was detected
according to manufacturer’s instruction by commercial CK2, ClK2,
and ALDH1A1 assay kits with CX-4945 as the positive control. As
shown in Figure S7, we take CK2 enzyme activity detection as an
example to briefly introduce the experimental process. 50 μL, 60 U/L
of CK2 enzyme reaction solution was added to each well of 96-well
plates after thawing, and then five gradient concentrations (500.0,
100.0, 20.0, 4.0, and 0.8 nM) of fresh compound solutions were added
to each well, respectively. In addition, the same solution without the
inhibitor was added to each well in the control group. The 96 well
plate was sealed with sealing film and incubated in 37 °C incubator
for 2 h. After that, the conjugate reagent (50 μL) was introduced to
every well, except for the control group, and incubated at 37 °C for
another 30 min. The color developing reagents A (50 μL) and B (50
μL) were successively added to each hole, the mixture was shaken
gently and mixed well, light was avoided for 15 min, and then the stop
buffer (50 μL) was added to each hole. The reaction mixtures were
incubated at room temperature for 10 min, and the value of O.D. was
measured via an enzyme-labeling instrument at 450 nm. The IC50
value of each compound is given as the mean SD and was obtained
by three parallel experiments.
Kinase Inhibition Assays. The potential of compound 1c as a
CK2 inhibitor was determined at 1 μmol/L concentration against a
panel of 208 kinases through the commercially available service
provided by ICE Bioscience Inc. (Beijing, China).
Molecular Docking. Molecular docking of 1c to CK2α (PDB ID:
6isj), Clk2 (PDB ID: 6khe), and ALDH1A1 (PDB ID: 4x4l) was
conducted via AutoDock Vina, where the Iterated Local Search
Globule Optimizer was employed as the optimization algorithm.
Ligand 1c was prepared in ChemBioDraw Ultra 14.0 and minimized
with ChemBio3D Ultra 14.0. Before the docking process, the natural
ligand along with the water molecules was deleted from the crystal
structure. Subsequently, all hydrogen atoms were added to the each
protein and ligand to be docked, and every coordinate file of the
protein, as well as the ligand was generated as a PDBQT file via
AutoDockTools-1.5.6. A grid box for binding site covered the
protein’s catalytic site. The box possessed a 1.0 Å grid spacing and was
centered at the geometric center of the protein. In every docking
experiment, we selected the best binding mode on the basis of the
binding affinity determined by the scoring function in AutoDock
Vina. Docking results were analyzed and visualized with PyMOL
(Schroedinger LLC, NY).
ALDH Activity Assay. The HCT-116 cells were inoculated on six-
well plates at 2 × 105 cells per well; grown at 37 °C in 5% CO2
overnight; and then treated with CX-4945 (5 μM), different
concentrations of 1c (5, 10, and 20 μM), and vehicle (DMSO),
respectively, for 24 h, to explore ALDH1A1 expression in HCT-116
cells. Aldefluor (Stem Cell Technologies, Vancouver, BC, Canada)
staining was assayed with the FITC channel of fluorescent activated
cell sorting as described by the manufacturer. To mark the base
fluorescence, pretreatment with the DEAB ALDH inhibitor was
employed as a negative control to mark the base fluorescence.
Quantitative Real-Time PCR Assay. TRIZOL was employed to
isolate total cellular RNA from HCT-116 or HCT-116AHDH+ cells
treated for 24 h with the tested compounds. RNA was reverse-
transcribed with the Luna universal one-step RT-qPCR (GeneAmp
9600, PERKIN ELMER), and the resulting cDNA was subsequently
run on a thermal cycler (ABI StepOnePlus, ABI). Thermal profile was
as follows: reverse transcription for 10 min at 55 °C and initial
denaturation for 1 min at 95 °C followed by 39 thermal cycles of
denaturation for 10 s at 95 °C and extension for 30 s at 60 °C. Primer
sequences are listed in Table S1. All PCRs included melting curve
analysis and template-free negative technical controls to verify specific
single-product amplification. β-Actin mRNA was employed as an
internal standard. The relative expression of mRNA is represented as
fold increase (2−ΔΔCt).42
CD44 and CD133 Expression Assay. The HCT-116ALDH+ cells
were planted in six-well plates at 2 × 105 cells per well; cultured
overnight under 37 °C and 5% CO2 conditions; and then treated with
vehicle (DMSO), BBI608 (20 μM), CX-4945 (20 μM), and 1c (20
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μM), respectively. After co-incubation for 24 h, we collected the cells
and stained them with PE-labeled anti-CD133 and FITC-labeled anti-
CD44 for 30 min as described by the manufacturer. The expression of
CD44+ along with CD133+ was detected with flow cytometry by the
Cell Quest software.
Cancer Stem-like Spheroid Formation Assay. A spheroid
formation assay was used to explore the stemness of HCT-116ALDH+
cells. Concisely, 800 cells/well were planted in the 24-well plates
(3473, Corning) in serum-free DMEM enriched with B27 (17504044,
Invitrogen, 1: 50), EGF (20 ng/mL, AF-100-15, Peprotech), and β-
FGF (10 ng/mL, 100-18B, Peprotech). Vehicle (DMSO), BBI608 (5
μM), CX-4945 (5 μm) along with 1C (5 μm) were added into each
well of the 24-well plate, respectively, and co-incubated with cells for
10 days. After that, the numbers and diameter of the tumorsphere
were measured in the microscopy images.
Transwell Invasion Assays. Transwell chambers with 8 mm
pores were employed to detect the invasion ability of cells. In the
assay, 1 × 105 HCT-116 cells in 200 μL of serum-free medium were
introduced into the upper compartment, and 500 μL of DMEM
enriched with 10% FBS as a chemoattractant was introduced into the
lower compartment. After inoculation with vehicle (DMSO), CX-
4945 (5 μM), and 1c (5 μM) for 24 h, a cotton swab was employed
to remove the HCT-116 cells on the upper surface of the inserted
membrane. Crystal violet staining was performed on the HCT-116
cells on the lower surface of the inserted membrane for 30 min. Lastly,
an inverted microscope was employed to image the number of cells
on the lower surface of the membrane.
Cell Apoptotic Assay. An Annexin V-FITC apoptotic detection
kit was employed to explore the apoptosis of HCT-116 cells induced
by vehicle (DMSO), CX-4945 (5 μM), and 1c (5, 10 and 20 μM).
We collected the HCT-116 cells in the log growth phase and
suspended them in DMEM enriched with 10% FBS. A total of 2 × 105
cells/well were inoculated in a six-well plate and left to adhere
overnight. Then, the original growth medium was discarded, and fresh
medium was added and treated with the compounds under study,
respectively, for 24 h at 37 °C. The cells were inoculated with
Annexin V-FITC followed by PI in the dark for 10 min at room
temperature. Afterward, the cells were assessed by flow cytometry,
and the apoptotic value was obtained by the Cell-Quest software (BD
Biosciences).
and a chemiluminescence reagent (Thermo Fisher Scientifics Ltd.)
was employed to visualize the protein bands.
Thermodynamic Solubility (Shake Flask Method). 1c was
added into 2 mL glass vials prior to the addition of pH 6.8 phosphate
buffer to obtain a nominal concentration of 2 mg/mL. The samples
were sonicated for 10 min and shaken overnight at 800 rpm (Titrimax
1000, Heidolph) at room temperature. A first phase separation of
supernatant and undissolved solid was performed by centrifugation at
5000 rpm for 20 min (Eppendorf Centrifuge 5804). The supernatant
was then transferred into a conical glass vial for a second
centrifugation. An adequate dilution was made using the particle-
free supernatant, and the concentration was quantified by LC-HRMS
using a six-point calibration curve (Vanquish coupled to an Exactive-
Plus, Thermo Scientific). The final determination of the solubility of
1c in phosphate buffer solution with pH = 6.8 was 0.89 mg/mL.
Pharmacokinetic Assay. The pharmacokinetic profile of 1c was
assessed in overnight-fasted male SD rats after a single oral dose (po).
1c was dissolved in 5% CMC-Na, and the positive control of CX-
4945·Na was a sodium salt formulated in phosphate buffer for the
rats. Six male SD rats (three for each leg) weighing about 250−270 g
were clustered randomly into two groups and administered with 1c
(25 mg/kg) and CX-4945·Na (25 mg/kg), respectively. We collected
blood samples from all the rats at 0.25, 0.50, 0.75, 1, 2, 3, 4, 6, 8, 12,
24, and 48 h post oral dosing. The blood samples were centrifuged at
4000 rpm for 10 min, and the plasma was collected and stored at −40
°C. LC−MS/MS was employed to analyze the sample. The data of
blood concentration (c)−time (T) measured after oral administration
were processed by the Das 2.0 software. The appropriate
mathematical model was selected to fit, and the pharmacokinetic
parameters were calculated.
Preparation of standard curves: 10 μL of blank plasma was added
to the standard solution of 1c or CX-4945·Na at different
concentrations (30, 60, 150, 300, 600, 1500, 3000, 6000, 15,000,
and 30,000 ng/mL), respectively, to prepare plasma samples
equivalent to ropivacaine with plasma mass concentrations of 3, 6,
15, 30, 60, 150, 300, 600, 1500, and 3000 ng/mL. The LC−MS/MS
technique was used to analyze the samples with different
concentrations (5 μL), the ratio Y of the peak area of each
component to the internal standard peak area was used to perform
linear regression operation on the plasma mass concentration X, and
the regression equation obtained was the standard curve. The
regression equations of 1c and CX-4945·Na are Y = 0.116X −
0.00140 (r = 0.9986) and Y = 0.704X + 0.000548 (r = 0.9968),
respectively, and the minimum quantitative limit is 3 ng/mL.
Antitumor Activity Assay. Animal studies were performed as per
the relevant laws and institutional guidelines set by the Jiangsu
Province Committee of Use and Care of Laboratory Animals, and all
protocols were approved by the Institutional Animal Care and Use
Committee of Southeast University. The antitumor efficacy of 1c was
investigated in HCT-116 xenograft tumor models in nude mice. A
total of 0.2 mL of 5 × 107 cell/mL cell suspension was taken and
injected into the right armpit of 5-week-old male BALB/c athymic
nude mice weighing about 16−18 g (purchased from Shanghai Sipo-
Bikai Laboratory Animal Co. Ltd. (Shanghai, China)). When the
tumors reached a volume of about 100 mm3 in all mice, they were
grouped randomly into four groups of five mice and treated via
intragastric administration with 0.1 mL 5% glucose injection (twice a
day for 4 weeks), CX-4945·Na (60 mg/kg, twice a day for 4 weeks),
and 1c (60 and 90 mg/kg, twice a day for 4 weeks). CX-4945·Na was
dissolved in 25 mM NaH2PO4 solution, and 1c was dissolved in 5%
CMC-Na. Tumor volumes along with body weights were recorded
twice a week. The calculation formula of tumor volume (TV, mm3) is
TV = 0.5 × length × width2. The average tumor volume within each
experimental group at the set time points was employed to plot
growth curves via Origin 9.0. We sacrificed 20 mice after the last
treatment, and then the tumor weight was determined as the
antitumor activity of the respective groups. The heart, liver, spleen,
lung, kidney, and tumor tissues were excised for HE staining. The
body weight along with physical state of the mice was determined
simultaneously as an indicator of systemic toxicity.
Cell Cycle Arrest Assay. The HCT-116 cells were diluted into 2
× 105 cells/mL mixed liquid with DMEM with 10% FBS, inoculated
in a six-well plate, and grown at 37 °C for 12 h. Then, the cancer cells
were treated with vehicle (DMSO), CX-4945 (20 μM), and 1c (20
μM) for 24 h. The cells in each group were collected into a 5 mL
centrifuge tube, about 500 μL of 70% ethanol was added, and the cells
were placed in a refrigerator at −20 °C for 12 h. Subsequently, the
supernatant was removed, treated with RnaseA (100 μg/mL) at 37 °C
for 20 min, and stained with 1 mg/mL PI for 30 min in the dark at 4
°C. The sample was assayed by flow cytometry, and the data were
analyzed with the FlowJo software (TreeStar, Inc.).
Western Blot Assay. The HCT-116 cells were diluted into 2 ×
105 cells/mL mixed liquid with DMEM with 10% FBS, planted in a
six-well plate, and grown at 37 °C for 12 h. The cells were treated with
CX-4945 (20 μM), 1c (20 μM), and vehicle (DMSO) for 24 h;
collected; centrifuged; and rinsed twice in PBS. Thereafter, the lysis
buffer was employed to lyse the cells for 30 min on ice. The cells were
then spun at 15,000 rpm for 15 min at 4 °C. The BCA protein assay
reagents (Imgenex, USA) were employed to quantify the proteins as
described by the manufacturer. Next, the proteins were fractionated
on 10% SDS-PAGE gels and blotted onto a PVDF membrane (GE
Healthcare). After that, 5% nonfat milk in the TBST buffer was
employed to block the membrane, which was subsequently co-
inoculated with primary antibodies (p-Akt1S129, Akt, p-Cdc37S13
,
Cdc37, p-GSK-3β(ser9), GSK-3β, DKK1, β-catenin, Cyt c, Bax, Bcl-2,
pro-caspase 9, cleaved caspase 9, pro-caspase 3, cleaved caspase 3,
total PARP, cleaved PARP, and β-actin) at 4 °C overnight. Thereafter,
the membranes were incubated with the HRP-labeled secondary
antibodies for 2 h and rinsed thrice in the TBST buffer for 15 min,
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Statistical Analysis. The data are given as means SD from at
least three independent experiments, each in triplicate samples for
individual treatment or dosage. Statistical analyses were conducted
using an unpaired, two-tailed Student’s t test. *P < 0.05 and **P <
0.01 signified statistical significance.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank the National Natural Science
Foundation of China (Project No. 22077015); Graduate
Research and Innovation Fund of Jiangsu Province, China
(Project No. KYCX19_0102); and Fundamental Research
Funds for the Central Universities (Project 2242020 K30059)
for financial aid to this work. The authors are thankful to ICE
Bioscience Inc. (Beijing) for undertaking the detection
experiment of kinase spectrum. Nanjing Biorn Life Science
Co. Ltd. is acknowledged for the in vivo tests.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00131.
Synthetic route of BTE and CX-4945, representative
flow cytometry figures from the Aldefluor assays,
docking of 1c into ALDH1A1 (PDB ID: 4x4l) kinase
X-ray crystal structure, Annexin CD44-FITC and
CD133-PI staining results of 1c in the HCT-116ALDH+
cells, HE staining internal organs and tumor tissues in
the animal xenograft model of HCT-116, the inhibitory
rate of the synthesized compounds on PC-3, HCT-116,
MCF-7, HT-29, T24, and LO2 cells at different
concentrations, the inhibitory rate of the synthesized
compounds on CK2 and Clk2 at different concen-
trations, schematic diagram of the inhibition activity
detection of compounds against CK2 and Clk2, table of
the percent inhibition of compound 1c against 208
kinases, table of the primer sequences used for qRT-
PCR, table of the in vivo blood concentration results of
CX-4945·Na and 1c, table of the inhibitory effects of the
measured samples on the HCT-116 cancer cell xenograft
mice model, figures of 1H and 13C NMR, HR-MS
spectra, and RP-HPLC chromatograms (PDF)
ABBREVIATIONS
■
AKT, protein kinase; ALDH1A1, aldehyde dehydrogenase
1A1; Bcl-2, B-cell lymphoma-2 protein; Bax, Bcl-2 associated X
protein; CSC, cancer stem cell; DIPEA, N,N-diisopropylethyl-
amine; DMEM, Dulbecco’s modified Eagle’s medium; CMC-
Na, sodium carboxymethyl cellulose; Cyt c, cytochrome C;
EGFR, epidermal growth factor receptor; FACS, fluorescence-
activated cell sorting; FBA, fetal bovine serum; FITC,
fluorescein isothiocyanate isomer; GSK-3β, glycogen synthase
kinase-3β; HCT-116, human colon cancer cell line; HT-29,
human colon cancer cell line; HR-MS, high-resolution mass
spectrometry; PC-3, human prostate cancer cell line; PI,
propidium iodide; PLK1, polo-like kinase 1; LO2, human
normal hepatocytes cell line; MCF-7, human breast cancer cell
line; PARP, poly ADP-ribose polymerase; qRT-PCR, quanti-
tative real-time PCR; RBITC, rhodamine B isothiocyanate;
RPMI-1640, Roswell Park Memorial Institute-1640;
SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electro-
phoresis; S100A4, S100 calcium binding protein A4; TMS,
tetramethylsilane; T24, human bladder cancer cell line;
ZAP70, 70-kDa zeta-associated protein.
Molecular formula strings (CSV)
PDB file of 1c-CK2α (PDB ID: 6isj) (PDB)
PDB file of 1c-Clk2 (PDB ID: 6khe) (PDB)
PDB file of 1c-ALDH1A1 (PDB ID: 4x4l) (PDB)
AUTHOR INFORMATION
Corresponding Author
■
REFERENCES
■
Shaohua Gou − Jiangsu Province Hi-Tech Key Laboratory for
Biomedical Research and Pharmaceutical Research Center
and School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, PR China;
orcid.org/0000-0003-0284-5480; Email: 2219265800@
qq.com
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Authors
Yuanjiang Wang − Jiangsu Province Hi-Tech Key Laboratory
for Biomedical Research and Pharmaceutical Research Center
and School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, PR China
Zhaodan Lv − Pharmaceutical Research Center and School of
Chemistry and Chemical Engineering, Southeast University,
Nanjing 211189, PR China
Feihong Chen − Jiangsu Province Hi-Tech Key Laboratory for
Biomedical Research and Pharmaceutical Research Center
and School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, PR China
Xing Wang − Pharmaceutical Research Center and School of
Chemistry and Chemical Engineering, Southeast University,
Nanjing 211189, PR China
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