Discovery of Novel Anti-Breast-Cancer Inhibitors by Synergistically Antagonizing Microtubule Polymerization and Aryl Hydrocarbon Receptor Expression

Article abstract of DOI:10.1021/acs.jmedchem.1c01099

A series of unreported dual-receptor inhibitors targeting both the tubulin colchicine site and AhR were designed and synthesized, and their anti-breast-cancer activities were evaluated. Compound 12 showed the strongest activity with an IC50 of 0.9 nM in M

Full text of DOI:10.1021/acs.jmedchem.1c01099

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Article
Discovery of Novel Anti-Breast-Cancer Inhibitors by Synergistically
Antagonizing Microtubule Polymerization and Aryl Hydrocarbon
Receptor Expression
Kun Wang,§ Hui Zhong,§ Na Li, Nairong Yu, Yujin Wang, Li Chen,* and Jianbo Sun*
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ABSTRACT: A series of unreported dual-receptor inhibitors
targeting both the tubulin colchicine site and AhR were designed
and synthesized, and their anti-breast-cancer activities were
evaluated. Compound 12 showed the strongest activity with an
IC50 of 0.9 nM in MCF-7 cell lines. Besides, 12 could significantly
inhibit cancer growth in MCF-7 xenograft tumor models with no
obvious toxic effects and was more effective than the positive
control (combretastatin A-4). With the in-depth study, it was found
that 12 could induce apoptosis in breast cancer cells by making
arrest in G2/M phase, depolarizing mitochondria and inducing
intracellular reactive oxygen generation. This evident anticancer
effect and the ability to inhibit cell migration were attributed to the
synergistic antagonism of 12 on tubulin and AhR. In general, 12 was
worthy of further research as an effective and safe anti-breast-cancer drug.
an innovative approach in breast cancer prevention and
therapy.10,11 Thus, based on the tubulin colchicine site and
the ligand characteristics of AhR, we used the fragment-based
design strategy to hybrid the scaffolds targeting the colchicine
site with the fragment that can target the AhR binding site to
develop duo-targeted inhibitors so as to inhibit the malignant
proliferation and metastasis of breast cancer cells.
With effective binding capacity at colchicine sites, quinoline
and quinazoline scaffolds are generally used as privileged
structures to design antimitotic agents and tubulin polymer-
ization inhibitors.12−15 As reported, many of the AhR
inhibitors involved privileged structures with a strong binding
affinity, including indole and other aromatic nuclei, such as
GNF351, PDM2, and StemRegenin 1.16,17 Therefore, we
hybridized quinazoline/quinoline scaffolds with indoles/
aromatic nucleus and with aminomethyl as the linker to
design and synthesize a series of dual-receptor inhibitors
targeting both the tubulin colchicine site and AhR (Figure 1).
The structure−activity relationship was investigated by
modifying the indole/aromatic nucleus. According to the
results of molecular docking, it could be inferred that the
1. INTRODUCTION
Breast cancer is the most prevalent cancer in females,
accounting for 30% of new cancer cases, making it the second
leading cause of cancer deaths in female patients.1 The
recurrence and metastasis of breast cancer are important
factors affecting the prognosis of breast cancer patients.2
Consequently, it is imminent to find safe and effective
therapeutic drugs to fight breast cancer, which is easy to
metastasize and invade and difficult to cure. The development
of new antimitotic drugs is currently an effective chemo-
therapeutic strategy in the treatment of breast cancer.3 Since
tubulin is the key structure of mitotic spindles, disruption of
tubulin dynamics by antimitotic drugs can arrest cell division,
thereby preventing cancer proliferation.4 Antitubulin therapy
has shown substantial therapeutic effects in clinical practice.
However, since tubulin is widely present in cancer cells and
normal cells, microtubule inhibitors have obvious toxicity in
normal tissues. Additionally, patients’ response rates are
different and unpredictable, possibly due to cancer metastasis.5
Cancer metastasis is closely associated with the tumor
microenvironment and is the cause of cancer death.6 Evidence
has shown that the aryl hydrocarbon receptor (AhR) is
involved in the control of breast cancer metastasis and
proliferation.7,8 Furthermore, AhR was reported to activate
members of the cytochrome P450 (CYP450) family, with
CYP1A1 being crucial for regulating breast cancer cell
proliferation and metastasis.8,9 Nowadays, the development
of the AhR/CYP1A1 signaling pathway inhibitors has become
Received: June 18, 2021
https://doi.org/10.1021/acs.jmedchem.1c01099
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reference 18. As shown in Table 1, the influence of the
substituents on the benzene ring for the activity was as follows:
4-N,N-diethylamino > 4-N,N-dimethylamino > 3,4-OCH2O >
3,4-OCH3 > CH3 > benzene > 3,4,5-OCH3/CF3. These results
showed that the electron-rich substituents contributed to the
increase of the antiproliferative activity, while the activity
decreased with the increase of electron-withdrawing groups.
Besides, when the benzene ring was replaced by indole, the
antiproliferative activities increased greatly, as the IC50 values
(MCF-7 cells) of compounds 9, 10, 11, and 12 were less than
4.5 nM and all better than CA-4 (IC50 = 4.8 nM). Moreover,
the activities were further improved by replacing the 3-bit
carbon atom of the quinoline ring with a nitrogen atom.
Furthermore, compound 12 (IC50 = 0.9 nM) showed
inhibitory activity on breast cancer cells greater than 11
(IC50 = 2.1 nM) as the 2-bit hydrogen on the indole ring was
replaced by methyl groups.
Human breast cancer adriamycin-resistant cells (MCF-7/
ADR) and two human normal cell lines including human
normal hepatocyte cells (L-O2) and human normal mammary
cells (MCF-10A) were chosen to further evaluate the reversal
of drug resistance and cytotoxicities of target compounds. The
cytotoxicities of most tested compounds in MCF-7/ADR were
at the nanomolar level (from 0.9 to 912 nM) and were weaker
in human normal cells than those in cancer cells.
Figure 1. Design strategy of target compounds.
inhibitors we designed could effectively bind to the colchicine
site. In this paper, the synthesis of target compounds, the
evaluation of their antitumor activities, and the clarification of
the antitumor mechanism for the most potential compound 12
have been reported.
2.2.2. Effects on Microtubule Networks of MCF-7 Cells. To
elucidate whether such dual-receptor inhibitors targeted the
microtubule, compound 12 was evaluated for its effect on
microtubule networks of MCF-7 cells. Colchicine, an effective
microtubule-destabilizing agent, was used as the control. As
exhibited in Figure 2, the tubulin networks in negative control
cells were normally arranged with plentiful and fibrous tubulins
surrounded by the cell nuclei. However, when treated with 12
and colchicine, the microtubule tissues in the cells were
severely contracted, indicating that the microtubule tissues
were damaged. With the increase in concentration, the damage
degree of microtubule tissues in MCF-7 cells also gradually
increased. Specifically, microtubules were less affected at 0.5
nM and the network structure was clear, with the effect of
colchicine produced being the same as 200 nM. When the
concentration reached 4 nM, only sporadic microtubule tissues
were found around the nucleus, indicating that 12 induced
2. RESULTS AND DISCUSSION
2.1. Chemical Synthesis. The synthetic sequence of dual-
receptor inhibitors has been described in Scheme 1. The
designed target products were obtained from 2-methyl-4-
chloroquinoline and 2-methyl-4-chloroquinazoline. First, 2-
methyl-4-chloroquinoline/2-methyl-4-chloroquinazoline and
aniline/indoleamine derivatives were mixed in the presence
of concentrated sulfuric acid to obtain the intermediates.
Subsequently, the target compounds were obtained from the
intermediates in the presence of sodium hydrogen and
iodomethane.
2.2. Biological Assessment. 2.2.1. In Vitro Antiprolifer-
ative Activity. To preliminarily determine the anti-breast-
cancer activity in vitro, the IC50 value of all target compounds
against MCF-7 cells was tested with microtubules overex-
pressed by the MTT assay. As a tubulin inhibitor,
combretastatin A-4 (CA-4) was included as a comparative
Scheme 1. Reagents and Conditions: (a) Aniline/Indoleamine Derivatives, Concentrated Sulfuric Acid, Isopropanol, Reflux, 4
h; (b) Sodium Hydride, Iodomethane, Dimethyl Formamide, rt, 12 h
B
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Table 1. Inhibitory Effects of Target Products on Cancer and Normal Cell Lines
a
IC50 (μM)
compounds
MCF-7
MCF-7/ADR
MCF-10A
L-O2
1
2
3
4
5
6
7
8
>1
0.912 0.036
0.029 0.001
0.054 0.003
0.046 0.001
0.859 0.031
0.817 0.020
0.033 0.001
0.267 0.020
0.0061 0.001
0.0022 0.0001
0.0021 0.0001
0.0009 0.00001
0.0031 0.0001
>1
4.121 0.816
2.172 0.680
1.527 0.196
2.497 0.120
3.839 0.698
3.447 0.749
0.227 0.019
0.031 0.001
0.010 0.007
0.0073 0.0005
0.0073 0.0006
0.0046 0.0001
0.0103 0.0004
0.097 0.004
0.103 0.019
0.054 0.001
>1
0.473 0.011
0.184 0.023
0.127 0.021
>1
>1
>1
0.943 0.046
0.025 0.009
0.0045 0.0003
0.0024 0.0001
0.0021 0.0002
0.0009 0.00006
0.0048 0.0002
0.049 0.001
0.012 0.001
0.024 0.001
0.010 0.001
0.016 0.001
0.0093 0.0008
0.099 0.003
9
10
11
12
CA-4
a
Values are expressed as mean SD of each compound from three independent experiments.
Figure 2. Effects of 12 on the cellular tubulin system intuitively observed by immunofluorescence.
destructions of the inner microtubule, which might ultimately
cause apoptosis.
2.2.3. Evaluation of Tubulin Polymerization In Vitro. 12
was then evaluated for its effect on microtubule dynamics in
MCF-7 cells. Paclitaxel and CA-4 were used as controls. As
exhibited in Figure 3, paclitaxel showed a significant effect of
the colchicine-binding site. As an alkylating reagent, EBI could
specifically cross-connect the Cys354 and Cys239 residues of
β-tubulin, which were key handles for the binding site of
colchicine.19 EBI could help materialize a β-tubulin adduct that
would be prevented by antimitotic agents occupying
colchicine-binding sites. As shown in Figure 4, 12 exerted
the same inhibitory effect as colchicine to antagonize the
generation of the β-tubulin adduct band. This result suggested
that 12 could inhibit microtubule polymerization by
combining with the colchicine site.
2.2.5. Docking Analysis of 12 with Microtubule. To further
describe the potential binding way of 12 to the microtubule,
the subunit of the tubulin complex with CA-4 (PDB: 5LYJ)
was selected as a template to simulate molecular docking
analysis.20 The result revealed that the indole ring penetrated
into the hydrophobic cavity surrounded with residues ASN-
258, MET-259, LYS-352, and THR-314 (Figure 5A). The 1-N
on the quinazoline scaffold of 12 and the crucial residue Cys
241 constituted a hydrogen bond. 12 and CA-4 shared a
remarkably similar combination with the colchicine-binding
cavity (Figure 5B). This result indicated that the quinoline
scaffold was a substitute for the 3,4,5-trimethoylphenyl
fragment and the indole ring substitutes the other benzene
ring upon binding to the colchicine site.
Figure 3. Effect of compound 12 on tubulin polymerization. The
experiments were performed three times.
promoting microtubule polymerization, whereas CA-4 pro-
duced a noteworthy promotion of microtubule depolymeriza-
tion. The effect of 12 was similar to CA-4, which indicated that
12 might act as a microtubule depolymerization agent by
binding to colchicine sites.
2.2.4. EBI Competition Assay. Consequently, the N,N′-
ethylenebis(iodoacetamide) (EBI) competition assay was
performed to determine whether 12 could bind directly to
2.2.6. 12 Induced G2/M Phase Arrest by Regulating the
Related Proteins. Since the majority of antimicrotubule drugs
are likely to disrupt cell-cycle distribution,21 propidium iodide
(PI) staining was adopted to detect the performance of 12 on
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Figure 4. Competition evaluation of 12 and colchicine with EBI on MCF-7 cells. **p < 0.01 vs negative control (mean SD, n = 3).
Figure 5. Molecular docking models for 12 binding with microtubule (PDB: 5LYJ). (A) 12 (white). (B) 12 (white) and CA-4 (pink).
the cell-cycle progression of MCF-7. As exhibited in Figure
6A,B, incubation of 12 resulted in G2/M phase arrest. In
comparison with the control, the incubation of MCF-7 cells as
the concentration of compound 12 increases resulted in the
cell percentage increasing from 28.79 to 46.87% in the G2/M
phase.
Cyclin B1, Cdc2, and Cdc25c were three important proteins
in the cell cycle associated with the G2/M phase. The
activation of Cdc2 kinase regulated mitosis in eukaryotic cells,
which includes Cdc25c phosphorylation and Cyclin B1
combination.22 To explore the action of cell-cycle arrest in
MCF-7 cells, the effect on cell-cycle-related proteins was
evaluated. As shown in Figure 6, 12 decreased the levels of
Cdc2 and Cdc25c proteins and increased the levels of Cyclin
B1 in a concentration-dependent manner, indicating that the
G2/M arrest induced by 12 might be related to the change in
expressions of Cyclin B1, Cdc2, and Cdc25c.
2.2.7. 12 Regulated Apoptosis-Associated Proteins to
Induce Apoptosis. The arrest on cell cycle of tubulin
polymerization inhibitors such as CA-4 could lead to cell
apoptosis.23 To determine whether 12 induced apoptosis, dual
staining with Annexin V-FITC and propidium iodide (PI) was
carried out in MCF-7 cells. As noted in Figure 7, after being
exposed to 0.5, 1, and 2 nM 12 for 24 h, the proportions of
total apoptotic (Q2 + Q3) MCF-7 cells were 4.45, 35.23, and
57.20%, respectively.
The regulation effects of 12 on apoptosis-related proteins
were then analyzed. As shown in Figure 7, 12 decreased the
level of Bcl-2 and improved the levels of Bax and Bad, and the
levels of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-
PARP were upregulated with the increase in concentration of
12. The result indicated that compound 12 induced cell
apoptosis by affecting the levels of apoptosis-related proteins.
2.2.8. Effect of 12 on Mitochondrial Membrane Potential
(Δψm). More and more evidence showed that mitochondria
played a critical role in the regulation of cell functions, and
mitochondrial dysfunction was implicated in apoptosis.24 To
investigate whether 12 could induce mitochondrial dysfunc-
tion, JC-1 staining of mitochondria was used to detect the
mitochondrial membrane potential in MCF-7. As presented in
Figure 8, after MCF-7 cells were administrated with varying
concentrations of 12 (0.5, 1, and 2 nM) for 24 h, the green
fluorescence intensity (JC-1 monomers, depolarized mitochon-
dria) raised up to 24.7% from 6.49% correspondingly,
indicating that 12 might disrupt mitochondrial function to
induce breast-cancer-cell apoptosis.
2.2.9. Action of 12 on Reactive Oxygen Species
Accumulation. The increase of intracellular reactive oxygen
species (ROS) in mitochondria could promote the depolariza-
tion of the mitochondrial membrane, thus activating the
apoptosis of cancer cells.25 Therefore, we tested whether 12
might stimulate the generation of ROS in MCF-7 cells. As
exhibited in Figure 9, the production of intracellular ROS was
induced in a concentration-dependent manner, and pretreat-
ment of 2.5 mM N-acetylcysteine (ROS scavenging agent)
inhibited the increase of ROS.
2.2.10. 12 Induced Cytostasis and Apoptosis and
Inhibited Cell Migration by the Regulation of AhR. Designed
as a dual-target inhibitor, compound 12 was expected to inhibit
the AhR/CYP1A1 signaling pathway, thus inducing cell
cytostasis. Therefore, Western blot analysis was utilized to
measure the levels of AhR and CYP1A1. MCF-7 cells were
incubated with varying concentrations (1, 2, 4 nM) of 12 for
24 h, and the AhR antagonist BAY 2416964 was used as a
positive control (PC). Then, the levels of AhR and CYP1A1
were detected. The result (Figure 10) showed that as the
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Figure 6. 12 induced G2/M phase arrest in MCF-7 cells. (A) Cells were acquired and then conducted with PI staining and flow cytometry
detection. (B) Histograms showing the distribution of the cell cycle (mean SD, n = 3). (C) Regulation effects of 12 on the G2/M-related
proteins. (D) Histograms showing the relative levels of Cdc2, Cyclin B1, and Cdc25c, to β-actin. *p < 0.05, **p < 0.01 vs the negative control
(mean SD, n = 3).
concentration increased, the levels of AhR and CYP1A1
continued to decrease, which indicated that 12 could inhibit
the AhR/CYP1A1 signaling pathway in a dose-dependent
manner.
groups were 2.5, 1.5, 27.0, and 51.6%, respectively. The
differences between DMBA+12 and 12 groups indicated that
the inhibition of AhR expression was involved in the induction
capability of 12 in the apoptosis of MCF-7 cells. Compound
12 could promote apoptosis by synergistically antagonizing
microtubule polymerization and AhR expression.
To investigate whether the regulation of 12 on the AhR/
CYP1A1 signaling pathway could inhibit the proliferation of
MCF-7 cells, the expression of AhR and proliferation of MCF-
7 cells after adding the exogenous AhR agonist 7,12-
dimethylbenz[a]anthracene (DMBA) and DMBA+12 were
investigated. The relative expression of AhR in the DMBA+12
group was notably lower than in the DMBA group (Figure
11A,B), indicating that 12 successfully interfered with the
enhanced AhR expression by DMBA. The inhibitory effects of
DMBA+12 on MCF-7 cell proliferation compared with the 12
group further indicated that 12 could inhibit cell proliferation
induced by AhR (Figure 11C).
To evaluate whether 12 promoted the apoptosis of MCF-7
cells by synergistically antagonizing microtubule polymer-
ization and AhR expression, the cells were harvested and
analyzed by a flow cytometric assay after Annexin V-FITC and
PI double staining. As exhibited in Figure 12, the total
apoptotic cells in the control, DMBA, DMBA+12, and 12
2.2.11. Effect of 12 on Cell Migration of MCF-7 Cells and
AhR Gene Silenced MCF-7 Cells. Compounds designed to
inhibit AhR in this experiment were primarily intended to
reduce metastatic transmission of breast cancer cells. Cell
migration is an important part of metastatic transmission of
tumor cells and the main cause of cancer recurrence.26
Consequently, we wanted to investigate experimentally
whether 12 could inhibit AhR-regulated MCF-7 cell migration
while inhibiting tumor growth. As demonstrated in Figure 13,
the migration rates of control, DMBA, DMBA+12, and 12
groups were 11.51, 12.18, 6.34, and 3.78%, respectively.
Compared with the DMBA group (12.18%), the migration rate
of the DMBA+12 group (6.34%) was decreased, which
suggested that AhR performed a regulating role in the
migration of MCF-7 cells and 12 could partially inhibit the
migration of MCF-7 cells through inhibition of AhR.
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Figure 7. 12 induced apoptosis of MCF-7 cells. (A) Cells were acquired and stained with Annexin V and PI following flow cytometric analysis. (B)
Percentage of apoptotic cells was calculated (mean SD, n = 3). (C) Regulation effects of 12 on apoptosis-related proteins. (D) Relative levels of
Bax, Bcl-2, Bad, cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP to GADPH. *p < 0.05, **p < 0.01 vs the negative control (mean SD, n
= 3).
2.2.12. Effect of 12 on the Growth of MCF-7 Xenografts in
Nude Mice. To further detect the in vivo anticancer potent of
12, human breast cancer xenograft was established by
subcutaneous inoculation of MCF-7 cells into the right side
of mice. The animal models were randomly divided into five
groups (negative control, paclitaxel, CA-4, low dose of 12, high
dose of 12) and administered intravenously every 2 days at the
indicated doses, with eight mice per group (Figure 14A).
Tumor volume and weight of the mice were measured every 2
days. As shown in Figure 14B,D, 12 (1 mg/kg) reduced the
tumor weight by 81.1%, which was comparable to PTX
(84.2%). Although CA-4 showed excellent inhibitory activity
against MCF-7 cells, its anticancer effect (inhibition rate:
37.5% at 20 mg/kg) was less impressive than 12. Moreover,
there was no obvious impact on body weight for 12 at two
different doses (Figure 14C). H&E staining of the spleen, lung,
liver, heart, and kidney demonstrated no noticeable major
organ damage (Figure 15).
Additionally, we observed the effect of 12 on tumor
microvessel density (MVD) in vivo stained with CD31 by
immunohistochemistry. As shown in Figure 16A,B, the MVD
was significantly reduced in the 12-treated group (1 mg/kg).
Furthermore, we explored the performance of 12 on apoptosis
of tumor cells by Tunel staining. As shown in Figure 16C,D,
the cells stained green increased obviously in the tumor treated
with 12 (1 mg/kg), which indicated that 12 induced the
apoptosis of cells in solid tumors.
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Figure 8. 12 affected the mitochondrial membrane potential of MCF-7 cells. (A) Cells were treated with 12 (0.5, 1, and 2 nM) and DMSO for 24 h
prior to staining with JC-1 and then detected by flow cytometry assay. (B) Percentage of cells with J-aggregates and J-monomers. *p < 0.05, **p <
0.01 vs the negative control (mean SD, n = 3).
3. CONCLUSIONS
4. EXPERIMENTAL SECTION
4.1. Chemistry. 4.1.1. General Methods. Target compounds were
purified by silica gel column chromatography (200−300 mesh,
Qingdao Ocean Chemical Co., Ltd, China). The structures of
In conclusion, 12 novel dual-receptor inhibitors targeting both
the tubulin colchicine site and AhR were synthesized and
evaluated for their antitumor effects. Compared with CA-4
(4.5 nM), compound 12 showed better antitumor ability with
an IC50 value of 0.9 nM. Furthermore, the inhibitory potent of
12 on microtubule polymerization was investigated by
microtubule polymerization, EBI competitive inhibition, intra-
cellular microtubule immunofluorescence, and molecular
docking analysis. These assays indicated that 12 significantly
inhibited tubulin polymerization by binding to the colchicine
site. Further research showed that 12 induced cell-cycle arrest
in the G2/M phase and cell apoptosis by regulating the levels
of G2/M-related and apoptosis-related proteins. In addition,
12 could depolarize the mitochondrial membrane potential of
MCF-7 cells and induce ROS production. Besides, 12 could
inhibit the proliferation of MCF-7 cells by synergistically
antagonizing microtubule polymerization and AhR expression.
The wound healing assay indicated that 12 could inhibit the
migration of MCF-7 cells by inhibiting AhR. Moreover, in vivo
antitumor activities of 12 were verified in MCF-7 breast cancer
xenograft mouse models. Most notably, 12 inhibited tumor
proliferation with a very small dose (1 mg/kg), while CA-4 had
a weak inhibitory effect with a high dose (20 mg/kg).
Collectively, these findings indicated that 12 was a novel dual-
receptor inhibitor with clinical potential for cancer treatment
and warrant further study.
1
products were elucidated by characterization with H NMR and 13C
NMR spectra (AV-300 spectrometer, Bruker Co., Ltd, Germany) in
the indicated solvents (CDCl3 or DMSO-d6). HRMS spectra were
detected on a Finnigan MAT 95 spectrometer (Finnigan, Germany).
The purity of all inhibitors 1−12 was characterized by HPLC analysis
(SHIMADZU Prominence-i LC-2030C HPLC system equipped with
a PDA detector) to be ≥95%.
4.1.2. General Procedure for the Synthesis of Compounds 1−12.
ArCl and aniline derivatives were added successively in isopropanol.
One drop of HClconc was added, and the composites were stirred at 80
°C for 4 h. The residue was washed and dried to obtain the
intermediate. The intermediate was mixed into a DMF (5 mL)
solution of NaH (3 equiv) at 0 °C. Then, CH3I (3 equiv) was added
at 0 °C and the composites were stirred for 12 h. Further treatment of
the crude products by silica gel column chromatography provided
target compounds 1−12.
4.1.3. N,2-Dimethyl-N-(3,4,5-trimethoxyphenyl)quinolone-4-
amine (1). 3,4,5-Trimethoxyaniline (103 mg, 0.565 mmol) was
mixed with 4-chloroquinoline (100 mg, 0.565 mmol) in 10 mL of
isopropanol. One drop of HClconc was added, and the solution was
stirred at 80 °C for 4 h. The residue was washed and dried to obtain
the intermediate (117 mg). The intermediate was added to a mixture
of NaH (26 mg) in DMF (5 mL) at 0 °C. The CH3I (67 μL) was
added dropwise at 0 °C, and the solution was stirred for 12 h. Further
treatment of the crude mixture by silica gel column chromatography
provided compound 1 (yellow powder, 91 mg, 48% yield). Mp
126.4−128.6 °C; 1H NMR (300 MHz, CDCl3) δH: 2.75 (3H, s), 3.47
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Figure 9. 12 induced ROS accumulation in MCF-7 cells. (A) Generation of ROS was quantified using DCFH-DA followed by FACScan flow
cytometry. (B) Relative levels of fluorescent DCF. *p < 0.05, **p < 0.01 vs the negative control (mean SD, n = 3).
Figure 10. Compound 12 reduced AhR and CYP1A1 expressions in MCF-7 cells. (A, B) Expression and relative levels of AhR/GAPDH and
CYP1A1/GAPDH. **p < 0.01, ***p < 0.001 vs the negative control (mean SD, n = 3).
Figure 11. Expression of AhR by adding DMBA (400 nM), DMBA (400 nM)+12 (2 nM) and 12 (2 nM) in MCF-7 cells. (A, B) Expression and
relative levels of AhR. (C) Cell growth curve of MCF-7 cells. **p < 0.01, ***p < 0.001 vs DMBA (mean SD, n = 3).
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Figure 12. Effects of DMBA (400 nM), DMBA (400 nM)+12 (2 nM), and 12 (2 nM) on apoptosis of MCF-7 cells. ***p < 0.001 vs the DMBA
group; ###p < 0.001 vs the DMBA+12 group (mean SD, n = 3).
Figure 13. Effect of DMBA (400 nM), DMBA (400 nM)+12 (2 nM), and 12 (2 nM) on the cell migration of MCF-7 cells. (A) Histograms
showing the width of the scratches (0 and 24 h). (B) Migration rate: calculate the area surrounded by cells before and after migration with ImageJ
software, and mobility = (area before migration − area after migration)/area before migration.
(3H, s), 3.70 (6H, s), 3.83 (3H, s), 6.14 (2H, s), 7.01 (1H, s), 7.61
(1H, t, J = 7.5 Hz), 7.69 (1H, d, J = 9.0 Hz), 8.03 (1H, d, J = 9.0 Hz);
13C NMR (75 MHz, CDCl3) δC: 161.2, 155.6 (C×2), 151.7, 151.4,
m), 7.06 (1H, s), 7.19 (1H, t, J = 7.5 Hz), 7.48−7.55 (2H, m), 7.81
(1H, d, J = 9.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 161.0, 155.8,
151.7, 151.1, 147.1, 146.0, 130.7 (C×2), 130.6, 126.4, 126.1, 123.9,
115.0, 114.2, 108.3, 58.1, 57.8, 44.5, 27.0. HRMS (ESI) calculated for
C19H20N2O2 [M + H]+ 309.1603, found 309.1601. Purity: 98.303%
(by HPLC).
147.8, 130.9 (C×2), 130.8, 126.5, 126.1, 124.5, 116.2, 100.1 (C×2),
62.6, 58.0 (C×2), 43.8, 27.0. HRMS (ESI) calculated for C20H22N2O3
[M + H]+ 339.1709, found 339.1706. Purity: 99.278% (by HPLC).
4.1.4. N,2-Dimethyl-N-(3,4-dimethoxyphenyl)quinoline-4-amine
(2). 3,4-Dimethoxyaniline (86 mg, 0.565 mmol) was mixed into a
mixture of 4-chloroquinoline (100 mg, 0.565 mmol) in 10 mL of
isopropanol. One drop of HClconc was added, and the solution was
stirred at 80 °C for 4 h. The residue was washed and dried to obtain
the intermediate (120 mg). The intermediate was added to a mixture
of NaH (29 mg) in DMF (5 mL) at 0 °C. The CH3I (76 μL) was
added at 0 °C, and the solution was stirred for 12 h. Further treatment
of the crude mixture by silica gel column chromatography provided
compound 2 (brown powder, 94 mg, 54% yield). Mp 99.8−101.4 °C;
1H NMR (300 MHz, DMSO-d6) δH: 2.61 (3H, s), 3.39 (3H, s), 3.65
4.1.5. N,2-Dimethyl-N-(4-methylphenyl)quinoline-4-amine (3).
4-Methylaniline (60 mg, 0.565 mmol) was mixed into a mixture of
4-chloroquinoline (100 mg, 0.565 mmol) in 10 mL of isopropanol.
One drop of HClconc was added, and the solution was stirred at 80 °C
for 4 h. The residue was washed and dried to obtain the intermediate
(105 mg). The intermediate was added to a mixture of NaH (31 mg)
in DMF (5 mL) at 0 °C. The CH3I (79 μL) was added at 0 °C, and
the solution was stirred for 12 h. Further treatment of the crude
mixture by silica gel column chromatography provided compound 3
1
(yellow powder, 89 mg, 60% yield). Mp 110.3−112.1 °C; H NMR
(300 MHz, DMSO-d6) δH: 2.21 (3H, s), 2.61 (3H, s), 3.38 (3H, s),
6.77 (2H, d, J = 9.0 Hz), 7.03 (2H, d, J = 9.0 Hz), 7.12 (1H, s), 7.21
(3H, s), 3.69 (3H, s), 6.31 (1H, dd, J = 9.0, 3.0 Hz), 6.75−6.79 (2H,
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Figure 14. 12 inhibited breast cancer xenograft growth in vivo. Negative control (1), paclitaxel (PTX) (10 mg/kg, 2), CA-4 (20 mg/kg, 3), 12 (0.5
mg/kg, 4), and 12 (1 mg/kg, 5). (A) Images of tumors at 3 weeks after initiation of drug administration. (B) Changes of compound administration
on tumor size in mice. (C) Changes of compound administration on mice body weight. (D) Weight of the excised tumors of each group. ***p <
0.001 vs control.
Figure 15. H&E staining of the heart, liver, spleen, lung, and kidney of mice.
(1H, t, J = 9.0 Hz), 7.50 (1H, d, J = 9.0 Hz), 7.56 (1H, d, J = 9.0 Hz),
7.84 (1H, d, J = 9.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 161.3,
155.9, 151.5, 149.5, 132.9, 131.6 (C×2), 130.9, 130.7, 126.3 (C×2),
124.6, 121.5 (C×2), 116.0, 43.7, 27.1, 22.2. HRMS (ESI) calculated
for C18H18N2 [M + H]+ 263.1548, found 263.1543. Purity: 99.347%
(by HPLC).
(3H, s), 3.35 (3H, s), 5.98 (2H, s), 6.29 (1H, dd, J = 9.0, 3.0 Hz),
6.68 (1H, d, J = 3.0 Hz), 6.75 (1H, d, J = 9.0 Hz), 7.08 (1H, s), 7.23
(1H, t, J = 7.5 Hz), 7.52−7.56 (2H, m), 7.83 (1H, d, J = 9.0 Hz); 13C
NMR (75 MHz, CDCl3) δC: 161.2, 155.8, 151.5, 150.2, 147.2, 144.9,
130.9, 130.6, 126.3, 126.2, 124.2, 115.4, 114.8, 110.2, 104.9, 103.0,
44.5, 27.1. HRMS (ESI) calculated for C18H16N2O2 [M + H]+
293.1290, found 293.1287. Purity: 98.459% (by HPLC).
4.1.7. N,2-Dimethyl-N-(4-trifluoromethylphenyl)quinoline-4-
amine (5). 4-Trifluoromethylaniline (91 mg, 0.565 mmol) was
mixed into a mixture of 4-chloroquinoline (100 mg, 0.565 mmol) in
10 mL of isopropanol. One drop of HClconc was added, and the
solution was stirred at 80 °C for 4 h. The residue was washed and
dried to obtain the intermediate (137 mg). The intermediate was
added to a mixture of NaH (33 mg) in DMF (5 mL) at 0 °C. The
CH3I (84 μL) was added at 0 °C, and the solution was stirred for 12
h. Further treatment of the crude mixture by silica gel column
chromatography provided compound 5 (white powder, 121 mg, 68%
4.1.6. N,2-Dimethyl-N-(3,4-methylenedioxyphenyl)quinoline-4-
amine (4). 3,4-Methylenedioxyaniline (77 mg, 0.565 mmol) was
mixed into a mixture of 4-chloroquinoline (100 mg, 0.565 mmol) in
10 mL of isopropanol. One drop of HClconc was added, and the
solution was stirred at 80 °C for 4 h. The residue was washed and
dried to obtain the intermediate (110 mg). The intermediate was
added to a mixture of NaH (28 mg) in DMF (5 mL) at 0 °C. The
CH3I (74 μL) was added at 0 °C, and the solution was stirred for 12
h. Further treatment of the crude mixture by silica gel column
chromatography provided compound 4 (brown powder, 81 mg, 49%
1
yield). Mp 71.8−73.2 °C; H NMR (300 MHz, DMSO-d6) δH: 2.61
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Figure 16. Tumor section staining. (A) Typical anti-CD31 immunohistochemistry images from four MCF-7 xenografts. (B) MVD measured by
immunohistochemical staining against CD31 in MCF-7 xenografts administrated with a negative control and 12 (1 mg/kg). **p < 0.01 vs the
negative control. (C) Representative images of TUNEL fluorescent staining from four tumor xenografts. DNA was stained blue, and the apoptotic
cells were stained green. (D) Ratio of positive cells by TUNEL fluorescent staining. ***p < 0.001 vs the negative control.
1
yield). Mp 129.6−132.4 °C; H NMR (300 MHz, DMSO-d6) δH:
2.66 (3H, s), 3.45 (3H, s), 6.77 (2H, d, J = 9.0 Hz), 7.39 (1H, s),
7.41−7.49 (3H, m), 7.62 (1H, d, J = 9.0 Hz), 7.70 (1H, t, J = 7.5 Hz),
7.99 (1H, d, J = 9.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 160.2,
152.7, 151.4, 149.9, 129.9 (C×2), 129.4 (C×2), 126.4, 126.4, 125.9,
123.6, 123.4, 118.8, 114.9 (C×2), 40.5, 25.3. HRMS (ESI) calculated
for C18H15F3O2 [M + H]+ 317.1266, found 317.1266. Purity: 99.485%
(by HPLC).
40.9 (C×2), 25.3. HRMS (ESI) calculated for C19H21N3 [M + H]+
292.1814, found 292.1812. Purity: 99.077% (by HPLC).
4.1.10. N,2-Dimethyl-N-(4-N,N-diethylaminophenyl)quinoline-4-
amine (8). N,N-Diethyl-1,4-phenylenediamine (93 mg, 0.565 mmol)
was mixed into a mixture of 4-chloroquinoline (100 mg, 0.565 mmol)
in 10 mL of isopropanol. One drop of HClconc was added, and the
solution was stirred at 80 °C for 4 h. The residue was washed and
dried to obtain the intermediate (138 mg). The intermediate was
added to a mixture of NaH (33 mg) in DMF (5 mL) at 0 °C. The
CH3I (84 μL) was added at 0 °C, and the solution was stirred for 12
h. Further treatment of the crude mixture by silica gel column
chromatography provided compound 8 (brown powder, 43 mg, 24%
4.1.8. N,2-Dimethyl-N-(4-biphenylyl)quinoline-4-amine (6). 4-
Biphenylamine (96 mg, 0.565 mmol) was mixed into a mixture of
4-chloroquinoline (100 mg, 0.565 mmol) in 10 mL of isopropanol.
One drop of HClconc was added, and the solution was stirred at 80 °C
for 4 h. The residue was washed and dried to obtain the intermediate
(140 mg). The intermediate was added to a mixture of NaH (33 mg)
in DMF (5 mL) at 0 °C. The CH3I (84 μL) was added at 0 °C, and
the mixture was stirred for 12 h. Further treatment of the crude
mixture by silica gel column chromatography provided compound 6
(yellow powder, 103 mg, 56% yield). Mp 135.8−137.4 °C; 1H NMR
(300 MHz, DMSO-d6) δH: 2.65 (3H, s), 3.47 (3H, s), 6.88 (2H, d, J =
9.0 Hz), 7.26−7.43 (5H, m), 7.53 (2H, d, J = 6.0 Hz), 7.58−7.65
(4H, m), 7.93 (1H, d, J = 9.0 Hz); 13C NMR (75 MHz, CDCl3) δC:
159.7, 153.8, 149.6, 148.8, 140.6, 133.7, 129.4, 128.7 (C×2), 127.8
(C×2), 126.7, 126.5 (C×2), 125.1, 124.3, 123.1, 118.5 (C×2), 115.9,
41.5, 25.3. HRMS (ESI) calculated for C23H20N2 [M + H]+ 325.1705,
found 325.1704. Purity: 99.161% (by HPLC).
4.1.9. N,2-Dimethyl-N-(4-N,N-dimethylaminophenyl)quinoline-
4-amine (7). N,N-Dimethyl-1,4-phenylenediamine (77 mg, 0.565
mmol) was mixed into a mixture of 4-chloroquinoline (100 mg, 0.565
mmol) in 10 mL of isopropanol. One drop of HClconc was added, and
the solution was stirred at 80 °C for 4 h. The residue was washed and
dried to obtain the intermediate (102 mg). The intermediate was
added to a mixture of NaH (26 mg) in DMF (5 mL) at 0 °C. The
CH3I (69 μL) was added at 0 °C, and the mixture was stirred for 12 h.
Further treatment of the crude mixture by silica gel column
chromatography provided compound 7 (brown powder, 64 mg,
1
yield). Mp 118.7−120.3 °C; H NMR (300 MHz, CDCl3) δH: 1.16
(6H, t, J = 6.0 Hz), 2.72 (3H, s), 3.33 (4H, d, J = 6.0 Hz), 3.41 (3H,
s), 6.62 (2H, d, J = 9.0 Hz), 6.84 (1H, s), 6.91 (2H, d, J = 9.0 Hz),
7.15 (1H, t, J = 7.5 Hz), 7.52 (1H, t, J = 7.5 Hz), 7.64 (1H, d, J = 9.0
Hz), 7.95 (1H, d, J = 9.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 159.0,
154.2, 144.8, 139.9, 130.9, 128.6, 128.5, 125.3, 124.1 (C×2), 123.8,
121.6, 113.2 (C×2), 110.13, 44.5 (C×2), 43.4, 25.4, 12.6 (C×2).
HRMS (ESI) calculated for C21H25N3 [M + H]+ 320.2127, found
320.2126. Purity: 98.652% (by HPLC).
4.1.11. N,2-Dimethyl-N-(1-methyl-7-azaindole-5-yl)quinoline-4-
amine (9). 7-Azazindole-5-amine (75 mg, 0.565 mmol) was mixed
into a mixture of 4-chloroquinoline (100 mg, 0.565 mmol) in 10 mL
of isopropanol. One drop of HClconc was added, and the solution was
stirred at 80 °C for 4 h. The residue was washed and dried to obtain
the intermediate (132 mg). The intermediate was added to a mixture
of NaH (35 mg) in DMF (5 mL) at 0 °C. The CH3I (90 μL) was
added at 0 °C, and the solution was stirred for 12 h. Further treatment
of the crude mixture by silica gel column chromatography provided
compound 9 (yellow powder, 29 mg, 17% yield). Mp 83.5−85.8 °C;
1H NMR (300 MHz, DMSO-d6) δH: 2.74 (3H, s), 3.51 (3H, s), 3.89
(3H, s), 6.32 (1H, d, J = 3.0 Hz), 6.95 (1H, s), 7.15 (1H, t, J = 7.5
Hz), 7.18 (1H, d, J = 3.0 Hz), 7.48−7.53 (2H, m), 7.60 (1H, d, J =
9.0 Hz), 7.96 (1H, d, J = 9.0 Hz), 8.20 (1H, d, J = 3.0 Hz); 13C NMR
(75 MHz, CDCl3) δC: 161.00 156.0, 146.8, 142.9, 141.2, 131.8, 130.8,
130.5, 126.6, 126.1, 123.6, 123.4, 122.3, 113.4, 111.8, 100.9, 45.6,
32.9, 27.2. HRMS (ESI) calculated for C19H18N4 [M + H]+ 303.1610,
found 303.1608. Purity: 99.533% (by HPLC). Solubility (H2O, 2.23
mg/mL).
1
39% yield). Mp 99.6−101.4 °C; H NMR (300 MHz, CDCl3) δH:
2.72 (3H, s), 2.93 (6H, s), 3.41 (3H, s), 6.68 (2H, d, J = 9.0 Hz), 6.86
(1H, s), 6.93 (2H, d, J = 9.0 Hz), 7.15 (1H, t, J = 7.5 Hz), 7.52 (1H, t,
J = 7.5 Hz), 7.61 (1H, d, J = 9.0 Hz), 7.96 (1H, d, J = 9.0 Hz); 13C
NMR (75 MHz, CDCl3) δC: 159.0, 154.2, 149.1, 147.4, 140.7, 128.7,
128.5, 125.2, 123.9, 123.8 (C×2), 121.6, 113.7 (C×2), 110.4, 43.3,
K
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4.1.12. N,2-Dimethyl-N-(1,2-dimethylindol-5-yl)quinoline-4-
amine (10). 5-Amino-2-methylindole (83 mg, 0.565 mmol) was
mixed into a mixture of 4-chloroquinoline (100 mg, 0.565 mmol) in
10 mL of isopropanol. One drop of HClconc was added, and the
solution was stirred at 80 °C for 4 h. The residue was washed and
dried to obtain the intermediate (127 mg). The intermediate was
added to a solution of NaH (32 mg) in DMF (5 mL) at 0 °C. The
CH3I (82 μL) was added at 0 °C, and the solution was stirred for 12
h. Further treatment of the crude mixture by silica gel column
chromatography provided compound 10 (yellow powder, 28 mg, 16%
lung cancer cells (A549), human normal mammary cells (MCF-10A),
and adriamycin-resistant human breast cancer cells (MCF-7/ADR)
were cultured in RPMI 1640 medium.
4.2.3. MTT Assay. The proliferation activity of human tumor cell
lines (MCF-7, HepG-2, A549, MCF-7/ADR) and human normal cells
(MCF-10A, L-O2) was measured using the MTT assay. Cells (5 ×
103 cells per well) were plated in 96-well plates. After being incubated
overnight, cells were administrated with target compounds at varying
concentrations and incubated for 48 h. Then, the plates were
incubated for 4 h after the MTT solution was added. After that, the
culture medium was replaced with 100 μL of dimethyl sulfoxide. The
absorbance was quantitatively detected by a microplate reader
(Biorad, Nazareth, Belgium) at 570 nm. All experiments were carried
out three times.
4.2.4. Immunofluorescent Staining. MCF-7 cells were incubated
in six-well plates (4 × 105 cells) and then given DMSO, 12 (0.5, 1, 2,
4 nM), and colchicine (200 nM) for 48 h. After being treated with
goat serum albumin (50−100 μL) for 20 min, cells were cultured with
anti-β-tubulin for 2 h. After fluorescent antibody staining and 4,6-
diamino-2-phenylindole (DAPI) labeling of the nucleus, the plates
were washed with PBS three times. Cells were subsequently
monitored by employing an LSM 570 laser confocal microscope
(Carl Zeiss, Germany).
4.2.5. Evaluation of Tubulin Polymerization Inhibition In Vitro.
In PEM buffer, 2 mg/mL tubulin (cytoskeleton) was resuspended.
Then, the suspension was preincubated on ice with target products or
the vehicle DMSO. Before monitoring the tubulin polymerization
reaction, PEG was added to make the ultimate concentration 3 mg/
mL. The absorbance was recorded every 5 min for 60 min at 37 °C by
a spectrophotometric operation at 350 nm.
4.2.6. EBI Competition Evaluation. MCF-7 cells were incubated in
six-well cell culture plates (5 × 105 cells) for 24 h. Then, cells were
first incubated with DMSO, 12 (1, 4 nM), and colchicine (10 μM) for
2 h, followed by processing with EBI (100 μM). After that, the cells
were collected and cell extracts were employed for the Western blot
assay.
4.2.7. Molecular Docking Analysis. The X-ray crystal structure of
the α,β-tubulin−CA-4 complex (PDB ID: 5LYJ) was obtained from
the Protein Data Bank. Discovery Studio modules were employed to
prepare the complex by removing the stathmin-like domain,
colchicine, water molecules, and subunits C and D. The mimic
procedure was carried out using the DOCK system in Discovery
Studio 3.0 software, and the generating graphics were acquired from
PyMOL software.
4.2.8. Cell-Cycle Assay. MCF-7 cells were incubated in six-well cell
culture plates for 24 h (4 × 105 cells). Then, DMSO and 12 (0.5, 1,
and 2 nM) were added to the plates. After that, cells were mixed with
ethyl alcohol (75%) at −20 °C for 12 h. Next, the plates were washed
with buffer A to remove ethanol. Staining was performed according to
the cell-cycle analysis kit, and then, analysis was performed with a flow
cytometric assay. The assay was carried out 3 times.
4.2.9. Cell Apoptosis Evaluation. MCF-7 cells were incubated in
six-well cell culture plates for 24 h (4 × 105 cells). Then, DMSO and
12 (0.5, 1, and 2 nM) were dropped into each well. After the next
culturation of 24 h, cells were collected and cleaned twice with PBS
(2000 rpm + 5 min). After that, the cells were resuspended in 500 μL
of binding buffer; then, 5 μL of annexin and 5 μL of propidium iodide
were added one by one. The mixed solution was incubated for 15 min
in the shade and detected by a flow cytometric assay. The assay was
carried out 3 times.
4.2.10. Mitochondrial Membrane Potential Evaluation. The
MCF-7 cell suspension was inoculated into six-well plates for 24 h (4
× 105 cells). Then, cells were cultured in a fresh medium with
dimethyl sulfoxide and 12 (0.5, 1, and 2 nM) for another 24 h. After
the next culturation of 24 h, cells were collected and cleaned twice
with PBS (2000 rpm + 5 min), followed by incubation with JC-1 (5
mg/mL) for 30 min shielded from light. After rinsing 3 times, the cells
were suspended in PBS, and the fluorescence of JC-1 was detected by
a flow cytometric assay. The assay was carried out 3 times.
1
yield). Mp 120.9−123.3 °C; H NMR (300 MHz, DMSO-d6) δH:
2.35 (3H, s), 2.62 (3H, s), 3.40 (3H, s), 3.62 (3H, s), 6.07 (1H, s),
6.84 (1H, dd, J = 9.0, 3.0 Hz), 6.98−7.01 (3H, m), 7.31 (1H, d, J =
9.0 Hz), 7.41−7.46 (2H, m), 7.76 (1H, d, J = 6.0 Hz); 13C NMR (75
MHz, CDCl3) δC: 158.9, 154.7, 143.7, 137.9, 134.6, 128.6 (C×2),
128.4, 125.4 (C×2), 123.8, 121.6, 117.4, 114.1, 110.1, 109.5, 99.7,
44.1, 29.5, 25.3, 12.7. HRMS (ESI) calculated for C21H21N3 [M +
H]+ 316.1814, found 316.1812. Purity: 99.694% (by HPLC).
4.1.13. N,2-Dimethyl-N-(1-methyl-7-azaindole-5-yl)quinazoline-
4-amine (11). 7-Azazindole-5-amine (75 mg, 0.562 mmol) was mixed
into a mixture of 4-chloroquinazoline (100 mg, 0.562 mmol) in 10
mL of isopropanol. One drop of HClconc was added, and the solution
was stirred at 80 °C for 4 h. The residue was washed and dried to
obtain the intermediate (145 mg). The intermediate was added to a
mixture of NaH (38 mg) in DMF (5 mL) at 0 °C. The CH3I (99 μL)
was added at 0 °C, and the solution was stirred for 12 h. Further
treatment of the crude mixture by silica gel column chromatography
provided compound 11 (brown powder, 40 mg, 24% yield). Mp
1
172.5−174.4 °C; H NMR (300 MHz, DMSO-d6) δH: 2.61 (3H, s),
3.58 (3H, s), 3.84 (3H, s), 6.46 (1H, d, J = 3.0 Hz), 6.86 (1H, d, J =
6.0 Hz), 6.97 (1H, t, J = 7.5 Hz), 7.56 (1H, t, J = 7.5 Hz), 7.61 (1H,
d, J = 3.0 Hz), 7.67 (1H, d, J = 6.0 Hz), 7.92 (1H, d, J = 3.0 Hz), 8.18
(1H, d, J = 3.0 Hz); 13C NMR (75 MHz, CDCl3) δC: 163.3, 161.9,
152.2, 146.1, 141.4, 138.6, 131.7, 130.7, 127.7, 126.1, 125.9, 124.1,
120.8, 114.5, 99.7, 43.6, 31.4, 26.4. HRMS (ESI) calculated for
C18H17N5 [M + H]+ 304.1562, found 304.1560. Purity: 96.734% (by
HPLC).
4.1.14. N,2-Dimethyl-N-(1,2-dimethylindol-5-yl)quinazoline-4-
amine (12). 5-Amino-2-methylindole (74 mg, 0.562 mmol) was
mixed into a mixture of 4-chloroquinazoline (100 mg, 0.562 mmol) in
10 mL of isopropanol. One drop of HClconc was added, and the
solution was stirred at 80 °C for 4 h. The residue was washed and
dried to obtain the intermediate (154 mg). The intermediate was
added to a mixture of NaH (38 mg) in DMF (5 mL) at 0 °C. The
CH3I (100 μL) was added at 0 °C, and the solution was stirred for 12
h. Further treatment of the crude mixture by silica gel column
chromatography provided compound 12 (white powder, 51 mg, 29%
1
yield). Mp 218.5−220.2 °C; H NMR (300 MHz, DMSO-d6) δH:
2.41 (3H, s), 2.59 (3H, s), 3.55 (3H, s), 3.69 (3H, s), 6.19 (1H, s),
6.84−6.91 (2H, m), 6.96−6.99 (1H, dd, J = 9.0, 3.0 Hz), 7.30 (1H,
s), 7.45 (1H, d, J = 9.0 Hz), 7.47−7.53 (1H, m), 7.61 (1H, d, J = 9.0
Hz); 13C NMR (75 MHz, DMSO-d6) δC: 162.8, 161.6, 152.2, 140.5,
139.3, 136.0, 132.0, 128.7, 127.8, 126.4, 124.1, 119.0, 116.9, 114.9,
111.1, 100.1, 43.5, 29.9, 26.6, 12.9. HRMS (ESI) calculated for
C20H20N4 [M + H]+ 317.1766, found 317.1764. Purity: 96.734% (by
HPLC).
4.2. Biological Evaluation. 4.2.1. Materials. EBI and the purified
tubulin polymerization kit were bought from Cytoskeleton Inc.
(Denver). The annexin V-FITC apoptosis detection kit, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT),
propidium iodide (PI), JC-1, DMEM, and RPMI 1640 medium were
bought from Nanjing KeyGen Biotech Co. Ltd. (Nanjing, China).
Primary antibodies against Cdc2, Cdc25c, Bax, Bad, Cyclin B1, Bcl-2,
cleaved-Caspase-3, cleaved-Caspase-9, cleaved-PARP, AhR, and
CYP1A1 were bought from Beyotime (Shanghai, China). Goat
antirabbit IgG/Alexa-Fluor 488 antibody was bought from Jackson
ImmunoResearch Inc. (West Grove).
4.2.2. Cell Lines and Cell Culture. Human breast cancer cells
(MCF-7), human normal hepatocyte cells (L-O2), and hepatocellular
carcinoma cells (HepG-2) were cultured in DMEM medium. Human
L
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4.2.11. Measurement of Intracellular ROS Generation. The
MCF-7 cell suspension was inoculated into six-well plates for 24 h (4
× 105 cells). Then, cells were cultured in a fresh medium with
dimethyl sulfoxide and 12 (0.5, 1, and 2 nM) for another 24 h. After
that, cells were cultured with DCFH-DA for 30 min shielded from
light. Then, cells were suspended in a medium with no fetal bovine
serum and detected by a flow cytometric assay. Cells were treated
with 2.5 mM N-acetyl-L-cysteine and then cultured with 2 nM 12 for
another 24 h. The assay was carried out 3 times.
4.2.12. Western Blotting Assay. The MCF-7 cell suspension was
inoculated into six-well plates for 24 h (4 × 105 cells). Then, cells
were cultured in a fresh medium with dimethyl sulfoxide and 12 (1, 2,
and 4 nM) for 24 h. Cells were incubated and lysed with the lysis
buffer, and proteins were extracted. After that, proteins were diluted
to 3 mg/mL based on the BCA protein assay kit (Beyotime, China).
Each sample (10 μL) was detected by SDS-PAGE (4−20% gel). The
assay was carried out 3 times.
4.2.13. Cell Migration Inhibition Assay. The MCF-7 cell
suspension was seeded into six-well cell plates for 24 h (5 × 105
cells). After cells were attached to the plate, a sterile pipette tip was
used to scrape the bottom of each hole to destroy the monolayer cells.
Then, the graphics were taken under an inverted fluorescence
microscope. After that, DMSO, DMBA, DMBA+12 and 12 (2 nM)
were added to each well. Photos were taken at fixed positions at 24 h,
and results were analyzed by ImageJ software.
4.2.14. Evaluation of Antitumor Activity In Vivo. The right side of
Bal b/c nude mice was inoculated subcutaneously with 6 × 106 MCF-
7 cells based on the protocols of the tumor transplantation analysis.
After culturation for 7 days, the weights of mice were recorded, and
then, the mice were randomly separated into five groups (eight
animals per group). Group 1 was administrated with 10% DMSO/5%
Tween 80/85% saline through intravenous injection (iv). Group 2
was administrated with PTX (10 mg/kg per 2 days) through
intravenous administration. Group 3 was administrated with CA-4
(20 mg/kg per 2 days) through iv. Group 4 was administrated with 12
(0.5 mg/kg every 2 days) through iv. Group 5 was administrated with
12 (1 mg/kg every 2 days) through iv. Tumor sizes and body weights
were detected every 2 days. All of the mice were killed after
administration for 21 consecutive days and then weighed. Tumor
volumes were calculated by employing the following formula: V = L ×
W2/2, with V (mm3) being the volume, W (mm) being the width, and
L (mm) being the length of the tumor. The tumors and organs were
fixed, paraffin-embedded, and sectioned. The tumor tissue sections
were stained with the TUNEL kit to observe the proportion of
apoptotic cells; pathologic staining of major tissues and organs was
performed using hematoxylin and eosin (HE) staining; CD31 staining
was used to calculate the mean microvascular density. All procedures
were carried out following institutional approval for the Care and Use
of Laboratory Animals.
Jianbo Sun − State Key Laboratory of Natural Medicines,
Department of Natural Medicinal Chemistry, School of
Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, China; Phone: +86-25-
83271415; Email: sunjianbo@cpu.edu.cn
Authors
Kun Wang − State Key Laboratory of Natural Medicines,
Department of Natural Medicinal Chemistry, School of
Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, China
Hui Zhong − Department of Pharmacology of Traditional
Chinese Medicine, School of Traditional Chinese Pharmacy,
China Pharmaceutical University, Nanjing 210009, China
Na Li − State Key Laboratory of Natural Medicines,
Department of Natural Medicinal Chemistry, School of
Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, China
Nairong Yu − State Key Laboratory of Natural Medicines,
Department of Natural Medicinal Chemistry, School of
Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, China
Yujin Wang − State Key Laboratory of Natural Medicines,
Department of Natural Medicinal Chemistry, School of
Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c01099
Author Contributions
§K.W. and H.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Natural Science
Foundation of China (31870325 and 31500277), the Six-talent
Peaks Project in Jiangsu Province (SWYY-071), and the
“Double First Class” Subject Innovation Team Construction
Project of China Pharmaceutical University (CPU2018GY12).
ABBREVIATIONS
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01099.
■
■
sı
Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X; Bad, Bcl-2
antagonist of cell death; CD31, platelet endothelial cell
adhesion molecule-1; Cdc2, cell division cycle 2; Cyclin B1,
cell division cyclin 25 homolog C; DAPI, 2-(4-amidinophen-
yl)-6-indolecarbamidine dihydrochloride; DCF-DA, 2′,7′-di-
chlorofluorescent yellow diacetate; DMSO, dimethylsulfoxide;
EBI, N,N′-ethylenebis(iodoacetamide); FITC, fluorescein
isothiocyanate; GAPDH, reduced glyceraldehyde-phosphate
dehydrogenase; GTP, guanosine triphosphate; H&E, hema-
toxylin−eosin; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-
imidacarbocyanine; MMP, mitochondrial membrane potential;
MTAs, microtubule-targeting agents; MTT, 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide; MVD, micro-
vessel density; PARP, poly ADP-ribose polymerase; PI,
propidium iodide; PTX, paclitaxel; iv, intravenous injection;
ROS, reactive oxygen species; TUNEL, terminal deoxynucleo-
tidyl transferase-mediated dUTP-biotin nick end labeling assay
Structural characterization of 1−12 (1H NMR, 13C
NMR, ESI/HRMS spectra); and HPLC analyses of 1−
12 (PDF)
Molecular string files for 1−12 (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Li Chen − State Key Laboratory of Natural Medicines,
Department of Natural Medicinal Chemistry, School of
Traditional Chinese Pharmacy, China Pharmaceutical
University, Nanjing 210009, China; orcid.org/0000-
0002-4655-9024; Phone: +86-25-83271447;
Email: chenli627@cpu.edu.cn
M
https://doi.org/10.1021/acs.jmedchem.1c01099
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
destabilizing tubulin: an approach to targeting tumor growth. J. Med.
Chem. 2016, 59, 8685−8711.
(19) Fortin, S.; Lacroix, J.; Coté, M. F.; Moreau, E.; Petitclerc, E.;
René, C. Quick and simple detection technique to assess the binding
of antimicrotubule agents to the colchicine-binding site. Biol. Proced.
Online 2010, 12, 113−117.
REFERENCES
■
(1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics, 2019. CA
Cancer J. Clin. 2019, 69, 7−34.
(2) Riggio, A. I.; Varley, K. E.; Welm, A. L. The lingering mysteries
of metastatic recurrence in breast cancer. Br. J. Cancer 2021, 124, 13−
26.
(3) Batran, R. Z.; Kassem, A. F.; Abbas, E. M.; Elseginy, S. A.;
Mounier, M. M. Design, synthesis and molecular modeling of new 4-
phenylcoumarin derivatives as tubulin polymerization inhibitors
targeting MCF-7 breast cancer cells. Bioorg. Med. Chem. 2018, 26,
3474−3490.
(4) Qi, C.; Wang, X.; Shen, Z.; Chen, S.; Yu, H.; Williams, N.; Wang,
G. Anti-mitotic chemotherapeutics promote apoptosis through TL1A-
activated death receptor 3 in cancer cells. Cell Res. 2018, 28, 544−
555.
́
̂
(20) Gaspari, R.; Prota, A. E.; Bargsten, K.; Cavalli, A.; Steinmetz, M.
O. Structural basis of cis- and trans-combretastatin binding to tubulin.
Chem 2017, 2, 102−113.
(21) Clarke, P. R.; Allan, L. A. Cell-cycle control in the face of
damage-a matter of life or death. Trends Cell Biol. 2009, 19, 89−98.
(22) Buolamwini, J. K. Cell Cycle Molecular targets in novel
anticancer drug discovery. Curr. Pharm. Des. 2000, 6, 379−392.
(23) Jordan, M. A.; Wilson, L. Microtubules and actin filaments:
dynamic targets for cancer chemotherapy. Curr. Opin. Cell Biol. 1998,
10, 123−130.
(24) Vander Heiden, M. G.; Chandel, N. S.; Williamson, E. K.;
Schumacker, P. T.; Thompson, C. B. Bcl-xL Regulates the membrane
potential and volume homeostasis of mitochondria. Cell 1997, 91,
627−637.
(25) Circu, M. L.; Aw, T. Y. Reactive oxygen species, cellular redox
systems, and apoptosis. Free Radical Biol. Med. 2010, 48, 749−762.
(26) Varga, I.; Hutóczki, G.; Petrás, M.; Scholtz, B.; Mikó, E.;
Kenyeres, A.; Tóth, J.; Zahuczky, G.; Bognár, L.; Hanzély, Z.; Klekner,
A. Expression of invasion-related extracellular matrix molecules in
human glioblastoma versus intracerebral lung adenocarcinoma
metastasis. Cent. Eur. Neurosurg. 2010, 71, 173−180.
(5) Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer
drugs. Nat. Rev. Cancer 2004, 4, 253−265.
(6) Mehlen, P.; Puisieux, A. Metastasis: a question of life or death.
Nat. Rev. Cancer 2006, 6, 449−458.
(7) Dietrich, C.; Kaina, B. The aryl hydrocarbon receptor (AhR) in
the regulation of cell−cell contact and tumor growth. Carcinogenesis
2010, 31, 1319−1328.
(8) Shan, A. Q.; Leng, L.; Li, J.; Luo, X. M.; Fan, Y. J.; Yang, Q. Y.;
Xie, Q. H.; Chen, Y. S.; Ni, C. S.; Guo, L. M.; Tang, H.; Chen, X.;
Tang, N. J. TCDD-induced antagonism of MEHP-mediated
migration and invasion partly involves aryl hydrocarbon receptor in
MCF7 breast cancer cells. J. Hazard. Mater. 2020, 398, No. 122869.
(9) Nebert, D. W.; Karp, C. L. Endogenous functions of the aryl
hydrocarbon receptor (AHR): intersection of cytochrome P450 1
(CYP1)-metabolized eicosanoids and AHR biology. J. Biol. Chem.
2008, 283, 36061.
(10) Al-Dhfyan, A.; Alhoshani, A.; Korashy, H. M. Aryl hydrocarbon
receptor/cytochrome P450 1A1 pathway mediates breast cancer stem
cells expansion through PTEN inhibition and β-Catenin and Akt
activation. Mol. Cancer 2017, 16, No. 1.
(11) Mulero-Navarro, S.; Pozo-Guisado, E.; Pérez-Mancera, P. A.;
Alvarez-Barrientos, A.; Catalina-Fernández, I.; Hernández-Nieto, E.;
Sáenz-Santamaria, J.; Martínez, N.; Rojas, J. M.; Sánchez-García, I.;
Fernández-Salguero, P. M. Immortalized mouse mammary fibroblasts
lacking dioxin receptor have impaired tumorigenicity in a subcuta-
neous mouse xenograft model. J. Biol. Chem. 2005, 280, 28731−
28741.
(12) Afzal, O.; Kumar, S.; Haider, M. R.; Ali, M. R.; Kumar, R.;
Jaggi, M.; Bawa, S. A review on anticancer potential of bioactive
heterocycle quinoline. Eur. J. Med. Chem. 2015, 97, 871−910.
(13) Mahal, K.; Resch, M.; Ficner, R.; Schobert, R.; Biersack, B.;
Mueller, T. Effects of the tumor-vasculature-disrupting agent
verubulin and two heteroaryl analogues on cancer cells, endothelial
cells, and blood vessels. ChemMedChem 2014, 9, 847−854.
(14) Zhou, Y. Y.; Yan, W.; Cao, D.; Shao, M. F.; Li, D.; Wang, F.;
Yang, Z.; Chen, Y.; He, L. H.; Wang, T. J.; Shen, M. S.; Chen, L. J.
Design, synthesis and biological evaluation of 4-anilinoquinoline
derivatives as novel potent tubulin depolymerization agents. Eur. J.
Med. Chem. 2017, 138, 1114−1125.
(15) Khelifi, I.; Naret, T.; Hamze, A.; Bignon, J.; Levaique, H.;
Alvarez, M. C. G.; Dubois, J.; Provot, O.; Alami, M. N, N-bis-
heteroaryl methylamines: Potent anti-mitotic and highly cytotoxic
agents. Eur. J. Med. Chem. 2019, 168, 176−188.
(16) Fang, Z. Z.; Krausz, K. W.; Nagaoka, K.; Tanaka, N.; Gowda,
K.; Amin, S. G.; Perdew, G. H.; Gonzalez, F. J. In vivo effects of the
pure aryl hydrocarbon receptor antagonist GNF-351 after oral
administration are limited to the gastrointestinal tract. Br. J.
Pharmacol. 2014, 171, 1735−1746.
(17) Marinelli, L.; Martin-Gallausiaux, C.; Bourhis, J. M.; Béguet-
̀
Crespel, F.; Blottiere, H. M.; Lapaque, N. Identification of the novel
role of butyrate as AhR ligand in human intestinal epithelial cells. Sci.
Rep. 2019, 9, No. 643.
(18) Pérez-Pérez, M. J.; Priego, E. M.; Bueno, O.; Martins, M. S.;
Canela, M. D.; Liekens, S. Blocking blood flow to solid tumors by
N
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