Discovery and Identification of Small Molecules as Methuosis Inducers with in Vivo Antitumor Activities

Article abstract of DOI:10.1021/acs.jmedchem.8b00753

Methuosis is a novel nonapoptotic mode of cell death characterized by vacuole accumulation in the cytoplasm. In this article, we describe a series of azaindole-based compounds that cause vacuolization in MDA-MB-231 cells. The most potent vacuole inducer,

Full text of DOI:10.1021/acs.jmedchem.8b00753

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Article
Discovery and Identification of Small Molecules as
Methuosis Inducers with In Vivo Antitumor Activities
Wei Huang, Xihuan Sun, Yunzhan Li, Zhixiang He, Li Li, Zhou Deng, Xiaoxing Huang, Shang
Han, Ting Zhang, Jiaji Zhong, Zheng Wang, Qingyan Xu, jianming zhang, and Xianming Deng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00753 • Publication Date (Web): 07 Jun 2018
Downloaded from http://pubs.acs.org on June 7, 2018
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Discovery and Identification of Small Molecules
as Methuosis Inducers with In Vivo Antitumor
Activities
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＃
＃
Wei Huang,†, ‡, Xihuan Sun,†, ‡, Yunzhan Li,†, ‡ Zhixiang He,†, ‡ Li Li,†, ‡ Zhou Deng,†, ‡
Xiaoxing Huang,†, ‡ Shang Han,†, ‡ Ting Zhang,†, ‡, Jiaji Zhong,†, ‡, § Zheng Wang,†, ‡ Qingyan
Xu,†, ‡ Jianming Zhang,⊥ Xianming Deng*, †, ‡
†State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network,
School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
‡Stateꢀprovince Joint Engineering Laboratory of Targeted Drugs from Natural Products, Xiamen
University, Xiamen, Fujian 361102, China.
§Medical College of Xiamen University, Xiamen, Fujian 361102, China.
⊥Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School,
Boston, MA 02129, USA.
ABSTRACT: Methuosis is a novel nonapoptotic mode of cell death characterized by vacuole
accumulation in the cytoplasm. In this report, we describe a series of azaindoleꢀbased
compounds that cause vacuolization in MDAꢀMBꢀ231 cells. The most potent vacuoleꢀinducer,
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compound 13 (Cmpd. 13), displayed differential cytotoxicities against a broad panel of cancer
cell lines, such as MDAꢀMBꢀ231, A375, HCT116, and MCFꢀ7, but it did not inhibit the growth
of the nontumorigenic epithelial cell line MCFꢀ10A. A mechanism study confirmed that the cell
death was caused by inducing methuosis. Furthermore, Cmpd. 13 exhibited substantial
pharmacological efficacy in the suppression of tumor growth in a xenograft mouse model of
MDAꢀMBꢀ231 cells without apparent side effects, which makes this compound the first example
of a methuosis inducer with potent in vivo efficacy. These results demonstrate that methuosis
inducers might serve as novel therapeutics for the treatment of cancer.
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INTRODUCTION
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Despite an increasing number of clinically available anticancer agents, intrinsic or acquired
resistance remains a great challenge in both chemotherapy and targeted therapy.1 Several
mechanisms have been implicated in drug resistance, including increases in drug efflux2 or DNA
repair capacity3 or altering apoptotic pathways.4ꢀ5 As many therapeutic agents work by triggering
intrinsic apoptosis,6 the discovery of alternative cell death pathways to kill these apoptotic
resistant tumor cells is of great interest.
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Several nonapoptotic cell death mechanisms,7ꢀ8 as exemplified by autophagy9 and necrosis,10
have been described. Recently, a new type of nonapoptotic cell death was named methuosis,
which is characterized by vacuole accumulation in the cytoplasm.11ꢀ12 These vacuoles are derived
from macropinocytotic vesicles showing impaired trafficking and recycling. As a result,
decreased metabolic activity and rupture of the cell membrane eventually lead to cell death.
Although the precise molecular mechanism is not clear,13ꢀ14 several methuosis inducers have
been reported. For example, indoleꢀbased chalcones such as MOMIPP (1) can effectively cause
methuosis in glioblastoma U251 cells.15ꢀ19 Moreover, MOMIPP showed similar activities against
both temozolomideꢀsensitive and temozolomideꢀresistant U251 cells. A quinoline derivative
named Vacquinolꢀ1 (2) also induces methuosis in glioblastomas.20 Recently, a ursolic acid
derivative (3) was reported to display cytostatic properties via methuosis against several cancer
cell lines with IC50 values of approximately 15 ꢁM.21 However, no in vivo pharmacological data
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have been reported for these methuosis inducers. To understand the pharmacological efficacy of
methuosis inducers as anticancer agents, we report the discovery and identification of small
molecule Cmpd. 13 as a potent in vitro methuosis inducer. More importantly, Cmpd. 13 exhibits
a remarkable tumorꢀsuppressing effect in a xenograft mouse model, making this compound the
first example of a methuosis inducer with potent in vivo efficacy.
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Figure 1. Structures of reported methuosis inducers.
RESULTS
Discovery of compound 13 with potent antitumor activity. As part of a largeꢀscale screen
for bioactive small molecules for cancer therapy, compound 12 caught our attention because it
effectively caused dramatic cytoplasmic vacuolization in MDAꢀMBꢀ231 cells at a concentration
of 10 ꢁM. However, the vacuolization effect caused by compound 12 decreased in a
doseꢀdependent manner to a negligible level at 1 ꢁM. To obtain more potent vacuolization
inducers, we started a systematic structureꢀactivity relationship (SAR) study using MOMIPP as a
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positive control (Table 1). First, the functionality of the A ring was explored. Replacing the
pyridinꢀ4ꢀyl moiety with a pyridinꢀ3ꢀyl afforded compound 13, which induced extensive
vacuolization even at a low concentration of 1 ꢁM (Figure S1). However, replacement of the
pyridinꢀ4ꢀyl moiety with phenyl, substituted phenyl, furanꢀ3ꢀyl or thiophenꢀ3ꢀyl moieties had
severe adverse effects. Almost no vacuolization could be observed when the cells were treated
with compounds 14–18, even at 10 ꢁM. These data suggest that there are key interactions
occurring between the pyridinꢀ3ꢀyl fragment and its target(s). Then, minor modifications of the
pyrrole unit of the scaffold were made. The methylated derivative, compound 19, dramatically
decreased vacuolization at 1 ꢁM. Next, we turned our attention to the aniline substituent at the
4ꢀposition of this scaffold. Compound 20, which lacked the oꢀmethyl substituent on the B ring,
showed a similar level of activity. When the orientation of the amide bond (linker L) was
reversed, resulting compounds 21–24 showed vacuoleꢀinducing capabilities at 10 ꢁM that were
similar to those of their counterparts 12, 13, 19 and 20, respectively. However, only compound
22 retained its activity at 1 ꢁM. The thiourea linkageꢀcontaining derivative, compound 25, also
lost its vacuoleꢀinducing activity at 1 ꢁM. Therefore, we decided to modify the C ring based on
the structure of compound 22. The addition of a hydrophobic group and a hydrophilic group at
the metaꢀposition resulted in compounds 26 and 27, respectively, which induced less vacuole
formation, even at 10 ꢁM. Replacing the trifluoromethyl group with trifluoromethoxy, methoxy,
methyl, and chloro groups resulted in compounds 28–31, which showed various degrees of
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activity at 1 ꢁM. The pyrazoleꢀsubstituted derivative, compound 32, was a potent
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vacuoleꢀinducer at 10 ꢁM but was inactive at 1 ꢁM. Compound 33, which possessed a
morpholine substituent, completely lost activity. The SAR exploration of 4,6ꢀdisubstituted
azaꢀindoles revealed that a free hydrogen (R1 = H), pyridinꢀ3ꢀyl (A ring), and 3ꢀtrifluoromethyl
phenyl (C ring) groups were key structural features required to achieve potent vacuoleꢀinducing
activity. The most active compound 13 (Cmpd. 13) induced greater than 99% vacuolization in
cells, even at 1 ꢁM.
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Table 1. Structure-activity Relationship Study of 4,6-Disubstituted Aza-indoles
Vacuolization ratio (%)a
Cmpd
R1 R2 A ring
L
C ring
1 ꢁM
1.46±1.37
99.75±0.56
0.00
10 ꢁM
96.34±1.10
100.00
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
12
13
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18
0.00
0.27±0.6
0.03±0.07
0.24±0.53
0.00
1.63±0.69
0.33±0.67
0.00
0.21±0.29
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Me Me
16.08±3.26 96.27±2.50
96.61±2.21 98.98±0.76
19
H
H
H
H
20
Me
Me
0.00
1.94±2.13
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70.29±11.52 99.42±0.86
3.63±1.11 70.96±15.72
22
Me Me
23
H
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H
3.78±0.95
1.26±0.92
0.00
80.23±7.52
87.3±6.45
0.00
24
Me
Me
Me
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Me
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Me
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26
0.62±0.87
0.00
27
38.57±8.58 99.66±0.77
76.86±10.97 100.00
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11.24±2.24 97.68±1.49
19.49±2.97 96.75±1.24
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31
2.48±1.11
0.00
95.24±1.44
0.00
32
33
7.01±1.35
96.47±3.38
MOMIPP (1)
aMDAꢀMBꢀ231 cells were treated with test compounds at concentrations of 1 ꢁM or 10 ꢁM for 24 h. Five
fields of cells were randomly selected and photographed, and then, the percentages of vacuolated cells were
quantitated. See EXPERIMENTAL SECTION for details.
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Cmpd. 13 was selected for further evaluation of its anticancer activities. As shown in Table 2,
Cmpd. 13 showed a broad spectrum of cytotoxicity against a panel of human cancer cell lines
with IC50 values in the micromolar range. A much lower cytotoxic effect was observed against
the nontumorigenic epithelial cell line MCFꢀ10A, representing normal human breast cells, and
the observed IC50 value was greater than 10 ꢁM. The antiproliferative activity of Cmpd. 13 was
further confirmed in a crystal violet staining assay, which revealed that the survival rate of
MDAꢀMBꢀ231 cells was reduced significantly in a doseꢀdependent manner (Figure 2B and C).
The morphological changes in MDAꢀMBꢀ231 cells treated with 2 ꢁM Cmpd. 13 were examined
using an optical microscope, which showed that vesicles accumulated and gradually fused to
form larger vacuoles after 12 h. Most cells were full of vacuoles after 24 h and lost viability after
48 h (Figure 2D). These data demonstrate that Cmpd. 13 is a potent and selective anticancer
agent.
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Table 2. IC50 Values of Cmpd. 13 and MOMIPP against Cancer and Normal Cellsa
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Cmpd. 13
IC50 (µM)
MOMIPP
IC50 (µM)
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Cell lines
A375
0.326±0.026
1.370±0.061
1.662±0.128
1.892±0.092
1.571±0.066
2.268±0.100
2.611±0.739
1.008±0.042
1.566±0.015
1.197±0.344
>10
5.020±0.08
8.156±0.247
>10
SKꢀMELꢀ28
SKꢀMELꢀ30
A549
>10
MDAꢀMBꢀ231
MDAꢀMBꢀ435S
MCFꢀ7
>10
>10
>10
HCT116
>10
BGC823
>10
HepG2
>10
MCFꢀ10A
>10
aCells were treated with Cmpd. 13 or MOMIPP for 48 h, and viability values were determined by MTS assays.
The IC50 values were calculated by GraphPad Prism 5 and are presented as the means ± SD of triplicate
samples.
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Figure 2. Cmpd. 13 induces extensive vacuolization and reduces the survival rate of MDAꢀMBꢀ231 cells. (A)
Structure of Cmpd. 13. (B) MDAꢀMBꢀ231 cells were stained with crystal violet after 48 h of incubation with
DMSO (control) or Cmpd. 13 at the indicated concentrations. (C) Quantification results for the crystal violet
staining. (***p < 0.001) (D) MDAꢀMBꢀ231 cells were treated with Cmpd. 13 at a concentration of 2 ꢁM, and
the morphological changes were observed by optical microscopy at the indicated time.
Identification of Cmpd. 13 as an effective methuosis inducer. As Cmpd. 13 inhibits cancer
cell proliferation by inducing full vacuolization in the cytoplasm, we sought to determine if the
cell death was caused by methuosis, which is nonapoptotic. Therefore, PARP cleavage and
caspaseꢀ3 activity were analyzed in MDAꢀMBꢀ231 cells treated with Cmpd. 13.22 As shown in
Figure 3A, cleaved PARP could not be detected after treatment with 0.5 ~ 4 ꢁM Cmpd. 13. The
increase in caspaseꢀ3 activity was not significant (Figure 3B). Moreover, when cells were
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pretreated with ZꢀVADꢀFMK, a panꢀcaspase inhibitor, for 1 h, neither cell death nor
vacuolization could be prevented (Figure 3C and D). Under the same conditions, cells treated
with staurosporine (STS) could be significantly rescued by treatment with ZꢀVADꢀFMK (Figure
3C). Additionally, the necrosis inhibitor, necrostatinꢀ1, could not prevent cellular vacuolization
or rescue cells from Cmpd. 13 (Figure 3E and F). These results clearly indicate that the cell death
caused by Cmpd. 13 is neither apoptosis nor necrosis.
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Figure 3. Cell death caused by Cmpd. 13 is distinct from apoptosis or necrosis. (A) MDAꢀMBꢀ231 cells were
treated with DMSO (control), Cmpd. 13 or STS for 24 h at the indicated concentrations. PARP and cleaved
PARP were analyzed by Western blot. (B) MDAꢀMBꢀ231 cells were treated with DMSO (control), Cmpd. 13
or STS for 24 h at the indicated concentrations. The cells were then lysed, and caspase 3 activity was analyzed
by a caspase 3 activity kit. (C) MDAꢀMBꢀ231 cells were treated with DMSO (control), 100 ꢁM ZꢀVADꢀFMK,
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2 ꢁM Cmpd. 13, 0.1 ꢁM STS or combinations of these as indicated for 48 h. Viability values were determined
by MTS assays and are presented as the means ± SD of triplicate samples (***p < 0.001). (D) Optical
microscopy images of MDAꢀMBꢀ231 cells treated with DMSO (control), 100 ꢁM ZꢀVADꢀFMK, 2 ꢁM Cmpd.
13, or their combination for 24 h. (E) MDAꢀMBꢀ231 cells were treated with DMSO (control), 50 ꢁM
necrostatinꢀ1, 2 ꢁM Cmpd. 13, or their combinations for 48 h. Viability values were determined by MTS
assays and are presented as the means ± SD of triplicate samples. (F) Optical microscopy images of
MDAꢀMBꢀ231 cells treated with DMSO (control), 50 ꢁM necrostatinꢀ1, 2 ꢁM Cmpd. 13, or their combination
for 24 h.
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Next, we investigated the relationship between Cmpd. 13ꢀinduced cell death and autophagy.8,
23 We first used Western blot analysis to determine whether the autophagosome marker LC3 was
elevated.24 As shown in Figure 4A, LC3ꢀII increased in timeꢀ and doseꢀdependent manners
according to the ratio of LC3ꢀII/βꢀActin, which suggests that Cmpd. 13 disrupts autophagic
flux.25 However, the autophagy inhibitor 3ꢀmethyladenine (3ꢀMA) could prevent neither
vacuolization nor cell death in MDAꢀMBꢀ231 cells treated with Cmpd. 13 (Figure 4B and C),
which excluded autophagy as the cause of cell death induced by Cmpd. 13.
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Figure 4. Cell death caused by Cmpd. 13 is distinct from autophagy. (A) MDAꢀMBꢀ231 cells were treated
with DMSO (control) or Cmpd. 13 for 24 h at the indicated concentrations or 2 ꢁM Cmpd. 13 for the indicated
time. LC3 was analyzed by Western blot. (B) MDAꢀMBꢀ231 cells were treated with DMSO (control), 1 mM
3ꢀMA, 2 ꢁM Cmpd. 13, or their combination for 48 h. Viability values were determined by MTS assays and
are presented as the means ± SD of triplicate samples. (C) Optical microscopy images of MDAꢀMBꢀ231 cells
treated with DMSO (control), 1 mM 3ꢀMA, 2 ꢁM Cmpd. 13, or their combination for 24 h.
We next turned our attention to the origin of the vacuoles. As shown in Figure 5A and B,
neither Mitoꢀtracker nor ERꢀtracker were incorporated into the vacuoles, suggesting that they did
not originate from the mitochondria or endoplasmic reticulum (ER). This conclusion was further
confirmed by transmission electron microscopy (Figure 5C), which clearly showed that these
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organelles remained integrated aside the vacuoles. In methuosis, the vacuoles are derived from
macropinosomes, which fuse with each other and take on some characteristics of late endosomes
but do not merge with lysosomes.12, 15 Therefore, we investigated whether the vacuoles induced
by Cmpd. 13 exhibited the same properties. As shown in Figure 6A, the fluidꢀphase tracer
Lucifer yellow (LY) accumulated in most of the vacuoles, which is consistent with what was
observed in macropinocytosis. Furthermore, the late endosomal marker Rab7 was detected on the
membranes of the vacuoles (Figure 6B), but most of the Lysoꢀtracker did not overlap with these
vacuoles (Figure 6C). Taken together, these observations demonstrated that the vacuoles induced
by Cmpd. 13 possessed the characteristics of methuosis, which suggests that Cmpd. 13 is a
potent methuosis inducer.
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Figure 5. Vacuoles induced by Cmpd. 13 did not originate from the mitochondria or endoplasmic reticulum
(ER). (A) Phaseꢀcontrast and fluorescent images of MDAꢀMBꢀ231 cells treated with DMSO (control) or 2 ꢁM
Cmpd. 13 for 12 h and then incubated with Mitoꢀtracker. Scale bar, 10 ꢁm. (B) Phaseꢀcontrast and fluorescent
images of MDAꢀMBꢀ231 cells treated with DMSO (control) or 2 ꢁM Cmpd. 13 for 12 h and then incubated
with ERꢀtracker. Scale bar, 10 ꢁm. (C) Electron microscopy images of MDAꢀMBꢀ231 cells treated with
DMSO (control) or 2 ꢁM Cmpd. 13 for 12 h. Red and yellow arrows indicate that the mitochondria and ER are
not swelling. Scale bar, 2 ꢁm for 1000X, 0.5 ꢁm for 5000X.
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Figure 6. The vacuoles induced by Cmpd. 13 are derived from macropinosomes and take on the characteristics
of endosomes but remain distinct from lysosomes. (A) Phaseꢀcontrast and fluorescent images of
MDAꢀMBꢀ231 cells treated with 2 ꢁM Cmpd. 13 for 12 h under Lucifer yellow incubation. Scale bar, 20 ꢁm.
(B) Phaseꢀcontrast and immunofluorescent images of MDAꢀMBꢀ231 cells treated with 2 ꢁM Cmpd. 13 for 12
h followed by staining with antiꢀRab7. Scale bar, 20 ꢁm. (C) Phaseꢀcontrast and fluorescent images of
MDAꢀMBꢀ231 cells treated with 2 ꢁM Cmpd. 13 for 12 h and then incubated with Lysoꢀtracker. Scale bar, 20
ꢁm.
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Cmpd. 13 inhibits tumor growth in vivo. To extend the potential preclinical application of
methuosis inducers, the pharmacokinetics of Cmpd. 13 were assessed in BALB/c mice at a dose
of 1 mg/kg via intravenous (IV) injection. Cmpd. 13 was found to have a halfꢀlife (T1/2) of 8.74
h, a clearance (Cl) of 36.5 mL/min/kg, and a volume of distribution (Vd) of 15.5 L/kg, which
represent moderate pharmacokinetic properties. The in vivo antitumor efficacy of Cmpd. 13 was
further evaluated using tripleꢀnegative breast cancer cells (MDAꢀMBꢀ231 cells) in a mouse
xenograft model. As shown in Figure 7, treatment with Cmpd. 13 led to substantial suppression
of tumor growth in a doseꢀdependent manner (Figure 7A and Figure S2), and the tumor growth
inhibition (TGI) values were 25.85±0.30% and 60.15±0.04% at doses of 5 mg/kg and 20 mg/kg,
respectively. In addition, Cmpd. 13 did not cause a significant loss of body weight during
treatment. Furthermore, H & E staining showed that numerous vacuoles were visible in
drugꢀtreated tumor tissues, indicating that Cmpd. 13 can induce methuosis in vivo (Figure 7C).
Treatment with Cmpd. 13 on healthy ICR mice at dose of 20 mg/kg for 12 days did not induce
vacuoles in normal tissues and had no effects on body weight and blood chemistry (Figure S3).
Together, these results demonstrate that Cmpd. 13 inhibits tumor growth in vivo with good
tolerability.
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Figure 7. Cmpd. 13 exhibited in vivo efficacy in a xenograft mouse model of MDAꢀMBꢀ231 cells. (A) and (B)
Tumor volume and body weight of mice during treatment. Cmpd. 13 was administered intravenously once
daily at 5 mg/kg or 20 mg/kg. Tumor volume and body weight were measured every day (*p < 0.05, **p <
0.01, ***p < 0.001). Error bars indicate mean tumor volume ± SEM and mean body weight ± SEM (n = 6
animals each group). (C) Hematoxylinꢀeosin staining (H & E) in tumors untreated or after 12 days of treatment
with Cmpd. 13. Red arrows indicate vacuoles. Scale bar, 25 ꢁm for and 10X and 100X, 50 ꢁm for 40X.
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CHEMISTRY
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As shown in Scheme 1, intermediate 6 was obtained from 4 and 5 through BuchwaldꢀHartwig
cross coupling,26ꢀ27 which gave intermediate 7 or final products 19 and 23 after Suzuki cross
coupling with boronic acids.28ꢀ30 Deprotection of the SEM group furnished final compounds
12ꢀ18, 20ꢀ22, 24 and 26ꢀ33.
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Scheme 1. Synthesis of azaindole-based compoundsa
aReagents and conditions: a. Xphos, Pd2(dba)3, K2CO3, tꢀBuOH, 60 °C. b. PdCl2(dppf)•CH2Cl2, Na2CO3,
1,4ꢀdioxane/H2O (V/V = 2/1), 100 °C. c. (1) TFA; (2) MeOH/THF/LiOH (1.0 M, aq.) (V/V/V = 1/1/1).
The synthesis of compound 25 is illustrated in Scheme 2. Intermediate 9 was obtained
through Pdꢀcatalyzed cross coupling reactions as described above. After reduction of the nitro
group and deprotection of the SEM, resulting aniline 11 was reacted with
1ꢀisothiocyanatoꢀ3ꢀ(trifluoromethyl)benzene to give compound 25.
Scheme 2. Synthesis of compound 25a
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aReagents and conditions: a. Xphos, Pd2(dba)3, K2CO3, tꢀBuOH, 60 °C. b. PdCl2(dppf)•CH2Cl2, Na2CO3,
1,4ꢀdioxane/H2O (V/V = 2/1), 100 °C. c. Zn, NH4Cl (aq.), EtOH, 60 °C. d. (1) TFA; (2) MeOH/THF/LiOH
(1.0 M, aq.) (V/V/V = 1/1/1). e. 1ꢀisothiocyanatoꢀ3ꢀ(trifluoromethyl)benzene, THF, 60 °C.
DISCUSSION AND CONCLUSIONS
Methuosis was first defined in cancer cells with ectopic expression of constitutively activated
Ras and was proven to be distinct from necrosis, autophagy and other nonapoptotic forms of cell
death.12, 31 These characteristics grant methuosis inducers great potential for the treatment of
cancers that are resistant to conventional therapies. Indeed, temozolomideꢀresistant U251 cells
remain sensitive to MOMIPP, an indoleꢀbased chalcone methuosis inducer.16 Herein, we
reported a series of 4,6ꢀdisubstituted azaꢀindoles that are structurally distinct from existing
methuosis inducers. Further structureꢀactivity relationship studies revealed the key factors for
methuosisꢀinducing activity. For example, the pyridinꢀ3ꢀyl moiety at the 2ꢀposition of this
scaffold and the hydrophobic group at the metaꢀposition of the C ring are critical for activity. A
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certain degree of flexibility in the pyrrole motif or B ring exists, which allows further
derivatization to improve potency or specificity.
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Tripleꢀnegative breast cancer (TNBC) accounts for approximately 15% of all breast cancers
and has a poor disease prognosis, a high risk of recurrence and a worse diseaseꢀfree survival rate
compared to other subtypes.32 Because TNBC does not express estrogen receptors or
progesterone receptors (PRs) and lacks human epidermal growth factor receptorꢀ2 (HER2)
amplification, it is insensitive to antihormonal and HER2 targeted therapies.33ꢀ34 Currently,
surgery and/or cytotoxic chemotherapy are the standard care for the treatment of TNBC, and
these methods do not show satisfying outcomes.35 Whether methuosis inducers can show
therapeutic benefits in vivo is of great interest. Cmpd. 13 induced vacuolization in both cancer
cells (MDAꢀMBꢀ231 cells) and nontumorigenic epithelial cells (MCFꢀ10A cells), and it
exhibited differential antiproliferative activities with IC50 values of 1.88 ꢁM and >10 ꢁM,
respectively. These results indicate that a sufficient therapeutic window may exist among tumor
and normal tissues. This hypothesis has been confirmed by the subsequent in vivo studies. Data
from an MDAꢀMBꢀ231 xenograft mouse model showed that Cmpd. 13 significantly suppressed
tumor growth in a doseꢀdependent manner, which demonstrates the acceptable therapeutic
benefits of this compound. Importantly Cmpd. 13 was tolerated in animals, and a significant
decrease in body weight was not observed during treatment. Further studies to identify the
cellular target(s) of this series of azaindoleꢀbased compounds and to elucidate the detailed
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molecular mechanism are needed to extend our understanding of the vulnerability of cancer cells
to these methuosis inducers.
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Taken together, our results demonstrate that the methuosis inducer Cmpd. 13 is effective and
tolerable in vivo, and thus, it can serve as a good starting point for the development of
methuosisꢀbased therapies against cancers for which there are no targeted therapeutics available,
such as TNBC.
EXPERIMENTAL SECTION
General Methods for Chemistry. Reagents and solvents were obtained from commercial
sources and were used without further purification. Reactions were monitored by thin layer
chromatography (TLC) on glass plates coated with silica gel with fluorescent indicator.
Compounds were purified either by chromatography on silica gel or by preparative high
performance liquid chromatography (HPLC). Silica gel chromatography was performed on
Teledyne ISCO CombiFlash system (RF200) eluting with petroleum ether/ethyl acetate (PE/EA)
or dichloromethane/methanol (DCM/MeOH). Preparative HPLC was conducted on Waters
autopurification system consisting of a 2767 sample manager, a 2545 binary gradient module, a
2489 UV detector, and a 3100 mass detector [SunFireTM C18 column, 5 ꢁM, 19 x 50 mm, 10 min
linear gradient from 10% solvent A (methanol with 0.035% trifluoroacetic acid) in solvent B
(water with 0.035% trifluoroacetic acid) to 100% A, then hold for 4 min at 100% A, 20 mL/min].
NMR spectra were recorded on a Bruker Ultrashield Plusꢀ600 (600 MHz) spectrometer and
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chemical shifts are reported in δ (ppm). 1H chemical shifts are reported as s (singlet), d (doublet),
dd (doublet of doublet), t (triplet), q (quartet), m (multiplet), and brs (broad singlet) and are
referenced to the residual solvent signal: DMSOꢀd6 (2.50), CDCl3 (7.26). 13C spectra are
referenced to the residual solvent signal: DMSOꢀd6 (39.52), CDCl3 (77.16). Mass Spectra were
recorded on Waters 3100 Mass Detector. High Resolution Mass Spectra were obtained on
Thermo Fisher Scientific LTQ FTICRꢀMS. The purity of all tested compounds were determined
to be > 95% by HPLC with UV detection at 254 nm [Waters LC/MS system, SunFireTM C18
column, 5 ꢁM, 4.6 x 50 mm, a 0.5 min hold at 5% solvent A (methanol with 0.035%
trifluoroacetic acid) in solvent B (water with 0.035% trifluoroacetic acid), then a 4 min linear
increase to 95% A, then to 100% A in 1 min, then a 4 min hold at 100% A, 1 mL/min].
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General procedure. N-(3-((6-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-
b]pyridin-4-yl)amino)-4-methylphenyl)-3-(trifluoromethyl)benzamide (6a) The mixture of
4,6ꢀdichloroꢀ1ꢀ((2ꢀ(trimethylsilyl)ethoxy)methyl)ꢀ1Hꢀpyrrolo[2,3ꢀb]pyridine (4a, 500 mg, 1.58
mmol), N-(3ꢀaminoꢀ4ꢀmethylphenyl)ꢀ 3ꢀ(trifluoromethyl)benzamide (5a, 464 mg, 1.58 mmol),
Xphos (56 mg, 0.12 mmol), Pd2(dba)3 (72 mg, 0.079 mmol) and K2CO3 (654 mg, 4.74 mmol)
were stirred in tꢀBuOH (15 mL) at 65 °C for 6 hours under N2, then filtered through celite. The
filtrate was concentrated and purified by CombiFlash purification system to give 6a as a
1
colorless oil (540 mg, 59.5%). H NMR (600 MHz, DMSOꢀd6) δ 10.50 (s, 1H), 8.76 (s, 1H),
8.29 (s, 1H), 8.26 (d, J = 7.9 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.77 (d,
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J = 2.1 Hz, 1H), 7.67 (dd, J = 8.3, 2.1 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 3.6 Hz, 1H),
6.58 (d, J = 3.6 Hz, 1H), 6.03 (s, 1H), 5.49 (s, 2H), 3.55 – 3.50 (m, 2H), 2.18 (s, 3H), 0.87 – 0.81
(m, 2H), ꢀ0.07 (s, 9H); 13C NMR (150 MHz, DMSOꢀd6) δ 164.5, 148.5, 147.8, 145.8, 138.3,
138.1, 136.2, 132.3, 131.6, 130.4, 130.2, 129.7 (q, J = 32.0 Hz), 128.6 (q, J = 3.5 Hz), 125.9,
124.7 (q, J = 3.9 Hz), 124.5 (q, J = 272.4 Hz), 119.1, 118.9, 107.3, 99.7, 97.6, 72.9, 65.7, 17.6,
ꢀ1.0； MS (ESI) m/z 575 [C28H30ClF3N4O2Si + H]+.
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N-(4-methyl-3-((6-(pyridin-3-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-b]py
ridin-4-yl)amino)phenyl)-3-(trifluoromethyl)benzamide (7a). The mixture of 6a (60 mg, 0.10
mmol), pyridinꢀ3ꢀylboronic acid (15 mg, 0.12 mmol), Na2CO3 (42 mg, 0.40 mmol) and
PdCl2(dppf)•CH2Cl2 (8 mg, 0.01 mmol) were stirred in 1,4ꢀdioxne/H2O (0.8/0.4 mL) at 100 °C
under N2 for 2 hours, then filtered through celite. The filtrate was concentrated and purified by
CombiFlash purification system to give 7a as a light yellow oil (52.0 mg, 80.8%). 1H NMR (600
MHz, DMSOꢀd6) δ 10.49 (s, 1H), 9.16 (dd, J = 2.4, 0.9 Hz, 1H), 8.53 (dd, J = 4.7, 1.6 Hz, 1H),
8.50 (s, 1H), 8.30 – 8.27 (m, 2H), 8.25 (d, J = 7.8 Hz, 1H), 7.96 – 7.93 (m, 1H), 7.88 (d, J = 2.2
Hz, 1H), 7.77 (t, J = 7.8 Hz, 1H), 7.58 (dd, J = 8.3, 2.2 Hz, 1H), 7.44 (ddd, J = 8.0, 4.8, 0.8 Hz,
1H), 7.38 (d, J = 3.6 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 6.85 (s, 1H), 6.59 (d, J = 3.6 Hz, 1H),
5.64 (s, 2H), 3.64 – 3.55 (m, 2H), 2.24 (s, 3H), 0.94 – 0.84 (m, 2H), ꢀ0.13 (s, 9H); 13C NMR (150
MHz, DMSOꢀd6) δ 164.5, 149.8, 149.3, 149.1, 148.1, 146.9, 139.1, 137.8, 136.2, 136.0, 134.1,
132.2, 131.4, 130.2, 129.6 (q, J = 32.0 Hz), 129.5, 128.6 (q, J = 3.4 Hz), 126.6, 124.7 (q, J = 3.9
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Hz), 124.4 (q, J = 272.5 Hz), 124.0, 118.3, 118.0, 108.2, 99.6, 96.6, 72.5, 65.8, 17.8, 17.6, ꢀ1.0;
MS (ESI) m/z 618 [C33H34ClF3N5O2Si + H]+.
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N-(4-methyl-3-((6-(pyridin-3-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)amino)phenyl)-3-(trifluor
omethyl)benzamide (13). Compound 7a (40.0 mg, 0.065 mmol) was stirred in TFA (1 mL) at
room temperature for 1 hour. The mixture was concentrated under vacuum and the residue was
dissolved in MeOH/THF (1/1 mL). To this solution was added 1 M aqueous LiOH solution (1
mL) and the resulting mixture were stirred at room temperature for another 1 hour before
diluting with water (30 mL). Then the mixture were extracted with EtOAc (20 mL × 3) and the
combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated
under reduced pressure. The residue was purified by CombiFlash purification system to give 13
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as a white solid (12.3 mg, 38.9%). H NMR (600 MHz, DMSOꢀd6) δ 11.51 – 11.48 (m, 1H),
10.48 (s, 1H), 9.11 (d, J = 2.3 Hz, 1H), 8.53 (dd, J = 4.7, 1.6 Hz, 1H), 8.37 (s, 1H), 8.30 – 8.28
(m, 1H), 8.28 – 8.23 (m, 2H), 7.98 – 7.95 (m, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.78 (t, J = 7.8 Hz,
1H), 7.59 (dd, J = 8.3, 2.2 Hz, 1H), 7.44 (dd, J = 8.0, 4.7 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.24
13
(dd, J = 3.4, 2.4 Hz, 1H), 6.79 (s, 1H), 6.55 (dd, J = 3.4, 2.0 Hz, 1H), 2.26 (s, 3H); C NMR
(150 MHz, DMSOꢀd6) δ 164.4, 150.5, 149.1, 149.1, 148.1, 146.6, 139.3, 137.8, 136.5, 136.3,
134.1, 132.3, 131.4, 130.2, 129.6 (q, J = 32.1 Hz), 129.3, 128.5 (q, J = 3.9 Hz) 124.7 (q, J = 3.9
Hz), 124.4 (q, J = 270.1 Hz), 124.0, 123.3, 118.0, 117.7, 108.2, 99.0, 96.0, 17.8. MS (ESI) m/z
488 [M + H]+; HRMS (DART): calcd for [C27H20F3N5O + H]+ 488.1693, found 488.1692.
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The other intermediates and final compounds were synthesized in similar ways and
characterization data are provided in the Supporting Information.
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Cell Culture. MDAꢀMBꢀ231, A375, SKꢀMELꢀ28, SKꢀMELꢀ30, BGC823, A549,
MDAꢀMBꢀ435S, MCFꢀ7, HCT116, HepG2 were grown in Dulbecco’s Modified Eagle Medium
(DMEM) or RPMIꢀ1640 medium containing 10% fetal bovine serum, penicillin (100 U/mL) and
streptomycin (100 ꢁg/mL). MCFꢀ10A cell line was cultured in F12/RPMI medium containing
5% horse serum, 2 mg/mL insulin, 1 mg/mL hydrocortisone, 100 ꢁg/mL EGF, 100 U/mL
penicillin and 100 ꢁg/mL streptomycin. All cell lines were maintained at 37°C in 5% CO2
humidified air.
Immunoblotting. After compound treatment, cells were harvested and lysed in cell lysis
buffer containing 50 mM TrisꢀHCl (pH 7.5), 150 mM NaCl, 1% (v/v) Triton Xꢀ100, 5%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 5 ꢁg/mL leupeptin, 1 mM sodium orthovanadate
and 50 mM sodium fluoride. Total protein concentrations were measured by BCA analysis
(Beyotime, Haimen, China). Equal amounts of protein were loaded for separation by
electrophoresis in a 10%ꢀ15% SDSꢀPAGE gel and then electrophoretically transferred to
ImmobilonꢀP PVDF membrane (Millipore, Billerica, MA, USA). Proteins were probed with
appropriate primary antibodies and peroxidaseꢀconjugated secondary antibodies. The membranes
were incubated with enhanced chemiluminescence (ECL) reagents and detected using a
ChemiDOC™ XRS+ Imaging system (BioꢀRad, Hercules, CA, USA). Antibodies against PARP,
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ClꢀPARP, LC3 and Rab7 were obtained from Cell Signaling Technology (Beverly, MA, USA).
AntiꢀβꢀActin was purchased from Sigma (St. Louis, MO, USA).
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Caspase 3 Activity Assay. After treatment as indicated for 24 h, MDAꢀMBꢀ231 cells were
harvested and lysed. Caspase 3 activity was detected using caspase 3 activity assay kit according
to the manufacturer’s instructions (Beyotime).
Cell Morphology and Viability. To determine the percentage of cells with cytoplasmic
vacuoles, phase contrast images of live cells were captured with a Zeiss OBSERVER A1/AX10
microscope. After treatments for 24 h, five fields of cells were randomly selected and
photographed. Cells containing one or more phaseꢀlucent vacuoles with diameters > 3 ꢁm or > 3
smaller vacuoles (0.5–3 ꢁm) were counted as positive. Cell viability was determined by MTS
assay. Cells were seeded at an appropriate density into 96ꢀwell plates and allowed to attach
overnight. Compounds were serially diluted with growth medium, and then added in triplicate
into 96ꢀwell plates and incubated for 48 h. Subsequently, viability was determined by MTS
(Promega, Madison, WI, USA), according to the manufacturer’s instructions and IC50 values
were calculated by software GraphPad Prism5.
Crystal Violet Staining Assay. MDAꢀMBꢀ231 cells were seeded into 6ꢀwellꢀplates and
cultured for 12 h. Then medium was refreshed and cells were exposed to 0.1% DMSO or
different concentrations of Cmpd. 13. After 48 h incubation, the cells were fixed with 4% (v/v)
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paraformaldehyde for 15 min at room temperature. Subsequently, the cells were stained with
crystal violet and observed by optical microscopy (AX20 OBSERVER A1, Zeiss, Germany).
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Live Cell Imaging. Mitoꢀtracker Red, ERꢀtracker and Lysoꢀtracker Red were purchased from
Beyotime (Haimen, China). Lucifer yellow CH was purchased from Molecular Probes (Eugene,
OR). MDAꢀMBꢀ231 cells were seeded in a 30 mm glass bottom culture dish (NEST
Biotechnology, China) and allowed to attach overnight. Cells were treated with 2 ꢁM Cmpd. 13
for 12 h and then incubated with MitoꢀTracker, ERꢀtracker and LysoꢀTracker Red according to
the manufacturer’s instructions, respectively. In Lucifer yellow (LY) staining, cells were treated
with Cmpd. 13 and LY simultaneously for 12 h, then washed with PBS and photographed. All
images were collected with a confocal microscope (Zeiss LSM 780).
Immunofluorescence Staining. Cells seeded on coverslips were fixed with 4% (v/v)
paraformaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton Xꢀ100
for 20 min, followed by blocked for 30 min at room temperature with 5% BSA. Cells were then
incubated for 1 h with antiꢀRab7 antibody (1:500 dilution; Cell Signaling Technology). After
washing for three times with PBS, the cells were further incubated for another 1 h with
secondary antibodies Alexa Fluor 488–conjugated anti–rabbit IgG (Life Technologies).
Subsequently, the cells were washed three times with PBS. All images were collected with a
confocal microscope (Zeiss LSM 780).
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