Journal of Medicinal Chemistry
Article
could provide precious information for a future lead
optimization.
EXPERIMENTAL SECTION
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General Methods. All of the reagents and solvents were purchased
from commercial suppliers and used without further purification unless
otherwise indicated. Reactions were cooled using a cryocooler or
external cooling baths (−78 °C or ice water 0 °C). Heating was
achieved using a silicone oil bath with heating controlled by an
electronic contact thermometer. Reaction courses were monitored by
thin-layer chromatography (TLC) on silica gel-precoated F254 Merck
plates. Purification of reaction mixtures was performed by column
chromatography using silica gel (100−200 mesh, 200−300 mesh,
Qingdao Marine Chemical Ltd., Qingdao, China). All of the
intermediates and target compounds were fully analyzed and
JM Downregulated PI3K/Akt Signaling Pathway in BC
Cells. The PI3K/Akt signaling pathway plays a central role in
intracellular signal transduction, which participates in regulating
various cell functions, including proliferation, survival, meta-
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2
stasis, metabolism, angiogenesis, etc. More recently, the
overexpression of oncogenic PTPs results in hyperactivating
the PI3K pathway in some human cancers, leading to the
53
promotion of tumor growth and development. For these
reasons, western blot analysis was carried out to check if the
inhibitory effect of JM on BC cells was mediated by the PTP1B/
PI3K/Akt pathway. The protein expression of PI3K p85 and Akt
in JM-treated BC cells were not altered compared to that of the
control group, but the phosphorylation levels of PI3K and Akt
were apparently inhibited in a concentration-dependent manner
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characterized by H and C NMR spectra, high-resolution mass
spectra (HRESI-MS), ultraviolet (UV) spectra, and infrared (IR)
spectra before biological screening. The following abbreviations were
used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q
= quartet, qn = quintet, m = multiplet, br = broad. All of the targeted
compounds were analyzed by high-performance liquid chromatography
(
Figure 9A−F). Consequently, our data suggested that JM
(
HPLC) with a UV−visible detector (2 μm, 50 mm × 2.0 mm, Agilent
YMC-UltraHT Pro C18), eluted at 0.35 mL/min with MeOH/water +
might block cell proliferation and induce cell apoptosis by
targeting PTP1B and deactivating the PI3K/Akt pathway.
JM-Suppressed MDA-MB-231 Xenograft Growth In
Vivo. Following the excellent selectivity of JM for TNBC in
vitro, we established an MDA-MB-231 xenograft mouse model
to evaluate the inhibitory efficacy of JM on TNBC in vivo. As
depicted in Figure 10A,B, the growth rate and tumor size in mice
were substantially retarded with increasing the dosage of JM.
Compared with the control group, the inhibiting values of tumor
growth inhibition (TGI, %T/C) at 15 and 30 mg/kg dosage of
JM for 20 days were up to 53.6 and 79.3%, respectively (Figure
5
% MeCN + 0.1% formic acid (gradient: 85−95% in 25 min). The
purities of compounds were confirmed as ≥95%.
Cell Culture and Cell Viability Assay. All of the cell lines used in
our study (obtained from the Chinese Academy of Science, Shanghai,
China) were cultured in Dulbecco’s modified Eagle’s medium
(
DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-
glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37
°C in a humidified 5% CO atmosphere incubator (Thermo Fisher
2
Scientific, Waltham, MA). The cytotoxicity and proliferation inhibition
were quantified by the MTT assay. The results were represented as
mean ± standard deviation (SD) from three independent experiments.
1
0C). Notably, there were no significant changes in body
The inhibition rate (%) = [A570 (control) − A (compound)]/A570
control) × 100%. The IC50 values were calculated by a nonlinear
570
weights among the three groups, which demonstrated no severe
toxicity of JM in mice (Figure 10D). Consistent with in vitro
study, our in vivo study indicated that the expression levels of
Bax, cleaved caspase-3, cleaved PARP, and p21 were
dramatically upregulated, whereas cyclin D3, cyclin E1, and
Bcl-2 expressions were downregulated in the JM-administered
mice (Figure 10E). Moreover, the PTP1B protein expression of
tumor tissue obviously decreased in JM-administered groups. As
a result, JM could target PTP1B and restrain the growth of
MDA-MB-231 tumor xenograft via inducing apoptosis and cell
cycle arrest.
(
regression analysis using SPSS 13.0 (SPSS, Inc., Chicago, IL).
Cluster Analysis Assay. Based on the Euclidean distance of the
IC50 values of test compounds against BC cells, cluster analysis was
performed to characterize their anti-BC potency and structural features
using the “ggplot2” package in the program R 4.0.2 (R Core Team,
2020).
Hoechst 33342 Staining. Cells stained with Hoechst 33342
Beyotime Biotechnology Co., Shanghai, China) were widely used to
(
evaluate nuclear morphological changes. MDA-MB-231 and MCF-7
4
cells (6 × 10 cells/well) were seeded in 24-well plates and treated with
different concentrations of JM (0 and 5.0 μM for MDA-MB-231; 0 and
1
5.0 μM for MCF-7) for 24 h. Subsequently, the cells were washed
CONCLUSIONS
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three times with phosphate-buffered saline (PBS) and stained with 1
mL suspension of Hoechst 33342 and PBS for 5 min in the dark at rt.
The changes in nuclear morphology were evaluated and photographed
by a fluorescence microscope (Leica, Wetzlar, HE, Germany).
In summary, we elaborated the total synthesis of jamunones and
deduced the primary SAR of anti-BC. The meaningful SARs can
be drawn as follow: (i) hydroxyl group at the C-7 position is
essential for activity; (ii) the length of alkyl side chain ranging
from 11 to 19 carbon atoms is favorable for anti-BC activity; (iii)
the 6,8-dimethyl substitution in chromanone or chromone
scaffold benefits to improve the selectivity toward TNBC cell.
JM was screened out as a highly selective candidate for treating
TNBC. The in-depth biological investigations revealed distinct
morphological changes, an excess ROS production, a downward
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Cell Apoptosis Assay. MDA-MB-231 and MCF-7 cells (3 × 10
cells/well) were seeded in six-well plates and exposed to compound JM
(0 and 5.0 μM for MDA-MB-231; 0 and 15.0 μM for MCF-7) for 24 h.
Afterward, the cells were collected, washed with cold PBS, and
resuspended in 195 μL of binding buffer. Fluorescein isothiocyanate
(FITC)-conjugated annexin-V reagent (1 μL) and PI (8 μL, BD,
Franklin Lakes, NJ) were added to the samples and mixed gently. After
15 min incubation in the dark at room temperature, the samples were
immediately run on an LSR-Fortessa flow cytometer and analyzed with
the Annexin V-FITC/PI apoptosis method. Cytographs were
performed using BD FACSDiva software (BD, Franklin Lakes, NJ).
Each experiment was conducted three times.
ΔΨ , apoptotic induction, and cell cycle blockage at the G0/G1
m
phase in JM-treated BC cells. The anti-BC feature of JM was
found to target PTP1B, which resulted in deactivating PI3K/Akt
pathway. Subsequently, the docking profile indicated that the
formation of multiple hydrogen bonds and hydrophobic tunnel
ensured the stable fit of JM in the catalytic domain of PTP1B,
further elucidating the targeting ability of JM and some
significant SARs. More importantly, JM dramatically inhibited
tumor growth in the MDA-MB-231 tumor xenografts without
obvious toxicity. All in all, these findings identified JM as an
attractive drug candidate for PTP1B-targeted TNBC therapy.
Reactive Oxygen Species (ROS) Assay. The formation of
intracellular ROS was assessed using a ROS Assay kit (Beyotime
Biotechnology Co., Shanghai, China). MDA-MB-231 and MCF-7 cells
4
(
6 × 10 cells/well) were seeded in 24-well plates with 5.0 and 15.0 μM
of M treatment for 24 h, respectively. After washing once with PBS,
these cells were stained with 10 μM dichlorodihydrofluorescein
diacetate (DCFH-DA) at 37 °C for 30 min in the dark, washed three
times with serum-free medium, and then resuspended in serum-free
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J. Med. Chem. 2021, 64, 6008−6020