Discovery of new fluorescent thiazole–pyrazoline derivatives as autophagy inducers by inhibiting mTOR activity in A549 human lung cancer cells

Article abstract of DOI:10.1038/s41419-020-02746-w

A series of fluorescent thiazole–pyrazoline derivatives was synthesized and their structures were characterized by ¹H NMR, ¹³C NMR, and HRMS. Biological evaluation demonstrated that these compounds could effectively inhibit the growt

Full text of DOI:10.1038/s41419-020-02746-w

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
https://doi.org/10.1038/s41419-020-02746-w
Cell Death & Disease
A R T I C L E
O p e n A c c e s s
Discovery of new fluorescent thiazole–pyrazoline
derivatives as autophagy inducers by inhibiting
mTOR activity in A549 human lung cancer cells
ZhaoMin Lin1, ZhaoYang Wang2, XueWen Zhou3, Ming Zhang1, DongFang Gao1, Lu Zhang1, Peng Wang1,
Yuan Chen1, YuXing Lin1, BaoXiang Zhao3, JunYing Miao2 and Feng Kong1,4
Abstract
1
A series of fluorescent thiazole–pyrazoline derivatives was synthesized and their structures were characterized by H
NMR, 13C NMR, and HRMS. Biological evaluation demonstrated that these compounds could effectively inhibit the
growth of human non-small cell lung cancer (NSCLC) A549 cells in a dose- and time-dependent manner in vitro and
inhibit tumor growth in vivo. The structure–activity relationship (SAR) of the compounds was analyzed. Further
mechanism research revealed they could induce autophagy and cell cycle arrest while had no influence on cell
necrosis. Compound 5e inhibited the activity of mTOR via FKBP12, which could be reversed by 3BDO, an mTOR
activator and autophagy inhibitor. Compound 5e inhibited growth, promoted autophagy of A549 cells in vivo.
Moreover, compound 5e showed good selectivity with no influence on normal vascular endothelial cell growth and
the normal chick embryo chorioallantoic membrane (CAM) capillary formation. Therefore, our research provides
potential lead compounds for the development of new anticancer drugs against human lung cancer.
Introduction
Nevertheless, studying the distribution and targets of
Cancer is still a major global health concern and a anticancer compounds in living cells poses a great chal-
leading cause of death all over the world. It is shown that lenge for researchers and great help to improve the
lung cancer remains the highest death rate in all cancer activity and selectivity. Fluorescigenic small molecules
deaths both in developed and developing countries1. Over provide a huge boost for determining their location and
the past decades, much attention has been paid to the targets in living cells. Fluorescent compounds have been
discovery of effective method to overcome cancer thor- used as powerful detection tools in cell biology. Currently,
oughly. Despite more and more anticancer therapies were due to the nature of high quantum yield and readily
developed, chemotherapy is still one of the most common synthetic process, some pyrazoline derivatives have been
cancer therapies to prolong the lifespan of cancer synthesized and used in fluorescence probes, for orien-
patients2,3. However, due to side effect and drug resis- tation4, detecting cation5–8, hydrazine9,10, thiols11–13, and
tance, it is an urgent issue to develop novel, selective DNA14. Moreover, their biological roles have been studied
anticancer agents.
in insecticidal function15–17, human monoamine oxidase
activity inhibition18,19
,
anti-inflammation20–22
,
anti-
microbial23,24, analgesia25. In addition, pyrazoline deriva-
tives could inhibit the proliferation of cancer cells with
Correspondence: Feng Kong (kongfeng@sdu.edu.cn)
1Institute of Medical Science, The Second Hospital of Shandong University,
Jinan 250033, PR China
satisfactory activity26,27
.
However, the anticancer
mechanism was little delineated.
Autophagy, an important process in eukaryotes through
which useless organelles were delivered to lysosomes for
2Shandong Provincial Key Laboratory of Animal Cells and Developmental
Biology, School of Life Science, Shandong University, Jinan 250100, PR China
Full list of author information is available at the end of the article
Edited by M Hamasaki
© The Author(s) 2020
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If
material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 2 of 12
degradation and reuse, plays double-edged roles in tumor Chloroquine (CQ) and Bafilomycin-A1 (Baf-A1) were
initiation and progression depending on different cell purchased from Sigma-Aldrich (St. Louis, MO, USA).
types and specific stages of tumor progression28,29. On the
one hand, autophagy deficiency has a positive effect on Preparation of chalcone compounds (3)
malignant transformation, indicating autophagy as a
In a flask, compound 1 (10 mmol) was dissolved in
tumor suppressor mechanism30,31. On the other hand, ethanol (10 ml), and sodium hydroxide solution in water
excessive autophagy could contribute to cell death in (8 ml, 2.5 M) was added. Then compound 2 dissolved in
certain cancer cell types which maintained the cellular ethanol (10 ml) was added to the above mixture. After
functions by triggering autophagy32,33. Considering the reaction completed (by TLC monitoring), yellow pre-
dual nature of autophagy in tumorigenesis and progres- cipitate was filtered, then the solid was washed with water
sion, more modulators of autophagy may provide a to make pH = 7. The yellow product was dried by infrared
powerful tool for cancer therapy.
lamp to give compound 3 in more than 80% yield.
Mechanistic target of rapamycin (mTOR [serine/
threonine kinase]/FK506-binding protein 12-rapamycin Preparation of 3,5-diaryl-4,5-dihydro-1H-pyrazole-1-
associated protein 1), regulates the maintenance of cell carbothioamide (4)
homeostasis, including cell growth, autophagy, and
Thiosemicarbazide (6 mmol), sodium hydroxide
cytoskeletal organization34,35. The dysregulated activity of (8 mmol), compound 3 and ethanol (30 ml) were added
mTOR involved in several human disorders, including into round-bottom flask. The reaction mixture was
cancers, such as lung cancer, breast cancer, and others36. refluxed for 3–8 h, it was monitored by TLC until com-
Due to the key role of proliferation in numerous malig- pletion. The mixture was cooled to room temperature.
nant cell types, there were many potential applications in Precipitate was filtered, and washed three times with
the therapy of various solid tumors and hematological water and ethanol. After dried with infrared lamp, cor-
malignancies by targeting the mTOR pathway37,38. How- responding product 4 was obtained.
ever, the expectations of more effective and less toxic
treatment with mTOR inhibitors have not realized.
In a continuation of an ongoing program aiming at
Preparation of compound 5
Compound 4 (1 mmol) was added into ethanol (25 ml),
finding novel fluorescent small molecules with anticancer then 2-bromo-1-(pyridin-4-yl)ethanone (1 mmol) was
activity39–41, a series of thiazole–pyrazoline derivatives added into the mixture. The reaction mixture was
were synthesized and their properties in A549 cells were refluxed for 4–9 h (monitored by TLC until completion).
evaluated. In this work, deep insights into the anti- The mixture was cooled to room temperature and filtered.
neoplastic activity and mechanism of pyrazoline deriva- The solid was washed with ethanol two times. The solid
tives were gained to provide a basis for the rational and was dissolved in dichloromethane. The mixture was
targetable design of fluorescent anticancer drug for clin- washed with saturated NaHCO3, followed by wash with
ical application.
saturated brine. Organic phase was dried over magnesium
sulfate. After desiccant was removed by suction filter,
organic phase was concentrated under reduced pressure
to give the target products 5. The spectroscopy data of
Materials and methods
Reagents and apparatus
All reagents were of analytical grade or chemically pure. compounds 5a–5j are loaded in the Supplementary file.
Thin-layer chromatography (TLC) was performed on
silica gel 60 F254 plates (Merck KGaA) and column Antibodies
chromatography was conducted over silica gel (mesh
Antibody for Light chain 3 beta (LC3B) (2775 S),
1
200–300). H NMR spectra were recorded on a Bruker EIF4EBP1 (9452), p-EIF4EBP1 (9459), RPS6KB1 (9202),
Avance 400 (400 MHz) spectrometer or Bruker Avance p-RPS6KB1 (9205), and p-mTOR (2971) were purchased
300 (300 MHz) spectrometer, using DMSO-d6 as solvent from CST. Antibody for β-actin (sc-47778), mTOR (sc-
and tetramethylsilane as an internal standard. Melting 8319), FKBP12, and horseradish peroxidase-conjugated
points were determined on an XD-4 digital micro melting secondary antibodies were bought from Santa Cruz.
point apparatus. IR spectra were recorded with an IR Secondary antibodies for immunofluorescence were
spectrophotometer Avtar 370 FT-IR (Termo Nicolet). MS donkey anti-rabbit IgG Alexa Fluor-488 (A10040), which
spectra were recorded on a Trace DSQ mass spectro- was purchased from life technology.
graph. Unless otherwise stated, all reagents were pur-
chased from J&K, Sinopharm Chemical Reagent Co. and Cell culture
Kermel and used without further purification. Twice-
All the cells utilized in the experiment were purchased
distilled water was used throughout all experiments. from the Cell Culture Bank of the Chinese Academy of
Rapamycin was from Calbiochem (Darmstadt, Germany). Sciences (http://www.cellbank.org.cn/). Human lung
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 3 of 12
cancer cell line A549 and H460, human liver carcinoma treatment. Cells were washed twice with ice-cold phos-
cell line HepG-2, human hormone-independent prostate phate-buffered saline (PBS), then lysed in protein lysis
carcinoma cell line PC3, human kidney clear cell adeno- buffer (Shanghai beyotime Co., China). The protein con-
carcinoma cell line 786-O, human breast carcinoma cell centration of the cells was measured by the Bradford
line 4T1, and human renal tubular epithelial cell HK-2 method. Following separation by SDS-PAGE and trans-
were cultured in RPMI-1640 medium with 10% (v/v) ferring to PVDF membrane (Millipore, USA), proteins
bovine calf serum and 80 U/ml penicillin/streptomycin. were incubated with primary antibodies, then incubated
Human glioblastoma cells U87 and human embryonic with horseradish peroxidase-linked secondary antibodies,
kidney cell 293T were grown in DMEM medium (Gibco, and finally probed by using an enhanced chemilumine-
USA) with 10% FBS, penicillin (50 U/ml), and strepto- sence detection kit (Thermo). Actin β was used as a
mycin (50 ug/mL) (Invitrogen, 10378-016). Human loading control. The relative quantity of proteins was
umbilical vein endothelial cells (HUVEC) were grown in analyzed by Image J software and normalized to loading
M199 medium (Gibco, 31100-035) with 10% (v/v) bovine controls.
calf serum and 8.4 IU/mL FGF2. All cell lines were cul-
tured in a humidified incubator with 5% CO2 at 37 °C.
RNA interference
A549 cells were transiently transfected with siRNA
duplex oligonucleotides targeting LC3B (GenePharmcon,
Cell morphology
Morphologic changes of A549 cells treated with com- Shanghai, China) using Lipofectamine 2000 (Invitrogen,
pounds at indicated concentration for 12, 24, and 48 h 11668-019). After 24-h transfection, cell lysates were
were examined by inverted phase-contrast microscope subjected for the western blot assay and cells were treated
(Eclipse TS-100; 21 Nikon, Tokyo).
with 5e or vehicle for an additional 24 h. Cell viability was
determined as described above. FKBP12 siRNA (sc-
35678) and scramble RNA (sc-37007) were obtained from
Cell viability assay
Cells were seeded onto 96-well plates for 24 h and then Santa Cruz Biotechnology. A549 cells at 50–60% con-
treated with 0.1% DMSO (v/v, as control), 5-fluorouracil fluence were transfected with 60-nM siRNA against
(5-FU, as positive group) or compounds 5a–5j at indi- FKBP25, FKBP12, and scramble siRNA with Lipofecta-
cated concentrations (0.1, 1, 5, 10 μM) for 24 and 48 h. mine 2000 according to the manufacturer’s instructions.
Cell viability was measured by sulforhodamine B (SRB) Then cells were harvested and analyzed by western blot.
assay, in accordance with the previous method42. The
intensity of light absorption was measured by using a Flow cytometric analysis of cell cycle distribution
SpectraMAX190 microplate spectrophotometer (GMI Co,
Following treated with compounds 5a, 5d, 5e, 5g and
USA) at the wavelength of 540 nm. In some experiments, 5h (10 μM) for 48 h, A549 cells were harvested and fixed
A549 cells were transfected with siRNAs and/or treated with 70% ice-cold ethanol, then stained with 50 mg/ml
with 5e. A549 cells were exposed to autophagy inhibitors propidium iodide containing 10 mg/ml RNase A at 4 °C
CQ (20 mM) and Baf-A1 (50 nM) for 2 h before treatment for 10 min. The stained cells were analyzed by using a flow
with 5e.
cytometer (ImageStreamX MarkII, Amnis, USA). The cell
cycle distribution was analyzed by IDEAS software
(Amnis, USA).
Lactate dehydrogenase (LDH) assay
Cell culture medium was gathered after 24-h treatment
with compounds 5a–5j (10 μM) or 0.1% DMSO (as con- Immunofluorescence assay
trol). LDH assay was conducted by using a LDH kit
Treated cells were fixed in 4% paraformaldehyde (w/v)
(Nanjing Jiancheng Co, China), according to the manu- for 30 min at room temperature and then incubated with
facturer’s description.
normal donkey serum (1:30) for 30 min and primary
antibodies (1:100) overnight at 4 °C. Cells were washed
with PBS times, and then three incubated with secondary
Co-localization imaging of cells
A549 cells were incubated with 5e (1 μM) for 1 h at antibodies (1:200) for 1 h at 37 °C. Fluorescence was
37 °C. Then, MitoTracker Deep Red (0.1 μM), Lyso Sensor detected by laser scanning confocal microscopy Zeiss
Green (0.3 μM), and ER Tracker Red (0.3 μM) were added LSM700 (Germany). Frozen sections of tumors formed on
and incubated for another 0.5 h and the confocal fluor- the chick embryo chorioallantoic membrane (CAM) were
escent images were captured.
fixed with cold acetone for 10 min and blocked with 10%
normal donkey serum (Solarbio, SL050) for 30 min at
room temperature. Then frozen sections of tumors were
Western blot analysis
Total proteins were obtained from A549 by using IP incubated with primary antibody (1:100; LC3B, Rabbit
lysis buffer (Shanghai beyotime Co., China) after different polyclonal antibody, Santa Cruz Biotechnology) at 4 °C
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 4 of 12
Scheme 1 Synthesis of compounds 5a–5j.
overnight and then corresponding secondary antibody software. Mean values were derived from at least three
(1:200) at 37 °C for 1 h. Frozen sections of tumors were independent experiments. Differences at p < 0.05 were
washed three times with 0.1-M PBST. DAPI (1:200) was considered statistically significant.
added to stain cell nucleus for 10 min and then the sec-
tions were washed three times with PBS. Fluorescence was Results
detected by confocal fluorescence microscopy Zeiss Chemistry
LSM700 (Germany).
The synthetic route of compounds 5a–5j has been
accomplished as shown in Scheme 143. An aryl aldehyde
(1) reacted with an aryl methyl ketone (2) to give a
Chick embryo CAM assay
Fertile chicken eggs (7–9 days old) were used to conduct chalcone (3). The chalcone (3) reacted with thiosemi-
the CAM assay. An amount of (1–10) × 106 A549 cells carbazide to afford 3,5-diaryl-4,5-dihydro-1H-pyrazole-1-
suspended in 20-μL RPMI-1640 was engraftment on the carbothioamide (4). Compound 4 reacted with 2-bromo-
CAM. Next, the egg shell was sealed with gas-permeable 1-(pyridin-4-yl)ethanone to produce target products, 2-
tape to avoid bacterial infection for another two days. (3,5-diaryl-4,5-dihydro-1H-pyrazol-1-yl)-4-(pyridin-4-yl)
Then, the eggs were treated with PBS (negative control, thiazole (5).
qod × 3), the compound 5e (25 and 50 μM, qod × 3) or 5-
Fu (50-μM positive group, qod × 3). After fixed by 4% In vitro antiproliferative activity and structure–activity
paraformaldehyde for 30 min, CAMs were separated from relationship (SAR) study
the eggs and photographed by a stereomicroscope (Japan).
In order to examine the anticancer activity of com-
pounds 5a–5j, we firstly observed the morphological
changes of A549 cells treated for 12, 24, or 48 h by a
Angiogenesis assay of CAM in vivo
Fertilized chicken eggs were incubated with 55% relative phase-contrast microscope. The data showed obvious
humidity at 37 °C. On embryonic day 7 or 8, 5e (25 and morphological changes in A549 cells treated with the
50 μM) soaked in the gelatin sponge was applied to the compounds 5a–5j in dose- and time-dependent manners
CAM and DMSO as the vehicle control for 48 h. Then, (Fig. S1). Compared with control group, the cell density
repeated the above operation for three times. At the end dramatically decreased and cells were elongated or
of the incubation, after fixed by 4% paraformaldehyde for formed triangle and arborization significantly treated for
30 min, the CAM zones around the gelatin sponge were 48 h (Fig. 1a, an excerpt of Fig. S1). SRB assay was con-
photographed and analyzed by using the Image-Pro Plus. ducted to investigate the effect of these compounds on
cell proliferation. The data indicated that these com-
Statistical analyses
pounds suppressed the growth of A549 cells in a dose-
Data were presented as means SE and analyzed by dependent manner after treatment with the compounds
SPSS software. Pictures were processed with Photoshop for 24 and 48 h (Fig. 1b). The IC50 (μM) values of the
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 5 of 12
Fig. 1 The viability of cells incubated with compounds 5a–5i. a Effects of compounds 5a–5i at 10 μM on changes in cell morphology for 48 h
(100×). These images were re-used from Fig. S1. b Effects of compounds 5a–5i on cell viability assessed by sulforhodamine B for 24 and 48 h. c The
effect of compound 5e on HUVECs cell viability assessed by sulforhodamine B for 24 and 48 h. Results were presented as mean SE; n = 3; *p < 0.05;
**p < 0.01. Bar = 20 μm.
compounds were totally <10 μM (Table 1). Our finding 4T1, J82, and human nontumorigenic cell lines HK-2 and
demonstrated that all tested derivatives exhibited con- 293T. As shown in Table S1, compound 5e also exhibited
siderable cell growth inhibition on A549 cells. Further- obvious growth inhibiting activity against other cancer
more, we explored the inhibitory effect of compound 5e cell lines, but did not inhibit the growth of normal cell
on human cancer cells lines H460, HepG-2, PC3, 786-O, lines. 5e had no influence on the growth of HUVECs (Fig.
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 6 of 12
1c), indicating that this compound showed good these compounds are mainly affected by aryl group in 5
selectivity. position of pyrazoline moiety. When substituent Ar is
Further, based on the above results, the SAR of the benzo[d][1,3]dioxol-5-yl (compounds 5g–5i), the inhibi-
compounds was analyzed. The antiproliferative activity of tion effect for cell growth is stronger. When Ar is 4-
methoxyl phenyl (5d–5f), compounds have also a higher
growth inhibitory effect. However, in the case of Ar is
phenyl, antitumor activity is poorer. Taken together,
compounds 5g, 5h were the most effective compounds in
suppressing A549 cell growth.
Table 1 Growth inhibitory properties (IC50, 48 h) of
compounds 5a–5i and 5-FU in A549 cells. Results are mean
SEM.
Compounds
IC50 (μM)
Compounds
IC50 (μM)
Continuous proliferation is the hallmark of cancer cells.
Because regulation of the cell cycle is critical for cell
growth, we investigated the effect of compounds 5 on cell
cycle progression using flow cytometry. The results
showed that compounds 5a, 5d, 5e, 5g, or 5h effectively
arrested the cell cycle at the G1 phase at the concentra-
tion of 10 μM for 48 h, which were in accordance with the
growth inhibitory effect of these compounds. Notably,
5-FU
5a
9.4 0.23
4.4 0.12
8.7 0.15
6.0 0.08
4.7 0.12
5e
5f
2.6 0.11
2.9 0.09
1.8 0.08
1.8 0.05
2.7 0.36
5b
5g
5h
5i
5c
5d
Fig. 2 The distribution of compound 5e in A549 cells. a A549 cells were treated with 10-μM compound 5e, followed by MitoTracker Deep Red
(0.1 μM, 0.5 h), Lyso Sensor Green (0.3 μM, 0.5 h), or ER Tracker Red (0.3 μM, 0.5 h). The average Pearson’s coefficient was shown in bar chart.
Compound 5e: λex = 405 nm, λem = 405–490 nm. MitoTracker Deep Red: λex = 635 nm, λem = 635–700 nm. Lyso Sensor Green: λex = 488 nm, λem
=
488–700 nm. ER Tracker Red: λex = 555 nm, λem = 555–700 nm. b A549 cells were treated by 1 μM, 5 μM, or 10 μM compound 5e for 24 h, then,
incubated with Lyso Sensor Green (0.3 μM, 0.5 h). The average Pearson’s coefficient was shown in bar chart. Compound 5e: λex = 405 nm, λem
405–490 nm. Lyso Sensor Green: λex = 488 nm, λem = 488–700 nm (200×). X-axis means mean intensity of compound 5e and y-axis means mean
intensity of different trackers. Numbers 1 and 2 mean respective regions and 3 means colocation region. Bar = 10 μm.
=
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 7 of 12
Fig. 3 (See legend on next page.)
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(see figure on previous page)
(
2
0
2
0
)
1
1
:
5
5
1
Page 8 of 12
Fig. 3 Compound 5e induced autophagy in a mTOR-dependent manner. a Western blot analysis of LC3B-I and LC3B-II in A549 cells treated with
compound 5e at 10 μM for indicated times and quantification of LC3B-II levels. b Images of EGFP-LC3B U87 cells were treated with compound 5e at
the concentration of 10 μM for 3, 6, 12, and 24 h (200×) and quantification of EGFP-LC3B dots. Bar = 10 μm. c Western blot analysis of LC3B-I and
LC3B-II in A549 cells treated with compound 5e (10 μM), Baf-A1 (50 nM), or both for 12 h and quantification of LC3B-II levels. d Western blot analysis
of RPS6KB1 and p-RPS6KB1 (S424/T421) in A549 cells treated with compound 5e at 10 μM for indicated times and quantification. e Western blot
analysis of EIF4EBP1 and p-EIF4EBP1 (S65/T70) in A549 cells treated with 5e at 10 μM for indicated times and quantification. β-actin was used as a
loading control. Results were presented as mean SE; n = 3; *p < 0.05; **p < 0.01.
treatment with 5e enhanced the G1 population by 33.8% in stably expressing EGFP-LCB3 U87 cells in a time-
(Fig. S2A). Necrosis, an unwanted side effect of cancer- dependent manner (Fig. 3b). Actually, all other com-
fighting agents, could be evaluated by the LDH assay. pounds could also induce autophagy (Fig. S4). In order to
LDH assay was performed on cells treated with the demonstrate whether compound 5e could induce intact
compounds and 0.1% DMSO. Our data revealed that autophagy flux, Baf-A1, a recognized inhibitor of vacuolar
these compounds had no influence on the release of LDH H+-ATPase, was used to block autophagy. As shown in
(Fig. S2B).
Fig. 3c, treatment with compound 5e further enhanced
the accumulation of LC3B-II induced by Baf-A1, implying
that compound 5e could induce complete autophagy flux.
Compound 5e distributed in lysosome in A549 cells
Given these compounds containing fluorescent group, To assess the impact of autophagic flux in 5e-induced cell
we detected the fluorescence in A549 cells by a fluor- death, we analyzed cell viability and cell death by pre-
escent microscope. The data indicated that these com- treating A549 cells with autophagy inhibitors, chlor-
pounds have good excellent water-solubility and oquine, and Baf-A1. The results showed that these two
membrane permeability, especially compound 5e had inhibitors both could increase the cell viabilities compared
good fluorescence at 0.1 μM (Fig. S3). Combining with 5e with cells treated with 5e solely (Fig. S5A,B). We further
showed good cell growth inhibition activity and high confirmed the role of autophagic flux in the action of 5e
selectivity, this compound was chosen for further by knockdown of specific autophagy-related LC3B gene.
mechanism research.
Reduction of LC3B by siRNA significantly alleviated
First, the intracellular distribution of compound 5e in cytotoxic activity of 5e (Fig. S5C,D). Taken together, our
A549 cells was explored according to good fluorescence. data clearly demonstrated that 5e activated an autophagic
We used commercial lysosome probe (Lyso Sensor flux and promoted autophagy-dependent cell death.
Green), mitochondria probe (mitochondria Deep Red), Compound 5e had no influence on the growth of
and endoplasmic reticulum probe (ER red) to co-stain HUVECs and could not induce autophagy in HUVECs
A549 cells. The result showed that compound 5e had (Fig. S6).
good co-localization with lysosome (Pearson’s coefficient
mTOR is a crucial molecular during the process of
0.903), but poor with mitochondria (Pearson’s coefficient autophagy. To understand whether compound 5e can
0.563) or endoplasmic reticulum (Pearson’s coefficient suppress the activity of mTOR, we examined the influence
0.733) (Fig. 2a), implying a preferential distribution of of compound 5e on the phosphorylation of RPS6KB1
compound 5e in lysosome. Furthermore, the concentra- (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and
tion of compound 5e had no significant effect on its cel- EIF4EBP1 (eukaryotic translation initiation factor 4E-
lular distribution (Fig. 2b).
binding protein 1), two essential substrates of mTOR36,37
.
Obviously, the levels of phosphorylation of RPS6KB1 and
Compound 5e induced autophagy of A549 cells
EIF4EBP1 were significantly decreased after treatment
Autophagy is a highly conserved lysosomal degradation with compound 5e for 3, 6, 12 and 24 h (Fig. 3d, e). The
pathway in which unnecessary byproducts and damaged levels of p-mTOR and mTOR were detected by immu-
organelles are engulfed into double-membrane vesicles nofluorescence staining based on the fluorescence char-
termed autophagosomes and transported to lyso- acteristics of compound 5e. The data showed that 5e
somes44,45. Due to compound 5e located in lysosomes, we could reduce the phosphorylation of mTOR (Fig. S7).
investigated the effect of this compound on autophagy. Therefore, compound 5e might induce autophagy in an
LC3B, an autophagy maker, was monitored by western mTOR-dependent manner.
blotting. The result showed that the levels of LC3B-II
were enhanced after incubation with 5e at 10 μM for 3, 6, Compound 5e targeted to FKBP12 and inhibited mTOR
12 and 24 h, indicating compound 5e induced autophagy
3-Benzyl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2
in a time-dependent manner (Fig. 3a). Moreover, autop- (3H)-one (3BDO), which was found by our group, could
hagic LC3B-II accumulation was dramatically enhanced activate mTOR by targeting FKBP12 (FK506-binding
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 9 of 12
Fig. 4 Compound 5e inhibited mTOR activity via FKBP12. Western blot analysis of RPS6KB1, p-RPS6KB1, EIF4EBP1, and p-EIF4EBP1. a A549 cells
were treated with compound 5e at indicated concentrations for 6 h along with pretreatment of 3BDO or not. The pretreatment time of 3BDO was
3 h and the concentration were 0.1, 1, and 30 μM respectively. b A549 cells were treated with compound 5e for 6 h in 10 μM solely or along with
SiFKBP12 in 60 nM for 24 h. β-actin was used as a loading control. Results were presented as mean SE; n = 3; *p < 0.05; **p < 0.01.
protein 1A)46. To understand how 5e inhibited mTOR, due to its immune-deficient environment47. As shown in
we examined the effect of 5e on RPS6KB1 and 4EBP1 Fig. 5a, compared with the DMSO-treated eggs, sig-
phosphorylation in the presence or absence of 3BDO. As nificant xenograft tumor remission was observed after
expected, levels of p-RPS6KB1 and p-EIF4EBP1 were 6 days in eggs treated with compound 5e. Notably,
decreased with 5e; however, 5e failed to decrease the compound 5e exerted a better antitumor effect than the
phosphorylation of RPS6KB1 and EIF4EBP1 in the pre- therapeutic drug 5-Fu. We further detected that com-
sence of 3BDO (Fig. 4A). Accordingly, the mTOR inhi- pound 5e had no significant toxicity on angiogenesis
bition of 5e was reversed by 3BDO. Then, SiFKBP12 was in vivo in contrast with 5-Fu which suppressed capillary
used to investigate the molecule target of 5e. The inter- formation (Fig. 5b). Therefore, 5e effectively inhibited
ference efficiency of SiFKBP12 was 56% in 60 nM (Fig. tumor growth in vivo without adverse effect on normal
S8). As shown in Figs. 4b and 5e could not inhibit the CAM angiogenesis.
activity of mTOR when FKBP12 was knockdown. These
results demonstrated that 5e inhibited the activity of Compound 5e induced A549 cells autophagy in vivo
mTOR via FKBP12.
To further investigate the mechanism by which com-
pound 5e inhibited tumor growth in vivo, we prepared
frozen sections of solid tumors formed on CAM. Immu-
Compound 5e inhibited tumor growth in vivo
Given compound 5e effectively inhibited the growth of nofluorescence experiment was performed on frozen
A549 cell and did not influence the growth of HUVECs sections of tumor. Data showed that compound 5e ele-
in vitro (Fig. 1c), we chose the chick embryo CAM model vated the level of LC3B-II in tumor tissue, which was in
for further research to evaluate the antitumor effect of 5e. accordance with the results in vitro (Fig. 5c). These data
The chick embryo CAM is extensively used for tumor demonstrated that 5e inhibited lung cancer growth
engraftment to evaluate the efficacy of anticancer drugs through inducing autophagy in vivo.
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 10 of 12
Fig. 5 Compound 5e inhibited the growth of tumor in vivo. a Biomicroscopy imaging of control and treated tumors. Bar = 1 mm. b
Biomicroscopy and quantification of capillary formation with and without compound 5e and 5-Fu treatment on gelatin sponge. Bar = 1 mm. c
Immunofluorescence of LC3B-II in the frozen sections of day 6 experimental tumors. Bar = 20 μm. Results were presented as mean SE; n = 3; *p <
0.05, **p < 0.01.
Discussion
that they all showed excellent ability compared with 5-FU.
Due to the painful side effects of chemotherapeutic Further SAR analysis showed that the antiproliferative
drugs, it is necessary for researchers to improve the activity of these compounds were mainly affected by aryl
selectivity of drugs. Understanding the distribution and group in 5 position of pyrazoline moiety. Further
target of drugs could be very helpful in improving selec- mechanism studied showed that these compounds could
tivity. In this study, a series of novel fluorescent com- arrest cell cycle at the G1 phase and had no influence on
pounds were synthesized though sample synthesis steps cell necrosis.
with high yield. The antiproliferative activity of these
Using its own fluorescence characteristics, our study
compounds against was examined. The results showed found that compound 5e could selectively accumulate in
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 11 of 12
Immunofluorescence experiment demonstrated that 5e
inhibited lung cancer growth through inducing autophagy
in vivo.
In conclusion, we found
a series of fluorescent
thiazole–pyrazoline derivatives, which could inhibit the
growth of A549 cells in vitro and inhibit tumor growth
in vivo. Compound 5e induced autophagy though inhibiting
the activity of mTOR via FKBP12, which could be reversed
by 3BDO, an mTOR activator and autophagy inhibitor.
Moreover, compound 5e showed no adverse influence of
normal cells and angiogenesis. Therefore, our research
provides potential lead compounds for the development of
new anticancer drugs against human lung cancer.
Acknowledgements
This work was supported by the National Natural Science Foundation of China
(81502948, 31871407, 31741083, and 31870831) and Natural Science
Foundation of Shandong Province (2018GSF118201).
Author details
Fig. 6 The mechanism of compound 5e in inhibiting cancer cell
survival. In A549 cells, compound 5e can inhibit the activity of mTOR
via FKBP12, which could be reversed by 3BDO. Then autophagy was
induced and cellsurvival was inhibited.
1Institute of Medical Science, The Second Hospital of Shandong University,
Jinan 250033, PR China. 2Shandong Provincial Key Laboratory of Animal Cells
and Developmental Biology, School of Life Science, Shandong University, Jinan
250100, PR China. 3Institute of Organic Chemistry, School of Chemistry and
Chemical Engineering, Shandong University, Jinan 250100, PR China.
4Department of Central Laboratory, Shandong Provincial Hospital affiliated to
Shandong University, Jinan 250021, PR China
lysosome. Lysosome decomposition is a very important
step of autophagy48. Autophagy is an indispensable cel-
lular process for protein and organelle quality control.
Autophagy is a double-edged sword. Moderate autophagy
could promote cell survival, while excessive autophagy
could induce cell death28,49. Previous studies have indi-
cated that autophagy suppression may be a therapeutic
strategy for cancer treatment29,50,51. We found that these
compounds could induce autophagy and complete
autophagy flux.
Conflict of interest
The authors declare that they have no conflict of interest.
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at (https://doi.org/
10.1038/s41419-020-02746-w).
mTOR has an essential role in different tissues and cells.
As mTOR-mediated signaling associated with many dis-
eases, pharmacological agents that modulate mTOR sig-
naling could be helpful in improving many diseases52.
mTOR also plays an important role in autophagy53. In this
study, phosphorylation of RPS6KB1 and EIF4EBP1 was
significantly decreased by 5e, which could be reversed by
3BDO, an mTOR activator, and autophagy inhibitor. 5e
failed to decrease the phosphorylation of RPS6KB1 and
EIF4EBP1 when FKBP12 was knockdown. Accordingly, we
demonstrated that 5e inhibited mTOR though targeting
FKBP12 (Fig. 6). FKBP12 targets mTOR in complex with
rapamycin54. 3BDO could dock on FKBP12 in the same
sites (TYR82A and ILE56A) as rapamycin46. The structure
of 5e is different with 3BDO, so we deduced that 5e might
occupy rapamycin binding sites more easily than 3BDO.
CAM model was chosen for further research to evaluate
the antitumor effect of 5e in vivo. Our results showed 5e
significantly inhibited tumor growth compared with 5-FU
and showed no adverse influence of angiogenesis.
Received: 2 December 2019 Revised: 28 June 2020 Accepted: 30 June 2020
References
1. Torre L. A. et al. Global cancer statistics, 2012. CA- Cancer J. Clin. 65, 87–108
(2015).
2. Hanahan, D. & Weinberg, R. A. Hallmarks of cancer: the next generation. Cell
144, 646–674 (2011).
3. Keefe, D. M. K. & Bateman, E. H. Tumor control versus adverse events with
targeted anticancer therapies. Nat. Rev. Clin. Oncol. 9, 98–109 (2012).
4. Eisinger, J., Boens, N. & Flores, J. Fluorescence polarization study of human
erythrocyte membranes with 1-phenyl-3-(2-naphthyl)-2-pyrazoline as orien-
tational probe. Biochim. Biophys. Acta 646, 334–343 (1981).
5. Li, M. M., Zhao, W. B. & Zhang, T. T. A new thiophenyl pyrazoline fluorescent
probe for Cu2+ in aqueous solution and imaging in live cell. J. Fluoresc. 23,
1263–1269 (2013).
6. Ahmed, M., Hameed, S., Ihsan, A. & Naseer, M. M. Fluorescent thiazol-
substituted pyrazoline nanoparticles for sensitive and highly selective sensing
of explosive 2,4,6-trinitrophenol in aqueous medium. Sens. Actuat. B-Chem.
248, 57–62 (2017).
7. Bozkurt, E. & Gul, H. I. A novel pyrazoline-based fluorometric “turn-off” sensing
for Hg2+. Sens. Actuat. B-Chem. 255, 814–825 (2018).
8. Xia S. et al. A novel sensitive fluorescent turn-on probe for rapid detection of
Al3+ and bioimaging. RSC Adv. 5, 5244–5249 (2015).
Official journal of the Cell Death Differentiation Association

Lin et al. Cell Death and Disease
(
2
0
2
0
)
1
1
:
5
5
1
Page 12 of 12
9. Wang L. et al. A novel pyrazoline-based fluorescent probe for detection of
hydrazine in aqueous solution and gas state and its imaging in living cells.
Sens. Actuat. B-Chem. 229, 441–452 (2016).
10. Zheng X. X. et al. Novel pyrazoline-based selective fluorescent probe for the
detection of hydrazine. Spectrochim. Acta A Mol. Biomol. Spectrosc. 138,
247–251 (2015).
11. Zhang R. R. et al. Novel pyrazoline-based fluorescent probe for detecting
thiols and its application in cells. Spectrochim. Acta A Mol. Biomol. Spectrosc.
137, 450–455 (2015).
30. Mulcahy Levy, J. M., Towers, C. G. & Thorburn, A. Targeting autophagy in
cancer. Nat. Rev. Cancer 17, 528–542 (2017).
31. Galluzzi, L., Bravo-San Pedro, J. M., Levine, B., Green, D. R. & Kroemer, G.
Pharmacological modulation of autophagy: therapeutic potential and per-
sisting obstacles. Nat. Rev. Drug Discov. 16, 487–511 (2017).
32. Amaravadi R. K. et al. Principles and current strategies for targeting autophagy
for cancer treatment. Clin. Cancer Res. 17, 654–666 (2011).
33. Kondo, Y. & Kondo, S. Autophagy and cancer therapy. Autophagy 2, 85–90
(2006).
12. Wang S. Q. et al. Novel pyrazoline-based fluorescent probe for detecting
glutathione and its application in cells. Biosens. Bioelectron. 55, 386–390
(2014).
13. Tan J. L. et al. A novel “off–on” colorimetric and fluorescent rhodamine-based
pH chemosensor for extreme acidity. Spectrochim. Acta A Mol. Biomol. Spec-
trosc. 140, 489–494 (2015).
34. Laplante, M. & Sabatini, D. M. mTOR signaling in growth control and disease.
Cell 149, 274–293 (2012).
35. Weichhart, T. Mammalian target of rapamycin: a signaling kinase for every
aspect of cellular life. Methods Mol. Biol. 821, 1–14 (2012).
36. Mamane Y. et al. mTOR, translation initiation and cancer. Oncogene 25,
6416–6422 (2006).
14. Li, J., Li, D., Han, Y., Shuang, S. & Dong, C. Synthesis of 1-phenyl-3-biphenyl-5-
(N-ethylcarbazole-3-yl)-2-pyrazoline and its use as DNA probe. Spectrochim.
Acta A Mol. Biomol. Spectrosc. 73, 221–225 (2009).
15. Silver, K. S. & Soderlund, D. M. Differential sensitivity of rat voltage-sensitive
sodium channel isoforms to pyrazoline-type insecticides. Toxicol. Appl. Phar-
macol. 214, 209–217 (2006).
16. Zhao P. L. et al. Synthesis, fungicidal, and insecticidal activities of beta-
methoxyacrylate-containing N-acetyl pyrazoline derivatives. J. Agric. Food
Chem. 56, 10767–10773 (2008).
17. Silver, K. & Soderlund, D. M. State-dependent block of rat Nav1.4 sodium
channels expressed in xenopus oocytes by pyrazoline-type insecticides. Neu-
rotoxicology 26, 397–406 (2005).
37. Zaytseva, Y. Y., Valentino, J. D., Gulhati, P. & Evers, B. M. mTOR inhibitors in
cancer therapy. Cancer Lett. 319, 1–7 (2012).
38. Alayev, A. & Holz, M. K. mTOR signaling for biological control and cancer. J. Cell
Physiol. 228, 1658–1664 (2013).
39. Zhang, J. F., Li, M., Miao, J. Y. & Zhao, B. X. Biological activities of novel pyrazolyl
hydroxamic acid derivatives against human lung cancer cell line A549. Eur. J.
Med. Chem. 83, 516–525 (2014).
40. Wei Q. et al. Discovery of novel HSP90 inhibitors that induced apoptosis and
impaired autophagic flux in A549 lung cancer cells. Eur. J. Med. Chem. 145,
551–558 (2018).
41. Li N. et al. Discovery of a new autophagy inducer for A549 lung cancer cells.
Bioorg. Med. Chem. 27, 2845–2856 (2019).
18. Mishra, N. & Sasmal, D. Development of selective and reversible pyrazoline
based MAO-B inhibitors: virtual screening, synthesis and biological evaluation.
Bioorg. Med. Chem. Lett. 21, 1969–1973 (2011).
42. Papazisis, K. T., Geromichalos, G. D., Dimitriadis, K. A. & Kortsaris, A. H. Opti-
mization of the sulforhodamine B colorimetric assay. J. Immunol. Methods 208,
151–158 (1997).
19. Chimenti F. et al. Synthesis and inhibitory activity against human monoamine
oxidase of N1-thiocarbamoyl-3,5-di(hetero)aryl-4,5-dihydro-(1H)-pyrazole deri-
vatives. Eur. J. Med. Chem. 45, 800–804 (2010).
20. Rathish I. G. et al. Synthesis and antiinflammatory activity of some new 1,3,5-
trisubstituted pyrazolines bearing benzene sulfonamide. Bioorg. Med. Chem.
Lett. 19, 255–258 (2009).
21. Chirumarry S. et al. Design, synthesis and surfactant properties of
perfluorobutyl-based fluorinated sodium alkanesulfonates. J. Fluor. Chem. 197,
111–117 (2017).
22. Sharma P. K. et al. Synthesis and biological evaluation of some pyrazolylpyr-
azolines as anti-inflammatory-antimicrobial agents. Eur. J. Med. Chem. 45,
2650–2655 (2010).
43. Zhang X. et al. New fluorescent pH probes for acid conditions. Sens. Actuat. B-
Chem. 206, 663–670 (2015).
44. Jaishy, B. & Abel, E. D. Lipids, lysosomes, and autophagy. J. Lipid Res. 57,
1619–1635 (2016).
45. Settembre C. et al. A lysosome-to-nucleus signalling mechanism
senses and regulates the lysosome via mTOR and TFEB. EMBO J. 31,
1095–1108 (2012).
46. Ge D. et al. Identification of a novel MTOR activator and discovery of a
competing endogenous RNA regulating autophagy in vascular endothelial
cells. Autophagy 10, 957–971 (2006).
47. Lokman, N. A., Elder, A. S. F., Ricciardelli, C. & Oehler, M. K. Chick chorioallantoic
membrane (CAM) assay as an in vivo model to study the effect of newly
identified molecules on ovarian cancer invasion and metastasis. Int. J. Mol. Sci.
13, 9959–9970 (2012).
23. Hu L. et al. Synthesis and antibacterial activity of C-12 pyrazolinyl spiro keto-
lides. Eur. J. Med. Chem. 45, 5943–5949 (2010).
24. Siddiqui, Z. N., Musthafa, T. N., Ahmad, A. & Khan, A. U. Thermal solvent-free
synthesis of novel pyrazolyl chalcones and pyrazolines as potential anti-
microbial agents. Bioorg. Med. Chem. Lett. 21, 2860–2865 (2011).
25. Marella A. et al. Pyrazolines: a biological review. Mini-Rev. Med. Chem. 13,
921–931 (2013).
48. Settembre, C., Fraldi, A., Medina, D. L. & Ballabio, A. Signals from the lysosome:
a control centre for cellular clearance and energy metabolism. Nat. Rev. Mol.
Cell Biol. 14, 283–296 (2013).
49. Rabinowitz, J. D. & White, E. Autophagy and metabolism. Science 330,
1344–1348 (2010).
26. Altintop M. D. et al. A novel series of thiazolyl-pyrazoline derivatives: synthesis
and evaluation of antifungal activity, cytotoxicity and genotoxicity. Eur. J. Med.
Chem. 92, 342–352 (2015).
50. Liu R. et al. Itraconazole suppresses the growth of glioblastoma through
induction of autophagy: 8 involvement of abnormal cholesterol trafficking.
Autophagy 10, 1241–1255 (2014).
27. Yang W. et al. Design, modification and 3D QSAR studies of novel naphthalin-
containing pyrazoline derivatives with/without thiourea skeleton as anticancer
agents. Bioorg. Med. Chem. 21, 1050–1063 (2013).
28. Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues. Cell
147, 728–741 (2011).
29. Fulda, S. & Kogel, D. Cell death by autophagy: emerging molecular
mechanisms and implications for cancer therapy. Oncogene 34, 5105–5113
(2015).
51. Nassour J. et al. Autophagic cell death restricts chromosomal instability during
replicative crisis. Nature 565, 659–663 (2019).
52. Zoncu, R., Efeyan, A. & Sabatini, D. M. mTOR: from growth signal integration to
cancer, diabetes and ageing. Nat. Rev. Mol. Cell Biol. 12, 21–35 (2011).
53. Yu L. et al. Termination of autophagy and reformation of lysosomes regulated
by mTOR. Nature 465, 942–946 (2010).
54. Huang, S., Bjornsti, M. A. & Houghton, P. J. Rapamycins: mechanism of action
and cellular resistance. Cancer Biol. Ther. 2, 222–232 (2003).
Official journal of the Cell Death Differentiation Association