XHL11, a novel selective EGFR inhibitor, overcomes EGFRT790M-mediated resistance in non-small cell lung cancer

Article abstract of DOI:10.1016/j.ejphar.2021.174297

The first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib significantly improved the therapeutic effect in non–small cell lung cancer (NSCLC) patients with EGFR mutation. However, the EGFR^T790M mutation occurs and results in acquired resistance. Consequently, mutant selective third-generation EGFR TKIs represented by AZD9291 (Osimertinib) have been developed to offer more effective therapeutic treatment, but the clinical application is limited by the acquired resistance and the high costs. A series of 5-chloropyrimidine-2,4-diamine derivatives were synthesized and screened for in vitro antitumor activity on H1975 and A431 cells. XHL11 showed the strongest antineoplastic activity. Compared to AZD9291, XHL11 suppressed cellular proliferation and colony formation and induced apoptosis in H1975 cells with EGFR^L858R/T790M mutation. In addition, XHL11 caused expression changes in EGFR and apoptosis-related pathways. Moreover, oral administration of XHL11 suppressed tumor progression in vivo in a H1975 subcutaneous xenograft model. These data demonstrated that XHL11 might be developed as a promising EGFR TKI for the therapeutic use of NSCLC patients.

Full text of DOI:10.1016/j.ejphar.2021.174297

European Journal of Pharmacology 907 (2021) 174297
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
XHL11, a novel selective EGFR inhibitor, overcomes EGFRT790M-mediated
resistance in non-small cell lung cancer
Yi Li1, Qing-Long Yu1, Tong-Fang Li, Ya-Ni Xiao, Li Zhang, Qiu-Yan Zhang**,
Chun-Guang Ren***, Hong-Lei Xie*
Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Institute of Materia Medica, Shandong, 264000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The first-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and
erlotinib significantly improved the therapeutic effect in non–small cell lung cancer (NSCLC) patients with EGFR
mutation. However, the EGFRT790M mutation occurs and results in acquired resistance. Consequently, mutant
selective third-generation EGFR TKIs represented by AZD9291 (Osimertinib) have been developed to offer more
effective therapeutic treatment, but the clinical application is limited by the acquired resistance and the high
costs. A series of 5-chloropyrimidine-2,4-diamine derivatives were synthesized and screened for in vitro anti-
tumor activity on H1975 and A431 cells. XHL11 showed the strongest antineoplastic activity. Compared to
AZD9291, XHL11 suppressed cellular proliferation and colony formation and induced apoptosis in H1975 cells
with EGFRL858R/T790M mutation. In addition, XHL11 caused expression changes in EGFR and apoptosis-related
pathways. Moreover, oral administration of XHL11 suppressed tumor progression in vivo in a H1975 subcu-
taneous xenograft model. These data demonstrated that XHL11 might be developed as a promising EGFR TKI for
the therapeutic use of NSCLC patients.
Non-small cell lung cancer
EGFRT790M
EGFR TKI
Apoptosis
1. Introduction
therapy. Unfortunately, the majority of patients eventually develop ac-
quired resistance after about 1 year of treatment (Kobayashi et al.,
Lung cancer is the most commonly diagnosed cancer (11.6% of the
total cases) and also the most frequent cause of cancerous mortality
worldwide. It accounts for 18.4% of all the cancer-related deaths (Bray
et al., 2018). EGFR mutations have been detected in the majority of
NSCLC patients and accelerate the progression of the disease (Engelman
and Janne, 2008; Heigener and Reck, 2011). Abnormal activation of
EGFR resulted in dysregulation of the downstream pathways, such as
MAPK and JNK pathways (Engelman and Janne, 2008; Tan et al., 2016),
and contributed to the fatality rate.
2005). A secondary mutation in exon 20 (T790M) improved the
ATP-binding capacity of the mutated protein and induced the resistance
in approximately 60% of these resistant cases (Pao et al., 2005).
Therefore, there remains an urgent requirement for an EGFR TKI which
could overcome T790M-mediated resistance.
The third generation EGFR TKIs, such as WZ4002 (Zhou et al., 2009),
CO-1686 (Walter et al., 2013) and AZD9291 (Janne et al., 2015) were
developed to overcome the EGFRT790M-mediated resistance and reduce
the unwanted side effects. AZD9291 which was approved by the FDA
performed the best, and improved the progression-free survival of
NSCLC patients with T790M mutation (Goss et al., 2016; Yu et al.,
2020). AZD9291 was reported to inhibit the phosphorylation of EGFR in
vitro, resulting in the suppression of EGFR downstream signal pathways
including Akt and ERK (Ku et al., 2016). However, the third-generation
EGFR TKI also developed new acquired resistance after an average of 9.6
EGFR has been reported to be one of the most successful targets for
NSCLC (Rosell et al., 2009). Patients with EGFR gene mutations, such as
the exon 19 deletion and L858R mutation, are sensitive to EGFR TKIs
(Camidge et al., 2014; Park et al., 2016). The first and the second gen-
erations EGFR TKIs, such as gefitinib, erlotinib and afatinib, regulated
EGFR pathways (Sequist et al., 2013) and participated in combination
* Corresponding author
** Corresponding author
*** Corresponding author
E-mail addresses: qyzhang@yimm.ac.cn (Q.-Y. Zhang), cgren@yimm.ac.cn (C.-G. Ren), qingteng51@163.com (H.-L. Xie).
1
These authors contribute equally.
https://doi.org/10.1016/j.ejphar.2021.174297
Received 1 February 2021; Received in revised form 23 June 2021; Accepted 28 June 2021
Available online 2 July 2021
0014-2999/© 2021 Elsevier B.V. All rights reserved.

Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
months of treatment (Murtuza et al., 2019). The primary cause of ac-
quired resistance to third-generation EGFR TKIs was EGFRC797S muta-
tion (Niederst et al., 2015), along with ERBB2 and MET amplifications
KRAS mutations (Ortiz-Cuaran et al., 2016) as possible mechanisms. The
C797S mutation influences the covalent binding of AZD9291 and EGFR
by changing the cysteine sidechain. The acquired resistance together
with the high price and several adverse reactions limited the prescrip-
tion of AZD9291. Therefore, further exploration and research are
needed to find more safe, effective and economical agents to overcome
therapeutic resistances caused by EGFRC797S mutation in lung cancer.
In this study, a series of 5-chloropyrimidine-2,4-diamine derivatives
were synthesized and identified as potent EGFR inhibitors. We discov-
ered a novel selective EGFR TKI, XHL11, which effectively inhibited the
EGFRL858R/T790M and EGFRL858R/T790M/C797S kinases activity. Molecular
docking of the new compounds into the EGFR was also performed. The
down-regulation of EGFR and the down-stream pathways by XHL11
contributed to the apoptosis and the regulation of cellular proliferation
in H1975 cells with EGFRL858R/T790M mutation. These results suggested
that XHL11 might be developed as a promising EGFR TKI for the
treatment of NSCLC patients.
phenyl)amino)pyrimidin-2-yl)amino)phenyl)acrylamide was obtained
as a light white solid. Yield: 77%. Purity 99.53% (Fig. S1B), ESI-MS: m/z
= 471.11 [M + H]. 1H NMR (400 MHz, DMSO‑d6): δ (ppm): 1.16 (s, 3H),
1.17 (s, 3H), 3.48–3.45 (m, J = 2 Hz, 1H), 5.72–5.71 (d, J = 2 Hz, 1H),
5.74–5.73 (d, J = 2 Hz, 1H), 6.26–6.25 (t, J = 1.6 Hz, 1H), 7.38 (d, J =
0.2 Hz, 1H), 7.42–7.40 (m, J = 1 Hz, 4H), 7.74 (m, J = 0.2 Hz, 1H),
7.85–7.84 (d, J = 2 Hz, 1H), 7.87–7.86 (m, J = 2 Hz,1H), 8.18 (s, 1H),
8.28 (s, 1H), 9.46 (s, 1H), 9.52 (s, 1H), 10.05 (s, 1H). 13C NMR (101
MHz, DMSO‑d6): δ (ppm): 15.39, 55.38, 100.00, 105.02, 114.34,
120.15, 120.54, 124.23, 126.91, 131.47, 132.52, 133.95, 135.31,
136.26, 138.56, 155.43, 155.80, 158.18, 163.28.
Compound XHL 11 b: N-(3-((5-chloro-4-((2-(isopropylsulfonyl)
phenyl)amino)pyrimidin-2-yl)amino)phenyl)-2-cyanoacetamide
was
obtained as a light white solid. Yield: 82%. Purity 99.19% (Fig. S1C),
ESI-MS: m/z = 484.11 [M + H]. 1H NMR (400 MHz, DMSO‑d6): δ (ppm):
1.17 (s, 3H), 1.19 (s, 3H), 3.47–3.44 (t, J = 4 Hz, 1H), 3.87 (s, 2H),
7.17–7.16 (d, J = 1.3 Hz, 1H), 7.20–7.18 (d, J = 4 Hz, 1H), 7.38–7.37
(m, J = 2 Hz, 2H), 7.73–7.71 (d, J = 4 Hz, 1H), 7.85–7.83 (d, J = 4 Hz,
2H), 8.30 (s, 1H), 8.67–8.64 (d, J = 6 Hz, 1H), 9.54 (s, 1H), 9.62 (s, 1H),
10.23 (s, 1H). 13C NMR (101 MHz, DMSO‑d6): δ (ppm): 15.40, 27.23,
55.47, 105.61, 111.50, 113.78, 116.11, 116.48, 124.06, 124.24, 129.23,
131.48, 135.40, 138.50, 138.98, 140.99, 144.36, 155.31, 155.63,
158.06, 161.32.
2. Materials and methods
2.1. Chemistry
5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(3-nitrophenyl)py-
2.2. Biology
rimidine-2,4-diamine:
A
mixture
of
2,5-dichloro-N-(2-(iso-
propylsulfonyl)phenyl)pyrimidin-4-amine (3.5 g, 10 mmol), 3-
nitroaniline (1.6 g, 12 mmol) and isopropyl alcohol 40 ml were added
to a 250 m three-necked round bottom flask. The solution was heated to
85 ◦C for 8 h and then cooled to room temperature. The precipitate was
filtered, washed with water, and then purified by column chromatog-
raphy (silica gel) to give target product (3.5 g, 79.5% yield) as a yellow
solid. ESI-MS: m/z = 447.89 [M + H].
2.2.1. Reagents
AZD9291 was purchased from MedChemExpress (MCE, NJ, USA),
Gefitinib was purchased from J&K Scientific Ltd (Beijing, China), MTT
was obtained from Sigma-Aldrich (St. Louis, MO, USA). The primary
antibodies against EGFR, phosphor-EGFR, ERK, phosphor-ERK, PARP
were purchased from Cell Signaling Technology (Danvers, MA, USA);
antibodies against Ki67 were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). The Annexin V-FITC Assay kit, Reactive Oxygen
Assay Kit, Crystal Violet Staining Solution and N-Acetylcysteine (NAC)
were purchased from Beyotime Institute of Biotechnology (Shanghai,
China).
N2-(3-aminophenyl)-5-chloro-N4-(2-(isopropylsulfonyl)phenyl)py-
rimidine-2,4-diamine:5-chloro-N4-(2-(isopropylsulfonyl)phenyl)-N2-(3-
nitrophenyl)pyrimidine-2,4-diamine (3.0 g, 6.7 mmol) was dissolved in
methanol 60 ml, Pd/C 0.3 g was added, and then reaction with H2. The
reaction mixture was stirred at room temperature for 6 h. The Pd/C was
filtered, washed with methanol, the filtrate was dried and obtained
product (2.6 g, 92.8% yield) as a yellow solid. ESI-MS: m/z = 417.91 [M
+ H].
2.2.2. Molecular docking
Crystal structure of EGFR (PDB ID: 6LUB) and inhibitors were both
pretreated by Discovery Studio 3.5. All docking runs were utilizing
LigandFit Dock protocol of Discovery Studio 3.5. The image files are
generated by the Accelrys DS visualizer 4.0 systems.
Compound XHL 11:
A mixture of N2-(3-aminophenyl)-5-chloro-N4-(2-(isopropylsulfonyl)
phenyl)pyrimidi-ne-2,4-diamine (2.4 g, 5.7 mmol), acryloyl chloride
(0.5 g, 6.2 mmol) and dichloromethane 20 ml were added to a 100 ml
three-necked round bottom flask. The reaction mixture was stirred at
room temperature for 2 h, and then dried and purified by column
chromatography (silica gel) to give target product (2.0 g, 75% yield) as a
light white solid. ESI-MS: m/z = 471.11 [M + H]. 1H NMR (400 MHz,
DMSO‑d6): δ (ppm):
2.2.3. Cell culture
Human NSCLC cell lines H1975 and A431 were obtained from
American Type Culture Collection (Manassas, VA), which were cultured
in RPMI1640 medium (Gibco, Grand Island, NY, USA). PC9 cell line
were purchased from Jiangsu Meimian industrial Co., Ltd (Jiangsu,
China), which were cultured in DMEM medium (Gibco, Grand Island,
NY, USA). All cultured mediums were supplemented with 10% fetal
bovine serum (FBS, Gibco) and 1% solution of antibiotics (Hyclone,
Logan, UT, USA) at 37 ◦C in an incubator with 5% CO2.
1.17 (s, 3H), 1.18 (s, 3H), 3.43–3.47 (m, J = 2 Hz, 1H), 5.73–5.72 (d,
J = 2 Hz, 1H), 5.76–5.75 (d, J = 2 Hz, 1H), 6.46–6.25 (t, J = 3 Hz, 1H),
7.22–7.20 (d, J = 2 Hz, 1H), 7.35–7.33 (m, J = 0.5 Hz, 3H), 7.81 (d, J =
0.2 Hz, 1H), 7.82 (d, J = 0.2 Hz, 1H), 7.94 (s, 1H), 8.30 (s, 1H),
8.70–8.68 (d, J = 2 Hz, 1H), 9.55 (s, 1H), 9.59 (s, 1H), 10.07 (s, 1H). 13
C
2.2.4. Kinase enzymatic activity assay
NMR (101 MHz, DMSO‑d6): δ (ppm): 15.34, 55.46, 105.51, 111.94,
114.17, 116.01, 123.98, 124.16, 124.55, 127.23, 129.10, 131.43,
132.50, 135.37, 138.53, 139.62, 140.82, 155.30, 155.69, 158.22,
163.56.
The Z′-Lyte Kinase Assay Kit, WT and mutated EGFR kinase enzyme
were purchased from Invitrogen. Kinase enzymatic activity assay was
performed in accordance with the instructions of the manufacturer. In
brief, prepare test compounds at four times the expected concentrations
HPLC purity 98.67% (0–3 min, A: 30% methanol (0.1% trifluoro-
acetic acid), B:70% water (0.1% trifluoroacetic acid); 4–15 min, A: 95%
methanol (0.1% trifluoroacetic acid), B:5% water (0.1% trifluoroacetic
acid); 16–18 min, A: 30% methanol (0.1% trifluoroacetic acid), B:70%
water (0.1% trifluoroacetic acid) (Fig. S1A).
in the 10 μl Kinase Reactions. The compound, ATP solution and kinase/
peptide mixture were mixed and incubated at the room temperature for
1 h. After the mixture of the development solution, the assay plate was
incubated at room temperature for 1 h. At last, stop reagent was added to
the plate. The activity was measured with a microplate reader (Molec-
ular Devices).
Compound XHL 11a: N-(4-((5-chloro-4-((2-(isopropylsulfonyl)
2

Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
2.2.5. Cell survival assay
intragastric administrated with 5% carboxymethyl cellulose sodium
(CMC-Na) twice a week. Tumor volume was measured three times a
week with a caliper (calculated volume = shortest diameter2 × longest
diameter/2). After 17 days treatment, tumor-bearing mice were eutha-
nized and tumor tissues were removed for immunohistochemistry
staining.
H1975 and A431 cells were incubated with various concentrations of
EGFR TKIs for 24, 48, 72 h. Tetrazolium dye (MTT) solution (5 mg/ml)
was added to each well. After at last 4 h incubation, 100 μl dimethyl
sulfoxide (DMSO) was added to each well to dissolve the formazan
crystals. The absorbance was read at a test wavelength of 570 nm with a
microplate reader (Molecular Devices). The IC50 values were calculated
using the SPSS software (SPSS Inc., Chicago, IL, USA).
2.2.11. Immunohistochemistry staining
Tumor samples were fixed in 10% formalin at room temperature and
paraffin embedded. Tumor sections were then deparaffinized and anti-
gen retrieval. After blocking for half an hour at room temperature, the
sections were incubated with the primary antibody overnight at 4◦C.
After washing three times with PBS, sections were then treated with
anti-rabbit secondary antibody for 1 h at room temperature, and then
developed by DAB reagent for 1 min. Some of the tumor sections were
subjected to TUNEL assay according to the manufacturer’s protocol
(Beyotime Institute of Biotechnology). Positive nuclei were stained with
brown colour. Images were taken by a Nikon Ti–U fluorescent micro-
scope with a CCD digital camera (magnification, × 200). Staining in-
tensity was scored as 0 (negative), 1 (weak), 2 (medium) and 3 (strong).
Extent of staining was scored as 0 (0%), 1 (1–25%), 2 (26–50%), 3
(51–75%) and 4 (76–100%), according to the percentage of the whole
carcinoma area which was positively stained. The product of the in-
tensity score and the extent score was expressed as the staining score.
2.2.6. Colony formation assay
H1975 and A431 cells were seeded into 6-well plates at a density of
200–300 cells per well. The medium was replaced by fresh media con-
taining different concentrations of XHL11. After incubated with series
concentration of XHL11 for 48 h, the cells were cultured with fresh
media for 10 d, and fixed by 4% paraformaldehyde. After being washed
by PBS, the cells were stained with Crystal Violet Staining Solution. The
colonies (≥50 cells) were counted and the typical images were
photographed.
2.2.7. Flow cytometry analysis
Annexin V-FITC apoptosis analysis was performed using the Annexin
V-FITC apoptosis detection kit. Briefly, cells were seeded into the 6-well
plate overnight, and treated with Gefitinib, AZD9291 and XHL11 for 24
h. Cells were harvested using trypsinization, washed by PBS, and re-
suspended in 500 μl buffer together with 5 μl of Annexin V-FITC and
5
μ
l of PI, and incubated for 30 min at room temperature, and analyzed
2.2.12. Statistical analysis
by a flow cytometer (BD Accuri C6, Franklin Lakes, NJ).
All the data are expressed as mean values ± standard deviation (S.
D.). Comparisons among multiple groups were made with a one-way
analysis of variance (ANOVA) followed by Dunnett’s test using SPSS
13.0 software and GraphPad Prism 6. P < 0.05 was used for statistical
significance.
2.2.8. Western blot analysis
About 5 × 106 H1975 cells were gathered after incubated with
XHL11 for 24 h and lysed with RIPA buffer together with protease in-
hibitor (PMSF) and phosphatase inhibitor (NaF). Equal amounts of
protein extracted from H1975 cells were electrophoresed by 8–10%
SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF)
membranes. After blocked with milk for 1 h at room temperature, the
membranes were incubated with primary antibodies and horseradish
peroxidase (HRP)-conjugated secondary antibodies, and the reacted
with ECL detection reagents (Thermo Scientific). The immune reactive
bands were visualized in Amersham Imager 680 (GE Healthcare, Buck-
inghamshire, UK).
3. Results
3.1. XHL11 was screened out as a novel mutant selective EGFR TKI
On the basis of AZD9291 functional group, we retained the 2,4,5-
substituted pyrimidine core, and chose preponderant EGFR TKI inhibi-
tion drug fragments reported in literature as substituents for 4-
substituted pyrimidine. The target EGFR TKIs were synthesized as
shown in Scheme 1 and evaluated for their antiproliferative activity in
H1975 cells with EGFRL858R/T790M mutation and in A431 cells with
EGFR high expression. As shown in Table 1, XHL11 showed the highest
2.2.9. Detection of intracellular reactive oxidative species production
H1975 and A431 cells were seeded into the 6-well plate overnight,
and treated with indicated compounds for 24 h, and Rosup compound
mixture for 30 min. The Rosup group was incubated with Rosup com-
pound mixture, which could activate the reactive oxidative species
generation within 30 min. After incubation for 30 min with diluted
antineoplastic activity with the IC50 of 0.055 ± 0.030 μM in H1975 cells,
and 0.034 ± 0.04 μM in A431 cells after 48 h treatment. Therefore, we
choose XHL11 for further research.
To further elucidate the binding pattern observed, molecular docking
of the most potent inhibitor XHL11 were performed in the binding site
cavity of EGFR. The binding modes of compound XHL11 and EGFR were
depicted in Fig. 1B, which revealed that the inhibitor is well occupied in
the binding pocket. As shown in Fig.1B, N-1 and 2-amino on the py-
rimidine and Met-393 contribute to the hydrogen bonding interaction
together, being a probable explanation for its activity. One hydrophobic
interaction is formed between the 3-aniline and Pro 794. The 5-chloride
on the pyrimidine and Thr864 contribute to the covalent bonding
interaction.
DCFH-DA (10 μM), the cells were washed with PBS and photographed by
microscopy (Ti–U, Nikon). Data were expressed as % of control.
2.2.10. Subcutaneous xenografts studies
Male nude mice were obtained from Gem Pharmatech Co., Ltd and
raised on a 12-h light-dark cycle. The animal experiment were approved
by the Committee on the Ethics of Animal Experiments of the Yantai
Institute of Materia Medica, and were performed in strict accordance
with the relevant institutional and national guidelines and regulations.
The ethical approval number is 20191011-1. Briefly, H1975 cells (2 ×
106 cells/200
μl) were subcutaneously injected to the left flank of mice.
3.2. XHL11 inhibited the EGFR kinase activity and the activation of EGFR
Mice were randomized into two groups (n = 5), when the average tumor
volume reached 100 mm3. It was reported that the antitumor efficacy in
xenograft mouse models of H1975 for the novel selective EGFR in-
hibitors against EGFRL858R/T790M resistance mutation was evaluated at
100 mg/kg once daily or 50 mg/kg twice daily orally (Hao et al., 2016).
Considering the oral absorption rate and first pass effect, the mice in the
treatment group were treated with XHL11 (60 mg/kg) twice a week by
oral gavage for 17 days. The mice in vehicle control group were
EGFRC797S mutation has been recently reported to contribute the
most to the AZD9291 acquired resistance in patients with relapse
(Thress et al., 2015). At the same time, the EGFRC797S mutation in a
majority of AZD9291-resistant patients shed light on the development of
novel EGFR inhibitors. The research and development of novel mole-
cules and strategies to overcome EGFRC797S mutation are urgently
warranted. As shown in Table 2, XHL11 was demonstrated to inhibit the
3

Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
Scheme 1. Synthetic routes. Reagents and conditions: a) isopropyl alcohol, 85◦C; b) Pd/C, MeOH, RT; c) DCM, RT.
MTT assay. It turned out that XHL11 inhibited the proliferation of A431
cells more potently than AZD9291 and Gefitinib. In addition, XHL11
exhibited relatively low inhibitory activity on A431 cells than that on
H1975 cells following a 72 h drug exposure.
Table 1
Antiproliferative activity of target compounds.
Compound
IC50(μM)
Colony formation assay was performed to further demonstrate the
anticancer activity of XHL11. In accordance with the MTT assay, XHL11
inhibited the density and size of the colony formed by H1975 and A431
cells as shown in Fig. 3B. These results revealed that XHL11 inhibited
proliferation of EGFR-mutant cell lines. Moreover, XHL11 could also
inhibit the proliferation of cell line having EGFR-sensitive mutation. As
shown in supplementary Fig. 2, XHL11 inhibited the proliferation of PC9
in a dose and time dependent manner, and inhibited the density and size
of the colony formed by PC9 cells.
H1975
A431
XHL11
0.055 ± 0.030
0.081 ± 0.024
0.40 ± 0.18
0.034 ± 0.04
0.037 ± 0.024
0.29 ± 0.08
1.29 ± 0.32
XHL11a
XHL11b
AZD9291
0.18 ± 0.02
IC50 values = mean ± SD of three independent assays.
EGFRL858R/T790M/C797S kinases activity with an IC50 of 71.9 ± 20.9 nm,
and inhibited the EGFRL858R/T790M activity with an IC50 of 155.2 ± 46.4
nM EGFRC797S mutation was the main cause of acquired resistance to
third-generation EGFR TKIs (Chabon et al., 2016). The selectivity of
3.4. XHL11 induced apoptosis in EGFR-mutant cell lines
EGFRL858R/T790M/C797S suggested that XHL11 might be
a novel
EGFRT790M mutant inhibitor with EGFRC797S inhibitory activity, which
might overcome the acquired resistance caused by EGFRT790M mutant.
The activation of EGFR was evaluated by Western blotting analysis
using H1975 cells with EGFR T790M. As shown in Fig. 2, XHL11 inhibited
the phosphorylation of EGFR in a dose- and time-dependent manner
with no impact on the total EGFR expression.
Annexin V-FITC/propidium iodide (PI) apoptosis analysis was used
to investigate the effect of XHL11 on the apoptosis of EGFR-mutant cell
lines. As shown in Fig. 4, XHL11 was found to induce apoptosis in both
H1975 and A431 cells. The early apoptosis rate induced by XHL11 was
31.0% and 29.1% at the concentration of 0.1 μM and 1 μM in H1975
cells. For the A431 cells, the early apoptosis rate was 31.4% and 36.9%
with the similar effect in H1975 cells. While, the late apoptosis rate
induced by XHL11 in H1975 was 9.9% (0.1 μM) and 8.0% (1 μM). In
3.3. XHL11 inhibited proliferation of EGFR-mutant cell lines
contrast, the late apoptosis rate induced by XHL11 in A431 was 31.4%
(0.1 μM) and 36.9% (1 μM). These results revealed that XHL11 induced
To compare the anti-proliferative activity of XHL11 with the other
EGFR TKIs, cytotoxic effect of XHL11 on EGFR-mutant cells was deter-
mined using MTT assay. As shown in Fig. 3A, XHL11 inhibited the
proliferation of H1975 (EGFRT790M positive cells) in a dose and time
dependent manner. Moreover, XHL11 inhibited the growth of EGFR-
mutant cell lines more potently than AZD9291 and Gefitinib with an
incubation of 24 h. Moreover, the antiproliferative activity of XHL11
strengthened with the passage of time, and could dramatically inhibit
cell viability at extremely low concentration (1 nM). The anti-
proliferative activity of XHL11 on A431 cells was also detected using
apoptosis in EGFR-mutant cell lines. Gefitinib and AZD9291 exhibited
little effect on the apoptosis of H1975 and A431 cells at the concen-
tration of 1 μM.
3.5. XHL11 regulated the apoptosis and EGFR signaling
To confirm the effect of XHL11 on the induction of apoptosis in
EGFR-mutant cell lines, the cleavage of PARP and the activation of
caspase-3 were detected by Western blotting analysis. In accordance
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Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
Fig. 1. XHL11 inhibits EGFR kinase activity. (A) Structure and EGFR kinase inhibitory activity of XHL11 (B) The proposed binding mode of compound XHL11 and
EGFR. XHL11 interactions with amino acid. XHL11 with carbon atoms colored black, oxygen atoms colored red, nitrogen atoms colored blue, chloride colored green,
sulphur colored yellow. Hydrogen bond colored purple, covalent bond colored dark yellow.
downstream signaling including Bcl-xL, AKT and ERK in H1975 cells in a
Table 2
dose- and time-dependent manner.
The effect of XHL11 on the tyrosine kinases activity of wild-type and mutated
EGFR.
IC50(nM)
EGFRWT
EGFRL858R/T790M
EGFRL858R/T790M/C797S
3.6. XHL11 induced reactive oxidative species generation in EGFR-
mutant cell lines
XHL11
352.4 ± 90.1
222.1 ± 99.5
155.2 ± 46.4
3.1 ± 1.1
71.9 ± 20.9
AZD9291
nd
It was reported that AZD9291 induced apoptosis via reactive oxygen
species generation in non-small cell lung cancer cells (Tang et al.,
2017a). Therefore, the levels of reactive oxidative species in H1975 and
A431 treated with Gefitinib, AZD9291 and XHL11 was detected. As
shown in Fig. 6, reactive oxidative species was increased by XHL11 and
AZD9291 in H1975 cells, but not in A431 cells. Gefitinib barely affected
nd means no results due to no inhibition.
with the Annexin V-FITC/propidium iodide (PI) apoptosis analysis,
PARP and caspase-3 activation was up-regulated by XHL11 in H1975
cells (shown in Fig. 5). Furthermore, XHL11 also down-regulated the
Fig. 2. XHL11 inhibits the activation of EGFR. (A) The expression of P-EGFR and EGFR were assessed by western blot analyses in H1975 cell line after the in-
cubation with XHL11 (1, 10, 100, 1000 nM) for 24 h. (B) The expression of P-EGFR and EGFR were assessed by western blot in H1975 cell line after the incubation
with XHL11 (0.1 μM) for 2, 4, 6, 12, 24 h. Data were represented as mean ± S.D. of triplicate determinations. *P < 0.05, **P < 0.01, significantly different compared
with the DMSO-treated control by one-way ANOVA followed by Dunnett’s test.
5

Fig. 3. Effects of XHL11 on the proliferation of EGFR-mutant cell lines. (A) Effects of XHL11 on cell viability of EGFR-mutant cell lines. H1975 and A431 cells were incubated with increasing concentrations of
XHL11 (from 0.001 to 100 μM) for 24, 48, 72 h. Cell viability was detected by MTT assay. The percentage of cell viability was calculated by the absorption of control cells (0.1% DMSO) as 100%. (B) Effects of XHL11 on
the colony forming ability of EGFR-mutant cell lines. Cells were incubated with XHL11 (1, 3, 10 nM) for 48 h, and then cultured with fresh media for 10 d. Data were represented as mean ± S.D. of triplicate independent
studies. *P < 0.05, significantly different compared with the DMSO-treated control by one-way ANOVA followed by Dunnett’s test.

Fig. 4. XHL11 induced apoptosis in EGFR-mutant cell lines. (A) Cells were incubated with Gefitinib (1 μM), AZD9291 (1 μM), XHL11 (0.1 and 1 μM) for 24 h, and stained by Annexin V staining and propidium iodide
(PI). (B) The percentage of PI+/Annexin + cells (late apoptosis) and PI-/Annexin + cells (early apoptosis). Data were represented as mean ± S.D. of triplicate independent determinations. *P < 0.05, ***P < 0.001,
significantly different compared with the DMSO-treated control by one-way ANOVA followed by Dunnett’s test.

Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
Fig. 5. XHL11 induces apoptosis and regulates apoptosis-related protein and EGFR signaling in a dose and time dependent manner. (A) H1975 cells were
assessed by western blot analyses after the incubation with XHL11 (1, 10, 100, 1000 nM) for 24 h. (B) The cleavage of PARP, Caspase 3, and the expression of Bcl-xL,
P-AKT and P-ERK were assessed by western blot in H1975 cell line after the incubation with XHL11 (0.1 μM) for 2, 6, 12, 24 h. Data were represented as mean ± S.D.
of triplicate independent studies. *P < 0.05, significantly different compared with the DMSO-treated control by one-way ANOVA followed by Dunnett’s test.
reactive oxidative species generation in both cells. To confirm the role of
reactive oxidative species generation on the apoptosis induction of
XHL11, the H1975 cells were pretreated with NAC, which is known as an
inhibitor of reactive oxidative species, and then incubated with XHL11
for 24 h. The pretreating of NAC (5 mM) not only reduced the produc-
tion of reactive oxidative species induced by XHL11 but also counteract
the effect of XHL11 on apoptosis. Therefore, we conjectured that reac-
tive oxidative species played an important role in the anticancer effect
and apoptosis induction of XHL11.
of Ki-67 which indicated that XHL11 suppressed tumor cell prolifera-
tion. Moreover, TUNEL staining of the tumor tissue showed that XHL11
induced apoptosis in vivo.
4. Discussion
Lung cancer is the most commonly diagnosed cancer (11.6% of the
total cases) and also the leading cause of cancerous mortality throughout
the world (18.4% of the total cancer deaths) with poor prognosis (Bray
et al., 2020). Remarkable achievement has been recognized in the
treatment of lung cancer over the past few years, among which EGFR
targeted therapy is the most prominent. Gefitinib as a representative of
the first generation reversible EGFR TKIs, is the standard first-line drug
for treatment of EGFR-mutant lung cancer. However, the acquired
resistance caused by the mutation of EGFRT790M spawned the third
generation EGFR TKIs, such as AZD9291, which was approved by the
FDA as a second-line treatment for lung cancer with resistance caused by
EGFRT790M mutant. Despite the high efficacy, AZD9291 was reported to
acquire resistance with EGFRC797S mutation (Kim et al., 2015), which
was the most common mechanism of AZD9291 resistance. Besides, the
high price and adverse reaction limited the prescription of the drug.
Therefore, more effort should be devoted to develop novel
3.7. XHL11 inhibited tumor growth in the H1975 subcutaneous xenograft
model in vivo
To further investigate the anti-tumor effect of XHL11 in vivo, a H1975
subcutaneous xenograft model was established in BALB/c nude mice.
The mice were intragastric administrated with XHL11 (60 mg/kg) twice
a week via oral gavage for 17 days. As shown in Fig. 7, XHL11 inhibited
tumor growth in the H1975 subcutaneous xenograft mice without sig-
nificant changes in body weight. To confirm the anti-proliferation
capability, we also investigated the expression of Ki67, P-EGFR, EGFR
by immunohistochemical analysis. As shown in Fig. 7C–D, XHL11
inhibited the EGFR phosphorylation and down-regulated the expression
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Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
Fig. 6. XHL11 induces ROS generation in EGFR-mutant cell lines. (A–B) Cells were treated with Gefitinib (1 μM), AZD9291 (1 μM), XHL11 (0.1 and 1 μM) for 24
h, and the Rosup compound mixture for 30 min, and labelled with DCFH-DA (10 μM) for 1 h before the detection. (C) H1975 cells were pretreated with NAC (5 mM)
and then incubated with XHL11 for 24 h, and labelled with DCFH-DA (10 μM) for 1 h before the detection. (D) The apoptosis was measured by the cleavage of PARP.
The photographs were taken at magnifications of × 200. Data were expressed as % of control and were represented as mean ± S.D. of triplicate independent studies.
*P < 0.05, **P < 0.01, ***P < 0.001, significantly different compared with the control group.
9

Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
Fig. 7. XHL11 inhibited tumor growth in the H1975 subcutaneous xenograft model. (A) The mice were orally administrated with vehicle, XHL11 (60 mg/kg)
twice a week for 17 days. Tumor volumes measured three times a week are expressed as mean values ± S.D. (n = 5). *P < 0.05, significantly different compared with
control group. (B) The body weight expressed as mean values ± S.D. (C) Representative photographs of tumor tissues stained with Ki67, P-EGFR, EGFR and TUNEL
staining of the tumor tissue. The photographs were taken at magnifications of × 200. Scale bar, 50 μm. (D) Quantification of Ki67 (proliferation index), apoptosis
index assessed by TUNEL, P-EGFR and EGFR. Data were represented as mean ± S.D. of triplicate independent studies. *P < 0.05, significantly different compared
with control group.
mutant-selective EGFR TKIs. Alternative drugs should be developed to
cut the costs and mitigate the side effects.
XHL11 against the secondary (T790M) EGFR mutations. Further study is
underway to find out the effect of XHL11 using an EGFRL858R/T790M/
C797S cell line. Moreover, XHL11 inhibited the phosphorylation of EGFR
and downstream pathways including Bcl-xL, AKT and ERK in a dose- and
time-dependent manner. Furthermore, XHL11 suppressed tumor volume
in the H1975 subcutaneous xenograft mice without significant changes
in body weight. In accordance with the in vitro results, XHL11 inhibited
the EGFR phosphorylation and the expression of Ki-67. Moreover,
XHL11 was detected to induce apoptosis in vivo by TUNEL staining.
These results demonstrated that XHL11 exhibited high antitumor ac-
tivity and could be developed as a promising EGFR TKI.
In this study, we aim to investigate whether XHL11 could inhibit the
growth of EGFRT790M-mutant NSCLC cells. It turned out that XHL11
inhibited the proliferation of H1975 cells more efficiently than
AZD9291, and induced apoptosis in EGFR mutant cell lines at the con-
centration 0.1 μM. While, AZD9291 couldn’t induce apoptosis of H1975
and A431 cells at the concentration of 1 μM. In this study, XHL11 was
demonstrated to inhibit the EGFRL858R/T790M and EGFRL858R/T790M/C797S
kinases activity. It indicated the potential that XHL11 might overcome
EGFRC797S mutation. However, this research was focused on the effect of
10

Y. Li et al.
European Journal of Pharmacology 907 (2021) 174297
There is a wide spread agreement that elevated reactive oxidative
Bureau, China (2019MSGY128).
species is one of the hallmarks of cancer cells. Contradictory evidence
has emerged during the research of reactive oxidative species. On the
one hand, cancer cells stimulated reactive oxidative species to facilitate
proliferation and accelerate the tumor progression. Reactive oxidative
species assists the cancer cells to maintain malignant phenotypes and
plays an important part in regulating the proliferation, differentiation,
and apoptosis in cancer cells [18]. On the other hand, oxidant stress
induced intracellular reactive oxidative species level to a toxicity con-
centration, which could be exploited in the development of a therapeutic
agent and could selectively kill cancer cells (Lampiasi et al., 2009). The
elevated level of reactive oxidative species devoted to the intracellular
dysfunction through damaging to DNA, arresting cell cycle and inducing
apoptosis. To sum up, tumor cells may die by the same sword they
benefit from (Zhang et al., 2021). In accordance with the previous re-
ports (Hu et al., 2020; Tang et al., 2017b), AZD9291 induced the gen-
eration of H2O2 in H1975 cells. In the present study, XHL11 was also
found to promote reactive oxidative species generation in H1975 but in
A431 cell line. It’s possible that H2O2 was not elicited in A431 because of
the cell type, the culture environment, the concentration and the incu-
bation time. Moreover, XHL11 was found to induce apoptosis of cancer
cells through the regulation of the EGFR pathway involving AKT and
ERK. Further study is underway to determine whether XHL11 promote
reactive oxidative species generation through the inhibition of EGFR.
In conclusion, this research evaluated XHL11 as a novel EGFR in-
hibitor which markedly inhibited the proliferation, induced apoptosis of
EGFRT790M-mutant NSCLC cells. Moreover, the down-regulation of
EGFR and down-stream signaling devoted to the apoptosis and the anti-
proliferation of XHL11. This study demonstrated XHL11 might serve as a
novel promising EGFR TKI for the treatment of NSCLC patients.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ejphar.2021.174297.
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CRediT authorship contribution statement
Yi Li: Formal analysis, Writing – original draft, designed the
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