Design, synthesis and evaluation of novel ErbB/HDAC multitargeted inhibitors with selectivity in EGFRT790M mutant cell lines

Article abstract of DOI:10.1016/j.ejmech.2021.113173

Acquired resistance leads to the failure of EGFR TKIs in NSCLC treatment. A novel series of hydroxamic acid-containing 4-aminoquinazoline derivatives as irreversible ErbB/HDAC multitargeted inhibitors for NSCLC therapy had been designed and synthesized, which displayed weak anti-proliferative activity in several EGFR wild-type cancer cell lines (NCI–H838, SK-BR-3, A549, A431) yet retained moderate activity to EGFR^T790M resistance mutation harboring NCI–H1975 cells. The mechanistic studies revealed that the representative compound 11e was able to inhibit the phosphorylation of EGFR, up-regulate hyperacetylation of histone H3 and even reduce the expression of EGFR and Akt in NCI–H1975 cells. In further assays, compound 11e also showed moderate anti-proliferative activity in other EGFR^T790M harboring tumor cell lines (NCI–H820, Ba/F3_EGFR_Del19-T790M-C797S) and low toxicities in normal cell lines (HL-7702, FHC). This selectivity of designed multitargeted compounds could serve as a potential strategy to circumvent multiple mechanisms of acquired resistance to EGFR-targeted therapy without severe toxicities and side effects resulting from broad inhibition.

Full text of DOI:10.1016/j.ejmech.2021.113173

European Journal of Medicinal Chemistry 213 (2021) 113173
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and evaluation of novel ErbB/HDAC multitargeted
inhibitors with selectivity in EGFRT790M mutant cell lines
,
,
,
Lei Zhao a b, Tingting Fan b c, Zhichao Shi a b, Chao Ding b, Cunlong Zhang b, Zigao Yuan b,
Qinsheng Sun b, Chunyan Tan b, Bizhu Chu e *, Yuyang Jiang a
,
, b, c, d, e, **
a Department of Chemistry, Tsinghua University, Beijing, 100084, PR China
b State Key Laboratory of Chemical Oncogenomics, Key Laboratory of Chemical Biology, Tsinghua Shenzhen International Graduate School, Tsinghua
University, Shenzhen, 518055, PR China
c Department of Chemistry Southern University of Science and Technology, Shenzhen, 518055, PR China
d Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, PR China
e Institute of Biomedical Health Technology and Engineering, Shenzhen Bay Laboratory, Shenzhen, 518132, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Acquired resistance leads to the failure of EGFR TKIs in NSCLC treatment. A novel series of hydroxamic
acid-containing 4-aminoquinazoline derivatives as irreversible ErbB/HDAC multitargeted inhibitors for
NSCLC therapy had been designed and synthesized, which displayed weak anti-proliferative activity in
several EGFR wild-type cancer cell lines (NCIeH838, SK-BR-3, A549, A431) yet retained moderate activity
to EGFRT790M resistance mutation harboring NCIeH1975 cells. The mechanistic studies revealed that the
representative compound 11e was able to inhibit the phosphorylation of EGFR, up-regulate hyper-
acetylation of histone H3 and even reduce the expression of EGFR and Akt in NCIeH1975 cells. In further
assays, compound 11e also showed moderate anti-proliferative activity in other EGFRT790M harboring
tumor cell lines (NCIeH820, Ba/F3_EGFR_Del19-T790M-C797S) and low toxicities in normal cell lines
(HL-7702, FHC). This selectivity of designed multitargeted compounds could serve as a potential strategy
to circumvent multiple mechanisms of acquired resistance to EGFR-targeted therapy without severe
toxicities and side effects resulting from broad inhibition.
Received 29 October 2020
Received in revised form
23 December 2020
Accepted 6 January 2021
Available online 18 January 2021
Keywords:
NSCLC
EGFRT790M mutant
HDAC
Multitargeted compounds
Drug design
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
resistance appeared[7]. The most common mechanism to this
resistance was EGFR threonine790 to methionine790 (T790M) mu-
Lung cancer is the leading cause of cancer death worldwide[1],
where non-small cell lung cancer (NSCLC) accounts for approxi-
mately 85%[2]. Epidermal growth factor receptor (EGFR, or ErbB1/
HER1) activating mutations (AMs), such as exon 19-deletions (Del-
19) and leucine858 to arginine858 (L858R) mutation, were detected
in a subset of NSCLC patients (~10%e50%)[3] and have been iden-
tified as oncogenic drivers for NSCLC[4]. The presence of AMs in-
dicates sensitivity for EGFR tyrosine kinase inhibitors (TKIs) in
NSCLC treatment[5]. First-generation ATP-competitive reversible
EGFR TKIs (such as gefitinib, Fig. 1) showed significant clinical
response rate in NSCLC patients with EGFR AMs[6] until acquired
tation[8]. The T790M mutation restores affinity of ATP to EGFR and
decreases sensitivity of EGFR to ATP-competitive reversible TKIs[9].
Based on the first-generation 4-aminoquinazoline scaffold, second-
generation EGFR TKIs (such as afatinib, Fig. 1) covalently bind to
cysteine797 in the ATP binding pocket of EGFR with the addition of
Michael acceptor such as an acrylamide group[10,11]. This irre-
versible strategy enhances the competitiveness of TKIs with ATP in
EGFR mutations including T790M. However, afatinib failed to
distinctly improve efficacy in the clinical trial[12], partly due to the
adverse effects caused by insufficient selectivity for EGFR mutant
[13,14]. Afterwards, several pyrimidine derivatives were developed
as the third-generation mutant-selective irreversible inhibitors
[15e18] (such as osimertinib, Fig. 1), the strong and selective ac-
tivity against EGFRT790M allowed these inhibitors to easily reach
sufficient therapeutic doses. Nevertheless, more complicated re-
sistances emerged including EGFR cysteine797 to serine797 (C797S)
mutation after using third-generation inhibitors[19e23]. Allosteric
inhibitors EAI045 and JBJ-04-125-02 show the potential to reduce
* Corresponding author. Institute of Biomedical Health Technology and Engi-
neering, Shenzhen Bay Laboratory, Shenzhen, 518132, PR China.
** Corresponding author. Department of Pharmacology and Pharmaceutical Sci-
ences, School of Medicine, Tsinghua University, Beijing, 100084, PR China.
E-mail addresses: chubz@szbl.ac.cn (B. Chu), jiangyy@sz.tsinghua.edu.cn
(Y. Jiang).
https://doi.org/10.1016/j.ejmech.2021.113173
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Fig. 1. The structures of relevant EGFR, HDAC or multitargeted inhibitors.
the incidence of drug resistance[24,25] yet some deficiencies
remain unsolved, such as Del-19 related resistance. In addition to
these "oncogene addiction" EGFR site mutations resulting in
resistance[26], numerous genetic and epigenetic signaling aberra-
tions including human epidermal growth factor receptor 2 (HER2,
or ErbB2/Neu) amplification and mesenchymal-epithelial transi-
tion factor (MET) amplification may also cause resistance in NSCLC
[27e29]. As a result, the identification of ever-increasing number of
de novo and acquired resistance mechanisms tempered the initial
enthusiasm to turn NSCLC with EGFR AMs into a chronic disease
through TKIs[27]. Therefore, how to adopt appropriate strategies to
circumvent multiple mechanisms of acquired resistance to EGFR-
targeted therapy without severe toxicities remains a formidable
challenge for TKIs design.
Many studies indicated that a synergistic crosstalk exists be-
tween EGFR/HER2 and histone deacetylase (HDAC)[30e32]. Inhi-
bition of HDAC effectively affects the activity of proteins that are
key regulators of receptor tyrosine kinases (RTKs). HDAC inhibitors
(such as vorinostat, Fig. 1) synergize with ErbB inhibitors in EGFR/
HER2 overexpression cancer cell lines, which lead to increased heat
shock protein 90 (HSP90) acetylation, EGFR/HER2 degradation and
cell death[33e35]. Thus, using multitarget-directed ligands
(MTDLs) approach to combine the pharmacophore of ErbB and
HDAC inhibitors should be useful in NSCLC therapy[36]. One
promising EGFR/HER2/HDAC multitargeted inhibitor CUDC-101
(Fig. 1) had been studied in Phase I clinical investigation[37e40],
with low IC50 values against EGFR, HDAC and HER2 of 2.4 nM,
4.4 nM and 15.7 nM, respectively. Our group also has developed a
series of multitargeted inhibitors (representative compound 6B in
Fig. 1) by combining the core pharmacophores of HDAC inhibitor
vorinostat and various kinase inhibitors with different 1,2,3-
triazole linked alkyl chain linkers[41,42]. Most of them worked
well in tumor cell lines such as A549 (EGFR overexpressed, K-RAS
mutation), A431 (EGFR overexpressed) and SK-BR-3 (HER2 over-
expressed), but less effective in EGFRT790M harboring cell lines. To
further improve the efficiency of our multitargeted compounds
against EGFRT790M-related resistance, a novel series of hydroxamic
acid-containing 4-aminoquinazoline derivatives were designed,
synthesized and evaluated for antitumor activity.
1.1. Design strategy
According to the second- and third-generation EGFR TKIs,
introducing the electrophilic acrylamide group as Michael acceptor
into the pharmacophore is an effective design strategy to enhance
the competition with ATP for EGFR AMs and T790M[43]. On the
other hand, the hydroxamic acid group is usually resided in the
developed HDAC inhibitors as zinc binding group (ZBG), which
interacts with the zinc ion at the conserved active site[36]. Based on
previous works, compound 6B performed well to inhibit EGFR/
HER2 and HDAC by combining hydroxamic acid fragment and
quinazoline core with appropriate 6-substituted triazole-contained
linker[42]. Since 6-position of quinazoline is a suitable site for the
introduction of the acrylamide group, bold modification was made
to enhance the binding ability of the compound to EGFR. As
depicted in Fig. 2, the substituted position of the linker was
changed from C-6 to C-7 on quinazoline ring, leaving space for the
Michael acceptor. The structure and length of the linker were
modified to reduce the potential steric hindrance and redesigned
(n ¼ 1, 2, 3) to identify the structure-activity relationship, respec-
tively. The R group of compounds exhibited superior activity in our
previous works, which were retained, including 3-chloro-2-
fluorophenyl, 3-chloro-4-fluorophenyl
and 3-chloro-4-[(2-
fluorobenzyl)oxy]phenyl. Docking studies suggested that our
designed compounds were capable of binding to target proteins in
high-scored poses. As shown in Fig. 3A, the nitrogen atom on the
2

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Fig. 2. Design strategy of ErbB/HDAC multitargeted inhibitors.
quinazoline ring of compound 11e forms a hydrogen bond with
Met793 in hinge area, while the 7-substituted linker forms another
hydrogen bond and directs the hydroxamic acid group to the hy-
drophilic region outside the binding pocket. Furthermore, the 3-
chloro-4-fluorophenyl (R group) of 11e occupies the hydrophobic
area of EGFRT790M ATP binding pocket and the 6-substituted
acrylamide group (Michael acceptor) is close enough to Cys797,
which provides a condition for the formation of covalent bond.
Another result in Fig. 3B shows that the hydroxamic acid group of
11e is induced to the active site of HDAC2 with appropriate linker
length, while the quinazoline ring on the other side stays at the
surface outside the active pocket. Thus, the design of multitargeted
inhibitors was reasonable and then nine new compounds were
synthesized.
fluorophenyl) showed moderate effect on HDAC1 and HDAC6
compared with vorinostat. A pattern can be observed in the data
that compounds generally inhibited tested targets more potently
when n ¼ 2, especially in HDAC1 and HDAC6, indicating that a
median n ¼ 2 linker length is appropriate for this series of multi-
targeted compounds. Compounds with larger substituent on the
quinazoline ring (11g-11i) showed low effect on HDAC1
(IC50 > 1000), and their effect on HDAC6 was limited either (IC50
was equal to 332 nM, 96 nM and 274 nM, respectively). In general,
all the compounds were more active against HDAC6, while the large
R group has been shown to decrease the HDAC activity of multi-
targeted compounds, similar to the results of our previous works
[41]. Additional docking study provided some explanation for the
low activity of 11g-11i on HDAC. As depicted in Fig. S1, 11h was
blocked at the binding entry by the larger substituents compared
with 11e. The substituent on the quinazoline ring of 11h failed to
form a hydrogen bond with the amino acid residue (Asp104) at the
entrance of the pocket, and the key hydroxamic acid group was not
deep enough to form hydrogen bonds with more amino acids
(Asp181, Gln265). This indicated that the hydroxamic acid group of
11h was difficult to reach the zinc binding site of HDAC1, which is
crucial for the activity. On the contrary, HDAC6 has a relatively loose
pocket structure, which was more accessible to the compounds
with larger cap group (Fig. S2). Therefore, the selectivity of com-
pounds in HDAC6 may due to the steric hindrance in interacting
with the active pocket of HDAC. Moreover, the activity of all com-
pounds against HDAC was somewhat lower. This should be due to
the modification of linker substitution site (from 6-substituted to 7-
substituted on quinazoline ring) of compounds that generate steric
hindrance at the active pocket of HDAC1, along with the large R
groups, affecting the inhibitory activity of HDAC1.
1.2. Chemistry
Compound 11a-11i were synthesized according to the synthetic
routes in Scheme 1. 4-Chloro-7-fluoro-6-nitroquinazoline (2) was
prepared via chlorination of commercially available 7-fluoro-6-
nitroquinazolin-4(3H)-one (1). Three kinds of anilines reacted
with 2 to produce compounds 3a-3c. Then, compounds 5a-5c were
obtained after nucleophilic substitution and reduction. Otherwise,
intermediates 8a-8c were synthesized from commercially available
6a-6c through azidation and amidation. Coupling 5ae5c with
8ae8c followed by substitution and acidification afforded the final
products 11ae11i.
2. Results and discussion
Firstly, enzyme inhibition activity of the nine newly synthetic
compounds was screened and the results were summarized in
Table 1. The inhibitory activity of all compounds against EGFR and
EGFRL858R was similar to or slightly less than afatinib. Compounds
11g-11i (R ¼ 3-chloro-4-[(3-fluorobenzyl)oxy]phenyl, n ¼ 1, 2, 3)
exhibited better inhibitory activity against HER2 compared with
afatinib. 11d-11f (R ¼ 3-chloro-4-fluorophenyl, n ¼ 1, 2, 3) dis-
played intermediate inhibitory activity against EGFRL858R/T790M
compared with afatinib (4-aminoquinazoline scaffold, IC50 ¼ 3 nM)
and osimertinib (pyrimidine scaffold, IC50 ¼ 0.43 nM). 11e showed
the strongest inhibition on EGFRL858R/T790M (IC50 ¼ 1.5 nM). In
addition, afatinib had a 10-fold inhibitory activity drop going from
EGFR WT to T790M, while 11e was about 4-fold. 11a-11f with
Next, compounds 11a-11i were evaluated for the anti-
proliferative activity against several tumor cell lines, including
A549 (NSCLC, EGFR WT, k-RAS mutation), NCIeH838 (NSCLC, EGFR
WT, k-RAS WT), SK-BR-3 (human breast cancer cells, HER2 over-
expressed), and A431 (human epithelioid squamous carcinoma,
EGFR overexpressed) using afatinib and vorinostat as reference
compounds. As shown in Table 2, afatinib and vorinostat showed
moderate to strong activity in these tumor cell lines, whereas most
of compounds showed poor activity. Such unexpected results
indicate that our aggressive structural modifications impair the
activity of compounds in these EGFR- or HER2-expressing tumor
cell lines, revealing the structural sensitivity of this kind of multi-
targeted compounds. Cai et al. mentioned that changing the
smaller
R
groups (3-chloro-2-fluorophenyl or 3-chloro-4-
3

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Fig. 3. Docking poses of compound 11e with EGFRT790M (A, PDB code: 4G5P) and HDAC2 (B, PDB code: 4LXZ).
substituted position of the oxyalkyl chain linkers from 6- to 7-
would reduce the target activity to some extent in the report of
CUDC-101[37]. In our previous works, a significant reduction in
anti-proliferative activity was also observed between compounds
with similar enzyme activity, simply as a result of the length
alteration of 6-substituted triazole-contained linkers[41,42]. It can
be inferred that the anti-proliferative profiles of our compounds are
easily affected by linker structure, and in this case, unfortunately,
the activity of the compounds was compromised via our structural
modifications.
Given the high cost of introducing the Michael acceptor at the 6-
position of the quinazoline ring, the effects of the compounds on
resistant cell lines are of concern. MTT assay manifested that 11a-
11i moderately inhibited NCIeH1975 (NSCLC, EGFRL858R/T790M
mutated) proliferation compared with reference drugs (Table 2).
The IC50 values reflected a b > c>a (e > f > d, h > i > g) pattern,
suggesting that compounds with the alkyl chain length of n ¼ 2
were more effective to NCIeH1975 cells than n ¼ 1 and n ¼ 3, which
is consistent with the results of enzymatic test. Similarly, 11a-11f
with small substituents on the phenyl ring were found to be more
potent than others. Among them, 11e showed the best anti-
proliferative activity, which was comparable to afatinib and vor-
inostat. Compared with previous data, this conspicuous anti-
proliferative activity on NCIeH1975 cells attracted our attention.
There was a report suggested that some triazole-tethered quina-
zoline derivatives with the same 7-substituted structure fragments
as our compounds have certain selectivity for EGFRT790M[44].
Apparently, it is not enough to explain our situation and further
research is required. We noticed that Neckers et al. had once re-
ported that HDAC inhibitors can achieve similar effect in cancer
cells as HSP90 inhibitors by keeping HSP90 acetylated and inhib-
iting its chaperone activity[45]. In addition, Wong and Shapiro et al.
4

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Scheme 1. Synthesis of 11a-11i. Reagents and conditions: (a) SOCl2, DMF(cat.), reflux, 5 h; (b) anilines, DCM, rt, 2 h; (c) propargyl alcohol, t-BuOK, THF, 0 oC-rt, 4 h; (d) NH4Cl, Fe,
EtOH/H2O, 70 ꢀC, 4 h; (e) NaN3, MeCN, reflux, 10 h; (f) NaOH, MeOH/H2O, 0 oC-rt, 8 h; (g) NH2OTHP, EDCI, DMAP, DCM, rt, 8 h; (h) Sodium ascorbate, CuSO4$5H2O, THF/H2O, 35 ꢀC,
1 h; (i) Acryloyl chloride, DIEA, THF, DCM, 0 ꢀC, 0.5 h; (j) HCl/dioxane, THF, rt, 1 h.
had pointed out that signal transduction in NCIeH1975 cells
dependent on multiple RTKs, irreversible EGFR inhibitor alone does
not fully suppress phosphorylation of other activated RTK and
result in residual activities of them. These T790M harboring cells
show substantial but incomplete dependence on EGFR, which are
sensitivie to Hsp90 inhibition[46]. Therefore, we hypothesized that
unusual activity of our multitargeted compounds on
NCIeH1975 cells were associated with the synergistic effect be-
tween related targets. Since the activity of compounds against each
target (EGFR, EGFRL858R, EGFRL858R/T790M, HER2, HDAC1 and HDAC6,
Table 1) was insufficient to account for the synergistic effect be-
tween targets, mechanistic studies were carried out on
NCIeH1975 cells with the most effective compound 11e to validate
the proposed hypothesis.
As can be seen in Fig. 4, 11e caused a dose-dependent reduction
of EGFR phosphorylation (Tyr1068 and Tyr1173) and up-regulation
of histone H3 (Lys27) hyperacetylation in NCIeH1975 cells, which
were consistent with the good inhibitory activity on EGFRT790M and
HDAC. Meanwhile, a small decrease in EGFR expression was
observed. As mentioned above, the inhibition of HDAC leads to
increased HSP90 acetylation[45] and consequently leads to degra-
dation of some client including EGFRT790M, hence these results
indicated that 11e is not only capable of modulating EGFR and
HDAC, but also influencing the function of HSP90 through the
mechanism reported by Neckers et al. To provide more evidence,
the expression of Akt in NCIeH1975 cells after exposure to 11e was
also investigated (Fig. 4). As a downstream signaling proteins of
EGFR and an HSP90 client, inhibition of EGFR alone leads to
reduction of Akt phosphorylation rather than Akt. However, a
reduction of Akt was indeed observed, indicating that Akt was
degraded due to HSP90 inhibition, in other words, HSP90 acetyla-
tion was maintained by 11e in NCIeH1975 cells. According to Wong
and Shapiro et al., EGFRT790M harboring NCIeH1975 cells are sen-
sitive to multitargeted inhibition[46]. Thus, although the anti-
proliferative activity of our multitargeted compounds against
several tumor cells without T790M were compromised due to
linker structural modification, the synergistic effect on multiple
targets allowed them to retain certain activity in NCIeH1975 cells.
Western blot analysis was carried on A431 cells in order to find
more clues. As depicted in Fig. 5, reduction of p-EGFR (Tyr1068 and
Tyr1173) and upregulation of histone H3 (Lys27) hyperacetylation
were observed after treating A431 cells with 11e. Compared with
NCIeH1975 cells at the same concentration of 11e, these effects
were less potent and the expression of EGFR and Akt were barely
decreased in these T790M-free tumor cells, which were consistent
with the weak anti-proliferative activity of 11e in A431 cells.
To further verify the synergistic effect of our multitargeted
compounds on HDAC and EGFR in NCIeH1975 cells. Western blot
assay of compound 11e and control compounds was carried out. As
shown in Fig. 6, compound 11e showed synergistic inhibition of
HDAC and EGFR in EGFRT790M mutant NCIeH1975 cells, which was
not observed in the control compound alone. Typical example is
that vorinostat did not reduce EGFR phosphorylation, or afatinib
did not reduce EGFR and Akt expression. Meanwhile, the control
experiment of compound 11e and reversible inhibitor gefitinib was
also carried out. In Fig. 7, the inhibition of 11e on EGFR-related
pathway protein expression in NCIeH1975 cells was stronger
than that of gefitinib, and it was more obvious with time. This
indicated that the strategy of introducing Michael acceptor
enhanced the ability of the compounds to inhibit EGFRT790M mu-
tation, although the modification of linker also reduced the activity
to some extent. By comparing the results of these control Western
blot assay, compound 11e showed that it had the effect of both
HDAC and EGFR inhibitors on EGFR-related pathway proteins in
EGFRT790M mutation NCIeH1975 cells.
Considering the special experimental results of our multi-
targeted compounds at cellular level, it is necessary to investigate
the activity of compound on T790M harboring cells other than
NCIeH1975. The anti-proliferative activity of 11e against NCIeH820
(NSCLC, EGFR E746_T751del_insI/T790M, E746-T751 deletion/I insertion/
T790M mutation) and Ba/F3_EGFR_Del19-T790M-C797S (exon 19
deletion/T790M mutation/C797S mutation) cells was evaluated.
Although the IC50 values listed inTable 3 demonstrated that the anti-
proliferative activity of 11e on these T790M harboring cells was
weaker than that of NCIeH1975 cells, it is still more potent than that
of A549, NCIeH838 and A431 cells. Additionally, compounds 11a-11i
weretested for toxicityon two normal cell lines (human normal liver
cell HL-7702 and human normal colon epithelial FHC). As shown in
5

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Table 1
Enzymatic activity of 11a-11i against EGFR, EGFRL858R, EGFRL858R/T790M, HER2, HDAC1 and HDAC6.
Cpds.
11a
R
n
1
2
IC50 (nM)
EGFR
EGFRL858R
1.1
EGFRL858R/T790M
8.3
HER2
13
HDAC1
44
HDAC6
26
0.34
11b
0.36
0.44
1.2
1.2
5.4
17
8.0
16
34
5.0
11c
3
132
43
11d
11e
1
2
0.33
0.35
1.1
1.1
2.3
5.4
5.7
93
61
1.5
75
12
11f
3
1
0.35
1.1
0.88
2.2
1.8
16
7.3
3.7
292
120
332
11g
>1000
11h
11i
2
3
1.8
1.5
2.9
2.6
15
14
3.7
3.8
>1000
>1000
96
274
Afatinib
Vorinostat
Osimertinib
0.32
NT
NT
0.88
NT
NT
3.0
NT
0.43
6.2
NT
NT
NT
16
NT
NT
13
NT
Notable IC50 values are represented in bold.
Table 2
Anti-proliferative activity of 11a-11i in A549, NCIeH838, SK-BR-3, A431 and NCIeH1975 cells.
Cpds.
IC50 (m
M)a
A549
NCIeH838
SK-BR-3
A431
NCIeH1975
11a
11b
11c
11d
11e
11f
11g
11h
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
30.80 7.18
19.97 3.16
24.77 2.52
20.24 3.00
15.97 4.31
5.56 1.69
2.13 1.10
14.96 2.99
16.94 4.32
15.57 4.56
22.05 1.02
20.65 5.37
14.98 1.23
16.29 3.00
19.91 9.26
18.80 8.88
0.59 0.27
1.47 0.42
6.61 3.66
2.80 1.43
4.18 2.92
4.45 0.95
1.82 0.50
2.50 1.34
21.01 3.10
12.01 1.08
14.51 2.22
0.74 0.28
2.04 0.31
11i
Afatinib
Vorinostat
>50
9.92 3.22
5.18 2.26
1.90 0.50
4.25 0.53
a
Data are expressed as the mean SD from the dose response curves of at least three independent experiments.
Table 4, reference compounds afatinib and vorinostat were lethal to
cells as usual, while our compounds were less harmful to cell sur-
vival. These results suggested that our multitargeted compounds are
quite insensitive to cells without T790M.
To investigate the effects of compound 11e on cell viability and
invasion of NCIeH1975 cells, colony formation assay and transwell
6

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Fig. 4. Western blot assay of compound 11e in NCIeH1975 cells.
Fig. 6. Western blot assay of compound 11e, afatinib and vorinostat in
NCIeH1975 cells.
normal cells.
3. Conclusion
In summary, a series of novel 4-aminoquinazoline derivatives as
multitargeted inhibitors were designed and synthesized. Most of
the compounds showed good inhibitory activity against various
enzymes (EGFR, EGFRL858R, EGFRL858R/T790M, HER2, HDAC1, HDAC6).
However, these compounds failed to inhibit the proliferation of
some EGFR WT tumor cell lines (A549, A431, SK-BR-3, NCIeH838)
well yet retained moderate anti-proliferative activity in
NCIeH1975 cells. A hypothetical explanation was proposed that our
multitargeted compounds caused synergistic effects in
NCIeH1975 cells according to previous reports. The mechanistic
studies provided evidence for our hypothesis that the representa-
tive compound 11e was able to inhibit the phosphorylation of EGFR,
up-regulate histone H3 hyperacetylation and even reduce the
expression of EGFR and Akt in NCIeH1975 cells. The Western blot
analysis on A431 cells and MTT assay on additional cell lines (Ba/
F3_EGFR_Del19-T790M-C797S, NCIeH820, HL-7702, FHC) of 11e
provided further validation. Thus, it may be a blessing in disguise
that our aggressive structural modification resulted in the loss of
cell inhibitory activity of the compounds. The certain activity
retained in some EGFRT790M harboring cell lines that are sensitive to
multitargeted inhibition, resulting in selectivity of multitargeted
compounds. These unexpected results indicated a potential strat-
egy to circumvent multiple mechanisms of acquired resistance to
EGFR-targeted therapy through multitarget inhibition without se-
vere toxicities and side effects resulting from broad inhibition,
hoping to alleviate the drug resistance problem in clinical treat-
ment of NSCLC.
Fig. 5. Western blot assay of compound 11e in A431 cells.
assay were conducted. Fig. 8A and B showed that compound 11e
reduced the cell viability and invasion of NCIeH1975 cells in a
concentration-dependent manner. Furthermore, the pro-apoptotic
effect of compound 11e in NCIeH1975 cells was evaluated by
conducting Annexin-V/FITC binding assay. As illustrated in Fig. 8C,
the percentage of early-stage apoptotic cells was increased from
6.67% to 31.38% and the late-stage apoptotic cells was increased
from 4.20% to 14.30% when treated with 2.5 mM of 11e. The possible
mechanism of 11e-induced apoptosis was also examined in
NCIeH1975 cells. In Fig. 8D, 11e was observed to increase active
(cleaved) caspase-8 levels. The subsequent activation of caspase-7,
caspase-3 and PARP were also detected. These results suggest that
our multitargeted compounds 11e has the ability to promote anti-
proliferative and proapoptotic effect in NCIeH1975 cells, which
may result from concerted inhibition of EGFRT790M, HER2, and
HDAC. Compared with TKIs that target only one resistance mech-
anism at a time, our multitargeted compounds may be more ad-
vantageous due to the potential to induce tumor cell apoptosis
through different mechanisms without excessive damage to
7

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Fig. 7. Western blot assay of compound 11e and gefitinib in NCIeH1975 cells.
Table 3
obtain the desired compound 3a as yellow solid, yield 95%. 1H NMR
(400 MHz, DMSO‑d6)
Anti-proliferative activity of 11e in NCIeH820 and Ba/F3_EGFR_Del19-T790M-C797S
cells.
d
9.69 (d, J ¼ 7.8 Hz, 1H), 8.77 (s, 1H), 7.94 (d,
J ¼ 12.0 Hz, 1H), 7.60 (ddd, J ¼ 8.3, 6.7, 1.6 Hz, 1H), 7.53 (ddd, J ¼ 8.3,
Cpds.
IC50 (m
M)a
6.8, 1.7 Hz, 1H), 7.35 (td, J ¼ 8.1, 1.3 Hz, 1H).
NCIeH820
Ba/F3_EGFR_Del19-T790M-C797S
4.1.2. N-(3-chloro-2-fluorophenyl)-6-nitro-7-(prop-2-yn-1-yloxy)
quinazolin-4-amine (4a)
11e
Afatinib
Vorinostat
1.75 0.81
0.55 0.15
1.43 0.62
7.61 1.32
0.89 0.21
0.59 0.26
General procedure for the synthesis of 4a-4c. A mixture of
compound 4a (1.52 g, 4.5 mmol) and propargyl alcohol (0.3 ml,
5 mmol) in THF (20 mL) was stirred at 0 ꢀC. Potassium tert-butoxide
(1.12 g, 10 mmol) was slowly added and then the reaction was
stirred for another 4 h. After the consumption of starting materials,
water (100 mL) and 1 M hydrochloric acid (20 mL) were added to
the mixture to form a precipitate. The mixture was filtered and the
filter cake was washed with water. The filter cake was dried to get
the desired compound 4a as yellow solid, yield 90%. 1H NMR
a
Data are expressed as the mean SD from the dose response curves of at least
three independent experiments.
Table 4
The cellular activity of 11a-11i in HL-7702 and FHC cell lines.
Cpds.
IC50 (m
M)a
HL-7702
FHC
(400 MHz, DMSO‑d6)
1H), 7.63e7.48 (m, 3H), 7.32 (td, J ¼ 8.1, 1.3 Hz, 1H), 5.22 (d,
J ¼ 2.4 Hz, 2H), 3.82 (m, 1H).
d
10.81 (s, 1H), 9.28 (d, J ¼ 4.3 Hz, 1H), 8.66 (s,
11a
11b
11c
11d
>25
>25
>25
>25
>25
>25
>25
>25
11e
11f
11g
11h
11i
Afatinib
Vorinostat
>25
>25
>25
>25
>25
1.62 0.37
5.45 2.65
>25
>25
>25
>25
>25
1.55 0.70
2.59 1.10
4.1.3. N4-(3-chloro-2-fluorophenyl)-7-(prop-2-yn-1-yloxy)
quinazoline-4,6-diamine (5a)
General procedure for the synthesis of 5a-5c. To a solution of
compound 4a (1.49 g, 4 mmol) and ammonium chloride (535 mg,
10 mmol) in 90% ethanol, iron powder (900 mg, 16 mmol) was
added. The suspension was stirred at 70 ꢀC for 4 h and then filtered
through celite. The filtrate was concentrated in vacuo, washed with
water and dried to form the desired compound 5a as yellow solid,
a
Data are expressed as the mean SD from the dose response curves of at least
three independent experiments.
yield 70%. 1H NMR (400 MHz, DMSO‑d6)
d 9.74 (s, 1H), 8.34 (s, 1H),
4. Experimental protocols
7.82 (d, J ¼ 13.9 Hz, 1H), 7.46e7.26 (m, 3H), 7.12 (d, J ¼ 24.2 Hz, 1H),
5.56 (s, 2H), 5.05 (s, 2H), 3.73 (s, 1H).
4.1. Chemistry
4.1.4. 3-Azidopropanoic acid (7a)
4.1.1. N-(3-chloro-2-fluorophenyl)-7-fluoro-6-nitroquinazolin-4-
amine (3a)
General procedure for the synthesis of 7a, 7b1 and 7c. To a
stirred solution of 3-bromopropionic (5.3 g, 35 mmol) in acetoni-
trile (30 mL) was added sodium azide (4.55 g, 70 mmol) and slowly
heated to reflux. After 8 h of reaction, the mixture was filtrated and
the filtrate was evaporated in vacuo to remove part of the aceto-
nitrile. The condensation was diluted in DCM (200 mL) and washed
three times with water (50 mL). The organic layer was dried over
anhydrous sodium sulfate, filtrated and evaporated to obtain the
desired compound 7a as colorless oil, yield 50%. 1H NMR (400 MHz,
General procedure for the synthesis of 3a-3c. Compound 1
(1.05 g, 5 mmol) was dissolved in sulfoxide chloride (7.26 mL,
100 mmol) followed by the addition of DMF (catalytic amount) and
the mixture was heated at reflux for 5 h. The excess sulfoxide
chloride was removed in vacuo and the solution of corresponding
aniline (5.1 mmol) in anhydrous DCM (20 mL) was added and
stirred at room temperature for 2 h. The precipitate was filtrated
and washed with DCM and water. The filter cake was dried to
CDCl3)
d
3.58 (t, J ¼ 6.5 Hz, 2H), 2.61 (t, J ¼ 6.5 Hz, 2H).
8

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
Fig. 8. 11e exerted cytotoxic activity in NCIeH1975 cells. (A) Cell viability, as determined by colony formation assay. (B) Cell invasion, as determined by transwell assay. (C)
Compound 11e induces the apoptosis of NCIeH1975 cells. (D) Possible mechanism of 11e-induced apoptosis, as determined by Western blot assay.
4.1.5. 4-Azidobutanoic acid (7b2)
to afford compound 9a as yellow solid, which was used directly in
the next step without further purification.
Sodium hydroxide (1.2 g, 30 mmol) was added to a mixture of
compound 7b1 (1.57 g, 10 mmol), water (15 mL) and methanol
(20 mL) at 0 ꢀC. After the reaction returned to room temperature
and stirred for 8 h, methanol was removed by evaporation. The pH
of the condensation was adjusted to 7 by 1 M HCl and then
extracted with DCM, washed with brine and dried over anhydrous
sodium sulfate. Filtration and evaporation to furnish the desired
compound 7b2 as colorless oil, yield 90%. 1H NMR (400 MHz, CDCl3)
4.1.8. N-(4-((3-chloro-2-fluorophenyl)amino)-7-((1-(3-oxo-3-
(((tetrahydro-2H-pyran-2-yl)oxy)amino)propyl)-1H-1,2,3-triazol-
4-yl)methoxy)quinazolin-6-yl)acrylamide (10a)
General procedure for the synthesis of 10a-10i. Acryloyl chloride
(130
mL, 1.6 mmol) was dissolved in anhydrous DCM (5 mL) and
added dropwise to a mixture of compound 9a (836 mg, 1.5 mmol)
d
11.53 (s, 1H), 3.38 (t, J ¼ 6.7 Hz, 2H), 2.48 (t, J ¼ 7.2 Hz, 2H), 1.91 (q,
and DIEA (330 mL, 2 mmol) in anhydrous THF (20 mL) under argon
protection at 0 ꢀC. After 0.5 h of reaction, the mixture was
concentrated in vacuo and purified by chromatography (dichloro-
methane/methanol, 20:1) to afford the desired compound 10a as
J ¼ 7.0 Hz, 2H).
4.1.6. 3-Azido-N-((tetrahydro-2H-pyran-2-yl)oxy)propanamide
(8a)
yellow solid, yield 35%. 1H NMR (400 MHz, DMSO‑d6)
d 11.01 (s, 1H),
General procedure for the synthesis of 8a-8c. EDCI (2.88 g,
15 mmol) was added to a solution of compound 7a (1.15 g,
10 mmol), O-(Tetrahydro-2H-pyran-2-yl)hydroxylamine (1.29 g,
11 mmol) and DMAP (122 mg, 1 mmol) in DCM (30 mL). After
stirred at room temperature for 8 h, the reaction mixture was
washed with water and extracted with DCM. The organic layer was
concentrated in vacuo and purified by chromatography (petroleum
ether/ethyl acetate, 3:1) to afford the desired compound 8a as
9.34 (s, 1H), 8.38 (s, 1H), 8.26 (s, 1H), 7.53 (m, 1H), 7.42 (m, 1H), 7.34
(s, 1H), 7.32 (s, 1H), 7.24 (m, 1H), 5.38 (s, 1H), 5.36 (s, 1H), 4.82 (s,
1H), 4.40 (t, J ¼ 6.3 Hz, 2H), 3.91 (m, 1H), 3.50 (m, 1H), 2.06 (m, 4H),
1.64 (m, 3H), 1.50 (m, 3H).
4.1.9. N-(4-((3-chloro-2-fluorophenyl)amino)-7-((1-(3-
(hydroxyamino)-3-oxopropyl)-1H-1,2,3-triazol-4-yl)methoxy)
quinazolin-6-yl)acrylamide (11a)
General procedure for the synthesis of 11a-11i. 1 M HCl in
dioxane (1 mL) was slowly added to the solution of compound 10a
(61 mg, 0.1 mmol) in anhydrous THF (5 mL) and the mixture was
stirred at room temperature for 0.5 h. The precipitate was filtered
off, washed with ethyl ether, isopropanol and DCM, and dried to
give the desired compound 11a as yellow solid, yield 70%. 1H NMR
white solid, yield 60%. 1H NMR (400 MHz, CDCl3)
d 8.55 (s, 1H), 4.98
(t, J ¼ 6.8 Hz, 1H), 3.73-3.58 (m, 2H), 2.39 (t, J ¼ 6.9 Hz, 2H). 1.83 (t,
J ¼ 6.8 Hz, 2H), 1.73-1.54 (m, 6H).
4.1.7. 3-(4-(((6-Amino-4-((3-chloro-2-fluorophenyl)amino)
quinazolin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-((tetrahydro-
2H-pyran-2-yl)oxy)propanamide (9a)
(400 MHz, DMSO‑d6)
d 11.48 (s, 1H), 10.57 (s, 1H), 9.99 (s, 1H), 9.20
General procedure for the synthesis of 9a-9i. Copper sulfate
pentahydrate (50 mg, 0.2 mmol) was added to the mixture of
compound 5a (684 mg, 2 mmol), compound 8a (428 mg, 2 mmol)
and sodium ascorbate (100 mg, 0.5 mmol), THF (15 mL) and water
(8 mL). The reaction was heated at 35 ꢀC for about 1 h. Part of the
THF was removed by evaporation after the consumption of 5a. The
condensation was mixed with water (30 mL), fully stirred and
filtered. The filter cake was washed with water and dried in vacuo
(s, 1H), 8.84 (s, 1H), 8.37 (s, 1H), 7.72 (s, 1H), 7.64 (ddd, J ¼ 8.3, 6.7,
1.7 Hz, 1H), 7.52 (ddd, J ¼ 8.3, 6.8, 1.7 Hz, 1H), 7.36 (td, J ¼ 8.1, 1.3 Hz,
1H), 6.79 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.34 (dd, J ¼ 17.0, 1.9 Hz, 1H),
5.85 (dd, J ¼ 10.1, 2.0 Hz, 1H), 5.76 (s, 1H), 5.54 (s, 2H), 4.61 (t,
J ¼ 6.7 Hz, 2H), 2.64 (t, J ¼ 6.8 Hz, 2H), 1.03 (d, J ¼ 6.1 Hz, 1H). 13
C
NMR (101 MHz, DMSO‑d6)
d 166.3, 164.3, 159.9, 156.1, 153.1 (d,
J ¼ 251.5 Hz), 151.1, 141.5, 131.9, 129.7 (d, J ¼ 4.0 Hz), 128.5, 128.0,
126.1, 125.8 (d, J ¼ 5.1 Hz), 120.9 (d, J ¼ 16.2 Hz), 117.2, 107.6, 102.4,
9

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
63.4, 55.4, 33.0. HRMS (ESI) m/z calculated for [MþH]þ 527.1358,
found 527.1356; mp 199e201 ꢀC. HPLC analysis: MeOHeH2O (0.1%
TFA, gradient elution, 0 min, 90:10, 25 min, 0:100), RT ¼ 17.28 min,
96.93% purity.
26.4. HRMS (ESI) m/z calculated for [MþH]þ 541.1515, found
541.1520; mp 255e257 ꢀC. HPLC analysis: MeOHeH2O (0.1% TFA,
gradient elution, 0 min, 90:10, 25 min, 0:100), RT ¼ 17.97 min,
93.31% purity.
4.1.10. 4-(4-(((6-Acrylamido-4-((3-chloro-2-fluorophenyl)amino)
quinazolin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-
hydroxybutanamide (11b)
4.1.14. 5-(4-(((6-Acrylamido-4-((3-chloro-4-fluorophenyl)amino)
quinazolin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-
hydroxypentanamide (11f)
1H NMR (400 MHz, DMSO‑d6)
d
11.57 (s, 1H), 10.48 (s, 1H), 10.02
1H NMR (400 MHz, DMSO‑d6)
d 11.48 (s, 1H), 10.47 (s, 1H), 10.05
(s, 1H), 9.22 (s, 1H), 8.86 (s, 1H), 8.48 (s, 1H), 7.77 (s, 1H), 7.64 (ddd,
J ¼ 8.2, 6.8, 1.7 Hz, 1H), 7.52 (td, J ¼ 7.5, 6.8, 1.7 Hz, 1H), 7.36 (td,
J ¼ 8.1, 1.3 Hz, 1H), 6.80 (dd, J ¼ 16.9, 10.2 Hz, 1H), 6.34 (dd, J ¼ 17.0,
1.9 Hz, 1H), 5.85 (dd, J ¼ 10.1, 1.9 Hz, 1H), 5.55 (s, 2H), 4.40 (t,
(s, 1H), 9.20 (s, 1H), 8.92 (s, 1H), 8.52 (s, 1H), 7.95 (dd, J ¼ 6.8, 2.5 Hz,
1H), 7.79 (s, 1H), 7.65 (td, J ¼ 4.5, 2.6 Hz, 1H), 7.56 (t, J ¼ 9.1 Hz, 1H),
6.79 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.34 (dd, J ¼ 17.0, 1.9 Hz, 1H), 5.85
(dd, J ¼ 10.1, 1.9 Hz, 1H), 5.54 (s, 2H), 4.39 (t, J ¼ 7.0 Hz, 2H), 1.99 (t,
J ¼ 6.7 Hz, 2H), 2.02 (ddt, J ¼ 15.6, 9.4, 4.9 Hz, 4H), 1.40 (s, 1H). 13
C
J ¼ 7.3 Hz, 2H), 1.80 (p, J ¼ 7.1 Hz, 2H), 1.44 (p, J ¼ 7.5 Hz, 2H). 13
C
NMR (101 MHz, DMSO‑d6)
d
168.5, 164.3, 160.0, 156.2, 153.1 (d,
NMR (101 MHz, DMSO‑d6) d 169.2, 164.3, 159.4, 156.4, 155.8 (d,
J ¼ 251.5 Hz), 150.8, 141.6, 131.9, 129.8 (d, J ¼ 4.0 Hz), 128.5, 128.1,
125.8 (2C), 120.9 (d, J ¼ 16.2 Hz), 117.2, 107.5, 102.0, 63.5, 49.6, 29.5,
26.4. HRMS (ESI) m/z calculated for [MþH]þ 541.1515, found
541.1511; mp 209e211 ꢀC. HPLC analysis: MeOHeH2O (0.1% TFA,
gradient elution, 0 min, 90:10, 25 min, 0:100), RT ¼ 17.43 min,
97.44% purity.
J ¼ 247.4 Hz), 150.3, 141.4, 137.7, 134.6 (d, J ¼ 4.0 Hz), 131.9, 129.7,
128.5, 127.4, 126.1, 126.0, 125.9, 119.7 (d, J ¼ 18.2 Hz), 117.8, 117.4 (d,
J ¼ 21.2 Hz), 107.7, 101.4, 63.4, 49.7, 32.0, 29.7, 22.53. HRMS (ESI) m/z
calculated for [MþH]þ 555.1671, found 555.1677; mp 264e266 ꢀC.
HPLC analysis: MeOHeH2O (0.1% TFA, gradient elution, 0 min,
90:10, 25 min, 0:100), RT ¼ 18.28 min, 92.95% purity.
4.1.11. 5-(4-(((6-Acrylamido-4-((3-chloro-2-fluorophenyl)amino)
quinazolin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-
hydroxypentanamide (11c)
4.1.15. N-(4-((3-chloro-4-((2-fluorobenzyl)oxy)phenyl)amino)-7-
((1-(3-(hydroxyamino)-3-oxopropyl)-1H-1,2,3-triazol-4-yl)
methoxy)quinazolin-6-yl)acrylamide (11g)
1H NMR (400 MHz, DMSO‑d6)
d
11.35 (s, 1H), 10.42 (s, 1H), 9.97
1H NMR (400 MHz, DMSO‑d6) d 11.37 (s, 1H), 10.62 (s, 1H), 10.02
(s,1H), 9.18 (s,1H), 8.80 (s,1H), 8.34 (s,1H), 7.67 (s,1H) 7.65-7.58 (m,
1H), 7.51 (t, J ¼ 7.3 Hz, 1H), 7.35 (t, J ¼ 8.0 Hz, 1H), 6.77 (m, 1H), 6.34
(dd, J ¼ 16.9, 1.9 Hz, 1H), 5.85 (dd, J ¼ 10.0, 1.9 Hz, 1H), 5.54 (s, 2H),
4.39 (t, J ¼ 6.9 Hz, 2H), 1.98 (t, J ¼ 7.3 Hz, 2H), 1.80 (h, J ¼ 7.5 Hz, 2H),
(s, 1H), 9.16 (s, 1H), 8.87 (s, 1H), 8.43 (m, 1H), 7.81 (d, J ¼ 2.5 Hz, 1H),
7.77e7.71 (m, 1H), 7.55 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.48 (td, J ¼ 8.0,
6.0 Hz, 1H), 7.36e7.30 (m, 3H), 7.19 (td, J ¼ 8.8, 2.6 Hz, 1H), 6.78 (dt,
J ¼ 14.5, 7.1 Hz, 1H), 6.34 (dd, J ¼ 17.0, 1.9 Hz, 1H), 5.85 (dd, J ¼ 10.1,
1.9 Hz, 1H), 5.52 (s, 2H), 5.30 (s, 2H), 4.60 (t, J ¼ 6.7 Hz, 2H), 2.65 (t,
1.46 (dd, J ¼ 9.3, 6.0 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 169.2,
164.3, 159.7, 156.0, 153.0 (d, J ¼ 251.5 Hz), 151.6, 141.8, 131.9, 129.5,
128.5, 127.9, 125.8 (d, J ¼ 4.0 Hz), 125.5, 120.9 (d, J ¼ 16.2 Hz), 117.0,
107.8, 103.1, 100.0, 63.48, 49.72, 32.0, 29.8, 22.6. HRMS (ESI) m/z
calculated for [MþH]þ 555.1671, found 555.1680; mp 218e220 ꢀC.
HPLC analysis: MeOHeH2O (0.1% TFA, gradient elution, 0 min,
90:10, 25 min, 0:100), RT ¼ 17.82 min, 94.10% purity.
J ¼ 6.6 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 166.3, 164.3, 162.7
(d, J ¼ 244.4 Hz),159.2,156.2,152.2,150.4,141.4,139.9 (d, J ¼ 8.1 Hz),
137.6, 131.9, 131.1 (d, J ¼ 9.1 Hz), 131.0, 129.5, 128.5, 127.0, 126.1,
125.2, 123.9 (d, J ¼ 3.0 Hz), 121.7, 117.9, 115.29 (d, J ¼ 21.2 Hz), 114.7,
114.5, 107.6, 101.5, 69.9, 63.3, 46.3, 33.0. HRMS (ESI) m/z calculated
for [MþH]þ 633.1777, found 633.1776; mp 195e197 ꢀC. HPLC
analysis: MeOHeH2O (0.1% TFA, gradient elution, 0 min, 90:10,
25 min, 0:100), RT ¼ 21.53 min, 95.76% purity.
4.1.12. N-(4-((3-chloro-4-fluorophenyl)amino)-7-((1-(3-
(hydroxyamino)-3-oxopropyl)-1H-1,2,3-triazol-4-yl)methoxy)
quinazolin-6-yl)acrylamide (11d)
4.1.16. 4-(4-(((6-Acrylamido-4-((3-chloro-4-((2-fluorobenzyl)oxy)
phenyl)amino)quinazolin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-
N-hydroxybutanamide (11h)
1H NMR (400 MHz, DMSO‑d6)
d 10.90 (s, 1H), 9.95 (s, 1H), 8.91 (s,
1H), 8.67 (s, 1H), 8.61 (d, J ¼ 8.7 Hz, 1H), 8.25 (s, 2H), 8.18 (d,
J ¼ 8.6 Hz,1H), 7.93e7.81 (m,1H), 7.47 (t, J ¼ 9.1 Hz,1H), 4.80 (s, 1H),
4.44 (t, J ¼ 6.9 Hz, 2H), 3.96e3.86 (m, 1H), 3.49 (d, J ¼ 11.2 Hz, 1H),
2.04e1.84 (m, 4H), 1.70e1.43 (m, 8H), 1.35e1.25 (m, 4H). 13C NMR
1H NMR (400 MHz, DMSO‑d6)
d 11.38 (s, 1H), 10.49 (s, 1H), 10.01
(s, 1H), 9.17 (s, 1H), 8.87 (s, 1H), 8.54e8.44 (m, 1H), 7.81 (d,
J ¼ 2.5 Hz, 1H), 7.78e7.71 (m, 1H), 7.55 (dd, J ¼ 8.9, 2.6 Hz, 1H), 7.48
(td, J ¼ 8.0, 6.0 Hz, 1H), 7.37e7.29 (m, 3H), 7.19 (td, J ¼ 8.6, 2.6 Hz,
1H), 6.78 (dd, J ¼ 17.0, 10.2 Hz, 1H), 6.34 (dd, J ¼ 17.0, 1.9 Hz, 1H),
5.85 (dd, J ¼ 10.1, 1.9 Hz, 1H), 5.53 (s, 2H), 5.30 (s, 2H), 4.40 (t,
J ¼ 6.6 Hz, 2H), 2.05 (dd, J ¼ 8.7, 4.5 Hz, 2H), 2.00 (q, J ¼ 6.6, 5.4 Hz,
(101 MHz, DMSO‑d6)
d 166.3, 164.3, 159.4, 156.3, 155.7 (d,
J ¼ 246.4 Hz), 150.4, 141.3, 137.9, 134.6 (d, J ¼ 3.0 Hz), 131.9, 129.60,
128.5, 127.3, 126.2, 126.0, 125.9, 119.7 (d, J ¼ 18.2 Hz), 117.9, 117.4 (d,
J ¼ 22.2 Hz), 107.7, 101.5, 63.3, 46.3, 33.0. HRMS (ESI) m/z calculated
for [MþH]þ 527.1358, found 527.1365; mp 242e244 ꢀC. HPLC
analysis: MeOHeH2O (0.1% TFA, gradient elution, 0 min, 90:10,
25 min, 0:100), RT ¼ 17.93 min, 95.97% purity.
2H). 13C NMR (101 MHz, DMSO‑d6)
d 168.5, 164.3, 162.7 (d,
J ¼ 244.4 Hz), 159.2, 156.3, 152.2, 150.4, 141.6, 139.9 (d, J ¼ 8.1 Hz),
137.5, 131.1 (d, J ¼ 8.1 Hz), 131.0, 129.5, 128.5, 127.1, 125.9, 125.3,
123.9 (d, J ¼ 3.0 Hz), 121.7, 117.9, 115.3 (d, J ¼ 21.2 Hz), 114.7, 114.5,
107.6, 101.4, 69.9, 63.4, 49.6, 29.5, 26.4. HRMS (ESI) m/z calculated
for [MþH]þ 647.1933, found 647.1929; mp 210e212 ꢀC. HPLC
analysis: MeOHeH2O (0.1% TFA, gradient elution, 0 min, 90:10,
25 min, 0:100), RT ¼ 21.63 min, 95.01% purity.
4.1.13. 4-(4-(((6-Acrylamido-4-((3-chloro-4-fluorophenyl)amino)
quinazolin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-N-
hydroxybutanamide (11e)
1H NMR (400 MHz, DMSO‑d6)
d 11.39 (s, 1H), 10.48 (s, 1H), 10.01
(s, 1H), 9.18 (s, 1H), 8.91 (s, 1H), 8.49 (s, 1H), 7.97-7.95 (m, 4H), 7.76
(s, 1H), 7.68-7.64 (m, 1H), 7.55 (t, J ¼ 9.0 Hz, 1H), 6.82-6.75(m, 1H),
6.34 (dd, J ¼ 1.8, 17.0 Hz, 1H), 5.85 (dd, J ¼ 1.8, 17.0 Hz, 1H), 4.40 (t,
J ¼ 6.6 Hz, 2H), 2.07-1.99 (m, 4H). 13C NMR (101 MHz, DMSO‑d6)
4.1.17. 5-(4-(((6-Acrylamido-4-((3-chloro-4-((2-fluorobenzyl)oxy)
phenyl)amino)quinazolin-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)-
N-hydroxypentanamide (11i)
d
168.5, 164.3, 159.3, 156.3, 155.7 (d, J ¼ 246.4 Hz), 150.7, 141.6, 134.7
1H NMR (400 MHz, DMSO‑d6)
d 11.29 (s, 1H), 10.44 (s, 1H), 10.00
(d, J ¼ 3.0 Hz), 131.9, 129.6, 128.5, 127.2, 125.9, 125.8, 119.7 (d,
(s, 1H), 9.16 (s,1H), 8.87 (s, 1H), 8.52-8.42 (m, 1H), 7.82 (d, J ¼ 2.5 Hz,
J ¼ 19.2 Hz), 117.7, 117.4 (d, J ¼ 22.2 Hz), 107.8, 102.0, 63.5, 49.6, 29.5,
1H), 7.77-7.68 (m, 1H), 7.60e7.43 (m, 2H), 7.32 (dd, J ¼ 7.6, 4.2 Hz,
10

L. Zhao, T. Fan, Z. Shi et al.
European Journal of Medicinal Chemistry 213 (2021) 113173
3H), 7.20 (td, J ¼ 8.6, 2.6 Hz, 1H), 6.77 (dd, J ¼ 16.9, 10.2 Hz, 1H), 6.33
(dd, J ¼ 17.0, 1.9 Hz, 1H), 5.85 (dd, J ¼ 10.1, 1.9 Hz, 1H), 5.53 (s, 2H),
5.30 (s, 2H), 4.39 (t, J ¼ 6.9 Hz, 2H), 1.98 (t, J ¼ 7.3 Hz, 2H), 1.86e1.74
(m, 1H), 1.80 (s, 1H), 1.45 (p, J ¼ 7.4 Hz, 2H). 13C NMR (101 MHz,
4.6. Annexin V-FITC apoptosis detection
NCIeH1975 cells were treated with 11e for 48 h. Then, the cell
apoptosis assay was measured by Annexin V-FITC/PI Apoptosis
Detection kit (Beyotime, China) according to the manufacturer’s
instructions in Flow cytometry of CytoFLEX (Beckman, America).
DMSO‑d6)
d
169.2, 164.2, 162.7 (d, J ¼ 244.4 Hz), 159.1, 156.2, 152.1,
141.6, 139.9 (d, J ¼ 8.1 Hz), 131.9, 131.2, 131.1, 129.4, 128.5, 126.9,
125.8, 125.1, 123.9 (d, J ¼ 3.0 Hz), 121.7, 117.9, 115.3 (d, J ¼ 21.2 Hz),
114.7, 114.5, 107.8, 101.9, 69.9, 63.4, 53.9, 29.7, 18.5, 17.2. HRMS (ESI)
m/z calculated for [MþH]þ 661.2090, found 661.2100; mp
222e224 ꢀC. HPLC analysis: MeOHeH2O (0.1% TFA, gradient
elution, 0 min, 90:10, 25 min, 0:100), RT ¼ 21.65 min, 93.03% purity.
4.7. Colony formation assay
The colony formation assays were performed as described[47].
NCIeH1975 cells (1 ꢁ 104/well) were seeded into 6-well plates and
cultured for 12 h. Then, NCIeH1975 cells were treated with 11e for
7 days and the colony formed by NCIeH1975 cells were fixed with
10% (wt/vol) buffered formalin for 1 h and then stained with crystal
violet for 1 h.
4.2. EGFR/EGFRL858R/EGFRL858R/T790M/HER2 kinase assays
In vitro kinase assays were carried out by Sundia Meditech
(Shanghai) Co. Ltd. using mobility shift assay. The general proced-
ures can be viewed in supporting information.
4.8. Transwell cell invasion assay
NCIeH1975 cells (1 ꢁ 104/well) were seeded with 0.1 mL serum-
free culture medium,in an upper chamber of a 24-well insert (BD
Falcon, 353097) that was put in a 24-well plate with 1 mL 10% FBS
culture medium added 11e at the indicated dilutions in the lower
chamber for 72 h. Subsequently, the noninvasive cells on the upper
chamber were removed with cotton swabs. The invasive cells on
the lower sides of the membranes were then fixed with 10% (wt/
vol) buffered formalin for 1 h, washed with PBS twice and stained
with crystal violet for 1 h. The lower sides of the membranes were
photographed randomly in three fields of views.
4.3. HDAC inhibition assays
In vitro HDAC assays were carried out by Sundia Meditech
(Shanghai) Co. Ltd. and the general procedures can be viewed in
supporting information.
4.4. Cytotoxicity assays
The HL-7702, A549, A431, SK-BR-3, NCIeH838 and
NCIeH1975 cell lines were kindly provided by Cell Banks, Chinese
Academy of Sciences, FHC cell line was purchased from BNBio Tech
Co., Ltd (Beijing, China), NCIeH820, Ba/F3_EGFR_Del19-T790M-
C797S cell line was purchased from CoBioer Biosciences CO., LTD
(Nanjing, China), which were cultured according to the in-
structions. The cytotoxic activity of all compounds was evaluated
with these cell lines by the standard MTT assay in vitro. Approxi-
mately 0.5e1 ꢁ 104 cells were suspended in the corresponding
culture mediums, moved to a 96-well plate and incubated in 5% CO2
at 37 ꢀC for 12 h. A series of compounds in different concentrations
were added to each well and continued for 72 h. After that, a ter-
minal concentration of 0.5 mg/mL MTT was added and waited for
4.9. Docking studies
Three-dimensional structures of EGFRT790M (PDB code: 4G5P)
and HDAC2 (PDB code: 4LXZ) were downloaded from RSCB Protein
Data Bank (HDAC2 not HDAC1 is due to the crystal structure of
HDAC2 with vorinostat has been parsed and HDAC2 have identical
active sites around the entrance of the channel with HDAC1). The
docking studies were achieved using SURFLEX-DOCK module of
SYBYL-X1.3 and the analysis of covalent binding were performed by
Steric-Clashes Alleviating Receptor (SCAR) Strategy [48].
4 h. The formazan crystals were dissolved in 100 mL DMSO after the
Declaration of competing interest
mediums were removed. These assays were performed in three
wells per condition and three independent experiments were
performed. The absorbance was measured by a microplate reader
(Tecan infinite M1000 Pro) at 490 nm.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4.5. Western blot assays
Acknowledgements
NCIeH975 and A431 cells were treated with 11e for 48 h, then,
cells were collected and dissolved by RIPA lysate buffer with pro-
tease and phosphatase inhibitor (Roche, Switzerland) in ice for
30 min, which were centrifugated at 14000 g for 5 min at 4 ꢀC to get
cell lysis. The concentrations of proteins were determined by BCA
Protein Assay Kit (Tiangen, China). The protein samples were mixed
with 5 ꢁ loading buffer, which was boiled at 100 ꢀC for 10 min and
stored at ꢂ20 ꢀC. The protein samples were separated using 10%e
12% SDS-PAGE and transferred from gel to PVDF membranes (Mil-
lipore, China). The membranes were blocked in QuickBlock™
Blocking Buffer for Western blot (Beyotime, China) for 2 h at room
temperature and incubated in primary antibody (CST, America)
dilution overnight at 4 ꢀC. After washing in TBST buffer for 3 times
(5 min per time), membranes were incubated in secondary anti-
bodies for 2 h at room temperature and washed with TBST buffer
again for 3 times. Then, the membranes were imaged in Chem-
iluminescence Gel Imaging System (Tanon-5200, China).
We appreciate the financial supports from Shenzhen Develop-
ment and Reform Committee (No. 20151961 and No. 2019156),
Department of Science and Technology of Guangdong Province (No.
2017B030314083) and Shenzhen Bay Laboratory Open Funding
(SZBL2019062801009).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113173.
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