Novel hydrazone moiety-bearing aminopyrimidines as selective inhibitors of epidermal growth factor receptor T790M mutant

Article abstract of DOI:10.1016/j.ejmech.2015.09.031

The epidermal growth factor receptor (EGFR) T790M mutant is found in approximately 50% of clinically acquired resistance to gefitinib among patients with non-small cell lung cancer (NSCLC). Here, a series of novel aminopyrimidines bearing a hydrazone moiety were identified as potent and selective EGFR inhibitors. Compounds 14a, 15g, and 15i potently inhibited all EGFR mutants including EGFR T790M/L858R, EGFR T790M/delE746-A750, and EGFR T790M while they showed weak effects on the wild type (WT) EGFR. In addition, these compounds effectively suppressed proliferation of gefitinib-resistant H1975 (EGFR T790M/L858R) cells but were less potent against A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing a high safety index. Therefore, 14a, 15g, and 15i might be promising candidates to overcome drug resistance mediated by the EGFR T790M mutant.

Full text of DOI:10.1016/j.ejmech.2015.09.031

Accepted Manuscript
Novel hydrazone moiety-bearing aminopyrimidines as selective inhibitors of epidermal
growth factor receptor T790M mutant
Mingze Qin, Tingting Wang, Boxuan Xu, Zonghui Ma, Nan Jiang, Hongbo Xie, Ping
Gong, Yanfang Zhao
PII:
S0223-5234(15)30275-0
10.1016/j.ejmech.2015.09.031
EJMECH 8128
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2015
Revised Date: 17 September 2015
Accepted Date: 24 September 2015
Please cite this article as: M. Qin, T. Wang, B. Xu, Z. Ma, N. Jiang, H. Xie, P. Gong, Y. Zhao, Novel
hydrazone moiety-bearing aminopyrimidines as selective inhibitors of epidermal growth factor receptor
T790M mutant, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.09.031.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel hydrazone moiety-bearing aminopyrimidines as selective
inhibitors of epidermal growth factor receptor T790M mutant
Mingze Qina, Tingting Wanga, Boxuan Xua, Zonghui Maa, Nan Jianga, Hongbo Xieb, Ping
Gonga,* and Yanfang Zhaoa,*
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical
University), Ministry of Education, 103 Wenhua Road, Shenyang 110016, PR China
bCollege of Bioinformatics Science and Technology, Harbin Medical University, Harbin 150086, PR
China
Corresponding author.
Tel: +86 24 2398 6429; Fax: +86 24 2398 6429;
E-mail address: yanfangzhao@126.com gongpinggp@126.com
;
Abstract
The epidermal growth factor receptor (EGFR) T790M mutant is found in
approximately 50% of clinically acquired resistance to gefitinib among patients with
non-small cell lung cancer (NSCLC). Here, a series of novel aminopyrimidines bearing a
hydrazone moiety were identified as potent and selective EGFR inhibitors. Compounds
14a, 15g, and 15i potently inhibited all EGFR mutants including EGFR T790M/L858R,
EGFR T790M/delE746_A750, and EGFR T790M while they showed weak effects on the
wild type (WT) EGFR. In addition, these compounds effectively suppressed proliferation
of gefitinib-resistant H1975 (EGFR T790M/L858R) cells but were less potent against
A549 (WT EGFR and k-Ras mutation) and HT-29 (non-special gene type) cells, showing
a high safety index. Therefore, 14a, 15g, and 15i might be promising candidates to
overcome drug resistance mediated by the EGFR T790M mutant.
Keywords: Aminopyrimidines; Synthesis; Antitumor activity; EGFR T790M mutant

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1. Introduction
The epidermal growth factor receptor (EGFR) belongs to the ErbB family of
receptor tyrosine kinases and plays a crucial role in cell proliferation, survival, and
differentiation via activation of downstream signaling pathways [1]. Overexpression or
mutation of EGFR is associated with poor prognosis in a number of cancers such as lung
and anal cancer [2,3]. Therefore, EGFR has become an attractive target for anticancer
therapeutic intervention.
Gefitinib (Fig. 1) and erlotinib are potent reversible inhibitors of EGFR kinase and
have yielded significant clinical benefits in patients with non-small cell lung cancer
(NSCLC). In particular, they are more effective in patients harboring the EGFR-activating
mutations such as delE746_A750 and L858R, which account for 90% of EGFR mutation
in NSCLC [4–6]. However, most patients develop drug resistance after 10–14 months of
gefitinib and erlotinib therapies. In approximately 50% of all resistant cases, a
substitution of threonine 790 with methionine (T790M mutation) at the “gatekeeper”
position of EGFR is detected, in combination with an activating mutation [7]. The T790M
mutation restores the affinity of EGFR for adenosine triphosphate (ATP), which reduces
the binding of ATP-competitive inhibitors to the kinase [8].
In order to overcome the resistance mechanism correlated with T790M mutation,
second-generation irreversible inhibitors have been developed including afatinib,
neratinib, and dacomitinib [9–11]. These compounds possess the same anilinoquinazoline
template as gefitinib but contain an acrylamide functionality that can undergo a Michael
addition reaction with Cys797 to achieve greater efficacy [12,13]. Unfortunately, their
poor kinase selectivity against EGFR T790M over the wild-type (WT) EGFR leads to
serious dose-limiting toxicity [14]. Moreover, these inhibitors have been proven
inefficient in gefitinib-resistant H1975 cell models [9,15].
In recent years, numerous irreversible EGFR inhibitors designed on the basis of an
aminopyrimidine scaffold have been introduced. WZ4002 (3), the representative
compound, exhibits a significantly improved kinase selectivity against EGFR T790M,
compared to quinazoline-based inhibitors, and shows potent activity in H1975 NSCLC
models [16]. A modified compound, CO-1686 has just advanced to phase II clinical trials,
and the results are eagerly awaited [17]. All the investigations suggest that the
aminopyrimidines may be a potential scaffold for identifying inhibitors that may
overcome acquired drug resistance mediated by the EGFR T790M mutant.
(Insert Fig. 1 here)
The purpose of our study was to identify novel selective inhibitors targeting the
EGFR T790M mutant based on the aminopyrimidine pharmacophore. All the irreversible
aminopyrimidine compounds reported so far were designed with a modified aryl moiety
linked directly to the amino group, and the aniline fragment which pointed to the outside
of the active pocket, was correlated with the physicochemical properties of the
compounds [16,18,19]. In this study, we reported the design, synthesis, and biological
evaluation of a series of novel aminopyrimidine derivatives bearing a hydrazone moiety
as selective EGFR inhibitors (Fig. 2), which demonstrated potent and selective inhibition

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of EGFR expressing T790M mutations including EGFR T790M/L858R, EGFR
T790M/delE746_A750, and EGFR T790M. In addition, these compounds potently
suppressed the proliferation of gefitinib-resistant H1975 cells harboring the EGFR
T790M/L858R.
(Insert Fig. 2 here)
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds (14a–14j and 15a–15o) containing a
hydrazone moiety is shown in Scheme 1. The intermediate 6 was prepared via a
regioselective coupling of the protected benzene-1,3-diamine 5 to the 4-position of 4
using a modified condition as previously reported [20]. Substitution of 6 with hydrazine
hydrate led to the intermediate 7, which was reacted with an appropriate aromatic
aldehyde or ketone under acid conditions to generate hydrazone 8 [21,22]. The key
intermediate 9 was obtained by the deprotection of 8 in the presence of trifluoroacetic
acid [20]. Acylation of 9 with acryloyl chloride conveniently produced compounds
14a–14j. On the other hand, condensation of
9
with commercial
(E)-4-(dimethylamino)but-2-enoic acid using HATU afforded compounds 15a–15o with
good yields.
(Insert Scheme 1 here)
The general steps for preparing the hydrazide analogs 16a and 16b are outlined in
Scheme 2. Intermediate 11 was obtained from intermediate 6 via a two-step reaction
using similar conditions as described for 9 and 14. Hydrazinolysis of the ester 12 yielded
the corresponding intermediate 13, which was reacted with 11 via an SN2 mechanism to
generate the target compounds.
(Insert Scheme 2 here)
The chemical structures of the target compounds were confirmed using infrared (IR),
nuclear magnetic resonance (NMR), and mass spectrometry (MS). Especially, the
configuration of the imino double bonds in compounds 14a–14j and 15a–15o were
determined using nuclear Overhauser spectroscopy (NOESY). Using compound 14b as
an example, a clear NOESY signal was observed between the proton of –NH–N= (δ 13.10,
singlet) and the proton of –N=CH– (δ 8.54, singlet), which only existed in the
E-configuration of the compound (supplementary data). The other related compounds
were assigned the same E-configuration by analogy.
2.2. Design strategy of target compounds
Taking compound 3 as the lead, we developed a series of novel, irreversible
inhibitors, which potently and selectively inhibited EGFR T790M. As part of the design
strategy, a modified aryl group was introduced to the aminopyrimidine core with a more

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flexible linker. We hypothesized that improved flexibility of the molecular skeleton might
be beneficial to the water-solubility of compounds, despite the removal of a hydrophilic
group (Fig. 2). Moreover, additional hydrogen bond donors and acceptors were expected
to contribute to the interaction with EGFR. Hydrazone and hydrazide moieties have been
widely applied in drug design, and as building blocks they could be found in several
therapeutic agents, such as PAC-1 and Isoniazid [24,25]. Accordingly, our process started
with compounds 14a, 16a, and 16b, which possessed a hydrazone or hydrazide group.
They were screened in two NSCLC cell lines harboring different forms of EGFR
including the A549 (WT EGFR and k-Ras mutation) and gefitinib-resistant H1975
(EGFR T790M/L858R). Interestingly, the hydrazone analog (14a) showed a favorable
activity on H1975 cells, with a half-maximal inhibitory concentration (IC50) of 0.43 ꢀM,
while the hydrazide compounds (16a and 16b) were less potent. Even more noteworthy
was that compound 14a exhibited much better inhibition of H1975 than A549 cells,
indicating good cellular selectivity (Table 1). Therefore, detailed modifications were
focused on the hydrazone analogs and a series of novel compounds (14b–14j) was
prepared by modifying the chemical structure of 14a. In addition, to further improve the
water-solubility of these compounds, a dimethylamino group was introduced, which
generated compounds 15a–15o.
(Insert Table 1 here)
2.3. Biological evaluation
2.3.1. Inhibitory activity against A549, H1975, and HT-29 cells
The antitumor activities of the target compounds were evaluated in A549 NSCLC
(WT EGFR and k-Ras mutation) and gefitinib-resistant H1975 NSCLC (EGFR
T790M/L858R) cells. In addition, these compounds were screened in HT-29 colon cancer
cells, which expressed a non-special gene type, to test their toxic effects.
(Insert Table 2. here)
As shown in Table 2, results of a biological analysis using a standard MTT assay
showed that the inhibitory activity of most compounds (14a–14i, 15a–15d, and 15g–15m)
was much more potent (IC50, 0.077–1.58 ꢀM) than that of gefitinib (positive control; IC50,
8.71 ꢀM) in inhibition of H1975 cells. However, they were less potent than WZ4002
(positive control; IC50, 0.058 ꢀM). In addition, the target compounds suppressed H1975
cells more effectively than they suppressed A549 cells, which suggests that the observed
potent efficacy might be attributable to selective inhibition of the mutant form of EGFR.
Further, no significant toxicity was shown against HT-29 cells by all the compounds at
concentrations ranging from 2.12–25.19 ꢀM, except for compound 15m, which
demonstrates that the target compounds might possess a high safety index.
Regarding compounds 14a, 14c, 14e, and 14f the introduction of a methyl group in
the hydrazone moiety (R1) induced a weak variation in inhibition of H1975 cells, which
indicates that the structure-activity relationship (SAR) in this region was limited. In
contrast, the substitution pattern on the terminal aryl moiety was closely related to

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potency. Compounds 14e and 14f, which contained an electron-withdrawing group on the
benzene ring, strongly inhibited the H1975 cells (IC50, 0.39 and 0.30 ꢀM, respectively).
However, decreased activity was observed for compounds 14g–14i (IC50, 0.74–1.19 ꢀM)
bearing an electron-donating group in the same region. Similar results were also observed
when 15b (IC50, 0.59 ꢀM) was compared with 15d (IC50, 1.58 ꢀM). Shifting the F atom
from the para position (14a IC50, 0.43 ꢀM) to the ortho position (14b IC50, 0.29 ꢀM)
slightly improved inhibition of H1975 cells, while a significant enhancement of inhibition
of A549 and HT-29 cells was also observed. The replacement of the terminal phenyl
group with a pyridinyl group obviously decreased the potency, as shown with compounds
14j, 15e, 15n, and 15o (IC50, 2.35–6.24 ꢀM). A dramatically adverse result was observed
with compound 15m, which exhibited outstanding potency against H1975 cells with an
IC50 of 0.077 ꢀM. However, it also showed strong toxicity against HT-29 cells (IC50, 0.12
ꢀM), illustrating an off-target effect, which might limit its further development.
Compound 15f with an indol group showed a sharply reduced efficacy against H1975
cells, with an IC50 of 5.45 ꢀM. Interestingly, the inhibitory activity was enhanced by
21-fold when an additional benzyl group was introduced on the N-position of the indol
fragment (compound 15g IC50, 0.26 ꢀM). Compound 9a (IC50, 9.92 ꢀM) exhibited a
significant loss of potency against H1975 cells compared to 14a (IC50, 0.43 ꢀM), which
demonstrated that the acrylamide fragment was essential for high activity. With an
additional dimethylamino group in the acrylamide moiety, compounds (15a–15d and
15g–15m) also possessed a high level of inhibitory activity, showing an attractive strategy
for optimizing the physicochemical property of these compounds, if necessary.
2.3.2. Inhibitory activity against EGFR kinase
To rationalize their appealing cellular inhibition, the optimized compounds (14a–14c,
14e–14g, 15a–15c, 15f–15i, 15m, and 15n) were evaluated using EGFR kinases with
different status including EGFR T790M/L858R, EGFR T790M/delE746_A750, EGFR
T790M, and WT EGFR. The results obtained using a well-established mobility shift assay
are summarized in Table 3, with gefitinib and WZ4002 as the positive controls.
(Insert Table 3 here)
Most compounds (14a, 14b, 14e, 14f, 15a–15c, and 15g–15m) significantly
inhibited EGFR T790M/L858R with IC50 values ranging from 0.68–2.52 ꢀM, which were
5.0- to 18.7-fold lower than that of gefitinib (IC50, 12.72 ꢀM). The inhibitory activities of
compounds 14a, 15a, 15b, 15g, and 15i were particularly more pronounced with IC50
values less than 1 ꢀM. As shown, the IC50 values of the compounds against EGFR
T790M/L858R were higher than that of WZ4002, but an obviously improved selectivity
was observed. All the tested compounds showed low-level inhibition of WT EGFR (IC50,
10.65–> 20 ꢀM) and among them, compounds 14a, 15a, 15g, and 15i were up to 20-fold
more selective against EGFR T790M/L858R than they were against WT EGFR. The
biological data also illustrated that the compounds potently inhibited EGFR
T790M/delE746_A750 and EGFR T790M with IC50 values that were lower than gefitinib.
Encouragingly, compounds 14a, 15g, and 15i efficiently suppressed all three kinases

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expressing the T790M mutation with IC50 values of 0.57–1.12, 0.76–0.88, and 0.72–1.25
ꢀM, respectively. In addition, it was noteworthy that compounds 14a, 15g, and 15i
possessed distinct substructures in their skeletons, highlighting a potential for further
investigation of this series. On the other hand, compounds 14g, 15f, and 15n exhibited
disappointing activity against EGFR T790M/L858R (IC50, 4.32–>20 ꢀM), which was
consistent with their weak inhibition of H1975 cells. In contrast to its excellent inhibition
of H1975 (IC50, 0.077 ꢀM), compound 15m only exhibited moderate activity against
EGFR T790M/L858R (IC50, 2.38 ꢀM), which suggested that other underlying
mechanisms might be involved [23].
3. Conclusion
In summary, an interesting series of aminopyrimidine derivatives bearing a
hydrazone moiety have been identified as potent and selective inhibitors targeting EGFR
harboring T790M mutants. Several of the compounds displayed more potent activity
against EGFR mutants (EGFR T790M/L858R, EGFR T790M/delE746_A750, and EGFR
T790M) than shown by gefitinib, and weak effects against WT EGFR. Compounds 14a,
15g, and 15i potently inhibited all three mutant forms of EGFR with IC50 values in the
low micromolar range. These compounds also strongly inhibited gefitinib-resistant H1975
cells expressing EGFR T790M/L858R. Importantly, they showed sharply decreased
activity against A549 cells and minimal toxicity against HT-29 cells, which indicated that
they might possess a favorable safety index. Our study provided a new strategy for the
discovery of selective EGFR inhibitors to overcome clinical drug resistance mediated by
T790M mutation. Compounds 14a, 15g, and 15i exhibited the most impressive activity in
the biological evaluation and have emerged as promising candidates for further
development. Extensive investigations of the pharmacokinetics and in vivo activity of 14a,
15g, and 15i are in progress, and the results will be reported in due course.
4. Experimental section
4.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without
further purification. The purity of the synthesized compounds was measured by high
performance liquid chromatography (HPLC, Agilent, USA). Flash chromatography was
performed using silica gel (300–400 mesh). All reactions were monitored by TLC on
silica gel plates. Melting points were determined on a Büchi Melting Point B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland). The 1H (1D and 2D) and 13C NMR
were recorded on Bruker ARX-400 or ARX-600 spectrometer (Bruker Bioscience,
Billerica, MA, USA) with TMS as an internal standard. Mass spectra (MS) were
measured in ESI mode on an Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA).
Elemental analysis was determined on a Carlo-Erba 1106 Elemental analysis instrument
(Carlo Erba, Milan, Italy). The IR spectra were recorded by means of the KBr pellet
technique on a Bruker FTS 135 spectrometer.
4.1.1. Tert-butyl 3-(2,5-dichloropyrimidin-4-ylamino)phenylcarbamate (6)

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To a solution of intermediate 5 (19.98g, 0.096 mol) in DMF (200 mL) were added
K2CO3 (19.90g, 0.144 mol) and compound 4 (17.46g, 0.096 mol). The reaction was
stirred at 50 °C for 3 h. After being cool to room temperature, the reaction mixture was
poured into water (1000 mL). The precipitate was filtered and dried to give a crude solid.
Intermediate 6 was generated as a pale-yellow solid (24.76g, 72%) using column
chromatography with petroleum ether and ethyl acetate (20:1 to 9:1, v/v) as the mobile
1
phase. mp 190.8–192.1 °C. H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H, NHCO), 9.44
(s, 1H, NH), 8.36 (s, 1H, ArH), 7.70 (s, 1H, ArH), 7.21–7.28 (m, 2H, ArH), 7.17 (dt, J =
7.1, 2.0 Hz, 1H, ArH), 1.48 (s, 9H, CH3). ESI-MS m/z: 355.1 [M+H]+.
4.1.2. Tert-butyl 3-(5-chloro-2-hydrazinylpyrimidin-4-ylamino)phenylcarbamate (7)
To a solution of 6 (5g, 0.014 mol) in MeCN (100 mL) was added 80% NH2NH2·H2O
(2.5 mL). The mixture was heated to reflux and stirred for 3 h, then being cooled to room
temperature. The precipitate was filtered and dried to give 7 as a gray solid (4.2g, 86%).
mp 165.0–166.3 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.30 (s, 1H, –NH–N), 8.55 (s, 1H,
NHCO), 8.30 (s, 1H, NH), 7.99 (s, 1H, ArH), 7.92 (s, 1H, ArH), 7.20–7.23 (m, 1H, ArH),
7.14 (d, J = 5.8 Hz, 2H, ArH), 4.23 (s, 2H, –NH–NH2), 1.48 (s, 9H, CH3). ESI-MS m/z:
351.0 [M+H]+.
4.1.3. General procedure for preparation of compounds (8a–8o)
A mixture of 7 (0.8g, 2.3 mmol), appropriate aromatic aldehyde or ketone (2.5mmol)
and a catalytic amount of glacial acetic acid was refluxed in i-PrOH for 6–8 h when TLC
showed the completion of the reaction. After being cooled to room temperature, the
precipitate was filtered, washed with Et2O and dried to afford compounds 8a–8o,
respectively, which were used without further purification.
4.1.3.1. (E)-Tert-butyl 3-(5-chloro-2-(2-(4-fluorobenzylidene)hydrazinyl)pyrimidin-4-yla
1
mino)phenylcarbamate (8a). A yellow solid. Yield: 71%. mp 188.3–190.0 °C. H NMR
(400 MHz, DMSO-d6) δ 11.41 (s, 1H, –NH–N), 9.35 (s, 1H, NHCO), 9.15 (s, 1H, NH),
8.16 (s, 1H, –N=CH), 8.09 (s, 1H, ArH), 7.83 (s, 1H, ArH), 7.75 (dd, J = 7.8, 6.0 Hz, 2H,
ArH), 7.51 (s, 1H, ArH), 7.22–7.28 (m, 3H, ArH), 7.18 (t, J = 8.0 Hz, 1H, ArH), 1.42 (s,
9H, CH3). ESI-MS m/z: 457.1 [M+H]+.
4.1.3.2. (E)-Tert-butyl 3-(5-chloro-2-(2-(2-fluorobenzylidene)hydrazinyl)pyrimidin-4-yla
1
mino)phenylcarbamate (8b). A yellow solid. Yield: 43%. mp 190.4–193.0 °C. H NMR
(400 MHz, DMSO-d6) δ 12.31 (s, 1H, –NH–N), 9.92 (s, 1H, NHCO), 9.48 (s, 1H, NH),
8.41 (s, 1H, ArH), 8.29 (s, 1H, –N=CH), 8.22 (s, 1H, ArH), 7.77 (s, 1H, ArH), 7.49 (dd, J
= 14.0, 6.4 Hz, 1H, ArH), 7.26–7.38 (m, 5H, ArH), 1.47 (s, 9H, CH3). ESI-MS m/z: 457.0
[M+H]+.
4.1.3.3. (E)-Tert-butyl 3-(5-chloro-2-(2-(2,4-difluorobenzylidene)hydrazinyl)pyrimidin-4-
ylamino)phenylcarbamate (8c). A wheat solid. Yield: 52%. mp 211.9–214.1 °C. 1H NMR
(400 MHz, DMSO-d6) δ 12.60 (s, 1H, –NH–N), 10.19 (s, 1H, NHCO), 9.51 (s, 1H, NH),
8.33–8.37 (m, 3H, ArH, –N=CH), 7.75 (s, 1H, ArH), 7.34–7.40 (m, 1H, ArH), 7.32 (d, J =
4.9 Hz, 2H, ArH), 7.26 (td, J = 8.6, 2.1 Hz, 2H, ArH), 1.47 (s, 9H, CH3). ESI-MS m/z:

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475.1 [M+H]+.
4.1.3.4. (E)-Tert-butyl 3-(5-chloro-2-(2-(4-methoxybenzylidene)hydrazinyl)pyrimidin-4-yl
1
amino)phenylcarbamate (8d). A pale-yellow solid. Yield: 60%. mp 166.6–169.4 °C. H
NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H, –NH–N), 9.48 (s, 1H, NHCO), 8.27 (s, 1H,
NH), 8.13 (s, 1H, –N=CH), 7.78–7.81 (m, 3H, ArH), 7.69 (s, 1H, ArH), 7.30 (d, J = 4.5
Hz, 2H, ArH), 7.02 (m, 3H, ArH), 3.80 (s, 3H, OCH3), 1.46 (s, 9H, CH3). ESI-MS m/z:
469.1 [M+H]+.
4.1.3.5. (E)-Tert-butyl 3-(5-chloro-2-(2-(pyridin-4-ylmethylene)hydrazinyl)pyrimidin-4-yl
1
amino)phenylcarbamate (8e). A pale-yellow solid. Yield: 67%. mp 211.9–213.4 °C. H
NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H, –NH–N), 9.37 (s, 1H, NHCO), 9.02 (s, 1H,
NH), 8.62 (d, J = 6.0 Hz, 2H, ArH), 8.20 (s, 1H, –N=CH), 8.07 (s, 1H, ArH), 7.90 (s, 1H,
ArH), 7.65 (d, J = 6.1 Hz, 2H, ArH), 7.59 (d, J = 7.3 Hz, 1H, ArH), 7.27 (t, J = 8.1 Hz,
1H, ArH), 7.17 (d, J = 7.9 Hz, 1H, ArH), 1.42 (s, 9H, CH3). ESI-MS m/z: 440.0 [M+H]+.
4.1.3.6. (E)-Tert-butyl 3-(2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-5-chloropyrimidin-4
-ylamino)phenylcarbamate (8f). A yellow solid. Yield: 45%. ESI-MS m/z: 478.1 [M+H]+.
4.1.3.7. (E)-Tert-butyl 3-(2-(2-((1-benzyl-1H-indol-3-yl)methylene)hydrazinyl)-5-chloropy
rimidin-4-ylamino)phenylcarbamate (8g). A yellow solid. Yield: 62%. ESI-MS m/z:
568.2 [M+H]+.
4.1.3.8. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-(4-fluorophenyl)ethylidene)hydrazinyl)pyrimidi
n-4-ylamino)phenylcarbamate (8h). A pale-yellow solid. Yield: 72%. mp 172.6–174.7 °C.
1H NMR (400 MHz, DMSO-d6) δ 11.53 (s, 1H, –NH–N), 10.13 (s, 1H, NHCO), 9.54 (s,
1H, NH), 8.29 (s, 1H, ArH), 8.09 (dd, J = 7.8, 5.9 Hz, 2H, ArH), 7.84 (s, 1H, ArH),
7.26–7.33 (m, 5H, ArH), 2.40 (s, 3H, CH3), 1.47 (s, 9H, CH3). ESI-MS m/z: 471.1
[M+H]+.
4.1.3.9. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-(2,4-difluorophenyl)ethylidene)hydrazinyl)pyri
midin-4-ylamino)phenylcarbamate (8i). A pale-yellow solid. Yield: 69%. mp
1
163.4–166.1 °C. H NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H, –NH–N), 10.10 (s, 1H,
NHCO), 9.56 (s, 1H, NH), 8.26 (s, 1H, ArH), 7.97 (dd, J = 15.9, 8.7 Hz, 1H, ArH), 7.88
(s, 1H, ArH), 7.28–7.39 (m, 4H, ArH), 7.20 (td, J = 8.5, 2.4 Hz, 1H, ArH), 2.39 (s, 3H,
CH3), 1.47 (s, 9H, CH3). ESI-MS m/z: 489.1 [M+H]+.
4.1.3.10. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-p-tolylethylidene)hydrazinyl)pyrimidin-4-yla
1
mino)phenylcarbamate (8j). A pale-yellow solid. Yield: 53%. mp 187.9–189.9 °C. H
NMR (400 MHz, DMSO-d6) δ 11.44 (s, 1H, –NH–N), 10.15 (s, 1H, NHCO), 9.51 (s, 1H,
NH), 8.26 (s, 1H, ArH), 7.91 (d, J = 8.0 Hz, 2H, ArH), 7.78 (s, 1H, ArH), 7.32–7.34 (m,
3H, ArH), 7.27 (d, J = 8.2 Hz, 2H, ArH), 2.36 (s, 3H, CH3), 2.35 (s, 3H, CH3), 1.48 (s, 9H,
CH3). ESI-MS m/z: 467.2 [M+H]+.
4.1.3.11. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)pyri
midin-4-ylamino)phenylcarbamate (8k). A pale-yellow solid. Yield: 61%. mp

ACCEPTED MANUSCRIPT
1
179.2–182.0 °C. H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, –NH–N), 9.36 (s, 1H,
NHCO), 9.10 (s, 1H, NH), 8.17 (s, 1H, ArH), 7.89 (s, 1H, ArH), 7.83 (d, J = 8.2 Hz, 2H,
ArH), 7.72 (s, 1H, ArH), 7.25 (t, J = 8.1 Hz, 1H, ArH), 7.16 (d, J = 7.9 Hz, 1H, ArH),
6.97 (d, J = 8.8 Hz, 2H, ArH), 3.81 (s, 3H, OCH3), 2.27(s, 3H, CH3), 1.45 (s, 9H, CH3).
ESI-MS m/z: 483.3 [M+H]+.
4.1.3.12. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-(3-methoxyphenyl)ethylidene)hydrazinyl)pyri
1
midin-4-ylamino)phenylcarbamate (8l). A pale-yellow solid. Yield: 58%. H NMR (400
MHz, DMSO-d6) δ 11.56 (s, 1H, –NH–N), 10.16 (s, 1H, NHCO), 9.55 (s, 1H, NH), 8.27
(s, 1H, ArH), 7.84 (s, 1H, ArH), 7.54–7.57 (m, 2H, ArH), 7.30–7.39 (m, 4H, ArH), 7.04
(dd, J = 8.2, 2.1 Hz, 1H, ArH), 3.83 (s, 3H, OCH3), 2.40 (s, 3H, CH3), 1.48 (s, 9H, CH3).
ESI-MS m/z: 483.2 [M+H]+.
4.1.3.13. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)pyrimidin
1
-4-ylamino)phenylcarbamate (8m). A yellow solid. Yield: 60%. mp 195.9–196.6 °C. H
NMR (400 MHz, DMSO-d6) δ 10.92 (s, 1H, –NH–N), 9.54 (s, 1H, NHCO), 9.47 (s, 1H,
NH), 8.59 (d, J = 4.4 Hz, 1H, ArH), 8.36 (d, J = 7.7 Hz, 1H, ArH), 8.27 (s, 1H, ArH), 7.94
(s, 1H, ArH), 7.85–7.90 (m, 1H, ArH), 7.55 (d, J = 5.6 Hz, 1H, ArH), 7.41 (dd, J = 6.6,
5.5 Hz, 1H, ArH), 7.24–7.30 (m, 2H, ArH), 2.43 (s, 3H, CH3), 1.45 (s, 9H, CH3). ESI-MS
m/z: 454.1 [M+H]+.
4.1.3.14. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-(pyridin-3-yl)ethylidene)hydrazinyl)pyrimidin
1
-4-ylamino)phenylcarbamate (8n). A yellow solid. Yield: 77%. mp 166.6–169.9 °C. H
NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H, –NH–N), 10.06 (s, 1H, NHCO), 9.55 (s, 1H,
NH), 9.35 (s, 1H, ArH), 8.72–8.75 (m, 1H, ArH), 8.67 (d, J = 7.9 Hz, 1H, ArH), 8.32 (s,
1H, ArH), 7.89 (s, 1H, ArH), 7.71 (dd, J = 7.4, 5.5 Hz, 1H, ArH), 7.40 (s, 1H, ArH), 7.33
(d, J = 6.4 Hz, 2H), 2.46 (s, 3H, CH3), 1.47 (s, 9H, CH3). ESI-MS m/z: 454.1 [M+H]+.
4.1.3.15. (E)-Tert-butyl 3-(5-chloro-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)pyrimidin
1
-4-ylamino)phenylcarbamate (8o). An orange solid. Yield: 52%. mp 174.8–176.4 °C. H
NMR (400 MHz, DMSO-d6) δ 10.88 (s, 1H, –NH–N), 9.45 (s, 1H, NHCO), 9.27 (s, 1H,
NH), 8.74 (d, J = 6.4 Hz, 2H, ArH), 8.27 (s, 1H, ArH), 8.06 (d, J = 6.3 Hz, 2H, ArH), 7.96
(s, 1H, ArH), 7.58 (d, J = 7.7 Hz, 1H, ArH), 7.29 (t, J = 8.1 Hz, 1H, ArH), 7.20 (d, J = 8.2
Hz, 1H, ArH), 2.35 (s, 3H, CH3), 1.41 (s, 9H, CH3). ESI-MS m/z: 454.2 [M+H]+.
4.1.4. General procedure for preparation of compounds (9a–9o)
TFA (20 mmol) was added to a solution of 8 (2 mmol) in CH2Cl2 (50 mL) at room
temperature. The reaction mixture was stirred for 2–7 h, and was monitored by TLC. The
mixture was extracted with water (2 × 100 mL), the combined aqueous layer was
neutralized with aqueous K2CO3 to pH 7–8. The precipitate was filtered and dried to
generate the compounds 9a–9o, respectively.
4.1.4.1. (E)-N1-(5-Chloro-2-(2-(4-fluorobenzylidene)hydrazinyl)pyrimidin-4-yl)benzene-1,
1
3-diamine (9a). A white solid. Yield: 66%. mp 230.1–233.3 °C. H NMR (400 MHz,
DMSO-d6) δ 10.99 (s, 1H,–NH–N), 8.45 (s, 1H, NH), 8.08 (s, 1H, –N=CH), 8.07 (s, 1H,
ArH), 7.71 (dd, J = 8.7, 5.7 Hz, 2H, ArH), 7.17–7.25 (m, 3H, ArH), 6.99 (t, J = 8.0 Hz,

ACCEPTED MANUSCRIPT
2H, ArH), 6.34 (dd, J = 7.8, 1.4 Hz, 1H, ArH), 4.97 (s, 2H, NH2). ESI-MS m/z: 356.9
[M+H]+.
4.1.4.2. (E)-N1-(5-Chloro-2-(2-(2-fluorobenzylidene)hydrazinyl)pyrimidin-4-yl)benzene-1,
1
3-diamine (9b). A white solid. Yield: 46%. mp 236.6.9–239.5 °C. H NMR (400 MHz,
DMSO-d6) δ 11.23 (s, 1H, –NH–N), 8.55 (s, 1H, NH), 8.31 (s, 1H, –N=CH), 8.13 (d, J =
1.9 Hz, 1H, ArH), 7.99 (t, J = 7.6 Hz, 1H, ArH), 7.40 (dd, J = 13.7, 6.4 Hz, 1H, ArH),
7.19–7.28 (m, 3H, ArH), 7.01 (td, J = 8.0, 2.1 Hz, 2H, ArH), 6.37 (d, J = 7.8 Hz, 1H,
ArH), 5.09 (s, 2H). ESI-MS m/z: 356.9 [M+H]+.
4.1.4.3. (E)-N1-(5-Chloro-2-(2-(2,4-difluorobenzylidene)hydrazinyl)pyrimidin-4-yl)benzen
1
e-1,3-diamine (9c). A gray solid. Yield: 65%. mp 243.6–245.8 °C. H NMR (400 MHz,
DMSO-d6) δ 11.19 (s, 1H, –NH–N), 8.53 (s, 1H, NH), 8.24 (s, 1H, –N=CH), 8.11 (s, 1H,
ArH), 8.01 (dd, J = 15.7, 8.4 Hz, 1H, ArH), 7.29–7.34 (m, 1H, ArH), 7.17 (td, J = 8.8, 2.1
Hz, 2H, ArH), 7.00 (dd, J = 10.7, 5.1 Hz, 2H, ArH), 6.36 (dd, J = 7.9, 1.3 Hz, 1H, ArH),
5.02 (s, 2H, NH2). ESI-MS m/z: 375.0 [M+H]+.
4.1.4.4. (E)-N1-(5-Chloro-2-(2-(4-methoxybenzylidene)hydrazinyl)pyrimidin-4-yl)benzene
1
-1,3-diamine (9d). A white solid. Yield: 51%. mp 229.4–232.7 °C. H NMR (400 MHz,
DMSO-d6) δ 10.84 (s, 1H, –NH–N), 8.42 (s, 1H, NH), 8.06 (s, 1H, –N=CH), 8.03 (s, 1H,
ArH), 7.79 (d, J = 8.8 Hz, 1H, ArH), 7.60 (d, J = 8.7 Hz, 2H, ArH), 7.21 (s, 1H, ArH),
7.04 (d, J = 8.8 Hz, 1H), 6.98 (d, J = 6.3 Hz, 2H), 6.34 (dd, J = 7.9, 1.3 Hz, 1H, ArH),
5.00 (s, 2H, NH2), 3.78 (s, 3H, OCH3). ESI-MS m/z: 369.0 [M+H]+.
4.1.4.5. (E)-N1-(5-Chloro-2-(2-(pyridin-4-ylmethylene)hydrazinyl)pyrimidin-4-yl)benzene
1
-1,3-diamine (9e). A white solid. Yield: 76%. mp 235.7–237.1 °C. H NMR (400 MHz,
DMSO-d6) δ 11.36 (s, 1H, –NH–N), 8.59 (s, 1H, NH), 8.58 (s, 2H, ArH), 8.15 (s, 1H,
–N=CH), 8.06 (s, 1H, ArH), 7.61 (d, J = 5.9 Hz, 2H, ArH), 7.17 (d, J = 6.2 Hz, 1H, ArH),
7.00–7.05 (m, 2H, ArH), 6.39 (dd, J = 7.9, 1.2 Hz, 1H, ArH), 5.16 (s, 2H, NH2). ESI-MS
m/z: 340.0 [M+H]+.
4.1.4.6. (E)-N1-(2-(2-((1H-Indol-3-yl)methylene)hydrazinyl)-5-chloropyrimidin-4-yl)benz
ene-1,3-diamine (9f). A white solid. Yield: 39%. mp 186.5–187.1 °C.ESI-MS m/z: 378.1
[M+H]+.
4.1.4.7. (E)-N1-(2-(2-((1-Benzyl-1H-indol-3-yl)methylene)hydrazinyl)-5-chloropyrimidin-
1
4-yl)benzene-1,3-diamine (9g). A yellow solid. Yield: 86%. H NMR (400 MHz,
DMSO-d6) δ 10.89 (s, 1H, –NH–N), 8.77 (s, 1H, NH), 8.35 (d, J = 9.2 Hz, 2H, ArH), 8.10
(s, 1H, –N=CH), 7.89 (s, 1H, ArH), 7.50 (d, J = 8.3 Hz, 2H, ArH), 7.13–7.34 (m, 9H,
ArH), 7.06 (t, J = 7.9 Hz, 1H, ArH), 6.84 (s, 1H, ArH), 6.43 (dd, J = 7.3, 0.6 Hz, 1H,
ArH), 5.44 (s, 2H, NH2). ESI-MS m/z: 468.1 [M+H]+.
4.1.4.8. (E)-N1-(5-Chloro-2-(2-(1-(4-fluorophenyl)ethylidene)hydrazinyl)pyrimidin-4-yl)b
1
enzene-1,3-diamine (9h). A gray solid. Yield: 71%. mp 177.8–178.3 °C. H NMR (400
MHz, DMSO-d6) δ 9.98 (s, 1H, –NH–N), 8.50 (s, 1H, NH), 8.13 (s, 1H, ArH), 7.92 (dd, J
= 8.3, 5.8 Hz, 2H, ArH), 7.31 (d, J = 7.6 Hz, 1H, ArH), 7.24 (t, J = 8.8 Hz, 2H, ArH),

ACCEPTED MANUSCRIPT
6.99–7.05 (dd, J = 14.6, 6.4 Hz, 2H, ArH), 6.37 (d, J = 7.6 Hz, 1H, ArH), 5.18 (s, 2H,
NH2), 2.28 (s, 3H, CH3). ESI-MS m/z: 370.9 [M+H]+.
4.1.4.9. (E)-N1-(5-Chloro-2-(2-(1-(2,4-difluorophenyl)ethylidene)hydrazinyl)pyrimidin-4-
yl)benzene-1,3-diamine (9i). A gray solid. Yield: 66%. mp 148.4–150.6 °C. 1H NMR (400
MHz, DMSO-d6) δ 9.99 (s, 1H, –NH–N), 8.47 (s, 1H, NH), 8.13 (s, 1H, ArH), 7.80 (dd, J
= 15.7, 8.5 Hz, 1H, ArH), 7.25–7.30 (m, 1H, ArH), 7.22 (d, J = 7.7 Hz, 1H, ArH),
7.12–7.18 (m, 1H, ArH), 7.07 (s, 1H, ArH), 6.96 (t, J = 7.9 Hz, 1H, ArH), 6.33 (d, J = 7.5
Hz, 1H, ArH), 5.01 (s, 2H, NH2), 2.29 (s, 3H, CH3). ESI-MS m/z: 389.0 [M+H]+.
4.1.4.10. (E)-N1-(5-Chloro-2-(2-(1-p-tolylethylidene)hydrazinyl)pyrimidin-4-yl)benzene-1
1
,3-diamine (9j). A white solid. Yield: 73%. mp 171.1–172.3 °C. H NMR (400 MHz,
DMSO-d6) δ 10.37 (s, 1H, –NH–N), 8.95 (s, 1H, NH), 8.16 (s, 1H, ArH), 7.82 (d, J = 8.2
Hz, 2H, ArH), 7.32 (s, 1H, ArH), 7.24 (d, J = 8.1 Hz, 2H, ArH), 7.06 (t, J = 8.0 Hz, 1H,
ArH), 6.99 (s, 1H, ArH), 6.44 (dd, J = 7.9, 1.3 Hz, 1H, ArH), 2.34 (s, 3H, CH3), 2.29 (s,
3H, CH3), (The active hydrogen of NH2 was disappeared). ESI-MS m/z: 367.2 [M+H]+.
4.1.4.11. (E)-N1-(5-Chloro-2-(2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)pyrimidin-4-y
1
l)benzene-1,3-diamine (9k). A gray solid. Yield: 65%. H NMR (400 MHz, DMSO-d6) δ
10.48 (s, 1H, –NH–N), 9.13 (s, 1H, NH), 8.17 (s, 1H, ArH), 7.87–7.90 (m, 3H, ArH), 7.07
(d, J = 8.0 Hz, 1H, ArH), 7.02–7.05 (m, 1H, ArH), 6.98 (d, J = 9.0 Hz, 2H, ArH), 6.45 (d,
J = 7.5 Hz, 1H, ArH), 3.81 (s, 3H, OCH3), 2.30 (s, 3H, CH3), (The active hydrogen of
NH2 was disappeared).
4.1.4.12. (E)-N1-(5-Chloro-2-(2-(1-(3-methoxyphenyl)ethylidene)hydrazinyl)pyrimidin-4-
yl)benzene-1,3-diamine (9l). A gray solid. Yield: 59%. mp 205.9–208.1 °C. 1H NMR (400
MHz, DMSO-d6) δ 9.89 (s, 1H, –NH–N), 8.41 (s, 1H, NH), 8.13 (s, 1H, ArH), 7.46 (dd, J
= 14.3, 7.6 Hz, 2H, ArH), 7.34 (dd, J = 14.8, 6.6 Hz, 2H, ArH), 6.99–7.02 (m, 2H, ArH),
6.94 (d, J = 7.3 Hz, 1H, ArH), 6.33 (d, J = 7.9 Hz, 1H, ArH), 4.95 (s, 2H, NH2), 3.79 (s,
3H, OCH3), 2.28 (s, 3H, CH3). ESI-MS m/z: 383.0 [M+H]+.
4.1.4.13. (E)-N1-(5-Chloro-2-(2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)pyrimidin-4-yl)be
1
nzene-1,3-diamine (9m). A gray solid. Yield: 70%. mp 104.5–107.4 °C. H NMR (400
MHz, DMSO-d6) δ 10.13 (s, 1H, –NH–N), 8.55 (d, J = 4.2 Hz, 1H, ArH), 8.53 (s, 1H,
NH), 8.22 (d, J = 8.1 Hz, 1H, ArH), 8.17 (s, 1H, ArH), 7.83 (td, J = 8.0, 1.7 Hz, 1H, ArH),
7.32–7.35 (m, 2H, ArH), 7.04–7.05 (m, 1H, ArH), 7.01 (d, J = 8.0 Hz, 1H, ArH), 6.37 (dd,
J = 7.9, 1.3 Hz, 1H, ArH), 5.02 (s, 2H, NH2), 2.37 (s, 3H, CH3). ESI-MS m/z: 353.9
[M+H]+.
4.1.4.14. (E)-N1-(5-Chloro-2-(2-(1-(pyridin-3-yl)ethylidene)hydrazinyl)pyrimidin-4-yl)be
1
nzene-1,3-diamine (9n). A white solid. Yield: 86%. H NMR (400 MHz, DMSO-d6) δ
10.14 (s, 1H, –NH–N), 9.06 (d, J = 1.8 Hz, 1H, ArH), 8.54 (dd, J = 4.7, 1.3 Hz, 1H, ArH),
8.50 (s, 1H, NH), 8.21–8.23 (m, 1H, ArH), 8.15 (s, 1H, ArH), 7.42–7.45 (m, 2H, ArH),
7.00–7.04 (m, 2H, ArH), 6.35 (dd, J = 7.9, 1.2 Hz, 1H, ArH), 5.01 (s, 2H, NH2), 2.32 (s,
3H, CH3). ESI-MS m/z: 353.9 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.4.15. (E)-N1-(5-Chloro-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)pyrimidin-4-yl)be
1
nzene-1,3-diamine (9o). A gray solid. Yield: 75%. mp 209.1–210.8 °C. H NMR (400
MHz, DMSO-d6) δ 10.44 (s, 1H, –NH–N), 8.72 (s, 1H, NH), 8.63 (d, J = 5.7 Hz, 2H,
ArH), 8.20 (s, 1H, ArH), 7.86 (d, J = 5.9 Hz, 2H, ArH), 7.33 (d, J = 7.9 Hz, 1H, ArH),
7.06 (dd, J = 10.5, 5.1 Hz, 2H, ArH), 6.45 (d, J = 7.6 Hz, 1H, ArH), 2.31 (s, 3H, CH3),
(The active hydrogen of NH2 was disappeared). ESI-MS m/z: 353.9 [M+H]+.
4.1.5. N1-(2,5-Dichloropyrimidin-4-yl)benzene-1,3-diamine (10)
TFA (15.96g, 0.14 mmol) was added to a solution of 6 (4.96g, 14 mmol) in CH2Cl2
(200 mL). The reaction mixture was stirred at room temperature for 1 h. The solvent was
evaporated under vacuum, which was then dissolved in water. The solution was alkalized
with aqueous K2CO3, the precipitate was filtered and dried to give compound 10 as a
pale-yellow solid (2.67g, 75%). ESI-MS m/z: 354.9 [M+H]+.
4.1.6. N-(3-(2,5-Dichloropyrimidin-4-ylamino)phenyl)acrylamide (11)
To a solution of 10 (2g, 7.9 mmol) and DIPEA (1.38 mL, 7.9 mmol) in CH2Cl2 (50
mL) at 0 °C, acryloyl chloride (0.64 mL, 7.9 mmol) was added dropwise. The mixture
was stirred for 1 h, then extracted with water (2 × 50 mL). The organic layer was dried
over anhydrous Na2SO4, and evaporated to give the crude residue. Intermediate 11 was
obtained as a pale-yellow solid (1.9g, 78%) after purification by silica gel column
1
chromatography (petroleum ether:ethyl acetate = 2:1, v/v). H NMR (400 MHz,
DMSO-d6) δ 10.26 (s, 1H, NHCO), 9.59 (s, 1H, NH), 8.39 (s, 1H, ArH), 7.91 (br, 1H,
ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.34 (t, J = 8.0 Hz, 1H, ArH), 7.28 (dd, J = 8.3, 1.4
Hz, 1H, ArH), 6.47 (dd, J = 17.0, 10.1 Hz, 1H, –CH=CH2), 6.27 (dd, J = 17.0, 2.0 Hz, 1H,
–CH=CH2), 5.77 (dd, J = 10.1, 2.0 Hz, 1H, –CH=CH2). ESI-MS m/z: 308.8 [M+H]+.
4.1.7. General procedure for preparation of compounds (13a–13b)
Compound 12 (0.013 mol) and 80% NH2NH2·H2O (5 mL) were added to EtOH (10
mL), the mixture was stirred under reflux for 5 h. After being cooled to room temperature,
the precipitate was obtained by filtration, and was dried to give the title compounds,
respectively.
4.1.7.1. 4-Fluorobenzohydrazide (13a). A white solid. Yield: 75%. mp 161.9–164.4 °C.
1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1H, –NH–N), 7.83–7.93 (m, 2H, ArH),
7.22–7.33 (m, 2H, ArH), 4.48 (s, 2H, –NH–NH2). ESI-MS m/z: 155.1 [M+H]+.
4.1.7.2. 4-Methoxybenzohydrazide (13b). A white solid. Yield: 69%. mp 137.9–139.0 °C.
1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H, –NH–N), 7.75–7.86 (m, 2H, ArH),
6.92–7.03 (m, 2H, ArH), 4.42 (s, 2H, –NH–NH2), 3.80 (s, 3H, OCH3). ESI-MS m/z:
167.2 [M+H]+.
4.1.8. General procedure for preparation of compounds (14a–14j)
Acryloyl chloride (1 mmol) was added dropwise to a solution of intermediate 9 (1
mmol) and DIPEA (1 mmol) in CH2Cl2 (5 mL) at 0 °C. The reaction was stirred for 1 h.
The target compounds 14a–14j were afforded after purification by silica gel column
chromatography.

ACCEPTED MANUSCRIPT
4.1.8.1. (E)-N-(3-(5-Chloro-2-(2-(4-fluorobenzylidene)hydrazinyl)pyrimidin-4-ylamino)p
henyl)acrylamide (14a). Flash column chromatography was performed using (petroleum
ether:ethyl acetate, 5:1 to 2:1 v/v). HPLC purity: 98.2%. A white solid. Yield: 32%. mp
1
225.8–227.2 °C. IR (KBr, cm-1): 3413.7 (NH), 3264.8 (RC=CH), 1657.9 (CO). H NMR
(400 MHz, DMSO-d6) δ 11.40 (s, 1H, –NH–N), 10.34 (s, 1H, NHCO), 9.06 (s, 1H, NH),
8.17 (s, 1H, –N=CH), 8.13 (br, 2H, ArH), 7.72 (dd, J = 8.6, 5.7 Hz, 2H, ArH), 7.58 (d, J =
53.2 Hz, 2H, ArH), 7.28 (dt, J = 17.7, 8.5 Hz, 3H, ArH), 6.53 (dd, J = 16.8, 10.1 Hz, 1H,
–CH=CH2), 6.26 (dd, J = 17.0, 1.9 Hz, 1H, –CH=CH2), 5.74 (dd, J = 10.3, 1.6 Hz, 1H,
13
–CH=CH2). C NMR (101 MHz, DMSO-d6) δ 164.18, 163.64, 161.74, 156.54, 139.56,
139.41, 132.54, 132.20, 128.97 (2C), 128.88, 128.80, 127.09, 118.27, 116.28 (2C), 116.06,
115.69, 114.44, 105.24. ESI-MS m/z: 411.2 [M+H]+. Anal. calcd. for C20H16ClFN6O (%):
C, 58.47; H, 3.93; N, 20.46; found (%): C, 58.52; H, 3.88; N, 20.37.
4.1.8.2. (E)-N-(3-(5-Chloro-2-(2-(2-fluorobenzylidene)hydrazinyl)pyrimidin-4-ylamino)p
henyl)acrylamide (14b). Flash column chromatography was performed using (petroleum
ether:ethyl acetate, 10:1 to 4:1 v/v). HPLC purity: 98.6%. A white solid. Yield: 43%. mp
1
222.7–223.8 °C. IR (KBr, cm-1): 3392.9 (NH), 3217.4 (RC=CH), 1667.2 (CO). H NMR
(600 MHz, DMSO-d6) δ 13.10 (s, 1H, –NH–N), 10.67 (s, 1H, NHCO), 10.27 (s, 1H, NH),
8.54 (s, 1H, –N=CH), 8.32 (d, J = 55.7 Hz, 3H, ArH), 7.75 (d, J = 5.9 Hz, 1H, ArH), 7.52
(dd, J = 13.3, 5.7 Hz, 1H, ArH), 7.39 (t, J = 7.9 Hz, 1H, ArH), 7.32 (dd, J = 16.3, 8.2 Hz,
3H, ArH), 6.63 (dd, J = 16.9, 10.2 Hz, 1H, –CH=CH2), 6.28 (d, J = 16.9 Hz, 1H,
–CH=CH2), 5.76 (d, J = 10.3 Hz, 1H, –CH=CH2). 13C NMR (101 MHz, DMSO-d6) δ
163.80, 162.47, 159.98, 157.93, 140.01, 137.70, 132.93, 132.40, 129.38, 127.38, 127.32,
125.26, 125.23, 121.67, 119.78, 117.41, 116.54, 116.33, 115.75, 106.51. ESI-MS m/z:
411.1 [M+H]+. Anal. calcd. for C20H16ClFN6O (%): C, 58.47; H, 3.93; N, 20.46; found
(%): C, 58.42; H, 3.97; N, 20.37.
4.1.8.3. (E)-N-(3-(5-Chloro-2-(2-(2,4-difluorobenzylidene)hydrazinyl)pyrimidin-4-ylamin
o)phenyl)acrylamide (14c). Flash column chromatography was performed using
(petroleum ether:ethyl acetate, 10:1 to 4:1 v/v). HPLC purity: 99.0%. A white solid. Yield:
52%. mp 263.6–265.2 °C. IR (KBr, cm-1): 3428.1 (NH), 3208.7 (RC=CH), 2953.0 (CH3),
1
1675.3 (CO). H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H, –NH–N), 10.16 (s, 1H,
NHCO), 8.94 (s, 1H, NH), 8.23 (s, 1H, –N=CH), 8.16 (s, 1H, ArH), 7.99 (s, 1H, ArH),
7.92 (dd, J = 15.5, 8.6 Hz, 1H, ArH), 7.73 (d, J = 6.5 Hz, 1H, ArH), 7.44 (d, J = 7.7 Hz,
1H, ArH), 7.31–7.35 (m, 2H, ArH), 7.13 (td, J = 8.8, 2.1 Hz, 1H, ArH), 6.46 (dd, J = 16.9,
10.1 Hz, 1H, –CH=CH2), 6.25 (dd, J = 17.0, 1.9 Hz, 1H, –CH=CH2), 5.75 (m, 1H,
–CH=CH2). ESI-MS m/z: 429.1 [M+H]+. Anal. calcd. for C20H15ClF2N6O (%): C, 56.02;
H, 3.53; N, 19.60; found (%): C, 55.93; H, 3.60; N, 19.72.
4.1.8.4. (E)-N-(3-(2-(2-((1-Benzyl-1H-indol-3-yl)methylene)hydrazinyl)-5-chloropyrimidi
n-4-ylamino)phenyl)acrylamide (14d). Flash column chromatography was performed
using (petroleum ether:ethyl acetate, 4:1 to 3:2 v/v). HPLC purity: 98.3%. A pale-yellow
1
solid. Yield: 30%. mp 163.3–165.5 °C. H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H,
–NH–N), 10.14 (s, 1H, NH), 8.74 (s, 1H, NH), 8.28 (s, 1H, –N=CH), 8.26 (d, J = 7.8 Hz,
1H, ArH), 8.10 (s, 1H, ArH), 7.86 (s, 1H, ArH), 7.81 (s, 1H, ArH), 7.46 (dd, J = 8.2, 3.3

ACCEPTED MANUSCRIPT
Hz, 2H, ArH), 7.21–7.33 (m, 7H, ArH), 7.15–7.19 (m, 1H, ArH), 7.07 (t, J = 7.4 Hz, 1H,
ArH), 6.42 (dd, J = 16.9, 10.3 Hz, 1H, –CH=CH2), 6.22 (dd, J = 16.9, 2.0 Hz, 1H,
–CH=CH2), 5.70 (dd, J = 10.2, 1.6 Hz, 1H, , –CH=CH2), 5.41 (s, 2H, CH2). ESI-MS m/z:
522.1 [M+H]+. Anal. calcd. for C29H24ClN7O (%): C, 66.73; H, 4.63; N, 18.78; found (%):
C, 66.65; H, 4.51; N, 18.86.
4.1.8.5. (E)-N-(3-(5-Chloro-2-(2-(1-(4-fluorophenyl)ethylidene)hydrazinyl)pyrimidin-4-yl
amino)phenyl)acrylamide (14e). Flash column chromatography was performed using
(petroleum ether:ethyl acetate, 10:3 to 5:3 v/v). HPLC purity: 97.9%. A white solid. Yield:
1
65%. mp 225.8–228.4 °C. H NMR (400 MHz, DMSO-d6) δ 10.49 (s, 1H, –NH–N),
10.28 (s, 1H, NHCO), 9.30 (s, 1H, NH), 8.22 (s, 1H, ArH), 8.10 (s, 1H, ArH), 7.93 (dd, J
= 7.3, 6.0 Hz, 2H, ArH), 7.71 (s, 1H, ArH), 7.48 (d, J = 8.4 Hz, 1H, ArH), 7.32–7.36 (m,
1H, ArH), 7.21–7.25 (m, 2H, ArH), 6.47 (dd, J = 16.9, 10.1 Hz, 1H, –CH=CH2), 6.25 (dd,
J = 16.9, 1.6 Hz, 1H, –CH=CH2), 5.74 (dd, J = 10.2, 1.3 Hz, 1H, –CH=CH2), 2.32 (s, 3H,
CH3). ESI-MS m/z: 425.3 [M+H]+. Anal. calcd. for C21H18ClFN6O (%): C, 59.37; H, 4.27;
N, 19.78; found (%): C, 59.26; H, 4.25; N, 19.83.
4.1.8.6. (E)-N-(3-(5-Chloro-2-(2-(1-(2,4-difluorophenyl)ethylidene)hydrazinyl)pyrimidin-
4-ylamino)phenyl)acrylamide (14f). Flash column chromatography was performed using
(petroleum ether:ethyl acetate, 10:1 to 3:1 v/v). HPLC purity: 98.6%. A white solid. Yield:
1
36%. mp 236.2–238.3 °C. H NMR (400 MHz, DMSO-d6) δ 10.15 (s, 1H, –NH–N),
10.02 (s, 1H, NHCO), 8.90 (s, 1H, NH), 8.18 (s, 1H, ArH), 8.10 (s, 1H, ArH), 7.78 (d, J =
7.7 Hz, 1H, ArH), 7.71 (td, J = 8.9, 7.0 Hz, 1H, ArH), 7.39 (dd, J = 8.4, 0.8 Hz, 1H, ArH),
7.24–7.31 (m, 2H, ArH), 7.09 (td, J = 8.6, 2.7 Hz, 1H, ArH), 6.45 (dd, J = 17.0, 10.1 Hz,
1H, –CH=CH2), 6.25 (dd, J = 17.0, 2.0 Hz, 1H, –CH=CH2), 5.74 (dd, J = 10.1, 2.0 Hz,
1H, –CH=CH2), 2.28 (s, 3H, CH3). ESI-MS m/z: 443.5 [M+H]+. Anal. calcd. for
C21H17ClF2N6O (%): C, 56.96; H, 3.87; N, 18.98; found (%): C, 60.07; H, 3.80; N, 19.11.
4.1.8.7. (E)-N-(3-(5-Chloro-2-(2-(1-p-tolylethylidene)hydrazinyl)pyrimidin-4-ylamino)ph
enyl)acrylamide (14g). Flash column chromatography was performed using (petroleum
ether:ethyl acetate, 2:1 to 5:3 v/v). HPLC purity: 99.1%. A white solid. Yield: 61%. mp
1
188.8–189.5 °C. H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H, –NH–N), 9.94 (s, 1H,
NHCO), 8.92 (s, 1H, NH), 8.17 (s, 1H, ArH), 8.07 (s, 1H, ArH), 7.92 (s, 1H, ArH), 7.74
(d, J = 7.8 Hz, 2H, ArH), 7.45 (dd, J = 8.1, 1.3 Hz, 1H, ArH), 7.31 (t, J = 7.8 Hz, 1H,
ArH), 7.19 (d, J = 7.9 Hz, 2H, ArH), 6.45 (dd, J = 16.9, 10.1 Hz, 1H, –CH=CH2), 6.26
(dd, J = 16.9, 1.9 Hz, 1H, –CH=CH2), 5.73 (dd, J = 10.3, 1.7 Hz, 1H, –CH=CH2), 2.32 (s,
3H, CH3), 2.26 (s, 3H, CH3). ESI-MS m/z: 421.5 [M+H]+. Anal. calcd. for C22H21ClN6O
(%): C, 62.78; H, 5.03; N, 19.97; found (%): C, 62.65; H, 4.96; N, 19.82.
4.1.8.8. (E)-N-(3-(5-Chloro-2-(2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)pyrimidin-4-
ylamino)phenyl)acrylamide (14h). Flash column chromatography was performed using
(petroleum ether:ethyl acetate, 5:1 to 3:2 v/v). HPLC purity: 98.7%. A white solid. Yield:
1
55%. mp 217.0–219.4 °C. H NMR (400 MHz, DMSO-d6) δ 11.55 (s, 1H, –NH–N),
10.36 (s, 1H, NHCO), 10.34 (s, 1H, NH), 8.29 (s, 1H, ArH), 7.96–7.80 (m, 3H, ArH),
7.58 (d, J = 7.6 Hz, 1H, ArH), 7.37–7.44 (m, 2H, ArH), 7.00 (d, J = 9.0 Hz, 2H, ArH),

ACCEPTED MANUSCRIPT
6.50 (dd, J = 17.0, 10.1 Hz, 1H, –CH=CH2), 6.28 (dd, J = 16.9, 1.8 Hz, 1H, –CH=CH2),
5.78 (dd, J = 10.1, 1.8 Hz, 1H, –CH=CH2), 3.82 (s, 3H, OCH3), 2.37 (s, 3H, CH3).
ESI-MS m/z: 437.2 [M+H]+. Anal. calcd. for C22H21ClN6O2 (%): C, 60.48; H, 4.85; N,
19.24; found (%): C, 60.53; H, 4.76; N, 19.07.
4.1.8.9. (E)-N-(3-(5-Chloro-2-(2-(1-(3-methoxyphenyl)ethylidene)hydrazinyl)pyrimidin-4-
ylamino)phenyl)acrylamide (14i). Flash column chromatography was performed using
(petroleum ether:ethyl acetate, 4:1 to 3:2 v/v). HPLC purity: 99.3%. A white solid. Yield:
1
29%. mp 198.2–200.1 °C. H NMR (400 MHz, DMSO-d6) δ 11.59 (s, 1H, –NH–N),
10.35 (s, 2H, NHCO, NH), 8.29 (s, 1H, ArH), 7.96 (s, 1H, ArH), 7.53–7.56 (m, 3H, ArH),
7.36–7.44 (m, 3H, ArH), 7.06 (dd, J = 8.0, 1.9 Hz, 1H, ArH), 6.48 (dd, J = 17.0, 10.1 Hz,
1H, –CH=CH2), 6.28 (dd, J = 17.0, 1.9 Hz, 1H, –CH=CH2), 5.79 (dd, J = 10.1, 1.9 Hz,
1H, –CH=CH2), 3.82 (s, 3H, OCH3), 2.39 (s, 3H, CH3). ESI-MS m/z: 437.2 [M+H]+.
Anal. calcd. for C22H21ClN6O2 (%): C, 60.48; H, 4.85; N, 19.24; found (%): C, 60.42; H,
4.73; N, 19.32.
4.1.8.10. (E)-N-(3-(5-Chloro-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)pyrimidin-4-yla
mino)phenyl)acrylamide (14j). Flash column chromatography was performed using
(petroleum ether:ethyl acetate, 1:1 to 1:3 v/v). HPLC purity: 98.6%. A yellow solid. Yield:
1
51%. mp 152.1–154.3 °C. H NMR (400 MHz, DMSO-d6) δ 10.31 (s, 1H, –NH–N),
10.22 (s, 1H, NHCO), 8.99 (s, 1H, NH), 8.56 (d, J = 5.5 Hz, 2H, ArH), 8.22 (s, 1H, ArH),
8.13 (s, 1H, ArH), 7.82 (d, J = 7.7 Hz, 1H, ArH), 7.73 (d, J = 5.5 Hz, 2H, ArH), 7.43 (d, J
= 7.6 Hz, 1H, ArH), 7.34 (t, J = 8.0 Hz, 1H, ArH), 6.45 (dd, J = 16.8, 10.1 Hz, 1H,
–CH=CH2), 6.24 (dd, J = 16.9, 0.5 Hz, 1H, –CH=CH2), 5.72 (dd, J = 10.7, 0.6 Hz, 1H,
–CH=CH2), 2.29 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 163.59, 158.48, 156.47,
155.19, 150.03 (2C), 146.30, 143.15, 139.65, 139.39, 132.41, 128.88, 127.22, 120.27
(2C), 118.23, 115.40, 114.30, 105.87, 13.00. ESI-MS m/z: 408.1 [M+H]+. Anal. calcd. for
C20H18ClN7O (%): C, 58.90; H, 4.45; N, 24.04; found (%): C, 58.82; H, 4.37; N, 24.15.
4.1.9. General procedure for preparation of compounds (15a–15o)
A solution of appropriate 9 (0.8 mmol), (E)-4-(dimethylamino)but-2-enoic acid (0.8
mmol), DIPEA (2 mmol) and HATU (0.8 mmol) in CH2Cl2 (5 mL) was stirred at room
temperature for 3–9 h. The solution was washed with aqueous K2CO3 (2 × 5 mL), then
brine (5 mL), and evaporated to give the crude residue. The target compounds 15a–15o
were obtained after purification by silica gel column chromatography.
4.1.9.1. (E)-N-(3-(5-Chloro-2-((E)-2-(4-fluorobenzylidene)hydrazinyl)pyrimidin-4-ylamin
o)phenyl)-4-(dimethylamino)but-2-enamide (15a). Flash column chromatography was
performed using (CH2Cl2:MeOH, 30:1 to 20:1 v/v). HPLC purity: 98.2%. A white solid.
Yield: 42%. mp 218.7–220.1 °C. IR (KBr, cm-1): 3413.6 (NH), 3243.6 (RC=CH), 2940.3,
1
2857.0 (CH2, CH3), 2776.1 (N–CH3), 1674.1 (CO). H NMR (400 MHz, DMSO-d6) δ
11.05 (s, 1H, –NH–N), 10.06 (s, 1H, NHCO), 8.85 (s, 1H, NH), 8.12 (s, 1H, –N=CH),
8.06 (s, 1H, ArH), 8.00 (s, 1H, ArH), 7.64–7.67 (m, 3H, ArH), 7.41 (d, J = 8.3 Hz, 1H,
ArH), 7.29 (t, J = 8.1 Hz, 1H, ArH), 7.19 (t, J = 8.8 Hz, 2H, ArH), 6.70 (dt, J = 15.3, 5.9
Hz, 1H, –CH=CH–CH2), 6.28 (d, J = 15.4 Hz, 1H, –CH=CH–CH2), 3.01 (d, J = 5.4 Hz,

ACCEPTED MANUSCRIPT
2H, –CH=CH–CH2), 2.13 (s, 6H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 163.63,
158.12, 156.38, 155.37, 141.76, 140.12, 139.63, 139.53, 132.46, 128.86, 128.57 (2C),
126.50, 118.09, 116.22 (2C), 116.00, 115.52, 114.47, 105.02, 60.20, 45.58 (2C). ESI-MS
m/z: 468.1 [M+H]+. Anal. calcd. for C23H23ClFN7O (%): C, 59.04; H, 4.95; N, 20.95;
found (%): C, 58.95; H, 4.77; N, 21.02.
4.1.9.2. (E)-N-(3-(5-Chloro-2-((E)-2-(2-fluorobenzylidene)hydrazinyl)pyrimidin-4-ylamin
o)phenyl)-4-(dimethylamino)but-2-enamide (15b). Flash column chromatography was
performed using (CH2Cl2:MeOH, 50:1 to 25:1 v/v). HPLC purity: 98.5%. A white solid.
Yield: 67%. mp 194.4–196.2 °C. IR (KBr, cm-1): 3422.6 (NH), 2925.8, 2852.2 (CH2,
1
CH3), 2779.5 (N–CH3), 1674.5 (CO). H NMR (400 MHz, DMSO-d6) δ 11.23 (s, 1H,
–NH–N), 10.16 (s, 1H, NHCO), 8.91 (s, 1H, NH), 8.30 (s, 1H, –N=CH), 8.17 (s, 1H,
ArH), 8.00 (s, 1H, ArH), 7.91 (t, J = 7.5 Hz, 1H, ArH), 7.73 (s, 1H, ArH), 7.45 (d, J = 7.6
Hz, 1H, ArH), 7.38 (dd, J = 13.3, 6.7 Hz, 1H, ArH), 7.31 (t, J = 8.0 Hz, 1H, ArH), 7.23
(dd, J = 16.2, 8.4 Hz, 2H, ArH), 6.73 (m, 1H, –CH=CH–CH2), 6.35 (d, J = 15.4 Hz, 1H,
–CH=CH–CH2), 3.29 (d, J = 5.2 Hz, 2H, –CH=CH–CH2), 2.37 (s, 6H, CH3). ESI-MS m/z:
468.1 [M+H]+. Anal. calcd. for C23H23ClFN7O (%): C, 59.04; H, 4.95; N, 20.95; found
(%): C, 59.10; H, 4.86; N, 20.99.
4.1.9.3. (E)-N-(3-(5-Chloro-2-((E)-2-(2,4-difluorobenzylidene)hydrazinyl)pyrimidin-4-yla
mino)phenyl)-4-(dimethylamino)but-2-enamide (15c). Flash column chromatography was
performed using (CH2Cl2:MeOH, 50:1 to 22:1 v/v). HPLC purity: 99.3%. A white solid.
Yield: 55%. mp 189.1–191.2 °C. 1H NMR (400 MHz, DMSO-d6) 11.45 (s, 1H, –NH–N),
10.55 (s, 1H, NHCO), 8.92 (s, 1H, NH), 8.30 (s, 1H, –N=CH), 8.19 (s, 1H, ArH), 8.17 (s,
1H, ArH), 7.92 (dd, J = 15.5, 8.5 Hz, 1H, ArH), 7.67 (s, 1H, ArH), 7.54 (d, J = 7.3 Hz, 1H,
ArH), 7.28–7.34 (m, 2H, ArH), 7.15 (td, J = 8.7, 2.0 Hz, 1H, ArH), 6.77 (m, 1H,
–CH=CH–CH2), 6.56 (d, J = 15.1 Hz, 1H, –CH=CH–CH2), 3.82 (d, J = 6.1 Hz, 2H,
–CH=CH–CH2), 2.69 (s, 6H, CH3). ESI-MS m/z: 486.1 [M+H]+. Anal. calcd. for
C23H22ClF2N7O (%): C, 56.85; H, 4.56; N, 20.18; found (%): C, 56.80; H, 4.61; N, 20.23.
4.1.9.4. (E)-N-(3-(5-Chloro-2-((E)-2-(4-methoxybenzylidene)hydrazinyl)pyrimidin-4-ylam
ino)phenyl)-4-(dimethylamino)but-2-enamide (15d). Flash column chromatography was
performed using (CH2Cl2:MeOH, 35:1 to 20:1 v/v). HPLC purity: 97.2%. A white solid.
1
Yield: 53%. mp 145.1–147.2 °C. H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H,
–NH–N), 10.08 (s, 1H, NHCO), 8.80 (s, 1H, NH), 8.10 (s, 1H, –N=CH), 8.01 (s, 1H,
ArH), 7.99 (s, 1H, ArH), 7.70 (s, 1H, ArH), 7.55 (d, J = 8.8 Hz, 2H, ArH), 7.42 (d, J = 7.7
Hz, 1H, ArH), 7.28 (t, J = 8.1 Hz, 1H, ArH), 6.93 (d, J = 8.8 Hz, 2H, ArH), 6.70 (dt, J =
15.4, 5.9 Hz, 1H, –CH=CH–CH2), 6.28 (d, J = 15.4 Hz, 1H, –CH=CH–CH2), 3.77 (s, 3H,
OCH3), 3.02 (d, J = 5.6 Hz, 2H, –CH=CH–CH2), 2.13 (s, 6H, CH3). 13C NMR (101 MHz,
DMSO-d6) δ 163.63, 160.36, 158.17, 156.29, 155.35, 141.60, 141.33, 139.71, 139.57,
128.85, 128.44, 128.10 (2C), 126.61, 117.95, 115.38, 114.66 (2C), 114.30, 104.61, 60.20,
55.67, 45.57 (2C). ESI-MS m/z: 480.1 [M+H]+. Anal. calcd. for C24H26ClN7O2 (%): C,
60.06; H, 5.46; N, 20.43; found (%): C, 59.89; H, 5.50; N, 20.28.
4.1.9.5. (E)-N-(3-(5-Chloro-2-((E)-2-(pyridin-4-ylmethylene)hydrazinyl)pyrimidin-4-ylam

ACCEPTED MANUSCRIPT
ino)phenyl)-4-(dimethylamino)but-2-enamide (15e). Flash column chromatography was
performed using (CH2Cl2:MeOH, 30:1 to 15:1 v/v). HPLC purity: 98.8%. A white solid.
1
Yield: 32%. mp 200.5–202.7 °C. H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H,
–NH–N), 10.17 (s, 1H, NHCO), 8.97 (s, 1H, NH), 8.54 (d, J = 5.2 Hz, 1H, ArH), 8.28 (d,
J = 3.1 Hz, 1H, ArH), 8.19 (s, 1H, –N=CH), 8.04–8.07 (m, 2H, ArH), 7.66 (d, J = 7.7 Hz,
1H, ArH), 7.54 (d, J = 5.2 Hz, 1H, ArH), 7.45 (d, J = 8.0 Hz, 1H, ArH), 7.32 (t, J = 8.3
Hz, 1H, ArH), 7.11 (dd, J = 8.0, 4.0 Hz, 1H, ArH), 6.71 (m, 1H, –CH=CH–CH2), 6.29 (d,
J = 15.1 Hz, 1H, –CH=CH–CH2), 3.02 (d, J = 3.6 Hz, 2H, –CH=CH–CH2), 2.14 (s, 6H,
CH3). ESI-MS m/z: 451.1 [M+H]+. Anal. calcd. for C22H23ClN8O (%): C, 58.60; H, 5.14;
N, 24.85; found (%): C, 58.48; H, 5.22; N, 24.72.
4.1.9.6. (E)-N-(3-(2-((E)-2-((1H-Indol-3-yl)methylene)hydrazinyl)-5-chloropyrimidin-4-yl
amino)phenyl)-4-(dimethylamino)but-2-enamide (15f). Flash column chromatography
was performed using (CH2Cl2:MeOH, 30:1 to 15:1 v/v). HPLC purity: 98.0%. A yellow
1
solid. Yield: 29%. mp 127.2–129.5 °C. H NMR (400 MHz, DMSO-d6) δ11.82 (s, 1H,
NH), 10.52 (s, 1H, –NH–N), 10.31 (s, 1H, NHCO), 9.76 (s, 1H, NH), 8.45 (s, 1H, ArH),
8.41 (d, J = 7.2 Hz, 1H, ArH), 8.26 (s, 1H, –N=CH), 7.92 (d, J = 8.1 Hz, 2H, ArH), 7.59
(d, J = 7.6 Hz, 1H, ArH), 7.45 (dd, J = 14.1, 7.6 Hz, 2H, ArH), 7.35 (s, 1H, ArH), 7.23
(dd, J = 16.6, 8.6 Hz, 2H, ArH), 6.76 (m, 1H, –CH=CH–CH2), 6.52 (d, J = 15.4 Hz, 1H,
–CH=CH–CH2), 3.23 (d, J = 5.6 Hz, 2H, –CH=CH–CH2), 2.81 (s, J = 3.5 Hz, 6H, CH3).
ESI-MS m/z: 489.1 [M+H]+. Anal. calcd. for C25H25ClN8O (%): C, 61.41; H, 5.15; N,
22.92; found (%): C, 61.46; H, 5.10; N, 22.90.
4.1.9.7. (E)-N-(3-(2-((E)-2-((1-Benzyl-1H-indol-3-yl)methylene)hydrazinyl)-5-chloropyri
midin-4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide
(15g).
Flash
column
chromatography was performed using (CH2Cl2:MeOH, 100:1 to 25:1 v/v). HPLC purity:
98.1%. A pale-yellow solid. Yield: 25%. mp 196.9–199.8 °C. IR (KBr, cm-1): 3422.4
1
(NH), 3237.4 (RC=CH), 2924.9, 2852.7 (CH2, CH3), 2780.8 (N–CH3), 1673.9 (CO). H
NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H, –NH–N), 10.05 (s, 1H, NHCO), 8.73 (s, 1H,
NH), 8.28 (s, 1H, –N=CH), 8.26 (d, J = 7.9 Hz, 1H, ArH), 8.10 (s, 1H, ArH), 7.85 (s, 1H,
ArH), 7.81 (s, 1H, NH), 7.45 (d, J = 8.2 Hz, 2H, ArH), 7.28–7.31 (m, 3H, ArH),
7.22–7.25 (m, 4H, ArH), 7.15–7.19 (m, 1H, ArH), 7.07 (t, J = 7.5 Hz, 1H, ArH), 6.69 (dt,
J = 15.3, 5.9 Hz, 1H, –CH=CH–CH2), 6.25 (d, J = 15.3 Hz, 1H, –CH=CH–CH2), 5.41 (s,
13
2H, CH2), 3.01 (d, J = 5.4 Hz, 2H, –CH=CH–CH2), 2.13 (s, 6H, CH3). C NMR (101
MHz, DMSO-d6) δ 163.67, 158.31, 156.45, 155.34, 141.59, 139.85, 139.75, 139.03,
139.02, 138.18, 137.29, 132.26, 129.06 (2C), 128.96, 127.96, 127.61 (2C), 126.57,
125.40, 123.09, 122.89, 120.95, 118.03, 115.02, 112.63, 110.81, 103.88, 60.17, 49.71,
45.58 (2C). ESI-MS m/z: 579.4 [M+H]+. Anal. calcd. for C32H31ClN8O (%): C, 66.37; H,
5.40; N, 19.35; found (%): C, 66.32; H, 5.38; N, 19.51.
4.1.9.8. (E)-N-(3-(5-Chloro-2-((E)-2-(1-(4-fluorophenyl)ethylidene)hydrazinyl)pyrimidin-
4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide
(15h).
Flash
column
chromatography was performed using (CH2Cl2:MeOH, 60:1 to 20:1 v/v). HPLC purity:
98.5%. A white solid. Yield: 63%. mp 151.9–153.0 °C. IR (KBr, cm-1): 3405.7 (NH),
1
2923.0, 2852.5 (CH2, CH3), 2784.9 (N–CH3), 1680.0 (CO). H NMR (400 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6) δ 10.47 (s, 1H, –NH–N), 10.03 (s, 1H, NHCO), 8.89 (s, 1H, NH), 8.18 (s, 2H,
ArH), 7.82–7.88 (m, 3H, ArH), 7.47 (d, J = 7.9 Hz, 1H, ArH), 7.32 (t, J = 8.1 Hz, 1H,
ArH), 7.20 (t, J = 8.8 Hz, 2H, ArH), 6.76 (dt, J = 14.3, 7.0 Hz, 1H, –CH=CH–CH2), 6.49
(d, J = 15.3 Hz, 1H, –CH=CH–CH2), 3.84 (d, J = 6.5 Hz, 2H, –CH=CH–CH2), 2.70 (s,
6H, CH3), 2.30 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 163.95, 162.48, 161.52,
158.75, 156.29, 155.21, 145.22, 139.80, 139.22, 135.78, 132.22, 128.92, 128.18 (2C),
118.18, 115.55, 115.30 (2C), 114.03, 105.18, 57.37, 42.56 (2C), 13.78. ESI-MS m/z:
482.1 [M+H]+. Anal. calcd. for C24H25ClFN7O (%): C, 59.81; H, 5.23; N, 20.34; found
(%): C, 59.77; H, 5.28; N, 20.35.
4.1.9.9. (E)-N-(3-(5-Chloro-2-((E)-2-(1-(2,4-difluorophenyl)ethylidene)hydrazinyl)pyrimi
din-4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide
(15i).
Flash
column
chromatography was performed using (CH2Cl2:MeOH, 50:1 to 30:1 v/v). HPLC purity:
99.1%. A white solid. Yield: 63%. mp 162.3–163.6 °C. IR (KBr, cm-1): 3415.8 (NH),
1
2943.2, 2821.4 (CH2, CH3), 2778.4 (N–CH3), 1675.3 (CO). H NMR (400 MHz,
DMSO-d6) δ 10.17 (s, 1H, –NH–N), 10.03 (s, 1H, NHCO), 8.89 (s, 1H, NH), 8.18 (s, 1H,
ArH), 8.11 (s, 1H, –N=CH, ArH), 7.68–7.76 (m, 2H, ArH), 7.39 (d, J = 7.8 Hz, 1H, ArH),
7.27 (ddd, J = 17.9, 12.8, 5.3 Hz, 2H, ArH), 7.09 (dt, J = 8.4, 2.0 Hz, 1H, ArH), 6.73 (dt,
J = 15.3, 6.2 Hz, 1H, –CH=CH–CH2), 6.33 (d, J = 15.1 Hz, 1H, –CH=CH–CH2), 3.22 (s,
2H, –CH=CH–CH2), 2.29 (s, 3H, CH3), 2.28 (s, 6H, CH3). 13C NMR (101 MHz,
DMSO-d6) δδ 163.31, 161.32, 161.30, 159.40, 158.71, 156.30, 155.25, 143.38, 139.69,
139.41, 131.24, 128.89, 117.88, 115.16, 113.90, 112.09, 105.53, 105.09, 104.83, 99.99,
59.41, 44.77 (2C), 17.19.
ESI-MS m/z: 500.3 [M+H]+. Anal. calcd. for C24H24ClF2N7O
(%): C, 57.66; H, 4.84; N, 19,61; found (%): C, 57.58; H, 4.90; N, 19.56.
4.1.9.10. (E)-N-(3-(5-Chloro-2-((E)-2-(1-p-tolylethylidene)hydrazinyl)pyrimidin-4-ylamin
o)phenyl)-4-(dimethylamino)but-2-enamide (15j). Flash column chromatography was
performed using (CH2Cl2:MeOH, 50:1 to 30:1 v/v). HPLC purity: 98.7%. A white solid.
1
Yield: 63%. mp 135.3–138.0 °C. H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H,
–NH–N), 9.95 (s, 1H, NHCO), 8.85 (s, 1H, NH), 8.17 (s, 2H, ArH), 7.89 (d, J = 7.3 Hz,
1H, ArH), 7.73 (d, J = 8.1 Hz, 2H, ArH), 7.49 (d, J = 7.9 Hz, 1H, ArH), 7.30 (t, J = 8.1
Hz, 1H, ArH), 7.19 (d, J = 8.0 Hz, 2H, ArH), 6.76 (dt, J = 14.8, 6.7 Hz, 1H,
–CH=CH–CH2), 6.47 (d, J = 15.3 Hz, 1H, –CH=CH–CH2), 3.12 (d, J = 7.2 Hz, 2H,
–CH=CH–CH2), 2.56 (s, 6H, CH3), 2.32 (s, 3H, CH3), 2.28 (s, 3H, CH3). ESI-MS m/z:
478.2 [M+H]+. Anal. calcd. for C25H28ClN7O (%): C, 62.82; H, 5.90; N, 20.51; found (%):
C, 62.75; H, 5.92; N, 20.52.
4.1.9.11. (E)-N-(3-(5-Chloro-2-((E)-2-(1-(4-methoxyphenyl)ethylidene)hydrazinyl)pyrimi
din-4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide
(15k).
Flash
column
chromatography was performed using (CH2Cl2:MeOH, 50:1 to 30:1 v/v). HPLC purity:
98.6%. A white solid. Yield: 67%. mp 131.1–132.4 °C. 1H NMR (400 MHz, DMSO-d6) δ
10.33 (s, 1H, –NH–N), 9.87 (s, 1H, NHCO), 8.87 (s, 1H, NH), 8.16 (s, 1H, ArH), 8.11 (d,
J = 2.4 Hz, 1H, ArH), 7.87 (br, 1H, ArH), 7.77 (d, J = 7.7 Hz, 2H, ArH), 7.46 (d, J = 7.3
Hz, 1H, ArH), 7.30–7.33 (m, 1H, ArH), 6.92 (d, J = 7.9 Hz, 2H, ArH), 6.73 (m, 1H,
–CH=CH–CH2), 6.43 (d, J = 15.3 Hz, 1H, –CH=CH–CH2), 3.79 (s, 3H, OCH3), 3.69 (d, J

ACCEPTED MANUSCRIPT
= 6.3 Hz, 2H, –CH=CH–CH2), 2.60 (s, 6H, CH3), 2.25 (s, 3H, CH3). ESI-MS m/z: 494.5
[M+H]+. Anal. calcd. for C25H28ClN7O2 (%): C, 60.78; H, 5.71; N, 19.85; found (%): C,
60.71; H, 5.76; N, 19.83.
4.1.9.12. (E)-N-(3-(5-Chloro-2-((E)-2-(1-(3-methoxyphenyl)ethylidene)hydrazinyl)pyrimi
din-4-ylamino)phenyl)-4-(dimethylamino)but-2-enamide
(15l).
Flash
column
chromatography was performed using (CH2Cl2:MeOH, 100:1 to 35:1 v/v). HPLC purity:
1
98.3%. A white solid. Yield: 45%. mp 88.2–90.9 °C. H NMR (400 MHz, DMSO-d6) δ
10.18 (s, 1H, –NH–N), 9.96 (s, 1H, NHCO), 8.88 (s, 1H, NH), 8.19 (s, 1H, ArH), 8.01 (m,
2H, ArH), 7.45 (d, J = 7.8 Hz, 1H, ArH), 7.40 (d, J = 7.9 Hz, 1H, ArH), 7.30–7.33 (m, 3H,
ArH), 6.93 (dd, J = 8.2, 1.7 Hz, 1H, ArH), 6.73 (dt, J = 14.1, 6.0 Hz, 1H, –CH=CH–CH2),
6.34 (d, J = 15.1 Hz, 1H, –CH=CH–CH2), 3.77 (s, 3H, OCH3), 3.42 (d, J = 6.1 Hz, 2H,
–CH=CH–CH2), 2.37 (s, 6H, CH3), 2.28 (s, 3H, CH3). 13C NMR (101 MHz, DMSO-d6) δ
163.16, 162.54, 159.72, 158.78, 156.30, 155.25, 146.05, 140.72, 139.87, 139.43, 129.66,
129.32, 128.91, 118.68, 117.81, 115.06, 113.77, 113.65, 112.32, 105.25, 59.07, 55.63
(2C), 44.41, 13.81. ESI-MS m/z: 494.3 [M+H]+. Anal. calcd. for C25H28ClN7O2 (%): C,
60.78; H, 5.71; N, 19.85; found (%): C, 60.79; H, 5.82; N, 19.80.
4.1.9.13. (E)-N-(3-(5-Chloro-2-((E)-2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)pyrimidin-4-
ylamino)phenyl)-4-(dimethylamino)but-2-enamide (15m). Flash column chromatography
was performed using (CH2Cl2:MeOH, 30:1 to 12:1 v/v). HPLC purity: 97.9%. A yellow
1
solid. Yield: 36%. mp 186.5–188.3 °C. H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H,
–NH–N), 9.71 (s, 1H, NHCO), 9.17 (s, 1H, NH), 8.56 (d, J = 4.6 Hz, 1H, ArH), 8.25 (s,
1H, ArH), 8.12–8.20 (m, 2H, ArH), 7.76–7.80 (m, 2H, ArH), 7.47 (dd, J = 7.9, 0.7 Hz, 1H,
ArH), 7.33–7.38 (m, 2H, ArH), 6.72 (m, 1H, –CH=CH–CH2), 6.44 (d, J = 15.2 Hz, 1H,
–CH=CH–CH2), 3.92 (d, J = 6.9 Hz, 2H, –CH=CH–CH2), 2.77 (s, 6H, CH3), 2.39 (s, 3H,
CH3). ESI-MS m/z: 465.3 [M+H]+. Anal. calcd. for C23H25ClN8O (%): C, 59.41; H, 5.42;
N, 24.10; found (%): C, 59.37; H, 5.43; N, 23.99.
4.1.9.14. (E)-N-(3-(5-Chloro-2-((E)-2-(1-(pyridin-3-yl)ethylidene)hydrazinyl)pyrimidin-4-
ylamino)phenyl)-4-(dimethylamino)but-2-enamide (15n). Flash column chromatography
was performed using (CH2Cl2:MeOH, 50:1 to 15:1 v/v). HPLC purity: 99.0%. A yellow
1
solid. Yield: 52%. mp 206.7–207.2 °C. H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H,
–NH–N), 10.20 (s, 1H, NHCO), 9.03 (d, J = 1.3 Hz, 1H, NH), 8.93 (s, 1H, ArH), 8.53 (d,
J = 4.5 Hz, 1H, ArH), 8.21 (s, 1H, ArH), 8.15–8.17 (m, 2H, ArH), 7.93 (d, J = 6.0 Hz, 1H,
ArH), 7.44 (d, J = 7.6 Hz, 1H, ArH), 7.40 (dd, J = 8.0, 4.8 Hz, 1H, ArH), 7.32 (t, J = 8.1
Hz, 1H, ArH), 6.74 (dt, J = 14.3, 7.0 Hz, 1H, –CH=CH–CH2), 6.46 (d, J = 15.4 Hz, 1H,
–CH=CH–CH2), 3.77 (d, J = 5.4 Hz, 2H, –CH=CH–CH2), 2.66 (s, 6H, CH3), 2.34 (s, 3H,
CH3). ESI-MS m/z: 465.3 [M+H]+. Anal. calcd. for C23H25ClN8O (%): C, 59.41; H, 5.42;
N, 24.10; found (%): C, 59.45; H, 5.37; N, 24.16.
4.1.9.15. (E)-N-(3-(5-Chloro-2-((E)-2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)pyrimidin-4-
ylamino)phenyl)-4-(dimethylamino)but-2-enamide (15o). Flash column chromatography
was performed using (CH2Cl2:MeOH, 35:1 to 13:1 v/v). HPLC purity: 98.5%. A yellow
1
solid. Yield: 26%. mp 237.3–238.7 °C. H NMR (400 MHz, DMSO-d6) δ 10.32 (s, 1H,

ACCEPTED MANUSCRIPT
–NH–N), 10.29 (s, 1H, NHCO), 8.94 (s, 1H, NH), 8.55 (d, J = 5.0 Hz, 2H, ArH), 8.22 (s,
1H, ArH), 8.19 (s, 1H, ArH), 7.72–7.76 (m, 3H, ArH), 7.46 (d, J = 7.6 Hz, 1H, ArH), 7.31
(t, J = 8.1 Hz, 1H, ArH), 6.71 (dt, J = 14.8, 5.7 Hz, 1H, –CH=CH–CH2), 6.33 (d, J = 15.2
Hz, 1H, –CH=CH–CH2), 3.06 (d, J = 5.1 Hz, 2H, –CH=CH–CH2), 2.30 (s, 3H, CH3),
2.16 (s, 6H, CH3). 13C NMR (101 MHz, DMSO-d6) δ 163.45, 162.56, 158.53, 156.40,
155.26, 150.16 (2C), 146.19, 143.19, 139.62, 139.60, 128.82, 127.71, 120.23 (2C),
117.94, 115.27, 114.10, 105.86, 59.62, 44.95 (2C), 13.11. ESI-MS m/z: 465.2 [M+H]+.
Anal. calcd. for C23H25ClN8O (%): C, 59.41; H, 5.42; N, 24.10; found (%): C, 59.38; H,
5.46; N, 24.16.
4.1.10. General procedure for preparation of compounds (16a–16b)
A mixture of 11 (0.5 mmol), 13 (0.5 mmol) and TFA (0.6 mmol) in i-PrOH (5 mL)
was stirred under reflux for 3 h. The reaction mixture was cooled to room temperature,
and the precipitate was filtered to give a crude product, which was recrystallized from
i-PrOH to generate the target compounds.
4.1.10.1. N-(3-(5-Chloro-2-(2-(4-fluorobenzoyl)hydrazinyl)pyrimidin-4-ylamino)phenyl)a
crylamide (16a). Flash column chromatography was performed using (petroleum
ether:ethyl acetate, 5:1 to 3:2 v/v)). HPLC purity: 98.7%. A white solid. Yield: 39%. mp
197.9–200.9 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, 1H, NH), 10.06 (s, 1H, NH),
8.97 (s, 1H, NHCO), 8.82 (s, 1H, NH), 8.09 (s, 1H, ArH), 7.87–7.93 (m, 3H, ArH), 7.46
(s, 1H, ArH), 7.30 (br, 3H, ArH), 6.63 (s, 1H, ArH), 6.45 (dd, J = 14.7, 10.5 Hz, 1H,
–CH=CH2), 6.25 (d, J = 16.6 Hz, 1H, –CH=CH2), 5.74 (d, J = 10.1 Hz, 1H, –CH=CH2).
ESI-MS m/z: 427.0 [M+H]+. Anal. calcd. for C20H16ClFN6O2 (%): C, 56.28; H, 3.78; N,
19.69; found (%): C, 56.31; H, 3.71; N, 19.82.
4.1.10.2. N-(3-(5-Chloro-2-(2-(4-methoxybenzoyl)hydrazinyl)pyrimidin-4-ylamino)phenyl
)acrylamide (16b). Flash column chromatography was performed using (petroleum
ether:ethyl acetate, 6:1 to 1:1 v/v)). HPLC purity: 99.3%. A white solid. Yield: 72%. mp
213.6–215.6 °C. 1H NMR (400 MHz, DMSO-d6) δ 10.79 (s, 1H, NH), 10.67 (s, 1H, NH),
10.49 (s, 1H, NH), 10.35 (s, 1H, NHCO), 8.29 (s, 1H, ArH), 8.03 (s, 1H, ArH), 7.93 (d, J
= 8.3 Hz, 2H, ArH), 7.62 (d, J = 7.4 Hz, 1H, ArH), 7.41 (t, J = 7.7 Hz, 1H, ArH), 7.34 (d,
J = 7.5 Hz, 1H, ArH), 7.08 (d, J = 8.3 Hz, 2H, ArH), 6.53 (dd, J = 17.0, 10.1 Hz, 1H,
–CH=CH2), 6.28 (d, J = 17.2 Hz, 1H, –CH=CH2), 5.78 (d, J = 10.6 Hz, 1H, –CH=CH2),
3.83 (s, 3H, OCH3). ESI-MS m/z: 439.5 [M+H]+. Anal. calcd. for C21H19ClN6O3 (%): C,
57.47; H, 4.36; N, 19.15; found (%): C, 57.80; H, 4.29; N, 19.12.
4.2. Pharmacology
4.2.1. In vitro enzymatic activity assay
The experiments were carried out by a well-established mobility shift assay, and
EGFR kinases (EGFR T790M/L858R, EGFR T790M/delE746_A750, EGFR T790M and
WT EGFR) were purchased from Invitrogen. The kinase base buffer was consist of 50
mM HEPES (pH 7.5), 0.0015% Brij35 and 2 mM DTT, while the stop buffer contained a
mixture of 100 mM HEPES (pH 7.5), 0.015% Brij-35, 0.2% Coating Reagent and 50 mM

ACCEPTED MANUSCRIPT
EDTA.
Initially, the tested compounds were diluted to 50-fold of the final desired highest
concentration in reaction by 100% DMSO. Subsequently, 30 ꢀL of the solution was
transferred to 60 ꢀL of 100% DMSO in the next well and so forth for a total of 5
concentrations. No compound and no enzyme controls were prepared by adding 100 ꢀL
DMSO to two empty wells in the same 96-well plate. Then, 10 ꢀL of compound was
transferred to a new 96-well plate, which was marked as the intermediate plate.
Additional 90 ꢀL of kinase buffer was added to each well of intermediate plate. The
mixture in intermediate plate was shaked for 10 min. The assay plate was prepared after
transferring 5 ꢀL of each well from the 96-well intermediate plate to a 384-well plate in
duplicates. The prepared enzyme solution (appropriate EGFR in kinase base buffer) was
added to the assay plate, which was then incubated at room temperature for 10 min,
followed by the addition 10 ꢀL of prepared peptide solution (FAM-labeled peptide and
ATP in kinase base buffer). The mixture was incubated at 28 °C for another 1 hour, then
25 ꢀL of stop buffer was added. The convertion data was copied from Caliper program,
and the values were converted to inhibition values. Percent inhibition =
(max-conversion)/(max-min) × 100.
4.2.2. MTT assay
The target compounds were screened in A549, H1975 and HT-29 cells by a standard
MTT assay. The cancer cell lines were cultured in minimum essential medium (MEM)
supplement with 10% fetal bovine serum (FBS).
Approximate 4 × 103 cells, suspended in MEM medium, were plated onto each well
of a 96-well plate and incubated in 5% CO2 at 37 °C for 24 h. The tested compounds were
added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT
was added to each well at a terminal concentration of 5 ꢀg/mL, and incubated with cells
at 37 °C for 4 h. The formazan crystals were dissolved in 100 ꢀL DMSO each well, and
the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the
reference wavelength) was measured with an ELISA reader. All of the compounds were
tested three times in each of the cell lines. The results expressed as IC50 (inhibitory
concentration 50%) were the averages of at least three determinations and calculated by
using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
This work was supported by Program for Innovative Research Team of the Ministry
of Education and Program for Liaoning Innovative Research Team in University. Also,
we were supported by grants from Science Foundation of Shenyang pharmaceutical
university (No. 51120219).
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ACCEPTED MANUSCRIPT
Legends
Figure 1. Representative EGFR inhibitors.
Figure 2. Design strategy of target compounds.
Scheme 1. Reagents and conditions: (a) K2CO3, DMF, 50 °C, 3 h; (b) NH2NH2·H2O,
MeCN, reflux, 3 h; (c) appropriate aromatic aldehyde or ketone, i-PrOH, reflux, 6–8 h; (d)
TFA, DCM, 25 °C, 2–7 h; (e) Acryloyl chloride, DIPEA, DCM, 25 °C, 1 h or
(E)-4-(dimethylamino)but-2-enoic acid, HATU, DIPEA, DCM, 25 °C, 3–9 h.
Scheme 2. Reagents and conditions: (a) TFA, DCM, 25 °C, 1 h; (b) Acryloyl chloride,
DIPEA, DCM, 25 °C, 1 h; (c) NH2NH2·H2O, EtOH, reflux, 5 h; (d) TFA, i-PrOH, reflux,
3 h.
Table 1. Cellular antiproliferative activities of compounds 14a, 16a and 16b
Table 2. Cellular antiproliferative activities of the hydrazone analogs
Table 3. In vitro enzymatic inhibition of compounds on different forms of EGFR

ACCEPTED MANUSCRIPT
Table 1. Cellular antiproliferative activities of compounds 14a, 16a and 16b
IC50 (µM)a
Compd
A549
H1975
10.44 ± 1.36
31.00 ± 3.13
25.99 ± 2.25
0.43 ± 0.17
5.31 ± 0.96
8.46 ± 0.77
14a
16a
16b
aThe biological data are generated from at least three independent experiments.

ACCEPTED MANUSCRIPT
Table 2. Cellular antiproliferative activities of the hydrazone analogs
IC50 (µM)a
Compd
Ar
R1
R2
H
A549
H1975
HT-29
4-F-Ph
4-F-Ph
H
H
13.68 ± 2.59
9.92 ± 0.76
12.32 ± 1.03
9a
10.44 ± 1.36
6.06 ± 0.54
11.25 ± 1.77
>100
0.43 ± 0.17
0.29 ± 0.07
0.38 ± 0.06
1.35 ± 0.39
0.39 ± 0.11
0.30 ± 0.12
0.74 ± 0.15
1.06 ± 0.37
1.19 ± 0.25
3.69 ± 1.07
0.44 ± 0.08
0.59 ± 0.16
0.17 ± 0.05
1.58 ± 0.29
4.13 ± 0.35
5.45 ± 0.37
0.26 ± 0.12
6.34 ± 1.59
3.50 ± 0.69
4.76 ± 0.66
11.03 ± 2.33
5.05 ± 0.71
4.36 ± 1.24
3.21 ± 0.67
4.56 ± 1.17
8.99 ± 1.22
12.80 ± 0.96
3.66 ± 0.68
3.30 ± 0.77
2.92 ± 0.08
4.82 ± 1.23
25.19 ± 2.59
9.68 ± 1.35
2.12 ± 0.37
14a
2-F-Ph
H
H
14b
14c
14d
14e
14f
14g
14h
14i
2,4-(F)2-Ph
H
4-F-Ph
2,4-(F)2-Ph
4-CH3-Ph
4-CH3O-Ph
3-CH3O-Ph
4-pyridyl
Me
Me
Me
Me
Me
Me
H
8.20 ± 0.38
1.63 ± 0.25
5.36 ± 1.68
7.71 ± 0.72
10.33 ± 2.24
28.71 ± 1.69
8.94 ± 1.26
6.77 ± 0.35
2.17 ± 0.23
6.24 ± 0.67
47.68 ± 3.54
28.68 ± 0.67
2.91 ± 0.16
14j
15a
15b
15c
15d
15e
15f
15g
2-F-Ph
4-F-Ph
H
2,4-(F)2-Ph
4-CH3O-Ph
pyridin-4-yl
indol-3-yl
H
H
H
H
H

ACCEPTED MANUSCRIPT
4-F-Ph
Me
Me
Me
8.96 ± 1.56
5.57 ± 0.76
10.8 ± 1.41
1.16 ± 0.26
0.48 ± 0.05
1.37 ± 0.27
10.2 ± 1.09
3.46 ± 0.32
11.8 ± 0.30
15h
15i
15j
2,4-(F)2-Ph
4-CH3-Ph
4-CH3O-Ph
Me
7.12 ± 0.33
0.99 ± 0.18
6.83 ± 0.57
15k
3-CH3O-Ph
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
Me
Me
Me
Me
11.38 ± 0.39
0.83 ± 0.12
17.29 ± 2.40
34.86 ± 1.12
0.91 ± 0.06
0.077 ± 0.005
2.35 ± 0.15
6.24 ± 0.36
3.87 ± 0.41
0.12 ± 0.06
9.66 ± 0.37
10.93 ± 0.59
15l
15m
15n
15o
9.86 ± 0.62
1.87 ± 0.37
8.71 ± 0.60
8.19 ± 0.68
2.95 ± 0.29
gefitinib
WZ4002
0.058 ± 0.022
aThe biological data are generated from at least three independent experiments.

ACCEPTED MANUSCRIPT
Table 3. In vitro enzymatic inhibition of compounds on different forms of EGFR
EGFRIC50 (µM)a
Compd
T790M/delE746
_A750
TLb/WT
T790M/L858R
T790M
WT
selectivity
0.86
1.51
11.57
1.68
2.27
5.67
0.79
0.68
2.52
>20
0.57
1.91
ND
1.12
4.74
ND
>20
>20
>23.3
>13.2
>1.7
>11.9
5.2
14a
14b
14c
14e
14f
14g
15a
15b
15c
15f
>20
1.29
ND
2.53
ND
>20
11.75
>20
ND
ND
>3.5
>25.3
15.7
>7.9
-
3.27
2.23
5.11
ND
1.56
1.49
2.51
ND
>20
10.65
>20
>20
0.88
0.76
0.79
>20
>22.7
15g
2.35
0.72
ND
1.25
ND
ND
0.82
ND
ND
4.27
ND
15.32
>20
6.5
>27.8
>8.4
>4.6
-
15h
15i
2.38
>20
15m
4.32
ND
>20
15n
12.72
10.62
0.022
gefitinib
0.031
ND
0.079
2.5
WZ4002
aThe biological data are means from at least two replicated experiments.
bTL represents EGFR T790M/L858R.
ND: not determined.