Identification of hydrazone moiety-bearing aminopyrimidines as potent antitumor agents with selective inhibition of gefitinib-resistant H1975 cancer cells

Article abstract of DOI:10.1016/j.cclet.2016.11.030

Based on our previous work, a series of hydrazone moiety-bearing aminopyrimidines were synthesized. The compounds were evaluated for inhibitory activities against EGFR T790M/L858R and antiproliferative activities against H1975 and A549 NSCLC cell lines harboring different forms of EGFR. Compounds 7f and 7k exhibited potent and selective activity in inhibition of gefitinib-resistant H1975 cancer cells (IC₅₀; 0.45, 0.2?μmol/L) while were much less active on A549 cancer cells (IC₅₀; 52.83, >100?μmol/L). Both compounds could be served as promising lead compounds for further investigation.

Full text of DOI:10.1016/j.cclet.2016.11.030

Accepted Manuscript
Title: Identification of hydrazone moiety-bearing
aminopyrimidines as potent antitumor agents with selective
inhibition of gefitinib-resistant H1975 cancer cells
Author: Ming-Ze Qin Lei Wang Shuang Yan Jun-Jie Ma Ye
Tian Yan-Fang Zhao Ping Gong
PII:
S1001-8417(16)30417-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2016.11.030
CCLET 3904
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
5-9-2016
30-10-2016
11-11-2016
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Original article
Identification of hydrazone moiety-bearing aminopyrimidines as potent antitumor
agents with selective inhibition of gefitinib-resistant H1975 cancer cells
Ming-Ze Qina, *, Lei Wanga, Shuang Yana, Jun-Jie Mab, Ye Tiana, Yan-Fang Zhaoa, Ping Gonga,*
aKey Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, Shenyang 110016, China
bSchool of Biomedical Sciences, Huaqiao University, Quanzhou 362021, China
 Corresponding authors.
E-mail addresses: qinmingze001@126.com (M.-Z. Qin), gongpinggp@126.com (P. Gong)
Graphical Abstract
Some aminopyrimidine analogues were identified as potent agents in inhibition of gefitinib-resistant H1975 NSCLC cells.
ABSTRACT
Based on our previous work, a series of hydrazone moiety-bearing aminopyrimidines were synthesized. The compounds were evaluated for
inhibitory activities against EGFR T790M/L858R and antiproliferative activities against H1975 and A549 NSCLC cell lines harboring different
forms of EGFR. Compounds 7f and 7k exhibited potent and selective activity in inhibition of gefitinib-resistant H1975 cancer cells (IC50; 0.45,
0.2 μmol/L) while were much less active on A549 cancer cells (IC50; 52.83, >100 μmol/L). Both compounds could be served as promising lead
compounds for further investigation.
Keywords:
Aminopyrimidines
EGFR T790M/L858R
Selectivity
Anti-resistance
Antiproliferative activity
1. Introduction
Lung cancer is the leading cause of death from cancer worldwide. Patients suffer a poor clinical outcome with five-year
survival of less than 15% [1]. Non-small-cell lung cancer (NSCLC) represents a major type which accounts for approximately
80–85% of all lung cancers [2,3].
The blockade of epidermal growth factor (EGF) signaling pathway has been identified a promising approach for intervention of
NSCLC, resulting in several therapeutic agents [4]. Gefitinib, a selective inhibitor targeting EGF receptor, has yielded great

clinical benefits in patients with NSCLC, especially those harboring the EGFR-activating mutations such as delE746_A750 and
L858R [5]. However, a secondary mutation T790M at the “gatekeeper” position of EGFR leads to drug resistance in much cases
after 10–14 months of gefitinib administration [6,7]. In order to overcome the resistance mechanism correlated with T790M
mutation, a mount of irreversible inhibitors such as rociletinib and osimertinib have been developed, in which a acrylamide
functionality was devised to undergo a Michael addition reaction with Cys797 [8–10] .
In our previous work, we reported a series of hydrazone moiety-bearing aminopyrimidines as selective inhibitors of EGFR
T790M mutant, from which we identified a potent compound (1) (Fig. 1) as the precusor [11]. As a continuous study, we initiated
a further optimization program on this promising series in order to discover compounds with more potent activity. Biological
evaluations led to the identification of compound 7f and 7k which showed significant and selective activity in inhibition of
gefitinib-resistant H1975 cancer cells.
2. Results and discussion
2.1. Chemistry
The synthesis of the target compounds (7a–k) is described in Scheme 1. The intermediate 4 was synthesized via a sequential
coupling of 2,4-dichloro-5-(trifluoromethyl)pyrimidine (2) with tert-butyl (3-aminophenyl)carbamate and hydrazine hydrate using
a modified condition as previously reported [11]. The condensation of intermediate 4 with an appropriate aromatic aldehyde or
ketone in an addition-elimination sequence generated intermediates 5a–k [12,13], which underwent a convenient deprotection
reaction to yield amines 6a–k [14]. Subsequently, amidation of 6 with commercial (E)-4-(dimethylamino)but-2-enoic acid using
HATU as a dehydrant furnished the desired compounds.
2.2. Design of new compounds
At beginning, compound 1 was docked in the active site of EGFR T790M (PDB: 3IKA) [15] to investigate the probable
interactions with kinase using Discovery Studio 3.1. The results reflected that it formed anticipated hydrogen bonds with Met793 in
hinge region. However, the functional acrylamide group adopted a reversed conformation, which prevented it from interacting
well with Cys797. Encouraged by these observations, we initiated a program to develop an additional series of compounds with
more suitable conformation to form desirable covalent bond with Cys797. In binding models of both 1 and WZ4002, a hydrophobic
interaction was observed between Cl atom with Met790. We speculated that the replacement of the Cl atom with a more
hydrophobic CF3 group might increase the interaction and more importantly, it could enforce the compound adopting a
significantly changed conformation as compared with compound 1. To rationalize this hypothesis, a molecular docking was
conducted using compound 7b. Figure 2 showed the putative binding model with EGFR T790M, which demonstrated that
apparent hydrogen-bond was formed between pyrimidine with Met793. Moreover, compound 7b adopted a very favorable
conformation close to Cys797. As a consequence, an additional series of hydrazone moiety-bearing aminopyrimidines were
synthesized and evaluated in both enzymatic and cellular levels with the aim to identify compounds with more potent activity.
2.3. Inhibitory activity against EGFR kinase
The newly synthesized compounds were screened in a double mutant EGFR kinase, namely T790M/L858R, as well as in wild
type (WT) EGFR. The enzymatic assay was carried out using a well-established mobility shift assay, and the results were shown
in Table 1. The biological data clearly demonstrated that all the target compounds suppressed EGFR T790M/L858R in
micromolar range (IC50, 0.75–5.39 μmol/L), and was more potent than gefitinib (positive control; IC50, 11.38 μmol/L). All the
target compounds were not active at 10 μmol/L against wild type EGFR, suggesting a favorable kinase selectivity. Compound 7b
exhibited significant activity against EGFR T790M/L858R, with IC50 value of 1.05 μmol/L, but no improvement was observed as
compared with the precusor 1 (IC50, 1.13 μmol/L). Further modification on terminal benzene ring indicated that electron-donating
groups in this region were better tolerated. Replacement of the fluorine atom with a methyl group led to compound 7d (IC50, 0.88
μmol/L), which was slightly more potent than 7b. Moreover, significantly increased potency could be observed when 7c (Ar =
2,4-(F)2-Ph; IC50, 5.39 μM) was compared with 7e (Ar = 2,4-(CH3O)2-Ph; IC50, 0.77 μmol/L). On the other hand, a substituent at
para-position was beneficial for high activity, as shifting the fluorine atom of compound 7b to ortho-position resulted in a 4.8-fold
decrease in activity (7a; IC50, 5.1 μmol/L). This effect could be explained by the putative binding model as described in Figure 2,
a hydrogen-bond was formed between fluorine atom with Lys716. The introduction of a methyl group in the hydrazone moiety (R1)
was not crucial for inhibition of EGFR T790M/L858R, which indicated that the structure-activity relationship (SAR) in this
region was limited. Interestingly, compound 7k containing a pyridine moiety instead of the benzene ring maintained favorable
potency (IC50, 3.32 μmol/L) as well as selectivity, which highlighted the potential for further optimization.
2.4. Inhibitory activity against H1975 and A549 NSCLC cells
Compounds 7a–k were screened in two type of NSCLC cancer cells including A549 (WT EGFR and K-Ras mutation) and
gefitinib-resistant H1975 (EGFR T790M/L858R). Notably, the attractive inhibitory activity against EGFR T790M/L858R
translated well into efficacy in inhibition of H1975 cancer cells (IC50; 0.2–1.06 μmol/L), except for compound 7b, and was much

more potent than that of gefitinib (IC50; 7.92 μmol/L). Also, the potency of the compounds was more pronounced against H1975
cells than against A549 cells, which correlated well with their kinase selectivity versus WT EGFR. Compound 7f demonstrated
potent activity in inhibition of H1975 cancer cells, with IC50 of 0.45 μmol/L. Taken the enzymatic profile together, compound 7f
was considered a promising lead in this chemical series. Also noteworthy is that compound 7k displayed the best activity in
inhibition of H1975 cells (IC50; 0.2 μmol/L), while was almost inactive against A549 cells at up to 100 μmol/L. Although it
possessed only moderate inhibition of EGFR T790M/L858R (IC50; 3.32 μmol/L), detailed optimization could be done to promote
the potency even further.
3. Conclusion
In this paper, we provided a valuable design strategy that aimed to improve the antitumor potency of compound 1, which was
identified previously as a selective inhibitor of EGFR T790M. The newly synthesized hydrazone moiety-bearing
aminopyrimidines maintained favorable kinase inhibitory activity and selectivity as compared to 1. More importantly, while these
compounds effectively suppressed proliferation of gefitinib-resistant H1975 cells, they showed weak effects on A549 cells,
indicating a favorable selectivity. The increasement in hydrophobicity accompanied with the introduction of the CF3 group might
partially account for the improved selectivity in cellular level. Compounds 7f and 7k potently and selectively inhibited
proliferation of H1975 cells, and were considered promising lead compounds for further investigation.
4. Experimental
4.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without further purification. All the reactions were
monitored by TLC using silica gel GF/UV 254. Flash chromatography was performed using silica gel (300–400 mesh). The purity
of the synthesized compounds was measured by high performance liquid chromatography (HPLC, Agilent, USA), and was
confirmed to be higher than 95%. Melting points were determined on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland). The 1H and 13C NMR spectra were recorded on Bruker AV-400 spectrometer at 400 MHz and
100 MHz, respectively, with TMS as an internal standard. The low resolution of ESI-MS was recorded on an Agilent 1100 LC-MS
spectrometer, and high resolution mass spectrometry was performed on a Biosystems Q-STAR Elite ESI-LC-MS/MS
spectrometer.
4.2. Biological evaluation
4.2.1. In vitro kinase assay
The experiments were carried out by a well-established mobility shift assay, and EGFR kinases (EGFR T790M/L858R and WT
EGFR) were purchased from Invitrogen. The kinase base buffer consists of 50 mmol/L HEPES (pH 7.5), 0.0015% Brij35 and 2
mmol/L DTT, while the stop buffer contained a mixture of 100 mmol/L HEPES (pH 7.5), 0.015% Brij-35, 0.2% Coating Reagent
and 50 mmol/L EDTA. Initially, the tested compounds were diluted to 50-fold of the final desired highest concentration in
reaction by 100% DMSO. Subsequently, 30 μL of the solution was transferred to 60 μL 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 μL DMSO to two empty
wells in the same 96-well plate. Then, 10 μL of compound was transferred to a new 96-well plate, which was marked as the
intermediate plate. Additional 90 μL 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 μL 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 μL 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 μL of stop
buffer was added. The conversion 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 cytotoxicity 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 μg/mL, and incubated with cells at 37 °C for 4 h. The formazan
crystals were dissolved in 100 μL 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.
Acknowledgment
This work was supported by grants from Science Foundation of Shenyang Pharmaceutical University (No. QNJJ2014502).

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Fig. 1. Structure of rociletinib, osimertinib, and the new compounds.

Fig. 2. Molecular docking of compound 7b with EGFR T790M. Hydrogen bond interactions were indicated with dashed lines in green.

Scheme 1. Reagents and conditions: (a) DIPEA, n-butyl alcohol, 25 °C, 3 h; (b) NH2NH2·H2O, pyridine, ethanol, 25 °C, 2 h; (c) appropriate aromatic
aldehyde or ketone, ethanol, reflux, 5–7 h; (d) TFA, DCM, 25 °C, 2–5 h; (e) (E)-4-(dimethylamino)but-2-enoic acid, HATU, DIPEA, DCM, 25 °C, 3–10
h.

Table 1. In vitro inhibitory profile of compounds 7a–k
EGFR IC50 (μmol/L)a
Cellular assay IC50 (μmol/L)b
Compound
Ar
R1
T790M/L858R
WT
H1975c
A549d
1.13 ± 0.12
5.10 ± 0.37
1.05 ± 0.13
5.39 ± 1.22
0.88 ± 0.20
0.77 ± 0.19
0.75 ± 0.07
2.21 ± 0.38
3.05 ± 0.65
2.83 ± 0.19
3.03 ± 0.38
3.32 ± 0.15
11.38 ± 1.23
0.019 ± 0.005
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.55 ± 0.09
0.96 ± 0.10
12.3 ± 0.57
0.51 ± 0.06
0.86 ± 0.13
0.47 ± 0.08
0.45 ± 0.12
0.57 ± 0.25
0.49 ± 0.09
0.59 ± 0.17
1.06 ± 0.22
0.20 ± 0.02
7.92 ± 0.93
0.076 ± 0.015
7.35 ± 1.17
48.44 ± 3.55
>100
1
7a
7b
7c
7d
7e
7f
7g
2-F-Ph
4-F-Ph
2,4-(F)2-Ph
4-Me-Ph
H
H
H
H
H
9.55 ± 0.86
17.26 ± 2.23
21.26 ± 3.32
52.83 ± 1.37
28.3 ± 1.59
18.52 ± 0.63
15.02 ± 1.95
9.26 ± 0.87
>100
2,4-(CH3O)2-Ph
3,4,5-(CH3O)3-Ph
2-F-Ph
H
Me
Me
Me
Me
Me
4-F-Ph
7h
7i
7j
2,4-(F)2-Ph
4-CH3O-Ph
Pyridin-2-yl
>10
>10
>10
7k
Gefitinib
Rociletinib
0.028 ± 0.009
0.312 ± 0.11
8.62 ± 0.26
44.68 ± 2.39
aEGFR inhibitory activities of compounds were evaluated using the mobility shift assay. Values are averages ± SD of at least two independent
experiments.
bInhibitory activities of compounds on cancer cells were determined by the MTT assay. Values are averages ± SD of at least three independent
experiments.
cH1975 is a human NSCLC cell line harboring EGFR T790M/L858R.
dA549 is a human NSCLC cell line harboring wild type EGFR and K-Ras mutation.