Novel hybrids of (phenylsulfonyl)furoxan and anilinopyrimidine as potent and selective epidermal growth factor receptor inhibitors for intervention of non-small-cell lung cancer

Article abstract of DOI:10.1021/jm400463q

A series of hybrids (12a-k) from (phenylsulfonyl)furoxan and anilinopyrimidine were synthesized and biologically evaluated as epidermal growth factor receptor (EGFR) inhibitors for intervention of non-small-cell lung cancer (NSCLC). Compound 12k exhibited

Full text of DOI:10.1021/jm400463q

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Article
Novel Hybrids of Phenylsulfonylfuroxan and Anilinopyrimidine
as Potent and Selective Epidermal Growth Factor Receptor
Inhibitors for Intervention of Non-Small Cell Lung Cancer
Chun Han, Zhangjian Huang, Chao Zheng, Ledong Wan, Lianwen
Zhang, Sixun Peng, Ke Ding, Hongbin Ji, Jide Tian, and Yihua Zhang
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 13 May 2013
Downloaded from http://pubs.acs.org on May 14, 2013
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Novel Hybrids of Phenylsulfonylfuroxan and
Anilinopyrimidine as Potent and Selective
Epidermal Growth Factor Receptor Inhibitors for
Intervention of NonꢀSmall Cell Lung Cancer
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Chun Han,a,b,# Zhangjian Huang,a,b,# Chao Zheng,c,# Ledong Wan,a,b Lianwen Zhang,d Sixun
Peng,a,b Ke Ding,d,* Hongbin Ji,c,* Jide Tian,e and Yihua Zhanga,b,*
aState Key Laboratory of Natural Medicines, bCenter of Drug Discovery, China Pharmaceutical
University, Nanjing 210009, P. R. China.
cState Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P. R. China.
dKey Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou
Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, P. R.
China.
eDepartment of Molecular and Medical Pharmacology, University of CaliforniaꢀLos Angeles, Los
Angeles, 90095, USA.
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ABSTRACT: A series of hybrids 12a−k from phenylsulfonylfuroxan and anilinopyrimidine
were synthesized and biologically evaluated as EGFR inhibitors for intervention of NSCLC.
Compound 12k exhibited strong and selective EGFR L858R/T790M inhibitory activity (IC50 =
0.047 ꢁM), and displayed antiproliferative effects on EGFR mutation NSCLC cell lines HCC827
(del E746_A750) and H1975 (L858R/T790M) with IC50s of 0.007 and 0.029 ꢁM, respectively.
Additionally, 12k released high levels of NO in H1975 cells but not in normal human cells, and
its activity was diminished by pretreatment with a NO scavenger. Furthermore, 12k induced
apoptosis of H1975 and HCC827 cells more strongly than WZ4002 (1), inhibited EGFR
downstream signaling in H1975 cells, and suppressed the NFꢀκB activation in H1975 cells while
1 had no significant effects under the same conditions. Finally, 12k substantially inhibited tumor
growth in a H1975 xenograft mouse model. Overall, 12k might be a promising candidate for the
treatment of NSCLC.
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INTRODUCTION
The epidermal growth factor receptor (EGFR) is an important mediator responsible for cell
proliferation, survival, adhesion, migration, and differentiation through downstream signaling
pathway activation.1 Dysregulation of EGFR caused by overexpression or mutation contributes to
malignant transformation,2 and is correlated with poor prognosis in a number of cancers,
including lung cancer,3 anal cancer4 and glioblastoma multiforme5. These features make EGFR a
good target for intervention of tumor.
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Lung cancer is the leading cause of death from cancer worldwide, with fiveꢀyear survival less
than 15%.6 Nonꢀsmall cell lung cancer (NSCLC) accounts for approximately 80−85% of lung
cancers.7 Overexpression of EGFR is found in the majority of NSCLCs,8 and EGFR mutations
occur in about 10% of NSCLC patients in the Caucasian population and 30–50% of NSCLC
patients from East Asian populations.9 Therefore, inhibition of EGFR has been recognized as an
attractive strategy for the treatment of NSCLC and validated in preclinical and clinical trials.10
Gefitinib11 and erlotinib12 (Figure 1), as the first generation ATPꢀcompetitive and reversible
EGFR inhibitors, were effective for NSCLC patients harbouring somatic EGFR mutations
L858R and delE746_A750 which account for 90% of all EGFR mutations in NSCLC.13
Unfortunately, due to acquired secondary mutations such as T790M missense in EGFR leading to
drug resistance in roughly 50% NSCLC patients, the ability of these EGFR inhibitors to
effectively treat NSCLC patients is shortꢀlived to subsequent treatments.14,15 EGFR T790M
mutation restores the affinity for ATP similar to that of wild type (WT) EGFR, and prevents the
reversible inhibitors from binding at higher ATP concentrations.16
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Second generation EGFR inhibitors such as canertinib17, afatinib18, neratinib19, and
dacomitinib20 (Figure 1) have been developed with intent to overcome the T790M mutation
related resistance.21 The molecules of these inhibitors are built based on a quinazoline or
quinoline core scaffold like gefitinib and erlotinib, but contain an electrophilic functionality that
can undergoes a Micheal addition reaction with a conserved cysteine residue present in EGFR
(Cys797) to achieve occupancy greater than that of the reversible inhibitors.22 However, since the
ATP affinity of EGFR T790M is similar to that of WT EGFR, these EGFR inhibitors at the
concentrations required to inhibit EGFR T790M will also effectively inhibit WT EGFR, leading
to the doseꢀlimiting toxicity such as diarrhoea and skin rash. Therefore, the clinical application of
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these inhibitors has been limited, especially in patients with gefitinib or erlotinibꢀresistant
NSCLC.17−20
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Recently, Gray et al. documented a novel series of substituted anilinopyrimidines as
irreversible EGFR inhibitors, which display good selectivity against EGFR T790M mutant over
the WT EGFR.23 The representative compound WZ400224 (1) shown in Figure 1 is 30 to 100ꢀ
fold more potent against EGFR T790M but up to 100ꢀfold less potent against WT EGFR than
irreversible quinazolineꢀbased inhibitors in vitro, and efficacious in murine cancer models of
T790M. Accordingly, the anilinopyrimidine is supposed to be an attracting scaffold for the
design of small molecule inhibitors against EGFR mutants.
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F
O
HN
N
Cl
H
N
N
O
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N
H
N
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N
Cl
N
O
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H
O
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afatinib
WZ4002 (1)
Figure 1. Representative reversible and irreversible EGFR inhibitors.
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Nitric oxide (NO) as a signaling and/or effector molecule plays a pivotal role in diverse
physiological and pathophysiological processes.25 It is wellꢀknown that high levels of NO
generated from NOꢀdonors can not only induce apoptosis and inhibit metastasis of tumor cells,
but also sensitize tumor cells to chemotherapy, radiation and immunotherapy in vitro and in
vivo26 by inhibiting nuclear factorꢀκB (NFꢀκB), hypoxiaꢀinducible factorꢀ1α (HIFꢀ1α), DNA
repair enzymes and drug efflux transporters, et al.27,28 As reported, NFꢀκB activation can suppress
the apoptotic potential of chemotherapeutic agents and contributes to resistance,29 whereas NO
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exerts negative feedback on iNOS expression by inhibiting the degradation of IκB (also known as
NFKBIA), thus preventing NFꢀκB activation.30 It has also been reported that suppressing NFꢀκB
can improve the response of NSCLC harboring EGFR mutations to EGFR inhibitors.31
As an important class of NO donors, furoxans are able to produce high levels of NO in vitro,
and inhibit the growth of tumors in vivo.32 Our group has developed a variety of
phenylsulfonylfuroxanꢀbased NO releasing compounds that exhibited selective antitumor activity
in vitro and in vivo.33−35 Based on these investigations, we hypothesized that hybridization of
such furoxan with anilinopyrimidine may release high levels of NO to exert synergistic antiꢀ
NSCLC effects with anilinopyrimidine. To test this hypothesis, a series of hybrids (12a−k) from
phenylsulfonylfuroxan and anilinopyrimidine were designed and synthesized. Their EGFR kinase
inhibitory activity, in vitro antiꢀNSCLC and apoptosis inductive activities, NOꢀreleaseing ability,
in vivo antitumor activity as well as the mechanism of action underlying their anticancer
activities were biologically evaluated.
RESULTS AND DISCUSSION
Chemistry. Synthesis of compounds 12a−k is illustrated in Scheme 1. Phenylpiperazine 2 was
prepared by amination at 5ꢀposition of 5ꢀfluoroꢀ2ꢀnitrophenyl methyl ether according to
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reference,36 followed by treatment with ethyl bromoacetate to give ester 3. Then the nitro group
of 3 was reduced to yield arylamine 4. Meanwhile, aryl ether 6 was obtained by regioselective
coupling of 3ꢀnitrophenol 5 to the 4ꢀposition of 2,4,5ꢀtrichloropyrimidine. Subsequent reduction
of nitro group of 6 afforded arylamine 7. Acylation of amino group of 7 led to acrylamide 8.
Coupling of 4 with 8 in the presence of trifluoroacetic acid offered anilinopyrimidine 9, which
was converted to corresponding acid 10 by hydrolysis. Finally, condensation of 10 with various
monophenylsulfonylfuroxans 11a−k, synthesized in a fourꢀstep sequence as described previously
by us,33 provided the target compounds 12a−k.
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Scheme 1. Synthetic route of 12a−k.
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Reagents and conditions: (a) piperazine, dioxane, reflux, overnight, 97%; (b) ethyl bromoacetate,
K2CO3, acetone, reflux, 4 h, 90%; (c) iron powder, NH4Cl, THF/H2O (1/1, v/v), reflux, 3 h, 99%
for 4, 80% for 7; (d) 2,4,5ꢀtrichloropyrimidine, K2CO3, DMF, rt, 2 h, 95%; (e) acryloyl chloride,
Et3N, anhydrous CH2Cl2, 0 ºC, 1 h, 90%; (f) TFA, 2ꢀBuOH, reflux, 3 h, 80%; (g) LiOH,
THF/H2O (1/1, v/v), 0 ºC, 1 h, 98%; (h) 11a−k, DCC/DMAP, anhydrous CH2Cl2, rt, overnight,
70%−85%.
Kinase Inhibitory Activity. The inhibitory activity of the target compounds 12a−k against
two mutant EGFRs (L858R and L858R/T790M) and WT EGFR were evaluated using a wellꢀ
established FRETꢀbased Z’ꢀLyte assay,37,38 with gefitinib and 1 employed as positive controls. As
shown in Table 1, all compounds, as 1 did, suppressed EGFR L858R/T790M more selectively
(IC50s = 0.006−0.047 ꢁM) relative to WT EGFR (IC50s = 0.039−0.292 ꢁM) and EGFR L858R
(IC50s = 0.040−0.305 ꢁM). Although less potent than 1 (IC50 = 0.0008 ꢁM), these compounds
were more active than gefitinib (IC50 = 0.941 ꢁM) against EGFR L858R/T790M.
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Table 1. In vitro enzymatic inhibitory activities of 12a−k against different status of EGFR
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EGFR IC50 (ꢁM)a
Compd.
L858R
T790M/L858R
WT
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gefitinib 0.0002 ± 0.0001 0.941 ± 0.073 0.0001 ± 0.0002
0.0054 ± 0.0008 0.0008 ± 0.0001 0.0032 ± 0.0006
1
0.063 ± 0.009
0.068 ± 0.014
0.040 ± 0.0004
0.066 ± 0.013
0.099 ± 0.004
0.083 ± 0.035
0.098 ± 0.010
0.189 ± 0.011
0.064 ± 0.009
0.113 ± 0.005
0.305 ± 0.008
0.014 ± 0.002
0.013 ± 0.001
0.006 ± 0.001
0.013 ± 0.003
0.020 ± 0.003
0.017 ± 0.001
0.023 ± 0.002
0.038 ± 0.001
0.016 ± 0.002
0.030 ± 0.004
0.047 ± 0.006
0.051 ± 0.004
0.059 ± 0.022
0.039 ± 0.005
0.042 ± 0.013
0.073 ± 0.012
0.059 ± 0.016
0.099 ± 0.007
0.174 ± 0.002
0.052 ± 0.002
0.101 ± 0.013
0.292 ± 0.018
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
aEGFR activity assays were performed using the FRETꢀbased Z'ꢀLyte assay according to the
manufacturer’s instructions. The compounds were incubated with the kinase reaction mixture for
1.5 h before measurement. The data were means ± SD of three independent experiments.
Inhibitory Activity on Gefitinib-sensitive HCC827 and -resistant H1975 Cells. The
antiproliferative effects of 12a−k on EGFR mutation NSCLC cell lines, gefitinibꢀsensitive
HCC827 bearing EGFR del E746_A750 and gefitinibꢀresistant H1975 harboring EGFR
L858R/T790M, were next determined by a MTT assay with gefitinib and 1 as controls. As can be
seen in Table 2, all the compounds except 12c (IC50 = 0.291 ꢁM) were potent in inhibition of
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HCC827 cells (IC50s = 0.006−0.041 ꢁM). Compounds 12a, 12f, 12h, 12i and 12k (IC50 = 0.009,
0.007, 0.006, 0.010 and 0.007 ꢁM, respectively) showed comparable inhibition to gefitinib (IC50
= 0.006 ꢁM) and 1 (IC50 = 0.008 ꢁM) on HCC827 cells. Additionally, consistent with their
kinase inhibition, most of these compounds displayed higher antiproliferative activity on H1975
cells (IC50s = 0.029−2.017 ꢁM) than gefitinib (IC50 = 6.917 ꢁM). In general, esters (12f−k)
exhibited better inhibitory effects than amides (12a−e) against H1975 cells. Among which, 12f,
12h, 12i and 12k (IC50 = 0.063, 0.069, 0.043 and 0.029 ꢁM, respectively) were as potent as 1
(IC50 = 0.046 ꢁM). Taken together, compounds 12f, 12h, 12i and 12k were potent to inhibit both
gefitinibꢀsensitive HCC827 and ꢀresistant H1975 cell lines.
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Effects on the Cell Lines Harboring WT EGFR. To examine how the target compounds
affected WT EGFR, we investigated the effects of these compounds on the cell lines harboring
WT EGFR, including NSCLC cells A549 possessing WT EGFR and kꢀRas mutation, human
epithelial carcinoma cells A431 with overexpressed WT EGFR, and human normal bronchial
epithelial cell lines BEASꢀ2B and 16HBE harboring WT EGFR. It can be seen from Table 2,
compounds 12a−k displayed much less inhibition on the growth of the cell lines harboring WT
EGFR than inhibition on H1975 and HCC827 cells. For instance, the active compounds 12h and
12k exhibited 60 to 7200ꢀfold weaker antiproliferative activity on A549 cells (IC50 = 17.23 and
>50 ꢁM) and A431 cells (IC50 = 4.067 and 5.666 ꢁM), respectively, than their inhibition against
the H1975 and HCC827 cells, suggesting that these compounds might possess a good selectivity
against NSCLC cells with EGFR mutants over the other cancer cells. In addition, 12h and 12k
also showed 40 to 6400ꢀfold less inhibition on human normal cells BEASꢀ2B (IC50 = 2.561 and
44.65 ꢁM) and 16HBE (IC50 = 4.153 and 23.86 ꢁM), respectively, than inhibition on the H1975
and HCC827 cells, suggesting the safety profile of these compounds. Notably, 12k was 5 to 25ꢀ
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fold less active against the cell lines harboring WT EGFR than 1, suggesting 12k might be a
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Table 2. Antiproliferative activities of 12a−k against cells harboring a different status of EGFR
IC50 (ꢁM)a
A431e
gefitinib 0.006 ± 0.002 6.917 ± 0.657 15.58 ± 2.143 4.485 ± 0.295 13.94 ± 1.992 12.71 ± 3.675
Compd.
HCC827b
H1975c
A549d
BEASꢀ2Bf
16HBEg
0.008 ± 0.003 0.046 ± 0.011 2.102 ± 0.584 1.108 ± 0.213 1.811 ± 0.253 1.355 ± 0.321
0.009 ±0.005 2.017 ± 0.283 2.259 ± 0.681 0.736 ± 0.088 1.531 ± 0.129 1.551 ± 0.573
0.021 ± 0.011 1.027 ± 0.375 2.484 ± 0.795 0.357 ± 0.067 2.445 ± 0.302 2.275 ± 0.990
0.291 ± 0.014 0.311 ± 0.136 2.118 ± 0.485 0.609 ± 0.153 1.893 ± 0.213 1.480 ± 0.742
0.033 ± 0.012 0.855 ± 0.218 4.249 ± 1.020 0.701 ± 0.098 2.200 ± 0.322 1.800 ± 0.593
0.038 ± 0.006 0.245 ± 0.182 1.793 ± 0.256 0.660 ± 0.050 0.988 ± 0.126 1.851 ± 0.922
0.007 ± 0.002 0.063 ± 0.015 2.562 ± 0.082 1.232 ± 0.275 1.349 ± 0.349 2.043 ± 0.379
0.022 ± 0.005 0.396 ± 0.029 2.927 ± 0.265 1.059 ± 0.364 2.982 ± 0.622 2.584 ± 0.290
0.006 ± 0.004 0.069 ± 0.014 17.23 ± 1.123 4.067 ± 0.986 2.561 ± 0.528 4.153 ± 1.533
0.010 ± 0.002 0.043 ± 0.008 5.528 ± 0.561 3.500 ± 1.310 0.959 ± 0.152 2.615 ± 1.112
0.041 ± 0.018 0.471 ± 0.165 4.447 ± 1.280 3.023 ± 1.203 2.944 ± 1.226 3.155 ± 1.905
1
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
11k
9
0.007 ± 0.004 0.029 ± 0.006
4.294 ± 0.980 5.931 ± 0.488 23.56 ± 3.119 3.378 ± 0.558 1.240 ± 0.405 3.401 ± 0.796
0.100 ± 0.027 1.514 ± 0.197 1.451 ± 0.173 >10 >50 0.707 ± 0.097
>50
5.666 ± 0.336 44.65 ± 3.221 23.86 ± 2.098
aThe inhibitory effects of individual compounds on the proliferation of cancer cell lines were
determined by the MTT assay. The data were means ± SD from at least three independent
b
c
experiments. HCC827 is a human lung cancer cell line (EGFR del E746_A750), H1975 is a
human lung cancer cell line (EGFR L858R/T790M), dA549 is a human lung cancer cell line (WT
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EGFR/kꢀRAS dependent), A431 is human epithelial carcinoma cell line (overexpressed WT
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EGFR), fBEASꢀ2B and g16HBE are normal human bronchial epithelial cell lines.
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Effects of NO on Antiproliferative Activity. Compounds 9 and 11k are anilinopyrimidine
and furoxan moieties of 12k, and their inhibitory effects on HCC827 and H1975 cells were
examined, respectively. As shown in Table 2, both 9 (IC50 = 0.100 and 1.514 ꢁM) and 11k (IC50
= 4.294 and 5.931 ꢁM) exhibited inhibitory activity but much less potent than 12k (IC50 = 0.007
and 0.029 ꢁM), respectively. These results suggest that the antiproliferative activity of 12k might
be attributed to the synergic effects of anilinopyrimidine and NO donor moieties.
To verify the contribution of NO to the inhibitory activity, the levels of NO released by the
tested compounds in vitro were detected. NSCLC cells H1975 and normal human bronchial
epithelial cells 16HBE were respectively exposed to 100 ꢁM of 12a, 12c, 12e, 12i−k, 11k and 1
for the same incubation time. The levels of NO generated in the cell lysates were determined and
presented as that of nitrite (Figure 2A) using a Griess assay.39 As expected, treatment with 1
resulted in little nitrite in both H1975 and 16HBE cells. In contrast, treatment with the tested
compounds generated variable levels of nitrite in H1975 cells. Interestingly, treatment with more
active compounds such as 12i and 12k produced higher levels of nitrite in the cells, whereas less
active compounds 12a and 11k produced lower levels of nitrite, indicating that the amounts of
NO released by these compounds were positively correlated with their inhibitions on H1975 cells
in vitro (R = 0.816, p < 0.05, determined by Pearson’s correlation analysis). It was also observed
that compounds 12i−k linked by an ester bond to anilinopyrimidine released higher levels of NO
and exhibited better inhibitory effects than amides 12a, 12c, and 12e against H1975 cells.
Additionally, the ester compounds, especially 12k, which exerted high selectivity towards EGFR
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L858R/T790M over WT EGFR, might have better ability to penetrate H1975 cells harboring
EGFR L858R/T790M, leading to high levels of NO released in H1975 cells but not in normal
human cells 16HBE bearing WT EGFR, which is in agreement with their strong selective
inhibition on proliferation of NSCLC cells harboring EGFR mutations in vitro.
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Next, 12k was tested on its antiproliferative effects in the presence or absence of a NO
scavenger, hemoglobin. H1975 and HCC827 cells were preꢀtreated with various concentrations
of hemoglobin for 1 h and then treated with 1 ꢂM of 12k. The effects of different treatments on
the growth of H1975 and HCC827 cells were determined by the MTT assay (Figure 2B). It was
observed that treatment with 12k alone remarkably inhibited the growth of H1975 and HCC827
cells and this inhibitory effect was diminished by pretreatment with hemoglobin in a doseꢀ
dependent manner. These results demonstrated that NO produced by 12k substantially
contributed to its inhibition on NSCLC cell proliferation in vitro.
Figure 2. Effects of NO production by the target compounds on NSCLC cell proliferation. A)
Variable levels of NO (presented as nitrite) produced by the indicated compounds. H1975 and
16HBE cells were treated with individual compounds at 100 ꢁM for 150 min, and the
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concentrations of nitrite in the cell lysates were determined by Griess assay. The individual
values were determined by measuring absorbance at 540 nm and calculated according to the
standard curve. Data were expressed as mean + SD of each compound in individual types of cells
from three experiments. **P < 0.01 and ***P < 0.001 vs. 16HBE group. B) Effects of hemoglobin
on the antiproliferative effect of 12k. H1975 and HCC827 cells were pretreated with the
indicated concentrations of hemoglobin (Hb) (0, 1, 5, 10 or 20 ꢂM) for 1 h and treated with 1 ꢂM
of 12k for 24 h. The results were expressed as percent of cell growth inhibition relative to control
cells. Data were mean value + SD obtained from three independent experiments.
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Induction of Apoptosis In Vitro. Since high levels of NO act as a tumor cell apoptosis
inducer,40 the ability of 12k as a NO donor to induce tumor cell apoptosis was tested and
compared with anilinopyrimidine compound 1. EGFRꢀmutant H1975 and HCC827 cells, as well
as EGFRꢀindependent H460 cells were treated with variable level of 12k, 1 or vehicle for 24 h,
respectively. The cells were harvested and stained with 7ꢀaminoactinomycin D (7ꢀAAD) and
annexinꢀV, and the percentages of apoptotic cells were determined by flow cytometry analysis. It
was found that treatment with 12k or 1 induced apoptosis in both H1975 and HCC827 cells in a
doseꢀdependent manner (Figure 3), but did not induce apoptosis of the cell line H460 with little
EGFR expression (see Figure S1 in the Supporting Information). Notably, the ability of 12k to
induce apoptosis was stronger than 1 in both H1975 and HCC827 cells, which may be attributed
to high levels of NO released by 12k.
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Figure 3. Compounds 12k and 1 induced H1975 (A) and HCC827 (B) cells apoptosis in vitro.
The cells were incubated with the indicated concentrations of 12k or 1 for 24 h and the cells were
stained with 7ꢀAAD and annexinꢀV, followed by flow cytometry analysis. The quantitative data
are expressed as mean + SD of the percentages of apoptotic cells from three independent
experiments. **P < 0.01 and ***P < 0.001 vs. 1 group.
Effects on EGFR Activation and Downstream Signaling. In order to get insight into the
mechanisms underlying the activity of these NOꢀreleasing EGFR inhibitors, we subsequently
examined the inhibitory effects of the active compound 12k on the EGFR activation and
downstream signaling in H1975 cells harboring EGFR L858R/T790M using 1 as a control. The
cells were treated with various concentrations of 12k and 1. The expression and activation of
EGFRꢀrelated signal events AKT, ERK and NFꢀκB were determined by immunoblotting, and the
results were summarized in Figure 4. It was observed that treatment with 12k significantly
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inhibited the phosphorylation of EGFR, AKT and ERK in a doseꢀ dependent manner but was less
potent than 1 in H1975 cells. However, 12k displayed obvious inhibition on phosphoꢀNFꢀκB in
H1975 cells while 1 had no significant change at increased concentrations, suggesting that
inhibition of NFꢀκB activation by 12k partially compensated its less inhibition on EGFR as
compared to 1, resulting in the significant activity of 12k in the growth inhibition and apoptosis
induction of the cell lines harboring EGFR mutations.
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Figure 4. Compounds 12k and 1 inhibited the activation of EGFR and downstream signaling in
H1975 NSCLC cells harboring EGFR L858R/T790M.
In Vivo Antitumor Activity. Compound 12k was further evaluated for its in vivo antitumor
efficacy in an EGFR L858R/T790Mꢀdriven human NSCLC xenograft mouse model of H1975
(Figure 5). Nude mice bearing established H1975 tumor xenografts were dosed intraperitoneally
with 12k (14 mg/kg) over a 14ꢀday period. Gefitinib (16 mg/kg) and 1 (8 mg/kg) were employed
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as reference drugs. Relative to the vehicle controls, 14 mg/kg of 12k was well tolerated,
displayed significant in vivo antitumor efficacy (p < 0.05) and inhibited tumor growth by 60%.
Treatment with gefitinib produced only a marginal effect on tumor growth while 1 had an
inhibition rate of 69%. During the experimental period, no significant body weight loss of mice
treated with 12k was observed relative to that of the vehicle controls.
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Figure 5. In vivo antitumor effect of compound 12k in a human NSCLC (H1975) xenograft nude
mouse model. The H1975 model was resistant to gefitinib and sensitive to 1. Tumor volumes
were measured every other day. Data are shown as means ± SD from each group of mice (n = 6).
Statistical significance (p < 0.05) for antitumor efficacy, based upon tumor growth relative to the
vehicle controls.
CONCLUSIONS
In summary, a series of hybrids of phenylsulfonylfuroxan and anilinopyrimidine 12a−k were
synthesized and biologically evaluated. All of the compounds showed strong and selective
inhibitory effects towards EGFR mutant L858R/T790M as 1 did, and superior to gefitinib. These
compounds also exhibited strong antiproliferative activity against HCC827 cells bearing EGFR
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del E746_A750 and H1975 cells harboring EGFR L858R/T790M. Among them, compounds 12f,
12h, 12i and 12k showed inhibition on HCC827 cells comparable with gefitinib and 1, and were
similar to 1 in the inhibition of H1975 cells. Importantly, these compounds showed lower to
moderate inhibition on the cancer and normal cells with WT EGFR (A549, A431, BEASꢀ2B and
16HBE cells), especially, 12k exhibited much less activity against these cell lines harboring WT
EGFR than 1. Interestingly, 12k released much higher levels of NO in H1975 cells than that in
the normal human 16HBE cells, and its antiproliferative activity in H1975 and HCC827 cells
was diminished by a NO scavenger hemoglobin in a doseꢀdependent manner. In addition, 12k
induced cell apoptosis of H1975 and HCC827 cells more potently than 1. Furthermore, 12k
inhibited EGFR activation and downstream signaling in H1975 cells. And 12k displayed
considerable suppression on the NFꢀκB activation in H1975 cells while 1 had no such effects
under the same conditions. Moreover, 12k exhibited significant tumor inhibition in the EGFR
L858R/T790Mꢀdriven human NSCLC xenograft mouse model of H1975. All these results
indicate that 12k showed certain advantages over 1 owing to hybridization of NO donor moiety
to the anilinopyrimidine scaffold, including selectivity against the cell lines harboring mutant
EGFR, and significant induction of apoptosis and inhibition of NFꢀκB in H1975 cells. Therefore,
our novel findings provide a proof of principle in design of NO releasing compounds as potent
and selective inhibitors of EGFR for intervention of NSCLC.
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EXPERIMENTAL SECTION
Chemistry. Melting points were measured on a MelꢀTEMP II melting point apparatus without
1
correction. H and 13C NMR spectral data were recorded with a Bruker Avance 300 MHz
spectrometer at 303 K using TMS as an internal standard. MS spectra were recorded on a
Mariner Mass Spectrum (ESI) and HRMS on Agilent technologies LC/MSD TOF. Analytical
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and preparative TLCs were performed on silica gel GF/UV 254. The chromatograms were
conducted on silica gel (100ꢀ200 mesh) and visualized under UV light at 254 and 365 nm. The
purities of the synthesized compounds were confirmed to be higher than 95% by HPLC analysis
performed with a dual pump Shimadzu LCꢀ20AT system, a SILꢀ20A autosampler, a SPDꢀ20A
UV/VIS detector, a CTOꢀ20A column oven and an ODSꢀC18 column (5 ꢁm, 4.6 × 150 mm) and
eluted with acetonitrileꢀwater (60 : 40 to 70 : 30). Compounds 11a−k were synthesized as
previously described.33
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Ethyl 2-(4-(3-methoxy-4-nitrophenyl)piperazin-1-yl)acetate (3). Compound 236 (0.5 g, 2.1
mmol), potassium carbonate (0.58 g, 4.2 mmol) and ethyl bromoacetate (0.23 mL, 2.1 mmol)
were added in 20 mL of acetone, and the resulting mixture was heated to reflux for 4 h. The
reaction mixture was cooled to room temperature and filtered. The filtrate was collected and
concentrated in vacuo. 0.61 g (90%) of the resulting yellow solid was obtained after purification
by flash chromatography with 1:1 (v/v) petroleum etherꢀethyl acetate. mp 76−77 °C Analytical
data for 3: ESIꢀMS: m/z 324 [M + H]+; 1H NMR (300 MHz, CDCl3): δ 1.21 (t, 3H, OCH2CH3, J
= 7.11 Hz), 2.67 (t, 4H, 2 × NCH2CH2N, J = 4.65 Hz), 3.21 (s, 2H, NCH2CO), 3.39 (t, 4H, 2 ×
NCH2CH2N, J = 4.74 Hz), 3.87 (s, 3H, OCH3), 4.13 (q, 2H, OCH2CH3, J = 7.08 Hz), 6.25 (d,
1H, ArH, J = 2.0 Hz), 6.33 (dd, 1H, ArH, J = 1.9, 9.3 Hz), 7.88 (d, 1H, ArH, J = 9.3 Hz); 13C
NMR (75 MHz, CDCl3): δ 169.95, 156.28, 155.54, 129.39, 128.91, 105.42, 97.02, 60.72, 59.03,
56.17, 52.24, 46.94, 14.20; ESIꢀHRMS (m/z): [M + H]+ calcd for C15H21N3O5 324.1559, obsd
324.1563, PPM error 1.2.
Ethyl 2-(4-(4-amino-3-methoxyphenyl)piperazin-1-yl)acetate (4). Compound 3 (0.469 g,
1.68 mmol) was dissolved in THF (50 mL) and water (50 mL). Iron powder (0.47 g, 8.4 mmol)
and ammonium chloride (0.45 g, 8.4 mmol) were then added, and the resulting mixture was
heated to 65 oC and stirred for 3 hours. The reaction mixture was cooled to room temperature and
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filtered through celite. The THF was removed in vacuo, and the resulting residue was basified
with sodium bicarbonate and extracted with ethyl acetate (20 mL) three times. The organic layer
was separated, dried using anhydrous sodium sulfate, concentrated and used without further
purification. As a yellow oil, 99% yield. Analytical data for 4: ESIꢀMS: m/z 294 [M + H]+; 1H
NMR (300 MHz, d6ꢀDMSO): δ 2.01 (t, 3H, OCH2CH3, J = 6.9 Hz), 3.45 (t, 4H, 2 × NCH2CH2N,
J = 4.5 Hz), 3.76 (t, 4H, 2 × NCH2CH2N, J = 4.5 Hz), 4.07 (s, 2H, NCH2CO), 4.56 (s, 3H,
OCH3), 4.92 (q, 2H, OCH2CH3, J = 6.9 Hz), 5.01 (s, 2H, ArNH2), 7.11 (dd, 1H, ArH, J = 1.8, 8.4
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Hz), 7.31 (d, 1H, ArH, J = 2.1 Hz), 7.36 (d, 1H, ArH, J = 8.4 Hz); C NMR (75 MHz, d6ꢀ
DMSO): δ 170.36, 147.48, 143.79, 131.51, 114.70, 109.23, 102.50, 60.28, 59.01, 55.69, 52.74,
50.86, 14.59; ESIꢀHRMS (m/z): [M + H]+ calcd for C15H23N3O3 294.1818, obsd 294.1822, PPM
error 1.4.
3-((2,5-Dichloropyrimidin-4-yl)oxy)aniline (7). The title compound was obtained staring
from 6,24 and the synthetic method was as same as compound 4. As a pale yellow solid, 80%
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yield. mp 103−105 C. Analytical data for 7: ESIꢀMS: m/z 256 [M + H]+; H NMR (300 MHz,
CDCl3): δ 6.48 (s, 1H, ArH), 6.54 (d, 1H, ArH, J = 8.1 Hz), 6.60 (d, 1H, ArH, J = 8.04 Hz), 7.20
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(t, 1H, ArH, J = 8.07 Hz), 8.44 (s, 1H, ArH); C NMR (75 MHz, d6ꢀDMSO): δ 165.38, 159.34,
156.79, 152.81, 149.55, 130.53, 117.08, 113.16, 109.45, 107.43; ESIꢀHRMS (m/z): [M + H]+
calcd for C10H7N3OCl2 256.0044, obsd 256.0048, PPM error 1.6.
N-(3-((2,5-Dichloropyrimidin-4-yl)oxy)phenyl)acrylamide (8). Acryloyl chloride (0.257 mL,
3.18 mmol) was added dropwise to a solution of compound 7 (0.814 g, 3.18 mmol) and
diisopropyethylamine (0.56 mL, 3.18 mmol) in methylene chloride (30 mL) at 0 oC. The reaction
was stirred for 1 h. 0.89 g (90%) of the resulting white solid was obtained after purification by
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flash chromatography with 2:1 (v/v) petroleum etherꢀethyl acetate. mp 199−201 C. Analytical
data for 8: ESIꢀMS: m/z 310 [M + H]+; 1H NMR (300 MHz, CDCl3): δ 5.79 (d, 1H, COCH=CH2,
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J = 10.23 Hz), 6.21 (dd, 1H, COCH=CH2, J = 10.17, 16.77 Hz), 6.44 (d, 1H, COCH=CH2, J =
16.8 Hz), 6.94 (d, 1H, ArH, J = 8.04 Hz), 7.30 (d, 1H, ArH, J = 8.16 Hz), 7.38 (t, 1H, ArH, J =
8.07 Hz), 7.42 (s, 1H, ArH), 7.74 (s, 1H, ArNHCO), 8.48 (s, 1H, ArH); 13C NMR (75 MHz, d6ꢀ
DMSO): δ 165.28, 163.85, 159.51, 156.68, 151.96, 140.89, 132.03, 130.50, 127.84, 117.66,
117.16, 116.92, 112.72; ESIꢀHRMS (m/z): [M + H]+ calcd for C13H9N3O2Cl2 310.0150, obsd
310.0155, PPM error 1.6.
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Ethyl-2-(4-(4-((4-(3-acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-methoxy-
phenyl)piperazin-1-yl)acetate (9). A flask was charged with compound 4 (196 mg, 0.70 mmol),
8 (217 mg, 0.70 mmol), TFA ( 0.08 mL, 1.05 mmol), 2ꢀBuOH (10 mL). The slurry was heated to
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100 C for 3 h. The reaction mixture was allowed to cool to room temperature and was
neutralized with a saturated aqueous sodium bicarbonate solution. The aqueous mixture was then
extracted with CH2Cl2 (20 mL) three times. The crude product was purified using flash
chromatography with 1:1 (v/v) petroleum etherꢀethyl acetate to afford 260 mg solid as a white
solid (80%). mp 168−170 oC. Analytical data for 9: ESIꢀMS: m/z 565 [M ꢀ H]ꢀ, 601 [M + Cl]ꢀ; 1H
NMR (300 MHz, CDCl3): δ 1.29 (t, 3H, OCH2CH3, J = 7.14 Hz), 2.75 (t, 4H, 2 × NCH2CH2N, J
= 4.59 Hz), 3.14 (t, 4H, 2 × NCH2CH2N, J = 4.92 Hz), 3.27 (s, 2H, NCH2CO), 3.81 (s, 3H,
OCH3), 4.21 (q, 2H, OCH2CH3, J = 7.11 Hz), 5.77 (d, 1H, COCH=CH2, J = 10.14 Hz), 6.24 (dd,
2H, COCH=CH2 and ArH, J = 10.14, 16.80 Hz), 6.43 (d, 2H, COCH=CH2 and ArH, J = 16.02
Hz), 6.97 (d, 1H, ArH, J = 8.1 Hz), 7.39−7.47 (m, 4H, 3 × ArH and ArNH), 7.63 (d, 2H, ArH
13
and ArNHCO, J = 7.71 Hz), 8.22 (s, 1H, ArH); C NMR (75 MHz, CDCl3): δ 170.27, 164.45,
163.48, 157.71, 157.47, 152.82, 148.69, 147.13, 139.02, 130.94, 129.84, 128.22, 121.76, 119.30,
118.17, 117.10, 113.90, 108.04, 100.30, 60.73, 59.46, 55.58, 53.04, 49.93, 14.26; ESIꢀHRMS
(m/z): [M + Na]+ calcd for C28H31ClN6O5 589.1942, obsd 589.1948, PPM error 1.0.
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2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-methoxyphenyl)pi-
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perazin-1-yl)acetic acid (10). To a solution of ethyl ester 9 (1 g, 1.76 mmol) in THF (15 mL)
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and water (15 mL), LiOH (0.723 g, 17.6 mmol) was added at 0 C. The resulting mixture was
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stirred at room temperature for 1 hour then cooled to 0 C and acidified to subacidity (pH 6−7)
with dilute hydrochloric acid. The precipitate was collected and used without further purification.
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As a yellow solid, 98% yield, mp 205−207 C. Analytical data for 10: ESIꢀMS: m/z 539.1 [M +
H]+; 1H NMR (300 MHz, d6ꢀDMSO): δ 2.85 (t, 4H, 2 × NCH2CH2N, J = 4.69 Hz), 3.15 (t, 4H, 2
× NCH2CH2N, J = 4.52 Hz), 3.33 (s, 2H, NCH2CO), 3.74 (s, 3H, OCH3), 4.66 (s, 1H, ArNH),
5.77 (d, 1H, COCH=CH2, J = 9.9 Hz), 6.25 (dd, 2H, COCH=CH2 and ArH, J = 10.14, 17.40 Hz),
6.49 (d, 1H, COCH=CH2, J = 15.02 Hz), 6.54 (s, 1H, ArH), 6.97 (d, 1H, ArH, J = 6.9 Hz), 7.28
(d, 1H, ArH, J = 7.5 Hz), 7.42 (t, 1H, ArH, J = 6.9 Hz), 7.60 (d, 1H, ArH, J = 6 Hz), 7.68 (s, 1H,
ArNHCO), 8.08 (s, 1H, ArH), 8.34 (s, 1H, ArH), 10.50 (s, 1H, COOH); 13C NMR (75 MHz, d6ꢀ
DMSO): δ 170.01, 164.02, 163.85, 158.22, 158.01, 152.18, 147.94, 140.33, 131.74, 129.81,
127.23, 122.82, 119.93, 116.76, 116.57, 112.85, 106.64, 104.06, 100.13, 58.36, 55.60, 51.83,
47.92; ESIꢀHRMS (m/z): [M + H]+ calcd for C26H27N6O5Cl 539.1810, obsd 539.1814, PPM error
0.7.
General Procedure for the Preparation of 12a−k. To a solution of compound 10 (4.54 g,
8.43 mmol) in dry CH2Cl2 (20 mL), dicyclohexylcarbodiimide (DCC) (1.74 g, 8.43 mmol),
11a−k (8.43 mmol) and 4ꢀdimethylaminopyridine (DMAP) (catalytic amount) were added, and
the mixture was stirred at room temperature for 12 h. After filtration, the filtrate was evaporated
to dryness in vacuo, and the crude product was purified by column chromatography with 1:1
(v/v) petroleum etherꢀethyl acetate.
4-(2-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetamido)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-
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oxide (12a). The title compound was obtained staring from 11a and 10. As a yellow solid, 83%
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yield. mp 194−196 °C. Analytical data for 12a: ESIꢀMS: m/z 806 [M + H]+, 828 [M + Na]+; H
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NMR (300 MHz, CDCl3): δ 2.75 (t, 4H, 2 × NCH2CH2N, J = 4.61 Hz), 3.14 (t, 4H, 2 ×
NCH2CH2N, J = 4.83 Hz), 3.16 (s, 2H, NCH2CO), 3.77 (s, 3H, OCH3), 3.82 (t, 2H,
NHCH2CH2O J = 5.19 Hz), 4.58 (t, 2H, NHCH2CH2O, J = 4.77 Hz), 5.74 (d, 1H, COCH=CH2, J
= 9.96 Hz), 6.17 (d, 1H, ArH, J = 8.31 Hz), 6.25 (dd, 1H, COCH=CH2, J = 9.99, 16.83 Hz), 6.41
(d, 2H, COCH=CH2 and ArH, J = 17.25 Hz), 6.95 (d, 1H, ArH, J = 8.07 Hz), 7.37 (t, 1H, ArH, J
= 8.07 Hz), 7.47 (s, 1H, ArNH), 7.50 (d, 1H, ArH, J = 8.19 Hz), 7.60 (t, 4H, 3 × ArH and
ArNHCO, J = 7.61 Hz), 7.74 (t, 3H, 2 × ArH and NHCO, J = 7.02 Hz ), 8.04 (d, 2H, ArH, J =
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7.77 Hz), 8.21 (s, 1H, ArH); C NMR (75 MHz, CDCl3): δ 170.76, 164.54, 163.70, 158.76,
157.73, 157.41, 152.87, 148.59, 146.85, 139.16, 137.67, 135.87, 131.02, 129.80, 129.74, 128.51,
128.09, 121.99, 119.06, 118.15, 116.93, 114.00, 108.07, 100.56, 70.16, 61.46, 59.34, 55.59,
53.69, 50.30, 37.82; ESIꢀHRMS (m/z): [M + Na]+ calcd for C36H36ClN9O9S 828.1943, obsd
828.1950, PPM error 0.8.
4-(3-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetamido)propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-
oxide (12b). The title compound was obtained staring from 11b and 10. As a yellow solid, 80%
yield. mp 141−143 °C. Analytical data for 12b: ESIꢀMS: m/z 820 [M + H]+; 1H NMR (300 MHz,
CDCl3): δ 2.16 (quint, 2H, CH2, J = 6.33 Hz), 2.73 (t, 4H, 2 × NCH2CH2N, J = 4.51 Hz), 3.12 (t,
4H, 2 × NCH2CH2N, J = 4.73 Hz), 3.13 (s, 2H, NCH2CO), 3.53 (q, 2H, NHCH2, J = 6.27 Hz),
3.81 ( s, 3H, OCH3), 4.51 (t, 2H, OCH2, J = 6.06 Hz), 5.75 (d, 1H, COCH=CH2, J = 9.63 Hz),
6.21 (d, 1H, ArH, J = 8.31 Hz), 6.25 (dd, 1H, COCH=CH2, J = 10.11, 16.86 Hz), 6.42 (d, 2H,
COCH=CH2 and ArH, J = 16.38 Hz), 6.99 (d, 1H, ArH, J = 8.16 Hz), 7.35−7.47 (m, 4H, 3 ×
ArH and ArNH), 7.57−7.66 (m, 5H, 3 × ArH, ArNHCO and NHCO), 7.76 (t, 1H, ArH, J = 7.47
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Hz ), 8.07 (d, 2H, ArH, J = 7.47 Hz), 8.23 (s, 1H, ArH); C NMR (75 MHz, CDCl3): δ 170.56,
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163.81, 162.83, 158.76, 157.75, 157.38, 152.84, 148.65, 146.84, 138.72, 137.55, 135.75, 131.03,
129.84, 129.76, 128.57, 128.12, 122.03, 119.10, 118.19, 116.97, 111.64, 108.05, 100.50, 69.24,
61.56, 55.62, 53.62, 50.25, 35.62, 28.81; ESIꢀHRMS (m/z): [M + Na]+ calcd for C37H38ClN9O9S
842.2099, obsd 842.2106, PPM error 0.8.
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4-(2-(4-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetyl)piperazin-1-yl)ethoxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole-2-oxide (12c). The title compound was obtained staring from 11c and 10. As a
yellow solid, 78% yield. mp 130−132 °C. Analytical data for 12c: ESIꢀMS: m/z 875 [M + H]+;
1H NMR (300 MHz, CDCl3): δ 2.56 (t, 2H, NCH2CH2N, J = 4.33 Hz), 2.65 (t, 6H, 3 ×
NCH2CH2N, J = 4.56 Hz), 2.88 (t, 2H, NCH2, J = 5.07 Hz), 3.09 (t, 4H, 2 × NCH2CH2N, J =
4.73 Hz), 3.25 (s, 2H, NCH2CO), 3.64 (t, 4H, 2 × NCH2CH2N, J = 4.65 Hz), 3.79 ( s, 3H,
OCH3), 4.57 (t, 2H, OCH2, J = 5.01 Hz), 5.75 (d, 1H, COCH=CH2, J = 9.99 Hz), 6.18 (d, 1H,
ArH, J = 8.31 Hz), 6.27 (dd, 1H, COCH=CH2, J = 9.99, 16.8 Hz), 6.42 (d, 2H, COCH=CH2 and
ArH, J = 16.47 Hz), 6.98 (d, 1H, ArH, J = 7.98 Hz), 7.41 (t, 1H, ArH, J = 7.77 Hz), 7.43 (s, 1H,
ArNH), 7.48 (s, 1H, ArH), 7.59 (t, 3H, 2 × ArH and ArNHCO, J = 7.74 Hz), 7.68 (d, 1H, ArH, J
= 8.07 Hz), 7.74 (t, 2H, ArH, J = 7.2 Hz ), 8.02 (d, 2H, ArH, J = 7.71 Hz), 8.22 (s, 1H, ArH); 13C
NMR (75 MHz, CDCl3): δ 167.83, 164.14, 163.82, 159.39, 158.72, 158.48, 152.62, 152.31,
148.75, 140.70, 137.79, 136.59, 132.20, 130.44, 130.27, 128.71, 127.64, 120.17, 117.17, 116.93,
113.24, 110.95, 107.01, 100.42, 69.83, 56.05, 53.57, 53.19, 52.90, 49.10, 45.99, 45.60; ESIꢀ
HRMS (m/z): [M + H]+ calcd for C40H43ClN10O9S 875.2702, obsd 875.2710, PPM error 0.9.
4-((1-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetyl)piperidin-4-yl)oxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole-2-oxide (12d). The title compound was obtained staring from 11d and 10. As a
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yellow solid, 80% yield. mp 128−130 °C. Analytical data for 12d: ESIꢀMS: m/z 846 [M + H]+;
1H NMR (300 MHz, CDCl3): δ 1.97ꢀ2.04 (m, 4H, CH2CCH2), 2.69 (t, 4H, 2 × NCH2CH2N, J =
4.56 Hz), 3.11 (t, 4H, 2 × NCH2CH2N, J = 4.73 Hz), 3.29 (s, 2H, NCH2CO), 3.80 ( s, 3H,
OCH3), 3.7ꢀ3.9 (m, 4H, CH2NCH2), 5.17 (m, 1H, OCH), 5.76 (dd, 1H, COCH=CH2, J = 9.96,
1.11 Hz), 6.19 (d, 1H, ArH, J = 8.31 Hz), 6.27 (dd, 1H, COCH=CH2, J = 9.96, 16.8 Hz), 6.43 (d,
2H, COCH=CH2 and ArH, J = 16.47 Hz), 6.98 (dd, 1H, ArH, J = 7.98, 1.32 Hz), 7.41 (t, 1H,
ArH, J = 8.25 Hz), 7.45 (s, 1H, ArNH), 7.50 (s, 1H, ArH), 7.62 (t, 4H, 2 × ArH and ArNHCO, J
= 7.89 Hz), 7.76 (t, 1H, ArH, J = 7.44 Hz ), 7.80 (s, 1H, ArH), 8.05 (d, 2H, ArH, J = 7.35 Hz),
8.22 (s, 1H, ArH); 13C NMR (75 MHz, CDCl3): δ 168.05, 164.48, 163.64, 157.90, 157.70,
157.44, 152.80, 148.68, 146.97, 139.21, 137.96, 135.76, 131.09, 129.74, 128.48, 128.06, 121.84,
119.20, 118.08, 117.13, 113.92, 110.46, 107.94, 100.39, 61.44, 55.62, 53.18, 50.12,42.12, 38.35,
30.84, 29.99; ESIꢀHRMS (m/z): [M + Na]+ calcd for C39H40ClN9O9S 868.2256, obsd 868.2265,
PPM error 1.0.
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4-((1-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetamido)propan-2-yl)oxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole-2-oxide (12e). The title compound was obtained staring from 11e and 10. As a
yellow solid, 85% yield. mp 105−107 °C. Analytical data for 12e: ESIꢀMS: m/z 820 [M + H]+,
842 [M + Na]+; 1H NMR (300 MHz, CDCl3): δ 1.47 (d, 3H, CHCH3, J = 6.24 Hz), 2.65 (t, 4H, 2
× NCH2CH2N, J = 4.56 Hz), 3.08 (t, 4H, 2 × NCH2CH2N, J = 4.73 Hz), 3.10 (s, 2H, NCH2CO),
3.50 (t, 1H, NHCH2, J = 6.63 Hz), 3.79 ( s, 3H, OCH3), 4.52 (t, 1H, NHCH2, J = 6.56 Hz), 5.15
(m, 1H, CHCH3), 5.72 (d, 1H, COCH=CH2, J = 9.96 Hz), 6.16 (d, 1H, ArH, J = 7.02 Hz), 6.25
(dd, 1H, COCH=CH2, J = 10.11, 16.74 Hz), 6.41 (d, 2H, COCH=CH2 and ArH, J = 16.47 Hz),
6.95 (d, 1H, ArH, J = 8.25 Hz), 7.37 (t, 1H, ArH, J = 8.43 Hz), 7.45 (s, 1H, ArNH), 7.58 (m, 5H,
3 × ArH, ArNHCO and NHCO), 7.71 (d, 1H, ArH, J = 6.36 Hz), 7.68 (t, 1H, ArH, J = 6.27 Hz),
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8.03 (d, 3H, ArH, J = 7.23 Hz), 8.20 (s, 1H, ArH); 13C NMR (75 MHz, CDCl3): δ 170.73,
164.53, 163.37, 158.74, 157.74, 157.41, 152.89, 148.68, 146.82, 139.11, 137.80, 135.82, 130..98,
129.78, 128.46, 128.12, 122.01, 119.02, 118.17, 116.93, 113.91, 110.46, 108.14, 100.58, 78.78,
61.47, 55.60, 53.72, 53.62, 50.22, 43.12, 17.20; ESIꢀHRMS (m/z): [M + Na]+ calcd for
C37H38ClN9O9S 842.2099, obsd 842.2106, PPM error 0.8.
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4-(2-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(12f). The title compound was obtained staring from 11f and 10. As a yellow solid, 75% yield.
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mp 123−125 °C. Analytical data for 12f: ESIꢀMS: m/z 807 [M + H]+; H NMR (300 MHz,
CDCl3): δ 2.80 (t, 4H, 2 × NCH2CH2N, J = 4.59 Hz), 3.16 (t, 4H, 2 × NCH2CH2N, J = 4.77 Hz),
3.38 (s, 2H, NCH2CO), 3.79 ( s, 3H, OCH3), 4.58 (t, 2H, OCH2CH2O J = 3.66 Hz), 4.68 (t, 2H,
OCH2CH2O, J = 3.03 Hz), 5.74 (d, 1H, COCH=CH2, J = 9.99 Hz), 6.27 (dd, 2H, COCH=CH2
and ArH, J = 9.99, 16.8 Hz), 6.42 (d, 2H, COCH=CH2 and ArH, J = 17.07 Hz), 6.96 (d, 1H,
ArH, J = 7.14 Hz), 7.39 (t, 1H, ArH, J = 8.55 Hz), 7.45 (s, 1H, ArNH), 7.58 (d, 3H, 2 × ArH and
ArNHCO, J = 6.54 Hz), 7.63 (t, 2H, ArH, J = 7.59 Hz), 7.76 (t, 1H, ArH J = 7.59 Hz ), 7.79 (s,
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1H, ArH), 8.07 (d, 2H, ArH, J = 7.71 Hz), 8.22 (s, 1H, ArH); C NMR (75 MHz, CDCl3): δ
170.05, 164.49, 163.61, 158.65, 157.69, 157.44, 152.82, 148.71, 147.02, 139.11, 137.83, 135.79,
130.98, 129.78, 129.74, 128.63, 128.18, 121.78, 119.33, 118.12, 117.09, 114.01, 108.01, 100.35,
68.81, 61.34, 59.20, 55.60, 52.98, 49.94; ESIꢀHRMS (m/z): [M + Na]+ calcd for C36H35ClN8O10S
829.1783, obsd 829.1790, PPM error 0.8.
4-(3-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetoxy)propoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-
oxide (12g). The title compound was obtained staring from 11g and 10. As a yellow solid, 72%
yield. mp 102−104 °C. Analytical data for 12g: ESIꢀMS: m/z 821 [M + H]+; 1H NMR (300 MHz,
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CDCl3): δ 2.25 (t, 2H, OCH2CH2CH2O, J = 5.67 Hz ), 2.73 (t, 4H, 2 × NCH2CH2N, J = 4.60 Hz),
3.12 (t, 4H, 2 × NCH2CH2N, J = 4.76 Hz), 3.31 (s, 2H, NCH2CO), 3.79 ( s, 3H, OCH3), 4.35 (t,
2H, OCH2CH2CH2O J = 5.49 Hz), 4.53 (t, 2H, OCH2CH2CH2O, J = 5.52 Hz), 5.75 (d, 1H,
COCH=CH2, J = 10.05 Hz), 6.25 (dd, 2H, COCH=CH2, ArH, J = 9.99, 17.4 Hz), 6.41 (d, 2H,
COCH=CH2 and ArH, J = 17.07 Hz), 6.97 (d, 1H, ArH, J = 7.35 Hz), 7.40 (t, 1H, ArH, J = 7.92
Hz), 7.43 (s, 1H, ArNH), 7.49 (s, 1H, ArH), 7.63−7.66 (t, 4H, 3 × ArH and ArNHCO, J = 7.08
Hz), 7.76 (t, 2H, ArH, J = 7.59 Hz ), 8.06 (d, 2H, ArH, J = 7.5 Hz), 8.21 (s, 1H, ArH); 13C NMR
(75 MHz, CDCl3): δ 170.21, 164.46, 163.68, 158.83, 157.66, 157.45, 152.77, 148.72, 147.07,
139.15, 137.91, 135.76, 131.00, 129.77, 128.53, 128.14, 121.73, 119.37, 118.08, 117.19, 113.96,
108.03, 100.30, 68.00, 60.58, 59.18, 55.59, 53.00, 49.90, 27.89; ESIꢀHRMS (m/z): [M + Na]+
calcd for C37H37ClN8O10S 843.1940, obsd 843.1947, PPM error 0.8.
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4-(4-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetoxy)butoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide
(12h). The title compound was obtained staring from 11h and 10. As a yellow solid, 78% yield.
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mp 98−100 °C. Analytical data for 12h: ESIꢀMS: m/z 835 [M + H]+; H NMR (300 MHz,
CDCl3): δ 1.88 (quint, 2H, CH2, J = 5.88, 7.2 Hz), 1.97 (quint, 2H, CH2, J = 5.67, 6.72 Hz), 2.74
(t, 4H, 2 × NCH2CH2N, J = 4.59 Hz), 3.13 (t, 4H, 2 × NCH2CH2N, J = 4.77 Hz), 3.30 (s, 2H,
NCH2CO), 3.79 ( s, 3H, OCH3), 4.25 (t, 2H, OCH2, J = 6.18 Hz), 4.47 (t, 2H, OCH2, J = 6.15
Hz), 5.75 (d, 1H, COCH=CH2, J = 10.17 Hz), 6.25 (dd, 2H, COCH=CH2, ArH, J = 10.08, 16.92
Hz), 6.42 (d, 2H, COCH=CH2 and ArH, J = 16.14 Hz), 6.97 (d, 1H, ArH, J = 8.07 Hz), 7.40 (t,
1H, ArH, J = 8.13 Hz), 7.43 (s, 1H, ArNH), 7.49 (s, 1H, ArH), 7.62 (t, 4H, 3 × ArH and
ArNHCO, J = 7.8 Hz), 7.76 (t, 2H, ArH, J = 7.83 Hz ), 8.05 (d, 2H, ArH, J = 7.56 Hz), 8.21 (s,
1H, ArH); 13C NMR (75 MHz, CDCl3): δ 170.31, 164.46, 163.66, 158.96, 157.68, 157.45,
152.79, 148.72, 147.08, 139.12, 137.95, 135.70, 130.98, 129.80, 129.71, 128.53, 128.16, 121.75,
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119.36, 118.11, 117.19, 113.96, 108.04, 100.32, 70.94, 63.91, 59.31, 55.60, 53.03, 49.92, 25.26,
25.04; ESIꢀHRMS (m/z): [M + Na]+ calcd for C38H39ClN8O10S 857.2096, obsd 857.2104, PPM
error 0.9.
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4-((4-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetoxy)but-2-yn-1-yl)oxy)-3-(phenylsulfonyl)-1,2,5-
oxadiazole-2-oxide (12i). The title compound was obtained staring from 11i and 10. As a yellow
solid, 83% yield. mp 185−187 °C. Analytical data for 12i: ESIꢀMS: m/z 831 [M + H]+ , 853 [M +
Na]+; 1H NMR (300 MHz, CDCl3): δ 2.75 (t, 4H, 2 × NCH2CH2N, J = 4.59 Hz), 3.13 (t, 4H, 2 ×
NCH2CH2N, J = 4.77 Hz), 3.34 (s, 2H, NCH2CO), 3.80 ( s, 3H, OCH3), 4.81 (s, 2H, OCH2), 5.10
(s, 2H, OCH2), 5.76 (d, 1H, COCH=CH2, J = 10.05 Hz), 6.25 (dd, 2H, COCH=CH2, ArH, J =
10.11, 16.98 Hz), 6.43 (d, 2H, COCH=CH2 and ArH, J = 16.14 Hz), 6.98 (d, 1H, ArH, J = 7.68
Hz), 7.41 (t, 1H, ArH, J = 8.07 Hz), 7.43 (s, 1H, ArNH), 7.49 (s, 1H, ArH), 7.64 (t, 4H, 3 × ArH
and ArNHCO, J = 7.47 Hz), 7.77 (t, 2H, ArH, J = 7.32 Hz ), 8.07 (d, 2H, ArH, J = 7.71 Hz), 8.22
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(s, 1H, ArH); C NMR (75 MHz, CDCl3): δ 169.41, 164.37, 163.48, 158.06, 157.72, 157.46,
152.84, 148.68, 147.02, 139.01, 137.95, 135.71, 130.97, 129.83, 129.75, 128.63, 128.21, 121.79,
119.27, 118.17, 117.06, 113.88, 108.02, 100.27, 83.77, 78.81, 59.08, 58.58, 55.60, 52.93, 51.97,
49.89; ESIꢀHRMS (m/z): [M + Na]+ calcd for C38H35ClN8O10S 853.1783, obsd 853.1790, PPM
error 0.8.
4-(2-(2-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetoxy)ethoxy)ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-
2-oxide (12j). The title compound was obtained staring from 11j and 10. As a yellow solid, 68%
yield. mp 91−93 °C. Analytical data for 12j: ESIꢀMS: m/z 851 [M + H]+ , 873 [M + Na]+; H
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NMR (300 MHz, CDCl3): δ 2.73 (m, 4H, 2 × NCH2CH2N), 3.11 (m, 4H, 2 × NCH2CH2N), 3.32
(s, 2H, NCH2CO), 3.78 ( s, 3H, OCH3), 3.82 (m, 2H, OCH2), 3.92 (m, 2H, OCH2), 4.34 (m, 2H,
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OCH2), 4.57 (m, 2H, OCH2), 5.74 (d, 1H, COCH=CH2, J = 9.84 Hz), 6.26 (dd, 2H, COCH=CH2,
ArH, J = 9.99, 16.77 Hz), 6.41 (d, 2H, COCH=CH2 and ArH, J = 16.14 Hz), 6.96 (d, 1H, ArH, J
= 7.29 Hz), 7.39 (t, 1H, ArH, J = 7.89 Hz), 7.43 (s, 1H, ArNH), 7.51 (s, 1H, ArH), 7.64 (t, 4H, 3
× ArH and ArNHCO, J = 7.17 Hz), 7.76 (t, 1H, ArH, J = 6.96 Hz ), 7.91 (s, 1H, ArH), 8.07 (d,
2H, ArH, J = 7.38 Hz), 8.20 (s, 1H, ArH); 13C NMR (75 MHz, CDCl3): δ 170.23, 164.47,
163.58, 159.03, 157.72, 157.47, 152.82, 148.71, 147.06, 139.04, 135.69, 130.96, 129.82, 129.70,
128.60, 128.22, 121.77, 119.33, 118.16, 113.90, 108.02, 100.30, 70.54, 69.37, 68.35, 63.46,
59.17, 55.60, 52.95, 49.91; ESIꢀHRMS (m/z): [M + Na]+ calcd for C38H39ClN8O11S 873.2045,
obsd 873.2053, PPM error 0.9.
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4-((6-(2-(4-(4-((4-(3-Acrylamidophenoxy)-5-chloropyrimidin-2-yl)amino)-3-
methoxyphenyl)piperazin-1-yl)acetoxy)hexyl)oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-
oxide (12k). The title compound was obtained staring from 11k and 10. As a yellow solid, 70%
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yield. mp 84−86 °C. Analytical data for 12k: ESIꢀMS: m/z 863 [M + H]+ , 885 [M + Na]+; H
NMR (300 MHz, CDCl3): δ 1.47 (quint, 4H, 2 × CH2, J = 6.39 Hz), 1.71 (quint, 2H, CH2, J =
6.66 Hz), 1.89 (quint, 2H, CH2, J = 6.27 Hz), 2.74 (t, 4H, 2 × NCH2CH2N, J = 4.57 Hz), 3.13 (t,
4H, 2 × NCH2CH2N, J = 4.75 Hz), 3.29 (s, 2H, NCH2CO), 3.79 ( s, 3H, OCH3), 4.17 (t, 2H,
OCH2, J = 6.57 Hz), 4.42 (t, 2H, OCH2, J = 6.42 Hz), 5.76 (d, 1H, COCH=CH2, J = 10.14 Hz),
6.25 (dd, 2H, COCH=CH2, ArH, J = 10.08, 16.86 Hz), 6.43 (d, 2H, COCH=CH2 and ArH, J =
15.93 Hz), 6.98 (d, 1H, ArH, J = 7.95 Hz), 7.40 (t, 1H, ArH, J = 8.13 Hz), 7.43 (s, 1H, ArNH),
7.48 (s, 1H, ArH), 7.62 (t, 4H, 3 × ArH and ArNHCO, J = 7.59 Hz), 7.76 (t, 2H, ArH, J = 7.2 Hz
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), 8.05 (d, 2H, ArH, J = 7.53 Hz), 8.21 (s, 1H, ArH); C NMR (75 MHz, CDCl3): δ 170.37,
164.55, 163.58, 159.03, 157.71, 157.45, 152.81, 148.70, 147.10, 139.04, 135.65, 130.96, 129.83,
129.67, 128.53, 128.22, 121.76, 119.30, 118.15, 117.10, 108.02, 100.28, 71.42, 64.52, 59.37,
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Journal of Medicinal Chemistry
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55.59, 53.03, 49.93, 28.49, 28.32, 25.49, 25.29; ESIꢀHRMS (m/z): [M + H]+ calcd for
C40H43ClN8O10S 863.2590, obsd 863.2597, PPM error 0.8.
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Cell Lines and Reagents. H1975 (NSCLC, EGFRL858R/T790M), HCC827 (NSCLC, EGFR
del E746_A750), A549 (NSCLC, EGFR wild type), H460 (NSCLC, EGFR independent), A431
(epidermoid carcinoma, EGFR overexpression), HLFꢀ1 (diploid human lung fibroblasts), BEASꢀ
2B and 16HBE (human bronchial epithelial) cells were obtained from ATCC. The cells were
maintained at 37 °C in a 5% CO2 incubator in DMEM or RPMI 1640 (Hyclone) containing 8%
fetal bovine serum (FBS, Biochrom, AG).
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In Vitro Enzymatic Activity Assay. Wild type (WT) and EGFR mutants (L858R,
L858R/T790M) and the Z’ꢀLyte Kinase Kit were purchased from Invitrogen. The experiments
were performed according to the instructions of the manufacturer. Briefly, the respective
concentration of different kinases determined by optimization experiments was: EGFR (PV3872,
Invitrogen) 0.287 ꢁg/ꢁL, EGFRꢀL858R (PV4128, Invitrogen) 0.054 ꢁg/ꢁL, EGFRꢀ
L858R/T790M (PV4879, Invitrogen) 0.055 ꢁg/ꢁL. Ten concentration gradients were set for all
the tested compounds from 5.1 × 10ꢀ9 M to 1 × 10ꢀ4 M in DMSO and a 4 × compound solution
was prepared. A ATP solution and a Kinase/Peptide Mixture were prepared right before use. The
10 ꢁL Kinase Reactions was consisted of 2.5 ꢁL compound solution, 5 ꢁL Kinase/Peptide
Mixture, and 2.5 ꢁL ATP solution. 100% phosphorylation control was provided by 5 ꢁL
phosphoꢀpeptide solution instead of Kinase/Peptide Mixture. 100% inhibition control was
prepared using 2.5 ꢁL Kinase Buffer instead of ATP solution, and 2.5 ꢁL 4% DMSO instead of
compound solution was used as 0% inhibition control. Mixed the plate thoroughly and incubated
for 1 h at room temperature. After 5 ꢁL Development Solution was added to each well, the plate
was incubated for 1 h at room temperature; the nonꢀphosphoꢀpeptides were cleaved in this time.
Finally, 5 ꢁL Stop Reagent was added to stop reaction. Plate was measured on EnVision
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