1,2,4-Oxadiazole derivatives targeting EGFR and c-Met degradation in TKI resistant NSCLC

Article abstract of DOI:10.1016/j.ejmech.2019.111607

Development of small-molecule agents with the ability to facilitate oncoprotein degradation has emerged as a promising strategy for cancer therapy. Since EGFR and c-Met are both implicated in oncogenesis and tumor progression, we initiated a screening program by using an in-house library to identify agents capable of inducing the concomitant suppression of EGFR and c-Met expression, which led to the identification of compound 1, a 1,2,4-oxadiazole derivative. Based on the scaffold of 1, we developed a series of derivatives to assess their efficacies in facilitating the downregulation of EGFR and c-Met, among which compound 48 represented the optimal agent. 48 showed equipotent antiproliferative activity against a panel of five NSCLC cell lines with different EGFR mutational status (IC₅₀ = 0.2–0.6 μM), while the same panel exhibited differential sensitivity to different EGFR kinase inhibitors tested. Cell cycle analysis indicated that the antiproliferative activity of 48 was associated with its ability to cause G2/M arrest and, to a lesser extent, apoptosis. Western blot and RT-PCR analyses revealed that 48 facilitated the downregulation of EGFR and c-Met at the protein level. In vivo data showed that oral administration of 48 was effective in suppressing gefitinib-resistant H1975 xenograft tumor growth in nude mice, and at a suboptimal dose, could sensitize H1975 tumors to gefitinib. Based on these findings, 48 represents a promising candidate for further development to target EGFR TKI-resistant NSCLC via dual inhibition of EGFR and c-Met oncoproteins.

Full text of DOI:10.1016/j.ejmech.2019.111607

European Journal of Medicinal Chemistry 182 (2019) 111607
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
1,2,4-Oxadiazole derivatives targeting EGFR and c-Met degradation in
TKI resistant NSCLC
,
,
,
c
,
, d, *
Eman M.E. Dokla a 1, Chun-Sheng Fang b 1, Khaled A.M. Abouzid a **, Ching S. Chen b
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, 11566, Egypt
b Institute of New Drug Development, China Medical University, Taichung, 40402, Taiwan
c Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Menoufia, Egypt
d Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, 40447, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Development of small-molecule agents with the ability to facilitate oncoprotein degradation has
emerged as a promising strategy for cancer therapy. Since EGFR and c-Met are both implicated in
oncogenesis and tumor progression, we initiated a screening program by using an in-house library to
identify agents capable of inducing the concomitant suppression of EGFR and c-Met expression, which
led to the identification of compound 1, a 1,2,4-oxadiazole derivative. Based on the scaffold of 1, we
developed a series of derivatives to assess their efficacies in facilitating the downregulation of EGFR and
c-Met, among which compound 48 represented the optimal agent. 48 showed equipotent anti-
proliferative activity against a panel of five NSCLC cell lines with different EGFR mutational status
Received 22 July 2019
Received in revised form
2 August 2019
Accepted 7 August 2019
Available online 9 August 2019
Keywords:
1,2,4-Oxadiazoles
Oncoprotein degradation
EGFR
c-Met
TKI resistant NSCLC
(IC50 ¼ 0.2e0.6
mM), while the same panel exhibited differential sensitivity to different EGFR kinase in-
hibitors tested. Cell cycle analysis indicated that the antiproliferative activity of 48 was associated with
its ability to cause G2/M arrest and, to a lesser extent, apoptosis. Western blot and RT-PCR analyses
revealed that 48 facilitated the downregulation of EGFR and c-Met at the protein level. In vivo data
showed that oral administration of 48 was effective in suppressing gefitinib-resistant H1975 xenograft
tumor growth in nude mice, and at a suboptimal dose, could sensitize H1975 tumors to gefitinib. Based
on these findings, 48 represents a promising candidate for further development to target EGFR TKI-
resistant NSCLC via dual inhibition of EGFR and c-Met oncoproteins.
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
represents a valuable approach to tackle cancer [2], such approach
was limited by the number of undruggable targets and the devel-
Despite extensive efforts directed toward developing effective
therapies to fight cancer in the past two decades, it remains a
threatening challenge worldwide. While conventional cancer
therapies are non-discriminative between normal and malignant
cells, targeted therapy acts by blocking essential biochemical
pathways or oncoproteins that are required for tumor cell growth
and survival, thereby selectively killing cancer cells over normal
cells [1]. Although targeting oncogenic proteins inhibition
opment of resistance. A more effective strategy was soon developed
to overcome these limitations by targeting oncoproteins degrada-
tion rather than merely inhibiting them [3,4]. This strategy involves
the use of small interfering RNA that acts on the gene expression
level or the use of chemical inducers that acts on post-translational
level [5,6].
Epidermal Growth Factor Receptor (EGFR) belongs to the ErbB
family of receptor tyrosine kinases [7] and it is over-expressed in
lung, colon and breast cancers [8]. On the other hand c-Met,
another receptor tyrosine kinase, can result in tumor growth and
progression in gastric, esophageal, colon and lung cancers [9].
Several small molecule tyrosine kinase inhibitors (TKIs) were
developed to inhibit both kinases, but due to the development of
resistance (e.g. EGFR mutation, c-Met over-expression), many of
these agents were rendered ineffective or of limited clinical use
[10]. Several approaches were proposed to target EGFR-TKI
* Corresponding author. Institute of New Drug Development, China Medical
University, Taichung, 40402, Taiwan.
** Corresponding author. Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Ain Shams University, Abbassia, Cairo, 11566, Egypt.
E-mail addresses: khaled.abouzid@pharma.asu.edu.eg (K.A.M. Abouzid),
cschencmu@gmail.com (C.S. Chen).
1
Both authors have equal contributions.
https://doi.org/10.1016/j.ejmech.2019.111607
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
resistant cancers [11], including design of new irreversible TKIs
[12,13], use of anti-EGFR antibodies [14,15], development of TKI-
combinations [16,17], or targeting EGFR and other related onco-
proteins degradation [18].
was achieved through the synthetic route outlined in Scheme 1.
Stirring nicotinoyl chloride hydrochloride (I) and the appropriate 4-
aminobenzonitrile (IIa-g) in acetonitrile and triethylamine at room
temperature furnished benzonitrile intermediates (IIIa-g) in
50e90% yield. Heating IIIa-g with hydroxylamine solution in
ethanol gave the amidoxime intermediates (IVa-g) in 80e100%
yield. Cyclization of IVa-g was achieved by reacting with 3-
nitrobenzoyl chloride in pyridine at 100 ꢀC to give the 1,2,4-
oxadiazole intermediates (Va-g) in 60e95% yield. Reduction of
Va-g was achieved using SnCl2$2H2O in EtOAc under refluxing
conditions to give the amino intermediates (VIa-g) in 40e80%
yield. Compound 1 was prepared in 64% yield by acylating the
amine derivative (VIc) with benzoyl chloride in pyridine. Com-
pounds 2e24 were synthesized by reacting the amine derivative
(VIa-g) with the respective sulfonyl chloride in pyridine at room
temperature to furnish the desired final compounds 2e24 in
20e81% yield. Compounds 25e50 were prepared in two steps by
heating the amine derivatives (VIc) with phosgene in dry THF to
give the corresponding isocyanate intermediates, which was used
immediately, without purification, to react with appropriate aniline
derivatives and N,N-diisopropylethylamine (DIPEA) in dry THF to
afford the desired final compounds 25e50 in 15e90% yield.
Several Hsp90 inhibitors (Fig. 1) were reported to target TKI
resistant cancer cell lines via promoting the degradation of several
kinases including EGFR and c-Met. A chalcone derivative 1f was
reported to inhibit Hsp90 with subsequent degradation of EGFR, c-
Met, Her2 and Akt kinases. 1f showed modest antiproliferative
activity against gefitinib-resistant NSCLC cell line (H1975)
(GI50 ¼ 48 mM) [19]. STA-9090 is another Hsp90 inhibitors devel-
oped by Synta pharmaceuticals. It is a triazole derivative (structure
not disclosed) that exhibited potent activity against several TKI
resistant human cancer cell lines at nanomolar concentrations via
inducing the degradation of several oncogenic proteins including
mutated EGFR and c-Met [20]. A thiochromeno[2,3-c]quinolin-12-
one derivative, TC-N19, was reported to target the degradation of
EGFR and c-Met in resistant NSCLC [21,22]. Recently an Hsp90 in-
hibitor; DPide was reported to exert a potent antiproliferative ac-
tivity against triple-negative breast cancer and NSCLC cell lines
(MDA-MB-231 and H1975 with a GI50 of 0.478 and 1.67 mM
respectively). Such activity is postulated to be mediated through
proteasomal degradation of EGFR, Her2, Met, Akt, c-Raf, and Cdk4
[23]. T315 is a pyrazole-based small molecule (Fig. 1) first reported
as an integrin-linked kinase (ILK) inhibitor [24]. In a subsequent
study, this compound was reported to inhibit EGFR-TKI resistant
lung adenocarcinoma via promoting EGFR degradation through the
ubiquitin-proteasome pathway [25]. These small molecules repre-
sent a proof of concept that targeting EGFR and c-Met oncoprotein
degradation can provide an alternative pathway to treat TKI resis-
tant cancers.
Till today, screening chemical libraries still provides an impor-
tant resource for identification of valuable lead compounds espe-
cially for new biological targets [26]. As targeting oncoprotein
degradation represents an attractive strategy for anti-cancer drug
discovery, we initiated a screening of our in-house compound li-
braries to identify agents capable of facilitating the degradation of
EGFR by using MDA-MB-231 breast cancer cells as a cell model. This
screening program led to the identification of an oxadiazole de-
rivative, 1 [N-(4-(5-(3-benzamidophenyl)-1,2,4-oxadiazol-3-yl)-3-
chlorophenyl)-nicotinamide] (Fig. 2), which exhibited a unique,
yet modest ability to concomitantly down-regulate the expression
of EGFR and another oncogenic receptor tyrosine kinase, c-Met
(Fig. 3). In this study, we developed a series of dual-target in-
hibitors, represented by compound 48, which exhibited high effi-
cacy in inducing the degradation of EGFR and c-Met oncoproteins.
2.2. Biological activities
2.2.1. In vitro antiproliferative screening in MDA-MB-231 cells
In light of the modest potency of 1 in suppressing EGFR and c-
Met expression in MDA-MB-231 cells (cell viability IC50, 12.8 mM
after 72 h; MTT assays), we conducted several optimization cycles
based on the modifications of ring A, ring B, and linker C, which
generated four series of derivatives consisting of a total of 49
compounds (Fig. 2), according to a synthetic scheme depicted in
Scheme 1. The antiproliferative activities of these compounds were
first evaluated in MDA-MB-231 cells (Fig. 2) by MTT assays to help
identify optimal agents. In series I, bioisosteric replacement of the
amide linker C with a sulfonamide, followed by adding various
substituents at position 3 and/or 4 on ring A gave inactive com-
pounds except for compound 8 (IC50, 2.5 mM). Subsequent optimi-
zation of 8 included the replacement of the 2-Cl moiety on ring B
with various substituents at position 2 or 3 and the acetamido
moiety on ring A with different urea functions, generating series II
(11e16) and III (17e24), respectively. Except for compound 18 (IC50
,
1.3 M), these modifications did not yield derivatives with
m
improved potency. Furthermore, replacement of the amide linker C
with a urea, followed by substitutions on ring A with various
functional groups at position 3 and/or 4 generated an array of de-
rivatives (series IV, 25e50) with a broad range of anti-proliferative
activity, especially noteworthy were those with IC50 values < 1
in our initial screening, including 39e43, 47 and 48.
mM
2. Results and discussion
2.1. Chemistry
2.2.2. Western blot analysis in MDA-MB-231 cells
The synthesis of the desired 1,2,4-oxadiazole derivatives 1e50
As part of the mechanistic validation, seven representative
Fig. 1. Chemical structures of reported small molecules targeting EGFR and c-Met Degradation via different mechanisms.

E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
3
Fig. 2. Structures of 1,2,4-oxadiazole-based compounds 1e50 and their respective antiproliferative IC50 values measured in MDA-MB-231 cells by MTT assays after 72 h of drug
treatment.
oxadiazole derivatives with varying antiproliferative potencies (29,
2.2.3. In vitro antiproliferative efficacy of compound 48 in a panel of
NSCLC cell lines with different EGFR mutational status
ꢀ
34, 39, 40, 43, 47, and 48) vis-a-vis compound 1 were tested for
their abilities to suppress the expression of EGFR and c-Met in
MDA-MB-231 cells. As shown in Fig. 3, among these seven de-
rivatives, only compounds 43, 47, and 48 exhibited a suppressive
effect on the expression of both oncoproteins with potencies par-
allel to the respective anti-proliferative activities. Although com-
pounds 29, 34, 39, and 40 were able to inhibit the expression of c-
Met, their suppressive effect on EGFR was less evident. As EGFR and
c-Met represent important therapeutic targets for lung cancer, we
used lung cancer cell lines with different EGFR mutational status to
elucidate the mode of antitumor mechanism of the optimal agent
48.
In this study, we used five NSCLC cell lines with different EGFR
mutational status to interrogate the effect of 48 on EGFR and c-Met
expression, including A549 (wild type), PC9 (DelE746-A750 [27]),
H1975 (L858R [27] þ T790M [28]), CL68 (DelE746-A750 þ T790M),
and CL97 (G719A [29] þ T790M). These NSCLC cell lines was first
characterized by their susceptibilities to four different TKIs by MTT
assays, including the first-generation TKIs gefitinib and erlotinib
and the irreversible TKIs afatinib and omisertinib. While PC9 cells
were extremely sensitive to all four TKIs, (IC50 values at 72 h: ꢁ
0.1
mM), the other cell lines examined (A549, H1975, CL68, and
CL97) were resistant to gefitinib and erlotinib (IC50 values at 72 h:

4
E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
Fig. 3. Western blot analysis of the effect of compound 1 and its derivatives, at indicated concentrations, on the expression of EGFR and c-Met in MDA-MB-231 cells after 48 h of
drug treatment.
Scheme 1. Synthesis of oxadiazole derivatives 1e50. Reagents and Conditions. (a) CH3CN, TEA, rt, overnight; (b) NH2OH (50% in water), EtOH, reflux, 24 h; (c) 3-Nitrobenzoyl
chloride, pyridine, 100 ꢀC, 16 h; (d) SnCl2$2H2O, EtOAc, reflux, 24 h; (e) Benzoyl chloride, THF, pyridine, rt, 4 h; (f) ArSO2Cl, pyridine, rt, 24 h; (g) Phosgene, THF, reflux, 4 h; (h)
ArNH2, THF, DIPEA, reflux, overnight.
5e20
proliferative potency among these five cell lines, with IC50 values
ranging from 0.3 to 0.6 M and 0.2e0.5 M after 48 and 72 h of
m
M) (Table 1). In contrast, 48 exhibited consistent anti-
RT-PCR analysis showed that the mRNA levels of EGFR and c-Met
remained unaltered in response to 48 at 0.2e0.6 M in H1975 and
m
m
m
A549 cells (Fig. 4C), indicating that the drug effect was mediated at
the protein level. Cell cycle analysis using propidium iodide showed
that the antiproliferative effect of 48 was mainly associated with
G2/M arrest and, to a lesser extent, apoptosis (Fig. 4D), which is
consistent with a reported finding that siRNA-mediated knock-
down of EGFR caused G2/M arrest in cancer cells [30].
treatment respectively (Table 1 and Fig. 4A).
2.2.4. Western blot, RT-PCR and flow cytometric analyses of the
effect of compound 48 on a panel of NSCLC cell lines
Western blot analysis confirmed the ability of 48, in the range of
0.2e0.6 mM after 48 h treatment, to suppress the expression of
EGFR and c-Met in these five cell lines irrespective of their muta-
tional status (Fig. 4B), which correlates with its ability to inhibit the
proliferation of these NSCLC cell lines. Apart from CL68 cells, this
downregulation of EGFR expression was accompanied by a parallel
decrease in Akt phosphorylation, suggesting differences in the
regulation of EGFR-mediated Akt activation in CL68 cells. Moreover,
2.2.5. In vivo efficacy of compound 48 in nude mice H1975
xenograft tumor model
Pursuant to the above findings, we tested the in vivo tumor-
suppressive efficacy of compound 48 in athymic nude mice
bearing subcutaneous H1975 xenograft tumors. H1975 cells are
resistant to the first-generation EGFR-TKIs (Table 1) because these

E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
5
Table 1
The antiproliferative efficacies (IC50 in mM) of four TKIs in comparison to compound 48 on five lung adenocarcinoma cell lines determined by MTT assays after 48e72 h
treatment.
Cell lines
EGFR mutation
Gefitinib
(1st gen. TKI)
Erlotinib
(1st gen. TKI)
Afatinib
(2nd gen. TKI)
Omisertinib
(3rd gen. TKI)
Compound 48
48 h
72 h
M)
72 h
M)
(
m
(m
M)
(
m
A549
PC9
H1975
CL68
CL97
WTa
20
0.1
10e20
5
20
0.1
10
5
>20
5e10
<0.01
1
0.1
0.1
5
0.3
0.6
0.3
0.5
0.6
0.2
0.5
0.3
0.4
0.5
delE746-A750b
L858Rc þ T790Md
<0.01
0.05
0.01
0.1
delE746-A750 þ T790M
G719Ae þ T790M
10e20
a
WT: wild-type.
b
delE746-A750: 747 to 750 frame deletion of amino acids in exon 19 [27].
c
d
e
L858R: single-point mutation of leucine to arginine at codon 858 in exon 21 [27].
T790M: threonine to methionine mutation at position 790 in exon 20 [28].
G719A: single-point mutation of glycine to alanine at codon 719 in exon 18 [29].
cells harbor the gatekeeper mutation T790M [31e33]. As shown,
daily oral administration of 48, at 50 or 150 mg/kg, versus vehicle
via gavage (n ¼ 8 for each group) exhibited a dose-dependent
suppression (29% and 60%, respectively) of H1975 xenograft tu-
mor growth relative to vehicle control at the end of the 20 day-
treatment (Fig. 5A, left panel; *P < 0.05). 48 was well tolerated by
tumor-bearing mice as no appreciable weight loss was noted in
both drug-treated groups, indicating that no acute toxicities were
associated with drug treatment (Fig. 5A, right panel). Western blot
analysis of tumor lysates showed that this tumor-suppressive ac-
tivity correlated with the ability of 48 to inhibit the expression of
EGFR and c-Met in a dose-dependent manner (Fig. 5B). In light of
the cross-activation between EGFR and c-Met in lung cancer cells,
dual targeting of EGFR and c-Met has been reported to be an
effective strategy against T790M EGFR-mediated TKI resistant lung
cancer [17]. Consequently, we assessed the ability of 48 at a sub-
optimal dose to sensitize H1975 xenograft tumors to gefitinib. As
shown, combination of 48 and gefitinib, each at 50 mg/kg daily via
oral gavage, gave rise to a higher extent of tumor suppression (72%)
relative to individual drugs alone (Fig. 5C; 48, 37%; gefitinib, 27%).
spectroscopic data of the intermediates and final compounds 1 to
50 is described in the Supplementary data. All commercially
available reagents were used without further purification unless
otherwise stated. Routine 1H and 13C nuclear magnetic resonance
spectra were recorded on Bruker AV300 or Bruker Ascend 400.
Samples were dissolved in deuterated chloroform (CDCl3) or
dimethyl sulfoxide (DMSO‑d6), and tetramethylsilane (TMS) was
used as a reference. Electrospray ionization mass spectrometry
analyses were performed with a Micromass Q-T of II high resolution
electrospray mass spectrometer at The Ohio State University
Campus Chemical Instrument Center. All compounds for bioassay
were identified with 1H NMR, 13C NMR, and HRMS, and in purity
higher than 95%. The purity of was confirmed by a Hitachi Elite
LaChrom HPLC system (comprised of a Versa Grad Prep 36 pump,
an L-2400 UV detector, an L-2200 auto sampler and a 150 ꢂ 4.6 mm
Agilent ZORBAX Eclipse XDB-C18 5m column; detection, 254 nm). A
linear solvent gradient with a mobile phase of 30% water in
methanol to 100% methanol in 20 min was used.
4.1.1. General synthetic procedures for compounds 1-50
3. Conclusion
Step a: To an ice-cold suspension of nicotinoyl chloride hydro-
chloride (I) (1.78 g, 10 mmol) and appropriate 4-
aminobenzonitrile (IIa-g) (10 mmol, 1.0 equiv) in acetonitrile
(50 ml) under nitrogen was added triethylamine (8.1 g, 11.15 ml,
40 mmol, 4.0 equiv) dropwise over 30 min. The resulting sus-
pension was stirred at 0 ꢀC for 1 h then at room temperature
overnight. The reaction mixture was evaporated, diluted with
EtOAc, washed with 10% HCl, brine, dried over anhydrous
Na2SO4 and concentrated in vacuo. The resulting solid was
washed with diethyl ether and dried to yield the desired com-
pounds (IIIa-g) in 50e90% yield.
Step b: To a solution of appropriate benzonitrile (IIIa-g)
(10 mmol, 1 equiv) in ethanol (100 ml) was added hydroxyl-
amine solution (50% in water, 2 ml, 30 mmol, 3.0 equiv) and the
reaction mixture was refluxed for 24 h. The reaction mixture
was evaporated, then dried under vacuum to give the desired
amidoxime derivatives (IVa-g) in 80e100% yield.
In this study we report the identification of a 1,2,4-oxadiazole-
based small molecule, compound 48, that exhibited a unique ability
to co-target EGFR and c-Met oncoprotein degradation. 48 showed
potent antiproliferative activity against MDA-MB-231 and a panel
of NSCLC cell lines. While the activities of four TKIs used for com-
parison were variable conforming to the type of EGFR mutation, the
efficacy of 48 was consistent irrespective of EGFR mutational status.
Moreover, 48 showed a dose-dependent tumor-suppressive activ-
ity in TKI resistant H1975 xenograft tumors and was effective in
sensitizing these tumors to gefitinib at
a suboptimal dose.
Accordingly, 48 provides a proof-of-concept that concomitant tar-
geting EGFR and c-Met degradation, in lieu of dual kinase activity
inhibition, might represent an alternative strategy to circumvent
EGFR mutation-mediated resistance, which is associated with even
the 3rd-generation irreversible TKIs [34]. Further optimization and
mechanistic validation of the exact molecular target of 48 will help
establish this series as a preclinical candidate to treat EGFR-TKI
resistant tumors.
Step c: To
a solution of appropriate amidoxime (IVa-g)
(10 mmol, 1.0 equiv) in pyridine (50 ml) was added 3-
nitrobenzoyl chloride (2.78 g, 15 mmol, 1.5 equiv) portion wise
with stirring. The reaction mixture was heated at 100 ꢀC for 16 h.
The solution was cooled and added to ice/water (500 ml) with
vigorous stirring, then kept in refrigerator overnight. The
precipitated solid was filtered, washed with water, diethyl ether
and dried in vacuo to give the desired 1,2,4-oxadiazole de-
rivatives (Va-g) in 60e95% yield.
4. Experimental
4.1. Chemistry
Detailed information on the syntheses and pertinent

6
E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
Fig. 4. (A) MTT assays of the time- and dose-dependent inhibitory effect of 48 on the viability of A549, PC9, H1975, CL68, and CL97 cells at different time intervals (-, 24 h; :,
48 h; ꢃ, 72 h). Data are presented as means S.D (n ¼ 6). (B) Western blot analysis of the dose-dependent effects of 48 on the expression/phosphorylation of EGFR, c-Met, and Akt
after 48 h treatment. (C) RT-PCR analysis of the effect of 48 on EGFR and c-Met mRNA expression in H1975 and A549 cells. (D) Flow cytometric analysis of the dose-dependent
effects of 48 on the cell cycle distribution after 24 and 48 h of treatment.
Step d: To a suspension of the appropriate nitro derivative (Va-g)
(5 mmol, 1.0 equiv) in EtOAc (100 ml) was added SnCl2$2H2O
(5.6 g, 25 mmol, 5.0 equiv) and the reaction mixture was
refluxed for 24 h. Upon completion of the reaction as indicated
by TLC, the mixture was cooled, washed with 1N NaOH, brine,
dried over anh. Na2SO4, filtered and concentrated in vacuo to
give the desired amino compounds (VIa-g) in 40e80% yield.
Step e: To an ice cold solution of N-[4-[5-(3-aminophenyl)-1,2,4-
oxadiazol-3-yl]-3-chlorophenyl]-3-pyridinecarboxamide (VIc)
(0.1 g, 0.26 mmol, 1 equiv) in THF (10 ml) and pyridine (0.04 g,
at room temperature for 4 h. Upon completion of the reaction as
indicated by TLC, the solution was concentrated and the residue
was purified by chromatography to give the desired final
products 1.
4.1.1.1. N-[3-chloro-4-[5-[3-[(benzoyl)amino]phenyl]-1,2,4-
oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide (1). Rf ¼ 0.25 (DCM/
MeOH 9:1). White solid, yield 64%. 1H NMR (300 MHz, DMSO‑d6)
d
10.88 (s, 1H), 10.62 (s, 1H), 9.14 (dd, J ¼ 2.3, 0.9 Hz, 1H), 8.85e8.71
41
44
m
m
l, 0.52 mmol, 2 equiv) was added benzoyl chloride (0.053 g,
l, 0.38 mmol, 1.5 equiv) and the reaction mixture was stirred
(m, 2H), 8.33 (dt, J ¼ 7.9, 2.0 Hz, 1H), 8.27e7.88 (m, 7H), 7.72e7.50
(m, 5H).

E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
7
4.1.1.4. N-[4-[5-[3-[[4-(aminosulfonyl)phenylsulfonyl]amino]
phenyl]-1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-pyridinecarboxamide
(4). Rf ¼ 0.25 (EtOAc/Hexane/MeOH 4:2:0.25). White solid, yield
69%. 1H NMR (300 MHz, DMSO‑d6)
d 10.98 (s, 1H), 10.87 (s, 1H), 9.14
(d, J ¼ 1.4 Hz, 1H), 8.80 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.33 (dt, J ¼ 8.0,
2.0 Hz, 1H), 8.22 (d, J ¼ 2.0 Hz, 1H), 8.17e7.93 (m, 7H), 7.88 (dt,
J ¼ 7.9, 1.3 Hz, 1H), 7.66e7.52 (m, 4H), 7.49e7.42 (m, 1H).
4.1.1.5. N-[3-chloro-4-[5-[3-[(4-methylphenylsulfonyl)amino]
phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
Rf ¼ 0.20 (EtOAc/Hexane/MeOH 4:4:0.25). White solid, yield 45%.
1H NMR (300 MHz, DMSO‑d6)
10.84 (s, 1H), 10.66 (s, 1H), 9.15 (dd,
(5).
d
J ¼ 2.4, 0.8 Hz, 1H), 8.80 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.33 (dt, J ¼ 8.0,
2.0 Hz,1H), 8.22 (d, J ¼ 2.0 Hz,1H), 8.04 (d, J ¼ 8.6 Hz,1H), 7.99e7.92
(m, 2H), 7.83 (dt, J ¼ 7.8, 1.3 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz, 2H),
7.65e7.49 (m, 2H), 7.49e7.41 (m, 1H), 7.37 (d, J ¼ 8.1 Hz, 2H), 2.33 (s,
3H).
4.1.1.6. N-[3-chloro-4-[5-[3-[[4-(trifluoromethyl)phenylsulfonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(6). Rf ¼ 0.35 (EtOAc/Hexane/MeOH 4:4:0.25). White solid, yield
63%. 1H NMR (400 MHz, DMSO‑d6)
d 10.88e10.82 (m, 2H), 9.14 (d,
J ¼ 2.2 Hz, 1H), 8.80 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.33 (dt, J ¼ 8.0, 2.1 Hz,
1H), 8.21 (d, J ¼ 2.1 Hz, 1H), 8.05e8.00 (m, 2H), 8.00e7.73 (m, 5H),
7.66e7.40 (m, 3H), 7.08 (d, J ¼ 8.9 Hz, 1H).
4.1.1.7. N-[3-chloro-4-[5-[3-[(3-methoxyphenylsulfonyl)amino]
Fig. 5. In vivo efficacy of compound 48, alone or in combination with gefitinib, in
suppressing H1975 xenograft tumors. (A) Effect of 48 at 50 and 150 mg/kg daily
(mpk) versus vehicle control, via oral gavage, on tumor volume (left) and body weight
(right) in H1975 xenograft model (n ¼ 8/each group) throughout a 20-day treatment
period. Points, means S.E. (n ¼ 8). (B) Immunoblotting analysis (left) of EGFR and c-
Met in tumor lysates of four representative tumors from each group and analysis of
relative expression values (right) of individual markers based on densitometric
quantitation of band intensities. (C) Effect of the combination of 48 and gefitinib versus
single agents, each at 50 mg/kg daily via oral gavage, on tumor volume in H1975
xenograft model (n ¼ 7/each group) after 20 days of treatment. The data are presented
as means S.E. (*P < 0.05, **P < 0.01).
phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
Rf ¼ 0.35 (EtOAc/Hexane/MeOH 4:3:0.25). White solid, yield 66%.
1H NMR (300 MHz, DMSO‑d6)
10.87 (s, 1H), 10.75 (s, 1H), 9.14 (s,
(7).
d
1H), 8.80 (d, J ¼ 4.8 Hz, 1H), 8.33 (d, J ¼ 8.0 Hz, 1H), 8.22 (s, 1H),
8.09e7.90 (m, 3H), 7.85 (d, J ¼ 6.6 Hz, 1H), 7.66e7.29 (m, 6H), 7.19
(d, J ¼ 8.3 Hz, 1H), 3.77 (s, 3H).
4.1.1.8. N-[4-[5-[3-[[4-(acetylamino)phenylsulfonyl]amino]phenyl]-
1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-pyridinecarboxamide
Rf ¼ 0.40 (EtOAc/Hexane/MeOH 4:1:0.25). White solid, yield 57%.
1H NMR (300 MHz, DMSO‑d6)
10.85 (s, 1H), 10.60 (s, 1H), 10.29 (s,
(8).
d
Step f: To a cooled solution of the appropriate N-[4-[5-(3-
1H), 9.15 (d, J ¼ 2.3 Hz, 1H), 8.81 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.34 (dt,
J ¼ 8.2, 1.9 Hz, 1H), 8.22 (d, J ¼ 1.9 Hz, 1H), 8.05 (d, J ¼ 8.6 Hz, 1H),
8.00e7.89 (m, 2H), 7.83 (d, J ¼ 8.0 Hz, 1H), 7.79e7.69 (m, 4H),
7.65e7.50 (m, 2H), 7.48e7.41 (m, 1H), 2.05 (s, 3H). 13C NMR
aminophenyl)-1,2,4-oxadiazol-3-yl]substituted
phenyl]-3-
pyridinecarboxamide (VIa-g) (0.26 mmol, 1 equiv) in pyridine
(10 ml) was added the appropriate sulfonyl chloride derivative
(0.38 mmol, 1.5 equiv) and the reaction mixture was stirred at
room temperature overnight. Upon completion of the reaction
as indicated by TLC, the solution was concentrated, stirred with
cold water and the resulting precipitate was filtered, dried and
purified by chromatography to give the desired final products
(2e24).
(75 MHz, DMSO‑d6)
d 174.19, 169.10, 166.83, 164.75, 152.58, 148.86,
143.45, 142.34, 139.07, 139.02, 135.70, 132.50, 132.39, 132.27, 130.76,
130.06, 128.10, 124.04, 123.63, 123.21, 121.35, 120.12, 118.75, 118.70,
118.35, 24.17. HRMS exact mass of C28H21ClN6O5S (M þ Na)þ:
611.0875 amu; found: 611.0884 amu.
4.1.1.9. N-[4-[5-[3-[[4-(acetylamino)-3-chlorophenylsulfonyl]amino]
phenyl]-1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-pyridinecarboxamide
(9). Rf ¼ 0.30 (DCM/MeOH 9.5:0.5). White solid, yield 29%. 1H NMR
4.1.1.2. N-[3-chloro-4-[5-[3-[(phenylsulfonyl)amino]phenyl]-1,2,4-
oxadiazol-3-yl]
(EtOAc/Hexane 3:1). White solid, yield 65%. 1H NMR (300 MHz,
DMSO‑d6)
phenyl]-3-pyridinecarboxamide
(2). Rf ¼ 0.30
(300 MHz, DMSO‑d6)
d 10.85 (s, 1H), 10.78 (s, 1H), 9.69 (s, 1H), 9.15
(d, J ¼ 2.3 Hz, 1H), 8.80 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.33 (dt, J ¼ 8.0,
d
10.84 (s, 1H), 10.74 (s, 1H), 9.15 (d, J ¼ 1.9 Hz, 1H), 8.80
2.0 Hz, 1H), 8.22 (d, J ¼ 2.0 Hz, 1H), 8.14e7.82 (m, 6H), 7.79e7.42 (m,
4H), 2.12 (s, 3H). 13C NMR (75 MHz, DMSO‑d6)
d 174.12, 169.25,
(dd, J ¼ 4.8, 1.5 Hz, 1H), 8.33 (dt, J ¼ 7.9, 1.9 Hz, 1H), 8.21 (d,
J ¼ 1.9 Hz, 1H), 8.04 (d, J ¼ 8.6 Hz, 1H), 7.99e7.90 (m, 2H), 7.87e7.80
(m, 3H), 7.69e7.49 (m, 5H), 7.49e7.38 (m, 1H).
166.83, 164.76, 152.59, 148.87, 142.36, 139.42, 138.58, 135.71, 135.06,
132.39, 132.29, 130.96, 130.06, 127.85, 126.20, 124.98, 124.90, 124.31,
124.19, 123.67, 123.62, 121.36, 120.10, 118.72, 118.65, 23.73.
4.1.1.3. N-[3-chloro-4-[5-[3-[(3-chlorophenylsulfonyl)amino]phenyl]-
1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
Rf ¼ 0.30 (EtOAc/Hexane/MeOH 4:4:0.25). White solid, yield 68%.
1H NMR (300 MHz, DMSO‑d6)
0.9 Hz, 1H), 8.80 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.33 (dt, J ¼ 8.0, 2.0 Hz, 1H),
8.22 (d, J ¼ 2.0 Hz, 1H), 8.05 (d, J ¼ 8.6 Hz, 1H), 7.99e7.82 (m, 4H),
7.79e7.69 (m, 2H), 7.67e7.52 (m, 3H), 7.46 (dt, J ¼ 8.6, 1.3 Hz, 1H).
(3).
4.1.1.10. N-[4-[5-[3-[[4-(acetylamino)-3-methylphenylsulfonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-
pyridinecarboxamide (10). Rf ¼ 0.30 (DCM/MeOH 9.5:0.5). White
d
10.87 (s, 2H), 9.14 (dd, J ¼ 2.4,
solid, yield 65%. 1H NMR (300 MHz, DMSO‑d6)
d 10.87 (s, 1H), 10.77
(s, 1H), 9.41 (d, J ¼ 4.6 Hz, 1H), 9.14 (d, J ¼ 2.2 Hz, 1H), 8.80 (d,
J ¼ 4.1 Hz, 1H), 8.33 (dt, J ¼ 8.1, 2.0 Hz, 1H), 8.22 (d, J ¼ 1.9 Hz, 1H),

8
E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
8.15e7.89 (m, 4H), 7.87e7.75 (m, 1H), 7.72e7.34 (m, 5H), 2.24 (s,
3H), 2.07 (s, 3H).
4.1.1.17. N-[4-[5-[3-[[4-[(aminocarbonyl)amino]phenylsulfonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-
pyridinecarboxamide (17). Rf ¼ 0.40 (DCM/MeOH 9:1). White solid,
yield 20%. 1H NMR (300 MHz, DMSO‑d6)
d 10.88 (s,1H),10.60 (s,1H),
4.1.1.11. N-[4-[5-[3-[[4-(acetylamino)phenylsulfonyl]amino]phenyl]-
9.15 (d, J ¼ 1.9 Hz, 1H), 9.01 (s, 1H), 8.81 (dd, J ¼ 4.9, 1.6 Hz, 1H), 8.34
(dt, J ¼ 8.0, 1.9 Hz, 1H), 8.22 (d, J ¼ 2.1 Hz, 1H), 8.06 (d, J ¼ 8.8 Hz,
1H), 7.99e7.90 (m, 2H), 7.83 (d, J ¼ 8.0 Hz, 1H), 7.72e7.59 (m, 3H),
7.58e7.49 (m, 3H), 7.47e7.37 (m, 1H), 6.08 (s, 2H). 13C NMR
1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
Rf ¼ 0.35 (EtOAc/Hexane/MeOH 4:1:0.25). White solid, yield 40%.
1H NMR (300 MHz, DMSO‑d6)
10.76 (s, 1H), 10.64 (s, 1H), 10.32 (s,
1H), 9.14 (d, J ¼ 1.3 Hz, 1H), 8.79 (dd, J ¼ 4.5, 1.5 Hz, 1H), 8.37e8.28
(m, 1H), 8.12e7.99 (m, 4H), 7.90 (s, 1H), 7.83 (d, J ¼ 7.2 Hz, 1H),
7.79e7.69 (m, 4H), 7.64e7.50 (m, 2H), 7.42 (d, J ¼ 9.3 Hz,1H), 2.04 (s,
3H).
(11).
d
(75 MHz, DMSO‑d6)
d 174.66, 167.25, 165.19, 155.91, 153.02, 149.27,
145.45, 142.75, 139.65, 136.13, 132.81, 132.72, 131.17, 130.73, 130.49,
128.51, 124.43, 124.07, 123.47, 121.76, 120.58, 120.57, 119.17, 118.54,
117.58. HRMS exact mass of C27H20ClN7O5S (M þ Na)þ: 612.0827
amu; found: 612.0845 amu.
4.1.1.12. N-[4-[5-[3-[[4-(acetylamino)phenylsulfonyl]amino]phenyl]-
1,2,4-oxadiazol-3-yl]2-chlorophenyl]-3-pyridinecarboxamide
Rf ¼ 0.20 (EtOAc/Hexane/MeOH 4:1:0.25). White solid, yield 53%.
1H NMR (300 MHz, DMSO‑d6)
10.51 (s, 1H), 10.32 (s, 1H), 9.16 (dd,
(12).
4.1.1.18. N-[3-chloro-4-[5-[3-[[4-[[(methylamino)carbonyl]amino]
phenylsulfonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
pyridinecarboxamide (18). Rf ¼ 0.20 (DCM/MeOH 9.5:0.5). White
d
J ¼ 2.3, 0.7 Hz, 1H), 8.81 (dd, J ¼ 4.8, 1.7 Hz, 1H), 8.38e8.31 (m, 1H),
8.18 (d, J ¼ 1.9 Hz, 1H), 8.10 (dd, J ¼ 8.4, 1.9 Hz, 1H), 7.97e7.90 (m,
2H), 7.83 (dt, J ¼ 7.7, 1.1 Hz, 1H), 7.79e7.68 (m, 4H), 7.61 (ddd, J ¼ 7.9,
4.9, 0.8 Hz, 1H), 7.53 (t, J ¼ 8.0 Hz, 1H), 7.46e7.40 (m, 1H), 2.03 (s,
solid, yield 36%. 1H NMR (300 MHz, DMSO‑d6)
d 10.88 (s, 1H), 10.59
(s, 1H), 9.15 (d, J ¼ 1.7 Hz, 1H), 9.02 (s, 1H), 8.81 (dd, J ¼ 4.9, 1.6 Hz,
1H), 8.34 (dt, J ¼ 8.1, 2.0 Hz, 1H), 8.22 (d, J ¼ 1.8 Hz, 1H), 8.05 (d,
J ¼ 8.7 Hz, 1H), 8.00e7.89 (m, 2H), 7.82 (d, J ¼ 7.9 Hz, 1H), 7.72e7.58
(m, 3H), 7.58e7.49 (m, 3H), 7.48e7.40 (m, 1H), 6.31e6.12 (m, 1H),
3H). 13C NMR (101 MHz, DMSO‑d6)
d 175.18, 168.99, 166.93, 164.20,
152.58, 148.82,143.30,139.36,137.64,135.60,132.76,130.62,129.44,
129.39,128.45,127.98,127.94,126.26,124.89,124.09,123.98,123.63,
123.01, 118.64, 118.41, 24.07. HRMS exact mass of C28H21ClN6O5S
(M þ Na)þ: 611.0875 amu; found: 611.0882 amu.
2.61 (d, J ¼ 4.6 Hz, 3H). 13C NMR (75 MHz, DMSO‑d6)
d 174.66,
167.25, 165.18, 155.66, 153.00, 149.27, 145.50, 142.76, 139.66, 136.12,
132.81, 132.70, 131.15, 130.58, 130.49, 128.52, 124.43, 124.26, 124.06,
123.45,121.77,120.57,119.17,118.56,117.45, 26.68. HRMS exact mass
of C28H22ClN7O5S (M þ Na)þ: 626.0984 amu; found: 626.0999 amu.
4.1.1.13. N-[4-[5-[3-[[4-(acetylamino)phenylsulfonyl]amino]phenyl]-
1, 2 , 4 - o x a d i a z o l - 3 - y l ] 2 - ( t r i fl u o r o m e t h y l ) p h e n y l ] - 3 -
4.1.1.19. N-[3-chloro-4-[5-[3-[[4-[[(ethylamino)carbonyl]amino]phe-
nylsulfonyl]amino] phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
pyridinecarboxamide
(13). Rf ¼ 0.40
(EtOAc/Hexane/MeOH
4:1:0.25). White solid, yield 55%. 1H NMR (300 MHz, DMSO‑d6)
pyridinecarboxamide
(19). Rf ¼ 0.20
(EtOAc/Hexane/MeOH
d
10.64 (d, J ¼ 8.7 Hz, 2H), 10.33 (s, 1H), 9.13 (d, J ¼ 2.2 Hz, 1H), 8.81
4:1:0.25). White solid, yield 35%. 1H NMR (300 MHz, DMSO‑d6)
(dd, J ¼ 4.8, 1.6 Hz, 1H), 8.47e8.37 (m, 2H), 8.31 (dt, J ¼ 8.0, 1.8 Hz,
1H), 7.93 (t, J ¼ 1.8 Hz, 1H), 7.87 (d, J ¼ 8.1 Hz, 2H), 7.78e7.69 (m,
4H), 7.62 (ddd, J ¼ 8.1, 4.8, 0.7 Hz, 1H), 7.55 (t, J ¼ 7.9 Hz, 1H),
7.48e7.43 (m, 1H), 2.03 (s, 3H).
d
10.87 (s, 1H), 10.58 (s, 1H), 9.14 (d, J ¼ 2.0 Hz, 1H), 8.92 (s, 1H), 8.80
(dd, J ¼ 4.6, 1.8 Hz, 1H), 8.33 (dt, J ¼ 8.1, 2.1 Hz, 1H), 8.22 (d,
J ¼ 2.2 Hz, 1H), 8.10e7.87 (m, 3H), 7.82 (dt, J ¼ 7.5, 1.5 Hz, 1H),
7.71e7.48 (m, 6H), 7.47e7.37 (m, 1H), 6.28 (t, J ¼ 5.3 Hz, 1H), 3.07 (p,
J ¼ 7.1 Hz, 2H), 1.01 (t, J ¼ 7.1 Hz, 3H). 13C NMR (75 MHz, DMSO‑d6)
4.1.1.14. N-[4-[5-[3-[[4-(acetylamino)phenylsulfonyl]amino]phenyl]-
1, 2 , 4 - o x a d i a z o l - 3 - y l ] 3 - ( t r i fl u o r o m e t h y l ) p h e n y l ] - 3 -
d
174.18, 166.78, 164.65, 154.48, 152.48, 148.78, 144.98, 142.26,
139.20,135.58,132.34,132.16,130.60,130.20,129.99,128.02,123.96,
123.84, 123.52, 122.96, 121.33, 120.12, 118.68, 118.17, 116.98, 33.96,
15.21. HRMS exact mass of C29H24ClN7O5S (M þ Na)þ: 640.1140
amu; found: 640.1160 amu.
pyridinecarboxamide
4:1:0.25). White solid, yield 70%. 1H NMR (300 MHz, DMSO‑d6)
11.01 (s, 1H), 10.68 (s, 1H), 10.32 (s, 1H), 9.17 (s, 1H), 8.82 (d,
(14). Rf ¼ 0.35
(EtOAc/Hexane/MeOH
d
J ¼ 4.9 Hz, 1H), 8.49 (d, J ¼ 1.9 Hz, 1H), 8.36 (dt, J ¼ 7.9, 1.8 Hz, 1H),
8.29 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.99 (d, J ¼ 8.6 Hz, 1H), 7.90 (t,
J ¼ 1.9 Hz, 1H), 7.82 (dt, J ¼ 7.7, 1.1 Hz, 1H), 7.78e7.69 (m, 4H),
7.66e7.59 (m, 1H), 7.54 (t, J ¼ 8.0 Hz, 1H), 7.46e7.40 (m, 1H), 2.04 (s,
3H).
4.1.1.20. N-[3-chloro-4-[5-[3-[[4-[[(diethylamino)carbonyl]amino]
phenylsulfonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
pyridinecarboxamide (20). Rf ¼ 0.30 (DCM/MeOH 9.5:0.5). White
solid, yield 26%. 1H NMR (300 MHz, DMSO‑d6)
d 10.88 (s, 1H), 10.58
(s, 1H), 9.14 (d, J ¼ 1.0 Hz, 1H), 8.80 (dd, J ¼ 4.3, 1.1 Hz, 1H), 8.56 (s,
1H), 8.33 (dt, J ¼ 7.9, 1.9 Hz, 1H), 8.22 (d, J ¼ 1.8 Hz, 1H), 8.05 (d,
J ¼ 8.6 Hz, 1H), 8.00e7.89 (m, 2H), 7.82 (d, J ¼ 7.6 Hz, 1H), 7.74e7.57
(m, 5H), 7.54 (t, J ¼ 8.0 Hz, 1H), 7.48e7.40 (m, 1H), 3.46e3.16 (m,
4.1.1.15. N-[4-[5-[3-[[4-(acetylamino)phenylsulfonyl]amino]phenyl]-
1,2,4-oxadiazol-3-yl]2-methoxyphenyl]-3-pyridinecarboxamide (15).
Rf ¼ 0.30 (EtOAc/Hexane/MeOH 4:1:0.25). White solid, yield 42%.
1H NMR (300 MHz, DMSO‑d6)
d 10.66 (s, 1H), 10.32 (s, 1H), 9.94 (s,
4H), 1.04 (t, J ¼ 6.9 Hz, 6H). 13C NMR (75 MHz, DMSO‑d6)
d 174.69,
1H), 9.12 (d, J ¼ 2.0 Hz, 1H), 8.78 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.32 (dt,
J ¼ 8.0, 1.8 Hz, 1H), 8.10 (d, J ¼ 8.2 Hz, 1H), 7.93e7.88 (m, 1H), 7.83 (d,
J ¼ 7.9 Hz, 1H), 7.79e7.66 (m, 6H), 7.61e7.49 (m, 2H), 7.46e7.41 (m,
1H), 3.98 (s, 3H), 2.04 (s, 3H).
167.28, 165.17, 154.26, 153.00, 149.26, 145.79, 142.76, 139.67, 136.09,
132.83, 132.68, 131.21, 131.13, 130.50, 128.01, 124.45, 124.38, 124.04,
123.49, 121.82, 120.62, 119.40, 119.20, 118.67, 41.11, 14.23.
4.1.1.21. N-[3-chloro-4-[5-[3-[[4-[[(cyclopentylamino)carbonyl]
amino]phenylsulfonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-
4.1.1.16. N-[4-[5-[3-[[4-(acetylamino)phenylsulfonyl]amino]phenyl]-
1,2,4-oxadiazol-3-yl]3-methoxyphenyl]-3-pyridinecarboxamide (16).
Rf ¼ 0.20 (EtOAc/MeOH 4:0.25). White solid, yield 81%. 1H NMR
3-pyridinecarboxamide
(21). Rf ¼ 0.30
(EtOAc/Hexane/MeOH
4:1:0.25). White solid, yield 45%. 1H NMR (300 MHz, DMSO‑d6)
(300 MHz, DMSO‑d6)
d
10.73 (s, 1H), 10.61 (s, 1H), 10.32 (s, 1H), 9.14
d
10.88 (s, 1H), 10.59 (s, 1H), 9.15 (dd, J ¼ 2.3, 0.9 Hz, 1H), 8.81 (dd,
(d, J ¼ 2.4 Hz, 1H), 8.79 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.33 (dt, J ¼ 7.9,
1.8 Hz, 1H), 7.96 (d, J ¼ 8.5 Hz, 1H), 7.88 (t, J ¼ 1.7 Hz, 1H), 7.84e7.69
(m, 6H), 7.68e7.55 (m, 2H), 7.52 (t, J ¼ 8.0 Hz, 1H), 7.44e7.38 (m,
1H), 3.91 (s, 3H), 2.04 (s, 3H).
J ¼ 5.1, 1.4 Hz, 1H), 8.73 (s, 1H), 8.34 (dt, J ¼ 8.1, 2.0 Hz, 1H), 8.22 (d,
J ¼ 2.0 Hz, 1H), 8.05 (d, J ¼ 8.6 Hz, 1H), 7.97 (dd, J ¼ 8.7, 2.0 Hz, 1H),
7.91 (t, J ¼ 1.9 Hz, 1H), 7.82 (d, J ¼ 8.3 Hz, 1H), 7.70e7.58 (m, 3H),
7.58e7.48 (m, 3H), 7.43 (ddd, J ¼ 8.2, 2.3, 1.1 Hz, 1H), 6.34 (d,

E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
9
J ¼ 7.2 Hz, 1H), 3.89 (p, J ¼ 6.6 Hz, 1H), 1.79 (dt, J ¼ 12.1, 5.9 Hz, 2H),
1.66e1.47 (m, 4H), 1.42e1.27 (m, 2H).
(d, J ¼ 7.6 Hz, 1H), 7.68 (d, J ¼ 8.3 Hz, 1H), 7.63e7.57 (m, 2H), 7.52 (d,
J ¼ 8.7 Hz, 2H), 7.34 (d, J ¼ 8.5 Hz, 2H).
4.1.1.22. N-[3-chloro-4-[5-[3-[[4-[(1-pyrrolidinylcarbonyl)amino]
phenylsulfonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
4.1.1.27. N-[4-[5-[3-[[[4-(aminosulfonyl)phenylamino]carbonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]-3-chlorophenyl]-3-
pyridinecarboxamide
(22). Rf ¼ 0.25
(EtOAc/Hexane/MeOH
pyridinecarboxamide
(27). Rf ¼ 0.30
(EtOAc/Hexane/MeOH
4:1:0.25). White solid, yield 60%. 1H NMR (300 MHz, DMSO‑d6)
4:1:0.25). White solid, yield 49%. 1H NMR (300 MHz, DMSO‑d6)
d
10.84 (s, 1H), 10.54 (s, 1H), 9.15 (s, 1H), 8.80 (d, J ¼ 4.9 Hz, 1H), 8.52
d
10.88 (s, 1H), 9.25 (s, 1H), 9.22 (s, 1H), 9.14 (dd, J ¼ 2.4, 0.9 Hz, 1H),
(s, 1H), 8.34 (d, J ¼ 8.0 Hz, 1H), 8.22 (s, 1H), 8.05 (d, J ¼ 8.6 Hz, 1H),
8.00e7.90 (m, 2H), 7.82 (d, J ¼ 7.4 Hz, 1H), 7.72e7.66 (m, 4H),
7.64e7.50 (m, 2H), 7.48e7.41 (m, 1H), 3.34 (d, J ¼ 5.6 Hz, 4H),
1.87e1.77 (m, 4H).
8.80 (dd, J ¼ 4.9, 1.6 Hz, 1H), 8.51 (t, J ¼ 1.9 Hz, 1H), 8.33 (dt, J ¼ 8.1,
1.9 Hz, 1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.09 (d, J ¼ 8.6 Hz, 1H), 7.97 (dd,
J ¼ 8.7, 2.1 Hz, 1H), 7.86e7.79 (m, 1H), 7.75 (d, J ¼ 8.7 Hz, 2H),
7.71e7.54 (m, 5H), 7.24 (s, 2H).
4.1.1.23. N-[3-chloro-4-[5-[3-[[4-[(1-piperidinylcarbonyl)amino]phe-
nylsulfonyl]amino] phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
pyridinecarboxamide (23). Rf ¼ 0.35 (DCM/MeOH 9.5:0.5). White
4.1.1.28. N-[3-chloro-4-[5-[3-[[(4-methylphenylamino)carbonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(28). Rf ¼ 0.30 (DCM/MeOH 9.5:0.5). White solid, yield 15%. 1H
solid, yield 45%. 1H NMR (300 MHz, DMSO‑d6)
d
10.88 (s, 1H), 10.60
NMR (300 MHz, DMSO‑d6) d 10.85 (s, 1H), 9.15 (s, 1H), 9.03 (s, 1H),
(s, 1H), 9.15 (s, 1H), 8.88 (s, 1H), 8.80 (d, J ¼ 3.5 Hz, 1H), 8.39e8.28
(m,1H), 8.22 (d, J ¼ 1.7 Hz,1H), 8.05 (d, J ¼ 8.6 Hz,1H), 8.01e7.89 (m,
2H), 7.85e7.78 (m, 1H), 7.73e7.49 (m, 6H), 7.47e7.39 (m, 1H),
3.50e3.18 (m, 4H), 1.64e1.36 (m, 6H). 13C NMR (75 MHz, DMSO‑d6)
8.80 (d, J ¼ 4.8 Hz, 1H), 8.65 (s, 1H), 8.48 (s, 1H), 8.34 (d, J ¼ 8.1 Hz,
1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H), 7.97 (d,
J ¼ 8.7 Hz, 1H), 7.78 (d, J ¼ 7.5 Hz, 1H), 7.71e7.52 (m, 3H), 7.37 (d,
J ¼ 8.1 Hz, 2H), 7.11 (d, J ¼ 8.0 Hz, 2H), 2.25 (s, 3H).
d
174.65, 167.25, 165.17, 154.54, 153.01, 149.28, 145.89, 142.76,
139.66, 136.12, 132.81, 132.70, 131.14, 131.01, 130.48, 128.13, 124.42,
124.30, 124.07, 123.46, 121.77, 120.56, 119.15, 119.02, 118.58, 45.16,
25.93, 24.45.
4.1.1.29. N-[3-chloro-4-[5-[3-[[[3-(trifluoromethyl)phenylamino]
carbonyl]amino] phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
pyridinecarboxamide (29). Rf ¼ 0.20 (DCM/MeOH 9:1). White
solid, yield 23%. 1H NMR (300 MHz, DMSO‑d6)
d 10.85 (s, 1H), 9.15
4.1.1.24. N-[4-[[[3-[3-[2-chloro-4-[(3-pyridinylcarbonyl)amino]
phenyl]-1,2,4-oxadiazol-5-yl]phenyl]amino]sulfonyl]phenyl]-4-
morpholinecarboxamide (24). Rf ¼ 0.35 (DCM/MeOH 9:1). White
(d, J ¼ 1.7 Hz, 1H), 9.00 (s, 1H), 8.80 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.56 (s,
1H), 8.47 (t, J ¼ 1.9 Hz, 1H), 8.23 (d, J ¼ 2.1 Hz, 1H), 8.33 (dt, J ¼ 8.1,
2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H), 7.96 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.77
(dd, J ¼ 7.6, 1.6 Hz, 1H), 7.71e7.50 (m, 3H), 7.39 (d, J ¼ 9.0 Hz, 2H),
6.89 (d, J ¼ 9.0 Hz, 2H), 3.73 (s, 3H). 13C NMR (75 MHz, DMSO- d6)
solid, yield 67%. 1H NMR (300 MHz, DMSO‑d6)
d 10.84 (s, 1H), 10.56
(s, 1H), 9.15 (s, 1H), 8.94 (s, 1H), 8.80 (d, J ¼ 5.0 Hz, 1H), 8.34 (d,
J ¼ 8.4 Hz, 1H), 8.26e8.18 (m, 1H), 8.05 (d, J ¼ 8.6 Hz, 1H), 8.01e7.89
(m, 2H), 7.82 (d, J ¼ 8.1 Hz, 1H), 7.75e7.48 (m, 6H), 7.48e7.41 (m,
1H), 3.64e3.52 (m, 4H), 3.47e3.37 (m, 4H).
d
174.78, 166.77, 164.62, 154.69, 152.65, 152.44, 148.76, 142.21,
141.00, 135.59, 132.37, 132.33, 132.16, 130.04, 129.99, 123.61, 123.51,
122.61, 121.32, 120.90, 120.37, 120.23, 118.65, 116.82, 113.96, 55.15.
Step g, h: To a solution of N-[4-[5-(3-aminophenyl)-1,2,4-
oxadiazol-3-yl]-3-chlorophenyl]-3-pyridinecarboxamide (VIc)
(0.2 g, 0.51 mmol, 1 equiv) in THF (20 ml) was added phosgene
(15 wt % in toluene, 1.7 ml, 2.55 mmol, 5.0 equiv) and the reac-
tion mixture was refluxed under N2 for 4 h. Upon the reaction
completion as indicated by TLC the solution was concentrated
under reduced pressure, flushed with dry toluene. To the
resulting residue was added THF (20 ml), the appropriate aniline
4.1.1.30. N-[3-chloro-4-[5-[3-[[(3-methoxyphenylamino)carbonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(30). Rf ¼ 0.30 (DCM/MeOH 9.5:0.5). White solid, yield 56%. 1H
NMR (300 MHz, DMSO‑d6)
d
10.85 (s, 1H), 9.15 (d, J ¼ 2.5 Hz, 1H),
9.06 (s, 1H), 8.85e8.74 (m, 2H), 8.47 (t, J ¼ 2.0 Hz, 1H), 8.34 (dt,
J ¼ 8.1, 2.0 Hz, 1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H),
7.97 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.80 (d, J ¼ 7.7 Hz, 1H), 7.72e7.52 (m,
3H), 7.26e7.14 (m, 2H), 6.97 (dd, J ¼ 7.7, 2.2 Hz, 1H), 6.58 (dd, J ¼ 8.1,
2.5 Hz, 1H), 3.75 (s, 3H).
(0.77 mmol, 1.5 equiv) and DIPEA (0.132 g, 178 ml, 1.02 mmol, 2
equiv) and the reaction mixture was refluxed under N2 over-
night. Upon completion of the reaction as indicated by TLC, the
solution was concentrated, and the residue was purified by
chromatography to give the desired final products (25e50).
4.1.1.31. N-[3-chloro-4-[5-[3-[[(4-methoxyphenylamino)carbonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(31). Rf ¼ 0.35 (DCM/MeOH 9.8:0.2). White solid, yield 80%. 1H
NMR (300 MHz, DMSO‑d6)
d 10.85 (s, 1H), 9.22 (s, 1H), 9.15 (s, 2H),
8.80 (d, J ¼ 4.8 Hz, 1H), 8.47 (s, 1H), 8.33 (d, J ¼ 8.2 Hz, 1H), 8.23 (d,
J ¼ 2.1 Hz, 1H), 8.13e7.92 (m, 3H), 7.82 (d, J ¼ 7.6 Hz, 1H), 7.71 (d,
J ¼ 8.7 Hz, 1H), 7.67e7.49 (m, 4H), 7.34 (d, J ¼ 7.7 Hz, 1H). 13C NMR
4.1.1.25. N-[3-chloro-4-[5-[3-[[(phenylamino)carbonyl]amino]
phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(25).
Rf ¼ 0.35 (DCM/MeOH 9.5:0.5). White solid, yield 26%. 1H NMR
(101 MHz, DMSO‑d6) d 174.73, 166.80,164.67, 152.49,148.78,142.25,
(300 MHz, DMSO‑d6)
d
10.85 (s, 1H), 9.15 (d, J ¼ 2.3 Hz, 1H), 9.08 (s,
140.50, 140.31, 135.62, 132.33, 132.21, 130.16, 130.01, 129.90, 129.68,
129.37, 129.06, 125.53, 123.67, 123.55, 123.06, 122.82, 122.11, 121.48,
121.32, 120.22, 118.70, 118.36, 117.19, 114.39. HRMS exact mass of
1H), 8.80 (dd, J ¼ 4.9, 1.6 Hz, 1H), 8.76 (s, 1H), 8.49 (s, 1H), 8.38e8.29
(m, 1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.09 (d, J ¼ 8.6 Hz, 1H), 7.97 (dd,
J ¼ 8.6, 2.1 Hz, 1H), 7.79 (d, J ¼ 7.6 Hz,1H), 7.71e7.53 (m, 3H), 7.49 (d,
J ¼ 8.1 Hz, 2H), 7.30 (t, J ¼ 7.8 Hz, 2H), 7.00 (t, J ¼ 7.4 Hz, 1H).
C
28H18ClF3N6O3 (M þ Na)þ: 601.0973 amu; found: 601.0989 amu.
4.1.1.32. N-[3-chloro-4-[5-[3-[[[4-chloro-3-(trifluoromethyl)phenyl-
amino]carbonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
pyridinecarboxamide (32). Rf ¼ 0.40 (DCM/MeOH 9.8:0.2). White
4.1.1.26. N-[3-chloro-4-[5-[3-[[(4-chlorophenylamino)carbonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(26). Rf ¼ 0.25 (DCM/MeOH 9.5:0.5). White solid, yield 63%. 1H
solid, yield 90%. 1H NMR (300 MHz, DMSO‑d6)
d 10.86 (s, 1H), 9.54
NMR (300 MHz, DMSO‑d6)
d
10.85 (s, 1H), 9.14 (s, 1H), 9.12 (s, 1H),
(s, 1H), 9.46 (s, 1H), 9.14 (dd, J ¼ 2.3, 0.9 Hz, 1H), 8.80 (dd, J ¼ 4.8,
1.7 Hz, 1H), 8.46 (t, J ¼ 1.9 Hz, 1H), 8.33 (dt, J ¼ 8.0, 2.0 Hz, 1H), 8.22
(d, J ¼ 2.0 Hz, 1H), 8.17 (s, 2H), 8.08 (d, J ¼ 8.6 Hz, 1H), 7.97 (dd,
8.91 (s, 1H), 8.80 (d, J ¼ 4.8 Hz, 1H), 8.47 (s, 1H), 8.33 (d, J ¼ 8.0 Hz,
1H), 8.22 (s, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H), 7.96 (d, J ¼ 8.7 Hz, 1H), 7.80

10
E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
J ¼ 8.6, 2.1 Hz, 1H), 7.83 (dt, J ¼ 7.7, 1.3 Hz, 1H), 7.74 (dd, J ¼ 2.3,
1.1 Hz, 1H), 7.67 (s, 1H), 7.65e7.55 (m, 2H).
(s, 1H), 8.80 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.48 (t, J ¼ 1.7 Hz, 1H), 8.34 (dt,
J ¼ 8.1, 2.0 Hz, 1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H),
7.96 (dd, J ¼ 8.6, 2.0 Hz, 1H), 7.80 (d, J ¼ 7.5 Hz, 1H), 7.71e7.52 (m,
3H), 7.41 (d, J ¼ 2.1 Hz, 1H), 7.31e7.18 (m, 2H), 6.90e6.80 (m, 1H),
3.00 (s, 3H).
4.1.1.33. N-[3-chloro-4-[5-[3-[[[3,5-bis(trifluoromethyl)phenyl-
amino]carbonyl]amino]phenyl]-1,2,4-oxadiazol-3-yl] phenyl]-3-
pyridinecarboxamide
(33). Rf ¼ 0.25
(EtOAc/Hexane/MeOH
4:4:0.25). White solid, yield 72%. 1H NMR (300 MHz, DMSO‑d6)
4.1.1.39. N-[3-chloro-4-[5-[3-[[[4-[(methylsulfonyl)amino]phenyl-
amino]carbonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
d
10.86 (s, 1H), 9.34 (s, 2H), 9.14 (d, J ¼ 1.7 Hz, 1H), 8.80 (dd, J ¼ 4.8,
1.6 Hz, 1H), 8.46 (t, J ¼ 1.9 Hz, 1H), 8.33 (dt, J ¼ 8.0, 1.9 Hz, 1H), 8.22
(d, J ¼ 2.0 Hz, 1H), 8.16e8.03 (m, 2H), 7.96 (dd, J ¼ 8.6, 2.1 Hz, 1H),
7.81 (d, J ¼ 7.7 Hz, 1H), 7.76e7.64 (m, 2H), 7.66e7.55 (m, 3H).
pyridinecarboxamide
(39). Rf ¼ 0.35
(EtOAc/Hexane/MeOH
4:2:0.25). White solid, yield 65%. 1H NMR (300 MHz, DMSO‑d6)
d
10.84 (s, 1H), 9.46 (s, 1H), 9.15 (d, J ¼ 2.0 Hz, 1H), 9.05 (s, 1H), 8.80
(dd, J ¼ 4.8, 1.6 Hz, 1H), 8.75 (s, 1H), 8.48 (t, J ¼ 1.9 Hz, 1H), 8.33 (dt,
J ¼ 8.0, 1.9 Hz, 1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H),
7.96 (dd, J ¼ 8.6, 2.0 Hz, 1H), 7.79 (d, J ¼ 7.6 Hz, 1H), 7.72e7.51 (m,
4.1.1.34. N-[3-chloro-4-[5-[3-[[[4-methoxy-3-(trifluoromethyl)phe-
nylamino]carbonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
pyridinecarboxamide
(34). Rf ¼ 0.20
(EtOAc/Hexane/MeOH
3H), 7.46 (d, J ¼ 8.8 Hz, 2H), 7.17 (d, J ¼ 8.7 Hz, 2H), 2.93 (s, 3H). 13
C
4:2:0.25). White solid, yield 68%. 1H NMR (300 MHz, DMSO‑d6)
NMR (75 MHz, DMSO‑d6)
d 174.83, 166.84, 164.76, 153.97, 152.55,
d
10.84 (s, 1H), 9.18e9.11 (m, 2H), 8.88 (s, 1H), 8.80 (dd, J ¼ 4.8,
148.86, 142.31, 140.88, 136.17, 135.71, 132.50, 132.38, 132.30, 130.22,
130.08, 123.68, 123.64, 122.79, 121.90, 121.35, 121.17, 120.25, 119.49,
1.7 Hz, 1H), 8.46 (t, J ¼ 2.0 Hz, 1H), 8.33 (dt, J ¼ 7.9, 2.0 Hz, 1H), 8.22
(d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H), 7.96 (dd, J ¼ 8.6, 2.1 Hz,
1H), 7.85 (d, J ¼ 2.7 Hz, 1H), 7.79 (dt, J ¼ 7.6, 1.3 Hz, 1H), 7.70 (dd,
J ¼ 8.7, 1.8 Hz, 1H), 7.66e7.54 (m, 3H), 7.23 (d, J ¼ 9.0 Hz, 1H), 3.86 (s,
118.73, 116.92, 38.84. HRMS exact mass of
C28H22ClN7O5S
(M þ Na)þ: 626.0984 amu; found: 626.0997 amu.
3H). 13C NMR (101 MHz, DMSO‑d6)
d
174.77, 166.79, 164.67, 152.71,
4.1.1.40. N-[3-chloro-4-[5-[3-[[[3-chloro-4-[(methylsulfonyl)amino]
phenylamino] carbonyl]amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-
152.50, 152.15, 148.79, 142.24, 140.76, 135.60, 132.33, 132.29, 132.21,
130.11, 130.01, 124.33, 123.63, 123.54, 122.93, 121.32, 121.22, 120.23,
118.70, 117.30, 117.24, 117.07, 116.88, 113.51, 56.24. HRMS exact mass
of C29H20ClF3N6O4 (M þ Na)þ: 631.1079 amu; found: 631.1089 amu.
3-pyridinecarboxamide
4:1:0.25). White solid, yield 28%. 1H NMR (300 MHz, DMSO‑d6)
10.84 (s, 1H), 9.32 (s, 1H), 9.21e9.11 (m, 2H), 9.02 (s, 1H), 8.80 (d,
(40). Rf ¼ 0.35
(EtOAc/Hexane/MeOH
d
J ¼ 3.9 Hz,1H), 8.47 (s, 1H), 8.33 (d, J ¼ 7.8 Hz,1H), 8.22 (d, J ¼ 1.9 Hz,
1H), 8.08 (d, J ¼ 8.6 Hz, 1H), 7.97 (d, J ¼ 8.2 Hz, 1H), 7.87e7.76 (m,
2H), 7.73e7.52 (m, 3H), 7.42e7.28 (m, 2H), 3.00 (s, 3H). 13C NMR
4.1.1.35. N-[3-chloro-4-[5-[3-[[(3-ethynylphenylamino)carbonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(35). Rf ¼ 0.30 (EtOAc/Hexane/MeOH 4:4:0.25). White solid, yield
(75 MHz, DMSO‑d6)
d 174.72, 166.79, 164.66, 152.47, 152.33, 148.77,
52%. 1H NMR (300 MHz, DMSO‑d6)
d
10.87 (s, 1H), 9.21e9.11 (m,
142.23, 140.49, 138.98, 135.58, 132.19, 132.13, 130.26, 130.14, 130.00,
129.36, 127.59, 123.66, 123.52, 122.99, 121.42, 121.32, 120.21, 118.92,
118.69, 117.71, 117.14, 40.69. HRMS exact mass of C28H21Cl2N7O5S
(M þ Na)þ: 660.0594 amu; found: 660.0608 amu.
2H), 8.91 (s, 1H), 8.80 (dd, J ¼ 4.9, 1.6 Hz, 1H), 8.48 (s, 1H), 8.33 (dt,
J ¼ 8.0, 2.0 Hz, 1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.09 (d, J ¼ 8.6 Hz, 1H),
7.96 (dd, J ¼ 8.7, 2.1 Hz, 1H), 7.80 (d, J ¼ 8.0 Hz, 1H), 7.74e7.52 (m,
4H), 7.50e7.40 (m, 1H), 7.31 (t, J ¼ 7.9 Hz, 1H), 7.11 (dt, J ¼ 7.6, 1.3 Hz,
1H), 4.19 (s, 1H). 13C NMR (75 MHz, DMSO‑d6)
d
174.74, 166.79,
4.1.1.41. N-[4-[5-[3-[[[3-bromo-4-[(methylsulfonyl)amino]phenyl-
amino]carbonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]-3-
chlorophenyl]-3-pyridinecarboxamide (41). Rf ¼ 0.35 (EtOAc/Hex-
ane/MeOH 4:1:0.25). White solid, yield 31%. 1H NMR (300 MHz,
164.66,152.48,152.43,148.77,142.23,140.63,139.64,135.58,132.32,
132.20, 130.13, 130.00, 129.18, 125.39, 123.65, 123.53, 122.89, 122.07,
121.33, 121.30, 121.23, 120.22, 119.17, 118.70, 117.06, 83.49, 80.40.
DMSO‑d6)
d 10.88 (s, 1H), 9.32 (s, 1H), 9.24 (s, 1H), 9.18e9.04 (m,
4.1.1.36. N-[3-chloro-4-[5-[3-[[(4-ethynylphenylamino)carbonyl]
amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-pyridinecarboxamide
(36). Rf ¼ 0.25 (EtOAc/Hexane/MeOH 4:4:0.25). White solid, yield
2H), 8.80 (d, J ¼ 4.7 Hz, 1H), 8.48 (s, 1H), 8.33 (d, J ¼ 7.9 Hz, 1H), 8.23
(s, 1H), 8.09 (d, J ¼ 8.5 Hz, 1H), 8.03e7.92 (m, 2H), 7.80 (d, J ¼ 7.5 Hz,
1H), 7.73e7.52 (m, 3H), 7.43e7.30 (m, 2H), 3.01 (s, 3H).
44%. 1H NMR (300 MHz, DMSO‑d6)
d 10.87 (s, 1H), 9.21e9.11 (m,
2H), 9.02 (s, 1H), 8.80 (dd, J ¼ 4.8, 1.7 Hz, 1H), 8.48 (t, J ¼ 1.9 Hz, 1H),
8.33 (dt, J ¼ 8.0, 2.0 Hz, 1H), 8.23 (d, J ¼ 2.1 Hz, 1H), 8.09 (d,
J ¼ 8.6 Hz, 1H), 7.96 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.80 (d, J ¼ 7.6 Hz, 1H),
7.70e7.54 (m, 3H), 7.51 (d, J ¼ 8.7 Hz, 2H), 7.41 (d, J ¼ 8.7 Hz, 2H),
4.07 (s, 1H).
4.1.1.42. N-[3-chloro-4-[5-[3-[[[4-[(methylsulfonyl)amino]-3-(tri-
fluoromethyl)phenyl-amino]carbonyl]amino]phenyl]-1,2,4-oxadiazol-
3-yl]phenyl]-3-pyridinecarboxamide (42). Rf ¼ 0.35 (EtOAc/Hexane/
MeOH 4:2:0.25). White solid, yield 18%. 1H NMR (300 MHz,
DMSO‑d6)
d
10.84 (s, 1H), 9.39e8.99 (m, 4H), 8.80 (dt, J ¼ 4.9, 1.7 Hz,
1H), 8.47 (t, J ¼ 1.9 Hz,1H), 8.34 (ddd, J ¼ 8.0, 2.3,1.7 Hz,1H), 8.23 (d,
J ¼ 2.1 Hz, 1H), 8.12e7.89 (m, 4H), 7.82 (dt, J ¼ 7.6, 1.4 Hz, 1H),
7.75e7.47 (m, 4H), 3.06 (s, 3H).
4.1.1.37. N-[4-[5-[3-[[[4-[(aminosulfonyl)amino]phenylamino]
carbonyl]amino]phenyl]-1,2,4-oxadiazol-3-yl]-3-chlorophenyl]-3-
pyridinecarboxamide
(37). Rf ¼ 0.30
(EtOAc/Hexane/MeOH
4:1:0.25). White solid, yield 21%. 1H NMR (300 MHz, DMSO‑d6)
4.1.1.43. N-[3-chloro-4-[5-[3-[[[4-[(cyclopropylsulfonyl)amino]phe-
nylamino]carbonyl] amino]phenyl]-1,2,4-oxadiazol-3-yl]phenyl]-3-
d
10.85 (s, 1H), 9.23e9.11 (m, 2H), 9.02 (s, 1H), 8.80 (dd, J ¼ 4.8,
1.7 Hz, 1H), 8.64 (s, 1H), 8.52e8.44 (m, 1H), 8.34 (dt, J ¼ 8.0, 2.0 Hz,
1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H), 7.96 (dd, J ¼ 8.7,
2.1 Hz, 1H), 7.78 (dt, J ¼ 7.5, 1.4 Hz, 1H), 7.71e7.51 (m, 3H), 7.39 (d,
J ¼ 8.8 Hz, 2H), 7.12 (d, J ¼ 8.8 Hz, 2H), 6.96 (s, 2H).
pyridinecarboxamide
(43). Rf ¼ 0.20
(EtOAc/Hexane/MeOH
4:1:0.25). White solid, yield 46%. 1H NMR (300 MHz, DMSO‑d6)
d
10.87 (s, 1H), 9.50 (s, 1H), 9.11 (d, J ¼ 18.6 Hz, 2H), 8.84e8.74 (m,
2H), 8.48 (s, 1H), 8.33 (d, J ¼ 7.5 Hz, 1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.08
(d, J ¼ 8.6 Hz, 1H), 7.96 (dd, J ¼ 8.7, 2.0 Hz, 1H), 7.78 (d, J ¼ 7.6 Hz,
1H), 7.70e7.50 (m, 3H), 7.45 (d, J ¼ 8.8 Hz, 2H), 7.19 (d, J ¼ 8.6 Hz,
2H), 2.61e2.48 (m, 1H), 0.95e0.86 (m, 4H). 13C NMR (75 MHz,
4.1.1.38. N-[3-chloro-4-[5-[3-[[[3-[(methylsulfonyl)amino]phenyl-
amino]carbonyl]amino]phenyl]-1,2,4-oxadiazol-3-yl] phenyl]-3-
pyridinecarboxamide
4:2:0.25). White solid, yield 59%. 1H NMR (300 MHz, DMSO‑d6)
10.85 (s, 1H), 9.72 (s, 1H), 9.15 (d, J ¼ 1.8 Hz, 1H), 9.03 (s, 1H), 8.88
(38). Rf ¼ 0.40
(EtOAc/Hexane/MeOH
DMSO‑d6) d 174.78, 166.78, 164.66, 152.48, 152.40, 148.77, 142.23,
140.82, 136.07, 135.58, 132.39, 132.32, 132.18, 130.11, 130.00, 123.63,
123.52, 122.72, 122.35, 121.32, 121.10, 120.23, 119.29, 118.68, 116.91,
d

E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
11
29.16, 4.82. HRMS exact mass of C30H24ClN7O5S (M þ Na)þ:
4.1.1.49. N-[3-chloro-4-[5-[3-[[[4-[(2-thienylcarbonyl)amino]-3-(tri-
fluoromethyl) phenylamino]carbonyl]amino]phenyl]-1,2,4-oxadiazol-
3-yl]phenyl]-3-pyridine-carboxamide (49). Rf ¼ 0.30 (EtOAc/Hex-
ane/MeOH 4:1:0.25). Light-pink solid, yield 44%. 1H NMR
652.1140 amu; found: 652.1158 amu.
4.1.1.44. N-[4-[5-[3-[[[4-(acetylamino)phenylamino]carbonyl]amino]
phenyl]-1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-pyridinecarboxamide
(44). Rf ¼ 0.35 (EtOAc/MeOH 4:0.25). Off-white solid, yield 24%. 1H
(300 MHz, DMSO‑d6)
d 10.87 (s, 1H), 10.07 (s, 1H), 9.26 (d,
J ¼ 12.8 Hz, 2H), 9.15 (d, J ¼ 2.3 Hz, 1H), 8.80 (d, J ¼ 2.9 Hz, 1H), 8.49
(s, 1H), 8.33 (d, J ¼ 8.1 Hz, 1H), 8.23 (d, J ¼ 2.2 Hz, 1H), 8.14e8.04 (m,
2H), 8.02e7.91 (m, 2H), 7.90e7.77 (m, 2H), 7.77e7.53 (m, 4H), 7.44
(d, J ¼ 8.6 Hz, 1H), 7.23 (t, J ¼ 4.2 Hz, 1H). 13C NMR (75 MHz,
NMR (300 MHz, DMSO‑d6)
d 10.88 (s, 1H), 9.86 (s, 1H), 9.15 (d,
J ¼ 2.3 Hz, 1H), 9.05 (s, 1H), 8.80 (dd, J ¼ 4.9, 1.7 Hz, 1H), 8.69 (s, 1H),
8.49 (t, J ¼ 1.9 Hz, 1H), 8.33 (dt, J ¼ 8.0, 2.0 Hz, 1H), 8.23 (d,
J ¼ 2.0 Hz, 1H), 8.09 (d, J ¼ 8.6 Hz, 1H), 7.96 (dd, J ¼ 8.7, 2.1 Hz, 1H),
7.78 (dt, J ¼ 7.4, 1.5 Hz, 1H), 7.68e7.53 (m, 3H), 7.50 (d, J ¼ 9.1 Hz,
2H), 7.40 (d, J ¼ 9.0 Hz, 2H), 2.02 (s, 3H).
DMSO‑d6)
d 175.22, 167.30, 165.15, 161.52, 153.01, 149.27, 142.73,
140.98, 139.59, 139.26, 136.08, 132.83, 132.69, 132.53, 132.14, 130.65,
130.49, 129.76, 129.12, 128.54, 127.55, 127.16, 125.72, 124.16, 124.02,
123.61, 122.92, 122.09, 122.01, 121.82, 120.71, 119.19, 117.76.
4.1.1.45. N-[4-[5-[3-[[[4-(acetylamino)-3-chlorophenylamino]
carbonyl]amino]phenyl]-1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-
4.1.1.50. N-[4-[5-[3-[[[4-[(aminocarbonyl)amino]-3-(tri-
fluoromethyl)phenylamino] carbonyl]amino]phenyl]-1,2,4-oxadiazol-
pyridinecarboxamide
4:1:0.25). White solid, yield 44%. 1H NMR (300 MHz, DMSO‑d6)
10.88 (s, 1H), 9.47 (s, 1H), 9.21e9.11 (m, 2H), 8.97 (s, 1H), 8.80 (dd,
(45). Rf ¼ 0.25
(EtOAc/Hexane/MeOH
3-yl]3-chlorophenyl]-3-pyridine-carboxamide
(EtOAc/MeOH 4:0.25). White solid, yield 26%. 1H NMR (300 MHz,
DMSO‑d6) 10.86 (s, 1H), 9.23e9.07 (m, 2H), 9.01 (s, 1H), 8.79 (dd,
(50). Rf ¼ 0.30
d
d
J ¼ 4.8, 1.7 Hz, 1H), 8.48 (t, J ¼ 2.0 Hz, 1H), 8.34 (dt, J ¼ 8.0, 2.0 Hz,
1H), 8.23 (d, J ¼ 2.0 Hz, 1H), 8.09 (d, J ¼ 8.6 Hz, 1H), 7.97 (dd, J ¼ 8.7,
2.1 Hz, 1H), 7.85e7.76 (m, 2H), 7.72e7.48 (m, 4H), 7.28 (dd, J ¼ 8.7,
2.3 Hz, 1H), 2.06 (s, 3H).
J ¼ 4.8, 1.7 Hz, 1H), 8.46 (t, J ¼ 2.0 Hz, 1H), 8.33 (dt, J ¼ 8.0, 1.9 Hz,
1H), 8.22 (d, J ¼ 2.0 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H), 8.01e7.91 (m,
2H), 7.84e7.47 (m, 7H), 6.24 (s, 2H). 13C NMR (75 MHz, DMSO‑d6)
d
174.80, 166.84, 164.75, 156.19, 152.62, 152.58, 148.86, 142.31,
140.70, 135.70, 135.12, 132.38, 132.31, 131.31, 130.22, 130.07, 127.30,
125.73, 123.69, 123.63, 123.03, 122.69, 122.11, 121.35, 120.74, 120.35,
120.25, 118.74, 117.12.
4.1.1.46. N-[4-[5-[3-[[[4-(acetylamino)-3-bromophenylamino]
carbonyl]amino]phenyl]-1,2,4-oxadiazol-3-yl]3-chlorophenyl]-3-
pyridinecarboxamide
4:1:0.25). White solid, yield 62%. 1H NMR (300 MHz, DMSO‑d6)
10.88 (s, 1H), 9.43 (s, 1H), 9.22e9.11 (m, 2H), 8.97 (s, 1H), 8.80 (dd,
(46). Rf ¼ 0.25
(EtOAc/Hexane/MeOH
4.2. Biological activity
d
J ¼ 4.7, 1.1 Hz, 1H), 8.48 (s, 1H), 8.33 (dt, J ¼ 7.9, 1.9 Hz, 1H), 8.23 (d,
J ¼ 2.0 Hz, 1H), 8.09 (d, J ¼ 8.6 Hz, 1H), 8.02e7.92 (m, 2H), 7.80 (d,
J ¼ 7.5 Hz, 1H), 7.72e7.52 (m, 3H), 7.43 (d, J ¼ 8.7 Hz, 1H), 7.33 (d,
J ¼ 9.1 Hz, 1H), 2.05 (s, 3H).
4.2.1. Cell lines, cell culture, reagents, and antibodies
The MDA-MB-231 breast cancer, A549 (wild-type) and H1975
(L858R/T790M) lung cancer cell lines were purchased from the
American Type Culture Collection (ATCC) (Manassas, VA). PC9 (exon
19 deletion, Del19) CL68 (Del19/T790M), and CL97 (G719A/T790M)
cell lines were kindly provided by Professor Pan-Chyr Yang (Insti-
tute of Biomedical Science, Academia Sinica, Taiwan) [35], among
which CL68 and CL97 cell lines were established from patients who
provided informed consent and with the approval of the institu-
tional review board (National Taiwan University Hospital Research
Ethics Committee). MDA-MB-231 and A549 cells were cultured in
DMEM medium (Life Technologies; Grand Island, NY), and H1975,
PC9, CL68, and CL97 cells were maintained in RPMI 1640 medium
(Life Technologies), both of which were supplemented with 10%
4.1.1.47. N-[3-chloro-4-[5-[3-[[[4-[(cyclopentylcarbonyl)amino]-3-
(trifluoromethyl) phenylamino]carbonyl]amino]phenyl]-1,2,4-
oxadiazol-3-yl]phenyl]-3-pyridine-carboxamide
(EtOAc/Hexane/MeOH 4:1:0.25). Light-pink solid, yield 49%. 1H
NMR (300 MHz, DMSO‑d6) 10.86 (s,1H), 9.39 (s,1H), 9.26e9.11 (m,
(47). Rf ¼ 0.30
d
3H), 8.80 (d, J ¼ 3.7 Hz, 1H), 8.47 (d, J ¼ 2.5 Hz, 1H), 8.33 (d,
J ¼ 7.3 Hz, 1H), 8.22 (d, J ¼ 2.2 Hz, 1H), 8.08 (d, J ¼ 8.6 Hz, 1H),
8.03e7.91 (m, 2H), 7.80 (d, J ¼ 7.6 Hz, 1H), 7.70 (d, J ¼ 8.2 Hz, 1H),
7.66e7.52 (m, 3H), 7.33 (d, J ¼ 8.7 Hz, 1H), 2.83 (p, J ¼ 7.9 Hz, 1H),
1.93e1.47 (m, 8H). 13C NMR (75 MHz, DMSO‑d6)
d 175.72, 175.20,
fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml
streptomycin (Life Technologies). Cells were incubated at 37 ꢀC in a
humidified incubator containing 5% CO2. Chemical agents and an-
tibodies used in this study were purchased from the following
sources. Chemical agents: gefitinib, erlotinib, afatinib and omi-
sertinib were purchased from Cayman Chemical (Ann Arbor, MI);
protease inhibitor, phosphatase inhibitor, MTT [3-(4,5-
167.28, 165.13, 152.98, 152.96, 149.27, 142.71, 141.00, 138.42, 136.06,
132.82, 132.66, 131.81, 130.60, 130.48, 129.91, 129.88, 125.75, 124.15,
124.00, 123.54, 122.80, 122.13, 121.93, 121.81, 120.71, 119.16, 117.70,
44.71, 30.25, 26.08. HRMS exact mass of C34H27ClF3N7O4 (M þ Na)þ:
712.1657 amu; found: 712.1669 amu.
4.1.1.48. N-[3-chloro-4-[5-[3-[[[4-[(cyclopropylcarbonyl)amino]-3-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide]
(trifluoromethyl) phenylamino]carbonyl]amino]phenyl]-1,2,4-
(Sigma-Aldrich; St. Louis, MO); BCA Protein Assay kit (Thermo
Fisher Scientific; Waltham, MA); Western Lighting Chem-
iluminescence Plus-ECL (PerkinElmer; Waltham, MA). Antibodies:
EGFR, GAPDH, HRP-conjugated anti-mouse, goat and rabbit sec-
ondary antibodies (Santa Cruz Biotechnology; Santa Cruz, CA); c-
Met, S473-Akt, Akt (Cell Signaling; Beverly, MA); Alexa Fluor 555-
and 488-conjugated goat anti-rabbit and anti-mouse IgG (Life
Technologies).
oxadiazol-3-yl]phenyl]-3-pyridine-carboxamide
(EtOAc/MeOH 4:0.25). Off-white solid, yield 65%. 1H NMR
(300 MHz, DMSO‑d6) 10.86 (s, 1H), 9.68 (s, 1H), 9.23 (s, 1H),
(48). Rf ¼ 0.30
d
9.19e9.09 (m, 2H), 8.79 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.47 (t, J ¼ 1.8 Hz,
1H), 8.32 (dt, J ¼ 7.9, 1.8 Hz, 1H), 8.22 (d, J ¼ 2.0 Hz, 1H), 8.08 (d,
J ¼ 8.6 Hz, 1H), 8.02e7.92 (m, 2H), 7.80 (dt, J ¼ 7.6, 1.0 Hz, 1H), 7.69
(ddd, J ¼ 8.3, 2.0, 0.9 Hz, 1H), 7.64e7.52 (m, 3H), 7.35 (d, J ¼ 8.9 Hz,
1H), 1.93e1.81 (m, 1H), 0.76 (d, J ¼ 4.4 Hz, 4H). 13C NMR (75 MHz,
DMSO‑d6)
d
174.79, 172.87, 166.85, 164.76, 152.57, 148.85, 142.31,
4.2.2. MTT cell viability assays
140.58, 137.94, 135.70, 132.38, 132.32, 131.29, 130.25, 130.07, 129.31,
125.32, 123.69, 123.64, 123.14, 122.40, 121.70, 121.53, 121.35, 120.25,
118.75, 117.24, 115.57, 115.49, 13.71, 7.04. HRMS exact mass of
To determine the drug effects on cell viability, cells were seeded
onto 96-well plates at a density of 5000 cells per well in the pres-
ence of 10% FBS. After overnight incubation, cells were exposed to
test agents or DMSO in the presence of 5% FBS for 48 h. After
C
32H23ClF3N7O4 (M þ Na)þ: 684.1344 amu; found: 684.1355 amu.

12
E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
treatment, cells were incubated with MTT for an additional 1 h. The
medium was then removed from each well and replaced with
DMSO to dissolve the reduced MTT dye for subsequent colorimetric
measurement of absorbance at 570 nm. Cell viabilities are
expressed as percentages of viable cells relative to the corre-
sponding vehicle-treated control group.
sterile water), or 48 at 50 mg/kg and 150 mg/kg. For the in vivo ef-
ficacy of combining 48 with gefitinib, mice were divided into the
following four groups, and treated once daily for 20 days by oral
gavage as follows. (A) Control group, the aforementioned vehicle,
(B) gefitinib at 50 mg/kg, (C) 48 at 50 mg/kg, and (D) 48 plus gefi-
tinib, 50 mg/kg each. Tumors were measured with calipers and the
volumes were calculated using V ¼ (width2 x length) x 0.52. Body
weight and tumor volumes were monitored twice a week. At the
end of the study, mice were sacrificed by CO2 asphyxiation and
tumor samples were analyzed by Western blot.
4.2.3. Cell lysis and immunoblotting
Drug-treated cells were collected by scrapping and then lysed in
SDS lysis buffer (1% SDS, 50 mM Tris-HCl pH8.0, 10 mM EDTA)
containing a commercial cocktail of protease inhibitors and phos-
phatase inhibitors. Lysates were sonicated and then centrifuged at
13,000 rpm for 10 min. Protein concentrations in the supernatants
were determined by the BCA Protein Assay kit, and equal amounts
of proteins were resolved via SDS-PAGE and transferred to a
nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh,
PA). The membrane was washed twice with Tris-buffered saline
containing 0.1% Tween-20 (TBST), blocked with TBST containing 5%
non-fat milk for 30 min, and then incubated with primary antibody
(1:1000 dilution) in TBST at 4 ꢀC overnight. After washing with
TBST, the membrane was incubated with goat anti-rabbit or anti-
mouse IgGeHRP conjugates (1:5000 dilution) for 1 h at room
temperature. The immunoblots were visualized by the Western
Lighting Chemiluminescence Reagent Plus-ECL.
4.2.7. Statistical analysis
In vitro experiments were performed at least three times and
data are presented as means SD. Group means were compared
using one-way ANOVA followed by Student's t tests. For the in vivo
experiments, differences in tumor volume were analyzed by Stu-
dent's t-test. Differences were considered significant at P < 0.05.
Author contributions
C.S.C. and K.A.M. conceived the study, and designed experi-
ments; E.M.E.D was responsible for the synthesis of all compounds;
C.S.F. performed biochemical experiments; C.S.C, C.S.F and E.M.E.D
wrote the manuscript and all authors revised it.
4.2.4. RNA extraction and RT-PCR analysis
Conflicts of interest
Total RNA was isolated from cells using TRIzol reagent (Thermo
Fisher Scientific) according to the manufacturer's protocol. One mg
The authors declare no competing financial interest.
Acknowledgment
RNA from each sample was reverse-transcribed into cDNA using the
iScript™ cDNA Synthesis Kit (Bio-Rad; Hercules, CA), and the cDNA
were separated by electrophoresis. PCR products were resolved by
electrophoresis in 2% agarose gels and visualized by ethidium
bromide staining. The sequences of primers used for RT-PCR were
as follow: EGFR, forward primer: 50- GGC ACT TTT GAA GAT CAT TTT
CTC-30; reverse primer: 50- CTG TGT TGA GGG CAA TGA G -30 (144
bp). c-Met, forward primer: 50- CAT GCC GAC AAG TGC AGT A -30;
reverse primer: 50- TCT TGC CAT CAT TGT CCA AC-30 (252 bp).
GAPDH, forward primer: 50- AAG CCC ATC ACC ATC TTC CAG-30;
reverse primer: 50- AGG GGC CAT CCA CAG TCT TCT -30 (361 bp).
PC9, CL68 and CL97 lung cancer cells were kindly provided by
Dr. Pan-Chyr Yang (Institute of Biomedical Science, Academia Sin-
ica, Taiwan). The authors are grateful to Fatma Almenier for her
kind help. This work was supported, in part, by the intramural
grants CMU-1065918B, 1075955A, and 1085955A from China
Medical University (C.S.C).
Appendix A. Supplementary data
4.2.5. Flow cytometry
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111607.
Cells were placed into 6-cm culture dishes at 3 ꢂ 105/well in
respective media overnight. After attachment, cells were treated
with compound 48 at indicated concentrations for 24 h and 48 h,
and the pellets were collected and fixed in 70% ethanol. Treated cell
References
[1] A. Sudhakar, History of cancer, ancient and modern treatment methods,
J. Cancer Sci. Ther. 01 (2009) ieiv.
[2] C.M. Croce, Oncogenes and cancer, N. Engl. J. Med. 358 (2008) 502e511.
[3] D. Ray, K.C. Cuneo, A. Rehemtulla, T.S. Lawrence, M.K. Nyati, Inducing onco-
protein degradation to improve targeted cancer therapy, Neoplasia 17 (2015)
697e703.
[4] H. Lei, W. Wang, Y. Wu, Targeting oncoproteins for degradation by small
molecules in myeloid leukemia, Leuk. Lymphoma 59 (2018) 2297e2304.
[5] K. Raina, C.M. Crews, Chemical inducers of targeted protein degradation,
J. Biol. Chem. 285 (2010) 11057e11060.
[6] D.P. Bondeson, C.M. Crews, Targeted protein degradation by small molecules,
Annu. Rev. Pharmacol. Toxicol. 57 (2017) 107e123.
[7] M.A. Olayioye, R.M. Neve, H.A. Lane, N.E. Hynes, NEW EMBO MEMBERS’ RE-
VIEW: the ErbB signaling network: receptor heterodimerization in develop-
ment and cancer, EMBO J. 19 (2000) 3159e3167.
[8] N. Normanno, C. Bianco, A. De Luca, M.R. Maiello, D.S. Salomon, Target-based
agents against ErbB receptors and their ligands: a novel approach to cancer
treatment, Endocr. Relat. Cancer 10 (2003) 1e21.
[9] J.A. Engelman, et al., MET amplification leads to gefitinib resistance in lung
cancer by activating ERBB3 signaling, Science 316 (2007) 1039e1043.
[10] R. Barouch-Bentov, K. Sauer, Mechanisms of drug resistance in kinases, Expert
Opin. Investig. Drugs 20 (2011) 153e208.
[11] A. Ahsan, Mechanisms of resistance to egfr tyrosine kinase inhibitors and
therapeutic approaches: an update, in: A. Ahmad, S. Gadgeel (Eds.), Lung
Cancer and Personalized Medicine. Advances in Experimental Medicine and
Biology, vol. 893, Springer, Cham, 2016.
pellets were stained with 40
mg/ml of propidium iodide (PI) (Sigma-
Aldrich) containing 20 g/ml of RNase A (Sigma-Aldrich) on ice in
m
the dark for 30 min before analysis. Cells were sorted and analyzed
using a FACSCalibur flow cytometry (BD Biosciences; Franklin
Lakes, NJ).
4.2.6. In vivo nude mice xenograft tumor model
Female Balb/c nude mice (Athymic Nude-Foxn1nu; 5 weeks of
age; National Animal Research Laboratory, Taipei, Taiwan) were
group-housed under constant photoperiod (12-h light/12-h dark)
with ad libitum access to sterilized food and water. All experimental
procedures were done according to protocols approved by the
Academia Sinica Animal Care Ethics Commission. To assess the ef-
fect of 48 on TKI resistant tumor growth, in vivo, H1975 cells were
harvested and subcutaneously injected in the right flank of mice
(106 cells/0.1 ml per mouse). When tumor volumes reached
approximately 50 mm3, mice were randomized to different groups
(n ¼ 8/each group). For the single agent in vivo efficacy study, mice
were treated once daily for 20 days by oral gavage with vehicle
solution (10% DMSO/0.1% Tween 80/0.5% methylcellulose [v/v] in

E.M.E. Dokla et al. / European Journal of Medicinal Chemistry 182 (2019) 111607
13
[12] P. Maione, et al., Irreversible EGFR inhibitors in the treatment of advanced
NSCLC, Curr. Pharmaceut. Des. 20 (2014) 3894e3900.
[13] A. Murtuza, et al., Novel third-generation EGFR tyrosine kinase inhibitors and
strategies to overcome therapeutic resistance in lung cancer, Cancer Res. 79
(2019) 689e698.
kinase inhibitor, J. Med. Chem. 54 (2011) 6364e6374.
[25] K.-Y. Huang, et al., Small molecule T315 promotes casitas B-lineage
lymphomaedependent degradation of epidermal growth factor receptor via
Y1045 autophosphorylation, Am. J. Respir. Crit. Care Med. 193 (2016)
753e766.
[14] H. Hasegawa, et al., Efficacy of afatinib or osimertinib plus cetuximab com-
bination therapy for non-small-cell lung cancer with EGFR exon 20 insertion
mutations, Lung Cancer 127 (2019) 146e152.
[15] K. Uchibori, et al., Brigatinib combined with anti-EGFR antibody overcomes
osimertinib resistance in EGFR-mutated non-small-cell lung cancer, Nat.
Commun. 8 (2017) 14768.
[16] Q. Wang, S. Yang, K. Wang, S.-Y. Sun, MET inhibitors for targeted therapy of
EGFR TKI-resistant lung cancer, J. Hematol. Oncol. 12 (2019) 63.
[17] Z. Tang, et al., Dual MET-EGFR combinatorial inhibition against T790M-EGFR-
mediated erlotinib-resistant lung cancer, Br. J. Canc. 99 (2008) 911e922.
[18] A. Kirisits, D. Pils, M. Krainer, Epidermal growth factor receptor degradation:
an alternative view of oncogenic pathways*, Int. J. Biochem. Cell Biol. 39
(2007) 2173e2182.
[19] C.-H. Jeong, H.B. Park, W.J. Jang, S.H. Jung, Y.H. Seo, Discovery of hybrid Hsp90
inhibitors and their anti-neoplastic effects against gefitinib-resistant non-
small cell lung cancer (NSCLC), Bioorg. Med. Chem. Lett. 24 (2014) 224e227.
[20] Y. Wang, J.B. Trepel, L.M. Neckers, G. Giaccone, STA-9090, a small-molecule
Hsp90 inhibitor for the potential treatment of cancer, Curr. Opin. Investig.
Drugs 11 (2010) 1466e1476.
[26] J. Hughes, S. Rees, S. Kalindjian, K. Philpott, Principles of early drug discovery,
Br. J. Pharmacol. 162 (2011) 1239e1249.
[27] T.J. Lynch, et al., Activating mutations in the epidermal growth factor receptor
underlying responsiveness of nonesmall-cell lung cancer to gefitinib, N. Engl.
J. Med. 350 (2004) 2129e2139.
[28] S. Kobayashi, et al., EGFR mutation and resistance of nonesmall-cell lung
cancer to gefitinib, N. Engl. J. Med. 352 (2005) 786e792.
[29] M. Roengvoraphoj, G.J. Tsongalis, K.H. Dragnev, J.R. Rigas, Epidermal growth
factor receptor tyrosine kinase inhibitors as initial therapy for non-small cell
lung cancer: focus on epidermal growth factor receptor mutation testing and
mutation-positive patients, Cancer Treat. Rev. 39 (2013) 839e850.
[30] C.-S. Kang, et al., Suppression of EGFR expression by antisense or small
interference RNA inhibits U251 glioma cell growth in vitro and in vivo, Cancer
Gene Ther. 13 (2006) 530e538.
[31] W. Zhou, et al., Novel mutant-selective EGFR kinase inhibitors against EGFR
T790M, Nature 462 (2009) 1070e1074.
[32] C.-H. Yun, et al., The T790M mutation in EGFR kinase causes drug resistance
by increasing the affinity for ATP, Proc. Natl. Acad. Sci. U. S. A 105 (2008)
2070e2075.
[21] D.-W. Wu, T.-C. Chen, H.-S. Huang, H. Lee, TC-N19, a novel dual inhibitor of
EGFR and cMET, efficiently overcomes EGFR-TKI resistance in non-small-cell
lung cancer cells, Cell Death Dis. 7 (2016) e2290ee2290.
[33] W. Pao, et al., Acquired resistance of lung adenocarcinomas to gefitinib or
erlotinib is associated with a second mutation in the EGFR kinase domain,
PLoS Med. 2 (2005) e73.
[22] H.S. Huang, H. Li, Composition containing thiochrome [2,3-c] quinolin-12-one
derivative for treating non-small cell lung cancer, Jpn. Tokyo Koho 17 (2018).
[23] Y. Oh, S. Park, Y. Seo, The targeted inhibition of Hsp90 by a synthetic small
molecule, DPide offers an effective treatment strategy against TNBCs, Oncol.
Rep. 39 (2007) 2173e2182, https://doi.org/10.3892/or.2018.6250.
[24] S.-L. Lee, et al., Identification and characterization of a novel integrin-linked
[34] K.S. Thress, et al., Acquired EGFR C797S mutation mediates resistance to
AZD9291 in nonesmall cell lung cancer harboring EGFR T790M, Nat. Med. 21
(2015) 560e562.
[35] H.-Y. Yen, et al., Effect of sialylation on EGFR phosphorylation and resistance
to tyrosine kinase inhibition, Proc. Natl. Acad. Sci. U. S.
A 112 (2015)
6955e6960.