Synthesis, anti-lung cancer activity and molecular docking study of 3-methylene-2-oxoindoline-5-carboxamide derivatives

Article abstract of DOI:10.1007/s00044-017-2050-3

A series of 3-methylene-2-oxoindoline-5-carboxamide derivatives were synthesized in appreciable yield by using 4-aminobenzoic acid as a starting material. The preliminary biological test results showed that most compounds displayed potent inhibitory activity against proliferation of human lung adenocarcinoma epithelial cell line (A549) in MTT assay. Compound 6l displayed the highest potency (IC₅₀ = 3.0 μM). The western blot analysis demonstrated a correlation between anti-proliferative activity of active compounds and blockade of the phosphorylation of extracellular signal-regulated kinases (ERK1/2). The docking study also provides new insights into further optimization of 3-methylene-2-oxoindoline-5-carboxamide derivatives for the discovery of more potent RAF/MEK/ERK pathway regulators as anti-lung cancer agents.

Full text of DOI:10.1007/s00044-017-2050-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2050-3
ORIGINAL RESEARCH
Synthesis, anti-lung cancer activity and molecular docking study of
3-methylene-2-oxoindoline-5-carboxamide derivatives
Juntao Ai1 Meng Lv1 Xiaohui Li2 Zhuo Chen1 Gaoyun Hu1 Qianbin Li1
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Received: 8 May 2017 / Accepted: 24 August 2017
© Springer Science+Business Media, LLC 2017
Abstract A series of 3-methylene-2-oxoindoline-5-car-
boxamide derivatives were synthesized in appreciable yield
by using 4-aminobenzoic acid as a starting material. The
preliminary biological test results showed that most com-
pounds displayed potent inhibitory activity against pro-
liferation of human lung adenocarcinoma epithelial cell line
(A549) in MTT assay. Compound 6l displayed the highest
potency (IC50 = 3.0 μM). The western blot analysis
Introduction
Deregulation of signaling pathways have been implicated to
promote carcinogenesis. The Ras signaling pathway occurs
downstream of most growth factor’s binding to their
receptors and is an essential signal transduction cascade that
controls cell survival, growth, differentiation and transfor-
mation (Downward 2003; Schubbert et al. 2007). The RAF/
MEK/ERK pathway represents one of the best-
characterized Ras pathways, which promotes cell cycle
progression and cell proliferation (Abdel-Rahman 2016;
Martinelli et al. 2017). Substantial evidence has supported
that deregulation of this pathway with downstream effectors
ERK1/2 constitutively active is involved in the oncogenesis
of various human cancers. About 30% of lung adenocarci-
nomas contain KRAS mutations (Pao et al. 2005), and
BRAF mutations exist in about 66% of malignant mela-
nomas (Davies et al. 2002). This pathway is also activated
to various degrees in many other malignant tumor, such as
thyroid cancer (Espinosa et al. 2007), colorectal cancer
(Douillard et al. 2013), which lead to excessive activation of
MEK1/2 and then ERK1/2. Therefore, RAF/MEK/ERK
signaling cascade has being treated as an important target
for anti-cancer drug discovery (Roberts and Der 2007).
3-(1H-pyrrol-2-yl) methylene-indolin-2-one analogs
have been widely applied in the development of anti-cancer
agents (Fig. 1). SU5416, SU5402 have been shown to be
specific inhibitors of the kinase activity of the fibroblast
growth factor receptor (FGFR) and vascular endothelial
growth factor receptor (VEGFR), respectively, whereas
SU6668 showed inhibitory activity against multi tyrosine
kinase, such as FGFR, VEGFR, and platelet-derived growth
factor (PDGFR) (Sun et al. 1998, 1999). Sunitinib
(SU11248) and toceranib (SU11654) are applied for the
treatment of renal cell carcinoma and imatinib-resistant
demonstrated
a correlation between anti-proliferative
activity of active compounds and blockade of the phos-
phorylation of extracellular signal-regulated kinases (ERK1/
2). The docking study also provides new insights into fur-
ther optimization of 3-methylene-2-oxoindoline-5-carbox-
amide derivatives for the discovery of more potent RAF/
MEK/ERK pathway regulators as anti-lung cancer agents.
●
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Keywords 2-Oxoindoline-5-carboxamide Synthesis
●
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Molecular docking Lung cancer RAF/MEK/ERK
pathway
* Qianbin Li
qbli@csu.edu.cn
1
Department of Medicinal Chemistry, Xiangya School of
Pharmaceutical Sciences, Central South University, 410013
Changsha, Hunan, China
2
Department of Pharmacology, Xiangya School of Pharmaceutical
Sciences, Central South University, 410013 Changsha, Hunan,
China

Med Chem Res
Fig. 1 Kinase inhibitors with 3-
(1H-pyrrol-2-yl)methylene-
indolin-2-one scaffold
gastrointestinal stromal tumor, respectively, because of their
broad inhibition against multi-tyrosine kinases, such as
PDGFR, VEGFR, KIT, RET, CSF-1R, and FLT3 (Gan
et al. 2009).
C5 or C6-substituted indolin-2-one derivatives are also
widely applied in the development of anticancer agents,
especially for Ser/Thr kinase inhibitors. NP603 was dis-
covered as a novel and potent inhibitor of FGFR1 tyrosine
kinase (Kammasud et al. 2007). GNF-5837, a potent,
selective, and orally bioavailable pan-TRKR inhibitor with
large substituent on C6 position resulted in the inhibition of
MEK/ERK pathway (Albaugh et al. 2012). NP506, an
analog of NP603, inhibited the activation of ERK, c-Jun-N-
terminal-kinase (JNK) and AKT after the rhFGF-2 stimu-
lation other than the tyrosine phosphorylation in FGFR,
VEGFR, and PDGFR. The introduction of the arylhy-
drazide motif on C5 position of the indolin-2-one scaffold
led to the inhibitory effect of ERK and JNK activation
(Kammasud et al. 2009).
Fig. 2 Design strategies for 3-methylene-2-oxoindoline-5-carbox-
amide derivatives
Materials and methods
Chemistry
All reagents and solvents were obtained from commercial
suppliers and used as received unless otherwise stated.
Reactions were monitored by thin layer chromatography
with 0.25 mm precoated silica gel plates (60GF-254) and
visualized with ultraviolet (UV) light at 254 nm, iodine stain
or by KMnO4. Flash column chromatography was per-
formed on silica gel (200–300 mesh) using solvents as
indicated. Melting points were determined uncorrected on
Herein, to find modulators of MEK/ERK pathway, 5-
carboxyl-indolin-2-one
derivatives,
3-(1H-furan-2-yl)
methylene-2-oxoindoline-5-carboxamide and 3-(1H-pyrrol-
2-yl)methylene-2-oxoindoline-5-carboxamide are designed
(Fig. 2) and synthesized based on our previous works
(Li et al. 2009, 2010). The target compounds were biolo-
gically evaluated in A549 human lung adenocarcinoma
epithelial cell for the inhibition of cell proliferation.
Structure–activity relationship was also studied for part I
and II. Western blot analysis was applied to preliminarily
demonstrate the influence of active compounds on the
components of RAF/MEK/ERK pathway, such as phospho-
ERK1/2 level, which is the substrate of dual-
phosphorylation kinase MEK1/2. Finally, the molecular
modeling studies were conformed to figure out the binding
mode of active compound to the possible kinases of RAF/
MEK/ERK pathway.
1
Boetius apparatus. H-nuclear amgnetic resonance (NMR)
and 13C-NMR spectra were recorded at 300 MHz (1H) and
at 75 MHz (13C) spectrometers (Bruker, Germany). The
measurements were made in dimethyl sulfoxide (DMSO)-d6
solutions, δ in parts per million and J in hertz, using tetra-
methylsilane as an internal standard. High resolution mass
spectra (HRMS) were recorded on MALDI-TOF/TOF-MS
spectrometer (Bruker, Germany). The purity of compounds
was determined on a Shimadzu LC-20AT HPLC instrument
using a Promosil C18 column (5 μm, 250 × 4.6 mm) with
UV detection (254 nm), eluted with 70% methanol/30%
water over 20 min and a flow rate of 1.0 mL/min.

Med Chem Res
2-Oxoindoline-5-carboxylic acid (4)
NH). 13C-NMR (75 MHz, DMSO-d6) δ: 109.0, 111.7,
115.8, 118.0, 120.0, 120.2, 121.1, 123.3, 125.0, 125.7,
127.0, 127.3, 128.0, 128.5, 128.5, 129.5, 139.4, 141.4,
165.5 (C=O), 169.5 (C=O). HRMS: m/z 330.1236 [M +
H]+ (calcd 330.1243).
To a solution of trichloracetic aldehyde (9.0 g, 61 mmol) in
120 mL of water were added anhydrous sodium sulfate (65
g, 458 mmol), 4-aminobenzoic acid (6.9 g, 51 mmol) and
4.3 mL of hydrochloric acid in 30 mL of water. Hydro-
xylamine hydrochloride (10.8 g, 152 mmol) in 50 mL of
water was added drop wise to the mixture mentioned above.
The resulting mixture was heated to 60–70 °C and kept
stirring for 2 h. The precipitate formed was collected by
filtration, washed with water and dried under vacuum. 14.8
g of intermediate compound 2 was obtained as a light yel-
low solid and was applied to the next reaction without any
purification.
To 32.6 mL of concentrated sulfuric acid at 60–70 °C
was added compound 2 (9.2 g, 44 mmol) in portions. The
mixture was then heated at 80 °C for 20 min and cooled to
room temperature, which was poured into 100 mL of ice-
water with stirring for 1 h. The precipitate was filtrated,
washed to neutral with water and added water (25 mL) and
80% hydrazine hydrate (25 mL, 62 mmol). The mixture was
then heated to 140 °C and kept stirring for 6 h. After cooling
to room temperature, the pH of the mixture was adjusted to
2 with hydrochloric acid, which yielded 8.0 g of 2-
oxoindoline-5-carboxylic acid (4) as a light yellow solid.
Mp >300 °C (Ogawa et al. 1988).
3-((1H-pyrrol-2-yl)methylene)-N-(2-methoxyphenyl)-2-
oxoindoline-5-carboxamide (6b) Yield 81%, yellow crys-
1
tals, mp: 232.1–234.2 °C. H-NMR (300 MHz, DMSO-d6)
δ: 3.86 (s, 3H, –CH3), 6.40 (s, 1H, Ar–H), 6.91–7.01 (m,
3H, Ar–H), 7.01–7.17 (m, 2H, Ar–H), 7.41 (s, 1H, Ar–H),
7.81 (d, 2H, Ar–H, J = 8.1 Hz), 7.90 (s, 1H, =CH), 8.30 (s,
1H, Ar–H), 9.26 (s, 1H, NH), 11.20 (s, 1H, NH), 13.26 (s,
1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 56.2 (–OCH3),
109.6, 111.8, 112.2, 116.3, 118.4, 121.0, 121.6, 124.5,
125.7, 125.8, 126.9, 127.2, 127.6, 127.7, 128.1, 130.0,
142.0, 151.7, 165.5 (C=O), 170.0 (C=O). HRMS: m/z
360.1348 [M + H]+ (calcd 360.1348).
3-((1H-pyrrol-2-yl)methylene)-N-(3-methoxyphenyl)-2-
oxoindoline-5-carboxamide (6c) Yield 71%, yellow crys-
1
tals, mp: 228.1–230.2 °C. H-NMR (300 MHz, DMSO-d6)
δ: 3.77 (s, 3H, –CH3), 6.39 (s, 1H, Ar–H), 6.54 (d, 1H,
Ar–H, J = 8.3 Hz), 6.93 (s, 1H, Ar–H), 6.99–7.02 (d, J =
8.1 Hz, 1H, Ar–H), 7.22–7.28 (m, 1H, Ar–H), 7.38–7.40
(m, 2H, Ar–H), 7.48 (s, 1H, Ar–H), 7.79–7.81 (m, 1H,
Ar–H), 7.89 (s, 1H, =CH), 8.26 (s, 1H, Ar–H), 10.11 (s,
1H, NH), 11.21 (s, 1H, NH), 13.26 (s, 1H, NH). 13C-NMR
(75 MHz, DMSO-d6) δ: 55.0 (–OCH3), 105.9, 108.8, 109.0,
111.7, 112.4, 115.8, 118.0, 121.1, 125.0, 126.4, 126.7,
127.3, 128.0, 129.3, 129.5, 140.6, 141.4, 159.4, 165.6
(C=O), 169.5 (C=O). HRMS: m/z 360.1348 [M + H]+
(calcd 360.1348).
General procedure for the synthesis of compounds 6 and 7
To a solution of 4 (1.77 g, 10 mmol) in 120 mL of
dichloromethane and 10 mL of DMF was added 4-
methylmorpholine or DIEA (10 mL), TBTU (4.81 g, 15
mmol) and aniline derivatives (11 mmol). The mixture was
stirred under room temperature for 18 h and washed in turn
with 5% sodium carbonate solution, 5% citric acid solution
and saturated sodium chloride solution. After dried by
anhydrous sodium sulfate, the organic layer was evaporated
to dryness. The residue was purified with flash chromato-
graphy (CH2Cl2:CH3OH, 50:1) to afford intermediates 5a–
5l, which were used in the next step in short time. To a
solution of compounds 5a–5l (1.0 mmol) in 10 mL metha-
nol was added piperidine (30 μL) and 1H-pyrrole-2-car-
baldehyde/furan-2-carbaldehyde (1.0 mmol). The mixture
was stirred at 60 °C for 5 h and cooled to room temperature.
After kept in fridge overnight, the target compounds 6a–6l
and 7a–7l were obtained in high yields.
3-((1H-pyrrol-2-yl)methylene)-N-(4-methoxyphenyl)-2-
oxoindoline-5-carboxamide (6d) Yield 92.0%, yellow
1
crystals, mp >300 °C. H-NMR (300 MHz, DMSO-d6) δ:
3.79 (s, 3H, –CH3), 6.40 (s, 1H, Ar–H), 6.93 (s, 1H, Ar–H),
7.00 (d, J = 8.1 Hz, 1H, Ar–H), 7.19 (m, 2H, Ar–H), 7.41
(s, 1H, Ar–H), 7.79–7.85 (m, 3H, Ar–H), 7.88 (s, 1H,
=CH), 8.26 (s, 1H, Ar–H), 10.18 (s, 1H, NH), 11.20 (s, 1H,
NH), 13.26 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ:
55.8 (–OCH3), 109.5, 112.2, 115.5, 115.8, 116.3, 118.5,
121.7, 122.4, 122.5, 125.5, 126.9, 127.4, 127.8, 128.4,
130.0, 136.3, 136.3, 142.0, 166.0 (C=O), 170.0 (C=O).
HRMS: m/z 360.1346 [M + H]+ (calcd 360.1348).
3-((1H-pyrrol-2-yl)methylene)-2-oxo-N-phenylindoline-5-
carboxamide (6a) Yield 83.3%, yellow crystals, mp >300
°C. H-NMR (300 MHz, DMSO-d6) δ: 6.40 (s, 1H, Ar–H),
6.94–7.12 (m, 3H, Ar–H), 7.34–7.42 (m, 3H, Ar–H),
7.78–7.86 (m, 3H, Ar–H), 7.89 (s, 1H, =CH), 8.28 (s, 1H,
Ar–H), 10.10 (s, 1H, NH), 11.21 (s, 1H, NH), 13.27 (s, 1H,
3-((1H-pyrrol-2-yl)methylene)-2-oxo-N-2-tolylindoline-5-
carboxamide (6e) Yield 78.0%, yellow crystals, mp
275.1–277.8 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 2.22 (s,
3H, –CH3), 6.38 (d, J = 3.39 Hz, 1H, Ar–H), 6.92 (s, 1H,
Ar–H), 7.01 (d, 1H, Ar–H, J = 2.46 Hz), 7.14–7.41 (m, 5H,
Ar–H), 7.82–7.86 (m, 2H, Ar–H), 8.30 (s, 1H, Ar–H), 9.75
1

Med Chem Res
(s, 1H, NH), 11.20 (s, 1H, NH), 13.27 (s, 1H, NH). 13C-
NMR (75 MHz, DMSO-d6) δ: 18.5 (–CH3), 109.5, 112.2,
116.4, 118.5, 121.6, 125.5, 126.2, 126.4, 126.8, 127.0,
127.4, 127.7, 128.2, 130.0, 130.8, 134.1, 137.2, 141.9,
165.8 (C=O), 170.0 (C=O). HRMS: m/z 344.1396 [M +
H]+ (calcd 344.1399).
(m, 2H, Ar–H), 7.72–7.85 (m, 2H, Ar–H), 7.89 (s, 1H,
=CH), 7.98 (s, 1H, Ar–H), 8.26 (s, 1H, Ar–H), 10.29 (s,
1H, NH), 11.23 (s, 1H, NH), 13.26 (s, 1H, NH). 13C-NMR
(75 MHz, DMSO-d6) δ: 109.6, 112.3, 116.2, 118.5, 118.9,
120.0, 121.7, 123.5, 125.5, 127.0, 127.6, 127.8, 128.1,
130.0, 130.8, 133.4, 141.4, 142.2, 166.3 (C=O), 170.0
(C=O). HRMS: m/z 364.0846 [M + H]+ (calcd 364.0853).
3-((1H-pyrrol-2-yl)methylene)-2-oxo-N-3-tolylindoline-5-
carboxamide (6f) Yield 80.2%, yellow crystals, mp
273.0–274.0 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 2.32 (s,
3H, –CH3), 6.39–6.40 (d, 1H, Ar–H, J = 3.6 Hz), 6.90–6.93
(m, 2H, Ar–H), 6.99 (d, 1H, Ar–H, J = 8.0 Hz), 7.24 (t, 1H,
Ar–H, J = 7.8 Hz), 7.41 (s, 1H, Ar–H), 7.60 (m, 2H, Ar–H),
7.79–7.82 (d, J = 8.4 Hz, 1H, Ar–H), 7.87 (s, 1H, =CH),
8.27 (s, 1H, Ar–H), 10.04 (s, 1H, NH), 11.20 (s, 1H, NH),
13.27 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 19.6
(–CH3), 109.5, 112.2, 116.3, 117.7, 121.4, 123.2, 125.5,
126.5, 127.8, 128.8, 129.0, 130.0, 134.4, 135.7, 137.8,
138.9, 140.8, 149.9, 164.6 (C=O), 169.8 (C=O). HRMS:
m/z 344.1393 [M + H]+ (calcd 344.1399).
3-((1H-pyrrol-2-yl)methylene)-N-(4-chlorophenyl)-2-
oxoindoline-5-carboxamide (6j) Yield 86%, yellow crys-
1
tals, mp >300 °C. H-NMR (300 MHz, DMSO-d6) δ: 6.40
(s, 1H, Ar–H), 6.94–7.02 (m, 2H, Ar–H), 7.38–7.43 (m, 3H,
Ar–H), 7.79–7.85 (m, 3H, Ar–H), 7.89 (s, 1H, =CH), 8.26
(s, 1H, Ar–H), 10.19 (s, 1H, NH), 11.21 (s, 1H, NH), 13.25
(s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 109.6,
112.2, 116.3, 118.5, 121.7, 122.2 (2C), 125.5, 126.9, 127.4,
127.5, 127.8, 128.2, 129.0 (2C), 130.0, 138.9, 142.1, 166.2
(C=O), 170.0 (C=O). HRMS: m/z 364.0853 [M + H]+
(calcd 364.0853).
3-((1H-pyrrol-2-yl)methylene)-N-(3-fluorophenyl)-2-oxoin-
doline-5-carboxamide (6k) Yield 75%, yellow crystals,
mp: 278.0–280.0 °C. 1H-NMR (300 MHz, DMSO-d6) δ:
6.40 (s, 1H, Ar–H), 6.90–6.94 (m, 2H, Ar–H), 7.01 (d, J =
8.1 Hz, 1H, Ar–H), 7.36–7.42 (m, 2H, Ar–H), 7.56–7.59
(m, 1H, Ar–H), 7.75–7.82 (m, 2H, Ar–H), 7.90 (s, 1H,
=CH), 8.26 (s, 1H, Ar–H), 10.33 (s, 1H, NH), 11.24 (s, 1H,
NH), 13.26 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ:
106.9, 109.1, 109.6, 111.8, 115.8, 115.8, 118.1, 121.2,
125.0, 126.5, 127.0, 127.5, 127.6, 129.5, 130.2, 141.3,
141.6, 163.7, 165.8 (C=O), 169.5 (C=O). HRMS: m/z
348.1156 [M + H]+ (calcd 348.1148).
3-((1H-pyrrol-2-yl)methylene)-2-oxo-N-4-tolylindoline-5-
carboxamide (6g) Yield 75%, yellow crystals, mp >300 °C.
1H-NMR (300 MHz, DMSO-d6) δ: 2.29 (s, 3H, –CH3), 6.39
(s, 1H, Ar–H), 6.93 (s, 1H, Ar–H), 7.00 (d, J = 8.0 Hz, 1H,
Ar–H), 7.16 (d, J = 8.4 Hz, 2H, Ar–H), 7.42 (s, 1H, Ar–H),
7.67 (d, J = 8.4 Hz, 2H, Ar–H), 7.80 (d, 1H, Ar–H, J = 8.0
Hz), 7.88 (s, 1H, =CH), 8.27 (s, 1H, Ar–H), 10.06 (s, 1H,
NH), 11.22 (s, 1H, NH), 13.27 (s, 1H, NH). 13C-NMR (75
MHz, DMSO-d6) δ: 20.8 (–CH3), 109.4, 112.1, 116.2, 118.3,
120.6 (2C), 121.5, 125.3, 126.7, 127.3, 127.6, 128.4, 129.3
(2C), 129.8, 132.6, 137.2, 141.7, 165.7 (C=O), 169.9
(C=O). HRMS: m/z 344.1371 [M + H]+ (calcd 344.1399).
3-((1H-pyrrol-2-yl)methylene)-N-(4-fluorophenyl)-2-oxoin-
doline-5-carboxamide (6l) Yield 76.7%, yellow crystals,
mp >300 °C. H-NMR (300 MHz, DMSO-d6) δ: 6.40 (s,
3-((1H-pyrrol-2-yl)methylene)-2-oxo-N-(3-(trifluoro-
1
methyl)phenyl)indoline-5-carboxamide (6h) Yield 71.5%,
1
yellow crystals, mp 258.1–260.2 °C. H-NMR (300 MHz,
1H, Ar–H), 6.93–7.02 (m, 2H, Ar–H), 7.20 (s, 2H, Ar–H),
7.42 (s, 1H, Ar–H), 7.69–7.81 (m, 3H, Ar–H), 7.89 (s, 1H,
=CH), 8.26 (s, 1H, Ar–H), 10.20 (s, 1H, NH), 11.22 (s, 1H,
NH), 13.26 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ:
109.0, 111.7, 115.0, 115.3, 115.8, 118.0, 121.2, 121.9,
122.0, 125.0, 126.4, 126.9, 127.3, 127.8, 129.5, 135.8,
141.5, 156.5, 165.5 (C=O), 169.5 (C=O). HRMS: m/z
348.1140 [M + H]+ (calcd 348.1148).
DMSO-d6) δ: 6.40 (s, 1H, Ar–H), 6.94 (s, 1H, Ar–H),
7.02–7.04 (d, J = 8.2 Hz, 1H, Ar–H), 7.43–7.46 (m, 2H,
Ar–H), 7.59–7.64 (m, 1H, Ar–H), 7.82–7.90 (m, 2H,
Ar–H), 8.09–8.12 (d, J = 8.3 Hz, 1H, Ar–H), 8.25–8.30 (m,
2H, Ar–H), 10.45 (s, 1H, NH), 11.25 (s, 1H, NH), 13.27 (s,
1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 109.1, 111.8,
115.7, 116.2, 118.0, 119.6, 121.2, 123.6, 125.0, 126.5,
127.1, 127.3, 127.4, 129.1, 129.5, 129.5, 129.8, 140.2,
141.7, 165.9 (C=O), 169.5 (C=O). HRMS: m/z 398.1089
[M + H]+ (calcd 398.1116).
3-(Furan-2-ylmethylene)-2-oxo-N-phenylindoline-5-car-
boxamide (7a) Yield 79.2%, yellow crystals, mp
300.6–301.1 °C. 1H-NMR (300 MHz, DMSO-d6) δ:
6.83–6.85 (m, 1H, Ar–H), 6.99 (d, J = 8.1 Hz, 1H, Ar–H),
7.07–7.12 (m, 1H, Ar–H), 7.33–7.38 (m, 3H, Ar–H), 7.44
(s, 1H, Ar–H), 7.79 (d, J = 8.0 Hz, 2H, Ar–H), 7.90 (d, J =
8.1 Hz, 1H, Ar–H), 8.19 (s, 1H, =CH), 8.92 (s, 1H, Ar–H),
10.25 (s, 1H, NH), 10.91 (s, 1H, NH). 13C-NMR (75 MHz,
3-((1H-pyrrol-2-yl)methylene)-N-(3-chlorophenyl)-2-
oxoindoline-5-carboxamide (6i) Yield 84%, yellow crys-
1
tals, mp 257.0–259.7 °C. H-NMR (300 MHz, DMSO-d6)
δ: 6.40 (s, 1H, Ar–H), 6.93 (s, 1H, Ar–H), 7.01 (d, J = 8.1
Hz, 1H, Ar–H), 7.16 (d, J = 7.1 Hz, 1H, Ar–H), 7.36–7.42

Med Chem Res
DMSO-d6) δ: 109.0, 113.7, 120.2 (2C), 120.3, 121.1, 121.4,
121.6, 123.3, 124.4, 128.4, 128.5 (2C), 129.5, 139.5, 145.1,
147.6, 150.5, 165.7 (C=O), 169.5 (C=O). HRMS: m/z
331.1082 [M + H]+ (calcd 331.1083).
126.4, 126.8, 128.6, 130.0, 130.8, 133.8, 137.2, 145.6,
148.0, 151.1, 166.0 (C=O), 170.0 (C=O). HRMS: m/z
345.1242 [M + H]+ (calcd 345.1239).
3-(Furan-2-ylmethylene)-2-oxo-N-3-tolylindoline-5-car-
boxamide (7f) Yield 81%, yellow crystals, mp
274.8–278.0 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 2.32 (s,
3H, –CH3), 6.83–7.00 (m, 3H, Ar–H), 7.20–7.25 (m, 1H,
Ar–H), 7.35 (s, 1H, Ar–H), 7.43 (s, 1H, Ar–H), 7.55–7.66
(m, 1H, Ar–H), 7.89 (s, 1H, Ar–H), 7.92 (s, 1H, Ar–H),
8.19 (s, 1H, =CH), 8.92 (s, 1H, Ar–H), 10.14 (s, 1H, NH),
10.89 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 21.7
(–CH3), 109.4, 114.3, 118.0, 120.8, 121.3, 121.6, 121.9,
122.1, 124.5, 124.9, 128.8, 128.9, 129.9, 138.1, 139.9,
145.6, 148.1, 151.0, 166.1 (C=O), 170.0 (C=O). HRMS:
m/z 345.1236 [M + H]+ (calcd 345.1239).
3-(Furan-2-ylmethylene)-N-(2-methoxyphenyl)-2-oxoindo-
line-5-carboxamide (7b) Yield 75.4%, yellow crystals, mp
268.2–271.1 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 3.87 (s,
3H, –CH3), 6.86 (s, 1H, Ar–H), 6.96–7.00 (m, 2H, Ar–H),
7.10–7.17 (m, 2H, Ar–H), 7.35 (s, 1H, Ar–H), 7.44 (s, 1H,
Ar–H), 7.90–7.94 (m, 2H, Ar–H), 8.14 (s, 1H, =CH), 8.93
(s, 1H, Ar–H), 9.32 (s, 1H, NH), 10.92 (s, 1H, NH). 13C-
NMR (75 MHz, DMSO-d6) δ: 55.9 (–OCH3), 109.2, 111.3,
113.8, 120.2, 120.4, 121.2, 121.4, 121.6, 123.1, 123.9,
125.0, 127.3, 127.8, 129.3, 145.2, 147.4, 150.6, 150.8,
164.9 (C=O), 169.5 (C=O). HRMS: m/z 361.1189 [M +
H]+ (calcd 361.1188).
3-(Furan-2-ylmethylene)-2-oxo-N-4-tolylindoline-5-car-
boxamide (7g) Yield 90%, yellow crystals, mp
292.0–294.0 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 2.29 (s,
3H, –CH3), 6.83–6.85 (m, 1H, Ar–H), 6.98 (d, J = 8.1 Hz,
1H, Ar–H), 7.15 (d, J = 8.3 Hz, 2H, Ar–H), 7.35 (s, 1H,
Ar–H), 7.44 (s, 1H, Ar–H), 7.68 (d, J = 8.3 Hz, 2H, Ar–H),
7.90 (d, J = 8.1 Hz, 1H, Ar–H), 8.18 (s, 1H, =CH), 8.91 (s,
1H, Ar–H), 10.17 (s, 1H, NH), 10.91 (s, 1H, NH). 13C-
NMR (75 MHz, DMSO-d6) δ: 20.5 (–CH3), 108.9, 113.7,
120.3 (2C), 121.1, 121.4, 121.6, 124.4, 128.5, 128.9 (2C),
129.4, 132.2, 133.0, 136.9, 145.0, 147.6, 150.5, 165.5
(C=O), 169.5 (C=O). HRMS: m/z 345.1238 [M + H]+
(calcd 345.1239).
3-(Furan-2-ylmethylene)-N-(3-methoxyphenyl)-2-oxoindo-
line-5-carboxamide (7c) Yield 68.8%, yellow crystals, mp
246.2–247.3 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 3.76 (s,
3H, –CH3), 6.66–6.69 (m, 1H, Ar–H), 6.84 (s, 1H, Ar–H),
6.97–7.00 (d, J = 7.7 Hz, 1H, Ar–H), 7.22–7.27 (m, 1H,
Ar–H), 7.35–7.49 (m, 4H, Ar–H), 7.88–7.90 (m, 1H,
Ar–H), 8.19 (s, 1H, =CH), 8.91 (s, 1H, Ar–H), 10.20 (s,
1H, NH), 10.90 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-
d6) δ: 55.0 (–OCH3), 106.0, 108.8, 109.0, 112.5, 113.8,
120.4, 121.1, 121.3, 121.6, 124.3, 128.4, 129.3, 129.5,
140.6, 145.1, 147.7, 150.5, 159.4, 165.7 (C=O), 169.5
(C=O). HRMS: m/z 361.1176 [M + H]+ (calcd 361.1188).
3-(Furan-2-ylmethylene)-N-(4-methoxyphenyl)-2-oxoindo-
line-5-carboxamide (7d) Yield 73%, yellow crystals, mp
309.0–310.0 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 3.75 (s,
3H, –CH3), 6.84 (s, 1H, Ar–H), 6.92–6.99 (m, 3H, Ar–H),
7.35 (s, 1H, Ar–H), 7.43 (s, 1H, Ar–H), 7.68–7.71 (m, 2H,
Ar–H), 7.88 (s, 1H, Ar–H), 8.19 (s, 1H, =CH), 8.91 (s, 1H,
Ar–H), 10.12 (s, 1H, NH), 10.90 (s, 1H, NH). 13C-NMR
(75 MHz, DMSO-d6) δ: 55.2 (–OCH3), 109.0, 113.6 (2C),
113.7, 120.3, 121.1, 121.4, 121.6, 121.9 (2C), 124.3, 128.5,
129.3, 132.5, 145.0, 147.6, 150.5, 155.4, 165.3 (C=O),
169.5 (C=O). HRMS: m/z 361.1184 [M + H]+ (calcd
361.1188).
3-(Furan-2-ylmethylene)-2-oxo-N-(3-(trifluoromethyl)phe-
nyl)indoline-5-carboxamide (7h) Yield 72%, yellow
crystals, mp 275.0–276.7 °C. H-NMR (300 MHz, DMSO-
1
d6) δ: 6.84 (s, 1H, Ar–H), 7.01 (d, J = 8.1 Hz, 1H, Ar–H),
7.35 (s, 1H, Ar–H), 7.45 (s, 2H, Ar–H), 7.57–7.60 (m, 1H,
Ar–H), 7.94 (d, J = 8.1 Hz, 1H, Ar–H), 8.03 (d, J = 7.9 Hz,
1H, Ar–H), 8.22 (s, 1H, =CH), 8.31 (s, 1H, Ar–H), 8.95 (s,
1H, Ar–H), 10.55 (s, 1H, NH), 10.94 (s, 1H, NH). 13C-
NMR (75 MHz, DMSO-d6) δ: 109.6, 114.3, 116.7, 120.1,
121.0, 121.7, 121.7, 122.3, 124.2, 124.9, 128.2, 129.7,
129.9, 130.1, 130.3, 140.8, 145.9, 148.3, 151.0, 166.6
(C=O), 170.0 (C=O). HRMS: m/z 399.0956 [M + H]+
(calcd 399.0957).
3-(Furan-2-ylmethylene)-2-oxo-N-2-tolylindoline-5-car-
boxamide (7e) Yield 80%, yellow crystals, mp
286.5–288.3 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 2.30 (s,
3H, –CH3), 6.83 (s, 1H, Ar–H), 6.98 (d, J = 6.6 Hz, 1H,
Ar–H), 7.26–7.34 (m, 3H, Ar–H), 7.35 (s, 1H, Ar–H),
7.42–7.43 (m, 2H, Ar–H), 7.90 (s, 1H, Ar–H), 8.11 (s, 1H,
=CH), 8.94 (s, 1H, Ar–H), 9.80 (s, 1H, NH), 10.89 (s, 1H,
NH). 13C-NMR (75 MHz, DMSO-d6) δ: 18.4 (–CH3),
109.6, 114.3, 120.8, 121.6, 121.9, 122.0, 124.8, 126.1,
N-(3-chlorophenyl)-3-(furan-2-ylmethylene)-2-oxoindo-
line-5-carboxamide (7i) Yield 74%, yellow crystals, mp
274.0–276.2 °C. 1H-NMR (300 MHz, DMSO-d6) δ:
6.84–6.85 (m, 1H, Ar–H), 7.00 (d, J = 7.9 Hz, 1H, Ar–H),
7.15 (d, J = 8.1 Hz, 1H, Ar–H), 7.36–7.42 (m, 2H, Ar–H),
7.45 (s, 1H, Ar–H), 7.70 (d, J = 8.1 Hz, 1H, Ar–H), 7.91 (d,
J = 7.9 Hz, 1H, Ar–H), 8.03 (s, 1H, Ar–H), 8.21 (s, 1H,
=CH), 8.93 (s, 1H, Ar–H), 10.42 (s, 1H, NH), 10.95 (s, 1H,

Med Chem Res
NH). 13C-NMR (75 MHz, DMSO-d6) δ: 109.0, 113.8,
118.6, 119.6 (2C), 120.5, 121.2, 121.7, 123.0, 124.4, 127.9,
129.5, 130.2 (2C), 132.9, 145.4, 147.7, 150.5, 165.9
(C=O), 169.5 (C=O). HRMS: m/z 365.0689 [M + H]+
(calcd 365.0693).
Selleck China. All reagents were prepared and used as
recommended by their suppliers.
Cell proliferation assays
A549 cells were seeded at a density of 1 × 105 cells (100 μL
per well) in 96-well plates and pretreated with active com-
pounds (0, 0.3, 1, 3, 10, 30, and 100 μM) for 48 h at 37 °C in
a fully humidified atmosphere containing 5% CO2, with
cisplatin and sunitinib as positive control (Xu et al. 2016).
Upon completion of the incubation, 1% of 0.5 mg/mL MTT
was added to each well and incubated for an additional 4 h.
Culture medium was removed by centrifuge and 100 mL of
DMSO/well was added. The formazan produced by reduc-
tion of MTT was determined by recording the absorbance of
each well at 570 nm using Multiskan Ascent 354 microplate
reader (Thermo Labsystems, Helsinki, Finland). Each
experiment was repeated at least three times to get the mean
values. The IC50 values were obtained from the
dose–response curves using GraphPad PrismTM 5.0 soft-
ware (GraphPad Software, Inc, La Jolla, California, USA).
N-(4-chlorophenyl)-3-(furan-2-ylmethylene)-2-oxoindo-
line-5-carboxamide (7j) Yield 87%, yellow crystals, mp
>300 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 6.83–6.85 (m,
1H, Ar–H), 6.99 (d, J = 8.1 Hz, 1H, Ar–H), 7.34–7.44 (m,
4H, Ar–H), 7.81–7.92 (m, 3H, Ar–H), 8.19 (s, 1H, =CH),
8.92 (s, 1H, Ar–H), 10.37 (s, 1H, NH), 10.91 (s, 1H, NH).
13C-NMR (75 MHz, DMSO-d6) δ: 109.5, 114.3, 120.9,
121.7, 121.8, 122.2 (2C), 124.9, 127.4, 128.5, 128.9 (2C),
129.4, 130.0, 138.9, 145.8, 148.2, 151.0, 166.3 (C=O),
170.0 (C=O). HRMS: m/z 365.0687 [M + H]+ (calcd
365.0693).
N-(3-fluorophenyl)-3-(furan-2-ylmethylene)-2-oxoindoline-
5-carboxamide (7k) Yield 80%, yellow crystals, mp:
1
>300 °C. H-NMR (300 MHz, DMSO-d6) δ: 6.84 (s, 1H,
Ar–H), 6.89–6.95 (m, 1H, Ar–H), 6.99–7.02 (m, 1H,
Ar–H), 7.35–7.45 (m, 3H, Ar–H), 7.57–7.77 (m, 1H,
Ar–H), 7.81–7.90 (m, 1H, Ar–H), 7.92–7.93 (m, 1H,
Ar–H), 8.20 (s, 1H, =CH), 8.93 (s, 1H, Ar–H), 10.44 (s,
1H, NH), 10.93 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-
d6) δ: 109.0, 113.8, 115.9, 120.4, 121.1, 121.2, 121.7,
124.4, 127.9, 129.5, 130.1, 130.2, 141.2, 141.3, 145.3,
147.7, 150.5, 163.6, 165.9 (C=O), 169.5 (C=O). HRMS:
m/z 349.0969 [M + H]+ (calcd 349.0988).
Western blot analysis
A549 cells (1 × 105/well) were pretreated with 6k, 6l, 7k,
and selumetinib (20 μM) for 4 h and stimulated with TGF-β
(10 ng/L) for 10 min. Protein sample preparation and
expression of phospho-ERK, total-ERK, and β-actin were
examined by western blot analysis as previously described
(Li et al. 2010). Briefly, whole-cell pellets were lysed in
radio immuno precipitation assay lysis buffer [50 mM Tris
(pH7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 1% NP-40, 1 mM sodium
orthovanadate, 1 mM EDTA, 1 mM sodium floride, 100
mg/mL phenylmethylsulfonylfluoride, 100 mg/mL dithio-
threitol (DTT)] at 4 °C for 30 min. The lysates were clarified
by centrifugation at 10,000×g for 10 min at 4 °C. Protein
concentration of the lysate samples was determined using
Bio-Rad Protein Assay kit. Protein samples were separated
by SDS-PAGE and transferred to polyvinylidene difluoride
membranes (Millipore). The membranes were then blocked
with 5% skimmed milk and incubated with primary anti-
bodies: phospho-p44/42 MAPK (Thr202/Tyr204) antibody
(1:1000; rabbit polyclonal; Cell Signaling, MA); p44/42
MAPK antibody (1:1000; rabbit polyclonal; Cell Signaling)
overnight followed by washing. Where indicated, the blots
were reprobed with antibodies against β-actin (Calbiochem)
to ensure equal loading and transfer of proteins. The reac-
tion was detected by IRDye® 800CW, conjugated goat
(polyclonal) anti-mouse or rabbit antibody (LI-COR Bios-
ciences, Lincoln, Nebraska USA) and visualized by Odys-
sey Scanner (LI-COR Biosciences, Lincoln, Nebraska
USA).
N-(4-fluorophenyl)-3-(furan-2-ylmethylene)-2-oxoindoline-5-
carboxamide (7l) Yield 76%, yellow crystals, mp: >300 °C.
1H-NMR (300 MHz, DMSO-d6) δ: 6.84 (s, 1H, Ar–H),
6.89–7.00 (m, 1H, Ar–H), 7.13–7.22 (m, 2H, Ar–H), 7.35 (s,
1H, Ar–H), 7.44 (s, 1H, Ar–H), 7.81–7.91 (m, 3H, Ar–H),
8.19 (s, 1H, =CH), 8.92 (s, 1H, Ar–H), 10.29 (s, 1H, NH),
10.90 (s, 1H, NH). 13C-NMR (75 MHz, DMSO-d6) δ: 109.0,
113.7, 114.9, 115.3, 120.4, 121.1, 121.3, 121.7, 121.9, 122.0,
122.1, 124.3, 128.2, 129.4, 135.8, 145.1, 147.7, 150.5, 165.6
(C=O), 169.5 (C=O). HRMS: m/z 349.0973 [M + H]+ (calcd
349.0988).
Biology
Human lung adenocarcinoma epithelial A549 cells were
obtained from American Type Culture Collection. A549
cells were cultured in RPMI-1640 medium supplemented
with 10% (v/v) of heat-inactivated fetal bovine calf serum at
37 °C in a fully humidified atmosphere (5% CO2). All
experiments were performed on logarithmically growing
cells. The MEK inhibitor selumetinib was purchased from

Med Chem Res
Molecular docking
5a–5l with 1H-pyrrole-2-carbaldehyde or furan-2-carbal-
dehyde, respectively, under Knoevenagel reaction condi-
tions (Kniess et al. 2009).
The molecular docking simulations were carried out using
crystal coordinates from the X-ray crystal structure of
MEK1 (PDB code: 1S9J), MEK2 (PDB code: 1S9I)
obtained from the Protein Data Bank (Ohren et al. 2004).
Compounds were built using the builder toolkit of MOE
2016 (Chemical Computing Group Inc, Canada) and the
energy was minimized by force field of MMFF94. The
protein crystal structures were prepared prior to docking in
order to remove waters, add hydrogen atoms, remove
atomic clashes and correct all structural items. Molecular
docking analysis was performed by the Docking module of
MOE package to confirm docking occurred between the
flexible ligand parts of the compound and enzyme. The
mode of construction for the docking was set to ligand.
Other default parameters were adopted in the MOE-docking
calculations. The binding energy involving H-bond inter-
actions, hydrophobic interactions and van der Waal’s
interactions was described by London dG Scoring. Inter-
actions between the compound and enzyme were explored
by distance measurements.
All tittled compounds obtained were fully characterized
by 1H NMR and 13C NMR, and high resolution mass
1
spectra (HRMS). The singlet protons in the H NMR at
around δ −10.20, −11.20 ppm in 7a–7l and δ −10.20,
−10.90 ppm in 6a–6l and were characteristic to NH proton
of isatin and amide, respectively. The singlet protons at
around δ −13.2 ppm in 6a–6l were attributed to pyrazoline.
δ −7.89 ppm (6a–6l) and -8.19 ppm (7a–7l) were assigned
1
to =CH protons. The H NMR peaks in the range of δ
6.40–8.26 ppm (6a–6l) and 6.84–8.93 ppm (7a–7l) were
due to different aromatic protons. In the 1H NMR spectra of
6b–6d and 7b–7d, the singlet proton at δ 3.78 ppm were
assigned to OCH3 proton, whereas the singlets at δ 2.30
ppm in the HNMR spectra of 6e–6g and 7e–7g were
attributed to methyl group on the phenyl ring. All com-
pounds have shown an excellent agreement between
experimentally obtained and calculated data for HRMS
analysis.
Structure–activity relationship
Results and discussion
Chemistry
A549 human lung carcinoma cell line obtained from
American Type Culture Collection (Rockville, MD, USA)
was used for the cell proliferation assay with cisplatin and
sunitinib as reference drugs. As shown in Table 1, com-
pounds 6a, 6c, 6d, 6j, 6k, 6l, 7c, 7g, 7h, 7i, and 7k dis-
played lower IC50 values than cisplatin in cell proliferation
assays. Compounds 6a, 6k, 6l, and 7k displayed equal
activity in comparison to sunitinib, with the IC50 value
range of 3.0–4.1 μM. Structurally, all compounds share
similar chemical scaffold of 3-methylene-2-oxoindoline-5-
carboxamide. The activity of compounds reported herein
was dependent on the nature of the moiety present at the
two different parts (Fig. 2). As for part I, the para-hydrogen
bond acceptors on the phenyl group in part II can reverse
the activity profiles for compounds 6 and 7, such as p-OCH3
(6d/7d), p-Cl (6j/7j), p-F (6l/7l).
Compounds 6a–6l and 7a–7l were synthesized following
the procedures described in Scheme 1. The isatin compound
2-oxoindoline-5-carboxylic acid (3) was constructed from
the starting material 4-aminobenzoic acid (1) by reacting
with trichloracetic aldehyde, hydroxylamine and con-
centrated sulfuric acid under the Sandmeyer isatin synthesis
conditions (Bursavich et al. 2007). The ketone on 3-position
of 3 was reduced to alkane (4) under Wolff-Kishner
reduction conditions (Mologni et al. 2010). Compound 4
was then applied for amidation with aniline derivatives in
the existence of TBTU as coupling agent to afford com-
pounds 5a–5l (de Candia et al. 2013). Final compounds 6a–
6l and 7a–7l were obtained in good yields via coupling of
Scheme 1 The synthesis of 3-
methylene-2-oxoindoline-5-
carboxamide derivatives 6a–6l
and 7a–7l. Reagent and
conditions: a Cl3CCHO,
NH2OH.HCl, Na2SO4, 60–70 °C,
5 h; b H2SO4, 80 °C, 1 h; c
NH2NH2, H2O, 140 °C, 8 h; d
DMF/DCM, TBTU, R-PhNH2,
4-methylmorpholine, rt, 16 h; e
furan-2-carbaldehyde or 1H-
pyrrole-2-carbaldehyde,
piperidine, 60 °C, 5 h

Med Chem Res
Table 1 The results of cell proliferation assay for compounds 6a–6l
and 7a–7l and reference drugs
with TGF-β only group (negative control), all three com-
pounds can inhibit TGF-β stimulated ERK 1/2 phosphor-
ylation significantly. 6k and 6l displayed equivalent
potency to selumetinib, which was also consistent with the
results in anti-proliferation assay. The preliminary results
indicated that 3-methylene-2-oxoindoline-5-carboxamide
derivatives reported herin may inhibit the proliferation of
A549 cell through regulation of phosphorylation of ERK1/
2, the substrate of dual-function kinase MEK1/2 in RAF/
MEK/ERK signaling cascade.
Compounds IC50 (μM)a A549 Compounds IC50 (μM)a A549
cell line
cell line
6a
3.9 1.3
27.1 1.7
7.9 0.5
8.5 1.7
15.3 1.2
30.0 2.4
19.8 0.7
13.3 1.3
14.7 0.9
9.9 0.5
3.3 1.4
3.0 0.5
10.2 0.8
7a
10.4 1.4
13.0 2.4
6.2 1.5
13.9 3.2
14.0 2.1
12.9 2.0
6.2 1.6
7.2 2.2
6.5 0.4
57.5 3.4
4.1 0.7
25.5 1.6
2.3 0.3
6b
6c
7b
7c
6d
6e
7d
7e
6f
7f
6g
7g
Molecular modeling studies
6h
6i
7h
7i
Since MEK1/2 is the kinase that regulates the phosphor-
ylation of ERK1/2, it would be of necessity to get further
insight into the binding mode of 6l with MEK1 and MEK2.
Compound 6l was built and docked into the Asp-Phe-Gly
(DFG) domain of MEK1 and MEK2 (Ohren et al. 2004). As
shown in Fig. 4, compound 6l fits well into the DFG-
domain of both MEK1 and MEK2 with the binding energy
of −23.6 and −21.6 kj/mol, which can anchor 6l to the
allosteric site of the enzyme. The amine group of indolin-2-
one interacts with the Asn82 residue of MEK2 through H-
bond interaction with distance of 2.12 Å. The aromatic parts
for indolin-2-one and aniline bind to MEK2 through π-
hydrogen interactions with Lys101, Asp212, and Phe213,
which is the DFG domain conserved in different sources of
MEK2. Furthermore, the fluorine atom on the phenyl group
of aniline form a weak bond with Val131 with distance of
3.60 Å, which would be of benefit to increase the affinity of
6l to enzymes. Similarly, the carbonyl group of indolin-2-
one and the NH of aniline interacts with Ile216 and Phe209
of MEK1 through H-bond interactions (2.41 and 2.40 Å),
respectively. And the phenyl moiety of indolin-2-one group
interact with Gly210 through π-hydrogen interaction.
Likewise, the fluorine atom also interacts with Gly128
through weak bonding with distance of 3.13 Å. The mole-
cular modeling studies also suggested that 3-methylene-5-
carboxyl-indolin-2-one is a potential scaffold for the dis-
covery of regulators of RAF/MEK/ERK pathway.
6j
7j
6k
6l
7k
7l
Cisplatin
Sunitinib
a Values are means of three experiments, standard deviations are given
For pyrrole-containing compounds (6a–6l), most com-
pounds displayed higher IC50 values than 6a (Table 1).
Structurally, there is no any substituent on the phenyl group
in part II for compound 6a (Fig. 2, R=H, X=NH). For
compounds 6b–6l, the functionalities and positions of group
attachment in this part displayed unfavorable effect. 6k and
6l are two exceptional compounds, which are also the two
most potent compounds in this work. The possible reason
lies in the special characteristics of fluorine atom, which has
been extensively applied in drug design and discovery
(Gillis et al. 2015).
For furan-containing compounds (7a–7l), compounds
with hydrogen bond donor or electron-withdrawing groups
attached on meta-position of phenyl group in Part II (Fig. 2)
displayed higher potency than those with non-substitution
(R=H, 7a), such as 7c (3-OCH3), 7h (3-CF3), 7i (3-Cl) and
7k (3-F). Additionally, the para-position of phenyl group in
part II need a methyl group to maintain the moderate
activity, such as 7g (4-CH3).
Effect of active compounds on the phosphorylation of
ERK1/2 in RAF/MEK/ERK pathway
Conclusions
In summary, a series of 3-methylene-2-oxoindoline-5-car-
boxamid derivatives were synthesized by keeping the car-
boxyl group on the 5-position of indolin-2-one. In vitro
biological assay indicated that both part I and part II are
important structural determinants for the inhibitory activity
against A549 cell proliferation, which also suggested a
relationship between the anti-proliferative activity and
blockade of the phosphorylation of ERK1/2. These results
encourage that 3-methylene-2-oxoindoline-5-carboxamide
To figure out whether the active compounds have the ability
to regulate the components of RAF/MEK/ERK pathway,
western blot analysis was applied to observe the p-ERK
level for compounds 6k, 6l, 7k, and selumetinib, a specific
inhibitor of MEK1/2. A549 cells were treated with target
compounds or selumetinib at 20 μM and then stimulated by
TGF-β to obtain high concentration of RAF/MEK/ERK
pathway components. As shown in Fig. 3, when compared

Med Chem Res
Fig. 3 The influence of
compound 6k, 6l, and 7k (20
μM, respectively) on the
phosphorylation of ERK1/2 with
selumetinib (20 μM) as positive
control. A549 cell line was
treated with compounds for 4 h
and then stimulated with TGF-β
(10 ng/L) for 10 min. p-ERK1/2,
phospho-ERK1/2; t-ERK, total-
ERK1/2
Fig. 4 The binding mode of
compound 6l to the DFG-
domain of MEK1 (left) and
MEK2 (right). The distance
labels are in red, compound 6l is
in green, the residues of protein
are in white. The molecular
surfaces of both proteins are
built with the color of
lipophilicity (hydrophilic surface
in cyan and lipophilic surface in
orange) (color figure online)
Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kos-
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of RAF/MEK/ERK pathway through inhibition of dual-
function kinase and provide a new strategy for the devel-
opment of anti-lung cancer agents.
Acknowledgements We would like to thank Dr. Dejian Jiang and
his groups (Drug safety and evaluation research center, Hunan, China)
for biological assays. This work was supported by National Nature
Science Foundation of China (Grant No. 21102184), the Ph.D. Pro-
grams Foundation of Ministry of Education of China (No.
20110162120033), Nature Science Foundation of Hunan (Grant No.
2016JJ2162).
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