Dual inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways: Design, synthesis and preliminary anticancer activity studies of 3-substituted-5-(phenylamino) indolone derivatives

Article abstract of DOI:10.1016/j.bmc.2019.01.028

The dysfunction and mutual compensatory activation of RAF-MEK-ERK and PI3K-PDK1-AKT pathways have been demonstrated as the hallmarks in several primary and recurrent cancers. The strategy of concurrent blocking of these two pathways shows clinical merits on effective cancer therapy, such as combinatory treatments and dual-pathway inhibitors. Herein, we report a novel prototype of dual-pathway inhibitors by means of merging the core structural scaffolds of a MEK1 inhibitor and a PDK1 inhibitor. A library of 43 compounds that categorized into three series (Series I–III) was synthesized and tested for antitumor activity in lung cancer cells. The results from structure-activity relationship (SAR) analysis showed the following order of antitumor activity that 3-hydroxy-5-(phenylamino) indolone (Series III) > 3-alkenyl-5-(phenylamino) indolone (Series I) > 3-alkyl-5-(phenylamino) indolone (Series II). A lead compound 9za in Series III showed most potent antitumor activity with IC₅₀ value of 1.8 ± 0.8 μM in A549 cells. Moreover, antitumor mechanism study demonstrated that 9za exerted significant apoptotic effect, and cellular signal pathway analysis revealed the potent blockage of phosphorylation levels of ERK and AKT in RAF-MEK-ERK and PI3K-PDK1-AKT pathways, respectively. The results reported here provide robust experimental basis for the discovery and optimization of dual pathway agents for anti-lung cancer therapy.

Full text of DOI:10.1016/j.bmc.2019.01.028

Accepted Manuscript
Dual Inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways: Design,
Synthesis and Preliminary Anticancer Activity Studies of 3-Substituted-5-(phe-
nylamino) indolone Derivatives
Zutao Yu, Zhuo Chen, Qiongli Su, Shiqi Ye, Hongbo Yuan, Mengni Kuai, Meng
Lv, Zhijun Tu, Xiaoping Yang, RangRu Liu, Gaoyun Hu, Qianbin Li
PII:
S0968-0896(18)32004-2
https://doi.org/10.1016/j.bmc.2019.01.028
BMC 14720
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 November 2018
21 January 2019
23 January 2019
Please cite this article as: Yu, Z., Chen, Z., Su, Q., Ye, S., Yuan, H., Kuai, M., Lv, M., Tu, Z., Yang, X., Liu, R.,
Hu, G., Li, Q., Dual Inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways: Design, Synthesis and
Preliminary Anticancer Activity Studies of 3-Substituted-5-(phenylamino) indolone Derivatives, Bioorganic &
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.028
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Dual Inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT pathways:
Design, Synthesis and Preliminary Anticancer Activity Studies of 3-
Substituted-5-(phenylamino) indolone Derivatives
Zutao Yua, Zhuo Chena, Qiongli Sub, Shiqi Yec, Hongbo Yuand, Mengni Kuaia,b, Meng Lva, Zhijun
Tua, Xiaoping Yangb, RangRu Liue, Gaoyun Hua, Qianbin Lia*
a Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South
University, Changsha 410013, Hunan, P. R. China;
b Department of Pharmacy, School of Medicine, Hunan Normal University, Changsha 410013,
Hunan, P. R. China;
c School of Medicine, Shenzhen University, Shenzhen 518060, Guangdong, P.R. China;
d Hunan Qianjin Xiangjiang Pharmaceutical Co.,Ltd, Changsha 410013, Hunan, P. R. China;
eKey Laboratory of Tropical Diseases and Translational Medicine of the Ministry of Education &
Hainan Provincial Key Laboratory of Tropical Medicine, Hainan Medical College, Haikou, China.
ABSTRACT
The dysfunction and mutual compensatory activation of RAF-MEK-ERK and PI3K-PDK1-AKT
pathways have been demonstrated as the hallmarks in several primary and recurrent cancers. The
strategy of concurrent blocking of these two pathways shows clinical merits on effective cancer
therapy, such as combinatory treatments and dual-pathway inhibitors. Herein, we report a novel
prototype of dual-pathway inhibitors by means of merging the core structural scaffolds of a MEK1
inhibitor and a PDK1 inhibitor. A library of 43 compounds that categorized into three series (Series
I-III) was synthetized and tested for antitumor activity in lung cancer cells. The results from
structure-activity relationship (SAR) analysis showed the following order of antitumor activity that
3-hydroxy-5-(phenylamino) indolone (Series III) > 3-alkenyl-5-(phenylamino) indolone (Series I) >
3-alkyl-5-(phenylamino) indolone (Series II). A lead compound 9za in Series III showed most
potent antitumor activity with IC50 value of 1.8±0.8 µM in A549 cells. Moreover, antitumor
mechanism study demonstrated that 9za exerted significant apoptotic effect, and cellular signal
pathway analysis revealed the potent blockage of phosphorylation levels of ERK and AKT in RAF-
MEK-ERK and PI3K-PDK1-AKT pathways, respectively. The results reported here provide robust
experimental basis for the discovery and optimization of dual pathway agents for anti-lung cancer
therapy.
Keywords
Dual inhibitor, RAF-MEK-ERK pathway, PI3K-PDK1-AKT pathway, Lung cancer, 5-
(phenylamino) indolone, Structure-activity relationship;

Abbreviations: SAR, structure activity relationship; NSCLC, non-small-cell lung cancer; CADD,
computer-aided drug design; FBDD, fragment-based drug design; MOE, molecular operating
environment; H460, NCI-H460 cancel cell line; FACS, fluorescence-activated cell sorting; PI,
propidium iodide; FBS, fetal bovine serum; EA, acetyl acetate; PE, petroleum ether.
*Corresponding authors: Department of Medicinal Chemistry, Xiangya School of Pharmaceutical
Sciences, Central South University, Changsha, Hunan, 410013, China
Email address: qbli@csu.edu.cn
1. Introduction
Nowadays, lung cancer becomes the second and first place in the rank of tumor morbidity and
mortality, respectively1. The advance in the understanding of disease mechanism accelerates the
establishment of molecularly targeted therapy that now has been implicated as the superior choice
for cancer therapy. RAF-MEK-ERK and PI3K-PDK1-AKT pathways play pivotal roles in the
process of tumor proliferation, differentiation, invasion and metastasis. Around 16% cancers show
pan-RAS mutations which result in consistent phosphorylation of ERK in RAF-MEK-ERK cascade
and AKT in PI3K-PDK-AKT cascade 2, 3. RAF mutants exist in approximately 70% of melanoma,
100% of hairy cell leukemia and 41% of hepatocellular carcinoma. Meanwhile, PI3K mutants, such
as PIK3CA and PIK3IR, have been identified as key therapeutic target in lung cancer, breast cancer
and colon cancer4. RAF-MEK-ERK and PI3K-PDK1-AKT pathways are over-activated in several
cancers, and they are considered as essential targets for anti-cancer drug discovery 5, 6
.
There are dozens of monotherapies targeting these two pathways in clinic use or clinical trial,
including inhibitors that blocking BRAF (Vemurafenib and Dabrafenib), MEK (PD0325901 and
4,
Selumetinib) and PI3K (Idelalisib and Buparlisib)
7. However, due to the crosstalk and
compensatory activation between these two pathways, monotherapy showed therapeutic efficacy
which limited to 6-8 months, and in soon, acquired drug resistance occurred. Also, the strategy of
combinational therapy of inhibitors targeting RAF-MEK-ERK and PI3K-PDK1-AKT pathways
showed reasonable delay of acquired drug resistance, but formula composition and drug-drug
interactions hinder the broad application to a certain extent 8, 9. More specifically, the over-activation
level and their relative ratio of these two pathways are heterogeneous among different patients.
Moreover, such ratio might vary a lot even for a patient but at the different disease stages. Because
cancer cells are so smart to evade normal therapeutics, in together with the complexity of
combinatory therapy, alternative strategy is required to tackle current challenges.

Pursuing dual inhibitor of RAF-MEK-ERK and PI3K-PDK-AKT cascades has been a hot topic in
the recent development of anticancer therapeutics. Ross et al. reported bifunctional MEK1/PI3K
inhibitor Comp. I through conjugating a PI3K inhibitor and a MEK inhibitor via a covalent linker,
which shows potent inhibitory effect on the activity of MEK1 and PI3K in enzymatic inhibition
assay and significant antitumor activity in cell assay (Figure 1) 10, 11. However, this simple covalent
conjugation generates big molecular size which might poise difficulty in cell uptake, and increasing
research interests are devoted in the design of small chemical scaffold. Zhang et al. firstly reported
dual inhibitors with small scaffold, i.e., Comp. II. Comp. II, which strongly downregulate the over-
activation of RAF-MEK-ERK and PI3K-PDK-AKT cascades and impede cell proliferation12.
Through in silico screening, Park et al. identified Comp. III and Comp. IV from a library of 23,000
molecules. In vitro assay results showed the IC50 value of 2.28.5 μM for MEK1, and the IC50 value
of 0.310.2 M for PI3Kα13. Beside these reports above, there is still a lack of diversity of chemical
scaffolds to sequester the effect of synergic blockade against RAF-MEK-ERK and PI3K-PDK-AKT
pathways, which could be potential as promising antitumor therapy with potential low recurrence
rate.
MEKi: PD0325901
PI3Ki: ZSTK474
H
O
N
O
O
F
O
O
N
O
H
N
N
N
N
N
N
I
F
F
N
N
Comp. I
F2HC
HO
HO
O
OH
N
N
S
S
OH
N
O
N
NH
N
NH
O
O
NH2
S
O
O
Comp. II
Comp. III
Comp. IV
Figure 1. Reported dual inhibitors of RAF-MEK-ERK and PI3K-PDK-AKT pathways.
For this purpose, we reported the design on dual inhibitor of RAF-MEK-ERK and PI3K-PDK-AKT
pathways and examined the efficacy as potential anticancer agents. A prototype of dual MEK/PDK1
inhibitor, 3-substitued 5-phenylamin-indolone, was settled by merging the core structural scaffolds
of PD0325901 (MEK inhibitor) and BX517 (PDK1 inhibitor). Over tuning the bond property of 3-
substituent on 5-phenylamin-indolone, three series of compounds in total 43 were synthesized,
including 3-alkenyl (Series I), 3-alkyl (Series II), and 3-hydroxy (Series III). To our knowledge, this
is the first report on the comparative study of three classes of 3-substituted-5-phenylamin-indolone.
Moreover, a convenient and high yield synthetic method to introduce -OH group at site 3 in indolone
was established. Preliminary anticancer activity and antitumor mechanism were studied in non-small
cell lung cancer (NSCLC) cell lines. Structure-activity relationship (SAR) analysis revealed the

following order of antitumor activity that 3-hydroxy (Series III) > 3-alkenyl (Series I) > 3-alkyl
indolone (Series II). Significantly, hit compound 9za in Series III showed IC50 of 1.8±0.8 µM in
A549 cells, and further studies confirmed that 9za exerts apoptotic effect through the blockage of
ERK and AKT phosphorylation in RAF-MEK-ERK and PI3K-PDK1-AKT pathways, respectively.
9za could be a lead scaffold as dual pathway inhibitor for the future development of anti-lung cancer
agents.
2. Results and discussion
2.1. Structural Design
MEK and PDK1 are the central components in RAF-MEK-ERK and PI3K-PDK1-AKT pathways
respectively. In the timeline of clinical development of MEK inhibitors, diphenylamine scaffold has
been well-characterized as a activity-indispensable scaffold responsible for the binding in the
allosteric site, such as PD0325901, pimasertib, selumetinib and trametinib 14. BX517 is a specific
PDK1 inhibitor with IC50 value less than 6.0 nM. It occupies the same binding site of the adenosine
triphosphate (ATP) which sets up three critical Hydrogen bonds with the backbone of the hinge
region 15, 16
.
Herein, we selected PD0325901 (MEK inhibitor) and BX517 (PDK1 inhibitor) as lead compounds
and designed a prototype of dual pathway inhibitors, 3-substituted-5-phenylamin-indolone. The
designed structures contain a diphenylamine scaffold for the binding to MEK allosteric pocket, and
at the same time, 3-substituted-indolone scaffold was designed to preserve PDK1 inhibitory potency.
Meanwhile, because bond-rotating property originating from large substituent poises critical
influence on protein binding affinity, we outlined three series of compounds based on the bond
variations of 3-sbustituent, i.e., 3-alkenyl-5-(phenylamino) indolone (Series I), 3-alkyl-5-
(phenylamino) indolone (Series II) and 3-hydroxy-5-(phenylamino) indolone (Series III) (Figure 2).
I
R2
F
F
H
N
R1
R2
-OMe, -Me, -H, -F, -Cl, -CF3
F
NH
H
N
O
R1
N
H
O
OH
O
OH
R2
H
N
PD0325901
MEK inhibitor
O
R1
N
H
R2
H
OH
N
O
O
H
N
N
H2N
H
O
R1
N
O
H
O
N
H
BX517
PDK1 inhibitor
Designed structures
3-Substitutents
Figure 2. Fragment-based drug design of three series of dual inhibitors of RAF-MEK-ERK and PI3K-PDK1-AKT

pathways.
To our best knowledge, this was the first report on the comparison on three kinds of 3-substituents in
one study, including 3-alkenyl, 3-alkyl and 3-hydroxy. Through literature studies, 3-alkenyl-5-
(phenylamino) indolone exhibited potent anticancer activity 17. After the reduction of 3-alkenyl bond
into 3-alkyl-5-(phenylamino) indolone, the latter scaffold led to a significant loss on anticancer
activity. Such a decrease in anticancer activity can be partially explained by the high free rotating
17
energy of 3-alkyl bond, which causes unstable binding in the enzyme allosteric pocket
.
Furthermore, the introduction of 3-hydroxy group has attracted increasing attentions in the
18
development of potent antitumor agents, such as TMC-95A, maremycin and arundaphine
.
Importantly, this research also enriched the structural diversity through introducing variable lengths
of the side chain, degrees of chemical bond saturation and steric shapes at 3-substituent site.
2.2. Chemistry
2-fluorine-4-bromonirobenzene (1) as the starting material was reacted with aniline through C-N
coupling reaction catalyzed by CuI (Supplementary S1) 19. Interestingly, it only gave substituted
aniline product in high yield, which can be explained that nitro group (-NO2) strongly activates 2-F
group. Of note, these unexpected results led to the development of a facile, one-pot synthetic method
of 1-Phenyl-1H-benzimidazole derivatives facilitated by Fe, and those results have been published
elsewhere (Supplementary S2) 20. After learned this lesson, we revised the synthetic route. Diethyl
malonate was firstly reacted with 1 to obtain 2 (Scheme 1). Followed by decarboxylation (3) and
esterification (4), 4 was obtained in up to 95% yield 21. Then it was coupled with various substituted
phenylamines (5) in high yield catalyzed by Pd(PPh3)4 under argon atmosphere. This reaction could
not proceed using CuI/N,N’-dimethylethylenediamine or CuI/proline as catalysis neither in the
absence nor presence of argon protection. The following step is one-pot synthesis to obtain critical
intermediate 6, through the sequential reaction of hydrogenation of –NO2 group and self-cyclization
of indolone heterocycle.

COOC2H5
Br
F
Br
b
Br
a
HOOC
O2N
C2H5OOC
O2N
O2N
3
2
1
H
N
Br
e
c
EtOOC
d
EtOOC
O2N
O2N
R1
4
5a-f
R2
O
H
N
R2
O
H
N
g
R1
N
H
R1
N
H
7a-j
Series I
8a-h
Series II
f
H
N
R1
R2
-OMe, -Me, -H, -F, -Cl, -CF3
O
N
H
R1
R2
O
f
H
N
HO
6a-f
R1
N
H
9a-zb
Series III
O
O
Scheme 1. Synthetic route of 5-phenylaminoindolone derivatives 7a-j (Series I), 8a-h (Series II)
and 9a-zb (Series III). Reagents and conditions: a. NaH, CH (COOC2H3)2, DMSO, 80-100℃; b. con.
HCl, CH3COOH, 120℃; c. ethanol, 90℃; d. R1PhNH2, Pd(P(Ph)3)4, Cs2CO3, 90-110℃; e. Pd/C, H2,
CH3COOH; f. RuCl3·xH2O, PPh3, NaOH, RCH2OH, 110℃, 1h for 7a-j and 5h for 9a-zb; g. H2,
Pd/C, ethanol, rt.
There are several approaches to introduce 3-alkenyl bond at indolone scaffold by reacting with
22, 23
carbonyl substrates, such as piperidine/EtOH, piperidine/THF and KF/Al2O3/microwave
.
However, these methods obtain cis-trans isomer mixture. Meanwhile, there was no report on the
introduction of 3-hydroxy substitution into indolone in simple way. Jensen et al. reported 3-alkylate
substitution catalyzed by RuCl3xH2O/PPh3 (toluene, 110℃, 20 h) with a yield of 71-92%, and they
proposed the formation of 3-alkenyl indolone intermediate as the reaction mechanism 24. We
followed their report but had minus adjustments. Intermediate 6 and benzyl alcohol were used as
starting materials. At 1 h, we detected the formation of 3-alkenyl-5-(phenylamino) indolone 7
(Series I) with the yield of 85%, which is consistent with the report (Scheme 2). Through structural
1
confirmation by H NMR and 2D-NOSY, Comp. 7 was identified as (E)-configuration. This
unexpected result would expand the synthesis of conformational selective product because there is
no report for the synthesis of optical pure configuration of (E)-3-alkenyl indolone (Supplementary
S3-S4).
Furthermore, when the reaction time elongated further, Comp.7 gradually converted to 9 instead of
8, which is dissimilar from previous report (Scheme 1). After 5-6h, Comp.9 reached the peak yield.
When the reaction time was longer than 10 to 15 h, 9 discomposed gradually. Taken together, we

reported the synthesis of 3-hydroxy indolone catalyzed by RuCl3·xH2O-PPh3, using substituted
indolone and organic alcohol as substrates 25. All target compounds (in total 43 compounds) were
structurally confirmed by HRMS, 1H NMR and 13C NMR, and the purities of which are higher than
95.0%.
2.3. Anti-proliferative activity in vitro
NSCLCs engage in approximately 85% of lung cancers, including squamous cell carcinoma,
adenocarcinoma and large cell carcinoma. In spite of progresses in the combinatorial therapy of
chemo- and radio-therapy, 1-year survival rate is around 44%, and the 5-year survival rate is lower
than 17% 26. Here, we evaluated all 43 compounds on anti-proliferative activity in two NSCLCs cell
lines, A549 and NCI-H460 (H460) using MTT method 27. Since most NSCLCs have been specified
as adenocarcinoma, the following SAR analysis were mainly focused on antiproliferative activity in
adenocarcinoma cell line A549.
Based on cell viability data shown in Table 1 and Table 2, SAR analysis were conducted. The
electrostatic effects were firstly studied at R1 site since we intentionally introduced diverse
substituents at this site. Results showed that the incorporation of electro-withdrawing group at R1
site was favorable for the anticancer activity, suggesting that preserving strong electronegative
substituent at R1 site was necessary (Figure 4A,B). More specifically, in Series I, when R2 was
cyclohexyl methylene or 3-benzodioxole-5-ylmethylene, p-Cl (7f, 2.4±0.6 µM) is 12-fold higher
inhibitory activity than p-Me (7b, 28.2±0.7 µM) (Figure 4A). Similar principle was also observed in
Series II and Series III. When R2 was saturate alkyl groups or different size of cyclic ring groups,
the increase of electronegativity was accompanied with the enhancement of inhibitory activity
(Figure 4A,B).
Table 1. Antiproliferative activity of Series I (7a–j) and Series II (8a–h) on A549 and H460 cell lines.
IC50±SD (M)
IC50±SD (M)
Comp.
R1
R2
Comp.
R1
R2
A549
H460
19.0±1.7
45.5±1.1
>100
A549
H460
55.3±1.4
>100
7a
7b
7c
7d
7e
7f
p-Me
p-Me
p-Me
p-F
3.6±1.1
28.2±0.7
16.7±1.0
26.3±0.8
7.4±0.2
2.4±0.6
15.0±0.1
3.1±0.4
8a
8b
8c
8d
8e
8f
p-Me
p-Me
p-Me
p-F
6.6±0.7
>30
O
O
O
O
18.9±0.5
>30
63.6±0.9
>100
O
O
O
>100
O
p-Cl
p-Cl
p-Cl
p-Cl
50.9±1.8
13.2±0.5
13.5±1.7
24.6±1.5
p-Cl
p-Cl
p-Cl
2.0±0.3
16.6±0.8
6.0±1.4
3.4±0.1
48.7±1.5
>100
O
O
7g
7h
8g
36.7±2.7
8.1±0.4
O
O
MK2206

AZD6244
5.2±0.3
>100
Table 2. Antiproliferative activity of Series III (9a–zb) on A549 and H460 cell lines.
IC50 ±SD (M)
IC50 ±SD (M)
Comp.
R1
R2
Comp.
R1
R2
A549
8.0±1.2
18.6±0.2
>30
H460
16.2±0.7
>100
A549
H460
O
O
9a
9b
9c
9d
9e
9f
p-OMe
p-OMe
p-OMe
p-Me
p-Me
p-Me
p-Me
p-Me
p-Me
p-F
9o
9p
9q
9r
p-F
p-Cl
24.9±1.8
2.8±1.1
1.9±1.2
2.7±0.9
5.4±0.5
3.1±2.3
3.0±0.6
2.3±1.7
2.2±0.6
4.2±0.7
2.8±0.1
2.0±0.9
1.8±0.8
2.3±0.2
80.4±0.8
24.4±0.7
40.8±1.4
25.0±2.5
30.3±4.3
20.2±2.8
26.6±6.5
12.0±3.9
16.3±2.9
17.2±4.6
18.8±3.7
9.1±2.1
>100
p-Cl
7.5±0.1
8.1±0.9
12.5±1.3
12.1±0.7
4.1±1.0
11.9±0.4
6.5±0.7
5.9±0.3
5.7±1.6
12.0±0.5
3.0±0.6
31.8±1.6
>100
p-Cl
9s
p-Cl
22.7±2.2
>100
9t
p-Cl
O
9g
9h
9i
9u
9v
9w
9x
9y
9z
p-Cl
O
31.6±0.3
14.7±1.0
32.5±2.5
>100
p-CF3
p-CF3
p-CF3
p-CF3
p-CF3
p-CF3
o-Cl
O
O
9j
9k
9l
p-F
p-F
16.8±0.6
50.7±2.8
18.1±3.5
O
O
9m
9n
p-F
9za
9zb
18.2±0.7
14.6±0.9
p-F
The steric effect of substituents was widely discussed in drug development. In terms of 3-hydroxy-5-
(phenylamino) indolone derivatives (Series III), the activities were negatively correlated with the
steric effect of R2 substituents either for saturated alkyl groups or for aromatic groups. For example,
when R1 is p-Me, the larger size of R2 with both alkyl and aromatic groups showed weak inhibitory
activity (Figure 4B).
Series I
Series II
A
B
C
30
20
10
0
30
20
10
0
30
20
10
p-OMe
p-Me
p-F
Series II
Series I
p-Me
p-Cl
Series III
p-Cl
p-CF3
0
-CH3 -CH3 -F -Cl -Cl
-Cl -Cl
R1
R2
R2
O
O
O
O
R2
O
O
O
O
O
O
O
O
Figure 4. SAR analysis among three series of target products. SAR analysis of Electro-effects in para-substituents in
Series I-II (A) and Series III (B). (C) Comparison study among Series I, II, and III.

As discussed in structural design, we devised three series of structures at 3-site of 5-(phenylamino)
indolone, i.e. 3-alkenyl bond (Series I), free rotatable 3-alkyl bond (Series II), and 3-hydroxy (Series
III). We wanted to study the unanimous rules of the difference among three series compounds
related with anticancer activity. Expectedly, there is a significant tendency when we apply inter-
series comparison. The 3-alkenyl bond in Series I was preferable than the freely rotated single bond
in Series II, which contributes unfavorably to the affinity of compound towards the target molecules
(Figure 4C). The underlying mechanism could be that fixed (E)-structure of Series I was in favor of
the interaction with enzyme active site, while freely rotate single bond in Series II devastates the
binding affinity 17, 28. Furthermore, regarding to the introduction of 3-hydroxy group, compounds in
Series III displayed highest inhibitory activity among three series. In consistent with computer
docking study, 3-hydroxy in Series III was critical responsible for inhibitory effect which contact
with Met219 residue through hydrogen bond interaction (Supplementary S4)18, 28. Similarly, in H460
cell lines, compounds of Series II exhibited lowest antiproliferative activity among whole library.
Taken these data together, SAR analysis from cell viability assay in two lung cancer cell lines
demonstrated the following order of antitumor activity that 3-hydroxy (Series III) > 3-alkenyl
(Series I) > 3-alkyl indolone (Series II). In addition, we screened two hit compounds, 9q and 9za,
with IC50 of 1.9±1.2 µM and 1.8±0.8 µM respectively in A549 cells, which showed higher anti-
proliferative activity than AZD6244 and MK2206.
2.4. Apoptosis assay
Mechanistic study such as apoptosis assay and signaling pathway assay could help to sequester the
anti-proliferative effect. Thus, apoptosis study using fluorescence-activated cell sorting (FACS)
analysis was performed firstly in A549 cell lines 29.

A. DMSO
B. 9q, 2.5 μM
C. 9q, 5.0 μM
Q1
2.31
Q2
2.57
Q1
1.42
Q2
0.76
Q1
1.82
Q2
5.65
5
4
5
4
5
4
10
10
10
10
10
10
3
3
3
10
0
10
0
10
0
Q4
Q3
Q4
Q3
Q4
Q3
83.2
11.9
90.6
7.19
72.3
20.3
3
4
5
10
3
4
10
5
10
3
4
5
0
10
10
0
10
0
10
10
10
FITC-A
FITC-A
FITC-A
D. 9za, 2.5 μM
E. 9za, 5.0 μM
Q1
1.70
Q2
7.57
Q1
2.25
Q2
3.39
5
4
5
4
40.0
30.0
20.0
10.0
0.0
10
10
***
***
10
10
***
***
3
3
10
0
10
0
Q4
Q3
Q4
Q3
O
M
M
M
M
S




0
65.3
25.5
82.7
11.6
5
0
5
M
.
.
.
.
5
D
2
5
2
,
,
q
,
,
a
a
q
z
z
9
9
9
3
4
5
10
9
3
4
10
5
10
0
10
10
0
10
FITC-A
FITC-A
Figure 5. Apoptosis assay using flow cytometry on A549 cells. (A-E) Representative flow cytometry scatter plots of
propidium iodide (PI) (Y axis) vs Annexin-FITC (X axis). Evaluation by Annexin V-FITC versus PI staining of A549
cells after 12 h incubation of 2.5 M and 5.0 M of 9q and 9za. DMSO as control group. The bottom left indicates
normal cells, the bottom right indicates early apoptotic, the top right is late apoptotic and the top left is necrotic cells
(numbers indicate the respective percentages of total cell populations). (F) Bar charts show quantitative data of
average of three independent flow cytometry experiments in A549 cells (*pꢀ<ꢀ0.05, **p<ꢀ0.01 compared with control
group).
According to the flow cytometric Annexin V-FITC/PI assay in A549 cells after 12 h treatment, 9q
and 9za exert a significant increase of apoptotic cell population (Figure 5). At the concentration of
2.5 M, 9q induced pro-apoptosis cell with population of 11.9% and necrotic cells (2.6%). After
increased the concentration of 9q to 5.0 M, there was similar activity on inducing apoptotic cells
(20.3% pro-apoptosis and 5.7% necrotic cells). Compared with 9q and control group, a more potent
antiproliferative activity was observed with 9za treatment. At the concentration of 2.5 M, the ratio
of pro-apoptosis percentage was 11.6%, without obvious increase in the proportion of necrotic cells
(only 3.4%). Interestingly, when the concentration of 9za increased to 5.0 M, there is a significant
increment of necrotic cells which took up 33.1%, with a similar level of pro-apoptosis cells (25.5%).
2.5. Anti-phosphorylation activity assay

To characterize the mechanism of 9za upon inhibitory effect on RAF-MEK-ERK and PI3K-PDK1-
AKT pathways, we performed kinase phosphorylation activity analysis with AZD6244 and MK2206
9
as positive control . We analyzed several central cellular components in these two pathways,
including p-ERK, t-ERK, p-AKT, t-AKT and β-actin.
0
0.1 0.5
1
2.5
AZD MK
A
B
C
1.5
1.0
0.5
0.0
1.5
1.0
0.5
0.0
p-ERK
8h
8h
p-AKT
t-ERK
t-AKT
β-actin
**
*
**
**
**
**
0
1
.
5
.
0
.
5
.
0
.
1
0
.
μM
0
1
5
.
0
5
.
0
.
0
μM
.
1
.
.
0
0
1
2
1
0
0
1
2
1
Figure 6. Anti-phosphorylation effect of 9za on RAF-MEK-ERK and PI3K-PDK1-AKT pathways in A549 cells. (A)
Representative western blot results of p-ERK, p-AKT, t-ERK and t-AKT. β-actin was included as a loading control.
The band density in western blot calculated the ratio of proteins p-ERK (B) and p-AKT (C) in comparison with β-
actin by using Image J software. Results are presented as the average of three independent experiments (*pꢀ<ꢀ0.05,
**p <ꢀ0.01 vs control).
After 8-hour treatment, AZD6244 showed 65% inhibition of p-ERK level at 1.0 M with no effect
on p-AKT, and MK2206 downregulated p-AKT level to 33% at 1.0 M which also shows slight
effect on RAF-MEK-ERK pathway (Figure 6). Promisingly, 9za inhibited both p-ERK and p-AKT
level significantly in dose-dependent manner. Although the inhibitory effect of 9za at higher
concentration of 2.5 M is similar to AZD6244 and MK2206 at lower concentration of 1.0 M in p-
ERK and p-AKT level, the synergic effect to block both RAF-MEK-ERK and PI3K-PDK1-AKT
pathways generated more potent therapeutic effects, which might be an explanation for the higher
activity of 9za in A549 and H460 cell lines compared with AZD6244 and MK2206. These results
highlighted that biological functions of the designed compounds are through down-regulation of
ERK phosphorylation in RAF-MEK-ERK pathway and AKT phosphorylation in PI3K-PDK1-AKT
pathway, followed by cell apoptotic effects and cell proliferative inhibition.
2.6. In silico preliminary docking study
A practical application of complex-based pharmacophore model is to determine interaction points to
trigger the optimization of binding affinity and selectivity. Harnessing the knowledge about the
rationality of the project design, we performed in silico docking study of representative compounds
with MEK1 kinase and PDK1 kinase by using Molecular operating environment (MOE) software30,
31
.

A
B
C
A
A
B
B
C
D
Figure 3. (A) The molecular docking model of PD0325901-MEK1 complexes (PDB: 3EQG)30. Left pocket shows
PD0325901 (purple) in MEK1 allosteric binding site and right pocket highlights ATP (red) in ATP-binding site. A
and B stand for two phenyl groups of PD0325901 respectively. (B) Docking study of three representative compounds
(blue) with MEK1 allosteric binding site. A stands for 5-phenyl group, B represents central indolone scaffold, and C
stands for 3-substituent. (C) The molecular docking model of BX517 (purple) binding in ATP-binding site in PDK1
(PDB: 2PE1)31. (D) Docking study of three representative structures in each series (blue) with PDK1. Before the
docking study, some necessary preparatory works have been done, including water removing and hydrogen addition
for protein and Powell energy gradient minimization.
PD0325901 is a non-ATP competitive inhibitor which represents one of the most potent and specific
inhibitors of MEK-ERK signaling. In PD0325901-MEK1 crystal structure (Figure 3A) (PDB:
3EQG)30., MEK consists of ATP-binding site (right side, red color) and allosteric site (left side,
purple color). The main core scaffold of PD0325901, diphenylamine localized at the center of the
allosteric pocket, while the side chain of PD0325901 which lies at the inlet of this pocket, suggesting
that the site chain is the favorable site for chemical modification 30. Thereafter, we docked all three
representative compounds into MEK1 allosteric pocket with PD0325901 in purple and design
compound in blue (Figure 3B). As shown in the docking study, the designed structures fitted to this
hydrophobic pocket in the way alike PD0325901 with interactions including Lys97, Val127 and
Met219 (Supplementary S5). The pocket inlet was occupied with 3-substituent, and the introduction
of extra interaction moiety might further enhance the binding affinity. Additionally, the forward
inner space of MEK1 pocket was relatively narrow, suggesting that a larger substituent at 5-phenyl
group should be avoided. Continually, based on the results of docking study on BX517-PDK1

(Figure 3C) (PDB: 2PE1)31 and designed structures with PDK1 (Figure 3D), key interactions
shared among designed structures and BX517 in ATP-binding site have been revealed, including
Val96, Ser160 and Ala162 (supplementary S6). Indolone scaffold lay inside of the pocket, and
further substitution at N-1 position might poise steric hindrance. At the same time, 3-side chain
occupied inlet and substituent at 5-phenyl group centered at outlet, which imply the potential
modification sites 31. According to CADD studies, 3-hydroxy group as hydrogen donor might form
hydrogen bond with the amino residue in the MEK allosteric pocket.
Results from docking study well explained SAR analysis and provided valuable experience for
future development of dual MEK1/PDK1 enzyme inhibitor. For example, 3-side chain modifications
such as introduction of hydrogen donor/acceptor would increase interaction between ligand with
both MEK1 inlet and PDK1 inlet 15. However, large substituents at N-1 site and large modification
group at 5-phenyl group would increase steric hindrance for PDK1 complexes and MEK1 complexes
respectively. Even though 9za was proved to target RAF-MEK-ERK and PI3K-PDK1-AKT dual
pathway, there is still lack of direct evidence to show the specific binding to MEK1 and PDK1, and
further experimental demonstration of enzyme binding activity must be required.
3. Conclusions
In addition to non-specific cytotoxic agents32 and selective cytotoxic agents33-35, molecularly
targeted therapy has become the research interests on cancer chemotherapy, especially in the era of
personalized precision medicine36, 37. The over-activation of RAF-MEK-ERK and PI3K-PDK1-AKT
pathways in cancer is considered as essential targets for effective cancer-targeting therapeutics.
However, the monotherapy of RAF-MEK-ERK pathway inhibitor and PI3K-PDK1-AKT pathway
inhibitor shows relatively short-term clinic efficacy, and mutual compensatory activations are partly
responsible for the acquired resistance4. Substantial efforts have been invested on co-targeting of
these two signaling pathways, which were believed to be a promising chemotherapeutic strategy in
effective cancer treatment8.
In summary, we studied three prototypes of bifunctional MEK/PDK1 inhibitors by merging a MEK1
non-ATP-competitive inhibitor with a PDK1 inhibitor. Through analyzing cell viability in lung
cancer cells, SAR analysis revealed the inhibitory activity order that 3-hydroxy-5-(phenylamino)
indolone (Series III) > 3-alkenyl-5-(phenylamino) indolone (Series I) > 3-alkyl-5-(phenylamino)
indolone (Series II). We identified lead compound 9za with IC50 value of 1.8±0.8 µM in A549 cell
line. Continually, mechanistic studies from flow cytometry assay and cellular signaling pathway
analysis demonstrated that 9za induced cancer cell apoptosis, which was triggered by potent
blockage of phosphorylation levels in RAF-MEK-ERK and PI3K-PDK1-AKT pathways. Current
research should provide useful experimental results for further development of dual pathway
inhibitor for targeted anticancer drug development.

4. Experimental
4.1. Chemical synthesis
4.1.1. General
Reagents and solvents were purchased from standard suppliers and used without further purification.
Hydrogen reduction synthesis was conducted by using BLT-2000 equipment (Beijing Jiawei Corp.).
Melting point was determined on a WRS-2 melting apparatus (Shanghai YiCe Apparatus &
Equipment co., LTD). Column chromatography purification was carried out on silica (80−100 mesh
and 300-400 mesh). The purity of all target compounds was confirmed to be >95% by HPLC
analysis conducted on an Agilent 20AT liquid chromatograph system. HPLC was carried out at
room temperature using a Promosil-C18 column (250 × 4.6 mm, 5 μm) in a speed of 1 ml/min with
1
methanol and water as mobile phase. The detection wavelength was set at 254 nm. H NMR, 13C
NMR and 2D-NOSY spectra were recorded on Bruker AVANCE III 500MHz and 400MHZ
spectrometer. The chemical shifts were presented in terms of parts per million with TMS as the
internal reference. In 1H NMR data, s means single peak, d is double peak, dd is di-double peak, t is
triple peak, q is quartet and m stands for multiple peak.
4.1.2. Synthesis of key examples (full information were listed in supplementary data file)
Diethyl 2-(5-Bromo-2-Nitrophenyl) Malonate (2). To a solution of 20 ml n-hexane, add 5.0 g 60% NaH (125 mmol)
and stir 2 min, then remove suspension. Slowly add 90 ml DMSO and 13.5 g diethyl malonate (100 mmol). Control
o
o
the temperature under 40 C. Then put mixture heated at 60 C for 15 min and cooled down to rt. Add 11.0 g 1 (50
o
mmol) by drop. The mixture was heated at 80 C for 4.5 h and then cooled to rt. Add 100 ml saturated NH4Cl
solution to stop the reaction. Three times extraction with acetyl acetate (EA) were performed and washed with
saturated NaCl solution, then dry with Na2SO4. Remove EA by evaporation and obtained 17.9 g Diethyl 2-(5-Bromo-
2-Nitrophenyl) Malonate (2) and proceed to next step without purification. Yellow solid，yield: 99.0%, mp. 70-72oC,
HPLC: 99.1%. HRMS (ESI) m/z calcd for [C13H14BrNO6+H]+ 381.9902; found, 381.9901 [M+Na]+, 383.9882
[M+Na]+.
5-Bromo-2-Nitrophenylacetic acid (3). To a solutuion of 60 ml 6 N HCl and 60 ml AcOH, dissove 17.9 g 2 (50
o
mmol) and stir at 110 C overnight. Romve all sovent and obtain 12.5 g 5-Bromo-2-Nitrophenylacetic acid (3), mp.
174.4-175.9 oC, yield 98%. Intermediate 3 proceed to next step without further purification. To a solution of 100 ml
dehydrated EtOH and 0.5 ml conc. H2SO4, add 12.9 g 3 (50 mmol).
Ethyl 2-(5-Bromo-2-Nitrophenyl)Acetate (4). Put the mixture in oil bath and reflux overnight. Cool down to rt and
o
place at -5 C overnight until the total precipitation occured. Filtrate white precipitate and obtain 12.5 Ethyl 2-(5-
o
1
Bromo-2-Nitrophenyl)Acetate (4), yield 99.2%. White solid, mp. 50.5-52.8 C, HPLC: 98.5%. H NMR (300 MHz,

DMSO-d6) δ: 8.06 (dd, J = 8.7, 3.3 Hz, 1H, Ar-H), 7.93 – 7.72 (m, 2H, Ar-H), 4.08 (d, J = 2.9 Hz, 4H), 1.22 – 1.08
(m, 3H).13C NMR (75 MHz, DMSO-d6) δ: 169.42, 136.17, 132.22, 131.70, 127.42, 126.80, 60.60, 38.29, 13.97.
Ethyl 2-(2-nitro-5-(p-tolylamino)phenyl)acetate (5b). To a 100 ml three neck bottle, add 2.9 g 4 (10 mmol), 6.5 g
Cs2CO3 (20 mmol), 0.11 g Pd (PPh3)4 (1 mmol), 45 ml DMF, and 1.4 g p-tolyamine (15 mmol). Ar protection and
o
heated at 90 C for 5 h. Cool down to rt, stop the reaction with 70 ml H2O. Adjust pH to 5.0–6.0 with 6 N HCl.
Followed by EA extration, sat. NaCl washing, and Na2 SO4 drying. Obtain 2.78 g Ethyl 2-(2-nitro-5-(p-
tolylamino)phenyl)acetate (5b). Proceed to next step without further purification.Yellow solid, yield: 98.2%, mp.
1
76.4-77.2oC, HPLC: 98.2%. H NMR (500 MHz, CDCl3) δ: 8.14 (d, J = 9.1 Hz, 1H), 7.28 (s, 1H), 7.21 (d, J = 8.0
Hz, 2H), 7.15 – 7.07 (m, 2H), 6.84 (dd, J = 9.1, 2.6 Hz, 1H), 6.69 (d, J = 2.6 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 3.95
(s, 2H), 2.38 (s, 3H), 1.29 (td, J = 7.1, 2.1 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ: 170.31, 149.96, 139.28, 136.59,
134.86, 133.40, 130.31, 128.54, 122.75, 117.46, 112.57, 61.10, 41.13, 20.93, 14.19.
Ethyl 2-(2-Nitro-5-(Phenylamino)Phenyl)Acetate (5c). Synthetic method refers to 5b. Yellow solid, yield: 97.2%,
1
mp. 74.5-76.1oC, HPLC: 97.2%. H NMR (300 MHz, DMSO-d6) δ: 9.26 (s, 1H), 8.16 – 8.04 (m, 1H, Ar-H), 7.37
(dd, J = 13.4, 7.1 Hz, 2H, Ar-H), 7.25 (d, J = 6.6 Hz, 2H, Ar-H), 7.09 (dd, J = 12.9, 6.6 Hz, 1H, Ar-H), 7.05 – 6.93
(m, 2H, Ar-H), 4.14 – 3.90 (m, 4H), 1.24 – 1.07 (m, 3H). 13C NMR (75 MHz, DMSO-d6) δ: 169.95, 149.69, 140.06,
138.02, 133.57, 129.45, 128.09, 123.26, 120.68, 117.52, 112.62, 60.16, 40.28, 14.05. HRMS (ESI) m/z calcd for
[C16H16N2O4+H]+ 300.1110; found, 301.1184 [M+H]+.
Ethyl 2-(2-nitro-5-(p-tolylamino)phenyl)acetate (6b)
To a hydrogen reduction equipment, add 2.86 g 5b (10 mmol), 0.14 g 10% Pd/C, and 45 ml AcOH. Stir overnight
at rt under the atmosphere of 0.4 MPa H2. Filtrate and recycle Pd/C. Remove solvent and perform silica colomn
purification with 200-300 mesh silica. Mobile phase is petrolum ether (PE): EA 2:1. Obtain 1.9 g Ethyl 2-(2-nitro-5-
(p-tolylamino)phenyl)acetate (6b). White solid, yield: 85.2%, mp. 194.5-196.1oC, HPLC: 97.2%.1H NMR (500 MHz,
CDCl3) δ 8.44 (s, 1H), 7.08 (d, J = 8.1 Hz, 2H), 7.02 (s, 1H), 6.92 (dd, J = 15.0, 5.3 Hz, 3H), 6.80 (d, J = 8.3 Hz, 1H),
5.49 (s, 1H), 3.53 (s, 2H), 2.31 (s, 3H). HRMS (ESI) m/z calcd for [C14H12N2O+H]+ 239.1184; found, 239.1189
[M+H]+.
5-(Phenylamino)Indolin-2-One (6c). Synthetic method refers to 6b. White solid, yield: 87.8%, mp. 185.5-186.6oC,
HPLC: 98.2%.1H NMR (300 MHz, DMSO-d6) δ: 10.21 (s, 1H), 7.84 (s, 1H), 7.16 (t, J = 7.9 Hz, 2H, Ar-H), 7.00 (s,
1H, Ar-H), 6.92 (d, J = 7.7 Hz, 3H, Ar-H), 6.71 (t, J = 7.9 Hz, 2H, Ar-H), 3.44 (s, 2H). 13C NMR (75 MHz, DMSO-
d6) δ: 176.09, 145.07, 137.51, 137.11, 129.03, 126.74, 118.27, 118.09, 116.34, 114.97, 109.41, 36.08. HRMS (ESI)
m/z calcd for [C14H12N2O+H]+ 224.0950; found, 247.0848 [M+Na]+.
(E)-3-Butylidene-5-(p-tolylamino)indolin-2-one (7a). To a 50 ml three neck flask, add 0.26 g 6a (1 mmol), 0.15 g
PPh3 (0.4 mmol), 40 mg NaOH (1 mmol), 42 mg RuCl3 H2O (0.2 mmol), and 15 ml tolune. Mixture well and add 0.3

g n-Butyl alcohol (5 mmol). Reflux at 110 oC for 5.5 h under the protection of Ar. Then add H2O to stop the reaction,
and extract with CH2Cl2, then washed by sat. NaCl and dryed by Na2SO4 for 5 h. Remove solvent and purified with
200-300 mesh silica colomn. Obtain 0.27 g (E)-3-Butylidene-5-(p-tolylamino)indolin-2-one (7a). Red solid, yield:
82.2%, mp. 166.1-168.2oC, HPLC: 98.7%.1H NMR (500 MHz, DMSO-d6) δ: 10.25 (s, 1H), 7.78 (s, 1H), 7.30 (d, J =
1.4 Hz, 1H, Ar-H), 7.00 (d, J = 8.2 Hz, 2H, Ar-H), 6.95 (dd, J = 8.3, 1.9 Hz, 1H, Ar-H), 6.89 – 6.85 (m, 2H, Ar-H),
6.80 – 6.72 (m, 2H, Ar-H), 2.55 (q, J = 7.5 Hz, 2H), 2.20 (s, 3H), 1.65 – 1.55 (m, 2H), 0.97 (t, J = 7.4 Hz, 3H). 13
C
NMR (125 MHz, DMSO-d6) δ: 168.45, 142.69, 140.68, 138.39, 136.08, 130.03, 129.27, 127.88, 123.28, 119.01,
116.20, 114.95, 110.53, 30.88, 21.93, 20.68, 14.30. HRMS (ESI) m/z calcd for [C19H20N2O+H]+ 293.1654; found,
293.1627 [M+H]+.
(E)-3-(Cyclohexylmethylene)-5-(p-tolylamino)indolin-2-one (7b).
Synthetic method refers to 7a. Red solid, yield: 89.5%, mp. 172.4-173.5oC, HPLC: 99.2%. 1H NMR (400 MHz,
DMSO-d6) δ: 10.25 (s, 1H), 7.82 (s, 1H), 7.27 (d, J = 1.8 Hz, 1H, Ar-H), 7.01 (d, J = 8.3 Hz, 2H, Ar-H), 6.94 (dd, J =
8.3, 2.1 Hz, 1H, Ar-H), 6.89 (d, J = 8.4 Hz, 2H, Ar-H), 6.74 (d, J = 8.3 Hz, 1H, Ar-H), 6.59 (d, J = 9.5 Hz, 1H), 2.79
(d, J = 9.8 Hz, 1H), 2.21 (s, 3H), 1.75 (dd, J = 20.0, 11.6 Hz, 4H), 1.67 (d, J = 10.3 Hz, 1H), 1.42 – 1.20 (m, 5H). 13
C
NMR (100 MHz, DMSO-d6) δ: 168.72, 145.22, 142.46, 138.56, 135.94, 130.02, 128.01, 127.68, 122.78, 118.85,
116.39, 114.25, 110.64, 37.74, 31.65, 25.75, 25.57, 20.70. HRMS (ESI) m/z calcd for [C22H24N2O+H]+ 333.1967;
found, 333.1959 [M+H]+.
(E)-3-(Benzo[d][1,3]dioxol-5-ylmethylene)-5-(p-tolylamino)indolin-2-one (7c).
Synthetic method refers to 7a. Red solid, yield: 86.5%, mp. 175.1-175.8oC, HPLC: 97.5%. H NMR (500 MHz,
DMSO-d6) δ: 10.34 (s, 1H), 7.75 (s, 1H), 7.49 (s, 1H), 7.43 (d, J = 2.0 Hz, 1H), 7.30 – 7.22 (m, 2H), 7.03 – 6.99 (m,
3H), 6.90 (dd, J = 8.3, 2.1 Hz, 1H), 6.83 (dd, J = 7.7, 5.9 Hz, 2H), 6.76 (d, J = 8.3 Hz, 1H), 6.10 (s, 2H), 2.20 (d, J =
5.9 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ: 169.26, 148.89, 148.06, 142.20, 138.27, 136.62, 136.07, 130.03,
128.79, 127.96, 127.38, 124.77, 122.01, 120.08, 116.54, 112.25, 111.02, 109.70, 109.11, 102.04, 20.71. HRMS (ESI)
m/z calcd for [C23H18N2O3+H]+ 371.1396; found, 371.13940[M+H]+.
3-Isopentyl-5-(p-tolylamino)indolin-2-one (8a). To a hydrogen reduction equipment, add 0.17 g 7a (0.5 mmol), 10
mg 10% Pd/C, and EtOH. Stir 1.5 h at rt under the atmosphere of 0.4 MPa H2. Filtrate and recycle Pd/C. Remove
solvent and perform silica colomn purification with 200-300 mesh silica. Mobile phase is PE: EA 2:1. Obtain 1.7 g 3-
Isopentyl-5-(p-tolylamino)indolin-2-one (8a). White solid, yield: 99.1%, mp. 185.1-185.8oC, HPLC: 98.5%. 1H NMR
(500 MHz, DMSO-d6) δ: 10.16 (s, 1H), 7.72 (s, 1H), 6.97 (d, J = 7.8 Hz, 3H, Ar-H), 6.86 (dd, J = 13.7, 5.1 Hz, 3H,
Ar-H), 6.71 (d, J = 8.2 Hz, 1H, Ar-H), 2.19 (s, 3H), 1.89 – 1.71 (m, 2H), 1.48 (dt, J = 13.2, 6.6 Hz, 1H), 1.14 – 1.07
(m, 2H), 0.83 (d, J = 6.6 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ: 179.09, 142.88, 138.34, 136.55, 131.15,
129.96, 127.66, 117.64, 116.05, 115.67, 109.93, 45.94, 34.60, 28.16, 27.97, 22.85, 20.66. HRMS (ESI) m/z calcd for

[C20H24N2O+H]+ 309.1967; found, 309.1943 [M+H]+.
3-(Cyclohexylmethyl)-5-(p-tolylamino)indolin-2-one (8b). Synthetic method refers to 8a. Red solid, yield: 98.4%,
1
mp. 171.2-172.8oC, HPLC: 97.5%. H NMR (500 MHz, DMSO) δ 10.15 (s, 1H), 7.74 (s, 1H), 7.00 (t, J = 11.0 Hz,
2H, Ar-H), 6.93 (s, 1H, Ar-H), 6.90 – 6.81 (m, 3H, Ar-H), 6.71 (d, J = 8.2 Hz, 1H, Ar-H), 3.42 (dt, J = 13.6, 4.1 Hz,
1H), 2.20 (s, 3H), 1.76 – 1.51 (m, 8H), 1.21 – 1.11 (m, 3H), 0.95 – 0.85 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ:
179.55, 142.80, 138.25, 136.23, 131.59, 129.97, 127.70, 117.42, 116.10, 115.82, 109.97, 43.59, 38.27, 34.65, 33.54,
32.84, 26.49, 26.27, 26.17, 20.68. HRMS (ESI) m/z calcd for [C22H26N2O+H]+ 335.2123; found, 335.2100 [M+H]+.
3-(Benzo[d][1,3]dioxol-5-ylmethyl)-5-(p-tolylamino)indolin-2-one (8c). Synthetic method refers to 8a. Red solid,
yield: 99.2%, mp. 194.5-195.8oC, HPLC: 98.5%. 1H NMR (500 MHz, DMSO-d6) δ: 10.16 (s, 1H), 7.64 (s, 1H), 6.96
(d, J = 8.2 Hz, 2H, Ar-H), 6.77 (dd, J = 7.7, 3.2 Hz, 3H, Ar-H), 6.73 (d, J = 8.3 Hz, 2H, Ar-H), 6.67 – 6.62 (m, 3H,
Ar-H), 5.93 (d, J = 4.5 Hz, 2H), 3.73 (dd, J = 8.1, 5.0 Hz, 1H), 3.23 (dd, J = 13.9, 4.9 Hz, 1H), 2.81 (dd, J = 13.9, 8.5
Hz, 1H), 2.20 (d, J = 4.4 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ: 178.37, 147.46, 146.08, 142.72, 138.09,
136.32, 132.42, 130.27, 129.94, 129.88, 127.54, 122.76, 118.14, 116.05, 115.74, 110.05, 109.82, 108.36, 101.12,
77.25, 47.25, 35.62, 20.68. HRMS (ESI) m/z calcd for [C23H20N2O3+H]+ 373.1552; found, 373.1526 [M+H]+
,
395.1340 [M+Na]+.
3-(Benzo[d][1,3]dioxol-5-ylmethyl)-5-(4-fluorophenylamino)indolin-2-one (8d). Synthetic method refers to 8a.
Red solid, yield: 99.5%, mp. 195.2-197.1oC, HPLC: 97.5%. 1H NMR (500 MHz, DMSO-d6) δ: 10.19 (s, 1H), 7.75 (s,
1H), 6.99 (dd, J = 15.2, 6.3 Hz, 2H, Ar-H), 6.78 (td, J = 8.7, 4.1 Hz, 5H, Ar-H), 6.64 (dd, J = 20.3, 7.6 Hz, 3H, Ar-
H), 5.93 (s, 2H), 3.73 (dd, J = 8.2, 5.0 Hz, 1H), 3.23 (dd, J = 13.9, 4.9 Hz, 1H), 2.81 (dd, J = 13.9, 8.4 Hz, 1H). 13
C
NMR (125 MHz, DMSO-d6) δ: 178.39, 147.45, 146.07, 141.92, 137.79, 136.77, 132.41, 130.37, 122.78, 118.57,
116.97, 116.91, 116.14, 116.00, 115.82, 110.13, 109.84, 108.37, 101.12, 47.26, 35.55. HRMS (ESI) m/z calcd for
[C22H17FN2O3+H]+ 377.1301; found, 377.1270 [M+H]+.
3-Hydroxy-5-(4-methoxyphenylamino)-3-pentylindolin-2-one (9a). The main difference of synthesis with Series I
is longer reaction time, which elongate from 1.5 h to 5.5 h. More specifically, to a 50 ml three neck flask, add 0.26 g
6a (1 mmol), 0.15 g PPh3 (0.4 mmol), 40 mg NaOH (1 mmol), 42 mg RuCl3 H2O (0.2 mmol), and 15 ml tolune.
Mixture well and add 0.3 g n-Butyl alcohol (5 mmol). Reflux at 110 oC for 5.5 h under the protection of Ar. Then add
H2O to stop the reaction, and extract with CH2Cl2, then washed by sat. NaCl and dryed by Na2SO4 for 5 h. Remove
solvent and purified with 200-300 mesh silica colomn. Obtain 0.27 g 3-Hydroxy-5-(4-methoxyphenylamino)-3-
pentylindolin-2-one (9a). White solid, yield: 75.1%, mp. 192.6-194.3oC, HPLC: 97.9%. 1H NMR (400 MHz, DMSO-
d6) δ: 9.98 (s, 1H), 7.63 (s, 1H), 6.94 (t, J = 6.3 Hz, 3H, Ar-H), 6.70 – 6.77 (m, 3H, Ar-H), 6.68 (t, J = 13.7 Hz, 1H,
Ar-H), 5.76 (s, 1H), 3.71 (s, 3H), 1.79 – 1.64 (m, 2H), 1.26 – 1.11 (m, 6H), 1.12 – 0.90 (m, 2H), 0.85 (t, J = 6.8 Hz,
3H).13C NMR (100 MHz, DMSO-d6) δ: 179.99, 153.45, 139.96, 138.19, 134.53, 133.58, 118.87, 116.87, 115.02,

113.89, 110.30, 76.45, 55.69, 38.37, 31.55, 29.22, 23.16, 22.39, 14.35. HRMS (ESI) m/z calcd for [C20H24N2O3+H]+
341.1865; found, 341.1414 [M+H]+.
3-Hexyl-3-hydroxy-5-(4-methoxyphenylamino)indolin-2-one (9b). Synthetic method refers to 9a. White solid,
1
yield: 76.4%, mp. 174.9-178.1oC, HPLC: 96.0%. H NMR (400 MHz, DMSO-d6) δ: 9.99 (s, 1H), 7.61 (s, 1H), 6.92
(t, J = 6.3 Hz, 3H, Ar-H), 6.88 – 6.77 (m, 3H, Ar-H), 6.67 (t, J = 13.7 Hz, 1H, Ar-H), 5.76 (s, 1H), 3.69 (s, 3H), 1.76
– 1.62 (m, 2H), 1.24 – 1.11 (m, 6H), 1.09 – 0.90 (m, 2H), 0.81 (t, J = 6.8 Hz, 3H).13C NMR (100 MHz, DMSO-d6) δ:
179.63, 153.44, 139.93, 138.19, 134.50, 133.58, 118.87, 116.87, 115.02, 113.89, 110.30, 76.45, 55.69, 38.37, 31.53,
29.22, 23.16, 22.39, 14.35. HRMS (ESI) m/z calcd for [C21H26N2O3+H]+ 355.2021; found, 355.2013 [M+H]+.
3-Benzyl-3-hydroxy-5-(4-methoxyphenylamino)indolin-2-one (9c). Synthetic method refers to 9a. White solid,
yield: 81.2%, mp. 194.3- 195.4oC, HPLC: 95.33%.1H NMR (400 MHz, DMSO-d6) δ: 9.87 (s, 1H), 7.55 (s, 1H), 7.20
– 7.10 (m, 3H, Ar-H), 7.05 – 6.93 (m, 2H, Ar-H), 6.87 – 6.78 (m, 4H, Ar-H), 6.77 (d, J = 2.2 Hz, 1H, Ar-H), 6.71
(dd, J = 8.2, 2.3 Hz, 1H, Ar-H), 6.50 (d, J = 8.2 Hz, 1H, Ar-H), 6.05 (s, 1H), 3.70 (s, 3H), 3.18 – 2.87 (m, 2H).13C
NMR (100 MHz, DMSO-d6) δ: 179.13, 153.30, 139.48, 138.29, 135.78, 134.55, 132.29, 130.66, 128.03, 126.76,
118.62, 117.86, 115.00, 114.51, 110.28, 77.27, 55.71, 44.04. HRMS (ESI) m/z calcd for [C22H20N2O3+H]+ 361.1552;
found, 361.1546 [M+H]+.
3-Butyl-3-hydroxy-5-(p-tolylamino)indolin-2-one (9d). Synthetic method refers to 9a. White solid, yield: 74.5%,
mp. 212.3-213.7oC, HPLC: 95.5%.1H NMR (500 MHz, DMSO-d6) δ: 10.03 (s, 1H), 7.77 (s, 1H), 6.99 (dd, J = 7.9,
5.1 Hz, 3H, Ar-H), 6.93 – 6.83 (m, 3H, Ar-H), 6.69 (d, J = 8.2 Hz, 1H, Ar-H), 5.80 (s, 1H), 2.20 (s, 3H), 1.80 – 1.67
(m, 2H), 1.26 – 1.15 (m, 2H), 1.09 – 0.89 (m, 2H), 0.79 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ:
179.68, 142.63, 138.74, 135.18, 133.56, 130.00, 127.87, 118.30, 116.28, 115.15, 110.30, 76.42, 38.14, 25.50, 22.79,
20.68, 14.34. HRMS (ESI) m/z calcd for [C19H22N2O2+H]+ 311.1759; found, 293.1647 [M-HO]+, 311.1751 [M+H]+,
333.1570 [M+Na]+.
3-Hydroxy-3-pentyl-5-(p-tolylamino)indolin-2-one (9e). Synthetic method refers to 9a. White solid, yield: 69.5%,
mp. 192.8-194.2oC, HPLC: 96.7%.1H NMR (400 MHz, DMSO-d6) δ: 10.02 (s, 1H), 7.75 (s, 1H), 6.99 (d, J = 8.4 Hz,
3H, Ar-H), 6.86 (t, J = 9.8 Hz, 3H, Ar-H), 6.68 (d, J = 7.9 Hz, 1H, Ar-H), 5.78 (s, 1H), 2.20 (s, 3H), 1.69 (d, J = 7.0
Hz, 2H), 1.13 (d, J = 32.9 Hz, 4H), 1.02 (d, J = 36.1 Hz, 2H), 0.78 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ:
179.67, 142.65, 138.72, 135.18, 133.54, 129.99, 127.85, 118.36, 116.27, 115.14, 110.30, 76.42, 38.29, 31.79, 22.85,
22.34, 20.68, 14.25. HRMS (ESI) m/z calcd for [C20H24N2O2+H]+ 325.1916; found, 307.1806 [M-HO]+, 325.1908
[M+H]+, 347.1726 [M+Na]+.
3-Hydroxy-3-isopentyl-5-(p-tolylamino)indolin-2-one (9f). Synthetic method refers to 9a. White solid, yield:
71.3%, mp. 223.7-225.5oC, HPLC: 94.9%.1H NMR (400 MHz, DMSO -d6) δ: 10.02 (s, 1H), 7.77 (s, 1H), 7.00 (s, 1H,
Ar-H), 6.99 – 6.95 (m, 2H, Ar-H), 6.87 (dd, J = 7.7, 5.3 Hz, 3H, Ar-H), 6.69 (d, J = 8.2 Hz, 1H, Ar-H), 5.79 (s, 1H),

2.20 (s, 3H), 1.80 – 1.64 (m, 2H), 1.41 (dt, J = 13.2, 6.6 Hz, 1H), 1.03 – 0.82 (m, 2H), 0.84 – 0.75 (m, 6H). 13C NMR
(100 MHz, DMSO-d6) δ: 179.62, 142.67, 138.69, 135.21, 133.49, 129.99, 127.84, 118.43, 116.21, 115.18, 110.32,
76.42, 36.26, 32.22, 28.10, 22.92, 22.86, 20.68. HRMS (ESI) m/z calcd for [C20H24N2O2+H]+ 325.1916; found,
307.1806 [M-HO]+, 325.1909 [M+H]+, 347.1727 [M+Na]+.
4.2. Cell line and culture conditions
A549 cells and H460 cells were purchased from ATCC (Manassas, USA). Cells were cultured in
DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 10 mg/ml
antibiotics (penicillin and streptomycin) at 37°C with 5% CO2 in humidified air. All other chemicals
were purchased from Sigma-Aldrich.
4.3. Anti-proliferative activity assay
2 × 105 monocytes were seeded into the 96-well plate containing 200 µL of culture medium 38. The
compounds were then individually added to the plates at 30 minutes after seeding. Plates were
incubated at 37℃ in 5% CO2 for 48 hours. At 4 hours before the end of incubation time, 20 µL 3-(4,
5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide solution (MTT, Sigma-Aldrich, 5 mg/ml)
was added to each well. After 4 hours of incubation, plates were centrifuged and culture medium
was removed. Precipitated formazan crystals were dissolved by adding 200 µL DMSO to each well.
The microplates were shaken at room temperature for 10 minutes and prepared for reading using a
microplate reader at the absorption wavelength of 570 nm. Statistical analysis was performed by
using two-way analysis of variance and Tukey multiple comparison posttests with significance
of p value less than 0.05.
4.4. Apoptosis analysis
Cancer cell apoptosis was detected by flow cytometry via the examination of altered plasma
membrane phospholipid packed with lipophilic dye Annexin V-FITC 38. Briefly, treated cells
were harvested by trypsin, washed twice with PBS, and then re-suspended in binding buffer at a
concentration of 1ꢀ×ꢀ106ꢀcells/mL according to the manufacturer’s instruction. Thereafter, 5 L of
Annexin V-FITC and 5ꢀL of propidium iodide (PI) solution were added into 100ꢀL of cell
suspension and incubated for 30ꢀminutes at room temperature in the dark. After the addition of
400ꢀL of binding buffer, labeled cells were counted by flow cytometry. All apoptotic cells at the
early stage (Annexin V-FITC–positive, PI–negative), necrotic/late apoptotic cells (double
positive), as well as living cells (double negative) were counted by flow cytometer and
subsequently analyzed by Cell Quest software (Becton Dickinson). Argon laser excitation
wavelength was 488ꢀnm, whereas emission data were acquired at wavelength 530ꢀnm (FL-1
channel) for FITC and 670ꢀnm (FL-3 c3 channel) for PI.

4.5. Western blot
Western blot assay was performed by following the manufactural procedure 38. Primary antibody
was added in BSA and allowed to incubate overnight at 4°C. After washed with TBS/0.05%
Tween-20, secondary antibody was added, and then incubated for an additional 1 hour at room
temperature. The membrane was again washed 3 times before adding Pierce Super Signal
chemiluminescent substrate (Rockford, IL) and then immediately imaged on Chemi Doc (Bio-
Rad, Hercules). The films were scanned using EPSON PERFECTION V500 PHOTO and
quantified by Image J.
4.6. Molecular docking
To get insight into the enzyme-compound interaction, flexible docking of ligand and protein binding
pocket was performed using Molecular operating environment (MOE, 2017 version). The crystal
structures of MEK1-PD0325901 (PDB ID 3EQG, 2.5 Å) and PDK1-BX-517 complexes (PDB ID
2PE1, 2.14 Å) were obtained from Protein Data Bank as the docking receptor 30, 31. The 3D
structures of the test set compounds were constructed using MOE builder tool and were energetically
minimized via MOE energy minimization algorithm [Gradient: 0.05, Force Field: MMFF94X]. The
predicted interactions of complex protein−ligand complexes were analyzed.
4.7. Statistical analysis.
GraphPad prism 5.0 was used for data analysis. Results for continuous variations presented as the
mean ± standard error. Two-group differences in continuous variations were assessed by the
unpaired t-test. P-values are two-tailed with confidence intervals of 95%.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (No. 21102184,
81773640), the Specialized Research Fund for the Doctoral Program of Higher Education of China
(No. 20110162120033), and Natural Science Foundation of Hunan Province (No. 2016JJ2162).
Hunan Province Strategic Emerging Industry Science and Technology Key Project, NO.
2016GK4028. Key research and development project of Hainan Province (project number:
ZDYF2017092), Haikou, China. We also would like to thank the Nuclear Magnetic Resonance
Laboratory of Advanced Research Center in Central South University for the technological
assistance in chemical characterization.
A. Supplementary data

Supplementary
data
associated
with
this
article
can
be
found
online
at
https://doi.org/10.1016/j.bmc.2019.xx.xxxx.
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Graphic
RTKs
N
IGFR
EGFR
G
F
PDGFR
Integrins
Extracellualr
NG
FR
P
Cytoplasm
P
P
PIP2
P
p85
PTEN
PI3K
P
RAS
p110
P
P
PIP3
P
P
RAF
O
O
PDK1
P
H
N
HO
P
P
MEK
F
F
F
O
N
H
9za
IC50=1.80.8 uM
(A549 cells)
P
P
AKT
P
ERK
Control cell transcription, proliferation, survival, migration, invation, apoptosis.