Discovery of indolin-2-one derivatives as potent PAK4 inhibitors: Structure-activity relationship analysis, biological evaluation and molecular docking study

Article abstract of DOI:10.1016/j.bmc.2017.04.047

Utilizing a pharmacophore hybridization approach, a novel series of substituted indolin-2-one derivatives were designed, synthesized and evaluated for their in vitro biological activities against p21-activated kinase 4. Compounds 11b, 12d and 12g exhibited the most potent inhibitory activity against PAK4 (IC₅₀?=?22?nM, 16?nM and 27?nM, respectively). Among them, compound 12g showed the highest antiproliferative activity against A549 cells (IC₅₀?=?0.83?μM). Apoptosis analysis in A549 cells suggested that compound 12g delayed cell cycle progression by arresting cells in the G2/M phase of the cell cycle, retarding cell growth. Further investigation demonstrated that compound 12g strongly inhibited migration and invasion of A549 cells. Western blot analysis indicated that compound 12g potently inhibited the PAK4/LIMK1/cofilin signalling pathways. Finally, the binding mode between compound 12g with PAK4 was proposed by molecular docking. A preliminary ADME profile of the compound 12g was also drawn on the basis of QikProp predictions.

Full text of DOI:10.1016/j.bmc.2017.04.047

Accepted Manuscript
Discovery of indolin-2-one derivatives as potent PAK4 inhibitors: Structure-
activity relationship analysis, biological evaluation and molecular docking study
Jing Guo, Mingyue Zhu, Tianxiao Wu, Chenzhou Hao, Kai Wang, Zizheng Yan,
Wanxu Huang, Jian Wang, Dongmei Zhao, Maosheng Cheng
PII:
S0968-0896(17)30848-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.04.047
BMC 13719
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 April 2017
26 April 2017
28 April 2017
Please cite this article as: Guo, J., Zhu, M., Wu, T., Hao, C., Wang, K., Yan, Z., Huang, W., Wang, J., Zhao, D.,
Cheng, M., Discovery of indolin-2-one derivatives as potent PAK4 inhibitors: Structure-activity relationship
analysis, biological evaluation and molecular docking study, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.04.047
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Discovery of indolin-2-one derivatives as potent PAK4 inhibitors:
Structure-activity relationship analysis, biological evaluation and molecular
docking study
Jing Guoa, Mingyue Zhua, Tianxiao Wua, Chenzhou Haoa, Kai Wanga, Zizheng Yana, Wanxu Huanga,b, Jian
Wanga, Dongmei Zhao*,a, Maosheng Cheng*, a
a
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, China. E-mail: mscheng@263.net, dongmeiz-67@163.com; Fax:
+86-24-23995043; Tel: +86-24-43520219
b School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, PR
China
Abstract
Utilizing a pharmacophore hybridization approach, a novel series of substituted indolin-2-one derivatives
were designed, synthesized and evaluated for their in vitro biological activities against p21-activated kinase 4.
Compounds 11b, 12d and 12g exhibited the most potent inhibitory activity against PAK4 (IC50 = 22 nM, 16 nM
and 27 nM, respectively). Among them, compound 12g showed the highest antiproliferative activity against A549
cells (IC50 = 0.83 μM). Apoptosis analysis in A549 cells suggested that compound 12g delayed cell cycle
progression by arresting cells in the G2/M phase of the cell cycle, retarding cell growth. Further investigation
demonstrated that compound 12g strongly inhibited migration and invasion of A549 cells. Western blot analysis
indicated that compound 12g potently inhibited the PAK4/LIMK1/cofilin signalling pathways. Finally, the binding
mode between compound 12g with PAK4 was proposed by molecular docking. A preliminary ADME profile of the
compound 12g was also drawn on the basis of QikProp predictions.
Keywords: p21-activated kinase; PAK4 inhibitor; Indolin-2-one; Structure-activity relationship; Molecular
docking
1. Introduction
The p21-activated kinases (PAKs), are a family of six serine/threonine kinases widely expressed across
tissues that phosphorylate a number of proteins associated with functions as diverse as proliferation, apoptosis, and
metastasis1-4. The six mammalian isoforms of PAKs are classified into two groups based on biochemical and
structural features: group I, including PAK1, PAK2 and PAK3, and group II, including PAK4, PAK5 and PAK65.
PAK1 activity may contribute to tumourigenesis in some disease contexts6, 7. Importantly, it has recently been
shown that acute cardiovascular toxicity result from the inhibition of PAK2, which may be enhanced by PAK1
inhibition, leading to doubts as to the validity of group I PAKs as drug targets8. PAK4 is the most extensively and
profoundly studied member among the group II PAKs. It is not only highly overexpressed in several cancer cells
but is also known to cause tumourigenesis1. Therefore, PAK4 is a promising target in the development of cancer
therapeutics.

Figure 1. Structures of potent and selective PAK4 inhibitors.
A few potent and selective PAK4 inhibitors have been disclosed (Figure 1). For example, Compound 1
(PF-3758309) was progressed into phase I clinical trials in patients with advanced/metastatic solid tumours.
Although designed to be a PAK4 inhibitor, the compound is, in fact, a pan-PAK inhibitor9. Unfortunately,
PF-3758309 was prematurely discontinued in a single clinical trial due to undesirable pharmacokinetic
characteristics10. Researchers from Genentech succeeded in identifying a highly potent and group II PAK-selective
inhibitor series with exquisite kinome selectivity11. Compound 2 displayed PAK4 activity and > 870-fold
selectivity over PAK1 ( PAK1 Ki = 2.9 μM, PAK4 Ki = 0.0033 μM )11. Recently, Karyopharm Therapeutics has
identified a new class of dual PAK4/nicotinamide phosphoribosyltransferase (NAMPT) inhibitors that show
anti-proliferative activity against several pancreatic cancer cell lines (IC50 < 250 nM)12, 13. Compound 3 (KPT-9274)
was progressed into phase I clinical trials in May 201614. Compound 4 (KY-04031) was discovered using
high-throughput screening. The unique molecular feature of KY-04031 can be exploited in designing new PAK4
inhibitors15, 16
.
Derivatives of the indolin-2-one scaffold are a prototypical class of protein kinase inhibitors17-23. Sunitinib
and Nintedanib (Figure 2) are the representative drugs that emerged from indolin-2-one (2-oxindole) and are in
clinical use for targeted anticancer therapies10. While it was reported that sunitinib was a weak inhibitor of the type
II PAKs (Ki is estimated in the range of 500 - 7000 nM for type II PAKs), it displayed selectivity for type II over
type I PAK family members24, 25. The selectivity of indolin-2-ones against particular kinases is mediated by
substituents on the indolin-2-one core. Particularly, the structural modifications at the C3- and C5-positions of the
indolin-2-one ring have led to many derivatives possessing increased antitumour activity26.
Figure 2. Representative examples of 3-alkenyl-indolin-2-one kinase inhibitors in clinical trials.
Guided by superposition of the crystal conformation of PF-3758309 (cyan) at the ATP-binding site of PAK4
with the predicted binding modes of Sunitinib (green) with the PAK4 (PDB code: 2x4z), we hypothesized that
further extending at the C5-position of Sunitinib into the ribose pocket and P-loop would improve potency (Figure
3). In the development of novel PAK4 inhibitors, our design process incorporated indolin-2-one nucleus as the
hinge binding group due to its planar geometrical conformation and possessing critical hydrogen bond donors and
acceptors to interact with the amino acid residues of the kinases hinge region. Furthermore, Our efforts were
focused on varying substituents at the 3, 5, and 6 positions of the indolin-2-one nucleus to capture additional
interactions (Figure 3). The diethylaminoethyl amide tail group of Sunitinib that is exposed to solvent without
specific contacts with protein was removed at present. On the basis of above strategy, we reported the synthesis,
structure-activity relationship (SAR) study and in vitro biological evaluation of 24 novel 3-heterocyclic-5-amide
substituted indolin-2-one derivatives as PAK4 inhibitors. The new compounds (11a-q, 12a-g) were profiled against
PAK4. Furthermore, we also explored the effects of selected compounds towards the cells proliferation, cells cycle
progression, apoptosis, cells migration/invasion and possible mechanism of inducing migration/invasion on A549
cells.

Figure 3. Rational design of the target compounds.
2. Results and discussion
2.1. Chemistry
The target compounds (11a-q, 12a-g) were prepared following the procedure reported in Scheme 1. For the
synthesis of 3-pyrrol indolin-2-one derivatives (11a–n), oxindole (7) was reacted with chloroacetyl chloride to
yield compound 8. Then, treatment of compound 8 with pyridine afforded an oxindole pyridinium salt, which was
subsequently hydrolysed under basic conditions to yield intermediate 9. Upon amidation with different amines
(R1NH2) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl) and
N-hydroxybenzotriazole (HOBt), intermediate 9 was converted to 5-carboxamides 10a-n. Finally, the target
compounds 11a-n were obtained by condensation of 5-substituted indolin-2-one 10a-n with
pyrrole-2-carboxaldehyde in the presence of piperidine as the base catalyst. For the preparation of 11o-q,
commercially available 6-bromo indolin-2-one (7d) was converted to 11n in four steps. Compound 11n, which
contains a reactive bromine at position 6, was further derivatized by Suzuki cross-coupling with a series of boronic
acids esters or by cross-coupling with zinc cyanide catalysed by palladium to obtain compounds 11o-q. Likewise,
intermediate 10b was converted to 3-substituted indolin-2-ones 12a-g by Knoevenagel condensation, employing
available aldehydes in the presence of piperidine as the base catalyst.
Scheme 1. Preparation of Compounds 11a-q, 12a-g.

Reagents and conditions: (a) chloroacetyl chloride, anhydrous AlCl3, 1,2-dichloroethane, r.t, 4.0 h. 80-92%. (b)
i.pyridine, 90 °C, 3 h; ii. 2.5N NaOH (aq), 100 °C, 3.0 h, 85-97%. (c) R1-NH2, EDCI, HOBt, DIPEA, anhydrous
DCM, r.t, 16 h, 63-88%. (d) appropriate aldehyde, piperidine, EtOH, sealed tube, 90 °C, 4.5 h, 59-90%. (e)
Zn(CN)2, Pd(PPh3)4, dry DMF, 85 °C, 46%. (f) R3 boronic ester, Pd(PPh3)4, K2CO3, toluene/EtOH, reflux, 16 h,
58-69%.
2.2. Assignment of configuration of synthesized compounds
The 3-substituted indolin-2-ones may exist as either the Z or E isomer depending on the characteristics of the
substituents at the C-3 position of the 3-substituted indolin-2-one (Scheme 2). Compounds containing a pyrrole
substituent at the C-3 position of the 3-substituted indolin-2-ones existed exclusively as the Z isomer. A diagnostic
feature is the intramolecular hydrogen bonding between the C-2 carbonyl oxygen atom of the indolin-2-one ring
and the proton on the N-1′ of the pyrrole ring27.
3-[(substituted furanyl)methylidenyl]indolin-2-ones (12a) appear to favour the E isomer form. This may be
due to the electrostatic repulsion between the C-2 carbonyl oxygen atom of the indolin-2-one and the O-1′ of furan
in the Z isomer. Indolin-2-ones having a 3-substituted thienyl group (12b) existed as Z/E mixtures although a sharp
prevalence of Z-isomer has been observed (see supplementary data). Compound 12c, which has a 3-substituted
pyridine group, existed as the E isomer, probably due to the unfavourable interaction between the lone pair of the
nitrogen of the pyridine ring and the carbonyl oxygen atom of the indolin-2-one ring in the Z-configured form.
Indolin-2-ones that have a 3-substituted imidazole or pyrazole group (12d, 12e, 12f) existed as Z/E mixture (Z
isomer is still the major form), probably due to imidazole or pyrazole tautomerization resulting in the loss of
intramolecular hydrogen bonding (see supplementary data for details). (the vinyl hydrogen for the E isomer
resonated downfield to the Z isomer by ~ 0.2-0.3 ppm due to deshielding effect of the carbonyl group of
indolin-2-one moiety.)
Scheme 2. Assignment of Configuration of 3-Substituted Indolin-2-ones.

2.3. In Vitro Activity against PAK4 Kinase and SAR Analyses.
The in vitro inhibition of PAK4 kinase activity of the newly synthesized compounds was evaluated using an
HTRF based kinase assay. One reported inhibitor of PAK4, compound 1, was included to validate the screening
conditions. Under the experimental conditions, compound 1 inhibited the activity of PAK4 with an IC50 value of
24 nM (Table 1), which was similar to previously reported data9.
Figure 4. (A) PF-3758309 taken from its crystal structure with PAK4 (PDB: 2x4z). (B)
3-(pyrrol-2-yl)methylene-2-indolinone scaffold (green) modeled with PAK4. The red arrow highlights the vector
off the 5-position of the 3-(pyrrol-2-yl)methylene-2-indolinone scaffold to grow toward the ribose pocket and
P-loop.
At the outset of this study, the indolin-2-one scaffold as well as the pyrrole moiety were fixed, and structural
variation was introduced by the synthesis of different amides groups (Table 1). In order to place a suitable
fragment mimicking the N-2-(dimethylamino)-1-phenylethyl of compound 1 (Figure 4), phenylglycinol was to be
grafted at the C5-positions of the indolin-2-one ring to develop compounds able to interact with the side chain
carbonyl group of Asp458 of the DFG motif and the P-loop. Therefore, we merged the
3-(pyrrol-2-yl)methylene-2-indolinone scaffold with D/L-phenylglycinol, which provided analogues 11a and 11b.
11a showed diminished enzymatic activity (IC50 = 3376 nM), while 11b had a beneficial effect (IC50 = 22 nM). The
S-enantiomer 11b was 153-fold more potent than the R-enantiomer 11a, emphasizing the importance of the
S-enantiomer being a better fit with the phenyl group inserted into the glycine rich loop. Hence, further
optimization work focused on S-enantiomer. Then, we investigated the substituent effects on the phenyl moiety of

phenylglycinol. Incorporation of a chlorine atom at the 4-position or 2-position of the phenyl ring provided 11c
and 11d, respectively, both of which displayed measurable decreases in potency (IC50 values of 391, 199 nM,
respectively). Furthermore, introduction of a fluorine atom at the 4-position of the phenyl ring afforded 11e, which
was also not tolerated (IC50 = 54 nM). Replacement of the phenyl group of 11b with a larger benzyl or smaller
i-propyl and t-butyl groups provided 11f, 11g, and 11h, with dramatically decreased enzyme activity (IC50 values
of 304, 2355 and 634 nM, respectively). Removal of the phenyl ring afforded 11i, which lost the most activity
(IC50 = 1255 nM). Compounds 11j and 11k which are homologues of 11i and 11b displayed either greater than
150-fold loss of potency or completely loss of potency (IC50 values of 3145, 5005 nM, respectively). The potency
loss of these analogues might be explained by the PAK4 P-loop pocket minor size and rigidity. Therefore, 11b with
an L-phenylglycinol substitution pattern was chosen as the compound for further optimization.
Table 1. Effect of R1 Substituents on PAK4 biochemical IC50 (nM).
Compd
11aa
R1
PAK4, IC50 (nM)c,d
Compd
11g
R1
PAK4, IC50 (nM)c,d
3376
2355
11bb
11c
11d
11e
11f
11h
11i
22
391
199
54
634
1255
3145
5005
24
11j
11k
PF-3758309e
304
-
a D-phenylglycinol, R-enantiomer. b L-phenylglycinol, S-enantiomer. c IC50 values are calculated based on the homogeneous
time-resolved fluorescence (HTRF) assay. d Reported data are the mean values from two independent experiments. e Used as a
positive control.
We next turned our attention to the pyrrole moiety by replacing it with other heterocyclics (Table 2). In vitro
kinase assays with compounds 12a, 12b, and 12c confirmed the importance of the pyrrole NH, which forms a
hydrogen bond with the PAK4 hinge region. Seeking alternatives to the pyrrole provided compounds 12d, 12e, and
12f respectively. Interestingly, when the imidazole nitrogen was moved to the 4'-position, the resulting compound
12d was more potent than the 3'- or 5'-position analogues (12e and 12f, respectively). These results implied that

the 4'-imidazole isomer interacted in a more favourable manner with the protein hinge residue. It's reported that the
alkyl substituents on the 3'- or 5'-position of the pyrrole could destabilize interactions with a subset of off-target
kinases28. Consistent with this idea, we prepared 12g, which was nearly equipotent to 11b in the enzymatic assay.
Table 2. Effect of R2 Substituents on PAK4 biochemical IC50 (nM).
Compd
12a
R2
PAK4, IC50 (nM)a,b
Compd
12e
R2
PAK4, IC50 (nM)a,b
802
281
12b
12c
12f
1290
379
27
12g
>10000
12d
PF-3758309c
16
24
-
a IC50 values are calculated based on the homogeneous time-resolved fluorescence (HTRF) assay. b Reported data are the mean
values from two independent experiments. c Used as a positive control.
The vector of the C6 position on the oxindole is in the direction of a lipophilic pocket beyond the “gatekeeper”
residue (Met-395 for PAK4). Access to this pocket affords enhancements in the activity and sometimes selectivity
of inhibitors and was our next area of investigation (Table 3). As a general trend, introduction of substituents
bearing hydrogen bond acceptors and their sizes was important for binding to the back pocket29-31
.
However, substitution at C6 with the small lipophilic F, Cl and Br substituents in compounds 11l, 11m and
11n, respectively, was not tolerated. Increasing the size of the substituent even moderately (11o) produced a
significant reduction in activity as well. Small-sized heterocycles with hydrogen bond acceptor capability such as
pyrazole analogues (11p-q) either abolished or greatly reduced activity, presumably due to deleterious steric
interactions with the gatekeeper residue Met-395, which prevented access to the lipophilic pocket.
Table 3. Effect of R3 Substituents on PAK4 biochemical IC50 (nM).
Compd
11l
R3
F
PAK4, IC50 (nM)a,b
Compd
11o
R3
PAK4, IC50 (nM)a,b
718
1544
CN

1423
1201
1454
1987
11m
11n
Cl
11p
11q
Br
a IC50 values are calculated based on the homogeneous time-resolved fluorescence (HTRF) assay. b Reported data are the mean
values from two independent experiments.
2.4. Antiviability Activities of Selected Compounds against Cancer Cell Lines.
The three compounds whose IC50 values were lower than 30 nM were further evaluated for in vitro anticancer
screening. Their IC50 values against MCF-7 cell line and A549 cell line, in which PAK4 has been found to be
overexpressed, were tested and listed in Tables 4. Meanwhile, the tumour cell line HT-1080, whose growth was
not dependent on PAK4, was used to test the potential off-target effects of the potent PAK4 inhibitors. As shown in
Table 4, the IC50 values of these compounds against different tumour cells ranged between 0.83 and 10.80 μM
except for compound 12d, to which MCF-7 cells are not sensitive to and therefore do not experience decrease cell
proliferation.
Table 4. Relative IC50 (μM) values for selected compounds against A549, MCF-7 and HT-1080 cells lines.
IC50 (μM)a
PAK4
Compound
(IC50, nM)
A549
2.37
3.70
0.83
MCF-7
6.56
HT-1080
0.98
22
16
27
11b
12d
12g
10.80
2.11
7.90
6.77
a
IC50: concentration of the compound (μM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by
the CCK-8 assay.
2.5. Effects of Compound 12g on Cell Apoptosis and Cell Cycle Progression.
The ability of compound 12g to induce tumour cell apoptosis was tested. The cells were harvested and stained
with 7-aminoactinomycin D (7-AAD) and annexin-V, and the percentage of apoptotic cells was determined by
flow cytometry analysis. As shown in Figure 4A, compound 12g induced apoptosis in cells in a
concentration-dependent manner. At a concentration of 5.0 μM, an apoptosis rate of 65.4% was observed after 72 h.
We further examined the influence of compound 12g on the cell cycle by using flow cytometry. When A549 cells
were treated with 12g at 1.0 μM or 5.0 μM, the number of cells in G2/M phase increased from 17.62% to 24.37%
and 33.75%, whereas cells in G0/G1 phase decreased from 44.99% to 35.7% and 27.78%, respectively (Figure 4B,
C). Taken together, these data demonstrated that compound 12g could effectively lead to cell apoptosis and G2/M
phase arrest in A549 cells.

Figure 4. (A). Compound 12g induced cell apoptosis. The cells were incubated with the indicated concentrations
of 12g for 72 h, and the cells were stained with 7-AAD and annexin-V, followed by flow cytometry analysis. (B).
Compound 12g induced G2/M cell cycle arrest of A549 cells. A549 cells were harvested after treatment with
various concentrations of compound 12g for 72 h. (C). Quantitative analysis of cell cycle.
2.6. Effects of Compound 12g on Tumor Cell Migration and Invasion
The effects of compound 12g on tumour cell migration and invasion were assessed using transwell assays. As
displayed in Figure 5A, after treatment of compound 12g at a concentration of 5.0 μM for 72 h, the migration of
A549 cells was significantly inhibited. The results from the transwell assays showed that 5.0 μM 12g potently
blocked the invasion ability of A549 cells. These results demonstrated that 12g could efficiently exhibit
antimetastatic potential against cancer cells.
Figure 5. (A). Compound 12g suppresses migration and invasion of A549 cells in transwell assays. The images
were captured using phase contrast microscopy after 72 h of treatment. Scale bar, 20 μm. (B). Quantitative analysis
of migration and invasion. (C). Compound 12g inhibits phosphorylation of PAK4 and downstream signalling in
A549 cells after 72 h treatments.

2.7. Inactivation of Key Signalling Proteins in A549 cells.
To further elucidate the mechanism of the inhibition of cancer cell migration and invasion, the expression of
cellular signalling molecules was determined by Western blot analysis. Previous work showed that the
PAK4/LIMK1/cofilin signalling pathway is correlated with cellular migration and invasion behaviour32. We
therefore investigated the effects of compound 12g on the PAK4/LIMK1/cofilin signalling pathway to validate its
strong PAK4 kinase inhibition. As shown in Figure 5C, compound 12g inhibited PAK4 phosphorylation in a
dose-dependent manner. As expected, we observed inhibition of PAK4 downstream signalling protein LIMK1 and
cofilin phosphorylation dose-dependently.
2.8. Binding mode analysis
To investigate potential binding modes, compound 12g was docked into the ATP-binding site of PAK4 (PDB
entry 2x4z) using Autodock 4.2. The predicted binding mode of 12g to PAK4 is shown in Figure 6, in which the
indolin-2-one scaffold forms two hydrogen bonds to the backbone carbonyl oxygen of Glu-396 and the NH of
Leu-398 in the hinge region. Meanwhile, the carbonyl oxygen atom of the amide attached to the 5-position of
indolin-2-one framework served as an H-bond acceptor for catalytic residue Lys-350, the conserved lysine in
kinases that also forms a salt bridge with Glu-366 of the C-helix in the active conformation. The primary hydroxyl
group interacted with the side chains of residue Asp-458 of the DFG motif. Beyond these H-bond interactions, the
phenyl group of 12g inserted into a small hydrophobic pocket under the P-loop and generated substantial
hydrophobic contacts with neighbouring residues.
Figure 6. Molecular docking of 12g in the ATP-binding site of PAK4. (A) Detailed interactions with the protein
residues. Hydrogen bonds are rendered as red dotted lines. (B) 2D interaction diagram showing compound 12g
docking pose interactions with the key amino acids in the PAK4 active site.
2.9. ADME profiling of 12g
In recent years, there has been considerable discussion on the importance of optimizing the absorption,
distribution, metabolism, and excretion (ADME) properties of drug candidates, in addition to their efficacy, to
increase the rate of drug discovery successes and to advance high quality candidates to clinical studies. To further
characterize the druglikeness of the most promising compounds 11b, 12d and 12g, several physicochemical
parameters were calculated, including lipophilicity (clogP), topological polar surface area (tPSA), molecular
weight (MW), ligand efficiency (LE), ligandlipophilicity efficiency (LLE)33, and ligand-efficiency-dependent
lipophilicity (LELP)34. The clogP value was in all cases close to 3, which is in agreement with the general
36
requirements for druglikeness and within the optimal range for orally administered drugs (clogP ≈ 3)35,
.
Additionally, the MW falls within the scope for optimal bioavailability (MW < 400 g mol-1)37. Moreover, the
estimated tPSA values were lower than 120. To evaluate the lipophilicity and structural contributions to in vitro
potency35, LE, LLE, and LELP were calculated using the pIC50 from in vitro PAK4 inhibition data. All compounds
displayed acceptable LE, LLE, and LELP values within the preferred range for good hits and leads34.

Table 5. Druglikeness of Compounds 11b, 12d and 12g.a
MW
tPSA
(Å)d
Entry
pIC50b
clogPc
LEe
LLEf
LELPg
(g/mol)
373.410
374.398
401.464
11b
12d
12g
7.66
7.79
7.56
2.84
1.93
3.47
106.88
121.74
106.41
0.38
0.39
0.35
4.82
5.86
4.09
7.42
4.95
9.83
aProperties determined using QikProp, version 4.3, Schrödinger, LLC, New York, NY, 2015. bpIC50 = -log[IC50 (PAK4, mol/L)].cPartition
coefficient. dTopological polar surface area. eLE = -1.4(log[IC50 (PAK4, mol/L)/number of heavy atoms]). fLLE = pIC50 - clogP. gLELP =
clogP/LE.
3. Conclusion
In this study, a novel series of PAK4 inhibitors bearing a 3,5,6-trisubstituted indolin-2-one scaffold were
designed and synthesized. Structure-activity relationships of these compounds were discussed based on enzymatic
activities. Several compounds showed high potencies in biochemical functional assays. The three most active
compounds were selected to be evaluated for their anticancer activity against A549, MCF-7 and HT1080 cells, of
which 12g exhibited the most potent anti-A549 cell activity. Further in-depth studies were carried out on 12g for
its biological assessment. The results showed that 12g could effectively block the migration and invasion of A549
cells and lead to apoptosis and G2/M phase arrest. Western blot indicated that compound 12g potently inhibited
PAK4/LIMK1/cofilin signaling pathways. Finally, the binding mode between compound 12g with PAK4 was
proposed by molecular docking. Drug-likeness predictions indicate the capability of drug development of
compound 12g. Further investigations on these advanced leads are ongoing and will be reported in due course.
4. Experimental section
4.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and used without further purification
unless otherwise indicated. Anhydrous solvents were dried and purified by conventional methods prior to use.
Column chromatography was carried out on silica gel (200-300 mesh). All reactions were monitored by thinlayer
chromatography (TLC) on silica gel plates with fluorescence F-254 and visualized with UV light. 1H NMR and 13
C
NMR spectral data were recorded in DMSO-d6 on a Bruker ARX-400 NMR or Bruker ARX-600 NMR
spectrometer with TMS as an internal standard. High resolution accurate mass spectrometry determinations
(HRMS) for all final target compounds were obtained on a Bruker Micromass Time of Flight mass spectrometer
equipped with an electrospray ionization (ESI) detector.
4.1. 1. General procedure for the synthesis of 5-(2-chloroacetyl)indolin-2-one derivatives (8a-d).
5-(2-chloroacetyl)indolin-2-one derivatives (8a-d) were synthesized according to previously reported
methods38.
4.1.2. General procedure for the synthesis of 2-oxoindoline-5-carboxylic acid derivatives (9a-d).
2-oxoindoline-5-carboxylic acid derivatives (9a-d) were synthesized according to previously reported
methods39.
4.1.3. General procedure for the synthesis of compounds (10a-n).
A solution of 9a-d (2 mmol), EDCI (3 mmol) and HOBt (3 mmol) in dry DCM (20 mL) was stirred at 0 °C
for 3.5 h. Then different substituted amines (2.2 mmol) and DIPEA (4 mmol) were added and the reaction was
stirred at r.t. for another 1.5 h. The organic layer was washed with water and brine and dried over Na2SO4.
Removal of the solvent gave a residue that was purified by column chromatography (silica gel, CH2Cl2-MeOH
100 : 1 as an eluent) to furnish 10a-n as white solids.
4.1.4. General procedure for the synthesis of target compounds (11a-n,12a-g).
To a solution of 10a-n (1.0 mmol) in ethanol (4 mL) was added the corresponding aldehydes (1.1 mmol) and

a catalytic amount of piperidine. The reaction mixture was stirred at reflux for 4.0 h (reaction monitored by TLC).
After cooling, the precipitate was filtered, washed with cold ethanol, and dried in air to provide the title
compounds 11a-n, 12a-g as brown yellow/brown/orange solids in moderate to excellent yields.
4.1.4.1.(3Z)-N-[(1R)-2-hydroxy-1-phenylethyl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindoline-5-carboxamide (11a).
Yellow solid, yield 83%; m.p.:251.5-253.0 °C; 1H NMR (400 MHz, DMSO) δ 13.28 (s, 1H), 11.15 (s, 1H), 8.53 (d,
J = 8.0 Hz, 1H), 8.24 (s, 1H), 7.86-7.68 (m, 2H), 7.41 (d, J = 7.6 Hz, 3H), 7.33 (t, J = 7.5 Hz, 2H), 7.24 (d, J = 7.2
Hz, 1H), 7.02-6.82 (m, 2H), 6.39 (s, 1H), 5.10 (d, J = 5.9 Hz, 1H), 4.96 (t, J = 5.8 Hz, 1H), 3.79-3.60 (m, 2H). 13
C
NMR (151 MHz, DMSO) δ 170.02, 166.59, 142.06, 141.62, 129.96, 128.55, 128.20, 127.48, 127.45, 127.34,
127.22, 126.77, 125.30, 121.42, 118.18, 116.59, 112.20, 109.41, 65.08, 56.45. HRMS calcd for C22H19N3O3,
[M+H]+, 374.1499; found 374.1498.
4.1.4.2. (3Z)-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindoline-5-carboxamide (11b).
Yellow solid, yield 78%; m.p.:249.3-250.9 °C; 1H NMR (400 MHz, DMSO) δ 13.28 (s, 1H), 11.14 (s, 1H), 8.54 (d,
J = 7.5 Hz, 1H), 8.24 (s, 1H), 7.87-7.68 (m, 2H), 7.41 (d, J = 7.3 Hz, 3H), 7.33 (t, J = 7.1 Hz, 2H), 7.24 (d, J = 6.9
Hz, 1H), 6.99-6.88 (m, 2H), 6.39 (s, 1H), 5.10 (d, J = 5.7 Hz, 1H), 4.96 (s, 1H), 3.81-3.60 (m, 2H). 13C NMR (151
MHz, DMSO) δ 169.58, 166.17, 141.59, 141.18, 129.52, 128.13, 127.81, 127.05, 126.98, 126.88, 126.81, 126.36,
124.86, 121.02, 117.68, 116.12, 111.78, 108.97, 99.56, 64.66, 55.94. HRMS calcd for C22H19N3O3, [M+H]+,
374.1499; found 374.1503.
4.1.4.3.(3Z)-N-[(1S)-1-(4-chlorophenyl)-2-hydroxyethyl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxami
1
de (11c). Yellow solid, yield 69%; m.p.:235.9-240.2 °C; H NMR (600 MHz, DMSO) δ 13.45 (s, 1H), 11.05 (s,
1H), 8.56 (dd, J = 19.8, 8.0 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H), 7.74 (dd, J = 17.0, 9.2 Hz, 2H), 7.38 (dq, J = 15.3,
8.1 Hz, 5H), 6.98-6.84 (m, 2H), 6.37 (s, 1H), 5.41-4.82 (m, 2H), 3.75-3.65 (m, 2H). 13C NMR (151 MHz, DMSO)
δ 170.05, 166.23, 140.72, 131.33, 129.58, 129.38, 128.98, 128.10, 128.04, 127.31, 127.04, 126.89, 126.76, 126.66,
126.13, 125.07, 120.70, 117.62, 111.67, 109.10, 64.34, 55.37. HRMS calcd for C22H18ClN3O3, [M+H]+, 408.1109;
found 408.1112.
4.1.4.4.(3Z)-N-[(1S)-1-(2-chlorophenyl)-2-hydroxyethyl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxami
1
de (11d). Yellow solid, yield 73%; m.p.:243.1-247.8 °C; H NMR (400 MHz, DMSO) δ 13.28 (s, 1H), 11.15 (s,
1H), 8.62 (d, J = 7.5 Hz, 1H), 8.26 (s, 1H), 7.83-7.73 (m, 2H), 7.58 (d, J = 7.5 Hz, 1H), 7.46-7.36 (m, 2H), 7.30
(ddd, J = 15.1, 11.8, 7.5 Hz, 2H), 6.99-6.89 (m, 2H), 6.39 (s, 1H), 5.48 (dd, J = 12.7, 7.5 Hz, 1H), 5.09 (t, J = 5.8
Hz, 1H), 3.83-3.52 (m, 2H). 13C NMR (151 MHz, DMSO) δ 170.01, 166.69, 141.70, 139.15, 132.66, 129.93,
129.62, 129.05, 128.79, 128.01, 127.68, 127.42, 127.38, 126.84, 125.30, 121.48, 118.14, 116.53, 112.24, 109.45,
63.38, 53.82. HRMS calcd for C22H18ClN3O3, [M+H]+, 408.1109; found 408.1112.
4.1.4.5.(3Z)-N-[(1S)-1-(4-fluorophenyl)-2-hydroxyethyl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxami
1
de (11e). Brown solid, yield 70%; m.p.:213.3-217.2 °C; H NMR (400 MHz, DMSO) δ 13.27 (s, 1H), 11.15 (s,
1H), 8.53 (d, J = 7.9 Hz, 1H), 8.22 (s, 1H), 7.75 (dd, J = 18.5, 11.7 Hz, 2H), 7.53-7.35 (m, 3H), 7.15 (t, J = 8.9 Hz,
2H), 6.98-6.89 (m, 2H), 6.39 (d, J = 3.4 Hz, 1H), 5.09 (dd, J = 13.6, 7.5 Hz, 1H), 5.00-4.91 (m, 1H), 3.76-3.59 (m,
2H). 13C NMR (151 MHz, DMSO) δ 170.53, 170.01, 166.81, 166.60, 162.44, 160.84, 144.37, 141.64, 138.32,
138.30, 138.22, 138.20, 130.13, 129.94, 129.43, 129.38, 128.15, 127.84, 127.43, 127.30, 126.82, 125.30, 122.10,
121.48, 118.75, 118.11, 116.52, 116.04, 115.33, 115.19, 112.22, 109.41, 64.93, 55.62, HRMS calcd for
C22H17FN3O3, [M+H]+, 392.1405; found 392.1409.
4.1.4.6.(3Z)-N-[(2S)-1-hydroxy-3-phenylpropan-2-yl]-3-(1H-pyrrol-2-ylmethylidene)-
2-oxoindoline-5-carboxamide (11f). Brown solid, yield 68%; m.p.:211.1-215.9 °C; 1H NMR (400 MHz, DMSO) δ
13.26 (s, 1H), 11.12 (s, 1H), 8.12 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.76 (s, 1H), 7.65 (dd, J = 8.1, 1.5 Hz, 1H),
7.40 (s, 1H), 7.35-7.21 (m, 4H), 7.16 (dd, J = 10.5, 4.2 Hz, 1H), 6.91 (d, J = 8.0 Hz, 2H), 6.38 (dd, J = 5.9, 2.3 Hz,
1H), 4.27 (t, J = 5.2 Hz, 1H), 4.23-4.13 (m, 1H), 3.47 (dtd, J = 16.7, 10.9, 5.7 Hz, 2H), 2.97 (dd, J = 13.7, 5.4 Hz,

1H), 2.82 (dd, J = 13.7, 8.8 Hz, 1H). 13C NMR (151 MHz, DMSO) δ 170.54, 169.99, 166.75, 166.40, 144.23,
141.49, 140.07, 139.99, 129.94, 129.54, 128.59, 128.54, 128.31, 128.19, 127.86, 127.31, 127.05, 126.78, 126.34,
125.25, 121.89, 121.44, 118.71, 118.02, 116.56, 116.06, 112.28, 112.18, 109.31, 109.12, 63.52, 63.45, 63.36,
53.81, 53.60, 41.95. HRMS calcd for C23H21N3O3, [M+H]+, 388.1656; found 388.1660.
4.1.4.7.(3Z)-N-[(2S)-1-hydroxy-3-methylbutan-2-yl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxamide
(11g). Yellow solid, yield 75%; m.p.:175.3-175.4 °C；1H NMR (600 MHz, DMSO) δ 13.28 (s, 1H), 11.13 (s, 1H),
8.19 (d, J =1.4 Hz, 1H), 7.80 (t, J = 4.4 Hz, 2H), 7.72 (dt, J = 10.0, 5.0 Hz, 1H), 7.39 (d, J = 1.0 Hz, 1H), 6.97-6.88
(m, 2H), 6.39 (dq, J = 3.7, 2.7 Hz, 1H), 4.60 (t, J = 5.4 Hz, 1H), 3.83 (dq, J = 8.8, 5.5 Hz, 1H), 3.52 (dd, J = 12.3,
7.2 Hz, 2H), 2.00-1.87 (m, 1H), 0.92 (dd, J = 14.4, 6.8 Hz, 6H). 13C NMR (151 MHz, DMSO) δ 170.02, 166.78,
141.44, 129.96, 128.61, 127.36, 127.28, 126.70, 125.21, 121.35, 118.08, 116.67, 112.17, 109.32, 61.87, 56.99,
29.08, 20.20, 19.32. HRMS calcd for C19H21N3O3, [M+H]+, 340.1656; found 340.1659.
4.1.4.8.(3Z)-N-[(2S)-1-hydroxy-3,3-dimethylbutan-2-yl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxamid
e (11h). Brown solid, yield 80%; m.p.:215.4-220.6 °C; 1H NMR (600 MHz, DMSO) δ 13.29 (s, 1H), 11.12 (s, 1H),
8.19 (s, 1H), 7.88-7.79 (m, 1H), 7.71 (dd, J = 30.4, 8.5 Hz, 2H), 7.40 (s, 1H), 6.94 (d, J = 8.3 Hz, 2H), 6.39 (s, 1H),
4.46 (t, J = 5.5 Hz, 1H), 4.02-3.84 (m, 1H), 3.73-3.63 (m, 1H), 3.57-3.47 (m, 1H), 0.94 (s, 9H). 13C NMR (151
MHz, DMSO) δ 170.03, 167.30, 141.38, 129.96, 128.89, 127.37, 126.68, 125.16, 121.35, 118.15, 116.69, 112.17,
109.27, 60.90, 59.77, 34.61, 27.55. HRMS calcd for C20H23N3O3, [M+H]+, 354.1812; found 354.1813.
4.1.4.9.(3Z)-N-(2-hydroxyethyl)-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxamide (11i). Yellow solid,
yield 71%; m.p.:235.3-236.0 °C; 1H NMR (400 MHz, DMSO) δ 13.25 (s, 1H), 11.13 (s, 1H), 8.28 (t, J = 5.4 Hz,
1H), 8.17 (d, J = 1.1 Hz, 1H), 7.79 (s, 1H), 7.70 (dd, J = 8.2, 1.4 Hz, 1H), 7.39 (s, 1H), 6.91 (dd, J = 9.6, 4.9 Hz,
2H), 6.38 (dd, J = 5.8, 2.3 Hz, 1H), 4.74 (t, J = 5.6 Hz, 1H), 3.52 (q, J = 6.0 Hz, 2H), 3.38-3.33 (m, 2H). 13C NMR
(151 MHz, DMSO) δ 170.00, 166.85, 141.52, 129.97, 128.21, 127.38, 126.96, 126.78, 125.34, 121.45, 118.04,
116.54, 112.17, 109.38, 60.38, 42.67. HRMS calcd for C16H15N3O3, [M+H]+, 298.1186; found 298.1191.
4.1.4.10.(3Z)-N-(2-hydroxypropyl)-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxamide (11j). Yellow solid,
1
yield 69%; m.p.:203.7-206.9 °C; H NMR (600 MHz, DMSO) δ 13.27 (s, 1H), 11.18 (s, 1H), 8.48-8.33 (m, 1H),
8.19 (s, 1H), 7.80 (s, 1H), 7.74-7.64 (m, 2H), 7.57 (s, 1H), 7.39 (s, 1H), 6.98-6.86 (m, 2H), 6.53-6.30 (m, 1H), 4.55
(s, 1H), 3.51-3.42 (m, 3H), 3.32-3.28 (m, 2H). 13C NMR (151 MHz, DMSO) δ 170.05, 166.23, 140.72, 131.33,
129.58, 129.38, 128.98, 128.10, 128.04, 127.31, 127.04, 126.89, 126.76, 126.66, 126.13, 125.07, 120.70, 117.62,
111.67, 109.10, 64.34, 55.37. HRMS calcd for C17H17N3O3, [M+H]+, 312.1343; found 312.1347.
4.1.4.11. (3Z)-N-[(1R)-3-hydroxy-1-phenylpropyl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindole-5-carboxamide
(11k). Yellow solid, yield 83%; m.p.:258.9-261.8 °C; 1H NMR (600 MHz, DMSO) δ 13.27 (s, 1H), 11.14 (s, 1H),
8.81-8.50 (m, 1H), 8.18 (d, J = 1.2 Hz, 1H), 7.79 (s, 1H), 7.73 (ddd, J = 23.8, 8.1, 1.5 Hz, 1H), 7.40 (d, J = 7.2 Hz,
3H), 7.33 (t, J = 7.7 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 6.96-6.89 (m, 2H), 6.50-6.33 (m, 1H), 5.18 (d, J = 6.0 Hz,
1H), 4.57 (t, J = 4.9 Hz, 1H), 3.54-3.37 (m, 2H), 1.99 (ddd, J = 20.0, 10.7, 5.8 Hz, 2H). 13C NMR (151 MHz,
DMSO) δ 169.56, 165.86, 144.35, 141.14, 129.50, 128.22, 127.82, 127.01, 126.73, 126.56, 126.35, 124.88, 121.05,
117.60, 116.06, 111.75, 108.95, 57.97, 50.23. HRMS calcd for C23H21N3O3, [M+H]+, 388.1656; found 388.1659.
4.1.4.12.
(3Z)-6-fluoro-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(1H-pyrrol-2-ylmethylidene)-
1
2-oxoindole-5-carboxamide (11l). Yellow solid, yield 67%; m.p.:164.7-168.6 °C; H NMR (400 MHz, DMSO) δ
13.11 (s, 1H), 11.23 (s, 1H), 8.42 (dd, J = 7.7, 2.9 Hz, 1H), 7.94 (d, J = 7.0 Hz, 1H), 7.87 (s, 1H), 7.40 (d, J = 7.6
Hz, 3H), 7.33 (t, J = 7.5 Hz, 2H), 7.25 (d, J = 7.1 Hz, 1H), 6.88 (s, 1H), 6.76 (dd, J = 10.7, 5.3 Hz, 1H), 6.39 (t, J =
12.6 Hz, 1H), 5.06-4.94 (m, 2H), 3.67 (t, J = 5.5 Hz, 2H). 13C NMR (151 MHz, DMSO) δ 169.53, 169.01, 162.99,
162.75, 159.33, 158.41, 157.69, 144.40, 144.31, 140.93, 140.85, 140.69, 140.60, 128.89, 127.55, 127.50, 126.72,
126.61, 126.35, 126.26, 126.19, 125.73, 125.14, 124.30, 122.79, 120.72, 120.56, 119.32, 119.30, 117.53, 116.66,
116.15, 116.05, 115.14, 115.05, 114.72, 114.30, 111.17, 111.07, 97.16, 96.97, 64.02, 55.18, 55.08. HRMS calcd for

C22H18ClN3O3, [M+H]+, 408.1109; found 408.1112.
4.1.4.13.(3Z)-6-chloro-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(1H-pyrrol-2-ylmethylidene)-
2-oxoindole-5-carboxamide (11m). Yellow solid, yield 71%; m.p.:281.0-283.8 °C; 1H NMR (400 MHz, DMSO) δ
13.20 (s, 1H), 11.13 (s, 1H), 8.69 (d, J = 8.1 Hz, 1H), 7.92 (s, 1H), 7.78 (s, 1H), 7.42 (d, J = 7.5 Hz, 3H), 7.34 (t, J
= 7.5 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H), 6.91 (d, J = 7.4 Hz, 2H), 6.42-6.36 (m, 1H), 4.94 (dt, J = 11.6, 6.3 Hz, 2H),
3.70-3.59 (m, 2H). 13C NMR (151 MHz, DMSO) δ 169.34, 166.28, 141.06, 140.08, 129.87, 129.58, 128.11,
128.02, 128.00, 127.19, 126.85, 126.81, 124.01, 121.56, 119.04, 114.99, 111.92, 110.16, 64.80, 55.88. HRMS
calcd for C22H18ClN3O3, [M+H]+, 408.1109; found 408.1114.
4.1.4.14. (3Z)-6-bromo-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(1H-pyrrol-2-ylmethylidene)-
2-oxoindole-5-carboxamide (11n). Brown solid, yield 59%; m.p.:276.7-280.8 °C; 1H NMR (600 MHz, DMSO) δ
13.21 (s, 1H), 11.11 (s, 1H), 8.69 (d, J = 8.2 Hz, 1H), 7.93 (s, 1H), 7.75 (s, 1H), 7.43 (dd, J = 8.1, 5.0 Hz, 3H),
7.35 (t, J = 7.7 Hz, 2H), 7.26 (t, J = 7.3 Hz, 1H), 7.07 (s, 1H), 6.91 (d, J = 1.6 Hz, 1H), 6.39 (dt, J = 3.7, 2.3 Hz,
1H), 5.02 (dd, J = 14.2, 7.2 Hz, 1H), 4.87 (t, J = 5.9 Hz, 1H), 3.71-3.61 (m, 2H). 13C NMR (151 MHz, DMSO) δ
169.56, 165.86, 144.35, 141.14, 129.50, 128.22, 127.82, 127.01, 126.73, 126.56, 126.35, 124.88, 121.05, 117.60,
116.06, 111.75, 108.95, 57.97, 50.23. HRMS calcd for C22H18BrN3O3, [M+H]+, 452.0604; found 452.0603.
4.1.4.15.(3E)-3-(furan-2-ylmethylidene)-N-[(1S)-2-hydroxy-1-phenylethyl]-2-oxoindole-5-carboxamide
(12a).
Yellow solid, yield 87%; m.p.:281.7-284.2 °C; 1H NMR (400 MHz, DMSO) δ 10.83 (s, 1H), 8.87 (s, 1H), 8.64 (d,
J = 8.0 Hz, 1H), 8.08 (s, 1H), 7.84 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 9.4 Hz, 3H), 7.33 (dd, J = 9.2, 5.5 Hz, 3H),
7.24 (t, J = 7.2 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.84 (dd, J = 3.3, 1.7 Hz, 1H), 5.07 (dd, J = 13.5, 7.8 Hz, 1H),
4.93 (t, J = 5.8 Hz, 1H), 3.75-3.63 (m, 2H). 13C NMR (151 MHz, DMSO) δ 170.04, 166.84, 151.04, 147.84,
145.25, 142.19, 129.62, 128.76, 128.58, 127.47, 127.26, 124.79, 122.01, 121.97, 121.44, 120.60, 114.26, 109.37,
65.16, 56.48. HRMS calcd for C22H18N2O4, [M+H]+, 375.1339; found 375.1344.
4.1.4.16. (3Z)-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(thiophen-2-ylmethylidene)-2-oxoindole-5-carboxamide (12b).
Yellow solid, yield 85%; m.p.:255.2-258.4 °C; 1H NMR (400 MHz, DMSO) δ 10.88 (s, 1H), 8.59 (dd, J = 47.8,
8.0 Hz, 1H), 8.27 (s, 1H), 8.18 (s, 1H), 8.02 (dd, J = 18.0, 4.0 Hz, 1H), 7.96-7.76 (m, 2H), 7.46-7.18 (m, 6H), 6.94
(dd, J = 20.9, 8.1 Hz, 1H), 5.19-4.83 (m, 2H), 3.80-3.58 (m, 2H). 13C NMR (151 MHz, DMSO) δ 170.05, 166.23,
140.72, 131.33, 129.58, 129.38, 128.98, 128.10, 128.04, 127.31, 127.04, 126.89, 126.76, 126.66, 126.13, 125.07,
120.70, 117.62, 111.67, 109.10, 64.34, 55.37. HRMS calcd for C22H18N2O3S, [M+H]+, 391.1111; found 391.1110.
4.1.4.17.(3E)-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(pyridin-2-ylmethylidene)-2-oxoindole-5-carboxamide
(12c).
1
Brown solid, yield 90%; m.p.:262.4-265.6℃; H NMR (400 MHz, DMSO) δ 10.89 (s, 1H), 9.56 (d, J = 1.2 Hz,
1H), 8.85 (d, J = 4.2 Hz, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.08-7.79 (m, 3H), 7.64 (s, 1H), 7.51 (dd, J = 6.8, 5.3 Hz,
1H), 7.42 (d, J = 7.4 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 5.20-4.86
(m, 2H), 3.90-3.52 (m, 2H). 13C NMR (151 MHz, DMSO) δ 169.62, 166.48, 153.03, 149.72, 145.86, 141.74,
137.41, 134.66, 130.45, 128.82, 128.65, 128.37, 128.15, 127.75, 127.03, 126.83, 124.48, 121.16, 109.01, 64.74,
55.86. HRMS calcd for C23H19N3O3, [M+H]+, 386.1499; found 386.1506.
4.1.4.18.(3Z)-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(3H-imidazol-4-ylmethylidene)-2-oxoindole-5-carboxamide
(12d). Yellow solid, yield 70%; m.p.:199.8-204.1 °C; 1H NMR (600 MHz, DMSO) δ 13.59 (s, 1H), 11.78 (s, 1H),
8.59 (d, 1H), 8.49-8.21 (m, 1H), 7.95 (dd, 2H), 7.76 (dd, 1H), 7.41 (d, 2H), 7.33 (dd, 2H), 7.23 (t, 1H), 6.90 (dd,
1H), 5.07 (ddd,2H), 3.70 (dt, 2H). 13C NMR (151 MHz, DMSO) δ 170.32, 166.91, 165.95, 143.80, 142.13, 141.88,
141.56, 138.65, 129.40, 128.15, 128.13, 128.11, 128.02, 127.71, 127.03, 127.01, 126.81, 126.75, 126.50, 124.21,
122.48, 120.55, 115.23, 109.15, 108.00, 69.80, 64.79, 64.65, 55.96, 55.78. HRMS calcd for C23H19N3O3, [M+H]+,
386.1499; found 386.1506. HRMS calcd for C21H18N4O3, [M+H]+, 375.1452; found 375.1470.
4.1.4.19.(3Z)-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(1H-imidazol-2-ylmethylidene)-2-oxoindole-5-carboxamide
(12e). Yellow solid, yield 76%; m.p.:208.3-212.8 °C; 1H NMR (600 MHz, DMSO) δ 13.98 (s, 1H), 10.87 (s, 1H),

9.87 (d, J = 1.2 Hz, 1H), 8.92-8.38 (m, 1H), 7.97-7.78 (m, 1H), 7.59 (s, 1H), 7.49-7.36 (m, 4H), 7.32 (dd, J = 12.8,
7.3 Hz, 2H), 7.23 (dd, J = 11.7, 7.2 Hz, 1H), 6.96 (dd, J = 56.3, 8.1 Hz, 1H), 5.30-4.92 (m, 2H), 3.92-3.54 (m, 2H).
13C NMR (151 MHz, DMSO) δ 170.26, 169.71, 167.13, 166.06, 145.16, 143.95, 143.32, 142.74, 142.31, 142.04,
141.03, 133.32, 129.66, 129.33, 129.00, 128.86, 128.63, 128.60, 128.54, 128.52, 128.42, 127.62, 127.45, 127.17,
125.35, 124.62, 124.09, 123.50, 122.10, 121.76, 121.61, 119.55, 118.72, 118.46, 110.08, 108.78, 71.69, 65.16,
65.09, 56.64, 56.46, 56.37, 48.40, 48.18. HRMS calcd for C21H18N4O3, [M+H]+, 375.1452; found 375.1457.
4.1.4.20.(3Z)-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(2H-pyrazol-3-ylmethylidene)-2-oxoindole-5-carboxamide (12f).
Brown solid, yield 77%; m.p.:251.9-255.5 °C; 1H NMR (600 MHz, DMSO) δ 13.67 (s, 1H), 10.81 (s, 1H), 8.53 (m,
2H), 7.97-7.73 (m, 2H), 7.58 (d, 1H), 7.32 (tt, J = 13.0, 7.6 Hz, 5H), 6.90 (m, 2H), 5.05 (m, 2H), 3.81-3.53 (m, 2H).
13C NMR (151 MHz, DMSO) δ 170.27, 167.06, 166.25, 145.19, 142.29, 141.96, 129.51, 129.36, 128.83, 128.59,
128.56, 127.50, 127.46, 127.27, 127.20, 124.85, 122.17, 108.78, 65.12, 65.10, 56.56, 56.41. HRMS calcd for
C21H18N4O3, [M+H]+, 375.1452; found 375.1477.
4.1.4.21.(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-N-[(1S)-2-hydroxy-1-phenylethyl]-2-oxoindoline-5-ca
1
rboxamide (12g). Orange solid, yield 85%; m.p.:274.3-275.5 °C; H NMR (400 MHz, DMSO) δ 13.34 (s, 1H),
11.07 (s, 1H), 8.74 (d, J = 7.4 Hz, 1H), 8.52 (s, 1H), 7.80-7.67 (m, 2H), 7.40 (d, J = 7.4 Hz, 2H), 7.31 (t, J = 7.5
Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.04 (s, 1H), 5.17 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.7,
7.3 Hz, 1H), 3.84-3.63 (m, 2H), 2.35 (d, J = 9.2 Hz, 6H). 13C NMR (151 MHz, DMSO) δ 170.22, 166.69, 142.29,
140.69, 136.79, 133.04, 128.44, 127.66, 127.49, 127.32, 127.05, 126.27, 126.06, 124.86, 117.98, 113.26, 112.45,
109.06, 64.96, 56.85, 14.05, 12.05. HRMS calcd for C24H23N3O3, [M+H]+, 402.1812; found 402.1816.
4.1.5.(3Z)-6-cyano-N-[(1S)-2-hydroxy-1-phenylethyl]-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindoline-5-carboxamid
e (11o)
A mixture of 11n (1.0 mmol), zinc cyanide (2.0 mmol), Pd(P(Ph3)4) (0.1 mmol) in DMF was heated at 185 °C
for 10 h. The reaction mixture was cooled to room temperature, diluted with ethyl acetate and filtered. The filtrate
was concentrated in vacuo, and the residue purified by column chromatography (silica gel, CH2Cl2-MeOH 100:1
as an eluent). The product was obtained as an orange solid, yield 54%; m.p.:205.7-207.6 °C. 1H NMR (400 MHz,
DMSO) δ 13.30 (s, 1H), 10.08 (s, 1H), 8.15 (s, 1H), 8.09 (s, 1H), 7.68 (s, 1H), 7.47 (s, 1H), 7.40 (d, J = 7.4 Hz,
2H), 7.31 (t, J = 7.5 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 6.96 (s, 1H), 6.42 (s, 1H), 5.56 (dd, J = 8.5, 5.8 Hz, 1H),
5.14 (t, J = 5.7 Hz, 1H), 4.46 (ddd, J = 22.6, 12.8, 7.5 Hz, 2H), 4.08 (dt, J = 11.1, 5.5 Hz, 1H). 13C NMR (151
MHz, DMSO) δ 169.86, 168.77, 160.01, 143.08, 139.45, 130.45, 130.25, 129.99, 129.75, 128.70, 128.16, 127.97,
127.52, 124.31, 123.02, 115.42, 113.11, 112.73, 104.02, 72.98, 63.51, 63.25, 61.19, 56.95. HRMS calcd for
C23H18N4O3 [M+H]+, 399.1452; found 399.1455.
4.1.6. General procedure for the synthesis of target compounds (11p-q)
A solution of 11n (1.0 mmol) in toluene : ethanol (volume 4:1,10 mL) was degassed by N2 bubbling for 5 min.
The corresponding boronic acid/ester (1.5 mmol) was added and the mixture was degassed for another 5 min.
Pd(P(Ph3)4) (0.1 mol) and aqueous potassium carbonate (2 M, 3.0 eq.) were added sequentially and the mixture
was further degassed for 5 min and then heated at 110 °C for 16 h. The mixture was quenched with water and
extracted with ethyl acetate (3 × 50 mL). The combined organic layer was washed with water, brine and dried over
sodium sulphate. The solvent was distilled off under reduced pressure, and the crude residue was purified by
column chromatography to yield the product.
4.1.6.1.(3Z)-N-[(1S)-2-hydroxy-1-phenylethyl]-6-(2-methylpyrazol-3-yl)-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindol
e-5-carboxamide (11p). Yellow solid, yield 59%; m.p.:136.5-140.2 °C; 1H NMR (400 MHz, DMSO) δ 13.31 (s,
1H), 11.25 (s, 1H), 8.59 (m, 1H), 7.93 (m, 2H), 7.46-7.18 (m, 7H), 6.95 (s, 1H), 6.80 (s, 1H), 6.40 (s, 1H), 6.18 (d,
J = 1.6 Hz, 1H), 5.02-4.79 (m, 2H), 3.66-3.54 (m, 1H), 3.52 (d, J = 11.5 Hz, 3H), 3.39 (s, 1H). 13C NMR (151
MHz, DMSO) δ 169.43, 167.75, 141.81, 141.06, 139.52, 137.43, 130.80, 129.67, 128.07, 128.05, 128.01, 127.25,

127.11, 127.08, 126.75, 126.71, 125.33, 121.50, 118.39, 115.56, 111.93, 111.57, 106.39, 72.57, 64.74, 64.57, 63.05,
62.83, 55.90, 36.68. HRMS calcd for C26H23N5O3 [M+H]+, 454.1874; found 454.1877.
4.1.6.2.(3Z)-N-[(1S)-2-hydroxy-1-phenylethyl]-6-(1-methylpyrazol-4-yl)-3-(1H-pyrrol-2-ylmethylidene)-2-oxoindol
e-5-carboxamide (11q). Yellow solid, yield 47%; m.p.:285.6-288.4 °C; 1H NMR (400 MHz, DMSO) δ 13.25 (s,
1H), 11.02 (s, 1H), 8.72 (d, J = 8.3 Hz, 1H), 7.82 (s, 1H), 7.68 (s, 1H), 7.58 (d, J = 14.6 Hz, 2H), 7.32 (dt, J = 8.3,
5.0 Hz, 6H), 6.90 (d, J = 18.4 Hz, 2H), 6.36 (d, J = 3.1 Hz, 1H), 5.11-4.89 (m, 2H), 3.86-3.42 (m, 5H). 13C NMR
(151 MHz, DMSO) δ 170.10, 141.46, 139.96, 137.82, 130.28, 130.13, 129.60, 129.18, 128.62, 127.67, 127.34,
127.03, 126.41, 123.26, 121.13, 120.71, 118.39, 116.60, 112.04, 108.89, 65.23, 56.16, 38.91. HRMS calcd for
C26H23N5O3 [M+H]+, 454.1874; found 454.1870.
4.2. Pharmacological assay
4.2.1. PAK4 HTRF Assays.
HTRF assays were performed in white 384 Well Small Volume plates with a total working volume of 20 μL.
Compounds were dispensed, with 4 μL per well, from a concentration stock of 20 mM in 100% DMSO and with
serial kinase reaction buffer dilutions. The IC50 measurements were performed in replicates. All HTRF reagents
were purchased from CisBio Bioassays and reconstituted according to the supplier protocols. For each assay 6 μL
of mix 1 (kinase + ATP + substrate S2) is added in the assay wells, containing previously dispensed inhibitors. The
assay wells were incubated at R.T. for 60 min, and terminated by adding 10 μL of mix 2 (Sa-XL665 +
STK-Antibody-Cryptate). After a final incubation (60 min at room temperature), HTRF signal was obtained by
reading the plate in Infinite® F500 microplate reader (Tecan, Switzerland). The fluorescence is measured at 620
nM (Cryptate) and 665 nM (XL665). A ratio is calculated (665/620) for each well. For IC50 measurements, values
were normalized and fitted with Prism (GraphPad software) using the following equation: Y = 100 / (1 + ((X /
IC50)^Hill slope)).
4.2.2. Cell Proliferation Assay.
Cells were seeded in 96-well cell culture plates. On the day of seeding, the cells were exposed to various
concentrations of compounds and further cultured for 72 h at 37 °C. Cell proliferation was then determined using
Cell Counts Kit-8 (CCK8) assay. The IC50 values were calculated by a concentration-response curve fitting using
the four-parameter method.
4.2.3. Cell-cycle analysis by flow cytometry.
A549 cells (5×105 cells) were incubated with the indicated concentrations of 12g for 72 h. After incubation,
cells were collected, washed with PBS, and suspended in a staining buffer (10 μg/mL propidium iodide, 0.5%
Tween-20, 0.1% RNase in PBS). The cells were analysed using a FACSVantage flow cytometer with the Cell
Quest acquisition and analysis software program (Becton Dickinson and Co., San Jose, CA).
4.2.4. Cell Migration and Invasion Assay.
Cell migration assays were evaluated in Transwell chambers (Corning Costar). Cell invasion assays were
evaluated in Matrigel invasion chambers (Corning Costar). Then, 5 × 105 cells in 100 μL serum-free DMEM
supplemented with 0.1% bovine serum were placed in the upper part of each chamber and the lower compartments
were filled with 600 μL DMEM containing 10% serum. After incubation for 24 h at 37 °C, the cells were fixed in
100% methanol and stained with 0.25% crystal violet; the cells that had not migrated or non-invaded from the top
surface of the filters were removed with cotton. Invaded/migrated cells were quantitated by counting cells in six
randomly selected fields on each filter under a microscope at 200 × magnification and graphed as the mean of
three independent experiments.
4.2.5. Western Blot Analysis.
A549 cells were treated with the indicated dose of 12g for 2 h and then lysed in 1x sodium dodecyl sulphate
(SDS) sample buffer. The cell lysates were subsequently resolved by 10% SDS-PAGE and transferred to

nitrocellulose membranes. The membranes were probed with the appropriate primary antibodies [phospho-PAK4,
PAK4, phospho-LIMK, LIMK, phospho-Cofilin, Cofilin, GAPDH (all from KeyGEN BioTECH, Nanjing,China)],
and then with horseradish peroxidase-conjugated antirabbit or antimouse IgG. The immunoreactive proteins were
detected using an enhanced chemiluminescence detection reagent (Thermo Fisher Scientific, Rockford, IL, USA).
5.3. Molecular docking study.
X-ray protein structure of PAK4 (PDB ID : 2X4Z) obtained from the Protein Data Bank was prepared with
AutoDockTools (ADT). Both the native ligand and the indicated compounds were built using 2D Sketcher and
were prepared with LigPrep with OPLS2005 force field, generating possible states at pH 7.0 ± 2.0 using Epik 2.0
module. The size of box was set to 60 × 60 × 60 units in number of grid points and grid spacing = 0.375 Å centred
in native ligand using AutoGrid4. Docking was performed using the Lamarckian genetic algorithm in AutoDock4.
Each docking experiment was performed 10 times, yielding 10 docked conformations and the most favourable
pose of 12g was displayed.
Acknowledgments.
We gratefully acknowledge the financial support from the National Natural Science Foundation of China
(Grant 81230077) and Program for Innovative Research Team of the Ministry of Education, and Program for
Liaoning Innovative Research Team in University.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version.
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Highlights




Twenty four indolin-2-one derivatives were synthesized.
All the compounds were screened for in vitro PAK4 inhibition assay.
Three of the compounds (11b, 12d, 12g) displayed promising enzymatic activity.
Compound 12g : Inducing apoptosis, G2/M phase arrest, Migration and Invasion inhibition
by regulating PAK4-LIMK1-cofilin signalling pathway.

Docking and ADME profiling of 12g are reported.