Design, synthesis, structure-activity relationships study and X-ray crystallography of 3-substituted-indolin-2-one-5-carboxamide derivatives as PAK4 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.05.051

We have previously described the identification of indolin-2-one-5-carboxamides as potent PAK4 inhibitors. This study expands the structure-activity relationships on our original series by presenting several modifications in the lead compounds, 2 and 3. A series of novel derivatives was designed, synthesized, and evaluated in biochemical and cellular assay. Most of this series displayed nanomolar biochemical activity and potent antiproliferative activity against A549 and HCT116 cells. The representative compound 10a exhibited excellent enzyme inhibition (PAK4 IC₅₀ = 25 nM) and cellular potency (A549 IC₅₀ = 0.58 μM, HCT116 IC₅₀ = 0.095 μM). An X-ray structure of compound 10a bound to PAK4 was obtained. Crystallographic analysis confirmed predictions from molecular modeling and helped refine SAR results. In addition, Compound 10a displayed focused multi-targeted kinase inhibition, good calculated drug-likeness properties. Further profiling of compound 10a revealed it showed weak inhibitory activity against various isoforms of human cytochrome P450.

Full text of DOI:10.1016/j.ejmech.2018.05.051

Accepted Manuscript
Design, synthesis, structure-activity relationships study and X-ray crystallography of
3-substituted-indolin-2-one-5-carboxamide derivatives as PAK4 inhibitors
Jing Guo, Fan Zhao, Wenbo Yin, Mingyue Zhu, Chenzhou Hao, Yu Pang, Tianxiao
Wu, Jian Wang, Dongmei Zhao, Haitao Li, Maosheng Cheng
PII:
S0223-5234(18)30475-6
10.1016/j.ejmech.2018.05.051
EJMECH 10462
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 March 2018
Revised Date: 26 May 2018
Accepted Date: 29 May 2018
Please cite this article as: J. Guo, F. Zhao, W. Yin, M. Zhu, C. Hao, Y. Pang, T. Wu, J. Wang, D. Zhao,
H. Li, M. Cheng, Design, synthesis, structure-activity relationships study and X-ray crystallography of 3-
substituted-indolin-2-one-5-carboxamide derivatives as PAK4 inhibitors, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.05.051.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, structure-activity relationships study and X-ray
crystallography of 3-substituted-indolin-2-one-5-carboxamide derivatives as
PAK4 inhibitors
Jing Guo‡a, Fan Zhao‡b, Wenbo Yina, Mingyue Zhua, Chenzhou Haoa, Yu Panga, Tianxiao Wua, Jian Wanga,
Dongmei Zhao*, a, Haitao Li*, b, 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 Tsinghua-Peking Joint Center for Life Sciences, Beijing Advanced Innovation Center for Structural Biology,
Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China. E-mail:
lht@tsinghua.edu.cn; Tel: +86-10- 62771392.
‡ These authors contributed equally to this work.
Keywords: p21-activated kinase 4; indolin-2-one; X-ray crystallography
Abstract
We have previously described the identification of indolin-2-one-5-carboxamides as potent PAK4 inhibitors.
This study expands the structure-activity relationships on our original series by presenting several modifications in
the lead compounds, 2 and 3. A series of novel derivatives was designed, synthesized, and evaluated in
biochemical and cellular assay. Most of this series displayed nanomolar biochemical activity and potent
antiproliferative activity against A549 and HCT116 cells. The representative compound 10a exhibited excellent
enzyme inhibition (PAK4 IC50 = 25 nM) and cellular potency (A549 IC50 = 0.58 µM, HCT116 IC50 = 0.095 µM).
An X-ray structure of compound 10a bound to PAK4 was obtained. Crystallographic analysis confirmed
predictions from molecular modelling and helped refine SAR results. In addition, Compound 10a displayed
focused multi-targeted kinase inhibition, good calculated drug-likeness properties. Further profiling of compound
10a revealed it showed weak inhibitory activity against various isoforms of human cytochrome P450.
1. Introduction
The p21-activated kinases (PAKs), members of the serine/threonine kinases family, were first identified as
effector proteins for Cdc42 and Rac1[1]. This kinase subfamily is comprised of six mammalian proteins that are
classified into group I (PAK1-3) and group II (PAK 4-6), based on structural homology and regulatory function[2].
Much of the interest in understanding the role of PAKs in human disease has been focused on cancer[3]. PAK4
plays an important role in oncogenic transformation and in cellular processes associated with transformation (e.g.,
cell survival, proliferation, cytoskeletal organization, invasion and migration)[4]. Importantly, increased PAK4
and/or activated PAK4 expression were associated with cancer metastasis, reduced patient survival, late stages, and
increased resistance to chemotherapy[5]. These reports strongly implicate PAK4 as an attractive drug target in
tumors that overexpress PAK4 or have PAK4 gene amplification.

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Figure 1. Structures of representative PAK4 inhibitors.
Despite this therapeutic potential, there have been very few disclosures of PAK4 inhibitors in the literature.
Most notable is the pioneering work from Pfizer, with reports in the patent literature stretching back to 2004[6]. A
representative compound from this effort, PF-3758309, is shown in Figure 1. PF-3758309 was progressed into
phase I clinical trials in patients with advanced/metastatic solid tumors. Although designed to be a PAK4 inhibitor,
the compound is, in fact, a multiple-target kinase inhibitor, which potently inhibited all group II PAK members,
PAK1, AMPK, CHK2, RSK, PKC, SRC et al. Unfortunately, PF-3758309 was prematurely discontinued in a
single clinical trial due to undesirable pharmacokinetic characteristics, no tumor responses, adverse events[7].
Researchers from Genentech succeeded in identifying a type I1/2 PAK4 inhibitors GNE-2861 with the common
structural feature of a tertiary propargyl alcohol[8]. GNE-2861 displayed PAK4 activity and > 870-fold selectivity
over PAK1 (PAK1 Ki = 2.9 µM, PAK4 Ki = 0.0033 µM). Our recently studies employing a structure-based design
approach identified CZh-226 as a potent inhibitor of PAK4 with 346-fold selectivity over PAK1 (PAK1 Ki = 3.11
µM, PAK4 Ki = 0.009 µM)[9]. Although high PAK4 potency, excellent permeability and satisfactory solubility,
GNE-2861 and CZh-226 showed large biochemical to cellular potency drop-off. Most recently, KPT-9274 has
identified a PAK4 allosteric inhibitors being currently under phase I clinical trials[10]. In addition to PAK4
inhibition, KPT-9274 was also shown to inhibit NAMPT (Nicotinamide phosphoribosyltransferase) activity.
Therefore, PAK4 in combination with conventional chemotherapy or other molecular targets could represent an
attractive approach for the treatment various human cancers.
We previously reported the discovery and optimization of 3-heterocyclic-5-amide substituted indolin-2-one
derivatives as potent PAK4 inhibitors through a scaffold hybridization strategy[11]. Representative indolin-2-one
derivatives 1-4 are shown in Figure 2A. However, they displayed modest ability to attenuate cancer cell growth.
We embarked upon a structure guided modification of 3 position of the indolin-2-one core. Based upon the
docking model of compound 2 bound to PAK4 (Figure 2B), it was reasonable to assume that the 4'- or 5'-position
on the imidazole and exocyclic alkene of the indolin-2-one core provided the appropriate vector to access the
lower-hinge region (highlighted in Magenta in Figure 2B) and hydrophobic region (highlighted in yellow in
Figure 2B), delimited by Leu447 and Val379, respectively. We envisioned that the introduction of appropriate size
substituents to the discussed above region to form new derivatives might improve potency and increase the cellular
permeability. To test our hypothesis, three series (series A, B and C) of derivatives were designed and synthesized
(Figure 2C). Herein, we report the detailed structure-activity relationship (SAR) and biological evaluation of these
compounds.

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Figure 2. A. The previously reported indolin-2-one based PAK4 inhibitors. B. Docking model for the binding of
compound 2 in the ATP-pocket of PAK4 cocrystal structure (PDB code 2x4z). Hydrogen bonds are shown as
yellow dots; the regions relevant for inhibitor binding are highlighted in the background: Hydrophobic region
(yellow), Lower hinge region (magenta). C. General structures of the designed PAK4 inhibitors in this work.
2. Chemistry
General procedures were developed for the synthesis of the 3-heterocyclic substituted indolin-2-one
derivatives 9a-e, 10a-q and 11a-c (Scheme 1). Namely, the intramolecular hydrogen bond between the carbonyl
oxygen of the indolin-2-one moiety and the NH of imidazole ring could stabilize compounds 9a-e, 10a-q and
11a-c in the Z-conformation. The preparation of intermediate 8 was performed starting from commercially
available indolin-2-one 5 as previously described (Scheme 1)[11]. Compounds 9a-e was prepared through the
reaction of intermediate 8 with 2-substituted-1H-imidazole-5-carbaldehyde 15a-e under optimized literature
conditions[12]. Synthetic routes of 2-substituted-1H-imidazole-5-carbaldehyde 15a-e are schematically illustrated
in Scheme 2. First, 2-substituted imidazoles 13, which were prepared by the condensation of the glyoxal 12 and
the appropriate aldehyde in the presence of ammonium hydroxide, were protected by the N, N-dimethylsulfamoyl
group using dimethylsulfamoyl chloride and sodium hydride as a base to produce the key intermediates 14. Second,
lithiation of 14 with n-BuLi followed by the addition of DMF and acidic aqueous workup afforded the
2-substituted imidazole aldehydes 15a-e [13].
Scheme 1. Preparation of Compounds 9a-e, 10a-q and 11a-c.

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H
N
O
O
H
N
HN
N
R1
HO
9a-e
H
N
CHO
R1
d
N
15a-e
H
N
H
N
H
N
O
O
H
O
O
O
Method e,f or g
O
c
N
O
O
HN
HO
O
H
7
6
5
OH
N
HN
HO
N
8
b
a
N
R2
10a-q
R2
H
N
16a-q
O
d
22a-c
Cl
H
N
H
N
O
O
H
N
R3
HN
HO
N
R4
11a-c
The reagents and conditions were as follows: (a) Chloroacetyl chloride, anhydrous AlCl3, 1,2-dichloroethane, r.t,
4.0 h; (b) i. pyridine, 90°C, 3 h; ii. 2.5 N NaOH (aq), 100°C, 3.0 h, 1N HCl (aq), pH = 2; (c)
(S)-(+)-2-Phenylglycinol, EDCI, HOBt, DIPEA, DCM, r.t., 16 h; (d) 15a-e and 22a-c, piperidine, EtOH, sealed
tube, 90°C, 4.5 h; (e) Pyrrolidine, EtOH, 105°C, 2d; (f) NH3, EtOH, 105°C, 2d. (g) Ti(OiPr)4, pyridine, THF, r.t. 4
h.
The synthesis of compounds 10a-q and 11a-c was carried out by the condensation of intermediate 8 and the
appropriate 2-benzoyl-(1H)-imidazoles under Knoevenagel condensation conditions in the presence of piperidine,
pyrrolidine[14], ammonia in ethanol[15], or Ti(OiPr)4/pyridine[16, 17] to yield the desired products after
chromatographic separation (Scheme 1). 2-benzoyl-(1H)-imidazoles 16a-q was prepared by heating imidazole
together with substituted benzoyl chloride in the presence of triethyl amine and pyridine[18] (Scheme 2). Although
both N- and C-acylation of the imidazole occurred, refluxing the diacylated intermediate with aqueous NaOH for 2
h led to hydrolysis at the N-acylated site, and yielded the desired C-acylated 2-substitutedbenzoyl-(1H)-imidazoles
16a-q. The corresponding 2-benzoyl-(1H)-imidazoles 22a-b and 2-benzoylbenzoimidazole 22c were also
synthesized in a similar manner. Reacting starting material 17 with thionyl chloride under nitrogen for one hour
yielded 18. Intermediate 18 was then converted to the imidazole 20 upon H2/Pd-C reduction and a reaction with
benzoyl chloride obtained the corresponding ketone 22b[19, 20].
Scheme 2. Synthesis of Intermediates 15a-e, 16a-q and 22a-c.

ACCEPTED MANUSCRIPT
Me
O
O
15a, R1= Methyl;
15b, R1= Ethyl;
15c, R1= i-propyl;
15d, R1= Phenyl;
15e, R1= 2-Cl phenyl
Cl
Me
N
O
N
N
H
N
S
N
CHO
Me
R1
H
H
R1
H
R1
O
O
Me
H
R1
N
N
H
j
N
h
i
O
S
O
O
N
Me
13
12
Me
14
H
N
16j, R2=2-Cl
16k, R2=2-Me
16a, R2=H
O
O
O
H
16b, R2=4-F
16c, R2=4-Cl
16d, R2=4-CN
16e, R2=4-OMe
16f, R2=3-F
16g, R2=3-Cl
16h, R2=3-OMe
16i, R2=2-F
N
SOCl2
k
16l, R2=2,4-diF
16m,R2=3,4-diF
16n, R2=2,4-diCl
16o, R2=3,4-diCl
16p, R2=3-Cl,4-F
16q, R2=3-F,4-Cl
N
l
HO
Cl
N
R2
R2
R2
16a-q
R2 = at least one group chosing
from H, F, Cl, OMe, CN, Me;
H
N
H
N
O
O
O
N
Me
N
Me
Cl
22a
H
19
H
N
H
H
N
Me
N
Me
HO
Me
Me
Me
N
n
m
·HCl
Cl
N
N
l
N
Me
N
22b
17
20
18
H
N
H
N
O
N
22c
N
21
The reagents and conditions were as follows: (h) NH4HCO3, CH3OH; (i) 60% NaH, DMF, 0 °C, 0.5 h;
Me2NSO2Cl, 0 - 25 °C, 2.0 h; (j) n-BuLi, DMF, -78 - 50 °C, 1 h; 1 M HCl, 0 - 25 °C, 2 h; NaHCO3 (aq), pH = 7;
(k) SOCl2, 1d DMF, DCM, 4.5 h; (l) Et3N, C5H5N, 0 -10 °C; r.t. 3 h; NaOH, H2O, reflux, 1 h. (m) SOCl2, r.t. 1 h.
(n) H2, Pd/C, EtOH, 60 °C, 4 h.
3. Results and discussion
3.1. In Vitro Activity against PAK4 Kinase and SAR Analyses
All newly synthesized compounds have been evaluated for inhibition of PAK4 activity using Homogeneous
Time-Resolved Fluorescence (HTRF) assays and the data is reported as IC50 values in Tables 1-3.
On the basis of the PAK4/compound 2 docking simulation, it seemed plausible that substituents at the
5'-position of the imidazole ring could increase PAK4 inhibitory activity and destabilize interactions with a subset
of off-target kinases by targeting the lower hinge region (Figure 2B). Compound 3 with 1', 4' -imidazole
substituted oxindole inhibited PAK4 with an IC50 of 16 nM. Hence, we attempted the synthesis of a variety of
substituents at the 5'-position of the 1', 4' -imidazole to investigate if higher potency and selectivity could be
obtained. Unfortunately, compared to 3, the analogues with alkyl substituents such as methyl, ethyl and i-propyl
(9a, 9b and 9c, respectively) and aromatic substituents such as phenyl and 2-chlorophenyl (9d, 9e) at the 5'-
position of the imidazole ring suffered from either greater than 7～40-fold loss of potency or a complete loss of
potency.
Table 1. Effect of R1 Substituents on PAK4 Biochemical IC50 (nM).

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Compd
3
R1
H
PAK4a, IC50 (nM)b,c
16
(14-27)
114
9a
9b
9c
9d
9e
Methyl
Ethyl
(76-178)
89
(56-121)
270
i-propyl
Phenyl
(213-326)
689
(529-815)
>10000
18
2-Cl phenyl
PF-3758309d
-
(12-27)
c
a Inhibitory activity against full-length human PAK4. b IC50 values were determined at KM ATP concentrations.
IC50 values are presented as the means of duplicate experiments with their 95% confidence intervals in
parentheses.
d Used as a positive control.
Docking of compound 2 to PAK4 led to a model that suggested placement of a phenyl ring at the substituted
alkene of the imidazole-oxindole core would provide an opportunity to interact with Leu447, Val379, in a
lipophilic environment (highlighted in yellow in Figure 2B). The addition of a phenyl group at the substituted
alkene provided compound 10a, which displayed an approximate 10-fold improvement in binding affinity for
PAK4 compared to its counterpart unsubstituted compound 2. We designated the two rings in 10a as rings A and B.
With 10a in hand, the SAR of substitution around the ring A was subsequently explored (Table 2). Introduction of
a fluoro group to the para position of the A ring provided compound 10b with a dramatic loss of activity.
Replacement of 4-fluoro with its congener Cl (as in compound 10c) also led to a loss of activity compared to that
of 10a, whereas it increased the activity compared to that of 4-F substituted analogue 10b. The 4-CN group
substituted compound 10d further improved the potency by approximately 8-fold compared to that of 10c.
Interestingly, introduction of a methoxy electron-donating group afforded compound 10e with approximately a
3-fold decreased inhibitory activity compared with that of 10a. Again, exploring substitution at the meta position
of A ring quickly revealed that 3-OMe was favored in the biochemical assays (10h vs 10f and 10g). Similar
potencies to 10a were exhibited by 10i, 10j, and 10k when the shift of the substituents at the A ring went from the
meta to ortho position. It was noted that 2-Cl substituent provided the better size to fit within the hydrophobic
pocket with a larger (Me, 10k) or smaller (F, 10i) substituent, resulting in a loss of potency.
Table 2. Effect of R2 Substituents on PAK4 Biochemical IC50 (nM).

ACCEPTED MANUSCRIPT
PAK4a, IC50
Compd
PAK4a, IC50
(nM)b,c
63
Compd
2
R2
-
R2
(nM)b,c
281
(224-335)
25
10i
10j
2-F
(40-113)
32
10a
10b
10c
10d
10e
10f
H
2-Cl
(18-34)
>10000
(29-64)
67
4-F
10k
10l
2-Me
(36-95)
706
4212
(3345-4989)
530
4-Cl
2,4-diF
3,4-diF
2,4-diCl
3,4-diCl
4-F, 3-Cl
3-F, 4-Cl
(623-834)
>10000
4-CN
4-OMe
3-F
10m
10n
10o
10p
10q
(485-628)
61
410
(40-102)
621
(328-519)
>10000
(543-714)
1112
>10000
10g
10h
3-Cl
(896-1457)
115
3285
(2589-3711)
18
3-OMe
(68-154)
PF-3758309d
-
(12-27)
a
c
Inhibitory activity against full-length human PAK4. b IC50 values were determined at KM ATP concentrations.
IC50 values are presented as the means of duplicate experiments with their 95% confidence intervals in
parentheses.
d Used as a positive control.
Next, we decided to explore the combination of F with Cl in compounds 10l - 10q. These bearing di-halogen
substitutions analogues were poorly active. It is worthwhile to note that 2, 4-disubstitution pattern exhibited a
tendency to retain potency compared to the 3, 4-disubstitution pattern. Our SAR results suggested that the A ring
likely interacts with a tight hydrophobic pocket of PAK4 protein. On the basis of the above results, thus compound
10a was selected for further optimization.
In another series, we further examined the modification at the C4 and C5 positions of the B ring that is
positioned adjacent to the lower hinge region of the protein, which is important for establishing selectivity between
kinases. Compounds 11a (C4 methyl substituted) and 11b (C4, C5 dimethyl substituted) had similar potencies and
were approximately 6-fold less active than 10a. The phenyl substituted 11c was 13-fold less potent than 10a
(Table 3). Combined with Table 1, we ascribed a lack of toleration of steric bulk in this region to the putative
binding site.

ACCEPTED MANUSCRIPT
Table 3. Effect of R3, R4 Substituents on PAK4 Biochemical IC50 (nM).
Compd
10a
R3
H
R4
H
PAK4 a, IC50 (nM) b,c
25
(18-34)
162
11a
11b
11c
H
Me
Me
(98-214)
139
Me
(87-194)
323
(264-391)
18
PF-3758309 d
(12-27)
c
a Inhibitory activity against full-length human PAK4. b IC50 values were determined at KM ATP concentrations.
IC50 values are presented as the means of duplicate experiments with their 95% confidence intervals in
parentheses.
d Used as a positive control.
3.2. Antiproliferative activity.
All new compounds with IC50 values below 200 nM (9a-b, 10a, 10e, 10h, 10i, 10j, 10k, and 11a-b) were
tested for their antiproliferative activity against A549 and HCT116 cell lines, in which PAK4 has been found to be
overexpressed, using the CCK-8 assay method and PF-3758309 as a reference. As illustrated in Table 4, five of the
compounds (10a, 10h, 10i, 10k and 11a) displayed favorable antiproliferative activity against A549 and HCT116
cell lines with IC50 values on the sub-micromolar scale, which was comparable to the reference compound
PF-3758309. Compounds 10e and 10j exhibited only weak cellular activity in the A549 cellular assay with IC50
values of 3.2 µM and 1.285 µM, respectively, and, corresponding values of 0.546 µM and 0.635 µM against the
HCT116 cell line. It is noteworthy that there was a large right shift between PAK4 biochemical potency and
cell-based potency for compounds 9a, 9b, 11b. For compounds 9a and 9b, their high polarity, poor cellular
permeability and/or high efflux rate might be part of the reason. However, we are curious but unable to definitively
explain the large shift in concentration between biochemical IC50 and cellular inhibition for compound 11b.
Table 4. Antiproliferative Activity and Calculated Properties (log P, QPPCaco) of the Selected Compounds.
Cellular inhibition
PAK4 IC50
(IC50, µM) a
Compd.
9a
clogP b
QPPCaco b, c
(nM)
A549
HCT116
114
(76-178)
89
56.092±3.674
4.09±0.299
2.19
131.7
29.825±2.342
0.579±0.132
2.865±0.358
0.095±0.012
2.59
3.42
155.7
227.6
9b
(56-121)
25
10a

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(18-34)
61
3.2±0.574
0.358±0.016
0.666±0.2
0.546±0.101
0.245±0.064
0.088±0.009
0.635±0.121
0.119±0.009
0.077±0.004
2.578±0.633
0.039±0.006
3.45
3.33
3.61
3.80
3.68
3.76
4.07
4.16
291.6
214.1
245.2
263.8
272.9
261.9
275.1
294.0
10e
10h
(40-102)
115
(68-154)
63
10i
(40-113)
32
1.285±0.098
0.768±0.041
0.511±0.026
17.741±0.671
0.463±0.04
10j
(29-64)
67
10k
(36-95)
162
11a
(98-214)
139
11b
(87-194)
18
PF-3758309d
(12-27)
a Each compound was tested in duplicate; the data are presented as the mean ± SD.
b Calculated by QikProp, version 4.3, Schrödinger, LLC, New York, NY, 2015.
c Predicted apparent Caco-2 cell permeability in nm/sec. Caco-2 cells are a model for the gut-blood barrier.
d Used as a positive control.
3.3. Kinase selectivity profile
The kinase selectivity of 10a was profiled against a panel of 47 kinases covering the major tumor progression,
angiogenesis, oncogenic activation, metastasis and mitogenic stimulation kinases of the human protein kinome at a
concentration of 1.0 µM. Reported 3-substituted indolin-2-one derivatives are usually known as multi-targeted
tyrosine kinase and angiogenesis inhibitors[21-26]. Various indolin-2-one based derivatives also exhibited
excellent inhibitory activity against c-Met kinase[27], PIM kinase[28], RET kinase[29], FLT3[30, 31], Aurora
kinases[32-34], GSK-3[35], cyclin-dependent kinases (CDKs)[36, 37], phosphoinositide-dependent kinase-1
(PDK1)[38], NEK2[12], Checkpoint kinase 1 (CHK1)[39], leucine-rich repeat kinase-2 (LRRK2)[40-42], p90
ribosomal S6 protein kinase 2 (RSK2)[43] and Polo-like kinase 4 (PLK4)[44, 45].
As expected, when screened against a wider panel of kinases, 10a exhibited more than 80% inhibition against
PAK4, BRAF, Receptor Tyrosine Kinases (PDGFRα, PDGFRβ, FLT3, CSF1R, VEGFR1-2, FGFR1, RET),
Non-Receptor Tyrosine Kinases SRC family (SRC, YES, and FYN), and cell cycle and mitogen activated protein
kinases (CDK2, MAPK3) but had little effect on KIT or IGF1R (Figure 3). Given that all major human cancers
seem to harbor not a single but several concomitant dysregulation of kinase pathways, multi-targeted kinase
inhibitors offer the opportunity not only to catch tumor progression more effectively but also to avoid mechanisms
of resistance commonly activated by the tumor to escape targeted therapies[46-51]. However, compound 10a may
also impart unwanted off-target side effects, which necessitates a further optimization and evaluation.

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Figure 3. The kinase profile of compound 10a tested at a concentration of 1.0 µM in a panel of 47 kinases
generated with the SelectScreen® Profiling Service from Thermo Fisher Scientific. Red columns denote > 80%,
yellow columns between 40 and 80%, and green columns < 40% inhibition.
3.4 X-ray Crystal Structure.
An X-ray structure of 10a bound to PAK4 at 1.83 Å resolution was obtained (Figure 4). The crystal structure
confirms earlier modeling conclusions that the 3-substituted-indolin-2-one scaffold interacts via a classic 3-point
donor-acceptor-donor hydrogen bond with the kinase hinge residues Glu396 and Leu398. The carbonyl oxygen
atom of the amide attached to the 5-position of the indolin-2-one framework forms two hydrogen bonds, one to the
side chain of Lys350, and one to the NH of main chain of Asp458. The hydroxyl of (S)-phenylglycinol interacts
with the carboxylate side chain of Asp458. A water molecule forms a H-bond to the imidazole nitrogen N3. In
addition, the alkene-linked phenyl (ring A) adopting an orthogonal conformation to the indolin-2-one core was
sandwiched in a narrow lipophilic cleft. This tight packing likely contributes to the high potency observed against
PAK4 in this series, and explains the intolerance to larger substituents on the ring A. The phenyl (ring C) of the
(S)-phenylglycinol occupies a small hydrophobic pocket under the P-loop.
Figure 4. Crystal structures of 10a bound to PAK4. Red dotted lines indicate hydrogen bonds. Red spheres
indicate water molecules. (PDB 5ZJW)
3.5. Physicochemical and In Vitro ADME Properties of 10a
Several physicochemical parameters, such as molecular weight (MW), Clog P, polar surface area (PSA),
number of hydrogen bond donors, number of hydrogen bond donors, and number of rotatable bonds, which are
associated with the absorption and distribution steps in the pharmacokinetic phase of a drug. The ADME properties

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of compound 10a were calculated using the Qikprop, and the results obtained are gathered in Figure 5A.
According to a recent perspective[52], small-molecule kinase inhibitors, 28 of which are approved by the US Food
and Drug Administration (FDA), show the following physicochemical characteristics: MW in the range of 300 -
600 g•mol-1, Clog P less than 5 with a preferred value approximately 2 - 5, total PSA in the range of 60 - 140 Å2,
no more than four hydrogen-bond donors (HBDs), no more than nine hydrogen-bond acceptors (HBAs), and the
number of rotatable bonds (NRBs) in the range of 3 - 9. The drug-like properties of compound 10a all fall within
the desired ranges of above properties (Figure 5B). (Predicted ADME properties of 10h, 10i, 10k and 11a were
summarized in table S1)
Figure 5. The drug-like properties of compound 10a. A. Calculated Physicochemical Properties of Compound 10a.
B. A radar plot representing the computed physicochemical parameters of 10a. Structural and physicochemical
properties of compound 10a are compared with the optimal blue area defined from the reference[52]. Values of
Compound 10a (turquoise line) all fall within the blue area. Calculated log P (Clog P); molecular weight (MW);
topological polar surface area (tPSA); hydrogen bond donors (HBD), were calculated by the total number of NH
and OH bonds; hydrogen bond acceptors (HBA), were calculated by the total number of nitrogen and oxygen
atoms; number of rotatable bonds (NRBs).
Compound 10a was next evaluated with a panel of cytochrome P450 isoforms (1A2, 2C9, 2C19, 2D6, and
3A4), where it showed only moderate inhibition of the CYP3A4 (IC50 = 3.03 µM) (Table 5). Compound 10a did
not significantly inhibit CYP1A2, CYP2C9, CYP2C19, or CYP2D6 metabolism, indicating minimal drug-drug
potential with compounds cleared predominantly via these metabolic pathways.
Table 5. CYP Inhibition Data for Compound 10a.a
CYP b IC50 (µM)
Isoforms
CYP1A2
> 50
CYP2C9
21.4
CYP2C19
> 50
CYP2D6
> 50
CYP3A4
3.03
a For experimental details, see Experimental Section.
b Assays were performed in pooled human liver microsomes.
4. Conclusion
In this study, a series of PAK4 inhibitors bearing a 3-substituted-indolin-2-one-5-carboxamides scaffold was
designed and synthesized. Structure-activity relationships of these compounds were discussed based on enzymatic
and cellular activities. The representative compound 10a exhibited excellent enzyme inhibition (PAK4 IC50 = 25
nM), cellular potency (A549 IC50 = 0.58 µM, HCT116 IC50 = 0.095 µM) and displayed focused multi-targeted
receptor tyrosine kinases inhibition. The X-ray crystal structure of compound 10a in PAK4 was helpful for
designing indolin-2-one scaffold-based kinase inhibitors. Further development of this class of compound will be
published in due course.
5. Experimental section
5.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and used without further purification

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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 thin layer
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-600 NMR spectrometer with TMS as an internal
standard. High-resolution accurate mass spectrometry (HRMS) determinations for all final target compounds were
obtained on a Bruker Micromass Time of Flight mass spectrometer equipped with an electrospray ionization (ESI)
detector.
General Procedure of Piperidine/EtOH Mediated Knoevenagel Condensations for the Synthesis of Target
Compounds (9a-e, 11a-c). (Method d)
The corresponding aldehydes 15a-e, 22a-c (1.1 mmol) and a catalytic amount of piperidine were added to a
solution of 8 (1.0 mmol) in ethanol (4 mL). The reaction mixture was stirred at reflux inside the sealed tube for 4.5
h (reaction monitored by TLC). After cooling to room temperature, the reaction mixture was diluted with
dichloromethane, and then washed with water and then brine successively. The organic phase was evaporated and
the residual oil was purified by silica gel chromatograph using CH2Cl2/MeOH (100:1) as an eluent to provide the
title compounds 9a-e and 11a-c as brown yellow/brown/orange solids, in moderate to excellent yields.
General Procedure of Pyrrolidine/EtOH Mediated Knoevenagel Condensations for the Synthesis of Target
Compounds (10a, 10c, 10e, 10f, 10g, 10h, 10o, 10q). (Method e)
The corresponding (1H-imidazol-2-yl) (phenyl)methanone (16a, 16c, 16e, 16f, 16g, 16h, 16o, 16p) (1.1 mmol)
and a catalytic amount of pyrrolidine was added to a solution of 8 (1.0 mmol) in ethanol (4 mL). The reaction
mixture was degassed by N2 bubbling for 5 min and then stirred at reflux inside a sealed tube for 48 h. After
cooling to room temperature, the reaction mixture was diluted with dichloromethane, and then washed with water
and brine sequentially. The organic phase was evaporated, and the residual oil was purified by silica gel
chromatograph using CH2Cl2/MeOH (120:1) as an eluent to provide the title compounds 10a, 10c, 10e, 10f, 10g,
10h, 10o, 10p as brown yellow/brown/orange solids, in moderate to excellent yields.
General Procedure of NH3/EtOH Mediated Knoevenagel Condensations for the Synthesis of Target Compounds
(10b, 10i, 10l, 10m, 10p). (Method f )
A solution of 8 (1.0 mmol) and the corresponding (1H-imidazol-2-yl) (phenyl)methanone (16b, 16i, 16l, 16m,
16p) (1.1 mmol) with ammonia in ethanol was introduced into a 10 mL sealed tube. The mixture was purged with
argon and stirred for 48 h at 105 °C. After cooling to room temperature, the reaction mixture was diluted with
dichloromethane, and then washed with water and brine sequentially. The organic phase was evaporated, and the
residual oil was purified by silica gel chromatograph using CH2Cl2/MeOH (120:1) as eluent to provide the title
compounds 10b, 10i, 10l, 10m, 10p as brown yellow/brown/orange solids, in moderate to excellent yields.
General Procedure of Ti(OiPr)4/Pyridine Mediated Knoevenagel Condensations for the Synthesis of Target
Compounds (10d, 10j, 10k, 10n). (Method g)
A mixture of 8 (1.0 mmol), the corresponding (1H-imidazol-2-yl) (phenyl)methanone (16d, 16j, 16k, 16o)
(1.1 mmol), and pyridine (2 mmol) in THF were stirred for 10 min followed by addition of titanium isopropoxide
(3 mmol). The resulting mixture was stirred at r.t. for 4 h. TLC showed that majority of the starting material was
converted to the product. The reaction mixture was diluted with EtOAc, and washed with 1N aqueous HCl,
NaHCO3(aq) and brine. The organic layer was dried over anhydrous Na2SO4, concentrated, and purified by
chromatography with CH2Cl2/MeOH (120:1) to provide the title compounds 10d, 10j, 10k, 10o as brown
yellow/brown/orange solids, in moderate to excellent yields.
General procedure for the synthesis of 2-substituted -1H-imidazole-5-carbaldehydes (15a-e).
An example of the synthetic procedure of 2-methyl-1H-imidazole-5-carbaldehyde (15a).
A suspension of NH4HCO3 (13.8 g, 174.6 mmol) and 40% glyoxal solution (10.9 mL, 87.3 mmol) in water

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(9 mL) was treated dropwise with the appropriate aldehyde (9.4 mL, 87.3 mmol) at 0 °C. The mixture was stirred
at 25 °C for 24 h. The mixture was concentrated under reduced pressure. The residue was diluted with CH2Cl2 and
washed with brine. The organic layer was dried over anhydrous MgSO4, filtered and concentrated. The product
was purified by column chromatography (CH2Cl2/MeOH = 80:1) to obtain the title compounds.
2-methyl-1H-imidazole (13a) (800 mg, 9.74 mmol) was added to a suspension of 60% NaH (700 mg,
29.22 mmol) in 20 mL DMF at 0 °C, and the mixture was stirred for 0.5 h. Then, N, N-dimethylsulfamoyl chloride
(1.68 g,11.69 mmol) was added dropwise and the reaction mixture was stirred for 2 h at room temperature.
Saturated NH4Cl solution was added, and the resulting mixture was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The residue
was purified by silica gel chromatography with petroleum ether/ethyl acetate (4:1) as an eluent to furnish the title
compound as a canary yellow solid.
n-Butyllithium
(2.49 mL,
5.71 mmol)
was
added
dropwise
to
a
solution
of
N,N-dimethyl-2-methyl-1H-imidazole-1-sulfonamide (14a) (900 mg, 4.76 mmol) in THF (20 mL) at -78 °C over a
period of 30 min. DMF (1.85 mL, 23.78 mmol) was added to the mixture. The resulting solution was stirred at -50 °
C for 30 min, and hydrogen chloride (11.9 mL, 11.9 mmol, 1M) was then added. The reaction mixture was stirred
for 2 h at room temperature. The pH of the solution was adjusted to 7 ~ 8 with a saturated sodium bicarbonate
solution. The resulting solution was extracted with 3 × 15 mL of ethyl acetate. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified by
silica gel chromatography (petroleum ether/ethyl acetate = 4:1) to furnish the title compound (15a) as yellow solid.
Other 2-substituted -1H-imidazole-5-carbaldehydes (15b-e) were prepared as described in the procedure for
synthesizing 15a.
General procedure for the synthesis of (1H-imidazol-2-yl) (phenyl)methanone (16a-q, 22a-c).
An example of the synthetic procedure of (1H-imidazol-2-yl) (phenyl)methanone (16a).
Imidazole (500 mg, 7.35 mmol) and triethylamine (2.0 mL) were added to pyridine (10.0 mL) at 0 °C and
stirred. Benzoyl chloride (2.07g, 14.7 mmol) was slowly added, and the temperature was slowly increased to room
temperature. The reaction mixture was stirred for another 3 hours. Upon the completion of the reaction, 6N NaOH
aqueous solution (5.0 mL) was added, and the reaction mixture was stirred for 1 hour at 100 °C. Cooled to room
temperature, the pH value was adjusted to 6 ~ 7 with 1N HCl, and the reaction mixture was extracted three times
with ethyl acetate and washed with water and dried over magnesium sulfate. The filtrate was distilled under
reduced pressure, and the concentrated residue was purified and separated by column chromatography.
5.1.1.1
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((2-methyl-1H-imidazol-5-yl)-methylene)-2-oxoindoline-5-carboxa
mide (9a). Orange solid, yield 75%; m.p.: 294.1-296.4 °C; 1H NMR (600 MHz, DMSO-d6): δ 13.37 (s, 1H), 11.20
(s, 1H), 8.52 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 7.79 (s, 1H), 7.75 (dd, J = 8.1, 1.4 Hz, 1H), 7.61 (s, 1H), 7.38 (d, J =
7.3 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.21 (t, J = 7.3 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 5.06 (dd, J = 13.5, 7.9 Hz,
1H), 4.92 (t, J = 5.9 Hz, 1H), 3.70 (t, J = 6.5 Hz, 2H), 2.45 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 170.84,
166.50, 147.40, 144.26, 141.97, 140.16, 137.20, 129.26, 128.57, 128.07, 127.47, 127.41, 127.26, 124.79, 122.79,
120.72, 118.63, 109.63, 108.76, 65.09, 56.39, 18.89. HRMS (+ESI) calcd for C22H20N4O3, [M+H]+, 389.1535;
found 389.1629.
5.1.1.2.
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((2-ethyl-1H-imidazol-5-yl)-methylene)-2-oxoindoline-5-carboxami
de (9b). Yellow solid, yield 78%; m.p.:265.2-267.5 °C; 1H NMR (600 MHz, DMSO-d6): δ 13.59 (s, 1H), 11.23 (s,

ACCEPTED MANUSCRIPT
1H), 8.55 (d, J = 7.3 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 7.84 (s, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.66 (s, 1H), 7.41 (d,
J = 7.3 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 6.92 (d, J = 55.3 Hz, 1H), 5.09 (dt, J = 11.8, 6.0
Hz, 1H), 4.98-4.92 (m, 1H), 3.75-3.66 (m, 2H), 2.84-2.74 (m, 2H), 1.33-1.27 (m, 3H). 13C NMR (150 MHz,
DMSO-d6): δ 170.85, 166.50, 144.25, 142.18, 142.11, 141.96, 129.26, 128.58, 128.50, 128.42, 128.10, 127.47,
127.27, 127.19, 124.83, 120.71, 118.64, 109.61, 108.63, 65.10, 56.39, 22.05, 12.24. HRMS (+ESI) calcd for
C23H22N4O3, [M+H]+, 403.1692; found 403.1818.
5.1.1.3
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((2-isopropyl-1H-imidazol-5-yl)-methylene)-2-oxoindoline-5-carbo
xamide (9c). Yellow solid, yield 67%; m.p.:210.3-213.9 °C; 1H NMR (600 MHz, DMSO-d6): δ 13.75 (s, 1H),
11.24 (s, 1H), 8.56 (d, J = 7.6 Hz, 1H), 8.25 (s, 1H), 7.86 (s, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.42 (d, J
= 7.5 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.24 (t, J = 7.3 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 5.10 (dd, J = 13.5, 7.7 Hz,
1H), 4.96 (d, J = 5.2 Hz, 1H), 3.81-3.56 (m, 2H), 3.17-3.05 (m, 1H), 1.33 (d, J = 7.1 Hz, 6H). 13C NMR (150 MHz,
DMSO-d6): δ 170.85, 167.50, 166.49, 156.31, 144.23, 142.06, 141.97, 136.68, 128.58, 128.47, 127.47, 127.27,
127.16, 124.85, 122.93, 120.68, 118.65, 109.69, 108.53, 65.10, 56.39, 28.48, 21.93, 21.88. HRMS (+ESI) calcd for
C24H24N4O3, [M+H]+, 417.1848; found 417.2090.
5.1.1.4.
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((2-phenyl-1H-imidazol-5-yl)-methylene)-2-oxoindoline-5-carboxa
mide (9d). Brown solid, yield 80%; m.p.:227.5-230.7 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.6 (s, 1H), 11.38 (s,
1H), 8.63-8.47 (m, 1H), 8.31 (s, 1H), 8.04-7.94 (m, 2H), 7.88-7.77 (m, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.55-7.39 (m,
5H), 7.37-7.20 (m, 4H), 6.96 (dd, J = 77.8, 8.0 Hz, 1H), 5.14 (dd, J = 24.9, 5.6 Hz, 1H), 5.05-4.92 (m, 1H),
3.78-3.67 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 170.07, 166.46, 149.15, 142.22, 141.97, 140.87, 130.36,
129.83, 129.54, 129.32, 129.26, 128.77, 128.58, 128.49, 127.55, 127.49, 127.27, 126.25, 125.66, 124.69, 123.90,
119.82, 119.01, 118.62, 109.89, 65.10, 56.40. HRMS (+ESI) calcd for C27H22N4O3, [M+H]+, 451.1692; found
451.1782.
5.1.1.5.
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((2-Chlorophenyl-1H-imidazol-5-yl)-methylene)-2-oxoindoline-5-c
arboxamide (9e). Orange solid, yield 61%; m.p.:295.4-297.8 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.61 (s, 1H),
11.33 (s, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.31-8.25 (m, 1H), 7.97 (s, 1H), 7.82 (dd, J = 8.1, 1.2 Hz, 1H), 7.67-7.64
(m, 1H), 7.52 (td, J = 6.9, 1.7 Hz, 1H), 7.42 (d, J = 7.4 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.25 (t, J = 7.3 Hz, 1H),
7.00 (d, J = 8.1 Hz, 1H), 5.11 (dd, J = 13.5, 7.9 Hz, 1H), 4.97 (t, J = 5.8 Hz, 1H), 3.76-3.66 (m, 2H). 13C NMR
(150 MHz, DMSO-d6): δ 170.28, 166.47, 146.55, 142.50, 141.93, 140.32, 132.47, 131.64, 131.37, 130.67, 130.50,
130.41, 128.73, 128.59, 128.45, 128.29, 128.05, 127.48, 127.44, 127.28, 124.51, 123.56, 120.43, 119.18, 109.87,
65.08, 56.40. HRMS (+ESI) calcd for C27H21ClN4O3, [M+H]+, 485.1302; found 485.1382.
5.1.1.6.
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((4-methyl-1H-imidazol-2-yl)(phenyl)methylene)-2-oxoindoline-5-c
1
arboxamide (11a). Yellow solid, yield 57%; m.p.:214.7-217.6 °C; H NMR(600 MHz, DMSO-d6): δ 14.90 (d, J =
21.5 Hz, 1H), 11.50 (s, 1H), 7.80 (s, 1H), 7.69 (dd, J = 8.1, 1.3 Hz, 1H), 7.52-7.48 (m, 2H), 7.47 (m, 1H),
7.34-7.23 (m, 8H), 6.91 (s, 1H), 5.96 (d, J = 28.7 Hz, 1H), 4.88 (d, J = 5.5 Hz, 2H), 3.58 (dd, J = 10.0, 5.6 Hz, 2H),
2.38 (s, 2H), 2.11 (s, 1H). 13C NMR (150 MHz, DMSO-d6): δ 170.07, 166.22, 144.93, 144.90, 143.43, 142.59,
141.82, 139.00, 131.82, 131.12, 130.12, 129.24, 128.80, 128.50, 128.30, 127.80, 127.71, 127.38, 127.23, 124.43,
123.94, 120.93, 120.25, 118.31, 109.27, 65.04, 56.10, 23.50. HRMS (+ESI) calcd for C28H24N4O3, [M+H]+,
465.1848; found 465.2075.
5.1.1.7.
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((4,5-dimethyl-1H-imidazol-2-yl)(phenyl)methylene)-2-oxoindoline

ACCEPTED MANUSCRIPT
-5-carboxamide (11b). Orange solid, yield 61%; m.p.:260.5-262.5 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.95 (s,
1H), 11.34 (s, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.4 Hz,
1H), 7.29 (t, J = 7.5 Hz, 2H), 7.24 (d, J = 7.3 Hz, 2H), 7.20 (d, J = 6.9 Hz, 3H), 6.87 (d, J = 8.1 Hz, 1H), 5.85 (s,
1H), 4.84 (dd, J = 13.3, 6.7 Hz, 2H), 3.54 (s, 2H), 2.28 (s, 3H), 2.02 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ
170.09, 166.30, 143.35, 142.89, 142.20, 141.83, 139.66, 139.12, 130.87, 129.63, 129.22, 129.18, 128.78, 128.74,
128.61, 128.50, 128.19, 127.71, 127.38, 127.32, 127.23, 124.65, 123.59, 119.03, 109.20, 65.04, 56.08, 12.93,
10.33. HRMS (+ESI) calcd for C29H26N4O3, [M+H]+, 479.2005; found 479.2105.
5.1.1.8.
(3Z)-N-((1S)-2-hydroxy-1-phenylethyl)-3-((1H-benzo[d]imidazol-2-yl)(phenyl)methylene)-2-oxoindoline-5-c
arboxamide (11c). Orange solid, yield 52%; m.p.:268.7-270.9 °C; 1H NMR (600 MHz, DMSO-d6): δ 13.85 (s, 1H),
11.32 (s, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.64 (dd, J = 23.3, 8.1 Hz, 2H), 7.52 (d, J = 5.9
Hz, 3H), 7.40-7.17 (m, 8H), 6.93 (d, J = 8.1 Hz, 1H), 6.59 (s, 1H), 4.93-4.85 (m, 2H), 3.58 (dt, J = 11.3, 6.4 Hz,
2H). 13C NMR (150 MHz, DMSO-d6): δ 168.72, 166.06, 150.69, 144.52, 144.31, 142.25, 141.85, 138.60, 134.47,
129.65, 129.62, 129.45, 129.22, 128.53, 128.47, 127.94, 127.38, 127.25, 124.39, 124.31, 123.25, 122.51, 120.05,
112.63, 109.49, 65.04, 56.19. HRMS (+ESI) calcd for C31H24N4O3, [M+H]+, 501.1848; found 501.2092.
5.1.1.9.
(3Z)-3-((1H-imidazol-2-yl)(phenyl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-carboxami
de (10a). Yellow solid, yield 65%; m.p.:256.5-258.1 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.89 (s, 1H), 11.52 (s,
1H), 7.85 (d, J = 7.7 Hz, 1H), 7.71 (dd, J = 8.1, 1.3 Hz, 1H), 7.60 (s, 1H), 7.54-7.43 (m, 3H), 7.35-7.21 (m, 8H),
6.92 (d, J = 8.1 Hz, 1H), 6.04 (s, 1H), 4.88 (t, J = 5.7 Hz, 2H), 3.58 (dd, J = 11.1, 5.7 Hz, 2H). 13C NMR (150
MHz, DMSO-d6): δ 170.02, 166.17, 145.53, 143.34, 142.89, 141.82, 139.04, 132.69, 129.21, 128.87, 128.79,
128.51, 128.38, 128.11, 127.38, 127.24, 124.25, 124.19, 121.84, 120.88, 109.32, 65.04, 56.12. HRMS (+ESI)
calcd for C27H22N4O3, [M+H]+, 451.1692; found 451.1870.
5.1.1.10.
(3Z)-3-((4-chlorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-ca
rboxamide (10c). Orange solid, yield 67%; m.p.:188.5-191.3 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.82 (s, 1H),
11.53 (s, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.77 (dd, J = 8.2, 1.4 Hz, 1H), 7.74-7.64 (m, 1H), 7.58 (d, J = 7.8 Hz, 3H),
7.33-7.27 (m, 5H), 7.25-7.19 (m, 2H), 6.93 (d, J = 8.1 Hz, 1H), 6.34 (s, 1H), 4.92 (dd, J = 13.5, 7.5 Hz, 1H), 4.87
(t, J = 5.8 Hz, 1H), 3.59 (dd, J = 12.7, 6.7 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): δ 169.88, 166.24, 145.27,
143.06, 141.80, 141.76, 137.77, 133.64, 132.15, 132.02, 131.01, 129.32, 129.14, 128.54, 128.41, 128.21, 127.38,
127.30, 127.23, 124.26, 124.05, 121.99, 109.44, 65.07, 56.23. HRMS (+ESI) calcd for C27H21ClN4O3, [M+H]+,
485.1302; found 485.1418.
5.1.1.11.
(3Z)-3-((4-methoxyphenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-
carboxamide (10e). Orange solid, yield 51%; m.p.:164.5-166.9 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.61 (s,
1H), 11.40 (s, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.71 (d, J = 7.4 Hz, 1H), 7.35-7.16 (m, 9H), 7.07 (d, J = 8.2 Hz, 2H),
6.91 (d, J = 8.1 Hz, 1H), 6.43 (s, 1H), 4.89 (dd, J = 12.0, 6.2 Hz, 2H), 3.80 (s, 3H), 3.57 (s, 2H). 13C NMR (150
MHz, DMSO-d6): δ 169.76, 166.39, 160.10, 143.03, 141.84, 133.46, 130.83, 130.13, 129.66, 129.14, 128.52,
128.31, 128.06, 127.38, 127.27, 127.21, 126.88, 124.42, 123.94, 114.71, 113.49, 109.28, 65.06, 56.75, 56.19.
HRMS (+ESI) calcd for C28H24N4O4, [M+H]+, 481.1798; found 481.1912.
5.1.1.12.
(3Z)-3-((3-fluorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-ca
1
rboxamide (10f). Yellow solid, yield 61%; m.p.:185.3-187.2 °C; H NMR (600 MHz, DMSO-d6): δ 14.89 (s, 1H),
11.55 (s, 1H), 8.04 (s, 1H), 7.76 (dd, J = 8.1, 1.3 Hz, 1H), 7.61 (s, 1H), 7.54 (dd, J = 14.0, 7.9 Hz, 1H), 7.34-7.27

ACCEPTED MANUSCRIPT
(m, 5H), 7.23 (dd, J = 10.3, 5.2 Hz, 2H), 7.16 (d, J = 9.4 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 6.93 (d, J = 8.1 Hz,
1H), 6.16 (s, 1H), 4.92 (dd, J = 13.5, 7.5 Hz, 1H), 4.86 (t, J = 5.5 Hz, 1H), 3.58 (dd, J = 12.0, 6.1 Hz, 2H). 13C
NMR (150 MHz, DMSO-d6): δ 169.99, 166.11, 163.71, 162.09, 145.18, 142.98, 141.86, 141.41, 141.16, 141.11,
132.79, 131.36, 130.13, 128.51, 128.21, 127.37, 127.23, 125.06, 124.37, 123.96, 121.87, 121.08, 115.77, 115.63,
109.37, 65.05, 56.15. HRMS (+ESI) calcd for C27H21FN4O3, [M+H]+, 469.1598; found 469.1690.
5.1.1.13.
(3Z)-3-((3-chlorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-carboxa
mide (10g). Orange solid, yield 63%; m.p.:220.3-223.4°C; 1H NMR (600 MHz, DMSO-d6): δ 14.87 (s, 1H), 11.56
(s, 1H), 8.14-7.90 (m, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.73-7.63 (m, 1H), 7.57-7.50 (m, 2H), 7.37 (s, 1H), 7.35-7.20
(m, 7H), 6.94 (d, J = 8.1 Hz, 1H), 6.18 (s, 1H), 4.96-4.82 (m, 2H), 3.59 (dd, J = 9.9, 5.7 Hz, 2H). 13C NMR (150
MHz, DMSO-d6): δ 169.94, 167.46, 145.16, 143.06, 141.78, 141.17, 140.93, 133.87, 132.15, 132.09, 132.02,
131.18, 130.13, 129.14, 128.83, 128.54, 128.32, 127.72, 127.33, 127.25, 123.93, 122.03, 109.49, 65.07, 56.16.
HRMS (+ESI) calcd for C27H21ClN4O3, [M+H]+, 485.1302; found 485.1460.
5.1.1.14.
(3Z)-3-((3-methoxyphenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-
1
carboxamide (10h). Orange solid, yield 67%; m.p.:164.5-167.2 °C; H NMR (600 MHz, DMSO-d6): δ 14.90 (s,
1H), 11.51 (s, 1H), 7.74-7.56 (m, 3H), 7.42 (t, J = 8.0 Hz, 1H), 7.35-7.27 (m, 4H), 7.26-7.18 (m, 2H), 7.04 (d, J =
7.4 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.83 (t, J = 3.9 Hz, 2H), 6.16 (s, 1H), 4.90 (dd, J = 13.8, 6.9 Hz, 2H), 3.72 (s,
3H), 3.59 (t, J = 6.4 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): δ 170.04, 167.46, 160.13, 145.40, 142.96, 142.88,
141.79, 140.20, 132.15, 132.02, 130.44, 129.82, 129.14, 128.53, 128.20, 127.34, 127.26, 124.11, 121.70, 120.96,
120.55, 114.53, 109.36, 65.08, 56.12, 55.73. HRMS (+ESI) calcd for C28H24N4O4, [M+H]+, 481.1798; found
481.1911.
5.1.1.15.
(3Z)-3-((3,4-dichlorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-
1
5-carboxamide (10o). Yellow solid, yield 59%; m.p.:254.1-256.3 °C; H NMR (600 MHz, DMSO-d6): δ 14.83 (s,
1H), 11.58 (s, 1H), 8.15 (s, 1H), 7.83-7.59 (m, 4H), 7.34-7.19 (m, 7H), 6.95 (d, J = 8.1 Hz, 1H), 6.38 (s, 1H), 4.94
(dd, J = 13.7, 7.4 Hz, 1H), 4.86 (t, J = 5.4 Hz, 1H), 3.63-3.54 (m, 2H). 13C NMR (150 MHz, DMSO-d6)：δ 169.88,
166.19, 145.03, 143.16, 141.77, 139.96, 139.46, 132.80, 132.03, 131.67, 131.50, 131.22, 129.62, 129.14, 128.56,
128.38, 127.29, 127.22, 124.27, 123.82, 122.09, 121.20, 109.55, 65.10, 56.21. HRMS (+ESI) calcd for
C27H20Cl2N4O3, [M+H]+, 519.0912; found 519.1033.
5.1.1.16.
(3Z)-3-((4-chloro-3-fluorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindo
line-5-carboxamide (10q) Yellow solid, yield 60%; m.p.:236.9-238.8 °C; 1H NMR (600 MHz, DMSO-d6)：δ 14.83
(s, 1H), 11.58 (s, 1H), 8.21 (d, J = 6.5 Hz, 1H), 7.81 (dd, J = 8.1, 1.2 Hz, 1H), 7.73 (t, J = 7.2 Hz, 1H), 7.61 (s, 1H),
7.44 (d, J = 8.9 Hz, 1H), 7.33-7.20 (m, 6H), 7.17 (dd, J = 8.2, 1.5 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 6.36 (s, 1H),
4.94 (dd, J = 13.6, 7.6 Hz, 1H), 4.86 (t, J = 5.9 Hz, 1H), 3.59 (dd, J = 11.7, 5.8 Hz, 2H). 13C NMR (150 MHz,
DMSO-d6): δ 169.90, 166.15, 158.83, 157.19, 144.98, 143.13, 141.79, 140.11, 139.90, 131.61, 128.54, 128.45,
128.34, 127.39, 127.31, 127.23, 126.59, 124.35, 123.79, 122.02, 120.12, 118.06, 109.51, 65.07, 56.19. HRMS
(+ESI) calcd for C27H20ClFN4O3, [M+H]+, 503.1208; found 503.1322.
5.1.1.17.
(3Z)-3-((4-fluorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-ca
rboxamide (10b). Yellow solid, yield 69%; m.p.:206.6-208.7 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.84 (s, 1H),
11.52 (s, 1H), 8.09 (d, J = 7.5 Hz, 2H), 7.76 (d, J = 7.7 Hz, 1H), 7.62-7.44 (m, 2H), 7.29 (ddd, J = 45.4, 25.4, 7.5
Hz,8H), 6.93 (d, J = 8.1 Hz, 1H), 6.23 (s, 1H), 4.90 (m, 2H), 3.58 (d, J = 4.2 Hz, 2H). 13C NMR (150 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6): δ 169.85, 166.20, 163.43, 161.80, 145.38, 143.06, 141.84, 135.15, 131.29, 131.25, 129.99, 129.27,
128.52, 128.39, 128.16, 127.41, 127.30, 127.22, 124.29, 124.14, 116.36, 116.22, 109.38, 65.07, 56.20. HRMS
(+ESI) calcd for C27H21FN4O3, [M+H]+, 469.1598; found 469.1722.
5.1.1.18.
(3Z)-3-((2-fluorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-ca
1
rboxamide (10i). Orange solid, yield 57%; m.p.:100.5-103.2 °C; H NMR (600 MHz, DMSO-d6): δ 14.85 (s, 1H),
11.60 (s, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.78 (ddd, J = 8.2, 2.6, 1.7 Hz, 1H), 7.62-7.59 (m, 1H), 7.56-7.50 (m, 1H),
7.40-7.18 (m, 9H), 6.95 (d, J = 8.1 Hz, 1H), 6.22 (s, 1H), 4.93-4.84 (m, 2H), 3.63-3.55 (m, 2H). 13C NMR (150
MHz, DMSO-d6): δ 169.76, 166.09, 144.63, 143.05, 141.80, 136.82, 136.71, 132.81, 131.34, 131.01, 128.72,
128.70, 128.52, 127.39, 127.25, 123.87, 123.83, 123.71, 123.68, 122.53, 121.22, 109.56, 109.54, 63.53, 56.20.
HRMS (+ESI) calcd for C27H21FN4O3, [M+H]+, 469.1598; found 469.1897.
5.1.1.19.
(3Z)-3-((2,4-difluorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5
1
-carboxamide (10l). Yellow solid, yield 71%; m.p.:129.7-131.3 °C; H NMR (600 MHz, DMSO-d6): δ 14.80 (s,
1H), 11.62 (d, J = 2.2 Hz, 1H), 8.26 (t, J = 7.5 Hz, 1H), 7.83 (dd, J = 8.1, 1.7 Hz, 1H), 7.56 (dd, J = 28.0, 20.6 Hz,
1H), 7.49-7.36 (m, 1H), 7.31 (d, J = 4.4 Hz, 2H), 7.28-7.18 (m, 4H), 6.96 (d, J = 8.1 Hz, 3H), 6.37 (d, J = 5.6 Hz,
1H), 4.99-4.81 (m, 2H), 3.66 – 3.55 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 169.69, 166.04, 144.60, 143.17,
141.86, 135.73, 132.82, 132.34, 130.13, 128.66, 128.52, 127.34, 127.32, 127.22, 123.80, 123.75, 122.91, 121.34,
112.86, 112.72, 109.59, 105.30, 105.18, 65.07, 56.27. HRMS (+ESI) calcd for C27H20F2N4O3, [M+H]+, 487.1503;
found 487.1667.
5.1.1.20.
(3Z)-3-((3,4-difluorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5
1
-carboxamide (10m). Orange solid, yield 68%; m.p.:231.1-233.8 °C; H NMR (600 MHz, DMSO-d6): δ 14.76 (s,
1H), 11.54 (s, 1H), 8.36 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.60 (dq, J = 17.4, 8.6 Hz, 4H), 7.35-7.28 (m,
4H), 7.27-7.20 (m, 2H), 6.96 (d, J = 8.1 Hz, 1H), 6.54 (s, 1H), 4.95 (dd, J = 13.6, 7.6 Hz, 2H), 3.63-3.55 (m, 2H).
13C NMR (150 MHz, DMSO-d6): δ 169.30, 166.06, 151.15, 149.60, 149.54, 149.45, 144.09, 143.81, 141.81,
135.12, 129.13, 128.52, 128.49, 127.30, 127.24, 126.62, 124.37, 123.28, 119.01, 118.94, 118.89, 118.83, 109.68,
65.04, 56.25. HRMS (+ESI) calcd for C27H20F2N4O3, [M+H]+, 487.1503; found 487.1611.
5.1.1.21.
(3Z)-3-((4-fluoro-3-chlorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindo
line-5-carboxamide (10p) Yellow solid, yield 55%; m.p.:237.9-239.6 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.83
(s, 1H), 11.56 (s, 1H), 8.20 (s, 1H), 7.80 (d, J = 7.5 Hz, 1H), 7.62-7.53 (m, 2H), 7.53-7.26 (m, 7H), 7.26 – 7.20 (m,
1H), 6.95 (d, J = 8.1 Hz, 1H), 6.31 (s, 1H), 4.99-4.83 (m, 2H), 3.58 (s, 2H). 13C NMR (150 MHz, DMSO-d6): δ
172.09, 168.43, 160.73, 159.09, 147.38, 145.33, 143.99, 142.47, 138.75, 138.73, 133.56, 132.20, 130.75, 130.51,
129.60, 129.48, 129.44, 126.49, 126.14, 124.54, 122.75, 122.63, 120.17, 120.02, 111.73, 65.73, 58.40. HRMS
(+ESI) calcd for C27H20ClFN4O3, [M+H]+, 503.1208; found 503.1315.
5.1.1.22.
(3Z)-3-((4-cyanophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-ca
rboxamide (10d). Orange solid, yield 65%; m.p.:209.6-211.5 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.84 (s, 1H),
11.59 (s, 1H), 8.18 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 6.9 Hz, 2H), 7.84-7.75 (m, 1H), 7.50 (t, J = 21.9 Hz, 3H),
7.36-7.15 (m, 6H), 6.94 (d, J = 8.1 Hz, 1H), 6.17 (s, 1H), 4.93 (dd, J = 13.6, 7.4 Hz, 1H), 4.86 (t, J = 5.3 Hz, 1H),
3.59 (d, J = 5.4 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): δ 169.86, 165.95, 144.84, 143.95, 143.19, 141.85,
140.76, 133.24, 130.26, 128.53, 128.39, 128.34, 127.32, 127.22, 124.37, 123.70, 121.93, 119.38, 111.60, 109.47,
65.06, 56.20. HRMS calcd for C28H21N5O3, [M+H]+, 476.1644; found 476.1750.

ACCEPTED MANUSCRIPT
5.1.1.23.
(3Z)-3-((2-chlorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-ca
1
rboxamide (10j). Yellow solid, yield 60%; m.p.:160.1-163.2 °C; H NMR (600 MHz, DMSO-d6): δ 14.88 (s, 1H),
11.61 (s, 1H), 8.11-8.01 (m, 1H), 7.82 – 7.76 (m, 1H), 7.66-7.58 (m, 2H), 7.54-7.45 (m, 2H), 7.36-7.20 (m, 7H),
6.95 (d, J = 8.1 Hz, 1H), 6.10 (s, 1H), 4.88 (ddd, J = 10.6, 8.8, 5.1 Hz, 2H), 3.63-3.54 (m, 2H). 13C NMR (150
MHz, DMSO-d6): δ 169.96, 165.98, 144.58, 143.00, 141.84, 139.83, 137.60, 132.87, 131.90, 131.71, 130.69,
130.46, 130.25, 128.71, 128.53, 128.39, 128.32, 127.38, 127.26, 123.81, 121.93, 121.17, 109.48, 65.02, 56.19.
HRMS (+ESI) calcd for C27H21ClN4O3, [M+H]+, 485.1302; found 485.1556.
5.1.1.24.
(3Z)-3-((2-methylphenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-5-ca
rboxamide (10k). Yellow solid, yield 65%; m.p.:158.7-160.7 °C; 1H NMR (600 MHz, DMSO-d6): δ 14.99 (s, 1H),
11.55 (s, 1H), 7.85-7.71 (m, 2H), 7.61 (s, 1H), 7.38-7.19 (m, 9H), 7.09 (t, J = 8.0 Hz, 1H), 6.93 (d, J = 8.0 Hz, 1H),
5.96 (s, 1H), 4.89 (dd, J = 7.2, 4.5 Hz, 2H), 3.64-3.55 (m, 2H), 1.94 (s, 3H). 13C NMR (150 MHz, DMSO-d6): δ
170.06, 166.12, 144.92, 142.87, 141.86, 138.67, 135.02, 130.83, 128.93, 128.64, 128.52, 128.29, 128.23, 128.14,
127.41, 127.39, 127.26, 127.06, 124.17, 123.72, 123.65, 121.49, 109.33, 65.03, 56.15, 19.50. HRMS (+ESI) calcd
for C28H24N4O3, [M+H]+, 465.1848; found 465.1949.
5.1.1.25.
(3Z)-3-((2,4-dichlorophenyl)(1H-imidazol-2-yl)methylene)-N-((1S)-2-hydroxy-1-phenylethyl)-2-oxoindoline-
1
5-carboxamide (10n). Yellow solid, yield 63%; m.p.:155.2-156.8 °C; H NMR (600 MHz, DMSO-d6): δ 14.81 (s,
1H), 11.64 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.90-7.78 (m, 2H), 7.58 (ddd, J = 21.3, 8.2, 2.0 Hz, 2H), 7.40 (dd, J =
8.2, 3.5 Hz, 1H), 7.34-7.29 (m, 4H), 7.26-7.18 (m, 2H), 6.97 (d, J = 8.1 Hz, 1H), 6.34 (s, 1H), 4.98-4.85 (m, 2H),
3.60 (qd, J = 11.1, 5.2 Hz, 2H). 13C NMR (150 MHz, DMSO-d6): δ 169.84, 165.91, 144.34, 143.15, 141.87,
138.43, 136.65, 134.38, 133.08, 132.89, 132.13, 129.86, 128.72, 128.61, 128.55, 128.48, 127.36, 127.32, 127.24,
123.85, 123.63, 122.11, 121.33, 109.56, 65.08, 56.26. HRMS (+ESI) calcd for C27H20Cl2N4O3, [M+H]+, 519.0912;
found 519.0976.
5.2. Pharmacological assay
5.2.1. PAK4 HTRF Assay
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 concentrated 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) was added in the assay wells, containing the 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 was measured at 620
nM (Cryptate) and 665 nM (XL665). A ratio was 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)).
5.2.2. 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 fit using the
four-parameter method.
5.3. Cytochrome P450 Inhibition Assay

ACCEPTED MANUSCRIPT
Cytochrome P450 inhibition was evaluated in human liver microsomes (0.25 mg/mL) using five specific
probe substrates (CYP1A2, 10 µM phenacetin; CYP2C9, 5 µM diclofenac; CYP2C19, 30 µM S-mephenytoin;
CYP2D6, 5 µM dextromethorphan; and CYP3A4, 2 µM midazolam) in the presence of multiple concentrations of
the test compound (0.05-50 µM). After pre-incubation at 37 °C for 10 min, the reaction was initiated by the
addition of 20 µL NADPH to a final concentration of 10 mM. The mixture was incubated at 37 °C for 10 min and
the reaction was terminated by the addition of 400 µL of cold stop solution (200 ng/mL tolbutamide and
200 ng/mL labetalol in acetonitrile). After the reactions were terminated the plates were centrifuged, and the
supernatants were analyzed by LC/MS/MS.
5.4. PAK4 Expression, Purification, and Crystallization.
The cDNA encoding the kinase domain (Resides 300 to 591) of human PAK4 was cloned into a modified
pET28b vector with an N-terminal 10xHis tag followed by a SUMO tag. The recombinant protein was
overexpressed in E. coli BL21 (DE3). After overnight induction with 0.2 mM isopropyl β-D-thiogalactoside
(IPTG) at 16 °C in LB medium, the cells were harvested and suspended in buffer (300 mM NaCl, 20 mM Tris, pH
7.5). Then, the cells were lysed with an Emulsiflex C3 (Avestin) high-pressure homogenizer. After centrifugation
at ×32,000 g, the supernatant was applied to a HisTrap column (GE Healthcare) and subjected to ion-exchange
chromatography (HiTrap Q HP). Then, the resultant protein was digested overnight by ULP1 protease. The
post-cleavage mixture was purified with a fresh HisTrap column to remove His-SUMO tags and further purified
using a Superdex 200 gel-filtration column (GE Healthcare). The purified protein was concentrated to 8 mg/mL
and stored at -80 °C for future use.
The PAK4 kinase domain and inhibitors 10a were mixed at a molar ratio of 1:2. After incubation at 4 °C for 1
h, crystallization screens were performed using an Art Robbins Gryphon crystallization robot by mixing equal
volumes of PAK4-inhibitor complex with different screening conditions from commercial crystallization kits.
Data collection and structure determination. The crystals were briefly soaked in a cryo-protectant composed
of reservoir solution supplemented with 20% ethylene glycol and flash frozen in liquid nitrogen for data collection
at 100 K. Data collection was performed at beamline BL19U1 at the Shanghai Synchrotron Radiation Facility. The
diffraction data were indexed, integrated and merged using the HKL2000 software package
(http://www.hkl-xray.com). The structures were solved using the molecular replacement method with the
published PAK4 kinase domain structure (PDB code 2J0I) as a search model. Structure refinement was performed
using PHENIX[53], and iterative manual model building was performed using COOT[54].
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.
The 1H NMR, 13C NMR, and HRMS spectra of compounds 9a-e, 10a-d, and 11a-c; detailed kinase selectivity
data of compound 10a; the crystallographic parameters of X-ray structures (co-crystal structures of 10a bound to
PAK4); predicted ADME properties of 10h, 10i, 10k and 11a.
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Highlights
‹
‹
A series of novel compounds bearing indolin-2-one-5-carboxamide scaffold was synthesized.
Most of this series displayed nanomolar biochemical activity and potent antiproliferative
activity against A549 and HCT116 cells.
‹
‹
‹
‹
Compound 10a was a multi-targeted kinase inhibitor.
An X-ray structure of 10a bound to PAK4 was obtained.
Predicted ADME properties of 10a was reported.
Compound 10a showed weak inhibitory activity against various isoforms of human
cytochrome P450.