Substituted indolin-2-ones as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors: Molecular docking simulation and structure-activity relationship analysis

Article abstract of DOI:10.1016/j.bmc.2013.01.047

A series of novel indolin-2-ones inhibitors against p90 ribosomal S6 protein kinase 2 (RSK2) were designed and synthesized and their structure-activity relationship (SAR) was studied. The most potent inhibitor, compound 3s, exhibited potent inhibition against RSK2 with an IC₅₀ value of 0.5 μM and presented a satisfactory selectivity against 23 kinases. The interactions of these inhibitors with RSK2 were investigated based on the proposed binding poses with molecular docking simulation. Four compounds and six compounds exhibited moderate anti-proliferation activities against PC 3 cells and MCF-7 cells, respectively.

Full text of DOI:10.1016/j.bmc.2013.01.047

Bioorganic & Medicinal Chemistry 21 (2013) 1724–1734
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Substituted indolin-2-ones as p90 ribosomal S6 protein kinase 2
(RSK2) inhibitors: Molecular docking simulation and
structure–activity relationship analysis
⇑
Ye Zhong , Mengzhu Xue , Xue Zhao , Jun Yuan, Xiaofeng Liu, Jin Huang, Zhenjiang Zhao ,
Honglin Li , Yufang Xu
⇑
⇑
Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology,
Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel indolin-2-ones inhibitors against p90 ribosomal S6 protein kinase 2 (RSK2) were
designed and synthesized and their structure–activity relationship (SAR) was studied. The most potent
inhibitor, compound 3s, exhibited potent inhibition against RSK2 with an IC50 value of 0.5 lM and pre-
sented a satisfactory selectivity against 23 kinases. The interactions of these inhibitors with RSK2 were
investigated based on the proposed binding poses with molecular docking simulation. Four compounds
and six compounds exhibited moderate anti-proliferation activities against PC 3 cells and MCF-7 cells,
respectively.
Received 13 December 2012
Revised 21 January 2013
Accepted 23 January 2013
Available online 30 January 2013
Keywords:
RSK2
Kinase inhibitor
Molecular docking
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
RSK2 and exhibits potent inhibition with an IC50 of 15 nM.18 How-
ever, only two crystal structures of RSK2 NTKD complexed with
The 90 kDa ribosomal S6 kinase 2 (RSK2) is a serine/threonine
kinase and belongs to the RSKs family, which possesses an N-ter-
minal kinase domain (NTKD) and a C-terminal kinase domain
(CTKD) connected by a linker region.1–9 Presently, RSK2 has been
verified to be a promising cancer therapeutic target due to its prin-
cipal performances in the regulation of diverse cellular process,
such as cell transformation, proliferation and invasion.3 Moreover,
overexpression of RSK2 is related to many human diseases includ-
ing breast cancer, prostate cancer, and some hematopoietic stem
cell carcinoma.10 These studies indicate the significance of devel-
oping novel and potent RSK2 inhibitors.
To date, dozens of potent RSK2 inhibitors have been reported
(Fig. 1). Among them, SL0101 is the first reported specific inhibitor
of RSK2, which was extracted from the tropical plant Forsteronia re-
fracta and showed an IC50 value of 89 nM.11–13 Some classical pro-
tein kinase inhibitors are also found to be active but nonspecific
against RSK2, such as PKC inhibitors GF109203X and Ro31–8220
obtained from kinase screenings,14 which may provide useful
information to develop RSK2 inhibitors as well.15–17 Moreover,
Fmk is an inhibitor designed to irreversibly bind to the CTKD of
similar inhibitors have been released recently,19 representing one
determinate binding mode of such inhibitors, but none of them
are used in the clinical trials. Therefore, there are growing needs
for developing more potent RSK2 inhibitors to ensure their clinical
applicability.
Using the ligand-based virtual screening program SHAFTS and
structure-based virtual screening protocols, our group discovered
a variety of compounds with potent or moderate inhibitory activ-
ities against RSK2, most of which have novel scaffolds different
from the reported RSK2 inhibitors.20–23 Among them, compounds
with an indolin-2-ones scaffold inspired us to perform structural
screening to improve the inhibitory activity and some of them
were reported as inhibitors against other kinases.24–27 In this
study, the designed novel indolin-2-ones compounds were synthe-
sized and their inhibitory activities against RSK2 were evaluated.
Most of these compounds showed inhibitory potential of RSK2,
and the most potent one, compound 3s showed a ꢀ7-fold greater
potency against RSK2 than the previous hit 3a, with an IC50 value
of 0.5 lM. Moreover, compounds 3s exhibited a satisfactory selec-
tivity against 23 kinases. The structure–activity relationship (SAR)
was analyzed and docking simulations were carried out to explore
the binding modes of these inhibitors. Furthermore, 9 potent com-
pounds were chosen for cellular studies. In the anti-proliferation
assays on PC 3 prostate cancer cells and MCF-7 breast cancer cells,
4 compounds and 6 compounds exhibited significant inhibitions
⇑
Corresponding authors. Tel.: +86 21 64250213 (H.L.); fax: +86 21 6425021.
E-mail addresses: zhjzhao@ecust.edu.cn (Z. Zhao), hlli@ecust.edu.cn (H. Li),
yfxu@ecust.edu.cn (Y. Xu).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.047

Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
1725
Table 2
on proliferations of PC 3 cells and MCF-7 cells, respectively, whilst
Compounds 3q–z with RSK2 inhibitory activities
four compounds could both suppress proliferations of the two cell
lines at the same digital level.
N
NH
O
R4
2. Results and discussion
2.1. Chemistry
R1
N
H
Synthesis of the compounds listed in Tables 1 and 2 is shown in
Scheme 1, which involved two steps: (1) in the presence of sodium
hydroxide, isatin or 5-substituted isatin was treated with hydra-
zine hydrate in ethanol at reflux temperature for 3–4 h to afford
indolin-2-one (2a–d);28 (2) purified indolin-2-one was dissolved
in ethanol with aldehydes,29,30 then the mixture was heated and
refluxed for 2–12 h. The precipitates were filtered to give crude
products 3a–z.28 The terminal products were separated and puri-
fied by recrystallization or silica gel chromatography and the struc-
tures were characterized by 1H NMR and HRMS (EI).
Compounds 4a–4m in Table 3 were prepared as described in
Scheme 2. The starting material was isatin or 5/7-subtitued isatin
with various 2-thioxothiazolidin-4-one/aromatic amines/hydra-
zones.31 The 5-(7-methyl-2-oxoindolin-3-ylidene)-2-thioxothiaz-
olidin-4-one (4a) and the 5-(7-ethyl-2-oxoindolin-3-ylidene)-2-
thioxothiazolidin-4-one (4b) were synthesized according to the
methods presented in the literature, whereas 4c–m were pur-
chased from a commercial reagent company.
a
Compound
R1
R4
Inhibitory rate (%)
IC50 (lM)
3q
3r
3s
3t
3u
3v
3w
3x
3y
3z
H-
H-
H-
F-
Ph-
79.48
38.56
93.36
78.43
<10
33.57
<10
33.25
44.46
<10
2.72
ND
0.5
8.59
ND
ND
ND
ND
ND
ND
4-FPh-
2-NO2Ph
Ph-
4-FPh-
Ph-
4-FPh-
Ph-
4-FPh-
4-ClPh-
F-
Br-
Br-
Cl-
Cl-
Cl-
a
IC50 values were measured if the inhibition rate at 10 lM was larger than 50%,
and ND represented not determined.
OH
HO
O
O
CH3
OAc
O
2.2. Structure–activity relationships
O
O
HO
O
OAc
N
H
Derived from the previously discovered hit 3a,22 structural opti-
mizations underwent three phases, as illustrated in Figure 2.
Firstly, the furan ring was reserved with the indolin-2-one scaffold,
and common substituent groups were introduced onto the furan
ring and the phenyl of the indolin-2-one separately or simulta-
neously. Secondly, the furan ring was replaced by its bioisostere
pyrazole, and then similar substituent groups were studied as
OH
SL0101
3a
O
O
HN
HN
N
NH
O
O
Table 1
N
Compounds 3a–p with RSK2 inhibitory activities
N
S
NH
N
R3
O
H2N
GF109203x
Ro31-8220
N
N
R1
O
H2N
N
F
N
H
OH
O
a
Compound R1
R3
Inhibitory rate (%) IC50
(
lM)
3a
3b
3c
3d
3e
3f
H-
H-
H-
H-
2-ClPh-
3-BrPh-
2,6-2Cl-4-CF3Ph-
69.59
84.75
87.35
16.83
21.89
<10
3.47b
Cl-
Br-
H-
H-
H-
2.02
1.51
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
2.49
ND
ND
Fmk
Figure 1. The reported specific RSK2 inhibitor and the hit compound 3a.
3g
3h
3i
3j
3k
3l
H-
O-PhthalimideCH2- 16.47
stated above. Finally, only the indolin-2-one was fixed, while var-
ious substituents were introduced onto the scaffold for diversity.
To evaluate the structure–activity relationship (SAR), the inhib-
itory activities against RSK2 for indolin-2-ones including synthe-
sized ones and commercial purchased ones were analyzed and
discussed (Tables 1–3). As a whole, 9 compounds (3a–3c, 3n, 3q,
3s, 3t, 4a, 4b) exhibited moderate inhibitory potencies against
Br-
Br-
Cl-
Cl-
F-
2-ClPh-
3-BrPh-
2-ClPh-
3-BrPh-
2-ClPh-
3-BrPh-
H-
22.28
<10
32.11
<10
17.12
26.29
100%
29.84%
25.63%
3m
3n
3o
3p
F-
NH2-
NO2-
H-
CH3O- H-
RSK2 with IC50 values less than 10
lM, and the inhibitory poten-
cies were substantially improved, especially compound 3s reached
a 10-fold (IC50 = 0.5 lM) improvement in potency compared to the
lead compound 3a.
a
IC50 values were measured if the inhibition rate at 10 lM was larger than 50%,
and ND represented not determined.
b
The IC50 value of 3a in previous report was 7.76 l
M.22

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Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
R2
CH
O
R1
R1
R1
a
b
O
O
O
NH
NH
NH
O
R3
R4
3a-p: R2
3q-z: R2
=
1
2a-d
=
N
NH
Scheme 1. Reagents and conditions: (a) Hydrazine hydrate/sodium hydroxide, EtOH/100 °C; (b) aldehydes, piperidine, EtOH/reflux.
Table 3
Compounds 4a–p with RSK2 inhibitory activities
R7
R1
R5
O
N
H
R6
R7
a
Compound
R1
H-
R5
H-
R6
Inhibitory rate (%)
92.36
IC50 (lM)
O
O
O
O
NH
4a
CH3-
1.68
17
S
S
NH
4b
4c
4d
H-
H-
H-
H-
Cl-
Cl-
CH3CH2-
CH3-
50.11
39.58
18.43
S
S
NH
ND
ND
S
S
NH
H-
NH
S
S
O
S
N
N
H
4e
4f
CH3-
H-
H-
H-
H-
41.07
<10
ND
ND
O
N
S
Cl-
N
O
O
H
N
N
H
4g
4h
F-
H-
H-
H-
H-
<10
ND
ND
H
N
O
Br
11.48
N
H
H
N
4i
H-
H-
H-
<10
ND
N
Cl
HN
O
H
N
O
O
4j
Br-
F-
H-
H-
H-
H-
<10
ND
ND
N
H
H
N
4k
30.05
N
H

Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
1727
M)
Table 3 (continued)
a
Compound
R1
H-
R5
H-
R6
H-
R7
Inhibitory rate (%)
<10
IC50
ND
(l
H
N
4l
NH2
O
S
H
N
N
H
Cl
4m
4n
CH3CH2-
H-
H-
H-
H-
<10
<10
ND
ND
O
H
N
F-
N
H
O
N
N
4o
H-
H-
H-
H-
H-
H-
<10
ND
ND
I
O
4p
13.42
N
N
O
a
IC50 values were measured if the inhibition rate at 10
lM was larger than 50%, and ND represented not determined.
R7
R3
O
R1
R1
R5
c
N
NH
O
O
N
O
R4
O
R5
N
H
H
R6
R6
R1
R1
Scheme 2. Reagents and conditions: (c) rhodanine/amines/hydrazones, EtOH/
reflux.
O
N
H
N
H
In detail, modifications were first focused on common substitu-
ent groups which were introduced onto the indolin-2-one and the
furan ring. Consequently comparing with 3b and 3c in Table 1, it
was inferred that a single substituent at the 5-position of the indo-
lin-2-one such as halogen could be accommodated which even
leads to a slight improvement on the inhibitory activity. Further-
more, an amino group substituent (3n) could also be tolerated at
the 5-position of the indolin-2-one, but the introduction of a nitro
group (3o) or a methoxy group (3p) would lead to the abortion of
inhibitory activities. While turning attention to the single substitu-
ent on the furan ring (3d–3g), we noticed that any large group at
the 2-position of the furan ring would lead to an obvious decrease
in the inhibitory activity against RSK2. And the deterioration in
activity would be sustained when substituents were introduced
onto both sites (3h–3m), especially, the inhibitory ratios would re-
duce more in the case that additional substituent groups were ex-
tended from 3-position instead of 2-positon at the phenyl
introduced onto the 2-position of the furan group (3i, 3k). But
the tendency would be reversed when it was a smaller fluorine
group compared with larger chlorine or bromine groups at the 5-
position of the indolin-2-one (3m), which was also observed in
the cases of hydrogen substituents 3d and 3e.
O
O
N
H
R7
R1
O
N
H
R5
R6
Figure 2. The structural modification progress derived from the hit 3a.
Then the furan ring was replaced by a pyrazole ring and a sim-
ilar investigation was undertaken. For 3q in Table 2, we found that
the replacement did not result in a big change but brought a slight
boost in the activity. And the phenyl here at the 3-position of the
pyrazole could be accepted, whereas additional substituents on
the phenyl affected activities of derived compounds 3r and 3s to
a larger extent. The observation indicated that electron-withdraw-
ing groups introduced onto the phenyl were accommodated,
but the 2-position might be superior for the derivation.
Moreover, when it was referred to 3t, 3v and 3x, the introduction
of an electron-withdrawing group like halogen into the 5-position

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Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
Table 4
The anti-proliferative activities of indolin-2-one compounds
Compound
Structure
IC50
(lM) against RSK2
IC50 in PC 3 cellsa
(l
M)
IC50 in MCF-7 cellsb
(lM)
O
3a
3.47
<10% at 100
l
l
M
M
30.07
O
N
H
H
S
S
N
O
O
S
4a
1.68
<10% at 100
41.46% at 100
lM
O
N
H
H
N
S
4b
17
40.89% at 100
lM
15.98% at 100
lM
O
N
H
N
NH
3q
2.72
6.19
8.55
O
O
N
H
NO2
N
NH
3s
0.5
9.32
14.84
N
H
O
3c
1.51
2.02
16.89
23.91
9.84
Br
Cl
O
O
N
H
O
3b
14.67
N
H
N
NH
3t
8.59
<10% at 100
l
M
<10% at 100 lM
F
O
N
H

Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
1729
M)
Table 4 (continued)
Compound
Structure
IC50
(l
M) against RSK2
IC50 in PC 3 cellsa
(
lM)
IC50 in MCF-7 cellsb
(l
O
3n
2.49
<10% at 100
1.74
l
M
15.54
1.96
H2N
O
N
H
Ro31-8220
a
All measurements were determined in PC 3 prostate cancer cells, IC50 values were measured if the inhibition rate at 100
All measurements were determined in MCF-7 breast cancer cells. IC50 values were measured if the inhibition rate at 100
l
l
M was larger than 50%.
M was larger than 50%.
b
of indolin-2-one here was not beneficial to the activity but the
Table 5
Selectivity of compound 3s versus a panel of 23 kinases
smaller fluorine group could affect less, which was different from
the observations in Table 1. As a result, with halogen groups simul-
taneously introduced into the 5-position of the indolin-2-one and
the 4-position of the phenyl, such derivatives as 3w, 3y and 3z, al-
most lost their inhibitory potencies against the enzyme.
b
Kinases
Inhibitory rate (%) with two compound
Control IC50 (nM)
concentrationsa
(lM)
10
3
RSK2
80%
13%
6.2%
71%
55%
49%
64%
4.7%
65%
65%
38%
70%
65%
55%
67%
61%
4.4%
6.7%
44%
31%
25%
55%
4.2%
44%
44%
20%
41%
39%
30%
43%
25%
12%
31%
7.8%
36%
37%
19%
31%
1.6
0.98
8.3
0.75
3.3
4.0
10
54
25
12
1.2
0.4
7.7
7.9
271
2.3
109
4.0
11
Lastly, as listed in Table 3, we tried other heterocyclic and non-
conjugate groups instead of the original furan group and inspected
substituents at other positions on the indolin-2-one scaffold. Gen-
erally speaking, non-conjugate groups derived compounds such as
4g, 4h, 4m and 4n significantly attenuated their inhibitory activi-
ties. Since kinase inhibitors like the indolin-2-ones usually bound
to the ATP site of a kinase, and mimicked the flat, hydrophobic nat-
ure of the adenine, it was supposed that the non-conjugate groups
might seriously break the coplanarity of the whole molecule and
thus hampered its inhibitory activity. Consequently, the 2-thioxo-
thiazolidin-4-one derivatives showed expected superior potencies.
For corresponding compounds 4a–4e, it was clear that a substitu-
ent not larger than a methyl at the 7-position of the scaffold could
be helpful, contrasting to a 10-fold decrease in the activity derived
from the ethyl substituent, whereas substituents at other positions
of the scaffold almost weakened activities.
PKCa
PDK1
p70S6K
LCK
Lyn A
cKit
PIM1
FGFR3
FGFR1
PDGFR
PDGFRb
KDR
a
SRC
ABL
Aurora B 34%
c-MET
MSK1
AKT1
JNK2
16%
33%
8.3%
71%
40%
27%
57%
5028
13
11
GSK3b
p38a
2.3. Anti-proliferative activity studies
ERK1
1146
a
Kinases selected for screening here were from Shanghai ChemPartner Co., Ltd.
Staurosporine and PI 103 were taken as the positive control for the kinase
For further study, 9 potent compounds from the above enzyme
assays were picked out to assess the anti-proliferation activities
against the human prostate cancer cell line PC 3 and the breast
cancer cell line, MCF-7. According to the data listed in Table 4
and 4 compounds and 6 compounds exhibited potent inhibitions
on proliferations of PC 3 cells and MCF-7 cells, respectively, whilst
4 compounds could both suppress proliferations of the two cell
lines with equivalent inhibitory activities. As to the structure, com-
pounds of furan and pyrazole series almost showed anti-prolifera-
tion activities on at least one cancer cell line except 3t, whereas 2-
thioxothiazolidin-4-one derivatives lost inhibitory activities at the
cellular level. Besides, the MCF-7 cells seemed to be more sensitive
to compounds of furan series, but various substituents on the indo-
lin-2-one scaffold didn’t affect the anti-proliferation activity
greatly.
b
screening study.
2.4. Kinase profiling study
The most potent compound 3s was further investigated for
selectivity versus a panel of 23 kinases at two concentrations (Ta-
ble 5). In details, at a lower concentration of 3 lM, the compound
exhibited satisfactory selectivity with inhibitory rate values almost
less than 50%, except for the one of 55% against c-Kit. But at a high-
er concentration of 10 lM, it turned to be promiscuous to multiple
kinases which showed inhibitory rate values more than 50% over
12 kinases. From the inhibitory activities against kinases of differ-
ent families based on the human protein kinases phylogenetic tree
Figure 3. Selectivity of compound 3s against 23 kinases at the concentration of
10
kinases phylogenetic tree by Manning et al. And they are exhibited in orders as:
RSK2, AKT1, PKC , p70S6K, MSK1, PDK1, Aurora B, PIM1, JNK2, p38 , ERK1, GSK3b,
ABL, LCK, Lyn A, SRC, c-MET, FGFR1, FGFR3, KDR, cKit, PDGFR , PDGFRb.
lM. The kinases were classified into four families based on the human protein
a
a
a

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Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
Figure 4. Binding models of the representative inhibitors. The proposed binding modes of compounds (stick, green carbon) 3c (A), 3n (B), 3q (C), 3s (D) and 4a (E) to the RSK2
NTKD homology model (wire, yellow carbon). The nitrogen and oxygen atoms are colored in blue and red, respectively. Hydrogen atoms are hidden for clarity. Potential
intermolecular hydrogen bonds are shown in red dashed lines. (C) The docked 3q and protein residues are shown in golden, while the furan substituent lead 3a in comparison
is colored in green. (D) The docked 3s is colored in green, whilst protein residues and 3q are shown in yellow.
by Manning et al. (Fig. 3),32 it is found that the compound is prone
to exhibit higher inhibitory activities versus members of AGC ki-
nase family such as RSK2 and p70S6K. Interestingly, it also shows
higher degree of inhibitory activities against members of TK and
CMGC kinase families which are far from the AGC kinase family
on the phylogenetic tree. In particular, the compound can result
in an inhibitory rate value of 70% to PDGFRb, but only 38% to the
2.5. Docking studies
To investigate the structural basis for the binding of the indolin-
2-ones to the RSK2 NTKD ATP-binding site, the compounds 3c, 3n,
3q, 3s and 4a were picked out for the docking simulation and the
binding poses of them were proposed, as illustrated in Figure 4.
Although there have been two crystal structures of RSK2 NTKD
complexed with inhibitors which have been released recently,
the ligands in the complexes are almost identical to each other
but very different from the indolin-2-one series here, which are
reported to induce the protein moiety to make unprecedented
homology PDGFRa. So it could be concluded that the affinity pro-
file might not be driven by kinase homology but greatly by the
chemotype of a compound, which is consistent with other reported
observations.33

Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
1731
structural rearrangements.19 In addition, in many other reports on
the indolin-2-one scaffold as kinase inhibitors, it was found that
the scaffold bound to kinases nearly in the same way as the inter-
actions of staurosporine to kinases.27,34–41 Thus we herein used the
previously reported RSK2 NTKD homology model complexed with
staurosporine.
In consistent with other RSK2 inhibitors we reported previ-
ously,22 3c binds to the enzyme in a similar manner (Fig. 4A), ex-
cept that the bromine at 5-position of the indolin-2-one extends
into a hydrophobic pocket constituted by Leu 147, Lys 100 and
Thr 210. In the case of 3n (Fig. 4B), it keeps the key hydrogen bond
interactions like 3c and formed an additional hydrogen bond with
Thr 210 as a donor. Thus the inhibitory activities of the nitro group
and the methoxy group substituent derivatives (3o and 3p) were
attenuated greatly. In addition, by the superimposition of 3a and
3q in Figure 4C, it should be noted that the furan ring located close
to the entrance of the ATP binding pocket, thus a large substituent
at a lower concentration of 3
l
M, but it showed inhibitory rate val-
ues more than 50% over 12 compounds at a higher concentration of
10 M. The proposed binding modes of these compounds were
l
subsequently explored to elucidate their SAR. Furthermore, cellular
assays confirmed that 4 compounds and 6 compounds exhibited
significant inhibitions on proliferations of PC 3 cells and MCF-7
cells, respectively, whilst 4 compounds could both suppress prolif-
erations of the two cell lines in the same digital level. To the best of
our knowledge, there are no public reports that indoline-2-ones
can act as RSK2 inhibitors, which exhibited moderate or potent
potencies. Therefore, the challenge remains to identify derivatives
which show greater potency and serve as novel lead compounds
for further development of therapeutics to treat cancer, especially
to suppress cell migration.
4. Experimental section
4.1. Chemistry
like a phenyl at the other
a position of the furan ring may make the
molecule stretch beyond the pocket. Thus such derivatives as 3d–
3m lost activities dramatically.
4.1.1. General methods
As for the pyrazole replaced 3q (Fig. 4C), the indolin-2-one scaf-
fold is found to form two hydrogen bonds with the hinge residues
Asp 148 and Leu 150 as it is in the lead 3a, and the pyrazole occu-
pies the same location but with different angles as the furan; more-
over, the extended phenyl here can contact the protein by VDW
interactions with Leu 200 which therefore is helpful to the activity.
Besides, noting that pyrazole derivatives (3q) exhibit superior pla-
narity to furan ones (3a) in Figure 4C, which probably reflects the
electronic conjugate profile in the former is more complete. Thus
substituent electron-withdrawing groups like halogen atoms in
pyrazole ones would reduce the electronic distribution in the
whole conjugate system efficiently to attenuate the key hydrogen
bond to Leu 150 and further result in a decrease in activities.
Accordingly, halogen substituents in the 5-position of indolin-2-
one here could impair binding affinities by intense electron-with-
drawing effects, especially Cl- and Br- ones (3x and 3v), compared
to those in furan series, might exhibit less electron-withdrawing
effects but more hydrophobic effects (3b and 3c). With respect to
the analogue 3s (Fig. 4D), it conserves the basic interaction profile
of 3q, but the nitro group at the 2-position of the phenyl can de-
stroy planarity of the whole molecule and form an additional
hydrogen bond with Lys 72, thus force the molecule to shift close
to the hinge with the corresponding hydrogen bond distances
shorten from 2.99 Å and 2.85 Å induced by 3q to 2.90 Å and
2.54 Å here (Fig. 4C), which may both make significant contribu-
tions to the great improvement in the activity for 3s.
All reagents were commercial quality without further purifi-
cation. Solvents were analytical grade, and were subsequently
purified and dried by standard methods according to reaction
conditions. 1H NMR and 13C NMR were measured on a Bruker
AV-400 spectrometer using TMS as internal standard. The chem-
ical shifts were reported as ppm (in DMSO-d6, CD3OD). Melting
points were measured on an X6 apparatus, which were uncor-
rected. High-resolution EI mass spectra were reported on
a
HPLC-Q–Tof MS (Micro) spectrometer. All the compounds were
visualized by UV illumination (254 nm). Analytical thin-layer
chromatography was performed with glass-baked silica gel
plates. HPLC was performed by using a Zopbax RX-C18 column
(packing: 5
l
m, 4.6 Â 150 mm) with a mobile phase consisting
of H2O/CH3CN at a flow rate of 1.0 mL/min. All stereochemistry
were confirmed according to synthesis methods reported by
Sun et al.28
4.1.2. Method A: General procedure for the preparation of
compounds 3a–o
The preparation was divided into two steps. Firstly, substituent
indoline-2,3-dione (1 equiv) was dissolved in ethanol (10 mL),
hydrazine hydrate (2 mL) was added. The reaction mixture was re-
fluxed with magnetic stirring for 3 h and cooled to 0 °C, then so-
dium hydroxide (3 equiv) was added, refluxed for 0.5 h and
cooled to room temperature, then 150 mL water was added. The
mixture was acidified by adding 2 N hydrochloric acid to pH 2,
and extracted twice with dichloromethane. The organic filtrate
was washed twice with brine, dried (Na2SO4), and then concen-
trated to give corresponding indolin-2-one by high-vacuum drying.
Secondly, a mixture of the corresponding indolin-2-one (1 equiv)
and furan-2-carbaldehyde (1 equiv) in ethanol was added two
drops of piperidine and refluxed with magnetic stirring for 5 h.
After the mixture cooled, the precipitate was filtered, washed with
cold ethanol, dried, and recrystallized from methanol to afford the
terminal product.
Similar to the above analysis, 4a binds to the enzyme in the
same region of the ATP binding site (Fig. 4E), noting that the
methyl at the 7-position of the indolin-2-one scaffold fits well into
the binding site to form VDW interactions and hydrophobic effects
with the residues Leu 147 and Val 131. Meanwhile, the hydropho-
bic sub-pocket comprised by the referred residues is found to be
small and hard to accommodate groups larger than a methyl, so
the ethyl substituent results in an intense decrease in the activity
of 4b.
3. Conclusions
4.1.2.1.
(3b).
(E)-6-Chloro-3-(furan-2-ylmethylene)indolin-2-one
Yellow powder (yield 45%), mp: 253–254 °C, 1H NMR
In summary, through structural optimizations of the previously
identified hit, a series of indolin-2-ones analogues were designed
and synthesized as potential RSK2 inhibitors. Most of these com-
pounds showed potential RSK2 inhibitory activity, and the most
potent one, compound 3s showed a ꢀ7-fold increased activity than
(400 MHz, DMSO-d6) d 10.87 (s, 1H), 8.31 (d, J = 2.0 Hz, 1H), 8.27
(d, J = 1.2 Hz,1H), 7.41 (s, 1H), 7.34 (d, J = 3.2 Hz, 1H), 7.32–7.30
(m, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.84 (dd, J1 = 1.6 Hz, J2 = 3.6 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) d 169.47, 150.96, 148.47,
141.74, 129.55, 125.81, 124.28, 123.35, 122.61, 121.44, 121.20,
114.25, 111.51; HRMS (EI) [M]+: Calcd for C13H8NO2Cl: 245.0244,
Found: 245.0244.
hit compound, with the IC50 value of 0.5 lM. In addition, the
compound 3s exhibited a satisfactory selectivity versus 23 kinases

1732
Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
4.1.2.2. (E)-6-Bromo-3-(furan-2-ylmethylene) indolin-2-one
(3c).
Yellow powder (yield 72%), mp: 260–261 °C, 1H NMR
(400 MHz, DMSO-d6) d 10.71 (s, 1H), 8.44 (d, J = 1.6 Hz, 1H), 8.27
(s, 1H), 7.45–7.42 (m, 1H), 8.27 (s, 1H), 7.34 (d, J = 3.6 Hz, 1H),
6.87–6.84 (m, 2H). HRMS (EI) [M]+: Calcd for C13H8NO2Br:
288.9738, Found: 288.9733.
J = 2.0 Hz, 1H), 8.11 (s, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.64 (d,
J = 8.8 Hz, 1H), 7.51–7.47 (m, 3H), 7.39 (s, 1H), 7.33 (dd,
J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 6.91 (d, J = 8.0 Hz, 1H); HRMS (EI)
[M]+: Calcd for C19H11NO2ClBr: 400.9641, Found: 400.9639.
4.1.2.9. (E)-5-Amino-3-(furan-2-ylmethylene) indolin-2-one
(3n).
The reduction product of 3o. Red powder (yield 80%),
4.1.2.3. 3-((5-(2-Chlorophenyl)furan-2-yl)methylene)indolin-2-
one (3d).
mp: 177.8–178.0 °C, 1H NMR (400 MHz, DMSO-d6) d 10.08 (s,
1H), 8.02 (s, 1H), 7.75 (d, J = 1.2 Hz, 1H), 7.23 (s, 1H), 7.20–7.19
(d, J = 3.6 Hz, 2H), 6.80–6.79 (m, 1H), 6.57–6.51 (m, 2H); 13C
NMR (100 MHz, DMSO-d6) d 169.60, 151.27, 146.94, 143.75,
133.75, 124.10, 122.25, 120.19, 118.89, 115.83, 113.85, 112.09,
110.27; HRMS (EI) [M]+: Calcd for C13H10N2O2: 226.0742, Found:
226.0743.
Orange powder (yield 90%), mp: 250–251 °C, 1H
NMR (400 MHz, DMSO-d6) d 10.60 (s, 1H), 8.39 (d, J = 7.6 Hz, 1H),
8.02–7.99 (m, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.61–7.57 (m, 1H),
7.52–7.48 (m, 1H), 7.45 (d, J = 3.6 Hz, 1H), 7.38–7.36 (m, 2H),
7.27 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.90 (d, J = 7.6 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) d 169.58, 153.69, 151.22,
142.93, 131.60, 130.98, 130.61, 130.42, 128.72, 128.45, 128.24,
124.48, 123.28, 123.12, 121.72, 119.20, 115.14, 110.31; HRMS
(EI) [M]+ Calcd for C19H12NO2Cl: 321.0557, Found: 321.0560.
4.1.2.10.
(3o).
(E)-3-(Furan-2-ylmethylene)-5-nitroindolin-2-one
Yellow powder (56%), mp: 305.8–306.3 °C, 1H
NMR(400 MHz, DMSO-d6) d 11.26 (s,1H), 9.10 (d, J = 2.0 Hz, 1H),
8.25 (s, 1H), 8.16 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 7.45 (s, 1H),
7.39 (d, J = 3.2 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 6.87–6.85 (m, 1H);
13C NMR (100 MHz, DMSO-d6) d 169.96, 150.84, 148.80, 148.43,
142.34, 126.31, 123.63, 122.24, 121.972, 120.26, 119.81, 114.57,
110.05; HRMS (EI) [M]+: Calcd for C13H8N2O4: 256.0484, Found:
256.0486.
4.1.2.4. 3-((5-(3-Bromophenyl)furan-2-yl)methylene)indolin-2-
one (3e).
Citrus red powder (yield 90%), mp: 256–257 °C, 1H
NMR (400 MHz, CD3OD) d 10.60 (s, 1H), 8.44 (d, J = 8.0 Hz, 1H),
8.12 (s, 1H), 7.91 (d, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.54–7.51 (m,
1H), 7.45 (d, J = 3.6 Hz, 1H), 7.41 (d, J = 3.6 Hz, 1H), 7.34 (s, 1H),
7.31(t, J = 7.6 Hz, 1H) 7.09 (t, J = 7.6 Hz, 1H), 6.92 (d, J = 8.0 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) d 169.60, 155.61, 151.52,
142.90, 132.01, 131.75, 130.42, 126.95, 123.96, 123,83, 123.32,
123.09, 122.82, 121.76, 121.51, 119.17, 111.82, 110.41; HRMS
(EI) [M]+: Calcd for C19H12NO2Br:365.0051, Found: 365.0055.
4.1.2.11. (E)-3-(Furan-2-ylmethylene)-5-methoxyindolin-2-one
(3p).
Brown powder (yield 60%), mp: 201.8–202.8 °C, 1H NMR
(400 MHz, DMSO-d6) d 10.37 (s, 1H), 8.22 (s, 1H), 8.00 (d, J = 2.0 Hz,
1H), 7.34 (s, 1H), 7.27 (d, J = 3.2 Hz, 1H), 6.87 (dd, J1 = 2.4 Hz,
J2 = 8.4 Hz, 1H), 6.82–6.78 (m, 2H), 3.79 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) d 169.77, 154.92, 151.14, 147.86, 136.81,
123.11, 122.54, 121.44, 119.92, 115.21, 114.01, 111.45, 110.39;
HRMS (EI) [M]+: Calcd for C14H11NO3: 241.0739, Found: 241.0736.
4.1.2.5. (Z)-5-Bromo-3-((5-(2-chlorophenyl)furan-2-yl)methy-
lene) indolin-2-one (3h).
Citrus red powder (yield 92%),
mp: 279–280 °C, 1H NMR (400 MHz, DMSO-d6) d 10.74 (s, 1H),
8.61 (d, J = 1.6 Hz, 1H), 8.07 (dd, J1 = 1.2 Hz, J2 = 8.0 Hz, 1H), 7.68
(d, J = 8.0 Hz, 1H), 7.60–7.56 (m, 1H), 7.52–7.50 (m, 2H), 7.45–
7.43 (m, 3H), 6.86 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) d 169.21, 154.16, 150.95, 141.88, 132.42, 131.63, 131.13,
130.65, 128.59, 128.43, 127.88, 126.71, 124.66, 123.77, 121.72,
120.44, 115.36, 113.53, 112.036; HRMS (EI) [M]+: Calcd for
4.1.2.12. (Z)-3-((3-Phenyl-1H-pyrazol-4-yl) methylene) indolin-
2-one (3q).
Light yellow powder (yield 87%), mp: 228–229 °C,
1H NMR (400 MHz, DMSO-d6) d 13.65 (br, 1H), 10.53 (s, 1H), 8.37
(br, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.59 (d, J = 6.8 Hz, 2H), 7.56–7.52
(m, 2H), 7.49–7.47 (m, 1H), 7.40 (s, 1H), 7.25–7.21 (m, 1H), 6.97–
6.94 (m, 1H), 6.88 (d, J = 7.6 Hz, 1H); HRMS (EI) [M]+: Calcd for
C18H13N3O:287.1059, Found: 287.1062.
C19H11NO2ClBr: 400.9641, Found: 400.9643.
4.1.2.6. 5-Bromo-3-((5-(3-bromophenyl)furan-2-yl)methylene)
indolin-2-one (3i). Citrus red powder (yield 89%), mp: 275–
276 °C, 1H NMR (400 MHz, DMSO-d6) d 10.74 (s, 1H), 8.63 (d,
J = 1.6 Hz, 1H), 8.14 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.65 (d,
J = 8.4 Hz, 1H), 7.54–7.46 (m, 4H),7.40 (s, 1H), 6.88 (d, J = 8.4 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) d 169.22, 156.40, 151.39,
141.79, 132.53, 132.35, 131.82, 131.51, 126.89, 126.26, 125.36,
123.87, 123.55, 123.35, 121.28, 120.43, 113.46, 112.16, 112.12;
HRMS (EI) [M]+: Calcd for C19H11NO2Br2: 444.9136, Found:
444.9144.
4.1.2.13. (Z)-3-((3-(2-Nitrophenyl)-1H-pyrazol-4-yl) methylene)
indolin-2-one (3s).
Light yellow powder (yield 90%), mp:
263–264 °C, 1H NMR (400 MHz, DMSO-d6) d 13.58 (br, 1H), 10.62
(s, 1H), 9.41 (s, 1H), 8.10–8.08 (m, 1H), 7.89–7.86 (m, 1H), 7.81–
7.75 (m, 1H), 7.70 (dd, J1 = 1.2 Hz, J2 = 7.6 Hz, 1H), 7.34 (d,
J = 7.2 Hz, 1H), 7.21–7.15 (m, 2H), 6.91 (t, J = 7.6 Hz, 1H), 6.84 (d,
J = 7.6 Hz, 1H); HRMS (EI) [M]+: Calcd for C18H12N4O3: 332.0909,
Found: 332.0909.
4.1.2.7. (Z)-5-Chloro-3-((5-(2-chlorophenyl)furan-2-yl)methy-
4.1.2.14. (Z)-5-Fluoro-3-((3-phenyl-1H-pyrazol-4-yl) methylene)
lene)indolin-2-one (3j).
Citrus red powder (yield 86%), mp:
indolin-2-one (3t).
Orange powder (yield 92%), mp: 259–
286–287 °C, 1H NMR (400 MHz, DMSO-d6) d 10.73 (s, 1H), 8.49
(d, J = 2.0 Hz, 1H), 8.07 (dd, J1 = 1.6 Hz, J2 = 7.6 Hz, 1H), 7.69 (dd,
J1 = 1.2 Hz, J2 = 7.6 Hz, 1H), 7.56–7.49 (m, 3H), 7.44 (s, 1H), 7.42
(d, J = 3.6 Hz, 1H), 7.32 (dd, J1 = 2.0 Hz, J2 = 8.4 Hz, 1H), 6.91 (d,
J = 8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 169.35, 154.20,
151.00, 141.53, 131.63, 131.22, 130.74, 129.65, 128.71, 128.30,
128.00, 125.85, 124.57, 124.11, 121.91, 120.50, 115.33, 111.52;
HRMS (EI) [M]+: Calcd for C19H11NO2Cl2: 355.0167, Found:
355.0168.
260 °C, 1H NMR (400 MHz, DMSO-d6) d 13.79 (br, 1H), 10.80 (s,
1H), 8.76 (s, 1H), 8.52 (br, 1H), 7.01–7.69 (m, 3H), 7.54–7.52 (m,
3H), 7.25–7.20 (m, 1H), 6.87 (dd, J1 = 4.0 Hz, J2 = 4.4 Hz, 1H); 13C
NMR (100 MHz, DMSO-d6) d 165.42, 159.15, 156.79, 150.87,
150.85, 141.43, 129.34, 129.07, 120.21, 119.98, 117.62, 117.53,
116.12, 115.87, 114.10, 111.98, 111.91; HRMS (EI) [M]+ Calcd for
C18H12N3OF: 305.0964, Found: 305.0969.
4.1.3. Method B: General procedure for the preparation of
compounds 4a–o
4.1.2.8. 3-((5-(3-Bromophenyl)furan-2-yl)methylene)-5-chloro-
Substituent indoline-2,3-dione (1 equiv) and 2-thioxothiazoli-
din-4-one (1 equiv) were dissolved in 5 mL ethanol, then refluxed
for 10 h. After cooling to the room temperature, the precipitate
indolin-2-one (3k).
Citrus red powder (yield 90%), mp: 289–
290 °C, 1H NMR (400 MHz, DMSO-d6) d 10.72 (s, 1H), 8.47 (d,

Y. Zhong et al. / Bioorg. Med. Chem. 21 (2013) 1724–1734
1733
was filtered, washed with ethanol, dried, and recrystallized from
methanol to afford the terminal product.
the default parameters, while the RSK2 NTKD homology model
was previously reported by our group elsewhere.22 Then GLIDE pro-
gram was chosen to perform the molecular docking simulation.
With the default parameter sets, the grid-enclosing box was cen-
tered on the centroid of the ligand in the RSK2 homology model
and defined so as to enclose residues within 20 Å around the ATP
binding site, and a scaling factor of 1.0 was set to van der Waals
(VDW) radii of those receptor atoms with the partial atomic charge
less than 0.25. The Extra-precision (XP) mode of GLIDE was taken to
ensure the docking, and the top 10 docked poses of each compound
were reserved for further analysis.
4.1.3.1. (Z)-5-(7-Methyl-2-oxoindolin-3-ylidene)-2-thioxothiaz-
olidin-4-one (4a).
Black powder (yield 53%), mp >300 °C, 1H
NMR (400 MHz, CD3OD)
d 8.77 (d, J = 8.0 Hz, 1H), 7.23 (d,
J = 8.0 Hz, 1H), 7.01–6.97 (m, 1H), 2.29 (s, 3H); HRMS (EI) [M]+:
Calcd for C12H8N2O2S2: 276.0027, Found: 276.0029.
4.1.3.2. (Z)-5-(7-Ethyl-2-oxoindolin-3-ylidene)-2-thioxothiaz-
olidin-4-one (4b).
Black powder (yield 65%), mp >300 °C, 1H
NMR (400 MHz, CD3OD) d 8.80 (dd, J1 = 0.4 Hz, J2 = 7.6 Hz, 1H),
7.27 (d, J = 7.2 Hz, 1H), 7.05–7.01 (m, 1H), 2.65 (q, J = 7.6 Hz, 2H)
1.24 (t, J = 7.6 Hz, 3H); HRMS (EI) [M]+: Calcd for C13H10N2O2S2:
290.0184, Found: 290.0185.
Acknowledgments
This work was supported by the Fundamental Research Funds
for the Central Universities, the National Natural Science Founda-
tion of China (Grants 21173076, 81102375, 10979072, 81230090,
81222046 and 81230076), the Special Fund for Major State Basic
Research Project (Grant 2009CB918501), the Shanghai Committee
of Science and Technology (Grants 09dZ1975700, 11DZ2260600
and 10431902600), the 863 Hi-Tech Program of China (Grant
2012AA020308) and the National S&T Major Project of China
(Grant 2011ZX09307-002-03). Honglin Li is also sponsored by Pro-
gram for New Century Excellent Talents in University (Grant NCET-
10-0378).
4.2. In vitro kinase inhibition assay
The kinase inhibition assay was performed as described previ-
ously.22 The ADP QuestÒ kinase assay kits were purchased from
DiscoveRx. The Kinase in 40
7.4, 20 mM NaCl, 1 mM EGTA, 0.02% Tween 20, 10 mM MgCl2,
and 0.1 mg/mL BGG (bovine- -globulins) containing 25 M S6
ll assay buffer (15 mM HEPES, pH
c
l
peptide substrate (AKRRRLSSLRA) was incubated for 20 min at
room temperature with varied concentrations of the compounds
to be tested. Reactions were started by the addition of 10
lL of
ATP (a final concentration of 10
60 min by the addition of 20
l
M). Reactions were stopped after
Supplementary data
l
l ADP Reagent A and 40 ll ADP Re-
agent B, respectively. Ro31-8220 was used as the positive control.
Compounds were prepared from stock in DMSO and diluted to dif-
ferent concentration with assay buffer. The fluorescence signal,
which detected the amount of ADP produced as a result of enzyme
activity was recorded on the Synergy™2 Multi-Mode Microplate
Reader (BioTek) at an excitation wavelength of 530 nm and an
emission wavelength of 590 nm at 30 min after the addition of
the ADP Reagent B. IC50 values were determined by using the
GraphPad Prism software with three independent determinations.
Supplementary data (the details of the testing process for kinase
profile study on 3s) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.047.
References and notes
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4.3. MTT assays
The anti-proliferative activity of RSK2 inhibitors picked out was
measured by the MTT assay as previously described with some
modification.22 Human breast cancer MCF-7 cells (5 Â 103) were
cultured in 96-well plates with 200 lL of DMEM plus 10% FBS
medium (Invitrogen) and incubated with compounds at various
concentrations. And human prostate cancer PC 3 cells (5 Â 103)
were seeded in 96-well plates with 200 lL of F-12K medium (Invit-
rogen) to be treated in the same way. Ro31-8220 was used as a po-
sitive comparison. After 48 h incubation, the cells were added with
10 lL of 5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra-
zolium bromide (MTT) at 37 °C in a humidified incubator with 5%
CO2 for another 4 h. Then the formazan crystals were dissolved
15. Nguyen, T. L. Anti-Cancer Agents Med. Chem. 2008, 8, 710.
16. Sapkota, G. P.; Cummings, L.; Newell, F. S.; Armstrong, C.; Bain, J.; Frodin, M.;
Grauert, M.; Hoffmann, M.; Schnapp, G.; Steegmaier, M. Biochem. J. 2007, 401,
29.
17. Cohen, M. S.; Zhang, C.; Shokat, K. M.; Taunton, J. Science 2005, 308, 1318.
18. Cohen, M. S.; Hadjivassiliou, H.; Taunton, J. Nat. Chem. Biol. 2007, 3, 156.
19. Utepbergenov, D.; Derewenda, U.; Olekhnovich, N.; Szukalska, G.; Banerjee, B.;
Hilinski, M. K.; Lannigan, D. A.; Stukenberg, P. T.; Derewenda, Z. S. Biochemistry
2012, 51, 6499.
in 150 lL DMSO, and the absorbance was measured at 570 nm
by using a Synergy 2 multimode microplate reader (BioTek, USA).
IC50 values were obtained from the results of three independent
experiments and calculated from the inhibition curves.
4.4. Kinase profiling study with compound 3s
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The details for the testing process are given in the Supplemen-
tary data.
4.5. Molecular docking
The 3D structures of the compounds were prepared by the
Ligprep module in Maestro (Schrödinger Inc., version 7.5) with

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