Discovery of novel indolin-2-one compounds as potent inhibitors of HsClpP for cancer treatment

Article abstract of DOI:10.1016/j.bioorg.2021.104820

Human caseinolytic protease proteolytic subunit (HsClpP) is a highly conserved serine protease that plays an essential role in cell homeostasis through removal of the damaged and/or misfolded proteins. Recently, due to its critical role in cancer prolifer

Full text of DOI:10.1016/j.bioorg.2021.104820

Bioorganic Chemistry 110 (2021) 104820
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of novel indolin-2-one compounds as potent inhibitors of HsClpP
for cancer treatment
a
,
1
a
,
1
a,
1
c,
1
a
d,*
Rao Song , Yang Yang , Jiasheng Huang , Wenliang Qiao , Baozhu Luo , Yuan Ju
Tao Yang
,
a
,
b
,
*
a,*
, Youfu Luo
a
State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan,
China
b
Laboratory of Human Diseases and Immunotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China
Lung Cancer Center, Laboratory of Lung Cancer, West China Hospital, Sichuan University, Chengdu, Sichuan, China
Sichuan University Library, Sichuan University, Chengdu, Sichuan, China
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
HsClpP
Human caseinolytic protease proteolytic subunit (HsClpP) is a highly conserved serine protease that plays an
essential role in cell homeostasis through removal of the damaged and/or misfolded proteins. Recently, due to its
critical role in cancer proliferation and metastasis, HsClpP has been considered as a promising target for the
cancer treatment. In this paper, through a random screening toward a library of 2086 bioactive chemicals, a
novel compound I, 3-(3,5-dibromo-4-hydroxybenzylidene) -5-iodoindolin-2-one, was identified as a potent
suppressor of HsClpP. Herein, a series of compound I derivatives were synthesized, and evaluated for their anti-
cancer activities on a variety of cancers cells. Through the preliminary biological assay in vitro, including MTT
assay and proteolytic activity assay, compound I was identified as the most potent inhibitor. Treatment with
compound I impaired the migration of Hela cells. In addition, compound I disrupted the mitochondrial function,
and reduced the level of the SDHB and induced the production of the ATF4. In general, compound I is a
promising probe of HsClpP for cancer treatment, and is a good lead compound for the development of novel anti-
cancer agent.
Inhibitors
Anti-tumor
Mitochondria
1
. Introduction
The mitochondrion is a vital organelle of energy metabolism, of
in the proliferation and metastasis of specific types of cancer, HsClpP has
been considered as an important anti-cancer drug target [4,7,8].
Chemical modulation of the proteolytic activity of HsClpP is a reli-
able therapeutic strategy to achieve cancer treatment. To date, HsClpP
activity was mostly inhibited through modification of Ser153 residues
located within its chamber by covalent inhibitors [5,9]. Three types of
compounds, β-lactones [10], phenyl esters [11,12] and boric acids
[13,14], were identified as HsClpP inhibitors (Fig. 1). β-lactones were
developed originally as potential anti-bacterial agents to inhibit Staph-
ylococcus aureus ClpP (SaClpP) [10]. Subsequently, the analogue A2-32-
01 showed potential activity to treat acute myeloid leukemia and was
the first discovered HsClpP inhibitor [4]. Phenyl esters were developed
which the proteostasis is sustained by some proteolytic machineries [1].
One of the proteolytic mechanism employs HsClpP in the mitochondrial
matrix which could remove proteins that are damaged and/or misfolded
[
2,3]. Besides, it is involved in the degradation and regulation of several
enzymes of the electron transport chain and other cellular metabolic
pathways [4]. In recent decades, HsClpP has been demonstrated to be
related to tumor cell proliferation and metastasis [4,5].For example, the
expression of HsClpP is upregulated in multiple cancers, such as lung,
liver, acute myeloid leukemia, breast and ovary [6]. Since its crucial role
Abbreviations: HsClpP, Human caseinolytic protease proteolytic subunit; SaClpP, Staphylococcus aureus ClpP; ABPP, activity-based protein profiling; FITC-casein,
fluorescein isothiocyanate-labeled casein; ROS, reactive oxygen species; ISR, integrated stress response.
*
Corresponding authors at: State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, and Laboratory of Human
Disease and Immunotherapies, West China Hospital, Sichuan University, Chengdu 610041, China (T. Yang, Y. Luo). Sichuan University Library, Sichuan University,
Chengdu 610041, China (Y. Ju).
E-mail addresses: yuan.ju@scu.edu.cn (Y. Ju), yangtao@wchscu.cn (T. Yang), luo_youfu@scu.edu.cn (Y. Luo).
1
Rao Song, Yang Yang, Jiasheng Huang and Wenliang Qiao contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104820
Received 12 December 2020; Received in revised form 18 February 2021; Accepted 6 March 2021
Available online 10 March 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
with improved activity and chemical stability over β-lactones. AV-167, a
representative compound of phenyl eaters, was screened from a library
of bacterial ClpP inhibitors, which also had inhibitory activity against
HsClpP [12]. Gel-free quantitative activity-based protein profiling
with methyl iodide to yield intermediate 13a, which was then reacted
with the 5-nitroindolin-2-one building block to afford compound D13a.
Compound E14 was obtained through the aldol reaction of 2-chloro-4-
hydroxybenzaldehyde (14) with intermediate E that was synthesized
by methylation of indolin-2-one (C).
(
ABPP) indicated the low selectivity of phenyl esters to many other
proteins [11]. Moreover, Tan et al. identified a series of -amino boronic
α
The Z/E configuration of some pivotal compounds were determined
by 2D NOESY NMR spectrum (Table 2 and Supporting Information). The
portion of Z/E configuration was judged by the co-signal of olefinic
hydrogen with hydrogen at C-4 position of indolin-2-one. For instance,
we found that the major peaks of the olefinic hydrogen and hydrogen at
C-4 position of indolin-2-one have a co-signal in 2D NOESY NMR
spectrum of compound C13, indicating (Z)-C13 accounted for more
(Fig. 4).
acids that covalently inhibited the HsClpXP complex through a virtual
screening platform [13]. Although, these aforementioned compounds
exerted inhibition activity against HsClpP, several drawbacks, such as
instability, poor effect, low selectivity and poor druggability, have
impeded their development.
Thus, in this work, aiming to discover novel HsClpP inhibitors as
anti-cancer agents, we screened an active compound library through the
peptidase activity assay, and identified the compound I as a potential
inhibitor of HsClpP. Based on the compound I, a series of analogues were
synthesized, of which the inhibitory activity toward HsClpP and the
anti-cancer activity in vitro were also investigated. Through the
structure-activity relationship study, it was demonstrated that hydroxyl
group on phenyl ring and hydrogen atom at N-1 position of indolin-2-
one was crucial to the inhibitory activity. The biological activity, such
as the inhibition of cell migration and the impact on mitochondrial
function, and the binding mode of compound I with HsClpP have also
been studied.
2.3. Inhibition of HsClpP activity in vitro
The synthesized compounds were initially evaluated for their inhi-
bition activity against HsClpP in a fluorescence-based AC-WLA-AMC
assay in independent triplicates (Table 1). Peptidase activity of HsClpP
after treatment with synthesized compounds was measured by moni-
toring release of fluorescent amino-methylcoumarin from AC-WLA-AMC
[16]. We started our investigation with modifying the indolin-2-one
with different substituents. As shown in Table 1, compounds A13,
B13, C13 and D13 showed good inhibitory activity to HsClpP with IC50
2
. Results and discussion
values of 0.79 ± 0.06, 0.76 ± 0.09, 1.69 ± 0.23 and 3.60 ± 0.49
μM,
respectively. These results implied that the phenyl ring of indolin-2-one
can tolerate a relative wide range of chemical space, such as hydrogen,
trifluoromethyl, bromine and nitro. Besides, E14, obtained by methyl-
ation at N-1 position of indolin-2-one, showed decreased inhibitory ac-
tivity, which indicated that the hydrogen atom of nitrogen in N-1
position was essential for the inhibitory activity. After the exploration
the SAR of phenyl ring of the indolin-2-one, the SAR of C-3 position of
the indolin-2-one was investigated. Among all the synthesized com-
pounds, A10, A13, A14, B10, B13, C1, C13, D1, D13 and D14 with
similar aromatic moieties containing hydrogen-bond donor have mod-
2
.1. Identification of the compound I as an HsClpP inhibitor
To discover hit compounds targeting HsClpP, we performed a
random screening toward an in-house library of 2086 bioactive chem-
icals (Fig. 2). In the primary screening, compounds were assayed at a
single-point concentration of 10 M, and ten initial hits (hit rate 0.48%)
μ
were selected for their good inhibitory activity on HsClpP cleavage to-
ward the substrate of AC-WLA-AMC (Fig. 3A). Then, in the secondary
screening, six of ten hits have been identified with IC50 values below 5
μ
M (Fig. 3B). The proteolytic activity of HsClpP can also be determined
erate to good inhibitory activity with IC50 values below 10 M. Thus,
μ
in vitro by monitoring the degradation of fluorescein isothiocyanate-
labeled casein (FITC-casein) substrate [15]. Using this assay, the
initial hits were tested for the inhibition of proteolytic activity at con-
substituents with hydrogen-bond donor were considered to be beneficial
to the inhibitory activity against HsClpP. However, C5 and D5 bearing
′
hydroxy on the 3 - position of phenyl group had significantly decreased
centration of 100
μ
M (Fig. 3C). Compound I, 3-(3,5-dibromo -4-
inhibitory activities with IC50 values greater than 10
μ
M, which implied
′
′
hydroxybenzylidene)-5-iodoindolin-2-one, exhibited the most potential
that hydrogen-bond donor substituted at 4 - position or 2 -position of the
phenyl group is critical. Otherwise, since the inhibitory activity of A9,
B9 and D16 towards HsClpP with hydrogen-bond donor was reduced,
the phenyl ring substituted at C-3 position of indolin-2-one was impor-
tant over 1H-pyrrole. To further confirm whether the hydrogen-bond
donor on the phenyl ring is beneficial for the inhibitory activity, the
compound D13a was obtained by methylation of hydroxyl group of
D13. Compound D13a exhibited weak activity against HsClpP, indi-
cating that the hydroxyl group of the phenyl ring is the key element of
these HsClpP inhibitors.
inhibitory activity toward HsClpP with IC50 value of 1.23 ± 0.11
μ
M on
peptidase activity assay (substrate: AC-WLA-AMC).
2
.2. Chemistry
As shown in Scheme 1, compound I and its 33 analogues were syn-
thesized through the aldol reaction of aromatic aldehyde with
substituted indolin-2-one building blocks in the presence of piperidine.
To make clear the importance of the hydroxyl group on the aromatic
ring and the hydrogen atom at N-1 position of the indolin-2-one scaffold,
compounds D13a and E14 were synthesized. As shown in Scheme 2, the
hydroxyl group of 3,5-dibromo-4-hydroxybenzaldehyde was methylated
Fig. 1. Representative inhibitors against HsClpP.
2

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
Fig. 2. Schematic illustration of chemical screening. Identification of novel HsClpP inhibitor hits in an in-house library of 2086 bioactive chemicals.
Fig. 3. Discovery of a novel HsClpP inhibitor from a bioactive chemical library screening. (A) A random screening of 2086 compounds identified ten primary hits
with good inhibitory effect at 10
FITC-casein substrate.
μ
M. (B) IC50 values of compounds A-J toward HsClpP. (C) Inhibition of HsClpP proteolytic activity by 100 M compounds A-J in the
μ
2
.4. Inhibition of HsClpP induces anti-tumor effects in vitro.
The expression of HsClpP is upregulated in multiple cancer cells,
compounds showed almost no inhibition activity on HsClpP-negative
MCF-7 cells at the concentration of 40 M, which demonstrated a
μ
good selectivity on HsClpP highly expression cell lines.
which is essential for the proliferation of cancer cells. To evaluate the
anticancer activities of synthesized compounds, four human cancer cell
lines (HepG2, Hela, HCT116 and Skov3) were treated with these com-
To assess the ability of blocking proteolytic activity of selected
compounds, HsClpP was activated via the ONC201 derived chaperone
mimic for inducing pore opening and digesting of FITC-casein [16].
(Fig. 4A). Using this assay, AV167 and the selected compounds towards
the inhibitory proteolytic activity of HsClpP were assessed at three
pounds (Table 3). Compounds with IC50 values below 10 M in pepti-
μ
dase activity assay were assessed for their anti-proliferation activity
using MTT assay. As shown in Table 2, compound I showed good
inhibitory activities against HepG2, Hela, HCT116 and Skov3 cells with
IC50 values of 14.42 ± 0.93, 11.81 ± 1.24, 18.99 ± 0.29, 37.47 ± 3.03
concentrations of 100
μM, 10
μ
M and 1 M in independent triplicates
μ
(Fig. 5A). Similarly to AV167, compounds I, A13 and B13 showed good
inhibition activity at the highest concentration, which demonstrated
that compounds I, A13 and B13 could efficiently block proteolysis
through inhibition of HsClpP.
μ
M, respectively. While the control AV167, exhibited almost no inhi-
bition activity against all four cell lines at the concentration of 40 M.
μ
The inhibition activity of A10, B10, C1 and D1 significantly decreased,
compared with that of compound I. Compound B13, with tri-
fluoromethyl group substituted at the indolin-2-one ring, and C13
inhibited the growth of HepG2, Hela, HCT116 and Skov3 cells with IC50
values comparable to that of compound I. Moreover, the synthesized
Previous investigation indicated that an increase of the expression of
HsClpP is crucial for proliferation and metastasis of certain cancer cell
lines, and CLPP knockdown in cancer cells resulted in inhibition of cell
migration [4]. In this work, migration of Hela cells was impaired after
incubation with compound I for 24 h (Fig. 5B).
3

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
◦
Scheme 1. Synthetic strategy of target compounds. Reagents and conditions: (a) piperidine, MeOH, 65 C.
◦
Scheme 2. Synthesis of compounds D13a and E14. Reagents and conditions: (a) K
2
CO
3
, CH
3
I, DMF, rt. (b) 5-nitroindolin-2-one, piperidine, MeOH, 65 C. (c)
◦
◦
(
CH
3
)
2
SO
4
, PhMe, 100 C. (d) 2-chloro-4-hydroxybenzaldehyde, piperidine, MeOH, 65 C.
2
.5. The binding mode of compound I with HsClpP
IC50 values of AV167 and compound I increased, indicative of a
competitive binding with HsClpP. In order to further define the binding
mode of compound I with HsClpP, molecular docking studies were
performed with Schrodinger. The structure of the HsClpP was obtained
from the Protein Data Bank. We generated 3D diagrams to show the
binding mode of compound I within the HsClpP (Fig. 6B). Although,
To explore the possible interaction modes of the selected compounds
with HsClpP, substrate competition assay was performed in the presence
of 200 M, 100 M or 50 M AC-WLA-AMC substrate in independent
triplicates (Fig. 6A). With the concentration of substrate increased, the
μ
μ
μ
4

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
Fig. 4. The 2D NOESY NMR spectrum (part) of compound C13. The portion of Z/E configuration was judging from the existing of co-signal of olefinic hydrogen with
hydrogen at C-4 position.
compound I did not directly bound to catalytic Ser97 residue, it is tightly
bound to residues surrounding the active site such as Pro66 and Gln138.
As shown in Fig. 5B, compound I binds to the HsClpP by hydrogen bonds
of Pro66 and Gln138 with NH and hydroxy, which is consistent with the
peptidase activity assay of D13a and E14.
hydroxybenzylidene)- -5-iodoindolin-2-one), was identified as HsClpP
inhibitor. Based on the compound I, a series of analogues have been
synthesized, some of which exerted promising inhibition activity on
peptidase and protease assay of HsClpP. Besides, they exhibited potent
anti-proliferation activity on HepG2, Hela, HCT116 and Skov3 cell lines,
while no obvious activity to HsClpP-negative MCF-7 cells. In addition to
the anti-proliferation activity, compound I inhibited the migration of
Hela cells. Also, treatment with compound I reduced the level of the
SDHB and induced the production of ATF4 in a dose-dependent manner
to impair the oxidative phosphorylation. Generally, compound I is
worth of further investigation toward the discovery of HsClpP inhibitor
as a valuable lead compound for oncotherapy.
2
.6. HsClpP inhibition by compound I and B13 leads to the mitochondria
impaired and induction of integrated stress response
We then studied the effects of HsClpP inhibition on mitochondrial
function. In cancer cells, the elevation of reactive oxygen species (ROS)
in mitochondria is important for facilitating and sustaining oncogenesis.
Inhibition of HsClpP increased the production of mitochondrial ROS that
will induce cell apoptosis [4]. Treatment with compound I and B13
increased the production of mitochondrial ROS in Hela cells, which is
4. Experimental section
4
.4-fold and 3.3-fold over the control group at the concentration of 10
4.1. Chemistry
μ
M for 48 h, respectively. (Fig. 7A). Respiratory chain subunit SDHB had
previously been considered as putative HsClpP substrates, and the in-
hibition of HsClpP may reduce the enzymatic activity of SDHB [4,5]. In
addition, influence of the function of HsClpP could induce an integrated
stress response (ISR) by the induction of ATF4 [5,15]. Further, we
treated Hela cells with compound I and B13 and found that both the
compound I and B13 reduced the level of the SDHB and induced the
production of ATF4 in a dose-dependent manner (Fig. 7B).
All reagents were directly used as purchased without further purifi-
cation. All solvents were dried according to standard methods before
use, solvents used in optical spectroscopic studies were HPLC grade. All
NMR spectrum were recorded on a Bruker Avance (Varian Unity Inova)
500 MHz spectrometer in DMSO‑d6 with tetramethylsilane (TMS) as
1
13
internal standard. H NMR and
C NMR spectra were recorded
1
13
respectively at 400 MHz and 100 MHz. H NMR and C NMR were
analyzed by MestReNova Software. High resolution mass spectrum
(HRMS) was performed on an Agilent LC/MSD TOF system G3250AA.
Analytical thin layer chromatography (TLC) was performed on silica gel
60 F254 precoated plates (0.25 mm) from Qingdao Haiyang Inc., and
3
. Conclusion
In summary,
a
novel compound I, (3-(3,5-dibromo-4-
5

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
Table 1
The inhibition activity of target compounds toward HsClpP in the AC-WLA-AMC assay.
Compd.
I
I
II
IC50
(
μ
M)
Compd.
C11
I
II
IC50( M)
μ
1.23 ± 0.11
>10
A3
A4
>10
>10
C12
C13
>10
1.69 ± 0.23
>10
A6
>10
C15
A7
A9
>10
>10
C17
C18
>10
>10
A10
A13
A15
3.11 ± 0.09
0.79 ± 0.06
>10
C19
D1
>10
2.43 ± 0.30
>10
D2
B3
B9
>10
>10
D3
D5
>10
>10
B10
B13
5.99 ± 0.30
0.76 ± 0.09
D12
D13
>10
3.60 ± 0.49
C1
9.42 ± 0.97
>10
D14
D15
1.76 ± 0.66
>10
C2
C5
>10
D16
>10
C6
C8
>10
>10
D13a
E14
>10
>10
6

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
Table 2
The Z/E configuration of some key compounds.
Compd.
I
Structure
Configuration
Compd.
C1
Structure
Configuration
E
Z/E≈1/9
A10
Z/E≈1/9
C13
Z/E≈4/1
A13
B10
B13
Z/E≈4/1
Z/E≈2/3
Z/E≈7/3
D1
Z/E≈4/1
Z/E≈4/1
Z/E≈1/4
D13
D14
give products.
Table 3
Anti-proliferation activity of selected compounds against HepG2, Hela, HCT116,
Skov3 and MCF-7 cells.
4
.2.1. 5-Bromo-3-(pyridin-2-ylmethylene)indolin-2-one (A3)
1
H NMR (400 MHz, DMSO) δ 10.76 (s, 1H), 9.28 (m, 1H), 8.89 (d, J
Compd.
IC50( M)
μ
=
4.1 Hz, 1H), 8.00 (dd, J = 7.6, 2.0 Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H),
13
HepG2
Hela
HCT166
Skov3
MCF-
7
7.63 (s, 1H), 7.54–7.44 (m, 1H), 6.84 (d, J = 8.0 Hz, 1H). C NMR (101
MHz, DMSO) δ 169.33, 153.30, 150.07, 143.20, 138.00, 135.77, 133.52,
A10
A13
>40
>40
>40
>40
>40
>40
>40
>40
130.85, 129.58, 128.70, 125.14, 124.04, 113.36, 111.96. HRMS (Q-
+
31.35 ±
36.18 ±
3.18
TOF): calculated for C14
H
9
2
BrN O
1
299.9898 [M + H] , found 300.9981.
0
.64
Purity ≥ 95.0%.
B10
B13
>40
>40
>40
>40
>40
>40
12.84 ±
10.66 ±
0.58
14.64 ±
1.31
27.51 ±
1.69
0
.65
4.2.2. 5-Bromo-3-(4-bromobenzylidene)indolin-2-one (A4)
1
C1
>40
>40
>40
>40
>40
>40
H NMR (400 MHz, DMSO) δ 10.79 (s, 1H), 7.76 (d, J = 8.4 Hz, 2H),
C13
11.61 ±
10.34 ±
0.65
13.75 ±
0.97
28.00 ±
2.65
7
1
1
.70–7.63 (m, 3H), 7.51 (d, J = 1.6 Hz, 1H), 7.42 (dd, J = 8.4, 2.0 Hz,
0
.94
13
H), 6.85 (d, J = 8.4 Hz, 1H). C NMR (101 MHz, DMSO) δ 168.45,
D1
>40
>40
>40
>40
>40
>40
42.68, 136.72, 133.77, 133.14, 132.33, 131.78, 127.70, 125.06,
D13
12.62 ±
11.25 ±
0.32
32.10 ±
0.65
>40
1
.36
123.86, 123.25, 113.18, 112.57. HRMS (Q-TOF): calculated for
+
D14
I
24.81 ±
.55
14.42 ±
.93
>40
16.56 ±
1.04
23.01 ±
1.87
27.99 ±
1.12
>40
>40
>40
C
15
H
9
Br
2
NO 376.9051 [M + Na] , found 399.8946. Purity ≥ 95.0%.
1
11.81 ±
1.24
18.99 ±
0.29
37.47 ±
3.03
4
.2.3. 5-Bromo-3-(3-methoxybenzylidene)indolin-2-one (A6)
0
1
AV167
>40
>40
>40
H NMR (400 MHz, DMSO) δ 10.74 (s, 1H), 7.71 (s, 1H), 7.65 (d, J =
7
1
.2 Hz, 1H), 7.57–7.50 (m, 1H), 7.44–7.38 (m, 2H), 7.20 (d, J = 8.0 Hz,
1
3
H), 7.11 (t, J = 7.2 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 3.87 (s, 3H).
C
components were visualized by ultraviolet light (254 nm). Silicycle sil-
NMR (101 MHz, DMSO) δ 168.59, 158.13, 142.32, 134.12, 132.70,
ica gel 300–400 (particle size 40–63
column chromatography experiments.
μ
m) mesh was used for all flash
1
1
3
32.62, 130.05, 126.94, 125.05, 123.72, 122.86, 120.71, 113.05,
12.36, 112.21, 56.09. HRMS (Q-TOF): calculated for C16
H
12BrNO
2
+
29.0051 [M + H] , found 330.0131. Purity ≥ 95.0%.
4
.2. General procedure for synthesis of compounds
To a solution of the corresponding starting materials (200 mg, 1 eq.)
4
.2.4. 5-Bromo-3-(2,4-dimethoxybenzylidene)indolin-2-one (A7)
1
3
H NMR (400 MHz, CDCl ) δ 8.02 (s, 1/4H), 7.99 (s, 3/4H), 7.82 (s,
3
1
5
1
1
/4H), 7.76 (d, J = 2 Hz, 1H), 7.66 (m, 1H), 7.33–7.27 (m, 1H), 6.74 (m,
H), 6.60 (m, 1H), 6.53 (d, J = 2.4 Hz, 3/4H), 6.45 (d, J = 2.4 Hz, 1/4H),
.30 (s, 1/4H), 3.90 (m, 6H).13C NMR (101 MHz, DMSO) δ 168.95,
63.62, 160.05, 141.99, 134.03, 132.09, 131.25, 124.72, 124.68,
in MeOH (2 mL), piperidine (1.5 eq.) and corresponding aromatic al-
◦
dehydes (1.2 eq.) were added. The reaction mixture was heated to 65 C
and stirred for 1–8 h, then cooled to room temperature. The mixture was
added with water (10 mL) and extracted with DCM (10 mL) for three
times, the combined organic layer was washed with brine and dried over
anhydrous sodium sulfate. Then purified by column chromatography to
24.07, 115.45, 112.99, 112.20, 106.00, 99.07, 56.28, 56.10. HRMS (Q-
+
TOF): calculated for C17
H
14BrNO
3
359.0157 [M + H] , found 360.0256.
7

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
Fig. 5. (A) FITC-casein degradation assay with HsClpP, ONC201 and inhibitors at three different concentrations. (B) Scratch test in Hela cells by treatment with
compound I at 5 M for 24 h.
μ
Fig. 6. The binding mode of compound I with HsClpP. (A) IC50 values of AV167 and compound I in the presence of 200
substrate. (B) 3D and 2D diagrams to show the binding mode through molecular docking study.
μ
M, 100
μ
M or 50 M AC-WLA-AMC
μ
Purity ≥ 95.0%.
.2.5. 5-Bromo-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one
4.2.7. 5-Bromo-3-(3,5-dibromo-4-hydroxybenzylidene)indolin-2-one
A13)
(
1
4
H NMR (400 MHz, DMSO) δ 10.43 (s, 1/5H), 10.35 (s, 4/5H), 8.74
(
A9)
(s, 8/5H), 7.84 (d, J = 1.6 Hz, 1/5H), 7.79 (s, 2/5H), 7.71 (d, J = 1.6 Hz,
4/5H), 7.51 (s, 4/5H), 7.37 (s, 1/5H), 7.27 (dd, J = 8.4, 2.0 Hz, 1/5H),
7.13 (dd, J = 8.4, 2.0 Hz, 4/5H), 6.79 (d, J = 8.0 Hz, 1/5H), 6.70 (d, J =
1
H NMR (400 MHz, DMSO) δ 13.38 (s, 1H), 10.87 (s, 1H), 8.02 (d, J
2 Hz, 1H), 7.69 (s, 1H), 7.22 (dd, J = 8.0, 2.0 Hz, 1H), 6.81 (d, J = 8.4
=
1
3
13
Hz, 1H), 6.04 (d, J = 2.0 Hz, 1H), 2.32 (d, J = 2.4 Hz, 6H). C NMR (101
8.0 Hz, 4/5H). C NMR (101 MHz, DMSO) δ 167.90, 139.32, 138.19,
MHz, DMSO) δ 169.58, 137.37, 137.34, 133.68, 128.87, 128.04, 127.41,
137.74, 135.17, 130.09, 127.51, 120.33, 117.94, 115.33, 112.83,
1
25.48, 121.21, 113.62, 113.46, 111.63, 111.39, 14.03, 11.79. HRMS
110.65. HRMS (Q-TOF): calculated for C15
H
8
Br
3
NO
2
470.8105 [M +
+
+
(
Q-TOF): calculated for C15
2
H13BrN O 316.0211 [M + H] , found
Na] , found 493.7997. Purity ≥ 95.0%.
3
17.0228. Purity ≥ 95.0%.
4
.2.8. 5-Bromo-3-((1-methyl-1H-indol-3-yl)methylene)indolin-2-one
4
.2.6. 5-Bromo-3-(5-bromo-2-hydroxybenzylidene)indolin-2-one (A10)
(A15)
1
1
H NMR (400 MHz, DMSO) δ 10.81–10.48 (m, 2H), 7.92 (s, 1/10H),
H NMR (400 MHz, DMSO) δ 10.65 (s, 1H), 9.43 (s, 1H), 8.31 (m,
1H), 8.26 (s, 1H), 8.19 (d, J = 1.6 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.30
7
.81 (d, J = 1.6 Hz, 1/10H), 7.72 (d, J = 2.4 Hz, 9/10H), 7.62 (s, 9/
1
3
1
0H), 7.53–7.35 (m, 2H), 6.96 (d, J = 8.8 Hz, 9/10H), 6.89 (d, J = 8.8
(m,3H), 6.79 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H). C NMR (101 MHz,
DMSO) δ 168.18, 138.50, 138.08, 137.06, 129.33, 129.21, 129.17,
128.57, 123.24, 121.95, 121.70, 119.40, 118.01, 113.27, 111.23,
1
3
Hz, 1/10H), 6.84 (d, J = 8.0 Hz, 9/10H), 6.79 (d, J = 8.0 Hz, 1/10H).
C
NMR (101 MHz, DMSO) δ 168.52, 156.24, 142.45, 134.50, 132.78,
1
32.72, 132.08, 127.33, 125.41, 123.68, 123.65, 118.79, 113.08,
111.18, 111.08, 34.02. HRMS (Q-TOF): calculated for C18
H13BrN
2
O
+
1
12.42, 110.13. HRMS (Q-TOF): calculated for C15
H
9
Br
2
N
1
O
2
392.9000
352.0211 [M + H] , found 353.0319.Purity ≥ 95.0%.
+
[
M + Na] , found 415.8867. Purity ≥ 95.0%.
8

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
Fig. 7. HsClpP inhibition by compound I and B13 impairs oxidative phosphorylation. (A) Effect of compound I and B13 treatment on mitochondrial reactive oxygen
species (ROS) production in Hela cells for 48 h. (B) Western blot of ATF4 and SDHB in Hela cells with compound I and B13-inducible for 24 h at the indicated
concentrations.
4
.2.9. 3-(Pyridin-2-ylmethylene)-6-(trifluoromethyl)indolin-2-one (B3)
10H).13C NMR (101 MHz, DMSO) δ 168.58, 167.45, 153.70, 152.90,
143.92, 141.27, 137.74, 137.10, 136.95, 134.12, 133.91, 130.99,
129.31, 129.13, 128.82, 128.71, 128.60, 126.74, 126.09, 124.93,
1
H NMR (400 MHz, DMSO) δ 10.99 (s, 1/2H), 10.91 (s, 1/2H), 9.26
(
d, J = 8.0 Hz, 1/2H), 8.94 (m, 1H), 8.73 (d, J = 4.0 Hz, 1/2H),
8
2
.06–7.87 (m, 2.5H), 7.74 (s, 1/2H), 7.57–7.50 (m, 1/2H), 7.44 (m, 1/
123.38, 122.87, 120.56, 118.46, 112.48, 112.33, 111.56, 106.03. HRMS
1
3
+
H), 7.36 (d, J = 8.0 Hz, 1H), 7.08 (d, J = 12.0 Hz, 1H). C NMR (101
(Q-TOF): calculated for C16
H
8
Br
2
F
3
NO
2
462.8851 [M + H] , found
MHz, DMSO) δ 169.42, 153.11, 150.23, 144.48, 137.91, 137.27, 130.62
463.2195. Purity ≥ 95.0%.
(
q, J = 31.7 Hz), 129.65, 128.95, 128.31, 125.74, 125.63, 125.24,
1
18.43 (q, J = 4.0 Hz), 106.11 (q, J = 3.9 Hz). HRMS (Q-TOF): calcu-
4.2.13. 3-(4-Hydroxybenzylidene)indolin-2-one (C1)
+
1
lated for C15
H
9
Br
3
N
2
O 290.0667 [M + H] , found 291.0740. Purity ≥
H NMR (400 MHz, DMSO) δ 10.50 (s, 1H), 10.11 (s, 1H), 7.69 (d, J
9
5.0%.
= 7.6 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.54 (s, 1H), 7.21 (td, J = 7.6,
1
3
0
.8 Hz, 1H), 6.96–6.84 (m, 4H). C NMR (101 MHz, DMSO) δ 169.52,
4
.2.10. 3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-(trifluoromethyl)
159.74, 142.96, 137.05, 132.28, 129.87, 125.49, 125.14, 122.51,
indolin-2-one (B9)
121.79, 121.47, 116.11, 110.40. HRMS (Q-TOF): calculated for
1
+
H NMR (400 MHz, DMSO) δ 13.44 (s, 1H), 11.03 (s, 1H), 7.94 (d, J
C
15
H11NO
2
237.0790 [M + H] , found 238.0869. Purity ≥ 95.0%.
=
7.6 Hz, 1H), 7.75 (s, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.09 (s, 1H), 6.09
13
(
d, J = 2.0 Hz, 1H), 2.34 (d, J = 4.8 Hz, 6H). C NMR (101 MHz, DMSO)
4.2.14. 3-(4-Methylbenzylidene)indolin-2-one (C2)
1
δ 169.78, 138.40, 138.26, 134.59, 130.49, 127.60, 126.58, 126.18,
H NMR (400 MHz, DMSO) δ 10.57 (s, 1H), 8.32 (d, J = 8.4 Hz, 1/
1
1
25.60 (d, J = 31.3 Hz), 118.65, 118.00, 113.88, 111.00, 105.83, 14.07,
10H), 7.76 (s, 1/10H), 7.71–7.55 (m, 19/5H), 7.34 (d, J = 8.0 Hz, 9/
5H), 7.28 (d, J = 8.4 Hz, 1/10H), 7.22 (m, 1H), 6.98 (td, J = 7.6 ,0.8 Hz,
+
1.81. HRMS (Q-TOF): calculated for C16
H
13
F
3
N
2
O 306.0980 [M + H] ,
1
3
found 307.1051. Purity ≥ 95.0%.
1/10H), 6.92–6.80 (m, 2H), 2.38 (m, 3H). C NMR (101 MHz, DMSO) δ
1
69.20, 143.32, 140.15, 136.43, 132.03, 130.44, 129.88, 129.81,
4
.2.11. 3-(5-Bromo-2-hydroxybenzylidene)-6-(trifluoromethyl)indolin-2-
127.42, 122.74, 121.54, 121.49, 110.56, 21.57. HRMS (Q-TOF): calcu-
+
one (B10)
lated for C16
95.0%.
H13NO
2
235.0997 [M + H] , found 236.1059. Purity ≥
1
H NMR (400 MHz, DMSO) δ 10.85 (s, 1H), 8.97 (d, J = 2.4 Hz, 2/
H), 8.10 (s, 2/5H), 7.79–7.71 (m, 8/5H), 7.53 (d, J = 8.0 Hz, 3/5H),
5
7
1
2
1
1
1
1
.48 (dd, J = 8.8, 2.4 Hz, 3/5H), 7.38 (d, J = 9.6 Hz, 2/5H), 7.29 (m,
H), 7.09 (s, 3/5H), 7.03 (s, 2/5H), 6.93 (d, J = 8.8 Hz, 3/5H), 6.79 (s,
/5H). 13C NMR (101 MHz, DMSO) δ 190.06, 168.65, 167.35, 160.37,
57.29, 156.34, 143.74, 141.53, 138.89, 135.64, 134.72, 134.30,
33.11, 132.06, 130.86, 130.03 (d, J = 31.9 Hz), 126.99, 125.79,
25.32, 124.48, 123.66, 123.29, 122.93 (d, J = 31.8 Hz), 120.64,
20.38, 118.79, 118.49, 118.08, 111.15, 110.31, 110.07, 106.61. HRMS
4.2.15. 3-(3-Hydroxy-4-methoxybenzylidene)indolin-2-one (C5)
1
H NMR (400 MHz, DMSO) δ 10.52 (s, 1H), 9.37 (s, 7/10H), 8.17 (d,
J = 2.0 Hz,3/10H), 7.83 (dd, J = 8.4, 2.0 Hz, 3/10H), 7.73 (d, J = 7.6
Hz, 7/10H), 7.69–7.63 (m, 3/5H), 7.49 (s, 7/10H), 7.26–7.13 (m, 27/
10H), 7.04 (m, 1H), 6.96 (m, 3/10H), 6.92–6.83 (m, 14/10H), 6.80 (d, J
1
3
= 7.6 Hz, 3/10H), 3.85 (s, 3H). C NMR (101 MHz, DMSO) δ 168.71,
144.81, 142.35, 138.79, 137.17, 131.27, 129.20, 126.85, 123.43,
(
Q-TOF): calculated for C16H9BrF3NO2 382.9769 [M + H]+, found
121.98, 119.60, 116.61, 114.94, 111.34, 109.25, 33.99. HRMS (Q-TOF):
+
3
84.3070. Purity ≥ 95.0%.
calculated for C16
H
13NO
3
267.0895 [M + H] , found 268.0975. Purity
≥
95.0%.
4
.2.12. 3-(3,5-Dibromo-4-hydroxybenzylidene)-6-(trifluoromethyl)
indolin-2-one (B13)
4.2.16. 3-(3-Methoxybenzylidene)indolin-2-one (C6)
1
1
1
H NMR (400 MHz, DMSO) δ H NMR (400 MHz, DMSO) δ 10.99 (s,
/10H), 10.95 (s, 3/10H), 8.84 (s, 7/5H), 7.95 (s, 3/5H), 7.92 (s, 7/
0H), 7.86 (d, J = 8.0 Hz, 7/10H), 7.66 (m, 3/5H), 7.37 (d, J = 8.0 Hz,
/10H), 7.28 (d, J = 8.0 Hz, 3/10H), 7.12 (s, 3/10H), 7.07 (s, 7/
H NMR (400 MHz, DMSO) δ 10.59 (s, 1H), 7.60 (s, 1H), 7.55 (d, J =
7
1
7
7.6 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.30–7.20 (m, 3H), 7.05 (dd, J =
8.4, 2.4 Hz, 1H), 6.87 (m, 2H), 3.81 (s, 3H). 13C NMR (101 MHz, DMSO)
δ 169.10, 158.10, 143.22, 132.22, 132.19, 130.37, 129.98, 127.79,
9

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
1
23.30, 122.73, 121.56, 121.50, 120.67, 112.00, 110.51, 56.03. HRMS
10H), 7.23 (m, 1H), 7.14 (d, J = 7.6 Hz, 3/10H), 7.00 (m, 7/10H), 6.91
(d, J = 7.6 Hz, 3/10H), 6.81 (m, 1H), 5.95 (d, J = 4.4 Hz, 7/10H), 3.91
(s, 21/10H), 3.80 (s, 9/10H). 13C NMR (101 MHz, DMSO) δ 168.42,
(
Q-TOF): calculated for
C
16
H
13NO
2
251.0946 [M + H]+, found
2
52.1028. Purity ≥ 95.0%.
1
58.64, 146.35, 143.84, 143.41, 138.39, 131.40, 130.76, 130.34,
4
.2.17. 3-(4-Fluorobenzylidene)indolin-2-one (C8)
129.71, 128.25, 127.83, 124.52, 123.14, 122.03, 120.80, 110.87,
1
H NMR (400 MHz, DMSO) δ 10.60 (s, 1H), 7.78 (m, 2H), 7.61 (s,
106.84, 56.25. HRMS (Q-TOF): calculated for C19
H
13ClN
2
O
2
336.0666
+
1
1
H), 7.50 (d, J = 7.6 Hz, 1H), 7.36 (t, J = 8.8 Hz, 2H), 7.24 (t, J = 7.6 Hz,
[M + Na] , found 359.0561 Purity ≥ 95.0%.
1
3
H), 6.93–6.81 (m, 2H). C NMR (101 MHz, DMSO) δ 169.03, 163.02
(
d, J = 249.1 Hz), 143.47, 135.15, 132.26, 132.17, 131.37 (d, J = 249.1
4.2.24. 3-((5-Bromofuran-2-yl)methylene)indolin-2-one (C19)
1
Hz), 130.68, 128.10, 122.77, 121.63, 121.23, 116.42, 116.20, 110.65.
H NMR (400 MHz, DMSO) δ 10.60 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H),
7.29 m, 2H), 7.04 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 3.6 Hz, 1H), 6.89 (d, J
+
HRMS (Q-TOF): calculated for C15
H10FNO 239.0746 [M + H] , found
1
3
2
4
4
40.0622. Purity ≥ 95.0%.
= 7.6 Hz, 1H). C NMR (101 MHz, DMSO) δ 169.53, 153.09, 143.30,
1
30.49, 126.94, 124.69, 123.23, 123.03, 121.85, 121.50, 118.61,
.2.18. 3-((5-Nitrofuran-2-yl)methylene)indolin-2-one (C11)
116.17, 110.40. HRMS (Q-TOF): calculated for C13
H
8
BrNO
2
288.9738
1
+
H NMR (400 MHz, DMSO) δ 10.91 (s, 1H), 8.19 (s, 1H), 8.16 (d, J =
[M + H] , found 289.9805. Purity ≥ 95.0%.
.4 Hz, 1H), 7.77 (d, J = 4.8 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.30 (td, J
=
7.6, 0.8 Hz, 1H), 7.05 (td, J = 7.6, 0.8 Hz, 1H), 6.90 (d, J = 7.6 Hz,
4.2.25. 3-(4-Hydroxybenzylidene)-5-nitroindolin-2-one (D1)
1
3
1
1
1
1
H). C NMR (101 MHz, DMSO) δ 167.76, 153.67, 143.46, 142.09,
H NMR (400 MHz, DMSO) δ 11.25 (s, 1H), 10.41 (s, 4/5H), 10.33 (s,
1/5H), 8.65 (d, J = 2.0 Hz, 4/5H), 8.50 (m, 2H), 8.20–8.08 (m, 9/5H),
7.76 (s, 1/5H), 7.70 (d, J = 8.4 Hz, 1/5H), 7.05 (d, J = 8.8 Hz, 1/5H),
6.99 (d, J = 8.4 Hz, 4/5H), 6.96 (d, J = 8.8 Hz, 2/5H), 6.89 (d, J = 8.8
Hz, 8/5/H). 13C NMR (101 MHz, DMSO) δ 169.70, 168.13, 161.61,
160.65, 148.52, 145.81, 142.39, 141.94, 141.85, 140.65, 136.15,
36.55, 130.90, 129.22, 127.59, 127.10, 123.77, 122.00, 121.14,
10.54. HRMS (Q-TOF): calculated for C13
H
N
8 2
O
4
256.0484 [M + H]+,
found 256.0964. Purity ≥ 95.0%.
′
4
.2.19. 3-([1,1 -Biphenyl]-4-ylmethylene)indolin-2-one (C12)
1
H NMR (400 MHz, DMSO) δ 10.63 (s, 1H), 8.51 (d, J = 8.4 Hz, 2H),
132.80, 126.88, 126.24, 125.83. HRMS (Q-TOF): calculated for
+
7
.85 (s, 1H), 7.83–7.71 (m, 5H), 7.50 (m, 2H), 7.41 (m, 1H), 7.22 (td, J
C
15
H
10
N
2
O
4
282.0641 [M + Na] , found 305.0541. Purity ≥ 95.0%.
=
7.6, 0.8 Hz, 1H), 7.00 (td, J = 7.6, 0.8 Hz, 1H), 6.84 (d, J = 7.6 Hz,
13
1
1
1
H). C NMR (101 MHz, DMSO) δ 167.66, 142.17, 141.25, 139.74,
4.2.26. 3-(4-Methylbenzylidene)-5-nitroindolin-2-one (D2)
1
36.65, 133.69, 133.17, 129.51, 129.45, 128.49, 127.22, 126.80,
H NMR (400 MHz, DMSO) δ 11.31 (m, 1H), 8.69 (d, J = 2.0 Hz, 3/
25.47, 121.54, 120.28, 109.84. HRMS (Q-TOF): calculated for
4H), 8.43 (d, J = 2.4 Hz, 1/4H), 8.39 (d, J = 8.0 Hz, 6/4H), 8.17 (m, 7/
4H), 7.81 (s, 1/4H), 7.69 (d, J = 8.0 Hz, 1/2H), 7.40 (d, J = 8.0 Hz, 1/
+
C
21
H
15NO 297.1154 [M + H] , found 298,1233. Purity ≥ 95.0%.
2
H), 7.33 (d, J = 8.0 Hz, 3/2H), 7.06 (d, J = 8.6 Hz, 1/4H), 7.01 (d, J =
13
4
.2.20. 3-(3,5-Dibromo-4-hydroxybenzylidene)indolin-2-one (C13)
8.6 Hz, 3/4H), 2.43 (s, 3/4H), 2.39 (s, 9/4H). C NMR (101 MHz,
1
H NMR (400 MHz, DMSO) δ 10.33 (m, 1H), 8.74 (s, 8/5H), 7.81 (s,
DMSO) δ 169.38, 148.89, 141.97, 141.32, 139.97, 131.27, 130.19,
2
/5H), 7.75 (m, 1/5H), 7.52 (d, J = 7.6 Hz, 4/5H), 7.44 (s, 4/5H), 7.34
129.98, 126.76, 125.44, 121.76, 117.74, 110.47, 21.64. HRMS (Q-TOF):
+
(
s, 1/5H), 7.13 (t, J = 7.6 Hz, 1/5H), 7.03 (t, J = 7.6 Hz, 4/5H),
calculated for C16
H
12
N
2
O
3
280.0848 [M + Na] , found 303.0741. Pu-
1
3
6
.95–6.83 (m, 6/5H), 6.77 (d, J = 7.6 Hz, 4/5H). C NMR (101 MHz,
rity ≥ 95.0%.
DMSO) δ 167.75, 152.98, 141.11, 136.54, 134.39, 129.39, 129.10,
1
26.52, 125.34, 121.66, 121.61, 120.10, 111.55, 109.91. HRMS (Q-
4.2.27. 5-Nitro-3-(pyridin-2-ylmethylene)indolin-2-one (D3)
392.9000 [M + Na]+, found
1
H NMR (400 MHz, DMSO) δ 11.33 (s, 1H), 10.11 (d, J = 2.4 Hz, 1H),
TOF): calculated for C15
H
9
Br
2
NO
2
4
15.8901. Purity ≥ 95.0%.
8.92 (d, J = 4.4 Hz, 1H), 8.25 (dd, J = 8.6, 2.4 Hz, 1H), 8.02 (m, 2H),
1
3
7
.75 (s, 1H), 7.62–7.49 (m, 1H), 7.06 (d, J = 8.6 Hz, 1H). C NMR (101
4
.2.21. 3-((1-Methyl-1H-indol-3-yl)methylene)indolin-2-one (C15)
MHz, DMSO) δ 170.01, 153.00, 150.12, 149.66, 142.34, 138.11, 137.13,
1
H NMR (400 MHz, DMSO) δ 10.51 (s, 1H), 9.40 (s, 1H), 8.22–8.17
130.00, 127.85, 127.55, 125.52, 124.14, 122.23, 110.15. HRMS (Q-
+
(
m, 1H), 8.12 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.61–7.55 (m, 1H),
TOF): calculated for C14
H
9
N
3
O
3
267.0644 [M + H] , found 268.0715.
7
.36–7.25 (m, 2H), 7.14 (td, J = 7.6, 1.2 Hz, 1H), 6.99 (td, J = 7.6, 0.8
Purity ≥ 95.0%.
1
3
Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 3.94 (s, 3H). C NMR (101 MHz,
DMSO) δ 168.54, 139.67, 137.41, 137.01, 129.17, 127.24, 127.01,
4.2.28. 3-(3-Hydroxy-4-methoxybenzylidene)-5-nitroindolin-2-one (D5)
1
1
1
2
26.13, 123.08, 121.52, 121.00, 119.47, 119.19, 119.05, 111.15,
H NMR (400 MHz, DMSO) δ 11.16 (s, 1H), 9.28 (s, 1H), 8.66 (d, J =
10.89, 109.45, 33.89. HRMS (Q-TOF): calculated for C18
H
14
N
2
O
2.4 Hz, 1/2H), 8.56 (d, J = 2.0 Hz, 1/2H), 8.27 (d, J = 2.0 Hz, 1/2H),
8.15 (m,1H), 8.07 (s, 1/2H), 7.94 (dd, J = 8.4, 2.0 Hz, 1/2H), 7.71 (s, 1/
+
74.1106[M + H] , found 275.1180. Purity ≥ 95.0%.
1
3
2
H), 7.29–7.23 (m, 1H), 7.15–6.97 (m, 2H), 3.89 (d, J = 2.0 Hz, 3H).
C
4
.2.22. 3-((5-Methylpyrazin-2-yl)methylene)indolin-2-one (C17)
NMR (101 MHz, DMSO) δ 169.64, 168.00, 151.71, 150.67, 148.66,
147.20, 146.30, 145.96, 142.43, 142.00, 141.96, 140.51, 127.56,
127.46, 126.77, 126.61, 126.38, 124.84, 123.78, 123.12, 122.00,
121.76, 119.72, 117.72, 116.85, 115.43, 112.59, 111.85, 110.35,
109.51, 56.21, 56.13. Purity ≥ 95.0%.
1
H NMR (400 MHz, DMSO) δ 10.64 (s, 1H), 8.96 (d, J = 0.8 Hz, 1H),
.87 (d, J = 7.6 Hz, 1H), 8.83 (s, 1H), 7.60 (s, 1H), 7.30 (td, J = 7.6, 0.8
8
Hz, 1H), 6.99 (td, J = 7.6, 0.8 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 2.59 (s,
1
3
3
H). C NMR (101 MHz, DMSO) δ 169.39, 154.26, 148.10, 146.60,
1
44.56, 144.19, 131.61, 130.87, 130.35, 128.23, 121.77, 110.22, 21.87.
+
′
HRMS (Q-TOF): calculated for C14
H
11
N
3
O 237.0902 [M + H] , found
4.2.29. 3-([1,1 -Biphenyl]-4-ylmethylene)-5-nitroindolin-2-one (D12)
1
2
38.0980. Purity ≥ 95.0%.
H NMR (400 MHz, DMSO) δ 11.36 (s, 1H), 8.73 (d, J = 2.0 Hz, 1/
2
H), 8.58 (d, J = 8.2 Hz, 1/2H), 8.46 (d, J = 2.0 Hz, 1/2H), 8.28 (s, 1H),
4
.2.23. 3-((2-Chloro-6-methoxyquinolin-3-yl)methylene)indolin-2-one
8.19 (td, J = 8.4, 2.4 Hz, 1H), 7.94–7.77 (m, 11/2H), 7.52 (dd, J = 13.2,
7.2 Hz, 2H), 7.43 (dd, J = 13.2, 7.2 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1/2H),
(
C18)
1
13
H NMR (400 MHz, DMSO) δ 10.74 (s, 3/10H), 10.23 (s, 7/10H),
7.03 (d, J = 8.6 Hz, 1/2H). C NMR (101 MHz, DMSO) δ 169.33,
8
.71 (s, 3/10H), 8.44 (s, 7/10H), 7.95 (d, J = 10.0 Hz, 3/10H), 7.87 (d, J
167.87, 149.03, 146.56, 143.10, 142.57, 142.05, 140.67, 139.57,
139.45, 133.73, 133.28, 133.25, 130.88, 129.60, 129.54, 128.69,
127.48, 127.31, 127.28, 126.95, 126.24, 126.18, 125.71, 124.89,
=
9.2 Hz, 7/10H), 7.62 (s, 3/10H), 7.56–7.52 (m, 6/10H), 7.48 (d, J =
2
.4 Hz, 7/10H), 7.44 (dd, J = 9.2, 2.8 Hz, 7/10H), 7.37 (d, J = 7.2 Hz, 7/
1
0

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
1
21.72, 117.96, 116.11, 110.60, 109.83. HRMS (Q-TOF): calculated for
4.3. Protein purification
+
C
21
H
14
2
N O
3
342.1004 [M + Na] , found 365.0903. Purity ≥ 95.0%.
HsClpP was expressed and purified as described previously. Wildtype
HsClpP (without mitochondrial targeting sequences) were expressed in
E. coli. To induce protein expression, bacteria, after reaching OD600 0.6
~ 0.8, were treated with 1 mM isopropyl-1-thio-B-D-galactopyranoside
4
.2.30. 3-(3,5-Dibromo-4-hydroxybenzylidene)-5-nitroindolin-2-one
(
D13)
1
H NMR (400 MHz, DMSO) δ 11.30 (m, 1H), 8.84 (s, 8/5H), 8.61 (d,
◦
J = 2.0 Hz, 4/5H), 8.42 (d, J = 2.0 Hz, 1/5H), 8.19 (dd, J = 8.6, 2.4 Hz,
for 4 h at 37 C, harvested by centrifugation, and disrupted in lysis buffer
1
7
/5H), 8.13 (dd, J = 8.6, 2.4 Hz, 4/5H), 8.05 (s, 4/5H), 8.01 (s, 2/5H),
.69 (s, 1/5H), 7.06 (d, J = 8.6 Hz, 1/5H), 7.00 (d, J = 8.6 Hz, 4/5H).
C NMR (101 MHz, DMSO) δ 168.02, 145.99, 142.43, 138.59, 137.43,
(25 mM Tris-HCl (pH 7.5), 0.5 M NaCl). Following cell lysis, the insol-
4
uble material was removed by centrifugation (1.4x10 rpm for 30 min)
1
3
and the supernatant was passed through a 5 mL Ni-sepharose high-
performance (GE) column pre-equilibrated with lysis buffer. The protein
was eluted with 400 mM imidazole, diluted with 25 mL of dialysis buffer
1
34.23, 126.51, 124.92, 115.36, 112.67, 112.21, 109.66. HRMS (Q-
+
TOF): calculated for C15
8
H Br
2
N
2
O
4
439.8830 [M + Na] , found
◦
4
62.8714. Purity ≥ 95.0%.
(25 mM Tris-HCl (pH 7.5), 0.1 M NaCl), and dialyzed overnight at 4 C
with light stirring into 4 L of dialysis buffer. The dialyzed material was
then passed through a second 5 mL Ni-column and the flow-through
solution containing untagged HsClpP was collected. All fractions were
analyzed by SDS-PAGE.
4
.2.31. 3-(2-Chloro-4-hydroxybenzylidene)-5-nitroindolin-2-one (D14)
1
H NMR (400 MHz, DMSO) δ 11.30 (s, 1H), 8.57 (d, J = 2.4 Hz, 1/
5
5
6
1
1
H), 8.49 (d, J = 8.8 Hz, 1/5H), 8.27–8.10 (m, 2H), 7.81–7.72 (m, 8/
H), 7.10–7.04 (m, 4/5H), 7.00 (d, J = 8.8 Hz, 1/5H), 6.94 (m, 4/5H),
1
3
.79 (dd, J = 8.8, 2.0 Hz, 1/5H). C NMR (101 MHz, DMSO) δ 169.17,
4.4. HsClpP enzymatic assays
61.24, 148.89, 142.04, 135.79, 135.53, 132.26, 126.87, 125.82,
22.60, 121.60, 117.87, 117.23, 115.25, 110.56. HRMS (Q-TOF):
Assay buffer consisted of 25 mM Tris-HCl, pH 7.5, 0.1 M NaCl pH 8
+
calculated for C15
H
9
2
ClN O
4
316.0251 [M + Na] , found 339.0143.
for AC-WLA-AMC assay. 1.0
buffer, incubated at 37C for 10 min, and mixed with increasing con-
centrations of synthesized compounds in 96 well plates at 80 L per well
in triplicate. Fluorescent tagged- substrates, AC-WLA-AMC (100 M),
μM HsClpP was dissolved in the assay
Purity ≥ 95.0%.
μ
4
.2.32. 3-((1-Methyl-1H-indol-3-yl)methylene)-5-nitroindolin-2-one
μ
(
D15)
were then added to each well and fluorescence was measured at 380/
1
◦
H NMR (400 MHz, DMSO) δ 11.23 (s, 1H), 9.48 (s, 1H), 8.95 (d, J =
460 nm every 5 min for 90 min at 37 C. Experiments were performed
2
7
.0 Hz, 1H), 8.54 (s, 1H), 8.41 (m, 1H), 8.09 (dd, J = 8.8, 2.4 Hz, 1H),
three times with triplicate wells in each experiment.
.61 (m, 1H), 7.41–7.27 (m, 2H), 7.02 (d, J = 8.4 Hz, 1H), 3.97 (s, 3H).
C NMR (101 MHz, DMSO) δ 169.47, 149.91, 146.94, 143.10, 136.94,
30.05, 127.36, 125.77, 122.82, 122.67, 121.67, 121.47, 116.70,
For the protease activity assay, the buffer solution consisted of 25
mM Hepes, pH 7.5, 20 mM MgCl2, 30 mM KCl, 0.03% Tween 20, 10%
1
3
1
1
3
glycerol, and 2 mM DTT. Each reaction contained 1
ONC201, and an ATP-regenerating system (4 mM ATP, 20 U/mL crea-
tine kinase, and 16 mM creatine phosphate). 4 M FITC-casein was then
μM HsClpP, 1
μM
12.56, 110.45, 56.11. HRMS (Q-TOF): calculated for C18
H
13
N
3
O
3
+
19.0957 [M + Na] , found 342.0851. Purity ≥ 95.0%.
μ
added to each well, and the fluorescence was measured at 485/535 nm
for 60 min. Experiments were performed three times with triplicate
wells in each experiment.
4
2
.2.33. 3-((5-(2-Fluorophenyl)-1H-pyrrol-3-yl)methylene)-5-nitroindolin-
-one (D16)
1
H NMR (400 MHz, DMSO) δ 12.27 (s, 1/5H), 12.11 (s, 4/5H), 11.15
(
d, J = 12.9 Hz, 1H), 8.85 (d, J = 2.4 Hz, 1/5H), 8.54 (d, J = 2.0 Hz, 4/
4.5. In vitro inhibitory activity
5
7
H), 8.32 (s, 4/5H), 8.21–8.15 (m, 1H), 8.09 (dd, J = 8.4, 2.0 Hz, 4/5H),
.78 (m, 7/5H), 7.61 (s, 4/5H), 7.39–7.26 (m, 3H), 7.21 (s, 1/5H), 7.03
Hela, HepG2, HCT116, MCF-7 and SKOV3 cells were seeded in 96-
m, 1H). 13C NMR (101 MHz, DMSO) δ 168.37, 145.26, 142.19, 135.31,
3
(
well plates at a density of 5 × 10 cells/well and cultured for 24 h,
1
1
1
30.52, 128.63 (d, J = 8.1 Hz), 127.23, 127.05 (d, J = 3.0 Hz), 126.90,
following the addition of different concentrations of compounds. The
cells were further cultured for 72 h, with 0.5% DMSO as the solvent
control group. Then cells were incubated for 1–4 h with 5 mg/ml of 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) so-
lution, and the formazan crystals were dissolved with 100 mL of DMSO.
After gentle shaking for 10 min, the optical density (OD) was measured
at 570 nm using a Spectra MAX M5 microplate spectrophotometer. Each
treatment was performed in triplicate. Results were analyzed with
GraphPad Prism 6 and data were shown as Mean ± SD.
25.31, 125.28, 123.72, 121.46, 120.18, 117.48, 116.90, 116.68,
14.28, 114.11 (d, J = 9.1 Hz), 109.30. HRMS (Q-TOF): calculated for
+
C
19
H
3
12FN O
3
349.0863 [M + Na] , found 372.0758. Purity ≥ 95.0%.
4
.2.34. 3-(3,5-Dibromo-4-methoxybenzylidene)-5-nitroindolin-2-one
(
D13a)
1
H NMR (400 MHz, DMSO) δ 11.41 (s, 1H), 8.82 (s, 2H), 8.65 (d, J =
.4 Hz, 1H), 8.19 (dd, J = 8.6, 2.4 Hz, 1H), 8.16 (s, 1H), 7.03 (d, J = 8.6
Hz, 1H), 3.88 (s, 2H).
2
1
3
C NMR (101 MHz, DMSO) δ 167.73, 155.65, 146.85, 142.62,
36.93, 134.16, 133.07, 126.44, 126.22, 125.69, 118.46, 117.66,
4.6. Molecular docking
1
1
10.10, 61.15. HRMS (Q-TOF): calculated for C16
H10Br
2
N
2
O
4
453.8987
Molecular docking studies were carried out using the Schrodinger
software. The protein was constructed based on the X-ray structure of
HsClpP, which was available through the RCSB Protein Data Bank (PDB
code: 1TG6). Both the compounds and the structure of HsClpP were
prepared and optimized using Schrodinger. The protein was prepared
with adding hydrogens, deleting water molecules. Compound I was
docked into the catalytic site using the Glide XP docking procedure.
+
[
M + Na] , found 476.3730. Purity ≥ 95.0%.
4
.2.35. 3-(2-Chloro-4-hydroxybenzylidene)-1-methylindolin-2-one (E14)
1
H NMR (400 MHz, DMSO) δ 8.91 (d, J = 1.6 Hz, 3/10H), 8.10 (m, 3/
1
2
0H), 7.75–7.64 (m, 2H), 7.61–7.54 (m, 7/5H), 7.27 (m, 1H), 6.99 (m,
1
3
7/10H), 6.88 (d, J = 8.4 Hz, 3/10H), 3.21 (d, J = 8.4 Hz, 3H). C NMR
(
101 MHz, DMSO) δ 168.06, 158.08, 144.05, 136.68, 134.60, 132.19,
1
30.72, 129.82, 125.10, 123.87, 122.06, 121.10, 118.91, 117.74,
4.7. Wound healing assay
1
09.21, 26.43. HRMS (Q-TOF): calculated for C16
H
12ClNO2 285.0557
+
[
M + Na] , found 308.0449. Purity ≥ 95.0%.
Hela cells were seed in 6 well plates and cultured until confluent.
Using a pipette tip makes a straight scratch to simulate a wound, 24 h
after treatment by compound I at 5 M. Cells were washed twice to
μ
1
1

R. Song et al.
Bioorganic Chemistry 110 (2021) 104820
remove detached cells and debris. Then, size of wounds were observed
and measured at the indicated times.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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This work was financially supported by grants from National Natural
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Appendix A. Supplementary material
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