Identification of Piperidine-3-carboxamide Derivatives Inducing Senescence-like Phenotype with Antimelanoma Activities

Article abstract of DOI:10.1021/acsmedchemlett.0c00570

This study evaluated the potential use of senescence-inducing small molecules in the treatment of melanoma. We screened commercially available small-molecule libraries with high-throughput screening and high-content screening image-based technology. Our findings showed an initial hit with the embedded N-arylpiperidine-3-carboxamide scaffold-induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. A focused library containing diversely modified analogues were constructed and examined to evaluate the structure-activity relationship of N-arylpiperidine-3-carboxamide derivatives starting from hit 1. This work identified a novel compound with remarkable antiproliferative activity in vitro and demonstrated the key structural moieties within.

Full text of DOI:10.1021/acsmedchemlett.0c00570

pubs.acs.org/acsmedchemlett
Letter
Identification of Piperidine-3-carboxamide Derivatives Inducing
Senescence-like Phenotype with Antimelanoma Activities
Sangmi Oh, Do Yoon Kwon, Inhee Choi, Young Mi Kim, Ji Young Lee, Jiyoung Ryu, Hangyeol Jeong,
Myung Jin Kim,* and Rita Song*
Cite This: ACS Med. Chem. Lett. 2021, 12, 563−571
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: This study evaluated the potential use of senescence-inducing small molecules in the treatment of melanoma. We
screened commercially available small-molecule libraries with high-throughput screening and high-content screening image-based
technology. Our findings showed an initial hit with the embedded N-arylpiperidine-3-carboxamide scaffold-induced senescence-like
phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. A focused library containing
diversely modified analogues were constructed and examined to evaluate the structure−activity relationship of N-arylpiperidine-3-
carboxamide derivatives starting from hit 1. This work identified a novel compound with remarkable antiproliferative activity in vitro
and demonstrated the key structural moieties within.
KEYWORDS: high-throughput screening, high-content screening, senescence, melanoma, piperidine-3-carboxamide,
structure−activity relationship
to a terminal nonproliferation state in cancer cells and/or
result in apoptosis. In addition, senescent cells could be
eliminated by CD4+T cells and macrophages, leading to an
apoptosis-like effect.19−21 The genes involved in senescence
and detailed mechanisms in anticancer therapies remain to be
fully elucidated, and many research groups have been focusing
on the identification of target proteins in the senescence
pathway or small molecules that can induce senescence
through library screening.13−16
Senescence-associated β-gal (SA-β-gal) activity of cells has
been widely used as a senescence marker in the screening for
the discovery of senescence inducers.14,16 However, it is
difficult to be applied to the high-throughput assay format due
to the use of bright-field microscopy. To efficiently conduct
high-throughput screening (HTS) and high-content screening
(HCS), a whole-cell-based approach was designed to select
compounds by analyzing the unique phenotype of senescent
ellular senescence is a cessation of cell division involving
1−3
Ccomplex signal transduction pathways within the cells.
Two types of cellular senescence have been described in the
literature: replicative senescence and premature senescence.
Replicative senescence has been observed in all metabolic
active cells, manifested as a spontaneous growth rate decline by
telomere shortening. Replicative senescence could be recov-
ered by ectopic expression of telomerase holoenzyme
(hTERT).4−6 Premature senescence can be induced by
oncogenes, chemicals, or oxidative stress.7,8 Senescent cells
are characterized by altered cell morphology, including
enlarged cell size, flattened cell shape, a single large nucleus,
and increased cytoplasmic granularity. They could be
distinguished from quiescent cells by their altered metabolic
and transcriptional activities.
Recently, senescence is emerging as a therapeutic target for
various diseases.9,10 Prosenescent and antisenescent therapies,
called senolytic, showed promising results in mouse models,
and human clinical trials are currently in progress. Some of the
most widely used senolytics are BCL-2 family inhibitors that
target apoptotic resistance of senescent cells.11,12 Conversely,
drugs that induce senescence (such as palbociclib and other
CDK4/6 inhibitors) have shown significant benefits as
anticancer agents.13−18 The induction of senescence can lead
Received: October 26, 2020
Accepted: March 17, 2021
Published: March 22, 2021
https://doi.org/10.1021/acsmedchemlett.0c00570
© 2021 American Chemical Society
ACS Med. Chem. Lett. 2021, 12, 563−571
563

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
cells with fluorescence dye.22 With this technology, we
screened ∼110,000 chemicals and selected compounds that
induced a senescence-like phenotype in human melanoma
A375 cells. With image-based HTS/HCS, we identified
compound 1 (Figure 1) as one of the initial hits to induce
were prepared by Suzuki coupling with 523 followed by
reductive amination with corresponding substituted benzalde-
hydes (12−14, 19−34) in the presence of NaBH(OAc)3.
Other derivatives with a modified linker part like carbonyl (15)
and sulfone (16) were prepared through substitution reactions
in the presence of base after Boc deprotection (route I in
Scheme 1).
On the other hand, after Boc deprotection of intermediate 4,
reductive amination with 3,4-difluorobenzaldehyde and Suzuki
coupling reaction with various boronic acid building blocks in
sequence described in route II were applied for the synthesis of
some derivatives having different heterocycles in the R3
position (35−38, 42, 47, 48).24
The preparation of other analogues, modified B ring and/or
R3 position (39−41, 43−46, 50−56), were done starting from
C−C bond formation between the B ring and R3 (route III in
Scheme 2). The key intermediates 10 were prepared by Suzuki
coupling reaction with corresponding heterocyclic boronic
acids except for 41 having a 2′-imidazolyl group. In this case,
the condensation of imidazole with aryldiazonium ion in situ
generated by 4-nitroaniline 9 in the presence of sodium nitrite,
followed by nitro reduction for the synthesis of intermediate
10.25 Amide coupling reaction with Boc-nipecotic acid and
then subsequent reductive amination led to the desired final
compounds (Scheme 2). In addition, compound 17, with a
reverse amide between aminopiperidine and the B ring, was
synthesized for the evaluation of amide linker via a Suzuki
coupling reaction followed by amide coupling reaction
(Scheme SI-5). The bicyclic benzothiazole 18 was generated
by the same reaction described in Scheme 2 except with Suzuki
coupling (Scheme SI-6). Detailed synthetic procedures
including the synthesis of the most potent analogue 54 are
described in Supporting Information. All final compounds
newly synthesized were characterized by melting point, low
Figure 1. Hit compound 1 and its derivatization for SAR study.
senescence-like phenotypic changes in human melanoma A375
cells without serious cellular toxicity (Table SI-1). We here
report the structure−activity relationship (SAR) study of N-
arylpiperidine-3-carboxamide derivatives starting from 1, which
led to the identification of 54, a novel compound with
remarkable antimelanoma activity in vitro.
In search of compounds with improved antiproliferative
activity as a senescence inducer, the initial hit 1 having the N-
arylpiperidine-3-carboxamide moiety was chemically derivat-
ized, as shown in Figure 1. General synthetic routes for N-
arylpiperidine-3-carboxamide analogues were classified into
three different ways, routes I−III, depending on the
modification sites described in Schemes 1 and 2. Boc-protected
N-bromophenyl heterocyclic carboxamides 4 were obtained by
the EDC coupling reaction of 4-bromoaniline (2) and
commercially available heterocyclic carboxylic acids (3). In
the case of final products with a modified A ring or ring size of
nitrogen-containing aliphatic cycle (12−16, 19−34, 51), they
1
resolution ESI mass, and H NMR.
To assess anticancer activities of synthetic derivatives against
melanoma cells, a phenomic assay was developed to measure
cellular morphological changes such as enlarged cytoplasm
containing a large nucleus and increased cytoplasmic granules
along with counting the number of cells in a well. Doxorubicin,
a
Scheme 1
a
Reagents and conditions: (A) EDC 1.2 equiv, HOBt 1.0 equiv, TEA 1.5 equiv, CH2Cl2, RT, 12 h; (B) 5 or R3-boronic acid 1.2 equiv, Pd(PPh3)4
0.05 equiv, Na2CO3 2.0 equiv, DME/H2O, 90 °C, 5 h; (C) 4 M HCl in dioxane, CH2Cl2, RT, 2 h; (D) [R = CH2] substituted benzaldehyde 1.2
equiv, NaBH(OAc)3 2.0 equiv, AcOH (cat.), CH2Cl2, RT, 12 h; [R = SO2 or CO, n = 1] 3,4-difluorobenzenesulfonyl chloride or 3,4-
difluorobenzoyl chloride 1.2 equiv, TEA 2.0 equiv, CH2Cl2, 0 °C, 3 h.
564
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
a
Scheme 2
a
Reagents and conditions: (A) R3-boronic acid 1.2 equiv, Pd(PPh3)4 0.05 equiv, Na2CO3 2.0 equiv, DME/H2O, 90 °C, 5 h; (B) 10% Pd/C, H2 gas,
MeOH, 0.5−2 h; (C) Boc-nipecotic acid 1.2 equiv, HATU 1.2 equiv, DIPEA 2.0 equiv, DMF, RT, 12 h; (D) TFA, CH2Cl2, RT, 1−3 h; (E)
substituted benzaldehyde 1.2 equiv, NaBH(OAc)3 2.0 equiv, AcOH (cat.), CH2Cl2, RT, 12 h; (F) NaNO2 1.0 equiv, aq HCl and then imidazole
1.0 equiv, NaOAc, 0 °C to RT, 48 h.
Figure 2. Assay validation with doxorubicin-treated cells. (A) Segmented images of negative control (0.5% DMSO) and positive control (37 nM
doxorubicin in 0.5% DMSO) for data analysis. (B) Dose-dependent changes of doxorubicin-treated cells. Measurement of cell growth and
senescence effect was analyzed by a customized algorithm. All experiments were duplicated. (C) Senescence effect of doxorubicin (37 nM in 0.5%
DMSO) in phenomic assay and SA-β-gal assay. Human melanoma A375 cells were treated with or without doxorubicin (37 nM) for 3 days. In the
phenomic assay, the cells were acquired images after staining with Hoechst 33342, LysoTracker Red DND-99, and CellMask (top panel). Under
the same condition, cells were performed SA-β-gal (bottom panel). Scale bar is 50 μm.
a topoisomerase II inhibitor, was used to validate the assay
(Figure 2) because it had been reported to induce DNA-
damage-mediated senescence in human cells.26−28 After 3 days
of compound treatment, human melanoma A375 cells were
imaged and analyzed by customized software. Prior to analyses,
the customized algorithm calibrated the segmentation
procedure of images (Figure 2A). Morphological properties
were captured and analyzed, including number of cells per well,
cell size, nucleus size, granule size, the distance of each granule,
cytoplasmic texture, volume of each organelle, etc. The EC50
and IC50 values were calculated from the dose−response
curves based on cell number and ratio of senescent cells per
well (Figure 2B). SA-β-gal assay, a traditional marker of
senescence, was also used to validate the senescence in
doxorubicin-treated cells (Figure 2C). Under the same
condition, doxorubicin-treated cells demonstrated highly
increased SA-β-gal activity. In vitro antimelanoma activities of
the synthetic compounds are summarized in Tables 1 and 2.
The hit compound 1 showed moderate senescence-inducing
activity (EC50 = 1.24 μM) and antiproliferative activity (IC50
=
0.88 μM) in A375 human melanoma cell line (Figure 1 and
Table 1). The piperidine-3-carboxamide moiety was important
in both regioisomeric aspects and ring size because the
regioisomer 12 with piperidine-4-carboxamide functionality
was found to be inactive, and also the replacement of
piperidine with smaller ones, pyrrolidine 13 or azetidine 14,
565
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
μM, much better than that of R-configuration 49 (19.9 μM),
while the EC50 of racemate 48 was 0.72 μM.
Table 1. Senescence-inducing Activity (EC50) and
Antiproliferative Activity (IC50) of N-Arylpiperidine-3-
carboxamide Derivatives
In regard to substituent effects of R1 in the A ring, the
antiproliferative activity related to the senescence-like
phenotype was strongly influenced by the presence of
electronegative atoms, such as fluorine or oxygen, and their
substitutional positions. Compounds without fluorine in C-2
and/or C-3, or regioisomers substituted in different positions
of the A ring resulted in a significant loss of antimelanoma
activities (21−26). Only 2,3,4-trifluoro-substituted 27 (EC50
=
1.26 μM) showed potency similar to that of initial hit 1, and
benzodioxole 34 (EC50 = 0.60 μM), which was an oxygen-
incorporated analogue at the position of C-2 and C-3,
exhibited activity better than that of 1. Therefore, this finding
indicated that the presence of hydrogen bonding acceptors at
the C-2 and C-4 position might be essential for their
antiproliferative activity against melanoma cancer cells. Other
compounds including bulkier substituents in the A ring or
pyridine instead of benzene were inactive (29−33).
To optimize R3 connected to the B ring, compounds 35−48
with various heterocycles were prepared and evaluated for their
biological activities. Replacement of the original thiazole by six-
membered aryl groups such as phenyl or pyridine (35−38) did
not show an improvement of activity. However, five-membered
heterocycles containing nitrogen or sulfur in the α-position
were able to enhance antimelanoma activities (39, 42, 46, and
48). Even though all four derivatives showed a similar range of
antiproliferative activities below 1.0 μM as senescence
inducers, we selected 2′-pyrrole as R3 for further modification
because it has higher metabolic stability than the others (Table
SI-2). Considering all of the structural factors detected in the
SAR study, S-isomers 50 and 51 were prepared and found to
have 10-fold increase in antimelanoma activities with
senescence-like phenotype (EC50 = 0.14 and 0.16 μM,
respectively). To be able to search for proper modification of
the B ring on the basis of 50, analogues 52−56 with different
substituents on the B ring or with pyridine instead of benzene
B ring were evaluated. A remarkable improvement in the
biological activity was achieved when benzene was replaced
with pyridine as the B ring, and 54 was found to be the most
potent compound in the focused library with an EC50 of 40
nM, which was comparable to that of reference drug,
doxorubicin (9 nM). Phase 1 metabolic stability studies of
some synthetic analogues including hit 1 and 54 were
performed for the future applications and confirmed that
they were stable enough to test in vivo (see Table SI-2).
To check the activity of N-arylpiperidine-3-carboxamide and
its derivatives on human melanoma A375 cells, the dose-
dependent responses were examined via a newly developed
phenomic assay. Compound 1 elicited phenomic changes and
decreased total cell numbers (Figure 3A). Compared to hit 1,
54 was more potent to induce cell growth inhibition and a
senescence-like phenotype (Figure 3B). To confirm the
senescence-like morphological change by compound 54, we
compared the morphological changes of 54-treated cells with
doxorubicin-treated ones. Compound 54-treated cells showed
a senescence-like phenotype which enlarged cell size and
nucleus size with increasing cytoplasmic granularity, similar to
doxorubicin-treated cells (Figure 3C).
a
b
Compound 1 was an already known molecule. EC50 means the
effective concentration of a compound that induces senescence in a
half population. IC50 represents the concentration of a compound
where the cell number is reduced by 50%.
c
displayed gradually decreasing activities (8.0 and >20 μM,
respectively). In addition to that, carbonyl (15) or sulfonyl
groups (16) instead of a methylene linker were not effective to
induce senescence. The reverse-amide analogue (17) of the
original scaffold and bicyclic benzothiazole 18 also proved to
be inactive.
In addition to evaluate the substituent effects of the A and B
rings, and replacement effect of R with different heteroaryls, a
series of compounds described in Table 2 were synthesized to
determine the enantioselectivity toward antimelanoma activity.
Notably, putative target molecules displayed a high level of
enantioselectivity for N-arylpiperidine-3-carboxamide deriva-
tives. S-Configuration 20 of the hit 1 showed an EC50 of 0.27
μM, better than that of racemic mixture 1 and a 15-fold
increase over that of R-configuration 19. This enantioselectiv-
ity also confirmed that S-configuration 50 had an EC50 of 0.14
A data set of 46 compounds in Tables 1 and 2 were used to
build a pharmacophore model based on their senescence-
inducing activity (EC50). All of the structures were built on
̈
Maestro, a module of Schrodinger (Maestro). Low-energy 3D
566
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Table 2. Senescence-inducing Activity (EC50) and Antiproliferative Activity (IC50) of N-Arylpiperidine-3-carboxamide
Derivatives
a
b
Compounds 1, 21, 29, and 32 are already known molecules. EC50 means the effective concentration of a compound that induces senescence in a
c
d
half population. IC50 represents the concentration of a compound where the cell number is reduced by 50%. One specific carbon in benzene was
replaced by nitrogen with the formation of pyridine.
567
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Figure 3. Identification of N-arylpiperidine-3-carboxamide derivatives to induce senescence-like phenotype in a dose-dependent manner. (A,B)
Quantification of compounds 1 (A) or 54-mediated cellular changes (B) in dose response. Human melanoma A375 cells were treated each
compound in 3-fold dilution. After 3 days, cellular images were acquired using Opera and then analyzed by customized software. Red line means
cell number, and black line means percentage of senescent cells per well. Z′ value of assay validation was about 0.8. (C) Cellular images treated with
doxorubicin and 54 in a dose-dependent manner. Scale bar is 20.0 μm. All experiments were duplicated.
conformations of compounds were processed with the LigPrep
program,29 and they were energy minimized with OPLS_2005
force field method. Next, the conformers were generated for
each compound using a torsional search method with
Confgen.30 A total of 100 conformers per rotatable bonds
were generated with the maximum number of conformers set
as 1000 per compound. Phase program was used to generate
the common pharmacophore hypothesis (CPH).31 The
activity data of the compound were taken as negative
logarithms of EC50 values. The activities of CPO compounds
were classified as follows: (1) active compounds were
considered as those with pEC50 values above −0.2, and (2)
inactive compounds were those with pEC50 values below −1.0.
Compounds were divided into both training and test sets based
on structural diversity and coverage of the activity range.
Pharmacophores from all conformations of compounds were
examined, and all possible pharmacophore models having
common features were identified. The minimum and
maximum number of sites that a CPH may have was set to
six. Also, it was set to match all tenactive compounds. Among
generated possible CPH, the best CPH was selected based on
scoring. As the Phase program also provided the option to
perform 3D-QSAR with the selected pharmacophore hypoth-
esis, the atom-based QSAR model was built with default values
for options.31
Out of twelve generated six featured hypotheses, the best
CPH was selected as AHPRRR.11, based on having a higher
survival score (survival score = 3.879) than others, as well as
better predictive ability (r2 = 0.884) from QSAR analysis. It
was a six-point pharmacophore hypothesis with one hydrogen-
bond acceptor (A), one hydrophobic group (H), one positively
ionizable (P), and three aromatic rings (R) as pharmacophoric
features. Active ligands were well-aligned with all six
pharmacophoric features such as the most active compound
54 (Figure 4A): (1) Rings with substituents R1−R3 are well-
represented by R11, R10, and R9 features, respectively; (2) the
carbonyl group of the 3-carboxamide was represented by an A2
feature; and (3) N-piperidine was represented by both H7 and
P8 features.
The best CPH clearly explained enantioselectivity for N-
arylpiperidine-3-carboxamide of the compounds. The S-
configuration 20 of the hit 1, which was more active than
that of racemic mixture 1, was better mapped to the CPH than
R-configuration 19, which had a more than 15-fold increase
than 20. Due to angular difference between S- and R-
configurations, the R11 feature was not mapped in the centroid
of the A ring, 2,3-difluoro, in 19 (Figure 4E,F). Another
notable mismapping was seen for the same R11 feature in 49,
where it is not flat but perpendicular to the planarity of R1
group. Also, R9 and R10 features of 49 were out-of-plane
against the rings in R2 and R3 groups. The A2 feature did not
map with the carboxamide (Figure 4G). On the other hand, all
pharmacophoric features mapped well with the active S-
configuration 50 of racemate 48 (Figure 4H). These analyses
clearly demonstrated structural differences of enantiomers
significantly affected biological activity.
Additionally, a 3D-QSAR study was performed to establish
relationship between the 3D pharmacophoric features and
senescence-inducing activities. An atom-based QSAR was
selected to better explain the SAR because it considered the
entire molecular structure by treating a molecule as a set of
overlapping van der Waals spheres.32 The 3D-QSAR results
were visualized by a generated pictorial contours, with the blue
cubes indicating favorable, while red cubes indicating
unfavorable regions for activity (Figure 4B,D). Blue cubes
surrounding amine in the linker indicated the inactivity of
reverse-amide analogue (17) of the original scaffold. For R3
substituents, blue cubes surrounding the 2-position of 2′-
pyrrole suggested an increase in activity, whereas 3- and 5-
positions were highlighted in the red cube, suggesting a
568
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
Figure 4. (A) Alignment of best fit compound 54 with the pharmacophore sites of the selected pharmacophore hypothesis, AHPRRR.11. Three
aromatic ring features (R9, R10, and R11), one H-bond acceptor feature with lone pairs of electrons (A2), one positive ionizable (P8) feature, and
one hydrophobic feature (H7) are shown in orange rings, pink spheres, a blue sphere, and a green sphere, respectively. (B) View of 3D-QSAR
model with the most potent compound, 54. The blue and red cubes show the structural features contributing positively and negatively to activity,
respectively. (C) S-Configuration 51 is mapped well with CPH. (D) View of 3D-QSAR model with the active compound, 51. (E) Less active R-
configuration 19 is not mapped well with CPH. (F) S-Configuration 20 is mapped well with CPH. (G) Inactive R-configuration 49 is not aligned
well with CPH. (H) Active S-configuration 50 is mapped well with CPH. All compounds are shown in atom-colored turquoise sticks.
decrease in activity such as with compounds 35 and 41. The
presence of electronegative atoms such as fluorine or oxygen,
and their substitutional positions in R1 in the A ring, had a
strong influence on the senescent phenotype. A large number
of blue cubes surrounded the 2,3,4-positions of the A ring and
overlapped with the oxygen atoms of 51 (Figure 4D) as well as
fluorine atoms of 54 (Figure 4B). The biological activity
dramatically improved when benzene was replaced with
pyridine in the B ring, and blue cubes were massed around
the 1N atom in the B ring (Figure 4B). There was no cube
representing the 2N atom in the B ring, which indicated a
decrease in activity like that with compound 55.
In order to understand structural requirements of molecules
as melanoma senescence inducers, ligand-based pharmaco-
phore model and atom-based 3D-QSAR studies were
performed on a series of PCA derivatives to correlate their
molecular architecture with senescence-inducing activity. The
pharmacophore model well-explained the difference in bio-
logical activities between S- and R-configurations. Also, the
detailed substituent in each group could be explained in
accordance with the activity. This derived model can provide
guidance for the rational design of novel potent senescence
inducers to treat melanoma.
As a cellular mechanism to avoid malignant transformation,
cellular senescence has been considered a potential therapeutic
target for cancer treatment.17,18,33 In order to discover a novel
candidate for the treatment of melanoma, a newly developed
whole-cell-based HTS and HCS assay was applied to pilot
screen a small-molecule library containing ∼110,000 diverse
compounds, and a hit compound with N-arylpiperidine-3-
carboxamide scaffold was discovered. To understand the
structural aspects of the initial hit, 45 analogues with diverse
modification in each moiety were designed and synthesized
based on the original structure. Among them, 54, a S-isomer
with a pyridine ring in the B ring position, and pyrrole in R3,
demonstrated improved antimelanoma activity (IC50 = 0.03
μM) in accordance with markedly induced senescence-like
morphological change (EC50 = 0.04 μM) in human melanoma
A375 cells. This compound also showed acceptable phase I
metabolic stability. The novel series of N-arylpiperidine-3-
569
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
(3) Schmitt, C. A. Senescence, apoptosis and therapycutting the
lifelines of cancer. Nat. Rev. Cancer 2003, 3, 286−295.
(4) Bodnar, A. G.; Ouellette, M.; Frolkis, M.; Holt, S. E.; Chiu, C.-
P.; Morin, G. B.; Harley, C. B.; Shay, J. W.; Lichtsteiner, S.; Wright,
W. E. Extension of life-span by introduction of telomerase into normal
human cells. Science 1998, 279, 349−352.
(5) Ozturk, N.; Erdal, E.; Mumcuoglu, M.; Akcali, K. C.; Yalcin, O.;
Senturk, S.; Arslan-Ergul, A.; Gur, B.; Yulug, I.; Cetin-Atalay, R.; et al.
Reprogramming of replicative senescence in hepatocellular carcino-
ma-derived cells. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 2178−2183.
(6) Gorbunova, V.; Seluanov, A.; Pereira-Smith, O. M. Expression of
human telomerase (hTERT) does not prevent stress-induced
senescence in normal human fibroblasts but protects the cells from
stress-induced apoptosis and necrosis. J. Biol. Chem. 2002, 277,
38540−38549.
(7) Chang, B. D.; Broude, E. V.; Dokmanovic, M.; Zhu, H.; Ruth, A.;
Xuan, Y.; Kandel, E. S.; Lausch, E.; Christov, K.; Roninson, I. B. A
senescence-like phenotype distinguishes tumor cells that undergo
terminal proliferation arrest after exposure to anticancer agents.
Cancer Res. 1999, 59, 3761−3767.
(8) Ewald, J. A.; Desotelle, J. A.; Wilding, G.; Jarrard, D. F. Therapy-
induced senescence in cancer. J. Natl. Cancer Inst. 2010, 102, 1536−
1546.
carboxamide derivatives proved the successful application of a
newly developed HTS/HCS assay based on senescence-
induced anticancer activities. Further studies including target
identification and in vivo efficacy study are undergoing.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00570.
Supporting Tables SI-1 and SI-2 for cytotoxicity and
phase I metabolic stability; experimental procedures for
biological assays, synthetic procedures, and character-
ization data for presented compounds, including the
1
spectral copies of H and 13C NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Myung Jin Kim − Functional Morphometry-I, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea; Phone: +82-2-
965-7977; Email: nanmolra1973@naver.com; Fax: +82-2-
965-7976
(9) Lee, S.; Lee, J.-S. Cellular senescence: a promising strategy for
cancer therapy. BMB Rep. 2019, 52, 35−41.
(10) Lee, S.; Schmitt, C. A. The dynamic nature of senescence in
cancer. Nat. Cell Biol. 2019, 21, 94−101.
(11) Yosef, R.; Pilpel, N.; Tokarsky-Amiel, R.; Biran, A.; Ovadya, Y.;
Cohen, S.; Vadai, E.; Dassa, L.; Shahar, E.; Ben-Porath, I. Directed
elimination of senescent cells by inhibition of BCL-W and BCL-XL.
Nat. Commun. 2016, 7, 11190.
Rita Song − Medicinal Chemistry Group, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea; orcid.org/0000-
0002-9992-7863; Phone: +82-2-577-1373;
Email: rsong1228@gmail.com; Fax: +82-70-8677-1373
Authors
(12) Zhu, Y.; Tchkonia, T.; Fuhrmann-Stroissnigg, H.; Dai, H. M.
M.; Ling, Y. Y. Y.; Stout, M. B.; Pirtskhalava, T.; Giorgadze, N.;
Johnson, K. O.; Giles, C. B.; Wren, J. D.; Niedernhofer, L. J.; Robbins,
P. D.; Kirkland, J. L. Identification of a novel senolytic agent,
navitoclax, targeting the Bcl-2 family of anti-apoptotic factors. Aging
Cell 2016, 15, 428−435.
(13) Tarunina, M.; Alger, L.; Chu, G.; Munger, K.; Gudkov, A.; Jat,
P. S. Functional genetic screen for genes involved in senescence: role
of Tid1, a homologue of the Drosophila tumor suppressor l (2) tid, in
senescence and cell survival. Mol. Cell. Biol. 2004, 24, 10792−10801.
(14) Bitler, B. G.; Fink, L. S.; Wei, Z.; Peterson, J. R.; Zhang, R. A
high-content screening assay for small-molecule modulators of
oncogene-induced senescence. J. Biomol. Screening 2013, 18, 1054−
1061.
Sangmi Oh − Medicinal Chemistry Group, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea; orcid.org/0000-
0003-0895-1110
Do Yoon Kwon − Functional Morphometry-I, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea
Inhee Choi − Medicinal Chemistry Group, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea
Young Mi Kim − Medicinal Chemistry Group, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea
Ji Young Lee − Functional Morphometry-I, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea
Jiyoung Ryu − Medicinal Chemistry Group, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea
(15) Lahtela, J.; Corson, L. B.; Hemmes, A.; Brauer, M. J.; Koopal,
S.; Lee, J.; Hunsaker, T. L.; Jackson, P. K.; Verschuren, E. W. A high-
content cellular senescence screen identifies candidate tumor
suppressors, including EPHA3. Cell Cycle 2013, 12, 625−634.
(16) Ewald, J. A.; Peters, N.; Desotelle, J. A.; Hoffmann, F. M.;
Jarrard, D. F. A high-throughput method to identify novel senescence-
inducing compounds. J. Biomol. Screening 2009, 14, 853−858.
(17) Wang, B.; Kohli, J.; Demaria, M. Senescent Cells in Cancer
Therapy: Friends or Foes? Trends Cancer 2020, DOI: 10.1016/
j.trecan.2020.05.004.
(18) Sherr, C. J.; Beach, D.; Shapiro, G. I. Targeting CDK4 and
CDK6: From Discovery to Therapy. Cancer Discovery 2016, 6 (4),
353−367.
(19) Xue, W.; Zender, L.; Miething, C.; Dickins, R. A.; Hernando,
E.; Krizhanovsky, V.; Cordon-Cardo, C.; Lowe, S. W. Senescence and
tumour clearance is triggered by p53 restoration in murine liver
carcinomas. Nature 2007, 445, 656−660.
Hangyeol Jeong − Functional Morphometry-I, Institut Pasteur
Korea, Gyeonggi-do 13488, South Korea
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00570
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Dr. Shawn Yu in NIH/NIAID for his critical
reading of this manuscript. This work was supported by the
National Research foundation of Korea (NRF) grant funded
by the Korea government (MSIP, Nos. 2007-00559 and 2010-
002495, and MSIT, NRF-2017M3A9G6068257), Gyeonggi-do
(No. K204EA000001-09E0100-00110), and KISTI.
(20) Rakhra, K.; Bachireddy, P.; Zabuawala, T.; Zeiser, R.; Xu, L.;
Kopelman, A.; Fan, A. C.; Yang, Q.; Braunstein, L.; Crosby, E.; et al.
CD4+ T cells contribute to the remodeling of the microenvironment
required for sustained tumor regression upon oncogene inactivation.
Cancer Cell 2010, 18, 485−498.
REFERENCES
■
(1) Kuilman, T.; Michaloglou, C.; Mooi, W. J.; Peeper, D. S. The
essence of senescence. Genes Dev. 2010, 24, 2463−2479.
(2) Rodier, F.; Campisi, J. Four faces of cellular senescence. J. Cell
Biol. 2011, 192, 547−556.
(21) Kang, T.-W.; Yevsa, T.; Woller, N.; Hoenicke, L.; Wuestefeld,
T.; Dauch, D.; Hohmeyer, A.; Gereke, M.; Rudalska, R.; Potapova, A.;
570
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571

ACS Medicinal Chemistry Letters
pubs.acs.org/acsmedchemlett
Letter
et al. Senescence surveillance of pre-malignant hepatocytes limits liver
cancer development. Nature 2011, 479, 547−551.
(22) Oh, S.; Lee, J. Y.; Choi, I.; Ogier, A.; Kwon, D. Y.; Jeong, H.;
Son, S. J.; Kim, Y.; Kwon, H.; Park, S.; Kang, H.; Kong, K.; Ahn, S.;
Nehrbass, U.; Kim, M. J.; Song, R. Discovery of 4H-Chromeno[2,3-
d]pyrimidin-4-one Derivatives as Senescence Inducers and Their
Senescence-Associated Antiproliferative Activities on Cancer Cells
using Advanced Phenotypic Assay. Eur. J. Med. Chem. 2021, 209,
112550.
(23) GENENTECH, Inc. Diazacarbazoles and methods of use.
Patent Appl. WO/2009/151598, 2009.
(24) Germain, A. R.; Carmody, L. C.; Dockendorff, C.; Galan-
Rodriguez, C.; Rodriguez, A.; Johnston, S.; Bittker, J. A.; MacPherson,
L.; Dandapani, S.; Palmer, M.; et al. Identification of small-molecule
inhibitors of Trypansoma cruzi replication. Bioorg. Med. Chem. Lett.
2011, 21, 7197−7200.
(25) Sharma, D.; Narasimhan, B.; Kumar, P.; Judge, V.; Narang, R.;
De Clercq, E.; Balzarini, J. Synthesis, antimicrobial and antiviral
evaluation of substituted imidazole derivatives. Eur. J. Med. Chem.
2009, 44, 2347−2353.
(26) Yang, M.-Y.; Lin, P.-M.; Liu, Y.-C.; Hsiao, H.-H.; Yang, W.-C.;
Hsu, J.-F.; Hsu, C.-M.; Lin, S.-F. Induction of cellular senescence by
doxorubicin is associated with upregulated miR-375 and induction of
autophagy in K562 cells. PLoS One 2012, 7, e37205.
(27) Bielak-Zmijewska, A.; Wnuk, M.; Przybylska, D.; Grabowska,
W.; Lewinska, A.; Alster, O.; Korwek, Z.; Cmoch, A.; Myszka, A.;
Pikula, S.; Mosieniak, G.; Sikora, E. A comparison of replicative
senescence and doxorubicin-induced premature senescence of
vascular smooth muscle cells isolated from human aorta. Biogerontol-
ogy 2014, 15, 47−64.
(28) Sliwinska, M. A.; Mosieniak, G.; Wolanin, K.; Babik, A.;
Piwocka, K.; Magalska, A.; Szczepanowska, J.; Fronk, J.; Sikora, E.
Induction of senescence with doxorubicin leads to increased genomic
instability of HCT116 cells. Mech. Ageing Dev. 2009, 130, 24−32.
̈
(29) LigPrep, version 3.0; Schrodinger, LLC: New York, 2014.
̈
(30) ConfGen, version 2.8; Schrodinger, LLC: New York, 2014.
̈
(31) Phase, version 3.9; Schrodinger, LLC: New York, 2014.
(32) Shah, U. A.; Deokar, H. S.; Kadam, S. S.; Kulkarni, V. M.
Pharmacophore generation and atom-based 3D-QSAR of novel 2-(4-
methylsulfonylphenyl) pyrimidines as COX-2 inhibitors. Mol.
Diversity 2010, 14, 559−568.
(33) Herranz, N.; Gil, J. Mechanisms and functions of cellular
senescence. J. Clin. Invest. 2018, 128, 1238−1246.
571
https://doi.org/10.1021/acsmedchemlett.0c00570
ACS Med. Chem. Lett. 2021, 12, 563−571