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

Article abstract of DOI:10.1016/j.ejmech.2020.112550

Current research suggests therapy-induced senescence (TIS) of cancer cells characterized by distinct morphological and biochemical phenotypic changes represent a novel functional target that may enhance the effectiveness of cancer therapy. In order to identify novel small-molecule inducers of cellular senescence and determine the potential to be used for the treatment of melanoma, a new method of high-throughput screening (HTS) and high-contents screening (HCS) based on the detection of morphological changes was designed. This image-based and whole cell-based technology was applied to screen and select a novel class of antiproliferative agents on cancer cells, 4H-chromeno[2,3-d]pyrimidin-4-one derivatives, which induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. To evaluate structure-activity relationship (SAR) study of 4H-chromeno[2,3-d]pyrimidin-4-one scaffold starting from hit 3, a focused library containing diversely modified analogues was constructed and which led to the identification of 38, a novel compound to have remarkable anti-melanoma activity in vitro with good metabolic stability.

Full text of DOI:10.1016/j.ejmech.2020.112550

Journal Pre-proof
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
Sangmi Oh, Ji Young Lee, Inhee Choi, Arnaud Ogier, Do Yoon Kwon, Hangyeol
Jeong, Sook Jin Son, Haejin Kwon, Seijin Park, Hwankyu Kang, Kwanghan Kong,
Sujin Ahn, Ulf Nehrbass, Myung Jin Kim, Rita Song
PII:
S0223-5234(20)30522-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112550
EJMECH 112550
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 March 2020
Revised Date: 3 June 2020
Accepted Date: 4 June 2020
Please cite this article as: S. Oh, J.Y. Lee, I. Choi, A. Ogier, D.Y. Kwon, H. Jeong, S.J. Son, H.
Kwon, S. Park, H. Kang, K. Kong, S. Ahn, U. Nehrbass, M.J. Kim, R. Song, 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, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112550.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
©
2020 Elsevier Masson SAS. All rights reserved.

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
a, b
c
a
d
c
Sangmi Oh , Ji Young Lee , Inhee Choi , Arnaud Ogier , Do Yoon Kwon , Hangyeol
c
c
a
e
e
e
Jeong , Sook Jin Son , Youngmi Kim , Haejin Kwon , Seijin Park , Hwankyu Kang ,
e
e
c
c, f, *
a, *
Kwanghan Kong , Sujin Ahn , Ulf Nehrbass , Myung Jin Kim
and Rita Song
a
Medicinal Chemistry Group, Institut Pasteur Korea, 16 Daewangpangyo-ro 712 beon-gil, Bundang-gu,
Seongnam-si, Gyeonggi-do13488, South Korea
b
Present address: Tuberculosis Research Section, Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,
United States
c
Functional Morphometry-I, Institut Pasteur Korea, 16 Daewangpangyo-ro 712 beon-gil, Bundang-gu,
Seongnam-si, Gyeonggi-do13488, South Korea
d
Cellular Differentiation and Toxicity Prediction, Institut Pasteur Korea, 16 Daewangpangyo-ro 712 beon-gil,
Bundang-gu, Seongnam-si, Gyeonggi-do13488, South Korea
e
Drug Metabolism and Pharmacokinetics, Institut Pasteur Korea, 16 Daewangpangyo-ro 712 beon-gil,
Bundang-gu, Seongnam-si, Gyeonggi-do13488, South Korea
f
Present address: New Drug and Bio Research Center, Handok Inc., A-dong, 2nd floor, KoreaBioPark, 700
Daewangpangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 13488, South Korea
∗
Co-corresponding author.
Tel.: +82-31-698-3832; fax: +82-31-698-3842; e-mail: rsong1228@gmail.com
Tel.: +82-2-527-5036; fax: +82-2-527-5020; e-mail: nanmolra1973@naver.com

Abstract
Current research suggests therapy-induced senescence (TIS) of cancer cells characterized by distinct
morphological and biochemical phenotypic changes represent a novel functional target that may
enhance the effectiveness of cancer therapy. In order to identify novel small-molecule inducers of
cellular senescence and determine the potential to be used for the treatment of melanoma, a new
method of high-throughput screening (HTS) and high-contents screening (HCS) based on the
detection of morphological changes was designed. This image-based and whole cell-based technology
was applied to screen and select a novel class of antiproliferative agents on cancer cells, 4H-
chromeno[2,3-d]pyrimidin-4-one derivatives, which induced senescence-like phenotypic changes in
human melanoma A375 cells without serious cytotoxicity against normal cells. To evaluate structure-
activity relationship (SAR) study of 4H-chromeno[2,3-d]pyrimidin-4-one scaffold starting from hit 3,
a focused library containing diversely modified analogues was constructed and which led to the
identification of 38, a novel compound to have remarkable anti-melanoma activity in vitro with good
metabolic stability.
Keywords
Structure-activity relationship
High-throughput screening
High-contents screening
Senescence
Antiproliferative agents
Melanoma

1
. Introduction
Melanoma is the most aggressive form of skin cancer that arises from melanocytes. It is estimated
that more than 192,310 new patients per year worldwide and consequently about 7,230 melanoma
related deaths occur in 2019 [1, 2]. In the case of early stage of melanomas (stage I and II), the tumor
can be surgically resected with high success rate. Whereas five-year survival rates for regional and
distant stage melanomas are 64 % and 23 %, respectively [1-4].
Two major drugs for the treatment of patients with metastatic melanoma, including monoclonal
antibody to cytotoxic T lymphocyte antigen 4 (CTLA4), ipilimumab, and a selective small molecule
inhibitor of V600E mutated BRAF kinase, vemurafenib, have been attributed to an improved
understanding of its immune microenvironment and molecular genetics of the disease, respectively [5-
7
]. However, these two treatments induced serious problems like autoimmune response of ipilimumab
[
8, 9] and acquired drug resistance to vemurafenib [10, 11]. Even though two new drugs having
slightly different mechanism of action, dabrafenib and trametinib, have been recently approved by the
FDA, they are only indicated for use in patients with whose tumors express the BRAF V600E gene
mutations [12]. Therefore, there is an urgent need to ikdentify novel therapeutic targets and develop
novel drug candidates to treat metastatic melanoma.
Over the past several years, acutely stress-responsive forms of cellular senescence have been
discovered that seem to play important roles during tumor development. Senescence can effectively
function as an in vivo tumor suppressor mechanism, limiting proliferation of damaged cells.
Mechanistically, senescence is being induced by a variety of pathways involving tumor suppressors
such as p53 and retinoblastoma protein (Rb) as well as increased expression of cyclin dependent
kinase (CDK) inhibitors including p21 (Cip1/Waf1), p16 (INK4a) and p27 (Kip1) [13]. Current
studies have discovered that therapy-induced senescence (TIS), a type of senescence caused by
ionizing radiation or genotoxic drugs, could play a critical role in anti-cancer therapy and will provide
insights into the development of powerful strategy for therapeutic benefits [14-17]. They have been
known to trigger senescence pathways so that senescent states have been observed in breast,
melanoma and lung cancers after drug treatment [18-20]. Interestingly, senescence induction followed

by chemotherapy has been correlated with a favorable patient outcome [21]. Furthermore, it was
reported that cellular senescence induced by p53 has been associated with tumor clearance and
contributed to the outcome of cancer therapy [22]. All these studies underscore the role of senescence
in limiting oncogene-initiated tumorigenesis and enhancing the antitumor effects of some cancer
drugs [23-27]. Previously, established anti-cancer therapies have been associated with the generation
of senescent states in vivo, as shown in patients with breast cancer [28] or non-small cell lung cancer
[
29] after neoadjuvant chemotherapy [17, 30]. Apparently therefore, the strategy of stopping
deregulated, malignant cell growth through activation of a senescence program appears feasible even
in the advanced stages of melanoma.
In the search for new modulating tools of cellular senescence, senescence-associated β-
galactosidase (SA-β-gal) activity of the cells has been generally used in the small-molecule screening
as a senescence marker [31, 32]. However, it has a limit for adaptation to the high-throughput assay
format due to the use of bright-field microscopy. In this study, we report newly designed image-based
assay system for high-throughput screening (HTS) and high-contents screening (HCS), and its
application to develop novel small-molecule candidates which induce senescence-like morphological
changes. In addition, we report structure-activity relationship (SAR) study of 4H-chromeno[2,3-
d]pyrimidin-4-one derivatives and then in vitro biological evaluation of senescence-associated anti-
melanoma activities, which chemical scaffold was selected and demonstrated by the novel phenotypic
assay for the discovery of senescence inducers. This image-based assay was designed for as a whole
cell-based approach that allows for the selection of a new set of compounds based on the unique
phenotypes of senescent cells. Focused library was constructed for the improvement of activity and
metabolic stability starting from initial hit 3, which was identified through developed phenotypic
screening of commercial library, and then the modified molecule, 38 was developed for in vivo study
as a consequence.

2
. Result and discussion
2
.1 Development of phenotypic assay to identify senescence-inducing compounds
To identify novel small molecules to induce cellular senescence and assess their antitumor activities
against melanoma cells, novel phenomic assay was developed to measure cellular morphological
changes with counting the number of cells in a well. For evaluating senescence-like morphological
changes, three different fluorescent dye were used for the cellular staining of nucleus DNA, plasma
membrane and lysosomes respectively after fixation of compound-treated cells and then phenotypic
changes due to cellular senescence such as enlarged cytoplasm containing a large nuclei and increased
cytoplasmic granules were detected. As shown in Fig. 1, this newly developed image-based assay was
validated by reference compound, doxorubicin, which is known as a topoisomerase II inhibitor and
has been reported to induce the DNA damage-mediated senescence in human cells [33-35]. After the
drug treatment for 3 days, stained images of human melanoma A375 cells were acquired and analyzed
by customized software which needs segmentation procedure of each stained image before analysis
(
Fig. 1A). This process has been developed for computational analysis of acquired cellular images to
assess diverse information such as number of cells per well, cell size, nucleus size, granule size, the
distance of each granules, cytoplasmic texture, and volume of each organelles. As a result, dose-
response curves to calculate EC and IC were generated based on the analytical data of the number
5
0
50
of cells as well as the ratio of senescent cells per well (Fig. 1B). In this study, ‘EC’ means the effective
concentration to induce cellular senescence phenotype, which represents dose-response curve to go
uphill. ‘IC’ means the cell growth inhibitory concentration to reduce cell number, so it is used to
express dose-response curve to go downhill. To verify senescence phenotype induced by doxorubicin,
A375 cells treated with doxorubicin were also detected with an enhanced activity of SA-β-gal which
is a commonly accepted and widely used senescence biomarker (Fig. 1C) [36].

Fig. 1. 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 were analyzed by
customized algorithm. All experiments were duplicated respectively. (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 phenomic assay, the cells were acquired images after staining with
TM
Hoechst 33342, LysoTracker® Red DND-99, and CellMask (Upper panel). Under the same condition, cells
were performed SA-β-gal (Lower panel) assay. Scale bar is 50 μm.
2
.2. Morphological and biochemical changes in human melanoma cells induced by initial hit 3
Compound 3 (Table 1) bearing 4H-chromeno[2,3-d]pyrimidin-4-one scaffold was one of initial hits
selected from the screening of ~110,000 compound library to induce senescence-like morphological
changes. The experimental evaluations of 3 were conducted on human melanoma cell line (A375) for
particular senescence events in the cell population including SA-β-gal activity, dose response
morphological changes, and counting the number of cells with customized image analysis software as
well as effects on colony formation and p53 pathway (Fig. 2). As shown in Fig. 2A, compound 3-
treated cells showed senescent phenotypes clearly such as enlarged and flattened morphology with
decreased cancer cell proliferation. As compared to the negative control (0.5 % DMSO),
morphologically changed A375 cells after the treatment of 3 were also detected with an enhanced
activity of SA-β-gal (Fig. 2A). Using customized software, cell images after compound treatment
taken from confocal microscopy were analyzed to detect cell number and morphological changes in

dose response. Whereas the number of total cells were decreasing in dose-dependent pattern (IC =
5
0
0
.09 µM), the number of morphological changed cells in well were increasing (EC = 0.11 µM, Fig.
50
2
B).
To check whether these events are irreversible, colony formation assay was performed after 3 days
incubation with 3. When melanoma cells initially treated with small molecule 3 were incubated with
fresh media for additional 4 days, they have not been increasing colony number or expanding colony
size in the concentration of 2.2 µM as EC100 (Fig. 2C).
As noted above, cellular senescence induced by chemotherapy has been known to be closely related
to not only the activation of p53 and p21 but also dephosphorylation of Rb. From the result of western
blotting experiment shown in Fig. 2D, we confirmed these elevations in the expression of p53 and p21
in compound-treated cells. In addition, another cell cycle arrest marker, Rb was appeared to be
dephosphorylated even at the concentration of 0.01 µM. This finding indicates that the activation of
p53 signaling pathway after compound treatment underlies the cell cycle arrest and may lead to the
cellular senescence.

Fig. 2. Activity of 3 in morphological and biochemical changes of human melanoma A375 cells. (A) Results of
negative control (0.5 % DMSO) and 3 (EC100, 2.2 µM) after 3 days’ incubation in SA-β-gal assay and
morphometric assay. (B) Dose-response analysis of 3. The dose response curves used to determine the EC50
(
black line) and IC (red line) values. Black line represents a percentage of senescent cell population in a well.
50
Red line means the number of cells in a well. (C) The effect of 3 in colony formation. Cells were incubated with
or without 3 for 72 hrs and then they were cultured in fresh media. Till the formation of colonies, cells were
changed media for every 3 days. (D) Biochemical changes of compound-treated cells in western blot. After 3
days treatment of 3, cells were blotted for p53, p21 and Rb.
2
.3. Cellular senescence independent of DNA-damage mechanism
DNA damage response (DDR) is a common feature for monitoring genome integrity [37]. The post-
translational modification of DDR factors plays prominent roles in controlling the formation of foci in
response to DNA-damaging agents. To define whether the cellular events of 3 are related in DNA-
damage mechanism, two different experiments were conducted for the DNA damage analysis: the
immunostaining of DNA damage markers, γ-H2AX and 53BP1, and the examination of DNA single-
strand break (SSB) formation (Fig. 3A and B).
When cells are exposed to DNA-damaging chemotherapeutic agents like doxorubicin, double-
stranded breaks (DSBs) are generated that rapidly result in the phosphorylation of histone H2A
139
variant H2AX. Because phosphorylation of H2AX at Ser (γ-H2AX) is relevant, and correlates well
with each DSB, it is the most sensitive marker that can be used to examine the DNA damage [38].
5
3BP1 is also known as a typical marker of DNA damage accomplishing its function in DNA repair
[
39]. As shown in Fig. 3A, compound 3 did not induce the recruitment of γ-H2AX and 53BP1 in
senescent cells, while doxorubicin-treated cells were generated and increased the γ-H2AX and 53BP1
foci in the nuclei, which induces the DNA damage-mediated senescence in human cells [33, 40, 41].
The formation of DNA SSB is a different type of DNA damage response, which is caused by
oxidative attack of free radicals generated from cell metabolism or chemotherapeutic agents [37]. In
comparison with control cells, however, compound 3 did not promote single-strand breaks in cells
(
Fig. 3B). In addition, compound effect on cell proliferation was evaluated by measuring the
distribution of the cells in the different phases of the cell cycle by flow cytometry. Compound 3
induced statistically significant cell cycle arrest in G2-M phase and Pre-G1 phase as well (Fig. 3C).
The ratio of apoptotic cells and senescent cells in sub-G1 fraction was dose-dependently investigated

by the detection of caspase-3 and morphological changes respectively. On the basis of this experiment,
we found that small part of cells underwent apoptosis at 72 hrs-treatment (Fig. 3D). We supposed that
apoptotic cells at 72 hrs came from fully senescent cells which underwent apoptosis due to continuous
stimuli by compound 3. These results indicated that 3 induced DNA damage-independent cellular
senescence through G2-M phase arrest.
Fig. 3. The effect of 3 in DNA damage mechanism and cell cycle arrest. (A) Recruitment of 53BP1 and γ-H2AX
1
39
(
Ser ) in A375 cells. A375 cells were incubated with or without 37 nM of doxorubicin (Dox) and 3 for 3 days.
After fixation, untreated cells, Dox- and 3-treated cells were incubated with anti-53BP1 and anti-γH2AX.
Fluorescent images were acquired by automated confocal microscopy (Opera). White arrows indicated foci in
nuclei and scale bar represents 20 μm. (B) Single strand break response of 3. (C) Cell cycle analysis in A375
cells with 20 μM of 3. (D) Dose response effect of 3 in apoptosis and cellular senescence. After 3 days treatment,
apoptosis and senescence of cell population per well were analyzed by caspase-3 and morphometric changes.
All experiments were duplicated respectively.
2
.4. The cell growth inhibition of 3 without any influence in BRAF and NRAS mutations
The major oncogenic mutations of melanoma are known to be frequently associated with BRAF
and/or NRAS kinases [42]. To profile the specificity of 3 against melanoma mutations, compound was
V600
Q61
tested in six different melanoma cell lines which have different mutations in BRAF
and NRAS

(
Fig. 4A). This result showed that compound 3 was more sensitive on some melanoma cell lines,
however, its activity was not limited to BRAF and NRAS mutations. Furthermore, compound 3 was
also tested in various types of cancer cells associated with different oncogenic mutations, and then
examined its specificity of cancer and genetic mutations. As shown in Fig. 3B, similar doses of 3 were
needed to inhibit different types of cancer cells with below 1 µM. Although common genetic factors
within tested cancer cells have not yet been identified, this finding may lead to the better
understanding of mechanism of action when the target protein of 3 might play an important role in a
specific signaling pathway related to anti-proliferation on a broad spectrum of cancer cell lines.
Fig. 4. Cell growth inhibition of 3 in different mutations and cancer types. (A) Growth inhibition of 3 in various
melanoma cells containing different mutations. All cells were incubated with 3 for 72 hrs in dose response and
then were counted cell numbers. (B) Activity profiling of 3 in various cancer types. All experiments were
performed in triplicate.
2
.5. Construction of focused library and their structure-activity relationship
To improve senescence-associated anti-melanoma activity and metabolic stability for in vivo study,
a series of 4H-chromeno[2,3-d]pyrimidin-4-one analogues were prepared. The synthesis of 4H-
chromeno[2,3-d]pyrimidin-4-one derivatives A and their isomers B with double bond migration were
conducted following the synthetic procedure which was previously reported (Scheme 1) [43, 44]. 2-
Iminocoumarin-3-carboxamides 2 were obtained by the reaction of salicylic aldehydes 1 with 2-
cyanoactamide in the presence of catalytic amount of piperidine. The interaction of intermediates 2
with aldehydes resulted in a mixture of isomers A and B in most cases. The ratio of isomers depended

on the structure of the starting aldehyde, the reaction temperature, and the reaction time as previously
reported [43, 44]. All final compounds newly synthesized (4−38) were characterized by low resolution
ESI mass and NMR, the melting points of some compounds were measured as well. Despite several
plausible tautomeric structures of final tricyclic ring, a singlet -CH - in pyran ring of B observed in
2
the range of near 4 ppm was clearly differentiated from -CH- of A type tautomers observed in the
downfield based on the proton NMR spectra. In vitro activities inducing senescence-like phenotype
(
EC ) and anti-proliferative activities (IC ) of synthetic compounds were summarized in Table 1.
50 50
Scheme 1. Synthesis for analogues 3−38. Reagents and conditions: (a) 2-cyanoacetamide, piperidine, EtOH; (b)
RCHO, piperidine, n-pentanol.
Table 1. Activity inducing senescence-like phenotype (EC ) and anti-proliferative activity (IC ) of synthetic
5
0
50
4
H-chromeno[2,3-d]pyrimidin-4-one derivatives
Chemical Structure
Activity (µM)
No.
1
2
3
4
a
b
Scaffold
R
R
R
R
R
EC50
IC50
3
4
5
6
7
8
9
A
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
4-hydroxyphenyl
phenyl
−(CH)
4
4
−
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.11
1.19
> 20
15.0
0.57
0.10
0.09
0.20
0.14
0.27
0.19
0.32
0.59
11.0
> 20
> 20
0.01
0.28
0.09
0.64
6.60
0.37
0.19
0.02
0.10
0.20
0.22
0.33
0.31
0.28
0.47
6.00
18.0
> 20
0.01
0.26
−(CH)
−
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
fluoro
methyl
ethyl
H
H
H
iso
propyl
H
tert
1
0
butyl
H
11
phenyl
H
1
1
1
1
1
1
1
1
2
2
3
4
5
6
7
8
9
0
3-furyl
H
methoxy
phenoxy
H
H
dimethylamino
morpholino
H
H
H
diethylamino
H
tert
tert
butyl
H
H
H
butyl
iso
propyl
H
H
iso
phenyl
propyl

iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
iso
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
benzyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
propyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
> 20
> 20
> 20
> 20
0.04
0.11
3.00
7.00
0.25
0.03
0.09
> 20
0.10
0.94
0.08
0.09
20.0
0.003
0.009
b
> 20
> 20
> 20
> 20
0.08
0.06
6.00
0.52
0.20
0.01
0.01
3.00
0.05
0.28
0.04
0.08
4.00
0.004
0.011
cyclohexyl
cyclopropyl
iso
propyl
2-thiophenyl
3-hydroxyphenyl
2-hydroxyphenyl
4-fluorophenyl
3-fluorophenyl
3-methoxyphenyl
4-toluyl
4-dimethylaminophenyl
3-fluoro-4-hydroxyphenyl
2-fluoro-4-hydroxyphenyl
4-hydroxy-3-methoxyphenyl
5-benzodioxol
2,3-dimethoxyphenyl
2,3-dimethylphenyl
Doxorubicin
a
EC means the effective concentration of a drug that induces senescence in a half population. IC represents
5
0
50
the dose of a compound where the cell number is reduced by 50 %.
To investigate structure-activity relationships, 36 analogues of initial hit 3 were synthesized and
evaluated their activities inducing senescence-like phenotype (EC ) and anti-proliferative activities
5
0
(
IC ). In this experiment, a known chemotherapeutic agent, doxorubicin, was used as a positive
50
control, which induces cellular senescence via inhibition of topoisomerase II and DNA damage under
low concentration [33, 41]. A comparison of scaffolds A and B had been a priority for the construction
of focused library. Compounds 4 (1.19 µM) and 20 (0.28 µM) bearing scaffold B, which are the
counterparts of initial hit 3 (0.11 µM) and 19 (0.01 µM) bearing scaffold A, respectively, showed
significant decrease of activities to induce senescence-like phenotype more than 10-fold. This finding
indicated that conformational change of main structure, 4H-chromeno[2,3-d]pyrimidin-4-one, caused
by double bond migration might influence on the binding event of putative target biomolecules. In
1
4
terms of substituent effects on tricyclic benzene ring (R −R ), the presence of electron donating and
2
hydrophobic alkyl or aryl substituent at R such as ethyl (8), isopropyl (9), and phenyl (11) was
necessary to retain activities so that they showed promising EC values (0.10, 0.09, and 0.14 µM,
5
0
1
2
respectively) similar to that of 3, which fused with benzene to make tetracyclic ring from R to R .

Whereas other derivatives with electronegative fluorine (6) or hydrophilic morpholine (16) at the
same position as well as the analogue without substituent (5) were significantly losing their activities
4
tert
or even inactive. Alkyl substituents at R was not good for the activity because 8 having two butyl
2
4
tert
2
groups at R and R was found to be inactive while 10 having one butyl group at R showed
3
promising activity with 0.26 µM. In addition, hydrophilic substituent at R like diethylamino group
2
(
17) was not effective for the improvement of activity while 15 having dimethylamino group at R
showed moderate activity with 0.59 µM.
To assess the importance of R, a series of compounds 19−38 were synthesized and evaluated their
activities to stimulate senescence-like morphological change. All analogues described here embedded
2
with isopropyl group at R instead of fused benzene because 9 showed improved activity in
comparison with initial hit 3 (Tables 1 and 2). As shown in Table 1, phenyl group in the position of R
was essential for maintaining activity due to the fact that synthetic analogues to have benzyl (21) or
the saturated hydrocarbons such as cyclohexyl (22), cyclopropyl (23), and isopropyl (24) were found
to be inactive. In the search for proper substituents on the phenyl ring in the R, various substituents
with different electronic effects, different hydrogen bonding properties, or different position were
applied in the phenyl ring, and then finally 38 bearing two methyl groups in ortho- and meta-positions
was selected as the most potent compound in the focused library with the activity of 3 nM, which was
an activity inducing senescence-like phenotype comparable to that of reference drug, doxorubicin (9
nM). As shown in Table 1, anti-proliferative activities of all synthetic analogues designated by IC50
were correlated well with the corresponding EC which were determined by image-based senescence-
5
0
inducing activities. In addition to the promising improvement of anti-proliferative activity related to
senescence, dimethyl substituted-phenyl embedded-molecule 38 was metabolically stable,
demonstrating significant improvement in the intrinsic clearance against both human and mouse liver
microsomes (Table 2).
Table 2. Metabolic stability of synthetic 4H-chromeno[2,3-d]pyrimidin-4-one derivatives
a
Compound
Metabolic Stability (T1/2 , min)

b
Phase I
Mouse
Phase II
Human
Human
Mouse
c
3
8
9
43.6
32.5
>60
39.7
>60
21.6
37.6
14.7
32.9
32.0
57.1
>60
32.5
51.6
9.30
7.97
13.0
11.8
30.1
6.56
6.65
8.47
14.7
6.44
13.2
20.2
18.6
54.9
N/T
N/T
N/T
N/T
N/T
17.4
N/T
N/T
N/T
N/T
N/T
N/T
2.88
N/T
>60
N/T
N/T
N/T
17.8
N/T
N/T
N/T
N/T
N/T
N/T
>60
N/T
>60
1
9
0
5
6
9
0
1
3
5
6
8
2
2
2
2
3
3
3
3
3
3
a
b
T1/2 means the time which the concentration of a drug is reduced by half for 1 hr incubation. Glucuronidation
c
only. N/T not tested.
Based on the data of metabolic stability, 38 was selected for mouse pharmacokinetics (PK) study.
As indicated in Table 3, 38 demonstrated modest oral bioavailability with acceptable half-lives in
blood circulation, suggesting strong possibility that a series of 4H-chromeno[2,3-d]pyrimidin-4-one
analogues could be developed as oral drugs. Even though the intraperitoneal (IP) administration gave
2
-fold higher AUC and prolonged t1/2 than oral (PO) administration, PK profile of 38 did not exhibit
in vivo efficacy which could be explained by in vitro EC correction with plasma protein binding.
5
0
Therefore, optimization of the series of compounds is required to improve drug-like properties,
maintaining their in vitro activities. Currently further optimization of compounds is underway to
improve oral PK parameters that are expected to show in vivo efficacy in xenograft model.
Table 3. Pharmacokinetic parameters for 38 in mice
a
b
d
e
C
(
0
/Cmax
c
AUClast
CL
g
h
f
Admin
Dose (mg/kg)
t
1/2 (h)
Vdss (mL/kg)
MRT (h)
F (%)
ng/mL)
(ng×h/mL)
(mL/min/kg)
i
IV
PO
IP
2
446
142
380
1.26
3.57
9.32
265
479
116
N/A
N/A
8714
N/A
N/A
0.870
3.78
4.95
N/A
10
10
36.1
105
1397
a
b
c
C means concentration at time 0 hr for IV. C means maximum concentration for PO and IP. t indicates
0
max
1/2
d
e
f
half-life. AUClast means area under the curve from 0 to 24 hr. CL means systemic clearance. V means
dss

g
h
i
volume of distribution at steady-state. MRT means mean residence time. F means oral bioavailability. N/A
not available.
2
.6. Pharmacophore modeling
To investigate SAR of 4H-chromeno[2,3-d]pyrimidin-4-one derivatives, total of eight common
pharmacophore hypotheses (CPH) featuring six pharmacophore sites were developed. Based on
survival score, AAHPRR.2 was selected as the best CPH having the highest score (8.105) than others.
It consists of two H-bond acceptors (A), one hydrophobic (H), two aromatic rings (R), and one
positive ionizable (P) features. Active ligands were well aligned with all six pharmacophoric features
such as the most active compound, 38 (Fig. 5A). The conformation difference of the main structure,
4
H-chromeno[2,3-d]pyrimidin-4-one, was clearly distinguished between scaffolds A and B. The
conformation of 3 with the scaffold A was puckered up above the plane at the saturated pyrimidine
ring. However, the centroid of P8 feature was not aligned with the nitrogen atom in the pyrimidine
ring in the scaffold B of 4 (10-fold less active) due to a flat conformation caused by double bond
migration (Fig. 5B). The contribution of phenyl group or aromaticity in the position of R in biological
activities were validated with the pharmacophore mapping results. Analogues 21 with benzyl and 24
with isopropyl substituents in the position of R were mapped with only five features of CPH. These
substituents of those inactive compounds failed to map with the aromatic ring feature, R10 (Fig. 5C).
2
In case of R position, the presence of electron donating and hydrophobic alkyl or aryl substituents at
2
R were very important for the good biological activity of the compounds. When there is no
substituent in this position like compound 5, the activity was significantly lost suggested by H7
feature not matching with 5 (Fig. 5D). Having a substituent in different position like a diethylamino
3
group in R of 17 was also not effective for the anti-proliferative activity. The orientation of most of
the features mapped with 17 (Fig. 5D) were moved away from the centroid.

Fig. 5. (A) The best CPH, AAHPRR.2, is superimposed with the best compound, 38 (yellow). Two aromatic
ring features (R9 and R10), two H-bond acceptor features with lone pairs of electron (A2 and A3), one positive
ionizable (P8) feature, and one hydrophobic feature (H7) are shown in orange rings, pink spheres, blue sphere,
and green sphere, respectively. The distances between pharmacophoric features are shown in magenta dotted
lines with values. (B) Conformational difference in scaffolds A and B are shown between compounds 3 (yellow)
and 4 (light blue). (C) The substituents in R position of inactive compounds 21 (yellow) and 24 (light blue) did
2
not map with R10 feature. (D) Inactive compounds, 5 (yellow) with no R substituent and 17 (light blue) with a
3
hydrophilic R substituent, did not map with H7 feature.
3
. Conclusion
Due to the fact that cellular senescence is characterized by a sustained growth arrest and is
commonly observed in premalignant lesions, it has been supporting the notion that senescence acts as
a potent tumor suppressor mechanism restricting cell proliferation [21]. A series of 4H-chromeno[2,3-
d]pyrimidin-4-one derivatives were identified as a novel class of anti-proliferative agents associated
with senescence. For the selection and evaluation of small-molecule hits, advanced image-based assay
was designed for high-throughput screening (HTS) and high-contents screening (HCS) as a whole
cell-based approach that could recognize the unique phenotypes of senescent cells. The hit compound,
3
was initially confirmed as an inducer of senescence-associated morphological changes in high-
throughput phenotypic screening, which was accompanied by anti-proliferative activity against
melanoma cells without DNA damage. The dose dependent senescence effect of 3 in melanoma cells
corresponded to a cell cycle arrest in G2-M phase and did not relate to BRAF and NRAS mutations.
In addition, cancer profiling data suggests that the sensitivity of cancer cells against 3 was
independent on cancer type. Together, our results provide the possibility of senescence as an

alternative anti-cancer therapy even though the mechanism should be investigated more. After
constructing a series of analogues derived from 3, analysis of the structure-activity relationship
iso
revealed that 2-phenyl and 5-alkyl substituents, especially
propyl, on 4H-chromeno[2,3-
d]pyrimidin-4-one tricyclic ring played a critical role to display anti-proliferative activity. In addition,
hydrophobic dimethyl substituents in 2-phenyl ring enhanced metabolic stability so that 38 was finally
selected for in vivo PK study. This advanced molecule was a potential candidate to be suitable for oral
administration with modest oral bioavailability and appropriate intrinsic clearance. Further
optimization of compounds is on-going to improve oral PK parameters and in vivo efficacy study in
xenograft model. According to the developed pharmacophore model, the best CPH generated,
AAHHRR.2, was found to be predictive in differentiating active and inactive 4H-chromeno[2,3-
d]pyrimidin-4-one analogues based on senescence-associated antitumor activity. It predicted the
aromatic ring (R10) as well as the hydrophobic (H7) features as important determinants in biological
activity. In addition, one positive ionizable (P8) and two H-bond acceptor features (A2 and A3) were
critical in sustaining the conformational difference between saturated and unsaturated main scaffold,
4
H-chromeno[2,3-d]pyrimidin-4-one.
Even though the molecular mechanism of senescence in particular of melanoma cells remains to be
examined in detail, it is no doubt that cellular senescence is an attractive therapeutic strategy to
prevent melanoma cell proliferation. In this regard, 4H-chromeno[2,3-d]pyrimidin-4-one derivatives
will be a good starting point to develop novel small-molecule probes exploring the potential targets
for pro-senescence drugs. A follow-up studies including optimization of in vivo efficacy and
elucidation of mechanism of action are currently underway.

4
. Experimental section
4
.1. General chemistry information
1
13
H and C NMR spectra were recorded on a Varian High Resolution FT-NMR Spectrometer-400,
and chemical shifts were measured in ppm relative to internal tetramethylsilane (TMS) standard or
specific solvent signal. Routine mass analyses were performed on Waters LC/MS ZQ2000 system
TM
equipped with a reverse phase column (XBridge C18 × 3.5 μm, 50 × 2.1 mm) and photodiode array
detector using electron spray ionization (ESI). The gradient mobile phase consisting of acetonitrile /
water with 0.1% formic acid and UV detection at 254 and 210 nm were used to confirm all final
products to be ≥95%. Melting point analyses were performed on BUCHI Melting point M-565. Most
reagents used in the synthetic procedure were purchased from Sigma-Aldrich, Alfa Aesar, and TCI.
The progress of reaction was monitored using thin-layer chromatography (TLC) (silica gel 60 F254
0
.25 mm), and the products were visualized by UV light (254 and 365 nm) or by ninhydrin staining
followed by heating. Silica gel 60 (0.040–0.063 mm) used in flash column chromatography was
purchased from Merck. Other solvents and organic reagents were purchased from commercial venders
and used without further purification unless otherwise mentioned.
4
.1.1. Synthetic procedure for 4H-chromeno[2,3-d]pyrimidin-4-one derivatives A and their isomers B
38)
All compounds 3−38 were obtained using the previously reported procedure with small
(3−
modification. Briefly, to a stirred solution of salicylic aldehydes 1 (1.0 eq.) and 2-cyanoacetamide (1.1
eq.) in ethanol, piperidine was added (0.05 eq.). The reaction mixture was stirred for about 5 hrs at
room temperature. After the reaction was completed, the solid was filtered and washed with ethanol
and dried in vacuo to give desired 2-iminocoumarin-3-carboxamide derivatives (2) with 60−80 %
yields. Compounds 2 was added to a stirring mixture of corresponding aldehyde (RCHO, 1.5 eq.) and
piperidine (2.0 eq.) in n-pentanol. The reaction mixture in the sealed bottle was heated (120−140 ˚C)
for about 10 min. After the reaction was completed, the reaction mixture cooled down to room
temperature. The solid was filtered and washed with ethanol and dried in vacuo to give desired

product. In case solid was not generated, the reaction mixture was evaporated and then purified by
flash column chromatography (methanol / CH Cl ) to give desired product.
2
2
1
9
-(4-Hydroxyphenyl)-7aH-benzo[5,6]chromeno[2,3-d]pyrimidin-11(10H)-one (3). Yellow solid; H
NMR (400 MHz, DMSO-d ) δ 9.45 (s, 1H, OH), 8.94 (s, 1H, NH), 8.63 (s, 1H, C-CH-C), 8.46 (d, J =
6
8
1
.4 Hz, 1H, Ar-H), 8.13 (d, J = 8.8 Hz, 1H, Ar-H), 8.00 (d, J = 8.0 Hz, 1H, Ar-H), 7.70 (t, J = 7.6 Hz,
H, Ar-H), 7.57 (t, J = 7.4 Hz, 1H, Ar-H), 7.42 (d, J = 8.8 Hz, 1H, Ar-H), 7.18 (d, J = 8.4 Hz, 2H, Ar-
+
H in R), 6.76 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.20 (s, 1H, N-CH-O); LC/MS (ESI) m/z 343 [M+H] .
9
-(4-Hydroxyphenyl)-10H-benzo[5,6]chromeno[2,3-d]pyrimidin-11(12H)-one (4). Yellow solid;
1
mp = 267.3 ˚C; H NMR (400 MHz, DMSO-d ) δ 8.06 (d, J = 8.8 Hz, 2H, Ar-H in R), 7.98–7.90 (m,
6
3
H, Ar-H), 7.66 (t, J = 7.6 Hz, 1H, Ar-H), 7.54 (t, J = 7.6 Hz, 1H, Ar-H), 7.38 (d, J = 8.8 Hz, 1H, Ar-
+
H), 6.89 (d, J = 8.8 Hz, 2H, Ar-H in R), 4.02 (s, 2H, -CCH C-); LC/MS (ESI) m/z 343 [M+H] .
2
1
2
-(4-Hydroxyphenyl)-3H-chromeno[2,3-d]pyrimidin-4(10aH)-one (5). Pale yellow solid; H NMR
(
400 MHz, DMSO-d ) δ 9.45 (s, 1H, OH), 8.91 (s, 1H, NH), 8.00 (s, 1H, Ar-H), 7.67 (dd, J = 7.6 and
6
1
.6 Hz, 1H, Ar-H), 7.53–7.49 (m, 1H, Ar-H), 7.23–7.18 (m, 2H, Ar-H), 7.14 (d, J = 8.4 Hz, 2H, Ar-H
+
in R), 6.74 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.13 (s, 1H, N-CH-O); LC/MS (ESI) m/z 293 [M+H] .
1
7
-Fluoro-2-(4-hydroxyphenyl)-3H-chromeno[2,3-d]pyrimidin-4(10aH)-one (6). Yellow solid; H
NMR (400 MHz, DMSO-d ) δ 9.47 (bs, 1H, OH), 8.99 (s, 1H, NH), 7.99 (s, 1H, Ar-H), 7.58 (dd, J =
6
8
.6 and 3.0 Hz, 1H, Ar-H), 7.37 (td, J = 8.6 and 3.2 Hz, 1H, Ar-H), 7.25–7.22 (m, 1H, Ar-H), 7.14 (d,
J = 8.4 Hz, 2H, Ar-H in R), 6.75 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.13 (s, 1H, N-CH-O); LC/MS (ESI)
+
m/z 311 [M+H] .
2
-(4-Hydroxyphenyl)-7-methyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (7). Yellow
1
solid; mp = 270.2 ˚C; H NMR (400 MHz, DMSO-d ) δ 9.45 (s, 1H, OH), 8.89 (s, 1H, NH), 7.94 (s,
6
1
H, Ar-H), 7.46 (d, J = 1.6 Hz, 1H, Ar-H), 7.32 (dd, J = 8.4 and 1.6 Hz, 1H, Ar-H), 7.13 (d, J = 8.8
Hz, 2H, Ar-H in R), 7.09 (d, J = 8.4 Hz, 1H, Ar-H), 6.74 (d, J = 8.8 Hz, 2H, Ar-H in R), 6.11 (s, 1H,
+
N-CH-O), 2.30 (s, 3H, CH3); LC/MS (ESI) m/z 307 [M+H] .
7
-Ethyl-2-(4-hydroxyphenyl)-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (8). Pale yellow
1
solid; H NMR (400 MHz, DMSO-d ) δ 9.45 (s, 1H, OH), 8.90 (s, 1H, NH), 7.95 (s, 1H, Ar-H), 7.51
6

(
d, J = 2.0 Hz, 1H, Ar-H), 7.36 (dd, J = 8.4 and 2.0 Hz, 1H, Ar-H), 7.14–7.10 (m, 3H, Ar-H), 6.74 (d,
J = 8.8 Hz, 2H, Ar-H in R), 6.11 (s, 1H, N-CH-O), 2.60 (q, J = 7.6 Hz, 2H, -CH CH ), 1.19 (t, J = 7.6
2
3
+
Hz, 3H, -CH CH ); LC/MS (ESI) m/z 321 [M+H] .
2
3
2
-(4-Hydroxyphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (9). Pale
1
yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.45 (s, 1H, OH), 8.89 (s, 1H, NH), 7.97 (s, 1H, Ar-
6
H), 7.55 (d, J = 1.6 Hz, 1H, Ar-H), 7.40 (d, J = 8.6 and 2.2 Hz, 1H, Ar-H), 7.12 (t, J = 8.0 Hz, 3H, Ar-
H), 6.74 (d, J = 8.6 Hz, 2H, Ar-H in R), 6.11 (s, 1H, N-CH-O), 2.93–2.85 (m, 1H, -CH(CH ) ), 1.21
3
2
+
(
d, J = 6.8 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 335 [M+H] .
3 2
7
-(tert-Butyl)-2-(4-hydroxyphenyl)-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (10). Pale
1
yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.44 (s, 1H, OH), 8.88 (s, 1H, NH), 8.01 (s, 1H, Ar-
6
H), 7.71 (d, J = 2.4 Hz, 1H, Ar-H), 7.40 (d, J = 8.8 and 2.4 Hz, 1H, Ar-H), 7.14–7.11 (m, 3H, Ar-H),
t
6
.74 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.11 (s, 1H, N-CH-O), 1.29 (s, 9H, Bu); LC/MS (ESI) m/z 349
+
[
M+H] .
1
2
-(4-Hydroxyphenyl)-7-phenyl-3H-chromeno[2,3-d]pyrimidin-4(10aH)-one (11). Yellow solid; H
NMR (400 MHz, DMSO-d ) δ 9.45 (s, 1H, OH), 8.95 (s, 1H, NH), 8.06 (s, 1H, Ar-H), 8.02 (d, J = 2.0
6
Hz, 1H, Ar-H), 7.82 (dd, J = 8.4 and 2.0 Hz, 1H, Ar-H), 7.69 (d, J = 8.4 Hz, 2H, Ar-H), 7.51–7.46 (m,
3
H, Ar-H), 7.41–7.37 (m, 1H, Ar-H), 7.29 (d, J = 8.4 Hz, 1H, Ar-H), 7.16 (d, J = 8.4 Hz, 2H, Ar-H in
+
R), 6.75 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.15 (s, 1H, N-CH-O); LC/MS (ESI) m/z 369 [M+H] .
7
-(Furan-3-yl)-2-(4-hydroxyphenyl)-3H-chromeno[2,3-d]pyrimidin-4(10aH)-one (12). Yellow
1
solid; mp = 241.8 ˚C; H NMR (400 MHz, DMSO-d ) δ 9.45 (s, 1H, OH), 8.95 (s, 1H, NH), 8.18 (s,
6
1
H, Ar-H), 7.96 (s, 1H, Ar-H), 7.93 (d, J = 2.0 Hz, 1H, Ar-H), 7.77–7.75 (m, 2H, Ar-H), 7.22 (d, J =
.4 Hz, 1H, Ar-H), 7.15 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.96 (s, 1H, Ar-H), 6.75 (d, J = 8.4 Hz, 2H,
8
1
3
Ar-H in R), 6.14 (s, 1H, N-CH-O); C NMR (100 MHz, DMSO-d ) δ 159.1, 157.6, 154.3, 152.8,
6
1
45.0, 139.8, 133.1, 133.0, 130.5, 128.4, 127.9, 126.7, 125.0, 119.8, 116.5, 115.6, 115.4, 109.1, 71.2;
+
LC/MS (ESI) m/z 359 [M+H] .
2
-(4-Hydroxyphenyl)-7-methoxy-3H-chromeno[2,3-d]pyrimidin-4(10aH)-one (13). Yellow solid;
1
mp = 267.7 ˚C; H NMR (400 MHz, DMSO-d ) δ 9.44 (s, 1H, OH), 8.91 (s, 1H, NH), 7.97 (s, 1H, Ar-
6

H), 7.28 (d, J = 2.8 Hz, 1H, Ar-H), 7.15–7.08 (m, 4H, Ar-H), 6.74 (d, J = 8.8 Hz, 2H, Ar-H in R), 6.11
1
3
(
s, 1H, N-CH-O), 3.77 (s, 3H, -OCH ); C NMR (100 MHz, DMSO-d ) δ 159.2, 157.6, 155.7, 154.5,
3 6
1
48.1, 133.31, 133.27, 127.9, 119.9, 119.8, 117.0, 115.6, 115.3, 113.3, 71.1; LC/MS (ESI) m/z 323
+
[
M+H] .
2
-(4-Hydroxyphenyl)-7-phenoxy-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (14). Pale
1
yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.46 (s, 1H, OH), 8.95 (s, 1H, NH), 8.00 (s, 1H, Ar-
6
H), 7.44 (d, J = 2.4 Hz, 1H, Ar-H), 7.39 (t, J = 8.0 Hz, 2H, Ar-H), 7.24–7.12 (m, 5H, Ar-H), 7.01 (d, J
1
3
=
8.8 Hz, 2H, Ar-H in R), 6.75 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.13 (s, 1H, N-CH-O); C NMR (100
MHz, DMSO-d ) δ 159.0, 157.6, 157.5, 154.3, 152.3, 150.1, 133.1, 132.9, 130.6, 127.9, 124.3, 123.8,
6
+
1
20.7, 119.9, 118.3, 117.6, 115.60, 115.58, 71.2; LC/MS (ESI) m/z 385 [M+H] .
7
-(Dimethylamino)-2-(4-hydroxyphenyl)-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (15).
1
Yellow solid; mp = 262.4 ˚C; H NMR (400 MHz, DMSO-d ) δ 9.44 (s, 1H, OH), 8.86 (s, 1H, NH),
6
7
.95 (s, 1H, Ar-H), 7.12 (d, J = 8.4 Hz, 2H, Ar-H in R), 7.05 (d, J = 9.2 Hz, 1H, Ar-H), 7.00 (d, J =
.8 Hz, 1H, Ar-H), 6.93 (dd, J = 8.8 and 2.8 Hz, 1H, Ar-H), 6.74 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.09
2
1
3
(
s, 1H, N-CH-O), 2.89 (s, 6H, -N(CH ) ); C NMR (100 MHz, DMSO-d ) δ 159.4, 157.5, 154.7,
3 2 6
1
47.7, 145.7, 134.1, 133.5, 127.8, 119.6, 118.0, 116.3, 115.5, 115.0, 112.3, 71.1, 41.0; LC/MS (ESI)
+
m/z 336 [M+H] .
2
-(4-Hydroxyphenyl)-7-morpholino-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one
(16).
1
Yellow solid; mp = 274.2 ˚C; H NMR (400 MHz, DMSO-d ) δ 9.44 (s, 1H, OH), 8.88 (s, 1H, NH),
6
7
.93 (s, 1H, Ar-H), 7.23 (d, J = 2.8 Hz, 1H, Ar-H), 7.17–7.08 (m, 4H, Ar-H ), 6.74 (d, J = 8.4 Hz, 2H,
Ar-H in R), 6.10 (s, 1H, N-CH-O), 3.76–3.74 (m, 4H, -NCH CH O-), 3.10–3.08 (m, 4H, -
2
2
+
NCH CH O-); LC/MS (ESI) m/z 378 [M+H] .
2
2
7
-(Diethylamino)-2-(4-hydroxyphenyl)-3H-chromeno[2,3-d]pyrimidin-4(10aH)-one (17). Yellow
1
solid; mp = 212.4 ˚C; H NMR (400 MHz, DMSO-d ) δ 9.41 (s, 1H, OH), 8.49 (s, 1H, NH), 7.81 (s,
6
1
2
6
H, Ar-H), 7.38 (d, J = 8.8 Hz, 1H, Ar-H), 7.11 (d, J = 8.4 Hz, 2H, Ar-H in R), 6.73 (d, J = 8.4 Hz,
H, Ar-H in R), 6.54 (d, J = 8.8 Hz, 1H, Ar-H), 6.38 (s, 1H, Ar-H), 6.04 (s, 1H, N-CH-O), 3.42 (q, J =
+
.8 Hz, 4H, -N(CH CH ) ), 1.13 (t, J = 6.8 Hz, 6H, -N(CH CH ) ); LC/MS (ESI) m/z 364 [M+H] .
2
3 2
2
3 2

7
,9-Di-tert-butyl-2-(4-hydroxyphenyl)-3H-chromeno[2,3-d]pyrimidin-4(10aH)-one (18). Yellow
1
solid; mp = 248.9 ˚C; H NMR (400 MHz, DMSO-d ) δ 9.46 (s, 1H, OH), 8.56 (s, 1H, NH), 7.98 (s,
6
1
H, Ar-H), 7.58 (d, J = 2.4 Hz, 1H, Ar-H), 7.44 (d, J = 2.4 Hz, 1H, Ar-H), 7.15 (d, J = 8.6 Hz, 2H,
t
Ar-H in R), 6.75 (d, J = 8.6 Hz, 2H, Ar-H in R), 6.12 (s, 1H, N-CH-O), 1.40 (s, 9H, Bu), 1.29 (s, 9H,
t
13
Bu); C NMR (100 MHz, DMSO-d ) δ 159.3, 157.6, 154.5, 150.3, 146.1, 135.8, 134.5, 133.4, 127.9,
6
+
1
27.7, 125.3, 119.4, 115.6, 114.2, 71.3, 35.0, 34.7, 31.5, 30.0; LC/MS (ESI) m/z 405 [M+H] .
1
7
-Isopropyl-2-phenyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (19). Yellow solid; H
NMR (400 MHz, DMSO-d ) δ 8.99 (s, 1H, NH), 8.00 (s, 1H, Ar-H), 7.57 (d, J = 2.0 Hz, 1H, Ar-H),
6
7
.42–7.32 (m, 6H, Ar-H), 7.13 (d, J = 8.4 Hz, 2H, Ar-H), 6.23 (s, 1H, N-CH-O), 2.94–2.87 (m, 1H, -
+
CH(CH ) ), 1.21 (d, J = 6.8 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 319 [M+H] .
3
2
3 2
1
7
-Isopropyl-2-phenyl-3,5-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (20). Pale yellow solid; H
NMR (400 MHz, DMSO-d ) δ 8.13 (d, J = 7.6 Hz, 2H, Ar-H), 7.60–7.52 (m, 3H, Ar-H), 7.17–7.12
6
(
m, 2H, Ar-H), 7.06 (d, J = 8.4 Hz, 2H, Ar-H), 3.73 (s, 2H, -CCH C-), 2.90–2.84 (m, 1H, -CH(CH ) ),
2 3 2
+
1
.21 (d, J = 7.2 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 319 [M+H] .
3 2
2
-Benzyl-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (21). Pale yellow solid;
1
H NMR (400 MHz, CD OD) δ 7.58 (s, 1H, Ar-H), 7.38 (d, J = 8.4 Hz, 1H, Ar-H), 7.27 (s, 1H, Ar-H)
3
7
.14–7.11 (m, 5H, Ar-H and NH), 7.07 (d, J = 8.4 Hz, 1H, Ar-H), 5.56 (t, J = 4.0 Hz, 1H, N-CH-O),
3
.07–3.02 (m, 2H), 2.91–2.87 (m, 1H, -CH(CH ) ), 1.15 (d, J = 6.8 Hz, 6H, -CH(CH ) ); LC/MS (ESI)
3
2
3 2
+
m/z 333 [M+H] .
2
-Cyclohexyl-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (22). Pale yellow
1
solid; mp = 253.2 ˚C; H NMR (400 MHz, DMSO-d ) δ 8.51 (s, 1H, NH), 7.85 (s, 1H, Ar-H), 7.50 (d,
6
J = 2.4 Hz, 1H, Ar-H), 7.38 (dd, J = 8.4 and 2.0 Hz, 1H, Ar-H), 7.12 (d, J = 8.4 Hz, 1H, Ar-H), 5.03
(
s, 1H, N-CH-O), 2.92–2.85 (m, 1H, -CH(CH ) ), 2.56–2.45 (m, 1H, cyclohexyl in R) 1.72–1.55 (m,
3 2
13
6
H, cyclohexyl in R), 1.26–0.86 (m, 4H, cyclohexyl in R), 1.20 (d, J = 6.8 Hz, 6H, -CH(CH ) ); C
3 2
NMR (100 MHz, DMSO-d ) δ 160.1, 154.8, 152.2, 144.5, 132.8, 131.4, 127.4, 119.3, 115.8, 115.5,
6
+
7
2.9, 45.6, 33.1, 27.7, 26.5, 26.2, 26.0, 25.9, 24.2; LC/MS (ESI) m/z 325 [M+H] .
2
-Cyclopropyl-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (23). Pale yellow

1
solid; mp = 224.8 ˚C; H NMR (400 MHz, DMSO-d ) δ 8.70 (s, 1H, NH), 7.88 (s, 1H, Ar-H), 7.52 (d,
6
J = 2.0 Hz, 1H, Ar-H), 7.39 (dd, J = 8.4 and 2.0 Hz, 1H, Ar-H), 7.11 (d, J = 8.4 Hz, 1H, Ar-H), 4.82
(
d, J = 6.4 Hz, 1H, N-CH-O), 2.93–2.86 (m, 1H, -CH(CH ) ), 1.20 (d, J = 6.8 Hz, 6H, -CH(CH ) ),
3 2 3 2
1
3
1
.16–1.10 (m, 1H, cyclopropyl in R), 0.45–0.33 (m, 4H, cyclopropyl in R); C NMR (100 MHz,
DMSO-d ) δ 159.6, 154.8, 152.2, 144.6, 133.1, 131.5, 127.5, 119.2, 115.8, 115.2, 71.2, 33.1, 26.7,
6
+
2
4.2, 19.6, 1.9, 1.6; LC/MS (ESI) m/z 283 [M+H] .
1
2
,7-Diisopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (24). Pale yellow solid; H
NMR (400 MHz, CD OD) δ 7.95 (s, 1H, Ar-H), 7.44–7.42 (m, 2H, Ar-H), 7.12 (d, J = 9.2 Hz, 1H,
3
Ar-H), 5.16 (dd, J = 3.4 and 1.0 Hz, 1H, N-CH-O), 2.98–2.91 (m, 1H, -CH(CH ) ), 2.04–1.96 (m, 1H,
3
2
-CH(CH ) in R), 1.27 (d, J = 6.8 Hz, 6H, -CH(CH ) ), 1.00 (d, J = 6.8 Hz, 3H, -CH(CH ) in R), 0.93
3 2 3 2 3 2
+
(
d, J = 6.8 Hz, 3H, -CH(CH ) in R); LC/MS (ESI) m/z 285 [M+H] .
3
2
7
-Isopropyl-2-(thiophen-2-yl)-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one
(25).
Pale
1
yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.21 (s, 1H, NH), 8.03 (s, 1H, Ar-H), 7.58 (d, J = 2.0
6
Hz, 1H, Ar-H), 7.48–7.43 (m, 2H, Ar-H), 7.20–7.15 (m, 2H, Ar-H), 7.02–7.00 (m, 1H, Ar-H), 6.52 (s,
1
H, N-CH-O), 2.95–2.88 (m, 1H, -CH(CH ) ), 1.21 (d, J = 6.8 Hz, 6H, -CH(CH ) ); LC/MS (ESI)
3 2 3 2
+
m/z 325 [M+H] .
2
-(3-Hydroxyphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (26). Yellow
1
solid; H NMR (400 MHz, DMSO-d ) δ 9.43 (s, 1H, OH), 8.96 (s, 1H, NH), 8.00 (s, 1H, Ar-H), 7.57
6
(
d, J = 2.0 Hz, 1H, Ar-H), 7.41 (dd, J = 8.4 and 2.4 Hz, 1H, Ar-H), 7.18–7.12 (m, 2H, Ar-H), 6.78–
.68 (m, 3H, Ar-H), 6.11 (s, 1H, N-CH-O), 2.94–2.87 (m, 1H, -CH(CH ) ), 1.21 (d, J = 7.2 Hz, 6H, -
6
3
2
+
CH(CH ) ); LC/MS (ESI) m/z 335 [M+H] .
3
2
2
-(2-Hydroxyphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (27). Pale
1
yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.66 (s, 1H, OH), 8.64 (s, 1H, NH), 7.94 (s, 1H, Ar-
6
H), 7.55 (d, J = 2.4 Hz, 1H, Ar-H), 7.38 (dd, J = 8.4 and 2.0 Hz, 1H, Ar-H), 7.15–7.09 (m, 3H, Ar-H),
6
.81–6.77 (m, 2H, Ar-H), 6.32 (s, 1H, N-CH-O), 2.93–2.87 (m, 1H, -CH(CH ) ), 1.21 (d, J = 7.2 Hz,
3 2
+
6
H, -CH(CH ) ); LC/MS (ESI) m/z 335 [M+H] .
3
2
2
-(4-Fluorophenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (28). Yellow

1
solid; H NMR (400 MHz, DMSO-d ) δ 8.99 (s, 1H, NH), 8.00 (s, 1H, Ar-H), 7.57 (d, J = 2.0 Hz, 1H,
6
Ar-H), 7.43–7.39 (m, 3H, Ar-H), 7.20 (t, J = 8.8 Hz, 2H, Ar-H), 7.13 (d, J = 8.4 Hz, 1H, Ar-H), 6.25
(
s, 1H, N-CH-O), 2.94–2.87 (m, 1H, -CH(CH ) ), 1.21 (d, J = 6.8 Hz, 6H, -CH(CH ) ); LC/MS (ESI)
3 2 3 2
+
m/z 337 [M+H] .
2
-(3-Fluorophenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (29). Pale
1
yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.02 (s, 1H, NH), 8.01 (s, 1H, Ar-H), 7.57 (s, 1H, Ar-
6
H), 7.43–7.41 (m, 2H, Ar-H), 7.23–7.13 (m, 4H, Ar-H), 6.26 (s, 1H, N-CH-O), 2.92–2.87 (m, 1H, -
+
CH(CH ) ), 1.21 (d, J = 6.8 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 337 [M+H] .
3
2
3 2
7
-Isopropyl-2-(3-methoxyphenyl)-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (30). Yellow
1
solid; H NMR (400 MHz, CD OD) δ 8.08 (s, 1H, NH), 7.47 (s, 1H, Ar-H), 7.45 (d, J = 8.8 Hz, 1H,
3
Ar-H), 7.32 (t, J = 7.8 Hz, 1H, Ar-H), 7.15 (d, J = 8.0 Hz, 1H, Ar-H), 6.99–6.95 (m, 2H, Ar-H), 6.91
(
dd, J = 8.0 and 2.4 Hz, 1H, Ar-H), 6.24 (s, 1H, N-CH-O), 3.80 (s, 3H, -OCH ), 3.00–2.93 (m, 1H, -
3
+
CH(CH ) ), 1.28 (d, J = 6.4 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 349 [M+H] .
3
2
3 2
1
7
-Isopropyl-2-(p-tolyl)-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (31). Yellow solid; H
NMR (400 MHz, DMSO-d ) δ 8.95 (s, 1H, NH), 7.98 (s, 1H, Ar-H), 7.56 (d, J = 2.4 Hz, 1H), 7.40 (dd,
6
J = 8.4 and 2.0 Hz, 1H, Ar-H), 7.23 (d, J = 8.4 Hz, 2H, Ar-H in R), 7.18 (d, J = 8.4 Hz, 2H, Ar-H in
R), 7.12 (d, J = 8.4 Hz, 1H, Ar-H), 6.18 (s, 1H, N-CH-O), 2.94–2.87 (m, 1H, -CH(CH ) ), 2.29 (s, 3H,
3
2
+
Ph-CH ), 1.21 (d, J = 7.2 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 333 [M+H] .
3
3 2
2
-(4-Dimethylaminophenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (32).
1
Yellow solid; H NMR (400 MHz, DMSO-d ) δ 8.87 (s, 1H, NH), 7.96 (s, 1H, Ar-H), 7.55 (d, J = 2.4
6
Hz, 1H), 7.40 (dd, J = 8.8 and 2.4 Hz, 1H, Ar-H), 7.14–7.11 (m, 3H, Ar-H), 6.70 (J = 8.8 Hz, 2H, Ar-
H in R), 6.10 (s, 1H, N-CH-O), 2.92–2.87 (m, 1H, -CH(CH ) ), 2.87 (s, 6H, -N(CH ) ), 1.21 (d, J =
3
2
3 2
+
7
.2 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 362 [M+H] .
3 2
2
-(3-Fluoro-4-hydroxyphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one
1
(
33). Yellow solid; H NMR (400 MHz, DMSO-d ) δ 8.92 (s, 1H, NH), 7.98 (s, 1H, Ar-H), 7.56 (d, J
6
=
2.4 Hz, 1H, Ar-H), 7.41 (dd, J = 8.8 and 2.4 Hz, 1H, Ar-H), 7.14–7.10 (m, 2H, Ar-H), 6.99–6.91 (m,
2
H, Ar-H), 6.14 (s, 1H, N-CH-O), 2.94–2.87 (m, 1H, -CH(CH ) ), 1.21 (d, J = 7.2 Hz, 6H, -
3
2

13
CH(CH ) ); C NMR (100 MHz, DMSO-d ) δ 159.3, 152.2, 145.1, 144.9, 144.7, 134.2, 133.8, 131.6,
3
2
6
+
1
27.6, 122.8, 119.3, 118.0, 115.9, 114.9, 114.7, 114.5, 70.5, 33.1, 24.2; LC/MS (ESI) m/z 353 [M+H] .
2
-(2-Fluoro-4-hydroxyphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one
1
(
34). Yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.96 (s, 1H, OH), 8.86 (s, 1H, NH), 7.99 (s, 1H,
6
Ar-H), 7.56 (d, J = 2.4 Hz, 1H, Ar-H), 7.41 (dd, J = 8.8 and 2.4 Hz, 1H, Ar-H), 7.17–7.11 (m, 2H, Ar-
H), 6.61–6.52 (m, 2H, Ar-H), 6.28 (s, 1H, N-CH-O), 2.94–2.87 (m, 1H, -CH(CH ) ), 1.21 (d, J = 7.2
3
2
+
Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 353 [M+H] .
3
2
2
-(4-Hydroxy-3-methoxyphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one
1
(
35). Pale yellow solid; H NMR (400 MHz, DMSO-d ) δ 9.01 (s, 1H, OH), 8.89 (s, 1H, NH), 7.97 (s,
6
1
1
2
1
H, Ar-H), 7.55 (s, 1H, Ar-H), 7.40 (d, J = 8.8 Hz, 1H, Ar-H), 7.12 (d, J = 8.4 Hz, 1H, Ar-H), 6.91 (s,
H, Ar-H), 6.77–6.72 (m, 2H, Ar-H), 6.13 (s, 1H, N-CH-O), 3.75 (d, J = 1.6 Hz, 3H, Ph-OCH )2.93–
3
1
3
.87 (m, 1H, -CH(CH ) ), 1.21(d, J = 7.2 Hz, 6H, -CH(CH ) ); C NMR (100 MHz, DMSO-d ) δ
3
2
3 2
6
59.3, 154.6, 152.2, 147.9, 146.8, 144.6, 133.8, 133.5, 131.6, 127.6, 119.3, 119.0, 115.9, 115.7, 115.0,
+
1
11.1, 71.2, 56.1, 33.1, 24.2; LC/MS (ESI) m/z 365 [M+H] .
2
-(Benzo[d][1,3]dioxol-5-yl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (36).
1
Yellow solid; H NMR (400 MHz, DMSO-d ) δ 8.91 (s, 1H, NH), 7.98 (s, 1H, Ar-H), 7.56 (s, 1H, Ar-
6
H), 7.41 (d, J = 8.4 Hz, 1H, Ar-H), 7.13 (dd, J = 8.4 and 1.6 Hz, 1H, Ar-H), 6.90–6.82 (m, 3H, Ar-H),
6
.15 (s, 1H, N-CH-O), 6.01 (d, J = 1.6 Hz, 2H, -OCH O-), 2.94–2.87 (m, 1H, -CH(CH ) ), 1.21 (d, J
2 3 2
+
=
6.8 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 363 [M+H] .
3 2
2
-(2,3-Dimethoxyphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one
(37).
1
Pale yellow solid; H NMR (400 MHz, DMSO-d ) δ 8.80 (s, 1H, NH), 7.98 (s, 1H, Ar-H), 7.56 (d, J =
6
2
7
2
.0 Hz, 1H, Ar-H), 7.39 (dd, J = 8.4 and 2.4 Hz, 1H, Ar-H), 7.11–7.01 (m, 3H, Ar-H), 6.82 (dd, J =
.6 and 2.0 Hz, 1H, Ar-H), 6.37 (s, 1H, N-CH-O), 3.81 (s, 3H, Ph-OCH ), 3.72 (s, 3H, Ph-OCH ),
3
3
+
.94–2.87 (m, 1H, -CH(CH ) ), 1.21 (d, J = 7.2 Hz, 6H, -CH(CH ) ); LC/MS (ESI) m/z 379 [M+H] .
3
2
3 2
2
-(2,3-Dimethylphenyl)-7-isopropyl-3,10a-dihydro-4H-chromeno[2,3-d]pyrimidin-4-one (38). Pale
1
yellow solid; mp = 231.6 ˚C; H NMR (400 MHz, DMSO-d ) δ 8.85 (s, 1H, NH), 7.99 (s, 1H, Ar-H),
6
7
.55 (d, J = 2.4 Hz, 1H, Ar-H), 7.39 (dd, J = 8.8 and 2.4 Hz, 1H, Ar-H), 7.14–7.03 (m, 4H, Ar-H),

6
1
1
2
.49 (s, 1H, N-CH-O), 2.93–2.86 (m, 1H, -CH(CH ) ), 2.29 (s, 3H, Ph-CH ), 2.27 (s, 3H, Ph-CH ),
3 2 3 3
1
3
.21 (d, J = 6.8 Hz, 6H, -CH(CH ) ); C NMR (100 MHz, DMSO-d ) δ 159.4, 154.2, 151.8, 144.2,
3
2
6
40.0, 136.8, 133.6, 133.3, 133.0, 127.2, 127.0, 123.3, 118.8, 114.5, 68.8, 32.7, 32.6, 23.9, 23.6, 20.3,
+
0.1, 14.3, 14.1; LC/MS (ESI) m/z 347 [M+H] .
4
.2. Cell lines and cell cultures
Human melanoma cell lines (A375, SK-MEL28, WM266-4 and SK-MEL2) and other cell lines
(
(
A549, MDA-MB231, DU145 and T47D) were purchased from American Type Culture Collection
ATCC, USA), and the other human melanoma cell lines (WM1366, WM3670) were obtained from
Coriell (Coriell Institute for Medical Research, USA). Human cell lines (A375, SK-MEL28, WM266-
, A549, MDA-MB231, DU145 and T47D) were maintained in DMEM high glucose (Gibco, USA)
supplemented with 10 % fetal bovine serum (FBS, Gibco, USA) and 1 % penicillin, streptomycin and
% sodium pyruvate (Gibco, USA). SK-MEL2 was cultured in ATCC-formulated Eagle’s Minimum
Essential Medium (EMEM, ATCC, USA) containing 10 % FBS. The other melanoma cell lines
WM1366, WM3670) were cultured in 4:1 mixture of MCDB153 (WelGENE Inc., Korea) and
Leibovitz L-15 (Gibco, USA), supplemented with 2 % FBS, 5 µg/mL of insulin (Gibco, USA) and
.68 mM of CaCl (Withlab Co., Ltd, Korea) in 37 ˚C, 5 % CO atmosphere at constant humidity.
4
1
(
1
2
2
WM1366 and WM3670 cells were cultured on bio-coated flask (BD Biosciences, USA). All cell lines
were mycoplasma free when periodically tested with the MycoAlert™ Mycoplasma Detection Kit
(
Lonza, USA).
4
.3. Senescence screening assay
The compounds were added to the assay plate (Evotec™ 384-well microplate, Germany) with 10
µL DMEM (Gibco, USA) supplemented with 1.0 mmol/L sodium pyruvate,
1
%
streptomycin/penicillin and 10 % heat-inactivated FBS using Hummingbird. A375 cells were
cultivated in DMEM (Gibco, USA) medium supplemented with 1.0 mmol/L sodium pyruvate, 1 %
streptomycin/penicillin and 10 % heat-inactivated FBS at 37 ˚C and 5 % CO . After compound
2

4
addition, cells (5 × 10 cells/mL) were diluted in DMEM supplemented with 1.0 mmol/L sodium
pyruvate, 1 % streptomycin/penicillin and 10 % heat-inactivated FBS, seeded in 40 µL per well using
WELLMATE (Thermo Scientific Matrix WellMate, USA) and incubated at 37 ˚C for 72 hrs.
Doxorubicin hydrochloride as the reference compound (positive control) at 37 nM as the EC100 and
0
.5 % DMSO (negative control) were used in all the plates for data normalization. After 3 days of
incubation, the assay plates were stained with Hoechst33342 (Invitrogen, USA), LysoTracker Red
DND-99 (Invitrogen, USA) and CellMask™ Deep Red Plasma membrane Stain (Invitrogen, USA)
for 1 hr. Microplates were read in an Opera High Content Screening System (PerkinElmer Inc.,
USA), enabling the determination of the senescence effect ratio by image analysis. An algorithm for
image analysis was developed to determine senescence effects and count cell number. With this
technique, the senescence was determined by 130 descriptions such as cell size, nucleus size, texture
of cells etc. The average senescent ratios from the positive and negative controls were normalized to
0
% and 100 % senescence effect, and the senescent ratio read from each compound activity was
proportionally distributed within this range. Z-factor was used for protocol validation and active
compound selection acceptance. Z-factor, calculated as 1-[3(σ +σ )/(μ -μ )], where μ , σ , μ and σ
n
p
n
p
n
p
p
n
are the means (μ) and standard deviations (σ) of both the positive (p) and negative (n) controls, and
other parameters, including DRC control for verification of the reference drug EC50 (accepted if
within the range of 3× higher or lower than a defined value from the literature), coefficient of
variation not higher than 10 % in the controls and edge effect evaluation, were used for screening
validation and hits selection.
4
.4. Double strand DNA and single strand DNA assay
For checking double DNA damage of cells, cells were seeded in 384-well plates (2 × 10 cells/well),
3
and exposed to dose response concentration (DRC) of a compound from 0 to 20 µM for 3 days. And
then cells were fixed in 4 % paraformaldehyde (WAKO, Japan) for 20 min at room temperature and
washed twice in PBS (WelGENE Inc., Korea) and permeabilized in 0.5 % Triton X-100 (Sigma, USA)
in PBS for 15 min. Cells were washed twice in PBS and blocked for 1 hr in 3 % BSA (Sigma, USA)

in PBS containing Tween-20 (PBST, Sigma, USA), after which they were incubated in anti-53BP1
Millipore, USA) and anti-γH2AX (Millipore, USA) in 1 % BSA-PBST buffer for overnight at 4 ˚C.
(
After 24 hrs, cells were washed three times in PBST and incubated in Alexa Fluor 488 goat anti-
mouse and Alexa Fluor 532 goat anti-rabbit secondary antibody (Invitrogen, USA) in 1 % BSA-
PBST for 1 hr at room temperature to label 53BP1 and γ-H2AX. Cells were washed three times for 10
min, stained with Hoechst (Invitrogen, USA) for 20 min, and then washed three times for 10 min. The
images were photographed at 20× water magnifications using Opera . To measure single-stranded
DNA, we purchased Quant-iT™ OliGreen ssDNA Assay Kit (Invitrogen, USA) and followed a
procedure in manual as described.
4
.5. Senescence-associated
β
-galactosidase (SA-β-gal) assay
2
A375 cells in 96-well plates (4 × 10 cells/well) were incubated with 0.5 % DMSO and 3 for 72 hrs.
After 3 days, the adherent cells were fixed for 20 min at room temperature in 0.2 % glutaraldehyde
Sigma, USA) and stained for 3 days at 37 ˚C freshly prepared X-Gal staining solutions (pH 6.0)
(
containing 1 mM MgCl , 0.1 mg/mL X-Gal (Invitrogen, USA), 5 mM potassium ferricyanide (Sigma,
2
USA), and 5 mM potassium ferrocyanide (Sigma, USA). The images were photographed at several
magnifications using a microscopy.
4
.6. Colony formation assay
Cells in 96-well plates (20 cells/well) were seeded and incubated with a compound for 3 days. And
then the fresh media was replaced for 4 days. Seven days after seeding, cells were fixed in 4 %
paraformaldehyde (WAKO, Japan) at room temperature for 20 min, washed twice with PBS. Next,
cells were stained with 0.1 % crystal violet (Sigma, USA) in PBS for 20 min at room temperature.
Stained surviving cells were photographed.
4
.7. Flow cytometry analysis of cell cycle