Discovery, design and synthesis of 6H-anthra[1,9-cd]isoxazol-6-one scaffold as G9a inhibitor through a combination of shape-based virtual screening and structure-based molecular modification

Article abstract of DOI:10.1016/j.bmc.2016.09.071

Protein lysine methyltransferase G9a is widely considered as an appealing antineoplastic target. Herein we present an integrated workflow combining shape-based virtual screening and structure-based molecular modification for the identification of novel G9a inhibitors. The shape-based similarity screening through ROCS overlay on the basis of the structure of UNC0638 was performed to identify CPUY074001 contained a 6H-anthra[1,9-cd]isoxazol-6-one scaffold as a hit. Analysis of the binding mode of CPUY074001 with G9a and 3D-QSAR results, two series compounds were designed and synthesized. The derivatives were confirmed to be active by in vitro assay and the SAR was explored by docking stimulations. Besides, several analogues showed acceptable anti-proliferative effects against several cancer cell lines. Among them, CPUY074020 displayed potent dual G9a inhibitory activity and anti-proliferative activity. Furthermore, CPUY074020 induced cell apoptosis in a dose-dependent manner and displayed a significant decrease in dimethylation of H3K9. Simultaneously, CPUY074020 showed reasonable in vivo PK properties. Altogether, our workflow supplied a high efficient strategy in the identification of novel G9a inhibitors. Compounds reported here can serve as promising leads for further study.

Full text of DOI:10.1016/j.bmc.2016.09.071

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery, design and synthesis of 6H-anthra[1,9-cd]isoxazol-6-one
scaffold as G9a inhibitor through a combination of shape-based
virtual screening and structure-based molecular modification
Wei-Lin Chen a, Zhi-Hui Wang a, Tao-Tao Feng a, Dong-Dong Li a, Chu-Hui Wang a, Xiao-Li Xu a,b
,
Xiao-Jin Zhang a,b,c, Qi-Dong You a,b, , Xiao-Ke Guo a,b,
⇑
⇑
a State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
c Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Protein lysine methyltransferase G9a is widely considered as an appealing antineoplastic target. Herein
we present an integrated workflow combining shape-based virtual screening and structure-based molec-
ular modification for the identification of novel G9a inhibitors. The shape-based similarity screening
through ROCS overlay on the basis of the structure of UNC0638 was performed to identify
CPUY074001 contained a 6H-anthra[1,9-cd]isoxazol-6-one scaffold as a hit. Analysis of the binding mode
of CPUY074001 with G9a and 3D-QSAR results, two series compounds were designed and synthesized.
The derivatives were confirmed to be active by in vitro assay and the SAR was explored by docking stim-
ulations. Besides, several analogues showed acceptable anti-proliferative effects against several cancer
cell lines. Among them, CPUY074020 displayed potent dual G9a inhibitory activity and anti-proliferative
activity. Furthermore, CPUY074020 induced cell apoptosis in a dose-dependent manner and displayed a
significant decrease in dimethylation of H3K9. Simultaneously, CPUY074020 showed reasonable in vivo
PK properties. Altogether, our workflow supplied a high efficient strategy in the identification of novel
G9a inhibitors. Compounds reported here can serve as promising leads for further study.
Ó 2016 Published by Elsevier Ltd.
Received 31 August 2016
Revised 29 September 2016
Accepted 29 September 2016
Available online xxxx
Keywords:
Histone methyltransferases
G9a inhibitor
Virtual screening
Structure–activity relationship
Anti-cancer
1. Introduction
knockdown of G9a inhibited cancer cell growth.11,14 It was later
found that G9a could methylate various histone15 and non-histone
Reversible methylation of histone lysine residues, which is reg-
ulated by histone methyltransferases (HMTs) and histone
demethylases (HDMs), plays an important role in epigenetic gene
expression.1 G9a (also known as KMT1C (lysine methyltransferase
1C) or EHMT2 (euchromatic histone methyltransferase 2)) was ini-
tially identified as a lysine methyltransferase (KMT) which was
responsible for catalyzing the mono-, di- and slowly trimethylation
of histone H3 lysine 9 (H3K9).2–7 Increasing evidence suggested
that G9a played crucial roles in many biological progresses includ-
ing gene expression, transcriptional regulation, cell differentiation,
proliferation, senescence and replication.2–8 The fact that G9a
mediates complex biological functions and is overexpressed in
human cancers including leukemia,9 prostate carcinoma,10,11 hep-
atocellular carcinoma12 and lung cancer.13 It has been shown that
proteins including the tumor suppressor p53, which was impli-
cated in over 50% of cancers.10 These observations suggest that
inhibition of G9a could be a validated therapeutic strategy against
cancers.
Till now, there are only three chemical types small molecular
inhibitors had been reported (Fig. 1), and these inhibitors of G9a
were mainly focused on quinazoline scaffold (UNC0638).16 Besides,
the discovery approaches can be just summarized as high through-
put screening (HTS) and optimization of known inhibitors such as
BIX-01294.17,18 Up to now, no inhibitor is advanced to clinical tri-
als, which may result of the poor in vivo pharmacokinetic proper-
ties of reported compounds. Therefore, it is urgent to discovery
novel chemical scaffolds for the study of therapeutic and biological
roles of G9a in various diseases.
Ligand-based virtual screening of large compound databases
has been proved to be an effective method in discovery novel
hits for a certain target.19–21 For instance, the OMEGA/ROCS
(OpenEye Scientific Software, Inc.) method represented an efficient
⇑
Corresponding authors. Tel./fax: +86 025 83271351.
E-mail addresses: youqidong@gmail.com (Q.-D. You), kexin95@126.com
(X.-K. Guo).
http://dx.doi.org/10.1016/j.bmc.2016.09.071
0968-0896/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Chen, W.-L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.071

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W.-L. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
2. Results and discussion
2.1. ROCS model generation based on UNC0638 and virtual
screening
ROCS represents heavy atoms by Gaussians with parametrized
decay constants according to the respective van der Waals radii.
This representation of atoms allows a fast shape comparison of
molecules due to the straightforward calculation of molecular
overlaps providing sufficient speed for virtual screening of large
database.25 Some studies also suggested that selecting of an appro-
priate query for shape-based screening was very important as it
affected the virtual screening performance.26,27 In most cases, the
query for the shape similarity search was generated from highly
potent small molecule inhibitors of the protein. These findings
encouraged us to employ this complementary methodology as a
screening model for identification of novel scaffolds that possessed
similar structures with UNC0638. UNC0638 was selected as a ref-
erence molecule in a shape similarity analysis to identify com-
pounds for three reasons: (1) UNC0638 was a potent inhibitor for
G9a and showed high activity in cells. (2) It also exhibited selectiv-
ity against G9a and GLP over a wide range of epigenetic and non-
epigenetic targets. (3) A high resolution co-crystal structure of
the G9a–UNC0638 complex has been obtained. The bioactive con-
formation of UNC0638 can promote to develop shape based
screening. The structure of UNC0638 was separated from the co-
crystal complex (PDB code: 3RJW) and directly used to generate
the ROCS query. The key scaffold of UNC0638 was defined by its
quinazoline core. The tertiary amine group was described as
cations. The polar atom such as the nitrogen of pyrimidine, 6-oxy-
gen atom and 7-oxygen atom were defined as hydrogen bond
acceptors. 4-Secondory amine group was defined as hydrogen
donor, respectively. The molecular shape of UNC0638 was depicted
in a cyan shadow (Fig. 3). Further exploration of database using the
model would lead to compounds with similar chemical property
and molecular shape to UNC0638.
Figure 1. Representative structures of known inhibitors of G9a.
strategy.22,23 To the best of our knowledge, no study has been pub-
lished about identification of G9a inhibitory compounds using vir-
tual screening (VS) method. Besides, several crystals of G9a have
been solved making a VS approach possible. With the aim of
acquiring novel scaffolds of G9a inhibitors, this present work, we
firstly developed
a virtual screening model on the basis of
UNC0638 by using the ROCS.22,23 The model was then applied to
screen the ChemDiv database, resulting in compound CPUY074001
with moderate inhibitory activity against G9a. Guided by the bind-
ing mode of CPUY074001 to G9a and 3D-QSAR results24, we
designed, synthesized two series of derivatives and evaluated
G9a inhibitory activity using Alphascreen assay. The representative
compounds were evaluated antitumor activities in multiple cancer
cell lines. Subsequently, compound CPUY074020, with promising
G9a inhibitory activity and anti-proliferative activity, was verified
to induce cell death through apoptosis. The cellular activity was
confirmed by analyzing the H3K9me2 and H3K9me3 methylation
in western blotting. What’s more, the pharmacokinetic parameters
of CPUY074020 in mice were reasonable. Thus, compound
CPUY074020 containing a 6H-anthra[1,9-cd]isoxazol-6-one scaf-
fold may represent a novel structure displaying inhibition activity
against G9a. The discovery workflow of the new scaffold was
shown in Figure 2.
2.2. Identification of the hit compound CPUY074001
To identify novel G9a inhibitors scaffold targeting the catalytic
site, a virtual screening procedure was employed and followed by
biological testing of the identified compounds. For the ChemDiv
database, the multiple conformations of different query molecules
were calculated using OMEGA with a maximum of 400 conforma-
tions.28,29 Initially, each molecule in these conformational database
was aligned with the query and scored for 3D shape overlap using
ROCS. Then, the 3D shape-based similarity of a given compound to
UNC0638 was ranked by a combined scoring method. The selection
was based on two criteria: (i) compounds were available at the
time of our study and (ii) showed sufficient structural diversity
among each other. Much to our delight, CPUY074001, with a 6H-
anthra[1,9-cd]isoxazol-6-one scaffold, showed 39.34% inhibition
against G9a under 10 lM and with IC50 value of 39.19 0.73 lM.
Figure 3. ROCS shape query derived from UNC0638. The green spheres illustrate
ROCS ring features, the red spheres illustrate hydrogen bond acceptors, the gray
sphere illustrates hydrogen bond donors, and the blue spheres illustrate cations.
Figure 2. Overview of the molecular modeling workflow.
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W.-L. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
The binding pattern of CPUY074001 with G9a was analyzed by
molecular docking using CDOKER module inbuilt in DS 3.0. The
bulk of CPUY074001 occupied the histone peptide binding site.
Two hydrogen bonds were observed: one was between the 6-car-
bonyl and Asp1088, a residue in the region of active site; another
was from the nitrogen atom and oxygen atom of isoxazole ring
to Asp 1078. The tetracyclic scaffold was surrounded by the
hydrophobic residues such as Ile1072, Ala1077, Leu108, Val1096
and Cys1098. The tetracyclic scaffold interacted with these resi-
dues by forming van der Waals force (Fig. 4A). The binding pattern
was also depicted by the ‘surface around ligand’ mode. It can be
easily observed that CPUY074001 can occupy the active cavity well
in a reasonable conformation and the tetracyclic scaffold occupied
the hydrophobic pocket. In addition to this, the 5-p-methoxyphe-
nyl side chain of it interacted with the lysine binding channel of
G9a (Fig. 4B). The binding mode analysis clearly explained the
activity of CPUY074001 and indicated it can serve as a proper hit
for further optimization.
2.3. Optimization of the hit compound CPUY074001
In order to investigate the effect of G9a inhibitory activities of
different substitutions on the phenyl ring and R2 groups, we
designed and synthesized series I compounds (CPUY074002–
CPUY074016). The synthesis route of this series of compounds
were outlined in Scheme 1. The 3,5-dibromo-6H-anthra[1,9-cd]
isoxazol-6-one (I-1) was obtained by the diazotization reaction.
The intermediate I-1 were substituted at the 5-position with vari-
ous aromatic amine to give arylamination of I-2a–I-2i. Then a sec-
ond amination was carried out at the 3-position using refluxing
acetonitrile to provide a moderate yield of target compounds
CPUY074002–16.30
The synthesized compounds CPUY074002–16 were initially
evaluated for inhibition of G9a activity at 10 lM using AlphaScreen
assay, while UNC0638 was used as a positive control for the
experiment. As shown in Table 1, most of the compounds pos-
sessed similar bioactivity to the hit CPUY074001. Replacement of
4-methoxylphenyl (CPUY074001, CPUY074002 and CPUY074003)
Figure 4. The binding pattern analysis of CPUY074001 to G9a. The key residues (A)
and the protein surface (B) around CPUY074001 were shown, respectively.
Compound CPUY074001 was shown as cylinders with carbon atoms in green. The
H-bonds were shown as red dot lines. The surface was colored by the electrostatic
state of the residues.
with
4-methylphenyl
(CPUY074006,
CPUY074007
and
CPUY074008) displayed equal inhibition activity against G9a. The
existence of electron withdrawing group (–CN, –F) at the 4-posi-
tion in phenyl moiety demonstrated superior inhibition against
G9a (CPUY074002 vs CPUY074009), (CPUY074003 vs
CPUY074012). While –OCF3 group at 4-position in phenyl moiety
diminished the activity (CPUY074005 vs CPUY074016). On the
whole, the modification of series I was less than satisfactory. None
of the compounds was superior to hit compound CPUY07001 a lot.
All of them displayed the moderate G9a inhibition. This result indi-
cated that introducing a bulk group at the 5-position was not tol-
erate. Thus, we proceeded to further optimize.
O
N
O
O
N
O
N
O
O
NH2
Br
R2
Br
c
Br
Br
b
a
O
HN
HN
R1
R1
O
Br
I-1
I-2a~I-2i
II-2a~II-2q
CPUY074002-16
CPUY074017-33
Scheme 1. Synthesis route of CPUY074002-16. Reagents and conditions (a) NaNO2/
H2SO4, NaN3, toluene, reflux, 16 h; (b) R1NH2, AlCl3, DCM, 40 °C, 12 h; (c) R2,
acetonitrile, 80 °C, 2 h.
Table 1
G9a inhibition profile of series I compounds
1
O
2
N
10
7
3
R2
9
8
4
6
5
HN
O
R1
Compd
R1
R2
G9a % inhibition @ 10 l
Ma
CPUY074001
CPUY074002
CPUY074003
CPUY074004
CPUY074005
CPUY074006
CPUY074007
CPUY074008
CPUY074009
CPUY074010
CPUY074011
CPUY074012
CPUY074013
CPUY074014
CPUY074015
CPUY074016
UNC0638
4-Methoxylphenyl
4-Methoxylphenyl
4-Methoxylphenyl
4-Methoxylphenyl
4-Methoxylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Cyanlphenyl
4-Chlorophenyl
2-Fluorophenyl
2-Fluorophenyl
3-Fluorophenyl
4-Fluorophenyl
2,4-Difluorophenyl
4-Trifluoromethoxyphenyl
—
4-Methylpiperazinyl
Dimethylamino
4-Ethylpiperazinyl
Pyrrolidinyl
4-Phenylpiperazinyl
Dimethylamino
4-Methylpiperazinyl
4-Ethylpiperazinyl
Dimethylamino
39.34
19.11
40.18
40.91
26.07
15.13
31.67
40.21
34.26
26.86
22.36
48.72
21.95
37.67
9.77
4-Methylpiperazinyl
Dimethylamino
4-Ethylpiperazinyl
4-Methylpiperazinyl
4-Methylpiperazinyl
4-Methylpiperazinyl
4-Phenylpiperazinyl
—
16.12
98.02
a
Values are means of at least two experiments.
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4
W.-L. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Figure 5. (A) The binding pattern of compound 33 with CoMSIA electrostatic field: contour maps of CoMSIA electrostatic field superimposed into the active binding site of
G9a and compound 33 was in the binding site of G9a. (B) The superimposition of compound 33 with CPUY074001. Compound 33 was shown as cylinders in green and
CPUY074001 in blue. (C) The superimposition of compound 33 with CPUY07017. Compound 33 was shown as cylinders in green and CPUY074017 in blue.
No significant inhibitory activity variation was observed when
changing the substitution in the phenyl ring, demonstrating a
structural tolerance at this position. Inspired by the docking result
of CPUY074001 (Fig. 4) and 3D-QSAR result (Fig. 5A),24 we specu-
lated this site was favorable for positively charged groups. Hence,
we kept the tetracyclic scaffold and firstly turned our attention
to the modification of the 5-position at the scaffold.
The series II compounds (CPUY074017–CPUY074033) were
prepared using the similar synthetic procedure of series I com-
pounds, with replacement of the aniline by corresponding aliphatic
amine II-2a–II-2q (Scheme 1). The inhibitory activity against G9a
at 10 lM were also evaluated using Alphascreen assay. The IC50
values were further determined from concentration–response
curve established by cumulatively applying ten escalating concen-
trations (0.1 nM–10 lM) of the test compounds. The IC50 was
determined from two independent experiments and reported as
mean SD. Interestingly, compounds of series II showed much
improved activities compared to compounds of series I (Table 2).
Replacing 4-methoxylphenyl at the 5-position with pyrro-
lidinylethyl
(IC50 = 39.19 0.73
group
significantly
M, CPUY074001 vs IC50 = 6.89 1.45
improved
the
activity
M,
l
l
CPUY074017). Compared with the hit compound, CPUY074017
exhibited a better superimposition to the quinazoline inhibitor
33 (Fig. 5B and C).24 This result validated our previous hypothesis
and enabled structure-based optimization of this novel scaffold
G9a inhibitors. In order to deeply understand the binding mode
of our compounds with G9a, the docking simulations were
performed to analyze the most active compounds CPUY074019,
CPUY074020 and CPUY074030 bound to G9a. As illustrated in
Figure 6, both of CPUY074019 and CPUY074020 showed the same
Figure 6. The binding pattern analysis of CPUY074019 (A and B), CPUY074020 (C
and D), CPUY074030 (E and F) to G9a. The key residues (A, C and E) and the protein
surface (B, D and F) were shown respectively. Compounds were shown as cylinders
with carbon atoms in green. The H-bonds were shown as red dot lines. The surface
was colored by the electrostatic state of the residues.
Table 2
G9a inhibition profile of series II compounds
Compd
R1
R2
G9a % inhibition @ 10
l
Ma
G9a (IC50, lM)
CPUY074017
CPUY074018
CPUY074019
CPUY074020
CPUY074021
CPUY074022
CPUY074023
CPUY074024
CPUY074025
CPUY074026
CPUY074027
CPUY074028
CPUY074029
CPUY074030
CPUY074031
CPUY074032
CPUY074033
UNC0638
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
Piperidinoethyl
4-Methylpiperazinyl
Dimethylamino
Pyrrolidinyl
62.93
69.21
92.02
91.52
70.58
70.01
87.14
63.92
64.31
81.51
60.98
63.69
60.99
79.01
64.28
72.21
71.54
98.02
6.89 1.45
3.87 1.92
1.22 0.13
2.18 0.013
3.48 0.36
4.57 0.64
3.22 0.14
3.96 0.54
3.47 0.31
3.75 0.24
5.97 0.32
4.84 0.38
3.84 0.035
1.87 0.19
2.49 0.13
2.68 0.089
2.98 0.22
0.61 0.17
Piperidino
4-Ethylpiperazinyl
4-(2-Pyrimidyl)piperazinyl
4-Phenylpiperazinyl
4-Methylhomopiperazinyl
Dimethylamino
Piperidino
Pyrrolidinyl
4-Methylpiperazinyl
4-(2-Pyrimidyl)piperazinyl
4-Methylpiperazinyl
4-Methylhomopiperazinyl
4-Phenylpiperazinyl
Dimethylamino
Piperidinoethyl
Piperidinoethyl
1-Pyrrolidinylpropyl
—
—
a
Values are means of at least two experiments.
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W.-L. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
binding pattern to G9a with CPUY074001, except for the difference
that the N-atom at the pyrrolidinylethyl of them formed electro-
static force with carboxyl groups of Asp1083 and Asp1088 (Fig. 6A
and C). This difference also appeared in piperidinoethyl of
CPUY074030 (Fig. 6E). These observations may explain the higher
G9a inhibition of series II compounds, revealing the flexible side
chains containing terminal tertiary amine in R1 were essential for
G9a inhibition activity. Molecular surface visualization showed
that all of them occupied the cavities well in a reasonable confor-
mation (Fig. 6B, D and F).
(IC50 = 1.87
as 4-methylhomopiperazinyl (CPUY074031, IC50 = 2.49
4-phenylpiperazinyl (CPUY074032, IC50 = 2.68 M), led to slightly
loss of activity, indicating that it was not suitable to introduce large
groups of R2 substituents. In this series, all of the compounds sig-
nificantly improved G9a inhibitory activity compared to the hit
compound and showed equal activity to the positive control
UNC0638.
l
M) compounds with bulky groups at 3-position, such
lM) and
l
2.4. Anti-proliferative activities evaluation of representative
compounds
We kept R2 group and changed R1 group from N,N-dimethy-
laminoethyl to pyrrolidinylethyl leading to CPUY074018 with
no significant variation (IC50 = 3.47
IC50 = 3.87 M, CPUY074018). In keeping with this, the
pyrrolidinylethyl substituent at R1 (CPUY074023, IC50 = 3.22 M)
showed similar inhibition with piperidinoethyl substituent
compound (CPUY074032, IC50 = 2.68 M). The binding mode also
demonstrated different steric modifications had no effect on elec-
trostatic force between carboxyl groups of Asp1083, Asp1088 and
N-atom at R1 substitution (CPUY074019 vs CPUY074030), suggest-
ing that the R1 position could bear different steric modifications.
With the aim to further investigate the effect of 3-position sub-
stituents, we replaced 4-methylpiperazinyl group (CPUY074017,
l
M, CPUY074025 vs
Anti-proliferative activities of representative potent compounds
were performed with cell survival being determined by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay against HCT116 (colon cancer), MCF-7 (breast
cancer) and HepG2 (liver cancer) cells. UNC0638 was used as a
positive control. As shown in Table 3, it was found that all the com-
pounds of series II showed moderate or potent anti-proliferative
activity against three cancer cell lines, while the compounds of ser-
ies I showed barely anti-proliferative activity. This might be
ascribed to their inferior aqueous solubility. The anti-proliferation
activities of these compounds were consistent with their target-
based data. To our delight, some compounds (CPUY074022,
CPUY074023, CPUY074029, CPUY074031 and CPUY074032)
showed comparable activities against MCF-7 cell line to the posi-
l
l
l
IC50 = 6.89
piperidino (CPUY074019, IC50 = 1.22
(CPUY074020, IC50 = 2.18 M) enhancing the inhibition against
G9a slightly. Meanwhile, the similar observation could also be
detected between CPUY074028 (IC50 = 4.84 M) and CPUY074025
(IC50 = 3.47 M), CPUY074026 (IC50 = 3.75 M), demonstrating
lM) with dimethylamino (CPUY074018, IC50 = 3.87
lM),
lM) and pyrrolidinyl
l
tive control with IC50 values ranging from 1.22 to 5.08 lM. It was
l
worth noting that CPUY074020 exhibited slightly improved
activity against both HCT116 and MCF-7 cell lines than
UNC0638. Taking all data into account, compound CPUY074020
was selected for further evaluation of the cellular activities.
l
l
the terminal tertiary amine substituents at 3- position were
not required for the activity. Compared to CPUY074030
Table 3
Cell anti-proliferative activity of the indicated compounds
1
O
2
N
10
9
3
R2
4
8
6
7
5
HN
O
R1
Compd
R1
R2
Cell viability IC50
MCF-7
(lM)
HCT116
HepG2
CPUY074001
CPUY074003
CPUY074004
CPUY074008
CPUY074012
CPUY074017
CPUY074018
CPUY074019
CPUY074020
CPUY074021
CPUY074022
CPUY074023
CPUY074024
CPUY074025
CPUY074026
CPUY074027
CPUY074028
CPUY074029
CPUY074030
CPUY074031
CPUY074032
CPUY074033
UNC0638
4-Methoxylphenyl
4-Methoxylphenyl
4-Methoxylphenyl
4-Methylphenyl
4-Methylpiperazinyl
4-Ethylpiperazinyl
Pyrrolidinyl
4-Ethylpiperazinyl
4-Ethylpiperazinyl
4-Methylpiperazinyl
Dimethylamino
Pyrrolidinyl
Piperidino
4-Ethylpiperazinyl
4-(2-Pyrimidyl)piperazinyl
4-Phenylpiperazinyl
4-Methylhomopiperazinyl
Dimethylamino
139.50 24.12
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
66.66 5.08
>200
>200
2-Fluorophenyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
Pyrrolidinylethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
N,N-Dimethylaminoethyl
Piperidinoethyl
6.23 1.15
6.99 1.05
7.46 0.21
3.18 1.33
4.34 1.12
3.36 0.41
4.28 0.72
3.20 0.14
30.68 2.64
25.43 1.38
52.85 17.18
18.24 1.87
4.39 1.02
4.56 0.39
3.96 0.38
5.32 1.10
11.34 1.47
3.35 1.21
13.92 1.11
27.99 7.11
10.78 1.15
4.52 0.93
20.78 3.61
4.06 0.61
3.77 0.42
8.26 1.05
16.58 6.71
10.29 1.23
17.64 4.93
10.83 0.41
4.31 0.30
8.37 0.36
5.08 0.91
1.22 0.15
8.61 0.31
6.99 0.79
27.79 1.08
24.85 2.41
24.95 2.03
7.93 1.40
8.32 0.98
3.10 0.14
10.75 2.25
9.35 1.16
12.26 1.73
15.55 0.91
20.49 4.97
28.58 2.62
9.14 0.36
8.34 1.04
6.21 0.16
10.36 3.01
16.94 1.76
3.70 2.06
Piperidino
Pyrrolidinyl
4-Methylpiperazinyl
4-(2-Pyrimidyl)piperazinyl
4-Methylpiperazinyl
4-Methylhomopiperazinyl
4-Phenylpiperazinyl
Dimethylamino
Piperidinoethyl
Piperidinoethyl
1-Pyrrolidinylpropyl
—
—
Please cite this article in press as: Chen, W.-L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.071

6
W.-L. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
2.5. Effects of CPUY074020 on cell apoptosis
In order to identify whether CPUY074020 induced cell death
through apoptosis, a morphological observation study was used
to evaluate its influence on cell skeleton and Hoechst 33258 stain-
ing was taken to evaluate nuclei condensation. Under the inverted
light microscope (Â200), incubation of 2
lM, 4 lM and 8 lM of
CPUY074020 for 24 h resulted in phenotypic changes of MCF-7
cells, such as distortion, membrane blebbing and shrinkage under
4
lM, and vast majority of cells became round in shape and necro-
sis at 8 M, while cells in untreated group grew well (Fig. 7A).
l
Meanwhile, Hoechst staining demonstrated the dose-dependent
apoptotic bodies appeared (arrow pointing) after treatment with
CPUY074020, (Fig. 7B) which is commonly accepted as a marker
of apoptosis. The annexin V staining assay also proved this result
(Fig. 7C). CPUY074020 induced MCF-7 cells apoptosis, confirming
our compounds may be promising novel anti-cancer agents.
Figure 8. Western blot analysis of H3K9me2 and H3K9me3 methylation after
treatment of MCF-7 cells with compound CPUY074020.
Table 4
2.6. Western-blot analysis of CPUY074020
In vivo PK properties of CPUY074020
PK parameters
po
iv
To further characterize 6H-anthra[1,9-cd]isoxazol-6-one scaf-
fold compounds as potential G9a inhibitors and evaluate the cellu-
AUCa
(
l
g/LÁh)
723.8 248.8
4.0 0.9
6.7 2.1
70.7 29.6
15.2 5.2
90.7 39.5
55.5
1304.9 317.6
6.9 0.8
a
t1/2 (h)
lar activity, MCF-7 cells were treated with 2.5
l
M, 5
lM and 10 lM
a
Tmax (h)
0.12 0.19
188.2 35.0
8.1 2.1
of compound CPUY074020 for 48 h and 2.5
lM of UNC0638 was
a
Cmax
(
l
g/L)
Cla (L/h/kg)
Va (L/kg)
F %
selected as positive control. Equivalent amounts of protein from
cell extracts were Western Blotted for H3K9me2 and H3K9me3,
using b-actin as a loading control, and DMSO as a negative control.
A significant decrease in dimethylation level of H3K9 was observed
80.2 20.2
a
Data are expressed as mean SD, n = 6.
after treatment with 2.5 lM of CPUY074020. The result showed
that CPUY074020 dose-dependently de-regulated H3K9 trimethy-
lation (Fig. 8). This data further verified the enzyme-based and cell-
based evaluation results and paved the way for in vivo study.
for evaluation of its in vivo PK properties in mice. The compound
exhibited reasonable PK properties, with an oral bioavailability of
55.5% and a T1/2 value of 4.0 h at an oral dose of 10 mg/kg (Table 4).
The compound exposure was also reasonable with an
2.7. Assessment of in vivo PK properties of CPUY074020
AUC = 723.8
l
g/LÁh. It had a similar PK properties at the i.v. dosing
protocol (Table 4). Taken together, these encouraging PK results
suggested that CPUY074020 may serve as a promising starting
point for further optimization leading to in vivo study.
In order to investigate the PK properties of these novel com-
pounds, CPUY074020, with good potencies in vitro, was selected
Figure 7. Morphologic changes of the whole cancer cells (A) and the nuclei (B) after treatment of MCF-7 with CPUY074020. The arrow pointed to apoptotic bodies. (C) Cell
apoptosis were analyzed by annexin V/staining.
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W.-L. Chen et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
3. Conclusions
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Acknowledgments
This work was supported by the Project Program of State Key
Laboratory of Natural Medicines, China Pharmaceutical University
(SKLNMZZCX201611), National Natural Science Foundation of
China (81230078, 81573346), National Major Science and Technol-
ogy Project of China (Innovation and Development of New Drugs)
(2013ZX09402102-001-005, 2014ZX09507002-005-015), Special-
ized Research Fund for the Doctoral Program of Higher Education
(20130096110002), Priority Academic Program Development of
Jiangsu Higher Education Institutions and National Natural Science
Foundation of China (81502915, youth foundation).
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.09.071.
Please cite this article in press as: Chen, W.-L.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.09.071