Design, synthesis and biological activities of pyrrole-3-carboxamide derivatives as EZH2 (enhancer of zeste homologue 2) inhibitors and anticancer agents

Article abstract of DOI:10.1039/c9nj04713a

Zeste enhancer homolog 2 (EZH2) is highly expressed in various malignant tumors, which could silence tumor suppressor genes via trimethylation of H3K27. Herein was first reported a novel series of pyrrole-3-carboxamide derivatives carrying a pyridone fragment as EZH2 inhibitors. By combining computational modeling, in vitro cellular assays and further rational structure-activity relationship exploration and optimization, compound DM-01 showed powerful inhibition towards EZH2. DM-01 was found to have significant ability to reduce the cellular H3K27me3 level in K562 cells in the Western blot test. Meanwhile, our data showed that knockdown EZH2 in A549 cells resulted in a decrease of cell sensitivity to DM-01 at 50 and 100 μM. DM-01 could also increase the transcription expression of DIRAS3 in a dose-dependent manner, a tumor suppressor in the downstream of EZH2, suggesting it was worth investigating further as a lead compound.

Full text of DOI:10.1039/c9nj04713a

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Design, Synthesis and Biological Activities of Pyrrole-3-
carboxamide Derivatives as EZH2 (Enhancer of Zeste Homologue
2) Inhibitors and Anticancer Agents
Received 00th January 20xx,
Accepted 00th January 20xx
†
†
Qifan Zhoua, , Lina Jiab, , Fangyu Dua, Xiaoyu Dongb, Wanyu Sunb, Lihui Wangb,**, Guoliang
DOI: 10.1039/x0xx00000x
Chena,*
Zeste enhancer homolog 2 (EZH2) is highly expressed in various malignant tumors, which could silence tumor suppressor
gene via trimethylation of H3K27. Herein was first reported a novel series of pyrrole-3-carboxamide derivatives carrying with
pyridone fragment as EZH2 inhibitors. By combining computational modeling, in vitro cellular assays and further rational
structure-activity relationship exploration and optimization, compound DM−01 showed powerful inhibition of EZH2. DM−01
was found to have a significant ability to reduce cellular H3K27me3 level in K562 cells in Western blot test. Meanwhile, our
data showed knockdown EZH2 in A549 cells resulted in the decrease of cell sensitivity to DM−01 at 50 and 100 μM. DM−01
could also increase the transcription expression of DIRAS3 in a dose-dependent manner, a tumor suppressor in downstream
of EZH2, suggesting it was worthy to investigate further as a lead compound.
In recent years, kinds of EZH2 inhibitors with different
scaffolds have already been reported. 12 Some of the specific
inhibitors of EZH2 have entered clinical trials for the treatment
1. Introduction
Disorders in epigenetic regulation, including methylation of
DNA and histones, have been recognized as a major feature of
malignant tumors. 1 Enhancer zeste of homolog 2 (EZH2), which
belongs to a class of methyltransferases, has been found in
various malignant cells and is involved in divergent biological
processes. 2,3 EZH2 is a catalytic subunit of polycomb repressive
complex 2 (PRC2, the other two subunits are VeFS domain
containing SUZ12 and WD40 repeat containing protein EED)
of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma
(FL), and SNF5/INI-1/SMARCB1 genetically defined solid tumors,
including GSK
−
−
126 (Phase I), 13 tazemetostat
1205 (Phase I/II) (Fig. 1)16. Notably, Epizyme Inc.
(EPZ−6438, Phase
II),14,15 and CPI
has submitted a listing application of tazemetostat to FDA for
treatment for metastatic/locally advanced epithelioid sarcoma
in May 2019. More recently, DS−3201 (valemetostat),
developed by Daiichi Sankyo, is undergoing Phase I clinical trial.
4
that acts as a transcriptional repressor. It catalyzes the
17,18
PF−06821497 has been pushed into clinical development
addition of three methyl groups from the S-adenosyl-L-
methionine (SAM) cofactor to lysine 27 of Histone H3 (H3K27),
one of the five main histone proteins involved in the structure
of chromatin in eukaryotic cells, which facilitates chromatin
compaction and gene silencing of tumor suppressor genes in
cancer cells. The functions of EZH2 are closely related to tumor
generation, 5 chemotherapy resistance, 6 tumor metastasis, 7,8
by Pfizer for the treatment of adult patients with relapsed or
refractory small cell lung cancer (SCLC), castration resistant
prostate cancer (CRPC), DLBCL and FL (Fig. 1). 19 Subsequently,
SHR−2554 (structure undisclosed), developed by Jiangsu
Hengrui, recently entered phase I clinical trial for the treatment
20
of hematologic-blood cancer.
However, there were some
126
defects in the clinical EZH2 inhibitors. For example, GSK
−
9,10
and maintain the characteristics of cancer stem cells.
was administered by injection in clinical trials due to its poor
Subsequent studies have found high expressing EZH2 in a
variety of malignancies, including head and neck cancer,
bladder cancer, colorectal cancer, and non-small cell lung
21
pharmacokinetic properties;
clinical dose of tazemetostat
was increased to 800 mg po bid. 22
To gain insight into the interactions between inhibitors and
EZH2 protein, molecular docking simulations were performed
(PDB code: 4W2R, 19 Fig. 1). EZH2 inhibitors in clinical trials all
have similar and unique pyridone moieties, which are necessary
to improve binding to the EZH2 SET domain in SAM-competitive
manner via forming two hydrogen bonds with Trp521. Additional
bonding with Arg582 or Tyr111 is also important. As we known,
pyrroles provide access to important subunits and precursors of
numerous biologically active compounds, natural products and
11
cancer. Due to this evidence, research on anti-cancer drugs
targeting EZH2 has attracted widespread attention.
a. Address here. Key Laboratory of Structure-Based Drugs Design & Discovery of
Ministry of Education, School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, No. 103 Wenhua Road, Shenhe District, Shenyang
110016, P.R. China. E-mail addresses: spucgl@163.com (G. Chen).
b. Department of Pharmacology, Shenyang Pharmaceutical University, No. 103
Wenhua Road, Shenhe District, Shenyang 110016, P.R. China
† Authors contributed equally to this work
Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
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Fig.1. Molecular docking model and chemical structures of several clinical EZH2 inhibitors. PDB code: 19 4W2R; GSK−126 13 (white), tazemetostat 14 (green), CPI−1205 16 (yellow),
PF−06821497 19 (blue).
pharmaceuticals. 23-25 Due to these evidences, we designed a the two methyl groups adjacent to the pyrrole N, limiting the
series of pyrrole-3-carboxamide derivatives according to the spatial conformation of N-substitutions. Meanwhile,
biosostere theory using CPI 1205 as the lead compound compounds DM
−
−0128 were obtained via scaffold hopping to
(Scheme 1). Compounds 0123 were designed by retaining find out the effect of the spatial conformation of the N-
D
−
Scheme 1. Schematic diagram depicting the procedure for the design of the target compounds.
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Scheme 2. Synthetic route to compounds D−01D−23
substitutions and the amide on EZH2 inhibitory activity. All rapidly followed by rearrangement reaction in the solvents of 4
inhibitors contained the key active pyridone fragment, as the N HCl under refluxing to form the desired pyridine 24. Following
previous work showed the modification of this structure was chlorination and nucleophilic reactions, 24 was assembled to
14
not conducive to activity. Consequently, these derivatives give 26. The cyano of 26 was transferred to Boc-NH in catalyst
were synthesized and evaluated for their antitumor activities. system of H2/Raney-Ni/Boc2O/TEA. Finally, 27 was transformed
Their structure-activity relationships were summarized and the to another pyridinone 21 in the solvents of 4 N HCl under
mechanism of action that the compounds exerted their EZH2 refluxing. Following a Knorr reaction and decarboxylation, tert-
inhibitory activities was examined by western-blot and butyl acetoacetate 28 was assembled to give the key
computational modeling.
intermediate 30
arylmethyl halogens to produce 31a-r, which was then
hydrolyzed and condensed with or 11 21 to furnish
compounds DM 01 DM 28
2.2 In vitro biological activity assay and structure-activity
relationships
.
30 Then compound 30 was reacted with several
3

2. Results and discussion
2.1 Chemistry
−

−
.
The strategy adopted for the synthesis of
outlined in Scheme 2. Briefly, cyanoacetamide
with acetylacetone to obtain 2-cyanopyridone
D
−
01
D−23 was
The preclinical data from non-Hodgkin lymphoma (NHL) and
selected solid tumors demonstrated that tazemetostat inhibit
cellular growth in vitro and in vivo, and reduce H3K27me3
expression, a mark of transcriptional repression. 14 Therefore,
our compounds were tested by comparing H3K27me3 protein
expression in treated cells with that in un-treated cells through
Western Blot. K562 cells (human chronic myeloid leukemia cell)
and A549 cells (Human lung adenocarcinoma epithelial cell lines)
were used for subsequent studies based on the protein levels of
EZH2 and H3K27me3 (Fig. S1).
1 was reacted
2
14
.
The cyano
group was transferred into amino via H2/Ni reduction to afford
the key intermediate . Ethyl acetate was reacted with Conc.
ammonia which was rapidly followed by a Hantzsch reaction
thus forming the desired pyrrole-3-carboxylate was
26 Then
hydrolyzed directly to give pyrrole-3-carboxylic acid . The
subsequent condensation of compounds with yielded
subsequently Compounds 6a were obtained by reactions of
3
4
5
.
5
8
8
3
D
−
06
.
−
v
5
with various halogen substituted materials, which were
hydrolyzed to form 7a . The targets 01 05 and
07 23 were obtained via condensation of with
compounds 7a , respectively.
The Western Blot analysis results on
D series compounds
−
v
D
−
D−
showed that only 16 19 could inhibit EZH2 at either 5 μM
D
−
~D−
D
−

D
−
3
or 10 μM, which resulted in decreased H3K27me3 levels in K562
cells (Fig. S2). However, their ability was much weaker than the
positive drug GSK−126. A549 cells with stable low expression of
EZH2 (A549 sh-EZH2) were established to measure the cell
sensitivity toward our compounds by MTT assay (Fig. S3). While
A549 sh-EZH2 cells were relatively less sensitive than control
cells (A549 sh-EV), which we defined as potential selectivity for
−
v
The general synthetic strategy of DM series compounds is
illustrated as Scheme 3. Initially, pyridinone intermediates
1120 were obtained using ketones and ethyl formates or ethyl
acetates as starting materials via condensation, 27,28 cyclization
and reduction. For another key intermediate 21, compound 23
was synthesized by condensation of malonylonitrile with 4-
methyleneoxetan-2-one in the presence of NaH, 29 which was
EZH2 of candidate compounds. Unfortunately,
D series
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Scheme 3. Synthetic route to compounds DM−01DM−28
compounds hardly exhibited potential cytotoxicity on either Meanwhile, the steric effect of the substituent on the pyrrole-N
A549 sh-EV cells or A549 sh-EZH2 cells (Table S1). was removed. Western Blot showed that DM 01 DM 16 could
DM series compounds were then obtained via transferring inhibit EZH2 at both two concentrations (5 and 10 μM), which
the methyl group at pyrrole from C5’ to C4’ position, in order to resulted in decreased H3K27me3 levels (Fig. 2B). It was
produce an angle between the amide and pyrrole relying on the consistent with the result of molecular docking, as shown in
steric hindrance of the two adjacent methyl group (Fig. 2A). Table S2. The DM series compounds and control drugs showed
−

−
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Fig. 2. (A) Modified process form D to DM series compounds; (B) The cell viability of A549 sh-EV and A549 sh-EZH2 cells after DM series compounds treatment at 10 μM for 48
h; (C) Western Blot analysis results of H3K27me3 and Histone3 for DM−01DM−16.
the better dock scores than that of D series compounds. Thus, compared with control cells, A549 sh-EZH2 cells showed less
it was preliminarily speculated that the methyl group at C4’ growth inhibition to DM
−01 (Fig. 2C).
position of pyrrole was essential for EZH2 inhibitory activity. It was then investigated the effects of pyrrole-N substituents
Notably, tazemetostat was used as a positive drug in on the cytotoxic activity of DM series compounds using MTT
subsequent experiments as it was proved to be more potent assay. For A549 sh-EV cells, compounds with electron-
than GSK
−
126 in WB analysis. At the concentration of 10 μM, as withdrawing substituents (e.g.,
−
CF3,
−
F) exhibited more
Table 1. Antiproliferative activities of compounds DM−01DM−16
IC50 (μΜ)±SEM a
A549 sh-EZH2
269.7
47.3±4.85
48.8±0.34
＞100
Compd.
cLogP c
A549 sh-EV
Selectivityb
K562
58.706±3.18
＞100
DM−01
DM−02
DM−03
DM−04
DM−05
DM−06
DM−07
DM−08
DM−09
DM−10
DM−11
DM−12
DM−13
DM−14
DM−15
DM−16
GSK−126
tazemetostat
72.748±5.45
79.83±7.62
43.832±1.66
＞100
3.7
0.6
1.1
--
0.3
--
--
--
--
--
3.72
2.99
3.70
2.46
2.72
2.47
3.50
3.48
2.96
2.83
4.11
2.86
2.87
2.10
4.01
3.27
3.87
3.45
＞100
＞100
＞100
75.978±4.81
22.803±0.73
＞100
＞100
＞100
＞100
＞100
＞100
＞100
64.338±1.43
＞100
61.924±5.65
30.551±1.54
4.902±0.82
6.59±0.83
10.387±1.53
＞100
64.89±2.28
95.415±3.19
＞100
39.92±1.87
＞100
--
＞100
＞100
＞100
＞100
＞100
＞100
67.66±2.68
80.621±6.33
＞100
＞100
＞100
16.1±0.054
＞100
0.1
0.1
--
--
--
＞100
51.931±3.56
36.3±1.00
＞100
2.3
--
17.94±1.82
59.161±0.55
a IC50: 50% inhibitory concentration (detemined by standard MTT assay for A549, CCK-8 assy for K562). Each experiment was carried out in triplicate. b Selectivity =
IC50 values on A549 sh-EZH2 / A549 sh-EV cells. c Octanol-water partition coefficient (LogP) was calculated according to Discovery Studio 3.0.
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sufficient cytotoxic effects, such as DM
−
01 (IC50 = 72.1 μM), particularly substituting at the para position, showed much
View Article Online
DOI: 10.1039/C9NJ04713A
DM
on IC50 values in Table 1. The sequent molecular docking micromolar (DM
showed that a special H-bond was alternatively formed selectivity index of 2.3, DM
between the Fluor of the compounds and the target protein, EZH2 with selectivity index of 3.7. Furthermore, DM
leading to an improving docking score. In contrast, compounds showed comparable inhibitory activity to tazemetostat (IC50
with electron-donating substituents (e.g., OMe, Me), 59.2 μΜ) with an IC50 value of 58.7 μΜ for K562 cells. Besides,
−02 (IC50 = 79.8 μM) and DM−03 (IC50 = 43.8 μM) depending weaker antiproliferative activity with IC values of over 100
50
−
06
,
DM−
08). As compared to GSK
01 exhibited highly selective for
01 also
−126 with
−
−
=
−
−
10
11
12
13
14
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16
17
18
19
20
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Table 2. Antiproliferative activities of target DM−17DM−31 compounds
IC50 (μΜ)±SEM a
Compd.
Pyridone Core
R6
H
cLogP b
1.752
K562
DM−17
47.303±2.13
43.642±1.29
DM−18
H
3.997
DM−19
DM−20
DM−21
DM−22
DM−23
H
H
H
H
H
47.303±1.39
82.29±1.40
>100
4.825
3.086
3.398
5.076
4.63
65.392±1.19
51.162±1.34
DM−24
H
48.233±1.32
4.453
DM−25
DM−26
DM−27
H
H
H
69.062±1.39
76.512±2.72
62.6±2.06
4.368
3.922
3.845
DM−28
CH3
--
77.35±1.01
4.222
3.45
tazemetostat
--
59.161±0.55
a IC50: 50% inhibitory concentration (detemined by standard CCK-8 assy for K562). Each experiment was carried out in triplicate. b Octanol-water partition coefficient
(LogP) was calculated according to Discovery Studio 3.0.
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Fig. 3. The potency of DM−01 as a selective EZH2 inhibitor. Cells were treated with
DM−01 (5, 10 μM) for 48 h. (A) The inhibitory effect of DM−01 on H3K27m3
expression in K562 cells. (B) The cell viability of A549 sh-EV and A549 sh-EZH2. (C)
The increasing effect of DM−01 on DIRAS3 transcription expression in A549 cells.
it was found that DM
drugs, ranging from 3~4, indicating a benign Pharmacokinetic
property. Further studies showed that DM 01 strongly
−01 had similar cLogP value with positive
Fig. 4. (A) Putative binding modes of DM−01 (blue) and CPI−1205 (purple) in 3D
representation from molecular docking; Hydrogen bond is shown in green dash
lines and small molecules shows as sticks in 3D representation. (B) Ligand 2D
interaction diagram for DM−01 with H-bonds underlined in purple, π−π
interactions underlined in green. (C) Surface interaction diagram of DM−01 (blue)
and CPI−1205 (purple). (D) Structure of CPI−1205 (purple), DM−01 (blue) and D−13
(green) in its active conformation.
−
inhibited the activity of EZH2 and resulted in abolished
H3K27me expression in K562 cells, as depicted in Fig. 3A.
Meanwhile, our data showed knockdown EZH2 in A549 cells
resulted in the decrease of cell sensitivity to DM
100 μM concentrations (Fig. 3B). Besides, DM
−
01 at 50 and
the pyridone fragment could form hydrogen bonding with
Trp521 at distances of 2.71 Å and 2.77 Å, respectively (Fig. 4A and
4C), and another important hydrogen bond interaction with
Tyr111 was formed at a distance of 2.71 Å. More importantly,
−
01 could also
increase the transcription expression of DIRAS3 in a dose-
dependent manner, a tumor suppressor in downstream of EZH2
(Fig. 3C). The above data suggested that DM
−
01 could inhibit
DM
−01 could enhance the interaction with receptor via binding
tumor growth through suppressing EZH2.
with His199. Additionally, the pyrrole and benzene ring of
Based on the excellent biological activity results, DM
−
01 was
DM
−
01 could also form hydrophobic interactions with Try111
,
further optimized by changing the substitutes of pyridon or
benzyl group to obtain its derivatives. Using tazemetostat as
the positive control, the synthesized targets DM−17DM−31
were evaluated for the cytotoxic activities in vitro against K562
cells, where EZH2 was highly expressed, and the results
expressed as IC50 values were summarized in Table 2. The
activities of most compounds were considerable to that of
Tyr558, and Phe562 (Fig. 4B). It was obvious that DM
−
01
interacted with EZH2 protein in the similar manner with
CPI
1205, a ligand contained in PDB structure 5LS6 16. As
described in Fig. 4D, the docking of compounds 13 DM 01
and CPI 1205 showed that a same torsional angle between the
aromatic ring and the amide carbonyl moiety. However, the
amide carbonyl group of 13, an isomer of DM 01, faced to
−
D
−
,
−
−
D
−
−
tazemetostat. Fused cycloalkane derivatives DM
−17 (IC50 = 47.3
reverse direction due to the steric hindrance of methyl group at
C3’ position, resulting in not able to bind with Tyr111. These
μM), DM 18 (IC50 = 43.6 μM) and DM 24 (IC50= 48.2 μM)
−
−
slightly improved the inhibitory activities than positive drug,
indicating that large hindered substituents were allowed at R4
and R5 positions. It was also confirmed by docking that the
modifications in pyridon part was tolerated, sharing similar
results explained why the EZH2 inhibitory activities of
D series
compounds were weaker or lost. A field point pattern analysis
docking scores with DM
DM 27 with methyl substitution at R5 position showed its
inhibitory with an IC50 value of 62.6 μM. However, when the
methyl group at R3 position was removed (DM 21, IC50 100
μM) or replaced by methoxy (DM 20, IC50 = 82.3 μM), the
inhibitory activity decreased. When methyl was introduced at
R6 (DM 28), the inhibitory activity decreased with an IC50 value
76.5 μM. These results showed that DM 01 had enough
−01 (Table S2). It was also proved that
−
−
＞
−
Fig. 5. (A) The field point pattern for the conformation of DM−01 and CPI−1205;
Negative field points (blue, like to interact with positives/H-bond donors on a
protein); Positive field points (red, like to interact with negatives/H-bond acceptors
on a protein); Van der Waals surface field points (yellow, describing possible
surface/vdW interactions). The size of the point indicates the potential strength of
the interaction. (B) The molecular electrostatic potential map at the 0.5 kcal/mol
contour of DM−01 (yellow) and CPI−1205 (grey); Regions explored in negative
electrostatics (blue) and regions explored in positive electrostatics (red).
−
−
modification space to improve the inhibitory activity.
2.3 Molecular interactions and docking simulations
To gain insight into the interactions between our compounds
and EZH2 protein, molecular docking simulations were
performed (PDB code: 4W2R). Taking DM−01 as an example,
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using this distribution of conformational propensities was also then electrically transferred onto polyvinylidene difluoride
View Article Online
performed (Fig. 5). DM−01 and CPI−
1205 shared the same (PVDF) membranes. Mouse or rabbit prDimOaI:r1y0.1a0n3t9i/bCo9dNiJe0s47a1n3dA
negative field points, positive field points and Van der Waals appropriate horseradish peroxidase (HRP)-conjugated
surface field with the similarity value of 0.829 (score was secondary antibodies were used to detect the designated
calculated by combining the field score and the shape score). proteins. The bound secondary antibodies on the PVDF
Besides, at the 0.5 kcal/mol contour, DM−01 and CPI−1205 membrane were reacted with ECL detection reagents (Pierce;
have similar negative electrostatics and positive electrostatics Rockford, USA) and exposed to X-ray films. Results were
in the molecular electrostatic potential map analysis (Fig. 5).
normalized to the internal control Histone3 31,32
.
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3.3 Molecular docking and field points
Molecular docking was performed as previously described. 33
The molecular docking study was performed by AutoDock 4.2.6.
The three dimensional structure of EZH2 was retrieved from the
Protein Data Bank (PDB code: 4W2R). The binding site was
3. Experimental
3.1 In vitro inhibitory activity on A549 sh-EV and A549 sh-
EZH2 cells assay
located on the SET region. The 3D structures of DM
−
01
, D−13
Lentiviruses were produced by transfecting HEK293T cells
with shRNA-EZH2-targeting-Plo.k1-plasmids and the helper
plasmids pCMV-VSVG, pMDLg-RRE (gag/pol), and pRSV-REV.
The cell supernatants were harvested 48 h after transfection. To
obtain stable cell lines, A549 cells were infected for 24 h with
normal culture medium in the presence of 5 μg/mL of polybrene.
After 48 h infection, cells were placed under puromycin
selection for one week and then passaged before use.
Puromycin was used at 1 μg/ml to maintain A549 cells.
A549 sh-EV and A549 sh-EZH2 cells (MSKCC, USA) plated in
RPMI 1640 (Roswell Park Memorial Institute, Gibco)
supplemented with 10% FBS (fetal bovine serum, Gibco), and
penicillin/streptomycin (HyClone, Shanghai, China) at 37 oC
under 5% CO2 until they were 70-80% confluent. The adherent
and CPI
−1205 were built by using Sybyl-X 2.0. Both the protein
and the ligands were prepared by adding polar hydrogen atoms
and partial charges with the assistance of AutoDockTools 1.5.6.
Grid points were set to 40 at the x, y, z axes. The conformational
search was carried out using the Lamarckian Genetic Algorithm.
All other parameters were set to default unless stated
otherwise. The field point pattern and molecular electrostatic
potential map were performed by using Forge 10.4 (Cresset,
USA). 34
Conclusions
Since EZH2 plays a pivotal role in cell proliferation and
cells were digested with 0.25% trypsin (containing EDTA 1 survival, it is recognized as one of the significant oncogenic
mmol/L) and suspended in culture medium. signaling pathways through improving H3K27me3 level. Some
Cell viability was analyzed by using 3-(4,5-dimethyl-2- of the specific EZH2 inhibitors have entered clinical trials for the
thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) (Sigmal- treatment of DLBCL, FL, and SNF5/INI-1/SMARCB1 genetically
aldrich, Shanghai, China), followed by manufacturer's defined solid tumors. Here were first reported a series of
instructions. The Cells were plated in 96-well plate and grown pyrrole-3-carboxamide derivatives as EZH2 inhibitors, leading to
for 24 h. Following the addition of different concentrations the discovery of DM
(100μM 50μM 10μM 5μM 1μM) of our compounds, the studies in K562 cells. Combining computational modeling and in
cells were further cultured for 48 h, with 0.5% DMSO as the vitro cellular assays, the structure-activity relationship was
−01 screened by H3K27me3 western blot
、
、
、
、
solvent control group. Then 10 μL MTT regent was added to summarized. It was found that DM
−
01 exhibited its function
o
each well and the culture plate was settled at 37 C incubation showing an obvious dependence on EZH2, which was worthy to
chamber for 3-5 h. The optical density (OD) at a wavelength of investigate further as a lead compound. Further development
450 nm was detected by using a microplate reader (Molecular of compound DM
Devices, USA). Cell viability was expressed as percentage of
−01 is in progress.
untreated controls. Each treatment was performed in triplicate.
The half inhibitory concentration IC50 values were calculated by
IBM SPSS Statistics 21.
Conflicts of interest
There are no conflicts to declare.
3
.
2
Western Blot assay
The human colon cancer cell line K562 cells were incubated
in presence of our compounds, GSK 126 or tazemetostat (in
Acknowledgements
−
0.5% DMSO) for 24 h, harvested, and rinsed with ice-cold PBS.
Total protein extracts were prepared by lysing cells in RIPA
buffer 50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl
sulfate (SDS), 1% NP-40, 0.5% sodium deoxycholate, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 100 M leupeptin, and 2
g/mL aprotinin (pH 8.0) supplemented with 0.2 trypsin
inhibitory units/ml aprotinin, 0.7 mg/mL pepstatin, and 1
mg/mL leupeptin. An equal amount of total protein extracts
This work was supported by the project 201602707,
20170540841 (Natural Science Foundation of Liaoning
Province), National Natural Science Foundation of China (No.
81572947, 81773216), Scientific Research Fund of Liaoning
Provincial Education Department (No. 2017LFW01) and Science
Foundation of Shenyang Pharmaceutical University (No.
DFJJ2018210).
from cultured cells were fractionated by 1015% SDS-PAGE and
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9
Wang, S. Wang, M. Wythes, S. Xin, S. Yamazaki, H. Zhu, J. Zhu,
L. Zehnder and M. Edwards, J. Med. Chem., 2018V,iew Article Online
DOI: 10.10396/1C,96N5J00476163A5.
20 B. Lu, X. D. Shen, M. X. He, D. Liu and M. S. Zhang, WO
2017/084494A1.
Notes and references
−
1
2
3
4
5
D. Hanahan and R. A. Weinberg, Cell, 2011, 144, 646
H. Heyn and M. Esteller, Cancer Cell, 2013, 33, 563 565.
J. A. Simon, Nat. Chem. Biol., 2012, , 875 876.
R. Margueron and D. Reinberg, Nature, 2011, 469, 343 349.
W. Cao, R. H. Younis, J. Li, H. Chen, R. Xia, L. Mao, W. Chen and
H. Ren, Cancer Prev. Res., 2011, , 1816 1824.
−674.
−
8
−
21 C. G. Nasveschuk, A. Gagnon, S. Garapaty-Rao, S.
Balasubramanian, R. Campbell, C. Lee, F. Zhao, L. Bergeron, R.
Cummings, P. Trojer, J. E. Audia, B. K. Albrecht and J-C. P.
−
4
−
Harmange, ACS Med. Chem. Lett., 2014, 5, 378−383.
6
E. E. Gardner, B. H. Lok, V. E. Schneeberger, P. Desmeules, L.
A. Miles, P. K. Arnold, A. Ni, I. Khodos, E. de Stanchina, T.
Nguyen, J. Sage, J. E. Campbell, S. Ribich, N. Rekhtman, A.
Dowlati, P. P. Massion, C. M. Rudin and J. T. Poirier, Cancer
22 A phase II, multicenter study of the EZH2 inhibitor
tazemetostat in adult subjects with INI1-negative tumors or
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
relapsed/refractory
synovial
sarcoma,
https://clinicaltrials.gov/ct2/show/NCT02601950?cond=Taze
metostat&draw=2&rank=11.
Cell, 2017, 31, 286−299.
7
D. Zingg, J. Debbache, S. M. Schaefer, E. Tuncer, S. C. Frommel,
P. Cheng, N. Arenas-Ramirez, J. Haeusel, Y. Zhang, M. Bonalli,
M. T. McCabe, C. L. Creasy, M. P. Levesque, O. Boyman, R.
Santoro, O. Shakhova, R. Dummer and L. Sommer, Nat.
23 P. Liu, Y. Yang, Y. Ju, Y. Tang, Z. Sang, L. Chen, T. Yang, Q. An,
T. Zhang and Y. Luo, Bioorg. Chem., 2018, 80, 422−432.
24 G. Meng, C. Liu., S. Qin, M. Dong, X. Wei, M. Zheng, L. Qin, H.
Wang, X. He and Z. Zhang, Res Chem Intermediat., 2015, 41
,
Commun., 2015, 6, 6051−6068.
N. Tiwari, V. K. Tiwari, L. Waldmeier, P. J. Balwierz, P. Arnold,
M. Pachkov, N. Meyer-Schaller, D. Schübeler, E. van
8941−8954.
8
9
25 G. Tarzia, A. Duranti, A. Tontini, G. Spadoni, M. Mor, S. Rivara,
P. V. Plazzi, S. Kathuriac and D. Piomellic, Bioorgan. Med.
Nimwegen and G. Christofori, Cancer Cell, 2013, 23, 768
K. Lund, P. D. Adams and M. Copland, Leukemia, 2014, 28
44 49.
−783.
Chem., 2003, 11, 3965−3973.
,
26 Y. Peng, J. Luo, Q. Feng and Q. Tang, Eur. J. Org. Chem., 2016,
2016, 5169 5179.
27 F. Liu, Y. Y. Hu, D. Li, Q. Zhou and J. M. Lu, Tetrahedron, 2018,
74, 5683 5690.
28 Y. Watanabe, S. C. Shim, T. Mitsudo, M. Yamashita and Y.
Takegami, B. Chem. Soc. Jpn., 1976, 49, 2302 2305.
−
−
10 C. J. Chang, J. Y. Yang, W. Xia, C. T. Chen, X. Xie, C. H. Chao, W.
A. Woodward, J. M. Hsu, G. N. Hortobagyi and M. C. Hung,
−
Cancer Cell. 2011, 19, 86−100.
11 M. Takawa, K. Masuda, M. Kunizaki, Y. Daigo, K. Takagi, Y. Iwai,
H. S. Cho, G. Toyokawa, Y. Yamane, K. Maejima, H. I. Field, T.
Kobayashi, T. Akasu, M. Sugiyama, E. Tsuchiya, Y. Atomi, B. A.
Ponder, Y. Nakamura and R. Hamamoto, Cancer Sci., 2011,
−
29 Z. K. Lu and R. J. Twieg, Tetrahedron, 2005, 61, 903-918.
30 G. Meng, C. Y. Liu, S. D. Qin, M. S. Dong, X. M. Wei, M. L. Zheng,
L. W. Qin, H. H. Wang, X. S. He and Z. G. Zhang, Res. Chem.
102, 1298
12 H. Ü . Kaniskan and J. Jin, Curr. Opin. Chem. Biol., 2017, 39
100 108.
−1305.
Intermediat., 2015, 41, 1−14.
,
31 L. Wang, H. Li, Y. Ren, S. Zou, W. Fang, X. Jiang, L. Jia, M. Li, X.
−
Liu, X. Yuan, G. Chen, J. Yang and C. Wu, Cell Death. Dis., 2016,
13 A study to investigate the safety, pharmacokinetics,
pharmacodynamics and clinical activity of GSK2816126 in
subjects with relapsed/refractory diffuse large B cell and
7
, e2063−2074.
32 L. Wang, G. Chen, K. Chen, Y. Ren, H. Li, X. Jiang, L. Jia, S. Fu, Y,
Li, X. Liu, S. Wang, J. Yang and C. Wu, Oncotarget, 2015,
9740 9755.
33 Q. Zhou, C. Peng, F. Du, L. Zhou, Y. Shi, Y. Du, D. Liu, W. Sun,
M. Zhang and G. Chen, Eur. J. Med. Chem., 2018, 151, 39 50.
34 T. Cheeseright, M. Mackey, S. Rose and J. G. Vinter, Expert.
Opin. Drug Discov., 2007, , 131 144.
6
,
transformed
follicularlymphoma,
−
http://clinicaltrials.gov/ct2/show/NCT02082977?term=NCT0
2082977&=1.
−
14 K. W. Kuntz, J. E. Campbell, H. Keilhack, R. M. Pollock, S. K.
Knutson, M. Porter-Scott, V. M. Richon, C. J. Sneeringer, T. J.
Wigle, C. J. Allain, C. R. Majer, M. P. Moyer, R. A. Copeland and
2
−
R. Chesworth, J. Med. Chem., 2016, 59, 1556−1564.
15 S. K. Knutson, S. Kawano, Y. Minoshima, N. M. Warholic, K.
Huang, Y. Xiao, T. Kadowaki, M. Uesugi, G. Kuznetsov, N.
Kumar, T. J. Wigle, C. R. Klaus, C. J. Allain, A. Raimondi, N. J.
Waters, J. J. Smith, M. Porter-Scott, R. Chesworth, M. P.
Moyer, R. A. Copeland, V. M. Richon, T. Uenaka, R. M. Pollock,
K. W. Kuntz, A. Yokoi and H. Keilhack, Mol. Cancer Ther., 2014,
13, 842−854.
16 R. G. Vaswani, V. S. Gehling, L. A. Dakin, A. S. Cook, C. G.
Nasveschuk, M. Duplessis, P. Iyer, S. Balasubramanian, F. Zhao,
A. C. Good, R. Campbell, C. Lee, N. Cantone, R. T. Cummings,
E. Normant, S. F. Bellon, B. K. Albrecht, J. C. Harmange, P.
Trojer, J. E. Audia, Y. Zhang, N. Justin, S. Chen, J. R. Wilson and
S. J. Gamblin, J. Med. Chem., 2016, 59, 9928
17 A Phase 1 Multiple Ascending Dose Study of DS-3201b in
Japanese Subjects With Lymphomas,
https://clinicaltrials.gov/ct2/show/NCT02732275?term=NCT
02732275&rank=1.
18 T. Watanabe, M. Yamagishi, O. Kanno, J. Watanabe, N. Adachi,
D. Honma and Y. Hamada, WO 2017/018499A1.
19 P. P. Kung, P. Bingham, A. Brooun, M. Collins, Y. L. Deng, D.
Dinh, C. Fan, K. S. Gajiwala, R. Grantner, H. J. Gukasyan, W. Hu,
B. Huang, R. Kania, S. E. Kephart, C. Krivacic, R. A. Kumpf, P.
Khamphavong, M. Kraus, W. Liu, K. A. Maegley, L. Nguyen, S.
Ren, D. Richter, R. A. Rollins, N. Sach, S. Sharma, J. Sherrill, J.
Spangler, A. E. Stewart, S. Sutton, S. Uryu, D. Verhelle, H.
−9941.
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DOI: 10.1039/C9NJ04713A
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Highlight
A series of pyrrole-3-carboxamide derivatives targeting on EZH2 were designed and synthesized.
Compound DM01 was found to possess significant inhibitory activities to reduce cellular
H3K27me3 level in K562 cells.
The structure–activity relationships of the synthesized compounds were summarized.
Molecular docking studies were performed.