Design and synthesis of (E)-1,2-diphenylethene-based EZH2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.126957

Enhancer of zeste homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repression complex 2 (PRC2), which is implicated in cancer progression metastasis and poor prognosis. Based on our EZH2 inhibitor SKLB1049 with low nanomolar activity, we e

Full text of DOI:10.1016/j.bmcl.2020.126957

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Design and synthesis of (E) -1,2-diphenylethene-based EZH2 inhibitors
Hualong He, Xi Hu, Fei Teng, Zhihao Liu, Qiangsheng Zhang, Zhanzhan Feng,
Qiang Feng, Luoting Yu
PII:
S0960-894X(20)30010-X
https://doi.org/10.1016/j.bmcl.2020.126957
BMCL 126957
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
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10 December 2019
2 January 2020
Please cite this article as: He, H., Hu, X., Teng, F., Liu, Z., Zhang, Q., Feng, Z., Feng, Q., Yu, L., Design and
synthesis of (E) -1,2-diphenylethene-based EZH2 inhibitors, Bioorganic & Medicinal Chemistry Letters (2020),
doi: https://doi.org/10.1016/j.bmcl.2020.126957
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Bioorganic & Medicinal Chemistry Letters
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Design and synthesis of (E) -1,2-diphenylethene-based EZH2 inhibitors.
Hualong Hea, Xi Hua, Fei Tenga, Zhihao Liua, Qiangsheng Zhanga, Zhanzhan Fenga, Qiang Fengband
Luoting Yua, 
a State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University and Collaborative Innovation
Center, Chengdu 610041, China
b College of Chemistry and Life Science, Chengdu Normal University, Chengdu 611130, China
———
ARTICLE INFO
ABSTRACT
 Corresponding author. Tel.: +86-28-8550-3817; fax: +86-28-8550-3817; e-mail: yuluot@scu.edu.cn
Article history:
Received
Revised
Accepted
Available online
Enhancer of zeste homolog 2 (EZH2) serves as the catalytic subunit of the polycomb repression
complex 2 (PRC2), which is implicated in cancer progression metastasis and poor prognosis.
Based on our EZH2 inhibitor SKLB1049 with low nanomolar activity, we extended the “tail”
region to get a series of (E)-1,2-diphenylethene derivatives as novel EZH2 inhibitors. SAR
exploration and preliminary assessment led to the discovery of the potent novel EZH2 inhibitor
9b (EZH2WT IC50=22.0 nM). Compound 9b inhibited the proliferation of WSU-DLCL2 and SU-
DHL-4 cell lines (IC50=1.61 µM and 2.34 µM, respectively). The biological evaluation showed
that 9b was a potent inhibitor for wild-type EZH2 and greatly reduced the overall levels of
H3K27me3 in a concentration-dependent manner. Further study indicated that 9b could
significantly induce apoptosis of SU-DHL-4 cells. These findings indicated that 9b would be an
attractive lead compound for further optimization and evaluation.
Keywords:
EZH2
PRC2
H3K27Me3
(E) -1,2-diphenylethene
SKLB1049
2019 Elsevier Ltd. All rights reserved.
Head
Head
Disorders of epigenetic regulation, including histone
methylation, have been implicated in many malignant tumors[1].
The histone methylation epigenetic regulator, enhancer Zeste of
homolog 2 (EZH2) is a catalytic subunit of polycomb repressive
complex 2 (PRC2) and it agglutinates chromosome structures
through trimethylation of lysine 27 on histone H3 (H3K27me3)
to achieve transcriptional inhibition of relevant target genes[2].
EZH2 amplification and functional alteration are frequently
found in castration-resistant prostate cancer and triple-negative
breast cancer with lower overall survival[3-5]. Subsequent studies
have found that high expression of EZH2 in a variety of
malignant tumors including head and neck cancer, bladder
cancer, colorectal cancer, and non-small cell lung cancer, which
are associated with poor prognosis[6]. Thus, EZH2 could be a
promising therapeutic target for cancer treatment and has
received extensive attention.
Neck
O
Neck
O
NH
NH
NH
O
NH
O
side
Tail
side
Tail
xposed
O
-e
t
xposed
HN
-e
t
solven
N
solven
N
Arm
N
Arm
N
N
Body
Body
O
GSK126
EPZ6438
NH
NH
NH
NH
O
NH
O
O
NH
O
O
NH
O
O
NH
O
N
N
N
N
N
O
N
N
N
N
N
N
N
N
N
N
N
N
SKLB1049
GSK503
UNC1999
Pfizer MP lead
NH
NH
O
NH
O
O
NH
O
N
N
N
N
N
O
N
N
N
Most EZH2 inhibitors are characterized by a pyridone moiety
as the main pharmacophore of SAM-competitive inhibitors (Fig.
1). Typically, they have a similar pyridone as the “head” region,
an amide linker as the “neck” region, different aromatic as the
“body” region, and variable “arm” and “tail” structures attached
to the top and bottom of the body, respectively[7]. Pyridone
inhibitors including GSK126, EPZ-6438 (Tazemetostat) and
SKLB1049 are highly efficacious in the suppression of tumor
growth [8,9] (Fig. 1).
GSK343
EPZ5687
Figure 1. Small-molecule inhibitors of EZH2 reported in recent years.
We then investigated the cocrystallized structure (PDB code
5WG6) to understand the interactions between GSK126 and
EZH2 (Figure 2) [6]. As a “head” region, the pyridin-2(1H)-one
could form two hydrogen bonds with Trp624. In addition, the
pyridin-2(1H)-one had a strong π-π interaction with the benzene

O
O
O
O
O
OH
O
OH
H
H
O
N
O
N
a
b
c
d
Where,A=
B=
Br
NO2
Br
NH2
Br
NO2
NO2
1
3
2
R1
NH
O
O
O
O
O
O
OH
g
f
e
Br
NH
Br
N
O
Br
N
Br
N
R1
O
NH
7
5
6
4
i
O
O
:R =A
O
1
7a
1
7b
:R =B
N
R2
O
O
R2
h
R2
B
Br
O
9
10
or
1
9
:R =A
1
10
:R =B
8
Scheme 1. The synthesis of compounds 9 or 10. Reagents and conditions: (a) 1,3-dibromo-5,5-dimethylhydantoin, sulfuric acid, RT; (b) MeI, Na2CO3, DMF,
60°C; (c) Fe, NH4Cl, MeOH/H2O, reflux; (d) sodium triacetoxyborohyride, acetic acid, dichloromethane, tetrahydro-4H-pyran-4-one, 0°C - RT; (e) sodium
triacetoxyborohyride, acetic acid, dichloromethane, acetaldehyde, 0°C - RT; (f) 3M NaOH, ethanol, 60°C, 3h, then adjust to pH 4-5 with 1N HCl; (g) EDC,
HOAT, NMM, DMSO, RT; (h) Bis(tri-tert-butylphosphine)palladium(0), DIEA, toluene, 90°C; (i) PdCl2(dppf)·CH2Cl2 , Na2CO3 , 4:1 dioxane/H2O, 95°C.
ring of Phe686. The “neck” region made of amide group could
form a hydrogen bond with Tyr111. Meanwhile, the “tail” region
consisting of pyridyl piperazine points outward into the solvent-
exposed side between the EZH2 and EED, which belongs to
PRC2 complex subunits. Notably, the “tail” region of EZH2
inhibitors points toward the solvent-exposed side between the
EZH2 and EED. This feature reminded us that the “tail” region
could be extended to acquire further structural optimization.
Hence, based on our previously developed EZH2 inhibitor
SKLB1049[10], we report the design, synthesis and biological
evaluation of (E)-1,2-diphenylethene derivatives having trans-
ethylene bonds as potent EZH2 inhibitors. (Fig. 3)
intermediate
tetrahydroisoquinolin-3(2H)-one and 3-(aminomethyl)-4-methyl-
5,6,7,8-tetrahydroquinolin-2(1H)-one were subjected to
condensation reaction with acids 6 afforded 7. Last, compounds 9
or 10 were gained by Suzuki coupling reaction of compound 7
and compound 8.
6.
4-(Aminomethyl)-1-methyl-5,6,7,8-
a
NH
NH
O
NH
O
O
NH
O

N
N
N
N
N
N
O
O
SKLB1049
9j
Figure 3. Schematic diagram showing the subgroups that were the focus of
structural modifications.
Table 1 provides the biological data of these compounds. All
synthetic derivatives were evaluated for its EZH2 inhibitory
activity in an AlphaLISA assay. The synthesis method of
pyridone A and B are provided in reference 12. It was obvious
that all the compounds with A structure exhibited better EZH2WT
inhibitory potency than the compounds with B structure. To
investigate the structure-activity relationships of the C5 position
of the “tail” region, we synthesized a total of 26 compounds with
different N-containing heterocyclic substituents. First, we
obtained compound 9j (EZH2WT IC50 = 39 nM) by modifying
SKLB1049 through the strategy shown in figure 3. Subsequent
SAR study of different substituents on the side-chain phenyl
group was further explored. Compound 9f (EZH2WT IC50 = 27 nM)
with 4-morpholinomethyl substituent appeared to be more active
Figure 2. Structure of PRC2 bound to GSK126 (PDB code: 5WG6).
Scheme 1 shows the synthesis route of the compounds
described in this paper. All the aryl-substituted pinacol
vinylboronates 8 were prepared by the Heck reaction[11]. The
intermediate 3 was obtained from 2-methyl-3-nitrobenzoic acid
by bromination, esterification, and reduction, respectively. Then,
a two-step reductive amination of 3 provided the compound 5,
and hydration of 5 with 3M NaOH in ethanol yielded the
than compound 9e (EZH2WT IC50
= 39 nM) with 3-
morpholinomethyl substituent. The activity of compound 9b was
improved nearly 2-fold by opening the morpholine ring as in 9e,
suggesting that substituents on this position might be tolerated.
Likewise, when two methyl groups were used to replace the o-
site of the oxygen on the morpholine ring of

Table 1. In Vitro Potencies of Newly Synthesized Inhibitors 9a -9g 和 10a-
10g.
H
H
O
O
O
N
O
N
9g
10g
9h
7.8
33
27.67
>30
>30
20.43
15.44
10.54
>30
4.36
12
N
N
H
H
O
N
O
N
F
F
X
N
O
X
N
O
10
9
O
O
O
O
N
EZH2WT
IC50
nM)
56
9.27
4.58
5.85
6.18
2.58
3.11
12.61
25.80
>30
21.10
24.28
>30
>30
>30
22.56
>30
>30
NT
a
(
cells IC50 (μM)
SU-DHL-4
>30
F
No.
X
N
N
N
10h
9i
161
60
WSU-
DLCL2
MV4-
11
EZH2WT
F
F
O
O
9a
10a
9b
44
>30
>30
4.56
5.69
2.80
3.98
>30
8.04
3.21
3.87
4.44
9.23
6.41
>30
NH
O
O
10i
9j
329
31
>30
189
22
25.68
2.34
F
NH
N
5.96
4.31
21.72
>30
N
1.61
20.93
>30
N
N
N
10j
9k
143
23
N
10b
9c
35
15.67
>30
N
N
N
O
O
O
O
110
>200
55
N
10k
9l
114
137
225
620
>1000
7.8
N
10c
9d
>30
>30
N
O
NTb
NT
N
O
O
S
S
O
O
O
27.70
17.54
>30
>30
O
O
N
O
10l
9m
10m
>30
NT
N
O
10d
9e
189
39
21.56
>30
N
21.25
NT
NT
NT
MeOOC
MeOOC
N
O
NT
NT
N
10e
9f
>200
27
>30
>30
O
O
SKLB
1049
1.64
17.79
1.78
14.34
>30
22.38
>30
>30
N
N
EPZ-
6438
0.34
O
10f
>200
>30
25.67
aData were represented as mean values ± SEM (n = 2).
bNot tested.

9f (EZH2WT IC50 = 27 nM), the activity of the compound 9c
(EZH2WT IC50 = 110 nM) was nearly 4-times lost due to the
increase of steric resistance. This conclusion is also supported by
compound 9d (EZH2WT IC50 = 55 nM).
to 2 and 4 μM, respectively. The results showed that 9b could
significantly induce apoptosis of SU-DHL-4 cells at 2μM (Fig. 6).
F atom can enhance the hydrophobic interaction between the
compound and the enzyme to improve the inhibitory activity (9g,
EZH2WT IC50 = 7.8 nM). Similarly, replacing the benzene ring
with the pyridine ring yielded compound 9k (EZH2WT IC50 = 23
nM). The maintenance of activity indicated that the structure has
a certain tolerance to some aromatic rings.
The introduction of the sulfonamide bond resulted in a less
potent compound (9l). A fuse ring substituent (9a, EZH2WT IC50
= 39 nM) did not help improve the activity. These results suggest
that flexible substituents are relatively preferred to rigid
substituents. Inserting a methylene bridge into the morpholine
ring led to some compounds(9h and 9i), which showed poor
activity due to the limitation of their ring’s flexibility.
Figure 6. Compound 9b induced apoptosis in EZH2-overexpressed SU-
DHL-4 cells. Cell apoptosis analysis (by flow cytometry) was assessed in SU-
DHL-4 cells during incubation with either vehicle or 0 μM, 1.0 μM, 2.0 μM,
4.0 μM 9b for 6 days.
It is worth noting that most compounds exhibited lower
inhibitory activities on cell proliferation against the EZH2-
overexpressed cell lines than the negative cell MV4-11. We
speculate as to the following reasons. Firstly, it was reported that
the inhibition of EZH2 reactivates TXNIP, inhibits thioredoxin
activity, and increases reactive oxygen species (ROS), leading to
apoptosis of acute myeloid leukemia cell lines [13]. Secondly, the
pyridone ring is the active fragment of many drugs, such as
Figure 4. Inhibition curve of compound 9b on EZH2WT at 1 μM SAM
concentration
, . Some compounds
BRD4 inhibitors[14] JAK2 inhibitors[15]
presented in this paper may have an off-target effect, leading to
superior inhibitory activity against MV4-11 cell line than EZH2-
overexpressed cell lines.
In this study, based on SKLB1049, a series of novel
compounds bearing (E)-1,2-diphenylethene were designed and
synthesized through the strategy of extending the side chain. But
all compounds except compound 9g showed decreased EZH2
inhibitory activities. For one thing, tetrahydroisoquinolinone
derivatives exhibited higher inhibitory potency than
tetrahydroquinolinone derivatives, which indicated that the
pyridone moiety has a more important impact on EZH2 inhibitors
than other regions. For another thing, there are many factors that
simultaneously affect the activity of inhibitors, such as steric
hindrance, hydrophobicity, and van der Waals force. The increase
of the steric hindrance from the extension of the side chain may
have a greater impact on enzyme activity than other forces,
resulting in that most compounds exhibited lower inhibitory
activities than SKLB1049.
Figure 5. Compound 9b reduced H3K27me3 in SU-DHL-4 cells in a
concentration-dependent manner.
We went on to study the inhibitory activities on cell
proliferation in vitro. Two EZH2-overexpressed cell lines WSU-
DLCL2 and SU-DHL-4 were selected, meanwhile, MV4-11 was
used as the negative control. Compound 9b demonstrated slightly
greater activity than other compounds against the three cell lines.
Based on the high inhibitory activities of enzyme and cells, 9b
was selected for subsequent studies to investigate the mechanism
of action. As shown in fig. 4, compound 9b bound to EZH2WT at
1 μM concentration SAM with an IC50 value of 22 nM. Western
blotting analysis was used to evaluate the ability of 9b to reduce
H3K27me3 in intact cells. Treatment with 9b at 5 μM for 6 days
potently abolished H3K27me3 mark in SU-DHL-4 cells in a
concentration-manner (Fig.5). Next, we performed flow
cytometry to understand the role of 9b in the inhibition of the
proliferation of lymphoma cells. When SU-DHL-4 cells were
exposed to 9b for 6 days, the proportion of early (Q3, only
Annexin V-positive) and late apoptotic cells (Q2, both Annexin
V- and PI-positive) remarkably increased from 36.81% to
73.42% and to 94.04% when the concentration increased from 1
In summary, preliminary optimization led to the discovery of
compound 9b with good inhibitory activities of enzyme and cells
which was selected for subsequent studies. The biological
evaluation indicated that 9b was a potent inhibitor for wild-type
EZH2. Moreover, 9b reduced the overall levels of H3K27me3 in
a
concentration-dependent manner without affecting the
expression of EZH2. Further study indicated that 9b could
significantly induce apoptosis of SU-DHL-4 cells. Therefore, this
study may contribute to the further design of novel scaffolds
against EZH2. However, several limitations need to be improved,
such as histone selectivity and inhibitory activity of mutant-type
EZH2. Further studies of the biological mechanisms and anti-
tumor research in vivo are in progress.

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9.
McCabe MT, Ott HM, Ganji G, Korenchuk S, et al. EZH2
inhibition as a therapeutic strategy for lymphoma with EZH2-
activating mutations. Nature. 2012, 492 (7427): 108-112.
Knutson SK, Warholic NM, Wigle TJ, Klaus CR. Durable tumor
regression in genetically altered malignant rhabdoid tumors by
inhibition of methyltransferase EZH2. Proc Natl Acad Sci U S A.
2013, 110 (19): 7922-7927.
Acknowledgments
This work was supported by Key research and development
program of Sichuan Provincial Science and Technology
Department, China (No. 2018SZ0007). The authors thank Shuhui
Xu of State Key Laboratory of Biotherapy (Sichuan University)
for acquiring NMR data
10. Zhang LD, Song XJ, Wang NY, et al. Design, synthesis and
biological evaluation of novel 1-methyl-3-oxo-2,3,5,6,7,8-
hexahydroisoquinolins as potential EZH2 inhibitors. RSC
Advances. 2015, 5: 25967-25978.
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Design and synthesis of (E) -1,2-diphenylethene-based EZH2 inhibitors.
Hualong He , Xi Hu , Fei Teng , Zhihao Liu , Qiangsheng Zhang , Zhanzhan Feng , Qiang Feng and Luoting
Yu.
NH
O
NH
O
N
N
O
9b
Compound
EZH2WT IC50 = 22 nM
WSU-DLCL2 IC50 = 1.61 M
SU-DHL-4 IC50 = 2.34 M
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