Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new bromodomain-containing protein 4 (BRD4) inhibitors

Article abstract of DOI:10.1016/j.cclet.2019.08.029

Targeting bromodomain-containing protein 4 (BRD4) has been proved to be an effective strategy for cancer therapy. To date, numerous BRD4 inhibitors and degraders have been identified, some of which have advanced into clinical trials. In this work, a focused library of new [1,2,4]triazolo[1,5-a]pyrimidine derivatives were discovered to be able to inhibit BRD4. WS-722 inactivated BRD4 (BD1/BD2), BRD2 (BD1/BD2) and BRD3 (BD1/BD2) broadly with the IC₅₀ values less than 5 μmol/L. Besides, WS-722 inhibited growth of THP-1 cells with an IC₅₀ value of 3.86 μmol/L. Like (+)-JQ1, WS-722 inhibited BRD4 in a reversible manner and enhanced protein stability. Docking studies showed that WS-722 occupied the central acetyl-lysine (Kac) binding cavity and formed a hydrogen bond with Asn140. In THP-1 cells, WS-722 showed target engagement to BRD4. Cellular effects of WS-722 on THP-1 cells were also examined, showing that WS-722 could block c-MYC expression, induce G0/G1 phase arrest and p21 up-regulation, and promote differentiation of THP-1 cells. BRD4 inhibition by WS-722 resulted in cell apoptosis and up-regulated expression of cleaved caspased-3/7 and PARP in THP-1 cell lines. The [1,2,4]triazolo[1,5-a]pyrimidine is a new template for the development of new BRD4 inhibitors.

Full text of DOI:10.1016/j.cclet.2019.08.029

G Model
CCLET 5180 No. of Pages 5
Chinese Chemical Letters xxx (2019) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Communication
Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new
bromodomain-containing protein 4 (BRD4) inhibitors
1
1
1
*
Shuai Wang , Dandan Shen , Lijie Zhao , Xiaohan Yuan, Jialing Cheng, Bin Yu ,
*
*
Yichao Zheng , Hongmin Liu
Key Laboratory of Technology of Drug Preparation, Ministry of Education of China & School of Pharmaceutical Sciences, Zhengzhou University,
Zhengzhou 450001, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Targeting bromodomain-containing protein 4 (BRD4) has been proved to be an effective strategy for
cancer therapy. To date, numerous BRD4 inhibitors and degraders have been identified, some of which
have advanced into clinical trials. In this work, a focused library of new [1,2,4]triazolo[1,5-a]pyrimidine
derivatives were discovered to be able to inhibit BRD4. WS-722 inactivated BRD4 (BD1/BD2), BRD2 (BD1/
Received 26 June 2019
Received in revised form 14 August 2019
Accepted 16 August 2019
Available online xxx
BD2) and BRD3 (BD1/BD2) broadly with the IC50 values less than 5
mmol/L. Besides, WS-722 inhibited
growth of THP-1 cells with an IC50 value of 3.86 mol/L. Like (+)-JQ1, WS-722 inhibited BRD4 in a
m
Keywords:
Epigenetic regulation
BRD4 inhibitor
[1,2,4]triazolo[1,5-a]pyrimidine
THP-1 cells
AML treatment
reversible manner and enhanced protein stability. Docking studies showed that WS-722 occupied the
central acetyl-lysine (Kac) binding cavity and formed a hydrogen bond with Asn140. In THP-1 cells, WS-
722 showed target engagement to BRD4. Cellular effects of WS-722 on THP-1 cells were also examined,
showing that WS-722 could block c-MYC expression, induce G0/G1 phase arrest and p21 up-regulation,
and promote differentiation of THP-1 cells. BRD4 inhibition by WS-722 resulted in cell apoptosis and up-
regulated expression of cleaved caspased-3/7 and PARP in THP-1 cell lines. The [1,2,4]triazolo[1,5-a]
pyrimidine is a new template for the development of new BRD4 inhibitors.
© 2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
As histone acetylation can be written by histone acetyltrans-
ferases (HATs) and erased by histone deacetylases (HDACs), the
reading process of histone acetylation marks is much less studied
until the discovery of potent and selective inhibitors of bromo and
extra-terminal (BET) domain proteins [1,2]. As a unique protein
domain, BET domain, a highly conserved domain composed of
about 110 amino acids, can recognize the histone acetylation
modification and mediate interaction of proteins. Proteins with
BET domain can be divided into nine protein families due to the
sequence similarity, of which BRD2, BRD3, BRD4 and BRDT are the
largest and most studied. All members of BET family have two
BRD active domains BD1 and BD2 and an extra-terminal (ET)
region, which bind to acetylated lysine located in H3 and H4 as
well as some other non-histone proteins [3,4]. Among the BET
family members, BRD4, abnormally expressed in many tumor
cancer cells, has been reported to be associated with various
diseases [5–10], which makes it an important epigenetic target
for disease treatment. To date, a large number of small-molecule
inhibitors and degraders targeting BET proteins have been
reported [11–19], some of which such as (+)-JQ1, OTX-015,
TEN-010, I-BET762, CPI-0610, I-BET151, PLX51107, ABBV-075,
AZD5153, BMS-986158 and INCB0543294 are currently undergo-
ing clinical assessment at different phases for cancer therapy
[20,21].
Our previous work has showed that the bicyclic triazole fused
pyrimidines have possessed interesting biological activities
[22–33]. Following our previous work, herein a focused library
of [1,2,4]triazolo[1,5-a]pyrimidine-based BRD4 inhibitors were
synthesized and evaluated for their inhibitory activity against
BRD4. Among these compounds, WS-722 effectively and reversibly
inhibited BRD4 (BD1) and BRD4 (BD2) with the IC50 values of 2.15
and 4.36 mmol/L, respectively. Cellular target engagement in THP-1
cells was confirmed by the thermal shift assay. Cellular effects were
also examined in THP-1 cells, indicating that WS-722 induced
differentiation of THP-1 cells, arrested cell cycle at G0/G1 phase
and resulted in obvious cell apoptosis. The results suggest that the
[1,2,4]triazolo[1,5-a]pyrimidine is a new starting point for the
development of BRD4 inhibitors.
* Corresponding authors.
E-mail addresses: yubin@zzu.edu.cn (B. Yu), yichaozheng@zzu.edu.cn (Y. Zheng)
, liuhm@zzu.edu.cn (H. Liu).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.cclet.2019.08.029
1001-8417/© 2019 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: S. Wang, et al., Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new bromodomain-containing
protein 4 (BRD4) inhibitors, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.08.029

G Model
CCLET 5180 No. of Pages 5
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Fig. 1. Modifications based on the score scaffold for SARs studies.
The general synthetic route of compounds D1ÀD29 is shown in
Scheme S1 (Supporting information). The synthesized compounds
are shown in Fig.1 with the core scaffold highlighted in red. Clearly,
modifications focused on the variations of substituents, generating
a focused library of [1,2,4]triazolo[1,5-a]pyrimidine derivatives.
Based on the established TR-FRET protocol (Fig. S1 in
Supporting information), we first screened the synthesized
compounds for their inhibitory activity against BRD4 (BD1) using
(+)-JQ1 as a positive control and the results are shown in Fig. S2
(Supporting information). The compounds showed varied inhibi-
tory activity against BRD4, indicating the importance of sub-
stituents for the activity. Particularly, nine of these compounds
(Fig. S2 in Supporting information) exhibited acceptable inhibitory
D12, introduction of the R group to the core scaffold did not
significantly improve the inhibitory activity. D24 inhibited BRD4
(BD1) and BRD4 (BD2) moderately with the IC50 values of 11.21 and
8.64
mmol/L, respectively.
In view of the acceptable potency of WS-722 toward BRD4
(Fig. 2A), we further evaluated its selectivity against BRD2 and
BRD3. As shown in Figs. 2B and C, WS-722 also moderately
inhibited BRD2 and BRD3, indicating that WS-722 is a pan-BRD
inhibitor in vitro. A dilution assay was then used to test the
reversibility using (+)-JQ1 as the control compound. We found that
100-fold dilution of the BRD4/WS-722 mixture resulted in the
recovery of BRD4 activity (Figs. 2D and E). Similarly, the BRD4
activity of (+)-JQ1 can also be recovered after dilution (Figs. 2D and
E). These results indicate that like (+)-JQ1, WS-722 reversibly
bound to BRD4 (BD1) and BRD4 (BD2) (Figs. 2D and E). The melting
temperature (Tm) is defined as the temperature at which half of the
protein is native and the other half denatured, and could be used to
describe the stability of a protein. The positive changes in Tm upon
ligand binding indicate thermal stabilization. To further evaluate
the binding property of WS-722 to BRD4, the protein thermal
activity against BRD4 (BD1) at 10 mmol/L with the inhibitory rates
over 39%. In view of their biochemical potency against BRD4 (BD1),
these nine compounds were then further evaluated against BRD4
(BD2), the IC50 values of these compounds were shown in Table 1.
Compound D7 (also named as WS-722) displayed the best potency
against BRD4 (BD1/BD2) with the IC50 values of 2.15 and
4.36
mmol/L, respectively. Compound D13 bearing the p-bromo-
benzyl group had comparable inhibitory activity with D7 against
BRD4. In contrast, compounds D1, D12, D14, D15, D18 and D19
showed decreased inhibitory activity against BRD4. Compared to
shift assay [34] was used to detect the
(BRD4 + 25 mol/L WS-722) value and the reference Tm
(BRD4 + DMSO) using the (+)-JQ1 as the control compound. As
indicated in Fig. 2F, for BRD4 (BD1) and BRD4 (BD2), the Tm
DTm between the Tm
m
D
increased upon WS-722 and (+)-JQ1 treatment. The results suggest
that (+)-JQ1 and WS-722 could enhance BRD4 protein stability.
Recent studies have shown that BRD4 has played key roles in
the maintenance of aberrant chromatin states in AML, acute
lymphoblastic leukemia (ALL), myeloma and lymphoma, and
treatment with BRD4 inhibitors could recapitulate anti-leukemic
effects in several AML cell lines [35–37]. Initially, the antiprolifer-
ative activity of WS-722 was evaluated against THP-1 cells. As
shown in Fig. 3A, after treatment for 7 days, WS-722 moderately
Table 1
Inhibitory activity of selected compounds against BRD4.
Compound
BRD4 (BD1) IC50
(m
mol/L)
BRD4 (BD2) IC50 (mmol/L)
D1
5.59 Æ 0.16
2.15 Æ 0.23
15.65 Æ 0.58
3.75 Æ 0.68
25.35 Æ 0.81
4.61 Æ 0.61
13.43 Æ 0.72
4.53 Æ 0.37
11.21 Æ 0.76
0.131 Æ 0.002
7.71 Æ 0.21
4.36 Æ 0.13
10.76 Æ 0.63
1.56 Æ 0.51
15.73 Æ 0.89
9.82 Æ 0.71
8.37 Æ 0.18
5.24 Æ 0.67
8.64 Æ 0.79
0.037 Æ 0.002
D7 (WS-722)
D12
D13
D14
D15
D18
D19
D24
(+)-JQ1
inhibited growth of THP-1 cells with an IC50 value of 3.86 mmol/L.
To confirm whether WS-722 could abrogate BRD4 activity in acute
leukemia cell lines, we used the cellular thermal shift assay to
study thermal stability of BRD4 upon WS-722 treatment in THP-1
Please cite this article in press as: S. Wang, et al., Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new bromodomain-containing
protein 4 (BRD4) inhibitors, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.08.029

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3
Fig. 2. Biochemical characterization of WS-722. (A, B, C) Inhibitory activity of WS-722 against BRD2/3/4 (BD1) and BRD2/3/4 (BD2); (D) Dilution assay of WS-722 to BRD4
(BD1) with TR-FRET assay, (+)-JQ1 was used as the positive control; (E) Dilution assay of WS-722 to BRD4 (BD2) with TR-FRET assay, (+)-JQ1 was used as the positive control;
(F) BRD4 (BD1) and BRD4 (BD2) recombinant protein thermal shift assays were performed when treated with WS-722 and (+)-JQ1. d(Fluorescence)/dTand
Data are the mean Æ SD. Each experiment was repeated three times. *** P < 0.001.
DTm was analyzed.
Fig. 3. WS-722 treatment decreases c-MYC and induces cell cycle in THP-1 cell line. (A) The effect of compound WS-722 on the viability of THP-1 cells for 7 days; (B)
Enhancement of thermal stability of BRD4 in THP-1 cells; (C) BRD4 and c-MYC expression in THP-1 cells when treated with WS- 722 for 24 h. GAPDH was used as the loading
control; (D) Expression of p21 when THP-1 cells were treated with WS-722 for 48 h. GAPDH was used as the loading control; (E) Effect of WS-722 on the cell cycle of THP-1
cells. * P < 0.05; ** P < 0.01; *** P < 0.001.
Please cite this article in press as: S. Wang, et al., Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new bromodomain-containing
protein 4 (BRD4) inhibitors, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.08.029

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Fig. 4. Effect of WS-722 on cell apoptosis and differentiation in THP-1 cell line. (A) THP-1 cell apoptosis when treated with WS-722 at doses from 0.3
mmol/L to 3.0 mmol/L for
48 h. (B) Cleaved caspase-3, caspase-7 and PARP expression when treated with WS-722 for 48 h. (C and D) Flow cytometry analysis of CD11b (C) and CD14 (D) in THP-1 cells
treated with WS-722. Data are the mean Æ SD. Each experiment was repeated three times. * P < 0.05; ** P < 0.01; *** P < 0.001.
cell line. THP-1 cells were treated with WS-722 and then heated for
3 min at 50 ꢀC. After freezing in liquid nitrogen and thawing on ice,
equal amounts of supernatant were removed and blotted with the
BRD4 antibody. Our results suggested that WS-722 stabilized BRD4
in a concentration-dependent manner, suggesting cellular target
engagement of WS-722 to BRD4 in THP-1 cells (Fig. 3B). It has been
reported that BET-bromodomain inhibition potently suppresses
MYC gene expression in leukemia by blocking its transcription
[38]. Hence, to further explore the biological role of our BRD4
inhibitor WS-722 in THP-1 cells, THP-1 cells were treated with
WS-722 and then subjected to western blotting analysis. As shown
in Fig. 3C, treatment with WS-722 dose-dependently decreased
expression of c-MYC, without impact on BRD4 expression (Fig. 3C).
To examine higher-order influences over biological networks
regulated by c-MYC, which can influence cell cycle and p21 (also
known as cyclin-dependent kinase inhibitor 1 or CDK-interacting
protein 1) expression [39]. Expression of cell cycle regulatory
protein p21 was also examined after treatment with WS-722 for
48 h. As shown in Fig. 3D, WS-722 treatment concentration-
dependently induced accumulation of p21 in THP-1 cells. p21 is an
universal cell-cycle inhibitor directly controlled by p53 and p53-
independent pathways [40,41]. Then, the cell cycle of THP-1 cells
was analyzed with flow cytometry after treatment with WS-722.
As shown in Fig. 3E, WS-722 arrested cell cycle of THP-1 cells at
G0/G1 phase in a concentration-dependent manner, which was
consistent with previous reports [42–44]. All these results
confirmed that pharmacological inhibition of BRD4 could
block c-MYC expression and then induce G0/G1 phase arrest
and p21 up-regulation.
To study the effect of WS-722 on cell apoptosis, THP-1 cells
were treated with WS-722 at doses from 0.3
for 48 h. We found WS-722 induced apoptosis at high concentra-
tion (3.0 mol/L) as detected by annexin V staining and PI uptake
mmol/L to 10 mmol/L
m
(Fig. 4A). Then samples were taken for the induction of apoptosis
by measuring caspase-3, caspase-7 and PARP activity. As shown in
Fig. 4B, after treatment of THP-1 cells with WS-722 for 48 h,
cleaved caspase 3, caspase 7 and PARP increased in a concentra-
tion-dependent manner. It has been reported that MYC (a family of
regulator genes and proto-oncogenes that code for transcription
factors) arrests the differentiation of embryonic stem cells and
various neoplasms [45]. So, we assessed whether WS-722 could
promote differentiation of THP-1 cells by quantifying the expres-
sion of two differentiation related markers: CD14 and CD11b. As
shown in Figs. 4C and D, WS-722 induced expression of CD11b and
CD14 in THP-1 cells, which promoted differentiation of leukemia
cells dose-dependently. To conclude, these results suggest that
WS-722 down-regulated expression of c-MYC, induced cell cycle
arrest, and promoted differentiation of THP-1 cells.
Please cite this article in press as: S. Wang, et al., Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new bromodomain-containing
protein 4 (BRD4) inhibitors, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.08.029

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S. Wang et al. / Chinese Chemical Letters xxx (2019) xxx–xxx
5
(+)-JQ1 features a novel thieno-triazolo-1,4-diazepine scaffold
and is a well characterized cell permeable BET family inhibitor.
(+)-JQ1 strongly blocks binding of a tetra-acetylated histone H4
peptide to BRD4 and potently inhibits the first and second
bromodomains (BD1 and BD2) of BRD4 with IC50 values of 77 and
33 nmol/L, respectively [46]. To explain the observed potency of
compound WS-722 against BRD4, an in silico docking simulation
was carried out using the MOE 2015.10 software package. The
crystal structure of the first bromodomain (BD1) of human BRD4 in
complex with (+)-JQ1 (PDB code: 3MXF) was therefore used as a
docking receptor for computational studies. Similar to interactions
observed in acetyl-lysine (Kac) complexes [47], (+)-JQ1 (Fig. S3A in
Supporting information) was fitted well into the hydrophobic Kac
binding site with an extraordinary shape complementarity [46]. As
revealed by the co-crystal structure of BRD4 (BD1)/(+)-JQ1
complex (Fig. S3C in Supporting information), the triazole ring
of (+)-JQ1 formed a hydrogen bond with surrounding residue
Asn140 in BRD4 (BD1), and the conserved BET residues stabilized
the binding of (+)-JQ1 through hydrophobic interactions. As
depicted in Figs. S3A and S3B (Supporting information), like
(+)-JQ1, WS-722 occupied the central acetyl-lysine (Kac) cavity and
formed a hydrogen bond with the key residue Asn140 as well
(Fig. S3D in Supporting information). The binding models may be
responsible for the observed potency of WS-722 against BRD4
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Nos. 81703326, 81773562, 81602961 and
81430085), Scientific Program of Henan Province (No.
182102310123), China Postdoctoral Science Foundation (Nos.
2018M630840 and 2019T120641).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in
the online version, at doi:https://doi.org/10.1016/j.cclet.2019.
08.029.
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WS-722 inhibited growth of THP-1 cells with an IC50 value of
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Please cite this article in press as: S. Wang, et al., Discovery of [1,2,4]triazolo[1,5-a]pyrimidine derivatives as new bromodomain-containing
protein 4 (BRD4) inhibitors, Chin. Chem. Lett. (2019), https://doi.org/10.1016/j.cclet.2019.08.029