Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and pyridone derivatives as BRD4 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2019.07.036

Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal (BET) family, has been recognized as an attractive candidate target for the treatment targeting gene transcription in several types of cancers. In this study, two types of novel compounds were designed, synthesized and evaluated as BRD4 inhibitors. Therein, pyridone derivatives were more effective against BRD4 protein and human leukemia cell lines MV4-11. Among them, compounds 11d, 11e and 11f were the most potential ones with IC₅₀ values of 0.55 μM, 0.86 μM and 0.80 μM against BRD4, and exhibited remarkable antiproliferative activities against MV4-11 cells with IC₅₀ values of 0.19 μM, 0.32 μM and 0.12 μM, respectively. Moreover, in western blot assay, compound 11e induced down-regulation of C-Myc, which is a significant downstream gene of BRD4. Cell cycle analysis assay also showed that compound 11e could block MV4-11 cells at G0/G1 phase. Taken together, our results suggested that compound 11e and its derivatives were a class of novel structural potential BRD4 inhibitors and could serve as lead compounds for further exploration.

Full text of DOI:10.1016/j.bmcl.2019.07.036

Accepted Manuscript
Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and pyri-
done derivatives as BRD4 inhibitors
Juan Rong, Zhan-Zhan Feng, Yao-Jie Shi, Jing Ren, Ying Xu, Ning-Yu Wang,
Qiang Xue, Kun-Lin Liu, Shu-Yan Zhou, Wei Wei, Luo-Ting Yu
PII:
S0960-894X(19)30492-5
https://doi.org/10.1016/j.bmcl.2019.07.036
BMCL 26577
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 April 2019
5 July 2019
22 July 2019
Please cite this article as: Rong, J., Feng, Z-Z., Shi, Y-J., Ren, J., Xu, Y., Wang, N-Y., Xue, Q., Liu, K-L., Zhou,
S-Y., Wei, W., Yu, L-T., Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and pyridone
derivatives as BRD4 inhibitors, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/
j.bmcl.2019.07.036
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Graphical Abstract
Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and pyridone
derivatives as BRD4 inhibitors
Juan Rong a, e, Zhan-Zhan Feng a, e, Yao-Jie Shi a, Jing Ren a, Ying Xu a, Ning-Yu Wang a, Qiang Xue a,
Kun-Lin Liu a, Shu-Yan Zhou a, Wei Wei a, Luo-Ting Yu a, 
a Department of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China.
Juan Rong
H
N
N
F
N
O
N
F
11e
Compound
IC50(BRD4)=860nM
IC50(MV-411)=320nM
———
 Corresponding author. Tel.: +86 28 8516 4063; fax: +86 28 8516 4060; e-mail: yuluot@scu.edu.cn
e These authors contributed equally to this work.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and
pyridone derivatives as BRD4 inhibitors
Juan Rong a, e , Zhan-Zhan Feng a, e, Yao-Jie Shi a, Jing Ren a, Ying Xu a, Ning-Yu Wang a, Qiang Xue a,
Kun-Lin Liu a, Shu-Yan Zhou a, Wei Wei a, Luo-Ting Yu a, 
a Department of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China.
———
ARTICLE INFO
ABSTRACT
 Corresponding author. Tel.: +86 28 8516 4063; fax: +86 28 8516 4060; e-mail: yuluot@scu.edu.cn
e These authors contributed equally to this work.
Article history:
Received
Bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal
(BET) family, has been recognized as an attractive candidate target for the treatment targeting
gene transcription in several types of cancers. In this study, two types of novel compounds were
designed, synthesized and evaluated as BRD4 inhibitors. Therein, pyridone derivatives were
more effective against BRD4 protein and human leukemia cell lines MV4-11. Among them,
compounds 11d, 11e and 11f were the most potential ones with IC50 values of 0.55 μM, 0.86 μM
and 0.80 μM against BRD4, and exhibited remarkable antiproliferative activities against MV4-
11 cells with IC50 values of 0.19 μM, 0.32 μM and 0.12 μM, respectively. Moreover, in western
blot assay, compound 11e induced down-regulation of C-Myc, which is a significant
downstream gene of BRD4. Cell cycle analysis assay also showed that compound 11e could
block MV4-11 cells at G0/G1 phase. Taken together, our results suggested that compound 11e
and its derivatives were a class of novel structural potential BRD4 inhibitors and could serve as
lead compounds for further exploration.
Revised
Accepted
Available online
Keywords:
BRD4 inhibitors
3,5-dimethylisoxazole derivatives
pyridone derivatives
2019 Elsevier Ltd. All rights reserved.
Lysine acetylation in histones is one of the most important epigenetic post-translational modifications,1 the disorder of which leads
to aberrant transcription, promoting the occurrence of cancers and other diseases. Bromodomains (BRDs) protein can recognize the
specific acetylated lysine (KAc) residues, and then mediate signaling transduction to regulate specific gene expression.2,3 Bromodomain
and extra terminal (BET) family (BRD2, BRD3, BRD4 and BRDT), is most extensively investigated. BRD4 is a well-studied member
of the BET family, it plays an essential role in gene epigenetics by binding KAc on chromatin structures as the ‘readers’ of lysine
acetylation state. BRD4 can recruit P-TEFb, one positive transcriptional elongation factor complex and essential in the regulation of
transcription by RNA polymerase II in eukaryotes.4 In addition, BRD4 was suggested to stimulate G1 gene transcription and promote
cell cycle progression to S phase.5 And it was able to regulate the expression of specific cancer-related genes, for instance, C-Myc and
Bcl-2,6,7 which are key regulators for the proliferation of tumor cells. Therefore, the inhibition against BRD4 can down-regulate the
expression of corresponding oncogenes. Furthermore, it was reported that BRD4 played a key roles in the proliferation of many types
of cancers, such as activated B-cell-like subtype of diffuse large B-cell lymphoma, lung adenocarcinoma.8 Therefore, BRD4 represents
a novel therapeutic target for antineoplastic drug development. BRD4 consists of two N-terminal tandem bromodomain (BD1 and
BD2) and an extra-terminal recruitment domain (ET). Each bromodomain comprises a four-helix bundle (helices αZ, αA, αB, and αC)
and two loop regions (ZA and BC loops).9 Helices αZ and αA, helices αB and αC form two hydrophobic loop, called ZA Loop and BC
Loop. Then ZA Loop, BC Loop and αZ together constitute a hydrophobic structure area, which is “ WPF shelf ”. 10,11 The entire spatial
structure is called KAc binding site.
At present, a mass of small molecule BET bromodomain inhibitors undergoing clinical trials with encouraging emerging data.12
Particularly, multiple BRD4 inhibitors with different scaffolds have been reported, and studied in clinical trials for cancer
treatment.13,14,15 As shown in Fig.1. (+)-JQ1(Compound X1), with a triazolothienodiazepine scaffold, was the first reported potent BRD4
inhibitor with an IC50 value of 77 nM in the Alpha-Screen assay,16 and was widely used to study the physiology function of BET
proteins in cancer.17 I-BET151 (Fig.1, compound X2) was the earliest BRD4 inhibitor bearing with 3,5-dimethylisoxazole scaffold
discovered by GSK, and expressed well efficacy in vivo against MLL-fusion leukemia.18 ABBV-075 (Fig.1, compound X3), another
potent BET family bromodomain inhibitor, was proved to have anti-proliferative activity in multiple cancer cell lines.19 Meanwhile,
RVX-208 (Fig.1, compound X4), as a specific BET bromodomain inhibitor for BD2, was under phase-Ⅱ clinical trial for the treatment
of coronary syndrome and atheros-clerosis.20 I-BET762 (Fig.1, compound X5), also recognized as a selective BET inhibitor, had entered
phase II clinical trial.21,22 Besides, PFI-1 (compound X6) was another BRD4 inhibitor with IC50 of 220nM.23

Cl
N
O
O
O
N
N
S
O
N
N
N
O
NH
N
N
X2: I-BET151
IC50=790nM
X1: JQ1
IC50=33nM
O
H
N
H
N
N
O
O
O
owing different
acetylated lysine
analogue fragment
F
S
N
O
N
O
O
O
H
S
X6: PFI-1
IC50=220nM
N
F
H
X3: ABBV-075
Ki≈2.2nM
Cl
O
O
OH
HO
HN
N
O
N
NH
O
O
X4: RVX-208
IC50=510nM
X5: I-BET726
IC50=22nM
O
Figure 1. The structures of reported BRD4 inhibitors (Compound X1-X6)
Although many reported effective inhibitors have made great progress, the pace of exploration will not stop. Novel inhibitors with
therapeutic potential still need to be discovered to provide more pharmacological tools in the investigation of BRD4’s functions and
selection of optimal structure.
I-BET151 and many other inhibitors contained a 3,5-dimethylisoxazole fragment, as an acetyl-lysine binding mimic,24 and the
Hewings team also verified that 3,5-dimethylisoxazole was an efficient acetylated lysine analogue fragment.25 Some BRD4 inhibitors
containing 3,5-dimethylisoxazole were listed in Fig.2A (left), and the IC50 of which were all at a low level. Fig.2A (right) showed that,
3,5-dimethylisoxazole fragment of compound Y4 interacted with KAc binding site in BRD4 protein. According to above-mentioned, we
retained 3,5-dimethylisoxazole fragment in the first structure.
O
NH
A
N
N
OH
N
N
O
NH2
O
N
O
Y2
N
O
Y1 I-BET151
BRD4(1) IC50=790nM
BRD4(1) IC50<50nM
HN N
O
N
O
N
H
N
O
N
H
O
N
HN
N
N
Y3
Y4
BRD4(1) IC50=12.9nM
BRD4(2) IC50=20.2nM
B
O
H
O
H
N
N
N
N
F
O
O
S
O
N
Y6
O
N
F
H
Y5 ABBV-075
BRD4(1)IC50=2
BRD4(1)IC50=400nM
nM
±0.5
Figure 2. (A) The structures of reported BRD4 inhibitors containing 3,5-dimethylisoxazole fragment, BRD4 (BD2) is shown to interact with Compound Y4.
(carbon atom in gray, oxygen in red, nitrogen in blue, sulfur in gold; BRD4 is present in a gray cartoon)26; (B) The structures of reported
BRD4 inhibitors containing 1-methylpyridin-2(1H)-one fragment, Inhibitor Y6 bound to BRD4 (BD1). 27
At the same time, we found that pyridone substructure was widely used in designs of BRD4 inhibitors (Fig.2B). The oxygen and
nitrogen atoms in pyridone fragment of compound Y6 bound to aspartic acid residue in KAc site as shown in Fig.2B (right). So we
decided to introduce 1-methyl-3-(methylamino)pyridin-2(1H)-one fragment, as acetyl-lysine binding mimic in the second structure.
In the design of the first type (Fig.3A), we retained the acetylated lysine analogue fragment, 3,5-dimethylisoxazole, and replaced
scaffold with an indazole according to scaffold hopping, one side of the chain was jointed with a substituted benzene ring by an N-H
bridge to increased its hydrophilicity and was expected to reach the ZA channel of KAc in BRD4. In the design of the second type, the
pyridone fragment was reserved, pyrrol ring in pyridone substructure was transformed into aliphatic hydrocarbon, N-hydrogen was still
reserved because of its function to form additional hydrogen bonds (Fig.3B).

A series of compounds containing 3,5-dimethylisoxazole fragment were synthesized, and the main steps were exhibited in Scheme 1.
All synthesized compounds were evaluated inhibitory activities against BRD4 protein and human leukemia cell line MV4-11.28 (Table
1)
Firstly, comparing 5a-5m with 5n-5p, when 3,5-dimethylisoxazole was in R1 position of the indazole ring , the activity potency was
relatively superior. A reasonable explanation was that R1 position was essential for the binding to KAc recognition site. Removing R4
substituent group from the indazole ring caused the reducing of inhibitory activity with 3~4 fold (compare 5a with 5b and 5m),
indicating that R4 substitution was important for inhibitory activity. Then, we fixed R4 position with a tetrahydropyrane ring,
compounds 5c-5f were prepared to explore the effects of the type and position of the substituent at side chain phenyl group in R3
position, the results indicated that poly-substitution or mono-substitution make no contribution to activity.
When tetrahydropyran ring in R4 position was replaced with benzene ring, there was no significant change for inhibitory activity.
Overall, substituents in R1 and R4 position were necessary for inhibitory activity. Noticeably, when 5-position of the indazole ring was
occupied by a methoxy group, compound 5m with 3,5-dimethylisoxanthine at the 6-position of indazole gained multiplied inhibitory
activity, which could be inferred that methoxy group was likely to facilitate the formation of hydrogen
bonds between the acetylated lysine residue mimic and BRD4 protein.
As shown in table 2, when the core scaffold was replaced with a pyrimidine ring, the inhibition rate of those compounds at 1μM was
less than 20%, so the activity of this type was not improved.
In summary, isoxazole derivatives (5a-5r) all showed poor activity both in protein and cell levels. Therefore, we gave up the further
research on this kind of structure but focused on the second type of structure, pyridone derivatives.
BRD4(1) IC50=37nM
H
N
BRD4(1) IC50=12.9nM
BRD4(1)IC50=400nM
BRD4(1) IC50=69nM
A
B
N
S
O
O
HN N
O
H
N
O
F
H
O
N
N
O
O
N
N
F
N
HN
N
H
R3'
or
N
R7'
HN
O
N
O
O
H
N
R5'
N
HN
N
R8
N
O
N
O
N
O
O
N
O
N
N
N
N
N
R6
R4
N
N
N
N
N
HN
O
O
O
O
O
N
NH
HN
BRD4(1) IC50=37nM
N
N
BRD4(1) IC50=790nM
Figure 3. (A) Design of 3,5-dimethylisoxazole derivatives; (B) Design of pyridone derivatives.29
I
I
N
N
Br
N
NH
N
NH
Br
Br
ii
R4
i
1
3
2
B(OH)2
R3
R3
O
N
O
N
N
N
N
Br
N
iv
iii
R4
R4
5
4
Scheme 1. Synthesis of 3,5-dimethylisoxazole derivatives. (i) I2, KOH, DMF; rt, 1~2h. 85%. (ii) 3,4-Dihydropyran or Benzyl bromide, p-toluenesulfonic
acid, DCM, rt, overnight. 75%. (iii) Pd(AcO)2, Xantphos, dry DMF, phenylamine, N2, 60℃, 6~7h. 40%. (iv) 3,5-dimethylisoxanthraquinone, Na2CO3,
dioxane and water, Pd(dppf)Cl2, N2, 90℃, 6~9h. 50%.

O
O
O
B
B
N
O
O
N
N
N
B
R8'
NH
N
Br
Br
O
v
R8'
vi
R7'
HN
8
N
6
7
N
O
N
viii
R7'
R8'
H2N
O
Br
HN
Br
11
N
vii
O
N
9
10
Scheme 2. Synthesis of pyridone derivatives. (v) Benzyl bromide or benzyl chloride, p-toluenesulfonic acid, DCM, rt, overnight. 75%. (vi) Potassium
acetate, Pd(dppf)Cl2, Bis(pinacolato)diboron, dioxane, N2, 95℃, 12h, 50%. (vii) HCHO or CH3CHO, methanol, acetic acid, DCM, rt, 0.5h, 65%. (viii)
Na2CO3, dioxane and water, Pd(dppf)Cl2, N2 , 90℃, 6~7h. 50%.
Table 1. Structure-activity relationship analysis of 3,5-dimethylisoxazole derivatives.
R3
R2
N
R1
N
R4

R1
R2
H
R3
R4
H
MV4-11,IC50 (µM)b
Compound
aInhibition at 1μM
F
O
N
N
H
5a
＜20%
7.80
2.80
33%
O
N
O
N
5b
5c
5d
5e
5f
H
H
H
Cl
O
O
＜20%
＜20%
＜20%
＜20%
9.30
9.90
8.50
7.40
O
N
F
F
N
H
O
O
O
O
N
N
H
H
F
O
O
N
H
H
N
H
Cl
O
N
N
H
F
＜20%
＜20%
ND
O
N
5g
5h
N
H
H
H
F3C
-10.30
O
N
N
H
NC
＜20%
5.80
O
N
N
5i
H
H
＜20%
＜20%
＜20%
45%
11.90
9.10
8.10
1.90
ND
O
F
O
N
5j
H
H
H
N
H
O
O
N
N
H
5k
5l
O
F
O
N
NH
O
N
5m
H
O
F
O
O
＜20%
O
N
N
H
5n
5o
H
H
＜20%
ND
17.50
ND
F
F
O
O
N
N
H
H
5p
O
O
O
N
N
H
JQ1
0.09
0.06
a represenTt inhibitory rate against BRD4 protein at 1μM; b Values are the average of three distinct runs.
ND represent the inhibitory activities are not performed.
a
b
le 2.

O
R5
N
N
N
N
R6
aInhibition at
MV4-11
IC50 (µM)
ND
Compound
R5
R6
1 μM
O
5q
＜20%
＜20%
F
OH
5r
ND
O
a represent inhibitory rate against BRD4 protein at 1μM; ND represent the inhibitory activities are not performed.
Then, thirteen compounds with 1-methyl-3-(methylamino) pyridin-2(1H)-one fragment were designed and synthesized (Fig.3B,
Scheme 2). Obviously, the inhibitory activity of pyridone derivatives were markedly improved compared with 3,5-dimethylisoxazole
derivatives.
As shown in Table 3. Firstly, to investigate the impact of 1-methyl-3-(methylamino)pyridin-2(1H)-one substructure, we replaced N-
methyl in amidogen of pyridine ring with N-ethyl, leading to 2~10 fold reduction in potency (compare 11i, 11j to 11a, 11d). It was
possible that N-methyl was necessary to anchor into the Kac binding pocket of BRD4. Then, we investigated the impact of R8 position.
When R8 position was mono-substitution phenyl, there was no
BRD4 (BD1,2)
MV4-11
significant difference among compounds 11a-11c with IC50 values
of 1.4~1.6 μM, indicating that electron-withdrawing group or
electron-donating group had no effect on the activity. when R8
position was disubstituted (3,5-dimethoxybenzyl, 3,4-difluoro-
benzyl and 4-fluoro-3-nitrobenzyl), compounds (11d, 11e and 11f)
showed leap-type improvement of inhibitory activity, with IC50 of
0.55, 0.86 and 0.8 μM against BRD4 protein., suggesting R8
preferred to disubstituted benzene and m-substitution. Finally, when
the substituents in R8 position were cyclohexyl, aliphatic chain
hydrocarbon and cyclopropyl, the inhibition rates of compounds
11k, 11l and 11m decreased slightly, so the position of R8 preferred
rigid flat phenyl.
Then, multiple tumor cell lines were utilized to further validate
the cellular proliferation inhibition effects of pyridone derivatives.
Compounds 11d, 11e and 11f exhibited reasonable anti-proliferative
activity, especially to cell line MV4-11 with IC50 value of 0.19, 0.32
and 0.13 μM, respectively (Table 4 and fig.5A), illustrating that
activity of pyridone derivatives on BRD4-sensitive cells(MV4-11)
was more significant than BRD4-independent cells (HCT-116,
SW480, DU145, Capan-1, BT474).
Compound
R8
R7
IC50(µM)a
1.40
IC50 (µM)b
2.39
NH
N
F
11a
O
O
O
NH
N
11b
11c
11d
1.60
1.60
0.55
2.70
3.28
0.20
O
NH
N
NH
N
O
O
O
NH
N
11e
11f
0.86
0.80
0..32
0.13
F
F
O
O
F
NH
N
O2N
Table 4. cellular proliferation inhibition effects of compound
11d, 11e and 11f in multiple tumor cell lines
NH
N
11g
11h
11i
2.40
1.40
2.25
4.37
4.98
5.65
F3C
O
O
b Values are the average of three distinct runs.
Table 3. Structure-activity relationship analysis of pyridone
derivatives.
NH
N
O
F
N
F
R7
N
HN
R8
O
N
Then we selected compound 11e, which was least cytotoxic to
human normal hepatocytes LO2 (Fig.4B), with relatively higher
O
O
11j
1.81
1.81
2.59
HN
O
Cpd
Tumor cell inhibitory activity (IC50/NµM)b
NH
11k
2.69
MV4-11
0.19
HCT116
2.90
SW480
7.40
DU145
Capan-1
BT474
O
N
11d
2.60
3.80
8.60
8.77
a AlphaScreen assay.
NH
O
11l
1.90
1.96
0.09
b Values are tOhe average of three distinct runs.
O
N
11e
11f
0.32
3.00
8.40
4.80
3.70
4.00
0.80
8.80
NH
11m
3.45
6.50
O
0.13
2.60
10.10
14.10
3.40
N
JQ1
0.06
1.70
JQ1
0.09
0.40
0.20

activity against cancer cell lines, to further evaluate its biological properties.
The anti-proliferative mechanism of 11e was investigated in MV4-11 cells , and C-Myc was selected to determine the inhibition of
11e against the transcription of BRD4 downstream genes. As shown in Fig.5, the expression of C-Myc was measured by western blot.
Results showed that the expression of C-Myc were decreased in a dose-dependent manner, indicating that compound 11e could affect
the transcription and expression of some downstream target genes.
H
N
H
N
N
N
H
N
N
O
F
N
N
NO2
F
N
A
O
N
O
N
F
O
N
O
11d
11e
11f
Compound 11d
Compound 11e
Compound 11f
B
Compound 11d
Compound 11e
Compound 11f
Figure 4. (A) Inhibition to MV4-11 cells by compounds 11d-11f. (B) The effects of 11d-11f on the normal human liver cell line LO2. MTT assays were used
after the treatment of drugs for 72h.
Figure 5. The expression of BRD4 and C-Myc was determined by Western blot assay. MV4-11 cells were treated with DMSO or the indicated concentrations of
compound 11e (0.1, 0.5 and 2.5 μM) for 48 hours and the expression of BRD4 and C-Myc were detected with the specific antibody. The expression of β-actin
was used as the internal control.
B
A
Figure 6. (A) MV4-11 cells were treated with the indicated concentrations of compound 11e (0.1, 0.5 and 2.5 μM) for 48 hours. The distribution of cell cycle
was analyzed by flow cytometry. (B) The quantification of cell cycle phase is shown in right panel (*P<0.05), the treatment group and the control group were
compared by t-test.

In the cell cycle arrest assay, MV4-11 cells were treated with different concentrations of compound 11e. As shown in Fig.6A and
6B, flow cytometry analysis results indicated that the number of cells in the G0/G1 phase obviously increased in a dose dependent
manner, showing that G0/G1 phase arrest might be associated with the effects of compound 11e in MV4-11 cells.
In this study, a new series of BRD4 inhibitors have been designed and synthesized through fragment-based strategy. Firstly, we
designed two kinds of new structure, isoxazole derivatives on the basis of reported BRD4 inhibitors owing 3,5-dimethylisoxazole
fragment, and pyridone derivatives based on pyridone substructure. Unfortunately, isoxazole derivatives showed an inferior inhibitory
efficacy against BRD4 protein and leukemia cell MV4-11. By contrast, pyridone derivatives with 1-methyl-3-(methylamino)pyridin-
2(1H)-one fragment could inhibit BRD4 protein with relatively higher efficiency, and exhibited remarkable anti-proliferative activities
toward MV4-11 cell at the same time. Structure-activity relationship study was performed and resulted in substantial improvement of
the inhibitory potency against BRD4 protein. Among them, compounds 11d, 11e and 11f were the most potent inhibitors against BRD4
with IC50 values of 0.55 μM, 0.86 μM and 0.80 μM, respectively. And compound 11e exhibited effective and selective inhibitory
activities in MV4-11 cell with IC50 value of 0.32 μM, and relatively lower toxicity to human hepatocytes LO2. In addition, 11e was also
verified to block cell cycle at G0/G1 phase. In the western blot assay, compound 11e could decrease the protein expressions of C-Myc
in MV4-11 cell. Further evaluation of these pyridone derivatives as BRD4 inhibitors and deeper SAR exploration will be completed in
due course to seek optimal inhibitor. In brief, these results indicated that this type of structure owing novel scaffold was promising to
become superior lead compounds, to explore potent BRD4 inhibitor as drug candidate in the therapy of BRD4 protein-related cancers.
Acknowledgments
This work was supported by Sichuan Provincial Science and technology program forꢀKey research and development, China (No.
2018SZ0007) and the National S&T Major Special Project on Major New Drug Innovations (2018ZX09201018). We thank Shu-Hui
Xu of State Key Laboratory of Biotherapy (Sichuan University) for NMR measurements.
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Graphical Abstract
Design, synthesis and biological evaluation of 3,5-dimethylisoxazole and pyridone
derivatives as BRD4 inhibitors
Juan Rong a, e, Zhan-Zhan Feng a, e, Yao-Jie Shi a, Jing Ren a, Ying Xu a, Ning-Yu Wang a, Qiang Xue a,
Kun-Lin Liu a, Shu-Yan Zhou a, Wei Wei a, Luo-Ting Yu a, 
a Department of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China.
———
 Corresponding author. Tel.: +86 28 8516 4063; fax: +86 28 8516 4060; e-mail: yuluot@scu.edu.cn
e These authors contributed equally to this work.
Juan Rong
H
N
N
F
N
O
N
F
11e
Compound
IC50(BRD4)=860nM
IC50(MV-411)=320nM