Structure-Based Discovery and Optimization of Benzo [d] isoxazole Derivatives as Potent and Selective BET Inhibitors for Potential Treatment of Castration-Resistant Prostate Cancer (CRPC)

Article abstract of DOI:10.1021/acs.jmedchem.8b00103

The bromodomain and extra-terminal (BET) family proteins have gained increasing interest as drug targets for treatment of castration-resistant prostate cancer (CRPC). Here, we describe the design, optimization, and evaluation of benzo[d]isoxazole-containing compounds as potent BET bromodomain inhibitors. Cocrystal structures of the representative inhibitors in complex with BRD4(1) provided solid structural basis for compound optimization. The two most potent compounds, 6i (Y06036) and 7m (Y06137), bound to the BRD4(1) bromodomain with K_d values of 82 and 81 nM, respectively. They also exhibited high selectivity over other non-BET subfamily members. The compounds potently inhibited cell growth, colony formation, and the expression of AR, AR regulated genes, and MYC in prostate cancer cell lines. Compounds 6i and 7m also demonstrated therapeutic effects in a C4-2B CRPC xenograft tumor model in mice. These potent and selective BET inhibitors represent a new class of compounds for the development of potential therapeutics against CRPC.

Full text of DOI:10.1021/acs.jmedchem.8b00103

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Structure-Based Discovery and Optimization of Benzo[d]isoxazole
Derivatives as Potent and Selective BET Inhibitors for Potential
Treatment of Castration-Resistant Prostate Cancer (CRPC)
Maofeng Zhang, Yan Zhang, Ming Song, Xiaoqian Xue, Junjian Wang, Chao Wang,
Cheng Zhang, Chenchang Li, Qiuping Xiang, Lingjiao Zou, Xishan Wu, Chun Wu,
Baijun Dong, Wei Xue, Yulai Zhou, Hongwu Chen, Donghai Wu, Ke Ding, and Yong Xu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00103 • Publication Date (Web): 22 Mar 2018
Downloaded from http://pubs.acs.org on March 23, 2018
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Structure-Based Discovery and Optimization of Benzo[d]isoxazole Derivatives as Potent and
Selective BET Inhibitors for Potential Treatment of Castration-Resistant Prostate Cancer (CRPC)
Maofeng Zhang,†,‡,# Yan Zhang,†,‡,# Ming Song, Xiaoqian Xue, Junjian Wang, Chao Wang,
†,#
†,‡
¶
†
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Cheng Zhang,†,§ Chenchang Li,†,§ Qiuping Xiang,†,‡ Lingjiao Zou,†,‡ Xishan Wu,†,‡ Chun Wu,†
‖
‖
§
¶
†
†,*
∇
Baijun Dong, Wei Xue, Yulai Zhou, Hongwu Chen, Donghai Wu, Ke Ding, and Yong Xu
†Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences,
Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou
510530; Guangzhou Medical University, Guangzhou 511436, China.
‡University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China.
§School of Pharmaceutical Sciences, Jilin University, No. 1266 Fujin Road, Chaoyang District,
Changchun, Jilin 130021, China.
¶Department of Biochemistry and Molecular Medicine, School of Medicine, University of
California, Davis, Sacramento, California 95817, USA.
‖Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University,
Shanghai 200127, China.
∇School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.
*Corresponding Author: Yong Xu, PhD, E-mail: xu_yong@gibh.ac.cn
#These authors contributed equally to this work.
KEYWORDS: BRD, Bromodomain, Benzo[d]isoxazole, Prostate Cancer, CRPC
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ABSTRACT
The bromodomain and extra-terminal (BET) family proteins have gained increasing interest as
drug targets for treatment of castration-resistant prostate cancer (CRPC). Here, we describe the
design, optimization and evaluation of benzo[d]isoxazole-containing compounds as potent BET
bromodomain inhibitors. Co-crystal structures of the representative inhibitors in complex with
BRD4(1) provided solid structural basis for compound optimization. The two most potent
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compounds, 6i (Y06036) and 7m (Y06137), bound to the BRD4(1) bromodomain with K values
d
of 82 and 81 nM, respectively. They also exhibited high selectivity over other non-BET
subfamily members. The compounds potently inhibited cell growth, colony formation, and the
expression of AR, AR regulated genes, and MYC in prostate cancer cell lines. Compounds 6i
and 7m also demonstrated therapeutic effects in a C4-2B CRPC xenograft tumor model in mice.
These potent and selective BET inhibitors represent a new class of compounds for the
development of potential therapeutics against CRPC.
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1
. INTRODUCTION
Prostate cancer (PCa) is the most common malignancy and the fifth leading cause of death from
malignancy in men.1-3 Since androgen receptor (AR) signaling has a pivotal role in PCa,
androgen deprivation therapy (ADT) with surgical or chemical castration is the standard
treatment with most patients going to remission.4-6 However, the disease progresses to an
incurable stage known as castration-resistant prostate cancer (CRPC). Most patients initially
benefit from and eventually develop resistance to second-generation therapies such as
enzalutamide (Enz) and abiraterone (Abi).7-9 Therefore, the development of new strategies to
tackle AR signaling in CRPC is urgently needed.
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Figure 1. Structures of representative BET bromodomain inhibitors including (+)-JQ1 (1), I-
BET762 (2), I-BET151 (3), and Y02224 (4). The IC50 values were obtained using different
methods and are given in italics.
Bromodomain and extra terminal domain (BET) family proteins, including BRD2, BRD3, BRD4,
and BRDT, bind to acetylated lysines via their tandem domains (BD1 and BD2) to regulate gene
transcription. These proteins have emerged as new therapeutic targets for human diseases and
conditions, including cancers10-15 and inflammation.16,17 There has been significant progress in
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the development of small molecule inhibitors targeting the BET bromodomains. These potent
1
8
19-21
and I-BET151 (3)10,22 (Figure
and selective inhibitors, such as (+)-JQ1 (1), I-BET762 (2),
1), can significantly inhibit tumor growth in xenograft models in mice. We previously reported
the design of a new class of BET bromodomain inhibitors containing a benzo[cd]indol-2(1H)-
one structure, exemplified by Y02224 (4, Figure 1), which exhibited reasonable antiproliferative
effect on leukemia cells.23
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Chinnaiyan and others have shown that BRD4 inhibitors can disrupt the AR–BD1 interactions,
induce down-regulation of AR-regulated genes, and inhibit tumor growth in CRPC xenograft
mouse models.12,13,19 BET family proteins have been gaining increasing interest as drug targets
for treatment of CRPC.24-26 Therefore, targeting BET proteins represents an alternative strategy
for treatment of CRPC. Despite the discovery of these BET inhibitors, chemicals that entered
clinical trials for CRPC are still limited. BET inhibitors 2, OTX-015, ZEN003694 and GS-5829,
are currently being evaluated in clinical trials in patients with CRPC as a single agent or in
combination with AR-antagonists (Enz or ARN-509).27-30 Thus, potent and selective BET
inhibitors with different chemotypes are still in high demand to enhance our understanding of the
therapeutic potential of BET inhibition.
In the present study, we report the structure-based design, synthesis, and biological evaluation of
a new class of potent and specific small molecular BET inhibitors, the benzo[d]isoxazoles, for
potential treatment of CRPC.
2. RESULTS AND DISCUSSION
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.1. Design and Optimization of Benzo[d]isoxazole Derivatives.
.1.1. Design of Benzo[d]isoxazole Scaffold.
2
To identify a novel scaffold, all available co-crystal structures of BET proteins and their
inhibitors were aligned to explore the key pharmacophore elements. From the structural analysis,
it is obvious that the ligands may form direct or indirect hydrogen bonds with Asn140 and Tyr97
and hydrophobic interactions with the WPF shelf. Some additional interactions near the BC and
ZA channels may also contribute to ligand binding. In this study, the representative BET
inhibitors 1, 2 and 3 were used to produce a new scaffold (3-methylbenzo[d]isoxazole) through a
hybridization approach (Figure 2). From the predicted binding mode analysis by molecular
docking, we can see that the 3-methylbenzo[d]isoxazole moiety fits snugly into the KAc binding
pocket and forms hydrogen bonds to a conserved water molecule and residue Asn140 (Figure 2).
Starting from this scaffold, various substituents, such as sulfonamide derivatives as disclosed in
our previous patent31 and conformation restrained benzimidazole derivatives as disclosed in
patents from Boehringer Ingelheim32-34 and GlaxoSmithKline , were designed to explore the
chemical space for potency improvement.
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35
(
+)-JQ1, 1
Extract
substructure
Hybridization
I-BET762, 2
I-BET151, 3
Novel scaffold
Solvent
region
D145
I146
Hybridization
W81
N140
Y97
P82
F83
WPF
shelf
WPF
shelf
BC channel
Benzimidazole derivatives
Sulfonamide derivatives
Docking-guided optimization
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Figure 2. Flowchart summarizing the discovery and optimization strategy for novel
benzo[d]isoxazole-containing BET inhibitors. Crystal structure of BRD4(1) with inhibitor 1
(PDB ID: 3MXF) were used for model construction.
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.1.2. SAR Studies of Sulfonamide Derivatives Occupying the WPF shelf.
To find potent benzo[d]isoxazole derivatives, we designed various substituents pointing to the
WPF shelf region with a sulfonamide linker to explore the chemical space (Figure 2). First,
lipophilic alkyl or cycloalkyl substituted compounds 5a–5e were synthesized (Table 1). Utilizing
the thermal shift assay (TSA) and the AlphaScreen assay, the compounds were evaluated for
their abilities to bind to the BRD4(1) bromodomain. The well characterized inhibitor 1 was
included as a reference compound to validate all the experimental procedures. Fortunately, these
compounds exhibited moderate potencies. Among them, compound 5e with a cyclohexyl group
1
at R position displayed a thermal shift of 4.5 °C and an IC50 value of 2.24 μM. Aromatic
derivatives were also synthesized to investigate the SAR. As shown in Table 1, the phenyl
derivative 5f exhibited a weaker potency compared to the corresponding aliphatic compound 5e.
However, regardless of the substitution of an electron withdrawing group -Cl (5h), -NO
2
(5i), or
electron-donating group -OCH (5j), substitutions at para-position of phenyl ring were
3
detrimental to BRD4(1) binding. The results suggested there was no chemical space at the para-
position of phenyl ring for affinity improvement. In contrast, compounds with phenyl ring ortho-
substitutions displayed encouraging results. For example, compound 5k exhibited a thermal shift
of 5.2 °C and an IC50 value of 1.59 μM. When the 2-chloro group at the ortho-position in 5k was
replaced with -Br, -COOCH
potencies.
3
or –COOH (5l–5n), the compounds showed slightly decreased
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Table 1. Structure−Activity Relationships of Compounds 5a−5v for BRD4 (1) Activitiesa
1
1
1
1
1
1
1
1
1
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2
2
2
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5
5
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5
6
0
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No.
R1
-
TSA ΔT
m
(C) AlphaScreen (μΜ)
1
11.5
3.0
4.5
5.0
5.0
4.5
2.0
2.0
1.0
0.5
1.5
5.2
4.4
3.0
3.8
0.12 ± 0.01
5a
5.30 ± 0.63
2.47 ± 0.13
2.55 ± 0.01
3.05 ± 0.54
2.24 ± 0.27
4.96 ± 1.18
9.82 ± 0.84
>20
5
b
5
c
5
d
5
e
5f
5g
5
h
5
i
>20
5
j
>20
5
k
1.59 ± 0.17
1.84 ± 0.11
6.93 ± 2.01
5.81 ± 1.34
5l
5m
5
n
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5
o
4.0
5.0
1.1
3.55 ± 0.49
2.35 ± 0.92
>20
5
5
p
q
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r
6.6
6.5
7.5
4.6
0.83 ± 0.01
1.05 ± 0.43
0.62 ± 0.09
2.13 ± 1.06
5
s
5t
5
u
5
v
2.0
10.68 ± 3.18
aThe IC50 of all compounds in the table were calculated from at least two independent
experiments. Values are given as the mean ± SD.
Considering the good potency of compound 5k, we kept the ortho chlorine and further added
various substituents at other positions in the phenyl ring. Thus, 5o–5q were designed and
synthesized. Among them, the 2,5-disubstituted compound (5p) showed a similar potency
compared to 5k. To better design potent compounds, we predicted the binding mode of 5p with
BRD4(1) using a molecular docking approach. When the structures of compound 5p and
compound 4 in complex with BRD4(1) were superimposed (Supporting Information, Figure
S1A), the phenyl groups overlapped very well. The 2-methoxy on the phenyl motif of 4 was
found to form a hydrogen bond with the 7th solvent water. This discovery encouraged us to
design 2-methoxy compounds. As expected, the 2-methoxy compound 5r displayed an increased
potency with a thermal shift of 6.6 °C and an IC50 value of 0.83 μM. When a chloro group was
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introduced at 5-position, the resulting compound (5s) achieved no improvement in potency.
Replacement of the 5-chloro (5s) with 5-bromo (5t) led to an increased activity, with a thermal
shift of 7.5 °C and an IC50 value of 0.62 μM. The size of the substituent at the 5-position may be
important for compound activity. When the 5-bromo group was replaced with a methoxy group
1
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(5v), the compound activity decreased approximately 20-fold as evaluated by the AlphaScreen
assay.
D145
D145
A
B
C
I146
WPF
WPF
I146
WPF
BC Channel
N140
BC
N140
⑦
W81
W81
W W8 18 1
P82
BC
P82
N140
P82
⑥
ZA
Channel
Channel
ZA
Channel
Channel
ZA
Channel
Q85
L92
L92
Y97
Y97
Q85
Y97
Q85
Figure 3. (A) X-ray crystal structure of 5t in complex with BRD4(1) (PBD ID: 5Y8Z, carbon:
gray); the electronic density of the ligand and water molecules are shown in the contour map. (B)
Overlaid X-ray crystal structures of 5t (PBD ID: 5Y8Z, carbon: gray) and compound 4 (PBD ID:
5
DX4, carbon: violet) in complex with BRD4(1). (C) Overlaid X-ray crystal structures of 5t
(PBD ID: 5Y8Z, carbon: gray) and diacetylated histone peptide (PBD ID: 3UVW, carbon: cyan)
in complex with BRD4(1).
To understand the structural basis for the high binding affinity of compound 5t to the BRD4(1)
protein, we determined the co-crystal structure of 5t in complex with BRD4(1) at 1.84 Å
resolution (Figure 3A). The overall binding mode was consistent with our initial docking
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prediction. The 3-methylbenzo[d]isoxazole motif resided deep in the KAc-binding pocket with
the oxygen atom forming a hydrogen bond with Asn140. The isoxazole ring nitrogen atom
formed a water-mediated hydrogen bond with Tyr97. The 3-methyl group fitted well into the
cavity formed by the remaining water molecules and occupied the sub-pocket that
accommodated the acetyl group of acetylated lysine. The sulfonamide linker formed a vector that
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
enabled the attached R -motif to occupy the WPF shelf. One of the sulfonamide linker oxygen
atoms formed a hydrogen bond interaction with the 6th water located in the ZA channel. This
solvent water presented a suitable geometry to enable the water-mediated hydrogen bond
network with the bottom conserved water molecules. As expected, the 2-methoxy and NH
formed hydrogen bonds with the 7th water molecule, which was consistent with that of
compound 4 (Figure 3B). This water molecule also formed additional hydrogen bond
interactions with the carbonyl group of Leu92 and another solvent water molecule.
2
.1.3. SAR Studies of Sulfonamide Derivatives towards the BC Channel.
The next region for SAR study was the BC channel. Previous studies by Filippakopoulos
reported the co-crystal structure of a diacetylated histone peptide bound to BRD4(1).36 The
peptide extended from the WPF shelf to the BC channel region. When the X-ray crystal structure
of 5t–BRD4(1) was overlaid with the co-crystal structure of the diacetylated histone peptide
H4K5AcK8Ac–BRD4(1) (PDB ID: 3UVW, Figure 3C),36 the 6-methyl group of
benzo[d]isoxazole scaffold pointed to the main chain of the histone H4 acetylated lysine mark
(
K5Ac). Inspired by this, we designed some peptide mimics to occupy the peptide binding groove
BC channel). Due to the hydrophilic and high electrostatic potential properties of the BC
(
channel, compounds 6a–6f were synthesized with amide linkages and polar substituents (Table
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2
). However, the compounds with longer and hydrophilic substituents showed moderate
2
inhibitory activities. It was obvious that shorter substitution at the R position (6a) exhibited
higher affinity (ΔT = 9.2 °C; IC50 = 0.23 μM). To understand the structural basis for further
m
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
structure-guided optimizations, compounds 6e and 6f were co-crystallized with BRD4(1). Due to
the flexibility of the substituent in 6e, the electron-density analysis indicated that a long
2
substituent at the R position exhibited an unstable conformation. The amide in 6f formed a
hydrogen bond with the 7th solvent water, which further bound to Leu92. The morpholine ring at
the end of the chain extended to the top of BC channel and showed fewer key interactions with
the protein (Supporting Information, Figure S1B).
Table 2. Structure−Activity Relationships of Compounds 6a, 6c−6n for BRD4(1) Activitiesa
No.
R1
R2
TSA ΔT
m
(C) AlphaScreen (μΜ)
6
a
-OH
9.2
6.2
5.1
5.4
4.8
6.0
0.23 ± 0.01
6
c
0.58 ± 0.03
1.60 ± 0.23
0.75 ± 0.01
2.27 ± 1.24
0.42 ± 0.01
6
d
6e
6
f
6
g
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2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
h
-OCH
2
CH
3
9.2
9.8
9.2
7.8
7.3
8.7
7.8
0.15 ± 0.05
0.13 ± 0.01
0.15 ± 0.04
0.22 ± 0.00
0.44 ± 0.03
0.16 ± 0.04
0.47 ± 0.03
6
i
-OCH
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6j
-NHCH
3
6
k
-OCH
-OCH
-OCH
-OCH
3
3
3
3
6
l
6
m
6
n
aThe IC50 of all compounds in the table were calculated from at least two independent
experiments. Values are given as the mean ± SD.
2
Based on the above analysis, we decided to focus on smaller R substituents, leading to the
synthesis of compounds 6g, 6h and 6i. The results demonstrated that the 6-methoxy group (6i)
was optimal for BRD4(1) binding potency. 6i was almost identical to that of reference compound
1
with an IC50 value of 0.13 μM. When a little larger group, such as propoxy group (6g), was
2
introduced at the R position, the compound potency decreased approximately 3-fold. Thus far,
2
the structure-activity relationships have established that the R -position favors small groups.
To understand the structural basis for the SAR, we determined the co-crystal structures of 6a, 6i,
and 6j bound to BRD4(1). As shown in Figures 4A and 4B, 6-OH derivative 6a and 6-OCH
3
derivative 6i formed hydrogen bond interactions with the 7th solvent water molecule.
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Importantly, the 6-OCH
3
in 6i occupied the position of the acetylated lysine (K5Ac) hydrophobic
chain. Similarly, the 6-NHCH
3
derivative 6j displayed similar binding affinity to the 6-OCH
3
derivative 6i. The crystal structure of 6j bound to BRD4(1) indicated that the solvent water-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mediated hydrogen bonds were important for the high potency (Figure 4C).
D145
D145
A
B
I146
I146
WPF
WPF
N140
N140
⑦
W81
W81
P82
P82
BC
Channel
BC
⑥
ZA
Channel
Channel
ZA
Channel
L92
L92
Y97
Q85
Y97
Q85
D145
D145
C
D
I146
I146
WPF
WPF
N140
N140
W81
ZA
W81
ZA
P82
P82
BC
Channel
BC
Channel
Channel
Channel
L92
L92
Y97
Q85
Y97
Q85
Figure 4. X-ray crystal structures of identified inhibitors in complex with BRD4(1). (A) 6a–
BRD4(1) (PDB ID: 5Y8W, carbon: green). (B) 6i–BRD4(1) (PDB ID: 5Y8Y, carbon: magenta).
(C) 6j–BRD4(1) (PDB ID: 5Y94, carbon: yellow). (D) 6m–BRD4(1) (PDB ID: 5Y8C, carbon:
pink). Structures are shown with an electrostatic potential surface view.
To validate the importance of the 6-OCH
3
substitution, analogs of compound 6i were prepared
1
1
with various R groups. Based on above established SAR from Table 1, representative R groups
acceptable for the WPF shelf were selected. The resulting compounds 6k–6n exhibited a thermal
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
shift of over 7 C and IC50 values ranging from 0.16 to 0.47 μM. They were more active than the
corresponding 6-CH -substituted compounds 5k, 5p, 5s and 5u. Replacement of 5-bromo (6i)
3
with 5-chloro (6m) on the phenyl ring led to a slightly decreased activity. The chlorine’s smaller
size may form weaker Van der Waals interaction with the hydrophobic WPF shelf (Figure 4D,
Supporting Information, Figure S2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
.1.4. SAR Studies of the Benzimidazole Derivatives.
Even though the sulfonamide derivatives exhibited good activities in the AlphaScreen and TSA
assays, more chemotypes are needed to explore the therapeutic potential of BET inhibition for
CRPC. In the above section, we demonstrated that the angle of the sulfonamide linker is
important and ensure that the attached substituents occupy the WPF shelf. To reduce the
molecular flexibility, a cyclization strategy was used at the sulfonamide position to maintain the
similar angle and key binding characteristic. By searching literatures and patents, we found that a
conformation restrained benzimidazole template was used as body of BRD4 inhibitors. Several
patents from Boehringer Ingelheim32-34 and GlaxoSmithKline35 described that benzimidazole
derivatives with a pyridone or bicyclic triazole as the acetyl lysine mimic can function as BRD4
inhibitors. Two structurally related benzimidazoles or bioisosteres of benzimidazoles reported by
Engelhardt in the patents are exemplified in Supporting Information, Figure S3.33,34 To
investigate whether the benzo[d]isoxazole may act as an effective KAc mimic in structurally
more diverse BRD4 inhibitors, we tried to introduce the benzimidazole template to the
benzo[d]isoxazole scaffold using a hybridization strategy. Thus, novel benzimidazole-containing
benzo[d]isoxazole derivatives were designed and synthesized.
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Table 3. Structure−Activity Relationships of Compounds 7a−7n for BRD4(1) Activitiesa
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
No.
R3
R4
H
R5
TSA ΔT
m
(C) AlphaScreen (μΜ)
7a
6.5
6.2
5.9
7.2
5.0
4.5
6.5
6.0
4.8
6.0
5.4
9.2
9.9
7.2
1.37 ± 0.78
7
b
Br
H
1.80 ± 0.07
1.16 ± 0.05
0.52 ± 0.04
4.42 ± 0.72
>20
7
c
7d
Br
H
7
e
7
f
Br
H
7g
1.41 ± 0.80
2.36 ± 0.45
3.62 ± 0.24
1.67 ± 0.36
0.18 ± 0.02
0.30 ± 0.06
0.18 ± 0.01
0.32 ± 0.04
7
h
Br
H
7
i
7
j
H
7
k
H
7
l
Br
H
7
m
7
n
Br
aThe IC50 of all compounds in the table were calculated from at least two independent
experiments. Values are given as the mean ± SD.
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
To investigate the possible binding mode, the designed compound 7a (Table 3) was first docked
into the bromodomain of BRD4(1) using the 6i-BRD4(1) complex structure (PDB ID: 5Y8Y) as
a template. The predicted binding mode indicated that compound 7a bound snugly in the acetyl
lysine binding pocket (Figure 5A). The benzo[d]isoxazole scaffold in 7a adopted the same
binding mode with that of 6i. The benzimidazole nitrogen atom attached substituent stretched to
the WPF shelf, and another nitrogen atom formed a hydrogen bond with the 6th water in the ZA
channel. The planar benzimidazole group formed π-π interaction with residue Trp81. The
morpholine, a commonly used group that was also adopted by Boehringer Ingelheim’s
patents,33,34 extended to the solvent region outside the binding pocket. Based on the above
analysis, we synthesized a series of benzimidazole-containing benzo[d]isoxazole derivatives and
performed extensive SAR studies (Table 3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
D145
D145
A
B
I146
I146
WPF
WPF
N140
N140
W81
W81
ZA
P82
P82
BC
BC
Channel
Channel
ZA
Channel
⑥
Channel
L92
L92
Y97
Q85
Y97
Q85
Figure 5. (A) Overlaid structures of compounds 6i (PBD ID: 5Y8Y, carbon: magenta) and 7a
docked pose, carbon: cyan) in complex with BRD4(1). (B) The docked pose of compound 7m
(
(carbon: green) in the BRD4(1) crystal structure (PDB ID: 5Y8Y).
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3
We initially introduced a hydrophobic propyl group (7a) at the R position of the benzimidazole
to occupy the WPF shelf. 7a demonstrated moderate potency against BRD4(1), with a ΔT
m
value
3
of 6.5 C and an IC50 value of 1.37 μΜ. We further investigated the contribution of the R group
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
to BRD4(1) binding potencies. Inspired by the substituents in previous work,23,33,34 the
hydrophobic groups such as flexible alkyl or cycloalkyls with 3–4 heavy atoms and benzyl group
3
were introduced at R position. The resulting compounds 7b–7j displayed comparable BRD4(1)
binding potencies to compound 7a. The investigation also revealed that a Br substitution was
4
tolerated at the R position. However, the effect of the Br substitution was sometimes beneficial
to BRD4(1) activity (e.g. 7c and 7d), and sometimes detrimental (e.g. 7a and 7b; 7e and 7f; 7g
3
and 7h). When a newly designed cyclohexylmethyl group was introduced at R position, the
resulting compounds 7k and 7l demonstrated improvement in their potencies with IC50 values of
0
.18 and 0.30 μΜ against BRD4(1), respectively. Finally, the morpholine ring in the solvent
region was replaced with a chiral (S)-3-methylmorpholine group (7m and 7n), and comparable
affinities were obtained.
To understand the possible binding mode of 7m to BRD4(1), molecular docking was performed
using the X-ray crystal structure of 6i-BRD4(1) complex (PDB ID: 5Y8Y). The
cyclohexylmethyl group of 7m occupied the WPF shelf and formed extensive Van der Waals
interactions with the hydrophobic residues Trp81, Pro82, and Ile146 (Figure 5B). The
benzimidazole nitrogen atom formed a hydrogen bond with the 6th water in ZA channel,
anchoring the benzimidazole group in a reasonable binding pose (Figure 5B). Taken together, the
benzo[d]isoxazole scaffold is an effective KAc mimic for designing BET inhibitors.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
.2. Evaluation of the ITC Binding Affinities and Bromodomain Selectivity.
The most potent compounds from the two series were chosen for binding affinity determination
using isothermal titration calorimetry (ITC) (Table 4, Figure 6A, 6B and Supporting Information,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure S4). All the tested compounds displayed high potencies with K
validated their strong binding affinities to BRD4(1). Compounds 6i and 7m bound to the
BRD4(1) bromodomain with K values of 82 and 81 nM, respectively. Overall, the results
d
< 200 nM, which further
d
indicated that compounds 6i and 7m displayed comparable binding activities to that of reference
compound 1.
Table 4. ITC Determination of K
d
for the Binding of the Representative Compounds to BRD4(1)
No. K
d
(nM)
92
N
ΔH (kcal/mol) TΔS (kcal/mol) ΔG (kcal/mol)
1
1.09
0.96
0.95
1.02
1.18
-9.3
0.32
-2.5
-6.0
-1.4
-2.2
-9.6
-9.7
-9.3
-9.3
-9.7
6
i
82
-12.2
-15.3
-10.7
-11.9
6
j
164
159
81
7
l
7
m
To investigate the selectivity profile, representative compounds were assessed by thermal
stability shift assay against the 12 representative bromodomain-containing proteins BRD4(1),
BRD2(1), BRD3(1), BRDT(1), BAZ2B, BRD1, BRD9, PCAF, TAF1(1), ASH1L, EP300 and
CREBBP. Most of the compounds showed good selectivity for the BET bromodomains over
other tested bromodomain-containing proteins (Figure 6C). For BRD4(1) protein, the thermal
shifts for all the tested compounds were over 9 °C. The sulfonamide derivatives 6i, and 6j
displayed weak activities for PCAF, EP300, and CREBBP, with thermal shifts less than 2.7 °C.
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As 6j displayed thermal shift of 2.3 °C against EP300, it was selected as a surrogate to validate
the binding affinity using ITC assay. The ITC result showed that no detectable binding affinity of
6j to EP300 was observed, which further demonstrated the good selectivity (Supporting
Information, Figure S4D). The benzimidazole derivatives 7l and 7m exhibited excellent
selectivity profiles for BET bromodomains over the other non-BET bromodomain-containing
proteins in the TSA assays.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To further confirm the selectivity profile from our TSA result, these two most potent BET
inhibitors 6i and 7m were also assessed by the commercial BROMOscan (DiscoverX) platform
to give a clearer indication of selectivity. The assay was performed in 32 representative
bromodomain modules at 5 µM concentration. BROMOscan profiling further confirmed the
excellent selectivity of both compounds for BET bromodomain over other bromodomain
subfamilies (Figure 6D and 6E, Supporting Information, Table S2).
A
Times (min)
B
Times (min)
C
CPD
1
6i
6j
7l
7m
BRD4(1) 11.5
9.8
9.2
9.2
9.9
BRD2(1)
BRD3(1)
8.7
11
5.3
8.1
5.6
0
5.3
6.5
5.1
0.3
-0.4
-0.8
2.6
-0.1
-0.1
2.3
2.1
6
6.3
8.7
8.9
4.2
-0.6
0
BRDT(1) 6.6
3.6
BAZ2B
BRD1
-0.3
0.5
-1
-0.6
0.3
-0.1
-0.9
2.7
0.1
0.3
2
BRD9
-0.9
-0.6
-0.6
-0.9
-0.3
0
-0.8
-0.6
-0.9
-0.5
0.3
PCAF
-0.1
-1
TAF1(1)
ASH1L
EP300
-1
6
i
7m
Kd = 81 nM
-0.4
Kd = 82 nM
CREBBP -0.6
1.4
0.3
Molar Ratio
Molar Ratio
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D
II
BRD2(2)
BRD3(2)
E
II
BRD2(2)
BRD3(2)
BRD3(1)
BRD2(1)
BRD3(1)
BRD2(1)
BRD4(2)
BRDT(2)
BRD4(2)
BRDT(2)
I
I
GCN5L2
PCAF
GCN5L2
PCAF
III
III
0
1
2
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0
1
2
3
4
5
6
7
8
9
0
BRWD1(2)
PHIP(2)
BRWD1(2)
PHIP(2)
PBRM1(1)
ASH1L
VIII
PBRM1(1)
ASH1L
VIII
BRWD3(2)
BAZ1B
BRWD3(2)
BAZ1B
SMARCA2
SMARCA4
SMARCA2
SMARCA4
IV BRD9
BRD7
BRPF3
IV BRD9
BRD7
BRPF3
PBRM1(5)
BRWD3(1)
PHIP(1)
PBRM1(5)
BRWD3(1)
PHIP(1)
PBRM1(6)
PBRM1(6)
BRPF1
ATAD2A
BRWD1(1)
PRKCBP1
TAF1(1)
TAF1L(1)
BRPF1
ATAD2A
BRWD1(1)
PRKCBP1
TAF1(1)
TAF1L(1)
TRIM66
% Ctrl @ 5μM
TRIM66
TRIM33
VI
0−5%
TRIM33
VI
TAF1(2)
VII
TAF1(2)
VII
5−20%
20−50%
50−70%
V
V
SP140L
SP140L
SP140
SP140
7m
6
i
Figure 6. Determinations of binding affinities and bromodomain selectivity. (A, B) ITC titration
curves for the binding of BRD4(1) to representative compounds 6i (A) and 7m (B). (C) Thermal
shift analysis for selected compounds against twelve proteins from seven distinct BRD sub-
families. The heat map shows the relative ΔT
m
, where red indicates a large ΔT
m
and yellow and
green indicate small ΔT values. (D, E) Bromodomain selectivity profile of compounds 6i (D)
m
and 7m (E) assessed by the BROMOscan (DiscoverX) platform at 5 μM concentration.
The %Ctrl = (test compound signal − positive control signal) / (negative control signal − positive
control signal) × 100%.
2
.3. Evaluation of the Inhibitory Effects on Cell Growth, Gene and Protein Expression in
Prostate Cancer Cells.
Given their high potencies and encouraging selectivity at the protein level, representative
compounds were assessed for their inhibitory effects on cell growth in a panel of human prostate
cancer cell lines such as LNCaP, C4-2B, 22Rv1 and VCaP (Figure 7). All the tested compounds
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exhibited reasonable potencies in these cell lines. The compounds 6i, 7l and 7m exhibited low
micromolar or nanomolar potencies (IC50: 0.29–2.6 μΜ) in the four AR-positive prostate cancer
cell lines LNCaP, C4-2B, 22Rv1 and VCaP. All the tested BET inhibitors were less potent than
compound 1 but more effective than second generation anti-androgen Enz in inhibiting cell
growth, demonstrating the promising therapeutic potential of BET inhibition on CRPC.
1
1
1
1
1
1
1
1
1
1
2
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0
1
2
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0
1
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0
1
2
3
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9
0
1
2
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0
1
2
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8
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0
A 1
25
LNCaP
B125
100
75
C4-2B
C 125
100
75
22Rv1
Enz
1
Enz
1
Enz
1
100
75
6i
6i
6i
6j
6j
6j
5
0
5
0
7l
50
7l
50
7l
7m
7m
7m
2
25
25
0
0
0.001 0.01 0.1
1
10 100
0.01
0.1
1
10
100
0.001 0.01 0.1
1
10 100
D
E
VCaP
LNCaP
C4-2B
22Rv1
IC50 (μΜ)
VCaP
IC50 (μΜ)
1
25
00
Enz
1
No.
IC50 (μΜ)
33.84
0.07
IC50 (μΜ)
17.96
0.20
1
Enz
1
36.66
0.07
1.50
6.73
50%@33
0.01
6i
7
5
6j
6i
6j
7l
1.06
2.62
0.63
50
25
0
7l
6.16
6.43
3.02
7m
0.57
0.47
0.85
0.84
0.64
0.70
0.50
0.29
7m
0.01
0.1
1
10
100
Figure 7. Cell viability, as measured by Cell-Titer GLO (Promega) of LNCaP (A), C4-2B (B),
2
2Rv1 (C) and VCaP (D) prostate cancer cells treated with the indicated concentrations of Enz, 1,
i, 6j, 7l, and 7m. Experiments were performed triplicate. (E) Half-maximum inhibitory
6
concentration (IC50) values for all compounds in each cell line are shown.
To evaluate cytotoxicity for various cell lines, compounds 6i and 7m were also evaluated in a
wide range of cancer cell lines and normal cell lines (Supporting Information, Figure S5).
Comparing with the AR-positive prostate cancer cell lines, 6i and 7m were less potent in AR-
negative prostate cancer cells DU145 (IC50 > 6 μΜ) and PC-3 (IC50 > 3 μΜ). The inhibitory
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effects of 6i and 7m in the AR-positive and AR-negative prostate cancer cell lines are consistent
1
2
with that of compound 1 reported by Asangani et al. Besides, the breast cancer cells MCF-7
and Hs578T also appeared to be sensitive to these compounds (IC50: 1–2.5 μΜ). BET
bromodomain inhibitors have previously shown antiproliferative effects in the AML MV4;11
cell line and human colon cancer HT-29 cell line reported by us and others.10,11,23,37,38 Thus, it
was not surprising to see that 6i and 7m demonstrated good inhibitory effect in MV4;11 and HT-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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6
7
8
9
0
1
2
3
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5
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8
9
0
1
2
3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
9 (0.4–1.7 μΜ). The studies showed that 6i and 7m displayed moderate (2.5–5 μΜ) or weak (>
μΜ) inhibitory effects in the rest of cancer cell lines. Compound 1 is a widely used probe and
5
exhibited stronger anti-proliferative effects in a range of cell lines compared to our compounds
(Supporting Information, Figure S5). However, in our cellular assay (Figure S5), 1 exhibited
very potent activities both in the cancer cell lines and in the normal lung fibroblast cell line HFL-
1
(IC50 = 0.29 μΜ). Our compounds 6i and 7m exhibited weak cytotoxicity in the normal lung
fibroblast cell line HFL-1 with IC50 values of 18.2 and 15.9 μΜ for 6i and 7m, respectively.
Overall, the compounds showed preference for both acute leukemia cell lines such as MV4;11
and the AR-positive prostate cancer cell lines.
To evaluate the long term cell growth inhibitory effects, colony formation assays were conducted
for the representative inhibitors 6i and 7m. Consistent with the cell viability assays, treatment of
C4-2B and 22Rv1 cells with 6i or 7m reduced colony formations in a dose-dependent manner
(Figure 8A). Colony formations were markedly suppressed by both compounds at 0.5 μM.
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A
Vehicle
0.5 μM
1 μM
2 μM
B
a
1
.5
DMSO
1
6i
7m
1.0
0.5
*
b
Vehicle 0.5 μM
1 μM
2 μM
**
**
*
1
1
1
1
1
1
1
1
1
1
2
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5
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5
5
5
5
5
6
0
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0
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2
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9
0
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9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
*
**
*
*
**
*
*
**
****
*
****
*
0.0
AR-FL
PSA
KLK2 TMPRSS2 C-MYC
LNCaP
C 1.5
D
1.2
PSA-luciferase
DMSO
1
6i
7m
1
0.0 μM
2
0
.0 μM
.4 μM
1.0
0.8
0.4
0.0
*
*
**
**
**
**
*
*
0
.5
.0
**
*
*
*
** **
**
**
**
**
*
***
** ** **
** **
**
*
** **
**
0
AR-FL
AR-V7
PSA
KLK2 TMPRSS2 ERG
C-MYC
DMSO
1
6i
7m
VCaP
Figure 8. (A) Compounds 6i (a) and 7m (b) inhibit the colony formations of C4-2B and 22Rv1
prostate cancer cells. Cells were cultured and treated with vehicle (DMSO), 0.5 μM, 1.0 μM or
2.0 μM of 6i or 7m for 14 days followed by staining. (B, C) qRT-PCR analysis for mRNA
expression in LNCaP cells (B) and VCaP cells (C) treated with vehicle (DMSO), 1 (5 µM), 6i (5
µM) or 7m (5 µM) for 48 h. (D) PSA transcriptional activity was evaluated by luciferase reporter
assays in LNCaP cells transfected with PSA-luc reporter plasmid. Cells were treated with vehicle
or with 0.4 μM, 2.0 μM or 10.0 μM of 1, 6i or 7m. Data are expressed as the mean ± s.e.m. (n =
3
). *p < 0.05 and **p < 0.01 by two-tailed Student’s t-test.
qRT-PCR analysis was performed to explore whether the selected compounds would have an
effect on AR, AR-regulated genes and other oncogenes expression in prostate cancer cells. As
shown in Figure 8B and 8C, the AR-regulated genes KLK2, PSA (also known as KLK3) and
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2
2
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2
2
2
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TMPRSS2 were suppressed at mRNA level upon 6i or 7m treatment in LNCaP and VCaP cells.
MYC, a known oncogene in many types of cancer including prostate cancer, was previously
identified as down-regulated by BET bromodomain inhibitors.12,19,39,40 c-Myc mRNA level was
significantly suppressed by 6i or 7m in both LNCaP and VCaP cells. ERG mRNA level was also
suppressed upon 6i or 7m treatment in VCaP cells. Besides, the tested compounds suppressed the
mRNA expression of full-length AR (AR-FL) or AR-V7 in VCaP cells. Furthermore, western
blot analysis indicated that treatment of 22Rv1 cells with compounds 6i and 7m resulted in
significant down regulation of both AR-FL and AR variants levels (Supporting Information,
Figure S6).
0
1
2
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8
9
0
1
2
3
4
5
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7
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9
0
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0
As prostatic specific antigen (PSA) is currently the only recognized biomarker for the prostate
cancer, a PSA promoter driven luciferase reporter assay was also designed as a surrogate to
further evaluate the potential impact of compounds on transcription of PSA. Both 6i and 7m
dose-dependently inhibited the PSA reporter activity and displayed comparable inhibitory effect
to that of the reference BET inhibitor (1) (Figure 8D). The results suggested that the BET
inhibitors 6i and 7m could effectively suppress cell growth and related gene expression in
prostate cancer cells.
2
.4. Pharmacokinetic Profiles of Compounds 6i and 7m.
The in vivo pharmacokinetic (PK) profiles were further evaluated for compounds 6i and 7m.
Pharmacokinetic studies were performed after administering Sprague-Dawley (SD) rats with an
intravenous (iv) dose of 2 mg/kg and an oral dose (po) of 10 mg/kg. As summarized in Table S3,
after an iv administration, compound 6i displays higher maximum concentration (Cmax = 1392
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μg/L) than that of 7m (Cmax = 807 μg/L). Both compounds exhibit reasonable exposure with
AUC values of 1169 μg/L·h and 881 μg/L·h. It is disappointing that both compounds display
poor drug exposure after an oral administration and results in low oral bioavailability.
Compounds 6i and 7m may be further optimized to generate orally available candidates for drug
development.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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0
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8
9
0
2
.5. Compounds 6i and 7m Inhibit Prostate Cancer Tumor Growth.
We next evaluated the effects of compounds 6i and 7m on CRPC tumor growth using a C4-2B
mouse xenograft model. Given the poor oral bioavailability, we adopted the intraperitoneal
injection as the administration route. The mice were randomized and intraperitoneally (i.p.)
treated with either vehicle or BET inhibitors 6i or 7m (50 mg/kg, five times per week) when the
3
tumor volume was approximately 100 mm . The efficacy data showed that both 6i and 7m
exhibited strong antitumor activities during the 25-day treatment period, with a tumor growth
inhibition (TGI) of 70% and 51%, respectively (Figure 9A). Both compounds were well tolerated
in the treated mice, based on the weight of the animal body and their general behavior (Figure
9
B).
A
B
25
800
Vehicle
i, 50mg/kg IP
6
2
0
5
7m, 50mg/kg IP
600
400
200
0
*
1
*
**
10
5
Vehicle
*
*
***
***
6i
7m
*
*
0
0
5
10
15
20
25
0
5
10
15
20
25
Days of drug treatment
Days of drug treatment
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5
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5
5
5
5
6
Figure 9. (A) Antitumor efficacy of compounds 6i and 7m in a C4-2B CRPC xenograft mouse
model. (B) Body weight of mice during treatment with 6i and 7m. Mice were treated with
intraperitoneal (i.p.) injections of vehicle or compounds 6i and 7m (50 mg/kg) five times per
week. Data are expressed as the mean tumor volume ± s.e.m. of the animals in each treatment
group. *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed Student’s t-test.
0
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0
3
. CHEMISTRY
The synthesis of novel benzo[d]isoxazole derivatives were illustrated in Schemes 1−4. In
Scheme 1, commercially available 8a was N-acetylated with acetic anhydride to give 9a, which
was converted to 10a via a Friedel-Crafts reaction. Compound 10a was used to prepare the
oxime precursor 11a, which was cyclized at high temperature to yield the benzo[d]isoxazole
scaffold 12a. Deprotection of the N-acetyl group of 12a with dilute hydrochloric acid generated
aniline 13a. This was then capped with sulfonyl chlorides to give the sulfonamide derivatives
5
a–5v. The synthesis of compounds 6i and 6k–6n followed the same procedures as mentioned
above with 8b as the starting material.
In Scheme 2, the methyl group of the intermediate 13b was removed using boron tribromide to
afford compound 14, which was further capped with sulfonyl chloride to give compound 6a. O-
alkylation of 6a using different haloalkanes gave 6b, 6g, and 6h. Subsequently, 6b was
hydrolyzed to acid 6c, followed by coupling with available amines to obtain target compounds
6
d–6f.
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In Scheme 3, the phenol derivative 15 was acetylated with acetic anhydride to afford compound
1
6. This was then converted to 17 under AlCl -mediated Fries rearrangement conditions. The
3
subsequent two steps were similar to Scheme 1, including oximation and cyclization, which
furnished compound 19. Treatment of 19 with fuming nitric acid gave the nitro-substituted
compound 20. 20 was then reacted with methylamine to afford the corresponding amine-
substituted derivative 21. Reduction and sulfonylation gave the target compound 6j.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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6
0
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7
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9
0
1
2
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7
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9
0
1
2
3
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In Scheme 4, starting from commercially available 23, the intermediate 25a was obtained
through a Fries rearrangement and an esterification reaction. Treatment of 25a with bromine
gave the Br-substituted compound 25b. Next, 27a and 27b were prepared in two steps from 25a
and 25b, respectively, following the same procedure as mentioned in Scheme 1. Deprotection of
2
7a and 27b gave the acid fragments 28a and 28b, respectively. Subsequently, 2-chloro-4-
fluoro-1-nitrobenzene 29 was reacted with different amines in two steps to give the amine-
substituted nitrobenzene intermediates 31a–31h. Reduction of the nitro groups led to the
corresponding anilines 32a–32h, which were coupled with the acid 28a or 28b, followed by a
ring closing reaction using acetic acid to give the final products 7a–7n.
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5
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5
5
6
Scheme 1. Synthesis of sulfonamide derivatives 5a–5v, 6i, and 6k–6na
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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9
0
aReagents and conditions: (a) acetic anhydride, Et
AlCl , CH Cl , 0−43 °C, 78−80%; (c) NH -OH·HCl, NaOAc, EtOH/H
DMF-DMA, 1,4-dioxane, 100 °C, 61−70%; (e) HCl/H
CH Cl , reflux, 11−80%.
N, CH
Cl , rt, 68−71%; (b) acetyl chloride,
3
2
2
3
2
2
2
2
O, 80 °C, 94−99%; (d)
1
2
O, reflux, 96−97%; (f) R SO
2
Cl, pyridine,
2
2
2
Scheme 2. Synthesis of O-linked R derivatives 6a–6ha
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aReagents and conditions: (a) BBr
, CH
Cl
, rt, 91%; (b) 5-bromo-2-methoxybenzene sulfonyl
3
2
2
6
chloride, pyridine, CH
2
Cl
2
, rt, 71%; (c) R X (X= Cl or I), K
2
CO , KI, acetone, 50 °C, 31−33%;
3
7
(d) NaOH, H
2
O, MeOH, rt, 76%; (e) R NH
2
, HATU, DIPEA, DMF, rt, 32−48%.
1
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Scheme 3. Synthesis of N-linked R derivative 6ja
2
aReagents and conditions: (a) acetic anhydride, H
c) NH -OH·HCl, NaOAc, EtOH/H O, 80 °C, 86%; (d) acetic anhydride, NaOAc, DMF, 100 °C,
0%; (e) HNO , H SO , 0 °C, rt, 82%; (f) CH NH , K CO , THF, 45 °C, 95%; (g) SnCl ·2H O,
HCl (conc.), acetic acid, 40 °C, 39%; (h) 5-bromo-2-methoxybenzenesulfonyl chloride, pyridine,
CH Cl , reflux, 71%.
SO
4
, CH Cl
2 2
, rt, 76%; (b) AlCl , 190 °C, 71%;
2
3
(
2
2
9
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