Discovery of Thieno[2,3- d]pyrimidine-Based Hydroxamic Acid Derivatives as Bromodomain-Containing Protein 4/Histone Deacetylase Dual Inhibitors Induce Autophagic Cell Death in Colorectal Carcinoma Cells

Article abstract of DOI:10.1021/acs.jmedchem.9b02178

Bromodomain-containing protein 4 (BRD4) and histone deacetylases (HDAC) are both attractive epigenetic targets in cancer and other chronic diseases. Based on the integrated fragment-based drug design, synthesis, and in vitro and in vivo evaluations, a series of novel thieno[2,3-d]pyrimidine-based hydroxamic acid derivatives are discovered as selective BRD4-HDAC dual inhibitors. Compound 17c is the most potent inhibitor for BRD4 and HDAC with IC₅₀ values at nanomolar levels, as well as the expression level of c-Myc, and increases the acetylation of histone H3. Moreover, 17c presents inhibitory effects on the proliferation of colorectal carcinoma (CRC) cells via inducing autophagic cell death. It also has a good pharmacokinetic profile in rats and oral bioavailability of 40.5%. In the HCT-116 xenograft in vivo models, 17c displays potent inhibitory efficiency on tumor growth by inducing autophagic cell death and suppressing IL6-JAK-STAT signaling pathways. Our results suggest that the BRD4-HDAC dual inhibition might be an attractive therapeutic strategy for CRC.

Full text of DOI:10.1021/acs.jmedchem.9b02178

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Article
Discovery of thieno[2,3-d]pyrimidine based hydroxamic acid derivatives
as bromodomain-containing protein 4/histone deacetylases dual
inhibitors induce autophagic cell death in colorectal carcinoma cells
Zhaoping Pan, Xiang Li, Yujia Wang, Qinglin Jiang, Li Jiang,
Min Zhang, Nan Zhang, Fengbo Wu, Bo Liu, and Gu He
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02178 • Publication Date (Web): 10 Mar 2020
Downloaded from pubs.acs.org on March 10, 2020
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Discovery of thieno[2,3-d]pyrimidine based hydroxamic acid
derivatives as bromodomain-containing protein 4/histone
deacetylases dual inhibitors induce autophagic cell death in
colorectal carcinoma cells
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Zhaoping Pan,†‡ Xiang Li,†‡ Yujia Wang,† Qinglin Jiang,#* Li Jiang, # Min Zhang, # Nan Zhang,†
Fengbo Wu,† Bo Liu,†* and Gu He†*
†
State Key Laboratory of Biotherapy and Department of Urology, West China Hospital, Sichuan
University and Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan 610041, PR
China
#
School of Pharmacy and Sichuan Province College Key Laboratory of Structure-Specific Small
Molecule Drugs, Chengdu Medical College, Chengdu 610500, PR China
‡
These authors contributed equally to this work.
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Abstract: BRD4 and HDAC are both attractive epigenetics targets in cancer and other
chronic diseases. Based on the integrated fragment-based drug design, synthesis, in
vitro and in vivo evaluations, a series of novel thieno[2,3-d]pyrimidine-based
hydroxamic acid derivatives are discovered as selective BRD4/HDAC dual inhibitors.
Compound 17c is the most potent inhibitor for BRD4 and HDAC with IC values at
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nanomolar levels, as well as the expression level of c-Myc, and increases the acetylation
of histone H3. Moreover, 17c presents inhibitory effects on the proliferation of
colorectal carcinoma cells via inducing autophagic cell death. It also has a good
pharmacokinetic profile in rats and oral bioavailability of 40.5%. In the HCT-116
xenograft in vivo models, 17c displays potent inhibitory efficiency on tumor growth by
inducing autophagic cell death and suppressing IL6-JAK-STAT signaling pathways.
Our results suggest the BRD4/HDAC dual inhibition might be an attractive therapeutic
strategy for colorectal carcinoma.
Keywords: BRD4; HDAC; Dual inhibitor; Colorectal carcinoma; Autophagy
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Introduction
Colorectal carcinoma (CRC) is one of the most common malignances worldwide
and remains the second and third leading causes of cancer-related deaths in male and
female adults, respectively.1, 2 Although improvements in diagnostic and effective
therapeutic methods have been developed for CRC, novel therapeutics for the treatment
of advanced CRC are still urgently needed.3-5 In addition, the clinically used molecular
targeted agents of CRC, such as epidermal growth factor receptor (EGFR) inhibitors,
cetuximab and bevacizumab, only appear to have limited activity in metastatic CRC
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(mCRC) patients, but they have dramatically increased therapeutic costs and have a
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heavy economic burden on the patient and medical system. The epigenetic alterations
of CRC are considered to be an important biomarker of mCRC, and among the
epigenetic targets, histone acetylation in tumor tissues is one of the most frequent
changes in mCRC.9-12 The histone acetylation level detected by immunohistochemical
(IHC) staining is strongly correlated with the stages, metastasis and prognosis of
CRC.13
In the process of histone acetylation, two types of protein families are considered
as potential drugable targets: bromodomain-containing protein 4 (BRD4) plays a role
as a histone acetylated lysine “reader” and histone deacetylases (HDAC) act as an
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acetylated lysine “eraser”. BRD4 is a member of the bromodomain and extra-terminal
(BET) protein family, and it acts as a central element in the recognition of histone or
non-histone substrates, which may regulate many molecular and cellular processes in
epigenetic modification and gene transcription.15-19 HDACs are actuators of
deacetylation reactions of lysine residues, which have been recently deemed as
potential therapeutic targets in human malignancies. 20-25 There are four main subgroups
containing 11 isoforms of HDAC that have been recently categorized: HDACs 1, 2, 3,
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and 8 are class I; HDACs 4, 5, 7, and 9 are class IIa; HDACs 6 and 10 are class IIb; and
HDAC11 is class IV.13, 26 In the past investigations on BRD4 and HDAC inhibition, a
number of structurally diverse inhibitors have been reported, and some HDAC
inhibitors were approved by the U.S. food and drug administration (FDA), or
discovered in various clinical evaluation or preclinical stages for cancer therapy, e.g.
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vorinostat, panobinostat, mocetinostat, trichostatin A, romidepsin and 9Z8,
etc. And some BRD4 inhibitors were evaluated in preclinical or clinical evaluation
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stages, such as JQ-1, I-BET151, I-BET726, I-BET762, XD14, RVX-208,
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PFI-1 , ZL0420, Mivebresib (ABBV-075), Alobresib (GS-5829) , PLX51107
and BIC1,44-47 etc. (Figure 1)
BRD4 and HDACs could synergistically down-regulate c-Myc expression and
inhibit the progress and metastasis of CRC. Moreover, Hu et al. reported that Protein
Phosphatase 1 alpha (PP1α) and class I histone deacetylase signaling pathways are
essential for releasing chromatin-bound BRD4 for positive transcriptional elongation
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factor b (P-TEFb) recruitment. Mishra et al. reported novel insights into the action
mechanisms of HDAC inhibition by eliciting BRD4 and c-Myc and inducing a subset
of gene expression in pancreatic ductal adenocarcinoma.49 Recently, Zeng et al.
reported that the BRD4 inhibitor JQ1 could sensitize breast cancer cells to HDAC
inhibition by the LIFR-JAK-STAT (Leukemia Inhibitory Factor Receptor - Janus
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Kinase - Signal Transducer and Activator of Transcription) signaling pathways. There
have been several reports about the combination of HDAC and BRD4 inhibitors as
potential synergistic therapeutics for various cancers, e. g. the suberoylanilide
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hydroxamic acid (SAHA) analogues of I-BET726 (DUAL946) , I-BET151 (Zhang et
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al.) and (+)-JQ1 (He et al.). The previous reports of our laboratory suggested that
the combination of RVX-208 and SAHA also resulted enhanced anti-proliferative
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effects in AML cells.54
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Shao et al. 2017
He et al. 2020
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Figure 1. Structures of some known BRD4 and HDAC inhibitors: A. Selection of
published BRD4 inhibitors with diverse scaffolds; B. Selection of published HDAC
inhibitors with diverse ZBG; C. Structures of some reported BRD4-HDAC dual-
inhibitors based on known BRD4 inhibitors.
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As a part of our continuous work on discovering histone epigenetic modification
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inhibitors as autophagic regulators, in the current study, we designed and synthesized
a series of novel BRD4-HDAC dual inhibitors and performed a panel of subsequent in
vitro and in vivo biological evaluations. Furthermore, compound 17c induced apoptosis
and autophagic cell death by downregulating c-Myc, deacetylation of histone H3,
inhibiting apoptosis inhibitor protein Bcl-2 and interfering the formation of
autophagolysosome in HCT-116 colorectal cancer cells. 17c mainly inhibited HDAC
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, 2, 3 and 6, while both western blotting and IHC analyses revealed that 17c potently
inhibited the deacetylation of histone H3. In addition, 17c displayed a good therapeutic
efficiency on the HCT-116 colorectal cancer xenograft mouse and increased the
cytotoxic T-cell infiltration via activation of IL6-JAK-STAT signaling pathways. In
summary, we reported a series of novel small molecule BRD4-HDAC dual inhibitors,
and 17c exhibited a superior anti-proliferative capacity on colorectal cancer compared
to those of RVX-208 and vorinostat, suggesting that 17c is a potential anticancer lead
compound via selective BRD4 and HDACs inhibition and subsequent autophagic cell
death.
Results and discussion
Tissue microarray (TMA) and IHC analyses
The expressions of BRD4 and HDAC2 were evaluated by three independent
pathologists to avoid diagnostic bias. The HDAC2 was used as a representative
biomarker to evaluate the expression level of HDACs. The relative expression levels of
BRD4 and HDAC2 in both tumor and matched normal tissues of each case are
illustrated in Figure 2. There were significant differences in both BRD4 and HDAC2
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expression levels between tumor and matched normal tissues. Moreover, there was
strong correlation between BRD4 and HDAC2 in tumor tissues. In the HDAC2 staining,
35.56% (16/45) of stage I-II CRC tumor tissues showed moderate to strong staining,
while in stage III-IV tissues, the percentage of moderate to strong staining was up to
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8.97% (23/39) (Figure 2D). Additionally, there were 48.88% (22/45) and 61.54%
(24/39) of tumor tissues that showed moderate to strong BRD4 staining in stage I-II
and III-IV tissues, respectively. In brief, the BRD4 and HDAC2 expressions were up-
regulated and correlated with the clinical stage and prognosis in CRC tissues, which
suggested that BRD4 and HDAC may be potential targets for combined therapeutics.
Figure 2. Identification of HDAC2 and BRD4 are significantly upregulated in CRC
clinical samples: A. Representative immunoreactivity intensities of HDAC2 and BRD4
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in CRC tissues and adjacent normal tissues; B. HDAC2 expression is upregulated in
CRC tissues; C. BRD4 expression is upregulated in CRC tissues; D. The upregulated
HDAC2 and BRD4 expression are related to the CRC stages.
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Discovery of novel BRD4-HDAC dual inhibitors
A four-step strategy was performed for the discovery of small-molecule BRD4-
HDAC dual-inhibitor candidates: the fragment-based design of the novel BRD4
inhibitory scaffold, the synthesis of BRD4 inhibitors and in vitro enzymatic evaluation,
the design and synthesis of a novel BRD4-HDAC inhibitor, and the systemic evaluation
of this novel BRD4-HDAC dual inhibitor as an anti-cancer lead compound both in vitro
and in vivo. The BRD4 protein has two bromodomains, an ubiquitin binding domain
and a carboxyl-terminal domain. A canonical BRD4 protein contains four left-handed
alpha helices and two loops (ZA and BC loops) to form the binding site of the acetylated
lysine (Kac) residue. Most of the BRD4 inhibitors were designed to mimic the
interaction modes of Kac, thus giving them the ability to disrupt the interaction between
an acetylated histone and the Kac binding site within a BRD protein. First, the
fragments generated from known BRD4 inhibitors were embedded into the fragment-
like library of the ZINC database and then individually docked into the two
bromodomains of the BRD4 protein by the Libdock program, a rigid docking method.
The top 200 hit fragments were subsequently screened by the Ligandfit program, a
flexible docking method considering induced-fit effects. The top ten scoring fragments
are shown in Figure 3, and there were three structural characteristics in the screened
fragments: the stable hydrogen bond interactions with the ZA-loop residues; the van-
der-walls and hydrogen bond interactions in the Kac binding pocket; and a hydrophobic
fragment to fulfill the steric requirement on the outside of the binding site. Secondly,
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based on these screened fragments, a focused library of twenty-four compounds was
synthesized, and the BRD4 inhibitory activities of the compounds were characterized
(Scheme 1 and 2, Table 1). The general synthetic route of the target molecules 4a-t, 9a-
d has been presented in Scheme 1 and Scheme 2, respectively. Commercially available
cyclohexanone, cyclohexanone and N-substituted piperidine 1a-e as the first starting
material were reacted with ethyl cyanoacetate and sulfur to give 2a-e, and then these
five compounds were reacted with commercially available cyanobenzene and 3,4,5-
substituted cyanobenzene (3a-d) to give 4a-t. Similarly, tert-butyl 4-oxopiperidine-1-
carboxylate was reacted with 2-cyanoacetamide and sulfur to give 5 by Gewald reaction,
and then 6 was obtained through acylation. After the removal of protection, ring-closure
reaction was completed under the catalysis of strong alkali to give 8. Finally,
compounds 9a-d were prepared by nucleophilic substitution.
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As shown in Table 1, the enzymatic assay results of compounds 4a-t demonstrated
that the substitutes on the phenyl groups at the 2-position of thieno[2,3-b]pyrimidin-4-
one played a key role for maintaining the BRD4 inhibitory activity. When the para-
hydroxyl group was removed, the BRD4 inhibitory capacity was totally lost; moreover,
methyl groups at the meta-position could obviously increase the potency of the inhibitor.
The 3,5-dimethylisoxazole fragment displayed comparable BRD4 inhibitory capacity
than that of 3,5-dimethyl-4-hydroxyl-phenyl fragment, the docked confirmations of 9a
were highly similar to that of 4l (Figure S1). In addition, larger substitutes on the outside
fragment could decrease the inhibitory activity.
Subsequently, compound 4l was chosen as a novel BRD4 inhibitor scaffold for
dual-inhibitor discovery. It was well known that a typical HDAC inhibitor contained
three fragments: a cap fragment, a hydrophobic linker and a zinc binding group (ZBG)
group. In the design of BRD4-HDAC dual-inhibitors, compound 4l was used as the cap
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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
fragment. There were two linkage sites in compound 4: the hydroxyl group on the 3,5-
dimethyl-4-hydroxyl-phenyl group and the N atom in the piperidine ring. According to
the previous report of our laboratory, the BRD4/HDAC dual inhibitor based on RVX-
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
08, the ZBG group could linked to the 3,5-dimethyl-4-hydroxyl-phenyl group. We
chose the N-hydroxyhexanamide and phthalate fragments as model groups to explore
the correct linking mode of the cap fragment, respectively. The model compounds were
synthesized and characterized for their BRD4/HDAC inhibition as well as cell
proliferation inhibitory activity in vitro (Scheme 3 and 4, Table 2). The N-
hydroxyhexanamide and phthalate fragments were also attached to the piperidine ring
of compound 9 to afford potential BRD4/HDAC dual inhibitor 21 and 22. It was
surprising that compound 20, 21 and 22 displayed none significant BRD4 inhibitory
capacity up to 10μM, and compound 17 was greatly superior to compound 20 in cell
proliferation assays. In addition, the HDAC2 and HDAC6 inhibitory activities of
compound 17c were moderately superior to that of compound 20 (58 nM to 136 nM,
7
3 nM to 129 nM), respectively. Then, the different linkages and ZBG fragments were
conjugated on the scaffold of compound 17 (Scheme 3, Table 2). The synthetic routes
of these dual-inhibitors are outlined in Scheme 3-5. Compounds 11a-f were reacted
with 3,5-dimethyl-4-hydroxybenzonitrile to give 12a-f, which were then hydrolyzed
with NaOH to give 13a-f. These compounds were treated with phthalates or NH OH to
2
give 14a-c or 15a-f. Compounds 14a-c were reacted with 2b to give 16a-c, and 15a-f
reacted with 2b to afford 17a-f. On the other hand, the co-crystallized structure of 4l to
BRD4 suggested another binding mode, the N-methylpiperidine moiety exposed out of
the binding pocket, providing another feasible linkage site for ZBG. As depicted in
Scheme 4 and 5, commercially available piperidin-4-one as the first starting material
was reacted with ethyl cyanoacetate and sulfur to give compound 18, which was reacted
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with methyl 6-bromohexanoate to give compound 19, and then 4-hydroxy-3,5-
dimethylbenzonitrile was reacted with the compound 19 in HCl/1,4-dioxane under
room temperature for 48h, the resultant was treated with hydroxylamine aqueous
solution in the mixed solvent CH OH and CH Cl to give compound 20. As described
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
3
2
2
before, compounds 21 and 22 were prepared by amination and amidation of 9b-d,
respectively.
Figure 3. Fragment based drug design protocols for the identification of the novel
BRD4-HDAC dual inhibitors (PDB ID: 4ZW1 for BRD4 BD1, PDB ID: 2YEM for
BRD4 BD2, PDB ID: 4LXZ for HDAC2, PDB ID: 5WGI for HDAC6 and PDB ID:
3C0Z for HDAC7).
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8
9
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
O
N
O
CN
( )n
R1
X
O
O
NH
a
b
X
( )n
+
R1
R4
R3
S
X
(
)n
NH2
R2
R4
R1
S
R3
1a-e
2a-e
3a-d
4
a-t
R2
Scheme 1. Reagents and conditions: (a) NCCH CO Et, S , Et N, EtOH, reflux, 12h;
2
2
8
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
(
b) HCl-1,4-dioxane, 25℃, overnight.
O
NH2
O
O
N
NH2
Boc
H
O
a
b
c
N
S
N
N
Boc
Boc
NH2
S
N
O
5
6
O
O
O
NH2
HN
R1
N
NH
e
NH
HN
d
H
O
N
S
N
S
S
N
N
O
N
O
N
9a-d
7
O
8
Scheme 2. Reagents and conditions: (a) NCCH CONH , S , Et3N, EtOH, reflux, 12h;
2
2
8
(
b) 3,5-dimethylisoxazole-4-carbonyl chloride, pyridine, 3h, 25℃; (c) 4M HCl/dioxane,
4
h, 25℃; (d) NaOH, EtOH, 24h, 100℃; (e) K CO , 1,4-dioxane, 25℃ or reflux, 6h.
2
3
Table 1. In vitro enzymatic and HCT-116 cell proliferation evaluations of compounds
4a-t, 9a-d.
BRD4 IC50
μM)
HCT-116
IC50 (μM)
>10
No.
n
X
R
1
R
2
R
3
R
4
(
4
a
0
0
0
0
1
CH
CH
CH
CH
CH
H
H
H
H
H
H
H
H
OH
H
H
H
>10
9.28±1.37
>10
4b
4c
4d
>10
CH
CH
H
3
3
CH
CH
H
3
3
>10
OH
H
2.64±0.54
>10
4.85±0.74
>10
4
e
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9
4
f
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
-
CH
CH
CH
N
N
N
N
N
N
N
N
N
N
N
N
-
H
H
H
CH
CH
H
OH
H
H
CH
CH
H
5.77±0.63
>10
9.73±3.16
>10
4
g
3
3
3
3
4h
4i
H
OH
H
1.81±0.23
>10
5.41±0.63
>10
CH
CH
CH
CH
3
3
3
3
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
4j
H
OH
H
H
6.31±1.08
>10
8.68±0.91
>10
4
k
CH
CH
H
3
3
CH
CH
H
3
3
4
l
OH
H
0.85±0.16
>10
1.33±0.52
>10
4
m
CH
CH
CH
CH
3
3
3
3
CH
CH
CH
CH
2
2
2
2
4
n
H
OH
H
H
>10
>10
4
o
p
q
CH
CH
H
3
3
CH
CH
H
3
3
>10
>10
4
OH
H
5.60±1.92
>10
8.91±1.97
>10
4
Bn
Bn
Bn
Bn
4
r
H
OH
H
H
>10
>10
4
s
CH
CH
-
3
3
CH
CH
-
3
3
>10
>10
4t
9a
9b
OH
-
>10
>10
CH
OOCCH
OOC(CH
3
2.41±0.31
6.68±1.08
5.93±0.56
3.62±0.53
9.77±1.05
16.21±2.04
12.70±1.09
17.03±2.58
-
-
CH
CH
CH
3
2
2
-
-
-
9
c
-
-
3
)
3
-
-
-
9
d
-
-
3
OOC(CH
-
2
)
5
-
-
-
RVX-
08
-
-
-
-
-
2.45±0.36
12.83±2.40
2
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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
NC
a
NC
NC
X
O
b
+
Br
X
O
X
OH
O
O
OH
NC
O
O
O
10
11a-f
12a-f
13a-f
O
N
N
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
NH
NH
NH2
c
e
e
H
X
N
S
S
NH2
O
H
N
X
O
O
1
4a-c
O
O
O
N
16a-c
NC
N
d
H
X
N
O
OH
H
N
O
X
O
OH
1
5a-f
1
7a-f
Scheme 3. Reagents and conditions: (a) HCl-1,4-dioxane, 25℃, 48 h; (b) 1,4-dioxane,
NaOH, 30 min; (c)DCC/HOBt, DCM, 24 h; (d) Ethyl chloroformate, N-
Methylmorpholine, 1,4-dioxane, 15 min, then NH OH, CH OH, 20 min; (e) compound
2
3
4l, HCl-1,4-dioxane, 25℃, overnight.
O
O
O
O
O
O
O
b
a
HN
HN
N
NH2
S
NH2
S
18
19
O
NH
O
c
N
HO
N
H
N
OH
S
2
0
Scheme 4. Reagents and conditions: (a) NCCH CO Et, S , Et N, EtOH, reflux, 12h;
2
2
8
3
(
b) methyl 6-bromohexanoate, DBU, THF, reflux, overnight; (c) 4-hydroxy-3,5-
dimethylbenzonitrile, HCl-1,4-dioxane, rt, 48h, then CH OH, CH Cl , HONH , 2h.
3
2
2
2
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HO
O
HN
O
N
(
)
N
n
NH
a
S
O
N
O
O
O
N
(
)
N
2
1a-c
n
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
NH
O
S
H N
2
HN
N
O
O
N
(
b
)
N
n
NH
9b-d
S
N
O
22a-b
Scheme 5. Reagents and conditions: (a) NaOH/CH OH, hydroxylamine aqueous
3
solution (50:50, w/w), 4M HCl/dioxane; (b) 2N NaOH, 4M HCl/dioxane and
thenbenzene-1,2-diamine, EDCI, HOBt, CH Cl , 12h, 25℃.
2
2
Table 2. In vitro enzymatic assays and colorectal carcinoma cell proliferation
evaluations of compounds 16a-c, 17a-f, 20, 21a-c and 22a-b.
IC50 (μM)
No.
X or n
BRD4
HDAC2
HDAC6
HDAC7
HCT-116
SW620
DLD1
1
6a
3.48±0.48
0.86±0.09
>10
>10
4.53±0.32
>10
>10
1
6b
6c
2.55±0.35
2.19±0.29
1.93±0.33
0.14±0.05
0.17±0.04
>10
>10
>10
>10
>10
4.32±0.39
6.93±0.78
5.49±0.75
5.49±0.40
9.87±0.71
7.83±0.96
>10
>10
1
1
7a
0.251±0.031
0.78±0.09
9.16±1.23
17b
17c
1.77±0.20
0.105±0.012
0.188±0.023
>10
1.07±0.05
3.87±0.66
6.24±0.59
0.71±0.09
1.17±0.21
0.058±0.004
0.16±0.04
0.073±0.005
0.25±0.09
>10
>10
0.45±0.06
2.08±0.33
1.78±0.31
3.83±0.59
2.11±0.38
9.72±0.89
1
7d
7e
1
1.19±0.18
0.097±0.011
0.085±0.006
0.202±0.016
0.159±0.008
>10
0.92±0.18
0.69±0.11
2.34±0.25
3.85±0.43
3.67±0.42
3.04±0.38
O
1
7f
0.98±0.07
>10
>10
>10
>10
>10
20
-
0.136±0.009
0.489±0.036
0.129±0.010
0.455±0.049
>10
15.82±1.43
19.60±1.58
>10
>
>
10
10
22.80±2.75
22.55±2.26
21a
21b
1
3
0.282±0.039
0.464±0.055
17.99±1.87
9.86±0.73
19.64±2.11
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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
>10
>10
0.135±0.011
0.352±0.045
0.112±0.014
0.156±0.016
0.391±0.058
0.196±0.014
15.29±2.02
28.29±3.75
15.23±2.09
8.47±0.59
15.25±2.23
11.08±1.25
16.79±1.62
15.22±1.62
14.83±2.10
2
1c
2a
2b
RVX-
08
5
1
5
>10
>10
>10
2
>
10
2
NDa
NDa
NDa
>10
12.83±2.46
>10
>10
>10
>10
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
-
-
2.45±0.36
NDa
2
SAHA
RVX-
0.035±0.004
0.047±0.007
6.94±0.78
2
08+S
-
2.54±0.28
0.031±0.006
0.049±0.005
>10
5.85±1.02
4.80±0.65
>10
AHA b
a
ND: Not determined.
b
The molar ratio of RVX-208 and SAHA was set to 1:1.
The synthesized dual-inhibitors were assayed for their inhibitory activities on
BRD4 as well as HDAC2, HDAC6 and HDAC7 by using RVX-208 and SAHA as
positive controls, respectively. Compounds 17a-f, which used hydroxamic acid as the
ZBG fragment and various types of linker group positions, had better HDAC inhibitory
activities than those of compounds 16a-c with o-phenylenediamine as the ZBG
fragment. Moreover, the inhibitory potencies of all of the compounds on HDAC2 and
HDAC6 were greatly superior to those on HDAC7; these results suggested that the
novel dual-inhibitors might selectively inhibit class I and IIb type HDACs rather than
class IIa. In addition, compounds 16a-c and 17a-f also displayed good BRD4 inhibitory
capacities at a sub-micromolar scale, which suggested that the ZBG and linker moiety
only slightly affected the BRD4 inhibitory effects. Intriguingly, the linkage of ZBG
moiety on the piperidine ring resulted in the totally loss of BRD4 inhibitory activities
in compounds 20-22. The most potent compound 17c, with IC values of 58 and 73
5
0
nM on HDAC2 and HDAC6, respectively, displayed good selectivity for HDAC 2/6
versus HDAC7. Compound 17c also displayed a moderate inhibitory capacity on BRD4,
with an IC value of 710 nM, which was better than that of the positive control RVX-
5
0
2
08.
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Table 3. HDACs and BRDs inhibitory profiling of compound 17c.
IC50 (μM)
Target
17c
JQ-1
Class I HDAC
NDa
SAHA
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
HDAC1
HDAC2
HDAC3
HDAC8
0.046±0.003
0.058±0.004
0.075±0.009
0.167±0.029
0.017±0.003
0.035±0.004
0.049±0.003
0.195±0.031
a
ND
a
a
ND
ND
Class IIa HDAC
a
HDAC4
HDAC5
HDAC7
HDAC9
>10
>10
>10
>10
ND
>10
>10
>10
>10
a
ND
a
ND
a
ND
Class IIb HDAC
a
HDAC6
0.073±0.005
0.923±0.005
ND
0.047±0.007
0.278±0.056
a
HDAC10
ND
Class IV HDAC
a
HDAC11
>10
ND
>10
BRDsb
a
a
a
a
a
a
a
BRD2(BD1)
BRD2(BD2)
BRD3(BD1)
BRD3(BD2)
BRD4(BD1)
BRD4(BD2)
16.4±1.13
>20
0.155±0.005
0.017±0.003
0.064±0.004
0.039±0.002
0.022±0.005
0.069±0.007
0.134±0.011
ND
ND
ND
ND
ND
ND
ND
>20
>20
1.10±0.14
2.05±0.71
>20
BRDT(BD1)
a
b
ND: Not determined; the BRDs inhibitory profiling were performed by an Alphascreen based
method reported in our previous study and using JQ-1 as a positive control.56
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Since compound 17c demonstrated both potent enzymatic and anti-proliferative
activity in vitro, it was further tested for its isoform selectivity on the other isoforms of
BRDs and HDACs. As shown in Table 3, compound 17c potently inhibited HDAC1-3
and HDAC6 with IC values from 45 to 482 nM and at the sub-micromolar level
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against HDAC8 and HDAC10, respectively. And there were almost no inhibitory
effects of compound 17c on class IIa and class IV HDAC isoforms up to a concentration
of 10 μM. Moreover, it was notable that compound 17c displayed better activities on
the BRD4 than on the other isoforms of BRD proteins by using the alphascreen assays,
compared to the positive control compound JQ-1, compound 17c showed higher
selectivity to BRD4 with moderate IC values. We then evaluated the anti-proliferative
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capacity of 17c on human colorectal carcinoma HCT-116, SW620 and DLD1 cells, and
7c displayed good activities with IC values of 0.45, 1.78 and 2.11 μM on HCT-116,
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SW620 and DLD1 cells, respectively. In summary, the above results suggested that 17c
deserved further development with potent BRD4-HDAC dual inhibition.
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Figure 4. The docked conformations and binding modes of 17c to BRD4 (A, PDB ID:
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ZW1), HDAC2 (B, PDB ID: 4LXZ) and HDAC6 (C, PDB ID: 5WGI), respectively.
The proteins were illustrated by solvent-accessible surface with charges (blue for
positive, red for negative), the binding site residues were highlighted.
Binding modes of 17c to BRD4, HDAC2 and HDAC6
The compound 17c was docked into the binding pocket of BRD4, HDAC2 and HDAC6
proteins, respectively. The docking results were illustrated in Figure 4. The hydroxamic
group of compound 17c stable chelated with the zinc atom in the binding sites of
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HDACs. Moreover, the thieno[2,3-d]pyrimidine fragment of compound 17c fitted into
the surface groove and occupied the cap region well. The carbonyl fragment of
hydroxamic group could form hydrogen bonds interaction with Tyr308 residues of
HDAC2 and Tyr745 residues of HDAC6, respectively. The hydroxylamine fragment
of 17c interacted with His145/His146 residues of HDAC2 and His614 residue of
HDAC6 via stable hydrogen bonds, respectively. And the phenyl substituted at 2-
position of thieno[2,3-d]pyrimidine formed a cation-π interaction with Arg275 residue
of HDAC2. In addition, the 3-nitrogen atom and 4-carbonyl group of thieno[2,3-
d]pyrimidine could form H-bond with Asn140 residues of BRD4. The N-
methylpiperidine group was placed between the WPF stack (Trp81, Pro82 and Phe83).
Furthermore, 17c derived supplementary activities by deploying the long alkanes chain
and hydroxamic group into the ZA channel. In general, the molecular docking results
displayed good agreement with the results of enzymatic assays.
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7c induces cell cycle arrest, apoptosis and autophagic cell death and in colorectal
carcinoma cells
The cytotoxicity of 17c was screened against a panel of colorectal carcinoma cells.
As shown in Figure 5, the IC of 17c was remarkably higher in CRC cells than in
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normal colorectal epithelial cells. Moreover, the cell cycles of HCT-116 cells after
being treated with 17c were determined by a flow cytometry method. As shown in
Figure 5B, the cell cycles results displayed obviously sub-G1 peaks in the 0.2 μM and
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.5 μM of compound 17c treated HCT-116 cells, which indicated that the apoptotic cell
death occurred. The death subroutines of 17c-treated HCT-116 cells were detected by
flow cytometry analysis using the Annexin V/PI staining kit (Figure 5C and D). The
percentages of Annexin V-positive apoptotic cells in the 17c treatment groups were
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3.2 ± 5.30% (0.2 μM) and 45.3 ± 6.8% (0.5 μM), which were significantly higher than
in the control (6.3 ± 0.4%, p < 0.05) group. However, there were significant differences
in apoptotic cells between the 0.2 μM and 0.5 μM of 17c treated groups. These results
suggest that 17c induced apoptotic death in a dose-dependent manner. The nuclei
morphology after Hoechst 33258 staining (Figure 5E) in 17c-treated HCT-116 cells
revealed that apoptotic cell death was apparent after 0.2 μM and 0.5 μM 17c treatments.
The colony formation of HCT-116 cells was also potently suppressed after 17c
incubation for 24 hours (Figure 5F).
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Figure 5. Compound 17c induced apoptotic cell death in HCT-116 cells: A. The cell
proliferation IC values were measured for compound 17c on a panel of colorectal
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carcinoma cells by MTT assay; B. The cell cycle analysis of HCT-116 cells after 0.2μM
and 0.5μM of 17c treated; C and D. The apoptosis assay of HCT-116 cells after 0.2μM
and 0.5μM of 17c treated by Annexin V/PI dual-staining; E. The nuclei morphology of
HCT-116 cells after 0.2μM and 0.5μM of 17c treated; F. The colony formation assay
of HCT-116 cells after 0.2μM and 0.5μM of 17c treated.
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To determine the effects of 17c, RVX-208 and SAHA on the expression level of
histone H3 as well as acetylated histone H3, the biomarkers of HDAC inhibition in
HCT-116 cells were determined via the western blotting method. As shown in Figure
6
, in agreement with their relative potencies in enzymatic assays, 17c and SAHA
upregulated Ac-H3 levels in a dosage-dependent manner, and the expression levels of
histone H3 were not affected, while both H3 and Ac-H3 were not disturbed by RVX-
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08 (Data not shown). Moreover, the expression levels of BRD4 were declined by the
addition of 17c or RVX-208, and 17c treatment suppressed the downstream c-Myc
proteins more efficiently than that of RVX-208. As expected, 17c induced more
cleavage of PARP, caspase-3 and caspase-9, which suggested more mitochondrial
apoptosis was induced by BRD4/HDAC dual inhibitor.
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Figure 6. Compound 17c induced mitochondrial apoptosis via BRD4-HDAC dual
inhibition. HCT-116 cells were treated with 0.2 or 0.5µM of compound 17c, positive
control RVX-208 or SAHA for 24 hours, respectively. Then the expression levels of
BRD4, c-Myc, Histone H3, Ac-H3, Bim, Bcl-2, Fas, FasL, PARP, Caspase-3 and
Caspase-9 were detected by western blot analysis.
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Figure 7. Compound 17c induces autophagic cell death in colorectal carcinoma cells.
(A) HCT-116 cells were incubated with 0.5 μM of 17c for 24 hours, then the cells were
fixed and imaged under transmitted electron microscopy; scale bar = 1 μm. (B) HCT-
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16 cells were transfected with a GFP-LC3 plasmid, followed by treatment with 0.5
μM 17c, the formation of autophagosomes were observed under a confocal
fluorescence microscope, scale bar = 6 μm. (C) After 17c incubation for 24 hours, the
protein expression of LC3, p62, and beclin-1 were detected by WB analysis. (D) HCT-
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viability was determined by MTT assay. ∗∗∗ indicated p < 0.001 and ∗∗ indicated p <
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.01. (E) HCT-116 cells were treated with 0.5 μM of 17c with or without Bafilomycin-
A1 for 24 hours, the protein expression of LC3 and p62 were detected by WB analysis.
F) HCT-116 cells were transfected with ATG5 siRNA or scramble siRNA,
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(
respectively. After RNA silence, with 0.5 μM of 17c or normal saline incubation, the
protein expression of ATG5, AMPKα, p-AMPKα, p62, and LC3 were determined by
WB analysis. (G) HCT-116 cells were treated with 0.5 μM 17c, 2.0 μM 4l or 10 μM
SAHA for the 24 hours, respectively. Then the protein expression of LC3 and p62 were
detected by WB analysis.
There were several reports suggested that BRD4 protein was an autophagy
suppressor in cancer cells, therefore inhibition of BRD4 might resulted in autophagy
initiation.55, 57-59 In current study, we observed 17c induced the formation of cytoplasm
autophagic vacuole in HCT-116 cells under transmission electron microscope (Figure
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A), and the increased LC3 puncta in GFP-LC3 transfected HCT-116 cells were also
observed after 0.5 μM of 17c incubation for 24 hours, these results suggested the
initiation of autophagy in HCT-116 cells (Figure 7B). The 17c-induced autophagy was
further confirmed by the WB analysis of autophagy protein markers, we found that 17c
incubation resulted in the declined expression of p62, upregulation of beclin-1 and
dosage-dependent cleavage of LC3-I to LC3-II in HCT-116 cells (Figure 7C). To
further validate the cytotoxic or cyto-protective effects of 17c-induced autophagy, 3-
MA (3-methyladenine), an inhibitor of type III phosphatidylinositol 3-kinases (PI3KIII)
was employed to block the initiation of autophagic flux. As depicted in Figure 7D, the
cytotoxicity of 17c were potently reversed by the addition of 500 μM 3-MA (p < 0.01).
In addition, an autophagolysosome inhibitor bafilomycin A1 (BafA1) was utilized to
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validate the effects of 17c on autophagic flux (Figure 7E). Both LC3-II and p62 were
accumulated after the combined treatment of BafA1 and 17c. These results suggest that
compound 17c induced autophagic cell death in HCT-116 colorectal carcinoma cells.
In recent reports, the BRD4-AMPK-mTOR-ULK signaling axis was suggested as an
upstream autophagy signaling pathway in cancer cells.55, 59, 60 To further determine
whether 17c-induced autophagy was depended on ATG5, the ATG5 mRNA was
interfered by a specific siRNA with or without the combination of 17c incubation
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(Figure 7F). In agreement with our previous report, the mRNA knockdown of ATG5
did not interfere the 17c-induced AMPK activation, however, the cleavage of LC3 and
degradation of p62 were obviously suppressed. Moreover, the autophagy induction
capacities of 17c and the corresponding BRD4i and HDACi fragments were detected
by WB analysis (Figure 7G). The high concentration of SAHA (10 μM) only led to
marginal autophagy, and 0.5 μM 17c incubation resulted stronger autophagy than that
of 2.0 μM 4l. Taken together, above results indicated that 17c induces autophagic cell
death via BRD4-AMPK signaling pathway.
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7c suppresses IL6-JAK-STAT pathway in colorectal carcinoma cells
Recent studies suggested that the activation of IL6-JAK-STAT signaling pathway
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might be a vital factor of drug resistant to HDAC inhibitors in cancer cells. And the
addition of BRD4 siRNA or inhibitor could reverse this drug resistance via suppressing
the activation of IL6 family cytokines. In the current study, we also observed the
transcriptional activation of four IL-6 family cytokines by RT-PCR, including
interleukin 6, oncostatin M(OSM), leukemia inhibitory factor (LIF) and cardiotrophin
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(CTF1) were stimulated by 10 μM SAHA incubation (Figure 8A). And the 4l
treatment did not change the expression of these cytokines, 17c treatment only slightly
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elevated these cytokines. The WB analysis of representative protein markers in IL6-
JAK-STAT signaling pathways also indicated that SAHA could activate IL6 family
cytokines and downstream JAK-STAT signaling axis, and these effects of HDAC
inhibitors were not observed in 17c treated group (Figure 8B). These results suggested
that these BRD4/HDAC dual inhibitors possibly overcome the drug resistance
mechanism of single HDAC inhibition.
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Figure 8. (A) The change of IL6, OSM, LIF and CTF1 mRNA levels in HCT-116 cells
after incubated with 0.5 μM 17c, 2.0 μM 4l or 10 μM SAHA for the 24 hours,
respectively. (B) HCT-116 cells were treated with 0.5 μM 17c, 5.0 μM 4l or 10 μM
SAHA for the 24 hours, respectively. Then the protein expression of OSMR, LIFR,
JAK1, STAT3 and phosphorylated STAT3 were detected by WB analysis.
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7c inhibits tumor growth in xenograft models
We subsequently determined that 17c bears potent inhibitory effects on the BRD4-
HDAC pathways in vivo, and then, HCT-116 human colorectal carcinoma xenograft
models were established to evaluate the efficacy of 17c. The mean tumor data after 19
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days of oral administration of SAHA or 17c are presented in Figures 9. Both 17c and
SAHA exhibited potent antitumor activity at the dosage tested, and the average
percentage of tumor growth inhibition in the 17c groups was 68.8%. Moreover, the 17c-
treated groups displayed a dose-dependent decrease in tumor volume compared with
the saline control (Figure 9B), with total treatment-to-control ratios (T/C) in the 17c 15
mg/kg and 30 mg/kg groups of 42.7% and 68.8%, respectively. As expected, there was
a significant difference between the 17c group and the SAHA group. Therefore, 17c
was effective in reducing the growth of HCT-116 tumors in a xenograft model in vivo.
Furthermore, to confirm whether the molecular mechanism of 17c was based on the
BRD4 and HDAC pathways in vivo, the changes in the expression levels of Ac-H3 and
c-Myc were determined by immunohistochemical methods as well as apoptosis marker
TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and Ki-67
staining. As shown in Figure 9D, the expression levels of Ac-H3 and c-Myc in HCT-
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16 xenograft tumor tissues in the vehicle- and 17c-treated groups were measured by
IHC analysis. The expression level of c-Myc was reduced in the 17c-treated group, and
Ac-H3 was elevated. Ki-67 is a proliferation marker with prognostic and predictive
potential in colorectal carcinoma. The results of the IHC analysis and statistical analysis
were also presented in Figure 9D. A significant reduction in the expression of Ki-67
was observed upon treatment with 17c. Histological assessment of apoptosis by
TUNEL staining revealed that TUNEL-positive apoptotic nuclei were significantly
increased by 17c. In agreement to the in vitro experiments, the autophagy marker LC3-
II was elevated in 4l and 17c treated groups. The protein expression of LIFR and
phosphorylated STAT3 were also activated in SAHA treated group, and these
phenomena were not observed in 17c treated group. In addition, there were no
significant changes observed in the H&E (hematoxylin and eosin) staining sections of
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the main organs as well as body weights of mice in the 17c-treated groups (Figure S2).
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Figure 9. In vivo anticancer effect of compound 17c on the HCT-116 xenograft models:
A. The tumor volume changes with oral administration 15 or 30 mg/Kg of compound
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7c, 30mg/Kg of 4l or SAHA, **: p < 0.01 compared to control group; B. The tumor
weights analysis after the end of therapy; C. The body weight changes of mice; D.
Representative images of immunohistochemical analysis of proliferative marker Ki-67,
c-Myc, Ac-H3, BRD4, LC3-II, LIFR, p-STAT3 and immunofluorescent analysis of
apoptosis marker TUNEL in different groups; scale bar = 50μm; *: p < 0.05 compared
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