Novel phenanthridin-6(5H)-one derivatives as potent and selective BET bromodomain inhibitors: Rational design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.06.067

Inhibition of BET family of bromodomain is an appealing intervention strategy for several cancers and inflammatory diseases. This article highlights our work toward the identification of potent, selective, and efficacious BET inhibitors using a structure-

Full text of DOI:10.1016/j.ejmech.2019.06.067

Accepted Manuscript
Novel phenanthridin-6(5H)-one derivatives as potent and selective BET bromodomain
inhibitors: Rational design, synthesis and biological evaluation
Yanle Zhi, Shu Wang, Wenhai Huang, Shenxin Zeng, Meihao Liang, Chixiao Zhang,
Zhen Ma, Zunyuan Wang, Zhimin Zhang, Zhengrong Shen
PII:
S0223-5234(19)30594-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.067
EJMECH 11470
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 May 2019
Revised Date: 12 June 2019
Accepted Date: 24 June 2019
Please cite this article as: Y. Zhi, S. Wang, W. Huang, S. Zeng, M. Liang, C. Zhang, Z. Ma, Z. Wang, Z.
Zhang, Z. Shen, Novel phenanthridin-6(5H)-one derivatives as potent and selective BET bromodomain
inhibitors: Rational design, synthesis and biological evaluation, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.067.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel phenanthridin-6(5H)-one derivatives as potent and
selective BET bromodomain inhibitors: rational design,
synthesis and biological evaluation
a
b
b
b
b
Yanle Zhi , Shu Wang , Wenhai Huang , Shenxin Zeng , Meihao Liang , Chixiao
b
b
b
b,*
b,*
Zhang , Zhen Ma , Zunyuan Wang , Zhimin Zhang ,Zhengrong Shen
a
College of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou
4
50046, Henan Province, P. R. China
b
Key Laboratory of Neuropsychiatric Drug Research of Zhejiang Province, Institute
of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou, 310013, P. R.
China
Correspondence
Zhimin Zhang, Institute of Materia Medica, Zhejiang Academy of Medical Sciences,
Hangzhou 310013, P. R. China Tel.: +86 571 88215626; fax: +86 571 88215625.
E-mail address: zzm996@126.com
Zhengrong Shen, Institute of Materia Medica, Zhejiang Academy of Medical Sciences,
Hangzhou 310013, P. R. China Tel.: +86 571 88215506; fax: +86 571 88215625.
E-mail address: shenzr601@163.com

ACCEPTED MANUSCRIPT
Novel phenanthridin-6(5H)-one derivatives as potent and
selective BET bromodomain inhibitors: rational design,
synthesis and biological evaluation
Abstract
Inhibition of BET family of bromodomain is an appealing intervention strategy for
several cancers and inflammatory diseases. This article highlights our work toward
the identification of potent, selective, and efficacious BET inhibitors using a
structure-based approach focused on improving potency. Our medicinal chemistry
efforts led to the identification of compound 24, a novel phenanthridin-6(5H)-one
derivative, as a potent (IC = 0.24 µM) and selective BET inhibitor with excellent
50
cancer cell lines inhibitory activities and favorable oral pharmacokinetic properties.
Keywords: Rational drug design; BET bromodomain inhibitor; antiproliferative
effect; apoptosis; pharmacokinetics
1
. Introduction
The bromodomain and extra-terminal (BET) family of proteins are epigenetic
readers which mediate the expression of therapeutically important genes such as
C-myc through the selective recognition of acetylated histones [1]. The BET proteins
consist of bromodomain containing protein 2 (BRD2), BRD3, BRD4, and the
testis-specific BRDT and have been positively recognized as promising therapeutic
targets for the treatment of cancer, inflammation [2], and viral infectious diseases [3,
4
]. BRD4, the most extensively studied member, has been shown to recruit the
positive transcription elongation factor b (P-TEFb), and regulated the phosphorylation

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of the RNA polymerase Ⅱ in the transition from transcription initiation to productive
elongation [5, 6]. BRD4 contains two N-terminal bromodomains (BD1 and BD2) that
selectively bind to acetylated lysine (KAc) in histones H3 and H4 [7]. More
specifically, the KAc residues bind in the active site of BRD4 with an evolutionarily
conserved asparagine (Asn140) that acts as a hydrogen bond donor, and through an
interaction between the acetyl carbonyl oxygen atom and the phenol of a conserved
tyrosine (Tyr97) via a structured water molecule [8]. Disrupting the protein-protein
interactions between bromodomain and KAc by inhibitors can be a viable therapeutic
target for many diseases [9, 10]. In the last few years, a number of high-affinity small
molecule BET inhibitors have been identified [11-16]. Till now, over 20 clinical trials
are underway for studying the effects of BET family inhibition as anti-cancer or
anti-inflammatory therapies [17, 18]. Most reported inhibitors act as pan-BET
bromodomain inhibitors due to the high sequence identity among BET proteins. JQ-1
(1) [19] and I-BET151 (2) (figure 1) [20] were first demonstrated the therapeutic
potential of BET bromodomain inhibitors and have been extensively employed to
evaluate the biological functions of BET bromodomain. Compounds 3-6 (figure 1) are
currently in phase I and phase II clinical trials for treatment of solid tumors and
hematological malignancies [16]. Compound 6 was initially described to upregulate
ApoA1 expression and increase the high-density lipoprotein mass and has recently
been reported to be a BET inhibitor [21, 22]. Considering these many phenotypes and
strategies of therapeutic relevance, novel chemotypes of BET inhibitors are being
intently pursued.

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Figure 1. Structures of typical BET bromodomain inhibitors JQ1 (1), I-BET151 (2), I-BET762 (3),
OTX-015 (4), TEN-010 (5), RVX-208 (6) and our previously reported compound 7.
In our ongoing efforts to identify potent and novel selective BET inhibitors, we
recently reported 4-chloro-2-methoxy-N-(2-oxo-2H-chromen-6-yl)benzenesulfon-
amide (7) as a potent and orally bioavailable BRD4 bromodomain inhibitor [23].
Compound 7 binds to BRD4 with nanomolar affinities and shows micromolar cell
growth inhibitory activities in four cell lines. Besides, compound 7 has a good
pharmacokinetic profile in rats. However, compared to some other reported inhibitors
[
24-27], both the BRD4 affinities and cell growth inhibitory activities of compound 7
are still remained to improve. We have therefore decided to pay our efforts toward the
generation of novel, highly potent, efficacious, and orally active BET bromodomain
inhibitors starting from compound 7.
By carefully analysis of the docking information of compound 7 with BRD4, we
identified an unoccupied hydrophobic region within the binding site. To the best of
our knowledge, few attentions have been paid to this hydrophobic region in designing
novel BET bromodomain inhibitors or improving the binding affinity of reported BET

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bromodomain inhibitors. In this article, focused on this significant but previously not
studied hydrophobic region, we reported the design, synthesis and biological
evaluation of phenanthridin-6(5H)-one derivatives as a novel class of BET
bromodomain inhibitors with promising therapeutic effects. The SAR investigation
and the pharmacological mechanisms, pharmacokinetics and in vitro anti-tumor
activity of selected compounds were also presented.
2
. Results and Discussion
2
.1 Binding Site Analysis and Design of Novel Phenanthridin-6(5H)-one Scaffold
Better understanding the structure of protein binding site is a cornerstone of
structure-based drug design. In our efforts to further improve the potency and increase
structural diversity of coumarin derivatives BET inhibitors, we examined the docking
formation of compound 7 in more detail with the aim of identifying areas where
additional binding interaction might be gained. As shown in figure 2A, the coumarin
moiety of 7 forms two hydrogen bonds with Asn140 and Tyr97 via a conserved water
molecule in the KAc binding site of BRD4. The 4-chloro-2-methoxybenzene group
occupies the hydrophobic WPF shelf and forms hydrophobic interactions with
Met149, Asp144, Asp145 and Ile146. The sulfonamide forms two additional
hydrogen bonds with two water molecules. In addition to these main interactions, a
large space was identified within the binding site that was not fully occupied by
compound 7. We refer to this space as the “S1 pocket” (highlighted by the red patch
in figure 2A). Furthermore, we applied SiteMap in the Schrödinger software suite
(Schrödinger, LLC: New York 2011) to take key steps toward efficient

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structure-based drug design by gaining insights into binding sites, which can be used
to suggest modifications to a ligand’s structure (both shape and physical properties)
[
28, 29]. The active site points and surfaces generated by SiteMap for BRD4 are
shown in figure 2B. These figures revealed that portions of the hydrophobic groups of
compound 7 occupy the hydrophobic regions, wherein the hydrogen bond donors and
acceptors of the ligand for the most part lie in or close to appropriate donor and
acceptor of BRD4.
Figure 2. (A) The docking model of compound 7 cbound to BRD4(1) (PDB entry 3MXF). The
red patch on the protein surface indicates a hydrophobic region. Compound 7 is shown in stick
with carbon atoms colored in cyan, oxygen atoms in red, sulphur atoms in yellow and nitrogen
atoms in blue. Water molecules are shown as blue spheres, and the hydrogen bonds were denoted
by golden dash lines. The figure was prepared using PyMOL. (B) SiteMap regions for the binding
of compound 7 to BRD4 displays 7 (cyan sticks) and the SiteMap site points (white spots) against
the background of the protein surface. The protein backbone is shown in ribbon. The figure
displays compound 7 in the context of the hydrophobic, ligand acceptor, and ligand donor maps
(green, red, and purple, respectively). The figure was prepared using Schrödinger software.
The previously unexplored S1 pocket was a hydrophobic region indicated by
Sitemap, which has greatly inspired our interest in this pocket. We postulated that an
improvement on binding affinity might be achieved by occupying S1 pocket through

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larger hydrophobic moieties. Hence, we combined a hydrophobic group (benzene ring)
on the coumarin scaffold (figure 3). Moreover, replacing of the O atom of coumarin
by NH might yield additional hydrogen-bond interactions for a hydrogen bond
acceptor (Asn140) close to the coumarin scaffold (figure 3). To test the above
hypothesis, we prepared the phenanthridin-6(5H)-ones derivatives and evaluated their
ability to disrupt the bromodomain−KAc interaction.
Figure 3. Flowchart summarizes the discovery and optimization strategy for novel
phenanthridin-6(5H)-one scaffold from coumarin scaffold.
2
.2 SAR Studies of the Linkers
Besides a KAc binding moiety, a typical BET inhibitor also contains a specific
linker to connect a group for reaching into the hydrophobic binding site known as the
WPF shelf pocket to gain essential potency and selectivity. It is believed that only the
specific linkers will allow the group to extend and reach the WPF subpocket. In order
to find out an optimal linker, we evaluated various sulfonamides, ureas and
carbamates derivatives attached with a phenyl group. As shown in Table 1, it was
found that when the linker was sulfonamide (compounds 11 and 12) it showed better
potency than those having linkers of ureas (compound 10) or carbamates (compounds
8
and 9). Moreover, addition of a carbon atom between the phenyl ring and sulfonyl
reduced the binding activity by almost 3-folds (in comparision of compound 11 with

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compound 12).
Table 1. Results of compounds 8-12 on BRD4 binding activities
a
Comp. No.
Linker
BRD4 IC (µM)
50
8
18.61±6.52
6.02±1.61
9.93±3.07
1.64±1.64
5.17±1.20
9
10
11
12
a
The IC50 in the table was calculated from two independent experimental measurements and
expressed as mean ± SD
To better understand the remarkable difference between these linkers, we docked
all the five compounds 8-12 to BRD4. As depicted in figure 4, only the sulfonamide
group (compound 11, carbon atoms colored in cyan) makes a turn to enable the
phenyl ring go into the WPF shelf.

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Figure 4. The docking model of compounds 8-13 bound to BRD4(1) (PDB entry 3MXF). 8 is
shown in stick with carbon atoms colored in yellow, 9 in green, 10 in blue, 11 in cyan and 12 in
pink. The figure was prepared using PyMOL.
2
.3 SAR Studies of Sulfonamide Derivatives Occupying the WPF Shelf
Taking advantage of the above knowledge, we next explored the SAR around the
WPF shelf while maintaining the sulfonamide as the linker (table 2). First, lipophilic
alkyl or cycloalkyl substituted compounds 13-16 were synthesized. However, no
matter the change of alky chain length or cycloalkane size, the BRD4 binding
activities of 13-16 reduced markedly compared with the phenyl group substituted
compound 11. This results probably due to a reduction of hydrophobic interactions
with WPF shelf. Subsequently, we focused our optimization back to the phenyl group
of compound 11 which preferably occupied the hydrophobic WPF shelf. An extensive
SAR exploration was then conducted, displaying that diverse substitutions at different
positions of the phenyl group in compound 11 can lead to various effects on the
BRD4 affinity. We preferentially evaluated the 2-chlorophenyl group and
2
-methoxyphenyl group. The biochemical activity results showed that

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ortho-substituents on the ring were very favorable. Chloro-substituent at
ortho-position was well tolerable and the corresponding compound 17 showed
comparable activity to compound 11, while compound 18, which bears an
ortho-position methoxy group, showed more potent with a 3-fold increase of activity.
We next investigated the para-position substituents (19-21). The para-chlorine
substituted analogue 20 was somewhat more potent than para-tertiary butyl
substituted analogue 19 and para-methoxyl substituted analogue 21, which may
indicate that electron-withdrawing groups at the para-position was favorable. We
further investigated the impact of meta-position substituents (compounds 22 and 23).
Interestingly, compound 22, which bears a meta-position cyan group showed
moderate activity, while, compound 23 bears a meta-position chlorine resulted in a
2
-fold increase of activity compared with compound 11. From the above SAR, it was
identified that chlorine at para-position (compound 23) or methoxy group substituent
at ortho-position (compound 18) possesses the favorable affinity on BRD4, so we
attempted to merge these two positions as bi-substituent. In line with our expectations,
the resulting 5-chloro-2-methoxy di-substituent compound 24 significantly improved
the activity with an IC of 0.24 µM, almost 7-fold increase compared with compound
5
0
1
1. For more understanding the SAR, we further replaced the phenyl ring of
compound 11 by a smaller size thienyl group, the resulting derivative 25 showed low
binding activity. The replacement by larger aromatic group also led to a reduction of
affinity (compounds 26 and 27).
Table 2. Results of compounds 13 -27 on BRD4 binding activities

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a
b
c
Comp. No.
R
BRD4 IC (µM)
cLogP
2.33
LE
5
0
0
.30
.31
13
14
15
16
17
6.71±2.66
5.54±3.58
6.17+2.06
5.92±6.02
1.25±0.25
0
2.85
2.74
3.30
3.44
0.29
0.28
0.32
1
1
8
9
0.48±0.13
1.13±0.56
2.57
4.72
0.33
0.29
2
2
0
1
0.85±0.22
1.32±0.39
3.74
3.05
0.33
0.30
2
2
2
2
2
4
1.79±0.61
0.79±0.26
0.24±0.27
2.86
3.74
3.39
0.30
0.33
0.34
2
2
2
5
6
7
6.74±1.81
7.23±2.44
13.12±3.69
2.67
4.07
0.30
0.25
3.19
4.82
0.24
0.33
JQ1
-
0.04±0.05
a
b
The IC50 was calculated from the AlphaScreen assay. cLogP values were calculated using
c
ChemBiodraw Ultra14.0. LE (Ligand Efficiency) = 1.4 (pIC /heavy atoms)
5
0

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2
.4 Docking Study of the Preferred Configuration of Compound 24
To elucidate the observed activity of compound 24, we performed a molecular
docking study starting from the crystal structure of the protein complexed with JQ1.
At first, self-docking procedure was successfully applied on JQ1, well reproducing
the crystallographic pose of JQ1 inside the binding site. By superimposing the
cocrystal structures of JQ1 to our docking structure of compound 24 bound to BRD4
(figure 5A), we found that these two molecule shared high overlaps. It is worth
pointing out that compound 24 fits the hydrophobic region better than JQ1. The
docking simulations (figure 5B) performed on compound 24 illustrated that the
phenanthridin-6(5H)-one establishes two hydrogen bonds to the conserved residue
Asn140 and a water-mediated hydrogen bond to Tyr97. The sulfonamide group made
a right angle to introduce the 5-chloro-2-methoxybenzene moiety toward the WPF
shelf. The moiety well occupied the hydrophobic WPF shelf and forms hydrophobic
interactions with Met149, Asp144, Asp145, and Ile146. Moreover, the sulfonamide
group itself forms two additional hydrogen bonds with two water molecules.
Figure 5. (A) Superimposed the cocrystal structure of BRD4 bound with JQ1 and the docking
model of compound 24 bound to BRD4 (PDB entry 3MXF). JQ1 and compound 24 are shown in

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stick with carbon atoms colored in yellow and magenta, respectively. (B) The docking model of
compound 24 bound to BRD4 (PDB entry 3MXF). Water molecules are shown as red spheres, and
the hydrogen bonds are denoted by blue dash lines. The figures were prepared using PyMOL.
2
.5 Evaluation of the Bromodomains Selectivity of Compound 24.
It is well established that the selectivity is critical for the success of drug discovery.
To investigate the selectivity profile, the compound 24 was chosen as a representative
and further evaluated against a panel of BET and non-BET bromodomains. The
AlphaScreen technology FRET assay was employed to test the binding tendency of
compound 24. As shown in figure 6, compound 24 showed excellent selectivity for
BET bromodomains (BRD2, BRD3, BRD4 and BRDT) over other non-BET
bromodomain-containing proteins. Compound 24 revealed high potency for BET
family members with an affinity of > 70% under 20 µM. In addition, moderate
activities for BFPF1B and BRD9 were observed, with the affinity of > 30%, which
suggests that compound 24 might be a promising starting point for developing
BFPF1B and BRD9 inhibitors.
Figure 6. Selectivity assessments of compound 24 against BRD families. The affinity percentages
were obtained at 20 µM.

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2
.6 Antiproliferative Activity of Compound 24 on Four Cell Lines
The representative compound 24 was next evaluated for its effects on the survival
of four cell lines with an MTT assay. The data obtained were summarized in Table 3
and the dose–response curves were provided in figure 7. Results indicated that,
consistent with its high affinity to BRD4, compound 24 potently inhibits the
proliferation in these four cell lines, with IC values of 0.75, 4.83, 6.70, and 5.35 µM,
5
0
respectively. It is noteworthy that, in the A549 cell line, compound 24 exhibited
excellent activity, which was worth in-depth study in its mechanism of anti-lung
adenocarcinoma action. Overall consideration of the data from the above assays,
compound 24 has good profiles for further evaluation.
Figure 7. Dose–response curves of compound 24 in incubation with cancer cell lines (mean ±SD,
n = 3).
Table 3. Anti-proliferation effects of compound 24 against four cell lines.
Cancer Type
Lung
Cell Line
A549
IC (µM)
50
0.75
4.83
adenocarcinoma
Hepatocellular
carcinoma
Pancreatic
carcinoma
Gastric
HepG2
PANC-1
6.70
5.35
SGC-7901

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adenocarcinoma
2
.7 Effects of Compound 24 on C-myc Protein Expression
Previous studies have shown that targeted inhibition of BET can effectively
down-regulate the transcription of C-myc, an oncogene frequently overexpressed in
various tumors [30, 31]. As shown in our above study, the A549 cell line is very
responsive to BET inhibitors, and accordingly we examined the effect of compound
2
4 in A549 cells on C-myc with JQ1 as the positive control (Figure 8). Consistent
with its high binding affinities to BET proteins, the western blot data showed that
compound 24 was very effective in inducing the downregulation of C-myc at a
concentration as low as 100 nM in a dose dependent manner.
Figure 8. Western blotting analysis of the C-myc protein in A549 cells treated with compound 24
or JQ1. A549 cells were treated for 48 h at the indicated concentrations. GAPDH was used as the
loading control.
2
.8 Effect of Compound 24 on Cell Apoptosis
Inducing apoptosis is considered as one of the major strategies for antitumor-drug
development. We employed flow cytometry analysis to detect the effect of compound
4 on apoptosis in A549 cells so as to further explore the anti-proliferative
2
mechanism of compound 24. Quantitative analysis of necrotic cells,
advanced-apoptotic cells and early-apoptotic cells was investigated through an

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Annexin V-FITC and PI assay. A549 cells were incubated with 0.3–5.0 µM
concentrations of compound 24 for 48 h with DMSO as the negative control. As
shown in Figure 9, compound 24 treatment preliminarily induced A549 cell apoptosis
at a concentration as low as 0.3 µM after 48 h incubation. Compound 24 was also
found to effectively induce apoptosis in a dose-dependent manner. Furthermore,
compound 24 significantly induced the apoptosis of A549 with the low dose of 1.0
µM (Figure 9). The apoptosis ratios of compound 24 measured at different
concentrations were 30.3% (0.3 µM), 53.9% (1.0 µM), 75.4% (3 µM), and 80.3% (5
µM).
Figure 9. Flow cytometry analysis of apoptosis induction by compound 24 in A549 cells at the
indicated concentrations for 48 h. Apoptosis was assessed by flow cytometry using Annexin V
and propidium iodine (PI) double staining.
2
.9 Effect of Compound 24 on Cell Cycle
We also employed flow cytometry analysis to investigate the ability of the
compound 24 to induce cell cycle arrest in A549 cell lines. A549 cells were incubated

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with 0.3–5.0 µM concentrations of compound 24 for 48 h with DMSO as the negative
control. The results shown in figure 10, indicated that the cell cycle spectrum clearly
changed as the compound’s concentrations increased. Compound 24 arrested the
cell-cycle progression of the cell line into the G1 and M phases compared with
vehicle treatment in a dose-dependent manner. The percentage of cells in G1 and M
phase after treatment by compound 24 under concentrations of 0.3, 1.0, 3.0 and 5.0
µM were 60.84%, 70.30%, 76.42% and 81.66%, respectively.
Figure 10. Effects of compound 24 on A549 cell cycle arrest at the indicated concentrations for
4
8 h.
2
.10 Microsomal Stability of Compound 24.
Our next attention was on assessing the selected compound 24 for its metabolic
stability in vitro using human liver microsomes (HLMs), a system extensively
employed to estimate the susceptibility to first-pass oxidative metabolism, a main
cause of metabolic degradation in vivo. Compound 24 was incubated with HLMs at
3
7 °C for 2 h. The regression line of concentration-time was summarized in figure 11.
The metabolic stability data indicated that compound 24 showed good microsomal

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stability with half-life of 4.17 h.
Figure 11. Metabolic stability of compound 24 on human liver microsomes.
2
.11 In vivo Pharmacokinetic (PK) Studies of Compound 24
The favorable in vitro metabolic stability prompted us to evaluate compound 24 in
vivo. Compound 24 was administered orally (p.o) or intravenously (i.v) to male
Sprague-Dawley rats at a dose of 10 mg/kg and 2.5 mg/kg, respectively. The plasma
concentrations were determined by liquid chromatography-tandem mass
spectrometric (LC-MS/MS). The mean arterial plasma concentration-time curves
were shown in Figure 12 and the pharmacokinetics parameters were concluded in
Table 6. Compound 24 achieves a Cmax of 52.3 µg/L and an AUC of 565.56µg/L*h
with oral administration and has an oral bioavailability (F %) of 36.6%. Compound 24
exhibited low clearance (CL) and high volume of distribution (V ). Collectively, the
d
results of this study indicated that compound 24 had favorable pharmacokinetic
properties.

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Figure 12. The plasma concentration-time curve of compound 24.
Table 4. Intravenous (i.v.) and oral (p.o) pharmacokinetic profiles of compound 24 in rats.
PK Parameters
AUC(0-t) (µg/L*h)
AUC(0-∞) (µg/L*h)
T1/2 (h)
i.v (2.5 mg/kg)
383.10
505.03
10.38
0.083
7.93
SD
43.54
85.83
0.77
0
p.o (10 mg/kg)
565.56
670.38
3.87
SD
140.89
15.71
1.01
Tmax (h)
5.33
1.15
MRT0-t (h)
0.56
0.86
10.92
9.36
-
7.52
1.80
CL (L/h/kg)
5.15
18.18
5.65
z
V (L/kg)
d
76.70
96.87
-
97.34
8.38
Cmax (µg/L)
52.37
5.67
F (%)
36.60%
2
.12 Chemistry
The syntheses of novel phenanthridin-6(5H)-one derivatives were concluded in
scheme 1. As depicted, The commercially available phenanthridin-6(5H)-one (I)
reacted with concentrated nitric acid in acetic acid under 100 Ⅱ to produce compound
II. Regrettably, the reaction of compound II under ammonium chloride and iron
powder system led to complicated mixtures, and a small quantity of the amino
intermediate III were obtained (yield < 20%). When we tried to conduct the reduction
reaction under hydrogen atmosphere, the amino intermediate III can be obtained with
comparatively high yield (46%). The target compounds (8-10) were obtained by the

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reactions between III and acyl chloride or phenyl isocyanate in CH Cl . The desired
2
2
sulfonamide products (11-27) were obtained by sulphonylation reactions between III
and various sulphonyl chlorides in pyridine, which act as solvent, as well as the base.
Scheme 1. Synthetic Route to Compounds 8-27
Reagents and conditions: (a) HNO /AcOH; (b) H , Pd/C; (c) CH Cl , Et N, acyl chloride / phenyl
3
2
2
2
3
isocyanate (d) pyridine, sulphuryl chloride, rt.
3
. Conclusion
In this research, a class of phenanthridin-6(5H)-one derivatives were designed and
synthesized with the goal of obtaining novel drug-like BET Bromodomain inhibitors.
Starting from our previously reported novel coumarin containing BRD4 inhibitors, a
hydrophobic interaction combined with an additional key hydrogen bond interaction
were identified leading to increased potency. Medicinal chemistry optimizations had
led to the discovery of a set of potent and highly efficacious BET inhibitors,
exemplified by compound 24. Compound 24 bound to BET proteins with an IC50
value of 240 nM and showed low micromolar to nanomolar potencies in four cancer
cell growth inhibition. Moreover, compound 24 also significantly arrested the cell
cycle distribution and induced down regulation of C-myc protein along with cell
apoptosis. Testing its binding activities against other 23 bromodomain containing

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proteins showed that compound 24 was also a highly selective BET inhibitor.
Compound 24 demonstrated favorable human microsomal stability in vitro and
acceptable oral pharmacokinetics in rats. Overall, the docking simulation study, along
with the in vitro assay results demonstrated compound 24 is a promising preclinical
candidate entity for the further development. More importantly, we anticipate that the
phenanthridin-6(5H)-one scaffold, together with the newfound hydrophobic pocket,
may stimulate other researchers to develop their own novel bromodomain inhibitors.
4
. Experimental Section
4
.1 Bromodomain Inhibition Assay
Assay format: The reader assay is a binding assay using AlphaScreen technology
FRET assay. The biotinylated peptide binding to the reader domain of His-tagged
protein is monitored by the singlet oxygen transfer from the Streptavidin-coated donor
beads to the AlphaScreen Ni-chelate acceptor beads. Reagent: Reaction buffer: 50
mM Hepes, pH7.5, 100 mM NaCl, 0.05% CHAPS, 0.1 % BSA, and 1% DMSO (the
final DMSO concentration may different depending on compound stock and test
concentrations). Bromodomain BRD4-Full Length (RBC Cat# RD-21-153):
Recombinant Human Bromodomain containing protein 4 (bromodomain 1 and 2; aa
2
-1362; Genbank Accesstion # NM_058243), expressed in Sf9 insect cells with an
N-terminal His-tag. MW=156.5 kDa. Ligand (C-term-Biotin) Histone H4 peptide
1-21) K5/8/12/16Ac-Biotin. Detection beads: PerkinElmer Donor beads:
(
Streptavidin-coated donor beads. Acceptor beads: AlphaScreen Ni acceptor beads
Reaction Procedure: 1. Deliver 2.5X BRD in wells of reaction plate except No BRD

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control wells. Add buffer instead. 2. Deliver compounds in 100% DMSO into the
BRD mixture by Acoustic technology (Echo550; nanoliter range). Spin down and
pre-incubation for 30 min. 3. Deliver 5× Ligand. Spin and shake. 4. Incubate for 30
min at room temperature with gentle shaking. 5. Deliver 5× donor beads. Spin and
shake. 6. Deliver 5× acceptor beads. Spin and shake. Then gentle shaking in the dark
for 60 min. 7. Alpha measurement (Ex/Em=680/520-620 nm) in Enspire.
4
.2 Cell Culture
Human lung carcinoma A549 cells, Human gastric carcinoma metastatic lymph
node SGC-7901 cells, human hepatocellular carcinoma HepG2 cells and human
pancreatic cancer PANC-1 cells were purchased from the Cell Center of the Chinese
Academy of Medical Sciences (Beijing, China). Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco, Gaithersburg, MD) or RPMI 1640
medium (Gibco, Gaithersburg, MD) supplemented with 10% foetal bovine serum
(
FBS) and incubated at 37 Ⅱ with 5% CO humidified atmosphere.
2
4
.3 Cell Growth Inhibition
Methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell survival rate.
4
Briefly, cells were seeded into a 96-well plate at a density of 2×10 cells/mL. Medium
containing a certain concentration of compound (0, 0.47, 0.94, 1.88, 3.75, 7.50, 15.0
and 30 µM) was added into each well in a volume of 100 µL for 48 h respectively.
The cell morphology was observed by Invert/phase contrast microscopy (Nikon
TE2000, Tokyo, Japan) (bar: 200 µm). Then 20 µL MTT solutions (Sigma, Shanghai,
China) was added into each well and followed by incubation at 37°C for 4 h. 150 µL

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of DMSO was further added after discarding culture medium. The crystals of
formazan product were then dissolved by oscillating for 10 min. The optical density
(OD) value was detected using a microplate reader (Bio-Rad, imark, USA) at a
wavelength of 570 nm. The experiments were performed in triplicate. Cell survival
rate (%) = (OD of administration group − OD ofblank group)/ (OD of control group −
OD of blank group) × 100%. The value of inhibitory concentration 50 (IC ) was
5
0
calculated by GraphPad Prism 5 software.
.4 Western Blot Analysis
To determine levels of C-myc, cells were seeded in a 6-well cell culture plate at a
4
density of 400000 cells per well for A549 in a total volume of 1800 µL and incubated
overnight in medium containing 10% fetal bovine serum (Life Technologies,
Rockville, MD). Then 200 µL of serially diluted compounds were added to each well
the next day. Cell lysates were harvested after 48 h and C-myc were quantified using
assay kits following the manufacturer’s protocols. Mouse monoclonal antibody for
c-Myc was from Cell Signaling Technology (Danvers, MA, USA).
4
.5 Cell Apoptosis Assay
The apoptosis of A549 cells was determined by Annexin V-FITC/PI assay. Annexin
V binds to phosphatidylserine, which is exposed on the cell membrane and is one of
the earliest indicators of cellular apoptosis. PI (Propidium Iodide) is used as a DNA
stain for both flow cytometry to evaluate cell viability or DNA content in cell cycle
analysis and microscopy to visualize the nucleus and other DNA containing
organelles. It can be used to differentiate necrotic, apoptotic and normal cells. Cells (2

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×
105) were seeded in 6-well plate and were treated with varying concentrations of
inhibitor for 48 h. A549 cells were collected and incubated with FITC-conjugated
Annexin V. The nuclei were then counterstained with PI. After the dual staining, the
cells were screened by a FAC Scan flow cytometer (FACS Calibun, Becton
Dickinson). The upper left corner of the quadrant represents debris, lower left are live
cells, upper right are advanced apoptotic or necrotic cells and lower right are
apoptotic cells.
4
.6 Cell Cycle Assay
Subconfluent cells were treated with test compounds at different concentrations for
8 h. The cultures were pulse-labeled with 10 µM 5-bromo-2’-deoxyuridine (BrdU)
4
for 30 min at 37°C prior to harvest. The cells were subsequently washed in PBS, fixed
with 70% ethanol, and denatured in 2 M HCl. Following neutralization, the cells were
stained with anti-BrdU fluoresceinlabeled antibodies, washed, stained with propidium
iodide, and analyzed by flow cytometry using a 488 nm laser (Cell Lab Quanta SC,
Beckman Coulter) 48. Cell cycle analysis was made using a FACScan cytometer
(FACSCalibun, Becton Dickinson).
4
.7 Microsomal Stability Studies
The metabolic stability was assessed using human liver microsomes (purchased
from Ltd Co (RILD), M008084). Briefly, 1 µM of compound 24 was incubated with
.7 mM cofactor β-NADPH and 0.5 mg/mL microsomes in 0.1 M phosphate buffer
pH = 7.4) containing 3.3 mM MgCl at 37 °C. The DMSO concentration was less
1
(
2
than 0.1 % in the final incubation system. At 0.025, 0.083, 0.25, 0.5, 1.0, 1.5 and 2 h

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ofincubation, an amount of 60 µL of reaction mixture was taken out, and the reaction
is stopped immediately by adding 3-fold excess of cold acetonitrile containing 100
ng/mL of internal standard for quantification. The collected fractions were centrifuged
at 30 000 rpm for 5 min to collect the supernatant for LC−MS/MS analysis, from
which the amount of compound remaining was determined.
4
.8 In vivo PK studies
SD female rats, 6−8 weeks old， were selected for dosing. Three mice were
randomly grouped per time point. Mice were received either a single intravenous
injection of 2.5 mg/kg compound or a single oral administration of 10 mg/kg
compound. Compounds were given as solutions in DMSO / PEG 200 / water. Blood
samples were collected from rats at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h and
were further processed to obtain plasma by centrifugation at 15 000 rpm for 10 min.
Plasma concentrations of the compounds were determined using the liquid
chromatography−tandem mass spectrometry (LC−MS/MS) method. The
pharmacokinetic parameters were calculated by WinNonlin. The study was approved
by the Zhejiang Academy of Medical Sciences Institutional Animal Care & Use
Committee
.9 Molecular Modeling
The X-ray crystal structure of BRD4 binding with JQ1 (PDB code: 3MXF) was
.
4
retrieved from the Protein Data Bank. The protein and compounds 7 and 24 were
prepared using the protein preparation wizard in Maestro with standard settings. Grids
of BRD4 was generated using Glide, version 10.2, following the standard procedure

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recommended by Schrodinger. The conformational ensembles were docked flexibly
using Glide with standard settings in both standard and extra precision mode. Only
poses with low energy conformations and good hydrogen bond geometries were
considered.
4
4
.10. Chemistry
.10.1. General Chemistry
Commercially available reagents and anhydrous solvents were used without further
purification. The crude reaction product was purified by Flash chromatography using
silica gel (300-400 mesh). All reactions were monitored by TLC, using silica gel
plates with fluorescence F254 and UV light visualization. If necessary, further
purification was performed on a preparative HPLC (Waters 2545) with a C18 reverse
1
phase column. Proton nuclear magnetic resonance ( H NMR) and carbon nuclear
1
3
magnetic resonance ( C NMR) spectra were recorded on a Bruker AV-400
spectrometer at 400 MHz. Coupling constants (J) are expressed in hertz (Hz). Each
signal is identified by its chemical shift δ expressed in parts per million (ppm) HRMS
analyzes were performed under ESI (electrospray ionization) using a TOF analyzer in
V mode with a mass resolution of 9000. Melting points (mp) were determined by an
M-565 melting-point apparatus with a microscope (BUCHI, made in Switzerland)
4
.10.2． Synthetic procedure of 2-nitrophenanthridin-6(5H)-one (II)
HNO (15 ml, 65%) was added into a solution of phenanthridin-6(5H)-one (3.0 g,
3
1
5 mmol) in acetic acid solution (15 mL) at 0℃, the reaction mixture was stirred at
room temperature for 10 min first , and then stirred at 90℃ for 4 h under nitrogen

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condition. After the reaction was completed, the reaction mixture was directly filtered
to obtain a filter cake, and the solid was washed with water for 3 to 4 times, and the
resulting crude product was yield of 2-nitrophenanthridin-6(5H)-one (II). Without
further purification, compound II was used directly to the next step according to 100%
yield.
4
.10.3 Synthetic procedure of 2-aminophenanthridin-6(5H)-one (
)
Palladium carbon (60 mg) and methanol solution (15 mL) were added to the flask
at room temperature, and then a solution of the compound II in methanol was added
under hydrogen atmosphere, and then the reaction solution was stirred at room
temperature for about 8 h. After purification by column chromatography, compound
1
Ⅲ
was obtained in a yield of 46% (pale brown solid); mp 286.0 - 287.4 ℃. H NMR
(400 MHz, DMSO-D6) δ 11.33 (s, 1H), 8.30 (d, J = 9.1 Hz, 1H), 8.22 (d, J = 8.1 Hz,
1
H), 7.82 (t, J = 6.9 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.48 (d, J = 2.3 Hz, 1H), 7.11
(d, J = 8.6 Hz, 1H), 6.82 (dd, J = 8.6, 2.3 Hz, 1H), 5.01 (s, 2H). HRMS m/z: calcd. for
+
C H N O [M+H] 211.0871, found 211.0874;
13
11
2
4
.10.4. General synthetic procedure of compound 8~10
A solution of 2-aminophenanthridin-6(5H)-one (0.30 g, 1 mmol), corresponding
sulphonyl chloride compound (1mmol) and pyridine (2 mL) in a flask was stired at
room temperature for 2 h. After the reaction, ethyl acetate and a dilute hydrochloric
acid solution were added for extraction, the organic layer was washed with dilute

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hydrochloric acid for 3 to 4 times, and then the organic layer was concentrated to
obtain the pure products.
4
.10.4.1 N-(6-oxo-5,6-dihydrophenanthridin-2-yl)benzamide (8) 73.2% yield (pale
1
gray solid); mp 273.2 - 273.6 ℃. H NMR (400 MHz, DMSO-D6) δ 11.73 (s, 1H),
1
0.46 (s, 1H), 8.82 (s, 1H), 8.39 – 8.27 (m, 2H), 8.05 (d, J = 6.9 Hz, 2H), 7.91 (dd, J
7.0, 4.0 Hz, 2H), 7.68 (t, J = 7.5 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.56 (t, J = 8.0
=
1
3
Hz, 2H), 7.39 (d, J = 8.8 Hz, 1H). C NMR (101 MHz, DMSO-D6) δ 165.9, 161.0,
1
1
4
35.3, 134.5, 133.4, 132.03, 128.9, 128.5, 128.1, 126.3, 123.6, 122.7, 117.9, 116.7,
+
15.0. HRMS m/z: calcd. for C H N O [M+H] 315.1134, found 315.1133
2
0
15
2
2
.10.4.2 phenyl (6-oxo-5,6-dihydrophenanthridin-2-yl)carbamate (9) 76.1% yield
1
(
white solid); mp 302.4 - 303.1 ℃. H NMR (400 MHz, DMSO-D6) δ 11.72 (s, 1H),
1
1.72 (s, 1H), 10.38 (s, 1H), 8.54 (s, 1H), 8.39 (d, J = 7.9 Hz, 1H), 8.30 (d, J = 8.2 Hz,
H), 7.92 (t, J = 8.1 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H), 7.50 (t,
1
1
3
J = 7.9 Hz, 2H), 7.40 (d, J = 8.8 Hz, 1H), 7.32 (t, J = 8.0 Hz, 3H). C NMR (101
MHz, DMSO-D6) δ 160.9, 152.5, 151.1, 134.3, 133.4, 129.9, 128.6, 128.1, 126.3,
+
1
25.9, 122.5, 118.1, 117.1. HRMS m/z: calcd. for C H N O [M+H] 331.1083,
2
0
15
2
3
found 331.1082
4
.10.4.3 1-(6-oxo-5,6-dihydrophenanthridin-2-yl)-3-phenylurea (10) 69.4% yield
1
(
white solid); mp 367.6 - 367.9 ℃. H NMR (400 MHz, DMSO-D6) δ 11.66 (s, 1H),
8
.78 (d, J = 1.9 Hz, 2H), 8.54 (s, 1H), 8.35 (t, J = 8.3 Hz, 2H), 7.90 (t, J = 7.6 Hz, 1H),
.67 (t, J = 7.5 Hz, 1H), 7.53 (d, J = 9.9 Hz, 3H), 7.33 (dd, J = 16.8, 9.2 Hz, 3H), 7.00
7
1
3
(
t, J = 7.3 Hz, 1H). C NMR (101 MHz, DMSO-D6) δ 160.8, 153.4, 140.2, 135.0,