Discovery of novel coumarin derivatives as potent and orally bioavailable BRD4 inhibitors based on scaffold hopping

Article abstract of DOI:10.1080/14756366.2019.1587417

The bromodomain and extra-terminal (BET) bromodomains, particularly BRD4, have been identified as promising therapeutic targets in the treatment of many human disorders such as cancer, inflammation, obesity, and cardiovascular disease. Recently, the discovery of novel BRD4 inhibitors has garnered substantial interest. Starting from scaffold hopping of the reported compound dihydroquinazolinone (PFI-1), a series of coumarin derivatives were designed and synthesised as a new chemotype of BRD4 inhibitors. Interestingly, the representative compounds 13 exhibited potent BRD4 binding affinity and cell proliferation inhibitory activity, and especially displayed a favourable PK profile with high oral bioavailability (F = 49.38%) and metabolic stability (T_1/2 = 4.2 h), meaningfully making it as a promising lead compound for further drug development.

Full text of DOI:10.1080/14756366.2019.1587417

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery of novel coumarin derivatives as potent
and orally bioavailable BRD4 inhibitors based on
scaffold hopping
Zhimin Zhang, Lili Gu, Beibei Wang, Wenhai Huang, Yanmin Zhang, Zhen Ma,
Shenxin Zeng & Zhengrong Shen
To cite this article: Zhimin Zhang, Lili Gu, Beibei Wang, Wenhai Huang, Yanmin Zhang, Zhen Ma,
Shenxin Zeng & Zhengrong Shen (2019) Discovery of novel coumarin derivatives as potent and
orally bioavailable BRD4 inhibitors based on scaffold hopping, Journal of Enzyme Inhibition and
Medicinal Chemistry, 34:1, 808-817, DOI: 10.1080/14756366.2019.1587417
To link to this article: https://doi.org/10.1080/14756366.2019.1587417
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 808–817
https://doi.org/10.1080/14756366.2019.1587417
RESEARCH PAPER
Discovery of novel coumarin derivatives as potent and orally bioavailable BRD4
inhibitors based on scaffold hopping
Zhimin Zhanga*, Lili Gua*, Beibei Wanga, Wenhai Huanga, Yanmin Zhangb, Zhen Maa, Shenxin Zenga and
Zhengrong Shena
aKey Laboratory of Neuropsychiatric Drug Research of Zhejiang Province, Institute of Materia Medica, Zhejiang Academy of Medical Sciences,
b
Hangzhou, PR China; School of Basic Science, China Pharmaceutical University, Nanjing, PR China
ABSTRACT
ARTICLE HISTORY
Received 3 January 2019
Revised 7 February 2019
Accepted 21 February 2019
The bromodomain and extra-terminal (BET) bromodomains, particularly BRD4, have been identified as
promising therapeutic targets in the treatment of many human disorders such as cancer, inflammation,
obesity, and cardiovascular disease. Recently, the discovery of novel BRD4 inhibitors has garnered substan-
tial interest. Starting from scaffold hopping of the reported compound dihydroquinazolinone (PFI-1), a ser-
ies of coumarin derivatives were designed and synthesised as a new chemotype of BRD4 inhibitors.
Interestingly, the representative compounds 13 exhibited potent BRD4 binding affinity and cell prolifer-
ation inhibitory activity, and especially displayed a favourable PK profile with high oral bioavailability
(F ¼ 49.38%) and metabolic stability (T1/2 ¼ 4.2 h), meaningfully making it as a promising lead compound
for further drug development.
KEYWORDS
Rational drug design; BRD4
bromodomain; synthesis;
biological evaluation
GRAPHICAL ABSTRACT
Introduction
therapeutic potential in a great many preclinical human disease
models15. Several JQ-1 analogues, such as OTX-01516–18 and CPI-
061019,20 have subsequently advanced into clinical trials for the
treatment of solid tumours, haematological malignancies and
other forms of human cancer. It was also worthy of attention that
PFI-1, a dihydroquinazolinone compound reported to be a potent
BRD inhibitor obtained through optimization of a fragment-
derived hit21. However, the solubility and pharmacokinetics of PFI-
1 are suboptimal22. Notwithstanding the above discovery, potent
BRD4 inhibitors with novel chemotypes are still in high demand
for only limited BRD4 inhibitors available in clinical trials.
The natural products, normally with unique chemical structure,
have long played an important role in drug discovery23. Coumarin
(benzo-a-pyrone) skeleton found in nature has been considered as a
privileged pharmacophore ascribed to the ability to exert noncova-
lent interactions (hydrogen bonds, hydrophobic, van der Waals force,
metal coordination, and electrostatic interactions, etc.), with the vari-
ous active sites in therapy targets24,25. Coumarins skeleton have
gained momentous attention in the last decades as a lead structure
The bromodomain and extra-terminal (BET) family proteins, mainly
including BRD2, BRD3, BRD4, and BRDT, are a kind of epigenetic
regulatory proteins that selectively bind to acetylated lysines (KAc)
of histone, thus translate chromatin status into transcription acti-
vation through RNA polymerase II and play critical roles in the
regulation of gene transcription1–3. Among the BET family pro-
teins, BRD4 has not just received a lot of attention, but been the
most extensively studied one. The KAc residues bind into a hydro-
phobic pocket of the BRD4 proteins specifically with conserved
asparagine (Asn140), also forming a water molecule-mediated
hydrogen bonding interactions with tyrosine (Tyr97)4,5. Small mol-
ecule BRD4 inhibitors can block the binding, which leads to pro-
found disruption of transcriptional programmes resulting in
therapeutic potential in several conditions, such as inflammation
and oncologic diseases6–10. In the last few years, a number of
high-affinity small molecule ligands of BRD have been identified
(Figure 1)11–14, wherein JQ-1 was the first reported potent BRD4
inhibitor and has been employed widely to evaluate its for the discovery of orally bioavailable anti-cancer, anti-HCV, anti-
CONTACT Zhengrong Shen
These authors contributed equally to this work.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
shenzr601@163.com
Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China
ꢀ
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
809
HIV, anti-Alzheimer, and anti-inflammatory agents26–29. Several cou- reveals that PFI-1 binds to BRD4 with three key hydrogen bonds
marin-based derivatives, such as Cloricromene30, Picumast31,
interactions, which are displayed in Figure 3(A). The carbonyl
Warfarin sodium32, and Difenacoum33 (Figure 2, coumarin skeleton
oxygen and NH group of the dihydroquinazolinone forms two
critical hydrogen bonds with the conserved residue Asn140.
Moreover, the carbonyl oxygen interacts with the conserved
residue Tyr97 via a water-mediated hydrogen bond. The anisole
group occupies the WPF (formed by residues W97, P98 and F99)
shelf and forms hydrophobic interaction with Asp145, Ile146,
and Met149. The sulphonamide forms two additional hydrogen
bonds with two water molecules. With this structural informa-
tion in mind, we hypothesised that rational replacement of the
dihydroquinazolinone core via coumarin skeleton based on scaf-
fold hopping would be tolerated (Figure 3(B)). Thus, a series of
novel coumarin derivatives were designed. As shown in Figure
3(C), the docking mode of coumarin derivative 1 binds to BRD4
was consistent with that of PFI-1. The alignment results depicted
in Figure 3(D) also indicated that 1 almost took the same inter-
action conformation as PFI-1 except that the hydrogen-bond
interaction between NH and Asn140 was lost.
marked in red) were approved for therapeutic purposes in clinic.
Scaffold hopping can not only provide an alternative strategy for
BRD4 inhibitor design, but also potentially lead the discovery of
potent BRD4 inhibitors with novel and diverse chemotypes. The
advantages of scaffold hopping, combining with the pharmacophore
property of coumarin and the skeleton modifiability of PFI-1 much
interest us to explore structurally novel BRD4 inhibitors. Herein, we
reported the rational design, syntheses, and structure-activity relation-
ship (SAR) exploration of novel coumarin heterocycle derivatives as
potent BRD4 inhibitors via the scaffold hopping of PFI-1. Furthermore,
in vitro anti-tumour test and in vivo preliminary pharmacokinetics
study of representative compound also were described in this article.
Results and discussion
Design of new BRD4 inhibitors containing a coumarin scaffold
Scaffold hopping is a powerful and promising strategy for drug
discovery34. It has been extensively used for the design of struc-
turally novel bioactive molecules35–38, which generally incorpo-
rates ring opening, ring closure, heterocycle replacement, and
shape/topology-based scaffold hopping39,40. Herein, we applied
the heterocycle replacement and shape-based scaffold hopping
strategy towards PFI-1, a BRD inhibitor developed by Pfizer
Worldwide R&D. First, we analysed the binding mode of PFI-1
binds to the BRD4 bromodomain. X-ray crystallographic analysis
SAR studies of coumarin derivatives
With the understanding of binding conformation, our next work is
to explore the SAR of the coumarin derivatives. We focused on
the hydrophobic WPF shelf to develop compounds with improved
affinity for BRD4. In order to optimise the interactions towards
WPF shelf, diverse substituents at the R position were designed to
investigate the chemical space for improving the activity. To
ensure the R group extends to the WPF shelf and forms hydro-
phobic interactions with residues located there, we maintained
the sulphamide linker. Thus, compounds 1 2 16 with R groups of
aromatic groups, alkyl or cycloalkyl were designed and synthes-
ised (Table 1). We preferentially evaluated the phenyl group.
Compound 1 characterised by phenyl group has shown moderate
BRD4 binding activity with an IC50 value of 6.59 lM in the
AlphaScreen assay. When alkyl or cycloalkyl groups were used to
occupy the WPF shelf, a > 3-fold decrease in affinity was found
(Table 1, compounds 2–5), probably due to a reduction of hydro-
phobic interactions with WPF shelf. Generally, the cycloalkyl group
compounds (compounds 2 and 3) were more tolerated than the
chain alkyl compounds (4 and 5). Next, we paid our attention
back to aromatic groups. An extensive SAR exploration was then
conducted: displaying that diverse substitution at different posi-
tions of the phenyl group in 1 may lead to various effects on the
BRD4 affinity. Our study showed that compounds bearing chlor-
ine, methoxy group or tertiary butyl group at the para-position
were tolerable (compounds 6–8). The inhibitory activities of 6–8
were similar to 1 with IC50 values of 2.97, 7.31, and 5.09 lM,
respectively. The para-chlorine substituted analogue 6 was some-
what more potent than 7 and 8, which indicated that electron-
Figure 1. Representative, previously reported bromodomain inhibitors.
Figure 2. Structures of approved coumarin-containing drugs.

810
Z. ZHANG ET AL.
Figure 3. (A) Crystal structure of BRD4 BD1 bound to PFI-1 (PDB ID: 4E96). The protein is shown as a light gray cartoon and PFI-1 is shown as sticks (carbon atoms in
green, oxygens in red, nitrogens in blue and sulfurs in brown). (B) Design concept of new BRD4 inhibitors. (C) The docking model of 1 with BRD4 BD1 (carbon atoms
in cyan). (D) Superimposition of PFI-1 (green carbon atoms) and 1 (cyan carbon atoms) in their putative bioactive conformations.
withdrawing groups at the para-position might be favourable. We ortho-position was favourable; we attempted to merge these two
further investigated the impact of cyano group on meta-position, positions as bi-substituent. The resulting compound 12 and 13
compound 9 maintained the similar potency at an IC50 value of displayed potent activity with IC50 values of 1.77 and 0.93 lM,
5.03 lM, which suggested that substitution at meta-position was respectively. It is interesting that compound 12 was slightly more
also feasible. Surprisingly, the enhanced potency was achieved potent than 10, while 13 was slightly more potency than 11,
when substitutions were introduced at the ortho-position (com- which may ascribe to greater lipophilicity because of the chlorine
pounds 10 and 11). The ortho-methoxy group analogue 11 atom. For example, the lipophilic efficiency (LipE, ꢁlg (IC50) ꢁ
showed significant increase with an IC50 value of 0.98 lM and is logP) of 13 and 11 was 3.2 and 3.9. For more understanding the
approximately 7-fold more potent than 1. Compound 11 was SAR, we further replaced the phenyl ring of 1 by thienyl group,
more potent than 10, which may due to the advisable sulphamide the resulting derivative 14 showed low binding activity. The
conformation caused by the larger methoxyl group on ortho-pos- replacement by larger aromatic group also led to a reduction of
ition. With the results that mono-substituent at para-position or affinity (compounds 15 and 16).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
811
Table 1. Structure-activity relationship of the R substitutions on synthesized
coumarin derivatives.
Unfortunately, 17 showed almost 5-fold decrease in potency
compared with 7.
Docking study of the preferred configuration of representative
compound 13
Comp. No.
1
R
BRD4 IC50 (lM)a
cLogPb
2.43
LEc
6.59
0.35
To disclose the structural basis for the high binding affinity of
compound 13 to the BRD4 protein, we conducted the docking
study of 13 in a complex with BRD4 (Figure 5). As expected, the
coumarin moiety of 13 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 sulphonamide forms two
additional hydrogen bonds with two water molecules.
2
3
19.02
17.9
2.83
2.73
0.31
0.33
4
5
6
38.53
30.04
2.97
1.86
2.39
2.55
0.34
0.33
0.34
7
8
7.31
5.09
3.18
4.26
0.33
Evaluation of the inhibitory effects on cell growth
The representative compound 13 was next evaluated for its
0.30 effects on the survival of human lung adenocarcinoma A549 cells,
hepatocellular carcinoma HepG2 cells, pancreatic carcinoma
PANC-1 cells, and gastric adenocarcinoma SGC-7901 cells with an
9
5.03
2.21
0.98
1.77
0.93
2.19
2.88
2.07
3.60
2.82
0.32
0.36
0.37
0.35
0.36
MTT assay. The data obtained were summarised in Table 2 and
dose–response curves were provided in Figure 6. Results showed
that 13 potently inhibits the proliferation in these four cell lines,
with IC50 values of 4.63, 4.75, 7.02, and 6.39 lM, respectively.
Overall consideration of the data from the above assays, 13 has
good profiles for further evaluation.
10
11
12
13
Effect of 13 on cells morphology
As shown in Figure 7, no abnormality was observed in the SGC-
7901 cells, PANC-1 cells, HepG2, and A549 cells from the control,
with oval or short fusiform, adhered to the wall and liked paving
stone shape. Cells in 13-treated groups, with increased of dose,
change became more obviously, contour was gradually clear, cell
diopter strengthened, cells gradually became smaller and round,
shrinking into the spherical, part of the cells was broken, and then
fell off or suspended.
14
15
9.26
2.16
3.60
0.35
0.28
12.23
16
18.42
2.68
0.28
JQ1
0.06
0.22
PFI-1d
Assessment of pharmacokinetic (PK) properties for 13
aThe IC50 values were tested by the AlphaScreen assay; bcLogP values were cal-
c
culated using ChemBiodraw Ultra14.0; LE (Ligand Efficiency) ¼ 1.4 (pIC50/heavy To assess the potential of this series of compound in vivo, we
atoms); dThe IC50 value was reported by Fish et al.22
.
evaluated 13 as a representative for its preliminary pharmacokin-
etics in Sprague-Dawley (SD) rats; the resulted data were listed in
Table 3. The plasma was collected after a single oral dose of
10 mg/kg or an i.v. dose of 2.5 mg/kg (Figure 8). Compound 13
demonstrated preferable PK properties with a T1/2 value of 4.2 h
and a Tmax of 5.87 h. In addition, in the oral administration mode,
13 displayed good drug exposure with an AUC value of
ꢀ
790.65 lg/L h and resulted in excellent oral bioavailability
(49.38%), which is higher than that of PFI-1 (32%)22.
Figure 4. The design and synthesis of reversed sulfonamide compound 17.
Chemistry
The x-ray crystal structure of PFI-1 bind to BRD4 revealed
that the sulphonamide linker provides a specific angle turn for
N-substitution to reach into WPF shelf. We were curious
about that whether a reversed sulphonamide could achieve the
same effect. We designed and synthesised a reversed sulphona-
The methodology employed for the synthesis of the coumarin
derivatives was demonstrated in Scheme 1. The commercially
available 2H-chromen-2-one (I) reacted with concentrated nitric
acid in sulphuric acid under 0–5 ꢂC to produce compound II,
which was then reduced in ammonium chloride and iron powder
mide compound 17 based on compound
7 (Figure 4).

812
Z. ZHANG ET AL.
Figure 5. The docking model (PDB ID: 4E96) of 13 with BRD4 BD1 (carbon atoms in blue). Water molecule is shown as purple sphere, and the hydrogen bonds are
denoted by gold dash lines.
Table 2. Anti-proliferation effects of 13 against four cell lines.
possesses a promising BRD4 inhibitory and orally bioavailable
characteristic, worth of making it as a lead compound for further
drug development.
Cancer type
Cell line
IC50 (lM)
Lung adenocarcinoma
Hepatocellular carcinoma
Pancreatic carcinoma
Gastric adenocarcinoma
A549
4.63
4.75
7.02
6.39
HepG2
PANC-1
SGC-7901
Experimental section
BRD4 binding assay
The binding affinity was evaluated by a using AlphaScreen tech-
nology 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: recombinant human bromodo-
main 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 ¼ 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 proced-
ure: (1) Deliver 2.5ꢃ BRD in wells of reaction plate except No BRD
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 ꢃ Ligand. Spin and shake. (4) Incubate for 30 min at
room temperature with gentle shaking. (5) Deliver 5ꢃ donor
beads. Spin and shake. (6) Deliver 5 ꢃ acceptor beads. Spin and
shake. Then gentle shaking in the dark for 60 min. (7) Alpha meas-
urement (Ex/Em ¼ 680/520–620 nm) in Enspire.
Figure 6. Dose–response curves of 13 in incubation with cancer cell lines
(mean SD, n ¼ 3).
system to give the amino intermediate III. The target compounds
were (1–16) were obtained by sulphonylation reactions between
III and various sulphonyl chlorides in pyridine, which act as solv-
ent, as well as the deacid reagent.
Conclusion
In this study, we have designed and synthesized a series of cou-
marin-containing compounds based on scaffold hopping with the
goal of improving the oral bioavailability of PFI-1 and obtaining
entirely new chemotype of BRD4 inhibitors. Our efforts have led
to the discovery of a representative compound 13 (4-chloro-2-
Cell culture
Human gastric carcinoma metastatic lymph node SGC-7901 cells,
human pancreatic cancer PANC-1 cells, human hepatocellular car-
cinoma HepG2 cells, and human lung carcinoma A549 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 ꢂC with 5%
methoxy-N-(2-oxo-2H-chromen-6-yl)benzenesulfonamide),
which
binds to BRD4 with nanomolar affinities and shows low micromo-
lar potencies in cell growth inhibition against four human cancer
cell lines. Significantly, it achieves excellent oral bioavailability
(F ¼ 49.38%) and is metabolically stable (T1/2 ¼ 4.2 h), which is pref-
erable than PFI-1 (F ¼ 32%). The obtained data suggested that 13 CO2 humidified atmosphere.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
813
Figure 7. Effect of 13 on A549, HepG2, PANC-1 and SGC-7901 cells morphology. Cells after treating with 13 under relevant concentrations for 24 h were observed by
invert/phase contrast microscopy.
group)ꢃ100%. The value of inhibitory concentration 50 (IC50) was
Table 3. Intravenous (i.v.) and oral (p.o) pharmacokinetic profiles of compounds
calculated by GraphPad Prism 5 software (La Jolla, CA).
13 in rats.
Parameters
i.v. (2.5 mg/kg)
SD
p.o (10 mg/kg)
SD
ꢀ
AUC(0–t) (lg/L h)
AUC(0–1) (lg/L h)
T1/2 (h)
Tmax (h)
MRT0–t (h)
CLz (L/h/kg)
Vd (L/kg)
Cmax(lg/L)
F (%)
400.87
607.74
–
0.083
7.9
4.46
84.28
96.87
–
32.41
226.07
–
0
0.81
790.65
816.46
4.2
5.87
8.5
12.30
74.28
61.7
55.47
64.99
0.29
0.95
0.66
10.93
5.19
4.6
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 centrifuga-
tion at 15,000 rpm for 10 min. Plasma concentrations of the com-
1.4
28.10
10.56
–
49.38
5.45
ꢀ
The statistical significance is meaningless due to the different dosages between
PO and IV.
pounds
were
determined
using
the
liquid
MTT assay
chromatography ꢁ tandem mass spectrometry (LC ꢁ MS/MS)
method. The pharmacokinetic parameters were calculated
WinNonlin. The study was approved by the Ethical Committee of
Zhejiang Academy of Medical Sciences on Use and Care of
Animals, in accordance with the recommendations in the Guide
for the Care and Use of Laboratory Animals.
Methyl thiazolyl tetrazolium (MTT) assay was used to detect the
cell survival rate. Briefly, cells were seeded into a 96-well plate at
a density of 2 ꢃ 104cells/mL. Medium containing a certain concen-
tration of compound (0, 0.47, 0.94, 1.88, 3.75, 7.50, 15.0, and
30 mM) was added into each well in a volume of 100 lL for 48 h,
respectively. The cell morphology was observed by Invert/phase
contrast microscopy (Nikon TE2000, Tokyo, Japan) (bar: 200 mm).
Then 20 lL MTT solutions (Sigma, Shanghai, China) was added
into each well and followed by incubation at 37 ꢂC for 4 h. An ali-
quot of 150 lL of DMSO was further added after discarding cul-
Computer aided drug design
The X-RAY crystal structure of BRD4 (PDB 4E96) were downloaded
from Protein Data Bank (PDB) and prepared using the Protein
ture medium. The crystals of formazan product were then Preparation Wizard in the Schro€dinger suite41. Due to its good
dissolved by oscillating for 10 min. The optical density (OD) value reproducibility of cocrystallised ligand conformations and accuracy
was detected using a microplate reader (Bio-Rad, imark, Hercules, in molecular docking and scoring42, Glide was selected as the
CA) at a wavelength of 570 nm. The experiments were performed molecular docking tool. A receptor grid was defined as the ligand-
in triplicate. Cell survival rate (%) ¼ (OD of administration binding site search region based on the cocrystallised ligand and
group ꢁ OD of blank group)/(OD of control group ꢁ OD of blank an enclosing box that was in similar in size to the cocrystallised

814
Z. ZHANG ET AL.
Figure 8. The plasma concentration-time curve of 13.
Scheme 1. The synthetic route of compounds. Reagents and conditions: (a) HNO3/H2SO4; (b) SnCl2/HCl; (c) pyridine, sulphuryl chloride, rt.
ligand were used to capture the compounds to be docked. The
cognate ligand of PDB 4E96 as well as the target compounds
were prepared by LigPrep module in Schro€dinger with the ioniza-
tion stage not changed and Generate tautomers not selected. For
each ligand, one low energy ring conformation was generated.
The prepared compounds were submitted for molecular docking.
Based on the Glide score and interactions formed between the
compounds and the active site, the best conformation of each
compound was conserved. Finally, the potential compounds were
flexibly docked into the binding site with standard precision (SP)
docking mode43. All the remaining parameters were kept as
default settings.
The solid was filtered off, and the filtrate was concentrated
under reduced pressure. The resulting crude product was yield 6-
nitro-2H-chromen-2-one (II) without purification.
Synthetic procedure and characterization of compound III
To a reaction mixture of Fe powder (1.7 g, 31 mmol) and NH4Cl
(0.8 g, 15 mmol) in EtOH (30 mL) and water (10 mL) was added 6-
nitro-2H-chromen-2-one (1.5 g, 7.8 mmol), then the reaction mix-
ture was stirred at 80 ꢂC for 2 h. After the reaction was completed,
the reaction mixture was cooled to room temperature (rt). The
reaction mixture was extracted with ethyl acetate (100 mL ꢃ 3).
The organic layer was washed with brine and dried over Na2SO4.
The solid was filtered off and the filtrate was concentrated under
reduced pressure. The resulting crude product was purified by sil-
ica gel chromatography with petroleum ether/ethyl acetate (5/1,
v/v) to yield 6-amino-2H-chromen-2-one (III) (1.04 g, yield 86.7%)
as a yellow solid. 1H NMR (400 MHz, DMSO) d 10.34 (s, 2H), 8.19
(d, J ¼ 9.6 Hz, 1H), 7.77 (s, 1H), 7.66 (dd, J ¼ 8.8, 2.2 Hz, 1H), 7.52 (d,
J ¼ 8.9 Hz, 1H), 6.58 (d, J ¼ 9.7 Hz, 1H). 13C NMR (101 MHz, DMSO)
Chemistry
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 (thin layer chromatography),
using silica gel plates with fluorescence F254 and UV light visual-
ization. If necessary, further purification was performed on a pre-
parative high-performance liquid chromatography (HPLC) (Waters
2545) with a C18 reverse phase column. Proton nuclear magnetic
resonance (1H NMR) and carbon nuclear magnetic resonance (13C
NMR) spectra were recorded on a Bruker AV-400 spectrometer at
d
159.98, 152.78, 143.91, 129.03, 127.01, 122.82, 119.74,
118.16, 117.81.
Synthetic procedure and characterization of compound 1–17
To a solution of 6-amino-2H-chromen-2-one (III) (80 mg, 0.5 mmol)
and corresponding sulphonyl chloride (0.5 mmol) in DCM (5 mL)
400 MHz. Coupling constants (J) was expressed in hertz (Hz). Each was added pyridine (0.3 mL). The mixture was stirred at rt for 2 h.
signal is identified by its chemical shift d expressed in parts per The reaction mixture was extracted with DCM (30 mL ꢃ 3). The
organic layer was washed with brine and dried over Na2SO4. The
solid was filtered off, and the filtrate was concentrated under
reduced pressure. The resulting crude product was purified by sil-
ica gel chromatography with petroleum ether/ethyl acetate (3/1,
v/v) to yield 1–17.
million (ppm). HRMS analyses were performed under ESI (electro-
spray ionization) using a TOF (time-of-flight) analyser in V mode
with a mass resolution of 9000.
Synthetic procedure of compound II
N-(2-oxo-2H-chromen-6-yl)benzenesulphonamide (1): White
1
To a solution of 2H-chromen-2-one (2.9 g, 20 mmol) in H2SO4
(10 mL) was added HNO3 (1.3 g, 20.1 mmol) at 0 ꢂC then the reac-
tion mixture was stirred at 0–5 ꢂC for 2 h. After the reaction was
completed, the reaction mixture was extracted with ethyl acetate
(200 mL ꢃ 3). The organic layer was washed with brine and dried
over Na2SO4.
solids (yield 89.6%). H NMR (400 MHz, DMSO) d 10.49 (s, 1H), 8.05
(d, J ¼ 9.6 Hz, 1H), 7.80 (d, J ¼ 7.6 Hz, 2H), 7.60 (dt, J ¼ 26.7, 7.3 Hz,
3H), 7.47 (s, 1H), 7.31 (s, 2H), 6.48 (d, J ¼ 9.6 Hz, 1H). 13C NMR
(101 MHz, DMSO) d 160.18, 150.79, 144.30, 139.61, 134.39, 133.51,
129.79, 127.14, 125.19, 119.92, 119.55, 117.70, 117.33. HRMS (ESI)
calcd. for C11H8N7O2S ([M þ H]þ) 302.0460, found 302.0480.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
815
N-(2-oxo-2H-chromen-6-yl)cyclohexanesulphonamide
(2): 120.59, 119.68, 117.87, 117.39, 113.08. HRMS (ESI) calcd. for
Yellow solids (yield 92.3%). 1H NMR (400 MHz, DMSO) d 9.97 (s, C16H11N2O4S ([M þ H]þ) 327.0440, found 327.0429.
2-chloro-N-(2-oxo-2H-chromen-6-yl)benzenesulphonamide
1H), 8.09 (d, J ¼ 9.6 Hz, 1H), 7.56 (d, J ¼ 2.4 Hz, 1H), 7.45 (dd,
(10): Light pink solids (Yield 91.0%). 1H NMR (400 MHz, DMSO) d
J ¼ 8.9, 2.5 Hz, 1H), 7.39 (d, J ¼ 8.9 Hz, 1H), 6.50 (d, J ¼ 9.6 Hz, 1H),
10.86 (s, 1H), 8.07 (m, 2H), 7.70 – 7.60 (m, 2H), 7.53 (t, J ¼ 6.1 Hz,
1H), 7.48 (s, 1H), 7.35 (m, 2H), 6.47 (d, J ¼ 9.6 Hz, 1H). 13C NMR
(101 MHz, DMSO) d 160.13, 150.63, 144.19, 136.68, 135.21, 133.78,
132.36, 132.08, 131.21, 128.21, 124.37, 119.55, 118.98, 117.76,
117.40. HRMS (ESI) calcd. for C11H7N7O2SCl ([M þ H]þ) 336.0070,
found 336.0091.
3.03 (t, J ¼ 10.2 Hz, 1H), 2.05 (d, J ¼ 11.5 Hz, 2H), 1.76 (d,
J ¼ 12.9 Hz, 2H), 1.59 (d, J ¼ 12.0 Hz, 1H), 1.43 (m, 2H), 1.18 (m, 3H).
13C NMR (101 MHz, DMSO) d 160.31, 150.34, 144.55, 135.38,
124.39, 119.63, 118.65, 117.74, 117.23, 59.50, 26.45, 25.19, 24.77.
HRMS (ESI) calcd. for C15H18NO4S ([M þ H]þ) 308.0957,
found 308.0952.
2-methoxy-N-(2-oxo-2H-chromen-6-yl)benzenesulphona-
mide (11): Light pink solids (Yield 84.9%). 1H NMR (400 MHz,
DMSO) d 10.20 (s, 1H), 8.01 (d, J ¼ 9.6 Hz, 1H), 7.78 (d, J ¼ 7.8 Hz,
1H), 7.56 (t, J ¼ 7.9 Hz, 1H), 7.42 (s, 1H), 7.31 (m, 2H), 7.18 (d,
J ¼ 8.4 Hz, 1H), 7.03 (t, J ¼ 7.6 Hz, 1H), 6.44 (d, J ¼ 9.6 Hz, 1H), 3.90
(s, 3H). 13 C NMR (101 MHz, DMSO) d 160.22, 156.81, 150.43,
144.33, 135.68, 134.70, 130.73, 126.50, 124.63, 120.59, 119.37,
119.10, 117.48, 117.24, 113.32. HRMS (ESI) calcd. for C16 H14 N
N-(2-oxo-2H-chromen-6-yl)cyclopentanesulphonamide
(3):
Pink solids (yield 85.9%). 1H NMR (400 MHz, DMSO) d 9.95 (s, 1H),
8.10 (d, J ¼ 9.6 Hz, 1H), 7.57 (d, J ¼ 2.3 Hz, 1H), 7.48–7.37 (m, 2H),
6.50 (d, J ¼ 9.7 Hz, 1H), 3.64–3.49 (m, 1H), 1.94–1.85 (m, 4H), 1.65
(m, 4H), 1.55 (m, 4H). 13C NMR (101 MHz, DMSO) d 160.31, 150.53,
144.52, 135.20, 124.93, 119.63, 119.25, 117.73, 117.23, 60.21, 27.77,
25.87. HRMS (ESI) calcd. for C14H16NO4S ([M þ H]þ) 294.0800,
found 294.0794.
N-(2-oxo-2H-chromen-6-yl)propane-1-sulphonamide (4): Pink O5 S ([M þ H]þ) 332.0593, found 332.0587.
solids (yield 88.4%). 1H NMR (400 MHz, DMSO) d 9.98 (s, 1H), 8.10
(d, J ¼ 9.5 Hz, 1H), 7.55 (s, 1H), 7.42 (q, J ¼ 8.9 Hz, 2H), 6.51 (d,
J ¼ 9.5 Hz, 1H), 3.23–3.00 (m, 2H), 1.72 (m, 2H), 0.96 (t, J ¼ 7.3 Hz,
3H) 13C NMR (101 MHz, DMSO) d 160.31, 150.51, 144.51, 135.14,
124.68, 119.65, 118.96, 117.78, 117.25, 52.86, 17.30, 12.99. HRMS
(ESI) calcd. for C12H14NO4S ([M þ H]þ) 268.0644, found 268.0635.
2,4-dichloro-N-(2-oxo-2H-chromen-6-yl)benzenesulphona-
mide (12): Yellow solids (Yield 83.1%). 1H NMR (400 MHz, DMSO)
d 10.92 (s, 1H), 8.05 (d, J ¼ 8.8 Hz, 2H), 7.87 (s, 1H), 7.61 (d,
J ¼ 10.4 Hz, 1H), 7.45 (s, 1H), 7.33 (s, 2H), 6.48 (d, J ¼ 9.6 Hz, 1H).
13 C NMR (101 MHz, DMSO) d 180.07, 150.82, 144.22, 139.20,
135.73, 133.45, 133.38, 132.45, 131.90, 128.47, 124.68, 119.82,
N-(2-oxo-2H-chromen-6-yl)butane-1-sulphonamide (5): White 119.38, 117.86, 117.45. HRMS (ESI) calcd. for C15H10NO4SCl2
solids (yield 93.1%). 1H NMR (400 MHz, DMSO) d 9.99 (s, 1H), 8.10 ([M þ H]þ) 369.9708, found 369.9694.
4-chloro-2-methoxy-N-(2-oxo-2H-chromen-6-yl)benzenesul-
(d, J ¼ 9.6 Hz, 1H), 7.56 (s, 1H), 7.42 (q, J ¼ 9.0 Hz, 2H), 6.51 (d,
phonamide (13): Pink solids (yield 81.9%). 1H NMR (400 MHz,
J ¼ 9.6 Hz, 1H), 3.18–3.07 (m, 2H), 1.75–1.61 (m, 2H), 1.45–1.30 (m,
2H), 0.85 (t, J ¼ 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO) d 160.29,
150.50, 144.49, 135.13, 124.66, 119.64, 118.96, 117.76, 117.24,
50.85, 25.57, 21.12, 13.88. HRMS (ESI) calcd. for C13H16NO4S
([M þ H]þ) 282.0800, found 282.0792.
DMSO) d 10.37 (s, 1H), 8.05 (d, J
¼
9.6 Hz, 1H), 7.71 (d,
J ¼ 2.6 Hz, 1H), 7.64 (dd, J ¼ 8.9, 2.5 Hz, 1H), 7.44 (s, 1H), 7.31 (s,
2H), 7.24 (d, J ¼ 8.9 Hz, 1H), 6.46 (d, J ¼ 9.6 Hz, 1H), 3.91 (s, 3H). 13C
NMR (101 MHz, DMSO) d 160.19, 155.71, 150.74, 144.32, 135.23,
134.16, 129.72, 128.03, 124.97, 124.15, 119.55, 117.58, 117.33,
4-chloro-N-(2-oxo-2H-chromen-6-yl)benzenesulphonamide
(6): Pink solids (yield 86.2%). H NMR (400 MHz, DMSO) d 10.55 (s, 115.49, 57.09. HRMS (ESI) calcd. for C16H13NO5SCl ([M þ H]þ)
1H), 8.06 (d, J ¼ 9.6 Hz, 1H), 7.76 (d, J ¼ 8.5 Hz, 2H), 7.64 (d, 366.0203, found 366.0193.
1
J ¼ 8.5 Hz, 2H), 7.46 (s, 1H), 7.31 (m 2H), 6.49 (d, J ¼ 9.6 Hz, 1H). 13C
N-(2-oxo-2H-chromen-6-yl)thiophene-2-sulphonamide (14):
Light pink solids (yield 90.5%). 1H NMR (400 MHz, DMSO) d 10.61
NMR (101 MHz, DMSO) d 160.16, 151.00, 144.31, 138.42, 134.03,
(s, 1H), 8.08 (d, J ¼ 9.6 Hz, 1H), 7.91 (d, J ¼ 4.9 Hz, 1H), 7.58 (d,
129.98, 129.10, 125.53, 120.36, 119.63, 117.81, 117.37. HRMS (ESI)
calcd. for C15H11NO4SCl ([M þ H]þ) 336.0097, found 336.0085.
J ¼ 3.5 Hz, 1H), 7.52 (s, 1H), 7.39–7.28 (m, 2H), 7.1–7.09 (m, 1H),
4-methoxy-N-(2-oxo-2H-chromen-6-yl)benzene
sulphona- 6.49 (d, J ¼ 9.6 Hz, 1H). 13C NMR (101 MHz, DMSO) d 160.19,
mide (7): Light pink solids (yield 72.8%). 1H NMR (400 MHz, 151.04, 144.33, 139.93, 134.11, 133.13, 128.18, 125.42, 120.28,
DMSO) d 10.34 (s, 1H), 8.05 (d, J
¼
9.6 Hz, 1H), 7.71 (d, 119.57, 117.64, 117.36. HRMS (ESI) calcd. for C13H10NO4S2
J ¼ 8.7 Hz, 2H), 7.46 (s, 1H), 7.35–7.21 (m, 2H), 7.06 (d, J ¼ 8.7 Hz, ([M þ H]þ) 308.0051, found 308.0044.
2H), 6.47 (d, J ¼ 9.6 Hz, 1H), 3.80 (s, 3H). 13 C NMR (101 MHz,
DMSO) d 162.98, 160.21, 150.66, 144.37, 134.69, 131.20, 129.39,
125.01, 119.57, 117.66, 117.28, 114.90, 56.00. HRMS (ESI) calcd. for
C16H14NO5S ([M þ H]þ) 332.0593, found 332.0586.
N-(2-oxo-2H-chromen-6-yl)naphthalene-2-sulphonamide
(15): Light pink solids (yield 83.5%). 1H NMR (400 MHz, DMSO) d
10.65 (s, 1H), 8.52 (s, 1H), 8.14 (t, J ¼ 8.9 Hz, 2H), 8.09–7.97 (m, 2H),
7.85 (d, J ¼ 8.7 Hz, 1H), 7.68 (m, 2H), 7.55 (s, 1H), 7.34 (m 2H), 6.47
4-(tert-butyl)-N-(2-oxo-2H-chromen-6-yl)benzene sulphona- (d, J ¼ 9.6 Hz, 1H). 13C NMR (101 MHz, DMSO) d 160.15, 150.77,
mide (8): Pink solids (yield 85.3%). 1H NMR (400 MHz, DMSO) d 144.24, 136.63, 134.75, 134.37, 132.00, 130.00, 129.70, 129.48,
10.48 (s, 1H), 8.04 (d, J ¼ 9.6 Hz, 1H), 7.72 (d, J ¼ 8.3 Hz, 2H), 7.57 128.54, 128.22, 125.16, 122.44, 119.90, 119.55, 117.70, 117.30.
(d, J ¼ 8.4 Hz, 2H), 7.48 (s, 1H), 7.31 (s, 2H), 6.47 (d, J ¼ 9.6 Hz, 1H), HRMS (ESI) calcd. for C19H14NO4S ([M þ H]þ) 352.0644,
1.25 (s, 9H). 13 C NMR (101 MHz, DMSO) d 160.19, 156.48, 150.60, found 352.0634.
N-(2-oxo-2H-chromen-6-yl)-2,3-dihydrobenzofuran-5-sul-
phonamide (16): Light pink solids (yield 91.9%). 1H NMR
(400 MHz, DMSO) d 10.28 (s, 1H), 8.05 (d, J ¼ 9.6 Hz, 1H), 7.63 (s,
144.36, 137.03, 134.62, 127.03, 126.65, 124.70, 119.50, 119.29,
117.73, 117.33, 35.32, 31.16. HRMS (ESI) calcd. for C19H20NO4S
([M þ H]þ) 358.1113, found 358.1109.
3-cyano-N-(2-oxo-2H-chromen-6-yl)benzene sulphonamide 1H), 7.53 (d, J ¼ 9.5 Hz, 1H), 7.44 (s, 1H), 7.34–7.24 (m, 2H), 6.86 (d,
(9): Light pink solids (Yield 87.9%). 1H NMR (400 MHz, DMSO) d J ¼ 8.5 Hz, 1H), 6.46 (d, J ¼ 9.6 Hz, 1H), 4.60 (t, J ¼ 8.8 Hz, 2H), 3.20
10.67 (s, 1H), 8.23 (s, 1H), 8.18 – 8.00 (m, 3H), 7.80 (t, J ¼ 7.9 Hz, (t, J ¼ 8.8 Hz, 2H). 13C NMR (101 MHz, DMSO) d 163.7, 160.24,
1H), 7.49 (d, J ¼ 2.2 Hz, 1H), 7.31 (dt, J ¼ 8.9, 5.6 Hz, 2H), 6.50 (d, 150.54, 144.40, 134.77, 131.21, 129.41, 128.67, 124.87, 124.54,
J ¼ 9.6 Hz, 1H). 13 C NMR (101 MHz, DMSO) d 160.14, 151.14, 119.46, 117.65, 117.27, 109.55, 72.69, 28.83. HRMS (ESI) calcd. for
144.27, 140.80, 137.18, 133.61, 131.57, 131.35, 130.72, 125.68, C17H14NO5S ([M þ H]þ) 344.0593, found 344.0586.

816
Z. ZHANG ET AL.
N-(4-methoxyphenyl)-2-oxo-2H-chromene-6-sulphonamide
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1
(17): Light purple solids (yield 77.2%). H NMR (400 MHz, DMSO) d
10.03 (s, 1H), 8.16 (d, J ¼ 9.6 Hz, 1H), 8.11 (s, 1H), 7.85 (d, J ¼ 7.0 Hz,
1H), 7.56 (d, J ¼ 8.7 Hz, 1H), 7.00 (d, J ¼ 8.8 Hz, 2H), 6.81 (d,
J ¼ 8.8 Hz, 2H), 6.60 (d, J ¼ 9.6 Hz, 1H), 3.67 (s, 3H). 13C NMR
(101 MHz, DMSO) d 159.60, 157.22, 156.17, 143.92, 135.89, 130.15,
128.08, 124.39, 119.29, 118.20, 117.96, 114.85, 55.62. HRMS (ESI)
calcd. for C16H13NO5NaS ([M þ Na]þ) 354.0412, found 354.0398.
Disclosure statement
The authors declare no conflict of interest.
Funding
This work was financially supported by The National Natural
Science Funds of China [81803372], Natural Science Foundation of
Zhejiang
Neuropsychiatric
Province
[LY17H310008],
Research
Key
Laboratory
of
Drug
of
Zhejiang
Province
[2019E10021], Doctoral Fund of Zhejiang Academy of Medical
Sciences [A11701Q] and Health Commission of Zhejiang Province
[2019RC141, XKQ-010–001 and WJK-ZJ-1918].
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