Optimization of 5-arylidene barbiturates as potent, selective, reversible LSD1 inhibitors for the treatment of acute promyelocytic leukemia

Article abstract of DOI:10.1016/j.bmc.2018.08.026

Histone lysine specific demethylase 1 (LSD1) is overexpressed in diverse hematologic disorders and recognized as a promising target for blood medicines. In this study, molecular docking-based virtual screening united with bioevaluation was utilized to identify novel skeleton of 5-arylidene barbiturate as small-molecule inhibitors of LSD1. Among the synthesized derivatives, 12a exhibited reversible and potent inhibition (IC₅₀ = 0.41 μM) and high selectivity over the MAO-A and MAO-B. Notably, 12a strongly induced differentiation effect on acute promyelocytic leukemia NB4 cell line and distinctly escalated the methylation level on histone 3 lysine 4 (H3K4). Our findings indicate that 5-arylidene barbiturate may represent a new skeleton of LSD1 inhibitors and 12a deserve as a promising agent for the further research.

Full text of DOI:10.1016/j.bmc.2018.08.026

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimization of 5-arylidene barbiturates as potent, selective, reversible
LSD1 inhibitors for the treatment of acute promyelocytic leukemia
Siyuan Xub,e, Chen Zhoua,c,e, Rongfeng Liud, Qihua Zhuc, Yungen Xuc,⁎, Fei Lanb,⁎
,
Xiaoming Zhaa,⁎
a
Department of Pharmaceutical Engineering, Department of Biomedical Engineering, Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing
210009, PR China
b
Liver Cancer Institute, Zhongshan Hospital, Fudan University, Key Laboratory of Carcinogenesis and Cancer Invasion, Ministry of Education, Key Laboratory of
Epigenetics and Metabolism, Ministry of Science and Technology, and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China
c
Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China
Shanghai ChemPartner Co. Ltd., Zhangjiang Hi-Tech Park, Shanghai 201203, PR China
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
LSD1
5-Arylidene barbiturate
Selective
Reversible
Leukemia
Histone lysine specific demethylase 1 (LSD1) is overexpressed in diverse hematologic disorders and recognized
as a promising target for blood medicines. In this study, molecular docking-based virtual screening united with
bioevaluation was utilized to identify novel skeleton of 5-arylidene barbiturate as small-molecule inhibitors of
LSD1. Among the synthesized derivatives, 12a exhibited reversible and potent inhibition (IC50 = 0.41 μM) and
high selectivity over the MAO-A and MAO-B. Notably, 12a strongly induced differentiation effect on acute
promyelocytic leukemia NB4 cell line and distinctly escalated the methylation level on histone 3 lysine 4 (H3K4).
Our findings indicate that 5-arylidene barbiturate may represent a new skeleton of LSD1 inhibitors and 12a
deserve as a promising agent for the further research.
1. Introduction
breast cancer.9–12 In addition to histones, LSD1 catalyzes the de-
methylation of p53, blocking p53 mutual effect with its coactivator
Lysine-specific demethylase 1 (LSD1), reported as the first histone
demethylase, is a flavin-dependent amine oxidase enzyme, ushering in
a new avenue in the chromatin remodeling.1 As an element of co-re-
pressor complexes, LSD1 facilitates target gene repression by removing
mono-and dimethyl marks from lysine 4 of histone H3 (H3K4).2
Nevertheless, LSD1-dependent demethylation of histone H3K9 results
in the activation of androgen receptor (AR) target gene expression.3
However, the mechanisms that dominate this dual personality are
poorly understood. LSD1 is a key histone demethylase that mediates the
epigenetic landscape. A flurry of studies has provided sights into the
particular biological roles of LSD1 and its potential relationships to
pathological processes including carcinogenesis.4–6 LSD1 is considered
to target cancerous cells rather than normal ones, which potentially
empowers the alternative targeting of cancer.7,8 High-level LSD1 ex-
pression has been associated with an alarming risk of acute myeloid
leukemia (AML), as well as prostate cancer, colon carcinoma, and
53BP1.13 Interestingly, elevated levels of LSD1 have a favorable effect
on muscle recovery and regeneration after injuries, due to direct reg-
ulation of key myogenic transcription factor genes.14 Owning to
abundant proof-of-concept studies forcefully back up its potential as a
druggable target, obtaining selective and potent LSD1 inhibitors has
emerged as a motivating and promptly evolving area of research.15
Up to now, plenty of LSD1 inhibitors with different scaffolds have
been reported (Fig. 1).16–18 The most widely investigated LSD1 in-
hibitors were mechanism-based agents which demethylate histone ly-
sine via redox reactions.19 Those agents are irreversible inhibitors
acting as FAD competitor situating at the LSD1 active site.20–23 Two
outstanding irreversible inhibitors,24–26 GSK2879552 (A) and RG-6016
(B) are currently undergoing advanced clinical trials for the treatment
of AML (Fig. 1).27,28 Based on the TCPA scaffold, Hoang et al. developed
a novel LSD1 inhibitor, CBB3001(C), which selectively inhibited ter-
atocarcinoma and embryonic carcinoma cells that express SOX2 and
Abbreviations: LSD1, histone lysine specific demethylase 1; lysine 4 of histone H3, H3K4; MAO, monoamine oxidase; FAD, flavin adenine dinucleotide; VS, virtual
screening; MST, microscale thermophoresis; ATRA, all-trans retinoic acid
⁎
Corresponding authors.
E-mail addresses: xyg64@126.com (Y. Xu), fei_lan@fudan.edu.cn (F. Lan), xmzha@cpu.edu.cn (X. Zha).
These authors contributed equally to this work.
e
https://doi.org/10.1016/j.bmc.2018.08.026
Received 9 July 2018; Received in revised form 17 August 2018; Accepted 20 August 2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Xu,S.,Bioorganic&MedicinalChemistry(2018),https://doi.org/10.1016/j.bmc.2018.08.026

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 1. Representative structures of reported LSD1 inhibitors.
OCT4.29 Other than mechanism-based inhibitors, a limited number of
potent reversible inhibitors of LSD1 have been developed, such as
aminotriazoles (D),30 pyridine containing compounds (E).31 Now-
otarski and co-workers have reported a series of polyamine analogs (F)
with thiourea moiety bearing potent inhibition to LSD1 activity.32 The
SAR analysis suggested that the ability of these analogues to promote
epigenetic changes was related to the length of the central chain. Very
recently, Vianello et al. employed a high-throughput screening (HTS)
strategy to get the compound G (IC50 = 2.9 µM) that upon further op-
timization yielded compound H with extremely high potency against
LSD1 (IC50 = 7.8 nM).33,34 The reversible LSD1 inhibitors display some
advantages over irreversible inhibitors, particularly in view of a safer
metabolic profile.
of the hit compounds. We introduced different aromatic rings bearing
one or more hydrogen bond donators to the N1 position of the 5-ar-
ylidene barbiturates scaffold. What’s more, benzene ring containing
different substituents was introduced to the C5 position of the 5-ar-
ylidene barbiturates scaffold, aiming to achieve substituent diversity
and explore the structure-activity relationships (SARs). Further ex-
perimental studies demonstrated that the derivative 12a significantly
inhibited acute promyelocytic leukemia cell proliferation. In conclu-
sion, we report herein 5-arylidene barbiturates as novel potent, re-
versible and selective LSD1 inhibitors, and they can provide us with
helpful structural clues to develop more powerful LSD1 inhibitors.
2. Results and discussion
The virtual screening (VS) has arised as a powerful computational
tool, which is commonly applied in drug discovery process.35,36 Apart
from its convenience, this technique can help identify leading com-
pounds with novel skeletons and provide structural clues for compound
optimization.37,38 So far, several successful docking-based virtual
screenings have been implemented with LSD1. Hazeldine et al. initiated
VS to gain a variety of low molecular weight amidoximes as reversible
LSD1 inhibitors of the most effective one (I) exhibited modest potency
against LSD1 (IC50 = 16.8 µM).39 Following our former success in this
area,40–42 herein we report the successful acquisition of 5-arylidene
barbiturate skeleton and design, synthesis and biological studies of a
new series of LSD1 inhibitors. We initiated a docking-based VS to hunt
for novel LSD1 inhibitors from the SPECS database and in vitro inhibi-
tion assaying campaign to identify potent and various lead compounds.
We examined these hits by interaction analyses and selected two hits for
structural optimization. Those two compounds were purchased for
further bioactivity testing (See Table 1). Excitingly, these two molecules
containing the same 5-arylidene barbiturates scaffold performed potent
inhibitory activity in the micromolar range. Through analysing binding
modes of the two compounds (Fig. 2), the hydrogen bond between
Trp756 and hydrogen bond donator of ligand is critical to the inter-
action. The docking score of XZ01 is lower than that of XZ02, which is
consistent with their respective inhibitory activity. Chemical optimi-
zation was conducted to acquire more structural analogues on the basis
2.1. Predicted binding patterns of the two hit compounds
The two hits show similar binding poses predicted by molecular
docking. As shown in Fig. 2, the binding poses of these two inhibitors
are quite similar, and both can form the hydrogen bond interaction with
Arg316, Ser287, Ser289. However, the C5 substitution of XZ02 does not
form H-bonding interactions with Trp756 which XZ01 does have. Thus,
according to the chemical structures of these two inhibitors, it may
explain why XZ01 showed three times inhibition activity than XZ02. It
seems that the addition of a hydrogen donator group at C5 substitution,
which formed a key H-bond with Trp756, may be beneficial for en-
hancing the inhibitory activity against LSD1.
2.2. Docking-based VS
In the present study, a docking-based VS strategy was used to
identify promising hits. Indicated by the AUC values, the Glide SP
scoring mode showed better discrimination power than the other two
docking tools. In addition to the docking score rankings, visual in-
spection of the docking poses was used to assess binding mode and
orientation. Taken together, this strategy identified a set of two hits.
They were procured and screened in the LSD1 biochemical assay.
Compound purity has been verified by vendors using nuclear magnetic
2

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 1
Structures of two hit compounds and their inhibition rates (IC50) to LSD1 in vitro.
Compound
Structures
SPECS ID
IC50 (μM) against LSD1
Glide Gscore (kj/mol)
XZ01
AG-690/11763270
6.42
−11.05
XZ02
AH-487/41938748
18.2
−10.15
solution of sodium alcohol under reflux, affording compounds 4a-4e.
With compounds 4a-4e in hand, we next introduced different aldehyde
substituents into the scaffold, aiming to achieve substituent diversity
and explore the structure-activity relationships. The reactions between
distinctive aldehydes and compounds 4a-4e proceeded smoothly and
efficiently under mild conditions, giving corresponding products 5a-5k
in good yields.
In order to further investigate the structure-activity relationships
(SARs), we also performed additional structural modifications by re-
placing the oxygen atom at C2 position with sulphur atom and the re-
duction of the double bond at C5 position. The synthesis of compound
6a could be completed in a single step, as shown in Scheme 1. Com-
pound 5k was treated with sodium borohydride to afford the desired
product 6a in 70.8% yield. The remaining target molecule 12a was
synthesized as outlined in Scheme 2. Treatment of 7 with the appro-
priate amiline 8a in the presence of benzoyl chloride under reflux af-
forded the target molecule 9a. 10a was prepared from 9a by treatment
with preheated solution of aqueous sodium hydroxide (5%). Then 10a
was treated similarly to 3a-3e and afforded the desired compound 12a
as shown in Scheme 2.
Fig. 2. The binding modes between XZ01, XZ02 and LSD1 (PDB id: 2Z5U), the
hydrogen bonds interactions are highlighted using red dash line, XZ01 is re-
presented as green sticks, XZ02 is represented as brown sticks, important re-
sidues are represented as blue sticks. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this
article.)
2.4. Biochemical activity of the candidate compounds against LSD1 and
SAR studies
resonance (NMR) experiments.
All the compounds synthesized in this study were examined for their
inhibitory effect on LSD1 activity in vitro. TCPA was chosen as a posi-
tive control. The results were summarized as Tables 2–5. The results of
compounds 5a-5f against LSD1 were determined initially and shown in
Table 2. All the compounds exhibit moderate to good potency with IC50
values ranging from 0.3 to 23 µM. Among them, compound 5c shows
the most potent activity to LSD1 (IC50 = 0.3 µM), which is 12 times
stronger than that of TCPA (IC50 = 3.6 µM). During the SAR studies, we
2.3. Chemistry
Compounds 5a-5k could be readily synthesized using the routes
described in Scheme 1. The phenylurea derivatives were synthesized
using the same method as described by Kurzer.43 Treatment of aniline
with sodium cyanate in the presence of glacial acetic acid in water gave
compounds 3a-3e, which then reacted with diethyl malonate in ethanol
Scheme 1. Synthesis of compounds 5a-k and 6a. Reagents and conditions: (a) AcOH, H2O, rt, 2 h; (b) diethyl malonate, Na, ethanol, reflux; (c) appropriate aromatic
aldehydes, ethanol, reflux, 2 h; (d) NaBH4, rt.
3

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 2. Synthesis of compound 12a. Reagents and conditions: (a) acetone, reflux; (b) 5% NaOH, 85 °C; (c) diethyl malonate, Na, ethanol, reflux; (d) 3,4-
dihydroxybenzaldehyde, ethanol, reflux, 2 h.
Table 2
Table 4
Structures of compounds 5a-5f and their inhibition data (IC50) against LSD1 in
The structure of compound 12a and the inhibition (IC50) against LSD1 in
vitro.
vitro.
Compound
R3
X
IC50 (μM)
Compound
X
R1
H
R2
H
R3
IC50 (μM)
12a
Br
0.41
5a
CH3
1.1
5b
5c
CH3
H
H
H
H
4.4
0.3
Br
Table 5
The structure of compound 6a and the inhibition (IC50) against LSD1 in
5d
5e
H
H
H
H
H
H
OCH3
CN
3.1
1.2
vitro.
Compound
R3
X
IC50 (μM)
6a
Br
> 75
5f
CH3
23
TCPA
3.6
found that the benzene ring substitution on the N1 was important for
the inhibitory activity: the 4-Br substitution derivative compound 5c
was more potent than the other benzene ring substitution derivative
compounds. Moreover, C5 substitution was also preferential: 3,4-di-
hydroxybenzy substitution derivative compound 5c (IC50 = 0.3 µM)
was more potent than 1H-indol-2-yl substitution derivative compound
5a (IC50 = 1.11 µM).
Table 3
Structures of compounds 5g-5k and their inhibition data (IC50) against LSD1 in
vitro.
To determine the inhibitory effect of compounds against LSD1 at
cellular level, acute premyeloid leukemia (APL) cell line NB4 was
chosen.44 NB4 is a cell line that has been used as a model to study APL.
Its differentiation can be partially detected by a cell surface marker
CD11b. Here, we used FACS to test how CD11b level change after the
drug treatment to explore the drug’s effect in vivo. 5a, 5b, 5c and 5e
were submitted to in vivo differentiation assay. We used two reported
irreversible LSD1 inhibitors, TCPA and RN-1 (IC50 = 8 nM) as con-
trol.45,46 The results were summarized as Fig. 3. At the concentration of
5 μM, our compounds 5a, 5c and 5e perform strong differentiation ef-
fect on NB4 cells, among them 5c and 5e perform better and are
stronger than RN-1 (Fig. 10).
Compound
R3
R4
R5
R6
R7
IC50 (μM)
5g
5h
5i
5j
5k
Br
Br
Br
Br
Br
H
H
H
OCH3
H
OCH3
OH
OCH3
OCH3
OCH3
OH
OCH3
OH
OCH3
OCH3
H
H
OCH3
H
H
0.87
3.6
0.55
3.6
2.8
3.6
TCPA
In view of the in vitro and in vivo inhibition assay of the first batch of
compounds 5a-5f, N1 substitution was locked as 4-Br. We also con-
sidered that the pyrocatechol part may be a false positive compound.
4

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 3. The in vivo differentiation assay of 5a, 5b, 5c and 5e.
Fig. 5. Kd value for compounds 12a, obtained by microscale thermophoresis
(MST) experiment.
IC50 value of 0.41 μM. However, due to the reduction of the double
bond in C5 position, compounds 6a did not exhibit LSD1 inhibitory
effect, compared to the corresponding compound 5k. These findings
indicate that the 5-arylidene barbituric scaffold is important to their
inhibitory activity.
2.5. In vitro inhibition properties of compound 12a against the recombinant
LSD1 and its homologies MAO-A and MAO-B.
Having identified compound 12a as a highly potent LSD1 inhibitor,
we further determined the dissociation constant (Kd) of 12a by micro-
scale thermophoresis (MST) experiment. MST assay shows the kinetic
binding affinity between protein and molecules. The Kd value for
compound 12a was 1.1 μM, indicating the robust binding affinity of
12a to LSD1 (Fig. 5).
To test the reversibility of compound 12a for LSD1, a dilution assay
was used. Our analysis suggested that dilution of the LSD1/12a mixture
by 100-fold resulted in the recovery of LSD1 activity, which indicated
noncovalent interaction with the enzyme. The in vitro IC50 of 12a to-
ward LSD1 is 0.41 μM, the recombinant LSD1 was incubated with the
compound at 4.1 μM for 1 h and then diluted 100-fold to test the LSD1
enzymatic activity. 98.19% of the enzymatic effect is restored, proving
that 12a is a reversible LSD1 inhibitor (Fig. 6).
Fig. 4. The predicted binding modes between compounds 5c, 5k and LSD1
(PDB id: 2Z5U). Compounds 5c and 5k were highlighted in green, grey re-
spectively. The hydrogen bonds interactions are highlighted using red dash line,
important residues are represented as blue sticks. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web
version of this article.)
Thus, we introduced different hydroxyl substituted benzene to the C5
position. Compounds 5g-5k were synthesized, and the results were
shown in Table 3. Replacing the pyrocatechol scaffold with substituted
benzene 5g-5k (Table 3) led to a lowered enzyme activity. 4-hydroxy-
2,3-dimethoxybenzyl was better than the other substitutions. 5j which
did not have phenol hydroxyl performed activity less than 3.0 μM.
Those results indicated the significance of the phenolic hydroxyl group
in retaining their activity. Furthermore, the predicted binding modes of
compounds 5c and 5k in the active site of between LSD1 are displayed
in Fig. 4. The compound 5c performed strong and various interactions
with LSD1. Gly287, Ser 289, Arg316 and Thr624 formed hydrogen
bonds with the 5-arylidene barbiturates scaffold. Besides, the OH of the
C5 substituent also formed hydrogen bonds with surrounding Glu308
and Ala309 residues. In contrast, the compound 5k stretched in an
opposite direction compared with 5c, which didn’t show interactions
with any key residues, such as Trp756 and Arg 316. The docking results
may explain the difference of the activity.
As LSD1 belongs to the monoamine oxidases (MAOs) family, the
inhibitory effects of 12a to its homologies MAO-A and MAO-B were also
examined. As shown in Fig 7, 12a showed slight effect on MAO-A and
MAO-B inhibitory activities. For MAO-A, the IC50 of MAO-A is
72.52 μM, 175 times to its effect on LSD1 and for MAO-B, the IC50 is out
of the detection range indicating > 487-fold to LSD1. These findings
Furthermore, the importance of barbituric scaffold of our com-
pounds on LSD1 inhibitory activity was explored. We replaced the
oxygen atom in C2 position with sulphur atom (Table 4). Benzyl pyr-
imidine-2,4,6-triones were also synthesized in order to evaluate the
importance of the double bond in C5 position (Table 5). As can be seen
from Table 4, substitution of the oxygen atom with a sulphur atom did
not much affect the inhibition activity. 12a showed potent activity with
Fig. 6. The reversibility of compound 12a to LSD1 activity was determined by
jump-dilution assay.
5

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 7. In vitro inhibitory activities of compound 12a against LSD1 and its homologies MAO-A and MAO-B.
showed moderate differentiation effect, while 5i performed weak dif-
ferentiation effect. Furthermore, we evaluated the differentiation effect
of 12a on other leukemia cell lines such as HL-60 and THP-1 cell lines.
As shown in Fig. 9, among three leukemia cells lines, 12a performed the
most robust differentiation effects on NB4 cell lines with the differ-
entiation rate of 89.7% at 10 μM comparing to 87% at 0.1 μM for ATRA
as positive control.47 (See Fig. 10).
To determine whether those compounds are cell active LSD1 in-
hibitors, the effects of six compounds on the methylation levels of LSD1
substrates H3K4 were analysed in human embryonic kidney 293 T cells
(HEK 293 T). 293 T cells were treated for 24 h with vehicle or 2.5, 5, or
10 μM of compound 12a for 24 h. We particularly selected the
H3K4me1 and H3K4me2 marks. The results are summarized in Fig. 11.
Among these compounds assayed, 12a displayed the most potent im-
provement of methylation on H3K4me1 and H3K4me2. Moreover, 12a
could elevate the methylation on H3K4me2 at 2.5 μM and particularly
significantly improved the methylation on H3K4me1 at 10 μM, which
may explain, compared to other conmpounds, why it owned more po-
tent effect of differentiation inducing. These results validated that the
compound 12a can decrease LSD1 activity at the cellular level.
Fig. 8. The differentiation assay of 5g, 5i and 12a on NB4 cells.
3. Conclusion
In this study, docking-based VS combined with bioassays was em-
ployed to identify two hits compounds bearing skeleton of 5-arylidene
barbiturate as LSD1 inhibitors in a micromolar range. After analysing
their binding modes with LSD1, optimizations of the hits were carried
out and 13 derivatives were synthesized. Among them, 12a, the most
potent compound (IC50 = 0.41 μM) displayed as a selective and re-
versible LSD1 inhibitor and exhibited strong differentiation inducing
effect on LSD1-overexpressed acute promyelocytic leukemia NB4 cells.
Furthermore, 12a also demonstrated potent improvement of methyla-
tion and dimethylation of H3K4. Taken togethor, 5-arylidene barbitu-
rates represent a new class of potent, selective and reversible LSD1
inhibitors and 12a deserves a lead compound for the future develop-
ment of reversible LSD1 inhibitors.
Fig. 9. The differentiation assay of 12a on NB4, HL-60 and THP-1 cell lines
(ATRA in 0.1 μM concentration).
indicated a good selectivity of 12a toward LSD1 in vitro.
The differentiation assay result of the second batch of compounds
5g, 5i and 12a was summarized in Fig. 8. 12a showed strong differ-
entiation effect on NB4 cells. At the concentrations of 2.5 μM and 5 μM,
the differentiation rates were 12.5% and 36.6%, respectively. 5g
Fig. 10. Chemical structures of control compounds,
RN-1 and ATRA.
6

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 11. Effect of six compounds including 12a on histone methylation.
4. Experimental section
solution of 4a-4e (5 mmol) in ethanol (20 mL) at rt. The reaction mix-
ture was heated at reflux for 1 h. The reaction mixture was cooled down
and the resulting precipitate was filtered and recrystallized from water
to give 5a-5k as yellow solids as a 1:1 mixture of geometrical isomers
(E/Z).
4.1. General
Reagents and solvents were purchased from commercial sources and
were used without further purification. NMR spectra were recorded on
a Bruker AMX-300 (1H at 300 MHz, 13C at 75 MHz) spectrometer.
Proton and carbon NMR chemical shifts are reported as values in ppm
with residual protic solvent as standard. All reagents were used as
supplied by the manufacturer. Analytical thin-layer chromatography
(TLC) was performed on pre-coated glass backed plates (Merck
Kieselgel F254).
4.1.3.1. (E/Z)-5-((1H-indol-2-yl)methylene)-1-(p-tolyl)pyrimidine-2,4,6
(1H,3H,5H)-trione (5a). Yellow solid, yield: 90%, M. p. > 250 °C. IR
(KBr): 3030.2, 1734.0, 1689.6, 1645.3, 1546.9, 1328.9, 1130.3,
871.8, 750.3 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ 12.47 (s, 1H,
indole –NH-), 12.11 (s, 1H, indole –NH-), 11.81 (s, 1H, –NH-), 11.68
(s, 1H, –NH-), 8.40 (s, 1H, –CH-), 8.34 (s, 1H, –CH-), 7.73–7.72 (m,
6H, ArH), 7.69–7.08 (m, 12H, ArH), 2.38 (s, 3H, –CH3-), 2.37 (s, 3H,
–CH3-). 13C NMR (75 MHz, DMSO‑d6) δ 163.33, 162.74, 150.22,
150.20, 142.36, 141.98, 139.95, 139.72, 137.79, 137.58, 132.88,
132.66, 132.16, 131.97, 129.35, 129.23, 128.88, 128.71, 127.75,
127.69, 127.59, 127.44, 122.52, 122.38, 122.01, 121.50, 121.06,
121.02, 114.48, 113.64, 113.42, 112.07, 20.70. HRMS m/z
([M + H]+) calcd for C20H16N3O3: 346.1186; found: 346.1188.
4.1.1. General procedure for the synthesis of substituted phenylureas (3a-
3e)
The phenylurea derivatives were synthesized according to the
method described by Kurzer. Briefly, the aniline (0.05 mol) was dis-
solved in glacial acetic acid (24 mL) and water (48 mL). To this solu-
tion, was slowly added under stirring the sodium cyanate (0.1 mol)
dissolved in water (45 mL). When a white precipitate appeared, the
solution was stirred for 1 additional hour, then it was allowed to stand
at rt for 2–3 h. The solution was cooled to 0 °C before filtration. The
resulting phenylurea was washed with cold water, and was used
without any further purification.
4.1.3.2. (E/Z)-5-((1H-indol-2-yl)methylene)-1-(o-tolyl)pyrimidine-2,4,6
(1H,3H,5H)-trione (5b). Yellow solid, yield: 82.0%, M. p. > 250 °C.
IR (KBr): 3066.8, 1726.3, 1683.9, 1651.1, 1533.4, 1338.6, 1107.1,
829.7, 718.7 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ 12.47 (s, 1H,
indole –NH-), 12.08 (s, 1H, indole –NH-), 11.88 (s, 1H, –NH-), 11.76
(s, 1H, –NH-), 8.43 (s, 1H, –CH-), 8.36 (s, 1H, –CH-), 7.75–7.70 (m,
6H, ArH), 7.43–7.29 (m, 10H, ArH), 7.17–7.08 (m, 2H, ArH), 2.14 (s,
3H, –CH3-), 2.11 (s, 3H, –CH3-). 13C NMR (75 MHz, DMSO‑d6) δ
163.36, 163.13, 162.90, 162.78, 149.71, 142.59, 142.39, 140.10,
139.83, 136.18, 136.02, 134.54, 134.33, 132.18, 131.98, 130.43,
130.32, 129.26, 129.13, 128.65, 128.55, 127.86, 127.81, 127.61,
127.44, 126.64, 126.49, 122.58, 122.42, 122.38, 121.77, 121.07,
113.74, 113.47, 111.97, 111.65, 16.98, 16.97. HRMS (ESI): m/z
[M + H]+ calcd for C20H15N3O3: 346.1186; found: 346.1194.
4.1.2. General procedure for the synthesis of pyrimidine-2,4,6-triones (4a-
4e)
Sodium (20 mmol) was added portionwise to ethanol (60 mL) at rt.
After dissolution of the sodium, urea (10 mmol) and diethyl malonate
(10 mmol) were added sequentially each in one portion to the solution
with stirring at rt and then heated at reflux for 5 h. Solvent was con-
centrated under reduced pressure and the remaining residue was dis-
solved in 1 M sodium hydroxide solution (20 mL). The aqueous layer
was washed with ethyl acetate (2 × 20 mL) and then acidified (pH = 1)
with 1 M aqueous hydrochloric acid solution and the resulting pre-
cipitate was filtered. The isolated solid material was recrystallized from
methanol to give 4a-4e as colorless solids.
4.1.3.3. (E/Z)-1-(4-bromophenyl)-5-(3,4-dihydroxybenzylidene)pyrimidi-
ne-2,4,6(1H,3H,5H)-trione (5c). Yellow solid, yield: 74.3%, M.
p. > 250 °C. IR (KBr): 3176.7, 1716.6, 1683.9, 1673.8, 1509.2,
1300.6, 1169.4, 827.5, 785.9 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ
11.57 (s, 1H, –NH), 11.46 (s, 1H, –NH), 10.42 (s, 2H, –OH), 9.50 (s, 2H,
–OH), 8.22–8.15 (m, 2H, –CH-, 2H, ArH), 7.68–7.56 (m, 6H, ArH), 7.30
4.1.3. General procedure for the synthesis of benzylidene pyrimidine-2,4,6-
triones (5a-5k)
Appropriate aldehyde (5 mmol) was added in one portion to a
7

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(d, J = 8.1 Hz, 4H, ArH), 6.86 (s, 1H, ArH), 6.83 (s, 1H, ArH). 13C NMR
(75 MHz, DMSO‑d6) δ 163.80, 163.23, 161.66, 161.42, 156.82, 156.58,
152.66, 152.56, 150.12, 150.05, 144.91, 135.15, 134.97, 131.98,
131.76, 131.74, 131.60, 131.41, 124.32, 124.19, 121.33, 121.04,
1335.9, 1147.5, 807.9, 789.1 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ
11.63 (s, 1H, –NH), 11.52 (s, 1H, –NH), 9.45 (s, 1H, –OH), 9.43 (s, 1H,
–OH), 8.26 (s, 1H, –CH-), 8.18 (s, 1H, –CH-), 8.14 (s, 2H, ArH),
7.75–7.64 (m, 6H, ArH), 7.33–7.28 (m, 4H, ArH), 7.10–7.03 (m, 2H,
ArH), 3.90 (s, 3H, –OCH3), 3.87 (s, 3H, –OCH3). 13C NMR (75 MHz,
DMSO‑d6) δ 163.58, 163.02, 161.49, 161.29, 156.26, 156.06, 153.07,
153.00, 150.07, 149.99, 145.81, 145.78, 135.06, 134.86, 131.72,
131.52, 131.34, 130.99, 130.38, 125.37, 125.25, 121.34, 120.32,
120.00, 115.31, 115.03, 111.29, 111.26, 55.77, 55.73. HRMS m/z:
([M + H]+) calcd for C18H13BrN2O5: 417.0081; found: 417.0086.
115.43, 113.96, 113.65. HRMS m/z: ([M + H]+
)
calcd for
C
17H11BrN2O5: 402.9924; found: 402.9931.
4.1.3.4. (E/Z)-5-(3,4-dihydroxybenzylidene)-1-(4-methoxyphenyl)pyrimi-
dine-2,4,6(1H,3H,5H)-trione (5d). Yellow solid, yield: 74.6%, M.
p. > 250 °C. IR (KBr): 3176.7, 1722.4, 1685.8, 1662.6, 1537.3,
1317.4, 1138.0, 825.5, 779.2 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ
11.51 (s, 1H, –NH-), 11.40 (s, 1H, –NH-), 10.39 (s, 2H, –OH), 9.51 (s,
2H, –OH), 8.25–8.15 (m, 2H, –CH-, 2H, ArH), 7.67–7.56 (m, 2H, ArH),
7.25–7.20 (m, 4H, ArH), 7.02–6.98 (m, 4H, ArH), 6.88–6.83 (m, 2H,
ArH), 3.80 (s, 6H, –OCH3). 13C NMR (75 MHz, DMSO‑d6) δ 164.02,
163.20, 161.93, 161.41, 158.84, 158.77, 156.69, 156.32, 152.50,
152.38, 150.43, 150.37, 144.85, 144.83, 140.50, 132.00, 131.30,
130.18, 129.99, 128.18, 127.95, 124.32, 124.18, 121.20, 120.88,
115.36, 114.06, 113.93, 113.87, 113.73, 55.31, 55.26. HRMS m/z:
([M + H]+) calcd for C18H14N2O6: 355.0925; found: 355.0925.
4.1.3.9. (E/Z)-1-(4-bromophenyl)-5-(4-hydroxy-2,3-dimethoxybenzylide-
ne)pyrimidine-2,4,6(1H,3H,5H)-trione (5i). Yellow solid, yield: 88.1%,
M. p. > 250 °C. IR (KBr): 3408.8, 1726.9, 1682.6, 1667.0, 1489.4,
1307.3, 1162.1, 832.3, 788.3 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ
11.65 (s, 1H, –NH), 11.54 (s, 1H, –NH), 10.06 (s, 2H, –OH), 8.38 (s, 1H,
–CH-), 8.28 (s, 1H, –CH-), 8.01 (s, 2H, ArH), 7.94 (s, 2H, ArH), 7.68 (d,
J = 8.2 Hz, 4H, ArH), 7.34–7.30 (m, 4H, ArH), 3.84 (s, 6H, –OCH3),
3.78 (s, 6H, –OCH3). 13C NMR (75 MHz, DMSO‑d6) δ 163.72, 163.16,
161.72, 161.63, 157.03, 156.80, 150.10, 149.97, 147.08, 142.72,
142.62, 135.09, 134.94, 131.80, 131.73, 131.55, 131.36, 122.78,
122.69, 121.35, 114.48, 114.32, 114.06, 113.86, 56.09, 56.01. HRMS
m/z: ([M + H]+) calcd for C19H15BrN2O6: 447.0186; found: 447.0189.
4.1.3.5. (E/Z)-4-(5-(3,4-dihydroxybenzylidene)-2,4,6-trioxotetrahydrop-
yrimidin-1(2H)-yl)benzonitrile (5e). Yellow solid, yield: 87.3%, M. p.
229 ∼ 230 °C. IR (KBr): 3184.5, 1734.3, 1697.4, 1681.9, 1506.4,
1338.6, 1136.1, 846.7, 785.9 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ
11.64 (s, 1H, –NH-), 11.53 (s, 1H, –NH-), 10.42 (s, 2H, –OH), 9.51 (s,
2H, –OH), 8.25–8.16 (m, 2H, –CH-, 2H, ArH), 7.98–7.95 (m, 4H, ArH),
7.67–7.56 (m, 6H, ArH), 6.89 (s, 1H, ArH), 6.82 (s, 1H, ArH). 13C NMR
(75 MHz, DMSO‑d6) δ 163.65, 163.21, 161.52, 161.39, 156.84, 156.74,
152.75, 152.65, 149.93, 149.84, 144.88, 140.17, 140.08, 132.88,
132.84, 132.20, 131.55, 130.74, 130.54, 124.22, 124.10, 121.26,
120.90, 118.45, 115.42, 113.76, 113.44, 111.08. HRMS m/z:
([M + H]+) calcd for C18H11N3O5: 350.0771; found: 350.0774.
4.1.3.10. (E/Z)-1-(4-bromophenyl)-5-(2,3,4-trimethoxybenzylidene)pyri-
midine-2,4,6(1H,3H,5H)-trione (5j). Yellow solid, yield: 89.1%, M.
p. > 250 °C. IR (KBr): 3448.1, 1735.2, 1697.5, 1664.6, 1493.9,
1377.3, 1100.1, 830.4, 789.8 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ
11.66 (s, 1H, –NH-), 11.51 (s, 1H, –NH-), 8.58 (s, 1H, –CH-), 8.54 (s,
1H, –CH-), 8.35 (d, J = 9.2 Hz, 1H, ArH), 8.15 (d, J = 9.2 Hz, 1H, ArH),
7.69–7.65 (m, 4H, ArH), 7.31–7.28 (m, 4H, ArH), 6.95 (d, J = 9.6 Hz,
1H, ArH), 6.85 (d, J = 9.3 Hz, 1H, ArH), 3.93 (s, 3H, –OCH3), 3.91 (s,
3H, –OCH3), 3.88 (s, 3H, –OCH3), 3.87 (s, 3H, –OCH3), 3.78 (s, 3H,
–OCH3), 3.76 (s, 3H, –OCH3). 13C NMR (75 MHz, DMSO‑d6) δ 163.42,
162.81, 161.19, 161.05, 158.10, 158.05, 154.95, 154.90, 150.11,
150.00, 149.95, 149.74, 140.65, 140.58, 134.94, 134.73, 131.68,
131.44, 131.33, 129.38, 129.24, 121.37, 121.31, 118.91, 118.81,
116.83, 116.68, 107.23, 107.07, 61.98, 60.48, 56.26, 56.21. HRMS
m/z: ([M + H]+) calcd for C20H17BrN2O6: 461.0341; found: 461.0347.
4.1.3.6. (E/Z)-5-(3-hydroxybenzylidene)-1-(p-tolyl)pyrimidine-2,4,6(1H,
3H,5H)-trione (5f). Yellow solid, yield: 70.1%, M. p. 249–250 °C. IR
(KBr): 3118.9, 1732.1, 1670.4, 1635.6, 1556.5, 1319.3, 1161.2, 894.9,
790.8 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ 11.68 (s, 1H, –NH), 11.54
(s, 1H, –NH), 9.71 (s, 1H, –OH), 9.65 (s, 1H, –OH), 8.28 (s, 1H, –CH-),
8.21 (s, 1H, –CH-), 7.63 (s, 2H, ArH), 7.45–7.17 (m, 12H, ArH),
6.97–6.91 (m, 2H, ArH), 2.36 (s, 6H, –CH3). 13C NMR (75 MHz,
DMSO‑d6) δ 163.19, 162.53, 161.09, 160.74, 156.82, 156.79, 155.51,
155.22, 150.26, 150.21, 137.61, 137.59, 133.77, 133.63, 132.83,
132.57, 129.21, 129.18, 129.10, 129.05, 128.81, 128.66, 125.02,
124.65, 119.69, 119.60, 119.27, 119.06, 119.00, 118.93, 20.71,
20.68. HRMS m/z: ([M + H]+) calcd for C18H14N2O4: 323.1026;
found: 323.1034.
4.1.3.11. (E/Z)-1-(4-bromophenyl)-5-(3,4-dimethoxybenzylidene)pyrimi-
dine-2,4,6(1H,3H,5H)-trione (5k). Yellow solid, yield: 80.5%, M.
p. > 250 °C. IR (KBr): 3447.1, 1735.2, 1691.1, 1667.1, 1504.4,
1338.2, 1149.6, 829.2, 792.1 cm-1. 1H NMR (300 MHz, DMSO‑d6) δ
11.66 (s, 1H, –NH), 11.56 (s, 1H, –NH), 8.39 (s, 1H, –CH-), 8.37 (s, 1H,
–CH-), 8.29 (s, 1H, ArH), 8.19 (s, 1H, ArH), 7.96–7.90 (m, 2H, ArH),
7.67 (d, J = 7.9 Hz, 4H, ArH), 7.33–7.28 (m, 4H, ArH), 7.15–7.08 (m,
2H, ArH), 3.90 (s, 3H, –OCH3), 3.87 (s, 3H, –OCH3), 3.83 (s, 3H,
–OCH3), 3.74 (s, 3H, –OCH3). 13C NMR (75 MHz, DMSO‑d6) δ 163.55,
162.97, 161.59, 161.53, 156.16, 155.89, 153.72, 153.69, 150.08,
149.96, 147.77, 135.04, 134.84, 131.76, 131.49, 131.33, 125.25,
125.13, 121.37, 117.03, 116.63, 115.56, 115.51, 111.08, 111.05,
4.1.3.7. (E/Z)-1-(4-bromophenyl)-5-(4-hydroxy-3-methoxybenzylidene)
pyrimidine-2,4,6(1H,3H,5H)-trione (5g). Yellow solid, yield: 69.5%,
M. p. > 250 °C. IR (KBr): 3434.5, 1725.5, 1696.9, 1617.6, 1488.6,
1338.7, 1168.2, 825.7, 790.4 cm-1 1H NMR (300 MHz, DMSO‑d6) δ
.
11.62 (s, 1H, –NH-), 11.51 (s, 1H, –NH-), 10.59 (s, 1H, –OH), 10.56 (s,
1H, –OH), 8.47 (s, 1H, –CH-), 8.34 (s, 1H, –CH-), 8.25 (s, 1H, ArH),
8.24 (s, 1H, ArH), 7.91–7.79 (m, 2H, ArH), 7.69–7.66 (m, 4H, ArH),
7.33–7.29 (m, 4H, ArH), 6.93–6.87 (m, 2H, ArH), 3.84 (s, 3H, –OCH3),
3.76 (s, 3H, –OCH3). 13C NMR (75 MHz, DMSO‑d6) δ 166.63, 166.59,
163.15, 161.62, 156.65, 156.37, 153.20, 151.33, 150.10, 149.99,
146.94, 134.90, 134.31, 132.62, 132.25, 131.83, 131.53, 131.35,
131.16, 124.18, 124.04, 121.45, 121.34, 118.46, 117.85, 115.32,
115.32, 114.23, 114.14, 55.63, 55.49. HRMS m/z: ([M + H]+) calcd
for C18H13BrN2O5: 417.0081; found: 417.0089.
55.84, 55.82, 55.52, 55.40. HRMS m/z: ([M + H]+
19H15BrN2O5: 431.0237; found: 431.024.
) calcd for
C
4.1.4. Synthesis of 1-(4-bromophenyl)-5-(3,4-dimethoxybenzyl)pyrimidine-
2,4,6(1H,3H,5H)-trione (6a)
Sodium borohydride (3 mmol) was added in one portion to a stirred
solution of 5 k (2 mmol) in ethanol (20 mL) at rt. The reaction mixture
was stirred at rt for 1 h. The reaction mixture was concentrated under
reduced pressure and the remaining residue was dissolved in water and
acidified with aqueous hydrochloric acid solution (pH = 1). The re-
sulting precipitate was filtered and recrystallized from water to give 6a
as white solids, yield: 70.8%, M. p. 186–188 °C. IR (KBr): 3231.1,
1701.3, 1516.9, 1321.9, 823.9, 824.6, 764.5 cm-1. 1H NMR (300 MHz,
DMSO‑d6) δ 11.59 (s, 1H, –NH-), 7.66 (d, J = 8.0 Hz, 2H, ArH),
4.1.3.8. (E/Z)-1-(4-bromophenyl)-5-(3-hydroxy-4-methoxybenzylidene)
pyrimidine-2,4,6(1H,3H,5H)-trione (5h). Yellow solid, yield: 78.4%,
M. p. > 250 °C. IR (KBr): 3510.7, 1736.4, 1696.6, 1664.0, 1501.1,
8

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
6.98–6.88 (m, 3H, ArH), 6.69–6.62 (m, 2H, ArH), 3.99 (s, 1H, CH), 3.73
(s, 3H, –OCH3), 3.70 (s, 3H, –OCH3), 3.30 (d, J = 3.3 Hz, 2H, –CH2-).
13C NMR (75 MHz, DMSO‑d6) δ 169.25, 169.16, 150.34, 148.34,
147.77, 134.05, 131.90, 130.85, 129.01, 121.52, 120.97, 112.59,
111.63, 55.42, 55.28, 50.63, 34.95. HRMS m/z: ([M + Na]+) calcd for
4.3. Preparing the ligand database
A Specs database containing 208 715 compounds was used as a li-
gand source. The three-dimensional conformations of the ligands were
generated with LigPrep in Schrodinger, and their protonation states
C
19H17BrN2O5: 455.0213; found: 455.021.
were determined at a target pH 7.0
2.0 with Epik.
4.4. Glide docking procedure
4.1.5. Synthesis of N-substituted thioureas (10a)
Benzoyl chloride (0.01 mol) was added over 5 min to a freshly
prepared solution of ammonium isothiocyanate (0.012 mol) in reagent
grade acetone and the mixture was heated under reflux for about
15 min. Heating was stopped and appropriate aniline in acetone was
added. The mixture was heated under reflux for 30 min and then
poured on to crushed ice. The resulting solid was collected, washed
with water, followed by cold mixture of water and methanol (1:1).
Suitably substituted benzoylthioureas were added to a preheated solu-
tion of aqueous sodium hydroxide (5%) and stirred. The mixture was
then poured into crushed ice containing hydrochloric acid (5%). The
benzoic acid separated was removed by treating the reaction mixture
with sodium carbonate. The product was collected, washed with water
and dried to afford 10a.
The Glide program was used to generate the grid file. The bounding
box of size 10 Å × 10 Å × 10 Å was centered on the FAD. Docking was
performed using Glide software (Glide, version 5.9; Schrödinger, LLC:
New York, NY, 2013) with standard precision (SP) mode. Top-ranked
compounds with docking scores lower than −12.0 kj/mol were re-
tained and filtered by the Linpiski rule. Two compounds that are
available from Specs were purchased and submitted to biological ex-
perimental.
4.5. GOLD docking procedure
Molecule docking experiments were performed using Genetic opti-
mization for ligand docking (GOLD) program version 5.0.48 All water
molecules were removed and missing hydrogen atoms were added. The
binding site is defined as a spherical region with a diameter of 12 Å and
centered on the native ligand. Docking calculations were performed
using Goldscore fitness function. The search efficiency was set as 90%.
4.1.6. Synthesis of thioxopyrimidine-4,6-diones (11a)
Sodium (20 mmol) was added portionwise to ethanol (60 mL) at rt.
After dissolution of the sodium, urea (10 mmol) and diethyl malonate
(10 mmol) were added sequentially each in one portion to the solution
with stirring at rt and then heated at reflux for 5 h. Solvent was con-
centrated under reduced pressure and the remaining residue was dis-
solved in 1 M sodium hydroxide solution (20 mL). The aqueous layer
was washed with ethyl acetate (2 × 20 mL) and then acidified (pH 1)
with 1 M aqueous hydrochloric acid solution and the resulting pre-
cipitate was filtered. The isolated solid material was recrystallized from
methanol to give 11a as colorless solid.
4.6. Cdocker docking procedure
Docking procedure was conducted by Discovery Studio (DS) v3.5
program.49 All water molecules were removed. Missing hydrogen atoms
were added at a pH of 7.0. The active site of the target protein was
created as a spherical region with a diameter of 10 Å and centered on
the FAD. Finally, the 10 top-ranking poses were saved for comparison
and analysis.
4.1.7. Synthesis of (E/Z)-1-(4-bromophenyl)-5-(3,4-dihydroxybenzylidene)-
6-thioxodihydropyrimidine-2,4(1H,3H)-dione (12a)
4.7. Validation of the performance of molecular docking
3,4-Dihydroxybenzaldehyde (5 mmol) was added in one portion to a
solution of 11a (5 mmol) in ethanol (20 mL) at rt. The reaction mixture
was heated at reflux for 1 h. The reaction mixture was cooled down and
the resulting precipitate was filtered and recrystallized from water to
give 12a as yellow solids as a 1:1 mixture of geometrical isomers (E/Z).
Yellow solid, yield: 75.9%, M. p. > 250 °C. IR (KBr): 3394.4, 3269.7,
1695.9, 1660.8, 1543.9, 1367.7, 1175.4, 784.2 cm-1. 1H NMR
(300 MHz, DMSO‑d6) δ 12.16 (s, 2H, –NH-), 10.59 (s, 2H, –OH), 9.59 (s,
2H, –OH), 8.28 (s, 2H, –CH-), 8.22 (s, 2H, ArH), 7.67–7.64 (m, 6H,
ArH), 7.28–7.26 (m, 4H, ArH), 6.86 (d, J = 8.4 Hz, 2H, ArH). 13C NMR
(75 MHz, DMSO‑d6) δ 179.12, 162.69, 161.40, 160.05, 159.05, 157.95,
157.58, 153.50, 153.40, 145.02, 138.87, 138.79, 132.94, 132.32,
131.78, 131.57, 131.41, 124.53, 124.37, 121.40, 121.22, 120.99,
Three different docking softwares were investigated, GOLD v5.0
(http://gold.ccdc.cam.ac.uk./index.php), Glide (standard precision)
and CDOCKER. 56 reported LSD1 inhibitors were used for docking
evaluation. 56, 51 and 39 molecules would be retrieved by Glide, Gold
and CDOCKER tools, respectively. To further examine the merits, ROC
analysis was performed. In general, the greater the AUC, the more ef-
fective the virtual screening workflow is in discriminating active from
active compounds. For the test sets considered, standard precision (SP)
of Glide outperformed GOLD, CDOCKER with ROC AUCs of 0.73.
4.8. In vitro LSD1 inhibition assay
The LSD1 screening biochemical assay was completed by and the
detailed protocol was presented as followed. The LSD1 (Enzo Life
Sciences) activity was monitored using the peroxidase-coupled assay
system as described. Briefly, the compounds in DMSO were added into
the LSD1 in the reaction buffer consisting of 50 mM Tris-HCl, pH 7.5,
and 1% DMSO, by using Acoustic technology (Echo 550) in nanoliter
range and incubated for 15 min at room temperature. The reactions
were initiated by adding peptide substrate (10 μM Histone H3 (1–21)
K4me2 peptide (AnaSpec)) to the reaction mixtures and incubated for
60 min at room temperature. The reactions were terminated by the
addition of the detection mixture consisting final concentration of 0.1
Unit/ml horseradish peroxidase (Sigma) and 10 μM Amplex® UltraRed
(Life Technologies) and fluorescence at Ex/Em = 535/590 nm was
measured with kinetic mode of 5 min interval in Envision for 30 min.
The signals after reaching plateau were taken for the data analysis. The
background subtracted signals were calculated to % activities relative
115.56, 114.10, 113.79. HRMS m/z: ([M + H]+
17H11BrN2O4S: 418.9696; found: 418.9683.
)
calcd for
C
4.2. Preparation of protein structure
The crystal structure of LSD1 resolved with the FAD cofactor was
retrieved from the Protein Data Bank (PDB id: 2Z5U). The water mo-
lecules were removed. The remaining protein structure was prepared
using the Protein Preparation Wizard module (Schrödinger, LLC, New
York, NY, 2013). In short, the hydrogen atoms were added to the
complexes. The module automatically optimize hydroxyl, Asn, Gln, and
His states using ProtAssign. Finally, the complex was submitted to a
restrained minimization using the OPLS2005 force field until the root-
mean-square deviation reached a maximum value of 0.2 Å. The other
parameters were set as default.
9

S. Xu et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
to DMSO controls, and analyzed using GrapgPad Prism 8 with “sig-
moidal dose-response (variable slope)”; 4 parameters with Hill Slope.
(2015) and the Priority Academic Program Development of Jiangsu
Higher Education Institutions (PAPD). F. L. thanks the financial support
by China ‘‘Thousand Youth Talents” Project (KHH1340001) and the
National Natural Science Foundation of China (91419306).
4.9. MST experiment
The LSD1 recombinant from Millipore was labeled with a red
Appendix A. Supplementary data
fluorescent dye using
a commercialized kit. The thermophoretic
movement of the fluorescently labeled protein in complex with selected
inhibitors was measured by monitoring the fluorescence distribution
inside the capillary. The concentration of the labeled protein was kept
constant at ∼200 nM, while the concentration of the compound was
varied. The samples were loaded into MST-grade glass capillaries. After
a short incubation period, the MST analysis was performed using the
Monolith NT.115.
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bmc.2018.08.026.
References
1. Shi YJ, Lan F, Matson C, et al. Cell. 2004;119:941–953.
2. Dawson MA, Kouzarides T. Cell. 2012;150:12–27.
3. Metzger E, Wissmann M, Yin N, et al. Nature. 2005;437:436–439.
4. Lizcano F, Garcia J. Pharmaceuticals. 2012;5:963–990.
4.10. MAO-A and MAO-B assays
5. Etani T, Suzuki T, Naiki T, et al. Oncotarg. 2015;6:2865–2878.
6. Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M. Nat Rev Drug Discovery.
2012;11:384–400.
7. He Y, Korboukh I, Jin J, Huang J. Acta Biochim Biophys Sin. 2012;44:70–79.
8. Jin L, Hanigan CL, Wu Y, Wang W, Park BH, et al. Biochem J. 2013;449:459–468.
9. Amente S, Lania L, Majello B. Biochim Biophys Acta, Gene Regul Mech.
2013;1829:981−986.
10. Harris WJ, Huang X, Lynch JT, et al. Cancer Cell. 2012;21:473–487.
11. Schenk T, Chen WC, Gollner S, Howell L, Jin LQ, et al. Nat Med. 2012;18:605–611.
12. Hayami S, Kelly JD, Cho HS, et al. Int J Cancer. 2011;128:574–586.
13. Kauffman EC, Robinson BD, Downes MJ, et al. Mol Carcinog. 2011;50:931–944.
14. Wang P, Fan F, Li X, et al. J Mol Cell Cardiol. 2018;115:115–129.
15. Helin K, Dhanak D. Nature. 2013;502:480–488.
The MAO-A and MAO-B is purchased from Active Motif
(Cat#31502, Cat#31503). Biochemical Kit is purchased from Promega
(MAO-Glo Assay, V1402). The inhibition of selectivity assay was pre-
formed in light of the manufacturer’s protocol. Luminescence is read on
Gen5 BioTek Spectrometer and data is processed using GraphPad Prism
8.
4.11. Reversibility assay
16. Zheng YC, Ma J, Wang Z, Li J, Jiang B, et al. Med. Res. Rev. 2015;35:1032–1071.
17. Stazi G, Zwergel C, Valente S, Mai A. Expert Opin Ther Pat. 2016;26:565–580.
18. Mould DP, McGonagle AE, Wiseman DH, Williams EL, Jordan AM. Med Res Rev.
2015;35:586–618.
19. Forneris F, Binda C, Battaglioli E, Mattevi A. Trends Biochem Sci. 2008;33:181–189.
20. Ueda R, Suzuki T, Mino K, et al. J Am Chem Soc. 2009;131:17536–17537.
21. Binda C, Valente S, Romanenghi S, et al. J Am Chem Soc. 2010;132:6827–6833.
22. Valente S, Rodriguez V, Mercurio C, et al. Eur J Med Chem. 2015;94:163–174.
23. Mimasu S, Sengoku T, Fukuzawa S, Umehara T, Yokoyama S. Biochem Biophys Res
Commun. 2008;366:15–22.
Using jump dilution analysis to determine the reversibility of com-
pounds. Briefly, the compound 12a at the concentrations of 10× their
biochemical IC50s were pre-incubated with high concentration of LSD1
protein (500 nM) for 1 h, and then diluted 100-fold by H3K4me2 pep-
tide solution. The data was plotted in GraphPad Prism 8.
4.12. Flow cytometry assay
24. Mimasu S, Umezawa N, Sato S, Higuchi T, Umehara T, Yokoyama S. Biochemistry.
2010;49:6494–6503.
FACS analyses were performed using an LSR Model II flow cyt-
ometer (BD Biosciences, Oxford, UK). Cell sorting experiments were
performed using either an Influx or a FACS Aria flow cytometer (both
from BD Biosciences). CD11b antibody is purchased from BD.
25. Zheng YC, Yu B, Chen ZS, Liu Y, Liu HM. Epigenomics. 2016;8:651–666.
26. Zheng YC, Yu B, Jiang GZ, et al. Curr Top Med Chem. 2016;16:2179–2188.
27. Mohammad HP, Smitheman KN, Kamat CD, et al. Cancer Cell. 2015;28:57–69.
28. Gallipoli P, Giotopoulo G, Huntly BJP. Ther Adv Hematol. 2015;6:103–119.
29. Hoang N, Zhang X, Zhang C, et al. Bioorg Med Chem. 2018;26:1523–1537.
30. Kumar V, Rosse G. ACS Med Chem Lett. 2016;7:132–133.
31. Wu F, Zhou C, Yao Y, et al. J Med Chem. 2016;59:253–263.
32. Nowotarski SL, Pachaiyappan B, Holshouser SL, et al. Bioorg Med Chem.
2015;23:1601–1612.
4.13. Western blot assay
33. Sartori L, Mercurio C, Amigoni F, et al. J Med Chem. 2017;60:1673–1692.
34. Vianello P, Sartori L, Amigoni F, et al. J Med Chem. 2017;60:1693–1715.
35. Young K, Lin S, Sun L, Lee E, Modi M, et al. Nat Biotechnol. 1998;16:946–950.
36. Hamasaki K, Rando RR. Anal Biochem. 1998;261:183–190.
37. Nussinov R, Wolfson HJ. Comb Chem High Throughput Screening. 1999;2:261–269.
38. Olson AJ, Goodsell DS. SAR QSAR Environ Res. 1998;8:273–285.
39. Hazeldine S, Pachaiyappan B, Steinbergs N, Nowotarski S, Hanson AS, et al. J Med
Chem. 2012;55:7378–7391.
40. Zha XM, Wu LM, Xu SY, et al. Med Chem Res. 2016;25:2822–2831.
41. Zhou C, Kang D, Xu YG, Zhang LY, Zha XM. Chem Biol Drug Des. 2015;85:659–671.
42. Xi JY, Xu SY, Wu LM, Ma TF, et al. Bioorg Chem. 2017;72:182–189.
43. In Kurzer F. Organic Synthesis. Wiley: New-York. 1964;4:49–50.
44. Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R. Blood.
1991;77:1080–1086.
H3K4me1 antibody (Active Motif 39297), H3K4me2 (Millipore 07-
030 (Crossreacting K4me1, should use 1:4000 above), H3K4me3(CS
9751S (use 1:5000 above) antibodies are used in the western blot assay.
5. Declaration of interest
F. L. is a shareholder of Constellation Pharmaceuticals Inc. as well as
a consultant of Active Motif. The other authors declare no competing
financial interests.
Acknowledgments
45. Lee HT, Choi MR, Doh MS, Jung KH, Chai YG. Oncol Rep. 2013;30:1587–1592.
46. Rivers A, Vaitkus K, Ruiz MA, Ibanez V, et al. Exp Hematol. 2015;43:546–553.
47. Liu TX, Zhang JW, Tao J, Zhang RB, Zhang QH, et al. Blood. 2000;96:1496–1504.
48. Mackey MD, Melville JL. J Chem Inf Model. 2009;49:1154–1162.
49. Konagurthu AS, Whisstock JC, Stuckey PJ, Lesk AM. Proteins. 2006;64:559–574.
X. Z. and Y. X. thank the financial support by the Natural Science
Foundation of Jiangsu Province (BK20161458), ‘‘Six Talent Peaks”
Project of Jiangsu Province (2016-YY-042), the Outstanding Scientific
and Technological Innovation Team of Jiangsu Province of China
10