Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design, synthesis and pharmacological evaluation

Article abstract of DOI:10.1016/j.ejmech.2016.10.062

NQO1 is a dimeric flavoprotein which intimately associated with cancer and overexpressed in the cytosol of numerous human tumor cells. Given that the cellular environment is quite dynamic and versatile, further investigation of the function of NQO1 depends on tools for specific detection of it. Currently, several activity-based assays have been developed to detect NQO1-expressing cancerous tissues. Herein, we report the development of a functional affinity-based small-molecule probe which is composed of a potent small-molecule NQO1 inhibitor 3d as the recognition group, a linker and the fluorophores group FITC. The probe exhibits good inhibitory activity of NQO1 and has been successfully used to label the protein in A549 cells at the micromolar level. These features make the probe favorable for mechanistic studies and cancer diagnostic biomarker. Based on these preliminary results, our laboratory will focus on the further development of fluorescent probe for NQO1, which could be anticipated to be applied in physiological and pathological studies of NQO1.

Full text of DOI:10.1016/j.ejmech.2016.10.062

European Journal of Medicinal Chemistry xxx (2016) 1e12
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Affinity-based small fluorescent probe for NAD(P)H:quinone
oxidoreductase 1 (NQO1). Design, synthesis and pharmacological
evaluation
,
,
, *
Jinlei Bian a 1, Xiang Li a 1, Lili Xu a, Nan Wang a, Xue Qian a, Qidong You a
,
Xiaojin Zhang a
, b, **
a State Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing,
210009, China
b Department of Organic Chemistry, China Pharmaceutical University, Nanjing, 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
NQO1 is a dimeric flavoprotein which intimately associated with cancer and overexpressed in the cytosol
of numerous human tumor cells. Given that the cellular environment is quite dynamic and versatile,
further investigation of the function of NQO1 depends on tools for specific detection of it. Currently,
several activity-based assays have been developed to detect NQO1-expressing cancerous tissues. Herein,
we report the development of a functional affinity-based small-molecule probe which is composed of a
potent small-molecule NQO1 inhibitor 3d as the recognition group, a linker and the fluorophores group
FITC. The probe exhibits good inhibitory activity of NQO1 and has been successfully used to label the
protein in A549 cells at the micromolar level. These features make the probe favorable for mechanistic
studies and cancer diagnostic biomarker. Based on these preliminary results, our laboratory will focus on
the further development of fluorescent probe for NQO1, which could be anticipated to be applied in
physiological and pathological studies of NQO1.
Received 18 September 2016
Received in revised form
27 October 2016
Accepted 28 October 2016
Available online xxx
Keywords:
Fluorescent probe
NQO1
Inhibitor
FITC
Dicoumarol
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
cancer therapy and several of NQO1 substrates are in multiple
clinical trials [4e7]. Considering that the cellular environment is
NAD(P)H:quinone oxidoreductase-1 (NQO1, DT-diaphorase) is a
cytosolic flavoenzyme, which is initially identified as a chemo-
preventive enzyme and now recognized plays of other biological
process including catalyze bioactivation of antitumor quinones and
act as chaperone protein, constitute attractive pharmacological
targets for development of anticancer therapeutics [1e3]. Indeed
NQO1 has been found to be a promising therapeutic target for
quite dynamic and versatile, the further exploration of function and
therapeutic potential of NQO1 under various condition is quite
urgent. These achievements depend on the tools for specific and
real-time detection of the NQO1 protein.
In the last decade, fluorescence-based techniques have under-
gone a huge development for studying pharmacological and
biochemical processes [8e10]. Given the sensitivity of fluorescence
detection and its simplicity, this fluorescent-based methodology
appears an important tool in many areas and is being employed for
discovery of gene-targeted drugs, molecular biological and protein-
protein interaction studies [11,12]. Actually, several activity-based
fluorescent probes which can be triggered by NQO1 have been
discovered recently (Probe I and Probe II, Fig. 1A) [13,14]. This type
of probes was designed based on NQO1-mediated redox property,
thus there are potential possibility for these probes triggered by
other quinone oxidoreductases. Therefore, small-molecule fluo-
rescent probe based on binding-signal output principle is needed to
be developed. In this work, we reported the first small-molecule
fluorescent probe based on affinity which can be used for and
Abbreviations: NQO1, NAD(P)H:quinone oxidoreductase-1; DIC, Dicoumarol;
b-
lap,
b-lapachone; DMSO, Dimethyl sulfoxide; HPLC, High performance liquid
chromatography; BSA, Bovine serum albumin; PDB, Protein data bank; equiv,
Equivalent; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NSCLC, Non-small cell lung cancer; DIC, Dicoumarol; MOE, Molecular operating
environment; FITC, Fluorescein isothiocyanate.
* Corresponding author.
** Corresponding author. State Key Laboratory of Natural Medicines and Jiangsu
Key Laboratory of Drug Design and Optimization, China Pharmaceutical University,
Nanjing, 210009, China.
E-mail addresses: youqd@163.com (Q. You), zxj@cpu.edu.cn (X. Zhang).
These two authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2016.10.062
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

2
J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
Fig. 1. (A) The structures of activity-based probe. (B) The principle of affinity-based probe and the structures of NQO1 inhibitors (DIC, ES936 and previous reported compound 1).
fluorescence polarization method and fluorescently visualize and
specifically detect NQO1 (Fig. 1B).
function of NQO1 in cells (Fig. 1A) [17]. Particularly, it should be
emphasized that ES936 is a mechanism-based suicide inhibitor
which is different with the competitive inhibitor DIC [13]. Thus,
competitive inhibitor DIC is more suitable than ES936 for devel-
oping affinity-based fluorescent probe. However, there are still
some disadvantages with DIC in that it is not specific towards NQO1
[18]. Therefore, to discover a suitable recognition group for the
affinity-based probe, we need to turn our attention to some other
competitive inhibitors.
To construct a useful probe, it is a pre-requisite to develop a
potent and specific inhibitor as the recognition group. A few of
compounds are known to inhibit the activity of NQO1 by competing
with NAD(P)H for binding to the enzyme [4]. These inhibitors
mainly include a series of flavones, coumarins and triazoloacridin-
6-ones [15,16]. Among them, dicoumarol (DIC) and ES936 are the
most commonly used as a pharmacological inhibitor to study the
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
3
Recently, we have been focused on discovering NQO1 inhibitors
with novel chemical scaffolds [17]. Among these novel NQO1 in-
hibitors, 1 (ChemDiv ID: K815-0286) showed the most potent
inhibitory activity against NQO1. In order to discover more potent
inhibitors, a series of compounds were designed based on 1
through rational drug designation. On the basis of more potent
inhibitor small-molecule ligand of NQO1, we then reported the
design, synthesis, and application of the affinity-based small probe
to fluorescently visualize and specifically detect NQO1, which can
be applied in further research of NQO1.
resulting product 6 was then reacted with aryladehydes to give the
target compounds (3a-3s, 3w-3z) in 34e75% yields, which
precipitated out from the boiling methanol solution after mixing 6
with corresponding aldehyde. Palladium-carbon catalyzed hydro-
genation of the double bond of 3a, 3d and 3s to provide the product
3t-3v in 35e86% yield. The structures of all compounds were
confirmed by 1H NMR and electrospray ionization mass spec-
trometry (ESI-MS). Before these compounds were used in biological
experiments, they were purified by silica gel column chromatog-
raphy and HPLC was used to determine their purity (all > 95%).
2. Result and discussion
2.2.2. Synthesis of the fluorescent probe
Starting with 4-hydroxy-3-(3-(4-hydroxyphenyl)acryloyl)-2H-
chromen-2-one, the probe III was synthesized in three steps
(Scheme 2). First, aminoalkanols (7) with carbon atom spacers of
six were Boc-protected to give intermediate 8 in excellent yields
(85%). Treatment 8 with tosyl chloride provided the product 9 in
81% yields. The condensation of compound 3d with 9 under the
base of K2CO3 affords compound 10 with the linker. By removing
the Boc protecting groups, we got the key intermediate 11. Finally,
the probe III was obtained through conjugating the fluorescein
isothiocyanate (FITC) group with compound 11.
2.1. Designation of derivatives of 1
In order to optimize the structure of 1, the docked posed of the
identified hit was analyzed within the NQO1 binding pocket
compared to DIC (Fig. 2). The general molecular orientation and the
spatial location of the chemical features of 1 were similar to DIC
(PDB ID: 2F1O). It was noted that, based in the docking pose, the
side chain was observed that it could occupy the additional pocket
formed by Tyr128, Phe232 and some other residues. As our previ-
ous work reported, residues that involved most prominent
conformation changes that occur in the presence of inhibitors of
NQO1 were Tyr128 and Phe232 [17]. Therefore, the fragment A
(region 1, Fig. 1B) was a possible modification site, and then the
para-dimethylaminophenyl was replaced by a series of substituted
aromatic ring. Moreover, substituents that can intercalate between
and form van der Waals interactions with Trp105 minipocket were
also favorable [19]. Thus, we also designed several compounds with
substituents at ring C (region 3, Fig. 1B). Representative compounds
2.3. Inhibitory activity on recombinant hNQO1 of all compounds
The 26 newly synthesized compounds were evaluated for their
ability to inhibit recombinant NQO1 in vitro. All of these inhibitors
were assayed in the presence of 0.14% (w/v) bovine serum albumin
(BSA), considering the effect of nonspecific protein binding [18,21].
The inhibition rates of these derivatives at 10 m
mol Lꢀ1 were shown
in Table 1. These compounds can be divided into three categories
(Fig. 1B). To investigate the A-ring substituent effects on the inhi-
bition of NQO1, nineteen compounds (3a-3s) with different sub-
stituent groups at A-ring were synthesized (region 1). In general,
most of these compounds possessed moderate to good inhibition
rates of NQO1, indicating that these compounds were potential
inhibitors for NQO1. Compounds 3d, 3e, 3m, 3n and 3s substituted
with electron-donating group (4-OH, 4-NH2, 2,3-OCH3, 4-OEt and
3-OCH3) at A-ring displayed excellent inhibition rates of NQO1
ranging from 81.7% to 92.7%. In addition, the presence of strong
electron-withdrawing group at A-ring, such as 3b, 3g, 3h and 3i
substituted with 4-NO2, 4-CN, 3-NO2 and 4-CF3 functional groups,
was unfavorable for inhibiting NQO1 activity. With regard to
naphthyl substitution (3q), the activity decreased sharply. This
phenomenon was possibly due to the fact that the binding pocket
were also reduced to investigate the role of
in inhibiting NQO1 (region 2, Fig. 1B).
a,b-unsaturated ketone
2.2. Chemistry
2.2.1. Synthesis of inhibitors
The common synthetic route of compounds 3a-3z was outlined
in Scheme 1. Most of the target compounds (3a-3v) were synthe-
sized using 4-hydroxy-2H-chromen-2-one (5) as the starting ma-
terial. As for compounds 3w-3z, the substituted 4-hydroxy-2H-
chromen-2-ones were preparing by base-mediated cyclization of
the corresponding 2-hydroxy acetophenones (4) with diethyl car-
bonate [20]. Compound
6 was prepared by reaction of 4-
hydroxycoumarine with POCl3 in the presence of acetic acid. The
Fig. 2. The crystal binding mode of DIC and docking-predicted binding mode of 1. (A) DIC and (B) 1 in the active site of NQO1 respectively, key residues were labeled in stick.
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

4
J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
Scheme 1. Reagents and conditions: (a) (EtO)2CO, NaH, 0 ꢁC to r.t., 0.5 h to 100 ꢁC, 2 h, 60e69%; (b) HOAc, POCl3, reflux, 75min, 32e58%; (c) aryladehydes, piperidine, CHCl3, reflux,
3e40 h, 34e75%. (d) H2, Pd/C, r.t., 10 h, 35e86%.
Scheme 2. Reagents and conditions: (a) DCM, r.t., 5min, 85%; (b) Et3N, DCM, 48 h; (c) K2CO3, DMF, 80 ꢁC, 8 h, 15%; (d) CF3COOH, DCM,, r.t., 20min, 80%; (e) DMF, r.t., 1 h.
was not large enough for naphthyl group binding. Among these A-
ring substituted derivatives (3a-3s), compound 3a with para-chloro
substitution exhibited the most potent inhibition rates of NQO1
inhibitory activity compared to DIC. Notably, compound 3z was
even slightly more potent than the positive control DIC with an IC50
of 0.35 0.13 m .
mol Lꢀ1
(91.1 4.3%) at 10
mM. As for the region 2 investigated compounds
3t-3v, which were obtained from reducing the double bonds of
a
,b
-
2.4. Molecular modeling
unsaturated ketone of compounds 3a, 3d and 3s, the inhibition
rates of these compounds were still retained though significantly
decreased, demonstrating that the olefinic bond may contribute to
the inhibitory activity. Compounds (3x-3z) with methyl or methoxy
groups at C-ring (region 3) showed equal or slightly better inhibi-
tion rates of NQO1 compared to the unsubstituted compounds (3a
and 3s), indicating that methyl- or methoxy-substitutions were
tolerable for NQO1 inhibitors. However, when chloric substituent
was introduced in the C-ring (3w), the inhibition rate was
dramatically reduced (see Table 2).
For purpose of investigating the binding mechanism of these
inhibitors, we then performed molecular docking experiments. All
of these compounds (3a-3z) were docked into the active site of
NQO1 using Gold 5.1 [22,23]. As shown in Table 1, ChemScore
values of most derivatives were similar to the control DIC,
explained that these derivatives possessed moderate to good in-
hibition rates of NQO1. Interestingly, according to the docking re-
sults, the electronic effects afforded by the additional functional
groups at A ring did impact the scores of the derivatives, com-
pounds (3a, 3d, 3m, 3n, 3o and 3s) with halogen, methoxy or
ethoxy substitution at the para position of benzene moiety pro-
vided better scores. As for compounds 3t-3v, their docking scores
were lower than other compounds. This could be ascribed to the
Representative compounds 3a, 3d, 3x and 3z that showed good
inhibition activities at the concentration of 10 mM were selected to
determine their IC50 values. DIC was also evaluated as a comparison
group (Fig. 3). All of the compounds revealed the same level
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
5
Table 1
Inhibition of hNQO1 by compounds 3a-3z and DIC.
Entry
Cpd
R1
R2
NQO1 IR (%) (10
m
M) with BSAa
Percentage of remaining A549 cells at 50
m
Mb
Chemscore
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
91.1 4.3
71.2 8.1
64.3 3.1
89.2 11
81.7 9.8
66.7 5.4
34.4 6.5
65.8 8.7
60.4 3.3
70.9 10
51.0 4.9
54.3 6.5
86.9 5.1
86.7 7.1
87.2 4.5
79.6 12
46.1 8.1
69.4 4.4
92.7 6.3
61.4 5.0
77.7 3.9
57.8 8.7
74.4 4.0
89.7 3.6
81.2 3.9
99.6 5.1
81.2 4.8
>80
96.46
92.42
93.66
99.51
91.83
91.70
93.17
91.74
87.64
92.54
93.84
93.31
96.98
94.13
95.73
89.89
88.40
96.44
95.03
93.76
91.90
90.77
91.18
96.23
94.31
99.05
98.00
4-NO2
4-CH3
4-OH
4-NH2
4-F
34 12
74 11
>80
>80
76 13
>80
43 11
>80
>80
>80
>80
>80
>80
>80
>80
71 12
>80
>80
>80
>80
4-CN
3-NO2
4-CF3
2-Cl
4-iPr
3-Cl
2,3-OCH3
4-OEt
2,4-Cl
4-Ph
3,4-C4H4
2-F
9
10
11
12
13
14
15
16
17
18
19
20b
21b
22b
23
24
25
26
27
H
H
H
H
H
H
6-Cl
6-OCH3
6,7-CH3
6,7-CH3
e
3s
3t
3-OCH3
4-Cl
3u
3v
3w
3x
3y
3z
DIC
4-OH
3-OCH3
3-OCH3
3-OCH3
3-OCH3
4-Cl
67 23
>80
>80
>80
>80
e
>80
a
The inhibitory ratio of each compound at 10 mM to inhibit purified hNQO1 activity in the presence of 0.14% (w/v) BSA.
The percentage of remaining A549 cells after incubation with compound at 50 mM after 48 h. Most of the derivatives showed non-toxic towards A549 cells and could be
b
employed as useful probe for studying the function of NQO1 in cells.
Table 2
The values of the concentration of selected inhibitors to cause 50% toxicity in A549 cells in the presence of 20
inhibit NQO1 activity in A549 cells.
mM b-lap. The results reflected the ability of the inhibitors to
Entry
1
Cpd
Structure
Concentration required to protect against
26 3.2
b-lap toxicity (m
M)a
3a
2
3
3d
3q
8.4 0.7
>100
5
4
3x
3z
18 3.8
3.6 1.2
6
DIC
11 1.6
a
Concentrations of each compounds that causes 50% toxicity in cells in the presence of 20 mM b-lap.
flexible of the conformation by reduction of olefinic bond, which
resulted in reduction of inhibitory rates of NQO1. Fig. 4 presented
the best-ranked pose of representative compounds 3a and 3z. The
the side chain of these compounds could fit deep into the pocket
and make the same -stacking interactions with Tyr128 and
Phe232 in the side binding hydrophobic pocket of NQO1. As for
p
coumarin ring made
p-stacking interaction with the isoalloxazine
compound 3z, the two methyls at the C ring can form additional
ring of the bound cofactor FAD, which was similar to the crystal
structures of DIC in complex with NQO1 (Fig. 4A and B). In addition,
CeH …
p
interaction with the adjacent Phe178 and Trp105 resi-
-stacking interaction
dues, which was observed for enhancing
p
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
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6
J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
the derivatives showed non-toxic towards A549 cells with more
than eighty percentage of remaining A549 cells at 50 M. Hence,
m
these compounds could be promising NQO1 recognition group for
further studies.
Moreover, in order to establish the potency and functional
ability of the novel series of compounds as inhibitors of NQO1 in
cells, A549 cells were then treated with 20
and varying concentrations of each representative derivatives (3a,
3d, 3q, 3x and 3z). -Lap was generally recognized as potential
substrates for NQO1 and the ability to modulate -lap toxicity can
mM b-lapachone (b-lap)
b
b
be regarded as surrogate measure if the pharmacological efficiency
of these compounds to act as inhibitors of NQO1 in cells (Fig. 5A).
Among the selected compounds, 3q was less active than other four
compounds according to the results of enzyme assays. 20 mM b-lap
alone results in more than 90% reduction in cell proliferation in
A549 cells. The concentration of the derivatives needed to inhibit
the toxicity of
ability of the compounds to inhibit NQO1 in cells. The results of
A549 cells exposed to -lap and the NQO1 inhibitors were shown in
b-lap by 50% can then be used as a measure of the
Fig. 3. The IC50 curves of DIC and the representative compounds (3a, 3d, 3x and 3z).
b
with surrounding residues, supporting 3z was the most potent
Fig. 5B. Except for compound 3q, which was selected as negative
inhibitor of NQO1.
control, other derivatives showed equal potency to DIC to protect
against 20
3d and 3z even appeared to be slightly more potent than DIC. The
ability of the representative compounds to protect against 20 M b-
mM b-lap toxicity in A549 cells. In addition, compounds
2.5. Evaluation of NQO1 inhibitors in A549 cell lines
m
lap toxicity in A549 cells was in line with the potency for inhibition
of the activity of recombinant NQO1 in the presence of BSA. In
addition, 3d and 3z were consistently more specific that DIC, sug-
gesting that they could be employed as more pharmacological
useful recognition group than DIC for developing fluorescent probe
to study the function of NQO1 in cells.
In this work, the main goal for discovering NQO1 inhibitors was
to develop fluorescent probe for NQO1 as tool to better understand
the pharmacological function of NQO1. Hence, compounds with
little to no cytotoxic effects are required. A549 (NQO1-rich) cell
lines were first treated for 48 h with 50
pounds to determine their cytotoxicity. As shown in Table 1, most of
m
mol Lꢀ1 of these com-
Fig. 4. The crystal binding mode of DIC and docking-predicted binding mode of 3a and 3z. (A) the superimposition conformations of DIC, 3a and 3z in the active site of NQO1 with a
surface colored by lipophilicity state; (B, C, D) DIC, 3a, 3z in the active site of NQO1 respectively, key residues were labeled in stick.
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
7
Fig. 5. (A) The role of NQO1 inhibitors was to protect the toxicity of NQO1 substrate (
b-lap). (B) A549 cells were treated for 2 h with 20
mM
b-lap and varying concentrations of
compounds 3a, 3d, 3q, 3x, 3z and DIC. The mortality was determined 72 h later by the MTT assay. The concentration of inhibitors to protect 50% toxicity in the presence of 20 mM b-
lap was used as the measure of inhibitory potency.
2.6. Design and evaluation of the fluorescent probe
relative to the plane of polarization, leading to the weak FP
response signal (a lower mP value) [24]. When the compound is
bound to a bigger molecule such as NQO1, the complex tumbles
much slower and the emitted light is polarized, resulting in the
enhanced FP response signal (a higher mP value) (Fig. 6A). There-
fore, the change of mP reflects the interaction between the
fluorescent-labeled compound (in the case probe III) and the pro-
tein (NQO1). To develop a fluorescence polarization assay, the
binding affinity of the probe to the protein should be high and the
binding range from maximum mP at saturation to minimal mP at
no protein should be large. According to the result, the mP differ-
ence value of the probe was about 150, which was in a reasonable
range (Fig. 6B). Then, we examined the binding affinity of the probe
with NQO1 using FP competition assay. As shown in Fig. 6C, the IC50
In order to obtain an efficient fluorescent probe, a potent and
specific inhibitor was needed. As described above, 3d and 3z were
the two most efficient inhibitors of NQO1. However, compound 3z
is difficult to obtain, and the deficiency of poor water-soluble also
restricted it serve as a tool for further investigation. Therefore,
taking both the efficacy and physiochemical property into account,
compound 3d was selected as the recognition group for the probe
in the preliminary studies. Subsequently, a new NQO1 fluorescent
probe with the general formula was designed on 3d bearing the
fluorescent moiety FITC linked to the N-alkyl chain (Fig. 5). Due to
the frequently presence of a long-chain alkyl group and FITC in
several fluorescent probe, we proposed that the preliminary
designed probe might be efficient. The probe was then evaluated
for its ability to inhibit recombinant NQO1. The inhibition rate of
in the FP assay was 3.14
activity test (IC50 ¼ 0.98
probe.
m
M, similar with the method of enzyme
mM), which indicated the effective of the
the probe at 10 m mM, still in the
mol Lꢀ1 was 77% and IC50 was 0.98
same range of 3d, which indicated that the introduction of the FITC
group maintained the NQO1 inhibitory activity (Table 3).
2.8. Fluorescently visualize cellular NQO1
Because of the good inhibitory activity, probe III would be a
suitable tool to fluorescently visualize cellular NQO1. In addition,
due to NQO1 is expressed in tumor tissue and is absent in normal
tissue, NQO1 may be regarded as a potential biomarker for tumors.
Accordingly, the imaging of NQO1 could provide useful information
for tumor detection and labeling. To highlight this potential,
immunofluorescence microscopy was achieved on the tumor cell
A549 with high levels of NQO1 expression. The imaging results
showed that the probe III was able to label NQO1 for visualization
(Fig. 7). As a negative control, the inhibition of NQO1 was imaged by
2.7. Development of NQO1 fluorescence polarization (FP) assay
As we know, the fluorescence polarization assay is very
powerful in measuring real-time protein-inhibitor interactions in
solution and quick used for high-throughput drug screening.
Hence, we tried to develop the NQO1 fluorescence polarization
assay using our obtained fluorescent probe III. The principle of
fluorescence polarization is according to the observation that when
a relatively small, fast-tumbling fluorescent-labeled compound is
excited with plane-polarized light, the emitted light is random
incubating the cells with 10 mM DIC together with the probe. In-
hibition of NQO1 by DIC resulted in a decrease of fluorescence in-
tensity. The results indicated that probe III display favorable
selectivity for NQO1 and could be used as a labeling toolkit for
NQO1 highly expressing tumor cells.
Table 3
Inhibitory Activity of the recognition group 3d, the probe III and the control DIC
against NQO1.
Cpd
NQO1 IR (%)a
(10 M) with BSA
IC50 (m
M)b
m
3. Conclusion
DIC
3d
Probe III
84 4.6
86 5.7
77 6.5
0.37 0.15
0.39 0.10
0.98 0.22
Herein, we reported the details of a study to identify a series of
novel NQO1 inhibitors and employed them for the designation of
the fluorescent probes. Molecular docking has been applied to
analysis the binding modes between the inhibitors and NQO1, and
thus to correlate their inhibition rates with predictions of the key
a
The inhibitory ratio of each compound at 10
m
M to inhibit purified hNQO1 ac-
tivity in the presence of 0.14% (w/v) BSA.
b
The concentration of each compound that inhibits the activity of purified
hNQO1 by 50% (IC50).
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

8
J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
Fig. 6. (A) The structure of probe III. (B) The curve of the probe III. (C) Dose-response inhibition curve of probe III determined by the FP-based binding assay.
interactions in the binding pocket of NQO1. Determination of the
ability for protecting against NQO1-mediated toxicity of -lap
4.1.1. 3-Acetyl-4-hydroxycoumarin (8)
To solution of 4-hydroxy-2H-chromen-2-one (3.0 g,
b
a
confirmed that these inhibitors may be specific for NQO1. In addi-
tion, using the potent small-molecule NQO1 ligand 3d as the
structure template, the functional group was attached to the linker
afforded the probe with FITC group. The probe exhibited good
inhibitory activity of NQO1 and has been successfully used to label
the protein in A549 cells at the micromolar level. Furthermore, the
preparation of the probe is also convenient. These features make
the probe favorable for mechanistic studies and cancer diagnostic
biomarker. Based on these preliminary results, our laboratory will
focus on the further development of fluorescent probe for NQO1,
which could be anticipated to be applied in physiological and
pathological studies of NQO1.
1.86 mmol) in acetic acid (16 mL) was added phosphorus oxy-
chloride (5.6 mL). The mixture was heated at reflux for 75 min.
After cooling to room temperature, the precipitate which separated
out was collected by filtration and recrystallized from ethanol to
give 3-acetyl-4- hydroxy-2H-chromen-2-one as light yellow nee-
dles (1.66 g, yield 43%). m.p. 134e136 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 17.7 (s, 1H), 7.98 (d, J ¼ 7.9 Hz, 1H), 7.70 (t, J ¼ 7.6 Hz, 1H),
7.37e7.26 (m, 2H), 2.79 (s, 3H). ESI-HRMS m/z [MþH]þ calculated
for C11H9O4: 205.1819, found: 205.1817.
4.1.2. General procedure for the preparation of compounds 3a-3z
3-acetyl-4-hydroxy-2H-chromen-2-one (0.031 mol) and the
substituted aromatic aldehyde (0.03 mol) were dissolved in 30 mL
of chloroform. A catalytic amount of piperidine (0.02 mol) was
added and the reaction mixture was refluxed till the reaction is
finished.
4. Experimental
4.1. Chemistry
4.1.2.1. (E)-4-Hydroxy 3-(3-(4-chlorophenyl)acryloyl)-2H-chromen-
2-one (3a). Following the procedure above, treatment 6 with 4-
chlorobenzaldehyde for 19 h under reflux gave the crude product
which recrystallized with methanol to afford 3a as yellow solid
All reagents were purchased from commercial sources. Organic
solutions were concentrated in a rotary evaporator (BüchiR-
otavapor) below 55 ꢁC under reduced pressure. Reactions were
monitored by thin-layer chromatography (TLC) on 0.25 mm silica
gel plates (GF254) and visualized under UV light. Melting points
were determined with a Melt-Temp II apparatus. The 1H NMR and
13C NMR spectra were measured on a Bruker AV-300 instrument
using deuterated solvents with tetramethylsilane (TMS) as internal
standard. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR
spectrometer using KBr film. EI-MS was collected on shimadzu
GCMS-2010 instruments. ESI-mass and high resolution mass
spectra (HRMS) were recorded on a Water Q-Tofmicro mass spec-
trometer. Analytical results are within 0.40% of the theoretical
values.
(0.27 g, 75%). m.p. 254e256 ꢁC. 1H NMR (300 MHz, DMSO)
d: 8.27
(d, J ¼ 15.9 Hz, 1H), 8.07 (m, 2H), 8.03 (d, J ¼ 15.7 Hz, 1H), 7.82 (d,
J ¼ 5.2 Hz, 2H), 7.58 (d, J ¼ 8.4 Hz, 2H), 7.47e7.43 (m, 2H). ESI-HRMS
m/z [MþH]þ calculated for C18H12ClO4: 327.0419, found: 327.0421.
4.1.2.2. (E)-4-Hydroxy-3-(3-(4-nitrophenyl)acryloyl)-2H-chromen-
2-one (3b). Following the procedure above, treatment 6 with 4-
nitrobenzaldehyde for 26 h under reflux gave the crude product
which recrystallized with ethanol to afford 3b as yellow solid
(0.24 g, 48%). m.p.197e199 ꢁC. 1H NMR (300 MHz, DMSO)
d: 8.36 (d,
J ¼ 15.0 Hz, 1H), 8.34e8.32 (m, 2H), 8.08e8.03 (m, 4H), 8.43 (t,
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
9
product which recrystallized with ethanol to afford 3e as yellow
solid (0.35 g, 50%). m.p. 235-137 ꢁC. 1H NMR (300 MHz, DMSO)
d
:
8.02e7.99 (m, 3H), 7.38e7.45 (m, 2H), 7.52 (d, J ¼ 8.7 Hz, 2H),
7.40e7.37 (m, 2H), 6.65 (d, J ¼ 8.6 Hz, 2H). IR (v, cmꢀ1): 3416, 1611,
1548, 898. ESI-HRMS m/z [MþH]þ calculated for C18H13NO4:
308.0917, found: 308.0925. HPLC (90% methanol in water):
tR ¼ 3.582 min, 96.3%.
4.1.2.6. (E)-4-Hydroxy-3-(3-(4-fluorophenyl)acryloyl)-2H-chromen-
2-one (3f). Following the procedure above, treatment 6 with 4-
fluorobenzaldehyde for 40 h under reflux gave the crude product
which was purified by column chromatography (eluent: Petro-
leumether/EtOAc 5:1) to afford the yellow solid 3f (0.12 g, 40%).
m.p. 197e200 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 8.41 (d, J ¼ 15.6 Hz,
1H), 8.14e8.03 (m, 2H), 7.78e7.70 (m, 3H), 7.40e7.32 (m, 2H), 7.16
(t, J ¼ 17.0 Hz, 2H). ESI-HRMS m/z [MþH]þ calculated for C18H12FO4:
311.0714, found: 311.0710. HPLC (90% methanol in water):
tR ¼ 4.046 min, 98.3%.
4.1.2.7. (E)-4-Hydroxy-3-(3-(4-cyanophenyl)acryloyl) -2H-chromen-
2-one (3g). Following the procedure above, treatment 6 with 4-
cyanobenzaldehyde for 26 h under reflux gave the crude product
which recrystallized with methanol to afford 3g as yellow solid
(0.32 g, 50%). m.p. 254e256 ꢁC. 1H NMR (300 MHz, DMSO)
d: 8.35
(d, J ¼ 15.8 Hz, 1H), 8.06 (d, J ¼ 7.2 Hz, 2H), 8.00 (s, 4H), 7.85 (d,
J ¼ 6.6 Hz, 1H), 7.48e7.45 (m, 2H). ESI-HRMS m/z [MþH]þ calcu-
lated for C19H12NO4: 318.3003, found: 318.3005. HPLC (90% meth-
anol in water): tR ¼ 3.240 min, 96.4%.
4.1.2.8. (E)-4-Hydroxy-3-(3-(3-nitrophenyl)acryloyl)-2H-chromen-
2-one (3h). Following the procedure above, treatment 6 with 3-
nitrobenzaldehyde for 26 h under reflux gave the crude product
which recrystallized with ethanol to afford 3h as yellow solid
Fig. 7. Fluorescence microscopic imaging of A549 cells incubated with 10
m
M or 50
m
m
M
M
probe III. The imaging of inhibition of NQO1 was accomplished by incubating 10
DIC with probe. A549 cells were incubated with each probe at 37 ꢁC for 10 min and
washed immediately. All images are representative of images obtained in three
separate experiments.
(0.32 g, 64%). m.p. 222e226 ꢁC. 1H NMR (300 MHz, DMSO)
d: 8.59
(s, 1H), 8.39e8.32 (m, 2H), 8.24 (d, J ¼ 7.5 Hz, 1H), 8.58e8.06 (m,
2H), 7.85e7.78 (m, 2H), 7.47 (d, J ¼ 7.5 Hz, 1H). ESI-HRMS m/z
[MþH]þ calculated for C18H12NO6: 338.0659, found: 338.0663.
HPLC (90% methanol in water): tR ¼ 3.326 min, 97.2%.
J ¼ 7.8 Hz, 1H), 7.47e7.43 (m, 2H). ESI-HRMS m/z [M ꢀ H]- calcu-
lated for C18H10NO6: 336.0514, found: 336.0516.
4.1.2.9. (E)-4-Hydroxy-3-(3-(4-(trifluoromethyl)phenyl)acryloyl)-
2H-chromen-2-one (3i). Following the procedure above, treatment
6 with 4-(trifluoromethyl) benzaldehyde for 16.5 h under reflux
gave the crude product which was purified by column chroma-
tography (eluent: Petroleumether/EtOAc 30:1) to afford yellow
4.1.2.3. (E)-4-Hydroxy-3-(3-(4-tolyl)acryloyl)-2H-chromen-2-one
(3c). Following the procedure above, treatment
6 with 4-
methylbenzaldehyde for 18 h under reflux gave the crude product
which was purified by column chromatography (eluent: Petro-
leumether/EtOAc 50:1) to afford the yellow solid 3c (0.4 g, 40%).
solid 3i (0.3 g, 34%). m.p. 257e260 ꢁC. 1H NMR (300 MHz, DMSO)
d:
m.p. 230e234 ꢁC. 1H NMR (300 MHz, DMSO)
d
: 8.26 (d, J ¼ 15.8 Hz,
8.36 (d, J ¼ 15.9 Hz, 1H), 8.08e8.03 (m, 2H), 7.99 (d, J ¼ 8.0 Hz, 2H),
7.87 (s, 1H), 7.83 (d, J ¼ 6.8 Hz, 2H), 7.47e7.43 (m, 2H). ESI-HRMS m/
z [MþH]þ calculated for C19H12F3O4: 361.0658, found: 361.3265.
HPLC (90% methanol in water): tR ¼ 3.359 min, 96.8%.
1H), 8.05 (m, 2H), 8.03 (t, J ¼ 15.7 Hz, 1H), 7.67 (d, J ¼ 7.9 Hz, 2H),
7.45e7.42 (m, 2H), 7.32 (d, J ¼ 7.9 Hz, 2H), 2.35 (s, 3H). ESI-HRMS m/
z [MþH]þ calculated for C19H15O4: 308.9080, found: 308.9078.
HPLC (90% methanol in water): tR ¼ 3.391 min, 98.7%. HPLC (90%
methanol in water): tR ¼ 3.246 min, 96.2%.
4.1.2.10. (E)-4-Hydroxy-3-(3-(2-chlorophenyl)acryloyl)-2H-chro-
men-2-one (3j). Following the procedure above, treatment 6 with
2-chlorobenzaldehyde for 18 h under reflux gave the crude product
which was purified by column chromatography (eluent: Petro-
leumether/EtOAc 10:1) to afford the yellow solid 3j (0.2 g, 40%).
4.1.2.4. (E)-4-Hydroxy-3-(3-(4-hydroxyphenyl)acryloyl)-2H-chro-
men-2-one (3d). Following the procedure above, treatment 6 with
4-hydroxybenzaldehyde for 3 h under reflux gave the orange solid
3d (0.43 g, 48%). m.p. 244e246 ꢁC. 1H NMR (300 MHz, DMSO)
d:
m.p. 270e273 ꢁC. 1H NMR (300 MHz, CDCl3)
d
: 8.48 (d, J ¼ 6.1 Hz,
10.40 (s, 1H), 8.14 (d, J ¼ 4.8 Hz, 1H), 8.01 (m, 2H), 7.79 (t, J ¼ 7.4 Hz,
1H), 7.67 (d, J ¼ 5.1 Hz, 2H), 7.42e7.41 (m, 2H), 6.88 (d, J ¼ 8.6 Hz,
2H). ESI-HRMS m/z [MþH]þ calculated for C18H13O5: 309.0757,
found: 309.0762. HPLC (90% methanol in water): tR ¼ 3.329 min,
98.0%.
2H), 7.76e7.70 (m, 1H) 7.49e7.46 (m, 1H), 7.40e7.28 (m, 4H). ESI-
HRMS m/z [MþH]þ calculated for C18H12ClO4: 327.0419, found:
327.0425. HPLC (90% methanol in water): tR ¼ 3.489 min, 97.2%.
4.1.2.11. (E)-4-Hydroxy-3-(3-(4-isopropylphenyl)acryloyl)-2H-chro-
men-2-one (3k). Following the procedure above, treatment 6 with
4-isopropylbenzaldehyde for 12 h under reflux gave the crude
product which was purified by column chromatography (eluent:
Petroleumether/EtOAc ¼ 10:1) to afford the yellow solid 3k (0.36 g,
4.1.2.5. (E)-4-Hydroxy-3-(3-(4-aminophenyl)acryloyl)-2H-chromen-
2-one (3e). Following the procedure above, treatment 6 with 3-
methoxybenzaldehyde for 10.5 h under reflux gave the crude
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

10
J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
45%). m.p. 288e290 ꢁC. 1H NMR (300 MHz, DMSO)
d
: 8.44 (d,
7.09e7.85 (m, 2H), 7.61 (s, 2H), 7.46 (d, J ¼ 8.2 Hz, 2H). ESI-HRMS m/
z [MþH]þ calculated for C22H15O4: 343.0965, found: 343.0965.
HPLC (90% methanol in water): tR ¼ 3.314 min, 94.0%.
J ¼ 15.6 Hz,1H), 8.13e8.07 (m, 2H), 7.71e7.68 (m, 3H), 7.38e7.33 (m,
2H), 7.30 (d, J ¼ 9.9 Hz, 2H), 2.98 (m,1H),1.28 (d, J ¼ 4.7 Hz, 6H). ESI-
HRMS m/z [MþNa]þ calculated for C21H18NaO4: 357.1097, found:
357.1101. HPLC (90% methanol in water): tR ¼ 3.949 min, 95.9%.
4.1.2.18. (E)-4-Hydroxy-3-(3-(2-fluorophenyl)acryloyl)-2H-chromen-
2-one (3r). Following the procedure above, treatment 6 with 2-
fluorobenzaldehyde for 18 h under reflux gave the crude product
which was purified by column chromatography (eluent: Petro-
leumether/EtOAc 10:1) to afford the yellow solid 3r (0.4 g, 64%).
4.1.2.12. (E)-4-Hydroxy-3-(3-(3-chlorophenyl)acryloyl)-2H-chro-
men-2-one (3l). Following the procedure above, treatment 6 with
3-chlorobenzaldehyde for 38.5 h under reflux gave the crude
product which was purified by column chromatography (eluent:
Petroleumether/EtOAc 10:1) to afford the yellow solid 3l (0.26 g,
m.p. 235e237 ꢁC. 1H NMR (300 MHz, DMSO)
d
: 8.40 (d, J ¼ 15.9 Hz,
1H), 8.06e8.01 (m, 2H), 7.85 (m, 2H), 7.60e7.50 (m, 1H), 7.47e7.36
(m, 4H). ESI-HRMS m/z [MþH]þ calculated for C18H12O4: 311.0714,
found: 311.0716. HPLC (90% methanol in water): tR ¼ 10.292 min,
96.4%.
45%). m.p. 216e218 ꢁC. 1H NMR (300 MHz, DMSO)
d: 8.30 (d,
J ¼ 15.5 Hz, 1H), 8.07e7.97 (m, 2H), 7.78e7.82 (m, 2H), 7.76 (d,
J ¼ 7.1 Hz, 1H), 7.57e7.47 (m, 2H), 7.45e7.43 (m, 2H). ESI-HRMS m/z
[MþNa]þ calculated for C18H11NaO4: 349.0238, found: 349.0239.
HPLC (90% methanol in water): tR ¼ 3.567 min, 96.4%.
4.1.2.19. (E)-4-Hydroxy-3-(3-(3-methoxyphenyl)acryloyl)-2H-chro-
men-2-one (3s). Following the procedure above, treatment 6 with
3-methoxybenzaldehyde for 10.5 h under reflux gave the crude
product which recrystallized with ethanol to afford 3s as yellow
4.1.2.13. (E)-4-Hydroxy-3-(3-(3,4-dimethoxyphenyl)acryloyl)-2H-
chromen-2-one (3m). Following the procedure above, treatment 6
with 3,4-dimethoxybenzaldehyde for 12.5 h under reflux gave the
crude product which recrystallized with ethanol to afford 3m as
yellow solid (0.45 g, 75%). m.p. 245e256 ꢁC. 1H NMR (300 MHz,
solid (0.35 g, 50%). m.p. 267e269 ꢁC. 1H NMR (300 MHz, DMSO)
d:
8.29 (d, J ¼ 13.3 Hz, 1H), 8.05e7.99 (m, 2H), 7.85 (t, J ¼ 6.0 Hz, 1H),
7.44e7.32 (m, 5H), 7.10 (d, J ¼ 6.0 Hz, 1H), 3.82 (s, 3H). ESI-HRMS m/
z [MþH]þ calculated for C19H15O5: 323.0915, found: 323.0907. HPLC
(90% methanol in water): tR ¼ 3.460 min, 96.0%.
DMSO)
d
: 8.03 (d, J ¼ 8.2 Hz, 2H), 7.45 (t, J ¼ 8.1 Hz, 2H), 7.48e7.43
(m, 4H), 7.13 (s, 1H), 2.67 (s, 6H). ESI-HRMS m/z [MþH]þ calculated
for C20H17O6: 353.102, found: 353.1018. HPLC (90% methanol in
water): tR ¼ 3.336 min, 97.7%.
4.1.2.20. 4-Hydroxy-3-(3-(4-chlorophenyl)propanoyl)-2H-chromen-
2-one (3t). As yellow solid (55% yield). m.p. 132e134 ꢁC. 1H NMR
4.1.2.14. (E)-4-Hydroxy-3-(3-(4-ethoxyphenyl)acryloyl)-2H-chro-
men-2-one (3n). Following the procedure above, treatment 6 with
4-ethoxybenzaldehyde for 7 h under reflux gave the crude product
which recrystallized with ethanol to afford 3n as yellow solid (0.4 g,
(300 MHz, DMSO) d: 8.04e8.01 (m, 1H), 7.85e7.81 (m, 1H),
7.46e7.42 (m, 2H), 7.36e7.27 (m, 4H), 3.41 (t, J ¼ 7.5 Hz, 2H), 2.92 (t,
J ¼ 7.5 Hz, 2H). IR (v, cmꢀ1): 3415, 1719, 1618, 991. ESI-HRMS m/z
[M ꢀ H]- calculated for C18H12ClO4 327.0424, found 327.0421. HPLC
(90% methanol in water): tR ¼ 9.717 min, 98.9%.
40%). m.p. 287e289 ꢁC. 1H NMR (300 MHz, DMSO)
d: 8.30 (d,
J ¼ 15.5 Hz, 1H), 8.05e8.03 (m, 2H), 7.83 (m, 1H), 7.75 (d, J ¼ 6.0 Hz,
2H), 7.44 (s, 2H), 7.05 (d, J ¼ 6.5 Hz, 2H), 4.10 (m, 2H), 1.34 (t,
J ¼ 6.6 Hz, 3H). ESI-HRMS m/z [MþNa]þ calculated for C20H16NaO5:
359.0890, found: 359.0896. HPLC (90% methanol in water):
tR ¼ 3.736 min, 98.5%.
4.1.2.21. 4-Hydroxy-3-(3-(4-hydroxyphenyl)propanoyl)-2H-chro-
men-2-one (3u). As yellow solid (63% yield). m.p. 170e173 ꢁC. 1H
NMR (300 MHz, DMSO) d: 11.75 (s, 1H), 9.20 (s, 1H), 8.04 (dd,
J1 ¼ 8.2 Hz, J2 ¼ 1.5 Hz, 1H), 7.87e7.81 (m, 1H), 7.48e7.43 (m, 2H),
7.06 (d, J ¼ 8.4 Hz, 2H), 6.68 (d, J ¼ 8.4 Hz, 2H), 3.80e3.75 (m, 2H),
2.82 (t, J ¼ 7.2 Hz, 2H). IR (v, cmꢀ1): 3415, 1733, 1612, 979. ESI-HRMS
m/z [M ꢀ H]- calculated for C18H13O5 309.0763, found 309.0722.
HPLC (90% methanol in water): tR ¼ 3.061 min, 98.2%.
4.1.2.15. (E)-4-Hydroxy-3-(3-(2,4-dichlorophenyl)acryloyl)-2H-chro-
men-2-one (3o). Following the procedure above, treatment 6 with
2,4-dichlorobenzaldehyde for 13 h under reflux gave the crude
product which recrystallized with ethanol to afford 3o as yellow
solid (0.3 g, 34%). m.p. 195e198 ꢁC. 1H NMR (300 MHz, DMSO)
d:
4.1.2.22. 4-Hydroxy-3-(3-(3-methoxyphenyl) propanoyl)-2H-chro-
8.30 (d, J ¼ 15.7 Hz, 1H), 8.16 (s, 1H), 8.08 (t, J ¼ 7.9 Hz, 1H), 7.93 (d,
J ¼ 8.6 Hz, 1H), 7.84e7.81 (m, 2H), 7.50 (d, J ¼ 7.9 Hz, 1H), 7.46e7.43
(m, 2H). ESI-HRMS m/z [MþH]þ calculated for C18H11Cl2O4:
361.0029, found: 361.0028. HPLC (90% methanol in water):
tR ¼ 3.925 min, 98.0%.
men-2-one (3v). As orange solid (65% yield). m.p. 110e112 ꢁC. 1H
NMR (300 MHz, DMSO)
d
: 8.06 (dd, J1 ¼ 8.2 Hz, J2 ¼ 1.5 Hz, 1H),
7.88e7.83 (m, 1H) 7.49e7.45 (m, 2H), 7.23 (t, J ¼ 8.1 Hz, 1H),
6.87e6.85 (m, 2H), 6.80e6.76 (m, 1H). IR (v, cmꢀ1): 3412, 1722,
1606, 902. ESI-HRMS m/z [M ꢀ H]- calculated for C19H15O5
323.0919, found 323.0947. HPLC (90% methanol in water):
tR ¼ 3419 min, 96.6%.
4.1.2.16. (E)-4-Hydroxy-3-(3-([1,1'-biphenyl]-4-yl)acryloyl)-2H-chro-
men-2-one (3p). Following the procedure above, treatment 6 with
[1,1'-biphenyl]-4-carbaldehyde for 13 h under reflux gave the crude
product which recrystallized with ethanol to afford 3p as yellow
4.1.2.23. (E)-3-(3-(4-chlorophenyl)acryloyl)-4-hydroxy-6,7-
dimethyl-2H-chromen-2-one (3w). As yellow solid (42% yield). m.p.
solid (0.45 g, 51%). m.p. 200e203 ꢁC. 1H NMR (300 MHz, DMSO)
d:
236e238 ꢁC. 1H NMR (300 MHz, DMSO)
d
: 8.22 (d, J ¼ 16.1 Hz, 1H),
8.32 (m, 2H), 8.12e8.05 (m, 2H), 7.91e7.84 (m, 4H), 7.76 (d,
J ¼ 7.3 Hz, 2H), 7.54e7.43 (m, 5H). ESI-HRMS m/z [MþH]þ calcu-
lated for C24H17O4: 369.1121, found: 369.1123. HPLC (90% methanol
in water): tR ¼ 4.096 min, 97.7%.
7.92 (d, J ¼ 15.9 Hz, 1H), 7.78e7.75 (m, 3H), 7.55 (d, J ¼ 8.6 Hz, 2H),
7.23 (s, 1H), 2.35 (s, 3H), 2.30 (s, 3H). IR (v, cmꢀ1): 3412, 1722, 1606,
902. ESI-HRMS m/z [M ꢀ H]- calculated for C20H14ClO4 353.0581,
found 531.0677. HPLC (90% methanol in water): tR ¼ 5.009 min,
97.7%.
4.1.2.17. (E)-4-Hydroxy-3-(3-(naphthalen-2-yl)acryloyl)-2H-chro-
men-2-one (3q). Following the procedure above, treatment 6 with
2-naphthaldehyde for 11.5 h under reflux gave the crude product
which recrystallized with ethanol to afford 3q as yellow solid (0.5 g,
4.1.2.24. (E)-3-(3-(3-methoxyphenyl)acryloyl)-4-hydroxy-6,7-
dimethyl-2H-chromen-2-one (3x). As yellow solid (41% yield). m.p.
197e199 ꢁC. 1H NMR (300 MHz, DMSO)
d
: 8.27 (d, J ¼ 15.9 Hz, 1H),
61%). m.p. 215e217 ꢁC. 1H NMR (300 MHz, DMSO)
d: 8.04 (t,
8.27 (d, J ¼ 16.1 Hz, 1H), 7.77 (s, 1H), 7.45e7.34 (m, 2H), 7.28 (d,
J ¼ 14.9 Hz, 2H), 8.20 (d, J ¼ 15.5 Hz, 1H), 8.08e7.96 (m, 4H),
J ¼ 7.2 Hz, 2H), 7.09 (d, J ¼ 8.3 Hz, 1H), 3.81 (s, 3H), 2.36 (s, 3H), 2.30
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
11
(s, 3H). IR (v, cmꢀ1): 3414, 1718, 1621, 987. ESI-HRMS m/z [MþH]þ
4.1.6. (E)-3-(3-(4-((6-aminohexyl)oxy)phenyl)acryloyl)-4-hydroxy-
2H-chromen-2-one (11)
calculated for C21H18O5 351.1232, found 351.0676. HPLC (90%
methanol in water): tR ¼ 4.421 min, 95.3%.
To a stirred solution of 11 (0.35 g) in CH2Cl2 was added a mixture
of CH2Cl2/CF3COOH (1: 1, 10 mL). The mixture was stirred at room
temperature for 20 min. After completion of the reaction, the pH
was adjusted to 12 using sodium hydroxide solution. Then the
precipitate was filtered to obtain the yellow solid 11 (0.23 g, 80%).
4.1.2.25. (E)-3-(3-(3-methoxyphenyl)acryloyl)-4-hydroxy-7-chloro-
2H-chromen-2-one (3y). As yellow solid (39% yield). m.p.
182e184 ꢁC. 1H NMR (300 MHz, DMSO)
d
: 8.24 (d, J ¼ 16.0 Hz, 1H),
8.06 (s, 1H), 8.01 (d, J ¼ 2.4 Hz, 1H), 7.88 (dd, J1 ¼ 9.0 Hz, J2 ¼ 2.3 Hz,
1H), 7.53e7.41 (m, 3H), 7.33 (s,1H), 7.14e7.11 (m,1H), 3.84 (s, 3H). IR
(v, cmꢀ1): 3415, 1718, 1622, 977. ESI-HRMS m/z [M ꢀ H]- calculated
for C19H12ClO5 355.0373, found 355.0365. HPLC (90% methanol in
water): tR ¼ 3.733 min, 96.9%.
4.1.6.1. (E)-1-(30,6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-
xanthen]-6-yl)-3-(6-(4-(3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-3-
oxoprop-1-en-1-yl)phenoxy)hexyl)thiourea (probe III). To a solution
of compound 11 (1.0 equ.) in DMF was added FITC (10 equ.). The
reaction mixture was stirred at 60 ꢁC for 8 h under nitrogen. The
crude product was then diluted in 20 mL of diethyl ether and stirred
for 30 min. Then the solution was filtered and the residue was
purified by column chromatography on silica gel (eluent: 15%
CH3OH in CH2Cl2), resulting in probe III as an orange solid (255 mg,
4.1.2.26. (E)-3-(3-(3-methoxyphenyl)acryloyl)-4-hydroxy-7-
methoxy-2H-chromen-2-one (3z). As yellow solid (50% yield). m.p.
206e208 ꢁC. 1H NMR (300 MHz, DMSO)
d: 9.05 (s, 1H), 6.67 (d,
40%). m.p. 286e288 ꢁC. 1H NMR (300 MHz, DMSO)
d: 10.78 (s, 1H),
J ¼ 15.7 Hz, 1H), 6.23e6.14 (m, 2H), 5.54e5.52 (m, 2H), 5.45 (s, 1H),
5.20 (s,1H), 5.14e5.08 (m, 2H), 2.11 (s, 3H), 2.03 (s, 3H). IR (v, cmꢀ1):
3412, 1717, 1618, 857. ESI-HRMS m/z [M ꢀ H]- calculated for
10.32e10.23 (m, 2H), 8.64 (brs, 1H), 8.41 (s, 1H), 7.93e7.80 (m, 2H),
7.55e7.48 (m, 4H), 7.21e7.13 (m, 3H), 6.98e6.96 (m, 2H), 6.98 (s,
2H), 6.57e6.56 (m, 4H), 4.01 (s, 2H), 3.47 (s, 2H), 2.87 (s, 1H), 2.70 (s,
1H), 1.73 (brs, 2H), 1.57 (brs, 2H), 1.42 (brs, 4H). IR (v, cmꢀ1): 3414,
1727, 1601, 1385, 1172, 1112, 830, 764, 601, 482. ESI m/z [MþH]þ
797.3.
C
20H15O6 351.0869, found 351.0697. HPLC (90% methanol in water):
tR ¼ 3.880 min, 97.7%.
4.1.3. N-Boc-6-aminohexan-1-ol (8)
Compound 7 (2.4 g,1 equ.) was dissolved in CH2Cl2 (10 mL), then
Boc2O (5.12 mL, 1.1 equ) was added drop wise under continuous
stirring condition and resulting solution was stirred at room tem-
perature for another 10 min. After completion of the reaction
(monitored by TLC) the excess solvent was removed under reduced
pressure. Added a few PE to obtain white product 8 (3.87, 85%). m.p.
4.2. Biology
4.2.1. NQO1 inhibition studies
All of the synthesized compounds were assayed for their ability
affinities to NQO1 protein, we performed the assay using the
methods as previous reported [14]. Briefly, NQO1 was diluted in
50 mmol Lꢀ1 phosphate buffer to give an enzyme activity that
would result in a change in optical absorbance of NQO1 substrate
46e48 ꢁC. 1H NMR (300 MHz, CDCl3)
d: 4.50 (brs, 1H), 3.64 (t,
J ¼ 6.5 Hz, 2H), 3.12 (t, J ¼ 6.5 Hz, 2H),1.57e1.46 (m, 6H),1.44 (s, 9H).
(b-lap) of approximately 0.1 per minute. The enzyme reaction was
4.1.4. 6-((Tert-butoxycarbonyl)amino)hexyl-4-
methylbenzenesulfonate (9)
started by adding 5 mL of this solution to 495 mL of 50 mmol Lꢀ1
phosphate buffer at pH 7.4 containing 200 mmol Lꢀ1 NADPH for
NQO1, with 0.14% (w/v) BSA, together with various concentrations
of the potential inhibitor dissolved in DMSO (final concentration
1.0% v/v). The DMSO concentration used was sufficiently small to
ensure minimal perturbation of hydrogen bonding networks in
aqueous NQO1 complexes. Reaction was initiated by automated
dispensing of the NADPH solution into the wells, and data was
recorded at 2 s intervals for 5 min at room temperature (22e25 ꢁC).
The oxidation of NADPH to NADPþ was monitored at 340 nm on a
Varioskan Flash (Thermo, Waltham, MA). Each measurement was
made in triplicate and the experiments carried out three times. The
IC50 curves were generated using Graphpad Prism 6.
Compound 8 (2.0 g, 1 equ.) was dissolved in CH2Cl2, para-
toluensulfonyl chloride (2.1 g, 1.1 equ.) in CH2Cl2 was added drop
wise into the solution. Then trimethylamine was added and the
reaction was stirred for another 48 h. After that, poured the solution
into water and washed with NaCl, dried by Na2SO4, the solvent was
removed under reduced pressure. The mixture was further purified
using silica gel column chromatography using 5e10% petroleum
ether to ethylacetate gradient solvent system to afford the desired
pure product with colorless oily matter 9 (1.02, 29%). m.p.
66e68 ꢁC. 1H NMR (300 MHz, CDCl3)
(d, J ¼ 8.0 Hz, 2H), 4.00 (t, J ¼ 6.5 Hz, 2H), 3.05 (brs, 2H), 2.44 (s, 3H),
2.04 (s, 1H), 1.42 (s, 9H), 1.34e1.22 (m, 6H).
d
: 7.77 (d, J ¼ 8.3 Hz, 2H), 7.34
4.2.2. Evaluation of NQO1 inhibitors in A549 cell lines
4.1.5. (E)- tert-butyl- (6-(4-(3-(4-hydroxy-2-oxo-2H-chromen-3-
yl)-3-oxoprop-1-en-1-yl)ph-enoxy)hexyl)carbamate (10)
Representative compounds were selected for their evaluation of
in A549 cell lines. Impact of NQO1 inhibition on b-lap toxicity was
determined by the MTT assay. Cells were plated in 96-well plates at
a density of 10 000 cells per mL and allowed to attach overnight
To a stirred solution of 3d (0.68 g, 1 equ.), K2CO3 (0.343 g, 1.1
equ.) in DMF (20 mL) under N2 atmosphere was added 9 (0.83 g, 1
equ.) in DMF. The mixture was then heated at 80 ꢁC for 8 h. The
excess solvent was then removed under reduced pressure and the
obtained residue was washed successively with water and brine,
and extracted with ethylacetate (3 ꢂ 10 mL). The organic layer was
dried over anhydrous Na2SO4 and concentrated under reduced
pressure. The crude was recrystallized using ethanol to obtain the
pure yellow solid 10 (0.35 g, 15%). m.p. 278e280 ꢁC. 1H NMR
(16 h). Cells were then given 30
m
mol Lꢀ1 DIC or the other com-
-lap for 2 h, removed, and
pounds with various concentrations of
b
replaced with fresh medium and the plates were incubated at 37 ꢁC
under a humidied atmosphere containing 5% CO2 for 72 h. MTT was
added and the cells were incubated for another 4 h. Medium/MTT
solutions were removed carefully by aspiration, the MTT formazan
crystals were dissolved in 100 mL of DMSO, and absorbance was
determined on a plate at 540 nm. Values of IC50 were calculated as
(300 MHz, DMSO)
d
: 7.88 (dd, J1 ¼ 7.7 Hz, J2 ¼ 1.5 Hz, 1H), 7.64 (d,
J ¼ 15.8 Hz, 1H), 7.52e7.49 (m, 2H), 7.28 (d, J ¼ 15.7 Hz, 1H),
7.18e7.10 (m, 2H), 6.94 (d, J ¼ 8.7 Hz, 2H), 6.81e6.79 (m,1H), 3.98 (t,
J ¼ 6.4 Hz, 2H), 2.91e2.87 (m, 2H), 1.72e1.68 (m, 2H), 1.36 (s, 9H),
1.30e1.22 (m, 6H).
the concentration of b
-lap, which in the presence of 30 mmol Lꢀ1
inhibitors, cause 50% cell kill. All toxicity experiments were
repeated on at least three technical replicates. Data were analyzed
and curves were generated using Graphpad Prism 6.
Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062

12
J. Bian et al. / European Journal of Medicinal Chemistry xxx (2016) 1e12
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4.2.3. Fluorescence polarization competition assay
The fluorescence polarization (FP) assay was performed on a
SpectraMax multi-mode microplate reader (Molecular Devices)
using the excitation and emission filters. The plates used for the FP
measurements were the black nonbinding surface Corning
3676 384-well plates loaded with 40
m
L of assay solution per well,
L HEPES
consisting 20 L various concentrations Probe III and 20
m
m
buffer to measure the mP value of the Probe III. As for the deter-
mination of the inhibition ability of the Probe III of NQO1, the plates
loaded woth 40
HEPES buffer, 10
m
L of assay solution per well, consisting 20
m
m
L
L
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emission filters were used. The IC50 of the Probe III was determined
from the plot %inhibition against inhibitor concentration analyzed
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4.2.4. Fluorescence microscopy imaging
The fluorescent imaging was performed on A549 cells. After the
culture medium was removed from the confocal dishes, cells were
washed with the corresponding culture medium without fetal
bovine serum. Probe III was dissolved in DMSO as a stock solution
(10 mM), which was further dilute with corresponding culture
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medium without fetal bovine serum. Subsequently, Probe III (10
mM
or 50 M) was incubated with A549 cells for 10 min, and then cells
m
were washed with the corresponding culture medium without fetal
bovine serum. Cells were then stained with fluorochrome dye DAPI
(Santa Cruz biotechnology, Santa Cruz, CA) to visualize the nuclei
and observed under
a laser scanning confocal microscope
(Olympus Fluoview FV1000, Japan).
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The imaging of A549 cells was also performed by co-incubation
with inhibitor (10
mM DIC) incubation together with the probe
(10 M or 50 M) at the same conditions, using A549 cells.
m
m
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Acknowledgements
[16] K.A. Nolan, H. Zhao, P.F. Faulder, A.D. Frenkel, D.J. Timson, D. Siegel, D. Ross,
T.R. Burke Jr., I.J. Stratford, R.A. Bryce, J. Med. Chem. 50 (2007) 6316e6325.
[17] J. Bian, X. Qian, B. Deng, X. Xu, X. Guo, Y. Wang, X. Li, H. Sun, Q. You, X. Zhang,
We are thankful for the financial support of the National Natural
Science Foundation of China (No. 81302636, 81603025, 81230078,
81573346), the Natural Science Foundation of Jiangsu Province (No.
BK20130656), the National Major Science and Technology Project of
China (No. 2013ZX09402102-001-005, 2014ZX09507002-005-015),
Specialized Research Fund for the Doctoral Program of Higher Ed-
ucation (No. 20130096110002), the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD), and
the Project Program of State Key Laboratory of Natural Medi-
cines,China Pharmaceutical University (No. SKLNMZZCX201611).
Discovery of NAD(P)H:quinone oxidoreductase
1 (NQO1) inhibitors with
novel chemical scaffolds by shape-based virtual screening combined with
cascade docking, RSC Adv. 5 (2015) 49471e49479.
[18] K.A. Scott, J. Barnes, R.C. Whitehead, I.J. Stratford, K.A. Nolan, Inhibitors of
NQO1: identification of compounds more potent than dicoumarol without
associated off-target effects, Biochem. Pharmacol. 81 (2011) 355e363.
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W.G. Holloway, J.M. Gerdes, M. Behforouz, H.D. Beall, Lavendamycin anti-
tumor agents: structure-based design, synthesis, and NAD(P)H:quinone
oxidoreductase 1 (NQO1) model validation with molecular docking and bio-
logical studies, J. Med. Chem. 51 (2008) 3104e3115.
[20] M. Sameiro, T. Goncalves, Fluorescent labeling of biomolecules with organic
probes, Chem. Rev. 109 (2009) 190e212.
Appendix A. Supplementary data
[21] K.A. Nolan, J.R. Doncaster, M.S. Dunstan, K.A. Scott, A.D. Frenkel, D. Siegel,
D. Ross, J. Barnes, C. Levy, D. Leys, R.C. Whitehead, I.J. Stratford, R.A. Bryce,
Synthesis and biological evaluation of coumarin-based inhibitors of NAD(P)H:
quinone oxidoreductase-1 (NQO1), J. Med. Chem. 52 (2009) 7142e7156.
[22] M. Faig, M.A. Bianchet, S. Winski, R. Hargreaves, C.J. Moody, A.R. Hudnott,
L.M. Amzel, Structure-based development of anticancer drugs: complexes of
Supplementary data related to this article can be found at http://
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Please cite this article in press as: J. Bian, et al., Affinity-based small fluorescent probe for NAD(P)H:quinone oxidoreductase 1 (NQO1). Design,
synthesis and pharmacological evaluation, European Journal of Medicinal Chemistry (2016), http://dx.doi.org/10.1016/j.ejmech.2016.10.062