Novel naphtho[2,1-d]oxazole-4,5-diones as NQO1 substrates with improved aqueous solubility: Design, synthesis, and in vivo antitumor evaluation

Article abstract of DOI:10.1016/j.bmc.2016.01.024

A new series of ortho-naphthoquinone analogs of β-lapachone were designed, synthesized and evaluated. The biological results indicated that most of our compounds were efficient substrates for NQO1. The new scaffold with water-soluble side chain resulted in greater solubility under acidic condition compared to β-lapachone. Thus avoiding the use of hydroxylpropyl β-cyclodextrin which would finally cause the rapid drug clearance from the blood and dose-limiting toxicity in the form of hemolytic anemia. The most soluble and promising compound in this series was 2-((4-benzylpiperazin-1-yl)methyl)naphtho[2,1-d]oxazole-4,5-dione (3k), which inhibited cancer cell (NQO1-rich A549 cell line) growth at IC₅₀ values of 4.6 ± 1.0 μmol·L^-1. Furthermore, compound 3k had in vivo antitumor activity in an A549 tumor xenografts mouse model comparable to the activity obtained with β-lapachone. The results indicated that these ortho-naphthoquinones could serve as promising leads for further optimization as novel substrates for NQO1.

Full text of DOI:10.1016/j.bmc.2016.01.024

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel naphtho[2,1-d]oxazole-4,5-diones as NQO1 substrates with
improved aqueous solubility: Design, synthesis, and in vivo
antitumor evaluation
Xiang Li a,b,y, Jinlei Bian a,y, Nan Wang a, Xue Qian a, Jing Gu a, Tong Mu a, Jun Fan a, Xiuwen Yang a,
Shangzhen Li a, Tingting Yang a, Haopeng Sun a,c, Qidong You a, , Xiaojin Zhang a,c,
⇑
⇑
a Jiangsu Key Laboratory of Drug Design and Optimization, and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China
b Department of Pharmaceutical Engineering, China Pharmaceutical University, Nanjing 210009, China
c 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:
A new series of ortho-naphthoquinone analogs of b-lapachone were designed, synthesized and evaluated.
The biological results indicated that most of our compounds were efficient substrates for NQO1. The new
scaffold with water-soluble side chain resulted in greater solubility under acidic condition compared to
b-lapachone. Thus avoiding the use of hydroxylpropyl b-cyclodextrin which would finally cause the rapid
drug clearance from the blood and dose-limiting toxicity in the form of hemolytic anemia. The most
soluble and promising compound in this series was 2-((4-benzylpiperazin-1-yl)methyl)naphtho[2,1-d]
oxazole-4,5-dione (3k), which inhibited cancer cell (NQO1-rich A549 cell line) growth at IC50 values of
Received 24 November 2015
Revised 13 January 2016
Accepted 14 January 2016
Available online xxxx
Keywords:
NQO1 substrates
b-Lapachone
Antitumor
ortho-Naphthoquinones
In vivo
4.6 1.0
l
molꢀLꢁ1. Furthermore, compound 3k had in vivo antitumor activity in an A549 tumor xeno-
grafts mouse model comparable to the activity obtained with b-lapachone. The results indicated that
these ortho-naphthoquinones could serve as promising leads for further optimization as novel substrates
for NQO1.
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
levels of the enzyme.7,8 Recently, several ortho-naphthoquinones
have been reported to be efficient substrates for NQO1.9–11 For
NAD(P)H:quinone oxidoreductase-1 (NQO1) is a homodimeric
flavoprotein that catalyzes the obligatory two-electron reduction
of quinones to hydroquinones using NADH or NADPH as cofac-
tor.1,2 Typically, this reduction detoxifies quinones, forming less
reactive hydroquinones. Glutathione S-transferase then detoxifies
hydroquinones, conjugating them with glutathione for secretion.3
While the two-electron reduction process has made some com-
pounds biological active. Moreover, the reduced hydroquinones
may be oxidized back to quinones followed by subsequent gener-
ation of reactive oxygen species (ROS) and oxidative stress.4 The
accumulated ROS via redox cycling can achieve selectivity toward
cancer cells due to the high basal redox levels uniquely present in
it.5,6
example, b-lapachone (b-lap), a natural ortho-naphthoquinone iso-
lated from the Bignoniaceae family and is currently in multiple
phase II clinical trials.12–14 While capable of killing cancer cells,
b-lap demonstrated limited water solubility. In the clinical studies,
the use of hydroxylpropyl b-cyclodextrin (HPbCD) (ARQ501 and
ARQ761) to formulate b-lap showed
a 400-fold increase in
solubility.15 However, rapid drug clearance from the blood and
dose-limiting toxicity in the form of hemolytic anemia due to
HPbCD were observed. These are the reasons why the clinical form
of ARQ501 and ARQ761 underwent unsuccessful clinical trials in
patients with several different cancers.16 Lately, we reported an
efficient NQO1 substrate (DDO-7101, 2),10 which possessed better
specificity and safety than b-lap. Despite the potency and
selectivity of 2 in killing NQO1-containing cancer cells in vitro,
demonstrated even lower water solubility of 2 (0.021 mg/ml) than
b-lap (0.043 mg/ml) which is limited back in its application in vivo.
Therefore, for in vivo evaluation studies, it was necessary to
improve the solubility of 2 (see Fig. 1).
Overexpression of NQO1 in cancer cells further makes it be a
promising therapeutic target for tumor-selective drug therapy
with reduced toxicities to normal cells that express little to no
⇑
Corresponding authors.
E-mail addresses: youqd@163.com (Q. You), zxj@cpu.edu.cn (X. Zhang).
These two authors contributed equally to this work.
Herein, as part of our ongoing program, we reported a series of
new scaffold of naphtho[2,1-d]oxazole-4,5-diones (3) with
y
http://dx.doi.org/10.1016/j.bmc.2016.01.024
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
X. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
O
O
O
O
O
O
O
O
Metabolism by NQO1 of ortho-naphthoquinones monitored by enzyme labeling
instrument at the absorbance of A340 nm and cytotoxicity toward A549 (NQO1-rich)
cell lines
N
H
N
R
O
O
O
F
β-lapachone (1)
DDO-7101 (2)
3
N
Figure 1. The structures of b-lapachone, DDO-7101 and the new designed structure
3.
R
O
3a-3l
Entry
R
Metabolism by NQO1
Cytotoxicity IC50
improved aqueous solubility, as novel efficient NQO1 substrates.
These ortho-naphthoquinones have been tested in vitro for their
antitumor activity against NQO1-rich cancer cell lines. Compound
3k, which was the most potent and drug-like, was further investi-
gated on the mechanism demonstrated that this compound was
selective toxic towards cancer cell line with high NQO1 activity
and had a capability to induce ROS. It was remarkably suppressed
tumor in the in vivo A549 tumor xenografts mouse model, which
was comparable to the activity obtained with b-lap. Thus, the com-
pound was deserved further optimization with the aim to obtain
antitumor agents through NQO1-dependent and ROS-mediated
pathways.
(
lmol NADPH/min/
(
l
molꢀLꢁ1
)
l
mol NQO1)
3a
3b
3c
3d
3e
3f
3g
3h
3i
H
Me
Me2N
1202 87
955 44
939 78
686 52
990 37
955 38
1063 45
968 76
1522 121
1391 78
1360 30
1139 42
1210 30
20 4.3
7.6 0.6
8.7 2.0
6.9 1.8
11 0.5
15 4.9
13 2.8
12 1.5
19 9.3
5.2 0.1
4.6 1.0
7.6 1.3
3.7 0.5
Morpholino
o-MeO-PhNH
m-MeO-PhNH
p-MeO-PhNH
p-Me-PhNH
p-Cl-PhNH
m-F-PhNH
4-Benzylpiperazin-1-yl
4-Phenylpiperazin-1-yl
—
3j
3k
3l
b-lap
2. Results and discussion
2.1. Chemistry
10
l
molꢀLꢁ1 was assessed. The quinone substrates were coincu-
bated with both human NQO1 as well as NADPH. It was capable
of quantifying NADPH oxidation to NADP+ by monitoring the
absorbance (A340 nm) on an enzyme labeling instrument.17 As
previously reported, the results of the quinone reduction were
The designed 12 ortho-naphthoquinone target compounds
3a–3l were synthesized according to the protocol outlined in
Scheme 1. The reaction of 4 with sodium azide at room tempera-
ture provided the product 5 in a yield of 83%. Treatment of 5 with
substituted aldehydes gave rise to 2-substituted 5-hydroxynaph-
tho[2,1-d]oxazoles 6–8 in moderate yields (Scheme 1). Generally,
these hydroxynaphtho[2,1-d]oxazoles could be simply concen-
trated in oil pump vacuum to remove traces of the aldehyde reac-
tants, leaving the products in purities of more than 95%. Oxidation
of 6–8 using the reagent Fremy’s salt provided the target
compounds 9, 3a and 3b in 72–85% yield. The other target
compounds 3c–3l that substituted with amine side chains were
synthesized by a nucleophilic substitution with corresponding
amines. All of the compounds were confirmed by 1H NMR, IR and
EI spectrum. The representative compounds 3a, 3d, 3g, 3k and 3l
were further confirmed by 13C NMR.
presented as
l
molꢀLꢁ1 NADPH oxidized minꢁ1
ꢀ
l
molꢁ1 NQO1.
In general, most of the ortho-naphthoquinones (3a–3l)
possessed moderate to good reduction rates by NQO1, indicating
that these compounds were good substrates for NQO1 (Table 1).
Among them, compounds 3i and 3j with halogen substitution at
the benzene moiety exhibited higher enzymatic conversion rates
(1391–1522 lmol NADPH/min/lmol NQO1) than other com-
pounds. Notably, compounds 3k and 3l with aromatic piperazinyl,
which is designed for the purpose of improving their drug-
like properties, showed efficient reduction rate by NQO1
(1139–1360
3i–3l exhibited comparable enzymatic conversion rate
(1139–1522 mol NADPH/min/ mol NQO1) compared to b-lap
lmol NADPH/min/lmol NQO1). In short, compounds
l
l
and thus them ranked as the efficient substrates for NQO1.
2.2. Enzyme studies
2.3. Molecular docking
All of the ortho-naphthoquinone derivatives were evaluated for
their ability to act as substrates for NQO1. The ability of NQO1 to
process the naphthoquinones in vitro at the concentration of
To elucidate the possible binding mode of NQO1 with the naph-
thoquinones, molecular docking of the representative compounds
O
O
HO
N
O
(i)
(ii)
1 = H
6
7
8
R
R
R1
1 = CH3
1
NH2
R = Cl
O
O
4
5
(iii)
O
(iii)
O
O
O
(iv)
N
N
R
Cl
O
O
3a-3l
9
Scheme 1. Reagents and conditions: (i) Sodium azide, THF/HCl, HOAc, rt, 7 h; (ii) R1CH2CHO, HBr, HAc, rt, 12–16 h; (iii) Fremy’s salt, 0 °C–rt, 1–3 h; (iv) DMF, KI, K2CO3,
45 °C,1–5 h. For the detailed R groups of compounds 3a–3l, see Table 1.
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X. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
Table 2
oxazole ring could form another hydrogen bond with Tyr128, pro-
viding a rationale for the better activity of these series of quinone
with oxazole scaffold. There existed an additional pocket formed
by Tyr128, Met154, Phe232, Phe236 and His194 for side chains
occupying. As depicted in Figure 2, for L-shaped compounds 3i
and 3k, the substituted amine side chains can occupy the
additional pocket, explained the better activity of the L-shaped
compounds compared to compounds without the side chains.
ChemScore and reduction rates of the representative compounds
O
O
5
N
R
O
Compound ChemScore C@O5ꢀ ꢀ ꢀNH5
Metabolism by NQO1
(lmol NADPH/min/lmol NQO1)
(Å)
Besides, forming p-stacking interactions with Tyr128 in the active
3a
3i
3k
b-lap
75.71
95.45
91.75
69.43
3.75
3.80
3.82
3.31
1202 87
1522 121
1360 30
1210 30
site of NQO1, explained the fact that the aromatic amine analogs
exhibited higher reduction rates by NQO1 than other compounds.
As for compound 3k, with larger side chain, which did not substan-
tially decrease the reduction rate compared to 3i. This result
indicated that the side pocket is sufficiently flexible so as to accom-
modate variety of side chains. Moreover, the C–Hꢀ ꢀ ꢀ
p interaction
3a, 3i, 3k and the control compound b-lap with NQO1 were
performed on the binding model based on the NQO1 complex
structure (PDB code 2F1O) using Gold 5.1.18,19 ChemSocre function
was explored during the docking experiments (Table 2). Higher
score represented better fit for the model. Of the selected
compounds studied, compound 3i was nicely bound into the active
site of NQO1 with highest score. The ChemScore values of these
compounds had the same trend as their reduction rates by
NQO1, which likely to prove the correlation between the reduction
rates and the binding affinity.
formed by piperazinyl with the Tyr128 and the benzyl in the side
chain with adjacent Phe232 residue, further explained the reason
for the considerable activity of compound 3i.
In addition, our recent molecular modeling studies have
demonstrated that the quinone substrates to be metabolized by
NQO1 required an appropriate hydride donor–acceptor distance
between the substrate (quinone carbonyl) and the FAD cofactor
(atom N5 which transfer the hydride).22 As illustrated in Figure 2,
all were within a reasonable distance (about 4 Å) for hydride trans-
fer from the reduced FAD (FADH2) to quinone substrates.
The binding model of the selected compounds with NQO1 com-
plex was carefully analyzed and the results were shown in Figure 2.
All of the ortho-naphthoquinones including b-lap oriented with
quinone ring above the isoalloxazing ring of the bound cofactor
2.4. Physicochemical properties
To verify our hypothesis that this series of compounds were
obtained with improved solubility and in an effort to identify
potential drug-like compounds prior to the time-consuming and
costly development to optimize derivatives that may ultimately
fail in in vivo efficacy experiments, then the representative com-
pounds with efficient enzymatic conversion rates were selected
to determine their solubility and LogD7.4 (Table 3). Two methods
were employed to determine the solubility. Their intrinsic aqueous
FAD as for hydride transfer by p-stacking interaction, which were
similar to the crystal structures of substrates or inhibitors in
complex with NQO1.20,21 Similar to our previously reported com-
pounds,10,11 the two carbonyl groups in the ortho-naphthoquinone
substrates and b-lap could bind firmly to the Tyr126 and Tyr128
residues by hydrogen bonding interactions. For compounds 3a, 3i
and 3k, compared to the control b-lap, the nitrogen atom in the
Figure 2. Docked conformation of compounds b-lap (A), 3a (B), 3i (C) and 3k (D) into active site of NQO1. The interaction mode was obtained through molecular docking (PDB
id: 2F1O) and depicted using MOE 2013.08. The carbon atoms of the compounds and the key residues in the active site of NQO1 were colored in yellow and purple,
respectively. The H-bonds were shown as black dot lines.
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X. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 3
Physicochemical properties of the selected compounds
Compound
Intrinsic solubility
g/mL)
Solubilitya
(pH = 3) (lg/mL)
LogD (pH 7.4)
(
l
3i
3j
3k
b-lap
38
44
125
43
<100
<100
>500
<100
2.65
3.18
1.88
2.85
a
Determined in diluted hydrochloric acid buffer at pH = 3.
solubility was determined on a Gemini Profiler instrument (pION)
by the ‘goldstandard’ Avdeef–Bucher potentiometric titration
method. For their intrinsic aqueous solubility, except for 3k with
three times more soluble (125 lg/mL) than b-lap, other com-
Figure 4. Rate of ROS production of 3 k in the presence and absence of the NQO1
pounds were on the same level with it. The other method was used
to determine their solubility under acidic condition, most of these
compounds (3c–3h, 3k–3l) (data not shown) showed dramatically
inhibitor (DIC, 25
l
molꢀLꢁ1) with NQO1 and rates are expressed as mean SD,
P < 0.001 versus control (n = 3). (Spectrophotometric assay with cytochrome c as
terminal electron acceptor (550 nm).)
increased solubility (solubility of >100 lg/mL in diluted hydrochlo-
ric acid buffer at pH = 3). The hydrophobicity parameters LogD7.4
are also summarized in Table 3. These compounds also showed
acceptable LogD7.4 values. Thus, compound 3k showed the best
physicochemical properties among the selected compounds
specificity) (Fig. 3). Meanwhile, compound 3k showed much less
toxic against H596 lung cancer cell line (NQO1-deficient), shifting
the IC50 > 12-fold. These results revealed that the efficient NQO1
substrate 3k was selective toxic towards cancer cell line with high
NQO1 activity. In addition, 3k was further evaluated for its toxicity
towards the human normal hepatic cell line L02, a superior safety
profile was confirmed. Because of the excellent antitumor activity,
selective toxic towards cancer cell lines and good solubility, 3k was
selected for further detailed analysis.
(LogD7.4 1.88, intrinsic solubility 125
lg/mL and solubility under
acidic condition >500 g/mL) and could be selected for further
l
pharmacological both in vitro and in vivo.
2.5. Cytotoxicity evaluation
Cytotoxicity studies were performed on the ortho-naphtho-
quinones with cell survival being determined by the MTT assay
against the human non-small cell lung cancer A549 cell line
(NQO1-rich). b-Lap was used as positive control. The results of
cytotoxic activity in vitro were expressed as the IC50 (Table 1).
As shown in Table 1, it was found that all the compounds
showed moderate to potent cytotoxicity against A549 cancer cell
2.6. Determination of ROS production
Compound 3k was selected as the representative compound to
investigate the ability of producing ROS. We investigated whether
the compound was able to generate ROS in the direct interaction of
NQO1. The production of the superoxide was measured by a spec-
trophotometric assay with cytochrome c as the terminal electron
line (NQO1-rich) with IC50 values ranging from 4.6 to 20
l
molꢀLꢁ1
.
acceptor.23 The initial rates (
lmol cytochrome c reduced/min/mg
Most of the analogs possessed comparative cytotoxicity to the pos-
itive control b-lap against A549 cancer cell line. Taken together of
the physicochemical properties and activity, the most potent and
drug-like compound 3k was further tested against H596 (NQO1-
deficient), L02 (normal hepatic cell lines) and A549 cell lines in
NQO1) were calculated from the liner portion (0–30 s) of the
reduction graphs. 3k showed slightly higher rates of ROS compared
to b-lap, and coincubation with dicoumarol (DIC) dramatically
reduced the rate of ROS production (Fig. 4). The result indicated
that compound 3k could generate a high amount of ROS through
NQO1-directed redox cycling.
the presence of the NQO1 inhibitor dicoumarol (DIC, 25
l
molꢀLꢁ1
)
to compare the cytotoxicity. Coincubation with DIC greatly pro-
tected A549 cells from the cell death mediated by 3k, shifting
the IC50 > 7-fold. (The fold is the ratio of the IC50 of cotreatment
with quinone and DIC to the IC50 of treatment with only quinone,
and a higher ratio indicates greater protection and greater NQO1
2.7. In vivo antitumor activity
Having obtained the excellent soluble and cytotoxic properties
of 3k in in vitro studies, we further evaluated the in vivo antitumor
efficacy of the compound in A549 tumor xenografts mouse model
(Fig. 5). Nude mice bearing established A549 tumor xenografts
were injection with compound 3k (15 mg/kg or 30 mg/kg daily
over a 21-day period). b-Lap (ARQ501, 30 mg/kg) was used as a
positive control drug. Tumor growth inhibition (TGI) and relative
tumor proliferation rate (T/C) were calculated to reveal the
antitumor effects in tumor weight and tumor volume, respectively.
Compared to the vehicle-treated control group, both of the
15 mg/kg and 30 mg/kg showed significant in vivo antitumor
efficacy (Fig. 5A) and produced no evident toxic signs in liver.
Compound 3k (30 mg/kg) demonstrated potent in vivo antitumor
activity with comparable TGI value (55.6%) and T/C value (41.4%)
to b-lap (TGI = 70.1%, T/C = 29.5%) (Fig. 5A and B). Consistent with
these results, the volume of tumors (440.8 105.9 mm3) was
obviously decreased when treated with 3k (30 mg/kg) compared
with the control group (1085.8 298.3 mm3) (Fig. 5C). Besides, in
the mice group treated by 3k, no significant body weight loss
Figure 3. Cell death of A549 cells treated with compound 3k in the presence and
absence of the NQO1 inhibitor (DIC, 25
l
molꢀLꢁ1). H596 (NQO1-deficient) and L02
(normal hepatic cell lines) were evaluated to compare the cytotoxicity with A549
cell lines (NQO1-rich cancer cell).
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X. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 5. Compound 3k retards the tumor growth in vivo in A549 tumor xenografted nude. (A) Tumor weight measurement (**p < 0.01). (B) Relative tumor proliferation rate
(T/C) measurement. T/C (%) = TRTV/CRTV ꢂ 100; RTV = VT/V0; TRTV represents the test group; CRTV represents the control group. (C) Tumor volume measurement. (D) Body
weight measurement.
was observed comparison with the vehicle control group (Fig. 5D).
Thus, our studies suggest that compound 3k would be promising
anticancer therapeutic for the treatment of cancer.
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 spec-
tra (HRMS) were recorded on a Water Q-Tofmicro mass spectrom-
eter. Analytical results are within 0.40% of the theoretical values.
3. Conclusions
4.1.1. 2-Amino-1,4-naphthoquinone (5)
As an alternative to the previously described ortho-quinone
substrates for NQO1, we described herein the design, synthesis
and evaluation of 12 naphtho[2,1-d]oxazole-4,5-diones as anti-
cancer agents. These ortho-naphthoquinones were characterized
and evaluated as efficient substrates for NQO1. This series of com-
pounds showed moderate to good solubility under acidic condi-
tion. Determination of ROS production and in vitro cytotoxicity
evaluation in the presence of the NQO1 inhibitor DIC confirmed
that these compounds exerted their antitumor activity through
NQO1-mediated ROS production via redox cycling. Moreover, high-
lighting compound 3k (named as DDO-7132) exhibited compara-
ble anticancer activity and superior safety profile as compared to
b-lap both in vitro and in vivo, which might be a promising and
novel candidate. Supported by these investigations, current efforts
in our group are focus on generation of novel ortho-quinone ana-
logs with enhanced and potent antitumor activity.
6.25 g NaN3 was dissolved in 15 mL H2O and acidified with 5 mL
glacial acetic acid. The NaN3 solution was added to a solution of
1,4-naphthoquinone (5 g, 29 mmol) dissolved in 100 mL of
THF/H2O (4: 1) and stirred at room temperature. After 8 h, the
reaction was poured into cold water and then the precipitate was
obtained. The reddish brown residue was recrystallized from etha-
nol to yield 4.0 g 5 (80% yield). Mp 105–106 °C, 1H NMR (300 MHz,
DMSO) d: 7.95–7.70 (m, 4H), 7.24 (s, 2H), 5.82 (s, 1H), EI-MS: 173.
4.1.2. 2-Methyl-5-hydroxynaphtho[2,1-d]oxazole (6)
2-Amino-1,4-naphthoquinone 5 (500 mg, 2.8 mmol) was dis-
solved in acetic acid (10 mL), after which a 33% solution of HBr
in acetic acid (0.5 mL) and the aldehyde (14.0 mmol) were added
dropwise. The reaction mixture was stirred at room temperature
for 6–12 h. After completion the reaction mixture was diluted with
water and extracted with diethyl ether. The combined organic
extracts were washed with aqueous sodium bicarbonate, aqueous
sodium sulfite and brine. Drying over MgSO4, followed by filtration
and concentration in vacuo gave the crude product. The product
was ready for the next step without the further purification.
4. Experimental
4.1. Chemistry
All reagents were purchased from commercial sources. Organic
solutions were concentrated in a rotary evaporator (BüchiRotava-
por) below 55 °C under reduced pressure. Reactions were moni-
tored 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
4.1.3. 2-Ethyl-5-hydroxynaphtho[2,1-d]oxazole (7)
2-Amino-1,4-naphthoquinone 5 (500 mg, 2.8 mmol) was dis-
solved in acetic acid (10 mL), after which a 33% solution of HBr
in acetic acid (0.5 mL) and the propionaldehyde (14.0 mmol) were
added dropwise. The reaction mixture was stirred at room temper-
ature for 6 h. After completion the reaction mixture was diluted
with water and extracted with diethyl ether. The combined organic
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X. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
extracts were washed with aqueous sodium bicarbonate, aqueous
sodium sulfite and brine. Drying over MgSO4, followed by filtration
and concentration in vacuo gave the crude product. The product
was ready for the next step without the further purification.
7.58 (t, J = 7.8 Hz, 1H), 3.83 (s, 2H), 2.45 (s, 6H). IR (m
, cmꢁ1):
3476, 3414, 2962, 1678, 1585, 1219, 1048, 911, 473. ESI-HRMS
m/z [M+NH4]+ calculated for C14H16N3O3: 274.1186, found:
274.2744.
4.1.4. 2-Chloromethyl-5-hydroxynaphtho[2,1-d]oxazole (8)
2-Amino-1,4-naphthoquinone 5 (5 g, 28 mmol) was dissolved
in acetic acid (20 mL), after which a 33% solution of HBr in acetic
acid (3 mL) and the chloroacetaldehyde (140 mmol) were added
dropwise. The reaction mixture was stirred at room temperature
for 12 h. After completion the reaction mixture was diluted with
water and extracted with diethyl ether. The combined organic
extracts were washed with aqueous sodium bicarbonate, aqueous
sodium sulfite and brine. Drying over MgSO4, followed by filtration
and concentration in vacuo gave the crude product. The product
was ready for the next step without the further purification.
4.1.6.2.
2-((Morpholino)methyl)naphtho[2,1-d]oxazole-4,5-
dione (3d). Yield: 53%. Red solid, mp 170–172 °C. 1H NMR
(300 MHz, CDCl3) d: 8.15 (d, J = 7.4 Hz, 1H), 7.77–7.69 (m, 2H),
7.57 (t, J = 7.1 Hz, 1H), 3.84 (s, 2H), 3.74 (s, 4H), 2.66 (s, 4H). 13C
NMR (75 MHz, CDCl3) d: 182.5, 178.4, 167.2, 135.1, 131.9, 130.9,
130.6, 130.4, 128.3, 125.4, 122.8, 67.6, 54.2, 52.8. IR (m
, cmꢁ1):
3474, 3415, 2926, 2361, 2342, 1690, 1402, 1108, 784. ESI-HRMS
m/z [M+H]+ calculated for C16H15N2O4: 299.1026, found: 299.1033.
4.1.6.3.
2-(((2-Methoxyphenyl)amino)methyl)naphtho[2,1-d]
oxazole-4,5-dione (3e). Yield: 41%. Dark red solid, mp 181–
183 °C. 1H NMR (300 MHz, CDCl3) d: 8.16 (d, J = 7.7 Hz, 1H),
7.74–7.71 (m, 2H), 7.60 (t, J = 6.9 Hz, 1H), 6.93–6.89 (m, 4H), 4.73
4.1.5. General procedure for the preparation of compounds 3a,
3b and 9
(s, 2H), 3.92 (s, 3H). IR (m
, cmꢁ1): 3418, 2924, 2850, 1672, 1604,
To a solution of naphthol (6–8, 1 mmol) in acetone (10 mL) was
1591, 1272, 1218, 1037, 910, 771, 473. ESI-HRMS m/z [M+H]+ cal-
added a solution of Fremy’s salt in KH2PO4 buffer (0.65 molꢀLꢁ1
,
culated for C19H15N2O4: 335.1026, found: 335.1031.
30 mL). The reaction mixture was stirred at room temperature
for 2–6 h. After that, the acetone was evaporated under reduced
pressure, giving a red precipitate. The precipitate was then col-
lected and purified by recrystallization from ethanol to give the
products (3a, 3b and 9).
4.1.6.4.
2-(((3-Methoxyphenyl)amino)methyl)naphtho[2,1-d]
oxazole-4,5-dione (3f). Yield: 34%. Dark red solid, mp 193–
194 °C. 1H NMR (300 MHz, CDCl3) d: 8.15 (d, J = 7.8 Hz, 1H),
7.71–7.68 (m, 2H), 7.56 (t, J = 5.3 Hz, 1H), 7.13 (t, J = 7.9 Hz, 1H),
6.36 (d, J = 8.2 Hz, 2H), 6.30 (s, 1H), 4.60 (s, 2H), 3.78 (s, 3H). IR
4.1.5.1. 2-Methylnaphtho[2,1-d]oxazole-4,5-dione (3a). Yield:
82%. Red solid, mp 180–181 °C. 1H NMR (300 MHz, CDCl3) d: 8.17
(d, J = 7.5 Hz, 1H), 7.72–7.70 (m, 2H), 7.60–7.54 (m, 1H), 2.67
(s, 3H). 13C NMR (75 MHz, CDCl3) d: 179.8, 175.1, 161.6, 139.3,
(m
, cmꢁ1): 3415, 2922, 2851, 1669, 1602, 1590, 1262, 1213, 1039,
910, 771, 469. ESI-HRMS m/z [M+H]+ calculated for C19H15N2O4:
335.1026, found: 335.1029.
137.5, 127.3, 124.2, 123.6, 122.7, 119.8, 119.3, 11.7. IR (
m
, cmꢁ1):
4.1.6.5.
2-(((4-Methoxyphenyl)amino)methyl)naphtho[2,1-d]
3415, 2361, 1683, 1638, 1592, 1561, 1228, 1065, 1002, 839, 581.
ESI-HRMS m/z [M+Na]+ calculated for C12H7NO3Na: 236.0318,
found: 236.0312.
oxazole-4,5-dione (3g). Yield: 30%. Dark red solid, mp 178–
180 °C. 1H NMR (300 MHz, CDCl3) d: 8.14 (d, J = 7.8 Hz, 1H), 7.68
(s, 2H), 7.57–7.55 (m, 1H), 6.79–6.73 (m, 4H), 4.57 (s, 2H), 3.74
(s, 3H). 13C NMR (75 MHz, CDCl3) d: 181.9, 179.2, 163.9, 152.5,
151.3, 139.8, 135.1, 130.9, 130.7, 129.1, 126.2, 125.2, 122.6,
4.1.5.2. 2-Ethylnaphtho[2,1-d]oxazole-4,5-dione (3b). Yield: 70%.
Dark red solid, mp 190–192 °C. d: 8.09 (d, J = 7.8 Hz, 1H), 7.63
(d, J = 3.8 Hz, 2H), 7.50–7.45 (m, 1H), 2.90 (q, J = 7.6 Hz, 2H), 1.39
114.5, 114.2, 55.2, 41.9. IR (m
, cmꢁ1): 3414, 2360, 2341, 1662,
1618, 1512, 1238, 1050, 911, 474. ESI-HRMS m/z [M+H]+ calculated
for C19H15N2O4: 335.1026, found: 335.1031.
(t, J = 7.6 Hz, 3H). IR (
m
, cmꢁ1): 3415, 1685, 1562, 1229, 1067,
838, 573. ESI-HRMS m/z [M+Na]+ calculated for C13H9NO3Na:
250.0475, found: 250.0470.
4.1.6.6.
2-(((4-Methyphenyl)amino)methyl)naphtho[2,1-d]
oxazole-4,5-dione (3h). Yield: 35%. Red solid, mp 151–153 °C.
1H NMR (300 MHz, DMSO) d: 7.96 (d, J = 8.6 Hz, 1H), 7.56
(t, J = 7.2 Hz, 1H), 7.67–7.59 (m, 2H), 6.90 (d, J = 8.2 Hz, 2H), 6.60
(d, J = 8.2 Hz, 2H), 4.53 (d, J = 6.3 Hz, 2H), 2.12 (s, 3H). IR
4.1.5.3.
2-Chloromethylnaphtho[2,1-d]oxazole-4,5-dione
(9). Yield: 75%. Red solid, mp 175–177 °C. 1H NMR (300 MHz,
DMSO) d: 8.02 (d, J = 7.4 Hz, 1H), 7.78 (s, 2H), 7.68–7.63 (m, 1H),
5.10 (s, 2H). IR (
m
, cmꢁ1): 3414, 3041, 2985, 2360, 2341, 1683,
(m
, cmꢁ1): 3414, 2919, 1678, 1638, 1521, 910, 468. ESI-HRMS m/z
1641, 1402, 1244, 1213, 1050, 781. ESI-HRMS m/z [M+Na]+ calcu-
[M+H]+ calculated for C19H15N2O3: 319.1077, found: 319.1083.
lated for C12H6ClNO3Na: 269.9928, found: 269.9931.
4.1.6.7. 2-(((4-Chlorophenyl)amino)methyl)naphtho[2,1-d]oxa-
zole-4,5-dione (3i). Yield: 38%. Red solid, mp 175–177 °C. 1H NMR
(300 MHz, DMSO) d: 7.96 (d, J = 7.7 Hz, 1H), 7.56 (t, J = 6.7 Hz, 1H),
7.67–7.59 (m, 2H), 7.11 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 8.6 Hz, 2H),
4.1.6. General procedure for the preparation of compounds
3c–3l
To a mixture of amine (0.48 mmol), KI (10 mg, 0.06 mmol),
K2CO3 (55 mg, 0.40 mmol) in DMF (5 mL) was added naphtho-
quinone 9 (100 mg, 0.40 mmol). The resulting mixture was stirred
at 55 °C for 1–2 h. After being cooled, the mixture was poured into
ice water and the resulting mixture was extracted with EtOAc. The
combined organic layer was washed with brine and dried over
anhydrous Na2SO4, filtered and concentrated to afford a crude
product, which was purified through column chromatography over
silica gel.
4.57 (d, J = 6.1 Hz, 2H). IR (m
, cmꢁ1): 3415, 3316, 2366, 1669,
1618, 1420, 1216, 1048, 912. ESI-HRMS m/z [M+H]+ calculated
for C18H12ClN2O3: 339.0531, found: 339.0532.
4.1.6.8. 2-(((3-Fluorophenyl)amino)methyl)naphtho[2,1-d]oxa-
zole-4,5-dione (3j). Yield: 35%. Red solid, mp 182–184 °C.
1H NMR (300 MHz, DMSO) d: 7.97 (d, J = 7.7 Hz, 1H), 7.62
(t, J = 6.4 Hz, 1H), 7.67–7.57 (m, 2H), 7.11–7.05 (m, 1H), 6.79
(t, J = 6.6 Hz, 1H), 6.52 (d, J = 7.8 Hz, 1H), 6.35 (t, J = 9.5 Hz, 1H),
4.1.6.1. 2-((Dimethylamino)methyl)naphtho[2,1-d]oxazole-4,5-
dione (3c). Yield: 75%. Red solid, mp 162–164 °C. 1H NMR
(300 MHz, CDCl3) d: 8.17 (d, J = 7.5 Hz, 1H), 7.80–7.72 (m, 2H),
4.60 (d, J = 6.3 Hz, 2H). IR (m
, cmꢁ1): 3415, 3326, 2923, 2361,
2342, 1671, 1617, 1212, 1159, 910, 771, 689, 486. ESI-HRMS m/z
[M+Na]+ calculated for C18H12FN2O3: 323.0826, found: 323.0817.
Please cite this article in press as: Li, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.024

X. Li et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
4.1.6.9. 2-((4-Benzylpiperazin-1-yl)methyl)naphtho[2,1-d]oxa-
zole-4,5-dione (3k). Yield: 42%. Red solid, mp 168–169 °C.
1H NMR (300 MHz, CDCl3) d: 8.16 (d, J = 7.5 Hz, 1H), 7.73
(d, J = 8.8 Hz, 2H), 7.58 (t, J = 8.8 Hz, 1H), 7.44–7.28 (m, 5H), 3.87
(s, 2H), 3.54 (s, 2H), 2.70–2.54 (m, 8H). 13C NMR (75 MHz, CDCl3)
d: 179.1, 171.8, 162.5, 158.9, 137.3, 135.1, 133.4, 130.9, 130.6,
128.8, 128.7, 127.7, 126.6, 125.3, 122.8, 62.4, 53.8, 52.4, 52.2. IR
an MMFF94 forcefield. For computational docking, GOLD 5.1 soft-
ware was used in combination with ChemScore scoring function.
The active site was defined as being any volume within 8 Å of
the scaffold of dicoumarol in its crystal pose in 2F1O. The number
of genetic algorithm (GA) run was set to 10, and scoring of the
docked poses was performed with the ChemScore scoring function.
Each GOLD run was saved and the strongest scoring binding pose
of each ligand (subject to a rmsd default distance threshold of
1.5 Å) was compared to that of the reference ligand position
observed in the crystal structure. The best output poses of the
ligands generated were analyzed on the basis of ChemScore, feasi-
bility of hydride transfer process, and H-bonding to the enzyme.
The best poses were visualized with MOE 2013.08.24
(m
, cmꢁ1): 3415, 2919, 2812, 1683, 1587, 1453, 1122, 697, 465.
ESI-HRMS m/z [M+H]+ calculated for C23H22N3O3: 388.1656, found:
388.1656.
4.1.6.10. 2-((4-Phenylpiperazin-1-yl)methyl)naphtho[2,1-d]oxa-
zole-4,5-dione (3l). Yield: 34%. Red solid, mp 174–176 °C. 1H NMR
(300 MHz, CDCl3) d: 8.06–7.98 (m, 1H), 7.90–7.82 (m, 2H), 7.64
(t, J = 6.7 Hz, 1H), 7.52 (t, J = 7.0 Hz, 1H), 7.33–7.23 (m, 2H), 7.01
(d, J = 8.0 Hz, 1H), 6.83 (t, J = 6.9 Hz, 1H), 4.98 (s, 2H), 3.55–3.35
(m, 8H). 13C NMR (75 MHz, CDCl3) d: 180.3, 177.8, 167.0, 150.5,
141.0, 133.0, 132.9, 131.3, 130.4, 129.3, 128.8, 127.8, 126.2,
4.2.4. Cell lines and culture
Human cell lines (NQO1-rich), NCI-H596 and human normal
liver cell L02 were obtained from the American Type Culture
Collection (ATCC, USA). All of these cells were cultured in Roswell
Park Memorial Institute (RPMI) 1640 medium, supplemented with
10% fetal bovine serum (FBS) at 37 °C and 5% CO2.
120.2, 116.1, 54.1, 49.9, 49.2. IR (
1584, 1497, 1228, 1017. ESI-HRMS m/z [M+H]+ calculated for
22H20N3O3: 374.1499, found: 374.1499.
m
, cmꢁ1): 3415, 1736, 1637,
C
4.2.5. Cell viability assay
4.2. Biology
Growth inhibition was determined by the MTT colorimetric
assay. Cells were plated in 96-well plates at
a density of
4.2.1. In vitro NQO1 assay
10,000 cells/mL and allowed to attach overnight (16 h). Cells were
then exposure to various concentrations of test compounds in
triplicate for 2 h, removed, and replaced with fresh medium, and
the plates were incubated at 37 °C under a humidified atmosphere
containing 5% CO2 for 72 h. MTT (50 lg) was added and the cells
were incubated for another 4 h. Medium/MTT solutions were
ortho-Naphthoquinones (0.1–50
l
molꢀLꢁ1) were monitored as
NQO1 substrates using an NADPH recycling assay and recombinant
NQO1 (DT-diaphorase, EC 1.6.5.5, human recombinant, Sigma), in
which NADPH oxidation to NADP+ was monitored by absorbance
(A340 nm) on a Varioskan Flash (Thermo, Waltham, MA). Com-
pounds in DMSO stock (2 lL) were added to a 96 plate. NADPH
removed carefully by aspiration, the MTT formazan crystals were
dissolved in 100 lL of DMSO, and absorbance was determined on
(400
l
molꢀLꢁ1) and NQO1 (1.4
l
g/mL) in 50 mmolꢀLꢁ1 potassium
phosphate buffer (pH = 7.4) were added to each well (198
lL).
a plate reader at 560 nm. When investigating the effect of dicou-
Once the 96-well plate was filled with the assay solutions, except
the NADPH solution, it was placed into the instrument and left to
sit for 3 min before starting the measurements. The enzyme reac-
tion 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 (22–25 °C). The linear portion of the absorbance
vs time graphs (the first 20 s to 1 min) were fitted, and the slops
were calculated (velocity). NADPH oxidation rates were compared
with reactions lacking compound. Initial velocities were calculated
marol, cells were cotreated with vehicle or 25
l
molꢀLꢁ1 dicoumarol
and compound 3k for 2 h with at least three technical replicates.
IC50 values (concentration at which cell survival equals 50% of
control) were determined from semilog plots of percent of control
versus concentration.
4.2.6. Superoxide generation assays
The reduction of ortho-naphthoquinones and the resulting was
monitored by a spectrophotometric assay in which the rate of
reduction of cytochrome c was quantified at 550 nm. Briefly, the
and data expressed as
lmol NADPH oxidized/min/lmol protein.
All reactions were carried out at least in triplicate.
assay mixture contained cytochrome c (30
l
molꢀLꢁ1), reduced
nicotinamide adenine dinucleotide (NADPH; 0.2 mmolꢀLꢁ1),
4.2.2. Physicochemical properties
recombinant human NQO1 (0.1–3.0 lg/mL) (DT-diaphorase, EC
At the neutral solution, the aqueous solubility and LogD7.4 were
determined on a Gemini Profiler instrument (pION) by the ‘gold-
standard’ Avdeef–Bucher potentiometric titration method.25 Under
acidic condition,26 the compounds were treated with the diluted
hydrochloric acid buffer solution, then shaken at 25 °C for 8 h. A
visual inspection was made as to whether the test substance was
fully in solution or not. Compounds which were not completely
1.6.5.5, human recombinant, Sigma), and naphthoquinones
(25
l
molꢀLꢁ1) in a final volume of 1 mL of Tris–HCl (25 mmolꢀLꢁ1
,
pH 7.4) containing 0.7 mg/mL bovine serum albumin (BSA) and
0.1% Tween-20. Reactions were carried out at room temperature
and started by the addition of NADPH. Rates of reduction were cal-
culated from the initial linear part of the reaction curve (0–30 s),
and results were expressed in terms of micromoles of cytochrome
c reduced per minute per milligram of NQO1 by use of a molar
extinction coefficient of 21.1 mMꢁ1ꢀcmꢁ1 for cytochrome c. All
reactions were carried out at least in triplicate.
soluble at 500 lg/mL were diluted to 100 lg/mL and the process
repeated until the compound dissolved.
4.2.3. Molecular modeling
A docking study was performed using the crystal structure of
the human NQO1 complex with dicoumarol (PDB code: 2F1O and
resolution 2.75 Å) and the structure was editor according to pro-
vide a monomer of the protein and protonated use GOLD 5.1. The
ligand was then removed to leave the receptor complex, which
was used for the subsequent docking studies. For preparation of
ligand structures, the selected analogs were energy minimized
using MOE 2013.08 with an MMFF94x forcefield using gas phase
calculations and a cutoff of 0.01. Charges were then fixed using
4.2.7. In vivo antitumor efficacy
Log-phase A549 cells (5 ꢂ 106) were injected into the flanks of
athymic nude mice (aged 7–8 weeks). Tumor sizes were regularly
measured using calipers, and volumes were calculated using the
following formula: volume (mm3) = a ꢂ b2/2, a and b represent
the length and width. Animals were randomized into four groups
(5 mice/group) when the average tumor size reached 100 mm3
for the following treatments: group 1, vehicle; group 2, 3k
(15 mg/kg); group 3, 3k (30 mg/kg), and group 4, b-lap (ARQ501,
Please cite this article in press as: Li, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.01.024

8
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Acknowledgements
We are thankful for the financial support of the National
Natural Science Foundation of China (No. 81302636), the Natural
Science Foundation of Jiangsu Province of China (No.
BK20130656), National Found for Fostering Talents of Basic Science
(NFFTBS) (No. J1030830), and the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD), the
Research Innovation Program for College Graduates of Jiangsu
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