2-Substituted 3-methylnaphtho[1,2-b]furan-4,5-diones as novel L-shaped ortho-quinone substrates for NAD(P)H:quinone oxidoreductase (NQO1)

Article abstract of DOI:10.1016/j.ejmech.2014.05.041

A series of L-shaped ortho-quinone analogs were designed by analyzing the binding mode with NQO1. Metabolic studies demonstrated that compounds 2m, 2n and 2q exhibited higher metabolic rates than β-lapachone. The docking studies, which supported the rationalization of the metabolic studies, constituted a prospective rational basis for the development of optimized ortho-quinone analogs. Besides, good substrates (2m, 2n and 2r) for NQO1 showed higher selective toxicity than β-lapachone toward A549 (NQO1-rich) cancer cells versus H596 (NQO1-deficient) cells. Determination of superoxide (O ₂^?-) production and in vitro cytotoxicity evaluation in the presence of the NQO1 inhibitor dicoumarol confirmed that the ortho-quinones exerted their antitumor activity through NQO1-mediated ROS production by redox cycling. It was suggested that the L-shaped quinone substrates for NQO1 possessed better specificity and safety than β-lapachone.

Full text of DOI:10.1016/j.ejmech.2014.05.041

Accepted Manuscript
2-Substituted 3-methylnaphtho[1,2-b]furan-4,5-diones as novel L-shaped ortho-
quinone substrates for NAD(P)H:quinone oxidoreductase (NQO1)
Jinlei Bian , Bang Deng , Lili Xu , Xiaoli Xu , Nan Wang , Tianhan Hu , Zeyu Yao ,
Jianyao Du , Li Yang , Yonghua Lei , Xiang Li , Haopeng Sun , Xiaojin Zhang ,
Qidong You
PII:
S0223-5234(14)00457-7
10.1016/j.ejmech.2014.05.041
EJMECH 6999
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2014
Revised Date: 12 May 2014
Accepted Date: 13 May 2014
Please cite this article as: J. Bian, B. Deng, L. Xu, X. Xu, N. Wang, T. Hu, Z. Yao, J. Du, L. Yang, Y.
Lei, X. Li, H. Sun, X. Zhang, Q. You, 2-Substituted 3-methylnaphtho[1,2-b]furan-4,5-diones as novel L-
shaped ortho-quinone substrates for NAD(P)H:quinone oxidoreductase (NQO1), European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.041.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
2-Substituted 3-methylnaphtho[1,2-b]furan-4,5-diones as novel L-shaped
ortho-quinone substrates for NAD(P)H:quinone oxidoreductase (NQO1)
a,c,1
c
a,c,1
a,c
a,c
a,c
c
c
Jinlei Bian
, Bang Deng
, Lili Xu , Xiaoli Xu , Nan Wang , Tianhan Hu , Zeyu Yao ,
a,c
a,c
a
a,c
a,d,
Jianyao Du , Li Yang , Yonghua Lei , Xiang Li , Haopeng Sun , Xiaojin Zhang *, Qidong
a,b,c
You
*
a
Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing, 210009,
China
b
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, 210009, China
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing, 210009,
c
China
d
Department of Organic Chemistry, School of Science, China Pharmaceutical University, Nanjing, 210009, China
1
These two authors contributed equally to this work.
*
Corresponding authors. Tel/fax: +86 2583271351 (Q. Y.); tel: +86 2583271216 (X. Z.).
E-mail:
youqidong@gmail.com (Q. You), zhangxiaojin@outlook.com (X. Zhang)
Abstract
A series of L-shaped ortho-quinone analogs were designed by analyzing the
binding mode with NQO1. Metabolic studies demonstrated that compounds 2m, 2n
and 2q exhibited higher metabolic rates than β-lapachone. The docking studies, which
supported the rationalization of the metabolic studies, constituted a prospective
rational basis for the development of optimized ortho-quinone analogs. Besides, good
substrates (2m, 2n and 2r) for NQO1 showed higher selective toxicity than
β-lapachone toward A549 (NQO1-rich) cancer cells versus H596 (NQO1-deficient)
•-
cells. Determination of superoxide (O ) production and in vitro cytotoxicity
2
evaluation in the presence of the NQO1 inhibitor dicoumarol confirmed that the
ortho-quinones exerted their antitumor activity through NQO1-mediated ROS
production by redox cycling. It was suggested that the L-shaped quinone substrates
for NQO1 possessed better specificity and safety than β-lapachone.
Keywords
NQO1 substrates, Ortho-quinones, Antitumor, β-Lapachone, Tanshione IIA
Abbreviations
NQO1, NAD(P)H:quinone oxidoreductase-1; FAD, flavin adenine dinucleotide; ROS,
reactive oxygen species; β-lap, β-lapachone; TSA, tanshinone IIA; MMC, mitomycin
C; STN, streptonigrin; DNQ, deoxynyboquinone; PDB, protein data bank; equiv,
equivalent; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe
nyltetrazolium bromide; NSCLC, non-small cell lung cancer; DIC, dicoumarol; cyt c
,
cytochrome c; MOE, molecular operating environment; SAR, structure-activity
relationship; RMSD, root mean square deviation.
1

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1
. Introduction
NAD(P)H:quinone oxidoreductase-1 (NQO1) is a dimeric flavoprotein that contains
a non-covalently bound molecule of flavin adenine dinucleotide (FAD), and uses the
reduced pyridine nucleotide NADH or NADPH as cofactor to catalyze the direct
two-electron reduction of a wide variety of quinones [1-2]. Structural biology studies
have revealed that NQO1 catalytically cycles using a ping-pong mechanism. In this
mechanism, NAD(P)H binds to NQO1, reduces the FAD cofactor to FADH and is
2
,
+
then released in its oxidized form NAD(P) , allowing the quinone substrate to bind to
the enzyme and to be reduced by FADH . NQO1-directed bioreduction can turn
2
certain quinone substrates into potent cytotoxic compounds through the formation of
unstable hydroquinones that are capable of either alkylating DNA or rapidly
generating reactive oxygen species (ROS) through redox cycling [3]. Due to the
dramatic elevation of NQO1 in many solid tumors and the little to no expression in
normal tissues [4-7], quinone substrates bioreduced by NQO1 to unstable
hydroquinones could be potent and selective antitumor agents, which identifies NQO1
as a promising therapeutic target for cancer therapy [8]. Several examples of NQO1
substrates with potent antitumor activity have already been reported in the literature,
including the alkylators mitomycin C (MMC), EO9, RH1 and AZQ [9-12] and the
redox cycling compounds streptonigrin (STN) [13-14], lavendamycin [15],
deoxynyboquinone (DNQ) [12], β-lapachone (β-lap) [16] and tanshinone IIA (TSA)
[
(
17] (Fig. 1). Structurally, these substrates fall into two main types: para-quinones
Fig. 1A) and ortho-quinones (Fig. 1B). Compared to the structurally diverse
2

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para-quinone substrates [9-15], the reported ortho-quinone substrates for NQO1 are
limited to two natural products, β-lap and TSA. Thus, it is urgent to develop novel and
efficient ortho-quinone substrates for NQO1 as potent NQO1-directed antitumor
agents.
β-Lap (Fig. 1B) is a natural tetrahydropyran-fused ortho-naphanoquinone isolated
from the Bignoniaceae family and is currently in multiple phase II clinical trials for
the treatment of pancreatic adenocarcinoma [18]. Unlike conventional
chemotherapeutic agents, β-lap has been reported to kill many human cancer cells
selectively through rapid ROS generation mediated by NQO1 bioreduction [19].
However, the pyran ring in β-lap is not considered stable as it tends to be hydrolyzed
to form ring-opening metabolic products [20], which could result in toxicity to normal
tissues and cause side effects. Recently, TSA (Fig. 1B), another natural ortho-quinone
isolated from Salvia miltiorrhiza (Danshen, in Chinese), has also been reported as the
substrate for NQO1 [20]. Its structure features a relatively more stable aromatic furan
ring fused to the ortho-naphanoquinone scaffold as compared to β-lap. In view of this,
we initially merged the two natural NQO1 substrates into a new structure of
3
-methylnaphtho[1,2-b]furan-4,5-dione (1) that retained the quinone pharmacophore
for NQO1 metabolism (Fig. 2A). This planar structure 1 was then used as a starting
point in the design of ortho-quinone analogs of β-lap as novel NQO1 substrates.
Subsequently, by analyzing the X-ray crystal structure of human NQO1 in complex
with its potent competitive inhibitor dicoumarol that obtained from the Protein Data
Bank (PDB ID code: 2F1O) [21], we found that the binding site for NQO1 substrates
3

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is a L-shaped pocket. Notably, the bound ligand dicoumarol also has a L-type
molecular shape that fits well with the L-shaped binding pocket of NQO1 (Fig. 2B).
However, the reported ortho-quinone substrates and the merged compound 1 are all
planar molecules. When docking compound 1 into the binding site of NQO1 using
GOLD 5.1 software [22], it has been shown that this planar scaffold only occupied the
bottom of the pocket parallel to FAD, but missed the side binding pocket that formed
by Tyr128, Phe232, Phe236 and His194 residues (Fig. 2C). In view of this, based on
the structural analysis of binding poses of 1 and dicoumarol, we are promoted to
introduce nitrogen-containing side chains at the C2 position of 1 using methylene as a
linker and to develop L-shaped ortho-quinone analogs 2a-2s of β-lap as novel NQO1
substrates (Fig. 2A). The designed L-shaped substrates are expected to have higher
specificity for NQO1 and better drug-like properties than the planar compound 1 and
β-lap. Herein, we now report the details of a study to identify these L-shaped
ortho-quinones 2a-2s as efficient NQO1 substrates and examine the effects of the
introduced side chains on the metabolism by NQO1 (Fig. 2A). Molecular docking has
been applied to analysis the binding modes between the ortho-quinone substrates and
NQO1, and thus to correlate their metabolic rates with predictions of the key
interactions in the catalytic pocket of NQO1. These ortho-quinone analogs of β-lap
have been tested in vitro for their antitumor activity against a pair of NQO1-rich and
NQO1-deficient cancer cell lines. In addition, we attempted to confirm whether the
ortho-quinone analogs exerted their antitumor activity through NQO1-dependent and
ROS-mediated pathways.
4

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2
. Result and discussion
.1 Chemistry
The designed 19 ortho-quinone target compounds 2a-2s were synthesized in 4-5
2
steps from commercially available, inexpensive starting materials (Scheme 1).
Treatment of 1,4-naphthoquinone with enamine 5, which prepared from
propionaldehyde 3 and morpholine, provided the adduct product 6 in 30% yield.
Compound 7 was obtained by heating intermediate 6 in the presence of diluted
hydrochloric acid in 78% yield. Oxidation of 7 using the regent Fremy’s salt provided
the key ortho-quinone intermediate 1 in 80% yield. The target compounds 2a-2i that
substituted with aliphatic amines were obtained directly through Mannich reaction by
the treatment of intermediate 1 with paraformaldehyde and the corresponding
aliphatic amines in the acetic acid. The target compounds 2j-2s that substituted with
aromatic amine side chains were synthesized by stepwise that involved a
chloromethylation of intermediate 1 to form compound 8 and followed by a
nucleophilic substitution with corresponding aromatic amines. All of the compounds
1
were confirmed by H NMR, IR and HRMS (ESI) spectra, and some representative
1
3
structures were also confirmed by C NMR spectrum.
2
.2 Measurement of metabolic rates of the ortho-quinone analogs by NQO1
All the ortho-quinone analogs of β-lap were evaluated for their ability to act as
substrates for NQO1. Although metabolic studies of various para-quinones by NQO1
5

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have been reported, there is little of systematic investigation on that of ortho-quinone
derivatives. To the best of our knowledge, the current work represents the first
detailed study of the metabolism of L-shaped ortho-quinones by NQO1. The ability of
NQO1 to process the ortho-quinone analogs of β-lap in vitro at the concentration of
-1
1
0 ꢀmol·L was assessed. In this assay, the quinone substrate was coincubated with
both human NQO1 as well as NADPH. It was capable of quantifying NADPH
+
oxidation to NADP by monitoring the absorbance (A nm) on an enzyme labeling
3
40
instrument [12]. This method gave initial rates of reduction that could judge directly
whether the compounds were good substrates for NQO1 (Fig. 3). Quinone reduction
was reversible with the decrease of NADPH, so the results (Table 1) were reported as
-1
-1
-1
ꢀ
mol·L NADPH oxidized min ꢀmol NQO1.
As shown in Table 1, generally, most of the L-shaped quinones (2a-2s) possessed
moderate to good metabolic rates by NQO1, indicating that these compounds were
good substrates for NQO1. The planar core scaffold (1) showed reduced metabolic
rate (765 ± 22 ꢀmol NADPH/min/ꢀmol NQO1) as compared to β-lap (1146 ± 22 ꢀmol
NADPH/min/ꢀmol NQO1). It was suggested that the replacement of pyran ring with
more stable furan ring was disadvantage of elevating the metabolic rate by NQO1.
However, most of the C2 substituted L-shaped compounds, such as compounds 2c, 2d,
2
e, 2g and 2k-2s, showed substantially increased metabolic rates by NQO1 relative to
the merged planar compound 1. Among them, compounds 2l, 2m, 2n and 2q showed
even more rapid metabolic rates than β-lap. It suggested that the L-shaped quinones
could have higher specificity for NQO1. These results validated our structure-based
6

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design ideas in one aspect. Besides, when compared to the aliphatic amine series
(
2a-2i) (438-988 ꢀmol NADPH/min/ꢀmol NQO1), the aromatic amine series (2j-2s)
697-1302 ꢀmol NADPH/min/ꢀmol NQO1) showed much higher metabolic rates in
(
general. This phenomenon was possibly due to the fact that the aromatic group served
as a more appropriate moiety to stabilize π-stacking interactions with the key amino
acid residues such as Tyr128 and Phe232 in the catalytic site of NQO1 [23]. Among
these aromatic amine series, interestingly, all of the compounds 2k-2s with halogen,
methyl or methoxy substitution at the benzene moiety exhibited higher metabolic
rates (930-1302 ꢀmol NADPH/min/ꢀmol NQO1) than the compound 2j (697 ± 23
ꢀmol NADPH/min/ꢀmol NQO1) that without any substitution. Furthermore,
compounds 2m and 2q with chloro substitution at the para or meta of the aromatic
amine moiety, showed higher metabolic rates (1248-1232 ꢀmol NADPH/min/ꢀmol
NQO1) than the ortho-substituted substrates 2s (930 ± 95 ꢀmol NADPH/min/ꢀmol
NQO1). The fluoro-substituted compound 2n (1302 ± 180 ꢀmol NADPH/min/ꢀmol
NQO1) was slightly better than the corresponding chloro–substituted compound 2m
(1248 ± 57 ꢀmol NADPH/min/ꢀmol NQO1), indicating that fluoro–substitution was a
better choice for NQO1 substrates. The compounds 2k, 2l and 2r with a methyl or a
methoxy group substituted at the ortho or para position (1021-1248 ꢀmol
NADPH/min/ꢀmol NQO1) showed better metabolic rates than the meta-substituted
compounds 2o and 2p (938-952 ꢀmol NADPH/min/ꢀmol NQO1). This is probably
due to the steric interaction effects of the substituents with the residues of the active
site. The measurement of metabolic rates of the ortho-quinone analogs supported our
7

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understanding of the preliminary structure-activity relationships (SAR) of these
L-shaped
para-fluorobenzylamino substitution exhibited the highest metabolic rate (1302 ± 180
mol NADPH/min/ꢀmol NQO1) by NQO1 and thus it ranked as the best substrate for
ortho-quinone
substrates.
Overall,
compound
2n
with
ꢀ
NQO1. Additionally, over the course of the assay for NQO1 metabolism, it was also
observed that each quinone substrate utilized greater than one equiv of NADPH
(Table 1), demonstrating the ability of the reduced hydroquinones to be oxidized back
to quinones. It suggested that these L-shaped ortho-quninoe analogs of β-lap were all
good substrates for NQO1 and had antitumor potential by ROS generation via redox
cycling (Fig. 3).
Representative compounds 2e, 2m and 2n that showed high metabolic rates at the
-1
concentration of 10 ꢀmol·L were selected to determine their detailed metabolic rates
-1
by NQO1 under various concentrations (0.1-50 ꢀmol·L ). β-Lap was also evaluated
as a comparison group. It was shown that the metabolic rates of these quinones by
NQO1 increased in a concentration-dependent manner (Fig. 4). According to the data
obtained at different concentrations, the corresponding Michaelis-Menten curves for
the four substrates were subsequently generated (Fig. 4), and their apparent catalytic
efficiencies were calculated. As illustrated in Fig. 4, it was observed that compound
6
2
n was a highly efficient substrate and redox cycler with a k /K value of 3.0 × 10
cat M
−
1
−1
6
−1 −1
M ·s , which was processed much faster than β-lap (k /K = 2.6 × 10 M ·s ).
cat
M
6
−1 −1
While compound 2m (k /K = 2.7 × 10 M ·s ) showed a slightly higher rate
cat
M
6
−1 −1
reduced by NQO1 than β-lap. However, for compound 2e (k /K = 2.2 × 10 M ·s )
cat
M
8

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with an aliphatic substitution, exhibited slightly slower metabolic rate than β-lap by
NQO1. Obviously, the trends of metabolic rates of the substrates observed in the
Michaelis-Menten curves were in accord with that obtained at one concentration of 10
-1
ꢀmol·L
2
.3 Molecular binding pattern study of ortho-quinone substrates with NQO1 by
molecular docking
Although several crystal structures of NQO1 in complex with its inhibitors or
para-quinone substrates were available [23-24], none of crystal structure of NQO1
complex that containing an ortho-quinone substrate has ever been reported. Thus, we
were prompted to use in silico modeling approach to gain insights into the structural
requirements for interactions between the ortho-quinone substrates and NQO1. In this
work, molecular docking was used to analysis the binding modes of the ortho-quinone
substrates with NQO1. The ortho-quinone compounds were docked into the active
site of NQO1 (PDB code 2F1O) using CCDCs software GOLD version 5.1 [25-26].
ChemScore function was explored during the docking experiments. The highest
scores represented the best fit for the model.
As shown in Table 2, ChemScore values of the L-shaped compounds were much
higher than the planar compounds 1 and β-lap. Among the L-shaped compounds, the
aromatic amine series (2j-2s) showed higher scores than the aliphatic amine series
(2a-2i). The docking scores were well consistent with the metabolism data, suggesting
that our model was suitable for the prediction of binding affinities between the
9

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ortho-quinone substrates and NQO1. All of these ortho-quinone compounds could be
docked well into the catalytic pocket of NQO1. It indicated that they were all
potential substrates for NQO1. The binding modes of these substrates with NQO1
were carefully analyzed and the results for representative compounds (2a, 2l, 2n and
2
p) were shown in Fig. 5. All of the ortho-quinones oriented with the quinone ring
above the isoalloxazine ring of the bound cofactor FAD as for hydride transfer by
π-stacking interaction, which were similar to the crystal structures of para-quinones
or inhibitors in complex with NQO1 [23-24]. It was found that the two carbonyl
groups in the ortho-quinone substrates could bind firmly to the Tyr126 and Tyr128
residues by hydrogen bonding interactions, which was different from the binding
mode of para-quinone substrates [14]. In addition, there existed an additional pocket
formed by Tyr128, Met 154, Phe232, Phe236 and His194 for the side chains
occupying. As illustrated in Fig. 5, for compounds 2a, 2l, 2n and 2p, the substituted
amine side chains fitted into the additional pocket and the –NH moiety could form
another hydrogen bond with S of Met154, providing a rationale for the better activity
of the L-shaped compounds compared to the planar structure 1. Furthermore, analogs
with side chains of aromatic amines could fit deep into the pocket and make
π-stacking interactions with Tyr128 and Phe232 in the side binding pocket of NQO1.
It explained the fact that the aromatic amine analogs exhibited higher metabolic rates
by NQO1 than the aliphatic amine analogs. Among the aromatic amine series, all of
the compounds 2k-2s with halogen, methyl or methoxy substitution at the benzene
moiety provided better scores than the compound 2j that without any substitution.
1
0

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This could be ascribed to the reduced electron cloud density of the aromatic ring by
introduction of halogens, which resulted in enhanced π-stacking interaction with
corresponding residues Phe232, Phe236 and Tyr128. As for introducing the methyl
group or the methoxy group at the para position of the aromatic amine moiety,
C-H...π interaction with the adjacent Phe236 residue was observed for enhancing
π-stacking interaction with surrounding residues [27]. Moreover, the meta-substituted
compounds 2o and 2p showed slightly worse metabolic rates than the compounds 2k,
2
l, 2r and 2s with substitutions at the ortho or para position, which was probably due
to the steric interaction effects of the substituents with the Met154 residue.
Besides of matching for the catalytic binding site of NQO1, 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) [28]. In the light of this, the corresponding distances between the
ortho-quinones and FAD were calculated and analyzed. As shown in Table 2, all were
within a reasonable distance (about 4
Å) for hydride transfer from the reduced FAD
(
FADH ) to quinone substrates. In addition, the hydrogen bonding interactions of
2
C=O4…Tyr128OH and C=O5…Tyr126OH were suggested to reduce the electron
cloud density of the carbonyl groups, thus promoting the hydride transfer from the
FADH to the ortho-quinone.
2
2
.4 In vitro cytotoxicity evaluation
Cytotoxicity studies were performed on all the ortho-quinone analogs of β-lap with
1
1

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cell
survival
being
determined
by
the
3-(4,
5
-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay
against the human non-small cell lung cancer (NSCLC) cell lines, A549 (NQO1-rich)
and H596 (NQO1-deficient) cells. β-Lap was used as positive control. The MTT assay
was dependent on the reduction of tetrazolium salt by the mitochondrial
dehydrogenase of viable cells to form a blue formazan product dissolved in DMSO
and measured at 560 nm. The results of cytotoxic activity in vitro were expressed as
the IC , and selectivity ratios were defined as IC value for the H596 cell line
5
0
50
divided by IC value for the A549 cell line (Table 3).
5
0
As shown in Table 3, it was found that all the compounds excepted for 2p showed
moderate to potent cytotoxicity against NQO1-rich A549 cell line with IC50 values
-1
ranging from 2.2 to 35 ꢀmol·L . Nearly half of the analogs possessed comparative
-1
cytotoxicity to the positive control β-lap with IC50 values less than 10 ꢀmol·L
against A549 cell line. On the contrary, generally, the compounds showed relative
poor cytotoxicity against NQO1-deficient H596 cell line. It suggested that these
NQO1 substrates were more sensitive to NQO1-rich cancer cells. Interestingly,
compound 1 with slower metabolic rate by NQO1 compared to β-lap also exhibited a
poorer selectivity (selectivity ratio = 1.4) than that of β-lap (selectivity ratio = 4.0).
Nevertheless, most of the L-shaped compounds showed better toxic selectivity to
NQO1-rich A549 cell line compared to the planar compound 1. Furthermore, it was
also observed that the aromatic amine series generally possessed higher selectivity
ratios than the aliphatic amine series. Especially, among the aromatic amine series,
1
2

ACCEPTED MANUSCRIPT
compounds 2m, 2n, 2q and 2r even showed better toxic selectivity to NQO1-rich
A549 cell line versus NQO1-deficient H596 cell line (selectivity ratio = > 5.3)
compared to the control β-lap (selectivity ratio = 4.0). It must be emphasized that
these four ortho-quinones were all efficient substrates for NQO1 with metabolic rates
more than 1000 ꢀmol NADPH/min/ꢀmol NQO1. Notably, compound 2n that showed
the highest metabolic rate also afforded the greatest selectivity to NQO1-rich A549
cell line (more than 8.3-fold). Compound 2n was as potent as the positive control
-1
β-lap against A549 cell line with an IC50 value of 6.0 ± 0.3 ꢀmol·L . Meanwhile,
compound 2n showed much less toxic against H596 cell line even at a high
-1
concentration of 50 ꢀmol·L . These results revealed that good substrates for NQO1
were selectivity toxic towards cell line with high NQO1 activity, which was consistent
with that reported previously in the literature [13]. In addition, it is worth noting that
the L-shaped ortho-quinones including 2n, 2m, 2q and 2r, which showed better
selectivity than β-lap against NQO1-rich cancer cells, were supposed to have a
superior safety profile since there was little or no NQO1 expressed in normal tissues.
These novel L-shaped ortho-quinones could be promising NQO1-directed antitumor
agents for further preclinical studies.
2
.5 Determination of ROS production
NQO1 substrates have been suggested to arise their antitumor activity through
rapid ROS generation [20]. We have found that each ortho-quinone substrate utilized
greater than one equiv NADPH (Table 1), demonstrating their ability of the redox
1
3

ACCEPTED MANUSCRIPT
cycling. Considering that the redox cycling could led to ROS generation (Fig. 3), we
thus selected representative compounds (2m, 2n and 2q) with efficient metabolic rates
to further identify their abilities to produce ROS. Menadione, a well-studied
para-quinone substrate for NQO1 was used as positive control [30]. The production
•-
of the superoxide (O ), the main constitute of ROS, was measured by a
2
spectrophotometric assay using cytochrome c as the terminal electron acceptor [31].
The initial rates (ꢀmol cytochrome c reduced/min/mg NQO1) were calculated from
the liner portion (0-30 s) of the reduction graphs. The highest value represented it can
produce maximum amount of ROS. As shown in Fig.6, the L-shaped ortho-quinones
2
m, 2n and 2q showed rapid rates of ROS generation, being much more efficient than
β-lap. Compound 2n with the highest metabolic rate by NQO1 (1302 ± 180 ꢀmol
NADPH/min/ꢀmol NQO1) also exhibited the highest rate of ROS production (1198 ±
2
0 ꢀmol cyt c/min/mg NQO1) (Fig. 6). In fact, the rate of ROS production for
compound 2n was comparable to that of the positive control menadione (1315 ± 25
mol cyt c/min/mg NQO1). The results indicated that these ortho-quinone substrates
could generate a high level of ROS via NQO1-directed redox cycling.
ꢀ
2
.6 Antitumor effect of 2n is NQO1-dependent and ROS-mediated
The best selective compound 2n was further tested in the presence of the NQO1
-1
inhibitor dicoumarol (DIC, 25 ꢀmol·L ) for its rate of ROS production and toxicity
against A549 cell line (Fig. 7). DIC has been widely used in studies of
NQO1-mediated cell death, as incubation of cells with the inhibitor was effective in
1
4

ACCEPTED MANUSCRIPT
blocking the enzymatic activity of NQO1 [12]. As illustrated in Fig. 7A, coincubation
with DIC dramatically reduced the rate of ROS production by 2n. Fig. 7B showed
that coincubation with DIC greatly protected A549 cells from the cell death mediated
by 2n, shifting the IC > 12-fold. (The fold is the ratio of the IC of cotreatment with
5
0
50
quinone and DIC to the IC50 of treatment with only quinone, and a higher ratio
indicates greater protection and greater NQO1 specificity). At the concentration of 10
-1
ꢀ
mol·L , 2n killed about half of the cells while scarcely dead cells were observed in
the presence of NQO1 inhibitor dicoumarol. The results suggested that the
ortho-quinone analogs exerted their antitumor activity through NQO1-dependent and
ROS-mediated pathways.
3
. Conclusion
In summary, a key scaffold of 3-methylnaphtho[1,2-b]furan-4,5-diones was merged
from two natural ortho-quinone products, β-lap and TSA. Based on the scaffold, a
series of L-shaped ortho-quinone analogs with substituted side chains at the position
of C2 were designed by analyzing the binding mode with NQO1. These
ortho-quinones have been characterized and evaluated as efficient substrates for
recombinant human NQO1. The C2-substituted L-shaped compounds showed
substantially increased metabolic rates by NQO1 relative to the planner structure 1
(
765 ± 22 ꢀmol NADPH/min/ꢀmol NQO1). Compounds 2m, 2n and 2q with halogen
substitution at the meta or para of aromatic amines exhibited higher metabolic rates
1248 to 1302 ꢀmol NADPH/min/ꢀmol NQO1) than the control β-lap (1146 ± 23
(
1
5

ACCEPTED MANUSCRIPT
ꢀmol NADPH/min/ꢀmol NQO1). Molecular modeling was used to analyze the
interactions between the ortho-quinone substrates and NQO1. Our docking studies
supported the rationalization of the metabolic study and constituted a prospective
rational basis for the development of optimal ortho-quinone analogs. Besides, good
substrates (2m, 2n and 2r, metabolism rates > 1000 ꢀmol NADPH/min/ꢀmol NQO1)
for NQO1 showed higher selective toxicity than β-lap toward A549 (NQO1-rich) cell
•-
line versus H596 (NQO1-deficient) cell line. Determination of superoxide (O )
2
production and in vitro cytotoxicity evaluation in the presence of the NQO1 inhibitor
dicoumarol confirmed that the ortho-quinone analogs of β-lap exerted their antitumor
activity through NQO1-mediated ROS production via redox cycling. Compound 2n
(named as YK-01), in which an encouraging improvement of better enzyme activity,
more selectivity and superior safety profile as compared to β-lap, can be considered as
a lead chemotype worth of further investigations toward the design and synthesis of
novel NQO1-directed antitumor ortho-quinones. These findings encourage further
investigations around this interesting antitumor chemotype.
4
. Experimental
4
.1 Chemistry
All reagents were purchased from commercial sources. Organic solutions were
concentrated in a rotary evaporator (BüchiRotavapor) 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
1
6

ACCEPTED MANUSCRIPT
1
13
determined with a Melt-Temp II apparatus. The H NMR and C 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 spectrometer. Analytical results are within
0
.40% of the theoretical values.
4
.1.1 (E)-4-(Prop-1-en-1-yl)morpholine (5).
To a stirred mixture of morpholine (26 mL, 0.294 mol) and anhydrous K CO (2.0
2
3
o
g, 14.7 mmol) was added dropwise propanal (9 mL, 0.125 mol) at 0 C. The
o
suspension was stirred at 0 C for 2 h and then warmed to room temperature in about
1
h. The reaction mixture was filtered and washed with dimethyl ether (2 × 20 mL).
The filtration was concentrated to give a dark color liquid, which was purified by
vacuum distillation to give the titled compound as a colorless liquid (7.0 g, 45%). b.p.
o
1
8
8-90 C/25 mm [32]. H NMR (300 MHz, DMSO) δ: 5.83 (d, 1H, J = 15 Hz), 4.38
(dq, 1H, J = 12 Hz, 9 Hz), 3.58-3.50 (m, 4H), 2.85-2.75 (m, 2H), 2.52-2.44 (m, 2H),
+
1
.60-1.53 (m, 3H). m/z (EI-MS): 127 [M] .
4
.1.2. 3-Methyl-2-morpholino-5-hydroxy-2,3-dihydronaphtho[1,2-b]furan (6).
To a solution of naphthoquinone 3 (7.3 g, 46.25 mmol) in dry dichloromethane
o
(
150 mL) was added 5 (5.9 g, 46.25 mmol) at 0 C. The reaction mixture was then
17

ACCEPTED MANUSCRIPT
warmed to room temperature and kept for 12 h. The precipitate was filtered, washed
with dichloromethane (2 × 10 mL), and dried to give the titled compound as a while
o
1
solid (4.0 g, 30%). m.p. 141-143 C. H NMR (300 MHz, DMSO) δ: 9.48 (s,1H), 8.03
(d, 1H, J = 7.8 Hz), 7.76 (d, 1H, J = 7.7 Hz), 7.44-7.35 (m, 2H), 6.70 (s, 1H), 5.11 (d,
1
2
H, J = 4.8 Hz), 3.54 (s, 4H), 3.43-3.40 (m, 1H), 2.82-2.75 (m, 2H), 2.57-2.53 (m,
+
H), 1.28 (d, 3H, J = 6.9 Hz). m/z (EI-MS): 285 [M] .
4
.1.3. 3-Methyl-5-hydroxynaphthol[1,2-b]furan (7).
To a solution of 6 (3.5 g, 12.37 mmol) in THF (20 mL) was added diluted
hydrochloric acid (5%, 90 mL). The reaction mixture was refluxed for 2 h, and then
cooled and extracted by ethyl acetate (3 × 30 mL). The organic layers were combined,
dried over anhydrous Na SO and concentrated to give a light pink solid, which was
2
4
further purified by recrystallization from dichloromethane to give the titled compound
o
1
as a white solid (1.9 g, 78%). m.p. 145-147 C. H NMR (300 MHz, CDCl ) δ:
3
8
.25-8.22 (m, 2H), 7.61 (t, 1H, J = 8.0 Hz), 7.53-7.47 (m, 2H), 6.94 (s, 1H), 5.06 (s,
+
1
H), 2.27 (s, 3H). m/z (EI-MS): 198 [M] .
4
.1.4. 3-Methylnaphtho[1,2-b]furan-4,5-dione (1).
To a solution of 7 (400 mg, 2.03 mmol) in acetone (10 mL) was added a 0.65 M
potassium dihydrogenphosphate aqueous solution (100 mL). The reaction mixture
was stirred at room temperature for 12 h. After that, the acetone was evaporated under
reduced pressure, giving a red precipitate. The precipitate was then collected and
1
8

ACCEPTED MANUSCRIPT
purified by recrystallization from ethanol to give the titled compound as a red solid
o
1
(
2.2 g, 80%). m.p. 170-172 C. H NMR (300 MHz, CDCl ) δ: 8.09 (d, 1H, J = 7.8
3
-1
Hz), 7.69-7.62 (m, 2H), 7.47 (t, 1H, J = 9.0 Hz), 2.31 (s, 3H). IR (ν, cm ): 3134, 1672,
+
1
590, 1539, 1020. ESI-HRMS m/z [M+Na] calculated for C H O Na: 235.0371,
1
3
8
3
found: 235.0374.
4
0
.1.5. General procedure for the preparation of compounds 2a-2i.
A mixture of naphthoquinone 1 (130 mg, 0.61 mmol), paraformaldehyde (73 mg,
.244 mmol) and the corresponding aliphatic amines (1.22 mmol) in acetic acid (10
mL) was stirred at room temperature until the initial reactant 1 disappeared by TLC
control. Then, the solvent was vacuum-evaporated and the residue was redissolved in
ethyl acetate. The organic layer was washed with saturated NaHCO aqueous and
3
brine, dried over anhydrous Na SO , filtrated and concentrated to afford a crude
2
4
product, which was purified through column chromatography over silica gel.
4
.1.5.1. 2-((Dimethylamino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2a).
Following the above procedure, treatment of 1 with dimethylamine for 12 h under
reflux, gave the crude product, which was purified by column chromatography (eluent:
Petroleum ether/EtOAc 10:1) to afford 2a as an orange solid (51 mg, 31%). m.p.
o
1
1
1
40-142 C. H NMR (300 MHz, DMSO) δ: 7.89 (d, J = 7.6 Hz, 1H), 7.69 (d, J =
1
3
0.2 Hz, 2H), 7.51 (d, J = 7.5 Hz, 1H), 3.48 (s, 2H), 2.18 (s, 9H). C NMR (75 MHz,
CDCl ) δ: 180.3, 175.1, 159.1, 150.5, 134.7, 129.8, 129.4, 128.3, 128.1, 122.0, 121.1,
3
1
9

ACCEPTED MANUSCRIPT
-1
1
18.8, 52.6, 44.6, 8.4. IR (ν, cm ): 2933, 1667, 1581, 1214, 906. ESI-HRMS m/z
+
[
M+H] calculated for C H NO : 270.1130, found: 270.1132.
16 16 3
4
.1.5.2. 2-((Diethylamino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2b).
Following the above procedure, treatment of 1 with dimethylamine for 12 h under
reflux, gave the crude product, which was purified by column chromatography (eluent:
Petroleum ether/EtOAc 2:1) to afford 2b as a red solid (65 mg, 36%). m.p. 149-151
o
1
C. H NMR (300 MHz, DMSO) δ: 7.90 (d, J = 7.4 Hz, 1H), 7.72-7.63 (m, 2H), 7.50
(t, J =7.5 Hz, 1H), 3.66 (s, 2H), 2.52 (dd, J = 6.7, 4.6 Hz, 4H), 2.19 (s, 3H), 1.05-1.00
1
3
(
m, 6H). C NMR (75 MHz, CDCl ) δ: 180.1, 175.1, 159.2, 151.0, 134.8, 129.8,
3
-1
1
29.4, 128.3, 128.1, 121.9, 121.0, 118.8, 58.7, 46.4, 11.2, 8.5. IR (ν, cm ): 2918, 1675,
+
1
581, 1216, 1109. ESI-HRMS m/z [M+H] calculated for C H NO : 298.1443,
1
8
20
3
found: 298.1439.
4
.1.5.3. 2-((Dipropylamino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2c).
Following the above procedure, treatment of 1 with dipropylamine for 6 h under
reflux, gave the crude product, which was purified by column chromatography (eluent:
o
Petroleum ether/EtOAc 4:1) to afford 2c as a red solid (54 mg, 27%). m.p. 153-155 C.
1
H NMR (300 MHz, CDCl ) δ: 8.07 (d, J = 7.7 Hz, 1H), 7.71-7.61 (m, 2H), 7.44 (t, J
3
=
7.5 Hz, 1H), 3.68 (s, 2H), 2.49-2.44 (m, 4H), 2.29 (s, 3H), 1.55 (dq, J = 14.7, 7.4 Hz,
1
3
4
H), 0.93 (t, J = 7.3 Hz, 6H). C NMR (75 MHz, CDCl ) δ: 180.4, 175.1, 159.1,
3
1
49.9, 134.8, 129.8, 129.3, 128.3, 128.1, 121.7, 121.2, 119.3, 55.3, 47.1, 19.5, 11.3,
2
0

ACCEPTED MANUSCRIPT
-1
+
8
.5. IR (ν, cm ): 3414, 2959, 1673, 1554, 1216, 1081, 905. ESI-HRMS m/z [M+H]
calculated for C H NO : 326.1756, found: 326.1752.
2
0
24
3
5
.1.5.4. 2-(Piperidin-1-ylmethyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2d).
Following the above procedure, treatment of 1 with piperidine for 12 h under reflux,
gave the crude product, which was purified by column chromatography (eluent:
Petroleum ether/EtOAc 10:1) to afford 2d as a red solid (38 mg, 20%). m.p. 154-157
o
1
C. H NMR (300 MHz, CDCl ) δ: 8.06 (d, J = 7.7 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H),
3
7
3
1
2
3
.63 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.71 (s, 2H), 2.64 (s, 4H), 2.30 (s,
1
3
H), 1.85 (s, 6H). C NMR (75 MHz, CDCl ) δ: 180.4, 175.1, 159.0, 150.4, 134.7,
3
-1
29.8, 129.3, 128.3, 128.2, 122.0, 121.2, 118.9, 53.7, 52.4, 25.3, 23.5, 8.6. IR (ν, cm ):
+
935, 1669, 1650, 1216, 907. ESI-HRMS m/z [M+H] calculated for C H NO :
19
20
3
10.1443, found: 310.1449.
4
.1.5.5. 2-(Morpholinomethyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2e).
Following the above procedure, treatment of 1 with morpholine for 12 h under
reflux, gave the crude product, which was purified by column chromatography (eluent:
o
Petroleum ether/EtOAc 4:1) to afford 2e as a red solid (57 mg, 30%). m.p. 170-171 C.
1
H NMR (300 MHz, CDCl ) δ: 8.05 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.62
3
(t, J = 4.8 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 3.73 (s, 4H), 3.58 (s, 2H), 2.54 (s, 4H),
1
3
2
1
.28 (s, 3H). C NMR (75 MHz, CDCl ) δ: 180.2, 174.9, 159.0, 149.6, 134.8, 129.8,
3
-1
29.4, 128.2, 128.0, 121.9, 121.1, 119.2, 66.3, 52.7, 52.0, 8.5. IR (ν, cm ): 3432,
2
1

ACCEPTED MANUSCRIPT
+
2
857, 1668, 1578, 1111, 908. ESI-HRMS m/z [M+Na] calculated for C H NO Na:
1
8
17
4
3
34.1055, found: 334.1057.
4
.1.5.6. 2-(Pyrrolidin-1-ylmethyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2f).
Following the above procedure, treatment of 1 with tetrahydropyrrole for 12 h
under reflux, gave the crude product, which was purified by column chromatography
eluent: Petroleum ether/EtOAc 4:1) to afford 2f as a dark red solid (36 mg, 20%).
(
o
1
m.p. 150-151 C. H NMR (300 MHz, CDCl ) δ 8.06 (d, J = 7.7 Hz, 1H), 7.76 (d, J =
3
7
4
1
.7 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.71 (s, 2H), 2.64 (s,
1
3
H), 2.30 (s, 3H), 1.85 (s, 4H). C NMR (75 MHz, CDCl ) δ: 180.2, 175.1, 159.2,
3
48.4, 134.8, 129.8, 129.5, 128.3, 128.0, 122.0, 121.1, 118.8, 53.3, 48.4, 23.0, 8.5. IR
-1
+
(
ν, cm ): 3221, 2757, 1678, 1578, 1032, 900. ESI-HRMS m/z [M+H] calculated for
C H NO : 296.1287, found: 296.1292.
18
18
3
4
.1.5.7. 2-((4-Methylpiperazin-1-yl)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione
2g).
Following the above procedure, treatment of 1 with N-methylpiperazine for 12 h
under reflux, gave the crude product, which was purified by column chromatography
(
(eluent: Petroleum ether/EtOAc 4:1) to afford 2g as a red solid (30 mg, 15%). m.p.
o
1
1
60-162 C. H NMR (300 MHz, CDCl ) δ: 8.06 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 7.0
3
Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 3.62 (s, 2H), 2.52-2.39 (m,
13
4
H), 2.32-2.29 (m, 7H), 2.10 (s, 3H). C NMR (75 MHz, CDCl ) δ: 180.2, 174.9,
3
2
2

ACCEPTED MANUSCRIPT
1
4
59.0, 150.0, 134.7, 129.8, 129.4, 128.2, 128.0, 121.9, 121.1, 119.1, 54.3, 52.1, 51.6,
-1
5.3, 8.5. IR (ν, cm ): 3423, 2795, 1667, 1577, 1292, 1216, 907, 786. ESI-HRMS m/z
+
[
M+H] calculated for C H N O : 325.1547, found: 325.1553.
1
9
21
2
3
4
.1.5.8.
2-((4-Acetylpiperazin-1-yl)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione
(
2h).
Following the above procedure, treatment of 1 with N-acetylpiperazine for 12 h
under reflux, gave the crude product, which was purified by column chromatography
(eluent: Petroleum ether/EtOAc 4:1) to afford 2h as a red solid (54 mg, 25%). m.p.
o
1
1
68-170 C. H NMR (300 MHz, CDCl ) δ: 8.07 (d, J = 8.9 Hz, 1H), 7.74 (d, J = 6.6
3
Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 6.6 Hz, 1H), 3.72-3.67 (m, 2H), 3.62 (s,
13
2
H), 3.51 (d, J = 4.5 Hz, 2H), 2.54 (t, J = 9.2 Hz, 4H), 2.28 (s, 3H), 2.10 (s, 3H). C
NMR (75 MHz, CDCl ) δ: 180.2, 174.9, 168.1, 159.0, 149.9, 134.7, 129.8, 129.4,
3
-1
1
28.2, 128.0, 121.9, 121.1, 119.1, 52.1, 51.6, 49.1, 20.8, 8.5. IR (ν, cm ): 3323, 2935,
+
1
700, 1569, 1216, 979. ESI-HRMS m/z [M+H] calculated for C H N O : 353.1501,
2
0
21
2
4
found: 353.1503.
4
.1.5.9. 2-((Cyclohexylamino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2i).
Following the above procedure, treatment of 1 with cyclohexylamine for 12 h
under reflux, gave the crude product, which was purified by column chromatography
eluent: Petroleum ether/EtOAc 4:1) to afford 2i as a dark red solid (55 mg, 28%).
(
o
1
m.p. 163-165 C. H NMR (300 MHz, CDCl ) δ: 8.06 (d, J= 7.2 Hz, 1H), 7.74 (d, J =
3
2
3

ACCEPTED MANUSCRIPT
6
.6 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 6.6 Hz, 1H), 3.87 (s, 2H), 2.50-2.49
13
(
m, 1H), 2.28 (s, 3H), 1.96-1.94 (m, 2H), 1.75-1.67 (m, 4H), 1.26-1.13 (m, 4H). C
NMR (75 MHz, CDCl ) δ: 180.4, 175.1, 159.0, 150.4, 134.7, 129.8, 129.3, 128.3,
3
-1
1
28.2, 122.0, 121.2, 118.9, 57.3, 43.5, 34.7, 25.9, 25.3, 8.6. IR (ν, cm ): 3233, 2915,
+
1
650, 1759, 1134, 988. ESI-HRMS m/z [M+H] calculated for C H NO : 324.1600,
2
0
22
3
found: 324.1601.
4
.1.6. 2-Chloromethyl-3-methylnaphtho[1,2-b]furan-4,5-dione (8).
Gaseous hydrogen chloride was filled into a solution of quinone 1 (100 mg, 0.47
mmol) and paraformaldehyde (138 mg, 4.7 mmol) in ethyl acetate (8 mL). The
o
reaction mixture was stirred at 45-50 C for 5 h. Then, the solvent was
vacuum-evaporated and the residue was redissolved in ethyl acetate. The organic
layer was washed with brine, dried Na SO , filtered and evaporated till dryness,
2
4
affording a crude product, which was purified through column chromatography over
o
1
silica gel to give 98 mg of the red product. Yield 80%, m.p. 158-159 C. H NMR
(
300 MHz, CDCl ) δ: 8.10 (d, J = 7.7 Hz, 1H), 7.77 (d, J = 7.3 Hz, 1H), 7.68 (t, J =
3
7
.5 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 4.79 (s, 2H), 2.34 (s, 3H). ESI-HRMS m/z
+
[
M+H] calculated for C H ClO : 261.0318, found: 261.0313.
1
4
10
3
4
.1.7. General procedure for the preparation of compounds 2j-2s.
To a mixture of aromatic amines (0.46 mmol), KI (10 mg, 0.06 mmol), K CO
2
3
(50 mg, 0.38 mmol) in DMF (5 mL) was added intermediate 8 (100 mg, 0.38 mmol).
2
4

ACCEPTED MANUSCRIPT
o
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 Na SO ,
2
4
filtered and concentrated to afford a crude product, which was purified through
column chromatography over silica gel.
4
.1.7.1. 2-((Phenylamino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione (2j).
o
Following the above procedure, treatment of 8 with aniline for 1 h at 55 C, gave
the crude product, which was purified by column chromatography (eluent: Petroleum
o
1
ether/EtOAc 10:1) to afford 2j as a dark red solid (60 mg, 50%). m.p. 178-179 C. H
NMR (300 MHz, CDCl ) δ: 8.05 (d, J = 6.2 Hz, 1H), 7.63-7.58 (m, 2H), 7.45-7.39 (m,
3
1
H), 7.25-7.19 (m, 2H), 6.78 (t, J = 6.6 Hz, 1H), 6.73-6.70 (m, 2H), 4.35 (s, 2H), 4.02
1
3
(
brs, 1H), 2.32 (s, 3H). C NMR (75 MHz, CDCl ) δ: 180.2, 175.1, 159.2, 151.0,
3
1
3
47.9, 134.8, 129.9, 129.5, 128.9, 128.3, 128.0, 121.7, 121.1, 120.5, 118.8, 113.5,
-1
8.9, 8.4. IR (ν, cm ): 3232, 2823, 2753, 1665, 1551, 1221, 914, 828, 775.
+
ESI-HRMS m/z [M+Na] calculated for C H NO Na: 340.0950, found: 340.0944.
2
0
15
3
4
.1.7.2. 2-(((4-Methoxyphenyl)amino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione
2k).
Following the above procedure, treatment of 8 with p-methoxyphenylamine for 1 h
(
o
at 55 C, gave the crude product, which was purified by column chromatography
eluent: Petroleum ether/EtOAc 10:1) to afford 2k as a dark red solid (29 mg, 22%).
(
2
5

ACCEPTED MANUSCRIPT
o
1
m.p. 185-187 C. H NMR (300 MHz, CDCl ) δ: 8.06 (d, J = 9.0 Hz, 1H), 7.67-7.62
3
(m, 2H), 7.46-7.41 (m, 1H), 6.83 (d, J = 8.1 Hz, 2H), 6.68 (d, J = 6.0 Hz, 2H), 5.12 (s,
1
3
1
H), 4.32 (s, 2H), 3.87 (s, 1H), 3.77 (s, 3H), 2.32 (s, 3H). C NMR (75 MHz, CDCl )
3
δ: 180.2, 174.8, 160.4, 158.5, 149.1, 143.0, 134.8, 129.9, 129.4, 128.3, 128.0, 121.9,
-
1
1
1
3
21.6, 118.7, 114.4, 114.1, 55.0, 39.7, 8.9. IR (ν, cm ): 3466, 2917, 1672, 1579, 1440,
+
246, 848, 771, 594. ESI-HRMS m/z [M+Na] calculated for C H NO Na:
2
1
17
4
70.1055, found: 370.1059.
4
.1.7.3. 2-(((4-Methylphenyl)amino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione
(2l).
Following the above procedure, treatment of 8 with p-methylphenylamine for 1 h at
o
5
5 C, gave the crude product, which was purified by column chromatography (eluent:
Petroleum ether/EtOAc 4:1) to afford 2l as a dark red solid (38 mg, 30%). m.p.
o
1
1
2
2
1
79-181 C. H NMR (300 MHz, CDCl ) δ: 8.05 (d, J = 7.5 Hz, 1H), 7.69-7.60 (m,
3
H), 7.46-7.41 (m, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.1 Hz, 2H), 4.34 (s,
1
3
H), 2.31 (s, 3H), 2.27 (s, 3H). C NMR (75 MHz, CDCl ) δ: 180.1, 174.9, 159.0,
3
50.4, 144.3, 134.8, 130.5, 129.9, 129.4, 129.3, 128.3, 128.0, 121.7, 121.2, 120.4,
-1
1
13.4, 39.3, 19.9, 8.3. IR (ν, cm ): 3308, 2920, 2356, 1655, 1518, 1221, 908, 773,
+
6
3
90. ESI-HRMS m/z [M+Na] calculated for C H NO Na: 354.1106, found:
21 17 3
54.1111.
4
.1.7.4. 2-(((4-Chlorophenyl)amino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione
2
6

ACCEPTED MANUSCRIPT
(2m).
Following the above procedure, treatment of 8 with p-chlorophenylamine for 2 h at 55
o
C, gave the crude product, which was purified by column chromatography (eluent:
Petroleum ether/EtOAc 4:1) to afford 2m as a dark red solid (33 mg, 25%). m.p.
o
1
1
2
2
1
83-184 C. H NMR (300 MHz, CDCl ) δ: 8.05 (d, J = 7.8 Hz, 1H), 7.70-7.62 (m,
3
H), 7.49-7.41 (m, 2H), 7.22 (d, J = 7.5 Hz, 2H), 6.81-6.78 (m, 1H) , 4.37 (s, 2H),
1
3
.29 (s, 3H). C NMR (75 MHz, CDCl ) δ: 180.6, 174.9, 159.1, 150.3, 144.0, 134.8,
3
30.0, 129.6, 128.8, 128.3, 128.1, 126.2, 121.7, 120.6, 118.9, 114.1, 38.9, 8.4. IR (ν,
-1
+
cm ): 3304, 1657, 1597, 1489, 1401, 1262, 908, 826, 771. ESI-HRMS m/z [M+H]
calculated for C H ClNO : 352.0735, found: 352.0734.
2
0
15
3
4
.1.7.5. 2-(((4-Fluorophenyl)amino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione
2n).
Following the above procedure, treatment of 8 with p-fluorophenylamine for 1 h at
(
o
5
5 C, gave the crude product, which was purified by column chromatography (eluent:
Petroleum ether/EtOAc 4:1) to afford 2n as a dark red solid (42 mg, 33%). m.p.
o
1
1
2
3
1
2
1
79-180 C. H NMR (300 MHz, CDCl ) δ: 8.05 (d, J = 7.5 Hz, 1H), 7.68-7.63 (m,
3
H), 7.48-7.42 (m, 1H), 6.97-6.90 (m, 2H), 6.69-6.65 (m, 2H), 4.32 (s, 2H), 2.32 (s,
1
3
1
H). C NMR (75 MHz, CDCl ) δ: 180.0, 174.9, 158.9, 156.0 ( J = 235.1 Hz),
3
CF
2
50.3, 142.8, 134.8, 129.9, 129.5, 128.3, 127.9, 121.6, 121.2, 117.8, 115.4 ( JCF
=
3
-1
2.5 Hz), 113.9 ( J = 7.6 Hz), 39.3, 8.5. IR (ν, cm ): 3332, 2924, 2853, 1663, 1511,
CF
+
221, 904, 818, 771. ESI-HRMS m/z [M+Na] calculated for C H FNO Na:
2
0
14
3
27

ACCEPTED MANUSCRIPT
3
58.0855, found: 358.0862.
4
.1.7.6. 2-(((3-Methylphenyl)amino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-dione
(
2o).
Following the above procedure, treatment of 8 with m-methylphenylamine for 2 h
o
at 55 C, gave the crude product, which was purified by column chromatography
eluent: Petroleum ether/EtOAc 4:1) to afford 2o as a dark red solid (38 mg, 30%).
(
o
1
m.p. 174-175 C. H NMR (300 MHz, CDCl ) δ: 8.05 (d, J = 7.5 Hz, 1H), 7.68-7.60
3
(m, 2H), 7.56-7.41 (m, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.64 (d, J = 7.2 Hz, 1H),
13
6
.57-6.55 (m, 2H), 4.35 (s, 2H), 2.33 (s, 3H), 2.31 (s, 3H). C NMR (75 MHz, CDCl )
3
δ: 180.2, 175.2, 159.1, 150.9, 145.0, 134.8, 134.7, 130.1, 129.9, 129.5, 128.8, 128.6,
-1
1
21.7, 121.2, 119.5, 117.5, 114.0, 110.3, 38.9, 20.9, 8.4. IR (ν, cm ): 3320, 2922,
+
2
842, 1670, 1589, 1219, 908, 691. ESI-HRMS m/z [M+Na] calculated for
C H NO Na: 354.1106, found: 354.1111.
21
17
3
4
.1.7.7. 2-(((3-Methoxyphenyl)amino)methyl)-3-methylnaphtho[1,2-b]furan-4,5-quin-
one (2p).
Following the above procedure, treatment of 8 with m-methoxyphenylamine for 2 h
o
at 55 C, gave the crude product, which was purified by column chromatography
eluent: Petroleum ether/EtOAc 4:1) to afford 2p as a dark red solid (44 mg, 33%).
(
o
1
m.p. 182-183 C. H NMR (300 MHz, CDCl ) δ: 8.04 (d, J = 7.5 Hz, 1H), 7.65-7.62
3
(m, 2H), 7.44-7.42 (m, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.34-6.31 (m, 2H), 6.28 (d, J =
2
8