Potent cytotoxicity of novel L-shaped ortho-quinone analogs through inducing apoptosis

Article abstract of DOI:10.3390/molecules24224138

Twenty-seven L-shaped ortho-quinone analogs were designed and synthesized using a one pot double-radical synthetic strategy followed by removing methyl at C-3 of the furan ring and introducing a diverse side chain at C-2 of the furan ring. The synthetic derivatives were investigated for their cytotoxicity activities against human leukemia cells K562, prostate cancer cells PC3, and melanoma cells WM9. Compounds TB1, TB3, TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC14, TC15, TC16, and TC17 exhibited a better broad-spectrum cytotoxicity on three cancer cells. TB7 and TC7 selectively displayed potent inhibitory activities on leukemia cells K562 and prostate cancer cells PC3, respectively. Further studies indicated that TB3, TC1, TC3, TC7, and TC17 could significantly induce the apoptosis of PC3 cells. TC1 and TC17 significantly induced apoptosis of K562 cells. TC1, TC11, and TC14 induced significant apoptosis of WM9 cells. The structure-activity relationships evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped ortho-quinone analogs.

Full text of DOI:10.3390/molecules24224138

molecules
Article
Potent Cytotoxicity of Novel L-Shaped
Ortho-Quinone Analogs through Inducing Apoptosis
Sheng-You Li 1,2, Ze-Kun Sun 1,2, Xue-Yi Zeng 2,3, Yue Zhang 1,2, Meng-Ling Wang 2,3
,
Sheng-Cao Hu 3, Jun-Rong Song 2,3, Jun Luo 2, Chao Chen 2,3,
*
, Heng Luo 2,3,* and
Wei-Dong Pan 1,2,3,
*
1
Guizhou University, Huaxi Avenue South, Guiyang 550025, China; 13118517665@163.com (S.-Y.L.);
zekunsun@163.com (Z.-K.S.); Zhangyueinsect@163.com (Y.Z.)
State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou Medical University, 3491
Baijin Road, Guiyang 550014, China; xueyizeng@126.com (X.-Y.Z.); menglingwang@yahoo.com (M.-L.W.);
18275365116@163.com (J.-R.S.); 18798628024@163.com (J.L.)
2
3
The Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of
Sciences, 3491 Baijin Road, Guiyang 550014, China; hushengcao0221@163.com
*
Correspondence: cc283818640@163.com (C.C.); luoheng@gzcnp.cn (H.L.); wdpan@163.com (W.D.P.);
Tel.: +86-15597724842 (C.C.); +86-0851-83876210 (H.L.); +86-18985130307 (W.D.P.)
Academic Editor: Qiao-Hong Chen
Received: 10 October 2019; Accepted: 11 November 2019; Published: 15 November 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Twenty-seven L-shaped ortho-quinone analogs were designed and synthesized using a
one pot double-radical synthetic strategy followed by removing methyl at C-3 of the furan ring and
introducing a diverse side chain at C-2 of the furan ring. The synthetic derivatives were investigated
for their cytotoxicity activities against human leukemia cells K562, prostate cancer cells PC3, and
melanoma cells WM9. Compounds TB1
TC15 TC16, and TC17 exhibited a better broad-spectrum cytotoxicity on three cancer cells. TB7 and
TC7 selectively displayed potent inhibitory activities on leukemia cells K562 and prostate cancer cells
PC3, respectively. Further studies indicated that TB3 TC1 TC3 TC7, and TC17 could significantly
induce the apoptosis of PC3 cells. TC1 and TC17 significantly induced apoptosis of K562 cells. TC1
, TB3, TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC14,
,
,
,
,
,
TC11, and TC14 induced significant apoptosis of WM9 cells. The structure-activity relationships
evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains
at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped
ortho-quinone analogs.
Keywords: ortho-quinones; antitumor activity; beta-lapachone; tanshione IIA
1. Introduction
Over several decades, cancer continues to be the most awful disease due to its uncontrolled cell
growth and the fact that it is a dominate killer of human beings worldwide [1]. Especially in China,
millions of deaths have been caused by tumor. The common cancer types in Chinese male, in 2018,
were lung, stomach, colorectum, liver, and esophageal cancer. Additionally, breast, lung, colorectum,
thyroid, and stomach cancer were the common types in Chinese female [
cancer in males and females has increased, however, the incidence of esophageal, stomach, and liver
cancer has decreased between 2000 and 2011 [ ]. Meanwhile, the incidence and mortality of prostate
cancer and bladder cancer in males, together with obesity and hormonal exposure-related cancers,
namely thyroid, breast, and ovarian cancer in females have shown a rising trend [ ]. However, the
2]. The incidence of colorectal
3
3
standardized treatments of cancer, including surgery, chemotherapy, and radiation therapy, show
many limitations, such as severe adverse effects, recurrence, and increasing drug resistance [4].
Molecules 2019, 24, 4138; doi:10.3390/molecules24224138
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4138
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Currently, phytochemicals have become a valuable source of anticancer drugs. Actually, over 75%
of nonbiological anticancer drugs approved are either plant-derived natural products or developed
based on these products [5]. Therefore, natural products have continued to be a hot research topic for
the development of new antitumor drugs [6–9].
Tanshinone IIA is a natural ortho-quinone isolated from the rhizome of Salvia miltiorrhiza Bunge with
antineoplastic activity, such as gastric cancer, breast cancer, osteosarcoma, etc. [10–13]. These various
properties demonstrate that tanshinone IIA is a potential antitumor drug candidate. Furthermore,
beta-lapachone is another natural ortho-quinone which has been reported to selectively kill many
human cancer cells [14], however, the pyran ring of beta-lapachone has been found to be unstable
during metabolism in the human body, and may led to side effects on normal tissues [15
studies have revealed that some tanshinone analogs show similar or stronger antitumor activity when
the ring-A is removed but the furan ring is retained [17 18]. You et al. [19 20] discovered that the binding
site for quinone oxidoreductase-1 (NQO1) substrates was an L-shaped pocket (Figure 1B) which binds
well with tanshinone analogs, and showed higher antitumor activities than the planar compound and
,16]. Recently,
,
,
1
beta-lapachone. Therefore, we surmise that removing methyl at C-3 of the furan ring is more suitable
for the binding site, and we anticipate that a novel L-shaped molecule without methyl at C-3 of the furan
ring could provide better antitumor activities. Considering that some nitrogen, oxygen-substituted,
and amino acid substrates can improve aqueous solubility and antitumor activities [21–25], we have
attempted to introduce a great diversity of oxygen-substituted, nitrogen-containing groups and amino
acids. Thus, in this work, we developed quinone-directed agents by removing methyl at C-3 of the
furan ring and introducing a diverse side chain at C-2 of the furan ring, culminating in the discovery of
a promising scaffold. The inhibitory activity was assessed in vitro using three cell lines including K562,
PC3, and WM9.
O
A)
O
O
O
O
O
O
-lapachone (1)
C-2
n
ce
hai
odu
intr
merge
e c
sid
H
3
3
2
remove methyl
at C-3
X
2
O
O
R
O
O
3
IC50 (A549) = 7.4 ± 1.6 µM
IC50 (H596) = 10 ± 3.5 µM
O
Tanshinone IIA (2)
Figure 1. (A) Structural design strategy and (B) l-shaped pocket. The figure is available in reference [19].

Molecules 2019, 24, 4138
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2. Discussion and Results
2.1. Chemistry
The synthesis of two substituted naphtho[1,2-b]furan-4,5-diones is outlined in Scheme 1. Briefly,
treatment of lawsone with allyl bromide followed by subsequent Claisen rearrangement afforded 7,
6
which was then cyclized to get the ortho-quinone 8 by using Lewis acid NbCl5 at room temperature [26].
O
O
O
OH
O
OH
K2CO3
NbCl5
DMF, 120 oC
DCM, 30 oC
O
6
O
8
O
7
NBS, AIBN
CCl4, 70 oC
O
O
O
O
Br
O
8a
O
8b
NBS, AIBN
CCl4, 70 oC
O
O
O
9
Br
30 oC-45 oC
R2NH2, K2CO3, THF
R1OH, K2CO3, THF
30 oC-45 oC
O
O
O
O
O
H
N
O
O
R2
TC 18 derivatives
R1
TB 9 derivatives
Scheme 1. Synthesis of L-shaped ortho-quinone analogs.
Initially, dealing
8
with N-bromosuccinimide (NBS) and 2,20-azobis(2-methylpropionitrile) (AIBN)
afforded only trace amounts of 8a. Another intermediate, 8b, was obtained by Nelson’s method [27
as shown in Scheme 1. Then, compound was obtained from 8b through a second radical reaction.
Considering the same reaction condition, we successfully got from through a bis-radical reaction.
The brominated intermediate was reacted with substituted phenol or amine to provide ortho-quinone
TC18, respectively. All the structures of ortho-quinone derivatives
]
9
9
8
9
derivatives TB1–TB9 and TC1–
were identified through 1H, 13C, and HRMS.
In summary, we successfully established an effective synthetic strategy, which removed the methyl
at C-3 of the furan ring and introduced diverse side chains at C-2 of the furan ring. In addition, we

Molecules 2019, 24, 4138
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replaced the bromide of
9
with a variety of oxygen-substituted, nitrogen-containing group, and amino
acid by a nucleophilic substitution.
2.2. In Vitro Cytotoxicity Assay
The cytotoxic activities of 5
determined by using three cancer cell lines (Table 1). The results revealed that compounds TB1
TB4 TB6 TC1 TC3 TC5 TC9 TC11 TC12 TC15 TC16, and TC17 showed a broad-spectrum potent
µmol/L of the synthesized l-shaped ortho-quinone analogs were
,
TB3
,
,
,
,
,
,
,
,
,
,
inhibitory activity on the proliferation of the cancer cell lines, with a more than 70% inhibition rate,
and TB7 showed better inhibitory activity on K562 cells as compared with other cells. Moreover, we
observed that TC7 inhibited the growth of PC3 cells more efficiently than other cells.
Table 1. The structures and inhibitory rates after treating cancer cell lines with 5 µmol/L of target
compounds, respectively. Data was presented as the mean ± SD of three independent experiments.
Inhibition (%)
Compounds
R
PC3
K562
WM9
TB1
TB2
92.078 ± 1.885
88.641 ± 3.055
80.287 ± 6.354
58.700 ± 29.309
20.642 ± 16.695
9.216 ± 2.449
TB3
TB4
91.861 ± 2.125
92.224 ± 1.811
83.613 ± 10.448
91.192 ± 1.479
54.153 ± 3.948
87.963 ± 2.430
TB5
TB6
TB7
30.673 ± 54.356
87.165 ± 1.953
−7.664 ± 10.055
49.127 ± 33.985
82.901 ± 1.484
81.835 ± 1.553
64.870 ± 2.894
89.843 ± 2.010
31.754 ± 6.422
TB8
TB9
9.692 ± 11.981
7.191 ± 4.705
15.334 ± 9.484
49.307 ± 3.791
−0.591 ± 1.055
−17.125 ± 11.619
TC1
91.027 ± 0.553
83.717 ± 3.469
84.117 ± 2.686
TC2
−0.437 ± 1.129
25.032±2.579
16.431±1.583
TC3
78.849 ± 13.221
83.518 ± 12.045
81.670 ± 0.994
TC4
TC5
TC6
24.242 ± 18.544
89.676 ± 0.331
43.133 ± 11.878
0.082 ± 15.067
81.950 ± 12.469
31.137 ± 16.045
15.453 ± 1.508
72.452 ± 8.039
63.384 ± 8.949
CO2Me
N
H
TC7
TC8
82.402 ± 4.585
44.842 ± 25.172
30.087 ± 20.307
28.448 ± 30.220
16.191 ± 2.699
0.468 ± 5.166

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Table 1. Cont.
Inhibition (%)
K562
Compounds
TC9
R
PC3
WM9
89.869 ± 1.839
81.545 ± 6.968
83.003 ± 2.806
TC10
TC11
TC12
−6.375 ± 8.094
90.214 ± 0.520
82.522 ± 9.039
−9.749 ± 17.889
85.766 ± 1.737
83.688 ± 8.252
1.005 ± 2.601
85.807 ± 3.752
61.839 ± 3.448
TC13
TC14
TC15
74.681 ± 2.470
4.453 ± 5.002
84.916 ± 1.761
84.251 ± 1.430
84.497 ± 0.876
84.185 ± 0.419
39.955 ± 6.425
90.717 ± 1.184
90.135 ± 1.602
TC16
78.720 ± 7.560
84.323 ± 1.273
90.520 ± 2.108
TC17
TC18
86.637 ± 2.482
83.999 ± 0.943
8.854 ± 12.236
72.021 ± 6.850
1.410 ± 5.093
−5.220 ± 13.979
tanshinone IIA
89.458 ± 1.987
82.215 ± 4.069
85.236 ± 3.654
Paclitaxel
81.589 ± 1.763
91.315 ± 2.467
78.369 ± 6.380
The concentration inhibition curves (Figure 2) were analyzed to calculate the IC50 values of the
selected active compounds. The results indicated there was a dose-dependent trend of the inhibitory
response of all active compounds on three cancer cells for treating 48 h. The IC50 values were
summarized in Table 2 and show that the cytotoxicity of compounds TB3, TC1, TC3, TC7, TC9, and
TC17 on PC3 were better (P < 0.05) than that of the positive control (tanshinone IIA and paclitaxel),
and another active compound exhibited similar activity to that of the positive control. The inhibition
activity of TC1 against the growth of K562 was better than that of the positive control, paclitaxel,
and tanshinone IIA. Compounds TB6, TC1, TC11, TC14, and TC15 inhibited the growth of WM9
better (P < 0.05) than that of tanshinone IIA and paclitaxel. In summary, most of novel L-shaped
ortho-quinone analogs exhibited relatively better cytotoxicity activity as compared with the two
positive controls, which indicated that the analogs containing L-shaped ortho-quinone as the core
structure, possessed stronger anticancer activity. This result provided a preliminary biological activity
basis for the investigation of anticancer candidate agents.

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K562
WM9
PC3
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
110
100
90
80
70
60
50
40
30
20
10
0
TB3
TC1
TC3
TC7
TC9
TC17
TB6
TC1
TB7
TC1
TC17
TC11
TC14
0
2
4
6
8
10
12
0
2
4
6
8
10
12
0
2
4
6
8
10
12
Concentration (μmol/L)
Concentration (μmol/L)
Concentration (μmol/L)
Figure 2. Growth inhibition induced by the active L-shaped ortho-quinone analogs on PC3, K562,
and WM9 cells by MTT assay. The IC50 values ( M) of the compounds were determined according
to these curves at different incubation times. The 100 L tested compounds were added to 96-well
104/well) were incubated for 48 h at 37
µ
µ
microculture plates and 100
µ
L cells (a final concentration of 5
×
◦C. Cell survival was evaluated by MTT assay. The inhibition ratio (%) was calculated as described in
the Methods section. Data was presented as mean ± SD of three independent experiments.
Table 2. IC50 values of selected compounds in vitro.
IC50/µM
K562
Compounds
PC3
WM9
TB1
TB3
TB4
TB6
TB7
2.809 ± 0.413
1.121 ± 0.731 **
3.348 ± 0.347
3.249 ± 0.464
NA
3.157 ± 0.947 **
2.580 ± 0.285 **
3.103 ± 0.702 **
2.964 ± 0.168 *
2.981 ± 0.368 **
4.841 ± 0.301
NA a
3.358 ± 0.297
2.774 ± 0.299 **
NA
TC1
TC3
TC5
TC7
TC9
TC11
TC12
TC13
TC14
0.347 ± 0.290 **
1.778 ± 0.835 **
3.018 ± 0.452
1.507 ± 0.369 **
0.469 ± 0.281 **
2.578 ± 0.957
2.963 ± 0.261
3.433 ± 0.444
NA
3.696 ± 0.492
3.874 ± 0.557
1.914 ± 0.224 **
3.162 ± 3.160
4.323 ± 0.929
0.379 ± 0.138
4.647 ± 0.647 **
3.448 ± 0.224 **
NA
0.406 ± 0.117 **
4.990 ± 0.360
NA
NA
4.194 ± 0.139 **
3.565 ± 0.344 **
4.157 ± 0.677 **
4.719 ± 0.984
3.100 ± 0.320 **
3.644 ± 0.524 **
2.640 ± 0.642 **
1.927 ± 0.414
4.638 + 1.270
2.149 ± 0.406
4.027 ± 0.341
2.127 ± 0.582 **
NA
NA
2.261 ± 0.111 **
3.050 ± 0.230 *
4.324 ± 0.292
NA
TC15
TC16
TC17
tanshinone IIA
Paclitaxel
4.261 ± 0.182
4.835 ± 0.359
Note: * represents p < 0.05 and ** represents p < 0.01, vs. the inhibition of the positive control to the cancer cell lines.
The data represented the average of three independent experiments.
2.3. Structure-Activity Relationships Study
To obtain two series of analogs, we successfully built an effective synthetic strategy by removing
the methyl at C-3 of the furan ring and introducing diverse side chains at C-2 of the furan ring. On the
basis of the cytotoxicity results (Tables 1 and 2), a preliminary structure-activity relationships could
be established. The TB series molecules bearing electron-withdrawing groups or multi-substituted
groups such as compounds TB3, TB4, and TB6 showed a better inhibitory effect on PC3 cell lines,
K562 cell lines, and WM9 cell lines, whereas the TB series molecules bearing alkane groups at the
2-position showed decreased cytotoxicity in PC3 cell lines, K562 cell lines, and WM9 cell lines, such as
compounds TB9. The TC series molecules with electron-withdrawing groups, saturate six-membered
rings, or multi-substituted groups emerged greater inhibitory effects on three cancer cell lines, such as
TC11, TC12, TC15, and TC16, whereas the TC series molecules bearing donating groups or alkane
groups at the 2-position showed reduced cytotoxicity in three cancer cell lines. The structure-activity
relationships evaluation also showed that removing methyl at C-3 of the furan ring and introducing

Molecules 2019, 24, 4138
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diverse side chains at C-2 of the furan ring were good strategies for improving the anticancer activity
of L-shaped ortho-quinone analogs.
2.4. Effects of Active Compounds on Cell Apoptosis
According to the above IC50 values of all active compounds, we selected six active compounds
(TB3
,
TC1
,
TC3
,
TC7
,
TC9, and TC17) for PC3, three active compounds (TB6, TC1, and TC17) for
K562, and four active compounds (TB6
,
TC1 TC11, and TC14) for WM9, based on their higher
,
activities than that of the positive control and better selectivity and, then, studied their effects on
cell apoptosis by microscope observation (Figure 3) and flow cytometry (Figure 4). The microscopic
observations (Figure 3A) showed that the number of PC3 cells was significantly reduced by treatments
with 2.5
µmol/L of TB3, TC1, TC3, and TC7; the apoptotic bodies and cell fragments were significantly
observed as compared with the control group. The PC3 cells treated with TC9 showed that the number
of cells were significantly reduced, while fewer cells died and there were no significant apoptotic bodies.
The PC3 cells treated with TC7 showed a significant decrease in the number of cells, meanwhile, some
cells died, apoptotic bodies appeared obviously, and the morphology of some cells became an irregular
shape of spindle length. Above all, the inhibitory activity of TB3, TC1, TC3, and TC7 may be through
inducing apoptosis, another two compounds may be through different types.
Figure 3. Cont.

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Figure 3. Effects of the active compounds on PC3 (
Cell number and morphological appearance of the two types of cell lines treated with 2.5
A
), K562 (
B
), and WM9 (
C
) cell growth and apoptosis.
mol/L of
µ
active compounds, then, it was observed by a fluorescent inverted microscope after 24 h. Scale bar =
100 µM in all images. All experiments were performed in triplicate.
Figure 4. Cont.

Molecules 2019, 24, 4138
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120
(D)
**
**
100
80
60
40
20
0
**
**
**
**
*
*
*
*
l
l
l
3
1
3
7
9
7
6
1
7
6
1
1
4
o
o
o
r
1
1
1
1
r
r
B
C
C
C
C
B
C
B
C
t
t
t
C
C
C
C
T
T
T
T
T
T
T
T
T
n
n
n
T
T
T
T
o
o
o
C
C
C
PC3
K562
WM9
Figure 4. Effects of the active compounds on PC3 (
A
), K562 (
B
),
and WM9 (
C
) cell growth and apoptosis.
Cell apoptosis induced by the compounds and tested by flow cytometry and the date was analyzed
with Origin Pro 9.0 ( ) and presented as means SEM from at least three independent experiments.
D
±
* p < 0.05, ** p < 0.01 (n = 3) as compared with the control.
The K562 cells treated with TC1 were obviously dead and dispersed, with the appearance of
apoptotic bodies as comparing with the control group (Figure 3B). The cells treated with TB6 had a
significantly reduced number of cells and most cells clumped growth similar to the control cells. For
the cells treated with TC17 we observed both dead cells and fewer clumps of cells. Furthermore, for the
WM9 cell lines treated with TB6, TC1, TC11, and TC14 (Figure 3C), we observed that the cells treated
with TC1 and TC11 were obviously dead with a large number of apoptotic bodies and dispersed cells;
the cells treated with TB6 showed a significant decrease in the number of cells and fewer dead cells.
Observation of the cells treated with TC14 showed that the number of cells was significantly reduced,
while some cells were obviously dead with apoptotic bodies appearing, and the morphology of some
cells also became an irregular shape of spindle length. The above results indicated that TC1 can induce
apoptosis for K562 and WM9 cells to inhibit the growth; TB6, TC11, TC14, and TC17 can jointly inhibit
the proliferation of cell through a variety of mechanisms.

Molecules 2019, 24, 4138
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Flow cytometry analyzed results (Figure 4) confirmed that TB3 (p < 0.01), TC1 (p < 0.01), TC3
(p < 0.01), TC7 (p < 0.01), and TC17 (p < 0.05) could significantly induce the apoptosis of PC3 cells
(Figure 4A), while TC9 did not. TC1 (p < 0.01) and TC17 (p < 0.05) significantly induced apoptosis
of K562 cells (Figure 4B), while TB6 had no significant effect on apoptosis of K562 and WM9 cells.
TC1 (p < 0.01), TC11 (p < 0.05), and TC14 (p < 0.01) could potently induce apoptosis for WM9 cells
(Figure 4C).
3. Materials and Methods
3.1. Instruments and Materials
High-resolution mass spectra (HRMS) were obtained on an electrospray ionization (ESI) mode on
a Bruker ESI-QTOF mass spectrometry. Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker Avance NEO (1H NMR, 600 MHz; 13C NMR, 150 MHz, Bruker, Switzerland) with TMS as an
internal standard. The IR spectra were recorded by using a FTIR Spectrometer (IR 200 Fourier Energy
Spectrum Technology Co., Ltd., TianJin, China) and the KBr disk method was adopted. The melting
points (mp) were determined on an WRX-4 microscope melting point apparatus. The column
chromatography was performed on silica gel (Qingdao, 200–300 mesh) and the thin-layer (0.25 mm)
chromatography (TLC) analysis was carried out on silica gel plates (Qingdao, China). Other reagents
were analytical grade or guaranteed reagent commercial product and used without further purification,
unless otherwise noted.
3.2. Methods of Synthesis
3.2.1. Synthesis of 2-Allyl-3-hydroxy-1,4-naphthoquinone (7)
A mixture of lawsone
6 (10.0 g, 57.42 mmol) and anhydrous K2CO3 (7.94 g, 57.42 mmol) in
anhydrous DMF (100 mL) were stirred for 15 min at room temperature. Allyl bromide (17.37 g,
143.55 mmol) in DMF (5 mL) was added dropwise and stirred for 15 min at 0 ◦C. The mixture was
◦
refluxed at 120 C for 3 h and then cooled to room temperature before it was poured into water
and extracted with EA. The organic phase was washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was purified by column chromatography on silica gel
(eluent: petroleum ether/EtOAc 15:1) to afford 7 (7.8 g, 63% yield) as a light yellow solid. Other data
was found in reference [26].
3.2.2. Synthesis of 2-Methyl-2,3-dihydrolnaphthol[1,2-b]furan-4,5-dione (8)
NbCl5 (18.92 g, 70.02 mmol) was added into (3.0 g, 14.00 mmol) in anhydrous DCM (50 mL)
7
at 0 ◦C. After stirring for 45 min at 30 ◦C, the mixture was poured into ice water and extracted with
DCM. The organic phase was washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The crude product was purified by column chromatography on silica gel (eluent: petroleum
ether/EtOAc 4:1) to afford 8 (2.16 g, 72% yield) as red solid. Other data was found in reference [26].
3.2.3. Synthesis of 2-Bromomethyl-naphtho[1-b]furan-4,5-dione (9)
A mixture of
2,20-azobis(2-methylpropionitrile) (114.98 mg, 1.40 mmol) in anhydrous CCl4 (50 mL) was stirred under
argon at 70 ◦C, until
were disappeared. Then, the mixture was cooled to room temperature, and
8 (1.5 g, 7.00 mmol), anhydrous N-bromosuccinimide (2.49 g, 1.40mmol), and
8
anhydrous N-bromosuccinimide (2.49 g, 1.40 mmol) and 2,20-azobis(2-methylpropionitrile) (114.98 mg,
1.40 mmol) were added and stirred at 70 ◦C for 2 h. The mixture was cooled to room temperature, and
poured into water, extracted with EA, washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The crude product was purified by column chromatography on silica gel (eluent: petroleum
ether/EtOAc 12:1) to afford
CDCl3) 8.09 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H),
9
(1.0 g, 67% yield) as red solid. Mp: 169–170 ◦C. 1H NMR (600 MHz,
δ

Molecules 2019, 24, 4138
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and 6.83 (s, 1H), 4.55 (s, 2H). 13C NMR (150 MHz, CDCl3)
δ
180.06, 174.13, 160.94, 153.46, 135.56, 130.74,
130.69, 129.03, 127.98, 122.69, 122.34, 108.04, and 21.87. IR (ν
, cm−1): 3118.33, 2920.28, 2339.32, 1670.84,
1551.16, and 691.42. HRMS (ESI) calcd. for [M + Na]+ C13H7O3BrNa+: 312.9471, found 312.9460.
3.2.4. Synthesis of TB1–9 and TC1–18
A mixture of the corresponding amines or alcohols (0.52 mmol), K2CO3 (142 mg, 1.03 mmol), and
9
(100 mg, 0.34 mmol) in THF (5 mL) was stirred at 30 ◦C to 50 ◦C for 4 h. After cooling, the mixture
was poured into water and extracted with EA. 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 on silica gel.
TB1: 2-(((4-methoxyphenyl)oxy)methyl)naphtho[1-b]furan-4,5-dione. Red solid, yield: 34%. Mp:
112–113 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.09 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.5
Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 6.94–6.92 (m, 2H), 6.87–6.85 (m, 2H), 6.83 (s, 1H), 5.04 (s, 2H), and
3.78 (s, 3H). 13C NMR (150 MHz, CDCl3)
180.34, 174.38, 160.88, 154.72, 153.82, 151.95, 135.47, 130.66,
130.50, 128.98, 128.28, 122.59, 122.12, 116.34, 114.81, 108.37, 63.03, and 55.73. IR (
, cm−1): 3445.32,
δ
ν
2358.40, 2339.32, 1671.47, 1598.48, 1253.20, 1219.81, 1161.58, and 1057.34. HRMS (ESI) calcd. for [M +
Na]+ C20H14O5Na+: 357.0733, found 357.0723.
TB2: 2-(((4-acetylphenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione. Light orange solid, yield: 40%.
Mp: 190–191 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.13 (d, J = 6.4 Hz, 1H), 8.00 (d, J = 8.9 Hz, 2H), 7.78 (d,
J = 9.1 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.06 (d, J = 8.9 Hz, 2H), 6.94 (s, 1H), 5.19
(s, 2H), and 2.60 (s, 3H). 13C NMR (150 MHz, CDCl3)
196.71, 180.20, 174.31, 161.58, 161.10, 152.60,
135.53, 131.28, 130.74, 130.70, 129.02, 128.11, 122.64, 122.08, 114.48, 108.98, 61.92, and 26.43. IR (
, cm−1):
δ
ν
3445.93, 2358.51, 2341.16, 1671.86, 1599.84, 1249.41, 1217.01, 1178.57, and 1008.47. HRMS (ESI) calcd.
for [M + Na]+ C21H14O5Na+: 369.0733, found 369.0721.
TB3: 2-(((4-propiophenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione. Orange solid, yield: 37%. Mp:
179–180 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.11 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 8.9 Hz, 2H), 7.77 (d,
J = 6.4 Hz, 1H), 7.71–7.68 (m, 1H), 7.53–7.50 (m, 1H), 7.06 (d, J = 8.9 Hz, 2H), 6.93 (s, 1H), 5.18 (s, 2H),
2.99 (q, J = 7.2 Hz, 2H), and 1.24 (t, J = 7.3 Hz, 3H). 13C NMR (150 MHz, CDCl3)
199.42, 180.20, 174.30,
161.40, 161.06, 152.68, 135.53, 130.98, 130.72, 130.68, 130.36, 129.01, 128.11, 122.63, 122.08, 114.47, 108.92,
δ
61.90, 31.53, and 8.38. IR (ν
, cm−1): 3447.03, 2358.62, 2341.16, 1669.65, 1601.07, 1222.31, 1180.35, and
1002.86. HRMS (ESI) calcd. for [M + Na]+ C22H16O5Na+: 383.0890, found 383.0876.
TB4: 2-(((4-nitrophenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 48%. Mp:
205–206 ◦C. 1H NMR (600 MHz, DMSO-d6)
δ
8.25 (d, J = 9.3 Hz, 2H), 7.96 (d, J = 7.6 Hz, 1H), 7.77–7.74
(m, 2H), 7.59–7.55 (m, 1H), 7.34 (d, J = 9.3 Hz, 2H), 7.18 (s, 1H), and 5.42 (s, 2H). 13C NMR (150 MHz,
DMSO-d6) 179.58, 174.56, 163.29, 160.07, 152.42, 141.83, 135.43, 130.84, 130.17, 129.83, 127.89, 126.41,
122.50, 122.36, 115.94, 110.32, and 62.45. IR (
δ
ν
, cm−1): 3445.75, 2358.70, 2341.16, 1681.37,1592.17, 1507.95,
1384.15, 1340.19, 1277.67, and 1110.13. HRMS (ESI) calcd. for [M + Na]+ C19H11O6NNa+: 372.0479,
found 372.0465.
TB5: 2-(((2-methoxyl-4-formyl)phenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield:
34%. Mp: 203–204 ◦C. 1H NMR (600 MHz, CDCl3)
δ
9.91 (s, 1H), 8.13 (d, J = 6.4 Hz, 1H), 7.77 (d, J = 7.6
Hz, 1H), 7.72–7.68 (m, 1H), 7.54–7.48 (m, 3H), 7.15 (d, J = 8.0 Hz, 1H), 6.96 (s, 1H), 5.28 (s, 2H), and
3.97 (s, 3H). 13C NMR (150 MHz, CDCl3)
190.88, 180.19, 174.31, 161.15, 152.50, 152.42, 150.30, 135.52,
131.28, 130.75, 129.67, 129.03, 128.10, 122.68, 122.09, 115.30, 112.93, 109.87, 109.40, 62.85, and 56.09. IR
δ
(ν
, cm−1): 3446.32, 2358.18, 2341.16, 1702.84, 1676.79, 1586.53, 1508.23, 1267.05, 1236.25, 1137.10, and
999.76. HRMS (ESI) calcd. for [M + Na]+ C21H14O6Na+: 385.0683, found 385.0668.
TB6: 2-(((4-formylphenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione. Orange solid, yield: 30%. Mp:
212–213 ◦C. 1H NMR (600 MHz, CDCl3)
δ
9.95 (s, 1H), 8.13 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 8.9 Hz, 2H),
7.78 (d, J = 7.8 Hz, 1H), 7.73–7.69 (m, 1H), 7.56–7.51 (m, 1H), 7.14 (d, J = 8.7 Hz, 2H), 6.96 (s, 1H), and
5.21 (s, 2H). 13C NMR (150 MHz, CDCl3)
190.71, 180.17, 174.30, 162.67, 161.14, 152.36, 135.53, 132.10,
130.82, 130.77, 130.74, 129.05, 128.09, 122.64, 122.08, 115.06, 109.11, and 61.99. IR (
, cm−1): 3446.21,
δ
ν

Molecules 2019, 24, 4138
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2358.24, 2337.30, 1687.41, 1671.45, 1598.48, 1253.19, 1219.95, 1161.49, and 1057.40. HRMS (ESI) calcd.
for [M + Na]+ C20H12O5Na+: 355.0577, found 355.0565.
TB7: 2-(((4-bromo-2-formyl)phenyl)oxy)methyl)naphtho[1,2-b]furan-4,5-dione. Light orange solid,
yield: 33%. Mp: 201–202 ◦C. 1H NMR (600 MHz, CDCl3)
δ
10.43 (s, 1H), 8.14 (d, J = 7.6 Hz, 1H), 7.99
(d, J = 2.7 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.73–7.69 (m, 2H), 7.57–7.51 (m, 1H), 7.06 (d, J = 8.7 Hz,
1H), 6.96 (s, 1H), and 5.24 (s, 2H). 13C NMR (150 MHz, CDCl3)
187.86, 180.06, 174.25, 161.30, 158.94,
151.80, 138.27, 135.59, 131.52, 130.87, 130.82, 129.07, 127.95, 126.84, 122.64, 121.99, 115.01, 114.95, 109.43,
δ
and 62.80. IR (ν
, cm−1): 3445.63, 2358.34, 2337.30, 1671.90, 1599.56, 1556.29, 1249.42, 1217.50, 1178.60,
and 1008.51. HRMS (ESI) calcd. for [M + Na]+ C20H11O5BrNa+: 432.9682, found 432.9673.
TB8: 2-(methoxymethyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 12%. Mp: 53–54 ◦C. 1H
NMR (600 MHz, CDCl3)
1H), 7.52–7.48 (m, 1H), 6.80 (s, 1H), 4.51 (s, 2H), and 3.46 (s, 3H). 13C NMR (150 MHz, CDCl3)
174.46, 160.91, 154.77, 135.45, 130.64, 130.44, 128.96, 128.34, 122.59, 122.05, 107.97, 66.02, and 58.32. IR
δ
8.11 (dd, J = 7.8, 1.5 Hz, 1H), 7.78 (dd, J = 7.7, 1.4 Hz, 1H), 7.70–7.66 (m,
δ
180.43,
(ν
, cm−1): 3446.10, 2358.55, 2337.96, 1677.60, 1276.65, 1216.21, 1153.22, and 1082.89. HRMS (ESI) calcd.
for [M + Na]+ C14H10O4Na+: 265.0471, found 265.0463.
TB9: 2-(ethoxymethyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 11%. Mp: 60–61 ◦C. 1H NMR
(600 MHz, CDCl3)
7.52–7.46 (m, 1H), 6.78 (s, 1H), 4.55 (s, 2H), 3.63 (q, J = 7.0 Hz, 2H), and 1.29 (t, J = 7.0 Hz, 3H). 13
NMR (150 MHz, CDCl3) 180.47, 174.45, 160.81, 155.23, 135.45, 130.61, 130.37, 128.90, 128.39, 122.59,
122.08, 107.66, 66.26, 64.25, and 15.10. IR (
δ 8.10 (dd, J = 8.2, 1.3 Hz, 1H), 7.77 (dd, J = 7.6, 1.2 Hz, 1H), 7.71–7.65 (m, 1H),
C
δ
ν
, cm−1): 3446.08, 2358.74, 2342.78, 1681.54, 1275.43, 1215.49,
1159.01, and 1083.47. HRMS (ESI) calcd. for [M + Na]+ C15H12O4Na+: 279.0628, found 279.0621.
TC1: 2-(diethylaminomethyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 51%. Mp: 78–79 ◦C. 1H
NMR (600 MHz, CDCl3)
(t, J = 7.6 Hz, 1H), 6.66 (s, 1H), 3.77 (s, 2H), 2.62 (q, J = 7.2 Hz, 4H), and 1.14 (t, J = 7.2 Hz, 6H). 13
NMR (150 MHz, CDCl3) 180.65, 174.55, 160.34, 156.44, 135.41, 130.55, 130.12, 128.74, 128.58, 122.45,
122.21, 107.11, 48.69, 47.10, and 12.01. IR (
δ 8.07 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.0 Hz, 1H), 7.46
C
δ
ν
, cm−1): 3445.24, 2953.88, 2358.48, 2339.23, 1700.91, 1676.52,
1216.10, and 1111.39. HRMS (ESI) calcd. for [M + Na]+ C17H18O3Na+: 284.1281, found 284.1271.
TC2: 2-(diisopropylaminomethyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 18%. Mp: 69–70 ◦C.
1H NMR (600 MHz, CDCl3)
δ
8.06 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 6.2 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H),
7.44 (t, J = 6.7 Hz, 1H), 6.66 (s, 1H), 3.72 (s, 2H), 3.13 (p, J = 6.5 Hz, 2H), and 1.08 (d, J = 6.7 Hz, 12H). 13
NMR (150 MHz, CDCl3) 180.88, 174.66, 161.17, 159.76, 135.37, 130.50, 129.81, 128.91, 128.65, 122.50,
122.09, 105.12, 49.12, 42.36, and 20.81. IR (
C
δ
ν
, cm−1): 3445.80, 2966.49, 2358.61, 2337.30, 1676.07, 1558.32,
1215.85, and 1149.51. HRMS (ESI) calcd. for [M + Na]+ C19H22O3Na+: 312.1594, found 312.1583.
TC3: 2-((l-methionine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 12%.
Mp: 52–53 ◦C. 1H NMR (600 MHz, CDCl3)
δ 8.08 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.66 (t, J
= 7.6 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 6.66 (s, 1H), 3.95 (d, J = 15.1 Hz, 1H), 3.79 (d, J = 15.1 Hz, 1H),
3.74 (s, 3H), 3.52 (dd, J = 8.4, 5.0 Hz, 1H), 2.64 (t, J = 7.2 Hz, 2H), 2.10 (s, 3H), 2.02–1.97 (m, 1H) and
1.89–1.82 (m, 1H). 13C NMR (150 MHz, CDCl3)
130.19, 128.80, 128.47, 122.35, 122.17, 106.00, 59.14, 52.14, 44.65, 32.68, 30.48, and 15.38. IR (
δ
180.55, 175.18, 174.44, 160.36, 157.00, 135.44, 130.58,
ν
, cm−1):
3446.14, 2923.56, 2358.62, 2335.37, 1698.61, 1670.40, 1215.45, and 1147.65. HRMS (ESI) calcd. for [M +
Na]+ C19H19O5NSNa+: 396.0876, found 396.0865.
TC4: 2-((l-alanine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 27%. Mp:
83–84 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.07 (d, J = 6.9 Hz, 1H), 7.73 (d, J = 4.9 Hz, 1H), 7.65 (d, J = 8.6
Hz, 1H), 7.46 (t, J = 6.6 Hz, 1H), 6.66 (s, 1H), 3.94 (d, J = 14.1 Hz, 1H), 3.81 (d, J = 14.9 Hz, 1H), 3.74
(s, 3H), 3.46 (q, J = 6.9 Hz, 1H), and 1.37 (d, J = 6.6 Hz, 3H). 13C NMR (150 MHz, CDCl3)
180.52,
175.63, 174.42, 160.36, 156.93, 135.40, 130.56, 130.18, 128.80, 128.46, 122.39, 122.18, 105.99, 55.69, 52.05,
δ
44.26, and 19.13. IR (ν
, cm−1): 3328.35, 2958.60, 2358.44, 2337.30, 1735.12, 1676.39, 1216.30, and 1139.72.
HRMS (ESI) calcd. for [M + Na]+ C17H15O5NNa+: 336.0842, found 336.0831.
TC5: 2-((l-isoleucinate methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 39%.
Mp: 112–113 ◦C. 1H NMR (600 MHz, CDCl3)
δ 8.07 (d, J = 7.7 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.67–7.62

Molecules 2019, 24, 4138
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(m, 1H), 7.48–7.43 (m, 1H), 6.65 (s, 1H), 3.92 (d, J = 15.1 Hz, 1H), 3.72 (d, J = 16.4 Hz, 4H), 3.17 (d, J = 5.8
Hz, 1H), 1.57–1.50 (m, 1H), 1.26–1.16 (m, 2H), and 0.93–0.88 (m, 6H). 13C NMR (150 MHz, CDCl3)
180.60, 175.16, 174.46, 160.27, 157.37, 135.45, 130.55, 130.15, 128.78, 128.52, 122.32, 122.19, 105.82, 65.31,
δ
51.64, 45.08, 38.47, 25.39, 15.66, and 11.49. IR (ν
, cm−1): 3445.72, 2924.70, 2358.43, 2341.16,1732.42,
1682.86, 1209.15, and 1150.10. HRMS (ESI) calcd. for [M + Na]+ C20H21O5NNa+: 378.1312, found
378.1299.
TC6: 2-((l-valine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 41%. Mp:
54–55 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.09 (d, J = 6.2 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.70–7.64 (m,
1H), 7.50–7.45 (m, 1H), 6.67 (s, 1H), 3.95 (d, J = 15.3 Hz, 1H), 3.77–3.72 (m, 4H), 3.11 (d, J = 5.8 Hz, 1H),
2.02–1.95 (m, 1H), and 0.98 (t, J = 6.3 Hz, 7H). 13C NMR (150 MHz, CDCl3)
180.62, 175.22, 174.49,
160.28, 157.39, 135.43, 130.59, 130.14, 128.82, 128.56, 122.32, 122.22, 105.83, 66.36, 51.70, 45.16, 31.75,
δ
19.28, and 18.35. IR (ν
, cm−1): 3425.42, 2923,62, 2358.56, 2337.30, 1698.57, 1670.37, 1187.10, and 1118.00.
HRMS (ESI) calcd. for [M + Na]+ C19H19O5NNa+: 364.1155, found 364.1141.
TC7: 2-((l-glycine methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 17%. Mp:
55–56 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.06 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 6.4 Hz, 1H), 7.68–7.62 (m,
1H), 7.49–7.43 (m, 1H), 6.67 (s, 1H), 3.93 (s, 2H), 3.75 (s, 3H), and 3.50 (s, 2H). 13C NMR (150 MHz,
CDCl3) 180.49, 174.42, 172.45, 160.49, 156.62, 135.44, 130.57, 130.25, 128.79, 128.39, 122.41, 122.14,
106.25, 52.03, 49.50, and 45.38. IR (
δ
ν
, cm−1): 3328.36, 2958.26, 2358.57, 2337.30, 1735.10, 1676.24, 1216.29,
and 1180.25. HRMS (ESI) calcd. for [M + Na]+ C16H13O5NNa+: 322.0686, found 322.0680.
TC8: 2-((l-leucinate methyl ester-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 19%.
Mp: 54–55 ◦C. 1H NMR (600 MHz, CDCl3)
δ 8.09 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.67 (t, J =
7.6 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 6.66 (s, 1H), 3.94 (d, J = 15.1 Hz, 1H), 3.77 (d, J = 15.1 Hz, 1H), 3.73
(s, 3H), 3.38 (t, J = 7.2 Hz, 1H), 1.82–1.77 (m, 1H), 1.52 (t, J = 7.4 Hz, 2H), 0.95 (d, J = 6.7 Hz, 3H), and
0.90 (d, J = 6.7 Hz, 3H). 13C NMR (150 MHz, CDCl3)
δ
180.59, 176.01, 174.47, 160.32, 157.18, 135.42,
130.59, 130.17, 128.84, 128.51, 122.32, 122.20, 105.94, 59.08, 51.88, 44.63, 42.75, 24.89, 22.78, and 22.09. IR
(ν
, cm−1): 3434.68, 2958.49, 2358.68, 2339.23, 1670.06, 1518.72, 1211.01, and 1107.62. HRMS (ESI) calcd.
for [M + Na]+ C20H21O5NNa+: 378.1312, found 378.1302.
TC9: 2-((4-boc-piperazin-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 26%. Mp: 56–57
◦C. 1H NMR (600 MHz, CDCl3)
δ
8.10 (d, J = 7.4 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H),
7.48 (t, J = 7.6 Hz, 1H), 6.70 (s, 1H), 3.68 (s, 2H), 3.49 (s, 4H), 2.52 (s, 4H), and 1.47 (s, 9H). 13C NMR
(150 MHz, CDCl3) 180.53, 174.48, 160.57, 155.01, 154.68, 135.44, 130.64, 130.29, 128.81, 128.46, 122.53,
122.17, 107.66, 79.88, 54.60, 52.60, and 28.42. IR (
δ
ν
, cm−1): 3434.73, 2358.58, 2339.23, 1669.96, 1518.77,
1211.18, and 1107.93. HRMS (ESI) calcd. for [M + H]+ C22H25O5N2+: 397.1758, found 397.1751.
TC10: 2-((pyrrolidin-1-yl)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 24%. Mp: 62–63 ◦C.
1H NMR (600 MHz, CDCl3)
δ
8.08 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 7.6 Hz, 1H),
7.46 (t, J = 7.6 Hz, 1H), 6.68 (s, 1H), 3.76 (s, 2H), 2.69–2.63 (m, 4H), and 1.89–1.83 (m, 4H). 13C NMR
(150 MHz, CDCl3) 180.64, 174.54, 160.39, 156.44, 135.40, 130.56, 130.14, 128.80, 128.56, 122.55, 122.24,
δ
106.71, 54.01, 51.84, and 23.53. IR (ν
, cm−1): 3388.30, 3110.62, 2358.68, 2337.30, 1660.65, 1510.49, 1222.52,
and 1091.51. HRMS (ESI) calcd. for [M + H]+ C17H16O3N+: 282.1125, found 282.1115.
TC11: 2-(morpholinomethyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 38%. Mp: 98–99 ◦C. 1H
NMR (600 MHz, CDCl3)
(m, 1H), 6.70 (s, 1H), 3.78–3.74 (m, 4H), 3.66 (s, 2H), and 2.61–2.55 (m, 4H). 13C NMR (150 MHz, CDCl3)
180.50, 174.46, 160.50, 155.01, 135.36, 130.59, 130.23, 128.86, 128.47, 122.46, 122.21, 107.64, 66.79, 54.93,
δ 8.09 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.69–7.64 (m, 1H), 7.51–7.45
δ
and 53.28. IR (ν
, cm−1): 3438.84, 2807.27, 2358.54, 2342.76, 1676.47, 1557.77, 1215.99, 1111.32, and
1006.63. HRMS (ESI) calcd. for [M + Na]+ C17H15O4NNa+: 320.0893, found 320.0886.
TC12: 2-(((4-fluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione. Dark red solid, yield: 51%.
Mp: 174–175 ◦C. 1H NMR (600 MHz, DMSO-d6)
δ
7.92 (d, J = 7.3 Hz, 1H), 7.73 (t, J = 7.5 Hz, 1H), 7.65
(d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H), 6.94 (t, J = 8.9 Hz, 2H), 6.74 (s, 1H), 6.73–6.68 (m, 2H), 6.20 (t,
J = 6.4 Hz, 1H), and 4.37 (d, J = 6.0 Hz, 2H). 13C NMR (150 MHz, DMSO-d6)
179.86, 174.62, 159.03,
157.32, 155.16 (d, J = 231.0 Hz), 145.06, 135.47, 130.37, 129.85, 129.69, 128.27, 122.42, 122.06, 115.77 (d,
δ

Molecules 2019, 24, 4138
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J = 21.0 Hz), 113.88 (d, J = 6.0 Hz), and 105.93. IR (
ν
, cm−1): 3378.05, 2923.56, 2358.52, 2335.37, 1662.70,
1514.18, 1215.45, and 1161.45. HRMS (ESI) calcd. for [M + Na]+ C19H12O3NFNa+: 344.0693, found
344.0681.
TC13: 2-(((3-fluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione. Dark red solid, yield: 45%.
Mp: 169–170 ◦C. 1H NMR (600 MHz, DMSO-d6)
δ
7.92 (d, J = 7.6 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.65
(d, J = 7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.10 (q, J = 8.0 Hz, 1H), 6.78 (s, 1H), 6.61 (t, J = 6.3 Hz, 1H),
6.54 (d, J = 8.2 Hz, 1H), 6.50 (d, J = 12.2 Hz, 1H), 6.35 (t, J = 8.4 Hz, 1H), and 4.41 (d, J = 6.2 Hz, 2H). 13
NMR (150 MHz, DMSO-d6) 179.83, 174.63, 163.90 (d, J = 238.5 Hz), 159.09, 156.86, 150.51 (d, J = 10.5
Hz), 135.46, 130.79 (d, J = 9.0 Hz), 130.40, 129.85, 129.73, 128.25, 122.42, 122.04, 109.34, 106.10, 102.99 (d,
C
δ
J = 21.0 Hz), and 99.24 (d, J = 27.0 Hz). IR (ν
, cm−1): 3390.68, 3105.83, 2360.44, 2337.30, 1665.28, 1618.38,
1220.09, and 1152.73. HRMS (ESI) calcd. for [M + Na]+ C19H12O3NFNa+: 344.0693, found 344.0688.
TC14: 2-(((2-fluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione. Dark red solid, yield: 46%.
Mp: 170–171 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.08 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.4 Hz, 1H), 7.66 (t,
J = 7.4 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.05–7.00 (m, 2H), 6.79 (t, J = 8.5 Hz, 1H), 6.75–6.72 (m, 1H), 6.71
(s, 1H), and 4.51 (d, J = 6.4 Hz, 2H). 13C NMR (150 MHz, CDCl3)
180.39, 174.38, 160.36, 155.98, 151.74
(d, J = 237.0 Hz), 135.45, 135.22 (d, J = 12.0 Hz), 130.63, 130.29, 128.79, 128.36, 124.67 (d, J = 4.5 Hz),
δ
122.26 (d, J = 15.0 Hz), 118.16 (d, J = 6.0 Hz), 114 (d, J = 19.5 Hz), 112.52, 112.50, 106.03, and 40.74. IR (ν,
cm−1): 3425.04, 2923.94, 2358.97, 2854,13, 1670.34, 1513.50, 1186.96, and 1117.57. HRMS (ESI) calcd. for
[M + Na]+ C19H12O3NFNa+: 344.0693, found 344.0684.
TC15: 2-(((2,4-difluorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 33%. Mp:
154–155 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.10 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.67 (t,
J = 7.4 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.14 (t, J = 8.1 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.71 (s, 2H), 6.59
(d, J = 8.2 Hz, 1H), and 4.46 (d, J = 6.4 Hz, 2H). 13C NMR (150 MHz, CDCl3)
180.36, 174.38, 160.42,
δ
155.83 (d, J = 9.0 Hz), 154.24 (d, J = 10.5 Hz), 151.95 (d, J = 12.0 Hz), 150.34 (d, J = 12.0 Hz), 135.49,
131.73 (d, J = 12.0 Hz), 130.52 (d, J = 46.5 Hz), 129.67, 128.55 (d, J = 73.5 Hz), 122.24 (d, J = 24 Hz),
115.33, 112.73 (dd, J = 12.0 Hz), 110.86 (dd, J = 25.5 Hz), 106.09, 103.90 (dd, J = 49.5 Hz), and 41.22. IR
(ν
, cm−1): 3434.72, 2358.46, 2337.30, 1689.86, 1518.53, 1210.93, 1107.68, and 957.02. HRMS (ESI) calcd.
for [M + Na]+ C19H11O3NF2Na+: 362.0599, found 362.0589.
TC16: 2-(((2,4,6-trimethylphenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione. Red solid, yield: 33%.
Mp: 173–174 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.10 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 3.8 Hz, 2H), 7.50–7.46
(m, 1H), 6.85 (s, 2H), 6.60 (s, 1H), 4.21 (s, 2H), 2.29 (s, 6H), and 2.25 (s, 3H). 13C NMR (150 MHz, CDCl3)
180.53, 174.47, 160.18, 157.50, 141.61, 135.47, 132.42, 130.65, 130.23, 130.13, 129.64, 128.84, 128.47,
δ
122.23, 122.18, 105.72, 44.94, 20.62, and 18.23. IR (ν
, cm−1): 3445.76, 2357.38, 2327.66, 1670.28, 1557.44,
1215.32, 1147.47, and 1025.94. HRMS (ESI) calcd. for [M + Na]+ C22H19O3NNa+: 368.1257, found
368.1250.
TC17: 2-(((4-chlorophenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione. Dark red solid, yield: 28%.
Mp: 205–206 ◦C. 1H NMR (600 MHz, DMSO-d6)
δ
7.92 (d, J = 6.8 Hz, 1H), 7.74 (t, J = 7.5 Hz, 1H), 7.65
(d, J = 7.6 Hz, 1H), 7.52 (t, J = 7.1 Hz, 1H), 7.12 (d, J = 8.9 Hz, 2H), 6.75 (s, 1H), 6.72 (d, J = 8.9 Hz, 2H),
6.49 (t, J = 6.3 Hz, 1H), and 4.40 (d, J = 6.2 Hz, 2H). 13C NMR (150 MHz, DMSO-d6)
: 179.82, 174.61,
159.06, 156.95, 147.33, 135.46, 130.39, 129.84, 129.72, 129.09, 128.24, 122.41, 122.05, 120.26, 114.42, and
δ
106.08. IR (ν
, cm−1): 3388.22, 3110.62, 2358.84, 2342.68, 1660.69, 1510.19, 1222.68 and 1118.65. HRMS
(ESI) calcd. for [M + Na]+ C19H12O3NClNa+, 360.0398, found 360.0384.
TC18: 2-(((4-methoxyphenyl)amino)methyl)naphtho[1,2-b]furan-4,5-dione. Dark solid, yield: 64%. Mp:
112–113 ◦C. 1H NMR (600 MHz, CDCl3)
δ
8.08 (d, J = 6.4 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.69–7.63 (m,
1H), 7.50–7.44 (m, 1H), 6.82 (d, J = 8.9 Hz, 2H), 6.70–6.67 (m, 3H), 4.42 (s, 2H), and 3.77 (s, 3H). 13
NMR (150 MHz, CDCl3) 180.49, 174.43, 160.22, 156.83, 152.98, 140.82, 135.43, 130.62, 130.21, 128.77,
C
δ
128.46, 122.27, 115.00, 114.74, 105.84, 55.77, and 42.13. IR (ν
, cm−1): 3370.45, 3105.23, 2358.89, 2337.30,
1660.16, 1514.41, 1234.80, and 1040.25. HRMS (ESI) calcd. for [M + Na]+ C20H15O4NNa+: 356.0893,
found 356.0883.

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3.3. In Vitro Cytotoxicity Assay
The human cancer cell lines, including prostate cancer cells PC3, leukemia cells K562, and melanoma
cells WM9, were stored in the biology laboratory of the Key Laboratory of Chemistry for Natural
Products of Guizhou Province and Chinese Academy of Sciences (Guiyang, China). All cells were
cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin
◦
(Sijiqing, Hangzhou, China) and incubated at 37 C under 5% CO2, 95% air, and 95% humidity.
Cytotoxicity was evaluated by performing the MTT assay [24]. Briefly, the cells were seeded in 96-well
microculture plates at a density from 4
concentrations of the assayed compounds for 48 h. Then, 20
to each well and incubated at 37 ◦C for an additional 4 h. The medium was then removed and 200
×
103 to 8
×
103 cells/well. Cells were then exposed to different
µL of MTT solution (5 mg/mL) was added
µL
Tris-DMSO solution was added. Plates were lightly shaking up to dissolve the dark blue formazan
crystals and the absorbance was measured in an ELISA plate reader at 570 nm.
3.4. Flow Cytometry Assay
Cell apoptosis was determined by an inverted fluorescence microscope observation and flow
cytometry as describe in our previous study [28]. Briefly, the cancer cells treated with compounds
were harvested for centrifugation at 1000 rpm for 5 min at room temperature, washed twice with
PBS and resuspended with binding buffer, and then PI (Sigma, St. Louis, MO, USA) was added to a
final concentration of 20 mg/mL. The cell lines were analyzed by flow cytometry (Becton Dickinson,
Franklin Lakes, NJ, USA).
3.5. Statistical Analysis
The IC50 values were calculated from the semilogarithmic dose-response curves. The data
were analyzed using SPSS 18.0 and reported as mean
±
SD of the number of experiments indicated.
For all measurements, one-way ANOVA followed by Student’s t-test was used to assess the statistical
significance of the difference between each group. The LSD method was used to assess the statistical
significance of the difference between the two groups. A statistically significant difference was
considered at the level of P < 0.05. The data are presented as the mean ± SEM of three assays.
4. Conclusions
In this study, 27 novel L-shaped ortho-quinone analogs were synthesized and evaluated for their
anti-cancer activities. Compounds TB1, TB3, TB4, TB6, TC1, TC3, TC5, TC9, TC11, TC12, TC14,
TC15 TC16, and TC17 possessed broad-spectrum potent cytotoxicity against PC3, K562, and WM9
,
cells. With more than a 70% inhibitory rate, TB7 showed better inhibitory activity of K562 cells
as compared with other cells. Moreover, we observed that TC7 inhibited the growth of PC3 cells
more efficiently than other cells. Some of the active compounds such as TB3, TC1, TC3, and TC7
inhibited cell proliferation mainly through inducing apoptosis. The structure-activity relationships
evaluation showed that removing methyl at C-3 of the furan ring and introducing diverse side chains
at C-2 of the furan ring is an effective strategy for improving the anticancer activity of L-shaped
ortho-quinone analogs.
Author Contributions: W.-D.P. and H.L. conceived and designed the experiments; S.-Y.L. and X.-Y.Z. performed
part of chemical experiment; Z.-K.S., Y.Z., M.-L.W. performed the biology experiment; S.-C.H., J.L., J.-R.S. and C.C.
contributed reagents and materials and revised the paper; S.-Y.L. and H.L. wrote the paper.
Funding: This work was supported by the Science and Technology Department of Guizhou Province (no. QKHJC
(2017)1412, QKHRC (2016)4037, QKHZC (2019)2757, QKHJC (2016)1099, and QKHPTRC (2017) 5737), the National
Science Foundation of China (NSFC no. 81660580 and 81702914), and Financial support from Guizhou Provincial
Engineering Research Center for Natural Drugs.
Conflicts of Interest: The authors declare no conflict of interest.

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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).