Design, synthesis and biological evaluation of novel plumbagin derivatives as potent antitumor agents with STAT3 inhibition

Article abstract of DOI:10.1016/j.bioorg.2020.104208

Based on the structure of signal transducer and activator of transcription 3 (STAT3), a series of 1,4-naphthoquinones derived from plumbagin (PL) with STAT3 inhibition potential were designed, synthesized, and biologically evaluated in vitro against several human cancer cell lines (MDA-MB-231, HepG2 and A549 cells) and three normal cells. The structure–activity relationship (SAR) and molecular docking result showed that the presence of hydroxyl group at C-5 of PL might interact with STAT3 in the form of hydrogen bonds, which is conducive to the binding of this kind structures with STAT3. Among the target compounds, 7a displayed the most potent inhibition against cancer cells and weaker cytotoxicity on normal cells than PL. The western bolting analysis showed that 7a could suppress the phosphorylation of STAT3 as well as the downstream genes instead of affecting its upstream tyrosine kinases (Src and JAK2) levels and p-STAT1 expression. Furthermore, molecular docking indicated that 7a bound to STAT3 more tightly than PL, and it could significantly induce the apoptosis of cancer cells in vitro. All these results may provide reference for the discovery of effective STAT3 inhibitors.

Full text of DOI:10.1016/j.bioorg.2020.104208

Bioorganic Chemistry 104 (2020) 104208
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel plumbagin derivatives
as potent antitumor agents with STAT3 inhibition
⁎
⁎⁎
Na Li, Jinfeng Ou, Na Bao, Cheng Chen, Zhixian Shi, Li Chen , Jianbo Sun
State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 24
Tong Jia Xiang, Nanjing 210009, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Based on the structure of signal transducer and activator of transcription 3 (STAT3), a series of 1,4-naphtho-
quinones derived from plumbagin (PL) with STAT3 inhibition potential were designed, synthesized, and bio-
logically evaluated in vitro against several human cancer cell lines (MDA-MB-231, HepG2 and A549 cells) and
three normal cells. The structure–activity relationship (SAR) and molecular docking result showed that the
presence of hydroxyl group at C-5 of PL might interact with STAT3 in the form of hydrogen bonds, which is
conducive to the binding of this kind structures with STAT3. Among the target compounds, 7a displayed the
most potent inhibition against cancer cells and weaker cytotoxicity on normal cells than PL. The western bolting
analysis showed that 7a could suppress the phosphorylation of STAT3 as well as the downstream genes instead
of affecting its upstream tyrosine kinases (Src and JAK2) levels and p-STAT1 expression. Furthermore, molecular
docking indicated that 7a bound to STAT3 more tightly than PL, and it could significantly induce the apoptosis
of cancer cells in vitro. All these results may provide reference for the discovery of effective STAT3 inhibitors.
Plumbagin
Antiproliferation
STAT3 inhibition
Apoptosis
Antitumor
1
. Introduction
Signal transducer and activator of transcription 3 (STAT3) is a
targeting STAT3-SH2 have been reported, such as S3l-20 l, STA-21 and
Stattic. However, none of them have been approved clinically due to
their defects on permeability, stability and/or side effect [16–22].
Therefore, discovery of novel STAT3-SH2 inhibitors is essential for the
elucidation of the structure–activity relationship and the development
of STAT3-SH2 inhibitors as precision anticancer agents.
member of the STAT family of transcription factors. Activated STAT3
can mediates the signal transduction which is closely related to cell
proliferations, differentiations and apoptosis [1–2]. Generally, the ac-
tivation of STAT3 is under careful control to ensure the proliferation of
normal cells. However, it is found that STAT3 is activated aberrantly in
several cancer types, such as breast, lung, prostate, ovarian [3–6].
Furthermore, introduction of antisense, dominant negative, and decoy
oligonucleotides against STAT3 lead to significant apoptosis of cancer
cells [7]. Therefore, inhibition of STAT3 activity has long been an at-
tractive strategy in cancer therapy [8–9].
Plumbagin (PL) is a natural 1,4-naphthoquinone isolated from the
plant Plumbago zeylanica [23]. 1,4-naphthoquinone1 is an important
class of small molecules for the the design of anticancer agents [24–25].
Previous studies reported that PL showed potential anticancer activity
against breast cancer, which was mainly due to the inhibition of STAT3
phosphorylation [26]. Moreover, PL shares a very similar structure with
STA-21 and LLL-3 which were efficient STAT3-SH2 inhibitors (Fig. 1A).
Therefore, it is very promising to optimize the structure of PL to de-
velopment novel STAT3 inhibitors based on the protein crystal struc-
ture of STAT3 and its pharmacophore model.
The STAT3 activation needs several procedures. Generally, cyto-
kines or growth factors or platelet-derived growth factor (PDGF) med-
iate the phosphorylation of STAT3 via its Src homology 2 (SH2) domain
[
10–11]. The phosphorylated STAT3 (p-STAT3) dimerizes through the
same domain and then translocate to the nucleus, where it initiates
transcription of its target genes [12–13]. These findings support the
hypothesis that phosphorylation of STAT3 on SH2 domain is a key
event in the STAT3 activation, which provides an attractive strategy in
the discovery of STAT3 inhibitor [14–15]. Currently, many inhibitors
2. Results and discussion
2.1. Design
The pTyr705 site and the side pocket (Fig. 1B) in STAT3-SH2
⁎
Corresponding author.
⁎
⁎
Corresponding author.
E-mail addresses: chenli627@cpu.edu.cn (L. Chen), sjbcpu@gmail.com (J. Sun).
https://doi.org/10.1016/j.bioorg.2020.104208
Received 4 May 2020; Received in revised form 15 August 2020; Accepted 25 August 2020
Available online 28 August 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Fig. 1. The analysis of various small-molecule STAT3 inhibitors (STAT3-SH2 site). (A) The structures of several STAT3 inhibitors. (B) Two main binding sites of
STAT3-SH2 (PDB ID: 1BG1). (C) The binding mode of PL with STAT3-SH2 domain (1BG1). (D) The structures of several STAT3 inhibitors.
domain were two hot binding sites of small molecule STAT3 inhibitors.
STAT3 protein ligands that can bind to the STAT3-SH2 domain gen-
erally contain three classic structural features: two aromatic rings (oc-
cupying the Site pTyr705 and Side pocket regions, respectively) and the
bridge chain that connects them. To increase the binding affinity of
planner scaffold (PL), we were promoted to introduce an aromatic ring
to occupy the side pocket. And the most suitable linker should allow
sufficient flexibility for PL and another aromatic ring to bind pTyr705
site and side pocket, respectively. According to the reported structure of
the potent small molecule STAT3 inhibitors, it was found that the lin-
2.2. Chemistry
The synthetic routes of target compounds (7a-9b) were shown in
Scheme 1. In brief, the syntheses began with the condensation between
β-alanine and differently substituted benzoyl chloride (1a-g) in the
presence of NaOH, which quickly yielded intermediates 2a-g. Ad-
ditionally, 5 was yielded by debenzylation of 3 and nicotinoyl chloride
(4). The intermediate 6 was obtained from 5 in the presence of Pd/C
and H . Compounds 7a-9b were obtained by Kochi-Anderson reaction
2
of PL or menadione using different intermediates [28]. In addition, the
yields of Kochi-Anderson reaction were low (products and a lot of re-
actant) in this study, which were due to the mild reaction conditions.
In order to further analyze the structure–activity relationship (SAR)
of these derivatives, compounds 11–13 were prepared (Scheme 2).
Kochi-Anderson reaction between PL and succinic acid yielded 10
which was then condensed with aniline in the presence of benzotriazol-
1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop) and
4-dimethylaminopyridine (DMAP) to produce 11. The oxidative addi-
3
2
kers could accommodate 0–5 sp or sp atoms and exist to be amide in
most cases. Therefore, seven linkers including amino or amide (P1-P7)
were chosen to form different compounds and were quickly screened
via computational docking for the final design. The binding mode of PL
and STAT3-SH2 domain were predicted using software Autodock Vina
1
.1.2 [27]. As shown in Fig. 1C, PL could embed in pTyr705 pocket by
forming a hydrogen bond with residue Ser611 and cation-π interaction
with residue Lys591, but it could not interact with the side pocket.
Various STAT3-SH2 inhibitors (such as STA-21, S3I-201 and carda-
monin, Fig. 1D) usually contain benzene, naphthoquinone or quinoline
to bind STAT3-SH2 domain. Based on the above analysis, the in-
troduction of an aromatic ring in PL may allow the new derivatives to
bind the two pockets simultaneously, thereby increasing the STAT3
binding affinity of PL.
tion reaction of PL with aniline catalyzed by Cu(AcO)
2
yielded 12.
Compound 13 was obtained from the ammonolysis of PL.
2.3. In vitro anti-proliferative activity
The inhibition rates of all the derivatives and their parent com-
Then, PL was connected to an aromatic ring via seven linkers
pounds (PL and menadione) were first evaluated against MDA-MB-231
by MTT assay (Table 1). The results showed that the PL derivatives
generally displayed a higher antiproliferative activity than the mena-
dione derivatives. For example, 7a-7d/9a (derived from PL) showed
higher activity (65.18–96.63%) than 8a-8g/9b (derived from mena-
dione, 16.76–46.38%), which suggesting that the hydroxyl groups at C-
(
Fig. 2A), which were docked to the STAT3 SH2 domain (PDB entry
1
BG1). As shown in Fig. 2B, P4 showed a higher STAT3-SH2 affinity
than the others. Thus, the linker in P4 was selected to introduce the
aromatic ring to PL. In addition, in order to investigate the effects of the
hydroxyl group (C-5) on the anti-proliferative activity of PL, menodione
was also selected as a parent compound to synthesize several new de-
rivatives (Fig. 3).
5
were essential for the activities of these compounds. Furthermore,
1
1–13 showed much lower activity (31.87%-48.34%) than 7a
(
91.86%), which indicated that the linker in 7a was suitable for the
2

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Fig. 2. Analysis of molecular docking of P1-P7. (A) Docking mode of P1-P7 with STAT3 (PDB ID). (B) Docking Scores of P1-P7.
O
O
O
which introduced benzene exhibited the most potential.
8
7
1
Next, 7a-7d and 9a with enhanced inhibitory activity than PL were
selected to determine their IC50 values against three cancer cell lines
2
R
R
pTyr 705 site
binding fragement
X
X
5
4
(
(
MDA-MB-231, HepG2 and A549). As shown in Table 2, compared to PL
OH
OH
O
O
IC50: 10.66–14.23 μM), increased activities were found in 7a, 7b and
PL
Linker
9a (IC50: 6.01–8.75 μM), which were selected for further investigation
of cytotoxicity against three normal human cell lines (MDA-MB-10A,
PBMCs and HFL-1 cells). It is worth mentioned that 7a showed a lower
cytotoxicity than 7b, 9a and PL. Combined with the high selective
index (SI) of 7a in breast cancer cells, 7a was preferentially selected for
further investigation in MDA-MB-231.
O
Side pocket
binding fragement
O
O
Menadione
Fig. 3. Design strategy of the new STAT3 inhibitors derived from PL and me-
nadione.
2.4. Compound 7a inhibited the phosphorylation of STAT3 at site pTyr705
promotion of the activity (Fig. 4A). In addition, we found that the
groups in benzene ring could affect the activity. As shown in Fig. 4B,
although electron-withdrawing groups substituted on benzene were
generally benefit for the inhibition (7b vs. 7e/7g), the compound (7a)
To further investigate whether the effects of 7a was related to
phosphorylation of STAT3, the level of p-STAT3 stimulated by IL-6 in
MDA-MB-231 was evaluated. As displayed in Fig. 5, 7a could effectively
decrease the level of p-STAT3 at site pTyr705 in a concentration-
Scheme 1. Reagents and conditions: (a) 10% NaOH, rt, 30 min. (b) DIPEA, dry DCM, 0 °C to r.t., 6 h. (c) H
ammonium persulfate (1.3 eq), aq·H O/CH CN (V/V = 3:4), 100 °C, 3–6 h.
2
, Pd/C. (d) 2a-g or 6 (3.0 eq), silver nitrate (0.3 eq),
2
3
3

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Scheme 2. Reagents and conditions: (a) succinic acid, AgNO
3
, (NH
4
)
2
S
O
2 8
, 30% CH
3
CN, 65–70 °C, 3 h, (b) aniline, PyBop, DMAP, TEA, rt, 12 h. (c) Cu(AcO) , AcOH,
2
6
0 °C, 4 h. (d) MeOH, NaN
3
, N
2
, NH , 50 °C, 5 h.
3
dependent manner and the function of 7a was stronger than that of PL.
However, the level of total STAT3 was not changed with the treatment
of 7a. These results indicated that 7a could inhibit the phosphorylation
of STAT3 at site pTyr705.
promote the survival of tumors, which is opposite to the function of
STAT3 [29]. Then, we detected whether 7a displayed an inhibition to p-
STAT1. As shown in Fig. 8, the level of p-STAT1 was slightly changed
with the treatment of 7a. This result indicated that 7a might show a
much higher inhibition on p-STAT3 than p-STAT1 in terms of sup-
pressing the survival of cancer cells.
2
.5. Compound 7a inhibited the expression of the downstream gene of
Combined with the expression of STAT3 and STAT1, it showed that
STAT3
7
a displayed the most effective concentration dependent inhibition of
STAT3, but has no effect on the expression of STAT1. This result in-
dicated that 7a might inhibited the survival of cancer cells through the
combination of STAT3, and did not show the characteristics of pan
active structures.
According to what has been mentioned above, compound 7a could
inhibit the level of STAT3. To further study the effects of 7a on the
STAT3 pathway, we examined the expressions of STAT3-targeted genes
(
Survivin and Mcl-1). As shown in Fig. 6, compound 7a down-regulated
the level of STAT3 downstream genes. These results preliminarily in-
dicated that 7a could inhibit the activity of STAT3.
2.8. Molecular docking of 7a to the STAT3 protein
2
.6. Compound 7a had no effects on the typical upstream kinases of STAT3
As mentioned above, 7a might bind to STAT3. To further speculate
the binding sites of 7a, molecular docking was adopted. 7a could
The phosphorylation of STAT3 also can be mediated by STAT3
embed into the key pockets related to the phosphorylation of STAT3
upstream tyrosine kinases, such as p-JAK2 and p-Src. To evaluate
whether the p-STAT3 inhibition of 7a brought about the regulation of
Src and JAK2, western blot was carried out. It was obvious in Fig. 7 that
the levels of p-Src and p-JAK2 as well as total proteins were not in-
fluenced by 7a, which illustrating that 7a might inhibit the phosphor-
ylation of STAT3 by binding to STAT3 protein.
(
Fig. 9). One hydrogen bond (between hydroxyl group and Arg609) and
two cation-π interactions (between the aromatic ring and Lys591; be-
tween the benzene and Arg595) might support 7a binding to the SH2
domain more tightly, thereby inhibiting the phosphorylation of STAT3.
Therefore, introduction of a suitable substituents at C-5 to from a new
interaction with STAT3 could enhance the combination of small mo-
lecular with STAT3 hopefully.
2.7. Compound 7a had no effects on the phosphorylation of STAT1
As a STAT isoform, inhibition of STAT1 phosphorylation can
Table 1
Preliminary inhibitory effects of the tested compounds at 20 μM on MDA-MB-231 cells.
Comp.
Inhibition rate % a
Comp.
Inhibition rate % a
Comp.
Inhibition rate % a
7
7
7
7
7
7
7
a
b
c
d
e
f
97.86 ± 6.35
93.63 ± 7.69
81.48 ± 7.59
65.18 ± 5.26
19.76 ± 1.44
23.13 ± 1.48
18.59 ± 1.25
8a
8b
8c
8d
8e
8f
16.76 ± 1.51
20.54 ± 1.95
35.65 ± 2.99
18.45 ± 1.02
32.21 ± 2.47
46.38 ± 3.59
23.54 ± 1.56
9a
96.63 ± 8.55
19.87 ± 1.73
31.87 ± 1.98
46.38 ± 3.26
48.34 ± 2.89
90.56 ± 8.41
36.33 ± 2.11
9b
11
12
13
PL
g
8g
menadione
a
MTT methods, cells were incubated with corresponding compounds for 48 h. Values are mean of three independent experiments.
4

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Fig. 4. Comparison of inhibition rates of partial target compounds. (A) Inhibition rates of 11–13 and 7a at 20 μM in MDA-MB-231 cells. (B) Inhibition rates of 7a, 7b,
e and 7g at 20 μM in MDA-MB-231 cells.
7
Table 2
IC50 values of the tested compounds against cancer cells and normal human cells.
Comp.
IC50 (μM)a
SIb
MDA-MB-231
HepG2
A549
MDA-MB-10A
PBMCs
HFL-1
MDA-MB-10A/MDA-MB-231
7
7
7
7
9
a
b
c
6.01 ± 0.22
11.19 ± 1.73
16.02 ± 2.21
19.36 ± 0.73
7.69 ± 0.77
14.23 ± 0.93
7.20 ± 0.52
10.95 ± 2.40
17.08 ± 2.62
19.56 ± 0.47
8.75 ± 0.97
10.66 ± 1.54
7.02 ± 0.46
11.98 ± 1.22
8.34 ± 1.45
12.85 ± 2.02
7.98 ± 0.35
12.40 ± 1.08
26.54 ± 1.29
20.14 ± 1.66
25.69 ± 1.68
23.52 ± 1.57
ND
12.52 ± 0.98
11.38 ± 1.01
ND
4.42
1.80
ND
c
ND
d
a
ND
ND
ND
ND
11.42 ± 0.74
9.12 ± 0.49
10.24 ± 0.84
10.12 ± 0.72
14.68 ± 1.23
3.21 ± 0.24
1.49
0.64
PL
a
b
c
MTT methods, cells were incubated with corresponding compounds for 48 h. IC50 (μM) values (means ± SD, n = 3).
Selective index;
Not determined.
2
.9. The morphological apoptosis induced by 7a
24.4% of the cells, which was much higher than that of parent com-
pound PL (13.3%) (Fig. 11B). These results suggested that the cap-
ability of inducing apoptosis of 7a was enhanced compared to PL.
The inhibition of STAT3 signaling pathway usually causes sig-
nificant apoptosis in cancer cells. The morphological apoptosis caused
by 7a was then detected by Hoechst 33,342 staining. It was clear that
the cells were killed obviously by the treatment of 7a. Simultaneously,
the apoptotic cells were stained with light blue (white arrows, Fig. 10).
These results illustrated that 7a could induce apoptosis of cancer cells.
3
. Conclusions
In summary, a series of compounds derived from PL or menadione
were designed, synthesized, and biologically evaluated with STAT3
inhibitory activity. SARs revealed that the hydroxyl group (C-5) was
important for the activity. According to the above results and the re-
ported inhibitors, we found that the formation of hydrogen bond be-
tween suitable substituents at C-5 and STAT3 might enhance the
combination of small molecular with STAT3. Thus, the presence of OH
at C-5 in PL derivatives could interact with STAT3 in the form of hy-
drogen bond, which was conducive to the binding of this kind structure
to STAT3. In addition, the introduction of additional groups in aromatic
2.10. Effects of 7a on cell apoptosis
In order to further quantitatively evaluated the apoptosis induced
by 7a, an annexin V-FITC/PI dual staining assay was used in MDA-MB-
2
31 cells. As shown in Fig. 11A, after 48 h of treatment with 7a,
apoptosis was induced in a dose-manner. Furthermore, at a con-
centration of 15 μM, the apoptotic cells induced by 7a accounted for
Fig. 5. Compound 7a inhibited STAT3 phosphorylation in MDA-MB-231. The MDA-MB-231 were serum-starved overnight, then left untreated or treated with PL
(
10.0 μM) and 7a (2.5, 5.0, 10 μM) for 6 h, followed by stimulation by IL-6 (25 ng/mL). The cells were harvested at 30 min and analyzed by Western blot assays.
5

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Fig. 6. Effects of compound 7a on expression of STAT3 downstream genes (Survivin and Mcl-1) in MDA-MB-231. The MDA-MB-231 were serum-starved overnight,
then treated with DMSO or 7a (2.5, 5.0, 10 μM) for 6 h, followed by stimulation by IL-6 (25 ng/mL). The cells were harvested at 30 min and analyzed by Western blot
assays.
genes without affecting the activated Src, JAK2 and STAT1.
Furthermore, the docking studies revealed that compound 7a could
bind to the SH2 domain of STAT3 protein more tight than PL. This
function allowed 7a induced a superior apoptosis against cancer cells
compared to PL. Together, these results warranted its potential to be a
promising STAT3 inhibitor in the cancer treatment.
4
. Experimental
.1. Chemistry
All reagents were purchased from commercial companies. H NMR
4
1
1
3
and C NMR spectra were recorded on a Bruker AVANCE instrument at
5 °C. The molecular weights were detected on HP 1100LC/MSD
spectrometer.
2
4
.1.1. Syntheses of intermediates and final compounds
4
.1.1.1. General procedures for the preparation of 2a-2g. Corresponding
Fig. 7. Effects of compound 7a on upstream kinases of STAT3 in MDA-MB-231.
MDA-MB-231 were serum-starved overnight, then treated with DMSO or 7a
acyl chloride (1a-1g, 5 mmol) was added to a solution of β-alanine
(489.5 mg, 5.5 mmol) in sodium hydroxide solution (5 M, 2 mL).
Sodium hydroxide solution (5 M) was added to the mixture to keep the
(
2.5, 5.0, 10 μM) for 6 h, followed by stimulation by IL-6 (25 ng/mL). The cells
were harvested at 30 min and analyzed by Western blot assays.
pH was 9–10. After 30 min, the reaction was washed with CH
2
Cl
2
(
5 mL) and the water layer was poured into 20 mL of ice water, the pH
ring and suitable linkers could also contribute to the activity. These
preliminary results might provide a theoretical basis for the further
development of STAT3 inhibitors. Among the target compounds, 7a
displayed the most potent inhibition. Western blotting showed that 7a
could inhibit the phosphorylation of STAT3 as well as the downstream
of mixture was changed to 2 with HCl (1 M), filtered, then washed with
excess cold water and dried. The intermediates were obtained as white
solids.
4.1.1.2. Syntheses of 6. DIPEA (645 mg, 5 mmol) was added to a
Fig. 8. Effects of compound 7a on STAT1 phosphorylation in MDA-MB-231. MDA-MB-231 were serum-starved overnight, then treated with DMSO or 7a (2.5, 5.0,
1
0 μM) for 6 h, followed by stimulation by IFN-γ (50 ng/mL). The cells were harvested at 30 min and analyzed by Western blot assays.
6

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Fig. 9. Docking model of 7a with the STAT3 SH2 domain. The protein (PDB ID 1BG1) was shown in a cartoon representation. Hydrogen bonds were shown as dashed
yellow lines and cation-π interactions shown as blue dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
solution of 3 (737 mg, 2.1 mmol) in dry CH
chloride (4, 356 mg, 2 mmol) was added slowly to the mixture after
0 min and stirred for 6 h at r.t. Then the reaction solution was added
into CH Cl (15 mL), washed by sodium hydroxide solution (0.5 M,
2
Cl
2
(5 mL) at 0 °C. Nicotinyl
4.1.1.3.2. N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
dihydronaphthalen-2′-yl)ethyl)-4-nitrobenzamide
(7b). Yellow
oil,
1
1
114 mg (38% yield). H NMR (300 MHz, CDCl
3
) δ
H
12.05 (s, 1H),
2
2
8.29 (d, J = 9.0 Hz, 2H), 7.90 (d, J = 9.0 Hz, 2H), 7.62 (m, 2H), 7.23
3
× 15 mL). The organic layer was dried with Na
2
SO and the solvent
4
(d, J = 1.8 Hz, 1H), 6.70 (s, 1H), 3.68 (t, J = 6.3 Hz, 2H), 3.04 (t,
1
3
was evaporated. The crude material was purified via column
J = 6.6 Hz, 2H), 2.28 (s, 3H). C NMR (75 MHz, CDCl
3
) δ 204.6,
C
chromatography (PE/EA = 1:3) to yield 5 (159 mg, 56%). Pd/C
161.3, 136.5, 130.9, 128.0, 124.1, 124.0, 119.3, 39.3, 26.2, 13.1. ESI/
+
(
100 mg) was added to a solution of 5 (1.8 mmol) in MeOH (15 mL).
HRMS (m/z) [M+H] 381.1080. Calcd for [C20
H
17
N
2
6
O ]: 381.1087.
The mixture was sonicated at 50 °C under H
2
for 12 h and then filtered
4.1.1.3.3. 2-fluoro-N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
with tripoli and the filtrate evaporated. The crude material was purified
dihydronaphthalen-2′’-yl)ethyl)benzamide (7c). Yellow oil, 127 mg (36%
1
via column chromatography (PE/EA = 1:3) to yield 6 (252 mg, 71%).
yield). H NMR (300 MHz, CDCl
3
) δ
H
12.15 (s, 1H), 8.07 (td,
J = 8.4 Hz, 1.8 Hz, 1H), 7.59 (m, 2H), 7.45 (m, 1H), 7.23 (m, 2H),
7
2
1
1
.11 (dd, J = 12.0 Hz, 7.5 Hz, 1H), 7.04 (s, 1H), 3.68 (q, J = 6.0 Hz,
4
.1.1.3. General procedures for the preparation of 7a-8g. Corresponding
13
H), 3.04 (t, J = 6.9 Hz, 2H), 2.27(s, 3H). C NMR (75 MHz, CDCl
3
) δ
C
intermediates (3 mmol, 3.0 eq), AgNO (47.3 mg, 0.28 mmol, 0.28 eq)
3
89.1, 183.1, 162.6, 160.2, 145.7, 142.5, 135.0, 132.4, 132.3, 131.0,
30.9, 123.8, 122.8, 118.0, 115.2, 114.8, 113.8, 38.0, 25.5, 12.0. ESI/
were successively added to a solution of PL (188 mg, 1 mmol, 1.0 eq) or
menadione (172 mg, 1 mmol, 1.0 eq) in aqueous acetonitrile (CH
3
CN/
+
HRMS (m/z) [M+H] 354.1136. Calcd for [C20
H
17FNO ]: 354.1142.
4
H
2
O = 4:3, 18 mL) at 60 °C. Then the reaction was heated to 70 °C.
4
.1.1.3.4. N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
Next, a solution of ammonium persulfate (0.8 mmol, 1.3 eq, CH
3
CN/
dihydronaphthalen-2′’-yl)ethyl)-3,5-bis(trifluoromethyl)benzamide
H
2
O = 4:3, 10 mL) was added dropwise over 30 min, and the resulting
1
(
7d). Yellow oil, 131 mg (28% yield). H NMR (300 MHz, CDCl
3
) δ
H
solution stirred at 65–70 °C for 3–4 h. On cooling, the mixture was
1
2.00 (s, 1H), 8.49 (d, J = 9.0 Hz, 3H), 8.08 (d, J = 8.7 Hz, 1H), 7.82
extracted with EtOAc (3 × 20 mL), and the organic layer was washed
(
m, 2H), 6.89 (s, 1H), 6.23 (s, 1H), 3.55 (t, J = 5.4 Hz, 2H), 3.04 (t,
with H
2
O (3 × 20 mL), saturated NaHCO
3
(4 × 20 mL), dried and
13
J = 5.1 Hz, 2H), 2.29(s, 3H). C NMR (75 MHz, CDCl
3
) δ 189.8,
C
solvent evaporated. The crude material was purified via column
1
1
63.6, 160.4, 146.1, 142.4, 135.4, 131.1, 126.1, 124.1, 123.9, 123.1,
chromatography (PE/EA = 10:1) to yield 7a-9b.
+
18.3, 38.4, 25.2, 12.0. ESI/HRMS (m/z) [M+H] 472.0972. Calcd for
4.1.1.3.1. N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
[
C
22
4
H
17FNO ]: 472.0984.
4
dihydronaphthalen-2′’-yl)ethyl)benzamide (7a). Yellow oil, 93 mg (28%
.1.1.3.5. N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
1
yield). H NMR (300 MHz, CDCl
3
) δ 12.12 (s, 1H), 7.72 (d, J = 8.4 Hz,
H
dihydronaphthalen-2′’-yl)ethyl)-4-methoxybenzamide (7e). Yellow oil,
2
6
3
1
H), 7.61 (m, 2H), 7.48 (m, 3H), 7.23 (dd, J = 7.8 Hz, 1.8 Hz, 1H),
.57 (s, 1H), 3.65 (q, J = 6.9 Hz, 2H), 3.02 (t, J = 6.6 Hz, 2H), 2.27(s,
1
1
09 mg (30% yield). H NMR (300 MHz, CDCl
3
) δ
H
12.06 (s, 1H),
7
.68 (m, 2H), 7.51 (t, J = 8.1 Hz, 2H), 7.17 (t, J = 8.7 Hz, 1H), 6.84 (d,
1
3
H). C NMR (75 MHz, CDCl
3
C
) δ 189.5, 166.7, 160.3, 145.9, 142.7,
J = 7.5 Hz, 2H), 6.44 (s, 1H), 3.78(s, 3H), 3.56 (d, J = 5.7 Hz, 2H),
35.2, 130.6, 127.6, 125.8, 125.8, 122.9, 118.2, 38.0, 25.5, 12.1. ESI/
13
2
.94 (t, J = 6.3 Hz, 2H), 2.19(s, 3H, CH
3
). C NMR (75 MHz, CDCl
3
) δ
C
+
HRMS (m/z) [M+H] 336.1227. Calcd for [C20
H
18NO ]: 336.1236.
4
Fig. 10. The morphological apoptosis induced by 7a in MDA-MB-321cells. MDA-MB-321 cells were treated with 7a or DMSO for 48 h. Fluorescence microscopy
images of MDA-MB-231 cells stained with Hoechst 33342.
7

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Fig. 11. Apoptotic effects of 7a and PL in
MDA-MB-231 cells. Treatment with DMSO
or 7a or PL for 48 h, MDA-MB-231 cells
were collected and stained with Annexin V/
PI, followed by flow cytometric analysis. (A)
Flow cytometry analysis of MDA-MB-231
cells treated with DMSO, 7a and PL. (B)
Representative histograms for the numbers
of living cells and apoptotic cells (%). The
values were presented as the mean ± SD
(
n = 3).
1
1
3
89.5, 183.1, 166.2, 160.3, 142.7, 135.2, 128.4, 127.6, 125.4, 122.9,
HRMS (m/z) [M+H]+ 338.1189. Calcd for [C20
H
17FNO ]: 338.1192.
3
+
18.1, 112.8, 112.7, 54.4, 38.0, 25.6, 12.1. ESI/HRMS (m/z) [M+H]
4.1.1.3.11. N-(2′-(3′’-methyl-1′’,4′’-dioxo-1,4-dihydronaphthalen-2′’-
66.1332. Calcd for [C21
H
20NO
5
]: 366.1341.
yl)ethyl)-3,5-bis(trifluoromethyl)Benzamide (8d). Yellow oil, 150 mg
1
4
.1.1.3.6. 4-fluoro-N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
(33% yield). H NMR (300 MHz, CDCl
3
) δ
H
8.15 (s, 2H), 8.02 (m,
dihydronaphthalen-2′’-yl)ethyl)benzamide (7f). Yellow oil, 141 mg (40%
2H), 7.92 (s, 1H), 7.66 (m, 2H), 7.03 (s, 1H), 3.61 (t, J = 5.7 Hz, 2H),
1
13
yield). H NMR (300 MHz, CDCl
3
) δ
H
12.10 (s, 1H), 7.75 (d, J = 5.4 Hz,
2.97 (t, J = 6.3 Hz, 2H), 2.24(s, 3H). C NMR (75 MHz, CDCl
) δ
3 C
2
6
3
1
2
H), 7.57 (m, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.10 (t, J = 6.9 Hz, 2H),
185.6, 183.7, 163.4, 144.8, 142.6, 135.1, 133.0, 132.7, 131.0, 126.2,
+
.57 (s, 1H), 3.64 (d, J = 5.4 Hz, 2H), 3.01 (t, J = 6.0 Hz, 2H), 2.26 (s,
125.5, 125.3, 124.0, 38.5, 25.3, 11.8. ESI/HRMS (m/z) [M+H]
1
3
H). C NMR (75 MHz, CDCl
3
) δ
C
190.6, 166.7, 161.3, 146.9, 143.7,
456.1033. Calcd for [C22
H
F
16 6
NO ]: 456.1034.
4
36.3, 132.1, 129.2, 129.1, 124.0, 119.2, 115.8, 115.5, 115.4, 39.1,
4.1.1.3.12. 4-methoxy-N-(2′-(3′’-methyl-1′’,4′’-dioxo-1,4-
+
6.5, 13.1. ESI/HRMS (m/z) [M+H]
354.1136. Calcd for
dihydronaphthalen-2′’-yl)ethyl)benzamide (8e). Yellow oil, 122 mg (35%
1
[
C
20
4
H
17FNO
4
]: 354.1142.
yield). H NMR (300 MHz, CDCl
3
H
) δ 8.01 (m, 2H), 7.65 (m, 4H), 6.85
.1.1.3.7. N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
(t, J = 6.9 Hz, 2H), 6.53 (s, 1H), 3.77 (s, 3H), 3.55 (q, J = 6.6 Hz, 2H),
13
dihydronaphthalen-2′’-yl)ethyl)-4-methylbenzamide (7 g). Yellow oil,
2.94 (t, J = 6.9 Hz, 2H), 2.21 (s, 3H). C NMR (75 MHz, CDCl
) δ
3 C
1
1
47 mg (42% yield). H NMR (300 MHz, CDCl
3
) δ
H
12.12 (s, 1H),
184.7, 183.9, 166.1, 144.5, 143.0, 132.6, 131.0, 130.9, 127.6, 125.4,
7
.60 (m, 4H), 7.23 (m, 3H), 6.53 (s, 1H), 3.64 (q, J = 6.6 Hz, 2H), 3.01
125.3, 123.4, 118.0, 112.7, 54.4, 38.1, 25.9, 11.9. ESI/HRMS (m/z) [M
1
3
+
(
t, J = 6.9 Hz, 2H), 2.39 (s, 3H), 2.26 (s, 3H). C NMR (75 MHz,
+H] 350.1388. Calcd for [C21
H
20NO ]: 350.1392.
4
CDCl
3
) δ
C
189.5, 183.1, 166.7, 160.2, 142.7, 141.0, 135.2, 131.1,
4.1.1.3.13. 4-fluoro-N-(2′-(3′’-methyl-1′’,4′’-dioxo-1,4-
1
28.9, 128.3, 125.8, 122.9, 118.2, 38.0, 25.5, 20.4, 12.1. ESI/HRMS
dihydronaphthalen-2′’-yl)ethyl)benzamide (8f). Yellow oil, 128 mg (38%
+
1
(
m/z) [M+H] 350.1388. Calcd for [C21
H20NO
4
]: 350.1392.
yield). H NMR (300 MHz, CDCl
3
H
) δ 8.02 (m, 2H), 7.66 (m, 4H), 7.02
4
.1.1.3.8. N-(2-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)
(t, J = 8.4 Hz, 2H), 6.64 (s, 1H), 3.56 (q, J = 6.6 Hz, 2H), 2.94 (t,
1
13
ethyl)benzamide (8a). Yellow oil, 143 mg (42% yield). H NMR
J = 6.6 Hz, 2H), 2.20(s, 3H), 2.26(s, 3H). C NMR (75 MHz, CDCl
3
) δ
C
(
300 MHz, CDCl
) δ
3 H
7.65 (d, J = 8.1 Hz, 2H), 7.55 (m, 2H), 7.38
185.9, 184.8, 166.5, 145.6, 143.9, 133.8, 133.6, 129.3, 129.1, 126.4,
+
(
m, 3H), 7.16 (dd, J = 7.8 Hz, 1.8 Hz, 2H), 6.50 (s, 1H), 3.58 (q,
126.3, 115.7, 115.4, 39.2, 26.8, 12.9. ESI/HRMS (m/z) [M+H]
1
3
J = 6.6 Hz, 2H), 2.95 (t, J = 6.9 Hz, 2H), 2.20(s, 3H). C NMR
338.1189. Calcd for [C20
H
17FNO ]: 338.1192.
3
(
75 MHz, CDCl
3
) δ
C
174.2, 142.9, 132.7, 132.5, 130.5, 127.6, 125.8,
4.1.1.3.14. 4-methyl-N-(2′-(3′’-methyl-1′’,4′’-dioxo-1,4-
+
1
3
25.4, 125.3, 123.3, 38.2, 25.8, 13.1. ESI/HRMS (m/z) [M+Na]
dihydronaphthalen-2′’-yl)ethyl)benzamide (8g). Yellow oil, 133 mg (40%
1
42.1095. Calcd for [C120
3
H17NNaO ]: 342.1106.
yield). H NMR (300 MHz, CDCl
3
H
) δ 8.01 (m, 2H), 7.59 (m, 4H), 7.14
4
.1.1.3.9. N-(2′-(3′’-methyl-1′’,4′’-dioxo-1,4-dihydronaphthalen-2-yl)
(d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 3.56 (q, J = 6.6 Hz, 2H), 2.94 (t,
1
13
ethyl)-4-nitrobenzamide (8b). Yellow oil, 157 mg (43% yield). H NMR
300 MHz, CDCl ) δ 8.08 (m, 2H), 7.72 (m, 4H), 7.45 (m, 2H), 6.70 (s,
H), 3.65 (q, J = 6.1 Hz, 2H), 3.03 (t, J = 6.9 Hz, 2H), 2.28 (s, 3H).
NMR (75 MHz, CDCl ) δ 186.4, 184.7, 165.4, 145.8, 143.8, 140.0,
34.0, 133.7, 128.1, 126.6, 126.4, 123.9, 39.5, 26.5, 12.9. ESI/HRMS
J = 6.6 Hz, 2H), 2.31(s, 3H), 2.20(s, 3H). C NMR (75 MHz, CDCl
3
) δ
C
(
3
H
184.6, 183.9, 166.7, 144.5, 142.9, 132.6, 131.1, 131.0, 128.2, 128.1,
1
3
+
1
C
125.8, 125.3, 125.2, 38.1, 25.9, 20.4, 11.9. ESI/HRMS (m/z) [M+H]
3
C
334.1430. Calcd for [C21
H
20NO ]: 334.1443.
3
1
4.1.1.3.15. N-(2′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
+
(
m/z) [M+H] 365.1128. Calcd for [C20
H
17
N
2
O
5
]: 365.1137.
dihydronaphthalen-2′’-yl)ethyl)isonicotinamide (9a). Yellow oil, 151 mg
1
4
.1.1.3.10. 2-fluoro-N-(2′-(3′’-methyl-1′’,4′’-dioxo-1,4-
(45% yield). H NMR (300 MHz, CDCl
3
) δ 12.07 (s, 1H), 8.75 (dd,
H
dihydronaphthalen-2′’-yl)ethyl)benzamide (8c). Yellow oil, 101 mg (38%
J = 4.5 Hz, 1.5 Hz, 2H), 7.51 (m, 4H), 7.16 (dd, J = 7.5 Hz, 2.1 Hz,
2H), 6.68 (d, J = 5.1 Hz, 1H), 3.59 (q, J = 6.9 Hz, 2H), 2.95 (t,
1
yield). H NMR (300 MHz, CDCl
3
H
) δ 8.08 (m, 3H), 7.70 (m, 2H), 7.46
1
3
(
dd, J = 13.2 Hz, 6.0 Hz, 1H), 7.24 (d, J = 7.5 Hz, 2H), 7.08 (s, 1H),
J = 6.9 Hz, 2H), 2.19(s, 3H). C NMR (75 MHz, CDCl
3
) δ 185.1,
C
1
3
3
.67 (d, J = 6.0 Hz, 2H), 3.04 (t, J = 6.9 Hz, 2H), 2.28(s, 3H). C NMR
) δ 184.0, 184.0, 161.2, 144.3, 142.7, 132.6, 132.5,
30.9, 130.9, 125.3, 123.8, 123.8, 115.1, 114.8, 38.1, 26.0, 12.0. ESI/
178.7, 162.2, 155.9, 150.7, 138.3, 130.8, 123.9, 121.3, 118.5, 113.8,
+
(
75 MHz, CDCl
3
C
109.4, 33.6, 21.1, 13.0. ESI/HRMS (m/z) [M+H] 337.1178. Calcd for
1
[C19H
17
N
2
O ]: 337.1188.
4
8

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
4
.1.1.3.16. N-(2′-(3′’-methyl-1′’,4′’-dioxo-1,4-dihydronaphthalen-2′’-
to yield the purple solid 13 (37 mg, 37%).
1
yl)ethyl)isonicotinamide (9b). Yellow oil, 134 mg(42% yield). H NMR
300 MHz, CDCl ) δ 8.67 (dd, J = 4.5 Hz, 1.8 Hz, 2H), 7.50 (m, 4H),
.16 (dd, J = 7.5 Hz, 2.1 Hz, 2H), 6.68 (d, J = 5.7 Hz, 1H), 3.59 (q,
4.1.1.6.1. 3-amino-5-hydroxy-2-methyl-1,4-naphthoquinone
1
(
3
H
(13). Orange powder, 37 mg (37% yield). M.p.108–110 °C. H NMR
(300 MHz, DMSO‑d ) δ 11.55 (1H, s), 7.76 (1H, d, J = 7.8 Hz), 7.48
(1H, d, J = 8.1 Hz), 7.24 (1H, d, J = 8.4 Hz), 5.84 (2H, brs), 2.01 (3H,
7
6
H
1
3
J = 6.6 Hz, 2H), 2.95 (t, J = 6.9 Hz, 2H), 2.19(s, 3H). C NMR
1
3
(
75 MHz, CDCl
3
) δ
C
180.1, 179.3, 158.1, 152.1, 139.9, 127.9, 123.7,
s). C NMR (75 MHz, DMSO‑d
6
) δ 186.6, 181.5, 160.7, 150.7, 137.8,
C
+
1
20.7, 33.5, 21.4, 15.9. ESI/HRMS (m/z) [M+H] 321.1189. Calcd for
133.9, 122.2, 117.9, 114.6, 103.0, 12.0; HREIMS m/z 204.0655 [M
+
[
C
19
H
17
N
2
O
3
]: 321.1239.
+H] (calcd for C11
H
10NO , 204.0655).
3
4
.1.1.4. Syntheses of 11. Succinic acids (495 mg, 4.5 mmol, 3.0 eq),
4.2. Biological Experiments.
4.2.1. Cytotoxic assay in vitro
AgNO
3
(76 mg, 0.45 mmol, 0.3 eq) were successively added to a
solution of PL (282 mg, 1.5 mmol, 1.0 eq) in 30% aqueous acetonitrile
(
20 mL) at 60 °C. when reaction system heated to 70 °C, a solution of
The antiproliferative activities of test compounds were investigated
by the MTT method. Briefly, human cancer cell lines such as MDA-MB-
231, A549 and HepG2 cells, or normal human cell line PBMCs and HFL-
ammonium persulfate (444.6 mg, 1.95 mmol, 1.3 eq) in 30% aqueous
acetonitrile (12 mL) was added dropwise over 30 min, and the resulting
solution stirred at 65–70 °C for 3–4 h. On cooling, the mixture was
extracted with EtOAc (3 × 30 mL), and the organic layer washed with
4
1 cells at a final density of 5.0 × 10 /ml were seeded into 96-well
plates and allowed to adhere for 24 h. After treated with or without
different concentrations of test compounds for 48 h, the cells were
exposed to MTT (5 mg/mL), and the plate was further incubated in
H
2
O (3 × 30 mL), dried (Na
2
SO4) and solvent evaporated. The crude
Cl
MeOH = 20:1) to yield the intermediate 10 (247 mg, 61%). PyBop
310 mg, 0.55 mmol), DMAP (72.5 mg, 0.55 mmol), TEA (101 mg,
mmol) were successively added to a solution of intermediate 10
135 mg, 0.5 mmol) in dry CH Cl (10 mL). A solution of aniline
material was purified via column chromatography (CH
2
2
/
37 °C CO incubator for another 4 h. Abandoned supernatant before
2
(
adding 100 µL DMSO to each well. Optical density (OD) was measured
1
using spectrophotometer at 570 nm. The number of viable cells was
determined from the absorbance. Assays were performed in triplicate
wells. Data are presented as the mean ± SD (n = 3).
(
(
2
2
116 mg, 1.3 mmol) in dry CH
2
Cl (2 mL). was added slowly and the
2
resulting solution stirred at room temperature for 24 h. The completion
of reaction was monitored by TLC. After completion, poured into 10 mL
4
.2.2. Molecular Modeling.
of CH
2
Cl , washed with 5% HCl (20 mL × 3) and saturated NaCl
2
The X-ray structure of the STAT3 (1BG1) was used. The molecular
(
20 mL × 2), dried (Na SO4) and solvent evaporated. The crude
2
docking software used in this study was Autodock Vina 1.1.2. The
parameters were set to “center x = 101.9; center y = 83.0; center
z = 66.2; size x = 20; size y = 20; size z = 0”.
material was purified via column chromatography (CH
MeOH = 100:1) to yield the orange powder (50 mg, 30%).
2
Cl /
2
4.1.1.4.1. 3′-(8′’-hydroxy-3′’-methyl-1′’,4′’-dioxo-1,4-
dihydronaphthalen-2′’-yl)-N-phenylpropanamide (11). Orange powder,
4
.2.3. Western blot analysis
1
5
1
0 mg (30% yield). M.p.102–104 °C. H NMR (300 MHz, CDCl
): δ
3 H
Cells were seeded in 10 cm plates and allowed to adhere overnight.
2.12 (s, 1H), 7.60 (m, 2H), 7.50 (d, J = 6.0 Hz, 2H), 7.32 (t,
The cells were serum-starved. The cells were then treated with DMSO or
J = 7.5 Hz, 3H), 7.22 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 7.11 (t, J = 7.5 Hz,
7
a. After 6 h, cells were stimulated by IL-6 (25 ng/mL) or IFN (25 ng/
1
3
1
H), 3.07 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 2.26(s, 3H).
C
mL). The cells were harvested after 30 min and analyzed by Western
blot.
NMR (75 MHz, CDCl
3
) δ
C
190.5, 161.3, 146.9, 143.7, 136.2, 131.6,
+
1
3
28.6, 126.8, 123.9, 119.2, 33.0, 20.5, 13.1. ESI/HRMS (m/z) [M+H]
36.1227. Calcd for [C20
H
17NO ]: 336.1236.
4
4
.2.4. Hoechst-33342 Staining.
5
MDA-MB-231 cells (4 × 10 cells) were incubated in 6 cm dish for
4
.1.1.5. Syntheses of 12. Cu(AcO)
2
(9.9 mg, 0.05 mmol), aniline
2
4 h. Then the cells were treated with the DMSO/7a at indicated
(
(
46.5 mg, 0.5 mmol) were successively added to a solution of PL
94 mg, 0.5 mmol) in glacial acetic acid (2 mL), and the mixture was
concentrations for 24 h. Cells were washed twice by PBS. Then cells
were incubated with Hoechst-33342 (10 μg/ml) for 5 min at room
temperature in the darkness. After incubation, stained cells were ob-
served under a fluorescent microscope (CORPORATION, Japan).
stirred at 60 °C for 4 h. Diluted with water (20 mL), extracted with
EtOAc (3 × 15 mL), and the organic layer washed with H O
2
(
3 × 15 mL) and saturated NaCl (15 mL × 1), dried (Na SO4) and
2
solvent evaporated. The crude material was purified via column
4
.2.5. Cell apoptosis analysis
chromatography (PE/EA = 2:1, v / v) to yield the purple solid 12
5
MDA-MB-231 cells (4 × 10 cells) were incubated in 6 cm dish for
(
44 mg, 32%).
4
8 h. Then DMSO/7a/PL was added to the cells with indicated con-
4
.1.1.5.1. 5-hydroxy-2-methyl-3-(phenylamino)naphthalene-1,4-dione
1
centrations. After another incubation of 48 h, the cells were harvested
softly and washed twice with cold PBS (2000 g for 5 min). Then the cells
were added to 250 μL binding buffer containing 2.5 μL PI and 2.5 μL
Annexin V (KeyGEN, China). The mixture was incubated at room
temperature for 15 min in the darkness and analyzed by flow cyto-
metry.
(
12). Orange powder, 44 mg (34% yield). M.p.113–116 °C. H NMR
(
300 MHz, CDCl
3
) δ
H
11.60 (s, 1H), 7.65 (m, 2H), 7.34 (t, J = 8.4 Hz,
3
H), 7.18 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 7.13 (t, J = 7.2 Hz, 1H), 6.97
1
3
(
d, J = 7.5 Hz, 2H), 1.73(s, 3H). C NMR (75 MHz, CDCl
3
) δ 189.5,
C
1
1
2
66.2, 145.8, 142.7, 135.2, 130.6, 127.6, 125.8, 125.8, 122.9, 118.2,
+
2.1. ESI/HRMS (m/z) [M+H] 280.0963. Calcd for [C17
H
14NO ]:
3
80.0974.
Declaration of Competing Interest
4
.1.1.6. Syntheses of 13. Dissolved PL (94 mg, 0.5 mmol) in MeOH
(
5 mL) under N
2
. NaN (3.5 mmol) was dissolved in water (2 mL) and
3
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
acidified to pH 4. To a solution of PL was added NaN solution and
3
slowly heated reaction to 50 °C and stirred for 5 h. Reaction was
quenched with water and extracted with ethyl acetate (10 mL × 3). The
organic layers were combined and washed with saturated NaCl
Acknowledgements
solution, dried over anhydrous Na
2
SO , and concentrated in vacuum.
4
The reaction was purified on silica gel eluting with PE/EA (10:1 to 5:1)
This research has been supported by the Academic Innovation
9

N. Li, et al.
Bioorganic Chemistry 104 (2020) 104208
Project of Jiangsu Department of Education (No. 1150021667) and
[14] O.S. Lee, W. Lou, K.M. Qureshi, F. Mehraein-Ghomi, D.L. Trump, A.C. Gao, RNA
interference targeting Stat3 inhibits growth and induces apoptosis of human pros-
tate cancer cells, Prostate. 60 (2004) 303–309.
“Double First Class” Subject Innovation Team Construction Project of
China Pharmaceutical University (CPU2018GY12).
[
15] L. Gao, L. Zhang, J. Hu, F. Li, Y. Shao, D. Zhao, D.V. Kalvakolanu, D.J. Kopecko,
X. Zhao, D.Q. Xu, Down-regulation of signal transducer and activator of tran-
scription 3 expression using vector-based small interfering rnas suppresses growth
of human prostate tumor in vivo, Clin Cancer Res. 11 (2005) 6333–6341.
16] H. Song, R. Wang, S. Wang, J. Lin, A low-molecular-weight compound discovered
through virtual database screening inhibits Stat3 function in breast cancer cells,
Proc. Natl. Acad. Sci. U.S.A. 102 (2005) 4700–4705.
Appendix A. Supplementary data
[
[
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104208.
17] L. Lin, D.M. Benson Jr., S. Deangelis, C.E. Bakan, P.K. Li, C. Li, J. Lin, A small
molecule, LLL12 inhibits constitutive STAT3 and IL-6-induced STAT3 signaling and
exhibits potent growth suppressive activity in human multiple myeloma cells, Int. J.
Cancer. 130 (2012) 1459–1469.
References
[
18] L. Lin, B. Hutzen, P.K. Li, S. Ball, M. Zuo, S. DeAngelis, E. Foust, M. Sobo,
L. Friedman, D. Bhasin, L. Cen, C. Li, J. Lin, A novel small molecule, LLL12, inhibits
STAT3 phosphorylation and activities and exhibits potent growth-suppressive ac-
tivity in human cancer cells, Neoplasia 12 (2010) 39–50.
[
[
1] J. Bromberg, J.E. Darnell, The role of STATs in transcriptional control and their
impact on cellular function, Oncogene. 19 (2000) 2468–2473.
2] T. Hirano, K. Ishihara, M. Hibi, Roles of STAT3 in mediating the cell growth, dif-
ferentiation and survival signals relayed through the IL-6 family of cytokine re-
ceptors, Oncogene. 19 (2000) 2548–2556.
[
[
[
19] S. Ball, C. Li, P.K. Li, J. Lin, The small molecule, LLL12, inhibits STAT3 phos-
phorylation and induces apoptosis in medulloblastoma and glioblastoma cells, PLoS
One. 6 (2011) e18820.
[
[
[
3] S. Haftchenary, M. Avadisian, P.T. Gunning, Inhibiting aberrant Stat3 function with
molecular therapeutics: a progress report, Anticancer Drugs. 22 (2011) 115–127.
4] B.D.G. Page, D.P. Ball, P.T. Signal transducer and activator of transcription 3 in-
hibitors: a patent review. Expert Opin. Ther. Pat. 21 (2011) 65–83.
5] S. Fletcher, J. Turkson, P.T. Molecular approaches towards the inhibition of the
signal transducer and activator of transcription 3 (Stat3) protein. Gunning. Chem.
Med. Chem. 3 (2008) 1159–1168.
20] D. Bhasin, K. Cisek, T. Pandharkar, N. Regan, C. Li, B. Pandit, J. Lin, P.K. Li, Design,
synthesis, and studies of small molecule STAT3 inhibitors, Bioorg Med Chem
Letters. 18 (2008) 391–395.
21] J. Turkson, D. Ryan, J.S. Kim, Y. Zhang, Z. Chen, E. Haura, A. Laudano, S. Sebti,
A.D. Hamilton, R. Jove, Phosphotyrosyl peptides block Stat3-mediated DNA
binding activity, gene regulation, and cell transformation, J Biol Chem. 276 (2001)
[
[
[
6] W. Yu, H. Xiao, J. Lin, C. Li, Discovery of novel STAT3 small molecule inhibitors via
in silico site-directed fragment-based drug design, J. Med. Chem. 56 (2013)
4
5443–45455.
[
22] J. Turkson, J.S. Kim, S. Zhang, J. Yuan, M. Huang, M. Glenn, E. Haura, S. Sebti,
A.D. Hamilton, R. Jove, Novel peptidomimetic inhibitors of signal transducer and
activator of transcription 3 dimerization and biological activity, Mol Cancer Ther. 3
4
402–4412.
7] P.K. Mandal, F. Gao, Z. Lu, Potent and selective phosphopeptide mimetic prodrugs
targeted to the Src homology 2 (SH2) domain of signal transducer and activator of
transcription 3, J. Med. Chem. 54 (2011) 3549–3563.
(
2004) 261–269.
[
[
23] Chinese Herbalism Editorial Board, State Administration of Traditional Chinese
Medicine of the People's Republic of China. Zhong Hua Ben Cao, 6, Shanghai
Science and Technology Press, (2000) 132.
8] L.B. Mora, R. Buettner, J. Seigne, J. Diaz, N. Ahmad, R. Garcia, T. Bowman, R.
Falcone, R. Fairclough, A. Cantor, C. Muro-Cacho, S. Livingston, J. Karras, J. Pow-
Sang, R. Constitutive activation of Stat3 in human prostate tumors and cell lines:
direct inhibition of Stat3 signaling induces apoptosis of prostate cancer cells. Jove.
Cancer Res. 62 (2002) 6659–6666.
24] A.A. Aly, E.M. El-Sheref, M.E.M. Bakheet, M.A.E. Mourad, A.B. Brown, S. Bräse,
M. Nieger, M.A.A. Ibrahim, Synthesis of novel 1,2-bis-quinolinyl-1,4-naphthoqui-
nones: ERK2 inhibition, cytotoxicity and molecular docking studies, Bioorganic
Chem. 81 (2018) 700–712.
[
9] B.E. Barton, J.G. Karras, T.F. Murphy, A. Barton, H.F. Huang, Signal transducer and
activator of transcription 3 (STAT3) activation in prostate cancer: Direct STAT3
inhibition induces apoptosis in prostate cancer lines, Mol. Cancer Ther. 3 (2004)
[
[
25] A.A. Aly, E.M. El-Sheref, M.E.M. Bakheet, M.A.E. Mourad, S. Bräse, M.A.A. Ibrahim,
M. Nieger, B.K. Garvalov, K.N. Dalby, T.S. Kaoud, Design, synthesis and biological
evaluation of fused naphthofuro[3,2-c] quinoline-6,7,12-triones and pyrano[3,2-c]
quinoline-6,7,8,13-tetraones derivatives as ERK inhibitors with efficacy in BRAF-
mutant melanoma, Bioorganic Chem. 82 (2019) 290–305.
1
1–20.
[
[
10] S. Fletcher, J.A. Drewry, V.M. Shahani, B.D. Page, P.T. Gunning, Molecular dis-
ruption of oncogenic signal transducer and activator of transcription 3 (STAT3)
protein, Biochem. Cell Biol. 87 (2009) 825–833.
26] W. Yan, B. Tu, Y.Y. Liu, T.Y. Wang, H. Qiao, Z.J. Zhai, H.W. Li, T.T. Tang,
Suppressive effects of plumbagin on invasion and migration of breast cancer cells
via the inhibition of STAT3 signaling and down-regulation of inflammatory cyto-
kine expressions, Bone Res. 1 (2013) 362–370.
11] B.D. Page, D.C. Croucher, Z.H. Li, S. Haftchenary, V.H. Jimenez-Zepeda,
J. Atkinson, P.A. Spaqnuolo, Y.L. Wong, R. Colaquori, A.M. Lewis, A.D. Schimmer,
S. Truder, P.T. Gunning, Inhibiting aberrant signal transducer and activator of
transcription protein activation with tetrapodal, small molecule src homology 2
domain binders: promising agents against multiple myeloma, J. Med. Chem. 56
[
[
27] O. Trott, A.J. Olson, AutoDock Vina: improving the speed and accuracy of docking
with a new scoring function, efficient optimization and multithreading, J. Comput.
Chem. 31 (2010) 455–461.
(
2013) 7190–7200.
[
[
12] S. Fletcher, B.D. Page, X. Zhang, P. Yue, Z.H. Li, S. Sharmeen, J. Singh, W. Zhao,
A.D. Schimmer, S. Truder, J. Turkson, P.T. Gunning, Antagonism of the Stat3–Stat3
protein dimer with salicylic acid based small molecules, Chem. Med. Chem. 6
28] G. Naturale, M. Lamblin, C. Commandeur, F.X. Felpin, J. Dessolin, Direct C-H al-
kylation of naphthoquinones with amino acids through a revisited Kochi-Anderson
radical decarboxylation: trends in reactivity and applications, Eur. J. Org. Chem. 44
(
2011) 1459–1470.
(
2013) 5774–5788.
13] H. Yu, R. Jove, The STATs of cancer-new molecular targets come of age, Nat. Rev.
Cancer. 4 (2004) 97–105.
[
29] Y. Zhang, Z.Y. Liu, STAT1 in cancer: friend or foe? Discov. Med. 24 (2017) 19–29.
10