Novel NO-releasing plumbagin derivatives: Design, synthesis and evaluation of antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2017.05.046

A series of plumbagin/NO donor hybrids were designed, synthesized and evaluated in vitro against triple negative breast cancer (MDA-MB-231), hepatocellular (HepG2) and lung (A549) carcinoma cells. Most furoxan-based plumbagin derivatives exhibited significantly superior potency compared to their parent compound. Noticeably, MDA-MB-231 cells are the most sensitive to these furoxan-based plumbagin derivatives as evidenced by IC₅₀ values ranging from 1.24 to 5.20 μM. Besides, NO released amounts detection of all hybrids suggested that in most cases, the antiproliferative activities were positively correlated with the levels of intracellular NO release in MDA-MB-231 cells. The most active compound (11a) also possessed higher chemical stability at different pHs (6.0, 7.4 and 8.0) than plumbagin. Together, the above promising results warrant the future potential of plumbagin/NO hybrids as the lead compounds against triple negative breast cancer deserving further research.

Full text of DOI:10.1016/j.ejmech.2017.05.046

Accepted Manuscript
Novel NO-releasing plumbagin derivatives: Design, synthesis and evaluation of
antiproliferative activity
Na Bao, Jinfeng Ou, Manyi Xu, Fuqin Guan, Wei Shi, Jianbo Sun, Li Chen
PII:
S0223-5234(17)30407-5
10.1016/j.ejmech.2017.05.046
EJMECH 9476
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 December 2016
Revised Date: 9 January 2017
Accepted Date: 21 May 2017
Please cite this article as: N. Bao, J. Ou, M. Xu, F. Guan, W. Shi, J. Sun, L. Chen, Novel NO-releasing
plumbagin derivatives: Design, synthesis and evaluation of antiproliferative activity, European Journal of
Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.05.046.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel NO-releasing Plumbagin Derivatives: Design, Synthesis and Evaluation of
Antiproliferative Activity
1
†
1
†
1
2
1
1,
Na Bao , Jinfeng Ou , Manyi Xu , Fuqin Guan , Wei Shi , Jianbo Sun *, and Li
1
,
Chen *
1
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24
Tong Jia Xiang, Nanjing 210009, China.
2
Institute of Botany, Jiangsu Province and Chinese Academy of Science, Nanjing
2
10014, China.
†
These authors contributed equally to this work.
Corresponding author: Tel: +86 83271415
*
E-mail address: sjbcpu@gmail.com (Jianbo Sun); chenli627@cpu.edu.cn (Li Chen)
ABSTRACT
A series of plumbagin/NO donor hybrids were designed, synthesized and evaluated in
vitro against triple negative breast cancer (MDA-MB-231), hepatocellular (HepG2)
and lung (A549) carcinoma cells. Most furoxan-based plumbagin derivatives
exhibited significantly superior potency compared to their parent compound.
Noticeably, MDA-MB-231 cells are the most sensitive to these furoxan-based
plumbagin derivatives as evidenced by IC50 values ranging from 1.24 to 5.20 µM.
Besides, NO released amounts detection of all hybrids suggested that in most cases,
the antiproliferative activities were positively correlated with the levels of
intracellular NO release in MDA-MB-231 cells. The most active compound (11a) also

ACCEPTED MANUSCRIPT
possessed higher chemical stability at different pHs (6.0, 7.4 and 8.0) than plumbagin.
Together, the above promising results warrant the future potential of plumbagin/NO
hybrids as the lead compounds against triple negative breast cancer deserving further
research.
Keywords
Plumbagin; NO-releasing; Cytotoxicity; Selectivity; Stability.

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1
. Introduction
Triple negative breast cancer (TNBC), which is characterized by the absence of
traditional receptors-estrogen receptor (ER), progesterone receptor, and Her-2/neu
(ErbB2), represent a very aggressive subtype of breast cancer. Due to its poor
prognosis, high incidence of local recurrence and distant metastases, the development
of effective anti-TNBC agents, particularly those obtained from natural products has
become a hotspot in breast cancer research [1, 2]. Quinones account for one of the
largest families of antitumor agents. A large number of natural products as well as
synthetic drugs containing quinone chromophor have exerted anticancer effects such
as Napabucasin (BBI608), β-lapachone (ARQ761) and STA-21 [3-6], some of them
have been successfully developed for clinical use for breast cancer treatment (Fig. 1).
The
1,4-naphthoquinone-based
compound,
plumbagin
(5-hydroxy-2-methyl-1,4-naphthoquinone) was isolated from Plumbago species,
which have been extensively used for the treatment of rheumatic arthralgia, abscess
and scrofula in Traditional Chinese Medicines (TCMs) (Fig. 1) [7]. In recent years,
plumbagin has been attracting a rising attention from cancer biologists due to its
attractive antitumor activity. Analyses of the sensitivity of the various cancer cell
lines indicated that breast, lung, cervical, leukemia and liver cancer cells are sensitive
to plumbagin (1) [8]. Notably, the naturally occurring plumbagin (1) was also found
to efficiently induce apoptosis in triple negative breast cancer MDA-MB-231 cells by
concomitant downregulation of Bcl-2 expression and NF-κB activity with no effect on
MCF-10A cells, the normal breast epithelial cells [9]. However, the development of

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plumbagin for cancer therapy was hampered largely by its moderate activity and poor
physico-chemical property, especially inferior stability in basic condition,
sublimability and penetrating odor etc.. Therefore, it is highly desirable to develop
novel chemically stable derivatives of plumbagin to improve its potency without
reducing its safety profile .
O
O
O
O
O
O
O
O
OH
O
O
O
O
OH
Plumbagin (1)
Napabucasin
β-Lapachone
STA-21
O
O
O
OH
O
H
N
NH2
OH
OH
O
O
HN
HN
OH
OH
O
O
O
OH
O
2
H N
OCH3
O
OH
O
N
NH
OH
N
H
OH
NH2
Doxorubicin
Mitoxantrone
Mitomycin C
Fig. 1. The structure of plumbagin, and several examples of
naphthoquinone-containing natural and synthetic antitumor agents.
Nitric oxide (NO) is a key signaling molecule involved in tumorigenesis. While high
level of NO can induce cell cycle arrest and apoptosis, particularly in tumor cells [10].
Indeed, numerous NO-based anti-cancer agents have been developed for the potential
application for cancer therapy. Furoxans, an important class of NO donors, are
thermally stable and able to produce high level of NO. Their excellent performance
makes them attractive for designing antitumor drugs [11]. In the past decade, a variety
of promising furoxan-based NO-releasing derivatives have been investigated as
anticancer drug candidates [12-14].

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Enlightened by these findings, together with our previous studies on the modification
of natural products, we decided to introduce varying carboxylic acid side chains into
the C-3 position of the quinone ring, and then designed and synthesized a series of
novel plumbagin/NO donor hybrids containing phenylsulfony-substituted furoxans to
improve the antiproliferative activity and stability of naturally-occuring plumbagin.
Herein, a total of 12 plumbagin/NO donor hybrids (10a-12d) have been synthesized
1
13
with their structure determined by IR, H NMR, C NMR and ESI/HRMS. The
cytotoxic activities of these derivatives on the proliferation of breast cancer
(MDA-MB-231) cells, hepatocellular (HepG2) and lung carcinoma (A549) cells were
determined by MTT method, respectively. We also detected the NO released amounts
of these derivatives by Griess assay to investigate the relationship between NO
released amounts and cytotoxic activities.
2
. Results and discussion
2
.1. Chemistry
3
,4-Diphenylsulfonyl-furoxan (
and the general route is outlined in Scheme 1. The starting material
benzenethiol ( ) was converted to 2-(phenylthio) acetic acid (4) by treatment
with chloroacetic acid ( ). Compound was oxidized by 30% H O aqueous
solution to give 2- (phenylsulfonyl) acetic acid (
HNO to form diphenylsulfonyl-furoxan ( ). Then the substituted furoxans
with corresponding diols. It
is noteworthy that low temperature (-15°C) and low ratio of NaOH (less than
6) was prepared as previously described [15],
2
3
4
2
2
5
) and reacted with fuming
6
3
(
7a-7d) were obtained from compound 6

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1
.5 eq.) are required for preparing substituted furoxans (7a-7d). Otherwise,
both of phenylsulfonyls would be substituted simultaneously.
O
O
O
S
O
S
OH
a
OH
b
SH
Cl
+
OH
O
5
2
3
4
7
2 2
a: R = (CH )
PhO2S
SO Ph
HORO
SO Ph
2
2
7b: R = (CH2)3
c
d
7
c: R = (CH2)4
N
N
N
N
O
O
O
O
7d: R = CH CH(CH )CH
2 3 2
6
7a-d
Scheme 1. Reagents and conditions (a) NaOH, r.t. 3 h, reflux 1 h (b) H O , r.t.
2
2
3
h
h (c) fuming HNO , 100°C 4 h (d) corresponding diols, 25% NaOH, -15°C 3
3
Scheme 2. Reagents and conditions: (a) HOOC(CH ) COOH (3.0 eq), silver
2
n
nitrate (0.3 eq), ammonium persulfate (1.3 eq), aq. 30% CH CN, 65-70°C, 3 h
3
(
b) 7a-7d, EDCI/DMAP, dry DCM, 0°C to r.t., 6h
The 1,4-dihydronaphthalenyl carboxylic acids (9a-9c) were prepared by
combining plumbagin ( ) with varying length of dicarboxylic acids ( ) through
1
8
oxidative decarboxylation and Kochi-Anderson addition following the
procedure of Salmon-Chemin et al [16].

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The plumbagin/furoxan hybrids (10a-12d) were synthesized in moderate to
high yields (28%-71%) by condensation of semisynthesized plumbagin
analogues (9a-9c) with intermediates (7a-7d). As shown in Scheme 2.
2
.2. In vitro cytotoxic activity
Table 1
Preliminary inhibitory effects of the tested compounds on MDA-MB-231 and HepG2
cells
Inhibition rate (%)
Inhibition rate (%)
at 10 µM*
at 10 µM*
Cpd.
Cpd.
MDA-MB-231 HepG2
MDA-MB-231
HepG2
1
0a
0b
0c
0d
1a
1b
1c
1d
97.3
97.7
96.3
97.3
97.6
96.8
97.6
97.4
93.3
97.2
87.2
96.6
95.7
95.1
95.4
73.4
12a
12b
12c
12d
PL
9a
97.9
95.1
1
95.3
92.7
1
92.7
89.2
1
94.4
91.5
1
32.8
41.0
1
2.4**
5.0**
6.1***
11.0**
20.3**
23.2***
1
9b
1
9c
*
MTT methods, cells were incubated with corresponding compounds at a
concentration of 10 µM for 72 h. Values are mean of three independent experiments.
*
* Inhibition rate (%) at 40 µM.
** Inhibition rate (%) at 20 µM.
*
We first detected the preliminary inhibitory effects of plumbagin (1) and all the
derivatives against two tumor cell lines (MDA-MB-231 and HepG2 cells) by MTT
method (Table 1). The antiproliferative activity of the plumbagin derivatives (9a-9c)
which varying carboxylic acids were introduced to the C-3 position of the quinone
ring were inferior compared with the parent compound plumbagin (1). Encouragingly,

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all plumbagin/NO donor hybrids displayed higher inhibition rate (73.4%-97.9%) than
plumbagin (32.8%-41.0%) at a concentration of 10 µM, which inspired us for further
assay of cytotoxic activities against MDA-MB-231, HepG2 and A549 cells. Their
IC50 Values were listed in Table 2. 1,4-naphthoquinone-based anticancer agent
Napabucasin and the parent compound plumbagin (PL) were selected as the positive
controls.
Almost all plumgabin/NO donor hybrids have exhibited significantly superior potency
compared to plumbagin (1), among which compound 11a possessed the highest and
selective cytotocixicy against MDA-MB-231 cells with IC50 of 1.21 µM despite
inferior than the positive control Napabucasin. Further analysis of the above results
also concluded that their inhibition effects remained some difference on these three
cancer cell lines. Unlike their parent compound (1) , nearly all NO hybrids exhibited
better selectivities for breast cancer cell (MDA-MB-231) than hepatocellular (HepG2)
and lung carcinoma (A549) cells.

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Table 2
Antiproliferative activity of all the tested compounds against three cancer cell lines
Cytotoxicity IC (µM)
Selectivity ratio IC50
(231)/IC50
Cytotoxicity IC (µM)
Selectivity ratio IC50
(231)/IC50
50
50
Cpd.
Cpd.
MDA-MB-
31
MDA-MB
-231
HepG2
A549
(HepG2)/IC (A549)
HepG2
A549
(HepG2)/IC50 (A549)
50
2
1
0a
0b
0c
0d
1a
1b
1c
3.27 ± 0.16
1.76 ± 0.03
5.20 ± 0.13
1.51 ± 0.07
6.11 ± 0.51
4.41 ± 0.12
7.72 ± 0.92
3.00 ± 0.19
18.58 ± 2.0
7.00 ± 0.50
>20
1: 1.9: 15.7
1: 2.5: 4.0
1: 1.5: >3.8.
1: 2.0: 3.9
11d
12a
12b
12c
2.67 ± 0.10
1.52 ± 0.11
2.42 ± 0.10
1.98 ± 0.28
6.69 ± 0.08
4.16 ± 0.57
4.32 ± 0.50
4.24 ± 0.23
18.17 ± 1.0
10.25 ± 0.29
13.94 ± 1.79
19.64 ± 4.30
1: 2.5: 6.8
1: 2.7: 6.7
1: 1.8: 5.8
1: 2.1: 10.0
1
1
1
5.83 ± 0.29
1
1.21 ± 0.14
4.31 ± 0.13
1.24 ± 0.10
2.98 ± 0.37
6.79 ± 0.59
3.33 ± 0.43
9.31 ± 0.58
>20
1: 2.5: 7.7
1: 1.6: >4.7
1: 2.7: 6.7
12d
2.11 ± 0.08
14.23 ± 0.93
0.34 ± 0.02
3.74 ± 0.36
10.66 ± 1.54
0.66 ± 0.16
12.82 ± 1.33
12.40 ± 1.08
0.74 ± 0.04
1: 1.8: 6.1
1: 0.7: 0.9
1: 1.8: 2.2
1
PL
b
1
8.31 ± 0.24
Nap
a
b
MTT methods, cells were incubated with corresponding compounds for 72 h. IC (µM) values (means ± SD, n = 3). Nap, Napabucasin
5
0
(BBI608), as positive control.

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Structure and activity relationships (SARs) revealed that mere introduction of
carboxylic acids to the C-3 position of the quinone ring (9a, 9b and 9c) could
diminish the antiproliferative activities, whereas the hybrids coupling with NO donor
moieties in general enhanced the cytotoxicities significantly. Furthermore, various
length of the carboxylic acids and diol linkers of theses hybrids had certain
differences to potency. Generally, when containing two or three carbons in carboxylic
acid linker (n = 2, 3), the compounds with ethoxy- (11a and 12a) and butoxy-furoxan
moieties (11c and 12c) manifested a bit higher potency than the propoxy- and
2
-methylpropoxy-furoxans (11b, 11d, 12b and 12d). Whereas the propoxy and
-methylpropoxy (10b and 10d) would be favored on the condition of one carbon in
2
carboxylic acid linker (10a and 10c).
.3. In vitro NO released amounts detection
2
Fig. 2. NO released amounts change curves of partial hybrids producing vs time
mean ± SD, n = 3).
(

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In an effort to test whether the cytotoxic activities were correlated with the
concentration of NO produced by these hybrides, we calculated the amounts of nitrate
and nitrite, the oxidative end products of NO using the Griess assay according to the
standard curve [17]. Then NO amounts change curves of the selected partial hybrids
(10c, 10d, 11b, 11d, 11c, 12b and 12d) producing with time in MDA-MB-231 cells
were shown in Figure 2. NO released amounts of compounds increased with time and
reached a high level after 24 h incubation and then maintained steady or peaked off.
Therefore, we further decided to evaluate the inhibition rate of all plumbagin/NO
donor hybrids at 2 µM and NO production for 24 h to study the correlation between
cytotoxic activities and NO released amounts.
Fig. 3. The correlation between the amounts of NO produced by all these hybrids
and cytotoxicities against MDA-MB-231 cells. Cells were cultured at a density of
4
6
.0 × 10 /ml, then treated in triplicate (3 × 100 µl) with each compound at 30 µM for
2
4 h. Cytotoxicities were represented by inhibition rate % at 2 µM.

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From the results illustrated from Figure 3, we can observe that the hybrids exhibited
NO-releasing properties at levels ranging from 23.5% to 46.9%. And the potent
compounds (10b, 11a, 11c and 12a) which exhibited high anti-proliferation activity
consistently produced high level of NO amounts. Thus, the correlation once again
supported the notion that high levels of NO positively exerted cytotoxic activities in
human carcinoma cells.
2
.4. In vitro chemical stability study
Considering the poor chemical stability of plumbagin, we further decided to select the
most active compound (11a) using an in vitro assay to investigate whether its
chemical stability is improved. The stability study was carried out under three
conditions (pHs 6.0, 7.4 and 8.0) in order to mimic the acidic duodenum (pH 4.0-6.0),
the neutral plasma (pH 7.4) environments and a basic condition (pH 8.0). Data shown
in Figure 4 suggested that the stability of plumbagin and compound 11a in acidic
condition were higher than in neutral and basic conditions.

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Fig. 4. In vitro chemical stability assay was performed by HPLC method, profile of
plumbagin (PL) and compound 11a in buffer (pH 6.0; 7.4 and 8.0).
1
,4-napthoquinone (1,4-Nap) was selected as internal standard.
Fig. 5. Concentration–time profiles of plumbagin (PL) and compound 11a in buffer
pH 7.4, 8.0) (Mean ± SD, n=3).
(
After being kept in for 24 h at pH 7.4 and 8.0, more than 95% of plumbagin degraded,
while in the conditions above, compound 11a degraded much less than plumbagin at
pH 8.0 and still retained more than 98% of the original content after 24 h in pH 7.4
buffer (Figure 5). The presence of hydroxy substituent on quinones is great limitation
for chemical stability at basic conditions. According to Borntrager’s reaction, it can be
postulated that the degradation of plumbagin starts with an attack from the acidic
phenolic hydroxyl and then undergoing keto-enol tautomerism (Figure 6).
Nevertheless, the plumbagin derivative (11a) was much more stable in the basic
condition, suggesting that the stability of plumbagin could be enhanced through
introducing to C-3 position of the quinone ring by alkyl groups.

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O
O
O
O
OH
OH
O
O
O
O
red color
O
O
O
O
pH<8.0 OH
O
O
O
(
)n
R
(
)n
R
( )n
R
OH
O
O
O
O
dark red color
Fig. 6. Chemical structure and possible degradation mechanism of plumbagin, where
-
the phenolic hydroxyl was attacked by OH and undergoing keto-enol tautomerism,
may play an important role in the chemical instability of plumbagin. While the
stability of plumbagin could be enhanced through introducing to C-3 position of the
quinone ring by alkyl groups.
3
. Conclusion
A total of 12 novel plumbagin/furoxans hybrids have been synthesized and their
biological functions were evaluated. Almost all these derivatives displayed improved
potency than the parent compound plumbagin, especially against triple negative breast
cancer (MDA-MB-231) and hepatocellular (HepG2) cells. SARs revealed that varying
length of carboxylic acid linkers and models of the NO donor moiety exerted different
cytotoxic effects on the above cell lines. Noticeably, all these hybrids showed greater
selectivity against MDA-MB-231 cells compared to HepG2 and A549 cells, which is
different from plumbagin, among which 10b, 10d, 11a, 11c and 12a have exerted
significantly potency with IC50 values below 2 µM. NO released amounts
measurement by Griess method revealed that the potent compounds which exhibited

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high anti-proliferation activities consistently produced high levels of NO in
MDA-MB-231, supporting the notion that high levels of NO positively exerted
cytotoxic activities in human carcinoma cells. Besides, the most active compound
(11a) also possess desired chemical stability at different pHs (6.0, 7.4 and 8.0). In
conclusion, all these promising results underlined the future potential of
plumbagin/furoxans hybrids as lead compounds against triple negative breast cancer.
4
. Experimental
4
.1. Chemistry
All reagents were purchased from commercial suppliers and used directly unless
1
otherwise stated. CH Cl was refluxed over P O for an hour and distilled. H NMR
2
2
2
5
1
3
and C NMR spectra were recorded on a Bruker AVANCE instrument at 25°C. The
IR spectra were measured on a fourier transform infrared spectroscopy Nicolet Impact
4
10. The molecular weights were detected on HP 1100LC/MSD spectrometer.
4
.1.1. Isolation and structure identification of plumbagin
Dried and powdered roots (2.2 kg) of P. zeylamca were successively and exhaustively
extracted with 95% ethanol at 50°C. The ethanol extracted solution was concentrated
under reduced pressure to give a dark brown residue. The residue was suspended in
water, and then partitioned successively with EtOAc to afford the residues of EtOAc
(180 g). The EtOAc soluble-extract was purified by a silica gel column
chromatography using petroleum ether (PE): EtOAc = 10:1 (v/v). Orange needle-like
crystals of plumbagin were obtained from the first fraction and the purity confirmed

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by HPLC was more than 95%. The total yield of plumbagin obtained after extraction
and purification by column chromatography was 0.081% (basing on the dried and
1
powdered roots (2.2 Kg) of P. zeylamca. H NMR (300 MHz, CDCl ) δ 11.98 (s, 1H,
3
H
-OH), 7.63 (dd, J = 8.5, 7.5 Hz, 1H, Ar-H), 7.61 (dd, J = 7.5, 2.5Hz, 1H, Ar-H), 7.25
1
3
(
dd, J = 8.5, 2.5 Hz, 1H, Ar-H), 6.82 (s, 1H, Ar-H), 2.20 (s, 3H, CH ). C NMR (75
3
MHz, CDCl ) δ 190.2, 164.7, 161.1, 149.6, 136.1, 135.4, 131.9, 124.1, 119.2, 115.0,
3
C
-
1
4
4
6.5. MS(ESI) m/z = 187.1 [M-H] [18]
.1.2. Syntheses of intermediates and final compounds
.1.2.1 General procedures for the preparation of 9a-9c
Dicarboxylic acids (3.0 eq), AgNO (50.7 mg, 0.3 mmol, 0.3 eq) were successively
3
added to a solution of Plumbagin (188.2 mg, 1 mmol, 1.0 eq) in 30% aqueous
acetonitrile (12 ml) at 60℃. when reaction system heated to 70℃, a solution of
ammonium persulfate (296.7 mg, 1.3 mmol, 1.3 eq) in 30% aqueous acetonitrile (10
ml) was added dropwise over 1 h, and the resulting solution stirred at 65-70°C for 3-4
h. On cooling, the mixture was extracted with EtOAc (3 × 20 ml), and the organic
layer washed with H O (3 × 20 ml), dried (Na SO ) and solvent evaporated. The
2
2
4
crude material was purified via column chromatography (DCM/MeOH = 20:1) to
yield the pure product.
4
.1.2.1.1. 2-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)acetic acid
9a)
Obtained as orange powder (27%); H NMR (300 MHz, CDCl ) δ 11.90 (s, 1H, OH),
(
1
3
H
7
.61 (m, 2H, Ar-H), 7.24 (m, 1H, Ar-H), 3.79 (s, 2H, CH -COOH), 2.22 (s, 3H, CH ).
2 3

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1
3
C NMR (75 MHz, CDCl ) δ 189.1, 171.1, 160.7, 140.7, 137.2, 132.2, 124.2, 119.2,
3
C
+
3
2.5, 13.2. MS (ESI) m/z = 269.1 [M+23]
4
.1.2.1.2. 3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)propanoic
acid (9b)
1
Obtained as orange powder (61%); H NMR (300 MHz, CDCl ) δ 12.09 (s, 1H, OH),
3
H
7
.59 (m, 2H, Ar-H), 7.22 (m,1H, Ar-H), 2.99 (t, J = 7.8 Hz, 2H, CH –COOH), 2.63 (t,
2
13
J = 7.5 Hz, 2H, CH -CH -CO) 2.22 (s, 3H, CH ). C NMR (75MHz, CDCl ) δ 189.6,
2
2
3
3
C
1
77.6, 161.3, 145.9, 144.5, 138.1, 136.1, 124.0 , 119.1 , 32.4, 22.0, 12.9. MS (ESI)
+
m/z = 283.1 [M+23]
4
.1.2.1.3. 4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoic acid
(9c)
1
Obtained as orange powder (60%); H NMR (300 MHz, CDCl ) δ 12.17(s, 1H, OH),
3
H
7
.61 (m, 2H, Ar-H), 7.25 (m,1H, Ar-H), 2.71 (t, J = 7.8 Hz, 2H, CH ), 2.48 (t, J = 7.8
2
13
Hz, 2H, CH ), 2.22 (s, 3H, CH ), 1.85 (m, 2H, CH -CH -CO). C NMR (75 MHz,
2
3
2
2
CDCl ) δ 189.8, 184.3, 179.1, 161.2, 145.9, 145.3, 135.9, 132.1, 123.9, 118.9, 114.8,
3
C
+
3
3.7, 25.6, 23.3, 12.8. MS (ESI) m/z = 297.1 [M+23]
4
.1.2.2. General procedures for the preparation of 10a-12d
Compound 9a (40mg, 0.16 mmol) in 8 ml of CH Cl was mixed with
2
2
N,N-dicyclohexylcarbodie (EDCI, 36 mg, 0.19 mmol) and 4-dimethylamino pyridine
DMAP, 8.6 mg, 0.064 mmol) by stirring at 0°C for 0.5 h, then reacting with
corressponding substituted furoxans (68.64 mg, 0.24 mmol) at room temperature for 6
(

ACCEPTED MANUSCRIPT
h with strring. The mixture was evaporated in vacuo to give the residue, subsequently
puried by column chromatography (PE/EA 7:1-5:1 v/v) to afford the title compounds.
4
4
3
.1.2.2.1.
-(2-(2-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)acetoxy)ethoxy)-
-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10a).
The title compound was obtained starting from 9a and 7a.Yellow powder, 31% yield.
-1
Analytical data for 10a: M.p.146-148℃; IR (KBr, cm ): 3550, 3413, 3131, 1731,
1
1
681, 1661, 1637, 1616, 1552, 1400, 1267, 1165, 1085, 1033, 745, 604; H NMR
(
300 MHz, CDCl ) δ 11.90 (s, 1H, OH), 8.05 (d, J = 8.0 Hz, 2H, Ar-H), 7.72 (d, J =
3 H
7
.5 Hz, 1H, Ar-H), 7.63 (m, 4H, Ar-H), 7.24 (m,1H, Ar-H), 4.68 (t, J = 3.7 Hz, 2H,
O-CH ), 4.60 (t, J = 3.7 Hz, 2H, O-CH ), 3.77 (s, 2H, CH -CO), 2.21 (s, 3H, CH ).
2
2
2
3
1
3
C NMR (75MHz, DMSO) δ 161.3, 136.3, 135.7, 129.7, 128.6, 124.1, 119.4, 114.1,
C
+
6
8.7, 62.0, 22.7, 14.1. ESI/HRMS (m/z) [M+NH4] 532.1025. Calcd for
[
C H N O S]: 532.1020.
23 22 3 10
4
4
.1.2.2.2.
-(3-(2-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)acetoxy)propoxy)
-
3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10b).
The title compound was obtained starting from 9a and 7b.Yellow powder, 29% yield.
-1
Analytical data for 10b: M.p.120-121°C; IR (KBr, cm ): 3552, 3413, 3131, 2360,
1
2
341, 1732, 1664, 1635, 1617, 1555, 1458, 1400, 1159, 745, 604; H NMR (300 MHz,
CDCl ) δ 11.83 (s, 1H, OH), 8.0 (d, J = 8.0 Hz, 2H, Ar-H), 7.64 (t, J = 7.5 Hz, 1H,
3
H
Ar-H), 7.64 (m, 4H, Ar-H), 7.24(m, 1H, Ar-H), 4.50 (t, J = 6.0 Hz, 2H, O-CH ), 4.35
2

ACCEPTED MANUSCRIPT
(
t, J = 6.0 Hz, 2H, O-CH ), 3.71 (s, 2H, CH -CO), 2.26 (m, 2H ,O-CH -CH ), 2.21 (s,
2
2
2
2
1
3
3
1
H, CH ) C NMR (75 MHz, CDCl ) δ 169.7, 137.1, 136.5, 130.5 , 128.7 , 124.3,
3 3 C
+
19.3, 68.4, 61.2, 32.5, 27.6, 13.4. ESI/HRMS (m/z) [M+NH ] 546.1178. Calcd for
4
[
C H N O S]: 546.1777.
24 24 3 10
4
4
3
.1.2.2.3.
-(4-(2-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)acetoxy)butoxy)-
-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10c)
The title compound was obtained starting from 9a and 7c. Red waxy solid, 34% yield.
-1
Analytical data for 10c: M.p.85-83°C; IR (KBr, cm ): 3550, 3413, 3131, 2360, 1721,
1
1
662, 1638, 1618, 1400, 1262, 1166, 601; H NMR (300 MHz, CDCl ) δ 11.96 (s,
3
H
1
H, OH), 8.07 (d, J = 8.5 Hz, 2H, Ar-H), 7.76 (d, J = 7.5 Hz, 1H, Ar-H), 7.65 (m, 4H,
Ar-H), 7.25 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 4.46 (t, J = 6.0 Hz, 2H, O-CH ), 4.26 (t, J
2
=
6.0 Hz, 2H, O-CH ), 3.74 (s, 2H, CH -CO), 2.23 (s, 3H, CH ), 1.94 (m, 2H,
2 2 3
1
3
O-CH -CH ), 1.85(m, 2H, O-CH -CH ). C NMR (75 MHz, DMSO) δ 136.3, 129.7,
2
2
2
2
C
+
1
5
4
4
28.6, 124.1, 119.3, 70.9, 64.6, 31.4, 30.2, 24.9. ESI/HRMS (m/z) [M+NH4]
60.1336. Calcd for [C H N O S]: 560.1333.
2
5
26
3
10
.1.2.2.4.
-(3-(2-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)acetoxy)-2-methy
lpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10d)
The title compound was obtained starting from 9a and 7d. Red solid, 28% yield.
-1
Analytical data for 10d: M.p.72-74°C; IR (KBr,cm ): 3550, 3413, 3133, 2360, 2341,
1
1
659, 1638, 1618, 1544, 1400, 1323, 1252, 1118, 1026, 609; H NMR (300 MHz,

ACCEPTED MANUSCRIPT
CDCl ) δ 11.81 (s, 1H, OH), 8.04 (d, J = 7.5 Hz, 2H, Ar-H), 7.63 (d, J = 7.5 Hz, 1H),
3
H
7
5
3
1
1
5
.62 (m, 4H, Ar-H), 7.24 (m, 1H, Ar-H), 4.34 (t, J = 5.7 Hz, 2H, O-CH ), 4.21 (t, J =
2
.7 Hz, 2H, O-CH ), 3.71 (s, 2H, CH -CO), 2.48 (m, 1H, O-CH -CH-CH ), 2.22 (s,
2
2
2
3
1
3
H, CH ), 1.14 (d, J = 7.0 Hz, 3H, CH-CH ). C NMR (75 MHz, DMSO) δ 169.7,
3
3
C
61.7, 160.6, 140.2, 137.2, 136.6, 130.5, 128.7, 124.3, 119.3, 71.5, 64.6, 30.8, 29.4,
+
4.4, 13.5. ESI/HRMS (m/z) [M+NH4] 560.1340. Calcd for [C H N O S]:
25
26
3
10
60.1333.
4
4
.1.2.2.5.
-(2-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)propanoyl)oxy)et
hoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (11a)
The title compound was obtained starting from 9b and 7a. Bright yellow powder,
-1
7
0% yield. Analytical data for 11a: M.p.133-136°C; IR (KBr, cm ): 3551, 3413,
1
3
132, 2360, 2341, 1746, 1637, 1618, 1550, 1460, 1400, 1262, 1165, 609; H NMR
(
300 MHz, CDCl ) δ 12.09 (s, 1H, OH), 8.06 (d, J = 8.0 Hz, 2H, Ar-H), 7.74 (d, J =
3 H
7
.5 Hz, 1H, Ar-H), 7.61 (m, 4H, Ar-H), 7.24 (m, 1H, Ar-H), 4.62 (t, J =3.9 Hz, 2H,
O-CH ), 4.54 (t, J = 3.9 Hz, 2H, O-CH ), 3.00 (t, J = 8.0 Hz, 2H, CH -CO), 2.63 (t, J
2
2
2
13
=
8.0 Hz, 2H, CH -CH -CO), 2.24 (s, 3H, CH ). C NMR (75 MHz, DMSO) δ 190.0,
2 2 3 C
1
1
5
84.2, 172.2, 160.6, 159.2, 145.5, 144.9, 137.6, 136.8, 136.5, 132.3, 130.4, 128.7,
+
24.0, 118.9, 115.1, 69.7, 62.0, 32.3, 22.1, 12.9. ESI/HRMS (m/z) [M+NH4]
46.1183. Calcd for [C H N O S]: 546.1177.
2
4
24
3
10

ACCEPTED MANUSCRIPT
4
4
.1.2.2.6.
-(3-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)propanoyl)oxy)p
ropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole 2-oxide (11b)
The title compound was obtained starting from 9b and 7b. Bright yellow powder,
-1
5
7% yield. Analytical data for 11b: M.p.126-128°C; IR (KBr, cm ): 3412, 3128,
1
2
360, 2342, 1731, 1636, 1615, 1458, 1400, 1260, 1084, 603; H NMR (300 MHz,
CDCl ) δ 12.11 (s, 1H, OH), 8.06 (d, J = 7.9 Hz, 2H, Ar-H), 7.76 (t, J = 7.3 Hz, 1H,
3
H
Ar-H), 7.61 (m, 4H, Ar-H), 7.24 (m, 1H, Ar-H), 4.50 (t, J = 5.9 Hz, 2H, O-CH ), 4.30
2
(
t, J = 5.9 Hz, 2H, O-CH ), 2.97 (t, J = 8.0 Hz, 2H, CH -CO), 2.58 (t, J = 8.0 Hz, 2H,
2
2
1
3
CH -CH -CO), 2.26 (m, 2H, O-CH -CH ), 2.24 (s, 3H, CH ). C NMR (75 MHz,
2
2
2
2
3
DMSO) δ 190.0, 184.3, 172.4, 160.6, 145.4, 145.1, 137.6, 136.9, 136.7, 132.3, 130.5,
C
1
28.7, 124.1, 118.9, 115.1, 68.8, 61.0, 32.4, 27.8, 22.1, 12.9. ESI/HRMS (m/z)
+
[
M+NH ] 560.1339. Calcd for [C H N O S]: 560.1333.
4
25 26
3
10
4
4
.1.2.2.7.
-(4-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)propanoyl)oxy)b
utoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (11c)
The title compound was obtained starting from 9b and 7c. Bright yellow powder, 71%
-1
yield. Analytical data for 11c: M.p.122-125°C; IR (KBr, cm ): 3552, 3413, 3128,
1
1
730, 1636, 1616, 1557, 1459, 1400, 1292, 1258, 1167, 1083, 734, 604; H NMR
(
300 MHz, CDCl ) δ 12.12 (s, 1H, OH), 8.06 (d, J = 7.8 Hz, 2H, Ar-H), 7.76 (d, J =
3 H
7
.2 Hz, 1H, Ar-H), 7.62 (m, 4H, Ar-H), 7.24 (m, 1H, Ar-H), 4.44 (t, J = 5.5 Hz, 2H,

ACCEPTED MANUSCRIPT
O-CH ), 4.20 (t, J = 5.5 Hz, 2H, O-CH ), 2.98 (t, J = 7.5 Hz, 2H, CH -CO), 2.58 (t, J
2
2
2
=
7.5 Hz, 2H, CH -CH -CO), 2.23 (s, 3H, CH ), 1.94 (m, 2H, O-CH -CH ), 1.84 (m,
2 2 3 2 2
1
3
2
1
2
5
4
4
2
H, O- CH -CH ). C NMR (75 MHz, DMSO) δ 190.0, 184.2, 172.4, 160.6, 159.3,
2 2 C
45.4, 145.1, 136.9, 136.6, 132.3, 130.5, 128.7, 124.1, 118.9, 71.5, 64.1, 32.4, 25.1,
+
4.9, 22.1, 12.9. ESI/HRMS (m/z) [M+NH ] 574.1490. Calcd for [C H N O S]:
4
26 28
3
10
74.1490.
.1.2.2.8.
-(3-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydro-naphthalen-2-yl)propanoyl)oxy)-
-methylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (11d)
The title compound was obtained starting from 9b and 7d. Bright yellow powder,
-1
7
0% yield. Analytical data for 11d: M.p.118-120°C; IR (KBr, cm ): 3409, 3128,
1
1
731, 1685, 1636, 1400, 1293, 1260, 1179, 1167, 603; H NMR (300 MHz, CDCl )
3
δH 12.09 (s, 1H, OH), 8.06 (d, J = 7.8 Hz, 2H, Ar-H), 7.76 (t, J = 7.5 Hz, 1H, Ar-H),
7
.62 (m, 4H, Ar-H), 7.22 (m, 1H, Ar-H), 4.37 (t, J = 5.0 Hz, 2H, O-CH ), 4.15 (t, J =
2
5
.0 Hz, 2H, O-CH ), 2.96 (t, J = 7.5 Hz, 2H, CH -CO), 2.58 (t, J = 7.5 Hz, 2H,
2
2
CH -CH -CO), 2.45 (m, 1H, O-CH -CH-CH ), 2.22 (s, 3H, CH ), 1.12 (d, J = 7.0 Hz,
2
2
2
3
3
1
3
3
1
1
5
H, CH-CH3). C NMR (75 MHz, CDCl ) δ 189.7, 184.16, 172.1, 161.3, 158.9,
3 C
45.8, 144.7, 136.1, 135.6, 129.7, 128.5, 123.9, 119.0, 72.4, 65.4, 32.6, 29.7, 22.1,
+
3.6, 12.9. ESI/HRMS (m/z) [M+NH4] 574.1495. Calcd for [C H N O S]:
26
28
3
10
74.1490.

ACCEPTED MANUSCRIPT
4
4
.1.2.2.9.
-(2-((4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoyl)oxy)eth
oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (12a)
The title compound was obtained starting from 9c and 7a.Yellow powder, 67% yield.
-1
Analytical data for 12a: M.p.132-134°C; IR (KBr, cm ): 3550, 3413, 3132, 1732,
1
1
637, 1561, 1450, 1400, 1293, 1260, 1166, 1149, 603; H NMR (300 MHz, CDCl )
3
1
H NMR (300 MHz, CDCl ) δ 12.15 (s, 1H, OH) , 8.06 (d, J = 7.5 Hz, 2H, Ar-H),
3
H
7
.75 (d, J = 7.5 Hz, 1H, Ar-H) , 7.61 (m, 4H, Ar-H), 7.24 (m, 1H, Ar-H), 4.66 (t, J
3.9 Hz, 2H, O-CH ), 4.59 (t, J =3.9 Hz, 2H, O-CH ), 2.73 (t, J = 8.0 Hz, 2H, CH ),
=
2
2
2
13
2
.52 (t, J = 8.0 Hz, 2H, CH ), 2.24 (s, 3H, CH ), 1.91(m, 2H, CH -CH -CO). C
2 3 2 2
NMR (75MHz, CDCl ) δ 189.9, 184.3, 161.2, 145.9, 145.3, 136.0, 135.6, 129.6,
3
C
+
1
5
4
4
28.6, 123.9, 118.9, 68.9, 61.2, 33.7, 25.6, 23.5, 12.8. ESI/HRMS (m/z) [M+NH4]
60.1339. Calcd for [C H N O S]: 560.1333.
2
5
26
3
10
.1.2.2.10.
-(3-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoyl)oxy)pro
poxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (12b)
The title compound was obtained starting from 9c and 7b. Yellow powder, 66% yield.
-1
Analytical data for 12b: M.p.119-121°C; IR (KBr, cm ):3550, 3414, 3132, 1732,
1
1
637, 1561, 1450, 1400, 1294, 1261, 1166, 762, 603; H NMR (300 MHz, CDCl ) δ
3
H
1
2.14 (s, 1H, OH), 8.05 (d, J = 8.0 Hz, 2H, Ar-H), 7.75 (t, J = 7.5 Hz, 1H, Ar-H), 7.61
(
m, 4H, Ar-H), 7.24 (m, 1H, Ar-H), 4.54 (t, J = 6.1 Hz, 2H, O-CH ), 4.31 (t, J = 6.1
2
Hz, 2H, O-CH ), 2.70 (t, J = 8.0 Hz, 2H, CH ), 2.47 (t, J = 8.0 Hz, 2H, CH ), 2.26 (m,
2
2
2

ACCEPTED MANUSCRIPT
1
3
2
H, O-CH -CH ), 2.22 (s, 3H, CH ), 1.85 (m, 2H, CH -CH -CO). C NMR (75 MHz,
2 2 3 2 2
CDCl ) δ 190.0, 184.3, 172.8, 162.2, 158.9, 145.9, 145.3, 138.0, 136.0, 135.6, 132.1,
3
C
1
29.7, 128.5, 123.9, 118.9, 68.1, 60.4, 33.7, 28.0, 25.6, 23.6, 12.8. ESI/HRMS (m/z)
+
[
M+NH ] 574.1497. Calcd for [C H N O S]: 574.1490.
4
26 28
3
10
4
4
.1.2.2.11.
-(4-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoyl)oxy)but
oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (12c)
The title compound was obtained starting from 9c and 7c. Yellow powder, 61% yield.
-1
Analytical data for 12c: M.p.112-114°C; IR (KBr, cm ): 3552, 3477, 3413, 3128,
1
1
723, 1634, 1620, 1567, 1452, 1400, 1295, 1251, 1163, 743, 606; H NMR (300 MHz,
CDCl ) δ 12.17 (s, 1H, OH), 8.07 (d, J = 7.5 Hz, 2H, Ar-H), 7.77 (t, J = 7.0 Hz, 1H,
3
H
Ar-H), 7.61 (m, 4H, Ar-H), 7.24 (m,1H, Ar-H), 4.48 (t, J = 6.0 Hz, 2H, O-CH ), 4.20
2
(
t, J = 6.0 Hz, 2H, O-CH ), 2.71 (t, J = 7.8 Hz, 2H, -CH ), 2.46 (t, J = 7.8 Hz, 2H,
2
2
CH ), 2.22 (s, 3H, CH ), 2.00-1.86 (m, 4H, O-CH -CH -CH ), 1.86 (m, 2H,
2
3
2
2
2
13
CH -CH -CO). C NMR (75 MHz, CDCl ) δ 190.0, 184.3, 173.0, 161.2, 158.9,
2
2
3
C
1
2
45.9, 145.3, 136.0, 135.6, 132.1, 129.6, 128.6, 123.9, 118.9, 70.9, 63.7, 33.8, 25.7,
+
5.3, 25.0, 23.6, 12.8. ESI/HRMS (m/z) [M+NH4] 588.1649. Calcd for
[
C H N O S]: 588.1646.
27 30 3 10
4
4
.1.2.2.12.
-(3-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoyl)oxy)-2-
methylpropoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (12d)

ACCEPTED MANUSCRIPT
The title compound was obtained starting from 9c and 7d. Yellow powder, 69% yield.
-1
Analytical data for 12d: M.p.107-110°C; IR (KBr, cm ): 3551, 3413, 3128, 2360,
1
1
723, 1716, 1620, 1567, 1452, 1400, 1259, 1163, 743, 607; H NMR (300 MHz,
CDCl ) δ 12.14 (s, 1H, OH), 8.06 (d, J = 7.5 Hz, 2H, Ar-H), 7.75 (t, J = 7.5 Hz, 1H,
3
H
Ar-H), 7.61 (m, 4H, Ar-H), 7.23 (m, 1H, Ar-H), 4.38 (t, J = 6.0 Hz, 2H, O-CH ), 4.17
2
(
t, J = 6.0 Hz, 2H, O-CH ), 2.70 (t, J = 7.8 Hz, 2H, CH ), 2.47 (t, J = 7.8 Hz, 2H,
2
2
CH ), 2.45 (m, 1H, O-CH -CH-CH ), 2.21 (s, 3H, CH ), 1.85 (m, 2H, CH -CH -CO),
2
2
3
3
2
2
1
3
1
1
3
.14 (d, J = 7.0 Hz, 3H, CH–CH ). C NMR (75 MHz, CDCl ) δ 190.0, 184.3, 172.8,
3 3 C
61.2, 159.0, 145.9, 145.3, 136.0, 135.6, 129.7, 128.5, 123.9, 118.9, 72.5, 65.1, 33.7,
+
2.7, 25.6, 23.6, 13.6, 12.8. ESI/HRMS (m/z) [M+NH ] 588.1644. Calcd for
4
[
C H N O S]: 588.1646.
27 30 3 10
4
4
.2. Biological Experiments.
.2.1. Cytotoxic assay in vitro
All Plumbagin/NO hybrids were evaluated for their cytotoxic activities against breast
carcinoma cells (MDA-MB-231), hepatocellular carcinoma cells (HepG2) and lung
4
carcinoma cells (A549) by MTT method. Briefly, 6.0×10 /ml (MDA-MB-231 cells),
4
5
.0×10 /ml (HepG2 and A549 cells), respectively, were seeded into 96-well plates
and allowed to adhere for 24 h. Each compounds was dissolved in DMSO and diluted
to 10 mM. After incubated in 37°C CO incubator for 72 h, MTT solution (5 mg/ml)
2
was added, and the plate was further incubated in 37°C CO incubator for another 4 h.
2
Abandoned supernatant before adding 100 µl DMSO to each well. Optical density
(OD) was measured using an enzyme-linked immunosorbent assay (ELISA) reader at

ACCEPTED MANUSCRIPT
5
70 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. NO released amounts detection
4
All hybrides were evaluated for their NO released amounts by Griess method.
4
Specifically, MDA-MB-231 cells were cultured at a density of 6.0×10 /ml, then
treated in triplicate (3×100 µl) treated with each compound at 30 µM (3×10 µl) for
different time, and then the contents of nitrate/nitrite in the cell lysates were
determined by Griess assay. The individual values were determined by measuring
absorbance at 540 nm and calculated according to the standard curve. Data were mean
of three independent experiments.
4
.2.3. In vitro stability study
In vitro chemical stability assay was performed by HPLC method. 1,4-Napthoquinone
was selected as internal standard. Compounds PL/1,4-napthoquinone,
1
1a/1,4-napthoquinone were separated using a Phenomenex Luna reverse-phase C18
(2)-HTS column. The mobile phase was 30:70 (water: methanol, v/v) and 20:80
(water: methanol, v/v), respectively. The injection volume was 5 µl and the flow rate
was 1 ml/min. The detector was set at 254 nm. For hydrolysis, compound PL, 11a and
,4-napthoquinone were diluted in methanol at 1000 µM, respectively. Then these
solution were diluted to appropriate concentration using three different PBS buffer
phosphate-buffered saline) to provide the following pHs: 6.0; 7.4 and 8.0. During the
1
(
assay, all samples were maintained at 37°C. Aliquots were taken from the solution at
the following times: 0, 3, 6 and 24 h. All analyses were conducted in triplicate, and

ACCEPTED MANUSCRIPT
the results were expressed as the averages of the concentrations in percentages (±
standard error of the mean [SEM]).
Acknowledgements
This work was financially supported by the Academic Innovation Project of Jiangsu
Department of Education (No. 1150021667)
References and notes
[
(
[
1] L. Carey, E. Winer, G. Viale, D. Cameron, L. Gianni, Nat. Rev. Clin. Oncol. 7
2010) 683-692.
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