European Journal of Medicinal Chemistry 154 (2018) 1e8

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Novel anticancer hybrids from diazen-1-ium-1,2-diolate nitric oxide

donor and ROS inducer plumbagin: Design, synthesis and biological
evaluations
Na Bao 1, Jinfeng Ou 1, Na Li, Pian Zou, Jianbo Sun*, Li Chen**
State Key Laboratory of Natural Medicines, 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

Article history: High levels of both nitric oxide (NO) and reactive oxygen species (ROS) could act as pro-apoptotic signals
Received 20 January 2018 in cancerous cells. In this study, we conjugated diazeniumdiolates (NONOates), an important class of NO
Received in revised form donors, with a natural occurring plumbagin (PL) which is primarily an excellent ROS inducer. Herein, a
12 April 2018
total of 12 novel plumbagin/NONOate hybrids have been synthesized and evaluated for their inhibitory
Accepted 22 April 2018
Available online 25 April 2018
effects on a panel of human cancer cell lines (MDA-MB-231, A549, HepG2 and HCT-116 cells) and two
normal human cells (HK-2 and WRL-68 cells). Among them, compounds 10a and 10b demonstrated
superior potencies compared to their parent compound (IC50 values of 3.48e6.68 mM) against the above
Keywords:
Nitric oxide
cancer cell lines but weak inhibitory effects on normal cells. In concordance with their selective cyto-
Reactive oxygen species toxicities, 10a and 10b released higher level of NO in cancer cells than normal cells. Besides, the potent
Plumbagin compound 10a induced apoptosis of A549 cells in a concentration-dependent manner and resulted in
Cytotoxicity more ROS generation compared with the parent compound plumbagin.
Selectivity © 2018 Elsevier Masson SAS. All rights reserved.

1. Introduction compounds may elicit effective but selective cytotoxic effects

Nitric oxide (NO), a cardinal signaling molecule, has emerged as
a mediator in many physiological and pathological processes [1]. It
has been shown that angiogenesis, proliferation and metastasis can
normally be stimulated or enhanced by lower levels of NO
(<100 nM). While higher concentrations of NO depress cancer
progression by inducing apoptosis, sensitizing tumors to chemo-,
radio-, or immunotherapy, reversing resistance to chemotherapy,
and retarding the angiogenic and metastatic cascades [2,3].
Accordingly, numerous NO-based anti-cancer agents have been
developed for the potential application for cancer therapy [4]. As
with NO, the cellular effects of reactive oxygen species (ROS) are
also concentration dependent [5]. Noticeably, most malignant cells
possess under varied contexts inherently higher ROS level
compared to normal cells [6,7]. This difference in the redox state
between cancer cells and non-cancer cells allows ROS inducing
* Corresponding author.
** Corresponding author.
E-mail addresses: sjbcpu@gmail.com (J. Sun), chenli627@cpu.edu.cn (L. Chen).
1
These authors contributed equally to this work.
against cancer cells [8,9]. And given that ROS has been involved in
regulation of NO synthases (NOSs) expressions and NO-mediated
signaling, and NO donors pretreatment in some types of cancer
cells have in turn been more sensitive to ROS inducing drugs such
as cisplatin and doxorubicin, coadministration of NO donor with
ROS inducers as medication for cancer treatment is of particular
interest in recent years [10e13].
Diazeniumdiolates (NONOates) as an important class of NO
donors, have abilities to release high levels of NO (two molecules of
NO from per molecule of diazeniumdiolate) under physiological
conditions (pH 7.4, 37
C) [14]. As the benefit of this excellent
performance, the utility of diazeniumdiolates has been an attrac-
tive application for designing antitumor drugs candidates [15e17].
The 1,4-naphthoquinone-based compound, plumbagin (5-
hydroxy-2-methyl-1,4-naphthoquinone) was isolated from Plum-
bago species, which have been extensively used for the treatment of
rheumatic arthralgia, abscess and scrofula in Traditional Chinese
Medicines (TCMs) [18]. Due to its attractive antitumor activity, it
has been attracting a rising attention from cancer biologists.
Although its exact mechanism of action varies in different systems,
the most prominent role is as a ROS inducer which overcome the

https://doi.org/10.1016/j.ejmech.2018.04.047
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
2 N. Bao et al. / European Journal of Medicinal Chemistry 154 (2018) 1e8
antioxidant threshold limit of the cancer cells, thus damaging
cellular components to cause cell death [19,20].
On the basis of the above-mentioned studies and our own
previous work on the modification of natural product plumbagin
[21], we decided to introduce varying carboxylic acid side chains
into the C-3 position of plumbagin, and then designed and syn-
thesized a series of novel plumbagin/NO donor hybrids containing
diazeniumdiolates with the aim to discover the promising de-
rivatives with improved efficiency, selectivity and safety compared
with their parent compound. Herein, a total of 12 plumbagin/NO
donor hybrids (9a-10c, 11a-16a) have been synthesized with their
structure determined by 1H NMR, 13C NMR and ESI/HRMS. Their
in vitro cytotoxicities, intracellular level of NO and ROS production,
and preliminary mechanism underlying their anticancer actions
were also investigated.
2. Results and discussion
2.1. Chemistry
The synthesis of the O2-protected diazeniumdiolates is depicted
in Scheme 1. The diazeniumdiolate sodium salts (3a-3c), were
synthesized according to a modified procedure reported by Leh-
mann [22], and then reacted with chloromethyl methyl sulfide and
subsequent treatment with sulfuryl chloride to furnish chlor-
omethyl NONOate (5a-5c) [23].
For the purpose of biological evaluation (see below). We also
performed derivatives (6a-6c) by the stepwise synthesis of 3a-3c
with bromides with different alkyl chain length (m ¼ 2, 3, 4), to
investigate metabolic capabilities of various ester bonds in the
presence of intracellular esterases.
The 1,4-dihydronaphthalenyl carboxylic acids (8a and 8b) were
prepared by combining plumbagin (1) with varying length of
dicarboxylic acids (7) through oxidative decarboxylation and
Scheme 1. Reagents and conditions (a) NO, 40 psi, MeOH/Ether, NaOMe (1 eq), r. t., 24 h (b) ClCH2SCH3 (1 eq), DMF, K2CO3 (0.5 eq), r. t., 3 h (c) Br(CH2)mBr (1.1 eq), Na2CO3(0.5 eq),
dry DMF, 0
C, 3 h (d) SO2Cl2(1.2 eq), CH2Cl2, 0
C, 3 h.
Kochi-Anderson addition following the procedure of Salmon-
Chemin et al. [24].
The plumbagin/NONOate hybrids (9a-10c, 11a-16a) were syn-
thesized in moderate yields by condensation of semisynthesized
plumbagin analogues (8a and 8b) with intermediates (5a-6c). As
shown in Scheme 2.
2.2. In vitro cytotoxic activity
We first evaluated the preliminary inhibitory effects of plum-
bagin (1) and compounds 9a-10c on three tumor cell lines (MDA-
MB-231, HepG2 and A549 cells) by MTT assay at 10 mM, As shown in
Table 1, compound 10a and 10b showed inhibitory activity (>75%
for all tested cell lines) superior to plumbagin (58.9e73.8%).
Given that various length of the linker in most case had certain
differences to inhibitory activities, we also detected inhibitory ac-
tivities of compounds 11a-16a with the same secondary amine in
the diazeniumdiolate moiety and different length of the carboxylic
acids and diol linkers. While it was found that except 11a which
showed modest inhibitory activity (30e35%), five other compounds
displayed compromised potency (<30% for all tested cell lines).
To comprehensively evaluate the cytotoxicities of these potent
compounds. We further selected the representative panel of human
cancer cell lines such as MDA-MB-231 (breast), A549 (lung), HepG2
(liver) and HCT-116 (colon) as a model for their anticancer activ-
ities. The human renal tubular epithelial cell line HK-2 and normal
human liver cell line WRL-68 were also chosen to determine their
nephrotoxicity and liver toxicity in vitro. As seen from the results
summarized in Table 2, compounds 10a and 10b displayed the
superior potent activities (IC50 values of 3.48e6.68 mM) in all the
cancer cell lines after incubation for 48 h. Noticeably, they had weak
inhibitory effects on normal HK-2 and WRL-68 cells, indicating
their effective and selective cytotoxicities against cancer cells.
Structure and activity relationships (SARs) revealed that
 N. Bao et al. / European Journal of Medicinal Chemistry 154 (2018) 1e8 3

Scheme 2. Reagents and conditions: (a) HOOC(CH2)nCOOH (3.0 eq), silver nitrate (0.3 eq), ammonium persulfate (1.3 eq), aq. 30% CH3CN, 65e70
C, 3 h (b) 5a-5c (1.5 eq), Et3N (1.0
eq), dry acetone, r. t., N2, 24 h (c) 6a-6c (1.5 eq), Et3N (1.0 eq), dry acetone, r. t., N2, 24 h.

Table 1 biological activities and lower toxicities than pyrrolidine moiety, as

Preliminary inhibitory effects of the tested compounds on MDA-MB-231, HepG2 and observed by comparing 10a and 10b with 10c. Additionally, the type
A549 cells.
of linker which connected the NO-donor moiety to the C-3 position
Cpd. Inhibition rate (%) at 10 mMa Cpd. Inhibition rate (%) at 10 mMa of plumbagin also affected the biological results. For example,
MDA-MB-231 HepG2 A549 MDA-MB-231 HepG2 A549 compounds 10a and 10b, with three carbons in carboxylic acid
linker (n ¼ 3) were more active than compounds 9a and 9b which
9a 37.2 31.3 40.3 14a 9.5 8.2 11.3
9b 40.1 35.1 45.0 15a 29.4 28.8 24.4 contain two carbons (n ¼ 2). Compounds 11a-16a, with the ethoxy-,
9c 31.6 29.8 32.9 16a 30.6 32.2 38.2 propoxy- and butoxy-diazeniundiolate moieties, exhibited reduced
10a 88.3 75.4 91.2 PL 73.8 58.9 60.7 potencies compared with compound 9a-10c, with the methoxy-
10b 83.6 80.3 86.7 5a 13.3 16.4 19.1
diazeniundiolate moiety. In this regard, it can be postulated that
10c 70.8 69.7 65.6 5b 11.4 17.6 10.8
11a 35.7 32.5 30.3 8a 8.7b 25.3b 12.3b
these potent compounds tend to be more easily metabolized to
12a 26.2 25.0 23.7 8b 9.9c 28.6c 8.5c release NO.
13a 28.6 28.0 26.9
a
MTT methods, cells were incubated with corresponding compounds at a con-
centration of 10 mM for 48 h. Values are mean of three independent experiments. 2.3. In vitro NO released amounts detection
b
Inhibition rate (%) at 40 mM.
c
Inhibition rate (%) at 20 mM. In an effort to test whether the cytotoxic activities were corre-
lated with the metabolism tendencies and NO released amounts
produced by these hybrids, we calculated the amounts of nitrate
and nitrite of the selected partial hybrids (9a-10c, 11a and 14a)
NONOates were crucial to the performance of theses hybrids. For using the Griess assay [25]. As shown in Fig. 1, NO released amounts
instance, mere introduction of carboxylic acids to the C-3 position of the 9a-10c producing with time in A549 cells while those of 11a
of the quinine ring (8a and 8b) could diminish the antiproliferative and 14a were almost trivial. And the compounds 10a and 10b which
activities compared with plumbagin, whereas further coupling exhibited high anti-proliferation activities consistently produced
with NO donor moieties in general enhanced the potencies at high level of NO amounts after 24 h incubation. Moreover, pre-
various extent. Moreover, the inhibitory effects of 10a and 10b were incubation of A549 cells with NO scavenger carboxy-PTIO (50 mM)
significantly higher than that of NO donor moieties 5a and 5b, remarkably attenuated 10a and 10b-induced NO generation and
which suggested that the improved antiproliferative activities on growth inhibitory effects. (Fig. 2A). All the results strongly indicated
cancer cells could be attributed to the synergic effects of plumbagin that the intracellular levels of NO released by such hybrids were
and NONOates moieties. Furthermore, the secondary amine in the quite well associated with their in vitro cytotoxicities.
diazeniumdiolate moiety was also of importance to these hybrids' Given that 10a and 10b displayed a selective cytotoxicity against
activities and toxicities. Morpholine and piperidine connected to cancer cell lines over normal cells HK-2 and WRL-68, it was
the N1-atom of the diazeniundiolate moiety exhibited better conceivable that they might produce different levels of NO in above

Table 2
Cytotoxicities of all the tested compounds.

Cpd. Potency IC50 (mM)a Toxicity IC50 (mM)a

MDA-MB-231 HepG2 A549 HCT-116 HK-2 WRL-68

9a 13.48 ± 0.36 16.52 ± 0.29 12.34 ± 0.30 15.55 ± 0.11 24.57 ± 0.89 19.30 ± 0.32
9b 11.67 ± 0.44 14.41 ± 0.12 10.79 ± 0.51 20.23 ± 0.56 27.68 ± 0.30 28.21 ± 0.11
9c 15.20 ± 0.39 17.82 ± 0.81 16.39 ± 0.12 17.19 ± 0.20 25.59 ± 0.51 19.48 ± 0.10
10a 4.12 ± 0.07 5.52 ± 0.19 3.48 ± 0.29 4.21 ± 0.18 23.48 ± 0.29 >40
10b 4.92 ± 0.14 6.68 ± 0.37 4.27 ± 0.58 5.26 ± 0.17 17.67 ± 0.74 >40
10c 6.97 ± 0.47 7.12 ± 0.51 9.24 ± 0.33 9.32 ± 0.66 18.14 ± 0.21 16.60 ± 0.38
PL 6.50 ± 0.13 9.17 ± 0.24 8.90 ± 0.10 9.80 ± 0.74 23.58 ± 0.35 15.36 ± 0.24
a
MTT methods, cells were incubated with corresponding compounds for 48 h. IC50 (mM) values (means ± SD, n ¼ 3).
4 N. Bao et al. / European Journal of Medicinal Chemistry 154 (2018) 1e8

2.5. Induction of apoptotic effects

Further, we also performed the FITC-Annexin V/PI staining assay

to examine whether the observed cytotoxicity of 10a is due to in-
duction of apoptosis of A549 cells. As shown in Fig. 4, after 48 h of
incubation, the percentage of annexin V þ apoptotic A549 cells
(Q2þQ3) gradually increased for those cells exposed to increasing
concentrations of 10a. And at the maximal tested concentration
(4 mM), 10a caused early and late apoptosis in 6.93% and 30.3% of
A549 cells, respectively, which clearly indicating its apoptotic ef-
fects in A549 cells is a concentration-dependent manner, especially
for late apoptosis.
Fig. 1. NO released amounts change curves of partial hybrids producing vs time
(mean ± SD, n ¼ 3).

cell lines. Thus, we evaluate the nitrite/nitrate production of those
two hybrids by means of the Griess assay. As observed in Fig. 2B, the
amounts of NO released in A549 cells were much higher than that
in HK-2 and WRL-68 cells, which verified partially the reduced
toxicities appear to be associated with the intrinsic NO release
capabilities.
2.4. ROS generation in A549 cells
To determine whether the ROS generation induced by the
plumbagin derivatives would be shifted or attenuated in the pres-
ence of NO donors, subsequently, we further used the membrane-
permeable fluorogenic probe 20 ,70 -dichlorodihydrofluorescein
diacetate (DCFH-DA) to detect the intracellular level of ROS induced
by plumbagin and 10a in A549 cells. Data obtained from flow
cytometry showed that incubation with 10a at 5 mM for 24 h
resulted in 1.64-fold increase in the ROS production relative to non-
treated cells, while the rise by plumbagin treatment was 1.34-fold.
This result obtained showed that coadministration with relatively
high level of NO amounts produced by plumbagin/NONOate hybrid
could enhance intracellular ROS level in A549 cells as compared
with parent compound plumbagin at the same concentration
(Fig. 3).
3. Conclusion
Combination of NO donors with ROS inducers is a promising
medication for cancer treatment. In this effort, we conjugated the
ROS inducer plumbagin with NONOates via various linkers. SARs
revealed that varying types of linker and models of the NO donor
moiety exerted different antiproliferative activities and toxicities
in vitro, and the improved potency on cancer cells could be attrib-
uted to the synergic effects of plumbagin and NONOates moieties.
In accordance with the SAR study, compounds 10a and 10b
exhibited substantial inhibitory efficacies on a panel of human
cancer cell lines (MDA-MB-231, A549, HepG2 and HCT-116 cells)
and low nephrotoxicity and liver toxicity in vitro. One plausible
explanation for these selective cytotoxicities could be presumed by
the Griess assay as 10a and 10b selectively released higher level of
NO in cancer cell lines than normal cells. Furthermore, 10a induced
relatively greater ROS generation as compared with the parent
compound plumbagin, which may also contribute to its superior
properties. Additionally, the mechanism of action of 10a detected
by flow cytometric analysis refers to the induction of apoptosis in
A549 cells in a concentration-dependent manner. Overall, the
plumbagin/NONOate hybrids described here represent a novel and
interesting class of ROS inducer hybrid of NO-donor potentially
useful in the anti-cancer treatment.

Fig. 2. NO release capabilities and in vitro cytotoxicities of compounds 10a and 10b (A) A549 cells were treated with 10a and 10b (5 mM) for 24 h or pre-incubation of A549 cells with
NO scavenger carboxy-PTIO (50 mM) for 1 h, followed by treatment with indicated the indicated compounds for 24 h. Cell proliferation and amounts of NO were determined by the
MTT and Griess assay, respectively. Data are means ± SD from three independent, **P < 0.05 vs 10a or 10b alone. (B) NO production of 10a and 10b (10 mM) in A549, HK-2 and WRL-
68 cells for 24 h. Data are expressed as mean ± SD from three independent, **P < 0.05 vs control, ***P < 0.01 vs control.
 N. Bao et al. / European Journal of Medicinal Chemistry 154 (2018) 1e8 5

Fig. 3. Plumbagin (PL) and Compound 10a induced ROS generations. A549 cells were incubated with PL and 10a at 5 mM for 24 h, and then cells were collected and loaded with
DCFH-DA. The mean fluorescence intensity was detected by flow cytometry, and results were expressed as fold of control. **p < 0.01 vs control.

Fig. 4. Compound 10a induced apoptosis in A549 cells. After 24 h of 10a treatment (0, 1, 2, 4 mM), A549 cells were collected and stained with FITC-Annexin V and PI, followed by
flow cytometric analysis. Data are expressed as means ± SD of the percentages of apoptotic cells from three independent experiments. *P < 0.01 vs control, **P < 0.05 vs control.

4. Experimental
4.1. Chemistry
All reagents were purchased from commercial suppliers and
used directly unless otherwise stated. CH2Cl2 was refluxed over
P2O5 for an hour and distilled. 1H NMR and 13C NMR spectra were
recorded on a Bruker AVANCE instrument at 25
C. The molecular
weights were detected on HP 1100LC/MSD spectrometer.
The parent compound plumbagin (1) was extracted and isolated
from P. zeylamca, as previously described [21]. Sodium dia-
zeniumdiolates 3a-3c were prepared according to the method
described previously [22].
4.1.1. Syntheses of intermediates and final compounds
4.1.1.1. General procedures for the preparation of 5a-5c.
Chloromethyl methyl sulfide (643 mL, 7.8 mmol) was added to a
slurry solution of K2CO3 (413 mg, 3.9 mmol) in DMF (18 mL) at room
temperature. Following 2 min of stirring, sodium dia-
zeniumdiolates 3a-3c (7.8 mmol) was added, and stirring was
continued for 3 h. The reaction was quenched by addition of ethyl
acetate (20 mL), and the solution was then filtered and subse-
quently washed with a 10% NaCl solution (5  20 mL). The organic
layer was then dried over Na2SO4 and evaporated to obtain the
crude product, which was further purified by column chromatog-
raphy using PE/EA ¼ 10:1 (v/v) to obtain a colorless oil. A solution of
the colorless oil above (0.5 mmol) dissolved in dichloromethane
(6 mL) was cooled to 15
C, and sulfuryl chloride (486 mL,
0.6 mmol) was added slowly, then the reaction mixture was
brought to room temperature. Reaction progress was monitored by
TLC. After 3 h, The reaction mixture was washed with water, dried
over Na2SO4, filtered, and evaporated to yield a colorless oil that
was then used immediately without further purification.
4.1.1.1.1. (Z)-2-(chloromethoxy)-1-morpholinodiazene 1-oxide
(5a). Obtained as a colorless oil (64%); 1H NMR (300 MHz, CDCl3)
dH 5.26 (s, 2H, O-CH2-Cl), 4.18 (t, J ¼ 5.2 Hz, 4H, CH2-O-CH2), 3.39 (t,
J ¼ 5.5 Hz, 4H, CH2-NH-CH2). MS (ESI) m/z ¼ 196.0 [Mþ1]þ.
4.1.1.1.2. (Z)-2-(chloromethoxy)-1-(piperidin-1-yl)diazene 1-
oxide (5b). Obtained as a colorless oil (68%); 1H NMR dH 5.26 (s,
2H, O-CH2-Cl), 3.77 (t, J ¼ 5.2 Hz, 4H, CH2-NH-CH2), 1.77 (t,
J ¼ 5.5 Hz, 4H, CH2-CH2-CH2-CH2-CH2), 1.53 (m, 2H, CH2-CH2-
CH2-CH2-CH2). MS (ESI) m/z ¼ 194.0 [Mþ1]þ.
6 N. Bao et al. / European Journal of Medicinal Chemistry 154 (2018) 1e8
4.1.1.1.3. (Z)-2-(chloromethoxy)-1-(pyrrolidin-1-yl)diazene 1-
oxide (5c). Obtained as a colorless oil (65%); 1H NMR dH 5.77 (s,
2H, O-CH2-Cl), 3.56 (t, J ¼ 6.7 Hz, 4H, CH2-NH-CH2), 1.91 (t,
J ¼ 7.0 Hz, 4H, CH2-CH2-CH2-CH2). MS (ESI) m/z ¼ 180.0 [Mþ1]þ.
4.1.1.2. General procedures for the preparation of 6a-6c.
Dibromoalkanes (260 mL, 2.2 mmol) was added to a solution of
Na2CO3 (106 mg, 1 mmol) in dry DMF (20 mL) at 0
C, and the re-
action mixture was stirred for 10 min 3a (338.2 mg, 2 mmol) was
added slowly to the reaction mixture. After the mixture was stirred
under nitrogen for 72 h, filtered and the filtrate was poured into
100 mL of cold water, the mixture was extracted with EtOAc
(4  50 mL), and the organic layer washed with H2O (5  50 mL),
dried (Na2SO4) and solvent evaporated. The crude material was
purified via column chromatography (PE/EA ¼ 2:1,v/v) to yield the
pure product.
4.1.1.2.1. (Z)-2-(2-bromoethoxy)-1-morpholinodiazene 1-oxide
(6a). Obtained as a light-yellow oil (40%); 1H NMR (300 MHz,
CDCl3) dH 4.41 (t, J ¼ 6.7 Hz, 2H, CH2-O), 3.77 (t, J ¼ 4.8 Hz, 4H,CH2-
O-CH2), 3.50 (t, J ¼ 6.7 Hz, 2H, CH2-Br), 3.37 (t, J ¼ 4.5 Hz, 4H, CH2-
NH-CH2). MS (ESI) m/z ¼ 252.0 [M  1]-.
4.1.1.2.2. (Z)-2-(3-bromopropoxy)-1-morpholinodiazene 1-oxide
(6b). Obtained as a light-yellow oil (45%); 1H NMR (300 MHz,
CDCl3) dH 4.29 (t, J ¼ 6.0 Hz, 2H, CH2-O), 3.77 (t, J ¼ 4.6 Hz, 4H,CH2-
O-CH2), 3.43 (t, J ¼ 6.5 Hz, 2H, CH2-Br), 3.36 (t, J ¼ 4.9 Hz, 4H, CH2-
NH-CH2), 2.23 (m, 2H, CH2-CH2-CH2). MS (ESI) m/z ¼ 266.0
[M  1]-.
4.1.1.2.3. (Z)-2-(4-bromobutoxy)-1-morpholinodiazene 1-oxide
(6c). Obtained as a light-yellow oil (58%); 1H NMR (300 MHz,
CDCl3) dH 4.18 (t, J ¼ 6.2 Hz, 2H, CH2-O), 3.77 (t, J ¼ 4.9 Hz, 4H,CH2-
O-CH2), 3.38 (t, J ¼ 3.2 Hz, 2H, CH2-Br), 3.54 (t, J ¼ 5.8 Hz, 4H, CH2-
NH-CH2), 1.89 (m, 4H, CH2-CH2-CH2-CH2). MS (ESI) m/z ¼ 280.0
[M  1]-.
4.1.1.3. General procedures for the preparation of 8a, 8b.
Dicarboxylic acids (3.0 eq), AgNO3 (50.7 mg, 0.3 mmol) were suc-
cessively added to a solution of plumbagin (1) (188.2 mg, 1 mmol)
in 30% aqueous acetonitrile (12 mL) at 60
C. when reaction system
heated to 70
C, a solution of ammonium persulfate (296.7 mg,
1.3 mmol) in 30% aqueous acetonitrile (10 mL) was added dropwise
over 1 h, and the resulting solution stirred at 65e70
C for 3e4 h.
On cooling, the mixture was extracted with EtOAc (3  20 mL), and
the organic layer washed with H2O (3  20 mL), dried (Na2SO4) and
solvent evaporated. The crude material was purified via column
chromatography (DCM/MeOH ¼ 20:1) to yield the pure product.
4 .1.1. 3 .1. 3 - ( 8 - H y d r o x y - 3 - m e t h y l - 1, 4 - d i o x o - 1, 4 -
dihydronaphthalen-2-yl)propanoic acid (8a). Obtained as orange
powder (61%); 1H NMR (300 MHz, CDCl3) dH 12.09 (s, 1H, OH), 7.59
(m, 2H, Ar-H), 7.22 (m, 1H, Ar-H), 2.99 (t, J ¼ 7.8 Hz, 2H, CH2-COOH),
2.63 (t, J ¼ 7.5 Hz, 2H, CH2-CH2-CO) 2.22 (s, 3H, CH3). 13C NMR
(75 MHz, CDCl3) dC 189.6, 177.6, 161.3, 145.9, 144.5, 138.1, 136.1124.0,
119.1, 32.4, 22.0, 12.9. MS (ESI) m/z ¼ 283.1 [Mþ23]þ.
4 .1.1. 3 . 2 . 4 - ( 8 - H y d r o x y - 3 - m e t h y l - 1, 4 - d i o x o - 1, 4 -
dihydronaphthalen-2-yl)butanoic acid (8b). Obtained as orange
powder (60%); 1H NMR (300 MHz, CDCl3) dH 12.17 (s, 1H, OH), 7.61
(m, 2H, Ar-H), 7.25 (m, 1H, Ar-H), 2.71 (t, J ¼ 7.8 Hz, 2H, CH2), 2.48 (t,
J ¼ 7.8 Hz, 2H, CH2), 2.22 (s, 3H, CH3), 1.85 (m, 2H, CH2-CH2-CO).
13
CNMR (75 MHz, CDCl3) dC 189.8, 184.3, 179.1, 161.2, 145.9, 145.3,
135.9, 132.1, 123.9, 118.9, 114.8, 33.7, 25.6, 23.3, 12.8. MS (ESI) m/
z ¼ 297.1 [Mþ23]þ.
4.1.1.4. General procedures for the preparation of 9a-16a.
Triethylamine (140 mL, 1 mmol) was added to a solution of 8a or 8b
(1 mmol) in dry acetone (10 mL), and the reaction mixture was
stirred for 30 min at room temperature. A solution of corresponding
substituted intermediates 5a-6c (1.5 mmol) dissolved in dry
acetone (10 mL) was added dropwise to the reaction mixture. After
the mixture was stirred under nitrogen for 24 h, the mixture was
evaporated in vacuo to give the residue, subsequently purified by
column chromatography (PE/EA ¼ 5:1, v/v) to afford the title
compounds.
4.1.1.4.1. (E)-1-(((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)propanoyl)oxy)methoxy)-2-
morpholinodiazene 1-oxide (9a). The title compound was obtained
starting from 8a and 5a. Yellow oil, 28% yield. Analytical data for 9a:
1
H NMR (300 MHz, CDCl3) dH 12.07 (s, 1H, OH), 7.61 (m, 2H, Ar-H),
7.22 (dd, J ¼ 7.9 Hz, 1.8 Hz, 1H, Ar-H), 5.81 (s, 2H, O-CH2-O), 3.83 (t,
J ¼ 4.7 Hz, 4H, CH2-O-CH2), 3.46 (t, J ¼ 4.9 Hz, 4H, CH2-NH-CH2),
2.97 (t, J ¼ 7.8 Hz, 2H, CH2-CH2-CO), 2.63 (t, J ¼ 7.9 Hz, 2H, CH2-CH2-
CO) 2.23 (s, 3H, Ar-CH3). 13C NMR (75 MHz, CDCl3) dC 220.8, 194.3,
174.1, 142.9, 135.1, 124.0, 123.4, 123.0, 118.1, 88.6, 66.7, 50.3, 33.5,
20.9, 11.9. ESI/HRMS (m/z) [MþNa]þ 442.1222. Calcd for
[C19H21N3O8Na]: 442.1227.
4.1.1.4.2. (E)-1-(((4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)butanoyl)oxy)methoxy)-2-
morpholinodiazene 1-oxide (10a). The title compound was ob-
tained starting from 8b and 5a. Yellow oil, 31% yield. Analytical data
for 10a: 1H NMR (300 MHz, CDCl3) dH 12.13 (s, 1H, OH), 7.61 (m, 2H,
Ar-H), 7.22 (d, J ¼ 7.9 Hz, 1H, Ar-H), 5.82 (s, 2H, O-CH2-O), 3.84 (t,
J ¼ 5.0 Hz, 4H, CH2-O-CH2), 3.48 (t, J ¼ 4.7 Hz, 4H, CH2-NH-CH2),
2.75 (t, J ¼ 7.7 Hz, 2H, CH2-CH2-Ar), 2.48 (t, J ¼ 7.1 Hz, 2H, CH2-CH2-
CO) 2.21 (s, 3H, Ar-CH3), 1.83 (m, 2H, CH2-CH2-CH2). 13C NMR
(75 MHz, CDCl3) dC 190.2, 161.1, 135.1, 135.0, 122.8, 118.0, 90.4, 60.8,
50.3, 30.4, 22.4, 21.5, 11.8. ESI/HRMS (m/z) [MþNa]þ 456.1371. Calcd
for [C20H23N3O8Na]: 456.1383.
4.1.1.4.3. (E)-1-(((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)propanoyl)oxy)methoxy)-2-(piperidin-1-yl)
diazene 1-oxide (9b). The title compound was obtained starting
from 8a and 5b. Yellow oil, 25% yield. Analytical data for 9b: 1H
NMR (300 MHz, CDCl3) dH 12.10 (s, 1H, OH), 7.60 (m, 2H, Ar-H), 7.23
(dd, J ¼ 7.7 Hz, 1.9 Hz, 1H, Ar-H), 5.77 (s, 2H, O-CH2-O), 3.56 (t,
J ¼ 6.9 Hz, 4H, CH2-N-CH2), 2.97 (t, J ¼ 7.8 Hz, 2H, CH2-CH2-CO), 2.63
(t, J ¼ 8.0 Hz, 2H, CH2-CH2-CO), 2.22 (s, 3H, Ar-CH3), 1.95 (m, 4H,
CH2-CH2-CH2), 0.85 (t, J ¼ 7.2 Hz, 2H, CH2-CH2-CH2). 13C NMR
(75 MHz, CDCl3) dC 160.2, 135.1, 123.4, 123.3, 123.0, 122.4, 118.1, 88.6,
66.7, 50.3, 33.5, 20.9, 11.9. ESI/HRMS (m/z) [MþH]þ 418.1614. Calcd
for [C20H24N3O7]: 418.1614.
4.1.1.4.4. (E)-1-(((4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)butanoyl)oxy)methoxy)-2-(piperidin-1-yl)
diazene 1-oxide (10b). The title compound was obtained starting
from 8b and 5b. Yellow oil, 30% yield. Analytical data for 10b: 1H
NMR (300 MHz, CDCl3) dH 12.16 (s, 1H, OH), 7.60 (m, 2H, Ar-H), 7.22
(dd, J ¼ 7.8 Hz, 1.8 Hz, 1H, Ar-H), 5.79 (s, 2H, O-CH2-O), 3.59 (t,
J ¼ 6.8 Hz, 4H, CH2-N-CH2), 2.68 (t, J ¼ 8.0 Hz, 2H, CH2-CH2-CO),
2.49 (t, J ¼ 7.1 Hz, 2H, CH2-CH2-Ar), 2.20 (s, 3H, Ar-CH3), 1.95 (m, 4H,
CH2-CH2-CH2), 1.84 (m, 2H, Ar-CH2-CH2-CH2), 0.86 (m, 2H, CH2-
CH2-CH2). 13C NMR (75 MHz, CDCl3) dC 170.6, 160.2, 144.9, 135.0,
122.8, 117.9, 86.4, 49.7, 32.7, 30.5, 29.2, 24.5, 21.9, 11.8. ESI/HRMS (m/
z) [MþNa]þ 454.1541. Calcd for [C21H25N3O7Na]: 454.1591.
4.1.1.4.5. (E)-1-(((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)propanoyl)oxy)methoxy)-2-(pyrrolidin-1-yl)
diazene 1-oxide (9c). The title compound was obtained starting
from 8a and 5c. Yellow oil, 26% yield. Analytical data for 9c: 1H NMR
(300 MHz, CDCl3) dH 12.08 (s, 1H, OH), 7.60 (m, 2H, Ar-H), 7.24 (dd,
J ¼ 7.8 Hz, 1.7 Hz, 1H, Ar-H), 5.81 (s, 2H, O-CH2-O), 3.40 (t, J ¼ 5.6 Hz,
4H, CH2-N-CH2), 2.97 (t, J ¼ 7.9 Hz, 2H, CH2-CH2-CO), 2.64 (t,
J ¼ 4.5 Hz, 2H, CH2-CH2-CO) 2.22 (s, 3H, Ar-CH3), 1.75 (m, 4H, CH2-
CH2-CH2-CH2). 13C NMR (75 MHz, CDCl3) dC 153.2, 135.1, 132.5,
123.0, 118.1, 100.8, 92.6, 51.2, 31.5, 23.6, 20.9, 11.9. ESI/HRMS (m/z)
[MþH]þ 404.1450. Calcd for [C19H22N3O7]: 404.1458.
 N. Bao et al. / European Journal of Medicinal Chemistry 154 (2018) 1e8
4.1.1.4.6. (E)-1-(((4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)butanoyl)oxy)methoxy)-2-(pyrrolidin-1-yl)
diazene 1-oxide (10c). The title compound was obtained starting
from 8b and 5c. Yellow oil, 30% yield. Analytical data for 10c: 1H
NMR (300 MHz, CDCl3) dH 12.19 (s, 1H, OH), 7.58 (m, 2H, Ar-H), 7.21
(dd, J ¼ 7.9 Hz, 2.6 Hz, 1H, Ar-H), 5.82 (s, 2H, O-CH2-O), 3.40 (t,
J ¼ 5.5 Hz, 4H, CH2-N-CH2), 2.69 (t, J ¼ 8.1 Hz, 2H, CH2-CH2-CO),
2.43 (t, J ¼ 7.1 Hz, 2H, CH2-CH2-Ar), 2.21 (s, 3H, Ar-CH3), 1.82 (m, 2H,
Ar-CH2-CH2-CH2), 1.60 (m, 4H, CH2-CH2-CH2-CH2). 13C NMR
(75 MHz, CDCl3) dC 200.5, 171.3, 135.0, 134.9, 122.8, 122.7, 118.0,
117.9, 117.8, 87.3, 45.5, 31.8, 23.5, 23.0, 22.5, 11.8. ESI/HRMS (m/z)
[MþH]þ 418.1614. Calcd for [C20H24N3O7]: 418.1614.
4.1.1.4.7. (E)-1-(2-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)propanoyl)oxy)ethoxy)-2-
morpholinodiazene 1-oxide (11a). The title compound was obtained
starting from 8a and 6a. Yellow oil, 16% yield. Analytical data for
11a: 1H NMR (300 MHz, CDCl3) dH 12.09 (s, 1H, OH), 7.60 (m, 2H, Ar-
H), 7.21 (d, J ¼ 2.0 Hz, 1H, Ar-H), 4.38 (t, J ¼ 4.3 Hz, 4H, O-CH2-CH2-
O) 3.89 (t, J ¼ 3.5 Hz, 4H, CH2-O-CH2), 3.43 (t, J ¼ 3.7 Hz, 4H,CH2-N-
CH2), 2.96 (t, J ¼ 5.9 Hz, 2H, CH2-CH2-CO), 2.59 (t, J ¼ 7.3 Hz, 2H,
CH2-CH2-CO), 2.22 (s, 3H, Ar-CH3). 13C NMR (75 MHz, DMSO) dC
187.9, 182.5, 170.5, 158.9, 143.8, 143.1, 128.7, 127.0, 122.3, 117.1, 68.0,
66.8, 60.2, 50.9, 30.6, 20.3, 11.2. ESI/HRMS (m/z) [MþNa]þ 456.1377.
Calcd for [C20H23N3O8Na]: 456.1383.
4.1.1.4.8. (E)-1-(2-((4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)butanoyl)oxy)ethoxy)-2-morpholinodiazene
1-oxide (12a). The title compound was obtained starting from 8b
and 6a. Yellow oil, 18% yield. Analytical data for 12a: 1H NMR
(300 MHz, CDCl3) dH 12.08 (s, 1H, OH), 7.53 (m, 2H, Ar-H), 7.15 (dd,
J ¼ 7.9 Hz, 2.0 Hz, 1H, Ar-H), 4.33 (t, J ¼ 4.8 Hz, 4H, O-CH2-CH2-O)
3.77 (t, J ¼ 4.7 Hz, 4H, CH2-O-CH2), 3.63 (t, J ¼ 4.5 Hz, 4H, CH2-N-
CH2), 2.61 (t, J ¼ 7.7 Hz, 2H, CH2-CH2-CO), 2.39 (t, J ¼ 7.1 Hz, 2H,
CH2-CH2-CO), 2.14 (s, 3H, Ar-CH3), 1.76 (m, 2H, CH2-CH2-CH2). 13C
NMR (75 MHz, CDCl3) dC 189.3, 183.7, 172.1, 160.6, 145.3, 144.8,
129.0, 128.0, 123.2, 118.4, 68.3, 64.6, 60.1, 50.3, 33.1, 25.0, 22.9, 12.2.
ESI/HRMS (m/z) [MþH]þ 448.1714. Calcd for [C21H26N3O8]:
448.1720.
4.1.1.4.9. (E)-1-(3-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)propanoyl)oxy)propoxy)-2-
morpholinodiazene 1-oxide (13a). The title compound was obtained
starting from 8a and 6b. Yellow oil, 15% yield. Analytical data for
13a: 1H NMR (300 MHz, CDCl3) dH 12.02 (s, 1H, OH), 7.53 (m, 2H, Ar-
H), 7.16 (d, J ¼ 7.8 Hz, 6.2 Hz, 1H, Ar-H), 4.22 (t, J ¼ 6.4 Hz, 2H, CO-O-
CH2), 4.16 (t, J ¼ 6.3 Hz, 2H, N-O-CH2), 3.77 (t, J ¼ 4.6 Hz, 4H, CH2-O-
CH2), 3.34 (t, J ¼ 4.8 Hz, 4H, CH2-N-CH2), 2.88 (t, J ¼ 7.9 Hz, 2H, CH2-
CH2-CO), 2.48 (t, J ¼ 7.7 Hz, 2H, CH2-CH2-CO), 2.15 (s, 3H, Ar-CH3),
2.02 (m, 2H, CH2-CH2-CH2). 13C NMR (75 MHz, DMSO) dC 188.9,
183.5, 171.7, 159.9, 144.7, 144.3, 129.7, 128.0, 123.3, 118.2, 68.1, 64.6,
60.3, 50.3, 31.6, 27.1, 21.3, 12.2. ESI/HRMS (m/z) [MþNa]þ 470.1541.
Calcd for [C21H25N3O8Na]: 470.1540.
4.1.1.4.10. (E)-1-(3-((4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)butanoyl)oxy)propoxy)-2-
morpholinodiazene 1-oxide (14a). The title compound was obtained
starting from 8b and 6b. Yellow oil, 18% yield. Analytical data for
14a: 1H NMR (300 MHz, CDCl3) dH 12.08 (s, 1H, OH), 7.52 (m, 2H, Ar-
H), 7.14 (dd, J ¼ 8.8 Hz, 1.6 Hz, 1H, Ar-H), 4.24 (t, J ¼ 6.3 Hz, 2H, CO-
O-CH2), 4.14 (t, J ¼ 6.3 Hz, 2H, N-O-CH2) 3.76 (t, J ¼ 4.6 Hz, 4H, CH2-
O-CH2), 3.34 (t, J ¼ 4.8 Hz, 4H, CH2-N-CH2), 2.60 (t, J ¼ 7.7 Hz, 2H,
CH2-CH2-CO), 2.36 (t, J ¼ 7.0 Hz, 2H, CH2-CH2-CO), 2.13 (s, 3H, Ar-
CH3), 2.05 (m, 2H, Ar-CH2-CH2-CH2), 1.75 (m, 2H, O-CH2-CH2-CH2).
13
C NMR (75 MHz, CDCl3) dC 188.7, 183.1, 171.7, 160.1, 144.8, 144.1,
128.5, 127.4, 122.7, 117.8, 66.9, 64.1, 59.2, 50.0, 32.6, 26.8, 24.5, 22.4,
11.6. ESI/HRMS (m/z) [MþH]þ 462.1871. Calcd for [C22H28N3O8]:
462.1876.
7
4.1.1.4.11. (E)-1-(4-((3-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)propanoyl)oxy)butoxy)-2-
morpholinodiazene 1-oxide (15a). The title compound was obtained
starting from 8a and 6c. Yellow oil, 15% yield. Analytical data for
15a: 1H NMR (300 MHz, CDCl3) dH 12.03 (s, 1H, OH), 7.50 (m, 2H, Ar-
H), 7.12 (d, J ¼ 7.8 Hz, 6.0 Hz, 1H, Ar-H), 4.21 (t, J ¼ 6.2 Hz, 2H, CO-O-
CH2), 4.16 (t, J ¼ 6.3 Hz, 2H, N-O-CH2) 3.76 (t, J ¼ 4.5 Hz, 4H, CH2-O-
CH2), 3.34 (t, J ¼ 4.6 Hz, 4H,CH2-N-CH2), 2.85 (t, J ¼ 7.8 Hz, 2H, CH2-
CH2-CO), 2.45 (t, J ¼ 7.7 Hz, 2H, CH2-CH2-CO), 2.14 (s, 3H, Ar-CH3),
1.29 (m, 4H, CH2-CH2-CH2-CH2). 13C NMR (75 MHz, DMSO) dC 188.5,
183.1, 171.2, 159.4, 144.3, 143.9, 129.3, 127.6, 122.9, 117.9, 70.3, 64.6,
63.0, 50.3, 31.3, 23.9, 23.7, 21.0, 11.8. ESI/HRMS (m/z) [MþH]þ
462.1871. Calcd for [C22H28N3O8]: 462.1876.
4.1.1.4.12. (E)-1-(4-((4-(8-hydroxy-3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)butanoyl)oxy)butoxy)-2-morpholinodiazene
1-oxide(16a). The title compound was obtained starting from 8b
and 6c. Yellow oil, 16% yield. Analytical data for 16a: 1H NMR
(300 MHz, CDCl3) dH 12.15 (s, 1H, OH), 7.59 (m, 2H, Ar-H), 7.22 (d,
J ¼ 8.0 Hz, 1H, Ar-H), 4.24 (t, J ¼ 5.6 Hz, 2H, CO-O-CH2), 4.12 (t,
J ¼ 6.3 Hz, 2H, N-O-CH2) 3.84 (t, J ¼ 4.4 Hz, 4H, CH2-O-CH2), 3.41 (t,
J ¼ 4.7 Hz, 4H, CH2-N-CH2), 2.68 (t, J ¼ 8.1 Hz, 2H, CH2-CH2-CO),
2.43 (t, J ¼ 7.0 Hz, 2H, CH2-CH2-CO), 2.20 (s, 3H, Ar-CH3), 1.81 (m,
6H, Ar-CH2-CH2-CH2, O-CH2-CH2-CH2-CH2-O). 13C NMR (75 MHz,
CDCl3) dC 188.7, 183.2, 171.8, 160.1, 144.8, 144.1, 128.5, 127.4, 122.7,
117.8, 69.8, 64.3, 62.7, 50.0, 32.7, 24.5, 24.1, 23.9, 22.5, 11.7. ESI/HRMS
(m/z) [MþH]þ 476.2029. Calcd for [C23H30N3O8]: 476.2042.
4.2. Biological experiments
4.2.1. Cytotoxic assay in vitro
The cytotoxicities of test compounds were investigated by the
MTT method. Briefly, human cancer cell lines such as MDA-MB-231,
A549, HepG2 and HCT-116 cells, or normal human cell line HK-2
and WRL-68 cells at a final density of 5.0  104/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
44 h the cells were exposed to MTT (5 mg/mL), and the plate was
further incubated in 37
C CO2 incubator for another 4 h. Aban-
doned supernatant before adding 100 ml DMSO to each well. Optical
density (OD) was measured 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 ¼ 3).
4.2.2. NO released amounts detection
NO levels in cells were determined by evaluating the content of
nitrite/nitrate using Griess assay with Nitrate/Nitrite Assay Kit
(Beyotime, China), according to the manufacturer's instructions.
Specifically, A549 cells were cultured at a density of 5.0  104/ml,
then treated in triplicate (3  100 mL) treated with the test com-
pound (9a-10c, 11a and 14a) at 5 mM (3  10 mL) 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. Reactive oxygen species (ROS) measurement assay
A549 cells (2  105/well) were plated into 12-well plates. Cells
were treated with plumbagin (1) and 10a at 5 mM for 24 h and then
were incubated with 10 mM DCFH-DA (keygenbio, Nanjing, China)
at 37
C for 25 min. The fluorescence of the cells from each well was
detected by flow cytometry (BD Accuri C6 flowcytometer, Becton &
Dickinson Company, Franklin Lakes, NJ).
8 N. Bao et al. / European Journal of Medicinal Chemistry 154 (2018) 1e8
4.2.4. Cell apoptotic analysis
A549 cells at 1.2  105 cells/well in a final volume of 2.5 mL were
seeded in six-well plates and allowed to adhere for 24 h. After in-
cubation with or without 10a at different concentrations, the pre-
pared cells were washed twice with cold PBS followed by re-
suspension in 500 mL binding buffer. Next, the samples were
added with 5 mL of annexin V and 5 mL of PI for 5 min at room
temperature in the dark. Finally, the cells were analyzed by flow
cytometry (BD Accuri C6 flowcytometer) within 1 h.
Author contributions
Na Bao and Jinfeng Ou contributed equally to this work. The
manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Acknowledgements
This research has been supported by the Academic Innovation
Project of Jiangsu Department of Education (No. 1150021667). We
also thank Public platform of State Key Laboratory of Natural
Medicines for assistance with fluorescence microscopy and flow
cytometry.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.04.047.
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