Design, synthesis, nitric oxide release and antibacterial evaluation of novel nitrated ocotillol-type derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.06.021

Abstract Nitric oxide (NO) and its auto-oxidation products are known to disrupt normal bacterial function and NO releasing molecules have the potential to be developed as antibacterial leads in drug discovery. We have designed and synthesized a series of

Full text of DOI:10.1016/j.ejmech.2015.06.021

European Journal of Medicinal Chemistry 101 (2015) 71e80
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis, nitric oxide release and antibacterial evaluation of
novel nitrated ocotillol-type derivatives
,
,
,
Yi Bi a 1, Xiao Yang b 1, Tingting Zhang a c, Zeyun Liu a, Xiaochen Zhang a, Jing Lu a,
Keguang Cheng c, Jinyi Xu d, Hongbo Wang a, Guangyao Lv a, Peter John Lewis b,
Qingguo Meng a *, Cong Ma b
,
, *
a School of Pharmacy, Yantai University, Yantai 264005, China
b Biological Sciences, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
c Key Laboratory for The Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin 541004, China
d State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitric oxide (NO) and its auto-oxidation products are known to disrupt normal bacterial function and NO
releasing molecules have the potential to be developed as antibacterial leads in drug discovery. We have
designed and synthesized a series of novel nitrated compounds by combining NO releasing groups with
ocotillol-type triterpenoids, which have previously demonstrated activity only against Gram-positive
bacteria. The in vitro NO release capacity and antibacterial activity were sequentially evaluated and
the data showed that most of the synthesized compounds could release nitric oxide. Compound 16a, 17a
and 17c, with nitrated aliphatic esters at C-3 position, displayed higher NO release than other analogues,
correlating to their good antibacterial activity, in which 17c demonstrated broad-spectrum activity
against both Gram positive and -negative bacteria, as well as excellent synergism at sub-minimum
inhibitory concentration when using with kanamycin and chloramphenicol. Furthermore, the epifluor-
escent microscopic study indicated that the ocotillol-type triterpenoid core may induce NO release on
the bacterial membrane. Our results demonstrate that nitrated substitutions at C-3 of ocotillol-type
derivatives could provide an approach to expand their antibacterial spectrum, and that ocotillol-type
triterpenoids may also be developed as appropriate carriers for NO donors in antibacterial agent dis-
covery with low cytotoxicity.
Received 10 February 2015
Received in revised form
6 June 2015
Accepted 8 June 2015
Available online 17 June 2015
Keywords:
Triterpenoid
Ocotillol
NO release
Antibacterial activity
Synergistic effect
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
Exogenous gaseous NO (gNO) was demonstrated to be bacte-
riostatic at 1.9 ppm [5], and bactericidal at relatively high concen-
As an important cellular signalling molecule in mammals, nitric
oxide (NO) is involved in numerous physiological and pathological
processes [1]. In addition to serving as a powerful vasodilator and
neurotransmitter, NO also takes part in the human immune system,
in which phagocytes generate NO through inducible nitric oxide
synthase (iNOS) [2]. As an immunological response, the resulting
free radicals together with the auto-oxidation products are toxic to
bacteria damaging their lipid membrane and DNA [3], as well as
affecting biofilm formation [4].
tration (120e200 ppm) [6,7], which suggested a high level of NO
production close to a membrane is critical for its therapeutic po-
tential for use against bacterial infections [3]. It has also been
shown that protein bound and nanoparticle fixed NO donors dis-
played antibacterial activity [8,9]. These results encouraged us to
explore the feasibility of hybrid compounds by linking NO releasing
agents to membrane targeting antibacterial agents, which may
exhibit improved membrane penetration and greater activity than
using solely NO or one membrane targeting antibacterial agent. In
fact, some reports already showed that NO hybrid drugs could
exhibit dual-action effects, the hybrid drugs of NO with NSAIDs
such as nitroaspirins could be used to inhibit inflammation without
causing gastric ulcer through protective functions of NO [10].
Ocotillol is a triterpenoid saponin isolated from Fouquieria
* Corresponding authors.
E-mail addresses: qinggmeng@163.com (Q. Meng), cma1@newcastle.edu.au
(C. Ma).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.06.021
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

72
Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
splendens engelm [11] (Fig. 1). It can be easily synthesized from
20(S)-protopanaxadiol (PPD), one of the main components of
Panax ginseng, which can also be isolated from other natural
sources [12]. Research showed that plant derived saponins
commonly participated in the plant defence system by perturbing
bacterial and viral membranes [13]. We recently synthesized a se-
ries of non-cytotoxic ocotillol-type derivatives, such as 3, D1 and
D2, which displayed excellent antibacterial activity against Gram-
positive bacteria including community-associated methicillin
resistant Staphylococcus aureus (CA-MRSA), and demonstrated
outstanding synergism with chloramphenicol (CHL) and kana-
mycin (KAN) [14,15]. In this study, we used the core of ocotillol to
combine with NO releasing agents, then tested their in vitro NO
release capacity and antibacterial activity.
chlorocarbonate in the presence of NaOH to furnish carbonate 5
(Scheme 1), which was subjected to the reaction of nitration to give
nitrate 6. After deprotection of carbonate group, nitrate 7 was ob-
tained with excellent overall yield (Scheme 1). With the same
starting material, substitution of phenol with dibromoethane pro-
vided bromide 8, which was transformed to nitrate 9 in the pres-
ence of silver nitrate (Scheme 1).
As shown in Scheme 2, after the esterification of 3 and 4 with
succinic anhydride, the resulting carboxylic acid 10 and 11 were
obtained with excellent yields. 10 and 11 then reacted with aro-
matic nitrates 7 and 9 in the presence of DMAP and EDCI to provide
hybrid ocotillol-type nitrate 12aeb and 13aeb, respectively.
Alternatively, esterification of 3 and 4 with bromoacetic acid, 5-
bromopentanoic acid or 6-bromohexanoic acid in the presence of
Et3N, DMAP and EDCI respectively furnished 14aec and 15aec
(Scheme 3), which were subjected to nitration with silver nitrate to
give 16aec and 17aec accordingly.
2. Synthetic chemistry
To study the mechanism of action of ocotillol-type compounds,
a fluorescent chemical probe was also synthesized through esteri-
fication using amine-diprotected lysine in the presence of DMAP
and EDCI followed by deprotection of the Boc group. The desired
compound 18 was obtained with 86% yield for 2 steps (Scheme 4).
As previously described [14], PPD was transformed to epimeric
triols 3 and 4 (24R-epimer) by a straightforward three-step pro-
cedure (Fig. 1): protection as diacetate by addition of acetic anhy-
dride in the presence of DMAP; epoxidation followed by
intramolecular nucleophilic addition in situ through mCPBA treat-
ment; and deprotection of acetate with aqueous KOH solution
furnished the desired products.
3. Results and discussion
To test our hypothesis on hybrid antibacterials, we decided to
start with the synthesis of a series of uncomplicated nitrates as NO
donors. 4-(2-hydroxyethyl)phenol was protected using ethyl
The in vitro release of nitric oxide was measured using Griess
reagent as nitrite (NO2-) and nitrate (NO3-) produced by the
Fig. 1. Ocotillol, PPD, 3, D1 and D2.

Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
73
Scheme 1. Synthesis of nitrate 7 and 9.
reaction of nitric oxide with oxygen and water could be detected by
the colourimetric azo coupling reaction with a sulphonamide and
an azo dye agent. Since discovery, Griess reaction has been widely
used in chemical and biological systems [16].
spectrum activity against Gram-positive and -negative bacteria at
16e32 g/ml, despite the fact its epimer 16c only had activity
against E. coli at 64 g/ml. The result suggested that the stereo-
chemistry at C-24 of epimeric 16c and 17c was critical for both NO
release capacity and antibacterial activity.
m
m
As typical NO donors, all of the ocotillol-type nitrate derivatives
displayed NO releasing capacity in a dose-dependent manner. As
shown in Fig. 2, NO release could be visualized by UV absorbance
after reaction with sulfanilamide and N-1-napthylethylenediamine
under acidic conditions. As a control, sodium nitroprusside (SNP)
released a significant quantity of NO after 30 min of reaction, which
is comprehensible as this inorganic compound is commonly used in
hospital for acute hypertension. In comparison, 6-nitrohexanoic
acid (6-NHA, SigmaeAldrich) can also release NO, but at a lower
level. In our case, most of the ocotillol-type compounds showed
In our previous studies [14,15], we demonstrated that these
ocotillol-type triterpenoids were narrow-spectrum antibacterials
that only possessed activity against Gram-positive bacteria. The
structure-activity relationship at present showed that hydrogen
donors at C-3, C-12 and C-24 are required for activity as shown in
Fig. 1 (3, D1 and D2), whereas non-hydrogen donor at C-3 and C-12
only led to loss of antibacterial activity. Our results again proved the
necessity of a hydrogen donor at C-3 which was lacking in our ni-
trate derivatives, as a result, 12ae13b and 17b didn't demonstrate
remarkable antibacterial activity. However, 16a maintained the
activity against Gram positive bacteria with a NO releasing group
substitution. Moreover, 16b, 16c, 17a and especially 17c surprisingly
acquired activity against Gram-negative bacteria, including natu-
rally drug-resistant species, A. baumannii and P. aeruginosa, which
are two particularly difficult organisms to treat in a clinical setting.
The activity of 17c broadly against Gram negative bacteria high-
lighted the promise for antibacterial development of its derivatives.
Comparing the in vitro antibacterial results, the antibacterial
activity of these compounds positively correlated with their in vitro
NO release capacity (Fig. 2): compound 16a, 17a and 17c demon-
strating greater NO release capacity displayed superior antibacte-
rial activity than other analogues, the resulting relationship of NO
concentration and antibacterial activity confirmed the previous
experiments [5e7]. Furthermore, the NO releasing curve (Fig. 2B)
showed that high NO concentration at early time was critical for
antibacterial activity (16a, 17a vs. 12a). The NO release capacity of
17c was significantly higher all along than other compounds, which
may explain its specific activity against A. baumannii and
P. aeruginosa. As an analogue of 17c, 15c with a bromide group
rather than nitrate didn't show any antibacterial activity, signifying
the activity of 17c was due to NO release. Lack of activity of 6-NHA
as the nitro side chain of 17c suggested that the ocotillol-type core
of 17c was also important to induce the antibacterial effect of NO.
To confirm the antibacterial activity of 17c that was triggered by
the ocotillol core as shown previously [14,15], we decided to test
these ocotillol-type nitrates in a synergistic antibacterial assay to
study the mechanism of action. Ocotillol-type nitrate 17c was added
similar NO release capability at 100
mM, however 16a, 17a and 17c
demonstrated superior NO release quantity (OD > 0.2) than other
analogues at 500
(Fig. 2A and B).
mM after 30 min and in a time-course experiment
Furthermore, 17c demonstrated a substantial augmentation on
NO release to 6-NHA as the side chain of 17c (Fig. 2A), suggesting
that the ocotillol-type triterpenoid core may facilitate NO release of
NO donor groups. Fig. 2B also showed that 17c could maximize the
NO release quantity of 6-NHA, as well as accelerate the NO release
rate converting 17c to a quick-acting NO donor agent than 6-NHA,
which has a stabilized NO release rate after 6 h of reaction.
The ocotillol-type nitrates were then tested for antibacterial
activity. Five representative bacteria were used: Gram-positive S.
aureus USA300, Bacillus subtilis 168 and Gram-negative Escherichia
coli DH5a, Acinetobacter baumannii ATCC 19606, Pseudomonas
aeruginosa PAO1.
As shown in Table 1, compound 12a,b and 13a,b only displayed
mild activity at 128 mg/ml against some of the bacteria being tested
(12a against P. aeruginosa; 12b against E. coli, 13a against B. subtilis
and 13b against both B. subtilis and E. coli), while compounds with
an aliphatic nitrate bound at the C-3 ester showed improved ac-
tivity: 16a inhibited the cell growth of Gram-positive S. aureus and
B. subtilis at 16
inhibit B. subtilis at 32
negative E. coli at 64 g/ml. Although epimeric 16b and 17b only
showed antibacterial activity against Gram-negative E. coli and
A. baumannii at 64e128 g/ml, the best activity obtained in the
antibacterial assay was with nitrate 17c, which showed broad-
mg/ml, while the epimeric compound 17a could only
mg/ml, but gained activity against Gram-
m
m

74
Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
Scheme 2. Synthesis of 12aeb and 13aeb.
to the bacterial cell culture at a sub-MIC level in the presence of
different concentrations of antibiotics (chloramphenicol or kana-
mycin). As shown in Table 2, 17c possessed synergistic activity when
used at sub-MIC levels in the presence of kanamycin or chloram-
phenicol, similar totheantibacterial derivatives describedpreviously
with H-donor at C-3 and C-12 [14,15]. When used in combination
with 17c, kanamycin and chloramphenicol prevented growth of CA-
previous studies [14,15], ocotillo-type triterpenoids may affect the
membrane of Gram-positive bacteria, however they appeared to be
ineffective against the lipopolysaccharides (LPSs) specific to the
Gram-negative bacterial outer membrane. The activity gained
against Gram-negative bacteria may be due to the membrane
damaging effect of nitric oxide on LPS. Furthermore, it is known
that NO donors are more efficient if they can be targeted to the
bacterial membrane [17]. As triterpenoid saponins function as
membrane perturbing agents, ocotillol-type nitrates containing the
ocotillol core structure probably bind at the same target of the
antibacterial derivatives described previously with H-donor at C-3
and C-12 [14,15], which places the NO donor groups in close
juxtaposition with the bacterial membrane helping maximize the
function of NO on inhibiting bacterial growth. We haven't tested
17c against A. baumannii and P. aeruginosa for synergism due to its
relatively high MIC, but the synergistic effect of an analogue with
MRSAUSA300 at 0.5 and 0.125
when used with 17c, inhibited the growth of B. subtilis (bacterio-
static) atonly 0.0078 g/ml, andbecamebactericidal at 0.0625 g/ml.
mg/ml, respectively. Chloramphenicol,
m
m
The FIC index [14] illustrated that 17c showed excellent synergistic
effects with FICIs of 0.03 and 0.004 when used with chloramphenicol
against CA-MRSA and B. subtilis, respectively (Table 2).
Furthermore, when 17c was used together with kanamycin or
chloramphenicol against Gram-negative E. coli, synergism was also
observed with an FIC index of 0.063e0.13. As we proposed in

Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
75
Scheme 3. Synthesis of 16aec and 17aec.
Scheme 4. Synthesis of 18.
improved activity against these two bacteria would be more
compelling in the future work.
Epifluorescent microscopy was then used to test our hypothesis
on the mechanism of action in live bacteria. B. subtilis strain BS125
comprises the green fluorescent protein (GFP) tagged p16.7 protein,
which is a membrane integrated protein [18], while B. subtilis strain
BS3 contains GFP tagged NusA protein, which is a transcription
factor distributed within the bacterial nucleoid [19]. As shown in
Fig. 3, GFP distribution reflected the membrane of B. subtilis in
BS125 and the nucleoids in BS3, respectively.

76
Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
As NO is known to affect bacterial and mammalian membranes
[3], the cytotoxicity of NO donor lead compounds should be
considered in drug discovery. We have tested the cytotoxicity of 17c
against human breast cancer (MCF-7), gastric cancer (MKN-45) and
proximal tubule epithelial (HK-2) cell lines as shown in Table 3.
Compared to doxorubicin as an anti-cancer drug, 17c didn't
demonstrated significant cytotoxicity. The selectivity index has also
been calculated using the half toxicity concentration (TC50) to hu-
man cell lines and half inhibitory concentration (IC50) to B. subtilis
or S. aureus, which indicated that 17c was relatively safe when
solely using against bacteria. Moreover, if 17c was synergistically
used at sub-MIC concentration with kanamycin or chloramphen-
icol, the selectivity index could be further improved.
4. Conclusions
We synthesized a series of ocotillol-type triperpenoid nitrates
from the natural plant extract PPD. These new derivatives have
been investigated for their in vitro NO release capacity, and the
results showed that all the compounds can release nitric oxide,
with compound 16a, 17a and 17c demonstrating more efficient
releasing capability than other compounds. Furthermore, the
in vitro antibacterial activity was tested and correlated to the NO
release capacity of compounds. 17c releasing most NO displayed
good activity against both Gram positive and -negative bacteria and
low cytotoxicity against three different human cell lines, whereas
15c lacking nitrate substituent and 6-NHA without the ocotillol-
type triterpenoid core didn't show any antibacterial activity, indi-
cating that the nitrate substituent and the triterpenoid core were
both required for activity. The epifluorescent microscopic study
also showed that fluorescent ocotillol-type triterpenoid 18c had
affinity to the bacterial membrane rather than the nucleoid, which
suggested the antibacterial activity of nitrated ocotillol-type tri-
perpenoids were due to the ocotillol-type triperpenoid core
directed nitric oxide release on the bacterial membrane. Further-
more, the synergistic study showed that ocotillol-type triperpe-
noids in combination with kanamycin or chloramphenicol
maintained the synergistic activity against Gram positive bacteria
as the ocotillol-type compounds reported previously [14,15], sug-
gesting they shared the same mechanism of synergism. Addition-
ally, the newly gained activity of 17c against clinically relevant
Gram negative bacteria demonstrated the potential of nitrate sub-
stitution in our ocotillol-type antibacterial discovery.
Although we discovered that the hydrogen donor substituents
at C-3 and C-12 positions of the ocotillol-type triterpenoid core
were essential for antibacterial activity against Gram positive bac-
teria [14,15], our study in this chapter demonstrated an exception
that some nitrated derivatives with non-hydrogen donor at C-3
could also exhibit antibacterial activity with broader spectrum
including Gram negative bacteria due to directed NO release on the
bacterial membrane. Based on the data shown above, an ideal
ocotillol-type antibacterial agent should possess hydrogen donors
at C-3 and C-12 positions and also a NO releasing group for superior
antibacterial activity and expansion of the antibacterial spectrum,
respectively. In the near future, the synthesis of novel nitrated
ocotillol-type tritepenoids containing both hydrogen donors and
nitrated substituents at C-3 position as new antibacterial leads
could be expected.
Fig. 2. A) NO release of 12ae17c, 6-NHA and sodium nitroprusside (SNP) at 100 and
500 mM after 30 min; B) Time course of NO release of 12ae17c at 500 mM.
We synthesized compound 18 composing the ocotillol-type
triterpenoid core, an H-donor at C-3 and a fluorescent Fmoc
group and measured the MIC against Gram positive bacteria at 8 g/
m
ml (Scheme 4). When normal B. subtilis strain 168 cells were treated
with compound 18 at sub-MIC concentration, as shown in Fig. 3
(middle), after 15 min of treatment, the fluorescence could be
observed with similar distribution to BS125, which indicated that
compound 18 was evenly distributed on the bacterial membrane
rather than within the nucleoid. This result suggested that the NO
release of 17c should be induced on the bacterial membrane due to
affinity of the ocotillol-type triterpenoid core to the membrane to
exhibit its antibacterial activity.
Table 1
In vitro antibacterial activity of ocotillol-type derivatives 12ae17c (MIC: mg/ml).
Strain
S. aureus
B. subtilis
E. coli
A. baumannii
P. aeruginosa
12a
12b
13a
13b
16a
16b
16c
17a
17b
17c
>128
>128
>128
>128
16
>128
>128
>128
>128
16
>128
>128
128
128
16
>128
>128
32
>128
16
>128
128
>128
128
>128
64
64
64
128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
128
>128
>128
>128
>128
>128
>128
>128
>128
32
5. Experimental
15c
>128
>128
1
>128
>128
0.25
>128
>128
1
>128
>128
1
>128
>128
8
6-NHAa
KANb
5.1. Chemistry
a
Most chemicals and solvents were analytical grade and, when
necessary, were purified and dried with standard methods. Melting
6-NHA: 6-nitrohexanoic acid.
KAN: kanamycin.
b

Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
77
Table 2
Synergistic effect of kanamycin and chloramphenicol with compounds 17c against S. aureus USA 300, B. subtilis 168 and E. coli DH5
a
.
petroleum ether: ethyl acetate) to provide 5 as a yellow oil (1.93 g,
9.2 mmol, 92%) and the spectrometric data of 5 are identical to the
literature [20].
A solution of 5 (10 mmol) and acetic anhydride (1 ml) in CH2Cl2
was cooled to 0 ꢁC, nitrosonitric acid (1 ml) was added slowly and
stirred for 4 h at room temperature. The solution was washed with
water (3 ꢂ 20 ml) followed by brine (3 ꢂ 10 ml), then dried over
anhydrous Na2SO4, filtered and evaporated in vacuo. Purification by
column chromatography (60:1 petroleum ether: ethyl acetate) gave
6 as a yellow oil (2.30 g, 9.0 mmol, 90%).
A solution of 6 (10 mmol) and cholamine (1 ml) in 95% ethanol
(25 ml) was stirred for 6 h at room temperature. The mixture was
neutralized by 10% HCl, and was added ethyl acetate. The organic
layer was washed with water and brine, dried over anhydrous
Na2SO4, filtered and evaporated. Purification by column chroma-
tography (50:1 petroleum ether: ethyl acetate) furnished 7 as a
yellow oil (1.57 g, 8.6 mmol, 86%) and the spectrometric data are
identical to the literature [21].
Fig. 3. Epifluorescent microscopic images of B. subtilis strain BS125 (left), strain 168
with compound 18 treatment (middle) and strain BS3 (right). Scale bar: 4 m.
m
points were taken on a WRS-13 micro melting point apparatus and
uncorrected. 1H NMR spectra were recorded with
Bruker
a
AVANCE-400 spectrometer in the indicated solvents (TMS as in-
ternal standard): the values of the chemical shifts are expressed in
d
values (ppm) and the coupling constants (J) in Hz. High-resolution
mass spectra were recorded using an Agilent QTOF 6520.
5.1.3. 4-[O-(2-nitro)ethyl]phenylethanol (9)
5.1.1. (20S, 24S)-Epoxy-dammarane-3b, 12b, 25-triol (3) and (20S,
To a solution of 2-(4-Hydroxyphenyl)ethanol (10 mmol) and
potassium carbonate (20.00 mmol) in THF (5 ml) was added 1,2-
dibromoethane (15 mmol) slowly. The resulting mixture was stir-
red for 5 h at room temperature, then washed with water and brine,
dried over anhydrous Na2SO4, filtered and evaporated. Purification
by column chromatography (30:1 petroleum ether: acetone) gave 8
as a yellow solid (2.17 g, 8.9 mmol, 89%).
24R)-Epoxy-dammarane-3 , 12 , 25-triol (4)
Compound 3 and 4 were prepared from PPD according to the
published procedures [14].
b
b
5.1.2. 4-[(2-nitroxy)ethyl]phenol (7)
A solution of 2-(4-Hydroxyphenyl)ethanol (10 mmol) in sodium
hydroxide solution (10 ml, 1 M) was cooled to ꢀ5 ꢁC. Then ethyl
chlorocarbonate (12 mmol) was added slowly and stirred for 5 h at
room temperature. After the pH of mixture was adjusted to 2 with
10% HCl, the solution was extracted by ethyl acetate and the organic
layer was washed with brine (3 ꢂ 10 ml), dried over anhydrous
Na2SO4 and the solvent was filtered and evaporated in vacuo. The
oily residue was purified by column chromatography (50:1
A solution of 8 (10 mmol), silver nitrate (20 mmol) in acetoni-
trile (12 ml) was stirred for 6 h at 70 ꢁC under protection from light.
The resulting mixture was washed with 10% HCl, water and brine
successively, dried over anhydrous Na2SO4, filtered and evaporated.
Purification by column chromatography (50:1 petroleum ether:
acetone) gave 9 as a yellow solid (1.93 g, 8.5 mmol, 85%). mp:
41e43 ꢁC. 1H NMR (CDCl3,
d
, 400 MHz): 2.80 (2H, t, J ¼ 6.6 Hz), 3.81
Table 3
Cytotoxicity and selectivity index of 17c against human cell line MCF-7, MKN-45 and HK-2.
TC50 M)
(
m
MCF-7 (breast cancer)
MKN-45 (gastric cancer)
HK-2 (human proximal tubule epithelial cell)
17c
Doxorubicin
Selectivity Index of 17c (TC50/IC50
45.43 0.86
9.99 1.21
3.60
52.00 0.54
15.83 2.32
4.12
97.74 5.82
18.47 3.41
9.05
)

78
Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
(2H, t, J ¼ 6.6 Hz), 4.22 (2H, t, J ¼ 4.6 Hz), 4.79 (2H, t, J ¼ 4.6 Hz), 6.85
5.1.9. 4-oxo-[(20S,24R)-Epoxy-dammarane-12b-hydroxy-3b-O]-1-
(2H, d, J ¼ 8.5 Hz), 7.15 (2H, d, J ¼ 8.5 Hz). 13C NMR (CDCl3,
d
,
[4-(2-nitro)ethyl]benzyl butyrate (13a)
100 MHz): 38.1, 63.6, 64.0, 71.0, 114.6, 130.1, 131.5, 156.5. HR-MS
(ESI, m/z) [M þ Na]þ calcd. for C10H13NO5, 250.0686, found:
250.0693.
Yellow solid, 90% yield, mp: 110e111 ꢁC. 1H NMR (CDCl3,
d,
400 MHz): 0.84 (3H, s), 0.86 (3H, s), 0.90 (3H, s), 1.01 (3H, s), 1.09
(3H, s), 1.22 (3H, s), 1.26 (3H, s), 1.28 (3H, s), 2.61 (4H, s), 2.87 (3H, t,
J ¼ 7.1 Hz), 3.51 (1H, dt, J ¼ 10.4 Hz, J ¼ 4.5 Hz), 3.85 (1H, dd,
J ¼ 8.7 Hz, J ¼ 6.7 Hz), 4.23 (2H, t, J ¼ 4.7 Hz), 4.25 (2H, t, J ¼ 7.2 Hz),
4.48 (1H, dd, J ¼ 10.3 Hz, J ¼ 5.9 Hz), 4.81 (2H, t, J ¼ 4.6 Hz), 6.83 (2H,
5.1.4. (20S, 24S)-Epoxy-3
12 , 25-diol (10)
Butanedioic anhydride (60 mg, 0.60 mmol) was added to a so-
b-O-(3-carboxy propionyl)-dammarane-
b
d, J ¼ 8.6 Hz), 7.13 (2H, d, J ¼ 8.5 Hz). 13C NMR (CDCl3,
d, 100 MHz):
lution of 3 (143 mg, 0.30 mmol) and DMAP (20 mg, 0.16 mmol) in
dry CH2Cl2 (8 ml). The reaction mixture was stirred at 35 ꢁC for 5 h,
then washed with 10% HCl, water and brine successively, dried over
anhydrous Na2SO4, concentrated and purified by silica gel column
chromatography (20:1 dichloromethane: methanol) to afford the
compound as a white solid (159 mg, 0.28 mmol, 92%).
15.3, 16.3, 16.4, 18.1, 23.6, 24.9, 26.1, 27.6, 27.8, 27.9, 28.5, 29.3, 29.4,
29.7, 31.2, 31.3, 32.6, 32.7, 34.7, 37.0, 37.9, 38.5, 39.7, 47.9, 49.3, 50.4,
51.9, 56.0, 70.1, 70.9, 73.1, 81.4, 85.4, 86.5, 121.8, 129.9, 133.6, 149.7,
170.9, 171.7. MS (ESI, m/z) [M þ H]þ: 742.5, HR-MS (ESI, m/z)
[M þ H]þ calcd. for C42H63NO10, 742.4525, found: 742.4536.
5.1.10. 4-oxo-[(20S,24R)-Epoxy-dammarane-12b-hydroxy-3b-O]-
5.1.5. (20S, 24R)-Epoxy-3
12 , 25-diol (11)
Butanedioic anhydride (63 mg, 0.63 mmol) was added to a so-
lution of 4 (151 mg, 0.32 mmol) and DMAP (17 mg, 0.14 mmol) in
dry CH2Cl2 (10 ml). The reaction mixture was stirred at 35 ꢁC for 5 h,
then washed with 10% HCl, water and brine successively, dried over
anhydrous Na2SO4, concentrated and purified by silica gel column
chromatography (30:1 dichloromethane: methanol) to afford the
compound as a white solid (164 mg, 0.28 mmol, 89%).
b
-O-(3-carboxy propionyl)-dammarane-
1-[4-O-(2-nitro)ethyl]phenethyl butyrate (13b)
b
Yellow solid, 89% yield, mp: 116e117 ꢁC. 1H NMR (CDCl3,
d,
400 MHz): 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10
(3H, s), 1.23 (3H, s), 1.25 (3H, s), 1.27 (3H, s), 2.73 (2H, t, J ¼ 6.3 Hz),
2.88 (2H, t, J ¼ 6.3 Hz), 3.01 (2H, t, J ¼ 7.0 Hz), 3.52 (1H, dt,
J ¼ 10.4 Hz, J ¼ 4.4 Hz), 3.84 (1H, dd, J ¼ 8.6 Hz, J ¼ 6.8 Hz), 4.52 (1H,
dd, J ¼ 10.4 Hz, J ¼ 6.0 Hz), 4.81 (2H, t, J ¼ 7.0 Hz), 6.83 (2H, d,
J ¼ 8.6 Hz), 7.13 (2H, d, J ¼ 8.5 Hz). 13C NMR (CDCl3,
d, 100 MHz):
15.3, 16.3, 16.4, 18.1, 23.6, 24.2, 24.9, 26.1, 27.6, 27.9, 28.5, 29.2, 29.5,
31.2, 31.3, 32.6, 34.1, 34.7, 37.0, 37.9, 38.6, 39.7, 47.9, 49.4, 50.4, 52.0,
56.0, 64.0, 65.2, 70.1, 70.9, 71.0, 73.2, 81.2, 85.4, 86.5, 114.6, 130.0,
130.8, 156.6, 171.9, 172.2. MS (ESI, m/z) [M þ H]þ: 786.5, HR-MS (ESI,
m/z) [M þ H]þ calcd. for C44H67NO11, 786.4787, found: 786.4809.
5.1.6. General procedure for the synthesis of 12aeb and 13aeb
To a solution of the appropriate nitrate (7 or 9) (0.15 mmol),
DMAP (0.12 mmol) and EDCI (0.084 mmol) in dry CHCl3 (8 ml),
compound 10 or 11 (0.1 mmol) was added. The mixture was stirred
for 6 h at 35 ꢁC. The organic solution was washed with 10% HCl,
water and brine successively, dried over anhydrous Na2SO4,
concentrated and purified by silica gel column chromatography to
provide compound 12aeb and 13aeb.
5.1.11. General procedure for the synthesis of 16aec and 17aec
To a solution of bromoacetic acid, 5-bromopentanoic acid or 6-
bromohexanoic acid (0.2 mmol), triethylamine (3 drops), DMAP
(0.12 mmol) and EDCI (0.084 mmol) in dry CHCl3 (8 ml), compound
3 or 4 (0.1 mmol) was added. The mixture was stirred for 4 h at
room temperature, then washed with 10% HCl, water and brine
successively, dried over anhydrous Na2SO4, concentrated and pu-
rified by silica gel column chromatography to provide the com-
pounds 14aec and 15aec. 15c was characterized for experiment
and comparison with 17c.
A solution of 14a (or 14b, 14c, 15a, 15b, 15c) (10 mmol), silver
nitrate (20 mmol) in acetonitrile (10 ml) was stirred for 6 h at 70 ꢁC
under the conditions of protection from light. The mixture was
washed with 10% HCl (20 ml ꢂ 3), water and brine successively,
dried over anhydrous Na2SO4, filtered and evaporated. Purification
by column chromatography to provide compound 16aec and
17aec.
5.1.7. 4-oxo-[(20S,24S)-Epoxy-dammarane-12b-hydroxy-3b-O]-1-
[4-(2-nitro)ethyl]benzyl butyrate (12a)
Yellow solid, 83% yield, mp: 112e113 ꢁC. 1H NMR (CDCl3,
d,
400 MHz): 0.84 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10
(3H, s), 1.23 (3H, s), 1.25 (3H, s), 1.27 (3H, s), 2.73 (2H, t, J ¼ 6.3 Hz),
2.88 (2H, t, J ¼ 6.3 Hz), 3.01 (2H, t, J ¼ 7.0 Hz), 3.51 (1H, dt,
J ¼ 10.5 Hz, J ¼ 4.4 Hz), 3.84 (1H, dd, J ¼ 8.6 Hz, J ¼ 6.8 Hz), 4.52 (1H,
dd, J ¼ 10.3 Hz, J ¼ 5.9 Hz), 4.62 (2H, t, J ¼ 7.0 Hz), 7.05 (2H, d,
J ¼ 8.4 Hz), 7.22 (2H, d, J ¼ 8.4 Hz). 13C NMR (CDCl3,
d, 100 MHz):
15.3, 16.3, 16.4, 18.1, 23.6, 24.9, 26.1, 27.5, 27.8, 27.9, 28.5, 29.3, 29.4,
29.6, 31.1, 31.2, 32.5, 32.6, 34.6, 37.0, 37.8, 38.5, 39.7, 47.8, 49.3, 50.3,
51.9, 56.0, 70.0, 70.8, 73.1, 81.3, 87.1, 87.4, 121.8, 129.8, 133.5, 149.6,
170.9, 171.7. MS (ESI, m/z) [MþH]þ: 742.5, HR-MS (ESI, m/z) [MþH]þ
calcd. for C42H63NO10, 742.4525, found: 742.4550.
5.1.12. (20S, 24R)-Epoxy-3
12 , 25-diol (15c)
Yellow oil, 92% yield, 1H NMR (CDCl3,
d, 400 MHz): 0.82 (3H, s),
b-O-(6-bromo hexanoyl)-dammarane-
b
5.1.8. 4-oxo-[(20S,24R)-Epoxy-dammarane-12
b
-hydroxy-3b-O]-1-
[4-O-(2-nitro)ethyl]phenethyl butyrate (12b)
0.83 (3H, s), 0. 88 (3H, s), 0.89 (3H, s), 0.90 (3H, s), 1.08 (3H, s), 1.25
(3H, s), 1.27 (3H, s), 2.32 (2H, t, J ¼ 7.4 Hz), 3.40 (2H, t, J ¼ 6.8 Hz),
3.51 (1H, td, J ¼ 10.5 Hz, J ¼ 4.5 Hz), 3.84 (1H, dd, J ¼ 8.8 Hz,
Yellow solid, 87% yield, mp: 114e115 ꢁC. 1H NMR (CDCl3,
d,
400 MHz): 0.83 (3H, s), 0.85 (3H, s), 0.91 (3H, s), 1.02 (3H, s), 1.10
(3H, s), 1.22 (3H, s), 1.25 (3H, s), 1.26 (3H, s), 2.61 (4H, s), 2.87 (2H, t,
J ¼ 7.1 Hz), 3.52 (1H, dt, J ¼ 10.2 Hz, J ¼ 4.7 Hz), 3.89 (1H, dd,
J ¼ 10.8 Hz, J ¼ 5.4 Hz), 4.23 (2H, t, J ¼ 4.6 Hz), 4.25 (2H, t, J ¼ 7.2 Hz),
4.49 (1H, dd, J ¼ 10.4 Hz, J ¼ 6.0 Hz), 4.62 (2H, t, J ¼ 4.7 Hz), 5.74 (1H,
J ¼ 6.7 Hz), 4.48 (1H, dd, 2H, t, J ¼ 6.6 Hz). 13C NMR (CDCl3,
d,
100 MHz): 15.3, 16.3, 16.4, 18.1, 23.7, 24.2, 24.9, 26.1, 26.5, 27.6, 27.9,
28.0, 28.5, 29.6, 31.1, 31.3, 32.4, 32.6, 33.5, 34.5, 34.7, 37.0, 37.8, 38.6,
39.7, 47.9, 49.3, 50.3, 51.9, 56.0, 70.0, 70.9, 80.6, 85.4, 86.4,173.2. HR-
MS (ESI, m/z) [M þ H]þ calcd. for C36H62BrO5, 653.3792, found:
653.3775.
s), 7.05 (2H, d, J ¼ 8.4 Hz), 7.23 (2H, d, J ¼ 8.4 Hz). 13C NMR (CDCl3,
d,
100 MHz): 15.4, 16.3, 16.4, 17.7, 18.1, 23.6, 24.2, 25.0, 27.9, 28.0, 28.5,
29.2, 29.5, 31.6, 31.7, 32.2, 34.1, 34.6, 37.0, 37.8, 38.5, 39.7, 48.8, 48.9,
50.1, 52.1, 55.9, 64.0, 65.2, 69.9, 70.1, 70.4, 71.0, 81.1, 87.1, 87.3, 114.4,
130.0, 130.8, 156.6, 171.8, 172.2. MS (ESI, m/z) [M þ H]þ: 786.5, HR-
MS (ESI, m/z) [M þ H]þ calcd. for C44H67NO11, 786.4787, found:
786.4826.
5.1.13. (20S, 24S)-Epoxy-3
12 , 25-diol (16a)
Yellow oil, 82% yield, 1H NMR (CDCl3,
0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.26
b-O-(2-nitrooxy acetyl)-dammarane-
b
d
, 400 MHz): 0.84 (3H, s),

Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
79
(3H, s),1.27 (3H, s), 3.57 (1H, td, J ¼ 10.3 Hz, J ¼ 4.7 Hz), 3.90 (1H, dd,
J ¼ 10.8 Hz, J ¼ 5.4 Hz), 4.65 (1H, dd, J ¼ 8.5 Hz, J ¼ 8.0 Hz), 4.89(2H,
s). 13C NMR (CDCl3,
, 100 MHz): 15.4, 16.3, 17.7, 18.1, 23.5, 24.2, 25.0,
J ¼ 6.6 Hz). 13C NMR (CDCl3,
d
, 100 MHz): 15.3, 16.4, 16.5, 18.1, 23.7,
24.5, 24.9, 25.2, 26.1, 26.5, 27.6, 27.89, 27.91, 28.5, 29.7, 31.2, 31.3, 32.6,
34.4, 34.7, 37.0, 37.8, 38.6, 39.7, 47.9, 49.3, 50.4, 51.9, 56.0, 70.0, 70.9,
72.9, 80.7, 85.4, 86.5,173.0. MS(ESI, m/z) [Mþ H]þ: 636.4, HR-MS(ESI,
m/z) [M þ H]þ calcd. for C36H61NO8, 636.4470, found: 636.4488.
d
27.9, 28.0, 28.5, 28.8, 29.3, 29.7, 31.6, 32.2, 34.6, 37.0, 37.9, 38.4, 39.7,
48.7, 48.8, 50.0, 52.1, 55.9, 67.4, 70.0, 70.4, 83.4, 87.1, 87.4, 165.6. MS
(ESI, m/z) [M þ H]þ: 580.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for
C
32H53NO8, 580.3844, found: 580.3862.
5.1.19. Procedure for the synthesis of 18
To
a solution of Boc-Lys(Fmoc)-OH (0.15 mmol), DMAP
5.1.14. (20S, 24S)-Epoxy-3
dammarane-12 , 25-diol (16b)
Yellow oil, 86% yield, 1H NMR (CDCl3,
b
-O-(5-nitrooxy pentanoyl)-
(0.12 mmol) and EDCI (0.084 mmol) in dry CHCl3 (8 mL) was added
compound 4 (0.1 mmol). The mixture was stirred for 6 h at 35 ꢁC,
then washed with 10% HCl, water and brine successively, dried over
anhydrous Na2SO4. The resulting solution was concentrated and
diluted by ethyl acetate-HCl (9 mL, 8:1). The mixture was stirred for
5 h at 40 ꢁC, then washed with 10% HCl, water and brine succes-
sively, dried over anhydrous Na2SO4. The resulting layer was
concentrated and purified by silica gel column chromatography
(200:1 dichloromethane: methanol) to provide 18 (71.2 mg,
0.086 mmol, 86%).
b
d, 400 MHz): 0.84 (3H, s),
0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.25
(3H, s), 1.27(3H, s), 2.34 (2H, t, J ¼ 6.8 Hz), 3.50 (1H, td, J ¼ 10.4 Hz,
J ¼ 4.8 Hz), 3.85 (1H, dd, J ¼ 10.8 Hz, J ¼ 5.4 Hz), 4.45 (1H, t,
J ¼ 6.0 Hz), 5.77 (1H, s). 13C NMR (CDCl3,
d, 100 MHz): 15.4, 16.3,
16.5, 17.7, 18.2, 24.2, 25.0, 26.2, 27.2, 28.0, 28.5, 28.8, 29.7, 31.60,
31.64, 31.9, 32.2, 33.9, 34.6, 37.1, 37.9, 38.5, 39.7, 48.8, 48.9, 50.1, 52.1,
55.9, 70.0, 70.5, 72.7, 80.9, 87.1, 87.4, 172.5. MS (ESI, m/z) [M þ H]þ:
622.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for C35H59NO8, 622.4313,
found: 622.4330.
5.1.20. (20S,24R)-Epoxy-3
dammarane-12 ,25-diol (18)
Yellow solid, mp. 132e134 ꢁC, 83% yield. 1H NMR (CDCl3,
d,
b
-O-{[2-amino-6-(N0-Fmoc)]hexanoly}-
b
5.1.15. (20S, 24S)-Epoxy-3
12 , 25-diol (16c)
Yellow oil, 84% yield, 1H NMR (CDCl3,
b
-O-(6-nitrooxy hexanoyl)-dammarane-
b
400 MHz): 0.83 (3H, s), 0.84 (3H, s), 0.87 (3H, s), 0.89 (3H, s), 0.91
(3H, s), 1.09 (3H, s), 1.24 (3H, s), 1.26 (3H, s), 3.21 (2H, t, J ¼ 5.6 Hz),
3.49 (1H, t, J ¼ 5.5 Hz), 3.51 (1H, td, J ¼ 10.4 Hz, J ¼ 4.7 Hz), 3.85 (1H,
dd, J ¼ 8.7 Hz, J ¼ 6.6 Hz), 4.38 (2H, d, J ¼ 6.9 Hz), 4.50 (1H, t,
J ¼ 8.2 Hz), 4.94 (1H, s), 5.60 (1H, s), 7.31 (2H, td, J ¼ 7.3, J ¼ 0.8 Hz),
7.40 (2H, td, J ¼ 7.4, J ¼ 0.8 Hz), 7.59 (2H, d, J ¼ 7.4), 7.76 (2H, d,
d, 400 MHz): 0.84 (3H, s),
0.85 (3H, s), 0.91 (3H, s), 1.01 (3H, s), 1.10 (3H, s), 1.23 (3H, s), 1.25
(3H, s), 1.27 (3H, s), 2.28 (2H, t, J ¼ 7.4 Hz), 3.52 (1H, td), 3.82 (1H,
dd, J ¼ 10.7 Hz, J ¼ 5.4 Hz), 4.43 (1H, dd, 2H, t, J ¼ 6.7 Hz). 13C NMR
(CDCl3, d, 100 MHz): 15.4, 16.3, 16.5, 17.7, 18.2, 23.7, 24.5, 25.0, 25.2,
26.5, 27.9, 28.0, 28.5, 28.8, 29.7, 31.6, 31.7, 32.2, 34.4, 34.7, 37.1, 37.9,
38.5, 39.7, 48.8, 48.9, 50.1, 52.1, 55.9, 70.0, 70.4, 72.9, 80.7, 87.1, 87.4,
173.0. MS (ESI, m/z) [M þ H]þ: 636.4, HR-MS (ESI, m/z) [M þ H]þ
calcd. for C36H61NO8, 636.4470, found: 636.4492.
J ¼ 7.6). 13C NMR (CDCl3,
d,100 MHz): 15.3,16.3,16.5,18.0, 23.7, 24.5,
25.0, 25.8, 26.1, 27.6, 27.9, 28.0, 28.5, 29.6, 31.3, 32.6, 34.0, 34.7, 37.0,
37.9, 38.5, 39.7, 40.7, 47.2, 47.9, 49.3, 50.3, 51.9, 52.6, 54.5, 55.9, 66.5,
70.1, 70.9, 81.5, 85.4, 86.4, 119.9, 125.0, 126.9, 128.8, 131.1, 141.2,
143.9, 156.4. HR-MS (ESI, m/z) [M þ H]þ calcd. for C51H75N2O7,
827.5588, found: 827.5569.
5.1.16. (20S, 24R)-Epoxy-3
12 , 25-diol (17a)
Yellow solid, 79% yield, mp. 157e158 ꢁC. 1H NMR (CDCl3,
b-O-(2-nitrooxy acetyl)-dammarane-
b
d
,
5.2. In vitro NO release evaluation assay
400 MHz): 0.83 (3H, s), 0.84 (3H, s), 0.88 (3H, s), 0.89 (3H, s), 0.98
(3H, s), 1.09 (3H, s), 1.26 (3H, s), 1.27 (3H, s), 3.54 (1H, td, J ¼ 10.4 Hz,
J ¼ 4.4 Hz), 3.86 (1H, dd, J ¼ 8.6 Hz, J ¼ 6.8 Hz), 4.63 (1H, dd,
In vitro NO release was measured as described [22]. Phosphate
buffer containing
a solution of KH2PO4 (269 mg), NaHPO4$7H2O (1831 mg) and
cysteine (218 mg). The final concentration of -cysteine in this PBC
L-cysteine (PBC) solution was prepared by mixing
J ¼ 8.8 Hz, J ¼ 7.6 Hz), 4.88 (2H, s). 13C NMR (CDCl3,
d, 100 MHz):
L-
15.3, 16.27, 16.30, 18.07, 18.11, 23.5, 24.9, 26.1, 27.5, 27.8, 28.5, 31.1,
31.3, 32.6, 34.6, 36.9, 37.9, 38.5, 39.7, 47.9, 49.3, 50.3, 51.9, 52.1, 55.9,
67.3, 70.0, 70.8, 83.4, 85.4, 86.4, 165.5. MS (ESI, m/z) [M þ H]þ:
580.4, HR-MS (ESI, m/z) [M þ H]þ calcd. for C32H53NO8, 580.3844,
found: 580.3860.
L
solution is 18 mM. Griess reagent was prepared by dissolving sul-
fanilamide (4.0 g) and N-napthylenediamine$2HCl (0.2 g) in a
mixture of 85% H3PO4 (10 ml) and distilled H2O (90 ml). A solution
of 5% dimethyl sulfoxide in PBC (5% DPBC solution) was prepared by
diluting dimethyl sulfoxide (2.5 ml) with PBC solution (7.5 ml). An
5.1.17. (20S, 24R)-Epoxy-3
dammarane-12 , 25-diol (17b)
Yellow oil, 85% yield, 1H NMR (CDCl3,
b
-O-(5-nitrooxy pentanoyl)-
aliquot of this 5% DPBC solution (200 ml) was used as a control.
b
Griess reagent (250 ml) was added, the mixture was maintained at
d, 400 MHz): 0.83 (3H, s),
37 ꢁC for 30 min with gentle shaking, and the solution's absorbance
at 540 nm was measured.
0.84 (3H, s), 0. 88 (3H, s), 0.90 (3H, s), 0.98 (3H, s), 1.09 (3H, s), 1.26
(3H, s), 1.27(3H, s), 2.37(2H, t, J ¼ 6.9 Hz), 3.54 (1H, td, J ¼ 10.4 Hz,
J ¼ 4.4 Hz), 3.86 (1H, dd, J ¼ 8.5 Hz, J ¼ 7.0 Hz), 4.50 (1H, dd, 2H, t,
A standard nitrite-absorbance concentration plot was prepared
as follows: A solution of NaNO2 (0.1 mM) in 5% DPBC solution
(2.4 ml) was prepared, and this solution was maintained at 37 ꢁC for
1 h with gentle shaking. Griess reagent (0.8 ml) was added and the
mixture was maintained at 37 ꢁC for 30 min with gentle shaking. A
solution of NaNO2 for use as a dilution solvent (SNDS) was prepared
by mixing Griess reagent (32 ml) with 5% DPBC solution (96 ml).
Dilutions of the NaNO2 containing solution with SNDS solution
were used to prepare the calibration curve from which nitrite
concentrations (absorbance at 540 nm) were calculated.
J ¼ 6.0 Hz). 13C NMR (CDCl3,
d, 100 MHz): 15.4, 16.4, 16.4, 18.1, 21.2,
23.7, 24.9, 26.1, 26.2, 27.6, 27.92, 27.98, 28.5, 29.7, 31.2, 31.3, 32.6,
33.9, 34.7, 37.0, 37.8, 38.6, 39.7, 47.9, 49.4, 50.4, 51.9, 56.0, 70.0, 70.9,
72.7, 80.9, 85.4, 86.5, 172.6. MS (ESI, m/z) [M þ Na]þ: 644.3, HR-MS
(ESI, m/z) [M þ H]þ calcd. for C35H59NO8, 622.4313, found:
622.4329.
5.1.18. (20S, 24R)-Epoxy-3
12 , 25-diol (17c)
Yellow oil, 81% yield, 1H NMR (CDCl3,
b-O-(6-nitrooxy hexanoyl)-dammarane-
b
d, 400 MHz): 0.83 (3H, s),
5.3. Antibacterial activity assay
0.84 (3H, s), 0.87 (3H, s), 0.89 (3H, s), 0.98 (3H, s),1.09 (3H, s),1.26 (3H,
s), 1.27 (3H, s), 2.34 (2H, t, J ¼ 7.3 Hz), 3.54 (1H, td, J ¼ 10.5 Hz,
J ¼ 4.5 Hz), 3.86 (1H, dd, J ¼ 8.8 Hz, J ¼ 6.7 Hz), 4.49 (1H, dd, 2H, t,
The antibacterial and synergistic activity assay was performed
as described previously [14,23].

80
Y. Bi et al. / European Journal of Medicinal Chemistry 101 (2015) 71e80
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The MTT assay was performed as described previously [24].
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
The authors are grateful to National Natural Science Foundation
of China (No. 81001358, 81473104 and 81202038); Promotive
Research Fund for Excellent Young and Middle-aged Scientists of
Shandong Province (No. BS2010YY073); Project of Shandong
Province Higher Educational Science and Technology Program
(J12LM53); Key Laboratory for the Chemistry and Molecular Engi-
neering of Medicinal Resources (Guangxi Normal University),
Ministry of Education of China (CMEMR2014-B07); Taishan Scholar
Project to Fenghua Fu and an Early Career Research Grant from the
University of Newcastle (CM) for financial support.
[18] D.H. Meredith, M. Plank, P.J. Lewis, Different patterns of integral membrane
protein localization during cell division in Bacillus subtilis, Microbiology 154
(2008) 64e71.
[19] K.M. Davies, A.J. Dedman, S. van Horck, P.J. Lewis, The NusA: RNA polymerase
ratio is increased at sites of rRNA synthesis in Bacillus subtilis, Mol. Microbiol.
57 (2005) 366e379.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.06.021.
[20] R. Barattin, T. Perrotton, D. Trompier, D. Lorendeau, A. Di Pietro, A. du Mou-
linet d’Hardemare, H. Baubichon-Cortay, Iodination of verapamil for a stron-
ger induction of death, through GSH efflux, of cancer cells overexpressing
MRP1, Bioorg. Med. Chem. 18 (2010) 6265e6274.
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activity and safety of hydrocortisone derivatives coupled with furoxans and
nitrates, Chin. J. Org. Chem. 28 (2008) 841e850.
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