Design, synthesis and in vitro NO-releasing activities of ocotillol-type furoxans

Article abstract of DOI:10.1691/ph.2015.4775

A series of novel ocotillol-type furoxan derivatives was synthesized by coupling various furoxans to 3-OH of 6-deoxy ocotillol, and their in vitro nitric oxide (NO) releasing capability was studied. The discharge of NO was examined after 30 min at two dif

Full text of DOI:10.1691/ph.2015.4775

ORIGINAL ARTICLES
1
2
School of Pharmacy , Yantai University, Shandong, China; Biological Sciences , School of Environmental and Life
Sciences, University of Newcastle, Callaghan, Australia; Key Laboratory for the Chemistry and Molecular Engineering
3
of Medicinal Resources , Guangxi Normal University, Guilin, China
Design, synthesis and in vitro NO-releasing activities of
ocotillol-type furoxans
1
1
2
1
1,3
1
1
1
Yi Bi , Jian Yang , Cong Ma , Ze-Yun Liu , Ting-Ting Zhang , Xiao-Chen Zhang , Jing Lu , Qing-Guo Meng
Received September 2, 2014, accepted October 30, 2014
Yi Bi, School of Pharmacy, Yantai University, Yantai, Shandong 264005, China
beeyee 413@163.com
Qing-guo Meng, School of pharmacy, Yantai University, Yantai, Shandong 264005, China
Pharmazie 70: 213–218 (2015)
doi: 10.1691/ph.2015.4775
A series of novel ocotillol-type furoxan derivatives was synthesized by coupling various furoxans to 3-OH of
-deoxy ocotillol, and their in vitro nitric oxide (NO) releasing capability was studied. The discharge of NO
6
was examined after 30 min at two different concentrations, the results showed that all of the compounds
tested could release NO in a dose-dependent manner. All of the synthesized compounds released similar
amounts of NO at 100 ␮M, whereas at 500 ␮M these compounds showed more difference, in which com-
pound II1, II3, II4, III2 displayed higher potency in releasing NO at this concentration. Analysis of the in vitro
data showed that the derivatives bearing the same furoxan group on different ocotillol cores possessed
various NO releasing capacity, suggesting that the structure of carrier of NO releasing groups may affect
the NO release. Indeed, except compound II2, 24(S)-6-deoxy ocotillol derivatives from compound 6 with
different furoxan substitutions at 3-OH and III2 displayed enhanced NO releasing capacity, compared to
other compounds derived from compounds 5 and 9. The results illustrated that the functional group and
the stereochemistry on the ocotillol structure may affect the NO release of furoxans.
1
. Introduction
(Fig. 1) are one of the NO releasing groups that have been used in
NO donor drug discovery (Gasco and Schoenafinger 2005), and
several reports showed that hybrid compounds by combination
of vasodilators, antihistamines and protein pump inhibitors to
furoxan groups improved the pharmacological effect of the core
drugs (Boschi et al. 2006; Coruzzi et al.2000; Sorba et al. 2003).
The triterpenoid saponin, ocotillol was isolated from Fouquieria
splendens Engelm (Warnhoff and Halls 1965) (Fig. 1). It can also
be synthesized from 20(S)-protopanaxadiol (PPD), one of the
main components of Panax ginseng (Fujita et al. 1995), which
contains mainly triterpenoid saponins and demonstrated various
beneficial effects (Kiefer and Pantuso 2003). In our previous
study, ocotillol-type triterpenoids derived from PPD dis-
played significant protective effect against myocardial ischemia
(Bi et al. 2011; Han et al. 2011; Wang et al. 2010) and excellent
antibacterial activity without cytotoxicity in human cell lines
(Zhou et al. 2013; Bi et al. 2014).
Nitric oxide (NO) is a simple and significant bioactive molecule
in mammals, which is involved in various physiological
and pathological processes (Moncada 1999). Known as the
endothelium-derived relaxing factor (EDRF), NO is produced
by nitric oxide synthase (NOS) enzymes and results in powerful
vasodilatation (Smits et al. 1995). Besides the effect on blood
vessels, NO is also engaged in central and peripheral nervous
systems as a neurotransmitter to modulate cGMP production
(Vincent 2010). Furthermore, NO is a bactericidal agent and can
be generated through phagocytes by the human immune system
responding to bacterial infection (Coleman 2001). Briefly, the
biological effects of nitric oxide and its autoxidation products
are highly complex and are still under investigation.
Currently NO is used to treat neonatal pulmonary diseases
(Finer and Barrington 2006) and adult hypoxemia (Griffiths and
Evans 2005). However, the gaseous form of NO renders the dose
inaccurate. The pulmonary toxicities associated with inspired
NO2 and the hypertension caused by acute withdrawal of NO
also limit the application of gaseous nitric oxide (Hayward
et al. 1999).
The research on NO donor drugs has drawn attention since
the last century. Three NO donor drugs are presently commer-
cially available: organic nitrates for treatment of angina, sodium
nitroprusside for hypertensive crisis and molsidomine as an
oral-active, long acting vasodilator (Megson and Webb 2002;
Rosenkranz et al. 2014), and variousother NO donor compounds
have also been discovered (Miller and Megson 2007). Furoxans
Mechanistically, plant-derived triterpenoid saponins are
employed in plant defense systems by perturbing the membrane
of bacteria and enveloped viruses (Hill and Connolly 2012;
Augustin et al. 2011), as well as improving the membrane
permeability of mammalian cells (Melzig et al. 2001). Conse-
quently, triterpenoid saponins possessing affinity to membranes
could be developed as appropriate carriers for NO releasing
functional groups. Furthermore, the hybrid compounds may
also improve the biological effect of ocotillol-type triterpenoids.
Inspiring this combinatorial strategy, we synthesized a series
of ocotillol-type furoxans and initially tested their in vitro NO
releasing capacity.
Pharmazie 70 (2015)
213

ORIGINAL ARTICLES
Fig. 1: 24(R)-Ocotillol, PPD and furoxans.
Scheme 1: Synthesis of furoxans A1-4 Reagents and conditions: a) NaNO2, HOAc, r.t., 1 h; b) (i) SOCl2, pyridine, CH2Cl2, r.t., 8 h; (ii) HOR1OH, K2CO3, KI, CH3CN, r.t., 3 h.
2
. Investigations and results
.1. Synthesis of compounds
Cinnamyl alcohol reacted with NaNO2 provided the furoxan
. Addition of thionyl chloride formed the corresponding
2
1
chlorinated compound, which was subjected to nucleophilic
substitution with para-(hydroxyalkyl)phenols to give phenol
ethers A1-4 (Chen et al. 2008; Fang et al. 2008).(Scheme 1).
As previously described, PPD was transformed to epimeric tri-
ols 3 and 4 by a straightforward three-step procedure: protection
as diacetate PA followed by epoxidation and base treatment fur-
nished the desired products. The reaction of compound 3 or
compound 4 with succinic anhydride in the presence of DMAP
provided carboxylic acids 5 and 6 (Scheme 2). PA was oxidized
to compound 7 by addition of chromium trioxide. After depro-
tection, lactone 8 was obtained in good yields. The reaction of
compound 8 with succinic anhydride in the presence of DMAP
provided carboxylic acid 9 (Scheme 2).
Fig. 2: NO-release of final products at 30 min at 100 and 500 ␮M.
same furoxan group discharged various amounts of nitric oxide,
which suggested that the NO releasing capacity of furoxan may
be affected by carrier’s structure. When different furoxan groups
were present, the comparison of NO release at 500 ␮M is more
convincing: II1, II3, II4 derived from compound 6 with 24(S)
configurationandIII2 from9possesssuperiorNOreleasecapac-
ity than other NO donor compounds synthesized from 5 and 9,
which suggested the stereochemistry and the functional group
at C-24 of ocotillol derivatives may affect the release of NO
even though the furoxan group substitutions situated at the other
extremity of the ocotillol core. As the reference, sodium nitro-
prusside (SNP) was similar to our compounds for NO release at
100 ␮M after 30 min, but multiplied its potency with a correla-
tion to the concentration at 500 ␮M. Since SNP is an inorganic
compound releasing NO in minutes for treatment of acute hyper-
tensive crises, the releasing effect of our compounds seem to
require longer acting time.
Many studies on the discovery of NO donor drugs focused on the
relationshipof drugstructures and their pharmacological effects,
the quantitative structure-activity relationship (QSAR) of hybrid
NO donor drugs demonstrated that the structural diversifica-
tion of NO donor compounds, including different NO releasing
groups and substitutions on the core drug could dramatically
affect the bioactivity (Gasco et al. 2008). However, our results
showedthatminorchangesinthestructureofocotillolcorecould
already cause differences in in vitro NO release in the presence
The coupling reaction between furoxans A1-4 and compounds
5
, 6 or 9 by esterification in the presence of DMAP and EDCI
yielded compounds I1-4, II1-4, III1-4, respectively (Scheme 3).
The structures of the aimed compounds were confirmed by H
NMR, C NMR, and HRMS or ESI-MS.
1
1
3
2
.2. Screening
The in vitro NO-releasing capacity was measured by the Griess
method (Coneski and Schoenfisch 2012), the azo dye formed
-
-
from NO2 and NO3 by reaction of released NO with oxygen
under aqueous conditions can be detected by UV absorbance.
All of the twelve compounds could significantly release nitric
oxide in 30 min in the presence of l-cysteine. As shown in Fig. 2,
most of the compounds showed similar NO releasing capability
at 100 ␮M, however at 500 ␮M, compounds II1, II3, II4, III2
discharged NO quantities superior to other analogues. Further-
more, all of the compounds showed higher NO release capability
at 500 ␮M than 100 ␮M.
NO release of the compounds with the four furoxan groups we
prepared, displayed similar results at 100 ␮M, while at 500 ␮M
the differences in NO release was more appropriate for anal-
ysis. The derivatives with different ocotillol cores bearing the
2
14
Pharmazie 70 (2015)

ORIGINAL ARTICLES
◦
Scheme 2: Synthesis of the derivatives from PPD (compounds 5, 6, 9) Reagents and conditions: a) (CH3CO)2O, DMAP, CHCl3, r.t. 12 h; b) m-CPBA, CH2Cl2, -3 C, 5 h; c)
◦
NaOH, H2O, CH3OH, reflux, 6 h; d) CrO3, CH3COOH, H2O, r.t, 3 h; e) Succinic anhydride, DMAP, CHCl3, 42 C, 6 h.
of the same furoxan group. As shown in Fig. 2, the NO release
at 500 ␮M of compound II2 improved when the hydroxyl tetra-
furan ring was replaced by a lactone in III2, which suggested
that the intramolecular influence amongst the functional groups
in drug compounds may affect the capability of NO release.
2.3. Conclusion
In summary, we designed and synthesized a series of ocotillol-
type furoxans. All of the compounds demonstrated NO releasing
capability in a dose-dependent manner. The NO release from
◦
Scheme 3: Synthesis of final products (compounds I1-4, II1-4, III1-4) Reagents and conditions: a) A1-4, DMAP, EDCI, 25 C, CH2Cl2, 6 h.
Pharmazie 70 (2015)
215

ORIGINAL ARTICLES
these compounds was similar at 100 ␮M clearly different at
00 ␮M. Compounds III2 showed superior NO-releasing quan-
29.50, 29.31, 29.06, 28.18, 28.02, 25.44, 24.26, 23.87, 18.42, 18.01, 16.70,
+
16.58, 15.71.HR-MS (ESI) m/z: 577.4104 [M + H] .
5
tity to other compounds, and most of the derivatives from
compounds 6 with a 24(S) configuration displayed relatively
greater NO-releasing capacity than those derived from com-
pounds 5 and 9, which suggested that the stereochemistry and
the functional group of ocotillol core may affect the NO release
of furoxans. The preliminary results illustrated that the carrier
structure of NO-releasing groups was important for the NO
release capability to some extent, which should be considered in
further NO donor drug design and pharmacological evaluation.
As the in vitro testing of NO release was promising, the pharma-
cological study and the further expansion of hybrid compound
library formed by NO releasing moieties bound to appropriate
carrier will be carried out in our laboratories.
3.2.3. 20S-Epoxy-3␤-O-(3-carboxy
propionyl)-12␤-hydroxy-dammarane-24-one (9)
1H NMR (CDCl3, 300 MHz) ␦ 4.41 (td, J = 17.3 Hz, 4.1 Hz, 1H), 3.79 (td,
J = 17.8 Hz, 4.1 Hz, 1H), 3.49 (t, J = 8.2 Hz, 7.6 Hz, 1H), 2.33-2.31 (m, 4H),
2.13-2.28 (m, 1H), 1.27 (s, 3H), 1.02 (s, 3H), 0.98(s, 3H), 0.93 (s, 3H), 0.87
(s, 3H), 0.85 (s, 3H).ESI-MS m/z 533.5 [M + H]+
3.3. Preparation of compunds I1-4, II1-4, III1-4
Compounds A1, A2, A3 or A4 (0.05 mmol) were added to a solution of
compounds 5, 6 or 9 (0.03 mmol) in CH2Cl2, then EDCI (0.05 mmol) and
DMAP (7 mg, 0.05 mmol) were added. The resulting mixture was stirred at
◦
2
5 C for 6 h. The reaction was quenched with 5% hydrochloric acid, washed
with saturated sodium bicarbonate, water and brine successively, dried over
anhydrous Na2SO4, concentrated in vacuum and the residue purified over
silica gel to provide compounds I1-4, II1-4, III1-4.
3
. Experimental
3
.3.1. 4-[(20S,24R)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
3
.1. General
oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-methoxy}
All chemicals and solvents were analytical grade and, when necessary, puri-
fied and dried using standard methods. TLC analyses were carried out on
silica gel G (Yantai Chemical group, Yantai, China). All chemicals were
purchased from the Tianjin Damao Chemical Factory (Tianjin, China), or
the Tianjin Basifu Chemical Factory (Tianjin, China). All solvents were
purchased from the Tianjin Bodi Chemical Factory (Tianjin, China). Silica
gels were produced by the Yantai Dexin Biological Science and Technology
Factory (Yantai, China).
Melting points were measured on an XT3A micro-melting point apparatus
and are uncorrected (Beijing Keyi Company, Beijing, China). H NMR
and C NMR spectra were recorded on a BRUKER-AV400 instrument or a
Bruker AV-300 (Bruker, Ettlingen, Germany) in the indicated solvents (TMS
as internal standard): the values of the chemical shifts expressed in δ values
benzyl alcohol ester (I )
1
1
Colorless, oleosus liquid, yield 71%, H NMR (400 MHz, CDCl3) ␦: 2.67-
2
4
2
.61 (m, 4H), 3.54-3.48 (td, 1H, J = 10.4 Hz, 4.4 Hz), 3.86-3.83 (m, 1H),
.51-4.46 (dd, 1H, J = 10.8 Hz, 5.6 Hz), 6.97 (d, 2H), 5.10 (s, 2H), 5.07 (s,
H), 7.31 (d, 2H), 7.58-7.49 (m, 3H), 7.85-7.83 (dd, 2H, J = 8.2 Hz, 6.6 Hz);
13
C NMR (400 MHz, CDCl3) ␦: 15.31, 16.32, 16.37, 18.08, 18.11, 23.56,
2
3
6
1
4.95, 26.08, 27.56, 27.86, 28.53, 29.22, 29.45, 29.64, 31.14, 31.27, 32.56,
4.67, 36.99, 37.82, 38.53, 39.68, 47.88, 49.32, 50.33, 51.94, 55.99, 58.31,
5.95, 70.03, 76.68, 70.87, 77.20, 81.16, 86.45, 111.92, 114.87, 126.05,
1
27.63, 129.33, 129.76, 131.25, 131.38, 156.88, 171.80, 172.12. HR-MS
1
3
+
(ESI) m/z: 857.497 [M + H] .
(
ppm)andthecouplingconstants(J)inHz. Massspectrawererecordedusing
3.3.2. 4-[(20S,24R)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
oxo-butyric acid{ 4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-methoxy}
phenethyl alcohol ester (I2)
an HP 1100 LC/MSD spectrometer (HP, Palo Alto, USA). High resolution
mass spectra were determined on an Agilent QTOF 6520 Accurate-Mass
Q-TOF LC/MS (Agilent, Palo Alto, USA). Absorbance was measured by a
SpectraMax® M3 multi-functional microplate reader (Molecular Devices,
Silicon Valley, USA).
Compounds1andA1-4 werepreparedfollowingpublishedprocedures(Chen
et al. 2008; Fang et al. 2008), 20(S)-protopanaxadiol (PPD) was isolated
from ginseng total saponin by the authors (Li et al. 2005), Compound 3, 4,
1
Colorless liquid, yield 71%, H NMR (400 MHz, CDCl3) ␦: 2.61 (s, 4H),
2.90-2.87 (t, 2H), 3.54-3.48 (td, 1H, J = 10.4 Hz, 4.4 Hz), 3.86-3.83(dd, 1H,
J = 8.6 Hz, 6.9 Hz), 4.28-4.24(t, 2H), 4.51-4.47(dd, 1H, J = 10.4 Hz, 6.0 Hz),
5
7
1
2
4
8
1
.08 (s, 2H), 6.94-6.92 (d, 2H), 7.17-7.13 (d, 2H), 7.58-7.50 (m, 3H), 7.86-
.84 (dd, 2H, J = 8.1 Hz, 6.6 Hz); C NMR (400 MHz, CDCl3) ␦: 15.35,
6.35, 16.40, 18.14, 23.61, 24.98, 26.11, 27.59, 27.89, 28.56, 29.47, 29.20,
9.67, 31.17, 31.30, 32.59, 34.15, 34.71, 37.03, 37.87, 38.56, 39.72, 47.92,
9.36, 50.37, 51.98, 56.02, 58.43, 65.17, 70.07, 70.91, 77.20, 81.17, 85.39,
6.49, 112.07, 113.87, 115.00, 126.05, 127.70, 129.34, 130.23, 131.37,
13
8
were prepared following published procedures (Zhou et al. 2013).
3
.2. Preparation of compounds 5,6,9
+
31.72, 155.71, 171.91, 172.23. HR-MS (ESI) m/z: 871.5121 [M + H] .
Butanedioic anhydride (60 mg, 0.60 mmol) was added to a solution of 3, 4,
8
(0.30 mmol) and DMAP (0.16 mmol) in dry CH2Cl2 (8 mL). The reaction
◦
3.3.3. 4-[(20S,24R)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-
mixture was stirred at 35 C for 5 h at room temperature, then the organic
solution was washed with 10% HCl (3 × 10 mL), water (3 × 20 mL) and
NaCl (3 × 10 mL) successively, dried over anhydrous Na2SO4.The mixture
was concentrated and purified by silica gel column chromatography (20:1
dichloromethane: methanol) to afford compounds 5, 6, 9.
3
-methoxy}-3-methoxy benzyl alcohol ester (I3)
◦
1
White, amorphous powder, yield 66%, m.p. 61-62 C; H NMR (400 MHz,
CDCl3) ␦: 2.70-2.62 (m, 4H), 3.54-3.48 (td, 1H, J = 10.4 Hz, 4.4 Hz), 3.84(s,
4
2
H), 4.50-4.46 (dd, 1H, J = 10.4 Hz, 5.9 Hz), 5.07 (s, 2H), 6.90-6.88 (d,
H), 5.13 (s, 2H), 7.00 -7.98 (d, 1H), 7.59-7.51(m, 3H), 7.94-7.92 (dd, 2H,
3
.2.1. (20S, 24R)-Epoxy-3␤-O-(3-carboxy
1
3
J = 8.0 Hz, 6.6 Hz). C NMR (400 MHz, CDCl3) ␦: 15.34, 16.33, 16.39,
8.11, 23.60, 24.97, 26.10, 27.58, 27.88, 28.55, 29.24, 29.46, 29.66, 31.16,
propionyl)-dammarane-12␤, 25-diol (5)
1
White solid, yield 87%,1H NMR (CDCl3, 400 MHz) ␦ 4.50 (t, J = 8.9 Hz,
31.29, 32.58, 34.69, 37.01, 37.85, 38.55, 39.71, 47.90, 49.34, 50.36, 51.96,
55.82, 56.02, 60.05, 66.22, 70.06, 70.90, 77.20, 81.21, 85.38, 86.48, 112.08,
112.43, 116.54, 121.06, 126.19, 127.93, 129.22, 131.26, 131.48, 150.46,
6
1
.9 Hz, 1H), 3.85 (t, J = 8.0 Hz, 7.3 Hz, 1H), 3.52 (dd, J = 10.1 Hz, 6.3 Hz,
H), 2.63-2.67 (m, 4H), 2.15-2.22 (m, 1H), 1.29 (s, 3H), 1.26 (s, 6H), 1.00
+
(s, 3H), 0.98(s, 3H), 0.90 (s, 3H), 0.88 (s, 3H), 0.84 (s, 3H); 13C NMR
157.27, 171.85, 172.15. HR-MS (ESI) m/z: 887.5078 [M + H] .
(CDCl3, 75 MHz) ␦ 177.16, 172.26, 86.93, 85.73, 81.70, 71.38, 70.72,
5
3
1
6.47, 52.43, 50.80, 49.68, 48.33, 40.16, 39.00, 38.30, 37.46, 35.14, 32.99,
1.63, 31.57, 29.81, 29.40, 28.98, 28.30, 28.23, 27.94, 26.45, 25.40, 24.01,
3
.3.4. 4-[(20S,24R)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
oxo-butyric acid{3-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-
8.54 (overlapping signal), 16.82, 16.74, 15.76.HR-MS (ESI) m/z: 577.4106
+
methoxy}benzyl alcohol ester (I4)
[
M + H] .
◦
1
White, amorphous powder, yield 69%, m.p. 65-66 C; H NMR (400 MHz,
CDCl3) ␦: 2.70-2.61 (m, 4H), 3.55-3.48 (td, 1H, J = 10.4 Hz, 4.4 Hz), 3.86-
3
.2.2. (20S, 24S)-Epoxy-3␤-O-(3-carboxy
3.83 (dd, 1H, J = 8.4 Hz, 6.8 Hz), 4.49-4.45 (dd, 1H, J = 16.3 Hz, 9.1 Hz),
propionyl)-dammarane-12␤, 25-diol (6)
5
.12 (s, 4H), 6.96-6.94 (d, 1H),7.02-7.00 (d, 2H), 7.32-7.28 (t, 1H), 7.59-
1
7.51 (m, 3H), 7.86-7.84 (d, 2H); 13C NMR (400 MHz, CDCl ) ␦: 15.33,
White solid, yield 83%, H NMR (CDCl3, 300 MHz) ␦ 4.47 (t, J = 17.7 Hz,
3
4
1
3
␦
4
.0 Hz, 1H), 3.86 (t, J = 17.9 Hz, 4.2 Hz, 1H), 3.52 (t, J = 8.2 Hz, 7.6 Hz,
H), 2.13-2.28 (m, 1H), 1.29 (s, 3H), 1.24 (s, 6H), 1.05 (s, 3H), 0.98(s,
H), 0.93 (s, 3H), 0.88 (s, 3H), 0.85 (s, 3H); 13C NMR (CDCl3, 75 MHz)
176.61, 172.12, 87.73, 87.43, 81.51, 70.84, 70.50, 56.30, 52.41, 50.37,
9.09, 48.89, 40.01, 38.82, 38.17, 37.33, 34.94, 32.48, 31.91, 31.69, 29.72,
16.32, 16.38, 18.10, 18.13, 23.57, 24.97, 26.10, 27.58, 27.88, 28.55, 29.21,
29.45, 31.17, 31.28, 32.58, 34.68, 37.00, 37.83, 38.51, 39.71, 47.91, 49.35,
50.32, 51.97, 55.96, 58.33, 65.89, 70.06, 70.90, 81.21, 85.38, 86.48, 111.94,
114.19, 114.71, 121.81, 126.10, 127.68, 129.36, 129.97, 131.39, 137.89,
+
157.06, 157.15, 171.86, 172.10. HR-MS (ESI) m/z: 857.4965 [M + H] .
2
16
Pharmazie 70 (2015)

ORIGINAL ARTICLES
3
.3.5. 4-[(20S,24S)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
3.3.10. 4-[20(S)-Dammarane-12␤-hydroxy-20,24-␥-lactone-3␤-O]-4-
oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-
methoxy}phenethyl alcohol ester (III2)
oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-methoxy}
benzyl alcohol ester (II1)
◦
1
◦
1
White, amorphous powder, yield 73%, m.p. 60-61 C; H NMR (400 MHz,
CDCl3) ␦: 2.68-2.62 (m, 4H), 3.49 (s, 2H), 3.56-3.49 (td, 1H, J = 10.1 Hz,
4
J = 11.1 Hz, 5.4 Hz), 5.07 (s, 2H), 5.10 (s, 2H), 6.99-6.97 (d, 2H), 7.33-
7
NMR (400 MHz, CDCl3) ␦: 15.43, 16.31, 16.42, 17.74, 18.13, 23.61, 24.22,
2
3
6
1
White, amorphous powder, yield 68%, m.p. 85-88 C; H NMR (400 MHz,
CDCl3) ␦: 2.52 (m, 6H), 2.86 (t, 2H, J = 7.0 Hz), 3.57 (td, 1H), 4.24 (t,
2H, J = 7.0 Hz), 4.47 (dd, 1H, J = 5.8 Hz, 10.4 Hz), 5.08 (2H, s), 6.92, 7.15
.2 Hz), 3.90-3.86 (dd, 1H, J = 10.8 Hz, 5.2 Hz), 4.52-4.48 (dd, 1H,
1
3
(4H, m), 7.70, 7.84 (m, 5H); C NMR (400 MHz, CDCl3) ␦: 13.69, 13.86,
14.08, 15.51, 16.12, 16.45, 17.57, 18.11, 19.15, 22.65, 23.57, 26.23, 26.44,
27.93, 28.57, 29.21, 29.32, 29.43, 29.48, 29.66, 30.55, 31.41, 31.70, 31.89,
34.17, 34.54, 36.96, 37.88, 38.53, 39.65, 48.88, 49.53, 49.89, 52.08, 55.90,
58.51, 61.86, 65.16, 65.53, 70.16, 70.49, 70.82, 72.47, 77.20, 81.10, 89.11,
112.06, 115.06, 126.18, 127.71, 128.81, 129.33, 130.22, 130.86, 131.36,
1
3
.3 (d, 2H), 7.57-7.50 (m, 3H), 7.86-7.84 (dd, 2H, J = 8.1 Hz, 1.5 Hz);
C
5.00, 27.90, 28.04, 28.51, 28.85, 29.25, 29.47, 29.67, 31.58, 31.66, 32.16,
4.63, 37.03, 37.85, 38.51, 39.71, 48.76, 48.87, 50.08, 52.12, 55.97, 58.34,
5.98, 69.97, 70.44, 77.20, 81.18, 87.12, 87.35, 111.94, 114.90, 126.07,
+
27.66, 129.35, 129.79, 130.28, 131.40, 156.90, 171.81, 172.16. HR-MS
131.75, 155.74, 171.87, 172.19, 176.04. ESI-MS m/z: 849.2 [M + K] .
+
(ESI) m/z: 857.4966 [M + H] .
3
3
3
.3.11. 4-[20(S)-Dammarane-12␤-hydroxy-20,24-␥-lactone-
␤-O]-4-oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-
-methoxy}-3-methoxy benzyl alcohol ester (III3)
3
.3.6. 4-[(20S,24S)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-
◦
1
White, amorphous powder, yield 72%, m.p. 88-91 C; H NMR (400 MHz,
CDCl3) ␦: 2.56 (m, 6H), 3.62 (td, 1H, J = 10.6 Hz, 5.0 Hz), 3.8 (s, 3H),
methoxy}phenethyl alcohol ester (II2)
◦
1
White, amorphous powder, yield 71%, m.p. 64-65 C; H NMR (400 MHz,
4
6
␦
2
4
1
.47 (dd, 1H, J = 10.5 Hz, 5.8 Hz), 5.06 (2H, s), 5.13 (2H, s), 5.30 (s, 1H),
.87, 6.98 (m, 3H), 7.50, 7.92 (m, 5H); C-NMR (400 MHz, CDCl3)
CDCl3) ␦: 2.61 (s, 4H), 2.90-2.87 (t, 2H), 3.56-3.50 (td, 1H, J = 10.0 Hz,
13
4
1
7
1
2
3
7
1
.3 Hz), 3.89-3.86 (dd, 1H, J = 10.2 Hz, 5.2 Hz), 4.52-4.48 (dd,
J
: 15.48, 16.11, 16.44, 17.56, 18.07, 23.54, 26.27, 26.43, 27.90, 28.55,
9.24, 29.32, 29.46, 29.66, 31.42, 31.63, 34.49, 36.92, 37.84, 38.48, 39.61,
8.79, 49.47, 49.84, 52.07, 55.82, 55.86, 60.06, 66.22, 70.80, 81.12, 89.12,
H, = 10.5 Hz, 5.8 Hz), 5.08 (s, 2H), 6.94-6.92 (d, 2H), 7.58-7.51 (m, 3H),
1
3
.18-7.15 (d, 2H), 7.86-7.85 (d, 2H); C NMR (400 MHz, CDCl3) ␦:
5.42, 16.30, 16.42, 17.73, 18.14, 23.63, 24.21, 25.00, 27.91, 28.02, 28.50,
8.85, 29.19, 29.46, 29.66, 31.57, 31.65, 32.16, 34.64, 37.03, 37.86, 38.51,
9.71, 48.77, 48.87, 50.08, 52.11, 55.98, 58.43, 65.16, 69.98, 70.43, 74.11,
7.19, 81.16, 87.11, 87.33, 112.05, 115.00, 126.13, 127.69, 129.33, 130.22,
12.10, 112.45, 116.56, 121.06, 126.18, 127.93, 129.22, 131.27, 131.48
+
(Ph), 146.17, 150.47, 171.84, 172.14, 176.07; ESI-MS m/z: 865.2 [M + K] .
31.36, 131.71, 155.70, 157.07, 171.87, 172.21. HR-MS (ESI) m/z: 871.512
3.3.12. 4-[20(S)-Dammarane-12␤-hydroxy-20,24-␥-lactone-3␤-O]-4-
oxo-butyric acid{3-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-methoxy}
benzyl alcohol ester (III4)
+
[
M + H] .
◦
1
White, amorphous powder, yield 64%, mp 81-83 C; H NMR (400 MHz,
CDCl3) ␦: 2.53 (m, 6H), 3.60 (td, 1H), 4.45 (dd, 1H, J = 9.8 Hz, 6.6 Hz),
5.10 (d, 4H), 5.30 (s, 1H), 6.94(s, 1H), 7.00(s, 1H), 7.28(s, 1H), 7.51, 7.84
(m, 5H); C NMR 400 MHz, CDCl3) ␦: 15.47, 16.09, 16.43, 17.55, 18.06,
23.52, 26.28, 26.44, 27.17, 27.89, 28.55, 29.20, 29.32, 29.44, 29.66, 31.42,
3
.3.7. 4-[(20S,24S)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-methoxy}-
-methoxy benzyl alcohol ester (II3)
Colorless, oleic-like liquid, yield 70%, 1H NMR (400 MHz, CDCl3) ␦:
1
3
3
3
5
1
1.63, 34.49, 36.90, 37.83, 38.43, 39.61, 48.79, 49.48, 49.80, 52.08, 55.80,
8.32, 61.84, 65.89, 70.80, 81.12, 89.13, 111.95, 114.17, 114.71, 121.79,
2
4
6
.69-2.58 (m, 4H), 3.54-3.49 (m, 1H), 3.84 (s,3H), 3.90-3.86 (m, 1H),
.51-4.47 (dd, 1H, J = 10.6 Hz, 5.4 Hz), 5.07 (s, 2H), 5.13 (s, 2H), 6.90-
.88 (d, 2H), 7.03-7.00 (d, 2H), 7.58-7.49 (m, 3H), 7.94-7.92 (dd, 2H,
26.09, 127.67, 129.37, 129.96, 131.40, 137.90, 157.16, 171.87, 172.11,
+
1
3
176.08; ESI-MS m/z: 835.2 [M + K] .
J = 8.0 Hz, 6.6 Hz); C NMR (400 MHz, CDCl3) ␦: 15.41, 16.29, 16.42,
1
2
4
8
1
8
7.73, 18.13, 22.66, 23.62, 24.19, 25.00, 27.90, 28.00, 28.50, 28.84, 29.25,
9.46, 29.66, 31.57, 31.65, 32.16, 34.63, 37.03, 37.85, 38.51, 39.71, 48.76,
8.86, 50.08, 52.11, 55.82, 55.98, 60.06, 66.22, 69.99, 70.43, 74.11, 81.22,
7.11, 87.33, 112.08, 112.44, 116.56, 121.06, 126.19, 127.94, 129.22,
31.26, 131.49, 146.17, 150.48, 157.26, 171.83, 172.15. HR-MS (ESI) m/z:
3
.4. In vitro nitric oxide-release assay
In vitro nitric oxide release was measured by determination of nitrite
-
-
(NO2 ) and nitrate (NO3 ) as described Griess reaction (Coneski and
+
Schoenfisch 2012). L-Cysteine in phosphate (PBS) buffer was prepared by
dissolving KH2PO4 (0.2694 g), K2HPO4 (1.8310 g), L-cysteine (218 mg)
in distilled water (100 mL) to a final concentration of 18 mM. The Griess
reagent was then prepared by dissolving sulfanilamide (4.0 g) and N-
napthylenediamine·2HCl (0.2 g) in a mixture of 85% H3PO4 (10 mL) and
distilled water (90 mL).
The test compounds (6 ␮mol) were first dissolved in 0.6 mL dimethyl sul-
foxide, then diluted into L-cysteine in PBS buffer to 500 ␮M and 100 ␮M,
respectively. A control blank was prepared by diluting equal amount of
dimethyl sulfoxide with PBS solution. Griess reagent (250 ␮L) was added
to the compound solution (200 ␮L) after 30 min, the mixture was then inoc-
87.5064 [M + H] .
3
.3.8. 4-[(20S,24S)-Epoxy-12␤,25-diol-dammarane-3␤-O]-4-
oxo-butyric acid{3-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-methoxy}
benzyl alcohol ester (II4)
◦
1
White, amorphous powder, yield 71%, mp 74-75 C; H NMR (400 MHz,
CDCl3) ␦: 2.72-2.63 (m, 4H), 3.57-3.50 (td, 1H, J = 10.2 Hz, 4.5 Hz), 3.90-
3
5
7
.86 (dd, 1H, J = 10.7 Hz, 5.3 Hz), 4.50-4.46 (dd, 1H, J = 16.4 Hz, 9.5 Hz),
.12 (s, 4H), 6.96-6.94 (dd, 1H, J = 8.0 Hz, 2.2 Hz), 7.01-7.00 (d, 2H), 7.32-
.28 (t, 1H), 7.58-7.50 (m, 3H), 7.86-7.84 (dd, 2H, J = 7.6 Hz, 1.2 Hz);
◦
1
3
ulated at 37 C for 30 min, the absorbance at 540 nm was measured and
repeated twice.
C
NMR (400 MHz, CDCl3) ␦: 14.10, 15.42, 16.28, 16.41, 17.73, 18.12, 23.59,
2
3
5
1
1
4.17, 25.01, 27.88, 28.00, 28.51, 28.84, 29.21, 29.44, 29.66, 31.57, 31.62,
2.17, 34.61, 37.00, 37.83, 38.46, 39.70, 48.75, 48.85, 50.04, 52.12, 55.91,
8.31, 65.90, 70.43, 77.20, 81.21, 87.12, 87.33, 111.94, 114.19, 114.70,
21.81, 126.09, 127.68, 129.36, 129.97, 131.39, 131.88, 157.06, 157.14,
Acknowledgments: The authors are grateful to National Natural Sci-
ence Foundation of China (No.81001358). Promotive research fund
for excellent young and middle-aged scientists of Shandong Province
+
71.85, 172.12; HR-MS (ESI) m/z: 857.4974 [M + H] .
(No. BS2010YY073), Shandong Provincial Natural Science Foundation
No.ZR2012HM036), Key Laboratory for the Chemistry and Molecular
(
Engineering of Medicinal Resources (Guangxi Normal University), Min-
istry of Education of China(CMEMR2014-B07), Research Fund for Youth
Scientists of Yantai University (No.YX13202), Taishan Scholar Project to
Fenghua Fu and 2014 Faculty Early Career Research Grant from the Uni-
versity of Newcastle for financial support.
3
.3.9. 4-[20(S)-Dammarane-12␤-hydroxy-20,24-␥-lactone-3␤-O]-4-
oxo-butyric acid{4-(4-phenyl-1,2,5-oxdiazole-2-oxides)-3-methoxy}
benzyl alcohol ester (III1)
◦
1
White, amorphous powder, yield 65%, mp 75-76 C; H NMR (400 MHz,
CDCl3) ␦: 2.57 (m, 6H), 3.62 (td, 1H, J = 4.9 Hz), 4.47 (dd, 1H, J = 10.7 Hz,
5
.5 Hz), 5.07 (s, 2H), 5.10 (s, 2H), 6.96, 7.30 (m, 4H), 7.50, 7.83 (m, 5H);
C NMR (400 MHz, CDCl3) ␦: 14.08, 15.51, 16.11, 16.45, 17.57, 18.10,
2.66, 23.55, 26.21, 26.44, 27.92, 28.58, 29.28, 29.33, 29.50, 29.67, 30.00,
1.41, 31.70, 31.90, 34.54, 36.96, 37.87, 38.53, 39.65, 48.87, 49.53, 49.88,
2.08, 55.90, 58.42, 65.97, 70.82, 81.11, 89.13, 114.95, 127.68, 129.35,
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