Synthesis and Anti-inflammatory Evaluation of (R)-, (S)-, and (±)-Sanjuanolide Isolated from Dalea frutescens

Article abstract of DOI:10.1021/acs.jnatprod.8b00596

The known chalcone (±)-sanjuanolide (1) can be isolated from Dalea frutescens. This study presents a convergent strategy for the first total synthesis of (R)-, (S)-, and (±)-sanjuanolide (1). The key step for synthesizing (R)- and (S)-1 was a Corey-Bakshi

Full text of DOI:10.1021/acs.jnatprod.8b00596

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Synthesis and Anti-inflammatory Evaluation of (R)‑, (S)‑, and
± )-Sanjuanolide Isolated from Dalea frutescens
(
†
,#
‡,#
§,#
†
†
†
†
Bo Fang, Zhongxiang Xiao, Yinda Qiu, Sheng Shu, Xianxin Chen, Xiaojing Chen, Fei Zhuang,
,
†
,†
,†
Yunjie Zhao,* Guang Liang,* and Zhiguo Liu*
†Chemical Biology Research Center at School of Pharmaceutical Sciences, Wenzhou Medical University, 1210 University Town,
Wenzhou, Zhejiang 325035, People’s Republic of China
‡
Department of Pharmacy, Affiliated Yueqing Hospital, Wenzhou Medical University, Wenzhou, Zhejiang 325035, People’s Republic
of China
§
College of Life and Environmental Science, Wenzhou University, Wenzhou, Zhejiang 325035, People’s Republic of China
*
S Supporting Information
ABSTRACT: The known chalcone (±)-sanjuanolide (1) can be isolated from Dalea frutescens. This study presents a
convergent strategy for the first total synthesis of (R)-, (S)-, and (±)-sanjuanolide (1). The key step for synthesizing (R)- and
(
S)-1 was a Corey−Bakshi−Shibata enantioselective carbonyl reduction to construct the C-2″ configuration. (R)-1 efficiently
inhibited the lipopolysaccharides (LPS)-induced expression of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6),
while (S)-1 produced no significant anti-inflammatory effect. (R)-1 also effectively inhibited the mRNA expression of several
inflammatory cytokines after the LPS challenge in vitro. The synthesis and biological properties of these compounds have
confirmed (R)-sanjuanolide and (±)-sanjuanolide as promising new leads for developing anti-inflammatory agents.
aturally occurring chalcones have been isolated from
several plant species and usually have significant and
diverse bioactivities. They have also proven to be a prolific
compound suitable for further modification during drug
1a
N
development, a new and efficient synthetic strategy has
been developed for the first total synthesis of (R)-1 and (S)-1
using the commercially available 2,4-dihydroxyacetophenone
as the starting material. In this approach, the C-2″
configuration was established using the Corey−Bakshi−
Shibata enantioselective carbonyl reduction as a key step.
This paper pursues the first total synthesis of (±)-sanjuanolide
source of small-molecular chemical entities for developing
1
clinical drugs. The incorporation of isoprenyl moieties in the
skeleton usually leads to a wide spectrum of biological
properties, including anti-inflammatory, anti-tumor, and anti-
2
diabetic activities. Sanjuanolide (1, Figure 1) possesses an
(
1) and both of its enantiomers [(R)-1, (S)-1] and the
isoprenylated chalcone skeleton and was isolated in 2016 from
3
evaluation of their in vitro anti-inflammatory activities.
The retrosynthetic analysis for synthesizing (R)-1 is
presented in Scheme 1. The target molecule, (R)-1, could be
constructed through an aldol condensation between the
the extracts of Dalea frutescens A. Gray (Leguminosae).
Further extensive spectroscopic studies have revealed the (2″-
R) absolute configuration. However, the specific rotation data
of (R)-1 indicated racemization after several weeks of frozen
4
3
protected acetophenone (R)-2 and benzaldehyde, followed
storage.
by the deprotection of both phenolic groups. The chirality in
In biological assays, (R)-1 exhibited a modest antiprolifer-
(
R)-2 would be installed unambiguously using the Corey−
ative action on PC-3 and DU 145 prostate cancer cells, with
3
Bakshi−Shibata stereospecific reduction of 3 with (S)-(+)-2-
IC values of 11.0 and 7.0 μM, respectively. Despite extensive
5
0
research on its isolation, biological activities, and mechanistic
studies, the total synthesis of sanjuanolide (1) has not yet been
reported. Since sanjuanolide (1) might be a valuable lead
Received: July 20, 2018
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.8b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Structures of sanjuanolide (1) and its enantiomers.
Scheme 1. Retrosynthetic Analysis of (R)-1
protection of the phenolic groups of 6, followed by NaBH4
reduction, provided 1-[2,4-bis(methoxymethoxy)phenyl]-
8
ethanol (7). The resulting alcohol (7) was protected with
tert-butyldimethylsilyl chloride (TBSCl) using a catalytic
amount of imidazole to obtain a 76% overall yield of
compound 8 in three steps. Treating 8 with n-butyllithium
(
n-BuLi) followed by dimethylformamide (DMF) at −78 °C
4b
provided benzaldehyde 5 in 78% yield. A Wittig olefination
of aldehyde 5 with methyltriphenylphosphonium bromide in
the presence of n-BuLi gave the anticipated phenylethylene 9.
9
When phenylethylene (9) was subjected to hydroboration
using BH ·SMe at 0 °C, it afforded the primary alcohol 10
3
2
10
after an oxidative workup in 70% overall yield. The primary
alcohol 10 was oxidized using Dess−Martin periodinane in
CH Cl , which smoothly generated the phenylacetaldehyde 4
2
2
in 80% yield. The addition of isopropenylmagnesium bromide
provided the key secondary alcohol 11 in a yield of 68%.
With the key intermediate 11 in hand, the chirality in ketone
methyloxazaborolidine, while compound 3 could be derived
from phenylacetaldehyde (4) via a Grignard reaction followed
by oxidation. Compound 4 would be derived from a protected
5
3
was introduced as depicted in Scheme 3. Thus, Dess−Martin
benzaldehyde (5), through olefination, hydroboration−oxida-
6
tion, and Dess−Martin oxidation. The protected aldehyde 5
oxidation of 11 in CH Cl afforded ketone 3 in 79% yield. (S)-
2
2
could be prepared from MOM (methoxymethyl ether)-
(+)-2-Methyloxazaborolidine was used as reagent due to its
protected 2,4-dihydroxyacetophenone (6), using Vilsmeier−
ability to provide defined configuration with a high
4
b,7
11
Haack formylation.
enantiomeric excess (ee). In this case, reduction with BH ·
3
The procedure for the synthesis of 11 from 2,4-
dihydroxyacetophenone (6) is illustrated in Scheme 2. MOM
SMe in the presence of catalytic (S)-(+)-2-methyloxazabor-
2
olidine provided (R)-12 in a yield of 72%.
Scheme 2. Synthesis of the Key Intermediate 11 from 2,4-Dihydroxyacetophenone (6)
B
DOI: 10.1021/acs.jnatprod.8b00596
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Journal of Natural Products
Article
Scheme 3. Synthesis of (R)-1 from Intermediate 11
Scheme 4. Synthetic Routes of (± )-Sanjuanolide and (S)-1
Acetylation of (R)-12 with Ac O/DMAP/Et N in CH Cl
2
significant that hydrolysis of the MOM protecting groups in
2
3
2
efficiently gave intermediate (R)-13 in a yield of 92%. TBS
deprotection of (R)-13 in the presence of tetrabutylammo-
nium fluoride (TBAF) proceeded smoothly, and subsequent
Dess−Martin oxidation produced the key acetophenone (R)-2
in an overall yield of 76%. Aldol condensation of the
acetophenone (R)-2 and benzaldehyde in ethanolic NaOH
solution afforded chalcone (R)-14 in a yield of 90%. It was
(
R)-14 under standard conditions (HCl in MeOH) failed to
give the target product. However, treating (R)-14 with Ac O
2
and catalytic amounts of DMAP under basic conditions
afforded the acetylated intermediate (R)-15, which was
effectively deprotected with HCl in MeOH to give target
(R)-1 (ee = 90.2%) in an overall yield of 57% in two steps.
12
C
DOI: 10.1021/acs.jnatprod.8b00596
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Figure 2. IC50 values (μM) of synthetic compounds for LPS-induced IL-6 and TNF-α. (A) IL-6 [(±)-1], (B) TNF-α [(±)-1], (C) IL-6 [(S)-1],
5
(
D) TNF-α [(S)-1], (E) IL-6 [(R)-1], (F) TNF-α [(R)-1]. Cells (5 × 10 /plate) were pretreated with vehicle or compounds at indicated
concentrations (1, 2.5, 5, and 10 μM) for 30 min. Then, cells were incubated with LPS (0.5 μg/mL) for 24 h. The inflammatory cytokine
production in the supernatants was measured by ELISA. The significance of the difference in mean values relative to the LPS group is indicated as
*p < 0.05, ***p < 0.001 vs LPS vehicle.
The NMR data of the synthetic (R)-1 (Table S1) and
μM, respectively (Figure 2A,B). Unexpectedly, (S)-1 exhibited
no inhibitory activity with these two cytokines (Figure 2C,D),
and its enantiomer (R)-1 exhibited similar activities to
(±)-sanjuanolide (1) with IC50 values of 1.1 μM (for IL-6)
and 1.2 μM (for TNF-α) (Figure 2E,F).
optical rotation data {observed [α]20 = −25.0 (c 0.2, MeOH);
D
reported [α]D = −27.0 (c 0.05, MeOH)} agreed well with
3
those reported for the natural product. Significantly, the
racemization reported for the naturally isolated (R)-1 (stored
3
in powder form) was not observed in the synthetic sample.
To further confirm the anti-inflammatory actions of these
synthetic compounds, the potency of (R)-1 for inhibiting
inflammatory gene expression at the mRNA level was
evaluated. Macrophages were treated with LPS (1.0 μg/mL)
for 6 h, then examined for the expression of pro-inflammatory
genes by a quantitative reverse transcription PCR (RT-qPCR)
study. Figure 3 shows that LPS induced a significant increase in
the mRNA expression of the pro-inflammatory cytokines,
including TNF-α, IL-6, IL-1β (interleukin-1β), ICAM-1
(intercellular cell adhesion molecule-1), MACP-1 (mitochon-
drial anion carrier proteins-1), and COX-2 (cyclooxygenase-2),
while pretreatment with (R)-1 and (±)-sanjuanolide (1)
significantly and effectively down-regulated mRNA expressions
of TNF-α (Figure 3A), IL-6 (Figure 3B), IL-1β (Figure 3C),
ICAM-1 (Figure 3D), MACP-1 (Figure 3E), and COX-2
(Figure 3F) in LPS-stimulated macrophages. These data
indicated that (R)-1 and (±)-sanjuanolide (1) were potent
inhibitors of LPS-induced mRNA overexpression of inflamma-
tory genes in LPS-stimulated macrophages, thus indicating that
the configuration of (R)-1 plays a key role in the observed anti-
inflammatory activity.
However, the specific rotation for (S)-1, dissolved in MeOH,
fell to almost zero after two months of frozen storage. Thus,
the presence of the polar protic solvent may be responsible for
its autoracemization.
A similar synthetic strategy was used to prepare (S)-1 (ee =
6.8%) but with (R)-(−)-2-methyloxazaborolidine as the chiral
auxiliary. Identical synthetic protocols were followed for the
8
successful synthesis of (±)-sanjuanolide (1) in six steps from
1
1 to give an overall yield of 7.3% (Scheme 4).
Pro-inflammatory cytokines, such as IL-6 and TNF-α, have
been recognized as critical regulators in inflammatory
1
3
14
diseases such as rheumatoid arthritis, inflammatory bowel
1
5
16
17
disease, asthma, and diabetic complications. Inhibiting
the release of pro-inflammatory cytokines is an important
mode of action for anti-inflammatory drugs. The reported
potent biological activity and anti-inflammatory potential of
18
isoprenylated chalcones therefore prompted the assessment
of the anti-inflammatory activities of synthesized (±)-sanjua-
nolide (1) and its enantiomers. The bioassay results are shown
in Figure 2. These results demonstrated that (±)-sanjuanolide
1) markedly reduced the production of lipopolysaccharide
LPS)-induced IL-6 and TNF-α with IC values of 1.1 and 1.6
(
(
In conclusion, a common route for the first total synthesis of
(±)-sanjuanolide (1), (R)-1, and (S)-1 has been established in
50
D
DOI: 10.1021/acs.jnatprod.8b00596
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Journal of Natural Products
Article
Figure 3. Inhibitory effects of compounds on the expression of inflammatory genes induced by LPS-stimulated macrophages. (A) TNF-α, (B) IL-6,
5
(
C) IL-1β, (D) ICAM-1, (E) MCP-1, (F) COX-2. The cells were plated at a density of 7.0 × 10 /plate, then incubated overnight at 37 °C in a 5%
2
CO atmosphere. The macrophages were pretreated with 2.5 and 10 μM compounds for 30 min, then incubated with LPS (1.0 μg/mL) for 24 h.
The cells were collected, and the total RNA was extracted. The mRNA levels of the inflammatory cytokines were detected by qPCR. The results are
presented as the percentage of the LPS control. Each bar represents the mean ± SEM from three independent experiments. The significance of the
difference in mean values relative to the LPS group are indicated as *p < 0.05, **p < 0.01, ***p < 0.001 vs LPS vehicle.
1
3
5 (racemic) and 17 (enantiomers) steps with overall yields of
.8%, 4.2%, and 7.3%, respectively. Biological studies have
h. The reaction progress was monitored by TLC. After completion of
the reaction, the resulting mixture was quenched with ice water and
diluted with EtOAc (50 mL). The organic layer was washed with H O
shown that the (2″R)-configuration is important for anti-
inflammatory activity. These results could be particularly useful
for further pharmaceutical development to treat LPS-induced
inflammatory damage. Further structural modifications and
evaluation of their biological potential are ongoing and will be
reported in due course.
2
(
3 × 50 mL), dried over MgSO , and concentrated in vacuo. The
4
residue was purified by flash column chromatography (petroleum
ether/EtOAc = 15:1) to provide 7.2 g (76%) of a colorless oil.
To a cooled (0 °C) solution of the oil (7.0 g, 29.1 mmol) in EtOH
(
50 mL) was added slowly NaBH (2.2 g, 58.3 mmol). The mixture
4
was warmed to room temperature and stirred for 3 h. The mixture
was quenched with saturated NH Cl solution, and the EtOH was
4
removed in vacuo. The aqueous layer was extracted with EtOAc (3 ×
EXPERIMENTAL SECTION
■
20 mL), and the combined organic layers were dried over MgSO and
4
General Experimental Procedures. Melting points were
determined on a Fisher-Johns melting point apparatus and are
uncorrected. Optical rotations were measured with a JASCO P-2000
digital polarimeter at ambient temperature. H and C NMR spectra
were recorded on a Bruker instrument at 400 or 500 MHz, and peak
positions are given in parts per million (δ) downfield from
concentrated in vacuo. The residue was purified by flash column
chromatography (petroleum ether/EtOAc = 5:1) to provide 7.1 g
1
(100%) of 7 as a colorless oil: H NMR (500 MHz, CDCl ) δ 7.28 (d,
3
1
13
J = 8.5 Hz, 1H), 6.80 (d, J = 2.3 Hz, 1H), 6.71 (dd, J = 8.5, 2.3 Hz,
1H), 5.21 (s, 2H), 5.15 (s, 2H), 5.10 (q, J = 6.5 Hz, 1H), 3.49 (s,
1
3
3H), 3.47 (s, 3H), 1.49 (d, J = 6.5 Hz, 3H); C NMR (125 MHz,
1
tetramethylsilane as internal standard. J values are given in Hz. H
CDCl ) δ 157.5, 155.0, 128.0, 126.7, 108.9, 103.5, 94.6, 94.6, 65.7,
3
+
NMR data are reported in the order chemical shift, multiplicity (s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet and/or multiple
resonances), number of protons, and coupling constant in Hz. High-
resolution measurements were made with a Synapt HDMS (Waters,
UK) instrument. Analytical HPLC analyses were performed at
ambient temperature on an Agilent 1260 liquid chromatograph and
equipped with a G1314A VWD detector. Unless noted otherwise, all
starting materials and reagents were obtained from commercial
suppliers and were used without further purification. Reaction flasks
were dried at 100 °C. Air- and moisture-sensitive reactions were
performed under an argon atmosphere. TLC was conducted on
Kieselgel 60 F254 plates, and flash column chromatography (MPLC)
purifications were performed using Merck silica gel 60 (230−400
mesh ASTM) (Merck KGaA, Darmstadt, Germany).
56.3, 56.0, 23.1; HRMS(ESI): calcd for C H O Na [M + Na] m/z
1
2
18
5
265.1052, found m/z 265.1017.
(±)-{1-[2,4-Bis(methoxymethoxy)phenyl]ethoxy}(tert-butyl)-
dimethylsilane (8). To a solution of 7 (3.0 g, 12.4 mmol) in
dichloromethane (DCM) (30 mL) was added imidazole (1.7 g, 24.8
mmol) at room temperature followed by TBSCl (3.7 g, 24.8 mmol),
and the mixture was stirred at this temperature for 6 h. The reaction
was quenched with saturated aqueous NH Cl (30 mL) and extracted
4
with EtOAc (3 × 30 mL). The combined organic layers were washed
with brine, dried over anhydrous MgSO , filtered, and concentrated in
4
vacuo. The residue was purified by flash chromatography on silica gel
(petroleum ether/EtOAc = 30:1) to give 3.6 g (100%) of 8 as a
1
colorless oil: H NMR (500 MHz, CDCl ) δ 7.42 (d, J = 8.5 Hz, 1H),
3
6.74 (s, 1H), 6.70 (d, J = 8.5 Hz, 1H), 5.18 (s, 2H), 5.17−5.13 (m,
(
±)-1-[2,4-Bis(methoxymethoxy)phenyl]ethanol (7). To a sol-
ution of 2,4-dihydroxyacetophenone (6) (6.0 g, 24.6 mmol) in
anhydrous tetrahydrofuran (THF) (50 mL) was slowly added NaH
3H), 3.49 (s, 3H), 3.48 (s, 3H), 1.35 (d, J = 6.1 Hz, 3H), 0.91 (s,
1
3
9H), 0.05 (s, 3H), −0.02 (s, 3H); C NMR (125 MHz, CDCl ) δ
3
156.9, 153.5, 129.7, 126.8, 108.8, 102.6, 94.7, 94.3, 65.0, 56.0, 56.0,
(
3.8 g, 157.7 mmol) at 0 °C. Chloromethyl methyl ether (6.4 g, 78.9
26.0, 25.9 × 3, 18.3, −4.9, −4.9; HRMS(ESI) calcd for C H O SiNa
18
32
5
+
mmol) was added, and the mixture stirred at room temperature for 4
[M + Na] m/z 379.1917, found m/z 379.1884.
E
DOI: 10.1021/acs.jnatprod.8b00596
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Journal of Natural Products
Article
1
(
± )-3-{1-[(tert-Butyldimethylsilyl)oxy]ethyl}-2,6-bis-
methoxymethoxy)benzaldehyde (5). To a solution of 8 (1.0 g, 2.8
mmol) in dry THF (15 mL) was added n-BuLi (2.5 M in n-hexane,
oil: H NMR (500 MHz, CDCl ) δ 9.66 (s, 1H), 7.44 (d, J = 8.7 Hz,
3
(
1H), 6.96 (d, J = 8.7 Hz, 1H), 5.22−5.07 (m, 3H), 4.90−4.87 (m,
2H), 3.72 (d, J = 7.7 Hz, 2H), 3.56 (s, 3H), 3.44 (s, 3H), 1.39 (d, J =
6.3 Hz, 3H), 0.89 (s, 9H), 0.05 (s, 3H), −0.04 (s, 3H); C NMR
(125 MHz, CDCl ) δ 200.3, 155.1, 153.7, 133.9, 126.7, 115.7, 110.4,
100.5, 94.6,65.7, 57.2, 56.2, 39.9, 26.3, 25.9 × 3, 18.2, −4.8, −4.9;
HRMS(ESI) calcd for C H O SiNa [M + Na] m/z 421.2023,
found m/z 421.2004.
(± )-1-{3-{1-[(tert-Butyldimethylsilyl)oxy]ethyl}-2,6-bis-
(methoxymethoxy)phenyl}-3-methylbut-3-en-2-ol (11). To a sol-
ution of 4 (0.5 g, 1.3 mmol) in dry THF (8 mL) was slowly added
isoprorenylmagnesium bromide (0.5 M solution in THF, 3.7 mL, 1.9
mmol) at −30 °C under argon. The mixture was stirred at 0 °C for 2
h. After completion of the reaction, the reaction was quenched with
saturated ice water (25 mL) and extracted with EtOAc (3 × 30 mL).
The combined organic layer was washed with brine (25 mL), dried
1
3
1
.6 mL, 3.9 mmol) slowly at −78 °C under argon. After 30 min, the
solution was allowed to warm to 0 °C and dry DMF (0.3 mL, 4.5
mmol) was then added dropwise. The mixture was stirred at room
temperature for 2 h. After completion of the reaction, the reaction was
3
+
2
0
34
6
quenched with saturated aqueous NH Cl and extracted with EtOAc
3 × 20 mL). The organic layer was washed with H O (3 × 10 mL),
dried over Mg SO , and concentrated in vacuo. The residue was
purified by flash column chromatography (petroleum ether/EtOAc =
5:1) to provide 0.8 g (78%) of 5 as a yellow oil: H NMR (500
MHz, CDCl ) δ 10.46 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.01 (d, J =
.8 Hz, 1H), 5.25−5.28 (m, 3H), 5.12 (d, J = 6.7 Hz, 1H), 5.00 (d, J
6.7 Hz, 1H), 3.58 (s, 3H), 3.52 (s, 3H), 1.37 (d, J = 6.3 Hz, 3H),
4
(
2
2
4
1
2
3
8
=
0
1
3
.90 (s, 9H), 0.06 (s, 3H), −0.03 (s, 3H); C NMR (125 MHz,
CDCl ) δ 189.4, 159.6, 154.6, 134.8, 133.4, 118.4, 111.0, 102.3, 95.1,
over MgSO , and concentrated in vacuo. The residue was purified by
3
4
6
5.1, 57.3, 56.6, 26.1, 25.8 × 3, 18.2, −4.9, −5.0; HRMS(ESI) calcd
flash column chromatography (petroleum ether/EtOAc = 20:1 to
5:1) to give 11 (0.4 g, 68%) as a yellow oil: H NMR (500 MHz,
+
1
for C H O SiNa [M + Na] m/z 407.1866, found m/z 407.1847.
19
32
6
(
±)-{1-[2,4-Bis(methoxymethoxy)-3-vinylphenyl]ethoxy}(tert-
CDCl ) δ 7.36 (d, J = 8.6 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 5.19 (d, J
3
butyl)dimethylsilane (9). To a suspension of Ph PCH Br (2.8 g, 6.5
= 2.4 Hz, 2H), 5.10 (q, J = 6.2 Hz, 1H), 5.03−4.99 (m, 3H), 4.82 (s,
1H), 4.35−4.30 (m, 1H), 3.62 (s, 3H), 3.49 (s, 3H), 3.03−2.99 (m,
1H), 2.94−2.89 (m, 1H), 1.85 (s, 3H), 1.38 (t, J = 6.3 Hz, 3H), 0.88
3
3
mmol) in THF (25 mL) was added n-BuLi (2.5 M in n-hexane, 3.3
mL, 8.3 mmol) slowly at −78 °C under argon. The mixture was
stirred at 0 °C for 1 h. A solution of 5 (1.0 g, 2.6 mmol) in THF (5
mL) was added dropwise to the reaction mixture. After stirring for 2
h, the reaction was quenched with saturated aqueous NH Cl (30 mL)
and extracted with Et O (3 × 30 mL). The combined organic layers
were dried over anhydrous MgSO , filtered, and concentrated in
vacuo. The residue was purified by chromatography (hexanes/EtOAc
15:1) to afford 9 (0.8 g, 82%) as a yellow oil: H NMR (500 MHz,
CDCl ) δ 7.37 (d, J = 8.5 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.79 (dd,
J = 18.0, 12.0 Hz, 1H), 5.99 (dd, J = 18.0, 2.4 Hz, 1H), 5.50 (dd, J =
2.0, 2.4 Hz, 1H), 5.26−5.16 (m, 3H), 4.97 (d, J = 5.9 Hz, 1H), 4.91
d, J = 5.8 Hz, 1H), 3.57 (s, 3H), 3.49 (s, 3H), 1.39 (d, J = 6.3 Hz,
H), 0.90 (s, 9H), 0.06 (s, 3H), −0.03 (s, 3H); C NMR (125 MHz,
CDCl ) δ 154.9, 152.4, 134.2, 128.5, 125.9, 120.3, 119.6, 111.1, 99.9,
1
3
(s, 9H), 0.04 (s, 3H), −0.05 (s, 3H); C NMR (125 MHz, CDCl ) δ
3
155.1, 153.7, 148.1, 133.8, 125.6, 121.1, 110.7, 109.9, 100.4, 94.8,
4
75.4, 65.8, 57.2, 56.2, 31.5, 26.3, 25.9 × 3, 18.2, 18.1, −4.8, −4.9;
+
2
HRMS(ESI) calcd for C H O SiNa [M + Na] m/z 463.2492,
23 40
6
4
found m/z 463.2489.
(± )-1-{3-{1-[(tert-Butyldimethylsilyl)oxy]ethyl}-2,6-bis-
(methoxymethoxy)phenyl}-3-methylbut-3-en-2-one (3). To a sol-
1
=
ution of 11 (0.4 g, 0.9 mmol) in CH
Dess−Martin periodinane (1.0 g, 2.4 mmol) at 0 °C. After stirring for
4 h, the reaction was quenched with saturated aqueous NaHCO (10
mL) and NaS (2 mol/L, 2.5 mL). The aqueous layer was extracted
with Et O (3 × 25 mL). The combined organic layers were washed
with NaHCO , dried over anhydrous MgSO , filtered, and
Cl (8 mL) was slowly added
3
2 2
1
(
3
3
O
2 3
1
3
2
3
3
4
9
4.9, 65.6, 57.3, 56.2, 26.3, 25.9 × 3, 18.2, −4.9, −5.0; HRMS (ESI)
calcd for C H O SiNa [M + Na] m/z 405.2074, found m/z
concentrated in vacuo. The residue was purified by chromatography
+
on silica gel (petroleum ether/EtOAc = 25:1) to afford 3 (0.3 g, 79%)
20
34
5
1
4
05.2053.
±)-2-{3-{1-[(tert-Butyldimethylsilyl)oxy]ethyl}-2,6-bis-
methoxymethoxy)phenyl}ethanol (10). To a solution of 9 (1.0 g,
.6 mmol) in dry THF (15 mL) was added BH /SMe (2.0 M in
as a yellow oil: H NMR (500 MHz, CDCl
3
) δ 7.38 (d, J = 8.7 Hz,
(
1H), 6.90 (d, J = 8.7 Hz, 1H), 6.05 (s, 1H), 5.76 (s, 1H), 5.16 (q, J =
6.2 Hz, 1H), 5.10 (q, J = 6.6 Hz, 2H), 4.85 (s, 2H), 4.08 (d, J = 4.3
Hz, 2H), 3.52 (s, 3H), 3.40 (s, 3H), 1.90 (s, 3H), 1.38 (d, J = 6.3 Hz,
(
2
3
2
1
3
Et O, 2.6 mL, 5.2 mmol) slowly at 0 °C under argon. The reaction
3H), 0.89 (s, 9H), 0.04 (s, 3H), −0.05 (s, 3H); C NMR (125 MHz,
2
mixture was allowed to warm to room temperature and stirred for 4 h
before 2 N NaOH (2.6 mL) and 30% H O (1.0 mL) were added.
CDCl ) δ 199.4, 154.8, 153.3, 144.4, 133.7, 125.8, 123.8, 118.5, 110.4,
3
2
2
100.2, 94.6, 65.7, 57.0, 56.0, 34.8, 26.4, 25.9 × 3, 18.2, 17.9, −4.8,
+
Stirring was continued for 3 h, the mixture was diluted with saturated
NH Cl (12 mL) and extracted with EtOAc (3 × 20 mL), and the
combined organic layers were washed with brine (50 mL), dried over
anhydrous MgSO , filtered, and concentrated in vacuo. The residue
was purified by chromatography on silica gel (petroleum ether/EtOAc
15:1 to 8:1) to afford 10 (0.4 g, 70%) as a colorless oil: H NMR
500 MHz, CDCl ) δ 7.35 (d, J = 8.7 Hz, 1H), 6.92 (d, J = 8.7 Hz,
−5.0; HRMS(ESI) calcd for C H O SiNa [M + Na] m/z
2
3
38
6
4
461.2336, found m/z 461.2322.
(2R)-1-{3-{1-[(tert-Butyldimethylsilyl)oxy]ethyl}-2,6-bis-
(methoxymethoxy)phenyl}-3-methylbut-3-en-2-ol, (R)-12. To a
solution of (S)-(+)-2-methyloxazaborolidine (S-CBS,1.0 M solution
4
1
=
(
in toluene, 0.1 mL, 27.7 mmol) in dry THF (2 mL) was added BH
/
3
SMe (2.0 M in Et O, 0.3 mL, 0.7 mmol) dropwise at 0 °C under
3
2
2
1
5
3
3
1
2
H), 5.19 (d, J = 4.5 Hz, 2H), 5.12 (d, J = 6.3 Hz, 1H), 4.98 (d, J =
.0 Hz, 1H), 3.84 (t, J = 6.3 Hz, 2H), 3.62 (s, 3H), 3.48 (s, 3H),
.04−2.95 (m, 3H), 1.38 (d, J = 6.3 Hz, 3H), 0.88 (s, 9H), 0.04 (s,
argon. After stirring for 30 min, a solution of 3 (0.2 g, 0.4 mmol) in
dry THF was slowly added dropwise to the mixture at −30 °C. The
reaction solution was allowed to warm gradually to 0 °C in 30 min.
After 2 h, the reaction was quenched with MeOH (10 mL) and
13
H), −0.05 (s, 3H); C NMR (125 MHz, CDCl ) δ 155.2, 153.7,
3
33.8, 125.5, 120.9, 110.6, 100.4, 94.7, 65.8, 62.6, 57.1, 56.2, 28.1,
diluted with saturated NH
with EtOAc (3 × 10 mL), and the combined organic layers were dried
over anhydrous MgSO , filtered, and concentrated in vacuo. The
4
Cl (20 mL). The mixture was extracted
6.3, 25.9 × 3, 18.2, −4.8, −4.9; HRMS(ESI) calcd for C H O SiNa
2
0
36
6
+
[
M + Na] m/z 423.2179, found m/z 423.2165.
4
(
±)-2-{3-{1-[(tert-Butyldimethylsilyl)oxy]ethyl}-2,6-bis-
residue was purified by chromatography (petroleum ether/EtOAc =
1
(methoxymethoxy)phenyl}acetaldehyde (4). To a solution of 10
15:1 to 8:1) to afford (R)-12 (0.1 g, 72%) as a colorless oil: H NMR
(
1.0 g, 2.5 mmol) in CH Cl (20 mL) was slowly added Dess−Martin
(500 MHz, CDCl ) δ 7.36 (d, J = 8.6 Hz, 1H), 6.93 (d, J = 8.6 Hz,
2
2
3
periodinane (2.1 g, 5.0 mmol) at 0 °C. The reaction mixture was
allowed to warm to room temperature and stirred for 4 h. The
mixture was quenched with saturated NaHCO (20 mL) and NaS O
3
1H), 5.19 (d, J = 2.4 Hz, 2H), 5.10 (q, J = 6.3 Hz, 1H), 5.03−4.96
(m, 3H), 4.82 (s, 1H), 4.35−4.30 (m, 1H), 3.62 (s, 3H), 3.49 (s, 3H),
3.03−2.98 (m, 1H), 2.94−2.88 (m, 1H), 1.85 (s, 3H), 1.38 (t, J = 6.3
3
2
1
3
(
4
2 mol/L, 4.0 mL). The aqueous layer was extracted with Et O (3 ×
Hz, 3H), 0.88 (s, 9H), 0.04 (s, 3H), −0.05 (s, 3H); C NMR (125
2
0 mL). The combined organic layers were washed with NaHCO3
MHz, CDCl ) δ 155.1, 153.7, 148.1, 133.8, 125.6, 121.0, 110.7, 109.9,
3
(
40 mL), dried over anhydrous MgSO , filtered, and concentrated in
100.4, 94.8, 75.4, 65.8, 57.2, 56.2, 31.5, 26.3, 25.9 × 3, 18.2, 18.1,
4
+
vacuo. The residue was purified by chromatography on silica gel
petroleum ether/EtOAc = 30:1) to give 4 (0.8 g, 80%) as a colorless
−4.8, −4.9; HRMS(ESI) calcd for C H O SiNa [M + Na] m/z
2
3
40
6
(
463.2492, found m/z 463.2489.
F
DOI: 10.1021/acs.jnatprod.8b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
The synthetic procedures, H and 13C NMR, and HRMS (ESI)
1
144.0, 134.8, 130.4, 129.7, 128.9 × 2, 128.4 × 2, 127.4, 126.2, 122.3,
data for (S)-12 were identical with those of (R)-12 described above.
110.0, 109.8, 101.7, 94.5, 75.1, 57.8, 56.4, 31.0, 18.2; HRMS(ESI)
+
(
S)-12: yield 73%, as a colorless oil.
2R)-1-{3-{1-[(tert-Butyldimethylsilyl)oxy]ethyl}-2,6-bis-
methoxymethoxy)phenyl}-3-methylbut-3-en-2-ylacetate, (R)-13.
To a solution of (R)-12 (0.5 g, 1.1 mmol) in CH Cl (10 mL)
calcd for C H O [M + Na] m/z 435.1784, found m/z 435.1763.
2
4
28
6
1
13
(
The synthetic procedures and H and C NMR and HRMS (ESI)
data for (S)-14 and racemic 14 were identical with those of (R)-14
described above. (S)-14: yield 89%, as a colorless oil. Racemic 14:
yield 92%, as a colorless oil.
(
2
2
were added Et N (0.5 mL, 3.4 mmol), Ac O (0.2 mL, 2.3 mmol), and
3
2
DMAP (0.02 g, 0.1 mmol). The mixture was stirred at room
temperature for 2 h. The reaction was quenched with a saturated
NH Cl solution (10 mL) and extracted with CH Cl (3 × 10 mL).
(R)-3′-(2″-Acetoxyl-3″-methylbut-3″-en-1-yl)-2′,4′-bis-
(methoxymethoxy)chalcone, (R)-15. To a solution of (R)-14 (0.2 g,
0.5 mmol) in CH Cl (8 mL) were added Et N (0.2 mL, 1.5 mmol),
4
2
2
2
2
3
The combined organic layer was washed with brine, dried over
Ac O (0.1 mL, 1.0 mmol), and DMAP (0.01 g, 0.1 mmol) at room
2
MgSO , and concentrated in vacuo. The residue was purified by flash
temperature. After 2 h, the reaction was quenched with a saturated
4
column chromatography (petroleum ether/EtOAc = 25:1) to provide
NH Cl solution and extracted with CH Cl (3 × 10 mL). The
4
2
2
1
.5 g (92%) of (R)-13 as a yellow oil: H NMR (500 MHz, CDCl ) δ
combined organic layer was washed with brine (30 mL), dried over
3
.34 (d, J = 8.7 Hz, 1H), 6.89 (d, J = 8.7 Hz, 1H), 5.62−5.57 (m,
H), 5.20−5.13 (m, 3H), 4.99 (q, J = 6.5 Hz, 1H), 4.95 (s, 1H),
.83−4.79 (m, 2H), 3.61 (s, 3H), 3.50 (s, 3H), 3.10−2.95 (m, 2H),
.93 (s, 3H), 1.79 (d, J = 4.4 Hz, 3H), 1.36 (t, J = 5.6 Hz, 3H), 0.88
MgSO , and concentrated in vacuo. The residue was purified by flash
4
column chromatography (petroleum ether/EtOAc = 10:1) to provide
1
0.2 g (92%) of (R)-15 as a bright yellow oil: H NMR (500 MHz,
CDCl ) δ 7.63 (d, J = 16.0 Hz, 1H), 7.61−7.59 (m, 2H), 7.50 (d, J =
3
1
3
8.7 Hz, 1H), 7.41−7.39 (m, 3H), 7.29 (d, J = 16.0 Hz, 1H), 6.97 (d, J
= 8.7 Hz, 1H), 5.65 (dd, J = 8.2, 5.8 Hz, 1H), 5.26 (s, 2H), 4.96−4.92
(m, 3H), 4.87 (s, 1H), 3.53 (s, 3H), 3.47 (s, 3H), 3.20 (dd, J = 13.4
Hz, 8.6 Hz, 1H), 3.09 (dd, J = 13.4, 5.7 Hz, 1H), 1.98 (s, 3H), 1.85 (s,
3
9
4.8, 76.2, 65.7, 57.1, 56.2, 28.8, 26.4, 25.9 × 3, 21.0, 18.2, 18.2, −4.8,
+
−
5.0; HRMS(ESI) calcd for C H O SiNa [M + Na] m/z
2
5
42
7
1
3
5
05.2598, found m/z 505.2582.
3H); C NMR (125 MHz, CDCl ) δ 192.3, 170.1, 159.2, 156.5,
3
The synthetic procedures and H and 13C NMR and HRMS (ESI)
1
143.8, 143.6, 135.0, 130.4, 129.8, 128.9 × 2, 128.4 × 2, 127.6, 126.4,
data for (S)-13 and racemic 13 were identical with those of (R)-13
described above. (S)-13: yield 95%, as a colorless oil. Racemic 13:
yield 92.5%, as a colorless oil.
120.8, 112.4, 109.6, 101.5, 94.5, 76.2, 57.9, 56.4, 28.3, 21.1, 18.2;
+
HRMS(ESI) calcd for C26
H
O
Na [M + Na] m/z 477.1890, found
30
7
m/z 477.1873.
The synthetic procedures and H and 13C NMR and HRMS (ESI)
data for (S)-15 and racemic 15 were identical with those of (R)-15
described above. (S)-15: yield 93%, as a colorless oil. Racemic 15:
yield 92%, as a colorless oil.
1
(R)-1-[3-Acetyl-2,6-bis(methoxymethoxy)phenyl]-3-methylbut-3-
en-2-ylacetate, (R)-2. To a solution of (R)-13 (0.4 g, 0.8 mmol) in
THF (6 mL) was slowly added TBAF (0.7 mL, 2.6 mmol) at room
temperature for 5 h. After completion of the reaction, it was quenched
with ice water (10 mL) and diluted with EtOAc (3 × 10 mL). The
organic layer was washed with H O (30 mL), dried over MgSO , and
(R)-2′,4′-Dihydroxy-3′-(2″-hydroxy-3″-methylbut-3″-en-1-yl)-
chalcone, (R)-1. To a solution of (R)-15 (0.2 g, 0.4 mmol) in MeOH
(6 mL) was added dropwise HCl (4.0 mol/L, 1.0 mL). The reaction
mixture was stirred at 70 °C for 1.5 h, and the reaction was quenched
2
4
concentrated in vacuo. The residue was purified by flash column
chromatography (petroleum ether/EtOAc = 10:1) to provide a
colorless oil. To a solution of the oil (0.4 g, 0.9 mmol) in CH Cl was
with a saturated aqueous NH
EtOAc (3 × 15 mL). The combined organic layers were washed with
brine (30 mL), dried over anhydrous MgSO , filtered, and
Cl solution (10 mL) and extracted with
4
2
2
added Dess−Martin periodinane (1.2 g, 2.9 mmol) slowly at 0 °C.
After stirring for 4 h, the reaction was quenched with saturated
NaHCO (10 mL) and the aqueous layer was extracted with Et O (3
4
concentrated in vacuo. The residue was purified by chromatography
on silica gel (petroleum ether/EtOAc = 5:1) to afford target
3
2
×
20 mL). The combined organic layers were washed with brine (60
2
0
mL), dried over anhydrous MgSO , filtered, and concentrated in
compound (R)-1 (0.09 g, 62%) as a bright yellow powder: [α]
D
4
1
vacuo. The residue was purified by chromatography on silica gel
= −25 (c 0.2, MeOH); H NMR (400 MHz, CDCl ) δ 13.83 (s, 1H),
3
(
petroleum ether/EtOAc = 20:1 to 5:1) to give (R)-2 (0.3 g, 76%) as
7.87 (d, J = 15.5 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.65−7.62 (m,
2H), 7.61 (d, J = 15.5 Hz, 1H), 7.43−7.42 (m, 3H), 6.54 (d, J = 8.9
Hz, 1H), 5.01 (s, 1H), 4.89 (s, 1H), 4.42 (d, J = 8.0 Hz, 1H), 3.23
(dd, J = 15.0, 1.6 Hz, 1H), 2.89 (dd, J = 15.0, 8.4 Hz, 1H), 1.88 (s,
1
a colorless oil: H NMR (500 MHz, CDCl ) δ 7.50 (d, J = 8.7 Hz,
3
1
4
3
3
1
1
H), 6.92 (d, J = 8.7 Hz, 1H), 5.59 (t, J = 6.5 Hz, 1H), 5.24 (s, 2H),
.97 (dd, J = 6.5 Hz, 5.9 Hz, 2H), 4.88 (s, 1H), 4.84 (s, 1H), 3.53 (s,
H), 3.50 (s, 3H), 3.16−3.14 (m, 1H), 3.07−3.05 (m, 1H), 2.55 (s,
1
3
3H); C NMR (125 MHz, CDCl ) δ 192.0, 164.5, 163.5, 146.8,
3
1
3
H), 1.95 (s, 3H), 1.82 (s, 3H); C NMR (125 MHz, CDCl ) δ
144.1, 135.0, 130.5, 130.0, 129.0 × 2, 128.5 × 2, 120.7, 113.7, 113.2,
3
99.3, 170.0, 159.5, 156.6, 143.5, 129.7, 127.4, 121.1, 112.4, 109.3,
110.4, 109.1, 77.6, 28.6, 18.5; HRMS(ESI) calcd for C20
H
20
O
4
Na [M
+
01.5, 94.5, 76.0, 57.8, 56.3, 29.9, 28.3, 21.0, 18.2; HRMS(ESI) calcd
+ Na] m/z 347.1260, found m/z 347.1225.
+
The synthetic procedures and H and 13C NMR and HRMS (ESI)
1
for C H O Na [M + Na] m/z 389.1577, found m/z 389.1549.
19
26
7
1
13
The synthetic procedures and H and C NMR and HRMS (ESI)
data for (S)-2 and racemic 2 were identical with those of (R)-2
described above. (S)-2: yield 81%, as a colorless oil. Racemic 2: yield
data for (S)-1 and racemic 1 [(±)-sanjuanolide] were identical with
2
0
D
those of (R)-1. (S)-1: yield 62.5%, as a colorless oil; [α]
0.2, MeOH). Racemic 1: yield 61%, as a colorless oil.
= +26 (c
8
2.5%, as a colorless oil.
R)-3′-(2″-Hydroxy-3″-methylbut-3″-en-1-yl)-2′,4′-bis-
methoxymethoxy)chalcone, (R)-14. To a solution of (R)-2 (0.2 g,
.5 mmol) and benzaldehyde (0.1 g, 1.1 mmol) in EtOH was added
Cells and Reagents. Lipopolysaccharide were purchased from
Sigma (St. Louis, MO, USA). Saline was prepared as a 0.9% NaCl
solution. eBioscience, Inc. (San Diego, CA, USA) was the source of
the mouse IL-6 enzyme-linked immunosorbent assay (ELISA) and
mouse TNF-α ELISA kits. Mouse RAW 264.7 macrophages were
obtained from the American Type Culture Collection (ATCC, U.S.).
RAW 264.7 macrophages were incubated in Dulbecco’s modified
Eagle’s medium (DMEM) medium (Gibco, Eggenstein, Germany)
supplemented with 10% fetal bovine serum (Hyclone, Logan, UT,
USA), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C
with 5% CO2.
(
(
0
KOH (0.05 g, 0.9 mmol) slowly. The reaction mixture was stirred at
room temperature for 6 h, diluted with H O (10 mL), and extracted
with EtOAc (3 × 10 mL). The combined organic layers were washed
with brine (30 mL), dried over anhydrous MgSO , filtered, and
concentrated in vacuo. The residue was purified by chromatography
on silica gel (petroleum ether/EtOAc = 15:1 to 3:1) to afford (R)-14
2
4
1
(
0.2 g, 90%) as a bright yellow oil: H NMR (500 MHz, CDCl ) δ
3
7
1
.65 (d, J = 16.0 Hz, 1H), 7.61−7.58 (m, 2H), 7.54 (d, J = 8.7 Hz,
H), 7.41−7.39 (m, 3H), 7.29 (d, J = 16.0 Hz, 1H), 7.00 (d, J = 8.7
Determination of TNF-α and IL-6. The TNF-α and IL-6 levels
in the medium and serum were determined by ELISA analysis as
13b
Hz, 1H), 5.28 (s, 2H), 5.03 (s, 1H), 4.99 (q, J = 6.3, 2H), 4.87 (s,
1
3
previously described. RAW 264.7 macrophages were seeded into
six-well plates at a density of 400 000 cells per well in DMEM
medium. Cells were incubated at 37 °C in 5% CO2 for 24 h.
H), 4.40−4.37 (m, 1H), 3.51 (s, 6H), 3.08−3.04 (m, 2H), 1.89 (s,
H); 13C NMR (125 MHz, CDCl ) δ 192.0, 159.0, 156.7, 148.0,
3
G
DOI: 10.1021/acs.jnatprod.8b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Macrophages were pretreated with compounds for 30 min, which was
followed by treatment with 0.5 μg/mL LPS. After treatment, the cells
were incubated for 24 h. ELISA was used to screen the inhibition of
all synthetic compounds for LPS-induced TNF-α and IL-6 release in
RAW 264.7 mouse macrophages.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (Grants No. 81773579, 21472142, and
■
2
1572166), the National Natural Science Foundation of
Real-Time Quantitative PCR. Cells were homogenized in a
TRIZOL kit (Invitrogen, Carlsbad, CA, USA) for extraction of RNA
according to the manufacturer’s protocol. Both reverse transcription
and quantitative PCR were carried out using a two-step M-MLV
Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen). An
Eppendorf Mastercycler ep Realplex detection system (Eppendorf,
Hamburg, Germany) was used for qPCR analysis. The primers of
genes including TNF-α, IL-6, IL-1β, ICAM-1, MCP-1, COX-2, and β-
actin were synthesized by Invitrogen. Details have been described
Zhejiang Province (LY17B020008), and the Wenzhou Public
Welfare Science and Technology Project (2018Y0465). We
thank Dr. P. Creed from Liwen Bianji, Edanz Group China, for
editing the English text of a draft of the manuscript.
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AUTHOR INFORMATION
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Corresponding Authors
(Y. Zhao) Tel: (+86)-577-86699892. Fax: (+86)-577-
6699892. E-mail: aabye1100@aliyun.com.
(G. Liang) Tel: (+86)-577-86699892. Fax: (+86)-577-
6699892. E-mail: wzmcliangguang@163.com.
15) (a) Kolesov, S. A.; Korkotashvili, L. V.; Yazykova, A. B.;
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8
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8
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Fedulova, E. N.; Tutina, O. A.; Tolkacheva, N. I. Clin. Lab. 2013, 59,
53−957. (b) Kleiner, G.; Zanin, V.; Monasta, L.; Crovella, S.;
Caruso, L.; Milani, D.; Marcuzzi, A. Exp. Ther. Med. 2015, 9, 2047−
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66, 2058−2061. (b) Wada, J.; Makino, H. Clin. Sci. 2013, 124, 139−
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(Z. Liu) Tel: (+86)-577-86699892. Fax: (+86)-577-
6699892. E-mail: lzgcnu@163.com.
ORCID
Zhiguo Liu: 0000-0001-8986-4427
Author Contributions
B. Fang, Z. Xiao, and Y. Qiu contributed to this work equally
(18) (a) Huang, Q. H.; Lei, C.; Wang, P. P.; Li, J. Y.; Li, J.; Hou, A.
J. Fitoterapia 2017, 122, 138−143. (b) Svouraki, A.; Garscha, U.;
Kouloura, E.; Pace, S.; Pergola, C.; Krauth, V.; Rossi, A.; Sautebin, L.;
Halabalaki, M.; Werz, O.; Gaboriaud-Kolar, N.; Skaltsounis, A. L. J.
Nat. Prod. 2017, 80, 699−706.
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and should be considered co-first authors.
Notes
The authors declare no competing financial interest.
H
DOI: 10.1021/acs.jnatprod.8b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX