Sesquiterpenes from Artemisia argyi: Absolute configurations and biological activities

Article abstract of DOI:10.1002/ejoc.201301445

Nine guaiane-type sesquiterpenes and one eudesmane-type one - namely argyinolides A-J (1-10) - as well as nine known analogues were isolated from the leaves of Artemisia argyi Levl. et Vant. Their structures were determined by interpretation of spectroscopic data (MS, 1D and 2D NMR). A combination of X-ray crystal diffraction, specific optical rotations, CD spectroscopy, ECD calculation, and Mosher ester methods was employed to resolve the absolute configurations of the isolated compounds. Biological investigations into their cytotoxicities and anti-inflammatory effects showed that 1, 13, 16, and 18 were remarkably cytotoxic against Bel-742 and/or A549 cells, with IC₅₀ values of 3.3-6.0 μM, whereas 7, 11-13, 16, and 18 exhibited sound inhibitory activity on LPS-stimulated NO production in BV-2 microglial cells, with IC ₅₀ values ranging from 3.2 to 8.6 μM. In addition, a brief discussion on the applicability of Geissman's rule for the sesquiterpene lactones, the probable reason for the presence of chlorine-containing sesquiterpenes, and preliminary structure-activity relationships (SARs) are included. Ten new sesquiterpenes - argyinolides A-J - together with nine known analogues were isolated from the leaves of Artemisia argyi. Their complete structures were elucidated by detailed NMR and MS analysis, single-crystal X-ray diffraction, CD spectroscopy, ECD calculations, and Mosher ester methods. The cytotoxic and NO inhibitory effects of the isolates are also presented. Copyright

Full text of DOI:10.1002/ejoc.201301445

FULL PAPER
DOI: 10.1002/ejoc.201301445
Sesquiterpenes from Artemisia argyi: Absolute Configurations and Biological
Activities
Shu Wang,[a,b] Jian Sun,[a] Kewu Zeng,[a] Xiaoguang Chen,[c] Wanqi Zhou,[c] Chen Zhang,[a]
Hongwei Jin,[a] Yong Jiang,*[a] and Pengfei Tu*[a]
Dedicated to Professor Xinsheng Yao on the occasion of his 80th birthday
Keywords: Natural products / Terpenoids / Configuration determination / Circular dichroism / Cytotoxicity / Nitric oxide
Nine guaiane-type sesquiterpenes and one eudesmane-type
one – namely argyinolides A–J (1–10) – as well as nine known
analogues were isolated from the leaves of Artemisia argyi
Levl. et Vant. Their structures were determined by interpret-
ation of spectroscopic data (MS, 1D and 2D NMR). A combi-
nation of X-ray crystal diffraction, specific optical rotations,
CD spectroscopy, ECD calculation, and Mosher ester meth-
ods was employed to resolve the absolute configurations of
the isolated compounds. Biological investigations into their
cytotoxicities and anti-inflammatory effects showed that 1,
13, 16, and 18 were remarkably cytotoxic against Bel-742
and/or A549 cells, with IC50 values of 3.3–6.0 μ , whereas 7,
11–13, 16, and 18 exhibited sound inhibitory activity on LPS-
stimulated NO production in BV-2 microglial cells, with IC50
values ranging from 3.2 to 8.6 μM. In addition, a brief dis-
cussion on the applicability of Geissman’s rule for the sesqui-
terpene lactones, the probable reason for the presence of
chlorine-containing sesquiterpenes, and preliminary struc-
ture–activity relationships (SARs) are included.
M
try. Many studies have confined themselves to the level of
relative configuration determination, and assignments of
the absolute configurations of sesquiterpene lactones have
often been based on indirect evidence,[4] such as the applica-
tion of the empirical Geissman rule to CD spectra.[5–7] Nev-
ertheless, as we know, the empirical rules do not always
work well, and it is better to confirm absolute configura-
tions further by more comprehensive methods such as X-
ray crystallography, chemical synthesis, NMR spectroscopy/
chiral derivatization (Mosher esters), ECD calculation, or
other chiroptical approaches.
We have recently reported a series of sesquiterpenes and
dimeric guaianolides from Artemisia species, together with
their anti-inflammatory effects and cytotoxicities.[8–11] Their
structural diversity and significant bioactivities prompted
us to research deeper into the active components of A. ar-
gyi, a traditional Chinese herb used for moxibustion and
for curing eczema, diarrhea, hemostasis, and menstruation-
related symptoms. As a result, ten new sesquiterpenes (1–
10, Figure 1), along with nine known derivatives 11–19,
were isolated and identified by interpretation of spectro-
scopic data. The absolute configurations were resolved with
the aid of a combination of single-crystal X-ray diffraction,
specific optical rotations, CD spectra, ECD calculation, and
the Mosher ester method. Moreover, the cytotoxic and anti-
inflammatory activities of the isolated compounds were
evaluated and are reported here.
Introduction
Sesquiterpenes, a group of naturally occurring 15-carbon
isoprenoid compounds, are mainly found in higher plants
and are characterized by enormous diversity in structure,
stereochemistry, biological function, and application.[1]
Over the last several decades, phytochemical investigations
of the Asteraceae (Compositae) family for chemically intri-
guing and medicinally significant sesquiterpenes have been
an attractive topic for natural product and synthetic chem-
istry studies.[2,3]
Scrutiny of the literature , however, reveals that there are
still some challenges facing the structural elucidation of ses-
quiterpenes, particularly for determination of stereochemis-
[a] State Key Laboratory of Natural and Biomimetic Drugs,
School of Pharmaceutical Sciences, Peking University Health
Science Center,
Beijing 100191, People’s Republic of China
E-mail: yongjiang@bjmu.edu.cn
pengfeitu@vip.163.com
http://sklnbd.bjmu.edu.cn/index.html
[b] Department of Medicinal Chemistry and Pharmaceutical
Analysis, Logistics College of Chinese People’s Armed Police
Forces,
Tianjin 300162, People’s Republic of China
[c] Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College,
Beijing 100050, People’s Republic of China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201301445.
Eur. J. Org. Chem. 2014, 973–983
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Y. Jiang, P. Tu et al.
FULL PAPER
Figure 1. Structures of compounds 1–19 isolated from Artemisia argyi.
On the basis of an accepted principle that 7-H always
has the α-orientation in natural guaianolides, 5-H, 6-H, and
8-H of 1 were deduced to be α-, β-, and β-oriented, respec-
Results and Discussion
Compounds 1–10 showed bands at 3489 to 3404, 1781 to
1691, and 1663 to 1623 cm–1 in their FT-IR spectra, sug-
gesting the presence of hydroxy and carbonyl groups and
double bonds. Compound 1 was shown to have a molecular
formula of C17H20O5, as indicted by the observed ion at m/z
327.1208 [M + Na]+ in its HRMS. The 1H NMR spectrum
(Table 1) exhibited a pair of signals typical of an exocyclic
methylene group conjugated to a γ-lactone ring at δH = 6.30
and 5.73 ppm. Two methyl resonances were evident at δH
= 2.16 and 1.96 ppm. The 13C NMR spectrum (Table 2)
indicated the presence of an acetoxy moiety at δC = 170.8
tively, from their large coupling constants (J5,6 = 9.5, J6,7
=
10.5, and J7,8 = 9.5 Hz) and the allylic coupling constants
4
(4J7,13a = 3.5 and J7,13b = 3.0 Hz) based on the Samek lac-
tone rule.[12] These postulated orientations were confirmed
by the 1D-NOESY correlations of 5-H/7-H and 6-H/8-H.
From the 3D molecular model, 9a-H (dd at δH = 2.83 ppm
with J8,9a = 5.0 Hz) was assigned the β-orientation, adopt-
ing a dihedral angle with 8-H of about 40°, whereas 9b-H
(dd at δH = 2.56 ppm with J8,9b = 3.5 Hz) was α-orientated,
leading to a dihedral angle of about 70°. The NOESY
cross-peaks of 1-H/5-H and 1-H/14a-H suggested the cis
relationship of 1-H and 5-H. The NOESY correlations of
2-H/6-H and 9b-H indicated the α-orientation for the 2-
hydroxy group. Furthermore, the preferred conformation
by geometry optimization (Figure 2), was easily able to ex-
plain the NOEs observed above and thus to support the
postulated configuration of 1.
and 21.6 ppm (C-1Ј, 2Ј), a lactone carbonyl group at δC
=
169.9 ppm (C-12), six olefinic carbons, and three oxygen-
bearing carbons. The above information, coupled with bio-
genetic considerations, implied that 1 was a sesquiterpene
lactone containing an acetoxy group.
The 1H-1H COSY and multiplicity-edited HSQC spectra
indicated the C-1, C-2, C-5, C-6, C-7, C-8, C-9, C-11, and
C-13 fragment sequences (Figure 2). The HMBC cross-
The absolute configuration at C-7 was assigned as R
peaks between 13-H2 and C-7/C-11/C-12 further verified from the negative Cotton effects (CEs) at 227 nm (Δε =
the existence of the α-methylene-γ-lactone moiety. The ob- –3.2) and 265 nm (Δε = –0.7) in the CD curves of 1, accord-
servable HMBC correlations from 15-Me to C-3/C-4/C-5 ing to the Geissman rule.[5–7] In parallel with the CD spec-
and from 14-H2 to C-1/C-9/C-10 established the molecular trum, the “in-NMR-tube” Mosher reaction[13] was applied
skeleton of 1 as that of a guaiane-type sesquiterpene. The to 1 for the assignment of the configuration at C-2. Analysis
1
positioning of the C-2 hydroxy group was also confirmed of H NMR chemical shift differences (Δδ = δS – δR) be-
from the HMBC correlations between 3-H and C-1/C-2. tween the (S)- and (R)-MTPA ester derivatives established
The esterification of 8-OH with acetic acid was secured by an S configuration for C-2 (Figure 3). Compound 1 was
the key HMBC correlation from a deshielded proton signal therefore characterized as (+)-(1R,2S,5R,6R,7R,8S)-8-acet-
at δH = 5.26 ppm (8-H) to δC = 170.8 ppm (C-1Ј). The mo- oxy-2-hydroxyguai-3,10(14),11(13)-trien-6,12-olide and as-
lecular framework of 1 was thus established.
signed the trivial name argyinolide A.
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Sesquiterpenes from Artemisia argyi: Absolute Configurations
1
Table 1. H NMR spectroscopic data (500 MHz) for compounds 1–10:[a] δ in ppm (J in Hz).
1
2
3
4
5
6
7
8
9
10[c]
1
2
3.32, dd
(9.5, 6.0)
5.14, m
2.83, d
(6.5)
3.64, dd
(12.0, 4.5)
α 2.25[b]
α 2.01, d
(15.0)
α 2.01, d
(15.0)
α 2.02, d
(15.0)
α 2.37, d
(16.5)
α 2.38, d
(16.5)
β 2.85, br.
d (16.5)
3.89, d
(4.5)
2.82, d
(10.5)
4.02, t
α 2.66, dd α 2.66, dd
(16.5, 8.0) (16.5, 8.0)
β 2.99, dd β 2.99, dd β 3.01, dd β 2.83, br.
(15.0, 5.0) (15.0, 5.0) (15.0, 5.0) d (16.5)
4.15, d
(5.0)
2.94, d
(9.5)
4.43, t
(9.5)
3.95, dddd 3.97, br. t
(10.5, 9.5, (9.5)
3.3, 3.0)
β 2.17[b]
β 2.17, dd
β 1.61, q
(16.5, 10.5) (12.0)
3
5
6
7
5.89, q
(1.4)
2.93, t
(9.5)
6.12, q
(1.5)
3.31, dd
(10.5, 6.5)
5.17, dd
4.15, d
(5.0)
2.94, d
(9.5)
4.43, t
(9.5)
4.16, d
(5.0)
2.96, d
(9.5)
4.43, t
(9.5)
4.03, br. t
(9.5)
3.86, d
(4.5)
4.08, dd
4.07, dd
5.24[b]
2.14[b]
(10.5, 8.0) (10.5, 8.0)
2.72, d
(10.5)
4.02, t
(10.5)
2.25, q
(10.5)
2.72, d
(10.5)
3.99, t
(10.5)
2.73, d
(10.5)
4.00, t
4.20, dd
4.09, t
(10.5)
2.57, dd
(12.0, 10.5)
(10.5, 9.5) (10.5, 9.0)
(10.5)
3.16, tt
(10.5, 3.0)
(10.5)
3.30[b]
3.30[b]
3.08, br. t
(10.5)
3.12, tt
(10.5, 3.0)
8
5.26, ddd
(9.5, 5.0,
3.5)
5.70, td
(11.0, 4.5)
5.17, dd
5.15, dd
5.24, dd
4.80, td
4.87, td
(10.5, 2.5)
4.94, dd
5.04, td
α 2.11[b]
(10.5, 4.5) (10.5, 4.5) (10.5, 4.5) (10.5, 2.5)
(10.5, 4.0) (10.5, 4.0)
β 1.62, q
(12.0)
α 1.37, td
9
β 2.83, dd β 2.48, dd
(14.5, 5.0) (13.5, 4.5)
α 2.56, dd α 1.85, dd
(14.5, 3.5) (13.5, 11.0)
5.60, d
(4.5)
5.57, d
(4.5)
5.65, d
(4.5)
α 2.48, dd
(14.0, 10.5) (14.0, 10.5) (m)
β 2.22, dd
(14.0, 2.5)
2.50, m
1.34, d
α 2.48, dd
2.31–2.39
α 2.40, dd
(15.0, 10.5) (13.5, 3.0)
β 2.32, dd
(14.0, 2.5)
β 2.47, dd
(15.0, 4.0)
β 2.11[b]
11
13 6.30, d
(3.5)
6.38, d
(3.4)
6.32, d
(3.3)
6.32, d
(3.3)
6.32, d
(3.4)
6.24, d
(3.2)
6.27, d
(3.3)
6.25, d
(3.2)
6.12, d
(3.2)
(7.0)
5.73, d
(3.0)
14 5.32, d
(2.0)
5.86, d
(3.0)
1.87, s
5.78, d
(3.0)
1.97, s
5.80, d
(3.0)
1.96, s
5.77, d
(3.0)
1.98, s
5.69, d
(3.0)
1.78, t (2.0) 1.80, d
(2.0)
5.82, d
(3.0)
1.71, br. s 1.72, s
5.73, d
(2.8)
5.45, d
(3.0)
0.86, s
5.11, d
(2.0)
15 1.96, br. s 2.21, br. s
1.89, s
1.89, s
1.90, s
6.21, q
1.58, s
2.10, s
1.61, s
2.15, s
1.28, s
1.28, s
5.23, s
5.03, s
2.28, m
2.15, m
2Ј 2.16, s
3Ј
2.17, s
2.27, m
2.15, m
2.44, m
1.74, dq
2.42, m
1.76, dq
6.20, br. q
(14.0, 7.5) (7.5)
1.52, dq
(14.0, 7.5)
(14.0, 7.0) (7.2)
1.50, dq
(14.0, 7.0)
4Ј
5Ј
0.99, d
(6.5)
0.99, d
(6.5)
0.94, t
(7.5)
1.19, d
(7.0)
2.03, br. d
(7.5)
1.94, br. s
0.95, t
(7.0)
1.21, d
(7.0)
2.02, br. d
(7.2)
1.92, br. s
0.99, d
(6.5)
0.99, d
(6.5)
[a] Diastereotopic methylene protons are referred to as Ha for the lower-field proton and Hb for the higher-field proton. Compounds 1
and 2 were examined in [D5]pyridine, others in CDCl3. [b] Overlapped signals are reported without designation of the multiplicity.
[c] Measured at 600 MHz.
A molecular formula of C17H20O6 was assigned to argyi- served NOEs (Figure S19 in the Supporting Information),
nolide B (2) on the basis of the [M + Na]+ ion peak in the are only possible if the seven-membered ring adopts a chair
1
HRMS (ESI). H and 13C NMR spectroscopic data for 2 geometry. The definite absence of NOE effect between 14-
(Table 1 and Table 2) showed some similarity to those of Me and 6-H or 8-H allowed 14-Me to be assigned as α-
compound 1, with two characteristic α-methylene-γ-lactone oriented with a more favored equatorial position, which
doublets and an acetoxy group. The esterification of 8-OH was also corroborated by its chemical shift at δC
=
could be established from the corresponding highly de- 31.3 ppm.[14] Similarly, the deshielding effects on 6-H and
shielded resonance at δH = 5.70 ppm,[16] even though the 8-H (δH = 5.17 and 5.70 ppm, respectively) supported a β-
HMBC cross-peak between 8-H and C-1Ј was invisible. The oriented axial 10-OH group.
combined analysis of 1H-1H COSY and HMBC corre-
The negative CE at 263 nm (Δε = –0.5) for 2 implied the
lations implied a planar structure of 2, as shown in the Sup- same configuration as in 1 for 7α-H (7R). A strong positive
porting Information (Figure S19). The NOE effect of 6-H/ CE at 226 nm (Δε = + 25.1) associated with the π–π* transi-
8-H showed them to be cis-oriented. Additionally, the vici- tion of an α,β-unsaturated cyclopentanone means the pres-
nal coupling constants between 8-H and 9b-H/9a-H (J = ence of a diene chromophore twisted in the sense of a right-
4.5 and 11.0 Hz, respectively), in combination with the ob- handed helix (P helicity). The helicity rule[15] was thus ap-
Eur. J. Org. Chem. 2014, 973–983
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Y. Jiang, P. Tu et al.
Table 2. 13C NMR spectroscopic data (125 MHz) for compounds recent approach increasingly applied for the determination
FULL PAPER
1–10:[a] δ in ppm.
of absolute configurations of natural products,[16] was used
to give further verification of the absolute configuration of
2. The overall predicted ECD of 2 was compared with the
experimentally measured one, and the results revealed a
good agreement between them (Figure 4). Argyinolide B (2)
was thus determined to be (+)-(1R,5R,6R,7R,8S,10S)-8-
acetoxy-10-hydroxy-2-oxoguai-3,11(13)-dien-6,12-olide.
1
2
3
4
5
6
7
8
9
10[b]
1
2
3
4
5
6
7
8
9
61.5 59.5 81.7 81.6 81.7 137.1 137.6 128.2 128.2 75.9
78.7 206.7 46.1 46.1 46.1 39.0 39.0 36.5 36.5 37.1
133.3 133.8 80.2 80.2 80.2 78.8 78.8 75.9 75.9 69.9
143.6 177.9 79.8 79.9 79.9 83.2 83.2 81.1 81.1 140.2
57.3 53.2 64.3 64.3 64.3 52.9 53.0 55.3 55.3 50.4
81.8 79.4 76.5 76.5 76.5 79.0 79.5 79.6 79.6 78.7
47.9 50.3 42.9 43.0 43.0 58.1 53.8 52.2 52.4 49.6
74.9 72.2 73.5 73.6 73.4 71.7 70.6 70.3 70.0 21.4
38.5 50.2 123.7 123.5 123.2 41.8 41.7 41.8 42.1 35.6
10 142.6 72.0 140.8 140.9 140.7 126.4 126.2 126.7 127.0 42.8
11 139.0 138.0 136.9 137.0 137.0 40.7 136.5 135.8 135.9 138.8
12 169.9 169.8 169.0 169.0 169.0 177.4 168.9 168.9 168.9 170.3
13 122.2 123.1 123.3 123.5 123.9 15.1 121.9 123.3 123.3 117.4
14 119.1 31.3 25.2 25.2 25.2 23.5 24.0 23.7 23.7 11.6
15 18.1 20.3 25.1 25.1 25.1 24.0 23.5 17.0 15.8 108.2
1Ј 170.8 170.3 172.7 176.3 167.1 169.9 169.8 175.5 166.5 172.7
2Ј 21.6 21.4 43.5 41.4 126.9 21.2 21.1 41.4 126.9 43.5
3Ј
4Ј
5Ј
25.6 26.5 140.7
22.4 11.6 16.0
22.4 16.5 20.5
26.3 140.1 25.8
11.8 15.9 22.4
15.8 20.5 22.3
[a] Compounds 1 and 2 were examined in [D5]pyridine, the others
in CDCl3. [b] Measured at 150 MHz.
Figure 4. Calculated and experimentally measured CD spectra of 2.
Argyinolides C–E (3–5) were each deduced to contain
one chlorine atom, from the relative abundance ratios of
3:1 for [M + Na]+ and [M + Na + 2]+ in their MS (ESI)
spectra. From their HRMS (ESI) spectra, compounds 3
and 4 were determined to share the same molecular formula
of C20H27O6Cl, whereas compound 5 was assigned the for-
mula C20H25O6Cl, two mass units lighter than 3 or 4. In-
spection of 1D-NMR spectroscopic data for 3–5 (Table 1
and Table 2) revealed that the gross framework of the three
compounds closely resembled that of 11,[17] with the only
differences being in the C-8 side chain: an acetoxy moiety
in compound 11 was replaced by an isovaleryloxy group in
3, a 2Ј-methylbutyryloxy system in 4, and an angeloyloxy
group in 5. The relative configurations of 3–5 were readily
established by the vicinal coupling constants, which were
identical to those observed in 11 (J2b,3 = 5.0, J5,6 = 9.5, J6,7
= 9.5, J7,8 = 10.5, and J8,9 = 4.5 Hz), which suggested 3β-
H, 5α-H, 6β-H, 7α-H, and 8β-H orientations, respectively.
From comparisons with literature data,[17] the signal at δH
= 2.01 ppm (d, J = 15.0 Hz) should be preferentially as-
cribed to 2α-H, and δH = 2.99 ppm (dd, J = 15.0, 5.0 Hz)
to 2β-H, in good agreement with the favorable conforma-
tion of 3 (Figure S103 in the Supporting Information).
The consistency of the CD data for 3–5 at around
260 nm with those for 1, due to the contribution of the α-
methylene lactone, suggested the same absolute configura-
tion at the C-7 stereocenter. In addition, the specific rota-
Figure 2. (a) 1H-1H COSY and key HMBC correlations of 1.
(b) Selected NOEs of geometry-optimized conformation for 1.
Figure 3. Δδ (δS – δR) values obtained from the 1H NMR spectra
of the MTPA esters of 1r and 1s.
plied to determine the R configurations for the C-1 and C- tions for 3–5 were found to be [α]2D1 = +82.2, [α]D21 = +88.0,
5 stereogenic centers, in accordance with the Geissman rule and [α]2D1 = +91.8, respectively: the same direction and sim-
prediction (Figure S23 in the Supporting Information). ilar magnitudes as in the case of 11 ([α]2D1 = +52.5). These
Moreover, the quantum chemical ECD calculation method data implied identical stereocenters in their core skeleton.
by time-dependent density functional theory (TDDFT), a Unfortunately, a limited sample amount of 4 prevented us
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Sesquiterpenes from Artemisia argyi: Absolute Configurations
from elucidating the configuration of the 2-methylbutyr-
yloxy moiety by chemical means. Compounds 3–5 were
hence established as (+)-(1S,3R,4R,5R,6S,7R,8S)-3-chloro-
1,4-dihydroxy-8-isovaleryloxyguai-9,11(13)-dien-6,12-olide
(argyinolide C, 3), (+)-(1S,3R,4R,5R,6S,7R,8S)-3-chloro-
1,4-dihydroxy-8-(2Ј-ξ-methylbutyryloxy)guai-9,11(13)-dien-
6,12-olide (argyinolide D, 4), and (+)-(1S,3R,4R,5R,
6S,7R,8S)-8-angeloyloxy-1,4-dihydroxy-3-chloroguai-
9,11(13)-dien-6,12-olide (argyinolide E, 5).
Argyinolide F (6) had a molecular formula of C17H24O6,
as elucidated from its HRMS (ESI) spectrum. Inspection of
1D-NMR spectroscopic data and the specific optical rota-
tion of 6 suggested that our sample corresponded exactly
to indicumolide B (6a), a previously reported guaianolide
from Chrysanthemum indicum.[18] The authors of ref.[18] had
proposed incorrect configurations at the C-3 and C-11 posi-
tions, however, and the structure should in both case be
revised to 8α-acetoxy-3α,4β-dihydroxyguai-11β-H-1(10)-en-
6α,12-olide.
Figure 6. ORTEP plot for the molecular structure of 6 drawn with
50% probability displacement ellipsoids (Note: a different number-
ing system is used for the structure in the text).
due to a high standard deviation (0.2).[19,20] Moreover, the
1D-TOCSY analysis was used to establish the assign- Bijvoet pair analysis of Hooft’s y = 0.3(2) also could not
ments of 11-H and 7-H, which had been oppositely as- enhance the precision and allow the safe assignment of the
signed in the original structural elucidation of 6a. In a 1D- absolute configuration.[21]
NOESY experiment (Figure 5) the cross-peaks of 6-H/11-H
In addition, preparation of the diastereomeric MTPA
and 7-H/13-Me, as well as 5H/15-Me, were clearly detected; and MPA esters of 6 failed to confirm the configuration,
meanwhile, irradiation of 15-Me faintly enhanced the signal due to identical signs for ΔδSR at both sides of the asymmet-
of 3-H, indispensable for defining 11-H as having the β- ric centers (Figure S54 and S59 in the Supporting Infor-
orientation and 15-Me the α-orientation. Furthermore, the mation). This anomalous behavior could be explained in
stereochemistry of C-3 could be tentatively established by terms of the steric hindrance of the axially oriented 3-OH
the expected Karplus-type relationship for vicinal coupling group modifying the conformation of the ideal Mosher es-
constants in combination with molecular modeling simula- ter.[22,23] In this case, the empirical lactone octant and sector
tions (Figure 5). A doublet at δH = 3.86 ppm (J = 4.5 Hz, rules were applied to determine the absolute configuration
3-H) suggested that 3-H should be equatorial (β-oriented) of the lactone group (Figure 7).[24] On the grounds of the
and flanked by two vicinal hydrogen atoms (2-H2): 2b-H at preferred conformation, a positive CE centered at 230 nm
δH = 2.37 ppm (d, J = 16.5 Hz) did not show a detectable (Δε = +0.2) was found for the n–π* transition of the lact-
coupling with 3-H, whereas a broadened 2a-H signal at δH one, indicting the R configuration at C-7. Additionally, the
= 2.83 ppm (br. d, J = 16.5 Hz) tended to, so the former absolute configuration of 6 was further checked by compar-
could be assigned as having an α-orientation. Finally, con- ing experimentally measured and calculated ECD spectra in
firmation of the proposed structure and evidence for the the same way as for compound 2. This comparison revealed
relative stereochemistry of 6 were confirmed by X-ray crys- proportional agreement between them, whereas the
tallography (Figure 6).
enantiomer of 6 showed the opposite result (Figure S63 in
Nevertheless, the refinement of the Flack parameter for the Supporting Information). Argyinolide F (6) was there-
6 [0.1(2)] led to an inconclusive result with regard to the fore reassigned as (–)-(2R,3R,5S,6S,7R,8S,11S)-8-acetoxy-
absolute structure and the handedness of the chiral centers 3,4-dihydroxyguai-11-H-1(10)-en-6,12-olide.
3
Figure 5. (a) Selected NOEs of geometry-optimized conformation of 6. (b) Karplus-type relationship analysis for JH,H couplings of the
C1–C4 moiety.
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Y. Jiang, P. Tu et al.
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Figure 7. Application of Klyne’s octant rule (a), viewed in the plane of the lactone along the bisectrix of the O–C–O angle and sector
lactone rule (b), viewed from above for prediction of the sign of the n–π* transition in the CD spectrum (230 nm) of 6.
The HRMS (ESI) spectrum of argyinolide G (7) gave a
Argyinolide I (9) had a molecular formula of C20H26O6,
1
sodiated molecular ion peak at m/z 345.1306, consistent established on the basis of HRMS (ESI, +). The H and
with a molecular formula of C17H22O6, two hydrogen atoms 13C NMR spectra of 8 and 9 displayed near-identical simi-
fewer than in 6. The NMR spectra of 6 and 7 (Tables 1 and larity for all signals, with the exception of the resonances
2) were nearly superimposable, with the main differences for the C-8 ester substituents. The methylbutyryloxy group
evident at C-11, C-13, and their immediate vicinity. The in 8 was replaced by the angeloyloxy group in 9, and this
existence of signals at δH = 6.24 and 5.69 ppm suggested was further confirmed by 2D-NMR spectra. Comparison
that 7 was an 11,13-exomethylene derivative, which was of the [α]2D1 value of 9 (+41.6) with that of 8 ([α]D21 = +23.0)
confirmed by the 2D-NMR spectroscopic data. On bioge- revealed that they shared the same stereogenic centers in
netic grounds the absolute configuration of the lactone moi- the structural skeletons. In the CD spectrum of 9, a strong
ety of 7 was believed to be identical to that of 6. To verify positive CE (Δε = +13.5) appeared at 222 nm for the π–π*
this assumption, quantum chemical ECD calculations were transition of the unsaturated ester moiety in position C-8,
undertaken. As shown in Figure S75 in the Supporting In- similar to that in the case of 5. In an extension of the Geiss-
formation, the overall pattern of the experimentally mea- man rule,[7] the absolute configuration of 9 could also be
sured CD of 7 only matched the ECD curve calculated for correlated through the C-8 chiral center, which was deduced
(3R,4R,5S,6S,7R,8S)-7. Argyinolide G (7) was thus estab- to be 8S from the positive CE in the 220–240 nm
lished as (+)-(3R,4R,5S,6S,7R,8S)-8-acetoxy-3,4-dihydroxy- range. Argyinolide I (9) was therefore established as
guai-1(10),11(13)-dien-6,12-olide.
The molecular formula of argyinolide H (8) was deter- 1(10),11(13)-dien-6,12-olide.
mined to be C20H28O6 by HRMS (ESI, +). Analysis of the Argyinolide J (10) had a molecular formula of C20H28O5,
(+)-(3S,4S,5S,6S,7R,8S)-8-angeloyloxy-3,4-dihydroxyguai-
1
compound’s NMR spectroscopic data (Table 1 and Table 2) as determined by HRMS (ESI, –). Comparison of its H
revealed a similar structure to 7. Compound 8 incorporated NMR spectroscopic data (Table 1) with literature reports
a 2Ј-methylbutyryloxy fragment instead of the acetoxy unit indicated that it closely resembled 3β-acetylridentin B, fea-
in 7. Meanwhile, there was an obvious discrepancy between turing an eudesmane-type sesquiterpene,[27] with a differ-
these two compounds in their C-2–C-4 moieties. A doublet ence in the C-3 substituent. The acetoxy moiety in the side
of doublets at δH = 4.08 ppm (J = 10.5, 8.0 Hz, 3-H) indi- chain of 3β-acetylridentin B was replaced in 10 by an isova-
cated that the proton 3-H in 8 was axially oriented, con- leroyloxy group, as was supported by 2D-NMR corre-
trary to the equatorial orientation in 7, and this was further lations. Model building of 10 by geometry optimization
confirmed by the NOESY cross-peak between 3-H and 5- provided a valuable insight into its stereochemical features,
H (Figure S103 in the Supporting Information). A positive and the NOESY correlations matched the constructed
CE at 261 nm (Δε = +0.4) in the CD spectrum indicated a model well (Figure S103 in the Supporting Information). A
7S configuration according to the Geissman rule, which did negative CE at 250 nm (Δε = –2.1) implied an α-configura-
not agree with biogenetic considerations. In this case, a tion for 7-H (7S configuration). Argyinolide J (10) was
practical and reliable method based on the CD data for therefore deduced to be (+)-(1R,3S,5S,6S,7S,10R)-1-
the Mo2(OAc)4 complex formed in situ was employed to hydroxy-3-isovaleroyloxyeudesman-4 (15),11(13)-dien-6,12-
determine the absolute configuration of the 3,4-diol moiety olide.
in 8.[25,26] According to Snatzke’s theory, the positive CEs
By comparison of their spectroscopic data with pre-
at 328 nm (Δε = +0.14, band IV) and 406 nm (Δε = +0.09, viously reported observations, the known compounds were
band II) in the CD spectrum (Figure 8), which correspond identified as 8α-acetoxy-3β-chloro-1α,4α-dihydroxyguai-
to a positive dihedral angle of the O–C–C–O moiety, indi- 9,11(13)-dien-6α,12-olide (11),[17] 8α-acetoxy-3α-chloro-
cated 3S and 4S configurations for 8 by the empirical 1α,4β-dihydroxyguai-9,11(13)-dien-6α,12-olide (12),[17] 8α-
helicity rule. The configuration of the 2-methylbutyryloxy acetoxy-3β-chloro-1α,4α-dihydroxyguai-10(14),11(13)-dien-
unit remained unassigned, due to limitations of sample 6α,12-olide (13),[17] deacetylmatricatin (14),[28] 14-deoxy-ac-
quantity. Argyinolide H (8) was hence characterized as (+)- tucin (15),[29] 8α-acetoxy-3α-chloro-1β,2β-epoxy-4β,10α-di-
(3S,4S,5S,6S,7R,8S)-3,4-dihydroxy-8-(2Ј-ξ-methylbutyryl- hydroxy-5α,7αH-guai-11(13)-en-12,6α-olide (16),[30] 3α-
oxy)guai-1(10),11(13)-dien-6,12-olide.
chloro-1β,2β-epoxy-4β,10α-dihydroxy-5α,7αH-guai-11(13)-
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Sesquiterpenes from Artemisia argyi: Absolute Configurations
Figure 8. CD spectrum of compound 8 in a DMSO solution of Mo2(OAc)4 with the inherent contribution subtracted and the O–C–C–
O dihedral in the compound 8–[Mo2]4+ complex.
en-12,6α-olide (17),[31] 3β-chloro-1α,2α-epoxy-4α,10α- but observable. This might result from the overlap of n–
dihydroxy-5α,7αH-guai-11(13)-en-12,6α-olide (18),[30] and π* transitions of the unsaturated ester at C-8 and lactone
ilicic acid (19).[32] The absolute configurations were chromophores, which could not be an indicator of the C-7
determined by comparison of the CD spectra (Figures configuration. The known compound 15 showed a split CE
S104–S111 in the Supporting Information) with those of (positive at 249 nm and negative at 272 nm with UV max-
1–10.
ima absorption at around 254 nm), due to the interaction
Determination of absolute configuration is one of the between the conjugated enone and lactone chromophores.
most challenging tasks in the sesquiterpene chemistry. It is The absolute configuration of 15 was therefore determined
a pity, however, that many studies have still left this problem by applying the exciton chirality CD method,[35] as shown.
unsolved or tacitly assumed absolute configurations on the Moreover, it is important to note that the empirical rule
basis of empirical rules.[33,34] For a long time, ECD spec- only worked for the lactones with α-methylene groups,
troscopy, an experimentally very sensitive and non-destruc- whereas the CD curve of 6, a saturated lactone, showed
tive technique, has been demonstrated to be a powerful tool only one weak but observable CE at 230 nm, which could
for the absolute configuration assignment of natural prod- conceivably be an indicator for the Klyne lactone octant
ucts. We have therefore made a brief summary of the CD and sector rule. In future studies of sesquiterpene lactones,
spectra characteristics for the isolated sesquiterpenes. All the validity of the Geissman rule correlation thus deserves
the eudesmanolide and guainolides bearing α-methylene-γ- more attention. In order to allow a safe assignment, the
lactone chromophores displayed negative CEs at around configuration should be checked by more comprehensive
220 nm and/or 260 nm in their CD spectra, the latter diag- methods, such as X-ray crystallography, formation of
nostic especially for the determination of configuration at Mosher esters, ECD calculation, or other chiroptical ap-
C-7, which is often assumed to be the 7α-H orientation on proaches.
the basis of the Geissman rule. As an extension of this rule,
Besides the stereochemistry of the sesquiterpenes, atten-
a second chiral indicator at around 220 nm for the lactones tion should also be paid to several chlorine-containing
with unsaturated ester moieties at C-8, such as compounds guaianolides (see 3–5, 11–13, 16–18); this has also been en-
5 and 9, also holds true. The empirical rule seems to be countered during the chemical investigation of some other
widely applicable, but in our study several exceptions were Artemisia species.[17,34,36] There are difficulties in explaining
observed and should be mentioned. Compounds 7 and 8, chlorine sources for the biosynthesis of chlorinated com-
for example, showed positive CE signs at around 260 nm, pounds in higher plants, and so it cannot be ruled out that
whereas further ECD calculation and Mo2(OAC)4-induced such chlorohydrins might be artifacts, generated during the
CD allowed the definite assignment of the same 7R config- isolation procedure, in which chlorinated solvents (CHCl3)
uration as in the other isolated sesquiterpenes. For com- might serve as sources of Cl– to attack corresponding sensi-
pound 9, a weak, broad, positive CE without a maximum tive epoxide derivatives,[37] which have been frequently re-
in the range from 250 to 280 nm was of little significance ported to be isolated from this genus plants.[17,38]
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Y. Jiang, P. Tu et al.
FULL PAPER
Of the isolated sesquiterpenes, most share the α-methyl-
ene-γ-lactone motif, a well-known Michael acceptor that
has often represented a wide spectrum of bioactivities in-
cluding antitumor, cytotoxicity, antimicrobial, and anti-in-
flammation.[39,40] In addition, prompted by the significant
cytotoxic and anti-inflammatory effects of previous isolates
from the genus Artemisia,[8–10] all sesquiterpenes obtained
here were screened for their cytotoxicity against five human
tumor cell lines, including lung adenocarcinoma (A549),
stomach cancer (BGC-823), colon cancer (HCT-8), hepa-
toma (Bel-7402), and ovarian cancer (A2780) cell lines, as
well as the inhibitory activity against LPS-stimulated NO
production in BV-2 microglial cells. Compounds 1 and 13
were selectively active against the Bel-7402 cell line with
IC50 values of 4.9 and 4.6 μm, respectively. Compounds 13,
17, and 18 showed strong inhibitory effect against the A549
cell line with IC50 values of 4.8, 6.0, and 3.3 μm, respec-
tively, whereas no significant positive results were observed
for other isolates (IC50 Ͼ 10 μm). The NO inhibitory effects
of the isolates are listed in Table 3. Of the compounds
screened, 7, 11–13, 15, and 18 exhibited marked potencies
in inhibiting NO production, comparable to that of the pos-
itive control indomethacin, and had no influence on LPS-
activated BV-2 cell viability. As expected, many isolates con-
taining the α-methylene lactone system exhibited sound in-
hibitory activities against NO production, whereas the ab-
sence of such a group significantly reduced the activity, as
is shown by comparison of 7 with 6. Additionally, the
chlorine-containing compounds (11–13, 17, and 18) showed
stronger inhibitory effects, and compounds 3–5 displayed
much less activity, probably due to the presence of a larger
group in place of the acetyl moiety at C-8. Although larger
ester groups can increase lipophilicity, these moieties, be-
yond a certain size limit, might give rise to steric hindrance
to the exocyclic methylene group, preventing it from ap-
proaching its target.[41] The structure–activity relationships
suggested that the chlorohydrin moiety and a “size opti-
mum” of lipophilic ester group as side chain in these α-
methylene lactones might contribute to the NO inhibitory
activities.
Conclusions
In summary, ten new sesquiterpenes, of the guaiane type
(1–9) and the eudesmane type (10), along with nine known
analogues (11–19) were isolated from the ethanolic extract
of A. argyi. Their complete structures, including their abso-
lute configurations, were elucidated by NMR and MS in-
terpretation, X-ray crystal diffraction, specific optical rota-
tions, CD spectroscopy, ECD calculation, and Mosher ester
analysis. The Geissman rule in CD spectra has proven to
be a general indicator for elucidation of the absolute stereo-
chemistry of α-methylene lactones, but the irregularities in
the sign of CE should be noticed. Deduction by compre-
hensive spectroscopic or chemical methods is thus to be
preferred. The cytotoxicities and anti-inflammatory activi-
ties of the isolated sesquiterpenes were evaluated in vitro,
and the preliminary SARs are also briefly discussed. Fur-
ther investigation of the mechanism(s) of action of these
sesquiterpenes is warranted, and additional studies are now
underway in our laboratory.
Experimental Section
General Experimental Procedures: Optical rotations were measured
with a Rudolph Autopol III automatic polarimeter. UV spectra
were recorded with a Shimadzu UV-2401 UV/Vis recording spec-
trophotometer. IR spectra were recorded with a Thermo Nicolet
Nexus 470 FT-IR spectrometer. CD spectra were measured with a
JASCO J-810 spectropolarimeter. NMR measurements were per-
formed with Varian INOVA-500 and Bruker Avance-600 FT NMR
spectrometers and use of solvent peaks as references. HRMS (ESI)
spectra were measured with Waters Xevo G2 Q-TOF, Shimadzu
LC-MS-ITTOF, and Bruker APEX IV FT-MS spectrometers. Mass
spectra were obtained with an Agilent 1200 Series LC/MSD ion-
trap mass spectrometer (ESI). Analytical HPLC was performed
with an Agilent 1100 system and Agilent Zorbax XDB-C18 column
(5 μm, 4.6ϫ250 mm), and semipreparative HPLC was conducted
with a Waters 600 instrument and Grace® Prevail column (5 μm,
10ϫ250 mm). Column chromatography was performed with silica
gel (200–300 mesh, Qingdao Marine Chemical Inc., China), ODS
(50 μm, Merck, Germany), and Sephadex LH-20 (Amersham Bio-
sciences, Sweden). TLC was carried out with precoated silica gel
GF254 glass plates. Spots were visualized under UV light or by
spraying with 2% vanillin-sulfuric acid.
Table 3. Inhibitory effects of bioactive compounds against LPS-
induced NO production in BV-2 cells.[a]
Plant Material: Dried leaves of A. argyi were purchased in Anguo
County, Hebei Province, China, in January 2010. The plant mate-
rial was authenticated by one of the authors (P. T.). A voucher spec-
imen (No. 20100119) was deposited at the Herbarium of the Peking
University Modern Research Center for Traditional Chinese Medi-
cine.
IC50 (μm)
Cell viability (%, 40 μm)[b]
6
17.9
5.3
3.2
6.9
4.2
22.2
8.6
6.4
20.9
7.1
99.7
99.5
88.6
83.5
97.0
100.0
99.8
100.0
96.7
100
7
11
12
Extraction and Isolation: Dried leaves (50 kg) of A. argyi were ex-
tracted with aqueous ethanol (95%, 400 Lϫ2.5 hϫ3). The con-
centrated extract (7.0 kg) was suspended in H2O (30 L) and parti-
tioned successively with petroleum ether (PE) and chloroform. The
CHCl3 extract (1500 g) was transferred to a silica gel column and
eluted with a stepwise gradient of PE/EtOAc (1:0, 9:1, 3:1, 1:1, and
0:1 v/v) to produce 10 fractions (F1–F10), which were examined by
TLC.
13
16
17
18
19
Indomethacin
[a] Only those compounds with strong or moderate inhibitory ef-
fects (IC50 Ͻ 40 μm) and low cytotoxities (cell viability Ͼ 80%) are
listed here. [b] Cell viability was expressed as a percentage (%) of
that of the LPS-only treatment group.
F9 (152 g) was chromatographed on a column of silica gel, with
elution with a gradient of PE/EtOAc (9:1, 4:1, 7:3, 3:2, and 1:1 v/
980
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Sesquiterpenes from Artemisia argyi: Absolute Configurations
v), to give fourteen subfractions (F9a–F9n). F9m (21 g) was chro-
matographed on a reversed-phase C18 silica gel column, with elu-
tion with MeOH/H2O (3:2, 4:1, 9:1, and 1:0 v/v), to yield seven
subfractions (F9m1–F9m7). F9m1 (4.1 g) was separated again on
a reversed-phase C18 silica gel column with elution with a gradient
of MeOH/H2O (3:2, 4:1, 9:1, and 1:0 v/v) to give ten subfractions
(F9m1a–F9m1j).
spectroscopy. The acylation reaction was found to be complete af-
ter 8 h. 1H NMR spectroscopic data for the (S)-MTPA ester deriva-
tive (1s) of 1 (500 MHz, [D5]pyridine): δ = 6.297 (d, J = 3.5 Hz, 1
H, 13a-H), 6.256 (m, 1 H, 2-H), 5.752 (d, J = 3.0 Hz, 1 H, 13b-H),
5.724 (q, J = 1.4 Hz, 1 H, 3-H), 5.259 (ddd, J = 9.5, 5.0, 3.5 Hz, 1
H, 8-H), 5.162 (d, J = 2.0 Hz, 1 H, 14a-H), 5.074 (d, J = 2.0 Hz,
1 H, 14b-H), 4.234 (dd, J = 10.5, 9.5 Hz, 1 H, 6-H), 3.369 (d, J =
9.5, 6.0 Hz, 1 H, 1-H), 3.290 (m, 1 H, 7-H), 2.948 (t, J = 9.5 Hz, 1
H, 5-H), 2.924 (dd, J = 14.5, 5.0 Hz, 1 H, 9β-H), 2.581 (dd, J =
14.5, 3.5 Hz, 1 H, 9α-H), 2.174 (s, 3 H, -OAc), 1.869 (s, 3 H, 15-
H) ppm.
F9m1e (840 mg) was repeatedly purified by semipreparative HPLC
(25% CH3CN/H2O, 2 mL min–1) to yield compound 15 (7.5 mg, tR
= 30 min). F9m1f (780 mg) was purified by semipreparative HPLC
(25% CH3CN/H2O, 2 mL min–1) to yield compounds 1 (4.1 mg, tR
= 23 min), 13 (2.0 mg, tR = 34 min), and 10 (5.1 mg, tR = 37 min).
F9m1d (800 mg) was purified by semipreparative HPLC (25%
CH3CN, 2 mLmin–1) to yield compounds 14 (1.7 mg, tR = 19 min),
16 (3.8 mg, tR = 25 min), and 2 (8.3 mg, tR = 28 min).
In the same manner, another portion of compound 1 (0.45 mg) was
treated with (S)-MTPA chloride (5 μL) in a second NMR tube at
room temperature for 8 h and in deuterated pyridine (0.5 mL) as
solvent, to afford the (R)-MTPA derivative of 1 (1r). 1H NMR
spectroscopic data for 1r (500 MHz, [D5]pyridine): δ = 6.305 (d, J
= 3.5 Hz, 1 H, 13a-H), 6.281 (m, 1 H, 2-H), 5.796 (q, J = 1.4 Hz,
1 H, 3-H), 5.756 (d, J = 3.1 Hz, 1 H, 13b-H), 5.248 (ddd, J = 9.5,
5.0, 3.5 Hz, 1 H, 8-H), 5.010 (d, J = 2.0 Hz, 1 H, 14a-H), 5.007 (d,
J = 2.0 Hz, 1 H, 14b-H), 4.229 (dd, J = 10.5, 9.5 Hz, 1 H, 6-H),
3.244 (d, J = 9.5, 6.0 Hz, 1 H, 1-H), 3.274 (m, 1 H, 7-H), 2.969 (t,
1 H, J = 9.5 Hz, 5-H), 2.928 (dd, J = 14.5, 5.0 Hz, 1 H, 9β-H),
2.557 (dd, J = 14.5, 3.5 Hz, 1 H, 9α-H), 2.173 (s, 3 H, -OAc), 1.926
(s, 3 H, 15-H) ppm.
F9l (23 g) was transferred to a reversed-phase C18 silica gel column
and eluted with MeOH/H2O (9:11, 3:2, 3:1, and 9:1 v/v) to produce
four subfractions (F9l1–F9l4). F9l1 (7.2 g) was further fractioned
on Sephadex LH-20 with elution with MeOH to give two subfrac-
tions (F9l1a and F9l1b); subfraction F9l1a was crystallized with
methanol to afford compound 17 (201 mg).
F9k (31 g) was chromatographed on a reversed-phase C18 silica gel
column, with elution with MeOH/H2O (2:3, 3:2, and 4:1 v/v) to
yield three subfractions (F9k1–F9k3). Compound 18 (490 mg) was
crystallized with methanol from subfraction F9k1 (10 g) during
evaporation at room temperature. F9k3 (7.3 g) was fractioned on
Sephadex LH-20 by isocratic elution with MeOH to give four
subfractions (F9k3a–F9k3d). F9k3b (1.7 g) was purified repeatedly
by semipreparative HPLC (55% CH3CN/H2O, 3 mL min–1) to af-
ford compounds 19 (8 mg, tR = 13 min), 3 (1.5 mg, tR = 20 min),
4 (1.0 mg, tR = 22 min), 5 (0.8 mg, tR = 23 min), and 10 (3.6 mg,
tR = 24 min).
Argyinolide B (2): White powder. [α]2D1 = +162.1 (c = 0.45, MeOH).
ECD (CH3CN): 227 (Δε = +25.1), 263 (Δε = –0.49), 322 (Δε =
–1.7). 1H NMR and 13C NMR spectroscopic data: see Table 1 and
Table 2. IR (KBr): νmax = 3436, 2964, 2931, 1767, 1721, 1691, 1628,
˜
1378, 1259, 1164, 1121, 1028, 1006 cm–1. HRMS (ESI, +): calcd.
for C17H20O6Na [M + Na]+ 343.1158; found 343.1152.
Argyinolide C (3): White powder. [α]2D1 = +82.2 (c = 0.22, MeOH).
ECD (MeOH): 212 (Δε = –1.1), 265 (Δε = –0.4). 1H NMR and 13
C
NMR spectroscopic data: see Table 1 and Table 2. IR (KBr): ν
˜
max
F10 (96 g) was chromatographed on a column of silica gel, with
elution successively with a gradient of PE/EtOAc (4:1, 7:3, 3:2, 1:1,
and 0:1 v/v), to give eight subfractions (F10a–F10 h). F10g (23 g)
was transferred to a reversed-phase C18 silica gel column and eluted
with MeOH/H2O (2:3, 3:2, and 4:1 v/v) to yield three subfractions
(F10g1–F10g3). During natural evaporation a white crystalline-like
powder precipitated from the mother liquor of F10g1. The residue
was further purified by semipreparative HPLC (20% CH3CN/H2O,
3 mLmin–1) to afford compounds 6 (10 mg, tR = 37 min) and 7
(2 mg, tR = 35 min). F10g2 (5.7 g) was purified repeatedly by semi-
preparative HPLC (CH3CN/H2O, 3 mL min–1) to afford com-
pounds 12 (3.6 mg, tR = 39 min, 25% CH3CN/H2O), 8 (2.0 mg, tR
= 42 min, 40% CH3CN/H2O), and 9 (3.0 mg, tR = 26 min, 45%
CH3CN/H2O).
= 3404, 2958, 2926, 1755, 1725, 1663, 1632, 1459, 1376, 1278, 1160,
1058, 1006 cm–1. HRMS (ESI, –): calcd. for C20H27O6Cl2
[M + Cl]– 433.1190; found 433.1199.
Argyinolide D (4): White powder. [α]2D1 = +88.0 (c = 0.16, MeOH).
ECD (MeOH): 227 (Δε = +2.9), 258 (Δε = –1.6). 1H NMR and
13C NMR spectroscopic data: see Table 1 and Table 2. IR (KBr):
ν
= 3432, 2927, 1753, 1722, 1626, 1459, 1383, 1250, 1151,
˜
max
1059 cm–1. HRMS (ESI, –): calcd. for C20H27O6Cl2 [M + Cl]–
433.1190; found 433.1201.
Argyinolide E (5): White powder. [α]2D1 = +91.8 (c = 0.24, MeOH).
1
ECD (MeOH): 223 (Δε = +18.2), 258 (Δε = –0.4). H NMR and
13C NMR spectroscopic data: see Table 1 and Table 2. IR (KBr):
ν
max = 3433, 2927, 1756, 1712, 1644, 1455, 1231, 1146, 1066, 1038,
˜
1008 cm–1. HRMS (ESI, –): calcd. for C20H25O6Cl2 [M + Cl]–
431.1034; found 431.1047.
Argyinolide A (1): Off-white amorphous powder. [α]2D1 = +118.0 (c
= 0.26, MeOH). ECD (MeOH): 227 (Δε = –3.2), 265 (Δε = –0.7).
1H NMR and 13C NMR spectroscopic data: see Table 1 and
Argyinolide F (6): White powder. [α]2D1 = –15.5 (c = 0.48, MeOH).
1
ECD (MeOH): 230 (Δε = +0.2). H NMR and 13C NMR spectro-
Table 2. IR (KBr): νmax = 3441, 2920, 1772, 1720, 1635, 1256, 1142,
˜
1033 cm–1. HRMS (ESI, +): calcd. for C17H20O5Na [M + Na]+
327.1203; found 327.1208, calcd. for C17H24NO5 [M + NH4]+
322.16490; found 322.16436.
scopic data: see Table 1 and Table 2. IR (KBr): ν
= 3489, 3390,
˜
max
2923, 1772, 1717, 1657, 1452, 1376, 1260, 1168, 1049, 994 cm–1.
HRMS (ESI, –): calcd. for C17H24O6Cl [M + Cl]– 359.1267; found
359.1256.
Preparation of (R)- and (S)-MTPA Esters of 1 by the “in-NMR-
tube” Mosher Ester Procedure: Compound 1 (0.45 mg) was trans-
ferred to a clean NMR tube and dried completely in a vacuum
drying oven. Deuterated pyridine (0.5 mL) and (R)-MTPA chloride
(5 μL) were added to the NMR tube immediately under a N2 gas
stream, and the NMR tube was then shaken rigorously to mix the
sample and MTPA chloride evenly. The NMR tube was permitted
to stand at room temperature and monitored every 2 h by 1H NMR
X-ray Crystallographic Analysis of 6: Upon crystallization from
methanol by use of the vapor diffusion method, colorless crystals
of 6 were obtained. A crystal (0.31ϫ0.38ϫ0.51 mm) was sepa-
rated from the sample and data were collected with a Rigaku
MicroMax 002+ diffractometer and use of Cu-Kα radiation and the
ω and κ scan technique to a maximum 2θ value of 144.48°. Crystal
data: C17H24O6, M = 324.37, monoclinic, P21, a = 10.160 (3) Å, b
Eur. J. Org. Chem. 2014, 973–983
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
981

Y. Jiang, P. Tu et al.
FULL PAPER
= 8.059 (9) Å, c = 11.068 (9) Å, α = 90°, β = 115.983(16)°, γ = 90°,
V = 814.6 (13) Å3, Z = 2, Dcalcd. = 1.322 gcm–3, 2892 reflections
independent, 2422 reflections observed (|F|2 Ն 2σ|F|2), R1 = 0.0404,
wR2 = 0.0888 (w = 1/σ|F|2), S = 1.042. The crystal structure was
solved by direct method with use of SHELXS-97, and all non-
hydrogen atoms were refined anisotropically by use of the least-
squares method. All hydrogen atoms were positioned by geometric
calculations. The Flack parameter is 0.1(2) and the Hooft param-
eter is 0.3(2).
simulated by use of the SpecDis-V151 software.[44] The final ECD
spectrum was obtained according to Boltzmann weighting of each
conformer.
Bioactivity Assays: The purities of the isolated compounds (Ͼ95%)
used for the biological assay in vitro were determined by 1H NMR
and HPLC techniques.
Cytotoxic Activity: Cell proliferation inhibition was determined by
the MTT method.[45,46] Taxol was used as the positive control
against HCT-8, Bel-7402, BGC-823, A549, and A2780 cell lines,
with IC50 values of 0.051, 0.006, 0.003, 0.016, and 0.008 μm, respec-
tively.
CCDC-926171 (for 6) contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
Argyinolide G (7): White powder. [α]2D1 = +43.1 (c = 0.44, MeOH).
ECD (MeOH): 217 (Δε = +2.0), 234 (Δε = –3.2), 264 (Δε = +0.4).
1H NMR and 13C NMR spectroscopic data: see Table 1 and
Inhibition of NO Production: The NO inhibitory activities of the
isolated compounds were evaluated against BV-2 microglial cells by
the MTT colorimetric method;[10] indomethacin was used as posi-
tive control. In addition, cell viabilities were also evaluated by MTT
assay.
Table 2. IR (KBr): νmax = 3484, 3389, 2926, 2898, 1762, 1716, 1655,
˜
Supporting Information (see footnote on the first page of this arti-
cle): Copies of IR, MS, 1D- and 2D-NMR, and ECD spectra for
compounds 1–10. ECD calculation details for compounds 2, 6, and
7. Mosher esters procedure and NMR spectroscopic data for com-
pound 6. CD and UV spectra of known compounds.
1396, 1375, 1254, 1162, 1049, 968 cm–1. HRMS (ESI, +): calcd. for
C17H22O6Na [M + Na]+ 345.1314; found 345.1306.
Argyinolide H (8): White powder. [α]2D1 = +23.0 (c = 0.24, MeOH).
ECD (MeOH): 261 (Δε = +0.43), 230 (Δε = –4.43); Mo2(OAc)4-
induced CD (DMSO): 328 (Δε = +0.14), 406 (Δε = +0.09). 1H
NMR and 13C NMR spectroscopic data: see Table 1 and Table 2.
Acknowledgments
IR (KBr): ν
= 3523, 2964, 2922, 2874, 1781, 1723, 1634, 1460,
˜
max
1376, 1265, 1183, 1160 cm–1. HRMS (ESI, +): calcd. for
This work was financially supported by grants from the National
Natural Science Foundation of China (NSFC) (grant numbers
30973629, 81222051, 81303253, and 81373294) and the National
Key Technology R&D Program “New Drug Innovation” of China
(grant numbers 2012ZX 09301002-002-002 and 2012ZX09304-
005).
C20H28O6Na [M + Na]+ 387.1778; found 387.1787.
Argyinolide I (9): White powder. [α]2D1 = +41.6 (c = 0.40, MeOH).
ECD (MeOH): 222 (Δε = +13.5), 260 (sh, Δε = +1.8). 1H NMR
and 13C NMR spectroscopic data: see Table 1 and Table 2. IR
(KBr): νmax = 3420, 2919, 2851, 1765, 1715, 1377, 1242, 1056 cm–1.
˜
HRMS (ESI, +): calcd. for C20H26O6Na [M + Cl]– 383.1627; found
383.1633.
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Received: September 23, 2013
Published Online: December 4, 2013
Eur. J. Org. Chem. 2014, 973–983
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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983