Antiviral spirooliganones A and B with unprecedented skeletons from the roots of Illicium oligandrum

Article abstract of DOI:10.1021/ol401992s

Two novel spirooliganones A (1) and (2), a pair of spiro carbon epimers, with a rare dioxaspiro skeleton were isolated from the roots of Illicium oligandrum. The structures were fully determined by spectroscopic analysis and chemical methods, especially modified Mosher's method, and X-ray diffraction analysis. Spirooliganone B was found to exhibit more potent activities against coxsackie virus B3 and influenza virus A (H3N2) (IC₅₀ 3.70-5.05 μM) than spirooliganone A. The biosynthetic pathway involving a hetero-Diels-Alder reaction of the epimers was proposed.

Full text of DOI:10.1021/ol401992s

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Antiviral Spirooliganones A and B with
Unprecedented Skeletons from the Roots
of Illicium oligandrum
Shuang-Gang Ma,† Rong-Mei Gao,‡ Yu-Huan Li,‡ Jian-Dong Jiang,†,‡
†
†
†
†
Ning-Bo Gong, Li Li, Yang Lu, Wen-Zhao Tang, Yun-Bao Liu, Jing Qu,
†
†
€
Hai-Ning Lu, Yong Li, and Shi-Shan Yu*
†
†
,†
€
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute
of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China, and Institute of Medicinal Biotechnology, Chinese
Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
yushishan@imm.ac.cn
Received July 16, 2013
ABSTRACT
Two novel spirooliganones A (1) and (2), a pair of spiro carbon epimers, with a rare dioxaspiro skeleton were isolated from the roots of Illicium
oligandrum. The structures were fully determined by spectroscopic analysis and chemical methods, especially modified Mosher’s method, and
X-ray diffraction analysis. Spirooliganone B was found to exhibit more potent activities against coxsackie virus B3 and influenza virus A (H3N2)
(IC50 3.70ꢀ5.05 μM) than spirooliganone A. The biosynthetic pathway involving a hetero-DielsꢀAlder reaction of the epimers was proposed.
Plants from the genus Illicium have demonstrated a
variety of antiviral activities. For example, essential oils
from Illicium verum show significant activity against the
herpes simplex virus type 2 (HSV-2).1 In view of the
potential of this genus to produce new antiviral compounds,
we chose to investigate the toxic shrub I. oligandrum, which
is used in Chinese folk medicine for the treatment of
rheumatoid arthritis. Previous investigations of the plant
resulted in the isolation of prenylated C6ꢀC3 compounds,
sesquiterpene lactones, neolignan glycosides, and phenolic
diglycosides.2 However, to this point no antiviral natural
products have been discovered from this species.
Our biological and chemical investigations of the lipo-
philic fraction of the CHCl3 extract from the roots extract
have now yielded two new compounds with an unprece-
dented dispiro skeleton: spirooliganones A (1) and B (2).
They exhibit potent activity against coxsackie virus B3
and influenza virus A/Hanfang/359/95 (H3N2) (IC50
3.70ꢀ33.33 μM). Herein, we present the isolation, struc-
ture elucidation, and proposed biosynthetic pathways of
1 and 2, as well as their antiviral activities.
† Institute of Materia Medica.
‡ Institute of Medicinal Biotechnology.
(1) Koch, C.; Reichling, J.; Schneele, J.; Schnitzler, P. Phytomedicine
2008, 15, 71–78.
(2) (a) Tang, W. Z.; Liu, Y. B.; Yu, S. S.; Qu, J.; Su, D. M. Planta
Med. 2007, 73, 484–490. (b) Zhu, Q.; Tang, C. P.; Ke, C. Q.; Wang, W.;
Zhang, H. Y.; Ye, Y. J. Nat. Prod. 2009, 72, 238–242. (c) Tang, W. Z.;
Ma, S. G.; Yu, S. S.; Qu, J.; Liu, Y. B.; Liu, J. J. Nat. Prod. 2009, 72,
1017–1021. (d) Tang, W. Z.; Ma, S. G.; Qu, J.; Yu, S. S.; Liu, Y. B.; Su,
D. M.; Liu, J. J. Nat. Prod. 2011, 74, 1268–1271. (e) Ma, S. G.; Tang,
W. Z.; Liu, Y. X.; Hu, Y. C.; Yu, S. S.; Zhang, Y.; Chen, X. G.; Qu, J.;
€
Ren, J. H.; Liu, Y. B.; Xu, S.; Liu, J.; Liu, Y. Y.; Li, Y.; Lu, H. N.; Wu,
Spirooliganone A (1) was obtained as a colorless oil.
The molecular formula, C25H34O4, was deduced from
HRESIMS and NMR (Table 1) data, indicating a degree
X. F. Phytochemistry 2011, 72, 115–125. (f) Ma, S. G.; Tang, W. Z.; Yu,
S. S.; Chen, X. G.; Liu, Y.; Wang, W. J.; Qu, J.; Xu, S.; Ren, J. H.; Li, Y.;
€
Lu, H. N. Carbohydr. Res. 2011, 346, 1165–1168.
r
10.1021/ol401992s
XXXX American Chemical Society

of unsaturation of nine. Infrared absorption bands at
3394 and 1643 cmꢀ1 implied the presence of hydroxyl
and carbonyl functionalities, respectively. The 13C NMR
and DEPT spectra showed 25 resonances attributable to
four methyls, eight methylenes, five methines, and eight
quaternary carbons (one R,β-unsaturated carbonyl). Thus,
33 hydrogen atoms could be accounted for; the remaining
one was likely from a hydroxyl group.
the first oxygen bridge between C-4 and C-12. In addition,
a pair of geminal methyl groups (CH3-13 and CH3-14) was
connected to the quaternary carbon C-12, as evidenced by
HMBC correlations from H3-13 to C-11, C-12, and C-14.
Thus, it could be deduced that a C5 prenyl unit (C10ꢀC14)
in a tetrahydrofuran ring system was connected to the
allylated cyclohexadienone moiety (a C6ꢀC3 unit, C1ꢀC9)
through an oxaspiro carbon (C-4). This was supported by
HMBC correlations from H2-10 to C-3 (δC 144.7) and C-5
(δC 167.4).
1
The H NMR spectrum of 1 displayed two methyl
singlets at δH 1.25 (H3-14) and 1.31(H3-13) and a de-
shielded singlet at δH 6.63 (H-3) suggesting a trisubstituted
1
1
double bond. Hꢀ H correlation spectroscopy (COSY)
and HSQC analysis further revealed six spin systems: a
(C7ꢀC9), b (C10ꢀC11), c (C15ꢀC16), d (C18ꢀC19), e
(C21ꢀC22), and f (C23ꢀC25) (drawn with thick bonds in
Figure 1).
13C NMR resonances at δC 185.4, 133.5, 144.7, 167.4,
and 110.0 suggested the presence of a cyclohexadienone
group containing an oxygenated olefinic carbon at the
β-position; this was confirmed by HMBC correlations
from H-3 to C-1, C-2, and C-5. Furthermore, the allyl
group (fragment a) was determined to be located at C-2 by
means of HMBC correlations from H2-7 to C-1 and C-2.
The molecular formula, and the presence of four oxyge-
nated quaternary carbons (δC 167.4, 77.7, 85.7, and 89.9),
implied the presence of two oxygen bridges in 1. HMBC
correlations from H-11 to C-4 (δC 77.7) and C-12 (δC 85.7)
and from H2-10 to C-12, as well as the fragment b, placed
1
Figure 1. 1Hꢀ H COSY and key HMBC correlations of 1.
The remaining 11 carbon resonances, including one oxyge-
nated quaternary carbon (δC 89.9), indicated the presence of
a monoterpene moiety (C16ꢀC25). Comparison of these 13C
NMR data with those of trans-sabinene hydrate (previously
synthesized from (ꢀ)-3-thujol)3 suggested that 1 possessed
a bicyclo[3.1.0]hexane moiety, which was confirmed by the
aforementioned two fragments d and e, as well as HMBC
correlations from H-18 to C-22, from H2-19 to C-17, C-20,
C-21, and from H2-21 to C-20. The isopropyl group
(fragment f) was assigned to C-20 based upon HMBC cross
peaks between H-23 and C-20, as well as between H2-19/
H2-21 and C-23. Furthermore, HMBC correlations from
H2-15 to C-1, C-5, and C-6, and from H2-16 to C-17 and
C-18, revealed that the bicyclo[3.1.0]hexane moiety was
attached to C-6 by the fragment c. Analysis of degrees of
unsaturation indicated that the bicyclo[3.1.0]hexane moiety
was also connected to C-5 through the second oxygen bridge,
forming another oxaspiro structure as shown in Figure 1.
Table 1. NMR Data for Spirooliganone A (1) in Acetone-d6
1
position
δC
δH (J in Hz)
1
185.4
133.5
144.7
77.7
167.4
110.0
33.8
137.0
116.3
44.3
78.8
85.7
28.0
24.0
17.6
28.6
89.9
30.3
11.4
2
3
6.63 (s)
4
5
6
7
2.96 (2H, d, 7.0)
8
5.84 (ddt, 17.0, 10.5, 7.0)
4.98 (d, 10.0)/5.04 (d, 17.0)
2.60 (dd, 12.5, 7.0)/2.03 (m)a
4.37 (m)
9a/9b
10a/10b
11
12
13
1.31 (s)
14
1.25 (s)
15a/15b
16a/16b
17
2.27 (2H, m)
1.78 (2H, m)a
18
1.35 (m)a
19a/19b
20
0.34 (dd, 5.0, 4.0)/0.49 (dd, 8.0, 5.5)
35.4
27.7
34.1
32.7
20.6
20.1
Figure 2. Key NOE correlations of 1 and 2.
21a/21b
22a/22b
23
1.73 (m)a/2.00 (dd like)
1.39 (m)a/1.75 (m)a
1.60 (sept, 6.5)
1.01 (d, 6.5)
The relative stereochemistry of 1 was deduced from
the NOESY spectrum and a series of NOE experiments.
The observation of NOESY correlations from H-3 to
24
25
0.86 (d, 6.5)
a Overlapped.
€
(3) Baeckstro, P.; Koutek, B.; Saman, D.; Vrkoc, J. Bioorg. Med.
Chem. 1996, 4, 419–421.
B
Org. Lett., Vol. XX, No. XX, XXXX

H-10b and H3-14, and from H-10b to H3-14, indicated that
H-10b and Me-14 were on the same face of the tetrahy-
drofuran ring. Moreover, a NOESY cross peak was also
observed between H-10a and H3-13, indicating that H-10a
and Me-13 were on the same face of the ring, opposite
H-10b and Me-14. Strong NOEs were observed for H-10a
(δH 2.60, dd, J= 12.5, 7.0 Hz) and H3-13 after irradiation of
H-11, but not for H-10b and H3-14, indicating that 11-OH
was on the same face as H-10b and Me-14. Further NOESY
correlations from H3-24 (δH 1.01, d) to H-10a and H-11,
and from H3-13 to H-21b (δH 2.00), indicated that the
tetrahydrofuran and cyclopentane rings were perpendicular
to the dihydropyranocyclohexadienone (DHPC) pseudo-
plane, as depicted in Figure 2. On the other hand, the relative
stereochemistry of two spiro skeletons in the molecule was
also unambiguously assigned by NOE experiments. Diag-
nostic NOEs for H3-24 and H3-13, but weak ones for H3-25
and H-23, were observed after irradiation of H-10a (see
Figure S11 in SI). This indicated the isopropyl group,
rather than the three-membered ring, was close to H-10a.
In addition, irradiation of H-11 enhanced the signals for
H3-24 and H-21b (δH 2.00), and irradiation of H-23 en-
hanced H-19b (δH 0.49) and H-10a. Collectively, these
NOEs suggested that C-10 and C-18 were on the same face
of the DHPC pseudoplane.
The absolute configuration at C-11 (R) was unambiguously
determined by the modified Mosher method (Figure 3).4
Based upon the assigned relative configuration, the absolute
configuration of 1 was established as 4R, 11R, 17R, 18R, 20S.
It is noteworthy that 1H NMR data for MTPA esters of 1 did
not provide convincing evidence of the absolute configuration
at C-11: both positive and negative ΔδSR values coexisted for
the same side of the C-11 stereocenter (see Table S2 in the
Supporting Information (SI)). Therefore, the diastereomeric
MPA (R-methoxyphenylacetic acid) esters of 1were prepared,
allowing the assignment of the absolute configuration of C-11
as R (see Table S3 in SI). This example confirms that MPA
esters are more reliable than MTPA esters for assignment of
the configuration of chiral carbons in sterically congested
spiro-ring environments.5
Figure 4. X-ray crystal structures of p-bromobenzoate deriva-
tives of 1 (upper) and 2 (below).
Because 1 possessed an unprecedented dispiro skeleton,
an X-ray diffraction experiment was necessary to confirm
its structure, especially the absolute configuration of C-17,
which had been determined by the above indirect methods
(Mosher mothod and NOEs). Because a single crystal of 1
could not be obtained, a p-bromobenzoate derivative of 1
was prepared by treatment with p-bromobenzoyl chloride,
a single crystal of which was obtained from acetoneꢀH2O.
Thus, the crystal structure of the derivative (Figure 4)
allowed unambiguous assignment of the absolute config-
uration of 1 (4R, 11R, 17R, 18R, 20S) based on the Flack
parameter (0.00(2)) for anomalous dispersion with Cu KR
radiation. This confirmed our assignment of the C-11
stereochemistry.
Spirooliganone B (2) was obtained as a colorless oil.
Its molecular formula, C25H34O4, was deduced from
HRESIMS and 1D NMR (Table S1 in SI), which was
the same as that of 1. The UV, IR, CD, and NMR spectral
data of 2 also resembled those of 1 (see Figures S24, S25,
S27, and S28 in SI). The structure was elucidated in the
1
same manner as for 1. Careful analysis of 1Hꢀ H COSY,
HSQC, and HMBC correlations, as well as other NMR
data, allowed us to conclude that its structure was similar
1
to 1. The H and 13C NMR chemical shifts from C-1 to
C-14 of 2 were nearly identical with those of 1, suggesting
that 2 shared the C-4 and C-11 absolute configurations
of 1. This was supported by the CD and NOE spectra
(Figure 2), as well as the Mosher method (see Tables S4
and S5 in SI). The NMR data from C-15 to C-25 of 2,
especially the spiro carbon C-17, were different from those
of 1, suggesting that 2 could be a C-17 diastereomer of 1,
which was supported by obvious NOEs for Hb-19 (δH
0.87, dd, 5.5, 3.0 Hz) and H3-13 (δH 1.33, s), instead of
H3-24 and H3-13, in the NOE spectra of 1. Finally, the
p-bromobenzoyl derivative of spirooliganone B (2) was
again prepared, crystallized, and subjected to X-ray dif-
fraction analysis (Figure 4), enabling unambiguous assign-
ment of the absolute configuration of spirooliganone B
(2) as shown (4R, 11R, 17S, 18R, 20S) based on the Flack
Figure 3. Selected Δδ1H NMR values (normal and red data
obtained in pyridine-d5 and acetone-d6, respectively) in ppm for
MPA (right) and MTPA (left) esters of 1.
(4) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kikisama, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(5) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Org.
Chem. 1991, 56, 1296–1298.
Org. Lett., Vol. XX, No. XX, XXXX
C

of 6 with monoterpene (ꢀ)-sabinene would afford the C-17
epimers 1 and 2.
Scheme 1. Proposed Biogenetic Pathway for 1 and 2
Compounds 1 and 2 were evaluated in vitro for activity
against coxsackie virus B3 (CVB3), coxsackie virus A16
(CVA16), influenza virus A/Hanfang/359/95 (H3N2), en-
terovirus 71 (EV71), and herpes simplex virus 1 (HSV-1).8
Compounds 1 and 2 showed obvious effect against CVB3
with IC50 values of 11.11 and 3.70 μM (see Table S6 in SI),
respectively, stronger than that of ribavirin, with their
selectivity index (SI) values (3.03 and 4.33, respectively)
being comparable to or stronger than that of ribavirin
(SI = 3.9). Additionally, compound 2 also showed poten-
tial activity against influenza A with an IC50 value of
5.05 μM, although its SI value of 4.57 was far less than
that of positive control oseltamivir (see Table S9 in SI).
However, Compounds 1 and 2 were inactive against EV71
(SHZH98, JS52, H, and BrCr), CVA16, and HSV-1 (see
Tables S6, S7, and S8 in SI).
In conclusion, we report two novel antiviral compounds
with a rare dioxaspiro skeleton, spirooliganones A and B,
from the roots of I. oligandrum. Their structures differ only
in the absolute configuration of the spiro carbon (C17).
This implies that 1 and 2 may be the products of Dielsꢀ
Alder cycloaddition where the π-face of (ꢀ)-sabinene’s
terminal double bond is attacked from either of two
directions. Futhermore, it is worthy of note that 2 shows
more potent antiviral activity against coxsackie virus
B3 and influenza virus A (H3N2) than 1, due to their
configuration difference. The potent antiviral activity and
structurally unique dispirocyclic nature of 2 makes it a
promising lead for the development of antiviral agents.
parameter of ꢀ0.023 (11) for anomalous dispersion with
Cu KR radiation.
Spirooliganones A (1) and B (2), which are epimeric at
C-17, feature two oxaspiro structural units which have
not previously been reported in a natural product: 7-allyl-
2,2-dimethyl-1-oxaspiro[4.5]deca-6,9-dien-8-one and
5-isopropyl-30,40-dihydrospiro[bicycle[3.1.0]hexane-2,20-
pyran]. A plausible biogenetic pathway for 1 and 2,
starting from 5-allylbenzene-1,2,4- triol (3), a common
biosynthetic precursor of prenylated C6ꢀC3 compounds,2e
is proposed in Scheme 1. Compound 3 may be converted to
intermediate 4 through prenylation, 1,3-σ migration6 of
prenyl, and oxidation steps, followed by a key epoxyprenyl
side-chain cyclization to generate oxaspiro intermediate 5.
Methylation of 5, followed by oxidation and dehydration,
would give 6. Subsequently, a hetero-DielsꢀAlder reaction7
Acknowledgment. Financial support from the Natural
Science Foundation of China (No. 21132009), the National
Mega-project for Innovative Drugs (No. 2012ZX09301002-
002), PCSIRT (No. IRT1007), and SRFDP (No.
20091106120035).
Supporting Information Available. Full experimental
details, including extraction and isolation, synthesis of
MTPA and MPA ester derivatives, X-ray crystal data for
p-bromobenzoyl derivatives, antiviral assays, MS, IR,
UV, CD, and 1D and 2D NMR spectra for 1 and 2. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(8) Li, Y. P.; Shan, G. Z.; Peng, Z. G.; Zhu, J. H.; Meng, S.; Zhang,
T.; Gao, L. Y.; Tao, R. M.; Li, Y. H.; Jiang, J. D.; Li, Z. R. Antiviral
Chem. Chemother. 2010, 20, 259–268.
(6) Oh, C. H.; Karmakar, S.; Park, H. S.; Ahn, Y. C.; Kim, J. W. J.
Am. Chem. Soc. 2010, 132, 1792–1793.
(7) Lawrence, A. L.; Adlington, R. M.; Baldwin, J. E.; Lee, V.;
Kershaw, J. A.; Thompson, A. L. Org. Lett. 2010, 12, 1676–1679.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX