Mollolide A, a diterpenoid with a new 1,10:2,3-disecograyanane skeleton from the roots of rhododendron molle

Article abstract of DOI:10.1021/ol401254e

Mollolide A (1), a diterpenoid featuring a new 1,10:2,3-disecograyanane skeleton, was isolated from the roots of Rhododendron molle. Its structure was elucidated through extensive MS, IR, and NMR spectroscopy analyses. The absolute configuration was determined by single-crystal X-ray diffraction of its p-bromobenzoate derivative (1b). Compound 1 exhibits a significant analgesic effect at a dose of 20 mg/kg and antiviral activity against the Coxsackie B3 virus with an IC₅₀ value of 27.7 μM.

Full text of DOI:10.1021/ol401254e

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Mollolide A, a Diterpenoid with a New
1,10:2,3-Disecograyanane Skeleton from
the Roots of Rhododendron molle
Yong Li,† Yun-Bao Liu,† Jian-Jun Zhang,† Yu-Huan Li,‡ Jian-Dong Jiang,†,‡
Shi-Shan Yu,*,† Shuang-Gang Ma,† Jing Qu,† and Hai-Ning Lv†
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 May 5, 2013
ABSTRACT
Mollolide A (1), a diterpenoid featuring a new 1,10:2,3-disecograyanane skeleton, was isolated from the roots of Rhododendron molle. Its structure
was elucidated through extensive MS, IR, and NMR spectroscopy analyses. The absolute configuration was determined by single-crystal X-ray
diffraction of its p-bromobenzoate derivative (1b). Compound 1 exhibits a significant analgesic effect at a dose of 20 mg/kg and antiviral activity
against the Coxsackie B3 virus with an IC50 value of 27.7 μM.
Grayanoids, a special type of diterpenoid, are toxic
compounds found exclusively in Ericaceae plants. Some
members of the grayanoids exhibit potent sodium channel
modulating,1 analgesic, sedative,2 and insect antifeedant
activities,3 which attracted great interest from both syn-
thetic and biological perspectives.4
Rhododendron molle G. Don (Ericaceae), a toxic plant in
China, has been used asananodyneand ananestheticsince
ancient times and is still used today to treat rheumatism
and relieve pain.5 Previous chemical studies of this plant
have shown that it is a rich source of grayanoids.6 How-
ever, the trace constituents have not yet been well studied.
In our present research, a trace amount of a grayanoid
(0.00008%) with a new 1,10:2,3-disecograyanane skeleton,
designated as mollolide A (1), was isolated from the roots
of Rhododendron molle (50 kg).7 Mollolide A (1) represents
the first example of a disecograyanane and was biosynthe-
tically formed from multiple ring cleavage of a grayanane
and anintramolecularesterification (Scheme 1). To the best
of our knowledge, it is the first time either the 1,10-seco or
2,3-seco pattern of 1 has been discovered in grayanoids. We
report herein the isolation, structure elucidation, postu-
lated biogenetic formation, and biological activities of
compound 1.
† State Key Laboratory of Bioactive Substance and Function of Natural
Medicines, Institute of Materia Medica.
‡ Institute of Medicinal Biotechnology.
(1) Maejima, H.; Kinoshita, E.; Seyama, I.; Yamaoka, K. J. Biol.
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(2) Wang, S. J.; Lin, S.; Zhu, C. G.; Yang, Y. C.; Li, S.; Zhang, J. J.;
Chen, X. G.; Shi, J. G. Org. Lett. 2010, 12, 1560–1563.
(3) Klocke, J. A.; Hu, M. Y.; Chiu, S. F.; Kubo, I. Phytochemistry
1991, 30, 1797–1800.
(4) (a) Borrelly, S.; Paquette, L. A. J. Am. Chem. Soc. 1996, 118, 727–
740. (b) Chow, S.; Kreß, C.; Albæk, N.; Jessen, C.; Williams, C. M. Org.
Lett. 2011, 13, 5286–5289.
(6) (a) Li, C. J.; Wang, L. Q.; Chen, S. N.; Qin, G. W. J. Nat. Prod.
2000, 63, 1214–1217. (b) Chen, S. N.; Zhang, H. P.; Wang, L. Q.; Bao,
G. H.; Qin, G. W. J. Nat. Prod. 2004, 67, 1903–1906. (c) Zhong, G. H.;
Hu, M. Y.; Wei, X. Y.; Weng, Q. F.; Xie, J. J.; Liu, J. X.; Wang, W. X.
J. Nat. Prod. 2005, 68, 924–926.
(5) Chen, J. S.; Zhen, S. Chinese Poisonous Plants; Science Press:
Beijing, 1987; p 232.
(7) For plant material, experimental procedures, and physicalꢀ
chemical properties for compounds 1, see the Supporting Information.
r
10.1021/ol401254e
XXXX American Chemical Society

C-14, and C-16. Meanwhile, protons of H-13, H-14, and
H-12 also showed critical HMBC correlations to an sp3
quaternary carbon at δC 80.6 (C-16). These HMBC corre-
lations indicated that a five-membered carbon ring (ring C,
Figure 1) was formed by C-15, C-8, C-14, C-13, and C-16.
Moreover, rings B and C comprised a bicyclo[3.2.1]octane
ring system, and C-8 and C-13 were bridged by C-14.
Furthermore, three oxygenated carbons in rings B and C
(δC 68.7, 79.3, and 80.6) were assigned to C-12, C-14, and
C-16, respectively, by the HMBC correlations from H2-11,
H-9, and H-13 to C-12 (δC 68.7); from H2-15 and H-13 to
C-14 (δC 79.3); and from H2-15, H-13, and H-12 to C-16
(δC 80.6). A tertiary methyl proton signal appearing at δH
2.34 showed strong HMBC correlations to the ketone
carbon (δC 211.5) and C-9, which not only suggested the
existence of an acetyl group but also placed it at C-9 of ring
B. Another tertiary methyl at δH 2.05 was connected
to C-16 of ring C as shown by HMBC correlations from
H3-17 to C-16, C-13, and C-15.
Compound 1, [R]20 þ 41.0 (c 0.08, MeOH), was
D
isolated as an amorphous powder. The molecular formula
was established as C20H32O8 by HRESI-MS with an m/z
423.2000 [M þ Na]þ (calcd 423.1989), corresponding to 5
degrees of unsaturation. The IR spectrum indicated the
presence of an ester carboxyl group (1757 cmꢀ1) and a
ketone moiety (1701 cmꢀ1). The 13C and DEPT NMR
spectra (Table 1) revealed four sp3 quaternary carbons
(two oxygenated at δC 81.4 and 80.6), five sp3 methines
(three oxygenated at δC 84.6, 79.3 and 68.7), five sp3
methylenes (one oxygenated at δC 57.8), four tertiary
methyls, and two sp2 quaternary carbons (one ketone
carbon at δC 211.5 and one ester carbonyl carbon at δC
181.9). Among the 20 carbons, two carbonyl carbons
accounted for 2 degrees of unsaturation, which suggested
that 1 is a diterpenoid possessing a tricyclic ring system.
The gross structure of 1 was initially deduced by com-
prehensive analysis of its 1D and 2D NMR spectra.
1
1
Figure 1. Selected Hꢀ H COSY and HMBC correlations for
1
According to the 1Hꢀ H COSY and HSQC spectra, three
compound 1.
spin systems [a: C(9)HꢀC(11)H2ꢀC(12)HꢀC(13)Hꢀ
C(14)H; b: C(6)HꢀC(7)H2; c: C(1)H2ꢀC(2)H2] were es-
tablished, as shown in Figure 1. In a, the protons of H-9
and H-14 showed significant HMBC correlations to an sp3
quaternary carbon at δC 50.9 (C-8), which suggested that
C-8, C-9, C-11, C-12, C-13, and C-14 constituted a six-
membered carbon ring (ring B, Figure 1). In contrast, the
protons of H2-15 showed HMBC correlations to C-8, C-9,
The abovespectral data establishedunitI (Figure 1) of1,
which resembles rings C/D of grayanane. However, the
remaining part (unit II, Figure 1) of 1 was quite different
from any known grayananes. The nine carbons of unit II
showed signals at δC 181.9 (C), 84.4 (CH), 81.4 (C), 57.8
(CH2), 48.3 (C), 35.7 (CH2), 32.6 (CH2), 22.1 (CH3), and
18.6 (CH3). The HMBC correlations from H3-18 and
H3-19 to C-3 (δC 181.9), C-4 (δC 48.3), and C-5 (δC 81.4)
indicated a fragment of ꢀC(5)(O)ꢀC(4)(CH3)2ꢀCOOꢀ.
Meanwhile, HMBC correlations from H2-1 and H2-2 to
C-5 established the connection of C-1 to C-5; HMBC
correlations from H-6 and H2-7 to C-5 suggested the direct
connection between C-6 and C-5. To fulfill the 5 degrees of
unsaturation, an additional ring (ring A, Figure 1) was
required in unit II of 1. Considering that C-6 (CH) (δC
84.4) shifted dramatically downfield comparedto a normal
hydroxylated methine, a lactone was likely present at this
position. The structure of unit II of 1 (Figure 1) was
thereby constructed, in which the 5/7-fused ring system of
grayanane was absent and replaced with a γ-lactone ring.
Finally, the connection of the two units (I and II) via C-7
and C-8 was confirmed by the key HMBC correlations
from H-6 and H-7 to C-8 (δC 50.9). As a result, the planar
structure of 1 was fully established, which is a new 1,10:2,3-
disecograyanane skeleton.
Table 1. NMR Data of Compound 1 in C5D5N (J in Hz)8
no.
1
δH
δC
no.
11
δH
δC
a 2.28 (dt,
14.5, 7.0)
b 2.18 (dt,
14.5, 7.0)
4.30 (m)
ꢀ
35.7
2.12 (m)
30.2
2
3
4
5
57.8
181.9
48.3
81.4
12
13
14
15
4.31 (m)
68.7
61.5
79.3
53.0
2.89 (brs)
5.29 (d, 6.0)
a 2.44 (d, 14.5)
b 2.00 (d, 14.5)
ꢀ
ꢀ
ꢀ
6
7
4.60 (brd, 10.0)
a 3.04 (brd,
15.5)
84.4
32.6
16
17
80.6
26.1
2.05 (s)
b 2.66 (dd,
15.5, 10.0)
ꢀ
8
50.9
53.2
211.5
18
19
20
1.44 (s)
1.57 (s)
2.34 (s)
22.1
18.6
30.5
9
3.26 (brd, 7.0)
ꢀ
10
The relative configuration of unit I was deduced by
NOESY experiments (Figure 2). A chair conformation
B
Org. Lett., Vol. XX, No. XX, XXXX

of ring B was indicated by the correlations of H-14/H-12
and H-12/H-13, which also defined the 1,3-diaxial orienta-
tion of H-12 and H-14 and the equatorial orientation
of H-13. Additionally, an NOE correlation between H-9
and H-15b suggested that H-9 was equatorial because C-15
and C-16 must adopt a 1,3-diaxial orientation to form a
bicyclo[3.2.1]octane ring system. In contrast, H3-17 corre-
lated with H-11, which meant that C-17 was in the
R-orientation. These findings led to the assignment of the
relative configuration of unit I.
Figure 3. (a) Conformations of C-6 and C-7. (b) Conformations
of C-7 and C-8. Boxes indicate conformations that agree
with the measured data and are energetically favored. Arrows
indicate the observed NOE correlations. For the designation of
H-7a and H-7b, see Figure 2.
attempts were made to structurally modify 1. Ultimately,
the primary alcohol of 1 was selectively converted to methyl
ether 1a, and then 1a was treated with p-bromobenzoyl
chloride in pyridine to obtain 1b. Crystallization of 1b from
4:1 acetone/H2O resulted in colorless crystals, which gave
an X-ray crystal structure with a Flack parameter of
ꢀ0.02(2). Thus, we were able to establish the relative
stereochemistry at C-5 and C-6 of the five-membered ring
as well as the absolute configuration of each chiral center
(5R, 6R, 8S, 9R, 12R, 13R, 14R, 16R) (Figure 4).10
Figure 2. Key NOESY correlations for compound 1.
However, the situation for C-5 and C-6 (unit II) is far
more complex because they are located in a five-membered
ring, whichisknowntoexistasa combination of numerous
puckered conformations. Each atom on this ring can assume
many relative positions.9 Thus, even though the correlation
between H-6 and H-1b was observed in the NOESY spec-
trum (Figure 2), the relative configuration of C-5 and C-6
still remains uncertain.
In addition, units I and II of 1 were connected through a
methylene, which should theoretically provide molecular
flexibility and further complicate the stereochemical ana-
lysis. However, bulky substituents at C-5 and C-8 restrict
the rotation of the C-6/C-7 bond and led to a preferential
conformation in which C-5 and C-8 were in the anti-posi-
tion (as shown in the Newman projections in Figure 3a).
This explained how a small 3JH6,H7a (J ≈ 3 Hz, measured
3
by peak half-width) and a large JH6,H7b (J = 10.0 Hz)
1
were observed in 1 (the H NMR spectra of 1 did not
change at 0, 20, and 60 °C; see Figure S21). Furthermore,
the NOE correlations of H-7a/H-15a, H-7b/H-9, H-7b/
H-14, H-6/H15a, and H-6/H-9 also suggested a restricted
rotation of the C-7/C-8 bond and placed C-6 and C-14 in
the anti-position (Figure 3b).
Based on the analyses described above, the relative stereo-
chemistry of 1 could not be confirmed entirely due to the
lack of evidence. To complete assignment, we attempted to
obtain an X-ray crystal structure. However, we found it
difficult to obtain a single crystal of 1 due to its poly-
hydric structure. To improve the crystallinity, a number of
Figure 4. X-ray structure of 1b.
(10) For reaction conditions, see the Supporting Information. Upon
crystallization from 4:1 acetoneꢀH2O using the vapor diffusion method,
colorless crystals of 1b were obtained. A crystal (2 mm ꢁ 0.03 mm ꢁ
0.02 mm) was separated from the sample and mounted on a glass fiber,
and data were collected using a Gemini E X-ray single crystal diffrac-
tometer Eos CCD area detector with a graphite monochromator and Cu
KR radiation at 101.7 K, μ(Cu KR) = 2.471 mmꢀ1; Crystal data:
C28H37BrO9, MW = 597.49, space group monoclinic, P21; unit cell
˚
˚
dimensions were determined to be a = 13.3229 (4) A, b = 6.1591 (2) A,
3
˚
˚
and c = 16.9032 (6) A; V = 1382.46 (8) A , Z = 2, Fcalcd = 1.435 mg/
mmꢀ3, F(000) = 624, 9233 reflections collected, 5308 independent
(Rint = 0.0419) which were used in all calculations. The final refinement
gave R1 = 0.0555 and wR2 = 0.1231. Crystallographic data have been
deposited at The Cambridge Crystallographic Data Centre and allo-
cated the deposition number CCDC 923268. The data can be obtained
free of charge via www.ccdc.cam.ac.uk/products/csd/ request.
(8) 1H and 13C NMR were measured at 500 and 125 MHz, repec-
tively. Overlapped signals were reported without designating
multiplicity.
(9) Wu, A.; Cremer, D.; Auer, A. A.; Gauss, J. J. Phys. Chem. A 2002,
106, 657–667.
Org. Lett., Vol. XX, No. XX, XXXX
C

an intramolecular esterification to generate compound 1
(Scheme 1).
Scheme 1. Proposed Biogenetic Pathway of Compound 1
Compound 1 was evaluated for analgesic, antiviral, and
cytotoxic activities.11 In the acetic acid induced writhing
test, at a dose of 20 mg/kg (i.p.), 1 exhibited an analgesic
effect with a 73% reduction in writhes compared with
the blank control. In the antiviral test, 1 demonstrated
moderate antiviral activity against the Coxsackie B3 virus
in African green monkey kidney cells (Vero cells) with
an IC50 value of 27.7 μM, and in the cytotoxic test, no
cytotoxicity against all of the assayed cell lines (HCT-8,
Bel-7402, BGC-823, A549, and A2780) was observed at a
concentration of 10 μM.
Acknowledgment. This work was supported by grants
from the Natural Science Foundation of China (No.
21132009), the National Science and Technology Project
of China (No. 2012ZX09301002-002), and PCSIRT (No.
IRT1007). The authors are grateful to the Department of
Instrumental Analysis of our institute for the UV, IR, NMR,
and MS spectra measurements and to the Instrumental
Analysis Center of Beijing University of Chemical Technol-
ogy for X-ray crystallographic measurement and analyses.
Supporting Information Available. Detailed experimen-
tal procedures; 1D and 2D NMR, MS, UV, IR, CD
spectra; and X-ray crystal data. This material is available
free of charge via the Internet at http://pubs.acs.org.
Compound 1 represents the first example of a grayanoid
with a new 1,10:2,3-disecograyanane skeleton. The bio-
genetic precursor of 1 could be plausibly traced back to
rhodojaponin VI, a common grayanoid in this plant.
Rhodojaponin VI could be initially transformed into 12-
hydroxyl rhodojaponin VI (i) by enzyme-catalyzed oxida-
tion. Intermediate i could undergo multiple ring cleavage
(between C-2 and C-3 and between C-1 and C-10) to form
intermediate iii. Then, the aldehyde group in iii could be
further oxidized to carboxylic acid iv, which could trigger
(11) (a) Chen, Q. Methodology for Pharmacological Study of Tradi-
tional Chinese Medicine; People's Health Press: Beijing, China, 1996; Vol.
661, pp 360ꢀ361. (b) 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. (c) Alley, M. C.;
Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine,
D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer
Res. 1988, 48, 589–601.
The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX