Ilelic acids A and B, two unusual triterpenes with a seven-membered ring from ilex latifolia

Article abstract of DOI:10.1021/ol301745b

Two unusual triterpenes, ilelic acids A (1) and B (2), together with their biosynthetic related compounds ilelic acids C (3) and D (4) were isolated from the leaves of Ilex latifolia. Their structures with absolute configurations were elucidated by spectroscopic analysis and modified Mosher's method. The plausible biogenetic pathway of 1 and 2 is proposed. These triterpenes exhibited a potent inhibitory effect on MCF-7 and MDA-MB-231 cells.

Full text of DOI:10.1021/ol301745b

ORGANIC
LETTERS
2012
Vol. 14, No. 16
4102–4105
Ilelic Acids A and B, Two Unusual
Triterpenes with a Seven-Membered Ring
from Ilex latifolia
Cun-Qin Wang,†,‡ Lei Wang,*,‡,§ Chun-Lin Fan,‡,§ Dong-Mei Zhang,‡,§
Xiao-Jun Huang,‡,§ Ren-Wang Jiang,‡,§ Liang-Liang Bai,‡,§ Jun-Min Shi,‡,§
Ying Wang,*,‡,§ and Wen-Cai Ye*,†,‡,§
Department of Phytochemistry, China Pharmaceutical University, Nanjing 210009,
People’s Republic of China, and Institute of Traditional Chinese Medicine & Natural
Products and Guangdong Province Key Laboratory of Pharmacodynamic Constituents
of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou
510632, People’s Republic of China
chywc@yahoo.com.cn; cpuwanglei@126.com; wangying_cpu@163.com
Received June 25, 2012
ABSTRACT
Two unusual triterpenes, ilelic acids A (1) and B (2), together with their biosynthetic related compounds ilelic acids C (3) and D (4) were isolated
from the leaves of Ilex latifolia. Their structures with absolute configurations were elucidated by spectroscopic analysis and modified Mosher’s method.
The plausible biogenetic pathway of 1 and 2 is proposed. These triterpenes exhibited a potent inhibitory effect on MCF-7 and MDA-MB-231 cells.
The leaves of Ilex latifolia (Aquifoliaceae) have long
been used as an herbal tea (Ku-Ding-Cha) in China for
adjuvant treatment of cold, headache, hypertension, and
diabetes.1 Previous phytochemical studies of this plant
had led to the isolation of more than 60 triterpenes and
triterpenoid saponins,2,3 which demonstrated antide-
pressant,4 antibacterial,5 and antitumor activities.6 As part
of a program to search for structurally unique and bio-
logically interesting natural products,7,8 ilelic acid A (1),
which represented a new type of triterpenoid with a seven-
membered ring, named as “Ilesane”, and a rare dubosane-
type triterpenoid,9 ilelic acid B (2), were isolated from the
leaves of I. latifolia. In addition, the biosynthetic related
compounds including three hexacyclic triterpenoids (3ꢀ5)
and two C-27 oxygenated triterpenoids (6ꢀ7) were obtained
† China Pharmaceutical University.
‡ Institute of Traditional Chinese Medicine & Natural Products, Jinan
University.
§ Guangdong Province Key Laboratory of Pharmacodynamic Constituents
of TCM and New Drugs Research, Jinan University.
(1) Chinese Materia Medica (Zhonghua Benchao); Editorial Com-
mittee of Chinese Materia Medica; Shanghai Science and Technology:
Shanghai, 1999; Vol. 13, pp 149ꢀ151.
(6) Fujiwara, Y.; Komohara, Y.; Kudo, R.; Tsurushima, K.;
Ohnishi, K.; Ikeda, T.; Takeya, M. Oncol. Rep. 2011, 26, 1533–1537.
(7) Shao, M.; Wang, Y.; Liu, Z.; Zhang, D. M.; Cao, H. H.; Jiang,
R. W.; Fan, C. L.; Zhang, X. Q.; Chen, H. R.; Yao, X. S.; Ye, W. C. Org.
Lett. 2010, 12, 5040–5043.
(8) Zhao, B. X.; Wang, Y.; Zhang, D. M.; Huang, X. J.; Bai, L. L.;
Yan, Y.; Chen, J. M.; Lu, T. B.; Wang, Y. T.; Zhang, Q. W.; Ye, W.-C.
Org. Lett. 2012, 14, 3096–3099.
(2) Ouyang, M. A.; Liu, Y. Q.; Wang, H. Q.; Yang, C. R. Phyto-
chemistry 1998, 49, 2483–2486.
(3) Huang, J.; Wang, X.; Ogihara, Y.; Shimizu, N.; Akiyama, T.;
Takeda, T. Chem. Pharm. Bull. 2001, 49, 239–241.
(4) Pemminati, S.; Gopalakrishna, H. N.; Venkatesh, V.; Rai, A.;
Shetty, S.; Vinod, A.; Yugandhar, B.; Shenoy, A. K. Res. J. Pharm., Biol.
Chem. Sci. 2011, 2, 431–437.
(9) Wafo, P.; Kamdem, R. S. T.; Ali, Z.; Anjum, S.; Khan, S. N.;
Begum, A.; Krohn, K.; Abegaz, B. M.; Ngadjui, B. T.; Choudhary, M. I.
Org. Lett. 2010, 12, 5760–5763.
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Pharmacol. 2011, 5, 1150–1156.
r
10.1021/ol301745b
Published on Web 07/26/2012
2012 American Chemical Society

from the title plant.10 Herein, we report the isolation and
structure elucidation of 1 and 2. The plausible biogenetic
pathway of 1 and 2 and the antitumor activities of these
triterpenoids are also described.
Table 1. NMR Data of 1 and 2 (in C5D5N, J in Hz)a
1
2
no.
1
δH
δC
δH
δC
R 1.14
β 1.92
R 1.82
β 1.82
40.1
R 1.20
β 1.96
R 1.82
β 1.82
40.2
2
28.4
28.3
3
4
5
6
3.40 (dd, 8.8, 7.2)
77.8
39.3
55.4
19.4
3.45 (dd, 9.0, 6.9)
ꢀ
77.9
39.4
55.3
19.5
ꢀ
0.90
0.92
R 1.34
β 1.55
R 1.33
β 2.01
ꢀ
R 1.36
β 1.58
R 1.34
β 2.06
ꢀ
7
42.3
42.1
The air-dried leaves of I. latifolia were pulverized and
extracted with water under reflux. The water extract was
subjected to a D101 macroporous resin column eluted
successively with H2O, 45%ꢀ95% EtOH. The fraction
eluted by 75% EtOH was purified by chromatography on
silica gel, reversed-phase C18 silica gel, and preparative
HPLC to yield compounds 1ꢀ9.
8
43.8
52.2
38.6
35.1
44.4
51.3
38.6
34.2
9
1.94
2.03
10
11
ꢀ
ꢀ
R 1.93
β 2.24
4.39 (m)
ꢀ
R 1.93
β 2.18
4.52 (m)
ꢀ
12
13
14
15
69.4
131.7
142.5
22.8
69.1
130.2
139.1
23.5
ꢀ
ꢀ
IlelicacidA (1) was obtainedasamorphouspowder. The
molecular formula of 1 was determinedasC30H48O4 onthe
basis of a quasi-molecular ion at m/z 495.3444 [MþNa]þ
(calcd 495.3444 for C30H48O4Na) in the HR-ESI-MS. The
IR spectrum showed the characteristic absorptions attrib-
R 2.15
β 2.45
R 1.94
β 2.19
ꢀ
R 2.31
β 2.38
R 1.99
β 1.99
ꢀ
16
24.2
25.7
17
18
19
49.9
58.4
39.0
45.5
45.2
41.7
utable to carboxyl (1708 cmꢀ1) and hydroxyl (3410 cmꢀ1
)
2.48 (d, 11.2)
1.22
2.95 (dd, 10.2, 3.3)
R 1.32
β 1.67
ꢀ
groups. The analysis of 1H and 13C NMR spectra revealed
that1 possessed 30carbons, including four tertiary methyls
[δH 1.21, 1.03, 0.94, and 1.12 (each 3H, s); δC 28.8, 17.0,
16.6, and 19.7], two secondarymethyls[δH 1.01(3H, d, J =
6.5 Hz) and 0.91(3H, d, J = 6.0 Hz); δC 18.0 and 20.3], two
oxygenated methines [δH 3.40 and 4.39 (each 1H); δC 77.8
and 69.4], and two olefinic carbons [δC 131.7 and 142.5] as
well as one carbonyl carbon [δC 180.6]. The above spectral
data suggested 1 could be a pentacyclic triterpenoid. With
20
21
1.06
38.7
31.1
30.7
34.8
R 1.48
β 1.48
R 1.82
β 2.04
1.21 (s)
1.03 (s)
0.94 (s)
1.12 (s)
R 3.24
β 2.66
R 1.26
β 1.43
R 1.80
β 2.05
1.24 (s)
1.03 (s)
0.95 (s)
1.09 (s)
R 3.23
β 2.40
ꢀ
22
34.8
30.7
23
24
25
26
27
28.8
17.0
16.6
19.7
49.1
28.8
16.5
17.5
20.4
44.5
1
1
the aid of Hꢀ H COSY, HSQC, HMBC, and ROESY
1
experiments, the H and 13C NMR signals of 1 were
assigned as shown in Table 1.
28
29
30
ꢀ
180.6
18.0
20.2
180.3
25.8
31.6
The 1H and 13C NMR signals assigned to rings A, B, and
E (Figure 1) were similar to those of ursolic acid (8)11
(Scheme 1). The presence of a methene and the absence
of an angular methyl in comparison with the usual
ursanes11,12 suggested that 1 should possess six methyls
and a seven-membered ring C, which was similar to the
skeleton of duboscic acid.9 The Hꢀ H COSY spectrum
revealed the presence of the spin-coupling systems in bold
as shown in Figure 1. The HMBC correlations between
H-27 (δH 2.66, 3.24) and C-11 (δC 35.1), C-13 (δC 131.7),
C-14 (δC 142.5), and C-18 (δC 58.4) confirmed that C-27
1.01 (d, 6.5)
0.91 (d, 6.0)
0.91 (s)
0.94 (s)
a Overlapped signals were reported without designating multiplicity.
1
1
(10) New compounds 3, 4 and known compounds 5ꢀ9 were also
isolated from I. latifolia. Their structures were elucidated by spectro-
scopic analysis and single-crystal X-ray diffraction (see Supporting
Information).
(11) Zhou, J. S.; Zhang, T. T.; Chen, J. J.; Wang, Q. Chin. J. Nat.
Med. 2009, 7, 108–110.
(12) Gnoatto, S. C. B.; Klimpt, A. D.; Nascimento, S. D.; Galera, P.;
Boumediene, K.; Gosmann, G.; Sonnet, P.; Moslemi, S. Eur. J. Med.
Chem. 2008, 43, 1865–1877.
1
Figure 1. Key 1Hꢀ H COSY and HMBC correlations of 1.
Org. Lett., Vol. 14, No. 16, 2012
4103

was inserted between C-12 and C-13 to form a seven-
membered ring C. The detailed interpretation of HMBC
correlations (Figure 1) allowed the establishment of the
planar structure of 1.
Ilelic acid B (2) showed the same molecular formula as 1
by its HR-ESI-MS (m/z 495.3448 [MþNa]þ; calcd for
C30H48O4Na: 495.3444). The IR spectrum of 2 was also
similar to that of 1. The 1H NMR spectrum of 2 exhibited
signals for six tertiary methyls at δH 1.24, 1.03, 0.95, 1.09,
0.91, and 0.94 (each 3H, s), as well as two oxygenated
methine protons at δH 3.45 (1H, dd, J = 9.0, 6.9 Hz) and
4.52 (1H, m). The 13C NMR and DEPT spectra displayed
30 signals for 6 methyls, 11 methylenes, 5 methines, and 8
quaternary carbons. Comparison of the NMR data of 2
with those of 1 (Table 1) revealed that most of signals were
similar, except for the signals due to ring E. In the HMBC
spectrum of 2, the correlations between Me-29 (δH 0.91)/
Me-30 (δH 0.94) and C-19 (δC 41.7), C-20 (δC 30.7),
and C-21 (δC 34.8) were observed, indicating that Me-29
and Me-30 were both connected to C-20. A comprehensive
In order to determine the relative configuration of 1, the
ROESY spectrum was extensively analyzed. The ROESY
correlations between H-1R and H-5, between H-5 and H-9,
between Me-25 and Me-26, and between H-18 and Me-29
indicated that 1 possessed the same A/B trans, B/C trans,
and D/E cis ring junctions as usual ursanes (Figure 2). In
addition, the ROESY correlations between H-3 and H-5,
between H-12 and Me-26, and between H-12 and H-27β
suggested the presence of 3β-OH and 12R-OH (Figure 2).
Subsequently, the modified Mosher’s method was applied
to determine the absolute configuration of 1.13,14 Compar-
ison of the 1H NMR chemical shifts between (S)- and (R)-
MTPA diesters of 1 (Figure 3) led to the assignment of S
configurations of C-3 and C-12, respectively. Thus, the
configurations of 1 were assigned as 3S, 5R, 8R, 9R, 10R,
12S, 17S, 18S, 19S, and 20R, respectively. Ilelic acid A (1)
represents a new class of pentacyclic triterpenoid derived
from ursolic acid. The name “Ilesane” was proposed for
this type of triterpenoid.
1
analysis of the 1Hꢀ H COSY, HSQC, HMBC, and ROESY
spectraallowed the assignment of NMR dataof2 asshown
in Table 1. The carbon skeleton of 2 belonged to a unique
dubosane-type triterpene. The first example of this skele-
ton, duboscic acid, had been recently reported with a
relative configuration.9 As in the case of 1, the result of
the modified Mosher’s method suggested that the absolute
configurations of C-3 and C-12 of 2 were both S (Figure 3).
Therefore, the structure of 2 was established, and the
absolute configurations were assigned as 3S, 5R, 8R, 9R,
10R, 12S, 17R, and 18S, respectively.
Oleanolic acid and ursolic acid are the most common
triterpenes occurring in natural plant populations. Based
on the plausible biogenetic pathway of duboscic acid,9 2
could be derived from oleanolic acid (9) by oxygenation
and migration of C-27 methyl, forming a seven-membered
ring C. Similarly, ilelic acid A (1) could be derived from
ursolic acid (8) by the same procedure (Scheme 1). It is
noteworthy that the key intermediates 6ꢀ7, which could
easily form the C-27 carbocations, and several biosynthetic
related hexacyclic triterpenoids (3ꢀ5) were also found in
the same plant,10 rationalized the proposed biogenetic
pathway. In addition, compound 4 could be formed by
β-attack of the incoming nucleophile, in which the partial
bond between C-12 and C-27 was broken under kinetic
control, while compounds 1 and 2 should be produced by
R-attack of the nucleophile with breaking of the partial
bond between C-12 and C-13 under thermodynamic
control.15
Figure 2. Key ROESY correlations of 1.
The growth inhibitory activities of these triterpenoids
(1ꢀ4, 6, and 7) were evaluated in human breast cancer cells
MCF-7 (estrogen receptor-positive) and MDA-MB-231
(estrogen receptor-negative). Compounds 1 and 2 showed
a growth inhibitory effect against MCF-7 cells with IC50
values of 29.51 ( 3.44 and 38.49 ( 3.16 μM, respectively.
Compound 7 was the most potent among all tested com-
pounds with IC50 values of 12.65 ( 0.94 and 4.58 ( 0.56 μM,
respectively, indicating that the p-(E)-coumaroyl moiety
at the C-27 position may contribute to improving the growth
inhibitory potential, when compared with the triterpene
with the p-(Z)-coumaroyl moiety (6) (see Supporting
Figure 3. Δδ values (ΔS ꢀ ΔR) for the MTPA diesters of 1 and 2.
(13) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512–519.
(14) Wang, L.; Yin, Z. Q.; Zhang, Q. W.; Zhang, X. Q.; Zhang, D. M.;
Liu, K.; Li, Y. L.; Yao, X. S.; Ye, W. C. Steroids 2011, 76, 238–243.
(15) Tadanier, J. J. Org. Chem. 1966, 31, 2124–2135.
Org. Lett., Vol. 14, No. 16, 2012
4104

Scheme 1. Plausible Biogenetic Route of 1 and 2
Information). In addition, all the tested triterpenoids
showed more potent growth inhibitory activity in MCF-
7 cells than in MDA-MB-231 cells, implying that their
growth inhibitory activities may be partly dependent on
the status of the estrogen receptor. It is necessary to
further study the role of estrogen receptor and the mo-
lecular mechanism underlying the cell death induced by
these triterpenoids.
(IRT0965), National Natural Science Foundation of
China (No. U0932004), and China Postdoctoral Science
Foundation (No. 201104369).
Supporting Information Available. Detailed description
of the experimental procedure; a listing of UV, IR, HR-
ESI-MS, HR-EI-MS, NMR spectral data of compounds
1ꢀ9; X-ray data for 4 (CIF); NMR data of MTPA diesters
of 1 and 2; and bioassay data. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This work was supported by the Team
Project of the Natural Science Foundation of Guangdong
Province (No. 8351063201000003), Program for Changjiang
Scholars and Innovative Research Team in University
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 16, 2012
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