Humulane-type sesquiterpenoids from Pilea cavaleriei subsp. crenata

Article abstract of DOI:10.1039/c3ob40872h

Nine new, uncommon humulane-type sesquiterpenoids (1, 2, 4, 6-11), together with two known derivatives, were isolated from extracts of the plant Pilea cavaleriei subsp. crenata. The structures of these compounds were fully elucidated by extensive analyses

Full text of DOI:10.1039/c3ob40872h

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Humulane-type sesquiterpenoids from Pilea cavaleriei
subsp. crenata†
Cite this: Org. Biomol. Chem., 2013, 11,
4840
Cang-Song Liao, Chun-Ping Tang, Sheng Yao and Yang Ye*
Nine new, uncommon humulane-type sesquiterpenoids (1, 2, 4, 6–11), together with two known deriva-
tives, were isolated from extracts of the plant Pilea cavaleriei subsp. crenata. The structures of these com-
pounds were fully elucidated by extensive analyses of spectroscopic data (MS, 1D- and 2D-NMR), use of
the Mosher method, and by X-ray crystallographic analysis, in combination with chemical conversions. An
ene reaction was discovered during the chemical transformations, which might provide an explanation
for the wide distribution of the allylic hydroperoxide group in natural products.
Received 27th April 2013,
Accepted 22nd May 2013
DOI: 10.1039/c3ob40872h
www.rsc.org/obc
Introduction
Sesquiterpenoids, derived from farnesyl pyrophosphate (FPP),
refer to a class of C-15 compounds comprised of three
isoprene units. Found mainly in higher plants, sesquiter-
penoids have an extensive range of complex structures1–3 with
a variable core ring system and various bioactivities, especially
anticancer activity.4
Found in plants,5–8 liverworts,9 and fungi,10 humulane-type
sesquiterpenoids represent an uncommon type of compound
possessing a characteristic 11-membered ring in the molecule,
which is very challenging for structural elucidation, particu-
larly for determination of the absolute configuration, due to
the flexible macrocycle. In 2009, Tang and co-workers11 identi-
fied three humulane-type sesquiterpenoids from Pilea cavaler-
iei subsp. crenata, belonging to the Urticaceae family, but the
absolute configuration of these compounds has not been fully
determined. Since plants of the genus Pilea are reported to
exhibit antidiabetic12 and antimicrobial13 activities, a systema-
tic investigation on the chemical constituents of Pilea cavaleriei
subsp. crenata was carried out.
Fig. 1 Structure of isolated and synthetic compounds.
Results and discussion
Herein, we report the isolation of nine new compounds
(1, 2, 4, 6–11), along with two known derivatives (3, 5),
and their structural elucidation using 1D- and 2D-NMR
spectroscopy, the Mosher method, and single-crystal X-ray
diffraction analysis, as well as chemical conversions (Fig. 1).
The aerial part of P. cavaleriei was extracted with ethanol. The
ethanol extract was suspended in water and partitioned with
petroleum ether to give a PE extract. The PE extract was again
partitioned with methanol to give a MeOH extract, which was
then isolated by repeated column chromatography over silica
gel, Sephadex LH-20 and MCI, and preparative HPLC to afford
pure compounds.
The molecular formula of 1 was determined to be C24H32O4
from the quasi-molecular ion peak at m/z 383.2238 [M − H]−
(calcd 383.2222) in the ESI-HRMS, corresponding to nine
degrees of unsaturation. The 13C NMR (Table 1) and DEPT
spectra showed 24 resonances ascribed to six quaternary,
nine methine, six methylene, and three methyl carbons.
The 1H NMR spectrum (Table 2) displayed signals of a
State Key Laboratory of Drug Research, and Department of Natural Products
Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, P. R. China.
E-mail: yye@mail.shcnc.ac.cn; Fax: +86-21-50807088; Tel: +86-21-50806726
†Electronic supplementary information (ESI) available: Spectroscopic data,
including 1H NMR, 13C NMR, 1H–1H COSY, HSQC, HMBC, ROESY, and
ESI-HRMS, for compounds 1, 1a, 2, 4, 6–11. CCDC 925951. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/c3ob40872h
4840 | Org. Biomol. Chem., 2013, 11, 4840–4846
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Table 1 13C NMR data for compounds 1, 1a, 2, 4, 6–11 in CDCl3 (δ in ppm)
Position
1
1a
2
4
6
7
8
9
10
11
1
2
3
4
5
6
7
8
127.7
29.7
36.3
156.3
71.9
45.0
43.8
70.4
46.3
131.6
32.7
127.1
29.6
36.2
156.4
71.8
48.0
45.1
67.9
49.8
131.8
32.6
28.6
29.3
17.9
111.9
127.3
29.6
36.2
156.1
72.0
44.9
43.5
70.3
46.1
131.5
32.7
127.5
30.1
37.6
152.4
85.4
40.4
43.6
70.1
46.2
131.7
32.4
127.9
25.3
39.5
134.3
124.5
39.5
42.6
72.7
46.2
131.7
33.7
27.0
31.0
18.2
16.1
166.7
117.8
143.8
127.9
132.2
115.2
157.1
126.8
24.8
38.0
60.8
62.1
36.2
43.6
70.1
46.7
131.3
32.4
27.6
29.7
17.5
126.9
24.8
38.0
60.7
62.0
36.2
43.4
69.7
46.6
131.2
32.3
27.5
28.6
17.7
62.4
24.9
37.0
134.2
123.7
43.3
43.2
72.0
45.3
59.2
33.6
24.1
33.7
20.5
63.2
24.0
37.2
134.0
123.3
37.5
45.6
68.2
48.3
59.9
32.8
28.2
29.7
18.1
63.2
24.2
37.5
134.2
123.5
37.7
46.1
68.8
48.7
60.0
33.1
28.6
30.0
18.0
9
10
11
12
13
14
15
1′
2′
3′
4′
28.1
29.0
17.4
28.0
29.6
17.6
27.8
28.7
17.4
112.1
166.9
116.3
144.2
127.5
131.6
116.0
157.7
112.2
166.3
117.6
143.6
127.8
131.5
115.2
157.1
115.0
166.1
117.8
143.5
127.5
132.4
115.1
156.9
16.9
16.9
15.9
15.6
15.8
167.2
115.0
144.6
126.7
131.3
115.5
158.2
166.1
117.2
143.9
127.0
132.3
115.0
157.1
166.8
115.7
144.4
127.0
132.4
115.8
157.8
165.9
117.4
143.5
127.3
132.4
114.9
156.7
167.1
116.1
144.5
127.4
130.1
116.0
157.7
5′/9′
6′/8′
7′
1
1,4-disubstituted phenyl group [δH 7.42 (2 H, d, J = 8.4 Hz), data. The H and 13C NMR data (Tables 1 and 2) of 2 showed
6.83 (2 H, d, J = 8.4 Hz)] and a double bond conjugated with a high similarities to those of 1, revealing that these two com-
carboxy group [δH 7.62 (d, J = 16.2 Hz), 6.26 (d, J = 16.2 Hz)] pounds shared a common sesquiterpenoid skeleton. The
which, in combination with the corresponding 13C resonances major differences were observed for H-2′ and H-3′ [δH 6.82 (d,
inferred from the HSQC spectrum, indicated the presence of J = 12.7 Hz), 5.74 (d, J = 12.7 Hz)], indicating a Z-2′-p-coumaroyl
an E-p-coumaroyl group.14 The remaining 1H and 13C NMR group instead of the E-isomer.14 This was also reflected in
data showed characteristic signals of an exocyclic double bond the chemical shifts of the aromatic protons [δH 7.64 (2 H, d, J =
(δC 112.1 and 156.3), a trisubstituted double bond (δC 127.7 8.6 Hz), 6.81 (2 H, d, J = 8.6 Hz)]. Thus, 2 was identified as the
and 131.6), and two oxygenated C–H moieties (δC 70.4 and C-2′ configurational isomer of 1. To further prove the proposed
71.9). Since the identified functionalities and segments con- structure, 2 was treated with LiAlH4, which yielded the identi-
tributed eight degrees of unsaturation, the one remaining was cal compound 1a to that obtained from 1. Accordingly, 2
ascribed to the existence of one additional ring. was identified as (1E,5R,8R)-8-O-[(Z)-p-coumaroyl]-5-hydroxy-
The structure of 1 was further established by extensive humula-1(10),4(15)-dien-8-ol.
HMBC analysis (Fig. 2). HMBC correlations from H-12 to C-6,
Compounds 3 and 4 possessed the same molecular formula
7, 11, and 13, from H-1 to C-2, 3, 10, and 14, from H-8 to C-7, of C24H32O5, with nine degrees of unsaturation. Their NMR
9, 10, and 11, and from H-15 to C-3, 4, and 5 constructed an data (Tables 1 and 2) suggested that 3 and 4 shared a common
11-membered ring, and the HMBC correlation from H-8 to C-1′ humulane-type skeleton, but were isomeric at the C-8 substitu-
allowed attachment of the p-coumaroyl group to C-8. Thus, ent, similar to the situation with 1 and 2. The NMR data of 3
1 was identified as a humulane-type sesquiterpenoid. The were almost superimposable with those of 1 except for the
E orientation of the 1,10-double bond was assigned from the chemical shift of H-5 (δ 3.94 in 1, δ 4.25 in 3), suggesting a
crosspeak of H-1/H-9 in the ROESY spectrum. Thus, the planar hydroperoxy rather than a hydroxy group located at C-5. This
structure of 1 was proved.
proposed structure was consistent with the molecular formula
Due to the flexible 11-membered ring in the molecule, the of 3, with one more oxygen atom than that of 1. In the event, 3
relative configuration of 1 was difficult to concretely establish was identified as a compound previously reported11 as 8-O-
based on ROESY experiments. Therefore, compound 1 was (p-coumaroyl)-5β-hydroperoxy-1(10)E,4(15)-humuladien-8α-ol with
treated with LiAlH4 to afford the 8-hydroxy derivative 1a, which the absolute configuration pending, and therefore 4 was
crystallized from methanol. Single-crystal X-ray diffraction ana- designated as its C-2′ configurational isomer.
lysis of 1a using Cu Kα radiation was then carried out and the
Taking the biogenesis into consideration, 3 was thought to
absolute configurations of C-5 and C-8 were both established have the same R configuration of C-5 and C-8 as compounds 1
as R (Fig. 3). Thus, the structure of compound 1 was fully veri- and 2. To verify this assumption, 3 was treated with NaBH4
fied as (1E,5R,8R)-8-O-[(E)-p-coumaroyl)]-5-hydroxyhumula-1 to yield a reduced product, which was identical to compound 1
(10),4(15)-dien-8-ol.
on the basis of the spectroscopic data. Thus, the structure of
Compound 2, an amorphous powder, gave the same mole- 3 was fully established, and renamed (1E,5R,8R)-8-O-[(E)-p-
cular formula of C24H32O4 as 1, according to the ESI-HRMS coumaroyl]-5-hydroperoxyhumula-1(10),4(15)-dien-8-ol.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4840–4846 | 4841

Table 2 1H NMR data for compounds 1, 1a, 2, 4, 6–11 in CDCl3 (δ in ppm, J in Hz)
Position
1
1a
2
4
6
7
8
9
10
11
1
2
3
5
6
5.36, br
5.27, dd (7.4, 5.9) 5.32, br
5.31, br s
4.97, m
5.13, m
5.13, m
3.12, d (10.9)
2.70, d (10.9)
2.74, d (10.9)
1.99, m 1.53, m
2.25, m
5.16, d (10.2)
2.29, m 1.78, d (14.6)
2.32, m 2.20, m 2.32, m 2.22, m
2.44, m 2.27, m 2.38, m 2.27, m
3.94, m
1.67, d (14.5)
1.32, m
2.32, m 2.20, m 2.30, m 2.21, m 2.32, m 2.12, m
2.44, m 2.27, m 2.52, m 2.51, m 2.18, m 2.06, m
2.35, m 2.12, m 2.35, m 2.12, m 2.08, m 1.47, m 1.97, m 1.49, m
2.15, m 1.17, m 2.15, m 1.17, m 2.34, m 2.26, m 2.25, m
2.60, d (5.0)
1.63, m 1.21, m 1.63, m 1.21, m 2.15, m 2.08, m 2.24, m 1.73,
d (14.6)
3.91, d (8.1)
1.55 1.32
3.94, br s
4.26, d (6.1)
1.45, m
4.89, m
2.06, m 1.80, m
2.59, d (3.8)
5.46, m
5.11, d (10.2)
1.70, d (14.5)
1.32, m
7
8
9
1.91, d (15.9)
1.57, m
4.81, br
1.92 1.28
1.91, dd (12.6,
12.7) 1.57, m
4.81, br
1.90, m 1.60, m 1.88, m 1.39,
dd (15.5, 7.9)
1.87, m 1.66, m 1.87, m 1.66, m 1.84, m 1.64, m 1.60, m 1.48, m
1.63, m 1.48, m
3.59, dddd (2.4,
4.6, 6.9, 9.3)
2.21, m 2.07, m
4.79, m
4.61, dd (8.6, 8.8) 4.68, br
4.68, br
2.16, m
5.27, m
4.68, dd (9.1, 7.7)
4.77, dd (9.1, 7.7)
2.23, m 2.07,
dd (11.8, 11.2)
0.88, s
2.23, m 2.07,
dd (11.8, 11.2)
0.88, s
2.29, m 2.07, m 2.18, m 2.04, m
2.16, m
1.98, m 1.55, m 2.07, d (13.1) 1.29, m 2.11, d (13.1) 1.35, m
12
1.01, s
0.83, s
0.88, s
0.86, s
0.86, s
0.94, s
0.84, s
0.89, s
13
14
1.05, s
1.76, s
1.03, s
1.76, s
1.05, s
1.76, s
0.96, s
1.72, s
0.96, s
1.70, s
1.04, s
1.88, s
1.04, s
1.88, s
0.99, s
1.31, s
0.95, s
1.45, s
1.01, s
1.47, s
15
2′
3′
5′/9′
6′/8′
5.10, s 4.99, s
6.26, d (16.2)
7.62, d (16.2)
7.42, d (8.4)
6.83, d (8.4)
5.05, s 4.94, s
5.10, s 4.99, s
5.74, d (12.7)
6.82, d (12.7)
7.64, d (8.6)
6.81, d (8.6)
5.17, s 5.14, s
5.74, d (12.7)
6.82, d (12.7)
7.64, d (8.6)
6.81, d (8.6)
1.49, s
1.20, s
1.20, s
1.64, s
1.63, s
1.68, s
5.79, d (12.7)
6.84, d (12.7)
7.59, d (8.6)
6.76, d (8.6)
6.26, d (16.2)
7.62, d (16.2)
7.42, d (8.4)
6.83, d (8.4)
5.74, d (12.7)
6.82, d (12.7)
7.64, d (8.6)
6.81, d (8.6)
6.26, d (16.2)
7.62, d (16.2)
7.42, d (8.4)
6.83, d (8.4)
5.74, d (12.7)
6.82, d (12.7)
7.64, d (8.6)
6.81, d (8.6)
6.28, d (16.2)
7.64, d (16.2)
7.46, d (8.4)
6.86, d (8.4)

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named
(1E,5E,8R)-8-O-[(Z)-p-coumaroyl]humula-1(10),4(5)-
dien-8-ol.
Compounds 7 and 8 had the same molecular formula of
C24H32O4, corresponding to nine degrees of unsaturation.
These two compounds were identified by 1H and 13C NMR
data (Tables 1 and 2) as a pair of C-2′ configurational isomers.
Their NMR spectra showed two oxygenated carbon resonances
[7: δC 60.8 (s), 62.1, (d); 8: δC 60.7 (s), 62.0 (d)] instead of the
olefinic C-4 (δC 134.6) and C-5 (δC 124.3) resonances of 511
which, in combination with the proton signal of one oxy-
genated C–H (7: δH 2.60; 8: δH 2.59), revealed the presence of
an epoxy group located at C-4 and C-5. The HMBC correlation
between H-5 and C-11 further confirmed this deduction.
Accordingly, compounds 7 and 8 were identified as the
respective epoxidation products of 5 and 6.
The relative configuration of 7 was tentatively established
on the basis of ROESY experiments. The crosspeak of H-8/H-12
indicated that these protons were on the same face, while H-5/
H-13 were on the other face on the basis of their crosspeak.
The configuration of C-8 would be R due to biosynthetic
reasoning and, consequently, C-4 and C-5 were both assigned
the R configuration. Thus, compound 7 was identified as
(1E,4R,5R,8R)-8-O-[(E)-p-coumaroyl]-4,5-epoxyhumula-1(10)-en-
Scheme 1 Chemical transformations of the isolated compounds.
from H-8 to C-10 and 11. Thus, compound 10 was deduced to
have the same planar structure as 9. In the ROESY spectrum,
the observed correlations of H-8/H-12(14) and H-5/H-1(13)
clearly suggested that H-1 and H-8 were not on the same
face; thus, the absolute configuration of both C-1 and C-10 was
proposed as R on the basis of the R-configured C-8 arising
from the biogenesis. Therefore, 10 was established
as (1R,4E,8R,10R)-8-O-[(Z)-p-coumaroyl]-1,10-epoxyhumula-4(5)-
en-8-ol. Consequently, 11 was identified as (1R,4E,8R,10R)-8-O-
[(E)-p-coumaroyl]-1,10-epoxyhumula-4(5)-en-8-ol.
To further confirm the structures of the isolated com-
pounds, especially the absolute configurations, chemical con-
versions were designed. As is apparent from Scheme 1, all of
the sesquiterpenoids containing an E-2′-p-coumaroyl group
could be derived from 5 (and those containing a Z-2′-p-coumar-
oyl group from 6). Epoxidation of compound 5 with m-CPBA in
CH2Cl2 gave a mixture of compounds 7, 9, and 11 in the ratio
87 : 11 : 2, as detected by HPLC. An ene reaction15,16 was per-
formed to introduce a hydroperoxide group into 5, yielding 3.
When oxygen was bubbled into a solution of 5 and methylene
blue in acetonitrile, the reaction occurred so fast that
both double bonds in 5 were peroxidated. With the use of air
as the oxygen source, however, compound 3 was successfully
obtained as the only oxidative product. Compound 3 was
further reduced, affording the relevant hydroxy derivative 1,
the absolute configuration of which was fully confirmed by
chemical transformation and single-crystal X-ray diffraction
analysis of 1a.
8-ol, and compound
8
was then deduced as being
(1E,4R,5R,8R)-8-O-[(Z)-p-coumaroyl]-4,5-epoxyhumula-1(10)-en-
8-ol.
The molecular formula of 9 was determined to be C24H32O4
1
from the ESI-HRMS data. A detailed analysis of its H and 13C
NMR data (Tables 1 and 2) revealed a humulane-type sesqui-
terpenoid skeleton with an E-p-coumaroyl substituent. The
signals at δH 3.12 (d, J = 10.9 Hz), δC 62.4 (d), and δC 59.2 (s)
suggested the presence of an epoxy group, while those at δH
5.46, δC 123.7 (d), and δC 134.2 (s) indicated a trisubstituted
double bond. These data suggested that 9 was also an epoxi-
dation product, but different from epoxides 7 and 8. HMBC
correlations from H-14 (δH 1.31) to C-9 (δC 45.3) and from H-9
(δH 1.55, 1.98) to C-1 (δC 62.4), C-10 (δC 59.2), and C-8 (δC 72.0)
revealed that the epoxy group was situated at C-1 and C-10,
while the HMBC correlations from H-6 (δH 2.08, 2.15) to C-4
(δC 134.2), C-5 (δC 123.7), and C-11 (δC 33.6) proved the location
of the double bond at C-4 and C-5. The ROESY correlations of
H-8/H-1 and H-8/H-5 suggested that these three protons were
in the same orientation. Thus, the absolute configuration was
assigned as 1S,8R,10S; finally, compound 9 was named as
(1S,4E,8R,10S)-8-O-[(E)-p-coumaroyl]-1,10-epoxyhumula-4(5)-en-
8-ol.
Compounds 10 and 11 had the same molecular formula of
C24H32O4, as determined from the respective ESI-HRMS data.
The 1H and 13C NMR data (Tables 1 and 2) showed that 10 and
11 were a pair of C-2′ configurational isomers. Similar to 9, the
signals for 10 at δH 2.70 (d, J = 10.9 Hz), δC 63.2 (d), and δC
59.9 (s) indicated the presence of an epoxy group, while those
Conclusions
at δH 5.11 (d, J = 10.2 Hz), δC 123.3 (d), and δC 134.0 (s) In summary, 11 humulane-type sesquiterpenoids, including
suggested a double bond in the sesquiterpenoid unit. The nine new derivatives, were identified from extracts of P. cavaler-
double bond was finally placed at C-4 and C-5 on the basis of iei. For the first time, the absolute configuration of this
the key HMBC correlations from H-6 to C-4, 5, and 11 and uncommon type of sesquiterpene was determined by X-ray
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crystallography, as well as by chemical conversions. The ene subjected to column chromatography (CC) over silica gel;
reaction with air as the oxidizing agent was discovered during elution with PE–acetone (10 : 1 to 0 : 1) in a stepwise manner
the chemical conversions, which gives an indication as to how gave four fractions (1–4). Fraction 2 was subjected to CC over
the allylic hydroperoxide group and related alcohols could be MCI gel (EtOH–H2O 70% to 95%) to yield five fractions (2a–
produced in plants. This reaction is more likely to occur in 2e). Fraction 2a was subjected to CC over Sephadex LH-20
nature than the conversion using oxygen, and might account (CHCl3–MeOH 1 : 1) to yield two subfractions (2a1 and 2a2).
for the prevalence of these functionalities not only in the Fraction 2a1 was purified further by preparative HPLC
isolated sesquiterpenoids but also other natural products. (CH3CN–H2O 40% to 70%) and yielded 7 (19 mg), 8 (9 mg), 9
Our findings also suggest that the Urticaceae family might (7 mg), 10 (5 mg), and 11 (1 mg). Fraction 2d was subjected to
be a rich source of natural sesquiterpenoids, and thus worthy CC over Sephadex LH-20 (CHCl3–MeOH 1 : 1) to give two sub-
of in-depth investigations.
fractions (2d1 and 2d2). Fraction 2d2 was then subjected to CC
over silica gel to afford 5 (2 g) and 6 (300 mg). Fraction 3 was
subjected to CC over MCI gel (EtOH–H2O 70% to 95%) to yield
three fractions (3a–3c). Fraction 3a was subjected to CC over
Sephadex LH-20 (CHCl3–MeOH 1 : 1) to yield two subfractions
(3a1 and 3a2). Compounds 3 (95 mg) and 4 (50 mg) were sepa-
rated from fraction 3a2 by preparative HPLC. Similarly, com-
pound 11 (3 mg) was purified from fraction 3b2 (obtained
from fraction 3b) by preparative HPLC. Fraction 4 was sub-
jected to CC over silica gel (CHCl3–MeOH 1 : 0, 100 : 1, 50 : 1,
0 : 1) to obtain four fractions (4a–4d). Fraction 4b was purified
by preparative HPLC, yielding 1 (210 mg) and 2 (90 mg).
Experimental section
General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polari-
meter. Melting points were determined on an XT-4 binocular
microscope (Beijing Tech Instrument Co., China) and are not
corrected. IR spectra were recorded on a Nicolet Magna FT-IR
750 spectrophotometer using KBr disks. NMR spectra were
recorded on Bruker AM-400 and INVOA-600 NMR spec-
trometers. The chemical shift (δ) values are given in ppm with
TMS as the internal standard, and coupling constants (J) are in
Hz. ESI-MS and ESI-HRMS data were recorded on Waters 2695-
3100 LC-MS and Waters Xevo TOF mass spectrometers. Silica
gel (Qingdao Marine Chemical Industrials) was used for flash
chromatography. MCI gel CHP20P (75–150 μm, Mitsubishi
Chemical Industries, Japan) and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden) were used for column chromato-
graphy (CC). TLC was carried out on precoated silica gel GF254
plates (Yantai Chemical Industrials) and the TLC spots were
viewed at 254 nm and visualized with 5% H2SO4 in EtOH con-
taining 10 mg mL−1 vanillin. Analytical HPLC was performed
on a Waters 2690 instrument with a 996 PAD (photodiode
array detector) and coupled with an Alltech ELSD 2000 detec-
tor. X-ray crystallographic analysis was carried out on a Bruker
APEX-II CCD diffractometer with graphite-monochromated
Cu Kα radiation (λ = 1.54718 Å).
Compound characteristics
(1E,5R,8R)-8-O-[(E)-p-coumaroyl]-5-hydroxyhumula-1(10),4-
(15)-dien-8-ol (1). [α]D25 +83.0 (c 0.1 in MeOH); IR (KBr)
νmax/cm−1 3415, 2952, 2857, 1704, 1671, 1631, 1604, 1585,
1515, 1440, 1369, 1328, 1280, 1189, 1168, 1141, 1099, 983, 968,
906, 833, 757, 520; 1H and 13C NMR: see Tables 1 and 2;
ESI-MS m/z 383 [M − H]−; ESI-HRMS m/z 383.2238 [M − H]−
(calcd for C24H31O4 383.2222).
(1E,5R,8R)-8-O-[(Z)-p-coumaroyl]-5-hydroxyhumula-1(10),4-
(15)-dien-8-ol (2). White powder; [α]2D5 +76.0 (c 0.1 in MeOH);
IR (KBr) νmax/cm−1 3417, 2929, 2859, 1683, 1631, 1604, 1587,
1513, 1446, 1367, 1326, 1166, 1101, 1049, 970, 908, 833,
516; 1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z 383
[M − H]−; ESI-HRMS m/z 383.2221 [M − H]− (calcd for
C24H31O4 383.2222).
(1E,5R,8R)-8-O-[(E)-p-coumaroyl]-5-hydroperoxyhumula-1(10),4-
(15)-dien-8-ol (3). 1H NMR (CDCl3) δ 7.60 (1 H, d, J = 15.9 Hz),
7.41 (2 H, d, J = 8.6 Hz), 6.84 (2 H, d, J = 8.6 Hz), 6.27 (1 H, d,
J = 15.9 Hz), 5.30 (1 H, d, J = 6.0 Hz), 5.18 (1 H, s), 5.15 (1 H, s),
4.78 (1 H, d, J = 8.8 Hz), 4.25 (1 H, d, J = 6.6 Hz), 2.50 (1 H, m),
2.10–2.34 (3 H, m), 2.06 (1 H, dd, J = 11.4, 11.4 Hz), 1.89 (1 H, d,
J = 15.4 Hz), 1.73 (3 H, s), 1.60 (1 H, dd, J = 15.6, 5.1 Hz), 1.43
(1 H, d, J = 8.6 Hz), 0.98 (3 H, s), 0.82 (3 H, s); ESI-MS m/z 399
[M − H]−.
Plant material
The whole plants of Pilea cavaleriei subsp. crenata were col-
lected in Guangxi Province, P. R. China in 2009, and identified
by Professor Jin-Gui Shen from the Shanghai Institute of
Materia Medica. A sample (20090903) was deposited at the
Herbarium of the Shanghai Institute of Materia Medica,
Chinese Academy of Sciences.
(1E,5R,8R)-8-O-[(Z)-p-coumaroyl]-5-hydroperoxyhumula-1(10),4-
Extraction and isolation
(15)-dien-8-ol (4). [α]D25 +34.2 (c 0.12 in MeOH); IR (KBr)
The dried and powered plants of P. cavaleriei (10 kg) were νmax/cm−1 3407, 2954, 2927, 2865, 1681, 1629, 1604, 1587,
extracted with 95% EtOH (3 × 40 L, 7 days each) at room temp- 1513, 1469, 1446, 1369, 1328, 1276, 1168, 1101, 979, 914, 833,
erature. After evaporation of the solvent, the obtained residue 518; 1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z 399
was dissolved in water (10 L) and extracted with petroleum [M − H]−; ESI-HRMS m/z 399.2162 [M − H]− (calcd for
ether (PE) (10 L × 3) and ethyl acetate (10 L × 3), successively. C24H31O5 399.2171).
The PE fraction was concentrated and extracted with 80%
MeOH in water (5 L × 3). The concentrated MeOH extract was (5). Powder; H NMR (CDCl3) δ 7.60 (1 H, d, J = 15.9 Hz), 7.41
(1E,5E,8R)-8-O-[(E)-p-coumaroyl]humula-1(10),4(5)-dien-8-ol
1
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(2 H, d, J = 8.6 Hz), 6.84 (2 H, d, J = 8.6 Hz), 6.27 (1 H, d, J = using the full-matrix least-squares method on F2, GOF = 1.093.
15.9 Hz), 4.86 (1 H, m), 4.85 (1 H, m), 4.63 (1 H, m), 2.11–2.25 The X-ray diffraction data have been deposited with the
(2 H, m), 2.06–2.14 (2 H, m), 1.73–1.84 (2 H, m), 1.63 (3 H, s), Cambridge Crystallographic Data Centre (CCDC 925951).
1.39 (2 H, dd, J = 15.5, 7.9 Hz), 1.46 (3 H, s), 1.07 (3 H, s),
Chemical transformations
0.90 (3 H, s); ESI-MS m/z 367 [M − H]−.
(1E,5E,8R)-8-O-[(Z)-p-coumaroyl]humula-1(10),4(5)-dien-8-ol
Compound 1a. To a solution of natural 1 (40 mg, 0.1 mmol)
(6). [α]2D5 +0 (c 0.1 in CHCl3); IR (KBr) νmax/cm−1 3376, 2954, dissolved in dried THF was added LiAlH4 (5 mg) at 0 °C, and
2921, 2856, 1704, 1675, 1604, 1513, 1440, 1367, 1278, 1166, the mixture was stirred at 0 °C for 30 min. Then, MeOH was
981, 831, 518; 1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z added to quench the reaction, and the mixture was diluted
367 [M − H]−; ESI-HRMS m/z 367.2266 [M − H]− (calcd for with water, then extracted with CH2Cl2. The combined organic
C24H31O3 367.2273).
layer was dried (anhydrous Na2SO4) and concentrated under
(1E,4R,5R,8R)-8-O-[(E)-p-coumaroyl]-4,5-epoxyhumula-1(10)- reduced pressure to give 1a (22 mg, 85%); colorless crystals;
en-8-ol (7). [α]2D5 −36.5 (c 0.2 in MeOH); IR (KBr) νmax/cm−1 mp 80–85 °C (MeOH); [α]2D5 +28.0 (c 0.1 in MeOH); IR (KBr)
3386, 3183, 1956, 2927, 2863, 1702, 1631, 1606, 1587, 1513, νmax/cm−1 3396, 2929, 2852, 1648, 1467, 1448, 1367, 1284,
1
1452, 1369, 1326, 1284, 1201, 1164, 1091, 985, 831, 518; 1161, 1068, 999, 900, 684, 530; H and 13C NMR: see Tables 1
1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z 383 and 2; ESI-MS m/z 221 [M + H − H2O]−; ESI-HRMS m/z
[M − H]−; ESI-HRMS m/z 383.2196 [M − H]− (calcd for 221.1916 [M + H − H2O]− (calcd for C15H25O 221.1905).
C24H31O4 383.2222).
Reduction of 3. To a solution of natural 3 (40 mg,
(1E,4R,5R,8R)-8-O-[(Z)-p-coumaroyl]-4,5-epoxyhumula-1(10)- 0.1 mmol) dissolved in MeOH (10 mL) was added NaBH4
en-8-ol (8). [α]D25 −30.5 (c 0.22 in MeOH); IR (KBr) νmax/cm−1 (5 mg, 0.13 mmol) at room temperature, and the mixture was
3394, 2956, 2867, 1706, 1683, 1631, 1604, 1587, 1513, 1488, stirred at 0 °C for 30 min. Then, saturated NaHCO3 (10 mL)
1
1369, 1326, 1278, 1166, 985, 943, 833, 516; H and 13C NMR: and CH2Cl2 (20 mL) were added, and the mixture was stirred
see Tables 1 and 2; ESI-MS m/z 383 [M − H]−; ESI-HRMS m/z for an additional 10 min. The organic layer was separated and
383.2231 [M − H]− (calcd for C24H31O4 383.2222).
the aqueous layer was extracted with CH2Cl2. All organic layers
(1S,4E,8R,10S)-8-O-[(E)-p-coumaroyl]-1,10-epoxyhumula-4(5)- were combined and dried (anhydrous Na2SO4), then finally
en-8-ol (9). White powder; [α]2D5 +16.5 (c 0.1 in MeOH); IR concentrated under reduced pressure to give 1 (35 mg 92%).
(KBr) νmax/cm−1 3382, 2958, 2927, 2871, 1702, 1631, 1604,
Compound 5a. To a solution of 5 (100 mg) dissolved in
1587, 1513, 1442, 1367, 1330, 1278, 1201, 1166, 1099, 983, 833, dried THF was added LiAlH4 (15 mg) at 0 °C, and the mixture
524; 1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z 383 was stirred at 0 °C for 30 min. Then, MeOH was added to
[M − H]−; ESI-HRMS m/z 383.2212 [M − H]− (calcd for quench the reaction, and the mixture was diluted with water,
C24H31O4 383.2222).
then extracted with CH2Cl2. The combined organic layer was
(1R,4E,8R,10R)-8-O-[(Z)-p-coumaroyl]-1,10-epoxyhumula-4(5)- dried (anhydrous Na2SO4) and then concentrated under
en-8-ol (10). [α]2D5 +18.5 (c 0.1 in MeOH); IR (KBr) νmax/cm−1 reduced pressure to give 5a (46 mg 75%); colorless oil; 1H
3382, 2958, 2927, 2871, 1702, 1631, 1604, 1587, 1513, NMR (CDCl3) δ 4.86 (1 H, m), 4.85 (1 H, m), 3.40 (1 H, m),
1442, 1367, 1330, 1278, 1201, 1166, 1099, 983, 833, 524; 2.11–2.25 (2 H, m), 2.06–2.14 (2 H, m), 2.05–2.13 (2 H, m),
1H and 13C NMR: see Tables 1 and 2; ESI-MS m/z 383 1.91–1.99 (2 H, m), 1.01–1.06 (2 H, m), 1.63 (3 H, s), 1.46 (3 H,
[M − H]−; ESI-HRMS m/z 383.2226 [M − H]− (calcd for s), 1.07 (3 H, s), 0.90 (3 H, s).
C24H31O4 383.2222).
Preparation of the (R)- and (S)-MTPA ester derivatives of
(1R,4E,8R,10R)-8-O-[(E)-p-coumaroyl]-1,10-epoxyhumula-4(5)- compound 5a. Compound 5a (2.0 mg) was added to two sepa-
en-8-ol (11). [α]D25 +20.0 (c 0.1 in CHCl3); IR (KBr) νmax/cm−1 rate NMR tubes, and dried under reduced pressure overnight
3349, 3006, 2923, 2854, 1708, 1606, 1587, 1515, 1465, 1278, at room temperature. Pyridine-d5 (0.5 mL) was transferred to
1
1166, 985, 833, 725, 632; H and 13C NMR: see Tables 1 and 2; each tube, to give a clear solution. (S)-(+)-α-Methoxy-α-(trifluoro-
ESI-MS m/z 383 [M − H]−; ESI-HRMS m/z 383.2219 [M − H]− methyl) phenylacetyl (MTPA) chloride (10 μL) was injected
(calcd for C24H31O4 383.2222).
into one NMR tube, and (R)-MTPA chloride (10 μL) into the
other, under N2 gas. The NMR tubes, with the reagents, were
sealed and stored overnight in a desiccator until the reactions
X-ray crystallography of 1a
1
1
1a: C16H30O3, M = 270, orthorhombic, colorless, crystal size were completed (monitored by H NMR spectroscopy). The H
0.25 × 0.22 × 0.18 mm, space group P21, a = 33.5085(13) Å, b = NMR chemical shifts of the (R)-MTPA ester and the (S)-MTPA
6.1892(2) Å, c = 8.0369(3) Å, V = 1666.78(10) Å3, Z = 4, Dcalcd
=
ester of 5a were recorded directly after each reaction.
1.078 mg m−3, F(000) = 600, reflections collected 2728, 2643 Ambiguous and overlapping signals were not used for the
unique (Rint = 0.0373), final R indices for I > 2σ(I), R1 = 0.0516, ΔδS–R calculation.
wR2 = 0.1472, R indices for all data R1 = 0.0526, wR2 = 0.1478,
Epoxidation of compound 5 with m-CPBA. A solution of
completeness to θ (64.98) 96.7%, maximum transmission compound 5 (183 mg, 0.50 mmol) and m-CPBA (90 mg,
0.9045, minimum transmission 0.8709, absolute structure para- 0.55 mmol) in CH2Cl2 (10 mL) was stirred under ice-bath
meter 0.1(4). The structure was solved by direct methods cooling for 1 h. The reaction mixture was washed successively
using the program SHELXS-97 (Sheldrick, 2008) and refined with Na2SO3 and NaHCO3, dried (anhydrous Na2SO4), and
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concentrated under reduced pressure to give crude residue
(200 mg, >100%). The residue was then subjected to prepara-
tive HPLC (CH3CN–H2O 40% to 70%) to afford compounds 7
(90 mg), 9 (10 mg), and 11 (1.5 mg).
Ene reaction of compound 5. A solution of compound 5
(18 mg, 0.05 mmol) and methylene blue (0.5 mg) in CH3CN
(5 mL) was stirred at room temperature for 6 h. The reaction
mixture was washed with saturated NaCl solution, dried
(anhydrous Na2SO4), and concentrated under reduced pressure
to give compound 3 (20 mg 98%).
4 Q.-F. Chen, Z.-P. Liu and F.-P. Wang, Mini-Rev. Med. Chem.,
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We thank the National Science
&
Technology Major
Project “Key New Drug Creation and Manufacturing Program”
(No. 2011ZX09307-002-03) for financial support. Our
thanks are also given to the National Natural Science Funds
for Distinguished Young Scholar (No. 30925043) and for grants
from the Ministry of Science and Technology (2007DFC31030,
2010DFA30980) and the Shanghai Commission of
Science and Technology (10DZ1972700, 11DZ1970700,
12JC1410300).
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