Machilusides A and B: Structurally unprecedented homocucurbitane glycosides from the stem bark of machilus yaoshansis

Article abstract of DOI:10.1021/ol200854k

Two structurally novel homocucurbitane triterpenoid glycosides, machilusides A (1) and B (2), possessing an unprecedented C₃₆ skeleton with a d-fructose moiety incorporated into a cucurbitane nucleus forming unique cage-like tricyclic ring moieties, were isolated from the stem bark of Machilus yaoshansis. Their structures were determined by spectroscopic methods. Both compounds exhibited nonselective cytotoxic activities against several human cancer cell lines. The biosynthetic pathway of 1 and 2 was postulated.

Full text of DOI:10.1021/ol200854k

ORGANIC
LETTERS
2
011
Vol. 13, No. 11
856–2859
Machilusides A and B: Structurally
Unprecedented Homocucurbitane
Glycosides from the Stem Bark
of Machilus yaoshansis
2
†
Mingtao Liu, Maoluo Gan, Sheng Lin, Yanling Zhang, Jiachen Zi, Weixia Song,
†,‡
†
†
†
†
†
†
†
Xiaona Fan, Ying Liu, Yongchun Yang, and Jiangong Shi
†
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, and
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal
Medicine, Ministry of Education, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic
of China, and Institute of Medicinal Biotechnology, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
shijg@imm.ac.cn
Received April 1, 2011
ABSTRACT
Two structurally novel homocucurbitane triterpenoid glycosides, machilusides A (1) and B (2), possessing an unprecedented C36 skeleton with a
D-fructose moiety incorporated into a cucurbitane nucleus forming unique cage-like tricyclic ring moieties, were isolated from the stem bark of
Machilus yaoshansis. Their structures were determined by spectroscopic methods. Both compounds exhibited nonselective cytotoxic activities
against several human cancer cell lines. The biosynthetic pathway of 1 and 2 was postulated.
Species of the genus Machilus (Lauraceae) are sources of
secondary metabolites with interesting chemical structures
chemical and biological diversity of several traditional
3
Chinese medicines, we investigated the stem barks of
Machilus yaoshansis S. Lee et F. N. Wei that is widely
distributed in the south of China. In our previous study,
two novel glycosidic triterpene alkaloids with cytotoxic
1
and significant bioactivities. Several plants of this genus
have long been used for the treatment of various diseases
including edema, abdominal distension, pain, and inflam-
2
mation in China. As part of a program to assess the
and TNF-R inhibitory activities, machilaminosides A and
4
B, were isolated from the H O soluble portion of the
2
†
Institute of Materia Medica.
‡
Institute of Medicinal Biotechnology.
1) For some examples, see: (a) Giang, P. M.; Son, P. T.; Matsunami,
K.; Otsuka, H. Chem. Pharm. Bull. 2006, 54, 308. (b) Cheng, M.-J.; Tsai,
I.-L.; Lee, S.-J.; Jayaprakasam, B.; Chen, I.-S. Phytochemistry 2005, 66,
180. (c) Park, E. Y.; Shin, S. M.; Ma, C. J.; Kim, Y. C.; Kim, S. G.
Planta Med. 2005, 71, 393.
2) Jiangsu New Medical College. In Dictionary of Traditional
(
(3) (a) Mo, S. Y.; Wang, S. J.; Zhou, G. X.; Yang, Y. C.; Li, Y.; Chen,
X. G.; Shi, J. G. J. Nat. Prod. 2004, 67, 823. (b) Wang, Y.; Wang, S. J.;
Mo, S. Y.; Li, S.; Yang, Y. C.; Shi, J. G. Org. Lett. 2005, 7, 4733. (c) Gan,
M. L.; Zhang, Y. L.; Lin, S.; Liu, M. T.; Song, W. X.; Zi, J. C.; Yang,
Y. C.; Fan, X. N.; Shi, J. G.; Hu, J. F.; Sun, J. D.; Chen, N. H. J. Nat.
Prod. 2008, 71, 647.
1
(
Chinese Medicine; Shanghai Science and Technology Publishing House:
Shanghai, 1977; pp 114, 1009, and 1423.
(4) Liu, M. T.; Lin, S.; Wang, Y. H.; He, W. Y.; Li, S.; Wang, S. J.;
Yang, Y. C.; Shi, J. G. Org. Lett. 2007, 9, 129.
1
0.1021/ol200854k r 2011 American Chemical Society
Published on Web 05/05/2011

EtOH extract of the stem bark of Machilus yaoshansis.
Continuing examination of the H O soluble portion of this
2
a
Table 1. NMR Data for Machilusides A (1) and B (2)
material led to the identification of two novel homocucur-
bitane glycosides, machilusides A (1) and B (2) (Figure 1).
They represent the first examples of C36 homocucurbitane
triterpenoid nuclei with unprecedented skeletons featuring
D-fructose moieties fused into unique cage-like tricyclic
ring systems in their side chain. Herein, we present the
isolation, structural elucidation, postulated biogenetic for-
b
1
2
no.
δ
H
δ
C
δ
H
δ
C
1
5.76 brs
120.6 d 5.76 brs
145.0 s
196.1 s
48.7 s
136.3 s
120.8 d
145.0 s
196.1 s
48.7 s
136.3 s
120.0 d
23.3 t
2
3
4
5
6
7
7
8
9
1
5
mation, and biological activity of 1 and 2.
5.70 brs
1.97 brd (19.8)
2.27 dd (19.8, 9.0)
1.92 brd (9.0)
120.0 d 5.73 brs
R
β
23.3 t 1.99 brd (17.0)
2.30 dd (17.0, 9.0)
41.1 d 1.93 brd (9.0)
48.0 s
34.3 d 3.66 brs
214.6 s
41.0 d
48.0 s
34.3 d
214.5 s
49.5 t
0
3.61 brs
11
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
2R 3.24 d (15.0)
2β 2.54 d (15.0)
3
4
5R 1.37 d (12.0)
5β 1.71 dd (12.0, 10.2)
49.1 t 3.31 d (15.0)
2.48 d (15.0)
51.3 s
47.2 s
43.8 t 1.39 d (12.0)
51.6 s
47.0 s
43.8 t
1.72 dd (12.0, 10.0)
6
7
8
9
0
1
2
3
4
5
6
7
8
9
4.45 m
2.36 d (6.0)
0.83 s
70.3 d 4.46 m
56.3 d 2.62 d (6.0)
19.4 q 0.86 s
19.6 q 0.86 s
74.8 s
24.4 q 1.27 s
122.7 s
62.7 d 3.17 d (2.5)
85.2 d 3.87 d (2.5)
84.9 s
28.0 q 1.10 s
23.8 q 1.31 s
20.4 q 1.19 s
27.1 q 1.19 s
17.2 q 1.33 s
74.3 t 3.57 d (12.0)
3.48 d (12.0)
70.0 d
54.5 d
19.2 q
19.6 q
75.2 s
24.8 q
114.1 s
46.6 d
84.8 d
83.4 s
26.4 q
22.6 q
20.4 q
27.2 q
17.2 q
66.2 t
0.84 s
1.25 s
3.45 d (8.4)
4.03 d (8.4)
1.22 s
1.25 s
1.16 s
1.16 s
1.29 s
Figure 1. Structures of machilusides A (1) and B (2).
0
0
Machiluside A (1) was obtained as a white amorphous
2
1 a 4.19 d (10.2)
0
0
1
2
3
4
5
6
6
1
b
3.68 d (10.2)
powder, [R] ꢀ38.6 (c 0.05, MeOH). IR absorptions at
D
0
0
0
0
0
0
ꢀ
1
88.0 s
83.2 d 3. 97 brs
76.5 s
74.3 d
77.0 d
3
410, 1687, and 1640 cm
implied the existence of
3.79 d (2.4)
3.41 ddd (8.0, 6.0, 2.4) 72.0 d 3.72 (8.0)
hydroxy, carbonyl, and double bond functional groups,
respectively. The (þ)-ESIMS of 1 exhibited quasi-
molecular ion peak at m/z 861. The molecular formula
of C H O was determined by HRESIMS at m/z
3.42 ddd (8.0, 6.0, 3.0) 69.0 d 3.79 ddd (8.0,6.0,3.0) 69.5 d
a
b
3.61 dd (12.0, 3.0)
3.40 dd (12.0, 6.0)
4.55 d (7.8)
63.2 t 3.71 dd (11.0, 3.0)
3.41 dd (11.0, 6.0)
98.6 d 4.55 d (8.0)
63.2 t
4
2
62 17
00
98.6 d
72.7 d
76.8 d
68.9 d
þ
8
The H NMR spectrum of 1 (Table 1 and Supporting
61.3861 [MþNa] with 12 degrees of unsaturation.
200
3.11 ddd (8.4, 7.8, 4.8) 72.7 d 3.13 dd (8.0, 8.5)
3.18 dd (8.4, 8.4)
3.26 dd (8.4, 8.4)
1
00
3
76.8 d 3.20 dd (8.0, 8.5)
68.9 d 3.28 dd (8.0, 8.5)
0
0
4
5
6
Information Table S1) in DMSO-d showed two olefinic
6
00
00
3.07 ddd (8.4, 4.0, 2.0) 76.9 d 3.08 ddd (8.5, 4.0, 2.0) 76.9 d
3.70 ddd (12.0, 5.4, 2.0) 60.0 t 3.72 dd (12.0, 2.0) 60.0 t
methines and ten exchangeable hydroxy signals in the
lower field region. In addition, it displayed eight methyl
singlets, three methylenes, and four methines, together
with several overlapping multiplets attributed to oxy-
methines and oxymethylenes, in addition to characteristic
signals due to a β-D-glucopyranosyl moiety (Table 1). The
presence of the β-D-glucopyranosyl moiety was supported
a
0
0
6 b 3.60 ddd (12.0,5.4, 4.0)
3.61 dd (12.0, 4.0)
a
1
at 600 MHz ( H) and 150 MHz
Data were recorded in DMSO-d
6
1
3
1
C) for 1 and at 500 MHz ( H) and 125 MHz ( C) for 2, respectively.
13
(
Coupling constants (J) in Hz were given in parentheses. The multiplicity
1
of C resonances was determined by the DEPT experiment. The assign-
3
1
1
b
ments were based on Hꢀ H COSY, HSQC, and HMBC. Data for
0
hydroxy protons of1: δ
H
4.87brs (OH-16), 4.53s (OH-20), 5.35 s (OH-2 ),
1
3
0
0
0
by the C NMR data (Table 1) and further confirmed by
enzymatic hydrolysis of 1 using the same methods as
4.46 d (6.0 Hz, OH-4 ), 4.77 brs (OH-5 ), 4.35 brs (OH-6 ), 5.17 d (4.8 Hz,
0
0
00
00
00
OH-2 ), 5.00 brs (OH-3 ), 4.87 brs (OH-4 ), 4.33 t (5.4 Hz, OH-6 ). For
data for 1 in MeOH-d , see Supporting Information Table S1.
3
c
13
4
described in the literature. The C NMR spectrum of
showed 42 carbon signals, and the DEPT experiment
differentiated them to be 8 ꢁ CH , 6 ꢁ CH , 16 ꢁ CH,
1
shift values, the 12 quaternary carbons were assigned to
2
be two carbonyls, two sp carbons (one oxygen bearing,
δ > 145 ppm), and eight sp carbons (four oxygen-
3
2
and 12 ꢁ C. (Table 1). On the basis of the chemical
3
bearing, δ > 74 ppm). All the above spectroscopic data
suggested that 1 is a highly oxygenated and unusual
cucurbitane glycoside.
(
5) For plant material, experimental procedures, and physicalꢀ
1 and 2, see: Supporting
chemical properties for compounds
Information.
4
,6
Org. Lett., Vol. 13, No. 11, 2011
2857

The structure of 1 was finally established by 2D NMR
experiments. The proton and protonated carbon signals in
the NMR spectra of 1 were unambiguously assigned by the
1
1
HSQC experiment. Extensive analysis of the Hꢀ H
COSY and HMBC spectroscopic data of 1 respectively
in MeOH-d and DMSO-d allowed the planar structure of
4
6
the nucleus with the tetracyclic ring system to be defined as
shown (Figure 2), which revealed unequivocally a cucur-
bita-1,5-diene-3,11-dione nucleus for 1 identical to that of
machilaminosides A and B isolated from the same material
4
before. In addition, HMBC correlations of C-16, C-20,
0
0
0
0
C-2 , C-4 , C-5 , and C-6 with respective hydroxy protons,
together with the chemical shift values of these carbons,
located a hydroxy group at each of the six carbons, res-
0
0
pectively. The connection of a hexose moiety (C-1 ꢀC-6 )
0
to C-23 via the quaternary carbon C-2 was clearly demon-
0
0
strated by the HMBC correlations from H-1 a and H-1 b
0
0
0
0
0
0
0
to C-2 and C-3 ; from H-3 to C-1 , C-2 , C-4 , C-5 , and
0
0
0
C-23; from OH-2 to C-1 , C-2 , and C-23; and from H-23
0
0
0
to C-1 and C-2 . Meanwhile, the key correlations of H-3
0
0
0
1
1
and C-24, H-24 and C-3 , and H-1 a/H-1 b and C-22, along
0
Figure 2. (a) Hꢀ H COSY (thick lines) and main HMBC (red
arrows) correlations of 1. (b) Key NOESY correlations (blue
arrows) for the side chain moiety of 1.
0
0
with the chemical shifts of C-1 (δ 74.3), C-2 (δ 88.0), C-3
δ 83.2), and C-24 (δ 85.2), unequivocally established the
(
0
0
linkage of C-3 and C-24, and C-1 and C-22 via an oxygen
atom to form two fused five-membered rings in the side
chain of the nucleus. Furthermore, the linkage of C-22
and C-25 via an oxygen atom to form another five-
membered ring was suggested by the chemical shifts of
C-22 (δ 122.7) and C-25 (δ 84.9) and the accurate assign-
ment of the ten exchangeable hydroxy signals, together
withthe absence ofOH-25 and OH-22. Thiswas confirmed
by the molecular formula of 1. Moreover, an HMBC
correlation from H-1 to C-2 indicated that the β-D-
glucopyranosyl moiety was located at C-2 of the nucleus.
Accordingly, the planar structure of 1 was determined as
depicted in Figure 2 a.
In addition, the presence of NOE correlations of OH-20/
H-16 and H -18 and H -21/H-12β and H-17 and the
absence of an NOE correlation of H-16/H -21 indicated
3
3
3
thatfreerotation ofthe single bondbetween C-17and C-20
was limited and possessed a major conformation in the
solution state as shown in Figure 2 b, supporting the 20R
configurationin1. Thisisalsoconsistent withthatof all the
6
,7
reported cucurbitane derivatives.
0
0
0
0
The NOE correlations of H-3 /H-23, H-24, and OH-2 ,
0
0
H-23/H-24, and H -26 and H-1 b/OH-2 unambiguously
3
revealed that these protons were cofacial and arbitrarily
0
defined as having a β-orientation. H -27 and H-1 a were
assigned to be R-configuration judging from the NOE
3
The configuration of 1 was elucidated from NOESY
correlations and J-based configurational analysis com-
bined with circular dichroism (CD) data. NOE correla-
0
0
0
correlations of H-1 a/H -27, H-4 , and OH-4 and H -27/
0
3
3
OH-4 . On the basis of the 20R configuration in 1, the
crucial NOE correlations of OH-20/H-23, H-24, and
H -26, and H -21/H-1 b and H-12β and the absence of
an NOE correlation of OH-20/H-1 b suggested that the
flexibility of the unique tricyclic ring system in the side
chain was severely limited and possessed a major confor-
mation in the solution state as shown in Figure 2 b, which
indicated a 22R,23S,24S,2 S,3 S configuration for 1. The
lowest-energy 3D conformation, obtained by Monte Carlo
searching with the MMFF molecular mechanics force field
tions of H-7β/H -19, H-8/H-15β, H-16, H -18, and H -19,
3
3
3
0
and H-16/H-15β and H -18 indicated that these protons
3
3
3
0
were oriented on the same side of the nucleus, whereas
NOE correlations of H-10/H -29 and H -30, H-15R/H-7R
3
3
and H -30, and H-12R/H-17 and H -30 revealed that they
3
3
were oriented on the other side of the ring system. These
data suggested that the configuration of the tetracyclic ring
system of 1 was identical to those of cucurbitane deriva-
0
0
4
,6,7
tives.
In the CD spectrum of 1, negative Cotton effects
8
at 335 (Δε ꢀ4.14) and 242 (Δε ꢀ3.85) nm and positive
Cotton effects at 298 (Δε þ4.48) and 275 (Δε þ6.68) nm
indicated that the configuration of the nucleus moiety was
using the SPARTAN 04 program, was consistent with
that assigned by the NOESY data (Supporting Informa-
0
0
0
0
0
tion, Figure S2). In the 4 ,5 ,6 -triolyl unit, the 3 ,4 -threo-
0
4
,7b
0
identical to that of the reported cucurbitane analogues.
4
of small J
,5 -erythro configuration was assigned by the magnitude
3
3
(2.4 Hz) and large J
0
0
0
0
(8.0 Hz)
H-3 ,H-4
H-4 ,H-5
9
coupling constants and the NOE correlations of H-1 a/
0
(
6) (a) Gamlath, C. B.; Gunatilaka, A. A. L.; Alvi, K. A.; Atta ur, R.;
Balasubramaniam, S.Phytochemistry 1988, 27, 3225. (b) Kanchanapoom,
T.; Kasai, R.; Yamasaki, K. Phytochemistry 2002, 59, 215.
0
0
0
0
0
0
0
H-4 and OH-4 , H-3 /H-5 and OH-5 , and H-4 /H -6 (for
2
(
7) (a) Chen, J. C.; Chiu, M. H.; Nie, R. L.; Cordellb, G. A.; Qiuc,
detailed J-based configuration analysis, see Supporting
S. X. Nat. Prod. Rep. 2005, 22, 386. (b) Yoshikawa, M.; Morikawa, T.;
Kobayashi, H.; Nakamura, A.; Matsuhira, K.; Nakamura, S.; Matsuda,
H. Chem. Pharm. Bull. 2007, 55, 428. (c) Restivo, R. J.; Bryan, R. F.;
Kupchan, S. M. J. Chem. Soc., Perkin Trans. 2 1973, 892.
10
Information Figure S3). This, combined with the 3 S
0
(8) Spartan ’04; Wavefunction, Inc.: Irvine, CA
Org. Lett., Vol. 13, No. 11, 2011
2
858

configuration as elucidated above, demonstrated that 1
0
0
had a 4 R,5 R-configuration. Therefore, the structure of
compound 1 was determined and designated as machilu-
side A.
Scheme 1. Plausible Biosynthetic Pathways of 1 and 2
The spectroscopic data of machiluside B (2) (Table 1 and
Supporting Information) indicated that it was an isomer of
1
1
1
. 2D NMR data ( Hꢀ H COSY, HSQC, and HMBC)
analysis revealed that 2 differed from 1 only in the replace-
0
ment of the oxygen-bridged linkage of C-22 and C-1 in
0
1
confirmed by the presence of a correlation from H-4
by that of C-22 and C-4 in 2. In particular, this was
0
0
to C-22 and the absence of the correlation from H -1 to
2
C-22 in the HMBC spectrum (Surpporting Information,
Figure S1). The similarity of the coupling constants and
CD data of 2 and 1 (Table 1 and Supporting Information)
suggested that the two compounds had the same config-
uration. Therefore, the structure of machiluside B (2) was
determined as shown in Figure 1.
The plausible biosynthetic pathways of machilusides A
1) and B (2) are postulated in Scheme 1. They may be
(
biosynthesized from an enzymatic catalyzed coupling of a
molecule of the co-occurring 2-O-β-D-glucopyranosyl-cu-
curbitacin I (3, an important precursor of several cucurbi-
4
,6a,11
tane derivatives
) with a molecule of D-fructose (4, a
rings with one tetrahydropyran ring syn-fused into a
contracted cagelike structure in 2) in the side chain, which
has never been described before in a natural product.
In a cytotoxic assay against human cancer cell lines
including the ovary (A2780), colon (HCT-8), hepatoma
common natural product). The precursors would be trans-
formed into key intermediates sequentially or simulta-
neouslyviaenzymatically catalyzedintermolecularnucleo-
philic addition followed by intramolecular nucleophilic
addition and then dehydration to yield 1 and 2. The
assigned configurations of 1 and 2 are supported by the
biosynthetic postulation.
(
Bel-7402), stomach (BGC-823), and lung (A549) by using
3
a
an MTT method, compounds 1 and 2 showed nonselec-
tive cytotoxic activities against the above cell lines with
IC50 values of 0.40ꢀ6.52 μM, while the positive control
The most structurally intriguing part of machilusides A
1) and B (2) is the triterpenoid nucleus that possesses a C36
(
camptothecin gave IC values of 0.28ꢀ12.5 μM.
skeleton with a D-fructose moiety incorporated into a
cucurbitane nucleus forming unique cagelike tricyclic ring
moieties (three tetrahydrofuran rings syn-fused into a
contracted cagelike structure in 1 and two tetrahydrofuran
50
Acknowledgment. Financial support from the National
Natural Sciences Foundation of China (NNSFC; Grant
Nos. 30825044 and 20932007) and the National Science
and Technology Project of China (Nos. 2009ZX09311-004
and 2009ZX09301-003-4-1) is acknowledged.
(
9) (a) Marshall, J. A.; Beaudoin, S. J. Org. Chem. 1994, 59, 6614. (b)
Bichard, C. J. F.; Bruce, I.; Hughes, D. J.; Girdhar, A.; Fleet, G. W. J.;
Watkin, D. J. Tetrahedron: Asymmetry 1993, 4, 1579. (c) Nakamura, M.;
Takayanagi, H.; Furuhata, K.; Ogura, H. Chem. Pharm. Bull. 1992, 40,
8
79. (d) McNicholas, P. A.; Batley, M.; Redmond, J. W. Carbohydr. Res.
Supporting Information Available. Plant material, ex-
perimental procedures, and physicalꢀchemical proper-
ties for compounds 1 and 2; and copies of MS, HRMS,
CD, IR, 1D and 2D NMR spectra of compounds 1 and 2.
1986, 146, 219.
(
10) (a) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
Tachibana, K. J. Org. Chem. 1999, 64, 866. (b) Tripathi, A.; Puddick, J.;
Prinsep, M. R.; Rottmann, M.; Tan, L. T. J. Nat. Prod. 2010, 73, 1810.
(
c) Cho, J. Y.; Kwon, H. C.; Williams, P. G.; Kauffman, C. A.; Jensen,
P. R.; Fenical, W. J. Nat. Prod. 2006, 69, 425.
11) (a) Hatam, N. A. R.; Whiting, D. a.; Yousif, N. J. Phytochemistry
0
0
Detailed J-based configuration analysis for C-3 ꢀC-5 of
. This material is available free of charge via the Internet
at http://pubs.acs.org.
(
1
1989, 28, 1268. (b) Hylands, P. J.; Mansour, E.-S. S. Phytochemistry 1982,
21, 2703.
Org. Lett., Vol. 13, No. 11, 2011
2859