Glucopyranosyloxybenzyl derivatives of (R)-2-benzylmalic acid and (R)-eucomic acid, and an aromatic glucoside from the pseudobulbs of Grammatophyllum speciosum

Article abstract of DOI:10.1016/j.tet.2012.11.082

Three new glucosyloxybenzyl derivatives of (R)-2-benzylmalic acid and of (R)-eucomic acid, grammatophyllosides A-C, and a new phenolic glucoside, grammatophylloside D, were isolated from the pseudobulbs of Grammatophyllum speciosum along with cronupapine, vandateroside II, gastodin, vanilloloside, orcinol glucoside, and isovitexin. The structure elucidations of these compounds were based on analyses of physical and spectroscopic data, as well as chemical evidence.

Full text of DOI:10.1016/j.tet.2012.11.082

Tetrahedron 69 (2013) 1031e1037
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Glucopyranosyloxybenzyl derivatives of (R)-2-benzylmalic acid
and (R)-eucomic acid, and an aromatic glucoside from the
pseudobulbs of Grammatophyllum speciosum
Poolsak Sahakitpichan a, Chulabhorn Mahidol a, Wannaporn Disadee a, Nitirat Chimnoi a,
,
,c,
*
Somsak Ruchirawat a b, Tripetch Kanchanapoom a
a Chulabhorn Research Institute and Chulabhorn Graduate Institute, Kamphaeng Phet 6, Talat Bang Khen, Lak Si, Bangkok 10210, Thailand
b Center of Excellence on Environment Health and Toxicology, CHE, Ministry of Education, Thailand
c Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 May 2012
Received in revised form 7 November 2012
Accepted 26 November 2012
Available online 1 December 2012
Three new glucosyloxybenzyl derivatives of (R)-2-benzylmalic acid and of (R)-eucomic acid, gramma-
tophyllosides AeC, and a new phenolic glucoside, grammatophylloside D, were isolated from the
pseudobulbs of Grammatophyllum speciosum along with cronupapine, vandateroside II, gastodin, va-
nilloloside, orcinol glucoside, and isovitexin. The structure elucidations of these compounds were based
on analyses of physical and spectroscopic data, as well as chemical evidence.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Grammatophyllum speciosum
Orchidaceae
(R)-2-Benzylmalic acid
(R)-Eucomic acid
Glucosyloxybenzyl derivatives
Grammatophyllosides AeD
Phenolic glucoside
1. Introduction
structures of previously isolated glucosyloxybenzyl eucomate de-
rivatives are discussed (Fig. 1).
Grammatophyllum speciosum Blume (Orchidaceae; Thai name:
Waan-Phet-Cha-hueng) is the largest orchid of the world, dis-
tributed in the tropical rainforests of Thailand, Malaysia, Indo-
nesia, and the Philippines. In Thai traditional medicine, the
decoction made from pseudobulbs is used to treat sore throats and
bronchitis, while the pseudobulb paste is applied externally to
relieve pain from scorpion stings. Phytochemical investigation has
not been reported for this species. However, the phytochemical
compositions and pharmacological properties of orchids have
been reviewed.1 As part of our continuing studies on Thai me-
dicinal plants, we investigated the polar constituents from this
plant. This paper describes the isolation and structure de-
termination of 10 polar compounds, including 5 glucosyloxybenzyl
derivatives of (R)-2-benzylmalic acid (1e3) and of (R)-eucomic
acid (4, 5), 4 phenolic glycosides (6e9), a flavone C-glucoside (10),
and a nucleotide (11) from the pseudobulbs of this plant. Also, the
2. Results and discussion
The methanolic extract of the fresh pseudobulbs of G. speciosum
was partitioned between Et2O and H2O. The aqueous soluble frac-
tion was separated by a combination of chromatographic methods
to provide 10 compounds.
Compound 1 was isolated as an amorphous powder, and its
molecular formula was determined to be C24H28O11 by negative HR-
APCI-TOF mass spectrometric analysis. The NMR spectroscopic and
the physical data of compound 1 were coincident with those of
cronupapine, first isolated from Cronura papirio.2 The structure was
confirmed by the respective HMBC correlations. However, the ab-
solute configuration at the C-2 position was not identified. Thus,
this compound was hydrolyzed in alkaline condition (see
Experimental section) to afford 2-benzylmalic acid (1a), as a white
powder, which was identified by 1H and 13C NMR spectroscopic
analysis, displaying an optical rotation [
Since the optical rotation value reported for (S)-2-benzylmalic acid
27
a]
ꢀ14.9 (c 0.48, H2O).
D
* Corresponding author. Tel.: þ66 43 362092; fax: þ66 43 202379; e-mail ad-
dress: trikan@kku.ac.th (T. Kanchanapoom).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.11.082

1032
P. Sahakitpichan et al. / Tetrahedron 69 (2013) 1031e1037
Fig. 1. Structures of compounds 1e5, 9, 1a, and 4a.

P. Sahakitpichan et al. / Tetrahedron 69 (2013) 1031e1037
1033
showed [
a
]
23 þ15.55 (c 2.9, H2O)3 and [
a
]
29 þ13.7 (c 1.0, H2O),4 the
(each 2H, d, J¼8.3 Hz) instead of the signals of the mono-
substituted aromatic ring systems of 1 and 2. Assignment of all
signals was based on the results of HMQC and HMBC experiments.
Since the signals of two AB-type methylene protons of H2-5 (dH 2.71
and 2.76) and one AB-type methylene proton of H-3 at dH 2.68 were
overlapped in DMSO-d6, another set of NMR data was acquired in
MeOH-d4 to provide resolved signals (Tables 1 and 2). In the HMBC
spectrum, significant correlations were found from dH 2.78 (H-5) to
dC 180.6 (C-1), 77.6 (C-2), 44.3 (C-3), 129.0 (C-6), 132.6 (C-7/11), and
dH 5.03 (H-7000) to dC 177.0 (C-4), indicating that the glucopyr-
anosyloxybenzyl moiety was connected through C-4. Alkaline hy-
D
D
absolute configuration of C-2 position was concluded to be R.
Compound 2 was obtained as an amorphous powder, and its
molecular formula was established as C24H28O11 by negative HR-
APCI-TOF mass spectrometric analysis. Inspection of the NMR
spectroscopic data indicated that this compound contained a 2-
benzylmaloyl moiety from the chemical shifts of five overlapping
proton signals at dH 7.13 (2H) and 7.20 (3H) together with two sets
of AB-type methylene protons at dH 2.41 and 2.71 (each d,
J¼14.4 Hz) and dH 2.83 and 2.89 (each d, J¼13.7 Hz). These proton
signals were in agreement with the relevant carbon signals of
a mono-substituted aromatic ring at dC 125.6 (1C), 127.3 (2C), and
130.6 (2C); and two methylene carbons at dC 43.7 and 44.5. The
remaining signals belonging to this part were two carboxyl carbons
at dC 170.6 and 176.8, an oxygenated quaternary carbon at dC 75.1,
and a quaternary carbon of an aromatic ring at dC 138.4. In addition,
the signals of the glucopyranosyloxybenzyl moiety were presented
as compared to 1, supported by the proton signals of a 1,4-
disubstituted aromatic ring at dH 7.30 and 7.00 (each 2H, d,
J¼8.5 Hz) and an anomeric proton at dH 4.86 (1H, d, J¼7.3 Hz). This
compound was an isomer of 1, differing by the location of the
glucopyranosyloxybenzyl moiety as connected through C-4 instead
of C-1. The conclusion was corroborated by the results of the HMBC
experiments (Fig. 2), for example, correlations between H-5 and C-
drolysis of 4 provided (R)-eucomic acid (4a) as a white powder,
26
identified by spectroscopic data and an optical rotation value [a]
D
ꢀ10.1 (c 1.79, MeOH), lit. [
a
]
D
28 ꢀ12.9 (c 0.50, MeOH).5 Therefore, the
structure of this new natural product was elucidated as shown,
namely grammatophylloside C. Interestingly, the same structure
had previously been assigned to vandateroside I, isolated from
Vanda teres,6 but the spectroscopic data obtained in the present
study clearly was not identical to the reported NMR data, especially
for the (R)-eucomoyl part. The structure of vandateroside I was
elucidated based on HMBC correlations, in particular from H2-5 (dH
2.77) to C-1 (dC 171.6) and from H-7000 dH 4.98) to C-4 (dC 173.7).6
(
However, as stated above the signals of H2-5 overlapped with one
proton signal of H-3 at dH 2.74 when measured in DMSO-d6, leading
to ambiguity in assigning C-1 and C-4. Even though vandateroside I
was not isolated in the present study, our results strongly suggest
that its structure should be revised.
Compound 5 was obtained as an amorphous powder, and its
molecular formula was established as C37H44O18 by negative HR-
APCI-TOF mass spectrometric analysis. The spectroscopic and phys-
ical data were identical to those of vandateroside II.6 However, pre-
viously the absolute configuration at C-2 was established as R only
based on comparison of its negative optical rotation with that of (R)-
eucomic acid. To provide stronger evidence, compound 5 was sub-
jected to alkaline hydrolysis to give a white amorphous residue. This
residue was identified to be (R)-eucomic acid (4a) with an optical
rotation value [
a
]
26 ꢀ15.1 (c 0.45, MeOH). Accordingly, the absolute
D
configuration at C-2 position was unambiguously assigned to be R.
Compound 9 was isolated as an amorphous powder. Its molec-
ular formula was established as C15H20O8 by negative HR-APCI-TOF
mass spectrometric analysis. The 1H NMR spectrum indicated the
presence of a 1,3,5-trisubstituted aromatic ring with signals at dH
6.24, 6.30 and 6.33 (each 1H, br s), one singlet methylene at dH 3.58
(2H), one acetyl group at dH 2.08 (3H) as well as one anomeric proton
Fig. 2. Significant HMBC correlations of grammatophylloside A (2).
1, C-2 and C-3, and between H-7000 and C-4. The absolute configu-
ration of C-2 was identified to be R by alkaline hydrolysis (see
Section 4), to obtain (R)-2-benzylmalic acid (1a), [
a
]
D
27 ꢀ18.4 (c 0.57,
H2O). Therefore, the structure of 2 was elucidated as 4-(4000-
b-glu-
copyranosyloxybenzyl)-(R)-2-benzylmalate, namely grammato-
phylloside A.
at dH 4.77 (1H, d, J¼7.5 Hz) for the
b C
-glucopyranosyl unit. In the 13
NMR spectrum, 15 carbon signals were observed, of which 9 were
assignable to two oxy-aryl carbons at dC 158.3 and 158.6, 3 aryl-
methines at dC 101.9, 108.6, and 110.3, 1 non-protonated aryl car-
bon at dC 136.7, 1 methylene at dC 50.0, and 2 carbon signals of an
acetyl group at dC 206.1 and 29.4 for the aglycone moiety. The
complete assignments were established by analyzing the 2D NMR
spectra including HMQC and HMBC. In the HMBC spectrum, the
correlations were found between (i) dH 3.58 (H-7) and dC 136.7 (C-1),
108.6 (C-2), 206.1 (C-8), and 29.4 (C-9); (ii) dH 4.77 (H-10 Glc) and dC
158.6 (C-3); and (iii) dH 9.45 (5-OH) and dC 158.3 (C-5), 101.9 (C-4),
and 110.3 (C-6), as illustrated in Fig. 3. Consequently, the structure of
compound 9 was elucidated as 1-(3,5-dihydroxyphenyl)propan-2-
Compound 3 was isolated as an amorphous powder. The
molecular formula was identified as
C37H44O17 by negative
HR-APCI-TOF mass spectrometric analysis. The NMR spectra were
closely related to those of compounds 1 and 2, except for the
observation of the additional signals for a second glucopyr-
anosyloxybenzyl moiety. The two glucopyranosyloxybenzyl moie-
ties were assigned to be connected through the two ester carbonyl
groups based on HMBC correlations from the overlapping signals
for H-70 and H-7000
respectively. Consequently, this compound was identified to be 1,4-
bis(40,4000-
-glucopyranosyloxybenzyl)-(R)-2-benzylmalate, namely
(dH 4.94) to C-1 (dC, 173.6) and C-4 (dC 169.8),
b
grammatophylloside B.
one 3-O-b-glucopyranoside, namely grammatophylloside D.
Compound 4 was isolated as an amorphous powder. Its molec-
ular formula was established as C24H28O12 by negative HR-APCI-
TOF mass spectrometric analysis. The 1H and 13C NMR spectra in-
dicated that this compound was an analogue of grammatophyllo-
side A (2), differing by the presence of one additional hydroxyl
function at C-9 of the 2-benzylmaloyl moiety. Consequently, the 1H
NMR spectrum showed a set of resonances corresponding to an
AA0BB0 aromatic ring system, comprising signals at dH 6.59 and 6.97
The remaining known compounds were identified as gastodin
(6),7 vanilloloside (7),8 orcinol glucoside (8),9 and isovitexin (10)10
by physical data and spectroscopic evidence.
3. Conclusions
The present study reported five glucosyloxybenzyl derivatives of
(R)-2-benzylmalic acid (1e3) and of (R)-eucomic acid (4, 5), four

1034
P. Sahakitpichan et al. / Tetrahedron 69 (2013) 1031e1037
Table 1
H NMR spectroscopic data of compounds 1e5 (300 MHz)
Position
3
1a
1b
2a
3a
2.13 (1H, d, J¼15.6 Hz)
2.46 (1H, d, J¼15.6 Hz)
2.78 (1H, d, J¼13.3 Hz)
2.86 (1H, d, J¼13.3 Hz)
7.06e7.12 (2H, m)c
2.45 (1H, d, J¼16.1 Hz)
2.81 (1H, d, J¼16.1 Hz)
2.90 (1H, d, J¼13.5 Hz)
2.96 (1H, d, J¼13.5 Hz)
7.08e7.12 (2H, m)c
2.41 (1H, d, J¼14.1 Hz)
2.71 (1H, d, J¼14.1 Hz)
2.83 (1H, d, J¼13.7 Hz)
2.89 (1H, d, J¼13.7 Hz)
7.10e7.16 (2H, m)c
2.49 (1H, d, J¼15.6 Hz)
2.86 (1H, d, J¼15.6 Hz)
2.87 (1H, d, J¼13.4 Hz)
2.93 (1H, d, J¼13.4 Hz)
7.08e7.12 (2H, m)c
5
7/11
8/10
7.15e7.21 (3H, m)c
7.16e7.20 (3H, m)c
7.16e7.22 (3H, m)c
7.17e7.21 (3H, m)c
9
20/60
30/50
70
7.19 (2H, d, J¼8.5 Hz)
6.99 (2H, d, J¼8.5 Hz)
4.91 (2H, s)
4.87 (1H, d, J¼7.1 Hz)
7.25 (2H, d, J¼8.6 Hz)
7.07 (2H, d, J¼8.6 Hz)
5.00 (2H, s)
4.93 (1H, d, J¼7.1 Hz)
7.24 (2H, d, J¼8.7 Hz)d
6.99 (2H, d, J¼8.7 Hz)d
4.94 (2H, s)
Glc-100
4.85 (1H, d, J¼6.9 Hz)d
200
300
3.13e3.23 (1H, m)c
3.23e3.33 (1H, m)c
3.42e3.50 (1H, m)c
3.40e3.48 (2H, m)c
3.20e3.28 (2H, m)c
400
500
600
3.13e3.23 (1H, m)c
3.11e3.18 (1H, m)c
3.28e3.34 (1H, m)c
3.41e3.48 (1H, m)c
3.63e3.70 (1H, m)c
7.20 (2H, d, J¼8.7 Hz)d
6.98 (2H, d, J¼8.7 Hz)d
4.94 (2H, s)
3.31e3.37 (1H, m)c
3.47e3.52 (1H, m)c
3.49 (1H, dd, J¼11.3, 5.0 Hz)
3.72 (1H, dd, J¼11.9, 4.6 Hz)
3.69 (1H, br d, J¼11.3 Hz)
3.90 (1H, dd, J¼11.9, 1.4 Hz)
2000/6000
3000/5000
7000
7.30 (2H, d, J¼8.5 Hz)
7.00 (2H, d, J¼8.5 Hz)
4.97 (2H, s)
4.86 (1H, d, J¼7.3 Hz)
Glc-10000
4.87 (1H, d, J¼6.9 Hz)d
20000
3.25e3.33 (1H, m)c
3.20e3.28 (2H, m)c
30000
40000
50000
60000
3.20e3.28 (1H, m)c
3.14e3.22 (1H, m)c
3.32e3.37 (1H, m)c
3.48e3.52 (1H, m)c
3.68 (1H, br d, J¼11.5 Hz)
3.11e3.18 (1H, m)c
3.28e3.34 (1H, m)c
3.41e3.48 (1H, m)c
3.63e3.70 (1H, m)c
Position
3
4a
4b
5a
2.38 (1H, d, J¼14.2 Hz)
2.59 (1H, d, J¼15.6 Hz)
3.00 (1H, d, J¼15.6 Hz)
2.78 (1H, d, J¼13.5 Hz)
2.95 (1H, d, J¼13.5 Hz)
7.09 (2H, d, J¼8.3 Hz)
6.68 (2H, d, J¼8.3 Hz)
d
2.48 (1H, d, J¼15.6 Hz)
2.88 (1H, d, J¼15.6 Hz)
2.77 (1H, d, J¼13.6 Hz)
2.83 (1H, d, J¼13.6 Hz)
6.92 (2H, d, J¼8.4 Hz)
6.61 (2H, d, J¼8.4 Hz)
d
2.68 (1H, d, J¼14.2 Hz)
2.71 (1H, d, J¼13.7 Hz)
2.76 (1H, d, J¼13.7 Hz)
6.97 (2H, d, J¼8.3 Hz)
6.59 (2H, d, J¼8.3 Hz)
d
5
7/11
8/10
9
20/60
30/50
70
7.26 (2H, d, J¼8.7 Hz)d
7.01 (2H, d, J¼8.7 Hz)d
4.97 (2H, s)
Glc-100
4.85 (1H, d, J¼6.9 Hz)d
200
3.21e3.29 (2H, m)c
300
400
500
600
3.14e3.23 (1H, m)c
3.30e3.38 (1H, m)c
3.42e3.50 (1H, m)c
3.66e3.74 (1H, m)c
7.22 (2H, d, J¼8.7 Hz)d
7.00 (2H, d, J¼8.7 Hz)d
4.97 (2H, s)
2000/6000
3000/5000
7000
7.29 (2H, d, J¼8.6 Hz)
7.01 (2H, d, J¼8.6 Hz)
4.94 (2H, s)
4.85 (1H, d, J¼7.3 Hz)
7.28 (2H, d, J¼8.4 Hz)
7.06 (2H, d, J¼8.4 Hz)
5.03 (2H, s)
4.93 (1H, d, J¼6.9 Hz)
Glc-10000
4.85 (1H, d, J¼6.9 Hz)d
20000
3.25e3.32 (1H, m)c
3.20e3.28 (1H, m)c
3.47e3.53 (1H, m)c
3.21e3.29 (2H, m)c
30000
3.40e3.48 (2H, m)c
3.44e3.52 (1H, m)c
40000
50000
3.18e3.25 (1H, m)c
3.30e3.36 (1H, m)c
3.14e3.23 (1H, m)c
3.30e3.38 (1H, m)c

P. Sahakitpichan et al. / Tetrahedron 69 (2013) 1031e1037
1035
Table 1 (continued)
Position
60000
4a
4b
5a
3.41e3.47 (1H, m)c
3.69 (1H, br d, J¼10.8 Hz)
3.69e3.76 (1H, m)c
3.88 (1H, br d, J¼11.9)
3.42e3.50 (1H, m)c
3.68e3.74 (1H, m)c
a
Measured in DMSO-d6.
Measured in MeOH-d4.
Chemical shifts was assigned by the results from COSY and HMQC.
Assignments with the same superscript may be reversed.
b
c
d
4. Experimental
4.1. General experimental procedures
Table 2
13C NMR spectroscopic data of compounds 1e5 (75 MHz)
Position
1a
1b
2a
3a
4a
4b
5a
1
2
3
4
5
6
174.9
76.0
44.1
174.9
45.0
136.7
130.3
127.7
126.2
129.4
129.4
116.1
157.1
65.2
176.7
77.8
45.7
178.7
46.0
137.2
131.5
129.0
127.7
131.0
131.1
117.6
159.0
67.6
176.8
75.1
43.7
170.6
44.5
138.4
130.6
127.3
125.6
173.6
75.6
43.0
169.8
44.7
135.7
130.6
128.0
126.8
129.2
129.9
116.3
157.4
66.2c
100.6
73.4
77.1
69.9
76.7
60.9
129.4
129.9
116.3
157.4
65.6c
100.6
73.4
177.0
75.2
43.5
170.7
43.8
128.4
131.4
114.3
155.5
180.6
77.6
44.3
173.2
45.5
129.0
132.6
115.6
156.8
173.6
75.5
42.6
169.6
43.8
125.7
131.4
114.7
156.1
129.1
129.6
116.2
157.3
65.9
NMR spectra were recorded in DMSO-d6 using a Bruker AV-300
spectrometer. MS data were obtained on a Bruker Micro TOF-LC
mass spectrometer. Optical rotations were measured with a Jasco
P-1020 digital polarimeter. For column chromatography (cc), Diaion
HP-20 (Mitsubishi Chemical Industries Co. Ltd.), silica gel 60
7/11
8/10
9
(70e230 mesh, Merck), and RP-18 (50 mm, YMC) were used. HPLC
(Jasco PU-980 pump) was carried out on an ODS column
(20ꢁ250 mm i.d., YMC) with a Jasco RI-2031 refractive index de-
tector. The flow rate was 6 mL/min. The spraying reagent used for
TLC was 10% H2SO4 in H2O/EtOH (1:1, v/v).
10
20/60
30/50
40
70
Glc-100
200
100.4
73.3
77.1
69.7
76.7
102.2
74.8
78.1
71.3
77.9
100.4
73.3
77.0
69.8
76.6
4.2. Plant material
300
The fresh pseudobulbs of G. speciosum Blume were collected in
March 2011 from the Herbal Paradise Garden, Chulabhorn Research
Institute. The identification of the plant was confirmed by Mr.
Nopporn Nontapa of Department of Pharmaceutical Botany and
Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen
University. A voucher specimen (TK-PSKKU-0068) has been de-
posited in the Herbarium of the Faculty of Pharmaceutical Sciences,
Khon Kaen University.
400
500
600
60.7
62.4
60.7
1000
129.8
129.4
116.1
157.1
64.7
100.4
73.2
77.1
129.9
129.4
116.1
157.1
64.6
100.4
73.3
77.1
131.3
130.7
117.6
158.8
66.8
102.0
74.7
78.0
129.2
129.6
116.2
157.3
65.4
100.4
73.3
77.0
2000/6000
3000/5000
4000
7000
Glc-10000
20000
30000
40000
50000
60000
77.1
69.9
76.7
60.9
4.3. Extraction and isolation
69.7
76.6
60.7
69.7
76.7
60.7
71.2
77.7
62.3
69.8
76.6
60.7
The fresh pseudobulbs of G. speciosum (11.6 kg) were extracted
with MeOH (30 L for 2 days, ꢁ3) at room temperature. The MeOH
extract was concentrated in vacuo to dryness. This residue (215.8 g)
was suspended in H2O (500 mL), and partitioned with Et2O
(3ꢁ1.0 L). The water-soluble fraction (189.4 g) was subjected to
a Diaion HP-20 column, and eluted with H2O, MeOH, and acetone,
successively. The fraction eluted with MeOH (35.4 g) was subjected
to silica gel cc using solvent systems EtOAc/MeOH (9:1, 7.0 L),
EtOAc/MeOH/H2O (40:10:1, 6.0 L), EtOAc/MeOH/H2O (70:30:3,
5.0 L), and EtOAc/MeOH/H2O (6:4:1, 9.0 L), respectively, to provide
six fractions (A to F). Fraction B (2.9 g) was applied to an RP-18
column using a gradient solvent system H2O/MeOH (9:1/1:4, v/
v) to provide five fractions. Fractions B-1 and B-2 were combined
and purified by preparative HPLC-ODS using solvent system H2O/
MeCN (9:1, v/v) to afford compounds 8 (1.3 g) and 9 (18.5 mg).
Fraction C (1.7 g) was subjected to an RP-18 column using solvent
system H2O/MeOH (9:1/1:4, v/v) to provide six fractions. Fraction
C-2 was purified by preparative HPLC-ODS with solvent system
H2O/MeCN (9:1, v/v) to yield compound 6 (34.8 mg). Fraction D
(19.2 g) was applied to an RP-18 column using solvent system, H2O/
MeOH (9:1/1:4, v/v) to afford 10 fractions. Fraction D-1 was
separated by preparative HPLC-ODS with solvent system H2O/
MeCN (9:1, v/v) to give compounds 1 (368.5 mg), 2 (343.4 mg), and
fraction D-1-1. Fraction D-1-1 was further purified by preparative
HPLC-Polyamine II with solvent system H2O/MeCN (1:9, v/v) to
provide compound 7 (11.3 mg). Fraction D-4 was purified by pre-
parative HPLC-ODS with solvent system H2O/MeCN (4:1, v/v) to
afford compound 10 (133.0 mg). Fraction D-5 was purified by
a
Measured in DMSO-d6.
Measured in MeOH-d4.
Assignments with the same superscript may be reversed.
b
c
Fig. 3. Significant HMBC correlations of grammatophylloside D (9).
phenolic glycosides (6e9), and a flavone C-glucoside (10) from the
polar fraction of the Thai orchid G. speciosum. Cronupapine (1),
grammatophyllosides AeC (2e4), vandateroside II (5) were the
glucopyranosyloxybenzyl moiety forming with (R)-2-benzylmalic
acid or (R)-eucomic acid through ester bonds, which were quite
specific type of compounds to obtain from the orchid family.2,6,11
These specific compounds provided further confirmation of the
typical profile of secondary metabolites found in this family, and
might be useful for further chemotaxonomic studies.

1036
P. Sahakitpichan et al. / Tetrahedron 69 (2013) 1031e1037
preparative HPLC-ODS with solvent system H2O/MeCN (4:1, v/v) to
provide compound 5 (105.6 mg). Fractions D-7 and D-8 were
combined and purified by preparative HPLC-ODS with solvent
system H2O/MeCN (7:3, v/v) to afford compound 3 (722.9 mg). Fi-
nally, fraction E (4.6 g) was similarly applied to an RP-18 column
using H2O/MeOH (9:1/1:4, v/v) to obtain seven fractions. Faction
E-1 was purified by preparative HPLC-ODS with solvent system
H2O/MeCN (9:1, v/v) to provide compound 4 (320.9 mg). Fraction E-
2 was purified by preparative HPLC-ODS with solvent system H2O/
MeCN (9:1, v/v) to afford compound 2 (316.0 mg). Fraction E-3 was
purified by preparative HPLC-ODS with solvent system H2O/MeCN
(9:1, v/v) to give compound 1 (107.9 mg).
(1H, d, J¼15.8 Hz, H-3b), or 1H NMR (MeOH-d4):
d 7.20e7.26 (5H, m,
AreH), 3.04 (1H, d, J¼13.4 Hz, H-5a), 2.97 (1H, d, J¼13.4 Hz, H-3a),
2.94 (1H, d, J¼15.8 Hz, H-5b), 2.59 (1H, d, J¼15.8 Hz, H-3b); 13C
NMR (DMSO-d6):
d 175.6 (C-1), 171.6 (C-4), 136.2 (C-6), 130.6 (C-7,
11), 127.8 (C-8, 10), 126.5 (C-9), 74.7 (C-2), 44.4 (C-5), 42.8 (C-3), or
13C NMR (MeOH-d4):
d
177.6 (C-1), 174.1 (C-4), 136.9 (C-6), 131.6 (C-
7, 11), 129.0 (C-8, 10), 127.8 (C-9), 76.7 (C-2), 46.1 (C-5), 43.8 (C-3);
negative HR-APCI-TOFMS, m/z: 223.0605 [MꢀH]ꢀ (calcd for
C11H11O5, 223.0612).
4.4.2. (R)-Eucomic acid (4a). Amorphous powder; IR (UATR) nmax
3460, 3030, 2607, 1731, 1515, 1370, 1192, 1135, 1070 cmꢀ1; 1H NMR
(DMSO-d6):
d
9.20 (1H, s, 9-OH), 6.99 (2H, d, J¼8.4 Hz, H-7,11), 6.63
27
4.3.1. Cronupapine (1). Amorphous powder; [
a]
ꢀ50.0 (c 0.48,
(2H, d, J¼8.4 Hz, H-8,10), 2.80 (1H, d, J¼13.4 Hz, H-5a), 2.74 (1H, d,
D
MeOH); IR (UATR) nmax 3364, 2920, 1727, 1583, 1512, 1400, 1230,
J¼13.4 Hz, H-5b), 2.70 (1H, d, J¼15.8 Hz, H-3a), 2.35 (1H, d,
1075, 1043 cmꢀ1 1H NMR (DMSO-d6 and MeOH-d4): Table 1; 13C
;
J¼15.8 Hz, H-3b), or 1H NMR (MeOH-d4):
7.06 (2H, d, J¼8.5 Hz, H-
d
NMR (DMSO-d6 and MeOH-d4): Table 2; significant HMBC corre-
lations: (i) H-3 and C-1,2,4,5, (ii) H-5 and C-1,2,3,6,7, (iii) H-70 and C-
1,10,20,60, (iv) H-100 and C-40; negative HR-APCI-TOFMS, m/z:
491.1554 [MꢀH]ꢀ (calcd for C24H27O11, 491.1559).
7,11), 6.68 (2H, d, J¼8.5 Hz, H-8,10), 2.95 (1H, d, J¼13.9 Hz, H-5a),
2.94 (1H, d, J¼16.0 Hz, H-3a), 2.85 (1H, d, J¼13.9 Hz, H-5b), 2.56
(1H, d, J¼16.0 Hz, H-3b); 13C NMR (DMSO-d6):
d 175.7 (C-1), 171.7
(C-4), 156.0 (C-9), 131.4 (C-7, 11), 126.2 (C-6), 114.6 (C-8, 10), 74.9 (C-
2), 43.7 (C-5), 42.6 (C-3), or 13C NMR (MeOH-d4):
d 177.7 (C-1), 174.2
26
4.3.2. Grammatophylloside A (2). Amorphous powder; [
a]
ꢀ32.4
(C-4), 157.4 (C-9), 132.6 (C-7, 11), 127.6 (C-6), 115.8 (C-8, 10), 76.8 (C-
2), 45.4 (C-5), 43.6 (C-3); negative HR-APCI-TOFMS, m/z: 239.0556
[MꢀH]ꢀ (calcd for C11H11O6, 239.0561).
D
(c 0.84, MeOH); IR (UATR) nmax 3369, 2920, 1726, 1599, 1513, 1400,
1342, 1226, 1072, 1043 cmꢀ1; 1H NMR (DMSO-d6): Table 1; 13C NMR
(DMSO-d6): Table 2; negative HR-APCI-TOFMS, m/z: 491.1554
[MꢀH]ꢀ (calcd for C24H27O11, 491.1559).
26
4.4.3. Grammatophylloside D (9). Amorphous powder; [
a]
ꢀ32.9
D
(c 0.49, MeOH); IR (UATR) nmax 3336, 2919, 1698, 1597, 1456, 1304,
26
4.3.3. Grammatophylloside B (3). Amorphous powder; [
a]
ꢀ52.2
1115, 1170, 1071, 1021 cmꢀ1 1H and 13C NMR (DMSO-d6): Table 3;
;
D
(c 0.61, MeOH); IR (UATR) nmax 3328, 2920, 1724, 1610, 1513, 1230,
negative HR-APCI-TOFMS, m/z: 363.0835 [MþCl]ꢀ (calcd for
1068, 1043 cmꢀ1
;
1H NMR (DMSO-d6): Table 1; 13C NMR (DMSO-
C
15H20Cl35O8, 363.0841).
d6): Table 2; negative HR-APCI-TOFMS, m/z: 795.2285 [MþCl]ꢀ
(calcd for C37H44Cl35O17, 795.2273).
27 ꢀ28.0
Table 3
4.3.4. Grammatophylloside C (4). Amorphous powder; [
a
]
D
1H and 13C NMR spectroscopic data of compound 9 (measured in DMSO-d6)
(c 0.79, MeOH); IR (UATR) nmax 3327, 2913, 1717, 1590, 1513, 1344,
1228, 1174, 1071, 1043 cmꢀ1 1H NMR (DMSO-d6 and MeOH-d4):
;
Position
dH (300 MHz)
dC (75 MHz)
Table 1; 13C NMR (DMSO-d6 and MeOH-d4): Table 2; significant
HMBC correlations: (i) H-3 and C-1,2,4,5, (ii) H-5 and C-1,2,3,6,7, (iii)
H-7000 and C-4,1000,2000,6000, (iv) H-10000 and C-4000; negative HR-APCI-
TOFMS, m/z: 507.1494 [MꢀH]ꢀ (calcd for C24H27O12, 507.1508).
1
2
3
4
5
6
7
8
136.7
108.6
158.6
101.9
158.3
110.3
50.0
6.30 (1H, br s)
6.33 (1H, br s)
6.24 (1H, br s)
3.58 (2H, s)
4.3.5. Vandateroside II (5). Amorphous powder; [
a
]
26 ꢀ30.6 (c 0.70,
D
206.1
29.4
DMSO); IR (UATR) nmax 3320, 2931, 1723, 1614, 1515, 1357, 1232,
9
2.08 (3H, s)
1068, 1043 cmꢀ1 1H NMR (DMSO-d6): Table 1; 13C NMR (DMSO-
;
5-OH
Glc-10
20
9.45 (1H, br s)
d6): Table 2; negative HR-APCI-TOFMS, m/z: 811.2235 [MþCl]ꢀ
4.77 (1H, d, J¼7.5 Hz)
3.22e3.28 (1H, m)a
3.12e3.17 (1H, m)a
3.22e3.28 (1H, m)a
3.17e3.22 (1H, m)a
3.47 (1H, dd, J¼11.5, 5.9 Hz)
3.68 (1H, dd, J¼11.5, 3.4 Hz)
100.4
73.3
77.0
69.6
76.7
60.6
(calcd for C37H44Cl35O18, 811.2222).
30
40
4.4. Alkaline hydrolysis of compounds 1e5
50
60
Compound 1 (60 mg, 0.12 mmol) was treated with sodium hy-
droxide solution (5 mL, 7% aq) and stirred at room temperature for
6 h. The reaction mixture was neutralized with hydrochloric acid
solution (5% aq) and extracted with EtOAc. The organic layer was
separated and the solvent was evaporated in vacuo to give (R)-
benzylmalic acid 1a (12.0 mg, 45%). By the same method, com-
pounds 2 (60 mg, 0.12 mmol) and 3 (100 mg, 0.13 mmol) provided
1a (23.4 mg, 87% and 24.5 mg, 83%, respectively). Compounds 4
(95 mg, 0.19 mmol) and 5 (60 mg, 0.08 mmol) yielded 4a (17.9 mg,
40% and 4.5 mg, 24%, respectively). The structures of 1a and 4a were
identified by 1D and 2D NMR spectroscopic analysis together with
comparison of the optical rotation data with the literature values.
a
Chemical shifts was assigned by the results from COSY and HMQC.
Acknowledgements
This research work was supported by the grant from the Thai-
land Research Fund (DBG5480007), Chulabhorn Research Institute;
and Center of Excellence Health and Toxicology, Science & Tech-
nology Postgraduate Education and Research Development Office
(PERDO), Ministry of Education.
References and notes
4.4.1. (R)-Benzylmalic acid (1a). Amorphous powder; IR (UATR)
ꢀ
nmax 3446, 2927, 1716, 1496, 1390, 1207, 1115, 1022 cmꢀ1 1H NMR
;
1. Gutierrez, R. M. P. J. Med. Plants Res. 2010, 4, 592e638.
2. Mochizuki, K.; Shizuri, Y.; Kosemura, S.; Miyoshi, E.; Yamamura, S. Chem. Lett.
1992, 1239e1242.
3. Spencer, H. K.; Khatri, H. N.; Hill, R. Bioorg. Chem. 1976, 5, 177e186.
(DMSO-d6):
d
7.19e7.28 (5H, m, AreH), 2.92 (1H, d, J¼13.4 Hz, H-
5a), 2.86 (1H, d, J¼13.4 Hz, H-5b), 2.72 (1H, d, J¼15.8 Hz, H-3a), 2.38

P. Sahakitpichan et al. / Tetrahedron 69 (2013) 1031e1037
1037
4. Tanaka, T.; Suda, H.; Naganawa, H.; Hamada, M.; Takeuchi, T.; Aoyagi, T.;
Umezawa, H. J. Antibiot. (Tokyo) 1984, 37, 682e684.
8. Ida, Y.; Satoh, Y.; Ohtsuka, M.; Nagasao, M.; Shoji, J. Phytochemistry 1993, 35,
209e215.
5. Okada, M.; Park, S.; Koshizawa, T.; Ueda, M. Tetrahedron 2009, 65, 2136e2141.
6. Simmler, C.; Antheaume, C.; Andre, P.; Bonte, F.; Lobstein, A. J. Nat. Prod. 2011,
74, 949e955.
9. Hu, H.; Fan, J. Bull. Korean Chem. Soc. 2009, 30, 703e706.
10. Peng, J.; Fan, G.; Hong, Z.; Chai, Y.; Wu, Y. J. Chromatogr., A 2005, 1074,
111e115.
ꢀ
ꢀ
7. Baek, N.-I.; Choi, S. Y.; Park, J. K.; Cho, S.-W.; Ahn, E.-M.; Jeon, S.-G.; Lee, B. Y.;
Bahn, J. H.; Kim, Y. K.; Shon, I. H. Arch. Pharm. Res. 1999, 22, 219e224.
11. Yoshikawa, K.; Otsu, M.; Ito, T.; Asakawa, Y.; Kawano, S.; Hashimoto, T. J. Nat.
Med. 2012, 1e5, http://dx.doi.org/10.1007/s11418-012-0653-z