American Journal of Plant Sciences, 2013, 4, 1954-1959
http://dx.doi.org/10.4236/ajps.2013.410242 Published Online October 2013 (http://www.scirp.org/journal/ajps)
Microtropins J-P: 6’-O-(2”S,3”R)-2”-Ethyl-2”,3”-
Dihydroxybutyrates of Phenolic Alcohol
 -D-Glucopyranosides from the Branches of Microtropis
japonica
Yuka Uemura1, Sachiko Sugimoto1, Katsuyoshi Matsunami1, Hideaki Otsuka1,2*, Yoshio Takeda2
1Department of Pharmacognosy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan;
2Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women’s University, Hiroshima, Japan.
Received July 26th, 2013; revised August 26th, 2013; accepted September 15th, 2013
Copyright © 2013 Yuka Uemura et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
From the branches of Microtropis japonica (Celastraceae), seven phenolic alcohol glucosides, named microtropins J-P
(1-7), were isolated. The 6-position of glucose was esterified with 2-ethyl-2,3-dihydroxybutyric acid. Microtropin K (2)
was hydrolyzed under a mild basic condition to give methyl (2S,3R)-2-ethyl-2,3-dihydroxybutyrate, whose absolute
structure was determined by the comparison of NMR data and the optical rotation value with that reported.
Keywords: Microtropis japonica; Celastraceae; Microtropin; (2S,3R)-2-Ethyl-2,3-Dihydroxybutyrate
1. Introduction
Celastraceous plants appeared in the spotlight after a po-
tent antileukemic ansa-type macrolide, maytansine, was
isolated from an Ethiopian shrub, Maytenus serrata (for-
merly M. ovatus) [1,2]. In continuous research on subtro-
pical resource plants, a Celestraceous plant, Microtropis
japonica, collected in Okinawa attracted our attention
and its constituents were investigated. In previous papers,
the isolation of ent-labdane diterpene glucosides, micro-
troipiosides A-F [3,4], from the leaves of M. japonica,
and the 2-ethyl-2,3-dihydroxybutyrate of nine aliphatic
glucosides, microtropins A-I [4], from its branches has
already been reported. Further extensive isolation work
on a MeOH extract of the branches of M. japonica re-
sulted in the isolation of seven new aromatic glucoside
(2S,3R)-2-ethyl-2,3-dihydroxybutyrates (1-7), named mi-
crotropins J-P, along with two known compounds, vanil-
lic (8) [5] and salicylic (9) acids (Figure 1). Isolation of
2-ethyl-2,3-dihydroxybutyrate may be interesting in a
chemotaxonomic point of view.
2. Results and Discussion
Seven new 2-ethyl-2,3-dihydroxybutyrates of various
phenolic glucosides, named microtropins J-P (1-7), were
isolated from the 1-BuOH-soluble fraction of a MeOH
extract of the branches of M. japonica by a combination
of various separation procedures. Their structures were
elucidated from spectroscopic evidence. Microtropin J
(1), [
 ]D
24 ‒59.8, was isolated as an amorphous powder
and its elemental composition was determined to be
C19H26O11 by high-resolution (HR)-electrospray ioniza-
tion (ESI) mass spectrometry (MS). The IR spectrum
exhibited absorption bands assignable to hydroxy (3380
cm‒1), ester carbonyl (1730 cm‒1), carboxylic acid (1705
cm‒1), aromatic ring (1606, and 1511 cm‒1), phenolic al-
cohol (1239 cm‒1), and aliphatic alcohols (1073 cm‒1).
The UV absorption band at 245 nm also supported the
presence of an aromatic ring. In the 13C-NMR spectra, six
signals assignable to 2-ethyl-2,3-dihydroxybutyrate were
observed together with ones assignable to glucose, which
had a substituent at the 6-position. The 1H-NMR spectrum
of the aglycone moiety comprised seven signals, which
included one for a para-substituted aromatic ring with a
carboxyl functional group. Acid hydrolysis liberated D-
glucose, which was identified by HPLC analysis with a
chiral detector. In the heteronuclear multiple bond corre-
lation spectrum (HMBC), the anomeric proton (δH 5.03)
showed a correlation peak with C-4 (δC 162.7), and H-6’
*Corresponding author.

Microtropins J-P: 6’-O-(2”S,3”R)-2”-Ethyl-2”,3”-Dihydroxybutyrates of Phenolic Alcohol
 -D-Glucopyranosides from the Branches of Microtropis japonica
1955
COO R2
RO
OCH3
RO OH
OCH3
OH
RO
OCH3
RO
OCH3
OH
OH
COO H
HO
OCH3
COO H
OH
1
2
3
R1
R1R2
H
OCH3
OCH3
H
H
CH3
456/
89
7
1
2
2
1
12
1
2
7
O
7
OH
HO
HO
O
O
OH
HO
1'
1"
2"
3"
4"
6"
R:
Figure 1. Structures of compounds isolated.
(δH 4.21 and 4.65) with the carbonyl signal at δC 176.2.
Therefore, the structure of microtropin J (1) was elucida-
ted to be p-hydroxybenzoic acid O-
 -D-glucopyranoside
6’-O-2”-ethyl-2”,3”-dihydroxybutyrate, as shown in Fig-
ure 1. The absolute configuration of the acyl moiety was
expected to be the same (2”S,3”R) as that of microtropin
A [4].
Microtropin K (2), [
 ]D
24 ‒50.6, was isolated as an
amorphous powder and its elemental composition was
determined to be C20H28O12 by HR-ESI-MS. The IR and
UV spectra showed similar absorption bands to those of
1, and in the NMR spectra, a methoxy signal [δH 3.90
(3H, s) on δC 56.8] was observed. The AA’BB’ type cou-
pled four protons observed in 1 were replaced by three
aromatic protons coupled in an ABX system. In the
HMBC spectrum, one of the aromatic protons [H-2, δH
7.625 (s)] showed correlation peaks with C-1 (δC 126.4),
C-4 (δC 151.8), and C-7 (δC 169.5), and the anomeric
proton (δH 5.03) with C-4. From the above evidence, the
structure of aglycone was determined to be vanillic acid
and the overall structure is shown as 2 in Figure 1. Since
microtropin K (2) was isolated in a good quantity, it was
hydrolyzed under a mild alkaline condition to give vanl-
lic acid  -D-glucopyranoside (2a) [5] and methyl 2-
ethyl-(2S,3R)-dihydroxybutyrate (2b) [4].
Microtropin L (3), [  ]D
23 ‒31.6, was isolated an amor-
phous powder and its elemental composition was deter-
mined to be C21H30O12 by HR-ESI-MS. Spectroscopic
data were almost superimposable on those of 2, except
for the presence of an ester methoxy group (δH 3.89 on δC
52.6), and the molecular weight was 14 mass units larger
than that of 2, which accounted for a labile hydrogen
atom being replaced by a methyl group. Therefore, the
structure of 3 was elucidated to be ester of 2, and it may
be an artifact produced during the extraction and isola-
tion procedures.
Microtropin M (4), [
 ]D
25 ‒96.7, was isolated as an
amorphous powder and its elemental composition was
determined to be C20H30O12 by HR-ESI-MS. The IR
spectrum exhibited a strong absorption band for a car-
bonyl functional group and two methoxy signals were
observed in the NMR spectra (δH 3.73 on δC 61.1 and
3.80 on δC 56.5). Judging from the NMR data, the aro-
matic ring had two meta-coupled protons [δH 6.27 (d, J =
2.7 Hz) and 6.32 (d, J = 2.7 Hz)] and thus was suggested
to have unsymmetrically substituted benzene, that is, four
electro-negative functional groups are substituted at the 1,
3, 4 and 5 positions. The relatively deshielded methoxy
signal (δC 61.1) was implied that it was located between
the substituents. The HMBC correlations from H-1' (δH
4.77) to δC 155.6 (C-1), those from H-2 and H-6 to δC
133.6, to which the methoxy signal at δH 3.73 on δC 61.1
also correlated, and those from H-2 and H-6 to C-6 and
C-2 respectively were observed (Figure 2). Therefore,
the structure of aglycone was expected to be 4,5-dime-
thoxybenzene-1,3-diol 1-O-β-D-glucopyranoside, which
was also supported by the NOESY spectrum in which the
anomeric proton showed significant correlation cross
peaks with H-2 and H-6. The structure was finally eluci-
dated to be 4 shown in Figure 1.
Figure 2.Microtropin N (5), [
 ]D
25 ‒11.5, was isolated
as an amorphous powder and its elemental composition
was determined to be C19H28O11. In the NMR spectra,
one methoxy carbon and three protons coupled in an
ABX system were observed in the aglycone region. Other
NMR spectral data for the aglycone were essentially the
same as those of isotachioside isolated from Isotachis ja-
ponica [6]. Therefore, the structure of 5 was elucidated to
be isotachioside 6’-O-(2”S,3”R)-2”-ethyl-2”,3”-dihydroxy-
butyrate, as shown in Figure 1.
Microtropin O (6) and microtropin P (7), [
 ]D
25 ‒32.1
and [
 ]D
26 ‒49.0, respectively, were isolated as amor-
phous powders and the elemental compositions of both
compounds were C22H34O12. In the 1H-NMR spectrum,
O
O
OH
HO
HO
O
O
OH OCH3
OCH3
OH
OH
HC
MHBC
Figure 2. HMBC correlations of 4. Dual arrow curve denote
HMBC correlations were observed in both directions.

Microtropins J-P: 6’-O-(2”S,3”R)-2”-Ethyl-2”,3”-Dihydroxybutyrates of Phenolic Alcohol
 -D-Glucopyranosides from the Branches of Microtropis japonica
1956
one methoxy signal and three protons couple in an ABX
system, one doublet methyl and two oxymethines were
observed. The methyl protons were coupled with one of
the oxymethine protons and the two oxymethine protons
were coupled with each other. Thus, the structure of the
aglycone moiety was expected to be 4-hydroxy-2-meth-
oxy or 4-hydroxy-3-methoxyphenylpropane-7,8-diol. In
the HMBC spectrum, H-2 and 6 (δH 7.03 and 6.87, re-
spectively) showed correlation peaks with C-7 (δC 79.9)
as well as C-4, to which also the anomeric proton (δH
4.87) was correlated. The key correlation from H-5 (δH
7.10) to C-3 (δC 151.0) established the structure of the
aglycone to be 4-hydroxy-3-methoxy-phenylpropane-
7,8-diol. Therefore, the structure of 6 was elucidated to
be as shown in Figure 1. From spectroscopic data, mi-
crotropin P (7) was expected to be a similar compound to
6. Although the 13C-NMR data of both compounds were
almost superimposable (Table 1), they were separated in
the same HPLC runs and exhibited significantly different
retention times. The stereochemistry of both side chains
has not fully been clarified yet, but, judging from the
Table 1. 13C NMR spectroscopic data for micotropins J-P
(1‒7) (CD3OD, 100 MHz).
C 1 2 3 4 5 6 7
1 125.9 126.4 125.8155.6 140.7 138.7138.7
2 132.7 114.7 114.599.1 152.5 112.7112.8
3 117.4 150.7 150.8151.9 102.0 151.0150.9
4 162.7 151.8 152.0133.6 155.2 147.3147.4
5 117.4 117.0 117.1154.8 107.6 118.3118.2
6 132.7 124.7 124.495.8 121.4 120.9120.9
7 169.6 169.5 168.3 79.979.9
8 72.972.9
9 19.419.3
2-OMe 56.6
3-OMe 56.8 56.9 56.856.8
4-OMe 61.1
5-OMe 56.5
-COOMe 52.6
1’ 101.6 104.9 101.9103.0 104.3 104.0103.0
2’ 74.8 75.2 74.8 74.9 75.1 75.075.0
3’ 77.8 77.8 77.7 77.8 77.7 77.777.7
4’ 71.5 71.3 71.5 71.6 71.6 71.5 71.5
5’ 75.6 75.2 75.6 75.5 75.5 75.675.6
6’ 65.4 65.1 65.3 65.4 65.3 65.465.4
1” 176.2 176.1 176.2176.2 176.1 176.2176.2
2” 82.9 82.9 82.9 83.0 82.9 82.982.9
3” 72.9 72.8 72.8 72.8 72.8 72.872.8
4” 16.9 16.8 16.8 16.8 16.8 16.916.9
5” 29.3 29.2 29.3 29.3 29.2 29.329.3
6” 8.4 8.4 8.4 8.3 8.3 8.4 8.4
fairly large coupling constants of H-7 and H-8 (6.7 Hz
for 6 and 6.9 Hz for 7) in the 1H-NMR, their aglycones
are in a threo-form, accordingly the stereochemistry of
the diol-part must be enantiomers, each other [7].
3. Material and Method
3.1. Plant Material
Branches of M. japonica Hallier f. (Celastraceae) were
collected in Kunigami Village, Kunigami County, Oki-
nawa, Japan, in July 1997, and a voucher specimen was
deposited in the Herbarium of Pharmaceutical Sciences,
Graduate School of Biomedical Sciences, Hiroshima
University (97-MJ-Okinawa-0716).
www.ramble-among-flora-of-miyazaki.com/sub73-46.html
3.2. General Experimental Procedures
Optical rotations were measured on a JASCO P-1030 di-
gital polarimeter. IR and UV spectra were measured on
Horiba FT-710 and JASCO V-520 UV/Vis spectropho-
tometers, respectively. 1H- and 13C-NMR spectra were
taken on a JEOL JNM α-400 at 400 MHz and 100 MHz,
respectively, with tetramethylsilane as an internal stan-
dard. Positive-ion HR-ESI-MS was performed with an
Applied Biosystems QSTAR XL NanoSprayTM System.
A highly-porous synthetic resin (Diaion HP-20) was
purchased from Mitsubishi Kagaku (Tokyo, Japan). Sil-
ica gel column chromatography (CC) was performed on
silica gel 60 (E. Merck, Darmstadt, Germany), and ODS
open CC on Cosmosil 75C18-OPN (Nacalai Tesque, Kyo-
to) [(Φ = 5 cm, L = 25 cm, H2O-MeOH (9:1) (2 L) →
H2O-MeOH (1:9) (2 L), linear gradient, 10-g fractions
being collected]. The droplet counter-current chromato-
graph (DCCC) (Tokyo Rikakikai, Tokyo, Japan) was
equipped with 500 glass columns (Φ = 2 mm, L = 40 cm),
the lower and upper layers of a solvent mixture of
CHCl3-MeOH-H2O-n-PrOH (9:12:8:2) being used as the

Microtropins J-P: 6’-O-(2”S,3”R)-2”-Ethyl-2”,3”-Dihydroxybutyrates of Phenolic Alcohol
 -D-Glucopyranosides from the Branches of Microtropis japonica
1957
stationary and mobile phases, respectively. Five-gram
fractions were collected and numbered according to their
order of elution with the mobile phase. HPLC was per-
formed on an ODS column (Inertsil; ODS-3, GL Science,
Tokyo, Japan; Φ = 6 mm, L = 250 mm, 1.6 mL/min), and
the eluate was monitored with a UV detector at 254 nm,
and a refractive index monitor.
3.3. Extraction and Isolation
Air-dried branches of M. japonica (13.0 kg) were ex-
tracted three times with MeOH (30 L × 3) at room tem-
perature for one week and then concentrated to 3 L in
vacuo. The concentrated extract was washed with n-he-
xane (3 L, 53.8 g), and then the MeOH layer was con-
centrated to a gummy mass. The latter was suspended in
water (3 L) and then extracted with EtOAc (3 L) to give
103 g of an EtOAc-soluble fraction. The aqueous layer
was extracted with 1-BuOH (3 L) to give a 1-BuOH-so-
luble fraction (40.9 g), and the remaining water-layer
was concentrated to furnish 107 g of a water-soluble
fraction. The 1-BuOH-soluble fraction (39.9 g) was sub-
jected to Diaion HP-20 CC (Φ = 50 mm, L = 40 cm), us-
ing H2O-MeOH (4:1, 2 L), (3:2, 2 L), (2:3, 2 L), and (1:4,
2 L), and MeOH (2 L), 500 mL-fractions being collected.
The residue (7.53 g out of 8.73 g) in fractions 7 - 10 was
subjected to silica gel (250 g) CC with increasing amounts
of MeOH in CHCl3 [CHCl3 (3 L), and CHCl3-MeOH
(49:1, 1.5 L), (24:1, 1.5 L), (23:2, 1.5 L), (9:1, 1.5 L),
(17:3, 1.5 L), (4:1, 1.5 L), (3:1, 1.5 L), and (7:3, 1.5 L)],
CHCl3-MeOH-H2O (35:15:2, 1.5 L), and MeOH (1.5 L),
300 mL-fractions being collected. The residue (0.962 g)
in fractions 23 - 29 was separated by ODS open CC and
the residue (108 mg) in fractions 81 - 87 was applied to
DCCC. The residue (4.03 mg) in fractions 51 - 61 was
purified by HPLC (H2O-MeOH, 3:1) to give 2.4 mg of 5
from the peak at 21 min. The residue (64.0 mg) in frac-
tions 99 - 105 obtained on ODS open CC was subjected
to DCCC and the residue (6.7 mg) in fractions 84 - 93
was purified by HPLC (H2O-MeOH, 13:7) to give 2.8
mg of 4 from the peak at 14 min. The residue (2.91 g out
of 3.10 g) in fractions 30 - 38 obtained on silica gel CC
was purified by ODS open CC to give three fractions.
The residue (306 mg) in the second lot of fractions, 81 -
93, was separated by DCCC and the residue (45.5 mg) in
fractions 33 - 35 was then purified by HPLC (H2O-
MeOH, 33:7) to give 3.96 mg of 7 and 10.1 mg of 6 from
the peaks at 46 min and 50 min, respectively. The residue
(1.67 g) in fractions 94 - 124 obtained on ODS open CC
was subjected to DCCC to give 1.21 g of 2 in fractions
63 - 86, 21.6 mg of 8 in fractions 142 - 164, and 10.1 mg
of 3 in fractions 165 - 183.
The residue (1.17 g out of 1.27 g) in fractions 39 - 47
obtained on silica gel CC was separated by ODS open
CC and the residue (351 mg) in fractions 121 - 144 was
applied to DCCC. The residue (86.6 mg out of 279 mg)
in fractions 30 - 46 was purified by HPLC (H2O-MeOH,
4:1) to yield 14.2 mg of 1 and 21.0 mg of 2 from the
peaks at 50 min and 65 min, respectively.
The residue (5.68 g out of 6.22 g) in fractions 11 - 14
obtained on Diaion HP-20 CC was subjected to silica gel
CC (180 g) with increasing amounts of MeOH in CHCl3
[CHCl3 (2 L), and CHCl3-MeOH (49:1, 1 L), (24:1, 1 L),
(23:2, 1 L), (9:1, 1 L), (17:3, 1 L), (4:1, 1 L), (3:1, 1 L),
and (7:3, 1 L)], CHCl3-MeOH-H2O (35:15:2, 1 L), and
MeOH (1 L), 200 mL-fractions being collected. The re-
sidue (1.30 g out of 1.41 g) in fractions 27 - 34 was se-
parated by ODS open CC to give 13.6 mg of 9 in frac-
tions 10 - 22.
3.4. Microtropin J (1)
Amorphous powder, [  ]D
24 ‒59.8 (c 0.92, MeOH); IR
max (film): 3380, 2934, 1730, 1705, 1606, 1511, 1454,
1239, 1073, 1016 cm‒1; UV

max (MeOH): 245 (4.10),
209 (3.90) nm (log
 ); 1H-NMR (CD3OD, 400 MHz) δ:
7.98 (2H, d, J = 8.7 Hz, H-2 and 6), 7.14 (2H, d, J = 8.7
Hz, H-3 and 5), 5.03 (1H, d, J = 7.7 Hz, H-1’), 4.65 (1H,
dd, J = 11.9, 2.1 Hz, H-6’a), 4.21 (1H, dd, J = 11.9, 6.3
Hz, H-6’b), 3.89 (1H, q, J = 6.4 Hz, H-3”), 3.72 (1H, ddd,
J = 9.5, 6.3, 2.1 Hz, H-5’), 3.51 - 3.40 (3H, m, H-2’, 3’
and 4’), 1.73 (1H, dq, J = 14.8, 7.4 Hz, H-5”a), 1.55 (1H,
dq, J = 14.8, 7.4 Hz, H-5”b), 1.15 (3H, d, J = 6.4 Hz,
H3-4”), 0.84 (3H, dd, J = 7.4, 7.4 Hz, H3-6”); 13C-NMR
(CD3OD, 100 MHz): Table 1; HR-ESI-MS (positive-ion
mode) m/z: 453.1367 [M+Na]+ (calcd for C19H26O11Na,
453.1367).
3.5. Microtropin K (2)
Amorphous powder, [  ]D
24 ‒50.6 (c 1.20, MeOH); IR
max (KBr): 3410, 2978, 1735, 1706, 1602, 1513, 1462,
1270, 1073, 1022; UV

max (MeOH): 285 (3.58), 250
(4.00), 214 (4.15) nm (log
 ); 1H-NMR (CD3OD, 400
MHz) δ: 7.626 (1H, d, J = 8.8 Hz, H-5), 7.625 (1H, s,
H-2), 7.19 (1H, d, J = 8.8 Hz, H-6), 5.03 (1H, d, J = 7.3
Hz, H-1’), 4.64 (1H, dd, J = 11.9, 2.1 H-6’a), 4.19 (1H,
dd, J = 11.9, 6.4 Hz, H-6’b), 3.90 (3H, s, -OCH3), 3.87
(1H, q, J = 6.4 Hz, H-3”), 3.69 (1H, ddd, J = 9.7, 6.4, 2.1
Hz, H-5’), 3.57 - 3.42 (3H, m, H-2’, 3’ and 4’), 1.70 (1H,
dq, J = 14.6, 7.4 Hz, H-5”a), 1.53 (1H, dq, J = 14.6, 7.4
Hz, H-5”b), 1.14 (3H, d, J = 6.4 Hz, H3-4”), 0.82 (3H, dd,
J = 7.4, 7.4 Hz, H3-6”); 13C-NMR (CD3OD, 100 MHz):
Table 1; HR-ESI-MS (positive-ion mode) m/z: 483.1474
[M+Na]+ (calcd for C20H28O12Na, 483.1472).
3.6. Microtropin L (3)
Amorphous powder, [  ]D
23 ‒31.6 (c 0.67, MeOH); IR

Microtropins J-P: 6’-O-(2”S,3”R)-2”-Ethyl-2”,3”-Dihydroxybutyrates of Phenolic Alcohol
 -D-Glucopyranosides from the Branches of Microtropis japonica
1958
max (film): 3392, 2938, 1733, 1716, 1512, 1458, 1274,
1115, 1073, 1028 cm‒1; UV 
max (MeOH): 335 (2.75),
287 (3.60), 248 (3.80), 220 (3.84) nm (log  ); 1H-NMR
(CD3OD, 400 MHz) δ: 7.62 (1H, d, J = 8.6 Hz, H-6),
7.61 (1H, s, H-2), 7.20 (1H, d, J = 8.6 Hz, H-5), 5.03 (1H,
d, J = 7.3 Hz, H-1’), 4.62 (1H, dd, J = 11.9, 2.2 Hz,
H-6’a), 4.19 (1H, dd, J = 11.9, 5.2 Hz, H-6’b), 3.90 (3H,
s, -OCH3), 3.89 (3H, s, -COOCH3), 3.87 (1H, m, H-3”),
3.69 (1H, ddd, J = 9.5, 5.2, 2.2 Hz, H-5’), 3.56 - 3.22 (3H,
m, H-2’, 3’ and 4’), 1.71 (1H, dq, J = 13.7, 7.4 Hz,
H-5”a), 1.53 (1H, dq, J = 13.7, 7.4 Hz, H-5”b), 1.13 (3H,
d, J = 6.6 Hz, H3-4”), 0.82 (3H, dd, J = 7.4, 7.4 Hz,
H3-6”); 13C-NMR (CD3OD, 100 MHz): Table 1;
HR-ESI-MS (positive-ion mode) m/z: 497.1630 [M+Na]+
(calcd for C21H30O12Na, 497.1630).
3.7. Microtropin M (4)
Amorphous powder; [  ]D
25 ‒96.7 (c 0.18, MeOH); IR
max (film): 3393, 2939, 1736, 1508, 1457, 1231, 1170,
1102, 1075, 1017 cm‒1; UV 
max (MeOH): 275 (3.18),
216 (3.79) nm (log  ); 1H-NMR (CD3OD, 400 MHz) δ:
6.32 (1H, d, J = 2.7 Hz, H-2), 6.27 (1H, d, J = 2.7 Hz,
H-6), 4.77 (1H, d, J = 7.5 Hz, H-1’), 4.64 (1H, dd, J =
11.9, 2.2 Hz, H-6’a), 4.21 (1H, dd, J = 11.9, 6.2 Hz,
H-6’b), 3.95 (1H, q, J = 6.4 Hz, H-3”), 3.80 (3H, s,
5-OCH3), 3.73 (3H, s, 4-OCH3), 3.62 (1H, ddd, J = 9.5,
6.2, 2.2 Hz, H-5’), 3.48 - 3.36 (3H, m, H-2’, 3’ and 4’),
1.75 (1H, dq, J = 13.9, 7.4 Hz, H-5”a), 1.52 (1H, dq, J =
13.9, 7.4 Hz, H-5”b), 1.16 (3H, d, J = 6.4 Hz, H3-4”),
0.84 (3H, dd, J = 7.4, 7.4 Hz, H3-6”); 13C-NMR (CD3OD,
100 MHz): Table 1; HR-ESI-MS (positive-ion mode)
m/z: 485.1633 [M+Na]+ (calcd for C20H30O12Na,
485.1629).
3.8. Microtropin N (5)
Amorphous powder; [  ]D
25 ‒11.5 (c 0.16, MeOH); IR
max (film): 3362, 2938, 1736, 1512, 1457, 1233, 1213,
1167, 1072, 1025 cm‒1; UV 
max (MeOH): 279 (3.30),
213 (3.71) nm (log  ); 1H-NMR (CD3OD, 400 MHz) δ:
6.96 (1H, d, J = 8.6 Hz, H-6), 6.46 (1H, d, J = 2.7 Hz,
H-3), 6.28 (1H, dd, J = 8.6, 2.7 Hz, H-5), 4.67 (1H, d, J =
7.7 Hz, H-1’), 4.63 (1H, dd, J = 11.8, 2.2 Hz, H-6’a),
4.15 (1H, dd, J = 11.8, 6.2 Hz, H-6’b), 3.88 (1H, q, J =
6.5 Hz, H-3”), 3.80 (3H, s, -OCH3), 3.50 (1H, ddd, J =
9.5, 6.2, 2.2 Hz, H-5’), 3.44 - 3.31 (3H, m, H-2’, 3’ and
4’), 1.66 (1H, dq, J = 13.9, 7.4 Hz, H-5”a), 1.50 (1H, dq,
J = 13.9, 7.4 Hz, H-5”b), 1.15 (3H, d, J = 6.5 Hz, H3-4”),
0.80 (3H, dd, J = 7.4, 7.4 Hz, H3-6”); 13C-NMR (CD3OD,
100 MHz): Table 1; HR-ESI-MS (positive-ion mode)
m/z: 455.1524 [M+Na]+ (calcd for C19H28O11Na,
455.1523).
3.9. Microtropin O (6)
Amorphous powder; [  ]D
25 ‒32.1 (c 0.34, MeOH); IR
max (film): 3366, 2932, 1736, 1512, 1457, 1265, 1241,
1167, 1073, 1038 cm‒1; UV

max (MeOH): 271 (3.35),
224 (3.82) nm (log
 ); 1H-NMR (CD3OD, 400 MHz) δ:
7.10 (1H, dd, J = 8.3, 0.4 Hz, H-5), 7.03 (1H, d, J = 1.9
Hz, H-2), 6.87 (1H, ddd, J = 8.3, 1.9, 0.4 Hz, H-6), 4.87
(1H, d, J = 7.5 Hz, H-1’), 4.63 (1H, dd, J = 11.9, 2.1 Hz,
H-6’a), 4.31 (1H, d, J = 6.7 Hz, H-7), 4.18 (1H, dd, J =
11.9, 5.6 Hz, H-6’b), 3.89 (1H, q, J = 6.4 Hz, H-3”), 3.87
(3H, s, -OCH3), 3.79 (1H, dq, J = 6.7, 6.4 Hz, H-8), 3.61
(1H, ddd, J = 9.5, 5.6, 2.1 Hz, H-5’), 3.50 - 3.38 (3H, m,
H-2’, 3’ and 4’), 1.71 (1H, dq, J = 14.9, 7.4 Hz, H-5”a),
1.53 (1H, dq, J = 14.9, 7.4 Hz, H-5”b), 1.15 (3H, d, J =
6.4 Hz, H3-4”), 0.98 (3H, d, J = 6.4 Hz, H3-9), 0.83 (3H,
dd, J = 7.4, 7.4 Hz, H-6”); 13C-NMR (CD3OD, 100 MHz):
Table 1; HR-ESI-MS (positive-ion mode) m/z: 513.1947
[M+Na]+ (calcd for C22H34O12Na, 513.1942).
3.10. Microtropin P (7)
Amorphous powder; [  ]D
26 ‒49.0 (c 0.13, MeOH); IR
max (film): 3362, 2933, 1736, 1512, 1457, 1266, 1231,
1165, 1073, 1025 cm‒1; UV

max (MeOH): 273 (3.44),
223 (3.94) nm (log
 ); 1H-NMR (CD3OD, 400 MHz) δ:
7.10 (1H, d, J = 8.4 Hz, H-5), 7.03 (1H, d, J = 1.9 Hz,
H-2), 6.87 (1H, dd, J = 8.4, 1.9 Hz, H-6), 4.87 (1H, d, J =
7.5 Hz, H-1’), 4.62 (1H, dd, J = 11.8, 2.2 Hz, H-6’a),
4.31 (1H, d, J = 6.9 Hz, H-7), 4.18 (1H, dd, J = 11.8, 6.1
Hz, H-6’b), 3.90 (1H, q, J = 6.4 Hz, H-3”), 3.87 (3H, s,
-OCH3), 3.79 (1H, dq, J = 6.9, 6.4 Hz, H-8), 3.61 (1H,
ddd, J = 9.5, 6.1, 2.2 Hz, H-5’), 3.52 - 3.30 (3H, m, H-2’,
3’ and 4’), 1.70 (1H, dq, J = 14.9, 7.5 Hz, H-5”a), 1.54
(1H, dq, J = 14.9, 7.5 Hz, H-5”b), 1.15 (3H, d, J = 6.4 Hz,
H3-4”), 0.98 (3H, d, J = 6.4 Hz, H3-9), 0.83 (3H, dd, J =
7.5, 7.5 Hz, H-6”); 13C-NMR (CD3OD, 100 MHz): Table
1; HR-ESI-MS (positive-ion mode) m/z: 513.1944
[M+Na]+ (calcd for C22H34O12Na, 513.1942).
3.11. Sugar Analysis
About 500 g of each of microtropins J-P (1-7) was hy-
drolyzed with 1M HCl (0.2 mL) at 90˚C for 2 h. The
reaction mixtures were partitioned with an equal amount
of EtOAc (0.2 mL), and then the water layers were ana-
lyzed with a chiral detector (JASCO OR-2090 plus) on
an amino column [Asahipak NH2P-50 4E, CH3CN-H2O
(3:1), 1 mL/min]. The hydrolyzates gave a peak for D-
glucose at 8.8 min with each positive optical rotation
sign. The peaks were identified by co-chromatography
with authentic samples.
3.12. Mild Alkaline Hydrolysis of 2
Microtropin K (2) (19.9 mg) in 450 L of MeOH was

Microtropins J-P: 6’-O-(2”S,3”R)-2”-Ethyl-2”,3”-Dihydroxybutyrates of Phenolic Alcohol
 -D-Glucopyranosides from the Branches of Microtropis japonica
1959
hydrolyzed with 50 L of 1M CH3ONa at 25˚C for 34
hrs. The reaction mixture was neutralized with Amberlite
IR-120B (H+) and filtered. The filtrate was evaporated
and the residue was purified by HPLC (H2O-MeOH, 17:3)
to give 14.1 mg of 2a from the peak at 3 min and 2.0 mg
of 2b from the peak at 21 min. Vanillic acid  -D-glu-
copyranoside (2a): amorphous powder; [  ]D
22 ‒61.8 (c
0.85, MeOH); IR max (film): 3334, 2937, 1603, 1559,
1413, 1383, 1261, 1216, 1073, 1026 cm‒1; UV 
max
(MeOH): 328 (3.02), 287 (3.51), 246 (3.85) nm (log  );
1H-NMR (CD3OD, 400 MHz) δ: 7.65 (1H, d, J = 1.9 Hz,
H-2), 7.57 (1H, dd, J = 8.4, 1.9 Hz, H-6), 7.13 (1H, d, J =
8.4 Hz, H-5), 4.96 (1H, d, J = 7.5 Hz, H-1’), 3.89 (3H, s,
-OCH3), 3.87 (1H, dd, J = 11.2, 2.0 Hz, H-6’a), 3.70 (1H,
dd, J = 11.2, 4.9 Hz, H-6’b), 3.34 - 3.54 (4H, m, H-2’, 3’,
4’ and 5’), 13C-NMR (CD3OD, 100 MHz): 173.0 (C-7),
150.7 (C-4), 150.1 (C-3), 130.9 (C-1), 124.2 (C-6), 116.5
(C-5), 114.7 (C-2), 102.4 (C-1’), 78.3 (C-5’), 77.9 (C-3’),
74.9 (C-2’), 71.4 (C-4’), 62.5 (C-6’), 56.7 (-OCH3);
HR-ESI-MS (positive-ion mode) m/z: 353.0848 [M+Na]+
(calcd for C14H18O9Na, 353.0843). Methyl 2-ethyl-
(2S,3R)-2,3-dihydroxybutyrate (2b): colorless liquid,
[  ]D
22 ‒3.2 (c 0.09, MeOH); IR max (film): 3411, 2978,
2940, 1735, 1558, 1511, 1457, 1381, 1172, 1078 cm‒1;
1H-NMR (CD3OD, 400 MHz) δ: 3.88 (1H, q, J = 6.5 Hz,
H-3), 3.75 (3H, s, -OCH3), 1.70 (1H, dq, J = 13.8, 7.4 Hz,
H-5a), 1.53 (1H, dq, J = 13.8, 7.4 Hz, H-5b), 1.16 ((3H, d,
J = 6.5 Hz, H3-4), 0.84 (3H, dd, J = 7.4, 7.4 Hz, H3-6);
13C-NMR (CD3OD, 100 MHz): 176.9 (C-1), 82.9 (C-2),
72.8 (C-3), 52.7 (-OCH3), 29.3 (C-5), 16.9 (C-4), 8.3
(C-6); HR-ESI-MS (positive-ion mode) m/z: 185.0786
[M+Na]+ (calcd for C7H14O4Na, 185.0784).
4. Acknowledgements
The authors are grateful for access to the superconduct-
ing NMR instrument (JEOL JNM  -400) at the Analyti-
cal Center of Molecular Medicine of the Hiroshima Uni-
versity Faculty of Medicine and an Applied Biosystem
QSTAR XL system ESI (Nano Spray)-MS at the Analy-
sis Center of Life Science of the Graduate School of
Biomedical Sciences, Hiroshima University. This work
was supported in part by Grants-in-Aid from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan (Nos. 22590006 and 23590130), the Japan So-
ciety for the Promotion of Science, and the Ministry of
Health, Labour and Welfare. Thanks are also due to the
Research Foundation for Pharmaceutical Sciences and
the Takeda Science Foundation for the financial support.
REFERENCES
[1] S. M. Kapchan, Y. Komoda, W. A. Court, G. J. Thomas,
R. M. Smith, A. Karim, C. J. Gilmore, R. C. Haltiwagner
and B. F. Bryan, “Maytansine, a Novel Antileukemic An-
sa Macrolide from Maytenus ovate,” Journal of American
Chemical Society, Vol. 94, No. 4, 1972, pp. 1354-1956.
http://dx.doi.org/10.1021/ja00759a054
[2] J. M. Cassady, K. K. Chan, H. G. Floss and E. Leistner,
“Recent Developments in the Maytansinoid Antitumor
Agents,” Chemical and Pharmaceutical Bulletin, Vol. 52,
No. 1, 2004, pp. 1-26. http://dx.doi.org/10.1248/cpb.52.1
[3] Y. Koyama, S. Sugimoto, K. Matsunami and H. Otsuka,
“Microtropiosides A-F: ent-Labdane Diterpenoid Gluco-
sides from the Leaves of Microtropis japonica (Celastra-
ceae),” Phytochemistry, Vol. 71, No. 5-6, 2010, pp. 67-
681. http://dx.doi.org/10.1016/j.phytochem.2010.01.004
[4] Y. Uemura, S. Sugimoto, K. Matsunami, H. Otsuka, Y.
Takeda, M. Kawahata and K. Yamaguchi, “Microtropins
A-I: 6’-O-(2”S,3”R)-2”-ethyl-2”,3”-dihydroxy-butyrates of
Aliphatic  -D-Glucopyranosides from the Branches of
Microtropis japonica,” Phytochemistry, Vol. 87, No. 1,
2013, pp. 140-147.
http://dx.doi.org/10.1016/j.phytochem.2012.11.007
[5] A. Sakushima, M. Coşkun and T. Maoka, “Hydroxy-Ben-
zoic Acids from Boreava orientalis,” Phytochemistry, Vol.
40, No. 1, 1995, pp. 257-261.
http://dx.doi.org/10.1016/0031-9422(95)00059-G
[6] Y. Asakawa, M. Toyota and L. Harrison, “Isotachin A
and Isotachin B, Two Sulphur-Containing Acrylates from
the Liverwort Isotachis japonica,” Phytochemistry, Vol.
24, No. 7, 1985, pp. 1505-1508.
http://dx.doi.org/10.1016/S0031-9422(00)81055-9
[7] B. A. A. A. Balboul, A. A. Ahmed, H. Otsuka and A. de
Adams, “4-Hydroxyphenylpropan-7,8-diols and Deriva-
tives from Narvalina domingensis,” Phytochemisty, Vol.
42, No. 4, 1996, pp. 1191-1193.
http://dx.doi.org/10.1016/0031-9422(96)00116-1