Phenolic compounds from the branches of eucalyptus maideni

Article abstract of DOI:10.1002/cbdv.201100021

Three new phenolic compounds, eucalmaidin F (1), (3S)-5-guaiacyl-3- hydroxypentanoic acid (2), and 8-β-C-glucopyranosyl-5,7-dihydroxy-2- isobutylchromone (3), were isolated from the branches of E. maideni, together with 30 known compounds, including four

Full text of DOI:10.1002/cbdv.201100021

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Phenolic Compounds from the Branches of Eucalyptus maideni
by Li-Wen Tiana)b), Min Xua), Yan Li a), Xing-Yao Li a)b), Dong Wanga), Hong-Tao Zhua),
Chong-Ren Yanga)c), and Ying-Jun Zhang*a)
a) State Key Laboratory of Phytochemistry and Plant Resources of West China, Kunming Institute of
Botany, Chinese Academy of Sciences, Kunming 650204, P. R. China
(phone: þ86-871-5223235; fax: þ86-871-5150124; e-mail: zhangyj@mail.kib.ac.cn)
b) Graduate University of the Chinese Academy of Sciences, Beijing 100049, P. R. China
c) Weihe Biotech Research and Development Center, Yuxi 653101, P. R. China
Three new phenolic compounds, eucalmaidin F (1), (3S)-5-guaiacyl-3-hydroxypentanoic acid (2),
and 8-b-C-glucopyranosyl-5,7-dihydroxy-2-isobutylchromone (3), were isolated from the branches of E.
maideni, together with 30 known compounds, including four phenylpropanoids, three lignans, four
phloroglucinol glucosides, five dihydroflavonoids, seven simple phenolic compounds, six terpenoids, and
glycerol. The new structures were established by spectroscopic studies (MS, and 1D- and 2D-NMR),
chemical degradation, and modified Mosherꢀs method. Compounds 3, guaiacylglycerol, 3-hydroxy-1-(4-
hydroxyphenyl)propan-1-one, caffeic acid, (2E)-3-(4-hydroxyphenyl)prop-2-enoic acid, (7’S,8R,8’R)-
lyoniresinol, (þ)-lyoresinol 3a-O-a-l-rhamnopyranoside, garcimangosone, phlorocetophenone 2’-glu-
copyranoside, (þ)-taxifolin 3a-O-a-l-rhamnopyranoside, (þ)-aromadendrin, (þ)-taxifolin, resveratrol,
piceatannol, 3,4,5-trihydroxyphenol. Tachiaside, gallic acid, macrocapals A und G, and oleuropeic acid
were evaluated for their cytotoxicities against five human cancer cell lines. Resveratrol, piceatannol,
gallic acid, and macrocapal G exhibited moderate inhibitory effects on human myeloid heukemia HL-60
cell, with IC50 values of 22.05, 22.05, 7.75, and 31.93 mm, respectively; and only macrocapal G showed
inhibitory effect on hepatocellular carcinoma SMMC-7721 cell, with an IC50 value of 26.75 mm.
Introduction. – The genus Eucalyptus (Myrtaceae), mainly occurring in the tropical
and subtropical areas of the world, is known to be a rich source of bioactive secondary
metabolites. A series of terpenoids, tannins, flavonoids, and phloroglucinol derivatives
with antiviral and antibacterial effects have been reported [1][2]. In our previous
works, five new (þ)-oleuropeic acid derivatives [3] and five new phloroglucinol
glycosides [4] were identified, respectively, from the fresh leaves and fresh fruits of
Eucalyptus maideni F. Muell., a tall timber tree growing widely in the southern parts of
China. To further study the chemical constituents of Eucalyptus trees and to search for
bioactive phenolics, the investigation on the air-dried branches of this species was
carried out. This led to the isolation of three new phenolic compounds, eucalmaidin F
(1), (3S)-5-guaiacyl-3-hydroxypentanoic acid (2), and 8-b-C-glucopyranosyl-5,7-dihy-
droxy-2-isobutylchromone (3; Fig. 1), together with 30 known compounds, including
four phenylpropanoids, three lignans, four phloroglucinol glucosides, four dihydro-
flavonoids, seven other phenolic compounds, six terpenoids, and glycerol. Here, we
report the isolation and structure elucidation of the new compounds. In addition,
several compounds were evaluated for their cytotoxicity against five human cancer cell
lines.
ꢁ 2012 Verlag Helvetica Chimica Acta AG, Zꢂrich

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Fig. 1. New compounds isolated from the branches of Eucalyptus maideni
Results and Discussion. – The 80% aqueous acetone extract of the branches of E.
maideni was partitioned between AcOEt and H2O. The AcOEt fraction was subjected
to various column chromatographies (CC) and semipreparative HPLC to yield 33
compounds. The known compounds were determined as guaiacylglycerol [5], 3-
hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one [6], caffeic acid, (2E)-3-(4-
hydroxyphenyl)prop-2-enoic acid [7], (7’S,8R,8’R)-lyoniresinol [8], (þ)-lyoniresinol
3a-O-a-l-rhamnopyranoside [9], (þ)-lyoniresinol 3a-O-b-d-xylopyranoside [10],
garcimangosone [11], phloracetophenone 2’-glucopyranoside [12], eucalmainosides
D and E [4], (þ)-taxifolin 3-O-a-l-rhamnopyranoside [13], engelitin [14], (þ)-
aromadendrin [14], eriodictyol [14], (þ)-taxifolin [14], (þ)-catechin [7], resveratrol
[15], piceatannol [16], 3,4,5-trihydroxyphenol [17], tachioside, gallic acid, 3-O-
methylellagic acid 3’-O-a-l-rhamnopyranoside [4], macrocapals A and G [18], (ꢀ)-
oleuropeic acid, b-sitosterol [7], icariside B4 [19], eucalmaidin A [3], and glycerol, on
the basis of detailed spectroscopic analyses, together with the comparison of their
spectroscopic and physical data with those in the literature. (þ)-Lyoniresinol 3a-O-a-l-
rhamnopyranoside, (þ)-lyoniresinol 3a-O-b-d-rhamnopyranoside, garcimangosone,
and phloroacetophenone 2’-glucopyranoside were reported from the genus Eucalyptus
for the first time.
Compound 1 was obtained as an amorphous powder. Its high-resolution (HR) ESI-
MS displayed a [MþCl]ꢁ ion peak at m/z 547.1954 (calc. for C25H36ClO1ꢁ1, 547.1946),
indicating the molecular formula C25H36O11. The 1H- and 13C-NMR spectra showed one
2-H singlet (d(H) 6.40 (s)) and signals of six symmetric aromatic C-atoms at d(C) 155.6
(C), 154.8 (2 C), 135.0 (C), and 96.5 (2 CH), arising from a symmetrically substituted
phloroglucinol moiety, a set of signals characteristic of a b-d-glucopyranosyl moiety
(anomeric H-atom signal at d(H) 4.85 (d, J¼7.7)), as well as signals of three MeO
groups (d(H) 3.79 (s, 6 H), 3.70 (s, 3 H)). Besides, the 13C-NMR and DEPT spectra
showed ten C-atom signals attributed to one COO group (d(C) 168.7), one

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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trisubstituted C¼C bond (d(C) 141.5, 131.1), two Me groups (d(C) 27.1, 26.4), three
CH2 groups (d(C) 28.5, 26.3, 24.4), one CH group (d(C) 45.6), and one O-bearing
quaternary C-atom (d(C) 72.8), arising from an oleuropeic acid unit. These data were
similar to those of cypellocarpin A, an oleuropeic acid derivative isolated from E.
cypellocarpa [20]. The positions of the oleuropeoyl ester and glycosidic linkages in 1
were established by 2D-NMR experiments. In the HMBC spectrum of 1, the glucosyl
CH2(6’) (d(H) 4.52, 4.23–4.21) and HꢁC(1’) (d(H) 4.85) correlated, respectively, with
the oleuropeoyl carboxylic C-atom C(7’’) (d(C) 168.7) and C(1) (d(C) 155.6) of the
phloroglucinol unit. In addition, the glucosyl anomeric H-atom (d(H) 4.85) showed
ROESY correlation with the phloroglucinol aromatic H-atoms (d(H) 6.40), confirming
the glycosidic linkage at C(1). Methanolysis of 1 with MeONa in MeOH afforded the
(ꢁ)-oleuropeic acid methyl ester ([a]D ¼ ꢁ32.9 (CHCl3)) [21]. Based on the above
evidence, the structure of 1 was determined to be (ꢁ)-3,4,5-trimethoxyphenol O-(6-O-
oleuropeoyl)-b-d-glucopyranoside1) and named eucalmaidin F.
Compound 2 was obtained as a pale amorphous powder. The molecular formula
C12H15O5 was elucidated from the HR-ESI-MS (m/z 239.0915 ([MꢁH]ꢁ , calc.
239.0919)). The 13C-NMR spectrum of 2 revelead the presence of a COO group (d(C)
179.7), six aromatic C-atoms (d(C) 148.8 (C), 145.4 (C), 135.2 (C), 121.8 (CH), 116.1
(CH), and 113.2 (CH)); an O-bearing CH group (d(H) 69.4), a MeO group (d(H)
56.3), and three CH2 group (d(C) 44.7, 42.7, 32.5). In the 1H-NMR spectrum, signals of
three aromatic H-atoms (d(H) 6.78 (d, J ¼ 1.6), 6.69 (d, J ¼ 8.0), and 6.63 (dd, J¼8.0,
1.6)), arising from a 1,3,4-trisubstitued benzene ring, of a MeO H-atom (d(H) 3.82 (s)),
and several aliphatic H-atom signals were observed. The ROESY correlation between
the MeO H-atom (d(H) 3.82) and HꢁC(2’) (d(H) 6.78) indicated the location of the
MeO group as C(3’), and the OH group was, accordingly, at C(4’) of the benzene ring.
From the HSQC and 1H,1H-COSY spectra of 2,
a partial structure of
ꢁCH2ꢁCH(OH)ꢁCH2ꢁCH2ꢁ from C(2) to C(5) could be elucidated. In the HMBC
spectrum of 2, correlations of the signals of HꢁC(2) and HꢁC(3) with that of the
carboxy C-atom, and of HꢁC(5) with those aromatic C-atoms C(1’), C(2’) and C(6’)
positioned the partial structure ꢁCH2ꢁCH(OH)ꢁCH2ꢁCH2ꢁ between the carboxy C-
atom and the aromatic ring (Fig. 2), similar to 3-hydroxy-5-phenylpentanoic acid [19].
The absolute configuration of C(3) was determined by applying the modified Mosherꢀs
method. Methylation of 2 with MeOH in AcCl afforded 2a. Treatment of 2a with (þ)-
(R)- and (ꢁ)-(S)-MTPA (MTPA¼3,3,3-trifluoro-2-methoxy-2-phenylpropanoic acid)
in the presence of N,N-dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyr-
idine (DMAP) provided mono ester derivatives, the Dd(H) (S–R) values (Scheme)
established the (S) configuration at C(3) of 2. Thus, compound 2 was determined to be
(3S)-5-guaiacyl-3-hydroxypentanoic acid.
Fig. 2. Key HMBC (H!C) and 1H,1H-COSY (—) correlations of compound 2
1
)
The absolute configuration of glucose was assumed to be d based on biogenetic considerations.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme. Determination of the Absolute Configuration of 2 by the MTPA Method
Compound 3, a pale yellow amorphous powder, had a molecular formula C19H23O9,
deduced from the HR-ESI-MS (m/z 395.1345 ([MꢁH]ꢁ , calc. 395.1342)). The 1H- and
13C-NMR spectra of 3, coupled with HSQC spectrum, exhibited two lower-field H-atom
signals at d(H) 6.06 and 6.23 (HꢁC(3) and HꢁC(6)); signals of a CH2 group at d(H)
2.58 and 2.50 (2dd, J¼6.5, 13.9), of a CH group at d(H) 2.21–2.23 (m) coupled with
two Me signals at d(H) 1.03 and 1.00 (3d, J¼6.6), and a set of aliphatic H-atom signals
arising from a b-glucopyranosyl moiety (anomeric H-atom signal at d(H) 4.60 (d, J¼
9.8)). These NMR features were similar to those of 8-b-C-glucopyranosyl-5,7-
dihydroxy-2-isopropylchromone [22]. However, instead of the signals of an i-Pr side
chain at C(2) of the known compound, compound 3 exhibited the C-atom signals (d(C)
44.1 (CH2), 28.2 (CH), 22.8 (Me) and 22.5 (Me)) attributed to an isobutyl group. In the
13C-NMR spectrum of 3, resonances ascribable to chromone and glucosyl moieties were
also observed. The HMBCs of the glucosyl anomeric H-atom with C(7) (d(C) 165.1),
C(8) (d(C) 105.6), and C(9) (d(C) 158.4) indicated the location of the b-C-
glucopyranosyl unit as C(8) of the chromone skeleton. This was confirmed by
comparison of the 13C-NMR data in (D6)DMSO of 3 (C(6) at d(C) 98.2) with those of
8-b-C-(C(6) at d(C) 98) and 6-b-C-glucopyranosyl-5,7-dihydroxy-2-isopropylchro-
mone (C(8) at d(C) 93) recorded in (D6)DMSO [22]. Accordingly, the structure of 3
was determined as 8-b-C-glucopyranosyl-5,7-dihydroxy-2-isobutylchromone.
Compound 3 and further 20 known compounds were evaluated for their
cytotoxicities against five human cancer cell lines using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method as reported in [23], with
cisplatin as positive control. As a result, only compounds resveratrol, piceatannol, gallic
acid, and macrocapal G exhibited moderate inhibitory effects on human myeloid
heukemia HL-60 cell line with IC50 values of 22.05, 22.05, 7.75, and 31.93 mm,
respectively, relative to 1.34 mm of cisplatin, and macrocapal G also showed inhibitory
effect on hepatocellular carcinoma SMMC-7721 cell line with an IC50 value of 26.75 mm
(cisplatin: 12.16 mm). All the tested compounds showed no activity to the breast cancer
MCH-7, colon cancer SW480, and lung cancer A-549 cell lines, at a concentration of
40 mm.
Conclusions. – The branches of Eucalyptus species are rich in diverse phenolic
compounds. Of them, oleuropeic acid derivatives may be evolved from terpineol, one
of the major chemical constituents of Eucalyptus oil [24]. The isolated dihydroflavo-
noid, (þ)-aromadendrin, and phenolic compounds, (þ)-catechin, piceatannol, gallic
acid, and 3-O-methylellagic acid 3’-O-a-l-rhamnopyranoside, also reported as the
chemical constituents of E. kino, a dark exudate when the active cambium is injured
[25], probably serve as the consistent preventive constituents of Eucalyptus maideni.
The occurrence of macrocapals in the branches of E. maideni is very surprising, since

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they are reported to be inducible second metabolites involved in hostꢁpathogen
interactions in the wounded wood of E. globulus and E. nitens [26]. Though some
chemical constituents isolated from Eucalyptus plants displayed potential anti-tumor-
promoting activities [20][27–29], only four compounds (see above) from this study
showed moderate inhibitory effects on HL-60 and SMMC-7721 cell lines. The
biological functions of these compounds need further studies.
Experimental Part
General. Column chromatography (CC): MCI-gel CHP-20P (75–150 mm; Mitsubishi Chemical Co.),
Sephadex LH-20 (25–100 mm; Pharmacia Fine Chemical Co. Ltd.), Toyopearl HW-40F (TOSOH,
Japan), RP-8 (40–63 mm; Merck), and silica gel (SiO2; 200–300 mesh; Qingdao Haiyang Chemical Co.
Ltd.). TLC: SiO2 plates; detection by anisaldehydeꢁH2SO4 reagent or 10% H2SO4 reagents, followed by
heating. Semiprep. reversed-phase (RP) HPLC: Waters 600 liquid chromatograph with a Zorbax SB-C18
column. Optical rotations: JASCO-20 polarimeter. UV Spectra: Shimadzu UV-2401A spectrometer;
MeOH solns.; lmax (log e) in nm. IR Spectra: Bio-Rad-FTS-135 spectrometer; KBr pellets; ˜n in cmꢁ1. 1D-
and 2D-NMR spectra: Bruker-AM-400 and -DRX-500 spectrometers; d in ppm rel. to Me4Si as internal
standard, J in Hz. MS: VG-Auto-Spec-3000 spectrometer with glycerol as matrix for FAB-MS; API-
QSTAR-Pulsar-1 spectrometer for ESI-MS and HR-ESI-MS; in m/z.
Plant Materials. The branches of E. maideni were collected in the Botanical Garden of Kunming
Institute of Botany, Chinese Academy of Sciences, Yunnan, China, during May 2007, and identified by
Prof. Xiao Cheng (Botanical Garden, Kunming Institute of Botany, Chinese Academy of Sciences). A
voucher specimen (KIB-ZL-200702) has been deposited with the State Key Laboratory of Phytochem-
istry and Plant Resoures in West China, Kunming Institute of Botany, Chinese Academy of Sciences.
Extraction and Isolation. The air-dried branches of E. maideni (20 kg) were extracted with 80% aq.
acetone at r.t. (3ꢂ30 l, each 1 week). After removal of org. solvents, the extracts were concentrated to a
smaller volume (5 l) and partitioned with AcOEt (5ꢂ2 l) after filtration of the precipitate. The AcOEt
fraction (220 g) was applied to CC (Sephadex LH-20, MeOH/H2O 0 :1–1:0) to afford seven fractions.
Fr. 1 (40 g) was subjected to CC (SiO2; CHCl3/MeOH/H2O 1:0 :0!6 :4 :1) to yield Frs. 1-1–1-5. Fr. 1-2
was applied to CC (MCI-gel CHP20P; MeOH/H2O 4 :6!7:3; RP-8; MeOH/H2O 4 :6!6 :4) to yield
icariside B4 (7 mg). Fr. 1-3 was subjected to CC (SiO2; CHCl3/MeOH, 95 :5!8.5 :1; MCI-gel CHP20P;
MeOH/H2O 2 :8!1:0; and RP-8; MeOH/H2O 4 :6!7:3) to yield 1 (5 mg), guaiacylglycerol (51 mg),
and (ꢀ)-oleuropeic acid (19 mg). Fr. 1–4 was subjected to CC (SiO2; CHCl3/MeOH 9 :1!7:3; MCI-gel
CHP20P; MeOH/H2O 0 :1!1:1; and Toyopearl HW-40F; MeOH/H2O 0 :1!2 :8) to yield tachioside
(10 mg), eucalmaidin A (37 mg), and glycerol (68 mg). Fr. 2 (8.2 g) was applied to CC (MCI-gel
CHP20P; MeOH/H2O 0 :1!1:0) to yield four fractions, Frs. 2-1–2-4. Fr. 2-2 was subjected to CC (SiO2;
CHCl3/MeOH 1:0!9 :1 and Toyopearl HW-40F; MeOH/H2O 1:1!8 :2) to yield 3-hydroxy-1-(4-
hydroxy-3-methoxyphenyl)propan-1-one (5 mg). Fr. 2-3 was subjected to CC (SiO2; CHCl3/MeOH
1:0!8.5 :1.5; MCI-gel CHP20P; MeOH/H2O 40!80%; Toyopearl HW-40F; MeOH/H2O 1:1!8 :2),
followed by semiprep. HPLC (35% MeOH/H2O) to yield 3 (14 mg), (þ)-lyoniresinol 3a-O-a-l-
rhamnopyranoside (9 mg), (þ)-lyoniresinol 3a-O-b-d-xylopyranoside (4 mg), eucalmainoside E (4 mg),
(þ)-taxifolin 3-O-a-l-rhamnopyranoside (4 mg), and (ꢀ)-oleuropeic acid (235 mg). Fr. 3 (18 g) was
purified by CC (MCI-gel CHP20P; MeOH/H2O 0!100%) to give three fractions, Frs. 3-1–3-3. Fr. 3-1
was subjected to CC (SiO2; CHCl3/MeOH 98 :2!8 :2; MCI-gel CHP20P; MeOH/H2O 2 :8!7:3; and
Toyopearl HW-40F; MeOH/H2O 30!80%) to afford 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)pro-
pan-1-one (22 mg), phloracetophenone 2’-glucopyranoside (6 mg), and eucalmainoside D (10 mg). Fr. 3-2
was chromatographed over MCI-gel CHP20P (MeOH/H2O 20!80%), SiO2 (CHCl3/MeOH/H2O
8.5 :1.5 :0.1!8 :2 :0.2) and RP-8 (MeOH/H2O 40!60%) to yield 2 (18 mg), (7’S,8R,8’R)-lyoniresinol
(17 mg), and garcimangasone (69 mg). Fr. 3-3 was subjected to CC (MCI-gel CHP20P; MeOH/H2O
3 :7!6 :4) to yield 3-O-methylellagic acid 3-O-a-l-rhamnopyranoside (16 mg). Fr. 4 (36 g) was
subjected to CC (MCI-gel CHP20P; MeOH/H2O 0 :1!1:1) to yield gallic acid (7 g). Fr. 5 (29 g) was

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subjected to CC (MCI-gel CHP20P; MeOH/H2O 0 :1!1:0; SiO2; CHCl3/MeOH 9 :1!7.5 :2.5; and
Toyopearl HW-40F; MeOH/H2O 1:1!1:0) to yield caffeic acid (300 mg), (2E)-3-(4-hydroxyphenyl)-
prop-2-enoic acid (22 mg), engelitin (14 mg), and (þ)-aromadendrin (22 mg). Fr. 6 (33 g) was
chromatographed over MCI-gel CHP20P (MeOH/H2O 0!100%) to yield five fractions, Frs. 6-1–6-5.
Frs. 6-1 and 6-2 were subjected to CC (SiO2; CHCl3/MeOH 9 :1!7:3; and MCI-gel CHP20P; MeOH/
H2O 4 :6!8 :2) to yield (þ)-catechin (77 mg) and resveratrol (24 mg). Fr. 6-3 was subjected to CC (SiO2;
CHCl3/MeOH 9 :1!7:3; and Toyopearl HW-40F; MeOH/H2O 3 :7!8 :2) to yield eriodictyol (230 mg),
and (þ)-taxifolin (4 mg). Frs. 6-4 and 6-5 were subjected to CC (MCI-gel CHP20P; MeOH/H2O 7:3!
1:0) to yield macrocarpal A (65 mg), macrocarpal G (50 mg), and b-sitosterol (6 mg). Fr. 7 (52 g) was
repeatedly subjected to MCI-gel CHP20P (70 to 100% MeOH/H2O) to yield piceattanol (5 mg), and
3,4,5-trihydroxyphenol (90 mg).
Eucalmaidin F (¼ 3,4,5-Trimethoxyphenyl 6-O-{[(4S)-4-(1-Hydroxy-1-methylethyl)cyclohex-1-en-1-
yl]carbonyl}-b-d-glucopyranoside; 1). Pale amorphous powder. [a]1D5 ¼ ꢁ45.4 (c¼0.2, MeOH). UV
(MeOH): 205 (4.65), 308 (3.38). IR: 3431, 2964, 2930, 1709, 1601, 1505, 1464, 1128, 1070. 1H-NMR
(500 MHz, CD3OD): 6.96–6.95 (m, HꢁC(2’’)); 6.40 (s, HꢁC(2/6)); 4.85 (d, J¼7.7, HꢁC(1’)); 4.52 (dd, J¼
2.0, 12.0, HꢁC(6’)); 4.23–4.21 (m, HꢁC(6’)); 3.79 (s, MeOꢁC(3), MeOꢁC(5)); 3.78–3.68 (m, HꢁC(5’));
3.70 (s, MeOꢁC(4)); 3.46 (t, J¼8.9, HꢁC(3’)); 3.42 (t, J¼7.7, HꢁC(2’)); 3.36 (t, J¼8.9, HꢁC(4’)); 2.46–
2.43 (m, HꢁC(6’’)); 2.31–2.30 (m, HꢁC(3’’)); 2.10–2.08 (m, HꢁC(6’’)); 2.00–1.97 (m, HꢁC(3’’/5’’));
1.52–1.51 (m, HꢁC(4’’)); 1.21–1.20 (m, HꢁC(5’’)); 1.17 (s, HꢁC(9’’/10’’)). 13C-NMR (125 MHz, CD3OD):
168.7 (C(7’’)); 155.6 (C(1)); 154.8 (C(3/5)); 141.5 (C(2’’)); 135.0 (C(4)); 131.1 (C(1’’)); 102.7 (C(1’)); 96.5
(C(2/6)); 77.7 (C(3’)); 75.5 (C(5’)); 74.8 (C(2’)); 72.8(C(8’’)); 71.8 (C(4’)); 64.8 (6’)); 56.7 (MeOꢁC(3),
MeOꢁC(5)); 61.3 (MeOꢁC(4)); 45.6 (C(4’’)); 28.5 (C(3’’)); 27.1(C(9’’)); 26.4 (C(10’’)); 26.3 (C(6’’)); 24.4
(C(5’’)). FAB-MS (neg.): 511 ([MꢁH]ꢁ ). HR-ESI-MS: 547.1954 ([MþCl]ꢁ , C25H36ClO1ꢁ1 ; calc.
547.1946).
(3S)-5-Guaiacyl-3-hydroxypentanoic Acid (¼(3S)-3-Hydroxy-5-(4-hydroxy-3-methoxyphenyl)pen-
tanoic Acid; 2). Pale amorphous powder. [a]2D5 ¼ ꢁ6.8 (c¼1.8, MeOH). UV (MeOH): 225 (3.66),
281(3.35). IR: 3422, 2935, 2856, 1710, 1602, 1517, 1399, 1273, 1033. 1H-NMR (400 MHz, CD3OD): 6.78 (d,
J¼1.6, HꢁC(2’)); 6.69 (d, J¼8.0, HꢁC(5’)); 6.63 (dd, J¼1.6, 8.0, HꢁC(6’)); 3.90–3.86 (m, HꢁC(3)); 3.82
(s, MeOꢁC(3’)); 2.70–2.65 (m, HꢁC(5)); 2.60–2.55 (m, HꢁC(5)); 2.42 (dd, J¼2.4, 11.6, HꢁC(2)); 2.36
(dd, J¼6.4, 11.6, HꢁC(2)); 1.74–1.72 (m, CH2(4)). 13C-NMR (100 MHz, CD3OD): 179.7 (C(1)); 148.8
(C(3’)); 145.4 (C(4’)); 135.2 (C(1’)); 121.8 (C(6’)); 116.1 (C(5’)); 113.2 (C(2’)); 69.4 (C(3)); 56.3 (MeO);
44.7 (C(2)); 42.7 (C(4)); 32.5 (C(5)). FAB-MS (neg.): 239 ([MꢁH]ꢁ ). HR-ESI-MS: 239.0915 ([Mꢁ
H]ꢁ , C12H15Oꢁ5 ; calc. 239.0919).
8-b-C-Glucopyranosyl-5,7-dihydroxy-2-isobutylchromone (¼(1S)-1,5-Anhydro-1-[5,7-dihydroxy-2-
(2-methylpropyl)-4-oxo-4H-1-benzopyran-8-yl]-d-glucitol; 3). Pale yellow amorphous powder. [a]D15
¼
ꢁ1.3 (c¼0.3, MeOH). UV (MeOH): 208 (4.37), 251 (4.28), 297 (3.83). IR: 3405, 2959, 2872, 1660, 1619,
1427, 1274, 1086, 1023. 1H-NMR (500 MHz, CD3OD): 6.23 (s, HꢁC(6)); 6.06 (s, HꢁC(3)); 4.60 (d, J¼9.8,
HꢁC(1’)); 4.09 (t, J¼9.0, HꢁC(2’)); 3.88 (d, J¼11.5, HꢁC(6’)); 3.66–3.64 (m, HꢁC(6’)); 3.48–3.45 (m,
HꢁC(3’)); 3.42–3.40 (m, HꢁC(4’/5’)); 2.58 (dd, J¼6.5, 13.9, HꢁC(11)); 2.50 (dd, J¼6.5, 13.9, HꢁC(11));
2.23–2.21 (m, HꢁC(12)); 1.03 (d, J¼6.6, Me(13)); 1.00 (d, J¼6.6, Me(14)). 13C-NMR (125 MHz,
CD3OD): 184.2 (C(4)); 171.5 (C(2)); 165.1 (C(7)); 162.7 (C(5)); 158.4 (C(9)); 109.2 (C(3)); 105.6
(C(8)); 105.1 (C(10)); 100.2 (C(6)); 82.6 (C(5’)); 80.1 (C(3’)); 75.1 (C(1’)); 72.9 (C(2’)); 69.8 (C(4’)); 62.2
(C(6’)); 44.1 (C(11)); 28.2 (C(12)); 22.8 (C(13)); 22.5 (C(14)). FAB-MS (neg.): 395 ([MꢁH]ꢁ ). HR-
ESI-MS: 395.1345 ([MꢁH]ꢁ , C19H23O9ꢁ ; calc. 395.1342).
Methanolysis of 1. A soln. of 1 (2 mg) in 0.02m MeONa in MeOH (1 ml) was kept standing at r.t. for
12 h. The soln. was then subjected to CC over MCI-gel CHP20P (1.5ꢂ14 cm); H2O, 60% and 100%
MeOH/H2O to give (ꢁ)-methyl 4-(1-hydroxy-1-methylethyl)cyclohex-1-ene-carboxylate (1a; (0.5 mg);
colorless oil. [a]D ¼ ꢁ32.9 (c¼0.063, CHCl3), identified by co-TLC with authentic sample.
Synthesis of Methyl 3-Hydroxy-5-(4-hydroxy-3-methoxyphenyl)pentanonate (2a). According to the
methodology described in [30], 5 ml of MeOH were added to AcCl (0.5 ml) dropwise, under stirring in
an ice bath, to which, compound 2 (6 mg) and molecular sieve desiccant were added. The mixture was
refluxed for 3.5 h (658), and then passed through MCI-gel CHP20P; eluting with H2O, 40% and 100%

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
129
MeOH/H2O, to give 2a (6 mg). Colorless oil. [a]D ¼ ꢁ22.1 (c¼0.1, CHCl3). FAB-MS (neg.): 253 ([Mꢁ
H]ꢁ ).
Preparation of (R)- and (S)-MTPA Esters of 2a. A soln. of 2a (3 mg), (R)-3,3,3-trifluoro-2-methoxy-
2-phenylpropanoic acid (MTPA) or (S)-MTPA (6 mg), N,N-dicyclohexylcarbodiimide (DCC; 4 mg),
and 4-(dimethylamino)pyridine (DMPA; 3 mg) in CH2Cl2 was stirred for 12 h at r.t. [31]. The mixture
was purified by CC (SiO2; CHCl3/MeOH 98 :2) to give (R)- or (S)-MTPA esters of 2a (2 mg). The
1
1
purified derivatives were dried and analyzed by means of H-NMR and H,1H-COSY spectroscopies.
Cytotoxicity Assay. Five human cancer cell lines, breast cancer MCH-7, hepatocellular carcinoma
SMMC-7721, human myeloid heukemia HL-60, colon cancer SW480, and lung cancer A-549 cells, were
used in the cytotoxic assay. All the cells cultured in RPMI-1640 or DMEM medium (Dulbeccoꢀs
Modified Eagle Medium; Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA)
in 5% CO2 at 378. The cytotoxicity assay was performed according to the MTT (¼ 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-2H-tetrazolium bromide) method in 96-well microplates. Briefly, 100 ml of adherent
cells was seeded into each well of 96-well cell-culture plates and allowed to adhere for 12 h before drug
addition, while suspended cells was seeded just before addition with an initial desity of 1ꢂ105 cells/ml.
Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.0625,
0.32, 1.6, 8, and 40 mm in triplicates for 48 h, with cisplatin (Sigma, USA) as positive controls. After
compound treatment, cell viability was detected, and a cell-growth curve was plotted. IC50 Values were
calculated by Reed and Muenchꢀs method.
We are grateful to the members of the Analytical Group in the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, for the recording of all
spectra. This work was supported by the NSFC 2008ZX09401-004 and 2011CB915503.
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Received January 18, 2011