Synthesis of the monoterpenoid esters cypellocarpin C and cuniloside B and evidence for their widespread occurrence in Eucalyptus

Article abstract of DOI:10.1016/j.carres.2010.07.029

Short syntheses of cuniloside B and cypellocarpin C, (+)-(R)-oleuropeic acid-containing carbohydrates, are reported. Also disclosed are syntheses of the noreugenin glycosides, undulatoside A and corymbosins K₁ and K ₂. Leaf extracts of 28 diverse eucalypts revealed cuniloside B to be present in all, and cypellocarpin C to be present in most, of the species examined. The widespread occurrence of these carbohydrate monoterpenoid esters supports their roles in essential oil biosynthesis or mobilization from sites of synthesis to secretory cavity lumena.

Full text of DOI:10.1016/j.carres.2010.07.029

Carbohydrate Research 345 (2010) 2079–2084
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of the monoterpenoid esters cypellocarpin C and cuniloside B
and evidence for their widespread occurrence in Eucalyptus
b
Zalihe Hakki a, Benjamin Cao a, Allison M. Heskes b, Jason Q. D. Goodger b, , Ian E. Woodrow ,
*
Spencer J. Williams a,
*
a Bio21 Molecular Science and Biotechnology Institute and School of Chemistry, The University of Melbourne, Victoria 3010, Australia
b School of Botany, The University of Melbourne, Victoria 3010, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2010
Received in revised form 15 July 2010
Accepted 16 July 2010
Available online 21 July 2010
Short syntheses of cuniloside B and cypellocarpin C, (+)-(R)-oleuropeic acid-containing carbohydrates, are
reported. Also disclosed are syntheses of the noreugenin glycosides, undulatoside A and corymbosins K1
and K2. Leaf extracts of 28 diverse eucalypts revealed cuniloside B to be present in all, and cypellocarpin C
to be present in most, of the species examined. The widespread occurrence of these carbohydrate mono-
terpenoid esters supports their roles in essential oil biosynthesis or mobilization from sites of synthesis to
secretory cavity lumena.
Keywords:
Eucalyptus
Ó 2010 Elsevier Ltd. All rights reserved.
Total synthesis
Monoterpenoids
Oleuropeic acid
Noreugenin
Essential oils
Comprising over 900 species, the genus Eucalyptus (family
Myrtaceae) dominates most Australian landscapes.1 Eucalypt leaves
are prodigious sources of a wide range of mono- and sesquiterpenes
(collectively essential oils), many of which are prized for their
perfumery and pharmaceutical applications.2 The essential oils of
eucalypts are localized in specialized extracellular structures
termed embedded secretory cavities. Recent investigations of the
contents of the foliar secretory cavities of three eucalypts,
Eucalyptus froggattii, Eucalyptus polybractea and Eucalyptus globulus,
revealed that in addition to the volatile essential oil constituents, a
substantial proportion of the cavity lumena is filled by a non-
volatile resinous material.3 Careful examination of the constituents
of this resinous material led to the identification of a novel carbohy-
isolated from bulk leaf extracts of various Eucalyptus species may
also be localized within the non-volatile fraction of foliar secretory
cavities.3 One such compound, cypellocarpin C (2) (isolated from
Eucalyptus cypellocarpa), has elicited special interest due to its
antitumour activity. Compound 2 inhibits Epstein-Barr virus early
antigen activation induced by 12-O-tetradecanoyl phorbol 13-ace-
tate and suppresses in vivo two-stage carcinogenesis induced with
nitric oxide and 12-O-tetradecanoyl phorbol 13-acetate in mice.5 In
order to better understand the roles of carbohydrate monoterpe-
noid esters in eucalypts, we report the synthesis of 1 and 2 and
several related natural products from woody plants and provide a
survey of their distribution in a range of Eucalyptus species.
The preparation of 2 required an efficient synthesis of the corre-
sponding glycoside, undulatoside A (7). Compound 7 is itself a natu-
ral product that was first isolated from Adina rubescens6 and has
since been isolated from a range of plants including Tecomella undu-
lata7 and Eucalyptus tereticornis.8 Previous approaches to the synthe-
drate monoterpenoid, cuniloside B (1), composed of D-glucopyra-
nose esterified to (+)-(R)-oleuropeic acid (Fig. 1).3 Unexpectedly,
given the long period under which eucalypts have been studied
for their natural product contents, the newly discovered 1 was
shown to be present at levels approaching those of the most
abundant volatile monoterpene, 1,8-cineole.3 A contemporaneous
report also identified 1 (therein named eucalmaidin E) from bulk
leaf extracts of Eucalyptus maidenii.4 It was proposed that many
other carbohydrate monoterpene esters that have previously been
sis of
7 utilized a silver carbonate-promoted Koenigs–Knorr
glycosylation of noreugenin by acetobromoglucose (5) and occurred
in poor to moderate yields, possibly owing to the poor solubility of
noreugenin in common organic solvents.9,10 Glycosylation of noreu-
genin under mild phase transfer conditions seemed a promising
alternative to improve the workup procedure, and given the highly
variable outcomes noted by others in glycosylation of phenols, we
surveyed a range of conditions.11 Treatment of noreugenin12 with
5 and 5 equiv of aqueous NaOH (0.14 M) was unsuccessful and TLC
analysis suggested that partial deacetylation was occurring. Using
* Corresponding authors. Tel.: +61 3 8344 7165; fax: +61 3 9347 5460 (J.Q.D.G.);
tel.: +61 3 8344 2422; fax: +61 3 9347 8124 (S.J.W.).
E-mail addresses: jgoodger@unimelb.edu.au (J.Q.D. Goodger), sjwill@unimelb.
edu.au (S.J. Williams).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.07.029

2080
Z. Hakki et al. / Carbohydrate Research 345 (2010) 2079–2084
10''
10''
OH
8''
9''
9''
2''
OH
8''
R
R
2''
OH
O
O
1''
1''
OH
7''
7''
R
O
O
O
O
O
1
1'
O
O
HO
HO
HO
HO
O
CH3
O
1'
1
HO
HO
2 cypellocarpin C
1 cuniloside B
OH
S
OH
S
O
O
O
O
HO
HO
O
HO
3 cuniloside A
Figure 1. Structures of cuniloside B (1), cypellocarpin C (2) and cuniloside A (3).
1 equiv of K2CO3 (0.03 M) the glycoside 6 was obtained in 32% yield.
However, using 5 equiv of K2CO3 (0.14 M) 6 was afforded in 73%
yield. Deacetylation using sodium methoxide in methanol smoothly
afforded 7 in 95% yield (Scheme 1).
We next examined the applicability of this glycosylation meth-
od for the synthesis of another recently reported noreugenin glyco-
OAc
O
OAc
O
AcO
AcO
HBr
OAc
AcO
AcO
AcOH, rt
Br
AcO
AcO
8
9
side, corymbosin K1 (11), an unusual b-
from the Chinese medicinal plant Knoxia corymbosa.13 Accordingly,
phase transfer glycosylation of noreugenin by -allopyranosyl
bromide (9), itself prepared from
-allose pentaacetate (8),14 affor-
ded the corresponding glycoside (10) in 19% yield (Scheme 2). The
lower yield of this reaction can be attributed to the known poorer
glycosylation ability of 9,15 and its lower stability, which in our
hands was found to be significantly less stable than the corre-
D-allopyranoside isolated
OH
O
OR
O
a-D
noreugenin
RO
D
O
O
CH3
aq K2CO3, CHCl3
50 °C
RO
RO
10: R = Ac, 19% from 8
11: R = H, corymbosin K1, 78%
NaOMe
MeOH
sponding
D-gluco bromide 5. Deprotection of 10 using sodium
methoxide in methanol afforded 11 in 78% yield, with spectral data
that matched the literature material, and with some variation ob-
served in melting point and optical rotation.13
Scheme 2. Synthesis of corymbosin K1.
The synthesis of (ꢀ)-(S)-oleuropeic acid has previously been re-
ported starting from (ꢀ)-b-pinene.16 However, (+)-b-pinene is not
readily available and so the synthesis of the enantiomeric (+)-(R)-
oleuropeic acid required an alternative starting material. According
to the reliable procedure of Brown and coworkers, (+)-a-pinene
OAc
O
OAc
O
was isomerized to (+)-b-pinene.17 The transformation of (+)-b-
pinene to (+)-(R)-oleuropeic acid faithfully followed the literature
for the enantiomeric series.16
AcO
AcO
AcO
AcO
HBr
OAc
AcO
AcO
AcOH, rt
Br
In order to establish that 7 exhibited the greatest reactivity at
the primary alcohol, we initially investigated its selective mono-
acetylation (Scheme 3). The product of this reaction is also a natu-
ral product isolated from K. corymbosa, namely, corymbosin K2
(12).13 Low temperature acetylation using acetyl chloride in the
presence of 2,4,6-collidine18 afforded 12 in 74% yield, thereby con-
firming the desired reactivity of the primary alcohol of undulato-
side A. The spectral data for 12 were consistent with those
reported for the natural product.13 Monoacylation of 7 by the
5
4
OH
O
OR
O
noreugenin
RO
RO
O
O
CH3
aq K2CO3, CHCl3
50 °C
RO
6: R = Ac, 73% from 4
7: R = H, undulatoside A, 95%
NaOMe
MeOH
a,b-unsaturated acid of (+)-(R)-oleuropeic acid to afford 2 proved
non-trivial. Attempted esterification under Mitsunobu conditions
(DIAD, Ph3P) was ineffective.19 Similarly, no product could be iso-
lated using DCC/DMAP. The more powerful acylation reagent HBTU
Scheme 1. Synthesis of undulatoside A.

Z. Hakki et al. / Carbohydrate Research 345 (2010) 2079–2084
2081
side reported from E. camaldulensis23 was later reassigned as 224),
E. cypellocarpa (0.03 mg/g dry wt)5 and the fruits of E. globulus
(0.02 mg/g dry wt).24 Of the six subgenera of Eucalyptus examined
here, subgenus symphyomyrtus (by far the largest subgenus with
approximately 500 species)1 generally exhibited the highest con-
centrations of 1 and 2. The only other report describing the co-
occurrence of 1 and 2 is a study of extracts from the leaves of E.
maidenii.25 In contrast to the trend observed here, the concentra-
tion of 2 (0.02 mg/g fresh weight; fr. wt) was found to be greater
than that of 1 (0.003 mg/g fr. wt). Recently, the same group iso-
lated 0.003 mg 1/g fr. wt from the fruits of E. maidenii, but did
not report any 2.26
OH
O
O
OAc
O
AcCl, collidine
-35 °C
HO
HO
O
CH3
HO
12: corymbosin K2, 74%
7
(+)-(R)-oleuropeic acid
HBTU, DMAP, NMM
2: cypellocarpin C, 56%
DMF, 50 °C
Abundant non-volatile compounds, particularly those bearing
an oleuropeic acid group such as 1 and 2, appear to be a common
constituent of leaves and likely the secretory cavities of a signifi-
cant proportion of members of the genus Eucalyptus. The role of
these non-volatile compounds remains to be determined, but they
may be involved in the biosynthesis and/or mobilization of mono-
terpene essential oils from their site of synthesis, presumably in
the epithelial cells lining the secretory cavity lumen, to the extra-
cellular lumen. The present work may therefore be of utility in the
identification of more abundant sources of these secondary
metabolites.
(+)-(R)-oleuropeic acid
HBTU, DMAP, NMM
1: cuniloside B, 7.2%
D-glucose
DMF, 50 °C
Scheme 3. Synthesis of corymbosin K2, cypellocarpin C and cuniloside B.
in the presence of DMAP was also ineffective, whereas HBTU–N-
methylmorpholine (NMM)20 afforded the benzotriazole ester of
oleuropeic acid. Ultimately, use of the combination HBTU–NMM–
DMAP provided 2 in 56% yield (Scheme 3). The requirement for
NMM and DMAP points to the need for a strong base for deproto-
1. Experimental
nation of the a,b-unsaturated acid to promote the formation of the
intermediate benzotriazole ester, and for a nucleophilic acylation
catalyst. The success of this reaction is remarkable given the com-
plexity of the two substrates, which between them possess every
type of alcohol (primary, secondary, tertiary and phenolic), yet
good selectivity is obtained for acylation of the primary alcohol
without needing to resort to the use of protecting groups.
The successful acylation of 7 by (+)-(R)-oleuropeic acid using
HBTU–NMM–DMAP prompted the investigation of a direct synthe-
sis of 1 from D-glucose and (+)-(R)-oleuropeic acid (Scheme 3).
1.1. General methods
Pyridine and collidine were distilled over NaOH before use. DMF
was dried over activated 4 Å molecular sieves. Flash chromatogra-
phy was performed according to the reported method using Merck
Silica Gel 60.27 Solvents were evaporated under reduced pressure
using a rotary evaporator. Melting points were obtained using a
Reichert–Jung hotstage microscope or Gallenkamp capillary appa-
ratus and are uncorrected. Optical rotations were obtained using a
Compound 1 represents a special challenge to synthesize using tra-
ditional protecting group approaches because of the sensitivity of
the ester, alkene and tertiary alcohol functional groups present in
JASCO DIP-1000 polarimeter (Melbourne, Australia). [
a
]
values
D
are given in 10ꢀ1 cm2 gꢀ1 1H and 13C NMR spectra were recorded
.
using Varian Inova 400 or 500 and Bruker 800 instruments (Mel-
bourne, Australia). Chemical shifts are expressed in parts per mil-
lion (d) using residual solvent (1H NMR d 7.26 ppm for CDCl3, d
this target. Treatment of D-glucose with 2 equivalents of (+)-(R)-
oleuropeic acid and HBTU–NMM–DMAP afforded a complex mix-
ture, which was purified by semi-preparative HPLC to afford 1 in
7% yield. While the yield of this procedure is low, it nonetheless
represents a very direct and protecting group free method for the
synthesis of 1.
2.50 ppm for DMSO-d6,
d d
3.31 for CD3OD-d4; 13C NMR
77.16 ppm for CDCl3, d 39.52 ppm for DMSO-d6, d 49.00 for
CD3OD-d4) or TMS as an internal standard (d 0.00 ppm). IR spectra
were obtained using a Perkin–Elmer Spectrum One FT-IR spec-
trometer with a zinc selenide/diamond Universal ATR sampling
Accessory as a thin film (Melbourne, Australia). High resolution
electrospray ionization mass spectra were obtained by Mr. Adrian
Lam on a Finnigan hybrid LTQ-FT mass spectrometer (Thermo Elec-
tron Corp.), The University of Melbourne, Australia. High perfor-
mance liquid chromatography was performed on an Agilent 1200
series instrument.
Using synthetic 1 and 2 as reference compounds, the EtOAc-sol-
uble fraction of acetone/water leaf extracts from 28 diverse euca-
lypts were analysed. Compound 1 was detected in all species
examined, with 2 also being found in 23 of the species (Table 1).
Compound 1 was present at low levels (<0.08 mg/g dry weight;
dry wt) in Eucalyptus halophila, Eucalyptus intermedia and Eucalyp-
tus olsenii, but ranged to over 28 mg/g dry wt in E. polybractea and
E. froggattii (Table 1). Compound 1 has previously been reported
from the leaves of E. polybractea and E. froggattii at the somewhat
lower mean concentrations of 4.6 mg/g dry wt and 7.7 mg/g dry
wt, respectively.3 A diastereoisomer of 1, cuniloside A (3) (contain-
ing (ꢀ)-(S)-oleuropeic acid), has been found at relatively low levels
in the leaves of Cunila spicata (0.08 mg/g dry wt).21 The isolation of
3 from the leaves of E. globulus (0.12 mg/g dry wt) was claimed;22
as discussed elsewhere this structural assignment is likely to be in
error and the actual compound isolated is most likely 13.
1.2. 50-Hydroxy-70-O-(2,3,4,6-tetra-O-acetyl-b-
D-glucopyranosyl)-
20-methylchromone (6)
A solution of HBr in AcOH (33% w/v, 3 mL) was added to D-glu-
cose pentaacetate (4) (1.00 g, 2.56 mmol) and the resulting solu-
tion was stirred at room temperature. After 3 h, the solution was
diluted with dichloromethane (10 mL) and the organic layer was
extracted with ice-water (3 ꢁ 10 mL), cold satd aq NaHCO3
(3 ꢁ 10 mL) and brine (3 ꢁ 10 mL). The organic layer was dried
In the 23 species where 2 was detected, it was consistently low-
er in abundance than 1 (Table 1). Compound 2 ranged from unde-
tectable levels in five of the examined species to a maximum of
1.38 mg/g dry wt in E. froggattii. Compound 2 has been found in
similarly low levels in the leaves of E. camaldulensis (camaldulen-
(MgSO4) and concentrated to provide tetra-O-acetyl-a-D-glucopyr-
anosyl bromide as a white foam (1.03 g). A two-phase mixture of
the crude bromide (1.03 g, 2.51 mmol), noreugenin12 (0.240 g,
1.25 mmol), Bu4NBr (0.403 g, 1.25 mmol), and K2CO3 (0.866 g,

2082
Z. Hakki et al. / Carbohydrate Research 345 (2010) 2079–2084
Table 1
Quantification of cuniloside B (1) and cypellocarpin C (2) in the leaves of representative species within the genus Eucalyptus
Subgenus
Species
Authority
1 (mg/g DW)
2 (mg/g DW)
Ratio 2:1
Alveolata
Corymbia
Corymbia
Eucalyptus
Eucalyptus
Eucalyptus
Eudesmia
Idiogenes
Eucalyptus microcorys
E. intermedia
E. eximia
E. gregsoniana
E. pauciflora
E. olsenii
E. erythrocorys
E. cloeziana
E. froggattii
E. polybractea
E. sideroxylon
E. dielsii
E. platypus
E. spathulata
E. halophila
F. Muell
R. Baker
Schauer
L.A.S. Johnson & Blaxell
Sieber ex Sprengel
L.A.S. Johnson & Blaxell
F. Muell
F. Muell
Blakely
3.796
0.028
0.126
0.335
0.098
0.021
0.098
0.097
29.496
28.565
2.015
2.619
0.628
2.065
0.072
9.633
7.822
6.104
6.120
17.025
0.916
6.235
0.500
0.191
0.413
0.472
0.057
1.069
0.016
0
0.001
0.015
0
0.008
0
0
0.004
0
0.009
0.045
0
0.380
0
0
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
Symphyomyrtus
1.375
0.749
0.218
0.046
0.165
0.174
0.044
0.394
0.020
0.140
0.062
0.344
0
1.123
0.008
0.020
0.050
0.099
0.030
0.221
0.047
0.026
0.108
0.017
0.263
0.084
0.613
0.041
0.003
0.023
0.010
0.020
0
0.180
0.016
0.107
0.120
0.209
0.520
0.206
R. Baker
A. Cunn. ex Woolls
C.A. Gardner
Hook
Hook
D.J. Carr & S.G.M. Carr
F. Muell
L.A.S. Johnson & K.D. Hill
F. Muell. ex Miq
Maiden & Blakely
Brooker
Luehm
Dehnh
Smith
F. Muell. ex Benth
L.A.S. Johnson
Labill.
Sims
Maiden
E. gracilis
E. loxophleba ssp. lissophloia
E. leptophylla
E. kochii
E. myriadena
E. torquata
E. camaldulensis
E. resinifera
E. cinerea
E. cypellocarpa
E. globulus
E. pulverulenta
E. dalrympleana
The EtOAc-soluble fraction of acetone/water extracts of the leaves of representative species within the genus Eucalyptus was quantified by LC–ESIMS using a standard series
prepared from the synthetic standards. Taxonomic classification is according to Brooker and Kleinig.1
6.27 mmol) in CHCl3 (45 mL) and H2O (45 mL) was stirred at 50 °C
for 18 h, and then neutralized with the addition of 1 M HCl. The or-
ganic layer was washed with brine, dried (MgSO4) and concen-
trated under reduced pressure. The residue was purified by flash
chromatography (1:1 EtOAc–pet. spirits) to provide 6 (0.476 g,
69.6, 73.1, 76.4, 77.1, 94.5, 99.5, 99.8 (C1,2,3,4,5,60,80), 105.1,
108.3, 157.4, 161.2, 162.9, 168.4 (C20,30,50,70,90,100), 182.0 (C40);
HRMS (ESI)+ m/z 355.1023 [C16H18O9 (M + H)+ requires 355.1029].
1.4. 50-Hydroxy-70-O-(2,3,4,6-tetra-O-acetyl-b-
D-allopyranosyl)-
73%): mp 166–168 °C (MeOH; lit.9 mp 168–171 °C); ½a 2D1
ꢂ
ꢀ42.1 (c
20-methylchromone (10)
0.16, CHCl3; lit.6 ꢀ37 (MeOH)); 1H NMR (500 MHz, CDCl3): d
2.04, 2.06, 2.11, 2.36 (4s, 15H, Me), 3.91 (m, 1H, H5), 4.20 (d, 1H,
J = 12.5 Hz, H6a), 4.27 (dd, 1H, J = 6.0, 12.5 Hz, H6b), 5.12–5.33
(2 m, 4H, H1,2,3,4), 6.05, 6.41, 6.46 (3s, 3 ꢁ 1H, H20,60,80), 12.35
(br s, 1H, C50–OH); 13C NMR (125 MHz, CDCl3): d 20.6, 20.7, 20.8
(5C, Me); 62.0, 68.4, 71.1, 72.6, 72.7 (5C, C2,3,4,5), 95.5, 98.3,
98.9, 106.8, 109.2, 157.9, 162.0, 162.5, 167.3 (8C,
C1,20,30,50,60,70,80,90,100), 169.3, 169.5, 170.3, 170.7 (4C, MeC@O),
182.8 (C40); HRMS (ESI)+ m/z 523.1446 [C24H26O13 (M + H)+ re-
quires 523.1446].
A solution of D
-allose pentaacetate (8)14 (0.95 g, 2.43 mmol) in
HBr in AcOH (33% w/v, 3 mL) was stirred at room temperature
for 3 h under nitrogen. The solution was diluted with dichloro-
methane (10 mL) and the organic layer extracted with ice water
(3 ꢁ 10 mL), cold saturated aqueous NaHCO3 (3 ꢁ 10 mL) and brine
(2 ꢁ 10 mL). The organic layer was dried (MgSO4) and concen-
trated to provide tetra-O-acetyl-a-D-allopyranosyl bromide as an
oil (0.90 g). The crude bromide (0.90 g, 2.19 mmol) was dissolved
in CHCl3 (20 mL), and then noreugenin12 (0.105 g, 0.548 mmol),
Bu4NHSO4 (0.186 g, 0.548 mmol), K2CO3 (0.379 g, 2.74 mmol) and
H2O (20 mL) were added. The resulting two-phase system was stir-
red at 50 °C for 48 h, and then neutralized with the addition of 1 M
HCl. The organic layer was washed with brine, dried (MgSO4) and
concentrated under reduced pressure. The residue was purified
by flash chromatography (3:2 EtOAc–pet. spirits) and recrystal-
lized from EtOH to provide the tetraacetate 10 (55.2 mg, 19%) as
1.3. 50-Hydroxy-70-O-(b- -glucopyranosyl)-20-methylchromone
D
(undulatoside A; 7)
Sodium metal (42 mg, 1.8 mmol) was added to a solution of the
tetraacetate 6 (0.476 g, 0.911 mmol) in MeOH (10 mL) at 0 °C. The
solution was stirred for 1 h until TLC indicated conversion of the
starting material into a single compound of higher polarity. After
neutralization with Dowex-50 resin (H+ form), the mixture was fil-
tered and the solvent was evaporated under reduced pressure to
a white powder: mp 121–123 °C; ½a D21
ꢂ
ꢀ30.1 (c 0.465, CHCl3); 1H
NMR (400 MHz, CDCl3): d 2.04, 2.05, 2.12, 2.18 (4s, 4 ꢁ 3H, Ac),
2.36 (s, 3H, C20-Me), 4.24–4.25, 4.28–4.31 (2 m, 3H, H2,4,5), 4.04
(dd, 1H, J = 2.8, 9.9 Hz, H6a), 5.16 (dd, 1H, J = 3.0, 8.2 Hz, H6b),
5.42 (d, 1H, J = 8.2 Hz, H1), 5.75 (t, 1H, J = 2.9 Hz, H3), 6.06 (s, 1H,
H20), 6.46 (d, 1H, J = 2.2 Hz, H60), 6.49 (d, 1H, J = 2.2 Hz, H80),
12.72 (s, 1H, C50–OH); 13C NMR (100 MHz, CDCl3): d 20.7, 20.8
(5C, Me), 62.4, 66.3, 68.5, 68.7, 71.0, 95.7, 96.6 (C1,2,3,4,5,6),
99.8, 106.6, 109.2, 157.8, 162.3, 162.4, 167.4 (8C,
C20,30,50,60,70,80,90,100), 169.1, 169.2, 169.8, 170.8 (MeC@O), 182.7
(C40); HRMS (ESI)+ m/z 523.1464 [C24H26O13 (M+H)+ requires
523.1446].
afford
a white solid. Recrystallization from MeOH gave 7
(0.306 g, 95%) as colourless needles: mp 248–250 °C (lit.6 mp
245–248 °C), ½a 2D2
ꢂ
ꢀ55.7 (c 1.0, C5H5N; lit.6 ꢀ50); IR (neat) mmax
3426, 1666, 1663, 1580, 1338, 1179, 1090, 1075 cmꢀ1 1H NMR
;
(500 MHz, CD3OD-d4): d 2.38 (s, 3H, Me), 3.38–3.42 (m, 1H),
3.47–3.52 (3H, m, H2,3,4,5), 3.70 (dd, 1H, J = 5.6, 12.1 Hz, H6a),
3.90 (dd, 1H, J = 2.3, 12.1 Hz, H6b), 5.00–5.04 (m, 1H, H1), 6.12
(d, 1H, J = 0.7 Hz, H30), 6.47 (d, 1H, J = 2.2 Hz, H60), 6.66 (d, 1H,
J = 2.2 Hz, H80); 13C NMR (125 MHz, DMSO-d6): d 20.0 (Me), 60.6,

Z. Hakki et al. / Carbohydrate Research 345 (2010) 2079–2084
2083
Prime
ax
00
00
00
1.5. 50-Hydroxy-70-O-(b-
D
-allopyranosyl)-20-methylchromone
2H, H3
; 5 ), 1.96–2.04 (m, 1H, H6 ), 2.17–2.24 (m, 1H, H3 ),
eq
ax
eq
(corymbosin K1; 11)
2.34–2.37 (m, 4H, C20 ꢀ CH3; H0e0q), 3.14–3.19 (m, 1H, H4), 3.25–
3.34 (m, 2H, H2,3), 3.77–3.80 (m, 1H, H5), 3.98 (dd, 1H, J = 7.9,
11.9 Hz, H6), 4.43 (dd, 1H, J = 2.0, 11.9 Hz, H6), 5.12 (d, 1H,
J = 7.5 Hz, OH), 5.24 (d, 1H, J = 4.8 Hz, OH), 5.34 (d, 1H, J = 5.4 Hz,
OH), 5.47 (d, 1H, J = 4.9 Hz, OH), 6.26 (d, 1H, J = 0.7 Hz, H30), 6.42
(d, 1H, J = 2.2 Hz, H60), 6.62 (d, 1H, J = 2.2 Hz, H80), 6.90–6.92 (m,
1H, H200), 12.84 (s, 1H, C50–OH); 13C NMR (125 MHz, DMSO-d6): d
20.5 (1C, C20–CH3), 23.4 (1C, C500), 25.3 (C600), 27.0, 27.5 (3C,
C300,900,1000), 44.1 (1C, C400), 64.1 (1C, C6), 70.6 (2C, C4800), 73.4 (1C,
C3), 74.2 (1C, C5), 76.5 (1C, C2), 94.9 (1C, C60), 99.77 (1C, C80),
99.80 (1C, C1), 105.6 (1C, C100), 108.8 (1C, C30), 129.8 (1C, C00),
140.7 (1C, C200), 157.9 (1C, C90), 161.7 (1C, C50), 163.1 (1C, C70),
166.7 (1C, C700), 168.8 (1C, C200), 182.5 (C40); HRMS (ESI)+ m/z
543.1829 [C26H32O11 (M+Na)+ requires 543.1842], 521.2009
[C26H33O11 (M+H)+ requires 521.2023]. The NMR data was consis-
tent with that reported.5
Sodium methoxide solution (1.0 M, 0.048 mmol, 48 lL) was
added to a solution of the tetraacetate 10 (12.6 mg, 0.0241 mmol)
in MeOH (3 mL) at 0 °C. The solution was stirred for 1 h until TLC
indicated conversion of the starting material to a single compound
of higher polarity. The mixture was neutralized with Dowex-50 re-
sin (H+ form), and the solvent was evaporated under reduced pres-
sure to afford 11 (6.6 mg, 78%) as a white solid: mp 188–191 °C
(lit.13 mp 196–198 °C); ½a 1D9
ꢂ
ꢀ91 (c 0.12, MeOH) (lit.13 ꢀ57.8); IR
(neat) mmax 3329, 1659, 1620, 1583, 1415, 1166, 1020,
1012 cmꢀ1 1H NMR (400 MHz, DMSO-d6): d 2.39 (s, 3H, Me),
;
3.32–3.48 (m, 3H, H2,4,6), 3.68 (dd, 1H, J = 4.4, 11.2 Hz, H6a),
3.75 (ddd, 1H, J = 2.0, 4.4, 11.2 Hz, H5), 3.92 (m, 1H, H3), 4.56 (t,
1H, J 5.6 Hz, H6–OH), 4.72 (d, J = 6.8 Hz, OH), 5.01 (br s, OH), 5.15
(d, J = 6.4 Hz, OH), 5.20 (d, 1H, J = 7.9 Hz, H1), 6.26 (s, 1H, H30),
6.39 (d, 1H, J = 2.1 Hz, H60), 6.60 (d, 1H, J = 2.1 Hz, H80), 12.83 (s,
1H, C50-OH); 13C NMR (100 MHz, DMSO-d6): d 20.0 (Me), 60.9,
66.9, 70.1, 71.4, 74.9 (C2,3,4,5,6), 94.4, 98.4, 99.4, 105.0, 108.3,
157.4, 161.2, 163.2, 168.4 (C1,20,30,50,60,70,80,90,100), 182.0 (C40);
HRMS (ESI)+ m/z 355.1025 [C16H18O9 (M+H)+ requires 355.1023].
The NMR data was consistent with that reported.13
1.8. 1,6-Di-O-[(R)-oleuropeyl]-b-D-glucopyranose (cuniloside B; 1)
(+)-(R)-Oleuropeic acid (102 mg, 0.555 mmol),
(50.0 mg, 0.277 mmol), HBTU (210 mg, 0.555 mmol), N-methyl-
morpholine (122 L, 1.11 mmol) and DMAP (68.0 mg, 0.555 mmol)
D-glucose
l
in dry DMF (2 mL) were stirred under nitrogen at 50 °C. The reac-
tion was monitored by TLC (17:2:1 EtOAc–MeOH–H2O) and
quenched by the addition of MeOH after 60 h. The crude reaction
mixture was co-evaporated under reduced pressure with toluene
(3 ꢁ 10 mL). The residue was purified by preparative HPLC to af-
ford 1 (10.2 mg, 7.2%; 98% HPLC purity) as an amorphous white
1.6. 50-Hydroxy-70-O-(6-O-acetyl-b-
methylchromone (corymbosin K2; 12)
D
-glucopyranosyl)-20-
Acetyl chloride (40.1 lL, 0.564 mmol) was added dropwise over
30 min to a solution of 7 (0.100 g, 0.282 mmol) in dry collidine
(1 mL) at ꢀ35 °C. The solution was stirred for 3 h at ꢀ35 °C until
TLC indicated conversion to a less polar compound. MeOH was
added and the reaction was allowed to attain rt. The crude reaction
mixture was co-evaporated under reduced pressure several times
with toluene. The residue was purified by flash chromatography
(17:2:1 EtOAc–MeOH–H2O) and recrystallized from EtOH to afford
12 (83.0 mg, 74%) as a white powder: mp 163–166 °C (lit.13 mp
powder: ½a 2D1
ꢂ
+63.1 (c 0.8, MeOH; lit.25 ꢀ1.3); IR (neat) mmax
3383, 1705, 1648, 1437, 1381, 1247, 1063, 1032, 917 cmꢀ1 1H
;
NMR (800 MHz, CD3OD-d4): d 1.18 (s, 12H, H90,100,900,1000), 1.21–
1.26 (m, 2H, H50ax), 1.54–1.56 (m, 2H, H40,400), 2.02–2.08 (m, 4H,
H30ax; 30a0x; 5e0 q; 500 ), 2.15–2.16 (m, 2H, Ha0 x; 600 ), 2.34–2.38 (m, 2H,
eq
ax
H3e0 q; 300 ), 2.49–2.56 (m, 2H, H6e0 q; 600 ), 3.38–3.40 (m, 2H, H2,4),
eq
eq
3.43–3.46 (m, 1H, H3), 3.60–3.62 (m, 1H, H5), 4.24–4.26 (dd, 1H,
J = 5.3, 11.9 Hz, H6a), 4.42 (d, 1H, J = 11.9 Hz, H6b), 5.51 (d, 1H,
J = 8.1 Hz, H1), 7.03, 7.15 (2s, 1H, H20,200); 13C NMR (200 MHz,
CD3OD-d4): d 24.521, 24.522 (2C, C50,500), 26.2, 26.3 (2C, C60,600),
26.4, 26.5 (2C, 90,900), 27.00, 27.04 (2C, C100,1000), 28.6, 28.7 (2C,
C30,300), 45.48, 45.52 (C40,400), 64.3 (1C, C6), 71.3 (1C, C4), 72.81,
72.83 (2C, C80,800), 73.9 (1C, C2), 76.2 (1C, C5), 78.0 (1C, C3), 95.7
(1C, C1), 130.6, 131.1 (2C, C10,100), 141.6, 143.1 (2C, C20,200), 167.2,
168.8 (2C, C70,700); HRMS (ESI)+ m/z 530.2958 [C26H40O10
(M+NH4)+ requires 530.2960]; HPLC conditions for cuniloside B;
164–166 °C), ½a 2D0
ꢂ
ꢀ108 (c 0.185, MeOH; lit.13 ꢀ68.7); IR (neat)
mmax 3350, 1728, 1659, 1622, 1174, 1042, 1076, 839 cmꢀ1 1H
;
NMR (500 MHz, DMSO-d6): d 2.02 (s, 3H, Ac), 2.36 (s, 3H, C20-
Me), 3.16–3.35 (m, 3H, H2,3,4), 3.69–3.73 (m, 1H, H5), 4.03 (dd,
1H, J = 7.4, 11.9 Hz, H6a), 4.30 (dd, 1H, J = 2.0, 11.5 Hz, H6b), 5.06
(d, 1H, J = 7.5 Hz, H1), 5.23 (d, 1H, J = 3.5 Hz, OH), 5.32 (d, 1H,
J = 5.0 Hz, OH), 5.45 (d, 1H, J = 4.5 Hz, OH), 6.25 (d, 1H, J = 1.0 Hz,
H30), 6.40 (d, 1H, J = 2.2 Hz, H60), 6.64 (d, 1H, J = 2.2 Hz, H80),
12.80 (s, 1H, C50–OH); 13C NMR (125 MHz, DMSO-d6): d 19.9,
20.5 (Me), 63.3, 69.8, 72.9, 73.8, 76.2 (C2,3,4,5,6), 94.6, 99.4, 99.6,
105.1, 108.3, 157.4, 161.1, 162.6, 168.3 (C1,20,30,50,60,70,80,90,100),
170.2 (C@OMe), 182.0 (C40); HRMS (ESI)+ m/z 419.0937
[C18H20O10 (M+Na)+ requires 419.0954]. The NMR data were con-
sistent with those reported.13
9.4 ꢁ 250 mm Agilent Eclipse XDB-C18 (5
lm particle size); 0–
80% 0.1% TFA in MeCN, linear gradient (80 min); wavelength
220 nm; room temperature. The NMR data were consistent with
those reported.4
1.9. Cypellocarpin C and cuniloside B survey
1.7. Cypellocarpin C (2)
Bulk leaf samples were collected in February 2010 from Euca-
lyptus trees growing in the Peter Francis Points Arboretum (Cole-
rain, Australia 37°36.570 S, 141°41.050 E). Dried leaves from each
species were ground to a fine powder and 200 mg was extracted
with acetone in water (70%, 20 mL) for 24 h at 50 °C. A 1 mL aliquot
of acetone extract was then extracted successively with petroleum
ether (60–80 °C fraction; 1 mL ꢁ 4) and EtOAc (1 mL ꢁ 4). The
combined EtOAc fraction was air-dried, redissolved in MeCN in
water (50%) and analysed by LC-ESIMS.
(+)-(R)-Oleuropeic acid (78.0 mg, 0.424 mmol),
0.424 mmol), HBTU (0.161 g, 0.424 mmol), N-methylmorpholine
(93 L, 0.85 mmol) and DMAP (0.103 g, 0.848 mmol) were stirred
7 (0.150 g,
l
in dry DMF (2 mL) under nitrogen at 50 °C. The reaction was
quenched by the addition of MeOH after 18 h. The crude reaction
mixture was co-evaporated under reduced pressure with toluene
(3 ꢁ 10 mL). The residue was purified by flash chromatography
(67:2:1 EtOAc–MeOH–H2O) and recrystallized from EtOH to afford
2 (123 mg, 56%) as a white solid: mp 216–218 °C (lit.5 mp 229–
LC–ESIMS was carried out on an Agilent 1200 series (Santa
Clara, CA, USA) with a triple-quadrupole mass spectrometer. The
230 °C); ½a 2D1
ꢂ
ꢀ113 (c 0.01, MeOH; lit.5 ꢀ138); IR (neat) mmax
3362, 1703, 1652, 1623, 1386, 1258, 1076, 824 cmꢀ1
;
1H NMR
analytical column used was a Gemini C18 (5
Phenomenex, Torrance, USA) eluted at a flow rate 0.5 mL/min with
l
m, 150 ꢁ 4.6 mm;
(500 MHz, DMSO-d6): d 1.027 (s, 3H, H900), 1.034 (s, 3H, H1000),
1.05–1.08 (m, 1H, H50a0x), 1.33–1.39 (m, 1H, H400), 1.84–1.92 (m,
a column temperature of 28 °C. The eluant system was a MeCN

2084
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gradient (acidified with 0.1% formic acid) from 30% to 45% over
2 min, followed by 45–65% over 8 min, then 65–100% over 4 min.
Compounds 1 and 2 eluted at retention times of 6.3 and 6.8 min,
respectively. The ESI source conditions were as follows: nebulizer
pressure of 45 psi, gas flow rate of 10 L/min, gas temperature
315 °C and capillary voltage of 4000 V.
Quantification of 1 and 2 for each species was carried out by
comparison of the response integral for the most abundant SRM
transition of each compound to standard series of synthesised 1
and 2 run under optimal conditions. The optimized conditions
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Acknowledgements
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This work was supported by grants from the Australian Re-
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the recipient of an ARC Australian Research Fellowship. J. Karas is
thanked for HPLC support.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2010.07.029.
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