Polyphenolic glycosides from african proteaceae

Article abstract of DOI:10.1021/np9902237

The phytochemical investigation of members of the genus Protea afforded a series of polyphenolic compounds (1-5) that were identified by 1D and 2D NMR experiments. Of these, 2-5 are new compounds. Chemical syntheses of 1-3 were performed in order to confirm the structures and to prepare additional material for biological evaluation.

Full text of DOI:10.1021/np9902237

1526
J . Nat. Prod. 1999, 62, 1526-1531
P olyp h en olic Glycosid es fr om Afr ica n P r otea cea e
L. Verotta,*,† F. Orsini,† F. Pelizzoni,† G. Torri,‡ and C. B. Rogers§
Dipartimento di Chimica Organica e Industriale, Universita` degli Studi di Milano, via Venezian 21, 20133 Milano, Italy,
Istituto di Chimica e Biochimica “G. Ronzoni”, via G. Colombo 81, 20133 Milano, Italy, and Department of Chemistry,
University Durban-Westville, Private Bag X54001, Durban 4000, Republic of South Africa
Received May 10, 1999
The phytochemical investigation of members of the genus Protea afforded a series of polyphenolic
compounds (1-5) that were identified by 1D and 2D NMR experiments. Of these, 2-5 are new compounds.
Chemical syntheses of 1-3 were performed in order to confirm the structures and to prepare additional
material for biological evaluation.
The Proteaceae or silk-oak family is one of evergreen
shrubs, trees, or sometimes subherbaceous perennial plants.
The more than 1000 species from approximately 55 genera
are found largely in the dry, Mediterranean-type, climatic
regions of the southern hemisphere, especially in South
Africa and Australia. Proteas were the most prominent
woody plants in the Cape Region of South Africa 300 years
ago and were used by the early European settlers for a
variety of their needs. The bark and leaves of most Protea
species may be regarded as astringent, and all have
probably been used in the Cape Region for tanning. As
early as 1720, “Syrupus Protea” was used as a cough syrup.
Nectar from P. nitida flowers was thickened into a nutri-
tious syrup, described as a tonic and cough syrup in the
old Cape Pharmacopoeia and sold as “Bessiestroop” until
about 100 years ago. The wood of most species has been
used as fuel, and the wood of P. nitida, the “Waboom”
(Afrikaans for “Wagon Tree”), was used extensively by the
early settlers to build wagons.1
side (rubropilosine) and 2-hydroxy-4-hydroxymethylphenyl-
(6′-O-benzoyl)-â-D-allopyranoside (pilorubrosine) were iso-
lated from P. rubropilosa.3 4-Hydroxyphenyl-(6′-O-benzoyl)-
O-â-D-glucopyranoside (1) (eximine) was isolated from P.
eximia and characterized by comparing its spectroscopic
data with those in the literature.4
P. neriifolia was submitted to a simplified extraction to
increase the yield of polyphenolic compounds. Leaves were
defatted with petroleum, then extracted with water-
saturated ethyl acetate. The ethyl acetate extracts were
evaporated, the residue washed with CH2Cl2, and then
submitted to reversed-phase medium-pressure chromatog-
raphy, followed by GFC [Sephadex LH20 and Toyopearl
HW40(S)] and counter current chromatography (CCC).
Four phenolic glycosides (2-5) were isolated.
Two classes of glycosides are found in Protea sp., the
C-glycosides (proteacin or leucodrin) and phenolic glyco-
sides, which have 3,4-dihydroxybenzyl alcohol as a common
aglycon. The known sugars involved in the â-glycosidic
linkage are (+)-D-glucose and (+)-D-allose.2,3 In this paper
we report the phytochemical investigation of 10 Protea
species. Four new polyphenolic glycosides are described
from P. neriifolia, together with known polyphenolic gly-
cosides from P. obtusifolia, P. rubropilosa, and P. eximia.
Resu lts a n d Discu ssion
A standardized extraction procedure was carried out on
leaves of 10 Protea species. The cytotoxicity of the extracts
was monitored through the brine shrimp lethality test.
Weak cytotoxic activity was found in ethyl acetate extracts
of P. obtusifolia, P. eximia, and P. neriifolia.
The dried plant materials were exhaustively extracted
with MeOH. The extracts were defatted, then extracted
with ethyl acetate to give the yields reported in the
Experimental Section. p-Hydroxybenzoic acid, protocath-
ecuic acid, methyl 3,5-dihydroxybenzyl ether, and 3,4-
dihydroxybenzyl alcohol were isolated from P. obtusifolia
and characterized by spectroscopic means and comparison
with reference samples or literature data. 2-Hydroxy-
4-hydroxymethylphenyl-(6′-O-cinnamoyl)-â-D-allopyrano-
Compounds 2-4 each showed a quasi-molecular peak at
m/z 391 [M - H]- in FABMS, indicating a molecular
formula of C19H20O9. The 1H and 13C NMR spectra of all
three products showed the presence of a benzoyl group
* To whom correspondence should be addressed. Tel.: +39 02 2363469.
Fax: +39 02 2364369. E.mail luisver@icil64.cilea.it.
† Dipartimento di Chimica Organica e Industriale.
‡ Istituto di Chimica e Biochimica “G. Ronzoni”.
§ Department of Chemistry.
10.1021/np9902237 CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/20/1999

Glycosides from Proteaceae
J ournal of Natural Products, 1999, Vol. 62, No. 11 1527
linked to the hydroxymethylene group. The characteristic
deshielding effects on 6′A and B protons and the HMBC
spectra confirmed this hypothesis. The sugar is linked to
a 1,2,4-trihydroxybenzene nucleus as a phenol glycoside,
as shown by the chemical shifts and the splitting pattern
dry DMF, followed by acidic aqueous work up, afforded 8
after simultaneous removal of the acetoxy protective
groups.11
The synthesis of glucoside 3 required a series of selective
protections and deprotections of the phenolic groups to give
2,4-dibenzyloxyphenol, which was transformed in three
steps into the glucoside 3.12 This approach also afforded
the nonnatural glucopyranoside 9.
1
in the H NMR spectra.
In compounds 2 and 3, the sugar moiety was identified
as â-D-glucopyranose, by self-consistent 2D NMR experi-
ments (HMQC and DQF-COSY) and comparison with
reference samples.5,6 In compounds 4 and 5, the sugar
moiety was identified as â-D-allopyranoside by both 1D and
2D NMR experiments and by methanolysis of 4, followed
by comparison of the resulting methyl alloside with a
reference sample of R- and â-D-methyl allopyranoside. The
proton and carbon chemical shifts of 3 and 4 show that
the same phenolic hydroxyl group is involved in the
â-glycosidic linkage, whereas a different hydroxyl group is
involved in this linkage in compound 2.
HMBC and 1D NOE experiments confirmed that the four
new compounds are 3,4-dihydroxyphenyl-(6′-O-benzoyl)-O-
â-D-glucopyranoside (2), 2,4-dihydroxyphenyl-(6′-O-ben-
zoyl)-O-â-D-glucopyranoside (3), 2,4-dihydroxyphenyl-(6′-
O-benzoyl)-O-â-D-allopyranoside (4), and 2,4 dihydroxy-
phenyl-[6′-O-(3-hydroxy, 3′-phenyl propanoyl)]-O-â-D-al-
lopyranoside (5). The trihydroxybenzene moiety and the
glycosylated positions were confirmed by permethylation,
followed by methanolysis of compounds 2-4. 3,4-Di-
methoxyphenol was isolated from 2 and its 1H NMR
spectrum compared with literature data; 2,4-dimethoxy-
phenol was isolated from 3 and 4 and compared with a
commercial sample. Compounds 1-3 were tested on human
fibroblast proliferation showing growth stimulation at
concentrations between 1 and 5 µg/mL.7
4-Hydroxyphenyl-(6′-O-benzoyl)-O-â-D-glucopyranoside
(eximine) (1) was synthesized directly from commercially
available arbutin by selective benzoylation of the primary
hydroxyl group. The best results were achieved with
benzoic acid in dry pyridine in the presence of BOP-Cl [bis-
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured on a Perkin-Elmer 241 polarimeter. Melting
points were measured on a Bu¨chi 570 mp apparatus and are
uncorrected. Kieselgel 60 F254 and RP18 (Merck) were used for
TLC. Most frequently used solvents were: CHCl3-MeOH-
H2O (5:5:3 or 5:3:2) (organic phase) for Si gel, MeOH-H2O (45:
55 or 1:1) for RP18. Spots were revealed by UV absorption (254
and 366 nm) and by spraying with H2SO4-MeOH (1:9),
followed by heating, or with FeCl3. Sephadex LH20 (Pharma-
cia) eluted with MeOH, at a flow rate of 25 mL/min and
Toyopearl HW40(S) (Tosohaas), eluted with EtOH at a flow
rate of 2 mL/min were used for GFC. Reversed-phase medium-
pressure chromatography was carried out using a Bu¨chi 681
pump and Bu¨chi 685 columns, home packed with LiChroprep
RP18 (40-63 µm, Merck) or LiChroprep RP8 (25-40 µm,
Merck). Counter current chromatography was carried out with
a CCC 1000 chromatograph (Pharmatech Research Corp.,
Baltimore, MD). The equipment consisted of three multilayer
coiled columns (1.6 mm i.d. PTFE, total volume capacity 350
mL), rotating at 1032 rpm, a liquid chromatography pump
(model 300, Scientific Systems Inc., State College, PA), a speed
controller, and an injection valve (Rheodyne). The separations
were carried out with the solvent system CHCl3-MeOH-H2O
(4:4:3). 1H and 13C NMR spectra were recorded at 200, 300,
and 500 MHz on Bruker spectrometers. 2D experiments
(HETCORR, COLOC, HMQC, HMBC) were run using default
parameters in the available Bruker software. Samples were
dissolved in DMSO-d6 or CDCl3 and TMS was used as internal
standard. Positive and negative FABMS were recorded on a
VG7070 EQ spectrometer, using NBA as matrix.
8,9
(2-oxo-3-oxazolidinyl)-phosphinic chloride]. Eximine (1)
was obtained with 82% regioselectivity with respect to the
secondary hydroxyl group, accompanied by a small amount
of the dibenzoyl derivative (6), easily separated by flash
chromatography.
The synthesis of the glucoside 2 was a priori formulated
in two key steps from 1,2,4-trihydroxybenzene: (a) regi-
oselective (at position 4) and stereoselective â-glucosidation
of the aglycon, and (b) regioselective benzoylation of the
primary hydroxyl group in the glucoside. Exclusive glu-
cosidation of position 4 was achieved through protection
of the ortho-dihydroxy moiety as a methylenedioxy group,
which is stable under the basic conditions used for the
glucosylation and which may be removed in nonacidic
conditions compatible with the glucosyl linkage. A further
advantage is that 3,4-methylenedioxyphenol (sesamol) is
commercially available.
Glucosylation of sesamol was performed under phase
transfer catalysis using R-bromo-2,3,4,6-tetra-O-acetyl-D-
glucose as the glucosyl donor in CHCl3-NaOH solution in
the presence of benzyltriethylammonium hydroxide6 and
afforded 7 in 68.5% yield, accompanied by starting material
that was easily separated by flash chromatography and
recycled. The reaction was stereoselective and afforded
exclusively the â-glucoside. Unsatisfactory results were,
however, obtained using phenyl 2,3,4,6-tetra-O-acetyl-D-
glucosyl sulfoxide as glucosyl donor, a procedure recom-
mended for the glucosylation of low reactive substrates
such as phenols.10 Treatment with sodium thioethoxide in
P la n t Ma ter ia ls. The locations, month of collection, and
the voucher numbers at Ward Herbarium, University of

1528 J ournal of Natural Products, 1999, Vol. 62, No. 11
Verotta et al.
Durban-Westville follow: P. wellwitschii Engl. (Protea Farm
Hillcrest, Durban, April 1994, R101), P. simplex L. (Durban,
April 1994, R102), P. obtusifolia Buek ex Meissner (National
Botanic Gardens [NGB], Pretoria, April 1994, R103), P.
susannae Phillips (NBG, Pretoria, April 1994, R104), P. repens
L. (NBG, Pretoria, April 1994, R105), P. neriifolia R. Br. (NBG,
Pretoria, April 1994, R106), P. neriifolia R. Br. (modified
extraction) (Protea Farm Hillcrest, Durban, J anuary 1997
R106A), P. eximia (Salisb. ex J . Knight) Fourc. (NBG, Pretoria,
April 1994, R107), P. rubropilosa Beard (NBG, Pretoria, April
1994, R108), P. lorifolia (Salisb. ex J . Knight) Fourc. (NBG,
Pretoria, April 1994, R109).
Extr a ction a n d Isola tion . P. obtusifolia: dried leaves
(74.3 g) were percolated at room temperature with 3 × 250
mL MeOH. The MeOH extract was concentrated, diluted with
H2O (150 mL), and extracted with petroleum (3 × 400 mL),
then with EtOAc (3 × 250 mL). Evaporation of the ethyl
acetate gave 7.0 g, 4.0 g of which was purified on Sephadex
LH20. Two-hundred fractions (10 mL each) were collected and
pooled according to their composition. Fractions 137-150 (372
mg) were submitted to reversed-phase MPLC, eluting with
MeOH-H2O (1:2) (300 mL), then 45:55 (800 mL), and then
on Si gel (mixtures of CHCl3-MeOH), obtaining 10 mg of 3,4-
dihydroxybenzyl alcohol and 38 mg of methyl 3,5-dihydroxy-
benzyl ether. Fractions 151-174 (1.075 g) were filtered on
SiO2, then purified by reversed-phase MPLC, eluting with
MeOH-H2O (3:7) (1 L), obtaining 49 mg of 3,4-dihydroxyben-
zoic acid and 96 mg of p-hydroxybenzoic acid. Caffeic acid and
quercetin were identified by comparison with reference stan-
dards.
P. rubropilosa: dried, minced leaves (229 g) were percolated
at room temperature with 5 × 450 mL of MeOH. The MeOH
extract was concentrated, diluted with H2O (450 mL), and
extracted with petroleum (1 × 600 mL), then with EtOAc
(3 × 200 mL). The ethyl acetate was evaporated to give 18.6
g, 9.12 of which was purified on Sephadex LH20 (5 cm i.d. ×
90 cm), 120 fractions (20 mL each). Fractions 96-120 (2 g)
were filtered on Si gel, then purified by reversed-phase MPLC
(RP8, MeOH-H2O 1:1, flow rate 10 mL/min). Fractions 50-
80 (306 mg) were partitioned in CHCl3-MeOH-H2O (5:3:2).
The CHCl3 layer afforded 123 mg of rubropilosine.3 Fractions
30-40 were partitioned in CHCl3-MeOH-H2O (5:3:2). The
CHCl3 layer afforded 29 mg of pilorubrosine.3
P. eximia: dried, minced leaves (130 g) were percolated at
room temperature with MeOH (3 × 300 mL). The MeOH
extract was concentrated, diluted with H2O (500 mL), and
extracted with petroleum (3 × 300 mL), then with EtOAc
(2 × 250 mL). Evaporation of the ethyl acetate gave 7.4 g that
was purified on Sephadex LH20 (5 cm i.d. × 50 cm). In all,
159 fractions (8 mL each) were collected and pooled according
to their composition. Fractions 80-89 (1 g) were purified by
reversed-phase MPLC (RP18, MeOH-H2O 2:3, flow rate 5 mL/
min), obtaining 134 mg of eximine (1).
Com p ou n d 1: mp 200- 201 °C (CHCl3-MeOH, lit.4 199°-
202 °C); [R]20 -47° (c 1,; lit.4 -48°); Rf 0.56 (CHCl3-MeOH-
D
H2O 5:5:3, organic phase); 1H NMR (DMSO-d6) δ 9.00 (1H, br
s, OH), 7.98 (2H, dd, J ) 7.0, 1.5 Hz, H-2′′,-6′′), 7.70 (1H, br t,
J ) 7 Hz, H-4′′), 7.55 (2H, br t, J ) 7.0 Hz, H-3′′,-5′′), 6.85
(2H, d, J ) 8.7 Hz, H-2,6), 6.56 (2H, d, J ) 8.7 Hz, H-3,-5),
5.25 (3H, br s, OH), 4.73 (1H, d, J ) 7 Hz, H-1′), 4.60 (1H, dd,
J ) 11.9, 1.5 Hz, H-6′A), 4.26 (1H, dd, J ) 11.9, 7.0 Hz, H-6′B),
3.68 (1H, br t, J ) 7.7 Hz, H-5′), 3.29 (3H, m, H-2′,-3′,-4′); 13
C
NMR (DMSO-d6) δ 165.6 (s, C-7′′), 152.3 (s, C-1), 150.1 (s, C-4),
133.4 (d, C-4′′), 129.8 (s, C-1′′), 2 × 129.2 (d, C-2′′,-6′′), 2 ×
128.8 (d, C-3′′,-5′′), 2 × 117.6 (C-3,-5), 2 × 115.4 (d, C-2,-6),
101.4 (d, C-1′), 76.5 (d, C-3′), 73.7 (d, C-5′), 73.3 (d, C-2′), 70.3
(d, C-4′), 64.4 (t, C-6′); FABMS (negative mode) m/z 375 [M -
H]- ; (positive mode) m/z 399 [M + Na]+, 377 [M + H]+.
P. neriifolia: dried, minced leaves (445 g) were defatted by
stirring with petroleum (750 mL) at room temperature for 2
h, then filtered. The vegetable material was later extracted
at room temperature for 2 h with water-saturated EtOAc
(7 × 750 mL). The ethyl acetate extract, on evaporation, gave
73.9 g that was washed with CH2Cl2 (2 × 800 mL) to leave 62
g.
From the above extract, 20.4 g was purified by reversed-
phase MPLC (RP8, MeOH-H2O 45:55, flow rate 15 mL/min);
214 fractions (6 mL each) were collected and pooled according
to their composition. Four fractions were obtained: 51-59
(3.446 g), 100-148 (4.561 g), 149-209 (3.597 g), 210-214
(2.168 g). Fractions 100-148 (1 g) were purified first on
Toyopearl HW 40(S), then by HSCCC (CHCl3-MeOH-H2O
4:4:3, aqueous phase as mobile phase), giving 190 mg of 2.
Fractions 149-209 (2.3 g) were purified on Sephadex LH20;
98 fractions (4 mL each) were collected and pooled according
to their composition. Fractions 47-50 were purified on Toyo-
pearl HW 40(S), giving 97 mg of 3. Fractions 51-64 (388 mg)
were purified by HSCCC (CHCl3-MeOH-H2O 4:4:3, aqueous
phase as mobile phase), giving 226 mg of 3, 89 mg of 4, and 7
mg of 5.
Com p ou n d 2: white powder, mp 91-93 °C (H2O-MeOH)
[R]20D -50° (c 1, MeOH); Rf ) 0.36 (CHCl3-MeOH-H2O 5:5:3
organic phase); 1H NMR (DMSO-d6, 500 MHz) δ 8.98 and 8.50
(2H, s, phenolic OH), 7.98 (2H, dd, J ) 7.5, 1.5 Hz, H-2′′,6′′),
7.69 (1H, t, J ) 7.5 Hz, H-4′′), 7.55 (2H, t, J ) 7.5 Hz, H-3′′,-
5′′), 6.52 (1H, d, J ) 8.1 Hz, H-5), 6.5 (1H, d, J ) 2.5 Hz, H-2),
6.33 (1H, dd, J ) 8.1, 2.5 Hz, H-6), 5.32 and 5.20 (2H, s, OH),
4.70 (1H, d, J ) 7 Hz, H-1′), 4.61 (1H, dd, J ) 11.7, 1.5 Hz,
H-6′A), 4.24 (1H, dd, J ) 11.7, 7.5 Hz, H-6′B), 3.68 (1H, dt,
J ) 7.5, 1.5 Hz, H-5′), 3.30 (1H, t, J ) 7.5 Hz, H-3′), 3.27 (1H,
m), 3.25 (1H, m); 13C NMR (DMSO-d6) δ 165.6 (s, C-7′′), 150.0
(s, C-1), 145.5 (s, C-3), 140.2 (s, C-4), 133.5 (d, C-4′′), 129.7 (s,
C-1′′), 2 × 129.3 (d, C-2′′,-6′′), 2 × 128.9 (d, C-3′′,-5′′), 115.3 (d,
C-5), 106.6 (d, C-6), 105.2 (d, C-2), 101.3 (d, C-1′), 76.3 (d, C-3′),
73.7 (d, C-5′), 73.2 (d, C-2′), 70.1 (d, C-4′), 64.5 (t, C-6′); FABMS
(negative mode) m/z 391 [M - H]-; (positive mode) m/z 415
[M + Na]+, 392 [M+].
r u br op ilosin e: white powder, mp 100 °C (lit.3 97-100 °C);
1
[R]25 -68° (c 1, MeOH) (lit.3 -67°, c 0.92); H NMR (DMSO-
D
d6+ D2O, 200 MHz) δ 7.70-7.50 (5H, m, H-2′′/6′′ + H-3′′/5′′ +
H-4′′), 7.66 (1H, d, J ) 16 Hz, H-9′′), 7.00 (1H, d, J ) 8 Hz,
H-6) 6.70 (1H, d, J ) 1.5 Hz, H-3), 6.65 (1H, d, J ) 16 Hz,
H-8′′), 6.50 (1H, dd, J ) 8.0, 1.5 Hz, H-5), 4.97 (1H, d, J ) 7.9
Hz, H-1′), 4.42 (1H, dd, J ) 11.8, 1.5 Hz, H-6′A), 4.30 (1H, s,
H-7), 4.30 (1H, m, H-6′B), 4.00 (1H, m, H-5′), 3.50 (3H, m,
H-2′,-3′,-4′); 13C NMR (DMSO-d6) δ 166.2 (s, C-7′′), 146.4 (s,
C-1), 144.8 (d, C-9′′), 144.1 (s, C-2), 137.3 (s, C-4), 134.0 (s,
C-1′′), 130.7 (d, C-4′′), 2 × 129.1 (d, C-3′′ + C-5′′), 2 × 128.4
(d, C-2′′ + C-6′′), 118.0 (d, C-5), 117.4 (d, C-8′′), 116.1 (d, C-3),
114.4 (d, C-6), 100.2 (d, C-1′), 71.7 (d, C-5′), 70.9 (d, C-2′), 70.2
(d, C-3′), 67.4 (d, C-4′), 64.7 (t, C-6′), 62.5 (t, C-7); FABMS
(negative mode) m/z 431 [M - H]-; (positive mode) m/z 455
[M + Na]+.
P ilor u br osin e: mp 170 °C (lit.3 167-169 °C); [R]25 -68 °
D
(c 1, MeOH) (lit.3 -66°, c 0.87); H NMR (DMSO-d6 + D2O) δ
1
8.00 (2H, dd, J ) 7.9, 1.5 Hz, H-2′′ + H-6′′), 7.68 (2H, d, J )
7.9 Hz H-3′′ + H-5′′), 7.55 (1H, t, J ) 7.9 Hz, H-4′′), 7.00 (1H,
d, J ) 8.5 Hz, H-6), 6.70 (1H, d, J ) 2 Hz, H-3), 6.50 (1H, dd,
J ) 8.5, 2 Hz, H-5), 5.00 (1H, d, J ) 8.0 Hz, H-1), 4.60 (1H,
dd, J ) 11.8, 1.5 Hz, H-6′A), 4.30 (1H, s, H-7), 4.30 (1H, dd,
J ) 11.8, 7.5 Hz, H-6′B), 4.00 (1H, m, H-5′), 3.50 (3H, m, H-2′,-
3′,-4′); 13C NMR (DMSO-d6) δ 165.7 (s, C-7′′), 146.4 (s, C-1),
144.1 (s, C-2), 137.3 (s, C-4), 133.5 (d, C-4′′), 129.7 (s, C-1′′),
2 × 129.3 (d, C-2′′,-6′′), 2 × 128.9 (d, C-3′′,-5′′), 117.2 (d, C-5),
115.9 (d, C-6), 114.3 (d, C-3), 100.2 (d, C-1′), 71.7 (d, C-5′), 70.9
(d, C-2′), 70.2 (d, C-3′), 67.5 (d, C-4′), 64.8 (t, C-6′), 62.5 (t, C-7);
FABMS (negative mode) m/z 405 [M - H]- ; (positive mode)
m/z 429 [M+Na]+.
Compound 2 (30 mg) was added to a suspension of Bu-t-
ONa (150 mg) and NaOH (30 mg) in 5 mL dry DMSO, under
N2; after 10 min, CH3I (2 mL) was added. The reaction mixture
was stirred, at room temperature, for 3 h. The solution was
poured onto ice and extracted with CH2Cl2. The organic phase
was dried and evaporated to dryness. The crude mixture was
purified on Si gel (n-hexane-EtOAc 7:3) giving 15 mg of 2a .
Com p ou n d 2a : 1H NMR (200 MHz, CDCl3) δ 6.78 (1H, d,
J ) 7.8 Hz, H-5), 6.70 (1H, d, J ) 3 Hz, H-2), 6.59 (1H, dd,
J ) 7.8, 3 Hz, H-6), 4.72 (1H,d, J ) 7.5 Hz, H-1′), 3.81, 3.79,
3.62, 3.60, 3.50, 3.35 (6 × 3H, s, -OCH3).

Glycosides from Proteaceae
J ournal of Natural Products, 1999, Vol. 62, No. 11 1529
Compound 2a was refluxed with 10% HCl-MeOH, under
N2, for 2 h. The solution was evaporated under N2, taken up
with H2O and extracted with CH2Cl2. After purification on Si
gel (n-hexane-EtOAc, 4:1), 11 mg of 3,4 dimethoxyphenol (2b)
were obtained: 1H NMR (200 MHz, CDCl3) δ 6.74 (1H, d, J )
8.7 Hz, H-5), 6.48 (1H, d, J ) 2.7 Hz, H-2), 6.35 (1H, dd, J )
8.7, 2.7 Hz, H-6), 5.03 (1H, s, -OH), 3.83 (6H, s, -OCH3).
Com p ou n d 3: white powder, mp 189-190 °C (EtOH-
CHCl3 ) [R]20D -48° (c 1, MeOH), Rf 0.41 (CHCl3-MeOH-H2O
5:5:3, organic phase); 1H NMR (DMSO-d6, 300 MHz) δ 8.94
and 8.40 (2H, s, phenolic OH), 8.00 (2H, dd, J ) 7.2, 1.5 Hz,
H-2′′,-6′′), 7.70 (1H, t, J ) 7.2 Hz, H-4′′), 7.57 (2H, t, J ) 7.2
Hz, H-3′′,-5′′), 6.86 (1H, d, J ) 8.7 Hz, H-6), 6.22 (1H, d, J )
2.7 Hz, H-3), 5.90 (1H, dd, J ) 8.7, 2.7 Hz, H-5), 5.60 (1H, s,
OH), 4.63 (1H, dd, J ) 11.9, 1.5 Hz, H-6′A), 4.56 (1H, d, J )
7.9 Hz, H-1′), 4.30 (1H, dd, J ) 11.9,7.7 Hz, H-6′B), 3.68 (1H,
dt, J ) 7.7, 1.5 Hz, H-5′), 3.29 (3H, m, H-2′,-3′,-4′); 13C NMR
(DMSO-d6) δ 165.7 (s, C-7′′), 153.4 (s, C-4), 147.7 (s, C-2), 138.3
(s, C-1), 133.6 (d, C-4′′), 129.7 (s, C-1′′), 2 × 129.3 (d,
C-2′′,-6′′), 2 × 128.9 (d, C-3′′,-5′′), 118.2 (d, C-6), 105.3 (d, C-5),
2 × 103.3 (d, C-3,-1′), 75.6 (d, C-3′), 73.9 (d, C-5′), 73.3 (d, C-2′),
70.3 (d, C-4′), 64.4 (t, C-6′); FABMS (negative mode) m/z 391
[M - H]-; (positive mode, NBA) m/z 415 [M + Na]+, 392 [M]+.
on RP18 (MeOH-H2O 45:55), giving 2 mg of racemic 3-hy-
droxy,3′-phenyl propanoic acid.
Syn th esis. 4-Hyd r oxyp h en yl-(6′-O-ben zoyl)-O-â-D-glu -
cop yr a n osid e (1). A solution of arbutin (4-hydroxyphenyl-
O-â-D-glucopyranoside; 0.5 g, 1.84 mmol) and benzoic acid (0.27
g, 2.22 mmol) in dry pyridine (30 mL) was distilled with
azeotropic removal of H2O to half volume (15 mL) and then
cooled. BOP-Cl (1.187 g, 4.67 mmol) was added, and the
resulting suspension was stirred at room temperature. The
reaction was monitored by TLC (Si gel, using as eluent the
organic phase of the biphasic system CHCl3-MeOH-H2O 5:5:
3). After 3 h the solvent was removed under reduced pressure,
and the residue was extracted with CHCl3-MeOH-H2O 5:5:3
(30 mL). The organic phase was dried, the solvent was removed
under reduced pressure, and the residue was chromatographed
on Si gel eluting with CHCl3-MeOH 9:1 to afford 4-hydroxy-
phenyl-(6′-O-benzoyl)-O-â-D-glucopyranoside (1) (0.137 g). The
aqueous phase was extracted with EtOAc (3 × 10 mL) and
gave a further 0.390 g of 1. The total amount of crude 1 (0.527
g, 76%) was crystallized from CHCl3-MeOH and afforded
0.420 g of a colorless compound identical in all respect to the
natural compound. As a side product 4-benzoyloxyphenyl-(6′-
O-benzoyl)-O-â-D-glucopyranoside (6) was obtained in 12.5%
yield.
Compound 3 (50 mg) was permethylated under the same
conditions used for 2. After purification, 22 mg of 3a were
Com p ou n d 6: mp 238 °C (CHCl3-MeOH); [R]20 ) -54°
D
1
(c 1, EtOH); Rf 0.58 (CHCl3-MeOH-H2O 5:5:3, organic phase);
1H NMR (DMSO-d6) δ 8.12 (2H, dd, J ) 7.1, 1.5 Hz, H-2′′′+
H-6′′′), 7.97 (2H, dd, J ) 7.0, 1.5 Hz, H-2′′+ H-6′′), 7.80-7.70
(2H, m, H-4′′+ H-4′′′), 7.70-7.50 (4H, m, H-3′′ + H-5′′ +
H-3′′′ + H- 5′′′), 7.10 (4H, m, H-2, -3, -5, -6), 5.52 (2H, s, OH),
5.38 (1H, s, OH), 4.93 (1H, d, J ) 7.1 Hz, H-1′), 4.62 (1H, dd,
J ) 12.0, 1.5 Hz, H-6′), 4.35 (1H, dd, J ) 12.0, 7.0 Hz, H-6),
3.82 (1H, ddd, J ) 7.0, 7.0, 1.5 Hz, H-5′), 3.45 (3H, m, H-2′,
-3′, -4′); 13C NMR (DMSO-d6) δ 165.6 (s), 164.8 (s), 154.9 (s),
145.1 (s), 134.1 (d), 133.4 (d), 130.2 (s), 130.1 (s), 2 × 129.8
(d), 2 × 129.2 (d), 2 × 129.0 (d), 2 × 128.8 (d), 2 × 117.7 (d),
2 × 115.5 (d), 101.5 (d), 73.8 (d), 76.4 (d), 73.3 (d), 70.2 (d),
64.2 (t); FABMS (negative mode) m/z 479 [M - H]-; (positive
mode) m/z 503 [M + Na]+, 481 [M + H]+; anal. C 64.7%, H
5.2%, calcd for C26H24O9, C 64.9%, H 5.1%.
obtained: H NMR (200 MHz, CDCl3); δ 7.07 (1H, d, J ) 8.7
Hz, H-6), 6.50 (1H, d, J ) 2.8 Hz, H-3), 6.38 (1H, dd, J ) 8.7,
2.8 Hz, H-5), 4.66 (1H, d, J ) 7.5 Hz, H-1′), 3.82, 3.70, 3.66,
3.56, 3.41, 3.39 (6 × 3H, s, -OCH3).
Com p ou n d 4: white powder, mp 115-116 °C (EtOH-
CHCl3) [R]20 -61.3° (c 1, MeOH); Rf 0.45 (CHCl3-MeOH-
D
H2O 5:5:3 organic phase); 1H NMR (DMSO-d6, 200 MHz) δ 8.91
and 8.40 (2H, s, phenolic OH), 8.00 (2H, d, J ) 7.0 Hz, H-2′′,-
6′′), 7.60 (2H, t, J ) 7.8 Hz, H-3′′,-5′′), 7.55 (1H, t, J ) 7.5 Hz,
H-4′′), 6.90 (1H, d, J ) 8.7 Hz, H-6), 6.21 (1H, d, J ) 2.7 Hz,
H-3), 5.95 (1H, dd, J ) 8.7, 2.7 Hz, H-5), 5.43 (1H, d, J ) 4.6
Hz, OH), 5.02 (1H, d, J ) 3.5 Hz, OH), 4.98 (1H, d, J ) 7.5
Hz, OH), 4.80 (1H, d, J ) 7.9 Hz, H-1′), 4.60 (1H, dd, J ) 11.8,
1.5 Hz, H-6′A), 4.30 (1H, dd, J ) 11.8, 7.5 Hz, H-6′B), 4.00
(1H, m, H-5′), 3.50 (3H, m, H-2′,-3′,-4′); 13C NMR (DMSO-d6)
δ 166.2 (s, C-7′′), 152.6 (s, C-4), 147.1 (s, C-2), 138.4 (s, C-1),
133.7 (d, C-4′′), 129.3 (s, C-1′′), 2 × 129.2 (d, C-2′′,-6′′), 2 ×
128.9 (d, C-3′′,-5′′), 117.6 (d, C-6), 105.6 (d, C-5), 103.4 (d, C-3),
100.4 (d, C-1′), 71.4 (d, C-5′), 70.8 (d, C-2′), 69.9 (d, C-3′), 67.4
(d, C-4′), 64.6 (t, C-6′); FABMS (negative mode) m/z 391 [M -
H]-, (positive mode) m/z 393 [M + H]+, 267 [M - aromatic
ring]+.
3,4-Meth ylen d ioxyp h en yl-2′,3′,4′,6′- tetr a a cetyl-O-â-D-
glu cop yr a n osid e (7). A solution of tetra-O-acetyl-R-bromo-
D-glucopyranose (2.97 g, 7.2 mmol) and benzyltriethylammo-
nium bromide (810 mg, 2.9 mmol) in CHCl3 (15.8 mL) was
added to a solution of sesamol (3,4-methylenedioxyphenol)
(1.04 g, 7.2 mmol) in NaOH (1.25 M, 8.8 mL). The reaction
was heated in an oil bath at 60 °C. The progress of the reaction
was monitored by TLC (Si gel, eluent n-hexane-ethyl acetate
7:3). After 16 h the reaction mixture was diluted with H2O
(50 mL) and extracted with CHCl3 (3 × 30 mL). The combined
organic phases were dried (Na2SO4) and the solvent removed
under reduced pressure. The crude material (4.46 g) was flash
chromatographed (Si gel, n-hexane-ethyl acetate 7:3) and
afforded 0.31 g of unreacted sesamol (29.8%) and 2.31 g of 7
(68.5%), which was crystallized from n-hexane-ethyl acetate
to afford 1.97 g of colorless crystals.
Compound 4 (30 mg) was permethylated under the same
conditions used for 2. After purification, 18 mg of 6a were
obtained. 2a : 1H NMR (200 MHz, CDCl3); δ 7.05 (1H, d, J )
8.7 Hz, H-6), 6.43 (1H, d, J ) 2.7 Hz, H-3), 6.32 (1H, dd, J )
8.7, 2.7 Hz, H-5), 5.10 (1H, d, J ) 7.5 Hz, H-1′), 3.74, 3.70,
3.62, 3.58, 3.40, 3.34 (6 × 3H, s, -OCH3)
Compound 4a (18 mg) was hydrolyzed under the same
conditions used for 2a , giving 10 mg of 2,4 dimethoxyphenol
1
(4b): H NMR (CDCl3, 200 MHz); δ 6.84 (1H, d, J ) 8.7 Hz,
Com p ou n d 7: mp 162 °C (n-hexane-ethyl acetate); [R]20
D
H-6), 6.50 (1H, d, J ) 2.8 Hz, H-3), 6.40 (1H, dd, J ) 8.7, 2.8
Hz, H5), 5.22 (1H, s, -OH); 3.87 (3H, s, -OCH3), 3.77 (2 ×
3H, s, -OCH3).
-68° (c 1, CHCl3) Rf 0.24 (n-hexane-EtOAc 7:3); 1H NMR
(CDCl3) δ 6.71 (1H, d, J ) 8.3 Hz, H-5), 6.60 (1H, d, J ) 2.3
Hz, H-2), 6.47 (1H, dd, J ) 8.3, 2.3 Hz, H-6), 5.30-5.15 (4H,
m, H-2′ + H-3′ + H-4′), 4.93 (1H, d, J ) 7.6 Hz, H-1′), 4.30
(1H, dd, J ) 12.2, 5.4 Hz, H-6′B), 4.18 (1H, dd, J ) 12.2, 6.0
Hz, H-6′A), 3.82 (1H, m, H-5′), 2.11 (3H, s), 2.09 (3H, s), 2.06
(3H, s), 2.05 (3H, s); 13C NMR (CDCl3) δ 170.4 (s), 170.1 (s),
169.3 (s), 162.2 (s), 151.9 (s), 148.0 (s), 143.5 (s), 109.5 (d), 107.8
(d), 101.3 (t), 100.4 (d), 100.2 (d), 72.6 (d), 71.8 (d), 70.9 (d),
68.1 (d), 61.8 (t), 4 × 20.5 (q); FABMS (negative mode) m/z
467 [M - H]- ; (positive mode) m/z 491 [M + Na]+, 469 [M +
H]+; anal. C 52.9%, H 5.3%, calcd for C21H24O12 C 53.8%, H
5.2%.
Com p ou n d 5: [R]25 -34.2° (c 0.53, MeOH); 1H NMR
D
(DMSO-d6) δ 8.90 (1H, s, OH), 8.42 (1H, s, OH), 7.50-7.30
(5H, m), 6.90 (1H d, J ) 8 Hz, H-6), 6.30 (1H, d, J ) 2 Hz,
H-3), 6.15 (1H, dd, J ) 8, 2 Hz, H-5), 5.00 (1H, t, J ) 7.0 Hz,
H-9′′), 4.45 (1H, d, J ) 7.9 Hz, H-1′), 4.35 (1H, dd, J ) 11.8,
1.5 Hz, H-6′A), 4.05 (1H, dd, J ) 11.8, 7.5 Hz, H-6′B), 2.16
(2H, d, J ) 7.0 Hz, H-8′′); 13C NMR (DMSO-d6) δ 170.5 (s,
C-7′′), 153.7 (s, C-4), 147.9 (s, C-2), 144.7 (s, C-1′′), 138.3 (s,
C-1), 2 × 128.2 (d, C-2′′,-6′′), 2 × 125.8 (d, C3′′,-5′′), 118.7 (d,
C-6), 105.5 (d, C-1′), 2 × 103.0 (d, C-3,5), 75.7 (d, C-3′), 73.9
(d, C-5′), 73.3 (d, C-2′), 70.1 (d, C-4′), 69.4 (d, C-9′′), 63.7 (t,
C-6′), 44.5 (t, C-8′′); FABMS (positive mode) m/z 459 [ M +
Na ]+.
3,4-Dih yd r oxyp h en yl-O-â-D-glu cop yr a n osid e (8). A 0.5
M solution of NaSEt in DMF was prepared by adding EtSH
(0.93 g, 1.1 mL, 15 mmol) to an ice-cooled and magnetically
stirred suspension of NaH (0.4 g of a 60% oil dispersion, 10
mmol) in DMF (10 mL) and stirring at room temperature for
Compound 5 was stirred with Ba(OH)2 (10 mg in 1 mL H2O),
under N2 for 1h. After acidification, the mixture was purified

1530 J ournal of Natural Products, 1999, Vol. 62, No. 11
Verotta et al.
15 min. Then the glucoside (7) (0.4 g, 0.87 mmol) was added,
and the resulting solution was heated in an oil bath at 80 °C.
The progress of the reaction was monitored by TLC (Si gel,
using as eluent the organic phase of the biphasic system
CHCl3-MeOH-H2O 5:5:3). After 10 h, the cooled reaction
mixture was acidified with 10% HCl (10 mL) to achieve a
neutral pH and extracted with BuOH (3 × 15 mL). The
combined organic extracts were washed with H2O and dried
(Na2SO4). Removal of the solvent under reduced pressure
afforded a residue (0.37 g) that was purified by chromatogra-
phy (MPLC, LiChroprep RP8, 25-40 µm, eluting with MeOH-
H2O 45:5). After crystallization (H2O-MeOH) 0.227 g (90.4%)
of 3,4-dihydroxyphenyl-O-â-D-glucopyranoside (8) was ob-
tained.
Com p ou n d 8: mp 123 °C; [R]20D -43° (c 1, MeOH); Rf 0.18
(CHCl3-MeOH-H2O 5:5:3, organic phase); 1H NMR (DMSO-
d6) δ 8.91 (1H, s, OH), 8.49 (1H, s, OH), 6.59 (1H, d, J ) 8.5
Hz, H-5), 6.47 (1H, d, J ) 2.7 Hz, H-2), 6.32 (1H, dd, J ) 8.5,
2.7 Hz, H-6), 5.23 (1H, d, J ) 4.4 Hz, OH), 5.02 (1H, d, J )
5.1 Hz, OH), 4.59 (1H, d, J ) 7.5 Hz, H-1′), 4.54 (1H, s, OH),
3.65 (1H, dd, J ) 11.0, 1.5 Hz, H-6′), 3.45 (1H, m, H-6′), 3.17
(4H, m, H-2′, -3′, -4′, -5′); 13C NMR (DMSO-d6) δ 150.8 (d), 145.6
(s), 140.3 (s), 115.5 (d), 106.8 (d), 105.4 (d), 101.8 (d), 76.9 (d),
76.7 (d), 73.4 (d), 69.8 (d), 60.8 (t); FABMS (negative mode)
m/z 287 [M - H]- ; (positive mode) m/z 311 [M + Na]+, 289
[M + H]+ ; anal. C 49.7%, H 5.4%, calcd for C12H16O8, C 50.0%,
H 5.6%.
11.0, 2.0 Hz, H-6′A), 2.08, 2.06, 2.05, 2.02 (4 × 3H, s, CH3),
3.78 (1H, m, H-5′); CIMS m/z 457 [M + H]+, 331 [C14H19O9]+.
2,4-Dih ydr oxyph en yl-O-â-D-glu copyr an oside (3). A 0.2-M
solution of sodium methoxide (1.5 mL) was added to a solution
of 2,4-dihydroxyphenyl-tetra-O-acetyl-â-D-glucopyranoside (0.020
g, 0.05 mmol) in MeOH (2 mL). The reaction was stirred at
room temperature and monitored by TLC (Si gel, eluting with
CHCl3-MeOH 8:2). After 2 h, DOWEX 50WX was added to
achieve a neutral pH. The suspension was filtered, and the
solvent was removed under reduced pressure to afford 2,4-
dihydroxyphenyl-O-â-D-glucopyranoside (3) in quantitative
yields: C12H16O8; 1H NMR (DMSO-d6 + D2O) δ 6.90 (1H d,
J ) 8.0 Hz, H-6), 6.24 (1H d, J ) 3 Hz, H-3), 6.12 (1H dd, J )
8.0, 3.0 Hz, H-5), 4.50 (1H d, J ) 7.5 Hz, H-1′), 3.70 (1H, m,
H-6′B), 3.50 (1H m, H-6′A), 3.20 (4H m, H-2′,3′,4′,5′); 13C NMR
(DMSO-d6) δ 153.7 (s), 148.1 (s), 138.4 (s), 119.1 (d), 105.5 (d),
104.2 (d), 103.3 (d), 77.2 (d), 76 (d), 73.5 (d), 69.9 (d), 60.9 (t);
CIMS m/z 288 [M]+.
2-Meth oxy-5-ben zyloxyp h en yl-tetr a -O-a cetyl-â-D-glu -
cop yr a n osid e. 2-Methoxy-4-benzyloxyphenol (0.3 g, 1.31
mmol) was treated with tetra-O-acetyl R-bromo-D-glucopyr-
anose (0.540 g) under phase-transfer catalysis as described
above for the preparation of 2,4-dibenzyloxyphenyl-tetra-O-
acetyl-â-D-glucopyranoside and afforded, after crystallization
from n-hexane-ethyl acetate, 0.35 g (50% yield) of pure,
colorless 2-methoxy-4-benzyloxyphenyl-tetra-O-acetyl-â-D-glu-
copyranoside: C28H32O12; mp 137.5 °C (n-hexane-EtOAc); 1H
NMR (CDCl3) δ 7.50-7.35 (5H, m), 6.81 (1H, d, J ) 9.0 Hz,
H-6), 6.78 (1H, d, J ) 2.5 Hz, H-3), 6.61 (1H, dd, J ) 9.0, 2.5
Hz, H-4), 5.05-5.30 (3H, m, H-2′,-3′,-4′), 4.98 (2H, s, -CH2-
Ph), 4.95 (1H, d, J ) 7.5 Hz, H-1′), 4.93 (1H, m, H-5′), 4.22
(1H, dd, J ) 10.0, 6.0 Hz, H-6′B), 4.12 (1H, dd, J ) 10.0, 2.8
Hz, H-6′A), 3.75 (3H, s, -CH3), 2.06, 2.04, 2.03, 2.00 (4 × 3H,
s, -CH3); EIMS m/z: 560 [M]+, 331 [C14H19O9]+, 230 [M -
3,4-Dih yd r oxyp h en yl-(6′-O-ben zoyl)-O-â-D-glu cop yr a n -
osid e (2). The glucoside 8 was benzoylated according to the
protocol given for arbutin. The crude material (0.107 g) was
purified by chromatography on Si gel (1:100, eluting with
CHCl3-MeOH 8:2) followed by crystallization from H2O-
MeOH) and afforded 0.47 g (69.1%) of 2 identical in all respects
to the natural compound.
C
14H18O9]+.
2,4-Diben zyloxyp h en yl-tetr a -O-a cetyl-â-D-glu cop yr a n -
osid e. A solution of tetra-O-acetyl R-bromo-D-glucopyranose
(0.10 g, 0.24 mmol) and benzyltriethylammonium bromide
(0.08 g, 0.27 mmol) in CHCl3 (3 mL) was added to a solution
of 2,4-dibenzyloxyphenol13 (0.073 g, 0.24 mmol) in NaOH (0.5
M, 1.5 mL). The reaction was heated at 60 °C and monitored
by TLC (Si gel, eluent n-hexane-ethyl acetate 7:3). After 13
h, the reaction mixture was diluted with H2O (10 mL) and
extracted with CHCl3 (3 × 10 mL). The combined organic
phases were dried (Na2SO4) and the solvent removed under
reduced pressure. The crude material was flash chromato-
graphed (Si gel, eluting with n-hexane-EtOAc 7:3) and
afforded, after crystallization from ethyl acetate-diisopropyl
ether, 0.084 g (55%) of pure, colorless 2,4-dibenzyloxyphenyl-
tetra-O-acetyl-â-D-glucopyranoside: C34H36O12; mp 141.5 °C
(ethyl acetate-diisopropyl ether); [R]20D -32.9° (c 1.08, CHCl3);
1H NMR (CDCl3) δ 7.50-7.35 (10H, m), 7.08 (1H, d, J ) 11.0
Hz, H-6), 6.62 (1H, d, J ) 3.0 Hz, H-3), 6.45 (1H, dd, J ) 10.0,
3.0 Hz, H-5), 5.3-5.1 (3H, m, H-2′,-3′,-4′), 5.05 (2H, s, -CH2-
Ph), 5.00 (2H, s, -CH2Ph), 4.90 (1H, d, J ) 7.6 Hz, H-1′), 4.28
(1H, dd, J ) 11.0, 5.6 Hz, H-6′B), 4.11 (1H, dd, J ) 11.0, 2.6
Hz, H-6′A), 3.70 (1H, m, H-5′), 2.08, 2.06, 2.05, 2.02 (4 × 3H,
s, -CH3); 13C NMR (CDCl3) δ 170.5 (s), 170.2 (s), 169.4 (s),
169.3 (s), 155.9 (s), 150.5 (s), 140.3 (s), 136.8 (s), 136.5 (s), 129.0
(d), 128.5 (d), 2 × 128.1 (d), 121.8 (d), 105.5 (d), 102.9 (d), 101.0
(d), 72.7 (d), 71.8 (d), 71.1 (d), 70.8 (t), 70.4 (t), 65.4 (d), 61.6
(t), 4 × 20.0 (q); EIMS m/z 636 [M]+, 331 [C14H19O9]+, 306
[M - C14H18O9]+, 91 [CH2C6H5]+.
2-H yd r oxy-5-b e n zyloxyp h e n yl-O-â-D -glu cop yr a n o-
sid e. 2-Methoxy-5-benzyloxyphenyl-tetra-O-acetyl-â-D-glu-
copyranoside (0.25 g) was treated with sodium thioethoxide
in DMF, as described above for the preparation of 2-methoxy-
5-benzyloxyphenol, and afforded, after chromatography (Si gel,
eluting with CHCl3-MeOH 9:1), 42% yield of pure 2-methoxy-
5-benzyloxyphenyl-O-â-D-glucopyranoside: C19H22O8, 1H NMR
(DMSO-d6) δ 6.85 (1H, d, J ) 2.8 Hz, H-3), 6.69 (1H, d, J )
10.0 Hz, H-6), 6.50 (1H, dd, J ) 10.0, 2.8 Hz, H-4), 4.97 (2H,
s, -CH2Ph), 4.63 (1H, d, J ) 8.0 Hz, H-1′), 3.70-3.45 (2H, m,
H-6′), 3.20 (4H m, H-2′,-3′,-4′,-5′); 13C NMR (DMSO-d6) δ 151.4
(s), 145.6 (s), 140.7 (s), 137.4 (s), 128.4 (d), 127.7 (d), 115.8 (d),
108.6 (d), 104.6 (d), 102.3 (d), 77.3 (d), 75.9 (d), 75.3 (d), 70.0
(d), 69.7 (t), 60.9 (t), CIMS m/z 378 [M]+
2,5-Dih yd r oxyp h en yl-O-â-D-glu cop yr a n osid e (9). 2-Hy-
droxy-5-benzyloxyphenyl-O-â-D-glucopyranoside (0.1 g, 0.262
mmol) in EtOH (4 mL) was treated with cyclohexene (2 mL)
and palladium hydroxide (0.04 g) as described above for the
preparation of 2,4-dihydroxyphenyl-tetra-O-acetyl-â-D-glucopy-
ranoside and afforded 2,4-dihydroxy-phenyl-O-â-D-glucopy-
ranoside (9) in quantitative yield: C12H16O8, 1H NMR (DMSO-
d6 + D2O) δ 6.59 (1H d, J ) 8 Hz, H-6), 6.42 (1H d, J ) 2.0
Hz, H-3), 6.25 (1H dd, J ) 8.0, 2.0 Hz, H-4), 4.55 (1H d, J )
7.0 Hz, H-1′), 3.5-3.7 (2H m, H-6′), 3.25 (4H m, H-2′,-3′,-4′,-
5′); 13C NMR (DMSO-d6) δ 150.1 (s), 145.6 (s), 139.4 (s), 115.8
(d), 109.0 (d), 105.0 (d), 102.6 (d), 77.1 (d), 75.8 (d), 73.3 (d),
69.7 (d), 60.7 (t); CIMS m/z 288 [M]+.
2,4-Dih yd r oxyp h en yl-tetr a -O-a cetyl-â-D-glu cop yr a n o-
sid e. To a solution of 2,4-dibenzyloxyphenyl-tetra-O-acetyl-â-
D-glucopyranoside (0.045 g, 0.07 mmol) in EtOH (3 mL),
cyclohexene (1.5 mL) and palladium hydroxide (0.03 g) were
added. The reaction mixture was vigorously stirred at reflux
for 3 h, filtered on a Celite pad, and the solvent removed under
reduced pressure to afford 2,4-dihydroxyphenyl-tetra-O-acetyl-
Ack n ow led gm en t. Work supported by MURST (40%
funds) and CNR of Italy.
Su p p or tin g In for m a tion Ava ila ble: Synthesis of 2,4-diben-
zyloxyphenol and synthetic schemes for compounds 1, 4, and 9.
This material is available free of charge via the Internet at http://
pubs.acs.org.
â-D-glucopyranoside as an oily compound (0.029 g, 90%):
1
C
20H24O12, H NMR (CDCl3) δ 6.78 (1H, d, J ) 9.0 Hz, H-6),
Refer en ces a n d Notes
6.45 (1H, d, J ) 3.0 Hz, H-3), 6.25 (1H, dd, J ) 9.0, 3.0 Hz,
H-5), 5.30-5.08 (3H, m, H-2′,-3′,-4′), 4.80 (1H, d, J ) 7.2 Hz,
H-1′), 4.27 (1H, dd, J ) 11.0, 5.6 Hz, H-6′B), 4.15 (1H, dd, J )
(1) Watt, J . M.; Breyer-Brandwijk, M. G. Medicinal and Poisonous Plants
of Eastern and Southern Africa, S. Livingstone: Edinburgh, p 1962;
pp 872-875.

Glycosides from Proteaceae
J ournal of Natural Products, 1999, Vol. 62, No. 11 1531
(2) Perold, G. W.; Beylis, P.; Howard, A. S. J . Chem. Soc., Perkin Trans
I 1973, 638-643.
(11) In an alternative approach, characterized by the protection of the
ortho-dihydroxy moiety in 1,2,4-trihydroxy benzene as a dimethyl
ether, the expected glucoside 4 was not obtained. This unexpected
outcome of the reaction is due to the failure of sodium thioethoxide
to demethylate a phenolic methyl ether in the presence of a free
phenolic group on the same aromatic ring. A free phenolic group is,
however, tolerated on an other aromatic ring in the same molecule.6
This approach, however, can be highly convenient for the preparation
of less polar monomethyl derivatives, useful for the evaluation of the
influence of the free hydroxyl groups on the biological activity.
(12) Direct benzylation of 1,2,4-trihydroxybenzene with benzyl bromide
in dimethylformamide afforded 2,4-dibenzyloxyphenol in 5% yield,
accompanied by the tribenzyl derivative (5%), by other monobenzyl
(35%) and dibenzyl derivatives (7%), with a 52% total recovery.
(13) The synthesis of 2,4-dibenzyloxyphenol is reported in the Supporting
Information.
(3) Perold, W. G.; Beylis, P.; Howard, A. S. J . Chem. Soc., Perkin Trans
I 1973, 643-649.
(4) Perold, G. W.; Rosenberg, M. E. K.; Howard, A. S.; Huddle, P. A. J .
Chem. Soc., Perkin Trans 1 1979, 239-243.
(5) Pelizzoni, F.; Verotta, L.; Rogers, C. B.; Colombo, R.; Pedrotti, B.;
Balconi, G.; Erba, E.; D’Incalci, M. Nat. Prod. Lett. 1993, 1, 273-
280.
(6) Orsini, F.; Pelizzoni, F.; Verotta, L.; Aburjai, T.; Rogers, B. C. J . Nat.
Prod. 1997, 60, 1082-1087.
(7) Cristoni, A. (Indena SpA, Milano-Italy) personal communication.
(8) Liguori, A.; Procopio, A. J . Chem. Soc., Perkin Trans. 1 1993, 1783-
1786.
(9) Transesterification with ethyl benzoate in the presence of alumina
(Rana, S. S.; Barlow, J . J .; Matta, K. L. Tetrahedron Lett. 1981, 22,
5007-5010), a useful procedure recommended for the regioselective
acetylation of primary hydroxy groups, did not afford the desired
compound.
(10) Kane, D.; Walker, S.; Cheng, Y.; Van Engen, D. J . Am. Chem. Soc.
1989, 111, 6881-6882.
NP9902237