Benzophenone O-glucoside, a biogenic precursor of 1,3,7-trioxygenated xanthones in Hypericum annulatum

Article abstract of DOI:10.1016/S0031-9422(01)00194-7

Two new benzophenones, hypericophenonoside (1) and annulatophenone (2) were isolated from the methanol extract of the herb of Hypericum annulatum. The structures of the benzophenones were established as 2′-O-β-D-glucopyranosyl-2,4,5′,6-tetrahydroxybenzophenone (1) and 2,3′,5′,6-tetrahydroxy-4-methoxybenzophenone (2) based on spectral and chemical evidence. Hypericophenonoside is the second benzophenone O-glycoside found in nature. Acid and enzymatic hydrolysis of (1) has led directly to the formation of 1,3,7-trihydroxyxanthone (gentisein). This fact confirmed the hypothesis that some xanthones could be formed in plants by dehydration of 2,2′-dihydroxybenzophenones, and the intermediate precursors appear to be benzophenone O-glycosides ortho to the carbonyl function.

Full text of DOI:10.1016/S0031-9422(01)00194-7

Phytochemistry 57 (2001) 1237–1243
www.elsevier.com/locate/phytochem
Benzophenone O-glucoside, a biogenic precursor of
1,3,7-trioxygenated xanthones in Hypericum annulatum
Gerassim M. Kitanov*, Paraskev T. Nedialkov
Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, Dunav str. 2, 1000 Sofia, Bulgaria
Received 22 January 2001; received in revised form 6 April 2001
Abstract
Two new benzophenones, hypericophenonoside (1) and annulatophenone (2) were isolated from the methanol extract of the herb of
Hypericum annulatum. The structures of the benzophenones were established as 20-O-b-d-glucopyranosyl-2,4,50,6-tetrahydroxy
benzophenone (1) and 2,30,50,6-tetrahydroxy-4-methoxybenzophenone (2) based on spectral and chemical evidence. Hyper-
icophenonoside is the second benzophenone O-glycoside found in nature. Acid and enzymatic hydrolysis of (1) has led directly to
the formation of 1,3,7-trihydroxyxanthone (gentisein). This fact confirmed the hypothesis that some xanthones could be formed in
plants by dehydration of 2,20-dihydroxybenzophenones, and the intermediate precursors appear to be benzophenone O-glycosides
ortho to the carbonyl function. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Hypericum annulatum; Guttiferae; Benzophenones; Annulatophenone; Hypericophenonoside; Transformation to 1,3,7-trihydroxy-
xanthone
1. Introduction
crude MeOH residue was dissolved in hot water and
partitioned subsequently with CHCl3 and EtOAc. The
EtOAc extract was fractionated by CC on polyamide
using H2O and H2O–EtOH mixtures as eluent. The
fraction containing compound 1 was rechromato-
graphed on polyamide and eluted with CHCl3–MeOH
mixtures. Sephadex LH-20 was used as final purification
step. The fraction rich in compound 2 was subjected to
gel filtration on Sephadex LH-20.
In previous studies of Hypericum annulatum Moris,
subsp. annulatum (Robson, 1993), also known as
Hypericum degenii Bornm. (Jordanovand Koz ucharov,
1970), the presence of several flavonols, biapigenin,
catechins, two hypericins and four xanthones was
demonstrated. Total hydrolysis of purified EtOH
extract from the aerial parts of this species has led to the
isolation of large amounts of 1,3,7-trihydroxyxanthone
(gentisein), but only low quantities of its 3-O-glucoside
(xanthohypericoside) were found in the plant (Kitanov
and Akhtardziev, 1979; Kitanov and Nedialkov, 2000).
This paper describes the isolation and structure eluci-
dation of two benzophenones and transformation of
one of them (hypericophenonoside) to gentisein by acid
and enzymatic hydrolysis.
Compound 1 was easily dissolved in water and gave a
dark brown color with ferric chloride. Quantitative acid
hydrolysis with HCl yielded an aglycone and d-glucose
in a ratio 1:1 (57.54 and 42.46%, respectively). The
1
aglycone was identified by UV, IR, EI-MS, H NMR
spectra and co-TLC as 1,3,7-trihydroxyxanthone (gen-
tisein) (1d), previously isolated from this species (Kita-
novand Akhtardzhiev, 1979). The main mystery was
the presence only of one band at 304 nm in the UV
spectrum of compound 1, while the aglycone showed
absorption maxima at 236, 260, 310 and 367 nm typical
for xanthones. The similar four maxima were also
observed for 3-O-b-d-glucoside of 1,3,7-trihydroxy-
xanthone found not so long ago in the same plant
(Kitanovand Nedialkov, 2000). Mild acid hydrolysis of
1 with 0.2–0.5 M HCl and TFA and treatment with b-
glucosidase similarly led to the obtaining of gentisein
and d-glucose. When monitored by TLC, mild and
2. Results and discussion
The dried aerial parts of H. annulatum were defatted
with n-hexan and then extracted with hot MeOH. The
* Corresponding author. Tel.: +359-2-987-6265; fax: +359-2-987-
9874.
E-mailaddress:gkitanov@mbox.pharmfac.acad.bg(G.M.Kitanov).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00194-7

1238
G.M. Kitanov, P.T. Nedialkov / Phytochemistry 57 (2001) 1237–1243
enzymatic hydrolysis of 1 showed presence of an inter-
mediate product, which could not be isolated because of
its instability.
C13H8O5 of gentisein 1d. In the FAB-MS spectrum the
[M+H]+ peak at m/z 425 was consistent with molecular
formula C19H20O11, the base peak at m/z 263 [Magl
+
The IR spectrum of 1 showed absorption bands at
.
H]+ (rel. int. 100%) with the formula C13H10O6 and an
ion peak at m/z 245 [Mgent+H]+ indicated the gentisein
moiety (Fig. 1). Gentisein 1d was obtained by dehydra-
tion of aglycone 1c (MaglÀMgent=18). The molecular
mass of 1 was also confirmed by elemental analysis
data: C=53.71 and H=4.82% respectively.
3302 (OH), 1643 (C¼O), 1601 and 1516 (C¼C) cmÀ1
The bathochromic shift of the band at 304 nm in the
UV spectrum with AlCl3 (+14 nm) and NaOAc (+33
nm) revealed the presence of free hydroxyls ortho and
para, respectively, to the carbonyl function. The HREI-
MS of 1 showed the highest peak at m/z 244.0383 (rel.
int. 100%) corresponding to the molecular formula
1
The H NMR spectrum of 1 in acetone-d6 (Table 1)
showed the presence of signals from two chelated
Fig. 1. Transformation of hypercophenonoside (1).

G.M. Kitanov, P.T. Nedialkov / Phytochemistry 57 (2001) 1237–1243
1239
Table 1
1H NMR spectral data (300 MHz) for 1, 1b and 2a
No.
1
(acetone-d6) (J)
1
(DMSO-d6) (J)
1b
(CDCl3) (J)
2
(acetone-d6) (J)
2
(DMSO-d6) (J)
1
–
–
–
–
–
2
10.96 br. s, OHb
5.91 sb
11.44 s, OHb
5.74 sb
3.68 s, OMeb
10.22 br. s, OHb
9.80 s, OHb
3
6.12 sb
3.84 s, OMe
6.12 sb
3.68 s, OMeb
6.02sb
3.81 s, OMe
6.02sb
5.90 sb
3.75 s, OMe
5.90 sb
9.80 s, OHb
4
8.26 br. s, OHb
5.91 sb
10.96 br. s, OHb
10.38 s, OH
5.74 sb
11.44 s, OHb
5
6
10.22 br. s, OHb
10
20
30
40
500
600
100
2
300
400
500
600
–
–
–
2.03 s, OAc
–
–
–
–
6.62 d, (2.2)b
6.65 d, (2.1)b
7.08 d, (8.8)
6.81 dd, (8.8, 3.0)
8.26 br. s, OHb
6.72 d, (3.0)
4.73 d, (7.7)
3.23 mc
3.31 mc
3.45 mc
3.66 br. tc
3.88 br. dc
7.0 d, (8.7)
6.70 dd, (8.7, 2.9)
9.15 s, OH
6.51 d, (2.9)
4.59 d, (7.2)
3.02–3.67 mc
Sugar
6.96 d, (8.8)
7.03 dd, (8.8, 3.0)
8.40 br. s, OHb
6.49 t, (2.2)
8.40 br. s, OHb
6.62 d, (2.2)b
9.50 s, OHb
6.35 t, (2.1)
9.50 s, OHb
6.65 d, (2.1)b
3.78 s, OMe
7.28 d, (3.0)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Protons
a
The chemical shifts are expressed in ppm and the coupling constants (J) in Hz/s. All protons were assigned by 1H–1H and HETCOR experi-
ments.
b
Overlapping.
Signal patterns are unclear due to overlapping.
c
hydroxyl protons at ꢀ 10.96 and a broad signal at ꢀ 8.26
(which were much more expressed in DMSO-d6 at ꢀ
11.44, 10.38 and 9.15) all disappearing after addition of
D2O. The presence of two hydroxyl groups ortho to the
carbonyl was confirmed by the appearance of signals
from two acetyl groups in lower field at ꢀ 2.30 and 2.35
after peracetylation of 1. The presence of five sub-
stituents of aromatic rings of 1 was confirmed by DEPT
experiment (Table 2). The signal at ꢀ 5.91 (s, 2H) indi-
cated a symmetrical three substituted phloroglucinol
ring A, which was confirmed by the presence of the
overlapping signals in 13C NMR at 165.86 ppm (C2 and
C6) and 96.48 ppm (C3 and C5) (Table 2). The signals of
three aromatic protons from ring B (acetone-d6)
appeared as two doublets and a quadruplet at ꢀ 7.08,
6.72 and 6.81 with J=8.8 and 3.0 Hz respectively. This
fact pointed to the ortho and meta positions of the pro-
tons and an asymmetrical structure of this ring.
UV spectra of 1a with AlCl3 (+19 and +70 nm,
respectively) indicated the presence of a free hydroxyl
group ortho to the carbonyl moiety.
Acetylation of 1a gave 1b with mp 121.5–123ꢀC. The
EI-MS spectrum of this compound showed a molecular
ion [M]+. at m/z 360 corresponding to the formula
C19H20O7 and a peak at m/z 318 [MÀAc +H]+, which
indicated 1b as a monoacetate. The IR spectrum showed
an absorption at 1769 cmÀ1 (–OAc group) and an
absence of characteristic band for free hydroxyl groups.
1
The H NMR spectrum (Table 1) indicated signals for
five aromatic protons, four methoxyl groups, two of
which are overlapping at ꢀ 3.68, and an acetyl group at ꢀ
2.03 (3H) ortho to the carbonyl function.
Thus the structure of 1 was established as 20-O-b-d-
glucopyranosyl-2,4,50,6-tetrahydroxybenzophenone,
named hypericophenonoside. It is a new natural com-
pound and the second benzophenone O-glycoside found
in the plants (Ferrari et al., 2000).
The signal at ꢀ 4.73 (d, J=7.7 Hz) and a multiplet at ꢀ
3.23–3.88 were assigned to the sugar residue with b-
configuration of the anomeric proton. The positions of
Compound 2 was isolated as pale yellow needles. It
reacted with ferric chloride to give a dark brown color.
The IR spectrum established the presence of a hydroxyl
(3302 cmÀ1), a conjugated carbonyl (1645 cmÀ1) and an
aromatic system (1582, 1520 cmÀ1). The bathochromic
shift of the UV maximum after addition of aluminum
chloride (+33 nm) and the absence of shift upon addi-
tion of sodium acetate revealed free hydroxyl sub-
stituents ortho to the carbonyl group. The presence of
four hydroxyls, two of which ortho to the carbonyl
function, was confirmed by the presence of two sharp
1
all protons were assigned by H–1H COSY and HET-
COR experiments. Results of 13C NMR and DEPT
experiments (Table 2) confirmed the structure of agly-
cone 1c as 2,20,4,50,6-pentahydroxybenzophenone.
The presence of hydroxyl groups para to the carbonyl
function and the symmetrical structure of ring A suggest
the location of sugar moiety in ring B. To determine the
position of the glucose, 1 was methylated and the resul-
tant compound was hydrolyzed by HCl (Fig. 1). The
bathochromic shift of the bands at 262 and 369 nm in
1
singlets at ꢀ 9.80 (2H) and 9.50 (2H) in the H NMR

1240
G.M. Kitanov, P.T. Nedialkov / Phytochemistry 57 (2001) 1237–1243
Table 2
13C NMR spectral data (75 MHz) for 1 and 2a
No.
1
1
2
2
Acetone-d6
DEPT
DMSO-d6
Acetone-d6
DEPT
DMSO-d6
1
107.09
165.86b
96.48b
167.11
96.48b
165.86b
136.81
148.66
119.95
118.31
153.95
115.38
104.93
75.51
–
105.04
163.71b
94.42b
165.26
94.42b
163.71b
134.66
146.46
117.73
116.18
151.83
113.36
102.59
73.48
106.96b
163.76b
95.12b
167.37
95.12b
163.76b
144.88
108.17b
159.56b
106.96b
159.56b
108.17b
–
–
106.97b
158.28b
92.83b
161.85
92.83b
158.28b
140.81
108.52b
157.52b
106.97b
157.52b
108.52b
–
2
–
–
3
94.99b
93.62b
4
–
–
5
94.99b
–
93.62b
6
–
10
–
–
20
–
106.68b
30
118.46
116.82
–
–
105.48
40
500
–
600
113.89
103.44
74.02
76.66
70.34
76.73
61.83c
–
106.68b
100
–
200
–
–
–
3
78.15
71.83
76.53
69.75
–
–
–
400
–
–
–
50
78.22
63.32
76.95
60.89
–
–
–
600
–
–
–
>C¼O
–OCH3
199.46
–
197.12
–
200.11
56.49
–
54.97
195.67
54.97
–
a
The chemical shifts are expressed in (ppm). All carbons were assigned by HETCOR and DEPT experiments.
Overlapping.
Signals are turned upside-down.
b
c
spectrum in DMSO-d6 (Table 1), both disappearing
after addition of D2O.
and Lee, 1989). The presence of simple benzophenones
in species of this genus and their co-occurrence with
simple xanthones may be of taxonomic and biogenetic
value.
The HREI-MS spectrum showed [M]+. at m/z 276.0642
consistent with the molecular formula C14H12O6 and ion
peaks at m/z 167 [C8H7O4] derived from ring A and at m/
z 137 [C7H5O3] from ring B (Fig. 2). The molecular
formula of 2 was confirmed by elemental analysis. The
data of 1H NMR and molecular ions of EI–MS support
the presence of two symmetrical hydroxyls and a meth-
oxyl group in ring A (phloroglucinol system) and two
symmetrical hydroxyls in ring B (resorcinol system).
1H NMR exhibits a signal of two protons from phloro
glucinol ring A at ꢀ 6.02. A doublet at ꢀ 6.62 (2H,
J=2.2) and a triplet at ꢀ 6.49 (1H) indicated a sym-
metric resorcinol structure of ring B. The methoxyl
group appeared as a singlet at ꢀ 3.81. The assignment of
The biosynthetic pathways to xanthones in higher
plants have been discussed in several reviews (Carpenter
et al., 1969; Sultanbawa, 1980; Bennett and Lee, 1989;
Peres and Nagem, 1997). It has been shown that poly-
hydroxybenzophenones could be intermediate pre-
cursors in the formation of xanthones. The biosynthesis
involves shikimate and acetate pathways formation of
intermediate benzophenones, which then react intramo-
lecularly to give xanthones. The mechanism for this
intramolecular reaction has been postulated involving
four possible pathways.
Two alternative pathways of xanthone biosynthesis in
cell cultures of Centaurium erythraea Rafn. and Hyper-
icum androsaemum L. were recently reported (Beerhues,
1996; Schmidt and Beerhues, 1997). In the first one, the
intermediate 2,30,4,6-tetrahydroxybenzophenone was
shown to be intramolecularly coupled to 1,3,5-trihy-
droxyxanthone, whereas in the second one it was coupled
to form the isomeric 1,3,7-trihydroxyxanthone. These
regioselective oxidative phenol couplings were catalyzed
by xanthone synthases, proposed to be cytochrome P450
oxydases (Peters et al., 1998).
1
the signals was confirmed by H–1H COSY, 13C NMR,
HETCOR and DEPT experiments (Table 2). Conse-
quently the structure of 2 was established as 2,30,50,6-
tetrahydroxy-4-methoxybenzophenone, named annula-
tophenone.
Up to now, only several benzophenone C-glucosides
were found in Mangifera indica L. (Tanaka et al., 1984)
and a benzophenone O-glucoside together with a C-
glucoside was recently reported in Gnidia involucrata
Steut. Ex A. Rich. (Ferrari et al., 2000). Although pre-
nylated benzophenones often occur in the Guttiferae,
simple benzophenones are rare in this family and have
not been found in species of genus Hypericum (Bennett
One of the pathways is dehydration between the
hydroxyl groups of the acetate and shikimate derived rings
(2,20-dihydroxybenzophenones) via intermediates such as

G.M. Kitanov, P.T. Nedialkov / Phytochemistry 57 (2001) 1237–1243
1241
Fig. 2. EI–MS fragmentation of annulatophenone (2).
O-pyrophosphates (Carpenter et al., 1969; Sultanbawa,
1980). Up to date however there is no evidence for in
vivo formation of polyhydroxyxanthones from benzo-
phenones by dehydration mechanism. The question of
the co-occurrence of suitable polyhydroxybenzo-
phenone intermediates and xanthones in plants has not
yet found a satisfactory answer.
the precursors appear to be benzophenones with O-gly-
cosilation ortho to the carbonyl function.
3. Experimental
3.1. General experimental procedures
In our case, in the herb of Hypericum annulatum we
found the co-occurrence of large amounts of hyper-
icophenonoside 1 (with 2,20-hydroxyl groups) together
with 1,3,7-trihydroxyxanthone (gentisein) 1d. The ben-
zophenone O-glucoside 1 was easily transformed into
gentisein by acid or enzymatic hydrolysis. This fact
supports the evidence that 2,4,50,6-tetrahydroxybenzo-
phenone-20-O-glucoside 1 is a precursor of 1,3,7-trihy-
droxyxanthone. The transformation is realized by
elimination of the glucose moiety and subsequent dehy-
dration involving hydroxyls from phloroglucinol ring A
and ring B, located ortho to the carbonyl group (Fig. 1).
These results pointed to the fact, that one of the bio-
synthetic pathways of xanthone formation in plants is
through an intermediate glycosidation at an earlier
stage of the benzophenone biosynthesis before cycliza-
tion of the two rings.
Mps uncorr. OR was measured on a Perkin-Elmer
241 polarimeter using MeOH as solvent. UV spectra
were run in MeOH and shift reagents on a Specord UV–
VIS and IR spectra were recorded in nujol on a Shi-
1
madzu FTIR-8101 M instruments. H and 13C NMR
spectra were obtained on a Bruker ARX 300 apparatus
at 300 and 75 MHz, respectively, in acetone-d6 and
DMSO-d6 (1 and 2) and CDCl3 (1b), using TMS as
internal standard. HREI- and FAB-MS analyses were
performed on a Varian MAT 711 mass spectrometer at
70 eV in positive ion mode. Open column chromato-
graphy (CC): polyamide S (Woelm, Germany) and
Sephadex LH-20 (Pharmacia, Sweden). TLC was per-
formed using silica gel 60 GF254 (Merck), CHCl3–
MeOH–H2O (70:20:2) (A), EtOAc–MeCOEt–HCO2H–
H2O) (25:10:2:4) (B), EtOAc–MeOH–H2O–AcOH (65:
15:15:20) (C) and cellulose (Merck), EtOAc-pyridine–
H2O (12:5:4) (D) mixtures were used as solvents.
Hydrolysis of hypericophenonoside 1 was carried out
with HCl and TFA (100ꢀC, 2h) and b-glucosidase
Finally, it can be concluded that some xanthones are
formed in vivo by dehydration of 2,20-dihydroxybenzo-
phenones. This is a spontaneous reaction which appears
to be regulated by deglucosilation of the precursor and

1242
G.M. Kitanov, P.T. Nedialkov / Phytochemistry 57 (2001) 1237–1243
(Koch-Light Laboratories) (25ꢀC, 48 h). Methylation of
1 (Me2SO4+K2CO3 in dry acetone) and acetylation of 1
and 1a (Ac2O–pyridine method) were performed by
usual procedures. Detection of benzophenones was by
The hydrolysis was monitored by TLC, system A, time
interval 5–10 min, for 2 h. After dilution with H2O
and cooling, the aglycone ppt. was filtered and
recrystallized from MeOH. The aq. solution was
evapd to dryness in vacuo. The residue was dis-
solved in 50% EtOH and examined for sugar by
TLC using solvent systems C and D. The aglycone
was identified as 1,3,7-trihydroxyxanthone (gentisein)
on the basis of co-TLC, UV, IR, EI–MS and 1H
NMR and the sugar was identified to be d-glucose.
The similar procedure was used for mild hydrolysis of
1 with 0.2 M, 0.5 M HCl and 2 M TFA in H2O–MeOH
(1:1).
spraying with Fast blue salt B (Riedel-De Haen, Ger-
¨
many) in MeOH–H2O (1:1) and of sugars by anizidine
phtalate reagent followed by heating at 110ꢀC for 3–5
min.
3.2. Plant material
The aerial parts of Hypericum annulatum (syn. H.
degenii) were collected during the flowering season from
wild habitat at the Central Rhodope Mountains in July
1997. A voucher specimen (No. 144296) has been
deposited at the Herbarium of Botany Institute of Sofia
(SOM).
3.6. Enzymatic hydrolysis of 1
Compound 1 (15 mg) was dissolved in H2O and 20
mg of b-glucosidase were added. The solution remained
for 48 h at 25ꢀC and then was heated at 80ꢀC for 30
min. The reaction mixture was extracted Â3 with
EtOAc to remove the aglycone part and both solutions
(H2O and EtOAc) were evaluated for identification of
the components.
3.3. Extraction and isolation
The air dried and powdered herb (1.7 kg) was defatted
with n-hexan (1 lÂ6) and extracted with hot MeOH (6
lÂ8). The crude MeOH residue was dissolved in hot
H2O (2 l), filtered and treated with CHCl3 (300 mlÂ10).
The aq. phase was fractionated with EtOAc (500 mlÂ7).
The EtOAc fraction (52 g) was chromatographed on a
polyamide column (150 g), using a 0–60% EtOH linear
gradient and gave mixture of 1 and other compounds
(H2O fraction, 5.5 g) and 2 (30–40% EtOH, 2.5 g). The
H2O fraction was rechromatographed (Â3) on poly-
amide (30 g, step gradient CHCl3–MeOH 85:15–80:20).
Subsequent Sephadex LH-20 gel filtration (MeOH) gave
pure compound 1 (2.1 g). The other fraction, rich in 2,
was further purified on Sephadex LH-20 (MeOH) and
by repeated recrystallization from H2O–EtOH, 383 mg
of this compound was obtained.
3.7. Methylation and hydrolysis of 1
Compound 1 (200 mg) was refluxed for 46 h with
Me2SO4 (6 ml) in dry acetone (150 ml), containing dry
K2CO3 (4 g). After filtration and evapn. to dryness the
residue was dissolved in 40 ml MeOH–4% HCl (1:1).
The mixture was refluxed on boiling water bath for 3 h
and MeOH was removed in vacuo. The aq. solution
was extracted with CHCl3 (30 mlÂ5), dryed and after
evapn. of the solvent the residue was purified on
Sephadex LH-20 (MeOH). The methylated aglycone
was crystallized from MeOH–C6H6 to give a solid (76
mg) of 1a.
3.4. Hypericophenonoside (1) (2.1g)
3.8. 20-Hydroxy-2,4,50,6-tetramethoxybenzophenone
(1a) (76 mg)
20
Pale yellow needles (from H2O), mp 156–158ꢀC. ½aꢁD
+46.11ꢀ (MeOH, c1.0300). UV lmMaexOH nm (log E): 206
(4.327), 225sh, 304 (4.149); +AlCl3 202, 220, 318;
+AlCl3/HCl 202, 220, 315; +NaOAc 337; +NaOAc/
H3BO3 308. IR ꢁmnuajxol cmÀ1: 3302 (chelated OH); 1643
Yellow flakes (from MeOH–C6H6), mp 177.5–179ꢀC.
MeOH
max
UV l
nm: 231, 262, 369; +AlCl3 250sh, 281, 439;
+AlCl3/HCl 262sh, 281sh, 369, 439sh; +NaOMe 231sh,
nujol
max
(>C¼O); 1601, 1516 (>C¼C<, aromatic). 1H and 13
C
NMR: see Tables 1 and 2. HREI–MS m/z (rel. int.):
262, 369. IR ꢁ
cmÀ1: 3300 (w, chelated OH), 1615
(>C¼O), 1608, 1587 (>C¼C<, aromatic). HREI-MS
+.
244.0383 [Mgent
]
[C13H8O5] (100), (calcd 244.1993).
m/z: 318.2896 (calcd. 318.3205) [C17H18O6].
FAB-MS m/z (rel. int.): 425 [M+H]+ (42), 263
[Magl+H]+ (100), 245 [Mgent+H]+ (16), 163 [(Mglu
À
3.9. Acetylation of 1 and 1a
H2O)+H]+ (4). Found: C, 53.71; H, 4.82%. C19H20O11
requires: C, 53.78; H, 4.75%.
Samples of 1 (30 mg) and 1a (20.4 mg) were treated
with C5H5N (0.6 ml) and Ac2O (2 ml) and refluxed for 3
h. The reaction mixtures were poured on ice and kept at
4ꢀC for 24 h. The ppts. were filtered, dried and crystal-
lized from EtOH to give 21 mg of 1-octaacetate and 16
mg of 1a-monoacetate (1b) derivatives.
3.5. Acid hydrolysis of 1
A sample of compound 1 (50 mg) was dissolved in 10
ml of 1 N HCl and refluxed on water bath (100ꢀC).

G.M. Kitanov, P.T. Nedialkov / Phytochemistry 57 (2001) 1237–1243
1243
3.10. 20-Acetoxy-2,4,50,6-tetramethoxybenzophenone
(1b) (16 mg)
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nujol
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ꢁ
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Acknowledgements
The authors are indebted to Professor Ivo Ivanov
(Sofia, Bulgaria), Dr. Ulrich Girreser (Kiel, Germany)
and to Dr. Joachim Opitz (Stuttgart, Germany) for
measurement and providing NMR an MS spectra. This
work was supported by a grant 21/1998 from Medicinal
Science Council (MSC) at Medical University of Sofia.
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