X.-H. Wu et al. / Fitoterapia 81 (2010) 617–620
619
2
-benzyl-4,6-dihydroxy benzoic acid (1, Fig. 1). An
Table 2
1
3C NMR (100 MHz) spectral data of 1–3 (in DMSO-d
6
).
amorphous powder (MeOH); negative HR-ESI-MS: 243.0660
−
-
, calc. 243.0658). 1H NMR (400 MHz)
(
[M−H] , C14
H
11
O
4
Position
Compound 1
Compound 2
Compound 3
1
3
and C NMR (100 MHz) data: see Tables 1 and 2.
1
2
3
4
5
6
112.1
146.4
110.9
163.4
101.7
158.9
172.6
142.3
128.7
128.9
126.3
128.9
128.7
38.0
–
110.6
145.6
109.4
162.6
100.2
160.9
172.4
142.2
128.6
128.7
126.3
128.7
128.6
37.8
110.9
145.7
109.1
165.5
100.6
158.1
172.5
142.3
128.7
128.8
126.3
128.8
128.7
37.7
2
-benzyl-4,6-dihydroxy benzoic acid-6-O-β-D-glucopyra-
noside (2, Fig. 1). White powder (MeOH); IR bands (KBr):
539, 3408, 1626, 1497, 1435, 1407, 1362, 1194, 1070, 856,
02 cm . Negative HR-ESI-MS: 405.1184 ([M − H]
H O
20 21 9
3
7
C
−
1
−
,
−
, calc. 405.1186). 1H NMR (400 MHz) and C NMR
13
2
1
-COOH
′
(
100 MHz) data: see Tables 1 and 2.
-benzyl-4,6-dihydroxy benzoic acid-4-O-β-D-glucopyra-
noside (3, Fig. 1). White powder (MeOH); IR bands (KBr):
2
2′
3′
4′
5′
−
1
3
600, 3478, 1597, 1506, 1441, 1318, 1205, 1061, 799, 721 cm
.
−
−
Negative HR-ESI-MS: 405.1192 ([M−H] , C20
H
21
O
9
, calc.
05.1186). H NMR (400 MHz) and C NMR (100 MHz) data:
see Tables 1 and 2.
6
7
′
1
13
4
′
Glc:1″
102.1
73.6
101.9
73.4
77.6
69.9
77.1
2
3
4
5
″
″
″
″
–
–
77.5
2
.4. Acid hydrolysis
–
70.1
–
77
Compounds 2 and 3 (2 mg each) were refluxed with 2N HCl
6″
–
61.1
60.9
for 3 h, respectively. The aglycone was extracted with EtOAc.
The aqueous layers of the acid hydrolysis of 2 and 3 were
3
neutralised with NaHCO and then concentrated. D-glucose
was identified in each aqueous layer by TLC on a silica G plate
comparison with authentic sample, respectively. The Rf values
suggestive of a 2,4,6-trisubstituted benzoic acid moiety. The 13
NMR spectrum showed signals corresponding to a carboxyl
carbon (δ 172.6), two aromatic methine carbons (δ 110.9,
101.7), four aromatic quaternary carbons (δ 112.1, 146.4,
163.4, 158.9) for benzoic acid moiety. Four aromatic carbons
signals (δ 142.3, 128.7, 128.9, 126.3) included six aromatic
C
were 0.12 and 0.14 with CHCl
n-BuOH–HOAC–H O abbreviated to BAW (4:1:5, upper
layer) as developing system, respectively. And by measur-
3
–MeOH–H
2
O (3:1:0.1) and
C
C
2
C
2
0
20
D
ing its optical rotation ([α]
D
+38.1 (2), [α]
+40.2 (3)).
C
Thus, D-glucose was identified.
carbons (a pair of them are equivalent) and one methylene
signal (38.0) for a nonsubstituted benzyl moiety. The spectral
data and HMBC correlations of compound 1 are similar to the
compounds reported in the literatures [8,9]. Thus, the structure
of compound 1 was established as 2-benzyl-4,6-dihydroxy
benzoic acid.
3
. Results and discussion
Compound 1 was obtained as a white powder (MeOH)
and its molecular formula, C14
H
12
O
4
, was also determined by
−
−
Compound 2 was obtained as a white amorphous powder
HR-ESI-MS ([M−H] , m/z 243.0660). (calc. C14
H
11
O
4
,
1
(
MeOH) and its molecular formula, C20
H
22
O
9
, was also deter-
2
43.0658). The H NMR spectrum exhibited two aromatic
−
mined by HR-ESI-MS ([M−H] , m/z 405.1184). (calc.
C
H Z H Z
protons at δ 6.43 (d, J3,5=2.4 H ), δ 6.35 (d, J5,3 =2.4 H )
−
, 405.1186). The 1H NMR spectrum of 2 showed
20
H
21
O
9
several features which are characteristic for trisubstituted
benzoic acid moiety. Two singlets at δ 6.45, and 6.42 were
Table 1
1
H
6
H NMR (400 MHz) spectral data of 1–3 (in DMSO-d ).
attributed to aromatic methine carbons C-3 and C-5,
respectively. The 13C NMR spectra of 2 were similar to those
of 1, apart from signals C-6 (160.9), C-1 (110.6) and C-5 (100.2)
chemical shifts instead of C-6 (158.9), C-1 (112.1) and C-5
Position
Compound 1
Compound 2
Compound 3
1
2
3
4
5
6
–
–
–
–
–
–
6.43 (d, 2.4)
–
6.45 (d, 2.4)
–
6.48 (d, 2.4)
–
(
101.7) as in 1, which indicated that it exists glycosidation
chemical shifts. The sugar moiety was determined as D-glucose
6.35 (d, 2.4)
–
6.42 (d, 2.4)
–
6.40 (d, 2.4)
–
2
0
(
D
2
[α] +38.1, c 0.1, H O) by acid hydrolysis, by comparison
2
1
2
3
4
5
6
7
-COOH
–
–
–
with an authentic sample. The connectivity of sugar unit was
deduced from the HMBC cross peaks between anomeric H-1′
(δ 4.92) and C-6 (δ 160.9). On the basis of the above
H C
observations, 2 was named as 2-benzyl-4,6-dihydroxy benzoic
acid-6-O-β-D-glucopyranoside.
′
′
′
′
′
′
′
–
–
–
7.06 (m)
7.14 (m)
7.08 (m)
7.14 (m)
7.06 (m)
2.81 (2H, m)
–
–
–
–
–
–
–
7.05 (m)
7.13 (m)
7.08 (m)
7.13 (m)
7.05 (m)
2.78 (2H, m)
4.92 (d, 8.3)
3.49 (o)
3.46 (o)
3.39 (o)
7.01 (m)
7.13 (m)
7.06 (m)
7.13 (m)
7.01 (m)
2.76 (2H, m)
4.88 (d, 8.6)
3.50 (o)
3.49 (o)
3.37 (o)
3.61 (m)
4.02 (d, 10.6)
3.74 (o)
Compound 3 was obtained as a white amorphous power
with a molecular formula of C20
22 9
H O as determined by
Glc:1″
HR-ESI-MS and NMR analysis. The molecular formula
a
a
2
3
4
5
6
″
″
″
″
″
1
a
a
20 22 9
C H O of 3 was the same as 2. The H NMR spectrum of 3
also showed several features which are characteristic for
trisubstituted benzoic acid moiety. Two aromatic protons at
3.63 (m)
4.09 (d, 10.3)
3.78 (o)
δ
2
H
6.48 (d, J3,5=2.4 H
,4,6-trisubstituted benzoic acid moiety. The C NMR spectra
Z H Z
), δ 6.40 (d, J5,3=2.4 H ) suggestive of a
13