Synthesis and anti-herpes simplex viral activity of monoglycosyl diglycerides

Article abstract of DOI:10.1016/j.phytochem.2003.09.008

Based on the discovery of antiviral β-galactosyl diglycerides from Clinacanthus nutans leaves, 19 monoglycosyl diglycerides were synthesized and examined for inhibitory effect on herpes simplex virus types 1 and 2 (HSV-1, HSV-2). A study of the structure-

Full text of DOI:10.1016/j.phytochem.2003.09.008

Phytochemistry 64 (2003) 1253–1264
www.elsevier.com/locate/phytochem
Synthesis and anti-herpes simplex viral activity of
monoglycosyl diglycerides
Wicharn Janwitayanuchita, Khanit Suwanboriruxa,*, Chamnan Patarapanicha,
Sunibhond Pummanguraa, Vimolmas Lipipuna, Tirayut Vilaivanb
aFaculty of Pharmaceutical Sciences, Chulalongkorn University Bangkok, 10330, Thailand
bFaculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand
Received 5 June 2003; received in revised form 20 August 2003
Abstract
Based on the discovery of antiviral b-galactosyl diglycerides from Clinacanthus nutans leaves, 19 monoglycosyl diglycerides were
synthesized and examined for inhibitory effect on herpes simplex virus types 1 and 2 (HSV-1, HSV-2). A study of the structure–
activity relationships of the synthetic monoglycosyl diglycerides indicated that the fatty acyl moieties were critical for inhibitory
action with higher activity displayed as the acyl groups became more olefinic in character. The sugar moiety was also important for
anti-HSVaction; however, the type of sugar (glucose or galactose) did not affect activity. The stereochemistry at C-2 of the glycerol
backbone displayed no significant effect on anti-HSVactivity. Among the compounds synthesized, 1,2- O-dilinolenoyl-3-O-b-d-
glucopyranosyl-sn-glycerol showed the highest inhibitory activity against HSV-1 and HSV-2 with IC50 values of 12.5ꢀ0.5 and
18.5ꢀ1.5 mg/ml, respectively.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Clinacanthus nutans; Acanthaceae; Synthesis; Anti-herpes simplex activity; Monoglycosyl diglycerides; 1,2-O-diacyl-3-O-b-d-glycopyr-
anosyl-glycerols
1. Introduction
treatment of herpes simplex, herpes zoster, skin pruritis
and insect bites. Previous pharmacological and clinical
studies of extracts from this plant showed both anti-
HSVactivity and anti-inflammatory action ( Kittisir-
ipornkul, 1984; Jayawasu et al., 1992). These findings
prompted us to investigate structure–activity relation-
ships of this class of compounds against HSV. Using
monoglycosyl diglycerides as a starting point. In order
to examine these analogs for anti-HSVactivity, a series
of mono-acid and mixed-acid 1,2-O-diacyl-3-O-b-glyco-
pyranosyl-rac-glycerols containing either a glucose or a
galactose moiety were synthesized, as well as studying the
effects of the stereochemistry at C-2 of the glycerol back-
bone of some selected compounds on anti-HSVactivity.
Galactosyl diglycerides are major constituents of the
stacked thylakoid membrane of chloroplasts and also
widely distributed in some bacterial membranes (Douce
and Joyard, 1980; Yagi and Maruyama, 1999).
Although various galactosyl diglycerides have been iso-
lated and characterized, the biological functions of these
compounds have not been fully elucidated (Kopitz,
1997). Studies on glycoglycerolipids reported them as
having antitumor-promoting (Shirahashi et al., 1996;
Colombo et al., 1998, 1999), oxygen scavenging
(Nakata, 2000), and virus neutralizing (Nakata et al.,
2000) activities. Recently, we reported that trigalactosyl
and digalactosyl diglycerides isolated from Clinacanthus
nutans leaves exhibited anti-herpes simplex virus (anti-
HSV) activity (Satakhun, 2001), with the latter plant
being a well-known medicinal plant in Thailand pro-
moted in primary and public health care units for the
2. Results and discussion
2.1. Synthesis
Glucosyl diglycerides and galactosyl diglycerides
bearing either saturated or unsaturated fatty acids were
* Corresponding author. Tel.: +662-2188363; fax: +662-2545195.
E-mail address: skhanit@chula.ac.th (K. Suwanborirux).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2003.09.008

1254
W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
synthesized as illustrated in Scheme 1. For the prepara-
tion of glucosyl diglycerides (7a–m), condensation of
commercially available acetobromo-a-d-glucose (1)
with d,l-a,b-isopropylideneglycerol (2) in the presence
of Ag2CO3 using Koenigs–Knorr conditions afforded
the peracetylated glucoside (3). The 1H-NMR signals of
anomeric protons at ꢀ 4.61, 4.63 (two overlapped
doublets, J=7.6 Hz) in 3 indicated a 1:1 ratio of dia-
stereomeric products, with the stereochemistry of the
new glycosidic bond being in the b-orientation. Then 3
was allowed to react with 60% acetic acid in aqueous
d-20,30,40,60-tetra-O-acetyl-glucopyranosyl)-rac-glycerols
(6a–g) were prepared by one step diacylation of 4 with 2
equivalents of the desired fatty acids in the presence of
dicyclohexylcarbodiimide (DCC) and 4-dimethylamino-
pyridine (DMAP) at room temperature, whereas the
desired mixed-acid 1,2-O-diacyl-3-O-(b-d-20,30,40,60-
tetra-O-acetyl-glucopyranosyl)-rac-glycerols
could be furnished by stepwise acylation of 4 with the
(6h–m)
ꢁ
required fatty acid at 0 C to provide 1-O-acyl-3-O-(b-
d-20,30,40,60-tetra-O-acetyl-glucopyranosyl)-rac-glycerols
(5a–d). The H-NMR spectra of 5a–d showed H2-1 and
1
ꢁ
solution at 60 C to remove the isopropylidene protect-
ing group, the diol intermediate (4) was obtained in
quantitative yield. The mono-acid 1,2-O-diacyl-3-d-(b-
H-2 signals in the range of 4.05–4.30 and 3.85–4.00
ppm, respectively, indicating that the fatty acyl groups
have attached to C-1 hydroxyl of the glycerol backbone.
Scheme 1. Reagent and conditions: (i) Ag2CO3, Drierite, CH2Cl2, rt; (ii) 60% CH3COOH, 60 ꢁC; (iii) fatty acid, DCC, DMAP, CH2Cl2, 0 ꢁC;
(iv) fatty acid, DCC, DMAP, CH2Cl2, rt; (v) NH2NH2, 85% EtOH, 40–60 ꢁC.

W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
1255
Subsequent acylation of the products obtained with the
second fatty acid at room temperature provided com-
pounds 6h–m. The difficult step in this synthesis was the
selective removal of the acetate moieties of 6a–m with-
out affecting the fatty acyl ester groups on the glycerol
backbone. Successful deacetylation of 6a–m was carried
out by hydrazine hydrate (3 moles per acetate group) in
85% aq. ethanol to give the corresponding 1,2-O-diacyl-
3-O-b-d-glucopyranosyl-rac-glycerols (7a–m).
dibenzyl-3-O-(b-d-20,30,40,60 -tetra-O-acetyl-glucopyr-
anosyl)-sn-glycerol (15), which was then debenzylated
by catalytic hydrogenolysis with 10% Pd/C in ethyl
acetate. The 3-O-(b-d-20,30,40,60-tetra-O-acetyl-gluco-
pyranosyl)-sn-glycerol (16) obtained was subsequently
acylated with the desired fatty acid to give 1,2-O-diacyl-
3-O-(b-d-20,30,40,60 -tetra-O-acetyl-glucopyranosyl)-sn-
glycerols (17a–b). Finally, 17a–b were reacted with
hydrazine to give the corresponding 1,2-O-diacyl-3-O-b-
d-glucopyranosyl-sn-glycerols (18a–b). The 1H- and
13C-NMR spectral data of 18a–b are listed in Table 3.
Galactosyl diglycerides (13a–d) were prepared from
acetobromo-a-d-galactose (8) following the procedure
for the synthesis of 1,2-O-diacyl-3-O-b-d-glucopyr-
1
anosyl-rac-glycerols as described above. The H-NMR
2.2. Biological evaluation
spectral data of these compounds (Table 1) showed sig-
nals attributable to glycosyl diglycerides. The proton
signals could be assigned by the correlations in the 2D
H–H COSY spectra. Interestingly, the anomeric pro-
tons of all peracetylated products were observed as 1:1
ratio of two overlapped doublets (J=7.5–7.8 Hz) indi-
cating 1:1 mixtures of the diastereomers, while the same
protons of the corresponding deacetylated final pro-
ducts appeared as a doublet. However, the 13C-NMR
spectra of these final products (Table 2), showing a 1:1
ratio of anomeric carbon signals separated by about
0.1–0.2 ppm, confirmed the 1:1 mixtures of the final
diastereomeric glycosyl diglycerides. In addition, the
observed carbons are less than the actual carbons due to
the overlapped methylene carbon signals of the fatty
acyl chains in the region of 29–30 ppm.
To study the effect of fatty acids on anti-HSVactivity,
a series of 1,2-O-diacyl-3-O-b-d-glycopyranosyl-rac-
glycerols bearing various fatty acyl moieties were deter-
mined. The data (Table 4) showed that glucosyl digly-
cerides bearing the same saturated fatty acyls (7a–d) and
different fatty acyls (7i–k) at C-1 and C-2 displayed little
or no inhibitory activity at 50 mg/ml, except for 7b
which contains a dilauroyl (C12:0) moiety. On the other
hand, compounds bearing the same unsaturated fatty
acyls (7e–g) and different fatty acyls (7h, 7l–m) exhibited
higher inhibitory activity than those bearing saturated
fatty acyls. By comparing anti-HSVactivity of four
compounds containing 18 carbon atom fatty acyls (7e–
g, 7m), the data showed that inhibitory activity of these
compounds followed the order 7g>7m>7f>7e. These
results suggested that more olefinic derivatives displayed
higher anti-HSVactivity.
For the synthesis of 1,2-O-diacyl-3-O-b-d-glucopyr-
anosyl-sn-glycerols (18a–b) (Scheme 2), 1 was allowed to
react with 1,2-O-dibenzyl-sn-glycerol (14) (prepared
from d-mannitol according to the method of Mannock
et al., 1987) in the presence of Ag2CO3 to give 1,2-O-
To study the effect of the sugar moiety on anti-HSV
activity, the selected galactosyl diglycerides bearing the
same fatty acyls (13a–b) and the different fatty acyls
Scheme 2. Reagent and conditions: (i) Ag2CO3, Drierite, CH2Cl2, rt; (ii) H2, 10% Pd/C, EtOAc; (iii) fatty acid, DCC, DMAP, CH2Cl2, rt;
(iv) NH2NH2, 85% EtOH, 40–60 ꢁC.

Table 1
1H-NMR spectral data for compounds 7a–m and 13a–d (in CDCl3)
H
7a
7b
7c
7d
7e
7f
7g
7h
7i
Glycerol moiety
1-Ha
4.08, dd, 4.13, dd, 4.08, dd, 4.12, dd, 4.06, dd, 4.12, dd, 4.07, dd, 4.14, dd, 4.07, dd, 4.14, dd, 4.06, dd, 4.12, dd, 4.07, dd, 4.13, dd, 4.07, dd, 4.14, dd, 4.09, dd, 4.15, dd,
12.0, 6.7
12.0, 6.7
12.0, 6.7
12.0, 6.7
12.0, 6.7
12.0, 6.7
12.0, 6.7
12.0, 6.7
12.0, 6.6
1-Hb
2
3-Ha
4.35, br d, 12.1
5.24, br
3.62, dd, 3.65, dd, 3.62, dd, 3.65, dd, 3.62, dd, 3.65, dd, 3.66, m
4.38, br d, 10.4
5.22, br
4.35, br d, 11.0
5.24, br
4.35, br d, 11.0
5.24, br
4.34, br d, 11.8
5.22, br
4.35, br d, 10.4
5.23, br
4.35, br d, 10.5
5.23, br
4.35, br d, 10.5
5.23, br
4.35, br d, 11.0
5.24, br
3.62, dd, 3.65, dd, 3.62, dd, 3.65, dd, 3.62, dd, 3.65, dd, 3.64, dd, 3.67, dd, 3.62, dd, 3.65, dd, 10.7, 6.4
10.7, 6.4
3.87, br d, 10.7
10.7, 6.4
3.85, br d, 10.5
10.7, 6.4
3.85, br d, 10.5
10.7, 6.4
3.86, br d, 10.6
10.7, 6.4
3.86, br d, 10.6
10.7, 6.4
3.85, br d, 10.5
10.7, 6.4
3.87, br d, 10.6
3-Hb
Sugar moiety
3.88, m
3.89, br d, 10.6
10
4.28, d, 7.8
3.35, m
3.57, m
3.53, m
3.28, m
3.80, br s
4.25, d, 7.7
3.36, m
3.58, m
3.55, m
3.29, m
3.79, br s
4.28, d, 7.8
3.39, m
3.60, m
3.57, m
3.32, m
3.79, br s
4.28, d, 7.8
3.31, m
3.48, m
3.44, m
3.24, m
3.80, br s
4.29, d, 7.5
3.33, m
3.54, m
3.50, m
3.26, m
3.79, br s
4.27, d, 7.6
3.33, m
3.56, m
3.52, m
3.26, m
3.79, br s
4.27, d, 7.5
3.32, m
3.56, m
3.52, m
3.25, m
3.80, br s
4.28, d, 7.8
3.38, m
3.57, m
3.55, m
3.30, m
3.79, br s
4.28, d, 7.8
3.38, m
3.55, m
3.53, m
3.30, m
3.79, br s
20
30
40
50
60-Methylene
Fatty acyl moiety
a-Methylene
b-Methylene
Bulk methylene
Allylic methylene
Methylenea
Olefinic
2.29, br t, 7.0
1.57, br
1.24, br
–
2.26, br t, 7.0
1.55, br
1.22, br
–
2.29, br t, 6.9
1.57, br
1.23, br
–
2.20, br t, 6.9
1.49, br
1.16, br
–
2.25, br t, 7.0
1.57, br
1.25, br
1.99, br
–
2.24, br t, 7.0
1.54, br
1.24, br
2.25, br t, 6.9
1.54, br
1.24, br
2.29, br t, 6.9
1.57, br
1.23, br
1.98, br
–
2.29, br t, 6.9
1.57, br
1.23, br
–
1.99, br
2.10, br
–
–
–
–
2.78, br t, 5.5
5.24–5.40, br
0.85, br t, 6.6
2.76, br t, 5.3
5.21–5.42, br
0.93, t, 7.5
–
–
0.85, br t, 6.7
–
0.81, br t, 6.7
–
0.84, br t, 6.7
–
0.76, br t, 6.5
5.27–5.36, br
0.85, br t, 6.7
5.28–5.32, br
0.85, br t, 6.5
–
0.85, br t, 6.8
Methyl
H
7j
7k
7l
7m
13a
13b
13c
13d
Glycerol moiety
1-Ha
4.07, dd, 4.13, dd, 4.08, dd, 4.15, dd, 4.07, dd, 4.14, dd, 4.07, dd, 4.13, dd, 4.11, dd, 4.15, dd, 4.08, dd, 4.12, dd, 4.09, dd, 4.14, dd, 4.08, dd, 4.13, dd,
12.0, 6.7
4.35, br d,
11.0
12.0, 6.7
4.35, br d, 12.1
12.0, 6.6
4.34, br d,
12.0
12.0, 6.6
4.35, br d,
12.0
12.0, 3.3
4.39, br d,
11.4
12.0, 3.3
4.35, br d,
10.4
12.0, 3.3
4.35, br d, 12.1
12.0, 4.4
4.38, br d, 10.4
1-Hb
2
3-Ha
5.27, br
5.23, br
5.23, br
5.23, br
5.23, br
5.21, br
3.62, dd, 3.65, dd, 3.62, dd, 3.65, dd, 3.62, dd, 3.65, dd, 3.62, dd, 3.65, dd, 3.63, dd, 3.70, dd, 3.63, dd, 3.70, dd, 3.68, dd, 3.71, dd, 3.65, dd, 3.70, dd,
5.25, br
5.28, br
10.7, 6.4
3.87, br d, 10.6
10.7, 6.4
3.88, br d, 10.6
10.7, 6.4
3.89, br d, 10.6
10.7, 6.4
3.87, br d, 10.6
10.9, 5.4
3.86, dd, 3.92, dd, 3.84, dd, 3.89, dd, 3.86, dd, 3.91, dd, 3.87, dd, 3.91, dd,
10.9, 5.4
11.0, 5.6
10.9, 5.3
3-Hb
10.9, 4.9
10.9, 4.9
10.9, 4.9
10.9, 4.9
Sugar moiety
10
4.29, d, 7.8
3.39, m
3.59, m
3.56, m
3.31, m
3.79, br s
4.28, d, 7.8
3.39, m
3.59, m
3.56, m
3.31, m
3.79, br s
4.28, d, 7.8
3.39, m
3.58, m
3.55, m
3.31, m
3.80, br s
4.27, d, 7.5
3.32, m
3.56, m
3.52, m
3.25, m
3.80, br s
4.22, d, 7.8
3.60, m
3.58, m
3.96, br s
3.52, m
3.80, br s
4.21, d, 7.5
3.61, m
3.58, m
3.96, br s
3.52, m
3.80, br s
4.21, d, 7.8
3.60, m
3.57, m
3.97, br s
3.53, m
3.81, br s
4.21, d, 7.5
3.61, m
3.58, m
3.98, br s
3.52, m
3.81, br s
20
30
40
50
60-Methylene
Fatty acyl moiety
a-Methylene
b-Methylene
Bulk methylene
Allylic methylene
Methylenea
Olefinic
2.29, br t, 7.0
1.57, br
1.23, br
–
2.29, br t, 7.0
1.57, br
1.23, br
–
2.29, br t, 6.9
1.57, br
1.23, br
1.98, br
–
2.25, br t, 7.0
1.54, br
1.24, br
2.24, br t, 7.0
1.52, br
1.24, br
–
2.28, br t, 7.0
1.58, br
1.30, br
2.29, br t, 7.0
1.57, br
1.23, br
–
2.31, br t, 7.0
1.60, br
1.28, br
2.10, br
2.02, br
2.02, br
–
–
2.76, br t, 5.5
5.21–5.42, br
0.85, br t, 6.7,
0.93, t, 7.5
–
2.76, br t, 5.5
5.21–5.30, br
0.85, br t, 6.7
–
2.78, br t, 5.5
5.22–5.40, br
0.85, br t, 7.0
0.95, t, 7.5
–
0.84, br t, 6.8
–
0.85, br t, 6.9
5.28–5.32, br
0.85, br t, 6.8
–
0.85, br t, 6.7
–
0.85, br t, 6.9
Methyl
a
Methylene lying between double bonds.

Table 2
13C-NMR spectral data for compounds 7a–m and 13a–d (in CDCl3)
C
7a
7b
7c
7d
7e
7f
7g
7h
Glycerol moiety
1
2
63.2, 63.3
70.5
68.6
63.2, 63.3
70.4
68.4
63.1, 63.2
70.4, 70.5
68.5
63.1, 63.2
70.4, 70.5
68.5
63.2, 63.3
70.4, 70.5
68.5
63.0, 63.1
70.4, 70.5
68.6
63.2, 63.3
70.5, 70.6
68.5
63.1, 63.2
70.5
68.6
3
Sugar moiety
10
103.7, 103.8
73.7, 73.8
69.8
76.7
76.2, 76.3
61.9
103.7, 103.8
73.6, 73.7
69.6
76.6
76.2
103.7, 103.9
73.7, 73.8
69.8
76.6
76.2
103.7, 103.9
73.7, 73.8
69.8
76.6
76.2
103.7, 103.8
73.6, 73.7
69.7
76.6, 76.7
76.2, 76.3
61.7
103.7, 103.8
73.7, 73.8
69.7
76.6
76.1
103.7, 103.8
73.7
69.7
76.9
76.2, 76.3
61.8
103.7, 103.9
73.8
69.8
76.6
76.2
20
30
40
50
60
Fatty acyl moiety
Carbonyl
61.7
61.9
61.9
62.1
61.8
173.6, 173.7,
173.9, 174.0
34.6, 34.8
25.4
23.2–32.4
–
–
–
173.7, 173.8,
174.1, 174.2
34.5, 34.6
25.2, 25.3
23.0–32.3
–
–
–
173.8, 173.9,
174.1, 174.2
34.6, 34.7
25.3
23.0–32.3
–
–
–
173.8, 173.9,
174.1, 174.2
34.6, 34.7
25.3
23.0–32.3
–
–
–
173.7, 173.8,
174.1, 174.2
34.6
25.2, 25.3
23.0–32.3
27.5, 27.7
–
173.8, 173.9,
174.1
34.5, 34.6
25.1, 25.2
22.9–32.3
27.5
26.0
128.3, 128.5,
130.4, 130.5
173.5, 173.6,
173.9, 174.0
34.6, 34.7
25.4
21.1–30.2
27.5, 27.6, 27.7
26.0, 26.1
127.4, 128.0,
128.5, 130.1,
130.4, 132.2
14.8
173.8, 173.9,
174.1, 174.2
34.5, 34.7
25.3
23.1–32.3
27.5, 27.6
–
a-Methylene
b-Methylene
Bulk methylenea
Allylic methylene
Methyleneb
Olefinic
130.1, 130.4
130.1, 130.4
Methyl
14.6
14.5
14.5
14.5
14.5
14.4
14.5
7i
7j
7k
7l
7m
13a
13b
13c
13d
Glycerol moiety
1
2
63.1, 63.2
70.5, 70.6
68.6
62.9, 63.0
70.5, 70.6
68.4, 68.5
63.2, 63.3
70.4, 70.5
68.5
62.9, 63.0
70.4, 70.5
68.7
63.2, 63.3
70.5, 70.6
68.5
63.4
70.6
68.5
63.1, 63.2
70.6
68.5, 68.6
63.2, 63.3
70.5, 70.6
68.5
63.2, 63.3
70.6
68.5, 68.6
3
Sugar moiety
10
103.7, 103.8
73.7
70.0
76.5
76.2, 76.3
62.0
103.8, 103.9
73.9, 74.0
70.0
76.9
76.3, 76.4
62.5
103.7, 103.8
73.5, 73.6
69.6
76.6
76.2
103.7, 103.9
73.7, 73.8
70.0
76.8
76.4
103.7, 103.8
73.7
69.7
76.9
76.5, 76.6
61.8
104.2, 104.3
71.5
73.6
69.2
74.9, 75.0
61.8
104.2, 104.4
71.5, 71.6
73.6
69.4
74.8, 74.9
62.3, 62.4
104.2, 104.4
71.4, 71.5
73.6
69.2
74.8, 74.9
61.9, 62.0
104.1, 104.3
71.6
73.7
69.4
74.8, 74.9
62.3
20
30
40
50
60
Fatty acyl moiety
Carbonyl
61.6
62.1
173.6, 173.7,
173.9
34.7, 34.8
25.4, 25.5
23.2–32.4
–
–
–
173.5,
173.6, 173.7
34.6, 34.8
25.4
23.2–32.4
–
–
–
173.8, 173.9,
174.2, 174.3
34.5, 34.7
25.3
23.1–32.3
–
–
–
173.9, 174.0,
174.2
34.5, 34.7
25.3
23.1–32.3
27.5, 27.6
–
173.5, 173.6,
173.9, 174.0
34.6, 34.7
25.4
21.1–30.2
27.5, 27.6, 27.7
26.0, 26.1, 26.2
127.4, 128.0,
128.5, 130.1,
130.4, 132.2
14.8
173.7, 173.8,
174.1
34.7, 34.8
25.5
23.2–32.4
–
–
–
173.8,
173.9, 174.0,
174.2, 174.3
34.6, 34.7
25.3
23.0–32.3
–
–
–
173.7, 173.8,
174.1, 174.2
34.6, 34.8
25.4
21.1–32.0
27.7
26.0, 26.2
127.4, 128.0,
128.2, 128.4,
130.3, 132.2
14.6, 14.7
173.9, 174.1
34.5, 34.6
25.3
22.2–31.2
27.3, 27.5
26.0
a-Methylene
b-Methylene
Bulk methylene
Allylic methylene
Methyleneb
Olefinic
130.1, 130.4
128.3, 128.5,
130.4, 130.6
Methyl
14.6
14.6
14.5
14.5
14.7
14.7
14.5
a
Overlapped signals.
Methylene lying between double bonds.
b

1258
W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
Table 3
1H and 13C-NMR spectral data for compounds 18a and 18b
Position
18a
18b
ꢀ13
C
ꢀ1H
ꢀ13
C
ꢀ1H
Glycerol moiety
1-Ha
1-Hb
2
63.2
4.15, dd, 11.9, 6.6
4.38, br d, 10.4
5.22, br
63.3
4.15, dd, 11.9, 6.6
4.38, br d, 10.4
5.22, br
70.5
68.5
70.6
68.5
3-Ha
3-Hb
3.66, dd, 10.8, 6.6
3.87, dd, 11.0, 5.2
3.66, dd, 10.8, 6.6
3.85, dd, 11.0, 5.2
Sugar moiety
10
103.9
73.8
69.8
76.6
76.2
61.9
4.25, d, 7.7
3.35, m
3.57, m
103.8
73.8
69.8
76.6
76.2
61.9
4.28, d, 7.7
3.36, m
3.58, m
20
30
40
3.53, m
3.28, m
3.72, br s
3.54, m
3.29, m
3.72, br s
50
60-Methylene
Fatty acyl moiety
Carbonyl
173.8, 174.2
34.5, 34.6
173.5, 174.0
34.6, 34.7
a-Methylene
b-Methylene
Bulk methylenea
Allylic methylene
Methyleneb
Olefinic
2.26, br t, 7.0
1.55, br
2.25, br t, 7.0
1.55, br
1.25, br
25.2, 25.3
29.5–32.3
25.4
29.6–32.0
1.22, br
–
–
27.5, 27.6, 27.7
26.0, 26.2
1.95–2.12, br
2.75, br t, 5.5
5.18–5.45, br
0.93, t, 7.5
–
–
–
14.5
–
0.81, br t, 6.7
127.4, 128.1, 128.5, 128.6, 130.4, 132.2
14.8
Methyl
a
Overlapped signals.
Methylene lying between double bonds.
b
(13c–d) based on the above-mentioned evidence were
synthesized and evaluated. Similar results were observed
in the series of galactosyl diglycerides. However, the
O-acetylated forms of glycosyl diglycerides displayed
no activity, indicating a requirement for free hydroxyl
groups in these glycosyl diglycerides for anti-HSV
action. In addition, neither glycoside derivatives lacking
fatty acyl moieties nor 1,2-diacylglycerol exhibited anti-
HSVactivity (data not shown). To evaluate the effect of
the stereochemistry at C-2 of the glycerol backbone, we
also synthesized and determined the anti-HSVactivity of
1,2-O-diacyl-3-O-b-d-glucopyranosyl-sn-glycerols (18a–b).
The IC50 and toxicity of 18a and 18b (Table 5) showed
that 18b displayed about two times the potency of 18a
and both compounds were not toxic to the Vero cell
lines at concentrations up to 100 mg/ml. The anti-HSV
activity of diastereomeric mixtures 7b compared with its
diastereomeric pure isomer 18a was not significantly
different. A similar result was observed in the case of
7g and its diastereomerically pure isomer 18b. This
observation suggests that the stereochemistry at C-2 of
the glycerol backbone of these compounds does not
influence anti-HSVactivity.
(Thormar et al., 1987). We might postulate that the
mechanism of action of these monoglycosyl diglycerides
conforms to that of monoglycerides to some extent.
In conclusion, the synthesis of 1,2-O-diacyl-3-O-b-d-
glycopyranosyl glycerols have been obtained. We have
examined the inhibitory action of all compounds against
HSV-1 and HSV-2, and some structure-activity relation-
ships were explored. The results showed that compounds
containing fatty acyl moieties bearing more olefinic char-
acter displayed higher activity, except for 7b which despite
having no olefinic character still showed activity. The
sugar moiety, whether it be glucose or galactose was
essential for activity and the stereochemistry at C-2 of the
glycerol backbone did not affect anti-HSVactivity.
Compound 18b displayed moderate anti-HSV-1 and
HSV-2 action with the IC50 12.5ꢀ0.5 and 18.5ꢀ1.5 mg/
ml, respectively. These results reveal the potential of these
compounds as a new type of anti-HSVagents, although
the mechanism of action must be further investigated.
3. Experimental
Even though the anti-HSVmechanism of these com-
pounds is still unknown, there was evidence that some
monoglycerides exhibited antiviral activities against
enveloped viruses by destruction of viral envelopes
3.1. General
1H-NMR and 13C-NMR spectra were obtained on a
Bruker AVANCE DPX-300 FT-NMR spectrometer in

W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
1259
Table 4
Anti-HSVactivity of the synthetic monoglycosyl diglycerides
dry dichloromethane (10 ml) was then added drop-
wise over a period of 1 h. The reaction mixture was
stirred overnight at room temperature and then fil-
tered. The residue was washed with dry dichloro-
methane, and the filtrate was evaporated in vacuo to
give a colorless syrupy mixture. The mixture was
purified by CC on silica gel, eluted with EtOAc–hex-
ane (1:8) to give the 1:1 mixtures of the two diaster-
eomers of 3 (or 9).
Compda
R1
R2
Inhibition (%)b
HSV-1
HSV-2
7a
7b
Decanoyl (C10:0)
Lauroyl (C12:0)
Stearoyl (C18:0)
Behenoyl (C22:0)
Oleoyl (C18:1)
Linoleoyl (C18:2)
Linolenoyl (C18:3)
Lauroyl
Decanoyl
Lauroyl
Stearoyl
Behenoyl
Oleoyl
n.a.c
n.a.
80ꢀ6
n.a.
n.a.
50ꢀ5
n.a.
n.a.
7c
7d
7e
n.a.
25ꢀ3
50ꢀ4
97ꢀ4
17ꢀ1
n.a.
7f
7g
Linoleoyl
Linolenoyl
Oleoyl
50ꢀ2
94ꢀ4
13ꢀ2
n.a.
3.2.1.1. 1,2-Isopropylidene-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-rac-glycerol (3). (470 mg,
50.8%); 1H-NMR (300 MHz, CDCl3): ꢀ 1.34, 1.42 (each
2ꢂ3H, s), 2.01, 2.03, 2.05, 2.11(each 2ꢂ3H, s), 3.60–4.33
(2ꢂ8H), 4.61 and 4.63 (each 1H, d, J=7.6 Hz), 5.01 and
5.03 (each 1H, dd (apparent t), J=9.8 and 7.5 Hz), 5.09
(2ꢂ1H, dd (apparent t), J=9.8 and 9.5 Hz), 5.22 and
5.23 (each 1H, dd (apparent t), J=9.4 and 9.4 Hz).
7h
7i
Stearoyl
Stearoyl
Lauroyl
Behenoyl
Lauroyl
Oleoyl
7j
n.a.
n.a.
n.a.
7k
7l
Behenoyl
Behenoyl
20ꢀ1
10ꢀ2
70ꢀ2
50ꢀ2
50ꢀ2
25ꢀ1
90ꢀ2
50ꢀ4
100ꢀ0
n.a.
7m
13a
13b
13c
13d
18a
18b
Linolenoyl
Lauroyl
Linoleoyl
Lauroyl
Linoleoyl
Lauroyl
Linoleoyl
Lauroyl
Linolenoyl
90ꢀ5
80ꢀ4
55ꢀ2
n.a.
Linoleoyl
Behenoyl
Linolenoyl
Lauroyl
Linolenoyl
100ꢀ0
80ꢀ8
100ꢀ0
3.2.1.2. 1,2-Isopropylidene-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-galactopyranosyl)-rac-glycerol (9). (458 mg,
49.7%); 1H-NMR (300 MHz, CDCl3): ꢀ 1.35, 1.43 (each
2ꢂ3H, s), 1.98, 2.01, 2.02, 2.13 (each 2ꢂ3H, s), 3.60–
4.20 (2ꢂ8H), 4.49 and 4.51 (each 1H, d, J=7.6 Hz),
5.01 (2ꢂ1H, br d, J=10.4 Hz), 5.18 (2ꢂ1H, br t, J=8.3
Hz), 5.38 (2ꢂ1H, br s).
a
Performed at concentration 50 mg/ml.
b
c
Values are expressed as meansꢀS.D. of three replicates.
n.a.=No activity.
3.2.2. General procedure for deacetonization
A solution of 3 (or 9) (400 mg) in 60% aq. acetic acid
(20 ml) was stirred at 60 C for 1.5 h. The mixture was
concentrated and co-evaporated with toluene three
times. The residue was then purified by silica gel CC,
eluted with EtOAc–hexane (5:1) to give a semisolid
substance 4 (or 10).
Table 5
IC50 for anti-HSVactivity and toxicity of compounds 18a and 18b
ꢁ
Compd
IC50 (mg/ml)a
Toxicity
(mg/ml)b
HSV-1
HSV-2
18a
18b
23.0ꢀ2.2
12.5ꢀ0.5
40.0ꢀ4.0
18.5ꢀ1.5
>100
>100
a
3.2.2.1. 3-O-(b-d-20,30,40,60-Tetra-O-acetyl-glucopyr-
anosyl)-rac-glycerol (4). (363 mg, 100%); 1H-NMR
(300 MHz, CDCl3): ꢀ 2.01, 2.04, 2.06, 2.10 (each 2ꢂ3H,
s), 3.52–3.95 (2ꢂ6H), 4.22 (2ꢂ2H, br s), 4.56 (2ꢂ1H, d,
J=7.8 Hz), 5.02 (2ꢂ1H, dd (apparent t), J=9.4 and 7.5
Hz), 5.09 (2ꢂ1H, dd (apparent t), J=9.4 and 9.5 Hz),
5.23 (2ꢂ1H, dd (apparent t), J=9.4 and 9.3 Hz).
Values are expressed as meansꢀS.D. of three replicates.
b
Concentration of the compound required to reduce the number of
viable Vero cells by 50%.
CDCl3 with tetramethylsilane as the internal standard.
FAB-MS were determined on a Jeol JMS-700 spectro-
meter. Reactions were monitored by TLC on silica gel
plates; spots were visualized by spraying with 2%
K2CrO4 in 30% aq. H2SO4 followed by heating.
3.2.2.2. 3-O-(b-d-20,30,40,60-Tetra-O-acetyl-galactopyr-
anosyl)-rac-glycerol (10). (360 mg, 99%); 1H-NMR
(300 MHz, CDCl3): ꢀ 1.98, 2.01, 2.02, 2.13 (each 2ꢂ3H,
s), 3.53–3.96 (2ꢂ6H), 4.14 (2ꢂ2H, br s), 4.48 (2ꢂ1H, d,
J=7.8 Hz), 5.01 (2ꢂ1H, dd, J=10.4 and 3.3 Hz), 5.18
(2ꢂ1H, dd, J=9.9 and 8.4 Hz), 5.38 (2ꢂ1H, br d, J=3.0
Hz).
3.2. Synthesis of 1,2-O-diacyl-3-O-ꢁ-d-glycopyranosyl-
rac-glycerols
3.2.1. General procedure for glycosylation
A mixture of d,l-a,b-isopropylideneglycerol (2) (2.0
mmol), Ag2CO3 (2.0 mmol) and fine Drierite (2.5 g) in
dichloromethane (40 ml) was stirred in the dark
under N2. After being stirred for half an hour, iodine
(0.2 mmol) was added and acetobromo-a-d-glucose
(1) [or acetobromo-a-d-galactose (8)] (2.0 mmol) in
3.2.3. General procedure for preparation of mono-acid
1,2-O-diacyl-3-O-(ꢁ-d-20,30,40,60-tetra-O-acetyl-glyco-
pyranosyl)-rac-glycerols
DCC (0.4 mmol) was added to a solution of 4 (or 10)
(0.3 mmol), fatty acid (0.7 mmol), and DMAP (0.03

1260
W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
mmol) in dry dichloromethane (8 ml), and stirred for 24
h at room temp. The solid was removed by filtration
and the solvent evaporated in vacuo. The residue was
purified by CC on silica gel, eluted with EtOAc–hexane
(1:3) to give the products 6a–g (or 12a–b). All com-
pounds were obtained as the 1:1 mixtures of two dia-
stereomers.
overlapped), 5.13 and 5.14 (each 1H, dd (apparent t),
J=9.6 and 7.3 Hz).
3.2.3.5. 1,2-O-Dioleoyl-3-O-(b-d-20,30,40,60-tetra-O-acetyl-
glucopyranosyl)-rac-glycerol (6e). (207.1 mg, 72.6%);
1H-NMR (300 MHz, CDCl3): ꢀ 0.85 (2ꢂ6H, br t, J=6.7
Hz), 1.30 (2ꢂ40H, br s), 1.51 (2ꢂ4H, br), 2.02 (2ꢂ8H,
overlapped), 2.01, 2.03, 2.05, 2.08 (each 2ꢂ3H, s), 2.29
(2ꢂ4H, br t, J=7.0 Hz), 3.60–3.72 (2ꢂ2H, m), 3.92
(2ꢂ1H, dd, J=10.9 and 4.9 Hz), 4.03–4.18 (2ꢂ2H, m),
4.19–4.33 (2ꢂ2H, m), 4.49 and 4.50 (each 1H, d, J=7.8
Hz), 4.96 and 4.97 (each 1H, dd (apparent t), J=9.5 and
7.5 Hz), 5.05 (2ꢂ1H, dd (apparent t), J=9.6 Hz), 5.12
(2ꢂ1H, overlapped), 5.13 and 5.14 (each 1H, dd (apparent
t), J=9.8 and 7.4 Hz), 5.28–5.38 (2ꢂ4H, br).
3.2.3.1. 1,2-O-Didecanoyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-rac-glycerol (6a). (198.2 mg,
90.5%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.80 (2ꢂ6H, br
t, J=6.9 Hz), 1.19 (2ꢂ24H, br s), 1.50 (2ꢂ4H, br), 1.90,
1.92, 1.94, 1.98 (each 2ꢂ3H, s), 2.21 (2ꢂ4H, br t, J=6.9
Hz), 3.61–3.72 (2ꢂ2H, m), 3.89 (2ꢂ1H, dd, J=10.9 and
4.9 Hz), 3.95–4.10 (2ꢂ2H, m), 4.12–4.28 (2ꢂ2H, m),
4.42 and 4.44 (each 1H, d, J=7.8 Hz), 4.88 and 4.89
(each 1H, dd (apparent t), J=9.5 and 7.9 Hz), 4.98
(2ꢂ1H, dd (apparent t), J=9.5 Hz), 5.12 (2ꢂ1H, over-
lapped), 5.11 and 5.13 (each 1H, dd (apparent t), J=9.4
and 7.2 Hz).
3.2.3.6. 1,2-O-Dilinoleoyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-rac-glycerol (6f). (198.3 mg,
69.9%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.87 (2ꢂ6H, br
t, J=6.8 Hz), 1.26 (2ꢂ28H, br s), 1.59 (2ꢂ4H, br), 2.02
(2ꢂ8H, overlapped), 2.02, 2.04, 2.06, 2.12 (each 2ꢂ3H,
s), 2.28 (2ꢂ4H, br t, J=7.0 Hz), 2.78 (2ꢂ4H, br t,
J=5.5 Hz), 3.60–3.74 (2ꢂ2H, m), 3.93 (2ꢂ1H, dd,
J=10.9 and 4.9 Hz), 4.02–4.17 (2ꢂ2H, m), 4.18–4.34
(2ꢂ2H, m), 4.50 and 4.51 (each 1H, d, J=7.8 Hz), 4.96
and 4.97 (each 1H, dd (apparent t), J=9.3 and 8.1 Hz),
5.06 (2ꢂ1H, dd (apparent t), J=9.6 Hz), 5.12 (2ꢂ1H,
overlapped), 5.13 and 5.14 (each 1H, dd (apparent t),
J=9.4 and 7.3 Hz), 5.22–5.38 (2ꢂ8H, br).
3.2.3.2. 1,2-O-Dilauroyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-rac-glycerol (6b). (212.8 mg,
90.2%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.79 (2ꢂ6H, br
t, J=6.9 Hz), 1.18 (2ꢂ32H, br s), 1.51 (2ꢂ4H, br), 1.90,
1.92, 1.94, 2.01 (each 2ꢂ3H, s), 2.20 (2ꢂ4H, br t, J=7.0
Hz), 3.53–3.68 (2ꢂ2H, m), 3.83 (2ꢂ1H, dd, J=10.9, 4.9
Hz), 3.97–4.10 (2ꢂ2H, m), 4.11–4.26 (2ꢂ2H, m), 4.43
and 4.45 (each 1H, d, J=7.8 Hz), 4.89 and 4.90 (each
1H, dd (apparent t), J=9.4 and 7.6 Hz), 4.99 (2ꢂ1H, dd
(apparent t), J=9.6 Hz), 5.09 (2ꢂ1H, overlapped), 5.10
and 5.11 (each 1H, dd (apparent t), J=9.6 and 7.4 Hz).
3.2.3.7. 1,2-O-Dilinolenoyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-rac-glycerol (6g). (158.5 mg,
1
56.1%); H-NMR (300 MHz, CDCl3): ꢀ 0.92 (2ꢂ6H, t,
3.2.3.3. 1,2-O-Distearoyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-rac-glycerol (6c). (223.5 mg,
78.1%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.85 (2ꢂ6H, br
t, J=6.9 Hz), 1.22 (2ꢂ56H, br s), 1.56 (2ꢂ4H, br), 1.91,
1.92, 1.94, 1.98 (each 2ꢂ3H, s), 2.23 (2ꢂ4H, br t, J=6.9
Hz), 3.58–3.72 (2ꢂ2H, m), 3.92 (2ꢂ1H, dd, J=11.0 and
4.9 Hz), 4.03–4.17 (2ꢂ2H, m), 4.19–4.32 (2ꢂ2H, m),
4.50 and 4.51 (each 1H, d, J=7.7 Hz), 4.96 and 4.97
(each 1H, dd (apparent t), J=9.5 and 7.9 Hz), 5.05
(2ꢂ1H, dd (apparent t), J=9.5 Hz), 5.10 (2ꢂ1H, over-
lapped), 5.11 and 5.12 (each 1H, dd (apparent t), J=9.4
and 7.2 Hz).
J=7.5 Hz), 1.29 (2ꢂ16H, br s), 1.56 (2ꢂ4H, br), 2.02
(2ꢂ8H, overlapped), 1.93, 2.01, 2.03, 2.08 (each 2ꢂ3H,
s), 2.25 (2ꢂ4H, br t, J=7.0 Hz), 2.75 (2ꢂ8H, br t,
J=5.5 Hz), 3.58–3.68 (2ꢂ2H, m), 3.90 (2ꢂ1H, dd,
J=10.7 and 4.6 Hz), 4.01–4.15 (2ꢂ2H, m), 4.16–4.30
(2ꢂ2H, m), 4.50 and 4.51 (each 1H, d, J=7.6 Hz), 4.95
and 4.96 (each 1H, dd (apparent t), J=9.3 and 7.8 Hz),
5.05 (2ꢂ1H, dd (apparent t), J=9.6 Hz), 5.12 (2ꢂ1H,
overlapped), 5.13 and 5.14 (each 1H, dd (apparent t),
J=9.4 and 7.3 Hz), 5.20–5.40 (2ꢂ12H, br).
3.2.3.8. 1,2-O-Dilauroyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-galactopyranosyl)-rac-glycerol (12a). (210.2 mg,
89.1%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.85 (2ꢂ6H, br t,
J=6.5 Hz), 1.29 (2ꢂ32H, br s), 1.51 (2ꢂ4H, br), 1.98, 2.01,
2.02, 2.14 (each 2ꢂ3H, s), 2.25 (2ꢂ4H, br t, J=7.0 Hz),
3.65 (2ꢂ1H, dd, J=10.8 and 5.5 Hz), 3.82–3.95 (2ꢂ2H,
m), 4.01–4.15 (2ꢂ3H, m), 4.27 (2ꢂ1H, br d, J=11.0 Hz),
4.44 and 4.46 (each 1H, d, J=7.6 Hz), 4.96 (2ꢂ1H, br d,
J=10.3 Hz), 5.12–5.22 (2ꢂ2H, br), 5.35 (2ꢂ1H, br s).
3.2.3.4. 1,2-O-Dibehenoyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-rac-glycerol (6d). (176.1 mg,
55.1%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.85 (2ꢂ6H, br
t, J=6.9 Hz), 1.25 (2ꢂ72H, br s), 1.51 (2ꢂ4H, br), 2.01,
20.2, 2.04, 2.08 (each 2ꢂ3H, s), 2.29 (2ꢂ4H, br t,
J=6.9 Hz), 3.61–3.72 (2ꢂ2H, m), 3.90 (2ꢂ1H, dd,
J=10.8 and 4.8 Hz), 4.05–4.18 (2ꢂ2H, m), 4.19–4.32
(2ꢂ2H, m), 4.50 and 4.51 (each 1H, d, J=7.8 Hz), 4.96
and 4.97 (each 1H, dd (apparent t), J=9.5 and 7.9 Hz),
5.05 (2ꢂ1H, dd (apparent t), J=9.7 Hz), 5.12 (2ꢂ1H,
3.2.3.9. 1,2-O-Dilinoleoyl-3-O-(b-d-20,30,40,60-tetra-O-
acetyl-galactopyranosyl)-rac-glycerol (12b). (149.1 mg,

W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
1261
52.7%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.85 (2ꢂ6H, br
t, J=6.8 Hz), 1.23 (2ꢂ28H, br s), 1.58 (2ꢂ4H, br), 1.92,
1.99, 2.00, 2.17 (each 2ꢂ3H, s), 2.02 (2ꢂ8H, over-
lapped), 2.23 (2ꢂ4H, br t, J=7.0 Hz), 2.76 (2ꢂ4H, br),
3.61 (2ꢂ1H, dd, J=10.8 and 5.5 Hz), 3.82–3.95 (2ꢂ2H,
m), 4.01–4.15 (2ꢂ3H, m), 4.27 (2ꢂ1H, br d, J=11.0
Hz), 4.42 and 4.44 (each 1H, d, J=7.6 Hz), 4.96 (2ꢂ1H,
br d, J=10.2 Hz), 5.10–5.18 (2ꢂ2H, br), 5.35 (2ꢂ1H,
overlapped), 5.20–5.40 (2ꢂ8H, br).
mg, 64.8%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.87
(2ꢂ6H, br t, J=6.7 Hz), 1.38 (2ꢂ64H, br s), 1.56
(2ꢂ4H, br), 2.01, 2.03, 2.05, 2.08 (each 2ꢂ3H, s), 2.30
(2ꢂ4H, br t, J=7.0 Hz ), 3.61–3.75 (2ꢂ2H, m), 3.92
(2ꢂ1H, dd, J=10.9 and 4.9 Hz), 4.02–4.19 (2ꢂ2H, m),
4.20–4.33 (2ꢂ2H, m), 4.50 and 4.51 (each 1H, d, J=7.8
Hz), 4.96 and 4.97 (each 1H, dd (apparent t), J=9.5 and
7.5 Hz), 5.07 (2ꢂ1H, dd (apparent t), J=9.6 Hz), 5.15
(2ꢂ1H, overlapped), 5.16 and 5.17 (each 1H, dd
(apparent t), J=9.8 and 7.4 Hz).
3.2.4. General procedure for preparation of mixed-acid
1,2-O-diacyl-3-O-(ꢁ-d-20,30,40,60-tetra-O-acetyl-glyco-
pyranosyl)-rac-glycerols
3.2.4.4. 1-O-Behenoyl-2-O-lauroyl-3-O-(b-d-20,30,40,60-
tetra-O-acetyl-glucopyranosyl)-rac-glycerol (6k). (194.2
mg, 69.9%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.88
(2ꢂ6H, br t, J=6.7 Hz), 1.25 (2ꢂ52H, br s), 1.54
(2ꢂ4H, br), 1.98, 2.01, 2.03, 2.07 (each 2ꢂ3H, s), 2.28
(2ꢂ4H, br t, J=7.0 Hz ), 3.60–3.72 (2ꢂ2H, m), 3.92
(2ꢂ1H, dd, J=10.9 and 4.9 Hz), 4.01–4.17 (2ꢂ2H, m),
4.18–4.32 (2ꢂ2H, m), 4.50 and 4.51 (each 1H, d, J=7.8
Hz), 4.96 and 4.97 (each 1H, dd (apparent t), J=9.5 and
7.5 Hz), 5.05 (2ꢂ1H, dd (apparent t), J=9.6 Hz), 5.14
(2ꢂ1H, overlapped), 5.15 and 5.16 (each 1H, dd
(apparent t), J=9.8 and 7.4 Hz).
To a solution of 4 (or 10) (0.3 mmol), the first fatty
acid (0.4 mmol) and DMAP (0.03 mmol) in dry di-
chloromethane (8 ml), DCC (0.4 mmol) was added and
ꢁ
stirred for 6 h at 0 C. The solid was filtered off and the
solvent was evaporated in vacuo. The residue was pur-
ified by CC on silica gel column, eluted with EtOAc–
hexane (1:3) to give the glycosyl monoglycerides (5a–d
or 11a–b) which were subsequently reacted with equi-
molar amounts of the second fatty acid, DCC and a lit-
tle amount of DMAP at room temperature overnight.
The solid was filtered off and the solvent was evapo-
rated in vacuo. The residue was purified by CC on silica
gel, eluted with EtOAc–hexane (1:3) to give the 1:1
mixtures of two diastereomers 6h–m (or 12c–d).
3.2.4.5. 1-O-Behenoyl-2-O-oleoyl-3-O-(b-d-20,30,40,60-
tetra-O-acetyl-glucopyranosyl)-rac-glycerol (6l). (205.8
mg, 68.1%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.87
(2ꢂ6H, br t, J=6.7 Hz), 1.25 (2ꢂ56H, br s), 1.58
(2ꢂ4H, br), 2.02 (2ꢂ4H, overlapped), 2.01, 2.03, 2.05,
2.10 (each 2ꢂ3H, s), 2.25 (2ꢂ4H, br t, J=7.0 Hz ),
3.60–3.73 (2ꢂ2H, m), 3.92 (2ꢂ1H, dd, J=10.9 and 4.9
Hz), 4.04–4.17 (2ꢂ2H, m), 4.19–4.33 (2ꢂ2H, m), 4.50
and 4.51 (each 1H, d, J=7.8 Hz), 4.96 and 4.97 (each
1H, dd (apparent t), J=9.5 and 7.5 Hz), 5.05 (2ꢂ1H, dd
(apparent t), J=9.6 Hz), 5.14 (2ꢂ1H, overlapped), 5.15
and 5.16 (each 1H, dd (apparent t), J=9.8 and 7.4 Hz),
5.27–5.35 (2ꢂ2H, br).
3.2.4.1. 1-O-Lauroyl-2-O-oleoyl-3-O-(b-d-20,30,40,60-
tetra-O-acetyl-glucopyranosyl)-rac-glycerol (6h). (187.5
mg, 72.0%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.84 (2ꢂ6H,
br t, J=6.7 Hz), 1.25 (2ꢂ36H, br s), 1.54 (2ꢂ4H, br), 2.02
(2ꢂ4H, overlapped) 1.95, 2.00, 2.02, 2.05 (each 2ꢂ3H, s),
2.25 (2ꢂ4H, br t, J=7.0 Hz), 3.60–3.70 (2ꢂ2H, m), 3.90
(2ꢂ1H, dd, J=10.9 and 4.9 Hz), 4.01–4.14 (2ꢂ2H, m),
4.15–4.30 (2ꢂ2H, m), 4.49 and 4.50 (each 1H, d, J=7.8
Hz), 4.95 and 4.96 (each 1H, dd (apparent t), J=9.5 and
7.5 Hz), 5.06 (2ꢂ1H, dd (apparent t), J=9.6 Hz), 5.14
(2ꢂ1H, overlapped), 5.15 and 5.16 (each 1H, dd (appar-
ent t), J=9.4 and 7.2 Hz), 5.28–5.32 (2ꢂ2H, br).
3.2.4.6. 1-O-Linolenoyl-2-O-linoleoyl-3-O-(b-d-
20,30,40,60-tetra-O-acetyl-glucopyranosyl)-rac-glycerol
(6m). (169.5 mg, 59.9%); 1H-NMR (300 MHz, CDCl3):
ꢀ 0.85 (2ꢂ3H, br t, J=6.7 Hz), 0.92 (2ꢂ3H, t, J=7.6
Hz), 1.29 (2ꢂ22H, br s), 1.56 (2ꢂ4H, br), 2.02 (2ꢂ8H,
overlapped), 1.93, 2.01, 2.03, 2.08 (each 2ꢂ3H, s), 2.25
(2ꢂ4H, br t, J=6.9 Hz), 2.75 (2ꢂ6H, br t, J=5.5 Hz),
3.58–3.68 (2ꢂ2H, m), 3.90 (2ꢂ1H, dd, J=10.9 and 4.9
Hz), 4.01–4.15 (2ꢂ2H, m), 4.16–4.30 (2ꢂ2H, m), 4.50
(2ꢂ1H, br d, J=7.6 Hz), 4.95 (2ꢂ1H, dd (apparent t),
J=9.3 and 8.1 Hz), 5.04 (2ꢂ1H, dd (apparent t), J=9.6
Hz), 5.12 (2ꢂ1H, overlapped), 5.15 (2ꢂ1H, dd (appar-
ent t), J=9.4 and 7.3 Hz), 5.20–5.38 (2ꢂ10H, br).
3.2.4.2. l-O-Stearoyl-2-O-lauroyl-3-O-(b-d-20,30,40,60-
tetra-O-acetyl-glucopyranosyl)-rac-glycerol (6i). (221.5
mg, 84.9%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.85
(2ꢂ6H, br t, J=6.7 Hz ), 1.27 (2ꢂ44H, br s), 1.51
(2ꢂ4H, br), 1.98, 2.01, 2.03, 2.06 (each 2ꢂ3H, s), 2.29
(2ꢂ4H, br t, J=7.0 Hz), 3.60–3.72 (2ꢂ2H, m), 3.92
(2ꢂ1H, dd, J=10.9 and 4.9 Hz), 4.01–4.18 (2ꢂ2H, m),
4.19–4.33 (2ꢂ2H, m), 4.49 and 4.50 (each 1H, d, J=7.8
Hz), 4.96 and 4.97 (each 1H, dd (apparent t), J=9.5 and
7.5 Hz), 5.05 (2ꢂ1H, dd (apparent t), J=9.6 Hz), 5.13
(2ꢂ1H, overlapped), 5.14 and 5.15 (each 1H, dd
(apparent t), J=9.8 and 7.4 Hz).
3.2.4.7. 1-O-Behenoyl-2-O-lauroyl-3-O-(b-d-20,30,40,60-
tetra-O-acetyl-galactopyranosyl)-rac-glycerol
(12c).
3.2.4.3. 1-O-Stearoyl-2-O-behenoyl-3-O-(b-d-20,30,40,60-
tetra-O-acetyl-glucopyranosyl)-rac-glycerol (6j). (196.3
(190.0 mg, 68.4%); 1H-NMR (300 MHz, CDCl3): ꢀ 0.85
(2ꢂ6H, br t, J=6.7 Hz), 1.29 (2ꢂ52H, br s), 1.51

1262
W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
(2ꢂ4H, br), 1.98, 2.01, 2.02, 2.14 (each 2ꢂ3H, s), 2.25
(2ꢂ4H, br t, J=7.0 Hz), 3.64 (2ꢂ1H, dd, J=10.8 and
5.5 Hz), 3.82–3.96 (2ꢂ2H, m), 4.01–4.17 (2ꢂ3H, m),
4.26 (2ꢂ1H, br d, J=11.0 Hz), 4.44 and 4.46 (each 1H,
d, J=7.6 Hz), 4.95 (2ꢂ1H, br d, J=10.4 Hz), 5.12–5.21
(2ꢂ2H), 5.35 (2ꢂ1H, br s).
3.2.5.5. 1,2-O-Dioleoyl-3-O-b-d-glucopyranosyl-rac-gly-
cerol (7e). (28.0 mg, 35.9%); for H-NMR (300 MHz,
CDCl3) and 13C-NMR (75 MHz, CDCl3) assignments,
see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
805.5820 (calc. 805.5805) for C45H82O10Na.
1
3.2.5.6. 1,2-O-Dilinoleoyl-3-O-b-d-glucopyranosyl-rac-
glycerol (7f). 25.7 mg, 33.0%); for 1H-NMR (300 MHz,
CDCl3) and 13C-NMR (75 MHz, CDCl3) assignments,
see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
801.5455 (calc. 801.5492) for C45H78O10Na.
3.2.4.8. 1-O-Linolenoyl-2-O-linoleoyl-3-O-(b-d-
20,30,40,60-tetra-O-acetyl-galactopyranosyl)-rac-glycerol
(12d). (151.5 mg, 53.5%); 1H-NMR (300 MHz,
CDCl3): ꢀ 0.85 (2ꢂ3H, br t, J=6.7 Hz), 0.95 (2ꢂ3H, t,
J=7.6 Hz), 1.29 (2ꢂ22H, br s), 1.51 (2ꢂ4H, br), 1.98,
2.01, 2.02, 2.14 (each 2ꢂ3H, s), 2.00 (2ꢂ8H, over-
lapped), 2.30 (2ꢂ4H, br t, J=6.9 Hz), 2.78 (2ꢂ6H, br t,
J=5.5 Hz), 3.64 (2ꢂ1H, dd, J=10.8 and 5.5 Hz), 3.82–
3.95 (2ꢂ2H, m), 4.01–4.15 (2ꢂ3H, m), 4.27 (2ꢂ1H, br d,
J=11.0 Hz), 4.44 and 4.46 (each 1H, d, J=7.6 Hz), 4.96
(2ꢂ1H, br d, J=10.4 Hz), 5.12–5.22 (2ꢂ2H), 5.35
(2ꢂ1H, overlapped), 5.30–5.40 (2ꢂ10H, br).
3.2.5.7. 1,2-O-Dilinolenoyl-3-O-b-d-glucopyranosyl-rac-
glycerol (7g). (23.5 mg, 30.4%); for 1H-NMR (300
MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) assign-
ments, see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
797.5190 (calc. 797.5179) for C45H74O10Na.
3.2.5.8. 1-O-Lauroyl-2-O-oleoyl-3-O-b-d-glucopyr-
anosyl-rac-glycerol (7h). (30.7 mg, 43.9%); for 1H-
NMR (300 MHz, CDCl3) and 13C-NMR (75 MHz,
CDCl3) assignments, see Tables 1 and 2; HR-FABMS
[M+Na]+ m/z: 723.5023 (calc. 723.5023) for
C39H72O10Na.
3.2.5. General procedure for selective removal of acetyl
protecting groups from 1,2-O-diacyl-3-O-(ꢁ-d-20,30,40,60-
tetra-O-acetyl-glycopyranosyl)glycerols
To a solution of 6a–m (or 12a–d) (0.1 mmol) in 85%
.
EtOH, NH2NH2 H2O (1.2 mmol) waꢁs added. Each
mixture was stirred for 4–6 h at 40–60 C, the mixture
poured into ice-cold water and extracted with CHCl3
(3ꢂ50 ml). The combined CHCl3 layers were dried
(anhydr Na2SO4) and individually evaporated to give a
syrupy mixture. Each mixture was purified by CC on
silica gel column, eluted with CHCl3–MeOH (8:1) to
give 1:1 mixtures of two diastereomers 7a–m (or 13a–d).
3.2.5.9. 1-O-Stearoyl-2-O-lauroyl-3-O-b-d-glucopyr-
anosyl-rac-glycerol (7i). (28.2 mg, 40.2%); for 1H-NMR
(300 MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3)
assignments, see Tables
:
1
725.5159 (calc. 725.5179) for
and 2; HR-FABMS
[M+Na]+ m/z
C39H74O10Na.
3.2.5.10. 1-O-Stearoyl-2-O-behenoyl-3-O-b-d-glucopyr-
anosyl-rac-glycerol (7j). (26.1 mg, 31.0%); for 1H NMR
(300 MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3)
3.2.5.1. 1,2-O-Didecanoyl-3-O-b-d-glucopyranosyl-rac-
glycerol (7a). (31.9 mg, 56.7%); for 1H-NMR (300
MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) assign-
ments, see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
585.3600 (calc. 585.3614) for C29H54O10Na.
assignments, see Tables
1
and 2; HR-FABMS
[M+Na]+ m/z: 865.6743 (calc. 865.6745) for
C49H94O10Na.
3.2.5.2. 1,2-O-Dilauroyl-3-O-b-d-glucopyranosyl-rac-
glycerol (7b). (31.6 mg, 51.1%); for 1H-NMR (300
MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) assign-
ments, see Tables 1 and 2; HR-FABMS [M+K]+ m/z:
657.3901 (calc. 657.3979) for C33H62O10K.
3.2.5.11. 1-O-Behenoyl-2-O-lauroyl-3-O-b-d-glucopyr-
anosyl-rac-glycerol (7k). (28.6 mg, 37.7%); for 1H-
NMR (300 MHz, CDCl3) and 13C-NMR (75 MHz,
CDCl3) assignments, see Tables 1 and 2; HR-FABMS
[M+Na]+ m/z: 781.5831 (calc. 781.5805) for
C43H82O10Na.
3.2.5.3. 1,2-O-Distearoyl-3-O-b-d-glucopyranosyl-rac-
glycerol (7c). (27.8 mg, 35.4%); for 1H-NMR (300
MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) assign-
ments, see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
809.6144 (calc. 809.6118) for C45H86O10Na.
3.2.5.12. 1-O-Behenoyl-2-O-oleoyl-3-O-b-d-glucopyr-
anosyl-rac-glycerol (7l). (26.8 mg, 31.9%); for 1H-NMR
(300 MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3)
assignments, see Tables
1
and 2; HR-FABMS
[M+Na]+ m/z: 863.6566 (calc. 863.6588) for
C49H92O10Na.
3.2.5.4. 1,2-O-Dibehenoyl-3-O-b-d-glucopyranosyl-rac-
glycerol (7d). (25.2 mg, 28.1%); for 1H-NMR (300
MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) assign-
ments, see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
921.7365 (calc. 921.7344) for C53H102O10Na.
3.2.5.13. 1-O-Linolenoyl-2-O-linoleoyl-3-O-b-d-gluco-
pyranosyl-rac-glycerol (7m). (24.0 mg, 30.9%)); for H
1
NMR (300 MHz, CDCl3) and 13C-NMR (75 MHz,

W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
1263
CDCl3) assignments, see Tables 1 and 2; HR-FABMS
[M+Na]+ m/z: 799.5366 (calc. 799.5336) for
C45H76O10Na.
(apparent t), J=9.4 and 7.5 Hz), 5.09 (1H, dd (apparent t),
J=9.4 Hz), 5.23 (1H, dd (apparent t), J=9.4 Hz).
3.3.3. 1,2-O-Dilauroyl-3-O-(ꢁ-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-sn-glycerol (17a)
3.2.5.14. 1,2-O-Dilauroyl-3-O-b-d-galactopyranosyl-rac-
glycerol (13a). (30.8 mg, 49.8%); for 1H-NMR (300
MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) assign-
ments, see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
641.4249 (calc. 641.4240) for C33H62O10Na.
17a was prepared from 16 according to the general
procedure for preparation of mono-acid 1,2-O-diacyl-3-
O-(b-d-20,30,40,60-tetra-O-acetyl-glycopyranosyl)-rac-glyc-
erols as described in Section 3.2.3 (210.2 mg, 89.1%);
1H-NMR (300 MHz, CDCl3): ꢀ 0.83 (6H, br t, J=6.9
Hz), 1.22 (32H, br s), 1.54 (4H, br), 1.94, 1.96, 1.98, 2.02
(each 3H, s), 2.23 (4H, br t, J=7.9 Hz), 3.57–3.72 (2H,
m), 3.89 (1H, dd, J=10.4 and 4.9 Hz), 4.05 (1H, dd,
(apparent t), J=6.2 Hz) 4.09 (1H, br s), 4.15 (1H, br d,
4.6 Hz) 4.24 (1H, dd, J=12.1 and 3.3 Hz), 4.48 (1H, d,
J=7.8 Hz), 4.93 (1H, dd (apparent t), J=9.4 and 8.1
Hz), 5.01 (1H, dd (apparent t), J=9.6 Hz), 5.12 (1H,
overlapped), 5.14 (1H, dd (apparent t), J=9.3 Hz).
3.2.5.15. 1,2-O-Dilinoleoyl-3-O-b-d-galactopyranosyl-
rac-glycerol (13b). (24.0 mg, 31.3%); for 1H-NMR (300
MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) assign-
ments, see Tables 1 and 2; HR-FABMS [M+Na]+ m/z:
801.5496 (calc. 801.5492) for C45H78O10Na.
3.2.5.16. 1-O-Behenoyl-2-O-lauroyl-3-O-b-d-galactopyr-
anosyl-rac-glycerol (13c). (30.2 mg, 39.8%); for 1H-
NMR (300 MHz, CDCl3) and 13C-NMR (75 MHz,
CDCl3) assignments, see Tables 1 and 2; HR-FABMS
[M+Na]+ m/z: 781.5866 (calc. 781.5805) for
C43H82O10Na.
3.3.4. 1,2-O-Dilinolenoyl-3-O-(ꢁ-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-sn-glycerol (17b)
17b was prepared from 16 according to the general
procedure for preparation of mono-acid 1,2-O-diacyl-3-
O-(b-d-20,30,40,60 -tetra-O-acetyl-glycopyranosyl)-rac-
glycerols as described in Section 3.2.3; (160.2 mg,
3.2.5.17. 1-O-Linolenoyl-2-O-linoleoyl-3-O-b-d-galacto-
1
pyranosyl-rac-glycerol (13d). (23.2 mg, 29.9%); for H-
NMR (300 MHz, CDCl3) and 13C-NMR (75 MHz,
CDCl3) assignments, see Tables 1 and 2; HR-FABMS
[M+Na]+ m/z: 779.5303 (calc. 779.5336) for
C45H76O10Na.
56.7%); H-NMR (300 MHz, CDCl3): ꢀ 0.92 (6H, t,
1
J=7.5 Hz), 1.25 (16H, br s), 1.53 (4H, br), 2.02 (8H,
overlapped), 1.93, 2.01, 2.03, 2.08 (each 3H, s), 2.25
(4H, br t, J=7.0 Hz), 2.76 (8H, br t, J=6.3 Hz), 3.58–
3.68 (2H, m), 3.90 (1H, dd, J=10.9 and 4.9 Hz), 4.05
(1H, dd (apparent t), J=6.3 Hz) 4.11 (1H, br s), 4.19
(1H, br d, J=4.6 Hz) 4.27 (1H, dd, J=12.0 and 3.6 Hz),
4.50 (1H, d, J=7.8 Hz), 4.93 (1H, dd (apparent t),
J=9.3 and 8.0 Hz), 5.03 (1H, dd (apparent t), J=9.7
Hz), 5.14 (1H, overlapped), 5.16 (1H, dd (apparent t),
J=9.3 Hz), 5.20-5.41 (12H, br).
3.3. Synthesis of 1,2-O-diacyl-3-O-ꢁ-d-glycopyranosyl-
sn-glycerols
3.3.1. 1,2-O-Dibenzyl-3-O-(ꢁ-d-20,30,40,60-tetra-O-
acetyl-glucopyranosyl)-sn-glycerol (15)
15 was prepared from 14 according to the general
procedure for glycosylation as described in Section
1
3.2.1; (618.2 mg, 51.6%); H-NMR (300 MHz, CDCl3):
3.3.5. 1,2-O-Dilauroyl-3-O-ꢁ-d-glucopyranosyl-sn-
glycerol (18a)
ꢀ 1.90, 1.96, 1.97, 2.01 (each 3H, s), 3.50–3.78 (5H, m),
3.86–3.97 (1H, m), 4.09 (1H, br d, J=12.1 Hz), 4.28
(1H, dd, J=12.2 and 4.3 Hz), 4.51 (1H, overlapped),
4.51 and 4.63 (each 2H, s), 4.97 (1H, dd (apparent t),
J=8.7 Hz), 5.01 (1H, dd (apparent t), J=9.6 Hz), 5.16
(1H, dd (apparent t), J=9.2 Hz), 7.30 (10H, br).
18a was prepared from 17a according to the general
procedure for selective removal of acetyl protecting
groups as described in Section 3.2.5; (29.8 mg, 48.2%);
for 1H-NMR (300 MHz, CDCl3) and 13C-NMR (75
MHz, CDCl3) assignments, see Table 3; HR-FABMS
[M+Na]+ m/z: 641.4247 (calc. 641.4240) for
C33H62O10Na.
3.3.2. 3-O-(ꢁ-d-20,30,40,60-Tetra-O-acetyl-
glucopyranosyl)-sn-glycerol (16)
A mixture of 15 (100 mg) and 10% Pd/C (45 mg) in
EtOAc (10 ml) containing 0.25 ml each of EtOH and
HOAc was vigorously shaken under H2 gas using a
Parr apparatus for 5 h. The catalyst was filtered off,
the filtrate was evaporated and purified by CC, eluted
with EtOAc–hexane (5:1) to give semisolid substance
16; (56 mg, 80.4%); 1H-NMR (300 MHz, CDCl3): ꢀ
2.01, 2.04, 2.06, 2.10 (each 3H, s), 3.52–3.95 (6H, m),
4.22 (2H, br s), 4.56 (1H, d, J=7.8 Hz), 5.02 (1H, dd
3.3.6. 1,2-O-Dilinolenoyl-3-O-ꢁ-d-glucopyranosyl-sn-
glycerol (18b)
18b was prepared from 17b according to the general
procedure for selective removal of acetyl protecting
groups as described in Section 3.2.5; (21.5 mg, 27.7%);
for 1H-NMR (300 MHz, CDCl3) and 13C-NMR (75
MHz, CDCl3) assignments, see Table 3; HR-FABMS
[M+Na]+ m/z: 797.5192 (calc. 797.5179) for
C45H74O10Na.

1264
W. Janwitayanuchit et al. / Phytochemistry 64 (2003) 1253–1264
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5% CO2 for 3 days. The viral plaques formed in the
Vero cell cultures were counted under an inverted
microscope. Antiviral activity was expressed as a score
of% inhibition and 50% inhibitory concentration
(IC50). Toxicity was evaluated by the ability of the
compounds to reduce the number of viable Vero cells by
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Financial support for this work was provided by The
Thailand Research Fund, grant No. RDG3/33/2543.
We thank the Pharmaceutical Research Instrument
Center, Faculty of Pharmaceutical Sciences, Chula-
longkorn University for providing the 300 MHz NMR
spectrometer, and Dr. Naoki Saito, Meiji Pharmaceutical
University, for his support in recording mass spectra.
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