2-O-β-d-Glucopyranosyl-sn-glycerol based analogues of sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting Epstein-Barr virus early antigen activation

Article abstract of DOI:10.1016/j.bmc.2009.06.064

New sulfoquinovosyldiacylglycerols derived from 2-O-β-d-glucopyranosyl-sn-glycerol, carrying acyl chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure in which a sulfonate is introduced at the C-6 positio

Full text of DOI:10.1016/j.bmc.2009.06.064

Bioorganic & Medicinal Chemistry 17 (2009) 5968–5973
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-O-b-D-Glucopyranosyl-sn-glycerol based analogues of
sulfoquinovosyldiacylglycerols (SQDG) and their role in inhibiting
Epstein-Barr virus early antigen activation
Milind Dangate a, Laura Franchini a, Fiamma Ronchetti a, Takanari Arai b, Akira Iida c,
Harukuni Tokuda d, Diego Colombo a,
*
a Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università di Milano, Via Saldini 50, 20133 Milano, Italy
b Kanazawa University Hospital, Kanazawa 920-8641, Japan
c Department of Agriculture, Kinki University, Nara 631-8505, Japan
d Department of Molecular Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
New sulfoquinovosyldiacylglycerols derived from 2-O-b-D-glucopyranosyl-sn-glycerol, carrying acyl
Received 16 March 2009
Revised 24 June 2009
Accepted 28 June 2009
Available online 3 July 2009
chains of various length on the glycerol moiety, were prepared through a convenient synthetic procedure
in which a sulfonate is introduced at the C-6 position of glucose by oxidation of a thioacetate in the pres-
ence of the unprotected secondary hydroxyl groups, and tested for their anti-tumor-promoting activity
using a short-term in vitro assay for Epstein-Barr virus early antigen (EBV-EA) activation. Our study
has allowed to ascertain the role of the 60-sulfonate group and the need of a free hydroxyl group on
the glycerol moiety in inhibiting the EBV activation promoted by the tumor promoter 12-O-tetradeca-
noylphorbol-13-acetate (TPA).
Keywords:
Glycoglycerolipids
Cancer chemoprevention
Sulfo-glycolipids
EBV-EA
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
with that of mono- and digalactosyl diacylglycerols (MGDG and
DGDG) isolated from the same micro-organism.6 The inhibitory ef-
Sulfoquinovosyldiacylglycerols (SQDG) are sulfo-glycolipids
mainly associated with photosynthetic organisms discovered in
microorganisms and higher plants by Benson in 19591 in which
fect of MGDG and DGDG was higher than SQDG but a sure struc-
ture–activity relationship of SQDG was not possible6 because
they were tested as a mixture of compounds carrying acyl chains
of different length, and because of the different nature of the sug-
ars present in these natural compounds (i.e., b-galactopyranosides
sulfoquinovose (6-deoxy-6-sulfo-glucose)2 is
a-linked to the sn-3
position of a diacylglycerol (Fig. 1). Even if their physiological func-
tions are still under investigation, SQDG are thought to be involved
in the maintenance of photosystem complexes function and to
substitute phospholipids under phosphate-limiting growth condi-
tions to maintain the total anionic lipids at a constant level in
the thylakoid membranes of chloroplasts.2,3 Concerning their
behavior in experiments testing the potential therapeutic use of
SQDG, recently reported4,5 biological activities, including inhibi-
tory effects on HIV-reverse transcriptase, mammalian DNA poly-
merase, proliferation of some cancer cell lines, angiogenesis
(especially when coupled with tumor radiotherapy), and also
apoptosis induction, make these compounds very attractive for
their use in cancer therapy.
in the case of MGDG and DGDG, vs
a-6-deoxy-glucopyranosides
for SQDG). During our search for new glycoglycerolipids active in
cancer chemoprevention, in recent years we have synthesized a
number of esters of 2-O-b-
shape, number and position of the acyl chain, and the type of sugar
and b glucose or galactose) were varied. These compounds were
D-glycosylglycerols in which the length,
(a
very active in inhibiting the tumor-promoting activity of TPA both
-
SO3
1
O
OR
H
HO
HO
2
Also, Shirahshi et al. reported the in vitro anti-tumor-promoting
activity of SQDG from Cyanobacterium Phormidium tenue together
R'O
O
OH
3
SQDG
R and R'= acyl chains
* Corresponding author. Tel.: +39 02 5031 6039; fax: +39 02 5031 6036.
E-mail address: diego.colombo@unimi.it (D. Colombo).
Figure 1.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.064

M. Dangate et al. / Bioorg. Med. Chem. 17 (2009) 5968–5973
5969
1
in in vitro and in in vivo tests, being such activities mainly influ-
enced by the changes of the acyl chains length.7 Thus, the above re-
ported promising biological activities of SQDG prompted us to plan
an easy synthesis of SQDG analogues based on the skeleton of
2-O-b-D-glucopyranosyl-sn-glycerol to which the previously tested
glycoglycerolipid analogues were related and to test their anti-
tumor-promoting activity.
acetylated sulfoquinovosides (diagnostic H NMR signals: H-40,
dd, at 4.65 ppm and CH3CO, s, at 2.08 ppm) occurred as a side reac-
tion, and the amphiphilic nature of the final compounds made their
work-up tedious (see Section 4) this procedure allowed us to ob-
tain the target sulfoquinovosides 1a–c in noteworthy 38–42%
yields avoiding protecting group manipulation.
Starting from the known 2-O-(2,3,4,6-tetra-O-chloroacetyl-b-
D
-
2.2. Biological evaluation
glucopyranosyl)-sn-glycerol (2),8 SQDG analogues 1a–c (Fig. 2),
namely 1,3-di-O-octadecanoyl (1a), -dodecanoyl (1b), and -hexa-
Epstein-Barr virus (EBV) is known to be activated by tumor pro-
moters to produce viral early antigens (EA), and an evaluation of its
inhibition is often used as a primary screening for in vitro anti-tu-
mor-promoting activities.11 The inhibitory effects of sulfonated
(1a–c) and de-sulfonated (4a–c) glucoglycerolipids were assayed
using a short-term in vitro assay for EBV-EA activation in Raji cells
induced by the tumor promoter TPA, as described in Refs. 6 and 12
(see Section 4). Table 1 shows the in vitro tumor inhibitory activity
of compounds 1a–c and 4a–c.
Only weak cytotoxicity against Raji cells was observed espe-
cially for compounds 1a–c at high concentration (50% viability at
1000 mol ratio/TPA vs 60% for compounds 4a–c Table 1) and, in
general, all compounds were active as indicated by their percent-
age to control (46.8–65.1% at 500 mol ratio/TPA, Table 1), though
previously tested glycoglycerolipids showed higher inhibitory ef-
fect on EBV activation.7 In Table 1 the in vitro inhibitory effects
noyl-2-O-b-
acyl chains of different length were linked to the primary hydrox-
yls of 2-O-b- -sulfoquinovopyranosyl-sn-glycerol), were prepared,
D-sulfoquinovopyranosyl-sn-glycerol (1c) (in which
D
exploiting chloroacetyls to temporary and selectively protect
glucose hydroxyls with respect to glycerol. As a preliminary check-
up of their biological behavior, in this paper the obtained com-
pounds were tested as anti-tumor-promoters using a short-term
in vitro assay for EBV-EA activation and, to get insight the influence
exerted by the 60-sulfonate, the corresponding 60-hydroxy syn-
thetic intermediates, 1,3-di-O-octadecanoyl (4a), -dodecanoyl
(4b), and -hexanoyl-2-O-b-D-glucopyranosyl-sn-glycerol (4c), were
also tested.
2. Results and discussion
2.1. Chemistry
of the already studied 1-O-hexanoyl-2-O-(6-O-hexanoyl-b-
D
-glu-
copyranosyl)-sn-glycerol (7)13 and 1-O-hexanoyl-2-O-(b-
D-galacto-
pyranosyl)ethylene glycol (8)14 are also reported for a comparison
2-O-(2,3,4,6-Tetra-O-chloroacetyl-b-D-glucopyranosyl)-sn-glyc-
of the structural requirements. It is interesting to note that the 1,3-
erol (2) was prepared from glucose pentaacetate and 1,3-dibenzyl-
glycerol as previously reported.8 Two acyl chains of the desired
length were then linked to the glycerol hydroxyls of 2 by treatment
with the proper acyl chloride (octadecanoyl, dodecanoyl or hexa-
noyl) in pyridine affording 1,3-diesters 3a–c in good yields (see
Section 4). Selective removal of the four chloroacetyls from the
sugar was then efficiently performed by hydrazine acetate treat-
diester,
1,3-di-O-hexanoyl-2-O-b-D-glucopyranosyl-sn-glycerol
(4c), is significantly less active than the corresponding 1,60-di-O-
hexanoyl isomer (7)13 in inhibiting the EBV-EA activation (IC50
327 vs 37.3, respectively, Table 1) suggesting that the double acyl-
ation of glycerol plays a negative role on the activity in these com-
pounds. These data in fact seem to support the reported
observation that the anti-tumor-promoting activity of these 2-O-
glycoglycerolipid analogues is closely related to the presence of a
free hydroxymethylene group on the glycerol-like aglycone moi-
ment8 to yield the 1,3-di-O-acyl-2-O-b-
D-glucopyranosyl-sn-glyce-
rols 4a–c. The main task of introducing a sulfonate at C-6 position
of glucose, was achieved by a strategy involving tosylation, substi-
tution by thioacetate and 60-SAc group oxidation.
ety.14 Actually, 1-O-hexanoyl-2-O-(b-
D-galactopyranosyl)ethylene
glycol (8)14 (in which glycerol is replaced by ethylene glycol as
the aglycone) shows an activity very similar to that of the 1,3-dies-
ter 4c (IC50 350 vs 327, respectively, Table 1). Concerning the pres-
ence of the sulfonate group, it seems to exert an additional
negative effect on the anti-tumor-promoting activity of glycoglyc-
erolipids in this in vitro experimental model, as evidenced by the
In particular, zinc bromide catalyzed the selective tosylation9 of
the primary 60-hydroxyl of 4a–c in pyridine at ꢀ20 °C yielding 1,3-
di-O-acyl-2-O-(6-O-tosyl-b-D-glucopyranosyl)-sn-glycerols 5a–c in
good yields, which further were converted into the corresponding
1,3-di-O-acyl-2-O-(6-deoxy-6-thioacetyl-b- -glucopyranosyl)-sn-
D
glycerols 6a–c by potassium thioacetate treatment in DMF. The
final oxidation step was carried out with OXONEÒ in acetic acid10
directly on the unprotected compounds 6a–c. The presence of free
secondary hydroxyls on these intermediates made the oxidation
step a challenging task. However, though the formation of 40-O-
Table 1
Inhibitory effects of 1a–c, 4a–c, and on TPA-induced EBV-EA activation
Concentration (mol ratio/TPA)
500 100
% to control SE (n = 3)a
IC50
1000
10
R'
1
OR
O
1a
1b
1c
4a
4b
4c
7c
25.1 1.6 (50)b
23.4 1.6 (50)
20.3 1.6 (50)
16.2 1.5 (60)
13.0 1.2 (60)
9.3 0.7 (60)
0.0 0.0 (70)
6.7 0.5 (70)
65.1 2.4 (70)
62.6 2.2 (70)
60.5 2.1 (70)
57.8 2.0 (70)
52.4 2.0 (70)
46.8 1.7 (70)
15.1 0.5 (80)
49.5 1.4 (80)
87.4 1.9 (80)
100 0.2 (80)
100 0.3 (80)
100 0.3 (80)
100 0.2 (80)
100 0.4 (80)
100 0.5 (80)
71.5 1.9(80)
100 0.2 (80)
632
581
539
484
402
327
37.3
350
2
"RO
O
H
85.2 1.7 (80)
84.3 1.9 (80)
82.8 1.8 (80)
80.5 1.8 (80)
76.9 1.8 (80)
34.6 1.2 (80)
78.5 2.5 (80)
"RO
3
OR"
OR
-
1: R= acyl, R'= SO3 , R"= H
2: R= H, R'= ClCH2COO, R"= ClCH2CO
3: R= acyl, R'= ClCH2COO, R"= ClCH2CO
4: R= acyl, R'= OH, R"= H
8d
a
Values are EBV-EA activation (%) in the presence of the test compound relative
5: R= acyl, R'= OTs, R"= H
to the control (100%). Activation was attained by treatment with TPA 32 pmol. IC50
represents the mol ratio to TPA that inhibits 50% of positive control (100%) activated
with 32 pmol TPA.
6: R= acyl, R'= SCOCH3, R"= H
a: acyl= octadecanoyl b: acyl= dodecanoyl
c: acyl= hexanoyl
b
Values in parentheses are viability percentages of Raji cells.
c
See Ref. 13.
See Ref. 14.
d
Figure 2.

5970
M. Dangate et al. / Bioorg. Med. Chem. 17 (2009) 5968–5973
comparison of the data reported in Table 1 for the 60-sulfo-diesters
CDCl3 fixed at 77.0 ppm (central line) or CD3OD at 49.00 ppm (cen-
tral line) for 13C NMR spectra; scalar coupling constants are re-
ported in hertz. Mass spectra were recorded in negative or
positive-ion electrospray (ESI) mode on a Thermo Quest Finnigan
1a–c (IC50 from 632 to 539, Table 1) and the 60-hydroxy 4a–c (IC50
from 484 to 327 Table 1). Nevertheless, the here reported 2-O-b-D-
analogues 1a–c show a significantly higher activity than the natu-
ral SQDG6 (20.3–25.1%, Table 1, vs 91.7% at 1000 mol ratio/TPA,
respectively). Finally, as further support to the data already re-
ported,7 also in this case shortening the acyl chains, from C18
(compounds of the a series) to C6 (compounds of the c series), re-
sulted in the increasing of the activity both in the case of sulfoqui-
novosides 1 and glucosides 4 (see Table 1).
TM
LCQ DECA ion trap mass spectrometer; the mass spectrometer
was equipped with a Finningan ESI interface; sample solutions
were injected with a ionization spray voltage of 4.5 kV or 5.0 kV
(positive and negative-ion mode, respectively), a capillary voltage
of 32 V or ꢀ15 V (positive and negative-ion mode, respectively),
and capillary temperature of 250 °C. Data were processed by Finn-
igan Xcalibur software system. TLC, 1H NMR and MS confirmed
purity of all synthesized compounds.
3. Conclusion
4.1.2. General procedure for the synthesis of diesters 3a–c
New sulfoquinovosyldiacylglycerols based on 2-O-b-D-gluco-
2-O-(2,3,4,6-Tetra-O-chloroacetyl-b-D-glucopyranosyl)-sn-glyc-
pyranosyl-sn-glycerol and their 60-hydroxy ‘de-sulfonated’ gluco-
syl analogues were prepared through an easy synthetic
procedure and some interesting conclusions on their in vitro
anti-tumor-promoting activity could be drawn from the obtained
results. In fact, the new compounds have allowed to ascertain
the influence of some till now unexplored structural features of
these glycoglycerolipids (i.e., presence of a sulfonate group on
the sugar moiety and double acylation of glycerol) on the tested
activity. In particular, the sulfonate group at the C-6 position of
glucose plays a negative role on the inhibition of EBV activation in-
duced by the tumor promoter TPA, also increasing the cytotoxicity.
In the same way, the double acylation of the glycerol moiety pro-
duces a negative effect, probably because of the masking of a pri-
mary hydroxyl group crucial for the activity. Basing on these
observations, sulfoquinovosylmonoacylglycerols (SQMG) might
be more active than the corresponding SQDG in the EBV-EA inhibi-
tion test. Due to the high interest related to bio-active natural sul-
fo-glycolipids, work is in progress to further characterize the
biological potential of these new synthetically available sulfoqui-
novosylacylglycerol analogues.
erol (2)8 (2.0 g, 3.57 mmol) was dissolved in dry CH2Cl2 (30 mL)
and cooled at ꢀ10 °C. The proper acyl chloride (8.8 mmol) as a
15% (v/v) CH2Cl2 solution and pyridine (22.0 mmol) as a 10% (v/
v) CH2Cl2 solution, were added in the order and the mixture stirred
at ꢀ10 °C under Ar atmosphere. The reaction was monitored by
TLC (petroleum ether:EtOAc, 70:30 v/v and CH2Cl2:CH3OH, 95:5
v/v) and stopped after 15–30 min diluting with dichloromethane
(160 mL). The solution was washed with 1 M HCl (80 mL), water
(80 mL), NaHCO3 saturated solution (80 mL), and water
(2 ꢁ 80 mL) in the order and the aqueous phases re-extracted with
dichloromethane (2 ꢁ 160 mL). The collected organic layers were
dried over Na2SO4, dried under reduced pressure and the crude res-
idue submitted to silica gel flash chromatography (petroleum
ether:EtOAc, from 75:25 to 80:20 v/v). In this way diesters 3a–c
were obtained.
4.1.2.1. 1,3-Di-O-octadecanoyl-2-O-(2,3,4,6-tetra-O-chloroace-
tyl-b-D-glucopyranosyl)-sn-glycerol (3a). Reaction time 15 min,
yield 80%; mp = 78 °C; ½a D20
ꢂ
¼ ꢀ2:5 (CHCl3); 1H NMR (CDCl3): d
0.86 (t, 6H, J = 6.7 Hz, 2CH3), 1.19–1.35 (m, 56H, 28CH2), 1.58 (m,
4H, 2CH2), 2.28 (m, 4H, 2CH2), 3.81 (m, 1H, H-50), 3.96 (s, 2H,
ClCH2), 3.99 (m, 2H, ClCH2), 4.02 (m, 2H, ClCH2), 4.03–4.25 (m,
5H, 2H-1, H-2, and 2H-3), 4.11 (s, 2H, ClCH2), 4.27 (dd, 1H,
4. Experimental
J6 a,5 = 2.3 Hz, J6 a,6 b = 12.4 Hz, H-60a), 4.35 (dd, 1H, J6 b,5 = 5.1 Hz,
4.1. Chemical procedures
4.1.1. Materials
0
0
0
0
0
0
H-60b), 4.70 (d, 1H, J1 ,2 = 7.9 Hz, H-10), 5.03 (dd, 1H, J2 ,3 = 9.5 Hz,
0
0
0
0
H-20), 5.12 (dd, 1H, J3 ,4 = 9.5 Hz, H-40), 5.30 (dd, 1H, H-30); 13C
NMR (CDCl3): d 14.09 (2CH3), 22.67 (2CH2), 24.82 (CH2), 24.85
(CH2), 29.00–30.00 (24CH2), 31.90 (2CH2), 34.02 and 34.06
(2CH2CO), 40.13, 40.22, 40.28 and 40.49 (4 CH2Cl), 62.71 (C1 or
C3), 62.74 (C1 or C3), 63.12 (C60), 69.62 (C40), 71.34 (C50), 72.27
(C20), 73.74 (C30), 75.90 (C2), 100.17 (C10), 165.90, 166.23, 166.90
and 167.01 (4ClCH2CO), 173.36 (2CO). ESI-MS (CH3OH, positive-
ion mode, relative intensity): m/z = 1115.6 [M+Na]+, 100%. Calcd
for C53H90O14Cl4, m/z 1092.5 [M].
0
0
2-O-(2,3,4,6-Tetra-O-chloroacetyl-b-
D-glucopyranosyl)-sn-glyc-
erol (2), was synthesized according to the literature procedure.8
Optical rotations were determined on a Perkin–Elmer 241 polarim-
eter in 1% CHCl3 or CHCl3:CH3OH:H2O (65:25:4) solutions at 20 °C,
in a 1 dm cell. Melting points were recorded on a Büchi 510 capil-
lary melting point apparatus and were uncorrected. All reagents
and solvents used were reagent grade and were purified before
use by standard methods. Dry solvents and liquid reagents were
distilled prior to use or dried on 4 Å molecular sieves. Column
chromatography was carried out on flash silica gel (Merck 230–
400 mesh). TLC analysis was carried out on silica gel plate (Merck
60F254) developing with 50% sulfuric acid or anisaldehyde based
reagent. Evaporation under reduced pressure was always effected
with a bath temperature below 40 °C. The structures of all the
new synthesized compounds were confirmed through full 1H and
13C NMR characterization and mass spectroscopy. 1H NMR analysis
4.1.2.2. 1,3-Di-O-dodecanoyl-2-O-(2,3,4,6-tetra-O-chloroacetyl-
b-D-glucopyranosyl)-sn-glycerol (3b). Reaction time 15 min,
yield 88%; mp = 57 °C; ½a D20
ꢂ
¼ ꢀ2:3 (CHCl3); 1H NMR (CDCl3): d
0.84 (t, 6H, J = 6.7 Hz, 2CH3), 1.17–1.32 (m, 32H, 16CH2), 1.57 (m,
4H, 2CH2), 2.27 (m, 4H, 2CH2), 3.81 (m, 1H, H-50), 3.95 (s, 2H,
ClCH2), 3.98 (m, 2H, ClCH2), 4.01 (m, 2H, ClCH2), 4.02–4.25 (m,
5H, 2H-1, H-2, and 2H-3), 4.11 (s, 2H, ClCH2), 4.26 (dd, 1H,
TM
J6 a,5 = 2.3 Hz, J6 a,6 b = 12.4 Hz, H-60a), 4.33 (dd, 1H, J6 b,5 = 5.1 Hz,
were performed at 500 MHz with a Bruker FT-NMR AVANCE
0
0
0
0
0
0
H-60b), 4.70 (d, 1H, J1 ,2 = 7.9 Hz, H-10), 5.02 (dd, 1H, J2 ,3 = 9.5 Hz,
0
0
0
0
DRX500 spectrometer using a 5 mm z-PFG (pulsed field gradient)
broadband reverse probe at 298 K unless otherwise stated, and
13C NMR spectra at 125.76 MHz were done of all the new com-
pounds. The signals were unambiguously assigned by 2D COSY
and HSQC experiments (standard Bruker pulse program). Chemical
shifts are reported as d (ppm) relative to residual CHCl3 or CH3OH
(when CDCl3:CD3OD:D2O solvent mixture was used) fixed at 7.24
and 3.30 ppm, respectively, for 1H NMR spectra and relative to
H-20), 5.11 (dd, 1H, J3 ,4 = 9.5 Hz, H-40), 5.30 (dd, 1H, H-30); 13C
NMR (CDCl3): d 14.05 (2CH3), 22.62 (2CH2), 24.77 (CH2), 24.81
(CH2), 29.00–30.00 (12CH2), 31.85 (2CH2), 33.98 and 34.03
(2CH2CO), 40.11, 40.20, 40.25 and 40.47 (4 CH2Cl), 62.67 (C1 or
C3), 62.70 (C1 or C3), 63.11 (C60), 69.60 (C40), 71.29 (C50), 72.24
(C20), 73.70 (C30), 75.86 (C2), 100.12 (C10), 165.87, 166.21, 166.88
and 167.00 (4ClCH2CO), 173.33 (2CO). ESI-MS (CH3OH, positive-ion
0
0

M. Dangate et al. / Bioorg. Med. Chem. 17 (2009) 5968–5973
5971
mode, relative intensity): m/z = 947.2 [M+Na]+, 100%. Calcd for
C41H66O14Cl4, m/z 924.3 [M].
positive-ion mode, relative intensity): m/z = 641.4 [M+Na]+, 100%.
Calcd for C33H62O10, m/z 618.4 [M].
4.1.2.3. 1,3-Di-O-hexanoyl-2-O-(2,3,4,6-tetra-O-chloroacetyl-b-
4.1.3.3. 1,3-Di-O-hexanoyl-2-O-b-D-glucopyranosyl-sn-glycerols
D
-glucopyranosyl)-sn-glycerol (3c). Reaction time 30 min, yield
(4c). Reaction time 18 h; yield 60%; oil; ½a D20
ꢂ
¼ ꢀ8:9 (CHCl3); 1H
70%; oil; ½a 2D0
ꢂ
¼ ꢀ2:1 (CHCl3); 1H NMR (CDCl3): d 0.85 (m, 6H,
NMR (CDCl3): d 0.87 (t, 6H, J = 7.0 Hz, 2CH3), 1.22–1.35 (m, 8H,
4CH2), 1.60 (m, 4H, 2CH2), 2.31 (m, 4H, 2CH2), 3.30–3.38 (m, 2H,
H-20 and H-50), 3.47–3.55 (m, 2H, H-30 and H-40), 3.76 (dd, 1H,
2CH3), 1.18–1.35 (m, 8H, 4CH2), 1.57 (m, 4H, 2CH2), 2.27 (m, 4H,
2CH2), 3.81 (m, 1H, H-50), 3.95 (s, 2H, ClCH2), 3.98 (m, 2H, ClCH2),
4.00 (m, 2H, ClCH2), 4.02–4.25 (m, 5H, 2H-1, H-2, and 2H-3), 4.10
J6 a,5 = 5.0 Hz, J6 a,6 b = 12.0 Hz, H-60a), 3.86 (dd, 1H, J6 b,5 = 2.8 Hz,
H-60b), 4.03 (m, 1H, H-2), 4.13–4.20 (m, 2H, H-1a and H-3a), 4.26
(dd, 1H, J1b,2 = 5.0 Hz, J1b,1a = 11.8 Hz, H-1b or H-3b), 4.31 (dd, 1H,
J3b,2 = 4.0 Hz, J3b,3a = 11.8 Hz, H-3b or H-1b), 4.39 (d, 1H,
0
0
0
0
0
0
(s, 2H, ClCH2), 4.26 (dd, 1H, J6 a,5 = 2.1 Hz, J6 a,6 b = 12.3 Hz, H-60a),
0
0
0
0
4.33 (dd, 1H, J6 b,5 = 5.1 Hz, H-60b), 4.70 (d, 1H, J1 ,2 = 7.9 Hz, H-
0
0
0
0
10), 5.02 (dd, 1H, J2 ,3 = 9.3 Hz, H-20), 5.11 (dd, 1H, J3 ,4 = 9.4 Hz,
H-40), 5.29 (dd, 1H, H-30); 13C NMR (CDCl3): d 13.81 (2CH3), 22.21
(2CH2), 24.41 (CH2), 24.45 (CH2), 31.18 (2CH2), 33.91 and 33.96
(2CH2CO), 40.12, 40.20, 40.24 and 40.47 (4 CH2Cl), 62.65 (C1 or
C3), 62.69 (C1 or C3), 63.10 (C60), 69.57 (C40), 71.24 (C50), 72.20
(C20), 73.66 (C30), 75.83 (C2), 100.07 (C10), 165.85, 166.19, 166.85
and 166.96 (4ClCH2CO), 173.29 (2CO). ESI-MS (CH3OH, positive-
ion mode, relative intensity): m/z = 779.2 [M+Na]+, 100%. Calcd
for C29H42O14Cl4, m/z 756.1 [M].
0
0
0
0
J1 ,2 = 7.7 Hz, H-10); 13C NMR (CDCl3): d 13.89 (2CH3), 22.27
(2CH2), 24.51 (CH2), 24.50 (CH2), 31.24 (2CH2), 34.07 and 34.12
(2CH2CO), 62.11 (C60), 63.12 (C1 or C3), 63.14 (C1 or C3), 69.92
(C30 or C40), 73.44 (C20), 75.83 (C2 and C50), 76.25 (C30 or C40),
103.25 (C10), 173.71 (CO), 174.07 (CO). ESI-MS (CH3OH, positive-
ion mode, relative intensity): m/z = 473.2 [M+Na]+, 100%. Calcd
for C21H38O10, m/z 450.2 [M].
0
0
4.1.4. General procedure for the synthesis of tosyl derivatives
5a–c
4.1.3. General procedure for the synthesis of diacylglucosyl-
glycerols 4a–c
To a solution of compound 4 (1.10 mmol) in dry pyridine
(33 mL) at ꢀ20 °C, zinc bromide (0.99 g, 4.4 mmol) was added
resulting into the formation of white precipitate. After the addition
of freshly recrystallized tosyl chloride (1.05 g, 5.5 mmol) reaction
mixture turned green and precipitate went on diminishing. The
reaction mixture was stirred at ꢀ20 °C for about 2 h (longer reac-
tion times reduced the yields) and monitored by TLC
(CH2Cl2:CH3OH, 97:3 v/v). After the completion of reaction, CH3OH
(65 mL) was added to quench excess tosyl chloride and the solvent
evaporated under vacuum at 35 °C. The residue was dissolved in
EtOAc (110 mL) and the organic solution was washed with 1 M
HCl (25 mL), H2O (25 mL), NaHCO3 saturated solution (25 mL),
H2O (2 ꢁ 25 mL), and the aqueous phases extracted again with
EtOAc (2 ꢁ 50 mL). The collected organic layers were dried over
Na2SO4, concentrated under vacuum and purified by flash column
chromatography using (CH2Cl2:CH3OH, 97:3 or 95:5 v/v) to obtain
the desired tosyl derivatives 5a–c.
Compound
3 (2.16 mmol) was dissolved in EtOAc:CH3OH
(56 mL, 1:1 v/v) and hydrazine acetate (3.0 g, 33 mmol) was added.
The reaction was stirred under Ar atmosphere at room tempera-
ture for 18 h and monitored by TLC (CH2Cl2:CH3OH, 95:5 and
petroleum ether:EtOAc, 70:30 v/v). The solvent was evaporated
under reduced pressure and the crude residue subjected to re-
peated flash column chromatography (CH2Cl2:CH3OH, 95:5 v/v)
followed by recrystallization from ethanol to remove hydrazine
impurities yielding pure diacylglucosylglycerols 4a–c.
4.1.3.1. 1,3-Di-O-octadecanoyl-2-O-b-D-glucopyranosyl-sn-glyc-
erol (4a). Reaction time 18 h; yield 68%; mp = 133 °C; ½a D20
¼ ꢀ4:1
ꢂ
(CHCl3); 1H NMR (CDCl3:CD3OD 95:5): d 0.82 (t, 6H, J = 7.0 Hz,
2CH3), 1.16–1.28 (m, 56H, 28CH2), 1.56 (m, 4H, 2CH2), 2.28 (m,
4H, 2CH2), 3.24 (dd, 1H, J1 ,2 = 7.7 Hz, J2 ,3 = 8.7 Hz, H-20), 3.28 (m,
0
0
0
0
1H, H-50), 3.36–3.45 (m, 2H, H-30 and H-40), 3.68 (dd, 1H,
J6 a,5 = 5.2 Hz, J6 a,6 b = 12.1 Hz, H-60a), 3.81 (dd, 1H, J6 b,5 = 3.1 Hz,
H-60b), 3.99 (m, 1H, H-2), 4.13–4.20 (m, 2H, H-1a and H-3a), 4.23
(dd, 1H, J1b,2 = 4.9 Hz, J1b,1a = 12.5 Hz, H-1b or H-3b), 4.26 (dd, 1H,
J3b,2 = 4.0 Hz, J3b,3a = 12.0 Hz, H-3b or H-1b), 4.34 (d, 1H, H-10);
13C NMR (CDCl3:CD3OD 95:5): d 14.02 (2CH3), 22.62 (2CH2),
24.79 (2CH2), 29.00–30.00 (24CH2), 31.86 (2CH2), 34.09 and
34.11 (2CH2CO), 62.12 (C60), 63.10 (C1 or C3), 63.15 (C1 or C3),
70.01 (C30), 73.41 (C20), 75.65 (C2), 75.94 (C50), 76.06 (C40),
103.29 (C10), 173.85 (CO), 174.10 (CO). ESI-MS (CH3OH, positive-
ion mode, relative intensity): m/z = 809.7 [M+Na]+, 100%. Calcd
for C45H86O10, m/z 786.6 [M].
0
0
0
0
0
0
4.1.4.1. 1,3-Di-O-octadecanoyl-2-O-(6-deoxy-6-tosyl-b-D-gluco-
pyranosyl)-sn-glycerol (5a). Reaction time 2 h; yield 73%;
mp = 90 °C; ½a 2D0
ꢂ
¼ ꢀ4:6 (CHCl3); 1H NMR (CDCl3): d 0.86 (t, 6H,
J = 7.0 Hz, 2CH3), 1.18–1.33 (m, 56H, 28CH2), 1.58 (m, 4H, 2CH2),
0
0
2.30 (m, 4H, 2CH2), 2.42 (s, 3H, CH3), 3.29 (dd, 1H, J1 ,2 = 7.7 Hz,
J2 ,3 = 9.0 Hz, H-20), 3.42–3.53 (m, 3H, H-30, H-40, H-50), 4.02 (m,
1H, H-2), 4.06–4.13 (m, 2H, H-1a and H-3a), 4.19 (dd, 1H,
J1b,2 = 5.3 Hz, J1b,1a = 11.5 Hz, H-1b or H-3b), 4.22 (dd, 1H,
0
0
J6 a,5 = 4.0 Hz, J6 a,6 b = 10.9 Hz, H-60a), 4.27 (dd, 1H, J6 b,5 < 1.0 Hz,
H-60b), 4.32 (d, 1H, H-10), 4.36 (dd, 1H, J3b,2 = 3.4 Hz, J3b,3a = 11.9 Hz,
H-3b or H-1b), 7.33 (d, 2H, J = 8.3 Hz, Ph), 7.77 (d, 2H, Ph); 13C NMR
(CDCl3): d 14.09 (2CH3), 21.64 (PhCH3), 22.67 (2CH2), 24.85 (2CH2),
28.50–30.10 (24CH2), 31.91 (2CH2), 34.04 and 34.18 (2CH2CO),
62.91 (C1 or C3), 63.00 (C1 or C3), 68.55 (C60), 69.28 (C40 or C50),
73.27 (C20), 73.66 (C40 or C50), 75.95 and 76.07 (C2 and C30),
103.09 (C10), 127.98 and 129.92 (4C, Ph), 132.70 and 145.02 (2C,
Ph), 173.42 (CO), 174.15 (CO). ESI-MS (CH3OH, positive-ion mode,
relative intensity): m/z = 963.6 [M+Na]+, 100%. Calcd for
C52H92O12S, m/z 940.6 [M].
0
0
0
0
0
0
4.1.3.2. 1,3-Di-O-dodecanoyl-2-O-b-D-glucopyranosyl-sn-glyce-
rols (4b). Reaction time 18 h; yield 60%; mp = 110 °C; ½a D20
¼ ꢀ6:0
ꢂ
(CHCl3); 1H NMR (CDCl3): d 0.86 (t, 6H, J = 7.0 Hz, 2CH3), 1.17–1.33
(m, 32H, 16CH2), 1.59 (m, 4H, 2CH2), 2.31 (m, 4H, 2CH2), 3.32 (m,
1H, H-20), 3.39 (m, 1H, H-50), 3.52 (m, 1H, H40), 3.54 (m, 1H, H-
30), 3.75 (dd, 1H, J6 a,5 = 4.7 Hz, J6 a,6 b = 12.1 Hz, H-60a), 3.89 (dd,
0
0
0
0
1H, J6 b,5 = 2.6 Hz, H-60b), 4.02 (m, 1H, H-2), 4.10–4.20 (m, 2H, H-
1a and H-3a), 4.25 (dd, 1H, J1b,2 = 5.1 Hz, J1b,1a = 11.6 Hz, H-1b or
H-3b), 4.36 (dd, 1H, J3b,2 = 3.6 Hz, J3b,3a = 11.8 Hz, H-3b or H-1b),
0
0
4.1.4.2. 1,3-Di-O-dodecanoyl-2-O-(6-deoxy-6-tosyl-b-D-gluco-
4.38 (d, 1H, J1 ,2 = 7.7 Hz, H-10); 13C NMR (CDCl3): d 14.11 (2CH3),
22.67 (2CH2), 24.84 (CH2), 24.86 (CH2), 28.9–29.8 (12 CH2), 31.90
(2CH2), 34.15 and 34.21 (2CH2CO), 62.58 (C60), 63.05 (C1 or C3),
63.20 (C1 or C3), 70.43 (C40), 73.57 (C20), 75.79 (C50), 76.13 (C2),
76.27 (C30), 103.35 (C10), 173.65 (CO), 174.12 (CO). ESI-MS (CH3OH,
0
0
pyranosyl)-sn-glycerol (5b). Reaction time 2 h, yield 70%;
mp = 114 °C; ½a 2D0
ꢂ
¼ ꢀ9:4 (CHCl3); 1H NMR (CDCl3): d 0.86 (t, 6H,
J = 7.0 Hz, 2CH3), 1.18–1.32 (m, 32H, 16CH2), 1.58 (m, 4H, 2CH2),
0
0
2.30 (m, 4H, 2CH2), 2.43 (s, 3H, CH3), 3.29 (dd, 1H, J1 ,2 = 7.7 Hz,
J2 ,3 = 9.0 Hz, H-20), 3.42–3.53 (m, 3H, H-30, H-40, H-50), 4.02 (m,
0
0

5972
M. Dangate et al. / Bioorg. Med. Chem. 17 (2009) 5968–5973
1H, H-2), 4.05–4.13 (m, 2H, H-1a and H-3a), 4.19 (dd, 1H,
4.1.5.2. 1,3-Di-O-dodecanoyl-2-O-(6-deoxy-6-thioacetyl-b-D-
J1b,2 = 5.3 Hz, J1b,1a = 11.5 Hz, H-1b or H-3b), 4.22 (dd, 1H,
glucopyranosyl)-sn-glycerol (6b). Reaction time 24 h; yield 71%;
J6 a,5 = 4.0 Hz, J6 a,6 b = 10.9 Hz, H-60a), 4.27 (dd, 1H, J6 b,5 <1.0 Hz,
H-60b), 4.32 (d, 1H, H-10), 4.37 (dd, 1H, J3b,2 = 3.4 Hz, J3b,3a = 11.9 Hz,
H-3b or H-1b), 7.33 (d, 2H, J = 8.3 Hz, Ph), 7.77 (d, 2H, Ph); 13C NMR
(CDCl3): d 14.10 (2CH3), 21.65 (PhCH3), 22.67 (2CH2), 24.84 (2CH2),
28.77–29.85 (12CH2), 31.89 (2CH2), 34.04 and 34.18 (2CH2CO),
62.91 (C1 or C3), 63.00 (C1 or C3), 68.53 (C60), 69.25 (C40 or C50),
73.26 (C20), 73.65 (C40 or C50), 75.93 and 76.10 (C2 and C30),
103.11 (C10), 127.98 and 129.93 (4C, Ph), 132.70 and 145.05 (2C,
Ph), 173.44 (CO), 174.18 (CO). ESI-MS (CH3OH, positive-ion mode,
relative intensity): m/z = 795.4 [M+Na]+, 100%. Calcd for
C40H68O12S, m/z 772.4 [M].
brown amorphous solid; ½a D20
ꢂ
¼ ꢀ39:0 (CHCl3); 1H NMR (CDCl3): d
0
0
0
0
0
0
0.86 (t, 6H, J = 7.0 Hz, 2CH3), 1.18–1.33 (m, 32H, 16CH2), 1.59 (m,
4H, 2CH2), 2.31 (m, 4H, 2CH2), 2.37 (s, 3H, –SCOCH3), 3.16 (dd,
1H, J6 a,5 = 3.4 Hz, J6 a,6 b = 14.6 Hz, H-60a), 3.26 (dd, 1H,
0
0
0
0
J3 ,4 = 9.3 Hz, J4 ,5 = 9.3 Hz, H-40), 3.33 (dd, 1H, J1 ,2 = 7.8 Hz,
0
0
0
0
0
0
J2 ,3 = 9.3 Hz, H-20), 3.37 (dd, 1H, J6 b,5 = 4.4 Hz, H-60b), 3.47 (m,
1H, H-50), 3.54 (dd, 1H, H-30), 4.02 (m, 1H, H-2), 4.07–4.16 (m,
2H, H-1a and H-3a), 4.21 (dd, 1H, J1b,2 = 5.5 Hz, J1b,1a = 11.6 Hz, H-
1b or H-3b), 4.36 (d, 1H, H-10), 4.37 (dd, 1H, J3b,2 = 3.6 Hz,
J3b,3a = 11.9 Hz, H-3b or H-1b); 13C NMR (CDCl3): d 14.11 (2CH3),
22.67 (2CH2), 24.85 (2CH2), 29.00–30.00 (12CH2), 30.46 (SCOCH3),
30.63 (C60), 31.90 (2CH2), 34.12 and 34.17 (2CH2CO), 63.06 (C1 or
C3), 63.14 (C1 or C3), 71.16 (C40), 73.37 (C20), 74.05 (C50), 75.21
(C30), 76.07 (C2), 103.12 (C10), 173.40 (CO), 174.00 (CO), 198.52
(–SCO). ESI-MS (CH3OH, positive-ion mode, relative intensity): m/
z = 699.5 [M+Na]+, 100%. Calcd for C35H64O10S, m/z 676.4 [M].
0
0
0
0
4.1.4.3. 1,3-Di-O-hexanoyl-2-O-(6-deoxy-6-tosyl-b-D-glucopyr-
anosyl)-sn-glycerol (5c). Reaction time 2 h, yield 72%;
mp = 137 °C; ½a 2D0
ꢂ
¼ ꢀ12:5 (CHCl3); 1H NMR (CDCl3): d 0.86 (t,
6H, J = 7.0 Hz, 2CH3), 1.20–1.34 (m, 8H, 4CH2), 1.58 (m, 4H,
2CH2), 2.30 (m, 4H, 2CH2), 2.42 (s, 3H, CH3), 3.29 (dd, 1H,
J1 ,2 = 7.7 Hz, J2 ,3 = 9.0 Hz, H-20), 3.40–3.54 (m, 3H, H-30, H-40, H-
50), 4.02 (m, 1H, H-2), 4.06–4.14 (m, 2H, H-1a and H-3a), 4.19
(dd, 1H, J1b,2 = 5.6 Hz, J1b,1a = 11.6 Hz, H-1b or H-3b), 4.21 (dd, 1H,
4.1.5.3. 1,3-Di-O-hexanoyl-2-O-(6-deoxy-6-thioacetyl-b-D-glu-
0
0
0
0
copyranosyl)-sn-glycerol (6c). Reaction time 24 h; yield 69%;
brown oil; ½a 2D0
ꢂ
¼ ꢀ34:3 (CHCl3); 1H NMR (CDCl3): d 0.87 (t, 6H,
J6 a,5 = 4.6 Hz, J6 a,6 b = 10.7 Hz, H-60a), 4.26 (dd, 1H, J6 b,5 = 1.2 Hz,
H-60b), 4.36 (d, 1H, H-10), 4.35 (dd, 1H, J3b,2 = 3.7 Hz, J3b,3a = 11.9 Hz,
H-3b or H-1b), 7.33 (d, 2H, J = 8.3 Hz, Ph), 7.77 (d, 2H, Ph); 13C NMR
(CDCl3): d 13.87 (2CH3), 21.62 (PhCH3), 22.24 (CH2), 22.28 (CH2),
24.49 (2CH2), 31.20 (CH2), 31.23 (CH2), 33.98 and 34.11 (2CH2CO),
62.87 (C1 or C3), 63.01 (C1 or C3), 68.67 (C60), 69.28 (C40 or C50),
73.26 (C20), 73.62 (C40 or C50), 75.92 (C30 and C2), 102.97 (C10),
127.96 and 129.92 (4C, Ph), 132.64 and 145.02 (2C, Ph), 173.45
(CO), 174.15 (CO). ESI-MS (CH3OH, positive-ion mode, relative
intensity): m/z = 627.3 [M+Na]+, 100%. Calcd for C28H44O12S, m/z
604.3 [M].
J = 7.0 Hz, 2CH3), 1.22–1.35 (m, 8H, 4CH2), 1.60 (m, 4H, 2CH2), 2.31
0
0
0
0
0
0
0
0
(m, 4H, 2CH2), 2.36 (s, 3H, –SCOCH3), 3.19 (dd, 1H, J6 a,5 = 3.3 Hz,
J6 a,6 b = 14.5 Hz, H-60a), 3.26 (dd, 1H, J3 ,4 = 9.1 Hz, J4 ,5 = 9.2 Hz, H-
0
0
0
0
0
0
40), 3.33 (dd, 1H, J1 ,2 = 7.7 Hz, J2 ,3 = 9.2 Hz, H-20), 3.34 (dd, 1H,
0
0
0
0
J6 b,5 = 4.7 Hz, H-60b), 3.45 (m, 1H, H-50), 3.53 (dd, 1H, H-30), 4.02
(m, 1H, H-2), 4.08–4.16 (m, 2H, H-1a and H-3a), 4.22 (dd, 1H,
J1b,2 = 5.5 Hz, J1b,1a = 11.6 Hz, H-1b or H-3b), 4.35 (dd, 1H,
J3b,2 = 3.7 Hz, J3b,3a = 11.9 Hz, H-3b or H-1b), 4.36 (d, 1H, H-10); 13C
NMR (CDCl3): d 13.87 (CH3), 13.89 (CH3), 22.26 (CH2), 22.29 (CH2),
24.51 (2CH2), 30.45 (SCOCH3), 30.65 (C60), 31.22 (CH2), 31.27
(CH2), 34.07 and 34.11 (2CH2CO), 63.06 (C1 or C3), 63.15 (C1 or
C3), 71.30 (C40), 73.39 (C20), 74.10 (C50), 75.28 (C30), 76.00 (C2),
103.09 (C10), 173.42 (CO), 174.00 (CO), 198.35 (–SCO). ESI-MS
(CH3OH, positive-ion mode, relative intensity): m/z = 531.2
[M+Na]+, 100%. Calcd for C23H40O10S, m/z 508.2 [M].
0
0
4.1.5. General procedure for the synthesis of thioacetates 6a–c
Compound 5 (0.74 mmol) was dissolved in dry DMF (14 mL)
and potassium thioacetate (0.382 g, 3.35 mmol) was added. The
mixture was stirred under Ar at room temperature and the chang-
ing of its color from blue to brown indicated the progress of reac-
tion that was monitored by TLC (CH2Cl2:CH3OH, 97:3 v/v). Owing
to co-elution of the starting and target compound, the reaction
was stopped after 24 h when the TLC spot color turned com-
pletely from green to brown (anisaldehyde based reagent). DMF
was then co-evaporated with cyclohexane at reduced pressure
at 45 °C and the following flash column chromatography
(CH2Cl2:CH3OH, 97:3 v/v) of the crude residue yielded the desired
derivative 6.
4.1.6. General procedure for the synthesis of sulfonates 1a–c
To a solution of compound 6 (0.37 mmol) in glacial acetic acid
(18 mL), potassium monopersulfate triple salt (OXONEÒ) (0.68 g,
1.1 mmol) and potassium acetate (1.50 g, 15.3 mmol) were added
in the order. The suspension was stirred at room temperature
and the reaction was monitored by TLC (CHCl3:CH3OH:H2O,
65:25:4). After disappearing of the starting, the solvent was evap-
orated under vacuum, the residue diluted with water (36 mL) and
extracted with CHCl3:CH3OH, (80:20 v/v) (5 ꢁ 36 mL). The com-
bined organic layers were washed with NaCl solution
(2 ꢁ 70 mL). The water layers were then extracted several times
following the same procedure checking by TLC the presence of
residual target compound. All collected organic layers were re-
duced to a convenient volume and submitted to centrifugation at
4500 rpm for 10 min. Water layer was gently separated by sharp
dropper, and the organic phase concentrated under reduced pres-
sure. The obtained crude was purified by flash column chromatog-
raphy (from CHCl3:CH3OH, 80:20 v/v to CHCl3:CH3OH:H2O,
65:25:4 v/v) to yield the pure sulfonate 1.
4.1.5.1. 1,3-Di-O-octadecanoyl-2-O-(6-deoxy-6-thioacetyl-b-D-
glucopyranosyl)-sn-glycerol (6a). Reaction time 24 h; yield 70%;
brown amorphous solid; ½a D20
ꢂ
¼ ꢀ27:5 (CHCl3); 1H NMR (CDCl3): d
0.86 (t, 6H, J = 7.0 Hz, 2CH3), 1.18–1.33 (m, 56H, 28CH2), 1.59 (m,
4H, 2CH2), 2.31 (m, 4H, 2CH2), 2.37 (s, 3H, –SCOCH3), 3.17 (dd,
1H, J6 a,5 = 3.4 Hz, J6 a,6 b = 14.5 Hz, H-60a), 3.26 (dd, 1H,
0
0
0
0
J3 ,4 = 9.0 Hz, J4 ,5 = 9.0 Hz, H-40), 3.33 (dd, 1H, J1 ,2 = 7.6 Hz,
0
0
0
0
0
0
J2 ,3 = 9.0 Hz, H-20), 3.37 (dd, 1H, J6 b,5 = 4.4 Hz, H-60b), 3.47 (m,
1H, H-50), 3.54 (dd, 1H, H-30), 4.02 (m, 1H, H-2), 4.07–4.17 (m,
2H, H-1a and H-3a), 4.21 (dd, 1H, J1b,2 = 5.5 Hz, J1b,1a = 11.6 Hz, H-
1b or H-3b), 4.36 (m, 2H, H-10 and H-3b or H-1b); 13C NMR (CDCl3):
d 14.11 (2CH3), 22.68 (2CH2), 24.85 (2CH2), 29.00–30.00 (24CH2),
30.50 (SCOCH3), 30.62 (C60), 31.92 (2CH2), 34.12 and 34.17
(2CH2CO), 63.07 (C1 or C3), 63.14 (C1 or C3), 71.14 (C40), 73.38
(C20), 74.06 (C50), 75.21 (C30), 76.09 (C2), 103.15 (C10), 173.40
(CO), 174.00 (CO), 198.56 (–SCO). ESI-MS (CH3OH, positive-ion
mode, relative intensity): m/z = 867.6 [M+Na]+, 100%. Calcd for
C47H88O10S, m/z 844.6 [M].
0
0
0
0
4.1.6.1. 1,3-Di-O-octadecanoyl-2-O-b-D-sulfoquinovopyranosyl-
sn-glycerol sodium salt (1a). Reaction time 6 h, yield 40%; white
amorphous solid; ½a D20
ꢂ
¼ þ1:2 (CHCl3:CH3OH:H2O, 65:25:4). 1H
NMR (CDCl3:CD3OD:D2O, 65:25:4, 312 K): d 0.84 (t, 6H, J = 7.0 Hz,
2CH3), 1.15–1.32 (m, 56H, 28CH2), 1.56 (m, 4H, 2CH2), 2.28 (m,
4H, 2CH2), 3.03 (dd, 1H, J6 a,5 = 7.0 Hz, J6 a,6 b = 14.5 Hz, H-60a),
0
0
0
0
3.18–3.25 (m, 2H, H-20 and H-40), 3.28 (dd, 1H, J6 b,5 = 3.5 Hz, H-
0
0
60b), 3.39 (dd, 1H, J2 ,3 = 9.1 Hz, J3 ,4 = 9.1 Hz, H-30), 3.70 (m, 1H,
0
0
0
0

M. Dangate et al. / Bioorg. Med. Chem. 17 (2009) 5968–5973
5973
H-50), 4.13–4.31 (m, 5H, 2H-1, 2H-3 and H-2), 4.41 (d, 1H,
37 °C for 48 h in 1 mL of a medium containing n-butyric acid
(4 mM), 32 pmol of TPA in DMSO, and a known amount of the test
compound in DMSO. The cells were stained by high titer EBV-posi-
tive sera from nasopharyngeal carcinoma patients and fluorescein-
isothiocyanate-labeled anti-human IgG. After staining, they were
detected by a conventional indirect immunofluorescence tech-
nique. The assays were performed in triplicate for each compound
in which at least 500 cells were counted. The average EBV-EA
inhibitory activity of the test compounds was compared to that
of control experiments (100%) with butyric acid (4 mM) and TPA
(32 pM) in which EBV-EA induction was typically around 30%.
The viability of the cells was assayed against treated cells using
the Trypan Blue staining method. For an accurate determination
of cytotoxicity, the cell viability was required to be more than
60% 2 days after treatment with the compounds.
J1 ,2 = 7.8 Hz, H-10); 13C NMR (CDCl3:CD3OD: D2O 65:25:4, 312 K):
d 14.26 (2CH3), 23.00 (2CH2), 25.22 (CH2), 25.24 (CH2), 29.40–
30.20 (24CH2), 32.28 (2CH2), 34.47 and 34.50 (2CH2CO), 53.85
(C60), 62.96 (C1 or C3), 63.94 (C1 or C3), 72.75 (C50), 73.72 (C20 or
C40), 73.84 (C20 or C40), 75.32 (C2), 76.43 (C30), 103.15 (C10),
174.74 (CO), 174.86 (CO). ESI-MS (CH3OH, negative-ion mode, rel-
ative intensity): m/z = 849.9 [M]ꢀ, 100%. Calcd for C45H85O12Sꢀ, m/z
849.6 [M]ꢀ.
0
0
4.1.6.2. 1,3-Di-O-dodecanoyl-2-O-b-D-sulfoquinovopyranosyl-
sn-glycerol sodium salt (1b). Reaction time 7 h, yield 42%; white
amorphous solid; ½a D20
ꢂ
¼ þ1:3 (CHCl3:CH3OH:H2O, 65:25:4). 1H
NMR (CDCl3:CD3OD:D2O 65:25:4, 312 K): d 0.84 (t, 6H, J = 7.0 Hz,
2CH3), 1.16–1.31 (m, 32H, 16CH2), 1.56 (m, 4H, 2CH2), 2.29 (m,
4H, 2CH2), 3.03 (dd, 1H, J6 a,5 = 7.0 Hz, J6 a,6 b = 14.5 Hz, H-60a),
0
0
0
0
3.18–3.25 (m, 2H, H-20 and H-40), 3.27 (dd, 1H, J6 b,5 = 3.2 Hz, H-
0
0
Acknowledgements
60b), 3.39 (dd, 1H, J2 ,3 = 9.1 Hz, J3 ,4 = 9.1 Hz, H-30), 3.69 (m, 1H,
0
0
0
0
H-50), 4.13–4.31 (m, 5H, 2H-1, 2H-3 and H-2), 4.41 (d, 1H,
This study was supported in part by the University of Milan
(Italy) and in part by Grants-in-Aid from the Ministry of Education,
Science and Culture and the Ministry of Health and Welfare
(Japan). The authors thank Professor Lucio Toma for helpful
discussion.
J1 ,2 = 7.8 Hz, H-10); 13C NMR (CDCl3:CD3OD: D2O 65:25:4, 312 K):
d 14.33 (2CH3), 23.04 (2CH2), 25.23 (2CH2), 29.20–30.20 (12CH2),
32.29 (2CH2), 34.44 and 34.49 (2CH2CO), 53.59 (C60), 62.86 (C1 or
C3), 63.90 (C1 or C3), 72.61 (C50), 73.47 (C20 or C40), 73.73 (C20 or
C40), 75.36 (C2), 76.24 (C30), 103.18 (C10), 174.82 (CO), 174.90
(CO). ESI-MS (CH3OH, negative-ion mode, relative intensity): m/
z = 681.6 [M]ꢀ, 100%. Calcd for C33H61O12Sꢀ, m/z 681.4 [M]ꢀ.
0
0
Supplementary data
Copies of 1H NMR and 13C NMR spectra of the target compounds
1a–1c and 4a–4c are available. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.06.064.
4.1.6.3. 1,3-Di-O-hexanoyl-2-O-b-D-sulfoquinovopyranosyl-sn-
glycerol sodium salt (1c). Reaction time 6 h, yield 38%; white
amorphous solid; ½a D20
ꢂ
¼ þ3:7 (CHCl3:CH3OH:H2O, 65:25:4). 1H
NMR (CDCl3:CD3OD:D2O 65:25:4, 312 K): d 0.85 (t, 6H, J = 7.0 Hz,
2CH3), 1.21–1.32 (m, 8H, 4CH2), 1.57 (m, 4H, 2CH2), 2.29 (m, 4H,
2CH2), 3.03 (dd, 1H, J6 a,5 = 7.1 Hz, J6 a,6 b = 14.5 Hz, H-60a), 3.18–
0
0
0
0
References and notes
3.25 (m, 2H, H-20 and H-40), 3.28 (dd, 1H, J6 b,5 = 3.5 Hz, H-60b),
0
0
3.39 (dd, 1H, J2 ,3 = 9.1 Hz, J3 ,4 = 9.1 Hz, H-30), 3.70 (m, 1H, H-50),
0
0
0
0
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0
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4.13–4.30 (m, 5H, 2H-1, 2H-3 and H-2), 4.41 (d, 1H, J1 ,2 = 7.8 Hz,
H-10); 13C NMR (CDCl3:CD3OD:D2O 65:25:4, 312 K):
d
13.98
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4.2.1. Short-term in vitro bioassay for anti-tumor promoters
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activation in Raji cells (obtained first from Professor G. Klein, Kar-
olinska Institute, Stockholm, Sweden) cultivated in RPMI 1640
medium containing 10% fetal calf serum, and induced by TPA as de-
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