Cyclic and branched acyl chain galactoglycerolipids and their effect on anti-tumor-promoting activity

Article abstract of DOI:10.1016/j.ejmech.2006.07.007

Fifteen new galactoglycerolipid analogues, in which one or two branched, alicyclic or aromatic acyl chains are linked to 2-O-β-d-galactosylglycerol (6′-position or 1,6′ positions), were prepared and tested for their anti-tumor-promoting activity using a s

Full text of DOI:10.1016/j.ejmech.2006.07.007

European Journal of Medicinal Chemistry 41 (2006) 1456e1463
http://www.elsevier.com/locate/ejmech
Original article
Cyclic and branched acyl chain galactoglycerolipids and
their effect on anti-tumor-promoting activity
a
b
a
d
Diego Colombo a, , Laura Franchini , Lucio Toma , Fiamma Ronchetti , Reiko Tanaka ,
*
Junko Takayasu c, Hoyoku Nishino c, Harukuni Tokuda c
a Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita` di Milano, Via Saldini 50, 20133 Milano, MI, Italy
b Dipartimento di Chimica Organica, Universita` di Pavia, Via Taramelli 10, 27100 Pavia, Italy
c Department of Molecular Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan
d Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan
Received 27 January 2006; received in revised form 18 July 2006; accepted 24 July 2006
Available online 22 September 2006
Abstract
Fifteen new galactoglycerolipid analogues, in which one or two branched, alicyclic or aromatic acyl chains are linked to 2-O-b-D-galacto-
sylglycerol (60-position or 1,60 positions), were prepared and tested for their anti-tumor-promoting activity using a short-term in vitro assay for
EpsteineBarr virus early antigen (EBV-EA) activation. All compounds were active in inhibiting the EBV activation promoted by the tumor pro-
moter 12-O-tetradecanoylphorbol-13-acetate (TPA), the branched compounds resulting in the most active glycoglycerolipid analogues of the
series. The branched 2-O-[6-O-(3-methylbutanoyl)-b-D-galactopyranosyl]-sn-glycerol (1a) and the structurally related alicyclic 2-O-[6-O-(2-cy-
clohexylethanoyl)-b-D-galactopyranosyl]-sn-glycerol (1d), when tested in an in vivo two-stage carcinogenesis test, exhibited inhibitory effects
on mouse skin tumor promotion.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Glycoglycerolipids; Cancer chemoprevention; EpsteineBarr virus
1. Introduction
and also to show in vitro anti-solid tumor activity [4]. Dur-
ing our search for new glycoglycerolipids active in cancer
chemoprevention we have found that apparently minor
structural changes of the lipophilic chain exert a positive ef-
fect on the anti-tumor-promoting activity of monoacyl- or
diacylderivatives of 2-O-galactosylglycerol [5]. Therefore
we have decided to synthesize a series of new analogues
carrying different, but structurally related, acyl chains, fo-
cusing our attention on branched, alicyclic and aromatic
structures. Here we describe the synthesis of galactoglycer-
olipid analogues 1aej and 2a, 2cef and their anti-tumor-
promoting activity based on the short-term in vitro assay
for the inhibition of EpsteineBarr virus early antigen
(EBV-EA) activation induced by TPA. The inhibitory effect
of compounds 1a and 1d on mouse skin tumor promotion in
an in vivo two-stage mouse skin carcinogenesis test will
also be discussed.
Cancer chemoprevention consists of administration of
synthetic or natural compounds to halt or inhibit the onset
of cancer and is based on the conventional multistage carci-
nogenesis model. According to this model, tumor promotion
is a long and reversible stage that can be efficiently sup-
pressed [1]. Besides their structural and functional roles in
the membranes of animals, plants and bacteria [2], glycogly-
cerolipids are known to inhibit the tumor-promoting activity
of the potent tumor promoter 12-O-tetradecanoylphorbol-13-
acetate (TPA) both in vitro and in vivo, their potential for
cancer chemoprevention being considered conspicuous [3],
* Corresponding author. Tel.: þ39 02 5031 6039; fax: þ39 02 5031 6036.
E-mail address: diego.colombo@unimi.it (D. Colombo).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.07.007

D. Colombo et al. / European Journal of Medicinal Chemistry 41 (2006) 1456e1463
1457
2. Results and discussion
effect of branched (1aec), alicyclic (1def) and aromatic
(1gej) monoesters and of diesters 2a, 2cef was 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.
[8,9]. Mouse skin tumor promotion inhibition activity of com-
pounds 1a and 1d was also evaluated in an in vivo two-stage
mouse skin carcinogenesis test as reported in Section 3.
Table 1 shows the in vitro tumor inhibitory activity of com-
pounds 1aej and 2a, 2cef together with that of the previously
studied [5] esters 1kem and 2kem for a comparison. Only
weak cytotoxicity against Raji cells was observed for all com-
pounds (70% viability at 1000 mol ratio/TPA and >80% at all
the other mol ratios/TPA, Table 1) that showed potent inhibi-
tory activity, as indicated by their percentage to control
(8.0e31.5% at 500 mol ratio/TPA, Table 1). Generally, the
branched analogues 1aec and 2a,c resulted in the most active
of the tested compounds, IC50 ranging from 21.5 to 30.6 (Table
1), with both the alicyclic 1def, 2def and the aromatic deriv-
atives 1gej being significantly less active (IC50 from 186 to
230, Table 1). In particular, a dramatic drop of activity could
be observed both for adding an aliphatic or an aromatic ring
to the acyl chain, e.g. 1k vs 1j (IC50 62.1 and 205, respec-
tively), and for cyclizing branched chains, e.g. 1a vs 1d or
1h (IC50 27.8, 205 or 223, respectively), 2a vs 2d (IC50 21.5
and 133, respectively), 1c vs 1g (IC50 30.6 and 230, respec-
tively), 1m vs 1e or 1f (IC50 26.5, 217 or 186, respectively)
and 2m vs 2e or 2f (IC50 19.4, 126 or 116, respectively). On
the contrary no significant effect was observed when alicyclics
2.1. Chemistry
Some of the desired compounds were prepared according to
a well established chemo-enzymatic approach starting from 1,3-
di-O-benzyl-2-O-b-D-(galactopyranosyl)-glycerol (3) or 2-O-b-
D-galactopyranosylglycerol (7) [6]. In particular, Pseudomonas
cepacia lipase (lipase PS) catalyzed transesterification of 3
yielded 45e90% of compounds 4a, 4cef after 20e180 h de-
pending on the acyl donor (see Section 3). Catalytic hydrogena-
tion afforded the 60-monoesters 1a, 1cef. The same enzymatic
procedure on 7 yielded, with lower yields (6e27%) and longer
reactions times (20e330 h), the 1,60-diesters 2a, 2cef (for the
assignment of the C-2 configuration see Section 3). Enzymatic
reactions were very slow with the cyclic acyl donors and did
not work in the case of the bulky 3,3-dimethyl-butanoyl acyl
chain b, even if conducted with a different enzyme (Candida
antarctica lipase) and solvents both on substrates 3 and 7.
Hence, only the monoesters 1b and 1gej (and not the corre-
sponding diesters 2b, 2gej) were prepared by the treatment of
the known 1,3-di-O-benzyl-2-O-(2,3,4-tri-O-benzyl-b-D-galac-
topyranosyl)-glycerol (5) [7] with the proper carboxylic acid to
give the intermediates 6b and 6gej followed by catalytic hydro-
genation.
1 : R=Acyl, R'=R''=R'''=H
OR
OR'''
2 : R=R'=Acyl, R''=R'''=H
3 : R'=R''=Bn, R=R'''=H
4 : R'=R''=Bn, R'''=H, R=Acyl
5 : R'=R''=R'''=Bn, R=H
6 : R'=R''=R'''=Bn, R=Acyl
7 : R=R'=R''=R'''=H
O
OR'
Table 1
Inhibitory effects of 1aej and 2a, 2cef on TPA-induced EBV-EA activation
R'''O
H
O
OR'''
OR''
Concentration (mol ratio/TPA)
IC50
1000 500
100
10
Acyl
% To control ꢀ SE (n ¼ 3)a
O
O
O
O
O
1a
1b
1c
0 ꢀ 0.4 (70)
0 ꢀ 0.3 (70)
0 ꢀ 0.4 (70)
0 ꢀ 0.6 (70)
0 ꢀ 0.8 (70)
0 ꢀ 0.6 (70)
0 ꢀ 0.6 (70)
0 ꢀ 0.3 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.5 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.2 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.0 (70)
0 ꢀ 0.2 (70)
9.9 ꢀ 0.8
29.6 ꢀ 1.0
28.4 ꢀ 1.1
31.4 ꢀ 1.3
72.4 ꢀ 2.2
73.9 ꢀ 2.1
70.2 ꢀ 1.9
75.7 ꢀ 0.7
74.6 ꢀ 1.3
73.1 ꢀ 0.5
71.6 ꢀ 1.0
43.6 ꢀ 1.0
30.1 ꢀ 0.9
28.9 ꢀ 1.5
25.5 ꢀ 1.2
29.7 ꢀ 1.4
54.2 ꢀ 1.8
52.8 ꢀ 1.9
51.6 ꢀ 1.3
47.6 ꢀ 1.2
32.3 ꢀ 1.1
23.5 ꢀ 1.1
67.7 ꢀ 1.5
66.3 ꢀ 1.4
68.9 ꢀ 1.4
95.3 ꢀ 0.6
96.4 ꢀ 0.7
94.3 ꢀ 0.5
96.3 ꢀ 1.7
95.9 ꢀ 0.2
94.0 ꢀ 0.8
92.8 ꢀ 1.6
78.2 ꢀ 1.1
67.8 ꢀ 1.2
67.0 ꢀ 2.1
63.6 ꢀ 2.0
70.1 ꢀ 1.9
94.9 ꢀ 0.5
94.6 ꢀ 0.3
93.3 ꢀ 0.4
80.5 ꢀ 1.3
69.5 ꢀ 1.8
62.0 ꢀ 2.0
27.8
9.2 ꢀ 0.9
25.6
30.6
a
e
b
c
d
O
11.5 ꢀ 0.8
28.3 ꢀ 1.3
29.6 ꢀ 1.4
24.9 ꢀ 1.4
30.9 ꢀ 1.4
30.5 ꢀ 1.1
30.1 ꢀ 0.1
30.0 ꢀ 0.5
20.4 ꢀ 0.8
10.7 ꢀ 0.1
9.2 ꢀ 0.9
O
1d
1e
205
217
186
230
223
213
205
1f
f
i
l
g
1g
1h
1i
O
O
O
O
1j
1kb
1lb
1mb
2a
2c
62.1
h
k
j
28.3
26.5
21.5
30.1
O
O
8.0 ꢀ 0.9
8.6 ꢀ 1.0
m
2d
2e
31.5 ꢀ 1.8
30.4 ꢀ 1.7
27.3 ꢀ 1.8
21.1 ꢀ 0.4
12.4 ꢀ 0.2
7.9 ꢀ 0.6
133
126
116
2f
2kb
2lb
2mb
a
75.2
32.2
19.4
2.2. Biological evaluation
Values are EBV-EA activation (%) in the presence of the test compound
relative to the control (100%). Activation was attained by treatment with
TPA 32 pmol. IC50 represents the mol ratio to TPA that inhibits 50% of pos-
itive control (100%) activated with 32 pmol TPA.
EpsteineBarr virus (EBV) is known to be activated by tu-
mor promoters to produce viral early antigens (EA), and an
evaluation of its inhibition is often used as a primary screening
for in vitro anti-tumor-promoting activities [1]. The inhibitory
b
See Ref. [5].

1458
D. Colombo et al. / European Journal of Medicinal Chemistry 41 (2006) 1456e1463
were substituted with aromatic rings, e.g. 1d vs 1h (IC50 205
and 223, respectively, Table 1) and 1e vs 1i (IC50 217 and
213, respectively, Table 1). Also, differently from literature
data for the gluco and galactoglycerolipids with linear acyl
chains [10,11], all the tested cyclic chain compounds were
almost equiactive regardless of their chain length (see Table 1
1gej and 1d,e). Concerning branching linear chains, this struc-
tural modification always afforded high activity of glycoglycer-
olipid analogues and the activity enhancement was especially
marked, as already observed [5], for the diester series (the in-
hibitory effects of 2a and 2c were, respectively, 71% and
60% higher than 2k, and 2m and 2c were, respectively, 40%
and 6% more active than 2l, see IC50 values, Table 1). In con-
clusion, the diester 2a is, together with 2m [5], the most potent
glycoglycerolipid analogue synthesized to date, for the inhibi-
tion of TPA promoted EBV-EA activation.
A
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
Weeks of promotion
B
10
8
For in vivo experiments monoesters 1a and 1d were chosen.
They are representative members of the branched and cyclic
series, respectively, and their structure is strictly related, the
cyclohexylacetyl chain in 1d resulting from the elongation
and cyclization of the isopropylacetyl chain of 1a. The diesters
2a and 2d, though appearing more active than the corresponding
monoesters in the in vitro experiment, were not tested in vivo
because 1-O-acyl and 1,60-di-O-acylgalactosylglycerols are
known to exhibit almost the same activity when assayed in
the in vivo two-stage mouse skin carcinogenesis test [5]. Thus
1a and 1d were submitted to an invivo two-stage carcinogenesis
test of mouse skin papillomas using DMBA as an initiator and
TPA as a promoter, in order to relate the inhibitory effect to their
different structures. After 20 weeks of promotion, there was no
statistically significant difference in body weights between con-
trol and any treated group. The activities, estimated by both the
incidence (percentage of mice bearing papillomas, Fig. 1A) and
the multiplicity (average number of papillomas per mouse,
Fig. 1B), were compared with those of the control group. In
the control group 100% of the mice bore papillomas at 10 weeks
of promotion (Fig. 1A), and 5 and 9 papillomas per mouse were
formed, respectively, after 10 and 20 weeks of promotion
(Fig. 1B). Both the tested compounds were able to inhibit the
tumor promotion in this in vivo assay, reducing the percentage
of mice bearing papillomas. In fact about 13% and 27% of mice
bore papillomas, respectively, in the 1a and 1d treated groups at
10 weeks of promotion, and 80% and 93% of mice bearing pap-
illomas resulted at 20 weeks of promotion (Fig. 1A). The differ-
ences in the incidence of papillomas in the treated cases with
respect to control, analyzed by the c2-test, appear to be signif-
icant after 10 weeks both for 1a (P < 0.005) and 1d (P < 0.005)
but not after 20 weeks. The decrease of the number of papillo-
mas per mouse (papilloma multiplicity) was statistically signif-
icant (P < 0.001, Student’s t-test for 1a and 1d) with respect to
the control, however, the differences among the two groups
were not significant in the lowered number of papillomas per
mouse, even if 1a is significantly more active than 1d in vitro
(Table 1). In fact 1.1 and 1.2 papillomas (20% and 22% with re-
spect to control) were formed by treatment, respectively, with
1a and 1d at 10 weeks of promotion, and 4.4 and 4.8 (47%
and 52% with respect to control) at 20 weeks of promotion
6
4
2
0
0
5
10
15
20
Weeks of promotion
Fig. 1. Inhibitory effects of compounds 1a and 1d (85 nmol) on DMBAeTPA
mouse skin carcinogenesis. All mice were initiated with DMBA (390 nmol)
and promoted with TPA (1.7 nmol) twice a week starting one week after ini-
tiation. A: percentage of mice with papillomas; B: averaged number of papil-
lomas per mouse (C, TPA alone; -, TPA þ 1a; , TPA þ 1d). At 20 weeks
A
of promotion the averaged number of papillomas per mouse was reduced, with
respect to the control group (9.3 ꢀ 1.3), to 4.4 ꢀ 0.6 for 1a (P < 0.001) and to
4.8 ꢀ 0.8 for 1d (P < 0.001).
(Fig. 1B). Discrepancy in the structureeactivity results between
in vivo and in vitro experiments was already observed for gly-
coglycerolipid analogues and might be due to different lipophi-
licities of the tested compounds [5].
In conclusion, 15 new galactoglycerolipid analogues have
been prepared which were active as anti-tumor-promoting
compounds. Structureeactivity relationship showed that while
branching of the acyl chains enhances the in vitro anti-
tumor-promoting activity, the presence of an aliphatic or aro-
matic ring exerts a negative effect. In particular the branched
analogues are very potent, the diester 2a being one of the
most active glycoglycerolipids discovered by our in vitro assay.
3. Experimental protocols
3.1. Chemistry
3.1.1. Materials
P. cepacia lipase (LPS, specific activity: 30.5 triacetin units/
mg solid), a generous gift from Amano Pharmaceutical Co

D. Colombo et al. / European Journal of Medicinal Chemistry 41 (2006) 1456e1463
1459
20
(Mitsubishi Italia), was supported on celite [12] and kept over-
night, under vacuum, prior to use. Pyridine was distilled from
calcium hydride. The trifluoroethyl esters were synthesized by
reacting the proper acid with trifluoroethanol and dicyclohexyl-
carbodiimide, and the vinyl esters from the acid and vinyl
neodecanoate (Aldrich) according to a literature procedure
[13]. 1,3-Di-O-benzyl-2-O-b-D-(galactopyranosyl)-glycerol (3),
1,3-di-O-benzyl-2-O-(2,3,4-tri-O-benzyl-b-D-galactopyranosyl)-
glycerol (5) and 2-O-b-D-galactopyranosylglycerol (7) were
synthesized according to the literature procedures [14,7].
Evaporation under reduced pressure was always effected with
a bath temperature below 40 ꢁC. All the new compounds were
characterized by 1H NMR analysis at 500 MHz with a Bruker
FT-NMR AVANCEÔ DRX500 spectrometer, and chemical
ionization mass spectrometry (CI-MS) [15] using a Thermo
Electron TRACE DSQÔ spectrometer through the rapid heating
filament direct-exposure probe (DEP) insertion mode. 13C NMR
spectra at 125.76 MHz were recorded for all the target com-
pounds, using 2D HSQC experiment (standard Bruker pulse
program) for the full assignment of the carbon resonances.
Chemical shifts are reported as d (ppm) relative to CHCl3,
CH3OH or pyridine, fixed at 7.24, 3.30 or 7.19 ppm, respectively,
for 1H NMR spectra and relative to CD3OD fixed at 49.00 ppm
(central line) for 13C NMR spectra. The elemental analyses of all
the new compounds were obtained with a PerkineElmer 2400
analyzer. Optical rotations were determined on a PerkineElmer
241 polarimeter in methanol or chloroform solutions (c ¼ 1.0)
75%; oil; ½aꢂD ¼ þ10:6 (CHCl3). 1H NMR (CDCl3): d 0.92e
0.96 (m, 6H, 2CH3), 1.43e1.53 (m, 4H, 2CH2), 2.10 (m, 1H,
COCH), 3.52e3.70 (m, 7H, H-30, H-50, 2H-3, 2H-1 and H20),
3.84 (d, 1H, J ¼ 3.0 Hz, H-40), 4.06 (m, 1H, H-2), 4.24 (dd,
1H, J6 a,5 ¼ 7.0 Hz, J6 a,6 b ¼ 11.0 Hz, H-60a), 4.36 (dd, 1H,
0
0
0
0
0
0
0
0
0
0
J6 b,5 ¼ 6.0 Hz, H-6 b), 4.39 (d, 1H, J1 ,2 ¼ 7.7 Hz, H-1 ),
4.46e4.56 (m, 4H, 2CH2Ph), 7.22e7.35 (m, 10H, Ph). CI-
MS m/z 550 [M þ NH4]þ. C29H40O9 calcd. C 64.40, H 7.57;
found C 64.30, H 7.49.
3.1.2.1.3. 1,3-Di-O-benzyl-2-O-[6-O-(2-cyclohexylethanoyl)-
b-D-galactopyranosyl]-sn-glycerol (4d). Reaction time 180 h,
20
1
yield 71%; oil; ½aꢂD ¼ þ9:6 (CHCl3). H NMR (CDCl3):
d 0.83e1.70 (m, 10H, 5CH2), 1.74 (m, 1H, CH), 2.16 (d,
2H, J ¼ 6.5 Hz, COCH2), 3.48e3.72 (m, 7H, H-30, H-50, 2H-
3, 2H-1 and H-20), 3.78 (d, 1H, J ¼ 3.0 Hz, H-40), 4.06 (m,
0
0
0
0
1H, H-2), 4.23 (dd, 1H, J6 a,5 ¼ 7.0 Hz, J6 a,6 b ¼ 11.2 Hz, H-
60a), 4.29 (dd, 1H, J6 b,5 ¼ 5.6 Hz, H-6 b), 4.37 (d, 1H,
0
0
0
0
0
0
J1 ,2 ¼ 7.7 Hz, H-1 ), 4.46e4.56 (m, 4H, 2CH2Ph), 7.23e7.36
(m, 10H, Ph). CI-MS m/z 576 [M þ NH4]þ. C31H42O9 calcd.
C 66.65, H 7.58; found C 66.50, H 7.64.
3.1.2.1.4. 1,3-Di-O-benzyl-2-O-[6-O-(3-cyclohexylpropa-
noyl)-b-D-galactopyranosyl]-sn-glycerol (4e). Reaction time
20
140 h, yield 83%; oil; ½aꢂD ¼ þ8:3 (CHCl3). 1H NMR
(CDCl3): d 0.80e1.74 (m, 13H, CH and 6CH2), 2.28 (m,
2H, COCH2), 3.52e3.70 (m, 7H, H-30, H-50, 2H-3, 2H-1
and H-20), 3.83 (d, 1H, J ¼ 3.0 Hz, H-40), 4.08 (m, 1H, H-
2), 4.23 (dd, 1H, J6 a,5 ¼ 6.3 Hz, J6 a,6 b ¼ 11.2 Hz, H-60a),
0
0
0
0
in a 1 dm cell at 20 ꢁC. Melting points were recorded on a Buchi
¨
4.32 (dd, 1H, J6 b,5 ¼ 6.3 Hz, H-60b), 4.39 (d, 1H,
0
0
0
0
0
510 capillary melting point apparatus and were uncorrected.
J1 ,2 ¼ 7.7 Hz, H-1 ), 4.46e4.56 (m, 4H, 2CH2Ph), 7.23e
7.36 (m, 10H, Ph). CI-MS m/z 590 [M þ NH4]þ. C32H44O9
calcd. C 67.11, H 7.74; found C 67.30, H 7.69.
3.1.2. General procedure for the synthesis of monoesters
1a, 1cef
3.1.2.1.5. 1,3-Di-O-benzyl-2-O-[6-O-(3-cyclopentylpropa-
noyl)-b-D-galactopyranosyl]-sn-glycerol (4f). Reaction time
20
3.1.2.1.
1,3-Di-O-benzyl-2-O-(6-O-acyl-b-D-galactopyrano-
90 h, yield 85%; oil; ½aꢂD ¼ þ7:6 (CHCl3). 1H NMR
syl)-sn-glycerols (4a, 4cef). 1,3-Di-O-benzyl-2-O-b-D-(galac-
topyranosyl)-glycerol (3) (0.255 g, 0.59 mmol) was dissolved in
dichloromethane (4.0 mL). The appropriate trifluoroethyl (4de
f) or vinyl (4a,c) ester (1.77 mmol) was added followed by LPS
(0.75 g) and the suspension was stirred at 45 ꢁC. The reaction
was monitored by TLC (CH2Cl2eMeOH 90:10 v/v) and
stopped after 20e180 h by filtering off the enzyme which was
washed with dichloromethane. The solvent was removed under
reduced pressure and the crude residue was submitted to silica
gel flash chromatography (CH2Cl2eMeOH from 98:2 to 95:5
v/v). In this way 60-O-acylderivatives were obtained.
(CDCl3): d 1.00e1.80 (m, 11H, CH and 5CH2), 2.30 (m,
2H, COCH2), 3.50e3.70 (m, 7H, H-30, H-50, 2H-3, 2H-1
and H-20), 3.81 (d, 1H, J ¼ 3.0 Hz, H-40), 4.07 (m, 1H, H-
2), 4.23 (dd, 1H, J6 a,5 ¼ 6.3 Hz, J6 a,6 b ¼ 11.2 Hz, H-60a),
0
0
0
0
4.30 (dd, 1H, J6 b,5 ¼ 6.3 Hz, H-60b), 4.38 (d, 1H,
0
0
0
0
0
J1 ,2 ¼ 7.7 Hz, H-1 ), 4.46e4.56 (m, 4H, 2CH2Ph), 7.23e
7.36 (m, 10H, Ph). CI-MS m/z 576 [M þ NH4]þ. C31H42O9
calcd. C 66.65, H 7.58; found C 66.49, H 7.68.
3.1.2.2.
2-O-(6-O-Acyl-b-D-galactopyranosyl)-sn-glycerols
(1a, 1cef). Compound 4 (0.40 mmol) was dissolved in meth-
anol (20 mL) and 10% palladium on activated carbon
(0.075 g) was added. The reaction mixture, monitored by
TLC (CH2Cl2eMeOH 80:20), was shaken under hydrogen at-
mosphere for 2e3 h, and then filtered through a celite bed af-
fording the debenzylated compounds.
3.1.2.1.1. 1,3-Di-O-benzyl-2-O-[6-O-(3-methylbutanoyl)-b-
D-galactopyranosyl]-sn-glycerol (4a). Reaction time 20 h,
20
1
yield 90%; oil; ½aꢂD ¼ þ11:4 (CHCl3). H NMR (CDCl3):
d 0.91 (d, 6H, J ¼ 6.5 Hz, 2CH3), 2.06 (m, 1H, CH), 2.16
(d, 2H, J ¼ 6.5 Hz, COCH2), 3.44e3.72 (m, 7H, H-30, H-50,
2H-3, 2H-1 and H-20), 3.75 (m, 1H, J ¼ 3.0 Hz, H-40), 4.05
(m, 1H, H-2), 4.260 (m, 2H, H-60a and H-60b), 4.35 (d, 1H,
3.1.2.2.1. 2-O-[6-O-(3-Methylbutanoyl)-b-D-galactopyrano-
20
0
0
syl]-sn-glycerol (1a). Yield 92%; oil; ½aꢂD ¼ þ9:3 (CH3OH).
J1 ,2 ¼ 7.7 Hz, H-1 ), 4.44e4.54 (m, 4H, 2CH2Ph), 7.22e
7.35 (m, 10H, Ph). CI-MS m/z 536 [M þ NH4]þ. C28H38O9
calcd. C 64.85, H 7.39; found C 64.72, H 7.33.
1H NMR (CD3OD): d 0.88 (d, 6H, J ¼ 6.5 Hz, 2CH3), 2.08 (m,
1H, CH), 2.22 (d, 2H, J ¼ 6.5 Hz, COCH2), 3.50 (dd, 1H,
J3 ,2 ¼ 10.0 Hz, J030,4 ¼ 3.0 Hz, H-30), 3.56 (dd, 1H,
0
0
0
3.1.2.1.2. 1,3-Di-O-benzyl-2-O-[6-O-(2-ethylbutanoyl)-b-D-
galactopyranosyl]-sn-glycerol (4c). Reaction time 144 h, yield
0
0
J2 ,1 ¼ 7.4 Hz, H-2 ), 3.60e3.70 (m, 4H, 2H-1 and 2H-3),

1460
D. Colombo et al. / European Journal of Medicinal Chemistry 41 (2006) 1456e1463
3.72 (m, 1H, H-2), 3.76 (m, 1H, H-50), 3.80 (d, 1H, H-40), 4.22
(m, 1H, H-50), 3.80 (d, 1H, H-40), 4.21 (dd, 1H, J6 a,5 ¼ 5.1 Hz,
0
0
(dd, 1H, J6 a,5 ¼ 4.4 Hz, J6 a,6 b ¼ 11.7 Hz, H-60a), 4.29 (dd,
J6 a,6 b ¼ 11.7 Hz, H-60a), 4.29 (dd, 1H, J6 b,5 ¼ 8.0 Hz, H-6 b),
4.37 (d, 1H, H-10). 13C NMR (CD3OD): d 26.05 (2CH2),
32.21 (CH2CH2CO), 33.38 (2CH2), 34.24 (CH2CO), 40.90
(CH), 62.78 (C1 or C3), 63.23 (C3 or C1), 64.68 (C60), 70.18
(C40), 72.51 (C20), 74.06 (C50), 74.48 (C30), 83.58 (C2),
105.00 (C10), 175.35 (C]O). CI-MS m/z 396 [M þ NH4]þ.
C17H30O9 calcd. C 53.96, H 7.99; found C 53.89, H 8.04.
0
0
0
0
0
0
0
0
0
1H, J6 b,5 ¼ 8.0 Hz, H-6 b), 4.37 (d, 1H, H-1 ). 13C NMR
(CD3OD): d 22.69 (2CH3), 26.77 (CH), 43.97 (CH2), 62.73
(C1 or C3), 63.17 (C3 or C1), 64.62 (C60), 70.16 (C40),
72.48 (C20), 74.03 (C50), 74.43 (C30), 83.45 (C2), 104.95
(C10), 174.50 (C]O). CI-MS m/z 356 [M þ NH4]þ.
C14H26O9 calcd. C 49.70, H 7.75; found C 49.81, H 7.89.
0
0
0
0
3.1.2.2.2. 2-O-[6-O-(2-Ethylbutanoyl)-b-D-galactopyrano-
20
syl]-sn-glycerol (1c). Yield 85%; oil; ½aꢂD
¼
þ9:4
3.1.3. General procedure for the synthesis of monoesters
1b, 1gej
1
(CH3OH). H NMR (CD3OD): d 0.86e0.92 (m, 6H, 2CH3),
1.48e1.68 (m, 4H, 2CH2), 2.25 (m, 1H, 0COCH), 3.50 (dd,
0
0
0
0
1H, J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz, H-3 ), 3.56 (dd, 1H,
3.1.3.1. 1,3-Di-O-benzyl-2-O-(2,3,4-tri-O-benzyl-6-O-acyl-b-
D-galactopyranosyl)-sn-glycerols (6b, 6ge6j). To a solution
of compound 5 (0.75 mmol) in dichloromethane (5.0 mL),
the appropriate carboxylic acid (0.83 mmol), dicyclo-
hexylcarbodiimide (0.83 mmol) and dimethylaminopyridine
(0.08 mmol) were added in order. The reaction mixture was
stirred at room temperature for 1e2 h and monitored by TLC
(petroleum ethereethyl acetate 70:30 v/v). After filtration of
the formed dicyclohexylurea, dichloromethane (20 mL) was
added and washed with 5% acetic acid (2 ꢃ 15 mL). The aque-
ous layers were extracted with dichloromethane (2 ꢃ 20 mL)
and the organic layers were dried over sodium sulfate. The sol-
vent was removed under reduced pressure, and the desired prod-
uct was obtained by silica gel flash chromatography (petroleum
ethereethyl acetate 75:25 or 70:30 v/v) of the residue.
0
0
0
J2 ,1 ¼ 7.4 Hz, H-2 ), 3.62e3.70 (m, 4H, 2H-1 and 2H-3),
3.72 (m, 1H, H-2), 3.76 (m, 1H, H-50), 3.80 (d, 1H, H-40),
4.24e4.30 (m, 2H, H-60a and H-60b), 4.38 (d, 1H, H-10). 13C
NMR (CD3OD): d 12.14 (2CH3), 26.07 (2CH2), 50.13 (CH),
62.71 (C1 or C3), 63.15 (C3 or C1), 64.71 (C60), 70.24
(C40), 72.51 (C20), 74.09 (C50), 74.47 (C30), 83.26 (C2),
104.97 (C10), 177.66 (C]O). CI-MS m/z 370 [M þ NH4]þ.
C15H28O9 calcd. C 51.13, H 8.01; found C 51.29, H 8.06.
3.1.2.2.3. 2-O-[6-O-(2-Cyclohexylethanoyl)-b-D-galacto-
20
pyranosyl]-sn-glycerol (1d). Yield 88%; oil; ½aꢂD ¼ þ7:6
(CH3OH). 1H NMR (CD3OD): d 0.86e1.82 (m, 11H, CH
and 5CH2), 2.21 (d, 2H, J ¼ 6.5 Hz, COCH2), 3.50 (dd, 1H,
J3 ,2 ¼ 10.0 Hz, J030,4 ¼ 3.0 Hz, H-30), 3.56 (dd, 1H,
0
0
0
0
0
J2 ,1 ¼ 7.4 Hz, H-2 ), 3.62e3.70 (m, 4H, 2H-1 and 2H-3),
3.72 (m, 1H, H-2), 3.76 (m, 1H, H-50), 3.80 (d, 1H, H-40),
3.1.3.1.1.
1,3-Di-O-benzyl-2-O-[2,3,4-tri-O-benzyl-
4.21 (dd, 1H, J6 a,5 ¼ 4.4 Hz, J6 a,6 b ¼ 11.0 Hz, H-60a), 4.28
6-O-(3,3-dimethylbutanoyl)-b-D-galactopyranosyl]-sn-glycerol
0
0
0
0
20
(dd, 1H, J6 b,5 ¼ 7.4 Hz, H-6 b), 4.37 (d, 1H, H-1 ). 13C
NMR (CD3OD): d 27.13 (2CH2), 27.23 (CH2), 34.06
(2CH2), 36.12 (CH), 42.76 (CH2CO), 62.77 (C1 or C3),
63.23 (C3 or C1), 64.68 (C60), 70.23 (C40), 72.53 (C20),
74.11 (C50), 74.50 (C30), 83.50 (C2), 105.02 (C10), 174.52
(C]O). CI-MS m/z 396 [M þ NH4]þ. C17H30O9 calcd. C
53.96, H 7.99; found C 53.71, H 8.11.
(6b). Yield 90%; oil; ½aꢂD ¼ þ5:3 (CHCl3). 1H NMR
0
0
0
0
(CDCl3): d 0.98 (s, 9H, 3CH3), 2.10 (s, 2H, CH2), 3.45e
3.52 (m, 2H, H-30 and H-50), 3.60e3.78 (m, 5H, 2H-1, 2H-3
and H-40), 3.82 (dd, J2 ,1 ¼ 7.7 Hz, J2 ,3 ¼ 10.0 Hz, 1H, H-
0
0
0
0
20), 4.01e4.08 (m, 2H, H-2 and H-60a), 4.18 (dd, 1H,
J6 b,5 ¼ 6.6 Hz, J6 a,6 b ¼ 11.2 Hz, H-60b), 4.57 (d, 1H, H-10),
4.44e5.04 (m, 10H, 5CH2Ph), 7.14e7.40 (m, 25H, Ph). CI-
MS m/z 820 [M þ NH4]þ. C50H58O9 calcd. C 74.79, H 7.28;
found C 74.66, H 7.42.
0
0
0
0
3.1.2.2.4. 2-O-[6-O-(3-Cyclohexylpropanoyl)-b-D-galacto-
20
pyranosyl]-sn-glycerol (1e). Yield 88%; oil; ½aꢂD ¼ þ6:3
(CH3OH). 1H NMR (CD3OD): d 0.80e1.80 (m, 13H, CH
and 6CH2), 2.35 (m, 2H, COCH2), 3.50 (dd, 1H,
3.1.3.1.2.
1,3-Di-O-benzyl-2-O-(2,3,4-tri-O-benzyl-6-O-
benzoyl-b-D-galactopyranosyl-sn-glycerol (6g). Yield 90%;
20
1
J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz, H-30), 3.57 (dd, 1H,
oil; ½aꢂD ¼ ꢄ10:6 (CHCl3). H NMR (CDCl3): d 3.53 (d,
0
0
0
0
0
0
0
0
0
0
0
0
0
0
J2 ,1 ¼ 7.4 Hz, H-2 ), 3.62e3.70 (m, 4H, 2H-1 and 2H-3 ),
1H, J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz, H3 ), 3.60e3.76 (m, 5H,
3.73 (m, 1H, H-2), 3.76 (m, 1H, H-50), 3.80 (d, 1H, H-40),
2H-1, 2H-3 and H-500), 3.81 (d, 1H, H-40), 3.86 (dd,
4.21 (dd, 1H, J6 a,5 ¼ 5.1 Hz, J6 a,6 b ¼ 11.0 Hz, H-60a), 4.29
J2 ,1 ¼ 7.7 Hz, 1H, H-2 ), 4.06 (m, 1H, H-2), 4.28 (dd, 1H,
0
0
0
0
0
0
(dd, 1H, J6 b,5 ¼ 7.4 Hz, H-6 b), 4.37 (d, 1H, H-1 ). 13C
NMR (CD3OD): d 27.34 (2CH2), 27.63 (CH2), 32.52
(CH2CO), 33.42 (CH2CH2CO), 34.11 (2CH2), 38.52 (CH),
62.81 (C1 or C3), 63.26 (C3 or C1), 64.69 (C60), 70.21
(C40), 72.55 (C20), 74.10 (C50), 74.52 (C30), 83.64 (C2),
105.06 (C10), 175.55 (C]O). CI-MS m/z 410 [M þ NH4]þ.
C18H32O9 calcd. C 55.09, H 8.22; found C 55.27, H 8.26.
3.1.2.2.5. 2-O-[6-O-(3-Cyclopentylpropanoyl)-b-D-galacto-
J6 a,5 ¼ 6.3 Hz, J6 a,6 b ¼ 11.2 Hz, H-60a), 4.42 (dd, 1H,
0
0
0
0
0
0
0
0
0
0
0
0
0
0
J6 b,5 ¼ 6.3 Hz, H-6 b), 4.60 (d, 1H, J1 ,2 ¼ 7.7 Hz, H-1 ),
4.44e5.04 (m, 10H, 5CH2Ph), 7.16e7.90 (m, 30H, Ph). CI-
MS m/z 826 [M þ NH4]þ. C51H52O9 calcd. C 75.72, H 6.48;
found C 75.58, H 6.43.
3.1.3.1.3. 1,3-Di-O-benzyl-2-O-[2,3,4-tri-O-benzyl-6-O-(2-
phenylethanoyl)-b-D-galactopyranosyl]-sn-glycerol (6h). Yield
20
1
85%; oil; ½aꢂD ¼ ꢄ2:6 (CHCl3). H NMR (CDCl3): d 3.42e
20
pyranosyl]-sn-glycerol (1f). Yield 86%; oil; ½aꢂD ¼ þ6:3
3.46 (m, 2H, H-30 and H-50), 3.50 (s, 2H, COCH2Ph), 3.60e
1
(CH3OH). H NMR (CD3OD): d 1.04e1.84 (m, 11H, CH and
3.74 (m, 5H, 2H-1, 2H-3 and H-40), 3.77 (dd, J2 ,1 ¼ 7.7 Hz,
0
0
0
0
0
0
0
5CH2), 2.36 (m, 2H, COCH2), 3.50 (dd, 1H, J3 ,2 ¼ 10.0 Hz,
J2 ,3 ¼ 10.0 Hz, 1H, H-2 ), 4.00 (m, 1H, H-2), 4.05 (dd, 1H,
0
0
J3 ,4 ¼ 3.0 Hz, H-3 ), 3.56 (dd, 1H, J2 ,1 ¼ 7.4 Hz, H-2 ),
J6 a,5 ¼ 6.3 Hz, J6 a,6 b ¼ 11.2 Hz, H-60a),0 4.18 (dd, 1H,
0
0
0
0
0
0
0
0
0
0
0
3.61e3.70 (m, 4H, 2H-1 and 2H-3), 3.74 (m, 1H, H-2), 3.76
J6 b,5 ¼ 6.3 Hz, H-6 b), 4.52 (d, 1H, H-1 ), 4.42e5.00 (m,

D. Colombo et al. / European Journal of Medicinal Chemistry 41 (2006) 1456e1463
1461
10H, 5CH2Ph), 7.16e7.35 (m, 30H, Ph). CI-MS m/z
840 þ NH4]þ. C52H54O9 calcd. C 75.89, H 6.61; found C 75
[M, H 6.69.
J ¼ 7.0 Hz, J ¼ 7.0 Hz, Ph), 7.60 (dd, 1H, J ¼ 7.0 Hz,
J ¼ 7.0 Hz, Ph), 8.04 (d, 2H, J ¼ 7.0 Hz, Ph). 13C NMR
(CD3OD): d 61.41 (C1 or C3), 61.76 (C3 or C1), 63.90
(C60), 68.92 (C40), 71.20 (C20), 72.79 (C50), 73.15 (C30),
82.01 (C2), 103.66 (C10), 128.20 (2CH, Ph), 129.25 (2CH,
Ph), 129.83 (C, Ph), 132.95 (CH, Ph), 166.46 (C]O). CI-
MS m/z 376 [M þ NH4]þ. C16H22O9 calcd. C 53.63, H 6.19;
found C 53.49, H 6.13.
3.1.3.1.4. 1,3-Di-O-benzyl-2-O-[2,3,4-tri-O-benzyl-6-O-(3-
phenylpropanoyl)-b-D-galactopyranosyl]-sn-glycerol
(6i).
20
1
Yield 87%; oil; ½aꢂD ¼ ꢄ2:8 (CHCl3). H NMR (CDCl3):
d 2.50 (m, 2H, CH2Ph), 2.86 (m, 2H, COCH2), 3.40 (dd,
1H, J5 ,6 a ¼ 6.0 Hz, J5 ,6 b ¼ 6.0 Hz, H-50), 3.46 (dd, 1H,
0
0
0
0
0
0
0
0
0
J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz, H-3 ), 3.60e3.76 (m, 5H,
3.1.3.2.3. 2-O-[6-O-(2-Phenylethanoyl)-b-D-galactopyrano-
20
2H-1, 2H-3 and H-40), 3.80 (dd, J2 ,1 ¼ 7.7 Hz, 1H, H-2 ),
syl]-sn-glycerol (1h). Yield 82%; oil; ½aꢂD ¼ þ6:2 (CH3OH).
0
0
0
4.01e4.06 (m, 2H, H-2 and H-60a), 4.15 (dd, 1H,
1H NMR (CD3OD): d 3.47 (dd, 1H, J3 ,2 ¼ 10.0 Hz,
0
0
J6 b,5 ¼ 6.3 Hz, J6 a,6 b ¼ 11.2 Hz, H-60b), 4.54 (d, 1H, H-10),
4.44e5.00 (m, 10H, 5CH2Ph), 7.10e7.36 (m, 30H, Ph). CI-
MS m/z 854 [M þ NH4]þ. C53H56O9 calcd. C 76.05, H 6.74;
found C 76.30, H 6.69.
J3 ,4 ¼ 3.0 Hz, H-3 ), 3.55 (dd, 1H, J2 ,1 ¼ 7.4 Hz, H-2 ),
0
0
0
0
0
0
0
0
0
0
3.60e3.68 (m, 6H, COCH2, 2H-1 and 2H-3), 3.69 (m, 1H,
0
H-2), 3.74 (dd, 1H, J5 ,6 a ¼ 4.4 Hz, J5 ,6 b ¼ 8.0 Hz, H-50),
0
0
0
3.76 (d, 1H, H-40), 4.23 (dd, 1H, J6 a,6 b ¼ 11.8 Hz, H-60a),
4.32 (dd, 1H, H-60b), 4.34 (d, 1H, H-10), 7.34e7.21 (m, 5H,
Ph). 13C NMR (CD3OD): d 41.79 (CH2), 62.76 (C1 or C3),
63.23 (C3 or C1), 65.16 (C60), 70.17 (C40), 72.53 (C20),
74.03 (C50), 74.46 (C30), 83.43 (C2), 104.96 (C10), 128.07
(CH, Ph), 129.54 (2CH, Ph), 130.39 (2CH, Ph), 135.51 (C,
Ph), 173.26 (C]O). CI-MS m/z 390 [M þ NH4]þ. C17H24O9
calcd. C 54.83, H 6.50; found C 54.74, H 6.59.
0
0
3.1.3.1.5. 1,3-Di-O-benzyl-2-O-[2,3,4-tri-O-benzyl-6-O-(4-
phenylbutanoyl)-b-D-galactopyranosyl]-sn-glycerol (6j). Yield
20
82%; oil; ½aꢂD ¼ ꢄ5:9 (CHCl3). 1H NMR (CDCl3): d 1.92 (m,
2H, CH2), 2.23 (m, 2H, CH2Ph), 2.64 (m, 2H, COCH2), 3.51
(dd, 1H, J5 ,6 a ¼ 6.0 Hz, J5 ,6 b ¼ 6.0 Hz, H-50), 3.54 (dd, 1H,
0
0
0
0
0
0
0
0
0
J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz, H-3 ), 3.64e3.82 (m, 5H,
2H-1, 2H-3 and H-40), 3.86 (dd, J2 ,1 ¼ 7.7 Hz, 1H, H-2 ),
0
0
0
4.05e4.12 (m, 2H, H-2 and H-60a), 4.20 (dd, 1H,
3.1.3.2.4. 2-O-[6-O-(3-Phenylpropanoyl)-b-D-galactopyra-
nosyl]-sn-glycerol (1i). Yield 80%; m.p. 125e126 ꢁC (amor-
J6 b,5 ¼ 6.3 Hz, J6 a,6 b ¼ 11.2 Hz, H-60b), 4.61 (d, 1H, H-10),
4.48e5.08 (m, 10H, 5CH2Ph), 7.13e7.40 (m, 30H, Ph). CI-
MS m/z 868 [M þ NH4]þ. C54H58O9 calcd. C 76.21, H 6.87;
found C 76.39, H 6.79.
0
0
0
0
20
phous solid); ½aꢂD ¼ þ5:9 (CH3OH). 1H NMR (CD3OD):
d 2.66 (m, 2H, CH2), 2.92 (m, 2H, CH2), 3.48 (dd, 1H,
J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz, H-30), 3.56 (dd, 1H,
0
0
0
0
0
0
0
0
J2 ,1 ¼ 7.4 Hz, H-2 ), 3.61e3.74 (m, 6H, 2H-1, 2H-3, H-5
and H-2), 3.76 (d, 1H, H-40), 4.21 (dd, 1H, J6 a,6 b ¼ 11.8 Hz,
0
0
3.1.3.2. 2-O-(6-O-Acyl-b-D-galactopyranosyl)-sn-glycerol (1b,
1gej). Compound 6 (0.6 mmol) was dissolved in methanol
(35 mL) and 10% palladium on activated carbon (0.09 g)
was added. The reaction mixture, monitored by TLC
(CH2Cl2eMeOH 80:20), was shaken under hydrogen atmo-
sphere for 3e5 h, and then filtered through a celite bed afford-
ing the debenzylated compounds.
0
0
0
0
0
0
J6 a,5 ¼ 4.4 Hz, H-6 a), 4.27 (dd, 1H, J6 b,5 ¼ 8.1 Hz, H-6 b),
4.34 (d, 1H, H-10), 7.14e7.29 (m, 5H, Ph). 13C NMR
(CD3OD): d 30.49 (CH2), 35.28 (CH2), 61.42 (C1 or C3),
61.85 (C3 or C1), 63.32 (C60), 68.75 (C40), 71.14 (C20),
72.68 (C50), 73.09 (C30), 82.24 (C2), 103.65 (C10), 125.90
(CH, Ph), 127.98 (2CH, Ph), 128.10 (2CH, Ph), 140.54 (C,
Ph), 172.97 (C]O). CI-MS m/z 404 [M þ NH4]þ. C18H26O9
calcd. C 55.95, H 6.78; found C 55.78, H 6.81.
3.1.3.2.1. 2-O-[6-O-(3,3-Dimethylbutanoyl)-b-D-galacto-
20
pyranosyl]-sn-glycerol (1b). Yield 85%; oil; ½aꢂD ¼ þ10:3
1
(CH3OH). H NMR (CD3OD): d 1.03 (s, 9H, 3CH3), 2.22
3.1.3.2.5. 2-O-[6-O-(4-Phenylbutanoyl)-b-D-galactopyrano-
20
0
0
0
0
(s, 2H, CH2), 3.50 (dd, 1H, J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz,
syl]-sn-glycerol (1j). Yield 78%; wax; ½aꢂD
¼
þ5:6
1
(CH3OH). H NMR (CD3OD): d 1.92 (m, 2H, CH2), 2.35 (m,
H-30), 3.56 (dd, 1H, J2 ,1 ¼ 7.4 Hz, H-2 ), 3.62e3.70 (m, 4H,
0
0
0
0
0
2H-1 and 2H-3), 3.73 (m, 1H, H-2), 3.76 (dd, 1H,
0
2H, CH2), 2.64 (m, 2H, CH2), 3.49 (dd, 1H, J3 ,2 ¼ 10.0 Hz,
0 0 0 0
0
0
0
0
0
0
0
J6 a,5 ¼ 4.4 Hz, J6 b,5 ¼ 7.4 Hz, H-5 ), 3.80 (d, 1H, H-4 ),
J3 ,4 ¼ 3.0 Hz, H-3 ), 3.56 (dd, 1H, J2 ,1 ¼ 7.4 Hz, H-2 ),
4.22 (dd, 1H, J6 a,6 b ¼ 11.0 Hz, H-60a), 4.26 (dd, 1H, H-60b),
4.38 (d, 1H, H-10). 13C NMR (CD3OD): d 30.00 (3CH3),
31.52 (C(CH3)3), 48.55 (CH2), 62.73 (C1 or C3), 63.20 (C3
or C1), 64.58 (C60), 70.28 (C40), 72.55 (C20), 74.16 (C50),
74.51 (C30), 83.30 (C2), 104.99 (C10), 173.76 (C]O). CI-
MS m/z 370 [M þ NH4]þ. C15H28O9 calcd. C 51.13, H 8.01;
found C 50.97, H 7.90.
3.60e3.70 (m, 4H, 2H-1 and 2H-3), 3.73 (m, 1H, H-2), 3.76
0
0
0
(dd, 1H, J5 ,6 a ¼ 4.4 Hz, J5 ,6 b ¼ 8.0 Hz, H-50), 3.79 (d, 1H,
0
0
0
H-40), 4.20 (dd, 1H, J6 a,6 b ¼ 11.8 Hz, H-60a), 4.29 (dd, 1H,
H-60b), 4.37 (d, 1H, H-10), 7.13e7.29 (m, 5H, Ph). 13C NMR
(CD3OD): d 27.76 (CH2), 34.24 (CH2), 36.05 (CH2), 62.79
(C1 or C3), 63.24 (C3 or C1), 64.72 (C60), 70.21 (C40), 72.55
(C20), 74.10 (C50), 74.51 (C30), 83.54 (C2), 105.02 (C10),
127.00 (CH, Ph), 129.41 (2CH, Ph), 129.52 (2CH, Ph),
142.82 (C, Ph), 174.98 (C]O). CI-MS m/z 418 [M þ NH4]þ.
C19H28O9 calcd. C 56.99, H 7.05; found C 57.12, H 7.19.
0
0
3.1.3.2.2. 2-O-(6-O-Benzoyl-b-D-galactopyranosyl)-sn-glyc-
erol (1g). Yield 83%; m.p. 152 ꢁC (from CHCl3eCH3OH);
20
½aꢂD ¼ þ15:6 (CH3OH). 1H NMR (CD3OD): d 3.54 (dd,
0
0
0
0
0
1H, J3 ,2 ¼ 10.0 Hz, J3 ,4 ¼ 3.0 Hz, H-3 ), 3.60 (dd, 1H,
0
0
0
J2 ,1 ¼ 7.4 Hz, H-2 ), 3.60e3.71 (m, 4H, 2H1 and 2H-3),
3.1.4. General procedure for the synthesis of diesters
2a, 2cef
2-O-b-D-Galactopyranosylglycerol (0.2 g, 0.79 mmol) was
dissolved in pyridine (3.0 mL); appropriate trifluoroethyl
3.75 (m, 1H, H-2), 3.90 (d, 1H, H-40), 3.93 (m, 1H,
J5 ,6 a ¼ 6.6 Hz, J5 ,6 b ¼ 6.6 Hz, H-50), 4.44 (d, 1H, H-10),
0
0
0
0
4.44e4.54 (m, 2H, H-60a and H-60b), 7.47 (dd, 2H,

1462
D. Colombo et al. / European Journal of Medicinal Chemistry 41 (2006) 1456e1463
(2def) or vinyl (2a,c) ester (3.95 mmol) and LPS (1.0 g) were
added in order and the suspension was stirred at 45 ꢁC. The re-
action was monitored by TLC (CH2Cl2eMeOH 80:20) and
stopped after 20e330 h by filtering off the enzyme which
was washed with pyridine. The solvent was removed under re-
duced pressure and the crude residue was submitted to re-
peated flash chromatographies (CH2Cl2eMeOH from 95:5 to
80:20 v/v) to obtain pure (2cef) or diastereomerically en-
riched (2a) 1,60-di-O-acylderivatives.
104.64 (C10), 174.51 (C]O), 174.65 (C]O). CI-MS m/z
520 [M þ NH4]þ. C25H42O10 calcd. C 59.74, H 8.42; found
C 59.60, H 8.57.
3.1.4.4. 1-O-(3-Cyclohexylpropanoyl)-2-O-[6-O-(3-cyclohexyl-
propanoyl)-b-D-galactopyranosyl]-sn-glycerol (2e). Reaction
20
time 260 h, yield 20%; oil; ½aꢂD ¼ þ4:0 (CHCl3). 1H
NMR (pyridine-d5): d 0.68e1.64 (m, 26H, 2CH and
12CH2), 2.31e2.43 (m, 4H, 2COCH2), 4.12e4.20 (m, 4H,
2H-3, H-30 and H-50), 4.37 (m, 1H, H-40), 4.43 (m, 1H,
H-20), 4.47 (m, 1H, H-2), 4.64e4.70 (m, 2H, H-1a and
3.1.4.1. 1-O-(3-Methylbutanoyl)-2-O-[6-O-(3-methylbutanoyl)-
b-D-galactopyranosyl]-sn-glycerol (2a). Reaction time 20 h,
yield 27%; oil; 90% diastereomeric purity (by careful integration
of the anomeric H-10 NMR signals: d 4.97 for the major 1-O-acyl
isomer and d 4.96 for the minor 3-O-acyl isomer). 1H NMR (pyr-
idine-d5): d0.82e0.88(m, 12H,4CH3),2.04e2.16(m, 2H, 2CH),
2.18e2.27 (m, 4H, 2COCH2), 4.11e4.20 (m, 4H, 2H-3, H-30 and
H-50), 4.36 (m, 1H, H-40), 4.42 (m, 1H, H-20), 4.45 (m, 1H, H-2),
0
0
0
0 0
H-1b), 4.76 (dd, 1H, J6 a,5 ¼ 4.9 Hz, J6 a,6 b ¼ 11.2 Hz, H-
60a), 4.89 (dd, 1H, J6 b,5 ¼ 7.7 Hz, H-6 b), 4.99 (d, 1H,
0
0
J1 ,2 ¼ 7.7 Hz, H-1 ). 13C NMR (CD3OD): d 27.36 (4CH2),
27.65 (2CH2), 32.58 (2CH2CO), 33.48 (2CH2CH2CO),
34.14 (4CH2), 38.54 (2CH), 63.24 (C3), 64.68 (C1 and
C60), 70.23 (C40), 72.28 (C20), 74.14 (C50), 74.58 (C30),
79.37 (C2), 104.70 (C10), 175.54 (C]O), 175.64 (C]O).
CI-MS m/z 548 [M þ NH4]þ. C27H46O10 calcd. C 61.11,
H 8.74; found C 61.02, H 8.69.
0
0
0
0
0
4.66 (m, 2H, H-1a and H-1b), 4.73 (dd, 1H, J6 a,5 ¼ 4.9 Hz,
0
J6 a,6 b ¼ 11.2 Hz, H-60a), 4.86 (dd, 1H, J6 b,5 ¼ 7.7 Hz, H-6 b),
0
0
0
0
4.97 (d, 1H, J1 ,2 ¼ 7.7 Hz, H-1 ). 13C NMR (CD3OD): d 22.73
(4CH3), 26.77 (CH), 26.85 (CH), 44.80 (2CH2), 63.19 (C3),
64.50 (C1), 64.65 (C60), 70.24 (C40), 72.25 (C20), 74.10 (C50),
74.54 (C30), 79.23 (C2), 104.67 (C10), 174.51 (C]O), 174.65
(C]O). CI-MS m/z 440 [M þ NH4]þ. C19H34O10 calcd. C
54.02, H 8.11; found C 54.30, H 8.28.
0
0
0
3.1.4.5. 1-O-(3-Cyclopentylpropanoyl)-2-O-[6-O-(3-cyclopen-
tylpropanoyl)-b-D-galactopyranosyl]-sn-glycerol (2f). Reac-
20
1
tion time 260 h, yield 27%; wax; ½aꢂD ¼ þ3:9 (CHCl3). H
NMR (pyridine-d5): d 0.88e1.72 (m, 22H, 2CH and
10CH2), 2.36 (m, 2H, COCH2), 4.12e4.20 (m, 4H, 2H-3, H-
30 and H-50), 4.37 (m, 1H, H-40), 4.43 (m, 1H, H-20), 4.47
(m, 1H, H-2), 4.64e4.72 (m, 2H, H-1a and H-1b), 4.76 (dd,
3.1.4.2. 1-O-(2-Ethylbutanoyl)-2-O-[6-O-(2-ethylbutanoyl)-b-
D-galactopyranosyl]-sn-glycerol (2c). Reaction time 330 h,
1H, J6 a,5 ¼ 4.9 Hz, J6 a,6 b ¼ 11.2 Hz, H-60a), 4.90 (dd, 1H,
0
0
0
0
20
1
0
0
0
0
0
0
yield 12%; oil; ½aꢂD ¼ þ7:6 (CHCl3). H NMR (pyridine-
d5): d 0.86e0.92 (m, 12H, 4CH3), 1.40e1.72 (m, 8H,
4CH2), 2.24e2.34 (m, 2H, 2COCH), 4.11e4.20 (m, 4H, 2H-
3, H-30 and H-50), 4.36 (m, 1H, H-40), 4.41 (m, 1H, H-20),
4.45 (m, 1H, H-2), 4.66 (dd, 1H, J1a,1b ¼ 11.2 Hz,
J1a,2 ¼ 5.6 Hz, H-1a), 4.76 (dd, 1H, J1b,2 ¼ 4.9 Hz, H-1b),
J6 b,5 ¼ 7.7 Hz, H-6 b), 4.99 (d, 1H, J1 ,2 ¼ 7.7 Hz, H-1 ).
13C NMR (CD3OD): d 26.07 (4CH2), 32.24 (CH2CH2CO),
32.29 (CH2CH2CO), 33.42 (4CH2), 34.31 (2CH2CO), 40.93
(2CH), 63.21 (C3), 64.65 (C1 and C60), 70.22 (C40), 72.26
(C20), 74.11 (C50), 74.56 (C30), 79.34 (C2), 104.67 (C10),
175.35 (C]O), 175.45 (C]O). CI-MS m/z 520 [M þ NH4]þ.
C25H42O10 calcd. C 59.74, H 8.42; found C 59.88, H 8.39.
4.78 (dd, 1H, J6 a,5 ¼ 4.9 Hz, J6 a,6 b ¼ 11.2 Hz, H-60a), 4.86
0
0
0
0
0
0
0
0
0
(dd, 1H, J6 b,5 ¼ 7.7 Hz, H-6 b), 4.98 (d, 1H, J1 ,2 ¼ 7.7 Hz,
H-10). 13C NMR (CD3OD): d 12.18 (4CH3), 26.03 (2CH2),
26.14 (2CH2), 50.24 (2CH), 63.19 (C3), 64.39 (C1), 64.75
(C60), 70.31 (C40), 72.25 (C20), 74.19 (C50), 74.56 (C30),
79.00 (C2), 104.58 (C10), 177.66 (C]O), 177.73 (C]O).
CI-MS m/z 468 [M þ NH4]þ. C21H38O10 calcd. C 55.99, H
8.50; found C 55.70, H 8.43.
3.1.5. Configuration assignment at C-2 for 2a, 2cef
The 1-O-acyl-sn-glycerols obtained by treatment of 2a,
2cef with b-galactosidase from Aspergillus oryzae were
reacted with acetone, 2,2-dimethoxypropane and p-toluene-
sulfonic acid. The obtained 1-O-acyl-2,3-O-isopropylidene-
sn-glycerols were analyzed on a chiral GLC-capillary col-
umn (dimethylpentyl-b-cyclodextrin, 25 m, ID 0.25 mm,
from MEGA e Italy) and compared with authentic analyti-
cal standards [6] under the following conditions: oven tem-
perature, from 120 to 200 ꢁC, 0.2 ꢁC/min; injector
temperature, 250 ꢁC; detector temperature, 295 ꢁC; helium
flow, 1 mL/min; split ratio 80:1. In the chromatogram, the
lower retention time peak corresponded to the 1-O-acyl-
2,3-O-isopropylidene-sn-glycerols and the higher retention
time peak to the 3-O-acyl-isomers. The following retention
times were found for the 3-methylbutanoyl, 2-ethylbutanoyl,
cyclohexylethanoyl, cyclopentylpropanoyl, and cyclohexyl-
propanoyl derivatives: 12.08/13.07, 17.22/17.43, 81.33/
82.13, 86.06/87.07 and 119.71/120.56 min.
3.1.4.3. 1-O-(2-Cyclohexylethanoyl)-2-O-[6-O-(2-cyclohexyle-
thanoyl)-b-D-galactopyranosyl]-sn-glycerol
(2d). Reaction
20
time 300 h, yield 6%; oil; ½aꢂD ¼ þ4:9 (CHCl3). 1H
NMR (pyridine-d5): d 0.84e1.84 (m, 22H, 2CH and
10CH2), 2.20e2.30 (m, 4H, 2COCH2), 4.10e4.20 (m, 4H,
2H-3, H-30 and H-50), 4.36 (m, 1H, H-40), 4.42 (m, 1H,
H-20), 4.47 (m, 1H, H-2), 4.63e4.72 (m, 2H, H-1a and
0
0
0
0 0
H-1b), 4.75 (dd, 1H, J6 a,5 ¼ 4.2 Hz, J6 a,6 b ¼ 11.2 Hz, H-
60a), 4.87 (dd, 1H, J6 b,5 ¼ 7.7 Hz, H-6 b), 4.98 (d, 1H,
0
0
J1 ,2 ¼ 7.7 Hz, H-1 ). 13C NMR (CD3OD): d 27.18 (4CH2),
27.28 (2CH2), 34.11 (4CH2), 36.13 (2CH), 42.85
(2CH2CO), 63.23 (C3), 64.55 (C1), 64.72 (C60), 70.28
(C40), 72.28 (C20), 74.16 (C50), 74.57 (C30), 79.21 (C2),
0
0
0

D. Colombo et al. / European Journal of Medicinal Chemistry 41 (2006) 1456e1463
1463
3.2. Biological evaluation
Acknowledgements
3.2.1. Short-term in vitro bioassay for anti-tumor promoters
Inhibition was tested using a short-term in vitro assay for
EBV activation in Raji cells cultivated in RPMI 1640 medium
containing 10% fetal calf serum, and induced by TPA as de-
scribed previously [8,9]. The assays were performed in tripli-
cate for each compound. 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 ordinarily around
30%. The viability of the cells was assayed against treated
cells using the Trypan Blue staining method. For the determi-
nation of cytotoxicity, the cell viability was required to be
more than 60% 2 days after treatment with the compounds
for an accurate result.
This study was supported in part by the Ministero
`
dell’Istruzione, dell’Universita e della Ricerca, Rome, Italy
(PRIN 2003: anti-tumoral natural and related synthetic com-
pounds) and 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),
and also supported by grant from the National Cancer Insti-
tute (CA 17625), USA.
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