Chemoenzymatic synthesis and antimicrobial activity evaluation of monogalactosyl diglycerides

Article abstract of DOI:10.1016/j.ejmech.2007.03.012

Monogalactosyl diglycerides with medium to long fatty acid acyl chains, were prepared and examined for antimicrobial activity against Gram positive, Gram negative bacteria and fungi. The study of their in vitro antimicrobial activity confirms the significant activity of some monogalactosyl diacylglycerol analogues and establishes for the galactose series that the 1,2-disubstitution and the octanoyl chain are the proper structural features for the maximum activity.

Full text of DOI:10.1016/j.ejmech.2007.03.012

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European Journal of Medicinal Chemistry 43 (2008) 210e221
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Laboratory note
Chemoenzymatic synthesis and antimicrobial activity
evaluation of monogalactosyl diglycerides
a
b
Francesca Cateni a, , Paolo Bonivento , Giuseppe Procida ,
Marina Zacchigna a, Luciana Gabrielli Favretto b,
Giuditta Scialino c, Elena Banfi c
*
a Department of Pharmaceutical Sciences, University of Trieste, P.zle Europa, 1, 34127 Trieste, TS, Italy
b Department of Materials and Natural Resources, University of Trieste, Via A. Valerio 6, 34127 Trieste, Italy
c Department of Biomedical Sciences, Microbiology Section, University of Trieste, Via A. Fleming 22, 34127 Trieste, Italy
Received 25 October 2006; received in revised form 5 March 2007; accepted 8 March 2007
Available online 2 April 2007
Abstract
Monogalactosyl diglycerides with medium to long fatty acid acyl chains, were prepared and examined for antimicrobial activity against Gram
positive, Gram negative bacteria and fungi. The study of their in vitro antimicrobial activity confirms the significant activity of some monog-
alactosyl diacylglycerol analogues and establishes for the galactose series that the 1,2-disubstitution and the octanoyl chain are the proper struc-
tural features for the maximum activity.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Synthesis; Monogalactosyl diglycerides; 1,2-O-Diacyl-3-O-b-D-galactopyranosyl-glycerols; Antimicrobial activity; Lipases
1. Introduction
activities [16]. The activity of such compounds seems strictly
related to the acyl chain length [10,11,16].
Glycoglycerolipids are common constituents of plant cell
membranes and bacterial cell walls [1e4]. Various glycolipids
have been isolated from plants [5,6], algae [7] and bacteria
[4,8].
Recently, we investigated the active principles from various
species of Euphorbiaceae and isolated monogalactosyl diacylgly-
cerols (MGDGs), monogalactosyl monoacylglycerols (MGMGs)
and digalactosyl diacylglycerols (DGDGs) as the active compo-
nents, after repeated chromatography [9]. MGDGs, MGMGs
and DGDGs, a class of glycolipids, have attracted much attention
in recent years because of their biological activities, such as
anti-tumor-promoting [5,6,8,10,11], oxygen scavenging [12],
anti-viral [13,14], anti-inflammatory [15] and anti-hyperlipemic
Previous pharmacological studies of methanol extracts
from Euphorbiaceae showed both antimicrobial and anti-
inflammatory action [17,18]. Antimicrobial monogalactosyl
diacylglycerols (MGDGs) have been isolated and character-
ized from the brown algae Sargassum stolonifolium Phang et
Yoshida [7]. However, glycoglycerolipids are usually available
from natural sources in only limited quantities and as hardly
separable mixtures in terms of their fatty acid compositions
so that their versatile synthesis seems to be of importance, es-
pecially for their biological investigation. Currently, different
synthetic studies of various analogues of natural glycoglycer-
olipids and their modified forms with unnatural structures are
intensively being developed [19e23]. Besides, a method for
identifying monoglycosyl diglycerides in lipid extracts by us-
ing combined gas chromatographyemass spectrometry was
reported [24].
* Corresponding author. Tel.: þ39 040 5583722; fax: þ39 040 52572.
E-mail address: cateni@univ.trieste.it (F. Cateni).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.03.012

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
211
On the basis of these evidences, which indicate the na-
2. Chemistry
ture of the lipophilic chain as a crucial structural feature
for the activity of many compounds, we decided to prepare
glycoglycerolipid analogues using monogalactosyl diglycer-
ides as a starting point in order to study the effect against
Gram positive, Gram negative bacteria and fungi. In this pa-
per, we report the synthesis of a series of mono-acid and
Galactosyl diglycerides 9aeh, 13aec and 14aec bearing
either saturated or unsaturated fatty acids were synthesized
as depicted in Schemes 1 and 2. Glucosidation of D,L-a,b-iso-
propylideneglycerol 3 under KoenigseKnorr conditions [14]
¨
by using tetra-O-acetyl-a-D-galactopyranosyl bromide 2 in di-
chloromethane in the presence of silver carbonate, gave the
desired peracetylated b-glucoside 4 [1H NMR signals of
anomeric protons: d 4.61, 4.63 (dd, J ¼ 8 Hz) indicated a 1:1
ratio of diastereomeric products] in 70% yield.
Removal of all the acyl protective groups of 4 by alkaline
hydrolysis afforded 5 which was subjected to protection of
the resulting free hydroxyl groups as levulinate esters 6.
Thus, successive treatment of 6 with 70% acetic acid in aque-
ous solution at 60 ꢀC in order to remove the isopropylidene
protecting group, provided the diol intermediate 7 in quantita-
tive yield (Scheme 1).
mixed-acid
1,2-O-diacyl-3-O-b-galactopyranosyl-rac-glyc-
erols 9aeh, 13aec and 14aec with medium to long
(from C8 to C18) acyl chains. In particular, as concerning
the mixed-acid 1,2-O-diacyl-3-O-b-galactopyranosyl-rac-
glycerols 13aec and 14aec, the effects on antimicrobial
activity of long-chain polyunsaturated fatty acids (PUFA)
located at the mid-position with medium chain fatty acids
(MCFA) at one of the end-positions of the glycerol back-
bone have been studied.
Besides, the compounds obtained in this study, in analogy
with the structured lipids, whose biological activity seems
directly influenced by the presence of determined fatty acids
in predetermined positions of the glycerol moiety, should
have an interesting application as nutraceutic compounds
[25e27].
The intermediate compound 7 represents the key synthon
for the synthesis of the mono-acid and mixed-acid 1,2-O-di-
acyl-3-O-b-galactosylglycerols 9a, b, 13aec and 14aec
(Scheme 2).
OAc
OAc
OAc
OAc
H
H
O
O
O
O
b
a
H
H
+
HO
OAc
AcO
AcO
H
H
OAc
Br
OAc
H
H
H
H
1
2
3
OH
OAc
H
OH
OAc
H
O
O
O
O
O
O
H
c
H
AcO
d
HO
O
O
H
OH
H
OAc
H
H
H
H
H
H
5
4
OLev
H
OLev
OLev
OLev
O
H
HO
OH
O
O
O
H
e
H
O
LevO
O
LevO
H
H
OLev
H
OLev
H
H
H
H
H
7
6
Lev = -CO(CH2)2COCH3
Lev = -CO(CH2)2COCH3
Scheme 1. Reagent and conditions: (a) HBr, CH3CO2H, r.t.; (b) Ag2CO3, CaSO4, I2, CH2Cl2, r.t.; (c) NaOCH3, CH3OH; (d) [CH3CO(CH2)2CO]2O, Py;
(e) CH3CO2H 70%, 60 ꢀC.

212
F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
OLev
OLev
OLev
OLev
H
H
O
O
HO
OH
R1O
OR1
H
H
LevO
a
O
LevO
O
H
H
OLev
OLev
H
H
H
H
H
H
7
8a-h
Lev = -CO(CH2)2COCH3
b
c
OLev
OLev
OH
OH
H
O
HO
OR1
H
O
H
R1O
OR1
H
O
LevO
H
O
HO
OLev
H
H
OH
H
H
H
H
H
10a, b
9a-h
10a : R1= -CO(CH2)6CH3
10b : R1 = -CO(CH2)8CH3
R1
d
8a, 9a
8b, 9b
8c, 9c
8d, 9d
8e, 9e
8f, 9f
-CO(CH2)6CH3
-CO(CH2)8CH3
-CO(CH2)10CH3
-CO(CH2)12CH3
-CO(CH2)16CH3
OLev
OLev
H
O
R2O
OR1
H
O
LevO
H
OLev
-CO(CH2)7CH=CH(CH2)7CH3
H
H
H
8g, 9g
8h, 9h
-CO(CH2)7(CH=CH-CH2)2(CH2)3CH3
-CO(CH2)7(CH=CH-CH2)3CH3
11a-c, 12a-c
e
OH
OH
H
O
R2O
OR1
H
O
HO
H
OH
H
H
H
13a-c, 14a-c
R2
-CO(CH2)7CH=CH(CH2)7CH3
R1
11a, 13a
-CO(CH2)6CH3
-CO(CH2)6CH3
-CO(CH2)6CH3
-CO(CH2)8CH3
-CO(CH2)8CH3
-CO(CH2)8CH3
-CO(CH2)7(CH=CH-CH2)2(CH2)3CH3
-CO(CH2)7(CH=CH-CH2)3CH3
-CO(CH2)7CH=CH(CH2)7CH3
11b, 13b
11c, 13c
12a, 14a
12b, 14b
-CO(CH2)7(CH=CH-CH2)2(CH2)3CH3
-CO(CH2)7(CH=CH-CH2)3CH3
12c, 14c
Scheme 2. Reagent and conditions: (a) fatty acid, DCCD, DMAP, CH2Cl2, r.t.; (b) NH2NH2 (1 M, PyeCH3CO2H 3:2); (c) M. miehei, vinyl caprylate (vinyl
capriate), 37 ꢀC; (d) fatty acid, DCCD, DMAP, CH2Cl2, r.t.; (e) NH2NH2 (1 M, PyeCH3CO2H 3:2).

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
213
The mono-acid 1,2-O-diacyl-3-O-(b-D-20,30,40,60-tetra-O-
3. Results and discussion
levulinyl-galactopyranosyl)-rac-glycerols 8aeh were pre-
pared by one step diacylation of 7 with 2 equivalents of
the desired fatty acids in the presence of dicyclohexylcarbo-
diimide (DCCD) and 4-dimethylaminopyridine (DMAP) in
dry dichloromethane at room temperature. We selected octa-
noate, caprate, laurate, myristate and stearate as introduction
acyl groups, because they are components of bioactive
analogues of natural glycoglycerolipids [19e23], and oleate,
linolate and linolenate that are, the major acyl components
of MGDGs, MGMGs and DGDGs isolated from Euphor-
biaceae [9]. Finally, the levulinyl protective groups of 8aeh
were removed by treatment with hydrazine hydrate
and mono-acid 1,2-O-diacyl-3-O-(b-D-galactopyranosyl)-rac-
glycerol derivatives with different acyl groups 9aeh were ob-
tained (Scheme 2).
All monogalactosyl diglycerides are stable compounds and
are found to be homogenous by thin layer chromatography.
3.1. Spectroscopy
The mass spectra of all compounds agreed with the pro-
posed structures. The series 9aeh, 13aec and 14aec have
1
the correct chemical shifts for the protons in the H NMR
spectra and for the carbons in the 13C NMR spectra. The
chemical shifts of the target compounds and of all the interme-
diates are given in Section 5.
4. Pharmacology
On the other hand, the synthesis of mixed-acid 1,2-O-di-
acyl-3-O-(b-D-galactopyranosyl)-rac-glycerols 13aec and
14aec requires full regioselectivity control and can hardly
be undertaken by synthetic organic chemistry methods without
multi-step protectionedeprotection processes [16]. Based on
their high regioselectivity, lipases are ideally suited as biocata-
lysts for the synthesis of structured lipids, by acting preferably
or exclusively at the primary positions of the glycerol moiety
[25e27]. Another important feature offered by lipases is the
mild conditions under which they act, which may become cru-
cial in hampering intramolecular acyl-migration side-reac-
tions. A chemoenzymatic approach was developed for the
synthesis of 2-O-b-D-glucosylglycerol derivatives through a di-
rect lipase catalyzed monoacylation of the substrate mediated
by Pseudomonas cepacia lipase (LPS) [20].
To get the intermediate adducts 10a, b (Scheme 2), com-
pound 7, obtained by standard procedures as depicted in
Scheme 1, was directly submitted to transesterification cata-
lyzed by Mucor miehei lipase in the presence of the appro-
priate octanoate 10a and caprate 10b vinyl esters in dry
dichloromethane as solvent. The reactions were completely
regioselective and afforded, through the expected acylation
of the primary hydroxyl group [25e27], the 1-O-acyl-3-
O-(b-D-20,30,40,60-tetra-O-levulinyl-galactopyranosyl)-rac-glyc-
erols 10a and b in good yields (95e98%) (Scheme 2).
There were no signs of any acyl-migration taking place
and subsequently, in the second step, pure oleic, linoleic
and linolenic fatty acids were chemically introduced to the
remaining mid-position by DCCD coupling agent to give
in excellent yields (58e87%) the intermediate products
11aec and 12aec with the hydroxyl groups of the galacto-
syl moiety temporarily protected as levulinates. Finally, the
levulinyl protective groups of 11aec and 12aec were re-
moved by treatment with hydrazine in acetic acidepyridine
mixture at room temperature and 1,2-O-diacyl-3-O-(b-D-gal-
actopyranosyl)-rac-glycerols 13aec and 14aec with me-
dium chain fatty acids (MCFAs) at the end-position and
long-chain polyunsaturated fatty acids (PUFA) located at
the mid-position of the glycerol back-bone were obtained.
The structures of the synthesized compounds were estab-
lished with the help of spectral analyses.
4.1. Preliminary antibacterial and antifungal evaluation
of monogalactosyl diglycerides 9aeh, 13aec and 14aec
All the synthesized 1,2-O-diacyl-3-O-b-D-galactopyrano-
syl-rac-glycerols 9aeh, 13aec and 14aec were submitted
for preliminary evaluation of their in vitro antibacterial activ-
ity against Staphylococcus aureus, Enterococcus faecalis,
Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa,
Mycobacterium tuberculosis, and for antifungal activity
against Candida albicans and Candida pseudotropicalis. Anti-
microbial activity was always evaluated by reference methods
[28e30]. Ciprofloxacin was chosen as a standard in antibacte-
rial activity measurements, as it has excellent activity against
most Gram-negative and Gram-positive bacteria, and is known
as an antibacterial drug in the treatment of a wide range of in-
fections [31]. Miconazole was chosen as a standard in antifun-
gal activity measurements. MIC was defined as the lowest
concentration of compound that inhibited bacterial/fungal
growth.
Table 1 shows MIC values of the synthesized compounds.
No inhibitory activity was exhibited by any compound against
Candida spp. As can be seen from the presented data galactosyl
diglycerides bearing the same saturated fatty acyls 9aee at C-1
and C-2 displayed no inhibitory activity, except for 9a and
b which contain a dicapryl (C8:0) and a dicaprinyl (C10:0)
moiety, respectively, and showed interesting MICs against
S. aureus and M. tuberculosis strains, respectively.
On the other hand, compounds bearing the same unsatu-
rated fatty acyls 9feh had little antimicrobial activity. Most
compounds bearing different fatty acyls 13aec and 14aec ex-
hibited an interesting inhibitory activity against mycobacteria
13aec and against S. aureus clinical isolate 13a, c and 14c. A
total of six compounds were active against a M. tuberculosis
reference and clinical strains with MICs ranging from 64 to
128 mg/ml.
The antibacterial activity of the compounds was lost by in-
creasing the length of the saturated acyl moiety 9cee. The
presence of a single double bond within the acyl chain
(18:1) in 9f did not modify the biological activity. Compound
9g, characterized by the presence of two linolate acyl groups
in the positions 1 and 2 of the glycerol moiety displayed

214
F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
inhibitory activity against M. tuberculosis reference and clini-
cal strains, B. subtilis and one clinical strain of S. aureus with
MIC of 128 mg/ml. Compound 9h bearing three unsaturations
within the acyl moiety showed inhibitory activity against S.
aureus (MIC of 128 mg/ml).
5. Experimental protocols
5.1. Chemical analysis
1H NMR and 13C NMR spectra were recorded with a Varian
Gemini 200 MHz spectrometer and a Varian Unity 400 MHz
spectrometer. 13C NMR spectra were recorded with
90.5 MHz, Gemini 200 spectrometer. NMR spectra were ob-
tained by using CDCl3 as solvent and chemical shifts are ex-
pressed as d units (ppm) relative to tetramethylsilane (TMS)
as internal standard. The abbreviations s, d, dd, t, q, m and
br s refer to singlet, doublet, doublet of doublet, triplet, quar-
tet, multiplet and singlet broad signal, respectively. The EI-MS
spectra were measured with a VG-ZAB 2F spectrometer. The
ionizing energy was 70 eV in all cases and compounds were
introduced by direct insertion. Electrospray analysis was car-
ried out with API Perkin Elmer (voltage þ 5600 with orifice
90 and/or 120).
The column chromatography was performed by using silica
˚
gel (Kieselgel 60, 230e400 Mesh, 60 A Merck). Analytical
thin layer chromatography (TLC) was carried out on Merck
60 F254 silica gel plates (0.25 mm thickness) and the spots
were detected by spraying 50% aqueous H2SO4 and heating
at 110 ꢀC. M. miehei (LipozymeÒ, specific activity 42 U/g)
was purchased from Fluka, Italy. Infrared spectra were re-
corded as film on a Jasco FT-IR-200 model spectrophotometer.
Reagents (Fluka, Italy) used were AR grade and all solvents
for synthesis, extraction and column chromatography were
distilled and dried before use. Evaporation under reduced pres-
sure was always effected with the bath temperature maintained
below 40 ꢀC. The elemental analyses were carried out on
a Carlo Erba model 1016 analyzer.
5.1.1. 2,3,4,6-Tetra-O-acetyl-a-D-galactopyranosyl
bromide 2
HBr in glacial acetic acid (33%) was added dropwise to
a solution of pentaacetylgalactose 1 (2 mmol) in acetic acid
(3 ml). The reaction mixture was stirred in the dark overnight
at room temperature, then ice-cold water (10 ml) was added
and the mixture was extracted with CH2Cl2 (3 ꢁ 20 ml). The
combined CH2Cl2 layers were washed with a saturated
NaHCO3 solution, H2O and brine, and the filtrate was evapo-
rated in vacuo to give a colorless syrupy mixture 2 recrystal-
lized from Et2O. Yield: 68%. Rf ¼ 0.84 (EtOAcehexane 1:1,
1
v:v). H NMR (CDCl3): d 1.97 (s, 3H, CH3CO), 2.0 (s, 3H,
CH3CO), 2.09 (s, 3H, CH3CO), 2.15 (s, 3H, CH3CO), 4.10e
4.40 (m, 2H, H2-6), 4.45 (m, 1H, H-5), 5.41 (dd,
J1 ¼ 10.2 Hz, J2 ¼ 3.9 Hz, 1H, H-3), 5.27 (dd, J1 ¼ 3.9 Hz,
J2 ¼ 10.2 Hz, 1H, H-2), 5.38 (dd, J1 ¼ 3.4 Hz, J2 ¼ 1.2 Hz,
1H, H-4), 6.12 (d, J1 ¼ 3.9 Hz, 1H, H-1). ESI-MS:

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
215
m/z ¼ 434 (M þ Na, 15%)þ. Anal. calcd. for C14H19BrO9: C,
was added. The mixture was extracted with chloroform
(3 ꢁ 30 ml) and the combined organic layers were extracted
with a 10% solution of sodium bicarbonate (2 ꢁ 50 ml) first
and then with water, dried over Na2SO4 and concentrated in
vacuo.
40.87; H, 4.62; Br, 19.46; found: C, 40.91; H, 4.40; Br, 19.90.
5.1.2. General procedure for glycosylation
A mixture of 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolan
3 (1.2 mmol), Ag2CO3 (1 mmol) and fine Drierite (2.0 g) in
dry dichloromethane (30 ml) was stirred in the dark under
N2. After being stirred for half an hour, iodine (0.1 mmol)
was added and acetobromo-a-D-galactose 2 (1 mmol) in dry
dichloromethane (10 ml) was added dropwise over a period
of 1 h. The reaction mixture was stirred overnight at room
temperature, filtered on Celite, and the filtrate was evaporated
in vacuo to give a colorless syrupy mixture.
5.1.4.1. 1,2-Isopropylidene-3-O-(20,30,40,60-tetra-O-levulinyl-b-
D-galactopyranosyl)-rac-glycerol 6. Yield: 75%. Rf ¼ 0.59
1
(CH2Cl2eMeOH 1:1, v:v). H NMR (CDCl3): d 1.22, 1.27
(2s, 6H, 2 ꢁ CH3), 2.10 (4s, 12H, 4 ꢁ CH3CO), 2.60 (br t,
8H, 4 ꢁ CH2OCO), 2.84 (br t, 8H, 4 ꢁ CH2COCH3), 3.60e
3.82 (m, 6H, 5H-glycerol, H-50), 4.0e4.35 (dd, 2H,
0
J ¼ 12.0 Hz, H2-60), 4.54 (d, 1H, J1 ,2 ¼ 7.9 Hz, H-1 ), 5.13
(dd, 1H, J ¼ 10.1, 3.9 Hz, H-20), 5.36 (dd, 1H, J ¼ 3.4,
1.3 Hz, H-40), 5.41 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-30). ESI-
MS: m/z ¼ 773 (M þ Na, 12%)þ. Anal. calcd. for
C32H46O16: C, 55.98; H, 6.70; found: C, 56.02; H, 6.68.
0
0
The mixture was purified by flash chromatography on silica
gel, eluted with EtOAcehexane (1:1, v:v) to give the 1:1 mix-
tures of the two diastereoisomers of 4.
5.1.2.1. 1,2-Isopropylidene-3-O-(b-D-20,30,40,60-tetra-O-acetyl-
galactopyranosyl)-rac-glycerol 4. Yield: 68%. Rf ¼ 0.59
5.1.5. General procedure for deacetonization
1
(EtOAcehexane 1:1, v:v). H NMR (CDCl3): d 1.38, 1.43
A solution of 6 (1.90 mmol) in 70% aqueous acetic acid
(10 ml) was stirred at 60 ꢀC for 4 h. The mixture was concen-
trated and co-evaporated with toluene four times. The residue
was then purified by silica gel flash chromatography, eluted
with CHCl3eMeOH (10:1, v:v) to give 7 as a semisolid.
(s, 6H, 2 ꢁ CH3), 1.97, 2.00, 2.09, 2.15 (s, 12H, 4 ꢁ COCH3),
3.60e4.10 (m, 5H, 5H-glycerol), 4.0e4.35 (m, 2H, H2-60),
4.46 (m, 1H, H-50), 4.61 (dd, J ¼ 8.0 Hz, 1H, H-10), 5.27
(dd, J1 ¼ 3.9, J2 ¼ 10.2 Hz, 1H, H-20), 5.38 (dd, J1 ¼ 3.4 Hz,
J1 ¼ 1.2 Hz, 1H, H-40), 5.40 (dd, J1 ¼ 10.2 Hz, J2 ¼ 3.9 Hz,
1H, H-30). ESI-MS: m/z ¼ 485 (M þ Na, 18%)þ. Anal. calcd.
for C20H30O12: C, 51.95; H, 6.49; found: C, 51.99; H, 6.52.
5.1.5.1. 3-O-(b-D-20,30,40,60-Tetra-O-levulinyl-galactopyranosyl)-
rac-glycerol 7. Yield: 80%. Rf ¼ 0.36 (EtOAceMeOH 10:1,
1
v:v). H NMR (CDCl3): d 2.10 (4s, 12H, 4 ꢁ CH3CO), 2.60
5.1.3. General procedure for removal of acetyl protecting
groups from 1,2-isopropylidene-3-O-(b-D-20,30,40,60-tetra-
O-acetyl-galactopyranosyl)-rac-glycerol 4
CH3ONa (10 mmol) was added to a solution of 4 (1 mmol)
in dry methanol (10 ml). The reaction mixture was stirred at
room temperature for 2 h, and the solution was neutralized
with Amberlist-15, filtered through Celite and concentrated.
The mixture was purified by flash chromatography on silica
gel, eluted with EtOAceMeOH (1:1, v:v) to give the 1:1 mix-
tures of the two diastereoisomers of 5.
(br t, 8H, 4 ꢁ CH2OCO), 2.84 (br t, 8H, 4 ꢁ CH2COCH3),
3.58e3.69 (m, 4H, H2-1, H2-3), 3.79 (m, 1H, H-2), 3.88 (dd,
1H, H-50), 4.0e4.35 (dd, 2H, J ¼ 12.0 Hz, H2-60), 4.54 (d,
0
0
0
J1 ,2 ¼ 7.9 Hz, 1H, H-1 ), 5.13 (dd, J ¼ 10.1, 3.9 Hz, 1H, H-
20), 5.36 (dd, J ¼ 3.4, 1.3 Hz, 1H, H-40), 5.41 (dd, J ¼ 10.þ1,
3.9 Hz, 1H, H-30). ESI-MS: m/z ¼ 669 (M þ Na, 50%) .
Anal. calcd. for C29H42O16: C, 53.54; H, 7.07; found: C,
53.60; H, 7.02.
5.1.6. General procedure for preparation of mono-acid 1,2-
O-diacyl-3-O-(b-D-20,30,40,60-tetra-O-levulinyl-
5.1.3.1. 1,2-Isopropylidene-3-O-(b-D-galactopyranosyl)-rac-
glycerol 5. Yield: 90%. Rf ¼ 0.40 (EtOAceMeOH 5:1, v:v).
1H NMR (CDCl3): d 1.39, 1.43 (s, 6H, 2 ꢁ CH3), 3.49e3.52
galactopyranosyl)-rac-glycerols
DCCD (0.4 mmol) was added to a solution of 7 (0.3 mmol),
fatty acid (0.7 mmol) and DMAP (0.03 mmol) in dry dichloro-
methane (10 ml), and stirred for 24 h at room temperature. The
solid was removed by filtration and the solvent was evaporated
in vacuo. The residue was purified by flash chromatography on
silica gel, eluted with EtOAcehexane (8:1, v:v) to give the
products 8aeh. All compounds were obtained as the 1:1 mix-
tures of two diastereoisomers.
(m, 2H, H-30, 50), 3.55 (dd, 1H, J2 ,1 ¼ 7.3 Hz, J2 ,3 ¼ 9.5 Hz,
H-20), 3.53e4.10 (m, 5H, 5H-glycerol), 3.73 (dd, 1H, H-60b),
0
0
0
0
3.84 (dd, 1H, J6 a,6 b ¼ 11.7 Hz, H-60a), 3.90 (d; 1H,
J ¼ 3.3 Hz, H-40), 4.95 (d, 1H, H-10). EI-MS (70 eV): m/
z ¼ 293 (M, 18%)þ, 179 (M ꢂ C6H11O2, 28%)þ, 113
(M ꢂ C6H11O6, 100%)þ. Anal. calcd. for C12H22O8: C,
48.98; H, 7.48; found: C, 49.01; H, 7.51.
0
0
5.1.6.1. 1,2-O-Dicaprilyl-3-O-(b-D-20,30,40,60-tetra-O-levulinyl-
galactopyranosyl)-rac-glycerol 8a. Yield: 73%. Rf ¼ 0.55
5.1.4. General procedure for preparation of levulinate
esters
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.90 (br t,
To a solution of 1,2-isopropylidene-3-O-(b-D-galactopyra-
nosyl)-rac-glycerol 5 (2.1 mmol) in dry pyridine (10 ml) at
room temperature, under an inert atmosphere, levulinic anhy-
dride (16 mmol) dissolved in dry pyridine (6 ml) was added
dropwise. The reaction was stirred for 24 h and ice water
6H, J ¼ 6.7 Hz), 1.22 (br s, 16H, CH2 aliph.), 1.55 (br t, 4H,
COCH2CH2), 2.10 (4s, 12H, 4 ꢁ CH3CO), 2.30 (br t, 4H,
COCH2), 2.60 (br t, 8H, 4 ꢁ CH2OCO), 2.84 (br t, 8H,
4 ꢁ CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz, H2-
3), 3.88 (dd, 1H, H-50), 4.0e4.13 (dd, 2H, J ¼ 11.9, 6.6 Hz,

216
F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
H2-1), 4.30e4.35 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.55 (d,
COCH2), 2.60 (br t, 8H, 4 ꢁ CH2OCO), 2.83 (br t, 8H,
4 ꢁ CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz, H2-3),
4.01 (dd, 1H, H-50), 4.0e4.15 (dd, 2H, J ¼ 12.0, 6.6 Hz,
0
0
0
0
0
J1 ,2 ¼ 8.0 Hz, 1H, H-1 ), 5.15 (dd, 1H, J ¼ 10.1, 3.9 Hz,
H-20), 5.24 (br s, 1H, H-2), 5.37 (dd, 1H, J ¼ 3.4 Hz, H-40),
5.41 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-30). ESI-MS: m/z ¼ 921
(M þ Na, 30%)þ. Anal. calcd. for C45H70O18: C, 60.13; H,
7.79; found: C, 60.09; H, 7.80.
H2-1), 4.30e4.37 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.58 (dd,
0
0
0
0
0
1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.19 (dd, 1H, J ¼ 9.5, 3.8 Hz,
H-20), 5.24 (br s, 1H, H-2), 5.39 (dd, 1H, J ¼ 3.5 Hz, H-40),
5.44 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30). ESI-MS: m/z ¼ 1201
(M þ Na, 41%)þ. Anal. calcd. for C65H110O18: C, 66.21; H,
9.34; found: C, 66.20; H, 9.37.
5.1.6.2.
1,2-O-Didecanoyl-3-O-(b-D-20,30,40,60-tetra-O-levu-
linyl-galactopyranosyl)-rac-glycerol 8b. Yield: 70%. Rf ¼ 0.57
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.90 (br t,
6H, J ¼ 6.7 Hz), 1.23 (br s, 24H, CH2 aliph.), 1.55 (br t, 4H,
COCH2CH2), 2.10 (4s, 12H, 4 ꢁ CH3CO), 2.31 (br t, 4H,
COCH2), 2.60 (br t, 8H, 4 ꢁ CH2OCO), 2.84 (br t, 8H,
4 ꢁ CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz,
H2-3), 3.87 (dd, 1H, H-50), 4.03e4.13 (dd, 2H, J ¼ 12.0,
5.1.6.6. 1,2-O-Dioleoyl-3-O-(b-D-20,30,40,60-tetra-O-levulinyl-
galactopyranosyl)-rac-glycerol 8f. Yield: 71%. Rf ¼ 0.66
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.91 (br t,
6H, 2 ꢁ CH3 term.), 1.24 (br s, 40H, CH2 aliph.), 1.62 (t,
4H, COCH2CH2), 2.05 (m, 8H, 4 ꢁ CH2CH]CH), 2.10 (4s,
12H, 4 ꢁ CH3CO), 2.40 (br t, 4H, COCH2), 2.61 (br t, 8H,
4 ꢁ CH2OCO), 2.83 (br t, 8H, 4 ꢁ CH2COCH3), 3.66e3.86
(dd, 2H, J ¼ 10.6, 6.3 Hz, H2-3), 4.01 (dd, 1H, H-50), 4.0e
6.6 Hz, H2-1), 4.30e4.37 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60),
0
0
0
0
0
4.55 (dd, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.16 (dd, 1H, J ¼ 10.0,
3.9 Hz, H-20), 5.24 (br s, 1H, H-2), 5.37 (dd, 1H, J ¼ 3.4 Hz,
H-40), 5.42 (dd, 1H, J ¼ 10.1, 3.9 Hz, H-30). ESI-MS:
m/z ¼ 977 (M þ Na, 26%)þ. Anal. calcd. for C49H78O18: C,
61.63; H, 8.18; found: C, 61.60; H, 8.21.
4.15 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd, 2H,
0
J6 a,6 b ¼ 12.0 Hz, H2-60), 4.58 (dd, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ),
5.18 (dd, 1H, J ¼ 9.5, 3.9 Hz, H-20), 5.24 (br s, 1H, H-2),
5.40 (dd, 1H, J ¼ 3.7 Hz, H-40), 5.42 (m, 4H, 2 ꢁ CH]CH ),
5.44 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30). ESI-MS: m/z ¼ 1198
(M þ Na, 83%)þ. Anal. calcd. for C65H106O18: C, 66.44; H,
9.03; found: C, 66.46; H, 9.00.
0
0
0
0
5.1.6.3. 1,2-O-Dilauroyl-3-O-(b-D-20,30,40,60-tetra-O-levulinyl-
galactopyranosyl)-rac-glycerol 8c. Yield: 68%. Rf ¼ 0.59
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.89 (br t,
6H, J ¼ 6.7 Hz), 1.24 (br s, 32H, CH2 aliph.), 1.54 (br t, 4H,
COCH2CH2), 2.11 (4s, 12H, 4 ꢁ CH3CO), 2.31 (br t, 4H,
COCH2), 2.60 (br t, 8H, 4 ꢁ CH2OCO), 2.83 (br t, 8H,
4 ꢁ CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.6, 6.4 Hz, H2-
3), 3.90 (dd, 1H, H-50), 4.02e4.15 (dd, 2H, J ¼ 11.9, 6.6 Hz,
5.1.6.7.
1,2-O-Dilinoleoyl-3-O-(b-D-20,30,40,60-tetra-O-levu-
linyl-galactopyranosyl)-rac-glycerol 8g. Yield: 54%. Rf ¼ 0.65
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.91 (br t,
6H, 2 ꢁ CH3 term.), 1.32 (br s, 28H, CH2 aliph.), 1.63 (br t,
4H, COCH2CH2), 2.02 (m, 8H, 4 ꢁ CH2CH]CH), 2.12 (4s,
12H, 4 ꢁ CH3CO), 2.35 (br t, 4H, COCH2), 2.60 (br t, 8H,
4 ꢁ CH2OCO), 2.83 (br t, 8H, 4 ꢁ CH2COCH3), 2.89 (m,
4H, 2 ꢁ ]CHCH2CH]), 3.65e3.84 (dd, 2H, J ¼ 10.6,
6.3 Hz, H2-3), 4.0 (dd, 1H, H-50), 4.0e4.14 (dd, 2H,
H2-1), 4.30e4.37 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.56
0
0
0
0
0
(dd, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.16 (dd, 1H, J ¼ 9.9, 3.9 Hz,
H-20), 5.24 (br s, 1H, H-2), 5.38 (dd, 1H, J ¼ 3.4 Hz, H-40),
5.43 (dd, 1H, J ¼ 10.0, 3.8 Hz, H-30). ESI-MS: m/z ¼ 1033
(M þ Na, 24%)þ. Anal. calcd. for C53H86O18: C, 62.97; H,
8.51; found: C, 63.00; H, 8.49.
0
0
J ¼ 11.8, 6.5 Hz, H2-1), 4.30e4.36 (dd, 2H, J6 a,6 b ¼ 12.0 Hz,
H2-60), 4.59 (dd, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.17 (dd, 1H,
J ¼ 9.5, 3.9 Hz, H-20), 5.23 (br s, 1H, H-2), 5.40 (dd, 1H,
J ¼ 3.8 Hz, H-40), 5.41 (m, 8H, 4 ꢁ CH]CH ), 5.44 (dd,
1H, J ¼ 10.0, 3.9 Hz, H-30),. ESI-MS: m/z ¼ 1194 (M þ Na,
70%)þ. Anal. calcd. for C65H102O18: C, 66.66; H, 8.72; found:
C, 66.91; H, 8.69.
0
0
0
5.1.6.4. 1,2-O-Dimyristoyl-3-O-(b-D-20,30,40,60-tetra-O-levu-
linyl-galactopyranosyl)-rac-glycerol 8d. Yield: 85%. Rf ¼ 0.65
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.90 (br t,
6H, J ¼ 6.7 Hz), 1.24 (br s, 40H, CH2 aliph.), 1.55 (br t, 4H,
COCH2CH2), 2.10 (4s, 12H, 4 ꢁ CH3CO), 2.31 (br t, 4H,
COCH2), 2.60 (br t, 8H, 4 ꢁ CH2OCO), 2.84 (br t, 8H,
4 ꢁ CH2COCH3), 3.66e3.85 (dd, 2H, J ¼ 10.7, 6.4 Hz, H2-3),
3.97 (dd, 1H, H-50), 4.0e4.15 (dd, 2H, J ¼ 11.9, 6.6 Hz,
5.1.6.8. 1,2-O-Dilinolenoyl-3-O-(b-D-20,30,40,60-tetra-O-levu-
linyl-galactopyranosyl)-rac-glycerol 8h. Yield: 45%. Rf ¼ 0.62
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.97 (br t,
H2-1), 4.30e4.37 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.57 (dd,
0
0
0
0
0
1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.18 (dd, 1H, J ¼ 9.8, 3.9 Hz,
H-20), 5.24 (br s, 1H, H-2), 5.38 (dd, 1H, J ¼ 3.4 Hz, H-40),
5.44 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30). ESI-MS: m/z ¼ 1089
(M þ Na, 48%)þ. Anal. calcd. for C57H94O18: C, 64.16; H,
8.82; found: C, 64.19; H, 8.76.
6H, 2 ꢁ CH3 term.), 1.32 (br s, 16H, CH2 aliph.), 1.63 (br t,
4H, COCH2CH2), 2.07 (m, 8H, 4 ꢁ CH2CH]CH), 2.11 (4s,
12H, 4 ꢁ CH3CO), 2.41 (br t, 4H, COCH2), 2.62 (br t,
8H, 4 ꢁ CH2OCO), 2.83 (br t, 8H, 4 ꢁ CH2COCH3), 2.90 (m,
8H, 2 ꢁ ]CHCH2CH]), 3.67e3.85 (dd, 2H, J ¼ 10.5,
6.2 Hz, H2-3), 4.0 (dd, 1H, H-50), 4.0e4.14 (dd, 2H, J ¼ 11.9,
1,2-O-Distearoyl-3-O-(b-D-20,30,40,60-tetra-O-levu-
6.6 Hz, H2-1), 4.30e4.36 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60),
0
0
5.1.6.5.
0
0
0
linyl-galactopyranosyl)-rac-glycerol 8e. Yield: 80%. Rf ¼ 0.68
4.60 (dd, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.18 (dd, 1H, J ¼ 9.6,
3.9 Hz, H-20), 5.24 (br s, 1H, H-2), 5.40 (dd, 1H, J ¼ 3.9 Hz,
H-40), 5.43 (m, 12H, 6 ꢁ CH]CH ), 5.45 (dd, 1H, J ¼ 10.0,
3.9 Hz, H-30),. ESI-MS: m/z ¼ 1190 (M þ Na, 72%)þ. Anal.
1
(EtOAcehexane 8:1, v:v). H NMR (CDCl3): d 0.89 (br t,
6H, J ¼ 6.6 Hz), 1.24 (br s, 56H, CH2 aliph.), 1.59 (br t, 4H,
COCH2CH2), 2.12 (4s, 12H, 4 ꢁ CH3CO), 2.40 (br t, 4H,

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
217
calcd. for C65H98O18: C, 66.89; H, 8.40; found: C, 66.92;
H, 8.44.
Rf ¼ 0.51 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3):
d 0.96 (br t, 6H, 2 ꢁ CH3 term.), 1.28 (br s, 16H, CH2 aliph.),
1.60 (br t, 4H, COCH2CH2), 1.98 (m, 4H, 2 ꢁ CH2CH]CH),
2.11 (4s, 12H, 4 ꢁ CH3CO), 2.40 (br t, 4H, COCH2), 2.61 (br
t, 8H, 4 ꢁ CH2OCO), 2.82 (br t, 8H, 4 ꢁ CH2COCH3), 2.90
(m, 4H, 2 ꢁ ]CHCH2CH]), 3.66e3.85 (dd, 2H, J ¼ 10.5,
6.2 Hz, H2-3), 4.02 (dd, 1H, H-50), 4.10e4.14 (dd, 2H,
5.1.7. General procedure for preparation of mixed-acid 1,2-
O-diacyl-3-O-(b-D-20,30,40,60-tetra-O-levulinyl-
galactopyranosyl)-rac-glycerols
To a mixture of 7 (1 mmol) and vinyl caprylate (or vinyl ca-
priate) (1.25 mmol) in dry dichloromethane (3.0 ml) was
added immobilized M. miehei lipase (0.06 g). The resulting
mixture was stirred at 37 ꢀC for 8 h. Then, additional lipase
(0.01 g) was added to the reaction mixture which was stirred
for additional 40 h at the same temperature. The lipase was
separated off by filtration and the solvent was removed in va-
cuo on a rotary evaporator. The residue was purified by flash
chromatography on silica gel column, eluted with EtOAce
hexane (8:1/v:v) to give the galactosyl monoglycerides 10a,
b which were subsequently reacted with equimolar amounts
of the second fatty acid, DCC and a little amount of DMAP
at room temperature overnight.
0
0
J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd, 2H, J6 a,6 b ¼ 12.0 Hz,
0
H2-60), 4.65 (dd, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.18 (dd, 1H,
J ¼ 9.6, 3.9 Hz, H-20), 5.23 (br s, 1H, H-2), 5.39 (m, 6H,
3 ꢁ CH]CH ), 5.42 (dd, 1H, J ¼ 3.9 Hz, H-40), 5.45 (dd,
1H, J ¼ 10.0, 3.9 Hz, H-30). ESI-MS: m/z ¼ 1055 (M þ Na,
90%)þ. Anal. calcd. for C55H84O18: C, 63.95; H, 8.14; found:
C, 63.91; H, 8.00.
0
0
5.1.7.4. 1-O-Caprinyl-2-O-oleoyl-3-O-(b-D-20,30,40,60-tetra-O-
levulinyl-galactopyranosyl)-rac-glycerol 12a. Yield: 36%.
Rf ¼ 0.51 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3):
d 0.96 (br t, 6H, 2 ꢁ CH3 term.), 1.28 (br s, 32H, CH2 aliph.),
1.62 (br t, 4H, COCH2CH2), 1.99 (m, 4H, 2 ꢁ CH2CH]CH),
2.10 (4s, 12H, 4 ꢁ CH3CO), 2.40 (br t, 4H, COCH2), 2.63 (br
t, 8H, 4 ꢁ CH2OCO), 2.83 (br t, 8H, 4 ꢁ CH2COCH3), 3.67e
3.85 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.01 (dd, 1H, H-50),
4.10e4.14 (dd, 2H, J ¼ 11.9, 6.7 Hz, H2-1), 4.30e4.36 (dd,
The solid was filtered off and the solvent was evaporated in
vacuo. The residue was purified by flash chromatography on sil-
ica gel column, eluted with EtOAcehexane (8:1, v:v) to give the
1:1 mixture of two diastereoisomers 11aec and 12aec.
5.1.7.1. 1-O-Caprylyl-2-O-oleoyl-3-O-(b-D-20,30,40,60-tetra-O-
levulinyl-galactopyranosyl)-rac-glycerol 11a. Yield: 39%.
Rf ¼ 0.52 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3):
d 0.95 (br t, 6H, 2 ꢁ CH3 term.), 1.28 (br s, 28H, CH2 aliph.),
1.62 (br t, 4H, COCH2CH2), 2.00 (m, 4H, 2 ꢁ CH2CH]CH),
2.10 (4s, 12H, 4 ꢁ CH3CO), 2.40 (br t, 4H, COCH2), 2.62 (br
t, 8H, 4 ꢁ CH2OCO), 2.82 (br t, 8H, 4 ꢁ CH2COCH3), 3.67e
3.84 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.02 (dd, 1H, H-50),
4.10e4.14 (dd, 2H, J ¼ 11.9, 6.6 Hz, H2-1), 4.30e4.36 (dd,
2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.64 (dd, 1H, J1 ,2 ¼ 8.0 Hz,
H-10), 5.19 (dd, 1H, J ¼ 9.6, 3.9 Hz, H-20), 5.23 (br s, 1H,
H-2), 5.39 (m, 2H, CH]CH ), 5.42 (dd, 1H, J ¼ 3.9 Hz,
H-40), 5.45 (dd, 1H, J ¼ 9.8, 3.9 Hz, H-30). ESI-MS: m/
z ¼ 1087 (M þ Na, 100%)þ. Anal. calcd. for C57H92O18: C,
64.28; H, 8.64; found: C, 64.32; H, 8.66.
0
0
0
0
5.1.7.5. 1-O-Caprinyl-2-O-linoleoyl-3-O-(b-D-20,30,40,60-tetra-
O-levulinyl-galacto-pyranosyl)-rac-glycerol 12b. Yield: 39%.
Rf ¼ 0.50 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3):
d 0.95 (br t, 6H, 2 ꢁ CH3 term.), 1.28 (br s, 28H, CH2 aliph.),
1.60 (br t, 4H, COCH2CH2), 2.00 (m, 4H, 2 ꢁ CH2CH]CH),
2.10 (4s, 12H, 4 ꢁ CH3CO), 2.40 (br t, 4H, COCH2), 2.63 (br
t, 8H, 4 ꢁ CH2OCO), 2.82 (br t, 8H, 4 ꢁ CH2COCH3), 2.90
(m, 2H, ]CHCH2CH]), 3.66e3.84 (dd, 2H, J ¼ 10.5,
6.2 Hz, H2-3), 4.0 (dd, 1H, H-50), 4.10e4.14 (dd, 2H,
2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.60 (dd, 1H, J1 ,2 ¼ 8.0 Hz,
H-10), 5.18 (dd, 1H, J ¼ 9.6, 3.9 Hz, H-20), 5.23 (br s, 1H,
H-2), 5.40 (dd, 1H, J ¼ 3.9 Hz, H-40), 5.43 (m, 2H, CH]CH ),
5.45 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30),. ESI-MS: m/z ¼ 1060
(M þ Na, 80%)þ. Anal. calcd. for C55H88O18: C, 63.71; H,
8.49; found: C, 63.75; H, 8.47.
0
0
0
0
5.1.7.2. 1-O-Caprylyl-2-O-linoleoyl-3-O-(b-D-20,30,40,60-tetra-
O-levulinyl-galactopyranosyl)-rac-glycerol 11b. Yield: 38%.
Rf ¼ 0.52 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3): d 0.93
(br t, 6H, 2 ꢁ CH3 term.), 1.28 (br s, 24H, CH2 aliph.), 1.61
(br t, 4H, COCH2CH2), 2.01 (m, 4H, 2 ꢁ CH2CH]CH), 2.10
(4s, 12H, 4 ꢁ CH3CO), 2.40 (br t, 4H, COCH2), 2.63 (br t, 8H,
4 ꢁ CH2OCO), 2.82 (br t, 8H, 4 ꢁ CH2COCH3), 2.90 (m, 2H,
]CHCH2CH]), 3.67e3.84 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3),
4.02 (dd, 1H, H-50), 4.10e4.14 (dd, 2H, J ¼ 11.9, 6.6 Hz,
0
0
J ¼ 11.8, 6.5 Hz, H2-1), 4.30e4.36 (dd, 2H, J6 a,6 b ¼ 12.0 Hz,
0
H2-60), 4.65 (dd, 1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.20 (dd, 1H,
J ¼ 9.7, 3.9 Hz, H-20), 5.24 (br s, 1H, H-2), 5.40 (m, 4H,
2 ꢁ CH]CH ), 5.43 (dd, 1H, J ¼ 3.9 Hz, H-40), 5.45 (dd,
1H, J ¼ 9.8, 3.9 Hz, H-30). ESI-MS: m/z ¼ 1086 (M þ Na,
75%)þ. Calcd. for C57H90O18: C, 64.41; H, 8.47; found: C,
64.38; H, 8.51.
0
0
H2-1), 4.30e4.36 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.64 (dd,
5.1.7.6. 1-O-Caprinyl-2-O-linolenoyl-3-O-(b-D-20,30,40,60-tetra-
O-levulinyl-galacto-pyranosyl)-rac-glycerol 12c. Yield: 42%.
Rf ¼ 0.51 (EtOAcehexane 8:1, v:v). 1H NMR (CDCl3):
d 0.93 (br t, 6H, 2 ꢁ CH3 term.), 1.29 (br s, 20H, CH2
aliph.), 1.61 (br t, 4H, COCH2CH2), 2.08 (m, 4H,
2 ꢁ CH2CH]CH), 2.10 (4s, 12H, 4 ꢁ CH3CO), 2.36 (br t,
4H, COCH2), 2.64 (br t, 8H, 4 ꢁ CH2OCO), 2.83 (br t,
8H, 4 ꢁ CH2COCH3), 2.90 (m, 4H, 2 ꢁ ]CHCH2CH]),
3.66e3.84 (dd, 2H, J ¼ 10.5, 6.2 Hz, H2-3), 4.02 (dd, 1H,
0
0
0
0
0
0
1H, J1 ,2 ¼ 8.0 Hz, H-1 ), 5.19 (dd, 1H, J ¼ 9.6, 3.8 Hz, H-2 ),
5.23 (br s, 1H, H-2), 5.39 (m, 4H, CH]CH ), 5.40 (dd, 1H,
J ¼ 3.9 Hz, H-40), 5.45 (dd, 1H, J ¼ 10.0, 3.9 Hz, H-30),. ESI-
MS: m/z ¼ 1058 (M þ Na, 80%)þ. Anal. calcd. for
C55H86O18: C, 63.83; H, 8.32; found: C, 63.85; H, 8.30.
5.1.7.3. 1-O-Caprylyl-2-O-linolenoyl-3-O-(b-D-20,30,40,60-tetra-
O-levulinyl-galacto-pyranosyl)-rac-glycerol 11c. Yield: 40%.

218
F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
H-50), 4.10e4.14 (dd, 2H, J ¼ 11.7, 6.4 Hz, H2-1), 4.30e
(M þ Na, 26%)þ. Anal. calcd. for C29H54O10: C, 61.92; H,
4.36 (dd, 2H, J6 a,6 b ¼ 12.0 Hz, H2-60), 4.64 (dd, 1H,
9.61; found: C, 61.87; H, 9.58.
0
0
0
0
0
0
0
J1 ,2 ¼ 8.0 Hz, H-1 ), 5.19 (dd, 1H, J2 ,3 ¼ 9.8, 3.7 Hz,
H-20), 5.24 (br s, 1H, H-2), 5.37 (m, 6H, 3 ꢁ CH]CH ),
5.42 (dd, 1H, J ¼ 3.9 Hz, H-40), 5.45 (dd, 1H,þ J ¼ 9.8,
3.9 Hz, H-30). ESI-MS: m/z ¼ 1084 (M þ Na, 87%) . Calcd.
for C57H88O18: C, 64.53; H, 8.30; found: C, 64.58; H, 8.33.
5.1.8.3. 1,2-O-Dilauroyl-3-O-(b-D-galactopyranosyl)-rac-glyc-
erol 9c. Yield: 79%. Rf ¼ 0.49 (CH2Cl2eMeOH 10:1, v:v). 1H
NMR (400 MHz, CDCl3): d 0.90 (br t, 6H, J ¼ 6.7 Hz, CH3
term.), 1.23 (br s, 32H, CH2 aliph.), 1.55 (br t, 4H, COCH2CH2),
2.36 (br t, 4H, J ¼ 6.9 Hz, COCH2), 3.53 (m, 1H, H-50), 3.58 (m,
1H, H-30), 3.60 (m, 1H, H-20), 3.62, 3.65 (dd, 1H, J ¼ 10.7,
6.4 Hz, Ha-3), 3.81 (br s, 2H, H2-60), 3.86 (br d, 1H,
J ¼ 10.6 Hz, Hb-3), 3.98 (m, 1H, H-40), 4.09, 4.14 (dd, 1H,
J ¼ 12.0, 3.3 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.8 Hz, H-10), 4.36
(br d, 1H, J ¼ 10.4 Hz, Hb-1), 5.24 (br s, 1H, H-2). 13C NMR
(100 MHz, CDCl3): d 173.6, 173.1 (COO), 104.2 (C-10), 74.8
(C-50), 73.6 (C-30), 71.4 (C-20), 70.6, 70.5 (C-2), 69.2 (C-40),
68.4 (C-3), 63.1, 62.9 (C-1), 61.9 (C-60), 33.8, 33.7 (COCH2),
32.3e23.0 (CH2 aliph.), 25.3, 25.2 (COCH2CH2), 14.5 (CH3).
ESI-MS: m/z ¼ 641 (M þ Na, 24%)þ. Anal. calcd. for
C33H62O10: C, 64.08; H, 10.03; found: C, 63.99; H, 10.01.
5.1.8. General procedure for selective removal of levulinyl
protecting groups from 1,2-O-diacyl-3-O-(b-D-20,30,40,60-
tetra-O-levulinyl-galactopyranosyl)-rac-glycerols
To a solution of 8aeh, 11aec and 12aec (0.1 mmol) in dry
pyridine, 1 M NH2NH2$H2O in pyridineeglacial acetic acid
(3:2, v:v) was added. Each mixture was stirred for 1 h at
room temperature, under inert atmosphere, the mixture was
poured into ice-cold water, extracted with CH2Cl2
(3 ꢁ 50 ml) and washed with 10% NaHCO3 and brine. The
combined CH2Cl2 layers were dried (anhydrous Na2SO4)
and individually evaporated to give a syrupy mixture. Each
mixture was purified by ‘flash chromatography’ on silica gel
column, eluted with CH2Cl2eMeOH (10:1) to give 1:1 mix-
tures of two diastereoisomers 9aeh, 13aec and 14aec.
5.1.8.4. 1,2-O-Dimyristoyl-3-O-(b-D-galactopyranosyl)-rac-
glycerol 9d. Yield: 80%. Rf ¼ 0.49 (CH2Cl2eMeOH 10:1,
v:v); 1H NMR (400 MHz, CDCl3): d 0.89 (br t, 6H,
J ¼ 6.6 Hz, CH3 term.), 1.23 (br s, 40H, CH2 aliph.), 1.55 (br
t, 4H, COCH2CH2), 2.33 (br t, 4H, J ¼ 7.0 Hz, COCH2), 3.53
(m, 1H, H-50), 3.57 (m, 1H, H-30), 3.61 (m, 1H, H-20), 3.62,
3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60),
3.85 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.98 (m, 1H, H-40), 4.09,
4.13 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.5 Hz,
H-10), 4.35 (br d, 1H, J ¼ 10.5 Hz, Hb-1), 5.22 (br s, 1H, H-2).
13C NMR (100 MHz, CDCl3): d 173.4, 173.3 (COO), 104.2
(C-10), 74.9 (C-50), 73.7 (C-30), 71.5 (C-20), 70.6, 70.5 (C-2),
69.4 (C-40), 68.6 (C-3), 63.3, 63.2 (C-1), 62.0 (C-60), 33.8, 34.2
(COCH2), 32.4e23.1 (CH2 aliph.), 25.4, 25.3 (COCH2CH2),
14.6 (CH3). ESI-MS: m/z ¼ 697 (M þ Na, 48%)þ. Anal. calcd.
for C37H70O10: C, 65.87; H, 10.38; found: C, 65.90; H, 10.40.
5.1.8.1.
1,2-O-Dicaprylyl-3-O-(b-D-galactopyranosyl)-rac-
glycerol 9a. Yield: 82%. Rf ¼ 0.48 (CH2Cl2eMeOH 10:1,
v:v). 1H NMR (400 MHz, CDCl3): d 0.90 (br t, 6H,
J ¼ 6.7 Hz, CH3 term.), 1.25 (br s, 16H, CH2 aliph.), 1.55
(br t, 4H, COCH2CH2), 2.30 (br t, 4H, J ¼ 7.0 Hz, COCH2),
3.52 (m, 1H, H-50), 3.57 (m, 1H, H-30), 3.60 (m, 1H, H-20),
3.63, 3.66 (dd, 1H, J ¼ 10.6, 6.3 Hz, Ha-3), 3.81 (br s, 2H,
H2-60), 3.87 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H-
40), 4.11, 4.15 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.22 (d, 1H,
J ¼ 7.8 Hz, H-10), 4.38 (br d, 1H, J ¼ 12.0 Hz, Hb-1), 5.22
(br s, 1H, H-2). 13C NMR (100 MHz, CDCl3): d 173.3,
173.2 (COO), 104.2 (C-10), 74.9 (C-50), 73.6 (C-30), 71.5
(C-20), 70.5 (C-2), 69.2 (C-40), 68.4 (C-3), 63.0, 62.9 (C-1),
61.8 (C-60), 33.8, 33.7 (COCH2), 32.3e23.2 (CH2 aliph.),
25.5, 25.4 (COCH2CH2), 14.5 (CH3). ESI-MS: m/z ¼ 529
(M þ Na, 25%)þ. Anal. calcd. for C25H46O10: C, 59.29; H,
9.09; found: C, 59.33; H, 9.12.
5.1.8.5.
1,2-O-Distearoyl-3-O-(b-D-galactopyranosyl)-rac-
glycerol 9e. Yield: 81%. Rf ¼ 0.51 (CH2Cl2eMeOH 10:1,
v:v). 1H NMR (400 MHz, CDCl3): d 0.88 (br t, 6H,
J ¼ 6.7 Hz, CH3 term.), 1.25 (br s, 56H, CH2 aliph.), 1.54
(br t, 4H, COCH2CH2), 2.39 (br t, 4H, J ¼ 7.0 Hz, COCH2),
3.52 (m, 1H, H-50), 3.57 (m, 1H, H-30), 3.61 (m, 1H, H-20),
3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H,
H2-60), 3.87 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.97 (m, 1H, H-
40), 4.08, 4.13 (dd, 1H, J ¼ 11.9, 4.4 Hz, Ha-1), 4.21 (d, 1H,
J ¼ 7.5 Hz, H-10), 4.35 (br d, 1H, J ¼ 11.2 Hz, Hb-1), 5.25
(br s, 1H, H-2). 13C NMR (100 MHz, CDCl3): d 173.8,
173.7 (COO), 104.1 (C-10), 74.9 (C-50), 73.6 (C-30), 71.5
(C-20), 70.6, 70.5 (C-2), 69.4 (C-40), 68.5 (C-3), 63.2, 63.1
(C-1), 62.0 (C-60), 34.2, 34.1 (COCH2), 32.3e23.0 (CH2
aliph.), 25.4, 25.3 (COCH2CH2), 14.6 (CH3). ESI-MS: m/
z ¼ 809 (M þ Na, 41%)þ. Anal. calcd. for C45H86O10: C,
68.70; H, 10.94; found: C, 68.73; H, 10.88.
5.1.8.2.
1,2-O-Dicaprinyl-3-O-(b-D-galactopyranosyl)-rac-
glycerol 9b. Yield: 79%. Rf ¼ 0.48 (CH2Cl2eMeOH 10:1,
v:v). 1H NMR (400 MHz, CDCl3): d 0.90 (br t, 6H,
J ¼ 6.7 Hz, CH3 term.), 1.24 (br s, 24H, CH2 aliph.), 1.54
(br t, 4H, COCH2CH2), 2.34 (br t, 4H, J ¼ 7.0 Hz, COCH2),
3.52 (m, 1H, H-50), 3.58 (m, 1H, H-30), 3.61 (m, 1H, H-20),
3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H,
H2-60), 3.85 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.97 (m, 1H, H-
40), 4.08, 4.12 (dd, 1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.21 (d, 1H,
J ¼ 7.5 Hz, H-10), 4.38 (br d, 1H, J ¼ 10.4 Hz, Hb-1), 5.23
(br s, 1H, H-2). 13C NMR (100 MHz, CDCl3): d 173.6,
173.1 (COO), 104.3 (C-10), 74.8 (C-50), 73.6 (C-30), 71.6
(C-20), 70.4 (C-2), 69.4 (C-40), 68.4 (C-3), 63.2, 63.1 (C-1),
61.9 (C-60), 33.8, 33.7 (COCH2), 32.4e23.0 (CH2 aliph.),
25.3, 25.2 (COCH2CH2), 14.6 (CH3). ESI-MS: m/z ¼ 585
5.1.8.6. 1,2-O-Dioleoyl-3-O-(b-D-galactopyranosyl)-rac-glyc-
erol 9f. Yield: 75%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1, v:v).

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
219
1H NMR (400 MHz, CDCl3): d 0.91 (br t, 6H, J ¼ 6.6 Hz, CH3
term.), 1.24 (br s, 40H, CH2 aliph.), 1.56 (br t, 4H,
COCH2CH2), 2.0 (br, 8H, 4 ꢁ CH2CH]CH), 2.35 (br t, 4H,
J ¼ 6.9 Hz, COCH2), 3.52 (m, 1H, H-50), 3.58 (m, 1H,
H-30), 3.60 (m, 1H, H-20), 3.62, 3.65 (dd, 1H, J ¼ 10.7,
6.4 Hz, Ha-3), 3.81 (br s, 2H, H2-60), 3.88 (br d, 1H,
J ¼ 10.6 Hz, Hb-3), 3.97 (m, 1H, H-40), 4.10, 4.14 (dd, 1H,
J ¼ 12.0, 4.4 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.8 Hz, H-10), 4.35
(br d, 1H, J ¼ 12.1 Hz, Hb-1), 5.24 (br s, 1H, H-2), 5.25e
5.40 (m, 4H, 2 ꢁ CH]CH ). 13C NMR (100 MHz, CDCl3):
d 173.9, 173.4 (COO), 130.3, 130.1 (CH]CH), 104.2 (C-
10), 74.8 (C-50), 73.7 (C-30), 71.6 (C-20), 70.5, 70.4 (C-2),
69.3 (C-40), 68.6 (C-3), 63.0, 62.9 (C-1), 61.9 (C-60), 33.8,
33.7 (COCH2), 32.3e22.9 (CH2 aliph.), 27.7, 27.5
(CH2CH]CH), 25.5, 25.4 (COCH2CH2), 14.5 (CH3). ESI-
MS: m/z ¼ 806 (M þ Na, 100%)þ. Anal. calcd. for
C45H82O10: C, 69.05; H, 10.48; found: C, 68.99; H, 10.52.
(CH2 aliph.), 27.7, 27.6, 27.5 (CH2CH]CH), 26.1, 26.0
(]CHCH2CH]), 25.5, 25.4 (COCH2CH2), 14.7 (CH3).
ESI-MS: m/z ¼ 798 (M þ Na, 100%)þ. Anal. calcd. for
C45H74O10: C, 69.77; H, 9.56; found: C, 69.91; H, 9.19.
5.1.8.9. 1-O-Caprylyl-2-O-Oleoyl-3-O-(b-D-galactopyranosyl)-
rac-glycerol 13a. Yield: 71%. Rf ¼ 0.49 (CH2Cl2eMeOH
1
10:1, v:v). H NMR (400 MHz, CDCl3): d 0.95 (br t, 6H,
J ¼ 6.8 Hz, CH3 term.), 1.23 (br s, 28H, CH2 aliph.), 1.56
(br t, 4H, COCH2CH2), 1.99 (br, 4H, 2 ꢁ CH2CH]CH),
2.39 (br t, 4H, J ¼ 7.0 Hz, COCH2), 3.52 (m, 1H, H-50),
3.58 (m, 1H, H-30), 3.60 (m, 1H, H-20), 3.62, 3.65 (dd, 1H,
J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60), 3.88 (br d,
1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H-40), 4.10, 4.15 (dd,
1H, J ¼ 12.0, 3.4 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.5 Hz, H-10),
4.35 (br d, 1H, J ¼ 12.1 Hz, Hb-1), 5.24 (br s, 1H, H-2),
5.20e5.40 (m, 2H, CH]CH ). 13C NMR (100 MHz,
CDCl3): d 174.2, 174.1 (COO), 130.4, 130.1 (CH]CH),
104.3 (C-10), 74.8 (C-50), 73.6 (C-30), 71.4 (C-20), 70.6, 70.5
(C-2), 69.4 (C-40), 68.5 (C-3), 63.0, 62.9 (C-1), 61.8 (C-60),
34.6, 34.5 (COCH2), 32.3e23.2 (CH2 aliph.), 27.6, 27.5
(CH2CH]CH), 25.3, 25.2 (COCH2CH2), 14.5 (CH3). ESI-
MS: m/z ¼ 668 (M þ Na, 100%)þ. Anal. calcd. for
C35H64O10: C, 65.22; H, 9.94; found: C, 65.19; H, 10.00.
5.1.8.7.
1,2-O-Dilinoleoyl-3-O-(b-D-galactopyranosyl)-rac-
glycerol 9g. Yield: 70%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1,
v:v). 1H NMR (400 MHz, CDCl3): d 0.91 (br t, 6H,
J ¼ 6.7 Hz, CH3 term.), 1.22 (br s, 28H, CH2 aliph.), 1.56
(br t, 4H, COCH2CH2), 1.99 (br, 8H, 4 ꢁ CH2CH]CH),
2.33 (br t, 4H, J ¼ 6.9 Hz, COCH2), 2.82 (br t, 4H,
J ¼ 5.5 Hz, 2 ꢁ ]CHCH2CH]), 3.52 (m, 1H, H-50), 3.58
(m, 1H, H-30), 3.60 (m, 1H, H-20), 3.63, 3.66 (dd, 1H,
J ¼ 10.7, 6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60), 3.89 (br d,
1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H-40), 4.09, 4.13 (dd,
1H, J ¼ 12.0, 3.3 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.8 Hz, H-10),
4.34 (br d, 1H, J ¼ 12.0 Hz, Hb-1), 5.23 (br s, 1H, H-2),
5.30e5.38 (m, 8H, 4 ꢁ CH]CH ). 13C NMR (100 MHz,
CDCl3): d 173.8, 173.4 (COO), 130.5, 130.4, 128.4, 128.3
(CH]CH), 104.3 (C-10), 74.8 (C-50), 73.7 (C-30), 71.6
(C-20), 70.5 (C-2), 69.2 (C-40), 68.6 (C-3), 63.3, 63.2 (C-1),
61.7 (C-60), 33.8, 33.7 (COCH2), 30.3e21.1 (CH2 aliph.),
27.5 (CH2CH]CH), 26.0 (]CHCH2CH]), 25.5, 25.4
(COCH2CH2), 14.4 (CH3). ESI-MS: m/z ¼ 802 (M þ Na,
100%)þ. Anal. calcd. for C45H78O10: C, 69.41; H, 10.02;
found: C, 69.37; H, 10.05.
5.1.8.10. 1-O-Caprylyl-2-O-linoleoyl-3-O-(b-D-galactopyrano-
syl)-rac-glycerol 13b. Yield: 72%. Rf ¼ 0.50 (CH2Cl2eMeOH
1
10:1, v:v). H NMR (400 MHz, CDCl3): d 0.93 (br t, 6H,
J ¼ 6.8 Hz, CH3 term.), 1.25 (br s, 24H, CH2 aliph.), 1.55 (br
t, 4H, COCH2CH2), 2.0 (br, 4H, 2 ꢁ CH2CH]CH), 2.30 (br t,
4H, J ¼ 7.0 Hz, COCH2), 2.87 (br t, 2H, J ¼ 5.6 Hz,
]CHCH2CH]), 3.52 (m, 1H, H-50), 3.58 (m, 1H, H-30), 3.61
(m, 1H, H-20), 3.63, 3.66 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3),
3.81 (br s, 2H, H2-60), 3.89 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.96
(m, 1H, H-40), 4.09, 4.14 (dd, 1H, J ¼ 12.0, 3.4 Hz, Ha-1),
4.21 (d, 1H, J ¼ 7.8 Hz, H-10), 4.34 (br d, 1H, J ¼ 12.0 Hz,
Hb-1), 5.23 (br s, 1H, H-2), 5.20e5.40 (m, 4H, 2 ꢁ CH]CH ).
13C NMR (100 MHz, CDCl3): d 173.5, 173.2 (COO), 130.6,
130.4, 128.4, 128.3 (CH]CH), 104.1 (C-10), 74.9 (C-50), 73.6
(C-30), 71.6 (C-20), 70.6, 70.5 (C-2), 69.3 (C-40), 68.5 (C-3),
63.3, 63.2 (C-1), 61.8 (C-60), 34.3, 34.1 (COCH2), 32.4e23.2
(CH2 aliph.), 27.6 (CH2CH]CH), 26.0 (]CHCH2CH]),
25.3, 25.2 (COCH2CH2), 14.7 (CH3). ESI-MS: m/z ¼ 666
(M þ Na, 100%)þ. Anal. calcd. for C35H62O10: C, 65.42; H,
9.65; found: C, 65.91; H, 9.69.
5.1.8.8. 1,2-O-Dilinolenoyl-3-O-(b-D-galactopyranosyl)-rac-
glycerol 9h. Yield: 70%. Rf ¼ 0.50 (CH2Cl2eMeOH 10:1,
v:v). 1H NMR (400 MHz, CDCl3): d 0.97 (br t, 6H,
J ¼ 6.8 Hz, CH3 term.), 1.23 (br s, 16H, CH2 aliph.), 1.54
(br t, 4H, COCH2CH2), 2.10 (br, 8H, 4 ꢁ CH2CH]CH),
2.35 (br t, 4H, J ¼ 6.9 Hz, COCH2), 2.90 (br t, 8H,
J ¼ 5.6 Hz, 4 ꢁ ]CHCH2CH]), 3.53 (m, 1H, H-50), 3.57
(m, 1H, H-30), 3.61 (m, 1H, H-20), 3.63, 3.66 (dd, 1H,
J ¼ 10.6, 6.3 Hz, Ha-3), 3.81 (br s, 2H, H2-60), 3.87 (br d,
1H, J ¼ 10.6 Hz, Hb-3), 3.96 (m, 1H, H-40), 4.10, 4.13 (dd,
1H, J ¼ 11.9, 3.3 Hz, Ha-1), 4.21 (d, 1H, J ¼ 7.5 Hz, H-10),
4.38 (br d, 1H, J ¼ 12.0 Hz, Hb-1), 5.22 (br s, 1H, H-2),
5.30e5.41 (m, 12H, 6 ꢁ CH]CH ). 13C NMR (100 MHz,
CDCl3): d 173.8, 173.4 (COO), 132.2, 130.4, 130.1, 128.6,
128.1, 127.5 (CH]CH), 104.2 (C-10), 74.9 (C-50), 73.7
(C-30), 71.5 (C-20), 70.5 (C-2), 69.3 (C-40), 68.7 (C-3), 63.2,
63.1 (C-1), 61.8 (C-60), 34.3, 34.1 (COCH2), 30.3e21.1
5.1.8.11. 1-O-Caprylyl-2-O-linolenoyl-3-O-(b-D-galactopyra-
nosyl)-rac-glycerol 13c. Yield: 69%. Rf ¼ 0.50 (CH2Cl2e
1
MeOH 10:1, v:v). H NMR (400 MHz, CDCl3): d 0.96 (br t,
6H, J ¼ 6.8 Hz, CH3 term.), 1.25 (br s, 16H, CH2 aliph.),
1.57 (br t, 4H, COCH2CH2), 2.10 (br, 4H, 2 ꢁ CH2CH]CH),
2.31 (br t, 4H, J ¼ 7.0 Hz, COCH2), 2.90 (br t, 4H, J ¼ 5.5 Hz,
2 ꢁ ]CHCH2CH]), 3.53 (m, 1H, H-50), 3.57 (m, 1H, H-30),
3.60 (m, 1H, H-20), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3),
3.80 (br s, 2H, H2-60), 3.85 (br d, 1H, J ¼ 10.5 Hz, Hb-3), 3.97
(m, 1H, H-40), 4.10, 4.13 (dd, 1H, J ¼ 11.9, 3.3 Hz, Ha-1), 4.21
(d, 1H, J ¼ 7.5 Hz, H-10), 4.35 (br d, 1H, J ¼ 10.5 Hz, Hb-1),

220
F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
5.22 (br s, 1H, H-2), 5.23e5.39 (m, 6H, 3 ꢁ CH]CH ). 13C
NMR (100 MHz, CDCl3): d 173.6, 173.5 (COO), 132.3,
130.4, 130.2, 128.5, 128.0, 127.6 (CH]CH), 104.2 (C-10),
74.9 (C-50), 73.7 (C-30), 71.5 (C-20), 70.6 (C-2), 70.0 (C-60),
69.4 (C-40), 68.6 (C-3), 63.1, 62.9 (C-1), 34.3, 34.2
(COCH2), 32.3e23.1 (CH2 aliph.), 27.7, 27.6, 27.5
(CH2CH]CH), 26.2, 26.0 (]CHCH2CH]), 25.5, 25.4
(COCH2CH2), 14.8 (CH3). ESI-MS: m/z ¼ 662 (M þ Na,
100%)þ. Anal. calcd. for C35H60O10: 65.62; H, 9.37; found:
C, 65.69; H, 9.40.
2.35 (br t, 4H, J ¼ 6.9 Hz, COCH2), 2.91 (br t, 4H, J ¼ 5.5 Hz,
2 ꢁ ]CHCH2CH]), 3.53 (m, 1H, H-50), 3.57 (m, 1H, H-30),
3.60 (m, 1H, H-20), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz, Ha-3),
3.80 (br s, 2H, H2-60), 3.88 (br d, 1H, J ¼ 10.6 Hz, Hb-3), 3.97
(m, 1H, H-40), 4.09, 4.15 (dd, 1H, J ¼ 12.0, 3.4 Hz, Ha-1), 4.21
(d, 1H, J ¼ 7.5 Hz, H-10), 4.35 (br d, 1H, J ¼ 12.1 Hz, Hb-1),
5.24 (br s, 1H, H-2), 5.20e5.40 (m, 6H, 3 ꢁ CH]CH ). 13C
NMR (100 MHz, CDCl3): d 173.6, 173.5 (COO), 132.3,
130.4, 130.2, 128.5, 128.0, 127.6 (CH]CH), 104.2 (C-10),
74.9 (C-50), 73.6 (C-30), 71.6 (C-20), 70.6, 70.5 (C-2), 70.0
(C-60), 69.4 (C-40), 68.5 (C-3), 63.3, 63.2 (C-1), 34.4, 34.2
(COCH2), 32.3e23.0 (CH2 aliph.), 27.7, 27.6, 27.5
(CH2CH]CH), 26.2, 26.0 (]CHCH2CH]), 25.4, 25.3
(COCH2CH2), 14.8 (CH3). ESI-MS: m/z ¼ 692 (M þ Na,
100%)þ. Anal. calcd. for C37H64O10: C, 66.47; H, 9.58; found:
C, 66.50; H, 9.62.
5.1.8.12. 1-O-Caprinyl-2-O-oleoyl-3-O-(b-D-galactopyrano-
syl)-rac-glycerol 14a. Yield: 75%. Rf ¼ 0.50 (CH2Cl2e
1
MeOH 10:1, v:v). H NMR (400 MHz, CDCl3): d 0.94 (br t,
6H, J ¼ 6.8 Hz, CH3 term.), 1.24 (br s, 32H, CH2 aliph.),
1.55 (br t, 4H, COCH2CH2), 1.99 (br, 4H, 2 ꢁ CH2CH]CH),
2.40 (br t, 4H, J ¼ 6.9 Hz, COCH2), 3.53 (m, 1H, H-50), 3.58
(m, 1H, H-30), 3.61 (m, 1H, H-20), 3.62, 3.65 (dd, 1H, J ¼ 10.7,
6.4 Hz, Ha-3), 3.80 (br s, 2H, H2-60), 3.85 (br d, 1H,
J ¼ 10.5 Hz, Hb-3), 3.98 (m, 1H, H-40), 4.09, 4.13 (dd, 1H,
J ¼ 12.0, 3.4 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.8 Hz, H-10), 4.38
(br d, 1H, J ¼ 10.4 Hz, Hb-1), 5.23 (br s, 1H, H-2), 5.20e
5.40 (m, 2H, CH]CH ). 13C NMR (100 MHz, CDCl3):
d 173.5, 173.4 (COO), 130.3, 130.2 (CH]CH), 104.1
(C-10), 74.9 (C-50), 73.6 (C-30), 71.6 (C-20), 70.5, 70.4 (C-2),
69.2 (C-40), 68.4 (C-3), 63.3, 63.2 (C-1), 61.9 (C-60), 34.1,
33.9 (COCH2), 32.3e23.0 (CH2 aliph.), 27.6, 27.5
(CH2CH]CH), 25.5, 25.4 (COCH2CH2), 14.5 (CH3). ESI-
MS: m/z ¼ 696 (M þ Na, 100%)þ. Anal. calcd. for
C37H68O10: C, 66.07; H, 10.12; found: C, 66.10; H, 10.09.
5.1.9. The antibacterial and antifungal activity of
compounds 9a, b, 13aec and 14aec
All the synthesized 1,2-O-diacyl-3-O-b-D-galactopyranosyl-
rac-glycerols 9aeh, 13aec, 14aec have been evaluated for their
in vitro activity against a number of Gram positive and Gram neg-
ative reference strains: S. aureus ATCC 25923, S. aureus ATCC
105487, E. faecalis ATCC 29212, B. subtilis ATCC 6633, E. coli
ATCC 25922, P. aeruginosa ATCC 27753, M. tuberculosis
H37Rv and against four different S. aureus, clinical strains
SA52, SA85, SA87 and two M. tuberculosis clinical isolates,
H160 and H190.
The antifungal activity of all compounds was evaluated
against two strains, clinical isolates, of C. albicans and C. pseu-
dotropicalis. Antibacterial activity was evaluated by a reference
agar dilution method [28]. MICs were determined twice in du-
plicate experiments. Antitubercular activity was evaluated by
a standard agar dilution method and by a recently developed col-
our assay[29]. MIC was defined as the lowest drug concentration
that prevented Resazurin colour change and was determined
twice in duplicate experiments. Ciprofloxacin was chosen as
a standard in antibacterial activity measurements. Antifungal
activity has been tested against Candida spp. clinical isolates
measuring MICs by a microdilution RPMI reference method,
in comparison with miconazole [30].
5.1.8.13. 1-O-Caprinyl-2-O-linoleoyl-3-O-(b-D-galactopyrano-
syl)-rac-glycerol 14b. Yield: 69%. Rf ¼ 0.50 (CH2Cl2eMeOH
1
10:1, v:v). H NMR (400 MHz, CDCl3): d 0.95 (br t, 6H,
J ¼ 6.7 Hz, CH3 term.), 1.24 (br s, 28H, CH2 aliph.), 1.54
(br t, 4H, COCH2CH2), 2.0 (br, 4H, 2 ꢁ CH2CH]CH), 2.33
(br t, 4H, J ¼ 7.0 Hz, COCH2), 2.88 (br t, 2H, J ¼ 5.6 Hz,
]CHCH2CH]), 3.52 (m, 1H, H-50), 3.58 (m, 1H, H-30),
3.60 (m, 1H, H-20), 3.62, 3.65 (dd, 1H, J ¼ 10.7, 6.4 Hz,
Ha-3), 3.81 (br s, 2H, H2-60), 3.89 (br d, 1H, J ¼ 10.6 Hz,
Hb-3), 3.97 (m, 1H, H-40), 4.10, 4.14 (dd, 1H, J ¼ 12.0,
4.4 Hz, Ha-1), 4.22 (d, 1H, J ¼ 7.8 Hz, H-10), 4.35 (br d, 1H,
J ¼ 10.5 Hz, Hb-1), 5.22 (br s, 1H, H-2), 5.22e5.40 (m, 4H,
2 ꢁ CH]CH ). 13C NMR (100 MHz, CDCl3): d 173.6, 173.4
(COO), 130.6, 130.4, 128.4, 128.3 (CH]CH), 104.3 (C-10),
74.8 (C-50), 73.6 (C-30), 71.5 (C-20), 70.6 (C-2), 70.0 (C-60),
69.2 (C-40), 68.5 (C-3), 63.2, 63.1 (C-1), 34.3, 33.6
(COCH2), 32.4e23.2 (CH2 aliph.), 27.6 (CH2CH]CH),
26.0 (]CHCH2CH]), 25.3, 25.2 (COCH2CH2), 14.7
(CH3). ESI-MS: m/z ¼ 694 (M þ Na, 100%)þ. Anal. calcd.
for C37H66O10: C, 66.27; H, 9.85; found: C, 66.31; H, 9.81.
6. Conclusions
In conclusion, a series of 1,2-O-diacyl-3-O-b-D-galactopyr-
anosyl glycerols were synthesized and characterized, and
some of them proved to be antibacterial agents. For all ob-
tained compounds, we have examined the antimicrobial and
antifungal activities and some structureeactivity relationships
have been explored. It was observed that the antimicrobial ac-
tivity in this class of compounds is dependent on the nature of
fatty acids. The octanoyl chain is the proper structural feature
for the maximum activity in 9a. This observation is confirmed
by the results showing that compounds containing the octanoyl
fatty acyl moiety at C-1 and unsaturated fatty acids at C-2 po-
sitions of glycerol 13aec displayed significant activity against
B. subtilis, S. aureus 87 and M. tuberculosis reference and
5.1.8.14. 1-O-Caprinyl-2-O-linolenoyl-3-O-(b-D-galactopyra-
nosyl)-rac-glycerol 14c. Yield: 66%. Rf ¼ 0.50 (CH2Cl2e
1
MeOH 10:1, v:v); H NMR (400 MHz, CDCl3): d 0.96 (br t,
6H, J ¼ 6.8 Hz, CH3 term.), 1.24 (br s, 20H, CH2 aliph.),
1.56 (br t, 4H, COCH2CH2), 2.10 (br, 4H, 2 ꢁ CH2CH]CH),

F. Cateni et al. / European Journal of Medicinal Chemistry 43 (2008) 210e221
[12] K. Nakata, J. Biochem. 127 (2000) 731e737.
221
clinical strains. Compounds 14aec, which differ from the de-
rivatives 13aec for the presence of a decanoyl chain at the C-1
position of glycerol, displayed an interesting activity against S.
aureus 14aec, E. faecalis 14b, B. subtilis 14a, b, but they de-
voided activity against M. tuberculosis. These results reveal
the potential of these compounds as a new type of antimicro-
bial agents, although the mechanism of action must be further
investigated.
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Acknowledgements
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