Synthesis and anti-tumor-promoting activity of glycoglycerolipid analogues lacking the glycerol backbone

Article abstract of DOI:10.1016/S0968-0896(02)00543-6

Glycoglycerolipid analogues lacking the glycerol backbone were prepared through a lipase catalyzed transesterification of β-D-galactosylethylene glycol. The inhibitory effect of the resultant isomeric hexanoates at the primary alcoholic positions, β-D-galactosylethylene glycol itself and nonyl β-D-galactopyranoside, was tested on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), as a primary screening test for inhibitors of tumor promotion. All the compounds assayed were found to be less active than the reference 2-O-β-D-galactopyranosylglycerol derivatives, of which they are simplified models, indicating that the anti-tumor-promoting activity is very closely related to the presence of a free hydroxymethylene group on the glycerol-like aglycone moiety.

Full text of DOI:10.1016/S0968-0896(02)00543-6

Bioorganic & Medicinal Chemistry 11 (2003) 909–912
Synthesis and Anti-tumor-promoting Activity of
Glycoglycerolipid Analogues Lacking the Glycerol Backbone
Diego Colombo,a,* Fiamma Ronchetti,a Antonio Scala,a
Lucio Toma,b Harukuni Tokudac and Hoyoku Nishinoc
a
`
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita di Milano, Via Saldini 50, 20133 Milan, Italy
b
`
Dipartimento di Chimica Organica, Universita di Pavia, Via Taramelli 10, 27100 Pavia, Italy
cDepartment of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-0841, Japan
Received 31 May 2002; accepted 22 October 2002
Abstract—Glycoglycerolipid analogues lacking the glycerol backbone were prepared through a lipase catalyzed transesterification
of b-d-galactosylethylene glycol. The inhibitory effect of the resultant isomeric hexanoates at the primary alcoholic positions, b-d-
galactosylethylene glycol itself and nonyl b-d-galactopyranoside, was tested on Epstein–Barr virus early antigen (EBV-EA) activa-
tion in Raji cells promoted by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), as a primary screening test for
inhibitors of tumor promotion. All the compounds assayed were found to be less active than the reference 2-O-b-d-galactopyr-
anosylglycerol derivatives, of which they are simplified models, indicating that the anti-tumor-promoting activity is very closely
related to the presence of a free hydroxymethylene group on the glycerol-like aglycone moiety.
# 2003Elsevier Science Ltd. All rights reserved.
Introduction
of 3 without the hydroxymethylene group, nonyl b-d-
galactopyranoside 4. Here we describe the synthesis of
compounds 2b–c and 4, and their anti-tumor promoting
activity based on the short term in vitro bioassay for the
inhibition of EBV-EA activation induced by TPA,
together with that of the non-acylated compound 2a.
In the course of a study aimed at clarifying the struc-
ture–anti-tumor-promoting activity relationship of gly-
coglycerolipids active in cancer chemoprevention,1ꢀ7
synthetic 1-O- and 60-O-hexanoyl derivatives 1b and 1c
of 2-O-b-d-galactopyranosylglycerol (1a) have shown a
remarkable inhibitory effect in vitro against Epstein–
Barr virus early antigen (EBV-EA) activation induced
by the tumor promoter 12-O-tetradecanoylphorbol-13-
acetate (TPA),4 and in vivo against mouse skin tumor
promotion in a two-stage carcinogenesis test.5
Results and Discussion
Chemistry
Compound 2a was prepared according to literature
procedures,9 whereas the two isomeric hexanoates 2b
and 2c were obtained in one step and with good yields
from 2a through an efficient chemoenzymatic synthesis
that made use of lipases of opposite regioselectivity. In a
first approach to the synthesis of 2b and 2c, we decided
to use the enzymatic method we had recently employed
to obtain compounds 1b and 1c. In that case, we had
managed, in a single experiment performed with Pseu-
domonas cepacia lipase (LPS),10 to simultaneously pro-
duce 1- and 60-monoesters in almost equimolar amounts
through the transesterification of 1a. However, when
b-d-galactopyranosylethylene glycol (2a) was under-
went to LPS catalyzed transesterification under the
To elucidate the minimal structural requirements nee-
ded for inhibitory activity we have planned the synthesis
of simple analogues of the glycoglycerolipids 1b and 1c,
namely the b-d-galactopyranosylethylene glycol hex-
anoates 2b and 2c, the aglycone in such hexanoates
lacking a hydroxymethylene group of glycerol and thus
being devoid of chirality. Moreover, given the sig-
nificant in vitro and in vivo activity8 of an alkane isoster
of 1b, compound 3, we also prepared a simplified model
*Corresponding author. Tel.: +39-02-5031-6045; fax: +39-02-5031-
6036; e-mail: diego.colombo@unimi.it
0968-0896/03/$ - see front matter # 2003Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00543-6

910
D. Colombo et al. / Bioorg. Med. Chem. 11 (2003) 909–912
Biological evaluation
Epstein–Barr virus (EBV) is activated by tumor pro-
moters to produce viral early antigens (EA), and EBV
inhibition is often evaluated in vitro as a primary
screening for anti-tumor-promoting activity.11 The pre-
sent study evaluated the inhibitory effect of compounds
2a–c and 4 using a short term in vitro assay for EBV-
EA activation in Raji cells induced by the tumor pro-
moter TPA.12,13 Each compound was assayed in tripli-
cate, and it was found that the cytotoxicity against Raji
cells was, for all the compounds, quite weak, even at a
concentration of 1Â103 mol ratio. Table 2 shows the in
vitro tumor inhibitory activity of compounds 2a–c and
4, and the reference compounds 1b–c4 and 3.8
same reaction conditions used for the acylation of 1a,
the reaction was found to be very regioselective (Table
1). The acylation of the primary hydroxyl of the sugar
gave 1-O-(6-O-hexanoyl-b-d-galactopyranosyl)ethylene
glycol (2c) as the main monoacylation product, and
only very small amounts of 2b being derived from the
acylation at the aglycone site, as ascertained by 1H
NMR analysis of the monoesters fraction.
Note that all the tested compounds (2a–c and 4) inhib-
ited the TPA promoted EBV activation (Table 2). The
four compounds, almost equiactive, showed small dif-
ferences in activity that appear to be mainly related to
the presence, or absence of a primary hydroxyl on the
aglycone moiety of the molecule. In fact, the hydroxyl
carrying compounds 2a and 2c were more active than 2b
and 4 that lack it, 2b and 4 not being able to completely
inhibit EBV activation even at 1000 mol ratio/TPA
(Table 2). Furthermore, as previously described,2,4 the
presence of a hexanoyl chain seemed to increase the
inhibitory activity. In fact, on comparing 2a and 2c,
both carrying the primary hydroxyl on the aglycone, 2a,
with no hexanoyl chain, was less active than 2c, with a
hexanoyl chain, this latter also being the most active
compound of this series of simple analogues (Table 2).
However, as evidenced by the data of Table 2, the
galactosylethylene glycol derivatives 2b–c and nonyl
b-d-galactopyranoside (4), the isoster of 2b, showed a
markedly lower inhibitory potency than the galacto-
sylglycerol derivatives 1b–c4 and the galactoside 3,8 the
isoster of 1b, to which they are referred.
To obtain the 1-hexanoate 2b in appreciable yields we
turned our attention to Candida antarctica lipase
(LCA), known to acylate 1a mainly at the glycerol
moiety.10 In fact, when, in the previously reported
reaction conditions, 2a was submitted to LCA catalyzed
transesterification, the reaction showed a regioselectivity
opposite that of the LPS catalyzed reaction, yielding,
almost exclusively, the desired 1-O-hexanoyl-2-O-(b-d-
galactopyranosyl)ethylene glycol (2b) (Table 1). The
two 1- and 60-hexanoyl derivatives were obtained pure
by flash chromatography of the respective monoester
fractions. Both the LPS and LCA catalyzed reactions
1
afforded some diester (Table 1), identified by H NMR
analysis as 1-O-hexanoyl-2-O-(6-O-hexanoyl-b-d-galac-
topyranosyl)ethylene glycol (2d). Lastly, the alkane iso-
ster, nonyl b-d-galactopyranosyde (4), was obtained
through a completely stereoselective glycosylation reac-
tion (no traces of the a anomer were detected) using b-
d-galactose pentaacetate as glycosyl donor, and 1-non-
anol as acceptor in the presence of boron trifluoride
diethyl etherate. The b configuration of the glycosidic
bond was indicated by the characteristic value (8.0 Hz)
of the trans-diaxial J1,2 coupling constant measured by
1H NMR analysis. The acetyl groups of the obtained
nonyl 2,3,4,6-tetra-O-acetyl-b-d-galactopyranoside were
removed by Zemplen reaction to afford 4.
In conclusion, the data indicate that a necessary requi-
site for significant anti tumor-promoting activity in gly-
coglycerolipid analogues is not only the presence of one
Table 2. Inhibitory effects of 1b–c, 2a–c, 3 and 4 on TPA-induced
EBV-EA activation
Mol ratio (test compound/TPA)a
1000
500
100
10
% to positive controlbÆSE (n=3) (% viability)
1bc
1cc
2a
2b
2c
3d
4
0.0Æ0.0 (70) 11.4Æ0.3( >80) 32.1Æ0.9 (>80) 63.4Æ1.3( >80)
0.0Æ0.0 (70) 10.7Æ0.1 (>80) 30.1Æ0.9 (>80) 67.8Æ1.2 (>80)
Table 1. Lipase catalyzed transesterification of 2aa
0.0Æ0.3(70) 46.2 Æ1.5 (>80) 76.0Æ2.2 (>80)
6.7Æ0.5 (70) 49.5Æ1.4 (>80) 78.5Æ2.5 (>80)
100Æ0.2 (>80)
100Æ0.2 (>80)
Enzyme
Time
(min)
Monoestersb
(%)
Diestersb
(%)
Monoester
ratioc
2b/2c
0.0Æ0.4 (70) 41.3Æ1.3( >80) 71.0Æ2.3( >80) 96.9Æ0.4 (>80)
0.0Æ0.5 (70) 20.3Æ1.3( >80) 38.5Æ1.5 (>80) 72.1Æ0.4 (>80)
12.4Æ0.3(70) 50.6 Æ1.7 (>80) 80.5Æ2.3( >80)
100Æ0.3( >80)
LPS
LCA
60
30
69
70
14
30
3:97
97:3
aTPA=32 pmol; Test compounds=32, 16, 3.2 and 0.32 nmol.
bValues represent EBV-EA activation (%) in the presence of test compound
relative to the positive control (TPA only).
cSee ref 4.
dSee ref 8.
aNo acylation took place in the absence of the enzyme.
bIsolated yield from flash chromatography.
cDetermined through 1H NMR analysis of the monoester fraction.

D. Colombo et al. / Bioorg. Med. Chem. 11 (2003) 909–912
911
hexanoyl chain, already suggested by previous results,2,4
but also integrity in the glycerol-like backbone, a key
structural feature being the presence of one hydro-
xymethylene group on the aglycone.
(m, 8H, 4CH2), 0.92 (m, 6H, 2CH3). 13C NMR
(CD3OD): d 175.5; 175.2; 105.1; 74.7; 74.0; 72.2; 70.3;
68.6; 64.7 (2C); 34.9 (2C); 32.4 (2C); 25.7 (2C); 23.4
(2C); 14.3(2C); MS: m/z 438 [M+NH4]+. Flash chro-
matography (dichloromethane–methanol 85:15, v/v) of
the above monoester fraction yielded 0.29 g (68%) of
20
pure 2b as a foam, mp: 55–56 ꢁC; ½aꢂD : ꢀ7.7 (c 1.0). H
1
Experimental
Chemistry
NMR (CD3OD): d 4.30–4.22 (m, 3H, H-1a, H-1b and
H-10), 4.06 (m, 1H, H-2a); 3.84 (d, 1H, J3 , 4 =3.0 Hz,
0
0
H-40), 3.80 (m, 1H, H-2b), 3.77–3.69 (m, 2H, H-60a and
P. cepacia lipase (LPS, lipase PS, specific activity 30.5
triacetin units/mg solid), a generous gift from Amano
Pharmaceutical Co (Mitsubishi Italia), was supported
on Celite14 and kept overnight under vacuum prior to
use. Candida antarctica lipase SP 435 L, (Novozym1
435, LCA, specific activity 9.5 PL units/mg solid), was
a generous gift from Novo Nordisk A/S. Pyridine was
distilled over calcium hydride prior to use. The acyl
carrier 2,2,2-trifluoroethyl n-hexanoate was synthesized
from hexanoyl chloride and 2,2,2-trifluoroethanol.15
b-d-Galactopyranosylethylene glycol (2a) was synthe-
sized according to literature procedures.9 b-d-Galac-
tose pentaacetate and 1-nonanol were purchased from
Fluka. 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 500.13MHz, 13C NMR at
125.76 MHz (Bruker AM500 spectrometer at 303 K)
and chemical ionization mass spectrometry (CI-MS) as
described in ref 15 by LC particle beam introduction
with a quadrupolar mass spectrometer Hewlett-Pack-
ard HP 5988A equipped with an interface PB 59980A
and a low pressure HPLC HP 1050. The elemental
analyses were consistent with the theoretical ones.
Optical rotations were determined on a Perkin-Elmer
241 polarimeter as methanol solutions, unless other-
wise stated, in a 1 dm cell at 20 ꢁC. Melting points
were recorded on a Buchi 510 capillary melting point
apparatus and were uncorrected.
H-60b), 3.56–3.50 (m, 2H, H-50 and H-20), 3.47 (dd, 1H,
0
0
0
J2 , 3 =9.0 Hz, H-3 ), 2.34 (m, 2H, CH2), 1.63(m, 2H,
CH2), 1.38–1.28 (m, 4H, 2CH2), 0.92 (m, 3H, CH3). 13
NMR (CD3OD): d 175.6; 105.2; 76.7; 74.9; 72.5; 70.3;
68.5; 64.8; 62.5; 34.9; 32.4; 25.7; 23.4; 14.3; MS: m/z 340
[M+NH4]+.
C
Flash chromatography (dichloromethane–methanol
85:15, v/v) of LPS catalyzed transesterification yiel-
ded 0.08 g (14%) of the diester 2d, 0.30 g (69%) of a
monoester fraction and 0.025 g (8%) of unreacted
2a. Flash chromatography (dichloromethane–metha-
nol 85:15, v/v) of the monoester fraction afforded
0.29 g (67%) of pure 2c, mp: 113–114 ꢁC (methylene
20
chloride–methanol); ½aꢂD : ꢀ0.7 (c 1.0); 1H NMR
0 0
4.30 (dd, 1H, J6 a, 6 b=11.0 Hz,
(CD3OD):
0
d
J5 , 6 a=7.0 Hz, H-60a), 4.26 (d, 1H, J1 , 2 =8.0 Hz,
0
0
0
H-10), 4.22 (dd, 1H, J5 , 6 b=5.0 Hz, H-60b), 30.91 (m,
0
0
0
0
1H, H-1a); 3.80 (d, 1H, J3 , 4 =3.0 Hz, H-4 ), 3.74
(dd, 1H, H-50), 3.72–3.63 (m, 3H, H-1b, H-2a and
0
0
0
H-2b), 3.55 (dd, 1H, J2 , 3 =9.0 Hz, H-2 ), 3.50 (dd,
1H, H-30), 2.34 (m, 2H, CH2), 1.63(m, 2H, C H2),
1.38–1.28 (m, 4H, 2CH2), 0.92 (m, 3H, CH3). 13C
NMR (CD3OD): d 175.2; 105.1; 74.6; 74.0; 72.5
(2C); 70.3; 64.7; 62.4; 34.9; 32.4; 25.7; 23.4; 14.3;
MS: m/z 340 [M+NH4]+.
Nonyl ꢀ-D-galactopyranoside (4). b-d-Galactose pentaa-
cetate (2.00 g, 5.12 mmol ) was dissolved in dry di-
chloromethane (40 mL) together with nonanol (2.7 mL,
15.5 mmol) and boron trifluoride diethyl etherate
(2.1 mL, 16.7 mmol) was added dropwise at 0 ꢁC. The
reaction mixture was kept under stirring overnight at
room temperature, and then quenched by addition of
saturated NaHCO3 solution (30 mL). The aqueous layer
was extracted with dichloromethane (3 Â 15 mL) and
the combined organic layers were then washed with
brine, dried, and evaporated under reduced pressure.
Flash chromatography (petroleum ether–ethyl acetate
75:25, v/v) of the crude reaction mixture yielded 1.30 g
(54%) of nonyl 2,3,4,6-tetra-O-acetyl-b-d-galactopyr-
1-O-Hexanoyl-2-O-(ꢀ-D-galactopyranosyl)ethylene gly-
col (2b) and 1-O-(6-O-hexanoyl-ꢀ-D-galactopyranosyl)e-
thylene glycol (2c). b-d-Galactopyranosylethylene
glycol (2a) (0.30 g, 1.34 mmol) was dissolved in pyridine
(5 mL) and 2,2,2-trifluoroethyl hexanoate (4.00 mmol)
and LCA (1.00 g) or LPS (1.50 g) were added in the
order. The mixture was stirred at 45 ꢁC and the reaction
was stopped, after 0.5 h for LCA and after 1.0 h for
LPS, by filtering-off the enzyme and washing with pyri-
dine. The solvent was removed under vacuum and the
crude product was submitted to flash chromatography.
Flash chromatography (dichloromethane–methanol
85:15, v/v) of LCA catalyzed transesterification yielded
0.17 g (30%) of the diester 2d, and 0.30 g (70%) of a
monoester fraction. Compound 2d, mp: 64–65 ꢁC
20
anoside, oil; ½aꢂD : ꢀ12.2 (c 1.0, chloroform). 1H NMR
0
1H, J1 , 2 =8.0 Hz, J2 , 3 =10.0 Hz, H-2 ), 4.96 (dd, 1H,
0
0
(CDCl3): d 5.33 (d, 1H, J3 , 4 =3.0 Hz, 0H-4 ), 5.14 (dd,
0
0
0
0
H-30), 4.40 (d, 1H, H-10), 4.13(dd, 1H, J6 a, 6 b=12.0 Hz,
0
0
20
(diisopropyl ether); ½ꢀꢂD : ꢀ0.5 (c=1.0); 1H NMR
J5 , 6 a=6.0 Hz, H-60a), 4.07 (dd, 1H, J5 , 6 b=7.0 Hz, H-
60b), 3.88–3.79 (m, 2H, H-50 and OCHa), 3.42 (m, 1H,
OCHb), 2.09 (s, 3H, CH3CO), 1.98 (s, 6H, 2CH3CO),
1.92 (s, 3H, CH3CO), 1.51 (m, 2H, CH2), 1.30–1.16 (m,
12H, 6CH2), 0.82 (m, 3H, CH3). 13C NMR (CDCl3): d
171.0; 170.9; 170.8; 170.0; 102.0; 71.6; 71.2; 70.9; 69.6;
67.7; 61.9; 32.5; 30.2; 30.0 (2C); 29.9; 26.4; 23.3; 21.3
(4C); 14.7; MS: m/z 492 [M+NH4]+.
0
0
0
0
0 0
4.30 (dd, 1H, J6 a, 6 b=11.0 Hz,
(CD3OD):
0
d
J5 , 6 a=7.0 Hz, H-60a), 4.28–4.23(m, 3H, H-1a, H-1b
0
and H-10), 4.21 (dd, 1H, J5 , 6 b=5.0 Hz, H-60b), 4.00 (m,
0
0
1H, H-2a); 3.84–3.76 (m, 2H, H-2b and H-40), 3.73 (dd,
1H, H-50), 3.53 (dd, 1H, J1 , 2 =8.0 Hz, J2 , 3 =9.0 Hz,
0
0
0
0
H-20), 3.47 (dd, 1H, J3 , 4 =3.0 Hz, H-3 ), 2.34 (m, 2H,
CH2), 2.33 (m, 2H, CH2), 1.62 (m, 4H, 2CH2), 1.39–1.28
0
0
0

912
D. Colombo et al. / Bioorg. Med. Chem. 11 (2003) 909–912
The obtained tetraacetyl galactoside (1.30 g, 2.76 mmol)
was dissolved in methanol (4 mL) and a solution
(12 mL, 0.70 M) of sodium methoxide in methanol was
added under stirring at room temperature. After 2 h the
reaction mixture was neutralized by adding an ion
exchange resin (Dowex 50 Â H+). The resin was fil-
tered off and the solvent removed under vacuum.
Crystallization of the crude residue (0.85 g) yielded
0.75 g (89%) of the desired nonyl b-d-galactopyrano-
Acknowledgements
This study was supported in part by the Ministero
dell’Universita e della Ricerca Scientifica e Tecnologica,
Rome, Italy (COFIN 2001: Antitumoral natural and
related synthetic compounds) 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).
20
side (4), mp: 147–148 ꢁC (acetone); ½aꢂD : ꢀ15.8 (c 1.0).
1H NMR (CD3OD): d 4.21 (d, 1H, J1 , 2 =7.4 Hz, H-
0
0
10), 3.88 (m, 1H, OCHa), 3.83 (d, 1H, J3 , 4 =3.0 Hz,
0
0
References and Notes
H-40), 3.75 (dd, 1H, J6 a, 6 b=11.0 Hz, J5 , 6 a=6.6 Hz,
0
0
0
0
H-60a), 3.73 (dd, 1H, J5 , 6 b=5.8 Hz, H-60b), 3.54 (m,
1H, OCHb), 3.52–3.47 (m, 2H, H-20 and H-50), 3.46
(dd, 1H, J2 , 3 =9.6 Hz, H-3 ), 1.62 (m, 2H, CH2), 1.44–
1.24 (m, 12H, 6CH2), 0.90 (m, 3H, CH3). 13C NMR
(CD3OD): d 105.0; 76.6; 75.1; 72.6; 70.8; 70.3; 62.5;
33.1; 30.8; 30.7 (2C); 30.5; 27.1; 23.7; 14.5; MS: m/z
324 [M+NH4]+.
0
0
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Short-term in vitro bioassay for anti-tumor promoters.
All the compounds were tested for their anti-tumor
promoting activity using a short-term in vitro assay for
EBV-EA activation in Raji cells, cultivated in RPMI
1640 medium containing 10% fetal calf serum, as
described in refs 12 and 13. Raji cells (1Â106/mL) were
incubated at 37 ꢁC for 48 h in 1 mL of a medium con-
taining n-butyric acid (4 mM), TPA (32 pmol) and a
known amount of the test compound. The inhibitory
activity of the test compounds was expressed by com-
parison of the average EBV-EA induction of each
experiment,
observed
by
indirect
immuno-
fluorescence,12,13 with that of positive control experi-
ments (100%) carried out with n-butyric acid (4 mM)
and TPA (32 pmol) only, in which EBV-EA induction
was ordinarily around 30%. In each assay at least 500
cells were counted and the assays were performed in
triplicate for each compound.
No sample exhibited significant toxicity against Raji
cells. The viability of the cells was tested against treated
cells using the Trypan blue staining method. For the
determination of cytotoxicity, the cell viability was
required to be more than 60% 2 days after treatment
with the compounds for an accurate result. The results
are reported in Table 2.
15. Colombo, D.; Ronchetti, F.; Scala, A.; Taino, I. M.;
Toma, L. Tetrahedron: Asymmetry 1996, 7, 771.