O-Methylglucogalloyl esters: Synthesis and evaluation of their antimycotic activity

Article abstract of DOI:10.1016/j.bmcl.2005.06.037

The two anomers of O-methyl gluco-2,3-digalloyl esters were synthesized and their antimycotic activity toward yeasts of biomedical importance was evaluated. When used at subinhibitory concentration and regardless of stereochemistry at the anomeric carbon,

Full text of DOI:10.1016/j.bmcl.2005.06.037

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4000–4003
O-Methylglucogalloyl esters: Synthesis and evaluation
of their antimycotic activity
Annalisa Romani,a Stefano Menichetti,b,* Panagiotis Arapitsas,a,b Cristina Nativi,b
Benedetta Turchettic and Pietro Buzzinic
a
c
`
Dipartimento di Scienze Farmaceutiche, Polo Scientifico Universita di Firenze, Via U. Schiff, 6, I-50019 Sesto Fiorentino (FI), Italy
b
`
Dipartimento di Chimica Organica, Polo Scientifico Universita di Firenze, Via della Lastruccia 13,
I-50019 Sesto Fiorentino (FI), Italy
`
Dipartimento di Biologia Vegetale e Biotecnologie Agroambientali, Universita di Perugia, Borgo XX Giugno 74, I-06121 Perugia, Italy
Received 22 December 2004; revised 7 June 2005; accepted 9 June 2005
Available online 6 July 2005
Abstract—The two anomers of O-methyl gluco-2,3-digalloyl esters were synthesized and their antimycotic activity toward yeasts of
biomedical importance was evaluated. When used at subinhibitory concentration and regardless of stereochemistry at the anomeric
carbon, these compounds enhance the antimycotic activity of Amphotericin B.
Ó 2005 Elsevier Ltd. All rights reserved.
Gallic acid (GA) and its derivatives are widespread nat-
ural molecules having an important role in food and
pharmaceutical industry.
Previous studies evaluated the biological activities of
some n-alkyl esters of gallic acid (propyl, octyl, etc.).
These non-natural molecules, currently used as antioxi-
dant additives, exhibited an antimycotic activity toward
some yeasts,1 and were noticed to be able to increase the
Figure 1. EGCG and O-methyl gluco-2,3-galloyl ester skeletons.
antimycotic activity of some antibiotics, such as
Amphotericin B (AmB),2 a polyenic molecule that, in
spite of its low stability, is currently used for the treat-
ment of human mycoses.3
form to more complex glucogalloyl and ellagitannins
derivatives and steric similarity with epigallocatechin
gallate (EGCG) (Fig. 1) that is known to exhibit a bio-
logical activity towards living yeast cell systems.6
Although the protecting action ofglucogalloyl derivatives
against cardiovascular diseases4,5 is a well-known phe-
nomenon, their antimycotic activity toward yeasts of bio-
medical relevance has never been critically evaluated.
The synthetic strategy started with the preparation of
suitable protected gallic acid 1. Commercially available
methyl gallate (MG) was reacted with dichlorodiphenyl
methane to give the expected cyclic dioxolane 2 that was
hydrolyzed to the corresponding acid 3 with LiOH in
MeOH/H2O followed by silylation with an excess of
TBDMSCl and DIPEA at both the acid and the residual
phenolic position.7 The silyl ester 4 was selectively
deprotected to 1 by acid hydrolysis with AcOH in
THF/H2O (Scheme 1).8
With this background, a chemical and biological pro-
gram was undertaken to synthesize and evaluate the
antimycotic activity of new glucogalloyl esters. The ini-
tial targets chosen were the O-methylgluco-2,3-digalloyl
esters for their relative low molecular weight, their lead-
Keywords: Glucogalloyl esters; Antimycotic activity; Amphotericin B.
*
As far as the sugar moiety is concerned, a 2:1 mixture of
a- and b-O-methylglucosides 5, protected on the posi-
Corresponding author. Tel.: +39 055 4573535; fax: +39 055
4573531; e-mail: stefano.menichetti@unifi.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.037

A. Romani et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4000–4003
4001
Scheme 1. Synthesis of galloyl acid derivative 1. Reagents and
conditions: (a) Ph2CCl2 180 °C, 83%; (b) LiOH, MeOH/H2O reflux,
97%; (c) TBDMSCl, DIPEA, DMF, 98%; (d) AcOH, THF/H2O, 80%.
tions 4 and 6 as benzylidene derivatives, were used. The
esterification reaction of 1 with 5 was initially carried
out by activating the acid ꢀviaꢁ the formation of the cor-
responding acyl chloride, but low yields (22%) of esters 6
were isolated. The poor result obtained prompted us to
carry on a modified Steglich esterification reaction.9
This procedure allowed the isolation of reasonable
yields of a mixture of galloyl esters 6 which were also
isolated as single anomers by skilled flash chromatogra-
phy10 (Scheme 2).
Scheme 2. Synthesis of a- and b-O-methylgluco-2,3-digalloyl esters 7.
Reagents and conditions: (a) DCC, DMAP, DMAPÆHCl, CH2Cl2, rt,
24 h, flash chromatography, [a-6 (26%), b-6 (16%), a/b-6 (13%)]; (b)
TBAF/AcOH, THF, rt, 5 h, 85%; (c) H2, Pd/C, THF, rt, 17 h (three
cycles), 27%.
The deprotection of the hydroxyl groups was achieved
in two steps by desilylation, with TBAF/AcOH to pre-
vent ester hydrolysis, followed by catalytic hydrogena-
tion of the dioxolane ring of both the aromatic and
the sugar moieties.11 Reverse phase preparative thin-
layer chromatography (TLC) allowed the purification
of a- and b-O-methylgluco-2,3-galloyl derivatives 7,
from a very small amount of partially protected interme-
diates, as reported in Scheme 2 where, for simplicity,
only the major a-anomer has been depicted.10
orientalis (teleomorphic state of Candida kruseii)
DBVPG 6782, a well-known yeast pathogen,15 was used
as target strain (Table 1). In addition, our studies indi-
cated that either mixture or pure a-7 or b-7 anomers
did not exhibit significantly different effects on the activ-
ity of AmB (Fig. 3).
The identification of the two anomers was performed by
NMR, UV–vis, and MS techniques.10
Concerning the possible mechanism(s) of such behavior,
some hypotheses can be proposed. Apparently, the more
significant may concern the well-known interaction be-
tween antioxidant molecules (such as galloyl derivatives)
and AmB.2,16
The HPLC-DAD analysis of galloyl derivatives 7, in
Figure 2, shows the different retention times, the high
purity (>98%), and the eventual separation of the two
anomers 6, performed by flash chromatography during
the synthetic pathway (Scheme 2, Fig. 2).
It is generally accepted that the damaging action of
AmB toward yeast cells originates from its binding to
sterols incorporated in cell membranes,3 even though
no clear relationship has been so far found between this
first step and the expression of biological activity.
Although some evidence suggests that yeast cell lethality
does not originate exclusively from changes in mem-
brane permeability, the binding of AmB to sterols re-
sults in a disorganization of cell membranes, through
the formation of specific pores constituted of small
aggregates of AmB and sterols, which causes a loss of
protons and monovalent cations.3
In preliminary tests aimed at evaluating the biological
properties of the glucogalloyl esters, a mixture of a-
and b-anomers 7 exhibited only a slight and temporary
antimycotic activity toward Candida glabrata DBVPG
3828.
When AmB13 was used in association with a subinhibi-
tory concentration of glucogalloyl compounds 7 (Table
1), an increased antimycotic activity of the above poly-
enic antibiotic was observed, under form of a clear de-
crease of its minimal inhibitory concentration (MIC).14
This effect was particularly relevant when Issatchenkia
Several reports indicate that oxidation-dependent events
may be involved in AmB antimycotic activity.2,16

4002
A. Romani et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4000–4003
Figure 2. HPLC-DAD chromatograms of a mixture of a-7and b-7 and the isolated pure compounds.
Table 1. MICs (lg/mL) of mixture a-7/b-7 alone, AmB alone, or AmB
in association with mixture a-7/b-7 (49.8 lg/mL), gallic acid (GA)
(34.0 lg/mL), and methyl gallate (MG) (36.8 lg/mL)
of glucogalloyl derivatives 7 on AmB molecule may be
hypothesized.
MICs
In conclusion, the present study shows that glucogalloyl
derivatives 7 could be used for enhancing the antimycot-
ic activity of AmB toward yeasts of biomedical rele-
vance. Accordingly, the use of reduced concentrations
of known antibiotics in association with subinhibitory
concentrations of glucogalloyl derivatives could be pro-
posed for the treatment of yeast infections, thereby
potentially reducing the effects of high dosage of antibi-
otic exposure. This finding is of interest for its potential
exploitation in pharmaceutical sciences.
a-7/b-7
AmB
AmB in association with
a-7/b-7
GA
MG
C. albicans12
C. glabrata12
I. orientalis12
1940
>4670
>4670
1.06
1.06
>100
0.05
0.05
2.05
0.09
1.00
8.00
0.09
0.09
45.0
References and notes
1. (a) Kubo, I.; Xiao, P.; Fujita, K. Bioorg. Med. Chem. Lett.
2001, 11, 347; (b) Fujita, K.; Kudo, I. Int. J. Food
Microbiol. 2002, 79, 193; (c) Fujita, K.; Kudo, I. J. Appl.
Microbiol. 2002, 92, 1035.
2. (a) Andrews, F. A.; Beggs, W. H.; Sarosi, G. A.
Antimicrob. Agents Chemother. 1977, 11, 615; (b) Beggs,
W. H.; Andrews, F. A.; Sarosi, G. A. Antimicrob. Agents
Chemother. 1978, 13, 266.
Figure 3. MICs (lg/mL) of AmB in association with increasing
concentrations of: mixture a/b-7 (A) or pure a-7 (B) or b-7 (C). Target
strain: C. glabrata DBVPG 3828.
3. (a) Brajtburg, J.; Powderly, W. G.; Kobayashi, G. S.;
Medoff, G. Antimicrob. Agents Chemother. 1990, 34, 183;
(b) Dix, S. P.; Wingard, J. R. Drugs Today 1996, 32, 411.
4. (a) Dufresne, C. J.; Farnworth, E. R. J. Nutr. Biochem.
2001, 12, 404; (b) Riemersma, R. A.; Rice-Evans, C. A.;
Tyrrell, R. M.; Clifford, M. N.; Lean, M. E. QJM Int. J.
Med. 2001, 94, 277.
Because AmB injury to yeast cells could be modulated
by the extracellular presence of compounds exhibiting
antioxidant action, it has been postulated that these
molecules could act as simple protective agents toward
polyene oxidation, thus resulting in an increased AmB
stability.2,16 Therefore, a possible explanation of the ob-
served synergistic (or additive) effect of glucogalloyl
molecules on AmB may be related to the presence of
tri-hydroxy, substituted aromatic rings in the structure
of glucogalloyl derivatives 7 which clearly can exert an
antioxidant action.
5. (a) Romani, A.; Coinu, R.; Carta, S.; Pinelli, P.; Galardi,
C.; Vincieri, F. F.; Franconi, F. Free Radic. Res. 2004, 38,
ˇ
ˇ
97; (b) Strlic, M.; Radovic, T.; Kolar, J.; Pihlar, B. J.
Agric. Food Chem. 2002, 50, 6313; (c) Aruoma, O. I.;
Murcia, A.; Butler, J.; Halliwell, B. J. Agric. Food Chem.
1993, 41, 1880.
6. Turchetti, B.; Pinelli, P.; Buzzini, P.; Romani, A.; Heimler,
D.; Franconi, F. Phytother. Res. 2005, 19, 44.
7. The different protection of the phenolic groups was chosen
to have, on the final galloyl esters, the possibility of an
oxidative coupling with formation of the biphenyl core of
ellagitannine derivatives.
8. Feldman, K. S.; Ensel, S. M. J. Am. Chem. Soc. 1993, 115,
1162.
9. Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394.
To verify the above hypothesis, MICs of AmB in asso-
ciation with either the mixture a-7/b-7 or with gallic acid
(GA) or methyl gallate (MG) (both at double molar
concentration, to have the same amount of antioxidant
moiety) were compared. Because both GA and MG re-
vealed an apparently lesser effect on AmB activity (un-
der form of higher MICs) (Table 1), the existence of
additional effect(s) (other than the antioxidant action)

A. Romani et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4000–4003
4003
10. Experimental procedure for the preparation of a-7 and b-7
is as follows: To a solution of methylglucoside 5, acid 1
(2.2 equiv), DMAP (1.1 equiv), and DMAPÆHCl (1.1
equiv) in dry CH2Cl2, a solution of DCC (2.2 equiv) in dry
CH2Cl2 was added. The mixture was purged with N2 and
stirred for 19 h at rt, then cooled at À18 °C for 30 min and
filtered through celite. The organic phase was washed with
HCl 3%, brine, and H2O, dried over Na2SO4, and
concentrated. Purification of the residue by flash chroma-
tography (hexane/ethyl acetate = 10:1) afforded pure pro-
tected galloyl derivatives a-6 (26%), b-6 (16%) and a
combination of a + b fraction (13%). To a solution of a-6
(or b-6) in THF, a 1/1 mixture of n-Bu4NF/acetic acid (3
equiv) was added and the mixture was stirred at rt for 5 h,
then diluted with EtOAc, and poured into 1 M H3PO3.
The organic phase was washed with H2O and brine, dried
over Na2SO4, filtered and concentrated to provide the
desilylated galloyl derivative which was hydrogenated over
Pd/C 10% in THF. The hydrogenation was repeated twice
to complete the deprotection. The gray–green glassy solid
obtained was triturated with three portions of Et2O and
hexanes and purified by a reverse phase C-18 preparative
TLC. a-7. 1H NMR (CD3OD, 400 MHz) d (ppm): 3.47 (s,
3H), 3.62–3.71 (m, 2H), 3.76–3.79 (m, 1H), 4.28 (td,
J = 0.8, 6.4 Hz, 1H), 4.90 (s, 8H), 5.01–5.07 (m, 2H, H),
5.65–5.72 (m, 1H), 7.00 (s, 2H), 7.06 (s, 2H). 13C NMR
(CD3OD, 100 MHz) d (ppm): 55.6, 67.9, 69.8, 73.6, 74.3,
98.6, 110.3, 110.4, 120.6, 121.4, 140.0, 140.1, 146.3, 146.4,
167.6, 168.2. MS m/z (%): 497(100, MÀ), 345(65), 169(60),
(s, 2H). 13C NMR (CD3OD, 100 MHz) d (ppm) 57.3, 62.3,
69.8, 73.4, 77.0, 78.1, 103.2, 110.26, 110.32, 120.9, 121.1,
139.8, 139.9, 146.3, 146.34, 167.2, 167.9. MS m/z (%):
497(100, MÀ), 345(65), 169(60), 125(9).
11. Feldman, K. S.; Lawlor, M. D.; Sahasrabudhe, K. J. Org.
Chem. 2000, 65, 8011.
12. Candida albicans DBVPG 6133, Candida glabrata DBVPG
3828, and Issatchenkia orientalis DBVPG 6782; the yeast
species used as target microorganisms are represented by
their type strain and are conserved in the DBVPG Collec-
tion of Industrial Yeasts of the University of Perugia, Italy,
www.agr.unipg.it/dbvpg. Working cultures were main-
tained on YEPG (yeast extract 1%, peptone 1%, glucose
2%, and agar 1.5%) slants at 4 °C until use. Preliminary tests
on antimycotic activity of glucogalloyl esters were evalu-
ated by the agar diffusion well bioassay (ADWB).
13. Amphotericin B was purchased from Sigma (Sigma–
Aldrich, St. Louis, Mo, USA).
14. (a) MIC determination was carried out in 96-well micro-
plates (Corning Inc., USA) by a broth microdilution
method in agreement with the NCCLS recommendations;
(b) National Committee for Clinical Laboratory Stan-
dards. Approved Standard NCCLS Document M27-A.
Wayne Pa. 1997.
15. (a) Buzzini, P.; Martini, A. Med. Mycol. 2001, 39, 479; (b)
Hazen, K. C. Clin. Microbiol. Rev. 1995, 8, 462.
16. (a) Beggs, W. H.; Andrews, F. A.; Sarosi, G. A. Res.
Commun. Chem. Pathol. Pharmacol. 1978, 20, 409; (b)
Andrews, F. A.; Sarosi, G. A.; Beggs, W. H. J. Antimicrob.
Chemother. 1979, 5, 173; (c) Beggs, W. H.; Andrews, F. A.;
Sarosi, G. A. J. Antimicrob. Chemother. 1980, 6, 291; (d)
Brajtburg, J.; Elberg, S.; Kobayashi, G. S.; Medoff, G. J.
Antimicrob. Chemother. 1989, 24, 333.
1
125(9). b-7. H NMR (CD3OD, 400 MHz) d (ppm): 3.54
(s, 3H), 3.65–3.71 (m, 1H), 3.76–3.84 (m, 2H), 4.67 (d,
J = 8.0 Hz, 1H), 4.91 (s, 8H), 5.11 (dd, J = 8.0, 9.6 Hz,
1H), 5.42 (dd, J = 9.2 and 9.6 Hz, 1H), 6.98 (s, 2H), 7.02