Synthesis and in vitro evaluation of antifungal and cytotoxic activities of eugenol glycosides

Article abstract of DOI:10.1007/s00044-013-0669-2

Six eugenol glycosides were prepared in order to assess their antifungal activity against Candida species. They were synthesized by glycosylation of eugenol with the appropriate glycosyl bromides followed by deacetylation with sodium methoxide in methanol and were evaluated in vitro for their antifungal activity through a Mueller-Hinton broth microdilution method. The peracetyl glycoside (derivative 4) was the most promising one since it was able to inhibit growth of C. albicans, C. tropicalis and C. glabrata with IC₅₀ values much lower than that of the prototype eugenol. Derivative 4 showed to be 160.0 and 3.4 times more potent than eugenol and fluconazole, respectively, against C. glabrata with low cytotoxity (selectivity index of 45). Moreover, it was possible to verify the positive effect of gluco configuration and lipophilicity on antifungal activity, since glucose peracetyl derivatives were more active than the free sugars of galacto configuration.

Full text of DOI:10.1007/s00044-013-0669-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0669-2
ORIGINAL RESEARCH
Synthesis and in vitro evaluation of antifungal and cytotoxic
activities of eugenol glycosides
•
Thiago Belarmino de Souza Marina Orlandi Luiz Felipe Leomil Coelho
•
•
•
Luiz Cosme Cotta Malaquias Amanda Latercia Tranches Dias
•
•
Roberta Ribeiro de Carvalho Naiara Chaves Silva Diogo Teixeira Carvalho
•
Received: 26 February 2013 / Accepted: 31 May 2013
Ó Springer Science+Business Media New York 2013
Abstract Six eugenol glycosides were prepared in order to
assess their antifungal activity against Candida species.
They were synthesized by glycosylation of eugenol with the
appropriate glycosyl bromides followed by deacetylation
with sodium methoxide in methanol and were evaluated
in vitro for their antifungal activity through a Mueller–Hin-
ton broth microdilution method. The peracetyl glycoside
(derivative 4) was the most promising one since it was able to
inhibit growth of C. albicans, C. tropicalis and C. glabrata
with IC50 values much lower than that of the prototype
eugenol. Derivative 4 showed to be 160.0 and 3.4 times more
potent than eugenol and fluconazole, respectively, against C.
glabrata with low cytotoxity (selectivity index of 45).
Moreover, it was possible to verify the positive effect of
gluco configuration and lipophilicity on antifungal activity,
since glucose peracetyl derivatives were more active than the
free sugars of galacto configuration.
Keywords Eugenol derivatives ꢀ Glycosides ꢀ
Anti-Candida activity
Introduction
Fungal diseases represent a serious challenge since ancient
times. At the beginning of the twentieth century, bacterial
epidemics were a global and important cause of mortality. In
contrast, fungal infections were almost not taken into
account. Since the late 1960s when antibiotic therapies were
developed, a drastic rise in fungal infections was observed,
and they currently represent a global health threat. This
increasing incidence of infection is influenced by the grow-
ing number of immunodeficient cases related to AIDS,
cancer, old age, diabetes, cystic fibrosis, organ transplants
and other invasive surgical procedures (Vandeputte et al.,
2012). Treating invasive fungal infections is becoming more
and more interesting. In the 1980s, our options to treat
patients with deadly fungal infections were restricted by the
number and toxicity of available compounds; since then
major efforts have resulted in more effective antifungal
drugs (Pasqualotto and Denning, 2008).
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0669-2) contains supplementary
material, which is available to authorized users.
T. B. de Souza ꢀ M. Orlandi
The use of known antimicrobial structural scaffolds to
afford new bioactive derivatives has not been the main
strategy employed, mainly for the high prevalence of
resistance to these agents. Thus, new chemical entities
design is the main focus when searching for new antibac-
terial and antifungal drug leads, oriented especially to new
molecular targets (Bush and Pucci, 2011; Moellering Jr.,
2011). In that context natural products, especially plant-
derived ones, represent an excellent source of new bioac-
tive compounds since they show innovative structures that
may be used as natural pharmacophores to structural
derivatization.
´
Centro Universitario de Lavras, Rua Padre Jose Poggel,
506 Lavras, MG, Brazil
´
L. F. L. Coelho ꢀ L. C. C. Malaquias ꢀ A. L. T. Dias ꢀ
R. R. de Carvalho ꢀ N. C. Silva
ˆ
Instituto de Ciencias Biomedicas, Universidade Federal de
Alfenas, Rua Gabriel Monteiro da Silva, 700 Alfenas, Brazil
´
D. T. Carvalho (&)
ˆ
ˆ
Faculdade de Ciencias Farmaceuticas, Universidade Federal
de Alfenas, Rua Gabriel Monteiro da Silva, 700 Alfenas,
MG, Brazil
e-mail: diogo.carvalho@unifal-mg.edu.br;
diogotcarv@gmail.com
123

Med Chem Res
Eugenol is a natural allylphenol used for several medical
purposes since many years and has been used alone or
associated to other constituents of clove oil, its most
important natural source (Pramod et al., 2010). Many
important biological activities have been attributed to
eugenol or to its close analogues, as analgesic and anti-
inflammatory (Daniel et al., 2009), anaesthetic (Guenette
et al., 2006), hypotensive (Lahloua et al., 2004), antioxi-
dant (Hidalgo et al., 2009), antitumoral (Manikandan et al.,
2010), anti-parasitic (Machado et al., 2011; Ueda-Na-
kamura et al., 2006) and antidepressive-like action (Kabuto
et al., 2007).
Results and discussion
Chemistry
The compounds were obtained following the synthetic
route depicted in Scheme 1.
The synthetic strategy involved the glycosylation of
eugenol by first reacting it with three peracetylglycosyl
bromides. This method is one of the most usual for this
purpose, since it is cheap and easily performed in aqueous
alkaline media, despite its variable reaction yields (Jacob-
son et al., 2006). Following, these intermediates obtained
(4–6) were deacetylated by methoxide/methanol solution,
The eugenol activities against microorganisms are also
known since many years. The mechanisms of antimicrobial
action have been well documented in bacteria and fungi
recently (Oyedemi et al., 2009; Devi et al., 2010). Ahmad
et al. (2010) studied the effects of eugenol in proton trans-
location through Candida cytoplasmatic membrane. They
showed eugenol is able to inhibit fungal ATPase enzyme
which is essential to maintain electrochemical proton gra-
dient across cell membrane necessary for nutrient uptake.
Recently, Carrasco and his colleagues (2012) have prepared
and tested a group of eugenol analogues against a panel of
fungal strains. They showed the ability of nitro derivatives in
disrupting fungal cytoplasmatic membrane without inter-
acting to ergosterol.
´
condition known as Zemplen method, which resulted in
final products 7–9 (Roy and Kim, 2003). To the best of our
knowledge, eugenol lactoside was not described previ-
ously. Eugenol galactoside synthesis was described in a
few studies focusing on its use as sanitary deodorant while
its glucose derivative, on the other hand, has been known
for a long time and was obtained both from synthetic way
and natural source extraction (Mulkens and Kapetanidis,
1988). Acetylated glycosides (4–6) were obtained in good
yields (ca. 55 %) as crystalline solids after recrystallisation
from isopropyl alcohol. It could be observed in IR spectra
ester C=O and vinyl C=C bands in 1,751–1,736 cm-1 and
1,642–1,636 cm-1, respectively. All three acetylated gly-
cosides were obtained as single b-anomers. This could be
illustrated by the coupling constant of 8 Hz for a singlet at
Considering these findings, we describe here our
attempts in enhancing eugenol antifungal ability by syn-
thesizing and evaluating some glycoside derivatives, i.e. its
peracetylated and deacetylated glucosides, galactosides and
lactosides. A series of biological activities has been
attributed to natural and synthetic glycosides, since gly-
cosylation has been pointed as an important method for
improving the physicochemical and biological properties
of the parental compound (Jung and Park, 2007; Zhang
et al., 2012). Although the saccharide unit itself is gener-
ally not active, the presence of carbohydrate moieties may
modify greatly the activity of bioactive compounds. This
may occur either by modulating solubility or enhancing
absorption via fungal membrane transporters for saccha-
rides or even optimizing affinity for receptors since it may
be involved in lectin–carbohydrate interactions.
1
d 4.8 ppm in H-NMR spectra of glycoside 5, typical to
this type of proton. Aromatic hydrogens signals could be
observed at d 7–6.6 ppm range for all products confirming
the 1,2,4-trissubstituted ring pattern of eugenol moiety.
Acetyl hydrogen signals were found at d 2.1–1.9 ppm for
tetra-acetylated (4 and 5) and hepta-acetylated derivatives
(6), while methoxy hydrogens signals could be verified
´
at d 3.8 ppm, for all compounds. Zemplen’s method of
deacetylation provided final glycosides in quantitative
yield as crystalline solids. IR spectra of compounds 7–9
showed broad O–H stretching bands in 3,360–3,290 cm-1
range and no signal relative to C=O acetyl groups. This
1
was certified by H and 13C NMR spectra of these sub-
stances which showed no acetyl carbon or hydrogen signal.
MeO
MeO
OAc
O
OH
R1
OAc
O
R1
R1
MeOK
O
Eugenol, LiOH
Acetone
R2
AcO
R2
AcO
R2
HO
O
O
MeOH
OAc
OAc
OH
Br
7: R1=H; R2=OH
8: R1=OH; R2=H
1: R1=H; R2=OAc
2: R1=OAc; R2=H
4: R1=H; R2=OAc
5: R1=OAc; R2=H
9: R1=H; R2=galactosyl
3: R1=H; R2=tetra-O-acetyl galactosyl
6: R1=H; R2=tetra-O-acetyl galactosyl
Scheme 1 Synthesis of eugenol derivatives
123

Med Chem Res
Compounds 4–9 were considered pure enough for the
biological evaluations described herein, since they gave
narrow melting ranges, single-spot in TLC analysis and
ascertained identities by IR and NMR spectroscopy tech-
niques. 1H and 13C NMR spectra of eugenol glycosides are
shown in Online Resource.
9 showed no activity up to the higher concentration tested.
The overall action of acetylated glycosides was higher than
that of deacetylated derivatives. This is not surprising since
they may be acting as high lipophilicity pro-drugs which
can more easily cross the fungal cell membrane, followed
by intracellular cleavage of the acetyl groups to the free
sugars. Deacetylated derivatives themselves are unable to
penetrate because they are too polar. Most of these glucose
and galactose eugenol derivatives presented fungistatic
action and only compounds 4 and 5 for C. krusei and
compound 4 for C. glabrata presented fungicidal activity.
The cytotoxic activities of the most active compounds
(4, 5 and 7) were tested in peripheral human blood
mononuclear cells obtained from healthy donors and the
results are shown in Table 2. The prototype molecule EUG
showed higher toxicity than their derivatives on these cells
with a CC50 equal to 90 lM. Eugenol derivatives 4, 5 and 7
showed an increase in cytotoxic concentration for 50 % of
cells values ranging from 1.9 to 3.3 times higher than EUG
CC50. These data indicate that the structural modifications
done on prototype have the potential to reduce the cyto-
toxic activity of EUG in eukaryotic cells. In accordance to
our findings, the midpoint cytotoxicity values of eugenol
solutions were reported in the region of 100–300 lM by
some researchers (Atsumi et al., 2000; Babich et al., 1993)
while Prashar and colleagues (2006) have demonstrated
that unsolubilized eugenol is highly cytotoxic in concen-
trations as low as 1.8 lM. It may suggest that the phenol
group masking seen in these glycosides could be respon-
sible for this lower toxicity. The selectivity index values of
\1 for some of tested microorganisms may indicate that
the observed antifungal activity may be a result of non-
specific toxicity. On the other hand, the selectivity index of
derivative 4 (45.3) showed the in vitro inhibitory potential
of this derivative to act as potential antifungal molecule to
treat C. glabrata infections. C. glabrata is considered a
In vitro bioassays
The prototype EUG and some of its derivatives presented
activity against the fungi (yeasts) evaluated. The results of
antifungal assays are reported in Table 1, along with those
of reference drug fluconazole.
The action of EUG was lower than the standard fungi-
static drug fluconazole. Eugenol peracetyl-glucoside 4 was
the only one that presented activity against all the yeasts
with prominent results against C. glabrata, with IC50 value
160 times lower than that of EUG. The glucosides 4 and 7
were both active against C. albicans and showed activities
3- and 2-fold greater than the natural prototype EUG,
respectively. For C. tropicalis, only the derivative 4 was
active and showed activity 3-fold greater than that of EUG.
The modifications carried out on the derivatives 4 and 5 did
not contribute to the enhancement of activity modulation
against C. krusei, since they were 1.1 and 1.67 times less
active than EUG, respectively. The same negative activity
modulation was seen with derivative 4 for C. parapsilosis.
On the other hand, derivative 4 were about 3.5 times more
active than fluconazole, showing that the modifications
carried out on this derivative in comparison to the proto-
type EUG were very important for positive activity mod-
ulation. Notably, eugenol peracetyl-galactoside was 80
times less potent than its close analogue 4. This may
suggest stereoselectivity in the drug actions on these fungi
since in all other cases the glucose derivatives were more
active than those of galacto configuration. Lactosides 6 and
Table 1 Antifungal activity of eugenol and its derivatives (in lM)
Compound Microorganism
C. albicans ATCC 10231 C. tropicalis ATCC 750 C. krusei ATCC 6258 C. parapsilosis ATCC 22019 C.glabrata ATCC 90030
IC50
IC100
IC50
IC100
IC50
IC100
IC50
IC100
IC50
IC100
a
4
121.5
–
–
202.0
–
–
–
–
–
–
–
–
202.0
306.7
–
202.0
202.0
–
–
–
–
–
–
–
–
3.8
306.7
–
202.0
5
–
–
–
–
–
–
–
–
306.7
–
–
–
–
–
–
–
–
6
–
–
–
–
–
–
–
–
–
7
184.0
–
–
–
–
–
8
–
–
–
–
9
–
–
–
–
–
EUG
FLCb
a
365.8
3.3
609.8
3.3
182.9
104.5
182.9
13.1
609.8
13.1
No significant activity
b
Fluconazole
123

Med Chem Res
Table 2 Cytotoxic activity
(CC50) and selectivity index for
antifungal compounds 4, 5 and 7
on test microorganisms
Compound CC50 Selectivity index
(lM)
C. albicans
ATCC 10231
C. tropicalis
ATCC 750
C. krusei C. parapsilosis
ATCC 6258 ATCC 22019
C. glabrata
ATCC 90030
4
172.0 1.42
262.0 NDa
300.0 1.63
90.0 0.25
0.85
ND
0.85
0.85
ND
0.85
ND
45.3
0.85
ND
5
7
ND
ND
EUG
0.15
0.49
0.49
0.15
a
Not determined
common commensal in gastrointestinal and genitourinary
tracts, but can turn into an opportunistic fungal pathogen in
immunocompromised patients. This species has both innate
and acquired resistance against antifungal drugs, due to its
ability to modify ergosterol biosynthesis, mitochondrial
function, or antifungal efflux. This resistance allows for its
relative overgrowth over other susceptible species and may
contribute to the recent emergence of C. glabrata infec-
tions in chronically immunocompromised populations (Li
et al., 2007).
monitored by thin-layer chromatography (TLC) on silica
gel-G TLC plates (Merck).
Synthesis of compounds
Synthesis of peracetylglycosyl bromides (1–3)
The a-D-glycosyl bromides (intermediates 1–3) were pre-
pared according to literature methods by reacting peracetyl
glucose, galactose or lactose with hydrobromide acid in
acetic anhydride at low temperature (Mitchell et al., 2001).
Conclusions
Synthesis of peracetylated glycosides (compounds 4–6)
A set of six eugenol glycosides were synthesized by con-
ventional methods and they were evaluated in vitro as
antifungal agents against Candida sp. Derivatives 4, 5 and
7 had variable anti-Candida activity. Lactoside derivatives
6 and 9 showed no activity up to the higher concentration
tested. The peracetylglucose derivative 4 presented the
most promising antifungal action since it was less cytotoxic
and more potent than eugenol in this antimicrobial evalu-
ation. Moreover, derivative 4 was 3.5 times more potent
than fluconazole against C. glabrata. Deacetylated ana-
logue 7 and galacto stereoisomer 5 were less active than
derivative 4 on Candida species suggesting the influence of
lipophilicity and configuration issues on observed activity.
Finally, all the derivatives were less cytotoxic than euge-
nol, indicating that glycosylation was effective in order not
to be harmful to eukaryotic cells.
A solution of the corresponding glycosyl bromide (6.15
mmol) in acetone (20 mL) was added to a solution of
eugenol (18 mmol) in 1.0 mol/L lithium hydroxide
(10 mL) and the solution stirred for 2 h at room tempera-
ture. The completion of reaction was monitored by TLC
technique, when acetone was removed by ventilation and
the resulting suspension was extracted with dichlorometh-
ane (3 9 50 mL). The crude product was washed with
10 % sodium hydroxide (3 9 30 mL), water and dried by
anhydrous sodium sulphate. After filtration and removal of
the solvent under reduced pressure, the crude product was
recrystallised from isopropyl alcohol, affording compounds
4–6 (Scheme 1, Table 1).
Synthesis of deacetylated glycosides (compounds 7–9)
The peracetylated glycosides (0.7 mmol) were solubilized
in a solution of MeOK in MeOH (20 mL, 1.0 mol/L) and
stirred at room temperature for 30 min. After the comple-
tion of the reaction, noticed by TLC, the mixture was
neutralized with IRA-120 resin. The resin was filtered off
and washed with methanol. The collected filtrate was
concentrated in vacuum to afford glycosylated derivatives
7–9 (Scheme 1; Table 1).
Materials and methods
Physical measurements
Melting point of the compounds was determined on Mi-
´
croquımica MOAs 301 apparatus and was uncorrected. IR
spectroscopy was performed by Spectrum One, Perkin-
1
Elmer. H-NMR and 13C-NMR spectra were obtained on
4-allyl-2-methoxyphenyl-(2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranoside) (4) White crystals, yield: 55 %; m.p.
117–119 °C; [a]D -16.0° (c 0.5 CH2Cl2); IR; tmax in
cm-1: 3050 (ar. C–H), 2969, 2901 (sp3 C–H), 1751 (C=O),
Bruker Avance DPX-200 or Avance-DRX-400. The spe-
cific optical rotation [a]D were measured on Perkin Elmer
341, at 20 °C. Reaction courses and product mixtures were
123

Med Chem Res
1636 (C=C), 1595, 1512, 1466 (ar. C=C); 1H NMR
(CDCl3, 200 MHz) d ppm: 7.0 (1H, Ar–H), 6.69–6.64 (m,
2H, Ar–H), 5.9–5.8 (1H, Allylic), 5.2–4.8 (m, 6H, sp3,
Allylic), 4.3–4.0 (m, 2H, sp3), 3.8–3.7 (m, 4H, sp3), 3.3
(2H, sp3), 2.05–2.01 (12H, sp3); 13C NMR (CDCl3,
50 MHz) d ppm: 170.4–169.1 (4C, C=O), 150.2 (1C, ar.),
144.1 (1C, ar.), 136.9 (1C, ar.), 136.5 (1C, allylic), 120.3
(1C, ar.), 120.0 (1C, ar.), 115.7 (1C, allylic), 112.7 (1C,
ar.), 100.7 (1C, sugar), 72.3 (1C, sugar), 71.6 (1C, sugar),
70.9 (1C, sugar), 68.1 (1C, sugar), 61.6 (1C, sugar), 55.6
(1C, sp3), 39.6 (1C, sp3), 20.3–20.3 (4C, sp3); Mol. for-
mula: C24H30O11; Mol. Weight: 494.4.
2975, 2917 (sp3 C–H), 1638 (C=C), 1595, 1512, 1465 (ar.
1
C=C); H NMR (DMSO, 200 MHz) d ppm: 7.0 (1H, Ar–
H), 6.8 (1H, Ar–H), 6.6 (1H, Ar–H), 6.0–5.8 (1H, Allylic),
5.1–4.8 (m, 6H, sp3, Allylic, OH), 4.5 (1H, OH), 3.7–3.1
(m, 10H, sp3, Allylic, OH); 13C NMR (DMSO, 50 MHz) d
ppm: 148.8 (1C, ar.), 144.8 (1C, ar.), 137.9 (1C, allylic),
133.4 (1C, ar.), 120.2 (1C, ar.), 115.5 (1C, allylic), 115.4
(1C, ar.), 112.8 (1C, ar.), 100.1 (1C, sugar), 76.9 (1C,
sugar), 76.8 (1C, sugar), 73.2 (1C, sugar), 69.6 (1C, sugar),
60.5 (1C, sugar), 55.5 (1C, sp3), 38.8 (1C, sp3); Mol. for-
mula: C16H22O7; Mol. Weight: 326.24.
4-allyl-2-methoxyphenylb-D-galactopyranoside(8) White
crystals, yield: 55 %; m.p. 163–165 °C; [a]D –34.6° (c 0.5
MeOH); IR; tmax in cm-1: 3350 (OH), 3001 (ar. C–H), 2975,
2938, 2903 (sp3 C–H), 1638 (C=C), 1594, 1514, 1464 (ar.
C=C); 1H NMR (DMSO, 200 MHz) d ppm: 7.0 (1H, Ar–H),
6.7 (1H, Ar–H), 6.6 (1H, Ar–H), 6.0–5.8 (1H, Allylic),
5.1–5.0 (m, 3H, sp3, OH), 4.87–4.80 (m, 2H, sp3, Allylic), 4.6
(1H, OH), 4.5 (1H, Allylic), 3.7–3.2 (m, 11H, sp3, Allylic);
13C NMR (DMSO, 50 MHz) d ppm: 148.9 (1C, ar.), 144.9
(1C, ar.), 137.9 (1C, allylic), 133.3 (1C, ar.), 120.2 (1C, ar.),
115.5 (1C, allylic), 115.4 (1C, ar.), 112.8 (1C, ar.), 100.7 (1C,
sugar), 75.4 (1C, sugar), 73.5 (1C, sugar), 70.2 (1C, sugar),
68.1 (1C, sugar), 60.3 (1C, sugar), 55.6 (1C, sp3), 39.1 (1C,
sp3); Mol. formula: C16H22O7; Mol. Weight: 326.24.
4-allyl-2-methoxyphenyl-(2,3,4,6-tetra-O-acetyl-b-D-galac-
topyranoside) (5)
White crystals, yield: 56 %; m.p.
125–127 °C; [a]D –11.5° (c 0.5 CH2Cl2); IR; tmax in cm-1
:
2972, 2881 (sp3 C–H), 1736 (C=O), 1642 (C=C), 1604,
1588, 1508, 1467 (ar. C=C); 1H NMR (CDCl3, 200 MHz) d
ppm: 7.0 (1H, Ar–H), 6.7–6.6 (m, 2H, Ar–H), 6.0–5.8 (1H,
Allylic), 5.5–5.4 (m, 2H, Allylic, sp3), 5.1–5.0 (m, 3H, sp3,
Allylic), 4.86 (1H, anomeric, J = 8 Hz), 4.2–4.1 (m, 2H,
sp3), 3.98–3.92 (1H, sp3), 3.8 (3H, sp3), 3.34 (2H, sp3),
2.1–2.0 (12H, sp3); 13C NMR (DMSO, 50 MHz) d ppm:
170.1–169.3 (4C, C=O), 150.2 (1C, ar.), 144.3 (1C, ar.),
136.9 (1C, ar.), 136.4 (1C, allylic), 120.3 (1C, ar.), 119.8
(1C, ar.), 115.7 (1C, allylic), 112.7 (1C, ar.), 101.3 (1C,
sugar), 70.56 (1C, sugar), 70.50 (1C, sugar), 68.4 (1C,
sugar), 66.6 (1C, sugar), 60.9 (1C, sugar), 55.7 (1C, sp3),
39.6 (1C, sp3), 20.5–20.3 (4C, sp3); Mol. formula:
C24H30O11; Mol. Weight: 494.4.
4-allyl-2-methoxyphenyl b-D-lactoside (9) Brown crys-
tals, yield: 50 %; m.p. 190–192 °C; [a]D -27.5° (c 0.5
MeOH); IR; tmax in cm-1: 3358 (OH), 2971, 2918 (sp3 C–
1
H), 1638 (C=C), 1593, 1512, 1460 (ar. C=C); H NMR
4-allyl-2-methoxyphenyl-(2,3,6,20,30,40,60-hepta-O-acetyl-
b-D-lactoside) (6) Pale yellow crystals, yield: 51 %; m.p.
51.2–53,7 °C; [a]D –19.7° (c 0.5 CH2Cl2); IR; tmax in
cm-1: 2972, 2901 (sp3 C–H), 1741 (C=O), 1638 (C=C),
1594, 1509 (ar. C=C); 1H NMR (CDCl3, 300 MHz) d ppm:
6.9 (1H, Ar–H), 6.7–6.6 (m, 2H, Ar–H), 5.9–5.8 (1H,
Allylic), 5.3–4.8 (m, 8H, sp3, allylic), 4.1–4.0 (m, 4H, sp3),
3.9–3.8 (m, 3H, sp3), 3.7–3.6 (m, 1H, sp3), 3.7 (s, 3H, sp3),
3.3 (2H, sp3, J = 6 Hz), 2.1–1.9 (21H, sp3); 13C NMR
(CDCl3, 75 MHz) d ppm: 170.6–169.3 (7C, C=O), 150.7
(1C, ar.), 144.6 (1C, ar.), 137.4 (1C, ar.), 136.9 (1C,
allylic), 120.8 (1C, ar.), 120.2 (1C, ar.), 116.2 (1C, allylic),
113.3 (1C, ar.), 101.2 (1C, sugar), 101.0 (1C, sugar), 76.5
(1C, sugar), 73.0 (1C, sugar), 72.9 (1C, sugar), 71.6 (1C,
sugar), 71.2 (1C, sugar), 70.9 (1C, sugar), 69.3 (1C, sugar),
62.2 (1C, sugar), 61.1 (1C, sugar), 60.6 (1C, sugar), 56.1
(1C, sp3), 39.9 (1C, sp3), 20.8 (7C, sp3); Mol. formula:
C36H46O19; Mol. Weight: 782.4.
(DMSO, 400 MHz) d ppm: 6.9 (1H, Ar–H), 6.7 (1H, Ar–
H), 6.5 (1H, Ar–H), 5.89–5.79 (1H, allylic), 5.23 (1H, sp3),
4.99–4.92 (4H, allylic, sp3), 4.83 (2H, OH), 4.66 (1H, OH),
4.56–4.40 (2H, sp3), 4.1 (1H, OH), 3.64 (3H, sp3),
3.53–3.19 (m, 14H, sp3, OH, allylic). 13C NMR (DMSO,
50 MHz) d ppm: 148.9 (1C, ar.), 144.7 (1C, ar.), 137.8 (1C,
ar.), 133.6 (1C, allylic), 120.2 (1C, ar.), 115.57 (1C,
allylic), 115.55 (1C, ar.), 112.9 (1C, ar.), 103.8 (1C, sugar),
99.8 (1C, sugar), 80.2 (1C, sugar), 75.5 (1C, sugar), 75.0
(1C, sugar), 74.8 (1C, sugar), 73.2 (1C, sugar), 73.0 (1C,
sugar), 70.5 (1C, sugar), 68.1 (1C, sugar), 60.4 (1C, sugar),
60.1 (1C, sugar), 55.6 (1C, sp3), 39.0 (1C, sp3); Mol. for-
mula: C22H32O12; Mol. Weight: 488.32.
In vitro bioassays
Antifungal activity evaluation
Eugenol (EUG) and its derivatives 4, 5, 6, 7, 8 and 9 were
evaluated in vitro for their antimicrobial activities against
the fungi through a Mueller–Hinton broth microdilution
method and with the methodology and interpretative
4-allyl-2-methoxyphenyl b-D-glucopyranoside (7) Brown
crystals, yield: 54 %; m.p. 109–110 °C; [a]D –38.4° (c 0.5
MeOH); IR; tmax in cm-1: 3295 (OH), 3077 (ar. C–H),
123

Med Chem Res
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DMSO 1 % at final concentration and tested at concen-
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(fungistatic action) and 100 % (IC100) (fungicidal action)
in lM and compared for each compound and microor-
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IC50 and IC100 values and to the growth control well were
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absence of growth confirmed the fungicidal action while
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gradient centrifugation. The cell suspension of PBMCs at a
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96-well plate, 90 lL in each well with 10 lL of test com-
pounds at different concentrations, incubated at 37 °C in an
incubator at 5 % CO2 for 48 h. After, it was added 10 lL of
MTT dye (5 mg/mL) and the cells were incubated again for
an additional 4-h period. Then, the medium was carefully
removed and added to 100 lL of DMSO for solubilization of
formazan crystals. The plates were shaken for 5 min and
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tometric microplate reader at 560 nm. The percentage of
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A and B are the absorbances of control and treated cells,
respectively. Data were analyzed using linear regression to
obtain values for CC50 (cytotoxic concentration for 50 %).
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