Structure-antifungal activity relationship of cinnamic acid derivatives

Article abstract of DOI:10.1021/jf0729098

A structure-antifungal activity relationship (SAR) study of 22 related cinnamic acid derivatives was carried out. Attention was focused on the antifungal activities exhibited against Aspergillus flavus, Aspergillus terreus, and Aspergillus niger. (E)-3-(4-Methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid (16) exhibited antifungal activity against A. niger, comparable to that of miconazole and a significant antifungal effect against A. flavus and A. terreus as well. A structure-activity relationship (SAR) study of related cinnamic acid derivatives has allowed a model to be proposed for the recognition of the minimal structural requirements for the antifungal effect in this series.

Full text of DOI:10.1021/jf0729098

J. Agric. Food Chem. 2007, 55, 10635–10640 10635
Structure-Antifungal Activity Relationship of
Cinnamic Acid Derivatives
FABRICIO BISOGNO,†,‡ LAURA MASCOTI,† CECILIA SANCHEZ,†,‡
FRANCISCO GARIBOTTO,† FERNANDO GIANNINI,† MARCELA KURINA-SANZ,†,‡ AND
RICARDO ENRIZ*,†
Departamento de Química, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de
San Luis, Chacabuco 917, 5700 San Luis, Argentina, and INTEQUI-CONICET-Departamento de
Química, Universidad Nacional de San Luis, Chacabuco 917, San Luis, Argentina
A structure-antifungal activity relationship (SAR) study of 22 related cinnamic acid derivatives was
carried out. Attention was focused on the antifungal activities exhibited against Aspergillus flavus,
Aspergillus terreus, and Aspergillus niger. (E)-3-(4-Methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic acid
(16) exhibited antifungal activity against A. niger, comparable to that of miconazole and a significant
antifungal effect against A. flavus and A. terreus as well. A structure–activity relationship (SAR) study
of related cinnamic acid derivatives has allowed a model to be proposed for the recognition of the
minimal structural requirements for the antifungal effect in this series.
KEYWORDS: Antifungal; aspergillosis; cinnamic acid derivatives; structure–activity relationship
INTRODUCTION
several animal species, including rodents, nonhuman primates,
and fishes (2). The toxicological data and extensive investiga-
tions on association between aflatoxin exposure and hepatocel-
lular carcinoma led to the classification of aflatoxin B1 as a
category 1 human carcinogen by the International Agency for
Research of Cancer (3–6).
The Aspergillus genus is composed of more than 180 different
species. Fortunately, only a few species are able to produce
infections. Three species, Aspergillus fumigatus, Aspergillus
flaVus, and Aspergillus terreus, cause about 95% of the
pathologic cases. Unfortunately, the available fungicides have
not been effective in the control of these fungi, and therefore
there is a real need for novel compounds against aspergillosis.
Aspergillus niger is less likely to cause human disease than some
other Aspergillus species, but if large amounts of spores are
inhaled, a serious lung disease, aspergillosis, can occur. As-
pergillosis is particularly frequent among horticultural workers
who inhale peat dust, which can be rich in Aspergillus spores.
A. niger is one of the most common causes of otomycosis
(fungal ear infections) which can cause pain, temporary hearing
loss, and, in severe cases, damage to the ear canal and tympani
membrane.
A major problem in corn and other cereal crops concerns
contamination with aflatoxins that are produced by the fungi
A. flaVus and Aspergillus parasiticus. These compounds con-
stitute a number of structurally related secondary metabolites,
which differ considerably in their biological effect. Aflatoxin
B1 is by far the most potent teratogen, mutagen, and hepato-
carcinogen of all aflatoxins (1). The carcinogenic potential of
aflatoxin B1 following oral administration has been shown in
The study of natural products as sources for lead structures
has enjoyed a resurgence of interest over the last 20 years. The
reasons for this are varied, but the success and the uniqueness
of natural products are probably the most important. Virtually
all plants share the problems of recognition and reaction in
defense mechanisms, and different processes have evolved for
combating pathological challenges (7). Natural compounds and
their derivatives could potentially serve as effective alternatives
to conventional antifungal agents. Conventional fungicides are
frequently perceived to present hazard to human health and
environment (8, 9). Several phenolic compounds and derivatives
have been reported possessing antifungal activity. Gallic acid
has been reported to prevent aflatoxin biosynthesis by A. flaVus
(10). Two new natural products [1-(3′-methoxypropanoyl)-2,4,5-
trimethoxybenzene and 2-(2Z)-(3-hydroxy-3,7-dimethylocta-2,6-
dienyl)-1,4-benzenediol] isolated from the root bark of Cordellia
alliodora have been reported as antifungal and larvicidal
compounds (11). More recently, Kim et al. (12) reported
antifungal effects of several phenolic compounds including
cinnamic acid (1), m-coumaric acid (12), p-coumaric acid (3),
and caffeic acid (6).
* Corresponding author: fax, 54-2652-431301; e-mail, denriz@
unsl.edu.ar.
The exudate of the aerial parts of the perennial shrub
Baccharis grisebachii has been shown to display activity toward
dermatophytic fungi and some bacteria (13). Gianello and
Giordano (14) reported the isolation of 3-(γ,γ-dimethylallyl)-
p-coumaric acid (14) from the aerial parts of B. grisebachii
† Departamento de Química, Facultad de Química, Bioquímica y
Farmacia, Universidad Nacional de San Luis.
‡ INTEQUI-CONICET-Departamento de Química, Universidad Na-
cional de San Luis.
10.1021/jf0729098 CCC: $37.00
2007 American Chemical Society
Published on Web 11/27/2007

10636 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Bisogno et al.
Preparation of (E)-Methyl 3-(3,4-Dimethoxyphenyl)acrylate (9).
Compound 8 (100 mg, 0.51 mmol) was dissolved in 20 mL of acetone,
255 mg (1.78 mmol) of K2CO3 and 122 µL (1.29 mmol) of Me2SO4
were added, and the solution was heated to reflux. After reaction
completion (3 h) the reaction mixture was filtered and rinsed with
EtOAc. Purification was carried out by column chromatography on Si
gel with n-hexane-EtOAc (7:3) as solvent, yielding 103 mg (90%) of
9 as pale yellow crystals; mp 63–66 °C. 1H NMR and 13C NMR data
were in agreement with literature data (16).
Preparation of (E)-3-(3,4-Dimethoxyphenyl)acrylic Acid (10). Com-
pound 9 (50 mg, 0.22 mmol) was dissolved in 5 mL of THF, and 100
µL of H2O was added. Then, 50 mg of KOH was added; the mixture
was stirred and heated to reflux for 1 h. After reaction completion the
mixture was brought to pH 4 by the addition of HCl and the solvent
evaporated in vacuo. The aqueous phase was extracted three times with
Et2O and the organic layer washed with H2O and dried with anhydrous
Na2SO4. The solvent was evaporated under reduced pressure. Purifica-
tion was carried out by column chromatography on Si gel with
n-hexane-EtOAc (7:3) as eluent, yielding 37 mg (78%) of 10 as white
crystals: mp 183–184 °C). The NMR spectra were in agreement with
reported data (19).
collected in the Province of Mendoza, Argentina. Antimicotic
effects toward the dermatophytes Microsporum canis, Epider-
mophyton floccosum, Trichophyton mentagraphytes, and Tri-
chophyton rubrum were reported for this compound. However,
compound 14 did not show antifungal activity against A.
fumigatus, A. flaVus, and A. niger (13).
Our principal goal is to obtain new compounds possessing
antifungal effects. In particular, we are interested in structures
with antifungal activity against fungi, such as A. flaVus, A.
terreus, and A. niger. In the present study we chose 3-(γ,γ-
dimethylallyl)-p-coumaric acid (14), known as drupanin, and
cinnamic acid (1) as the starting structures. To perform a
structure–activity relationship study, several derivatives of
compounds 1 and 14 were prepared and tested. Thus, we report
here the preparation of a series of cinnamic acid derivatives,
their antifungal effects, acute toxicity, and a structure–activity
relationship (SAR) study performed in this series.
MATERIALS AND METHODS
Preparation of (E)-Methyl 3-(4-Hydroxy-3-methoxyphenyl)acrylate
(11). Compound 8 (100 mg, 0.51 mmol) was methylated with an Et2O
solution of CH2N2 at 0 °C for 2 h. Purification by column chromatog-
raphy on Si gel using n-hexane-EtOAc (5:5) as solvent yielded 74
mg (69%) of 11 as white crystals: mp 49–51 °C. 1H NMR and 13C
NMR data were in agreement with literature data (17).
General Methods. Solvents were distilled and dried prior to use.
1H NMR and 13C NMR spectra were obtained on Bruker AC-200 and
AC-400 (200 and 400 MHz and 50.2 and 100 MHz, respectively)
spectrometers in CDCl3; chemical shifts (δ) in parts per million are
relative to TMS; coupling constants (J) are in Hertz. Analytical TLC
was performed on Merck precoated silica gel 60 F254 plates. Solvents
for TLC were n-hexane-EtOAc mixtures. A. niger ATCC 11394 was
purchased from the American Type Culture Collection, while A. flaVus
UBA 294 and A. terreus INM 031783 were obtained from the Micoteca
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
Aires, and Instituto “Carlos Malbran”, Argentina, respectively. Fungi
were maintained on PDA-slants. Spore suspensions were obtained
according to reported procedures (15) and adjusted to 107 spores
with colony forming ability per milliliter in PBS with added Tween
20 (5 µL/mL).
Compounds. Cinnamic acid (1), methyl cinnamate (2), (E)-3-(4-
hydroxyphenyl)acrylic acid (p-coumaric acid) (3), (E)-3-(3,4-dihydroxy-
phenyl)acrylic acid (caffeic acid) (6), (E)-3-(4-hydroxy-3-methoxyphe-
nyl)acrylic acid (ferulic acid) (8), and (E)-3-(3-hydroxyphenyl)acrylic acid
(m-coumaric acid) (12) were purchased from Sigma-Aldrich Chemical Co.,
Buenos Aires, Argentina.
Isolation of Drupanin (14). (E)-3-(4-Hydroxy-3-(3-methylbut-2-
enyl)phenyl)acrylic acid was isolated as colorless crystals (mp 147-148
°C) from aerial parts of B. grisebachii Hieron. Its chemical identity
was confirmed by spectroscopic data, compared with previous reports
(14).
Preparation of (E)-Methyl 3-(3-Hydroxyphenyl)acrylate (13). Com-
pound 12 (100 mg, 0.61 mmol) was dissolved in 20 mL of MeOH,
and two drops of HCl (35%) was added. The mixture was stirred and
heated to reflux overnight. After reaction completion it was neutralized
with solid Na2CO3 and partitioned with EtOAc. Purification was carried
out by column chromatography on Si gel with n-hexane-EtOAc (5:5)
1
as solvent, yielding 92 mg (85%) of 13. H NMR and 13C NMR data
were in agreement with literature data (17).
Preparation of (E)-Methyl 3-(4-Methoxy-3-(3-methylbut-2-enyl)-
phenyl)acrylate (15). Compound 14 (100 mg, 0.43 mmol) was dissolved
in 20 mL of acetone, 208 mg (1.5 mmol) of K2CO3 and 122 µL (1.29
mmol) of Me2SO4 were added, and the solution was heated to reflux.
After reaction completion (3 h) the reaction mixture was filtered, and
20 mL of EtOAc was added. Then, the solvent was evaporated under
reduced pressure, and the crude residue was subjected to column
chromatography on Si gel with n-hexane-EtOAc (7:3) as eluent,
yielding 68 mg (61%) of 15 as a colorless oil. The NMR spectrum
was in agreement with reported data (20).
Preparation of (E)-3-(4-Methoxy-3-(3-methylbut-2-enyl)phenyl)acrylic
Acid (16). Compound 15 (100 mg, 0.38 mmol) was dissolved in 5 mL
of THF, and 100 µL of H2O was added. Then, 50 mg of KOH was
dissolved, and the mixture was stirred and heated to reflux for 1 h.
After reaction completion the mixture was brought to pH 4 by the
addition of HCl and the solvent evaporated in vacuo. The aqueous phase
was extracted three times with Et2O and the organic layer washed with
H2O and dried with anhydrous Na2SO4. The solvent was evaporated
under reduced pressure. Purification was carried out by column
chromatography on Si gel with n-hexane-EtOAc (5:5) as eluent,
yielding 72 mg (76%) of 16. 1H NMR and 13C NMR data were in
agreement with literature data (21).
Preparation of (E)-Methyl 3-(4-Hydroxy-3-(3-methylbut-2-enyl)phe-
nyl)acrylate (17). Compound 14 (100 mg, 0.43 mmol) was methylated
with an Et2O solution of CH2N2 at 0 °C for 2 h. Purification by column
chromatography on Si gel using n-hexane-EtOAc (6:4) as eluent
yielded 76 mg (72%) of 17 as pale yellow crystals: mp 81–84
°C (22, 23). 1H NMR and 13C NMR data were in agreement with those
previously reported (20).
Preparation of (E)-3-(4-Acetoxy-3-(3-methylbut-2-enyl)phenyl)acrylic
Acid (18). Compound 14 (100 mg, 0.43 mmol) was dissolved in 2 mL
of dry pyridine, and 2 mL of acetic anhydride was added. The mixture
was kept at room temperature overnight. After all of the starting material
was consumed (TLC control), the mixture was poured into a beaker
containing ice and 30 mL of Et2O. The organic layer was washed with
an aqueous solution of 5% CuSO4 to remove pyridine, followed by a
Preparation of (E)-Methyl 3-(4-Hydroxyphenyl)acrylate (4). Com-
pound 3 (100 mg, 0.61 mmol) was methylated with an Et2O solution
of CH2N2 at 0 °C for 2 h. Purification by column chromatography on
Si gel using n-hexane-EtOAc (5:5) as solvent yielded 80.4 mg (74%)
1
of 4. H NMR and 13C NMR data were in agreement with literature
data (16, 17).
Preparation of (E)-Methyl 3-(4-Methoxyphenyl)acrylate (5). Com-
pound 3 (100 mg, 0.61 mmol) was dissolved in 20 mL of acetone, 294
mg (2.14 mmol) of K2CO3 and 122 µL (1.29 mmol) of Me2SO4 were
added, and and the solution was heated to reflux. After reaction
completion (3 h) the reaction mixture was filtered and rinsed with
EtOAc. Purification was carried out by column chromatography in Si
gel with n-hexane-EtOAc (7:3) as solvent, yielding 79.5 mg (68.%)
1
of 5. H NMR and 13C NMR data were in agreement with literature
data (16).
Preparation of (E)-Methyl 3-(3,4-Dihydroxyphenyl)acrylate (7).
Compound 6 (100 mg, 0.52 mmol) was dissolved in MeOH, and two
drops of HCl (35%) was added. The mixture was stirred and heated to
reflux overnight; after reaction completion it was neutralized with solid
Na2CO3 and partitioned with EtOAc. Purification was carried out by
column chromatography on Si gel with n-hexane-EtOAc (4:6) as
solvent, yielding 86.4 mg (80%) of 7: white crystals; mp 161–163 °C
(18).

Antifungal Activity
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10637
5% solution of NaHCO3 and water. After drying over anhydrous
Na2SO4, the organic solvent was removed in vacuum, and the residue
was subjected to Si gel column chromatography using n-hexane-EtOAc
(7:3) to yield 95.5 mg (81%) of 4-acetyl-3-prenyl-p-coumaric acid (18):
colorless crystals; mp 141 °C. Spectroscopic and spectrometric data
were in agreement with those previously reported (14).
H-4), and 7.92 (dd, 1, J ) 7.4 Hz, J ) 1.7 Hz, H-6); 13C NMR (CDCl3)
δ 23.79 (CH3), 25.83 (CH3), 29.74 (C-3), 71.56 (COH(CH3)2), 90.31
(C-2), 108.77 (C-7), 123.72 (C-9), 127.11 (C-4), 127.56 (C-5), 131.66
(C-6), 165.56 (C-8), and 166.65 (COOH); MS [m/z (%)] 222 (47, M),
204 (37), 189 (37), 164 (100), 119 (60), and 91 (36). These data were
in agreement with those reported for anodendroic acid (27).
Acute Toxicity Test. The toxic effect of compounds 1-22 was
evaluated using a toxicity test on fish and amphibians. The static
technique recommended by the U.S. Fish and Wildlife Service
Columbia National Fisheries Research Laboratory (28) was modified
in order to use a lower amount of test compounds (29). Fish of the
species Poecilia reticulata were born and grown in our laboratory until
they reached a size of 0.7-1 cm (20 days old). Amphibians of
embryo-larval stage XII (30) of Bufo arenarum were also used. In
the toxicity test, at least 10 specimens were exposed to each of the
three concentrations tested per drug (ranged from 10 to 2.5 µg/mL) in
2000 mL wide-mouthed jars containing 1000 mL of test solutions. The
test began upon initial exposure to the potentially toxic agent and
continued for 96 h. The number of dead organisms in each test chamber
was recorded, and the dead organisms were removed every 24 h.
General observations on the conditions of the test organism were also
recorded at this time. However, the percentage of mortality was recorded
at 96 h. Each experiment was performed two times with three replicates
each.
Preparation of Methyl 3-(4-Hydroxy-3-isopentylphenyl)propanoate
(19). To 17 (100 mg, 0.40 mmol) dissolved in EtOAc (50 mL) was
added a catalytic amount of 5% Pd/C. The suspension was stirred
vigorously under hydrogen (1 atm). When the consumption of H2
subsided, the mixture was filtered through a thin layer of Si gel over
Celite and concentrated under reduced pressure. The residue was
subjected to column chromatography on Si gel with n-hexane-EtOAc
(7:3) as eluent to yield 84.7 mg (87%) of 19 as pale yellow crystals:
mp 172–175 °C. Spectroscopic data were in agreement with those
reported by Carrizo et al. (24).
Preparation of 3-(4-Hydroxy-3-methoxyphenyl)propanoic Acid (20).
To 8 (100 mg, 0.51 mmol) dissolved in EtOAc (50 mL) was added a
catalytic amount of 5% Pd/C. The suspension was stirred vigorously
under hydrogen (1 atm). When the consumption of H2 subsided, the
mixture was filtered through a thin layer of Si gel over Celite and
concentrated under reduced pressure. The residue was subjected to
column chromatography on Si gel with mixtures of n-hexane-EtOAc
1
(7:3) as eluent to yield 85 mg (84%) of 20. H NMR and 13C NMR
data were in accordance with literature (25).
Preparation of Methyl 3-(4-Hydroxyphenyl)propanoate (21). To 4
(100 mg, 0.51 mmol) dissolved in EtOAc (50 mL) was added a catalytic
amount of 5% Pd/C. The suspension was stirred vigorously under
hydrogen (1 atm). When the consumption of H2 subsided, the mixture
was filtered through a thin layer of Si gel over Celite and concentrated
under reduced pressure. The residue was subjected to column chro-
matography on Si gel with mixtures of n-hexane-EtOAc (7:3) as eluent
to yield 83 mg (82%) of 21 as a colorless oil. 1H NMR and 13C NMR
data were in agreement with those previously reported (20).
Preparation of 3-(3-Hydroxyphenyl)propanoic Acid (22). Compound
12 (100 mg, 0.61mmol) was dissolved in EtOAc and treated as
described in the preparation of compound 8 to yield 89 mg (88%) of
22. Spectroscopic data were in agreement with literature values (22).
Antifungal Assays. The agar dilution method was used for the
antifungal evaluations, according to reported procedures (13, 23).
Sabouraud dextrose media were used, and stock solutions of pure
compounds in H2O/DMSO, 24:1 (1000 µg/mL), were added to give
final dilutions ranged from 250 to 1.95 µg/mL. The final DMSO
concentration in the media did not exceed 2%. An inoculum of 105
spores/mL from each agar well was used. The antifungal agent
myconazole (Laboratorio Saporiti, Argentina) was included as positive
control. The wells were incubated for 48 h at 28 °C. MIC was defined
as the lowest compound concentration showing no visible fungal growth
after incubation time. MICs g250 µg/mL were considered as
inactive.
Biotransformation Experiments. A two-step procedure adapted
from Orden et al. (26) was used. Spore suspensions from A. terreus
were incubated in liquid soybean meal-glucose medium (glucose, 20 g;
yeast extract, 5 g; soybean meal, 5 g; NaCl, 5 g; K2HPO4, 5 g; distilled
water to 1000 mL, pH 7) at 28 °C on a rotatory shaker at 180 rpm.
Forty-eight-hour-old cultures (5 mL) were subcultured in 125 mL
Erlenmeyer flasks containing 30 mL of fresh culture medium each.
After 24 h, substrates (5 mg/batch), dissolved in ethanol (50 µL), were
added to the fungal cultures. Biotransformation progress was monitored
daily by TLC. After 8 days of incubation, the fermentation broth was
filtered, and the filtrate was extracted with EtOAc (4 × 50 mL), dried
over anhydrous Na2SO4, and evaporated under reduced pressure. Blank
assays without substrates and without fungi were carried out in parallel.
Each experiment was performed three times with three replicates
each.
RESULTS AND DISCUSSION
Kim et al. (12) reported that coumaric acids (3 and 12) were
less inhibitory to fungal growth than cinnamic acid (1) whereas
caffeic acid (6) showed no antifungal activity at any concentra-
tions. Our MIC values (Table 1) are in agreement with those
results; however, it should be noted that, in our bioassays,
compound 1 displays only a moderate antifungal activity. Our
results indicate that compound 14 showed no antifungal activity
against A. terreus and A. flaVus and only a moderate effect
against A. niger (125 µg/mL). These results are in agreement
with those previously reported by Feresin et al. (13). To study
the structure–activity relationships, different types of structures
and the effects of structural changes in different regions of the
molecules were considered: elimination of the double bond of
the side chain, giving the general structure type B, and changes
of the R1, R2, and R3 substituents in the general structure types
A and B according to Table 1, which gives the MIC values
obtained for the different compounds of this series. From these
results, it is clear that compounds without the double bond in
the side chain (compounds possessing the general structure type
B) were inactive. This result is not unexpected; the presence of
such double bond confers particular conformational and elec-
tronic characteristics to these compounds. The most active
compounds in this series were compounds 8 and 16, which
possess a COOH group in the side chain. However, it is clear
that the presence of this group seems to be necessary but not
by itself sufficient to produce the antifungal effect. The lack of
activity of compounds 3, 6, 10, 12, and 14 illustrates this
situation very well. In general, it seems that a COOH group is
preferred to a COOCH3 group (compare MICs of compound 1
with 2, 8 with 11, and 16 with 15). However, this situation is
not clear when the substituents at R2 and/or R3 are OH groups
(compare MICs of 3 with 4, 6 with 7, and 12 with 13).
Nevertheless, these compounds displayed only moderate or
marginal antifungal effects.
Compound 16 was the most active compound in this series,
displaying a MIC of 1.95 against A. niger, which is comparable
to that of miconazole, and also possessing very interesting
activities against A. terreus and A. flaVus as well. The lack of
activity obtained for compounds 14, 15, and 17 is noteworthy
even though they possess a closely related structure to that of
Biotransformation of 14. Organic extracts were pooled and purified
by preparative TLC using EtOAc as eluent, yielding 2-(2-hydroxypro-
pan-2-yl)-2,3-dihydrobenzofuran-5-carboxylic acid (23) as pale yellow
1
crystals: mp 156–158 °C; H NMR (CDCl3) δ 1.25 (s, 3, CH3), 1.37
(s, 3, CH3), 3.20 (d, 2, J ) 8.5 Hz, H-2), 3.60 (br s, 1, OH), 4.70 (t, 1,
J ) 8.5 Hz, H-1), 6.82 (d, 1, J ) 7.4 Hz, H-7), 7.90 (d, 1, J ) 1.5 Hz,

10638 J. Agric. Food Chem., Vol. 55, No. 26, 2007
Bisogno et al.
Table 1. Structural Features and Minimal Inhibitory Concentrations (MICs)
of Compounds 1–22
chemically with the fungi in a different way. Thus, in an attempt
to explain the difference in bioactivity of compounds 14 and
16 toward A. terreus, both compounds were used as substrates
in whole cell biotransformation procedures. The inactive natural
product 14 was metabolized into a more polar metabolite, 23
(Figure 1), after 8 days of incubation in a two-step biotrans-
formation procedure. Compound 23 was recovered from the
culture media as the sole biotransformation product; meanwhile,
the substrate was completely consumed.
MIC (µg/mL)
Its 1H NMR spectrum shows two singlets at high fields (1.25
and 1.37 ppm) assigned to the methyl groups on the side chain
at C-1. In the HSQC spectrum both singlets show a strong cross-
peak with two carbons at 23.79 and 25.83 ppm, respectively.
The HMBC shows the long-range interactions of both methyl
groups with two oxygenated carbons, a singlet at 71.56 ppm
and a doublet at 90.31 ppm. The doublet at 3.20 ppm in the 1H
NMR, which integrates for two hydrogens, is coupled with a
triplet of a proton at 4.70 ppm located on an oxygenated carbon
(90.31 ppm). These data are supported with the correlation
observed between both signals in the COSY spectrum. In the
HSQC they show correlations with C-2 at 29.73 ppm. On the
other hand, the HMBC spectrum shows the long-range correla-
tions between the methyl and the methylene signals with the
oxygenated C-2 at 90.31 ppm. In the same spectra the methylene
group shows a cross-peak with aromatic carbon C-4 at 127.56
ppm, oxygenated carbon C-2 at 90.31 ppm, and a cross-peak
with the singlet at 71.56 ppm attributable to a quaternary
alcoholic carbon. At low fields, in the 1H NMR spectrum, three
signals integrating for one proton each, corresponding to the
aromatic hydrogens, are evident. The doublet (J ) 7.4 Hz) at
6.82 ppm is coupled with a double doublet at 7.92 ppm (J )
7.4 and 1.5 Hz), indicating that the hydrogens present an ortho
coupling. Meanwhile, the former signal is coupled with the other
doublet (J ) 1.5 Hz), indicative of meta coupling. These data
are in complete agreement with the strong correlations observed
in the COSY spectra. In addition, the HSQC relates the doublet
at 6.82 ppm with carbon C-7 at 108.77 ppm and the other
aromatic signals with carbon C-6 at 131.66 ppm and C-4 at
127.11 ppm, respectively. The described data indicate that the
aromatic ring is substituted at C-5 with a carboxylic acid group
(164.35 ppm). These data are consistent with the structure of
the bicyclic metabolite 2-(2-hydroxypropan-2-yl)-2,3-dihy-
drobenzofuran-5-carboxylic acid which was previously reported
as anodendroic acid from the species Anodendron affine
(Apocynaceae) and Eriodictyon sessilifolium (Hydrophyllaceae)
(30).
A.
A.
A.
compd
type R1
R2
R3
terreus niger
flavus
1
2
3
4
5
6
7
8
A
A
A
A
A
A
A
A
A
A
A
A
A
A
H
CH3
H
CH3
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
H
H
H
H
H
OH
OH
H
H
250
>250
>250
125
125
>250
125
250
>250
>250
125
>250
>250
>250
31.25
>250
>250
>250
>250
125
OH
OH
125
OCH3 >250
>250
>250
125
OH
OH
OH
>250
125
>250
OCH3
OCH3
OCH3
OCH3
OH
OH
γ,γ-
dimethylallyl
γ,γ-
dimethylallyl
γ,γ-
dimethylallyl
γ,γ-
dimethylallyl
γ,γ-
dimethylallyl
62.5
9
OCH3 >250
OCH3 >250
>250
>250
>250
>250
125
10
11
12
13
14
OH
H
H
OH
>250
>250
125
>250
125
>250
15
16
17
18
A
A
A
A
CH3
H
OCH3 125
>250
>250
62.5
250
OCH3
OH
31.25
125
1.95
CH3
H
>250
OAc >250
62.5 >250
19
20
21
22
B
B
B
B
CH3
H
CH3
H
isopentyl
OCH3
H
OH
OH
OH
H
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
OH
miconazole
3.9
15.62
15.62
(control +)
16. The replacement of COOH by COOCH3 in the side chain
of 15 and 17 could explain, at least in part, the lack of activity
(or the only marginal effect on some fungi) obtained for these
compounds. However, this is not the case for compound 14. It
is interesting to note that the only structural difference between
compounds 14 and 16 is the replacement of OH by OCH3 in
R3. Whereas compound 16 was the most active molecule in
this series, compound 14 was inactive. There are several
explanations for the lack of activity obtained for compound 14.
One possibility is that compounds 14 and 16 could interact
A probable route for the biotransformation is depicted in
Figure 1 and could be rationalized, as cleavage of acetate from
Figure 1. Drupanin biotransformation pathway.

Antifungal Activity
J. Agric. Food Chem., Vol. 55, No. 26, 2007 10639
the unsaturated side chains is one of the most common metabolic
pathways of cinnamates in fungi. The possible mechanism was
depicted by Rosazza et al. (31), who proposed water addition
to the double bond, a subsequent oxidation, and a further
removal of an acetate unit to yield a benzylic acid moiety. The
requirements of cofactors by this process suggest the involve-
ment of a classical ꢀ-oxidation process like fatty acid degrada-
tion. In addition, the dihydrofuran ring formation could be
rationalized through the enzymatic oxidation of the side chain
double bond catalyzed by a monooxygenase and the subsequent
nucleophilic attack of the phenolic hydroxyl group on the less
substituted carbon of the oxirane moiety. The pathway described
is in agreement with the existence of several pulvinone
derivatives in A. terreus cultures reported by Ojima et al. (32),
since it supposes the expression of the necessary enzymes to
catalyze the proposed reactions in this fungal species. This
experiment does not offer enough evidence to demonstrate
which of the two side chain degradation routes is occurring first.
However, these data are irrelevant for the purpose of the present
study, which was to find out if A. terreus has the ability of
metabolizing the compound that was inactive in the antifungal
assay.
the low toxicity gives a hope for the future development of
nontoxic new antifungal agents. A SAR study on these
compounds has allowed a model to be proposed for the
recognition of the minimal structural requirements for the
antifungal effect.
ACKNOWLEDGMENT
R.E. and M.K.-S. are members of the CONICET staff.
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Received for review October 1, 2007. Revised manuscript received
October 5, 2007. Accepted October 10, 2007. This work was supported
by grants from CONICET (PIP 6228) and Agencia Nacional de
Promociones Cientificas y Tecnológicas de la Argentina (PICTR-260).
F.B. thanks CONICET for a doctoral fellowship, and F.M.G. thanks
ANPCyT for a fellowship (PICTR-260).
JF0729098