Antifungal activity and studies on mode of action of novel xanthoxyline-derived chalcones

Article abstract of DOI:10.1002/ardp.200400929

Chalcones and chalcone-like compounds, most of them new ones, prepared by base-catalyzed condensation of appropriate aldehydes and xanthoxyline, were tested for antifungal properties against a panel of yeasts, hialohyphomycetes as well as dermatophytes with the agar dilution assay. Results indicate that neither the sole presence of a xanthoxyline-like substitution pattern nor a 2′-OH substituent on ring A are sufficient for these compounds to have antifungal properties. The chalcone 3-(2-chlorophenyl)-1- (2′-hydroxy-4′,6′-dimethoxyphenyl)prop-2-en-1-one, with a Cl atom in the ortho position of benzene ring B showed the best antifungal activity against standardized strains of Trichophyton rubrum (MIC = 12.5 μg/mL) and inhibited all of the ten clinical isolates of T. rubrum tested (MIC at which 50% [MIC₅₀] and 90% [MIC₉₀] of the isolates were inhibited = 12.5 and 25 μg/mL). Regarding its mode of action, the Neurospora crassa assay showed a blotchy appearance in the inhibition halo produced by this chalcone, strongly suggesting that it could act by inhibiting the fungal cell wall. This chalcone seems to be an hyphal malformation inducer, since a clear curling of the hypha was observed in this hazy zone at a magnification of X 400. This work strongly contributes to the knowledge of the antifungal properties of hydroxy-chalcones.

Full text of DOI:10.1002/ardp.200400929

Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
DOI 10.1002/ardp.200400929
87
Antifungal Activity and Studies on Mode of Action
of Novel Xanthoxyline-Derived Chalcones
Paula Boecka, Paulo C. Leala, Rosendo A. Yunesa, Valdir Cechinel Filhob,
c
c
c
c
´
´
Silvia Lopez , Maximiliano Sortino , Andrea Escalante , Ricardo L. E. Furlan , and
Susana Zacchinoc
a
´
´
Departamento de Quımica, Universidade Federal de Santa Catarina, Florianopolis, Brasil
b
´
´
ˆ
´
´
Nucleo de Investigaco˜es Quımico-Farmaceuticas, Universidade do Vale do Itajaı, Itajaı, Santa Catarina,
¸
Brasil
c
´
´
Farmacognosia, Facultad Ciencias Bioquımicas y Farmaceuticas, Universidad Nacional de Rosario,
Rosario, Argentina
Chalcones and chalcone-like compounds, most of them new ones, prepared by base-catalyzed conden-
sation of appropriate aldehydes and xanthoxyline, were tested for antifungal properties against a panel
of yeasts, hialohyphomycetes as well as dermatophytes with the agar dilution assay. Results indicate
that neither the sole presence of a “xanthoxyline-like” substitution pattern nor a 2Ј-OH substituent
on ring A are sufficient for these compounds to have antifungal properties. The chalcone 3-(2-chloro-
phenyl)-1-(2Ј-hydroxy-4Ј,6Ј-dimethoxyphenyl)prop-2-en-1-one, with a Cl atom in the ortho position of
benzene ring B showed the best antifungal activity against standardized strains of Trichophyton rubrum
(MIC ϭ 12.5 μg/mL) and inhibited all of the ten clinical isolates of T. rubrum tested (MIC at which
50% [MIC50] and 90% [MIC90] of the isolates were inhibited ϭ 12.5 and 25 μg/mL). Regarding its
mode of action, the Neurospora crassa assay showed a blotchy appearance in the inhibition halo
produced by this chalcone, strongly suggesting that it could act by inhibiting the fungal cell wall. This
chalcone seems to be an hyphal malformation inducer, since a clear curling of the hypha was observed
in this hazy zone at a magnification of ϫ 400. This work strongly contributes to the knowledge of the
antifungal properties of hydroxy-chalcones.
Keywords: Antifungal drugs; Dermatophytes; Chalcones; Fungal cell-wall inhibitors
Received: August 4, 2004; Accepted: January 10, 2005 [FP929]
Introduction
marginal activities when tested against Cladosporium
cucumerinum [8]. The majority of the studies about the anti-
fungal properties of hydroxy-chalcones reported to date
have focused on their activity against Cladosporium cucum-
erinum or yeasts like Cryptococcus neoformans and Candida
spp. [8Ϫ13].
Chalcones represent an important group of natural com-
pounds with a variety of biological actions including anti-
fungal activity [1Ϫ5].
Various reports about antifungal chalcones suggest that the
presence of -OH groups on the aromatic rings plays a sig-
nificant role, though it is not clear yet how the exact lo-
cation of those groups correlates with the observed activity
[1]. So, the evaluation of a series of chalcones against differ-
ent Candida species suggested that a OH in the 2-position
is required for antifungal activity [6]. However, a further
report of same authors [7] showed that a 2-hydroxy group
does not always confer antifungal activity. Another study
concluded that among a series of chalcones tested, a 4Ј-
hydroxychalcone was the most active [1]. However, two
2Ј,4Ј-diOH chalcones isolated from Myrica serrata showed
Additional studies of the antifungal properties of hydroxy-
chalcone derivatives seem to be necessary, and will provide
information on the structure-activity relationships of this
kind of compounds.
As part of our continuing work on the isolation of bioactive
compounds from South-American plants, we previously iso-
lated 2Ј-OH- 4Ј,6Ј-diOMe acetophenone (xanthoxyline) in
high yield from leaves and stems of Sebastiania schottiana
[14], and then, we easily synthesized it by methylation of
phloroacetophenone [15], rendering enough available
amounts of this compound for its further use.
Correspondence: Susana A. Zacchino, Farmacognosia, Facultad
We describe here the synthesis of a series of chalcones and
analogues possessing a unique OH in position 2Ј, by the
base-catalysed condensation of appropriate aldehydes with
xanthoxyline 1 as the starter ketone.
´
´
Ciencias
Bioquımicas
y
Farmaceuticas,
Universidad
Nacional de Rosario, Suipacha 531-(2000) Rosario, Argentina.
Phone: ϩ54 341 4375315, Fax: ϩ54 341 4375315, e-mail:
szaabgil@citynet.net.ar
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

88
Zacchino et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
These xanthoxyline derived-chalcones were tested against a
panel of 12 human pathogenic opportunistic fungi, includ-
ing 5 dermatophyte species in order to have a view about
their spectra of antifungal action.
for compounds 6, 8 and 12 (Tables 1 and 2 show the spectro-
scopic data of novel compounds).
In addition, considering that some chalcones have showed
to be inhibitors of the fungal cell-wall [16], the most active
compound was evaluated with the whole-cell Neurospora
crassa assay [17, 18], an agar diffusion method which allows
a macroscopical detection of fungal cell wall inhibitors.
Since fungal but not mammalian cells possess a wall, its
inhibition represents an ideal mode of action for antifungal
drugs. The malformations of N. crassa hypha produced by
this chalcone are reported too.
Antifungal evaluation
The antifungal properties of compounds 3Ϫ19 were evalu-
ated using the agar dilution method against a panel of
twelve fungal species: three human opportunistic patho-
genic yeasts, three hialohyphomycetes and five dermato-
phytes. To carry out the antifungal evaluation, concen-
trations of compounds up to 250 μg/mL were incorporated
to growth media according to reported procedures [23]. Am-
photericin B, terbinafine and ketoconazole were used as
positive controls.
Results and discussion
Chemistry
In order to evaluate the antifungal activity of these chal-
cones and analogues, the effect of different structural modi-
fications were considered: (a) replacement of the benzene
ring B by alternative aromatic systems such as naphthalene
or furane; (b) variations of the substitution pattern of the
benzene ring B and (c) variations on the ring A by substi-
tution with bromine in the 3Ј-position.
Chalcones and chalcone-like compounds were prepared by
base-catalysed condensation of the appropriate aldehyde
and either xanthoxyline 1 or 3-bromo xanthoxyline 2
(Scheme 1). After purification, chalcones were obtained in
16Ϫ89% yields. Compounds 3, 6, 7, 8, 11, and 17 have been
reported in the literature [15, 19Ϫ22]. Structures 4, 5, 9, 10,
12-16, 18 and 19 are new compounds.
Results showed that none of the compounds tested was ef-
fective against the yeasts C. albicans, S. cerevisiae or C. neo-
formans nor against the filamentous fungi A. niger, A. fumi-
gatus or A. flavus. In contrast, different antifungal activities
were observed for the compounds of the series against der-
matophytes (Table 3).
Structures of the products were established using IR, UV
and NMR spectroscopies, and elemental analysis. Inspec-
1
tion of the H NMR spectra suggested that chalcones were
geometrically pure (E isomer, JHα-Hβ ϭ 15Ϫ16 Hz), except
Scheme 1. Synthetic route to xanthoxyline-derived chalcones and analogues. Reagents: a) CHO-Ph-R, NaOH/EtOH;
b) 2Ϫnaphthaldehyde, NaOH/EtOH; c) 2-furaldehyde, NaOH/EtOH; d) Br2/ AcOH. (*) New compounds.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
Antifungal properties of xanthoxyline-derived chalcones
89
1
Table 1. H NMR Data (δ in ppm, CDCl3) of compounds: 4, 5, 9, 10, 12Ϫ16, 18, 19†.
4
5
9
10
12
13
14
15
16
18
19
H3·
H5·
Hα
Hβ
5.99 d;
J ϭ 2.2 Hz
6.13 d;
J ϭ 2.2 Hz
7.47Ϫ7.99 m 7.57 d;
5.99 d;
5.96 d;
Jϭ 2.2 Hz
6.11 d;
J ϭ 2.2 Hz
7.68 d;
6.91 d;
J ϭ 2.2 Hz
6.08 d;
J ϭ 2.2 Hz
7.01Ϫ7.42 m 7.98 d;
6.17 d;
Jϭ 2.2 Hz
6.42 d;
6.0 d;
J ϭ 2.2 Hz
6.15 d;
J ϭ 2.2 Hz
7.91 d;
5.97 d;
J ϭ 2.2Hz
6.14 d;
J ϭ 2.2 Hz
7.62 d;
5.98 d;
Jϭ 2.2Hz
6.12 d;
J ϭ 2.2Hz
7.74 d;
6.09 d;
J ϭ 2.2Hz
6.12 d;
J ϭ 2.2Hz
7.81 d;
Ϫ
Ϫ
J ϭ 2.2 Hz
6.03Ϫ d;
J ϭ 2.2 Hz
6.05 s
7.81 d;
6.10 s
J ϭ 2.2 Hz
8.48Ϫ8.60 m
8.48Ϫ8.60 m
J ϭ 15.37 Hz J ϭ 15.51 Hz
J ϭ 15.51 Hz J ϭ 15.67 Hz J ϭ 15.60 Hz J ϭ 15.68 Hz J ϭ 15.49 Hz J ϭ 15.58 Hz
7.01Ϫ7.42 m 8.26 d; 8.19 d; 7.85 d; 7.98 d; 8.44 d; 8.17 d;
J ϭ 15.51 Hz J ϭ 15.67 Hz J ϭ 15.60 Hz J ϭ 15.68 Hz J ϭ 15.49 Hz J ϭ 15.58 Hz
7.47Ϫ7.99 m 7.79 d;
7.87 d;
J ϭ 15.37 Hz J ϭ 15.51 Hz
H1
H2
7.47Ϫ7.99 m
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
7.47Ϫ7.51 m 7.01Ϫ7.42 m 6.75 d
J ϭ 8.85 Hz
7.47Ϫ7.51 m 7.01Ϫ7.42 m 7.25 d
J ϭ 8.85 Hz
7.43Ϫ7.51 m
8.48Ϫ8.60 m
H3
H4
H5
H6
7.47Ϫ7.99 m 6.50 d;
J ϭ 8.4 Hz
7.47Ϫ7.99 m 6.67 t;
J ϭ 8.4 Hz
7.47Ϫ7.99 m 7.51 d;
J ϭ 8.4 Hz
7.30Ϫ7.75 m
7.30Ϫ7.75 m
Ϫ
Ϫ
8.46 s
7.96 d;
J ϭ 7.87 Hz
7.47Ϫ7.81 m 7.28Ϫ7.70 m 8.48Ϫ8.60 m
7.28Ϫ7.70 m
Ϫ
Ϫ
Ϫ
8.22 d;
J ϭ 7.75 Hz
7.47Ϫ7.51 m 7.01Ϫ7.42 m 7.25 d
J ϭ 8.85 Hz
7.47Ϫ7.51 m 7.01Ϫ7.42 m 6.75 d
J ϭ 8.85 Hz
7.30Ϫ7.75 m 7.43Ϫ7.51 m 7.84Ϫ7.88 m 7.47Ϫ7.81 m 7.28Ϫ7.70 m 7.43Ϫ7.51 m
7.47Ϫ7.99 m
Ϫ
7.30Ϫ7.75 m 7.43Ϫ7.51 m 7.84Ϫ7.88 m 7.47Ϫ7.81 m 7.28Ϫ7.70 m 8.48Ϫ8.60 m
H7
H8
7.47Ϫ7.99 m
7.47Ϫ7.99 m
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
OCH3 3.95 s
OCH3 3.85 s
3.91 s
3.83 s
3.91 s
3.84 s
3.83 s
3.79 s
3.97 s
3.93 s
3.94 s
3.88 s
3.92 s
3.84 s
3.94 s
3.85 s
3.93 s
3.84 s
3.98 s
3.98 s
4.03 s
4.01 s
†
Measured at 200 MHz. J ϭ Coupling constants.
Table 2. 13C NMR Data (δ in ppm, CDCl3) of compounds 4, 5, 9, 10, 12Ϫ16, 18 and 19†.
4
5
9
10
12
13
14
15
16
18
19
CϭO
C1Ј
C2Ј
C3Ј
C4Ј
C5Ј
C6Ј
Cα
193.25
107.07
163.20
94.50
169.14
91.99
166.93
124.41
143.17
128.44
134.86
124.41
129.29
128.44
127.35
127.83
129.29
134.11
133.77
56.60
192.74
106.99
163.21
94.41
169.09
91.88
166.85
129.63
145.32
Ϫ
152.93
113.20
116.11
145.32
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
56.45
56.24
Ϫ
193.00
106.99
163.15
94.10
169.13
92.03
167.07
128.34
141.47
135.21
130.36
132.79
124.88
132.79
130.36
Ϫ
193.05
106.91
163.12
94.45
169.08
91.94
166.93
128.12
141.69
132.48
130.73
116.43
161.94
116.43
130.73
Ϫ
192.21
107.21
160.05
94.41
169.51
89.52
163.08
123.82
139.05
147.22
131.24
122.44
149.55
122.44
131.24
Ϫ
193.01
106.99
163.17
94.49
169.14
91.99
167.08
127.65
138.55
136.05
134.53
130.94
131.35
128.49
130.73
Ϫ
191.96
106.22
162.45
93.81
168.47
91.37
166.53
129.22
139.23
135.69
130.83
133.75
133.12
132.04
129.67
Ϫ
191.78
106.20
162.45
93.84
168.46
91.37
166.65
129.78
138.68
137.43
122.08
148.71
123.99
130.51
134.01
Ϫ
199.66
106.00
162.43
93.57
167.15
90.60
167.15
122.43
149.67
133.94
128.93
129.00
125.50
132.00
133.94
Ϫ
193.25
106.00
162.90
92.80
163.93
87.80
163.92
127.72
139.49
136.21
134.33
131.06
131.62
128.60
130.22
Ϫ
199.99
107.02
162.39
92.12
163.14
87.16
163.09
124.81
140.20
134.85
121.00
148.30
122.55
130.40
131.00
Ϫ
Cβ
C1
C2
C3
C4
C5
C6
C7
C8
Ϫ
Ϫ
Ϫ
56.57
56.30
Ϫ
Ϫ
Ϫ
Ϫ
56.52
56.26
Ϫ
Ϫ
Ϫ
Ϫ
56.26
56.18
Ϫ
Ϫ
Ϫ
Ϫ
56.56
56.30
Ϫ
Ϫ
Ϫ
Ϫ
55.94
55.64
Ϫ
Ϫ
Ϫ
Ϫ
55.82
55.56
Ϫ
Ϫ
Ϫ
Ϫ
55.44
55.30
172.00
Ϫ
Ϫ
Ϫ
57.08
56.85
Ϫ
Ϫ
Ϫ
Ϫ
56.76
56.18
Ϫ
C9
C10
OCH3
OCH3
COOH
56.30
Ϫ
†
Measured at 50 MHz (compounds 4, 5, 9, 10, 13Ϫ16 and 18) and at 100 MHz (compounds 12 and 19).
The lack of activity of chalcones 3, 6Ϫ8, 14 and 19 indicates
that the presence of a 2Ј-OH in the chalcone is not enough
for these compounds to have antifungal properties.
(5), the structure possessed marginal activity, this result
differing from previous reports for an analogous structure
[1].
Regarding the influence of different B-ring systems alterna-
tive to benzene B, chalcone 4 with naphthalene as B-ring
was devoid of antifungal activity. When B-ring was a furane
Concerning the influence of substituents on the benzene
ring B, some interesting conclusions could be extracted:
compounds holding electron withdrawing groups (EWGs)
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

90
Zacchino et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
Table 3. MIC values (μg/mL) of xanthoxyline-derived chalcones and analogues acting against dermatophytes†.
Cp
Type
R3Ј
R2
R3
R4
E. f.
T. m.
T.r.
M.c.
M.g.
3
4
A
B
H
Ϫ
Ϫ
H
H
H
H
H
H
H
H
H
H
H
Br
Br
Br
H
Ϫ
Ϫ
H
H
H
H
H
H
H
Cl
H
H
COOH
H
H
Ϫ
Ϫ
H
H
H
Ϫ
Ϫ
>250
>250
125
>250
250
>250
50
62.5
100
62.5
100
>250
250
125
25
>250
>250
250
>250
250
>250
50
62.5
100
62.5
50
>250
>250
250
50
50
>250
0.3
>250
>250
250
>250
250
>250
50
62.5
125
100
12.5
250
125
250
25
>250
>250
250
>250
250
>250
50
100
125
250
100
>250
>250
125
50
50
>250
>250
>250
>250
250
>250
250
>250
>250
>250
250
>250
>250
250
250
250
>250
6.25
6.25
0.04
5
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
6
OCH3
CH3
OϪCH2ϪO
7
8
9
H
H
H
H
H
Cl
NO2
H
H
H
Br
F
Cl
NO2
H
Cl
H
H
10
11
12
13
14
15
16
17
18
19
Amp
Ket
Terb
H
H
H
Cl
H
25
50
>250
25
15
0.01
NO2
>250
6.25
12.5
0.004
>250
>50
15
25
0.04
0.01
†
Abbreviations: M.c. Ϫ Microsporum canis C 112, M.g. Ϫ Microsporum gypseum C 115, T.r. Ϫ Trichophyton rubrum C113, T.m. Ϫ Trichophy-
ton mentagrophytes ATCC 9972; E.f. ϪEpidermophyton floccosum C 114. Amp Ϫ Amphotericin, Ket Ϫ Ketoconazole, Terb Ϫ Terbinafine.
such as bromine, fluorine, chlorine, or a nitro group in the
4-position (compounds 9Ϫ12) improved the antifungal
properties compared with the non ring B-substituted chal-
cone 3. In contrast, compounds with electron-donating sub-
stituents such as methoxy, methyl, and methylenedioxy
groups in the same position of B-ring (chalcones 6, 7, and
8 respectively) were all inactive.
Modification of ring A by introducing a bromine substitu-
ent in the 3Ј-position of chalcone 3 significantly improved
the antifungal properties: compound 17 is 5Ϫ10 times more
active against four different species of fungi. However, the
effect of such substitution was not significant for other com-
pounds (compare activities of compounds 13 vs. 18, and 15
vs. 19).
The effect of the position of the EWG on the antifungal
activity could be observed within the group of chlorinated
compounds 11, 13, and 14 where the antifungal activities
follow the order 2-Cl (13) > 4-Cl (11) > 3, 4-di Cl (14).
Similarly the 4-NO2 chalcone 12 showed better activities
than chalcone 15 with a nitro group in the 3-position. The
observed effect of EWGs on the para-position of ring B in
a chalcone structure is in agreement with a previous report
on the antifungal activities of non-hydroxylated chalcones
[16]. However, the increased activities showed by com-
pounds holding a EWG in the o-position of ring B are
somehow contradictory to the previously observed corre-
lation between planarity of chalcones and their antifungal
activity [16]. This fact deserves further studies.
The selective activity of compound 13 against T. rubrum is
particularly interesting since this fungus is responsible of
approximately 80-93% of chronic and recurrent dermato-
phyte infections in human beings [24]. It is the ethiological
agent of tinea unguium (producer of invasive nail infec-
tions), tinea manuum (palmar and interdigital areas of the
hand infections) and tinea pedis (AthleteЈs foot), the last
one being the most prevalent fungal infection in developed
countries, and the first one accounting for 50% and 90% of
all fingernail and toenail infections respectively [24].
In order to gain insight into the capacity of compound 13
in acting not only against standardized strains, but against
clinical isolates of T. rubrum, we tested it against ten clinical
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
Antifungal properties of xanthoxyline-derived chalcones
91
Table 4. MIC values of chalcone 3-(2-Chlorophenyl)-1-(2Ј-hy-
droxy-4Ј,6Ј-dimethoxyphenyl)prop-2-en-1-one (13), against
10 different clinical strains of T. rubrum from Centro de
´
Referencia Micologica (CEREMIC), Facultad de Ciencias
´
´
Bioquımicas y Farmaceuticas, Universidad Nacional Rosario.
Voucher specimen
MIC [μg/mL]
T. rubrum C 110
T. rubrum C 133
T. rubrum C 134
T. rubrum C 135
T. rubrum C 136
T. rubrum C 137
T. rubrum C 138
T. rubrum C 139
T. rubrum C 140
T. rubrum C 141
12.5
12.5
25.0
25.0
50.0
25.0
12.5
25.0
12.5
12.5
Figure 1. A. Blotchy appearance of the inhibition halo produ-
ced by chalcone [3-(2-Chlorophenyl)-1-(2Ϫhydroxy-4,6-di-
methoxyphenyl)prop-2-en-1-one] (13) in the Neurospora
crassa assay. B. Clear halo produced by ketoconazole.
strains of T. rubrum isolated from skin infections of different
immunocompromised patients. Results showed (Table 2)
that chalcone 13 inhibited all the clinical strains of T. rub-
rum tested, with MICs between 12.5-50 μg/mL being the
minimum concentration that inhibits 50% (MIC50) and
90% (MIC90) of the strains tested ϭ 12.5 and 25 μg/mL
respectively (Table 4).
fusion method which allows a macroscopical detection of
fungal cell wall inhibitors. Neurospora crassa usually grows
as long hypha in a diffuse or branched way. When it grows
in the presence of certain inhibitors of its wall, the hyphal
growth is inhibited, and fungi grow as protoplasts. Macro-
scopically they can be seen as a blotchy or hazy appearance
around the paper disk. The microscopic observation of this
hazy zone usually shows morphological changes of the hy-
pha [26].
Considering our previous finding [16] that non-hy-
droxylated chalcones were inhibitors of the fungal cell wall,
we tested structure 13 for its capacity of inhibiting the fun-
gal cell-wall. Since mammalian cells do not possess a wall
[25], this is the intrinsic target to develop highly selective
and useful drugs in medicinal investigation, and antifungal
structures acting by this mechanism are actively sought.
Results showed that chalcone 13 produced a blotchy zone
around the paper disk (Figure 1A and B), indicating that
the mode of action could be associated with the inhibition
Amongst methods that deal with this mode of action, we
have chosen the Neurospora crassa assay [17] an agar dif-
Figure 2. Microscopic appearance of Neurospora crassa hypha in the absence (A) or in the presence (B) of chalcone [3-(2-
chlorophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)prop-2-en-1-one] (13) observed under a light microscope at magnification
ϫ400.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

92
Zacchino et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
Elemental analyses were obtained on a Perkin Elmer 2400 (Thermo-
quest Italia, Milano, Italia). Percentages of C and H were in agree-
ment with the product formula (within 0.4% of theoretical values).
of cell wall polymers or assembly [18]. The microscopic ob-
servation of this hazy zone showed a marked curling of the
hypha (Figure 2A and B), a defect in the directionality of
hypha growth, this giving a new sign that chalcone 13 could
act inhibiting the synthesis or assembly of the fungal cell
wall polymers. It is important to take in account that some
hyphal malformation inducers have proved to be inhibitors
of (1,3)β-glucan synthase [27], the enzyme that catalyzes the
synthesis of (1,3)β-glucan, main polymer of the fungal cell
wall.
The purity of the synthesized substances was monitored by thin-
layer chromatography (TLC) using silica (Macherey-Nagel GmbH,
Inc-USA) pre-coated aluminum plates (Alugram® SIL G/UV254
layer 0.2 mm) with several solvent systems of different polarity.
Compounds were visualized with ultraviolet light (254 nm) and
using ferric chloride (Ridel-DE Hae¨n AG, Seelze-Hannover-Ger-
many) solution and purified by recrystallization from ethyl ether or
ˆ
˜
hexane (Dinamica-Sao Paulo-Brasil).
All the aromatic aldehydes used are commercially available (Sigma-
´
Aldrich Quımica, S.A., Alcobendas, Madrid). Xanthoxyline (1) was
isolated from leaves and stems of Sebastiania schottiana according
to a reported procedure [14]. 2-Hydroxy-3-bromo-4,6-dimethoxy-
acetophenone (2) was prepared from 1 according to a literature pro-
cedure [15].
Conclusion
This work strongly contributes to the knowledge of the anti-
fungal properties of hydroxy-chalcones by testing the anti-
fungal activity of a series of 17 synthetic chalcones and
analogues based in the natural product xanthoxyline (1).
Results showed that the sole presence of an OH in the 2Ј-
position is not enough for hydroxy-chalcones to possess
antifungal activity. Nevertheless, certain members of the
series showed moderate activity against dermatophytes.
Chalcone 13, with a Cl- in the o-position of ring B, showed
the lowest MIC against T. rubrum and inhibited all of the
ten clinical isolates of T. rubrum tested. Regarding its mode
of action, it is interesting to note that the most active mem-
ber of the series produced a blotchy appearance of the inhi-
bition halo in the Neurospora crassa assay, clearly suggest-
ing that its mode of action could be the inhibition of the
synthesis or assembly of the polymers of the fungal cell wall.
The microscopic appearance of this hazy zone showed curl-
ing of the hypha. Since fungal but not mammalian cells
possess a wall, results obtained in targeted assays with chal-
cone 13 open the possibility of using this compound as a
starting point for the development of safe antifungal agents,
since it would attack selectively the fungi without inhibiting
any biochemical system of the host.
Synthesis of chalcones 3Ϫ16
A solution of xanthoxyline 1 (0.18 g; 0.92 mmol), EtOH (15 mL),
NaOH (0.1 g; 2.5 mmol, containing a minimum amount of H2O),
and the appropriate aldehyde (0.95 mmol) was stirred at room tem-
perature for 1Ϫ23 h. The crude product was isolated by acidification
of the cool diluted solution, and recrystallized from ethyl ether or
hexane.
1-(2Ј-Hydroxy-4Ј,6Ј-dimethoxyphenyl)-3-(2-naphthyl)prop-2-en-1-
one (4)
Yellow solid, mp. ϭ 110Ϫ112°C; UV λmax 336 (4.05); IR (KBr)
1
1638 (CϭO), 1586 (CϭC) cmϪ1. H NMR (CDCl3) δ 14.35 (s, 1H,
OH), 7.47Ϫ7.99 (m, 9H, Hβ, Hα, H1, H3, H4, H5, H6, H7, H8),
6.13 (d, 1H, J ϭ 2.26 Hz, H5Ј), 5.99 (d, 1H, J ϭ 2.26 Hz, H3Ј),
3.95 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C NMR (CDCl3) δ
193.25 (CϭO), 169.14 (C4Ј), 166.93 (C6Ј), 163.20 (C2Ј) , 143.17
(Cβ), 134.86 (C2), 134.11 (C9), 133.77 (C10), 129.29 (C4, C8),
128.44 (C1, C5), 127.83 (C7), 127.35 (C6), 124.41 (C3, Cα), 107.07
(C1Ј), 94.50 (C3Ј), 91.99 (C5Ј), 56.30, 56.60 (OCH3). C21H18O4
Calc. C 75.43, H 5.43, Found C 75.52, H 5.47. Yield ϭ 30%.
3-(2-Furyl)-1-(2Ј-hydroxy-4Ј,6Ј-dimethoxyphenyl)prop-2-en-1-one
(5)
Yellow solid; mp. ϭ 92-94°C; UV λmax 363 (3.42); IR (KBr) 1626
(CϭO), 1586 (CϭC) cmϪ1 1H NMR (CDCl3) δ 7.79 (d, 1H, J ϭ
.
These data together with previous results obtained by us
[16] clearly indicate that hydroxy and non-hydroxy chal-
cone-like compounds possess antifungal properties with a
mode of action related unless in part, to the inhibition to
the synthesis or ensamble of polymers of the fungal cell-
wall.
15.37 Hz, Hβ), 7.57 (d, 1H, J ϭ 15.37 Hz, Hα), 7.51 (d, 1H, J ϭ
8.4 Hz, H5), 6.67 (t, 1H, J ϭ 8.4 Hz, H4), 6.50 (t, 1H, J ϭ 8.4 Hz,
H3), 3.91 (s, 3H, OCH3), 3.83 (s, 3H, OCH3). 13C NMR (CDCl3) δ
192.74 (CϭO), 169.09 (C4Ј), 166.85 (C6Ј), 163.21 (C2Ј), 152.93
(C2), 145.32 (Cβ, C5), 129.63 (Cα), 116.11 (C4), 113.20 (C3), 106.99
(C1Ј), 94.41 (C3Ј), 91.88 (C5Ј), 56.45 (OCH3), 56.24 (OCH3).
C
15H14O5 Calc. C 65.69, H 5.1, Found C 64.76, H 5.18. Yield ϭ
52%.
Experimental
3-(4-Bromophenyl)-1-(2Ј-hydroxy-4Ј,6Ј-dimethoxyphenyl)prop-2-en-
1-one (9)
General experimental procedures
Orange solid; mp. ϭ 150Ϫ151°C; UV λmax 338 (4.08); IR (KBr)
1
Melting points were determined with a Microquimica MG APF-301
1632 (CϭO), 1588 (CϭC) cmϪ1. H NMR (CDCl3) δ 14.21 (s, 1H,
apparatus (Palhoca, Santa Catarina, Brasil) and are uncorrected.
¸
OH), 7.87 (d, 1H, J ϭ 15.51 Hz, Hβ), 7.68 (d, 1H, J ϭ 15.51 Hz,
Hα), 7.47Ϫ7.51 (m, 4H, H2, H3, H5, H6), 6.11 (d, 1H, J ϭ 2.2 Hz,
H5Ј), 5.96 (d, 1H, J ϭ 2.2 Hz, H3Ј), 3.91 (s, 3H, OCH3), 3.84 (s,
3H, OCH3). 13C NMR (CDCl3) δ 193 (CϭO), 169.13 (C4Ј), 167.07
(C6Ј), 163.15 (C2Ј), 141.47 (Cβ), 135.21 (C1), 132.79 (C3, C5),
130.36 (C2, C6), 128.34 (Cα), 124.88 (C4), 106.99 (C1Ј), 94.1 (C3Ј),
Infrared (IR) spectra were recorded with a FT Perkin Elmer 16 PC
spectrometer (Connecticut, USA). Nuclear magnetic resonance (1H
and 13C NMR) spectra were recorded on a Bruker AC-200 F
(Rheinstetten, Germany) instrument with tetramethylsilane as in-
ternal standard.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
Antifungal properties of xanthoxyline-derived chalcones
93
92.03 (C5Ј), 56.57 (OCH3), 56.30 (OCH3). C17H15 BrO4 Calc. C
56.22, H 4.16, Found C 56.04, H 4.21. Yield ϭ 38%.
15.68 Hz, Hα), 6.12 (d, 1H, J ϭ 2.2 Hz, H5Ј), 5.98 (d, 1H, J ϭ 2.2
Hz, H3Ј), 3.94 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C NMR
(CDCl3) δ 191.78 (CϭO), 168.46 (C4Ј), 166.65 (C6Ј), 162.45 (C2Ј),
148.71 (C3), 138.68 (Cβ), 137.43 (C1), 134.01 (C6), 130.51 (C5),
129.78 (Cα), 123.99 (C4), 122.08 (C2), 106.20 (C1Ј), 93.84 (C3Ј),
91.37 (C5Ј), 55.82 (OCH3), 55.56 (OCH3). C17H15NO6 Calc. C
62.00, H 4.59, N 4.25, Found C 60.08, H 4.55, N 4.26. Yield ϭ 57%.
3-(4-Fluorophenyl)-1-(2Ј-hydroxy-4Ј,6Ј-dimethoxyphenyl)prop-2-en-
1-one (10)
Yellow solid; mp. ϭ 140-141°C; UV λmax 339 (4.00); IR (KBr) 1632
(CϭO), 1572 (CϭC) cmϪ1. 1H NMR (CDCl3) δ 13.72 (s, 1H, OH),
7.01Ϫ7.42 (m, 6H, Hβ, Hα, H2, H3, H5, H6), 6.08 (d, 1H, J ϭ
2.22 Hz, H5Ј), 6.91 (d, 1H, J ϭ 2.22 Hz, H3Ј), 3.83 (s, 3H, OCH3),
3.79 (s, 3H, OCH3). 13C NMR (CDCl3) δ 193.05 (CϭO), 169.08
(C4Ј), 166.93 (C6Ј), 163.12 (C2Ј), 161.94 (C4), 141.69 (Cβ), 132.48
(C1), 130.73(C2, C6), 128.12 (Cα), 116.43 (C3, C5), 106.91 (C1Ј),
94.45 (C3Ј), 91.94 (C5Ј), 56.52 (OCH3), 56.26 (OCH3). C17H15FO4
Calc. C 67.54, H 5.00, Found C 67.50, H 5.04. Yield ϭ 87%.
2-[-3-(2Ј-Hydroxy-4Ј,6Ј-dimethoxyphenyl)-3-oxoprop-1-enyl]-
benzoic acid (16)
Yellow solid; mp. ϭ 160Ϫ161°C; UV λmax 291 (3.82) IR (KBr) 1640
(CϭO), 1560 (CϭC) cmϪ1. 1H NMR (CDCl3) δ 13.99 (s, 1H, OH),
8.44 (d, 1H, J ϭ 15.49 Hz, Hβ), 7.81 (d, 1H, J ϭ 15.49 Hz, Hα),
7.96 (d, 1H, J ϭ 7.87 Hz, H3), 7.47Ϫ7.81 (m, 3H, H4, H5, H6),
6.12 (d, 1H, J ϭ 2.2 Hz, H3Ј), 6.09 (d, 1H, J ϭ 2.2 Hz, H5Ј), 3.93
(s, 3H, OCH3), 3.84 (s, 3H, OCH3). 13C NMR (CDCl3) d 199.66
(CϭO), 167.15 (C6Ј, C4Ј), 162.43 (C2Ј), 149.67 (Cβ), 133.94 (C1,
C6), 132 (C5), 129 (C3), 128.93 (C2), 125.50 (C4), 125.04 (C4, C6),
122.43 (Cα), 106 (C1Ј), 93.57 (C3Ј), 90.60 (C5Ј), 55.44 (OCH3),
55.30 (OCH3). C18H16O6 Calc. C 65.85, H 4.91, Found C 65.00, H
4.84. Yield ϭ 44%.
1-(2Ј-Hydroxy-4Ј,6Ј-dimethoxyphenyl)-3-(4-nitrophenyl)prop-2-en-
1-one (12)
Orange solid; mp. ϭ 295Ϫ296°C; UV λmax 379 (3.80) R (KBr) 1664
(CϭO), 1584 (CϭC) cmϪ1 1H NMR (CDCl3) δ 8.26 (d, 1H, J ϭ
.
15.51 Hz, Hβ), 7.98 (d, 1H, J ϭ 15.51 Hz, Hα), 7.25 (d, 2H, J ϭ
8.85 Hz, H3, H5), 6.75 (d, 2H, H2, H6), 6.42 (d, 1H, J ϭ 2.2 Hz,
H5Ј), 6.17 (d, 1H, J ϭ 2.2 Hz, H3Ј), 3.97 (s, 3H, OCH3), 3.93 (s,
3H, OCH3). 13C NMR (CDCl3) δ 192.21 (CϭO), 169.51 (C4Ј),
163.08 (C6Ј), 160.05 (C2Ј), 149.55 (C4), 147.22 (C1), 139.05 (Cβ),
131.24 (C2, C6), 123.82 (Cα), 122.44 (C3, C5), 107.21 (C1Ј), 94.41
(C3Ј), 89,52 (C5Ј), 56.26 (OCH3), 56.18 (OCH3).C17H15NO6 Calc.
C 62.00, H 4.59, N 4.25, Found C 61.98, H 4.56, N 4.23. Yield ϭ
16%.
Synthesis of chalcones 18, 19
A solution of 2-hydroxy-3-bromo-4,6-dimethoxyacetophenone 2
(0.2 g; 0.73 mmol), EtOH (15 mL), NaOH (0.1 g; 2.5 mmol, con-
taining a minimum amount of H2O), and the appropriate aldehyde
(0.95 mmol) was stirred at room temperature for 2Ϫ3 h. The crude
product, isolated by acidification of the cool diluted solution, was
recrystallized from ethyl ether and hexane
3-(2-Chlorophenyl)-1-(2Ј-hydroxy-4Ј,6Ј-dimethoxyphenyl)prop-2-en-
1-one (13)
1-(3Ј-Bromo-2Ј-hydroxy-4Ј,6Ј-dimethoxyphenyl)-3-(2-chlorophen-
yl)prop-2-en-1-one (18)
Yellow solid; mp. ϭ 136Ϫ137°C; UV λmax 336 (3.68); IR (KBr)
1
1630 (CϭO), 1556 (CϭC) cmϪ1. H NMR (CDCl3) δ 14.25 (s, 1H,
Orange solid; mp. ϭ 210Ϫ212 °; UV λmax 283 (4.12); IR (KBr)
1
1624 (CϭO), 1556 (CϭC) cmϪ1. H NMR (CDCl3) δ 14.67 (s, 1H,
OH), 8.19 (d, 1H, J ϭ 15.67 Hz, Hβ), 7.91 (d, 1H, J ϭ 15.67 Hz,
Hα), 7.30Ϫ7.75 (m, 4H, H3, H4, H5, H6), 6.15 (d, 1H, J ϭ 2.2 Hz,
H5Ј), 6.0 (d, 1H, J ϭ 2.2 Hz, H3Ј), 3.88 (s, 3H, OCH3), 3.94 (s, 3H,
OCH3). 13C NMR (CDCl3) δ 193.01 (CϭO), 169.14 (C4Ј), 167.08
(C6Ј), 163.17 (C2Ј), 138.55 (Cβ), 136.05 (C1), 134.53 (C2), 131.35
(C4), 130.94 (C3), 130.73 (C6), 128.49 (C5), 127.65(Cα), 106.99 (C
1Ј), 94.49 (C3Ј), 91.99 (C5Ј), 56.56 (OCH3), 56.30 (OCH3).
OH), 8.17 (d, 1H, J ϭ 15.58 Hz, Hβ), 7.81 (d, 1H, J ϭ 15.58 Hz,
Hα), 7.28Ϫ7.70 (m, 4H, H3, H4, H5, H6), 6.05 (s, 1H, H5Ј), 3.98
(s, 6H, OCH3). 13C NMR (CDCl3) δ 193.25 (CϭO), 163.93 (C4Ј),
163.92 (C6Ј), 162.90 (C2Ј), 139.49 (Cβ), 136.21 (C1), 134.33 (C2),
131.62 (C4), 131.06 (C3), 130.22 (C6), 128.60 (C5), 127.72 (Cα),
106 (C1Ј), 92.80 (C3Ј), 87.80 (C5Ј), 57.08 (OCH3), 56.85 (OCH3).
C17H14BrClO4 Calc. C 51.35, H 3.55, Found C 51.45, H 3.58.
Yield ϭ 47%.
C
17H15ClO4 Calc. C 64.06, H 4.74, Found C 63.87, H 4.80.
Yield ϭ 89%.
3-(3,4-Dichlorophenyl)-1-(2Ј-hydroxy-4Ј, 6Ј-dimethoxyphenyl)prop-
2-en-1-one (14)
1-(3Ј-bromo-2Ј-hydroxy-4Ј,6Ј-dimethoxyphenyl)-3-(3-nitrophenyl)-
prop-2-en-1-one (19)
Yellow solid; mp. ϭ 120Ϫ123°C; UV λmax 343 (3.80); IR (KBr)
Orange solid; mp. ϭ 264°C; UV λmax 288 (3.25)IR (KBr) 1632 (Cϭ
1
1622 (CϭO), 1586 (CϭC) cmϪ1. H NMR (CDCl3) δ 14.14 (s, 1H,
O), 1558 (CϭC) cmϪ1 1H NMR (CDCl3) δ 14.55 (s, 1H, OH),
.
OH), 7.85 (d, 1H, J ϭ 15.60 Hz, Hβ), 7.62 (d, 1H, J ϭ 15.60 Hz,
Hα), 7.43Ϫ7.51 (m, 3H, H2, H5, H6), 6.14 (d, 1H, J ϭ 2.2 Hz,
H5Ј), 5.97 (d, 1H, J ϭ 2.2 Hz, H3Ј), 3.92 (s, 3H, OCH3), 3.84 (s,
3H, OCH3). 13C NMR (CDCl3) δ 191.96 (CϭO), 168.47 (C4Ј),
166.53 (C6Ј), 162.45 (C2Ј), 139.23 (Cβ), 135.69 (C1), 133.75 (C3),
133.12 (C4), 132.04 (C5), 130.83(C2), 129.22 (Cα), 106.22 (C1Ј),
93.81 (C3Ј), 91.37 (C5Ј), 55.94 (OCH3), 55.64 (OCH3). C17H14Cl2O4
Calc. C 57.81, H 4.00, Found C 57.32, H 4.09. Yield ϭ 47%
8.48Ϫ7.60 (m, 6H, Hβ, Hα, H2, H5, H4, H6), 6.10 (s, 1H, H5Ј),
4.03 (s, 3H, OCH3), 4.01 (s, 3H, OCH3). 13C NMR (CDCl3) δ
199.99 (CϭO), 163.14 (C4Ј), 163.09 (C6Ј), 162.39 (C2Ј), 148.30
(C3), 140.20 (Cβ), 134.85 (C1), 131.00 (C6), 130.40 (C5), 124.81
(Cα), 122.55 (C4), 121 (C2), 107.02 (C1Ј), 92.12 (C3Ј), 87.16 (C5Ј),
56.76 (OCH3), 56.18 (OCH3). C17H14BrNO Calc. C 50.02, H 3.46,
N 3.43, Found C 50.10, H 3.44, N 3.40. Yield ϭ 71%.
Biological evaluation
1-(2Ј-Hydroxy-4Ј, 6Ј-dimethoxyphenyl)-3-(3-nitrophenyl)prop-2-en-
1-one (15)
Microorganisms and media
Orange solid; mp. ϭ 171Ϫ172°C; UV λmax 335 (3.74); IR (KBr)
The microorganisms used for the fungistatic evaluation were pur-
chased from the American Type Culture Collection (ATCC, Rock-
ville, MD, USA) or were clinical isolates and were kindly provided
1
1640 (CϭO), 1580 (CϭC) cmϪ1. H NMR (CDCl3) δ 14.09 (s, 1H,
OH), 8.46 (s, 1H, H2), 8.22 (d, 1H, J ϭ 7.75 Hz, H4), 7.98 (d, 1H,
J ϭ 15.68 Hz, Hβ), 7.84Ϫ7.88 (m, 2H, H5, H6), 7.74 (d, 1H, J ϭ
´
by Centro de Referencia Micologica (C, CEREMIC, Facultad de
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

94
Zacchino et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95
´
´
Ciencias Bioquımicas
y
Farmaceuticas, Suipacha 531, (2000)
tion of glycerol:H2O 15:85. Spore suspension free of hypha was pre-
pared as described in Microorganisms and media.
Rosario, Argentina: Candida albicans ATCC 10231, Saccharomyces
cerevisiae ATCC 9763, Cryptococcus neoformans ATCC 32264,
Aspergillus flavus ATCC 9170, Aspergillus fumigatus ATCC 26934,
Aspergillus niger ATCC 9029 and Trichophyton mentagrophytes
ATCC 9972. Strains were grown on Sabouraud-chloramphenicol
agar slants for 48 h at 30 °C. Cell suspensions in sterile distilled
water were adjusted to give a final concentration of 106 viable yeast
cells/mL [28]. Microsporum canis C 112, Trichophyton rubrum C 113,
110, 133, 134, 135, 136, 137, 138, 139, 140 and 141; Epidermophyton
floccosum C 114, Microsporum gypseum C 115, and Candida
tropicalis C 131.
Acknowledgements
This work was supported by grants to SAZ (Agencia de
´
´
Promociones Cientıficas y Tecnologicas de la Argentina
PICTR 260 and OEA); to SNL (Grantee IFS F/3114-2F),
to RLEF (Antorchas) and it is part of the Iberoamerican
Project X.7 PIBEAFUN (Search and development of new
antifungal agents), part of the Iberoamerican Program of
Science and Technology for the Development (CYTED-X).
Collaboration from Iberoamerican Network on Natural
Products for Medicinal Use (RIPRONAMED, X.A) of
CYTED is gratefully acknowledged. VCF and RAY thank
CNPq (Brazil) for financial support.
The strains were maintained on slopes of Sabouraud-dextrose agar
(SDA, Oxoid) and subcultured every 15 days to prevent pleo-
morphic transformations. Spore suspensions were obtained accord-
ing to reported procedures [28] and adjusted to 106 spores with col-
ony forming ability/mL.
Antifungal assays
The antifungic activities of chalcones were evaluated with the agar
dilution method by using Sabouraud-chloramphenicol agar for
yeasts and filamentous fungi according with reported procedures
[23, 29Ϫ31]. Stock solutions of pure compounds were diluted in
DMSO to give serial decreasing dilutions ranging from 0.10 to 250
μg/mL. The final concentrations of DMSO in the assay did not
exceed 2%. Using a micropipette, an inoculum of 5 μL of the yeast,
cell or spore suspensions was added to each Sabouraud-chloram-
phenicol agar tube. The antifungal agents ketoconazole (Janssen
Pharmaceutical, Beerse, Belgium), amphotericin B (Sigma Chemical
Co, St Louis, MO, USA) and terbinafine (Novartis, Buenos Aires,
Argentina) were included in the assays as positive controls for all
fungi. Drug-free solution was also used as a blank control. The
tubes were incubated for 24, 48 or 72 h at 30°C (according to the
control fungus growth) up to 15 days for dermatophyte strains.
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Neurospora crassa assay [17]
Thirty mL of a medium containing 0.5% proteose-peptone # 3 (Bri-
tania cat. B # 02-0700), yeast extract (Britania cat # 01-006-05),
4.0% sucrose (reagent grade) and 1.5% agar (Merck cat. # 1613)
was autoclaved (115°C, 15 min), inoculated with 30 μL of spore
inoculum of Neurospora crassa (IM70 ATCC 9279) at 40°C and
then layered on a petri dish (diameter 9 cm). After the medium has
solidified, 1/4" filter disks (Baxter cat # F-2882-1) were applied to
media. DMSO solutions of samples were spotted on disks (25 μg/
disk). Disks containing DMSO as negative controls were included
in the assay. 1.25 μL cilofungin (20 mg/mL) and miconazole (Sigma
cat # M-3512) were spotted on disks for positive control and to
produce a clear zone respectively. Zones of inhibition were examined
macroscopically for hazy or mottled appearance following incu-
bation of the plates at room temperature for 24 h with light. In
those cases where hazy zones were observed, the microscopic ap-
pearances of the fungi were observed. For preparing spore inocu-
lum, N. crassa M70, ATCC 9279 was grown in a medium containing
0.25% (w/w), 0.25% (w/w) yeast extract (w/w), 1% sucrose (w/w)
and 1.5% agar (w/w). 4-5days of incubation at rom temperature and
light produced an orange mat of hyphal growth with spores. These
were harvested with a buffer containing 0.075 g/100 mL K2HPO4
(reagent grade), 0.10 g/100 mL KH2PO4 (reagent grade) in a solu-
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