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,
Silvia Lópezc, Maximiliano Sortinoc, Andrea Escalantec, Ricardo L. E. Furlánc, and
Susana Zacchinoc

a
Departamento de Quı́mica, Universidade Federal de Santa Catarina, Florianópolis, Brasil
b
Núcleo de Investigações Quı́mico-Farmacêuticas, Universidade do Vale do Itajaı́, Itajaı́, Santa Catarina,
Brasil
c
Farmacognosia, Facultad Ciencias Bioquı́micas y Farmacéuticas, 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 2⬘-OH substituent
on ring A are sufficient for these compounds to have antifungal properties. The chalcone 3-(2-chloro-
phenyl)-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 ⫻ 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-
Chalcones represent an important group of natural com-
fungal properties of hydroxy-chalcones reported to date
pounds with a variety of biological actions including anti-
have focused on their activity against Cladosporium cucum-
fungal activity [1⫺5].
erinum or yeasts like Cryptococcus neoformans and Candida
Various reports about antifungal chalcones suggest that the spp. [8⫺13].
presence of -OH groups on the aromatic rings plays a sig-
Additional studies of the antifungal properties of hydroxy-
nificant role, though it is not clear yet how the exact lo-
chalcone derivatives seem to be necessary, and will provide
cation of those groups correlates with the observed activity
information on the structure-activity relationships of this
[1]. So, the evaluation of a series of chalcones against differ-
kind of compounds.
ent Candida species suggested that a OH in the 2-position
is required for antifungal activity [6]. However, a further As part of our continuing work on the isolation of bioactive
report of same authors [7] showed that a 2-hydroxy group compounds from South-American plants, we previously iso-
does not always confer antifungal activity. Another study lated 2⬘-OH- 4⬘,6⬘-diOMe acetophenone (xanthoxyline) in
concluded that among a series of chalcones tested, a 4⬘- high yield from leaves and stems of Sebastiania schottiana
hydroxychalcone was the most active [1]. However, two [14], and then, we easily synthesized it by methylation of
2⬘,4⬘-diOH chalcones isolated from Myrica serrata showed 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
Ciencias Bioquı́micas y Farmacéuticas, Universidad
Nacional de Rosario, Suipacha 531-(2000) Rosario, Argentina.
analogues possessing a unique OH in position 2⬘, by the
Phone: ⫹54 341 4375315, Fax: ⫹54 341 4375315, e-mail: base-catalysed condensation of appropriate aldehydes with
szaabgil@[Link] xanthoxyline 1 as the starter ketone.

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88 Zacchino et al. Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95

These xanthoxyline derived-chalcones were tested against a for compounds 6, 8 and 12 (Tables 1 and 2 show the spectro-
panel of 12 human pathogenic opportunistic fungi, includ- scopic data of novel compounds).
ing 5 dermatophyte species in order to have a view about
their spectra of antifungal action.
In addition, considering that some chalcones have showed Antifungal evaluation
to be inhibitors of the fungal cell-wall [16], the most active
compound was evaluated with the whole-cell Neurospora The antifungal properties of compounds 3⫺19 were evalu-
crassa assay [17, 18], an agar diffusion method which allows ated using the agar dilution method against a panel of
a macroscopical detection of fungal cell wall inhibitors. twelve fungal species: three human opportunistic patho-
Since fungal but not mammalian cells possess a wall, its genic yeasts, three hialohyphomycetes and five dermato-
inhibition represents an ideal mode of action for antifungal phytes. To carry out the antifungal evaluation, concen-
drugs. The malformations of N. crassa hypha produced by trations of compounds up to 250 μg/mL were incorporated
this chalcone are reported too. to growth media according to reported procedures [23]. Am-
photericin B, terbinafine and ketoconazole were used as
positive controls.
Results and discussion
In order to evaluate the antifungal activity of these chal-
Chemistry cones and analogues, the effect of different structural modi-
Chalcones and chalcone-like compounds were prepared by fications were considered: (a) replacement of the benzene
base-catalysed condensation of the appropriate aldehyde ring B by alternative aromatic systems such as naphthalene
and either xanthoxyline 1 or 3-bromo xanthoxyline 2 or furane; (b) variations of the substitution pattern of the
(Scheme 1). After purification, chalcones were obtained in benzene ring B and (c) variations on the ring A by substi-
16⫺89 % yields. Compounds 3, 6, 7, 8, 11, and 17 have been tution with bromine in the 3⬘-position.
reported in the literature [15, 19⫺22]. Structures 4, 5, 9, 10,
Results showed that none of the compounds tested was ef-
12-16, 18 and 19 are new compounds.
fective against the yeasts C. albicans, S. cerevisiae or C. neo-
Structures of the products were established using IR, UV formans nor against the filamentous fungi A. niger, A. fumi-
and NMR spectroscopies, and elemental analysis. Inspec- gatus or A. flavus. In contrast, different antifungal activities
tion of the 1H NMR spectra suggested that chalcones were were observed for the compounds of the series against der-
geometrically pure (E isomer, JHα-Hβ ⫽ 15⫺16 Hz), except matophytes (Table 3).

Scheme 1. Synthetic route to xanthoxyline-derived chalcones and analogues. Reagents: a) CHO-Ph-R, NaOH/EtOH;
b) 2⫺naphthaldehyde, NaOH/EtOH; c) 2-furaldehyde, NaOH/EtOH; d) Br2/ AcOH. (*) New compounds.

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Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95 Antifungal properties of xanthoxyline-derived chalcones 89

Table 1. 1H NMR Data (δ in ppm, CDCl3) of compounds: 4, 5, 9, 10, 12⫺16, 18, 19†.

4 5 9 10 12 13 14 15 16 18 19

H3· 5.99 d; 5.99 d; 5.96 d; 6.91 d; 6.17 d; 6.0 d; 5.97 d; 5.98 d; 6.09 d; ⫺ ⫺
J ⫽ 2.2 Hz J ⫽ 2.2 Hz J⫽ 2.2 Hz J ⫽ 2.2 Hz J⫽ 2.2 Hz J ⫽ 2.2 Hz J ⫽ 2.2Hz J⫽ 2.2Hz J ⫽ 2.2Hz
H5· 6.13 d; 6.03⫺ d; 6.11 d; 6.08 d; 6.42 d; 6.15 d; 6.14 d; 6.12 d; 6.12 d; 6.05 s 6.10 s
J ⫽ 2.2 Hz J ⫽ 2.2 Hz J ⫽ 2.2 Hz J ⫽ 2.2 Hz J ⫽ 2.2 Hz J ⫽ 2.2 Hz J ⫽ 2.2 Hz J ⫽ 2.2Hz J ⫽ 2.2Hz
Hα 7.47⫺7.99 m 7.57 d; 7.68 d; 7.01⫺7.42 m 7.98 d; 7.91 d; 7.62 d; 7.74 d; 7.81 d; 7.81 d; 8.48⫺8.60 m
J ⫽ 15.37 Hz J ⫽ 15.51 Hz J ⫽ 15.51 Hz J ⫽ 15.67 Hz J ⫽ 15.60 Hz J ⫽ 15.68 Hz J ⫽ 15.49 Hz J ⫽ 15.58 Hz
Hβ 7.47⫺7.99 m 7.79 d; 7.87 d; 7.01⫺7.42 m 8.26 d; 8.19 d; 7.85 d; 7.98 d; 8.44 d; 8.17 d; 8.48⫺8.60 m
J ⫽ 15.37 Hz J ⫽ 15.51 Hz J ⫽ 15.51 Hz J ⫽ 15.67 Hz J ⫽ 15.60 Hz J ⫽ 15.68 Hz J ⫽ 15.49 Hz J ⫽ 15.58 Hz
H1 7.47⫺7.99 m ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
H2 ⫺ ⫺ 7.47⫺7.51 m 7.01⫺7.42 m 6.75 d ⫺ 7.43⫺7.51 m ⫺ ⫺ ⫺ 8.48⫺8.60 m
J ⫽ 8.85 Hz
H3 7.47⫺7.99 m 6.50 d; 7.47⫺7.51 m 7.01⫺7.42 m 7.25 d 7.30⫺7.75 m ⫺ 8.46 s 7.96 d; 7.28⫺7.70 m
J ⫽ 8.4 Hz J ⫽ 8.85 Hz J ⫽ 7.87 Hz
H4 7.47⫺7.99 m 6.67 t; ⫺ ⫺ ⫺ 7.30⫺7.75 m ⫺ 8.22 d; 7.47⫺7.81 m 7.28⫺7.70 m 8.48⫺8.60 m
J ⫽ 8.4 Hz J ⫽ 7.75 Hz
H5 7.47⫺7.99 m 7.51 d; 7.47⫺7.51 m 7.01⫺7.42 m 7.25 d 7.30⫺7.75 m 7.43⫺7.51 m 7.84⫺7.88 m 7.47⫺7.81 m 7.28⫺7.70 m 7.43⫺7.51 m
J ⫽ 8.4 Hz J ⫽ 8.85 Hz
H6 7.47⫺7.99 m ⫺ 7.47⫺7.51 m 7.01⫺7.42 m 6.75 d 7.30⫺7.75 m 7.43⫺7.51 m 7.84⫺7.88 m 7.47⫺7.81 m 7.28⫺7.70 m 8.48⫺8.60 m
J ⫽ 8.85 Hz
H7 7.47⫺7.99 m ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
H8 7.47⫺7.99 m ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
OCH3 3.95 s 3.91 s 3.91 s 3.83 s 3.97 s 3.94 s 3.92 s 3.94 s 3.93 s 3.98 s 4.03 s
OCH3 3.85 s 3.83 s 3.84 s 3.79 s 3.93 s 3.88 s 3.84 s 3.85 s 3.84 s 3.98 s 4.01 s

†
Measured at 200 MHz. J ⫽ Coupling constants.

13
Table 2. C NMR Data (δ in ppm, CDCl3) of compounds 4, 5, 9, 10, 12⫺16, 18 and 19†.

4 5 9 10 12 13 14 15 16 18 19

C⫽O 193.25 192.74 193.00 193.05 192.21 193.01 191.96 191.78 199.66 193.25 199.99
C1⬘ 107.07 106.99 106.99 106.91 107.21 106.99 106.22 106.20 106.00 106.00 107.02
C2⬘ 163.20 163.21 163.15 163.12 160.05 163.17 162.45 162.45 162.43 162.90 162.39
C3⬘ 94.50 94.41 94.10 94.45 94.41 94.49 93.81 93.84 93.57 92.80 92.12
C4⬘ 169.14 169.09 169.13 169.08 169.51 169.14 168.47 168.46 167.15 163.93 163.14
C5⬘ 91.99 91.88 92.03 91.94 89.52 91.99 91.37 91.37 90.60 87.80 87.16
C6⬘ 166.93 166.85 167.07 166.93 163.08 167.08 166.53 166.65 167.15 163.92 163.09
Cα 124.41 129.63 128.34 128.12 123.82 127.65 129.22 129.78 122.43 127.72 124.81
Cβ 143.17 145.32 141.47 141.69 139.05 138.55 139.23 138.68 149.67 139.49 140.20
C1 128.44 ⫺ 135.21 132.48 147.22 136.05 135.69 137.43 133.94 136.21 134.85
C2 134.86 152.93 130.36 130.73 131.24 134.53 130.83 122.08 128.93 134.33 121.00
C3 124.41 113.20 132.79 116.43 122.44 130.94 133.75 148.71 129.00 131.06 148.30
C4 129.29 116.11 124.88 161.94 149.55 131.35 133.12 123.99 125.50 131.62 122.55
C5 128.44 145.32 132.79 116.43 122.44 128.49 132.04 130.51 132.00 128.60 130.40
C6 127.35 ⫺ 130.36 130.73 131.24 130.73 129.67 134.01 133.94 130.22 131.00
C7 127.83 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
C8 129.29 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
C9 134.11 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
C10 133.77 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺
OCH3 56.60 56.45 56.57 56.52 56.26 56.56 55.94 55.82 55.44 57.08 56.76
OCH3 56.30 56.24 56.30 56.26 56.18 56.30 55.64 55.56 55.30 56.85 56.18
COOH ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ 172.00 ⫺ ⫺

†
Measured at 50 MHz (compounds 4, 5, 9, 10, 13⫺16 and 18) and at 100 MHz (compounds 12 and 19).

The lack of activity of chalcones 3, 6⫺8, 14 and 19 indicates (5), the structure possessed marginal activity, this result
that the presence of a 2⬘-OH in the chalcone is not enough differing from previous reports for an analogous structure
for these compounds to have antifungal properties. [1].

Regarding the influence of different B-ring systems alterna- Concerning the influence of substituents on the benzene
tive to benzene B, chalcone 4 with naphthalene as B-ring ring B, some interesting conclusions could be extracted:
was devoid of antifungal activity. When B-ring was a furane compounds holding electron withdrawing groups (EWGs)

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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 R3⬘ R2 R3 R4 E. f. T. m. T.r. M.c. M.g.

3 A H H H H >250 >250 >250 >250 >250

4 B ⫺ ⫺ ⫺ ⫺ >250 >250 >250 >250 >250
5 C ⫺ ⫺ ⫺ ⫺ 125 250 250 250 >250
6 A H H H OCH3 >250 >250 >250 >250 >250
7 A H H H CH3 250 250 250 250 250
8 A H H O⫺CH2⫺O >250 >250 >250 >250 >250
9 A H H H Br 50 50 50 50 250
10 A H H H F 62.5 62.5 62.5 100 >250
11 A H H H Cl 100 100 125 125 >250
12 A H H H NO2 62.5 62.5 100 250 >250
13 A H Cl H H 100 50 12.5 100 250
14 A H H Cl Cl >250 >250 250 >250 >250
15 A H H NO2 H 250 >250 125 >250 >250
16 A H COOH H H 125 250 250 125 250
17 A Br H H H 25 50 25 50 250
18 A Br Cl H H 25 50 50 50 250
19 A Br H NO2 H >250 >250 >250 >250 >250
Amp 6.25 0.3 25 >50 6.25
Ket 12.5 25 15 15 6.25
Terb 0.004 0.04 0.01 0.01 0.04

†
Abbreviations: M.c. ⫺ Microsporum canis C 112, M.g. ⫺ Microsporum gypseum C 115, T.r. ⫺ Trichophyton rubrum C113, T.m. ⫺ Trichophy-
ton mentagrophytes ATCC 9972; E.f. ⫺Epidermophyton floccosum C 114. Amp ⫺ Amphotericin, Ket ⫺ Ketoconazole, Terb ⫺ Terbinafine.

such as bromine, fluorine, chlorine, or a nitro group in the Modification of ring A by introducing a bromine substitu-
4-position (compounds 9⫺12) improved the antifungal ent in the 3⬘-position of chalcone 3 significantly improved
properties compared with the non ring B-substituted chal- the antifungal properties: compound 17 is 5⫺10 times more
cone 3. In contrast, compounds with electron-donating sub- active against four different species of fungi. However, the
stituents such as methoxy, methyl, and methylenedioxy effect of such substitution was not significant for other com-
groups in the same position of B-ring (chalcones 6, 7, and pounds (compare activities of compounds 13 vs. 18, and 15
8 respectively) were all inactive. vs. 19).

The effect of the position of the EWG on the antifungal The selective activity of compound 13 against T. rubrum is
activity could be observed within the group of chlorinated particularly interesting since this fungus is responsible of
compounds 11, 13, and 14 where the antifungal activities approximately 80-93 % of chronic and recurrent dermato-
follow the order 2-Cl (13) > 4-Cl (11) > 3, 4-di Cl (14). phyte infections in human beings [24]. It is the ethiological
Similarly the 4-NO2 chalcone 12 showed better activities agent of tinea unguium (producer of invasive nail infec-
than chalcone 15 with a nitro group in the 3-position. The tions), tinea manuum (palmar and interdigital areas of the
observed effect of EWGs on the para-position of ring B in hand infections) and tinea pedis (Athlete⬘s foot), the last
a chalcone structure is in agreement with a previous report one being the most prevalent fungal infection in developed
on the antifungal activities of non-hydroxylated chalcones countries, and the first one accounting for 50 % and 90 % of
[16]. However, the increased activities showed by com- all fingernail and toenail infections respectively [24].
pounds holding a EWG in the o-position of ring B are
somehow contradictory to the previously observed corre- In order to gain insight into the capacity of compound 13
lation between planarity of chalcones and their antifungal in acting not only against standardized strains, but against
activity [16]. This fact deserves further studies. clinical isolates of T. rubrum, we tested it against ten clinical

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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-(2⬘-hy-

droxy-4⬘,6⬘-dimethoxyphenyl)prop-2-en-1-one (13), against
10 different clinical strains of T. rubrum from Centro de
Referencia Micológica (CEREMIC), Facultad de Ciencias
Bioquı́micas y Farmacéuticas, Universidad Nacional Rosario.

Voucher specimen MIC [μg/mL]

T. rubrum C 110 12.5

T. rubrum C 133 12.5
T. rubrum C 134 25.0
T. rubrum C 135 25.0
T. rubrum C 136 50.0
T. rubrum C 137 25.0
T. rubrum C 138 12.5
T. rubrum C 139 25.0
T. rubrum C 140 12.5
T. rubrum C 141 12.5

Figure 1. A. Blotchy appearance of the inhibition halo produ-

ced by chalcone [3-(2-Chlorophenyl)-1-(2⫺hydroxy-4,6-di-
methoxyphenyl)prop-2-en-1-one] (13) in the Neurospora
strains of T. rubrum isolated from skin infections of different crassa assay. B. Clear halo produced by ketoconazole.
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
fusion method which allows a macroscopical detection of
90 % (MIC90) of the strains tested ⫽ 12.5 and 25 μg/mL
fungal cell wall inhibitors. Neurospora crassa usually grows
respectively (Table 4).
as long hypha in a diffuse or branched way. When it grows
Considering our previous finding [16] that non-hy- in the presence of certain inhibitors of its wall, the hyphal
droxylated chalcones were inhibitors of the fungal cell wall, growth is inhibited, and fungi grow as protoplasts. Macro-
we tested structure 13 for its capacity of inhibiting the fun- scopically they can be seen as a blotchy or hazy appearance
gal cell-wall. Since mammalian cells do not possess a wall around the paper disk. The microscopic observation of this
[25], this is the intrinsic target to develop highly selective hazy zone usually shows morphological changes of the hy-
and useful drugs in medicinal investigation, and antifungal pha [26].
structures acting by this mechanism are actively sought.
Results showed that chalcone 13 produced a blotchy zone
Amongst methods that deal with this mode of action, we around the paper disk (Figure 1A and B), indicating that
have chosen the Neurospora crassa assay [17] an agar dif- the mode of action could be associated with the inhibition

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
⫻400.

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92 Zacchino et al. Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95

of cell wall polymers or assembly [18]. The microscopic ob- Elemental analyses were obtained on a Perkin Elmer 2400 (Thermo-
quest Italia, Milano, Italia). Percentages of C and H were in agree-
servation of this hazy zone showed a marked curling of the
ment with the product formula (within ±0.4 % of theoretical values).
hypha (Figure 2A and B), a defect in the directionality of
hypha growth, this giving a new sign that chalcone 13 could The purity of the synthesized substances was monitored by thin-
layer chromatography (TLC) using silica (Macherey-Nagel GmbH,
act inhibiting the synthesis or assembly of the fungal cell
Inc-USA) pre-coated aluminum plates (Alugram® SIL G/UV254
wall polymers. It is important to take in account that some layer 0.2 mm) with several solvent systems of different polarity.
hyphal malformation inducers have proved to be inhibitors Compounds were visualized with ultraviolet light (254 nm) and
of (1,3)β-glucan synthase [27], the enzyme that catalyzes the using ferric chloride (Ridel-DE Haën AG, Seelze-Hannover-Ger-
synthesis of (1,3)β-glucan, main polymer of the fungal cell many) solution and purified by recrystallization from ethyl ether or
hexane (Dinâmica-São Paulo-Brasil).
wall.
All the aromatic aldehydes used are commercially available (Sigma-
Aldrich Quı́mica, S.A., Alcobendas, Madrid). Xanthoxyline (1) was
isolated from leaves and stems of Sebastiania schottiana according
Conclusion to a reported procedure [14]. 2-Hydroxy-3-bromo-4,6-dimethoxy-
acetophenone (2) was prepared from 1 according to a literature pro-
This work strongly contributes to the knowledge of the anti- cedure [15].
fungal properties of hydroxy-chalcones by testing the anti-
fungal activity of a series of 17 synthetic chalcones and Synthesis of chalcones 3⫺16
analogues based in the natural product xanthoxyline (1).
A solution of xanthoxyline 1 (0.18 g; 0.92 mmol), EtOH (15 mL),
Results showed that the sole presence of an OH in the 2⬘- NaOH (0.1 g; 2.5 mmol, containing a minimum amount of H2O),
position is not enough for hydroxy-chalcones to possess and the appropriate aldehyde (0.95 mmol) was stirred at room tem-
antifungal activity. Nevertheless, certain members of the perature for 1⫺23 h. The crude product was isolated by acidification
series showed moderate activity against dermatophytes. of the cool diluted solution, and recrystallized from ethyl ether or
hexane.
Chalcone 13, with a Cl- in the o-position of ring B, showed
the lowest MIC against T. rubrum and inhibited all of the
1-(2⬘-Hydroxy-4⬘,6⬘-dimethoxyphenyl)-3-(2-naphthyl)prop-2-en-1-
ten clinical isolates of T. rubrum tested. Regarding its mode one (4)
of action, it is interesting to note that the most active mem-
Yellow solid, mp. ⫽ 110⫺112 °C; UV λmax 336 (4.05); IR (KBr)
ber of the series produced a blotchy appearance of the inhi-
1638 (C⫽O), 1586 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 14.35 (s, 1H,
bition halo in the Neurospora crassa assay, clearly suggest- OH), 7.47⫺7.99 (m, 9H, Hβ, Hα, H1, H3, H4, H5, H6, H7, H8),
ing that its mode of action could be the inhibition of the 6.13 (d, 1H, J ⫽ 2.26 Hz, H5⬘), 5.99 (d, 1H, J ⫽ 2.26 Hz, H3⬘),
synthesis or assembly of the polymers of the fungal cell wall. 3.95 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C NMR (CDCl3) δ
The microscopic appearance of this hazy zone showed curl- 193.25 (C⫽O), 169.14 (C4⬘), 166.93 (C6⬘), 163.20 (C2⬘) , 143.17
(Cβ), 134.86 (C2), 134.11 (C9), 133.77 (C10), 129.29 (C4, C8),
ing of the hypha. Since fungal but not mammalian cells 128.44 (C1, C5), 127.83 (C7), 127.35 (C6), 124.41 (C3, Cα), 107.07
possess a wall, results obtained in targeted assays with chal- (C1⬘), 94.50 (C3⬘), 91.99 (C5⬘), 56.30, 56.60 (OCH3). C21H18O4
cone 13 open the possibility of using this compound as a Calc. C 75.43, H 5.43, Found C 75.52, H 5.47. Yield ⫽ 30 %.
starting point for the development of safe antifungal agents,
since it would attack selectively the fungi without inhibiting 3-(2-Furyl)-1-(2⬘-hydroxy-4⬘,6⬘-dimethoxyphenyl)prop-2-en-1-one
any biochemical system of the host. (5)

Yellow solid; mp. ⫽ 92-94 °C; UV λmax 363 (3.42); IR (KBr) 1626
These data together with previous results obtained by us (C⫽O), 1586 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 7.79 (d, 1H, J ⫽
[16] clearly indicate that hydroxy and non-hydroxy chal- 15.37 Hz, Hβ), 7.57 (d, 1H, J ⫽ 15.37 Hz, Hα), 7.51 (d, 1H, J ⫽
cone-like compounds possess antifungal properties with a 8.4 Hz, H5), 6.67 (t, 1H, J ⫽ 8.4 Hz, H4), 6.50 (t, 1H, J ⫽ 8.4 Hz,
H3), 3.91 (s, 3H, OCH3), 3.83 (s, 3H, OCH3). 13C NMR (CDCl3) δ
mode of action related unless in part, to the inhibition to
192.74 (C⫽O), 169.09 (C4⬘), 166.85 (C6⬘), 163.21 (C2⬘), 152.93
the synthesis or ensamble of polymers of the fungal cell- (C2), 145.32 (Cβ, C5), 129.63 (Cα), 116.11 (C4), 113.20 (C3), 106.99
wall. (C1⬘), 94.41 (C3⬘), 91.88 (C5⬘), 56.45 (OCH3), 56.24 (OCH3).
C15H14O5 Calc. C 65.69, H 5.1, Found C 64.76, H 5.18. Yield ⫽
52 %.

Experimental 3-(4-Bromophenyl)-1-(2⬘-hydroxy-4⬘,6⬘-dimethoxyphenyl)prop-2-en-
1-one (9)
General experimental procedures
Orange solid; mp. ⫽ 150⫺151 °C; UV λmax 338 (4.08); IR (KBr)
Melting points were determined with a Microquimica MG APF-301 1632 (C⫽O), 1588 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 14.21 (s, 1H,
apparatus (Palhoça, Santa Catarina, Brasil) and are uncorrected. OH), 7.87 (d, 1H, J ⫽ 15.51 Hz, Hβ), 7.68 (d, 1H, J ⫽ 15.51 Hz,
Infrared (IR) spectra were recorded with a FT Perkin Elmer 16 PC Hα), 7.47⫺7.51 (m, 4H, H2, H3, H5, H6), 6.11 (d, 1H, J ⫽ 2.2 Hz,
spectrometer (Connecticut, USA). Nuclear magnetic resonance (1H H5⬘), 5.96 (d, 1H, J ⫽ 2.2 Hz, H3⬘), 3.91 (s, 3H, OCH3), 3.84 (s,
and 13C NMR) spectra were recorded on a Bruker AC-200 F 3H, OCH3). 13C NMR (CDCl3) δ 193 (C⫽O), 169.13 (C4⬘), 167.07
(Rheinstetten, Germany) instrument with tetramethylsilane as in- (C6⬘), 163.15 (C2⬘), 141.47 (Cβ), 135.21 (C1), 132.79 (C3, C5),
ternal standard. 130.36 (C2, C6), 128.34 (Cα), 124.88 (C4), 106.99 (C1⬘), 94.1 (C3⬘),

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Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95 Antifungal properties of xanthoxyline-derived chalcones 93

92.03 (C5⬘), 56.57 (OCH3), 56.30 (OCH3). C17H15 BrO4 Calc. C 15.68 Hz, Hα), 6.12 (d, 1H, J ⫽ 2.2 Hz, H5⬘), 5.98 (d, 1H, J ⫽ 2.2
56.22, H 4.16, Found C 56.04, H 4.21. Yield ⫽ 38 %. Hz, H3⬘), 3.94 (s, 3H, OCH3), 3.85 (s, 3H, OCH3). 13C NMR
(CDCl3) δ 191.78 (C⫽O), 168.46 (C4⬘), 166.65 (C6⬘), 162.45 (C2⬘),
3-(4-Fluorophenyl)-1-(2⬘-hydroxy-4⬘,6⬘-dimethoxyphenyl)prop-2-en- 148.71 (C3), 138.68 (Cβ), 137.43 (C1), 134.01 (C6), 130.51 (C5),
1-one (10) 129.78 (Cα), 123.99 (C4), 122.08 (C2), 106.20 (C1⬘), 93.84 (C3⬘),
91.37 (C5⬘), 55.82 (OCH3), 55.56 (OCH3). C17H15NO6 Calc. C
Yellow solid; mp. ⫽ 140-141 °C; UV λmax 339 (4.00); IR (KBr) 1632 62.00, H 4.59, N 4.25, Found C 60.08, H 4.55, N 4.26. Yield ⫽ 57 %.
(C⫽O), 1572 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 13.72 (s, 1H, OH),
7.01⫺7.42 (m, 6H, Hβ, Hα, H2, H3, H5, H6), 6.08 (d, 1H, J ⫽ 2-[-3-(2⬘-Hydroxy-4⬘,6⬘-dimethoxyphenyl)-3-oxoprop-1-enyl]-
2.22 Hz, H5⬘), 6.91 (d, 1H, J ⫽ 2.22 Hz, H3⬘), 3.83 (s, 3H, OCH3), benzoic acid (16)
3.79 (s, 3H, OCH3). 13C NMR (CDCl3) δ 193.05 (C⫽O), 169.08
(C4⬘), 166.93 (C6⬘), 163.12 (C2⬘), 161.94 (C4), 141.69 (Cβ), 132.48 Yellow solid; mp. ⫽ 160⫺161 °C; UV λmax 291 (3.82) IR (KBr) 1640
(C1), 130.73(C2, C6), 128.12 (Cα), 116.43 (C3, C5), 106.91 (C1⬘), (C⫽O), 1560 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 13.99 (s, 1H, OH),
94.45 (C3⬘), 91.94 (C5⬘), 56.52 (OCH3), 56.26 (OCH3). C17H15FO4 8.44 (d, 1H, J ⫽ 15.49 Hz, Hβ), 7.81 (d, 1H, J ⫽ 15.49 Hz, Hα),
Calc. C 67.54, H 5.00, Found C 67.50, H 5.04. Yield ⫽ 87 %. 7.96 (d, 1H, J ⫽ 7.87 Hz, H3), 7.47⫺7.81 (m, 3H, H4, H5, H6),
6.12 (d, 1H, J ⫽ 2.2 Hz, H3⬘), 6.09 (d, 1H, J ⫽ 2.2 Hz, H5⬘), 3.93
1-(2⬘-Hydroxy-4⬘,6⬘-dimethoxyphenyl)-3-(4-nitrophenyl)prop-2-en- (s, 3H, OCH3), 3.84 (s, 3H, OCH3). 13C NMR (CDCl3) d 199.66
1-one (12) (C⫽O), 167.15 (C6⬘, C4⬘), 162.43 (C2⬘), 149.67 (Cβ), 133.94 (C1,
C6), 132 (C5), 129 (C3), 128.93 (C2), 125.50 (C4), 125.04 (C4, C6),
Orange solid; mp. ⫽ 295⫺296 °C; UV λmax 379 (3.80) R (KBr) 1664 122.43 (Cα), 106 (C1⬘), 93.57 (C3⬘), 90.60 (C5⬘), 55.44 (OCH3),
(C⫽O), 1584 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 8.26 (d, 1H, J ⫽ 55.30 (OCH3). C18H16O6 Calc. C 65.85, H 4.91, Found C 65.00, H
15.51 Hz, Hβ), 7.98 (d, 1H, J ⫽ 15.51 Hz, Hα), 7.25 (d, 2H, J ⫽ 4.84. Yield ⫽ 44 %.
8.85 Hz, H3, H5), 6.75 (d, 2H, H2, H6), 6.42 (d, 1H, J ⫽ 2.2 Hz,
H5⬘), 6.17 (d, 1H, J ⫽ 2.2 Hz, H3⬘), 3.97 (s, 3H, OCH3), 3.93 (s, Synthesis of chalcones 18, 19
3H, OCH3). 13C NMR (CDCl3) δ 192.21 (C⫽O), 169.51 (C4⬘),
163.08 (C6⬘), 160.05 (C2⬘), 149.55 (C4), 147.22 (C1), 139.05 (Cβ), A solution of 2-hydroxy-3-bromo-4,6-dimethoxyacetophenone 2
131.24 (C2, C6), 123.82 (Cα), 122.44 (C3, C5), 107.21 (C1⬘), 94.41 (0.2 g; 0.73 mmol), EtOH (15 mL), NaOH (0.1 g; 2.5 mmol, con-
(C3⬘), 89,52 (C5⬘), 56.26 (OCH3), 56.18 (OCH3).C17H15NO6 Calc. taining a minimum amount of H2O), and the appropriate aldehyde
C 62.00, H 4.59, N 4.25, Found C 61.98, H 4.56, N 4.23. Yield ⫽ (0.95 mmol) was stirred at room temperature for 2⫺3 h. The crude
16 %. product, isolated by acidification of the cool diluted solution, was
recrystallized from ethyl ether and hexane
3-(2-Chlorophenyl)-1-(2⬘-hydroxy-4⬘,6⬘-dimethoxyphenyl)prop-2-en-
1-one (13) 1-(3⬘-Bromo-2⬘-hydroxy-4⬘,6⬘-dimethoxyphenyl)-3-(2-chlorophen-
yl)prop-2-en-1-one (18)
Yellow solid; mp. ⫽ 136⫺137 °C; UV λmax 336 (3.68); IR (KBr)
1630 (C⫽O), 1556 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 14.25 (s, 1H, Orange solid; mp. ⫽ 210⫺212 °; UV λmax 283 (4.12); IR (KBr)
OH), 8.19 (d, 1H, J ⫽ 15.67 Hz, Hβ), 7.91 (d, 1H, J ⫽ 15.67 Hz, 1624 (C⫽O), 1556 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 14.67 (s, 1H,
Hα), 7.30⫺7.75 (m, 4H, H3, H4, H5, H6), 6.15 (d, 1H, J ⫽ 2.2 Hz, OH), 8.17 (d, 1H, J ⫽ 15.58 Hz, Hβ), 7.81 (d, 1H, J ⫽ 15.58 Hz,
H5⬘), 6.0 (d, 1H, J ⫽ 2.2 Hz, H3⬘), 3.88 (s, 3H, OCH3), 3.94 (s, 3H, Hα), 7.28⫺7.70 (m, 4H, H3, H4, H5, H6), 6.05 (s, 1H, H5⬘), 3.98
OCH3). 13C NMR (CDCl3) δ 193.01 (C⫽O), 169.14 (C4⬘), 167.08 (s, 6H, OCH3). 13C NMR (CDCl3) δ 193.25 (C⫽O), 163.93 (C4⬘),
(C6⬘), 163.17 (C2⬘), 138.55 (Cβ), 136.05 (C1), 134.53 (C2), 131.35 163.92 (C6⬘), 162.90 (C2⬘), 139.49 (Cβ), 136.21 (C1), 134.33 (C2),
(C4), 130.94 (C3), 130.73 (C6), 128.49 (C5), 127.65(Cα), 106.99 (C 131.62 (C4), 131.06 (C3), 130.22 (C6), 128.60 (C5), 127.72 (Cα),
1⬘), 94.49 (C3⬘), 91.99 (C5⬘), 56.56 (OCH3), 56.30 (OCH3). 106 (C1⬘), 92.80 (C3⬘), 87.80 (C5⬘), 57.08 (OCH3), 56.85 (OCH3).
C17H15ClO4 Calc. C 64.06, H 4.74, Found C 63.87, H 4.80. C17H14BrClO4 Calc. C 51.35, H 3.55, Found C 51.45, H 3.58.
Yield ⫽ 89 %. Yield ⫽ 47 %.

3-(3,4-Dichlorophenyl)-1-(2⬘-hydroxy-4⬘, 6⬘-dimethoxyphenyl)prop- 1-(3⬘-bromo-2⬘-hydroxy-4⬘,6⬘-dimethoxyphenyl)-3-(3-nitrophenyl)-

2-en-1-one (14) prop-2-en-1-one (19)
Yellow solid; mp. ⫽ 120⫺123 °C; UV λmax 343 (3.80); IR (KBr) Orange solid; mp. ⫽ 264 °C; UV λmax 288 (3.25)IR (KBr) 1632 (C⫽
1622 (C⫽O), 1586 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 14.14 (s, 1H, O), 1558 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 14.55 (s, 1H, OH),
OH), 7.85 (d, 1H, J ⫽ 15.60 Hz, Hβ), 7.62 (d, 1H, J ⫽ 15.60 Hz, 8.48⫺7.60 (m, 6H, Hβ, Hα, H2, H5, H4, H6), 6.10 (s, 1H, H5⬘),
Hα), 7.43⫺7.51 (m, 3H, H2, H5, H6), 6.14 (d, 1H, J ⫽ 2.2 Hz, 4.03 (s, 3H, OCH3), 4.01 (s, 3H, OCH3). 13C NMR (CDCl3) δ
H5⬘), 5.97 (d, 1H, J ⫽ 2.2 Hz, H3⬘), 3.92 (s, 3H, OCH3), 3.84 (s, 199.99 (C⫽O), 163.14 (C4⬘), 163.09 (C6⬘), 162.39 (C2⬘), 148.30
3H, OCH3). 13C NMR (CDCl3) δ 191.96 (C⫽O), 168.47 (C4⬘), (C3), 140.20 (Cβ), 134.85 (C1), 131.00 (C6), 130.40 (C5), 124.81
166.53 (C6⬘), 162.45 (C2⬘), 139.23 (Cβ), 135.69 (C1), 133.75 (C3), (Cα), 122.55 (C4), 121 (C2), 107.02 (C1⬘), 92.12 (C3⬘), 87.16 (C5⬘),
133.12 (C4), 132.04 (C5), 130.83(C2), 129.22 (Cα), 106.22 (C1⬘), 56.76 (OCH3), 56.18 (OCH3). C17H14BrNO Calc. C 50.02, H 3.46,
93.81 (C3⬘), 91.37 (C5⬘), 55.94 (OCH3), 55.64 (OCH3). C17H14Cl2O4 N 3.43, Found C 50.10, H 3.44, N 3.40. Yield ⫽ 71 %.
Calc. C 57.81, H 4.00, Found C 57.32, H 4.09. Yield ⫽ 47 %
Biological evaluation
1-(2⬘-Hydroxy-4⬘, 6⬘-dimethoxyphenyl)-3-(3-nitrophenyl)prop-2-en-
1-one (15) Microorganisms and media

Orange solid; mp. ⫽ 171⫺172 °C; UV λmax 335 (3.74); IR (KBr) The microorganisms used for the fungistatic evaluation were pur-
1640 (C⫽O), 1580 (C⫽C) cm⫺1. 1H NMR (CDCl3) δ 14.09 (s, 1H, chased from the American Type Culture Collection (ATCC, Rock-
OH), 8.46 (s, 1H, H2), 8.22 (d, 1H, J ⫽ 7.75 Hz, H4), 7.98 (d, 1H, ville, MD, USA) or were clinical isolates and were kindly provided
J ⫽ 15.68 Hz, Hβ), 7.84⫺7.88 (m, 2H, H5, H6), 7.74 (d, 1H, J ⫽ by Centro de Referencia Micológica (C, CEREMIC, Facultad de

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94 Zacchino et al. Arch. Pharm. Chem. Life Sci. 2005, 338, 87−95

Ciencias Bioquı́micas y Farmacéuticas, Suipacha 531, (2000) tion of glycerol:H2O 15:85. Spore suspension free of hypha was pre-
Rosario, Argentina: Candida albicans ATCC 10231, Saccharomyces pared as described in Microorganisms and media.
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
Acknowledgements
agar slants for 48 h at 30 °C. Cell suspensions in sterile distilled This work was supported by grants to SAZ (Agencia de
water were adjusted to give a final concentration of 106 viable yeast
Promociones Cientı́ficas y Tecnológicas de la Argentina
cells/mL [28]. Microsporum canis C 112, Trichophyton rubrum C 113,
110, 133, 134, 135, 136, 137, 138, 139, 140 and 141; Epidermophyton PICTR 260 and OEA); to SNL (Grantee IFS F/3114-2F),
floccosum C 114, Microsporum gypseum C 115, and Candida to RLEF (Antorchas) and it is part of the Iberoamerican
tropicalis C 131. Project X.7 PIBEAFUN (Search and development of new
The strains were maintained on slopes of Sabouraud-dextrose agar
antifungal agents), part of the Iberoamerican Program of
(SDA, Oxoid) and subcultured every 15 days to prevent pleo- Science and Technology for the Development (CYTED-X).
morphic transformations. Spore suspensions were obtained accord- Collaboration from Iberoamerican Network on Natural
ing to reported procedures [28] and adjusted to 106 spores with col- Products for Medicinal Use (RIPRONAMED, X.A) of
ony forming ability/mL.
CYTED is gratefully acknowledged. VCF and RAY thank
CNPq (Brazil) for financial support.
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, 29⫺31]. Stock solutions of pure compounds were diluted in References
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© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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