A new class of antifungal agents. Synthesis and antimycotic activity of disubstituted N-azolylamines

Article abstract of DOI:10.1002/1521-4184(20009)333:9<299::AID-ARDP299>3.0.CO;2-F

In this study we extended our exploration of the N-azolylamine moiety for its antifungal activity. We prepared a number of N-azolylamino derivatives. The synthetic sequence includes the preparation of aminoazole Schiff bases, and the reduction and the alk

Full text of DOI:10.1002/1521-4184(20009)333:9<299::AID-ARDP299>3.0.CO;2-F

Antifungal Agents
299
A New Class of Antifungal Agents. Synthesis and Antimycotic Activity
of Disubstituted N-Azolylamines
a)
a)
b)
b)
Sabrina Castellano , Giorgio Stefancich *, Chiara Musiu , and Paolo La Colla
a) Dipartimento di Scienze Farmaceutiche, Università di Trieste, P.le Europa 1, 34127 Trieste, Italy
b) Dipartimento di Biologia Sperimentale, Sezione di Microbiologia, Università di Cagliari, Cittadella Universitaria, SS 554, Km 4,500,
09042 Monserrato, Cagliari, Italy
Key Words: Antifungal agents; aminoazole antifungal agents
amines 1–23, which are structurally related to the previously
described compounds and contain the most important and
Summary
[3]
typical portions of the azole and non-azole antifungals
(Scheme 1). Moreover, we thought that this preliminary in-
vestigation could help us to find a lead compound for the
development of a new class of antifungal agents.
In this study we extended our exploration of the N-azolylamine
moiety for its antifungal activity. We prepared a number of N-
azolylamino derivatives. The synthetic sequence includes the
preparation of aminoazole Schiff bases, and the reduction and the
alkylation of the corresponding secondary amines. The title com-
pounds were evaluated in vitro against several pathogenic fungi
responsible for human disease. The most potent antimicrobial
compound was the N-(biphenyl-4-yl)methyl-N-(2,4-dichloro-
phenyl)methyl-1H-imidazol-1-ylamine (21), which was found to
be active against yeasts and dermatophytes; its potency and selec-
tivity were comparable to those of miconazole.
R'
Y
X
N
N
N
R
1-23
Introduction
No.
X
Y
R
R'
During the past 20 years an increase of invasive fungal
infections has been observed, particularly in immunosup-
pressed patients, which are now causes of morbidity and
mortality. Autopsy data in fact indicate that more than half of
the patients who die with malignancies are infected with
Candida spp and increasing numbers with other fungi. Since
the discovery of amphotericin B a number of different classes
of antifungal agents have been discovered. However, there is
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CH CH
N
CH
CH CH
N
CH
CH CH
N
CH
N
CH
N
CH
N
CH
D
D
D
F
F
F
C
C
C
C
C
C
C
C
C
C
C
B
B
B
B
A
E
E
A
A
E
E
E
A =
B =
C =
D =
E =
F =
CH
N
CH
N
E
E
E
B
B
D
D
E
E
E
A
A
A
A
D
D
D
still a critical need for new antifungal agents to treat life-
[1]
threatening invasive mycoses
.
CH
N
CH
N
CH
N
CH
N
The route by which fungal cells synthesize ergosterol cer-
tainly offers a number of possible targets. One of them,
lanosterol C-14α-demethylase, has continued to be inten-
sively studied because of the success use of its inhibitors, the
azoles. Furthermore, one of the very early enzymes in the
pathway of ergosterol synthesis, squalene epoxidase, is the
target of other antimycotics, namely the allylamines and the
[2]
benzylamines .
CH CH
Recently, we reported the synthesis and the antimycotic
activity of compounds which simultaneously contain the
structural characteristics of both azole and allylamine (or
benzylamine) antifungals. These substances were devoid of
activity against yeasts; on the other hand, however, some
1-aminoazole derivatives inhibited the growth of dermato-
phytes, showing an activity comparable with that of nafti-
N
CH
N
CH
CH CH
CH
N
CH
N
Cl
Cl
N
CH
CH
N
[3]
S
fine .
In order to explore the importance of the N-azolylamine
moiety for antifungal activity, we here report the synthesis
and the antimycotic activity of the disubstituted N-azolyl-
Scheme 1. Structure of the disubstituted N-azolylamines 1–23.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/0909/0299 $17.50 +.50/0

300
Castellano, Stefancich, Musiu, and La Colla
mans), dermatophytes (Trichophyton verrucosum, Tricho-
phyton rubrum, Microsporum gypseum), and molds (Asper-
gillus fumigatus). Miconazole was used as reference drug.
The title compounds were also tested for in vitro cytotoxity
in a lymphoid cell line (MT-4). Cytotoxicity evaluation was
performed in order to determine whether test compounds
were endowed with selective antimicrobial activity.
Chemistry
As shown in Scheme 2, tertiary amines 1–23 were prepared
starting from the secondary amines 24–37 and the appropriate
alkyl halides, using three different methods according to the
substrate and the reagent: (a) anhydrous THF in the presence
[4]
of NaH ; (b) anhydrous diethyl ether in the presence of
[5]
potassium tert-butoxide and 18-crown-6 ; (c) dichloro-
methane/50% sodium hydroxide aqueous solution in the pres-
ence of tetrabutylammonium hydrogen sulfate . Secondary
amines 24–37 were generated by reducing the corresponding
None of the compounds was active against the mold strain
tested (A. fumigatus).
[6]
All triazole derivatives were inactive against yeasts and
dermatophytes. On the other hand, under the same experi-
mental conditions imidazole derivatives were inhibitory to
the growth of yeasts and dermatophytes, although generally
with lower potency than that of miconazole. These results are
in agreement with literature. In a study on the antifungal
activity of aromatic ethers of 1-aryl-2-(1H-azolyl)ethanol
(which are analogs of miconazole) Y. Wahbi and coworkers
also found that derivatives with an imidazole portion were
more potent than triazolic counterparts. This may be related
to the difference between the lipophilic parameters of the two
Schiff bases 38–51 with NaBH in methanol. All the imida-
4
zole and triazole Schiff bases but N-(biphenyl-4-yl)-
methylene-1H-imidazol-1-ylamine (40) (see Experimental
part), were prepared according to a procedure previously
[3]
reported .
No.
X
Y
R
N
X
24, 38
25, 39
26, 40
27, 41
28, 42
29, 43
30, 44
41, 45
32, 46
33, 47
34, 48
35, 48
36, 50
37, 51
CH CH
CH CH
CH CH
CH CH
F
R
Y
[7]
N
X
A
D
E
A
A
D
D
E
E
B
B
F
i, ii
groups . Among the imidazole derivatives, the best antifun-
N
N
N
H
Y
gal activity was obtained with compound 21, which contains
the 4-biphenyl and 2,4-dichlorophenyl portions, and whose
potency and selectivity were comparable to those of micona-
zole (Table 1).
Compound 4 was also found to inhibit the growth of yeasts
and dermatophytes, although with low potency. Compounds
16, 7, and 1 (data not shown) follow in order of decreasing
potency.
The substitution of R or R′ in compound 21 turned out to
seriously decrease the activity of the parent compound. In
particular, compound 1, containing the 4-biphenyl and 4-tert-
butylphenyl portions, proved to be inactive against yeasts and
poorly active against dermatophytes. It may be assumed that
the steric hindrance of the substituent at position 4 might play
an important role in the activity.
To conclude, in our opinion 1H-imidazol-1-ylamine could
represent a pharmacophore group for antimycotic activity.
Although from the results obtained it is difficult to establish
a general relationship between chemical structure and anti-
fungal activity, some preliminary conclusions can be the
following: a) the presence of the 4-biphenyl portion seems to
be a determinant factor for the observed activity; b) in these
derivatives, increase in size of the substituent at position 4 of
the second aryl portion, appears to have a marked negative
X = CH
Y = CH, N
38-51
N
CH
N
CH
N
CH
N
iii
CH
N
R'
CH
N
R
Y
iv
CH
N
X
N
N
X
N
N
N
NH
CH
N
Y
R
CH
N
CH
N
24-37
1-23
CH
F
Scheme 2. Synthesis of tertiary amines 1–23. Reagents and reaction condi-
tions: (i) NH2OSO3H, water, NaHCO3; (ii) RCHO, ethanol, HCl; (iii)
NaBH4, methanol; (iv) method a (for compounds 2, 12, 17, 22): HalCH2R′,
NaH, THF; method b (for compounds 1, 4): HalCH2R′, 18-crown-6, diethyl
t
ether, BuOK; method c (for compounds 3, 5–11, 13–16, 18–21, 23):
HalCH2R′, dichloromethane/water, NaOH, TBAHS. Hal = Br (for com-
pounds 1–15), Cl (for compounds 16–20, 22, 23), I (for compound 21).
Results and Discussion
The title compounds were evaluated in vitro against several
pathogenic fungi including representatives of yeasts (Can-
dida albicans, Candida parapsilosis, Criptococcus neofor- effect on biological activity.
Table 1. In vitro antifungal activity of compounds 4, 21 and miconazole.
—————————————————————————————————————————————————————————————–
Compound
MIC a)
C. neoformans
—————————————————————————————————————————————————————————————–
C. albicans
C. parapsilosis
T. verrucosum
T. rubrum
M. gypseum
4
67
67
67
25 (1.6)
50
6 (4.8)
21
2.5 (19)
5 (3.6)
1.2 (40)
2.5 (7)
2.5 (19)
1.2 (15)
0.3 (160)
0.15 (120)
0.15 (320)
0.15 (120)
1.5 (32)
1.2 (15)
miconazole
—————————————————————————————————————————————————————————————–
a) Minimum inhibitory concentration (µM). Values in brackets represent the CC50/MIC ratio (selectivity index). Data represent mean values from three
independent determinations.
Arch. Pharm. Pharm. Med. Chem. 333, 299–304 (2000)

Antifungal Agents
301
Table 2. Structure, synthetic and analytical data of the disubstituted N-azolylamines 1–23.
—————————————————————————————————————————————————————————————–
No.
X
Y
R
R′
Mp (°C)
Recrystallization
solvent
Formula
(MW)*
MS: m/z
Method
Yield
(**)
[M+]
—————————————————————————————————————————————————————————————–
1
CH
CH
CH
N
D
D
D
F
C
C
C
C
C
C
C
C
C
C
C
B
B
B
B
A
E
E
A
A
E
E
E
114–116
C27H29N3
395
396
396
325
326
326
b
a
c
b
c
c
c
c
c
c
c
a
c
c
c
c
a
c
c
c
c
a
c
67
benzene/petroleum ether (395.54)
(I)
2
N
131
C26H28N4
(396.54)
73
benzene/cyclohexane
(II)
76
3
CH
CH
N
102–103
ligroin
C26H28N4
(396.54)
(III)
49
4
CH
CH
N
66
C19H23N3S
(325.47)
benzene/ligroin
glass
(I)
5
F
C18H22N4S
(326.45)
85
(II)
90
6
CH
CH
N
F
oil
C18H22N4S
(326.45)
(III)
20
.
7
CH
CH
N
E
E
E
B
B
D
D
E
E
E
A
A
A
A
D
D
D
153–154
C21H23N3Cl2 (CO2H)2
(478.37)
387
[M+-(CO2H)2]
ethanol/diethyl ether
(I)
8
125
C20H22N4Cl2
(389.32)
388
70
benzene/ligroin
(II)
59
9
CH
N
64
C20H22N4Cl2
(389.32)
388
346
346
366
366
358
358
ligroin
(III)
65
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CH
N
129
C22H26N4
(346.47)
benzene/ligroin
(II)
60
CH
N
61
C22H26N4
(346.47)
petroleum ether
(III)
78
CH
N
164
C24H22N4
(366.46)
benzene/ligroin
(II)
74
CH
N
133
C24H22N4
(366.46)
ligroin
125
(III)
69
CH
N
C18H16N4Cl2
benzene/petroleum ether (359.25)
(II)
43
CH
CH
N
88
C18H16N4Cl2
(359.25)
ligroin
(III)
25
.
CH
CH
N
125–127
C21H17N3Cl2 (CO2H)2
(472.32)
381
[M+-(CO2H)2]
ethanol/diethyl ether
151
(I)
C20H16N4Cl2
382
17
toluene/petroleum ether (383.27)
(II)
64
CH
N
120
C20H16N4Cl2
382
364
364
407
408
408
ligroin
(383.27)
(III)
74
CH
N
215
C24H20N4
(364.44)
ethanol
(II)
47
CH
CH
N
135–136
benzene/ligroin
117
C24H20N4
(364.44)
(III)
32
CH
CH
N
C23H19N3Cl2
(408.33)
benzene/ligroin
178
(I)
C22H18N4Cl2
(409.31)
26
benzene/ligroin
155–156
ligroin
(II)
88
CH
C22H18N4Cl2
(409.31)
(III)
—————————————————————————————————————————————————————————————–
* Analytical results for C, H, N were within ±0.4% of the calculated values; ** Chromatographic conditions: I = silica/ethyl acetate, II = alumina/petro-
leum ether-chloroform (3:7), III = alumina/petroleum ether-chloroform (7:3).
Arch. Pharm. Pharm. Med. Chem. 333, 299–304 (2000)

302
Castellano, Stefancich, Musiu, and La Colla
Table 3. Structure, synthetic and analytical data of the methylazolylamines 24–37.
————————————————————————————————————————————————————
No.
X
Y
R
Mp (°C)
Formula
(MW)*
MS: m/z
[M+]
Recryst. solvent
————————————————————————————————————————————————————
24
25
26
27
28
29
30
41
32
33
34
35
36
37
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
F
66
C8H9N9S
(179.23)
179
223
249
241
224
224
250
250
242
242
200
200
180
180
toluene/petroleum ether
102–103[lit.102–103] [3]
A
D
E
A
A
D
D
E
E
B
B
F
C14H13N3
(223.28)
toluene/petroleum ether
125
C16H15N3
(249.30)
toluene/petroleum ether
89–90
C10H9N3Cl2
(242.09)
toluene/petroleum ether
173–175 [lit.171] [8]
ethanol
88–89 [88–89] [3]
cyclohexane
170–172
C13H12N4
(224.26)
CH
N
C13H12N4
(224.26)
CH
N
C15H14N4
(250.30)
toluene
CH
N
137
C15H14N4
(250.30)
ethanol
CH
N
155
C9H8N4Cl2
(243.09)
toluene
CH
N
102–103
C9H8N4Cl2
(243.09)
ethanol
CH
N
127
C11H12N4
(200.24)
ethanol
CH
N
105–106
C11H12N4
(200.24)
ethanol
CH
N
103–104
C7H8N4S
(180.22)
toluene
CH
F
77
C7H8N4S
(180.22)
cyclohexane
————————————————————————————————————————————————————
* Analytical results for C, H, N were within ±0.4% of the calculated values.
were determined on a V6-Micromass 7070H mass spectrometer. Silica gel
Further structural modifications of compound 21 are in
progress in order to define the structural requirements for
optimal activity of this new class of antifungal agents.
chromatography was performed using Merck silica gel 60 (0.015–
0.040 mm); alumina chromatography was performed using Merck aluminum
oxide 90 (0.063–0.200 mm). Petroleum ether refers to petroleum ether
(40–60 °C).
Acknowledgements
General Procedure for the Preparation of Compounds 38–39, 41
M U R S T ( Ministero dell’Università e della Ricerca Scientifica e
Tecnologica) is gratefully acknowledged for its financial support (Pro-
grammi di ricerca di rilevante interesse nazionale – cofinanziamento 1997).
A solution of hydroxylamine-O-sulfonic acid (11.3 g, 100 mmol) in water
(100 ml) was made neutral with NaHCO3 and imidazole (13.6 g, 200 mmol)
was added. After stirring for 20 h at room temperature, the mixture was
acidified with 2N hydrochloric acid and then was evaporated under reduced
pressure. To a stirring suspension of the residue in 150 ml of absolute ethanol,
a solution of the appropriate aldehyde (100 mmol) in the same solvent
(50 ml) was added and the reaction mixture was stirred at room temperature
for 24 h. The solvent was evaporated under reduced pressure and the residue
was taken up with water (200 ml). The insoluble portion was filtered off and
the filtrate was extracted with diethyl ether (2 × 100 ml). Organic phase was
eliminated while aqueous solution was basified with NaHCO3 and extracted
with chloroform (3 × 100 ml). The combined extracts were dried over
Na2SO4, chloroform was evaporated and the residue was crystallized from
suitable solvent. Yields, melting points, analytical and spectroscopic data are
reported in Table 4.
Experimental Part
Chemistry
Melting points were determined in open capillary tubes with a Büchi
apparatus and are uncorrected. Elemental analyses (C, H, N) were performed
by Dr. Emilio Cebulec at the Chemistry Department of the University of
Trieste. IR spectra were obtained on a Jasco FT/IR-200 spectrophotometer
(KBr). All compounds showed appropriate IR, which are not reported. The
1H-NMR spectra were determined on a Varian 200 instrument. 1H-NMR data
were in agreement with the proposed structure and the full set of data is in
the PhD thesis of S. Castellano, University of Milan, 1998. Mass spectra data
Arch. Pharm. Pharm. Med. Chem. 333, 299–304 (2000)

Antifungal Agents
303
Table 4. Structure, synthetic and analytical data of the methylenazolylamines 38–51.
————————————————————————————————————————————————————
No.
X
Y
R
Yield
(%)
Mp (°C)
Recryst. solvent
Formula
(MW)*
MS: m/z
[M+]
————————————————————————————————————————————————————
38
39
40
41
42
43
44
45
46
47
48
49
50
51
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
F
53
38
50
33
13
13
7
84
C8H7N3S
177
221
247
239
222
222
248
248
240
240
198
198
178
178
benzene/petroleum ether (177.23)
A
D
E
A
A
D
D
E
E
B
B
F
87–88 [lit.87–88] [3]
cyclohexane
150–151
C14H11N3
(221.26)
C16H13N3
ethanol
(247.29)
141
C10H7N3Cl2
toluene/petroleum ether (240.09)
143 [lit. 142–143] [9]
C13H10N4
(222.24)
ethanol
CH
N
89 [lit.89] [3]
petroleum ether
200 [lit. 210–212] [10]
methanol
C13H10N4
(222.24)
C15H12N4
(248.28)
CH
N
CH
N
22
13
15
10
15
11
12
133–134
C15H12N4
(248.28)
cyclohexane
170 [lit. 171–172] [9]
toluene/cyclohexane
157–158
CH
N
C9H6N4Cl2
(241.07)
CH
N
C9H6N4Cl2
(241.07)
ethanol
186–187 [lit. 190] [10]
CH
N
C11H10N4
(198.22)
toluene
CH
N
97
C11H10N4
(198.22)
ethanol
CH
N
120
C7H6N4S
(178.21)
toluene
CH
F
94–95
C7H6N4S
(178.21)
cyclohexane
————————————————————————————————————————————————————
* Analytical results for C, H, N were within ±0.4% of the calculated values.
Preparation of N-(Biphenyl-4-yl)methylene-1H-imidazol-1-ylamine (40)
triazole derivatives were recovered. Yields, melting points, analytical and
spectroscopic data are reported in Table 4.
Prepared from imidazole, hydroxylamine-O-sulfonic acid, and bipheny-4-
carboxaldehyde following the procedure described above. After stirring for
24 h at room temperature, solvent was evaporated under reduced pressure
and the residue was taken up with water (200 ml) and filtered. The solid was
washed with diethyl ether, treated with boiling ethanol and then filtered. The
filtrate was concentrated under reduced pressure, taken up with water,
basified with NaHCO3 and extracted with chloroform (3 × 100 ml). The
combined extracts were dried over Na2SO4, chloroform was evaporated and
the residue was crystallized from suitable solvent. Yield, melting point,
analytical and spectroscopic data are reported in Table 4.
General Procedure for the Preparation of Compounds 24–37
A cooled solution of Schiff base (0.050 mol) in methanol (200 ml) was
treated portionwise with an excess of sodium borohydride powder until the
starting material disappeared (TLC), then the solvent was removed under
reduced pressure. The crude residue was taken up with water (200 ml) and
extracted with chloroform. The organic phase was dried over Na2SO4 and
the solvent was evaporated to give a solid which was crystallized from
suitable solvent. The yield of the reduction reaction wasquantitative. Melting
points, analytical and spectroscopic data are reported in Table 3.
General Procedure for the Preparation of Compounds 42–51
General Procedure for the Preparation of Compounds 1–23
General Method a
Prepared from 1H-1,2,4-triazole (13.8 g, 200 mmol) following the proce-
dure described above. After stirring for 24 h at room temperature, the solvent
was evaporated under reduced pressure and the residue was taken up with
water (200 ml). The reaction mixture was basified with NaHCO3 and ex-
tracted with chloroform (3 × 100 ml). The combined extracts were dried over
Na2SO4, chloroform was evaporated and the residue was separated by
alumina column chromatography (chloroform-petroleum ether 1:1). First
eluates were discarded, then1H-1,2,4-triazole derivatives andafter4H-1,2,4-
Sodium hydride (0.96 g, 40.0 mmol) was added to a solution of the
appropriate secondary amine (20.0 mmol) in anhydrous tetrahydrofuran
(200 ml) under dry nitrogen at room temperature. The mixture was stirred
for 24 h then a solution of the appropriate halide (22.0 mmol) in anhydrous
tetrahydrofuran (70 ml) was added dropwise over a period of 20 min. After
stirring under nitrogen at room temperature for 24 h, methanol (15 ml) was
added. The solvent was evaporated and the residue was taken up with water
Arch. Pharm. Pharm. Med. Chem. 333, 299–304 (2000)

304
Castellano, Stefancich, Musiu, and La Colla
broth using a glass homogenizer. Glycerol, final concentration 10%, was
added as a cryoprotective agent to both yeast and dermatophyte suspension,
aliquots of which were then stored in liquid nitrogen. Test tubes were
inoculated with 103 blastospores or colony forming units (CFU)/tube. The
minimal inhibitory concentration (MIC) was determined by serial dilutions
of compound using Sabouraud dextrose broth (pH 5.7) and incubating at
37 °C. The growth control for yeast was read after 1 day and for dermato-
phytes after 3 days (5 days for Cryptococcus neoformans). The MIC was
defined as the compound concentration at which no macroscopic signs of
fungal growth were detected. The minimal germicidal concentration (MGC)
was determined by subcultivating negative test tubes in Sabouraud dextrose
agar.
(100 ml) and extracted with chloroform (3 × 50 ml). The combined organic
solution was washed with brine and dried over Na2SO4. The solvent was
removed by evaporation under reduced pressure and the residue was purified
by chromatography.
General Method b
Potassium tert-butoxide (2.24 g, 20.0 mmol) was added to a solution of
18-crown-6 (0.53 g, 2.00 mmol) in diethylether (50 ml), then the appropriate
secondary amine (20.0 mmol) was added in a single portion. After stirring
for 15 min at room temperature under nitrogen, a solution of the appropriate
halide (20.0 mmol) in diethylether (30 ml) was added dropwise to the cooled
reaction mixture over a period of 20 min. Stirring was continued for 4 h then
water (100 ml) was added and the layers were separated. Aqueous phase was
extracted with diethyl ether (3 × 50 ml) and the combined extracts were
washed with brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was purified by chromatography.
The cytotoxity evaluation of compounds was based on the viability of
mock-infected cells, as monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method [11]
.
References
General Method c
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A solution of the secondary amine (10.0 mmol) and tetrabutylammonium
hydrogen sulfate (3.39 g, 10.0 mmol) in dichloromethane (50 ml) was treated
with the appropriate halide (15.0 mmol) and 50% sodium hydroxide aqueous
solution (30 ml). The reaction mixture was stirred overnight at room tem-
perature, then treated with water (60 ml) and dichloromethane (60 ml) and
shaken. The aqueous layer was extracted with dichloromethane (3 × 50 ml).
Organic extracts were collected and the solvent was removed by evaporation
under reduced pressure. The residue was taken up with ethyl acetate (150 ml),
washed with brine and then dried over Na2SO4. The solvent was removed by
evaporation under reduced pressure and the residue was purified by chroma-
tography.
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Microbiology
[8] H.G.O. Becker, H. Timpe, J. Prakt. Chem. 1969, 311, 9–14.
Compounds. Test compounds were dissolved in DMSO at an initial
concentration of 200 mM and then were serially diluted in culture medium.
Cells. Cell lines were from American Type Culture Collection (ATCC);
fungal strains were clinical isolates (obtained from Clinica Dermosifilopa-
tica, University of Cagliari) or collection strains from ATCC.
[9] M. Mazza, L. Montanari, F. Pavanetto, Farmaco-Ed. Sc. 1969, 31,
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Antimycotic Assays. Yeast blastospores were obtained from a 30 h old
shaken culture incubated at 30 °C in Sabouraud dextrose broth. The dermato-
phyte inoculum was scraped aseptically with a spatula from a 7 day-old
culture on agar and the macerate was finely suspended in Sabouraud dextrose
[11] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Sholds, P. Herdewijn,
J. Deshyter, E. De Clercq, J. Virol. Methods 1998, 20, 309–321.
Received: June 2, 2000 [FP494]
Arch. Pharm. Pharm. Med. Chem. 333, 299–304 (2000)