Azole antifungal agents related to naftifine and butenafine

Article abstract of DOI:10.1002/1521-4184(20006)333:6<162::AID-ARDP162>3.0.CO;2-S

The methyl group of naftifine (1) and butenafine (2) was replaced by an azolic nucleus to obtain the new compounds 3-8 which exhibit the characteristics of both allylamine (or benzylamine) and azole antifungals. The title compounds were evaluated in vitro against several pathogenic fungi responsible for human disease. Among these, compounds 5, 6, and 8 were found to inhibit the growth of dermatophytes with a potency comparable to that of naftifine. The synthetic sequence includes the preparation of aminoazole Schiff bases, reduction, and alkylation of the corresponding secondary amines.

Full text of DOI:10.1002/1521-4184(20006)333:6<162::AID-ARDP162>3.0.CO;2-S

162
Castellano, La Colla, Musiu, and Stefancich
Azole Antifungal Agents Related to Naftifine and Butenafine
a)
b)
b)
a)
Sabrina Castellano , Paolo La Colla , Chiara Musiu , and Giorgio Stefancich *
a) Dipartimento di Scienze Farmaceutiche, Università di Trieste, P.le Europa 1, 34127 Trieste, Italy
b) Dipartimento di Biologia Sperimentale, Università di Cagliari, S.P. Monserrato-Sestu, Km. 0,700, 09042 Monserrato (CA), Italy
Key Words: Azole naftifine analogues; azole butenafine analogues; aminoazole antifungal agents
The new compounds 3–8 would thus contain structural ele-
Summary
ments of both allylamine (or benzylamine) and azole antifun-
gals.
The methyl group of naftifine (1) and butenafine (2) was replaced
by an azolic nucleus to obtain the new compounds 3–8 which
exhibit the characteristics of both allylamine (or benzylamine) and
azole antifungals. The title compounds were evaluated in vitro
against several pathogenic fungi responsible for human disease.
Among these, compounds 5, 6, and 8 were found to inhibit the
growth of dermatophytes with a potency comparable to that of
naftifine. The synthetic sequence includes the preparation of ami-
noazole Schiff bases, reduction, and alkylation of the correspond-
ing secondary amines.
This approach might lead to a mechanistically ideal drug in
that it might inhibit two enzymes in a single linear biosyn-
thetic pathway. This property, usually achieved only by drug
combination, results in a synergy which enhances activity and
makes it more difficult for pathogens to acquire resistance by
alteration of the target.
Chemistry
Tertiary amines 3–8 were prepared starting from the secon-
dary amines 9–11 and the appropriate alkyl bromides. Tria-
zole derivatives 4, 5, 7, and 8 were obtained in anhydrous
tetrahydrofuran in the presence of sodium hydride. Imidazole
amine 9 did not react under the same conditions. Therefore
potassium tert-butoxide/18-crown-6 in anhydrous diethyl
ether was used in this case. Secondary amines 9–11 were
produced by reduction of the corresponding Schiff bases
12–14 with sodium borohydride in methanol. Schiff base 12
was synthesised by adding imidazole to a neutral solution of
hydroxylamine-O-sulfonic acid in water. The crude residue
obtained after acidification and elimination of the water was
subsequently treated with a solution of naphthalene-1-carbal-
dehyde in ethanol.
Introduction
Only one target, the cytochrome P-450 dependent lano-
sterol C-14α-demethylase, can be considered a major success
in antifungal chemotherapy. Inhibitors of this enzyme, the
imidazole and triazole antimycotics, include the most impor-
tant and successful of today’s antifungal drugs endowed with
clinical efficacy. On the other hand, an enzyme at the very
early stage in the pathway of ergosterol synthesis, squalene
epoxidase, is the target of two antimycotics of the allylamine
and benzylamine groups, namely naftifine (1) and butenafine
[1]
(2)
.
When 1H-1,2,4-triazole was used instead of imidazole, a
mixture of 4H-1,2,4-and 1H-1,2,4-triazole derivatives 13 and
14 was obtained and chromatographic separation of isomers
was necessary.
In the present work we report the synthesis of novel struc-
tural analogues of naftifine and butenafine, in which the
methyl moiety has been substituted with an azolic nucleus.
Attempts to obtain tertiary amines 6–8 by reduction of the
amides 15–17 by various methods were unsuccessful. Amine
6, however, could be obtained (yield: 10%) by reduction of
4-tert-butylbenzamide 15 with lithium borohydride in the
[2]
presence of chlorotrimethylsilane . Deacylated amine 9 and
4-tert-butylbenzyl alcohol were also recovered.
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/0606/0162 $17.50 +.50/0

Azole Antifungal Agents
163
Table 1. Antimycotic activity of compounds 3–8 against dermathophytes.
——————————————————————————————
Compd. aCC50
MT-4
.
bMIC
T. rubrum
T. verrucosum
M. gypseum
——————————————————————————————
3
4
5
6
7
8
>200
51
200
100
100
>200
>200
>200
59
6 (10)
3 (9)
>200
3 (20)
3 (9)
>200
6 (10)
3 (9)
>200
28
37
154
3 (51)
6 (26)
3 (51)
Amides 15–17 were prepared in anhydrous tetrahydrofuran
from secondary amines 9–11 and 4-tert-butyl-benzoyl chlo-
ride in the presence of triethylamine.
Naftifine >200
2.4 (>83)
0.15 (120)
7.4 (>27)
0.15 (120)
2.4 (>83)
1.2 (15)
Micon-
azole
18
——————————————————————————————
a)
Results
Compound concentration (µM) required to reduce the viability of MT4
cells by 50%.
The title compounds were evaluated in vitro against several
pathogenic fungi responsible for human disease. Test patho-
gens included representatives of yeasts (Candida albicans,
Candida parapsilosis, Criptococcus neoformans), dermato-
phytes (Tricophyton verrucosum, Tricophyton rubrum, Mi-
crosporum gypseum), and molds (Aspergillus fumigatus).
Naftifine and miconazole were used as reference drugs. The
title compounds were also tested for antibacterial activity
against representative Gram positive and negative bacteria
and for in vitro cytotoxicity in a lymphoid cell line (MT-4).
Cytotoxicity evaluation was performed in order to determine
whether test compounds were endowed with selective antimi-
crobial activity.
All test compounds were inactive against yeasts (MIC >
200 µM). In the same tests, the azole antifungal miconazole
showed a good activity against tested strains (MICs = 1.2–
5.0 µM) and the allylamine naftifine was active only against
Candida parapsilosis (MIC = 12.0 µM).
On the other hand, imidazole derivative6 and 1H-1,2,4-tria-
zole derivatives 5, 8 inhibited the growth of dermatophytes
similarly to naftifine, the imidazole analogous 3 of naftifine
was moderately active, whereas the 4H-1,2,4-triazole deriva-
tives 4 and 7 were inactive (Table 1).
b) Minimum inhibitory concentration (µM). Values in parenthesis represent
the CC50/MIC ratio (selectivity index). Data represent mean values from
three independent determinations.
In the attempt to improve the antimicotic effect of naftifine
and butenafine, the methyl group was replaced with an azolic
ring, to obtain a simultaneous effect on both fungal 14α-de-
methylase and squalene epoxidase enzymes.
Interestingly, derivatives 5, 6, and 8 showed an antifungal
activity against dermatophytes comparable to that of nafti-
fine, although a systematic variation of individual structural
elements in naftifine had demonstrated that substitution of
methyl group seems to be limited to the N-ethyl analogue,
which is still highly active in vitro and in vivo. The N-cyclo-
hexyl, the N-phenyl, and the N-benzyl analogues were com-
pletely devoid of activity
[4]
.
Although showing an activity restricted to dermatophytes,
the title compounds seem to act similarly to the azole antifun-
gals. This hypothesis is supported by the observation that
4H-1,2,4-triazole derivatives 4 and 7 were inactive. Triazole
compounds in fact are reported to inhibit the binding of the
natural substrate lanosterol to demethylase by coordination
of the unhindered N-4 ring nitrogen atom to the sixth coordi-
nation position of the iron atom of the enzyme protoporphyrin
The presence of a carbonyl group in the amide 15 and 17
abates activity (MIC > 200 µM). All compounds were inac-
tive when used against the mold strain tested (Aspergillus
fumigatus) and the representative of Gram positive (Staphy-
[5]
system . Furthermore, the introduction of a carbonyl group
completely abates the activity in the amides 15 and 17. On
lococcus aureus) and Gram negative (Salmonella spp.) bac- the other hand, the 1-naphthamide obtained from naftifine
teria.
When evaluated in vitro for antiproliferative activity (Ta-
ble 1), compounds 3–8 inhibited the growth of MT-4 cells
modified at the amino group, displayed a good activity in
[4]
vitro and to a somewhat lesser extent in vivo
.
It is noteworthy that the imidazole analogue 3 of naftifine
was less active than the triazole 5. According to some authors
, the triazole ring should show increased specificity for
with a range of CC in between that of miconazole (18 µM)
and that of naftifine (>200 µM).
50
[6]
fungal enzymes and apparent increased potency (itraconazole
versus ketoconazole). However, more recently, the use of
comparative molecular field analysis in the molecular mod-
elling of azole antifungals revealed the preference of an
imidazole over a triazole group in the active site. This result
was also supported by the observed in vitro inhibitory activity
Discussion
Replacement of 3-phenyl-2-propenyl moiety in the nafti-
fine structure with a 4-tert-butylbenzyl group have led to
butenafine that exhibits enhanced activity against a range of
values, which consistently show that imidazole derivatives
human pathogenic fungi, but is moderately effective against
[3]
[7]
Candida species
.
are more active than the triazoles
.
Arch. Pharm. Pharm. Med. Chem. 333, 162–166 (2000)

164
Moreover, we assume that the title compounds did not give
synergistic effects because the azolyl group that replaced the
methyl group in naftifine and butenafine may be too hindered
to fit properly into the active site of the squalene epoxidase.
Our efforts to define the importance of the N-azolylamine
portion in antimycotic chemotherapy are the subject of fur-
ther investigations.
Castellano, La Colla, Musiu, and Stefancich
N-(Naphthalen-1-ylmethylene)-4H-1,2,4-triazol-4-amine (13)
Yield 13%, mp 143 °C (ethanol) (ref.[8]142–143 °C). 1H-NMR: 7.55–7.75
(m, 3H, aromatic H), 7.90–8.11 (m, 3H, aromatic H), 8.72 (s, 2H, H triazole),
8.75–8.85 (m, 1H, aromatic H), 9.17 (s, 1H, CHN). Anal. (C13H10N4) C, H,
N.– MS; m/z = 222 [M+].
N-(Naphthalen-1-ylmethylene)-1H-1,2,4-triazol-1-amine (14)
Yield 13%, mp 89 °C (petroleum ether). 1H-NMR: 7.54–7.74 (m, 3H,
aromatic H), 7.90–8.17 (m, 4H, aromatic H and 5-H triazole), 8.49 (s, 1H,
3-H triazole), 8.77–8.86 (m, 1H, aromatic H), 9.84 (s, 1H, CHN). Anal.
(C13H10N4) C, H, N.– MS; m/z = 222 [M+].
Acknowledgements
MURST (Ministero dell’Università e della Ricerca Scientifica e Tec-
nologica) is gratefully acknowledged for financial support (Programmi di
ricerca di rilevante interesse nazionale - cofinanziamento 1997).
General Procedure for the Preparation of Secondary Amines 9–11
A cooled solution of appropriate Schiff base (50.0 mmol) in methanol
(200 ml) was treated portionwise with an excess of sodium borohydride
powder until the reaction was completed (TLC), then the solvent was
removed under reduced pressure. The residue was taken up with water
(200 ml) and extracted with chloroform. The organic phase was dried with
sodium sulfate and the solvent was evaporated to give a solid which was
recrystallized.
Experimental
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 and were within ±0.4% of the calculated values. 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 using deuteriochloroform as solvent.
Chemical shifts are given in δ values downfield from tetramethylsilane as
internal standard; coupling constants are given in Hertz. Mass spectra data
were determined on a V6-Micromass 7070H mass spectrometer. Silica gel
chromatography was performed using Merck silica gel 60 (0.015–
0.040 mm); alumina chromatography was performed using Merck alu-
minium oxide 90 (0.063–0.200 mm). Petroleum ether refers to petroleum
ether (40–60 °C).
N-(1H-Imidazol-1-yl)-N-(naphthalen-1-ylmethyl)-amine (9)
Yield 99%, mp 102–103 °C (toluene-petroleum ether). 1H-NMR: 4.70 (d,
J = 5.0 Hz, 2H, CH2), 5.05 (t, J = 5.0 Hz, 1H, exchanges with deuterium
oxide, NH), 6.96 (s, 2H, 5-H and 4-H imidazole), 7.24–8.20 (m, 8H, aromatic
H and 2-H imidazole). Anal. (C14H13N3) C, H, N.– MS; m/z = 223 [M+].
N-(Naphthalen-1-ylmethyl)-4H-1,2,4-triazol-4-amine (10)
Yield 99%, mp 173–175 °C (ethanol) (ref. [9] 171 °C). 1H-NMR: 4.69 (d,
J = 5.0 Hz, 2H, CH2), 5.56 (t, J = 5.0 Hz, 1H, exchanges with deuterium
oxide, NH), 7.16–7.67 (m, 4H, aromatic H), 7.80–7.95 (m, 2H, aromatic H),
7.99 (s, 2H, H triazole), 8.03–8.16 (m, 1H, aromatic H). Anal. (C13H12N4)
C, H, N.– MS; m/z = 224 [M+].
N-(Naphthalen-1-ylmethyl)-1H-1,2,4-triazol-1-amine (11)
N-(1H-Imidazol-1-yl)-N-(naphthalen-1-ylmethylene)-amine (12)
Yield 99%, mp 88–89 °C (cyclohexane) 1H-NMR: 4.81 (d, J = 4.4 Hz, 2H,
CH2), 5.47 (t, J = 4.4 Hz, 1H, exchanges with deuterium oxide, NH), 7.23–
7.97 (m, 8H, aromatic H, 5-H triazole and 3-H triazole), 8.20–8.30 (m, 1H,
aromatic H). Anal. (C13H12N4) C, H, N.– MS; m/z = 224 [M+].
A solution of hydroxylamine-O-sulfonic acid (11.3 g, 100 mmol) in water
(100 ml) was made neutral with sodium hydrogen carbonate 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 naphthalene-1-carbaldehyde (15.6 g,
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). The organic phase was eliminated while the aqueous
solution was rendered basic with sodium hydrogen carbonate and extracted
with chloroform (3×100 ml). The combined extracts were dried with sodium
sulfate, the chloroform was evaporated off, and the residue was crystallized
from cyclohexane. Yield 38%, mp 87–88 °C. 1H-NMR: 7.22 (s, 1H, 5-H
imidazole), 7.51–8.07 (m, 8H, aromatic H, 4-H and 2-H imidazole), 8.73–
8.82 (m, 1H, aromatic H), 9.08 (s, 1H, CHN). Anal. (C14H11N3) C, H, N.–
MS; m/z = 221 [M+].
General Procedure for the Preparation of Tertiary Amines 3 and 6
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 diethyl ether (50 ml), then N-(1H-imida-
zol-1-yl)-N-(naphthalen-1-ylmethyl)-amine (9) (4.47 g, 20.0 mmol) was
added in a single portion under stirring. After stirring for 15 min at room
temperature under nitrogen, a solution of the opportune bromide (20.0 mmol)
in diethyl ether (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 a
saturated sodium chloride solution, dried over sodium sulfate and concen-
trated under reduced pressure. The residue was purified by chromatography.
N-(1H-Imidazol-1-yl)-N-(naphthalen-1-ylmethyl)-(3-phenylallyl)-amine (3)
Procedure for the Preparation of N-(Naphthalen-1-ylmethylene)-
1,2,4-triazolamine 13 and 14
Chromatography: silica (chloroform). Yield 61%, oil. 1H-NMR: 3.91 (d,
J = 7.0 Hz, 2H, CH2CH), 4.58 (s, 2H, CH2Ar), 6.21 (dt, J = 15.8/7.0 Hz, 1H,
CH2CH), 6.52 (d, J = 15.8 Hz, 1H, CHAr), 6.95 (s, 1H, 5-H imidazole),
7.13–7.89 (m, 13H, aromatic H, 4-H imidazole and 2-H imidazole), 8.22–
8.29 (m, 1H, aromatic H). Anal. (C23H21N3) C, H, N.– MS; m/z = 339 [M+].
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 rendered basic with sodium
hydrogen carbonate and extracted with chloroform (3×100 ml). The com-
bined extracts were dried with sodium sulfate, the chloroform was evapo-
rated, and the residue was separated by alumina column chromatography
(chloroform-petroleum ether 1:1). First eluates were discarded, then com-
pound 14 and subsequently compound 13 were recovered.
N-(1H-Imidazol-1-yl)-N-(naphthalen-1-ylmethyl)-(4-tert-butylbenzyl)-
amine (6)
From N-(1H-imidazol-1-yl)-N-(naphthalen-1-ylmethyl)-amine (9), as re-
ported in the General Procedure.
Arch. Pharm. Pharm. Med. Chem. 333, 162–166 (2000)

Azole Antifungal Agents
165
Chromatography: alumina (cyclohexane-chloroform 2:8). Yield 46%, mp
126 °C (benzene-petroleum ether). 1H-NMR: 1.29 (s, 9H, C(CH3)3), 4.24 (s,
2H, CH2), 4.56 (s, 2H, CH2), 6.90 (s,1H, 5-H imidazole), 7.01 (s, 1H, 4-H
imidazole), 7.10–8.14 (m, 12H, 2-H imidazole and aromatic H). Anal.
(C25H27N3) C, H, N.– MS; m/z = 369 [M+].
General Procedure for the Preparation of Amides 15–17
A solution of 4-tert-butyl-benzoyl chloride (2.16 g, 11.0 mmol) in anhy-
drous tetrahydrofuran (30 ml) was added to a solution of secondary amine
(10.0 mmol) and triethylamine (1.11 g, 11.0 mmol) in the same solvent
(60 ml), then the mixture was refluxed for 4 h. After cooling, the mixture was
filtered, the filtrate was evaporatedand the residue dissolved with chloroform
(100 ml). The organic layer was washed with a saturated sodium chloride
solution, dried with anhydrous sodium sulfate and concentrated. After puri-
fication by alumina chromatography (chloroform as eluent) the amides were
recrystallized from suitable solvent.
From N-(1H-imidazol-1-yl)-N-(naphthalen-1-ylmethyl)-(4-tert-butyl)-
benzamide (15)
To a solution of lithium borohydride in tetrahydrofuran (8.0 ml, 2 M
solution, 16.0 mmol) was added a solution of chlorotrimethylsilane (3.48 g,
32.0 mmol) in anhydrous tetrahydrofuran (10 ml). After 2 min, a solution of
N-(1H-imidazol-1-yl)-N-(naphthalen-1-ylmethyl)-4-tert-butyl)-benzamide
(15) (1.53 g, 4.00 mmol) in anhydrous tetrahydrofuran (30 ml) was added
dropwise over a period of 5 min and the reaction mixture was stirred for 24 h
at room temperature. The reaction was treated cautiously with methanol
(10 ml) and the solvent was evaporated under reduced pressure. The residue
was taken up with chloroform and the resulting inorganic precipitate was
filtered. The filtrate was then concentrated under reduced pressure and the
residue was separated by alumina column chromatography (cyclohexane-
chloroform 4:6). First eluates gave 4-tert-butylbenzyl alcohol, then com-
pound 6 (yield 10%) and after desacylamine 9 were recovered.
N-(1H-Imidazol-1-yl)-N-(naphthalen-1-ylmethyl)-(4-tert-butyl)-benzamide
(15)
Yield 97%, mp 197 °C (ethanol). 1H-NMR: 1.21 (s, 9H, C(CH3)3); 5.54
(s, 2H, CH2); 6.51 (s, 1H, 5-H imidazole); 6.74 (s, 1H, 4-H imidazole); 6.79
(s, 1H, 2-H imidazole); 7.06–8.22 (m, 11H, aromatic H). Anal. (C25H25N3O)
C, H, N.– MS; m/z = 383 [M+].
General Procedure for the Preparation of Tertiary Amines 4, 5, 7, and 8
N-(Naphthalen-1-ylmethyl)-N-(4H-1,2,4-triazol-4-yl)-(4-tert-butyl)-benz-
amide (16)
Sodium hydride (0.96 g, 40.0 mmol) was added to a solution of the
opportune triazole amine (4.49 g, 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 bromide (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
(100 ml) and extracted with chloroform (3×50 ml). The combined organic
solution was washed with a saturated sodium chloride solution and dried with
sodium sulfate. The solvent was removed by evaporation under reduced
pressure and the residue was purified by chromatography.
Yield 38%, mp 197 °C (ethanol). 1H-NMR: 1.22 (s, 9H, C(CH3)3); 5.54
(s, 2H, CH2); 7.02–8.12 (m, 13H, aromatic H and H triazole). Anal.
(C24H24N4O) C, H, N.– MS; m/z = 384 [M+].
N-(Naphthalen-1-ylmethyl)-N-(1H-1,2,4-triazol-4-yl)-(4-tert-butyl)-benz-
amide (17)
Yield 77%, mp 145 °C (ethanol). 1H-NMR: 1.20 (s, 9H, C(CH3)3); 5.60
(br. s, 2H, CH2); 6.85 (s, 1H, 5-H triazole); 7.05–8.20 (m, 12H, aromatic H
and 3-H triazole). Anal. (C24H24N4O) C, H, N.– MS; m/z = 384 [M+].
N-(Naphthalen-1-ylmethyl)-N-(3-phenylallyl)-4H-1,2,4-triazol-4-amine (4)
Chromatography: alumina (cyclohexane-chloroform 1:1). Yield 79%, mp
131–132 °C (benzene-ligroin). 1H-NMR: 3.95 (d, J = 6.9 Hz, 2H, CH2CH),
4.66 (s, 2H, CH2Ar), 6.24 (dt, J = 15.8/6.9 Hz, 1H, CH2CH), 6.56 (d,
J = 15.8 Hz, 1H, CHAr), 7.10–7.38 (m, 7H, aromatic H), 7.49–7.65 (m, 2H,
aromatic H), 7.78–7.91 (m, 2H, aromatic H), 8.10 (s, 2H, H triazole),
8.14–8.22 (m, 1H, aromatic H). Anal. (C22H20N4) C, H, N.– MS; m/z = 340
[M+].
Microbiology
Compounds. Test compounds were dissolved in DMSO at an initial
concentration of 200 mM and then were serially diluted in culture medium.
Cells. Cell line were from American Type Culture Collection (ATCC);
bacterial and fungal strains were either clinical isolates (obtained from
Clinica Dermosifilopatica, University of Cagliari) or collection strains from
ATCC.
Antibacterial Assays. Staphylococcus aureus, group D Streptococcus,
Shighella, and Salmonella spp. were recent clinical isolates. Tests were
carried out in nutrient broth, pH 7.2, with an inoculum of 103cells/tube. MICs
were determined after 18 h incubation at 37 °C in the presence of serial
dilutions of the test compounds.
N-(Naphthalen-1-ylmethyl)-N-(3-phenylallyl)-1H-1,2,4-triazol-1-amine (5)
Chromatography: alumina (cyclohexane-chloroform 7:3). Yield 71%, mp
77–78 °C (ligroin). 1H-NMR: 4.08 (d, J = 6.9 Hz, 2H, CH2CH), 4.70 (s, 2H,
CH2Ar), 6.21 (dt, J = 15.8/6.9 Hz, 1H, CH2CH), 6.53 (d, J = 15.8 Hz, 1H,
CHAr), 7.08–7.38 (m, 8H, aromatic H and 5-H triazole), 7.46–7.64 (m, 2H,
aromatic H), 7.70–7.90 (m, 3H, aromatic H and 3-H triazole); 8.20–8.28 (m,
1H, aromatic H). Anal. (C22H20N4) C, H, N.– MS; m/z = 340 [M+].
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
broth using a glass homogenizer. Glicerol, 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
using Sabouraud dextrose broth (pH 5.7) and incubating at 37 °C. The growth
control for yeast was read after 1 day and for dermatophytes after 3 days
(5 days for Cryptococcus neoformans). The MIC was defined as the com-
pound 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.
N-(4-tert-Butylbenzyl)-N-(naphthalen-1-ylmethyl)-4H-1,2,4-triazol-
4-amine (7)
Chromatography: alumina (cyclohexane-chloroform 4:6). Yield 92%, mp
152 °C (toluene-cyclohexane). 1H-NMR: 1.28 (s, 9H, C(CH3)3), 4.32 (s, 2H,
CH2), 4.64 (s, 2H, CH2), 7.08–7.61 (m, 8H, aromatic H), 7.74–8.08 (m, 5H,
aromatic H and H triazole). Anal. (C24H26N4) C, H, N.– MS; m/z = 370 [M+].
N-(4-tert-Butylbenzyl)-N-(naphthalen-1-ylmethyl)-1H-1,2,4-triazol-
4-amine (8)
Chromatography: alumina (cyclohexane-chloroform 1:1). Yield 85%, mp
107 °C (petroleum ether). 1H-NMR: 1.28 (s, 9H, C(CH3)3), 4.41 (s, 2H,
CH2), 4.72 (s, 2H, CH2), 7.08–7.34 (m, 7H, aromatic H and 5-H triazole),
7.44–7.62 (m, 2H, aromatic H), 7.70 –7.90 (m, 3H, aromatic H and 3-H
triazole), 8.08–8.18 (m, 1H, aromatic H). Anal. (C24H26N4) C, H, N.– MS;
m/z = 370 [M+].
The cytotoxicity evaluation of compounds was based on the viability of
mock-infected cells, as monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT) method [10]
.
Arch. Pharm. Pharm. Med. Chem. 333, 162–166 (2000)

166
Castellano, La Colla, Musiu, and Stefancich
[6] S. M. Grant, S. P. Clissold, Drugs 1989, 37, 310–344.
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Received: February 11, 2000 [FP456]
Arch. Pharm. Pharm. Med. Chem. 333, 162–166 (2000)