Synthesis and biological evaluation of azole derivatives, analogues of bifonazole, with a phenylisoxazolyl or phenylpyrimidinyl moiety

Article abstract of DOI:10.1016/S0014-827X(01)01117-X

A series of azole derivatives, isoxazole or pyrimidine analogues of the antifungal drug bifonazole, were synthesized and tested in vitro against representative human pathogenic fungi (Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus). They were also evaluated as antibacterial agents against Staphylococcus aureus and Salmonella spp. Only 5-(imidazol-1-yl-phenylmethyl)-2,4-diphenyl-pyrimidine 7c showed weak antimicrobial activity (MIC=66 μM) against C. albicans, C. neoformans and S. aureus. Results of biological tests proved, therefore, that replacement of the biphenyl portion of the bifonazole with a phenylisoxazolyl or phenylpyrimidinyl moiety is not profitable for antimicrobial properties. Copyright

Full text of DOI:10.1016/S0014-827X(01)01117-X

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 633–640
Synthesis and biological evaluation of azole derivatives, analogues
of bifonazole, with a phenylisoxazolyl or phenylpyrimidinyl moiety
Giulia Menozzi a,*, Luisa Mosti a, Paola Fossa a, Chiara Musiu b, Chiara Murgioni b,
Paolo La Colla b
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Geno6a, Viale Benedetto XV 3, I-16132 Geno6a, Italy
b Dipartimento di Biologia Sperimentale, Uni6ersita` di Cagliari, Viale Regina Margherita 45, I-09124 Cagliari, Italy
Received 13 February 2001; accepted 8 March 2001
Abstract
A series of azole derivatives, isoxazole or pyrimidine analogues of the antifungal drug bifonazole, were synthesized and tested
in vitro against representative human pathogenic fungi (Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus).
They were also evaluated as antibacterial agents against Staphylococcus aureus and Salmonella spp. Only 5-(imidazol-1-yl-phenyl-
methyl)-2,4-diphenyl-pyrimidine 7c showed weak antimicrobial activity (MIC=66 mM) against C. albicans, C. neoformans and S.
aureus. Results of biological tests proved, therefore, that replacement of the biphenyl portion of the bifonazole with a
phenylisoxazolyl or phenylpyrimidinyl moiety is not profitable for antimicrobial properties. © 2001 Elsevier Science S.A. All
rights reserved.
Keywords: Azole derivatives; Antifungal agents; Bifonazole analogues
potency and selectivity. In particular, bioisosteric re-
placements of either or both benzene rings, constituting
the biphenyl portion of the molecule, have been studied
[2].
In a previous paper [3], we reported the synthesis and
biological activity of pyrazole analogues (II) of bifona-
zole, in which the biphenyl portion was replaced with a
phenylpyrazolyl moiety, bearing an aliphatic or aro-
matic substituent at C-5 of the heterocyclic ring (Fig.
1).
1. Introduction
The continuously changing epidemiology of invasive
fungal infections results in the need for an expanded
armamentarium of antifungal therapies. On the other
hand, the increased use of antimicrobial agents in re-
cent years has caused the development of resistance to
available drugs. For these reasons, a constant effort
toward the synthesis of new antifungal agents has been
made in the last few years.
In pursuing our research in the area of heterocyclic
analogues of bifonazole, we planned the synthesis of
new azole derivatives, containing an isoxazole (III) or a
pyrimidine nucleus (IV).
In support of our approach, the literature reported
some isoxazolecarboxanilides [4] and phenylisoxazole
derivatives [5] endowed with fungicidal activity. More-
over, pyrimidine nucleus is a basic component of well-
known chemotherapeutic drugs, like sulfanylamides,
pyrimethamine, trimethoprim and 5-fluorocytosine, and
it is also present in the structure of voriconazole [6], a
new azole drug active against resistant yeasts and
In particular, the class of azoles (imidazole and tria-
zole derivatives) has supplied many effective antifungal
drugs currently in clinical use, and newer azoles with
expanded spectrum of activity are at the moment in
continuous development [1].
After the discovery of bifonazole (I), an imidazole
derivative used currently for topic therapy of mycoses
and dermatomycoses, many modifications were carried
out on its structure so as to improve its antifungal
* Corresponding author.
E-mail address: menozzi@unige.it (G. Menozzi).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01117-X

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G. Menozzi et al. / Il Farmaco 56 (2001) 633–640
Fig. 1. Bifonazole (I), pyrazole (II), isoxazole (III) and pyrimidine (IV) analogues; X=H, N.
molds, which is in phase III of clinical trials. Finally,
natural compounds with antifungal properties,
bacimethrin [7] and blasticidin S [8], are pyrimidine
derivatives.
In this paper we present the synthesis and biological
evaluation of some bifonazole-like azole derivatives (3,
4, 7b–g, 10b,c,e): besides the antifungal activity, both
antibacterial and anti-human immunodeficiency virus-1
(HIV-1) properties were tested.
(38–82%), imidazole derivatives 3 and 7d–g, respec-
tively; 6a–c, in the same reaction conditions, did not
give the desired compounds, but gave intermediate
imidazole-N-carboxylic esters (8a,c) or a carbonic di-
ester (9), not behaving as benzyl alcohols should [11].
Therefore, in order to obtain the required imidazole
derivatives from 6a–c, we followed another route:
treatment of carbinols with PBr3 and successive reac-
tion of crude bromo derivatives with imidazole in the
presence of triethylamine. Finally 6b,c gave, although
in low yields (17 and 25%, respectively), the imidazole
derivatives 7b,c, whereas 6a did not afford the desired
compound.
2. Chemistry
By analogous reaction of carbinols 2, 6b,c with 1,2,4-
triazole via bromo derivatives, were obtained, in very
low yields (17, 7 and 15%, respectively), triazoles 4,
10b,c; chlorination of 6e with SOCl2 and successive
reaction of crude chloro derivative with 1,2,4-triazole in
the presence of triethylamine afforded 10e in 29% yield.
No effort was carried out to transform carbinols 6f,g
into the corresponding triazole derivatives owing to the
The title azole derivatives were prepared from isoxa-
zole carbinol 2 or pyrimidine carbinols 6a–g, as de-
picted in Schemes 1 and 2. 2 and 6a–g were obtained
by a sodium borohydride reduction of the known ke-
tones 1 [9], 5a–d [10] and new ketones 5e–g, which
were prepared similar to 5a–d.
The reaction of carbinols 2 and 6d–g with N,N%-car-
bonyldiimidazole (CDI) afforded, with variable yields
Scheme 1.

G. Menozzi et al. / Il Farmaco 56 (2001) 633–640
635
Scheme 2.
easy cleavage of the ether function by the required
halogenation agents, while all attempts made starting
from 6a,d were unsuccessful.
MHz); chemical shifts are reported as l (ppm) relative
to TMS as internal standard (J in Hz). Elemental
analyses were performed using a Carlo Erba Elemental
Analyzer Model 1106 and results were within 90.3%
of the calculated values.
3. Experimental
3.1.1. Phenyl(2-substituted
3.1. Chemistry
4-phenylpyrimidin-5-yl)methanones (5e–g)
5e–g were prepared as described previously for 5a–d
[10], namely by cyclization of 2-dimethylamino-
methylene-1,3-diphenylpropane-1,3-dione with 1,1-
dimethylguanidine or O-methylisourea or S-methyliso-
thiourea, respectively.
Melting points were determined with a Fisher–Johns
apparatus and are uncorrected. IR spectra were regis-
tered on a Perkin–Elmer 398 spectrophotometer and
1
are expressed in cm−1. H NMR spectra were regis-
tered on a Hitachi Perkin–Elmer R-600 instrument (60

636
G. Menozzi et al. / Il Farmaco 56 (2001) 633–640
5e: Yield 88%, m.p. 138–139 °C from 95% ethanol.
give a liquid residue which was purified by bulb-to-bulb
distillation in vacuo (b.p. 177–180 °C (0.2)), followed
by crystallization from anhydrous diethyl ether–
petroleum ether (b.p. 40–70 °C), m.p. 69–70 °C, yield
1.85 g (74%). IR (CHCl3, cm−1): w(OH) 3580 and 3370.
1H NMR (CDCl3): l 3.36 (d, 1H, J=3.5 Hz, OH;
disappears with D2O), 5.97 (d, 1H, J=3.5 Hz, CHO;
becames s with D2O), 7.2–7.95 (m, 10H, 2C6H5), 8.10
(s, 1H, H-3). Anal. C16H13NO2 (C, H, N).
1
IR (CHCl3, cm−1): w(CO) 1642. H NMR (CDCl3): l
3.34 (s, 6H, (CH3)2N), 7.1–7.85 (m, 10H, 2C6H5), 8.64
(s, 1H, H-6). Anal. C19H17N3O (C, H, N).
5f: Yield 52%, m.p. 97–98 °C from anhydrous di-
ethyl ether–petroleum ether (b.p. 40–70 °C). IR
1
(CHCl3, cm−1): w(CO) 1660. H NMR (CDCl3): l 4.19
(s, 3H, CH3O), 7.1–7.85 (m, 10H, 2C6H5), 8.74 (s, 1H,
H-6). Anal. C18H14N2O2 (C, H, N).
5g: Yield 77%, m.p. 99–100 °C from anhydrous
diethyl ether. IR (CHCl3, cm−1): w(CO) 1658. 1H NMR
(CDCl3): l 2.69 (s, 3H, CH3S), 7.15–7.9 (m, 10H,
2C6H5), 8.70 (s, 1H, H-6). Anal. C18H14N2OS (C, H,
N).
3.1.3. General procedure for the preparation of
phenyl(2-substituted 4-phenylpyrimidin-5-yl)methanols
(6a–g)
A solution of sodium borohydride (0.38 g, 10 mmol)
in water (6 ml) was added dropwise to a solution of
5a–g (10 mmol) in tetrahydrofuran (220 ml). The mix-
ture was refluxed for 1 h (3 h in the case of 6e), cooled
at r.t. and diluted with water (70 ml). After concentra-
tion to a small volume under reduced pressure, the
solution was extracted with chloroform (3×20 ml).
Organic extracts were washed with water, dried
(MgSO4) and evaporated under reduced pressure to
give a residue, which was purified by chromatography
on Florisil (diethyl ether as eluent), followed by a
recrystallization from a suitable solvent.
3.1.2. Phenyl(5-phenylisoxazol-4-yl)methanol (2)
A solution of sodium borohydride (0.38 g, 10 mmol)
in water (6 ml) was added dropwise to a cooled (0 °C)
solution of phenyl(5-phenylisoxazol-4-yl)methanone (1)
(2.50 g, 10 mmol) in tetrahydrofuran (180 ml). The
mixture was stirred at 0 °C for 1 h and, then, at room
temperature (r.t.) for 3 h. After cooling at 0 °C, water
(60 ml) was added and the solution was stirred for few
minutes, concentrated to a small volume under reduced
pressure and extracted with chloroform (3×20 ml).
Organic extracts were washed with water, dried
(MgSO4) and evaporated under reduced pressure to
1
Yields, m.p. values, recrystallization solvents and H
NMR spectral data are reported in Table 1. IR spectral
Table 1
Yields, physical, analytical and 1H NMR data of methanols 6a–g
Comp.
6a
R
Yield (%)
m.p. (°C)
181–182 a
98–99 b
Molecular
formula
Analyses
1H NMR (CDCl3), l (ppm)
H
78
76
C17H14N2O
C18H16N2O
C, H, N
C, H, N
ꢀ3.6 (very br s, 1H, OH, disappears with D2O), 6.03 (s,
1H, CHꢀPh), 7.1–7.35 (m, 5H, C6H5), 7.47 (s, 5H, C6H5),
8.94 (s, 1H, H-6), 9.10 (s, 1H, H-2)
2.63 (s, 3H, CH3), 4.58 (d, Jꢀ3 Hz, 1H, OH, disappears
with D2O), 5.89 (d, Jꢀ3 Hz, 1H, CHꢀPh, became s with
D2O), 6.95–7.3 (m, 5H, C6H5), 7.39 (s, 5H, C6H5), 8.71 (s,
1H, H-6)
6b
CH3
6c
6d
6e
C6H5
NH2
98
60
138–139 b
186–187 a
95–96 b
C23H18N2O
C17H15N3O
C19H19N3O
C, H, N
C, H, N
C, H, N
3.32 (br s, 1H, OH, disappears with D2O), 6.03 (s, 1H,
CHꢀPh), 7.27 (s, 5H, C6H5), 7.35–7.8 (m, 8H, ArH),
8.35–8.75 (m, 2H, ArH), 8.97 (s, 1H, H-6)
5.3–5.55 (m, 1H, OH, disappears with D2O), 5.6–5.95 (m,
3H, CHꢀPh+NH2, 2H, disappear with D2O), 7.27 (s, 5H,
C6H5), 7.35–7.75 (m, 5H, C6H5), 8.41 (s, 1H, H-6)
2.26 (d, Jꢀ4 Hz, 1H, OH, disappears with D2O), 3.23 (s,
6H, (CH3)2N), 5.96 (d, Jꢀ4 Hz, 1H, CHꢀPh, became s with
D2O), 7.31 (s, 5H, C6H5), 7.4–7.8 (m, 5H, C6H5), 8.40 (s,
1H, H-6)
N(CH3)2 72
6f
OCH3
SCH3
86
81
155–156 a
134–135 b
C18H16N2O2
C18H16N2OS
C, H, N
C, H, N
3.20 (d, Jꢀ4 Hz, 1H, OH, disappears with D2O), 4.02 (s,
3H, CH3O), 6.00 (d, Jꢀ4 Hz, 1H, CHꢀPh, became s with
D2O), 7.1–7.35 (m, 5H, C6H5), 7.4–7.6 (m, 5H, C6H5), 8.63
(s, 1H, H-6)
2.56 (s, 3H, CH3S), 2.95 (br s, 1H, OH, disappears with
D2O), 5.97 (s, 1H, CHꢀPh), 7.0–7.7 (m, 10H, 2C6H5), 8.67
(s, 1H, H-6)
6g
a From ethyl acetate.
b From anhydrous diethyl ether–petroleum ether (b.p. 40–70 °C).

G. Menozzi et al. / Il Farmaco 56 (2001) 633–640
637
Table 2
Yields, physical, analytical and 1H NMR data of imidazoles 7b–g
Comp.
R
Yield (%)
m.p. (°C)
Molecular
formula
Analyses
1H NMR (CDCl3), l (ppm)
7b
7c
7d
CH3
C6H5
NH2
17
25
66
137–138 a
162–163 a
196–197 b
C21H18N4
C26H20N4
C20H17N5
C, H, N
C, H, N
C, H, N
2.82 (s, 3H, CH3), 6.60 (s, 1H, CHꢀPh), 6.75–7.7 (m, 13H,
2C6H5+3H imidazole), 8.43 (s, 1H, H-6 pyrimidine)
6.67 (s, 1H, CHꢀPh), 6.75–7.7 (m, 16H, 13ArH+3H
imidazole), 8.45–8.75 (m, 3H, 2ArH+H-6 pyrimidine)
5.78 (s, 2H, NH2, disappears with D2O), 6.43 (s, 1H,
CHꢀPh), 6.75–7.7 (m, 13H, 2C6H5+3H imidazole), 8.06 (s,
1H, H-6 pyrimidine)
7e
7f
N(CH3)2 82
176–177 c
175–176 b
172–173 a
C22H21N5
C, H, N
C, H, N
C, H, N
3.24 (s, 6H, (CH3)2N), 6.46 (s, 1H, CHꢀPh), 6.8–7.65 (m,
13H, 2C6H5+3H imidazole), 8.10 (s, 1H, H-6 pyrimidine)
4.09 (s, 3H, CH3O), 6.60 (s, 1H, CHꢀPh), 6.8–7.7 (m, 13H,
2C6H5+3H imidazole), 8.31 (s, 1H, H-6 pyrimidine)
2.60 (s, 3H, CH3S), 6.57 (s, 1H, CHꢀPh), 6.8–7.7 (m, 13H,
2C6H5+3H imidazole), 8.29 (s, 1H, H-6 pyrimidine)
OCH3
SCH3
60
38
C21H18N4O
C21H18N4S
7g
a From anhydrous diethyl ether.
b From ethyl acetate.
c From ethyl acetate–petroleum ether (b.p. 40–70 °C).
data are consistent with the proposed structures and are
not reported.
drous diethyl ether–petroleum ether (b.p. 40–70 °C).
1
IR (CHCl3, cm−1): w(CO) 1760. H NMR (CDCl3): l
6.8–7.6 (m, 13H, 2C6H5+3H imidazole), 8.15 (s, 1H,
CHꢀO), 9.02 (s, 1H, H-6 pyrimidine), 9.34 (s, 1H, H-2
pyrimidine). Anal. C21H16N4O2 (C, H, N).
3.1.4. General procedure for the preparation of
4-[1H-imidazol-1-yl-(phenyl)methyl]-5-phenylisoxazole
(3) and 2-substituted 5-[1H-imidazol-1-
yl-(phenyl)methyl]-4-phenylpyrimidines (7d–g)
8c: Yield 3.35 g (78%), m.p. 167–168 °C from anhy-
drous diethyl ether–petroleum ether (b.p. 40–70 °C).
1
IR (CHCl3, cm−1): w(CO) 1760. H NMR (CDCl3): l
N,N%-Carbonyldiimidazole (1.78 g, 11 mmol) was
added to a solution of the proper carbinol 2 or 6a–g
(10 mmol) in dry toluene (120 ml) and the mixture was
refluxed for 4 h. After being cooled, the supernatant
solution was decanted and evaporated under reduced
pressure and the residue was dissolved in chloroform
(100 ml). The organic solution was washed with water
(3×20 ml), dried (MgSO4) and evaporated under re-
duced pressure to give a crude oil, which was purified
by chromatography on Florisil, using diethyl ether (3,
7f,g) or ethyl acetate (7d,e) as eluent. The solid residue,
obtained after evaporation of the solvent, was recrystal-
lized from a suitable solvent.
7.0–7.8 (m, 16H, 13 ArH+3H imidazole), 8.16 (s, 1H,
CHꢀO), 8.4–8.8 (m, 2H, ArH), 9.03 (s, 1H, H-6 pyrim-
idine). Anal. C27H20N4O2 (C, H, N).
9: Yield 1.0 g (35%), m.p. 188–189 °C from anhy-
1
drous diethyl ether. IR (CHCl3, cm−1): w(CO) 1743. H
NMR (CDCl3): l 2.79 (s, 6H, 2CH3), 6.80 (s, 2H,
2CHꢀO), 6.95–7.6 (m, 20H, 4C6H5), 8.82 (s, 2H, 2H-6
pyrimidine). Anal. C37H30N4O3 (C, H, N).
3.1.5. General procedure for the preparation of
2-substituted 5-[1H-imidazol-1-yl-(phenyl)methyl]-
4-phenylpyrimidines (7b,c) and of 2-substituted
4-phenyl-5-[phenyl-(1H-1,2,4-triazol-1-yl)methyl]
pyrimidines (10b,c)
A solution of phosphorous tribromide (1.9 g, 7
mmol) in anhydrous diethyl ether (30 ml) was added
dropwise to a stirred solution of 6b,c (10 mmol) in the
same solvent (200 ml). The mixture was stirred at r.t.
for 2 h, then diluted with chloroform (80 ml) and
further stirred for 2 h. The clear solution so obtained
was washed with 5% CH3COONa and with saturated
NaCl solution, dried (MgSO4) and evaporated under
reduced pressure. The residue was dissolved in dry
acetonitrile (50 ml) and added dropwise into a stirred
solution of the proper azole (10 mmol) and triethy-
lamine (1.0 g, 10 mmol) in the same solvent (20 ml).
3: Yield 1.43 g (48%), m.p. 109–110 °C from anhy-
1
drous diethyl ether. H NMR (CDCl3): l 6.63 (s, 1H,
CHꢀPh), 6.9–7.7 (m, 13H, 2C6H5+3H imidazole),
8.03 (s, 1H, H-3 isoxazole). Anal. C19H15N3O (C, H,
N).
1
Yields, m.p. values, recrystallization solvents and H
NMR spectral data of compounds 7d–g are reported in
Table 2. IR spectral data are consistent with the pro-
posed structures and are not reported.
In the case of carbinols 6a–c, instead of expected
imidazole derivatives, from the reaction mixture were
isolated side compounds which, by analytical and spec-
tral data, resulted to be the imidazole N-carboxylic
esters 8a,c and the carbonic diester 9 (Scheme 2).
8a: Yield 2.08 g (59%), m.p. 107–108 °C from anhy-

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G. Menozzi et al. / Il Farmaco 56 (2001) 633–640
The mixture was heated at 60 °C for 24 h, concentrated
to a small volume under reduced pressure, diluted with
toluene and washed with saturated NaCl solution. The
organic layer was dried (MgSO4) and evaporated under
reduced pressure to give a sticky oil, which was chro-
matographed on silica gel, using first a mixture of ethyl
acetate–petroleum ether (b.p. 40–70 °C) 1:1 and, then,
ethyl acetate, as eluents. The solid residue, obtained
from ethyl acetate eluate, was recrystallized from a
suitable solvent.
10H, 2C6H5), 8.10 (s, 1H, H-3 triazole), 8.15 (s, 1H,
H-5 triazole), 8.29 (s, 1H, H-3 isoxazole). Anal.
C19H15N3O (C, H, N).
3.1.7. N,N-dimethyl-4-phenyl-5-[phenyl(1H-1,2,4-
triazol-1-yl)methyl]pyrimidin-2-amine (10e)
A solution of 6e (3.04 g, 10 mmol) and SOCl2 (11 ml)
was stirred at r.t. for 20 h and then evaporated to
dryness, under reduced pressure. The viscous oily
residue was dissolved in dry acetonitrile (20 ml) and
added dropwise into a stirred solution of 1,2,4-triazole
(0.69 g, 10 mmol) and triethylamine (3.03 g, 30 mmol)
in the same solvent (10 ml). The mixture was stirred at
reflux temperature for 24 h and evaporated under re-
duced pressure to give a residue, which was dissolved in
chloroform. The organic solution was washed with
saturated NaCl solution, dried (MgSO4) and evapo-
rated under reduced pressure. The residue was chro-
matographed on Florisil column (diethyl ether as
eluent). Evaporation of the eluates gave a solid, which
was recrystallized from anhydrous diethyl ether.
Yield, m.p. value and 1H NMR spectral data are
reported in Table 3.
1
Yields, m.p. values, recrystallization solvents and H
NMR spectral data of compounds 7b,c and 10b,c are
reported in Tables 2 and 3, respectively. IR spectral
data are consistent with the proposed structures and are
not reported.
3.1.6. 1-[Phenyl(5-phenylisoxazol-4-yl)methyl]-
1H-1,2,4-triazole (4)
A solution of phosphorous tribromide (1.9 g, 7
mmol) in anhydrous diethyl ether (30 ml) was added
dropwise to a stirred solution of 2 (2.51 g, 10 mmol) in
the same solvent (80 ml). The mixture was stirred at
0 °C for 1 h and at r.t. for 3 h. The solution was then
washed with 5% CH3COONa and with saturated NaCl
solution, dried (MgSO4) and evaporated under reduced
pressure. The white solid residue was dissolved in dry
acetonitrile (30 ml) and added dropwise into a cooled
(0 °C) solution of 1,2,4-triazole (0.69 g, 10 mmol) and
triethylamine (1.0 g, 10 mmol) in the same solvent (20
ml). The mixture was stirred at 0 °C for 3 h and at r.t.
overnight, then concentrated to a small volume under
reduced pressure, diluted with chloroform (50 ml) and
washed with saturated NaCl solution. The organic layer
was dried (MgSO4) and evaporated under reduced pres-
sure to give a yellow oil, which was chromatographed
on silica gel (ethyl acetate–petroleum ether, (b.p. 40–
70 °C), 1:1 as eluent). The solid product, isolated from
the last fractions, was recrystallized from anhydrous
3.2. Biological assays
All compounds were evaluated in vitro for antimicro-
bial activity against representative human pathogenic
fungi (Candida albicans, Cryptococcus neoformans, As-
pergillus fumigatus), Gram positive (Staphylococcus au-
reus) and Gram negative (Salmonella spp.) bacteria.
Bifonazole and streptomycin were used as reference
drugs in antifungal and antibacterial assays, respec-
tively. Test compounds were also evaluated for an-
tiretroviral activity in MT-4 cells infected with HIV-1.
Cytotoxicity against MT-4 cells, carried out in paral-
lel with anti-HIV-1 activity, was evaluated to determine
whether the compounds were endowed with selective
antimicrobial/antiviral activity.
1
diethyl ether, m.p. 139–140 °C, yield 0.51 g (17%). H
NMR (CDCl3): l 6.88 (s, 1H, CH-Ph), 7.0–7.7 (m,
Table 3
Yields, physical, analytical and 1H NMR data of triazoles 10b,c,e
Comp.
10b
R
Yield (%)
m.p. (°C)
122–123 a
220–221 b
145–146 a
Molecular
formula
Analyses
1H NMR (CDCl3), l (ppm)
CH3
C6H5
7
C
20H17N5
C, H, N
C, H, N
C, H, N
2.81 (s, 3H, CH3), 6.84 (s, 1H, CHꢀPh), 6.9–7.7 (m, 10H,
2C6H5), 7.90 (s, 1H, H-3 triazole), 8.05 (s, 1H, H-5 triazole),
8.56 (s, 1H, H-6 pyrimidine)
6.78 (s, 1H, CHꢀPh), 6.9–7.75 (m, 13H, 13ArH), 8.06 (s,
2H, H-3+H-5 triazole), 8.4–8.8 (m, 3H, 2ArH+H-6
pyrimidine)
3.23 (s, 6H, (CH3)2N), 6.73 (s, 1H, CHꢀPh), 6.9–7.65 (m,
10H, 2C6H5), 7.93 (s, 1H, H-3 triazole), 8.05 (s, 1H, H-5
triazole), 8.24 (s, 1H, H-6 pyrimidine)
10c
15
C25H19N5
10e
N(CH3)2 29
C
21H20N6
a From anhydrous diethyl ether.
b From ethyl acetate.

G. Menozzi et al. / Il Farmaco 56 (2001) 633–640
639
3.2.1. Materials and methods
HIV-1 suspension containing 100 CCID50 were then
added. After 4 days of incubation at 37 °C, the number
of viable cells was determined by the 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
method [13,14]. Cytotoxicity of compounds, based on
the viability of mock-infected cells as monitored by the
MTT method, was evaluated in parallel with their
antiviral activity.
3.2.1.1. Compounds. Test compounds were dissolved in
DMSO at an initial concentration of 200 mM and were
then serially diluted in culture medium.
3.2.1.2. Cells. Cell lines were from American Type
Culture Collection (ATCC); bacterial and fungal strains
were either clinical isolates (obtained from Clinica Der-
mosifilopatica, University of Cagliari) or collection
strains from ATCC. H9/IIIB, MT-4 and C8166 cells
(grown in RPMI 1640 containing 10% fetal calf serum
(FCS), 100 UI/ml penicillin G and 100 mg/ml strepto-
mycin) were used for anti-HIV-1 assays. Cell cultures
were checked periodically for the absence of my-
coplasma contamination with a Myco Tect Kit (Gibco).
3.2.1.5. Antibacterial assays. S. aureus and Salmonella
spp. were recent clinical isolates. Assays were carried
out in nutrient broth, pH 7.2, with an inoculum of 103
bacterial cells/tube. Minimum inhibitory concentrations
(MIC) were determined after 18 h incubation at 37 °C
in the presence of serial dilutions of test compounds.
3.2.1.6. Antimycotic assays. Yeast inocula were ob-
tained by properly diluting cultures incubated for 30 h
at 37 °C in Sabouraud dextrose broth to obtain 5×103
cells/ml. On the contrary, dermatophyte inocula were
obtained from cultures grown at 37 °C for 5 days in
Sabouraud dextrose broth by finely dispersing clumps
3.2.1.3. Viruses. HIV-1 IIIB strain was obtained from
supernatants of persistently infected H9/IIIB cells. HIV-
1 stock solutions had a titer of 5×107 cell culture
infectious dose fifty (CCID50)/ml.
3.2.1.4. Anti6iral assays. Activity against the HIV-1
multiplication in cells infected acutely was based on
inhibition of virus-induced cytopathogenicity in MT-4
cells [12]. Briefly, 50 ml of RPMI 10% FCS containing
1×104 cells were added to each well of flat-bottomed
microtiter trays containing 50 ml of medium and serial
dilutions of test compounds. Twenty microliters of an
with a glass homogenizer before diluting to 0.05 OD590/
ml. Then, 20 ml of the above suspensions were added to
each well of flat-bottomed microtiter trays containing
80 ml of medium with serial dilutions of test com-
pounds, and were incubated at 37 °C. Growth controls
were determined visually after 2 days (yeasts) or 3 days
(dermatophytes). MIC was defined as the compound
Table 4
In vitro biological activity of compounds 3, 4, 7b–g, 10b,c,e
MIC c/MGC d
C. albicans
a
b
Comp.
CC50
EC50
MT-4
HIV-1
C. neoformans
A. fumigatus
S. aureus
Salmonella spp.
3
7b
7c
7d
7e
7f
7g
4
76
\200
26
\76
\200
\26
In e
In
66/\200
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
66/\200
66/\200
134
\134
\58.7
\177.5
\61.7
\200
\200
\200
97
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
In
58.7
177.5
61.7
\200
\200
\200
10b
10c
1In0e
In
In
50/100
\97
AZT
Streptomycin
Bifonazole
150
\200
28
0.01
3.1/3.1
6.2/6.2
15/15
3.7/3.7
3.7/3.7
a Compound concentration (mM) required to induce the viability of MT-4 cells by 50%, as determined by the MTT method.
b Compound concentration (mM) required to achieve 50% protection of MT-4 cells from the HIV-1 induced cytopathogenicity, as determined
by the MTT method.
c Minimum inhibitory concentration (mM).
d Minimum germicidal concentration (mM).
e Inactive up to MIC and MGC \200 mM.

640
G. Menozzi et al. / Il Farmaco 56 (2001) 633–640
concentration at which no macroscopic sign of fungal
growth was detected. The minimal germicidal concen-
trations (MBC or MFC) were determined by subculti-
vating samples from cultures with no apparent growth
in Sabouraud dextrose agar.
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lower than the cytotoxic ones.
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Acknowledgements
We would like to thank CNR and the Ministero
dell’Universita` e della Ricerca Scientifica e Tecnologica
for their financial support.
.