Synthesis and biological evaluation of [α-(1,5-disubstituted 1H-pyrazol-4-yl)benzyl]azoles, analogues of bifonazole

Article abstract of DOI:10.1016/S0014-827X(99)00036-1

A series of pyrazole analogues of bifonazole, an antifungal drug used in clinical practice, 2a-h and 4a-h were synthesized and tested in vitro against Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, with no significant results. Imidazoles 2a-h were also tested in vivo for antiarrhythmic and antihypertensive activities; two of these compounds showed moderate activity against ventricular fibrillation caused by aconitine in rats. The above compounds were prepared by reaction of phenyl-[5 substituted 1-phenyl (or 1-methyl)-1H-pyrazol-4-yl]methanols with N,N'-carbonyldiimidazole (2a-h) or of the respective chloro derivatives with 1H-1,2,4-triazole (4a-h).

Full text of DOI:10.1016/S0014-827X(99)00036-1

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 416–422
Synthesis and biological evaluation of [a-(1,5-disubstituted
1H-pyrazol-4-yl)benzyl]azoles, analogues of bifonazole
Giulia Menozzi a,*, Luisa Mosti a, Olga Bruno a, Eleonora Lo Presti a, Chiara Musiu b,
Silvia Longu b, Paolo La Colla b, Walter Filippelli c, Giuseppe Falcone c,
Brunella Piucci c
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Geno6a, Viale Benedetto XV 3, I-16132 Genoa, Italy
b Dipartimento di Biologia Sperimentale, Uni6ersita` di Cagliari, Viale Regina Margherita 45, I-09124 Cagliari, Italy
c Istituto di Farmacologia e Tossicologia, Facolta` di Medicina e Chirurgia, II Uni6ersita` di Napoli, Via S. Andrea delle Dame 8,
I-80138 Naples, Italy
Received 20 November 1998; accepted 10 March 1999
Abstract
A series of pyrazole analogues of bifonazole, an antifungal drug used in clinical practice, 2a–h and 4a–h were synthesized and
tested in vitro against Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, with no significant results. Imidazoles
2a–h were also tested in vivo for antiarrhythmic and antihypertensive activities; two of these compounds showed moderate
activity against ventricular fibrillation caused by aconitine in rats. The above compounds were prepared by reaction of phenyl-
[5 substituted 1-phenyl (or 1-methyl)-1H-pyrazol-4-yl]methanols with N,N%-carbonyldiimidazole (2a–h) or of the respective chloro
derivatives with 1H-1,2,4-triazole (4a–h). © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Azole derivatives; Antifungal agents; Bifonazole analogues; Antiarrhythmic agents
1. Introduction
phenylpyrazolyl moiety with aliphatic or aromatic sub-
stituents on the heterocyclic ring (2a–h) (Fig. 1). Fur-
ther structural modifications involved the replacement
of the imidazole by a triazole ring (4a–h).
In this paper we present the synthesis and biological
evaluation of antimycotic, antibacterial and anti-HIV-1
activities of the above bifonazole analogues as well as
some cardiovascular properties elicited by imidazoles
2a–h.
Since the discovery of bifonazole, an antifungal imi-
dazole derivative developed by Bayer AG and currently
used for topic therapy of mycoses and dermatomycoses,
various attempts have been made to modify its struc-
ture in order to improve its antifungal potency and
selectivity. In particular, the biphenyl portion of the
molecule (Fig. 1) has been subjected to modifications
consisting of bioisosteric replacements of either or both
benzene rings with different aromatic or heteroaromatic
moieties. Among the various derivatives, bifonazole
analogues, in which one of the phenyl rings had been
replaced with a pyrrole nucleus, turned out to be en-
dowed with high antifungal activity in vitro [1–4].
In order to develop new active bifonazole derivatives
and to contribute to the SAR studies of this class, we
synthesized a number of compounds containing a
2. Chemistry
Imidazoles 2a–h and triazoles 4a–h were prepared
from carbinols 1b–g, recently used by us as intermedi-
ates for the synthesis of pyrazole aminoethers with
antinociceptive activity [5], and 1a,h, as described in
Scheme 1.
Compound 1a was synthesized in the same way as
the 5-substituted analogues 1b–g, starting from 4-
formyl-1-phenylpyrazole and phenylmagnesium bro-
* Corresponding author. Tel.: +39-010-353 8361; fax: +39-010-
353 358.
E-mail address: menozzi@unige.it (G. Menozzi)
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00036-1

G. Menozzi et al. / Il Farmaco 54 (1999) 416–422
417
whereas the synthesis of triazoles 4a–h was achieved by
chlorination of 1a–h to 3a–h and subsequent reaction
of the latter with 1H-1,2,4-triazole.
3. Experimental
3.1. Chemistry
Melting points were determined with a Fisher–Johns
apparatus and are uncorrected. The IR spectra were
registered on a Perkin–Elmer 398 spectrophotometer in
CHCl3 as solvent. The 1H NMR spectra were registered
on a Hitachi Perkin–Elmer R-600 instrument (60
MHz); chemical shifts are reported as l (ppm) relative
to TMS as internal standard; J in Hz; CDCl3 as sol-
vent. The elemental analyses were performed using a
Carlo Erba elemental analyser Model 1106 and the
results were within 90.3% of the calculated values.
Fig. 1. Bifonazole and new pyrazole analogues (2a–h: X=CH; 4a–h:
X=N; R=H, alkyl, phenyl).
mide [6]. 1h was obtained by sodium borohydride re-
duction of ketone 5a [7]; similarly the reduction of 5b
[7] provided the same carbinol 1g already prepared by
Grignard’s reaction [5], following an alternative route
(Scheme 1). The correspondence of chemical and physi-
cal characteristics and IR and 1H NMR spectral data of
the two compounds, prepared in two different ways,
gave further confirmation of the supposed structures.
The reaction of carbinols 1a–h with N,N%-car-
bonyldiimidazole afforded imidazole derivatives 2a–h,
3.1.1. Intermediates
Most of the carbinols used as intermediates for the
synthesis of the title compounds were known (1a) [6],
(1b–g) [5].
Scheme 1.

418
G. Menozzi et al. / Il Farmaco 54 (1999) 416–422
3.1.1.1. Phenyl-(1-methyl-5-phenyl-1H-pyrazol-4-yl)-
methanol (1h). A solution of 4-benzoyl-1-methyl-5-
phenyl-1H-pyrazole (2.62 g, 10 mmol) in THF (220 ml)
and water (8 ml) was treated with sodium borohydride
(0.38 g, 10 mmol) and refluxed for 1 h. After cooling to
room temperature, water (60 ml) was added to the
solution and the mixture was stirred for some minutes,
concentrated to a small volume and extracted with
chloroform (3×20 ml). The organic layer was washed
with water, dried (MgSO4), filtered and evaporated to
give a crude residue, which was purified by recrystal-
lization from anhydrous diethyl ether/petroleum ether
(b.p. 40–70°C), m.p. 110–111°C, yield 1.35 g (51%). IR
1
(CHCl3): wmax 3585 and 3270 (OH) cm−1. H NMR
(CDCl3): l 3.47 (d, J=4, 1H, OH; disappears with
D2O), 3.65 (s, 3H, CH3), 5.60 (d, J=4, 1H, CHO;
becomes s with D2O), 7.1–7.6 (m, 11H, 2C6H5+H-3).
Anal. (C, H, N) for C17H16N2O.
3.1.2. General procedure for the synthesis of imidazoles
2a–h
N,N%-Carbonyldiimidazole (1.78 g, 11 mmol) was
added to a solution of the proper carbinol 1b–g (10
mmol) in dry toluene (130 ml) or 1a,h (10 mmol) in dry
benzene (130 ml) and the mixture was refluxed for 4 h.
The solvent was removed under reduced pressure and
the residue was dissolved in chloroform (200 ml). The
organic solution was washed with water (3×50 ml),
dried (MgSO4) and evaporated under reduced pressure
to give crude 2a–h, which were purified by chromatog-
raphy on Florisil (diethyl ether as eluent) and recrystal-
lized from a suitable solvent.
1
Yields, m.p. values, recrystallization solvents and H
NMR spectral data are reported in Table 1. IR spectral
data are consistent with the proposed structures and are
not reported.
3.1.3. General procedure for the synthesis of triazoles
4a–h
A solution of the proper carbinol 1a–h (10 mmol) in
thionyl chloride (20 ml) was stirred at room tempera-
ture for 24 h and then evaporated under reduced pres-
sure. The reddish oily residue obtained was
chromatographed on Florisil, eluting with diethyl ether/
chloroform (1:1) to give a yellow, sticky oil, which was
dissolved in dry acetonitrile (20 ml) and added drop-
wise into a solution of 1H-1,2,4-triazole (0.69 g, 10
mmol) and triethylamine (2.02 g, 20 mmol) in the same
solvent (20 ml). The mixture was refluxed for 24 h and
then evaporated under reduced pressure to give a
brownish oil, which was dissolved in chloroform (200
ml). The organic solution was washed with saturated
NaCl solution (3×30 ml), dried (MgSO4) and evapo-
rated under reduced pressure. The crude residue ob-
tained was purified by recrystallization from a suitable
solvent.

G. Menozzi et al. / Il Farmaco 54 (1999) 416–422
419
1
Yields, m.p. values, recrystallization solvents and H
NMR spectral data are reported in Table 2. IR spectral
data are consistent with the proposed structures and are
not reported.
3.2. Biological assays
All compounds were evaluated in vitro for antimicro-
bial activity against representative human pathogenic
fungi (C. albicans, C. neoformans, A. fumigatus), Gram
positive (Staphylococcus aureus) and Gram negative
(Salmonella spp.) bacteria. Bifonazole and streptomycin
were used as reference drugs in antifungal and antibac-
terial assays, respectively. Test compounds were also
evaluated for antiretroviral activity in MT-4 cells in-
fected 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.
3.2.1. Material and methods
3.2.1.1. Compounds. Test compounds were dissolved in
DMSO at an initial concentration of 200 mM and then
were 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% foetal 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 myco-
plasma contamination with a Myco Tect Kit (Gibco).
3.2.1.3. Viruses. Human immunodeficiency virus type-1
(HIV-1, IIIB strain) was obtained from supernatants of
persistently infected H9/IIIB cells. HIV-1 stock solu-
tions had a titre of 5×107 cell culture infectious dose
fifty (CCID50)/ml.
3.2.1.4. Anti6iral assays. Activity against the HIV-1
multiplication in acutely infected cells was based on
inhibition of virus-induced cytopathogenicity in MT-4
cells [8]. 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. A total of 20 ml of an
HIV-1 suspension containing 100 CCID50 was then
added. After 4 days incubation at 37°C, the number of
viable cells was determined by the 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl-tetrazolium
bromide
(MTT)
method [9,10]. Cytotoxicity of compounds, based on

420
G. Menozzi et al. / Il Farmaco 54 (1999) 416–422
Table 3
Activity of compounds 2a–h against ventricular fibrillation caused by aconitine in rats a
Comp.
Dose (mg/kg p.o.)
Appearance time (s9SE) of extrasystoles
Death time (s9SE)
b
Control (aconitine HCl)
Quinidine
2a
2b
2c
182914.7
359916.3 c
212916.1
224919.7
307911.6 c
251913.7 d
227918.1
198912.5
215913.8
321921.5 c
288915.9 c
241921.3 d
209915.4
209918.2
658921.3
1003911.6 c
698917.1
693922.5
760919.7 c
687916.5 d
661919.3
665915.9
689916.8
739914.7 c
706919.3 c
672920.4 d
685920.2
723918.1 d
25
50
50
50
25
12.5
50
2d
2e
2f
50
50
25
12.5
50
2g
2h
50
a Groups of ten animals (250–300 g) of both sexes, pregnant females excluded.
b 15 mg/kg/i.v./min until death.
c Statistically significant value calculated in comparison with the test performed with aconitine only (PB0.01).
d Statistically significant value calculated in comparison with the test performed with aconitine only (PB0.05).
the viability of mock-infected cells as monitored by the
MTT method, was evaluated in parallel with their
antiviral activity.
3.3. Pharmacology
Preliminary tests on the imidazole derivative 2b (as
hydrochloride) carried out by Panlabs Inc. (Bothell,
WA, USA) have shown significant antiarrhythmic and
hypotensive activities at doses of 100 mg/kg (in in vivo
tests in mice) or at a concentration of 100 mM (in in
vitro tests). Subsequently, the presence of these phar-
macological activities was further investigated at the
Istituto di Farmacologia e Tossicologia (Universita` di
Napoli) on all the imidazoles 2a–h.
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
Antihypertensive and antiarrhythmic (Table 3) activi-
ties were evaluated by standard procedures.
ED50 of the most active compounds were determined
by the administration of three dosages: 12.5, 25 and 50
mg/kg.
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 visually determined after 2 days (yeasts) or 3 days
(dermatophytes). MIC was defined as the compound
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.
4. Results and discussion
4.1. Microbiology
As shown in Table 4, none of the compounds was
capable of protecting MT-4 cells from the cytopathic
effect induced by HIV-1, at least at concentrations
which were lower than the cytotoxic ones.
When tested against yeast and mould representatives,
2d–g were active against C. neoformans at concentra-
tions ranging between 22 and 66 mM; only 2g also
showed activity against C. albicans (MIC=22 mM) and
S. aureus (MIC=66 mM) whereas 4g showed activity
only against S. aureus (MIC=50 mM). Triazoles 4a–f,
h were devoid of antimicrobial activity.
3.2.1.7. Linear regression analysis. Viral growth at each
drug concentration was expressed as a percentage of
untreated controls and the concentration resulting in
50% (EC50) growth inhibition was determined by linear
regression analysis.
In conclusion, replacement of the biphenyl portion
with a phenyl–pyrazolyl moiety afforded bifonazole

G. Menozzi et al. / Il Farmaco 54 (1999) 416–422
421
analogues with low antimycotic activity. Among these
compounds the most active turned out to be 2g, which
is characterized by a pyrazole nucleus bearing a double
aromatic substitution at positions 1 and 5. Among
derivatives in which the second substituent on the hete-
rocycle is an alkyl chain (2b–f), the antifungal activity
was directly related to the presence of a bulky alkyl
radical.
When the imidazole moiety was replaced by a triazole
ring (4a–h), the weak antimycotic activity shown by 2d–g
vanished. This observation is in agreement with the results
of a recent molecular modelling study carried out on azole
antifungal agents active against C. albicans [4].
Preliminary in vitro (at a 100 mM concentration)
and in vivo (at 100 mg/kg i.p. dose on mice) tests
specific for the antiarrhythmic activity, performed on
2b as hydrochloride by Panlabs Inc. (data not re-
ported in this paper) pointed out significant results.
Further investigation of the antiarrhythmic activity
was carried out on rats at lower doses (50, 25 and
12.5 mg/kg) of all derivatives 2a–h. The results are
reported in Table 3. The most active compounds
turned out to be 2c, f, which showed a moderate
protection against ventricular extrasystoles caused by
aconitine, with an ED50 in delaying the appearance of
extrasystoles of 40.78 (32.44–51.25) and 29.55 (24.79–
35.24) mg/kg, respectively. Moreover, the ED50 in
protracting death time resulted \50 mg/kg in both
cases.
4.2. Pharmacology
Preliminary in vitro tests regarding calcium antago-
nism, angiotensin I inhibition, angiotensin II antagonism,
performed by Panlabs Inc. on imidazole 2b as hydrochlo-
ride at a 100 mM concentration (data not reported in this
paper), suggested a possible hypotensive activity. There-
fore all imidazoles 2a–h were later evaluated in vivo on
spontaneously hypertensive rats at the dose of 50 mg/kg
p.o., at the laboratories of Istituto di Farmacologia e
Tossicologia of the University of Napoli, but none of
them showed a significant antihypertensive activity.
The results which emerged from the pharmacological
tests performed on imidazoles 2 discouraged us from also
extending the investigation to their triazole analogues 4.
Acknowledgements
We would like to thank CNR and the Ministero
dell’Universita` e della Ricerca Scientifica e Tecnolog-
ica for their financial support.
Table 4
In vitro biological activity of compounds 2a–h and 4a–h
a
b
Comp.
CC50
EC50
MIC c/MGC d
C. albicans
MT-4
HIV-1
C. neoformans
A. fumigatus
S. aureus
Salmonella spp.
2a
2b
2c
2d
2e
2f
2g
2h
4a
4b
4c
4d
4e
4f
4g
4h
38.2
72.2
53.4
40.3
41.3
30
22.2
56
105.5
162
54.8
55
\38.2
\72.2
\53.4
\40.3
\41.3
\30
\22.2
\56
\105.5
\162
\54.8
\55
In e
In e
In e
In e
In e
In e
22/\200
In e
In e
In e
In e
In e
In e
In e
In e
In e
–
200/200
200/\200
200/200
66/66
66/66
66/66
22/22
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
–
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
In e
–
In e
In e
200/200
200/\200
200/200
66/\200
In e
In e
In e
In e
In e
In e
In e
In e
In e
44
43.5
41
\44
\43.5
\41
In e
In e
In e
In e
In e
50/100
\200
\200
In e
In e
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 MT4 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.

422
G. Menozzi et al. / Il Farmaco 54 (1999) 416–422
aminoalkyl ethers of phenyl-(5-substituted 1-phenyl-1H-pyrazol-
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