Synthesis, antimicrobial activity and molecular modeling studies of halogenated 4-[1H-imidazol-1-yl(phenyl)methyl]-1,5-diphenyl-1H-pyrazoles

Article abstract of DOI:10.1016/j.bmc.2004.07.029

Halogenated diphenylpyrazole analogues of bifonazole were prepared and their microbiological properties were investigated. During the course of our studies in the azole antifungals area, we synthesized a number of 1,5-disubstituted 4-[1H-imidazol-1-yl(phenyl)methyl]-1H-pyrazoles, analogues of bifonazole. 1,5-Diphenyl-1H-pyrazole 3 showed weak antimycotic and antibacterial activities in vitro against Candida albicans, Cryptococcus neoformans and Staphylococcus aureus. In order to increase these properties, given that the halo substitution was found to be capable of enhancing antifungal effects, we prepared a series of fluoro and chloro derivatives of 3. The microbiological evaluation carried out on newly synthesized compounds included in vitro assays for antifungal, antibacterial and antimycobacterial activities. Among the tested compounds, some dichloro and trichloro-derivatives showed interesting antimicrobial properties. In particular, compounds 10j,k,l produced inhibitory effects against pathogen representatives of yeast (C. albicans, C. neoformans) and Gram positive bacteria (S. aureus) similar or superior to those of bifonazole. In addition, their activity against Mycobacterium tuberculosis was superior to that of clotrimazole and econazole, which were used as reference drugs. The replacement, in these compounds, of chlorine with fluorine atoms led to inactive derivatives.Docking studies were carried out on the most active compounds, in order to rationalize the pharmacological results.

Full text of DOI:10.1016/j.bmc.2004.07.029

Bioorganic & Medicinal Chemistry 12 (2004) 5465–5483
Synthesis, antimicrobial activity and molecular modeling studies
of halogenated 4-[1H-imidazol-1-yl(phenyl)methyl]-
1,5-diphenyl-1H-pyrazoles
a
a
a
a
Giulia Menozzi,a, Luisa Merello, Paola Fossa, Silvia Schenone, Angelo Ranise,
*
Luisa Mosti,a Francesco Bondavalli,a Roberta Loddo,b Chiara Murgioni,b
Valeria Mascia,b Paolo La Collab, and Elena Tamburini
b
*
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
bDipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche,
`
Universita di Cagliari, S.S. 554, 09042 Monserrato (CA), Italy
Received 19 March 2004; accepted 13 July 2004
Available online 1 September 2004
Abstract—During the course of our studies in the azole antifungals area, we synthesized a number of 1,5-disubstituted 4-[1H-imid-
azol-1-yl(phenyl)methyl]-1H-pyrazoles, analogues of bifonazole. 1,5-Diphenyl-1H-pyrazole 3 showed weak antimycotic and anti-
bacterial activities in vitro against Candida albicans, Cryptococcus neoformans and Staphylococcus aureus. In order to increase
these properties, given that the halo substitution was found to be capable of enhancing antifungal effects, we prepared a series of
fluoro and chloro derivatives of 3. The microbiological evaluation carried out on newly synthesized compounds included in vitro
assays for antifungal, antibacterial and antimycobacterial activities. Among the tested compounds, some dichloro and trichloro-
derivatives showed interesting antimicrobial properties. In particular, compounds 10j,k,l produced inhibitory effects against patho-
gen representatives of yeast (C. albicans, C. neoformans) and Gram positive bacteria (S. aureus) similar or superior to those of
bifonazole. In addition, their activity against Mycobacterium tuberculosis was superior to that of clotrimazole and econazole,
which were used as reference drugs. The replacement, in these compounds, of chlorine with fluorine atoms led to inactive derivatives.
Docking studies were carried out on the most active compounds, in order to rationalize the pharmacological results.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
crease in morbidity and mortality. Therefore, new thera-
peutic options are required.
Fungi usually cause infections on body surfaces,
although occasionally they may cause systemic infec-
tions. The latter generally occur in immunosuppressed
subjects (AIDS patients or patients receiving anticancer
therapy) who are especially susceptible because their im-
mune system is not functioning normally.
At the same time, Mycobacterium tuberculosis is a re-
emerging pathogen both in developed and developing
countries. In some areas, the human immunodeficiency
virus (HIV) infection is primarily responsible for the
global resurgence of tuberculosis. In fact, 50% of peo-
ple living with HIV/AIDS in Africa are affected by
tuberculosis and about one third of the 40million
HIV-infected people worldwide are co-infected with
M. tuberculosis.1 This increased risk of developing the
infection and the emergence of multi-drug resistant mu-
tants are dictating new approaches in the treatment of
mycobacterial disease.
In recent years the widespread use of antifungal agents
has resulted in the development of resistance to these
drugs by pathogenic microorganisms, causing an in-
Keywords: Azoles; Bifonazole analogues; Antifungals; Antimycobac-
terials.
The potential benefit of drugs endowed with both
antimycobacterial and antifungal properties repre-
sents a valuable goal, since the association between
*
Corresponding authors. Tel.: +39-010-3538397; fax: +39-010-
3538358 (G.M.); tel.: +39-070-6754147; fax: +39-010-3538358
(P.L.C.); e-mail addresses: menozzi@unige.it; placolla@unica.it
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.029

5466
G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
mycobacterial and mycotic infections often occurs in
immunocompromised patients.
Bifonazole (2) derives from the structure of clotrimazole
but, unlike the parent compound, it contains no halogen
atoms. It is a broad-spectrum antimycotic, employed in
the therapy of various dermatomycoses. Recent studies
performed on some imidazole antifungal drugs dis-
played other biological properties of bifonazole, such
as antiproliferative action on melanoma cells7 and an
inhibitory effect on prostanoid biosynthesis in various
cell systems.8
In recent years the class of azoles (imidazoles and triaz-
oles) has supplied many effective antifungal agents,
which are currently in clinical use; drugs such as flucon-
azole and voriconazole can be used to treat generalized
systemic mycoses. The mechanism of their antifungal ac-
tion includes the inhibition of cytochrome P450 51
(CYP51), which is essential for ergosterol biosynthesis
at the step of lanosterol-14-demethylation.2
Since the discovery of bifonazole, many modifications
on its structure were carried out, so as to increase its
antifungal potency and selectivity and to improve its
chemical physical properties. Among other changes, sev-
eral bioisosteric replacements of either or both benzene
rings, constituting the biphenyl moiety of the molecule,
have been investigated.9
Recent studies have demonstrated that selected azole
drugs are very potent antimycobacterial agents, which
show inhibitory concentrations in the nanomolar range.
A soluble protein from M. tuberculosis (MT-CYP51),
similar in sequence to CYP51 isozymes, has been cloned,
expressed and purified. Several azole compounds with
known antifungal activity proved to bind MT-CYP51
with high affinity and to impair the growth of Myco-
bacterium bovis and Mycobacterium smegmatis, two
mycobacterial species which closely resemble M. tuber-
culosis.2 Clotrimazole and econazole, in particular, were
reported to be more effective than rifampin and isonia-
zid against M. smegmatis.3
Several pyrazole,10 isoxazole and pyrimidine11 heteroan-
alogues of bifonazole were synthesized and tested for
their antifungal and antibacterial activities, in previous
research carried out in our laboratory; among these,
the 1,5-diphenylpyrazole 3 (Fig. 1) showed weak anti-
fungal and antibacterial properties.10
Given that in many studies the halo substitution was
found to be capable of enhancing both antifungal and
antimycobacterial activities,12–14 we decided to synthe-
size a number of fluoro and chloro derivatives of com-
pound 3, extending the biological investigation to the
antimycobacterial activity too. Fluorine or chlorine
atoms are present as common substituents on aromatic
rings, in the most potent antifungal azoles, as flucon-
azole, voriconazole, ketoconazole, itraconazole and
others.
More recently, the expression, characterization and
crystallization of a novel cytochrome P450 (CYP121)
from M. tuberculosis has been reported.4,5 MT-
CYP121 binds commercially available azole drugs with
a higher affinity than MT-CYP51. P450 drug-binding
titrations and gene-knockout studies suggested that
MT-CYP121 may be the true target for the antimyco-
bacterial activity of the azoles in vivo, even though its
physiological role remains unclear.6
These studies may lead to the development of a new gen-
eration of azole antimycotic compounds that may prove
effective against multi-drug resistant strains of the tuber-
culosis pathogen.3,5
Thus, in this paper we are presenting the synthesis,
preliminary microbiological evaluation (antimycotic,
antibacterial and antimycobacterial activities) and
molecular modeling studies (docking into the active site
of cytochrome P450 from M. tuberculosis MT-CYP121)
of halogenated pyrazole analogues of bifonazole 10e–u.
The high affinity of the azole antifungals as clotrimazole
(Fig. 1: 1) for MT-CYP121 appears to be determined
primarily by their bulky, polycyclic structure, giving
favourable interactions with the hydrophobic residues
in the largely apolar active site of the enzyme.3
2. Results and discussion
2.1. Chemistry
Compounds 10e–u were synthesized as depicted in
Schemes 1 and 2.
N
N
1,5-Diarylpyrazoles 10e–o were obtained following the
same synthetic pathway described for the compound
310 (Scheme 1): treatment of ethyl aroylacetates 4a–d15
with N,N-dimethylformamide dimethylacetal provided
a-dimethylaminomethylene b-ketoesters 5a–d,16 which
were condensed with appropriated arylhydrazines to af-
ford pyrazole esters 6e–o. Following reduction by lith-
ium aluminium hydride in diethyl ether (6k,n) or by
diisobutylaluminium hydride (DIBAL-H) in toluene
(6e–j,l,m,o) gave alcohols 7e–o, which were oxidized to
the corresponding aldehydes 8e–o by pyridinium chloro-
chromate (PCC) in dichloromethane. GrignardÕs reac-
N
N
N
N
N
N
Cl
1
2
3
Figure 1. Structures of chlotrimazole (1), bifonazole (2) and 1,5-
diphenylpyrazole analogue of bifonazole (3).

G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
5467
COOC2H5
NHNH2
C
C
CHN(CH3)2
O
COOC2H5
CH2OH
COCH2COOC2H5
(CH3)2NCH(OCH3)2
N
N
N
N
R1
DIBAL-H
or LiAlH4
PCC
R2
R2
R2
R2
4a-d
5a-d
R1
R1
6e-o
7e-o
R2
a
b
c
2-Cl
4-Cl
2-F
d
4-F
N
CHOH
CH
R2
R1
CHO
N
e
f
g
h
i
j
k
l
m
n
o
H
H
H
H
2-Cl
4-Cl
2-F
N
N
N
N
N
N
C6H5MgBr
CDI
4-F
2-Cl
2-Cl
2-Cl
4-Cl
4-Cl
2-F
4-F
4-F
R2
R2
R2
2,4-Cl2
4-Cl
2,4-Cl2
2-F
R1
R1
R1
4-F
2,4-F
8e-o
9e-o
10e-o
2
Scheme 1. Synthesis of compounds 10e–o.
NHNH2
O
C
O
C
(CH3)2NCH(OCH3)2
R1
CH2
C
CHN(CH3)2
C
C
O
O
12
11
CH
N
O
C
CH OH
N
N
LiAlH4
CDI
N
N
N
R1
N
N
p
q
r
s
2-Cl
4-Cl
2,4-Cl2
R1
2-F
R1
13p-u
Scheme 2. Synthesis of compounds 10p–u.
R1
9p-u
t
4-F
u
2,4-F
2
10p-u
tion of 8e–o with phenylmagnesium bromide led to car-
binols 9e–o, which, by reaction with N,N0-carbonyldi-
imidazole (CDI), yielded final imidazole compounds 10e–o.
nols 9p–u. Finally, the reaction of 9p–u with CDI affor-
ded imidazole derivatives 10p–u.
The aim of treating esters 6e–j,l,m,o with DIBAL-H was
to obtain directly corresponding aldehydes 8,18 but the
reduction, even if carried out at a temperature of
ꢀ78ꢁC and under nitrogen atmosphere, led to alcohols
7e–j,l,m,o, which were easily isolated from the reaction
mixture with high yields. On the contrary, the treatment
of the same esters with lithium aluminium hydride pro-
duced unsatisfactory results.
1-Aryl-5-phenylpyrazole derivatives 10p–u were pre-
pared by a shorter pathway (Scheme 2), from 2-dimethyl-
aminomethylene-1,3-diphenylpropane-1,3-dione 12, an
intermediate already used by us to synthesize other pyr-
azoles.17 By condensation of 12 with suitable substituted
phenylhydrazines, ketones 13p–u were obtained, which,
by lithium aluminium hydride reduction, led to carbi-

5468
G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
1
It is interesting to emphasize that the H NMR spectra
of the ethyl esters 6e,i,j and of the carbinols 7e,i,j, having
an o-chlorophenyl group at C5 of the pyrazole ring,
show complex signals in the d ranges of 4.05–4.30 and
4.42–4.54ppm, respectively (Tables 1, 2). These signals
correspond to methylene protons belonging to ethoxy-
carbonyl (esters 6e,i,j) or hydroxymethyl (carbinols
7e,i,j) group at C4 of the pyrazole ring, respectively.
On the contrary, ethyl CH2 protons of the other esters
6 resonate as a quartet, while the hydroxymethyl CH2
protons of the other carbinols 7 as a singlet or a doublet
(if coupled with OH), as expected (Tables 1, 2).
In vitro cytotoxicity in MT-4 cells, carried out in parallel
with anti-HIV-1 activity, was performed in order to
determine whether the compounds were endowed with
selective antimicrobial/antiviral activity.
Miconazole, bifonazole and amphotericin B were used
as reference compounds in antimycotic assays. Strepto-
mycin and AZTwere used in antibacterial and antiretro-
viral assays, respectively.
Clotrimazole and econazole were used as reference
drugs in antimycobacterial assays.
1
Moreover, H NMR spectra of alcohols 9e,i,j (Table 5)
and of imidazole derivatives 10e,i,j (Table 6) show dou-
ble signals corresponding to:
The results of biological tests are summarized in Tables
7 and 8; compounds completely devoid of antimicrobial
properties are not reported.
ꢁ pyrazole H-3, methyne proton and hydroxyl proton
of 9e,i,j;
ꢁ imidazole H-2 and methyne proton of 10e,i,j.
None of the tested compounds was capable of protect-
ing MT-4 cells from the cytopathic effect induced by
HIV-1, at least in concentrations, which were lower than
the cytotoxic ones.
These data are in agreement with the existence, in all
these cases, of stable conformers, rotationally con-
strained, due to the presence of both o-chlorine atom
on the C5 phenyl substituent and bulky groups at pyraz-
When tested against yeast and mold representatives,
chloro derivatives 10j–l showed an appreciable
activity against both C. albicans (MIC = MFC values,
ranging between 11.1 and 66.6lM) and C. neoformans
(ratio MIC/MFC = 11.1/33.3lM for all three com-
pounds) (Table 7). Derivative 10l displayed the
best activity, three times superior to that of bifonazole
and twice that of the parent compound 3, with a
ratio MIC/MFC = 11.1/11.1lM towards C. albicans
and with a ratio MIC/MFC = 11.1/33.3lM towards
C. neoformans. Compound 10r was active only against
C. neoformans with a ratio MIC/MFC = 33.3/33.3.
None of the tested compounds showed significant
inhibitory activity against A. fumigatus, as well as bifo-
nazole.
1
ole C4. In fact, H NMR spectra of analogues 2-fluor-
ophenyl derivatives (6g,m, 7g,m, 9g,m, 10g,m) (Tables
1, 2, 5, 6) show regular signals, as well as those of alde-
hydes 8e,i,j (Table 3), in which is evident that the formyl
group at pyrazole C4 does not hinder the free rotation of
the o-chlorophenyl ring at pyrazole C5.
Currently, crystallographic studies are in progress on
some significant 2-chlorophenyl derivatives so as to de-
fine their spatial arrangement, while preliminary Ôin sil-
icoÕ conformational studies on compounds 6e,i,j, 7e,i,j
confirm the hypothesis of two stable conformers, rota-
tionally constrained (Fig. 6).
With regard to antibacterial activity, compounds 10j,k,l
turned out to be weakly efficacious against representa-
tive Gram positive bacteria (S. aureus) whereas other
tested derivatives showed no activity; all compounds
were inactive towards representative Gram negative bac-
teria (Salmonella spp).
2.2. Biological properties
The activities of the title compounds were evaluated
in vitro against:
(a) Representative human pathogenic fungi (Candida
albicans, Cryptococcus neoformans, Aspergillus
fumigatus);
The results of the in vitro antimycobacterial activity are
reported in Table
8 (derivatives 10i,p were not
(b) Gram positive (Staphylococcus aureus) and Gram
negative (Salmonella spp.) bacteria;
cited, because lacking in any antimycobacterial effect). In
particular, 10j emerged as the most potent derivative,
also against M. tuberculosis strains resistant to drugs
commonly employed in the antitubercular therapy,
showing MIC values ranging between 6.7 and 57.7lM.
All compounds, except 10j, were inactive against M. for-
tuitum, as well as clotrimazole and econazole, used as
reference azole drugs. Among M. tuberculosis strains
used for biological assays, ATCC 27294 (wt) was gener-
ally the most susceptible to the majority of tested com-
pounds: the lowest values of MIC towards this strain
were shown by the three derivatives 10j–l (MIC = 6.7,
6.1, 6.7lM, respectively) (Table 8), which showed the
highest activity in antimycotic assays too. Compounds
3, 10e,r exhibited moderate activity against all M. tuber-
culosis strains.
(c) Several strains of Mycobacteria: M. smegmatis
ATCC 19420, M. fortuitum (clinical isolate), M.
tuberculosis ATCC 27294 (wild type), M. tuberculo-
sis ATCC 35820 (Str.R), M. tuberculosis ATCC
35822 (Ison.R), M. tuberculosis ATCC 35828
(Pyr.R), M. tuberculosis ATCC 35837 (Eth.R).
Only antimycobacterial activity of the parent compound
3 was investigated here; its antifungal and antibacterial
properties had formerly been evaluated.10
All title compounds were also evaluated at random for
antiretroviral activity in a lymphoid cell line (MT-4) in-
fected with HIV-1.

Table 1. Chemical, physical, IR and 1H NMR spectral data of compounds 6e–o
COOC2H5
N
N
R2
R1
Compound
R1
R2
Yield
(%)
Mp (ꢁC)
Molecular
formula
Analyses%
calcd/found
IR (cmꢀ1
(CHCl3)
)
1H NMR, d (CDCl3)
C
H
N
6e
6f
H
2-Cl
4-Cl
2-F
83
83
93
89
91
91
87
89
88
84
88
80–81a
C18H15ClN2O2
C18H15ClN2O2
C18H15FN2O2
C18H15FN2O2
C18H14Cl2N2O2
C18H13Cl3N2O2
C18H14Cl2N2O2
C18H13Cl3N2O2
C18H14F2N2O2
C18H14F2N2O2
C18H13F3N2O2
66.16
66.18
66.16
66.13
69.67
69.61
69.67
69.41
59.85
60.00
54.64
54.79
59.85
59.68
54.64
54.90
65.85
65.70
65.85
65.71
62.43
62.68
4.63
4.52
4.63
4.84
4.87
4.71
4.87
4.81
3.91
3.98
3.31
3.20
3.91
3.92
3.31
3.01
4.30
4.11
4.30
4.25
3.78
NDe
8.57
8.76
8.57
8.78
9.02
9.00
9.02
8.95
7.75
7.77
7.08
7.18
7.75
7.76
7.08
7.17
8.53
8.55
8.53
8.69
8.09
8.13
1708
1715
1712
1712
1710
1705
1708
1708
1708
1705
1707
1.15 (t, J = 7.1, 3H, CH3), 4.05–4.3 (m, 2H, CH2), 7.2–7.45
(m, 9H, ar), 8.21 (s, 1H, H-3 pyr)
H
102–103a
79–80b
1.26 (t, J = 7.1, 3H, CH3), 4.23 (q, J = 7.1, 2H, CH2),
7.15–7.4 (m, 9H, ar), 8.18 (s, 1H, H-3 pyr)
6g
6h
6i
H
1.20 (t, J = 7.1, 3H, CH3), 4.20 (q, J = 7.1, 2H, CH2),
7.0–7.5 (m, 9H, ar), 8.22 (s, 1H, H-3 pyr)
H
4-F
114–115b
83–84b
1.25 (t, J = 7.1, 3H, CH3), 4.22 (q, J = 7.1, 2H, CH2),
6.95–7.1 (m, 2H, ar), 7.15–7.35 (m, 7H, ar), 8.19 (s, 1H, H-3 pyr)
1.14 (t, J = 7.1, 3H, CH3), 4.05–4.3 (m, 2H, CH2),
7.1–7.45 (m, 8H, ar), 8.25 (s, 1H, H-3 pyr)
2-Cl
2,4-Cl2
4-Cl
2,4-Cl2
2-F
2-Cl
2-Cl
4-Cl
4-Cl
2-F
6j
102–103a
130–131c
103–104d
85–86b
1.14 (t, J = 7.1, 3H, CH3), 4.05–4.3 (m, 2H, CH2),
7.15–7.45 (m, 7H, ar), 8.25 (s, 1H, H-3 pyr)
6k
6l
1.25 (t, J = 7.1, 3H, CH3), 4.22 (q, J = 7.1, 2H, CH2),
7.1–7.4 (m, 8H, ar), 8.17 (s, 1H, H-3 pyr)
1.26 (t, J = 7.1, 3H, CH3), 4.24 (q, J = 7.1, 2H, CH2),
7.15–7.35 (m, 6H, ar), 7.43 (s, 1H, ar), 8.21 (s, 1H, H-3 pyr)
1.20 (t, J = 7.1, 3H, CH3), 4.21 (q, J = 7.1, 2H, CH2), 6.95–7.45 (m, 8H, ar),
8.26 (s, 1H, H-3 pyr)
6m
6n
6o
4-F
4-F
110–111d
78–79b
1.25 (t, J = 7,1, 3H, CH3), 4.22 (q, J = 7,1, 2H, CH2), 6.95–7.1 (m, 4H, ar),
7.15–7.3 (m, 4H, ar), 8.17 (s, 1H, H-3 pyr)
2,4-F2
4-F
1.24 (t, J = 7,1, 3H, CH3), 4.23 (q, J = 7,1, 2H, CH2), 6.75–7.05 (m, 4H, ar),
7.2–7.45 (m, 3H, ar), 8.20 (s, 1H, H-3 pyr)
a From anhydrous diethyl ether.
b From anhydrous diethyl ether/petroleum ether (bp 40–60ꢁC).
c From 95% ethanol.
d From petroleum ether (bp 40–60ꢁC).
e ND = not determined.

Table 2. Chemical, physical, IR and 1H NMR spectral data of compounds 7e–o
CH2OH
N
N
R2
R1
Compound
R1
R2
Yield
(%)
Bp (ꢁC/mmHg)
or mp (ꢁC)
Molecular
formula
Analyses%
calcd/found
IR (cmꢀ1
(CHCl3)
)
1H NMR, d (CDCl3)
C
H
N
7e
7f
H
H
H
2-Cl
4-Cl
2-F
96
98
90
165–170/0.25
140–141a
77–78a
C16H13ClN2O
C16H13ClN2O
C16H13FN2O
67.49
67.31
67.49
67.20
71.63
71.54
4.60
4.92
4.60
4.79
4.88
5.02
9.84
3605, 3380
3605, 3350
3610, 3380
1.82 (s, 1H, OH, ex), 4.42 and 4.52 (2d, J = 12.5, 2H, CH2),
7.3–7.5 (m, 9H, ar), 7.85 (s, 1H, H-3 pyr)
ꢂ1.9 (very br s, 1H, OH, ex), 4.53 (s, 2H, CH2), 7.15–7.35
(m, 9H, ar), 7.79 (s, 1H, H-3 pyr)
9.50
9.84
9.82
10.44
10.56
7g
1.73 (t, J = 5.6, 1H, OH, ex), 4.52 (d, J = 5.6, 2H, CH2,
became s with D2O), 7.0–7.45 (m, 9H, ar), 7.86 (s, 1H,
H-3 pyr)
7h
H
4-F
71
99–100a
C16H13FN2O
71.63
71.40
4.88
5.10
10.44
10.53
3610, 3370
1.73 (t, J = 5.1, 1H, OH, ex), 4.54 (d, J = 5.1, 2H, CH2,
became s with D2O), 6.95–7.1 (m, 2H, ar), 7.15–7.4 (m,
7H, ar), 7.81 (s, 1H, H-3 pyr)
7i
7j
7k
7l
2-Cl
2-Cl
2-Cl
4-Cl
4-Cl
83
88
92
92
84–86b
C16H12Cl2N2O
C16H11Cl3N2O
C16H12Cl2N2O
C16H11Cl3N2O
60.20
60.34
54.34
54.24
60.20
60.24
54.34
54.25
3.79
4.00
3.13
3.19
3.79
3.77
3.13
3.15
8.78
8.69
7.92
7.90
7.78
7.84
7.92
8.07
3600, 3370
3595, 3360
3610, 3370
3595, 3330
1.92 (s, 1H, OH, ex), 4.45 and 4.54 (2d, J = 12.5, 2H,
CH2), 7.1–7.4 (m, 8H, ar), 7.88 (s, 1H, H-3 pyr)
1.73 (br s, 1H, OH, ex), 4.46 and 4.53 (2d, J = 12.6, 2H,
CH2), 7.15–7.45 (m, 7H, ar), 7.90 (s, 1H, H-3 pyr)
ꢂ1.9 (very br s, 1H, OH, ex), 4.52 (s, 2H, CH2), 7.1–7.4
(m, 8H, ar), 7.79 (s, 1H, H-3 pyr)
2,4-Cl2
4-Cl
85–87b
135–136b
118–119a
2,4-Cl2
1.82 (br s, 1H, OH, ex), 4.56 (s, 2H, CH2),
7.16 (d, J = 8.4, 2H, ar), 7.27 (s, 2H, ar), 7.30
(d, J = 8.4, 2H, ar), 7.43 (s, 1H, ar), 7.84 (s, 1H, H-3 pyr)
1.58 (t, J = 5.3, 1H, OH, ex), 4.55 (d, J = 5.3,
2H, CH2, became s with D2O), 6.5–6.8 (m, 8H, ar),
7.91 (s, 1H, H-3 pyr)
7m
7n
7o
2-F
2-F
4-F
4-F
74
92
92
83–85b
C16H12F2N2O
C16H12F2N2O
C16H11F3N2O
67.13
66.92
4.23
4.01
9.78
9.83
3605, 3380
3600, 3360
3600, 3370
4-F
112–113a
82–83a
67.13
67.20
4.22
4.40
9.78
9.64
1.89 (t, J = 4.7, 1H, OH, ex), 4.52 (d, J = 4.7, 2H, CH2),
6.9–7.1 (m, 4H, ar), 7.15–7.3 (m, 4H, ar), 7.78 (s, 1H,
H-3 pyr)
2,4-F2
63.16
62.85
3.64
NDc
9.21
9.14
1.73 (br s, 1H, OH, ex), 4.56 (s, 2H, CH2), 6.75–7.1 (m, 4H,
ar), 7.15–7.25 (m, 2H, ar), 7.3–7.45 (m, 1H, ar),
7.84 (s, 1H, H-3 pyr)
a From anhydrous diethyl ether/petroleum ether (bp 40–60ꢁC).
b From anhydrous diethyl ether.
c ND = not determined.

Table 3. Chemical, physical, IR and 1H NMR spectral data of compounds 8e–o
CHO
N
N
R2
R1
Compound
R1
R2
Yield (%)
Mp (ꢁC)
Molecular
formula
Analyses%
calcd/found
IR (cmꢀ1
(KBr)
)
1H NMR, d (CDCl3)
C
H
N
8e
8f
H
2-Cl
4-Cl
2-F
88
88
95
93
97
91
95
92
91
98
85
107–108a
124–125b
104–105b
149–150b
116–117b
163–164a
134–135c
137–138b
141–142a
117–118c
98–99a
C16H11ClN2O
C16H11ClN2O
C16H11FN2O
C16H11FN2O
C16H10Cl2N2O
C16H9Cl3N2O
C16H10Cl2N2O
C16H9Cl3N2O
C16H10F2N2O
C16H10F2N2O
C16H9F3N2O
67.97
67.74
67.97
67.86
72.17
72.40
72.17
72.01
60.59
60.90
54.65
54.46
60.59
60.53
54.65
54.72
67.60
67.67
67.60
67.41
63.58
63.55
3.92
3.92
3.92
4.01
4.16
4.31
4.16
3.92
3.18
3.20
2.58
2.56
3.18
3.27
2.58
2.44
3.54
3.30
3.54
3.60
3.00
NDd
9.90
1680
1675
1672
1678
1680
1682
1676
1678
1683
1677
1680
7.2–7.5 (m, 9H, ar), 8.26 (s, 1H, H-3 pyr), 9.65 (s, 1H, CHO)
7.2–7.45 (m, 9H, ar), 8.24 (s, 1H, H-3 pyr), 9.77 (s, 1H, CHO)
7.05–7.55 (m, 9H, ar), 8.27 (s, 1H, H-3 pyr), 9.73 (s, 1H, CHO)
7.05–7.45 (m, 9H, ar), 8.24 (s, 1H, H-3 pyr), 9.76 (s, 1H, CHO)
7.2–7.5 (m, 8H, ar), 8.29 (s, 1H, H-3 pyr), 9.68 (s, 1H, CHO)
7.2–7.5 (m, 7H, ar), 8.29 (s, 1H, H-3 pyr), 9.67 (s, 1H, CHO)
7.15–7.5 (m, 8H, ar), 8.22 (s, 1H, H-3 pyr), 9.75 (s, 1H, CHO)
7.15–7.5 (m, 7H, ar), 8.27 (s, 1H, H-3 pyr), 9.79 (s, 1H, CHO)
7.0–7.55 (m, 8H, ar), 8.30 (s, 1H, H-3 pyr), 9.76 (s, 1H, CHO)
7.0–7.35 (m, 8H, ar), 8.22 (s, 1H, H-3 pyr), 9.75 (s, 1H, CHO)
6.8–7.55 (m, 7H, ar), 8.26 (s, 1H, H-3 pyr), 9.77 (s, 1H, CHO)
9.85
9.90
10.03
10.52
10.38
10.52
10.63
8.83
9.04
7.97
7.99
8.83
8.99
7.97
7.93
9.85
9.89
9.85
9.82
9.27
9.29
H
8g
8h
8i
H
H
4-F
2-Cl
2,4-Cl2
4-Cl
2,4-Cl2
2-F
2-Cl
2-Cl
4-Cl
4-Cl
2-F
8j
8k
8l
8m
8n
8o
4-F
4-F
2,4-F2
4-F
a From anhydrous diethyl ether.
b From anhydrous diethyl ether/petroleum ether (bp 40–60ꢁC).
c From 95% ethanol.
d ND = not determined.

Table 4. Chemical, physical, IR and 1H NMR spectral data of compounds 13p–u
O
C
N
N
R1
Compound
R1
Yield (%)
Mp (ꢁC)
Molecular formula
Analyses%
calcd/found
IR (cmꢀ1) (CHCl3)
1H NMR, d (CDCl3)
C
H
N
13p
13q
13r
13s
13t
13u
2-Cl
62
79
70
68
81
68
115–116a
155–156b
157–158b
145–146b
144–145a
130–131b
C22H15ClN2O
C22H15ClN2O
C22H14Cl2NO
C22H15FN2O
C22H15FN2O
C22H14F2N2O
73.64
73.76
73.64
73.45
67.19
67.03
77.18
77.17
77.18
76.81
73.33
73.66
4.21
4.39
4.21
4.34
3.59
3.57
4.41
4.48
4.41
4.21
3.92
3.63
7.81
7.88
7.81
7.78
7.12
7.23
8.18
8.14
8.18
8.13
7.77
7.89
1643
1645
1640
1642
1640
1643
7.15–7.55 (m, 12H, ar), 7.83 (d, J = 6.8, 2H, ar),
8.11 (s, 1H, H-3 pyr)
4-Cl
7.15–7.55 (m, 12H, ar), 7.80 (d, J = 6.8, 2H, ar),
8.05 (s, 1H, H-3 pyr)
2,4-Cl2
2-F
7.15–7.55 (m, 11H, ar), 7.82 (d, J = 6.8, 2H, ar),
8.10 (s, 1H, H-3 pyr)
6.95–7.1 (m, 2H, ar), 7.2–7.6 (m, 10H, ar), 7.81
(d, J = 6.8, 2H, ar), 8.04 (s, 1H, H-3 pyr)
6.95–7.1 (m, 2H, ar), 7.2–7.6 (m, 10H, ar), 7.81
(d, J = 6.8, 2H, ar), 8.02 (s, 1H, H-3 pyr)
6.8–7.0 (m, 2H, ar), 7.2–7.6 (m, 9H, ar), 7.81
(d, J = 6.8, 2H, ar), 8.09 (s, 1H, H-3 pyr)
4-F
2,4-F2
a From anhydrous diethyl ether/petroleum ether (bp 40–60ꢁC).
b From anhydrous diethyl ether.

G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
5473
As reported in Table 8, MIC values of tested compounds
appear higher than those related to the common antitu-
bercular agents, used as reference drugs, but lower or
comparable to values of the azole antifungal drug econa-
zole. Clotrimazole, furthermore, was active only towards
the strain M. tuberculosis ATCC 27294 (wt), moreover
with a MIC value higher than those of 10j–l,q,r.
10j, the most active antimycobacterial agent, endowed
with a significant antifungal activity. The compound
was docked into the active site of cytochrome P450 from
M. tuberculosis,19 MT-CYP121, using FlexX.20 FlexX is
a widely used docking algorithm in drug design whose
ability in predicting a conformation of the ligand very
close to its X-ray structure has been extensively reviewed
in literature.21
With regard to in vitro cytotoxycity assays, all chloro
derivatives were cytotoxic for MT-4 cells, at concentra-
tions near the active doses.
According to the results of our docking experiments,
compound 10j could bind to the cytochrome catalytic
site orienting the nitrogen in position 2 of pyrazole ring
towards the iron atom of the haem group. Previous
docking studies reported by Munro et al. for the binding
of miconazole and clotrimazole with MT-CYP121, put
in evidence a steric clash that could be present between
the ligand and the Arg386 side chain6 if the azole
nucleus was positioned perpendicularly to the iron atom.
On the contrary, FlexX proposes a binding model where
10j is shifted so as to avoid any negative interaction with
Arg386, while a favourable p–p interaction is present be-
tween the phenyl ring of substituent on position 4 and
Phe280 (Fig. 2A). Since Munro described a positive
interaction between clotrimazole and side chain of
Phe280, this residue seems to play an important role in
the binding of azole drugs. Only site-directed mutagen-
esis experiments, however, will be able to confirm this
hypothesis.
All fluoro derivatives, on the contrary, showed very low
cytotoxicity (CC50 > 100lM), but produced poor bio-
logical effects. As already observed in other similar
cases,22 replacing chlorine with fluorine atoms led to a
dramatic decrease in activity. All fluoro derivatives were
inactive against the fungi or bacteria tested, but 10m–o
and 10s–u produced a moderate inhibition of the growth
of M. smegmatis ATCC 19420 and M. tuberculosis
ATCC 27294 (wt) (MIC values ranging from 48.9 to
88.2lM and from 21.1 to 65.3lM, respectively).
The results of the preliminary biological tests thus
pointed out that the introduction of halo substituents
on the phenyl rings linked to the pyrazole nucleus of
compound 3, yielded modifications in the microbiologi-
cal profile. In particular, derivatives bearing 2 or 3 chlo-
rine atoms emerged as the most interesting for
antifungal, antibacterial and antimycobacterial activi-
ties, in accordance with literature.12–14 Moreover, as
already observed,12 the number of chloro substituents
rather than their position on phenyl rings is the major
determinant for antimicrobial activities. Monochloro
and all fluoro derivatives are inactive as antimycotics,
whereas a number of them showed some antimycobacte-
rial activity. In particular, the monochloro derivative
10e exhibited appreciable inhibitory effects against all
Mycobacteria strains tested, except M. fortuitum, but
it also proved to be highly toxic in the assay on the
MT-4 cells.
Interestingly, in the case of docking studies performed
with 10j and MT-CYP51,23 which shares a significative
structure similarity with C. albicans CYP51 (CA-
CYP51),24 the binding model obtained was fully compa-
rable to the bioactive conformation of fluconazole into
the crystallized complex, thus demonstrating the reli-
ability of the selected computational methodology.
With the aim of investigating which molecular charac-
teristics could affect the differences in biological
responses of chlorinated versus fluorinated derivatives, a
docking study was also performed on 10o, one of the less
active fluorinated compounds. Figure 2B shows the dif-
ferent pose of 10o into the catalytic pocket, in compar-
ison with 10j. A superimposition of FlexX solutions
for 10j and 10o underlined that conformers of 10j occu-
py in the catalytic area different regions than conformers
2.3. Molecular modeling studies
In order to further rationalize the biological results, a
molecular docking study was performed on compound
Figure 2. Binding mode proposed for 10j (A) and 10o (B) into the catalytic site of MT-CYP121. For the sake of clarity, only few amino acids of the
macromolecule are displayed as sticks.

5474
G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
Figure 3. A superimposition of the best FlexX docking solution for 10j
(magenta) and for 10o (yellow).
Figure 4. Isopotential contour maps for 10o (left) and 10j (right). Only
negative potentials have been represented, coloured in blue. The cut
has been done at ꢀ4.00 level.
of 10o, due to their different conformational freedom. In
Figure 3 the best FlexX solutions for 10j and 10o are de-
picted. This finding could suggest that the rotationally
constrained 10j could have some specific interactions
with MT-CYP121, and thus a different degree of activ-
ity. In addition, isopotential contour maps evaluation
(Fig. 4), highlights higher values of negative charge for
fluorine atom, in comparison with chlorine one. This
marked negative potential could be responsible of un-
favourable interactions with the macromolecule and,
consequently, of the reduced activity of fluorinated com-
pounds.
do not convert one into the other. Conformers I (namely
Ia and Ib) and conformers II (namely IIa and IIb) could
instead easily convert one into the other. In the case of
compounds 7e,i,j, 9e,i,j and 10e,i,j, the absolute minima,
located in a large isoenergetical area of the plot, are sep-
arated from a specular large isoenergetical area, the rel-
ative minima, by an energy barrier of about 95kJ/mol.
In Figure 6 are reported, as an example, the relevant
conformers and the energy profiles for the rotation of
s1 and s2 in 6j and 7j.
These calculations support the hypothesis of two stable
and distinct conformations at room temperature for
compounds 6e,i,j, 7e,i,j, 9e,i,j and 10e,i,j. However, only
experimental data from crystallographic studies (in pro-
gress) will fully elucidate what are the conformers ratio
at room temperature and the structure of the crystal of
the above mentioned compounds.
Furthermore, so as to support the presence of two stable
conformers for compounds 6e,i,j, 7e,i,j, 9e,i,j and 10e,i,j
1
such as proposed by the H NMR data, a conforma-
tional analysis based on a systematic molecular mechan-
ics search was carried out on these structures (Fig. 5).
The energy profiles for the rotation of the angles s1 and
s2 were calculated and the absolute minima plus local
minima were found for all mentioned compounds. s1,
s2 and corresponding energy values are reported in
Table 9.
3. Conclusion
In conclusion, we have synthesized and evaluated in
vitro the antimicrobial activity of chloro and fluoro
derivatives 10e–u, in order to improve the antimycotic
and antibacterial activities of 4-[1H-imidazol-1-yl(phe-
nyl)methyl]-1,5-diphenyl-1H-pyrazole 3, heteroanalogue
of bifonazole.
Ramachandran plots for compounds 6e,i,j identify two
couples of conformers, I and II, separated by an energy
barrier of about 95kJ/mol (around 25kcal/mol), which
OH
O
C H
2 5
*
*
*
*
Cl
_
O
CH
CH OH
CH
N
2
*
*
*
*
*
N
Cl
Cl
Cl
*
•
*
•
*
•
•
•
•
N
N
N
N
*
*
*
*
•
•
•
•
•
•
•
•
N
N
N
N
•
•
R
R
R
R
1
1
1
1
10
6
7
9
Figure 5. Torsional angles s1 ( ) and s2 (d) selected for the conformational analysis.
ꢃ

Table 5. Chemical, physical, IR and 1H NMR spectral data of compounds 9e–u
CHOH
N
N
R2
R1
Compound
R1
R2
Yield
(%)
Mp (ꢁC)
Molecular
formula
Analyses%
calcd/found
IR (cmꢀ1
(KBr)
)
1H NMR, d (CDCl3)
C
H
N
9e
9f
9g
9h
9i
H
2-Cl
4-Cl
2-F
71
84
95
88
89
153–154a
216–218a
177–178a
197–198a
158–159a
C22H17ClN2O
C22H17ClN2O
C22H17FN2O
C22H17FN2O
C22H16Cl2N2O
73.23
73.25
73.23
73.26
76.73
76.48
76.73
76.72
66.85
66.69
4.75
4.62
4.75
4.76
4.97
5.02
4.97
4.88
4.08
3.94
7.76
7.80
7.76
7.86
8.13
8.24
8.13
8.04
7.09
7.05
3295
3260
3275
3265
3310
ꢂ2.5 (very br s, 1H, OH, ex), 5.63 and 5.65 (2s, 1H, CH-Ph),
7.15–7.4 (m, 14H, ar), 7.59 and 7.77 (2s, 1H, H-3 pyr)
ꢂ2.3 (very br s, 1H, OH, ex), 5.70 (s, 1H, CH-Ph), 7.15–7.45
(m, 14H, ar), 7.64 (s, 1H, H-3 pyr)
H
H
ꢂ2.2 (very br s, 1H, OH, ex), 5.71 (s, 1H, CH-Ph), 7.0–7.45
(m, 14H, ar), 7.70 (s, 1H, H-3 pyr)
H
4-F
ꢂ2.1 (very br s, 1H, OH, ex), 5.69 (s, 1H, CH-Ph), 6.95–7.1
(m, 2H, ar), 7.15–7.45 (m, 12H, ar), 7.63 (s, 1H, H-3 pyr)
2.19 and 2.32 (2d, J = 3, 1H, OH, ex), 5.65–5.75 (m, 1H,
CH-Ph, became s with D2O), 7.1–7.45 (m, 13H, ar), 7.65 and
7.82 (2s, 1H, H-3 pyr)
2-Cl
2-Cl
9j
2,4-Cl2
4-Cl
2-Cl
4-Cl
67
70
146–147b
158–159c
C22H15Cl3N2O
C22H16Cl2N2O
61.49
61.75
3.52
3.62
6.52
6.52
3270
3285
2.32 and 2.39 (2d, J = 3, 1H, OH, ex), 5.6–5.7 (m, 1H, CH-Ph
became s with D2O), 7.05–7.4 (m, 12H, ar), 7.65 and 7.81 (2s,
1H, H-3 pyr)
9k
66.85
66.70
4.08
4.08
7.09
7.04
2.29 (d, J = 4, 1H, OH, ex), 5.68 (d, J = 4, 1H, CH-Ph, became
s with D2O), 7.1–7.2 (m, 4H, ar), 7.25–7.45 (m, 9H, ar), 7.64 (s,
1H, H-3 pyr)
9l
2,4-Cl2
2-F
4-Cl
2-F
4-F
4-F
76
51
69
68
197–198b
174–175d
144–145c
145–146b
C22H15Cl3N2O
C22H16F2N2O
C22H16F2N2O
C22H15F3N2O
61.49
61.57
72.92
73.05
72.92
72.73
69.47
69.66
3.52
3.33
4.45
4.77
4.45
NDe
3.97
NDe
6.52
6.61
7.73
7.80
7.73
7.69
7.36
7.45
3280
3265
3295
3305
ꢂ1.6 (very br s, 1H, OH, ex), 5.74 (s, 1H, CH-Ph), 7.1–7.2 (m,
2H, ar), 7.25–7.45 (m, 10H, ar), 7.70 (s, 1H, H-3 pyr)
ꢂ2.2 (br s, 1H, OH, ex), 5.74 (s, 1H, CH-Ph), 6.95–7.4 (m, 13H,
ar), 7.73 (s, 1H, H-3 pyr)
9m
9n
9o
4-F
2.25–2.35 (m, 1H, OH, ex), 5.69 (d, J = 3, 1H, CH-Ph, became
s with D2O), 6.9–7.4 (m, 13H, ar), 7.62 (s, 1H, H-3 pyr)
2.29 (d, J = 4, 1H, OH, ex), 5.73 (d, J = 4, 1H, CH-Ph, became
s with D2O), 7.1–7.2 (m, 2H, ar) 7.25–7.45 (m, 10H, ar),
7.69 (s, 1H, H-3 pyr)
2,4-F2
9p
9q
9r
2-Cl
H
H
H
91
55
93
170–171a
200–201a
160–161d
C22H17ClN2O
C22H17ClN2O
C22H16Cl2NO
73.23
72.97
73.23
73.58
66.85
67.13
4.75
5.05
4.75
4.84
4.08
3.92
7.76
7.67
7.76
7.89
7.09
7.20
3255
3310
3310
2.22 (d, J = 4, 1H, OH, ex), 5.79 (d, J = 4, 1H, CH-Ph, became
s with con D2O), 7.2–7.5 (m, 14H, ar), 7.70 (s, 1H, H-3 pyr)
2.24 (d, J = 4, 1H, OH, ex), 5.72 (d, J = 4, 1H, CH-Ph, became
s with con D2O), 7.1–7.45 (m, 14H, ar), 7.65 (s, 1H, H-3 pyr)
2.35 (d, J = 4, 1H, OH, ex), 5.77 (d, J = 4, 1H, CH-Ph, became
s with con D2O), 7.15–7.45 (m, 13H, ar), 7.69 (s, 1H, H-3 pyr)
4-Cl
2,4-Cl2
(continued on next page)

5476
G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
Dichloro and trichloro derivatives 10j,k,l emerged as the
most interesting compounds, confirming the effective-
ness of chloro substituents to enhance antifungal activ-
ity.10–12 These compounds produced inhibitory effects
against pathogen representatives of yeast (C. albicans,
C. neoformans) and Gram positive bacteria (S. aureus)
similar or superior to those of bifonazole. In addition,
they showed an antimycobacterial activity against M.
tuberculosis superior to that of clotrimazole and econa-
zole, which were used as reference drugs.
Computational studies suggest an inhibition of CA-
CYP51 and MT-CYP121 as possible mechanism of ac-
tion for these compounds.
4. Experimental
4.1. Chemistry
Melting points were determined with a Fisher–Johns
apparatus and are uncorrected. IR spectra were regis-
tered on a Perkin–Elmer 398 spectrophotometer and
are expressed in cmꢀ1. 1H NMR spectra were registered
on a Varian Gemini 200 (200MHz) spectrometer; chem-
ical shifts are reported as d values (ppm) relative to TMS
as internal standard; coupling constants (J) are ex-
pressed in Hertz (Hz). The following NMR abbrevia-
tions are used: br (broad), s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), ex (exchangeable
with D2O), ar (aromatic proton), pyr (pyrazole), imid
(imidazole). Microanalyses for C, H, N were performed
using a Carlo Erba Elemental Analyzer Model EA 1110
and results agree within 0.4% with calculated values.
4.1.1. General procedure for the synthesis of compounds
6e–o and 13p–u. The appropriate arylhydrazine hydro-
chloride (11mmol) was added to a stirred solution of
5a–d or 12 (10mmol) in absolute ethanol (50mL). The
mixture was heated to reflux for 2h and evaporated
under reduced pressure. The residue was taken up in
chloroform and the organic solution was washed with
water, dried (magnesium sulfate), filtered and evapo-
rated in vacuo. The solid residue was purified by recrys-
tallization from suitable solvent.
1
Yields, mp values, recrystallization solvents, IR and H
NMR spectral data are reported in Table 1 (6e–o) and in
Table 4 (13p–u).
4.1.2. General procedure for the synthesis of compounds
7k,n. A solution of 6k,n (20mmol) in anhydrous diethyl
ether (150mL) was slowly added to a stirred solution of
lithium aluminium hydride (1.52g, 40mmol) in the same
solvent (100mL). The mixture was refluxed with stirring
for 7h, cooled at 0ꢁC and treated in succession with
water (2mL), 10% sodium hydroxide solution (2.5mL)
and water (8mL). The supernatant organic solution
was decanted, the insoluble residue was treated with
3N hydrochloric acid (pH ꢂ 1) and the mixture was
then thoroughly extracted with diethyl ether. The com-
bined organic extracts were washed with water, dried

Table 6. Chemical, physical and 1H NMR spectral data of compounds 10e–u
N
CH
N
N
N
R2
R1
Compound
R1
R2
Yield
(%)
Mp (ꢁC) or
bp (ꢁC/mm Hg)
Molecular
formula
Analyses%
calcd/found
1H NMR, d (CDCl3)
C
H
N
10e
10f
10g
10h
10i
H
2-Cl
4-Cl
2-F
4-F
2-Cl
2-Cl
4-Cl
4-Cl
2-F
4-F
4-F
H
65
42
47
64
55
67
68
39
53
80
60
74
70
72
75
––a
C25H19ClN4
C25H19ClN4
C25H19FN4
C25H19FN4
C25H18Cl2N4
C25H17Cl3N4
C25H18Cl2N4
C25H17Cl3N4
C25H18F2N4
C25H18F2N4
C25H17F3N4
C25H19ClN4
C25H19ClN4
C25H18Cl2N4
C25H19FN4
73.07
73.17
73.07
72.97
76.12
75.99
76.12
75.79
67.42
67.52
62.58
62.28
67.42
67.41
62.58
62.37
72.80
72.70
72.80
73.05
69.76
69.50
73.07
72.92
73.07
73.10
67.42
67.39
76.12
76.24
4.66
4.91
4.66
4.78
4.85
5.07
4.85
5.03
4.07
4.23
3.17
3.43
4.07
4.02
3.57
3.32
4.40
NDd
4.40
4.26
3.98
3.71
6.66
4.51
4.66
4.34
4.07
3.92
4.85
4.80
13.63
13.46
13.63
13.59
14.20
14.10
14.20
14.10
12.58
12.62
11.68
11.33
12.58
12.64
11.68
11.46
13.58
13.59
13.58
13.59
13.01
12.79
13.63
13.80
13.63
13.68
12.58
12.64
14.20
14.30
6.16 and 6.21 (2s, 1H, CH-Ph), 6.85–7.4 (m, 16H, ar and imid), 7.44 and
7.61 (2s, 1H, H-2 imid), 7.53 (s, 1H, H-3 pyr)
6.22 (s, 1H, CH-Ph), 6.85–7.4 (m, 16H, ar and imid), 7.49 (s, 2H, H-2
imid and H-3 pyr)
H
156–157b
113–114b
156–157b
128–129b
205–215/0.38
134–135b
166–167c
140–141b
141–142b
121–122e
131–132e
122–123b
152–153b
126–127b
H
6.26 (s, 1H, CH-Ph), 6.85–7.4 (m, 16H, ar and imid), 7.46 (s, 1H, H-2
imid), 7.53 (s, 1H, H-3 pyr)
H
6.24 (s, 1H, CH-Ph), 6.9–7.4 (m, 16H, ar and imid) 7.51 (s, 1H, H-3 pyr),
7.57 (s, 1H, H-2 imid)
2-Cl
2,4-Cl2
4-Cl
2,4-Cl2
2-F
6.20 and 6.23 (2s, 1H, CH-Ph), 6.85–7.45 (m, 15H, ar and imid), 7.49 and
7.64 (2s, 1H, H-2 imid), 7.54 (s, 1H, H-3 pyr)
6.18 and 6.21 (2s, 1H, CH-Ph), 6.85–7.4 (m, 14H, ar and imid), 7.46 and
7.64 (2s, 1H, H-2 imid), 7.54 (s, 1H, H-3 pyr)
6.21 (s, 1H, CH-Ph), 6.85–7.4 (m, 15H, ar and imid), 7.49 (s, 2H, H-2 imid
and H-3 pyr)
10j
10k
10l
6.25 (s, 1H, CH-Ph), 6.8–7.45 (m, 14H, ar and imid), 7.50 (s, 1H, H-2 imid),
7.53 (s, 1H, H-3 pyr)
10m
10n
10o
10p
10q
10r
10s
6.30 (s, 1H, CH-Ph), 6.9–7.4 (m, 15H, ar and imid), 7.46 (s, 1H, H-2 imid),
7.58 (s, 1H, H-3 pyr)
4-F
6.21 (s, 1H, CH-Ph), 6.9–7.4 (m, 15H, ar and imid), 7.47 (s, 2H, H-2 imid
and H-3 pyr)
2,4-F2
2-Cl
4-Cl
2,4-Cl2
2-F
6.24 (s, 1H, CH-Ph), 6.75–7.45 (m, 14H, ar and imid), 7.49 (s, 1H, H-2 imid),
7.53 (s, 1H, H-3 pyr)
6.30 (s, 1H, CH-Ph), 6.9–7.45 (m, 16H, ar), 7.52 (s, 1H, H-2 imid), 7.55 (s,
1H, H-3 pyr)
H
6.24 (s, 1H, CH-Ph), 6.9–7.4 (m, 16H, ar and imid), 7.49 (s, 1H, H-2 imid),
7.51 (s, 1H, H-3 pyr)
H
6.29 (s, 1H, CH-Ph), 6.9–7.45 (m, 15H, ar and imid), 7.50 (s, 1H, H-2 imid),
7.55 (s, 1H, H-3 pyr)
H
6.25 (s, 1H, CH-Ph), 6.9–7.4 (m, 16H, ar and imid), 7.50 (s, 2H, H-2 imid
and H-3 pyr)
(continued on next page)

5478
G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
(magnesium sulfate), filtered and evaporated. The crude
product was then recrystallized from suitable solvent.
1
Yields, mp values, recrystallization solvents, IR and H
NMR spectral data are reported in Table 2.
4.1.3. General procedure for the synthesis of compounds
7e–j,l,m,o. Diisobutylaluminium hydride (DIBAL-H)
(1.5moldmꢀ3 in toluene, 13mL, 20mmol) was added
dropwise, under dry nitrogen, to a solution of 6e–
j,l,m,o (10mmol) in anhydrous toluene, cooled to
ꢀ78ꢁC. After 45min of stirring at ꢀ78ꢁC, the mixture
was quenched by cautious addition of 2N hydrochloric
acid (70mL) and thoroughly extracted with ethyl ace-
tate. The combined organic extracts were washed with
water, dried (magnesium sulfate), filtered and evapo-
rated under reduced pressure. The crude product was
then recrystallized from suitable solvent. Crude com-
pounds 7e and 7i were obtained as liquid residues, which
were purified by silica gel chromatography, using ethyl
acetate–petroleum ether (bp 40–60ꢁC) 1:1 as eluant, fol-
lowed by bulb-to-bulb distillation in vacuo (7e) or by
recrystallization from anhydrous diethyl ether (7i).
Yields, bp and mp values, recrystallization solvents, IR
and H NMR spectral data are reported in Table 2.
1
4.1.4. General procedure for the synthesis of compounds
8e–o. Compounds 7e–o (20mmol) dissolved in anhydrous
dichloromethane (100mL) were added to a magnetically
stirred suspension of pyridinium chlorochromate (6.46g,
30mmol) in the same solvent (50mL). The resulting mix-
ture was stirred at room temperature for 24h and, there-
fore, diluted with anhydrous diethyl ether (50mL). The
supernatant organic solution was decanted from black
gum and the insoluble residue was washed with anhy-
drous diethyl ether (3 · 30mL). The combined organic
solution was filtered on florisil and the solvent was re-
moved by distillation. The crude residues were purified
by recrystallization from suitable solvent.
1
Yields, mp values, recrystallization solvents, IR and H
NMR spectral data are reported in Table 3.
4.1.5. General procedure for the synthesis of compounds
9e–o. Compounds 8e–o (10mmol) dissolved in anhy-
drous diethyl ether (8e,g–i,k,l,n,o) or tetrahydrofuran
(8f,j,m) (50mL) were slowly added with stirring to an
ice-cooled solution of phenylmagnesium bromide, pre-
pared from magnesium turnings (0.73g, 30mmol) and
bromobenzene (3.60g, 43mmol) in the same solvent
(20mL). The mixture was refluxed for 1h, cooled at
0ꢁC and treated with 15% sulfuric acid (pH ꢂ 1). The
organic layer was separated from the acid phase, which
was extracted thoroughly with diethyl ether. The com-
bined organic solution was washed with saturated
sodium hydrogen carbonate solution and with water,
dried (magnesium sulfate), filtered and evaporated to
give a white solid residue, which was purified by recrys-
tallization from a suitable solvent.
1
Yields, mp values, recrystallization solvents, IR and H
NMR spectral data are reported in Table 5.

G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
Table 7. In vitro antifungal and antibacterial activity of compounds 3, 10j–l,r
5479
Compound
CC50a
EC50b
MICc/MFC (MBC)d
MT-4
HIV-1
C. albicans
C. neoformans
A. fumigatus
S. aureus
Salmonella spp.
3e
10j
22.2
33
>22.2
>33
>34
>12
>32
22/>100
33.3/33.3
66.6/66.6
11.1/11.1
Inf
22/22
Inf
66/>100
33.3/33.3
25/100
33.3/33.3
Inf
Inf
Inf
Inf
Inf
Inf
P11.1/33.3
11.1/33.3
11.1/33.3
33.3/33.3
––
Inf
10k
10l
34
12
Inf
Inf
10r
32
Inf
AZT>100
Streptomycin
Bifonazole
Miconazole
Amphotericin B
0.008
––
––
––
––
––
>100
>100
18
––
––
––
––
––
––
Inf
3.1/3.1
––
––
10/23.6
––
––
33.3/33.3
6.0/10.6
3.7/3.7
33.3/100
3.7/3.7
1.2/3.7
27.6/44.4
11.1/11.1
>100
––
––
a Compound concentration (lM) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by the MTT method.
b Compound concentration (lM) required to achieve 50% protection of MT4 cells from the HIV-1 induced cytopathogenicity, as determined by the
MTT method.
c Minimum inhibitory concentration (lM).
d Minimum fungicidal (bactericidal) concentration (lM).
e Ref. 10.
f Inactive up to MIC P 100lM.
4.1.6. General procedure for the synthesis of compounds
9p–u. A solution of 13p–u (10mmol) in anhydrous diethyl
ether (50mL) was slowly added with stirring to a solution
of lithium aluminium hydride (0.38g, 10mmol) in the
same solvent (20mL). The mixture was refluxed with stir-
ring for 4h, cooled at 0ꢁC and treated in succession with
water (0.5mL), 10% sodium hydroxide solution (0.5mL)
and water (2mL). The supernatant organic solution was
decanted and the insoluble residue was treated twice with
diethyl ether. The combined organic extracts were
washed with water, dried (magnesium sulfate), filtered
and evaporated. The crude product was then recrystal-
lized from suitable solvent.
in culture medium. Cell lines were from American Type
Culture Collection (ATCC). H9/IIIB, MT-4 and C8166
cells [grown in RPMI 1640 containing 10% foetal calf
serum (FCS), 100UI/mL penicillin G and 100lg/mL
streptomycin] were used for anti-HIV-1 assays. Cell cul-
tures were checked periodically for the absence of myco-
plasma contamination with a MycoTect Kit (Gibco).
Human immunodeficiency virus type-1 (HIV-1, IIIB
strain) was obtained from supernatants of persistently
infected H9/IIIB cells. HIV-1 stock solutions had a titre
of 5 · 107 cell culture infectious dose 50 (CCID50)/mL.
Bacterial and fungal strains were either clinical isolates
(obtained from Clinica Dermosifilopatica, University
of Cagliari) or collection strains from ATCC. M.
smegmatis 19420 and M. tuberculosis 27294, 35820,
35822, 35828, 35837 strains were purchased from Amer-
ican Type Culture Collection (ATCC), whereas M. for-
tuitum and all bacterial strains were clinical isolates
provided by University Hospitals of Cagliari, Italy.
1
Yields, mp values, recrystallization solvents, IR and H
NMR spectral data are reported in Table 5.
4.1.7. General procedure for the synthesis of compounds
10e–u. N,N0-Carbonyldiimidazole (1.78g, 11mmol) was
added to a solution of 9e–u (10mmol) in dry toluene
(60mL) and the mixture was refluxed for 4h. The sol-
vent was removed under reduced pressure and the
residue was dissolved in diethyl ether (200mL). The
organic solution was washed with water, dried (magne-
sium sulfate), filtered and evaporated. The crude residue
was then purified by chromatography on florisil, using
diethyl ether for 10k,l,q, or diethyl ether–petroleum
ether (bp 40–60ꢁC) 1:1 for 10g,j,m–p,r,t,u, or ethyl ace-
tate–petroleum ether (bp 40–60ꢁC) 1:1 for 10i,f as elu-
ent, and recrystallization from a suitable solvent.
4.2.1. Antiviral assays. Activity against the HIV-1 multi-
plication in acutely infected cells was based on inhibition
of virus-induced cytopathogenicity in MT-4 cells.25
Briefly, 50lL of RPMI/10% FCS containing 1 · 104
cells were added to each well flat-bottomed microtitre
trays containing 50lL of medium and serial dilutions
of test compounds. HIV-1 suspension (20lL) containing
100 CCID50 were then added. After a 4days incubation
at 37ꢁC, the number of viable cells was determined
by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-
lium bromide (MTT) method.26,27 Cytotoxicity of com-
pounds, based on the viability of mock-infected cells as
monitored by the MTT method, was evaluated in paral-
lel with their antiviral activity.
1
Yields, bp and mp values, recrystallization solvents, H
NMR spectral data are reported in Table 6. IR spectral
data are consistent with the proposed structures and are
not reported, because lacking of characteristic absorp-
tion values.
4.2.2. Antibacterial assays. S. aureus and Salmonella spp.
were recent clinical isolates. Assays were carried out in
nutrient broth (DIFCO), pH7.2, with an inoculum of
103 bacterial cells/tube. Minimum inhibitory concentra-
tions (MICs) were determined after incubation at 37ꢁC
4.2. Biological assays
Test compounds were dissolved in DMSO at an initial
concentration of 100lM and then were serially diluted

Table 8. In vitro antimycobacterial activity of compounds 3, 10e–h,j–o,q–u
Compound
CC50a
MIC50b/MIC90c
MT-4
M. smegmatis
M. fortuitum
M. tuberculosis
M. tuberculosis
ATCC 35820 (Str.R)
M. tuberculosis
ATCC 35822 (Ison.R)
M. tuberculosis
ATCC 35828 (Pyr.R)
M. tuberculosis
ATCC 35837 (Eth.R)
ATCC 19420
(Cl. is.)
ATCC 27294 (wt)
3
10e
22.2
11
Ind
Ind
69.9/>100
56.3/>100
25.3/90.0
Ind
43.5/>100
41.1/>100
Ind
43.3/86.6
44.1/87.4
Ind
30.0/>100
27.3/89.2
Ind
33.0/>100
41.1/>100
Ind
90.0/>100
Ind
Ind
10f
10g
26
Ind
>100
>100
33
50.4/>100
55.3/>100
29.8/>100
16.2/67.3
Ind
Ind
Ind
Ind
Ind
Ind
10h
10j
Ind
Ind
Ind
Ind
Ind
Ind
57.7/>100
Ind
6.7/26.2
6.1/27.3
6.7/25.3
47.2/>100
Ind
22.4/100
Ind
23.9/>100
Ind
15.6/60.2
Ind
36.2/>100
Ind
10k
10l
34
12
Ind
Ind
91.7/>100
Ind
73.1/>100
Ind
Ind
10m
10n
>100
>100
>100
P 100
32
88.2/>100
50.0/P 100
59.0/>100
Ind
Ind
Ind
Ind
Ind
Ind
Ind
Ind
Ind
10o
10q
Ind
21.1/>100
10.7/84.0
11.7/29.3
47.0/>100
65.3/>100
57.5/>100
0.7/3.0
Ind
Ind
Ind
Ind
Ind
Ind
Ind
Ind
Ind
10r
10s
Ind
Ind
60.8/98.3
Ind
Ind
Ind
61.2/99.6
Ind
Ind
Ind
48.9/88.5
Ind
Ind
Ind
64.2/>100
Ind
Ind
Ind
>100
>100
>100
>100
60
51.6/>100
50.0/P 100
48.9/P 100
2.0/7.6
Ind
10t
10u
Ind
Ind
Ofloxacin
Ciprofloxacin
Isoniazid
Streptomycin
Rifampin
Ethambutol
Clotrimazole
Econazole
1.1/7.2
2.7/15.2
Ind
0.6/2.1
0.4/1.1
0.07/1.1
Ind
1.6/3.4
1.3/3.2
Ind
0.9/2.8
0.7/2.4
0.1/2.4
0.1/0.3
0.6/1.1
4.3/24.2
Ind
1.1/2.9
0.9/2.4
0.1/0.4
0.1/0.3
0.4/1.0
Ind
1.0/4.0
2.3/8.1
0.4/3.0
0.2/1.1
>100
>100
>100
>100
>100
>100
0.2/0.36
3.2/11.7
0.96/2.6
42.2/>100
19.6/62.3
15.7/>100
0.7/5.9
Ind
Ind
Ind
0.01/0.06
0.1/0.3
2.7/6.6
0.08/0.3
0.6/1.0
5.0/>100
Ind
0.2/1.1
2.3/9.7
Ind
20.4/61.2
12.5/52.1
Ind
13.1/30.9
18.2/30.7
13.7/28.3
20.0/32.3
a Compound concentration (lM) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined by the MTT method.
b Minimum inhibitory concentration (lM) required to reduce the number of viable mycobacteria by 50%, as determined by the MTT method.
c Minimum inhibitory concentration (lM) required to reduce the number of viable mycobacteria by 90%, as determined by the MTT method.
d Inactive up to MIC50 P 100lM.

G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
5481
Table 9. Absolute and local minima calculated for 6e,i,j, 7e,i,j, 9e,i,j,
10e,i,j
3days (dermatophytes). MIC was defined as the com-
pound concentration at which no macroscopic sign of
fungal growth was detected. The minimal germicidal
concentrations (MBC or MFC) were determined by sub-
cultivating in Sabouraud dextrose agar samples from
cultures with no apparent growth.
Conformer
Angle s1 (ꢁ)
Angle s2 (ꢁ)
E (kJ/mol)
6e_Ia
6e_Ib
6e_IIa
6e_IIb
6i_Ia
6i_Ib
6i_IIa
6i_IIb
6j_Ia
6j_Ib
6j_IIa
6j_IIb
7e_Ia
7e_IIa
7i_Ia
7i_IIa
7j_Ia
7j_IIa
9e_a
0
190
0
260
259
100
110
241
240
98
154.7
159.1
155.0
158.2
176.6
179.0
177.4
180.6
144.6
146.9
149.3
150.0
196.2
196.2
220.0
215.4
186.0
188.0
194.3
196.1
213.3
220.5
183.7
189.0
197.0
199.1
216.5
222.9
186.2
191.6
172
5
4.2.5. Linear regression analysis. Viral and cell growth at
each drug concentration was expressed as percentage of
untreated controls and the concentrations resulting in
50% (EC50, CC50) growth inhibition were determined
by linear regression analysis.
194
0
168
0
104
260
258
90
180
0
4.3. Molecular modeling
159
142
217
264
240
90
118
245
115
247
69
Molecular structures of ligands were built and energy
minimized within MacroModel.28 Conformational anal-
ysis was carried out using the AMBER* force field, as
included in MacroModel. The initially minimized struc-
ture was used as a starting point to perform a systematic
search using the DRIVE option implemented in Macro-
Model. Torsional angles s1 and s2 (Fig. 5) were in-
creased by 5ꢁ increments in the range 0ꢁ–360ꢁ and the
obtained geometries were fully relaxed. Since a third tor-
sional angle should be taken into account for a complete
conformational analysis of these compounds, a more
accurate exploration of the conformational space was
done by random search, using the Monte Carlo option
implemented in MacroModel. Starting from one of the
lowest energy conformations retrieved with the previous
strategy, several Monte Carlo cycles were run. For each
cycle the following parameters were used: automatic set-
up, maximum number of search interactions 1000,
energy cut-off set differently to 5, 10, 20, 30kcal/mol. Only
in the case of an energy cut-off set to 30kcal/mol, the
high energy barrier could be transpassed and from con-
formation ÔaÕ, namely the absolute minimum, conforma-
tion ÔbÕ could be found among the possible results for all
compounds examined.
290
60
260
100
269
79
180
171
169
171
168
171
279
80
9e_b
9i_a
9i_b
9j_a
259
81
9j_b
249
160
73
10e_a
10e_b
10i_a
10i_b
10j_a
10j_b
90
90
162
60
276
80
158
289
for 18h in the presence of serial dilutions of test com-
pounds.
4.2.3. Antimycobacterial assays. Mycobacterial strains
were grown in Middlebrook 7H9 medium with 0.5%
glycerol and 10% albumine-dextrosecatalase (ADC)
enrichment (Microbiol diagnostici, Italy). MICs were
assessed in microtitre plates by adding 20lL aliquots
of a culture suspension [whose turbidity was equal to
that of a No 1 McFarland standard containing 108
colony forming units (CFU)/mL] to 80lL of the same
medium containing various concentrations of test com-
pounds. Plates were then incubated for 1day (M.
smegmatis and M. fortuitum) or 9days M. tuberculosis)
at 37ꢁC. At the end of incubation, the number of viable
mycobacteria was determined by the MTT method, as
already reported.25
For all compounds, the resulting geometries of all the
lower energy conformers were re-optimized with semi-
empirical quantum mechanic calculations, using the
Hamiltonian AM1 as implemented in Spartan.29 In the
case of compounds 10j and 10o atomic charges calcu-
lated with AM1 were used to evaluate the corresponding
isopotential contour surfaces.
The crystallographic coordinates of CYP121 and
CYP51 from M. tuberculosis were obtained from the
Brookhaven Protein Databank, entries 1N4O19 and
1EA1.24
4.2.4. Antimycotic assays. Yeast inocula were obtained
by properly diluting cultures incubated at 37ꢁC for
30h in Sabouraud dextrose broth to obtain
5 · 103 cells/mL. On the contrary, dermatophyte inocula
were obtained from cultures grown at 37ꢁC for 5days in
Sabouraud dextrose broth by finely dispersing clumps
with a glass homogenizer and then diluting to 0.05
OD590/mL. Then, 20lL of the above suspensions were
added to each well of flat-bottomed microtitre trays con-
taining 80lL of medium with serial dilutions of test
compounds, and were incubated at 37ꢁC. Growth con-
trols were visually determined after 2days (yeast) or
The structural models of selected compounds, were
docked into the active site of the cytochrome using
FlexX.20 Preparation of the protein for FlexX requires
definition of the binding pocket in terms of Ôinteraction
pointsÕ. In this work the active sites were defined as all
˚
atoms within a distance of 10A from the iron atom of
the haem. The specific distance was determined in order
to ensure a significant portion of the active site for the
docking experiments. The results were evaluated in
terms of total estimated binding energy (FlexX total

5482
G. Menozzi et al. / Bioorg. Med. Chem. 12 (2004) 5465–5483
Figure 6. Ramachandran plots of compound 6j, 7j and their identified conformers. Different colours define different energetical areas. Colour code
(kJ/mol): violet 154.0, dark-blue 172.0, green-blue 190, turquoise 208.0, light-green 226.0, dark-green 244.0, yellow 262.0, orange 280.0, brown 298.0,
red 316.0.
P. M.; Reid, G. A.; Chapman, S. K.; Walkinshaw, M. D.
Acta Cryst. 2002, D58, 704.
6. Munro, A. W.; McLean, K. J.; Marshall, K. R.; Warman,
A. J.; Lewis, G.; Roitel, O.; Sutcliffe, M. J.; Kemp, C. A.;
Modi, S.; Scrutton, N. S.; Leys, D. Biochem. Soc. Trans.
2003, 31, 625.
7. Penso, J.; Beitner, R. Eur. J. Pharmacol. 1998, 342,
113.
8. Koefeler, H. C.; Fauler, G.; Windischhofer, W.; Leis, H. J.
Br. J. Pharmacol. 2000, 130, 1241.
score) and lipophilicity interactions (FlexX lipophilicity
score). Since the macromolecule has a high degree of
hydrophobic residues into the catalytic core,3 this latest
parameter, decoding for hydrophobic interactions, has
been taken into particular account. All selected com-
plexes underwent to a final energy minimization, before
the model would be achieved within Charmm as imple-
mented in INSIGHT-II.30
All calculations were carried out on SGI O2 worksta-
tions and on a standard personal computer running
under Linux.
9. Di Santo, R.; Massa, S.; Costi, R.; Simonetti, G.; Retico,
A.; Apuzzo, G.; Troccoli, F. Farmaco 1994, 49, 229 (and
references cited therein).
10. Menozzi, G.; Mosti, L.; Bruno, O.; Lo Presti, E.; Musiu,
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C.; La Colla, P. Farmaco 2001, 56, 633.
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Acknowledgements
We would like to thank the ÔMinistero dellÕIstruzione,
`
dellÕUniversita e della RicercaÕ for its financial support.
13. Rossello, A.; Bertini, S.; Lapucci, A.; Macchia, M.;
Martinelli, A.; Rapposelli, S.; Herreros, E.; Macchia, B.
J. Med. Chem. 2002, 45, 4903.
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