Synthesis, antifungal and antimycobacterial activities of new bis-imidazole derivatives, and prediction of their binding to P45014DM by molecular docking and MM/PBSA method

Article abstract of DOI:10.1016/j.bmc.2007.07.023

New bis-imidazole derivatives have been synthesized and their antifungal and antimycobacterial activity was determined. Almost all compounds exhibited a moderate to good activity against two clinical isolates of Candida albicans 3038 and Candida glabrata

Full text of DOI:10.1016/j.bmc.2007.07.023

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 15 (2007) 7444–7458
Synthesis, antifungal and antimycobacterial activities of new
bis-imidazole derivatives, and prediction of their binding
to P45014DM by molecular docking and MM/PBSA methodq
Daniele Zampieri,a Maria Grazia Mamolo,a,* Luciano Vio,a Elena Banfi,b
Giuditta Scialino,b Maurizio Fermeglia,c Marco Ferronec and Sabrina Priclc
aDepartment of Pharmaceutical Sciences, Piazzale Europa 1, University of Trieste, 34127 Trieste, Italy
bDepartment of Biomedical Sciences, Microbiology Section, Via A. Fleming 32, University of Trieste, 34127 Trieste, Italy
cDepartment of Chemical, Environmental and Raw Materials Engineering, Piazzale Europa 1, University of Trieste,
34127 Trieste, Italy
Received 6 February 2007; revised 12 June 2007; accepted 10 July 2007
Available online 22 August 2007
Abstract—New bis-imidazole derivatives have been synthesized and their antifungal and antimycobacterial activity was determined.
Almost all compounds exhibited a moderate to good activity against two clinical isolates of Candida albicans 3038 and Candida glab-
rata 123. The same compounds showed an interesting killing activity against Mycobacterium tuberculosis H37Rv reference strain.
Docking procedures combined with molecular dynamics simulations in the MM/PBSA framework of theory were applied to predict
the binding mode of all compounds in the binding pocket of the cytochrome P450 14a-sterol demethylase (14DM) of C. albicans, for
which no ligand–protein crystal structure is currently available. The results obtained in silico showed that the active compounds may
interact at the active site of protein, and that their binding free energy values are in agreement with the corresponding experimental
activity values.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
inhibiting concentrations (MICs) to those of two
reference drugs, miconazole and amphotericin B,
respectively.
The increasing incidence of severe systemic mycoses fre-
quently represents a significant cause of morbidity and
mortality in immunocompromised patients, such as
those affected by AIDS, cancer, or who received an or-
gan transplant. Azoles are the most widely studied and
currently used class of antifungal agents. However, the
emergence of azole resistant strains has spurred the
search for new antimycotic compounds. With the aim
of obtaining new antifungal compounds, we synthesized
the series of the bis-imidazole derivatives 1a–m (Table
1). The in vitro activities of derivatives 1a–m were tested
against two clinical strains of Candida albicans 3038 and
Candida glabrata 123, by comparing their minimal
The cytochrome P450-dependent lanosterol 14a-
demethylase (P45014DM, CYP51) is the target enzyme
for azole antifungal agents in the ergosterol biosynthesis
pathway. Azoles inhibit P45014DM causing accumulation
of methylated sterols, depletion of ergosterol, and inhi-
bition of cell growth. Because it was established, using
genomic DNA from Mycobacterium tuberculosis (MT)
H37Rv strain, that (i) a CYP-like gene encodes a bacte-
1
rial sterol 14a-demethylase (MT P45014DM
)
which
binds 14a-methyl sterols and azole inhibitors2 of
P45014DM
,
3,4 and (ii) many imidazole derivatives showed
antimycobacterial activity associated with antifungal
activity,5–8 we tested compounds 1a–m also for their
possible antimycobacterial activity against a strain of
M. tuberculosis H37Rv. In this case, rifampicin was used
as a reference compound.
Keywords: Bis-imidazole; Antifungal and antimycobacterial activity;
Docking; Free energy of binding.
q
A preliminary account of this work was presented at Second Joint
Italian–Swiss Meeting on Medicinal Chemistry, Modena, September
12–16, 2005.
Corresponding author. Fax: +39 04052572; e-mail: mamolo@units.it
This series of new inhibitors also offers a significant com-
putational challenge to quantitatively evaluate the bind-
*
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.023

D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
Table 1. Yields and physical data of the synthesized compounds 1a–m
7445
O
C
N
N
N
R
N
1 a-h
Compound
R
Mp (°C)
Yield (%)
Yield Microwave (%)
Rf (CHCl3–EtOH) (8:2)
Anal. (C, H, N)
MW
1a
1b
1c
1d
1e
1f
H
Br
212–5
>270
>270
>270
240–2
222–4
232–4
237–9
10
11
8
32
50
39
44
42
69
71
43
0.18
0.20
0.17
0.20
0.25
0.38
0.34
0.40
C16H16N4O
C16H15BrN4O
C16H15ClN4O
280.33
359.22
314.77
298.32
294.35
310.35
325.35
356.43
Cl
F
7
C16H15FN4O
C17H18N4O
C17H18N4O2
C16H15N5O3
C22H20N4O
CH3
OCH3
NO2
Ph
13
36
24
11
1g
1h
O
S
N
N
R
N
N
1 i-k
Compound
R
Mp (°C)
Yield (%)
Yield Microwave (%)
Rf (CHCl3–EtOH) (8:2)
Anal. (C, H, N)
C14H14N4OS
MW
1i
H
258–60
>270
249–51
18
5
55
32
36
0.25
0.21
0.20
286.36
365.25
320.80
1j
1k
Br
Cl
C14H13BrN4OS
C14H13ClN4OS
9
OH
N
N
R
N
N
1 l-m
Mp (°C)
R
Compound
Yield (%)
Rf (CHCl3–EtOH) (8:2)
Anal. (C, H, N)
MW
4-Br
4-OCH3
1l
1m
30
36
180–2
155–7
0.26
0.23
C16H17BrN4O
C17H20N4O2
361.24
312.37
ing affinity to its target receptor. Although all molecules
share a common scaffold, the substituents attached to the
scaffold are quite diverse. Accordingly, in this work we
calculate the free energy of binding of the synthesized
inhibitors to cytochrome P450 14a-sterol demethylase
(14DM) of C. albicans using the so-called Molecular
Mechanics/Poisson–Boltzmann Surface Area (MM/
PBSA) method.9 This method is one of a class of ap-
proaches that attempts to include free energy terms di-
rectly into
a molecular simulation. Basically, it
combines explicit solvent molecular dynamics (MD) sim-
ulations with implicit solvation models, Poisson–Boltz-
mann (PB) analysis,10 and solvent accessible surface
area-dependent nonpolar solvation free energy calcula-

7446
D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
tions11 to estimate free energies. A set of snapshots along
the MD trajectory for each drug/enzyme complex are
saved as representative conformations of the macromol-
ecule/ligand system. The set of structures is then postpro-
cessed with the explicit solvent replaced by a continuum
solvent model. The free energy of the system then con-
sists of the molecular mechanics potential energy of the
complex, solvation free energy, and an entropy term
for the complex. The solvation free energy, in turn, is
composed of an electrostatic (or polar) portion, esti-
mated by solving the PB equation, and a nonpolar solva-
tion contribution, associated with the formation of a
protein-sized cavity in the solvent, and van der Waals
interactions between the complex and the solvent. There-
fore, we have two further goals for this paper. First, we
calculate free energies of association, relate these values
to the corresponding experimental MIC, and provide in-
sights into the interaction mechanism of the active site of
P45014DM and the synthesized compounds. Second, we
exploit all relevant information and explore modifica-
tions of these compounds that might result in a second-
generation of new, more potent inhibitors.
substituted acetophenones with paraformaldehyde
and the corresponding secondary amines in acetic
acid according to published procedures.12a–c Under
these reaction conditions the intermediate 1-aryl-2-
[(dimethylamino)methyl]-propenones 2a–h and 2-
[(dimethylamino)methyl]-1-(thiophene-2-yl)-propenones
2i–k were prepared as hydrochlorides (Table 2) by
treating the corresponding substituted arylmethylke-
tones and thienylmethylketones with paraformalde-
hyde and dimethylamine hydrochloride in acetic
acid. The 1-aryl-3-(1H-imidazol-1-yl)-2-[(1H-imidazol-
1-yl)methyl]-propan-1-one derivatives 1a–h and 3-
(1H-imidazol-1-yl)-2-[(1H-imidazol-1-yl)methyl]-1-(thiophen-
2-yl)-propan-1-one derivatives 1i–k (Table 1) were
prepared (Scheme 1) from propenone derivatives 2a–
h and 2i–k by nucleophilic attack of imidazole both
on carbon bearing the dimethylamino group and
a,b-unsaturated ketone moiety via a Michael type
reaction. Compounds were obtained in higher yields
(Table 1) by microwave (MW) irradiation of the re-
agent mixture in EtOH–H2O at room temperature.
The optimal conditions were found to be 2 min of
irradiation in open vials at 450 W for 5 times corre-
sponding to a total time of 10 min. The conventional
synthetic procedure required heating under reflux for
reaction times varying from 12 to 36 h and, as al-
ready pointed out, the corresponding yields were
quite lower (Table 1).
2. Chemistry
1-Aryl-2-[(dialkylamino)methyl]-propenones can be
prepared by Mannich reaction of the corresponding
Table 2. Yields and physical data of the synthesized compounds 2a–k
O
C
CH3
N
. HCl
CH2
CH3
R
2 a-h
Compound
R
Mp (°C)
Yield (%)
Rf (CHCl3–EtOH) (8:2)
Anal. (C, H, N)
MW
2a(a)12b
2b(a)13
2c(a)12b
2d
H
Br
156–9
180–2
176–9
170–2
140–2
149–51
200–02
182–4
48
41
53
47
55
54
65
62
0.18
0.20
0.17
0.20
0.25
0.38
0.34
0.40
C12H16ClNO
C12H15BrClNO
C12H15Cl2NO
C12H15ClFNO
C13H18ClNO
C13H18ClNO2
C12H15ClN2O3
C18H20ClNO
225.71
304.61
260.17
243.71
239.76
255.75
270.72
301.82
Cl
F
2e(a)13
2f(a)12b
2g
CH3
OCH3
NO2
Ph
2h(a)14
O
C
CH3
S
N
. HCl
R
CH2
CH3
2 i-k
Compound
R
Yield (%)
Mp (°C)
Rf (CHCl3–EtOH) (8:2)
Anal. (C, H, N)
MW
2i
H
69
73
48
150–2
186–8
185–7
0.25
0.21
0.40
C10H14ClNOS
C10H13BrClNOS
C10H13Cl2NOS
231.74
310.64
266.19
2j
2k
Br
Cl
(a)These compounds have been previously synthesized but not fully characterized on the basis of spectral data.

D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
7447
O
O
R
R = H, Br, Cl, F,CH3,
R = H, Br, Cl
OCH3, NO2, Ph
R
S
CH3
CH3
[CH2O]n
[CH2O]n
AcOH
AcOH
H
N
H
N
H3C
CH3
H3C
C
H3
O
C
O
C
CH3
CH3
N
N
* HCl
* HCl
S
CH2
2 i-k
CH3
CH2
CH3
R = H, Br, Cl
R
R
R = H, Br, Cl, F,CH3,
OCH3, NO2, Ph
2a-h
NH
1. EtOH-H2O
2. MW 250W
NH
1. EtOH-H2O
2. MW 250W
N
N
O
N
O
N
N
N
N
N
S
R
R = H, Br, Cl, F,CH3,
OCH3, NO2, Ph
R
R = H, Br, Cl
NaBH4
N
N
1 i-k
1 a-h
OH
N
R = Br, OCH3,
N
R
N
N
1 l-m
Scheme 1.
The reduction of the above obtained ketone derivatives
1 b and 1f with NaBH4 produced the secondary alcohols
1l,m (Table 1, Scheme 1).
which the biphenylyl moiety is present, exhibited a good
inhibitory activity against the clinical strain of C. albi-
cans 3038, with MIC values of 2 and 4 lg/mL after
24 h and 48 h, respectively. The activity was higher than
that of the reference drug miconazole and similar to the
activity of amphotericin B. The antifungal activity of the
corresponding derivatives 1a–g and 1i–k against the
strain of C. albicans 3038 was moderate, with MIC val-
ues ranging from 16 to 128 lg/mL and 32 to 128 lg/mL
after 24 h and 48 h, respectively. The antifungal activi-
ties of compounds 1a–k against the clinical strain of
C. glabrata 123 were characterized by a similar profile,
3. Results and discussion
A series of bis-imidazole derivatives 1a–m (Table 1) have
been synthesized with the aim to evaluate their anti-
fungal and antimycobacterial activities. The results of
the in vitro evaluation of antifungal activity of com-
pounds are reported in Table 3. The derivative 1h, in

Table 3. Activity of compounds 1a–m against Candida albicans 3038, Candida glabrata 123, clinical strains and against Mycobacterium tuberculosis reference strain H37Rv
O
C
OH
CH
O
C
N
N
N
N
N
N
S
N
N
R
R
N
R
N
N
N
1 l-m
1 a-h
1 i-k
Compound
R
MIC (lg/mL)
Compound
R
H
MIC (lg/mL)
Compound
R
MIC (lg/mL)
C. albicans
C. glabrata
M. tuberc.
C. albicans
C. glabrata
M. tuberc.
H37Rv
C. albicans
C. glabrata
M. tuberc.
H37Rv
3038
123
H
37Rv
3038
123
3038
123
24 h 48 h 24 h
48 h
24 h 48 h 24 h
48 h
24 h 48 h 24 h
48 h
1a
1b
1c
1d
1e
1f
H
64
16
16
64
64
64 128
32 32
32 32
128
16
16
16
16
8
2i
128 128 128
128
128
128
16
16
16
1l
1m
Br 128
OCH3 128
>128 128
>128 128
>128
>128
8
64
Br
Cl
64
64
2j
2k
Br 64
Cl 64
64
64
64
64
F
CH3
128 128
128 128
128 128
>128 128
128
128
128
>128
8
OCH3 64
16
64
8
1g
1h
NO2
Ph
128
2
4
4
Amphotericin B
Miconazole
1
2.5
2
5
0.5
1.25
2 Rifampicin Amphoteric.B
2.5 1 mg/disk Miconazole
1
2.5
2
5
0.5
1.25
2 Rifampicin Amphoteric.B
2.5 1 mg/disk Miconazole
1
2.5
2
5
0.5
1.25
2 Rifampicin
2.5 1 mg/disk

D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
7449
˚
and only compound 1h exhibited a remarkable anti-
fungal activity against the tested strain, reaching MIC
values of 4 lg/mL and 8 lg/mL after 24 h and 48 h,
respectively. These MIC values were not very dissimilar
from those of the reference drugs (Table 3).
inhibitor is equal to 0.08 A. In the light of these
blank-test results, and of the fact that this computa-
tional ansatz was already successfully applied by us to
predict the activity of allosteric inhibitors of HCV
RNA-dependent RNA-polymerase,19 and of other series
of 14DM inhibitors,8,20 the conceived modeling/docking
procedure was applied for predicting the binding mode
of the new compounds 1a–m to 14DM of C. albicans.
Compounds 1l and 1m, with the hydroxylic function
replacing the carbonyl group, were weakly active after
24 h, and devoid of antifungal activity on both Candida
tested strains after 48 h. In summary, only compound
1h, substituted with a phenyl group at position 4 of
the benzene ring characterizing compounds 1a–h, exhib-
ited a considerable antifungal activity.
The Ca RMSD of each complex from the docked struc-
˚
ture for each MD simulation ranges from 0.48 to 1.09 A.
The all-atom RMSD of compounds 1a–m in the com-
plex from the initial docked models are in the range of
˚
1–1.5 A, which indicates that the initial conformations
Compounds 1a–m were also tested for antitubercular
activity against the reference strain of M. tuberculosis
H37Rv, in comparison with rifampicin. The results of
the in vitro antimycobacterial activity of compounds
are reported in Table 3. All compounds exhibited a
moderate activity, with MIC values in the range 8–
64 lg/mL.
of these molecules generated according to the computa-
tional recipe described above are reasonable. Finally,
the RMSD of the common inhibitor scaffold (–C–CH–
˚
(CH2-imidazole)2) is less than 1 A. Accordingly, all these
indications speak further in favor of the quality of the
docking procedure adopted also for this set of new com-
pounds (as well as for miconazole, see below).
Accordingly to the results obtained for other imidazole
derivatives 8,15–17 the newly synthesized imidazole deriv-
atives showed both in vitro antifungal and antimycobac-
terial properties. Particularly, the antimycobacterial
activity of compound 1h was comparable to its anti-
fungal activity towards the employed Candida strains,
suggesting the presence of similar enzymatic interaction
sites. To support this hypothesis, we performed a com-
putational study of the molecular bases of the interac-
tions between the synthesized compounds and the
active site of the enzyme P45014DM with the additional,
ultimate goal of exploiting this information in the design
of further, more active inhibitors.
One underlying assumption in our computational recipe
is that both 14DM and its inhibitors undergo only a lim-
ited conformational change in going from the unbound
to the bound state. The fluconazole blank-test reported
and discussed above strongly supports this hypothesis.
To verify the confined conformational changes of inhib-
itors 1a–m upon binding to 14DM, however, we per-
formed an MD simulation of 1h in a box of water. In
this case, we applied particle mesh Ewald summation21
˚
to treat long-range electrostatics, and with a 9 A cutoff
for the short-range nonbonded interactions. The simula-
tion time was selected equal to the MM/PBSA produc-
tion time, i.e. 400 ps. We verified that, during the MD
˚
run, the all-atom average RMSD is equal to 1.5 A, as
Generally speaking, there are two key requirements for
the computer-aided structure-based drug design meth-
ods: (i) the generation of correct conformations of
docked ligands, and (ii) the accurate prediction of bind-
ing affinity. To check whether our procedure complied
with requirement (i), we modeled and docked fluconaz-
ole, for which the crystallographic structure in complex
with 14DM of M. tuberculosis is available,18 as a refer-
ence system. This was basically done by removing the
inhibitor from the enzyme allosteric site, building a
new molecular model for this compound, applying the
conformational procedure described in details in Section
5 for the synthesized compounds, and finally docking it
back into the protein binding pocket. The best docked
structure, which is the configuration with the lowest
docking energy in a prevailing cluster, was then com-
pared with the corresponding crystal structure. Figure
1(a) and (b) shows a comparison between the co-crystal-
lized conformation of fluconazole into the allosteric
binding site of 14DM from M. tuberculosis and the
docked conformation obtained upon application of the
computational strategy adopted in this work.
could be expected for molecules like these, characterized
by a relatively rigid structure (see Fig. 2). Therefore, we
think that our original assumption of minimal confor-
mational change was utterly reasonable.
Interestingly, all compounds 1a–m and miconazole were
characterized by a similar docking mode in the active
site of 14DM from C. albicans (see Fig. 3(a) and (b)).
One of the two imidazole rings (1IR) of the inhibitors,
and the corresponding moiety of miconazole, lies almost
perpendicular to the heme group, with a ring nitrogen
atom just above and coordinating the metal component
of the prosthetic group.
The analysis of the MD trajectories reveals that the
average dynamic distance (ADD) distance between
N3(4) of the azole ring and the heme iron is
˚
2.06 0.5 A, in good agreement with that found in the
crystal structure of P450cam complexed with azole
inhibitors.22 Further stabilizing nonbonded interactions
are established between the same azole ring (1IR) of
each drug and the side chains of residues G254, M257,
G258, and T262. The phenyl group of all compounds,
as well as one of the aromatic moieties of miconazole,
locate in a hydrophobic subsite above the prosthetic
group. This subsite is delimited by the side chains of
the ceiling residues F186, P187, and H261 on one side,
At a first glance it can be seen that the agreement be-
tween the two structures is excellent: the root-mean-
square deviation (RMSD) between the docked configu-
ration and the relevant crystal structures of this test

7450
D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
Figure 1. Comparison between the co-crystallized conformation of fluconazole into the active site of 14a-sterol demethylase from Mycobacterium
tuberculosis (a), and the corresponding docked conformation of fluconazole in the same enzyme pocket obtained upon application of the
computational strategy adopted in this work (b). The inhibitor molecules are shown as a stick model, and the atom color coding is as follows: carbon,
gray; nitrogen, blue, oxygen, red; and chlorine, green. The heme group is also represented in sticks, with the iron ion depicted as a pink ball.
Hydrogen atoms and water molecules are omitted for clarity.
miconazole, are both stabilized by favorable dispersion
forces with the side chains of K98, L354, V355, P357,
and Y359. Interestingly, all structures of each 14DM/
drug complex, as resulting from our validated docking/
optimization procedure on similar enzyme/compound
series,8,20 did not predict any complex configuration fea-
turing 2IR in coordination with the heme ion. We found
that a sensible, neat reason for lies in the unfavorable
steric reconfiguration the inhibitors must undergo to
accommodate the CH2-2IR moiety in a productive posi-
tion in the active site (data not shown).
Finally, we verified that both the carbonyl (1a–h and 1i–
k) and hydroxyl oxygen atom (1l–m) of the synthesized
compounds form a hydrogen bond with the –OH group
of the T262 side chain, with an average dynamic dis-
˚
tance of 2.82 A.
Figure 2. Superposition of the average structures of 1h in water (pink)
and in complex with 14DM (green).
Further insights and more quantitative information
about the forces involved in substrate binding can be ob-
and V460, V461, and L462 on the other. In an analo-
gous fashion, the second imidazole ring (2IR), common
to all inhibitors, and the halogenated phenyl ring of
tained by analyzing the values of the free energy of bind-
ing DGBIND and its components, which are listed in
Table 4 for the entire ligand family.

D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
7451
DGPOLÞ contribution for all molecules reported in Table
4. The DGAPOL contributions to DGBIND for the 1i–k/
14DM complexes are less favorable (from 3 to 6 kcal/
mole) than for the remaining complexes, because of a
less negative van der Waals term. The analysis of the
MD trajectories and the relevant 3D models reveals that
the presence of the thiophene ring induces a non-optimal
sterical configuration of the global molecules for bind-
ing, ultimately resulting in the lowest energy contribu-
tions of both electrostatic and van der Waals terms
DGBIND. Interestingly, however, the desolvation penal-
ties upon binding of these inhibitors are also lower.
Thereby, the net electrostatic contributions to 14 DM
binding for 1i–k are not significantly unfavorable, and
their DGBIND values are comparable to those of the
other molecules of this series.
Compound 1h (see Fig. 3(a)) exhibits the highest affinity
toward 14DM of C. albicans, with DGBIND = ꢀ7.36 k-
cal/mol. Besides the several van der Waals and hydro-
phobic interactions this molecule can form with the
residues making up the enzyme binding site, common
to all inhibitors, Figure 3(a) reveals a further, important
p–p stabilizing interaction occurring between both aro-
matic rings of 1h and the phenyl side chain of F186,
resulting from the parallel-displaced geometry of these
residues. The electronic nature of the p–p interactions
indeed favors the stacking of aromatic rings either by
parallel-displaced (off-center) or edge-on (T-stacking)
geometries, while the face-to-face geometry is unfavor-
able (particularly in environments where there is a low
effective dielectric constant), since the dominant interac-
tion is p-electron repulsion. The average dynamic dis-
tances of the F186 aromatic ring from the two phenyl
˚
substituents of 1h are 3.7 A for the aromatic moiety di-
˚
rectly linked to the scaffold and 4.7 A for the aromatic
ring as a substituent in 1h only, respectively.
Finally, although we cannot directly correlate the calcu-
lated binding energies to the experimental MIC values,
the trend exhibited by our DGBIND values and the
MIC is in good agreement, with a correlation coefficient
equal to 0.92. Importantly, the calculated free energy for
miconazole confirms that this reference drug is the tigh-
ter binder of 14DM as expected.
Figure 3. Equilibrated molecular dynamics snapshot of the docked
compound 1h (a), and the reference compound miconazole (b) in the
active site of 14DM of C. albicans. The heme group and the amino
acids pertaining to the binding site or contacting the ligands are shown
in atom-colored sticks, and the ligands are in orange- (1h, (a)) and
green-colored (miconazole, (b)) stick representation. Water molecules
and hydrogen atoms are omitted for clarity.
As we can see from Table 4, both the intermolecular van
der Waals and the electrostatics are important contribu-
tions to the binding. However, the electrostatic desolva-
These results allowed us to be confident that the tech-
niques and procedures adopted in this work for docking
compounds 1a–m to the active site of 14DM, and esti-
mating their free energy of binding, were accurate en-
ough to be used in a multistep virtual screening
protocol. Under this perspective, we used the PROFEC
suite of programs23 to study how the most active inhib-
itor of this series, 1h, could be modified to further im-
prove its binding affinity toward 14DM. PROFEC is a
set of software tools for carrying out and displaying
extrapolative free energy calculations. Specifically, the
PROFEC software suite calculates the free energy for
inserting a specified test particle at a grid of points near
the ligand of interest. A weighted electrostatic potential
is also calculated for each position on this grid. These
two (van der Waals and electrostatic) grids can be visu-
alized and overlaid on a three-dimensional structure of
SOLV
tion penalty ðDG
Þ
offsets the favorable (i.e.,
ELE
negative) intermolecular electrostatics, yielding an unfa-
vorable net electrostatic contribution to the compound
affinities for the enzyme. As discussed above, the com-
mon molecular scaffold forms only a single, persistent
hydrogen bond with the surrounding residues; the
intermolecular electrostatic interactions are accordingly
weak. Therefore, as previously verified for similar series
of compounds,8,20 the association between 1a–m and
cytochrome P45014DM is mainly driven by more
favorable
nonpolar
interactions
in
the
complex than in solution. This result was better evi-
denced by comparing the van der Waals/nonpolar
SOLV
NP
MM
vDW
ðDE
þ DG
¼ DGAPOLÞ and ðDEEMLME þ DGESOLLEV
¼

7452
D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
Table 4. Free energy components and total binding free energies for compounds 1a–m
SOLV
SOLV
MM
ELE
MM
vDW
Compound
DE
DE
DEMM
DG
ELE
DGNP
DGSOLV
TDS
DGBIND
1a
1b
1c
1d
1e
1f
ꢀ34.48
ꢀ35.98
ꢀ36.41
ꢀ36.06
ꢀ34.01
ꢀ35.02
ꢀ36.49
ꢀ35.87
ꢀ31.18
ꢀ31.72
ꢀ31.51
ꢀ36.99
ꢀ36.06
ꢀ32.01
ꢀ32.64
ꢀ33.11
ꢀ33.00
ꢀ31.05
ꢀ32.17
ꢀ30.91
ꢀ34.01
ꢀ27.51
ꢀ28.54
ꢀ28.17
ꢀ31.06
ꢀ31.59
ꢀ66.49
ꢀ68.62
ꢀ69.52
ꢀ69.06
ꢀ65.06
ꢀ67.19
ꢀ67.40
ꢀ69.88
ꢀ58.69
ꢀ60.26
ꢀ59.68
ꢀ68.05
ꢀ67.65
49.45
51.71
53.43
52.65
49.56
52.44
53.65
50.93
43.05
44.77
44.01
53.72
53.91
ꢀ3.53
ꢀ3.25
ꢀ3.19
ꢀ3.57
ꢀ3.31
ꢀ3.47
ꢀ3.57
ꢀ3.18
ꢀ3.54
ꢀ3.21
ꢀ3.44
ꢀ3.15
ꢀ3.24
45.92
48.46
50.24
49.08
46.25
48.97
50.08
47.75
39.51
41.56
40.57
50.57
50.67
14.67
14.06
12.74
13.98
13.09
12.56
13.01
14.77
14.72
13.31
13.84
13.45
12.78
ꢀ5.90
ꢀ6.10
ꢀ6.54
ꢀ6.00
ꢀ5.72
ꢀ5.66
ꢀ4.31
ꢀ7.36
ꢀ4.46
ꢀ5.39
ꢀ5.27
ꢀ4.03
ꢀ4.20
1g
1h
1i
1j
1k
1l
1m
Miconazole
ꢀ42.45
ꢀ47.18
ꢀ89.63
68.31
ꢀ3.45
64.86
14.89
ꢀ9.88
All energies are in kcal/mol.
the ligand or ligand–receptor complex to suggest posi-
tions where modifications to the ligand would improve
binding. The main strengths of PROFEC are its unbi-
ased evaluation of possible modifications and the ability
to explicitly include the effect of solvation in the analy-
sis. By postprocessing the MD trajectories of 1h in solu-
tion and in complex with 14MD, PROFEC estimated
the free energy cost of adding a test group to the inhib-
itor. One of the most interesting modifications resulting
from running PROFEC is in the para and one ortho
positions of the phenyl ring substituent of 1h, as shown
in Figure 4(a).
As suggested by PROFEC analysis, both p-H and o-H
should be replaced with two hydroxyl groups: in fact,
as seen in Figure 4(b), the new p-OH group can
potentially form a hydrogen bond with the nitrogen
atom of the peptidic bond between V461 and L462,
whilst the o-OH will eventually be stabilized by a
bifurcated H-bond with the carbonyl oxygen of the
CONH moiety linking P187 and N188, and with the
CO of the aminoacidic bond between again P187
and F186, respectively. Therefore, by applying the
RESP fitting scheme,24 we kept all charges of the
inhibitor fixed except for the two, new modified
groups. Next, we carried out a new molecular dynam-
ics simulation of the new designed compound 1n in
complex with 14DM by applying the same computa-
tional protocol described above. Table 5 lists the free
energy of binding for 1n.
It is well evident that, when p-H and o-H are replaced by
two –OH groups, both the intermolecular van der Waals
and electrostatic interactions become more favorable,
although, as expected from the introduction of two po-
lar groups, more desolvation penalty has to be paid.
Accordingly, the increase in favorable DE with re-
spect to 1h (2.47 kcal/mol, see Tables 4 and 5) is more
MM
ELE
Figure 4. (a) Model of compound 1h showing the potential sites for
modification as suggested by PROFEC. (b) Details of compound 1n
resulting from design according to PROFEC indications (see text for
details). The new, potential hydrogen bonds are represented by green
lines.
SOLV
ELE
is increased
than cancelled out by the unfavorable DG
MM
vDW
(5.70 kcal/mol); on the other hand, DE
favorably by 4.31 kcal/mol. Overall, however, the bind-
ing free energy is improved by 1.42 kcal/mol; therefore,
as a result of the PROFEC calculations, it seems that
the –OH substitutions at the p-H and o-H positions of
1h ought to yield a tighter binder and, hence, a stronger
inhibitor of 14DM.

D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
Table 5. Free energy components and total binding free energies for compound 1n
7453
SOLV
ELE
SOLV
MM
ELE
MM
vDW
Compound
DE
DE
DEMM
ꢀ76.66
DG
DG
NP
DGSOLV
TDS
DGBIND
ꢀ8.78
1n ꢀ38.34
All energies are in kcal/mol.
ꢀ38.32
56.63
ꢀ3.56
53.07
14.81
4. Conclusions
found 63.72; H calcd, 7.14; found 7.21; N calcd,
6.21%; found 6.33.
Almost all synthesized bis-imidazole derivatives de-
scribed for the first time in this work exhibited some de-
gree of antifungal and antimycobacterial activity,
compound 1h being the most active antifungal deriva-
tive in the series. The application of combined dock-
ing/MM-PBSA free energy of binding calculations
allowed us to rationalize the interactions between the
two series of inhibitors and the active site of the
14DM both from a qualitative and, most importantly,
from a quantitative point of view. Particularly in the last
case, we verified an agreement between the calculated
DGBIND values of all compounds and the correspondent
experimental evidences, expressed in terms of MIC.
Accordingly, the models and procedures proposed will
be used in our laboratories for targeted computer-as-
sisted drug design and subsequent prediction of activity
of the new, potential inhibitors in reasonable length of
computer and human time.
Compounds 2b–k were obtained similarly. Yields and
melting points are reported in Table 2.
5.1.1.2. 1-(4-Bromophenyl)-2-[(dimethylamino)methyl]-
1-propenone hydrochloride (2b). IR (Nujol, cmꢀ1): 1788.
1H NMR (DMSO-d6): 2.75 (s, 6H, (CH2)3N), 4.07 (s,
2H, CH2), 6.19(s, 1H, @CH2), 6.78 (s, 1H, @CH2),
7.67–7.81 (m, 4H, arom.), 11.07 (br s, 1H, NH+ disap-
pearing in D2O). Anal. (C12H15BrClNO) C, H, N: C
calcd 47.32, found 47.53; H calcd, 4.96, found 5.10; N
calcd, 4.60, found 4.48.
5.1.1.3. 1-(4-Chlorophenyl)-2-[(dimethylamino)methyl]-
1-propenone hydrochloride (2c). IR (Nujol, cmꢀ1): 1783.
1H NMR (DMSO-d6): 2.77 (s, 6H, (CH2)3N), 4.10 (s,
2H, CH2), 6.20 (s, 1H, @CH2), 6.80 (s, 1H, @CH2),
7.62 (d, 2H, arom., J = 8.54 Hz), 7.82 (d, 2H, arom.,
J = 8.54 Hz), 11.15 (br s, 1H, NH+ disappearing in
D2O). Anal. (C12H15Cl2NO) C, H, N: C calcd 55.40,
found 55.62; H calcd, 5.81, found 5.93; N calcd, 5.38,
found 5.22.
5. Experimental
Melting points were determined with a Buchi 510 capil-
lary apparatus and are uncorrected. Infrared spectra in
Nujol mulls were recorded on a Jasco FT 200 spectro-
photometer. Proton nuclear magnetic resonance (1H
NMR) spectra were determined on a Varian Gemini
200 spectrometer, chemical shifts are reported as d
(ppm) in DMSO-d6 solution. Reaction courses and
product mixtures were routinely monitored by thin-layer
chromatography (TLC) on silica gel precoated F254
Merck plates. ESI-MS spectra were obtained on a PE-
API I spectrometer by infusion of a solution of the sam-
ple in MeOH. Elemental analyses (C, H, N) were per-
formed on a Carlo Erba analyzer and were within
0.3 of the theoretical value.
5.1.1.4. 2-[(Dimethylamino)methyl]-1-(4-fluorophenyl)-
propenone hydrochloride (2d). IR (Nujol, cmꢀ1): 1785.
1H NMR (DMSO-d6): 2.78 (s, 6H, (CH2)3N), 4.10 (s,
2H, CH2), 6.19 (s, 1H, @CH2), 6.77 (s, 1H, @CH2),
7.40–7.90 (m, 4H, arom.), 11.06 (br s, 1H, NH+ disap-
pearing in D2O). Anal. (C12H15ClFNO) C, H, N: C
calcd 59.14, found 59.28; H calcd, 6.20, found 6.27; N
calcd, 5.75, found 5.68.
5.1.1.5. 2-[(Dimethylamino)methyl]-1-(4-methylphenyl)-
propenone hydrochloride (2e). IR (Nujol, cmꢀ1): 1788.
1H NMR (DMSO-d6): 2.41 (s, 3H, CH3), 2.77 (s, 6H,
(CH2)3N), 4.10 (s, 2H, CH2), 6.16 (s, 1H, @CH2), 6.73
(s, 1H, @CH2), 7.37 (d, 2H, arom., J = 7.93 Hz), 7.73
(d, 2H, arom., J = 7.93 Hz), 10.90 (br s, 1H, NH+ disap-
pearing in D2O). Anal. (C13H18ClNO) C, H, N: C calcd
65.13, found 65.22; H calcd, 7.57, found 7.61; N calcd,
5.84, found 5.80.
5.1. Synthesis
5.1.1. General method for synthesis of compounds 2a–k
5.1.1.1.
2-[(Dimethylamino)methyl]-1-phenyl-prope-
none hydrochloride (2a). To a solution (3.0 g, 25 mmol)
of acetophenone in 50 ml of acetic acid, 1.5 g (50 mmol)
of paraformaldehyde and 2.0 g (25 mmol) of dimethyl-
amine hydrochloride were added. The mixture was
heated under stirring for 16 h. Thereafter, the solvent
was removed under reduced pressure. To remaining oil
70 ml of acetone was added and the formed precipitate
was filtered to give 2.73 g (48%) of the chromatograph-
ically pure compound 2a. Mp 156–159 °C. Rf = 0.37
5.1.1.6. 2-[(Dimethylamino)methyl]-1-(4-methoxyphe-
nyl)-propenone hydrochloride (2f). IR (Nujol, cmꢀ1):
1783. 1H NMR (DMSO-d6): 2.81 (s, 6H, (CH2)3N),
3.80 (s, 3H, OCH3), 3.91 (s, 2H, CH2), 6.15(s, 1H,
@CH2), 6.70 (s, 1H, @CH2), 7.18 (d, 2H, arom.,
J = 8.79 Hz), 8.20 (d, 2H, arom., J = 8.79 Hz), 11.30
(br s, 1H, NH+ disappearing in D2O). Anal.
(C13H18ClNO2) C, H, N: C calcd 61.05, found 61.20;
H calcd, 7.09, found 6.97; N calcd, 5.48, found 5.52.
1
(CHCl3–EtOH 9:1). IR (Nujol, cmꢀ1): 1780. H NMR
(DMSO-d6): 2.76 (s, 6H, (CH2)3N), 4.12 (s, 2H, CH2),
6.18 (s, 1H, @CH2), 6.81 (s, 1H, @CH2), 7.50–7.85 (m,
5H, arom.), 11.20 (br s, 1H, NH+ disappearing in
D2O). Anal. (C12H16ClNO) C, H, N: C calcd 63.85;
5.1.1.7. 2-[(Dimethylamino)methyl]-1-(4-nitrophenyl)-
propenone hydrochloride (2g). IR (Nujol, cmꢀ1): 1786.
1H NMR (DMSO-d6): 2.80 (s, 6H, (CH2)3N), 4.11 (s,

7454
D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
2H, CH2), 6.25 (s, 1H, @CH2), 6.88 (s, 1H, @CH2), 8.00
(d, 2H, arom., J = 9.15 Hz), 8.36 (d, 2H, arom.,
J = 9.15 Hz), 11.00 (br s, 1H, NH+ disappearing in
D2O). Anal. (C12H15ClN2O3) C, H, N: C calcd 53.24,
found 53.32; H calcd, 5.58, found 5.64; N calcd, 10.35,
found 10.42.
Mp 212–5 °C. Rf = 0.18 (CHCl3–EtOH 8:2). IR (Nujol,
1
cmꢀ1): 1784. H NMR (DMSO-d6): 4.20 (dd, 2H, HA,
upfield H of CH2; JAB = 13.91 Hz, JAX = 5.86 Hz),
4.35 (dd, 2H, HB, downfield H of CH2; JBA = 13.91 Hz,
JBX = 8.05 Hz), 4.76 (m, 1H, –CH(CH2)2), 6.79 (s, 2H,
2H5 imidazole), 7.12 (s, 2H, 2H4 imidazole), 7.40–7.90
(m, 7H, 5H arom., 2H, 2H2 imidazole). MS: m/z 281
[MH+]. Anal. (C16H16N4O) C, H, N: C calcd 68.55,
found 68.42; H calcd, 5.75, found 5.93; N calcd, 19.99,
found 20.12.
5.1.1.8. 1-(4-Biphenylyl)-2-[(dimethylamino)methyl]-1-
propenone hydrochloride (2h). IR (Nujol, cmꢀ1): 1780.
1H NMR (DMSO-d6): 2.79 (s, 6H, (CH2)3N), 4.12 (s,
2H, CH2), 6.26 (s, 1H, @CH2), 6.75 (s, 1H, @CH2),
7.41–7.96 (m, 9H, arom.), 10.75 (br s, 1H, NH+ disap-
pearing in D2O). Anal. (C18H20ClNO) C, H, N: C calcd
71.63, found 71.72; H calcd, 6.68, found 6.64; N calcd,
4.64, found 4.62.
Compounds 1b–k were prepared similarly. Yields and
melting points are reported in Table 1.
5.1.2.2. 1-(4-Bromophenyl)-3-(1H-imidazol-1-yl)-2-[(1H-
imidazol-1-yl)methyl]-1-pro-pan-1-one (1b). IR (Nujol,
1
5.1.1.9. 2-[(Dimethylamino)methyl]-1-(thiophen-2-yl)-
propenone hydrochloride (2i). Mp 150–2 °C. Rf = 0.13
cmꢀ1): 1780. H NMR (DMSO-d6): 4.20 (dd, 2H, HA,
upfield H of CH2; JAB = 13.18 Hz, JAX = 5.86 Hz),
4.33 (dd, 2H, HB, downfield H of CH2; JBA = 13.18 Hz,
JBX = 8.05 Hz), 4.78 (m, 1H, –CH(CH2)2), 6.77 (s, 2H, 2
H5 imidazole), 7.12 (s, 2H, 2H4 imidazole), 7.52 (s, 2H,
2H2 imidazole), 7.65 (d, 2H, arom., J = 8.79 Hz), 7.77
(d, 2H, arom., J = 8.79 Hz). MS: m/z 359 [MH+], 361
[MH++2]. Anal. (C16H15BrN4O) C, H, N: C calcd
53.50, found 53.33; H calcd, 4.21, found 4.34; N calcd,
15.60, found 15.73.
1
(CHCl3–EtOH 9:1). IR (Nujol, cmꢀ1): 1786. H NMR
(DMSO-d6): 2.74 (s, 6H, (CH2)3N), 4.07 (s, 2H, CH2),
6.45 (s, 1H, @CH2), 6.66 (s, 1H, @CH2), 7.30 (m, 1H,
H4 thiophene, J = 3.90, 4.88 Hz), 7.92 (d, 1H, H3 thio-
phene, J = 3.90 Hz), 8.15 (d, 1H, H5 thiophene,
J = 4.88 Hz) 10.92 (br s, 1H, NH+ disappearing in
D2O). Anal. (C10H14ClNOS) C, H, N: C calcd 51.83,
found 51.64; H calcd, 6.09, found 6.16; N calcd, 6.04,
found 5.92.
5.1.2.3. 1-(4-Chlorophenyl)-3-(1H-imidazol-1-yl)-2-[(1H-
imidazol-1-yl)methyl]-1-pro-pan-1-one (1c). IR (Nujol,
5.1.1.10. 1-(5-Bromothiophen-2-yl)-2-[(dimethylamino)-
methyl]-propenone hydrochloride (2j). IR (Nujol, cmꢀ1):
1788. 1H NMR (DMSO-d6): 2.72 (s, 6H, (CH2)3N),
4.03 (s, 2H, CH2), 6.45 (s, 1H, @CH2), 6.66 (s, 1H,
@CH2), 7.46 (d, 1H, H4 thiophene, J = 3.90 Hz), 7.76
(d, 1H, H3 thiophene, J = 3.90 Hz), 11.08 (br s, 1H,
NH+ disappearing in D2O). Anal. (C10H13ClBrNOS)
C, H, N: C calcd 38.66, found 38.47; H calcd, 4.22,
found 4.30; N calcd, 4.51, found 4.55.
1
cmꢀ1): 1780. H NMR (DMSO-d6): 4.20 (dd, 2H, HA,
upfield H of CH2; JAB = 14.03 Hz, JAX = 6.10 Hz),
4.33 (dd, 2H, HB, downfield H of CH2; JBA = 14.03 Hz,
JBX = 7.93 Hz), 4.80 (m, 1H, –CH(CH2)2), 6.77 (s, 2H, 2
H5 imidazole), 7.14 (s, 2H, 2H4 imidazole), 7.50 (d, 2H,
arom., J = 8.54 Hz), 7.53 (s, 2H, 2 H2 imidazole), 7.86
(d, 2H, arom., J = 8.54 Hz). MS: m/z 315 [MH+], 317
[MH++2]. Anal. (C16H15ClN4O) C, H, N: C calcd
61.05, found 61.22; H calcd, 4.80, found 4.88; N calcd,
17.80, found 18.03.
5.1.1.11. 1-(5-Chlorothiophen-2-yl)-2-[(dimethylamino)-
methyl]-propenone hydrochloride (2k). IR (Nujol, cmꢀ1):
1783. 1H NMR (DMSO-d6): 2.62 (s, 6H, (CH2)3N), 4.13
(s, 2H, CH2), 6.40 (s, 1H, @CH2), 6.61 (s, 1H, @CH2),
7.42 (d, 1H, H4 thiophene, J = 3.90 Hz), 7.72 (d, 1H,
H3 thiophene, J = 3.90 Hz), 11.08 (br s, 1H, NH+ disap-
pearing in D2O). Anal. (C10H13Cl2NOS) C, H, N: C
calcd 45.12, found 45.23; H calcd, 4.92, found 5.06; N
calcd, 5.26, found 5.18.
5.1.2.4. 1-(4-Fluorophenyl)-3-(1H-imidazol-1-yl)-2-
[(1H-imidazol-1-yl)methyl]-1-pro-pan-1-one (1d). IR
1
(Nujol, cmꢀ1): 1786. H NMR (DMSO-d6): 4.19 (dd,
2H, HA, upfield
JAX = 5.49 Hz), 4.36 (dd, 2H, HB, downfield
H
of CH2; JAB = 14.03 Hz,
H
of CH2; JBA = 14.03 Hz, JBX = 7.93 Hz), 4.80 (m, 1H,
–CH(CH2)2), 6.77 (s, 2H, 2 H5 imidazole), 7.13 (s, 2H,
2H4 imidazole), 7.27 (m, 2H, arom.) 7.52 (s, 2H, 2H2
imidazole), 7.93 (m, 2H, arom.). MS: m/z 299 [MH+].
Anal. (C16H15FN4O) C, H, N: C calcd 64.42, found
64.21; H calcd, 5.07, found 4.94; N calcd, 18.78, found
18.67.
5.1.2. General method for synthesis of compounds 1a–k
5.1.2.1.
methyl]-1-phenyl-propan-1-one (1a). To 1.0 g (4.4 mmol)
of 2-[(dimethylamino)methyl]-1-phenyl-propenone
3-(1H-imidazol-1-yl)-2-[(1H-imidazol-1-yl)-
hydrochloride 2a in 10 ml water–ethanol (8:2), 1.21 g
(17.7 mmol) of imidazole was added. The mixture was
irradiated in open vial using a domestic Daewoo
KOR-63D7 microwave oven at 250 W for a total time
of 6 min (2 min for 3 times). Thereafter, the solvent
was removed under reduced pressure, the remaining oil
was dissolved in 50 ml of CHCl3, and the solution was
washed with water until neutrality was achieved. The or-
ganic phase was dried over CaCl2 and filtered. The sol-
vent was evaporated in vacuo and the residue was
recrystallized from AcOEt to obtain 0.40g (32%) of 1a.
5.1.2.5. 3-(1H-imidazol-1-yl)-2-[(1H-imidazol-1-yl)-
methyl]-1-(4-methylphenyl)-propan-1-one (1e). IR (Nujol,
1
cmꢀ1): 1785. H NMR (DMSO-d6): 2.32 (s, 3H, CH3),
4.17 (dd, 2H, HA, upfield H of CH2; JAB = 14.28 Hz,
JAX = 5.49 Hz), 4.31 (dd, 2H, HB, downfield H
of CH2; JBA = 14.28 Hz, JBX = 8.24 Hz), 4.73 (m, 1H,
–CH(CH2)2), 6.77 (s, 2H, 2 H5 imidazole), 7.11 (s, 2H,
2H4 imidazole), 7.25 (d, 2H, arom., J = 7.79 Hz), 7.51
(s, 2H,
2 H2 imidazole), 7.75 (d, 2H, arom.,
J = 7.79 Hz). MS: m/z 295 [MH+]. Anal. (C17H18N4O)

D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
7455
C, H, N: C calcd 69.37, found 64.21; H calcd, 6.16,
found 6.14; N calcd, 19.03, found 19.21.
JAX = 6.59 Hz), 4.30 (dd, 2H, HB, downfield
H
of CH2; JBA = 13.18 Hz, JBX = 8.24 Hz), 4.61 (m, 1H,
–CH(CH2)2), 6.80 (s, 2H, 2 H5 imidazole), 7.17 (s, 2H,
2H4 imidazole), 7.31 (d, 1H, H4 thiophene, J = 3.67
Hz), 7.56 (s, 2H, 2H2 imidazole), 7.77 (d, 1H, H3 thio-
5.1.2.6. 3-(1H-imidazol-1-yl)-2-[(1H-imidazol-1-yl)me-
thyl]-1-(4-methoxyphenyl)-propan-1-one (1f). IR (Nujol,
cmꢀ1): 1781. 1H NMR (DMSO-d6): 3.79 (s, 3H,
phene, J = 3.67 Hz). MS
:
m/z 365 [MH+], 367
OCH3), 4.15 (dd, 2H, HA, upfield H of CH2; JAB
=
[MH++2]. Anal. (C14H13BrN4OS) C, H, N: C calcd
46.04, found 46.22; H calcd, 3.59, found 3.69; N calcd,
15.34, found 15.58.
13.91 Hz, JAX = 5.12 Hz), 4.32 (dd, 2H, HB, downfield
H of CH2; JBA = 14.28 Hz, JBX = 8.24 Hz), 4.73 (m,
1H, –CH(CH2)2), 6.76 (s, 2H, 2 H5 imidazole), 6.92 (d,
2H, arom., J = 8.79 Hz), 7.11 (s, 2H, 2H4 imidazole),
7.49 (s, 2H, 2 H2 imidazole), 7.82 (d, 2H, arom.,
J = 8.79 Hz). MS: m/z 311 [MH+]. Anal. (C17H18N4O2)
C, H, N: C calcd 65.79, found 65.96; H calcd, 5.85,
found 5.90; N calcd, 18.05, found 18.17.
5.1.2.11. 1-(5-Chlorothiophen-2-yl)-3-(1H-imidazol-1-
yl)-2-[(1H-imidazol-1-yl)methyl]-1-propan-1-one (1k). IR
1
(Nujol, cmꢀ1): 1787. H NMR (DMSO-d6): 4.20 (dd,
2H, HA, upfield
JAX = 6.35 Hz), 4.32 (dd, 2H, HB, downfield
H
of CH2; JAB = 14.16 Hz,
H
of CH2; JBA = 14.16 Hz, JBX = 8.79 Hz), 4.62 (m, 1H,
–CH(CH2)2), 6.80 (s, 2H, 2 H5 imidazole), 7.17 (s, 2H,
2H4 imidazole), 7.22 (d, 1H, H4 thiophene, J = 3.90
Hz), 7.58 (s, 2H, 2H2 imidazole), 7.84 (d, 1H, H3 thio-
5.1.2.7. 3-(1H-imidazol-1-yl)-2-[(1H-imidazol-1-yl)me-
thyl]-1-(4-nitrophenyl)-propan-1-one (1g). IR (Nujol,
1
cmꢀ1): 1779. H NMR (DMSO-d6): 3.53 (m,1H, Hx)
3.96 (dd, 1H, upfield H of CH2; JAB = 10.25 Hz,
JAX = 9.50 Hz), 4.06 (dd, 1H, downfield H of CH2;
JBA = 10.25 Hz, JBX = 8.05 Hz) 4.25 (d, 2H, CH2,
J = 7.30 Hz), 4.27 (m, 1H, –CH(CH2)2), 6.70 (s, 2H,
2H5 imidazole), 7.00 (s, 2H, 2 H4 imidazole), 7.53 (s,
2H, 2H2 imidazole), 7.57 (d, 2H, arom., J = 8.79 Hz),
8.15 (d, 2H, arom., J = 8.79 Hz). MS: m/z 326 [MH+].
Anal. (C16H15N5O3) C, H, N: C calcd 59.07, found
58.91; H calcd, 4.65, found 4.50; N calcd, 21.53, found
21.66.
phene, J = 3.90 Hz). MS :
m/z 321 [MH+], 323
[MH++2]. Anal. (C14H13ClN4OS) C, H, N: C calcd
52.42, found 52.33; H calcd, 4.08, found 4.20; N calcd,
17.46, found 17.68.
5.1.3. General method for synthesis of compounds 1l–m
5.1.3.1. 1-(4-Bromophenyl)-3-(1H-imidazol-1-yl)-2-[(1H-
imidazol-1-yl)methyl]-1-propan-1-ol (1l). To a solution
of 0.50g (1.39 mmol) of 1-(4-bromophenyl)-3-(1H-imi-
dazol-1-yl)-2-[(1H-imidazol-1-yl)methyl]-propan-1-one
1b in 20 ml methanol, 0.06g (1.39 mmol) of NaBH4
were added and the reaction mixture was stirred for
3 h at room temperature. Thereafter, the solvent was
completely evaporated under reduced pressure, the ob-
tained oil was dissolved in 10 ml of CHCl3 and the
solution was washed with cold water until neutrality.
The organic phase was dried (CaCl2) and filtered.
The filtrate was evaporated to dryness to give a resi-
due which was re-crystallized from dichloromethane
to give 0.15 g (30%) of 1l. Mp 180–2 °C. Rf = 0.26
(CHCl3–EtOH 8:2). IR (Nujol, cmꢀ1): 3130. 1H
NMR (DMSO-d6): 3.71 (dd, 2H, HA, upfield H of
CH2; JAB = 13.91 Hz, JAX = 5.86 Hz), 3.85 (m, 1H,
Hx, –CH(CH2)2),4.08 (dd, 2H, HB, downfield H of
CH2; JBA = 13.91 Hz, JBX = 8.05 Hz), 4.20 (m, 1H,
CHOH), 5.77 (d, 1H, OH, J = 4.02 Hz, disappearing
in D2O), 6.69–7.64 (m, 10H, arom. and imidazole).
MS: m/z 361 [MH+], 363 [MH+ + 2]. Anal.
(C16H17BrN4O) C, H, N: C calcd 53.20, found
52.98; H calcd, 4.74, found 4.62; N calcd, 15.51,
found 15.39.
5.1.2.8. 1-(4-Biphenylyl)-3-(1H-imidazol-1-yl)-2-[(1H-
imidazol-1-yl)methyl]-1-propan-1-one (1h). IR (Nujol,
1
cmꢀ1): 1784. H NMR (DMSO-d6): 4.22 (dd, 2H, HA,
upfield H of CH2; JAB = 14.03 Hz, JAX = 5.49 Hz),
4.36 (dd, 2H, HB, downfield H of CH2; JBA = 14.03
Hz, JBX = 7.93 Hz), 4.83 (m, 1H, –CH(CH2)2), 6.78 (s,
2H, 2 H5 imidazole), 7.16 (s, 2H, 2H4 imidazole),
7.40–8.00 (m, 11H, 9H arom. and 2H, H2 imidazole).
MS: m/z 357 [MH+]. Anal. calcd for C22H20N4O (MW
356.43): C, 74.14; H, 5.66; N, 15.72%; found: C, 74.33;
H, 5.54; N, 15.90%.
Anal. (C22H20N4O) C, H, N: C calcd 74.14, found 74.33;
H calcd, 5.66, found 5.54; N calcd, 15.72, found 15.90.
5.1.2.9. 3-(1H-imidazol-1-yl)-2-[(1H-imidazol-1-yl)me-
thyl]-1-(thiophen-2-yl)-propan-1-one (1i). Mp 212–5 °C.
Rf = 0.25 (CHCl3–EtOH 8:2). IR (Nujol, cmꢀ1): 1788.
1H NMR (DMSO-d6): 4.19 (dd, 2H, HA, upfield H of
CH2; JAB = 14.16 Hz, JAX = 6.35 Hz), 4.32 (dd, 2H,
HB, downfield H of CH2; JBA = 14.16 Hz, JBX
=
8.79 Hz), 4.63 (m, 1H, –CH(CH2)2), 6.77 (s, 2H, 2 H5
imid.), 7.14 (m, 3H, 2H4 imid. and H4 tiophene), 7.54
(s, 2H, 2H2 imid.), 7.93 (d, 1H, H3 tiophene,
J = 3.41 Hz), 8.01 (d, 1H, H5 tiophene, J = 4.88 Hz).
MS: m/z 287 [MH+]. Anal. (C14H14N4OS) C, H, N: C
calcd 58.72, found 58.94; H calcd, 4.93, found 5.12; N
calcd, 19.57, found 19.38.
Compound 1m was prepared similarly. Yield and melt-
ing point are reported in Table 1.
5.1.3.2. 3-(1H-imidazol-1-yl)-2-[(1H-imidazol-1-yl)-
methyl]-1-(4-metoxyphenyl)-propan-1-ol (1m). IR (Nujol,
1
cmꢀ1): 3144. H NMR (DMSO-d6): 3.68 (dd, 2H, HA,
upfield H of CH2; JAB = 13.42 Hz, JAX = 6.10 Hz),
3.73 (m, 3H, OCH3), 3.88 (m, 1H, Hx, –CH(CH2)2),4.05
(dd, 2H, HB, downfield H of CH2; JBA = 13.42 Hz,
JBX = 7.93. Hz), 4.36 (m, 1H, CHOH), 5.80 (d, 1H,
OH, J = 4.20 Hz, disappearing in D2O), 6.80–7.60 (m,
10H, arom. and imidazole). MS: m/z 313 [MH+]. Anal.
5.1.2.10. 1-(5-Bromothiophen-2-yl)-3-(1H-imidazol-1-
yl)-2-[(1H-imidazol-1-yl)methyl]-1-propan-1-one (1j). IR
1
(Nujol, cmꢀ1): 1785. H NMR (DMSO-d6): 4.19 (dd,
2H, HA, upfield
H
of CH2; JAB = 13.18 Hz,

7456
D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
(C17H20N4O2) C, H, N: C calcd 65.37, found 65.21; H
calcd, 6.45, found 6.34; N calcd, 17.94, found 18.07.
again using 10ꢀ4 kcal/(mol A) as a convergence crite-
˚
rion).19,30–33For each compound 1a–m, only the struc-
tures corresponding to the minimum energy were
selected for further modeling. The atomic partial
charges for the geometrically optimized compounds
were obtained via the RESP protocol ,24,34 producing
the electrostatic potentials by single-point quantum
mechanical calculations at the Hartree–Fock level with
a 6–31G* basis set, using the Merz–Singh–Kollman
van der Waals parameters.35,36
5.2. Microbiology
Two clinical isolates of Candida species, C. glabrata 123
and C. albicans 3038, were selected for testing the anti-
fungal activity of the title compounds.
The susceptibility of the Candida spp. isolates to the
newly synthesized compounds was determined measur-
ing MIC by a microdilution RPMI reference method,
according to the recommendations of the National
Committee for Clinical Laboratory Standards (NCCLS,
1997). Stock solutions of chemicals were prepared in
DMSO at a concentration of 4 mg/mL; miconazole
and amphotericin B were used as reference test com-
pounds. Each MIC was determined twice in duplicate
experiments after 24 and 48 h incubation time, respec-
tively. The in vitro antitubercular activity was evaluated
measuring MIC by MRA, a microdilution resazurin as-
say performed in liquid medium within 8 days of incuba-
tion on M. tuberculosis H37Rv.25 Briefly, twofold
dilutions of each compound were prepared from stock
solutions in 96-well plates in complete 7H9 broth, final
compound concentrations being 128–0.125 lg/mL.
Twenty microliters of each bacterial suspension was
added to 180 lL of drug-containing culture medium.
The plates were sealed and incubated for 7 days at
37 °C; 5 lL of esazurin (Sigma-Aldrich, 10 mg/mL ster-
ile water stock solution) was added per well, coloring
blue; plates were allowed to incubate at 37 °C for addi-
tional 24 h. Plates were finally read by visual inspection
for color change from blue to pink in wells containing
live Mycobacteria. MIC was defined as the lowest drug
concentration that prevented resazurin color change;
MIC was determined twice in duplicate experiments
and was considered bactericidal by viable counts from
blue wells when 99% cfu was inhibited. Rifampicin was
always included as positive control in each experiment;
DMSO was also evaluated and was always devoid of
inhibiting activity up to the concentration of 2% (v/v).
The optimized structures of the antifungal molecules
were docked into the 14DM active site according to a
procedure already successfully employed for this enzyme
and similar compounds.8,20 Accordingly, it will only
briefly described below. The software AutoDock 3.037
was employed to estimate the possible binding orienta-
tions of all compounds in the receptor. This popular
program performs automated docking of flexible ligands
to a receptor by rapid energy evaluation achieved by
precalculating atomic affinity potentials for each atom
type. The protocol employed in this work8,20 was based
˚
on the following settings: (i) grids were extended 60 A
from the enzyme binding site; (ii) a grid spacing of
˚
0.375 A and 120 grid points were applied in each Carte-
sian direction so as to calculate mass-centered grid
maps; (iii) AMBER 12–6 and 12–10 Lennard-Jones
parameters were used in modeling van der Waals inter-
actions and hydrogen bonding (N–H, O–H, and S–H),
respectively. In the generation of the electrostatic grid
maps, the distance-dependent relative permittivity of
Mehler and Solmajer37 was applied. For the docking
of each compound to the protein, three hundred Monte
Carlo/Simulated Annealing (MC/SA) runs were per-
formed, with 100 constant temperature cycles for simu-
lated annealing. For these calculations, the GB/SA
implicit water model38,39 was used to mimic a solvated
environment. The rotation of the angles / and u, and
the angles of side chains were set free during the calcu-
lations. All other parameters of the MC/SA algorithm
were kept as default. Following the docking procedure,
all structures of compounds 1a–m were subjected to
˚
cluster analysis with a tolerance of 1 A for an all-atom
5.3. Molecular modeling
root-mean-square (RMS) deviation from a lower-energy
structure representing each cluster family. In the absence
of any relevant crystallographic information, the struc-
ture of each resulting complex characterized by the low-
est interaction energy was selected for further
evaluation.
All calculations were carried out on a cluster of Silicon
Graphics Octane R12K computers and were based on
a series of well-validated procedures.8,20 The starting
point of all simulations was our high quality homology
model of the cytochrome P450 sterol 14a-demethylase
(14DM) from C. albicans.8 All inhibitor molecules (plus
miconazole and fluconazole, used as reference com-
pounds, see Results and discussion) were generated
using the Biopolymer module of InsightII26 energy min-
imized using the Sander module of the AMBER 7.0 suite
of programs,27,28 with the Parm94 version29 of the AM-
BER force field (FF), and a convergence criterion equal
Each best substrate/14DM complex resulting from the
automated docking procedure was further refined in
the AMBER suite using the quenched molecular dynam-
ics method (QMD).40–42 In this case, 100 ps MD simula-
tion at 300 K was employed to sample the
conformational space of the substrate–enzyme complex
in the GB/SA continuum solvation environment.38,39
The integration step was equal to 1 fs. After each pico-
second, the system was cooled to 0 K, the structure
was extensively minimized and stored. To prevent global
conformational changes of the enzyme, the backbone of
the protein binding site was constrained by a harmonic
to 10ꢀ4 kcal/(mol A). A conformational search was car-
˚
ried out using a combined molecular mechanics/molecu-
lar dynamics simulated annealing (MDSA) protocol
(i.e., five repeated temperature cycles—from 300 to
1000 K and back—in the constant volume/constant tem-
perature ensemble, followed by energy minimization,
˚
force constant of 100 kcal/A, whereas the amino acid

D. Zampieri et al. / Bioorg. Med. Chem. 15 (2007) 7444–7458
7457
side chains and the ligands were allowed move without
any constraint.
The solvation free energy, DGSOLV, can in turn be ex-
pressed as the sum of an electrostatic component,
SOLV
NP
DGESOLLEV, and a nonpolar contribution, DG
:
The best energy configuration of each complex result-
ing from the previous step was solvated by adding a
sphere of TIP3P water molecules with a 3 nm radius
SOLV
ELE
SOLV
NP
DGSOLV ¼ DG
was calculated by solving the finite-difference
þ DG
ð3Þ
43
SOLV
ELE
DG
from the mass center of the ligand with the use of the
cap option of the Leap module of AMBER 7.0 The
protein complex was neutralized adding a suitable
number of counterions (Na+ and Clꢀ) in the positions
of largest electrostatic potential, as determined by the
module Cion of the AMBER platform. The counteri-
Poisson–Boltzmann equation using the Delphi pack-
age,47 with the Parse atomic radii,48 and Cornell et al.
charges,29 with interior and exterior dielectric constants
˚
equal to 1 and 80, respectively. A grid spacing of 2/A,
extending 20% beyond the dimensions of the solute,
was employed. The nonpolar component in Eq. 3 was
obtained from the solvent accessible surface area
˚
ons having distances larger than 25 A from the active
(SASA)
using
the
following
relationship48:
site were fixed in space during all simulations to avoid
artifactual long-range electrostatic effects on the calcu-
lated free energies. After energy minimization of the
water molecules for 1500 steps, and MD equilibration
of the water sphere with fixed solute for 20 ps, further
unfavorable interactions within the structures were re-
lieved by progressively smaller positional restraints on
DGSOLV ¼ cSASA þ b, in which c = 0.00542 kcal/
NP
˚
2
(mol A ), b = 0.92 kcal/mol, and the surface area evalu-
ated by means of the MSMS software.11
Different approaches are available to estimate the con-
figurational/conformational entropy component TDS
in Eq. 1.49–52 Considering that normal mode has been
successfully applied in estimating the binding entropy
for several biological systems,53 including the same en-
zyme and structurally related compounds,8,20 this meth-
od was the one selected in this work to evaluate TDS
2
˚
the solute (from 25 to 0 kcal/(mol A )) for a total of
4000 steps. Each system was gradually heated to
300 K in three intervals, allowing a 5 ps interval per
each 100 K, and then equilibrated for 50 ps at 300 K,
followed by 400 ps of data collection runs, necessary
for the estimation of the free energy of binding (vide
infra). After the first 20 ps of MD equilibration, addi-
tional TIP3P water molecules were added to the 3 nm
water cap to compensate for those who were able to
diffuse into gaps of the enzyme. The MD simulations
were performed at 300 K using the Parm94 FF29 with
the heme model parameters of Paulsen and Ornstein,44
the Berendsen coupling algorithm,45 an integration
time step of 2 fs, and the applications of the SHAKE
algorithm46 to constrain all bonds to their equilibrium
values, thus removing high frequency vibrations. Long-
range nonbonded interactions were truncated by using
˚
values. In the first step of this calculation, an 8 A sphere
around the ligand was cut out from an MD snapshot for
each ligand–protein complex. This value was shown to
be large enough to yield converged mean changes in sol-
ute entropy. On the basis of the size-reduced snapshots
of the complex, we generated structures of the uncom-
plexed reactants by removing the atoms of the protein
and ligand, respectively. Each of those structures was
minimized, using a distance dependent dielectric con-
stant e = 4r, to account for solvent screening, and its en-
tropy was calculated using classical statistical formulas
and normal mode analysis. To minimize the effects due
to different conformations adopted by individual snap-
shots we averaged the estimation of entropy over 10
snapshots.
˚
a 30 A residue-based cut-off.
For the calculation of the binding free energy between
14DM and all inhibitors in water, a total of 400 snap-
shots were saved during the MD data collection period
described above, one snapshot per each 1 ps of MD
simulation.
Acknowledgment
This research was carried out with the financial support
of the Italian M.U.R.S.T. (60%) (University of Trieste
2001).
The binding free energy DGBIND of each complex in
water was estimated via the widely used MM/PBSA
(Molecular Mechanics/Poisson–Boltzmann Surface
Area) methodology, originally proposed by Peter Koll-
man and coworkers.9 According to this approach,
DGBIND can be calculated from computational analysis
of a single simulation of each ligand-bound protein,
and includes explicit computed components correspond-
ing to ‘gas-phase’ protein/ligand interactions (DEMM),
conformational entropy (TDS), and solvation contribu-
tions (DGSOLV):
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