Synthesis, biological evaluation and structure-activity correlation study of a series of imidazol-based compounds as Candida albicans inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.07.001

A new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivatives was synthesized. The antifungal activity was evaluated in vitro against different fungal species. The biological results show that the most active compounds possess an antifungal activity comparable or higher than Fluconazole against Candida albicans, non-albicans Candida species, Cryptococcus neoformans and dermathophytes. Because of their racemic nature, the most active compounds 5f and 6c were tested as pure enantiomers. For 6c the (R)-enantiomer resulted more active than the (S)-one, otherwise for 5f the (S)-enantiomer resulted the most active. To rationalize the experimental data, a ligand-based computational study was carried out; the results of the modelling study show that (S)-5f and (R)-6c perfectly align to the ligand-based model, showing the same relative configuration. Preliminary studies on the human lung adenocarcinoma epithelial cells (A549) have shown that 6c, 5e and 5f possess a low cytotoxicity.

Full text of DOI:10.1016/j.ejmech.2014.07.001

European Journal of Medicinal Chemistry 83 (2014) 665e673
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and structureeactivity correlation
study of a series of imidazol-based compounds as Candida albicans
inhibitors
,
,
, b
Francesca Moraca d 1, Daniela De Vita a 1, Fabiana Pandolfi a, Roberto Di Santo a
,
Roberta Costi a b, Roberto Cirilli e, Felicia Diodata D’Auria c, Simona Panella c,
,
Anna Teresa Palamara c, Giovanna Simonetti c, Maurizio Botta d **, Luigi Scipione a
,
, *
a Department of “Chimica e Tecnologie del Farmaco”, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy
b “Istituto Pasteur-Fondazione Cenci Bolognetti”, Department of “Chimica e Tecnologie del Farmaco”, Sapienza University of Rome,
Piazzale Aldo Moro, 5, 00185 Rome, Italy
c
ꢀ
Department of “Sanita Pubblica e Malattie Infettive”, Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy
d
ꢀ
Department of “Biotecnologie, Chimica e Farmacia”, “Universita degli Studi di Siena”, Via A. Moro 2, 53100 Siena, Italy
“Dipartimento del Farmaco”, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome, Italy
e
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 2-(1H-imidazol-1-yl)-1-phenylethanol derivatives was synthesized. The antifungal ac-
tivity was evaluated in vitro against different fungal species. The biological results show that the most
active compounds possess an antifungal activity comparable or higher than Fluconazole against Candida
albicans, non-albicans Candida species, Cryptococcus neoformans and dermathophytes. Because of their
racemic nature, the most active compounds 5f and 6c were tested as pure enantiomers. For 6c the (R)-
enantiomer resulted more active than the (S)-one, otherwise for 5f the (S)-enantiomer resulted the most
active. To rationalize the experimental data, a ligand-based computational study was carried out; the
results of the modelling study show that (S)-5f and (R)-6c perfectly align to the ligand-based model,
showing the same relative configuration. Preliminary studies on the human lung adenocarcinoma
epithelial cells (A549) have shown that 6c, 5e and 5f possess a low cytotoxicity.
Received 25 April 2014
Received in revised form
25 June 2014
Accepted 1 July 2014
Available online 1 July 2014
Keywords:
Antifungal
Azole derivatives
Enantioselective synthesis
Ligand-based drug design
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
The largest family of antifungal drugs is represented by the azole
compounds i.e. imidazoles (Miconazole, Econazole, Clotrimazole,
Fungi infect billions of people every year and millions contract
diseases that kill at least as many people as tuberculosis or malaria
[1]. The highly fatal fungal systemic infections are supported
mainly by Candida albicans, Candida species non albicans, Crypto-
coccus neoformans and Aspergillus spp. Superficial mucosal and
cutaneous infections are mainly supported by Candida spp. and
dermatophytes.
Despite the antifungal drugs used in clinical treatments appear
to be diverse and numerous, to date few classes of antifungal agents
are currently available to treat mucosal or systemic infections [2].
and Ketoconazole) and triazoles (Fluconazole, Itraconazole, and the
latest agents, Voriconazole and Posaconazole) (Chart 1) [3e5].
Azoles block fungal membrane ergosterol biosynthesis in the cell
by inhibiting the activity of the lanosterol 14a-demethylase, the
enzyme necessary to convert lanosterol to ergosterol. The active
binding site of lanosterol demethylase contains a heme domain.
Azoles bind with a nitrogen atom to the iron atom of the heme,
preventing the demethylation of lanosterol [6]. The depletion of
ergosterol and accumulation of 14a-methylated sterols disrupt the
structure and many functions of fungal membrane leading to in-
hibition of fungal growth [7].
However, the treatment of invasive fungal diseases still remains
unsatisfied as mortality rate, is unacceptable high [8]. Moreover,
non-invasive infections, as onychomycoses, are often recurrent,
chronic, and generally require long-term treatment with antifungal
agents [9]. Hence, there is considerable urgency to discover new
antimicrobial agents.
* Corresponding author.
** Corresponding author.
E-mail addresses: maurizio.botta@unisi.it (M. Botta), Luigi.scipione@uniroma1.it,
luigi.scipione@fastwebnet.it (L. Scipione).
1
These authors equally contribute to the work.
http://dx.doi.org/10.1016/j.ejmech.2014.07.001
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
Fig. 1. Contour maps of the ligand-based model generated according to GRID MIFs. A: Cyan surface represents the shape that active compounds should match; B: N1 ¼ blue lone
pair nitrogen counter maps; C: HAL1 ¼ grey halogen counter map; AeA2 ¼ orange aromatic counter maps; HYD1eHYD3 ¼ blue marine mesh hydrophobic counter maps. In yellow
sticks is represented compound (S)-5f. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. A; contour maps of the LBM2 model are the same as described in Fig. 1. In yellow stick is represented compound (S)-5f and in orange stick compound (R)-9. Non-polar
hydrogen atoms are omitted. B; structure of the most active compounds (S)-5f and (R)-9. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
We have previously reported the synthesis and in vitro evalua-
tion of antifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-
phenylethanol derivatives [10]. Given the azole nature of those
compounds, we hypothesized CYP51 as the target and focused on
the design and synthesis of new imidazole derivatives, modifying
the side chain of 2-(1H-imidazol-1yl)-ethyl carbamates or esters
previously reported, in order to improve their antifungal activity
[10]. All the new synthetized compounds were tested in vitro to
evaluate the antifungal activity against different fungal strains and
some of them showed high inhibitory activity. Up to date, there is
no three dimensional structural information available on C. albicans
(CACYP51) or other fungal CYP51 enzymes. Therefore,
computational techniques were used to rationalize the experi-
mental data. A ligand-based approach helped the discrimination
between active and inactive compounds in fully agreement with
the in vitro data, giving useful information about the functional
groups that could be responsible for the antifungal activity.
2. Results and discussion
2.1. Chemistry
The racemic 2-(1H-imidazol-1-yl)-1-phenylethanols 1aec, the
esters 2a,b and 3aeg and the carbamates 4aec were prepared as

F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
667
R = H: Econazole
R = Cl: Miconazole
Ketoconazole
Clotrimazole
Voriconazole
Fluconazole
Chart 1. Main azole drugs used for the treatment of candidiasis.
b
(5a-g)
a: R1= F, R2= 2-yl-3-nitropyridin
b: R1= Cl, R2= furan-2-ylcarbonyl
c: R1= F, R2= 4-nitrophenyl
d: R1= Cl, R2= 4-nitrophenyl
e: R1= F, R2= 4-chlorophenyl
f: R1= Cl, R2= 4-chlorophenyl
g: R1= F, R2= furan-2-ylcarbonyl
a
b
(1b-c) b: R1 = F
c: R1 = Cl
(6a-c) a: R1= F, R2= ethylsulfonyl
R1= F, R2= furan-2-ylcarbonyl
R1= F, R2= 4-chlorophenyl
Scheme 1. Preparation of compounds 5aeg and 6aec. Reagents and conditions: (a) dry CH3CN, triphosgene, rt, overnight; (b) TEA, selected amine, rt, overnight.

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F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
described in the literature [10]. The pure enantiomers of 1b,c have
been prepared using the procedure described in our previous letter
[11]. The synthesis of 5aeg and 6aec was carried out using tri-
phosgene in dry CH3CN to obtain activation of the eOH group as
chloroformate of the appropriate 2-(1H-imidazol-1-yl)-1-
phenylethanol; then the mixture was added with TEA and the
selected amine and stirred overnight as described in Scheme 1.
Commercial available amines have been used to synthesize the
compounds 5beg. The side chains for the synthesis of 5a is pre-
pared as reported in Scheme 2, by condensation of the piperazine
with 2-chloro-3-nitropyridine. The required amines for the syn-
thesis of the side chains of compounds 6aec were prepared as
described in Scheme 3. The synthesis was carried out by conden-
sation of 1-fluoro-4-nitrobenzene with the appropriate commercial
piperazine in CH3CN under reflux condition for 2 h. Then the nitro
group was converted to amino group by reduction with H2ePd/C.
The obtained amines were used immediately.
a
b
(7a-c)
(8a-c)
a: R= ethylsulfonyl
a: R= ethylsulfonyl
b: R= furan-2-ylcarbonyl
c: R= 4-chlorophenyl
b: R= furan-2-ylcarbonyl
c: R= 4-chlorophenyl
Scheme 3. Preparation of the side chains of 6aec. Reagents and conditions: (a) CH3CN,
reflux, 2 h; (b) H2, Pd/C, 50 psi, 4 h, rt.
respectively of 0.20, 0.25, 0.17 and 3.17
m
g mLꢀ1. Moreover, (S)-5f
2.2. In vitro antifungal activity and cytotoxicity
was found more active than Flu against C. albicans, non-albicans
Candida species and dermathophytes with GM MIC values respec-
The antifungal activity of the synthesized compounds was
evaluated by CLSI broth microdilution methods against different
strains of C. albicans, non-albicans Candida species, C. neoformans
and dermathophytes. Fluconazole (Flu) was used as reference drug.
The antifungal activity was expressed as MIC50
yeasts (the lowest concentration that showed ꢁ50% growth inhi-
bition compared with the control) and as MIC80
g mLꢀ1) for the
dermatophytes (the lowest concentration that showed ꢁ80%
growth inhibition compared with the control). For each fungal
strain the results were reported as the median of the MIC values of
five replicates and for all the strains within to C. albicans, non-
albicans Candida species, C. neoformans and dermatophytes the
geometric mean (GM) value was reported in Table 1.
tively of 0.27, 1.33 and 3.61 m .
g mLꢀ1
The antifungal activity of 5f, (S)-5f, 6c and (R)-6c, was also
evaluated towards some Fluconazole resistant Candida species and
the activity was reported as geometric mean of MIC (GM-MIC
(m
g mLꢀ1) for the
m
g mLꢀ1) in Table 1. The obtained results indicate that all the new
compounds were more active than Flu on the resistant fungal
strains; particularly (R)-6c showed a GM MIC value of 2.97
g mLꢀ1
and (S)-5f a GM MIC value 4.56
g mLꢀ1, resulting about fifteen and
tenfold more active than Flu (GM MIC 44.51
g mLꢀ1).
(m
m
m
m
Preliminary studies carried out on 5e, 5f and 6c, have shown
that the above compounds possess a low cytotoxicity on the human
lung adenocarcinoma epithelial cells (A549) with CC50 values
respectively of 78.4, 82.2 and 171.8 m
g mLꢀ1 (Table 2).
The data in Table 1 show that the compounds 5e, 5f and 6c
possess an interesting antifungal activity. In particular, we found
that 5e and 6c show GM MIC values of 1.87 and 1.81
against C. albicans, comparable to Flu, otherwise 5f shows a GM MIC
value of 0.67
g mLꢀ1 resulting more active than Flu. Furthermore,
m ,
g mLꢀ1
2.3. Structureeactivity correlation based on GRID MIFs
m
Given the structural similarity of the compounds presented here
to known antifungal CYP51 inhibitors, this enzyme has been pro-
posed as the target. However, there are no three dimensional
structural information about C. albicans CYP51, hence it has been
developed a ligand-based structureeactivity correlation study by
means of GRID MIFs (Molecular Interaction Fields) on the 27 azole-
based compounds (Chart 2), some of which previously reported
[10], evaluated in vitro against C. albicans (Table S6 supporting info),
in order to help the rationalization of experimental data. The
ligand-based model is able to help the discrimination between
active and inactive compounds, in fully agreement with the in vitro
data, giving useful insights for the understanding of the functional
groups possibly responsible for the antifungal activity. In a previous
work [12] we reported a pharmacophore hypothesis (HYPO1)
derived from a 3D-QSAR study on 1-[(aryl)[4-aryl-1H-pyrrol-3-yl]-
1H-imidazole as C. albicans CYP51 inhibitors, described elsewhere
[12]. The HYPO1 showed an aromatic feature with a lone pair ni-
trogen in the ring plane and three aromatic features. According to
HYPO1, an ideal CYP51 inhibitor should show (i) an aromatic ni-
trogen with an accessible lone pair; (ii) a diarylmethyl moiety on
the azole N-1, which is preferred over a 1,2-diarylethyl; (iii) one of
the two aryls on the methyl group could be better a phenyl ring
with a para lypophilic substituent; (iv) the second aryl group
should be made up of two coplanar aromatic rings. Qualitatively,
our ligand-based model (thereafter called LBM) shows similarities
to the previously reported HYPO1, supporting our hypothesis of
CYP51 as the target enzyme of the azole-based compounds
5e, 5f and 6c also resulted more active than Flu against non-
albicans Candida species with GM MIC values respectively of 2.06,
1.83 and 0.51
Flu towards C. neoformans (GM MIC 0.22
dermathophytes (GM MIC 6.35
g mLꢀ1). Moreover, compound 5f
show a GM MIC value of 3.70
g mLꢀ1 against dermathophytes and
resulted more potent than Flu.
m
g mLꢀ1. Compound 6c also resulted more active than
m
g mLꢀ1) and towards
m
m
In the Tables S1eS4 of the supplementary information the
detailed results obtained for each strain of C. albicans, non-albicans
Candida species, C. neoformans and dermathophytes are reported.
Because of their racemic nature, the most active compounds 5f
and 6c were tested as pure (R)- and (S)-enantiomers to evaluate if
they possess a different antifungal activity. (R)-6c resulted more
active than Flu against C. albicans, non-albicans Candida species,
C. neoformans, and dermathophytes with GM MIC values
a
(7d)
Scheme 2. Preparation of side chain of 5a (7d). Reagents and conditions: (a) CH3CN,
piperazine dihydrochloride hydrate, TEA, reflux, 2 h.

F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
669
Table 1
In vitro antifungal activity of studied compounds against Candida albicans, Candida species, Cryptococcus neoformans and dermathophytes.
Cmp
C. albicansa GM
MIC (
g mLꢀ1
Candida speciesb GM
MIC (
g mLꢀ1
C. neoformansc GM
Dermathophytesd GM
MIC (
g mLꢀ1
Candida species resistant strainse GM MIC ( g mLꢀ1
m )
m
)
m
)
MIC (
m
g mLꢀ1
)
m
)
5a
5b
5c
5d
5e
5f
(R)-5f
(S)-5f
5g
6a
6b
6c
(R)-6c
(S)-6c
Flu
11.31
58.69
17.75
13.22
1.87
0.67
5.86
0.27
85.92
128.00
111.43
1.81
0.20
40.09
1.27
11.99
128.00
32.00
34.90
2.06
1.83
15.54
1.33
50.80
128.00
128.00
0.51
0.25
3.89
4.49
90.51
21.98
12.34
3.00
3.17
3.36
2.52
98.70
16.95
16.00
0.22
0.17
1.41
27.43
128
50.79
34.56
13.03
3.7
7.03
3.61
109.73
128
118.51
6.35
nd
nd
nd
nd
nd
7.25
nd
4.56
nd
nd
nd
5.38
2.97
nd
3.17
118.51
7.2
2.91
1.89
44.51
Data represent the geometric mean of minimal inhibitory concentration (GM MIC) of five replicates.
a
C. albicans strains: ATCC24433, ATCC3153, ATCC10231, ATCC76615, ATCC10261, ATCC90028, ATCC20891, PMC1011.
b
Candida species strains: C. krusei DSM6128 and PMC0613, C. tropicalis PMC0910 and DSM11953, C. parapsilosis DSM11224 and ATCC22019, C. glabrata PMC0805 and
PMC0807.
c
C. neoformans strains: PMC2123, PMC2136, PMC2115, PMC2103, PMC2107, PMC2102, DSM11959, PMC2111.
Dermathophytes strains: T. mentagrophytes DSM4870, PMC6515, PMC6531, PMC6509, PMC6503 and PMC6552, M. gypseum PMC7303, PMC7331 and DSM3824.
Candida species resistant strains: C .albicans PMC1040R, PMC1041R and PMC1042R, C. glabrata PMC0850R, PMC0851R, PMC0852R and PMC0853R; nd: not determined.
d
e
reported in this paper. As in HYPO1, LBM shows a blue contour map
representing a region that leads to the enhancement of activity
with a lone pair nitrogen, possibly able to interact with the iron
atom of the heme prosthetic group; two orange contour maps and
two green contour maps that are representative of favourable re-
gions for aromatic and hydrophobic groups respectively. The dif-
ferences to the previous HYPO1 are the presence of a grey contour
map that represents a region favourable for halogen substituents
and a cyan contour map that represents the shape that an active
compound should match (Fig. 1).
the HAL contour map, emphasizing the importance of the presence
of halogens in this region and/or the presence of more hydrophobic
moieties aligned to the HYD3 contour map. The inactive com-
pounds 1a, 1b and 1c represent a fragment of the most active
compound (S)-5f, hence a good starting point for the synthesis of
more potent compounds, on the basis of the study reported here. To
give more value to this study and to propose a general SAR for the
further design of highly potent antifungal agents, we built a second
ligand-based model (LBM2) combining information taken from this
study and the already reported one [12]. Hence, LBM2 derives from
the alignment of the antifungal drugs bifonazole, miconazole, flu-
conazole, our most active compounds (S)-5f and the most active
compounds (R)-9 taken from the previous study [12]. LBM2 sum-
marizes the essential characteristics that an active compound to-
wards C. albicans CYP51 should possess. It shows i) a lone pair
nitrogen contour map, of pivotal importance for the interaction
with the heme iron of the prosthetic group; ii) two aromatic and
two hydrophobic contour maps, of which A1 and HYD1 correspond
to an aromatic or an aliphatic ring that enhance hydrophobic in-
teractions in this region, A2 and HYD2 to the imidazolic ring
coordinating the heme iron; iii) a halogen contour map and a
characteristic shape (Fig. 2). LBM2 is also in agreement with the
refined pharmacophoric model proposed by Di Santo et al. (2005)
(MOD3) [14]. Indeed, MOD3 shows i) a feature for unsubstituted
aromatic nitrogen; ii) a feature for aromatic rings; iii) two features
for hydrophobic moieties. In addition, it shows two excluded vol-
umes indicating the portion of space that an active compound
should not match, slightly recalling the shape proposed by our
LBM2 model.
The active azole-based compounds presented show most of
their functional groups within the above described contour maps,
that can explain the reason of their higher activity. It is worth
noting the highest activity of compound (R)-5f with respect to
its eS enantiomer and the other active compounds. The reason of
the decrease of their activity with respect to the most active (S)-5f
is not evident from the ligand-based model but, assuming CYP51 as
the target enzyme, it is possible that those compounds show a
worst coordination at the heme iron with respect to (R)-5f. To show
a good coordination at the heme prosthetic group, a compound
should place its heme-coordinating group perpendicularly to the
heme iron, as reported by Holtje et al. and subsequently by us
[11,13]. After the alignment of compounds, it is evident that the
imidazolic ring of the less active (R)-5f has a slightly different
orientation with respect to the imidazolic ring of the other active
compounds, that could support this hypothesis. All the active
compounds show the alignment to the A1 and HYD1 contour maps,
highlighting the importance of non-polar interactions in this re-
gion. Compound (S)-6c that does not align to LBM, is much less
active thaneR enantiomer. In addition, it is evident from our data
that compounds possessing a fluorine atom in correspondence to
the HAL contour map are less active than compounds that show a
chlorine substituent. Compounds 3e, 3g and 3f, are the only actives
that do not show a halogen substituent alignment to the HAL
contour map, but are the only that show two phenyl rings directly
linked together (as in the antifungal bifonazole) that in LBM are
aligned to the HYD3 contour map, emphasizing the importance of
non-polar interactions in this region. This is another useful infor-
mation to understand the difference in the activity of compounds
that show a similar scaffold. All the inactive compounds lose
functional groups aligned to the A1 and HYD1 contour maps and to
3. Conclusion
The biological in vitro data show that some of the synthesized
compounds possess an interesting antifungal activity versus
Candida species and other fungal species. The structural modifica-
tions of the side chain allowed us to obtain some new compounds
with high antifungal activity. In particular 5e, 5f and 6c showed
activity similar to or higher than Flu against C. albicans and Candida
species, while they resulted up to 15 fold more active than Flu
against resistant strains of Candida species. All these three com-
pounds possess the common 4-chloro-phenylpiperazino moiety

670
F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
Table 2
aligned to the A2 and HAL contour maps of the ligand-based model,
that appears to be particularly important for the antifungal activity
and to try to face the problem of drug resistance against Candida
species. Experimental data show that (S)-5f is more active than its
(R)-enantiomer and (R)-6c is more active than its (S)-enantiomer.
Taking into account modelling results, it is not surprising since (S)-
5f and (R)-6c perfectly align to the model, showing the same
relative configuration (even if the absolute configuration is
reversed). This because 6c has a eNHe in plus with respect to 5f
that allows a movement leading to the alignment of (R)-6c N-[4-[4-
(4-chlorophenyl)piperazin-1-yl]phenyl]carbamate moiety to (S)-5f
4-(4-chlorophenyl)piperazine-1-carboxilate group. Hence, from
the modelling study we can speculate that to exert an antifungal
activity, the relative configuration of compounds is of great
Effect of 5e,f and 6c on the human lung adeno-
carcinoma epithelial cells (A549) growth.
CC50
(m )
g mLꢀ1
5e
5f
6c
78.4 2.2
82.2
171.8 14.2
5
CC50: concentration (
m
g mLꢀ1) required to reduce
50%. Data represent
standard deviation from two indepen-
cell
viability
by
means
dent experiments, each performed in triplicate.
N
N
N
N
N
N
OH
R2
O
O
Ph
O
O
O
(3a-g)
a: R1 = H, R2 = 3-(trifluoromethyl)-phenyl
b: R1 = F, R2 = 3-(trifluoromethyl)-phenyl
c: R1 = CF3, R2 = 3-(trifluoromethyl)-phenyl
d: R1 = Cl, R2 = 3-(trifluoromethyl)-phenyl *
e: R1 = H, R2 = 4-(trifluoromethyl)-phenyl
f: R1 = H, R2 = 4-biphenyl
(1a-c)
(2a,b)
a: R1 = H
b: R1 = F
c: R1 = Cl
a: R1 = F
b: R1 = Cl
R1
R1
R1
g: R1 = F, R2 = 4-biphenyl
h: R1 = Cl, R2 = 4-biphenyl
N
N
N
R2
N
N
NHR2
O
O
N
O
(5a-g)
O
a: R1= F, R2= 2-yl-3-nitropyridin
b: R1= Cl, R2= furan-2-ylcarbonyl
c: R1= F, R2= 4-nitrophenyl
d: R1= Cl, R2= 4-nitrophenyl
e: R1= F, R2= 4-chlorophenyl
f: R1= Cl, R2= 4-chlorophenyl
g: R1= F, R2= furan-2-ylcarbonyl
(4a-d)
R1
a : R1 = H, R2 = 4-(propan-2-yl)-phenylamino
b: R1 = F, R2 = 4-(propan-2-yl)-phenylamino
c: R1 = Cl, R2 = 4-(propan-2-yl)-phenylamino
R1
d: R1 = Cl, R2 = 2,6-dichloropyridin-4-yl-amino
N
N
N
N
H
O
N
N
O
N
(9)
(6a-c)
N
R2
a: R1= F, R2= ethylsulfonyl
b: R1= F, R2= furan-2-ylcarbonyl
c: R1= F, R2= 4-chlorophenyl
R1
Chart 2. compounds described in this study.

F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
671
importance, since only those in which the imidazole moiety aligns
4.2.1.3. 1-(4-Chlorophenyl)-4-(4-nitrophenyl)piperazine
Yield 43%, as yellow solid. 1H NMR (DMSO-d6):
(7c).
8.08 (d, 2H,
to the blue contour map and the 4-chloro-phenylpiperazino moiety
aligns to the A2 and HAL contour maps of the ligand-based model
show a high activity in vitro. The confirmation of the target of the
synthesized azole-based compounds and their further optimization
through synthetic strategies at the light of the information ob-
tained by molecular modelling, will be the theme of the future
work.
d
J ¼ 9.3), 7.26 (d, 2H, J ¼ 8.3), 7.08 (d, 2H, J ¼ 8.8), 7.00 (d, 2H, J ¼ 8.3),
3.63 (m, 4H); 4H piperazine overlap.
4.2.2. Synthesis of 1-(3-nitro-2-pyridyl)piperazine (7d)
3 mmol of piperazine dihydrochloride hydrate was added of
10 mL of CH3CN, TEA (6 mmol) and 2-chloro-3-nitropyridine
(1 mmol). After 2 h, at reflux, the mixture was evaporated under
reduced pressure and the residue was treated with H2O (5 mL) and
ethylacetate (5 mL). The aqueous phase was separated and
extracted twice with ethylacetate (2 ꢂ 5 mL). The combined organic
phases were dried on Na2SO4, filtered and evaporated under
reduced pressure. The crude residue was purified by silica gel col-
umn chromatography, using dichloromethane/methanol (9:1) as
eluent. Yield 62%, as yellow solid. 1H NMR (DMSO-d6): 8.44 (dd, 1H,
J ¼ 4.5 Hz, J ¼ 1.6 Hz), 8.29 (dd, 1H, J ¼ 8.1 Hz, J ¼ 1.6 Hz), 6.94 (dd,
1H, J ¼ 8.1 Hz, J ¼ 4.5 Hz), 3.56 (m, 4H), 2.80 (m, 4H).
4. Experimental
4.1. Chemistry
All reagents and solvents were of high analytical grade and were
purchased from SigmaeAldrich (Milano, Italy). 2-(1H-imidazol-1-
yl)-1-phenylethanol derivatives 1aec, 2a,b, 3aeg, 4aec were pre-
pared according to the literature procedure [10]. The pure enan-
tiomers of 1b,c have been prepared as previously described [11] and
used for the synthesis of the enantiomeric pure carbamates;
analytical HPLC analysis of these final compounds were performed
using the commercially available 250 mm ꢂ 4.6 mm I.D. Chiralcel
OD column (Chiral Technologies Europe, Illkirch, France). HPLC
grade ethanol was purchased from Aldrich (St. Louis, Missouri USA).
The analytical HPLC apparatus consisted of a Perkin Elmer (Nor-
walk, CT, USA) 200 LC pump equipped with a Rheodyne (Cotati, CA,
USA) injector, a 20 mL sample loop, an HPLC Dionex CC-100 oven
(Sunnyvale, CA, USA) and a Jasco (Jasco, Tokyo, Japan) Model CD
2095 Plus UV/CD detector. All the enantiomeric final compounds
showed an optical purity higher than 99.9% e.e.. Melting points
were determined on Tottoli apparatus (Buchi) and are uncorrected.
Infrared spectra were recorded on a Spectrum One ATR Perkin
Elmer FT-IR spectrometer. 1H NMR and 13C NMR spectra were ac-
quired on a Bruker AVANCE-400 spectrometer at 9.4 T, in CDCl3,
CD3OD or DMSO-d6 at 27 ꢃC; chemical shift values are given in
4.2.3. General procedure for the preparation of amines 8aec
1 mmol of the nitrocompound (7aec) was suspended in 90 mL
of MeOH and then reduced by hydrogenation at 50 psi in the
presence of 10% Pd/C (10 mg) as catalyst, for 4 h at room temper-
ature. The catalyst was removed by filtration, the solution was
evaporated under reduce pressure to give 8aec immediately used
for the subsequent reaction.
4.2.4. General procedure for the synthesis of (1H-imidazol-1yl)-
ethyl carbamates 5aef, 6aec
2-(1H-imidazol-1-yl)-1-phenylethanol (1 mmol) was sus-
pended in 5 mL of anhydrous CH3CN, then triphosgene (0.5 mmol)
was added. After 12 h at r.t. the reaction mixture was treated with
Et2O obtaining a white precipitate, subsequently the solvent was
removed and the precipitate was suspended in anhydrous CH3CN
and added with TEA (2.0 mmol) and 0.8 mmol of the selected
amine. After 12 h at r.t., the solvent was removed under reduced
pressure and the residue was treated with CH2Cl2 and extracted
with saturated aqueous Na2CO3. The organic layer was dried over
Na2SO4 and evaporated under vacuum to give a crude residue
which was purified by silica gel column chromatography using
CH2Cl2/MeOH (8:2) as eluent.
d
(ppm) relatively to TMS as internal reference. Coupling constants
are given in Hz. Mass spectrometric experiments were carried out
with a 2000 Q TRAP instrument (Applied Biosystems), a commer-
cial hybrid triple-quadrupole linear ion-trap mass spectrometer
(Q1q2QLIT), equipped with an ESI source and a syringe pump have
been used. The examined compounds were previously dissolved in
methanol (10ꢀ5 M) and aqueous HCl was added just before the
injection. The molecular peaks (m/z) have been observed as
[MþH]þ. Elemental analyses were obtained by a PE 2400 (Per-
kineElmer) analyzer and the analytical results were within 0.4%
of the theoretical values for all compounds.
4.2.4.1. [1-(4-Fluorophenyl)-2-imidazol-1-yl-ethyl]4-(2-nitro-3-
pyridyl)piperazine-1-carboxylate (5a). Yield 65%, as yellow solid.
Mp: 143e5. 1H NMR (CD3OD):
d
8.40 (d, 1H, J ¼ 4.4 Hz), 8.25 (d, 1H,
J ¼ 8.0 Hz), 7.81 (s, 1H), 7.40 (m, 2H), 7.23 (s, 1H), 7.14 (m, 2H), 7.07
(s, 1H), 6.95 (dd, 1H, J ¼ 4.4 Hz, J ¼ 8.0 Hz), 6.01 (m, 1H), 4.50 (m,
4.2. Synthesis of carbamates 5aef, 6aec
2H), 3.74e3.43 (m, 8H). 13C NMR (CD3OD):
d
162.8 (J ¼ 243.0 Hz),
154.0, 152.6, 151.5, 137.7, 135.3, 133.9, 133.4, 128.1 (J ¼ 8.0 Hz), 127.5,
4.2.1. General procedure to obtain nitrocompounds 7aec
120.0,115.1 (J ¼ 22.0 Hz),114.3, 75.3, 51.1, 43.4, 43.1. IR: ʋ 1686 cmꢀ1
.
1 mmol of 1-substituted piperazine was dissolved in 5 mL of
CH3CN, then 2.5 mmol of 1-fluoro-4-nitrobenzene were added. The
mixture was refluxed for 2 h, then the solvent was removed and the
crude residue was purified by silica gel column chromatography,
using dichloromethane/methanol (9:1) as eluent.
MS, m/z: 441.00 [MþH]þ.
4.2.4.2. [1-(4-Chlorophenyl)-2-imidazol-1-yl-ethyl]4-(furan-2-
carbonyl)piperazine-1-carboxylate (5b). Yield 60%, as waxy solid. 1H
NMR (CDCl3):
J ¼ 8.3 Hz), 6.75 (s, 1H), 5.93 (t,1H, J ¼ 6.0 Hz), 4.29 (m, 2H), 3.63 (m,
4H), 3.10 (m, 4H). 13C NMR (DMSO-d6):
158.9, 153.3, 147.2, 145.4,
d 7.28e7.13 (m, 4H), 7.03 (m, 3H), 6.83 (d, 2H,
4.2.1.1. 1-(Ethylsulfonyl)-4-(4-nitrophenyl)piperazine
64%, as yellow solid. 1H NMR (CDCl3):
(7a). Yield
8.11 (d, 2H, J ¼ 9.0 Hz), 6.86
d
d
136.7, 136.4, 133.6, 129.1, 128.6, 122.9, 121.4, 116.4, 111.8, 74.3, 52.6,
43.9, 43.9 IR: ʋ 1701-1622 cm-1. MS, m/z: 428. 98 [MþH]þ.
(d, 2H, J ¼ 9.0 Hz), 3.48 (m, 8H), 3.00 (q, 2H, J ¼ 7.3 Hz), 1.40 (t, 3H,
J ¼ 7.3 Hz).
4.2.4.3. [1-(4-Fluorophenyl)-2-imidazol-1-yl-ethyl] 4-(4-
4.2.1.2. Furan-2-yl[4-(4-nitrophenyl)piperazin-1-yl]methanone (7b).
nitrophenyl)piperazine-1-carboxylate (5c). Yield 70%, as a yellow
Yield 53%, as yellow solid. 1H NMR (CDCl3):
d
8.17 (d, 2H, J ¼ 9.3 Hz),
solid. Mp: 168e9. 1H NMR (CDCl3):
d
8.13 (d, 2H, J ¼ 8.9 Hz), 7.84 (s,
1H), 7.22 (m, 2H), 7.07 (m, 3H), 6.81 (m, 3H), 5.98 (t, 1H, J ¼ 5.2 Hz),
4.39 (m, 2H), 3.67 (m, 4H), 3.42 (m, 4H). 13C NMR (CDCl3)
: 162.9
7.53 (s, 1H), 7.11 (d, 1H, J ¼ 3.4 Hz), 6.86 (d, 2H, J ¼ 9.3 Hz), 6.53 (m,
1H), 4.03 (m, 4H), 3.55 (m, 4H).
d

672
F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
(J ¼ 247 Hz), 154.4, 153.5, 139.3, 137.5, 132.4 (J ¼ 3 Hz), 128.2, 128.0
(J ¼ 8 Hz), 125.9, 119.8, 116.1 (J ¼ 22 Hz), 113.2, 77.2, 74.9, 52.1, 46.8.
IR: ʋ 1704 cm-1. MS, m/z: 439.93 [MþH]þ.
7.55 (s, 1H), 7.40 (m, 2H), 7.29e7.15 (m, 7H), 7.01 (d, 2H, J ¼ 8.8 Hz),
6.93 (d, 2H, 8.2 Hz), 6.85 (s, 1H), 5.94 (m, 1H), 4.41 (m, 2H), 3.26 (m,
4H, partially obscured by HDO signal, it appears after D2O ex-
change), 3.18 (m, 4H). 13C NMR (CD3OD):
d
162.7 (J ¼ 243 Hz), 153.2,
4.2.4.4. [1-(4-Chlorophenyl)-2-imidazol-1-yl-ethyl] 4-(4-
nitrophenyl)piperazine-1-carboxylate (5d). Yield 80%, as a yellow
151.4, 150.1, 133.5 (J ¼ 2.5 Hz), 128.7, 128.5, 128.0 (J ¼ 8 Hz), 127.4,
124.4, 120.1, 119.9; 117.4, 116.9, 116.3, 115.0 (J ¼ 21 Hz), 74.2, 51.3,
49.8, 49.4. IR: ʋ 1721 cmꢀ1. MS, m/z: 520.03 [MþH]þ.
solid. Mp: 188e190. 1H NMR (CDCl3):
d
8.14 (d, 2H, J ¼ 9.3 Hz), 7.45
(s, 1H), 7.34 (m, 2H), 7.14 (d, 2H, J ¼ 10.5 Hz),7.04 (s, 1H) 6.81 (m,
3H), 5,94 (t, 1H, J ¼ 5.5 Hz), 4.33 (m, 2H), 3.67 (m, 4H), 3.42 (m, 4H).
4.3. Organisms
13C NMR (CDCl3)
d: 154.4, 153.5, 139.2, 137.6, 135.2, 135.0, 129.2,
129.1, 127.5, 125.9, 119.7, 113.1, 75.0, 51.7, 46.8, 43.3. IR: ʋ 1704 cmꢀ1
.
For the antifungal evaluation, strains obtained from the Amer-
ican Type Culture Collection (ATCC, Rockville, MD, USA), the
German Collection of Microorganisms (DSMZ, Braunschweig, Ger-
many) and the Pharmaceutical Microbiology Culture Collection
(PMC, Department of Public Health and Infectious Diseases, “Sapi-
enza” University, Rome, Italy) were tested. The strains were:
C. albicans (ATCC24433, ATCC10231, ATCC76615, ATCC10261,
ATCC90028,
MS, m/z: 455.97 [MþH]þ.
4.2.4.5. [1-(4-Fluorophenyl)-2-imidazol-1-yl-ethyl] 4-(4-
chlorophenyl)piperazine-1-carboxylate (5e). Yield 92%, as a waxy
solid. 1H NMR (DMSO-d6):
d
7.56 (s, 1H), 7.38 (dd, 2H, J ¼ 5.5 Hz,
J ¼ 7.8 Hz), 7.24e7.13 (m, 5H), 6.95 (d, 2H, J ¼ 8.9 Hz), 6.84 (s, 1H),
5.83 (m, 1H), 4.36 (m, 2H), 3.37 (m, 4H), 3.04 (m, 4H). 13C NMR
(DMSO-d6)
d
: 161.8 (J ¼ 243 Hz), 153.2, 149.6, 137.9, 134.2 (J ¼ 2 Hz),
ATCC20891,
3153,
PMC1011,
PMC1040R,
PMC1041R,
128.7, 128.4 (J ¼ 9 Hz), 128.1, 122.9, 120.0, 117.4, 115.3 (J ¼ 22 Hz),
PMC1042R), Candida krusei (DSM6128 and PMC0613), Candida
tropicalis (DSM11953 and PMC0910), Candida parapsilosis
(ATCC22019, DSM11224), Candida glabrata (PMC0805, PMC0807,
PMC0850R, PMC0851R, PMC0852R, PMC0853R), C. neoformans
(DSM11959, PMC2136, PMC2123, PMC2115, PMC2103, PMC2107,
PMC2102, and PMC2111), Trichophyton mentagrophytes (DSM 4870,
PMC6515, PMC6531, PMC6503, PMC6509, PMC6552), Microsporum
gypseum (DSM3824, PMC7331 and PMC7303). All of the strains
were stored and grown in accordance with the procedures of the
Clinical and Laboratory Standards Institute (CLSI) [15,16].
74.7, 50.6, 48.1, 43.2. IR: ʋ 1703 cmꢀ1. MS, m/z: 428.89 [MþH]þ.
4.2.4.6. 1-(4-Chlorophenyl)-2-imidazol-1-yl-ethyl] 4-(4-
chlorophenyl)piperazine-1-carboxylate (5f). Yield 88%, as a white
solid. Mp: 113e6. 1H NMR (DMSO-d6):
d 7.53 (s, 1H), 7.45 (d, 2H,
J ¼ 8.3 Hz), 7.37 (d, 2H, J ¼ 8.3 Hz), 7.25 (d, 2H, J ¼ 8.9 Hz), 7.13 (s,
1H), 6.97 (d, 2H, J ¼ 8.9 Hz), 6.84 (s, 1H), 5.86 (m, 1H), 4.38 (m, 2H),
3.65 (m, 4H), 3.09 (m, 4H). 13C NMR (DMSO-d6):
d 153.8, 149.9,
137.8, 136.2, 134.1, 128.7, 128.6, 127.7, 127.4, 124.7, 120.1, 117.7, 75.1,
51.1, 48.9, 43.4 IR: ʋ 1702 cmꢀ1. MS, m/z: 444.87 [MþH]þ.
4.4. Antifungal susceptibility assays
4.2.4.7. [1-(4-Fluorophenyl)-2-imidazol-1-yl-ethyl]4-(furan-2-
carbonyl)piperazine-1-carboxylate (5g). Yield 78%, as a waxy solid.
In vitro antifungal susceptibility was evaluated for all com-
pounds, using the CLSI broth microdilution methods [15,16]. Flu-
conazole and Amphotericin B were used as reference drug. The final
1H NMR (CD3OD):
d
7.71 (s, 1H), 7.41e7.38 (m, 2H), 7.14e7.08 (m,
5H), 6.97 (s, 1H), 6.61 (s, 1H), 5.99 (t, 1H, J ¼ 6.0 Hz), 4.49e4.42 (m,
2H), 3.90e3.40 (m, 8H). 13C (CD3OD):
d
164.2 (J ¼ 244 Hz), 161.2,
concentration ranged from 0.125 to 64 m
g mLꢀ1 for all compounds.
155.3, 148.2, 146.1, 139.2, 134.7 (J ¼ 3 Hz), 129.6 (J ¼ 8 Hz), 129.0,
121.5, 118.0, 116.5 (J ¼ 22 Hz), 112.5, 76.8, 73.5, 55.3, 52.5. IR: ʋ 1699,
1621 cmꢀ1. MS, m/z: 412.99 [MþH]þ.
The compounds were dissolved previously in dimethyl sulfoxide at
concentrations 100 times higher than the highest desired test
concentration and successively diluted in test medium in accor-
dance with the procedures of the CLSI [17].
4.2.4.8. [1-(4-Fluorophenyl)-2-imidazol-1-yl-ethyl]N-[4-(4-
Microdilution trays containing 100 mL of serial two-fold di-
ethylsulfonylpiperazin-1-yl)phenyl]carbamate (6a). Yield 37%, as a
lutions of compound in RPMI 1640 medium (Sigma Aldrich, St.
Louis, Missouri, U.S.A) were inoculated with an organism suspen-
sion adjusted to attain a final inoculum concentration of 1.0 ꢂ 103
white solid. Mp: 113e5. 1H NMR (DMSO-d6):
d 9.64 (s broad, 1H),
7.55 (s, 1H), 7.40 (m, 2H), 7.28e7.15 (m, 5H), 6.90 (d, 2H, J ¼ 8.9 Hz),
6.85 (s, 1H), 5.94 (m, 1H), 4.41 (m, 2H), 3.30 (m, 4H, partially
obscured by HDO signal, it appears after D2O exchange), 3.09 (m,
and 1.5
ꢂ
103 cells mLꢀ1 for yeasts and 0.4
ꢂ
104 and
5 ꢂ 104 CFU mLꢀ1 for dermatophytes. The panels were incubated at
35 ꢃC and observed for the presence of growth at 48 h (Candida
spp.) and 72 h (C. neoformans and dermatophytes).
The MIC defined as, for yeasts, the lowest concentration that
showed ꢁ50% growth inhibition compared with the growth control
and, fordermatophytes, the lowest concentration that showed ꢁ80%
growth inhibition compared with the growth control was evaluated
for all compounds. The results were expressed as the median of the
MIC values of five replicates, and the geometric mean (GM) values.
6H), 1.23 (t, 3H, J ¼ 7.5 Hz). 13C NMR (CDCl3):
162.8 (J ¼ 246.0 Hz),
d
152.5, 147.2, 138.1, 132.8, 131.4, 128.2, 127.9 (J ¼ 8.0 Hz), 120.2, 120.0,
117.7, 115.8 (J ¼ 22.0 Hz), 73.9, 52.1, 50.2, 45.8, 44.0, 7.8. IR: ʋ
1707 cmꢀ1. MS, m/z: 501.93 [MþH]þ.
4.2.4.9. [1-(4-Fluorophenyl)-2-imidazol-1-yl-ethyl] N-[4-[4-(furan-
2-carbonyl)piperazin-1-yl]phenyl]carbamate (6b). Yield 71%, as a
white solid. Mp: 89e91. 1H NMR (DMSO-d6):
d 9.62 (s broad, 1H),
7.86 (s, 1H), 7.54 (s, 1H), 7.39 (m, 2H), 7.27e7.15 (m, 5H), 7.02 (d, 1H,
J ¼ 2.9 Hz), 6.90, (d, 2H, J ¼ 8.4 Hz), 6.84 (s,1H), 6.64 (s, 1H), 5.94 (m,
1H), 4.41 (m, 2H), 3.80 (m, 4H), 3.19 (m, 4H). 13C NMR (CDCl3):
4.5. Cell viability assay
d
162.7 (J ¼ 247 Hz), 159.1, 152.5, 147.9, 143.8, 138.1, 132.7 (J ¼ 3 Hz),
The cytotoxicity of tested new compounds was evaluated
on human lung adenocarcinoma epithelial cell line (A549) ob-
tained from American Type Culture Collection (ATCC CCL-185,
Rockville, MD, USA) by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) reduction assay. The cells
(2 ꢂ 104 cells/well) were seeded into 96-well plates containing
131.0, 127.9 (J ¼ 8 Hz), 120.4, 120.3, 120.0, 117.4, 117.3, 116.7, 115.8
(J ¼ 21 Hz), 111.4, 73.9, 72.3, 52.1, 50.2. IR: ʋ 1714, 1602 cmꢀ1. MS, m/
z: 504.00 [MþH]þ.
4.2.4.10. [1-(4-Fluorophenyl)-2-imidazol-1-yl-ethyl] N-[4-[4-(4-
chlorophenyl)piperazin-1-yl]phenyl]carbamate (6c). Yield 74%, as a
100 ml of supplemented RPMI 1640 (Invitrogen, San Diego, CA,
white solid. Mp: 209e211. 1H NMR (DMSO-d6):
d
9.62 (s broad, 1H),
USA) without phenol red, supplemented with 10% foetal bovine

F. Moraca et al. / European Journal of Medicinal Chemistry 83 (2014) 665e673
673
serum (Invitrogen, San Diego, CA, USA),
L
-glutamine
files and InChiKeys of the most important compounds described in
this article.
(0.3 mg mLꢀ1), penicillin (100 mLꢀ1), and streptomycin
U
(100
m
g mLꢀ1) (EuroClone, Celbio, Milan, Italy), and they were
cultured at 37 ꢃC in 5% CO2. The cells were exposed to the indi-
cated compounds (dissolved in dimethyl sulfoxide, DMSO) at the
m
g mLꢀ1. Each con-
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selected automatically by the software. For the compounds experi-
mentally separated, both (R)- and (S)-enantiomers where intro-
duced in the alignment procedure. Compounds were automatically
cat into a training set and a test set according to GRID MIFs in a ratio
of 1:1. The computation of the ligand-based model was performed
with an accuracy of 150% applying the dissimilarity penalty and was
validated by means of the leave-one-out method. Potential contour
maps were identified by default GRID probes.
[16] Clinical and Laboratory Standards Institute, Reference Method for Broth
Dilution Antifungal Susceptibility Testing of Filamentous Fungi, 2008.
Approved Standard M38 A2, Wayne, PA.
Acknowledgements
[17] Clinical and Laboratory Standards Institute, Reference Method for Broth
Dilution Antifungal Susceptibility Testing of Yeasts, 2008. Informational
Supplement M27S3, Wayne, PA.
This work was supported by the Project PON 01_01802. We are
indebted to Molecular Discovery for access to the FLAP code.
€
[18] Maestro, Version 9.2, Schrodinger, LCC, New York, NY, 2011.
€
[19] LigPrep, Version 2.5, Schrodinger, LCC, New York, NY, 2011.
€
[20] Epik, Version 2.2, Schrodinger, LCC, New York, NY, 2011.
Appendix A. Supplementary data
[21] M. Baroni, G. Cruciani, S. Sciabola, F. Perruccio, J.S. Mason, A common refer-
ence framework for analyzing/comparing proteins and ligands. Fingerprints
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.001. These data include MOL