Synthesis and antifungal activity of a new series of 2-(1H-imidazol-1-yl)- 1-phenylethanol derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.01.034

A new series of aromatic ester and carbamate derivatives of 2-(1H-imidazol-1-yl)-1-phenylethanol were synthesized and evaluated for their antifungal activity towards Candida albicans and non-albicans Candida species strains. The aromatic biphenyl ester derivatives 6a-c were more active than the reference compound fluconazole. 6c possesses a MIC mean values of 1.7 ± 1.4 μg mL^-1 vs C. albicans and 1.9 ± 2.0 μg mL ^-1 vs non-albicans Candida species strains. The racemic mixtures of 6a, b were purified to afford the pure enantiomers. The (-) isomers were up to 500 times more active than (+) isomers. (-)-6a and (-)-6b were thirty and ninety times more active than fluconazole towards C. krusei strain respectively. The racemates of 6a-c showed low cytotoxicity against human monocytic cell line (U937) with 6a demonstrating a CC₅₀ greater than 128 μg mL ^-1.

Full text of DOI:10.1016/j.ejmech.2012.01.034

European Journal of Medicinal Chemistry 49 (2012) 334e342
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-
phenylethanol derivatives
,
a
a
a
Daniela De Vita a, Luigi Scipione a , Silvano Tortorella , Paolo Mellini , Barbara Di Rienzo ,
Giovanna Simonetti b, Felicia Diodata D’Auria b, Simona Panella b, Roberto Cirilli c, Roberto Di Santo a,
Anna Teresa Palamara b
*
a Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco “Sapienza” Università di Roma, P.le Aldo Moro 5, 00185 Roma, Italy
b Dipartimento di Sanità Pubblica e Malattie Infettive, “Sapienza” Università di Roma, P.le Aldo Moro 5, 00185 Roma, Italy
c Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
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 aromatic ester and carbamate derivatives of 2-(1H-imidazol-1-yl)-1-phenylethanol were
synthesized and evaluated for their antifungal activity towards Candida albicans and non-albicans
Candida species strains. The aromatic biphenyl ester derivatives 6aec were more active than the refer-
Received 22 June 2011
Received in revised form
17 January 2012
Accepted 17 January 2012
Available online 26 January 2012
ence compound fluconazole. 6c possesses a MIC mean values of 1.7 ꢀ 1.4
m
g mLꢁ1 vs C. albicans and
1.9 ꢀ 2.0
m
g mLꢁ1 vs non-albicans Candida species strains. The racemic mixtures of 6a, b were purified to
afford the pure enantiomers. The (ꢁ) isomers were up to 500 times more active than (þ) isomers. (ꢁ)-6a
and (ꢁ)-6b were thirty and ninety times more active than fluconazole towards C. krusei strain
respectively.
Keywords:
Antifungal imidazoles
Enantiomers separation
Candida albicans species
Non-albicans Candida species
Human monocytic cell line
The racemates of 6aec showed low cytotoxicity against human monocytic cell line (U937) with 6a
demonstrating a CC50 greater than 128 m .
g mLꢁ1
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
The main drugs used for the treatment of invasive candidiasis
belong to the classes of azoles, polyenes (amphotericin B), echi-
nocandines (caspofungin) and fluoropyrimidines (5-flucytosine),
used alone or associated in combination therapy [6,7] (Fig. 1). The
azole derivatives including triazoles (e.g. fluconazole) and imidaz-
oles (e.g. econazole, miconazole, clotrimazole, and ketoconazole)
inhibit the biosynthesis of fungal sterols through the inhibition of
CYP51 and are commonly used as first line drugs to treat Candida
infections.
In fungal species ergosterol plays an important role in the
maintenance of membrane organization and functions. 14
a-Lano-
sterol demethylase (CYP51, P45014DM) is a key enzyme in the
pathway of ergosterol biosynthesis. It is a member of the cyto-
chrome P450 superfamily containing an iron protoporphyrin unit
The increasing incidence of serious fungal infections is a well-
recognized problem. Such infections, predominantly affecting
immunocompromised individuals, including HIV-1 infected, organ-
transplanted, and those undergoing cancer chemotherapy, require
prompt and appropriate therapy. Candida species are the main
agent responsible for nosocomial fungal infections [1]. Candida
albicans, which is commensal in healthy individuals [2], is the most
common pathogen isolated in invasive candidiasis with about
30e40% of mortality. Furthermore, an increasing rates of invasive
candidiasis caused by non-albicans Candida species have been re-
ported worldwide; these species, typically less sensitive to the
principal antifungal drugs, include Candida tropicalis, Candida
glabrata, Candida parapsilosis, and Candida krusei [3e5]. For these
reasons, the development of new and more potent antifungal drugs
becomes even more urgent.
located in its active site catalyzing the oxidative removal of the 14
a-
methyl group of lanosterol to give
D
14,15-desaturated intermediates.
Azole drugs bind the CYP51 active site through coordination to
the heme iron by the N-3 imidazole nitrogen (or N-4 triazole) thus
preventing the binding of steroidic substrates [8,9]. The depletion
of ergosterol and accumulation of 14a-methylated sterols alter
membrane fluidity which reduce the activity of the enzymes
associated with membrane and in turn increase the permeability.
* Corresponding author. Tel.: þ390649913966; fax: þ390649913133.
E-mail addresses: luigi.scipione@uniroma1.it, luigi.scipione@fastwebnet.it
(L. Scipione).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.034

D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
HO
335
OH
OH
OH
O
O
HO
O
OH OH
OH OH
COOH
OH
OH
O
HO
O
O
NH
O
OH
O
H2N
O
Amphotericin B
OH
HO
NH2
NH
NH
HO
O
O
NH2
NH
F
OH
N
H
N
NH
O
O
N
H
OH
Caspofungin
H2N
5-Flucytosine
N
N
N
N
Cl
H
O
Cl
N
N
N
N
OH
N
O
O
N
N
O
Cl
R
F
N
Cl
N
N
Cl
F
Cl
O
Fluconazole
Ketoconazole
R = H : Econazole
R = Cl : Miconazole
Clotrimazole
Fig. 1. Main drugs used for the treatment of invasive candidiasis.
Ultimately resulting inhibition of fungal growth and replication
[10,11].
specific features of the pharmacophore described in the above
theoretical models: the iron-binding azole group (A) and the
aromatic group adjacent to it (B). The data reported in literature
encouraged our efforts to synthesize new derivatives, precisely
aromatic esters and carbamates, introducing a second hydrophobic
or aromatic moiety thus satisfying the required interaction features
to allow a more effective interaction with the active site of CYP51.
Wahbi and coll. reported that some alkyl esters of 2-(1H-imi-
dazol-1-yl)-1-phenylethanols were characterized by lower anti-
fungal activity than reference drugs [19]. While Walker and coll.
reported that some aryl and arylalkyl esters are slightly more active
than Miconazole towards some C. albicans strains [20], indicating
the importance of the second aromatic region (C). In this context, it
is relevant to point out that some azole compounds containing
biphenyl moiety inhibit Trypanosoma cruzi and T. brucei CYP51
reducing parasite cells growth [21,22]. These results encouraged us
Fluconazole is the first line azole-antifungal drug and is
considered the gold standard in the treatment of infections by
C. albicans and Cryptococcus neoformans, but its extensive clinical
use has resulted in resistance especially with respect to C. albicans
[12].
The increasing incidence of Candida species infections coupled
with the emergence of azole-resistant fungal strains have stimu-
lated the search for alternative antifungal drugs.
The crystal structure of several bacterial CYP51 cytochromes,
such as Mycobacterium tuberculosis [13] and Trypanosoma brucei
[14] have been published. Unfortunately, there is no three-
dimensional structural information available on C. albicans (CA-
CYP51) or other fungal CYP51 enzymes. However, the rational
design of new antifungal molecules can take advantage of the
homology modeling and pharmacophore modeling techniques
[15e17].
Iron
According to these models, schematically depicted in Fig. 2, the
main azole drugs possess at least three principal pharmacophoric
groups: (A) iron coordinating group, consisting of imidazole or
triazole ring, able to interact with the heme iron, (B) a first
hydrophobic moiety (typically aromatic) near the iron coordinating
group and (C) a second aromatic region. Certain active compounds
present an additional hydrophobic area referred as region (D).
In this paper, we report the synthesis and in vitro evaluation of
antifungal activity of a new series of 2-(1H-imidazol-1-yl)-1-
phenylethanol derivatives. We started from the observation that
some phenyl-substituted 2-(1H-imidazol-1-yl)-1-phenylethanols
described in literature are characterized by a weak antifungal
activity [18]. Such compounds represent an interesting starting
point to develop new antifungal drugs since they possess two
coordinating
second
group
N
C
aromatic
H
group
A
O
N
O
D
additional
hydrophobic
group
O
Cl
N
N
aromatic
group
Cl
B
O
Ketoconazole
Fig. 2. Schematic representation of the pharmacophore of CA-CYP51 binding azoles.

336
D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
to synthesize the biphenyl esters derivatives of 2-(1H-imidazol-1-
yl)-1-phenylethanol.
N
N
N
Karakurt and coworkers reported the lack of antifungal activity,
against C. albicans and non-albicans Candida species, of some 2-
(1H-imidazol-1-yl)-1-(naphthalen-2-yl)ethanol alkyl and aryl
esters [23]. These results suggest to us not to increase the size of the
aromatic ring (B), since this could lead to a reduction in antifungal
activity.
N
O
OH
O
a; b
O
b: X = F
c: X = Cl
The literature data [19,20] also showed that a double substitu-
tion of hydrogen atom at positions 2 and 4 of the aromatic ring (B)
with chlorines does not appear to be determinant for activity. For
this reason, in the first instance, we decided to investigate only the
replacement of H in position 4 with a halogen atom, i.e. fluorine and
chlorine.
X
X
2b,c
7b,c
Scheme 2. (a) NaH, CH3CN, rt, 2 h; (b) phenoxyacetyl chloride, over night, rt.
The separation was performed by HPLC on the Chiralcel OD
2. Results and discussion
chiral stationary phase (CSP) using the binary mixture n-hexane-
ethanol 50:50 (v/v) as eluent (Fig. 3). The optimized analytical
conditions were easily scaled-up to semipreparative level
employing a 1 cm I.D. Chiralcel OD column. Multi-mg amounts of
enantiomerically pure samples (ee > 99%) were isolated and
submitted to polarimetric measurements. The first eluting enan-
tiomers were dextrorotatory in ethanol solution at 589 nm wave-
length and the more retained ones were levorotatory in the same
experimental conditions.
2.1. Chemistry
2-(1H-imidazol-1-yl)-1-phenylethanols (2aed) were prepared,
in two steps as described in literature [18]. Treatment of substituted
2-bromo-1-phenylethanones with 1H-imidazole in dime-
thylformamide afforded 2-(1H-imidazol-1-yl)-1-phenylethanones,
which were subsequently reduced by methanolic sodium borohy-
dride to give rise to the alcohols 2 (2aed).
The racemic alcohols 2aed were treated with sodium hydride in
anhydrous acetonitrile followed by the addition of the appropriate
acylchloride to afford the final desired products (Schemes 1 and 2).
Likewise, carbamates 8aec were prepared by adding 4-(2-propyl)-
phenylisocyanate to an acetonitrile suspension of the alcoholates
obtained from 2aec (Scheme 3).
The isocyanate of 2,6-dichloro-4-aminopyridine was generated
using triphosgene and TEA in anhydrous benzene and reacted with
alcohol 2c to afford the carbamate 9c (Scheme 4).
All compounds were tested as racemic mixtures to evaluate the
antifungal and cytotoxic activities, and only active racemates were
resolved. Racemates 6a,b were separated to generate (ꢁ)-6a,b and
(þ)-6a,b which were tested for the antifungal activity.
2.2. In vitro antifungal activity
The in vitro antifungal activity of imidazole derivatives 2aec,
3aed, 4bec, 5a, 6aec, 7bec, 8aec and 9c was evaluated against
four strains of C. albicans and seven strains of non-albicans Candida
species. The obtained data, expressed as the mean of minimal
inhibitory concentration (MIC) values, MIC50, MIC90 and MIC range
are reported in Table 1 for C. albicans and in Table 2 for non-albicans
Candida species.
MIC values obtained for the alcohols 2aec confirm their previ-
ously observed weak antifungal activity [18] and are in agreement
with above mentioned pharmacophoric models, which suggest that
azole drugs need at least three points of binding to have an optimal
interaction with CA-CYP51. 2aec possess only the iron-binding
imidazole and the aromatic group (B) adjacent to it. According to
this observation, the esterification of alcoholic function of 2aec
with aromatic groups produced, in most cases, an enhancement
in antifungal activity. The presence of a second single aromatic
moiety produced a noticeable, but not particularly pronounced
effect, as indicated from the observed activity values of the 3-
trifluorobenzoyl ester 3b. 3b is one of the most active benzoyl
N
N
R3
R2
N
N
O
OH
a, b
O
R1
derivatives, with MIC mean values of 27.7
ꢀ
49.4 and
52.9 ꢀ 61.7
m
g mLꢁ1 vs C. albicans and non-albicans Candida species
X
X
N
N
N
2a-d
3a-d, 5a, 6a-c
N
H
OH
O
N
a , b
3a: R1=R3=H, R2=CF3, X=H
3b: R1=R3=H, R2=CF3, X=F
3c: R1=R3=H, R2=CF3, X=Cl
3d: R1=R3=H, R2=CF3, X=CF3
O
2a : X=H
2b : X=F
2c : X=Cl
2d : X=CF3
X
X
a : X = H
b : X = F
c : X = Cl
2a-c
8a-c
Scheme 1. (a) NaH, CH3CN, rt, 2 h; (b) 3aec: 3-(trifluoromethyl)benzoyl chloride, 24 h,
rt. 3d: 3-(trifluoromethyl)benzoyl chloride, 48 h, rt. 5a: 4-(trifluoromethyl)benzoyl
chloride, 24 h, rt. 6aec: biphenyl-4-carbonyl chloride, 48 h, rt.
Scheme 3. (a) NaH, CH3CN, rt, 2 h; (b): 1-Isocyanato-4-(propan-2-yl)benzene, 48 h, rt.

D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
337
Table 1
N
In vitro antifungal activity for studied compounds against Candida albicans strains.
NH2
N
N
Compound
MICꢀ SD
g mLꢁ1
MIC50
g mLꢁ1
MIC90
g mLꢁ1
Range
g mLꢁ1
)
H
N
(
m
)
(
m
)
(
m
)
(
m
a , b , c
O
Cl
2a
2b
2c
3a
3b
3d
4b
4c
5a
6a
6b
6c
7b
7c
8a
8b
106.7 ꢀ 33.0
101.3 ꢀ 42.5
112.0 ꢀ 39.2
91.4 ꢀ 34.2
27.7 ꢀ 49.4
91.4 ꢀ 34.2
34.3 ꢀ 46.1
46.3 ꢀ 56.5
33.1 ꢀ 46.7
3.7 ꢀ 5.1
128
128
128
64
8
64
8
8
8
1
4
1
64
64
64
16
64
128
1
128
128
128
128
16
128
64
16
64
8
8
4
128
128
128
64
128
128
16
64e128
32e128
32e128
64e128
2e128
64e128
8e128
4e128
O
N
Cl
Cl
Cl
Cl
9c
8e128
Scheme 4. (a) TEA, benzene, 35 ꢂC; (b) triphosgene, 12 h, rt; (c) 2c, 48 h, rt.
0.125e16
0.25e8
0.125e4
8e128
2e128
32e128
1e128
4e128
64e128
0.25e16
4.4 ꢀ 3.1
1.7 ꢀ 1.4
76.6 ꢀ 52.6
67.7 ꢀ 59.8
77.7 ꢀ 36.3
39.6 ꢀ 48.1
60.0 ꢀ 52.1
118.8 ꢀ 24.2
4.9 ꢀ 6.9
strains respectively. The corresponding hydrochloride 4b showed
MIC mean values of 34.3 ꢀ 46.1
g mLꢁ1 vs C. albicans and
g mLꢁ1 vs non-albicans Candida species strains, indi-
m
22.6 ꢀ 20.1
m
8c
9c
cating that the protonation of imidazole ring do not produce
a significant modification in antifungal activity.
Fluconazole
Replacement of the ester function with a carbamate moiety led
to compounds 8aec and 9c. Despite the presence of a second
aromatic moiety the compounds of this sub-series were not very
active. In particular, the most active carbamate 8b showed MIC
Antifungal activity was determined according to CLSI guidelines (CLSI document
M27-A3,2008). MIC arithmetic mean of minimal inhibitory concentration;
SD ¼ standard deviation. MIC50 and MIC90: MICs at which 50% and 90% of strains,
respectively, are inhibited. Data represent MIC values of three separate experiments
in triplicate. Additional data are given in supplemental Table S1.
¼
mean values of 39.6 ꢀ 48.1
m
g mLꢁ1 vs C. albicans strains and
68.4 ꢀ 59.2
m
g mLꢁ1 vs non-albicans Candida species strains, only
slightly better than those found for alcohols 2aec. Compound 9c, in
which a 2,6-dichloropyridine moiety was introduced as the second
aromatic moiety, was found to be the least active of the carbamate
sub-series.
species in 90% of strains tested. Compounds 6a, 6b and 6c showed
MIC90 values of 8, 8 and 4
8 and 4
g mLꢁ1 towards non-albicans Candida strains compared to
16
g mLꢁ1 for fluconazole (Tables 1 and 2). Furthermore, 6c
possesses a MIC mean values of 1.7 ꢀ 1.4
g mLꢁ1 vs C. albicans and
1.9 ꢀ 2.0
g mLꢁ1 vs non-albicans Candida species strains, lower
than fluconazole which exhibited MIC values of 4.9 ꢀ 6.9
g mLꢁ1
and 7.9 ꢀ 15.8
g mLꢁ1 vs C. albicans and non-albicans Candida
species, respectively (Tables 1 and 2).
m
g mLꢁ1 towards C. albicans strains and 4,
m
The esterification of alcoholic function of 2aec with biphenyl-4-
carbonyl chloride gave rise to the most active compounds of this
series (6aec). These analogs were found to be more active than
fluconazole towards both C. albicans and non-albicans Candida
m
m
m
m
m
The biphenyl esters 6aec were found to be active towards
C. krusei, a Candida species intrinsically resistant to fluconazole. In
Table 2
In vitro antifungal activity of studied compounds against non-albicans Candida
species.
Compound
MICꢀ SD
g mLꢁ1
98.5 ꢀ 33.2
MIC50
g mLꢁ1
64
128
128
64
8
128
16
32
32
0.5
1
1
MIC90
g mLꢁ1
Range
g mLꢁ1
)
(
m
)
(
m
)
(
m
)
(
m
2a
2b
2c
3a
3b
3d
4b
4c
5a
6a
6b
6c
7b
7c
8a
8b
128
128
128
128
128
128
32
128
128
4
64e128
16e128
16e128
8e128
4e128
32e128
1e64
0.5e128
4e128
0.25e16
1e8
0.125e8
1e128
2e128
64e128
4e128
104.0 ꢀ 45.0
108.0 ꢀ 38.1
85.0 ꢀ 49.0
52.9 ꢀ 61.7
100.0 ꢀ 39.8
22.6 ꢀ 20.1
43.8 ꢀ 49.5
55.5 ꢀ 50.2
2.1 ꢀ 3.9
2.5 ꢀ 2.9
8
4
1.9 ꢀ 2.0
61.1 ꢀ 63.7
51.3 ꢀ 58.1
106.7 ꢀ 32.0
68.4 ꢀ 59.2
114.7 ꢀ 40.0
120.9 ꢀ 21.3
7.9 ꢀ 15.8
4
128
128
128
128
128
128
16
16
128
4
128
128
2
8c
9c
8e128
64e128
0.5e64
Fluconazole
Antifungal activity was determined according to CLSI guidelines (CLSI document
M27-A3,2008). MIC arithmetic mean of minimal inhibitory concentration;
¼
Fig. 3. Typical chromatograms illustrating the resolution of 6a,b. Column, Chiralcel OD
SD ¼ standard deviation. MIC50 and MIC90: MICs at which 50% and 90% of strains,
respectively, are inhibited. Data represent MIC values of three separate experiments
in triplicate. Additional data are given in supplemental table S1.
(250 mm ꢃ 4.6 mm I.D.); eluent, n-hexane/ethanol 50:50 (v/v); flow-rate, 1 mL minꢁ1
;
detector, UV (black) and CD (gray) at 254 nm; temperature, 40 ꢂC.

338
D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
Table 3
Table 5
MIC mean values for compounds 6aec towards various non-albicans Candida
Effect of 6a, 6b and 6c on the human monocytic cells (U937) growth and their
species.
calculated selectivity index values (SI).
MICꢀSD (
m
g mLꢁ1
)
Compound
CC50
(
m
g mLꢁ1
)
SI
6a
6b
6c
> 128
> 44.1
12.0
49.1
Compound
C. parapsilosis
C. tropicalis
C. krusei
C. glabrata
41.5 ꢀ 5.1
6a
6b
6c
0.70 ꢀ 0.74
1.30 ꢀ 0.96
1.05 ꢀ 0.62
1.60 ꢀ 0.55
0.50 ꢀ 0.39
4.08 ꢀ 3.47
0.89 ꢀ 0.67
3.25 ꢀ 1.4
4.70 ꢀ 5.70
2.25 ꢀ 2.02
3.70 ꢀ 2.60
26.0 ꢀ 23.71
4 ꢀ 0
1 ꢀ 0
88.3 ꢀ 10.8
1 ꢀ 0
CC50: concentration (m
g mLꢁ1) required to reduce cell viability by 50%; SI: defined as
Fluconazole
0.87 ꢀ 0.25
the ratio between CC50 and mean value of MIC (the concentration of the drug
needed to reduced fungal cell viability to 50%) observed towards all Candida species.
Data represent means ꢀ standard deviation from three independent experiments,
each performed in triplicate.
fact, compounds 6a, 6b and 6c showed MIC mean values for
C. krusei of 4.70 ꢀ 5.70, 2.25 ꢀ 2.02 and 3.70 ꢀ 2.60
g mLꢁ1. Five to
eleven times lower than that observed for fluconazole
g mLꢁ1, Table 3).
The presence of a halogen atom at 4 position of the aromatic
ring of the phenylethyl moiety does not markedly affect the anti-
fungal activity, as evidenced by MIC mean values of compounds
6aec.
The antifungal activity of the pure separated enantiomers of
esters 6a, b was preliminarily assessed against strains of C. albicans
(ATCC24433), C. parapsilosis (DSM11224), C. krusei (PMC0613) and
C. tropicalis (DSM11953). The data are summarized in Table 4, which
also reports the activities of the corresponding racemates and
fluconazole.
m
indicated by CC50 values of fluoro derivative 6b (41.5 ꢀ 5.1
m )
g mLꢁ1
and chloro derivative 6c (88.3 ꢀ 10.8
m
g mLꢁ1).
(26.0 ꢀ 23.71
m
3. Conclusions
The results obtained for this series of 2-(1H-imidazol-1-yl)-1-
phenylethanolesters and are in agreement with the previously
developed theoretical pharmacophoric models. The presence of an
additional extended aromatic biphenyl moiety was essential to
enhance the activity as demonstrated by compounds 6aec. The
presence of a halogen atom at 4 position of the aromatic ring of the
phenylethyl moiety proved to be a minor factor.
In the 90% of tested strains of C. albicans and non-albicans
Candida species, compounds 6aec were more active than fluco-
nazole. These compounds showed an interesting activity against
Candida species strains with lower sensitivity to fluconazole
These data indicate that the levorotatory enantiomers were
more active than the dextrorotatory ones. (ꢁ)-6b was found to be
about 500 times more active than (þ)-6b towards C. albicans,
C. parapsilosis and C. krusei strains and about 120 times towards
C. tropicalis.
(>8 m
g mLꢁ1). (Table S1, Supplementary material).
Cytotoxicity studies showed that compounds 6aec presented
a low toxicity on cultured human monocytic cell line (U937). This
activity was negatively influenced by the presence of a halogen
atom at 4 position of the aromatic ring of the phenylethyl moiety.
Preliminary data on pure enantiomers showed a markedly
higher antifungal activity of (ꢁ)-6a and (ꢁ)-6b with respect to
dextrorotatory isomers, thus suggesting that the former are able to
better satisfy the pharmacophore features to establish optimal
interactions with CA-CYP51. Further studies are in progress to
determine the absolute configuration of the (ꢁ)-isomers and to
assess how these compounds overlap with computational phar-
macophoric models.
In conclusion, the antifungal activity and preliminary toxicity
data obtained for esters 6aec, in particular for their levorotatory
enantiomers, may suggest that these compounds represent an
interesting starting point to develop new antifungal azole drugs
characterized by higher potency and an improved safety profile.
(ꢁ)-6a and (ꢁ)-6b were found to be more active than flucona-
zole towards strains of C. albicans, C. parapsilosis, C. tropicalis and up
to 30e90 times towards strains of C. krusei.
2.3. In vitro cytotoxic activity
The in vitro toxicity of compounds 6aec were assessed analyzing
the dose related effects towards the growth of cultured human
monocytic cells (U937) and calculating the corresponding CC50, i.e.
the concentration (m
g mLꢁ1) required to reduce cell viability by 50%
(Table 5, Fig. S1 Supplementary material); the selectivity index (SI),
i.e. the ratio between CC50 and mean value of MIC observed towards
all Candida species, was also reported. The toxicity test showed that
compound 6a presents the highest value of CC50 (>128 m )
g mLꢁ1
and good selectivity towards fungi. In this context, it can be pointed
out that the presence of a halogen atom, at 4 position of the
aromatic ring of the phenylethyl moiety, influenced the toxicity of
compounds 6aec towards cultured human cells (U937), as
4. Experimental section
4.1. Chemistry
Table 4
MIC mean values of racemates 6a, b and their pure enantiomers against four strains
of Candida species.
4.1.1. Materials and methods
MICꢀSD (
m
g mLꢁ1
)
All reagents and solvents were of high analytical grade and were
purchased from SigmaeAldrich (Milano, Italy). Substituted 2-(1H-
imidazol-1-yl)-1-phenylethanols (2aed) were prepared according
to literature procedure [18]. Melting points were determined on
Tottoli apparatus (Buchi) and are uncorrected. Infrared spectra
were recorded on a Spectrum One ATR PerkineElmer FT-IR spec-
trometer. 1H NMR spectra were acquired on a Bruker AVANCE-400
spectrometer at 9.4 T, in DMSO-d6 or CDCl3 at 27 ꢂC; chemical shift
values are given in
ence. Coupling constants are given in Hz.
Mass spectra were recorded on an API-TOF Mariner by
Perspective Biosystem (Straford, Texas, USA), samples were
Compound
C. albicans
ATCC24433
C. parapsilosis
DSM11224
C. krusei
PMC0613
C. tropicalis
DSM11953
(ꢀ) 6a
0.37 ꢀ 0.22
33.70 ꢀ 12.50
0.15 ꢀ 0.09
1.5 ꢀ 0.87
64 ꢀ 0
0.33 ꢀ 0.14
26.67 ꢀ 9.24
0.125 ꢀ 0
0.67 ꢀ 0.29
21.33 ꢀ 9.24
0.37 ꢀ 0.22
0.83 ꢀ 0.29
64 ꢀ 0
0.58 ꢀ 0.38
26.67 ꢀ 9.24
0.15 ꢀ 0.09
2.17 ꢀ 1.76
32 ꢀ 0
(þ) 6a
(ꢁ) 6a
(ꢀ) 6b
0.42 ꢀ 0.14
35.55 ꢀ 17.50
0.08 ꢀ 0.04
1 ꢀ 0
(þ) 6b
(ꢁ) 6b
0.125 ꢀ 0
1 ꢀ 0
0.125 ꢀ 0
12 ꢀ 4.4
0.27 ꢀ 0.22
2.5 ꢀ 1.6
d (ppm) with respect to TMS as internal refer-
Fluconazole
Antifungal activity was determined according to CLSI guidelines (CLSI document
M27-A3,2008). MIC arithmetic mean of minimal inhibitory concentration;
SD ¼ standard deviation.
¼

D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
339
injected by an Harvard pump using a flow-rate of 5e10
infused in the electrospray system.
Elemental analyses were obtained by a PE 2400 (PerkineElmer)
analyzer and the analytical results were within ꢀ0.4% of the
theoretical values for all compounds.
m
l minꢁ1
,
Methanol/dichloromethane (0.4 : 9.6) was used as eluent for the
chromatographic purification. 5a was obtained in 48% yield as
a white solid, mp 70e2 ꢂC (from ethylacetate).1H NMR (CDCl3):
d
8.14 (d, 2H, J ¼ 7.6 Hz), 7.72 (d, 2H, J ¼ 7.6 Hz), 7.38e7.32 (m, 6H),
7.00 (s,1H), 6.84 (s,1H), 6.22 (dd,1H, J ¼ 6.7 Hz, J ¼ 5.0 Hz), 4.44 (dd,
1H, J ¼ 14.1 Hz, J ¼ 6.3 Hz), 4.39 (dd, 1H, J ¼ 14.1 Hz, J ¼ 5.0 Hz). IR:
n
4.1.2. General procedure for the synthesis of 3aed, 5a and 6aec
4.1.2.1. Example: synthesis of (ꢀ)2-(1H-imidazol-1-yl)-1-phenylethyl-
3-(trifluoromethyl)benzoate (3a). To a stirred suspension of 2-(1H-
imidazol-1-yl)ethanol (2a) (0.53 mmol) in dry acetonitrile (5 mL)
was added sodium hydride (0.53 mmol). The reaction mixture was
stirred for 2 h at room temperature (rt) and then treated with 3-
(trifluoromethyl)benzoyl chloride (0.75 mmol). The reaction
mixture was stirred for 24 h at rt. The solvent was removed under
reduced pressure and the residue was dissolved in an adequate
volume of dichloromethane (5e25 mL) and washed with aqueous
saturated potassium carbonate.
1726 cmꢁ1. MS, m/z: 361 [M þ H]þ.
4.1.2.6. Synthesis of (ꢀ)2-(1H-imidazol-1-yl)-1-phenylethyl biphenyl-
4-carboxylate (6a). 6a was prepared as described above for 3d
using the alcohol 2a and biphenyl-4-carbonyl chloride. Methanol/
ethylacetate (0.25 : 9.75) was used as eluent for the chromato-
graphic purification. 6a was obtained in 40% yield as a white solid,
mp 110e2 ꢂC (from ethylacetate). 1H NMR (CDCl3):
d 8.15e8.08 (m,
2H), 7.55e7.68 (m, 2H), 7.69e7.60 (m, 2H), 7.50e7.29 (m, 9H), 7.02
(s, 1H), 6.84 (s, 1H), 6.24 (t, 1H, J ¼ 5.4 Hz), 4.49e4.40 (m, 2H). IR:
n
1718 cmꢁ1. MS, m/z: 369 [M þ H]þ.
The separated organic layer was dried over anhydrous sodium
sulphate and after filtration was evaporated under reduced pres-
sure. The obtained crude yellow oil was purified by column chro-
matography on silica gel using methanol/dichloromethane (1 : 9) as
eluent to give 80 mg of 3a as colorless oil (yield 58%).
4.1.2.7. Synthesis of (ꢀ)1-(4-fluorophenyl)-2-(1H-imidazol-1-yl)
ethyl biphenyl-4-carboxylate (6b). 6b was prepared as described
above for 6a using the alcohol 2b. After addition of biphenyl-4-
carbonyl chloride the reaction mixture was refluxed for 3 h.
Methanol/ethylacetate (0.25 : 9.75) was used as eluent for the
chromatographic purification, and the more retained fraction was
furtherly purified by a column chromatography on alumina with
the same eluent. 6b was obtained in 75% yield as colorless waxy
solid.
1H NMR (CDCl3):
d
8.29 (s, 1H), 8.21 (d, 1H, J ¼ 7.8 Hz), 7.86 (dd,
1H, J ¼ 7.8 Hz, J ¼ 0.8 Hz), 7.63 (t, 1H, J ¼ 7.8 Hz), 7.41e7.38 (m, 4H),
7.34e7.30 (m, 2H), 7.04 (s, 1H), 6.86 (s, 1H), 6.21 (dd, 1H, J ¼ 6.6 Hz,
J ¼ 4.7 Hz), 4.49 (dd, 1H, J ¼ 6.6 Hz, J ¼ 16.6 Hz). 4.43 (dd, 1H,
J ¼ 4.7 Hz, J ¼ 16.6 Hz). IR:
n
1726 cmꢁ1. MS, m/z: 361 [M þ H]þ.
1H NMR (CDCl3):
d
8.13 (d, 2H, J ¼ 8.3 Hz), 7.71 (d, 2H, J ¼ 8.9 Hz),
4.1.2.2. Synthesis of (ꢀ)1-(4-fluorophenyl)-2-(1H-imidazol-1-yl)
ethyl 3-(trifluoromethyl)benzoate (3b). 3b was prepared as
described above for 3a using the alcohol 2b. Refluxing conditions
were used during 2 h period. Methanol/dichloromethane (0.4 : 9.6)
was used as eluent for the chromatographic purification. 3b was
7.69e7.61 (m, 2H), 7.55e7.46 (m, 3H), 7.44e7.40 (m, 1H), 7.33e7.25
(m, 2H), 7.09e7.01 (m, 3H), 6.88 (s, 1H), 6.25 (t, 1H, J ¼ 5.4 Hz), 4.46
(d, 2H, J ¼ 5.4 Hz). IR:
n
1713 cmꢁ1. MS, m/z: 387 [M þ H]þ.
4.1.2.8. Synthesis of (ꢀ)1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)
ethyl biphenyl-4-carboxylate (6c). 6c was prepared as described
above for 6a using the alcohol 2c. Methanol/ethylacetate (0.25 :
9.75) was used as eluent for the chromatographic purification. 6c
was obtained in 56% yield as a colorless waxy solid. 1H NMR
obtained in 54% yield as a colorless oil. 1H NMR (CDCl3):
d 8.30 (s,
1H), 8.23 (d, 1H, J ¼ 7.8 Hz), 7.87 (d, 1H, J ¼ 7.8 Hz), 7.63 (t, 1H,
J ¼ 7.8 Hz), 7.36 (s, 1H), 7.29e7.26 (m, 2H), 7.07e7.02 (m, 3H), 6.87
(s, 1H), 6.21 (t, 1H, J ¼ 6.6 Hz), 4.46 (dd, 1H, J ¼ 14.6 Hz, J ¼ 6.6 Hz),
4.40 (dd, 1H, J ¼ 14.6 Hz, J ¼ 4.8 Hz). IR:
n
1729 cmꢁ1. MS, m/z: 379
(CDCl3):
d
8.12 (d, 2H, J ¼ 8.4 Hz), 7.70 (d, 2H, J ¼ 8.4 Hz), 7.67e7.60
[M þ H]þ.
(m, 2H), 7.51e7.41 (m, 4H), 7.35 (d, 2H, J ¼ 8.4 Hz), 7.24 (d, 2H,
J ¼ 8.4 Hz), 7.04 (s, 1H), 6.86 (s, 1H), 6.22 (t, 1H, J ¼ 5.4 Hz), 4.43 (d,
4.1.2.3. Synthesis of (ꢀ)1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)
ethyl 3-(trifluoromethyl)benzoate (3c). 3c was prepared as
described above for 3a using the alcohol 2c. Methanol/dichloro-
methane (0.4 : 9.6) was used as eluent for the chromatographic
purification. 3c was obtained in 56% yield as a colorless oil. 1H NMR
2H, J ¼ 5.4 Hz). IR:
n
1718 cmꢁ1. MS, m/z: 403 [M þ H]þ.
4.1.3. Preparation of the hydrochlorides 4b, c
3b was dissolved in methanol and the solution was saturated
with gaseous HCl for 1 h and cooled at 0 ꢂC. The solvent was
removed under reduced pressure to give the hydrochloride 4b as
a white solid. The solid was washed with diethylether and crys-
tallized from methanol/diethylether. 4c was prepared using the
same procedure.
(CDCl3):
d
8.29 (s, 1H), 8.21 (d, 1H, J ¼ 7.8 Hz), 7.87 (d, 1H, J ¼ 7.8 Hz),
7.64 (t, 1H, J ¼ 7.8 Hz), 7.39e7.33 (m, 3H), 7.28e7.21 (m, 2H), 7.04 (s,
1H), 6.86 (s, 1H), 6.20 (t, 1H, J ¼ 5.3 Hz), 4.48e4.41 (m, 2H). IR:
n
1729 cmꢁ1. MS, m/z: 395 [M þ H]þ.
4b. Yield 100% as a white solid, mp 175e7 ꢂC 1H NMR (DMSO-
d6): 8.93 (s, 1H), 8.34 (d, 1H, J ¼ 7.7 Hz), 8.27 (s, 1H), 8.09 (d, 1H,
J ¼ 7.7 Hz), 7.80 (t, 1H, J ¼ 7.7 Hz), 7.76 (s, 1H), 7.61e7.53 (m, 3H),
7.29 (t, 2H, J ¼ 8.9 Hz), 6.36 (dd, 1H, J ¼ 8.9 Hz, J ¼ 3.3 Hz), 4.87 (dd,
4.1.2.4. Synthesis of (ꢀ)1-(4-trifluorophenyl)-2-(1H-imidazol-1-yl)
ethyl 3-(trifluoromethyl)benzoate (3d). 3d was prepared as
described above for 3a using the alcohol 2d. After addition of 3-
(trifluoromethyl)benzoyl chloride the reaction mixture was stirred
for 48 h. Methanol/dichloromethane (0.4 : 9.6) was used as eluent
for the chromatographic purification. 3d was obtained in 25% yield
1H, J ¼ 14.4 Hz, J ¼ 8.9 Hz), 4.73 (dd, 1H, J ¼ 14.4 Hz, J ¼ 3.3 Hz). IR:
n
1723 cmꢁ1
4c. Yield 100% as a white solid, mp 156e8 ꢂC (from methanol).
1H NMR (DMSO-d6):
.
as a colorless oil. 1H NMR (CDCl3):
d
8.30 (s, 1H), 8.22 (d, 1H,
d
9.08 (s, 1H), 8.34 (d, 1H, J ¼ 7.7 Hz), 8.27 (s,
J ¼ 7.8 Hz), 7.87 (d, 1H, J ¼ 7.8 Hz), 7.67e7.60 (m, 3H), 7.42 (d, 2H,
J ¼ 8.1 Hz), 7.37 (s, 1H), 7.04 (s, 1H), 6.86 (s, 1H), 6.27 (t, 1H,
J ¼ 5.5 Hz), 4.51 (dd, 1H, J ¼ 13,7 Hz, J ¼ 4.2 Hz), 4.46 (dd, 1H,
1H), 8.09 (d, 1H, J ¼ 7.7 Hz), 7.79e7.72 (m, 2H), 7.61 (s, 1H),
7.57e7.50 (m, 4H), 6.41, (dd, 1H, J ¼ 8.6 Hz, J ¼ 3.4 Hz), 4.88 (dd, 1H,
J ¼ 14.3 Hz, J ¼ 8.6 Hz), 4.77 (dd, 1H, J ¼ 14.3 Hz, J ¼ 3.4 Hz). IR:
n
J ¼ 13,7 Hz, J ¼ 2.9 Hz). IR:
n
1730 cmꢁ1. MS, m/z: 429 [M þ H]þ.
1727 cmꢁ1
.
4.1.2.5. Synthesis of (ꢀ)2-(1H-imidazol-1-yl)-1-phenylethyl 4-(tri-
fluoromethyl)benzoate (5a). 5a was prepared as described above for
3a using the alcohol 2a and 4-(trifluoromethyl)benzoyl chloride.
4.1.4. General procedure for the synthesis of 7b, c
4.1.4.1. Example: Synthesis of (ꢀ)1-(4-fluorophenyl)-2-(1H-imidazol-
1-yl)ethyl phenoxyacetate (7b). To a stirred suspension of 1-(4-

340
D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
fluorophenyl)-2-(1H-imidazol-1-yl)ethanol (2b) (0.53 mmol) in
dry acetonitrile (5 mL) was added sodium hydride (0.53 mmol). The
reaction mixture was stirred for 2 h at rt, then phenoxyacetyl
chloride (1.59 mmol) was dropwise added and the reaction mixture
was over night stirred at rt. The solvent was removed under
reduced pressure and the obtained residue was dissolved in
dichloromethane and washed with aqueous saturated potassium
carbonate.
5.98e5.90 (m, 1H), 4.45e4.37 (m, 2H), 2.84e2.76 (m, 1H), 1.14 (d,
6H, J ¼ 6.8 Hz). IR:
n
1722 cmꢁ1. MS, m/z: 384 [M þ H]þ.
4.1.5.4. Synthesis of (ꢀ)1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)
ethyl(2,6-dichloropyridin-4-yl)carbamate (9c). 2,6-Dichloro-4-
aminopyridine (1.23 mmol) was dissolved in 30 mL of anhydrous
benzene at 30 ꢂC, then triethylamine (3.09 mmol) was added. The
solution was stirred at rt and 5 mL of anhydrous benzene con-
taining 0.75 mmol of triphosgene were added dropwise. The yellow
suspension was stirred for 12 h at rt and then 1.23 mmol of 2c were
added. The reaction mixture was stirred for 48 h at rt. Afterward
30 mL of aqueous saturated sodium carbonate was added under
vigorous stirring; the organic layer was separated and the alkaline
solution was extracted with 3 portions (10 mL each) of dichloro-
methane. The combined organic fractions were dried over anhy-
drous sodium sulphate and evaporated under reduced pressure to
give a residue which was purified on silica gel column chroma-
tography using methanol/dichloromethane (1 : 9) as eluent. The
The separated organic layer was dried over anhydrous sodium
sulphate and evaporated under reduced pressure. The solid residue
was crystallized from ethylacetate to give pure 7b in 95% yield (mp
106e8 ꢂC).
1H NMR (DMSO-d6):
d 7.54 (s, 1H), 7.39e7.33 (m, 2H), 7.29e7.18
(m, 4H), 7.13 (s, 1H), 6.96 (t, 1H, J ¼ 7.4 Hz), 6.87 (s, 1H), 6.84e6.75
(m, 2H), 6.15e6.08 (m, 1H), 4.90 (d, 1H, J ¼ 16.7 Hz), 4.81 (d, 1H,
J ¼ 16.7 Hz), 4.48e4.41 (m, 2H). IR:
[M þ H]þ.
n
1752 cmꢁ1. MS, m/z: 341
4.1.4.2. Synthesis of (ꢀ)1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)
ethyl phenoxyacetate (7c). 7c was prepared as described above for
7b using the alcohol 2c. 7c was obtained in 95% yield as a white
solid, mp 103e5 ꢂC (from ethylacetate).
purification provided a white solid that was washed with
dichloromethane and crystallized from methanol. Yield 25%, mp:
227-9 ꢂC.
1H NMR (DMSO-d6):
d 10.81 (s, broad,1H), 7.54 (s, 1H), 7.50e7.41
1H NMR (DMSO-d6):
d
7.54 (s, 1H), 7.43 (d, 2H, J ¼ 8.5 Hz), 7.35
(m, 4H), 7.38 (d, 2H, J ¼ 8.5 Hz), 7.14 (s, 1H), 6.86 (s, 1H), 6.03 (t, 1H,
(d, 2H, J ¼ 8.5 Hz), 7.27 (dd, 2H, J ¼ 8.5 Hz, J ¼ 7.4 Hz), 7.13 (s, 1H),
6.97 (t, 1H, J ¼ 7.4 Hz), 6.86e6.77 (m, 3H), 6.11 (t, 1H, J ¼ 5.5 Hz),
4.91 (d, 1H, J ¼ 16.7 Hz), 4.83 (d, 1H, J ¼ 16.7 Hz), 4.42 (d, 2H,
J ¼ 5.7 Hz), 4.48 (d, 2H, J ¼ 5.7 Hz). IR:
n
1722 cmꢁ1. MS, m/z: 412
[M þ H]þ.
J ¼ 5.5 Hz). IR:
n
1751 cmꢁ1. MS, m/z: 357 [M þ H]þ.
4.1.6. Enantioselective HPLC
Analytical HPLC analysis of 6a, b were performed using the
commercially available 250 mm ꢃ 4.6 mm I.D. Chiralcel OD column
(Chiral Technologies Europe, Illkirch, France). HPLC grade ethanol
was purchased from Aldrich (St. Louis, Missouri USA). Semi-
preparative HPLC enantioseparations were performed using the
commercially available 250 mm ꢃ 10 mm I.D. Chiralcel OD column.
The analytical HPLC apparatus consisted of a PerkineElmer
(Norwalk, 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.
The semipreparative separation was carried out with a Per-
kineElmer (Norwalk, CT, USA) 200 LC pump equipped with
a Rheodyne (Cotati, CA, USA) injector, a 1 mL sample loop, a Per-
kineElmer LC 101 oven and a Waters 484 detector (Waters
Corporation, Milford, MA, USA). The signal was acquired and pro-
cessed by Clarity software (DataApex, Prague, The Czech Republic).
4.1.5. General procedure for the synthesis of 8aec
4.1.5.1. Example: Synthesis of (ꢀ)2-(1H-imidazol-1-yl)-1-phenylethyl
[4-(propan-2-yl)phenyl]carbamate (8a). To a stirred suspension of
2-(1H-imidazol-1-yl)ethanol (0.53 mmol) in dry acetonitrile
(10 mL), was added sodium hydride (0.53 mmol). The reaction
mixture was stirred for 2 h at rt, then 1-isocyanate-4-(propan-2-yl)
benzene (0.79 mmol) was added and the reaction mixture was
stirred for 24 h at rt. The solvent was removed under reduced
pressure and the obtained residue was washed with methanol
(2 ꢃ 3 mL). The mother liquor was dried and purified by silica gel
column chromatography using 9 : 1 dichloromethane/methanol as
eluent, to obtain a white solid that was washed with diethylether
and crystallized from ethylacetate (yield 53%, mp: 190e2 ꢂC).
1H NMR (DMSO-d6):
d 9.74 (s, broad, 1H), 7.56 (s, 1H), 7.40e7.30
(m, 7H), 7.17 (s, 1H), 7.16e7.09 (m, 2H), 6.85 (s, 1H), 5.99e5.91 (m,
1H), 4.44e4.37 (m, 2H), 2.85e2.78 (m, 1H), 1.15 (d, 6H, J ¼ 6.9 Hz).
IR:
n
1719 cmꢁ1. MS, m/z: 350 [M þ H]þ.
4.1.7. Polarimetry
4.1.5.2. Synthesis of (ꢀ)1-(4-fluorophenyl)-2-(1H-imidazol-1-yl)-1-
phenylethyl [4-(propan-2-yl)phenyl]carbamate (8b). 8b was
Specific rotations were measured at 589 nm by a PerkineElmer
polarimeter model 241 equipped with a Na/Hg lamp. The volume of
the cell was 1 mL and the optical path was 10 cm. The system was
set to 20 ꢂC.
prepared as described for 8a using the alcohol 2b. Methanol/
dichloromethane (1 : 9) was used as eluent for the chromato-
graphic purification. 8b was obtained in 45% yield as a white solid,
The specific rotation values of the enantiomers of 6a, b were:
mp 200e2 ꢂC (from ethylacetate). 1H NMR (DMSO-d6):
d
9.74 (s,
(þ)-6a, ½a 2D0
ꢄ
þ31 (c ¼ 0.2, EtOH); (ꢁ)-6a, ½a D20
ꢄ
ꢁ32 (c ¼ 0.2, EtOH);
broad, 1H), 7.55 (s, 1H), 7.45e7.39 (m, 2H), 7.31 (d, 2H, J ¼ 8.5 Hz),
7.26e7.18 (m, 2H), 7.15 (s,1H), 7.13 (d, 2H, J ¼ 8.5 Hz) 6.84 (s, 1H),
5.94 (t,1H, J ¼ 5.4 Hz), 4.47e4.40 (m, 2H), 2.83e2.77 (m, 1H),1.16 (d,
(þ)-6b, ½a 2D0
ꢄ
þ31 (c ¼ 0.2, EtOH); (ꢁ)-6b, ½a D20
ꢄ
ꢁ31 (c ¼ 0.2, EtOH).
4.2. In vitro antifungal activity
6H, J ¼ 6.9 Hz). IR:
n
1715 cmꢁ1. MS, m/z: 368 [M þ H]þ.
4.2.1. Organisms
4.1.5.3. Synthesis of (ꢀ)1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)
ethyl[(4-propan-2-yl)phenyl]carbamate (8c). 8c was prepared as
described for 8a using the alcohol 2c. Methanol/dichloromethane
(1 : 9) was used as eluent for the chromatographic purification. 8c
was obtained in 30% yield as a white solid, mp 162e4 ꢂC (from
For the antifungal evaluation, strains obtained from the Amer-
ican Type Culture Collection (ATCC, Rockville, MD, USA), from the
German Collection of Microorganisms (DSMZ, Braunschweig, Ger-
many) and from Pharmaceutical Microbiology Culture Collection
(PMC, Department of Public Health and Infectious Diseases, Sapi-
enza, Rome, Italy) were tested. The strains obtained from the ATCC
were: C. albicans ATCC10231, C. albicans ATCC3153, C. albicans
ATCC76615, C. albicans ATCC24433 (tested as reference strain),
ethylacetate). 1H NMR (DMSO-d6):
d 9.75 (s, broad, 1H), 7.55 (s, 1H),
7.43 (d, 2H, J ¼ 8.4 Hz), 7.36 (d, 2H, J ¼ 8.4 Hz), 7.30 (d, 2H,
J ¼ 8.4 Hz), 7.15 (s, 1H), 7.12 (d, 2H, J ¼ 8.4 Hz), 6.84 (s, 1H),

D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
341
C. parapsilosis ATCC22019 (tested as quality control strain). The
strains obtained from the DSMZ were: C. tropicalis DSM11953,
C. krusei DSM6128, C. parapsilosis DSM11224. The strains obtained
from PMC were: C. krusei PMC0613, C. tropicalis PMC0910,
C. glabrata PMC0805. All the strains were stored at ꢁ70 ꢂC in 15%
glycerol solution, as indicated in Clinical and Laboratory Standards
Institute (CLSI) document M27-A3 [24]. Yeasts were grown on
sabouraud dextrose agar (SigmaeAldrich, St. Louis, Missouri, U.S.A)
plate for 24 h at 35 ꢂC, in accordance with the CLSI procedures [24].
concentration of the drug needed to reduced fungal cell viability to
50% (MIC).
Antifungal activity was determined according to CLSI guidelines
(CLSI document M27-A3,2008). MIC ¼ arithmetic mean of minimal
inhibitory concentration; SD ¼ standard deviation.
Acknowledgments
This research was financially supported by grants from Sapi-
enza, Universit di RomaProgetti di Ricerca di Ateneo, and by Min-
4.2.2. Antifungal susceptibility testing
istero dell’Istruzione, dell’Università
e della Ricerca (Progetti
The tests were conducted in accordance with the CLSI M27-A3
broth microdilution methods [24]. Yeast inoculum suspensions
were prepared as described in the CLSI protocols. The antifungal
reference fluconazole (SigmaeAldrich, St. Louis, Missouri, U.S.A.)
Speciali “Fondi per gli Investimenti sulla Ricerca di Base”- Reti FIRB
RBPR05NWWC_006).
Elemental and mass analysis were performed by Dr. M. Zancato,
Dipartimento di Scienze Farmaceutiche, Università di Padova, Italia.
was also tested (range 0.0078e128
m
g mLꢁ1). The final concentra-
g mLꢁ1 for new compounds.
l of serial two-fold dilutions of
tions ranged from 0.125 to 64
m
Appendix. Supplementary material
Microdilution trays containing 100
m
each substance in RPMI 1640 medium (SigmaeAldrich, St. Louis,
Missouri, U.S.A) were inoculated with an organism suspension
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2012.01.034. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
adjusted to attain
a
final inoculum concentration of
1.0 ꢃ 103e1.5 ꢃ 103 cells mLꢁ1. The panels were incubated at 35 ꢂC
and observed for the presence of growth at 48 h. Quality control
was performed by testing the CLSI-recommended strain
C. parapsilosis ATCC22019 as indicated in CLSI document M27-S3
[25]. MICs were determined three times in triplicate. The MICs of
new compounds and fluconazole were read as the lowest concen-
tration at which a significant decrease in turbidity (ꢅ50%) was
discerned compared to that of the growth control.
References
[1] M.A. Pfaller, D.J. Diekema, Epidemiology of invasive mycoses in North Amer-
ica, Cri. Rev. Microbiol. 36 (2010) 1e53.
[2] A. Puel, C. Picard, S. Cypowyj, D. Lilic, L. Abel, J.L. Casanova, Inborn errors of
mucocutaneous immunity to Candida albicans in humans: a role for IL-17
cytokines? Curr. Opin. Immunol. 22 (2010) 467e474.
[3] M.A. Pfaller, D.J. Diekema, Epidemiology of invasive candidiasis: a persistent
public health problem, Clin. Microbiol. Rev. 20 (2007) 133e163.
[4] E. Concia, A.M. Azzini, Epidemiology, incidence and risk factors for invasive
candidiasis in hight-risk patients, Drugs 69 (Suppl.1) (2009) 5e14.
[5] M.C. Arendrup, Epidemiology of invasive candidiasis, Curr. Opin. Crit. Care 16
(2010) 445e452.
The minimum concentrations of the drug that inhibited 90% and
50% of the isolates were defined as the MIC90 and MIC50
,
respectively.
4.3. In vitro cytotoxicity test
[6] M. Pennisi, M. Antonelli, Clinical aspects of invasive candidiasis in critically Ill
patients, Drugs 69 (Suppl. 1) (2009) 21e28.
4.3.1. Cell viability assay
[7] S.C. Chen, E.G. Playford, T.C. Sorrell, Antifungal therapy in invasive fungal
infection, Curr. Opin. Pharmacol. 10 (2010) 522e530.
[8] N. Strushkevich, S.A. Usanov, Structural basis of human CYP51 inhibition by
antifungal azoles, J. Mol. Biol. 397 (2010) 1067e1078.
[9] F. Odds, A.J.P. Brown, N.A.R. Gow, Antifungal agents:mechanism of action,
Trends Microbiol. 11 (2003) 272e279.
[10] J.L. Adams, B.W. Metcalf, Therapeutic Consequences of the inhibition of sterol
metabolism, in: C. Hansch, P.G. Sammes, J.B. Taylor (Eds.), Comprehensive
Medicinal Chemistry, vol. 2, Pergamon Press, Oxford, England, 1990, pp.
333e364.
[11] Y.Q. Zhang, S. Gamarra, G. Garcia-Effron, Requirement for ergosterol in V-
ATPase function underlies antifungal activity of azole drugs, PLOS Pathogenes
6 (6) (June 2010) e1000939.
[12] M.A. Pfaller, D. Andes, D.J. Diekema, Wild-type MIC distributions, epidemio-
logical cutoff values and species-specific clinical breakpoints for fluconazole
and Candida: time for harmonization of CLSI and EUCAST broth microdiluition
methods, Drug Resist. Updates 13 (2010) 180e195.
[13] L.M. Podust, T.L. Poulos, M.R. Waterman, Crystal structure of cytochrome P450
14R-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex
with azole inhibitors, Proc. Natl. Acad. Sci. U.S.A 98 (6) (2001) 3068e3073.
[14] G.I. Lepesheva, H. Park, T.Y. Hargrove, B. Vanhollebeke, Z. Wawrzak, J.M. Harp,
M. Sundaramoorthy, W.D. Nes, E. Pays, M. Chaudhuri, F. Villalta,
M.R. Waterman, Crystal structures of Trypanosoma brucei sterol 14-
demethylase and Implications for selective treatment of human infections,
J. Biol. Chem. 285 (3) (2010) 1773e1780.
The cytotoxicity of tested new compounds was evaluated on
human monocytic cell line (U937) obtained from American Type
Culture Collection (ATCC CRL1593.2, Rockville, MD, USA) by using
a
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) reduction assay [26]. The cells (2 ꢃ 104 cells wellꢁ1) were
seeded into 96-well plates containing 100
mL of supplemented
RPMI 1640 (Invitrogen, San Diego, CA, USA) without phenolred,
supplemented with 10% fetal bovine serum (Invitrogen, San Diego,
CA, USA),
streptomycin (100
L
-glutamine (0.3 mg mLꢁ1), penicillin (100 U mLꢁ1), and
m
g mLꢁ1) (EuroClone, Celbio, Milan, Italy), and
they were cultured at 37 ꢂC in 5% CO2. The cells were exposed to the
indicated compounds (dissolved in dimethyl sulfoxide, DMSO) at
the final concentration ranging from 8 to 128
m
g mLꢁ1. Each
concentration and control was assayed in three replicates with at
least five concentrations. The cells were cultured at 37 ꢂC and 5%
CO2 for 48 h. MTT solution (SigmaeAldrich, St. Louis, Missouri,
U.S.A.) was added to each well in an amount equal to 10% of the
culture volume, and the plates were incubated for 3e4 h at 37 ꢂC in
5% CO2. MTT solvent (SigmaeAldrich, St. Louis, Missouri, U.S.A.) was
successively added to dissolve the intracellular crystal. The plates
were then incubated at 37 ꢂC in 5% CO2, and the optical density of
each well was measured spectrophotometrically at 570 nm. The
cytotoxicity of the compounds was calculated as percentage
reduction in viable cells with respect to the control culture (cells
[15] C. Sheng, W. Wang, X. Che, G. Dong, S. Wang, H. Ji, Z. Miao, J. Yao, W. Zhang,
Improved model of lanosterol 14
a-demethylase by ligand-supported
homology modeling: Validation by virtual screening and azole optimization,
Chem. Med. Chem. 5 (2010) 390e397.
[16] R. Di Santo, A. Tafi, R. Costi, M. Botta, M. Artico, F. Corelli, M. Forte, F. Caporuscio,
L. Angiolella, A.T. Palamara, Antifungal agents. 11. N-Substituted derivatives of
1-[(aryl)(4-aryl-1H-pyrrol-3-yl)methyl]-1H-imidazole:
synthesis,
anti-
Candida activity, and QSAR studies, J. Med. Chem. 48 (2005) 5140e5153.
treated with DMSO only). The 50% cytotoxic concentration (CC50
)
[17] G. La Regina, F.D. D’Auria, A. Tafi, F. Piscitelli, S. Olla, F. Caporuscio, L. Nencioni,
R. Cirilli, F. La Torre, N. Rodrigues De Melo, S.L. Kelly, D.C. Lamb, M. Artico,
M. Botta, A.T. Palamara, R. Silvestri, 1-[(3-Aryloxy-3-aryl)propyl]-1H-imidaz-
oles, new imidazoles with potent activity against Candida albicans and
Dermatophytes. Synthesis, structure-activity relationship, and molecular
modeling studies, J. Med. Chem. 51 (2008) 3841e3855.
was evaluated as the drug concentration required to reduce human
cell viability by 50% compared to the drug-free control. The Selec-
tivity Index (SI) was defined as the ratio of the concentration of the
drug that reduced human cell viability to 50% (CC50) to the

342
D. De Vita et al. / European Journal of Medicinal Chemistry 49 (2012) 334e342
[18] G.C. Porretta, R. Fioravanti, M. Biava, R. Cirilli, N. Simonetti, A. Villa, U. Bello,
P. Faccendini, B. Tita, Research on antibacterial and antifungal agents. X.
Synthesis and antimicrobial activity of 1-phenyl-2-(1H-azol-1-yl) ethane
derivatives. Anticonvulsivant activity of 1-(4-methylphenyl)-2-(1H-imidazol-
1-yl) ethanol, Eur. J. Med. Chem. 28 (1993) 749e760.
[19] Y. Wahbi, C. Tournaire, R. Caujolle, M. Payard, M.D. Lynas, J.P. Seguela,
Aliphatic ethers and esters of 1-(2,4-dichlorophenyl)-2-(1H-imidazolyl)-
ethanol: study of antifungal activity yeast and hydrophobic character, Eur. J.
Med. Chem. 29 (1994) 701e706.
[22] G.I. Lepesheva, H.W. Park, T.Y. Hargrove, B. Vanhollebeke, Z. Wawrzak,
J.M. Harp, M. Sundaramoorthy, W.D. Nes, E. Pays, M. Chaudhuri, F. Villalta,
M.R. Waterman, Structural insights into inhibition of Sterol 14a-demethylase in
the human pathogen Trypanosoma cruzi, J. Biol. Chem. 285 (2010) 25852 25590.
[23] A. Karakurt, M. Ozalp, S.I. sik, J.P. Stables, S. Dalkara, Synthesis, anti-
convulsivant and antimicrobial activities of some new 2-acetylnaphtalene
derivatives, Bioorg. Med. Chem. 18 (2010) 2902e2911.
[24] CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts Approved Standard-Third Edition. CLSI document M27eMA3, Clinical
and Laboratory Standards Institute, Wayne, PA, 2008.
[25] CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts Third Informational Supplement. CLSI document M27eMS3, Clinical
and Laboratory Standards Institute, Wayne, PA, 2008.
[20] K.A.M. Walker, D.R. Hirschfeld, M. Marx, Antimycotic imidazole. 2. Synthesis
and antifungal properties of 1-[2-Hydroxy(mercapto)-2-phenylethyl]-1H-
imidazoles, J. Med. Chem. 21 (1978) 1335e1338.
[21] G.I. Lepesheva, R.D. Ott, T.Y. Hargrove, Y.Y. Kleshchenko, I. Schuster, W.D. Nes,
G.C. Hill, F. Villalta, M.R. Waterman, Sterol 14
target for antitrypanosomal therapy: enzyme inhibition and parasite cell
growth, Chem. Biol. 14 (2007) 1283e1293.
a
-demethylase as a potential
[26] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assay, J. Immunol. Methods 65
(1983) 55e63.