New azoles with potent antifungal activity: Design, synthesis and molecular docking

Article abstract of DOI:10.1016/j.ejmech.2009.05.018

In response to the urgent need for novel antifungal agents with improved activity and broader spectrum, computer modeling was used to rational design novel antifungal azoles. On the basis of the active site of lanosterol 14α-demethylase from Candida albicans (CACYP51), a series of new azoles with substituted-phenoxypropyl piperazine side chains were rational designed and synthesized. In vitro antifungal activity assay indicates that the new azoles show good activity against most of the tested pathogenic fungi. Interestingly, the designed compounds are also active against an azole-resistant clinical strain. Compared to fluconazole and itraconazole, several compounds (such as 12i, 12j and 12n) show higher antifungal activity and broader spectrum, which are promising leads for the development of novel antifungal agents.

Full text of DOI:10.1016/j.ejmech.2009.05.018

European Journal of Medicinal Chemistry 44 (2009) 4218–4226
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New azoles with potent antifungal activity: Design, synthesis
and molecular docking
,
a
a
a
b
Xiaoying Che a, Chunquan Sheng a , Wenya Wang , Yongbing Cao , Yulan Xu , Haitao Ji ,
**
Guoqiang Dong a, Zhenyuan Miao a, Jianzhong Yao a, Wannian Zhang a
,
*
a School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
b Department of Chemistry, Department of Biochemistry, Molecular Biology and Cell Biology and Center for Drug Discovery and Chemical Biology,
Northwestern University, Evanston, IL 60208-3113, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 April 2009
Received in revised form 17 May 2009
Accepted 19 May 2009
Available online 24 May 2009
In response to the urgent need for novel antifungal agents with improved activity and broader spectrum,
computer modeling was used to rational design novel antifungal azoles. On the basis of the active site of
lanosterol 14a-demethylase from Candida albicans (CACYP51), a series of new azoles with substituted-
phenoxypropyl piperazine side chains were rational designed and synthesized. In vitro antifungal activity
assay indicates that the new azoles show good activity against most of the tested pathogenic fungi.
Interestingly, the designed compounds are also active against an azole-resistant clinical strain. Compared
to fluconazole and itraconazole, several compounds (such as 12i, 12j and 12n) show higher antifungal
activity and broader spectrum, which are promising leads for the development of novel antifungal
agents.
Keywords:
Azole
Rational design
Molecular docking
Antifungal activity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
antifungal spectrum and better toleration. However, its use is
hampered by variable oral absorption and low bioavailability. This
During the past two decades, fungal infection has become an
important complication and a major cause of morbidity and
mortality in immunocompromised individuals such as patients
undergoing anticancer chemotherapy or organ transplants and
patients with AIDS [1]. Clinically, candidosis, aspergillosis and
cryptococcosis are the three major fungal infections in immuno-
compromised individuals [2,3]. The common antifungal agents
currently used in clinic are azoles (such as fluconazole, ketocona-
zole and itraconazole) [4], polyenes (such as amphotericin B [5]
and nystatin [6]), echinocandins (such as caspofungin and mica-
fungin) [7] and allylamines (such as naftifine and terbinafine) [8].
Among those, azoles are widely used in antifungal chemotherapy.
Fluconazole is preferred as first-line antifungal therapy with good
antifungal activity and relatively low toxicity [9]. However, fluco-
nazole is not effective against invasive aspergillosis and has
suffered severe drug resistance [10,11]. Itraconazole is an
situation has led to an ongoing search for new azoles and several
novel azoles have been developed with improved profiles. The
second generation of azoles (Fig. 1), such as voriconazole [12],
posaconazole [13], ravuconazole [14] and albaconazole [15], is
marketed or currently in the late stages of clinical trials. They are
noted for their broad antifungal spectrum, low toxicity and
improved pharmacodynamic profiles.
Azole antifungals act by competitive inhibition of the lanosterol
14
a
-demethylase (CYP51), the enzyme that catalyzes the oxidative
-methyl group of lanosterol to give 14,15-
removal of the 14
a
D
desaturated intermediates in ergosterol biosynthesis [16]. In yeast
and fungi, CYP51 is the key enzyme in sterol biosynthesis. Selective
inhibition of CYP51 would cause depletion of ergosterol and
accumulation of lanosterol and other 14-methyl sterols resulting in
the growth inhibition of fungal cells [17]. Eukaryotic CYP51s are
membrane associated proteins and solving their crystal structures
remains a challenge. In our continual interest in rational antifungal
drug design, we have constructed three-dimensional (3D) models
of CYP51 from Candida albicans (CACYP51) and Aspergillus fumi-
gatus (AFCYP51) through homology modeling [18,19] on the basis
of the crystal coordinates of CYP51 from Mycobacterium tubercu-
losis (MTCYP51) [20]. The overall structures of the models are
similar to that of the template structure (MTCYP51), which
remains the core structural motif characteristic for cytochrome
improvement of fluconazole in terms of having
a broader
* Corresponding author. Tel./fax: þ86 21 81871243.
** Corresponding author. Tel./fax: þ86 21 81871233.
E-mail addresses: shengcq@hotmail.com (C. Sheng), zhangwnk@hotmail.com
(W. Zhang).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.05.018

X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
4219
O
N
N
N
1
OH
3
N
O
CH2
Cl
O
N
N
N
N
N
N
F
O
N CH2
2
N
N
N
F
Cl
Itraconazole
Fluconazole
O
N
N
N
OH
CH3
CH2
O
O
N
N
OH
N
N
N
N
CH2
N
N
N
F
N
F
F
F
F
Voriconazole
Posaconazole
CH3
O
N
CH3
OH
OH
N
N
N
N
N
F
N
CN
N
N
S
F
Cl
F
F
Albaconazole
Ravuconazole
Fig. 1. Chemical structures of azole antifungal agents.
P450 proteins. The structurally and functionally important regions,
such as the heme environment, the substrate access channel, and
the active site have been recognized accurately. The binding mode
of azoles with CACYP51 has been investigated by flexible molec-
ular docking [18,21]. Important residues involved in azole binding
are validated by site-directed mutagenesis [22]. The information
obtained from molecular modeling greatly facilitates the process
of rational antifungal drug design. On the basis of the results from
molecular modeling, novel non-azole CACYP51 inhibitors [23] and
highly potent new azoles [21] have been discovered successfully,
which demonstrates that the utilization of structural information
of fungal CYP51s can accelerate the discovery of novel antifungal
agents.
the presence of potassium carbonate and DMF at 80 ꢀC to afford
side chains 7a–c. The target compounds 8a–c were obtained as
racemates by treating epoxide 4 with side chains 7a–c in the
presence of triethylamine and ethanol at 80 ꢀC with moderate to
high yields. The target compounds 12a–n (racemates) were
obtained by a similar procedure (Scheme 2). The nitro groups on
the phenyl ring of 12h, 12m and 12n were reduced to amino
groups in the presence of Raney Ni and hydrazine hydrate to give
12o, 12p and 12q, respectively (Scheme 3). Compound 12h was
converted to 12r and 12s by reacting with corresponding acyl
chloride in the presence of triethylamine and CH2Cl2 at room
temperature (Scheme 4).
In the present study, a series of new azoles with substituted-
phenoxypropyl piperazine side chains were designed and synthe-
sized on the basis of the active site of CACYP51. The new azoles
reveal excellent in vitro antifungal activity with broad spectrum. The
structure–activity relationships (SARs) of the azoles were analyzed
by molecular docking.
3. Pharmacology
In vitro antifungal activity was measured by means of the MIC
using the serial dilution method in 96-well microtest plates. Test
fungal strains were obtained from the ATCC or were clinical
isolates. The MIC determination was performed according to the
National Committee for Clinical Laboratory Standards (NCCLS)
recommendations with RPMI 1640 (Sigma) buffered with
0.165MMOPS (Sigma) as the test medium. The MIC value was
defined as the lowest concentration of test compounds that resul-
ted in a culture with turbidity less than or equal to 80% inhibition
when compared with the growth of the control. Test compounds
were dissolved in DMSO serially diluted in growth medium. The
yeasts were incubated at 35 ꢀC and the dermatophytes at 28 ꢀC.
2. Chemistry
As a key intermediate of our designed triazole antifungals, the
oxirane compound 4 was synthesized by our reported procedure
(Scheme 1) [21]. The phenoxyalkylpiperazine side chains 7a–c
were synthesized via three steps. Excess dibromoalkanes 5a–c
were treated with phenol to give bromoalkyloxybenzenes 6a–c.
Compounds 6a–c were subsequently reacted with piperazine in

4220
X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
O
O
N
Cl
N
O
F
N
N
b
c
N
.
F
N
CH3SO3H
F
F
F
F
F
F
2
4
1
3
R
e
d
Br (CH2)n Br
(CH2)n
O(CH2)n
O
Br
N
NH
5a-c
6a-c
7a-c
OH
F
N
f
(CH2)nO
N
N
N
N
F
8a n = 3
8b n = 4
8c n = 5
Scheme 1. Reagents and conditions: a. ClCH2COCl, AlCl3, CH2Cl2, 40 ꢀC, 3 h, 50%; b. triazole, K2CO3, CH2Cl2, rt, 24 h, 70.0%; c. (CH3)3SOI, NaOH, toluene, 60 ꢀC, 3 h, 62.3%; d. phenol,
K2CO3, DMF, rt, 24 h, 91.8–92.3%; e. piperazine, K2CO3, EtOH, 70 ꢀC, 8 h, 90.5–95.2%; f. 4, Et3N, EtOH, reflux, 9 h, 29.8–40.1%.
Growth MIC was determined at 24 h for Candida species, at 72 h for
Cryptococcus neoformans, and at 7 days for filamentous fungi.
agents binds to the S2 pocket through the formation of a coordi-
nation bond with iron of heme group. The difluorophenyl or
dichlorophenyl group is located in the S3 pocket and forms
hydrophobic interaction with Ala114, Phe126, Leu139, Ile304 and
Met306. In the crystal structure of MTCYP51 in complex with flu-
conazole [20], the active site is filled with water molecules and
fluconazole interacts with at least three water molecules. From our
previous docking models [21], the hydroxyl group attached to C2
was supposed to interact with the S1 pocket through the hydrogen-
bonding interaction with His310 mediated by crystal waters as
found in MTCYP51–fluconazole complex. Different side chains
attached to C3 are located in the S4 pocket and form hydrophobic
and van der Waals interactions. Because most of the recent efforts
on the optimization of azole antifungal agents have been focused
on the modification of the side chain attached to C3 [21,24–26], the
triazoyl ring, difluorophenyl group and C2 hydroxyl group were
retained as essential pharmacophoric elements in the present
study. Considering the hydrophobic nature of the C4 pocket of
CACYP51, a hydrophobic side chain is necessary, while an additional
4. Results and discussion
4.1. Rational design of the new azoles
In our previous studies, azole–CACYP51 interactions have been
explored by flexible molecular docking [18,21]. Moreover, the active
site of CACYP51 has been characterized by multiple copy simulta-
neous search (MCSS) [23]. As discussed earlier [23], the active site
of CACYP51 can be divided into four pockets. The S1 pocket
represents the hydrophilic hydrogen bond binding site. The S2
pocket is above the heme ring representing the core hydrophobic
area. The S3 pocket represents the narrow and hydrophobic cleft
(facing BC loop). The S4 pocket represents a hydrophobic hydrogen
bond binding site (facing FG loop), which is important for the
optimization of the C3-side chain of azole antifungal agents. Our
docking studies indicate that the triazoyl ring of azole antifungal
R
R
R
a
c
b
OH
OH
O
Br
O(CH2)3
N
NH
9a-s
10a-s
11a-s
12a R = 4-CH3
12b R = 3-CH3
12c R = 2-CH3
12d R = 3-Br
12e R = 4-F
12f R = 2-Cl
12g R = 4-Br
12h R = 4-NO2
12i R = 4-C(CH3)3
12j R = 4-Cl
N
N
F
(CH )
2 3O
N
N
N
R
12k R = 2-Br
12l R = 4-OCH2Ph
12m R = 2-NO2
12n R = 3-NO2
F
12a-n
Scheme 2. Reagents and conditions: a. substitute phenol, K2CO3, DMF, rt, 24 h, 90.8–94.2%; b. piperazine, K2CO3, EtOH, 70 ꢀC, 8 h, 90.1–93.6%; c. 4, Et3N, EtOH, reflux, 9 h,
29.8–40.1%.

X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
4221
OH
NH2
OH
F
N
NO2
(CH2)3
O
N
N
N
F
N
N
Ni / NH2NH2
EtOH
(CH2)3
O
N
N
F
N
N
F
12o R = 4-NH2
12p R = 2-NH2
12q R = 3-NH2
12h R = 4-NO2
12m R = 2-NO2
12n R = 3-NO2
Scheme 3.
hydrogen-bonding interaction will further improve the affinity and
specificity of the inhibitors. In the present study, Ser378 was
selected as a hydrogen-bonding target, because it was well vali-
dated in our previous inhibitor design [21,23]. Moreover, Ser378 is
conserved across the fungal CYP51 enzymes, but the corresponding
residue in human is Ile379. Therefore, using Ser378 as a hydrogen-
bonding target may also improve the selectivity of the inhibitors
toward fungal CYP51. On the basis of the above hypothesis, we
designed compound 8a with a phenoxypropyl piperazine side
chain. Besides the hydrophobic interactions, the oxygen atom of the
side chain was supposed to form a hydrogen bond with Ser378. In
order to validate the hypothesis, compound 8a was docked into the
active site of CACYP51. The binding mode of compound 8a revealed
that the phenoxypropyl piperazine side chain was oriented into the
S4 pocket with an extended conformation (Fig. 2). The propylpi-
perazinyl group formed hydrophobic and van der Waals interac-
tions with Leu376, Ile379, Leu461 and Val519. Hydrogen-bonding
interaction between the oxygen atom and Ser378 was observed and
the terminal phenyl group interacted with surrounding hydro-
phobic residues such as Phe72 and Met374. Compared to flucona-
zole, compound 8a bound to CACYP51 with a lower value of
interaction energy (Table 1). In order to test the importance of the
propyl group, compounds 8b–c were synthesized. Moreover,
compounds 12a–s were designed to investigate the effect of various
substitutions attached to the terminal phenyl group on the anti-
fungal activity.
its activity is comparable to that of voriconazole. Fluconazole is not
effective against A. fumigatus, while our compounds show moderate
activity. The MIC80 values of compounds 12g, 12i and 12l against
A. fumigatus are in the range of 1–2 mg/mL. Moreover, the designed
compounds also show good activity against dermatophytes (i.e. Tri-
chophyton rubrum and Microsporum gypseum). For example,
compounds 12i and 12j are 4–32 fold more potent against T. rubrum
than fluconazole, itraconazole and voriconazole. In clinic, azole
antifungal agents have suffered serious drug resistance [27] and it is
of great significance to find new azoles with activity against resistant
clinical isolates. Most of the designed compounds show good
inhibitory activity against an azole-resistant strain with their MIC80
values in the range of 0.25–0.125 mg/mL. Compound 12i exhibited
the highest activity with broad antifungal spectrum, which is a good
candidate for further evaluation.
4.3. SARs of the azoles
We first varied the alkyl length attached to the piperazinyl
group. When the propyl group (compound 8a) is replaced by butyl
group (compound 8b) or pentyl group (compound 8c), the anti-
fungal activity is decreased. From the docking model, the oxygen
atom of phenyl group of compound 8a forms a hydrogen bond with
Ser378 of CACYP51. The hydrogen bond will be broken if the propyl
group of compound 8a is extended, which results in the decrease
of the antifungal activity. Then, the effect of the substitutions at
the phenol group on the antifungal activity was evaluated. The
in vitro antifungal activity assay indicates that 4-substituted and
3-substituted derivatives show higher antifungal activity compared
to 2-substituted derivatives. The docking model shows that the
active site of CACYP51 at position 2 of the bound compound is not
large enough to accommodate an additional group. However,
positions 3 and 4 of the phenol group contain an extra small pocket
defined by Met374, Tyr69 and Phe72. Among the 4-substituted
derivatives, halogen and alkyl substituted compounds (e.g.
compounds 12a,12g, and 12i) show lower interaction energies with
CACYP51 than unsubstituted compound 8a, because these substi-
tutions formed additional hydrophobic interactions with Met374,
4.2. In vitro antifungal activities
In vitro antifungal activity assay (Table 2) indicates that all the
synthesized compounds show moderate to excellent activity against
all the tested fungal pathogens. The target compounds revealed the
highest activity against C. albicans and Candida parapsilosis. Most of
the compounds are more potent than fluconazole and itraconazole
with their minimal inhibitory concentration (MIC80) values in the
range of 0.06–0.016
mg/mL. On the C. neoformans strain, most of the
compounds show higher activity than fluconazole. The MIC80 value
of compound 12i against C. neoformans is 0.06 mg/mL, indicating that
OH
OH
N
N
N
NHCOR
N
N
N
F
(CH2)3O
NH2
acyl chloride
N
N
F
N (CH2)3O
N
Et3N, CH2Cl2
F
F
12o
12r R = Ph
12s R = CH3
Scheme 4.

4222
X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
Fig. 2. Stereoview of the docking conformation of compound 8a (stick model) in the active site of CACYP51. The residues interacting with compound 8a are shown and hydrogen
bonds are displayed through red dotted lines.
Tyr69 and Phe72. Although 4-halogen or 4-alkyl substituted
derivatives show the same MIC value against C. albicans as
compound 8a, the broader antifungal spectrum of these
compounds is observed. Moreover, these compounds show higher
activity against C. albicans from a fluconazole-resistant isolate than
compound 8a. This result is in good agreement with the previous
observation that the additional hydrophobic interaction serves to
stabilize the binding of these azoles to the mutated CYP51 proteins,
which is important for the reservation of the activity against clinical
resistant isolates [28]. If these hydrophobic groups are replaced by
a more hydrophilic nitro group (compound 12h) or amino group
(compound 12o), the antifungal activity is decreased. For the
compounds with large substitutions at position 4 (i.e. compounds
12l, 12r and 12s), their antifungal activities are not as good as those
of 8a, possibly because of the steric crowding. The docking model
reveals that the terminal phenyl group of 8a is near the entrance of
substrate access channel (FG loop), the side chains of compounds
12l, 12r and 12s are too long to be accommodated in the active site.
a series of new azoles with substituted-phenoxypropyl piperazine
side chains were designed and synthesized. Besides the hydro-
phobic and van der Waals interactions with CACYP51, a new
hydrogen-bonding interaction with Ser378 was introduced into our
designed compounds to improve the affinity and specificity. In vitro
antifungal activity assay indicates that the new azoles show good
activity against important fungal pathogens. Interestingly, the
compounds are also active against azole-resistant clinical strain.
Several compounds (such as 12i, 12j and 12n) show high in vitro
antifungal activity with broad spectrum, which were valuable for
further evaluation. The structure–activity relationships (SARs) of
the compounds were explained by flexible molecular docking. The
propyl of the compounds is found important for the maintaining
the hydrogen-bonding interaction. A small hydrophobic group is
favorable at the position 3 or position 4 of the terminal phenyl
group, possibly because of the additional hydrophobic interaction
with CACYPY51. Our structure-based inhibitor design for CACYP51
reveals the robustness and efficiency of computer modeling in
structural optimization of azole antifungal agents.
5. Conclusion
6. Experimental protocols
In summary, computer modeling was used to rational design
novel antifungal azoles. On the basis of the active site of CACYP51,
6.1. General procedure for the synthesis of compounds
Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker 500 spectrometer with TMS as an internal standard and
CDCl3 as solvent. Chemical shifts (
d values) and coupling constants
Table 1
Calculated interaction energies (kcal/mol) for the complexes of representative
compounds with the active site of CACYP51.
(J values) are given in ppm and Hz, respectively. ESI mass spectra
were performed on an API-3000 LC–MS spectrometer. Elemental
analyses were performed with a MOD-1106 instrument and were
consistent with theoretical values within ꢁ0.4%. TLC analysis was
carried out on silica gel plates GF254 (Qindao Haiyang Chemical,
China). Silica gel column chromatography was performed with
Silica gel 60G (Qindao Haiyang Chemical, China). Commercial
solvents were used without any pretreatment.
Compounds
Evdw
Eelect
Etotal
8a
12a
12g
12h
12i
12o
Fluconazole
ꢂ77.3
ꢂ81.5
ꢂ78.1
ꢂ70.4
ꢂ87.6
ꢂ71.3
ꢂ54.8
ꢂ1.2
ꢂ1.4
ꢂ0.8
ꢂ0.6
ꢂ0.7
ꢂ2.3
ꢂ3.2
ꢂ78.5
ꢂ82.9
ꢂ78.9
ꢂ71.0
ꢂ88.3
ꢂ73.6
ꢂ58.0

X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
4223
Table 2
Antifungal in vitro activities of the compounds (MIC80
, m
g mLꢂ1).a
Compounds
C. albicans
C. albicans (R)b
C. neoformans
C. tropicalis
T. rubrum
A. fumigatus
M. gypseum
8a
8b
0.0156
0.25
64
32
1
1
0.0625
0.25
0.25
1
32
8
4
4
8c
0.0625
0.0156
0.0156
0.0625
0.0156
0.0156
0.0625
0.0156
0.0625
0.0156
0.0156
0.0625
0.0625
0.25
0.0625
0.25
0.25
1
0.25
0.25
1
0.125
0.0625
4
1
0.25
1
1
0.25
1
0.0625
0.0625
0.0625
0.0625
0.0156
0.0625
0.0625
0.0156
0.0625
0.0156
0.0156
0.0156
0.0625
0.25
0.0156
1
0.25
1
1
0.25
1
0.125
0.0625
0.25
0.0625
0.25
0.25
0.0625
0.25
0.25
0.0625
0.0625
0.0156
0.0156
0.25
0.0625
1
0.0625
4
1
4
16
0.25
4
0.125
0.0625
32
8
16
32
4
32
32
1
32
2
4
0.25
1
1
0.25
1
1
0.25
1
0.0625
0.25
1
0.0625
4
0.25
16
16
64
64
4
16
0.25
0.0625
12a
12b
12c
12d
12e
12f
12g
12h
12i
0.12
0.125
0.125
16
32
16
0.25
0.125
0.125
0.125
8
1
0.25
0.25
0.0625
0.25
1
0.25
4
0.25
4
4
16
16
1
12j
12k
12l
4
16
2
32
2
12m
12n
12o
12p
12q
12r
12s
FLZ
ITZ
64
32
64
64
>64
>64
>64
>64
2
0.25
0.25
0.5
0.25
0.5
2
>64
>64
>64
1
0.125
0.0625
VOR
0.5
a
Abbreviations: C. albicans, Candida albicans; C. neoformans, Cryptococcus neoformans; C. tropicalis, Candida tropicalis; T. rubrum, Trichophyton rubrum; A. fumigatus,
Aspergillus fumigatus; M. gypseum, Microsporum gypseum; FLZ, Fluconazole; ITZ, Itraconazole; VOR, Voriconazole.
b
Clinical isolate resistant to FLZ, ITZ and VOR.
6.1.1. Chemical synthesis of 1-(3-bromopropoxy)-4-methylbenzene
(10a)
The residue was purified by silica gel column chromatography
(CH2Cl2:MeOH 100:2, v/v) to give 12a as pale yellow oil, 0.54 g
A solution of 1,3-dibrompropane (20.19 g, 0.10 mol) in 80 mL
DMF was added dropwise to a stirred mixture of p-cresol (5.41 g,
0.05 mol), K2CO3 (10.35 g, 0.075 mol) and DMF (20 mL) at room
temperature. The mixture was stirred at the same temperature for
24 h, diluted with ether (200 mL) and washed by H2O (200 mL ꢃ 3).
The organic layer was separated, dried with Na2SO4, and concen-
trated under reduced pressure. The residue was purified by column
chromatography (hexane) to give 10a as yellow oil: 10.57 g, (92.3%).
(38.2%). 1H NMR
d (ppm): 8.13 (s, 1H, TriazC3–H), 7.78 (s, 1H, Tri-
azC5–H), 6.75–7.55 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼5.2 Hz,
J2 ¼ 14.3 Hz, C1–H), 3.93 (t, 2H, J ¼ 6.3 Hz, OCH2), 3.07 (d, 1H,
J ¼ 13.6 Hz, C3–Ha), 2.66 (d, 1H, J ¼ 13.6 Hz, C3–Hb), 2.45 (t, 2H,
J ¼ 7.4 Hz, piperazine–CH2), 2.37 (br, 8H, piperazine–H), 2.27 (s, 3H,
PhCH3), 1.89 (br, 2H, CH2CH2CH2). 13C NMR
d (ppm): 161.51, 157.74,
156.62, 150.77, 144.45, 129.63, 129.14, 126.04, 114.16, 111.32, 104.06,
71.76, 65.90, 62.10, 56.17, 56.76, 54.12, 52.98, 26.51, 20.24. MS (ESI)
m/z: 472 (M þ 1). Anal. calcd. for C25H31F2N5O2: C, 63.68; H, 6.63; N,
14.85. Found: C, 63.76; H, 6.62; N, 14.82.
The synthetic methods for the target compounds 8a–c and 12b–
n were similar to the synthesis of compound 12a.
1H NMR
d
(ppm): 6.80–7.09 (m, 4H, Ar–H), 4.07 (t, 2H, J ¼ 5.8 Hz,
OCH2), 3.60 (t, 2H, J ¼ 6.5 Hz, CH2Br), 2.30–2.34 (m, 2H,
CH2CH2CH2), 2.28 (s, 3H, CH3). MS (ESI) m/z: 229 (M). The synthetic
methods for the following compounds 6a–c and 10b–n were
similar to the synthesis of compound 10a.
6.1.4. Chemical synthesis of 3-(4-(3-(4-
6.1.2. Chemical synthesis of 1-(3-(p-tolyloxy)propyl)piperazine (11a)
A solution of compound 10a (2.29 g, 0.01 mol) in EtOH (20 mL)
was added dropwise to a stirred mixture of piperazine (1.04 g,
0.012 mol), K2CO3 (2.08 g, 0.015 mol) and EtOH (10 ml). Then, the
mixture was heated to reflux for 8 h. After filtration, the solvent was
evaporated under reduced pressure to give 11a as solid, 1.86 g,
(79.49%). The product was used in the next step without further
aminophenoxy)propyl)piperazin-1-yl)-2-(2,4-difluorophenyl)-1-
(1H-1,2,4-triazol-1-yl)propan-2-ol (12o)
Catalytic amount of Raney Ni was added to the solution of 12h
(0.2 g, 0.0004 mol) in 5 mL alcohol in the presence of hydrazine
hydrate (1 mL). The mixture was stirred at room temperature for
5 h. The solid was separated, and the filtrate was evaporated under
reduced pressure. The residue was purified by column chroma-
tography (CH2Cl2:MeOH 100:5, v/v) to give 12o as brown oil, 0.18 g
purification. 1H NMR
d (ppm): 6.77–7.08 (m, 4H, Ar–H), 3.97 (t, 2H,
J ¼ 6.2 Hz, OCH2), 2.64–3.12 (m, 8H, piperazine–H), 2.57
(t, 2H, J ¼ 7.1 Hz, piperazine–CH2), 2.28 (s, 3H, CH3), 1.92–1.96 (m,
2H, CH2CH2CH2). MS (ESI) m/z: 235 (M þ 1). The synthetic methods
for the following compounds 7a–c and 11b–n were similar to the
synthesis of compound 11a.
(92.3%). 1H NMR
d (ppm): 8.13 (s, 1H, TriazC3–H), 7.78 (s, 1H, Tri-
azC5–H), 6.60–7.54 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼3.8 Hz,
J2 ¼ 14.2 Hz, C1–H), 3.88 (t, 2H, J ¼ 6.3 Hz, OCH2), 3.06 (d, 1H,
J ¼ 13.5 Hz, C3–Ha), 2.67 (d, 1H, J ¼ 13.6 Hz, C3–Hb), 2.48 (t, 2H,
J ¼ 6.0 Hz piperazine–CH2), 2.39 (br, 8H, piperazine–H), 1.87 (br, 2H,
CH2CH2CH2). 13C NMR
d (ppm): 162.64, 159.79, 157.82, 152.64,
6.1.3. Chemical synthesis of 2-(2,4-difluorophenyl)-3-(4-(3-(p-
tolyloxy)propyl) piperazin-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-
ol (12a)
150.79, 144.65, 129.22, 126.19, 117.38, 114.70, 111.44, 104.16, 71.68,
66.86, 62.25, 56.30, 54.90, 54.13, 53.03, 26.59. MS (ESI) m/z: 471
(M ꢂ 1). Anal. calcd. for C24H30F2N6O2: C, 61.00; H, 6.40; N, 17.79.
Found: C, 61.06; H, 6.39; N, 17.76.
A
solution of epoxide 4 (1.00 g, 0.003 mol), 11a (0.94 g,
0.004 mol), triethylamine (2 mL) and EtOH (20 mL) was heated to
reflux for 12 h. The solvent was evaporated under reduced pressure.
The synthetic methods for the target compounds 12p and 12q
were similar to the synthesis of compound 12o.

4224
X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
6.1.5. Chemical synthesis of 3-(4-(3-(4-
benzamidophenoxy)propyl)piperazin-1-yl)-2-(2,4-difluorophenyl)-
1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12r)
C1–H), 3.97 (t, 2H, J ¼ 6.0 Hz, OCH2), 3.08 (d, 1H, J ¼ 13.5 Hz, C3–Ha),
2.69 (d, 1H, J ¼ 13.5 Hz, C3–Hb), 2.46 (t, 2H, J ¼ 7.3 Hz, piperazine–
CH2), 2.37 (br, 8H, piperazine–H), 2.18 (s, 3H, PhCH3), 2.00 (br, 2H,
CH2CH2CH2). MS (ESI) m/z: 472 (M þ 1). Anal. calcd. for
C25H31F2N5O2: C, 63.68; H, 6.63; N, 14.85. Found: C, 63.62; H, 6.64;
N, 14.87.
A solution of benzoyl chloride (0.007 g, 0.046 mmol) in 5 mL
methanol was added dropwise to the solution of 12o (0.02 g,
0.042 mmol), triethylamine (1 mL), and CH2Cl2 (5 mL) at 0 ꢀC. The
mixture was stirred at room temperature for 5 h. The solvent was
evaporated under reduced pressure. The residue was purified by
column chromatography (CH2Cl2:MeOH 100:2, v/v) to give 12r as
6.1.11. 3-(4-(3-(3-Bromophenoxy)propyl)piperazin-1-yl)-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12d)
yellow oil, 0.02 g (90.9%). 1H NMR
d
(ppm): 8.31 (s,1H, TriazC3–H), 7.86
1H NMR
d (ppm): 8.13 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–
(s, 1H, TriazC5–H), 7.85 (s, 1H, NHCO), 6.80–7.79 (m, 13H, Ar–H), 4.54
(dd, 2H, J1 ¼3.8 Hz, J2 ¼14.3 Hz, C1–H), 4.00 (t, 2H, J ¼ 6.1 Hz, OCH2),
3.09 (d,1H, J ¼ 13.5 Hz, C3–Ha), 2.72 (d,1H, J ¼ 13.3 Hz, C3–Hb), 2.52 (br,
10H, piperazine–H, piperazine–CH2), 2.08 (br, 2H, CH2CH2CH2). MS
(ESI) m/z: 577 (M þ 1). Anal. calcd. for C31H34F2N6O3: C, 64.57; H, 5.94;
N, 14.57. Found: C, 64.51; H, 5.96; N, 14.62.
H), 6.78–7.26 (m, 7H, Ar–H), 5.29 (s, 1H, 2-OH), 4.51 (dd, 2H,
J1 ¼5.6 Hz, J2 ¼14.0 Hz, C1–H), 3.94 (t, 2H, J ¼ 6.2 Hz, OCH2), 3.07
(d, 1H, J ¼ 13.5 Hz, C3–Ha), 2.67 (d, 1H, J ¼ 13.5 Hz, C3–Hb), 2.46
(t, 2H, J ¼ 7.4 Hz, piperazine–CH2), 2.39 (br, 8H, piperazine–H),
1.90 (br, 2H, CH2CH2CH2). 13C NMR
d (ppm): 162.43, 159.46,
158.61, 150.63, 144.36, 130.21, 129.05, 125.92, 123.37, 122.45,
117.43, 113.24, 111.22, 103.86, 71.70, 65.98, 62.02, 56.07, 54.44,
52.87, 26.22. MS (ESI) m/z: 537 (M þ 1). Anal. calcd. for
C24H28BrF2N5O2: C, 53.74; H, 5.26; N, 13.06. Found: C, 53.79; H,
5.24; N, 13.04.
The synthetic method for compound 12s was similar to the
synthesis of compound 12r.
6.1.6. 2-(2,4-Difluorophenyl)-3-(4-(3-phenoxypropyl)piperazin-1-
yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (8a)
1H NMR
d
(ppm): 8.13 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.1.12. 2-(2,4-Difluorophenyl)-3-(4-(3-(4-fluorophenoxy)
6.77–7.55 (m, 8H, Ar–H), 5.30 (br, 1H, OH), 4.52 (dd, 2H, J1 ¼ 2.1 Hz,
J2 ¼14.7 Hz, C1–H), 3.96 (t, 2H, J ¼ 6.3 Hz, OCH2), 3.07 (d, 1H,
J ¼ 13.5 Hz, C3–Hb), 2.66 (d, 1H, J ¼ 13.5 Hz, C3–Ha), 2.47 (t, 2H,
J ¼ 7.8 Hz, piperazine–CH2), 2.37 (br, 8H, piperazine–H), 1.91 (br, 2H,
CH2CH2CH2). MS (ESI) m/z: 458 (M þ 1). Anal. calcd. for C26H33F2N5O2:
C, 63.00; H, 6.39; N, 15.31. Found: C, 62.95; H, 6.41; N, 15.30.
propyl)piperazin-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12e)
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.78–7.55 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼7.3 Hz, J2 ¼ 14.2 Hz, C1–
H), 3.92 (t, 2H, J ¼ 6.3 Hz, OCH2), 3.06 (d, 1H, J ¼ 13.5 Hz, C3–Ha),
2.66 (d, 1H, J ¼ 13.6 Hz, C3–Hb), 2.44 (t, 2H, J ¼ 7.4 Hz, piperazine–
CH2), 2.35 (br, 8H, piperazine–H), 1.87 (br, 2H, CH2CH2CH2). 13C
NMR
d (ppm): 163.67, 161.69, 161.59, 156.13, 150.89, 144.55, 129.22,
6.1.7. 2-(2,4-Difluorophenyl)-3-(4-(4-phenoxybutyl)piperazin-1-yl)
126.16, 115.53, 111.44, 104.28, 71.84, 66.60, 52.17, 56.27, 54.76, 54.18,
53.05, 26.53. MS (ESI) m/z: 476 (M þ 1). Anal. calcd. for
C24H28F3N5O2: C, 60.62; H, 5.94; N, 14.73. Found: C, 60.67; H, 5.93;
N, 14.69.
-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (8b)
1H NMR
d (ppm): 8.15 (s, 1H, TriazC3–H), 7.79 (s, 1H, TriazC5–H),
6.78–7.53 (m, 8H, Ar–H), 5.30 (br, 1H, OH), 4.50 (dd, 2H, J1 ¼1.5 Hz,
J2 ¼ 12.2 Hz, C1–H), 3.96 (t, 2H, J ¼ 6.2 Hz, OCH2), 3.06 (d, 1H,
J ¼ 13.8 Hz, C3–Ha), 2.62 (d, 1H, J ¼ 13.5 Hz, C3–Hb), 2.55 (t, 2H,
J ¼ 7.5 Hz, piperazine–CH2), 2.36 (br, 8H, piperazine–H), 1.74–7.77
(m, 2H, CH2CH2CH2CH2), 1.62 (br, 2H, CH2CH2CH2CH2). MS (ESI) m/
z: 472 (M þ 1). Anal. calcd. for C31H34F2N6O3: C, 63.68; H, 6.63; N,
14.85. Found: C, 63.62; H, 6.64; N, 14.87.
6.1.13. 3-(4-(3-(2-Chlorophenoxy)propyl)piperazin-1-yl)-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12f)
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.79–7.54 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼5.3 Hz, J2 ¼ 14.2 Hz, C1–
H), 4.04 (t, 2H, J ¼ 6.2 Hz, OCH2), 3.06 (d, 1H, J ¼ 13.6 Hz, C3–Ha),
2.66 (d, 1H, J ¼ 13.6 Hz, C3–Hb), 2.51 (t, 2H, J ¼ 7.6 Hz, piperazine–
CH2), 2.37 (br, 8H, piperazine–H), 1.95 (br, 2H, CH2CH2CH2). MS
(ESI) m/z: 492 (M þ 1). Anal. calcd. for C24H28ClF2N5O2: C, 58.59; H,
5.74; N, 14.24. Found: C, 58.64; H, 5.73; N, 14.23.
6.1.8. 2-(2,4-Difluorophenyl)-3-(4-(5-phenoxypentyl)piperazin-1-yl)-
1-(1H-1,2,4-triazol-1-yl)propan-2-ol (8c)
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.76–7.58 (m, 8H, Ar–H), 5.29 (br, 1H, OH), 4.47 (dd, 2H, J1 ¼4.8 Hz,
J2 ¼ 10.5 Hz, C1–H), 3.92 (t, 2H, J ¼ 6.4 Hz, OCH2), 3.06 (d, 1H,
J ¼ 13.5 Hz, C3–Ha), 2.65 (d, 1H, J ¼ 13.8 Hz, C3–Hb), 2.36 (br, 8H,
piperazine–H), 2.25 (t, 2H, J ¼ 7.6 Hz, piperazine–CH2), 1.74–1.78
(m, 2H, CH2CH2CH2CH2CH2), 1.45 (br, 4H, CH2CH2CH2CH2CH2). MS
(ESI) m/z: 486 (M þ 1). Anal. calcd. for C26H32F2N6O3: C, 64.31; H,
6.85; N, 14.42. Found: C, 64.28; H, 6.86; N, 14.42.
6.1.14. 3-(4-(3-(4-Bromophenoxy)propyl)piperazin-1-yl)-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12g)
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.73–7.56 (m, H, Ar–H), 5.29 (s, 1H, 2-OH), 4.51 (dd, 2H, J1 ¼6.7 Hz,
J2 ¼ 14.2, C1–H), 3.92 (t, 2H, J ¼ 6.2 Hz, OCH2), 3.06 (d, 1H, J ¼ 13.5 Hz,
C3–Ha), 2.66 (d, 1H, J ¼ 13.5 Hz, C3–Hb), 2.43 (t, 2H, J ¼ 7.1 Hz, piper-
azine–CH2), 2.35 (br, 8H, piperazine–H), 1.87 (br, 2H, CH2CH2CH2).
6.1.9. 2-(2,4-Difluorophenyl)-3-(4-(3-(m-tolyloxy)
13CNMR
d(ppm):163.23,161.29,157.73,150.53,144.29,131.79,129.00,
propyl)piperazin-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12b)
125.91, 115.96, 112.28, 111.20,103.86, 71.74, 65.95, 62.02, 56.00, 54.40,
53.97, 52.81, 26.19. MS (ESI) m/z: 537 (M þ 1). Anal. calcd. for
C24H28BrF2N5O2: C, 53.74; H, 5.26; N, 13.06. Found: C, 53.69; H, 5.27;
N, 13.09.
1H NMR
d (ppm): 8.13 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.66–7.55 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼5.4 Hz, J2 ¼ 14.0, C1–H),
3.94 (t, 2H, J ¼ 6.2 Hz, OCH2), 3.06 (d,1H, J ¼ 13.5 Hz, C3–Ha), 2.66 (d,
1H, J ¼ 13.5 Hz, C3–Hb), 2.45 (t, 2H, J ¼ 7.4 Hz, piperazine–CH2), 2.37
(br, 8H, piperazine–H), 2.32 (s, 3H, PhCH3),1.89 (br, 2H, CH2CH2CH2).
MS (ESI) m/z: 472 (M þ 1). Anal. calcd. for C25H31F2N5O2: C, 63.68; H,
6.63; N, 14.85. Found: C, 63.63; H, 6.64; N, 14.88.
6.1.15. 2-(2,4-Difluorophenyl)-3-(4-(3-(4-
nitrophenoxy)propyl)piperazin-1-yl)-1-(1H-1,2,4-triazol-1-
yl)propan-2-ol (12h)
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.1.10. 2-(2,4-Difluorophenyl)-3-(4-(3-(o-tolyloxy)
6.78–8.18 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼9.7 Hz, J2 ¼ 14.2, C1–H),
4.06 (t, 2H, J ¼ 6.2 Hz, OCH2), 3.07 (d, 1H, J ¼ 13.5 Hz, C3–Ha), 2.67
(d, 1H, J ¼ 13.5 Hz, C3–Hb), 2.46 (t, 2H, J ¼ 6.4 Hz, piperazine–CH2),
2.37 (br, 8H, piperazine–H), 1.94 (br, 2H, CH2CH2CH2). MS (ESI) m/z:
propyl)piperazin-1-yl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12c)
1H NMR
d
(ppm): 8.15 (s, 1H, TriazC3–H), 7.80 (s, 1H, TriazC5–H),
6.76–7.53 (m, 7H, Ar–H), 4.53 (dd, 2H, J1 ¼3.2 Hz, J2 ¼ 14.1 Hz,

X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
4225
501 (M ꢂ 1). Anal. calcd. for C24H28F2N6O4: C, 57.36; H, 5.62; N,
6.1.21. 2-(2,4-Difluorophenyl)-3-(4-(3-
(3-nitrophenoxy)propyl)piperazin-1-yl)-1-(1H-1,2,4-triazol-1-
yl)propan-2-ol (12n)
16.72. Found: C, 57.42; H, 5.61; N, 16.70.
6.1.16. 3-(4-(3-(4-Tert-butylphenoxy)propyl)piperazin-1-yl)-2-
1H NMR
d (ppm): 8.13 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12i)
6.79–7.81 (m, 7H, Ar–H), 5.29 (s, 1H, OH), 4.51 (dd, 2H, J1 ¼6.8 Hz,
J2 ¼ 14.25 Hz, C1–H), 4.05 (t, 2H, J ¼ 6.3 Hz, OCH2), 3.07 (d, 1H,
J ¼ 13.6 Hz, C3–Ha), 2.67 (d, 1H, J ¼ 13.6 Hz, C3–Hb), 2.47 (t, 2H,
J ¼ 6.1 Hz, piperazine–CH2), 2.38 (br, 8H, piperazine–H), 1.93 (br,
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.79–7.55 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼5.2 Hz, J2 ¼14.3 Hz, C1–
H), 3.94 (t, 2H, J ¼ 6.3 Hz, OCH2), 3.04 (d, 1H, J ¼ 13.5 Hz, C3–Ha),
2.66 (d, 1H, J ¼ 13.5 Hz, C3–Hb), 2.45 (t, 2H, J ¼ 6.7 Hz, piperazine–
CH2), 2.36 (br, 8H, piperazine–H), 1.88 (br, 2H, CH2CH2CH2), 1.28 (s,
2H, CH2CH2CH2). 13C NMR
d (ppm): 162.45, 161.38, 158.47, 150.67,
148.90, 144.38, 129.61, 129.08, 126.06, 121.30, 115.30, 111.21, 108.50,
103.96, 71.76, 66.50, 62.60, 56.10, 54.30, 54.04, 52.91, 26.12. MS (ESI)
m/z: 503 (M þ 1). Anal. calcd. for C24H28F2N6O4: C, 57.36; H, 5.62; N,
16.72. Found: C, 57.40; H, 5.61; N, 16.70.
9H, C(CH3)3). 13C NMR
d (ppm): 166.58, 163.65, 161.66, 157.875,
150.96, 144.58, 129.262, 126.09, 113.87, 111.47, 104.20, 71.81, 65.92,
62.21, 56.32, 54.90, 54.30, 53.13, 33.96, 31.45, 26.70. MS (ESI) m/z:
514 (M þ 1). Anal. calcd. for C28H37F2N5O2: C, 65.48; H, 7.26; N,
13.64. Found: C, 65.41; H, 7.27; N, 13.65.
6.1.22. 3-(4-(3-(3-Aminophenoxy)propyl)piperazin-1-yl)-2-(2,4-
difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12p)
6.1.17. 3-(4-(3-(4-Chlorophenoxy)propyl)piperazin-1-yl)-2-
1H NMR
d (ppm): 8.12 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12j)
6.20–7.54 (m, 7H, Ar–H), 4.52 (dd, 2H, J1 ¼3.4 Hz, J2 ¼ 14.1 Hz, C1–
H), 3.92 (t, 2H, J ¼ 6.1 Hz, OCH2), 3.07 (d, 1H, J ¼ 13.4 Hz, C3–Ha),
2.68 (d, 1H, J ¼ 13.4 Hz, C3–Hb), 2.45 (t, 2H, J ¼ 6.2 Hz, piperazine–
CH2), 2.37 (br, 8H, piperazine–H),1.91 (br, 2H, CH2CH2CH2). MS (ESI)
m/z: 471 (M ꢂ 1). Anal. calcd. for C24H30F2N6O2: C, 61.00; H, 6.40; N,
17.79. Found: C, 60.94; H, 6.42; N, 17.81.
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.77–7.55 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼6.5 Hz, J2 ¼14.2 Hz, C1–
H), 3.93 (t, 2H, J ¼ 6.3 Hz, OCH2), 3.06 (d, 1H, J ¼ 13.6 Hz, C3–Ha),
2.67 (d, 1H, J ¼ 13.6 Hz, C3–Hb), 2.45 (t, 2H, J ¼ 6.4 Hz, piperazine–
CH2), 2.37 (br, 8H, piperazine–H), 1.89 (br, 2H, CH2CH2CH2). 13C
NMR
d (ppm): 163.70, 159.74, 157.49, 150.49, 144.58, 129.19, 126.18,
115.69, 111.47, 104.21, 71.86, 66.30, 62.19, 56.30, 54.74, 54.23, 53.10,
26.50. MS (ESI) m/z: 492 (M þ 1). Anal. calcd. for C24H28ClF2N5O2: C,
58.59; H, 5.74; N, 14.24. Found: C, 58.62; H, 5.73; N, 14.26.
6.1.23. 3-(4-(3-(2-Aminophenoxy)propyl)piperazin-1-yl)-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12q)
1H NMR
d (ppm): 8.12 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.67–7.54 (m, 7H, Ar–H), 4.52 (dd, 2H, J1 ¼3.6 Hz, J2 ¼ 14.2 Hz, C1–
H), 3.99 (t, 2H, J ¼ 6.1 Hz, OCH2), 3.07 (d, 1H, J ¼ 13.4 Hz, C3–Ha),
2.68 (d, 1H, J ¼ 13.4 Hz, C3–Hb), 2.45 (t, 2H, J ¼ 6.4 Hz, piperazine–
CH2), 2.38 (br, 8H, piperazine–H), 1.92 (br, 2H, CH2CH2CH2). MS
(ESI) m/z: 471 (M ꢂ 1). Anal. calcd. for C24H30F2N6O2: C, 61.00; H,
6.40; N, 17.79. Found: C, 60.97; H, 6.41; N, 17.81.
6.1.18. 3-(4-(3-(2-Bromophenoxy)propyl)piperazin-1-yl)-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12k)
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.79–7.55 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼5.4 Hz, J2 ¼ 14.1 Hz C1–
H), 4.03 (t, 2H, J ¼ 6.2 Hz, OCH2), 3.06 (d, 1H, J ¼ 13.6 Hz, C3–Ha),
2.66 (d, 1H, J ¼ 13.6 Hz, C3–Hb), 2.52 (t, 2H, J ¼ 7.2 Hz, piperazine–
CH2), 2.36 (br, 8H, piperazine–H), 1.94 (br, 2H, CH2CH2CH2). 13C
6.1.24. 2-(2,4-Difluorophenyl)-3-(4-(3-(4-
acetamidophenoxy)propyl)piperazin-1-yl)-1-(1H-1,2,4-triazol-1-
yl)propan-2-ol (12s)
NMR
d (ppm): 163.62, 161.65, 157.85, 150.92, 144.57, 133.21, 129.25,
128.30, 126.20, 121.69, 113.21, 112.12, 111.45, 104.18, 71.81, 67.08,
62.19, 56.30, 54.70, 53.11, 26.44. MS (ESI) m/z: 537 (M þ 1). Anal.
calcd. for C24H28BrF2N5O2: C, 53.74; H, 5.26; N, 13.06. Found: C,
53.79; H, 5.24; N, 13.05.
1H NMR
d (ppm): 8.13 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
7.02 (s, 1H, NHCOCH3), 6.78–7.54 (m, 7H, Ar–H), 4.52 (dd, 2H,
J1 ¼3.8 Hz, J2 ¼14.1 Hz, C1–H), 3.94 (t, 2H, J ¼ 6.1 Hz, OCH2), 3.07 (d,
1H, J ¼ 13.5 Hz, C3–Hb), 2.67 (d, 1H, J ¼ 13.3 Hz, C3–Ha), 2.39 (br, 10H,
piperazine–H, piperazine–CH2), 2.15 (s, 3H, CH3), 1.93 (br, 2H,
CH2CH2CH2). MS(ESI)m/z: 515 (M þ 1). Anal. calcd. for C26H32F2N6O3:
C, 60.69; H, 6.27; N, 16.33. Found: C, 60.60; H, 6.28; N, 16.37.
6.1.19. 3-(4-(3-(4-(Benzyloxy)phenoxy)propyl)piperazin-1-yl)-2-
(2,4-difluoro phenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12l)
1H NMR
d (ppm): 8.11 (s, 1H, TriazC3–H), 7.78 (s, 1H, TriazC5–H),
6.77–7.42 (m, 12H, Ar–H), 5.00 (s, 2H, PhCH2OPh), 4.52 (dd, 2H,
J1 ¼6.2 Hz, J2 ¼14.3 Hz, C1–H), 3.92 (t, 2H, J ¼ 6.1 Hz, OCH2), 3.07 (d,
1H, J ¼ 13.8 Hz, C3–Ha), 2.68 (d, 1H, J ¼ 13.7 Hz, C3–Hb), 2.45 (t, 2H,
J ¼ 7.0 Hz, piperazine–CH2), 2.38 (br, 8H, piperazine–H), 1.92 (br,
2H, CH2CH2CH2). MS (ESI) m/z: 564 (M þ 1). Anal. calcd. for
C31H35F2N5O3: C, 66.06; H, 6.26; N, 12.43. Found: C, 66.00; H, 6.27;
N, 12.46.
6.2. Flexible docking analysis
The 3D structures of the designed azoles were built by the
Builder module within InsightII 2000 software package [29]. Then,
the flexible ligand docking procedure in the Affinity module within
InsightII was used to define the lowest energy position for the
substrate using a Monte Carlo docking protocol. All the atoms
within a defined radius (8 Å) of the substrate were allowed to move.
The solvation grid supplied with the affinity Program was used. If
the resulting substrate/enzyme system was within a predefined
energy tolerance of the previous structure, the system was sub-
jected to minimization. The resulting structure was accepted on the
basis of energy check, which used the Metropolis criterion, and also
a check of RMS distance of the new structure versus the structure
found so far. The final conformation was obtained through a simu-
lation annealing procedure from 500 to 300 K, and then 5000
rounds of energy minimization were performed to reach a conver-
gence, where the resulting interaction energy values were used to
define a rank order.
6.1.20. 2-(2,4-Difluorophenyl)-3-(4-(3-
(2-nitrophenoxy)propyl)piperazin-1-yl)-1-(1H-1,2,4-triazol-1-
yl)propan-2-ol (12m)
1H NMR
d (ppm): 8.14 (s, 1H, TriazC3–H), 7.79 (s, 1H, TriazC5–H),
6.80–7.81 (m, 7H, Ar–H), 4.51 (dd, 2H, J1 ¼4.4 Hz, J2 ¼ 14.2, C1–H),
4.12 (t, 2H, J ¼ 6.1 Hz, OCH2), 3.06 (d,1H, J ¼ 13.5 Hz, C3–Ha), 2.65 (d,
1H, J ¼ 13.5 Hz, C3–Hb), 2.49 (t, 2H, J ¼ 7.1 Hz, piperazine–CH2), 2.36
(br, 8H, piperazine–H), 1.94 (br, 2H, CH2CH2CH2). 13C NMR
d (ppm):
162.70,159.61,152.29,150.96,144.64,139.98,133.96,129.33,126.22,
125.49, 120.19, 114.49, 111.54, 104.24, 71.92, 67.67, 62.27, 56.37,
54.36, 53.13, 26.28. MS (ESI) m/z: 503 (M þ 1). Anal. calcd. for
C24H28F2N6O4: C, 57.36; H, 5.62; N, 16.72. Found: C, 57.30; H, 5.63;
N, 16.74.

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X. Che et al. / European Journal of Medicinal Chemistry 44 (2009) 4218–4226
[15] J. Capilla, C. Yustes, E. Mayayo, B. Fernandez, M. Ortoneda, F.J. Pastor, J. Guarro,
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This work was supported in part by the National Natural Science
Foundation of China (Grant No. 30430750) and Shanghai Leading
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