Novel conformationally restricted triazole derivatives with potent antifungal activity

Article abstract of DOI:10.1016/j.ejmech.2010.09.070

In continuation of our work on azole antifungal agents, a series of new conformationally restricted triazole derivatives possessing benzylpiperidin-4-yl methyl amino side chains were designed and synthesized. All the new azoles showed moderate to excellent in vitro antifungal activity against most of the tested pathogenic fungi. Several compounds (such as 12e, 12f, 12h and 12n) showed higher antifungal activity against Candida albicans than fluconazole. Moreover, compounds 12g-i also showed good activity against Aspergillus fumigatus with their MIC₈₀ on the level of 1 μg/mL. Flexible molecular docking was used to analyze the binding mode of the designed compounds. They interact with CACYP51 through hydrophobic and van der Waals interactions.

Full text of DOI:10.1016/j.ejmech.2010.09.070

European Journal of Medicinal Chemistry 45 (2010) 6020e6026
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel conformationally restricted triazole derivatives with potent
antifungal activity
Wenya Wang, Shengzheng Wang, Yang Liu, Guoqiang Dong, Yongbing Cao, Zhenyuan Miao,
*
**
Jianzhong Yao, Wannian Zhang , Chunquan Sheng
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
In continuation of our work on azole antifungal agents, a series of new conformationally restricted tri-
azole derivatives possessing benzylpiperidin-4-yl methyl amino side chains were designed and
synthesized. All the new azoles showed moderate to excellent in vitro antifungal activity against most of
the tested pathogenic fungi. Several compounds (such as 12e, 12f, 12h and 12n) showed higher anti-
fungal activity against Candida albicans than fluconazole. Moreover, compounds 12gei also showed good
Received 29 July 2010
Received in revised form
29 September 2010
Accepted 29 September 2010
Available online 7 October 2010
activity against Aspergillus fumigatus with their MIC80 on the level of 1 mg/mL. Flexible molecular docking
was used to analyze the binding mode of the designed compounds. They interact with CACYP51 through
hydrophobic and van der Waals interactions.
Keywords:
Azole
Rational design
Ó 2010 Elsevier Masson SAS. All rights reserved.
Conformationally restricted analogues
Molecular docking
Antifungal activity
1. Introduction
Azole antifungals have been shown to competitively inhibit
lanosterol 14a-demethylase (CYP51), leading to block fungal
During the past two decades, the incidence of life-threatening
invasive and systemic fungal infections has increased significantly in
the immunocompromised patients due to AIDS, organ trans-
plantation and chemotherapy [1]. Serious fungal infections are
caused mostly by Candida albicans (C. albicans) [2], Cryptococcus
neoformans (C. neoformans) and Aspergillus fumigatus (A. fumigatus)
[3]. For the treatment of these infections, only four major classes of
antifungalagentsare availableinclinicaluse. Theseareazoles(suchas
fluconazole and itraconazole) [4], polyene macrolides (amphotericin
B) [5], flucytosine (5-fluorocytosine) and echinocandins (such as
caspofungin and micafungin) [6]. Among them, azoles are the most
widely used antifungal agents in clinic because of their generally
broad antifungal spectrum, high potency and low toxicity [4].
Unfortunately, massive use of azoles has led to the emergence of
severe resistance [7], showing an urgent need of searching for new
azoles. Second generation azole antifungal drugs, such as vor-
iconazole [8], posaconazole [9], ravuconazole [10] and albaconazole
[11,12], areavailableinthemarketorarecurrentlyunderclinicaltrials.
biosynthesis of ergosterol which is a key constituent of the fungal
cell membrane, thereby preventing fungal growth [13]. Due to
membrane associated proteins, it is difficult to solve the crystal
structures of eukaryotic CYP51s. In our previous studies, our group
has constructed three-dimensional (3D) models of CYP51 from
C. albicans (CACYP51), C. neoformans (CNCYP51) and A. fumigatus
(AFCYP51) using homology modeling methods [14e16] on the basis
of the crystal coordinates of CYP51 from Mycobacterium tuberculosis
(MTCYP51) [17,18]. The binding modes of azoles were investigated
by flexible molecular docking [16,19] and site-directed mutagenesis
[20]. The results from molecular modeling provided important
information for rational inhibitor design and led to the discovery of
novel azole and non-azole CYP51 inhibitors [19,21e26].
Recently, we have designed and synthesized a series of novel
azoles with substituted phenoxyalkyl C-3 side chains [23e25].
These compounds showed excellent in vitro antifungal activity
against most of the tested pathogenic fungi, representing a prom-
ising lead for further optimization. In order to extend their struc-
tureeactivity relationships (SARs), a series of new conformationally
restricted triazole derivatives possessing benzylpiperidin-4-yl side
chains were designed and synthesized, which revealed potent
antifungal activity and broad spectrum. The binding mode of the
azoles was investigated by molecular docking.
* Corresponding author. Tel./fax: þ86 21 81870243.
** Corresponding author. Tel./fax: þ86 21 81871233.
E-mail addresses: zhangwnk@hotmail.com (W. Zhang), shengcq@hotmail.com
(C. Sheng).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.070

W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 6020e6026
6021
O
O
N
N
Cl
O
F
N
N
a
c
b
N
.
F
N
CH3SO3H
F
F
F
F
2
F
F
4
1
3
OH
OH
N
N
e
d
N
N
N3
F
N
N
NH2
F
F
F
5
6
Scheme 1. Reagents and conditions: a. ClCH2COCl, AlCl3, CH2Cl2, 40 ꢀC, 3 h, 50%; b. triazole, K2CO3, CH2Cl2, r.t., 24 h, 70.0%; c. (CH3)3SOI, NaOH, toluene, 60 ꢀC, 3 h, 62.3%; d. NaN3,
NH4Cl, MeOH, reflux, 8 h, 98%; e. H2, PdeC, EtOH, 4 h, 99.1%.
2. Chemistry
or chloride in the presence of K2CO3 in methanol with moderate to
high yields (Scheme 2).
We have reported the synthetic procedure (Scheme 1) of the key
intermediate 6 with the total yield of 21.2% [24]. Piperidin-4-one
hydrochloride 7 was treated with excess di-tert-butyl dicarbonate
in the presence of N,N-diisopropylethylamine (DIEA) in 1,4-
dioxane/H2O (4:1) to give compound 8. Starting from compound 8,
2-(2,4-difluorophenyl)-1-{methyl[1-(tert-butoxycarbonyl) piper-
idin-4-yl]amino}-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (compound
10) was synthesized via two steps of reductive amination. First,
intermediate 6 was reacted with tert-butyl 4-oxopiperidine-1-
carboxylate (compound 8) and sodium cyanoborohydride in
methanol at room temperature to give 9. Then compound 9 was
reacted with formaldehyde according to the same protocol
described for 9 to afford 10. Compound 10 was subsequently
treated with trifluoroacetic acid at room temperature over night to
afford 11. The target compounds 12aen were obtained as racemates
by treating compound 11 with various substituted benzyl bromide
3. Pharmacology
In vitro antifungal activity was measured by means of the
minimum inhibitory concentration (MIC) using the serial dilution
method in 96-well microtest plates. Fluconazole was used as the
reference drug. 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 Stan-
dards (NCCLS) recommendations with RPMI 1640 (Sigma) buffered
with 0.165 M MOPS (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.
Scheme 2. Reagents and conditions: a. Boc2O, DIEA, 1,4-dioxane-H2O, r.t., 24 h, 80.2%; b. 6, NaBH3CN, AcOH/MeOH, r.t., 16 h, 99.0%; c. formaldehyde, NaBH3CN, AcOH/MeOH, r.t.,
24 h, 92.6%; d. CF3COOH, CH2Cl2, 12 h, 95.1%; e. substituted benzyl bromide, K2CO3, MeOH, r.t., 16 h, 45.5%e81.2%.

6022
W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 6020e6026
OH CH3
OH CH3
R
R
N
N
N
N
F
N CH2
N
N
F
O
N
N
F
F
Lead structure
12 a-n
Fig. 1. Design rationale of the new azoles with benzylpiperidin-4-yl side chains.
Growth MIC was determined at 24 h for Candida species, at 72 h for
C. neoformans, and at 7 days for filamentous fungi.
CACYP51 with an extended conformation, which is similar to that in
our reported docking models [19,24e26]. The triazolyl ring, diflur-
ophenyl group and C2 hydroxyl group are essential pharmacophoric
elements of the azole antifungal agents. The triazolyl ring of the
compounds binds to CACYP51 through the formation of a coordina-
tion bond with iron of heme group. The difluorophenyl group is
located in a hydrophobic pocket and interacts with Phe126, Met306
and Phe145. The N-methyl group forms hydrophobic and van der
Waals interactions with Tyr118. The piperidyl group interacts with
the surrounding hydrophobic residues lined with Ile379 and Val509.
The terminal benzyl group binds to substrate access channel 2 [19]
(FG loop) through the hydrophobic and van der Waals interactions
with Leu461, Leu376, Leu403, Met372, and Met374.
4. Results and discussion
4.1. Design rationale
As a part of our continual effort in azoles optimization, we have
designed a series of novel azoles with substituted phenoxyalkyl C-3
side chains [24e26]. In vitro antifungal assay indicated that they
displayed excellent antifungal activity with broad spectrum, which
could serve as a good starting point for the discovery of novel anti-
fungal agents. Therefore, the extension of SARs of these potent
antifungal azoles is of great importance. We chose the N-methyl
derivative (Fig. 1) as a starting point. In the present study, N-methyl
group was retained as the linker, and the propoxy group was
replaced by the piperidin-4-yl group to restrict the conformation of
the C3-side chain and form stronger hydrophobic and van der Waals
interactions with CACYP51. Then a series of conformationally
restricted triazole derivatives was designed. In order to validate the
binding mode of the designed compounds, compound 12n, a repre-
sentative derivative, was docked into the active site of CACYP51
using the Affinity module within Insight II 2000 software package
[27]. Fig. 2 shows that compound 12n binds to the active site of
4.2. In vitro antifungal activity
In vitro antifungal activity of the synthesized compounds is lis-
ted in Table 1. The MIC80 values of all the targeted compounds were
determined against seven important fungal pathogens (such as
C. albicans, C. neoformans and A. fumigatus) and compared with
fluconazole. The assay indicated that the synthesized compounds
12aen showed moderate to excellent activity against all the tested
fungal pathogens. Most of the compounds were more potent than
fluconazole. In general, compounds 12eei exhibited potent anti-
fungal activity and a broad spectrum. On the C. albicans strain, the
most common cause of life-threatening fungal infections, most of
the compounds showed higher antifungal activity than fluconazole
(MIC80 ¼ 0.25
0.0625 g/mL. In particular, compound 12n displayed the highest
activity (MIC80 ¼ 0.0156 g/mL), which was 16 fold more potent
mg/mL) with their MIC80 values on the level of
m
m
than fluconazole. Moreover, these compounds also revealed
excellent inhibitory activity against other Candida spp., such as
C. tropicalis, Candida parapsilosis and Candida kefyr with their MIC80
values in the range of 1e0.0625 mg/mL. For the dermatophytes
Table 1
Antifungal in vitro activity of the compounds (MIC80, m
g mLꢁ1).a
Compd
C. alb.
C. neo.
C. tro.
C. par.
C. kef.
T. rub.
A. fum.
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
FLZ
0.25
1
1
0.25
1
1
1
1
0.25
0.25
1
0.0625
0.25
0.25
0.25
0.25
0.25
0.25
1
1
64
4
4
4
4
4
1
1
1
16
16
64
64
16
>64
0.0625
0.0625
0.25
0.0625
0.0625
0.0625
0.0625
0.0625
0.25
0.25
0.25
0.0625
0.0156
0.25
0.25
0.25
0.25
0.25
0.25
0.0625
0.0625
0.25
0.25
0.25
0.25
0.25
0.25
0.0625
0.25
0.25
0.25
0.0625
0.0625
0.25
0.25
0.0625
0.25
0.25
0.25
0.25
0.25
1
0.0625
0.0625
0.0625
0.0625
0.0625
0.0625
0.25
0.0625
0.25
0.25
0.25
0.25
1
0.25
0.25
0.25
0.0625
0.25
1
1
1
1
0.25
1
0.25
0.25
0.25
0.25
a
Abbreviations: C. alb. Candida albicans; C. neo. Cryptococcus neoformans; C. tro.
Candida tropicalis; C. par. Candida parapsilosis; C. kef. Candida kefyr; T. rub. Tricho-
phyton rubrum; A. fum. Aspergillus fumigatus; FLZ: Fluconazole.
Fig. 2. The docking conformation of compound 12n in the active site of CACYP51.
Important residues interacting with the compounds are shown.

W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 6020e6026
6023
(e.g., T. rubrum), most compounds showed higher activity than
fluconazole. Especially, the MIC80 value of compound 12h is
a Kratos-concept mass spectrometer under electron impact ioni-
zation (EI) conditions. 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.
0.0625
nazole. However, most of the compounds were inferior to fluco-
nazole against C. neoformans (MIC80 ¼ 0.0625 g/mL) with their
MIC80 values on the level of 0.25 g/mL and only compounds 12g
mg/mL, indicating that it is 16 fold more potent than fluco-
m
m
and 12h were comparable to that of fluconazole. Interestingly,
fluconazole is not effective against A. fumigatus, while most of our
synthesized compounds show moderate antifungal activity. For
6.1.1. Chemical synthesis of tert-butyl 4-oxopiperidine-1-
carboxylate (8)
example, the MIC80 values of compounds 12gei are 1
m
g/mL.
DIEA (32.31 g, 0.25 mol, 2.5 equiv) was added to a solution of
piperidin-4-one hydrochloride 7 (13.5 g, 0.10 mol,1.0 equiv) in 200 mL
1,4-dioxane and H2O (v/v, 4/1). Subsequently, di-tert-butyl dicarbonate
(32.74 g, 0.15 mol, 1.5 equiv) was added dropwise to the reaction
mixture over 1 h, and the resulting solution was stirred at room
temperature for 24 h. Then the solvent was evaporated under reduced
pressure, and the residue was poured into a 5% citric acid solution, then
extracted with dichloromethane (100 mL ꢂ 3). The organic layer was
separated, dried with anhydrous Na2SO4, and concentrated to give
crude solid, which was recrystallized from cyclohexane to afford 8 as
Among the synthesized azoles, compounds 12h and 12n exhibited
strong in vitro antifungal activity with broad antifungal spectrum,
which were worthy of further evaluation.
4.3. Structureeactivity relationships
According to the in vitro antifungal activity data, preliminary
SARs of the synthesized compounds were obtained. Compared with
compound 12a, the introduction of various substitutent groups on
the terminal benzyl group could significantly improve the anti-
fungal activity. The electrostatic property of the substitutions had
little effect on the antifungal activity. For the type of the substitu-
tions, halogen, nitro, cyano and iso-propyl are favorable for the
antifungal activity (e.g., compounds 12g, 12b, 12h and 12n). Among
the halogen substituted derivatives, fluorine substituted
compounds (12jek) are less active than bromine or chlorine
substituted compounds (e.g., compounds 12c, 12g and 12i). In
comparison with the mono-chlorine substituted compounds (12d
and 12g), the di-substituted derivatives (12eef) showed improved
antifungal activity. For the position of halogen, the 4-substituted
and 3-substituted derivatives exhibit higher inhibitory activity than
2-substituted derivatives. When the halogen was replaced by alkyl
group, such as methyl and methoxy group, similar antifungal
activity was observed. The good antifungal activity of 12h and 12n
highlighted the importance of the hydrophobic and van der Waals
interactions of the substituent with CACYP51. In addition, 4-cyano,
3-choloro and 3-bromo derivatives displayed the highest inhibitory
activity against A. fumigatus which was not sensitive to fluconazole.
white needle: 15.96 g, (80.2%, yield). 1H NMR
d (ppm): 3.60 (t, 4H,
J ¼ 6.2 Hz, piperidin-2,6-CH2), 2.34 (t, 4H, J ¼ 6.2 Hz, piperidin-3,5-
CH2), 1.43 (s, 9H, C(CH3)3). MS (ESI) m/z: 200 (M þ 1).
6.1.2. Chemical synthesis of 2-(2,4-difluorophenyl)-1-[1-(tert-
butoxycarbonyl)piperidin-4-yl]amino-3-(1H-1,2,4-triazol-1-yl)
propan-2-ol (9)
Compound 8 (3.0 g, 0.015 mol, 1 equiv) was added to a solution
of intermediate 6 (3.8 g, 0.015 mol, 1 equiv) in methanol 100 mL
and acetic acid 2.0 mL. Then sodium cyanoborohydride (1.1 g,
0.018 mol, 1.2 equiv) was added under nitrogen atmosphere. The
reaction mixture was stirred at room temperature for 16 h and the
reaction was almost completed. The solvent was evaporated under
reduced pressure, and the residue was diluted with 25 mL H2O,
extracted with dichloromethane (30 mL ꢂ 3). The organic layer was
separated, dried with anhydrous Na2SO4 and concentrated under
reduced pressure to give 9 as yellow oil: 6.5 g, (99.0%, yield). The
product was used in the next step without further purification.
6.1.3. Chemical synthesis of 2-(2,4-difluorophenyl)-1-{methyl[1-
(tert-butoxycarbonyl)piperidin-4-yl]amino}-3-(1H-1,2,4-triazol-1-
yl)propan-2-ol (10)
5. Conclusion
In conclusion, a series of conformationally restricted triazole
derivatives with substituted benzylpiperidin-4-yl methyl amino
side chains was rational designed and synthesized. Flexible
molecular docking studies revealed that the designed compounds
interacted with CACYP51 mainly through hydrophobic and van der
Waals interactions. Most of the synthesized compounds showed
good antifungal activity against both systemic pathogenic fungi and
dermatophytes. Several compounds were found to be more potent
than fluconazole. Compounds 12gei were most potent against
Formaldehyde (1.2 mL, 0.015 mol, 1 equiv) was added to a solu-
tion of compound 9 (6.6 g, 0.015 mol, 1 equiv) in methanol 100 mL
and acetic acid 2.0 mL. Then sodium cyanoborohydride (1.1 g,
0.018 mol, 1.2 equiv) was added under nitrogen atmosphere. The
reaction mixture was stirred at room temperature for 24 h and the
reaction was almost completed. The solvent was evaporated under
reduced pressure, and the residue was diluted with 25 mL H2O,
extracted with dichloromethane (30 mL ꢂ 3). The organic layer was
separated, dried with anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (CH2Cl2: MeOH, 100:5, v/v) to give 10 as yellow
A. fumigatus with their MIC80 value on the level of 1
mg/mL. In
particular, compounds 12h and 12n exhibited strong antifungal
activity and broad spectrum, suggesting that they are promising
leads for further structural optimization.
oil: 6.3 g, (92.6%, yield). 1H NMR
d (ppm): 8.16 (s, 1H, TriazC3eH),
7.80 (s, 1H, TriazC5eH), 6.78e7.60 (m, 3H, AreH), 5.30 (s, 1H, OH),
4.50 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 4.11 (br, 2H, piperidin-2-CH2),
3.02 (d, 1H, J ¼ 13.3 Hz, C3eHa), 2.72 (d, 1H, J ¼ 13.5 Hz, C3eHb),
2.51 (br, 2H, piperidin-6-CH2), 2.25 (br, 1H, piperidin-4-CH), 1.97 (s,
3H, NCH3), 1.57e1.59 (m, 2H, piperidin-3-CH2), 1.45 (s, 9H, C(CH3)3),
1.22e1.25 (m, 2H, piperidin-5-CH2). MS (ESI) m/z: 452 (M þ 1).
6. Experimental protocols
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
(J values) are given in ppm and Hz, respectively. ESI mass spectra
were performed on an API-3000 LCeMS spectrometer. High-reso-
lution mass spectrometry measurements were performed on
6.1.4. Chemical synthesis of 2-(2,4-difluorophenyl)-1-[methyl
(piperidin-4-yl)]amino-3-(1H-1,2,4-triazol-1-yl)propan-2-ol
trifluoroacetate (11)
Trifluoroacetic acid (2.32 g, 0.020 mol, 4 equiv) was added to
a solution of 10 (2.26 g, 0.005 mol, 1 equiv) in dichloromethane

6024
W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 6020e6026
(50 mL), and the resulting solution was stirred at room temperature
for 24 h. Then the solution was evaporated to dryness under
reduced pressure to give 11 as yellow oil: 2.21 g, (95.1%, yield). The
product was used in the next step without any further purification.
6.1.8. 3-{[1-(2-Chlorobenzyl)piperidin-4-yl](methyl)amino]-2-(2,4-
difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12d)
Pale yellow solid: 0.23 g (76.7%, yield), mp 78e79 ꢀC. IR (KBr,
cmꢁ1): 3406, 2942, 2854, 1612, 1501, 1468, 1270, 1137, 1047, 961. 1H
NMR
d (ppm): 8.17 (s, 1H, TriazC3eH), 7.79 (s, 1H, TriazC5eH),
6.1.5. Chemical synthesis of 3-[(1-benzylpiperidin-4-yl)(methyl)
amino]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-
ol (12a)
6.80e7.57 (m, 7H, AreH), 5.30 (s, 1H, OH), 4.50 (d, 2H, J ¼ 14.2 Hz,
C1eHaHb), 3.54 (br, 2H, AreCH2), 3.00 (d, 1H, J ¼ 13.7 Hz, C3eHa),
2.90e2.99 (m, 2H, piperidin-2-CH2), 2.82 (d, 1H, J ¼ 13.7 Hz,
C3eHb), 2.12e2.18 (m, 1H, piperidin-4-CH), 1.97 (s, 3H, NCH3),
1.91e1.94 (m, 2H, piperidin-6-CH2), 1.57e1.60 (m, 2H, piperidin-3-
A suspension of 11 (0.46 g, 1.0 mmol, 1 equiv), benzyl bromide
(0.26 g, 1.5 mmol, 1.5 equiv) and anhydrous K2CO3 (0.83 g, 6 mmol,
6 equiv) in methanol (15 mL) was stirred at room temperature for
16 h. Then the mixture was filtrated, and the filtrate was
concentrated under reduced pressure. The residue was diluted
with 10 mL H2O and extracted with dichloromethane (20 mL ꢂ 3).
The organic layer was separated, dried with anhydrous Na2SO4
and evaporated under reduced pressure. The residue was purified
by silica gel column chromatography (CH2Cl2: MeOH 100:2, v/v) to
give 12a as yellow oil: 0.20 g (45.5%, yield). IR (KBr, cmꢁ1): 3424,
2942, 2855, 1617, 1500, 1454, 1272, 1137, 1049, 963. 1H NMR
CH2), 1.39e1.45 (m, 2H, piperidin-5-CH2). 13C NMR
d (ppm): 162.65,
158.92, 150.88, 144.72, 136.08, 134.14, 130.48, 129.35, 128.01, 126.93,
126.54, 111.40, 104.15, 71.10, 62.77, 58.93, 58.43, 56.56, 53.07, 39.28,
28.63, 27.61. HRMS (m/z): calcd. for C24H28ClF2N5O: 475.1590;
found: 475.1600.
6.1.9. 3-{[1-(2,4-Dichlorobenzyl)piperidin-4-yl](methyl)amino]-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12e)
Pale yellow solid: 0.25 g (78.1%, yield), mp 101e102 ꢀC. IR (KBr,
cmꢁ1): 3404, 2930, 2854, 1612, 1494, 1466, 1270, 1139, 1053, 960. 1H
d
(ppm): 8.16 (s, 1H, TriazC3eH), 7.78 (s, 1H, TriazC5eH), 6.77e7.56
(m, 8H, AreH), 5.30 (s, 1H, OH), 4.50 (d, 2H, J ¼ 14.2 Hz, C1eHaHb),
3.48 (br, 2H, AreCH2), 3.01 (d, 1H, J ¼ 13.8 Hz, C3eHa), 2.90 (br, 2H,
piperidin-2-CH2), 2.81 (d, 1H, J ¼ 13.8 Hz, C3eHb), 2.13 (s, 1H,
piperidin-4-CH), 1.97 (s, 3H, NCH3), 1.86 (br, 2H, piperidin-6-CH2),
1.57e1.60 (m, 2H, piperidin-3-CH2), 1.25 (br, 2H, piperidin-5-CH2).
NMR d (ppm): 8.17 (s, 1H, TriazC3eH), 7.80 (s, 1H, TriazC5eH),
6.78e7.58 (m, 6H, AreH), 5.30 (s, 1H, OH), 4.50 (d, 2H, J ¼ 14.3 Hz,
C1eHaHb), 3.49 (br, 2H, AreCH2), 3.00 (d, 1H, J ¼ 13.8 Hz, C3eHa),
2.99 (br, 2H, piperidin-2-CH2), 2.82 (d, 1H, J ¼ 13.8 Hz, C3eHb),
2.12e2.15 (m, 1H, piperidin-4-CH), 1.97 (s, 3H, NCH3), 1.91e1.93 (m,
2H, piperidin-6-CH2),1.45e1.50 (m, 2H, piperidin-3-CH2), 1.25e1.41
13C NMR
d (ppm): 162.60, 158.89, 150.85, 144.66, 138.11, 129.35,
129.02, 128.14, 127.00, 111.35, 104.11, 71.11, 62.76, 58.56, 56.58,
52.91, 39.17, 28.40, 27.56. HRMS (m/z): calcd. for C24H29F2N5O:
441.2340; found: 441.2352.
The target compounds 12cen were synthesized according to the
same protocol described for 12a.
(m, 2H, piperidin-5-CH2). 13C NMR
d (ppm): 162.71, 158.91, 150.92,
144.77, 134.67, 132.95, 131.25, 129.37, 129.11, 126.90, 111.44, 104.20,
71.13, 62.71, 58.39, 56.54, 53.07, 39.30, 28.65, 27.60. HRMS (m/z):
calcd. for C24H27Cl2F2N5O: 509.1561; found: 509.1570.
6.1.10. 3-{[1-(3,4-Dichlorobenzyl)piperidin-4-yl](methyl)amino]-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12f)
Yellow oil: 0.26 g (71.2%, yield). IR (KBr, cmꢁ1): 3432, 2925,
6.1.6. Chemical synthesis of 3-{[1-(4-nitrobenzyl)piperidin-4-yl]
(methyl)amino]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)
propan-2-ol (12b)
2855, 1617, 1500, 1467, 1272, 1136, 1050, 963. 1H NMR
d (ppm): 8.16
A suspension of 11 (0.30 g, 0.64 mmol, 1 equiv), 4-nitrobenzyl
chloride (0.16 g, 0.96 mmol, 1.5 equiv) and triethylamine (1 mL) in
methanol 15 mL was stirred at room temperature for 16 h. Then the
solvent was evaporated and the residue was diluted with 10 mL
H2O and extracted with dichloromethane (20 mL ꢂ 3). The organic
layer was separated, dried with anhydrous Na2SO4 and evaporated
under reduced pressure. The residue was purified by silica gel
column chromatography (CH2Cl2: MeOH 100:2, v/v) to give 12b as
yellow oil: 0.15 g (48.4%, yield). IR (KBr, cmꢁ1): 3455, 2923, 2853,
(s,1H, TriazC3eH), 7.80 (s,1H, TriazC5eH), 6.78e7.60 (m, 6H, AreH),
5.30 (s, 1H, OH), 4.50 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.37 (br, 2H,
AreCH2), 3.00 (d, 1H, J ¼ 13.1 Hz, C3eHa), 2.82 (br, 2H, piperidin-2-
CH2), 2.80 (d, 1H, J ¼ 13.1 Hz, C3eHb), 2.12 (br, 1H, piperidin-4-CH),
1.98 (s, 3H, NCH3), 1.82 (br, 2H, piperidin-6-CH2), 1.57 (br, 2H,
piperidin-3-CH2), 1.44e1.47 (m, 2H, piperidin-5-CH2). HRMS (m/z):
calcd. for C24H27Cl2F2N5O: 509.1561; found: 509.1573.
6.1.11. 3-{[1-(3-Chlorobenzyl)piperidin-4-yl](methyl)amino]-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12g)
Yellow oil: 0.24 g (80.0%, yield). IR (KBr, cmꢁ1): 3429, 2943,
1616, 1520, 1462, 1273, 1138, 1049, 964. 1H NMR
d (ppm): 8.15e8.18
(m, 2H, AreH,TriazC3eH), 7.80 (s,1H, TriazC5eH), 6.78e7.59 (m, 6H,
AreH), 5.30 (s, 1H, OH), 4.50 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.51 (br,
2H, AreCH2), 3.01 (d, 1H, J ¼ 13.0 Hz, C3eHa), 2.82 (br, 2H, piper-
idin-2-CH2), 2.80 (d, 1H, J ¼ 13.0 Hz, C3eHb), 2.20 (br, 1H, piperidin-
4-CH), 1.98 (s, 3H, NCH3), 1.87 (br, 2H, piperidin-6-CH2), 1.58 (br, 2H,
piperidin-3-CH2), 1.25 (br, 2H, piperidin-5-CH2). HRMS (m/z): calcd.
for C24H28F2N6O3: 486.2191; found: 486.2177.
2856, 1617, 1500, 1467, 1272, 1137, 1049, 964. 1H NMR
d (ppm): 8.16
(s,1H, TriazC3eH), 7.80 (s,1H, TriazC5eH), 6.77e7.58 (m, 7H, AreH),
5.30 (s, 1H, OH), 4.50 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.42 (br, 2H,
AreCH2), 3.01 (d, 1H, J ¼ 14.1 Hz, C3eHa), 2.83 (br, 2H, piperidin-2-
CH2), 2.80 (d, 1H, J ¼ 14.1 Hz, C3eHb), 2.13 (br, 1H, piperidin-4-CH),
1.98 (s, 3H, NCH3), 1.84 (br, 2H, piperidin-6-CH2), 1.57e1.60 (m, 2H,
piperidin-3-CH2), 1.40e1.48 (m, 2H, piperidin-5-CH2). HRMS (m/z):
calcd. for C24H28ClF2N5O: 475.1950; found: 475.1963.
6.1.7. 3-{[1-(4-Bromobenzyl)piperidin-4-yl](methyl)amino]-2-(2,4-
difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12c)
Pale yellow solid: 0.25 g (75.8%, yield), mp 126e127 ꢀC. IR (KBr,
6.1.12. 3-{[1-(4-Cyanobenzyl)piperidin-4-yl](methyl)amino]-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12h)
Yellow oil: 0.13 g (46.4%, yield). IR (KBr, cmꢁ1): 3424, 2922, 2853,
cmꢁ1): 3436, 2924, 2854, 1610, 1492, 1466, 1270, 1136, 1059, 959. 1H
NMR
d (ppm): 8.16 (s, 1H, TriazC3eH), 7.79 (s, 1H, TriazC5eH),
6.77e7.57 (m, 7H, AreH), 5.30 (s, 1H, OH), 4.49 (d, 2H, J ¼ 14.1 Hz,
C1eHaHb), 3.37 (br, 2H, AreCH2), 3.00 (d, 1H, J ¼ 13.4 Hz, C3eHa),
2.82 (br, 2H, piperidin-2-CH2), 2.80 (d, 1H, J ¼ 13.4 Hz, C3eHb), 2.12
(br, 1H, piperidin-4-CH), 1.97 (s, 3H, NCH3), 1.80 (br, 2H, piperidin-
6-CH2), 1.47e1.55 (m, 2H, piperidin-3-CH2), 1.36e1.39 (m, 2H,
piperidin-5-CH2). HRMS (m/z): calcd. for C24H28BrF2N5O: 519.1445;
found: 519.1434.
1615, 1501, 1460, 1273, 1138, 1049, 963. 1H NMR
d (ppm): 8.17 (s, 1H,
TriazC3eH), 7.80 (s, 1H, TriazC5eH), 6.78e7.60 (m, 7H, AreH), 5.30
(s, 1H, OH), 4.50 (d, 2H, J ¼ 14.1 Hz, C1eHaHb), 3.47 (br, 2H,
AreCH2), 3.01 (d, 1H, J ¼ 13.0 Hz, C3eHa), 2.83 (br, 2H, piperidin-2-
CH2), 2.80 (d, 1H, J ¼ 13.0 Hz, C3eHb), 2.14 (br, 1H, piperidin-4-CH),
1.98 (s, 3H, NCH3), 1.86 (br, 2H, piperidin-6-CH2), 1.58 (br, 2H,
piperidin-3-CH2), 1.39e1.49 (m, 2H, piperidin-5-CH2). 13C NMR

W. Wang et al. / European Journal of Medicinal Chemistry 45 (2010) 6020e6026
6025
d
(ppm): 162.66, 158.85, 150.88, 144.76, 144.20, 132.06, 129.19,
104.11, 71.13, 62.95, 60.52, 58.50, 56.56, 53.08, 39.23, 28.73, 27.64,
19.10. HRMS (m/z): calcd. for C25H31F2N5O: 455.2497; found:
455.2779.
126.81, 118.87, 111.44, 110.88, 104.18, 71.15, 62.56, 62.14, 58.40,
56.46, 53.05, 39.20, 28.44, 27.47. HRMS (m/z): calcd. for
C25H28F2N6O: 466.2293; found: 466.2279.
6.1.18. 3-{[1-(4-Isopropylbenzyl)piperidin-4-yl](methyl)amino}-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12n)
Pale yellow solid: 0.14 g (46.7%, yield), mp 75e76 ꢀC. IR (KBr,
cmꢁ1): 3432, 2923, 2854, 1612, 1498, 1464, 1269, 1135, 1053, 960. 1H
6.1.13. 3-{[1-(3-Bromobenzyl)piperidin-4-yl](methyl)amino]-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12i)
Yellow oil: 0.23 g (69.7%, yield). IR (KBr, cmꢁ1): 3431, 2925,
2855, 1617, 1500, 1465, 1272, 1137, 1049, 963. 1H NMR
d
(ppm): 8.16
NMR d (ppm): 8.15 (s, 1H, TriazC3eH), 7.78 (s, 1H, TriazC5eH),
(s,1H, TriazC3eH), 7.80 (s,1H, TriazC5eH), 6.78e7.57 (m, 7H, AreH),
4.49 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.39 (br, 2H, AreCH2), 3.01 (d,
1H, J ¼ 13.7 Hz, C3eHa), 2.83 (br, 2H, piperidin-2-CH2), 2.80 (d, 1H,
J ¼ 13.7 Hz, C3eHb), 2.10 (br,1H, piperidin-4-CH),1.97 (s, 3H, NCH3),
1.82 (br, 2H, piperidin-6-CH2), 1.57 (br, 2H, piperidin-3-CH2),
1.40e1.50 (m, 2H, piperidin-5-CH2). HRMS (m/z): calcd. for
C24H28BrF2N5O: 519.1445; found: 519.1429.
6.77e7.56 (m, 7H, AreH), 5.30 (s, 1H, OH), 4.49 (d, 2H, J ¼ 14.1 Hz,
C1eHaHb), 3.49 (br, 1H, AreCH(CH3)2), 3.41 (br, 2H, AreCH2), 3.00
(d, 1H, J ¼ 13.7 Hz, C3eHa), 2.87e2.92 (m, 2H, piperidin-2-CH2),
2.81 (d, 1H, J ¼ 13.7 Hz, C3eHb), 2.12 (br, 1H, piperidin-4-CH), 1.97
(s, 3H, NCH3), 1.81 (br, 2H, piperidin-6-CH2), 1.55e1.58 (m, 2H,
piperidin-3-CH2), 1.41e1.48 (m, 2H, piperidin-5-CH2), 1.23e1.26 (m,
6H, eCH(CH3)2). HRMS (m/z): calcd. for C27H35F2N5O: 483.2810;
found: 483.2822.
6.1.14. 3-{[1-(2-Fluorobenzyl)piperidin-4-yl](methyl)amino]-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12j)
Yellow oil: 0.21 g (72.4%, yield). IR (KBr, cmꢁ1): 3432, 2943,
6.2. Flexible docking analysis
2858, 1689, 1498, 1454, 1273, 1136, 1084, 965. 1H NMR
d (ppm): 8.16
The 3D structures of the designed azoles were built by the
Builder module within Insight II 2000 software package. Then, the
flexible ligand docking procedure in the Affinity module within
Insight II was used to define the lowest energy position for the
substrate using a Monte Carlo docking protocol. The detailed
docking parameters were from our previous studies [19].
(s,1H, TriazC3eH), 7.78 (s,1H, TriazC5eH), 6.77e7.56 (m, 7H, AreH),
5.30 (s, 1H, OH), 4.49 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.53 (br, 2H,
AreCH2), 2.99 (d, 1H, J ¼ 13.7 Hz, C3eHa), 2.88 (br, 2H, piperidin-2-
CH2), 2.80 (d, 1H, J ¼ 13.7 Hz, C3eHb), 2.15 (br, 1H, piperidin-4-CH),
1.96 (s, 3H, NCH3), 1.84e1.89 (m, 2H, piperidin-6-CH2), 1.57e1.60
(m, 2H, piperidin-3-CH2), 1.42e1.50 (m, 2H, piperidin-5-CH2).
HRMS (m/z): calcd. for C24H28F3N5O: 459.2246; found: 459.2233.
Acknowledgement
6.1.15. 3-{[1-(4-Fluorobenzyl)piperidin-4-yl](methyl)amino]-2-
(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12k)
Yellow oil: 0.20 g (69.0%, yield). IR (KBr, cmꢁ1): 3428, 2925,
This work was supported by the National Natural Science
Foundation of China (Grant No. 30930107), Shanghai Rising-Star
Program (Grant No. 09QA1407000) and Shanghai Leading
Academic Discipline Project (Project No. B906).
2855, 1613, 1505, 1452, 1272, 1136, 1052, 963. 1H NMR
d (ppm): 8.16
(s,1H, TriazC3eH), 7.79 (s,1H, TriazC5eH), 6.78e7.57 (m, 7H, AreH),
5.30 (s, 1H, OH), 4.49 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.39 (br, 2H,
AreCH2), 3.00 (d, 1H, J ¼ 13.7 Hz, C3eHa), 2.82 (br, 2H, piperidin-2-
CH2), 2.79 (d, 1H, J ¼ 13.7 Hz, C3eHb), 2.12 (br, 1H, piperidin-4-CH),
1.97 (s, 3H, NCH3), 1.80 (br, 2H, piperidin-6-CH2), 1.56 (br, 2H,
piperidin-3-CH2), 1.37e1.40 (m, 2H, piperidin-5-CH2). HRMS (m/z):
calcd. for C24H28F3N5O: 459.2246; found: 459.2260.
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1614, 1497, 1460, 1270, 1138, 1047, 964. 1H NMR
d (ppm): 8.16 (s, 1H,
TriazC3eH), 7.79 (s, 1H, TriazC5eH), 6.77e7.56 (m, 7H, AreH), 4.49
(d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.80 (s, 3H, OCH3), 3.41 (br, 2H,
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1.97 (s, 3H, NCH3), 1.81 (br, 2H, piperidin-6-CH2), 1.41e1.47 (m, 2H,
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(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (12m)
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1616, 1499, 1461, 1272, 1138, 1048, 963. 1H NMR
d (ppm): 8.16 (s, 1H,
TriazC3eH), 7.78 (s, 1H, TriazC5eH), 6.79e7.57 (m, 7H, AreH), 5.29
(s, 1H, OH), 4.49 (d, 2H, J ¼ 14.2 Hz, C1eHaHb), 3.37 (br, 2H,
AreCH2), 2.99 (d, 1H, J ¼ 13.8 Hz, C3eHa), 2.83e2.86 (m, 2H,
piperidin-2-CH2), 2.81 (d, 1H, J ¼ 13.8 Hz, C3eHb), 2.32 (s, 3H,
AreCH3), 2.13 (br, 1H, piperidin-4-CH), 1.96 (s, 3H, NCH3), 1.82e1.84
(m, 2H, piperidin-6-CH2), 1.45 (br, 2H, piperidin-3-CH2), 1.32e1.38
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144.70, 137.29, 136.65, 130.17, 129.55, 129.38, 126.90, 125.40, 111.39,
d (ppm): 163.63, 159.01, 150.93,

6026
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