Discovery of highly potent triazole antifungal derivatives by heterocycle-benzene bioisosteric replacement

Article abstract of DOI:10.1016/j.ejmech.2013.04.025

On the basis of our previously discovered triazole antifungal lead compounds, heterocycle-benzene bioisosteric replacement was used to improve their pharmacokinetic profile. The designed new triazole derivatives have good antifungal activity toward a wide range of pathogenic fungi. Their binding mode with the target enzyme was clarified by molecular docking. The MIC value of the highly potent compound 8f against Candida albicans, Candida tropicalis, and Cryptococcus neoformans is 0.016 μg/mL, 0.004 μg/mL, and 0.016 μg/mL, respectively. Moreover, preliminary pharmacokinetic studies revealed that it showed improved oral absorption as compared to the lead compound iodiconazole and deserved for further evaluations.

Full text of DOI:10.1016/j.ejmech.2013.04.025

European Journal of Medicinal Chemistry 64 (2013) 16e22
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Discovery of highly potent triazole antifungal derivatives by
heterocycle-benzene bioisosteric replacement
Zhigan Jiang 1, Yan Wang 1, Wenya Wang, Shengzheng Wang, Bo Xu, Guorong Fan,
**
Guoqiang Dong, Yang Liu, Jianzhong Yao, Zhenyuan Miao, Wannian Zhang ,
*
Chunquan Sheng
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:
On the basis of our previously discovered triazole antifungal lead compounds, heterocycle-benzene
bioisosteric replacement was used to improve their pharmacokinetic profile. The designed new tri-
azole derivatives have good antifungal activity toward a wide range of pathogenic fungi. Their binding
mode with the target enzyme was clarified by molecular docking. The MIC value of the highly potent
Received 11 October 2012
Received in revised form
27 February 2013
Accepted 10 April 2013
Available online 17 April 2013
compound 8f against Candida albicans, Candida tropicalis, and Cryptococcus neoformans is 0.016
mg/mL,
0.004 g/mL, and 0.016 g/mL, respectively. Moreover, preliminary pharmacokinetic studies revealed
m
m
that it showed improved oral absorption as compared to the lead compound iodiconazole and deserved
for further evaluations.
Keywords:
Triazole
Antifungal activity
Bioisosteric replacement
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
generation of triazole antifungal agents. Numerous efforts have
been made to design and synthesize novel antifungal triazoles and
The incidence and mortality of invasive fungal infections are
rising dramatically due to the increase in the number of immuno-
compromised or immunosuppressed individuals including patients
receiving cancer chemotherapy or organ transplantation, and pa-
tients infected with human immunodeficiency virus [1,2]. The three
most common species of human fungal pathogens are Candida
albicans (mortality rate: 20%e40%) [3], Cryptococcus neoformans
(mortality rate: 20%e70%) [4] and Aspergillus fumigatus (mortality
rate: 50%e90%) [3]. Clinically, antifungal agents for the treatment of
invasive fungal infections include polyenes (e.g. amphotericin B)
[5], triazoles (e.g. fluconazole and itraconazole, see Fig. 1) [6] and
candins (e.g. caspofungin and micafungin) [7]. Among them, tri-
azoles are the most widely used antifungal agents because of their
high therapeutic index, broad spectrum of activity and more
favorable safety profile [8]. However, broad application of them also
led to severe drug resistance, which has significantly reduced their
clinical efficacy. Therefore, it is highly desirable to develop new
the progress in this field can be found in recent reviews [9e11]. Two
of them, isavuconazole and albaconazole (Fig. 1), are candidate new
drugs under clinical trials.
Triazole antifungal agents act by competitive inhibition of lan-
osterol 14a-demethylase (CYP51), a key enzyme in sterol biosyn-
thesis of fungi [12]. In our previous studies, three-dimensional (3D)
models of fungal CYP51s were constructed by homology modeling
[13e16]. Moreover, highly active triazole analogs were designed
and synthesized by our group [15,17e21]. Among them, triazole
derivatives shown in Fig. 2 exhibited excellent in vitro activity with
broad spectrum [17]. However, these triazoles showed low oral
bioavailability due to poor water solubility. The heterocycle-
benzene bioisosteric replacement is proved to be
a useful
approach to improve the solubility and pharmacokinetic profile of
the lead compound [22e24]. Herein, a series of heterocyclic analogs
of the triazoles in Fig. 2 were designed and synthesized. Several
target compounds showed excellent antifungal activity with
improved oral absorption, which present promising leads for the
development of novel antifungal agents.
* Corresponding author. Tel./fax: þ86 21 81871239.
** Corresponding author. Tel./fax: þ86 21 81870243.
E-mail addresses: zhangwnk@hotmail.com (W. Zhang), shengcq@hotmail.com
(C. Sheng).
2. Chemistry
The first step of chemical synthesis is to prepare various chlor-
omethyl heterocycles or bromomethyl heterocycles according to
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.025

Z. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 16e22
17
CH3
O
N
CH3
1
OH
3
OH
N
N
OH
N
N
N
N
N
F
N
N
N
N
2
N
N
N
N
F
F
Cl
F
F
F
F
Fluconazole
Albaconazole
Voriconazole
Cl
N
O
CH3
N
CN
CH3
CH3
OH
O
N
O
OH
F
N
H
N
N
N
F
I
N
CH3
N
H3C
O
N
N
S
F
F
Isavuconazole
Iodiconazole
Fig. 1. Chemical structures of representative triazole antifungal agents.
the reported procedures [25,26]. Then, the heterocyclic in-
termediates were reacted with methylamine or piperazine to give
various side chains 5 and 7 (Scheme 1). Finally, the target com-
pounds were obtained as racemates by the ring-open reaction of
the oxirane intermediate 4 [18] with intermediates 5 and 7 in the
presence of EtOH and a base (Et3N or K2CO3). The isomers of
compound 8f were obtained by chiral HPLC.
4. Results and discussion
4.1. Design rationale
In our previous studies, we reported a series of new azoles with
tertiary amine or piperazine side chains (Fig. 2) [17]. These com-
pounds showed good antifungal activity with a broad spectrum.
Among them, iodiconazole was developed as a topical antifungal
agent for the treatment of dermatomycosis, which is currently
under phase III clinical trial [27e29]. Although iodiconazole has
excellent in vitro antifungal activity, its oral bioavailability was low
mainly because of its poor water solubility. Thus, it is highly
desirable to improve its oral absorption and develop novel orally
active antifungal agent. In the present investigation, the terminal
phenyl group of the lead structures was replaced by various het-
erocyclic groups to afford target compounds 6aee and 8aef. This
type of bioisosteric replacement was based on the following ra-
tionales: (1) the solubility and pharmacokinetic profiles of the
compounds should be improved. The heterocycle-benzene ex-
change is a well-validated approach for the improvement of ADME
properties [22]. In particular, this method has been successfully
used in the discovery of novel triazole antifungal agents. For
example, the replacement of the triazole ring of fluconazole with
fluoropyrimidine led to the discovery of voriconazole. Moreover,
heterocyclic group seems to be necessary in many marketed or
emerging triazole antifungal agents (e.g. voriconazole, isavucona-
zole, albaconazole, see Fig. 1). The calculated LogP values of com-
pounds 6aee and 8aef are in the range of 1.92e3.09, suggesting
their potential as orally active drugs [30]. (2) Key interactions be-
tween the triazole lead and CYP51 should be retained for the bio-
isosteric replacement. Molecular docking studies revealed that the
C3 side chain of the lead compounds (Fig. 2) mainly formed hy-
drophobic and van der Waals interactions with the S4 pocket of C.
albicans CYP51 (CACYP51) [15]. Analysis of the docking model
indicated that five-member or six-member heterocycles and ben-
zoheterocycles can be well accommodated in the active site of
CACYP51.
3. Microbiology
In vitro antifungal activity was measured according to the Na-
tional Committee for Clinical Laboratory Standards (NCCLS) rec-
ommendations. Serial dilution method in 96-well microtest plate
was used to determine the minimum inhibitory concentration
(MIC) of the target compounds [18]. Tested fungal strains were
obtained from the ATCC or clinical isolates. Briefly, the MIC value
was defined as the lowest concentration of tested compounds that
resulted in a culture with turbidity less than or equal to 80% inhi-
bition when compared with the growth of the control. Tested
compounds were dissolved in DMSO serially diluted in growth
medium. The yeasts were incubated at 35 ꢀC and the dermato-
phytes at 28 ꢀC. Growth MIC was determined at 24 h for Candida
species, at 72 h for C. neoformans, and at 7 days for A. fumigatus.
CH3
OH
OH
R
N
N
N
N
N
N
N
N
N
F
3
3
1
2
1
R
2
F
F
F
Lead Compounds
J Med Chem, 2006, 49: 2512-2525.
Improve Solubility
Improve ADME
CH3
OH
OH
N
N
N
N
N
N
N
N
F
Heterocycle
3
1
3
1
2
Heterocycle
2
N
F
4.2. In vitro antifungal activities
In vitro antifungal activity of the target compounds is reported in
Table 1. Fluconazole was used as a reference drug. In general, most
of the target compounds showed moderate to good inhibitory
F
F
Fig. 2. Design rationale of the target compounds.

18
Z. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 16e22
O
F
O
N
N
Cl
O
F
N
N
a
b
c
N
N
F
.
F
CH3SO3H
F
F
F
2
F
3
1
4
OH
CH3
N
F
N
N
R
CH3
HN
N
d
f
CH2Cl
Heterocycle
Heterocycle
5
F
R = Heterocycle
6a-e
6a R =
6b R =
6c R =
S
O
N
N
6d R =
6e R
=
N
N
H
OH
N
N
N
F
N
R
e
N
f
CH2Cl
HN
N
Heterocycle
Heterocycle
7
F
R = Heterocycle
8a-f
8a R =
8b R =
8c R =
S
N
N
N
N
S
N
8f R =
8d R =
8e R
=
N
H
O
Reagents and conditions a. ClCH2COCl, AlCl3, CH2Cl2, 40 , 3h, 50%; b. triazole,
K2CO3, CH2Cl2, rt, 24h, 70.0%; c. (CH 3)3SOI, NaOH, toluene, 60 , 3h, 62.3%; d.
CH3NH2, Et3N, EtOH, r.t., 12h, ~ 100%; e. piperazine, Et 3N, nBuOH, r.t.~80 , 5h,
95.5%~98.0%; f. 4, Et3N, EtOH, 80 , 9h, 38.8% ~ 76.6%.
Scheme 1.
activity against the tested fungi, especially for Candida species and
C. neoformans. Candida species rank the fourth among the causes of
bacterial/fungal nosocomial infectious diseases. The MIC range for
Table 1
C. albicans was 4
and 8f were more active than fluconazole. Among them, compound
g/mL) showed the best activity against C. albicans.
mg/mLe0.016 mg/mL. Compounds 6b, 8a, 8c, 8e
Calculated LogP value and in vitro antifungal activities of the target compounds
(MIC80, m
g mLꢁ1).a
8f (MIC ¼ 0.016
m
Compd.
LogPb
C. alb.
C. tro.
C. neo.
T. rub.
A. fum.
The target compounds showed better inhibitory activity against
Candida tropicalis than C. albicans with MIC values in the range of
1 mg/mLe0.004 mg/mL. Moreover, all of them were more potent
against C. tropicalis than fluconazole. Particularly, compounds 8e
and 8f showed excellent inhibitory activity with MIC value of
0.004 mg/mL. C. neoformans is the leading cause for cryptococcal
meningitis, which kills more than 650,000 people per year world-
wide. Good activity of the target compounds was also observed for
C. neoformans and most compounds had MIC values ranging from
6a
6b
6c
6d
6e
8a
8b
8c
2.60
2.04
3.26
1.92
2.57
2.56
3.22
1.88
2.52
2.54
3.09
e
1
0.125
0.016
1
0.0625
0.25
0.016
0.016
1
0.016
0.0625
0.25
0.25
16
0.25
0.0625
1
1
1
4
4
>64
4
0.25
1
16
64
>64
>64
>64
16
2
16
>64
0.25
64
0.0625
4
1
4
0.0625
1
0.0625
4
0.0625
0.016
0.25
8d
8e
8f
1
16
64
0.004
0.004
4
0.0625
0.016
2
0.25
0.0625
1
1.0
remaining compounds were 2e125 fold more active than flucon-
g/mL) revealed the best
mg/mLe0.016 mg/mL. Except compounds 6e and 8d, the
Fluconazole
>64
azole. Compounds 6a and 8f (MIC ¼ 0.016
m
a
Abbreviations: C. alb. Candida albicans; C. tro. Candida tropicalis; C. neo. Crypto-
coccus neoformans; T. rub. Trichophyton rubrum; A. fum. Aspergillus fumigatus.
activity against C. neoformans. In contrast, the target compounds
showed decreased activity against Trichophyton rubrum. Only
b
The LogP values were calculated by Discovery Studio 3.0 software package.

Z. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 16e22
19
compounds 8e (MIC ¼ 0.25
m
g/mL) and 8f (MIC ¼ 0.0625
m
g/mL)
CACYP51 active site, but their interactions with CACYP51 were
different. The furan ring of compound 6b formed face to edge
were 4e16 fold more potent than fluconazole (MIC ¼ 1
m
g/mL). A.
pep
fumigatus is one of the most common causes of mold infections of
humans with high mortality. Fluconazole is inactive against
A. fumigatus, while several target compounds showed moderate to
good activity (MIC range: 0.25
compound 8e was the most active one with MIC value of 0.25
mL. Among the synthesized triazoles, compounds 8e and 8f
showed excellent antifungal activities with a broad spectrum,
which represent promising candidates for further pharmacological
and pharmacokinetic evaluations. In order to further investigate
the influence of the chirality on the antifungal activity, isomers of
compound 8f were prepared by chiral HPLC. The MIC values for
interaction with Tyr118 and hydrophobic interactions with Phe380
and Leu121. The side chain of compound 6b is longer than that of
compound 8f. The piperazine group formed hydrophobic and van
der Waals interactions with Tyr118 and Met508. The terminal
benzothiazole group interacts with surrounding residues Leu121,
Phe233, Phe380, and Met508 through hydrophobic contacts.
Because compound 8f (GOLD fitness score: 81.92) had larger
docking scores than compound 6b (GOLD fitness score: 69.23),
compound 8f might have higher binding affinity with CACYP51 and
thus showed better inhibitory activity against C. albicans.
m
g/mLe16
mg/mL). In particular,
mg/
isomer (ꢁ)-8f were lower than 0.016
C. neoformans, whereas the MIC values for isomer (þ)-8f were
0.0625 g/mL and 0.0625 g/mL, respectively. The results indicated
mg/mL against C. albicans and
4.4. Preliminary pharmacokinetic profile of compound 8f
m
m
In order to investigate the pharmacokinetic profile of the
designed compounds, compound 8f was orally administrated to SD
rats at the dose of 15 mg/kg and iodiconazole was used as a refer-
ence drug. Blood sample from orbital venous plexus was obtained
at different time point and subjected to LC/MS/MS analysis
(detailed analysis and experimental protocols to be published).
Preliminary pharmacokinetic parameters revealed that compound
8f had better oral absorption than iodiconazole, which further
validated the feasibility of the design rationale. The Cmax and AUC0e
that isomer (ꢁ)-8f had better antifungal activities than isomer
(þ)-8f.
4.3. Structureeactivity relationships and molecular docking
From the antifungal activity data, preliminary SARs can be ob-
tained. In general, the amine linker was important for the antifungal
activities. Substituted piperazine derivatives were comparable or
superior to the corresponding N-methyl derivatives. In the N-methyl
derivatives, the furan derivative 6b showed the best antifungal ac-
tivity. Moreover, the furan, thiophene or pyridine group was more
favorable for the antifungal activity than the benzoheterocycles
(such as benzimidazole and quinoline). For the substituted pipera-
zine derivatives, the SAR of the heterocyclic substitutions was
different. Two benzoheterocyclic derivatives, 8e (benzoxazole) and
8f (benzothiazole), showed the best antifungal activity. Decreased
antifungal activity was observed for the benzimidazole derivative
8d. In addition, the thiophene group (compound 8a) was more
favorable than the pyridine group (compound 8c).
In order to investigate the binding mode of the designed com-
pounds with CYP51, two highly potent compounds, 6b and 8f, were
docked into the homologous model of CACYP51 [13,16]. As shown
in Fig. 3, the triazole ring of compounds 6b and 8f interacted with
the heme group through the formation of a coordination bond with
the iron atom. Their difluorophenyl group formed hydrophobic
interactions with Phe126, Met306, and Tyr132. The side chain of
the two compounds was extended into the S4 pocket of the
value of compound 8f were 508.25 ꢂ 101.25 ng/mL and
N
1421.8 ꢂ 406.98 ng h/mL, respectively, while the two parameters
for iodiconazole was
297.11
ꢂ
112.73
ng/mL and
545.79
ꢂ
177.96 ng h/mL. The half-time of compound 8f
(Tmax ¼ 0.8 ꢂ 0.2 h, T1/2 ¼ 1.27 ꢂ 0.5 h) was similar to that of
iodiconazole (Tmax ¼ 0.9 ꢂ 0.1 h, T1/2 ¼ 1.38 ꢂ 0.91 h). Further
pharmacokinetic and pharmacological evaluations are in progress.
5. Conclusion
Heterocycle-benzene exchange was used to improve the phar-
macokinetic profile of our previously identified triazole antifungal
agents. The designed triazole derivatives showed good antifungal
activity with a broad spectrum. The binding mode of them was
analyzed by molecular docking. Several compounds, such as 8e and
8f, showed significantly higher antifungal activity than fluconazole,
which represent promising leads for the development of
novel antifungal agents. Preliminary pharmacokinetic profile of
compound 8f was consistent with the design rationale and it
Fig. 3. The binding mode of compound 6b (A) and 8f (B) in the active site of CACYP51.

20
Z. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 16e22
showed better oral absorption than the lead compound
iodiconazole.
75.80, 62.75, 57.00, 56.20, 43.90. IR (neat) 3156, 3103, 2839, 2788,
1613, 1518, 1500, 1452, 1279, 1133, 1126, 1142, 1037, 854, 720, 700,
678 cmꢁ1. ESI-MS (m/z): 365.38 [M þ 1].
6. Experimental protocols
6.1.4. 2-(2,4-Difluorophenyl)-1-((furan-2-ylmethyl)(methyl)
amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6b)
6.1. General procedure for the synthesis of compounds
Yellow oil: 1.14 g, yield 65.3%. 1H NMR (500 MHz, CDCl3, TMS):
Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker 300 or 500 spectrometer with TMS as an internal standard
and CDCl3 as solvent. Chemical shifts (d values) and coupling con-
d
2.17 (s, 3H), 2.80 (d, J ¼ 13.9 Hz, 1H), 2.90 (d, J ¼ 14.9 Hz, 1H), 3.53
(d, J ¼ 14.0 Hz, 2H), 4.46 (d, J ¼ 14.3 Hz,1H), 4.52 (d, J ¼ 14.3 Hz, 1H),
5.64 (s, 1H), 6.18e7.56 (m, 6H), 7.71 (s, 1H), 8.27 (s, 1H). 13C NMR
stants (J values) are given in ppm and Hz, respectively. ESI mass
spectra were performed on an API-3000 LCeMS spectrometer. IR
was performed on Thermo FT-IR (Nicolet 380). Melting points were
carried out on OptiMelt MPA100. TLC analysis was carried out on
silica gel plates GF254 (Qingdao Haiyang Chemical, China). Silica gel
column chromatography was performed with Silica gel 60 G
(Qingdao Haiyang Chemical, China). Commercial solvents were
used without any pretreatment. The conditions for chiral separa-
(125 MHz, CDCl3): d 162.60, 158.92, 152.35, 151.80, 142.75, 130.40,
130.32, 126.45, 111.10, 110.60, 109.10, 104.15, 75.20, 62.15, 55.80,
54.70, 44.08. ESI-MS (m/z): 349.18 [M þ 1].
6.1.5. 2-(2,4-Difluorophenyl)-1-(methyl(quinolin-2-ylmethyl)
amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6c)
Pale yellow oil: 1.13 g, yield 55.3%. 1H NMR (500 MHz, CDCl3,
TMS):
d
2.21 (s, 3H), 2.89 (d, J ¼ 14.0 Hz, 1H), 3.24 (d, J ¼ 14.0 Hz,
tion were as follows: Chiralpak AD-3 50 ꢃ 4.6 mm I.D.; 3
m
m Mobile
1H), 3.94 (d, J ¼ 15.7 Hz, 2H), 4.48 (d, J ¼ 14.0 Hz, 1H), 4.59 (d,
J ¼ 14.0 Hz, 1H), 5.30 (s, 1H), 6.78e8.14 (m, 9H), 7.57 (s, 1H), 8.16 (s,
phase: methanol (0.05% DEA) in CO2 from 5% to 40%; flow rate:
4 mL/min; wavelength: 220 nm.
1H). 13C NMR (125 MHz, CDCl3):
d 161.42, 158.50, 157.83, 150.70,
147.01, 144.60, 137.08, 130.41, 130.06, 128.31, 127.64, 127.26, 126.65,
126.1, 120.45, 111.42, 103.92, 73.84, 64.36, 62.26, 56.75, 45.36. ESI-
MS (m/z): 410.29 [M þ 1].
6.1.1. Chemical synthesis of N-methyl-1-(thiophen-2-yl)
methanamine (5a)
To a cooled solution of methylamine in EtOH (50 mL) at 0 ꢀC was
added dropwise a solution of 2-(chloromethyl)thiophene (3.32 g,
0.025 mol) in EtOH (20 mL). Then the reaction mixture was stirred
at room temperature overnight. After the reaction was complete,
the solvent was removed under reduced pressure to provide a
yellow solid, which was used in next step without further purifi-
6.1.6. 2-(2,4-Difluorophenyl)-1-(methyl(pyridin-2-ylmethyl)
amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6d)
Yellow solid: 1.08 g, yield 60.1%, mp 97e98 ꢀC. 1H NMR
(500 MHz, CDCl3, TMS):
d
2.13 (s, 3H), 2.83 (d, J ¼ 13.95 Hz, 1H), 3.17
(d, J ¼ 13.95 Hz, 1H), 3.66 (d, J ¼ 14.6 Hz, 1H), 3.75 (d, J ¼ 14.6 Hz,
1H), 4.43 (d, J ¼ 14.1 Hz, 1H), 4.57 (d, J ¼ 14.1 Hz, 1H), 6.79e7.68 (m,
7H), 7.69 (s, 1H), 8.13 (s, 1H), 8.53 (m, 1H). 13C NMR (125 MHz,
cation. 1H NMR (300 MHz, CDCl3, TMS):
d 2.48 (s, 3H), 3.95 (s, 2H),
6.96e7.23 (m, 3H).
The synthetic procedure for other N-methyl heterocyclic analogs
was similar to the synthesis of compound 5a.
CDCl3): d 162.35, 159.85, 159.10, 150.75, 148.75, 145.55, 137.05,
130.25, 126.75, 122.65, 122.30, 111.05, 104.00, 75.05, 64.15, 63.05,
56.05, 42.25. IR (neat) 3065, 3033, 3019, 2845, 2807, 2788, 1611,
1597, 1573, 1505, 1460, 1432, 1420, 1272, 1137, 963, 760, 680,
655 cmꢁ1. ESI-MS (m/z): 360.31 [M þ 1].
6.1.2. Chemical synthesis of 1-(thiophen-2-ylmethyl)piperazine
(7a)
A solution of 2-(chloromethyl)thiophene (3.0 g, 0.023 mol) in n-
butanol (30 mL) was added dropwise to the suspension of anhy-
drous piperazine (12.0 g, 0.94 mmol), K2CO3 (4.0 g, 29.0 mmol) and
n-butanol (30 mL) at 0 ꢀC. The resulting mixture was stirred at room
temperature for 3 h and then heated to 80 ꢀC for another 2 h. The
reaction mixture was cooled down and filtered. After evaporation,
the resulting residue was partitioned between EtOAc and H2O. The
organic layer was dried and evaporated to give a yellow oil, which
was used in next step reaction without further purification (4.0 g,
6.1.7. 1-(((1H-Benzo[d]imidazol-2-yl)methyl)(methyl)amino)-2-
(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6e)
Yellow solid: 0.91 g, yield 45.5%, mp 101e102 ꢀC. 1H NMR
(500 MHz, CDCl3, TMS):
d
2.20 (s, 3H), 2.96 (d, J ¼ 13.9 Hz, 1H), 3.17
(d, J ¼ 13.9 Hz, 1H), 3.47 (d, J ¼ 14.5 Hz, 1H), 3.66 (d, J ¼ 14.5 Hz, 1H),
4.57 (d, J ¼ 14.0 Hz, 2H), 6.75e7.76 (m, 7H), 7.72 (s, 1H), 8.08 (s, 1H).
13C NMR (125 MHz, CDCl3):
d 162.10, 159.65, 152.75, 150.65, 145.75,
143.00, 134.35, 130.03, 126.15, 122.25, 121.65, 118.45, 111.50, 111.30,
104.25, 75.10, 63.00, 56.45, 56.02, 44.60. IR (neat) 3400, 3123, 2954,
1615, 1497, 1455, 1418, 1271, 1135, 964, 849, 743, 676 cmꢁ1. ESI-MS
(m/z): 399.29 [M þ 1].
yield 95.5%). 1H NMR (300 MHz, CDCl3, TMS):
d 2.48 (m, 4H), 2.84
(m, 4H), 3.60 (s, 2H), 6.83 (d, J ¼ 7.5 Hz, 1H), 6.86 (t, J ¼ 7.5 Hz, 1H),
7.15 (t, J ¼ 7.5 Hz, 1H).
The synthetic procedure for other piperazine analogs was
similar to the synthesis of compound 7a.
6.1.8. 2-(2,4-Difluorophenyl)-1-(4-(thiophen-2-ylmethyl)
piperazin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8a)
6.1.3. 2-(2,4-Difluorophenyl)-1-(methyl(thiophen-2-ylmethyl)
amino)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6a)
A solution of epoxide 4 (1.67 g, 0.005 mol), 1-(thiophen-2-
ylmethyl)piperazine (1.09 g, 0.006 mol) and triethylamine
(2.0 mL) in anhydrous EtOH (20 mL) were heated to reflux for 8 h.
The solvent was evaporated under reduced pressure. The residue
was purified by silica gel column chromatography (eluents: pe-
troleum ether:EtOAc ¼ 1:1, v/v) to give a yellow oil (1.22 g, yield
A solution of epoxide 4 (1.67 g, 0.005 mol), intermediate 5a
(0.76 g, 0.006 mol) and triethylamine (3.0 mL) in EtOH (30 mL)
were heated to reflux for 9 h. The solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluents: petroleum ether:EtOAc ¼ 1:1, v/v) to
give a pale yellow solid (0.94 g, yield 51.8%): mp 86e87 ꢀC. 1H NMR
58.2%). 1H NMR (500 MHz, CDCl3, TMS):
d 2.36 (s, 8H), 2.65 (d,
J ¼ 13.5 Hz, 1H), 3.07 (d, J ¼ 13.5 Hz, 1H), 3.65 (dd, J1 ¼ 13.8 Hz,
J2 ¼ 21.9 Hz, 2H), 4.50 (dd, J1 ¼14.3 Hz, J2 ¼ 18.0 Hz, 2H), 5.30 (s,1H),
6.77e7.54 (m, 6H), 7.77 (s, 1H), 8.14 (s, 1H). 13C NMR (125 MHz,
(500 MHz, CDCl3, TMS):
d
2.10 (s, 3H), 2.80 (d, J ¼ 13.5 Hz, 1H), 3.08
(d, J ¼ 13.5 Hz, 1H), 3.62 (d, J ¼ 14.0 Hz, 2H), 4.46 (d, J ¼ 14.2 Hz, 1H),
4.52 (d, J ¼ 14.2 Hz, 1H), 5.20 (s, 1H), 6.76e7.61 (m, 6H), 7.75 (s, 1H),
CDCl3):
d 162.04, 159.61, 151.01, 144.64, 141.12, 129.25, 126.41,
8.10 (s, 1H). 13C NMR (125 MHz, CDCl3):
d
161.90, 159.85, 150.80,
126.23, 126.11, 125.08, 111.61, 104.26, 71.83, 62.16, 56.87, 56.38,
145.20, 142.25,130.02,126.75,126.30, 125.90, 125.58, 111.00,104.35,
54.27, 52.68. ESI-MS (m/z): 420.24 [M þ 1].

Z. Jiang et al. / European Journal of Medicinal Chemistry 64 (2013) 16e22
21
6.1.9. 2-(2,4-Difluorophenyl)-1-(4-(quinolin-2-ylmethyl)piperazin-
6.1.15. (ꢁ)-1-(4-(Benzo[d]thiazol-2-ylmethyl)piperazin-1-yl)-2-
(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol ((ꢁ)-8f)
1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8b)
Yellow oil: 1.45 g, yield 62.3%. 1H NMR (500 MHz, CDCl3, TMS):
1H NMR (500 MHz, CDCl3):
d 2.33e2.63 (m, 8H), 2.70 (d,
d
2.34e2.44 (m, 8H), 2.65 (d, J ¼ 13.5 Hz, 1H), 3.09 (d, J ¼ 13.5 Hz,
J ¼ 13.6 Hz, 1H), 3.13 (d, J ¼ 13.6 Hz, 1H), 3.87e3.97 (m, 2H), 4.49e
4.60 (m, 2H), 5.23 (br s, 1H), 6.77e6.86 (m, 2H), 7.35e7.41 (m, 1H),
7.43e7.49 (m, 1H), 7.53e7.60 (m, 1H), 7.81 (s, 1H), 7.86 (d, J ¼ 8.0 Hz,
1H), 7.97 (d, J ¼ 8.0 Hz, 1H), 8.17 (s, 1H). IR (neat) 3425, 3130, 2934,
2820,1615,1596, 1510,1495,1422,1360,1270,1153, 1134, 1120,1011,
1H), 3.78 (dd, J1 ¼13.8 Hz, J2 ¼ 17.4 Hz, 2H), 4.51 (t, J ¼ 15.6 Hz, 2H),
5.30 (s, 1H), 6.75e8.06 (m, 9H), 8.08 (s, 1H), 8.11 (s, 1H). 13C NMR
(125 MHz, CDCl3):
d 162.70, 159.52, 159.04, 151.00, 147.61, 144.65,
136.35, 129.39, 129.30, 129.04, 127.48, 127.32, 126.30, 126.19, 120.99,
111.60, 104.21, 71.88, 64.93, 62.22, 56.35, 54.28, 53.31. ESI-MS (m/z):
465.30 [M þ 1].
964, 854, 756, 688, 655 cmꢁ1. ESI-MS (m/z): 470.20 [M þ 1].
20
[a
]
¼ ꢁ16.0 (C ¼ 0.1, DMF), 97.2% ee.
D
6.1.10. 2-(2,4-Difluorophenyl)-1-(4-(pyridin-2-ylmethyl)piperazin-
1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8c)
6.2. Molecular docking
Pale yellow oil: 1.24 g, yield 60.1%. 1H NMR (500 MHz, CDCl3,
Our previous study indicated that GOLD [31] is an accurate
method to dock CYP51 inhibitors [8]. Homology model of CACYP51
[15] from our group was used for molecular docking and the docking
parameters were defined the same with our previous reports [8].
TMS):
d
2.30e2.39 (m, 8H), 2.65 (d, J ¼ 13.5 Hz, 1H), 3.08 (d,
J ¼ 13.5 Hz, 1H), 3.62 (ded, J1 ¼ 13.5 Hz, J2 ¼ 17.7 Hz, 2H), 4.51 (t,
J ¼ 4.4 Hz, 2H), 6.75e7.78 (m, 7H), 7.92 (s, 1H), 8.15 (s, 1H). 13C NMR
(125 MHz, CDCl3):
d 162.03, 160.69, 158.15, 151.05, 149.33, 144.67,
136.36, 129.35, 126.26, 123.19, 122.10, 111.64, 104.26, 71.87, 64.37,
62.26, 56.38, 54.25, 53.30. ESI-MS (m/z): 415.10 [M þ 1].
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant 30930107, 81222044), Key Project of
Science and Technology of Shanghai (No.10431902100), the 863 Hi-
Tech Program of China (Grant 2012AA020302), Shanghai Rising-
Star Program (grant 12QH1402600) and Shanghai Municipal
Health Bureau (Grant XYQ2011038).
6.1.11. 1-(4-((1H-Benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-2-
(2,4-difluoro phenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8d)
Yellow solid: 1.04 g, yield 45.7%, 134e135 ꢀC. 1H NMR (500 MHz,
CDCl3, TMS):
d
2.42e2.47 (m, 8H), 2.66 (d, J ¼ 13.5 Hz, 1H), 3.09 (d,
J ¼ 13.5 Hz, 1H), 3.80 (dd, J1 ¼ 14.7 Hz, J2 ¼ 15.6 Hz, 2H), 4.52 (dd,
J1 ¼14.4 Hz, J2 ¼ 18.6 Hz, 2H), 6.75e7.79 (m, 7H), 7.81 (s, 1H), 8.17 (s,
1H). 13C NMR (125 MHz, CDCl3):
d 163.25, 160.80, 158.20, 152.00,
References
151.05, 145.25, 130.35, 126.45, 121.80, 110.10, 104.10, 75.00, 64.05,
56.00, 54.35, 53.10. IR (neat) 3280, 3053, 2702, 1559, 1385, 1305,
1182, 1050, 909, 705 cmꢁ1. ESI-MS (m/z): 454.24 [M þ 1].
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6.1.12. 1-(4-(Benzo[d]oxazol-2-ylmethyl)piperazin-1-yl)-2-(2,4-
difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8e)
Brown oil: 1.64 g, yield 72.1%. 1H NMR (500 MHz, CDCl3, TMS):
d
2.43e2.49 (m, 8H), 2.66 (d, J ¼ 13.2 Hz, 1H), 3.09 (d, J ¼ 13.2 Hz,
1H), 3.82 (ded, J1 ¼14.4 Hz, J2 ¼ 15.6 Hz, 2H), 4.48 (dd, J1 ¼13.8 Hz,
J2 ¼ 17.1 Hz, 2H), 5.27 (s, 1H), 6.76e7.72 (m, 7H), 7.78 (s, 1H), 8.14 (s,
1H). 13C NMR (125 MHz, CDCl3):
d 162.67, 162.35, 159.55, 1051.06,
150.87, 144.63, 140.89, 129.34, 126.14, 125.16, 124.41, 120.08, 111.70,
110.73, 104.30, 72.02, 62.25, 60.37, 55.02, 54.09, 53.05. ESI-MS (m/
z): 455.10 [M þ 1].
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difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8f)
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CDCl3, TMS):
d
2.43e2.54 (m, 8H), 2.68 (d, J ¼ 13.2 Hz, 1H), 3.12 (d,
J ¼ 13.2 Hz, 1H), 3.90 (dd, J1 ¼ 15.0 Hz, J2 ¼ 16.5 Hz, 2H), 4.52 (dd,
J1 ¼14.1 Hz, J2 ¼ 17.7 Hz, 2H), 5.31 (s, 1H), 6.76e7.96 (m, 7H), 7.79 (s,
1H), 8.15 (s, 1H). 13C NMR (125 MHz, CDCl3):
d 171.41, 162.21, 159.76,
153.23, 151.10, 144.68, 135.34,129.36, 126.28, 125.88, 124.95,122.85,
121.66, 111.60, 104.30, 72.02, 62.22, 60.00, 56.29, 54.29, 53.30. ESI-
MS (m/z): 471.30 [M þ 1].
6.1.14. (þ)-1-(4-(Benzo[d]thiazol-2-ylmethyl)piperazin-1-yl)-2-
(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol ((þ)-8f)
1H NMR (500 MHz, CDCl3):
d 2.32e2.62 (m, 8H), 2.70 (d,
J ¼ 13.6 Hz, 1H), 3.13 (d, J ¼ 13.6 Hz, 1H), 3.86e3.98 (m, 2H), 4.49e
4.61 (m, 2H), 5.23 (br s, 1H), 6.77e6.86 (m, 2H), 7.34e7.42 (m, 1H),
7.43e7.51 (m, 1H), 7.52e7.61 (m, 1H), 7.81 (s, 1H), 7.86 (d, J ¼ 7.8 Hz,
1H), 7.97 (d, J ¼ 8.0 Hz, 1H), 8.17 (s, 1H). IR (neat) 3374, 2937, 2816,
1615, 1596, 1497, 1456, 1420, 1333, 1270, 1159, 1135, 1010, 963, 849,
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20
[
a]
¼ þ14.0 (C ¼ 0.1, DMF), 98.6% ee.
D

22
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