New triazole derivatives containing substituted 1,2,3-triazole side chains: Design, synthesis and antifungal activity

Article abstract of DOI:10.1016/j.cclet.2016.11.027

In order to discover new generation of triazole antifungal agents, a series of novel antifungal triazoles were designed and synthesized by structural simplification of our previously identified triazole-piperdine-heterocycle lead compounds. Several target

Full text of DOI:10.1016/j.cclet.2016.11.027

Accepted Manuscript
Title: New triazole derivatives containing substituted
1,2,3-triazole side chains: design, synthesis and antifungal
activity
Author: Hai-Jiang Chen Yan-Juan Jiang Yong-Qiang Zhang
Qi-Wei Jing Na Liu Yan Wang Wan-Nian Zhang Chun-Quan
Sheng
PII:
S1001-8417(16)30414-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2016.11.027
CCLET 3901
To appear in:
Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
29-9-2016
3-11-2016
15-11-2016
Please cite this article as: Hai-Jiang Chen, Yan-Juan Jiang, Yong-Qiang Zhang, Qi-Wei
Jing, Na Liu, Yan Wang, Wan-Nian Zhang, Chun-Quan Sheng, New triazole derivatives
containing substituted 1,2,3-triazole side chains: design, synthesis and antifungal
activity, Chinese Chemical Letters http://dx.doi.org/10.1016/j.cclet.2016.11.027
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Original article
New triazole derivatives containing substituted 1,2,3-triazole side chains: design, synthesis and antifungal activity
Hai-Jiang Chen 1, 2, §, Yan-Juan Jiang1, 2, §, Yong-Qiang Zhang3, §, Qi-Wei Jing , Na Liu , Yan Wang , Wan-Nian Zhang , Chun-
2
2
2
2
Quan Sheng1
, 2,
*
1
2
3
. School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China
. School of Pharmacy, Second Military Medical University, Shanghai 200433, China
. School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China

Corresponding author.
E-mail address: shengcq@hotmail.com
§
These authors contributed equally to this article.
ARTICLE INFO
Article history:
Received 29 September 2016
Received in revised form 3 November 2016
Accepted 15 November 2016
Available online

Graphical Abstract
A series of new triazole antifungal derivatives were designed and synthesized. Compound 7l showed potent in vitro and in vivo antifungal
activity.
ABSTRACT
In order to discover new generation of triazole antifungal agents, a series of novel antifungal triazoles were designed and synthesized by structural
simplification of our previously identified triazole-piperdine-heterocycle lead compounds. Several target compounds showed good antifungal activity
with a broad spectrum. In particular, compound 7l was highly active against C. albicans and C. glabrata. Moreover, compound 7l showed potent in
vivo antifungal efficacy in the C. elegans–C. albicans infection model.
Keywords: Triazole derivatives, Antifungal activities, Click reaction, Molecular Docking, CYP51
1
. Introduction
Recently, the incidence of invasive fungal infections (IFIs) and associated mortality has been increasing rapidly mainly due to the
large number of immunocompromised patients and limited antifungal agents [1]. Most life-threatening IFIs are caused by Candida
albicans, Cryptococcus neoformans and Aspergillus fumigatus, whose mortality rate ranging from 20% to 90% [2, 3]. However, there
are only three classes of antifungal agents (i.e. polyenes, triazoles and echinocandins) available for the treatment of IFIs [4, 5]. Clinical
application of polyene antifungal agent amphotericin B is limited in some severe infections because of serious nephrotoxicity and
many other side effects [6, 7]. Echinocandins (e.g., caspofungin and micafungin) have fungicidal activity, but they cannot be orally
administrated. Clinically, triazole antifungal agents (e.g., fluconazole, voriconazole, itraconazole, and posaconazole) are widely used as
the first-line antifungal therapy for the prevention and treatment of IFIs (Fig. 1). However, broad application of the triazoles has caused
severe drug resistance, which significantly reduced the clinical efficacy [8]. Thus, there is still an urgent need for the discovery and
development of new generation of triazole antifungal agents [9-13]. For example, isavuconazole [14] was marketed in 2015 for
treatment of invasive aspergillosis and invasive mucormycosis and albaconazole [15] are under late stages of clinical evaluations (Fig.
1
).
Lanosterol 14-demethylase (CYP51), a key enzyme in fungal membrane ergosterol biosynthesis, is the target of triazole antifungal
agents. Due to the difficulties in solving structures of membrane-bound proteins, only one crystal structure of fungal CYP51
Saccharomyces cerevisiae CYP51) has been reported [16]. However, the lack of high-resolution structural information for CYP51
(
from invasive fungal pathogens limited the structural optimization of the triazoles. Previously, we constructed three-dimensional
models of C. albicans CYP51 (CACYP51), C. neoformans CYP51 (CNCYP51), and A. fumigatus CYP51 (AFCYP51) by homology
modeling [17-19]. Guided by the binding modes of triazole antifungal agents [17, 20, 21], we rationally designed a number of highly
potent new triazoles [21-34]. Among them, triazole 5 showed excellent in vitro antifungal activity with a broad antifungal spectrum
(
Fig. 2). Inspired by the results, further lead optimization was focused on improving the metabolic stability and in vivo antifungal

potency. Herein a series of new 1,2,3-triazole containing triazole derivatives were designed and synthesized, which showed potent in
vitro and in vivo antifungal activity.
2
. Results and discussion
2
.1 Design Rationale and Molecular Docking
In our previous studies, triazole 5 containing a benzyloxypiperidinyl side chain was identified as a potent antifungal agent with a
broad spectrum (Fig. 2) [30]. However, it was metabolically unstable because of the benzyl alcohol substructure. Thus, lead compound
was further optimized by replacing the benzyl alcohol substructure with substituted heterocycles such as 1,2,3-triazole [31], 1,2,4-
oxadiazole and 1,3,4-oxadiazole [34] (Fig. 2). Excellent antifungal activity was retained for these piperidinyl heterocylic derivatives
31, 34]. Inspired by the results, we envisioned that the piperidinyl group can be further removed to reduce molecular weight and
5
[
increase water solubility. Thus, a series of new traizole derivatives containing substituted 1,2,3-triazole side chains were synthesized
and assayed because of its synthetic accessibility and usefulness in antifungal drug discovery [35-37].
In order to investigate whether the designed triazoles can bind well with the active site of CACYP51, the binding mode of
compound 7l was explored by molecular docking [31, 34]. As shown in Fig. 3, the interactions between compound 7l and CYP51 are
similar to those observed in our previous studies [34]. The triazole ring formed a coordination bond with the Fe atom of the heme
group and the difluorophenyl group was located into a hydrophobic pocket lined with Phe126 and Tyr132. The 1,2,3-triazole ring
formed - interaction with Tyr118. Finally, the terminal cyclopropyl group interacted with Leu376, Phe380 through hydrophobic and
Van der Waals interactions.
2
.2 In vitro Antifungal Activity
The inhibitory activity of the target compounds against clinically important pathogenic fungi was determined according to the
protocols from National Committee for Clinical Laboratory Standards (NCCLS).The results revealed that most compounds generally
showed moderate to excellent activity against the tested fungal pathogens (Table1). Particularly, compounds 7f (MIC = 0.125g/mL)
and 7l (MIC = 0.125 g/mL) were highly active against C.albicans, which were more active than fluconazole (MIC = 0.5 g/mL). In
contrast, the target compounds generally showed improved activity against C.glabrata, whereas they were less potent against C.
parapsilosis. For example, the activity of compounds 7j, 7k, 7l, 8aand 8b (MIC range: 0.125 ~ 0.5 g/mL) against C. glabrata were
comparable or superior to that of fluconazole (MIC = 0.25 g/mL). For C. neoformans, most compounds showed moderate activity
except compound 8a (MIC = 0.25 g/mL), which was 8 fold more potent than fluconazole (MIC = 2 g/mL). However, all the
compounds as well as fluconazole were inactive against A. fumigatus (MIC > 64 g/mL). For dematophytes (T. rubrum and M.
gypseum), the target compounds also showed moderate to good activity. In particular, compound 7d (MIC = 0.25 g/mL) revealed
better activity against T. rubrum than fluzonazole (MIC = 0.5 g/mL).
2
.3 Structure-activity Relationship
On the basis of the antifungal activity, preliminary structure-activity relationships (SARs) were obtained. For the phenyl derivatives
(7a-f), 3-substitutions (7d, 7e and 7f) were more favorable than the 4-substitutions (7b, 7c). Among the 3-substituted derivatives, 3-
bromo substitution (7f) was more active than the 3-methyl substitution (7d). However, 3-chloro substitution (7e) had little effect on the
antifungal activity as compared to the unsubstituted compound 7a. When the phenyl group of compound 7a was replaced by elctron-
rich thiophene (7h, 7i), the antifungal activity was significantly improved. In contrast, electron-deficient pridinyl group (7g) led to
substantial decrease of the antifungal activity. Replacement of the phenyl group of compound 7a by acycloalkyl group, namely
cyclopentyl (7i), cyclohexyl (7j) and cyclopropyl (7k), resulted in the improvement of antifungal activity. In contrast, compound with
the tert-butyl substitution (7m) only showed moderate activity. Good antifungal activity was retained when the substitution was
attached on the N1 position of 1,2,3-triazole (8a and 8b).
2
.4 In vivo antifungal activity of compound 7l
Finally, the in vivo antifungal activity of compound 7l was evaluated in a Caenorhabditis elegans–C. albicans infection model [38].
Nematodes were infected with C. albicans for 4 h and then moved to pathogen-free liquid medium in the presence of compound 7l,
fluconazole or DMSO. Dead worms were counted and removed daily. C. elegans’ survival was examined by using the Kaplan-Meier
method and differences were determined by using the log-rank test. Interestingly, compound 7l showed potent in vivo antifungal
efficacy, which could effectively protect C. elegans from C. albicans infection (Fig. 4A). At the concentration of 16 g/mL, C. elegans
survival rate of compound 7l was about 70%, which was higher than that of fluconazole (60% survival rate at the concentration of 32
g/mL, Fig. 4B).
3
. Conclusion
In summary, a series of newtriazole antifungal derivatives were designed and synthesized by structural simplification of the triazole-
piperdine lead compound. Several target compounds were highly active against a variety of fungal pathogens. In particular, compound
7
l showed potent in vitro and in vivo antifungal activity, which can serve as a good lead compound for further optimization.
4
. Experimental

Nuclear magnetic resonance (NMR) spectra were generated on a Bruker AVANCE300 and AVANCE500 spectrometer (Bruker
Company, Germany), using CDCl as the reference standard or DMSO-d . Chemical shifts (δ values) and coupling constants (J values)
3
6
are expressed in ppm and Hz, respectively. ESI mass spectra were gathered on an API-3000 LC-MS spectrometer. High-resolution
mass spectrometry data were collected on a Kratos Concept mass spectrometer. TLC analysis was carried out on silica gel plates
GF254 (Qindao Haiyang Chemical, China). Silica gel column chromatography was performed with Silica gel 60 G (Qindao Haiyang
Chemical, China). Commercial solvents were used without any pretreatment.
The synthetic route of the target compounds is outlined in Scheme 1. The ring-open reaction between oxirane intermediate 9 [21]
and NaN
various alkynes in H
afforded intermediate 12, which reacted with RN
3
afforded the azide compound 10. Then, target compounds 7a-m was synthesized by click reaction of intermediate 10 with
t
2
O/ BuOH in the presence of CuSO
4
and sodium ascorbate. Propargylation of ketone 11 by propargyl bromide
3
to give target compounds 8a-b using a similar click reaction procedure.
4
.1 Synthesis of 2-(2,4-difluorophenyl)-1-(4-phenyl-1H-1,2,3-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7a)
To a solution of compound 10 (200 mg, 0.71 mmol, 1 equiv) and phenylacetylene (72.0 mg, 0.71 mmol, 1 equiv) in the mixed
solvent tert-butyl alcohol/H O (5:1, 24 mL) was added a mixture of CuSO aqueous solution (0.005 mol/L, 1.4 mL) and sodium
ascorbate aqueous solution (0.01 mol/L, 7.1 mL). The reaction mixture was stirred at room temperature overnight under the N
atmophere. Then, tert-butyl alcohol was removed under reduced pressure and the residue was diluted with CH Cl (50 mL) and washed
by saturated saline (20 mL×3). The organic layers were dried over Na SO , filtrated and evaporated under reduced pressure. The crude
product was purified by chromatography using CH Cl /MeOH as eluents (50:1 - 30:1) to give target compound 7a as a white solid: 150
): δ 8.10 (s, 1H), 7.88 (s, 1H), 7.85 (s, 1H), 7.80-7.75 (m, 2H), 7.49-7.43 (m, 1H), 7.41 (t,
H, J = 7.6 Hz), 7.34 (t, 1H, J = 7.4 Hz), 6.86-6.80 (m, 1H), 6.80-6.75 (m, 1H), 4.91 (dd, 2H, J = 20.2, 14.4 Hz), 4.78 (d, 1H, J = 14.4
2
4
2
2
2
2
4
2
2
1
mg, yield 56%. H NMR (600 MHz, CDCl
2
Hz), 4.39 (d, 1H, J = 14.3 Hz). C NMR (151 MHz, CDCl
3
1
3
3
): δ 164.07 (d, J = 12.2 Hz), 162.40 (d, J = 12.5 Hz), 159.41 (d, J = 11.6
Hz), 157.77 (d, J = 11.6 Hz), 151.70 (s), 147.73 (s), 144.56 (s), 130.11 (s), 128.89 (s), 128.39 (s), 125.73 (s), 121.81 (s), 112.22 (d, J =
+
+
1
8.8 Hz), 104.43 (t, J = 26.5 Hz), 75.33 (d, J = 4.4 Hz), 56.04 (s), 54.65 (s). MS (ESI) m/z: 383.92 (M+H). HR-MSESI : [M + H]
calcd. for C19 O, 383.2411; found, 383.2411.
17 2 6
H F N
4
.2 The synthetic procedure for compounds 7b-m and 8a-b was similar to the synthesis of compound 7a.
-(2,4-Difluorophenyl)-1-(4-(4-fluorophenyl)-1H-1,2,3-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7b): H NMR (600 MHz,
CDCl ) δ 8.02 (s, 1H ), 7.86 (s, 1H), 7.84 (s, 1H), 7.76 (m, 2H), 7.45 (m, 1H), 7.11 (t, 1H, J = 8.6 Hz), 6.80 (m, 2H), 5.51 (brs, 1H),
.94 (d, 1H, J = 14.3 Hz), 4.90 (d, 1H, J = 14.3 Hz), 4.74 (d, 1H, J = 14.3 Hz), 4.32 (d, 1H, J = 14.3 Hz). MS (ESI) m/z: 401.25 (M+H)
1
2
3
4
1
2
3
-(2,4-Difluorophenyl)-1-(4-(p-tolyl)-1H-1,2,3-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7c): H NMR (600 MHz, CDCl ):
δ 8.01 (s, 1H ), 7.84 (s, 2H), 7.67 (d, 2H, J = 7.9 Hz), 7.45 (m, 1H), 7.22 (d, 2H, J = 7.9 Hz), 6.79 (m, 2H), 5.49 (brs, 1H), 4.91 (d, 1H,
J = 14.4 Hz), 4.87 (d, 1H, J = 14.4 Hz), 4.74 (d, 1H, J = 14.3 Hz), 4.32 (d, 1H, J = 14.4 Hz). MS (ESI) m/z: 397.38 (M+H).
1
2
-(2,4-Difluorophenyl)-1-(4-(m-tolyl)-1H-1,2,3-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7d):
H NMR (600 MHz,
CDCl
1
1
3
): δ 8.03 (s, 1H ), 7.86 (s, 1H), 7.84 (s, 1H), 7.64 (s, 1H), 7.56 (d, 1H, J = 7.6 Hz), 7.45 (m, 1H), 7.30 (t, 1H, J = 7.6 Hz), 7.15 (d,
H, J = 7.4 Hz), 6.79 (m, 2H), 5.49 (brs, 1H), 4.92 (d, 1H, J = 14.0 Hz), 4.87 (d, 1H, J = 14.0 Hz), 4.75 (d, 1H, J = 14.0 Hz), 4.32 (d,
H, J = 14.0 Hz). 13C NMR (151 MHz, CDCl
): δ 164.07 (d, J = 12.5 Hz), 162.40 (d, J = 12.5 Hz), 159.38 (d, J = 11.8 Hz), 157.75 (d,
3
J = 12.0 Hz), 152.15 (s), 147.88 (s), 138.60 (s), 130.16 (dd, J = 9.3, 5.0 Hz), 130.01 (s), 129.15 (s), 128.78 (s), 126.41 (s), 122.83 (s),
21.75 (s), 112.24 (d, J = 18.8 Hz), 104.42 (t, J = 26.5 Hz), 75.41 (d, J = 4.1 Hz), 56.02 (d, J = 3.6 Hz), 54.65 (d, J = 2.3 Hz), 21.41 (s).
1
+
+
MS (ESI) m/z: 397.34 (M+H). HRMS-ESI : [M + H] calcd. for C20
H
19
F
2
N
6
O, 397.3658; found, 397.3658.
1
1
-(4-(3-Chlorophenyl)-1H-1,2,3-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7e): H NMR (600 MHz,
3
CDCl ): δ 8.09 (s, 1H), 7.94 (s, 1H), 7.87 (s, 1H), 7.80 (t, 1H, J = 1.7 Hz), 7.69 (dt, 1H, J = 7.6, 1.3 Hz), 7.46 (td, 1H, J = 9.0, 6.4 Hz),
7
4
1
1
.36 (t, 1H, J = 7.8 Hz), 7.33-7.30 (m, 1H), 6.86-6.82 (m, 1H), 6.81-6.77 (m, 1H), 4.96 (d, 1H, J = 14.4 Hz), 4.92 (d, 1H, J = 14.3 Hz),
.77 (d, 1H, J = 14.4 Hz), 4.35 (d, 1H, J = 14.3 Hz). C NMR (151 MHz, CDCl ): δ 164.12 (d, J = 12.5 Hz), 162.45 (d, J = 12.5 Hz),
3
1
3
59.35 (d, J = 11.9 Hz), 157.72 (d, J = 11.7 Hz), 151.81 (s), 146.52 (s), 144.47 (s), 134.88 (s), 131.97 (s), 130.18 (s), 128.34 (s),
25.82 (s), 123.79 (s), 122.17 (s), 112.31 (d, J = 20.7 Hz), 104.48 (t, J = 26.5 Hz), 75.38 (d, J = 4.4 Hz), 56.07 (d, J = 3.7 Hz), 54.50
+
+
(
d, J = 5.6 Hz). MS (ESI) m/z: 417.47 (M+H). HRMS-ESI : [M + H] calcd. for C19
-(4-(3-Bromophenyl)-1H-1,2,3-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7f): H NMR (600 MHz,
CDCl ): δ 8.05 (s, 1H ), 7.94 (s, 1H), 7.93 (s, 1H), 7.86 (s, 1H), 7.73 (d, 1H, J = 7.6 Hz), 7.45 (m, 2H), 7.29 (t, 1H, J = 7.6 Hz), 6.80
m, 2H), 5.51 (brs, 1H), 4.95 (d, 1H, J = 14.2 Hz), 4.95 (d, 1H, J = 14.2 Hz), 4.90 (d, 1H, J = 14.0 Hz), 4.75 (d, 1H, J = 14.0 Hz), 4.32
d, 1H, J = 14.0 Hz). MS (ESI) m/z: 461.24 (M+H).
-(2,4-Difluorophenyl)-1-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7g): 1H NMR (600 MHz,
): δ 8.97 (s, 1H), 8.57 (d, 1H, J = 4.2 Hz), 8.17 (d, 1H, J = 7.9 Hz), 8.02 (s, 1H ), 7.98 (s, 1H), 7.84 (s, 1H), 7.46 (m, 1H), 7.37
dd, 1H, J = 5.0, 7.8 Hz), 6.82 (m, 2H), 5.58 (brs, 1H), 4.95 (d, 1H, J = 14.4 Hz), 4.93 (d, 1H, J = 14.4 Hz), 4.77 (d, 1H, J = 14.4 Hz),
2 6
H16ClF N O, 417.1023; found, 417.1023.
1
1
3
(
(
2
CDCl
(
3
4
.31 (d, 1H, J = 14.4 Hz). MS (ESI) m/z: 384.22 (M+H).
1
2
-(2,4-Difluorophenyl)-1-(4-(thiophen-2-yl)-1H-1,2,3-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7h): H NMR (600 MHz,
CDCl
3
): δ 8.00 (s, 1H ), 7.83 (s, 1H), 7.81 (s, 1H), 7.44(m, 1H), 7.35 (d, 1H, J = 3.3 Hz), 7.29 (d, 1H, J = 5.0 Hz), 7.06 (dd, 1H, J =
3
=
.3, 5.0 Hz), 6.80 (m, 2H), 5.50 (brs, 1H), 4.92 (d, 1H, J = 14.5 Hz), 4.87 (d, 1H, J = 14.5 Hz), 4.71 (d, 1H, J = 14.5 Hz), 4.31 (d, 1H, J
14.5 Hz). MS (ESI) m/z: 389.38 (M+H).

1
2
-(2,4-Difluorophenyl)-1-(4-(thiophen-3-yl)-1H-1,2,3-triazol-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7i): H NMR (600 MHz,
DMSO-d ): δ 8.39 (s, 1H), 8.23 (s, 1H), 7.86 (s, 1H), 7.83-7.80 (m, 1H), 7.63 (dd, 1H, J = 4.9, 3.0 Hz), 7.48 (dd, 1H, J = 5.0, 1.0 Hz),
.27 (t, 1H, J = 10.5 Hz), 7.21 (dd, 1H, J = 15.9, 8.8 Hz), 6.89 (t, 1H, J = 8.4 Hz), 6.56 (s, 1H), 5.03 (d, 1H, J = 14.4 Hz), 4.80 (d, 1H,
6
7
1
3
J = 14.5 Hz), 4.72 (d, 1H, J = 14.4 Hz), 4.66 (d, 1H, J = 14.5 Hz). C NMR (151 MHz, DMSO-d
d, J = 12.6 Hz), 160.26 (d, J = 12.4 Hz), 158.62 (d, J = 12.1 Hz), 151.32 (s), 145.66 (s), 142.72 (s), 132.35 (s), 130.21 (dd, J = 9.0, 5.8
Hz), 127.41 (s), 126.14 (s), 122.72 (s), 121.11 (s), 111.36 (d, J = 20.8 Hz), 104.45 (t, J = 26.9 Hz), 74.27 (d, J = 4.5 Hz), 55.95 (d, J =
6
): δ 163.31 (d, J = 12.3 Hz), 161.68
(
+
+
3
3
15 2 6
.9 Hz), 55.26 (d, J = 4.5 Hz). MS (ESI) m/z: 389.27 (M+H). HRMS-ESI : [M + H] calcd. for C17H F N OS, 389.1066; found,
89.1066.
1
-(4-Cyclopentyl-1H-1,2,3-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7j): 1H NMR (600 MHz,
CDCl
14.4 Hz), 4.68 (d, 1H, J = 14.4 Hz), 4.35 (d, 1H, J = 14.0 Hz), 3.12 (m, 1H), 2.04 (m, 2H), 1.65 (m, 6H). 13C NMR (151 MHz,
CDCl ): δ 164.01 (d, J = 12.6 Hz), 162.35 (d, J = 12.3 Hz), 159.38 (d, J = 11.8 Hz), 157.75 (d, J = 11.8 Hz), 152.74 (s), 151.93 (s),
30.17 (dd, J = 9.2, 5.3 Hz), 121.88 (s), 112.07 (d, J = 21.0 Hz), 104.27 (t, J = 26.5 Hz), 75.39 (d, J = 3.9 Hz), 55.82 (d, J = 3.5 Hz),
3
): δ 8.06 (s, 1H ), 7.84 (s, 1H), 7.43 (m, 1H), 7.30 (s, 1H), 6.80 (m, 2H), 5.41 (brs, 1H), 4.85 (d, 1H, J = 14.0 Hz), 4.75 (d, 1H, J
=
3
1
5
+
+
4.67 (d, J = 2.6 Hz), 36.55 (s), 33.12 (d, J = 9.4 Hz), 25.04 (s). MS (ESI) m/z: 375.30 (M+H). HRMS-ESI : [M + H] calcd. for
O, 375.1745; found, 375.1745.
-(4-Cyclohexyl-1H-1,2,3-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7k): 1H NMR (600 MHz,
CDCl ): δ 8.05 (s, 1H), 7.81 (s, 1H), 7.39 (dd, 1H, J = 15.5, 8.9 Hz), 6.81-6.72 (m, 2H), 5.47 (s, 1H), 4.83 (d, 1H, J = 14.4 Hz), 4.71 (q,
H, J = 14.3 Hz), 4.35 (d, 1H, J = 14.3 Hz), 2.70-2.64 (m, 1H), 1.94 (t, 2H, J = 11.0 Hz), 1.74 (d, 2H, J = 12.5 Hz), 1.68 (d, 1H, J =
18 21 2 6
C H F N
1
3
2
1
1
2
1
3
3.3 Hz), 1.40-1.26 (m, 4H), 1.25-1.19 (m, 1H). C NMR (151 MHz, CDCl
3
): δ 162.33 (d, J = 12.6 Hz), 159.37 (d, J = 12.0 Hz),
57.78 (s), 156.23 (s), 153.62 (s), 151.81 (s), 144.61 (s), 130.16 (dd, J = 9.4, 5.5 Hz), 121.61 (s), 112.07 (d, J = 18.7 Hz), 104.27 (t, J =
+
+
6.2 Hz), 75.37 (d, J = 4.6 Hz), 55.82 (d, J = 4.1 Hz), 54.65 (d, J = 5.6 Hz), 35.02 (s), 32.86 (s), 25.97 (s). HRMS-ESI : [M + H] calcd.
O, 389.1909; found, 389.1909.
-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7l): 1H NMR (600 MHz,
CDCl ): δ 8.09 (s, 1H), 7.85 (s, 1H), 7.44 (dt, 1H, J = 15.3, 7.7 Hz), 7.32 (s, 1H), 6.81 (tdd, 2H, J = 10.9, 8.3, 2.3 Hz), 4.87 (d, 1H, J =
4.3 Hz), 4.78 (d, 1H, J = 14.3 Hz), 4.66 (d, 1H, J = 14.3 Hz), 4.33 (d, 1H, J = 14.2 Hz), 1.91 (ddd, 1H, J = 17.0, 8.5, 5.0 Hz), 0.94
dd, 2H, J = 8.4, 2.2 Hz), 0.81-0.77 (m, 2H). 13C NMR (151 MHz, CDCl
): δ 164.04 (d, J = 12.1 Hz), 162.37 (d, J = 12.3 Hz), 159.36
d, J = 12.0 Hz), 157.73 (d, J = 11.6 Hz), 151.72 (s), 150.26 (s), 130.17 (dd, J = 9.3, 5.3 Hz), 121.95 (s), 112.17 (d, J = 20.8 Hz),
04.35 (t, J = 26.5 Hz), 75.33 (d, J = 4.5 Hz), 55.81 (d, J = 4.0 Hz), 54.64 (d, J = 5.2 Hz), 7.80 (d, J = 10.7 Hz), 6.52 (s). MS (ESI)
23 2 6
for C19H F N
1
3
1
(
(
3
1
+
+
m/z: 347.22 (M+H). HRMS-ESI : [M + H] calcd. for C16
H
17
F
2
N
6
O, 347.1498; found, 347.1498.
1
-(4-(tert-Butyl)-1H-1,2,3-triazol-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7m): 1H NMR (600 MHz,
DMSO-d
1
6
): δ 8.36 (s, 1H), 7.84 (s, 1H), 7.54 (s, 1H), 7.20 (dt, 2H, J = 16.0, 10.1 Hz), 6.89 (t, 1H, J = 8.5 Hz), 6.43 (s, 1H), 4.89 (d,
H, J = 14.3 Hz), 4.78 (d, 1H, J = 14.4 Hz), 4.64 (d, 1H, J = 14.4 Hz), 4.59 (d, 1H, J = 14.5 Hz), 1.20 (s, 9H). 13C NMR (151 MHz,
DMSO-d
6
): δ 163.36 (d, J = 12.7 Hz), 161.73 (d, J = 12.7 Hz), 160.26 (d, J = 12.5 Hz), 158.62 (d, J = 12.4 Hz), 156.10 (s), 151.34 (s),
1
3
45.65 (s), 130.34 (dd, J = 9.2, 5.8 Hz), 121.24 (s), 111.28 (d, J = 21.0 Hz), 104.40 (t, J = 26.9 Hz), 74.43 (d, J = 4.5 Hz), 55.87 (d, J =
.5 Hz), 55.39 (d, J = 4.9 Hz), 30.62 (s). MS (ESI) m/z: 363.26 (M+H). HRMS-ESI : [M + H] calcd. for C17H F N O, 363.1788;
21 2 6
+
+
found, 363.1788.
-(1-Cyclopentyl-1H-1,2,3-triazol-4-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8a): 1H NMR (600 MHz,
CDCl ): δ 8.15 (s, 1H ), 7.80 (s, 1H), 7.39 (m, 1H), 7.18 (s, 1H), 6.72 (m, 2H), 5.54 (brs, 1H), 4.80 (m, 1H), 4.71 (d, 1H, J = 14.4 Hz),
1
3
1
3
4
.58 (d, 1H, J = 14.3 Hz), 3.15 (d, 1H, J = 14.9 Hz), 3.15 (d, 1H, J = 14.9 Hz), 2.17 (m, 2H), 1.68-1.95 (m, 6H). C NMR (151 MHz,
3
CDCl ): δ 163.41 (d, J = 12.2 Hz), 161.76 (d, J = 12.2 Hz), 159.38 (d, J = 11.7 Hz), 157.74 (d, J = 11.8 Hz), 151.27 (s), 142.45 (s),
1
3
3
30.19 (dd, J = 9.1, 5.8 Hz), 120.95 (s), 111.36 (d, J = 22.9 Hz), 103.90 (t, J = 26.6 Hz), 75.20 (d, J = 4.6 Hz), 61.84 (s), 57.06 (d, J =
.6 Hz), 33.75 (d, J = 4.3 Hz), 29.68 (s), 23.93 (s). MS (ESI) m/z: 376.00 (M+H). HRMS-ESI : [M + H] calcd. for C18H F N O,
21 2 6
+
+
75.2012; found, 375.2012.
1
-(1-Cyclohexyl-1H-1,2,3-triazol-4-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8b): 1H NMR (600 MHz,
CDCl
1
1
3
): δ 8.24 (s, 1H), 7.82 (s, 1H), 7.38 (dd, 1H, J = 15.6, 8.9 Hz), 7.19 (s, 1H), 6.76-6.67 (m, 2H), 4.72 (d, 1H, J = 14.0 Hz), 4.60 (d,
H, J = 14.4 Hz), 4.30 (tt, 1H, J = 11.8, 3.8 Hz), 3.44 (d, 1H, J = 14.9 Hz), 3.14 (d, 1H, J = 14.9 Hz), 2.07 (dd, 2H, J = 23.9, 12.5 Hz),
.86 (d, 2H, J = 13.9 Hz), 1.72 (d, 1H, J = 13.3 Hz), 1.67-1.56 (m, 2H), 1.45-1.35 (m, 2H). 13C NMR (151 MHz, CDCl
): δ 163.41 (d,
3
J = 12.2 Hz), 161.76 (d, J = 12.6 Hz), 159.36 (d, J = 11.7 Hz), 157.73 (d, J = 11.8 Hz), 142.20 (s), 130.17 (dd, J = 9.2, 5.8 Hz), 124.99
d, J = 9.8 Hz), 120.22 (s), 111.37 (d, J = 22.8 Hz), 103.90 (t, J = 26.6 Hz), 75.19 (d, J = 4.5 Hz), 60.09 (s), 57.23 (s), 33.76 (d, J = 4.2
(
+
+
23 2 6
Hz), 33.37 (d, J = 7.1 Hz), 29.69 (s), 25.03 (s). MS (ESI) m/z: 389.69 (M+H). HRMS-ESI : [M + H] calcd. for C19H F N O, 389.1864;
found, 389.1864.
4
.3 In vitro antifungal activity assay
In vitro antifungal activity was measured according to the protocols from National Committee for Clinical Laboratory Standards
NCCLS). Serial dilution method in 96-well microtest plate was used to determine the minimum inhibitory concentration (MIC) of the
(
target compounds. 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% inhibition 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 mold and dermatophytes at 28 °C. Growth MIC was determined at 24 h for Candida species, at 72 h for
Cryptococcus neoformans, and at 7 days for Aspergillus fumigatus.
4
.4 In vivo antifungal activity assay
C. elegans was first infected by C. albicans. Briefly, C. elegans glp-4; sek-1 adult nematodes were added to the center of C.
albicans SC5314 lawns on BHI kanamycin (45 µg/mL) agar plates and incubated at 25°C for 4 h to allow infections. Worms were
washed four times with sterile M9. Thirty worms were then pipetted into each well of 12-well tissue culture plates (Corning, USA)
containing 2 ml of liquid medium (80% M9, 20% BHI) and kanamycin (45 µg/mL). For compounds treatment groups, compound was
added at 16 µg/mL. 32 µg/mL FLC treatment group was set as the positive control, and the DMSO solvent group was set as the
negative control. Worms were scored daily and dead worms were removed from the assay. Survival was examined by using the
Kaplan-Meier method and differences were determined by using the log-rank test (STATA 6; STATA, College Station, TX). A P value
of, 0.05 was considered statistically significant.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 81573283, 21502224), the 863 Hi-Tech
Program of China (No. 2014AA020525), and the Shanghai “ShuGuang” Project (No. 14SG33).

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Fig. 1 Structures of triazole antifungal agents

Fig. 2 Design rationale of the target compounds.

Fig. 3 The binding mode of compound 7l in the active site of CACYP51.
Fig. 4 Compound 7l and fluconazole prolong the survival of C. elegans glp-4; sek-1 nematodes infected by C. albicans SC5314. A P value of < 0.05 was
considered statistically significant.

3 4 4 2 4
Scheme 1. (a) NaN /NH Cl, MeOH, reflux, overnight;(b) CuSO , sodium ascorbate, t-BuOH, H O, overnight;(c) Zn, DMF, THF, rt, 5h; (d) CuSO , sodium
ascorbate, H O, overnight.
2

Table 1
a
In vitro antifungal activities of the target compounds (MIC80, g/mL)
Compds C. alb. C. gla C. para. C. neo. A. fum. T. rub. M. gyp.
7
7
7
a
b
c
8
0.5
8
8
>64
>64
>64
4
>64
>64
>64
16
64
8
16
64
32
64
8
2
16
7
7
7
7
7
7
7
7
7
7
8
8
d
e
f
0.5
8
0.5
0.5
0.5
16
64
8
64
8
>64
>64
>64
>64
>64
>64
0.25
2
>64
2
0.125
64
2
8
8
>64
>64
8
g
h
i
64
2
32
8
64
2
0.5
0.5
0.5
0.5
4
16
2
4
j
0
0
.25
0.25
0.25
4
4
8
8
>64
>64
2
2
8
8
k
l
.25
0
2
.125
0.25
2
2
1
>64
>64
4
8
m
a
b
32
32
16
>64
1
1
0
0.25
0.125
0.25
1
1
1
0.25
1
>64
>64
>64
2
8
4
2
2
FLZ
.5
2
0.5
a
Abbreviations: C. alb. Candida albicans; C. gla. Candida glabrata; C. para. Candida parapsilosis; C. neo. Cryptococcus neoformans; A. fum. Aspergillus
fumigatus; T. rub. Trichophyton rubrum; M. Gyp. Microsporum gypseum.