Design, synthesis and antifungal activity of novel triazole derivatives containing substituted 1,2,3-triazole-piperdine side chains

Article abstract of DOI:10.1016/j.ejmech.2014.05.079

Due to increasing incidence of invasive fungal infections and severe drug resistance to triazole antifungal agents, a series of novel antifungal triazoles with substituted triazole-piperidine side chains were designed and synthesized. Most of the target compounds showed good inhibitory activity against a variety of clinically important fungal pathogens. In particular, compounds 8t and 8v were highly active against Candida albicans and Cryptococcus neoformans with MIC values in the range of 0.125 μg/mL to 0.0125 μg/mL. They represent promising leads for the development of new generation of triazole antifungal agents. Molecular docking studies revealed that the target compounds interacted with CACYP51 mainly through hydrophobic and Van der Waals interactions.

Full text of DOI:10.1016/j.ejmech.2014.05.079

Accepted Manuscript
Design, Synthesis and Antifungal Activity of Novel Triazole Derivatives Containing
Substituted 1,2,3-Triazole-piperdine Side Chains
Zhigan Jiang , Julin Gu , Chen Wang , Shengzheng Wang , Na Liu , Yan Jiang ,
Guoqiang Dong , Yan Wang , Yang Liu , Jianzhong Yao , Zhenyuan Miao , Wannian
Zhang , Chunquan Sheng
PII:
S0223-5234(14)00511-X
10.1016/j.ejmech.2014.05.079
EJMECH 7037
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2013
Revised Date: 30 May 2014
Accepted Date: 31 May 2014
Please cite this article as: Z. Jiang, J. Gu, C. Wang, S. Wang, N. Liu, Y. Jiang, G. Dong, Y. Wang, Y.
Liu, J. Yao, Z. Miao, W. Zhang, C. Sheng, Design, Synthesis and Antifungal Activity of Novel Triazole
Derivatives Containing Substituted 1,2,3-Triazole-piperdine Side Chains, European Journal of Medicinal
Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.079.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Design, Synthesis and Antifungal Activity of Novel Triazole
Derivatives Containing Substituted 1,2,3-Triazole-piperdine Side
Chains
§
§
Zhigan Jiang , Julin Gu , Chen Wang*, Shengzheng Wang, Guoqiang Dong, Yan
Wang, Yang Liu, Jianzhong Yao, Zhenyuan Miao, Wannian Zhang* and Chunquan
Sheng*
A series of novel antifungal triazoles with substituted triazole-piperdine side chains
were designed and synthesized. Most of the target compounds showed good inhibitory
activity against a variety of clinically important fungal pathogens.

ACCEPTED MANUSCRIPT
Design, Synthesis and Antifungal Activity of Novel Triazole
Derivatives Containing Substituted 1,2,3-Triazole-piperdine Side
Chains
1
,2,§
3,§
4,
1
1
1
Zhigan Jiang , Julin Gu , Chen Wang *, Shengzheng Wang , Na Liu , Yan Jiang ,
1
1
1
1
1
Guoqiang Dong , Yan Wang , Yang Liu , Jianzhong Yao , Zhenyuan Miao , Wannian
1
,
1,
Zhang * and Chunquan Sheng *
1
2
3
4
. School of Pharmacy, Second Military Medical University, 325 Guohe Road,
Shanghai 200433, China
. WuXi AppTec (Shanghai) Co., Ltd.,288 FuTe ZhongLu Road, Shanghai
200131, China
. Department of Dermatology and Mycology Center, Changzheng Hospital,
Second Military Medical University, Shanghai 20003, China
. Changhai Hospital of Traditional Chinese Medicine, Second Military Medical
University, 800 Xiangyin Road., Shanghai 200433, China
§
These two authors contributed equally to this work.
*
8
8
8
To whom correspondence should be addressed. For C. Q. Sheng: Phone/Fax,
6-21-81871239, E-mail, shengcq@hotmail.com. For W. N. Zhang: /Fax,
6-21-81870243, E-mail, zhangwnk@hotmail.com. For C. Wang: Phone,
6-21-3116-1966, Fax, 86-21-8187-3143, E-mail, wangchenchina@126.com
Abstract
Due to increasing incidence of invasive fungal infections and severe drug
resistance to triazole antifungal agents, a series of novel antifungal triazoles with
substituted triazole-piperdine side chains were designed and synthesized. Most of the
target compounds showed good inhibitory activity against a variety of clinically
important fungal pathogens. In particular, compounds 8t and 8v were highly active
against Candida albicans and Cryptococcus neoformans with MIC values in the range
of 0.125 µg/mL to 0.0125 µg/mL. They represent promising leads for the development
of new generation of triazole antifungal agents. Molecular docking studies revealed
that the target compounds interacted with CACYP51 mainly through hydrophobic and
Van der Waals interactions.
Keywords: Triazole derivatives; Antifungal activities; Click reaction.
1

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1
. Introduction
The incidence of invasive fungal infections (IFIs) has significantly increased over the
past two decades due to increasing number of organ transplant recipients and stem
cell transplantation, broad use of aggressive chemotherapy and prevalence of human
immunodeficiency virus (HIV) infection [1]. Candidosis, aspergillosis, and
cryptococcosis represent the three most common IFIs, whose mortality rate is high
(e.g. about 50% for candidosis and nearly 100% for invasive aspergillosis). However,
effective and safe antifungal agents are very limited. Clinically available antifungal
agents (Fig. 1) for IFIs mainly include amphotericin B, triazoles, echinocandins, and
5
-fluorocytosine [2, 3]. Among them, triazole antifungal agents (i.e. fluconazole,
voriconazole, itraconazole and posaconazole) are widely used as the first-line
antifungal therapy. However, broad use of them has caused severe drug resistance [4].
Thus, the development of new generation of triazole antifungal agents is urgently
needed. New triazoles with improved pharmacological and pharmacokinetic profiles
are emerging rapidly [5-9]. For example, isavuconazole and albaconazole are under
late stages of clinical evaluations [10].
Triazole antifungal agents act by inhibition of fungal lanosterol 14α-demethylase
(CYP51), which is a key enzyme in ergosterol biosynthesis. The crystal structure of
fungal CYP51 has not been solved up to date. In our previous studies, we built
three-dimensional models of CYP51 from Candida albicans (CACYP51),
Cryptococcus neoformans (CNCYP51), and Aspergillus fumigatus (AFCYP51) by
homology modeling [11-13]. Binding modes of triazole antifungal agents were
2

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investigated by molecular docking and molecular dynamics simulations [11, 14, 15].
Guided by the results from molecular modeling, a number of new triazoles were
rationally designed and synthesized by our group [15-24]. Among them, triazoles with
benzyloxypiperidinyl side chains showed good antifungal activity with a broad
spectrum (Fig. 1) [24]. In order to investigate their structure-activity relationship
(SAR) and discover highly active antifungal lead compound, herein a series of
triazole-piperdine derivatives were designed and synthesized. Most of the target
compounds showed excellent antifungal activity against a variety of fungal pathogens.
2
. Chemistry
The oxirane intermediate 5 was synthesized according to our previously reported
procedure (Scheme 1) [15]. As a key intermediate of the designed triazoles, the azide
compound 7 was synthesized by the NaN displacement reaction with mesylate of
3
intermediate 6 [25]. The target compounds were obtained by click reaction of
t
intermediate 7 with various alkynes in H O/ BuOH in the presence of CuSO .5H O
2
4
2
and sodium ascorbate.
Microbiology
3
.
In vitro antifungal activity was measured according to the protocols from National
Committee for Clinical Laboratory Standards (NCCLS). Serial dilution method in
9
6-well microtest plate was used to determine the minimum inhibitory concentration
(MIC) of the target compounds [15]. 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
3

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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. Results and Discussion
4.1 Design Rationale
Triazole, difluorophenyl and tertiary alcohol are essential pharmacophore for triazole
antifungal agents. Most of the structural optimization studies were focused on
variation of the C3-side chain. In our previous studies, new triazoles containing
benzyloxypiperidinyl side chain were designed by structure-based lead fusion, which
showed excellent antifungal activity [24]. Thus, it is highly desirable to obtain more
SAR information for this promising class of antifungal lead compound. Click reaction
is an efficient approach to the synthesis of drug-like molecules which has been widely
applied in drug discovery [26]. Herein, 1,2,3-triazole was used to replace the benzyl
alcohol group and various alkyl or aromatic groups were also introduced to form
additional interactions with CYP51 (Fig. 2).
4
.2 In vitro Antifungal Activity
In vitro antifungal activity of the target compounds is listed in Table 1. Fluconazole
and itraconazole were used as reference drugs. In general, most of the target
compounds showed good inhibitory activity against the tested fungal pathogens,
especially for Candida albicans and Cryptococcus neoformans. There are thirteen
4

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compounds (8a, 8b, 8c, 8e, 8l, 8q-x) that showed better inhibitory activity against
Candida albicans than fluconazole and itraconazole (MIC range: 0.125 µg/mL ~
0
.0125 µg/mL). Particularly, compounds 8t (MIC = 0.0625 µg/mL) and 8v (MIC =
.0125 µg/mL) were highly active Candida albicans inhibitors. In contrast, the target
0
compounds as well as positive drugs showed decreased inhibitory activity against
other Candida species including Candida parapsilosis and Candida glabrata.
Eventhough, three compounds (8e, 8t, 8x) still revealed good activity with MIC
values in the range of 0.25 µg/mL to 0.5 µg/mL. On the Cryptococcus neoformans
strain, ten compounds (8b, 8e, 8l, 8q, 8r, 8s, 8t, 8v, 8w and 8x) were more active
(MIC range: 0.25 µg/mL ~ 0.5 µg/mL) than fluconazole and itraconazole.
Fluconazole is inactive against Aspergillus fumigatus (MIC > 64 µg/mL), while
several target compounds showed moderate activity (MIC range: 8 µg/mL ~ 32
µg/mL). However, all of them were less active than itraconazole (MIC = 1 µg/mL).
Moreover, several compounds also showed good activity against dermatophytes
Trichophyton rubrum and Microsporum gypseum. For example, the MIC values of
compounds 8e, 8p, 8s, 8v, 8w and 8x against Trichophyton rubrum were in the range
of 0.25 µg/mL to 0.5 µg/mL, whose activity was comparable or superior to that of
itraconazole and fluconazole. Among the synthesized triazole derivatives, compounds
8
e, 8t, 8v and 8x were highly active with a broad spectrum.
4
.3 Structure-Activity Relationships
Compounds with ethyl (8a), propyl (8b) and cyclopropyl (8c) substitutions on the
1
,2,3-triazole ring showed broad-spectrum antifungal activity. In contrast, the
5

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cyclopentyl derivative 8d was less active. Moreover, all the hydroxylalkyl derivatives
(8f-8k) showed significantly decreased antifungal activity. Most of them were
inactive against Candida parapsilosis, Cryptococcus neoformans, Aspergillus
fumigatus, Trichophyton rubrum and Microsporum gypseum. Interestingly, the
methylation of compound 8f led to obvious improvement of the antifungal activity.
Compound 8e is one of the most active compounds in this series. For the compounds
with ester and carboxylic acid substitutions, the butyrate (8m) and butyric acid (8n)
derivatives were more potent than the corresponding formate (8o) and formic acid (8p)
derivatives. All the aromatic derivatives 8q-8x were proven to be excellent fungal
inhibitors. The replacement of the phenyl group of compound 8q by a pyridinyl group
(8r) resulted in slight increase of the activity against Cryptococcus neoformans, but
its inhibitory activity against dermatophytes was reduced. For the substitutions on the
phenyl ring of compound 8q, the introduction of 4-acetyl (8t, MIC = 0.0625 µg/mL)
and 4-trifluoromethoxy (8v, MIC = 0.0125 µg/mL) group was favorable for the
anti-Candida albicans activity. In contrast, the addition of 4-N,N-dimethyl group (8u)
led to slight decrease of the antifungal activity. Moreover, the addition of the 2-methyl
substitution (8x) had positive effects on inhibitory activity against Candida
parapsilosis and Candida glabrata.
4
.4 Binding Mode of Compounds 8t and 8v with the Active Site of CACYP51
In order to investigate the binding mode of the designed triazoles, two highly active
compounds, 8t and 8v, were docked into the active site of CACYP51. As shown in
Fig. 3, the triazole ring and difluorophenyl group of compounds 8t and 8v formed
6

ACCEPTED MANUSCRIPT
coordination bond and hydrophobic interactions with heme group (Fe ion) and the
hydrophobic pocket (Phe126, Ile131 and Tyr132), respectively. The side chains of the
two compounds were extended into the S4 pocket [13, 27] of the active site of
CACYP51. The triazoyl and piperdinyl groups interacted with Leu376, Met508,
Leu121, Phe380 and Tyr118 through hydrophobic and Van der Waals interactions. The
substitutions on the 1,2,3-triazole ring were located into a hydrophobic pocket lined
with Tyr505, Phe233, Pro230 and Tyr64. The magnitude of hydrophobic interactions
of various substitutions with this pocket played an important role for the binding
affinity and antifungal activity. For example, compounds with hydrophilic hydroxyl
group (8f-8k) at this site showed weak antifungal activity.
5
. Conclusion
In summary, a series of novel antifungal triazoles with substituted triazole-piperdine
side chains were designed and synthesized. Several compounds showed excellent
antifungal activity against a variety of clinically important fungal pathogens.
Molecular docking studies revealed that the target compounds interacted with
CACYP51 mainly through hydrophobic and Van der Waals interactions. Compounds
8
e, 8t, 8v and 8x were identified as highly active fungal inhibitors, which represent
promising lead compounds for the development of new generation of triazole
antifungal agents. Further pharmacological and pharmacokinetic evaluation of them is
in progress.
6
. Experimental Section
6
.1 General procedure for the synthesis of compounds
7

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Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 400
spectrometer with TMS as an internal standard. Chemical shifts (δ values) and
coupling constants (J values) are given in ppm and Hz, respectively. High-resolution
mass spectrometry data were collected on a Kratos Concept mass spectrometer.
Infrared spectra (IR) were recorded on a Brucker Vector II instrument. 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.
6
.1.1
1-(4-Azidopiperidin-1-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)
propan-2-ol (7)
Methanesulfonyl chloride (1.88 g, 1.1 eq) was added dropwise to a solution of
compound 6 (5.07 g, 15 mmol) and triethylamine (1.1 mL, 4 eq) in anhydrous CH Cl
2
2
(50 mL) at 0 ℃ . The reaction mixture was stirred for 1.5 h at this temperature. Then,
water (30 mL) was added to quench the reaction and the mixture was extracted with
CH Cl for 3 times. After drying and evaporation, the crude product was used in the
2
2
next step without further purification. The above intermediate was dissolved in DMF
30 mL) and was added sodium azide (2.93 g, 3 eq). The reaction solution was stirred
at 40 ℃ for 6 h. Then, water (20 mL) and CH Cl (50 mL) was added and the
(
2
2
aqueous layer was extracted with CH Cl . The combined organic layer was dried over
2
2
Na SO , filtered and evaporated under reduced pressure. The crude product was
2
4
purified by silica gel column chromatography (eluents: hexane : EtOAc = 2:1, v/v) to
8

ACCEPTED MANUSCRIPT
1
afford compound 7 as yellow oil (3.93 g, 76.2% yield of two steps). H NMR (400
MHz, CDCl )℃ δ:℃ 8.16 (s, 1H), 7.81 (s, 1H), 7.45-7.66 (m, 1H), 6.73-6.97 (m, 2H),
3
5
.19 (brs., 1H), 4.44-4.61 (m, 2H), 3.38 (brs, 1H), 3.08 (d, J = 13.55 Hz, 1H), 2.68 (d,
J = 13.80 Hz, 1H), 2.51-2.60 (m, 1H), 2.41-2.51 (m, 1H), 2.27-2.38 (m, 1H), 2.18 (t, J
9.41 Hz, 1H), 1.70-1.91 (m, 2H), 1.45-1.60 (m, 2H). MS (ESI) m/z: 363.9 (M+1).
=
6
.1.2
2-(2,4-Difluorophenyl)-1-(4-(4-ethyl-1H-1,2,3-triazol-1-yl)piperidin-1-yl)-
3
-(1H-1,2,4-triazol-1-yl)propan-2-ol (8a)
To a solution of compound 7 (40 mg, 0.11 mmol) and but-1-yne (8 mg, 1.3 equiv) in
tert-butyl alcohol (1 mL) was added a mixture of CuSO .5H O (20 mol %) and
4
2
sodium ascorbate (40 mol %) in H O (0.5 mL). The reaction mixture was stirred at
2
5
0 ℃ overnight. Then, water (10 mL) was added and was extracted with EtOAc (2×
2
0 mL). The combined organic layers were washed with water (10 mL), dried over
Na SO , and evaporated under reduced pressure. The crude product was purified by
2
4
flash chromatography using EtOAc/heptane as eluents (1:5) to give the target
1
compound as a white solid: 20 mg, yield 43.3%, 53.0-55.0 ℃. H NMR (400 MHz,
CDCl )δ: 1.26 (t, J = 7.53 Hz, 3H), 1.81 - 2.04 (m, 3H), 2.08 - 2.15 (m, 1H), 2.21 -
3
2
1
7
1
5
1
.30 (m, 1H), 2.46 - 2.59 (m, 2H), 2.73 (q, J = 7.45 Hz, 4H), 3.12 (d, J = 13.55 Hz,
H), 4.29 - 4.38 (m, 1H), 4.48 - 4.60 (m, 2H), 6.76 - 6.87 (m, 2H), 7.24 (s, 1H), 7.52 -
1
3
.61 (m, 1H), 7.80 (s, 1H), 8.14 (s, 1 H). C NMR (100 MHz, CDCl ): δ 162.31,
3
59.52, 150.54, 147.82, 143.90, 129.28, 125.45, 117.97, 111.85, 103.56, 72.47, 62.24,
7.34, 56.60, 54.55, 52.34, 32.48, 31.53, 25.62, 12.78. IR (KBr) 3256, 2843, 2360,
-1
617, 1540, 1500, 1420, 1395, 1255, 1201, 1152, 1034, 835, 812, 632 cm . HRMS
9

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+
calcd for C H F N O [M + H] : 418.2445, found: 418.2448.
2
0
25
2
7
The synthetic procedure for compounds 8b-x was similar to the synthesis of
compound 8a.
6
3
.1.3 2-(2,4-Difluorophenyl)-1-(4-(4-propyl-1H-1,2,3-triazol-1-yl)piperidin-1-yl)-
-(1H-1,2,4-triazol-1-yl)propan-2-ol (8b)
1
White solid: 26 mg, yield 47.8%, 50.0-51.5 ℃. H NMR (400 MHz, CDCl ) δ: 0.95
3
(
t, J = 7.40 Hz, 3H), 1.63-1.71 (m, 2H), 1.82-2.05 (m, 3H), 2.08-2.16 (m, 1 H), 2.25
td, J = 11.80, 2.26 Hz, 1H), 2.45-2.60 (m, 2H), 2.61-2.79 (m, 4H), 3.12 (d, J = 13.55
(
Hz, 1H), 4.27-4.39 (m, 1H), 4.46-4.64 (m, 2H), 6.68-6.92 (m, 2H), 7.23 (s, 1H),
1
3
7
.47-7.62 (m, 1H), 7.80 (s, 1H), 8.14 (s, 1H).
C NMR (100 MHz, CDCl ): δ
3
1
62.35, 159.22, 150.85, 147.43, 143.34, 130.02, 125.78, 118.03, 112.12, 103.43,
2.57, 62.27, 57.45, 56.22, 54.34, 52.44, 32.86, 32.28, 31.23, 24.36, 13.98. IR (KBr)
312, 2825, 2798, 2361, 1615, 1534, 1499, 1450, 1268, 1200, 1143, 1056, 1021, 858,
7
3
-1
+
8
10, 766, 698, 670, 622 cm . HRMS calcd for C H F N O [M + H] : 432.2521,
21 27 2 7
found: 432.2519.
6
.1.4
1-(4-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)piperidin-1-yl)-2-(2,4-
difluorophenyl) -3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8c)
1
Colorless oil: 35 mg, yield 45.3%. H NMR (400 MHz, CDCl )δ: 0.78-0.87 (m, 2H),
3
0
.88-0.98 (m, 2H), 1.24-1.26 (m, 1H), 1.83-1.99 (m, 3H), 2.06-2.13 (m, 1H), 2.25 (td,
J = 11.80, 2.26 Hz, 1H), 2.45-2.60 (m, 2H), 2.68-2.77 (m, 2H), 3.11 (d, J = 13.55 Hz,
1
2
H), 3.72 (q, J = 7.03 Hz, 1H), 4.25-4.38 (m, 1H), 4.47-4.61 (m, 2H), 6.76-6.89 (m,
1
3
H), 7.19 (s, 1H), 7.56 (td, J = 9.03, 6.78 Hz, 1H), 7.80 (s, 1H), 8.14 (s, 1H).
C
1
0

ACCEPTED MANUSCRIPT
NMR (100 MHz, CDCl ): δ 162.44, 159.85, 152.44, 151.23, 144.58, 129.46, 125.46,
3
117.53, 111.67, 104.55, 72.67, 62.24, 57.34, 56.46, 54.56, 52.79, 33.38, 32.63, 10.22,
9
.58. IR (KBr) 3276, 2874, 2812, 2360, 1622, 1540, 1500, 1462, 1420, 1285, 1223,
-1
1
145, 1065, 986, 856, 732, 688, 650, 594 cm . HRMS calcd for C H F N O [M +
21 25 2 7
+
H] : 430.2056, found: 430.2053.
.1.5 1-(4-(4-Cyclopentyl-1H-1,2,3-triazol-1-yl)piperidin-1-yl)-2-(2,4-
6
difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8d)
1
White solid: 80 mg, yield 60.3%, 58.0-60.0 ℃. H NMR (400 MHz, CDCl ) δ: 8.13
3
(s, 1H), 7.77 (s, 1H), 7.46-7.60 (m, 1H), 7.22 (s, 1H), 6.71-6.87 (m, 2H), 5.12 (s, 1H),
4
2
.44-4.63 (m, 2H), 4.22-4.36 (m, 1H), 3.03-3.18 (m, 2H), 2.65-2.77 (m, 2H),
1
3
.40-2.53 (m, 2H), 2.17-2.29 (m, 1H), 1.80-2.14 (m, 6H), 1.49-1.78 (m, 6H).
C
NMR (100 MHz, CDCl ): δ 162.32, 159.72, 152.43, 151.07, 144.68, 129.36, 125.88,
3
117.33, 111.60, 104.31, 72.37, 62.04, 57.09, 56.10, 54.35, 52.89, 36.76, 33.18, 32.83,
3
1
2.33, 25.11. IR (KBr) 3128, 2954, 2868, 2808, 2359, 1617, 1547, 1499, 1452, 1421,
-1
383, 1272, 1206, 1138, 1050, 1011, 965, 850, 818, 724, 679, 652 cm . HRMS calcd
+
for C H F N O [M + H] : 458.2474, found: 458.2483.
2
3
29
2
7
6
.1.6
2-(2,4-Difluorophenyl)-1-(4-(4-(methoxymethyl)-1H-1,2,3-triazol-1-yl)
piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8e)
1
Colorless oil: 40 mg, yield 51.0%. H NMR (400 MHz, CD OD)δ:8.38 (s, 1H),
3
8.00 (s, 1H), 7.78 (s, 1H), 7.46-7.59 (m, J = 6.78, 8.78, 8.78 Hz, 1H), 6.96 (ddd, J =
2.51, 9.10, 11.73 Hz, 1H), 6.88 (dt, J = 2.26, 8.41 Hz, 1H), 4.60-4.78 (m, 2H), 4.52 (s,
2H), 4.41-4.50 (m, 1H), 3.37 (s, 3H), 3.07 (d, J = 13.80 Hz, 1H), 2.95 (d, J = 11.80
1
1

ACCEPTED MANUSCRIPT
Hz, 1H), 2.88 (d, J = 14.05 Hz, 1H), 2.70-2.80 (m, 1H), 2.50-2.61 (m, 1H), 2.33-2.44
1
3
(
m, 1H), 2.06-2.19 (m, 2H), 1.93-2.05 (m, 2H). C NMR (100 MHz, CD OD): δ
3
1
7
2
8
4
6
62.20, 159.60, 149.68, 144.67, 144.03, 129.52, 125.46, 121.81, 110.72, 103.50,
3.73, 64.86, 62.76, 57.90, 56.96, 55.98, 53.98, 53.15, 32.22, 31.95. IR (KBr) 3406,
932, 2817, 2359, 1618, 1499, 1455, 1421, 1338, 1272, 1138, 1100, 1050, 1010, 965,
-1
+
52, 819, 791, 723, 679, 619, 601 cm HRMS calcd for C H F N O [M + H] :
.
20 25
2
7
2
34.2269, found: 434.2263.
.1.7 2-(2,4-Difluorophenyl)-1-(4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)
piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8f)
1
White solid: 20 mg, yield 33.3%, 68.5-69.0 ℃. H NMR (400 MHz, DMSO-d ) δ:
6
8
.30 (s, 1H), 7.95 (s, 1H), 7.74 (s, 1H), 7.34-7.46 (m, 1H), 7.16 (ddd, J = 2.26, 9.29,
11.80 Hz, 1H), 6.96 (dt, J = 2.26, 8.53 Hz, 1H), 5.69 (br s, 1H), 5.12 (t, J = 5.65 Hz,
1
1
4
1
5
1
H), 4.57 (s, 2H), 4.46 (d, J = 5.52 Hz, 2H), 4.28-4.40 (m, 1H), 2.90 (d, J = 13.80 Hz,
H), 2.84 (d, J = 11.80 Hz, 1H), 2.69-2.79 (m, 2H), 2.27-2.37 (m, 2H), 1.81-1.91 (m,
1
3
H). C NMR (100 MHz, DMSO-d ): δ 162.45, 159.72, 148.25, 142.88, 142.05,
6
30.15, 124.85, 121.80, 110.95, 102.40, 72.93, 64.66, 54.86, 56.28, 53.65, 52.26,
4.20, 31.82, 31.55. IR (KBr) 3405, 2928, 2822, 2360, 2030, 1499, 1422, 1386, 1272,
-1
206, 1137, 1102, 1056, 1010, 966, 872, 822, 791, 728 cm . HRMS calcd for
+
C H F N O [M + H] : 420.1954, found: 420.1963.
19
23
2
7
2
6
.1.8
2-(2,4-Difluorophenyl)-1-(4-(4-(1-hydroxyethyl)-1H-1,2,3-triazol-1-yl)
piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8g)
1
White solid: 46 mg, yield 50.5%, 64.0-65.0 ℃. H NMR (400 MHz, CD OD) δ: 1.53
3
1
2

ACCEPTED MANUSCRIPT
(d, J = 6.53 Hz, 3H), 1.96-2.15 (m, 4H), 2.39 (td, J = 11.29, 3.51 Hz, 1H), 2.55 (td, J
=
11.36, 3.14 Hz, 1H), 2.75 (d, J = 11.80 Hz, 1H), 2.88 (d, J = 13.80 Hz, 1H), 2.95 (d,
J = 11.80 Hz, 1H), 3.07 (d, J = 13.80 Hz, 1H), 4.37-4.50 (m, 1H), 4.62-4.76 (m, 2H),
4
.94-5.01 (m, 1H), 6.83-6.92 (m, 1H), 6.96 (ddd, J = 11.67, 9.03, 2.38 Hz, 1H),
1
3
7
.48-7.58 (m, 1H), 7.78 (s, 1H), 7.89 (s, 1H), 8.38 (s, 1H). C NMR (100 MHz,
CD OD): δ 162.21, 159.46, 152.01, 149.72, 144.71, 129.55, 126.86, 119.35, 110.74,
3
1
3
1
4
6
03.51, 73.72, 62.76, 62.26, 57.86, 55.99, 53.92, 53.19, 32.28, 32.00, 22.25. IR (KBr)
405, 2926, 2853, 2360, 2025, 1709, 1618, 1500, 1421, 1384, 1272, 1206, 1138, 1102,
-1
+
011, 965, 892, 851, 808, 723, 679 cm HRMS calcd for C H F N O [M + H] :
.
20 25
2
7
2
34.2305, found: 434.2312.
.1.9 2-(2,4-Difluorophenyl)-1-(4-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl)
piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8h)
1
Gray solid: 60 mg, yield 56.2%, 63.0-64.0 ℃. H NMR (400 MHz, CD OD)δ:
3
1
.93-2.14 (m, 4 H), 2.34-2.44 (m, 1 H), 2.54 (td, J = 11.17, 3.76 Hz, 1 H), 2.75 (d, J =
1.80 Hz, 1 H), 2.85-2.97 (m, 4 H), 3.07 (d, J = 13.80 Hz, 1 H), 3.80 (t, J = 6.65 Hz, 2
H), 4.42 (tt, J = 10.45, 5.24 Hz, 1 H), 4.64-4.78 (m, 2 H), 6.88 (td, J = 8.41, 2.26 Hz,
H), 6.96 (ddd, J = 11.80, 9.03, 2.26 Hz, 1 H), 7.47-7.57 (m, 1 H), 7.79 (d, J = 10.04
1
1
1
3
Hz, 2 H), 8.38 (s, 1 H). C NMR (100 MHz, CD OD): δ 162.20, 162.26, 152.09,
3
1
5
1
7
47.09, 131.98, 128.20, 123.09, 113.15, 105.88, 76.14, 65.13, 63.04, 60.15, 58.34,
6.30, 55.59, 34.62, 34.35, 30.89. IR (KBr) 3310, 3113, 2957, 2855, 2803, 2360, 2026,
615, 1515, 1498, 1420, 1384, 1306, 1274, 1140, 1098, 1051, 996, 965, 860, 805, 777,
-1
.
+
37, 680, 655, 625, 588, 533 cm HRMS calcd for C H F N O [M + H] :
20 25
2
7
2
1
3

ACCEPTED MANUSCRIPT
4
34.2318, found: 434.2323.
.1.10 3-(1-(1-(2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)
6
piperidin-4-yl)-1H-1,2,3-triazol-4-yl)propan-1-ol (8i)
1
Yellow oil: 30 mg, yield 36.2%. H NMR (400 MHz, CDCl ) δ:℃ 8.13 (s, 1H), 7.79 (s,
3
1
H), 7.55 (dt, J = 6.65, 8.97 Hz, 1H), 7.20-7.34 (m, 1H), 6.69-6.89 (m, 2H), 5.09 (br.
s., 1H), 4.45-4.60 (m, 2H), 4.23-4.37 (m, 1H), 3.68 (t, J = 6.02 Hz, 2H), 3.10 (d, J =
3.55 Hz, 1H), 2.79 (t, J = 7.40 Hz, 2H), 2.69-2.75 (m, 2H), 2.46-2.58 (m, 2H), 2.25
1
1
3
(
dt, J = 2.13, 11.73 Hz, 1H), 1.84-2.12 (m, 6H). C NMR (100 MHz, CDCl ): δ
3
1
7
3
1
62.24, 159.69, 151.16, 147.30, 144.72, 129.38, 125.83, 118.70, 111.75, 104.38,
2.43, 62.03, 61.88, 57.24, 56.11, 54.35, 52.94, 32.83, 32.35, 31.92, 22.17. IR (KBr)
405, 2932, 2857, 2360, 2341, 2025, 1617, 1551, 1499, 1420, 1384, 1272, 1207, 1137,
-1
056, 965, 851, 816, 723, 679, 654, 619, 590 cm HRMS calcd for C H F N O
2
.
21 27
2
7
+
[
M + H] : 448.2267, found: 448.2259.
6
.1.11 4-(1-(1-(2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)
piperidin-4-yl)-1H-1,2,3-triazol-4-yl)butan-1-ol (8j)
1
Yellow oil: 56 mg, yield 54.6%. H NMR (400 MHz, CDCl ) δ: 1.60-1.67 (m, 2H),
3
1
2
4
1
1
5
.72-1.80 (m, 2H), 1.82-2.17 (m, 6H), 2.26 (td, J = 11.80, 2.76 Hz, 1H), 2.48-2.60 (m,
H), 2.68-2.81 (m, 4H), 3.13 (dd, J = 13.55, 1.25 Hz, 1H), 3.68 (t, J = 6.40 Hz, 2H),
.29-4.39 (m, 1H), 4.50-4.60 (m, 2H), 6.77-6.87 (m, 2H), 7.26 (s, 1H), 7.53-7.62 (m,
13
H), 7.81 (s, 1H), 8.15 (s, 1 H). C NMR (100 MHz, CDCl ): δ 162.33, 159.71,
3
51.14, 147.80, 144.70, 129.28, 125.77, 118.43, 111.63, 104.36, 72.36, 62.40, 62.03,
7.16, 56.09, 54.36, 52.94, 32.84, 32.36, 32.10, 25.52, 25.28. IR (KBr) 3381, 2936,
1
4

ACCEPTED MANUSCRIPT
2
8
4
6
861, 2359, 2026, 1708, 1616, 1550, 1499, 1421, 1384, 1272, 1208, 1138, 995, 965,
-1
+
52, 817, 723, 679, 655, 620, 590, 514 cm HRMS calcd for C H F N O [M + H] :
.
22 29
2
7
2
62.2424, found: 462.2421.
.1.12 2-(2,4-Difluorophenyl)-1-(4-(4-(hydroxy(phenyl)methyl)-1H-1,2,3-triazol-1
-yl) piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8k)
1
White solid: 50 mg, yield 62.1%, 65.0-67.0 ℃. H NMR (400 MHz, CDCl ) δ: 8.11
3
(
s, 1H), 7.75 (s, 1H), 7.48-7.58 (m, 1H), 7.39-7.46 (m, 2H), 7.27-7.38 (m, 3H), 7.20 (s,
H), 6.74-6.84 (m, 2H), 6.00 (s, 1H), 4.96-5.16 (m, 1H), 4.45-4.56 (m, 2H), 4.22-4.34
m, 1H), 3.82 (br. s., 1H), 3.08 (d, J = 13.55 Hz, 1H), 2.68 (d, J = 13.55 Hz, 2H),
1
(
1
3
2
.41-2.56 (m, 2H), 2.15-2.26 (m, 1H), 1.81-2.08 (m, 4H). C NMR (100 MHz,
CDCl ): δ 162.08, 159.64, 151.18, 144.69, 141.97, 129.37, 128.59, 127.97, 126.35,
3
1
3
1
4
6
25.82, 119.93, 111.80, 104.37, 72.43, 69.07, 62.02, 57.46, 56.02, 54.30, 52.88, 32.76,
2.25. IR (KBr) 3423, 2923, 2360, 2026, 1618, 1498, 1420, 1384, 1272, 1137, 1047,
-1
+
013, 965, 850, 679, 619, 561 cm HRMS calcd for C H F N O [M + H] :
.
25 27
2
7
2
96.2167, found: 496.2255.
.1.13 1-(4-(4-Benzyl-1H-1,2,3-triazol-1-yl)piperidin-1-yl)-2-(2,4-difluorophenyl)
3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8l)
White solid: 42 mg, yield 50.0%, 70.0-71.5 ℃. H NMR (400 MHz, CDCl ) δ:
-
1
3
8
7
.39-8.44 (m, 2H), 8.24 (s, 1H), 8.14 (s, 1H), 7.81 (s, 1H), 7.56-7.65 (m, 2H),
.46-7.54 (m, 2H), 6.78-6.89 (m, 2H), 5.01 (br. s., 1H), 4.53-4.67 (m, 2H), 4.45-4.52
(m, 1H), 3.13 (d, J = 13.55 Hz, 1H), 2.73-2.85 (m, 2H), 2.54-2.64 (m, 2H), 2.25-2.36
1
3
(
m, 1H), 2.22 (d, J = 12.30 Hz, 1H), 2.03-2.15 (m, 2H), 1.89-2.01 (m, 1H). C NMR
1
5

ACCEPTED MANUSCRIPT
(
100 MHz, CDCl ): δ 162.18, 159.60, 150.48, 146.21, 140.66, 128.27, 127.39, 127.27,
3
1
3
1
26.54, 126.22, 118.24, 112.30, 105.70, 71.72, 61.62, 57.46, 56.02, 54.30, 52.88,
3.24, 32.76, 32.25. IR (KBr) 3288, 2964, 2817, 2361, 2028,1736, 1609, 1499, 1422,
-1
378, 1135, 1041, 965, 850, 799, 770, 728, 677, 588, 531 cm . HRMS calcd for
+
C H F N O [M + H] : 480.2341, found: 480.2344.
25
27
2
7
6
.1.14 Methyl 4-(1-(1-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperidin-4-yl)-1H-1,2,3-triazol-4-yl)butanoate (8m)
1
Yellow oil: 30 mg, yield 33.2%. H NMR (400 MHz, CDCl ) δ: 1.90-2.02 (m, 4H),
3
2
2
4
1
1
5
.03-2.17 (m, 2H), 2.23-2.31 (m, 1H), 2.38 (t, J = 7.40 Hz, 2H), 2.49-2.60 (m, 2H),
.69-2.79 (m, 4H), 3.13 (d, J = 13.55 Hz, 1H), 3.67 (s, 3H), 4.29-4.39 (m, 1H),
.50-4.61 (m, 2H), 6.79-6.88 (m, 2H), 7.28-7.30 (m, 1H), 7.53-7.62 (m, 1H), 7.81 (s,
1
3
H), 8.16 (s, 1 H). C NMR (100 MHz, CDCl ): δ 176.85, 162.44, 159.25, 151.88,
3
48.12, 144.32, 129.50, 126.02, 119.10, 112.05, 105.18, 72.78, 61.85, 57.26, 56.72,
4.65, 52.92, 51.79, 32.85, 32.41, 31.92, 28.72, 20.24. HRMS calcd for C H F N O
3
2
3
29
2
7
+
[
M + H] : 490.2158, found: 490.2162.
6
.1.15 4-(1-(1-(2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)
piperidin-4-yl)-1H-1,2,3-triazol-4-yl)butanoic acid (8n)
1
Yellow oil: 40 mg, yield 45.0%. H NMR (400 MHz, CD OD) δ: 1.97-2.12 (m, 2H),
3
2
.38-2.71 (m, 6H), 2.89-2.98 (m, 2H), 3.61 (br. s., 2H), 3.85-4.29 (m, 3H), 5.03-5.22
(m, 3H), 7.00-7.09 (m, 1H), 7.18 (ddd, J = 11.73, 8.97, 2.38 Hz, 1H), 7.52-7.64 (m,
1
3
1
H), 8.54 (br. s., 1H), 8.74 (s, 1H), 9.86 (s, 1 H). C NMR (100 MHz, CD OD): δ
3
1
78.65, 162.74, 159.33, 151.51, 148.02, 144.65, 123.22, 126.32, 119.52, 112.19,
1
6

ACCEPTED MANUSCRIPT
1
06.08, 72.08, 61.05, 58.13, 56.14, 54.35, 51.89, 32.47, 33.02, 31.54, 28.99, 21.79.
+
HRMS calcd for C H F N O [M + H] : 476.2216, found: 462.2230.
2
2
27
2
7
3
6
.1.16 Ethyl 1-(1-(2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl) piperidin-4-yl)-1H-1,2,3-triazole-4-carboxylate (8o)
1
Colorless oil: 36 mg, yield 52.1%. H NMR (400 MHz, CDCl ) δ: 8.12 (s, 1H),
3
8.05 (s, 1H), 7.79 (s, 1H), 7.52-7.59 (m, 1H), 6.77-6.85 (m, 2H), 5.01 (br. s., 1H),
4.49-4.59 (m, 2H), 4.37-4.44 (m, 3H), 3.11 (d, J = 13.80 Hz, 1H), 2.70-2.79 (m, 2H),
2.52-2.59 (m, 2H), 2.23-2.31 (m, 1H), 2.13-2.19 (m, 1H), 1.98-2.06 (m, 2H), 1.90 (dd,
1
3
J = 3.51, 12.05 Hz, 1H), 1.38 (t, J = 7.15 Hz, 3H). C NMR (100 MHz, CDCl ): δ
3
1
7
61.82, 159.64, 151.21, 144.70, 140.06, 129.35, 125.77, 125.60, 111.64, 104.37,
2.62, 61.99, 61.31, 57.80, 55.94, 54.15, 52.76, 32.76, 32.24, 14.29. HRMS calcd for
+
C H F N O [M + H] : 462.2060, found: 462.2057.
21
25
2
7
3
6
.1.17
1-(1-(2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)
piperidin-4-yl)-1H-1,2,3-triazole-4-carboxylic acid (8p)
1
White solid: 51 mg, yield 56.2%, 80.5-82.0 ℃. H NMR (400 MHz, CD OD) δ: 9.81
3
(s, 1H), 8.54-8.81 (m, 2H), 7.52-7.69 (m, 1H), 7.19 (t, J = 9.41 Hz, 1H), 6.94-7.12 (m,
1
H), 5.08-5.16 (m, 1H), 5.02 (br s, 1H), 4.91 (br s, 1H), 3.86-4.35 (m, 3H), 3.54 (d, J
1
3
=
11.29 Hz, 1H), 3.69 (br s, 1H), 3.37 (s, 1H), 2.35-2.70 (m, 4H). C NMR (100
MHz, CD OD): δ 166.45, 161.35, 159.24, 150.88, 141.70, 139.86, 129.55, 122.45,
3
1
21.50, 110.25, 103.85, 71.62, 61.65, 55.80, 56.25, 53.18, 52.21, 32.56, 32.25.
+
HRMS calcd for C H F N O [M + H] : 434.1747, found: 434.1749.
1
9
21
2
7
3
6
.1.18 2-(2,4-Difluorophenyl)-1-(4-(4-phenyl-1H-1,2,3-triazol-1-yl)piperidin-1-yl)
17

ACCEPTED MANUSCRIPT
-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8q)
1
White solid: 43 mg, yield 46.5%, 85.0-86.0 ℃. H NMR (400 MHz, CDCl ) δ:
3
8
.17 (s, 1H), 7.80-7.85 (m, 3H), 7.75 (s, 1H), 7.57-7.64 (m, 1H), 7.41-7.47 (m, 2H),
.32-7.38 (m, 1H), 6.82-6.89 (m, 2H), 5.11 (br. s., 1H), 4.53-4.63 (m, 2H), 4.42-4.49
7
(m, 1H), 3.16 (d, J = 13.55 Hz, 1H), 2.75-2.83 (m, 2H), 2.56-2.64 (m, 2H), 2.28-2.36
1
3
(
m, 1H), 1.97-2.23 (m, 4H). C NMR (100 MHz, CDCl ): δ 162.12, 159.66, 151.58,
3
1
1
2
8
46.05, 144.28, 131.25, 128.87, 127.55, 126.54, 125.22, 123.03, 116.88, 110.99,
05.26, 72.30, 62.80, 56.20, 55.95, 54.16, 52.45, 32.41, 32.02. IR (KBr) 3128, 2962,
814, 2359, 2232, 2025,1730, 1617, 1500, 1421, 1379, 1207, 1137, 1039, 965, 851,
-1
02, 777, 736, 723, 679, 620, 590, 531 cm HRMS calcd for C H F N O [M +
.
24 25
2
7
+
H] : 466.2161, found: 466.2183.
.1.19 2-(2,4-Difluorophenyl)-1-(4-(4-(pyridin-3-yl)-1H-1,2,3-triazol-1-yl)
6
piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8r)
1
White solid: 52 mg, yield 43.6%, 98.0-100.0 ℃. H NMR (400 MHz, CDCl ) δ:
3
8
7
1
2
.97 (br s, 1H), 8.58 (br s, 1H), 8.20 (d, J = 7.78 Hz, 1H), 8.15 (s, 1H), 7.82 (s, 2H),
.55-7.63 (m, 1H), 7.38 (dd, J = 4.89, 7.65 Hz, 1H), 6.80-6.88 (m, 2H), 4.57 (q, J =
4.31 Hz, 2H), 4.41-4.50 (m, 1H), 3.15 (d, J = 13.55 Hz, 1H), 2.72-2.84 (m, 2H),
1
3
.55-2.65 (m, 2H), 2.31 (t, J = 11.04 Hz, 1H), 1.95-2.23 (m, 4H). C NMR (100 MHz,
CDCl ): δ 162.30, 159.78, 151.20, 149.23, 146.93, 144.72, 132.99, 129.42, 126.75,
3
1
25.73, 117.85, 111.74, 104.37, 72.56, 62.07, 57.68, 56.02, 54.29, 52.91, 32.85, 32.37.
IR (KBr) 3423, 2955, 2360, 2026, 1618, 1499, 1420, 1384, 1272, 1138, 1272, 1138,
-1
+
9
66, 851, 808, 709, 679, 620 cm HRMS calcd for C H F N O [M + H] : 467.2181,
. 23 24 2 8
1
8

ACCEPTED MANUSCRIPT
found: 467.2190.
.1.20 1-(4-(4-(4-Bromophenyl)-1H-1,2,3-triazol-1-yl)piperidin-1-yl)-2-(2,4-
6
difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8s)
1
White solid: 60 mg, yield 62.0%, 88.0-90.5 ℃. H NMR (400 MHz, CDCl ) δ: 8.14
3
(s, 1H), 7.80 (s, 1H), 7.72 (s, 1H), 7.66 (d, J = 8.53 Hz, 2H), 7.48-7.61 (m, 3H),
6
1
1
1
5
2
1
.76-6.88 (m, 2H), 5.07 (br s, 1H), 4.48-4.62 (m, 2H), 4.34-4.46 (m, 1H), 3.12 (d, J =
3.55 Hz, 1H), 2.68-2.82 (m, 2H), 2.50-2.62 (m, 2H), 2.28 (t, J = 10.92 Hz, 1H),
1
3
.90-2.21 (m, 4H). C NMR (100 MHz, CDCl ): δ 162.02, 159.38, 151.21, 146.53,
3
44.70, 131.98, 129.77, 127.14, 125.82, 122.03, 117.44, 111.70, 104.36, 72.51, 62.35,
7.50, 56.03, 54.26, 52.89, 32.82, 32.35. IR (KBr) 3529, 3403, 3114, 3098, 3077,
961, 2938, 2857, 2810, 1615, 1495, 1477, 1241, 1305, 1272, 1210, 1139, 1084, 1069,
-1
011, 995, 964, 876, 849, 826, 736, 719, 719, 678, 653, 622, 607, 530, 510, 471 cm
.
+
HRMS calcd for C H BrF N O [M + H] : 544.1875, found: 544.1872.
2
4
24
2
7
6
.1.21
1-(4-(1-(1-(2-(2,4-Difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)
propyl)piperidin-4-yl)-1H-1,2,3-triazol-4-yl)phenyl)ethanone (8t)
1
White solid: 52 mg, yield 56.5%, 95.5-97.0 ℃. H NMR (400 MHz, DMSO-d )
6
δ:8.80 (s, 1H), 8.30 (s, 1H), 7.95-8.03 (m, 4H), 7.75 (s, 1H), 7.37-7.45 (m, 1H),
7
.14-7.21 (m, 1H), 6.97 (dt, J = 2.26, 8.41 Hz, 1H), 5.72 (s, 1H), 4.58 (s, 2H),
.40-4.49 (m, 1H), 2.85-2.97 (m, 2H), 2.80 (d, J = 11.80 Hz, 1H), 2.73 (d, J = 13.80
4
1
3
Hz, 1H), 2.58 (s, 3H), 2.36 (t, J = 11.29 Hz, 2H), 1.83-2.01 (m, 4H). C NMR (100
MHz, CDCl ): δ 162.60, 159.85, 151.26, 146.48, 144.74136.55, 135.01, 129.31,
3
1
29.05, 125.76, 118.36, 111.85, 104.41, 72.58, 62.10, 57.63, 56.04, 54.29, 52.93,
1
9

ACCEPTED MANUSCRIPT
3
1
6
5
6
2.88, 32.40, 26.63. IR (KBr) 3544, 3405, 3083, 2963, 2940, 2859, 2812, 2359, 1679,
613, 1508, 1494, 1422, 1309, 1273, 1211, 1141, 1012, 963, 875, 848, 835, 799, 774,
-1
+
87, 17, 594, 536 cm . HRMS calcd for C H F N O [M + H] : 508.2567, found:
2
6
27
2
7
2
08.2573.
.1.22 2-(2,4-Difluorophenyl)-1-(4-(4-(4-(dimethylamino)phenyl)-1H-1,2,3-triazol
-1-yl)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8u)
1
Brown solid: 30 mg, yield 30.4%, 92.0-93.5 ℃. H NMR (400 MHz, CDCl ) δ: 8.17
3
(
s, 1H), 7.83 (s, 1H), 7.69 (d, J = 8.78 Hz, 2H), 7.61 (s, 2H), 6.81-6.91 (m, 2H), 6.78
d, J = 8.53 Hz, 2H), 4.52-4.63 (m, 2H), 4.36-4.46 (m, 1H), 3.15 (d, J = 13.80 Hz,
(
1
1
5
6
H), 3.01 (s, 6H), 2.73-2.84 (m, 2H), 2.53-2.67 (m, 2H), 2.30 (t, J = 10.79 Hz, 1H),
+
.96-2.22 (m, 4H). HRMS calcd for C H F N O [M + H] : 509.2583, found:
26
30
2
8
09.2582.
.1.23
2-(2,4-Difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-(4-(4-(4-
(trifluoromethoxy) phenyl)-1H-1,2,3-triazol-1-yl)piperidin-1-yl)propan-2-ol (8v)
1
White solid: 53 mg, yield 58.0%, 86.0-88.0 ℃. H NMR (400 MHz, DMSO-d ) δ:
6
8.69 (s, 1H), 8.30 (s, 1H), 7.94 (d, J = 8.78 Hz, 2H), 7.75 (s, 1H), 7.37-7.47 (m, 3H),
7.13-7.21 (m, 1H), 6.92-7.03 (m, 1H), 5.71 (s, 1H), 4.52-4.62 (m, 2H), 4.37-4.49 (m,
1H), 2.85-2.96 (m, 2H), 2.79 (d, J = 11.54 Hz, 1H), 2.72 (d, J = 13.55 Hz, 1H), 2.35 (t,
1
3
J = 11.04 Hz, 2H), 1.83-2.01 (m, 4H). C NMR (100 MHz, CDCl ): δ 163.54, 159.48,
3
151.21, 148.91, 146.31, 129.30, 126.98, 125.85, 121.36, 117.52, 111.62, 104.37,
72.44, 62.02, 57.52, 56.04, 54.28, 52.87, 32.84, 32.36. IR (KBr) 3415, 3134, 3072,
3052, 2498, 2927, 2824, 2360, 2089, 1618, 1498, 1455, 1419, 1378, 1329, 1255, 1224,
2
0

ACCEPTED MANUSCRIPT
-1
1
172, 1140, 1112, 1015, 995, 876, 823, 783, 736, 678, 622 cm HRMS calcd for
.
+
C H F N O [M + H] : 550.2214, found: 550.2217.
25
24
5
7
2
6
.1.24
2-(2,4-Difluorophenyl)-1-(4-(4-(3-methoxyphenyl)-1H-1,2,3-triazol-1-yl)
piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8w)
1
White solid: 36 mg, yield 52.8%, 89.5-90.0 ℃. H NMR (400 MHz, CDCl ) δ:
3
1
3
6
7
1
.96-2.24 (m, 4H), 2.32 (t, J = 11.29 Hz, 1H), 2.53-2.66 (m, 2H), 2.72-2.85 (m, 2H),
.17 (d, J = 13.05 Hz, 1H), 3.89 (s, 3H), 4.45 (br. s., 1H), 4.53-4.64 (m, 2H),
.81-6.92 (m, 3H), 7.30-7.37 (m, 2H), 7.45 (d, J = 1.76 Hz, 1H), 7.57-7.64 (m, 1H),
1
3
.74 (s, 1H), 7.84 (s, 1H), 8.18 (s, 1 H). C NMR (100 MHz, CDCl ): δ 162.06,
3
59.76, 151.20, 147.45, 144.75, 131.86, 129.70, 129.39, 125.82, 118.03, 117.60,
114.26, 111.75, 110.73, 72.43, 62.06, 57.45, 56.07, 55.36, 54.34, 52.94, 32.86, 32.38.
IR (KBr) 3405, 2963, 2360, 2026, 1618, 1500, 1457, 1420, 1384, 1284, 1270, 1138,
-
1
+
1
4
6
049, 959, 874, 775, 682, 618, 518 cm HRMS calcd for C H F N O [M + H] :
. 25 27 2 7 2
96.2267, found: 496.2276.
.1.25 2-(2,4-Difluorophenyl)-1-(4-(4-(o-tolyl)-1H-1,2,3-triazol-1-yl)piperidin
-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (8x)
1
White solid: 45 mg, yield 57.2%, 84.0-84.5 ℃. H NMR (400 MHz, CDCl ) δ: 8.18
3
(s, 1H), 7.84 (s, 1H), 7.70-7.78 (m, 1H), 7.55-7.67 (m, 2H), 7.28 (s, 3H), 6.79-6.93 (m,
2
1
4
1
H), 5.12 (br. s., 1H), 4.52-4.65 (m, 2H), 4.41-4.51 (m, 1H), 3.16 (d, J = 13.55 Hz,
H), 2.73-2.85 (m, 2H), 2.55-2.67 (m, 2H), 2.48 (s, 3H), 2.28-2.38 (m, 1H), 2.03 (s,
1
3
H). C NMR (100 MHz, CDCl ): δ 162.55, 159.35, 151.77, 145.95, 144.68, 131.31,
3
30.88, 127.99, 127.25, 126.95, 126.76, 125.35, 123.13, 116.95, 111.24, 105.55,
2
1

ACCEPTED MANUSCRIPT
7
2
2.36, 62.15, 57.20, 56.24, 54.66, 53.05, 32.88, 32.42, 20.26. IR (KBr) 3129, 2952,
360, 2352, 2236, 1729, 1616, 1499, 1408, 1380, 1217, 966, 852, 781, 681, 679, 585
-1
+
cm HRMS calcd for C H F N O [M + H] : 480.2318, found: 480.2324.
.
25 27
2
7
6
.2 Molecular Docking
Homology model of CACYP51 were obtained from our previous studies [12].
GOLD [28] was used for molecular docking and the docking parameters were defined
the same with our previous reports [29].
Acknowledgements
This work was supported in part by National Natural Science Foundation of China
(grants 81222044), Key Project of Science and Technology of Shanghai (grant
1
3431900301), the 863 Hi-Tech Program of China (grant 2014AA020525), the
National Basic Research Program of China (grant 2014CB541800), and Shanghai
Municipal Health Bureau (grant XYQ2011038) for financial support.
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Figure Captions
Figure 1. Chemical structures triazole antifungal agents and lead compounds.
Figure 2. Design rationale of the target compounds.
Figure 3. The binding mode of compounds 8t (A) and 8v (B) in the active site of
CACYP51.
2
6

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Table 1. In vitro antifungal activities of the target compounds (MIC , µg/mL)
8
0
C. alb.
C. par.
C. neo.
C. gla.
A. fum.
T. rub
M. gyp
Comd.
8
a
0.125
0.125
0.125
0.5
1
1
1
0.5
1
0.5
0.25
0.25
4
64
16
1
1
8
4
8b
8
c
1
64
2
8
8d
8
8
>64
16
16
0.5
>64
>64
>64
>64
64
32
1
64
2
8
e
0.125
8
0.5
>64
>64
>64
>64
32
8
0.25
64
0.25
32
16
16
16
8
8
f
>64
>64
>64
>64
>64
16
>64
>64
>64
>64
>64
>64
4
8g
8
64
8h
4
>64
>64
32
8
i
8
8
j
4
8k
2
32
8
8
l
0.125
0.25
0.5
1
0.5
4
1
32
8m
4
2
64
4
8
8n
8
4
4
>64
>64
>64
>64
64
4
32
>64
>64
4
8
o
p
q
8
>64
64
1
>64
16
32
8
>64
>64
0.5
4
8
4
8
0.125
0.125
0.125
0.0625
0.25
0.0125
0.125
0.125
1
0.25
0.125
0.125
0.125
1
1
8
r
1
1
16
2
8
s
1
0.5
0.5
2
>64
64
0.25
1
8
t
0.5
2
2
8u
8
2
4
8v
2
0.125
0.125
0.25
2
0.5
2
8
0.5
4
4
8w
4
>64
>64
1
32
64
2
8
x
0.5
4
0.5
1
0.5
0.5
8
ITZ
FCZ
0.25
2
1
1
>64
32
a
Abbreviations: C. alb. Candida albicans; C. par. Candida parapsilosis; C. neo. Cryptococcus
neoformans; C. gla. Candida glabrata; A. fum. Aspergillus fumigatus; T. rub.
Trichophyton rubrum; M. gyp. Microsporum gypseum; FCZ: Fluconazole; ITZ:
Itraconazole.
27

ACCEPTED MANUSCRIPT
Figure 1. Chemical structures triazole antifungal agents and lead compounds.
2
8