Discovery of highly potent triazole antifungal agents with piperidine-oxadiazole side chains

Article abstract of DOI:10.1039/c4md00505h

Due to increasing incidence and mortality of invasive fungal infections, discovery and development of new generations of antifungal agents represents a challenging task. On the basis of our previously reported triazole-benzyloxypiperidinyl lead compound, a series of novel triazole antifungal agents containing piperidine-oxadiazole side chains were rationally designed and synthesized. Most of the target compounds showed excellent inhibitory activity against clinically important fungal pathogens. In particular, compounds 6g (MIC = 0.031 μg mL^-1) and 11b (MIC = 0.016 μg mL^-1) were highly active against Candida albicans including fluconazole-resistant strains. Moreover, they showed inhibitory activity against hyphal formation with low toxicity, which were promising leads for further development. This journal is

Full text of DOI:10.1039/c4md00505h

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Discovery of Highly Potent Triazole Antifungal Agents with
Piperdine-oxadiazole Side Chains
Xiaomeng He§, Yan Jiang§, Yongqiang Zhang, Shanchao Wu, Guoqiang Dong, Na
Liu, Yang Liu, Jianzhong Yao, Zhenyuan Miao, Yan Wang*, Wannian Zhang* and
Chunquan Sheng*
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai
200433, China
§These authors contributed equally to this work.
* To whom correspondence should be addressed. For C. Sheng: Phone/Fax,
86ꢀ21ꢀ81871239, Eꢀmail, shengcq@hotmail.com. For W. Zhang: Phone/Fax,
86ꢀ21ꢀ81870243, Eꢀmail, zhangwnk@hotmail.com. For Y. Wang: Phone/Fax,
86ꢀ21ꢀ81871359, Eꢀmail, wangyansmmu@126.com.
1

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Abstract
Due to increasing incidence and mortality of invasive fungal infections, discovery and
development of new generations of antifungal agents represents a challenging task.
On the basis of our previously reported triazoleꢀbenzyloxypiperidinyl lead compound,
a series of novel triazole antifungal agents containing piperdineꢀoxadiazoleside chains
were rationally designed and synthesized. Most of the target compounds showed
excellent inhibitory activity against clinically important fungal pathogens. In
particular, compounds 6g (MIC = 0.031 ꢀg/mL) and 11b (MIC = 0.016 ꢀg/mL) were
highly active against Candida albicans including fluconazoleꢀresistant strains.
Moreover, they showed inhibitory activity against hyphal formation with low toxicity,
which were promising leads for further development.
2

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Introduction
There has been a substantial increase in the incidence and mortality of invasive fungal
infections (IFIs) over the past two decades.1 Most patients infected with IFIs are
immunocompromised, such as patients with human immunodeficiency virus (HIV)
infection and recipients of the organ transplantation, stem cell transplantation, or
aggressive antitumor chemotherapy. Candida albicans (mortality rate: 20%–40%),
Cryptococcus neoformans (mortality rate: 50%–90%) and Aspergillus fumigatus
(mortality rate: 50%–90%) are the three most common species of lifeꢀthreatening IFIs.
2, 3
Because the immune system of the immunocompromised host cannot effectively
clear off the infection, the success of treatment of IFIs is largely dependent on the
efficacy of the antifungal agent. Unfortunately, effective antifungal agents are rather
limited, particularly in comparison to the number of clinically available antibacterial
agents.
Triazole antifungal agents, including fluconazole, voriconazole, itraconazole and
posaconazole (Fig. 1), are widely used as the firstꢀline antifungal drugs for treatment
and prophylaxis of IFIs. Triazole antifungals are inhibitors of lanosterol
14αꢀdemethylase (CYP51), which is a key enzyme in the biosynthesis of ergosterol
from lanosterol in the fungal cell membrane.4 Despite broad use of the triazoles in
clinic, they only have achieved modest success in reducing the high mortality rates
associatedwith IFIs. Limitations of triazole antifungal agents mainly include severe
resistance, narrow antifungal spectrum and therapeutic window, nonꢀoptimal
pharmacological and pharmacokinetic profiles, drugꢀrelated toxicity and undesirable
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drugꢀdrug interactions.5 Thus, discovery and development of new generations of
antifungal agents is an active research area. A number of new triazoles with improved
profiles have been reported.6ꢀ10 Among them, two candidate drugs, namely
isavuconazole and albaconazole, are under late stages of clinical trials.11
Fungal CYP51s from fungal pathogens are transmembrane proteins, whose crystal
structures have not been solved up to date. Previously, we constructed
threeꢀdimensional models of fungal CYP51s by homology modeling12ꢀ14 and
12, 15, 16
investigated the binding modes of substrate and triazole antifungal agents
.
Moreover, structural information of CYP51s facilitated the identification of a number
of new antifungal triazoles.17ꢀ19 Among them, the triazoleꢀbenzyloxypiperidinyl
derivative (Fig. 2) showed good antifungal activity with a broad spectrum,20
providing a good lead compound for further optimization. Herein, a series of novel
triazole derivatives with piperdineꢀoxadiazoleside chains were rationally designed and
synthesized. Most of the target compounds showed excellent antifungal activity
against a variety of fungal pathogens.
Chemistry
As
shown
in
Scheme
1,
treatment
of
1ꢀtertꢀbutyl
4ꢀmethyl
by
piperidineꢀ1,4ꢀdicarboxylate (1) with hydrazine hydrate afforded
2
a
hydrazinolysis reaction. Then, it was reacted with various acylchlorides to give
intermediates 3a-o. Ring closure reaction of intermediates 3a-o in presence of
4ꢀmethylbenzenesulfonic acid (TsCl) and triethylamine (Et3N) in THF yielded
1,3,4ꢀoxadiazole intermediates 4a-o. After deprotection of 4a-o, 1,3,4ꢀoxadiazoles
4

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5a-o were further reacted with the oxirane intermediate prepared by our previous
protocol16 to afford the target compounds 6a-o via ringꢀopen reactions. Chemical
synthesis of 1,2,4ꢀoxadiazole target compounds 11a-j is outlined in Scheme 2. The
construction of 1,2,4ꢀoxadiazole ring was different from that of 1,3,4ꢀoxadiazole.
Reaction of hydroxylamine with various nitriles afforded 8a-j, which was further
condensed with 1ꢀ(tertꢀButoxycarbonyl)isonipecoticacidin in the presence of
1ꢀethylꢀ3ꢀ(3ꢀdimethyllaminopropyl)carbodiimide
hydrochloride
(EDCI),
N,Nꢀdiisopropylethylamine (DIPEA) and 1ꢀhydroxybenzotriazole (HOBt) to give
1,2,4ꢀoxadiazoles 9a-j. Finally, target compounds 11a-j were obtained by a similar
protocol as described in Scheme 1.
Results and Discussion
Design Rationale
Currently, structural optimization of triazole antifungal agents was focused on
variation of the C3ꢀside chains because triazole, difluorophenyl and tertiary alcohol
are regarded as essential pharmacophores.21 Lead compound in this study had a
benzyloxypiperidinyl side chain (Fig. 2), which was fitted well with the active site of
C. albicans CYP51 (CACYP51).20 However, the lead compound has the unstable
benzyl ether substructure. Pharmacokinetic study revealed that its in vivo half time
(T1/2) was only about 1 h. Thus, it is highly desirable to design more stable triazoles
retaining good antifungal activity. Our previous study revealed that the benzyl ether
substructure of the lead compound could be replaced by the substituted
1,2,4ꢀtriazoles.17 Inspired by the results, herein substituted 1,2,4ꢀoxadiazoles and
5

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1,3,4ꢀoxadiazoles were used as the biosteres of the benzyl ether group to afford target
compounds 6a-o and 11a-j (Fig. 2). The selection of oxadiazole was based on the
following considerations. First, oxadiazole is a drugꢀlike privileged structure in many
therapeutic drugs and natural products.22 Second, oxadiazole can be used as a flat,
aromatic linker to place substituents in the appropriate orientation23 and thus favors
the interaction of C3ꢀside chain with CYP51. Third, introduction of 1,3,4ꢀoxadiazole
ring was proven to be an effective strategy to modulate lipophilicity and improve the
characteristics of the in vivo metabolic profile.22
In Vitro Antifungal Activity
The target compounds were assayed for inhibitory activity against clinically important
pathogenic fungi. Fluconazole was used as the reference drug. As shown in Table 1,
all of the title compounds were highly active against the tested fungal pathogens.
There are seven compounds (6b-d, 6g-h, 11a-b) proven to be more potent than
fluconazole against most of the tested fungal strains. Particularly, compounds 6g
(MIC = 0.031 ꢀg/mL) and 11b (MIC = 0.016 ꢀg/mL) revealed excellent activity
against C. albicans, which were 8 to 16 fold more active than fluconazole. In contrast,
the target compounds generally showed decreased activity against C. parapsilosis as
well as C. glabrata. Cryptococcosis, particularly Cryptococcal meningitisis, is one of
the most important fungal infections of humans with high mortality.24 To our delight,
compounds 6c, 6d, 6h, 6n, 11a showed better activity (MIC ≤ 0.125 ꢀg/mL) than
fluconazole (MIC = 0.25 ꢀg/mL). Invasive aspergillosis is another devastating illness
with mortality rates in some patient groups reaching as high as 90%.25However,
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fluconazole is inactive against A. fumigatus (MIC > 64 ꢀg/mL). Interestingly, most
compounds exhibited moderate to good activity against A. fumigatus. In particular,
compounds 11a and 11b revealed the best results (MIC = 2 ꢀg/mL). Furthermore, the
target compounds also showed good activity against dematophytes (T. rubrum and M.
gypseum) with MIC values ranging from 0.5 ꢀg/mL to 64 ꢀg/mL, which was
significantly more potent than fluconazole. Among the synthesized compounds, 6c, 6g,
6h, 11a and 11b showed excellent antifungal activity with a broad spectrum, which
were promising leads for the development of new generations of triazole antifungal
agents.
Notably, compounds 6g and 11b also had potent activity against
fluconazoleꢀresistant strains (Table 2). Fluconazoleꢀresistant C. albicans strains 103,
805 and 0710922 were used in this study. Compounds 6g and 11b exhibited
antifungal effect, with MIC50 values ranging from 0.0625 to 0.25 ꢀg/mL and MIC80
values ranging from 4 to 16 ꢀg/mL. In contrast, the MIC50 values of fluconazole
ranged from 256 to 512 ꢀg/mL, and the MIC80 of fluconazole is more than 1024
ꢀg/mL. The results indicated that they might have therapeutic potential to overcome
triazole resistance.
Structure-activity Relationships
From the antifungal activity data, preliminary structureꢀactivity relationships (SARs)
for both series were obtained. Generally, both of the substituted 1,2,4ꢀoxadiazoles and
1,3,4ꢀoxadiazoles showed broad–spectrum antifungal activity, validating the
effectiveness of incorporating oxadiazoles in the side chain of triazole antifungal
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agents. For the substitutions on oxadiazole, there was obvious difference between
1,2,4ꢀoxadiazole and 1,3,4ꢀoxadiazole series. For the 1,2,4ꢀoxadiazole derivatives, the
replacement of the phenyl group of compound 6b with pridinyl group (6a and 6l) led
to decrease of the antifungal activity. In contrast, the corresponding furan derivative
(6n) showed comparable or superior activity to compound 6b. Substitutions on the
phenyl group of 6b had various effects on the antifungal activity. Introduction of
4ꢀmethoxyl (6f), 4ꢀtrifluoromethyl (6g), and 4ꢀfluoro (6h) substitutions were
favorable for the antifungal activity. Particularly, the 4ꢀtrifluoromethyl derivative 6g
showed the best activity against C. albicans (MIC = 0.031 ꢀg/mL). Interestingly, the
other two fluorineꢀcontaining compounds (6c and 6d) also showed excellent
antifungal activity. In contrast, introduction of 4ꢀchloro (6j), 4ꢀethyl (6k) and 4ꢀnitro
(6o) substitutions resulted in decreased antifungal activity. Similarly, incorporating
2ꢀmethyl (6e) and 3ꢀmethyl (6i) groups or replacing the phenyl group with more steric
naphthyl (6m) group also had negative effects on the antifungal activity. For the
1,3,4ꢀoxadiazole derivatives, compounds with heterocyclic substitutions were
generally more active than the substituted phenyl derivatives with the exception of
compound 11b. The 4ꢀmethoxylphenyl derivative 11b was the most active C. albicans
inhibitor (MIC = 0.016 ꢀg/mL) among the synthesized compounds. Unlike the
1,2,4ꢀoxadiazole derivatives, the fluorineꢀcontaining derivatives (11f, 11i and 11j)
only showed moderate antifungal activity. Among the heterocyclic analogues, the
pyridinyl derivative 11a was the most active one with a broad spectrum.
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Binding Mode of Compounds 6g and 11b with the Active Site of CACYP51
In order to investigate the binding mode of the designed triazoles, two highly active
compounds, 6g and 11b, were docked into the active site of CACYP51. As shown in
Fig. 3, the triazole ring and ifluorophenyl group of compounds 6g and 11b formed
coordination bond and hydrophobic interactions with the heme group and the
hydrophobic pocket (Phe126, Ile131 and Tyr132), respectively. The side chains of the
two compounds were extended into the S4 pocket of the active site of CACYP51. The
oxadiazoyl and piperdinyl groups interacted with Leu376, Met508, Leu121, Ile379
and Tyr118 through hydrophobic and Van der Waals interactions. The substitutions on
the oxadiazole ring were located into a hydrophobic pocket lined with Tyr505, Gly65
and Tyr64. The magnitude of hydrophobic interactions of various substitutions with
this pocket played an important role for the binding affinity and antifungal activity.
Anti-hyphal Activity and Toxicity Assay
We further investigated the activity of 6g and 11b against yeastꢀtoꢀhypha transition of
C. albicans with fluconazole as the control drug. At 1 ꢀg/mL or higher concentrations,
compounds 6g and 11b exhibited mild activity against hyphal formation, with less
hyphae and more yeasts or pseudohyphal cells compared with the group without drug
treatment (Fig. 4). No obvious difference was observed between 6g, 11b and
fluconazole.
Caenorhabditis elegans were used to evaluate the toxicity of compounds 6g and
11b at the concentrations ranging from 1 ꢀg/mL to 160 ꢀg/mL. The results revealed
that no toxicity was observed at any concentration tested (Fig. 5). More specifically,
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no more worm was found dead after drug treatment for 6 days compared with the
drug free control group.
Conclusion
In summary, a series of novel triazole antifungal agents were rationally designed and
synthesized by incorporating oxadiazole in the C3ꢀside chain. Most of the target
compounds showed excellent antifungal activity with a broad spectrum. In particular,
compounds 6g and 11b were highly active against a variety of fungal pathogens,
which can be used as good lead compounds for further development. Molecular
docking studies revealed that the oxadiazole derivatives interacted with CACYP51
mainly through hydrophobic and Van der Waals interactions. Further evaluation and
optimization of the highly potent compounds are in progress.
Experimental Section
General procedure for the synthesis of compounds
1
All of the H and 13C nuclear magnetic resonance (NMR) spectra were recorded on
aBruker 300 or 600 spectrometer with TMS as the internal standard. Chemical shifts
(δvalues) and coupling constants (J values) are given in ppm and Hz, respectively.
ESI mass spectra were performed on a Trap LCꢀMS spectrometer. Highꢀresolution
mass spectrometrydata were collected on anAgilent Technologies QꢀTOFLCꢀMS
spectrometer.TLC analysis was carried out onsilica gel plates GF254 (Qingdao
Haiyang Chemical, China). Silica gelcolumn chromatography was performed with
Silica gel 60 G(Qingdao Haiyang Chemical, China). Commercial solvents were used
without any pretreatment. The target products were analyzed by HPLC (C18 column,
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460*250 mm, MeOH : H2O = 70: 30, 254 nm, 0.8 mL/min) and their purity was
larger than 96%.
tert-Butyl 4-(2-nicotinoylhydrazinecarbonyl)piperidine-1-carboxylate (3a)
1ꢀtertꢀButyl 4ꢀmethyl piperidineꢀ1,4ꢀdicarboxylate (2.000 g, 8.23 mmol, 1 eq) was
dissolved in the mixed solution of hydrazine hydrate and ethanol (1:1, 30 mL) and
then stirred overnight at room temperature. After evaporating the ethanol, the residue
was poured into water, extracted with DCM (3 × 50 mL), dried with Na2SO4, filtered
and evaporated under reduced pressure to give 2 as white solid (1.80 g, 95%). Then,
product 2 (1.800 g, 7.40 mmol, 1 eq) and Et3N (1.120 g, 11.10 mmol, 1.5 eq) were
dissolved in DCM (180 mL), and was slowly added nicotinoyl chloride (1.980 g,
11.10 mmol, 1.5 eq) under ice bath. The reaction solution was stirred at room
temperature for 2 h. After evaporation, the crude product was purified by silica gel
column chromatography (eluents: EtOAc : MeOH = 50:1ꢀ30:1) to afford compound
1
3a as white solid (1.80 g, 70%). HNMR (600 MHz, d6ꢀDMSO) δ 10.53 (s, 1H,
NHNH), 9.88 (s, 1H, NHNH), 9.01 (d, J = 2.2 Hz, 1H, pyridineꢀH), 8.75 (dd, J = 1.6
Hz, 4.8 Hz, 1H, pyridineꢀH), 8.20 (dt, J = 1.7 Hz, 7.9 Hz, 1H, pyridineꢀH), 7.54 (dd, J
= 4.8 Hz, 7.9 Hz, 1H, pyridineꢀH), 3.96 (m, 2H, piperidinꢀ2ꢀCH2), 2.79 (m, 2H,
piperidinꢀ 6ꢀCH2), 2.47 (m, 1H, piperidinꢀ4ꢀCH), 1.73 (m, 2H, piperidinꢀ5ꢀCH2), 1.48
(m, 2H, piperidinꢀ3ꢀCH2), 1.40 (s, 9H, C(CH3)3); MS (ESI) m/z: 347.3 (MꢀH).
tert-Butyl 4-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)piperidine-1-carboxylate (4a)
Compound 3a (1.800 g, 5.17 mmol, 1 eq) and Et3N (3.140 g, 31.02 mmol, 6 eq) were
dissolved in dry THF (100 mL), and were added pꢀtoluene sulfonyl chloride (2.960 g,
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15.51 mmol, 3 eq). The mixture was stirred overnight at 50 ℃. The crude product
was purified by silica gel column chromatography (eluents: EtOAc : MeOH =
1
80:1ꢀ50:1) to give 4a as faint yellow solid (1.200 g, 71%). HNMR (600 MHz,
d6ꢀDMSO) δ 9.15 (s, 1H, pyridineꢀH), 8.79 (dd, J = 4.8 Hz, 1H, pyridineꢀH), 8.35 (d,
J = 8.0 Hz, 1H, pyridineꢀH), 7.62 (dd, J = 4.8 Hz, 8.0 Hz, 1H, pyridineꢀH), 3.93 (m,
2H, piperidinꢀ2ꢀCH2), 3.29 (m, 1H, piperidinꢀ4ꢀCH), 3.00 (m, 2H, piperidinꢀ6ꢀCH2),
2.07 (m, 2H, piperidinꢀ3ꢀCH2), 1.68 (m, 2H, piperidinꢀ5ꢀCH2), 1.41 (s, 9H, C(CH3)3);
HRMS (ESI) calcd for C17H22N4O3 [M+H]+ = 331.1765, found 331.1765.
2-(2,4-Ddifluorophenyl)-1-(4-(5-(pyridin-3-yl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl
)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6a)
A solution of intermediate 4a (0.240 g, 0.72 mmol, 1 eq) and trifluoroacetic acid (3.0
mL) in DCM (50 mL) were stirred at room temperature for 10 h. Then, the reaction
mixture was neutralized by adding aqueous K2CO3 (3.000 g) slowly. The DCM layer
was separated, and the water layer was extracted with DCM. The organic layer was
combined, dried over anhydrous Na2SO4 and filtrated. Compound 5a was obtained by
evaporating under reduced pressure (yellow solid, 0.15 g, 91%). The product was
used directly in the next step without further purification. A solution of 5a (0.140 g,
0.61 mmol, 1 eq), oxirane intermediate（0.20 g, 1 eq）and Et3N (1 mL) in EtOH were
refluxed at 78℃ for 5 h. The solvent was removed under the reduced pressure and
the residue was purified by silica gel column chromatography (eluents: DCM : MeOH
= 30:1) to afford target product 6a (yellow powder, 0.15 g, 52.8%). 1HNMR (300
MHz, CDCl3) δ 9.22 (d, J = 1.5 Hz, 1H, pyridineꢀH), 8.76 (dd, J = 1.5, 4.8 Hz, 1H,
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pyridineꢀH), 8.32(dt, J = 1.7, 7.9 Hz, 1H, pyridineꢀH),8.16 (s, 1H, TriazC3ꢀH), 7.80 (s,
1H, TriazC5ꢀH), 7.51 ꢀ 7.64 (m, 1H, pyridineꢀH), 7.45 (dd, J = 4.8, 8.2 Hz, 1H, ArꢀH),
6.83 (m, 2H, ArꢀH), 4.56 (d, J = 14.6 Hz, 1H, C1ꢀHa), 4.50 (d, J = 14.6 Hz, 1H,
C1ꢀHb), 3.12 (d, J = 13.8 Hz, 1H, C3ꢀHa), 2.89 – 3.00 (m, 1H, C3ꢀHb), 2.69 (d, J =
13.8 Hz, 2H, piperidinꢀCH2), 2.38 ꢀ2.58 (m, 2H, piperidinꢀCH2), 2.23 (t, J = 11.3 Hz,
1H, piperidinꢀ4ꢀCH), 2.07 (t, J = 11.6 Hz, 1H, piperidinꢀCH2), 1.72ꢀ2.00 (m, 3H,
piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 169.0, 162.7 (1JCF = 248.2 Hz), 162.6,
158.9 (1JCF = 247.1 Hz), 152.4, 151.1, 147.7, 144.7, 134.1, 129.3, 126.1, 123.8, 120.4,
111.6, 104.3, 72.1, 62.5, 56.1, 54.5, 53.4, 32.7, 29.7, 29.2; MS (ESI) m/z: 468.38
(M+H). HRMS (ESI) calcd for C23H23F2N7O2 [M+H]+ = 468.1962, found 468.1960.
The synthetic procedure for compounds 6b-o was similar to the synthesis of
compound 6a.
2-(2,4-Difluorophenyl)-1-(4-(5-phenyl-1,3,4-oxadiazol-2-yl)piperidin-1-yl)-3-(1H-
1,2,4-triazol-1-yl)propan-2-ol (6b)
1HNMR (300 MHz, CDCl3) δ 8.16 (s, 1H, TriazC3ꢀH), 8.00 (dd, J = 2.1, 7.8 Hz, 2H,
ArꢀH), 7.79 (s, 1H, TriazC5ꢀH), 7.39ꢀ7.65 (m, 4H, ArꢀH), 6.82 (m, 2H, ArꢀH), 4.56 (d,
J = 14.6 Hz, 1H, C1ꢀHa), 4.50 (d, J = 14.6 Hz, 1H, C1ꢀHb), 3.11 (d, J = 13.6 Hz, 1H,
C3ꢀHa), 2.91 (d, J = 4.5 Hz, 1H, C3ꢀHb), 2.69 (d, J = 12.2 Hz, 2H, piperidinꢀCH2),
2.47 (m, 2H, piperidinꢀCH2), 2.22 (t, J = 10.1 Hz, 1H, piperidinꢀ4ꢀCH), 1.78ꢀ2.11 (m,
4H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 168.4, 164.6, 162.8 (1JCF = 246.0
Hz), 158.9 (1JCF = 246.0 Hz), 151.0, 144.6, 131.6, 129.3, 129.0, 126.7, 126.1, 123.8,
111.6, 104.3, 72.1, 62.5, 56.3, 54.5, 53.4, 32.6, 29.6, 29.2; MS (ESI) m/z: 489.3
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(M+Na). HRMS (ESI) calcd for C24H24F2N6O2 [M+H]+ = 467.1933, found 467.1932.
2-(2,4-Difluorophenyl)-1-(4-(5-(2,6-difluorophenyl)-1,3,4-oxadiazol-2-yl)piperidin
-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6c)
1HNMR (300 MHz, CDCl3) δ 8.15 (s, 1H, TriazC3ꢀH), 7.79 (s, 1H, TriazC5ꢀH), 7.72
(m, 1H, ArꢀH), 7.56 (m, 1H, ArꢀH), 7.21 (t, J = 5.9 Hz, 2H, ArꢀH), 6.82 (m, 2H, ArꢀH),
5.20 (brs, 1H, OH), 4.56 (d, J = 14.6 Hz, 1H, C1ꢀHa), 4.50 (d, J = 14.6 Hz, 1H,
C1ꢀHb), 3.11 (d, J = 13.2 Hz, 1H, C3ꢀHa), 2.95 (m, 1H, C3ꢀHb), 2.69 (d, J = 13.2 Hz,
2H, piperidinꢀCH2), 2.47 (t, J = 9.8 Hz, 2H, piperidinꢀCH2), 2.21 (t, J = 9.8 Hz, 1H,
piperidinꢀ4ꢀCH), 1.76 ꢀ 2.11 (m, 4H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ
169.1, 162.4 (1JCF = 249.6 Hz), 160.6, 158.9 (1JCF = 247.6 Hz), 158.3 (1JCF = 244.7
Hz), 155.9 (1JCF = 254.5 Hz), 151.0, 144.6, 129.3, 126.1, 120.0, 118.4, 115.9, 113.4,
111.6, 104.3, 72.1, 62.5, 56.2, 54.5, 53.4, 32.5, 29.6, 29.1; MS (ESI) m/z: 525.3
(M+Na). HRMS (ESI) calcd for C24H22F4N6O2 [M+H]+ = 503.1724, found 503.1724.
2-(2,4-Difluorophenyl)-1-(4-(5-(2-fluorophenyl)-1,3,4-oxadiazol-2-yl)piperidin-1-
yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6d)
1HNMR (300 MHz, CDCl3) δ 8.15 (s,1H, TriazC3ꢀH), 8.01 (t, J = 7.3 Hz, 1H, ArꢀH),
7.78(s,1H, TriazC5ꢀH), 7.53 (m, 2H, ArꢀH), 7.25 (m, 2H, ArꢀH), 6.80 (m, 2H, ArꢀH),
4.56 (d, J = 14.6 Hz, 1H, C1ꢀHa), 4.50 (d, J = 14.6 Hz, 1H, C1ꢀHb), 3.10 (d, J = 13.3
Hz, 1H, C3ꢀHa), 2.93 (m, 1H, C3ꢀHb), 2.68 (d, J = 13.8 Hz, 2H, piperidinꢀCH2), 2.47
(m, 2H, piperidinꢀCH2), 2.22 (t, J = 11.6 Hz, 1H, piperidinꢀ4ꢀCH), 2.07 (d, J = 11.6
Hz, 1H, piperidinꢀCH2), 1.76 ꢀ 2.11 (m, 3H, piperidinꢀCH2); 13CNMR (75 MHz,
CDCl3): δ 168.8, 162.8 (1JCF = 248.5 Hz), 161.5, 159.8 (1JCF = 251.0 Hz), 158.9 (1JCF
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= 244.8 Hz), 151.0, 144.6, 133.4, 129.7, 129.3, 126.0, 124.6, 116.9 (2JCF = 20.2 Hz),
112.3, 111.5, 104.3, 72.0, 62.5, 56.2, 54.5, 53.4, 32.5, 29.5, 29.1; MS (ESI) m/z: 507.3
(M+Na). HRMS (ESI) calcd for C24H23F3N6O2 [M+H]+ = 485.1825, found 485.1824.
2-(2,4-Difluorophenyl)-1-(4-(5-(o-tolyl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl)-3-(1H
-1,2,4-triazol-1-yl)propan-2-ol (6e)
1HNMR (300 MHz, d6ꢀDMSO) δ 8.18 (s, 1H, TriazC3ꢀH), 7.86 (d, J = 7.7 Hz, 1H,
ArꢀH), 7.79 (s, 1H, TriazC5ꢀH), 7.66 (m, 1H, ArꢀH), 7.27 ꢀ 7.48 (m, 3H, ArꢀH), 6.85
(m, 2H, ArꢀH), 5.66(brs, 1H, OH), 4.52 (m, 2H, C1ꢀH), 2.86 ꢀ 3.32 (m, 4H, C3ꢀH,
piperidinꢀCH2), 2.68 (s, 1H, piperidinꢀ4ꢀCH), 2.37 ꢀ 2.61 (m, 2H, piperidinꢀCH2), 2.14
ꢀ 2.37 (m, 2H, piperidinꢀCH2), 1.88 ꢀ 2.13 (m, 3H, CH3) ; MS (ESI) m/z: 481.4 (M+1).
HRMS (ESI) calcd for C25H26F2N6O2 [M+H]+ = 481.2142, found 481.2142.
2-(2,4-Difluorophenyl)-1-(4-(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)piperidin-
1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6f)
1HNMR (300 MHz, d6ꢀDMSO) δ 8.31 (s,1H, TriazC3ꢀH), 7.90 (d, J = 8.8 Hz, 2H,
ArꢀH), 7.75 (s, 1H, TriazC5ꢀH), 7.41 (m, 1H, ArꢀH), 7.20 (m, 1H, ArꢀH), 7.12 (d, J =
8.8 Hz, 2H, ArꢀH), 6.97 (t, J = 8.5 Hz, 1H, ArꢀH), 5.66 (brs, 1H, OH), 4.58 (m, 2H,
C1ꢀH), 3.84 (s, 3H, OCH3), 2.88 (d, J = 15.3 Hz, 2H, C3ꢀH), 2.80 (d, J = 10.9 Hz,
1H, piperidinꢀH), 2.71 (d, J = 13.8 Hz, 2H, piperidinꢀH), 2.29 (t, J = 8.7 Hz, 2H,
piperidinꢀH), 1.91 (m, 2H, piperidinꢀH), 1.68 (q, J = 10.9, 2H, piperidinꢀH);13CNMR
(75 MHz, CDCl3): δ 167.9, 164.5, 162.7 (1JCF = 249.8 Hz), 162.2, 158.8 (1JCF = 244.3
Hz), 151.0, 144.9, 129.3, 128.5, 126.1, 116.4, 114.4, 111.5, 104.4, 72.1, 62.6, 56.3,
55.4, 54.6, 53.4, 32.7, 29.6, 29.2; MS (ESI) m/z: 497.3 (M+1). HRMS (ESI) calcd for
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C25H26F2N6O3 [M+H]+ = 497.2022, found 497.2022.
2-(2,4-Difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-(4-(5-(4-(trifluoromethyl)pheny
l)-1,3,4-oxadiazol-2-yl)piperidin-1-yl)propan-2-ol (6g)
1HNMR (300 MHz, CDCl3) δ 8.14 (s, 2H, ArꢀH), 8.11 (s, 1H, TriazC3ꢀH), 7.78 (s, 1H,
TriazC5ꢀH), 7.74 (d, J = 8.3 Hz, 2H, ArꢀH), 7.56 (m, 1H, ArꢀH), 6.81 (m, 2H, ArꢀH),
5.18 (brs, 1H, OH), 4.53 (m, 2H, C1ꢀH), 3.11 (d, J = 13.6 Hz, 1H, C3ꢀHa), 2.94 (m,
1H, C3ꢀHb), 2.70 (d, J = 13.6 Hz, 2H, piperidinꢀCH2), 2.48 (m, 2H, piperidinꢀCH2),
2.23 (t, J = 10.8 Hz, 1H, piperidinꢀ4ꢀCH), 2.08 (m, 1H, piperidinꢀCH2), 1.76 ꢀ 1.99 (m,
3H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 169.0, 163.5, 162.6 (1JCF = 249.5
Hz), 158.7 (1JCF = 248.3 Hz), 151.0, 144.7, 133.3, 129.3, 127.1, 126.1, 125.3, 121.7,
111.6, 104.2, 71.9, 62.2, 56.1, 54.4, 53.3, 32.7, 29.6, 29.2; MS (ESI) m/z: 535.3
(M+H). HRMS (ESI) calcd for C25H23F5N6O2 [M+H]+ = 535.1821, found 535.1821.
2-(2,4-Difluorophenyl)-1-(4-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)piperidin-1-
yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6h)
1HNMR (300 MHz, CDCl3) δ 8.14 (s, 1H, TriazC3ꢀH), 7.94ꢀ8.05 (m, 2H, ArꢀH), 7.78
(s, 1H, TriazC5ꢀH), 7.55 (m, 1H, ArꢀH), 7.16 (t, J = 8.4 Hz, 2H, ArꢀH), 6.73 ꢀ 6.87 (m,
2H, ArꢀH), 4.52 (m, 2H, C1ꢀH), 3.09 (d, J = 13.2 Hz, 1H, C3ꢀHa), 2.90 (m, 1H, C3ꢀHb),
2.68 (d, J = 13.4 Hz, 2H, piperidinꢀCH2), 2.46 (m, 2H, piperidinꢀCH2), 2.20 (t, J =
11.2 Hz, 1H, piperidinꢀ4ꢀCH), 2.05 (m, 1H, piperidinꢀCH2), 1.74 ꢀ 1.94 (m, 3H,
piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 168.4, 164.7 (1JCF = 252.4 Hz), 163.8,
162.7 (1JCF = 250.2 Hz), 159.0 (1JCF = 247.3 Hz), 151.0, 144.6, 129.3, 129.1, 128.9,
126.1, 120.2, 116.5, 116.2, 111.6, 104.3, 72.1 , 62.4, 56.2, 54.5, 53.4, 32.6, 29.6, 29.2;
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MS (ESI) m/z: 485.3 (M+H). HRMS (ESI) calcd for C24H23F3N6O2 [M+H]+ =
485.1825, found 485.1825.
2-(2,4-Difluorophenyl)-1-(4-(5-(m-tolyl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl)-3-(1
H-1,2,4-triazol-1-yl)propan-2-ol (6i)
1HNMR (300 MHz, CDCl3) δ 8.14 (s, 1H, TriazC3ꢀH), 7.81(s, 1H, TriazC5ꢀH), 7.78 (s,
2H, ArꢀH), 7.55 (q, J = 9.0 Hz, 1H, ArꢀH), 7.33 (m, 2H, ArꢀH), 6.80 (m, 2H, ArꢀH),
4.52 (m, 2H, C1ꢀH), 3.10 (d, J = 14.0 Hz, 1H, C3ꢀHa), 2.90 (m, 1H, C3ꢀHb), 2.69 (d, J
= 12.7 Hz, 2H, piperidinꢀCH2), 2.46 (m, 2H, piperidinꢀCH2), 2.40 (s, 3H, CH3), 2.21
(t, J = 11.8 Hz, 1H, piperidinꢀ4ꢀCH), 2.06 (m, 1H, piperidinꢀCH2), 1.77 ꢀ 1.99 (m, 3H,
piperidinꢀCH2) ; 13CNMR (75 MHz, CDCl3): δ 168.2, 164.8, 162.6 (1JCF = 249.1 Hz),
158.8 (1JCF = 246.8 Hz), 151.0, 144.7, 138.9, 132.4, 129.3, 128.9, 127.3, 125.9, 123.9,
123.7, 111.6, 104.3, 72.0, 62.4, 56.3, 54.5, 53.4, 32.4, 29.5, 29.1, 21.3; MS (ESI) m/z:
481.4 (M+H). HRMS (ESI) calcd for C25H26F2N6O2 [M+H]+ = 481.2131, found
481.2131.
1-(4-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl)-2-(2,4-difluorophen
yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6j)
1HNMR (300 MHz, CDCl3) δ 8.18 (s, 1H, TriazC3ꢀH), 7.94 (d, J = 8.8 Hz, 2H, ArꢀH),
7.80 (s, 1H, TriazC5ꢀH), 7.56 (q, J = 9.4 Hz, 1H, ArꢀH), 7.46 (d, J = 8.5 Hz, 2H, ArꢀH),
6.81 (m, 2H, ArꢀH), 4.53 (m, 2H, C1ꢀH), 3.12 (d, J = 13.6 Hz, 1H, C3ꢀHa), 2.92 (m,
1H, C3ꢀHb), 2.71 (d, J = 12.8 Hz, 2H, piperidinꢀCH2), 2.48 (m, 2H, piperidinꢀCH2),
13
2.23 (m, 1H, piperidinꢀ4ꢀCH), 1.82ꢀ2.08 (m, 4H, piperidinꢀCH2); CNMR (75 MHz,
CDCl3): δ 168.5, 164.3, 162.6 (1JCF = 248.7 Hz), 159.0 (1JCF = 248.7 Hz), 150.9,
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144.7, 137.9, 129.4, 129.3, 128.0, 126.0, 122.3, 111.6, 104.3, 72.0, 62.4, 56.3, 54.5,
53.4, 32.6, 29.6, 29.2; MS (ESI) m/z: 501.4 (M+H). HRMS (ESI) calcd for
C24H23ClF2N6O2 [M+H]+ = 501.1523, found 501.1523.
2-(2,4-Difluorophenyl)-1-(4-(5-(4-ethylphenyl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl
)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6k)
1HNMR (300 MHz, CDCl3) δ 8.44 (s, 1H, TriazC3ꢀH), 7.91 (d, J = 8.1 Hz, 1H, ArꢀH),
7.70 (s, 1H, TriazC5ꢀH), 7.53 (d, J = 7.2 Hz, 1H, ArꢀH), 7.31 (d, J = 8.0 Hz, 2H, ArꢀH),
7.04 (s, 1H, ArꢀH), 6.82 (t, J = 8.8 Hz, 2H, ArꢀH), 4.57 (m, 2H, C1ꢀH), 3.17 (m, 1H,
C3ꢀHa), 2.98 (m, 1H, C3ꢀHb), 2.72 (d, J = 7.4 Hz, 2H, piperidinꢀCH2), 2.43 (m, 2H,
piperidinꢀCH2), 2.33 (m, 1H, piperidinꢀ4ꢀCH), 2.22 (m, J = 7.4 Hz, 2H, CH2CH3),
1.91 ꢀ 2.06 (m, 4H, piperidinꢀCH2), 1.17 (m, 3H, CH2CH3); MS (ESI) m/z: 495.5
(M+H). HRMS (ESI) calcd for C24H23F3N6O2 [M+H]+ = 495.2217, found 495.2217.
2-(2,4-Difluorophenyl)-1-(4-(5-(pyridin-4-yl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl)-
3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6l)
1HNMR (300 MHz, CDCl3) δ 8.81 (d, J = 6.4 Hz, 2H, pyridineꢀH), 8.28 (s, 1H,
TriazC3ꢀH), 7.88 (d, J = 6.0 Hz, 2H, pyridineꢀH), 7.83 (s, 1H, TriazC5ꢀH), 7.55 (m,
1H, ArꢀH), 6.83 (m, 2H, ArꢀH), 4.56 (m, 2H, C1ꢀH), 3.15 (d, J = 13.6 Hz, 1H, C3ꢀHa),
2.98 (m, 1H, C3ꢀHb), 2.73 (m, 2H, piperidinꢀCH2), 2.53 (m, 2H, piperidinꢀCH2), 2.08
(s, 1H, piperidinꢀ4ꢀCH), 1.92 ꢀ 2.04 (m, 4H, piperidinꢀCH2); MS (ESI) m/z: 468.4
(M+H). HRMS (ESI) calcd for C23H23F2N7O2 [M+H]+ = 468.1936, found 468.1936.
2-(2,4-Difluorophenyl)-1-(4-(5-(naphthalen-1-yl)-1,3,4-oxadiazol-2-yl)piperidin-1
-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6m)
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1HNMR (300 MHz, CDCl3) δ 9.16 (d, J = 8.4 Hz, 1H, naphtalinumꢀH),8.19 (s, 1H,
TriazC3ꢀH), 8.10 (d, J = 7.4 Hz, 1H, naphtalinumꢀH), 8.01 (d, J = 8.4 Hz, 1H,
naphtalinumꢀH), 7.92 (d, J = 7.9 Hz, 1H, naphtalinumꢀH), 7.80 (s, 1H, TriazC5ꢀH),
7.66 (t, J = 7.5 Hz, 1H, ArꢀH), 7.56 (q, J = 7.9 Hz, 3H, naphtalinumꢀH), 6.81 (m, 2H,
ArꢀH), 4.53 (m, 2H, C1ꢀH), 3.13 (d, J = 13.6 Hz, 1H, C3ꢀHa), 2.99 (m, 1H, C3ꢀHb),
2.71 (d, J = 13.6 Hz, 2H, piperidinꢀCH2), 2.50 (m, 2H, piperidinꢀCH2), 2.25 (t, J =
11.5 Hz, 1H, piperidinꢀ4ꢀCH), 1.86 ꢀ 2.13 (m, 4H, piperidinꢀCH2); 13CNMR (75 MHz,
CDCl3): δ 168.0, 164.6, 162.7 (1JCF = 249.4 Hz), 158.7 (1JCF = 245.9 Hz), 151.0,
144.7, 133.8, 132.5, 130.0, 129.3, 128.6, 128.2, 128.1, 126.7, 126.1, 126.0, 124.7,
120.5, 111.6, 104.3, 72.1, 62.5, 56.3, 54.6, 53.5, 32.6, 29.7, 29.3; MS (ESI) m/z: 517.4
(M+H). HRMS (ESI) calcd for C28H26F2N6O2 [M+H]+ = 517.2138, found 517.2138.
2-(2,4-Difluorophenyl)-1-(4-(5-(furan-2-yl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl)-3-
(1H-1,2,4-triazol-1-yl)propan-2-ol (6n)
1HNMR (300 MHz, CDCl3) δ 8.24 (s, 1H, TriazC3ꢀH), 7.81 (s, 1H, TriazC5ꢀH), 7.55
(m, 2H, furanꢀH), 7.12 (s, 1H, ArꢀH), 6.82 (s, 2H, ArꢀH), 6.57 (s, 1H, furanꢀH), 4.53
(m, 2H, C1ꢀH), 3.13 (d, J = 12.7 Hz, 1H, C3ꢀHa), 2.92 (m, 1H, C5ꢀHa), 2.69 (s, 2H,
piperidinꢀCH2), 2.49 (s, 2H, piperidinꢀCH2), 2.23 (s, 1H, piperidinꢀ4ꢀCH), 1.75 ꢀ 2.11
(m, 4H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 167.6, 162.6 (1JCF = 249.6 Hz),
158.6 (1JCF = 249.0 Hz), 157.5, 150.9, 145.6, 144.8, 139.3, 129.3, 125.9, 113.8, 112.1,
111.6, 104.3, 71.9, 62.5, 56.3, 54.4, 53.4, 32.4, 29.6, 29.0; MS (ESI) m/z: 457.4
(M+H). HRMS (ESI) calcd for C22H22F2N6O3 [M+H]+ = 457.1725, found 457.1725.
2-(2,4-Difluorophenyl)-1-(4-(5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)piperidin-1-yl
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)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6o)
1HNMR (300 MHz, CDCl3) δ 8.80 (s, 1H, ArꢀH), 8.37 (d, J = 7.2, 1H, ArꢀH), 8.15 (s,
1H, TriazC3ꢀH), 7.79 (s, 1H, TriazC5ꢀH), 7.71 (t, J = 8.1 Hz, 1H, ArꢀH), 7.57 (q, J =
8.9 Hz, 1H, ArꢀH), 6.81 (m, 2H, ArꢀH), 4.53 (m, 2H, C1ꢀH), 3.13 (d, J = 13.4 Hz, 1H,
C3ꢀHa), 2.92 (m, 1H, C3ꢀHb), 2.71 (d, J = 12.6 Hz, 2H, piperidinꢀCH2), 2.49 (m, 2H,
piperidinꢀCH2), 2.23 (m, 1H, piperidinꢀ4ꢀCH), 1.83 ꢀ 2.10 (m, 4H, piperidinꢀCH2);
13CNMR (75 MHz, CDCl3): δ 169.3, 162.8, 162.7 (1JCF = 249.6 Hz), 158.8 (1JCF
=
245.6 Hz), 151.0, 148.6, 144.7, 132.3, 130.4, 129.3, 126.0, 126.0, 125.5, 121.6, 111.5,
104.3, 72.1, 62.5, 56.2, 54.5, 53.4, 32.7, 29.6, 29.2; MS (ESI) m/z: 512.4 (M+H).
HRMS (ESI) calcd for C24H23F2N7O4 [M+H]+ = 512.1843, found 512.1843.
N'-hydroxynicotinimidamide (8a)
A mixture of nicotinonitrile (0.67 g, 6.44 mmol, 1 eq) and NH2OH⋅HCl (1.11 g, 16.10
mmol, 2.5 eq) in ethanol (20 mL) were refluxed for 8h. After cooling, white solid was
precipitated. After filtration, the residue was recrystallized from waterꢀisopropanol
1
(1:9) to give 8a (0.79 g, 89%). HNMR (600 MHz, d6ꢀDMSO) δ 9.88 (s,1H, OH),
8.88 (d, J = 2.3 Hz, 1H, pyridineꢀH), 8.57 (dd, J = 1.6 Hz, 4.8 Hz, 1H, pyridineꢀH),
8.03 (dt, J = 1.7 Hz, 8.0 Hz, 1H, pyridineꢀH), 7.41 (dd, J = 4.8 Hz, 8.0 Hz, 1H,
pyridineꢀH), 6.00 (brs, 2H, NH2); MS (ESI) m/z: 138.2 (M+H).
tert-Butyl 4-(3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl)piperidine-1-carboxylate (9a)
1ꢀ(tertꢀButoxycarbonyl)piperidineꢀ4ꢀcarboxylic acid (1.33 g, 5.80 mmol, 1 eq), HOBt
(0.94 g, 6.96 mmol, 1.2 eq), EDCI (1.67 g, 8.70 mmol, 1.5 eq) and
N,Nꢀdiisopropylethylamine (1.50 g, 11.60 mmol, 2 eq) were dissolved in dioxane
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and stirred at room temperature for 15 min. Subsequently, the solution was added 8a
(0.79 g, 5.80 mmol, 1 eq) and the resulting mixture was stirred for about 10 h at
100℃. After evaporating the solvent, the residue was dissolved in ethyl acetate and
washed with water (20 mL × 2), aqueous NaOH (20 mL × 2), saturated NaCl (20 mL
× 2), dried over Na2SO4, and filtrated. The solvent was evaporated under reduced
pressure and the crude product was purified by silica gel column chromatography
(eluents: DCM : MeOH = 80:1ꢀ50:1) to give 9a as a white solid (0.58 g, yield 31%).
1HNMR (600 MHz, d6ꢀDMSO) δ 9.16 (d, J = 2.2 Hz, 1H, pyridineꢀH), 8.78 (d, J =
1.7 Hz, 4.8 Hz, 1H, pyridineꢀH), 8.35 (dt, J = 1.8 Hz, 8.5 Hz, 1H, pyridineꢀH), 7.61
(dd, J = 4.8 Hz, 8.0 Hz, 1H, pyridineꢀH), 3.96 (m, 2H, piperidinꢀCH2); 3.39 (m, 1H,
piperidinꢀ4ꢀCH), 3.00 (m, 2H, piperidinꢀCH2), 2.09 (m, 2H, piperidinꢀCH2), 1.68 (m,
2H, piperidinꢀCH2), 1.42 (s, 9H, C(CH3)3); MS (ESI) m/z: 331.4 (M+H).
2-(2,4-Difluorophenyl)-1-(4-(3-(pyridin-3-yl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)-
3-(1H-1,2,4-triazol-1-yl)propan-2-ol (11a)
The synthetic protocol of target compounds 11a-j was similar to that of compound 6a.
11a (yellowish pink powder, 0.18 g, 43% for two steps) was obtained from 9a (0.30 g,
0.89 mmol, 1 eq). 1HNMR (600 MHz, CDCl3) δ 9.28 (d, J = 2.2 Hz, 1H, pyridineꢀH),
8.73 (dd, J = 1.8 Hz, 5.0 Hz, 1H, pyridineꢀH), 8.32 (dt, J = 1.8 Hz, 1H, pyridineꢀH),
8.15 (s, 1H, TriazC3ꢀH), 7.80 (s, 1H, TriazC5ꢀH), 7.58 (m, 1H, ArꢀH), 7.41 (dd, J = 4.8
Hz, 8.0 Hz, 1H, pyridineꢀH), 6.83 (m, 2H, ArꢀH), 5.20 (brs, 1H, OH), 4.57 (d, J =
14.1 Hz, 1H, C1ꢀHa), 4.52 (d, J = 14.1 Hz, 1H, C1ꢀHb), 3.11 (d, J = 13.9 Hz, 1H,
C3ꢀHa), 2.96 (m, 1H, piperidinꢀCH2), 2.71 (d, J = 13.9 Hz,1H, C3ꢀHb), 2.69 (m, 1H,
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piperidinꢀCH2), 2.49 (m, 2H, piperidinꢀCH2), 2.25 (t, J = 11.4 Hz, 1H,
piperidinꢀ4ꢀCH), 1.80ꢀ2.09 (m, 4H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ
181.9, 166.2, 162.7 (1JCF = 248.8 Hz), 158.9 (1JCF = 245.3 Hz), 151.9, 151.0, 148.6,
144.6, 134.6, 129.2, 126.0, 123.6, 123.0, 111.7, 104.6, 72.1, 62.4, 56.2, 54.5, 53.4,
33.6, 29.6, 29.2; MS (ESI) m/z: 468.4 (M+H). HRMS (ESI) calcd for C23H23F2N7O2
[M+H]+ = 468.1917, found 468.1917. The synthetic procedure for compounds 11b-j
was similar to the synthesis of compound 11a.
2-(2,4-Difluorophenyl)-1-(4-(3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (11b)
1HNMR (300 MHz, CDCl3) δ 8.21 (s, 1H, TriazC3ꢀH), 7.97 (d, J = 9.1 Hz, 2H, ArꢀH),
7.80(s, 1H, TriazC5ꢀH), 7.55 (q, J = 9.4 Hz, 1H, ArꢀH), 6.96 (d, J = 8.5 Hz, 2H, ArꢀH),
6.82 (m, 2H, ArꢀH), 5.20 (brs, 1H, OH), 4.53 (m, 2H, C1ꢀH), 3.85 (s, 3H, OCH3), 3.11
(d, J = 14.1 Hz, 1H, C3ꢀHa), 2.91 (m, 1H, C3ꢀHb), 2.66 (m, 2H, piperidinꢀCH2), 2.47
(t, J = 10.5 Hz, 2H, piperidinꢀCH2), 2.22 (t, J = 10.5 Hz, 1H, piperidinꢀ4ꢀCH), 1.74 ꢀ
13
2.11 (m, 4H, piperidinꢀCH2); CNMR (75 MHz, CDCl3): δ 181.0, 167.9, 162.7 (1JCF
= 248.9 Hz), 161.8, 158.8 (1JCF = 248.9 Hz), 150.8, 144.7, 129.3, 128.9, 126.02, 119.2,
114.2, 111.6, 104.3, 71.9, 62.4, 56.3, 55.3, 54.5, 53.4, 33.6, 29.7, 29.3; MS (ESI) m/z:
497.50 (M+H). HRMS (ESI) calcd for C25H26F2N6O3 [M+H]+ = 497.2034, found
497.2034.
2-(2,4-Difluorophenyl)-1-(4-(3-(furan-2-yl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)-3-
(1H-1,2,4-triazol-1-yl)propan-2-ol (11c)
1HNMR (300 MHz, CDCl3) δ 8.08 (s, 1H, TriazC3ꢀH), 7.70 (s, 1H, TriazC5ꢀH), 7.51
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(m, 2H, furanꢀH), 7.02 (d, J = 3.4 Hz, 1H, ArꢀH), 6.74 (m, 2H, ArꢀH), 6.46 (m, 1H,
furanꢀH), 5.03 (brs, 1H, OH), 4.46 (m, 2H, C1ꢀH), 3.02 (d, J = 13.6 Hz, 1H, C3ꢀHa),
2.84 (m, 1H, C3ꢀHb), 2.61 (d, J = 13.6 Hz, 2H, piperidinꢀCH2), 2.39 (m, 2H,
piperidinꢀCH2), 2.14 (t, J = 11.1 Hz, 1H, piperidinꢀ4ꢀCH), 1.65 ꢀ 2.04 (m, 4H,
piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 181.4, 162.6 (1JCF = 249.9 Hz), 161.0,
158.8 (1JCF = 248.2 Hz), 150.9, 145.0, 144.6, 142.1, 129.3, 126.0, 113.5, 111.7, 111.5,
104.2, 72.1, 62.4, 56.1, 54.4, 53.3, 33.5, 29.6, 29.2; MS (ESI) m/z: 457.4 (M+H).
HRMS (ESI) calcd for C22H22F2N6O3 [M+H]+ = 457.1712, found 457.1712.
2-(2,4-Difluorophenyl)-1-(4-(3-(thiophen-3-yl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl
)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (11d)
1HNMR (300 MHz, CDCl3) δ 8.15 (s, 1H, TriazC3ꢀH), 7.79 (s, 1H, TriazC5ꢀH), 7.75
(d, J = 3.5 Hz, 1H, thiopheneꢀH), 7.57 (q, J = 7.5 Hz, 1H, thiopheneꢀH), 7.49 (d, J =
4.9 Hz, 1H, thiopheneꢀH), 7.14 (t, J = 4.0 Hz, 1H, ArꢀH), 6.83 (m, 2H, ArꢀH), 5.20 (br
s, 1H, OH), 4.53 (m, 2H, C1ꢀH), 3.11 (m, 1H, C3ꢀHa), 2.92 (m, 1H, C3ꢀHb), 2.70 (m,
2H, piperidinꢀCH2), 2.47 (m, 2H, piperidinꢀCH2), 2.22 (m, 1H, piperidinꢀ4ꢀCH), 1.72
ꢀ 2.13 (m, 4H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 181.2, 164.3, 162.7
(1JCF = 248.2 Hz), 158.8 (1JCF = 248.2 Hz), 151.0, 144.6, 129.4, 129.3, 129.2, 128.3,
127.9, 125.9, 111.6, 104.3, 72.1, 62.4, 56.2, 54.5, 53.4, 33.6, 29.6, 29.2; MS (ESI) m/z:
473.3(M+H). HRMS (ESI) calcd for C22H22F2N6O2S [M+H]+ = 473.1522, found
473.1522.
2-(2,4-Difluorophenyl)-1-(4-(3-(thiophen-2-yl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl
)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (11e)
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1HNMR (300 MHz, CDCl3) δ 8.15 (s, 1H, TriazC3ꢀH), 8.01 (m, 1H, thiopheneꢀH),
7.78 (s, 1H, TriazC5ꢀH), 7.56 (m, 2H, thiopheneꢀH), 7.40 (m, 1H, ArꢀH), 6.81 (m, 2H,
ArꢀH), 5.20 (brs, 1H, OH), 4.53 (m, 2H, C1ꢀH), 3.09 (d, J = 13.0 Hz, 1H, C3ꢀHa), 2.90
(m, 1H, C3ꢀHb), 2.67 (d, J = 13.3 Hz, 2H, piperidinꢀCH2), 2.46 (m, 2H,
piperidinꢀCH2), 2.21 (t, J = 10.1 Hz, 1H, piperidinꢀ4ꢀCH), 1.79 ꢀ 2.09 (m, 4H,
piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 181.1, 164.6, 162.6 (1JCF = 250.9 Hz),
158.8 (1JCF = 245.6 Hz), 151.1, 144.7, 129.3, 128.2, 127.5, 127.0, 126.1, 126.0, 111.6,
104.3, 72.0, 62.4, 56.2, 54.5, 53.4, 33.5, 29.6, 29.2; MS (ESI) m/z: 473.3(M+H).
HRMS (ESI) calcd for C22H22F2N6O2S [M+H]+ = 473.1522, found 473.1522.
2-(2,4-Difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)-3-(4-(3-(4-(trifluoromethyl)pheny
l)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)propan-2-ol (11f)
1HNMR (300 MHz, CDCl3) δ 8.17 (s, 1H, TriazC3ꢀH), 8.14 (s, 2H, ArꢀH), 7.79 (s, 1H,
TriazC5ꢀH), 7.72 (d, J = 8.1 Hz, 2H, ArꢀH), 7.56 (m, 1H, ArꢀH), 6.82 (m, 2H, ArꢀH),
4.53 (m, 2H, C1ꢀH), 3.10 (d, J = 12.9 Hz, 1H, C3ꢀHa), 2.94 (m, 1H, C3ꢀHb), 2.68 (d, J
= 13.6 Hz, 2H, piperidinꢀCH2), 2.47 (m, 2H, piperidinꢀCH2), 2.22 (t, J = 11.3 Hz, 1H,
piperidinꢀ4ꢀCH), 1.79 ꢀ 2.10 (m, 4H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ
181.8, 174.6, 167.2, 162.6 (1JCF = 249.9 Hz), 158.9 (1JCF = 249.9 Hz), 151.1, 144.7,
132.8, 130.2, 129.3, 127.7, 126.0, 125.8, 125.5, 121.9, 111.6, 104.3, 72.0, 62.4, 56.2,
54.5, 53.4, 33.5, 29.7, 29.3; MS (ESI) m/z: 535.4(M+H). HRMS (ESI) calcd for
C25H23F5N6O2 [M+H]+ = 535.1835, found 535.1835.
2-(2,4-Difluorophenyl)-1-(4-(3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl)piperidin-
1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (11g)
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1HNMR (300 MHz, CDCl3) δ 8.13 (s, 1H, TriazC3ꢀH), 7.76 (s, 1H, TriazC5ꢀH), 7.61
(d, J = 7.8 Hz, 1H, ArꢀH), 7.54 (m, 2H, ArꢀH), 7.34 (m, 1H, ArꢀH), 7.00 (m, 1H,
ArꢀH), 6.83 (m, 2H, ArꢀH), 4.50 (m, 2H, C1ꢀH), 3.83 (s, 3H, OCH3), 3.07 (d, J = 13.4
Hz, 1H, C3ꢀHa), 2.90 (m, 1H, C3ꢀHb), 2.66 (d, J = 13.7 Hz, 2H, piperidinꢀCH2), 2.45
(m, 2H, piperidinꢀCH2), 2.20 (t, J = 11.3 Hz, 1H, piperidinꢀ4ꢀCH), 1.72 ꢀ 2.10 (m, 4H,
piperidinꢀCH2); MS (ESI) m/z: 497.4(M+H). 13CNMR (75 MHz, CDCl3): δ 181.2,
168.1, 162.6 (1JCF = 249.5 Hz), 159.8, 158.9 (1JCF = 247.0 Hz), 151.1, 144.7, 129.9,
129.6, 129.3, 127.9, 119.8, 117.6, 112.0, 111.7, 104.3, 72.0, 62.4, 56.2, 55.4, 54.5,
53.4, 33.5, 29.6, 29.2; HRMS (ESI) calcd for C25H26F2N6O3 [M+H]+ = 497.2019,
found 497.2019.
2-(2,4-Difluorophenyl)-1-(4-(3-(p-tolyl)-1,2,4-oxadiazol-5-yl)piperidin-1-yl)-3-(1H
-1,2,4-triazol-1-yl)propan-2-ol (11h)
1HNMR (300 MHz, CDCl3) δ 8.15 (s, 1H, TriazC3ꢀH), 7.92 (d, J = 8.1 Hz, 2H, ArꢀH),
7.79 (s, 1H, TriazC5ꢀH), 7.56 (m, 1H, ArꢀH), 7.28 (s, 1H, ArꢀH), 7.25 (s, 1H, ArꢀH),
6.82 (m, 2H, ArꢀH), 4.50 (m, 2H, C1ꢀH), 3.11 (d, J = 13.4 Hz, 1H, C3ꢀHa), 2.94 (d, J =
4.7 Hz, 1H, C3ꢀHb), 2.68 (d, J = 13.2 Hz, 2H, piperidinꢀCH2), 2.46 (m, 2H,
piperidinꢀCH2), 2.40 (s, 3H, CH3), 2.22 (t, J = 11.0 Hz, 1H, piperidinꢀ4ꢀCH), 1.72 ꢀ
2.09 (m, 4H, piperidinꢀCH2); 13CNMR (75 MHz, CDCl3): δ 181.1, 168.2, 162.7 (1J CF
= 247.9 Hz), 158.8 (1JCF = 246.6 Hz), 151.1, 144.7, 141.4, 129.5, 129.3, 127.3, 126.1,
123.9, 111.6, 104.3, 72.0, 62.4, 56.2, 54.5, 53.4, 33.6, 29.7, 29.3, 21.5; MS (ESI) m/z:
481.4(M+H). HRMS (ESI) calcd for C25H26F2N6O2 [M+H]+ = 481.2136, found
481.2135.
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2-(2,4-Difluorophenyl)-1-(4-(3-(4-fluorophenyl)-1,2,4-oxadiazol-5-yl)piperidin-1-
yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (11i)
1HNMR (600 MHz, CDCl3) δ 8.15 (s, 1H, TriazC3ꢀH), 8.04 (dd, J = 5.3 Hz, 8.7 Hz,
2H, ArꢀH), 7.79 (s, 1H, TriazC5ꢀH), 7.56 (dd, J = 8.2 Hz, 15.4 Hz, 1H, ArꢀH), 7.14 (t,
J = 8.5 Hz, 2H, ArꢀH), 6.83 (m, 2H, ArꢀH), 4.56 (d, J = 14.1 Hz, 1H, C1ꢀHa), 4.50 (d,
J = 14.1 Hz, 1H, C1ꢀHb), 3.11 (m, 1H, C3ꢀHa), 2.93 (m, 1H, C3ꢀHb), 2.69 (m, 2H,
piperidinꢀCH2), 2.47 (m, 2H, piperidinꢀCH2), 2.22 (m, 1H, piperidinꢀ4ꢀCH), 2.05
(m,1H, piperidinꢀCH2), 1.94 (m, 2H, piperidinꢀCH2), 1.81 (m, 1H, piperidinꢀCH2);
MS (ESI) m/z: 485.4 (M+H). HRMS (ESI) calcd for C24H23F3N6O2 [M+H]+ =
485.1822, found 485.1822.
2-(2,4-Difluorophenyl)-1-(4-(3-(2-fluoro-4-(trifluoromethyl)phenyl)-1,2,4-oxadiaz
ol-5-yl)piperidin-1-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (11j)
1HNMR (600 MHz, CDCl3) δ 8.17 (t, J = 7.4 Hz, 1H, ArꢀH), 8.14 (s, 1H, TriazC3ꢀH),
7.79 (s, 1H, TriazC5ꢀH), 7.57 (t, J = 8.4 Hz, 1H, ArꢀH), 7.54 (d, J = 8.4 Hz, 1H, ArꢀH),
7.50 (d, J = 10.2 Hz, 1H, ArꢀH), 6.82 (m, 2H, ArꢀH), 5.14 (br s, 1H, OH), 4.56 (d, J =
14.1 Hz, 1H, C1ꢀHa), 4.51 (d, J = 14.1 Hz, 1H, C1ꢀHb), 3.10 (d, J = 14.0 Hz, 1H,
C3ꢀHa), 2.97 (m, 1H, C3ꢀHb), 2.70 (m, 2H, piperidinꢀCH2), 2.48 (m, 2H,
piperidinꢀCH2), 2.22 (m, 1H, piperidinꢀ4ꢀCH), 1.79 ꢀ 2.09 (m, 4H, piperidinꢀCH2);
MS (ESI) m/z: 553.3 (M+H). HRMS (ESI) calcd for C25H22F6N6O2 [M+H]+ =
553.1712, found 553.1712.
In Vitro Antifungal Activity Assay
In vitro antifungal activity was measured according to the protocols from National
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Committee for Clinical Laboratory Standards (NCCLS) with a few modifications26.
Serial dilution method in 96ꢀwell microtest plate was used to determine the minimum
inhibitory concentration (MIC) of the target compounds. Briefly, for yeasts, the initial
concentration of fungal suspension in RPMI 1640 medium was 103 CFU/ml. For
filamentous fungi, the initial concentration of fungal suspension in RPMI 1640
medium was 5 × 103 CFU/ml. Tested compounds were dissolved in DMSO serially
diluted in growth medium. The final concentrations of each compound ranged from
0.125 to 64 ꢀg/mL. The yeasts were incubated at 35 °C and the filamentous fungi
were incubated at 37 °C. Growth MIC was determined at 48 h after incubation. The
growth inhibition was determined by spectrophotometer. Optical density was
measured at 630 nm, and background optical densities were subtracted from that of
each well. Each strain was tested in triplicate.The growth control wells contained 100
µl of the diluted fungal suspension. Sterility control wells contained 100 µl of RPMI
1640. The MICs at which 50% and 80% of the isolates were inhibited were
determined (MIC50 and MIC80 respectively).
Hyphal Formation Assay
To evaluate the activity of the compounds 6g and 11b against yeastꢀtoꢀhypha
morphological transition, hyphal formation assay was carried out.27 Briefly, C.
albicans cells were harvested by centrifugation (3,000 rpm, 5 min) and washed three
times with PBS. The C. albicans suspension was then adjusted to 1 × 106 cells/ml
with Spider medium. The C. albicans suspension was divided into the wells in
12ꢀwell plates with different concentrations of 6g/11b added. Then the 12ꢀwell plates
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were incubated in 37°C. After 3ꢀh incubation, the cellular morphology was
photographed.
Toxicity assay using C. elegans.
The assay was carried out as described previously28 with slight modification.
Synchronized C. elegans grown at 25°C for 3 days on NGM plates with OP50 as food.
Adult worms were carefully collected and washed using sterile M9 buffer into a
15ꢀml conical tube. Thirty worms were then pipetted into a single well of a 12ꢀwell
tissue culture plate containing 2 ml of liquid medium (80% M9 and 20% BHI). Six
concentrations of 6g and 11b were tested, including 1, 5, 20, 40, 80, and 160 ꢁg/ml.
Worms were kept at 25°C and observed everyday to record dead worms. Two
independent experiments were carried out. Photos were taken after incubation for 6
days.
Molecular Docking
Homology model of CACYP51 were obtained from our previous studies.13 GOLD
4.1.2 as well as Goldscore function was used for molecular docking and the docking
parameters were set as default.29 The docking conformation was selected according to
the docking score and visual inspection.
Acknowledgements
This work was supported in part by National Natural Science Foundation of China
(grants 81222044), Science and Technology Commission of Shanghai Municipality
(grant 13431900301), the 863 HiꢀTech Program of China (grant 2014AA020525).
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