English

• 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).

  图 1

  

  图 1  Structures of triazole antifungal agents.

  Figure 1.  Structures of triazole antifungal agents.

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  Lanosterol 14α-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 threedimensional 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.

  图 2

  

  图 2  Design rationale of the target compounds.

  Figure 2.  Design rationale of the target compounds.

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  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 5 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 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 π–π interaction with Tyr118. Finally, the terminal cyclopropyl group interacted with Leu376, Phe380 through hydrophobic and Van der Waals interactions.

  图 3

  

  图 3  The binding mode of compound 7l in the active site of CACYP51.

  Figure 3.  The binding mode of compound 7l in the active site of CACYP51.

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  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 (Table 1). Particularly, compounds 7f (MIC = 0.125 μg/mL) and 7l (MIC = 0.125 μg/mL) were highly active against C. albicans, which were more active than fluconazole (MIC = 0.5 μg/mL). In contrast, the target compounds generally showed improved activity against Candida glabrata, whereas they were less potent against Candida parapsilosis. For example, the activity of compounds 7j, 7k, 7l, 8a and 8b (MIC range: 0.125– 0.5 μg/mL) against C. glabrata were comparable or superior to that of fluconazole (MIC = 0.25 μg/mL). For C. neoformans, most compounds showed moderate activity except compound 8a (MIC = 0.25 μg/mL), which was 8 fold more potent than fluconazole (MIC = 2 μg/mL). However, all the compounds as well as fluconazole were inactive against A. fumigatus (MIC > 64 μg/mL). For dematophytes (Trichophyton rubrum and Microsporum gypseum), the target compounds also showed moderate to good activity.In particular, compound 7d (MIC = 0.25 μg/mL) revealed better activity against T. rubrum than fluzonazole (MIC = 0.5 μg/mL).

  表 1

  

  表 1  In vitro antifungal activities of the target compounds (MIC₈₀, μg/mL).^a

  Table 1.  In vitro antifungal activities of the target compounds (MIC₈₀, μg/mL).^a

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  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 μg/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 μg/mL, Fig. 4B).

  图 4

  

  图 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.

  Figure 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.

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  3.   Conclusion

  In summary, a series of new triazole 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 7l 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 CDCl₃ as the reference standard or DMSO-d₆. Chemical shifts (δ values) and coupling constants (J values) are expressed in ppm and Hz, respectively. ESI mass spectra were gathered on an API-3000 LC–MS spectrometer. Highresolution 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 NaN₃ afforded the azide compound 10. Then, target compounds 7a–m were synthesized by click reaction of intermediate 10 with various alkynes in H₂O/^tBuOH in the presence of CuSO₄ and sodium ascorbate. Propargylation of ketone 11 by propargyl bromide afforded intermediate 12, which reacted with RN₃ to give target compounds 8a–b using a similar click reaction procedure.

  Scheme 1

  

  图 Scheme 1  (a) NaN₃/NH₄Cl, MeOH, reflux, overnight; (b) CuSO₄, sodium ascorbate, t-BuOH, H₂O, overnight; (c) Zn, DMF, THF, r.t., 5 h; (d) CuSO₄, sodium ascorbate, H₂O, overnight.

  Scheme 1.  (a) NaN₃/NH₄Cl, MeOH, reflux, overnight; (b) CuSO₄, sodium ascorbate, t-BuOH, H₂O, overnight; (c) Zn, DMF, THF, r.t., 5 h; (d) CuSO₄, sodium ascorbate, H₂O, overnight.

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  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/H₂O (5:1, 24 mL) was added a mixture of CuSO₄ 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 N₂ atmosphere. Then, tert-butyl alcohol was removed under reduced pressure and the residue was diluted with CH₂Cl₂ (50 mL) and washed by saturated saline (20 mL × 3). The organic layers were dried over Na₂SO₄, filtrated and evaporated under reduced pressure. The crude product was purified by chromatography using CH₂Cl₂/ MeOH as eluents (50:1–30:1) to give target compound 7a as a white solid: 150 mg, yield 56%. ¹H NMR (600 MHz, CDCl₃): 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, 2H, 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 Hz), 4.39 (d, 1H, J = 14.3 Hz). ¹³C NMR (151 MHz, CDCl₃): 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 = 18.8 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 C₁₉H₁₇F₂N₆O, 383.2411; found, 383.2411.

  The synthetic procedure for compounds 7b-m and 8a-b was similar to the synthesis of compound 7a.

  2-(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): ¹H NMR (600 MHz, CDCl₃): 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), 4.94 (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).

  2-(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): ¹H NMR (600 MHz, CDCl₃): 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).

  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): ¹H NMR (600 MHz, CDCl₃): 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, 1H, 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, 1H, J = 14.0 Hz). ¹³C NMR (151 MHz, CDCl₃): 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, 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), 121.75 (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). MS (ESI) m/z: 397.34 (M+H). HRMS-ESI⁺: [M +H]⁺ calcd. for C₂₀H₁₉F₂N₆O, 397.3658; found, 397.3658.

  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): ¹H NMR (600 MHz, CDCl₃): 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), 7.36 (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), 4.77 (d, 1H, J = 14.4 Hz), 4.35 (d, 1H, J = 14.3 Hz). ¹³C NMR (151 MHz, CDCl₃): δ 164.12 (d, J = 12.5 Hz), 162.45 (d, J = 12.5 Hz), 159.35 (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), 125.82 (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 C₁₉H₁₆ClF₂N₆O, 417.1023; found, 417.1023.

  1-(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): ¹H NMR (600 MHz, CDCl₃): 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-(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, CDCl₃): 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), 4.31 (d, 1H, J = 14.4 Hz). MS (ESI) m/z: 384.22 (M+H).

  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): ¹H NMR (600 MHz, CDCl₃): 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).

  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): ¹H NMR (600 MHz, DMSO-d6): 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), 7.27 (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, J = 14.5 Hz), 4.72 (d, 1H, J = 14.4 Hz), 4.66 (d, 1H, J = 14.5 Hz). ¹³C NMR (151 MHz, DMSO-d6): d 163.31 (d, J = 12.3 Hz), 161.68 (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 = 3.9 Hz), 55.26 (d, J = 4.5 Hz). MS (ESI) m/z: 389.27 (M + H). HRMS-ESI⁺: [M+H]⁺ calcd. for C₁₇H₁₅F₂N₆OS, 389.1066; found, 389.1066.

  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, CDCl₃): 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 = 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, CDCl₃): 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), 130.17 (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), 54.67(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 C₁₈H₂₁F₂N₆O, 375.1745; found, 375.1745.

  1-(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, CDCl₃): 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, 2H, 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 = 13.3 Hz), 1.40–1.26 (m, 4H), 1.25–1.19 (m, 1H). ¹³C NMR (151 MHz, CDCl₃): δ 162.33 (d, J = 12.6 Hz), 159.37 (d, J = 12.0 Hz), 157.78 (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 = 26.2 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). HRMSESI⁺: [M+H]⁺ calcd. for C₁₉H₂₃F₂N₆O, 389.1909; found, 389.1909.

  1-(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, CDCl₃): 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 = 14.3 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, CDCl₃): 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), 104.35 (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) m/z: 347.22 (M+H). HRMS-ESI⁺: [M+H]⁺ calcd. for C₁₆H₁₇F₂N₆O, 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-d₆): 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, 1H, 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-d6): 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), 145.65 (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 = 3.5 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 C₁₇H₂₁F₂N₆O, 363.1788; found, 363.1788.

  1-(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, CDCl₃): 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), 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). ¹³C NMR (151 MHz, CDCl₃): d 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), 130.19 (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 = 3.6 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 C₁₈H₂₁F₂N₆O, 375.2012; 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, CDCl₃): 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, 1H, 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), 1.86 (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, CDCl₃): δ 163.41 (d, 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 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 C₁₉H₂₃F₂N₆O, 389.1864; found, 389.1864.

  4.2   In vivo 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 ℃ and the mold and dermatophytes at 28 ℃. Growth MIC was determined at 24 h for Candida species, at 72 h for C. neoformans, and at 7 days for A. fumigatus.

  4.3   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 ℃ 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 mg/ 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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• 

  Figure 1  Structures of triazole antifungal agents.

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  Figure 2  Design rationale of the target compounds.

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  Figure 3  The binding mode of compound 7l in the active site of CACYP51.

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  Figure 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.

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  Scheme 1  (a) NaN₃/NH₄Cl, MeOH, reflux, overnight; (b) CuSO₄, sodium ascorbate, t-BuOH, H₂O, overnight; (c) Zn, DMF, THF, r.t., 5 h; (d) CuSO₄, sodium ascorbate, H₂O, overnight.

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  Table 1.  In vitro antifungal activities of the target compounds (MIC₈₀, μg/mL).^a

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•