Discovery of highly potent antifungal triazoles by structure-based lead fusion

Wenya Wang; Shengzheng Wang; Guoqiang Dong; Yang Liu; Zizhao Guo; Zhenyuan Miao; Jianzhong Yao; Wannian Zhang; Chunquan Sheng

doi:10.1039/C1MD00103E

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DOI: 10.1039/C1MD00103E (Concise Article) Med. Chem. Commun., 2011, 2, 1066-1072

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Discovery of highly potent antifungal triazoles by structure-based lead fusion†

Wenya Wang , Shengzheng Wang , Guoqiang Dong , Yang Liu , Zizhao Guo , Zhenyuan Miao , Jianzhong Yao , Wannian Zhang * and Chunquan Sheng *
Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai, 200433, China. E-mail: shengcq@hotmail.com.; Fax: (+86)21-81870243; Tel: (+86)21-81870243; zhangwnk@hotmail.com; Fax: (+86)21-81871239; Tel: (+86)21-81871239

Received 16th April 2011 , Accepted 15th August 2011

First published on 9th September 2011

Abstract

By means of structure-based lead fusion, a series of novel drug-like azoles possessing 4-(benzyloxy)piperidin-1-yl side chains were rationally designed and synthesized. Flexible molecular docking studies indicated that the newly synthesized azoles took advantages of the key interactions between the two lead structures and CACYP51. As a result, they revealed improved antifungal activity and broader spectrum. All the new azoles showed good to excellent in vitro antifungal activity against all of the tested pathogenic fungi (MIC₈₀ range: 2.33–0.01 μM). Compounds 10a, 10g and 10h, the most active azoles toward Candida albicans (MIC₈₀ = 0.01 μM) are 82 fold more potent than fluconazole. In particular, all the compounds also showed good activity against Aspergillus fumigatus (MIC₈₀ = 2.33–0.55 μM) that is not sensitive to fluconazole.

Introduction

There is a tendency that the incidence of invasive and systemic fungal infections, such as invasive candidiasis, cryptococcosis and aspergillosis, has increased dramatically worldwide during the last decades.¹ Furthermore, the mortality (per 100,000 population) associated with invasive mycosis increased by 3.4-fold from 1980 to 1997.² It is mainly caused by the growing number of immunocompromised individuals due to AIDS, organ transplantation and chemotherapy.³Candida albicans (C. albicans), Cryptococcus neoformans (C. neoformans) and Aspergillus fumigatus (A. fumigatus) are the most frequent pathogens isolated from clinical practice.⁴ Moreover, various moulds and yeasts such as Mucor, Fusarium and Zygomucetes have also emerged as new opportunistic fungi threatening patients' life. Currently, clinically available drugs against these fungal infections contain four major types: azoles (e.g. fluconazole and itraconazole),^5,6 polyene macrolides (e.g. amphotericin B),⁷ allyamines (e.g. terbinafine)⁸ and echinocandins (e.g. caspofungin and micafungin).^9,10 They vary in chemical structures and modes of action in different biological pathways. Clinically, azoles, especially triazoles, are first-line agents in treating fungal infections due to their advantageous properties (e.g. broad antifungal spectrum, high activity, oral applicability and chemical stability). However, their extensive use has led to the occurrence of resistant strains which greatly limited the therapeutic options.¹¹ Hence, there is an emergent demand for the discovery of new antifungal azoles. Now, newer triazoles (e.g. voriconazole and posaconazole)^12,13 have been marketed. Other triazole candidates (e.g. ravuconazole and albaconazole)^14,15 are currently under development.

Antifungal azoles target the ergosterol biosynthesis pathway by inhibiting lanosterol 14α-demethylase (CYP51) which removes the methyl group at position C-14 of precursor sterols and thus prevents the synthesis of ergosterol, a major component of fungal cell membrane.¹⁶ Podust et al. reported the crystal structure of a prokaryotic sterol CYP51 from Mycobacterium tuberculosis (MTCYP51).¹⁷ On the basis of the MTCYP51 structure, our group constructed three-dimensional (3D) models of CYP51 from C. albicans (CACYP51), C. neoformans (CNCYP51) and A. fumigatus (AFCYP51) using homology modeling methods^18–20 and investigated the binding modes of azoles by flexible molecular docking^20,21 and site-directed mutagenesis.²² Several key residues, such as Tyr118 and Ser378, were found to play an important role in azole binding. All of these results facilitated the rational design of novel azole and non-azole CYP51 inhibitors.^21,23–28

Recently, we reported a series of potent antifungal azoles with substituted phenoxyalkyl C-3 side chains as well as their conformationally restricted derivatives.^24–26,29 In an attempt to improve the antifungal activity, spectrum and drug-like properties, structure-based lead fusion was used to design a new type of side chain containing 4-(benzyloxy)piperidin-1-yl group. As compared with the lead structures, the synthesized azoles showed improved antifungal activity and broader spectrum.

Results and discussion

Chemistry

The chemical synthesis of compounds 10a–i is outlined in Scheme 1. The synthetic procedure of oxirane compound 4 was according to our reported protocol.²¹ The 4-(substituted benzyloxy)piperidin-1-yl side chains 9a–i were synthesized via four steps. First, piperidin-4-one hydrochloride 5 was treated with excess di-tert-butyl dicarbonate in the presence of N,N-diisopropylethylamine (DIEA) in 1,4-dioxane/H₂O (4 [thin space (1/6-em)]

1) to give compound 6. Subsequently, compound 6 was reduced by potassium borohydride in methanol to afford tert-butyl 4-hydroxypiperidine-1-carboxylate (compound 7). Then, compound 7 was reacted with various substituted benzyl bromide in the presence of sodium hydride in N,N-dimethylformamide to give compounds 8a–i. Finally, compounds 8a–i were treated with trifluoroacetic acid in dichloromethane at room temperature overnight to afford the side chains 9a–i. The target compounds 10a–i were obtained as racemates by treating epoxide 4 with side chains 9a–i in the presence of triethylamine in ethanol at 80 °C with moderate to high yields.


	Scheme 1 Reagents and conditions: a) ClCH₂COCl, AlCl₃, CH₂Cl₂, 40 °C, 3 h, 50%; b) triazole, K₂CO₃, CH₂Cl₂, RT, 24 h, 70.0%; c) (CH₃)₃SOI, NaOH, toluene, 60 °C, 3 h, 62.3%. d) Boc₂O, DIEA, 1,4-dioxane-H₂O, RT, 24 h, 80.2%; e) KBH₄, MeOH, 65 °C, 0.5 h, 100%; f) substituted benzyl bromide, NaH, DMF, 0 °C∼RT, 24 h, 29.5%–41.7%; g) CF₃COOH, CH₂Cl₂, RT, 12 h, 90.1%; h) 4, Et₃N, EtOH, reflux, 9 h, 28.0%–60.6%.

Design rationale

In our previous studies, we have designed a series of novel azoles with substituted phenoxyalkyl C-3 side chains (lead structure 1, Fig. 1)^25–27 and their conformationally restricted derivatives possessing benzylpiperidin-4-yl side chains (lead structure 2, Fig. 1).²⁹ The lead series exhibited good antifungal activity against most of the tested pathogenic fungi, but their potency toward A. fumigatus was weak. Starting from the two lead structures, we aimed to design new antifungal triazoles with improved antifungal activity, broader antifungal spectrum and proper physico-chemical properties. Herein, a new idea of design rationale called structure-based lead fusion was used in the lead optimization process (Fig. 1). The fundamental idea of structure-based lead fusion is to take advantages of key interactions of different lead structures with the receptor and merge them into a new structure that possesses improved binding affinity and drug-like properties. Before structure fusion, the binding mode of two lead structures should be fully investigated.


	Fig. 1 Design rationale of the new azoles with 4-(benzyloxy)piperidin-1-yl side chains by structure-based lead fusion.

Previous molecular modeling and structure–activity relationship (SAR) studies showed that lead structure 1 interacted with CACYP51 through hydrophobic, van der Waals and hydrogen-bonding interactions.^24,25 In particular, the hydrogen-bonding interaction between its side chain oxygen atom with Ser378 of CACYP51 was important for the antifungal activity. Lead structure 2 was conformationally restricted and formed stronger hydrophobic interactions with CACYP51, but it lost hydrogen interaction with Ser378.²⁹ On the basis of their binding mode, the side chains of the two lead structures were merged into a new chemotype, namely O-substituted piperidin-4-ol. The new side chain has several advantages: (1) The piperidin-4-ol group is conformationally restricted and the oxygen atom can function as hydrogen bond acceptor to interact with CACYP51; (2) The piperidin-4-ol group is one of the most frequent scaffolds in marketed oral drugs with good drug-like properties;³⁰ (3) Benzyl substituted piperidin-4-ol side chains can form strong hydrophobic, van der Waals and hydrogen-bonding interactions with CACYP51; (4) The designed molecules have decreased Log P values. By the method of Wang's group,³¹ the Log P value of compound 10a, lead structure 1 and lead structure 2 turned out to be 2.74, 3.22 and 3.22, respectively. It means that the designed azoles may have improved water-solubility, suggesting their potential as orally active antifungal agents. The comparison of physicochemical properties of compound 10a with azole antifungal agents indicates that the designed compound has good drug-like properties (Table 1 in the ESI†).

In order to verify our hypothesis, a representative derivative, compound 10h, was docked into the active site of CACYP51 using the Affinity module within InsightII 2000 software package.³² Fig. 2 shows that compound 10h binds to the active site of CACYP51 with an extended conformation. The triazolyl ring of the compound coordinates with iron of heme group, while the difluorophenyl group interacts with Phe126, Met306 and Phe145 though hydrophobic interaction. The piperidyl group forms hydrophobic and van der Waals interactions with Ile379 and Val509. Hydrogen-bonding interaction was observed between the oxygen atom attached to the piperidyl group and Ser378. It is worth noting that π–π stacking interaction was found between the terminal benzyl group and Tyr118 which further improved the affinity and specificity of the inhibitors. In addition, the terminal benzyl group binds to substrate access channel 2 (FG loop)²¹ through the hydrophobic and van der Waals interactions with Phe380, Phe228 and Leu121.


	Fig. 2 The docking conformation of compound 10h in the active site of CACYP51. Important residues interacting with the compound are shown.

In vitro antifungal activity

In vitro antifungal activities of the synthesized compounds were shown in Table 1. All the synthesized compounds revealed good activity against seven common fungal pathogens. In particular, compound 10a was more active than lead structures 1 and 2. C. albicans has a worldwide distribution and is the most common cause of life-threatening fungal infections. All the compounds showed higher antifungal activity toward C. albicans (MIC₈₀ range: 0.14–0.01 μM) than fluconazole (MIC₈₀ = 0.82 μM). Particularly, the MIC₈₀ values of compounds 10a, 10g and 10h were 0.01 μM, indicating that they were 82 fold more potent than fluconazole. Moreover, these compounds also displayed excellent inhibitory activity against other Candida spp., such as C. tropicalis, C. parapsilosis and C. kefyr with their MIC₈₀ values in the range of 0.56 to 0.01 μM. For the dermatophyte (i.e. T. rubrum), most compounds were superior to fluconazole and their MIC₈₀ values ranged from 0.15 μM to 0.03 μM. On the C. neoformans strain, most compounds showed higher antifungal activity than fluconazole (MIC₈₀ = 0.20 μM) with their MIC₈₀ values on the level of 0.03 μM. A. fumigatus is the leading cause of mortality in patients infected with invasive fungal pathogens. Improving the activity of azoles against A. fumigatus is a challenging task. Fluconazole is almost inactive toward A. fumigatus, whereas the two lead structures only reveal weak inhibitory activity. In contrast, all the designed compounds exhibit improved activity (MIC₈₀ range: 2.33–0.55 μM). Among them, compound 10f was the most active azole with its MIC₈₀ value of 0.55 μM. Among these azoles, compounds 10a, 10g and 10h exhibited the highest in vitro antifungal activity with broad antifungal spectrum, which were good drug candidates for further evaluation.

Table 1 In vitro antifungal activities of the compounds (MIC₈₀, μM)^a

Compd	C. alb.	C. neo.	C. tro.	C. par.	C. kef.	T. rub.	A. fum.
a Abbreviations: C. alb. = Candida albicans; C. neo. = Cryptococcus neoformans; C. tro. =Candida tropicalis; C. par. = Candida parapsilosis; C. kef. = Candida kefyr; T. rub. = Trichophyton rubrum; A. fum. = Aspergillus fumigatus; FLZ = Fluconazole.
10a	0.01	0.04	0.04	0.04	0.15	0.15	2.33
10b	0.03	0.03	0.14	0.03	0.54	0.14	2.16
10c	0.13	0.03	0.13	0.03	0.50	0.13	2.01
10d	0.13	0.03	0.13	0.03	0.50	0.03	2.01
10e	0.14	0.03	0.14	0.03	0.54	0.03	2.16
10f	0.03	0.03	0.14	0.01	0.55	0.03	0.55
10g	0.01	0.12	0.12	0.12	0.49	0.12	1.97
10h	0.01	0.03	0.03	0.03	0.56	0.14	2.24
10i	0.14	0.03	0.03	0.03	0.56	0.03	2.24
Lead 1	0.04	0.15	0.04	0.15	0.60	0.60	>153.68
Lead 2	0.57	2.26	2.26	0.57	2.26	2.26	144.95
FLZ	0.82	0.20	3.26	0.82	3.26	3.26	>208.97

Structure–activity relationships

On the basis of the in vitro antifungal activity assay, preliminary SARs of the synthesized compounds were obtained. Because all the synthesized compounds exhibited good antifungal activity, the SARs are not so obvious. In general, the introduction of various substituent groups on the terminal benzyl group did not show significant effect on improving the antifungal activity. When the terminal benzyl group was substituted, the antifungal activities against C. neoformans and A. fumigatus were maintained, while the activities against C. tropicalis and C. kefyr. were decreased. For the position of the substitutions on the terminal phenyl group, the 2-substitited and 3-substituted derivatives (e.g. compounds 10g and 10h) are more potent than 4-substituted derivatives. Compared to the compounds substituted by mono-chlorine group (e.g. compounds 10b and 10e), di-chloro substituted derivatives (e.g. compounds 10c and 10d) exhibit lower inhibitory activity. When the halogen atom was replaced by a cyano group (e.g. compound 10f), the antifungal activity against A. fumigatus was increased by 4 fold.

Conclusions

In the present investigation, we provide a new idea, namely structure-based lead fusion, for azole optimization. The 4-(benzyloxy)piperidin-1-yl side chain was designed by taking advantages of the lead structures' key interactions with CACYP51. Flexible molecular docking studies revealed that the designed compounds showed improved binding affinity and interacted with CACYP51 mainly through hydrogen bonding (Ser378), π–π stacking, hydrophobic and van der Waals interactions. As compared with the lead compounds, the azoles reported here have several advantages: (1) They showed improved antifungal activity with MIC₈₀ values against Candida spp. and C. neoformans in the range of 0.56–0.01 μM. Several compounds, such as 10a, 10f, 10g and 10h, are worth of in vivo antifungal efficacy testing and evaluating their potential as drug candidates. (2) The antifungal spectrum of the synthesized azoles is expanded. Particularly, they showed good activity toward A. fumigatus that was not sensitive to fluconazole. (3) The designed azoles showed good drug-like properties. Piperidin-4-ol is a drug-like scaffold and the compounds have suitable Log P values as orally active drugs. The results of present studies can support the hypothesis that structure-based lead fusion could be developed into a useful approach in lead optimization.

Experimental section

Chemistry

General methods. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 500 spectrometer with TMS as an internal standard and CDCl₃ as solvent. Chemical shifts (δ values) and coupling constants (J values) are given in ppm and Hz, respectively. ESI mass spectra were performed on an API-3000 LC-MS spectrometer. High-resolution mass spectrometry measurements were performed on an Agilent 6538 UHD Accurate-Mass 2-TOF LC–MS spectrometer under electrospray ionization (ESI) conditions. 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.

tert-Butyl 4-oxopiperidine-1-carboxylate (6). N,N-Diisopropylethylamine (32.31 g, 0.25 mol, 2.5 equiv) was added to a solution of piperidin-4-one hydrochloride 5 (13.5 g, 0.10 mol, 1.0 equiv) in 200 mL 1,4-dioxane and H₂O (v/v, 4/1). Subsequently, di-tert-butyl dicarbonate (32.74 g, 0.15 mol, 1.5 equiv) was added dropwise to the reaction mixture over 1 h, and the resulting solution was stirred at room temperature for 24 h. Then the solvent was evaporated under reduced pressure, and the residue was poured into a 5% citric acid solution, then extracted with dichloromethane (100 mL × 3). The organic layer was separated, dried with anhydrous Na₂SO₄, and concentrated to give crude solid, which was recrystallized from cyclohexane to afford 6 as white needle (15.96 g, 80.2%): mp 72 °C; ¹H-NMR (500 MHz, CDCl₃): δ 3.60 (t, 4H, J = 6.2 Hz, piperidin-2,6-CH₂), 2.34 (t, 4H, J = 6.2 Hz, piperidin-3,5-CH₂), 1.43 (s, 9H, C(CH₃)₃); MS (ESI) m/z: 200 [M + H]⁺.

tert-Butyl 4-hydroxypiperidine-1-carboxylate (7). Potassium borohydride (0.54 g, 0.010 mol, 1 equiv) was added to a solution of compound 6 (2.99 g, 0.015 mol, 1.5 equiv) in methanol 30 mL. The reaction mixture was heated to reflux for 0.5 h. Then the solvent was evaporated under reduced pressure, and the residue was diluted with 100 mL ethyl acetate, washed with H₂O (30 mL × 3). The organic layer was separated, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to give 7 as white solid (2.01 g, 100%): R_f 0.36 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); mp 68 °C; ¹H-NMR (500 MHz, CDCl₃): δ 3.60 (t, 4H, J = 6.2 Hz, piperidin-2,6-CH₂), 3.31 (s, 1H, piperidin-4-CH), 2.34 (t, 4H, J = 6.2 Hz, piperidin-3,5-CH₂), 1.43 (s, 9H, C(CH₃)₃); MS (ESI) m/z: 202 [M + H]⁺. The product was used in the next step without further purification.

tert-Butyl 4-(benzyloxy)piperidine-1-carboxylate (8a). Sodium hydride (60% dispersion, 1.20 g, 0.030 mol, 1.5 equiv) was added to a solution of compound 7 (4.02 g, 0.020 mol, 1 equiv) in 80 mL dried DMF which was cooled in an ice bath to 0 °C. The reaction mixture was stirred at room temperature for 2 h, then cooled to 0 °C again, and a solution of benzyl bromide (4.45 g, 0.026 mol, 1.3 equiv) in 20 mL dried DMF was added dropwise to the mixture. The resulting mixture was stirred at room temperature for 24 h and then diluted with 200 mL ethyl acetate, washed with H₂O (50 mL × 3). The organic layer was separated, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:EtOAc, 20 [thin space (1/6-em)]

1, v/v) to give 8a as colorless oil (1.72 g, 29.5%): R_f 0.32 (hexane/EtOAc, 20 [thin space (1/6-em)]

1); ¹H-NMR (500 MHz, CDCl₃): δ 7.25∼7.35 (m, 5H, Ar-H), 4.56 (s, 2H, CH₂O), 3.75∼3.79 (m, 2H, piperidin-2-CH₂), 3.31 (s, 1H, piperidin-4-CH), 3.07∼3.13 (m, 2H, piperidin-6-CH₂), 1.83∼1.87 (t, 2H, piperidin-3-CH₂), 1.56∼1.60 (t, 2H, piperidin-5-CH₂), 1.45 (s, 9H, C(CH₃)₃); MS (ESI) m/z: 292 [M + H]⁺. Compounds 8b–i were synthesized according to the same protocol described for 8a.

4-(Benzyloxy)piperidine trifluoroacetate (9a). Trifluoroacetic acid (0.91 g, 2.0 mmol, 4 equiv) was added to a solution of 8a (0.58 g, 2.0 mmol, 1 equiv) in dichloromethane (25 mL), and the resulting solution was stirred at room temperature for 12 h. Then the solution was evaporated to dryness under reduced pressure to give 9a as yellow oil (0.55 g, 90.1%). The product was used in the next step without any further purification. Compounds 9b–i were synthesized according to the same protocol described for 9a.

3-[4-(Benzyloxy)piperidin-1-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10a). A solution of epoxide 4 (1.67 g, 0.005 mol), 7a (2.57 g, 0.006 mol), triethylamine (3 mL) and EtOH (30 mL) was heated to reflux for 9h. The solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂: MeOH 100 [thin space (1/6-em)]

2, v/v) to give 10a as pale yellow solid (0.60 g, 28.0%): R_f 0.28 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); mp 89–90 °C; ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.77 (s, 1H, TriazoleC₅-H), 6.77∼7.57 (m, 8H, Ar-H), 4.50 (s, 2H, C₁-HaHb), 4.47 (s, 2H, OCH₂), 3.36 (br, 1H, piperidin-4-CH), 3.04 (d, 1H, J = 12.5 Hz, C₃-Ha), 2.65 (d, 1H, J = 13.1 Hz, C₃-Hb), 2.47∼2.53 (m, 2H, piperidin-2-CH₂), 2.13∼2.25 (m, 2H, piperidin-6-CH₂), 1.77 (br, 2H, piperidin-3-CH₂), 1.59 (br, 2H, piperidin-5-CH₂); ¹³C-NMR (500 MHz, CDCl₃): δ 162.71, 158.91, 150.98, 144.63, 138.69, 129.32, 128.34, 127.42, 126.38, 111.50, 104.19, 72.98, 71.72, 69.75, 62.19, 56.49, 52.50, 51.97, 31.19, 29.66 ppm; HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₆F₂N₄O₂: 429.2102, found: 429.2101. The target compounds 10b–i were synthesized according to the same protocol described for 10a.

3-[4-(2-Chlorobenzyloxy)piperidin-1-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10b). Pale yellow solid (1.20 g, 51.9%); R_f 0.26 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); mp 76–77 °C; ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.80 (s, 1H, TriazoleC₅-H), 6.79∼7.46 (m, 7H, Ar-H), 5.30 (s, 1H, OH), 4.55 (s, 2H, OCH₂), 4.50 (d, 2H, J = 14.5 Hz, C₁-HaHb), 3.42 (br, 1H, piperidin-4-CH), 3.05 (d, 1H, J = 13.4 Hz, C₃-Ha), 2.66 (d, 1H, J = 13.6 Hz, C₃-Hb), 2.49∼2.55 (m, 2H, piperidin-2-CH₂), 2.14∼2.28 (m, 2H, piperidin-6-CH₂), 1.80 (br, 2H, piperidin-3-CH₂), 1.62 (br, 2H, piperidin-5-CH₂); ¹³C-NMR (500 MHz, CDCl₃): δ 162.69, 158.90, 150.98, 144.61, 136.41, 132.62, 129.29, 129.14, 128.78, 128.48, 126.73, 126.36, 111.47, 104.18, 73.70, 71.73, 66.90, 62.19, 56.46, 52.45, 51.93, 31.20 ppm; HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₅ClF₂N₄O₂: 463.1712, found: 463.1715.

3-[4-(2,4-Dichlorobenzyloxy)piperidin-1-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10c). Yellow oil (1.13 g, 45.6%); R_f 0.27 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.78 (s, 1H, TriazoleC₅-H), 6.77∼7.56 (m, 6H, Ar-H), 5.30 (s, 1H, OH), 4.52 (d, 2H, J = 14.4 Hz, C₁-HaHb), 4.49 (s, 2H, OCH₂), 3.40 (br, 1H, piperidin-4-CH), 3.05 (d, 1H, J = 13.2 Hz, C₃-Ha), 2.66 (d, 1H, J = 13.6 Hz, C₃-Hb), 2.48∼2.54 (m, 2H, piperidin-2-CH₂), 2.15∼2.26 (m, 2H, piperidin-6-CH₂), 1.79 (br, 2H, piperidin-3-CH₂), 1.60 (br, 2H, piperidin-5-CH₂); HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₄Cl₂F₂N₄O₂: 497.1323, found: 497.1320.

3-[4-(3,4-Dichlorobenzyloxy)piperidin-1-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10d). Pale yellow solid (1.13 g, 45.6%); R_f 0.27 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); mp 77–78 °C; ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.78 (s, 1H, TriazoleC₅-H), 6.77∼7.56 (m, 6H, Ar-H), 5.30 (s, 1H, OH), 4.52 (d, 2H, J = 14.0 Hz, C₁-HaHb), 4.49 (s, 2H, OCH₂), 3.35 (br, 1H, piperidin-4-CH), 3.04 (d, 1H, J = 13.3 Hz, C₃-Ha), 2.65 (d, 1H, J = 13.6 Hz, C₃-Hb), 2.46∼2.53 (m, 2H, piperidin-2-CH₂), 2.13∼2.25 (m, 2H, piperidin-6-CH₂), 1.76 (br, 2H, piperidin-3-CH₂), 1.56 (br, 2H, piperidin-5-CH₂); ¹³C-NMR (500 MHz, CDCl₃): δ 162.77, 158.91, 151.00, 144.62, 139.10, 132.03, 131.25, 130.29, 129.29, 129.10, 126.40, 111.50, 104.20, 73.59, 71.83, 68.36, 62.18, 56.42, 52.42, 51.89, 31.17 ppm; HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₄Cl₂F₂N₄O₂: 497.1323, found: 497.1325.

3-[4-(3-Chlorobenzyloxy)piperidin-1-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10e). Yellow oil (1.32 g, 57.1%); R_f 0.28 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.78 (s, 1H, TriazoleC₅-H), 6.79∼7.56 (m, 7H, Ar-H), 4.50 (d, 2H, J = 14.2 Hz, C₁-HaHb), 4.44 (s, 2H, OCH₂), 3.36 (br, 1H, piperidin-4-CH), 3.04 (d, 1H, J = 13.5 Hz, C₃-Ha), 2.65 (d, 1H, J = 13.5 Hz, C₃-Hb), 2.47∼2.54 (m, 2H, piperidin-2-CH₂), 2.12∼2.27 (m, 2H, piperidin-6-CH₂), 1.76 (br, 2H, piperidin-3-CH₂), 1.54∼1.60 (m, 2H, piperidin-5-CH₂); HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₅ClF₂N₄O₂: 463.1712, found: 463.1716.

3-[4-(4-Cyanobenzyloxy)piperidin-1-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10f). Pale yellow solid (1.37 g, 60.6%); R_f 0.28 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); mp 140–141 °C; ¹H-NMR (500 MHz, CDCl₃): δ 8.14 (s, 1H, TriazoleC₃-H), 7.78 (s, 1H, TriazoleC₅-H), 6.79∼7.62 (m, 7H, Ar-H), 4.52 (s, 2H, OCH₂), 4.50 (d, 2H, J = 14.2 Hz, C₁-HaHb), 3.38 (br, 1H, piperidin-4-CH), 3.04 (d, 1H, J = 13.9 Hz, C₃-Ha), 2.66 (d, 1H, J = 13.6 Hz, C₃-Hb), 2.47∼2.54 (m, 2H, piperidin-2-CH₂), 2.12∼2.27 (m, 2H, piperidin-6-CH₂), 1.76 (br, 2H, piperidin-3-CH₂), 1.54∼1.60 (m, 2H, piperidin-5-CH₂); ¹³C-NMR (500 MHz, CDCl₃): δ 162.65, 158.88, 150.94, 144.57, 144.31, 132.07, 129.26, 127.40, 126.32, 118.68, 111.35, 104.13, 73.81, 71.82, 68.77, 62.14, 56.35, 52.31, 51.81, 31.09 ppm; HRMS-ESI: m/z [M + H]⁺ calcd for C₂₄H₂₅F₂N₅O₂: 454.2055, found: 454.2054.

3-[4-(3-Bromobenzyloxy)piperidin-1-yl]-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10g). Yellow oil (0.97 g, 38.3%); R_f 0.28 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.78 (s, 1H, TriazoleC₅-H), 6.77∼7.56 (m, 7H, Ar-H), 5.30 (s, 1H, OH), 4.50 (d, 2H, J = 14.2 Hz, C₁-HaHb), 4.43 (s, 2H, OCH₂), 3.35 (br, 1H, piperidin-4-CH), 3.04 (d, 1H, J = 13.6 Hz, C₃-Ha), 2.66 (d, 1H, J = 13.6 Hz, C₃-Hb), 2.47∼2.53 (m, 2H, piperidin-2-CH₂), 2.14∼2.27 (m, 2H, piperidin-6-CH₂), 1.76 (br, 2H, piperidin-3-CH₂), 1.54∼1.59 (m, 2H, piperidin-5-CH₂); HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₅BrF₂N₄O₂: 507.1207, found: 507.1210.

2-(2,4-Difluorophenyl)-3-[4-(2-fluorobenzyloxy)piperidin-1-yl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10h). Pale yellow solid (0.79 g, 35.4%); R_f 0.27 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); mp 77–78 °C; ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.78 (s, 1H, TriazoleC₅-H), 6.79∼7.56 (m, 7H, Ar–H), 5.30 (s, 1H, OH), 4.53 (s, 2H, OCH₂), 4.50 (d, 2H, J = 14.2 Hz, C₁-HaHb), 3.39 (br, 1H, piperidin-4-CH), 3.04 (d, 1H, J = 13.7 Hz, C₃-Ha), 2.65 (d, 1H, J = 13.6 Hz, C₃-Hb), 2.54 (br, 2H, piperidin-2-CH₂), 2.14∼2.26 (m, 2H, piperidin-6-CH₂), 1.78 (br, 2H, piperidin-3-CH₂), 1.55∼1.61 (m, 2H, piperidin-5-CH₂); HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₅F₃N₄O₂: 447.2008, found: 447.2010.

2-(2,4-Difluorophenyl)-3-[4-(4-fluorobenzyloxy)piperidin-1-yl]-1-(1H-1,2,4-triazol-1-yl)propan-2-ol (10i). Pale yellow solid (0.88 g, 39.5%); R_f 0.27 (CH₂Cl₂/MeOH, 100 [thin space (1/6-em)]

2); mp 89–90 °C; ¹H-NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, TriazoleC₃-H), 7.78 (s, 1H, TriazoleC₅-H), 6.79∼7.56 (m, 7H, Ar–H), 5.30 (s, 1H, OH), 4.50 (d, 2H, J = 14.4 Hz, C₁-HaHb), 4.42 (s, 2H, OCH₂), 3.35 (br, 1H, piperidin-4-CH), 3.04 (d, 1H, J = 13.9 Hz, C₃-Ha), 2.65 (d, 1H, J = 13.7 Hz, C₃-Hb), 2.46∼2.53 (m, 2H, piperidin-2-CH₂), 2.11∼2.27 (m, 2H, piperidin-6-CH₂), 1.75 (br, 2H, piperidin-3-CH₂), 1.55∼1.58 (m, 2H, piperidin-5-CH₂); ¹³C-NMR (500 MHz, CDCl₃): δ 162.66, 162.21, 158.90, 150.98, 144.61, 134.44, 129.31, 129.02, 126.29, 115.15, 111.42, 104.17, 73.17, 71.79, 69.05, 62.20, 56.45, 52.49, 51.95, 31.16, 29.63 ppm; HRMS-ESI: m/z [M + H]⁺ calcd for C₂₃H₂₅F₃N₄O₂: 447.2008, found: 447.2006.

In vitro antifungal activity assays

In vitro antifungal activity was measured by means of the minimum inhibitory concentration (MIC) using the serial dilution method in 96-well microtest plates. Fluconazole was used as the reference drug. Test fungal strains were obtained from the ATCC or were clinical isolates. The MIC determination was performed according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations with RPMI 1640 (Sigma) buffered with 0.165M MOPS (Sigma) as the test medium. The MIC value was defined as the lowest concentration of test compounds that resulted in a culture with turbidity less than or equal to 80% inhibition when compared with the growth of the control. Test compounds were dissolved in DMSO serially diluted in growth medium. The yeasts were incubated at 35 °C and the dermatophytes at 28 °C. Growth MIC was determined at 24 h for Candida species, at 72 h for C. neoformans, and at 7 days for filamentous fungi.

Flexible docking analysis

The 3D structures of the designed azoles were built by the Builder module within InsightII 2000 software package. Then, the flexible ligand docking procedure in the Affinity module within InsightII was used to define the lowest energy position for the substrate using a Monte Carlo docking protocol. The detailed docking parameters were from our previous studies.²¹

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant Nos. 30930107), Science and Technology Commission of Shanghai (Grant Nos. 10431902100), Shanghai Rising-Star Program (Grant Nos. 09QA1407000) and Shanghai Leading Academic Discipline Project (Project Nos. B906).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c1md00103e

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Discovery of highly potent antifungal triazoles by structure-based COMPOUND LINKSRead more about this on ChemSpiderDownload mol file of compoundlead fusion†

Abstract

Introduction

Results and discussion

Chemistry

Design rationale

In vitro antifungal activity

Structure–activity relationships

Conclusions

Experimental section

Chemistry

In vitro antifungal activity assays

Flexible docking analysis

Acknowledgements

References

Footnote

Discovery of highly potent antifungal triazoles by structure-based lead fusion†