Design, Synthesis and Antifungal Activity of Novel Benzofuran-Triazole Hybrids

A series of novel benzofuran-triazole hybrids was designed and synthesized by click chemistry, and their structures were characterized by HRMS, FTIR and NMR. The in vitro antifungal activity of target compounds was evaluated using the microdilution broth method against five strains of pathogenic fungi. The result indicated that the target compounds exhibited moderate to satisfactory activity. Furthermore, molecular docking was performed to investigate the binding affinities and interaction modes between the target compound and N-myristoyltransferase. Based on the results, preliminary structure activity relationships (SARs) were summarized to serve as a foundation for further investigation.


Introduction
Fungal infections have posed a continuous and serious threat to human health and life during the past two decades, especially among hosts, such as patients undergoing anticancer chemotherapy or organ transplants, and patients with AIDS [1,2]. Clinically, available antifungal drugs have several drawbacks, such as drug-related toxicity, non-optimal pharmacokinetics, and the emergence of drug resistance [3,4]. Therefore, the development of new antifungal drugs with novel modes of action is required.
Encouraged by the results above, we attempted to design and synthesize a series of benzofurantriazole hybrids to evaluate the in vitro antifungal activity.
Encouraged by the results above, we attempted to design and synthesize a series of benzofuran-triazole hybrids to evaluate the in vitro antifungal activity.

Chemistry
The synthetic route to target compounds was outlined in Scheme 1. The reaction of 2 1 ,6 1dihydroxyacetophenone with the corresponding 2-bromoacetophenone in a modified Rap-Stormer reaction condition [22] gave the benzofuran scaffold (1). Alkylation of the hydroxyl group of 1a,b with propargyl bromide gave terminal alkyne derivatives (2a,b). The aromatic azides (3a-i) were prepared from the corresponding anilines following the Sandmeyer conditions [27,31]. Finally, employing click chemistry, compounds 2a,b were cyclized with 3a-i, respectively, to give target compounds 4a-r in good yields.

Antifungal Activity
The in vitro antifungal activity of the target compounds was measured by means of the minimal inhibitory concentrations (MICs) with fluconazole as the control drug. The results are summarized in Table 1. Against fluconazole-resistant Trichophyton rubrum, many target compounds (4e,f, 4h and 4b-r) showed better activity than fluconazole (128 µg¨mL´1) in the range of 32 to 64 µg¨mL´1, and some compounds (4b, 4d, 4g, and 4i-l) showed equivalent activity to fluconazol. Except compounds 4a and 4c, most of the compounds showed antifungal activity against Cryptococcus neoformans in concentrations ranging from 32 to 128 µg¨mL´1. The target compounds (4f, 4h, 4m, 4p and 4r) showed antifungal activity against Candida zeylanoides at the concentration of 64 µg¨mL´1. Some compounds showed weak activity merely against Candida albicans (4f, 4h, 4m, 4o and 4r) and Rhodotorula rubra (4d, 4f, 4h, 4o and 4q,r) at the concentration of 128 µg¨mL´1. synthetic route to target compounds was outlined in Scheme 1. The reaction droxyacetophenone with the corresponding 2-bromoacetophenone in a modified Rap-Storm condition [22] gave the benzofuran scaffold (1). Alkylation of the hydroxyl group of 1a,b wi yl bromide gave terminal alkyne derivatives (2a,b). The aromatic azides (3a-i) were prepar corresponding anilines following the Sandmeyer conditions [27,31]. Finally, employing cli y, compounds 2a,b were cyclized with 3a-i, respectively, to give target compounds 4a-r lds. eme 1. Synthesis of target compounds 4a-r. Reagents and conditions: Scheme 1. Synthesis of target compounds 4a-r. Reagents and conditions:

Antifungal Activity
The in vitro antifungal activity of the target compounds was measured by means of the minimal inhibitory concentrations (MICs) with fluconazole as the control drug. The results are summarized in Table 1. Against fluconazole-resistant Trichophyton rubrum, many target compounds (4e,f, 4h and 4b-r) showed better activity than fluconazole (128 μg•mL −1 ) in the range of 32 to 64 μg•mL −1 , and some compounds (4b, 4d, 4g, and 4i-l) showed equivalent activity to fluconazol. Except compounds 4a and 4c, most of the compounds showed antifungal activity against Cryptococcus neoformans in concentrations ranging from 32 to 128 μg•mL −1 . The target compounds (4f, 4h, 4m, 4p and 4r) showed antifungal activity against Candida zeylanoides at the concentration of 64 μg•mL −1 . Some compounds showed weak activity merely against Candida albicans (4f, 4h, 4m, 4o and 4r) and Rhodotorula rubra (4d, 4f, 4h, 4o and 4q,r) at the concentration of 128 μg•mL −1 . Observing the antifungal assay results, it can be noticed that the derivatives with a di-fluorinesubstituted phenyl ring at the benzofuran C-2 side chain (ring A) are more effective than the mono-fluorine ones (e.g., 4f vs. 4o). Meanwhile, the substituted groups on the phenyl ring linked to the triazole (ring B) also had an impact on the activity. The alkyl-substituted compounds are more potent than the halogenated derivatives (e.g., 4b vs. 4f) and the ortho-substituted derivatives are more potent than the para isomers (e. g., 4e vs. 4f, 4j vs. 4k). The preliminary structure activity relationships (SARs) were supported by the outstanding bioactivities of 4o and 4r among all the target compounds.

Molecule Docking
In an attempt to investigate the action modes of the target compounds, 4o was docked into the crystal structure of NMT from C. albicans (CaNMT, PDB ID: 1IYL) using Discovery Studio 3.0. The docking results are illustrated in Figure 2. The benzofuran ring was located at the center of the active site, surrounding some hydrophobic residues, such as Tyr225, Tyr354 and Leu394, and forming a pi-pi interaction with Tyr225. The di-fluorine phenyl fragment formed a hydrophobic interaction with Phe115, Phe240 and Phe339. The phenyl triazole side chain stretched into the hydrophobic pocket constituted by Phe117, Tyr119 and Phe176. The oxygen atom of the benzofuran ring formed a hydrogen bond with His227. The interaction mode between 4o and the receptor is similar to the co-crystal ligand. On the other hand, no hydrogen bond was observed between triazole and Leu451, which is formed between the C-4 secondary amine of the co-crystal ligand. This hydrogen bond is important to the antifungal activity [34]. The docking result is supported by the weak antifungal activity against C. albicans of the target compounds.

Molecule Docking
In an attempt to investigate the action modes of the target compounds, 4o was docked into the crystal structure of NMT from C. albicans (CaNMT, PDB ID: 1IYL) using Discovery Studio 3.0. The docking results are illustrated in Figure 2. The benzofuran ring was located at the center of the active site, surrounding some hydrophobic residues, such as Tyr225, Tyr354 and Leu394, and forming a pi-pi interaction with Tyr225. The di-fluorine phenyl fragment formed a hydrophobic interaction with Phe115, Phe240 and Phe339. The phenyl triazole side chain stretched into the hydrophobic pocket constituted by Phe117, Tyr119 and Phe176. The oxygen atom of the benzofuran ring formed a hydrogen bond with His227. The interaction mode between 4o and the receptor is similar to the co-crystal ligand. On the other hand, no hydrogen bond was observed between triazole and Leu451, which is formed between the C-4 secondary amine of the co-crystal ligand. This hydrogen bond is important to the antifungal activity [34]. The docking result is supported by the weak antifungal activity against C. albicans of the target compounds.

Chemistry
The 1 H-and 13 C-NMR spectra were recorded respectively on a Bruker AV-600 spectrometer and a Bruker AV-400 spectrometer. Chemical shifts were reported in parts per million (ppm, δ) downfield from TMS as an internal standard. High-resolution mass spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 in ESI mode (Agilent, Santa Clara, CA, USA). FTIR spectra were recorded on a Bruker IFS 55 spectrometer (Bruker Co., Karlsruhe, Germany). Melting points (m.p.) were determined on an X-4 microscope melting point apparatus (Beijing Tech instrument Co., Ltd., Beijing, China) without calibration.

Chemistry
The 1 H-and 13 C-NMR spectra were recorded respectively on a Bruker AV-600 spectrometer and a Bruker AV-400 spectrometer. Chemical shifts were reported in parts per million (ppm, δ) downfield from TMS as an internal standard. High-resolution mass spectra (HRMS) were measured with an Agilent Accurate-Mass Q-TOF 6530 in ESI mode (Agilent, Santa Clara, CA, USA). FTIR spectra were recorded on a Bruker IFS 55 spectrometer (Bruker Co., Karlsruhe, Germany). Melting points (m.p.) were determined on an X-4 microscope melting point apparatus (Beijing Tech instrument Co., Ltd., Beijing, China) without calibration.

General Procedure for the Synthesis of Compounds 2a,b
To a solution of compound 1a or 1b (10.0 mmol) in DMF (20 mL), propargyl bromide (1.30 g, 10.9 mmol) and potassium carbonate (1.70 g, 12.3 mmol) were added. The reaction mixture was stirred at room temperature for 5 h, and was then diluted with ethyl acetate (80 mL) and washed with water (2ˆ100 mL). The organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude products were used without purification.    The compounds 4b-r were synthesized using the same operation procedure of compound 4a.