Synthesis and Bioactivity Evaluation of Novel 2-Salicyloylbenzofurans as Antibacterial Agents

In order to discover new antibacterial agents, series of 2-salicyloylbenzofuran derivatives were designed, synthesized and evaluated for their antibacterial activities against three Gram-(+) strains (methicillin-sensitive Staphylococcus aureus (MSSA) ATCC 29213, methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300, and Streptococcus faecalis (S. faecalis) ATCC 29212) and one Gram-(−) strain (Escherichia coli (E. coli) ATCC 25922). The 2-salicyloylbenzofuran heterocycles were generated by Rap–Stoermer condensation of salicylaldehydes with phenacyl bromides and then converted to diverse O-ether derivatives by Williamson synthesis. The targeted products were screened for in vitro qualitative (zone of inhibition) and quantitative (MIC) antibacterial activities by agar well diffusion assay and agar dilution method. Amongst the compounds, those bearing carboxylic acid functional group were found to exhibit reasonable activity against Gram-(+) bacterial strains including S. faecalis, MSSA and MRSA with the most potent antibacterial agent 8h (MICs = 0.06–0.12 mM). Besides, the 2-salicyloylbenzofurans partly displayed inhibitory activity against MRSA with the best MICs = 0.14 mM (8f) and 0.12 mM (8h). Finally, the antibacterial results preliminarily suggested that the substituent bearing carboxylic acid group at salicyloyl-C2 and the bromine atoms on the benzofuran moiety seem to be the functionality necessary for antibacterial activities.


Introduction
Drug resistant infections are complex problems that have been threatening the health care with related morbidity and mortality being on the rise worldwide. Moreover, infectious diseases have been extremely difficult to treat due to antibiotic resistant problem resulting in high extra in-hospital costs [1][2][3][4]. Therefore, the prevention and control of the resistance threats have been identified as global public health priorities that require actions across all government sectors and society [1][2][3][4]. discovery and development of new classes of antibacterial agents as well as exploring possible new drugs from existing classes of antibiotics that can effectively against the multidrug-resistant bacterial strains [1, 2,4].

Chemistry
The O-ether derivatives of 5,7-dibromo-2-salicyloyl benzofuran 7a-h and 8f-h were prepared according to the synthetic Scheme 1. Bromination of starting material 2-methoxyacetophenone 1 using NBS as bromine source in the presence of p-toluenesulfonic acid (PTSA) gave 2-methoxyphenacyl bromide 2 [17] that was reacted with 3,5-dibromosalicyladehyde via Rap-Stoermer condensation reaction, using 4-dimethylaminopyridine (DMAP) as catalyst, to obtain 5,7-dibromo-2-(2-methoxybenzoyl)benzofuran 4 [18]. Demethylation of compound 4 by anhydrous AlCl 3 in dried dichloromethane afforded 5,7-dibromo-2-salicyloylbenzofuran 5 as key intermediate in good yield [19,20]. O-ether derivatives of compound 5 were synthesized by Williamson reaction at 2-OH group of the salicyloyl moiety with diverse halogenated materials 6a-h (for details see Section 3.2.4) using K 2 CO 3 as a base in acetone gave the desired compounds 7a-h [21]. Alkaline hydrolysis of esters 7f-h in non-aqueous condition furnished the products 8f-h as their respective carboxylic acid derivatives [22]. K2CO3 as a base in acetone gave the desired compounds 7a-h [21]. Alkaline hydrolysis of esters 7f-h in non-aqueous condition furnished the products 8f-h as their respective carboxylic acid derivatives [22]. To investigate the role of the substituents on the benzofuran moiety on the antibacterial activities, series of 2-(2-(benzofuran-2-carbonyl)phenoxy)ethanoic acid derivatives 14a-f were synthesized according to the synthetic Scheme 2. The key intermediate 11 was prepared by O-etherification at 2-OH group of 2-hydroxyacetophenone 9 with ethyl chloroacetate 6f [21] and then brominated by NBS in the presence of PTSA as catalyst [17]. The Rap-Stoermer condensation reaction was applied to generate the benzofuran heterocycles 13a-f [18] that was subjected to alkaline hydrolysis in non-aqueous condition to obtain the final products 14a-f [22]. To investigate the role of the substituents on the benzofuran moiety on the antibacterial activities, series of 2-(2-(benzofuran-2-carbonyl)phenoxy)ethanoic acid derivatives 14a-f were synthesized according to the synthetic Scheme 2. The key intermediate 11 was prepared by O-etherification at 2-OH group of 2-hydroxyacetophenone 9 with ethyl chloroacetate 6f [21] and then brominated by NBS in the presence of PTSA as catalyst [17]. The Rap-Stoermer condensation reaction was applied to generate the benzofuran heterocycles 13a-f [18] that was subjected to alkaline hydrolysis in non-aqueous condition to obtain the final products 14a-f [22]. K2CO3 as a base in acetone gave the desired compounds 7a-h [21]. Alkaline hydrolysis of esters 7f-h in non-aqueous condition furnished the products 8f-h as their respective carboxylic acid derivatives [22]. To investigate the role of the substituents on the benzofuran moiety on the antibacterial activities, series of 2-(2-(benzofuran-2-carbonyl)phenoxy)ethanoic acid derivatives 14a-f were synthesized according to the synthetic Scheme 2. The key intermediate 11 was prepared by O-etherification at 2-OH group of 2-hydroxyacetophenone 9 with ethyl chloroacetate 6f [21] and then brominated by NBS in the presence of PTSA as catalyst [17]. The Rap-Stoermer condensation reaction was applied to generate the benzofuran heterocycles 13a-f [18] that was subjected to alkaline hydrolysis in non-aqueous condition to obtain the final products 14a-f [22]. In this study, the Rap-Stoermer condensation reaction was applied for the efficient generation of benzofuran heterocycle in 57-87% yields. The Williamson reaction was used to synthesize desired 2-salicyloylbenzofuran derivatives bearing diverse O-ether moieties in rather good yields (60-87%). It was found that the most preferable alkylating agents for ether formation are primary α-halogenated materials since the Williamson reaction using ethyl 2-bromopropanoate 6g, a secondary α-halogenated material, gave ether product 7g with the lowest yield (60%). The structures of synthesized 2-salicyloylbenzofurans were characterized by 1 H-NMR, 13 C-NMR and HRMS. The obtained results showed consistency with the expected structures and formulas of the targeted products. The 1 H-NMR spectra of 2-salicyloylbenzofurans displayed a single signal at the range of δ 7.70-7.30 ppm, which confirmed the formation of benzofuran heterocycle.

Evaluation of Antibacterial Activity
The newly synthesized compounds were preliminarily screened for in vitro qualitative (zone of inhibition) and quantitative (MIC) antibacterial activities against three Gram-(+) strains (MSSA ATCC 29213, MRSA ATCC 43300, and S. faecalis ATCC 29212) and one Gram-(-) strain (E. coli ATCC 25922) by slight modified agar well diffusion assay [23,24] and agar dilution method [25]. Dimethyl sulfoxide (DMSO) and potent antibacterial drugs ampicillin, cefuroxime and vancomycin were used as solvent control and standards, respectively. The diameters of the inhibition zone (mm) corresponding to the MICs (mM) are presented in Table 1. The 2-methoxybenzoylbenzofuran 4 showed week antibacterial activity against E. coli and S. faecalis with inhibition zone diameters of 9.5 mm. Similarly, the respective phenolic compound, 2-salicyloylbenzofuran 5, showed week antibacterial activity against three Gram-(+) bacterial strains with inhibition zone diameters in the range of 9-13 mm (MICs > 2.59 mM) while completely lost activity against E. coli. These preliminary results showed that compound 4 and 5 were considered poor antibacterial agents. From the literature, we found that the substituent at the aroyl-C2 moiety was identified as the important characteristic feature for antibacterial activity [7][8][9][10][11]. Therefore, in order to improve the antibacterial activity of 2-salicyloylbenzofurans, we focused on the replacing of 2-OCH 3 and 2-OH group by different ether residues. Amongst the obtained ethers, compounds 7a-f except 7c showed some slight improvements of antibacterial activity with inhibition zone diameters in the range of 8-18 mm (MICs > 1.84 mM) against Gram-(+) and/or Gram-(-) strains. Meanwhile, the 2-salicyloylbenzofuran derivatives 8f-h bearing carboxylic acid group on the ether residue significantly increased their antibacterial activities against three Gram-(+) bacterial strains (S. faecalis, MSSA and MRSA) as compared to the parent derivatives 4 and 5 with the MIC values = 0.06-0.55 mM. Compound 8h showed the most potent antibacterial activity with MIC values = 0.06-0.12 mM against three Gram-(+) bacterial strains. In addition, these 2-salicyloylbenzofurans also displayed inhibitory activity against MRSA with the best MICs = 0.14, 0.27, and 0.12 mM for compound 8f-h, respectively. However, compounds 8f-h were completely inactive against Gram-(-) E. coli at the tested concentration. It was found from these results that the carboxylic acid group seems to be an essential part of the pharmacophore required for inhibitory activity against Gram-(+) bacterial strains since the others including nitrile 7c, amide 7e, and ester 7f generally possessed very poor or no antibacterial activity. It was also suggested that the -COOH group keeps an important role for a potential hydrogen bonding as hydrogen bond donor functions with hydrophilic properties.
We next investigated the potential impact of substituents on the benzofuran heterocycle by replacing bromine atoms with different substituents (14a-f). The antibacterial activity showed compounds 14a-f decreased their bioactivity with MIC values ≥ 0.70 mM as compared to compound 8f. These results indicated that bromine atoms may serve as potential halogen bond donor functions with lipophilic properties that are consistent with other research [7]. Besides, most of these derivatives were also inactive against E. coli (except compound 14d, MIC = 3.10 mM) that again confirmed that the presence of carboxylic acid group on the salicyloyl moiety seems to be favor for inhibitory activity against Gram-(+) strains. Overall, the results of this study suggested that the 5,7-dibromo-2-salicyloylbenzofuran bearing carboxylic group via ether linker moiety (Figure 2) may become the promising scaffold for the follow-up research to discover novel antibacterial agents, particularly against Gram-(+) bacterial strains including MRSA, a main cause of a variety of community and healthcare-associated infections. poor antibacterial agents. From the literature, we found that the substituent at the aroyl-C2 moiety was identified as the important characteristic feature for antibacterial activity [7][8][9][10][11]. Therefore, in order to improve the antibacterial activity of 2-salicyloylbenzofurans, we focused on the replacing of 2-OCH3 and 2-OH group by different ether residues. Amongst the obtained ethers, compounds 7a-f except 7c showed some slight improvements of antibacterial activity with inhibition zone diameters in the range Compound 8h showed the most potent antibacterial activity with MIC values = 0.06-0.12 mM against three Gram-(+) bacterial strains. In addition, these 2-salicyloylbenzofurans also displayed inhibitory activity against MRSA with the best MICs = 0.14, 0.27, and 0.12 mM for compound 8f-h, respectively. However, compounds 8f-h were completely inactive against Gram-(-) E. coli at the tested concentration. It was found from these results that the carboxylic acid group seems to be an essential part of the pharmacophore required for inhibitory activity against Gram-(+) bacterial strains since the others including nitrile 7c, amide 7e, and ester 7f generally possessed very poor or no antibacterial activity. It was also suggested that the -COOH group keeps an important role for a potential hydrogen bonding as hydrogen bond donor functions with hydrophilic properties. We next investigated the potential impact of substituents on the benzofuran heterocycle by replacing bromine atoms with different substituents (14a-f). The antibacterial activity showed compounds 14a-f decreased their bioactivity with MIC values ≥ 0.70 mM as compared to compound 8f. These results indicated that bromine atoms may serve as potential halogen bond donor functions with lipophilic properties that are consistent with other research [7]. Besides, most of these derivatives were also inactive against E. coli (except compound 14d, MIC = 3.10 mM) that again confirmed that the presence of carboxylic acid group on the salicyloyl moiety seems to be favor for inhibitory activity against Gram-(+) strains. Overall, the results of this study suggested that the 5,7dibromo-2-salicyloylbenzofuran bearing carboxylic group via ether linker moiety ( Figure 2) may become the promising scaffold for the follow-up research to discover novel antibacterial agents, particularly against Gram-(+) bacterial strains including MRSA, a main cause of a variety of community and healthcare-associated infections.

Chemicals and Instruments
All starting materials were purchased from commercial sources such as Acros Organics-Thermo Fisher Scientific (Geel, Belgium), TCI Chemicals (Tokyo, Japan) and used without further purification. Thin-layer chromatography (TLC) was carried out on aluminum-supported silica gel plates (Merck 60F 254, Darmstadt, Germany) with visualization of components by UV light (254 nm). Column chromatography was carried out on silica gel (0.04-0.063 mm, 230-400 mesh ASTM, Merck, Darmstadt, Germany). The melting points were recorded on a Gallenkamp apparatus (Sanyo Gallenkamp, Southborough, UK) and were uncorrected. NMR spectra were recorded in CDCl3 or DMSO-d6 using a Bruker Avance 500 MHz spectrometer (Bruker Corporation, Billerica, MA, USA). Chemical shifts (δ) are reported in ppm related to internal tetramethylsilane (TMS) and coupling

Chemicals and Instruments
All starting materials were purchased from commercial sources such as Acros Organics-Thermo Fisher Scientific (Geel, Belgium), TCI Chemicals (Tokyo, Japan) and used without further purification. Thin-layer chromatography (TLC) was carried out on aluminum-supported silica gel plates (Merck 60F 254, Darmstadt, Germany) with visualization of components by UV light (254 nm). Column chromatography was carried out on silica gel (0.04-0.063 mm, 230-400 mesh ASTM, Merck, Darmstadt, Germany). The melting points were recorded on a Gallenkamp apparatus (Sanyo Gallenkamp, Southborough, UK) and were uncorrected. NMR spectra were recorded in CDCl 3 or DMSO-d 6 using a Bruker Avance 500 MHz spectrometer (Bruker Corporation, Billerica, MA, USA). Chemical shifts (δ) are reported in ppm related to internal tetramethylsilane (TMS) and coupling constant (J) are reported in Hertz (Hz). Mass spectra were recorded on a Shimadzu LCMS-IT-TOF Mass spectrometer (Shimadzu Scientific Instruments, Kyoto, Japan).
Three Gram-(+) strains (MSSA ATCC 29213, MRSA ATCC 43300, S. faecalis ATCC 29212) and one Gram-(-) strain (E. coli ATCC 25922) were preserved and activated at our Department of Microbiology and Parasitology. The bacterial culture media were purchased from Merck (Darmstadt, Germany) in which Tryptic Soy Agar (TSA) was used to isolate and preserve bacterial strains, Tryptic Soy Broth (TSB) was used as bacteria-activating medium and Muller-Hinton Agar (MHA) was used for antimicrobial susceptibility testing. Dimethyl sulfoxide (DMSO) from Merck and potent antibacterial drugs ampicillin, cefuroxime, and vancomycin (Sigma-Aldrich Pte. Ltd., St. Louis, MO, USA) were used as solvent control and standards, respectively.

General Procedure for the Synthesis of Phenacyl Bromides 2, 11
The round-bottom flask containing acetophenone derivative 1, 10 (4 mmol) and PTSA (0.076 g, 0.4 mmol) was heated to 60 • C to turn the reaction mixture into a paste and NBS (0.854 g, 4.8 mmol) then added slowly. After 15 min, the reaction mixture was cooled to room temperature and water (20 mL) added. The crude product was extracted with dichloromethane (2 × 20 mL), dried over anhydrous Na 2 SO 4 and purified by crystallization from n-hexane-dichloromethane to give pure product. This result showed consistency with the data in literature [26].

Measurement of MIC Values
The quantitative (MIC) antibacterial activity against three Gram-(+) strains (MSSA ATCC 29213, MRSA ATCC 43300, S. faecalis ATCC 29212) and one Gram-(-) strain (E. coli ATCC 25922) was determined by agar dilution method as per CLSI recommendation on MHA containing 2-fold serial dilutions of antibiotics and selectively active 2-salicyloylbenzofurans [25]. The compounds in the test medium were prepared at the required quantities of 1024, 512, 256, 128, 64, 32, 16, and 8 µg/mL concentrations and the standard drugs at 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 µg/mL with MHA. The final inoculum size was 10 4 CFU/mL for the antibacterial assay. All samples were tested in duplicate. The test plates were incubated at 37 • C for 24 h. The MIC (expressed in µg/mL) was taken as the minimum concentration of the dilution with no growth of microorganisms. The concentration of the solvents used in the following assays was maintained at 2-5% so that no inhibition of microorganisms or interference occurred.

Conclusions
In summary, we designed, synthesized twenty five 2-salicyloylbenzofuran derivatives and evaluated for their antibacterial activities against three Gram-(+) strains (MSSA ATCC 29213, MRSA ATCC 43300, S. faecalis ATCC 29212) and one Gram-(-) strain (E. coli ATCC 25922). Most of the 2-salicyloylbenzofurans were found to possess weak to moderate activities against Gram-(+) and/ or Gram-(-) bacteria with the inhibition zone diameters of 8-19 mm and MIC values ≥ 0.06 mM. Amongst the 2-salicyloylbenzofurans bearing carboxylic acid functional group 8f-h and 14a-f displayed the stronger bioactivity and showed the strain-specific to Gram-(+) bacteria with the most potent antibacterial agent 8h (MICs = 0.06-0.12 mM). In addition, the 2-salicyloylbenzofuran scaffold was found to exhibit activity against MRSA with the best MIC values = 0.14 mM (8f) and 0.12 mM (8h). The antibacterial results suggested that the substituent bearing carboxylic acid group at salicyloyl-C2 and the bromine atoms on the benzofuran moiety might be the functional necessary for antibacterial activity. Finally, although the antibacterial activities of 2-salicyloylbenzofurans in this study were not very significant, these results initially provide some necessary information for further chemical optimization of this scaffold in the future.