Synthesis and Biological Evaluation of Benzo[b]thiophene Acylhydrazones as Antimicrobial Agents against Multidrug-Resistant Staphylococcus aureus

The benzo[b]thiophene nucleus and the acylhydrazone functional group were combined to prepare three new series of compounds for screening against Staphylococcus aureus. The reaction of substituted benzo[b]thiophene-2-carboxylic hydrazide and various aromatic or heteroaromatic aldehydes led to a collection of 26 final products with extensive structural diversification on the aromatic ring and on position 6 of the benzo[b]thiophene nucleus. The screening lead to the identification of eight hits, including (E)-6-chloro-N’-(pyridin-2-ylmethylene)benzo[b]thiophene-2-carbohydrazide (II.b), a non-cytotoxic derivative showing a minimal inhibitory concentration of 4 µg/mL on three S. aureus strains, among which were a reference classical strain and two clinically isolated strains resistant to methicillin and daptomycin, respectively.


Introduction
Antimicrobial resistance (AMR) is a public health issue that will continue to worsen in the years to come. At least 700,000 deaths are attributed each year to drug-resistant pathogens, but this could rise to 10,000,000 deaths per year by 2050 [1][2][3]. Among those pathogens, some are widely spread, such as vancomycin-resistant Enterococcus faecium or methicillin-resistant Staphylococcus aureus (MRSA) [4]. The development of new antibiotics targeting antibioresistant bacteria is therefore critical. Substituted benzo [b]thiophenes are interesting compounds in medicinal chemistry [5] that display a broad range of activity including antimicrobial [6,7], anticancer [8], anti-diabetic [9], anti-depressant [10], antiinflammatory and analgesic agents [11,12]. As part of an ongoing research program aiming at the discovery of new potential antibiotics targeting multidrug-resistant Staphylococcus aureus strains, we combined the benzo[b]thiophene nucleus with the acylhydrazone functional group, which is also relevant in bioactive molecules design [13]. We focused our efforts on the synthesis and biological evaluation of a collection of acylhydrazones built from various aromatic or heteroaromatic aldehydes and benzo[b]thiophene-2-carboxylic hydrazide, readily accessible from benzo[b]thiophene-2-carboxylic acid. This sequence allowed an easy 2.1.5. General Procedure for Benzo[b]thiophene-2-carboxylic Acids (1) To a solution of ethyl 6-chlorobenzo[b]thiophene-2-carboxylate (14.1 mmol, 1 eq.) in EtOH (15 mL) was added a solution of NaOH 3N (28.2 mmol, 2 eq.). The solution was stirred at room temperature overnight, concentrated under vacuum, diluted with H 2 O (75 mL), acidified with HCl 1 N, extracted with EtOAc, dried over Na 2 SO 4 and concentrated under vacuum. (Adapted from Patent WO2018/122232 [15]).
Step 2: The crude material was diluted in MeOH (10 mL) before the addition of the corresponding substituted benzaldehyde (2 mmol, 2 eq.) at room temperature. Reflux for 2 h was performed, and the final compound crystallized from the reaction mixture. Then, the reaction mixture was cooled to 0 • C, and the solid was filtered off and washed with cold MeOH. [1,3] Intermediate 2a (938 mg) was deprotected under the reported acidic conditions (TFA) to produce, after an aqueous workup with NaHCO 3 and extractions with EtOAc, a white solid (615 mg), which was directly engaged in the presence of 2-pyridinecarboxaldehyde (6.40 mmol, 2.0 eq.) in MeOH (20 mL). After 2 h reflux, the reaction mixture was concentrated in vacuo to give a brown viscous oil, which was purified by recrystallization in EtOAc to give product. White powder (292 mg, 32% yield). 1   Yellowish solid (110 mg, 30% yield). 1   Intermediate 2b was deprotected under the reported acidic conditions (TFA) to afford a white solid, which was engaged without further purification for the next step. White Intermediate 2b was deprotected under the reported acidic conditions (TFA) to afford, after an aqueous workup with NaHCO 3 and extractions with EtOAc, a white solid, which was engaged without further purification for the next step. White solid (52 mg, 30% yield). 1 Intermediate 2c was deprotected under the reported acidic conditions (TFA) to afford, after an aqueous workup with NaHCO 3 and extractions with EtOAc, a white solid, which was engaged without further purification for the next step. White solid (23 mg, 24% yield). 1 Intermediate 2b was deprotected under the reported acidic conditions (TFA) to afford, after an aqueous workup with NaHCO 3 and extractions with EtOAc, a white solid, which was engaged without further purification for the next step. Beige powder (90 mg, 48% yield). 1 Intermediate 2b was deprotected under the reported acidic conditions (TFA) to afford a white solid, which was engaged without further purification for the next step. Beige Intermediate 2b was deprotected under the reported acidic conditions (TFA) to afford a white solid, which was engaged without further purification for the next step. Beige powder (94 mg, 46% yield). 1 Intermediate 2e was deprotected under the reported acidic conditions (TFA) to afford, after an aqueous workup with NaHCO 3 and extractions with EtOAc, a white solid, which was engaged without further purification for the next step. White powder (63 mg, 44% yield). 1 Intermediate 2d was deprotected under the reported acidic conditions (TFA) to afford, after an aqueous workup with NaHCO 3 and extractions with EtOAc, a white solid, which was engaged without further purification for the next step. White powder (64 mg, 44% yield). 1 Under H 2 atmosphere, a solution of (E)-N'-(pyridin-2-ylmethylene)benzo[b]thiophene-2-carbohydrazide (0.12 mmol, 1 eq.) and palladium on carbon 10% (0.019 mmol, 15 mol%) in 5 mL of EtOAc and 1 mL of MeOH was stirred at room temperature for 7 h. The mixture was filtered on Celite ® and washed with EtOAc, and the residue was concentrated under vacuum and purified by chromatography (eluent: EtOAc/MeOH 98:2). (Adapted from Kim et al. [22]).   (6) To a solution of pyridine-2-carboxaldehyde (1.87 mmol, 1 eq.) and hydroxylamine hydrochloride (2.33 mmol, 1.25 eq.) in water (5 mL) was added dropwise a solution of sodium bicarbonate (2.33 mmol, 1.25 eq.) in water (10 mL), and the mixture was stirred for 3 h at room temperature. The solution was then extracted with EtOAc and the organic layer dried over Na 2 SO 4 Under a dried and inert atmosphere (N 2 ), a solution of 6-chlorobenzothiophene-2carboxylic acid (1.06 mmol, 1.3 eq.) and pyridine 2-aldoxime (0.82 mmol, 1.0 eq.) in anhydrous DCM (3 mL) was cooled to 0 • C. Then, DMAP (0.12 mmol, 0.15 eq.) and DCC (1.06 mmol, 1.3 eq.) were gradually added to the mixture. The mixture was stirred at room temperature overnight and then filtered on Celite ® and washed with DCM. The filtrate was then concentrated, and the residue was purified by column chromatography (eluent: pentane/EtOAc 2:1).
Orange powder (166 mg, 64% yield). 1  MICs were evaluated in CaMHB (cation-adjusted Mueller-Hinton broth) by the method of microdilution in liquid medium, which follows the CLSI recommendations (Clinical and Laboratory Standards Institute (CLSI), methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard seventh edition. Clinical and Laboratory Standards Institute, Wayne, PA, USA). Evaluated compounds were diluted in DMSO with a concentration of 5 mg/mL and then further diluted in CaMHB. The 0.5 MacFarland bacterial suspensions were made from colonies previously grown on blood agar plate (COS, Biomérieux) in a saline solution (0.45% NaCl). They were diluted in CaMHB (1/100) before addition into 96-well microplates. MICs were carried out in triplicate and determined after 18 h of incubation at 37 • C. The median values were reported.

Cytotoxicity Assay
Cytotoxicity was evaluated using a propidium iodide assay. Briefly, human lung adenocarcinomic A549 cells were plated on black flat 96-well plates (Greiner) at a concentration of 0.5 × 10 6 cells/mL in DMEM GlutaMAX (Gibco) supplemented with 10% fetal bovine serum for 24 h in a humidified atmosphere at 37 • C and 5% CO 2 . The medium was then removed and fresh medium with 2.5 µg/mL of propidium iodide and tested compounds (128 µg/mL of II.b and 0.2 µg/mL for recombinant Hla) was added. Absorbance at 618 nm was measured every 10 min over 7 h. Each concentration was made in triplicate and mean values are displayed with standard deviation.

Synthesis and Biological Evaluation of Series I-Benzo[b]thiophene-2-Acylhydrazones
Benzo[b]thiophene-2-acylhydrazones I derived from various aldehydes were first investigated ( Figure 1). then removed and fresh medium with 2.5 µ g/mL of propidium iodi pounds (128 µ g/mL of II.b and 0.2 µ g/mL for recombinant Hla) was at 618 nm was measured every 10 min over 7 h. Each concentration wa and mean values are displayed with standard deviation.

Synthesis and Biological Evaluation of Series I-Benzo[b]thiophene-2-A
Benzo[b]thiophene-2-acylhydrazones I derived from various alde vestigated ( Figure 1). The synthetic route to these compounds was achieved by the con ophene-2-carboxylic acid 1a into tert-butyl 2-(benzothiophene-2-carb boxylate 2a in 88% yield using DCC as a coupling reagent. This meth the substitution of methyl benzothiophene-2-carboxylate with hydr mixture of products difficult to purify [25]. Then, the tert-butyl carb removed under acidic conditions using TFA to give a crude material gaged without purification for reaction with diversely substituted aro matic aldehydes under acidic and refluxing conditions to form the acylhydrazone (Scheme 1). The synthetic route to these compounds was achieved by the conversion of benzothio phene-2-carboxylic acid 1a into tert-butyl 2-(benzothiophene-2-carbonyl)hydrazinecarboxylate 2a in 88% yield using DCC as a coupling reagent. This method was preferred to the substitution of methyl benzothiophene-2-carboxylate with hydrazine, leading to a mixture of products difficult to purify [25]. Then, the tert-butyl carboxylate group was removed under acidic conditions using TFA to give a crude material which was then engaged without purification for reaction with diversely substituted aromatic or heteroaromatic aldehydes under acidic and refluxing conditions to form the imine bond of the acylhydrazone (Scheme 1).
Biomolecules 2022, 11, x FOR PEER REVIEW 11 of 20 Scheme 1. Synthesis of set I derivatives.
All the synthesized derivatives I were conveniently obtained in satisfactory yields by spontaneous precipitation in pure form in the reaction medium after two hours of reflux. Compounds such as the 2-pyridinyl derivative I.c required the careful removal of all traces of acid after TFA deprotection, by a basic aqueous work-up and extraction with ethyl acetate before reaction with 2-pyridinecarboxaldehyde.
Structural analysis showed the presence of mixtures of geometric isomers for each final compound, as 1 H and 13 C NMR signals were split in two (spectra available in Supplementary Materials, Figure S1). Theoretically, each N-acylhydrazone derivative could Scheme 1. Synthesis of set I derivatives.
All the synthesized derivatives I were conveniently obtained in satisfactory yields by spontaneous precipitation in pure form in the reaction medium after two hours of reflux. Compounds such as the 2-pyridinyl derivative I.c required the careful removal of all traces of acid after TFA deprotection, by a basic aqueous work-up and extraction with ethyl acetate before reaction with 2-pyridinecarboxaldehyde.
Structural analysis showed the presence of mixtures of geometric isomers for each final compound, as 1 H and 13 C NMR signals were split in two (spectra available in Supplementary Materials, Figure S1). Theoretically, each N-acylhydrazone derivative could present four different isomers due to (E)/(Z) and s-cis/s-trans isomerism. Previous studies have shown that only the more stable (E)-isomer is generated due to the steric hindrance and under such thermodynamic conditions, and that s-cis-and s-trans-isomers (rotamers) were identified by 1 H NMR spectroscopy [13,26,27]. To demonstrate the presence of rotamers in the case of the piperonyl derivative I.a, the temperature of 1 H NMR experiments was gradually increased, and the expected coalescence phenomenon was observed at 330 K and at higher temperatures ( Figure 2).

Scheme 1. Synthesis of set I derivatives.
All the synthesized derivatives I were conveniently obtained in satisfactory yields by spontaneous precipitation in pure form in the reaction medium after two hours of reflux. Compounds such as the 2-pyridinyl derivative I.c required the careful removal of all traces of acid after TFA deprotection, by a basic aqueous work-up and extraction with ethyl acetate before reaction with 2-pyridinecarboxaldehyde.
Structural analysis showed the presence of mixtures of geometric isomers for each final compound, as 1 H and 13 C NMR signals were split in two (spectra available in Supplementary Materials, Figure S1). Theoretically, each N-acylhydrazone derivative could present four different isomers due to (E)/(Z) and s-cis/s-trans isomerism. Previous studies have shown that only the more stable (E)-isomer is generated due to the steric hindrance and under such thermodynamic conditions, and that s-cis-and s-trans-isomers (rotamers) were identified by 1 H NMR spectroscopy [13,26,27]. To demonstrate the presence of rotamers in the case of the piperonyl derivative I.a, the temperature of 1 H NMR experiments was gradually increased, and the expected coalescence phenomenon was observed at 330 K and at higher temperatures ( Figure 2).  This first set of compounds was then tested on three strains of Staphylococcus aureus, including a reference (ATCC29213) and two clinically isolated strains, resistant to methicillin (SF8300) and daptomycin (ST20171643), respectively. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the benzo[b]thiophene-derived acylhydrazones. The results are presented in Table 1. This first set of compounds was then tested on three strains of Staphylococcus aureus, including a reference (ATCC29213) and two clinically isolated strains, resistant to methicillin (SF8300) and daptomycin (ST20171643), respectively. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the benzo[b]thiophene-derived acylhydrazones. The results are presented in Table 1.  This first set of compounds was then tested on three strains of Staphylococcus aureus, including a reference (ATCC29213) and two clinically isolated strains, resistant to methicillin (SF8300) and daptomycin (ST20171643), respectively. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the benzo[b]thiophene-derived acylhydrazones. The results are presented in Table 1.  This first set of compounds was then tested on three strains of Staphylococcus aureus, including a reference (ATCC29213) and two clinically isolated strains, resistant to methicillin (SF8300) and daptomycin (ST20171643), respectively. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the benzo[b]thiophene-derived acylhydrazones. The results are presented in Table 1.  This first set of compounds was then tested on three strains of Staphylococcus aureus, including a reference (ATCC29213) and two clinically isolated strains, resistant to methicillin (SF8300) and daptomycin (ST20171643), respectively. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the benzo[b]thiophene-derived acylhydrazones. The results are presented in Table 1.  This first set of compounds was then tested on three strains of Staphylococcus aureus, including a reference (ATCC29213) and two clinically isolated strains, resistant to methicillin (SF8300) and daptomycin (ST20171643), respectively. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the benzo[b]thiophene-derived acylhydrazones. The results are presented in Table 1.  This first set of compounds was then tested on three strains of Staphylococcus aureus, including a reference (ATCC29213) and two clinically isolated strains, resistant to methicillin (SF8300) and daptomycin (ST20171643), respectively. The purpose of this experiment was to establish the minimum inhibitory concentration (MIC), which is the minimum concentration of compound needed to prevent the growth of a standardized bacterial inoculum, for the benzo[b]thiophene-derived acylhydrazones. The results are presented in Table 1. In this series, the two most active compounds are the piperonyl derivative I.a, which shows a MIC of 32 µ g/mL for ATCC29213 and a range from 16 to 64 µ g/mL for the two strains of antibiotic-resistant S. aureus, and the pyridinyl compound I.c, which shows a MIC of 16 µ g/mL for every strain tested, making it the most potent molecule in this series. Interestingly, the position of the nitrogen atom in position 2 of the pyridine moiety in I.c was found to be important, as the other pyridinyl compounds I.d and I.e, in which the nitrogen is, respectively, in position 3 and 4, were found to be inactive. In this set, another position of interest in the phenyl ring is position 4 (I.i, I.l and, to a lesser extent, I.a), with interesting MICs for the products, while the others had no biological activity.
To examine the effect of the reduction of the imine bond and its consequences on free rotation and conjugation, the pyridinyl compound I.c was reduced using palladium over carbon under a H2 atmosphere with a 60% yield (Scheme 2). Reduced compound I.p exhibited a MIC four times higher than the one of I.c (Table 1) In this series, the two most active compounds are the piperonyl derivative I.a, which shows a MIC of 32 µ g/mL for ATCC29213 and a range from 16 to 64 µ g/mL for the two strains of antibiotic-resistant S. aureus, and the pyridinyl compound I.c, which shows a MIC of 16 µ g/mL for every strain tested, making it the most potent molecule in this series. Interestingly, the position of the nitrogen atom in position 2 of the pyridine moiety in I.c was found to be important, as the other pyridinyl compounds I.d and I.e, in which the nitrogen is, respectively, in position 3 and 4, were found to be inactive. In this set, another position of interest in the phenyl ring is position 4 (I.i, I.l and, to a lesser extent, I.a), with interesting MICs for the products, while the others had no biological activity.
To examine the effect of the reduction of the imine bond and its consequences on free rotation and conjugation, the pyridinyl compound I.c was reduced using palladium over carbon under a H2 atmosphere with a 60% yield (Scheme 2). Reduced compound I.p exhibited a MIC four times higher than the one of I.c (Table 1) In this series, the two most active compounds are the piperonyl derivative I.a, which shows a MIC of 32 µ g/mL for ATCC29213 and a range from 16 to 64 µ g/mL for the two strains of antibiotic-resistant S. aureus, and the pyridinyl compound I.c, which shows a MIC of 16 µ g/mL for every strain tested, making it the most potent molecule in this series. Interestingly, the position of the nitrogen atom in position 2 of the pyridine moiety in I.c was found to be important, as the other pyridinyl compounds I.d and I.e, in which the nitrogen is, respectively, in position 3 and 4, were found to be inactive. In this set, another position of interest in the phenyl ring is position 4 (I.i, I.l and, to a lesser extent, I.a), with interesting MICs for the products, while the others had no biological activity.
To examine the effect of the reduction of the imine bond and its consequences on free rotation and conjugation, the pyridinyl compound I.c was reduced using palladium over carbon under a H2 atmosphere with a 60% yield (Scheme 2). Reduced compound I.p exhibited a MIC four times higher than the one of I.c (Table 1) In this series, the two most active compounds are the piperonyl derivative I.a, which shows a MIC of 32 µ g/mL for ATCC29213 and a range from 16 to 64 µ g/mL for the two strains of antibiotic-resistant S. aureus, and the pyridinyl compound I.c, which shows a MIC of 16 µ g/mL for every strain tested, making it the most potent molecule in this series. Interestingly, the position of the nitrogen atom in position 2 of the pyridine moiety in I.c was found to be important, as the other pyridinyl compounds I.d and I.e, in which the nitrogen is, respectively, in position 3 and 4, were found to be inactive. In this set, another position of interest in the phenyl ring is position 4 (I.i, I.l and, to a lesser extent, I.a), with interesting MICs for the products, while the others had no biological activity.
To examine the effect of the reduction of the imine bond and its consequences on free rotation and conjugation, the pyridinyl compound I.c was reduced using palladium over carbon under a H2 atmosphere with a 60% yield (Scheme 2). Reduced compound I.p exhibited a MIC four times higher than the one of I.c (Table 1). It is therefore suggested that In this series, the two most active compounds are the piperonyl derivative I.a, which shows a MIC of 32 µg/mL for ATCC29213 and a range from 16 to 64 µg/mL for the two strains of antibiotic-resistant S. aureus, and the pyridinyl compound I.c, which shows a MIC of 16 µg/mL for every strain tested, making it the most potent molecule in this series. Interestingly, the position of the nitrogen atom in position 2 of the pyridine moiety in I.c was found to be important, as the other pyridinyl compounds I.d and I.e, in which the nitrogen is, respectively, in position 3 and 4, were found to be inactive. In this set, another position of interest in the phenyl ring is position 4 (I.i, I.l and, to a lesser extent, I.a), with interesting MICs for the products, while the others had no biological activity.
To examine the effect of the reduction of the imine bond and its consequences on free rotation and conjugation, the pyridinyl compound I.c was reduced using palladium over carbon under a H 2 atmosphere with a 60% yield (Scheme 2). Reduced compound I.p exhibited a MIC four times higher than the one of I.c (Table 1). It is therefore suggested that preventing the free rotation of the pyridine ring and allowing conjugation due to the tautomeric form of the amide bond is important for the activity of I.c. An effect of hydrophobicity is also observed, decreasing the LogP from 3.13 for I.c to 2.33 for I.p.
Biomolecules 2022, 11, x FOR PEER REVIEW 14 of 2 preventing the free rotation of the pyridine ring and allowing conjugation due to the tau tomeric form of the amide bond is important for the activity of I.c. An effect of hydropho bicity is also observed, decreasing the LogP from 3.13 for I.c to 2.33 for I.p. Scheme 2. Synthesis of compound I.p.

Synthesis and Biological Evaluation of Series II-6-Halogenobenzo[b]thiophene-2-Acylhydrazones
Based on this first set of results, we selected the two compounds I.a and I.c (bearing a 5-piperonyl and a 2-pyridinyl moiety, respectively) as hits for further structural modu lation. With 6-halogenobenzo[b]thiophene rings, being easily accessible using 2-fluoro-4 halogenobenzaldehyde as precursor, we prepared a series of four compounds, namely two piperonyl or pyridinyl compounds bearing a fluorine or chlorine atom, respectively (Figure 3).

II-6-Halogenobenzo[b]thiophene-2-Acylhydrazones
Based on this first set of results, we selected the two compounds I.a and I.c (bearing a 5-piperonyl and a 2-pyridinyl moiety, respectively) as hits for further structural modulation. With 6-halogenobenzo[b]thiophene rings, being easily accessible using 2-fluoro-4-halogenobenzaldehyde as precursor, we prepared a series of four compounds, namely two piperonyl or pyridinyl compounds bearing a fluorine or chlorine atom, respectively ( Figure 3).

Synthesis and Biological Evaluation of Series II-6-Halogenobenzo[b]thiophene-2-Acylhydrazones
Based on this first set of results, we selected the two compounds I.a and I.c (be a 5-piperonyl and a 2-pyridinyl moiety, respectively) as hits for further structural m lation. With 6-halogenobenzo[b]thiophene rings, being easily accessible using 2-fluo halogenobenzaldehyde as precursor, we prepared a series of four compounds, na two piperonyl or pyridinyl compounds bearing a fluorine or chlorine atom, respect ( Figure 3). In order to synthesize the acylhydrazones, 6-chloro-and 6-fluorobenzo[b]thioph 2-carboxylic acids 1b and 1c were thus prepared. First, 2-fluoro-4-halogenobenzalde reacted with ethyl thioglycolate by nucleophilic substitution followed by an intramo lar cyclization to obtain compounds 4a and 4b, as described in the literature [14]. T esters were then saponified with sodium hydroxide to give the desired carboxylic 1b and 1c. The general procedure used for the first set was applied to the latter to o compounds II.a-d with similar yields, as shown in Scheme 3. In order to synthesize the acylhydrazones, 6-chloro-and 6-fluorobenzo[b]thiophene-2carboxylic acids 1b and 1c were thus prepared. First, 2-fluoro-4-halogenobenzaldehyde reacted with ethyl thioglycolate by nucleophilic substitution followed by an intramolecular cyclization to obtain compounds 4a and 4b, as described in the literature [14]. These esters were then saponified with sodium hydroxide to give the desired carboxylic acids 1b and 1c. The general procedure used for the first set was applied to the latter to obtain compounds II.a-d with similar yields, as shown in Scheme 3. These compounds were also tested to establish their MIC against S. aureus with the same method used for set I, and the results are presented in Table 2. These compounds were also tested to establish their MIC against S. aureus with the same method used for set I, and the results are presented in Table 2. These compounds were also tested to establish their MIC against S. aureus with the same method used for set I, and the results are presented in Table 2. This short series allowed us to identify the chloropyridinyl compound II.b as significantly more active compared with its non-halogenated counterpart I.c, reaching 4 µ g/mL for the three strains. The presence of a fluorine atom in position 6 of the benzothiophene for the pyridinyl compound did not change the activity. Both halogenated compounds 4.85 >256 >256 >256

II.b
Scheme 3. Synthesis of set II and III derivatives.
These compounds were also tested to establish their MIC against S. aureus with the same method used for set I, and the results are presented in Table 2. This short series allowed us to identify the chloropyridinyl compound II.b as significantly more active compared with its non-halogenated counterpart I.c, reaching 4 µ g/mL for the three strains. The presence of a fluorine atom in position 6 of the benzothiophene for the pyridinyl compound did not change the activity. Both halogenated compounds 3.79 4 4 4

II.c
Scheme 3. Synthesis of set II and III derivatives.
These compounds were also tested to establish their MIC against S. aureus with the same method used for set I, and the results are presented in Table 2. This short series allowed us to identify the chloropyridinyl compound II.b as significantly more active compared with its non-halogenated counterpart I.c, reaching 4 µ g/mL for the three strains. The presence of a fluorine atom in position 6 of the benzothiophene for the pyridinyl compound did not change the activity. Both halogenated compounds 4 These compounds were also tested to establish their MIC against S. aureus with the same method used for set I, and the results are presented in Table 2. This short series allowed us to identify the chloropyridinyl compound II.b as significantly more active compared with its non-halogenated counterpart I.c, reaching 4 µ g/mL for the three strains. The presence of a fluorine atom in position 6 of the benzothiophene for the pyridinyl compound did not change the activity. Both halogenated compounds This short series allowed us to identify the chloropyridinyl compound II.b as significantly more active compared with its non-halogenated counterpart I.c, reaching 4 µg/mL for the three strains. The presence of a fluorine atom in position 6 of the benzothiophene for the pyridinyl compound did not change the activity. Both halogenated compounds derived from the piperonyl compound I.a were deprived of biological activity (II.a and II.c).

Synthesis and Biological Evaluation of Series III-Structural Modifications of II.b
Attempts to improve the activity of the chloropyridinyl compound II.b were then undertaken by replacing the pyridine ring with three five-bond rings, namely imidazole, furan and 5-hydroxymethylfuran. The procedure used was the same as the one used for chlorinated derivatives II.a and II.b (Scheme 3). As shown in Table 3, the activity remains better for compound II.b, confirming the importance of the 2-pyridinyl group.
Then, the acylhydrazone was replaced by an acyloxime moiety, leading to a more flexible molecule and losing the s-cis/s-trans isomerism. Compound III.d was obtained through the conversion of pyridine-2-carboxaldehyde into the corresponding oxime 6, followed by the acylation from 6-chlorobenzo[b]thiophene-2-carboxylic acid 1b (Scheme 4).
iomolecules 2022, 11, x FOR PEER REVIEW 16 of 20 derived from the piperonyl compound I.a were deprived of biological activity (II.a and II.c).

Synthesis and Biological Evaluation of Series III-Structural Modifications of II.b
Attempts to improve the activity of the chloropyridinyl compound II.b were then undertaken by replacing the pyridine ring with three five-bond rings, namely imidazole furan and 5-hydroxymethylfuran. The procedure used was the same as the one used for chlorinated derivatives II.a and II.b (Scheme 3). As shown in Table 3, the activity remains better for compound II.b, confirming the importance of the 2-pyridinyl group.
Then, the acylhydrazone was replaced by an acyloxime moiety, leading to a more flexible molecule and losing the s-cis/s-trans isomerism. Compound III.d was obtained through the conversion of pyridine-2-carboxaldehyde into the corresponding oxime 6, followed by the acylation from 6-chlorobenzo[b]thiophene-2-carboxylic acid 1b (Scheme 4). Compound III.d showed no anti-staphylococcal activity at concentrations lower or equal to 256 µ g/mL (Table 3). Therefore, the rigidity imposed by the acylhydrazone moiety is critical to the biological activity.
Then, the effect of the hydrophobicity of the compounds on the antibacterial activity was investigated using the benzo[b]furan counterpart of II.b (Scheme 5). First, 4-chloro-2hydroxybenzaldehyde 3d was converted into the ester 4d using diethyl 2-bromomalonate as described [18]. Then, the synthetic route was the same as described before for benzo[b]thiophene acylhydrazone derivatives. Compound III.d showed no anti-staphylococcal activity at concentrations lower or equal to 256 µg/mL (Table 3). Therefore, the rigidity imposed by the acylhydrazone moiety is critical to the biological activity. MIC values ≥ 128 µ g/mL. Therefore, the hydrophobicity of the benzo[b]thiophene ring is critical for the biological activity. As expected, the replacement of the sulfur by an oxygen atom decreases the overall LogP of the compound (3.79 for II.b and 3.15 for III.e). Finally, the chlorine atom was replaced by a bioisostere, a trifluoromethyl group, using the same synthetic strategy (Scheme 3), leading to compound III.f. Even if the MICs are interesting (Table 3), the chlorinated derivative II.b remains the most active compound.

Cytotoxicity Assay of II.b
Due to its interesting antistaphylococcal activity, the potential mammalian cytotoxicity of the chloropyridinyl derivative II.b was examined. Therefore, a 7 h propidium iodide (PI)-based assay was used on adenocarcinomic human alveolar basal epithelial cells (A549), with recombinant S. aureus α-hemolysin as a positive control. As depicted in Figure 4, no cytotoxicity was found for II.b after 7 h at a concentration of 128 µ g/mL (32 times the MIC). Then, the effect of the hydrophobicity of the compounds on the antibacterial activity was investigated using the benzo[b]furan counterpart of II.b (Scheme 5). First, 4-chloro-2-hydroxybenzaldehyde 3d was converted into the ester 4d using diethyl 2bromomalonate as described [18]. Then, the synthetic route was the same as described before for benzo[b]thiophene acylhydrazone derivatives.

Synthesis and Biological Evaluation of Series III-Structural Modifications of II.b
Attempts to improve the activity of the chloropyridinyl compound II.b were then undertaken by replacing the pyridine ring with three five-bond rings, namely imidazole, furan and 5-hydroxymethylfuran. The procedure used was the same as the one used for chlorinated derivatives II.a and II.b (Scheme 3). As shown in Table 3, the activity remains better for compound II.b, confirming the importance of the 2-pyridinyl group.
Then, the acylhydrazone was replaced by an acyloxime moiety, leading to a more flexible molecule and losing the s-cis/s-trans isomerism. Compound III.d was obtained through the conversion of pyridine-2-carboxaldehyde into the corresponding oxime 6, followed by the acylation from 6-chlorobenzo[b]thiophene-2-carboxylic acid 1b (Scheme 4). Compound III.d showed no anti-staphylococcal activity at concentrations lower or equal to 256 µ g/mL (Table 3). Therefore, the rigidity imposed by the acylhydrazone moiety is critical to the biological activity.
Then, the effect of the hydrophobicity of the compounds on the antibacterial activity was investigated using the benzo[b]furan counterpart of II.b (Scheme 5). First, 4-chloro-2hydroxybenzaldehyde 3d was converted into the ester 4d using diethyl 2-bromomalonate as described [18]. Then, the synthetic route was the same as described before for benzo[b]thiophene acylhydrazone derivatives.  Table 3, compound III.e showed a weak activity against S. aureus with MIC values ≥ 128 µg/mL. Therefore, the hydrophobicity of the benzo[b]thiophene ring is critical for the biological activity. As expected, the replacement of the sulfur by an oxygen atom decreases the overall LogP of the compound (3.79 for II.b and 3.15 for III.e).

As shown in
Finally, the chlorine atom was replaced by a bioisostere, a trifluoromethyl group, using the same synthetic strategy (Scheme 3), leading to compound III.f. Even if the MICs are interesting (Table 3), the chlorinated derivative II.b remains the most active compound.

Cytotoxicity Assay of II.b
Due to its interesting antistaphylococcal activity, the potential mammalian cytotoxicity of the chloropyridinyl derivative II.b was examined. Therefore, a 7 h propidium iodide (PI)-based assay was used on adenocarcinomic human alveolar basal epithelial cells (A549), with recombinant S. aureus α-hemolysin as a positive control. As depicted in Figure 4, no cytotoxicity was found for II.b after 7 h at a concentration of 128 µg/mL (32 times the MIC).

Conclusion
In this study, benzo[b]thiophene-based acylhydrazones were shown to be interesting compounds acting as anti-staphylococcal agents. More precisely, (E)-6-chloro-N'-(pyridin-2-ylmethylene)benzo[b]thiophene-2-carbohydrazide II.b showed an equal minimum inhibitory concentration of 4 µ g/mL for the three strains of S. aureus, including two clinically isolated strains of drug-resistant S. aureus. Moreover, II.b showing no cytotoxicity on A549 cells; this suggests that the chloropyridinyl benzothiophene acylhydrazone structure is pertinent for future work on chemical diversification and the understanding of the mode of action.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1

Conclusions
In this study, benzo[b]thiophene-based acylhydrazones were shown to be interesting compounds acting as anti-staphylococcal agents. More precisely, (E)-6-chloro-N'-(pyridin-2-ylmethylene)benzo[b]thiophene-2-carbohydrazide II.b showed an equal minimum inhibitory concentration of 4 µg/mL for the three strains of S. aureus, including two clinically isolated strains of drug-resistant S. aureus. Moreover, II.b showing no cytotoxicity on A549 cells; this suggests that the chloropyridinyl benzothiophene acylhydrazone structure is pertinent for future work on chemical diversification and the understanding of the mode of action.