Exploiting thiol-functionalized benzosiloxaboroles for achieving diverse substitution patterns – synthesis, characterization and biological evaluation of promising antibacterial agents

Benzosiloxaboroles are an emerging class of medicinal agents possessing promising antimicrobial activity. Herein, the expedient synthesis of two novel thiol-functionalized benzosiloxaboroles 1e and 2e is reported. The presence of the SH group allowed for diverse structural modifications involving the thiol-Michael addition, oxidation, as well as nucleophilic substitution giving rise to a series of 27 new benzosiloxaboroles containing various polar functional groups, e.g., carbonyl, ester, amide, imide, nitrile, sulfonyl and sulfonamide, and pendant heterocyclic rings. The activity of the obtained compounds against selected bacterial and yeast strains, including multidrug-resistant clinical strains, was investigated. Compounds 6, 12, 20 and 22–24 show high activity against Staphylococcus aureus, including both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) strains, with MIC values in the range of 1.56–12.5 μg mL−1, while their cytotoxicity is relatively low. The in vitro assay performed with 2-(phenylsulfonyl)ethylthio derivative 20 revealed that, in contrast to the majority of known antibacterial oxaboroles, the plausible mechanism of antibacterial action, involving inhibition of the leucyl-tRNA synthetase enzyme, is not responsible for the antibacterial activity. Structural bioinformatic analysis involving molecular dynamics simulations provided a possible explanation for this finding.


Introduction
5][16] Recent intensive efforts resulted in the preparation of over 10 000 benzoxaborole derivatives.][25][26][27][28] The introduction of the SiMe 2 group to the oxaborole ring in place of the methylene group resulted in increased Lewis acidity and lipophilicity, which may be beneficial for biological activity. 2325][26][27]29 Our preliminary microbiological studies show that simple fluorinated benzosiloxaboroles are active against selected yeast strains, 23 whereas more extended systems demonstrate potent antibacterial activity, especially against Gram-positive cocci, including multidrug-resistant clinical strains. 26,27Some derivatives were also found to be effective inhibitors of KPC-2/AmpC β-lactamases responsible for drug resistance in bacteria (Fig. 1b). 25he sulfide group is widely used in medicinal chemistry as a linker and many benzoxaboroles containing this moiety have been already reported. 30,31However, thiol-functionalized benzoxaboroles themselves have not been widely exploited so far.Only two S-functionalized benzoxaboroles were synthesized through direct transformation of a thiol group at the benzoxaborole (Fig. 2).These compounds were investigated as potential anti-Wolbachia agents, however they did not exhibit significant potency. 32Considering the growing potential of benzosiloxaboroles in medicinal chemistry we decided to extend the library of these compounds by utilization of a thiol substituent.The proposed general concept is practical due to the high reactivity of the thiol group, which can be easily converted into other sulfur-based groups, such as thioether, thioester, sulfonamide, etc.Thus, it was successfully validated by synthesis of 27 new functionalized benzosiloxaboroles followed by comprehensive evaluation of antimicrobial activity and cytotoxicity.This work was complemented by a study on the plausible mechanism of action of one of the obtained compounds.

Synthesis
The approach to fluorinated thiol-functionalized benzosiloxaboroles 1e and 2e involved a general four-step protocol (Scheme 1) starting with the preparation of appropriate halogenated thiophenols from inexpensive starting materials.The synthesis of thiophenol 1b was accomplished through deprotonative lithiation of 1-bromo-3,5-difluorobenzene with LDA in THF at −78 °C followed by addition of sulfur and hydrolytic workup.The respective disulfide 1b_D was also formed as a byproduct (ca.5%).Notably, when the reaction mixture was warmed to room temperature prior to hydrolysis, bis((4-bromo-2,6-difluorophenyl)thio)methane 1b_CH2 was formed to a significant extent (ESI, Scheme S1).The structures of 1b_D and 1b_CH2 were confirmed by single-crystal X-ray diffraction.The extensive formation of 1b_CH2 is intriguing but not fully clear.It was rationalized by the reaction of 1b_D with lithium enolate formed from LDA-induced cleavage of THF according to the mechanism proposed for a similar transformation 33 (ESI, † Scheme S2).Thiophenol 2b was synthesized by the reduction of 4-bromo-2-fluorobenzene-1sulfonyl chloride with PPh 3 .Compounds 1b and 2b were converted to respective TBDMS thioethers 1c and 2c which were subjected to deprotonation with LDA in THF at −78 °C followed by trapping of corresponding aryllithium intermediates with chlorodimethylsilane. 23 In both cases, the reaction occurred regioselectively at the position between fluorine and bromine atoms in agreement with a strong cumulated ortho-acidifying effect of those two halogen substituents. 34,35Finally, the conversion of functionalized arylsilanes 1d and 2d to respective benzosiloxaboroles 1e and 2e was performed as described by us previously. 23,25It involved a Br/Li exchange reaction using t-BuLi in Et 2 O and subsequent boronation of resultant aryllithiums with B(OMe) 3 at very low temperatures (≤95 °C) followed by Fig. 2 Pleuromutilin-functionalized benzoxaboroles obtained from respective thiol precursors. 32ydrolysis.The simultaneous Si-H bond cleavage and deprotection of the thiol group was cleanly performed under alkaline conditions.After acidification, 1e and 2e were isolated as white powders.
The incorporation of various side chains into the structure of benzosiloxaboroles was achieved through the thiol-Michael addition reaction with 1e and 2e as effective S-nucleophiles (Table 1).The thiol-Michael addition is broadly applicable and usually proceeds under mild conditions; moreover, it can be regarded as a "click chemistry" method owing to 100% atom economy. 36,37The syntheses involving selected Michael acceptors proceeded smoothly under relatively mild conditions (temperature range from 0 to 25 °C) and resulted in preparation of 19 S-linked functionalized benzosiloxaboroles with good yields (>70%).
In the case of compound 3, the reaction occurred in water without the need for the use of a base.All other syntheses required the use of a base to generate more effectively active anionic forms of 1e and 2e.The wide representation of Michael acceptors used in the syntheses of benzosiloxaboroles 3-21 includes α,β-unsaturated ketones, esters, nitriles, amides, imides and sulfones.Reactions leading to compounds 3, 4, 6, 7, 12, 13, 20 and 21 utilized readily available substrates.Protocols for the preparation of the Michael acceptors used in the syntheses of 5, 9-11, 14-19 are available in the ESI.† We assumed that the introduction of pendant substituents containing various polar end groups to the benzosiloxaborole may result in specific interactions with targeted biomolecules which would be beneficial for antibacterial activity.Moreover, benzosiloxaboroles 7-12 containing an amide group constitute an interesting group as it seems that bioconjugates of benzosiloxaboroles with amino acids or peptides could be obtained analogously.Benzosiloxaboroles 12-17 possess a pendant succinimide ring where the presence of a nitrogen atom enables further functionalization.Compounds 18 and 19 are specific as they can be regarded as ionic liquids (ILs) which might potentially improve solubility and the drug delivery process. 38erivatives 20 and 21 feature a pendant phenylsulfonyl moiety and thus show structural similarity to benzosiloxaboroles decorated with arylsulfonate and arylsulfonamide groups which exhibit potent activity against Gram-positive cocci. 26,27enzosiloxaboroles bearing side chains linked via the thioether group can also be obtained by treatment of 1e or 2e with electrophilic partners comprising reactive C-Hal (Hal = Cl, Br) or P-Cl bonds.Thus, respective products 22-25 were isolated using α-bromoketones, 4-(trifluoromethyl)benzoyl chloride and diethyl chlorophosphate (Scheme 2).
The obtained compounds are white solids that are well soluble in most organic solvents.They were characterized by multinuclear NMR ( 1 H, 13 C, 11 B, 19 F, 31 P) spectroscopy and

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HRMS analyses.The molecular structures of selected benzosiloxaboroles 1e, 6, 11, 12, 14, 15, 20, 22, 24, 26, 27 and 29 were determined by X-ray crystallography (see Fig. S3 in the ESI †) showing that the geometric parameters of benzosiloxaborole cores are similar as in structures reported previously. 45ince acidity is an important parameter in medicinal chemistry, the pK a values of the benzosiloxaborole precursors 1e and 2e, as well as the selected promising compounds 6, 20 and 22-24 were measured (Table 2) by potentiometric titration with aq.0.1 M NaOH in H 2 O/MeOH (1 : 1 v/v).In the case of 1e and 2e possessing an acidic thiol group, both apparent pK a1 and pK a2 values were obtained.Since the acidic properties of the aromatic thiol group 46 and siloxaborole 23 moiety are comparable, it is likely that pK a1 values represent the formation of an equilibrium mixture of both possible monoanions, i.e., the species with the deprotonated SH group and the boronate anion resulting from the coordination of OH − to the boron atom.Difluoro-substituted benzosiloxaboroles 1e, 6, 20 and 24 are characterized by approximately one unit lower pK a values than their monofluoro counterparts 2e, 22 and 23, which is consistent with previous findings. 23Overall, pK a values of mono-and difluoro derivatives are very similar within each of these groups indicating that the type of pendant sulfur-based moiety plays a minor role.All studied compounds are sufficiently acidic to exist in respective anionic forms under standard physiological conditions (pH = 7.4), which should be beneficial for solubility in biological systems.

Antimicrobial activity
][27] Thus, in this study, we tested the ability of all obtained compounds to inhibit the growth of selected standard strains of bacteria (6 Gram-positive strains and 11 Gram-negative strains) and yeasts (7 strains).All obtained results are presented in Table 3 and Tables S1 and S2 in the ESI.† Most compounds displayed moderate to weak activity against Gram-positive cocci, with MICs ranging from 12.5 to >400 μg mL −1 (Table 3).However, derivatives 6, 12, 20, and 22-24 were highly active against standard staphylococci, including MRSA, with MIC = 1.56-6.25 μg mL −1 .MRSA strains are of great clinical concern.Not only are they resistant to almost all β-lactams (except ceftaroline and ceftobiprole), but they are often resistant to many other antibiotic classes (macrolides, tetracyclines, aminoglycosides, fluoroquinolones), which severely limits therapeutic options. 47,48Consequently, they entered the WHO list of the most dangerous high-priority pathogens, for which the search for new antibiotics is urgently needed. 49Thus, in this study, agents highly active against the standard MRSA strain were subsequently tested against five clinical MRSA strains (Table 4).Compound 23 bearing the benzoylmethylthio functionality displayed the highest potency (MIC = 1.56-3.12μg mL −1 ), followed by 2-(phenylsulfonyl)ethylthio-and acetylmethylthio derivatives 20 and 22, respectively (MRSA: MIC = 3.12-6.25μg mL −1 ).Their activity was comparable or only 3-to 6-fold lower than our reference agent linezolid, which is an antibiotic indicated in infections caused by multi-drug resistant Gram-positive cocci, including MRSA. 48nterestingly, their MICs for S. aureus were comparable with linezolid breakpoints (according to CLSI, strains are classified as sensitive when linezolid MIC is ≤ 4 μg mL −1 and as resistant when MIC is ≥8 μg mL −1 ). 50Thus, 20, 22, and 23 are promising anti-MRSA agents comparable with the previously described N-methyl arylsulfonamide benzosiloxaboroles (MRSA: MIC = 3.12-6.25μg mL −1 ). 27It should be noted that the structural homologue of 22, namely compound 3, bearing the additional methylene spacer between the sulfur atom and the carbonyl group, is much less active.
The minimal bactericidal concentrations (MBCs) of most tested agents for staphylococci were established at 2-16 × MIC, whereas for enterococci these values were usually above the highest tested concentration (>400 μg mL −1 ) (Table 3).The lowest MBC values against S. aureus strains were obtained for compounds 1e, 2e and 25 (MBCs 25-50 μg mL −1 ).Interestingly, for 4, 6, 20, 23, and 24 the Eagle effect (also known as the paradoxical growth) 51,52 was observed in the case of S. aureus ATCC 6538P and five MRSA clinical strains, which is in line with previous results obtained for benzosiloxaboroles decorated with arylsulfonate 26 and N-methyl arylsulfonamide 27 groups.Consequently, two MBC values were determined (Tables 3 and 4).According to CLSI recommendations, MBC is the lowest concentration that kills at least 99.9% of bacteria. 54In our study, the first MBCs were 2-or 4-fold higher than the MICs.However, a progressive increase in the number of surviving bacteria was observed at concentrations beyond it, followed by a subsequent decrease at 100->400 μg mL −1 .If the bacterial population was reduced again to the MBC threshold, a second MBC value was reported.
Furthermore, only agents 12 and 13 displayed weak activity against Gram-negative rods with MICs ranging from 25 to >400 μg mL −1 (ESI, † Table S1).8][59] It turned out that the activity of agents 12 and 13 against Enterobacterales is affected by the efflux phenomenon, as their MICs are reduced at least 4-fold in the presence of PAβN.Compounds 4, 20, and 21 are also actively extruded from bacterial cells, whereas for other compounds the efflux assay confirmed the lack of any activity.

RSC Medicinal Chemistry Research Article
The highest activity against Gram-positive bacteria indicated by the low MIC values (≤ 6.25 μg mL −1 ) is shown in boldface.(-):the inhibition zone was not observed in the disc-diffusion method.The diameter of the paper discs was 9 mm.a Only the MBC values ≤400 μg mL −1 are presented.b The growth type of B. subtilis in MHB medium prevented reading the MIC values of tested agents.c The MIC and MBC values of the agent were determined up to 200 μg mL −1 .In the table, only MBC values ≤200 μg mL −1 are presented.The tested agent dissolved in DMSO precipitated after implementation into MHB medium at a concentration above 200 μg mL −1 .d The MIC and MBC values of the agent were determined up to 100 μg mL −1 .In the table, only MBC values ≤100 μg mL −1 are presented.The tested agent dissolved in DMSO precipitated after implementation into MHB medium at a concentration above 100 μg mL −1 .e The MIC and MBC values of the agent were determined up to 50 μg mL −1 .In the table, only MBC values ≤50 μg mL −1 are presented.The tested agent dissolved in DMSO precipitated after implementation into MHB medium at a concentration above 50 μg mL −1 .f The Eagle effect was observed during the determination of the MBC value of the same tested agents against S. aureus strains. 51,52The Eagle effect is shown in italic face.g LIN, linezolid, was used as a reference agent active against Gram-positive bacteria.The diameter of a commercial disc containing 0.03 mg of linezolid was 6 mm; the MIC of linezolid was determined according to the CLSI recommendations.
The highest activity against S. aureus strains indicated by the low MIC values (≤6.25 μg mL −1 ) is shown in boldface.a The Eagle effect was observed when determining the MBC value of the same tested agents against S. aureus strains. 51,52The Eagle effect is shown in italic face.b LIN, linezolid, was used as a reference agent, active against S. aureus strains.The MIC of linezolid was determined according to the CLSI recommendations. 53

Cytotoxicity studies
To evaluate the cytotoxic effect of the tested compounds, an MTT-based assay was performed.Human normal lung fibroblasts MRC-5 were treated with representative compounds including the most active ones in the concentration range of 6.25-400 μg mL −1 for 48 h.All viability data are summarized in Table S3 (ESI †).Whenever possible, the respective IC 50 values were calculated and summarized in Table 5.The representative plots demonstrating sigmoidal dose-response curves for the tested compounds are shown in the ESI † (Fig. S1).Overall, the results indicate rather weak cytotoxicity as for most compounds (except for 16 and 28), IC 50 values are above 100 μg mL −1 .Moreover, based on available IC 50 and MIC (for standard and clinical S. aureus MRSA strains) values, the selectivity index (SI) was calculated for several compounds as the IC 50 /MIC ratio (Table 5). 60ithin the group of most active compounds (6, 12, 20,  22-24), SI values were generally higher than 10, indicating their potential as antibacterial agents with respect to S. aureus MRSA.

Studies on the mechanism of antibacterial activity
We have undertaken research devoted to determining the most probable mechanism of antibacterial action.Numerous reports indicate that related benzoxaboroles exhibit antimicrobial activity through the oxaborole tRNA trapping (OBORT) mechanism 10,[61][62][63][64][65] involving inhibition of leucyl-tRNA synthetase (LeuRS).Considering structural similarity to benzoxaboroles we assumed that benzosiloxaboroles are active through this mechanism.Our working hypothesis was supported by the fact that benzosiloxaboroles are rather bacteriostatic than bactericidal, 26,27 which is consistent with the specificity of the OBORT mechanism.The enzyme inhibition assay was performed for compound 20 as it features high activity against S. aureus MRSA strains and the respective favorable SI = 19.5 (Table 5).Moreover, due to the presence of the SO 2 group in the pendant substituent, it shows structural analogy to previously reported benzosiloxaboroles bearing sulfonate and sulfonamide groups which also exhibit high activity against S. aureus. 26,27e obtained S. aureus MRSA LeuRS in the E. coli expression system.From 400 mL culture, approximately 20 mg of 6× His tagged LeuRS (with a molecular mass of 92.3 kDa) was purified to more than 90% homogeneity, which was confirmed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Fig. S2, ESI †).Next, we used an in vitro aminoacylation assay to investigate the ability of 20 to inhibit S. aureus MRSA LeuRS activity consistent with affecting the transfer of the 14 C-radiolabeled leucine to a total tRNA.We found that 20 did not show the expected activity (residual activity of 92.9% at 25 μM), compared to reference arylsulfonamide-substituted benzoxaborole PT662 known as a potent S. aureus MRSA LeuRS inhibitor 10 (residual activity 30.8% at 25 μM).
Subsequent bioinformatic analysis provided possible explanations for this disclosure.Compound 20 possesses a high degree of structural similarity to potent antibacterial compound 1- 65 AceSPhO_BOB, which has been crystallized in complex with Streptococcus pneumoniae leucyl-tRNA synthetase (Fig. 3a, right).By using the crystallized complex as a reference, we generated a putative S. aureus MRSA LeuRS/(20 bound to AMP) complex through docking.A comparison of binding modes of both compounds (Fig. 3b) revealed that although the oxaborole moiety of 20 occupies a similar space within the protein as for AceSPhO_BOB, 65 the pendant groups of both compounds are oriented very differently.In the case of 20, the phenylsulfonyl group forms tight interactions with LeuRS, penetrating the cavity between structured regions of the protein and a loop (Fig. 3c).Clearly, it is not surprising as in  Residues in proximity of the ligands are colored according to the residue property (whitenonpolar, greenpolar, bluenegatively charged, redpositively charged).Labels were added to selected residues adjacent to the ligand for reference; (c) contacts between the pendant group of both compounds and its interaction with a polar LeuRS loop.The loop is colored according to the residue property (the same color code as above).20 the large substituent is attached at the para position with respect to the boron atom, while for AceSPhO_BOB (ref.65)  an analogous moiety is located at the meta position (Fig. 3a).
It should be stressed that the loop comprises mostly polar and negatively charged residues (Fig. 3c).Such an environment is highly unfavorable for the primarily hydrophobic pendant group of 20 which would likely aggravate binding of this compound.
To further evaluate the stability of the generated complex, we have carried out extensive (5 × 1 μs) unbiased MD simulation of the generated ligand, i.e., (20 bound to AMP)/ LeuRS complex.Importantly, as parameters for silicon atoms are not available within the utilized simulation forcefield, this atom in the ligand structure was replaced with a carbon atom (as it shares multiple chemical properties with silicon atoms).Unbiased simulations were performed in order to observe changes in the structure of the complex, which could help rationalize the lack of affinity of studied compounds towards LeuRS.The relative positions of the protein and atoms in the benzosiloxaborole core and the pendant moiety were plotted to monitor the stability of the ligands (Fig. 4a).Intriguingly, we found that in each of the replicates both fragments assume distances characteristic to unbound conformations.A more detailed analysis of one of the unbinding events (Fig. 4b) observed in the unbiased simulations reveals the following sequence of events: in the initial frames the polar loop quickly unbinds (likely due to unfavorable interactions with the ligand).This is followed by destabilization of the pendant group, which later leads to full unbinding and displacement of 20.The observed unbinding event, preceded by the opening of the polar loop, would suggest that 20 does not interact with LeuRS as the largely polar and electronegative environment of the loop cannot accommodate the hydrophobic and electronegative pendant moiety of the studied ligand.
The lack of activity of 20 towards LeuRS indicates a different mechanism of antibacterial activity.Bioinformatic analysis suggests that this inability might be linked with the placement of its bulky sulfur-linked moiety in the para position with respect to the boron atom.Importantly, this would also suggest that none of the compounds within this series, which have a large hydrophobic/negatively charged pendant group in the para position, would inhibit LeuRS.Although LeuRS remains the most frequently identified molecular target for antimicrobial organoboron compounds, few examples of differently acting boron-based antimicrobial agents are also known.For instance, in the case of bis(indolyl)methylboronic acid derivatives, it was found that their antibacterial activity might be linked with a strong binding affinity to the peptidoglycan layer of the Grampositive bacteria cell wall. 67On the other hand, some benzoxaboroles were identified as potent inhibitors of other bacterial enzymes, such as NADH dehydrogenase, 9 enoyl acyl carrier protein FabI 68 or carbonic anhydrases. 69These findings are in line with our disclosure and prove that benzosiloxaboroles, like other boron-based antimicrobials, could utilize another antibacterial mechanism than OBORT as well.

Conclusions
Thiol-functionalized benzosiloxaboroles proved to be very useful precursors for the preparation of a wide variety of novel derivatives.Most of them were obtained by the thiol-Michael addition reaction, which turned out to be a convenient tool for the preparation of many structurally diverse benzosiloxaboroles.Some of them were also synthesized through the nucleophilic substitution reaction of thiolates with appropriate electrophilic partners as well as through chemoselective SH-oxidation and subsequent transformations.We intended to decorate new derivatives with extended substituents containing multiple polar groups as we assumed that such functionalizations may result in a stronger and specific binding to biological targets by means of polar interactions, e.g., hydrogen bonds.Regardless of the substitution pattern at the sulfur atom, the studied benzosiloxaboroles display a comparable acidity (pK a of 4.7-6.4 in H 2 O/MeOH, 1 : 1).Compounds 6, 12, 20 and 22-24 show strong bacteriostatic activity, especially against S. aureus, including multidrug-resistant clinical strains (S. aureus MRSA).Overall, moderate activity against Grampositive bacteria is common.Furthermore, SAR analysis indicates that the antibacterial properties are enhanced for difluorinated derivatives.Following the initial premises, the antibacterial activity of the studied benzosiloxaboroles seemed to be based on the tRNA-targeting OBORT mechanism.However, experimental investigation revealed that the studied agent 20 does not effectively inhibit LeuRS as IC 50 > 200 μM.Hence, it can be concluded that benzosiloxaboroles have a different molecular target in a bacterial cell than benzoxaboroles.The lack of affinity to LeuRS was also confirmed by bioinformatic structural analysis.To summarize, the potential of SH-substituted benzosiloxaboroles for the synthesis of a diverse library of structurally extended derivatives was amply demonstrated and represents a significant progress in the field.Selected results of antimicrobial activity screening are promising.Hopefully, the presented findings will be followed by future research, eventually leading to development of new boronbased antibiotics.

General comments
Solvents used for reactions were dried by heating to reflux with sodium/benzophenone and distilled under argon.Starting materials and other reagents including halogenated benzenes, alkyllithiums, diisopropylamine, trimethyl borate, tert-butyl(chloro)dimethylsilane, or chlorodimethylsilane were used as received without further purification.Reactions in which organometallic compounds were used were carried out under an argon atmosphere.Detailed procedures for the synthesis of Michael acceptors used in reactions leading to the formation of compounds 5, 9-11, 14-19 as well as α-bromoketones used in syntheses of 22 and 23 are given in ESI, † section S1. 1 H, 13 C, 19 F and 31 P NMR spectra were recorded on an Agilent NMR 400 MHz DDR2 spectrometer. 11B NMR spectra were recorded on a Bruker AVANCE III 300 MHz spectrometer.In the 13 C NMR spectra the resonances of boron-bound carbon atoms were not observed in most cases as a result of their broadening by a quadrupolar boron nucleus. 1 H and 13 C NMR chemical shifts are given relative to TMS using residual solvent resonances. 11B and 19 F NMR chemical shifts are given relative to BF 3 •Et 2 O and CFCl 3 , respectively. 31 Synthesis 4-Bromo-2,6-difluorobenzenethiol (1b).A solution of 1-bromo-3,5-difluorobenzene (58.0 g, 0.301 mol) in THF (90 ml) was added dropwise to a stirred solution of LDA (0.316 mol) freshly prepared from n-BuLi (9.6 M in hexane, 32.9 mL, 0.316 mol) and diisopropylamine (46.3 ml, 33.45 g, 0.331 mol) in THF (300 ml) at −78 °C.The resultant brown solution was stirred for 30 minutes, followed by an addition of sulfur (9.61 g, 0.301 mol).The mixture was stirred overnight at −78 °C.Then it was acidified at −78 °C using 1.5 M aq.H 2 SO 4 until the pH = 1.The resulting orange slurry was then slowly warmed up to room temperature.During warming, gradual dissolution of the pale precipitate was observed.The organic phase was separated and the aqueous phase extracted with Et 2 O (3 × 30 mL).The combined organic solutions were dried over anhydrous MgSO 4 and evaporated to give a yellow colored residue which was distilled under reduced pressure (b.p. 40-45 °C, ca. 10 −3 mbar) to give 1b as a pale yellow oil.Yield 34.21 g (51%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.13-7.02(m, 2H), 3.56 (s, 1H) ppm. 13C NMR (101 MHz, CDCl 3 ) δ 159.1 (d, J = 248.9Hz), 159.0 (d, J = 249.1 Hz), 117.7 (t, J = 11.8Hz), 115.5-115.1 (m), 108.9-107.8(m) ppm. 19F NMR (376 MHz, CDCl 3 ) δ −107.22 to −107.32 (m) ppm.

Determination of acidity constants (pK a values)
The acidity constants of benzosiloxaboroles 1e, 2e, 6, 20 and 22-24 were determined by standard pH titration with 0.05 M aq.NaOH.The measurements were performed using a thermostated (25.0 °C) glass vessel equipped with a magnetic stirrer and a 10 mL burette.Prior to the measurement, the pH glass electrode was calibrated using borax and phosphate buffers.A solution of the analyzed compound (ca.20-40 mg) in a mixture of MeOH/H 2 O (v/v 1 : 1, 30 mL) was titrated until the pH of the solution exceeded the value of 12.0.

Antimicrobial activity
Direct antimicrobial activity was determined against the following standard strains: (1)

Fig. 1
Fig. 1 (a) Examples of biologically active benzoxaboroles already introduced into clinical use (both marketed and under clinical trials); (b) examples of benzosiloxaboroles showing antimicrobial activity.

Scheme 3
Scheme 3 Oxidation of 1e to the corresponding sulfonyl chloride derivative 26 and subsequent conversion to sulfonamides 27, 28 and 29.

Fig. 3
Fig. 3 Structural determinants of LeuRS inhibition by oxaborole derivatives; (a) 2D representations of ligands.Since known LeuRS oxaborole inhibitors work by forming complexes with tRNA nucleotides, both compounds have been represented as AMP adducts.The studied compound 20 is depicted on the left and a known potent inhibitor (AceSPhO_BOB (ref.65)) is depicted on the right; (b) comparison of the binding modes of AMP adducts of both compounds with LeuRS.On the left, the binding mode of compound 20 obtained through docking (using MOE (ref.66)) into a homology model of the S. aureus MRSA LeuRS, and on the right, the binding mode of AceSPhO_BOB (ref.65) as seen in the crystal structure [PDB code: 7BZJ].Residues in proximity of the ligands are colored according to the residue property (whitenonpolar, greenpolar, bluenegatively charged, redpositively charged).Labels were added to selected residues adjacent to the ligand for reference; (c) contacts between the pendant group of both compounds and its interaction with a polar LeuRS loop.The loop is colored according to the residue property (the same color code as above).

Fig. 4
Fig. 4 MD evaluation of interactions between the ligand -AMP adduct of benzosiloxaborole 20 and S. aureus MRSA LeuRS; (a) initial structure of the (AMP adduct of 20)/LeuRS complex subjected to 5 × 1 μs of unbiased MD simulations.The distances between the protein and the benzosiloxaborole core (blue) and the pendant group (yellow) are depicted as lines, and their evolution across the simulations is plotted; (b) evolution of the position of the AMP adduct of 20 across one unbiased simulation run.The loop initially interacting with the ligand is colored based on the residue property (whitenonpolar, greenpolar, rednegatively charged).

Table 1 (
continued) Scheme 2 Synthesis of benzosiloxaboroles 22-25 through treatment of 1e or 2e with electrophiles featuring reactive C-Hal or P-Cl bonds.

Table 3
The antibacterial activity of tested agents against standard Gram-positive strains 53