Development of structurally extended benzosiloxaboroles – synthesis and in vitro biological evaluation

The synthesis of potassium 6-hydroxy-7-chloro-1,1-dimethyl-3,3-difluorobenzo-1,2,3-siloxaborolate 5b from readily available 4-bromo-2-chlorophenol was developed. This compound proved useful in various derivatizations resulting in a wide range of O-functionalized benzosiloxaboroles. Reactions of 5b with selected substituted benzoyl chlorides gave rise to a series of respective derivatives with 6-benzoate side groups attached to the benzosiloxaborole core. Furthermore, treatment of 5b with substituted benzenesufonyl chlorides afforded several benzosiloxaboroles bearing functionalized benzenesulfonate moieties at the 6 position. The synthesis of related chloropyridine-2-yloxy substituted benzosiloxaboroles was accomplished by a standard approach involving silylation/boronation of appropriate heterodiaryl ethers. Investigation of biological activity of obtained compounds revealed that some benzoate and most benzenesulfonate derivatives exhibit high activity against Gram-positive cocci such as methicillin-sensitive Staphylococcus aureus ATCC 6538P as well as methicillin-resistant S. aureus ATCC 43300 with the MIC values in the range of 0.39–3.12 mg L−1. Some benzenesulfonate derivatives showed also potent activity against Enterococcus faecalis ATCC 29212 and E. faecium ATCC 6057 with MIC = 6.25 mg L−1. Importantly, for the most promising cocci-active benzenesulfonate derivatives the obtained MIC values were far below the cytotoxicity limit determined with respect to human normal lung fibroblasts (MRC-5). For those derivatives, the obtained IC50 values were higher than 12.3 mg L−1. The results of antimicrobial activity and cytotoxicity indicate that the tested compounds can be considered as potential antibacterial agents.


Introduction
Recently, organoboron compounds have attracted increased attention as a subject of studies in the area of medicinal chemistry. [1][2][3] Numerous compounds were found to exhibit biological activity, mostly as anti-cancer, anti-inammatory and anti-microbial agents. From the structural point of view, selected classes of organoboranes seem to be especially suitable for such applications. These include boron-rich cluster compounds, i.e., carborane derivatives considered for the use in Boron Neutron Capture Therapy. 4 There are also numerous examples of biologically active boronated peptide derivatives. [5][6][7] Currently, arylboronic acids are highly popular synthetic reagents which found also applications in other elds, e.g., as potent receptors of saccharides and other diols with a special emphasis on nucleosides and catechol derivatives. 8 Their antimicrobial activity was recognized in the 1930's. 9 Benzoxaboroles are a specic class of cyclic arylboronic hemiesters which were obtained the 1950's. 10,11 However, they became highly popular only 50 years later when their potent antimicrobial activity was discovered. 12 Extensive studies resulted in the preparation of numerous derivatives which were further evaluated from the point of view of medicinal chemistry. [13][14][15] Those efforts have already met with success as two benzoxaboroles including antifungal agent Tavaborole I (trade name Kerydin) (Fig. 1), and anti-inammatory Crisaborole II (trade name Eucrisa) (Fig. 1), were approved by FDA for the clinical use. 16 The mechanism of action of benzoxaboroles relies on their physicochemical spec-ity based on enhanced electron-decient character of the boron atom. In fact, benzoxaboroles are stronger Lewis acids than the corresponding arylboronic acids. 17 In general, the boron centre plays the key role in the binding to the biological targets through formation of strong covalent bonds. However, the binding can be substantially enhanced by additional interactions occurring with participation of various functional groups or larger structural fragments. Thus, a specic activity can be achieved by proper structural design, therefore intensive efforts resulted in elaboration of synthetic protocols for the preparation of thousands of functionalized benzoxaboroles. 15 For example, 7-(cyanophenyl) benzoxaboroles III (Fig. 1) showed antituberculosis potency 18 whereas 3-aminomethyl derivatives were active against Gram-negative bacteria. 19 In another work, the series of compounds bearing substituted phenyl groups connected to benzoxaborole core at the 6 position through various linkages ( Fig. 1) were prepared and tested as potential antitrypanosomal agents. 20 It was demonstrated that compounds with sulfone IV, sulfonamide V and amide VI linkages were most promising, and therefore it was concluded that their improved activity is connected to enhanced hydrogenbond-acceptor character of these linkage groups.
Based on the concept of bioisosterism, 21 we decided to prepare some benzoxaborole congeners. Thus, we developed synthetic routes to pyridoxaboroles 22 where the benzene rings replaced with the pyridine one. However, we have put our major efforts to benzosiloxaboroles 23,24 where the silicon atom serves as the bioisostere of the carbon atom in the oxaborole ring. Despite the close analogy resulting from the location of carbon and silicon in the same group of periodic table, the chemical properties of those elements are quite different. From the point of view of biological activity it is important to note that Lewis acidity of the boron atom is increased when comparing benzosiloxaboroles to benzoxaboroles which may be attributed to increased p-acceptor ability of silicon vs. saturated carbon atom. 25 In addition, one can expect that lipophilicity will be increased when the methylene group is replaced with the larger SiMe 2 fragment. As a consequence, antimicrobial activity of respective benzoxa-and benzosiloxaboroles is different. We have already succeeded in preparation and comprehensive characterization of various functionalized benzosiloxaboroles VII (Fig. 2). It was found that simple uorinated benzosiloxaboroles are potent antifungal agents whereas other diboron derivatives VIII-IX were identied as inhibitors of KPC-2 b-lactamase. 26 We have also observed that replacement of uorines with chlorines at 6 and 7 positions was benecial for antibacterial activity. Therefore, we decided to check whether introduction of larger substituents adjacent to chlorine will further enhance antibacterial potency. To some extent, this concept was inspired by the fact that diverse biological activity of benzoxaboroles is observed or improved due to attachment of various pendant aryl substituents as demonstrated by examples shown in Fig. 1. Thus, in this work we report new family of structurally expanded benzosiloxaboroles with a special focus on derivatives with arylsulfonate side groups which showed the most promising antibacterial activity, especially towards various strains of Staphylococcus aureus. Clinical strains of methicillin-resistant S. aureus have been a serious problem in both hospital and open treatment for many years. S. aureus MRSA strains are resistant to almost all b-lactams and oen resistant to antibiotics of other classes. Recently, an increase in the number of isolates resistant to one of the newer group of antibiotics, i.e., glycopeptides, has been observed. 27 Therefore, it is necessary to search for new groups of compounds active against these bacteria, preferably with a new mechanism of action.

Synthesis
The general synthetic approach to nal targeted benzosiloxaboroles started with inexpensive 4-bromo-2-halophenols 1a-1b   26 Position numbering scheme is additionally provided for the general structure VII (note that it is different between benzoxa-and benzosiloxaboroles).
(Scheme 1). The hydroxyl groups were protected with chloro(tert-butyl)dimethylsilane (TBDMSCl) and the resulting silyl ethers 2a-2b were subjected to deprotonation with LDA in THF at À78 C followed by trapping of corresponding aryllithium intermediates with Me 2 Si(H)Cl in accordance with a general protocol reported by us previously. 23 The reactions occurred regioselectively at the position between two halogens in accordance with a strong cumulated ortho-acidifying effect of those two substituents. 28,29 The functionalized arylsilanes 3a-3b were converted to respective benzosiloxaboroles 4a-4b aer some optimization of reaction conditions. Thus, the most effective approach involved Br/Li exchange with t-BuLi in THF at À78 C followed by immediate trapping with B(OiPr) 3 present in a reaction mixture ("in situ quench" technique 30 ). The hydrolysis effected with water resulted in cleavage of Si-H bond which occurs rapidly under alkaline conditions due to ortho-assistance of the anionic boronate group. 31 The benzosiloxaboroles 4a-4b bearing silyloxy groups at the 6-position have been obtained in good yields as white solids soluble in common organic solvents (Scheme 1). In a subsequent step, 4a-4b were subjected to deprotection of OTBDMS groups with KHF 2 in MeOH/H 2 O. Unfortunately, in the case of 4a, the reaction resulted in a mixture of potassium salts of benzosiloxaborolate and aryl-triuoroborate anions 5a, 5a 0 , respectively; the latter product formed due to subsequent cleavage of siloxaborole ring in 5a. The attempts to isolate the desired product 5a in a pure form were unsuccessful. In contrast, the salt 5b was formed selectively as it was not prone to subsequent ring opening. It was isolated as a non-stoichiometric DMF solvate as this solvent was used for the nal extraction of 5b from a crude product containing substantial amounts of inorganic uoride salts. The solvent could not be quantitatively removed even by prolonged heating under reduced pressure (10 À3 mbar). However, the presence of DMF does not disturb subsequent derivatization of 5b as it was also carried out using this solvent.
The presence of free hydroxyl group in 5b was utilized in various derivatization reactions through initial generation of anionic phenolate species. Various bases including K 2 CO 3 / acetone, NaOH/EtOH and DIPEA (Hünig's base)/THF were tested but they proved ineffective which can be attributed to the poor solubility or degradation of 5b under such conditions. Finally, the use of sodium hydride in anhydrous DMF gave satisfactory results allowing for clean and effective deprotonation of the 6-OH group. Subsequent nucleophilic substitution reactions with MeI, Et 2 NCOCl, benzoyl, and benzenesulfonyl chlorides as electrophilic partners proceeded smoothly under mild conditions (temperature range of 0-25 C) giving rise to a series of functionalized benzosiloxaboroles 6, 7, 8a-8g, and 9a-9r, respectively (Scheme 2). In addition, we attempted to use the mixture 5a/5a 0 using the protocol developed for derivatization of 5b but the results were not satisfactory as we were unable to isolate 7-uoro analogues of aforementioned products.
We have also used dichloropyridines 10a-10b as electrophiles in order to attach the pyridine ring through the ether linkage. Unfortunately, the reactions did not proceed under conditions described above whereas at higher temperatures a tarry mixture was obtained indicating that degradation of starting materials occurred during heating. Thus, we have changed the reactions sequence leading to targeted products 13a-13b (Scheme 3). In the rst step, 10a-10b were subjected to Scheme 1 Synthesis of hydroxy-substituted benzosiloxa(difluoro)borolates 5a-5b.
S N 2Ar reactions with the phenolate anion generated from 1b using NaOH/DMSO at 100 C. 32 The obtained halogenated phenoxypyridines 11a-11b were converted to respective dimethylsilyl derivatives 12a-12b followed by nal transformation to benzosiloxaboroles 13a-13b; both steps were carried out using a protocol described for preparation of 4a-4b from 2a-2b.

Compound characterization
All nal benzosiloxaboroles were obtained and fully characterized by multinuclear NMR spectroscopy and HRMS analysis. The 11 B NMR spectrum of the salt 5b in DMSO-d 6 showed a broadened resonance at 5.5 ppm consistent with the presence of tetracoordinate boron atom whereas the respective 19 F NMR spectrum showed a signal at À133.60 ppm indicating the attachment of uorides. In addition, X-ray diffraction analysis of the salt 5b conrmed the tetrahedral arrangement of the boron atom (Fig. 3b) whereas the geometry of the entire boracyclic ring in the benzosiloxaborolate anion is slightly different than that in neutral benzosiloxaboroles, 23 mainly due to elongation of the B-O distance. The structural formulation of selected benzosiloxaboroles 4b, 6, 8a, 9a, 9h and 13a was also conrmed by single-crystal X-ray diffraction analyses ( Fig. 3a and c-g). The metric features of ve-membered boracyclic rings in all studied structures are similar to those found previously in analogous compounds (Table S3, see ESI †). 23 In most cases, the molecules tend to form centrosymmetric dimers due to formation of intermolecular hydrogen bonds between BOH groups (Fig. S84, ESI †). Exceptionally, in the case of 13b, the dimer is formed by O-H/N hydrogen bonds between B(OH) hydroxyl group and pyridine nitrogen atom (Fig. S85, ESI †). Furthermore, the acidity (pK a values) of selected derivatives was determined by potentiometric titration with 0.05 M aq. NaOH in water/methanol solution (1 : 2). The results (see Table 1) indicate that the benzenesulfonate derivatives (9a, 9c, 9k, 9o) exhibit the highest acidity in the studied series (pK a in the range 5.4-6.1) which depends to some extent on the structure of the pendant aryl substituent. This is in agreement with the strong electron-withdrawing effect of the benzenesulfonate group. The benzoate derivatives are slightly weaker acids (pK a in the range 6.4-6.6) whereas acidity of 4b (pK a ¼ 7.6) is decreased due to strong electron-donating character of TBDMSO group. Overall, the obtained pK a values indicate that the most of studied compounds tend strongly to exist as corresponding anions under standard physiological conditions (pH ¼ 7.4), which should enhance their solubility.

Antibacterial activity
It has been shown recently, that sulfonamide-substituted benzoxaboroles have high activity against S. aureus including methicillin-resistant S. aureus ATCC BAA-1762 strain. 33 The MIC  values from 0.4 to 6.25 mg L À1 were obtained for the most active compounds. Besides, we have previously presented the antibacterial activity, also against Gram-positive cocci, of the several benzosiloxaboroles. 26 In this study we have investigated antimicrobial activity of the following groups of the newly synthesized benzosiloxaboroles: Group I (TBDMSO derivatives 4a-4b), Group II (benzoyloxy derivatives 8a-8g), Group III (benzenesulfonyloxy 9a-9r) and Group IV (chloropyridin-2-yloxy derivatives 13a-13b). All obtained data for the new derivatives of benzosiloxaboroles and the reference agents are presented in the ESI in Tables S4-S6 †. In general, the compounds from the Group III (9a-9r) showed the highest antibacterial activity towards cocci of the Staphylococcus genus, especially S. aureus. It is worth to underline, that study was carried out with methicillin-sensitive S. aureus ATCC 6538P and methicillinresistant S. aureus ATCC 43300. 14 out of 16 well soluble compounds from the Group III showed the high activity against methicillin-sensitive as well as methicillin-resistant S. aureus strains, with the MIC values in the range of 0.39-3.12 mg L À1 ( Table 2, for full data set see Table S4 †). Interestingly, compounds 9k, 9q and 9r showed relatively high activity also against other Gram-positive cocci such as Enterococcus faecalis ATCC 29212 and E. faecium ATCC 6057, with the MIC value of 6.25 mg L À1 (Tables 2 and S4 †). The activity of new groups of compounds against Enterococcus sp. is rarely observed. It is worth emphasizing that E. faecalis and E. faecium used in our research belong to two species of the genus Enterococcus responsible for frequent human infections, including nosocomial infections. 34 Compounds from the remaining three groups (4a-4b, 8a-8g and 13a-13b) showed lower activity against Gram-positive bacteria as the MIC range was 12.5-400 mg L À1 whilst diameters of the growth inhibition zones ranged from 18-24 mm (Tables 2 and S4 †). Thus, the substitution of benzosiloxaboroles with benzenesulfonate substituents is necessary to achieve high activity against staphylococci and enterococci. In this study, linezolidone of the relatively new group of antibacterial drugs belonging to the oxazolidinones, was used as the reference substance. The indications for linezolid treatment are infections caused by multi-drug resistant cocci including both methicillin-resistant staphylococci and glycopeptide-resistant enterococci strains. 27, 34 We have found that ve compounds from the Group III were more active than linezolid against MSSA and MRSA strains. The obtained MIC range of these compounds (9d, 9h, 9k, 9q and 9r) was 0.39-0.78 mg L À1 for the MSSA strain (linezolid: MIC ¼ 1 mg L À1 ) and 0.39-1.56 mg L À1 for the MRSA strain (linezolid: MIC ¼ 2 mg L À1 ) ( Table 2). The high activity of these compounds is due to the presence of chloro or tri-uoromethyl groups at the para position or two such groups at the meta and para positions of the benzenesulfonate substituent. Also the presence of three methyl groups at the 2,4,6 positions of the benzenesulfonate substituent results in the high activity of 9k. Contrary to staphylococci, no potency of the obtained sulfonate-substituted benzosiloxaboroles comparable to linezolid was observed against enterococci. In the case of nine derivatives of the parent compound 9a, the activity against E. faecalis and E. faecium increased from 2-to 8-fold indicative of positive effect of substituents at the benzenesulfonate scaffold. The analysis of the relationship between the activity and the structure of the tested compounds revealed that the presence of two Cl (9q) or Cl and CF 3 groups (9r) as well as the presence of three Me groups (9k) is necessary to achieve the highest activity against enterococci. However, the activity of these compounds was still 3-fold weaker than that of linezolid.
Examining the antibacterial activity of new compounds, the minimum bactericidal concentration (MBC) can be determined aer establishing the MIC value. For most compounds of Groups I, II and IV, the MBC values were high $ 200 mg L À1 . Interestingly, in the case of the tested compounds from Group III, a paradoxical growth effect was observed during the determination of bactericidal activity. This so-called Eagle effect has previously been reported for several antibiotics, such as some blactams, glycopeptides, aminoglycosides, quinolones and polymyxins. 35 This phenomenon was rst published for S. aureus. 36 According to the EUCAST and CLSI denitions, the MBC value is the lowest concentration of a agent that kills 99.9% of bacteria. 37,38 For 11 out of 16 well soluble compounds (9a, 9c, 9d, 9g, 9i, 9j, 9m, 9n, 9p-9r) the two MBC values for both S. aureus strains were observed (Table S4 †). Following the CLSI guidelines, 38 the results were read as the low MBC values in range 0.78-12.5 mg L À1 for S. aureus MSSA and 1.56-25 mg L À1 for S. aureus MRSA. However unusually, on the plates with samples taken from the wells containing progressively increasing the agent concentrations (from 2-to 4-fold over the rst MBC values), a signicant increase in the number of growing colonies, as a paradoxical growth effect, was observed. Finally the second MBC value (in the range 25-400 mg L À1 ) was obtained. So far, the mechanisms causing paradoxical bacterial growth with increasing concentrations of antibiotics are not fully elucidated. However, there have also been several in vivo studies in animal models to support the occurrence of the Eagle effect. 35 In addition, two case reports of the Eagle effect observation during the treatment of human bacterial infections have been described. The reduction in the doses of antibiotics resulted in therapeutic success and correlated with a reduction of the bacteria survived in the bloodstream. 35 Overall, all the studied groups of the newly synthesized benzosiloxaboroles showed no signicant activity against Gramnegative rods (Table S5 †). Only a few compounds from the Group II and III of benzosiloxaboroles derivatives showed weak activity against Stenotrophomonas maltophilia strains (MICs 200-400 mg L À1 ) and Bordetella bronchiseptica (MICs 50-400 mg L À1 ). As in our previous publications, we have investigated the contribution of efflux pumps to the resistance of Gram-negative bacilli to the new synthesized compounds. 23,26 We used the well-known RND efflux pump inhibitor, Phe-Arg-bnaphthylamide (PAbN). 39,40 It inhibits the activity of efflux systems found in all Gram-negative rods, like Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Proteus mirabilis, Pseudomonas aeruginosa, S. maltophilia and Acinetobacter baumannii. [39][40][41][42][43] According to the recent publications, we have used lower concentration of PAbN, i.e., 20 mg L À1 because the destabilization of bacterial cell covers was observed at higher concentration of this inhibitor. [44][45][46] In order to minimize the inuence of PAbN on cell covers, the tests were conducted also in the presence of 1 mM MgSO 4 . 44 Only in the case of six compounds, we showed a signicant (4-fold) decrease in the MIC value of the studied compound in the presence of PAbN. These results conrm the lack of activity of the tested benzosiloxaborole derivatives against Gram-negative rods.

Antifungal activity
Inspired by our previous report about high activity of few benzosiloxaborole derivatives against yeast-like fungi, Candida tropicalis and C. guilliermondii, 23,26 we have investigated the activity of the newly synthesized benzosiloxaboroles against 5 species of Candida and Saccharomyces cerevisiae ATCC 9763. For all but one compounds, the antifungal activity determination was performed using the disk diffusion method as well as by an evaluation of the MIC and MFC values. The results of antifungal activity of the newly synthesized benzosiloxaboroles agents are presented in the ESI (Table S6 †). The collection strains of Candida species which most commonly cause infections in humans were selected for the study. In most cases, they are responsible for opportunistic infections. However, they can cause also nosocomial infections, including severe infections, and cause death, mainly of immunocompromised patients. 47,48 Compounds from the Group II demonstrated the highest activity against all tested Candida species, especially against C. krusei and C. tropicalis. The presence of methyl group at the para position of the phenyl ring (compound 8d) led to 2-4-fold Table 3 The viability of human normal lung fibroblasts, MRC-5 after 72 h treatment with the tested compounds. Linezolid was used as a reference. The concentration (IC 50 ) that causes a response half way between the maximal (top) response and the maximally inhibited (bottom) response was calculated using MTT-based assay data and an equation increase in the activity against all tested Candida strains. The lowest MIC values 3.12-6.25 mg L À1 were observed for two strains of C. tropicalis. Other studied benzosiloxaboroles showed relatively high activity only against S. cerevisiae, a species that is not clinically signicant.

Cytotoxic activity
To evaluate the cytotoxic effect of the tested compounds, MTTbased assay was performed. Human normal lung broblasts MRC-5 were treated with the newly synthesized compounds at the concentrations range of 0.78 to 50 mg L À1 for 72 h. IC 50 values describing half inhibitory concentrations of each tested compound were calculated and summarized in Table 3. The representative plots demonstrating sigmoidal dose response curves for the tested compounds were shown in the ESI (Fig. S86-S91 †). The obtained IC 50 values for compounds 9a-9r, 8a-8g, 13a-13b are in the range of 3.19 to above 50 mg L À1 , with the lowest value for the 4-chloro-3-nitrobenzenesulfonate derivative 9p, and the highest values for compounds 9a, 9g, 5b, 8f and 8g.