Review pubs.acs.org/CR

Recent Developments in the Chemistry and Biological Applications of Benzoxaboroles Agnieszka Adamczyk-Woźniak,* Krzysztof M. Borys, and Andrzej Sporzyński Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland Corresponding Author Notes Biographies Acknowledgments Abbreviations Used References

T T T U U V

1. INTRODUCTION Benzoxaboroles, first obtained by Torssell in 1957,1 remained underestimated for a long time. The new beginning for these remarkable compounds took place in 2006, when the exceptional sugar-binding properties of 1 (Figure 1) at CONTENTS 1. Introduction 2. Physicochemical Properties of Benzoxaboroles 2.1. Lewis Acidity 2.2. Structural and Spectral Properties 3. Applications 3.1. Binding of Biomolecules 3.1.1. Binding of Biomolecules by Simple Benzoxaboroles 3.1.2. Diols Binding by Complex Benzoxaboroles and Benzoxaborole-Containing Materials 3.2. Medicinal Applications 3.2.1. Antifungal Activity 3.2.2. Antibacterial Activity 3.2.3. Antiviral Activity 3.2.4. Anti-Inflammatory Activity 3.2.5. Antiprotozoal Activity 3.2.6. Other Medicinal Aspects 3.3. Synthetic Utility of Benzoxaboroles 3.3.1. Reactions Aimed at Functionalizing Benzoxaboroles 3.3.2. Reactions Aimed at Complexation of Benzoxaboroles 3.3.3. Reactions Leading to the Loss of the Benzoxaborole Function 3.3.4. Potential Catalytic Application of Benzoxaboroles 4. Synthesis of Benzoxaboroles 4.1. Synthetic Methods Utilizing Boronic Pinacol Esters 4.2. Synthetic Methods Utilizing Aromatic Bromides 4.3. Synthetic Methods Utilizing 2-Formylphenylboronic Acid 5. Summary and Perspectives Author Information © XXXX American Chemical Society

A B B B D D D Figure 1. Unsubstituted benzoxaborole (1), with atom numbering scheme, and compounds in clinical development: AN2690 (2), AN2728 (3), AN2898 (4), and SCYX-7158/AN5568 (5).

D G G G I J L N N

physiological conditions were described.2,3 However, the vast majority of benzoxaboroles have been reported over the last 5 years. The increasing interest in these derivatives of phenylboronic acids is connected first of all to their biological activity, with 5-fluoro-substituted benzoxaborole (2, AN2690, tavaborole) being the most spectacular example of antifungal action.4−6 Very recently, a topical solution of 2 (KERYDIN) was approved by the FDA for treatment onychomycosis.7 The discovery of its mechanism of action enabled rational design of other biologically active benzoxaboroles,8 with 3 (AN2728)9 and 4 (AN2898)10 currently under clinical trials for psoriasis. Another benzoxaborole that entered clinical trials is 5 (SCYX7158/AN5568), tested as treatment for human African trypanosomiasis (Figure 1).11 Benzoxaboroles (a) combine structural features of boronic acids (b) and cyclic boronic esters (c) (Figure 2). The presence of a free hydroxyl group, as well as a relatively strong Lewis acidic center, on the heterocyclic boron atom results in the exceptional properties of benzoxaboroles (Figure 2). Introduction of additional substituents or incorporation of a

N O P P Q Q R S T T

Received: November 10, 2014

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physicochemical properties of boronic molecules.24 Benzoxaboroles generally display higher acidity than the corresponding phenylboronic acids,25 which is explained by the ring strain generated in the five-membered heterocyclic ring. The strain of the benzoxaborole ring can be reduced upon addition of the electron-rich nucleophiles (like OH−), generating anionic, tetrahedral species (Scheme 1).

Figure 2. General structure of benzoxaboroles (a), phenylboronic acids (b), and their cyclic esters (c).

Scheme 1. Acidic Properties of Benzoxaboroles benzoxaborole structural motif into various materials enables multidendate interactions that result in improved selectivity or binding. The fast development of benzoxaboroles’ chemistry follows undiminished interest in various aspects of the properties and applications of boronic acids.12−14 A comprehensive review covering the synthesis, properties, and applications of benzoxaboroles was published in 2009.15 A reasonable number of papers (over 140) as well as patents (almost 20) dealing with these compounds has been published since. Among other small boron-containing molecules, benzoxaboroles have been recognized as promising substances of medicinal importance.16 Benzoxaboroles were found, for example, to selectively inhibit several enzymes and be potent antiprotozoal drugs,17 as well as promising drug delivery agents.18 Selected medicinal aspects of boron-containing small molecules including benzoxaboroles have been analyzed in a recent review by Del Rosso and Plattner,19 who also highlighted their clinical relevance in dermatological applications and the drug development process. Various classes of boron-based chemotherapeutics, including benzoxaboroles, have been reviewed by Baker and co-workers.20 A review on boroncontaining inhibitors of synthetases, along with a comprehensive description of the discovery of AN2690 and its mechanism of action, has also been published by Baker et al.21 In the recent review by Benkovic and co-workers, particular classes of bioactive benzoxaboroles have been discussed with the emphasis put on the mechanisms of action and medicinal applications.22 Identification of the biological activity of benzoxaboroles received positive feedback from synthetic chemists, who developed novel strategies for the synthesis of a variety of benzoxaboroles. A detailed report on the recent advances in the synthesis of benzoxaboroles has been given by Zhang and co-workers23 and recently updated by Liu et al.22 The present review covers broad aspects of the chemistry, molecular structures, and reported applications of benzoxaboroles published or patented since 2009. It focuses on the physicochemical properties of benzoxaboroles and a great variety of novel structures (more than 500 described in scientific papers over the last 5 years, whereas only 65 had been described in 2009). General ideas of plausible synthetic methods are given, rather than a description of all the reported procedures. The present review clearly identifies structures of the promising leads for new drugs, giving a hint of future synthetic targets. It also describes the issues of application of benzoxaboroles in medicinal and materials chemistry.

For that reason, benzoxaboroles form dative bonds more readily than the corresponding boronic acids. It is a remarkable feature as the acid−base interactions play a crucial role in phenylboronic chemistry.26−28 Moreover, formation of the socalled “spiro” form has been postulated as the mechanism of benzoxaboroles’ action, like some enzyme inhibitors.4,29 Investigation of the influence of the structure of benzoxaboroles on their pKa has been recently reported,30 showing a good correlation with the σ Hammett values of the substituents on the phenyl ring (Figure 3). Moreover, the pKa values of benzoxaboroles were shown to be higher in a 50% (v/v) ethanol−aqueous solution in comparison with that observed in pure water.30

Figure 3. pKa values of benzoxaboroles.30,31

Interestingly, the introduction of an amino group into the heterocyclic oxaborole ring in 11−13 does not influence the pKa,31 whereas two methyl groups at the C-3 position in 14 considerably decrease the Lewis acidity of the boronic center (Figure 3).30 The enhanced Lewis acidity of benzoxaboroles in comparison with phenylboronic acids results in about 50% of the anionic form being present in aqueous solution at physiological pH, which leads to their higher water solubility25 and significantly better pharmacokinetic properties than those of phenylboronic acids.32 2.2. Structural and Spectral Properties

Structural characteristics of benzoxaboroles by X-ray crystallography were detailed in a recent review.15 Molecular and crystal structures of several benzoxaboroles, including 2, 12, and 15− 18 (Figure 4), have been determined since.33−38 Similarly to phenylboronic acids, most of the described benzoxaboroles display dimeric interactions in the solid state. The dimers are formed by medium-strong intermolecular hydrogen bonds (Figure 5). Benzoxaboroles containing addi-

2. PHYSICOCHEMICAL PROPERTIES OF BENZOXABOROLES 2.1. Lewis Acidity

Similarly to phenylboronic acids and their diol esters, benzoxaboroles behave as Lewis acids rather than Brønsted acids. Lewis acidity is one of the most important B

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solid state to show the influence of solvents with different donor properties on the formation of hydrogen bonds.39 Benzoxaboroles 1 and 2 were investigated by multinuclear solid-state NMR (1H, 11B, 13C, and 19F NMR),38 and the solution and solid-state NMR spectra were discussed in view of DFT calculations of the NMR parameters. The NMR spectroscopy is also a useful tool in the investigations of benzoxaboroles chemistry in solution. All the signals in 1H and 13 C NMR spectra of 1, 15 and 16 in deuterated acetone were assigned on the basis of 1H−1H COSY, HSQC, and HMBC measurements.35 Signals assignments were also carried out for the spectra of 1 and 2 in DMSO solutions on the basis of the 1 H−13C HMQC experiments.38 The 13C NMR spectra of all of the investigated benzoxaboroles displayed the characteristic lack of the signal of carbon atom adjacent to boron atom.35,38 The most characteristic 1H NMR signals in the spectra of 1, 15, and 16 correspond to the aromatic proton adjacent to the boronic group as well as the benzylic protons (Figure 8).35 The latter observation has been used in the study of the reactivity of aromatic amines with 2-formylphenylboronic acid.40

Figure 4. Benzoxaboroles with recently investigated crystal structures.

Figure 5. Dimeric interactions in the crystal state of some benzoxaboroles.

tional hydrogen bond donors or acceptors tend to form chains, as in structures 1635 and 17 (Figure 6).36 The highly biologically active bis(benzoxaborole) 18 cocrystallized with DMSO, forming intermolecular hydrogen bonds with the solvent molecules (Figure 7).37

Figure 8. Characteristic signals in 1H NMR spectra of 1, 15, and 16 recorded in (CD3)2CO.

Figure 6. Chain motifs in the crystal structures of benzoxaboroles.

The 11B signal corresponding to the benzoxaborole boron atom appears around 32 ppm in (CD3)2CO solution.33−35 17O NMR characterization of benzoxaboroles 1, 6, and 19 in CDCl3 solution was recently published (Figure 9).41

Figure 7. Molecular structure of bis(benzoxaborole) 18 cocrystallized with DMSO. Figure 9. Benzoxaboroles studied by 17O NMR in CDCl3 solution.

Two polymorphs of 1 have been described.35 The polymorphic behavior is important for molecules of pharmaceutical interest, such as benzoxaboroles. A detailed insight into the crystal structures of 1 and 2, including intermolecular interactions and anisotropic thermal expansion as well as polymorphism issues, has been recently given.38 Density functional theory (DFT) calculations in vacuo have been used to estimate the binding energy of the dimeric forms of 1 and 11,39 indicating the strength of the intermolecular hydrogen bonds. Atoms in molecules (AIM) and electron localization (ELF) theories were applied to give insight into the electronic structure of 1 and 11.39 On the basis of theoretical calculations carried out for 15, it was shown that the formation of hydrogen-bonded dimers results in remarkable changes in the electron density distribution within the heterocylic ring.35 DFT modeling of 1, 2,38 and 1535 as dimers in vacuum has been carried out together with calculation of the infrared (IR) vibration modes. The IR and Raman spectroscopy measurements were performed for 1 and 11 both in solution and in the

Experimental data have been compared with the results of calculations. The 17O NMR technique was found to be a potent tool to investigate the known42 tautomeric equilibrium of 2formylphenylboronic acids leading to 3-hydroxybenzoxaboroles (Scheme 2). An equimolar mixture of both tautomers has been observed in the 0.1 M solution of 3-fluoro-2-formylphenyboronic acid in deuterated acetone.41 Scheme 2. Tautomeric Equilibrium Leading to 3Hydroxybenzoxaboroles

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3. APPLICATIONS As mentioned in the Introduction, benzoxaboroles first gained attention as sugar-binding agents with improved solubility in aqueous solutions and enhanced diol binding at neutral pH.2,3 These properties have been investigated further in recent years, involving a broader range of biomolecules as well as incorporation of benzoxaboroles into materials like nanoparticles and copolymers. However, the major development of benzoxaboroles’ chemistry was connected with their biological action. Large libraries of novel compounds were prepared for bioactivity studies, at the same time enhancing the number of methods for their synthesis. Simultaneously, the synthetic utility of benzoxaboroles emerged in the fields of drugs and natural products synthesis as well as the preparation of molecular receptors and metal complexes.

Scheme 4. Formal Presentation of Diol Binding by Benzoxaboroles

noted that only values determined at analogous and strictly controlled conditions can be compared.2,18,31,46 The association constants of unsubstituted benzoxaborole (1) with several analytes (D-fructose, N-acetylneuraminic acid, glucose, methyl-β-D-galactopyranoside, and methyl-α-D-mannopyranoside) in solution have been determined by the ARS binding assay at pH 7.4. All the values are considerably higher in comparison with those of the phenylboronic acid (PBA), yet they are still relatively low (Table 1).46 The binding constants have been also determined by 1H NMR spectroscopy, resulting in different values but ones also reasonably higher for benzoxaborole 1 than for PBA (Table 1).18 The binding constants of 1 and 11−13 with glucose, fructose, and galactose have been evaluated by the competition binding assay with ARS and compared with those of their boronic acid analogs (11_PBA−13_PBA).31 The introduction of an amino group into the benzoxaborole ring in 11−13 results in higher apparent binding constants values, which remain however low. Moreover, the determined K values follow the usual order characteristic for most of the phenylboronic acids containing a single boronic group (KFru > KGal > KGlu), which means stronger binding of fructose than galactose or glucose (Table 1). The influence of pKa of benzoxaboroles on binding to AMP has also been reported.30 Recently, benzoxaborole 1, along with arylboronic acids, was shown to form adducts with sulfenic acids in aqueous solution (Scheme 5).47 The techniques used for the investigation of the adduct formation (and the equilibria involved) included UV/vis spectrophotometry, isothermal titration calorimetry (ITC), electrospray ionization time-of-flight mass spectrometry (ESITOF-MS), and 11B NMR. In relation to this, boronic species were evaluated as inhibitors of an iron-based Rhodococcus erythropolis nitrile hydratase. NHases catalyze hydration of nitriles to amides, which allows for their use in industrial production of acrylamide and nicotinamide.48 They can also be applied in bioremediation processes like waste treatment and herbicide degradation that involve degradation of nitrile-based materials. Importantly, the iron-based NHases require cysteine-derived sulfenic acid at its active site to retain the catalytic activity.22,47 In an enzymatic assay conducted with photoactivated R. erythropolis NHase, benzoxaborole showed a superior capacity for inhibition compared to investigated arylboronic acids. These findings provide an exciting opportunity for new applications of benzoxaboroles, based on the interactions with sulfenic acidbearing biological targets. 3.1.2. Diols Binding by Complex Benzoxaboroles and Benzoxaborole-Containing Materials. Benzoxaborole-containing materials have activity superior to that of the parent benzoxaboroles, which has been assigned to the advantage of multivalent interactions. The importance of multivalent interactions in sensing was confirmed by several research groups.18,46,49−52 Along that line, weak binding in solution does not preclude usefulness of the given benzoxaborole in selective sensing. For example, the ITC experiments confirmed enhanced binding of

3.1. Binding of Biomolecules

3.1.1. Binding of Biomolecules by Simple Benzoxaboroles. The basis for the action of benzoxaboroles as biomolecular receptors is a stepwise formation of cyclic esters with diols (Scheme 3). The mechanism of the binding of Scheme 3. Reactivity of Benzoxaboroles with Diols

benzoxaboroles with ARS (Alizarin Red S) as a model aromatic diol was studied in detail by Tomsho and Benkovic.43 The results of this study have been generalized for other diols, yet the mechanism of binding depends also on the pKa of the diol, which is considerably lower for ARS than for sugars. There is, however, currently a general agreement that the neutral, trigonal form of benzoxaboroles is the predominant reacting form in the binding of diols (Scheme 3).21,22,44 In comparison with phenylboronic acids, the binding takes place at lower pH (∼7), which enables sugar sensing at conditions close to physiological ones. In contrast to phenylboronic acids, benzoxaboroles display the ability to bind to hexopyranosides. This property makes benzoxaboroles potential targeting agents for cell-surface oligosaccharides.3 Due to those exceptional binding properties, benzoxaboroles have been classified as small-molecule-based boronolectins, i.e., relatively simple boron compounds that mimic the function of natural, highly specific sugar-binding proteins called lectins.45 The affinity of a diol for phenylboronic compounds is evaluated by determining the apparent binding or association constants (K) of the esterification reaction formally presented in Scheme 4. The well-established competition assay with ARS,31,46 as well as the NMR method,2,18 was applied to study the complexation of benzoxaboroles with sugars. It should be noted that the determined K values strongly depend on the applied conditions (e.g., type of buffer or cosolvent used) as well as the applied determination method, and great care should be taken when comparing data from various sources (Table 1). It should be D

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Table 1. Apparent Binding Constants [K (M−1)] of 1, 11−13, and the Corresponding Phenylboronic Acids (PBA) with ARS and Sugars K of phenylboronic compd diol ARS fructose galactose glucose Neu5Ac (sialic acid) Me-β-D-galactopyranoside Me-α-D-mannopyranoside

PBA

1 46,d

31,c

1307 , 659 33618,a, 66446,d, 6062,b, 5031,c 1931,c 2818,a, 172,b, 2146,d, 313,d, 431,c 4318,a, 16046,d 2446,d 2146,d

46,d

31,c

1260 , 1300 12818,a, 16046,d, 79 − 518,a, 0 2,b, 646,d 1318,a, 2146,d not measurable46,d not measurable46,d

2,b

1131,c

11_PBA31,c

1231,c

12_PBA31,c

1331,c

13_PBA31,c

1750 395 78 10 − − −

2900 120 100 13 − − −

2556 263 17 14 − − −

720 55 5 2 − − −

2315 930 58 33 − − −

3209 449 48 32 − − −

a Determined in 0.1 M aqueous phosphate buffer (pH 7.4) containing 2% of D2O by the 1H NMR method. bDetermined in 0.1 M phosphatebuffered (pH 7.4) D2O solution by the 1H NMR method. cDetermined in the mixture of methanol and 0.1 M aqueous phosphate buffer (pH 7.4) in the v/v ratio of 1:1 + 1 vol % DMSO by the competition binding assay with ARS. dDetermined in 0.1 M aqueous phosphate buffer (pH 7.4) by the competition binding assay with ARS.

solution to release the benzoxaborole−glycerol adduct for fluorescence measurements conducted in solution. This way the glycoprotein terminal glycosylation could be detected and semiquantified. Appropriately functionalized benzoxaboroles (21−26) have been used for the construction of peptidoboronolectin hybrids of peptides and phenylboronic compounds (Figure 11).57

Scheme 5. Reversible Formation of an Anionic Adduct between a Benzoxaborole and Fries Acid, Which Bears a Sulfenic Acid Moiety47

the nanoparticle-immobilized benzoxaborole with fructose, compared to that observed for the benzoxaborole itself.53 Moreover, as evidenced by the surface plasmon resonance (SPR) measurements, a benzoxaborole-functionalized oligomer (25% of benzoxaborole) displayed about 10 times higher affinity for the HIV envelope glycoprotein (HIVBaL gp120) than the unsubstituted benzoxaborole (1) investigated in solution.54 A benzoxaborole-based method for a naked-eye visual assessment of the terminal glycosylation state of glycoproteins has been developed.55 Glycosylation of a protein influences many of its properties and functions, including folding, stability, activity, interactions, and mobility.56 Abnormal changes in glycosylation have been associated with pathogenic states like inflammatory diseases, metabolic disorders, and cancer, which makes new methods for glycosylation analysis highly desirable. The reported benzoxaborole-based method comprised four major steps. First, the terminal monosaccharide was cleaved from the glycoprotein by means of a specific glycosidase. Second, hydroxylamine-functionalized glass beads were used to capture the released monosaccharide in the form of an oxime. Third, the saccharide-bearing beads were incubated with a solution of benzoxaborole 20 (Figure 10). The formation of benzoxaborole−galactose spiroadducts resulted in turning the beads red. The labeled beads were washed with a glycerol

Figure 11. 3-Substituted benzoxaboroles designed for the synthesis of peptidoboronolectins.57

Several dipeptidyl benzoxaboroles and their peptide mimetics have been obtained by Fu and co-workers (27−50, Figure 12).58 A library of 400 peptidic benzoxaboroles containing two amido benzoxaborole units has been designed, obtained, and screened for activity as the tumor marker TF antigen in water.59

Figure 10. Fluorescent benzoxaborole investigated by Sørensen and co-workers.55

Figure 12. Dipeptidyl benzoxaboroles and peptide mimetics. E

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which has been correlated with the protein concentration. Since benzoxaborole 56 bound more tightly with the mannose surface and could only be partially displaced by the protein, compound 54 has been used as a component of an electrochemical displacement sensor for the detection of Escherichia coli.63 The amino derivatives of benzoxaboroles 57−66 (Figure 15) should be particularly useful in material derivatization. The

As this antigen is present in over 90% of cancers, a small molecular receptor that can recognize it will have wide application potential.60,61 The design was based on carboxybenzoxaborole amide 51 displaying enhanced binding to Meα-D-galactoside in comparison with 1 (dissociation constant Kd = 14 and 33 mM, respectively).3,59 The structure of the most potent receptor (52) with an IC50 value of 20 μM is presented in Figure 13.

Figure 13. Structure of the carboxybenzoxaborole amide 51 and the most potent benzoxaborole tumor marker TF antigen 52.59

The boronic acid analog of 52 has been shown to be less favorable for complexing hexapyranosides (IC50 value of 54 μM). Interestingly, the analogous amidopeptide, lacking any boronic unit, also binds the Gal-β-1,3-GalNAc disaccharide with an IC50 value of 100 μM. The results confirm the importance of interactions like hydrogen bonding or hydrophobic packing with the peptide pattern as contributing significantly to the binding.59 Recently, a set of fluorescent benzoxaborole-containing adamantane lectin mimetics (53, 53a, 53b, and 54) was synthesized and studied in terms of their multivalent binding properties (Figure 14).62 Binding evaluation was performed by

Figure 15. Amino and amido derivatives of benzoxaboroles.

commercially available amino benzoxaborole (58) has been used for the functionalization of polymeric nanoparticles that displayed sugar-binding properties at physiological pH.64 Its acrylamido derivative (64) has been synthesized and applied in preparation of molecularly imprinted polymers capable of fructose recognition at pH 7.4.65 Aminobenzoxaborole 58 was also used for the surface functionalization of magnetic core/shell Fe3O4/poly(acrylic acid) (PAA) microspheres via amide formation.66 The obtained nanocomposites were applied for a highly specific enrichment of glycoproteins, which was successfully carried out under physiological conditions. Noteworthy, crude biological samples like E. coli lysate or fetal bovine serum could be used. The carboxylic benzoxaborole 67 (Scheme 6) has been applied for the derivatization of a monolithic column for selective enrichment and separation of cis-diol-containing biomolecules.67,68 Scheme 6. Derivatization of a Monolithic Column with 67.67

Figure 14. Adamantane62 and ferrocene63 benzoxaboroles and polybenzoxaboroles.62

using carbohydrate arrays immobilized on glass chips and carried out by means of fluorescence spectroscopy. The study showed that all of the investigated lectin mimetics preferentially bound to galactosides (compared with other mono- and disaccharides). The ferrocene benzoxaborole derivatives 55 and 56 (Figure 14) have been bound to a mannose-modified gold electrode.63 Upon reaction of the saccharide-binding protein (concanavalin A) with mannose, the benzoxaboroles were displaced and washed off, resulting in a decrease of the electrochemical signal,

A controlled polymerization of benzoxaborole 68 with a PEG-based chain transfer agent resulted in well-defined monosaccharide-responsive block copolymers (Figure 16). The reported polymers may find applications in the development of sensors and drug delivery agents.69 Shortly afterward, a series of works on the saccharideresponsive benzoxaborole-containing copolymers was reported. The sugar responsiveness of the block copolymer poly(NF

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applications. Readers interested in further insights into the benzoxaboroles’ mechanisms of action, broader biological context, as well as progress in drug development are advised to consult the available reviews.19−22 For detailed information, such as methods for bioactivity evaluation or structure−activity relationship (SAR) analyses, it is recommended to see the original works cited in the particular subchapters. 3.2.1. Antifungal Activity. The mechanism of action of the leading fungicidal benzoxaborole AN2690 (2) is based on the inhibition of fungal leucyl-tRNA synthetase.4 LeuRS belongs to aminoacyl-tRNA synthetases, a class of enzymes crucial for gene translation. Their role during translation is to attach the correct amino acid (leucine, in the case of LeuRS) to the corresponding tRNA. Blocking the enzyme’s editing site can be therefore a potential mechanism of action of antimicrobial therapeutics. This proved to be the case for AN2690, found to block protein synthesis in fungi by inhibiting LeuRS. The X-ray studies of a complex of AN2690 with cytoplasmic yeast LeuRS revealed that AN2690 forms a spiro ester adduct with the enzyme-bound tRNALeu at the LeuRS editing site, precisely with the 3′-terminal adenosine of tRNA. As a result of such binding, the synthetase’s catalytic cycle is blocked and so is the correct protein synthesis within the fungus. Recently, several benzoxaboroles (1, 2, 11, 13, and 18) have been tested as agents against E. coli, Staphylococcus aureus, Mycobacterium luteum, Aspergillus niger, and Candida tenuis.5 Compound 18, containing two benzoxaborole units, displayed the highest activity against M. luteum and both of the investigated fungi. A straightforward synthesis of bis(benzoxaborole) 18 has been developed starting from piperazine and 2-formylphenylboronic acid.37 The superiority of the benzoxaborole scaffold over phenylboronic acid in terms of biological activity was shown by Adamczyk-Woźniak and co-workers, who compared the fungicidal activity of 3-piperazine-bis(benzoxaborole) 18 with its bis(phenylboronic acid) analog.80 In the evaluation studies carried out against five filamentous fungiA. terreus, Fusarium dimerum, Fusarium solani, Penicillium ochrochloron, and A. nigerthe bis(benzoxaborole) 18 had inhibitory activity even higher than the standard antibiotic amphotericin B, while the corresponding bis(phenylboronic acid) was found to be inactive. This way the presence of the benzoxaborole system was demonstrated to be crucial for the antifungal activity of the examined compounds, though the mechanism of action of compound 18 has not been investigated yet. Benzoxaboroles mixed with α- or β-hydroxy carboxylic acids were claimed as stable formulations for the treatment of onychomycosis and tinea infections.81,82 Hydroxy acids played the role of antifungal agent carriers, so the whole system could be considered as an antifungal drug delivery. The fungicidal properties of benzoxaboroles and their O-alkylated, O-arylated, and O-acylated derivatives were found to be useful for plant protection, and such applications were patented recently.83−85 These compounds could also serve as antimicrobial agents for the protection of both plants and meats.85 3.2.2. Antibacterial Activity. One of the biological targets for antibacterial benzoxaboroles are β-lactamases, enzymes that make bacteria resistant to β-lactam antibiotics. The key catalytic role in their active sites is played by a serine residue. A number of boronic acid β-lactamase inhibitors have been developed, with their mechanism of action based on the interaction between an empty p orbital of boron and the hydroxyl group of

Figure 16. Vinyl benzoxaborole (68) and its homopolymers (69−73) and block copolymers (74−78).69

isopropylacrylamide)-block-poly(5-acrylamido-1,2-benzoboroxole) (PNIPAAm-b-PAAmBOB) takes advantage of controlling its micelles’ inversion kinetics in water.70 Copolymer poly(Nisopropylacrylamide-co-5-acrylamido-1,2-benzoboroxole) (P(NIPAAm-co-AAmBOB)) was utilized as a saccharide-responsive agent in the investigation of how the stereoisomerism of the glycopolymers’ sugar units affects hydrogelation.71 Statistical copolymers poly(N-isopropylacrylamide-stat-5-methacrylamido-1,2-benzoxaborole) [P(NIPAAm-st-MAAmBOB)] were found to be responsive to glucose and so were the hydrogels and nanogels formed by mixing poly(N-isopropylacrylamidestat-5-methacrylamido-1,2-benzoxaborole) [P(NIPAAm-stMAAmBOB)] with well-defined glycopolymers.72 Very recently, a hydrogel formed from poly(N-isopropylacrylamide-co5-acrylamido-1,2-benzoboroxole) [P(NIPAAm-co-MAAmBOB)] and poly(3-gluconamidopropylmethacrylamide) (PGAPMAAm) was investigated.73 The gel was loaded with o-nitrobenzaldehyde, which served as a photoacid generator for a photoinduced proton transfer. Upon ultraviolet (UV) irradiation, a spatiotemporally controlled gel−sol transition could be achieved owing to the decrease in pH inside the gel, resulting in breaking up the benzoxaborole−saccharide bindings. According to a recent patent application, benzoxaboroles can serve as cross-linkers for polysaccharides like guar.74 Such systems could be applied for example, for the thickening of wellbore fluids. 3.2. Medicinal Applications

Boron-containing biologically active compounds are known to bind to the active sites of enzymes by esterification as well as by strong noncovalent interactions such as dative bonds.21,51,75 Also for benzoxaboroles, the Lewis acidity of boron and diolbinding properties constitute the basis for their molecular mechanism of action. Upon specific binding of benzoxaboroles to the active site of an enzyme, its biological functions are impaired. As yet, the most remarkable molecular targets for benzoxaboroles have been found to be leucyl-tRNA synthetases. Benzoxaboroles inhibiting fungal,4 bacterial,76 and protozoal77 LeuRS synthetases were developed. Among other classes of enzymes, benzoxaboroles showed selective activity in inhibiting β-lactamases,32 HCV NS3/4A serine protease,78 PDE4 nucleotide phosphodiesterase,10 and D,D-carboxypeptidase.79 These enzymes are often key for different diseaserelated pathogenic processes to occur, which makes benzoxaboroles attractive candidates for enzyme-inhibition-based therapeutics. The following subsections will focus on the particular types of bioactivity of benzoxaboroles, showing the diversity of benzoxaborole libraries, briefly describing the proposed mechanisms of biological action, and discussing the medicinal G

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a serine present in the catalytic site.75 This results in the reversible formation of a tetragonal boronate adduct, a transition state analogue inactivating β-lactamase. In the search for novel β-lactamase inhibitors, a series of 6-hydroxybenzoxaborole derivatives was investigated (79−107, Figure 17).32

A promising example of an antibacterial oxaborole-based lead was the chiral benzoxaborole AN3365/GSK2251052 (109) (Figure 18). It proved to be an effective agent against Gramnegative bacteria, including E. coli and Pseudomonas aeruginosa (MIC values in the range of 0.5−4 μg/mL), with its mechanism of action based on the inhibition of bacterial LeuRS (E. coli LeuRS IC50 value of 0.31 μM).76 Compound 109 was found to bind to E. coli LeuRS not only via spiro ester formation by the oxaborole fragment, but also via hydrogen bonds between the 3-aminomethyl group and amino acids in the enzyme’s editing site (methionine and two aspartic acid residues).76 This showed that functionalized benzoxaboroles may additionally benefit from the possibility of multidendate interactions with the target site, e.g., formation of multiple hydrogen bonds. A combination of benzoxaborole 109 with norvaline and/or other amino acids was used to manage the development of resistance in bacteria exposed to benzoxaboroles.87 Detailed in vitro studies of its antibacterial activity against a broad range of anaerobic bacteria were reported,88,89 followed by the in vivo investigations of the pharmacokinetics of 109 in humans.90,91 The compound reached phase II clinical trials, which were, however, discontinued due to the development of bacterial resistance in a number of patients.92 Benzoxaborole ZCL039 (83, Figure 18) was found to be another antibacterial agent, being particularly active against Gram-positive Streptococcus pneumoniae, known as pneumococcus (MIC value of 5 μg/ mL).93 The detailed study also highlighted its mechanism of action as an inhibitor of LeuRS, with a S. pneumoniae IC50 value of 1.73 μM. Novel antibacterial agents were obtained by the incorporation of benzoxaborole motifs into three well-known antibiotics: vancomycin (derivatives 111−114, Figure 19) and eremomycin (115, Figure 19), as well as teicoplanin aglycon (116−122, Figure 20).94 The obtained conjugates proved to be

Figure 17. Benzoxaboroles tested as β-lactamase inhibitors.

The study showed the carboxylpyrazine-functionalized benzoxaborole 106 to be the most potent one, with low nanomolar Ki values for class C β-lactamases AmpC and CMY. Importantly, the use of compound 106 allowed the restoration of the activity of ceftazidime antibiotic toward β-lactamase-expressing bacterial strains. In another study inspired by serine proteases’ inhibition by boronic compounds, the unsubstituted benzoxaborole (1) was shown to inhibit D,D-carboxypeptidase R39, with the enzyme’s residual activity being 45% at 1 μM concentration of 1.79 The chiral benzoxaborole 108 (Figure 18) served as the model inhibitor in a reversible, continuous fluorescence assay for inhibition of E. coli LeuRS.86 The assay was successfully applied for the investigation of the inhibition kinetics, allowing for the measurement of the rate constants for the inhibition onset as well as the residence time of the inhibitor.

Figure 18. Chiral benzoxaborole 108 used in the LeuRS inhibition assay, its biologically active 7-substituted derivative 109 (AN3365/ GSK2251052), and the antipneumococcal benzoxaborole 83 (ZCL039).

Figure 19. Benzoxaborole-functionalized derivatives of vancomycin and eremomycin. H

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Figure 22. Benzoxaborole derivatives of danoprevir and vaniprevir.78 Figure 20. Benzoxaborole-functionalized derivatives of teicoplanin aglycon.

docked into HCV NS3 protease suggested that the benzoxaborole moiety can form polar interactions with threonine and lysine residues, rather than covalently bind to the active site of the enzyme.78 Acylsulfamoyl benzoxaborole analogs of danoprevir (123 and 124, Figure 22) and vaniprevir (125−127, Figure 22) have been obtained and their activity as HCV NS3/4A serine protease inhibitors evaluated. Though all the synthesized derivatives displayed inhibition activity in the HCV NS3/4A 1a enzyme assay (IC50 values of 0.3−0.8 nM), they were equipotent with danoprevir (IC 50 value of 0.4 nM). Interestingly, only a minimal influence of the position of the acylsulfamoyl substituent (4-, 5-, or 6-substitution in benzoxaborole ring) on the inhibitory activity was observed. In vivo tests in rats showed low absorption of the benzoxaborole derivatives and consequently their poor oral bioavailability. In order to improve the in vivo absorption, a series of nonmacrocyclic benzoxaborole-containing inhibitors (128− 139, Figure 23) has been obtained and studied as potential HCV NS3 protease inhibitors.100 Compounds 131 and 139 gave the best results in terms of their inhibition potency, with nanomolar activity in both an enzyme assay and cell-based replicon assay. In vivo studies in rats showed that the best absorption and oral bioavailability was exhibited by compound

effective against Gram-positive bacteria. Compound 111 showed even higher activity than the parent antibiotic (MIC values for seven vancomycin-susceptible Staphylococcus and Enterococcus strains in the range of 0.25−2 μg/mL, compared to 1−16 μg/mL for vancomycin). Interestingly, introduction of two benzoxaborole rings in compound 114 resulted in a substantially decreased activity. It is also worth noticing that teicoplanin aglycons 120−122 (Figure 20), with linkers between the glycopeptide and 6-aminobenzoxaborole fragments, were found to be active against vancomycin-resistant Enterococcus strains (MIC values in the range of 4−16 μg/mL, compared to >32 μg/mL for vancomycin, eremomycin, and teicoplanin aglycon). The study indicated that the spatial distance between glycopeptide and benzoxaborole may have an effect on the antibacterial activity of the conjugate. Extensive libraries of benzoxaboroles were patented as antibacterial agents, and their general structures (P1−P6) are presented in Figure 21.95−98

Figure 21. General structures of antibacterial benzoxaboroles P1,95 P2−P4,96 P5,97 and P6,98 claimed in patent applications in the recent years.99

3.2.3. Antiviral Activity. Benzoxaboroles have also been studied as antiviral agents, particularly against hepatitis C virus (HCV).78,100,101 The targeted biomolecule was HCV NS3/4A serine protease, an enzyme crucial for HCV replication. In this case, the strategy of drug development was based on functionalizing the already known HCV serine proteases inhibitors, like danoprevir and vaniprevir (Figure 22), with benzoxaborole units. The incorporation of boron was expected to lead to new compounds with improved potency. Modeling studies of a benzoxaborole inhibitor derived from danoprevir

Figure 23. Benzoxaboroles studied as HCV NS3 protease inhibitors. I

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134. A conclusion was drawn that the lack of a linker between benzoxaborole and the rest of the molecule was beneficial to bioavailability, as it decreased the molecular weight and polar surface area of the compound. Apart from benzoxaboroles, aliphatic α-amino cyclic boronates were also used for derivatization of both macrocyclic102 and nonmacrocylic103 HCV NS3 protease inhibitors. Extensive synthetic and biological studies yielded further libraries of potent macrocyclic HCV NS3 protease inhibitors with benzoxaborole scaffolds (140−155, Figure 24).101

Figure 25. Structures and compositions of the multivalent benzoxaborole-containing polymers.

tion-related cytokines’ release. PDE4 catalyzes hydrolysis of cyclic adenosine monophosphate (cAMP), a second messenger regulating immune responses. Inhibition of PDE4 results in accumulation of cAMP, activation of a metabolism-regulating protein kinase A, and inhibition of cytokines production.10 Since the excessive production of cytokines promotes inflammatory and immune responses, inhibition of their release is therapeutically beneficial.105 A series of phenoxybenzoxaboroles (3, Figure 1; 158−173, Figure 26) was synthesized and evaluated as inhibitors of PDE4

Figure 26. Phenoxybenzoxaboroles studied as PDE4 and cytokine release inhibitors.9 Figure 24. Macrocyclic benzoxaboroles studied as inhibitors of HCV protease.

as well as inhibitors of the release of proinflammatory cytokines, including tumor-necrosis factor α (TNF-α), interleukin-2 (IL-2), and interferon-γ (IFN-γ).9 The study revealed AN2728 (3, Figure 1) as the most promising lead for treatment of the inflammatory skin disorders psoriasis and atopic dermatitis. In 2014, it entered phase III clinical trials as a topical treatment of atopic dermatitis. AN2728 was found to inhibit PDE4 with IC50 value of 0.49 μM.9 The SAR studies showed that a 5-phenoxy group with an electron-withdrawing group (cyano or trifluoromethyl) at the para position was crucial to the high inhibitory potency. An additional cyano group installed at the meta position of the 5-phenoxy substituent additionaly enhanced the inhibitory activity toward PDE4 and cytokines release.10 The dicyano compound AN2898 (4, Figure 1) has also been in clinical trials (phase IIa results in 2011) for treatment of atopic dermatitis. X-ray crystallography studies of AN2898 (4) cocrystallized with the catalytic domain of PDE4B allowed the investigation of the binding mode of boron to the PDE4 active site.10 The crystal structure showed tetrahedral boron, datively bound to a water molecule held and activated between zinc and magnesium cations of the PDE4 bimetallic center. Within the

Interestingly, the product of oxidative deboronation of 155 has been also reported to display inhibitory activity toward HCV protease and gave rise to a new series of urea-based macrocyclic inhibitors.104 Another antiviral application of benzoxaboroles is their use as components of a polymeric entry inhibitor of the human immunodeficiency virus (HIV).46,54 Multivalent polymer 156 (Figure 25a), including copolymerized water-soluble N-(2hydroxypropyl)methacrylamide (HPMA), showed antiviral activity at nanomolar concentrations. The material was developed as a promising gp120 glycan targeted HIV entry inhibitor.54 The benzoxaborole-modified polymeric material was further functionalized by introduction of the sulfonated regions (157, Figure 25b), which resulted in an improved performance as an HIV entry inhibitor.46 3.2.4. Anti-Inflammatory Activity. The mechanism of action of the majority of the developed anti-inflammatory benzoxaboroles is based on the inhibition of cyclic nucleotide phosphodiesterase 4 (PDE4) and the inhibition of inflammaJ

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same work, kinetic studies on the PDE4 inhibition were carried out, showing that AN2728 (3) and AN2898 (4) are reversible, substrate-competitive inhibitors. A common clinical problem with anti-inflammatory therapeutics based on PDE4 inhibition is posed by their systemic side effects, including emesis. In the search for novel, potent, benzoxaborole-based PDE4 inhibitors with low emectic activity, a series of carboxylic derivatives of 5-phenoxybenzoxaboroles and 5-pyridyloxybenzoxaboroles (168, 174−177, and 178− 184, Figure 27) was synthesized and evaluated in comparison

Figure 27. Analogs of AN2728 tested as PDE4 inhibitors106 (168− 184) and the anti-inflammatory benzoxaborole 185 (AN6414).107

with the anti-inflammatory lead AN2728 (3).106 Replacement of the cyano group with carboxylic esters or nitriles improved the inhibitory activity, whereas carboxylic acids were much less active. Pyridyloxy derivatives (178−184) were found to be better PDE4 inhibitors than the corresponding phenoxy compounds (168 and 174−177), except for the carboxylic acid derivatives. The most potent compound in the study was found to be 5-pyridyloxybenzoxaborole 179, with PDE4 IC50 value of 47 nM. The rapid hydrolysis of 179 in plasma (in vitro) as well as in mice (in vivo) afforded the practically inactive carboxylic acid 180. That observation, along with its low emectic activity confirmed in emesis tests in mice, made benzoxaborole 179 a good anti-inflammatory drug candidate with limited systemic side effects. The substituted 5-pyridyloxybenzoxaborole 185 (AN6414, Figure 27) was found to be another promising antiinflammatory lead with a mechanism of action based on the inhibition of PDE4 (IC50 value of 5.2 nM) and cytokine release (TNF-α IC50 value of 2.5 nM).107 Following the growing interest in the in vivo metabolite identification in early drug discovery, a detailed LC/MS/MS study of the in vivo rat metabolites of AN6414 was carried out.107 The major metabolic transformations were found to be oxidative deboronation, protodeboronation, oxidation, and sulfation. Such metabolic studies give a deeper insight into the tested compound’s metabolism, providing hints to possible modifications for stability improvement and supporting early safety assessment. A series of ether and thioether 6-substituted derivatives of benzoxaboroles (Figure 28, up to compound 203) was studied as cytokine release-inhibiting anti-inflammatory agents.108 Benzoxaboroles 198 and 202 were identified as the most potent inhibitors of cytokines, with the TNF-α, IL-1β, and IL-6 IC50 values in the range of 33−83 nM. Moreover, compound 202 showed a favorable pharmacokinetic profile, and it was regarded as a promising lead for the development of novel antiinflammatory agents. While AN3485 (202) inhibited proinflammatory cytokines, it was not found to inhibit PDE4 or other typical biological targets of anti-inflammatory action.108 Although further insights into the in vivo anti-inflammatory activity of AN3485 (202) confirmed its possible applications for the treatment of inflammatory diseases like rheumatoid arthritis, psoriasis, and atopic dermatitis,109 the mechanism of

Figure 28. Ether and thioether 6-substituted derivatives of benzoxaboroles,108 along with substituted 6-(benzoylamino)benzoxaboroles and their analogs,110 all studied as anti-inflammatory agents.

its action still needs to be investigated. The mechanism is also unclear for the anti-inflammatory agents identified within a series of 6-(benzoylamino)benzoxaboroles and analogs (86, 204−224, Figure 28).110 The most potent cytokine release inhibitor of the series, compound 221 (AN4161), showed TNF-α, IL-1β, and IL-6 IC50 values in the range of 0.19−0.50 μM. Libraries of benzoxaboroles showing cytokine-release-related anti-inflammatory activity (P7−P11, Figure 29) were also

Figure 29. General structures of anti-inflammatory benzoxaboroles P7111 and P8−P11,112 claimed in patent applications in the recent years.99

described in patent applications.111,112 The inflammatory diseases that could be potentially treated with those benzoxaboroles include psoriasis, atopic dermatitis, rheumatoid dermatitis, inflammatory bowel disease, asthma, and chronic obstructive pulmonary disease. A set of benzoxaboroles, consisting mainly of 5- and 6(aminomethylphenoxy)benzoxaboroles, was subjected to a kinome-wide screening for kinase inhibitors (225−233, Figure 30).113 The study revealed several benzoxaboroles capable of K

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Figure 30. Benzoxaboroles studied as potential ROCK inhibitors.113 Figure 31. Library of benzoxaboroles and their B-substituted analogs screened for possible HAT treatment.

inhibiting Rho-activated kinases (ROCK), enzymes known to strongly affect inflammation. The potent compounds (226, 227, and 230−232) showed sub-micromolar ROCK1 and ROCK2 IC50 values. The X-ray crystallography studies of benzoxaborole 227 (AN3484) cocrystallized with ROCK2 showed a novel binding mode for benzoxaboroles. The oxaborole fragment formed two hydrogen bonds with methionine and glutamic acid residues at the active site, with boron staying in its trigonal planar form. The next two hydrogen bonds were formed between the aminomethyl group of 227 and the asparagine and aspartic acid residues.113 Taking into account the role of ROCK in both smooth muscle contraction and inflammation processes, benzoxaborole-based ROCK inhibitors can be of potential use for the treatment of asthma.113 3.2.5. Antiprotozoal Activity. A comprehensive review, highlighting advances in benzoxaboroles as potential treatments for human African trypanosomiasis, malaria, and Chagas disease, was published in 2011.17 Hence, only a short introduction is given here, followed by the description of the works published since. Human African trypanosomiasis (HAT), also known as African sleeping sickness, belongs to the so-called neglected tropical diseases.114 It is caused by the parasite Trypanosoma brucei transmitted by the tsetse flies. Due to several limitations of the known therapies, there is a great need for new, orally active drugs to safely treat HAT that are effective against all known strains of the parasite, especially in the second stage of HAT. Identification of several lead compounds and subsequent screening of the library of benzoxaboroles (Figure 31, up to compound 275) as well as their B-substituted analogs (Figure 31, 276−284) resulted in 5 and 273 as promising agents for HAT treatment.29 Compound 5 (SCYX-7158, AN5568), identified as a preclinical candidate for HAT treatment,11,115,116 was developed for phase I clinical trials in March 2012.117 Additional experiments showed that the presence of the heterocyclic boron atom is essential for the antiparasitic activity, as the corresponding carbon analogs and acyclic boronic acids are inactive against T. brucei (Figure 32).29

Figure 32. Analogs of benzoxaboroles inactive against T. brucei.

Several other 6-substituted benzoxaboroles (82, 83, 86, 88, and 285−289) have been studied against T. brucei (Figure 33). The highest inhibitory activity was showed by sulfonamide compound 287, with the reported T. brucei growth inhibition IC50 value of 0.02 μg/mL.118

Figure 33. 6-Substituted benzoxaboroles studied as agents against T. brucei.118

Chalcone−benzoxaborole hybrid molecules have been synthesized and investigated as potential anti-T. brucei agents. This project constituted a notable example of synergy between distinct classes of bioactive molecules, as separate antiprotozoal benzoxaboroles and chalcones had been known earlier on. Among over 40 studied species (290−335, Figure 34), L

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compound 334 displayed the highest activity with a T. brucei IC50 value of 10 ng/mL.119

Figure 36. C-6 and C-7 substituted benzoxaboroles investigated as potential LeuRS inhibitors.77

Benzoxaboroles 5, 213, and 382 (Figure 37) have been active against Trypanosoma cruzi, a pathogen causing Chagas disease, another neglected tropical disease.17

Figure 34. Chalcone−benzoxaborole hybrid molecules studied as antiT. brucei agents.119

Ding et al. showed that the antitrypanosomal benzoxaboroles can inhibit T. brucei LeuRS.77 By analogy with inhibition of fungal and bacterial LeuRS, the role of boron in the benzoxaborole system is to form a spiro ester adduct with tRNALeu at the T. brucei LeuRS editing site, thus impairing the protein synthesis within this protozoal pathogen. A binding pocket structure guided design of a series of 6-hydroxybenzoxaborole derivatives (338−373, Figure 35) led to the

Figure 37. Benzoxaboroles active against T. cruzi.17

Some benzoxaboroles display activity against Plasmodium falciparum, which causes malaria transmitted by mosquitoes.120 Screening of compounds 383−402 (Figure 38) revealed the

Figure 38. Benzoxaboroles screened for antimalarial activity,121 along with isotopically labeled species.123 Figure 35. Benzoxaboroles screened as potential LeuRS inhibitors.

high activity of 383, 397, and 400.121 7-Carboxyethylbenzoxaborole 383 (AN3661), with a P. falciparum IC50 value of 26 nM, has been identified as a preclinical candidate for treatment of malaria, and as many as four different strategies for its synthesis have been proposed (Scheme 21).122 Isotopically labeled benzoxaboroles 383a and 383b have been designed and obtained to support preclinical development studies of 383 as a potential antimalarial agent (Figure 38).123 The 13C- and D-labeled derivative 383a was used as an LC/ MS/MS internal standard in the pharmacokinetic studies of 383, while the 14C-labeled derivative 383b was used for an in vivo mass balance study of 383.

preparation of a library of potential benzoxaborole-containing antitrypanosomals.77 Alkyl ester and ketone substitutions at the 6-hydroxy group were favorable for the activity as T. brucei LeuRS inhibitors, while amide functionalization resulted in reduced potency. The highest inhibitory activity was found for compound 352, with a T. brucei LeuRS IC50 value of 1.6 μM. A comparative study showed that compounds substituted at C-7 (378-381, Figure 36) were in most cases less active as T. brucei LeuRS inhibitors than their C-6 substituted analogs (374−377, Figure 36). M

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Further studies of benzoxaboroles as antimalarial agents covered compounds 403−421 (Figure 39). Interestingly, the

Figure 41. Benzoxaborole-containing phenylalanines.25

Benzoxaboroles may be also applied as target delivery agents, since they display high affinity for pyranose sugars abundant in the glycocalyx covering mammalian cells. Bovine pancreatic RNase A functionalized with 6-aminobenzoxaborole (58, Figure 15) displayed enhanced affinity toward ganglioside GD3, a sialic acid-containing glycosphingolipid overexpressed on the surface of cancer cells. Moreover, the protein− benzoxaborole conjugate was found to exhibit a 4−5-fold increased cellular uptake (compared to nonboronated RNase A) into the cytosole of mammalian cells.18 Benzoxaborole 427 (Figure 42) has been used for the surface modification of an Figure 39. Chemical structures of benzoxaboroles screened against P. falciparum.

most active compounds were found to be fluorine-containing compounds 410, 415, and 421, with the highest inhibitory activity found for 4-fluoro-substituted benzoxaborole 421 (P. falciparum IC50 value of 26 nM).124 The effect of the presence of fluorine in pharmaceutical substances is complex and covers, among others, such aspect as its impact on acidity and liphophilicity, as well as the mechanism of action.125 It is worth mentioning that about 25% of the currently available drugs contain a fluorine atom,126 and so does KERYDIN (2), the first benzoxaborole-based drug. The antiprotozoal applications of a series of benzoxaboroles (P12−P14) and their O-functionalized derivatives (P15) were the subject of several patent applications (Figure 40).127−130

Figure 42. Benzoxaborole used for the surface functionalization of a nanotube carrier.131

amine-functionalized nanotube. Its introduction enabled penetration of the nanocarrier into human epithelial carcinoma cells and response to intracellular ATP.131 Along with the continuous development of benzoxaborolebased therapeutics, studies on their safety assessment have been conducted. Four enzyme-inhibiting benzoxaborolesantifungal AN2690 (2), anti-inflammatory AN2728 (3) and AN2898 (4), and antibacterial AN3365/GSK2251052 (109)were subjected to three in vitro and in vivo genotoxicological assays.132 The tests were negative, meaning no genetic toxicity was observed. AN2690 was additionally tested in an extended, 2-year in vivo bioassay, in which it also did not show any genetoxic effect. The results of the study indicate that there is no inherrent genetic toxicity associated with benzoxaboroles. Novel hybrid organic−inorganic formulations for the benzoxaborole drugs were very recently proposed.133 The materials were obtained by intercalation of 1 and 2 into a Mg− Al layered double hydroxide and characterized by powder X-ray diffraction and multinuclear (11B, 27Al, 13C, 19F, 25 Mg, and 1H) solid-state NMR. The maximum loading capacity toward benzoxaboroles and the optimal storage conditions, as well as release kinetics in simulated physiological media, were also determined.133

Figure 40. General structures of antiprotozoal benzoxaboroles P12,127 P13,128 and P14129 and the derivatives P15,130 claimed in patent applications in the recent years.99

3.2.6. Other Medicinal Aspects. Although to date there have been no reports on their anticancer activity, benzoxaboroles can potentially find applications in boron neutron capture therapy (BNCT). A series of benzoxaborole analogs of pboronophenylalanine, a boronated amino acid with high affinity for tumor cells and documented BNCT applications, has been synthesized (422−426, Figure 41).25 Replacement of the phenylboronic fragment of p-boronophenylalanine with a benzoxaborole was proposed to overcome the poor solubility of p-boronophenylalanine in water at physiological pH. The developed benzoxaborole-containing phenylalanine analogs 424 and 426 indeed showed improved solubility, which makes them potentially useful for BNCT applications.

3.3. Synthetic Utility of Benzoxaboroles

3.3.1. Reactions Aimed at Functionalizing Benzoxaboroles. Many novel benzoxaboroles have been reported as semiproducts in the synthesis of their biologically active derivatives.124 Their structures have been presented here to pertain to a variety of reported benzoxaboroles (390, 428−436, Figure 43). N

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Scheme 9. Reduction of 437 and Solution Equilibrium of the Resulting Product134

The formylbenzoxaborole 437, useful in all the presented methodologies, has been obtained by Ye et al., according to two different strategies (Scheme 10).134 Figure 43. Benzoxaboroles useful in the synthesis of bioactive derivatives.124

Scheme 10. Synthesis of the 7-Formylbenzoxaborole (437)a

Several research groups reported 7-formylbenzoxaborole (437) to be exceptionally useful in the synthesis of many benzoxaboroles (Scheme 7).124,134 Compound 437 has been further functionalized by the nucleophilic addition to the formyl group, followed by other transformations (Schemes 8124 and 9134). Scheme 7. Synthetic Utility of 7-Formylbenzoxaborole (437) a

Reagents and conditions: (a) ethylene glycol, TsOH, toluene; (b) NaBH4, MeOH; (c) MOMCl, DIPEA, CH2Cl2; (d) (1) n-BuLi, (2) B(i-PrO)3, (3) HCl, 44% overall yield; (e) DHP, TsOH, DMF; (f) (1) n-BuLi, (2) B(i-PrO)3, (3) HCl; (g) PCC/Celite, CH2Cl2, 56% overall yield.

Interestingly, 4-formylbenzoxaborole121 (452) and its regioisomers 453121 and 454118,121,134 have not been utilized that often so far (Figure 44).

Figure 44. Isomeric formylbenzoxaboroles.

More reactions aimed at functionalizing benzoxaboroles have been included in section 4 on the synthesis of benzoxaboroles. 3.3.2. Reactions Aimed at Complexation of Benzoxaboroles. The molecular structure of the aluminum− benzoxaborole complex 455 (Scheme 11) has been reported by Ma et al.135 Other complexes of benzoxaborole 1 with group 13 metalsaluminum (456), gallium (457, 458), and indium (459)have been recently obtained and their molecular and crystal structures measured by XRD.136 Magnesium and calcium complexes of 1 have been also prepared.133 An interesting example of exploiting benzoxaboroles’ reactivity is the selective and sensitive method for their (as well as of other organoboron species) qualitative and quantitative fluorescence-based detection.137 The method is based on disrupting the excited-state intramolecular proton transfer (ESIPT) photophysical process of 10-hydroxybenzo[h]quinolone (HBQ) upon complexation with boronic compounds. Noteworthy, HBQ solution was successfully implemented as a stain for TLC analysis of organoboron

Scheme 8. Synthesis of Phosphonate Benzoxaborolesa,124

Reagents and conditions: (a) CH2[P(O)(OEt)2]2, NaH, THF, 0 °C to rt, 10 h; (b) H2, 10% Pd/C, rt, 1 h; (c) KI, Me3SiCl, MeCN, rt, 10 h. a

Upon reduction, 437 transforms into 451, which displays an interesting equilibrium in CD3OD as well as D2O solution. No exchange was observed in DMSO (Scheme 9).134 O

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Scheme 13. Suzuki Coupling of Benzoxaborole 460140

Scheme 11. Reactivity of 1 with Organoaluminum, Organogallium, and Organoindium Compounds135,136

Scheme 14. Bis(benzoxaborole) 461 as Starting Material for Preparation of Chiral Auxiliaries and Ligandsa,142,143

compounds, highly selectively affording brightly fluorescent spots upon UV visualization after staining and drying. A very important advancement in the synthetic chemistry of benzoxaboroles was made by Raines and co-workers, who proposed 1-dimethylamino-8-methylaminonaphthalene as a protecting group for benzoxaboroles (Scheme 12).138,139 The

a

Reagents and conditions: (a) PhI, Pd(PPh3)4, Na2CO3, DME, H2O, EtOH, reflux, 51%; (b) H2O2, NaOH, H2O, EtOH, 30%; (c) 1cyclooct-1-enyl triflate, Pd(dppf)Cl2, DME, EtOH, Na2CO3, reflux, 21%; (d) Pd/C, EtOH, rt, quant.

Scheme 12. Protection of Benzoxaborole with 1Dimethylamino-8-methylaminonaphthalene

steps in assembling the building blocks for the synthesis of unprecedented dibenzo-9a-azaphenalenes (Scheme 15), useful for preparation of nitrogen-doped polycyclic aromatic hydrocarbons.141,144 Scheme 15. Benzoxaboroles Suzuki Couplings in the Synthesis of Azaphenalenesa,141 complexes were formed straightforwardly upon azeotropic removal of water. The protected benzoxaboroles were stable under basic conditions and moderately stable in the presence of anhydrous acids. The complexes were fluorescent, making them easily visualized under UV light (365 nm wavelength), as well as chromatographically stable. The protecting group showed good functional group compatibility and high stability under a range of synthetic conditions, including Suzuki−Miyaura and Buchwald−Hartwig palladium-catalyzed couplings and peptide synthesis, as well as with the use of strong reducing agents. This kind of protection is, however, not suitable for oxidative reactions, which is attributed to the susceptibility of diaminonaphthalene toward oxidation. The deprotection is achieved under aqueous acidic conditions. The 1-dimethylamino-8-methylaminonaphthalene protecting group can significantly extend the scope of transformations carried out on benzoxaboroles, therefore increasing their synthetic and application potential. 3.3.3. Reactions Leading to the Loss of the Benzoxaborole Function. Benzoxaboroles undergo transformations typical for other phenylboronic compounds, including Suzuki coupling140−143 and oxidation.142 Compound 460 has been applied in a Suzuki coupling with a vinyl iodide, resulting in the corresponding styryl sulfoxide in high yields (Scheme 13).140 Bis(benzoxaborole) 461 was obtained in the course of a study on bidirectional metalation of hydrobenzoin and subsequently used for the synthesis of chiral auxiliaries and ligands (Scheme 14).142,143 Suzuki coupling reactions involving benzoxaborole 1 or compound 462, containing two oxaborole units, were the key

a

Reagents and conditions: (a) (Pd(PPh3)4, K2CO3, toluene, EtOH.

Finally, reduction of the 3-amino-substituted benzoxaboroles affords Wulff-type receptors, as exemplified by the reactivity of the morpholinyl derivative 11 (Scheme 16).145 3.3.4. Potential Catalytic Application of Benzoxaboroles. Benzoxaborole 463 and its phenylboronic acid analogue have been examined as potential catalysts for the intraP

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4.1. Synthetic Methods Utilizing Boronic Pinacol Esters

Scheme 16. Reactivity of 3-Amino-Substituted Benzoxaboroles under Reducing Conditions145

The use of a boronic group in the form of a pinacol ester is a common case for the synthesis of benzoxaboroles. Four different neighboring groups have been used as hydroxymethyl group surrogates: formyl (method A), carboxymethyl (method B), acetoxy (method C), and methoxymethyl (method D) (Scheme 18). Scheme 18. Synthetic Methods for Benzoxaboroles Utilizing Boronic Pinacol Esters

molecular, ring-closing Michael reaction (Scheme 17). The investigated phenylboronic acid displayed high activity at Scheme 17. Boronic Compound as Potential Catalysts of Intramolecular Michael Reaction

Method A is the most common way to introduce a benzoxaborole moiety. It has been applied in the synthesis of 464,121 465,101,121 466−470,101,118 and 471−475124 (Scheme 19).

applied conditions (MeCN, 80 °C, 24 h, yield 99%), whereas no such activity was detected for 463 (yield