Accepted Manuscript Review The Unique Chemistry of Benzoxaboroles: Current and Emerging Applications in Biotechnology and Therapeutic Treatments C. Tony Liu, John W. Tomsho, Stephen J. Benkovic PII: DOI: Reference:

S0968-0896(14)00339-3 http://dx.doi.org/10.1016/j.bmc.2014.04.065 BMC 11560

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

4 March 2014 21 April 2014 30 April 2014

Please cite this article as: Tony Liu, C., Tomsho, J.W., Benkovic, S.J., The Unique Chemistry of Benzoxaboroles: Current and Emerging Applications in Biotechnology and Therapeutic Treatments, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.04.065

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The Unique Chemistry of Benzoxaboroles: Current and Emerging Applications in Biotechnology and Therapeutic Treatments C. Tony Liu†,§, John W. Tomsho⊥,§,*, Stephen J. Benkovic†,*

†

Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802

600 S. 43rd Street, Philadelphia, PA 19104, Department of Chemistry & Biochemistry, University of Sciences, Philadelphia, PA 19104-4495 ⊥

§

These authors contributed equally to this work.

* Authors to whom the correspondence should be addressed: (JWT) 215-596-7395, [email protected]; (SJB) [email protected].

Contents: 1. Introduction 2. Recent Progress in Synthesis 3. Molecular recognition and biotechnology 3.1 Materials applications 4. Therapeutic Applications 4.1. Leucyl tRNA synthetase inhibition 4.2. Other biological targets and applications 4.3. Trapping cysteine-sulfenic acids 5. Conclusions 6. Acknowledgments 7. References

Abstract Benzoxaboroles have garnered much attention in recent years due to their diverse applications in bio-sensing technology, material science, and therapeutic intervention. Part of the reason arises from the benzoxaboroles’ unique chemical properties, especially in comparison to their acyclic boronic acid counterparts. Furthermore, the low bio-toxicity combined with the high target specificity associated with benzoxaboroles make them very attractive as therapeutic agents. Herein, we provide an updated summary on the current knowledge of the fundamental chemical reactivity of benzoxaboroles, followed by highlighting their major applications reported to date.

1. Introduction Benzoxaboroles have utility as scaffolds important for molecular recognition, biotechnology, and various therapeutic applications. Benzoxaborole (1,3-dihydro-1-hydroxy-2,1benzoxaborole; CAS# 5735-41-1), less commonly benzoboroxole, consists of a benzene ring fused with an oxaborole heterocycle (Figure 1). The first synthesis of this compound was reported by Torssell in 1957[1]and subsequent studies revealed that this compound is water soluble and highly resistant to hydrolysis.[2] Until recently, this class of compounds has been largely ignored. This review will focus on the developments in the one year since a recent publication[3] focused on the areas of the synthesis and medicinal chemistry of this class of compounds (Section 2) and in the general field over the five years since the previous broad review.[4]

Figure 1. Compound structures and terminology.

Benzoxaboroles were briefly mentioned as potential mono-alcohol binding agents[5] but their general utility was not widely recognized until the Hall group discovered their ability to bind diols, especially sugars such as glucose and fructose.[6] They found that benzoxaborole has

higher affinities for the diol motif than other common boronic acid-based compounds such as phenylboronic acids or Wulff-type o-aminomethylphenylboronic acids (Figure 1).[7] Additional benefits of this class of compounds included good water solubility and tight binding to sugars which is maintained in aqueous solutions at neutral pH. This affinity and specificity for binding diol motifs has been extensively exploited for applications including sugar sensing (Section 3), the enrichment of glycosolated proteins (Section 3.1), and therapeutics (Section 4.1) to name a few. All boronic acids, with their empty p-orbitals, are Lewis acids where the neutral form adopts a trigonal planar geometry while the conjugate base is tetrahedral with the negative charge formally localized on the boron atom itself (Figure 2). This addition of water with the accompanying loss of a proton is responsible for their acid/base properties. The primary physicochemical difference observed between benzoxaborole and simple phenylboronic acid is the pKa; benzoxaborole has a pKa of 7.3 while phenylboronic acid is 8.7.[8] The source of this difference is the strain that is induced by the five-membered oxaborole ring when the boron atom has trigonal planar geometry. Upon water addition ring strain is relieved thus providing for the observed pKa depression.[9]This conclusion was supported by studies that examined the effects of both aromatic ring substitutions and oxaborole ring expansion and substitution, on measured pKa’s and sugar binding.[8]

Figure 2. Acid-base equilibrium of benzoxaborole.

Of central interest is benzoxaboroles’ ability to efficiently bind diols such as those found in sugars (i.e. ribose, fructose, and glucose)[6, 7] and 1,2 aromatic diols (i.e. catechol)[10] in aqueous media at neutral pH. With both reactions, the underlying chemistry consists of a sequential two-step process (Figure 3) that yields the cyclic boronate ester. The initial step is an intermolecular esterification followed by intramolecular ring closure with concurrent formation of a spiro adduct. The current consensus in the field is that the neutral, trigonal species is the predominant reactant for both phenylboronic acids and benzoxaboroles.[11]

OH + OH

OH B O

H2O

OH O

O B

H

O

O B O

Figure 3. Formation of a spiro adduct between a benzoxaborole and a diol. Due to the pKa diffrences, benzoxaboroles show an optimal pH for diol binding near neutral whereas the phenylboronic acids generally show increased binding as the pH increases to 10-11. Initially, it was proposed that the spiro adduct resulting from this diesterification could undergo acid- or base-dependent oxaborole ring opening to account for this narrow range of optimal pH for binding.[7] Recent studies have examined the reaction mechanism in more detail and have found no evidence of oxaborole ring opening.[10] The findings of the latter work are sumarized in Figure 4. The preferred reaction occurs at pH ~7 between species A1 and B1 yielding the ester P2, which can subsequently generate P4 with the rate of ring closure/opening on the order of seconds. At lower and higher solution pH, the reaction rates determined were much slower and the associated equilibria layed further towards reactants. A separate study, however, suggested that the adduct exists primarily in the tetrahedral form (P4) in water.[12] A linear relationship correlated the adduct formation constant (boron-diol adducts) and the pKa of the reacting species.

O

O

O Slow

OH O3S

A0

OH P1

OH

pK1 = 6.0

O B

O HO B

H+

pK1 = 4.6 H+ O

B1 Fast

O B

B O

O P2 O

O

O A1

P4 -OH

pK = 7.3

pK2 = 9.5 -OH

OH O3S

O

O

Slow

O HO B HO

Very Slow

O O B OH O

B2

P3

Figure 4. Proposed reaction scheme for the complexation between benzoxaborole and Alizarin Red S.[10]

2.Recent Progress in Synthesis The synthesis of benzoxaboroles has been reviewed in detail elsewhere.[3, 4] Briefly, positions 4- through 7-substituted benzoxaboroles (substitution on the aromatic ring; Figure 1) are most often synthesized from a corresponding substituted ortho-bromobenzaldehyde as illustrated in Scheme 1. The first route for boronic acid installation utilizes n-butyl lithium and triisopropyl borate followed by an acidic deprotection step for boronic acid installation. The second route involves formation of the C-B bond via direct Miyaura coupling[13] of bis(pinacolato)diboron to the aryl halide. In both cases, the oxaborole ring closure occurs spontaneously upon sodium borohydride reduction of the aldehyde to the corresponding alcohol when in the presence of the free boronic acid.[14] Scheme 1. A general synthetic scheme for preparing 4-7 substituted benzoxaboroles.

Of interest in the synthesis of 3-substituted benzoxaboroles is recent work by Kumar, et al.[15] With a number of different reaction protocols, the authors produced a large variety of novel benzoxaboroles from ortho-boronoaldehydes (Scheme 2) in synthetically useful yields (~40-80%). In this work, the susceptibility of aldehydes to nucleophilic attack to produce an αsubstituted alcohol was exploited by in-situ production of various nucleophilic carbon species. The highest yielding reactions utilized the Barbier allylation protocol for the coupling of allyl bromides (top). The middle reaction illustrates the production of α-amido benzoxaboroles with isonitriles via the Passerini reaction. Lastly, the Baylis-Hillman reaction, with a stoichiometric amount of DABCO base, was used to achieve production of an allyl-type substitution. Some aldol reactions were also pursued for the production of β-keto substituted benzoxaboroles but were deemed to be not synthetically useful due to the large amount of unreacted starting material recovered in all cases. Scheme 2. Alternative synthetic scheme for preparing 3-substituted benzoxaboroles.

Introduction of the benzoxaborole heterocycle has historically been positioned late in multistep reaction schemes due to the inherent reactivity of boron’s empty p-orbital and complications in isolation and purification. Recently, the Raines group has developed a divalent, charge-neutral protecting group designed specifically for benzoxaboroles.[16] Using easily prepared 1-dimethylamino-8-methylaminonaphthalene, protected benzoxaboroles (Figure 5) are synthesized, in high yields, after azeotropic water removal. The resulting complexes were found to be readily cleaved via treatment by aqueous acid, yet stable under basic and strongly reducing conditions. Additional benefits of this protecting group include compatibility with chromatographic purification, in part due to the neutral character of the complex, and visible fluorescence upon long wavelength UV illumination.

Figure 5. 1-Dimethylamino-8-methylaminonaphthalene derivative of benzoxaborole.

The use of benzoxaboroles and the related oxaboroles in synthetic methodologies has only recently begun to be explored. For example benzoxaboroles as metal ligands were recently investigated.[17] By the exposure of benzoxaborole to trialkyls of Al, Ga, and In, metal benzoxaborolates were prepared and characterized. Crystallographic analysis of these

compounds has revealed that the benzoxaboroles form two different types of complexes depending on the metal. The aluminum complex formed an M2B2O4 eight-membered bidentate ring system while the gallium and indium metals yielded an M2O2 four-membered ring similar to that formed with alcohols and other monodentate ligands (Figure 6). In both cases, the boron center maintained a trigonal planar geometry.

Figure 6. Metal containing benzoxaborole complexes. Moreover, the oxaborole heterocycle has been found as an intermediate in the synthesis of meroterpenoids.[18] The “borono-sclareolide” intermediate prepared by Dixon, et. al. is one in which the native lactone ring of sclareolide was replaced with the oxaborole heterocycle. This intermediate was then used in the direct formation of the desired product, chromazonarol (Figure 7) under strongly oxidizing conditions. This result was unexpected given the lability of boronic acids under oxidizing conditions and mechanistic studies to understand this process are currently underway.

Figure 7. Borono-sclareolide intermediate for the synthesis of chromazonarol.

3.Molecular recognition and biotechnology Many of the applications of benzoxaboroles in molecular recognition involve the development of improved carbohydrate sensors. In general, researchers are taking advantage of

benzoxaborole’s high affinity for sugar molecules at physiological pH. Some of the more promising results focus on multi-valency, where two or more binding units are arrayed with a specific geometry. The Hall group applied their discovery of efficient saccharide binding by benzoxaboroles to the construction of a peptidyl bis-benzoxaborole library (Figure 8) that would be used as a synthetic receptor.[19]The receptor was targeted against a disaccahride unit (Gal-β1,3-GalNAc) that is found on the surface of many tumor cells, the Thomsen-Friedenreich (TF) antigen. A 400-member library was assembled using standard solid phase peptide and split-pool synthetic methods. At a late stage of library production, 6-carboxy-benzoxaborole was coupled to the free amine from the diaminopropionic acid groups incorporated during backbone synthesis. The best candidate exhibited high selectively for TF-antigen with a 0.9 mM Kd, similar to the values reported with some naturally-occuring lectins. O B

O OH HO B

O

O

HN O H2N

(CH2CH2O)3CH2CH2

N H

HN O

H N O

N H

R'

O

H N O

N H

R"

Figure 8. Peptidyl benzoxaborole TF-antigen disaccharide receptor library that contains a soluble polyethylene glycol linker and an assortment of spacers (R’, 20 different substituted natural and unnatural amino acids) and capping groups (R”, 20 different (mostly aromatic) carboxylic acids).

The Maison group utilized an adamantanyl core to provide a rigid, tetrahedral scaffold for the synthesis of a trimeric benzoboroxole-based lectin mimic.[20] Figure 9shows the essential structure of these constructs. The linkers consisted of carboxylic acids connected to the adamantane via alkyl chains of various lengths. The carboxylic acids were then coupled with Lazidohomoalanine which allows the installation of 3-alkynyl-benzoxaborole via copper-catalyzed “click chemistry”. This scaffold has additional potential diversity due to the ability to couple other functional molecules via the adamantanyl amine and/or the remaining carboxyl groups on the linkers. A fluorescent molecule, PromoFluor, and a short peptide were coupled to these groups respectively as proof-of-principle to demonstrate further functionalization potential.

Figure 9. Trimeric benzoxaborole with an adamantanyl core.

In an attempt to increase the binding ability of synthetic receptors, Schumacher et al. introduced high multivalency via surface decoration of a nanoparticle with benzoxaborole groups.[21] Styrene and vinylbenzyl chloride monomers were polymerized to produce latex nanoparticles. Nucleophilic substitution reactions on the benzyl chloride groups with 3-aminophenylboronic acid and 6-amino-benzoxaborole were utilized for the installation of the binding groups. Isothermal titration calorimetry measurements were done to measure saccharide binding to free building blocks and the prepared and functionalized nanoparticles in aqueous solutions at pH = 7.4. It was determined that the free 6-amino-benzoxaborole bound fructose with a KB of 460 M-1 while 3-amino-phenylboronic acid’s KB was 210 M-1, approximately a two-fold increase. Also both types of decorated nanoparticles, benzoxaborole and arylboronic acid, bound fructose two times better than their free counterparts (KB = 1150 M-1 and 590 M-1 respectively) due primarily to multivalent binding effects. In addition to providing multiple binding groups randomly arrayed on a surface, binding affinity and specificity can be increased by arranging these groups in specific spatial orientations. This can be achieved by using the target molecule as a template during macromocular assembly. With D-fructose serving as the template, a benzoxaborole-containing monomer (Figure 10) was polymerized in the presence of the template molecule.[22] After removal of fructose from the polymer matrix via extensive washing, the imprinted polymer was assessed for its ability to bind fructose. At pH = 7.4, the benzoxaborole-polymer showed significantly improved binding capacity (~50% greater) over an analogously prepared phenylboronic acid control polymer. This polymer also showed a good selectivity for fructose over glucose or sucrose.

Figure 10. Benzoxaborole-containing monomer synthesized for the construction of a molecularly-imprinted polymer.

Recently a variety of 3-amino substituted benzoxaboroles (Figure 11) was prepared, characterized, and examined for their ability to complex with various diols.[23] It was observed that these compounds had pKa = 7.4, approximately equal to that of unsubstituted benzoxaborole and one unit lower than those known for other 3-susbtituted benzoxaboroles. The study showed that although the Lewis acidity of these compounds is equal to that of the parent benzoxaborole, their binding affinities for fructose, galactose, and glucose are generally weaker than those observed for benzoxaborole. Thus, the stability of the sugar-benzoxaborole adducts depends on more factors (e.g. steric effect and π-π interactions) than just the acidity of the reacting species.

Figure 11. 3-Amino substituted benzoxaboroles show a pKa of ~7.4 which is significantly lower than that of other 3-substituted benzoxaboroles.

3.1 Materials Applications Material scientists have also begun to take advantage of the high affinity of benzoxaboroles for sugars and other diols under neutral aqueous conditions. Liu and co-workers have reported a method to append benzoxaboroles to the surface of a monolithic capillary column for the chromatographic separation of various diols.[24] In their previous work with boronate affinity chromatography (BAC), the authors were frustrated with the need for alkaline conditions when using phenylboronic acid as a surface functionality.[25] Subsequently, 6carboxy-benzoxaborole was used to functionalize methylene bisacrylamide/glycidyl methacrylate polymer capillary monoliths via amide bond formation (Figure 12A). The columns prepared provided efficient chromatographic separation of a variety of nucleosides as well as efficient retention of model glycoproteins at neutral pH. These columns may also be useful in the selective enrichment of nucleosides and glycosylated proteins.

Figure 12. Benzoxaborole incorporated for A. affinity chromatography on monolithic capillary and B. glycoprotein enrichment with magnetic core-shell microspheres. A nearly identical approach has been applied to the rapid (10 min) enrichment of proteins that have been post-translationally glycosylated.[26] Beginning with a magnetic microsphere core coated with a shell of cross-linkedpoly (acrylic acid), standard solid-phase amide bond formation chemistry was used to functionalize the bead surface with a simple 6-aminobenzoxaborole (Figure 12B). Once prepared, these beads allowed the facile enrichment of model glycoproteins from various complex biological media. The glycoproteins are bound to these beads during a simple incubation step carried out at pH 7.4. Due to their magnetic properties, washing and recovery of these beads is efficient. Finally, by taking advantage of the reversible nature of the adduct formation between sugars and benzoxaboroles, the bound proteins may be released from the beads by lowering the pH of the solution. These applications mark significant improvements over previous approaches where phenylboronic acid[25] and Wulff-type boronate[27] moieties were used to attempt glycoprotein enrichment. “Smart” materials can respond to environmental cues with changes in their physical properties. Besides the above applications in chromatography and glycoprotein binding, benzoxaboroles have also been called upon for the development of these smart materials. Kim et al. were able to develop a stimuli-responsive polymer where the stimuli of interest was the presence of simple sugars.[28] Synthesis of a 6-vinyl substituted benzoxaborole enabled the direct incorporation of the benzoxaborole group into the polymer itself via reversible additionfragmentation and chain transfer (RAFT) polymerization. The block copolymers thus produced were found to have unique properties including sugar binding and self-assembly into macromolecular vesicle-like structures called polymersomes. Furthermore, it was found that the presence of sugars at neutral pH triggered the disassembly of these structures. The authors then proceeded to encapsulate insulin within these polymersomes and found that they could release

the enclosed insulin in response to the presence of sugar. Materials such as this may prove useful drug-delivery systems for sugar-related diseases. Another material has been reported where, in addition to a benzoxaborole-containing polymer, there is a co-mixed glyco-polymer.[29] It is thought that upon mixing, the benzoxaborole groups on chain spontaneously cross-link to sugar molecules appended to another polymer, the glycol-polymer. This cross-linking results in a controllable gelation of the polymers. These gels were found to be responsive to different types of external stimuli, specifically glucose concentration, pH, and temperature. When the gel is exposed to excess glucose or low pH, the inter-chain cross-links are disrupted and the gel dissociates.

4. Therapeutic applications Benzoxaboroles have emerged as a novel class of small molecule therapeutic agents, with a rapidly growing number of literature examples reporting their diverse potential applications, many of which are actively being explored by pharmaceutics. The cyclic five-membered oxaborole heterocycle of benzoxaboroles endows this class of compounds with unique bioactivities that are often absent in acyclic boronic acids or boric acids. As will become apparent in the following sections, small modifications to the benzoxaborole core structure can lead to potent therapeutic candidates for various human diseases, including fungal, bacterial, and viral infections, inflammation, cancer, and even for drug delivery.[4, 11, 30-33] Multiple academic investigations and clinical trials have shown that the benzoxaborole core itself has a low intrinsic toxicity.. Furthermore, benzoxaboroles and boronic acids are considered green or environmentally-friendly compounds because they ultimately decompose into boric acid whose toxicity is similar to table salt.[11, 32] Nonetheless, comprehensive metabolite identification is especially important in drug discovery and development because it helps to guide further optimizations such as improving the stability of the compound, aiding the planning of appropriate in vitro tests, and assessing potential toxic effects. A new LC/MS/MS analytic protocol was developed for rapid examination of the in vivo metabolites of some pharmaceutically interesting benzoxaboroles given to rats.[34] The results from that particular study confirmed again that the major metabolites of a benzoxaborole, being evaluated for its anti-inflammatory effect, to be boric acid and the oxidative deboronation products. In a separate study, it was found that boron-containing compounds have very low toxicity, specifically neurotoxicity. Very high doses were needed (>100mg/kg) to impair motor function in mice, again supporting the safety of boron-containing compounds as suitable therapeutic agents.[35] Studies have also shown that several benzoxaboroles in current clinical development have no genetic toxicology liability.[36] Collectively these studies demonstrate that the benzoxaborole moiety does not confer additional toxicity liability to the compounds that contain it, suggesting that the benzoxaborole core is a suitable building block for designing drug candidates. The major

targets and clinical applications of this unique class of compounds are discussed below. It should be noted that the realization of benzoxaboroles’ medicinal potential is relatively recent and most of the advancements have occurred within the last decade.

4.1. Leucyl tRNA synthetase inhibition Most examples of benzoxaboroles for medicinal applications are based on their ability to interfere with protein synthesis. Onychomycosis is a fungal infection which targets the nail plate or nail bed.[37, 38] The dermatophytes Trichophyton rubrum and Trichophyton mentagrophytes are the main pathogens as they are estimated to be the cause of 80-90% of reported onychomycosis cases.[38] Untreated, the infection leads to gradual deterioration of the nail plate and its separation from the nail bed. It has been estimated that onychomycosis affects ~10% of the worldwide population, and the occurrence rate doubles (~20%) for the >60 years old population while jumping to ~50% for people aged >70 years.[38-40] While onychomycosis is often considered a cosmetic problem, it can become a source of infectious lesions in other parts of the body in addition to causing serious complications when occurring in combination with other existing health issues such as diabetes.[41] Furthermore, onychomycosis can negatively impact both the physical and mental wellbeing of patients, leading to poor quality of life.[42] An individual suffering from onychomycosis can also pose a public health threat by acting as a fungal reservoir for spreading the infection to others. One of the most effective treatments against onychomycosis is benzoxaborole AN2690 (5-fluoro-1,3-dihydro-1-2,1-benzoxaborole; Tavaborole; Figure 13) which exhibits a broadspectrum antifungal activity and can penetrate nail plates easily. The ability to penetrate nail plates and the nail bed allows for the topical application of AN2690, distinguishing it from other available treatments for this disease.[43, 44] AN2690 has been shown to be effective against T. rubrum and T. mentagrophytes by targeting the fungal leucyl tRNA synthetase (LeuRS), which is necessary for the faithful biosynthesis of proteins.[31, 44] In studies, many benzoxaboroles have shown MIC (minimum inhibitory concentration) values in the low µg/ml against pathogenic organisms, including T. mentagrophytes and T. rubrum, while AN2690 is one of the more effective antifungal compounds.[30, 31, 44, 45]

Figure 13. AN2690 and AN2718. The mechanism of inhibition has been elucidated from crystallographic, biochemical, and chemical studies. Aminoacyl tRNA synthetases (RS) are a family of enzymes that are

responsible for attaching specific amino acids to the appropriate tRNAs. The ribosome then transfers the amino acids from the tRNAs onto the protein being synthesized. Leucyl tRNA synthetase (LeuRS) transfers leucine to the 3’-terminal nucleotide of tRNALeu. This RS enzyme has two activities, synthesis and proofreading, that occur in separate synthetic and editing sites (Figure 14). AN2690 has been shown to be a slow-tight binding inhibitor that selectively targets the editing domain of LeuRS, leading to mis-incorporation of amino acids into proteins and subsequent failure of normal protein production needed for cell survival.[46] X-ray crystallographic data have shown that AN2690 forms a stable spiro complex with the cis-diols of the terminal adenosine of tRNALeu bound to the tetrahedral boron center in the editing site of LeuRS (Figure 14). With a mechanism analogous to the general diol-benzoxaborole adduct formation described above, the boron atom forms a bidentate complex with the 2’ and 3’-cis diols of the terminal ribose of the tRNA, thus preventing the tRNA from moving into the synthetic domain of LeuRS, where the aminoacylation process takes place.[31] This complex is further stabilized by hydrogen bonds with the conserved threonine-rich peptide and a water molecule to generate a tight enzyme-benzoxaborole complex. This binding conformation is important for the enzyme inhibition, and it is often referred to as the oxaborole tRNA trapping (OBORT) mechanism.[11] It is well known that benzoxaboroles can form adducts with the cisdiols moiety of sugars,[6, 7, 12] and the bidentate tRNA-AN2690 complex shown in Figure 14 can only be derived from non-aminoacylated tRNA. Once formed, the inhibition complex is quite stable and has a half-life of approximately seven hours inside the active site.[31]

Figure 14. X-Ray crystal structure (PDB 2V0G)[31] depicting AN2690 forming a spiro complex with tRNALeu inside the editing site of T. thermophilus LeuRS (Left). The tRNA is in blue, the LeuRS is in pink, and the adenosine-AN2690 portion highlighted by the red arrow. Close up view of the adenosine-AN2690 adduct inside the editing active site (Right).

Structure-activity studies have shown that the five-membered ring structure around the boron is critical for the pharmaceutical activity of benzoxaboroles. Analogous 6-membered ring benzoxaborins or acyclic boronic acids exhibit significantly reduced antifungal activity in comparative biochemical assays.[31] The unique chemical reactivity of the 5-membered ring benzoxaborole is curious considering that both acyclic boronic acids and benzoxaboroles are

known to form adducts with monoalcohols and polyols in aqueous solution and the adduct formation constant is primarily based on the pKa values of the reacting compounds, not the structural architecture.[12] While benzoxaboroles are intrinsically more acidic than comparable acyclic boronic acids,[8] the pKa differences are relatively small while a large discrepancy in antifungal potency exists when comparing compounds with similar pKa values. Also, in neutral aqueous medium and without LeuRS, both benzoxaboroles and boronic acids can rapidly exchange monoalcohol and diol ligands due to the weak apparent association constants.[10, 12, 47]Thus, it is believed that the hydrophobic active site of the editing domain in LeuRS selectively stabilizes the benzoxaborole-tRNALeu complex by preventing the hydrolysis of borondiol bonds that would normally take place in bulk water.[44] However, the active site of LeuRS is less able to stabilize the cyclic boronate ester adduct formed with an acyclic boronic acid. Further structure activity studies also showed that small halogen substituents at the 5-position can improve the antifungal activity of the benzoxaborole compounds, presumably by increasing the boron-diol association constant.[44] Large substituents around the aryl ring of the benzoxaborole parent structure resulted in reduced inhibitory efficacy, possibly due to unfavourable steric/electrostatic restrictions in the crowded binding site. In early 2013, AN2690, being developed by Anacor Pharmaceuticals, received positive Phase III clinical trial results on ~600 patients, and later the same year the New Drug Application for this compound was accepted by the FDA for review.[48] The chloro-substituted derivative (AN2718; Figure 13) also exhibits good inhibitory effects against T. mentagrophytes and T. rubrum, while showing similar nail plate penetration as AN2690. Phase I trial data showed that AN2718 is a promising treatment for fungal infections of the skin due to its low irritation profile.[48] Further testing is expected in near future. The unique ability of benzoxaboroles to inhibit the function of LeuRS from other organisms allowed them to be pursued as a novel class of antibiotics. For example, GSK2251052 (Figure 15) was designed to target the LeuRS in Gram-negative bacteria, but it has also been shown to be effective against many Gram-positive bacteria.[45, 49] The mode of inhibition is the same OBORT mechanism described above, and the X-ray crystal structures of different benzoxaboroles bound to the editing site of bacteria LeuRS have been published.[50, 51] The 3aminomethyl moiety was found to yield significant improvement for the inhibitory efficacy, and this was attributed to favourable hydrogen bonding interactions with the polar residues (Asp342, Asp345, and Met336) in the proximity of E. coli LeuRS editing active site (Figure 15).[51] The MIC values determined for the antibacterial effect of GSK2251052 on Enterobacteriaceae are typically in the low ug/ml range. One of the advantages is that benzoxaboroles avoid a common antibiotic resistance mechanism that is associated with β-lactamases. GSK2251052 also exhibited no significant drug-related adverse events in healthy volunteers, but unfortunately its development was halted after a phase II clinical trial for treatment of urinary tract infections that revealed a small number of patients developing bacterial resistance to this drug.[48]

HO

O

OH B O

F GSK2251052

NH2

Figure 15. GSK 2251052 (left). Structure of a GSK2251052 analog (GSK2251052 without the F atom) bound as a spiro adduct inside E. coli. LeuRS (right; PDB 3ZJV).

Benzoxaboroles also offer new opportunities for treating other important and often neglected diseases. These are health issues that disproportionally impact people living in poorer and less privileged countries. Unfortunately due to the lack of financial incentives, developing treatments for these neglected diseases are a low priority for the pharmaceutical industry.[52] For example, human African trypanosomiasis (HAT), also referred to as sleeping sickness, is commonly caused by protozoan parasites Trypansoma brucei through the tsetse fly of the genus Glossina.[53] About 36 countries between latitudes 14o north and 29o south in the sub-Saharan Africa region are at risk of HAT, which is almost always fatal when untreated.[53] SAR screening has shown that many benzoxaboroles are potent inhibitors of T. brucei LeuRS, making them promising new antitrypanosomal agents.[15, 54-56] Guided by the knowledge of the T. brucei LeuRS active site structure, a series of 6-substituted benzoxaboroles (Figure 16) were designed and found to exhibit low µM IC50 (half maximal inhibitory concentration) values for T. brucei LeuRS. The SAR data were consistent with the structural analysis, which suggested that placing a substituent at the 6-position of the benzoxaborole aromatic ring should improve the binding affinity of the inhibitor by providing additional favourable electrostatic interactions in a well-defined binding pocket within the T. brucei LeuRS active site.[54] While the initial screening showed that alkyl ester and ketone substitutions at the 6-position yielded better inhibition than an amide-based substitution, SCYX-7158[56] (Figure 16) was found to be a very potent antitrypanosomal agent. SCYX-7158 (Figure 16) also exhibited desirable drug-suitability traits in pre-clinical studies, and it is currently in Phase I clinical trial.[48] A similar antitrypanosomal compound was prepared as the chalcone-benzoxaborole hybrid, where the chalcone moiety was inserted at the 6-position (Figure 16).[57] This is an interesting approach because many chalcones independently exhibit good antiprotozoal activity, and this approach linked two antitrypanosomal structures (benzoxaborole and chalcone) together to achieve greater efficacy.

Figure 16. Structures of 6-substituted benzoxaboroles (R = alkyl, amide, ketone), SCYX-7158, and a chalcone-benzoxaborole hybrid.

Malaria is another important tropical parasitic disease and it is caused by Plasmodium falciparum transmitted through mosquitoes. It is responsible for close to a million deaths each year, and the fatalities occur predominately in young children.[52, 58] Several benzoxaboroles with 7-carboxyethyl substituents (Figure 17) have been reported to have very potent antimalarial properties with IC50 values in the nM range.[59] Again, the boron atom is absolutely essential for the antimalarial effect, and replacing boron with carbon leads to the loss of inhibition. AN3661 currently is being developed as a new treatment for malaria.[48] It should be noted that further inhibition against Plasmodium falciparum can be achieved by introducing a single fluorine atom at the X, Y, or Z positions of AN3661 shown in Figure 17. As shown by the representative examples above, growing evidence suggests that benzoxaboroles can be effective for treating many widespread, deadly diseases that are often being overlooked.

Figure 17. Structures of AN3661 and its derivatives.

4.2 Other medicinal targets and applications The biological activity of benzoxaboroles is not limited to the inhibition of leucyl tRNA synthetase. They have also been shown to inhibit other enzymes, such as phosphodiesterases,[6062], β-lactamases,[63] and kinases.[64] Several substituted 6-phenoxybenzoxaboroles (Figure 18) were found to be potent class C β-lactamase inhibitors with low to sub micromolar Ki (inhibition constant) values.[63] The pyrazine derivative was found to be the most potent compound for inhibiting bacteria growth, but it appears that the carboxyl group might be a bigger contributor for the enhanced antibiotic activity. Furthermore, it was found that by inhibiting the

β-lactamase function in bacteria expressing β-lactamases (P99AmpC and CMY-2), the presence of the tested benzoxaborole restored sensitivity to ceftazidime.[63] Another approach for extending the antibacterial application of benzoxaboroles is to conjugate anti-bacterial benzoxaboroles with existing glycopeptides antibiotics. This approach is similar to the chalconebenzoxaborole hybrids discussed above. Printsevskaya et al. prepared 12 new hybrid antibiotics linking benzoxaboroles with vancomycin, eremomycin, or teicoplanin aglycone.[65] Overall, these new entities all exhibited excellent antibacterial properties against Gram-positive bacteria. More importantly, the teicoplanin aglycone-benzoxaborole conjugate was able to overcome the resistance of Gram-positive bacteria to vancomycin.

Figure 18. Substituted 6-phenoxybenzoxaboroles as class C β-lactamase inhibitors.

Moreover, various boronic acids have been found to inhibit the Staphylococcus aureus NorA efflux pump.[66] Efflux pumps provide a common resistance mechanism employed by bacteria where membrane transporters actively push toxins (e.g. antibiotics) out of the cell. Efflux pump based resistance is becoming a concern for the development of antibiotic resistance because, by lowering the cellular concentration of an antibiotic down to sub-lethal levels, the microorganism is more predisposed to develop mutations to overcome the specific antibiotic.[67] Fontaine et al. were able to show that several boronic acids are able to restore the antibiotic property of ciprofloxacin in a Staphylococcus aureus strain that over-expresses the NorA transporter.[66] While benzoxaboroles were not examined in that particular study, it is reasonable to think that they would also be good candidates to screen for efflux pump inhibition. Phosphodiesterase 4 (PDE4) is responsible for catalyzing the breakdown of 3’,5’adenosine cyclic monophosphate (cAMP) and it is ubiquitously expressed in inflammatory cells. Inhibition of PDE4 function has been shown to suppress the activity of human inflammatory cells, making PDE4 an attractive target for fighting various inflammatory diseases, such as asthma, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.[68] Psoriasis is a skin disorder caused by inflammatory cell infiltration of the dermis and epidermis. A series of phenoxy-substituted benzoxaboroles was found to exhibit good PDE4 inhibition,[60] and AN2728 (Figure 19) is among the most effective compounds identified for this purpose. It has passed Phase II clinical trials for topical treatment of psoriasis and atopic dermatitis.[48] The phenoxy-substituted benzoxaboroles tested exhibited good cell membrane permeability and effective inhibition of PDE4 as well as reducing inflammation-related cytokine release.[60] Kinetic studies revealed that these are competitive and reversible PDE4 inhibitors. Crystal structures showed that a water molecule is held in the bimetallic active site by the zinc and

magnesium ions while also coordinating to the boron atom thus generating a tetrahedral boron center. This type of interaction is different from other existing catechol-based PDE4 inhibitors such as Roflumilast (Figure 19) providing new avenues for further inhibitor optimization and design.[62] Phase III trials for the use of AN2728 on atopic dermatitis are expected to start in 2014.[48] In 2013, a series of 4-benzoylamino-substituted benzoxaboroles (Figure 20), which also show antiprotozoal activity, were reported to be good anti-inflammatory agents, presumably via the inhibition of PDE4 and cytokine release.[61] One of the compounds, AN4161, was found to significantly improve collagen-induced arthritis in mice. Another potential therapeutic application related to inflammation comes from several benzoxaboroles being identified as competitive inhibitors of Rho-activated kinases, which have been implicated in the inflammation and regulation of smooth muscle contraction in the vasculature and lungs.[64, 69] However, the utility of benzoxaborole-based Rho kinase inhibitors is unclear at this moment.

Figure 19. PDE4 inhibitors: AN2728 and Roflumilast.

Figure 20. 4-benzoylamino-substituted benzoxaboroles and AN4161.

Another important medical application of boronic acids/benzoxaboroles is for enhancing drug delivery into cells. This approach takes advantage of boronic acids’ affinity for diols and saccharides,[32, 70] especially those that coat the surface of mammalian cells. For example, the addition of a pendant boronic acid functional group to polyethylenimine has been shown to improve DNA transfection into cells.[71] More recently, Ellis et al. have demonstrated that pendant benzoxaboroles can be utilized to enhance the cytosolic delivery of polar macromolecule into mammalian cells.[72] The authors made an RNase A-benzoxaborole conjugate (Figure 21), which increased the cellular uptake of RNase A by 4-5 fold. It was also shown that the pendant boronate does not exert any cytotoxicity effect. Similarly, a supramolecular complex of a ~800 kDa GroEL mutant in a nanotubular structure functionalized with benzoxaborole groups showed

enhanced cell permeability.[73] These nanotubes were found to lack cell permeability until their surfaces were functionalized with benzoxaborole moieties.

Figure 21. Proposed mechanism for the conjugation between a benzoxaborole with RNase A, as well as the subsequent complexation step between the boron-RNase A adduct with cell surface glycan. It should be noted that there are 11 carboxyl groups on RNase A and each RNase can have multiple benzoxaboroles attached.

Besides facilitating the delivery of a biomolecule into the cell, Wang et al. utilized bovine serum albumin-poly(N-3-acrylamidophenylboronic acid) derived nanoparticles to significantly increase the accumulation of doxorubicin inside tumor cells by 16-fold when compared with free doxorubicin.[74] Doxorubicin, a DNA intercalator used in cancer chemotherapy, was loaded into the boronic acid decorated nanoparticles. The authors showed that this drug delivery strategy not only provided superior inhibition of tumor growth, it also induced distinct shrinkage and apoptosis of the tumor. In other words, the study showed that once a drug is loaded into nanoparticles, the efficiency of drug delivery into the cell can be significantly improved by functionalizing the surface of the carriers (nanoparticles) with boronic acids, presumably to increase the surface interaction between the carrier and the cell. This enhanced surface interaction can also be utilized to prevent entry of pathogens into cells. Boronic acid functionalized iron-, silica-, and diamond-derived nanoparticles were tested for their ability to block viral entry into hepatocyte cells.[75] While the boronated-nanoparticles showed only modest viral (hepatitis C virus) infection (~40% boost), they did show a reduction in cellular toxicity of the nanoparticle. Nonetheless, the authors were encouraged by the results and postulated that such an approach can be used to inhibit the infectivity of other pathogens that feature cell wall glycoproteins that are essential to their life cycle. The utility of this strategy will depend on the boronated nanoparticles’ ability to selectively bind and cover the surface of the pathogens, creating additional repulsion between the covered pathogens and healthy cells (Figure 22). In principle, benzoxaborole-decorated nanoparticles should work just as well, if not better, for the above purpose.

Figure 22. Potential use of boronated-nanoparticle to prevent pathogens from entering cell.

In fact, the use of a benzoxaborole coating to inhibit pathogen entry has been tested recently. It is thought that macromolecules binding to the glycoproteins of the viral coat may act as entry inhibitors thus preventing infection by viruses such as HIV. Polymers that specifically target the glycans that coat individual viral particles may be able to act in this fashion. The Kiser laboratory has recently synthesized[76] and evaluated the effectiveness[77] of a benzoxaborolecontaining polymer for potential use as a topical microbicide for HIV. The benzoxaborole monomer was assembled by coupling 6-amino-benzoxaborole and methacrylic acid via standard peptide coupling chemistry. Copolymers of varying benzoxaborole loading were achieved with a corresponding variation in the different monomer ratios in the feedstock during free radical polymerization. Polymers thus formed were found to have minimal toxicity, efficient carbohydrate binding at physiological pH, and ~1 nM EC50 for HIV entry inhibition. Other emerging potential applications of benzoxaboroles include their use as antiviral agents by inhibiting proteases[78-80] and in boron neutron capture therapy for cancer treatments[81, 82]. The antiviral applications are currently being explored by several pharmaceutical companies and research groups in terms of the possibility of using benzoxaboroles/boronic acids for inhibiting hepatitis C virus NS3 serine protease.

4.3. Trapping cysteine-sulfenic acids. Only recently has endogenous redox signalling become recognized as a prevalent signalling pathway that is responsible for cell proliferation and survival.[83-85] In the presence of reactive oxygen species, cysteine residues are prone to undergoing reversible oxidation first into sulfenic acids (Figure 23), which can influence various biological processes, such as signal transduction, balancing the intracellular redox state, catalysis, and gene transcription.[83, 85-90] Cysteine based sulfenic acids can also undergo further chemical modifications,[83, 90] to provide another level of control on biological activities. While sulfenic acids are typically

reactive, biological sulfenic acids can be very stable inside a protective protein microenvironment that shields the sulfenic acid from solvent molecules and nucleophiles.[83, 89] The rapidly growing number of examples in the literature suggest that the reversible oxidation of cysteine is an important post-translational modification, similar to protein phosphorylation. In fact, the formation of biological sulfenic acids has been implicated to play an important role in many health issues, such as cancer,[91, 92] heart disease,[93] and scurvy.[94] Thus, biological sulfenic acids represent a novel pharmaceutical target with great potential.

Figure 23. General structure of sulfenic acid, and the structures of dimedone and Fries acid.

Dimedone-based[83] (Figure 23) or analogous probes, such as β-ketoesters,[95] have become very useful tools for detecting biological cysteine-based sulfenic acids in vivo, and demonstrating the importance of cysteine redox chemistry in various biological functions. Despite the growing awareness of the vital regulatory roles cysteine-sulfenic acids can play, strategies for trapping sulfenic acids are otherwise currently scarce. It has been found that sulfenic acids can form reversible complexes with boronic acids/benzoxaboroles in aqueous media under physiologically relevant conditions.[96] Spectrophotometric, 11B-NMR, mass spectroscopy, and isothermal calorimetry data show that in the presence of a boronic acid/benzoxaborole, a stable sulfenic acid (Fries acid; Figure 23) predominately forms an anionic tetrahedral boronate species with a RSO-B bond (Figure 24). The experimental data also suggest that the neutral trigonal adduct level is very minor under physiological conditions. In the case of acyclic arylboronic acids, it seems that the pKa of the boronic acid has very little influence on the formation constant of the sulfenic acid-boronic acid adduct. This is different from the diolboronic acid adduct, where the pKa value of the boronic acid has a much more significant effect on the adduct formation constant.[70] Interestingly, this discrepancy might potentially be useful for a specific boron-compound to discriminate between diol/alcohol and sulfenic acid targets.

Figure 24. Proposed equilibria for the complexation of sulfenic acid with a benzoxaborole. Only one of the stereoisomers is shown for simplicity.

As proof-of-principle for the utility of using boronic acids to trap biological cysteinesulfenic acid, the catalytic activity of an iron-containing nitrile hydratase (NHase; EC 4.2.1.84) from Rhodococcus erythropolis was tested. NHase is an important biocatalyst for the mass commercial production of acrylamide and nicotinamide. The catalytic activity of the Fecontaining NHase depends on the presence of a cysteine-sulfenic acid (αCys114-OH) and overoxidation of the cysteine at this position into αCys114-O2 resulted in an inactive entity. It has been shown that the presence of the αCys113-OH sulfenic acid is essential for the enzymatic reaction. The catalytic role of the αCys113-OH has been proposed to either undergo the direct nucleophilic attack on the nitrile[97] or act as a general base for a nucleophilic water molecule.[87, 98]A simple enzyme activity assay showed that boronic acids and benzoxaboroles could inhibit NHase’s catalytic activity. More importantly, similar to the trend observed in the case of LeuRS,[31] the five-membered cyclic benzoxaboroles exhibit greater inhibition over acyclic arylboronic acids. Subsequently, Martinez et al. showed that two simple boronic acids, 1butaneboronic acid and phenylboronic acids, are extremely effective at inhibiting the catalytic activity of the Co-containing NHase from Pseudonocardia thermophila JCM 3095 (PtNHase).[97] For phenylboronic acids, they observed a slow-binding inhibition process that is reminiscent of the inhibition property of benzoxaborole with LeuRS.[31] Unlike the case with LeuRS, boronic acids are found to be competitive inhibitors of NHases, because they compete with the substrates for the same binding target, the αCys113-OH sulfenic acid in the active site. Once the boronic acid/benzoxaborole forms a covalent bond with the catalytic αCys113-OH, the sulfenic acid is no longer available to carry out the necessary step for the hydration of nitriles. Finally, very recent X-ray crystal data, refined at 1.2-1.6 Å resolution, confirmed that boronic acids do form a covalent bond with the αCys113-OH of the S-O-B nature inside the active site of wild-type PtNHase.[97] While NHase is not a clinically important target, this proof-of-principle demonstration, plus the low toxicity of boron-containing compounds, demonstrates that benzoxaboroles are a promising class of inhibitors for targeting sulfenic-acid containing proteins. It should be noted that cysteine sulfenic acids can be further oxidized into sulfinic acids, which also appear to conjugate with boronic acids/benzoxaboroles.[96] However, the biological

significance of cysteine-based sulfinic acids is not well understood and may simply be a product of over-oxidation of proteins.

5. Conclusion. Benzoxaboroles have been proven to be a unique class of compounds with very distinct chemical reactivity from acyclic aryl boronic acids. The diverse utility of benzoxaboroles has only been recognized recently, especially regarding their bio-analytic and medicinal applications. They have already been utilized for detecting biomolecules and for treating various health issues. The most exciting aspect of benzoxaboroles’ therapeutic potential is that they are very safe and thus provide a novel therapeutic pharmacophore for use against diseases where resistance is emerging to existing approaches. While the fundamental chemical reactivity between benzoxaboroles and diols/alcohols has been extensively investigated, there are still many unexplored potential interactions (e.g. sulfinic acid and nitrosothiols) which could warrant further studies. For example, the recent realization that benzoxaboroles can form reversible adducts with sulfenic acids suggest that benzoxaboroles might be useful inhibitors for biological targets that use cysteine redox chemistry to regulate their functions. The anticipated rapid expansion of benzoxaboroles’ applications should be of great interest.

6. Acknowledgement. C.T.L. would like to acknowledge postdoctoral fellowships from The Natural Sciences and Engineering Research Council of Canada.

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