Hemiboronic heterocyclic compounds and uses thereof

Abstract

Herein are provided compounds of Formula I in which X is O or NR3 where R3 is H, alkyl, optionally substituted aromatic, or optionally substituted heteroaromatic. Also provided are uses of the disclosed compounds as anti-infective agents, in particular as antibacterial or antifungal agents. In preferred embodiments, the compound is used for treating C. albicans or S. aureus in a subject.

Description

HEMIBORONIC HETEROCYCLIC COMPOUNDS AND USES THEREOFFIELD

[0001] The present disclosure relates generally to hemiboronic heterocyclic compounds and uses thereof. In particular, the present disclosure relates to antimicrobial agents, including antibacterials and antifungals.BACKGROUND

[0002] Identification of druggable chemotypes is needed to fight against challenging and neglected disease areas, including the ever-evolving threat of bacterial and fungal resistance.SUMMARY

[0003] A compound of Formula I:(Formula I) wherein:X is O or NR3; each R1 is independently H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, amine, -CN, -NO2, -B(OH)2,, optionally substituted aromatic, or optionally substituted heteroaromatic, and / or two adjacent R1 together forming a cycloalkyl, a heterocycloalkyl, an aromatic, or a heteroaromatic group;R2is H, alcohol, amine, thiol, -CN, -NO2, -B(OH)2, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, optionally substituted aromatic, or optionally substituted heteroaromatic;R3is H, alkyl, optionally substituted aromatic, or optionally substituted heteroaromatic;R4is H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, amine, -CN, NO2, or B(OH)2; andn is 0 to 5.

[0004] The compounds may be for use as antiinfective agents, such as antimicrobial, antibacterial, or antifungal agents. Also provided are uses of the compounds as antimicrobial agents, and for inhibiting C. albicans or S. aureus. Methods of treating infection may include administering to a subject a compound herein disclosed.BRIEF DESCRIPTION OF THE FIGURES

[0005] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures as described herein.

[0006] FIG. 1 is a chart showing the key differences between prototypic benzoxazaborine 1a and benzodiazaborine 2a and the four scaffold subclasses l-IV examined in this study.3(1 :1 CH3CN / aq. phosphate buffer)

[0007] FIG. 2 is an image showing tavaborole-adenosine complex bound to fungal leucyl-tRNA synthetase (2V0C). A) View into the binding pocket from the solvent. B) Focused view displaying the limited space around the oxaborole ring.DETAILED DESCRIPTION

[0008] Generally, the present disclosure describes expanding the role of boron in new drug chemotypes: properties, chemistry, pharmaceutical potential of hemiboronic naphthoids. Studies have shown that boron displays relatively low toxicity and can be integrated into drug development programs. The recent success of benzoxaborole drugs, their properties and diverse mechanisms of action, motivates the development of novel classes of heterocyclic hemiboronic acids. The compounds referred to as hemiboronic naphthoids herein may also be more generally referred to as hemiboronic heterocycles or benzo-fused hemiboronic heterocycles.

[0009] "Alkyl" refers to a group formed by removing one hydrogen from an alkane chain and is described by the formula CnH2n+i. The alkyl group may be a straight or branched chain. As used herein, “alkyl” may include alkane or alkene chains, or optionally substituted alkane chains including those substituted by alkenes or alkynes. The alkyl chains herein may be, for example CMO alkyl.

[0010] “Antiinfective” means useful in preventing infection.

[0011] “Antimicrobial” including antibiotics, antivirals, antifungals and antiparasitics - are medicines used to prevent and treat infections in a subject.

[0012] "Ar group" or “aryl group” refers to any functional group or substituent derived from an aromatic ring, usually an aromatic hydrocarbon, such as phenyl or naphthyl.

[0013] "Aromatic" refers to a chemical compound that consists of conjugated planar ring systems accompanied by delocalized pi-electron clouds in place of individual alternating double and single bonds. These are also called arenes. Aromatic compounds usually refer to organic compounds with a chemistry typified by benzene.

[0014] "Cx.y" refers to a group or compound having x to y number of carbon atoms. For example “C O” refers to a group or compound having 1 to 10 number of carbon atoms.

[0015] "Compound" refers to a chemical substance composed of many identical molecules containing atoms from more than one chemical element held together by chemical bonds.

[0016] "Cycloalkyl" refers to a univalent group derived from cycloalkane by removal of a hydrogen atom from a ring carbon atom and has the general formula of -CnH2n-i. A cycloalkyl is a cyclic version of an alkyl. Cycloalkyl is not aromatic. The cycloalkyl may be monocyclic, bicyclic, or multicyclic. The cycloalkyls herein may be, for example, C3-10 cycloalkyl.

[0017] "Double bond" refers to a chemical bond in which two pairs of electrons are shared between two atoms.

[0018] "Heteroalkyl" refers to a straight or branched chain including at least one carbon atom and at least one heteroatom (e.g. O, N, P, Si, or S). The heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. If the heteroatom is nitrogen or sulfur, the heteroatom may optionally be oxidized, or the nitrogen heteroatom may optionally be quaternized. Heteroalkyl is an uncyclized chain. The heteroalkyl may include one heteroatom, two or three heteroatoms, or more. If the heteroalkyl contains more than one heteroatom, the heteroatoms may be the same or different. The heteroalkyls herein may be, for example, C- O heteroalkyls.

[0019] "Heteroaromatic" refers to a compound or group with a ring structure which is both heterocyclic and aromatic. In other words, the heteroaromatic has the characteristics of an aromatic compound whilst having at least one non-carbon atom (e.g. O, N, S) in the ring. Heteroaromatics may include, but are not limited to, furan, pyrrole, thiophene, imidazole, oxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, quinoline, or isoquinoline.

[0020] "Heterocycloalkyl" refers to a cyclic version of a heteroalkyl. A heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule, or another position within the ring. Heterocycloalkyls may include, but are not limited to, piperidine, pyrrolidine, or tetra hydrofuran. The heterocycloalkyls herein may be, for example, C1.10 heterocycloalkyls.

[0021] "Pseudohalide" refers to a compound or ion that is not a halide, but which resembles a halide in its charge and reactivity. Examples of pseudohalides include but are not limited to triflate (OTf).

[0022] "Substituted" refers to an atom, group, or molecule which has a substituent. The substituent is one or a group of atoms that replaces (one or more) atoms, thereby becoming a moiety in the resultant molecule. The term substituent may be used interchangeably with functional group, side chain, or pendant group.

[0023] "Unsubstituted" refers to an atom, group, or molecule which does not have a substituent. In the case of an unsubstituted aromatic, for example, the aromatic group is defined as above, except that it has no substituent groups.

[0024] Herein provided are compounds of Formula I:(Formula I).

[0025] In Formula I, X may be O or NR3, where R3is H, alkyl, optionally substituted aromatic, or optionally substituted heteroaromatic. X may be O. X may be NH. X may be N-alkyl. X may be N-aryl. X may be N-heteroaryl.

[0026] In Formula I, each R1 is independently H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, amine, -CN, -NO2, -B(OH)2,optionally substituted aromatic, or optionally substituted heteroaromatic, and / or two adjacent R1 together forming a cycloalkyl, a heterocycloalkyl, an aromatic, or a heteroaromatic group, n is 0 to 5. In some embodiments, R1 may be H, methyl, ethyl, isopropyl, tert-butyl, methoxy, ethoxy, iso-propoxy, tert-butoxy, SMe, F, Cl, NO2, B(OH)2, OTf, or CF3.

[0027] In Formula I, R2is H, alcohol, amine, thiol, -CN, -NO2, -B(OH)2, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, optionally substituted aromatic, or optionally substituted heteroaromatic. In some embodiments, R2may be H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, optionally substituted aromatic, or optionally substituted heteroaromatic. In some embodiments, R2may be H, alkyl, cycloalkyl, optionally substituted aromatic, or optionally substituted heteroaromatic. R2may be H. R2may be alcohol (OH). R2may be amine (NH2, NRH, NR2). R2may be thiol (SH). R2may be alkyl. R2may be haloalkyl (such as CF3). R2may be heteroalkyl, cycloalkyl or heterocycloalkyl. R2may be an alkyl, heteroalkyl, aromatic or heteroaromatic group optionally substituted by alcohol, amine, thiol, -CN, -NO2, -B(OH)2, halide, pseudohalide, alkyl, heteroalkyl, halide, or pseudohalide. R2may be optionally substituted aromatic, such as an aromatic group optionally substituted by alkyl, heteroalkyl, halide, or pseudohalide. R2may be optionally substituted heteroaromatic, such as optionally substituted furan, pyrrole, thiophene, imidazole, oxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, quinoline, or isoquinoline.

[0028] In Formula I, R3is H, alkyl, optionally substituted aromatic, or optionally substituted heteroaromatic.

[0029] In Formula I, R4is H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, amine, -CN, NO2, or B(OH)2.

[0030] In some embodiments, the compound may be selected from the following formulae:

[0031] The compound may be a compound herein disclosed, such as a compound selected from the compounds of Tables 6, 7, 8, and 9.

[0032] The compound may be a compound selected from the following formulae:

[0033] The compound may be a compound selected from the following formulae:

[0034] The compound may be for use as an antiinfective. The compound may be for use as an antimicrobial agent, such as an antibacterial agent or antifungal agent. The compound may be for use in inhibiting C. albicans or S. aureus. The compound may be for use in treating a C. albicans or S. aureus infection in a subject.

[0035] Also disclosed herein is the use of the compound as an antiinfective. Use of the compound as an antimicrobial agent, such as an antibacterial agent or antifungal agent. Use of the compound for inhibiting C. albicans or S. aureus. Use of the compound for treating a C. albicans or S. aureus infection in a subject. The use of the compound may be in the manufacture of a medicament. The use of the compound may be in the manufacture of a medicament for treating an infection. The use of the compound may be in the manufacture of a medicament for treating a microbial (such as bacterial or fungal) infection (such as a C. albicans or S. aureus infection) in a subject.

[0036] Also provided are methods of treating an infection. The method may be a method of treating a microbial (such as bacterial or fungal) infection (such as a C. albicans or S. aureus infection) in a subject. The method may include administering to the subject a therapeutically effective amount of a compound herein disclosed.

[0037] Though specifically mentioned, C. albicans and S. aureus are not the only microbial infections the present compounds may be useful for. Other species within the genus Candida and Staphylococcus may be inhibited by a compound herein disclosed. Other strains of interest may be, for example, C. auris, or A. baumannii.

[0038] Method of the invention are conveniently practiced by providing the compounds and / or compositions used in such method in the form of a kit. Such kitpreferably contains the composition. Such a kit preferably contains instructions for the use thereof.

[0039] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in anyway.

[0040] EXAMPLES

[0041] EXAMPLE 1 : Expanding the Role of Boron in New Drug Chemotypes: Properties, Chemistry, Pharmaceutical Potential of Hemiboronic Naphthoids

[0042] 1. INTRODUCTION

[0043] The World Health Organization considers ever-evolving antimicrobial resistance to be a significant global health threat, for which there is a lack of innovative new antimicrobials. The development of new classes of boron heterocycle-based antimicrobials, with unique modes of action, could be an important step towards combating resistance. Compounds such as tavaborole, a topical antifungal, acoziborole, an antiprotozoal compound in late-stage clinical trials for the treatment of African tryptanosomiasis, and DNDI-6148, an antiprotozoal compound that successfully completed phase I clinicals trials for the treatment of leishmaniasis, are compelling benzoxaborole-based examples of antimicrobials. However, metabolic instability and high renal clearance have been common challenges in the development of boron-containing therapeutics, for example tavaborole is limited to topical applications due to metabolic instability, leaving significant room for improvement of the existing scaffolds.

[0044] Though much less studied than other hemiboronic heterocycles, the boronaphthoids 1-2 (Figure 1) may offer new opportunities as non-classical drug chemotypes. In a recent comprehensive study, our laboratory showed that benzoxazaborine 1a is chemically related to benzoxaboroles owing to its moderate acidity (pKa5.5 for 1a vs 7.4 for benzoxaborole) along with a boron-containing ring devoid of appreciable aromatic character. In contrast, the aza analog 2a displays partial aromatic character and a significantly higher pKa, making it distinct from benzoxaborole, and more akin to a naphthoid isostere (cf. Figure 1).

[0045] The potential of naphthoid boroheterocycles to expand upon the success of benzoxaboroles and offer new chemical space in drug discovery has been underexplored. In this study, we explore four subclasses of benzoborines and develop small model libraries of derivatives. The entire library was screened for antifungal and antibacterial activity.

[0046] 2. RESULTS

[0047] To encompass a range of structural features and physical properties, four general subclasses of hemiboronic naphthoids were selected for development in this study. Both oxaza (1a) and diaza (2a) scaffolds are further subdivided into aldimine (I and III) and ketimine (II and IV) subclasses (cf. Figure 1).

[0048]

[0049] Aldimine derivatives 1a and 2a were synthesized as described before through a condensation of 2-formylphenylboronic acid with hydroxylamine or hydrazine, whereas heterocycles 6a and 7a (phenyl ketimine analogs of 1a and 2a, respectively) were synthesized by following a three-step procedure (Scheme 1).

[0050] Scheme 1 . Three-step procedure for the synthesis of model compounds 6a and 7a.

[0051] 2.1 Mimetics of isoleucyl-tRNA synthetase inhibitor tavaborole

[0052] Tavaborole is an antifungal agent used clinically for the treatment of onychomycosis, an infection of the nail bed. Its unique mode of action involves the formation of a covalent tetracoordinate adduct between the boron center of the oxaborole moiety and the diol of a tRNA 3’ terminal adenosine. This complex, which inhibits the fungal leucyl-tRNA synthetase by blocking the enzyme’s editing site essential for protein synthesis, is crucial to antifungal activity. Accordingly, the pKaof the hemiboronic acid moiety, which describes the propensity of the boron center to become tetracoordinate, is a key characteristic of a replacement scaffold. A moderate acidity enables access to both the neutral and ionized species under biological conditions. For this reason, the benzoxazaborine subclass I, with its pKaof 5.5, was expected to be a more suitable replacement of the benzoxaborole system compared to benzodiazaborine, which has a pKa> 14. To this end, the 4-fluoro tavaborole analogs 1b and 2b (subclasses I and III), along with the benzoketimines 6g and 7b (subclasses II and IV), were synthesized by an early-stage approach (cf. Scheme 2 and 3, vide infra).

[0053] To assess the suitability of oxazaborine and diazaborine ring systems as oxaborole isosteres in the context of tavaborole mimicry, the analogs were assessed for antifungal activity against C. albicans (Table 1). As expected from their stronger boron acidity, the benzoxazaborine analogs 1b and 6g (subclasses I and III) maintained better antifungal activity (MIC = 8-16 pM) in comparison to the benzodiazaborine analogs (subclasses II and IV) (MIC > 128 pM). However, the oxazaborine analogs remain inferior to tavaborole, which displays an MIC of 1 pM. This small loss of activity may be due to destabilizing electrostatic interactions caused by the additional N atom, or to limited space in the enzyme's binding pocket to accommodate the increased steric demand of the 6-membered oxazaborine ring (see Figure 2). Nonetheless, this comparative study of tavaborole mimetics confirms the key importance of boron's Lewis acidity.

[0054] Table 1 . Antifungal Activity of Tavaborole Analogs Against C. albicans

[0055] 2.2 Early-stage synthetic approach for benzoborine analogs

[0056] In the early-stage approach towards functionalized analogs, a three-step procedure similar to that discussed above was employed (cf. Scheme 1). Specifically, most of the aldoxime derivatives (subclasses I and III) were synthesized from commercially available 2-bromobenzaldehyde precursors whereas all the ketimine analogs (subclasses II and IV) were prepared from substituted 2-bromobenzophenones. Several derivatives with functional groups ortho, meta, or para to the boronyl substituent were synthesized and resulted in a good to moderate yield over three steps (Scheme 3 and 4). Following a Pd-catalyzed borylation and condensation with hydroxylamine or hydrazine, aldoxime derivatives 1a-1e and ketimine derivatives 6a-6i and 17 were isolated. Some analogs (1a, 1b, 1e, 17, 2a, and 2b) were prepared from commercially available arylboronic acids with the appropriate ortho-formyl or ortho-acyl substituent. Ofnote, the fluorine-substituted analogs 1b and 2b are the tavaborole isosteres of subclasses I and III. The hydroxy-substituted analogs (1d and 2c) were synthesized from the commercially available 5-hydroxy-2-bromobenzaldehyde. A crisaborole analog 1c was prepared by first performing an SnAr reaction between 5-hydroxy-2- bromobenzaldehyde and 4-fluorobenzonitrile, followed by the usual sequence of borylation, deprotection, and hydroxylamine condensation. As described in the next subsection, functionalized analogs, such as 1a, 1b, 1d, 6c, 6f, 6i, 17, 2c, 2d, 7c, and 7d, were further utilized to prepare other derivatives via a late-stage synthetic strategy.

[0057] Scheme 2. Library of analogs of 1a (subclasses I and II) prepared using an early-stage approach. Yields are reported over three steps, unless stated otherwise.

[0058] Scheme 3. Library of analogs of 2a (subclasses III and IV) prepared using an early-stage approach. Yields are reported over three steps, unless stated otherwise.

[0059] 2.3 Late-stage synthetic approach for benzoborine analogs

[0060] Late-stage derivatization is an important strategy in drug discovery since it can produce a library of compounds in a highly divergent manner from just one judiciously functionalized precursor. In the context of heterocycle subclasses l-IV, late-stage derivatization implies any modification performed post borylation and heterocycle formation. For example, a late-stage approach was explored to achieve a chemoselective reduction of the oxime moiety leading to saturated ring analogs. Other conventional reactions, such as amidation, chemoselective Chan-Lam, and Suzuki-Miyaura crosscoupling were also selected to demonstrate the versatility and diversification of the benzoborine scaffolds.

[0061] It was envisioned that hydrogenation of the oxime moiety in 1a could provide a new, saturated scaffold that may demonstrate properties akin to benzoxaboroles. After an extensive screening of hydrogenation reagents and catalysts, Luche reduction conditions were initially identified (Figure 11 A). In order to confirm that the reaction resulted in the formation of the desired reduction product, the amide derivative 20, prepared from 19, was recrystallized (Figure 11 B). X-ray crystallographic analysis of a suitable crystal confirmed hydroxylamine 19 as the reduction product, thus excluding the possibility of N-0 cleavage products.

[0062] Scheme 4. A) Luche reduction of 1a and 17a. B) Synthesis of amide 20 from 19.

[0063] Alternative oxime reduction reactions were explored, including the use of an Ir catalyst for the hydrogenation of oximes to hydroxylamines. Catalyst 21 was found to be very efficient for the chemoselective reduction of 1a, generating hydroxylamine 22 as a methanesulfonate salt with nearly quantitative conversion (Scheme 5). Other substrates such as 17a and 1b (tavaborole analog) also yielded the respective reduction products (23 and 24).

[0064] Scheme 5. Hydrogenation of oximes 1a, 17, and 1 b using Ir catalyst 21 .

[0065] An amide group was introduced in subclass ll / IV benzoborines by reacting the corresponding amino derivatives (6i, 7c, and 7d), with various acyl chlorides in NMP, which acts both as a mild base and as a solvent (Scheme 6 and Scheme 7). The acyl chlorides that were selected embody a variety of functional groups, such as halogens, methoxy, nitro, trifluoromethyl groups, and heterocycles. Furthermore, since fluorine is increasingly recognized as an important element in drug discovery, acyl chlorides containing fluoroaryl groups were also chosen.

[0066] Scheme 6. Amidation reaction of the benzoxazaborine aniline precursor 6i, along with additional examples employing both early- and late-stage approaches.

[0067] A few analogs of acoziborole (25j, 25k, and 26) were also synthesized by combining early-stage approach and late-stage reduction of the corresponding nitroarene substrate followed by the amidation reaction.

[0068] Scheme 7. Amidation reaction of benzodiazaborine aniline precursors 7c and 7d, along with additional examples employing both early- and late-stage approaches.

[0069] The same amide functionalization chemistry was applied to subclass IV benzodiazaborines (Scheme 7). Thus, anilide derivatives 27a-27d were prepared from 7c, while derivatives 27e and 27f were synthesized from 7d. As with the benzoxazaborines (Scheme 6), a few analogs of acoziborole (28g-28i) were synthesized by following the analogous route of late-stage nitro reduction of the corresponding heterocycle followed by the amidation reaction. In this case, the reduction of the nitro group of precursor 2d (cf. Scheme 3) was accomplished with classical H2 / Pd-C conditions.

[0070] Chemoselective Chan-Lam coupling was performed on the hydroxy derivative 2c. To this end, the reaction of 2c with arylboronic acids in the presence of CU(OAC)2and Et3N resulted in the formation of ether containing analogs 29 of subclass III heterocycles (Scheme 8). A small subset of arylboronic acids with various para substituents were examined as coupling partners. Both electron donating (29b) and electron-withdrawing groups (29c-29e) were tolerated under the reaction conditions. Notably, the crisaborole analog 29c discussed above was synthesized using this strategy.

[0071] Scheme 8. Chan-Lam reaction between 4-hydroxy benzodiazaborine 2c and arylboronic acids.

[0072] An analog was prepared by treating a methoxy-functionalized analog of benzoxazaborine 6f with a strong Lewis acid, BBr3, which resulted in clean conversion to hydroxy product 30 without affecting the boroheterocycle (Scheme 9). A Chan-Lam reaction was performed on 30 with 4-cyanophenylboronic acid, which resulted in the formation of the desired ether product (31).

[0073] Scheme 9. Ether cleavage of 6f with BBr3, followed by Chan-Lam reaction to afford crisaborole analog 31.

[0074] The Suzuki-Miyaura cross-coupling between organoborons and organic halides is a powerful transformation enabling the mild and selective assembly of carboncarbon bonds. A chemoselective Suzuki-Miyaura cross-coupling procedure was developed for the synthesis of a variety of biaryl analogs of subclass l / lll benzoborines by reacting their corresponding triflate precursors 32 and 34 with arylboronic acids (Scheme 10 and 11. The coupling of 32 (a subclass I substrate) with a boronic acid proceeded sluggishly. In contrast, the Suzuki-Miyaura cross-coupling of subclass III benzodiazaborine 34 was cleaner. To assemble a small sublibrary of biaryl derivatives, arylboronic acids were chosen that contained a variety of functional substituents, such asether, fluoro, and chloro that are generally found in bioactive molecules. Arylboronic acids containing electron-withdrawing or electron-donating groups were all well tolerated and resulted in the formation of products in modest to good yields. Heterocyclic boronic acids were included, resulting in the desired products (33i, 33j, 35i, 35j). In the end, two diverse sublibraries of biaryl analogs of subclass l / lll heterocycles were prepared and readied for biological activity screening.

[0075] Scheme 10. Suzuki-Miyaura cross-coupling of triflate-functionalized benzoxazaborine 32 with arylboronic acids.

[0076] Scheme 11. Suzuki-Miyaura cross-coupling of triflate-functionalized benzodiazaborine 34 with arylboronic acids.

[0077] 2.4 Biological evaluation of prototypic libraries for antimicrobial activity

[0078] Derivatives of all four subclasses l-IV were assembled using a diversity of transformations, and a preliminary assessment of the potential of these hemiboronic naphthoids to display biological activity was planned.

[0079] The goal of this preliminary study, therefore, is to assess the suitability of the benzoxaza- and benzodiazaborine scaffolds l-IV as new classes of antimicrobials. To this end, the compound library was screened for antimicrobial activity against C. albicans, methicillin-resistant S. aureus (MRSA), and E. coli. Microbes were selected to encompass a broad spectrum of potential antimicrobial activity, and for their relevance in the clinic or to existing boron-containing drugs. Thus, the fungi C. albicans is relevant as a known target of tavaborole, whereas methicillin-resistant S. aureus (MRSA) was selected as a drug resistant and highly virulent example of Gram-positive bacteria. E. coli was selected as a representative Gram-negative example for its prevalence and history of GSK2251052 resistance. Library members were initially screened at 100 pM and assessed based on percent inhibition as well as modified Z-score. Based on this assessment, compounds were classified as hits (> 80% activity or Mi > 2.5), partial hits (50-80% activity), or inactive (< 50% activity). Hit compounds were then advanced to minimum inhibitory concentration (MIC) determination and select examples are discussed below. Since none of the compounds screened showed any activity against E. coli, only results of C. albicans and MRSA screening are discussed herein.

[0080] The escalating drug resistance of S. aureus is widely recognized as a growing problem with significant human cost. Methicillin was introduced as a S. aureus treatment in the 1960s in response to developing penicillin resistance. However, methicillin resistance began to emerge within a year. S. aureus strains resistant to methicillin and other p-lactam based drugs are now common in the clinic. Vancomycin is currently the preferred treatment for MRSA, and it was selected for the positive control accordingly. A selection of assay results are highlighted in Table 2. Broadly speaking, arylketoxime derivatives of 6a (subclass II) performed best against S. aureus. For example, 6g is more potent than 1b, and 27f is more potent than 28i. The methoxysubstituted derivative 6f was found to be the most potent library member against S. aureus, with an MIC of 8 pM (i.e., half the potency of vancomycin). Comparisons between subclass II compounds and the related subclass IV compounds illustrate the importance of boron’s Lewis acidity to antibiotic activity. For example, 6g displays an MIC of 64 pM, whereas the analogous benzodiazaborine structure 7b was found to be inactive at 100 pM. These results agree with the existing literature demonstrating that boronic and hemiboronic acids usually interact with biological targets in their conjugate base form (cf.Figure 2B).24Analogous to the trends seen for tavaborole analogs vs C. albicans (Section 2.3.3), the fluorinated analog 6g was more active vs S. aureus than the parent scaffold 6a. The current model library includes relatively few subclass II derivatives, however a comparison to compounds such as 6c and S6 (see S.l.) suggests that combining alkoxy substituents and derivatization of the ketoxime’s pendant aryl group may improve the potency (compound S6 was synthesized by a late-stage bromination reaction, see S.l. for details). Hence, future efforts to optimize potency via structure-activity relationship studies will focus on derivatization of the ketoxime aryl group.

[0081] Table 2. Minimum Inhibitory Concentrations (MIC) Against S. aureus for Select Library Members

[0082] A highlight of C. albicans screening with the most active library members is shown in Table 3. In contrast to the results of S. aureus screening, the most potent compounds are the subclass I aldoxime derivatives. The moderate Lewis acidity of the benzoxazaborine scaffold confers higher activity against C. albicans than the less acidic benzodiazaborine counterpart (for example, 1a vs 2a and 33g vs 35g), furtheremphasizing the importance of a tetracoordinate boron atom in target binding. Furthermore, as observed with S. aureus testing, tavaborole analogs were also more active against C. albicans than their non-fluorinated counterparts (for example 1a vs 1b and 6a vs 6g), further supporting that these heterocycles may be suitable mimics of benzoxaboroles. Contrasting the results of S. aureus testing, the pendant phenyl group of ketoxime subclass II derivatives appears to be detrimental to the inhibition of C. albicans (for example 1b vs 6g and 1a vs 6a). Amongst all the compounds tested, 33g was identified as the most potent, with an MIC of 125 nM approximately ten times more effective than tavaborole. The results of series 33 demonstrate the amenability to aryl substitution at the 3-position opposite the boron atom, including heterocycles (e.g., 33a- g). These exploratory results will guide future optimization with a focus on derivatization of the aryl group, including alternate substitution patterns.

[0083] Table 3. Minimum Inhibitory Concentrations Against C. albicans for Select Library Members

[0084] 2.5 DMPK profiling

[0085] To assess the suitability of these benzoborine heterocycles for drug development, the parent molecules representing the four subclasses l-IV scaffolds, compounds 1a, 2a, 6a, and 7a, were subjected to a panel of DMPK (drug metabolism andpharmacokinetics) assays. The isosteric replacement of a C-C bond for a B-X bond is known to increase aqueous solubility and reduce lipophilicity due to the addition of a polar bond with atoms that can participate in hydrogen bonding. Thus, basic assays such as lipophilicity, solubility, and plasma protein binding were conducted to assess the overall drug likeness of the compounds. Membrane permeability and stability in the presence of human hepatocytes were selected as additional assays given that common challenges in the development of boron-containing drugs are poor oral bioavailability and rapid metabolism. The rapid metabolism of boron-containing drugs is generally attributed to the lability of the C-B bond, either by oxidative deboronation or protodeboronation. Accordingly, none on the boron-containing drugs currently approved by the US FDA are administered orally.

[0086] Results of the DMPK assays are summarized in Table 4, with comparison to controls and literature data for tavaborole where available. Overall, all four compounds showed promising profiles for consideration as bona fide drug chemotypes. When comparing compound subclasses l / lll to 11 / IV, a clear relationship between logD, logP, and pKacan be seen. Though all compounds demonstrate moderate lipophilicity in an unbuffered environment (logP) comparable to tavaborole, at a fixed pH of 7.4 (logD) compound 1a is much more hydrophilic than 2a. This difference arises because at a neutral pH, with a pKaof 5.5, compound 1a exists mostly in its water-soluble ionized conjugate base form. The representative subclass III ketoxime compound 6a finds a middle ground between 1a and 2a, with a low pKathat favors the ionized form at pH 7.4, but an extra phenyl ring to boost lipophilicity. All four compounds demonstrated good aqueous solubility approaching the assay’s upper limit of 400 pM, which is in agreement with the high aqueous solubility reported for tavaborole. As expected, the more lipophilic compounds 2a and 6a display relatively high plasma protein binding (~90%), whereas the more hydrophilic compound 1a demonstrates low plasma binding (~30%) highlighting the significant tunability of properties within the benzoxazaborine scaffolds (i.e., subclasses I and II). In comparison, tavaborole has a more moderate reported plasma protein binding of 66%, which may be a reflection of its intermediate pKaand lipophilicity in comparison to benzoborine subclasses l-IV. Compounds with high plasma protein binding tend to show reduced tissue distribution because the concentration of free compound at any given time is smaller. This behavior in turn means there is less compound available to interact with the target. On the other hand, since there is less of the free compound available to be metabolized, high plasma protein binding can lead to higher half-life for in vivo models.

[0087] To assess metabolic stability, the compounds were treated with human hepatocytes. Results are expressed as half-life (ti / 2), in vitro clearance (Cl,nwfra), and percentage hepatic blood flow (%Qh). The latter is a particularly revealing indicator, since it takes into account species-specific factors, and can be used to compare results across species. In the current study, compound 1a showed a relatively low metabolic stability typical of hemiboronic acids such as tavaborole, with a %Qh greater than 75%. Compounds 2a and 6a however demonstrated remarkable metabolic stability with %Qh below the assay’s lower limit of 36.1%, maintaining stability for over three hours in the presence of human hepatocytes. Compound 7a showed an intermediate level of stability, further suggesting that the DMPK parameters of these heterocycles are not defined predominantly by the boron moiety and its pKa, but they can also be modulated by other structural determinants. Overall, these results demonstrate the variability in properties between the subclass l / ll benzoxazaborine scaffolds and provide encouraging signs for the exploration of oral drug dosing in future studies..

[0088] As an additional investigation of parameters relating to oral bioavailability, Caco-2 membrane permeability was determined as a marker of intestinal uptake. Of note, all compounds showed high recovery (83-94%) in these assays. The subclass III representative, compound 2a, demonstrated no active efflux despite relatively high rates of bidirectional permeability (Papp= 30 x106cm / s, efflux ratio of 1), suggesting that intestinal uptake of the compound would be high. The high permeability of benzodiazaborine 2a, paired with its high metabolic stability, makes for a strong profile of drug-like properties. Subclass l / ll benzoxazaborine representatives 1a and 6a both showed relatively low basolateral-to-apical rates (Papp= 4 x106cm / s) but high apical-to- basolateral rates (Papp= 35 xio6cm / s) resulting in an efflux ratio of ~0.1 . This observation indicates that the intrinsic permeability is moderate but that the compounds may be subject to a high rate of active influx. This situation would correspond to high intestinal uptake. Though there are boron-containing examples of efflux pump inhibitors, the apparent active intestinal influx of boron-containing compounds suggested in this work appears to be unique. Once again, the significant difference in Lewis acidity between subclass l / ll benzoxazaborines and subclass lll / IV benzodiazaborines can be taken into consideration. Benzoxazaborines such as 1a and 6a (pKa~ 5.5) may be subject to active transport in their anionic form, possibly mediated by a member of the organic anion transporter (OAT) or organic anion transporting polypeptide (OATP) family, which would explain why the model benzodiazaborines 2a and 7a (pKa> 14) are not subject to such active transport. The benzoxazaborines (1a, 6a) may also be subject totransport by OATs in a conjugated form, such as sulfate, glutathione, or cysteine conjugates. Clearly the membrane permeability of both benzoxazaborines 1a and 6a warrant further investigation to fully characterize the details of their unique active influx.

[0089] Table 4. Key DMPK Data for Representative Compounds of Subclass l-IV Scaffolds

[0090] 3. CONCLUSION

[0091] The identification of novel druggable functional groups and chemotypes can open new chemical space to address unmet therapeutic needs and the ongoing threat of bacterial multidrug resistance. Inspired by the successful benzoxaborole chemotype, this comprehensive study explored the suitability of four overlooked subclasses of the pseudo-aromatic hemiboronic naphthoids, benzoxaza- and benzodiazaborines. The lower pKaof benzoxazaborine subclasses l / ll makes those chemotypes more benzoxaborole-like compared to benzodiazaborine subclasses lll / IV. Facile ionization of benzoxazaborines l / ll in neutral aqueous medium favors high solubility and low logD, and it led to more hits in a preliminary antimicrobial screening evaluation possibly due to the enhanced ability of these moderately Lewis acidic boron heterocycles to interact in multiple ways with biological targets. However, the tendency ofbenzoxazaborines to ionize and coordinate Lewis bases creates adverse issues in the development of orthogonal transformations to derivatize and purify these subclass l / ll scaffolds. In contrast, the non-acidic nature of benzodiazaborine subclass lll / IV compounds greatly facilitated their orthogonal transformations in late-stage strategies.

[0092] Table 5. Summary of Key Properties and Drug Profile for Subclasses l-IV Benzoxaza- and Benzodiazaborines

[0093] To enable the orthogonal derivatization of subclass I— IV scaffolds through both early- and late-stage strategies, a series of synthetic reactions popular in drug discovery was developed along with effective purification procedures. It was found that late-stage amidation, Chan-Lam coupling, and Suzuki-Miyaura cross-coupling of suitably functionalized heterocycles provided modest to high yields of products with none or minimal cross-reactivity at the potentially susceptible B-C bond in the case of benzodiazaborine subclasses lll / IV. Due to their acidity and facile activation of the boronatom, reactions of benzoxazaborine subclasses l / ll provided lower, yet serviceable yields. Using these transformations, diverse libraries of functionalized analogs were assembled and tested against three model pathogens; the fungi C. albicans, with methicillin-resistant S. aureus (MRSA) and E. coll as respective models of Gram positive and Gram negative bacteria. This preliminary evaluation of the antibacterial potential of subclass I— IV chemotypes unveiled a number of promising hits with low micromolar MICs. Precisely, a number of subclass l / ll library members were active against MRSA with one analog as low as 8 pM MIC, whereas a 4-arylated series of subclass I benzoxazaborines displayed high potency with submicromolar activity greatly superior to tavaborole against the fungi C. albicans. These preliminary results provide clear directions towards future structureactivity relationship studies. Lastly, select DMPK assays for representative compounds of each subclass suggested promising drug-like behavior, with high metabolic stability, which is unique for boron-containing compounds, and excellent membrane permeability. Moreover, membrane permeability testing of benzoxazaborines suggests active influx and predicts a high intestinal uptake. Overall, the presence or absence of Lewis acid character in subclasses l-IV was found to exert a determinant influence on much of their properties and reactivity. In the case of subclasses l / ll, it enables the formation of the unique dihydroxyboryl functional group, which appears to favor target engagement. In closing, this study validates the merits of devoting efforts in fundamental medicinal chemistry research for the identification of novel chemotypes, especially those based on unconventional functional groups or atoms like boron.

[0094] Supplementary Information

[0095] 1. Protocols for Antimicrobial Work

[0096] 1.1 General

[0097] 96-well plates and lids were obtained from Fisher Scientific (Greiner Bio-One, 655161 and 656101). Control compounds were obtained from Millipore Sigma (Vancomycin HCI, V2002-100MG) (Colistin H2SO4, C4461) (Fluconazole, F8929). Cation- adjusted Mueller Hinton broth (Millipore, 90922) and Yeast Nitrogen Base (BD Difco, 233520) were obtained from Fisher Scientific and prepared as directed. Optical density was measured using a Molecular Devices SpectraMax i3x. Data analysis was completed as described using Excel 2016.

[0098] 1.2 One-Point Screening ProtocolPreparing test compounds: a. 100 mM stock solution: prepared in DMSO. Stored in the freezer when not in use. b. 1 mM working solution: dilute 10 pL of the 100 mM solution in 990 pL of water.c. 100 pM per well: add 20 pL of the 1 mM solution per well, and dilute in 180 pL of bacteria / fungi for a total volume of 200 pL.Preparing bacteria: a. Bacteria stock solutions are cultured in cation-adjusted Mueller Hinton broth (CAMHB) at 37 °C (incubate overnight, then store in the fridge). b. Dilute the stock solution 40-fold in fresh CAMHB and incubate at 37 °C for ~2.5 h until the bacterial growth is in the mid-log phase (OD600 ~0.5). c. Dilute the bacteria solution to 5.1 x 105CFU / mL in CAMHB, using the following calculation to determine the dilution factor.d. Add 180 pL of the bacteria solution per well, for a final density of 5 x 105CFU / mL and final volume of 200 pL.Preparing fungi (C. albicans, UAMH 3489): a. Fungi are kept on Yeast Extract-Peptone Dextrose (YPD) agar. b. Before the assay, fungi are freshly cultured on YPD agar at 30 °C for 3 days. c. Prepare media: combine 14 mL of water, 2 mL of 10x Yeast Nitrogen Base (YNB), 2 mL of 20% dextrose, and 2 mL of 0.1 M ammonium sulfate to create supplemented YNB media (YNB + 2% dextrose + 10 mM ammonium sulfate). d. Stock solution: gather a colony from the new agar plate and suspend it in 5 mL of media, then measure the OD530. Based on the OD, dilute the fungi to 2.8 x 103CFU / mL in media, using the following calculation:.S x 103CFV / mL'' add _ un / 3.67 x 107 CFU / mL.AUX OD530e. Add 180 pL of the fungi per well, for a final density of 2.5 x 103CFU / mL and final volume of 200 pL.Preparing positive controls: Vancomycin vs S. aureus (ATCC 43300) a. 100 mM stock solution: dissolve the 100 mg Vancomycin HCI (Sigma, V2002-100MG) standard in 673 pL DMSO. Stored as aliquots at -20 °C. b. 0.1 mM working solution: dilute 10 pL of the 100 mM solution in 990 pL of water (100x) for a 1 mM solution. Dilute 100 pL of the 1 mM solution in 900 pL of water (10x) for a 0.1 mM solution. c. 2 pM per well: add 4 pL of the 0.1 mM solution per well, and dilute in 196 pL of bacteria for a total volume of 200 pL.Preparing positive controls: Colistin H2S04vs E. coli (ATCC 25922) a. 100 mM stock solution: dissolve the 100 mg Colistin H2S04(Sigma, C4461) standard in 714 pL of water. Stored as aliquots at -20 °C. b. 0.1 mM working solution: dilute 10 pL of the 100 mM solution in 990 pL of water (100x) for a 1 mM solution. Dilute 100 pL of the 1 mM solution in 900 pL of water (10x) for a 0.1 mM solution. c. 1 pM per well: add 2 pL of the 0.1 mM solution per well, and dilute in 198 pL of bacteria for a total volume of 200 pL.Preparing positive controls: Fluconazole vs C. albicans (UAMH 3489) a. 100 mM stock solution: dissolve the 100 mg Fluconazole (Sigma, F8929) standard in 3.265 mL of DMSO. Stored as aliquots at -20 °C. b. 0.1 mM working solution: dilute 10 pL of the 100 mM solution in 990 pL of water (100x) for a 1 mM solution. Dilute 100 pL of the 1 mM solution in 900 pL of water (10x) for a 0.1 mM solution. c. 2 pM per well: add 4 pL of the 0.1 mM solution per well, and dilute in 196 pL of fungi for a total volume of 200 pL.Preparing the plates: a. Add the test compounds or positive controls to the plate as described above, along with the appropriate fungi / bacteria. Everything is done in triplicate. Final well volumes are 200 pL. b. Negative controls are just fungi / bacteria. c. Blanks are just media (CAMHB or supplemented YNB).d. Cover the plate and incubate without shaking at 37 °C for 18 h for bacteria and 24 h for fungi. e. Remove the plate from the incubator and read the OD600 (bacteria) or OD530 (fungi), with a 5 s orbital shake before reading.Note: C. albicans does not form a uniform suspension in the 96-well plates, even after orbital shaking. To obtain more accurate OD readings, the OD was measured with 9 points per well, distributed in an even 3x3 grid. The average of these 9 OD values was taken, and that average was used as the representative OD for the well in further calculations.Statistical analysis:34a. MAD (median absolute deviation): determine the median of the data set (OD600 / 530 values), calculate the absolute values of the deviations from the median, then determine the median of those absolute deviations. b. Mi (modified Z-score): Mt=x is the OD600 / 530 of a given well;Mi > |2.5| is considered active. c. % inhibition: %inhib = fl -Xl~pos) . ioo; a value above 80% is k NEG-POSj considered active, and from 50-80% is considered partially active.

[0099] 1.3 Minimum Inhibitory Concentration (MIC) DeterminationProtocolThe procedure remains the same as for the one-point screening, except for the following deviation in plating to achieve a range of concentrations for the compounds of interest:

[0100] A. Combine 51.2 pL of the 1 mM working solution and 348.8 pL of bacteria / fungi for a 128 pM solution in the first well.

[0101] B. Dilute the 128 pM well serially 1 :1 in bacteria / fungi for a final volume of 200 pL and final concentrations of 128, 64, 32, 16, 8, 4, 2, 1 , 0.5, 0.25, 0.125, and 0.0625 pM.

[0102] C. Each series of concentrations is done in triplicate.

[0103] 2. Antimicrobial Screening Results

[0104]

[0105] Table 6. Results for Compound Screening Versus S. aureus.

[0107] Table 7. Results for Compound Screening Versus C. albicans.EXAMPLE 2: C. albicans MIC Data

[0108] All compounds were synthesized following methods analogous to those previously disclosed.

[0109] All compounds were screened for antifungal activity against C. albicans(UAMH 3489), following methods analogous to those previously disclosed.

[0110] Table 8: C. albicans MIC Data for New Compounds

[0111] EXAMPLE 3: S. aureus MIC Data

[0112] All compounds were synthesized following methods analogous to those previously disclosed.

[0113] All compounds were screened for antimicrobial activity against S. aureus(ATCC 43300), following methods analogous to those previously disclosed.

[0114] Table 9: S. aureus MIC Data for New Compounds

[0115] The embodiments described herein are intended to be examples only.Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

[0116] All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0117] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modification as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula I:(Formula I) wherein:X is O or NR3; each R1 is independently H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, amine, -CN, -NO2, -B(OH)2,toptionally substituted aromatic, or optionally substituted heteroaromatic, and / or two adjacent R1 together forming a cycloalkyl, a heterocycloalkyl, an aromatic, or a heteroaromatic group;R2is H, alcohol, amine, thiol, -CN, -NO2, -B(OH)2, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, optionally substituted aromatic, or optionally substituted heteroaromatic;R3is H, alkyl, optionally substituted aromatic, or optionally substituted heteroaromatic;R4is H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, amine, -CN, NO2, or B(OH)2; and n is 0 to 5.

2. The compound of claim 1 wherein X is O.

3. The compound of claim 1 wherein X is NH.

4. The compound of any one of claims 1 to 3, wherein R1 is H, methyl, ethyl, iso-propyl, tert-butyl, methoxy, ethoxy, iso-propoxy, tert-butoxy, SMe, F, Cl, NO2, B(OH)2, OTf, or CF3.

5. The compound of any one of claims 1 to 4, wherein R2is H, halide, pseudohalide, alkyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, optionally substituted aromatic, or optionally substituted heteroaromatic.

6. The compound of any one of claims 1 to 5, wherein R2is an alkyl, heteroalkyl, aromatic or heteroaromatic group optionally substituted by alcohol, amine, thiol, -CN, - NO2, -B(OH)2, halide, pseudohalide, alkyl, heteroalkyl, halide, or pseudohalide.

7. The compound of claim 1 , selected from the following formulae:

8. The compound of claim 1 , selected from the compounds of Tables 6, 7, 8, and 9.

9. The compound of claim 1 , selected from the following formulae:

10. The compound of claim 1 , selected from the following formulae:11 . A pharmaceutical composition comprising the compound according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient, diluent, or carrier.

12. The compound according to any one of claims 1 to 10 or the composition of claim 11 , for use as an antimicrobial agent, such as an antibacterial agent or antifungal agent.

13. The compound according to any one of claims 1 to 10 or the composition of claim 11 , for use in inhibiting C. albicans or S. aureus.

14. The compound according to any one of claims 1 to 10 or the composition of claim 11 , for use in treating a C. albicans or S. aureus infection in a subject.

15. Use of the compound according to any one of claims 1 to 10 or the composition of claim 11 as an antimicrobial agent, such as an antibacterial agent or antifungal agent.

16. Use of the compound according to any one of claims 1 to 10 or the composition of claim 11 for inhibiting C. albicans or S. aureus.

17. Use of the compound according to any one of claims 1 to 10 or the composition of claim 11 for treating a C. albicans or S. aureus infection in a subject.

18. A Method of treating a microbial (such as bacterial or fungal) infection (such as a C. albicans or S. aureus infection) in a subject comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 10 or the composition of claim 11 .

19. Use of the compound according to any one of claims 1 to 10 or the composition of claim 11 in the manufacture of a medicament for treating a microbial (such as bacterial or fungal) infection (such as a C. albicans or S. aureus infection) in a subject.

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