ABSTRACT The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis . Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

A novel series of isoxazoline benzoxaborole small molecules was designed and synthesized for a structure–activity relationship (SAR) investigation to assess the ectoparasiticide activity against ticks and fleas. The study identified an orally bioavailable molecule, (S)-3,3-dimethyl-5-(5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)benzo[c][1,2]oxaborol-1(3H)-ol (38, AN8030), which was long lasting in dogs (t1/2 = 22 days). Compound 38 demonstrated 97.6% therapeutic effectiveness within 24 h of treatment, with residual efficacy of 95.3% against American dog ticks (Dermacentor variabilis) on day 30% and 100% against cat fleas (Ctenocephalides felis) on day 32 after a single oral dose at 50 mg/kg in dogs.

A series of 6-hetaryloxy benzoxaborole compounds was designed and synthesized for a structure-activity relationship (SAR) investigation to assess the changes in antimalarial activity which result from 6-aryloxy structural variation, substituent modification on the pyrazine ring, and optimization of the side chain ester group. This SAR study discovered highly potent 6-(2-(alkoxycarbonyl)pyrazinyl-5-oxy)-1,3-dihydro-1-hydroxy-2,1-benzoxaboroles (9, 27-34) with IC50s = 0.2-22 nM against cultured Plasmodium falciparum W2 and 3D7 strains. Compound 9 also demonstrated excellent in vivo efficacy against P. berghei in infected mice (ED90 = 7.0 mg/kg).

Structure-activity relationships of 6-(benzoylamino)benzoxaborole analogs were investigated for the inhibition of TNF-α, IL-1β, and IL-6 from lipopolysaccharide stimulated peripheral blood mononuclear cells. Compound 1q showed potent activity against all three cytokines with IC50 values between 0.19 and 0.50μM, inhibited LPS-induced TNF-α and IL-6 elevation in mice and improved collagen-induced arthritis in mice. Compound 1q (AN4161) is considered to be a promising lead for novel anti-inflammatory agent with an excellent pharmacokinetic profile.

Gram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms in Escherichia coli and Pseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, including Enterobacteriaceae bearing NDM-1 and KPC carbapenemases, as well as P. aeruginosa. This novel boron-based antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious against E. coli and P. aeruginosa in murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.

Benzoxaboroles are a novel class of drug-like compounds that have been rich sources of novel inhibitors for various enzymes and of new drugs. While examining benzoxaborole activity in phenotypic screens, our attention was attracted by the (amino-methylphenoxy)benzoxaborole family, which potently inhibited Toll-like receptor-stimulated cytokine secretion from leukocytes. After considering their structure-activity relationships and the central role of kinases in leukocyte biology, we performed a kinome-wide screen to investigate the members of the (amino-methylphenoxy)benzoxaborole family. This technique identified Rho-activated kinase (ROCK) as a target. We showed competitive behavior, with respect to ATP, and then determined the ROCK2-drug cocrystal structure. The drug occupies the ATP site in which the oxaborole moiety provides hydrogen bond donors and acceptors to the hinge, and the aminomethyl group interacts with the magnesium/ATP-interacting aspartic acid common to protein kinases. The series exhibits excellent selectivity against most of the kinome, with greater than 15-fold selectivity against the next best member of the AGC protein kinase subfamily. Medicinal chemistry efforts with structure-based design resulted in a compound with a K-i of 170 nM. Cellular studies revealed strong enzyme inhibition rank correlation with suppression of intracellular phosphorylation of a ROCK substrate. The biochemical potencies of these compounds also translated to functional activity, causing smooth muscle relaxation in rat aorta and guinea pig trachea. The series exhibited oral availability and one member reduced rat blood pressure, consistent with ROCK's role in smooth muscle contraction. Thus, the benzoxaborole moiety represents a novel hinge-binding kinase scaffold that may have potential for therapeutic use.

The macrocyclic urea 2, a byproduct in the synthesis of benzoxaborole 1, was identified to be a novel and potent HCV protease inhibitor. We further explored this motif by synthesizing additional urea-based inhibitors and by characterizing them in replicase HCV protease-resistant mutants assay. Several compounds, exemplified by 12, were found to be more potent in HCV replicon assays than leading second generation inhibitors such as danoprevir and TMC-435350. Additionally, following oral administration, inhibitor 12 was found in rat liver in significantly higher concentrations than those reported for both danoprevir and TMC-435350, suggesting that inhibitor 12 has the combination of anti-HCV and pharmacokinetic properties that warrants further development of this series.

Pro-inflammatory cytokines play a critical role in the development of autoimmune and inflammatory diseases. Targeting the cytokine environment has proven efficient for averting inflammation. In this study, we reported that 6-[4-(aminomethyl)-2-chlorophenoxyl]benzo[c][1,2]oxaborol-1(3H)-ol (AN3485), a benzoxaborole analog, inhibited TLR2-, TLR3-, TLR4-, and TLR5-mediated TNF-α, IL-1β, and IL-6 release from human PBMCs and isolated monocytes with IC50 values ranging from 18 to 580 nM, and the inhibition was mediated at the transcriptional level. Topical administration of AN3485 significantly reduced PMA-induced contact dermatitis and oxazolone-induced delayed-type hypersensitivity in mice, indicating its capability of penetrating skin and potential topical application in skin inflammation. Oral administration of AN3485 showed dose-dependent suppression of LPS-induced TNF-α and IL-6 production in mice with an ED90 of 30 mg/kg. Oral AN3485, 35 mg/kg, twice a day, suppressed collagen-induced arthritis in mice over a 20-day period. The potent anti-inflammatory activity in in vitro and in vivo disease models makes AN3485 an attractive therapeutic lead for a variety of cutaneous and systemic inflammatory diseases.

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.

There is an increasing interest in in vivo metabolite identification in early drug discovery in order to (i) give a more complete picture of metabolic profile in investigational animal models, (ii) propose phase I and phase II metabolites using the same pharmacokinetic/toxicokinetic study samples, (iii) expose metabolically labile groups where chemical modifications could improve stability, and (iv) enable early safety assessment of metabolites. In the early discovery stage of our anti-inflammatory program, one novel benzoxaborole, AN6414, exhibiting both PDE4 enzyme and TNFα inhibition activities, became our primary candidate for further investigation. The traditional metabolite identifications usually require high dosed samples with long data scans and analysis. In this study, we conducted quick and more selective core-structure related precursor scans followed by daughter ion scans and identified a total of 10 major phase I and phase II metabolites using rat plasma samples from a toxicokinetic study at an oral dosing of 30mg/kg. Plasma samples were treated with solid phase extraction (SPE) prior to LC/MS/MS. An AB SCIEX API 4000 QTRAP mass spectrometer coupled with a Shimadzu LC system was used for LC/MS/MS analysis. We found the major metabolites of AN6414 to be oxidative deboronation, protodeboronation, oxidation products and their sulfate-conjugated species. This analysis drove analoging efforts which improved the pharmacokinetic profile, namely, lowering clearance and increasing exposure relative to AN6414. Toxicity predictions by the software program DEREK suggest the identified potential metabolites to be safe.

We have synthesized and evaluated a new series of acyclic P4-benzoxaborole-based HCV NS3 protease inhibitors. Structure–activity relationships were investigated, leading to the identification of compounds 5g and 17 with low nanomolar potency in the enzymatic and cell-based replicon assay. The linker-truncated compound 5j was found to exhibit improved absorption and oral bioavailability in rats, suggesting that further reduction of molecular weight and polar surface area could result in improved drug-like properties of this novel series.

PDE4 inhibitors are a validated approach as anti-inflammatory agents but are limited by systemic side effects including emesis. We report a soft-drug strategy incorporating a carboxylic ester group into boron-containing PDE4 inhibitors leading to the discovery of a series of benzoxaborole compounds with good potency (for example IC(50)=47 nM of compound 2) and low emetic activity. These compounds are intended for dermatological use further limiting possible systemic side effects.

A sensitive and high throughput off-line μElution 96-well solid-phase extraction (SPE) followed by strong cation exchange (SCX) liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for determination of cefepime has been developed and validated in mouse plasma. Using the chemical analog, ceftazidime as an internal standard (IS), the linear range of the method for the determination of cefepime in mouse plasma was 4–2048ng/mL with the lower limit of quantitation level (LLOQ) of 4ng/mL. The inter- and intra-assay precision and accuracy of the method were below 9.05% and ranged from 95.6 to 113%, respectively, determined by quality control (QC) samples at five concentration levels including LLOQ. After μElution SPE, 71.1% of cefepime was recovered. The application of the validated assay for the determination of cefepime in mouse pharmacokinetics (PK) samples after intravenous (IV) and subcutaneous (SC) doses was demonstrated.

HCV NS3/4A serine protease is essential for the replication of the HCV virus and has been a clinically validated target. A series of HCV NS3/4A protease inhibitors containing a novel acylsulfamoyl benzoxaborole moiety at the P1′ region was synthesized and evaluated. The resulting P1–P3 and P2–P4 macrocyclic inhibitors exhibited sub-nanomolar potency in the enzymatic assay and low nanomolar activity in the cell-based replicon assay. The in vivo PK evaluations of selected compounds are also described.

Due to severe chelating effect caused by N-hydroxylpyridone group of ciclopirox, there is no published direct HPLC or LC/MS/MS method for the determination of ciclopirox in any in vitro or in vivo matrix. Instead, the time-consuming pre-column derivatization methods have been adapted for indirect analysis of ciclopirox. After overcoming the chelating problem by using K(2)EDTA coated tubes, a direct, sensitive and high-throughput LC/MS/MS method was successfully developed and validated to determine the amount of ciclopirox that penetrated across the nail plate during in vitro nail penetration studies. The method involved adding a chemical analog, chloridazon as internal standard (IS) in K(2)EDTA coated tubes, mixing IS with ciclopirox in a 96-well plate and then proceeding to LC/MS/MS analysis. The MS/MS was selected to monitor m/z 208.0-->135.8 and 221.8-->77.0 for ciclopirox and IS, respectively, using positive electrospray ionization. The method was validated over a concentration range of 8-256 ng/mL, yielding calibration curves with correlation coefficients greater than 0.9991 with a lower limit of quantitation (LLOQ) of 8 ng/mL. The assay precision and accuracy were evaluated using quality control (QC) samples at three concentration levels. Analyzed concentrations ranged from 101% to 113% of their respective nominal concentration levels with coefficients of variation (CV) below 10.6%. The average recovery of ciclopirox from nail matrix was 101%. The validated method was successfully used to analyze the ciclopirox formulation and in vitro nail penetration samples.

A high throughput off-line μElution 96-well solid phase extraction (SPE) followed by liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for the determination of N-acetyl retigabine in dog plasma has been developed and validated. The method involves the use of μElution 96-well SPE for the simultaneous extraction of N-acetyl retigabine and rapid removal of its N-glucuronide metabolite that has shown to be problematic due to its instability using other clean-up methods. The μElution SPE technology eliminates the need for post-extraction solvent evaporation and greatly reduces sample preparation time consequently improving assay efficiency.