crossmark

In Vitro and In Vivo Characterization of the Novel OxabicyclooctaneLinked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase Christopher M. Tan,a Charles J. Gill,a Jin Wu,a Nathalie Toussaint,a Jingjun Yin,b Takayuki Tsuchiya,c Charles G. Garlisi,a David Kaelin,a Peter T. Meinke,b Lynn Miesel,a David B. Olsen,c Armando Lagrutta,c Hideyuki Fukuda,d Ryuta Kishii,d Masaya Takei,d Kouhei Oohata,d Tomoko Takeuchi,d Taku Shibue,d Hisashi Takano,d Akinori Nishimura,d Yasumichi Fukuda,d Sheo B. Singha* Merck Research Laboratories, Kenilworth, New Jersey, USAa; Merck Research Laboratories, Rahway, New Jersey, USAb; Merck Research Laboratories, West Point, Pennsylvania, USAc; Kyorin Pharmaceutical Co., Ltd., Tochigi, Japand

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, and in vivo characterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli and displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergence in vitro at concentrations 16- to 32fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activity in vitro and in vivo.

N

ovel bacterial type II topoisomerase inhibitors (NBTIs) represent a new class of antimicrobial agents that display potent broad-spectrum antibacterial activity (1–11). NBTIs bind to and inhibit the essential bacterial enzymes DNA gyrase and topoisomerase IV at sites distinct from the fluoroquinolone-binding site; therefore, they retain activity against clinically relevant pathogens resistant to quinolones and other antibiotic agents. We have been systematically studying the NBTI class of DNA gyrase inhibitors and have recently reported several oxabicyclooctane-linked 1,5-naphthyridinyl-pyridoxazone NBTIs, including AM-8085 (Fig. 1, compound 1) and AM-8191 (Fig. 1, compound 2) (12), as well as several hydroxy tricyclic series of NBTIs (13, 14). However, development of NBTIs in general and this series in particular has been hindered due to existence of significant human ether-à-go-go-related gene (hERG) potassium channel activity. The molecules that inhibit hERG potassium channel lead to QT interval prolongation and sudden death. In the previous reports we have described modifications on all three structural moieties of NBTIs: left-hand side (LHS) (15, 16), right-hand side (17), and linker (18). We found that maximal modulation of the hERG activity was achieved from modifications of the DNA binding motif of the left-hand-side moiety, particularly introduction of a hydroxy tricyclic group (e.g., Fig. 1, compound 3) which encompasses an N-alkylated-1,5-naphthyridone unit. To understand the structural requirements of the LHS moiety, we prepared and profiled a simpler N-hydroxyethyl-3-chloro-1,5-naphthyridone NBTI, AM8722 (Fig. 1, compound 4). We report the in vitro and in vivo characterization of AM8722 and comparison with a hydroxy tricyclic compound

4830

aac.asm.org

(Fig. 1, compound 3) (13) as well as an amino-piperidine linked hydroxy tricyclic 1,5-naphthyridone (Fig. 1, compound 5) (19). The data demonstrate that AM-8722 specifically inhibited DNA replication and showed potent inhibitory activities against bacterial type II topoisomerases. AM-8722 was active against a panel of Gram-positive and Gram-negative organisms and exhibited favorable in vivo activity against Staphylococcus aureus in a systemic infection model, providing further proof of principle for the N-alkylated-1,5-naphthyridone LHS moietycontaining NBTI class of inhibitors as promising new scaffold antibacterial agents.

Received 17 March 2016 Returned for modification 16 April 2016 Accepted 25 May 2016 Accepted manuscript posted online 31 May 2016 Citation Tan CM, Gill CJ, Wu J, Toussaint N, Yin J, Tsuchiya T, Garlisi CG, Kaelin D, Meinke PT, Miesel L, Olsen DB, Lagrutta A, Fukuda H, Kishii R, Takei M, Oohata K, Takeuchi T, Shibue T, Takano H, Nishimura A, Fukuda Y, Singh SB. 2016. In vitro and in vivo characterization of the novel oxabicyclooctane-linked bacterial topoisomerase inhibitor AM-8722, a selective, potent inhibitor of bacterial DNA gyrase. Antimicrob Agents Chemother 60:4830 –4839. doi:10.1128/AAC.00619-16. Address correspondence to Christopher M. Tan, [email protected], or Sheo B. Singh, [email protected]. * Present address: Sheo B. Singh, SBS Pharma Consulting LLC, Edison, New Jersey, USA. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.00619-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Antimicrobial Agents and Chemotherapy

August 2016 Volume 60 Number 8

AM-8722, a Potent Bacterial DNA Gyrase Inhibitor

MATERIALS AND METHODS Synthetic procedure for AM-8722. (i) Synthesis of tert-butyl-(E)-(1-(2(3-chloro-6-methoxy-1,5-naphthyridin-4-yl)vinyl)-2-oxabicyclo [2.2.2]octan-4-yl)carbamate (compound 8) (step i). To a solution of 6-methoxy-4-bromo-3-chloro-1,5-naphthyridine (compound 6) (6.00 g) in benzene (73 ml) were added Boc-protected-vinyl oxabicyclooctane (compound 7) (5.56 g), silver carbonate (3.63 g), and palladium acetate (493 mg). The mixture was stirred at 100°C (bath temperature) for 3 days. After the insoluble materials were filtered off, the filtrate was concentrated under reduced pressure. The residue was triturated with ethyl acetate and filtered to give compound 8 (3.84 g) as a powder. The filtrate was concentrated under reduced pressure and flash chromatographed (ratio of silica, hexane to ethyl acetate ⫽ 3:1) to afford additional product (3.37 g; total, 7.21 g; 72%). 1H nuclear magnetic resonance (NMR) (400 MHz, CDCl3) ␦ 1.44 (s, 9H), 1.90 to 2.04 (m, 4H), 2.08 to 2.27 (m, 4H), 4.10 (s, 2H ⫹ 3H), 4.34 (brs, 1H), 7.07 (d, J ⫽ 9.2 Hz, 1H), 7.20 (d, J ⫽ 16.5 Hz, 1H), 7.38 (d, J ⫽ 17.1 Hz, 1H), 8.16 (d, J ⫽ 9.2 Hz, 1H), 8.62 (d, J ⫽ 2.4 Hz, 1H); MS (ESI⫹) m/z 446 (MH⫹); HRMS (ESI⫹) for C23H29ClN3O4 (MH⫹): calculated, 446.18446; found, 446.18499. (ii) Synthesis of tert-butyl-(1-(2-(3-chloro-6-methoxy-1,5-naphthyridin-4-yl)ethyl)-2-oxabicyclo[2.2.2]octan-4-yl)carbamate (compound 9) (step ii). A suspension of compound 8 (3.00 g) and 5% platinum on carbon (1.20 g) in methanol (330 ml) was stirred at room temperature for 3 h under an H2 atmosphere (3 kg/cm2). After completion of the reaction, insoluble materials were removed by filtration and filtrate was concentrated under reduced pressure. Flash chromatography (ratio of silica and hexane to ethyl acetate ⫽ 3:1) of the residue gave compound 9 (2.14 g; 71%) as a powder. 1H NMR (400 MHz, DMSO-d6) ␦ 1.36 (s, 9H), 1.53 to 1.62 (m, 2H), 1.69 to 2.02 (m, 8H), 3.18 to 3.27 (m, 2H), 3.78 (s, 2H), 4.02 (s, 3H), 6.60 (brs, 1H), 7.27 (d, J ⫽ 9.2 Hz, 1H), 8.26 (d, J ⫽ 9.2 Hz, 1H), 8.72 (s, 1H); MS (ESI⫹) m/z 448 (MH⫹); HRMS (ESI⫹) for C23H31ClN3O4 (MH⫹): calculated, 448.20031; found, 448.20072. (iii) Synthesis of tert-butyl-(1-(2-(3-chloro-6-hydroxy-1,5-naphthyridin-4-yl)ethyl)-2-oxabicyclo[2.2.2]octan-4-yl)carbamate (compound 10) (step iii). A solution of compound 9 (5.60 g) in 1,4-dioxane (60 ml) and 6 N hydrochloric acid (60 ml) was stirred at 60°C for 40 h. The mixture was adjusted to pH 12 by addition of sodium hydroxide while cooling in an ice bath. Boc2O (2.73 g) was added to the resulting mixture, and the mixture was stirred at 60°C for 6 h. The reaction was allowed to cool to the ambient temperature, and the resulting precipitates were collected by filtration and washed with ethyl acetate to give compound 10 (2.64 g) as a powder. The filtrate was extracted with chloroform (100 ml ⫻ 2), and the organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. Flash chromatography (ratio of silica and hexane to ethyl acetate ⫽ 1:2) of the residue gave the additional product (738 mg; total, 3.38 g; 62%) as a powder. 1H NMR (400 MHz, DMSO-d6) ␦ 1.34 (s, 9H), 1.43 to 1.56 (m, 2H), 1.62 to 2.03 (m, 8H), 2.93 to 3.07 (m, 2H), 3.82 (s, 2H), 6.61 (brs, 1H), 6.75 (d, J ⫽ 9.8 Hz, 1H), 7.90 (d, J ⫽ 9.8 Hz, 1H), 8.44 (s, 1H), 11.30 (s, 1H); MS (ESI⫹) m/z 434 (MH⫹); HRMS (ESI⫹) for C22H29ClN3O4 (MH⫹): calculated, 434.18466; found, 434.18422. (iv) Synthesis of tert-butyl-(1-(2-(3-chloro-5-(2-hydroxyethyl)-6oxo-5,6-dihydro-1,5-naphthyridin-4-yl)ethyl)-2-oxabicyclo[2.2.2] octan-4-yl)carbamate (compound 11) (step iv). To a solution of compound 10 (3.37 g) in N,N-dimethylformamide (78 ml) were added potassium carbonate (1.50 g) and 1-bromo-2-chloroethane (0.78 ml). The mixture was stirred at 50°C for 4 h. After compound 10 disappeared (by thin-layer chromatography [TLC]), the mixture was stirred at 100°C for 8 h and then concentrated under reduced pressure. Flash chromatography (ratio of silica and hexane to ethyl acetate ⫽ 1:4) of the residue gave compound 11 (2.10 g, 57%) as a powder. 1H NMR (400 MHz, CDCl3) ␦ 1.43 (s, 9H), 1.71 to 1.80 (m, 2H), 1.84 to 1.96 (m, 4H), 2.02 to 2.18 (m, 4H), 3.01 to 3.12 (m, 2H), 3.99 (s, 2H), 4.06 (t, J ⫽ 6.4 Hz, 1H), 4.16 (q, J ⫽ 5.9 Hz, 2H), 4.26 to 4.33 (m, 3H), 6.88 (d,

August 2016 Volume 60 Number 8

J ⫽ 9.8 Hz, 1H), 7.85 (d, J ⫽ 9.8 Hz, 1H), 8.45 (s, 1H); MS (ESI⫹) m/z: 478 (MH⫹); HRMS (ESI⫹) for C26H28ClN6O5 (MH⫹): calculated, 478.21087; found, 478.21036. (v) Synthesis of 6-(((1-(2-(3-chloro-5-(2-hydroxyethyl)-6-oxo5,6-dihydro-1,5-naphthyridin-4-yl)ethyl)-2-oxabicyclo[2.2.2]octan4-yl)amino)methyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (compound 4; AM-8722) (step v). To a solution of compound 11 (2.10 g) in dichloromethane (20 ml) was added trifluoroacetic acid (20 ml) with cooling in an ice bath. The mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. After the residue was diluted with water, the mixture was adjusted to pH 9 by addition of 10% aqueous sodium hydroxide solution. The resulting precipitates were collected by filtration to give free amine (1.18 g). The filtrate was extracted with dichloromethane-methanol (4/1). The organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and then concentrated in vacuo to give the additional free amine product (446 mg; total, 1.63 g; 98%) as a powder. 1H NMR (400 MHz, CDCl3) ␦ 1.64 to 1.81 (m, 6H), 1.92 to 2.10 (m, 4H), 3.05 to 3.13 (m, 2H), 3.67 (s, 2H), 4.17 (t, J ⫽ 6.1 Hz, 2H), 4.31 (t, J ⫽ 6.1 Hz, 2H), 6.88 (d, J ⫽ 9.2 Hz, 1H), 7.85 (d, J ⫽ 9.8 Hz, 1H), 8.45 (s, 1H). To a solution of the free amine (1.17 g) and pyridoxazinone-aldehyde (compound 12) (552 mg) in N,N-dimethylformamide (16 ml) was added acetic acid (3.9 ml), and the mixture was stirred at room temperature for 2 h. To the resulting mixture was added sodium triacetoxyborohydride (1.31 g); the mixture was stirred at room temperature for 4 h and concentrated in vacuo. The residue was diluted with saturated sodium carbonate solution, and the mixture was extracted with chloroform-methanol (4/1; 50 ml ⫻ 4). The organic extracts were washed with brine, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure and chromatographed by flash chromatography (ratio of silica and chloroform to methanol ⫽ 20:1) to give the desired NBTI (compound 4; AM-8722) (1.38 g; 82%) as a powder. 1H NMR (400 MHz, DMSO-d6) ␦ 1.58 to 1.74 (m, 8H), 1.76 to 1.92 (m, 2H), 3.12 to 3.21 (m, 2H), 3.54 to 3.67 (m, 6H), 4.38 (t, J ⫽ 6.1 Hz, 2H), 4.59 (s, 2H), 4.77 (t, J ⫽ 5.5 Hz, 1H), 6.83 (d, J ⫽ 9.2 Hz, 1H), 7.01 (d, J ⫽ 8.6 Hz, 1H), 7.27 (d, J ⫽ 8.6 Hz, 1H), 7.84 (d, J ⫽ 9.8 Hz, 1H), 8.50 (s, 1H); MS (ESI⫹) m/z: 540 (MH⫹); HRMS (ESI⫹) for C27H31ClN5O5 (MH⫹): calculated, 540.20137; found, 540.20171. (vi) Preparation of 4.HCl (step vi). To a solution of compound 4 (1.345 g) in dichloromethane (30 ml) and ethanol (20 ml) was added a solution of hydrochloric acid in 1,4-dioxane (4 M, 0.75 ml) under cooling with ice. The mixture was stirred at room temperature for 2 h. The resulting precipitates were collected by filtration and washed with ethanol to give hydrochloride salt of AM-8722 (4.HCl) (1.29 g; 90%) as a powder. 1H NMR (400 MHz, DMSO-d6) ␦ 1.69 to 1.73 (m, 2H), 1.76 to 1.84 (m, 2H), 1.90 to 2.11 (m, 6H), 3.16 to 3.20 (m, 2H), 3.57 to 3.66 (m, 2H), 3.94 (s, 2H), 4.05 to 4.14 (m, 2H), 4.38 (t, J ⫽ 5.8 Hz, 2H), 4.68 (s, 2H), 4.83 (brs, 1H), 6.84 (d, J ⫽ 9.7 Hz, 1H), 7.25 (d, J ⫽ 8.5 Hz, 1H), 7.45 (d, J ⫽ 8.5 Hz, 1H), 7.85 (d, J ⫽ 9.7 Hz, 1H), 8.51 (s, 1H), 9.39 (brs, 1H), 11.32 (s, 1H); MS (ESI⫹) m/z: 540 (MH⫹); HRMS (ESI⫹) for C27H31ClN5O5 (MH⫹): calculated, 540.20137; found, 540.20103. Analysis (C27H30ClN5O5·HCl) was conducted for C, H, and N; calculated, C, 56.25; H, 5.42; N, 12.15%; found, C, 54.66; H, 5.28; N, 11.71%. Inhibition of macromolecular synthesis. The assay was performed as previously described (12, 20). Enzyme activity. The supercoiling activity of DNA gyrase, decatenation activity of topoisomerase IV, and decatenation activity of human topoisomerase II were determined. The inhibitory effect of compounds was assessed by determining the concentration required to inhibit 50% of the enzyme (IC50) (12). MICs. The bacterial isolates used in the antibacterial activity assays were obtained from the wild-type culture collection of Kyorin and Merck and were selected to represent commonly occurring bacterial organisms of clinical relevance. Escherichia coli TG1 [⌬(lac-pro)supE thi hsd⌬5/ F=traD36 proAB⫹ lacIqlacZ⌬M15] and KAM3 were provided from

Antimicrobial Agents and Chemotherapy

aac.asm.org

4831

Tan et al.

Okayama University (21). Pseudomonas aeruginosa PAO969 and PAO6006 were a gift from D. Haas (22). P. aeruginosa PAO4009, KH4013E, and KH4014a and quinolone-resistant S. aureus and Streptococcus pneumoniae were generated in our Kyorin laboratories (23–26). S. aureus RN4220 NBTI mutant strains APNBTI-1R (gyrA) and APNBTI-2R (gyrA grlA) were sequentially selected by plating bacterial suspension on Mueller-Hinton agar with 4-aminopiperidine-linked NBTI. Levofloxacin and vancomycin were obtained from Sigma-Aldrich. Linezolid was synthesized at Kyorin. MICs were determined by agar or broth dilution methods as described in the Clinical and Laboratory Standards Institute (CLSI) guidelines (27). The MIC was determined to be the lowest concentration of the test article that showed no visible growth after incubation at 37°C for 18 to 24 h. To compare the relative antibacterial activity of AM-8722 with those of comparator controls, a panel of 225 Gram-positive and Gram-negative isolates was tested to determine the MIC required to inhibit growth in 50% (MIC50) or 90% (MIC90) of the isolates. To ascertain the impact of exogenous DNA or human serum on the antibacterial activity of AM-8722, antibacterial activity assays were conducted according to CLSI guidelines in the presence of salmon sperm DNA (500 ␮g/ml) or human serum (50%). Time-kill study. Precultured S. aureus ATCC 29213 was diluted into fresh cation-adjusted Mueller-Hinton II broth (CAMHB; Becton Dickinson) and was incubated in a water bath at 35°C with constant shaking. After 1 h, a final concentration of 1/2, 1, 2, 4, 8, and 16 times the MIC of each compound was added to the bacterial culture (approximately 5 ⫻ 105 CFU/ml). Bacterial counts were determined at 0, 2, 4, 6, 8, and 24 h after adding the compound (12). Frequency of resistance (FOR). Overnight cultures of S. aureus ATCC 29213 grown on Mueller-Hinton broth were centrifuged and resuspended in fresh broth media. The mutant strains were selected by plating the bacterial suspension (approximately 108 CFU per plate for each concentration of compound) on Mueller-Hinton agar containing compounds at 1/2 to 256 times the MIC. The selection plates were incubated at 35°C for 72 h before being scored for the number of bacterial colonies. The incidence of the appearance of resistant strains was calculated as the ratio of the number of colonies that emerged to the number of bacteria inoculated (in CFU). hERG potassium channel assay. To determine the in vitro test article effects on cardiac ion channel current (IKr), the rapidly activating, delayed rectifier potassium current was studied using Chinese hamster ovary (CHO) cells stably expressing the hERG channel encoded by human ether-à-go-go-related gene. Briefly, channel activity was functionally assessed using a high-throughput planar voltage-clamp technology (PatchXpress 7000A). Signal amplitudes were quantified at steady state during control (vehicle) treatment and in the presence of (i) a fixed concentration or (ii) increasing concentrations of the test article. Concentration-dependent changes of signal in response to the test article were expressed as percent block relative to control (predrug/⫺trigger) and are reported as the means ⫾ standard errors of the means (SEM) of the indicated number of tested cells per wells. IC50s were determined where applicable by fitting the averaged concentration-response data (means ⫾ SEM) with a Hill equation (12). In vivo formulation feasibility studies. Intravenous (i.v.) and oral (p.o.) formulations were screened for adequate solubility and stability for dosing AM-8722 in CD-1 mice. The AM-8722 i.v. and p.o. dosing solutions were prepared in 30% (wt/wt) Captisol (purchased from Cydex) in water at a dosing concentration up to 50 mg/kg of body weight. In vivo bacteremia models. To determine the in vivo efficacy of AM8722, immunocompetent female CD-1 mice were inoculated intraperitoneally with ⬃1.0 ⫻ 103 CFU of a methicillin-susceptible strain of Staphylococcus aureus (Smith) resuspended in 5% gastric hog mucin. AM-8722 was administered once by the oral or intravenous (tail vein) route 1 h postinoculation at fixed concentrations of 50, 20, 8, and 3.2, mg/kg. Twenty-four hours postinoculation, mice were euthanized and kidneys were harvested, homogenized, and serially plated to enumerate colonies

4832

aac.asm.org

for CFU per gram tissue. The dose (in milligrams per kilogram) that achieved 50%, 1-log, 2-log, and 3-log reductions (ED50, ED90, ED99, ED99.9, respectively) in bacterial counts was determined using nonlinear regression curve fitting. Blood samples were obtained from the high-dose (50 mg/kg) and low-dose (8 mg/kg) groups (using satellite, infected animals) to determine blood exposure levels of AM-8722. In vivo studies were approved and conducted in accordance with the Merck Institutional Animal Care User Committee. Rodent PK. Pharmacokinetic (PK) studies were carried out with infected female CD-1 mice in the in vivo bacteremia infection study described above, following i.v. and oral dosing at 8 mg/kg and 50 mg/kg. A PK study was also conducted with normal male Wistar Han rats with an i.v. dose of 5 mg/kg and an oral dose of 10 mg/kg. AM-8722 was formulated in 30% (wt/wt) Captisol in water for both i.v. and p.o. administration. Following dosing of animals, blood collection was performed at predetermined time points. Serial mouse blood samples (20 ␮l) from the tail vein were added to 60 ␮l of 0.1 M trisodium citrate buffer and stored at ⫺80°C until liquid chromatography-tandem mass spectrometry (LCMS/MS) analysis. Rat blood was collected in EDTA tubes, and rat plasma was then obtained by centrifugation of the blood. Mouse whole blood and rat plasma samples were analyzed by LC-MS/MS to quantify AM-8722 concentrations. Pharmacokinetic parameters of AM-8722 were calculated using Watson LIMS software (version 7.4.1; Thermo Fisher Scientific, Philadelphia, PA). Plasma protein binding. The extent of plasma protein binding was determined in CD-1 mouse, Wistar Han rat, beagle dog, and human plasma by equilibrium dialysis with a 96-well HTDialysis plate (HTDialysis LLC, Gales Ferry, CT). AM-8722 was tested at 2.5 ␮M for all species. The dialysis chambers for plasma and buffer were separated by a semipermeable membrane (10-kDa cutoff). After 4 h of incubation at 37°C with 5% CO2, compound concentrations in plasma (Cp) and buffer (Cb) were determined by LC-MS/MS. The plasma protein binding was calculated as percent bound ⫽ [(Cp ⫺ Cb)/(Cp)] ⫻ 100. Permeability and Pgp assays. The bidirectional transport of AM-8722 (1 ␮M) was evaluated in LLC-PK1 cells and in LLC-PK1 cells stably transfected with a human MDR1, rat Mdr1a, or mouse Mdr1a cDNA, which encodes the human, rat, or mouse multidrug resistance (MDR) P-glycoprotein (Pgp), respectively. The transfected cell lines are called human LLC-MDR1, rat LLC-Mdr1a, and mouse LLC-Mdr1a, respectively. LLCPK1-derived cell lines can form a tight monolayer and therefore can be used to assess vectorial transport of compounds from basolateral to apical (B¡A) and from apical to basolateral (A¡B). Briefly, LLC-PK1, human LLC-MDR1, rat LLC-Mdr1a, and mouse LLC-Mdr1a cell lines were cultured in 96-well Transwell culture plates. AM-8722 (final concentration, 1 ␮M [150 ␮l]) was added to either the apical or the basolateral compartment of the culture plate, and buffer (150 ␮l; Hanks’ balanced salt solution [HBSS] plus 10 mM HEPES, pH 7.4) was added to the compartment opposite to that containing the compound. At 3 h, 50-␮l samples were removed from both sides of monolayers dosed with AM-8722 and placed in 96-well plates, and 50 ␮l of acetonitrile containing the internal standard (1 ␮M labetalol) was added to the samples. After centrifugation, supernatants were analyzed quantitatively by LC-MS/MS. Apparent permeability across MDCKII monolayers. The apparent permeability (Papp) of AM-8722 could not be accurately measured in the LLC-PK1 cells due to endogenous transporters. Therefore, the Papp of AM-8722 was measured across MDCKII monolayers. Briefly, MDCKII cells were cultured in 96-well Transwell culture plates. AM-8722 (final concentration, 1 ␮M) was prepared in HBSS with 10 mM HEPES, pH 7.4, plus 10 ␮M cyclosporine (CsA). Substrate solution (150 ␮l) was added to either the apical or the basolateral compartment of the culture plate, and buffer (150 ␮l; HBSS plus 10 mM HEPES, pH 7.4) was added to the compartment opposite to that containing the compound. At 3 h, 50 ␮l of sample was taken out from both sides, and 50 ␮l of acetonitrile containing the internal standard (1 ␮M labetalol) was added to the samples. Samples were analyzed quantitatively by LC-MS/MS.

Antimicrobial Agents and Chemotherapy

August 2016 Volume 60 Number 8

AM-8722, a Potent Bacterial DNA Gyrase Inhibitor

FIG 1 Chemical structure of AM-8722 and related NBTIs.

RESULTS AND DISCUSSION

AM-8722 (6-(((1-(2-(3-chloro-5-(2-hydroxyethyl)-6-oxo-5,6dihydro-1,5-naphthyridin-4-yl)ethyl)-2-oxabicyclo[2.2.2]octan4-yl)amino)methyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one) is an oxabicyclooctane-linked NBTI containing a pyridoxazinone as a right-hand-side moiety and an N-hydroxyethyl-3-chloro-1,5naphthyridone as a left-hand-side moiety while maintaining the required 8-atom linker with a basic NH at linker position 7 (Fig. 1). A route depicted in Fig. 2 was used to synthesize AM-8722. The synthesis started with a Heck reaction of 6-methoxy-8-bromo-3chloro-1,5-naphthyridine (compound 6) (13) and the Boc-protected vinyl oxabicyclooctane (compound 7) (12), affording

E-olefin (compound 8). The olefin was reduced by catalytic hydrogenation to yield the saturated compound 9. Acid hydrolysis of compound 9 followed by Boc protection of the liberated amine gave hydroxy-naphthyridine (compound 10), which upon alkylation with 2-bromo-ethyl chloride followed by solvolysis afforded N-hydroxy-1,5-naphthyridone (compound 11). Boc deprotection followed by reductive amination with pyridoxazinone-aldehyde (compound 12) (12) furnished the desired NBTI compound 4 (AM-8722), which was converted to hydrochloride salt (4.HCl, AM-8722.HCl) by treatment with anhydrous HCl. Reagents and conditions. Reagents and conditions were as follows: (i) Pd(OAc)2, Ag2CO3, and benzene, with reflux, for 3 days;

FIG 2 Chemical synthesis of AM-8722. Reagents and conditions were as follows: (i) Pd(OAc)2, Ag2CO3, and benzene, with reflux, for 3 days; (ii) 5% Pt/C, 3 kg/cm2, and methanol; (iii) (a) 6 M HCl and dioxane at 70°C for 40 h and (b) Boc2O, aqueous NaOH, and THF at 60°C; (iv) 2-bromo-ethyl-chloride, K2CO3, and DMF at 50°C for 4 h or 100°C for 8 h; (v) (a) TFA and dichloromethane and (b) pyridoxazine aldehyde, AcOH, DMF, and NaBH(OAc)3; and (vi) 4 M HCl-dioxane and dichloromethane.

August 2016 Volume 60 Number 8

Antimicrobial Agents and Chemotherapy

aac.asm.org

4833

Tan et al.

FIG 3 Macromolecular labeling of AM-8722 (HCl salt).

(ii) 5% Pt/C, 3 kg/cm2, and methanol; (iii) (a) 6 M HCl and dioxane at 70°C for 40 h and (b) Boc2O, aqueous NaOH, and tetrahydrofuran at 60°C; (iv) 2-bromo-ethyl-chloride, K2CO3, and dimethylformamide at 50°C for 4 h or 100°C for 8 h; (v) (a) trifluoroacetic acid and dichloromethane and (b) pyridoxazine aldehyde, AcOH, DMF, and NaBH(OAc)3; and (vi) 4 M HCldioxane and dichloromethane. AM-8722 is a selective and potent inhibitor of bacterial DNA gyrase and topoisomerase IV. The effect of AM-8722 on macromolecular synthesis was determined via macromolecular labeling (MML) studies with S. aureus. The results demonstrated that AM8722 selectively inhibited DNA synthesis (Fig. 3). The AM-8722 IC50 for DNA synthesis was 1.10 ␮g/ml, whereas the IC50 for RNA, protein, cell wall, and lipid synthesis was ⬎3 ␮g/ml. The control antibiotics rifampin, linezolid, vancomycin, and triclosan exhibited their expected inhibitory activities on RNA, protein, cell wall, and lipid synthesis, respectively (data not shown). The IC50 for inhibition of DNA synthesis was lower than the MIC for S. aureus killing (0.031 ␮g/ml [see Table 2]). These findings demonstrated that AM-8722 selectively inhibited DNA replication. To confirm enzymatic potency, a cell-free assay was used to assess AM-8722 inhibitory activity against bacterial DNA gyrase and topoisomerase IV from both S. aureus and E. coli (Table 1). AM-8722 displayed potent inhibitory activity against DNA gyrase (IC50 for S. aureus, 0.83 ␮M; IC50 for E. coli, 0.14 ␮M) and topoisomerase IV (IC50 for S. aureus, 12.8 ␮M; IC50 for E. coli, 0.22 ␮M). Importantly, AM-8722 had no effect on human topoisomerase II up to 500 ␮M, the highest concentration tested (Table 1). While levofloxacin displayed activity against DNA gyrase (IC50 for S. aureus, 35.4 ␮M; IC50 for E. coli, 0.25 ␮M) and topoisomerase IV (IC50 for S. aureus, 21.3 ␮M; IC50 for E. coli, 5.09 ␮M), its potency was 1.7to 43-fold lower than that of AM-8722 across the four enzymes. Thus, these data demonstrate that AM-8722 is a selective inhibitor of DNA synthesis and a specific inhibitor of DNA gyrase and topoisomerase IV with greater intrinsic potency than that of levofloxacin. AM-8722 exhibits potent antibacterial activity: MICs. AM8722 was profiled for antibacterial activity across a selected set of organisms (Table 2) to understand its antibacterial spectrum and mechanism of action. AM-8722 exhibited potent activity against the wild-type Gram-positive strains S. aureus Smith (MIC ⫽ 0.031 ␮g/ml) and S. pneumoniae IID554 (MIC ⫽ 0.25 ␮g/ml) compared to that against Enterococcus faecalis ATCC 29212 (MIC ⫽ 2 ␮g/ ml). Against wild-type Gram-negative organisms, AM-8722

4834 aac.asm.org

showed MICs, in micrograms per milliliter, in the single digits for E. coli ATCC 25922 (MIC ⫽ 2 ␮g/ml), P. aeruginosa PAO1 (MIC ⫽ 8 ␮g/ml), and Haemophilus influenzae ATCC 49247 (MIC ⫽ 1 ␮g/ml), with MICs below 1 ␮g/ml versus Acinetobacter baumannii IID876 (MIC ⫽ 0.25 ␮g/ml) and Moraxella catarrhalis ATCC 25238 (MIC ⫽ 0.063 ␮g/ml). AM-8722 was further characterized and showed potent activity against the clinical methicillin-resistant S. aureus strain OITI 1-971 (MIC ⫽ 0.031 ␮g/ml), whereas it was less active against the vancomycin-resistant E. faecium clinical isolate A2373 (MIC ⫽ 2 ␮g/ml), in line with results for E. faecalis (see above). Studies with E. coli and an efflux pump mutant showed that AM-8722 was an efflux substrate, as AM8722 antibacterial activity (compared to that against wild-type E. coli TG1 [MIC ⫽ 1 ␮g/ml]) was significantly reduced when tested in the efflux-deficient strain E. coli KAM3 (MIC ⫽ 0.004 ␮g/ml). The efflux pump sensitivity was corroborated with P. aeruginosa, for which the MIC activity was further reduced ⱖ2-fold when tested in OprJ-, OprN-, or OprM-overexpressing P. aeruginosa strains (Table 2). We employed the popular class of 4-aminopiperidine-linked NBTI, APNBTI (6-({1-(⫾)-2-(3-chloro-6-methoxy[1,5]naphthyridin-4-yl)-2-hydroxy-ethyl-piperidin-4-ylamino}methyl)-4H-pyrido[3,2-b][1,4]oxazin-3-one-dichloride) (28), for resistance selection and produced single-step (APNBTI-1 resistance mutation in gyrA) and double-step (APNBTI-2 resistance mutation in gyrA and parC) S. aureus resistant mutants to assess AM-8722 binding to DNA gyrase. Compared to wild-type S. aureus RN4220 (MIC ⫽ 0.125 ␮g/ml), AM-8722 antibacterial activity was significantly reduced (MIC ⫽ 2 ␮g/ml; 16-fold) against the RN4220 mutant APNBTI-1R (gyrA), and even more reduced against the double mutant APNBTI-2R (MIC ⫽ ⬎16 ␮g/ml; ⬎128fold), confirming that the AM-8722 was on-target and binding at the same binding site as APNBTI-1 and APNBTI-2 (i.e., gyrA and parC). We confirmed AM-8722 activity against quinolone-resistant S. aureus and S. pneumoniae strains. AM-8722 exhibited excellent potency against wild-type S. aureus MS5935 (MIC ⫽ 0.063 ␮g/ml) and its isogenic, highly quinolone-resistant mutant harboring point mutations at grlA (S80F), gyrA (S84L), grlA (E84K), and gyrA (E88V) (29) (MIC ⫽ 0.25 ␮g/ml). Additionally, AM8722 activity in the quinolone-resistant S. pneumoniae strain NC9971 (parC gyrA) was unaltered (MIC ⫽ 0.5 ␮g/ml) compared to that against wild-type S. pneumoniae IID553 (MIC ⫽ 0.25 ␮g/ml). We assessed AM-8722 and controls ciprofloxacin, novobiocin, and ethidium bromide for antibacterial activity against S. aureus RN4220 in the absence or presence of excess DNA (500 ␮g/ml of salmon sperm) or serum (50% human serum). Ciprofloxacin inhibited S. aureus at 0.5 ␮g/ml; as anticipated, the ciprofloxacin

TABLE 1 Inhibitory effects of AM-8722 or levofloxacin on supercoiling activity of DNA gyrase, decatenation activity of topoisomerase IV, and decatenation activity of human topoisomerase IIa IC50 (␮M) for: S. aureus

E. coli

Compound

DNA gyrase

Topo IV

DNA gyrase

Topo IV

Human Topo II

AM-8722 Levofloxacin

0.83 35.4

12.8 21.3

0.14 0.25

0.22 5.09

⬎500 2,880

a

The concentration required to inhibit 50% of the enzyme reaction (IC50) is indicated. AM-8722 was used as a dihydrochloride salt. Topo, topoisomerase.

Antimicrobial Agents and Chemotherapy

August 2016 Volume 60 Number 8

AM-8722, a Potent Bacterial DNA Gyrase Inhibitor

TABLE 2 AM-8722 antibacterial activitya MIC (␮g/ml) Organism

Strain

Phenotype or description

AM-8722

Compound 5

Compound 3b

S. aureus S. pneumoniae E. faecalis E. coli P. aeruginosa A. baumannii M. catarrhalis H. influenzae MRSA E. faecium

Smith IID554 ATCC 29212 ATCC 25922 PAO1 IID876 ATCC 25238 ATCC 49247 OITI 1-971 A2373

Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Clinical isolate vanA

0.031 0.25 2 2 8 0.25 0.031 1 0.031 2

0.125 0.5 8 4 8 4 NT 0.5 0.5 8

0.25 0.25 4 4 16 2 0.25 1 0.25 8

E. coli

TG1 KAM3

Derivative of K-12d Efflux deficient

1 0.004

2 0.063

2 0.016

P. aeruginosa

PAO4009 KH4013E (nfxB) KH4014a (nfxc) PAO969 PAO6006 (nalB)

MexCD-OprJ overexp. MexEF-OprN overexpression Wild type MexAB-OprM overexpression

8 ⬎16 16 8 16

8 ⬎64 64 8 32

8 ⬎16 ⬎16 8 ⬎16

S. aureus

RN4220 APNBTI-1R gyrA (D83N) APNBTI-2R gyrA (D83N) parC (D79N) MS5935 MS5935 grlA (S80F) gyrA (S84L) grlA (E84K) gyrA (E88V)

Wild type APNBTIc-resistant 1st-step mutant APNBTI-resistant 2nd-step mutant Wild type Quinolone resistant

0.125 2 ⬎16 0.063 0.25

0.5 8 ⬎64 0.25 1

1 16 ⬎16 0.5 2

S. pneumoniae

IID553 NC9971 parC (79Y) gyrA (S81F)

Wild type Quinolone resistant

0.5 0.5

0.5 0.5

2 2

a

MICs were determined against a panel of Gram-positive and Gram-negative microorganisms. Part of the MIC data for compound 3 has been reported earlier by Singh et al. (13). c APNBTI, 6-({1-(⫾)-2-(3-chloro-6-methoxy-[1,5]naphthyridin-4-yl)-2-hydroxy-ethyl-piperidin-4-ylamino}-methyl)-4H-pyrido[3,2-b][1,4]oxazin-3-one-dichloride (22). d E. coli TG1 [⌬(lac-pro)supE thi hsd⌬5/F=traD36 proAB⫹ lacIqlacZ⌬M15]. b

MIC was unaffected by excess DNA or serum (Table 3). In contrast, the MICs of novobiocin and ethidium bromide, agents known to be impacted by serum and DNA, respectively, were significantly right shifted (novobiocin alone, 0.125 ␮g/ml; novobiocin plus 50% human serum, 16 ␮g/ml [128-fold shift]; ethidium bromide alone, 2 ␮g/ml; ethidium bromide plus 500 ␮g/ml of salmon sperm DNA, 32 ␮g/ml [16-fold shift]). As expected, the potencies of novobiocin and ethidium bromide were unaffected by the presence of DNA and serum, respectively (Table 3). AM8722 retained its potent antibacterial activity (MIC ⫽ 0.125 ␮g/ ml) in the presence of excess salmon sperm DNA and 50% human serum, similar to ciprofloxacin (Table 3). Based on its in vitro antimicrobial profile, AM-8722 was

TABLE 3 AM-8722 antibacterial activity against S. aureus RN4220 MIC (␮g/ml)a Compound

Alone

With DNA

With serum

AM-8722 Ciprofloxacin Novobiocin Ethidium bromide

0.125 0.5 0.125 2

0.125 (1) 0.5 (1) 0.125 (1) 32 (16)

0.125 (1) 0.5 (1) 16 (128) 1 (0.5)

a MICs were determined for the compounds indicated in the presence of salmon sperm DNA (500 ␮g/ml) or human serum (50%). Values in parentheses indicate n-fold changes.

August 2016 Volume 60 Number 8

tested for antibacterial activity against a broader set of recently identified, geographically diverse clinical isolates. We initially focused on a broad range (⬃200 isolates in total) of Grampositive organisms and selected Gram-negative pathogens to understand the predictive value of the in vitro panel (Table 2) and to compare AM-8722 activity versus standard-of-care agents, levofloxacin, linezolid, and vancomycin. MICs required to inhibit growth in 50% (MIC50) or 90% (MIC90) of the isolates were determined for AM-8722, levofloxacin, linezolid, and vancomycin (Table 4). AM-8722 displayed potent in vitro activity and a narrow range of MICs (range, ⱕ0.015 to 0.12 ␮g/ ml) against the broad set of Staphylococcus isolates (MIC50, 0.03 ␮g/ml; MIC90, 0.06 ␮g/ml) and was significantly more active than levofloxacin (MIC50, 8 ␮g/ml; MIC90, ⬎32 ␮g/ml), linezolid (MIC50, 2 ␮g/ml; MIC90, 8 ␮g/ml), and vancomycin (MIC50, 1 ␮g/ml; MIC90, 2 ␮g/ml). AM-8722 displayed similar potent in vitro activity against coagulase-negative Staphylococcus (CoNS) (MIC50, 0.06 ␮g/ml; MIC90, 0.12 ␮g/ml), superior to those of linezolid (MIC50, 1 ␮g/ml; MIC90, 1 ␮g/ml), levofloxacin (MIC50, 0.5 ␮g/ml; MIC90, 8 ␮g/ml), and vancomycin (MIC50, 2 ␮g/ml; MIC90, 2 ␮g/ml). Antimicrobial agent rank order potency was similar against Enterococcus (AM-8722 ⬎ linezolid ⬎ levofloxacin ⬃ vancomycin) (Table 4). AM-8722 displayed marked in vitro activity against a collection of Acinetobacter isolates (MIC50, 0.25 ␮g/ml; MIC90, 0.25 ␮g/ml) resistant to all other agents (levofloxa-

Antimicrobial Agents and Chemotherapy

aac.asm.org

4835

Tan et al.

TABLE 4 AM-8722 antibacterial activitya MIC (␮g/ml)

Organism

Drug

Total no. of isolates tested

Range

Mode

50%

90%

S. aureus

AM-8722 Levofloxacin Linezolid Vancomycin

53 53 53 53

ⱕ0.015–0.12 0.12–⬎32 0.5–32 0.5–⬎64

0.03 8 2 1

0.03 8 2 1

0.06 ⬎32 8 2

CoNS

AM-8722 Levofloxacin Linezolid Vancomycin

20 20 20 20

ⱕ0.015–1 0.12–32 0.5–1 0.5–2

0.06 0.25 1 2

0.06 0.5 1 2

0.12 8 1 2

E. faecalis

AM-8722 Levofloxacin Linezolid Vancomycin

20 20 20 20

0.03–0.5 1–⬎32 0.25–2 1–⬎64

0.25 ⬎32 1 ⬎64

0.12 32 1 ⬎64

0.5 ⬎32 2 ⬎64

E. faecium

AM-8722 Levofloxacin Linezolid Vancomycin

17 17 17 17

0.12–4 1–⬎32 1–2 0.25–⬎64

0.12 ⬎32 1 ⬎64

0.5 ⬎32 1 ⬎64

2 ⬎32 2 ⬎64

S. pneumoniae

AM-8722 Levofloxacin Linezolid Vancomycin

20 20 20 20

0.03–0.5 0.5–16 0.25–1 0.12–0.5

0.25 0.5 1 0.25

0.12 1 1 0.25

0.25 16 1 0.5

S. agalactiae

AM-8722 Levofloxacin Linezolid Vancomycin

15 15 15 15

0.25–4 0.5–2 0.5–1 0.25–0.5

2 0.5 1 0.5

2 0.5 1 0.5

4 1 1 0.5

S. pyogenes

AM-8722 Levofloxacin Linezolid Vancomycin

15 15 15 15

0.06–0.5 0.25–2 0.5–1 0.25–0.25

0.25 0.5 1 0.25

0.25 0.5 1 0.25

0.25 1 1 0.25

A. baumannii

AM-8722 Levofloxacin Linezolid Vancomycin

17 17 17 17

ⱕ0.015–1 0.06–32 ⬎64–⬎64 64–⬎64

ⱕ0.015 0.06 ⬎64 ⬎64

0.03 4 ⬎64 ⬎64

0.25 32 ⬎64 ⬎64

H. influenzae

AM-8722 Levofloxacin Linezolid Vancomycin

15 15 15 15

0.06–2 0.015–0.03 4–16 16–⬎64

0.5 0.015 16 64

0.5 0.015 16 64

1 0.03 16 ⬎64

M. catarrhalis

AM-8722 Levofloxacin Linezolid Vancomycin

11 11 11 11

ⱕ0.015–0.03 0.015–0.06 4–4 32–64

ⱕ0.015 0.03 4 64

ⱕ0.015 0.03 4 64

0.03 0.06 4 64

a

MICs required to inhibit growth in 50% (MIC50) or 90% (MIC90) of the isolates were determined for AM-8722, levofloxacin, linezolid, and vancomycin.

cin [MIC50, 4 ␮g/ml; MIC90, 32 ␮g/ml], linezolid [MIC50, ⬎64 ␮g/ml; MIC90, ⬎64 ␮g/ml], and vancomycin [MIC50, ⬎64 ␮g/ml; MIC90, ⬎64 ␮g/ml]) as well as excellent in vitro activity against H. influenzae (MIC50, 0.5 ␮g/ml; MIC90, 1 ␮g/ml) and M. catarrhalis (MIC50, ⬍0.015 ␮g/ml; MIC90, 0.03 ␮g/ml) clinical isolates (Table 4). AM-8722 is bactericidal: time-kill kinetics. Time-kill curves for AM-8722.HCl and levofloxacin were performed with S. aureus ATCC 29213 (Fig. 4; see also Fig. S1 in the supplemental material).

4836

aac.asm.org

S. aureus in the absence of compound exhibited growth from 5.6 log CFU at 0 h to 9.5 log CFU at 24 h. In the presence of 2 ␮g/ml (16⫻ MIC) or 4 ␮g/ml (32⫻ MIC), AM-8722.HCl (MIC ⫽ 0.125 ␮g/ml) displayed rapid bactericidal activity, with no regrowth up to 24 h (Fig. 4). At lower MIC multiples (1/2⫻ MIC to 2⫻ MIC [corresponding to 0.063 ␮g/ml to 0.25 ␮g/ml]), S. aureus growth was rapidly reduced (⬃2.5 to 3 log CFU/ml) to levels near the limit of detection (assay limit: 3 log CFU/ml) and thereafter (8 h) exhibited growth back to near control (no drug) levels (Fig. 4). In the

Antimicrobial Agents and Chemotherapy

August 2016 Volume 60 Number 8

AM-8722, a Potent Bacterial DNA Gyrase Inhibitor

FIG 4 S. aureus ATCC 29213 (⬃5 ⫻ 105 CFU/ml) was incubated at 35°C with constant shaking. After 1 h, a final concentration of 1/2, 1, 2, 4, 8, or 16 times the MIC of AM-8722 was added to the bacterial culture. Bacterial counts were determined at the specified time points after compound addition.

presence of 1 ␮g/ml (8⫻ MIC), AM-8722.HCl was rapidly bactericidal and S. aureus regrowth starting after 8 h was still below initial inoculum levels by 24 h (0 h, 5.9 log CFU/ml; 24 h, 4.2 log CFU/ml). The data with S. aureus ATCC 29213 demonstrate that AM-8722.HCl exhibits potent and rapid bactericidal activity comparable to that of levofloxacin (see Fig. S1). AM-8722 FOR. In vitro frequency of resistance (FOR) emergence was determined for AM-8722.HCl and levofloxacin by incubating S. aureus ATCC 29213 with various MIC multiples for 72 h and enumerating colonies (Table 5). Concentrations of AM8722.HCl equivalent to 32⫻ and 64⫻ MIC (4 to 8 ␮g/ml) were shown to display an FOR emergence of 1.6 ⫻ 10⫺8 to 7.9 ⫻ 10⫺9; in comparison, a levofloxacin concentration equivalent to 2⫻ MIC (0.25 ␮g/ml) displayed an FOR emergence of ⬍2.6 ⫻ 10⫺9. Lower AM-8722.HCl concentrations (1/2⫻ to 16⫻ MIC) were associated with higher FOR emergence (⬃10⫺6 to 10⫺7) at 72 h. AM-8722 has an improved cardiac potassium ion channel (hERG) profile. In the potassium channel hERG functional assay, AM-8722 hydrochloride salt inhibited hERG current with an IC50 of 3.8 ␮M, whereas the activity of the free base was significantly attenuated, showing hERG current inhibition of 12% at 30 ␮M. This difference could be attributed to poor solubility of the free base compared to the salt. The hERG profile is far superior than for AM-8085 (IC50 ⫽ 0.6 ␮M) but not as good as those of AM8191 (IC50 ⫽ 18 ␮M) and the hydroxy tricyclic compound 3 (IC50 ⫽ 174 ␮M) (12, 13). As a reference, GSK2140944, the most advanced compound in the clinic, showed an hERG IC50 of 1.4 mM (30). AM-8722 pharmacokinetics. The pharmacokinetics of AM8722 was evaluated in infected female CD-1 mice and normal male Wistar Han rats. Multiple intravenous (i.v.) and oral (p.o.) formulations were screened for AM-8722. Captisol (30%, wt/wt)

FIG 5 Pharmacokinetic studies with infected female CD-1 mice following i.v. and oral dosing at 8 mg/kg and 50 mg/kg. AM-8722 was formulated in 30% (wt/wt) Captisol in water for i.v. and oral administration. Blood collection was performed at predetermined time points following dosing for PK analysis as described in Materials and Methods.

in water was chosen as the best formulation based on the solubility and stability assessment. AM-8722 was formulated in 30% captisol and administered intravenously and orally at both 8 mg/kg and 50 mg/kg in infected female CD-1 mice (n ⫽ 3 per group). The concentration-versus-time curves at 8 mg/kg i.v. and 50 mg/kg p.o. are shown in Fig. 5. AM-8722 displayed moderate to high clearance (54.5 to 74.2 ml/min/kg), a large steady-state volume of distribution (5.2 to 7.7 liters/kg), a long terminal half-life (10 to 16 h), and moderate oral bioavailability (21 to 27%) in mice (see Table S1 in the supplemental material). Pharmacokinetic results

TABLE 5 Frequencies of resistance to AM-8722 and levofloxacina Compound

Frequency of resistance at the following multiples of the MIC: MIC (␮g/ml) 1/2⫻ 1⫻ 2⫻ 4⫻ 8⫻

AM-8722.HCl 0.125 Levofloxacin 0.125

16⫻

32⫻

64⫻

128⫻

256⫻ MIC

⬎1.3 ⫻ 10⫺6 ⬎1.3 ⫻ 10⫺6 ⬎1.3 ⫻ 10⫺6 6.7 ⫻ 10⫺7 7.8 ⫻ 10⫺7 1.2 ⫻ 10⫺7 1.6 ⫻ 10⫺8 7.9 ⫻ 10⫺9 ⬍2.6 ⫻ 10⫺9 ⬍2.6 ⫻ 10⫺9 ⬎1.3 ⫻ 10⫺6 ⬎1.3 ⫻ 10⫺6 ⬍2.6 ⫻ 10⫺9 ⬍2.6 ⫻ 10⫺9 ⬍2.6 ⫻ 10⫺9 ⬍2.6 ⫻ 10⫺9 NT NT NT NT

a

Overnight cultures of S. aureus ATCC 29213 on Mueller-Hinton broth were centrifuged and resuspended in fresh broth media. The mutant strains were selected by plating the bacterial suspension (approximately 108 CFU per plate for each concentration of compound) on Mueller-Hinton agar with compounds. The selection plates were incubated at 35°C for 72 h before being scored for the number of bacterial colonies. The incidence of the appearance of resistant strains was calculated as described in Materials and Methods. NT, not tested.

August 2016 Volume 60 Number 8

Antimicrobial Agents and Chemotherapy

aac.asm.org

4837

Tan et al.

FIG 6 Mice (5/group) were challenged intraperitoneally with S. aureus Smith

(⬃1.4 ⫻ 103 CFU/mouse) in 5% hog gastric mucin. Mice were treated p.o. or i.v. with 1 total dose of AM-8722. Kidneys were aseptically collected at 24 h after challenge, weighed, homogenized, and plated to determine CFU remaining per gram of kidney.

of AM-8722 in rats demonstrated a shorter half-life (3.9 h) but in general were in line with mouse results, with a plasma clearance of 52.2 ml/min/kg, a volume of distribution of 2.6 liters/kg, and an oral bioavailability of 35% (see Table S1). AM-8722 showed moderate plasma protein binding, with 19.5%, 10.0%, 3.1%, and 9.7% unbound in human, mouse, rat, and dog plasma, respectively. It exhibited high permeability in MDCKII cells (Papp ⫽ 23.6 ⫻ 10⫺6 cm/s). AM-8722 showed BA/AB ratios of ⬎27.1, ⬎21.5, and 24.7 in human LLC-MDR1, rat LLC-Mdr1a, and mouse LLC-Mdr1a cells, respectively, indicating it was a substrate for human, rat, and mouse Pgp. Pgp is an efflux transporter extensively distributed and expressed in the intestinal epithelium, which could contribute to the limited oral bioavailability observed in the mouse and rat for AM-8722. AM-8722 in vivo efficacy. The efficacy of AM-8722 compared to linezolid in vivo was assessed using a murine methicillin-susceptible S. aureus (MSSA) model of disseminated infection. CD-1 female mice were challenged with 1.4 ⫻ 103 CFU of S. aureus Smith intraperitoneally (reconstituted in 5% hog gastric mucin), and AM-8722 or linezolid was administered once by either the oral or intravenous route 1 h postinoculation at set concentrations of 50, 20, 8.0, and 3.2 mg/kg. Twenty-four hours postchallenge, mice were euthanized, kidneys were harvested, homogenized, and serially plated, and colonies enumerated to determine CFU remaining per gram of tissue. Blood samples were obtained from mice administered 50- and 8-mg/kg doses of each compound to determine exposure levels. The dose that achieved 50%, 1-log, 2-log, and 3-log reductions (ED50, ED90, ED99, and ED99.9, respectively) in bacterial counts was determined using nonlinear regression curve fitting. AM-8722 was highly efficacious in this assay when dosed intravenously (Fig. 6; see also Table S2 in the supplemental material), achieving an ED50 of 2.1 mg/kg. AM-8722 achieved 1-, 2-, and 3-log reductions (ED90, ED99, and ED99.9) in bacterial burden at 6.3, 11.5, and 17.0 mg/kg, respectively. In comparison, AM-8722 efficacy via the oral route was less pronounced, with an ED50, ED90, ED99, and ED99.9 of 20.6, 31.6, 42.7, and 52.8 mg/kg, respectively. This was consistent with the 21 to 27% oral bioavailability of AM-8722 observed in mice. In summary, AM-8722 was shown to be a selective and potent inhibitor of bacterial DNA gyrase and topoisomerase IV by cellular macromolecular labeling and direct enzymatic assessment. As

4838

aac.asm.org

seen with this class of compounds, AM-8722 exhibited potent whole-cell activity across selected wild-type susceptible and resistant Gram-positive and Gram-negative organisms. AM-8722 was shown to retain its antibacterial activity in the presence of excess serum and DNA, and it displayed broad activity against a large panel of clinically relevant isolates. AM-8722 is rapidly bactericidal in vitro, and importantly, AM-8722 analogs demonstrate improved hERG activity. While a high FOR was observed for AM8722, prevention of resistance emergence was shown to be improved at high multiples of MIC, suggesting that an improved resistance profile could be achievable. Finally, AM-8722 was efficacious in a murine model of disseminated S. aureus infection. AM-8722 represents a new class of novel bacterial topoisomerase inhibitor agents that show promise to combat bacterial resistance. FUNDING INFORMATION This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

REFERENCES 1. Bax BD, Chan PF, Eggleston DS, Fosberry A, Gentry DR, Gorrec F, Giordano I, Hann MM, Hennessy A, Hibbs M, Huang J, Jones E, Jones J, Brown KK, Lewis CJ, May EW, Saunders MR, Singh O, Spitzfaden CE, Shen C, Shillings A, Theobald AJ, Wohlkonig A, Pearson ND, Gwynn MN. 2010. Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature 466:935–940. http://dx.doi .org/10.1038/nature09197. 2. Wiener JJ, Gomez L, Venkatesan H, Santillan A, Jr, Allison BD, Schwarz KL, Shinde S, Tang L, Hack MD, Morrow BJ, Motley ST, Goldschmidt RM, Shaw KJ, Jones TK, Grice CA. 2007. Tetrahydroindazole inhibitors of bacterial type II topoisomerases. Part 2: SAR development and potency against multidrug-resistant strains. Bioorg Med Chem Lett 17:2718 –2722. 3. Black MT, Stachyra T, Platel D, Girard AM, Claudon M, Bruneau JM, Miossec C. 2008. Mechanism of action of the antibiotic NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II topoisomerases. Antimicrob Agents Chemother 52:3339 –3349. http://dx.doi.org/10.1128 /AAC.00496-08. 4. Black MT, Coleman K. 2009. New inhibitors of bacterial topoisomerase GyrA/ParC subunits. Curr Opin Investig Drugs 10:804 – 810. 5. Gomez L, Hack MD, Wu J, Wiener JJ, Venkatesan H, Santillan A, Jr, Pippel DJ, Mani N, Morrow BJ, Motley ST, Shaw KJ, Wolin R, Grice CA, Jones TK. 2007. Novel pyrazole derivatives as potent inhibitors of type II topoisomerases. Part 1: synthesis and preliminary SAR analysis. Bioorg Med Chem Lett 17:2723–2727. 6. Miles TJ, Axten JM, Barfoot C, Brooks G, Brown P, Chen D, Dabbs S, Davies DT, Downie DL, Eyrisch S, Gallagher T, Giordano I, Gwynn MN, Hennessy A, Hoover J, Huang J, Jones G, Markwell R, Miller WH, Minthorn EA, Rittenhouse S, Seefeld M, Pearson N. 2011. Novel aminopiperidines as potent antibacterials targeting bacterial type IIA topoisomerases. Bioorg Med Chem Lett 21:7489 –7495. http://dx.doi.org/10.1016/j .bmcl.2011.09.117. 7. Miles TJ, Barfoot C, Brooks G, Brown P, Chen D, Dabbs S, Davies DT, Downie DL, Eyrisch S, Giordano I, Gwynn MN, Hennessy A, Hoover J, Huang J, Jones G, Markwell R, Rittenhouse S, Xiang H, Pearson N. 2011. Novel cyclohexyl-amides as potent antibacterials targeting bacterial type IIA topoisomerases. Bioorg Med Chem Lett 21:7483–7488. http://dx .doi.org/10.1016/j.bmcl.2011.09.114. 8. Mitton-Fry MJ, Brickner SJ, Hamel JC, Brennan L, Casavant JM, Chen M, Chen T, Ding X, Driscoll J, Hardink J, Hoang T, Hua E, Huband MD, Maloney M, Marfat A, McCurdy SP, McLeod D, Plotkin M, Reilly U, Robinson S, Schafer J, Shepard RM, Smith JF, Stone GG, Subramanyam C, Yoon K, Yuan W, Zaniewski RP, Zook C. 2013. Novel quinoline derivatives as inhibitors of bacterial DNA gyrase and topoisomerase IV. Bioorg Med Chem Lett 23:2955–2961. http://dx.doi.org/10.1016 /j.bmcl.2013.03.047. 9. Miles TJ, Hennessy AJ, Bax B, Brooks G, Brown BS, Brown P, Cailleau N, Chen D, Dabbs S, Davies DT, Esken JM, Giordano I, Hoover JL, Huang J, Jones GE, Sukmar SK, Spitzfaden C, Markwell RE, Minthorn

Antimicrobial Agents and Chemotherapy

August 2016 Volume 60 Number 8

AM-8722, a Potent Bacterial DNA Gyrase Inhibitor

10.

11.

12.

13.

14.

15.

16.

17.

EA, Rittenhouse S, Gwynn MN, Pearson ND. 2013. Novel hydroxyl tricyclics (e.g., GSK966587) as potent inhibitors of bacterial type IIA topoisomerases. Bioorg Med Chem Lett 23:5437–5441. http://dx.doi.org/10 .1016/j.bmcl.2013.07.013. Reck F, Alm R, Brassil P, Newman J, Dejonge B, Eyermann CJ, Breault G, Breen J, Comita-Prevoir J, Cronin M, Davis H, Ehmann D, Galullo V, Geng B, Grebe T, Morningstar M, Walker P, Hayter B, Fisher S. 2011. Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II: broad-spectrum antibacterial agents with reduced hERG activity. J Med Chem 54:7834 –7847. http://dx.doi.org/10 .1021/jm2008826. Reck F, Alm RA, Brassil P, Newman JV, Ciaccio P, McNulty J, Barthlow H, Goteti K, Breen J, Comita-Prevoir J, Cronin M, Ehmann DE, Geng B, Godfrey AA, Fisher SL. 2012. Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II with reduced pK (a): antibacterial agents with an improved safety profile. J Med Chem 55:6916 – 6933. http: //dx.doi.org/10.1021/jm300690s. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Meinke PT, Olsen D, Lagrutta A, Bradley P, Lu J, Patel S, Rickert KW, Smith RF, Soisson S, Wei C, Fukuda H, Kishii R, Takei M, Fukuda Y. 2014. Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad spectrum antibacterial agents. ACS Med Chem Lett 5:609 – 614. http://dx.doi .org/10.1021/ml500069w. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Black T, Nargund R, Meinke PT, Olsen DB, Lagrutta A, Lu J, Patel S, Rickert KW, Smith RF, Soisson S, Sherer E, Joyce LO, Wei C, Peng X, Wang X, Fukuda H, Kishii R, Takei M, Takano H, Shibasaki M, Yajima M, Nishimura A, Shibata T, Fukuda Y. 2015. Tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broadspectrum antibacterial agents-SAR of left-hand-side moiety (part-2). Bioorg Med Chem Lett 25:1831–1835. http://dx.doi.org/10.1016/j.bmcl .2015.03.044. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Gill C, Black T, Nargund R, Meinke PT, Olsen DB, Lagrutta A, Wei C, Peng X, Wang X, Fukuda H, Kishii R, Takei M, Takeuchi T, Shibue T, Ohata K, Takano H, Ban S, Nishimura A, Fukuda Y. 2015. Hydroxy tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of RHS moiety (part-3). Bioorg Med Chem Lett 25:2473–2478. http://dx.doi.org/10.1016/j.bmcl.2015.04 .063. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Meinke PT, Olsen DB, Lagrutta A, Wei C, Peng X, Wang X, Fukuda H, Kishii R, Takei M, Shibata T, Ohata K, Takano H, Kurasaki H, Takeuchi T, Nishimura A, Fukuda Y. 2015. Structure activity relationship of substituted 1,5naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (part-4). Bioorg Med Chem Lett 25:2409 –2415. http://dx.doi.org/10.1016/j.bmcl .2015.04.002. Singh SB, Kaelin DE, Meinke PT, Wu J, Miesel L, Tan CM, Olsen DB, Lagrutta A, Fukuda H, Kishii R, Takei M, Takeuchi T, Takano H, Ohata K, Kurasaki H, Nishimura A, Shibata T, Fukuda Y. 2015. Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (part-5). Bioorg Med Chem Lett 25: 3630 –3635. http://dx.doi.org/10.1016/j.bmcl.2015.06.061. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Meinke PT, Olsen DB, Lagrutta A, Wei C, Liao Y, Peng X, Wang X, Fukuda H, Kishii R, Takei M, Yajima M, Shibue T, Shibata T, Ohata K, Nishimura A, Fukuda Y.

August 2016 Volume 60 Number 8

18.

19.

20.

21.

22.

23.

24. 25. 26.

27.

28. 29.

30.

2015. Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (part-6). Bioorg Med Chem Lett 25:3636 –3643. http://dx.doi.org/10.1016/j.bmcl.2015.06.057. Singh SB, Kaelin DE, Wu J, Miesel L, Tan CM, Meinke PT, Olsen DB, Lagrutta A, Wei C, Liao Y, Peng X, Wang X, Fukuda H, Kishii R, Takei M, Shibata T, Takeuchi T, Ohata K, Nishimura A, Fukuda Y. 2015. C1–C2-linker substituted 1,5-naphthyridine analogues of oxabicyclooctane-linked NBTIs as broad-spectrum antibacterial agents (part 7). Medchemcomm 6:1776 –1780. Voight EA, Yin H, Downing SV, Calad SA, Matsuhashi H, Giordano I, Hennessy AJ, Goodman RM, Wood JL. 2010. Target-directed synthesis of antibacterial drug candidate GSK966587. Org Lett 12:3422–3425. http: //dx.doi.org/10.1021/ol101235f. Onishi HR, Pelak BA, Gerckens LS, Silver LL, Kahan FM, Chen MH, Patchett AA, Galloway SM, Hyland SA, Anderson MS, Raetz CR. 1996. Antibacterial agents that inhibit lipid A biosynthesis. Science 274:980 – 982. http://dx.doi.org/10.1126/science.274.5289.980. Morita Y, Kodama K, Shiota S, Mine T, Kataoka A, Mizushima T, Tsuchiya T. 1998. NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli. Antimicrob Agents Chemother 42:1778 –1782. Rella M, Haas D. 1982. Resistance of Pseudomonas aeruginosa PAO to nalidixic acid and low levels of beta-lactam antibiotics: mapping of chromosomal genes. Antimicrob Agents Chemother 22:242–249. http://dx.doi .org/10.1128/AAC.22.2.242. Hirai K, Suzue S, Irikura T, Iyobe S, Mitsuhashi S. 1987. Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa. Antimicrob Agents Chemother 31:582–586. http://dx.doi.org/10.1128/AAC.31.4 .582. Fukuda H, Hosaka M, Hirai K, Iyobe S. 1990. New norfloxacin resistance gene in Pseudomonas aeruginosa PAO. Antimicrob Agents Chemother 34:1757–1761. http://dx.doi.org/10.1128/AAC.34.9.1757. Fukuda H, Hiramatsu K. 1999. Primary targets of fluoroquinolones in Streptococcus pneumoniae. Antimicrob Agents Chemother 43:410 – 412. Fukuda H, Hori S, Hiramatsu K. 1998. Antibacterial activity of gatifloxacin (AM-1155, CG5501, BMS-206584), a newly developed fluoroquinolone, against sequentially acquired quinolone-resistant mutants and the norA transformant of Staphylococcus aureus. Antimicrob Agents Chemother 42:1917–1922. Cockerill FR, Wikler MA, Alder J, Dudley MN, Eliopoulos GM, Ferraro MJ, Hardy DJ, Hecht DW, Hindler JA, Patel JB, Powell M, Swenson JM, Thomson RB, Traczewski MM, Turnidge JD, Weinstein MP, Zimmer BL. 2012. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard—ninth edition. MA07-A9. Clinical and Laboratory Standard Institute, Wayne, PA. Axten JM, Brooks G, Brown P, Davies D, Gallagher TF, Markwell RE, Miller WH, Pearson ND, Seefeld M. 2004. Antibacterial agents. Patent WO2004/058144. Singh SB, Dayananth P, Balibar CJ, Garlisi CG, Lu J, Kishii R, Takei M, Fukuda Y, Ha S, Young K. 2015. Kibdelomycin is a bactericidal broadspectrum aerobic antibacterial agent. Antimicrob Agents Chemother 59: 3474 –3481. http://dx.doi.org/10.1128/AAC.00382-15. Bouchillon SK, Hackel M, Miller LA, Scangarella-Omen NE. 2013. In vitro activity of GSK2140944, a novel topoisomerase inhibitor, against isolates associated with lower respiratory tract and skin infections, abstr. F-1216. Abstr 53rd Intersci Conf Antimicrob Agents Chemother.

Antimicrobial Agents and Chemotherapy

aac.asm.org

4839