The research presented in this manuscript complies with all relevant ethical regulations. This includes ethical approval for all the animal studies employed in this work, and the informed consent and approvals associated with the Phase I clinical trial presented in this work.
Ethics approval for preclinical animal studies
Dose finding studies and efficacy of BWC0977 in the neutropenic murine thigh infection model, pharmacokinetics of BWC0977 in neutropenic mouse thigh model infected with Pseudomonas aeruginosa NCTC 13921, and epithelial lining fluid concentration determination in neutropenic mouse thigh model infected with Pseudomonas aeruginosa NCTC 13921 were conducted in the laboratory of Professor William Hope by the Antimicrobial Pharmacodynamics and Therapeutics Group (Department of Molecular and Clinical Pharmacology), University of Liverpool, United Kingdom. These experiments were conducted under UK Home Office project License PAC022930 which was renewed in 2022 as PP3585942. These licenses and all experiments conducted under them are approved by the University of Liverpool Animal Welfare Ethics Review Board.
Pharmacokinetics of BWC0977 in plasma and pulmonary epithelial lining fluid in a neutropenic lung infection model in rats, dose fractionation and dose response studies of BWC0977 in rat lung infection model, and determination of PK-PD index for BWC0977 in the rat lung infection model were conducted at TheraIndx Lifesciences Private Limited, Bangalore, after obtaining permission from the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India and approval from the Institutional Animal Ethics Committee (IAEC). Approval numbers: IAEC/08/2018/078, IAEC/13/2020/159, IAEC/22/2022/254, IAEC/22/2022/255, and IAEC/24/2023/269.
The dog pharmacokinetic study, rat and dog safety and toxicokinetic studies were performed in accordance with the agreed Protocol and with Covance Laboratories Limited standard operating procedures. Aspects of the studies performed at Covance, Harrogate were conducted in compliance with the United Kingdom Good Laboratory Practice (GLP) Monitoring Authority, Medicines and Healthcare Products Regulatory Agency (MHRA) Good Laboratory Practice Regulations 1999, Statutory Instrument 1999 No. 3106, as amended by the Good Laboratory Practice (GLP) - (Codification Amendments Etc.), Regulations 2004, Statutory Instrument 2004 No. 994 and the Organisation for Economic Co-operation and Development (OECD) Principles of Good Laboratory Practice ENV/MC/CHEM (98) 17 (revised 1997, issued January 1998).
The pharmacokinetic studies in rats & mice, and the bile duct cannulation (BDC) study in rats were performed at Syngene International, India, were carried out on naïve animals as per the provisions of the Institutional Animal Ethics Committee (IAEC) approvals, viz., SYNGENE/IAEC/800/01-2017, SYNGENE/IAEC/937/05-2018, SYNGENE/IAEC/1005/11-2018, and SYNGENE/IAEC/1147/02-2020.
Guinea pig pharmacokinetic study in male Guinea pigs (BWR-001 / PKMGP-001) was conducted under the approved protocol PK-PRO-715-06 by Suven Life Sciences Institutional Animal Ethics Committee Meeting dated 18-May-2019 and facility is registered for research and breeder; registration number as 769/PO/RcBi/SL/03/CPCSEA; dated 14-Jan-2016. The studies were approved by the Animal Welfare Ethical Review Board (A WERB) from the Suven Life Sciences Limited, Pashamylaram, India.
Ethics Approval for First in human studies
The two first in human trials were conducted in CMAX, Adelaide, Australia.
- 1.
C001-2020-01 (https://clinicaltrials.gov/study/NCT05088421): A randomized, double-blind, placebo-controlled, Phase 1 study of the safety, tolerability and pharmacokinetics of single and multiple ascending doses of BWC0977 in healthy adult volunteers.
- 2.
C002-2023-01 (https://clinicaltrials.gov/study/NCT05942820): A randomized, double-blind, placebo-controlled, Phase 1 study of the safety, tolerability, and pharmacokinetics of single and multiple ascending doses of BWC0977 in healthy adult volunteers.
These human studies were initiated following approval by the Bellberry Human Research Ethics Committee (HREC) of the trial design, protocol and the key criteria specified for the study. The Bellberry Human Research Ethics committee (HREC) has scientifically and ethically reviewed this study. This Bellberry HREC is constituted and operates in accordance with the National Health and Medical Research Council’s National Statement on Ethical Conduct in Human Research (2007, incorporating all updates). Bellberry Human Research Ethics Committee does not disclose personal details of its reviewing members. Please note that the Principal Investigator and Co-Investigators were not members of the Bellberry Human Research Ethics Committee that reviewed this study.
Determination of the Minimum Inhibitory Concentration (MIC)
MIC was determined based on the Clinical Laboratory Standards Institute (CLSI) guidelines24 in cation-adjusted Mueller Hinton broth (MHB) using broth microdilution methodology in 96-well microtitre plates (ThermoFisher Scientific, Catalogue No. 130188). Briefly, the inoculum used for all the experiments was derived from a single seed lot, maintained as a glycerol (20%) stock at −80 oC. To revive the cultures, it was first sub-cultured on LB plates for isolated colonies and a single colony inoculated into LB broth, grown for 16–18 h at 37 oC and appropriately diluted in cation-adjusted Mueller Hinton broth (3–7 × 105 CFU/ml). BWC0977 stocks (4 mg/ml) and serial dilutions were prepared in dimethylsulfoxide (DMSO). To 147 μl of bacterial culture, 3 μL of compound from each of the dilutions was added to the wells of 96-well microtitre plates. A 10-concentration range was set up with a start concentration of 4 μg/ml, and two fold serial dilutions resulting in final concentrations of 2 μg/ml, 1μg/ml, 500 ng, 250, 125, 62.5, 31.25, 15.6 and 7.8 ng/ml. For reference inhibitors, the specific solvents used are listed in Supplementary Table13. Media control, culture control and appropriate reference drug controls were included in each assay. The plates were incubated at 37 oC for 16–18 h. Growth was monitored by checking the absorbance at 600 nm (A600). MIC is the concentration that resulted in ≥80% growth inhibition. MIC90 (µg/ml) values of BWC0977 against multiple bacterial isolates were determined using a total of 8591 isolates comprising 7187 Gram-negative and 1404 Gram-positive organisms. This global collection comprised of geographically diverse set of clinical isolates and was tested in different laboratories [St. John’s Medical College & Hospital and Narayana Health, Bangalore India, JMI Laboratories, International Health Management Associates (IHMA), Colorado State University, University of Michigan, Seattle Children Hospital, University of Alabama, Birmingham, University of Texas Health Science Center at San Antonio / National Institute of Allergy and Infectious Diseases (NIAID), Walter Reed Army Institute of Research (WRAIR), Maryland, USA and United States Military Research Institute of Infectious Diseases (USAMRIID)]. In addition, a CARB-X sponsored MIC90 study of both Gram-positive and Gram-negative bacteria (total of 2,945 isolates) was conducted at IHMA.
Determination of the spontaneous resistance frequency of BWC0977
The spontaneous resistance frequency of BWC0977 was determined and the resistant colonies of Gram-negative bacteria were characterized using microbiology and molecular techniques. Towards this aim, mid-logarithmic phase cultures of Gram-negative bacteria (109 CFU/mL) were plated on Luria-Bertani agar plates containing BWC0977 equivalent to 2x, 4x, 8x and 16x MIC. Plates were incubated for 24–36 h at 37 oC, and the resistance frequency determined by counting the number of colony forming units (CFUs) on agar plates46. BWC0977 MICs for the colonies isolated from drug containing plates were determined and the gyrA and parC gene sequenced to confirm any target site mutations. The resistance frequency of ciprofloxacin was determined in parallel using the same methodology.
Killing kinetics assay
Killing kinetics was determined by enumerating the number of bacterial survivors at various time points following compound exposure. Approximately 107 CFU/ml cells (A600 of 0.1) were treated with various concentrations (2-fold serial dilutions, 9 concentrations, with start of either 256 to 1 μg/ml, 64 to 0.25 μg/ml, 32 to 0.125 μg/ml, 16–0.06 μg/ml, 8 to 0.03 μg/ml, 4 to 0.015 μg/ml, or 2 to 0.008 μg/ml μg/ml) of compounds in a 96-well microtitre plate47. At various time intervals (0,1,3,6,9,12,24 h), 30 μl/150 μl of treated cultures was serially diluted and plated for bacterial survivors on LB agar plates. The plates were incubated at 37 oC for 16–18 h, and the bacterial colonies enumerated. Multiple wells of each concentration with total volume of 150μl (column-wise) was prepared in the same plate. Sampling was done for the same concentration every time from a different well for the various time points. The extent of bactericidality was monitored against a panel of susceptible and MDR strains of E. coli, A. baumannii, K. pneumoniae, P. aeruginosa and E. cloacae. The viable colony forming units (CFUs) were enumerated, expressed as Δlog10CFU for each drug treatment per timepoint in comparison to start log10CFU and the plots generated using GraphPad Prism.
Dose finding studies and efficacy of BWC0977 in the neutropenic murine thigh infection model
These studies were conducted in the laboratory of Professor William Hope by the Antimicrobial Pharmacodynamics and Therapeutics Group (Department of Molecular and Clinical Pharmacology), University of Liverpool, United Kingdom. For the dose finding study, BWC0977 was administered by subcutaneous (SC) injection at 40 mg/kg every 6 h, 80 mg/kg every 12 h or 160 mg/kg every 24 h using a 26 h neutropenic murine dual thigh infection model infected with P. aeruginosa NCTC 13921. Polymyxin B (SC, 25 mg/kg q6h) served as a comparator for this study. Mice were infected intramuscularly with P. aeruginosa NCTC 13921 two hours prior to treatment with BWC0977. Inoculum was adjusted to ~ 1 × 107 CFU /ml and BWC0977 (in 10% ascorbic acid, pH adjusted to 4.6 with sodium bicarbonate) was administered subcutaneously at fractionated dose levels, 40 mg/kg q6h x 4, 80 mg/kg q12 x 2 or 160 mg/kg q24h x1. Animals were euthanized at 2 h post-infection (pre-treatment controls) or serially at 8, 14 and 26 h post-infection (untreated controls and all BWC0977-treated groups); polymyxin B treated group was euthanized at 26 hours post-infection only.
To investigate the efficacy of BWC0977 in the 26 hour neutropenic murine dual thigh infection model following infection with different Gram-negative bacteria, multiple doses (Supplementary Table14) were administered every 8 h (q8h). Mice (male CD-1, n = 3 per group) were infected intramuscularly with a Gram-negative bacterial inoculum. To make the inoculum, bacteria was revived from −80 °C by streaking onto Mueller Hinton agar and incubating for 16–24 h. Single colonies were picked from the agar plate, emulsified into 30 ml Mueller Hinton broth, and placed on shaking incubator overnight. The overnight culture was centrifuged at 4000 x g for 5 min to produce a bacterial pellet. The supernatant was removed, and the pellet resuspended in sterile PBS and adjusted to the correct OD450 (determined from bacterial growth curve studies). Further dilutions were made in PBS to reach the target CFU/mL (as determined by inoculum finding studies). Two hours post-infection, treatment with BWC0977 was administered subcutaneously (SC) at doses (Supplementary Table14) q8h and comparator control. Animals were euthanized at 2-hour post infection (pre-treatment controls), or 26 h post-infection (vehicle control and all treated groups) except for animals that reached their clinical endpoints as follows: one animal in the vehicle control group at 20.5 h post-infection, and one animal at 10 mg/kg at 23 h post-infection. Data was collected from all animals at the time of euthanasia. All animals were monitored, and health scored throughout, increasing in frequency, as needed, when clinical observations started to develop e.g. piloerection, hunched posture etc. All laboratory animal experiments were conducted under UK Home Office project License PAC022930 which was renewed in 2022 as PP3585942. These licenses and all experiments conducted under them were approved by the University of Liverpool Animal Welfare Ethics Review Board.
Plasma protein binding assay
Protein binding was measured using the equilibrium dialysis method48. Compound was added to 10% plasma giving a concentration of 10 µM and dialysed with isotonic buffer for 18 h at 37 oC. The plasma and buffer solutions were analysed using generic LC-UV-MS and the first apparent binding constant for the compound derived. The binding constant was then used to determine the % free fraction in 100% plasma.
The percentage of plasma bound/unbound fraction preparation was calculated as follows:
$$\%{Unbound}=100 \,\ast \,\frac{{F}_{C}}{{T}_{C}}$$
$$\%{Recovery}=100 \,\ast \,\frac{\left({F}_{C}+{T}_{C}\right)}{{T}_{O}}\,$$
Wherein,
Tc = Total plasma concentration was determined by the calculated concentration on the plasma side of the chamber
Fc = Total plasma concentration was determined by the calculated concentration on the buffer side of the chamber
To = Total compound concentration determined before analysis
Inhibition of supercoiling activity of E. coli gyrase
The E. coli gyrase supercoiling activity assays were performed using reagents obtained from Inspiralis Limited, Norfolk. UK). Test compounds were pre-incubated with 1 nM enzyme at 24 oC for 10 min. Reaction was initiated by the addition of 60 ng relaxed pBR322 DNA, and the incubation was continued at 37 oC for 40 min. Reactions were terminated using a mixture of Proteinase K (3 μL 2% SDS + 0.8 μL 20 mg/mL Proteinase K), followed by 30 min incubation at 37 °C. Samples were mixed with 4 μL STEB [40 % (w/v) sucrose, 100 mM Tris-HCl (pH 8), 10 mM EDTA, 0.5 mg/mL bromophenol blue], and resolved by gel electrophoresis (2.5 V/cm for 3 h) on 0.8 % agarose gel in 1X TAE buffer. Gels were stained with 0.8 µg/mL ethidium bromide (EtBr) for 10 min and the DNA bands were imaged using a gel documentation system. The band intensities were quantified using the Quantity One basic software. The compound IC50 values were determined using non-linear regression, four parameter curve-fit using GraphPad Prism software.
Inhibition of decatenation activity of E. coli topoisomerase IV
The E. coli topoisomerase IV decatenation reactions were performed using 2.5 nM enzyme and 60 ng kinetoplast DNA (kDNA) obtained from Inspiralis. The assay protocol followed was like the above-described supercoiling assays.
Homology Modelling of E. coli Gyrase and Topo IV Complexes
The sequences of E. coli gyrA, gyrB, parC and parE were extracted from UniprotKB. The NBTI class of oxabicyclooctane derivative bound SaGyrase crystal structure (PDB ID: 5BS3) was utilized to build EcGyrase complex. In the case of EcTopoIV, A. baumannii TOPO IV crystal structure (PDB ID: 2XKK) served as a template. SWISS-MODEL49 web server was utilized to build the homology models of independent chains and superimposition protocols of PyMOL software were employed to construct the tetrameric complexes. The coordinates of dsDNA molecules extracted from template crystal structures was used to generate the complete complexes.
Input Compound Structure Preparation
BWC0977 was considered for molecular docking, the 2-D structure was sketched using MarvinSketch program and exported in a.SDF file format. The ligand molecules were prepared in LigPrep50 module of the Schrodinger suite. The structure was expected to be at or near a local energy minimum, but not necessarily either a global minimum or the optimal conformation for binding to the target. The prepared ligands by LigPrep (output) were considered for multi-conformation generation by Macromodel conformation search module i.e., Mixed-torsional/Low-mode sampling (MTLM). This model combines a Monte Carlo method of exploring torsional space that efficiently locates the widely separated minima on the potential energy surface with a low-mode conformational search method along the energetically “soft” degrees of freedom. The OPLS_2005 force field was used, and energy minimization was 500 steps of the TNCG method. The energy window for saving structures was set to 21 kJ/mol. The redundancy threshold was 0.5 Å RMSD, and any redundant conformers were removed. The maximum number of conformers to be generated was set to 25 and default parameters were used for the remaining options.
Glide Molecular Docking
The Glide molecular docking51 of Schrodinger suite was utilized to predict the binding mode of BWC0977 with homology modelled EcGyrase and EcTopoIV protein 3-D structures. Bound co-crystal NBTI ligand (PDB ID: 5BS3) was superposed into the modelled complexes of EcGyrase and EcTopoIV to specify the NBTI binding pocket. The docking grid was restricted to NBTI pocket based on the extracted NBTI bound ligand. Before starting the Glide grid generation for molecular docking, homology model structures were processed using the protein preparation wizard. The Glide-SP (standard precision) algorithm utilizes pre-computed grids generated using receptor sites defined by centroids of the crystallographic ligands. The docking protocol starts with the systematic conformational expansion of the ligand, followed by placement at the receptor site. Minimization of ligand in the field of receptor was carried out using the OPLS-AA force field with the default distance-dependent dielectric parameters. The lowest energy poses are then subjected to a Monte Carlo procedure that samples nearby torsional minima. Different compounds can then be ranked using GlideScore, a modified version of the ChemScore function that includes terms for steric clashes and buried polar groups. Default Van der Waal’s scaling was used (1.0 for the receptor and 0.8 for the ligand). Advanced settings were edited to increase the pose sampling. A total of 10,000 poses (default 5000) per ligand were set for the initial phase of docking and the poses per ligand per energy minimization raised to 1000 from 400. The output from the conformation generation method (25 conformers) was considered as the input for molecular docking. A total of 10 poses per ligand were saved as output for post-docking analyses.
Molecular Dynamics
Initial binding orientations of BWC0977 generated by molecular docking approach with modelled structures of EcGyr and EcTopoIV were considered further for Molecular Dynamics simulations. Three completely independent replicas were launched for each system. The individual pieces of the system i.e., protein, water molecule (TIP3P), Mg2+ and Na+Cl− ions at a concentration of 0.15 mM were built and assembled. Proteins and ions were described by the ff99SB52 and the parameter for BWC0977 was obtained with the GAFF253. The energy minimization, equilibration and MD protocol was carried out with the PMEMD program of AMBER18 MD package54. After initial energy minimization, successive steps of NVT and NPT (300 K, 1 bar) MD were performed, with progressive removal of position restraints applied to the protein atoms. Then, simulations were run for 100 ns (NPT ensemble, 300 K, 1 bar) and the first 50 nanoseconds were considered as equilibration and discarded; the last 50 nanoseconds of each replica were retained for analysis. A time step of 2 femtoseconds was used in the production phase and PME (Particle Mesh Ewald)55 was employed for the treatment of long-range electrostatic interactions, with the application of a switch function between 1.0 and 1.2 nm. Temperature was kept at 300 K by the Nose-Hoover scheme56,57 using a time constant for coupling of 1 picosecond. Pressure was maintained at 1 bar by a semi-isotropic Parrinello-Rahman barostat58, with coupling time constant of 5 picoseconds and compressibility of 4.5 × 10−5 bar−1. All bonds to hydrogen atoms were constrained by the LINCS algorithm59. CPPTRAJ module of AmberTools19 program was used for trajectory analysis. Before analysis, ions, and water molecules beyond 5 Å of the ligand position were removed.
Pharmacokinetics of BWC0977 in neutropenic mouse thigh model infected with Pseudomonas aeruginosa NCTC 13921
The objective of this study was to describe the concentrations of BWC0977 in mouse plasma in a neutropenic murine, P. aeruginosa strain NCTC 13921 infected thigh model. Male CD-1 mice were infected with P. aeruginosa NCTC 13921 in both thighs and BWC0977 administered via a single subcutaneous (SC) injection at 10, 40, 80, or 120 mg/kg. Terminal blood samples (3/time-point) were collected for concentration analysis of BWC0977 at 0.5, 1, 2, 4, 6, 8, and 24 h post-dosing. Using the plasma protein binding value of 87%, the free plasma levels were calculated. BWC0977 concentration analysis in mouse plasma was conducted using LC/MS/MS, with a lower limit of quantitation (LLOQ) of 0.25 μg/mL for plasma. Descriptive parameters were calculated (mean and median concentrations) of which dose-proportionality and other descriptive characteristics were determined.
Epithelial lining fluid concentrations in neutropenic mouse thigh model infected with Pseudomonas aeruginosa NCTC 13921
BWC0977 was administered subcutaneously at 10, 40, 80 and 120 mg/kg, q24h in neutropenic CD-1 mice infected intramuscularly with P. aeruginosa NCTC 13921 in the thigh. The ELF was obtained by instilling sterile saline into the lungs and removing saline from the lungs at 0, 0.5, 1, 2, 4, 6, 8 h post-dosing.
Pharmacokinetics of BWC0977 in plasma and pulmonary epithelial lining fluid in a neutropenic lung infection model in rats
100 mg/kg of BWC0977 was administered intravenously for 60 min once to neutropenic rats (male, Wistar) infected with P. aeruginosa ATCC27853 (MIC − 0.25 µg/ml) and plasma samples were taken at 0.5 and 1 hour (during infusion) and post-infusion at 1.25, 1.5, 2.0, 3.0, 5.0, 9.0 and 25 h. Similarly, the epithelial lining fluid (ELF) was obtained by instilling 2 ml of sterile saline into the lungs and removing saline from the lungs. PK data analysis was done using the WinNonlin® software.
Dose fractionation and dose response studies of BWC0977 in rats
For efficacy studies, prior to the start of the infection process, all animals were divided into groups of 2 or 3 animals each. Four days and one day before the date of infection, for inducing neutropenia, each rat was dosed intra-peritoneally with cyclophosphamide equivalent to 150 mg/kg and100 mg/kg respectively. Rats were then placed into an induction chamber and anaesthesia induced by exposing the animals to 3–5% isoflurane in an oxygen flow set at approximately 1 liter per minute. On the day of the infection, a 16−18 h Casein Soybean Digest (CSD) broth culture, was centrifuged and the cells resuspended in sterile normal saline to obtain ~ 109 CFU/ml. Infection was initiated by instilling 35 μl containing ~107 CFU/rat of the inoculum into each nostril of the anesthetized animal with the following strains: A. baumannii ATCC19606, A. baumannii SAC002 (a recent clinical isolate from Bangalore, India; ciprofloxacin & meropenem-resistant), E. coli ATCC BAA-2469 (ciprofloxacin & meropenem-resistant), E. coli ATCC BAA-2471 (ciprofloxacin & meropenem-resistant), E. coli SEC-015 (a recent clinical isolate from Bangalore, India; ciprofloxacin & meropenem-resistant), K. pneumoniae SKB067 (a recent clinical isolate from Bangalore, India; colistin, ciprofloxacin & meropenem-resistant), K. pneumoniae ATCC 13883, K. pneumoniae KPNIH1 (ciprofloxacin & meropenem-resistant), K. pneumoniae MKP103 (ciprofloxacin-resistant), P. aeruginosa ATCC 27853, P. aeruginosa SPA041 (a recent clinical isolate from Bangalore, India; ciprofloxacin-resistant). Four hours post-infection, animals were treated at a constant rate infusion with doses of either BWC0977 {4% L-Ascorbic acid + 1.8 % lactic acid + 2.5% niacinamide in WFI, pH 4.5 with sodium bicarbonate, 3 mg/kg, 10 mg/kg, 30 mg/kg, 100 mg/kg} by constant rate intravenous infusion over 2 h or meropenem (in saline,15 mg/kg bid, intravenous bolus dose) or ciprofloxacin (in MilliQ water, pH 4. 5 adjusted with HCl, 30 mg/kg bid, intravenous bolus dose). Rats were anaesthetized using ketamine 70 mg/kg IP + xylazine 20 mg/kg IP at a dose volume of 10 ml/kg, at the rate of 1 ml/hr, except untreated controls. Animals were euthanized at 4 h post-infection (pre-treatment controls), or 28 h post-infection (vehicle control and all treated groups). The euthanized animals were dipped into 70% ethanol for surface decontamination, lung muscles aseptically excised, weighed, placed into 2 ml broth, and homogenized. Serial ten-fold dilutions of the lung homogenates were prepared in sterile broth, plated onto agar plates and CFUs enumerated following 16−18 h of incubation at 37 °C. The individual log10CFU/g lung values were plotted against C/MIC, AUC/MIC and %T > MIC (24 h) and the relationship was assessed with the appropriate pharmacodynamic model using the GraphPad Prism software. The neutropenic rats were monitored throughout the study for general clinical signs.
Determination of PK-PD index for BWC0977 in the rat lung infection model
Dose-fractionation studies were carried out to identify the PK-PD index that correlates best with the efficacy of BWC0977 in the rat lung infection model using different Gram-negative pathogens. Neutropenic rats were infected intranasally with ~7 × 107 CFU/animal of P. aeruginosa ATCC27853. Four hours post-infection, animals were treated with varying total doses of 450, 400, 350, 300, 150, 75, 40, 2 0,1 0 & 5 mg/kg fractionated as q24h, q12h, or q8h over a 24 h period and administered as a 2 h intravenous infusion, over a period of 24 h. Meropenem (total dose of 30 mg/kg), administered as 15 mg/kg, i.v. bolus or twice daily served as a positive control to validate the study. The mean log10CFU/g lung was estimated in each group. The individual log10CFU/g lung values were plotted against C/MIC, AUC/MIC and %T > MIC (24 h) and the relationship was assessed using the appropriate pharmacodynamic model using the GraphPad Prism software.
Reaction phenotyping
To determine the major human CYP P450 isozymes (1A2, 2C9, 2C19, 2D6 and 3A4) responsible for metabolism of the test compound, using the method was reported earlier60. Working stock solutions (50 µM) of test compound (4 µL) was spiked to 356 µL of CYP isozymes to obtain a final test compound concentration of 0.5 µM and pre-incubated for 10 min at 37 oC. After pre-incubation, 45 µL of the pre-incubation mixture was precipitated with 200 µL of ice-cold acetonitrile containing internal standard (0 min sample) and 5 µL of 10 mM NADPH was added to the mixture. To the remaining mixture, 35 µL master stock solution of NADPH (10 mM) was added to the remaining pre-incubation mixture and incubated on a shaking water bath for 60 min at 37 oC. At each time point (0, 3, 6, 9, 12, 15, 20, 30, 45, and 60 min), 50 µL of incubation mixture was precipitated with 200 µL of ice-cold acetonitrile containing the internal standard. Samples were vortexed for 10 min and centrifuged at 1800 x g for 10 min. After centrifugation, 100 µL of supernatant was diluted with 100 µL of water and analysed by LC-MS/MS analysis.
Similarly, for without co-factor incubations, instead of 10 mM NADPH, 100 mM potassium phosphate buffer was added to the samples and incubated for 60 min at 37 oC. The 0 minute samples (50 µL) were precipitated immediately with 200 µL of acetonitrile containing the internal standard. After 30 and 60 min of incubation time, 50 µL of sample was precipitated with 200 µL of acetonitrile containing the internal standard. After precipitation, the samples were treated like the co-factor samples and analysed using LC-MS/MS.
In vitro - in vivo extrapolation (IVIVE) of clearance in humans and prediction of human pharmacokinetic parameters
The well-stirred model61 was used for predicting human CL using human hepatocyte Clint and free fraction (fu) in human plasma. Liver blood flow rates, liver weights, hepatocellularity and in vitro - in vivo correlation / extrapolation (IVIVC / E) templates were routinely employed.
Briefly, cryo-preserved hepatocytes were thawed and transferred into pre-warmed (maintained at 37 oC) buffer medium, and hepatocytes mixed by gently inverting the tube three times. The cell suspension was centrifuged at 50 x g at room temperature for 5 min. The supernatant was discarded, and cell pellet loosened by gently swirling the centrifuge tube and the hepatocytes resuspended in 2 mL of pre-warmed buffer. The total cell count was determined, and the number of viable cells enumerated by trypan blue dye exclusion method. The acceptable cell viability at the beginning of the assay was ~85%. The hepatocyte suspension was diluted with the buffer to attain a final concentration of 1 million cells/mL (1 × 106 cells/mL). The stock (10 mM) solution of test compounds and positive controls were prepared in DMSO. Subsequently, sub-stock (1 mM) solutions were prepared by diluting 10 µL of 10 mM stock solution with 90 µL of DMSO. The final working stock (1 µM) solution was prepared by diluting 2 µL of sub-stock solution with 1998 µL of incubation media. For the assay, working stock solution (1 µM) was spiked into hepatocyte incubation mixture to obtain a final concentration of 0.5 µM. The final organic content in the assay was <0.1%.
The stability assay was conducted in duplicate (n = 2). Manually, 200 µL of diluted hepatocyte suspension (1 × 106 cells/mL) was added to each well of a 48-well plate. 200 µL of test compound (1 µM) prepared was added in the incubation medium to each of the wells containing hepatocytes. Final concentration of hepatocytes and test compound in the assay were 0.5 × 106 cells/mL (0.5 million cells/mL) and 0.5 µM, respectively. The 48-well plate was placed in an incubator maintained at 37 oC, 5% CO2 atmosphere and 95% relative humidity. The hepatocyte mixture was incubated for 120 min with constant shaking at 250 rpm. At each time-point (0, 5, 10, 15, 30, 60, 90, and 120 min), 50 µL aliquot of hepatocyte mixture was added into 96-deep well plate and precipitated with 200 µL of acetonitrile containing an internal standard. The samples were mixed well in a vortex mixer and centrifuged for 10 min at 2000 x g. After centrifugation, the supernatant (100 µL) was separated and transferred to a fresh 96-well plate and diluted with 100 µL of water. The samples were analyzed using LCMS/MS method.
The metabolic stability was expressed as the percentage of parent remaining and calculated from the peak area ratio of NCE remaining after incubation (tx) compared to the time zero (t0) incubation. The percentage of parent test compound remaining at each time point was calculated by comparing the peak area ratio of test compound after incubation (tx) with peak area of time zero (t0) incubation. Similarly, half-life (t1⁄2) was calculated using the following equation:
$${In}-{vitro}\,{T}_{1/2}=\frac{0.693}{{K}_{{el}}}$$
The intrinsic clearance (Clint) was calculated using the following equations:
$${{CL}}_{{\mathrm{int}}}=\frac{0.693}{{K}_{{el}}}x\frac{{{\rm{\mu }}}{L\,of\,incubation}}{{K}_{{el}}x\,{Number\; of\; cells}/{incubation}}x\,{no}.\,{of}\frac{{cells}}{{gram}}{liver}$$
Scaling factors to represent hypocellularity million cells/gm liver and liver/kg body weight.
P-gp, BCRP and BSEP inhibition and substrate determination
Membrane vesicles were diluted in incubation medium and added to a 96-well incubation plate. To evaluate the test article as an inhibitor - solvent control, test article or inhibitors in DMSO were added to the membrane vesicles (1% v/v of the final reaction volume) and were pre-incubated for 15 min at 37 ± 2 °C. After pre-incubation, the incubation was initiated by the addition of probe substrate and MgATP (4 mM) or MgAMP (4 mM) in incubation medium and incubated for the designated time.
To evaluate the test article as a substrate - solvent control, or inhibitors in DMSO were added to the membrane vesicles (1% v/v of the final reaction volume) and were preincubated for 15 min at 37 ± 2 oC. After pre-incubation, the incubation was initiated by the addition of the probe substrate or the test article and MgATP (4 mM) or MgAMP (4 mM) in incubation medium and incubated for the designated time. At the end of incubation period, an aliquot was collected for recovery measurement. The final protein concentration was 50 μg / incubation. The incubation reaction was terminated by the addition of chilled washing mix and samples filtered using a filter plate. The filtered samples were washed five times with chilled washing mix and dried at room temperature for approximately two hours. Experimental conditions are summarized in Supplementary Tables15, 16. For samples incubated with radiolabeled substrate, the substrate was extracted from the filtered vesicles with scintillation fluid and analyzed using a MicroBeta2 liquid scintillation counter. For samples incubated with unlabeled substrate, the substrate was extracted from the filtered vesicles with 50:50 v/v methanol:water containing internal standard and analyzed by LC MS/MS.
OATP, OAT, OCT and MATE substrate and Km / Vmax determination
Cells were plated onto standard 24-well tissue culture plates in cell culture medium 1 to 3 days prior to the experiment. OATP1B1, OATP1B3, MATE1, MATE2-K and control cells were incubated with butyric acid (10 mM) for 24 h prior to the experiment to induce transporter gene expression. Incubation of HEK293 cells were carried out in HBSS buffer containing sodium bicarbonate (4 mM) and HEPES (9 mM), pH 7.4 (OATP, OAT and OCT) or pH 8.5 (MATE).
Prior to the experiment, cell culture plates (transporter-expressing and control cells) were removed from the incubator, the cell culture medium was removed, and incubation medium was added to the plate to rinse the cell culture medium from the cells. To evaluate BWC0977 as a substrate, incubation medium was replaced with incubation medium containing the inhibitor or solvent control, and the plates were preincubated for 15 or 30 min. After pre-incubation, incubation medium was replaced with incubation medium containing the inhibitor or solvent control and BWC0977 or positive control substrate. Samples were incubated at 37 ± 2 °C in triplicate for the designated time. After incubation, incubation medium was removed with an aliquot collected to measure recovery, and cells were rinsed once with chilled PBS containing 0.2% w/v BSA and twice with chilled PBS. For samples incubated with radiolabeled substrates, the PBS was removed, and sodium hydroxide (0.1 M) was added with pipette mixing to extract the compound from the cells. An aliquot of the medium was added to a 96-well plate, diluted with scintillation fluid, and analyzed on a MicroBeta2 scintillation counter. The concentration of protein in incubations was determined with a BCA Protein Assay Kit. Experimental conditions are summarized in Supplementary Table17.
For samples incubated with unlabeled substrates, the PBS was removed, and 50:50 v/v methanol:water containing an internal standard was added with pipette mixing to extract the compound from the cells for analysis using a LC-MS/MS. When the accumulation of unlabeled substrate was measured, the protein concentration in representative wells was measured as protein concentration that cannot be determined after extraction with organic solvent.
Samples were analyzed by multiple reaction monitoring LC-MS/MS methods developed at the testing facility. Analysis was performed with an appropriate SCIEX or Waters mass spectrometer equipped with a Shimadzu Nexera, Shimadzu Prominence or Waters Acquity LC system interfaced by electrospray ionization.
Pharmacokinetic studies in mice and rats
Intravenous (i.v) formulations for pharmacokinetic (PK) studies were formulated in different vehicles and excipients to achieve the desirable solubility for intravenous route of administration. An appropriate amount of the test compound was weighed and dissolved in the required volume of vehicle, followed by vortexing for a few seconds to dissolve the compound. Then the solution (or) suspension was sonicated at room temperature for 5 min to obtain a visually clear solution and or homogenous suspension. All the formulations were prepared freshly at room temperature before dosing. All these formulations were observed to be stable at room temperature for more than 24 h. The formulation details of each of the compound tested is shown in Supplementary Table18.
The rodent PK studies were carried out in male Sprague-Dawley (SD) rats (8–12 weeks of age, weighing 280 ± 20 gm at the time of dosing) and CD1 mice (8−12 weeks of age, weighing 30–35 gm body weight at the time of dosing) to estimate the plasma clearance, VD and terminal half-life, area under curve (AUC) and peak plasma concentration (Cmax) and time of peak plasma concentration (Tmax) following intravenous routes of administration.
Rats were anaesthetized by using isoflurane. The jugular and femoral veins of rat were cannulated, and the study was performed 48 h post-cannulation.
At each time point, about 100 µL of rat blood was collected from the jugular vein into a labelled microfuge tube containing 200 mM K2EDTA solution (20 µL per mL of blood) and equivalent volume of heparinized saline was replaced following sample collection. Similarly, 25 µL of mice blood was collected from the saphenous vein into a labelled microfuge tube containing 200 mM K2EDTA solution. Serial blood sampling method was used for blood collection. Blood samples were collected at pre-dose, 0.25 h, 0.5, 1, 2, 4, 6, 8 and 24 h post-dosing. The blood samples were processed to obtain the plasma samples within 30 min of the scheduled sampling time. All plasma samples were stored at −70 °C until bioanalysis was performed.
Bile duct cannulation (BDC) study in rats
The rat BDC study was carried out in male Sprague-Dawley (SD) rats (8–12 weeks of age, weighing 280 ± 20 gm at the time of dosing) to estimate the plasma clearance, VD and terminal half-life, AUC and Cmax and Tmax following an intravenous route of administration. Rats were anaesthetized using isoflurane anaesthesia. The bile duct, jugular and femoral veins of rat were cannulated, and the study was performed 48 h post-cannulation. At each time point, about 100 µL of rat blood was collected from the jugular vein into a labelled microfuge tube containing 200 mM K2EDTA solution (20 µL per mL of blood) and equivalent volume of heparinized saline was replaced following sample collection. Bile, urine and faeces were collected at 0–2, 2–4, 4–8, 8–12 and 12–24 h post-dose administration. The blood samples were processed to obtain the plasma samples within 30 min of scheduled sampling time and other matrices (bile, urine and faeces) as well. All samples were stored at −70 °C until the bioanalysis was performed.
Guinea pig pharmacokinetic study
The formulation details of the compound tested is shown in Supplementary Table18.The study was performed in fasted, male Dunkin Hartley guinea pigs. A total of 6 animals were used and were catheterized to the jugular vein for infusion and the femoral artery for blood collection. Test item formulations were prepared on the day of treatment. Blood samples were collected through catheterized femoral artery at pre-dose (0 min) and 0.25, 0.5, 1 (at the end of infusion), 1.083, 3, 5, 7, 9 and 24 h post-dose. At each time point, 0.3 mL of blood was withdrawn and transferred into pre-labelled 0.5 mL micro centrifuge tubes containing 10 μL of 1000 IU/mL heparin sodium as anticoagulant and mixed gently to facilitate mixing of anticoagulant with the blood. Blood samples were kept on ice bath until centrifugation. The collected blood samples were centrifuged at 2000 x g for 10 min at 4 °C. Plasma was separated and transferred into pre-labelled tubes and stored at −20 °C until analysis was performed.
Non-rodent (dog) pharmacokinetic study
The formulation details of the compound tested is shown in Supplementary Table18. The dog PK study was carried out in male Beagle dogs of minimum 10 kg body weight to estimate the plasma clearance, VD and terminal half-life, area under curve (AUC) and peak plasma concentration (Cmax), time of peak plasma concentration (Tmax) following an intravenous infusion route of administration. Briefly, compound was administered to a group of 3 non-naïve male dogs. The first set of 3 animals received an intravenous infusion and following a washout period of 4 days, received a higher dose administration. Blood samples (1 mL) was collected from the jugular vein by venepuncture into tubes containing K2EDTA anticoagulant at the following sampling times: Pre-dose, 0.083 (5 min), 0.25, 0.5, 1, 2, 4, 8 and 24 h post-dosing. Immediately following collection, blood samples were inverted to ensure mixing with anticoagulant and placed on wet ice. As soon as practically possible, samples were centrifuged (2000 x g, 10 min, at 4 °C) and the resultant plasma decanted into appropriately labelled polypropylene tubes in 96-well plate format and stored in a freezer set to maintain a temperature of ≤ −65 °C, until analysis was performed.
Bioanalysis of plasma samples
Plasma samples (mice/rat/guinea pig/dog) were analysed using a fit-for purpose LC-MS/MS method. For guinea pig sample analysis (range was 40.1−20100 ng/mL) for mice, rats, and dogs (range was 1–3000 ng/mL).
Pharmacokinetic data analysis
Pharmacokinetic parameters were estimated using Phoenix® WinNonlin® version 6.4 or higher (Certara USA, Inc., Princeton, New Jersey). A non-compartmental approach consistent with the intravenous infusion route of administration was used for parameter estimation. The individual plasma concentration-time data was used for pharmacokinetic calculations. In addition to parameter estimates for individual animals, descriptive statistics (e.g., mean, standard deviation, coefficient of variation, median, min, max) have been reported, as appropriate.
hERG screening data
BWC0977 was tested for inhibition of the human ether-a-go-go related gene (hERG) K+ channel using QPatch HTX automated electrophysiology62. BWC0977 was solubilised to 100 mM in DMSO before dilution in HBPS to 300 μM. A 6-point concentration-response curve was generated using 3.16-fold serial dilutions from the top test concentration. Electrophysiological recordings were made from a Chinese hamster ovary cell line stably expressing the full-length hERG potassium channel. Single cell ionic currents were measured in whole-cell patch clamp configuration at room temperature (21−23 oC) using the QPatch HTX platform (Sophion). Intracellular solution contained (in mM): 120 KF, 20 KCl, 10 EGTA,10 HEPES and was buffered to pH7.3. The extracellular solution (HEPES-buffered physiological saline, HBPS) contained (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 % glucose buffered to pH7.4. Cells were clamped at a holding potential of −80mV. Cells were stepped to +20 mV for 2 seconds then −40 mV for 3 seconds before returning to the holding potential. This sweep was repeated 10 times at 10 second intervals. hERG currents were measured from the tail step and referenced to the holding current. Compounds were then incubated for 2 min prior to a second measurement of ion channel current using an identical pulse train. The IC50 values were obtained from a 4-parameter logistic fit of the concentration-response data. Reference compound (cisapride) values were consistent with those reported in the literature.
Prediction of human PK parameters based on simple allometry of pre-clinical PK parameters estimated from PK modelling of combined species mixed effect model approach
The human PK profile was predicted based on four species data using Non-Linear Mixed Effect (NLME) model63. Specifically, total PK data from mice (0.023 kg), rat (0.282 kg), guinea pig (0.43 kg) and dog (10.5 kg) studies were extracted, and the combined species data were fit together to different 1, 2 and 3 compartment IV bolus/infusion PK models, respectively, following different weighting scheme. The best fit PK model among the fitting was a two-compartment IV bolus/ infusion PK model with a 1/Y2 predicted weighting scheme. Individual species data was fit to a two-compartment intravenous model and PK parameters such as V1, CL, V2, CLD2 were estimated. For all four species, individual PK parameters were individually scaled as per simple allometry and respective allometric exponents and coefficients were determined.
Combined species data was fit using NLME allometric model using Phoenix NLME (ver 6.4). Human PK parameters such as V1, CL, V2 and CLD2 were estimated along with corresponding exponents and allometric coefficients.
$${{CL}}_{{ind}}=a \,\ast\, {{BW}}_{{ind}}^{b}$$
(1)
$${V1}_{{ind}}=c \,\ast\, {{BW}}_{{ind}}^{d}$$
(2)
$${V2}_{{ind}}=e \,\ast\, {{BW}}_{{ind}}^{d}$$
(3)
$${{CLD}2}_{{ind}}=g \,\ast\, {{BW}}_{{ind}}^{b}$$
(4)
Following the 2-compartment model fitting and using predicted human PK parameters human profile (plasma concentrations vs time) the PK profile following different 80mg–1050mg single dose via IV infusion administration for 120 min was predicted.
First-in-human study of BWC0977 administered intravenously for 2 h as single ascending doses (SAD)
A Phase 1, randomized, double-blind, placebo-controlled, single dose escalation study to evaluate the safety, tolerability and PK of BWC0977 administered through IV infusion to healthy adult subjects has been completed in CMAX, Adelaide, Australia. The first in human trial was initiated following approval by the Human Research Ethics Committee (HREC) of the trial design, protocol and the key criteria specified for the study. Within each cohort of 8 subjects, efforts were made to randomize approximately equal numbers of males and females to either active or placebo (including both genders).
In single ascending dose (SAD) escalation study, 40 subjects in five dose cohorts of 8 subjects each (6 active, 2 placebo) were randomized to receive single IV infusion doses of BWC0977 or placebo infused over 120 ( ± 10 min) (Supplementary Table19). Five dose levels of BWC0977 were assessed according to an ascending single-dose regimen (120 mg, 240 mg, 480 mg, 720 mg, 1050 mg). The starting dose is based on safety results from non-clinical studies.
All details pertaining to formulation, screening of subjects, written informed consent, admission and duration of hospitalization, evaluation parameters, inclusion and exclusion criteria, study endpoints, PK monitoring, statistical analysis and safety aspects of the trial are available at ClinicalTrials.gov ID: NCT05088421.
Chemistry: General chemical methods
All commercial reagents and solvents were used without further purification. Analytical thin-layer chromatography (TLC) was performed on SiO2 plates on alumina. Visualization was accomplished by UV irradiation at 254 and 220 nm. Purity of all the final derivatives for biological testing was confirmed to be >95%.
Evaporations were carried out by rotary evaporation in vacuo and work-up procedures were carried out after removal of residual solids by filtration; temperatures are quoted as °C; operations were carried out at room temperature, that is typically in the range 18−26 °C and without the exclusion of air unless otherwise stated, or unless the skilled person would otherwise work under an inert atmosphere; column chromatography (by the flash procedure) was used to purify compounds and was performed on Merck Kieselgel silica (Art. 9385) unless otherwise stated.
In general, the course of reactions was followed by TLC, HPLC, or LC/MS and reaction times are given for illustration only; yields are given for illustration only and are not necessarily the maximum attainable. The structure of the end products was generally confirmed by NMR and mass spectral techniques. Proton magnetic resonance spectra were generally determined in DMSO d6 unless otherwise stated, using a Bruker DRX 300 spectrometer or a Bruker DRX-400 spectrometer, operating at a field strength of 300 MHz or 400 MHz, respectively. In cases where the NMR spectrum is complex, only diagnostic signals are reported. Chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane as an external standard (δ scale) and peak multiplicities are shown, thus: s - singlet; d - doublet; dd - doublet of doublets; dt - doublet of triplets; dm - doublet of multiplets; t - triplet; m - multiplet; br - broad. Fast atom bombardment (FAB) mass spectral data were generally obtained using a Platform spectrometer (upplied by Micromass) run in electrospray and, where appropriate, either positive ion data or negative ion data were collected using Agilent 1100 series LC/MS equipped with Sedex 75ELSD. The lowest mass major ion is reported for molecules where isotope splitting results in multiple mass spectral peaks (for example when chlorine is present). Reverse-phase HPLC was carried out using YMC Pack ODS AQ (100×20 mmID, S 5 Å particle size, 12 nm pore size) on Agilent instruments; each intermediate was purified to the standard required for the subsequent stage and was characterized in sufficient detail to confirm that the assigned structure was correct; purity was assessed by HPLC, TLC, or NMR and identity was determined by infrared spectroscopy (IR), mass spectroscopy or NMR spectroscopy as appropriate. HRMS data was acquired using an Agilent 6520, Quadrupole-time of flight tandem mass spectrometer (Q-Tof MS/MS) coupled with an Agilent 1200 series HPLC system.
Synthesis of compound 1-9
Compound 1 (CAS: 2156619-18-8; 6-[5-(3-Aminopropyl)−2-oxo-3-oxazolidinyl]−2H-pyrido[3,2-b]−1,4-oxazin-3(4H)-one)
Compound 1 (CAS: 2156619-18-8) was synthesized as reported earlier in the patent WO2017199265A164. 1H NMR (400 MHz, DMSO-d6): δ 7.71 (bs, 1H), δ 7.59 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 4.68-4.75 (m, 1H), 4.61 (s, 2H), 4.22 (dd, J = 8.4, 10 Hz, 1H), 3.70 (dd, J = 6.6 Hz, 10 Hz, 1H), 2.85 (t, J = 8.0 Hz, 2H), 1.73-1.81 (m, 2H), 1.61-1.71 (m, 2H); LCMS calculated for C13H16N4O4, 292.30 Observed = 293.2; HRMS calculated for C13H16N4O4, 292.30, Observed = 293.1298;
Compound 2 (CAS: 2156618-99-2; 6-[5-[3-[[(6-Fluoro-2,3-dihydro-1-methyl-2-oxo-1H-indol-7-yl) methyl] amino]propyl]−2-oxo-3-oxazolidinyl]−2H-pyrido[3,2-b]−1,4-oxazin-3(4H)-one)
Compound 2 (CAS: 2156618-99-2) was synthesized as reported earlier in the patent WO2017199265A1. 1H NMR (400 MHz, MeOD): δ 8.44 (bs, 1H), 7.70 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.30–7.34 (m, 1H), 6.87–6.92 (m, 1H), 4.74-4.76 (m, 1H), 4.63 (s, 2H), 4.39 (s, 2H), 4.31–4.36 (m, 1H), 3.84–3.88 (m, 1H), 3.67–3.70 (m, 2H), 3.54–3.58 9 m, 5H), 3.08–3.10 (m, 2H), 1.84–1.90 (m, 4H); LCMS calculated for C23H24FN5O5, 469.47 Observed = 470.0; HRMS calculated for C23H24FN5O5, 469.47 Observed = 470.3601; HPLC = 87.33% (Zorbax Eclipse plus C18 RRHD(50×2.1) mm,1.8μ; Mobile phase A:0.1%TFA in WaterB:Acetonitrile);
Compound 3 (CAS: 2156619-07-5; 6-[(5S)−5-[2-[[2-(6-fluoro-2,3-dihydro-1-methyl-2-oxo-1H-indol-7-yl)ethyl]amino]ethyl]−2-oxo-3-oxazolidinyl]−2H-pyrido[3,2-b]−1,4-oxazin-3(4H)-one)
Compound 3 (CAS: 2156619-07-5) was synthesized as reported earlier in the patent WO2017199265A1. 1H NMR (400 MHz, DMSO-D6): δ 11.23 (s, 1H), 7.59 (d, 1H, J = 8.4 Hz), 7.44 (d, 1H, J = 8.8 Hz), 7.18–7.15 (m, 1H), 6.86–6.82 (m, 1H), 4.79–4.76 (m, 1H), 4.62 (s, 2H), 4.26–4.22 (m, 1H), 3.79–3.75 (m, 1H), 3.53 (s, 2H), 3.12–3.10 (m, 2H), 2.96 (s, br, 4H), 2.03 (s, br, 2H). LCMS Calc. for Calc. for C23H24FN5O5, 469.47; Obs 470.0; [M+ + H]. HRMS Calc. for C23H24FN5O5, 469.47; Obs 470.1190; HPLC Purity = 95.07% (HPLC Column: Atlantis dC18 (250*4.6) mm 5 µm, Mobile Phase A: 0.1% TFA in water, Mobile Phase B: Acetonitrile.).
Compound 4: (S)−6-(5-(((2-(6-fluoro-1-methyl-2-oxoindolin-7-yl) ethyl) amino) methyl)−2-oxooxazolidin-3-yl)−2H-pyrido[3,2-b] [1,4] oxazin-3(4H)-one
Compound 4 was synthesized using the procedure for compound 3 as reported earlier in the patent WO2017199265 A1 and WO2018225097 A1.
1H NMR (400 MHz, DMSO-D6): δ 11.20 (s, 1H), 7.60 (d, 1H, J = 8.8 Hz), 7.44 (d, 1H, J = 8.8 Hz), 7.12–7.09 (m, 1H), 6.82–6.77 (m, 1H), 4.77 (s, br, 1H), 4.62 (s, 2H), 4.15–4.11 (m, 1H), 3.87–3.83 (m, 1H), 3.49 (s, 2H), 3.38 (m, 3H), 3.03–3.01(m, 2H), 2.99–2.96 (m, 2H), 2.84–2.82 (m, 2H). LCMS Calc. for Calc. for C22H22FN5O5, 455.45; Obs :456.2; [M + + H]; HRMS Calc. for Calc. for C22H22FN5O5, 455.45; Obs :456.1166; HPLC Purity = 98.13% (HPLC Column: Atlantis dC18 (250*4.6) mm 5 µm, Mobile Phase A: 0.1% TFA in water, Mobile Phase B: Acetonitrile, RT = 8.90 min.
Compound 5 (CAS: 2254566-42-0; 6-[(5S)−5-[[[2-(7-Fluoro-1,2-dihydro-1-methyl-2-oxo-8-quinolinyl)ethyl]amino]methyl]−2-oxo-3-oxazolidinyl]−2H-pyrido[3,2-b]−1,4-oxazin-3(4H)-one)
Compound 5 (CAS: 2254566-42-0) was synthesized as reported earlier in patent WO2018225097 A165.
1H-NMR (400 MHz, DMSO-d6): δ 11.19 (brs,1H), 7.84 (d, J = 9.16 Hz, 1H), 7.64–7.58 (m, 2H), 7.42 (d, J = 8.76 Hz, 1H), 7.13 (t, J = 9.12 Hz, 1H), 6.53 (d, J = 9.28 Hz, 1H), 4.70 (s, 1H), 4.61 (s, 2H), 4.11 (t, J = 9.40 Hz, 1H), 3.86–3.82 (m, 1H), 3.71 (s, 3H), 3.13–2.84 (m, 2H), 2.80–2.67 (m, 4H). HRMS Calc. for C23H22FN5O5, 467.46; Obs.: 468.1665. LCMS Calc. for C23H22FN5O5, 467.46; Obs.: 468.3 [M+ + H] +.
Compound 6 (CAS: 2254566-55-5); 2-[(5S)−5-[[[2-(7-Fluoro-1,2-dihydro-1-methyl-2-oxo-8-quinolinyl)ethyl]amino]methyl]−2-oxo-3-oxazolidinyl]−6H-pyrimido[5,4-b][1,4]oxazin-7(8H)-one)
Compound 6 (CAS: 2254566-55-5) was synthesized as reported earlier in WO2018225097 A1 patent. 1H-NMR (400 MHz, DMSO-d6): δ 8.22 (s, 1H), 8.16 (s, 1H), 7.85 (d, J = 9.20 Hz, 1H), 7.64 (t, J = 6.80 Hz, 1H), 7.15 (t, J = 8.80 Hz, 1H), 6.55 (d, J = 9.20 Hz, 1H), 4.72 (s, 3H), 4.13 (t, J = 9.20 Hz, 1H), 3.89–3.85 (m, 1H), 3.73 (s, 3H), 3.17–3.15 (m, 2H), 2.92-2.84 (m, 4H).LC_MS: Calc. for C22H21FN6O5, 468.45; Obs. 469.1 [M + H] +.
Compound 7 (CAS: 2254566-91-9; 6-[(5S)−5-[[[2-(6-Fluoro-3,4-dihydro-4-methyl-3-oxo-5-quinoxalinyl)ethyl]amino]methyl]−2-oxo-3-oxazolidinyl]−2H-pyrazino[2,3-b]−1,4-oxazin-3(4H)-one)
Compound 7 (CAS: 2254566-91-9) was synthesized as reported earlier in WO2018225097 A1. 1H-NMR (400 MHz, DMSO-d6): δ 11.61 (s, 1H), 8.38 (s, 1H), 8.16 (s, 2H), 7.72 (t, J = 6.64 Hz, 1H), 7.25 (t, J = 9.28 Hz, 1H), 4.87 (s, 2H), 4.77 (brs, 1H), 4.07 (t, J = 8.92 Hz, 1H), 4.05–3.75 (m, 4H), 3.17–3.10 (m, 2H), 2.91–2.85 (m, 4H). LCMS Calc. for C21H20FN7O5, 469.43; Obs.: 468.0 [M+-H]. HRMS Calc. for C21H20FN7O5, 469.43; Obs.: 470.0989. HPLC Purity = 93.26% (HPLC Column: Atlantis dC18 (250*4.6) mm 5 µm, Mobile Phase A: 0.1% AcOH in water, Mobile Phase B: Acetonitrile.
Compound 8 (CAS: 2254566-53-3; 6-[(5 R)−5-[[[2-(7-Fluoro-1,2-dihydro-1-methyl-2-oxo-8-quinolinyl)ethyl]amino]methyl]−2-oxo-3-oxazolidinyl]−2H-pyrazino[2,3-b]−1,4-oxazin-3(4H)-one)
Compound 8 (CAS: 2254566-53-3) was synthesized as reported earlier in WO2018225097 A1. 1H NMR (400 MHz, DMSO-d6): δ 11.62 (s, 1H), 9.16 (brs, 1H), 8.40–8.38 (m,1H), 7.89–7.86 (m, 1H), 7.73–7.69 (m, 1H), 7.22–7.17 (m, 1H), 6.59–6.55 (m, 1H), 5.01 (brs, 1H), 4.88–4.87 (m, 2H), 4.23–4.19 (m, 1H), 3.83–3.79 (m, 1H), 3.73–3.72 (m, 3H),3.40–3.34 (m, 4H), 3.14 brs, 2H). LCMS Calc. for C22H21FN6O5, 468.45; Obs.: 467.1 [M+-H]. HRMS Calc. for C21H20FN7O5, 468.45; Obs.: 469.0989. HPLC Purity = 96.38% (HPLC Column: Atlantis dC18 (250*4.6) mm 5 µm, Mobile Phase A: 0.1% TFA in water, Mobile Phase B: Acetonitrile).
Compound 9 (CAS: 2254567-00-3; 6-[(5S)−5-[[[2-(7-Fluoro-1,2-dihydro-1-methyl-2-oxo-8-quinolinyl)ethyl]amino]methyl]−2-oxo-3-oxazolidinyl]−2H-pyrazino[2,3-b]−1,4-oxazin-3(4H)-one)
Compound 9 (CAS: 2254567-00-3) was synthesized as reported earlier in WO2018225097 A1. 1H NMR (400 MHz, DMSO- d6): δ 11.61 (bs, 1H), 8.38–8.37 (d, 1H, J = 3.2 Hz), 8.14 (s, 1H), 7.84–7.82 (d, 1H, J = 9.2 Hz), 7.64–7.60 (dd,1H, J1 = 8.8, J2 = 6.4 Hz), 7.15–7.10 (t, 1H, J = 8.8 Hz), 6.54–6.52 (d, 1H, J = 9.2 Hz), 4.85 (s, 2H), 4.79–4.76 (m, 1H), 4.11-4.06 (m, 1H), 3.83–3.79 (m, 1H), 3.71 (s, 3H), 3.16–3.13 (m, 2H), 2.95–2.91 (m, 2H), 2.89–2.82 (m, 2H). LCMS Calc. for C22H21FN6O5, 468.45; Obs.: 469.1; [M+ + H]. HRMS Calc. for C22H21FN6O5, 468.45; Obs.: 469.1077; HPLC Purity = 97.52%, Column: Atlantis dC18 (250 × 4.6) mm, 5μm, Mobile Phase A: 0.1% TFA in water, Mobile Phase B: Acetonitrile.
Reporting summary
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