Spirocycle MmpL3 Inhibitors with Improved hERG and Cytotoxicity Profiles as Inhibitors of Mycobacterium tuberculosis Growth

With the emergence of multi-drug-resistant strains of Mycobacterium tuberculosis, there is a pressing need for new oral drugs with novel mechanisms of action. A number of scaffolds with potent anti-tubercular in vitro activity have been identified from phenotypic screening that appear to target MmpL3. However, the scaffolds are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-à-go-go related gene (hERG) and IKr potassium current modulation. The spirocycle compound 3 was reported to target MmpL3 and displayed promising efficacy in a murine model of acute tuberculosis (TB) infection. However, this highly lipophilic monobasic amine was cytotoxic and inhibited the hERG ion channel. Herein, the related spirocycles (1–2) are described, which were identified following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis. The novel N-alkylated pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterion series, exemplified by lead 29, with decreased HepG2 cytotoxicity as well as limited hERG ion channel inhibition. Strains with mutations in MmpL3 were resistant to 29, and under replicating conditions, 29 demonstrated bactericidal activity against M. tuberculosis. Unfortunately, compound 29 had no efficacy in an acute model of TB infection; this was most likely due to the in vivo exposure remaining above the minimal inhibitory concentration for only a limited time.


■ INTRODUCTION
Mycobacterium tuberculosis, 1 the causative agent of tuberculosis (TB), can be fatal if not properly treated and disproportionately affects the poor in developing countries. In 2015, TB became the world's most deadly infectious disease, killing 1.4 million people (1.2 million HIV-negative and 0.3 million HIV-positive) in 2019. 2 The current 6 month treatment results in high default rates, increased transmission, and drug resistance. 3−5 In order to reduce treatment length, TB treatments working through novel mechanisms are needed. 6−8 However, identifying novel drugs remains a significant challenge, and high-quality leads are still urgently required. 9,10 Target-directed TB drug discovery programs have historically been largely unsuccessful in delivering high-quality late-stage leads. 7 To address this issue, cell-based phenotypic screening became a focus for identifying active starting points. Many of the most potent phenotypic hits target membrane proteins such as DprE1 and MmpL3 that are involved in cell wall biosynthesis. 11 MmpL3 is required for the export of trehalose monomycolates (TMM) to the periplasmic space and outer membrane of M. tuberculosis. A number of structurally diverse putative MmpL3 inhibitor series have been reported. 12−29 Several of these series inhibit MmpL3-mediated TMM export but may also have pleiotropic effects targeting the proton motive force. 13 The scaffolds of MmpL3 inhibitors are typically lipophilic, which facilitates partitioning into hydrophobic membranes, and several contain basic amine groups. Highly lipophilic basic amines are typically cytotoxic against mammalian cell lines and have associated off-target risks, such as inhibition of human ether-a-go-go related gene (hERG) and IKr potassium current modulation. One particular spirocyclic series has been reported as a potential MmpL3 inhibitor following a phenotypic screen of the GSK library against Mycobacterium bovis. 19 Although further exploration of this original hit identified compounds with excellent in vivo activity against M. tuberculosis (3), the series was discontinued because of concerns over safety related to the lipophilicity and basic nature of the scaffold. 30 Of note, the original authors highlighted that further exploration may be able to design around the series liabilities while retaining the remarkable in vivo potency 30 Herein, we report on a novel pyrazole spirocyclic amine series (1 and 2) with a putative MmpL3 mechanism of action that is structurally related to 3 ( Table 1). The novel pyrazole portion offered improved physicochemical properties, and optimization led to identification of a zwitterionic series, with improved selectivity over both HepG2 cytotoxicity and hERG inhibition. The zwitterionic series retained potent M. tuberculosis whole cell activity, with large shifts against MmpL3 mutant strains. Unfortunately, the series representative with the best overall properties, 29, failed to show efficacy in an acute model of TB infection. As such, further work on this series was put on hold. We feel that the approach presented indicates useful insights into mechanisms for reduction of metabolism and hERG liabilities while highlighting the challenge within drug discovery of balancing these properties with potency.
■ RESULTS AND DISCUSSION Structure−Activity Relationship. To identify novel antitubercular agents, an aerobic whole cell phenotypic screen was undertaken, evaluating the Eli Lilly corporate screening deck against M. tuberculosis strain H37Rv. One of the outcomes of this screen was the identification of a cluster of pyrazolecontaining spirocyclic amine analogues; the spirocyclic portion of the molecule showed a clear structural resemblance to 3, a previously reported spirocycle series (Table 1). 19,30 The initial hits had good anti-tubercular activity with a minimum inhibitory concentration (MIC) of 0.22 and 0.14 μM for 1 and 2, respectively ( Table 1). As the original spirocycle compound was considered to be an MmpL3 inhibitor, we tested the activity of these two compounds against a strain containing a mutation in MmpL3 (F255L) and observed a large shift in the MIC confirming the likely on-target activity ( Table  1).
According to the literature, 3 had not been developed further because "it suffered from a high clog P value, with the consequent potential liabilities for further development". 19,30,31 Compounds 1 and 2 offered an attractive alternative to 3 because the central aromatic ring in both was a pyrazole not a phenyl; this modification was predicted to reduce the clog D pH7.4 by around 1 log unit. The reduced lipophilicity translated into a better solubility forecast index and M. tuberculosis-derived LLE, 32−35 providing optimism for improving off-target liabilities, metabolic stability, as well as solubility profile. Unfortunately, as a result of the basic nature of the piperidine group, hERG inhibition remained an issue for 1 and 2; moreover, despite the lower clog D pH7. 4 , both showed unexpectedly high mouse microsomal clearance. Initial emphasis for expansion of the confirmed hits 1 and 2 was placed on understanding the scope for improving hERG channel inhibition and microsomal stability. The pyrazole of 1 and the spirocycle of 2 were combined to afford 4, which showed marginally improved selectivity over the hERG channel, although the mouse microsomal clearance remained high (Tables 1 and 2). In addition, the human microsomal metabolic stability was higher for 4 than the published molecule  3, although both values were lower than the equivalent mouse data. As a first step to attempt to reduce metabolism of 4, a mouse microsomal metabolite identification study was carried out to evaluate why the pyrazole series had worse microsomal stability than 3 despite a lower clog D pH7.4 (Table 1). This showed that 4 was metabolized rapidly to four main metabolites primarily involving hydroxylation. After a 3 min incubation in mouse microsomes, only 29% of the parent ion remained, while 61% of the detectable ions were associated with three metabolites that resulted from hydroxylation associated with regions of the molecule around the spirocycle group (Table S1; Figures S1 and S2). Because phenyl groups are known to be prone to hydroxylation, which can be prevented by fluorination, 36 the phenyl substituent on the spirocycle was replaced by a series of different fluoro-substituted phenyl groups (Table 2). Unfortunately, compounds that retained good MIC activity remained very unstable in mouse microsomes, while the compounds with improved metabolic stability had significantly decreased potency (Table 2). To examine the metabolic stability further, isoform-specific cytochrome P450 studies were carried out on 4, evaluating CYP3A4 and CYP2D6 as the two most abundant human CYP450 enzymes. Pyrazole 4 was more rapidly cleared by CYP2D6 bactosomes (0.34 min −1 ) than CYP3A4 bactosomes (0.07 min −1 ). As the CYP2D6-active site is known to be smaller in comparison to CYP3A4, 37 it was proposed that increasing the size of substituents on either the 5-methyl pyrazole or the phenyl in 4 could potentially lead to a decrease in CYP2D6 metabolism and thereby improve the metabolic stability to be more in line with the larger compound 3.
To test the above hypothesis, the structure−activity relationship (SAR) around the methyl and phenyl substituents was expanded. Initial modifications to the 5-methyl pyrazole had little effect on MIC, hERG channel inhibition, or metabolic stability (11−14), and so further exploration of this substituent was put on hold. For the phenyl substituted compounds 15− 17, there was a trend toward improved metabolic stability, in particular with the bulky OCF 3 blocking group of 17 despite it having a high clog D pH7.4 (Table 3). In an attempt to reduce or maintain as low a clog D pH7.4 as possible, the SAR around a 1pyridyl substituent was explored. For the pyridyl-substituted compounds 18−20, again there was a trend toward improved metabolic stability, in particular with bulkier and more lipophilic groups such as the CF 3 in 20 (Table 3).
As an alternative phenyl substitution, the addition of a carboxylic acid group was explored (Table 3). Such a modification adds bulk, introduces polarity, and the presence of a zwitterion has been shown previously to overcome hERG channel inhibition. 38 While the ortho-acid 21 was not tolerated, the modest M. tuberculosis whole cell activity of 22 and 23 was encouraging especially because the para-acid 23 showed a dramatic improvement in the compound's liabilities including hERG channel inhibition, mouse metabolic stability, and HepG2 cytotoxicity. Although the overall properties of 23 were an improvement on 4, this came at a significant reduction in antibacterial potency (∼40 fold). To continue optimization of 23, we reassessed modifications of the 5-methyl pyrazole as these had been tolerated previously on 4. In general, substitutions 29−34 were well tolerated, apart from cyclic amines 33 and 34, with good profiles in relation to hERG inhibition, mouse metabolic stability, and HepG2 cytotoxicity. Substituted alkyl as well as fluoroalkyl resulted in notable improvements in the whole cell potency for cyclopropyl 29, trifluoromethyl 28, ethyl 24, and ethoxy 32 (Table 3).
Biological Profiling. Compound 29 was not cytotoxic to HepG2 cells, grown in either glucose or galactose media, nor the THP-1 macrophage-like cell line (data not shown). 29 had good potency against intracellular bacteria in THP-1 cells [IC 50 = 1.5 ± 1.3]. Compound 29 was also shown to retain good activity against clinical samples from the four main lineages and  strains containing resistance mutations to either isoniazid or rifampicin (Table 4). Minimum bactericidal concentration (MBC) and kill kinetics were evaluated under both aerobic (replicating) and starvation (nonreplicating) conditions. Under replicating conditions, 29 showed concentration-dependent kill of M. tuberculosis with an MBC of 3 μM ( Figure 1A). At later stages, outgrowth was observed because of either compound instability over long incubation periods or the appearance of resistant mutants. Under nonreplicating conditions, 29 showed no bactericidal effect against M. tuberculosis ( Figure 1B).
Compound 29 showed a 10-fold decrease in activity against the MmpL3 F255L strain of M. tuberculosis, indicating that it retained an MmpL3-related mechanism of action. An impact on this pathway was also indicated using a hypomorph strain (P606-5C-mmpL3) that underexpresses MmpL3 when grown in the absence of anhydrotetracycline ( Figure 2). Initially, it was confirmed that SQ109, a known inhibitor of MmpL3, was more active against the hypomorph strain with a four-fold improvement in MIC 50 , while ethambutol, a cell wall inhibitor targeting a different pathway (arabinosyl transferases), was equally effective against both the wild-type and hypomorph strains. We tested two representatives from the series (4 and 23); both showed significant shifts in potency, with the MmpL3 hypomorph being at least eight-fold more sensitive. These data support the conclusion that, as shown in the literature for 3, the series works through an MmpL3-related mechanism.
In Vivo Analysis. Based on the hit to lead SAR, representative compound 29 was selected for follow-up. A fluorinated derivative, 35, was also prepared as such modifications had been shown previously (Table 2) to not have a detrimental effect on MIC activity but could potentially improve metabolic stability and in vivo exposure. As expected, 35 had a similar MIC activity to 29 (Table 5). Although there was no significant change in metabolic stability, neither mouse nor human, the fluorination did result in an unexpected increase in activity against the hERG ion channel. The in vivo exposure of both compounds was evaluated in female C57BL/6 mice (n = 3/dose level) ( Table 5). Both compounds had moderate in vivo blood clearance, moderate volume of distribution, moderate half-life, and low bioavailability. Although the  Based on the results from the PK studies, 29 was assessed in an acute mouse model of TB infection using Balb/c mice in a direct comparison with the previously reported compound 3. 30 As expected, 3 was very active in the study promoting a >2.5 log 10 reduction in colony forming units (CFUs), reducing lung burdens in infected mice near to or below the limit of detection ( Figure 3). This was similar to the control drug rifampicin given at a dose of 20 mg/kg ( Figure 3). In contrast, 29 showed no appreciable reduction in lung CFUs relative to the untreated control in this experiment. Plasma samples for PK from these infected animals were taken during steady state at 1 and 24 h after dosing. Although both compounds showed free plasma concentrations well above MIC at 1 h post-dosing, only 3 remained above MIC for the full 24 h period. Thus, one potential explanation for the difference in efficacy was inadequate drug exposure above MIC for 29 compared to 3. ■ CHEMISTRY Synthetic Routes. Synthesis of related spirocyclic amines has been previously reported. 19,39 The general synthetic routes employed for the synthesis of pyrazole containing spirocyclic amines are shown below (Scheme 1). The appropriately substituted R1 aryls or heteroaryls were reacted with 1-[(4methoxyphenyl)methyl]piperidin-4-one to form 48, which was then deprotected to form amine 49. An alternative route involved reacting the appropriately substituted R1 aryls or heteroaryls with the protected piperidin-4-one to form diols 40−43. Cyclic dehydration afforded 44−47, which were deprotected to afford amines 50−53. Substituted pyrazole aldehydes 70−77 were prepared from the esters 63−69 (Scheme S1 in Supporting Information) by reduction to the alcohol and then oxidation to the aldehydes. R2-and R3substituted pyrazole esters 63−69 were prepared in three main ways: from R3-substituted pyrazole 60, using copper-mediated coupling of suitable R2 boronic acids, by cyclo-dehydration of ethyl 4-(R3)-2,4-dioxo-butanoates 55−59 with R2-substituted hydrazines, and by reaction of dimethyl but-2-ynedioate with R2-substituted hydrazines. In the final step, reductive amination with appropriate R2-and R3-substituted pyrazole aldehydes 70−77 afforded target compounds 1−14, 17, 18, 20, and 21. Further Chan−Lam coupling of the NH pyrazole 78 with appropriate boronic acids afforded target compounds 15, 16, and 19.
For compounds containing a carboxylic acid moiety on the phenyl ring attached to the pyrazole core, a different approach was needed. For these compounds, some could be synthesized by carrying the carboxylic acid through the synthesis protected as an oxazoline (Scheme 2) and some by a late-stage conversion from the bromide (Scheme 3). Carboxylic acid substituted phenyl hydrazines could be reacted with suitable triketo compounds to afford R3-substituted pyrazole esters 87, 88, and 90−92. Because of carrying an ester in these molecules, ester protection of the acid was not a plausible route at this stage, and so to synthetically distinguish the different functional groups, the acids were protected as oxazolines. Amide coupling with ethanolamine followed by chlorination and cyclization easily provided oxazolines 93−98, which could then be carried through the synthesis without problem. Reduction to aldehydes 99−104 as before followed by reductive amination to 105−109 and simple deprotection with 2 M HCl afforded the target compounds 22, 23, and 26−28. Compound 24 followed the majority of this route; however, the carboxylate 95 was synthesized via reaction of 4-aminobenzoic acid with ethyl 2chloro-3-oxo-butanoate and subsequent cyclization to the pyrazole. Alternatively, when the substituted phenyl hydrazine could be reacted with R3-substituted diethoxy diketo compounds 81−83, then only one synthetic step was needed to afford compounds 25, 29, 30, and 35.
The carboxylic acid compounds could also be realized from the bromides. Reaction of 4-bromophenylhydrazines with dimethyl but-2-ynedioate afforded pyrazole 110. Where the desired R group was an alkoxy group, the hydroxyl moiety could be simply alkylated. Where R3 was an amine, this was introduced via a Vilsmeier reaction, followed by S N Ar and removal of the aldehyde, to form compounds 114−117. As in the previous schemes, reduction to aldehydes 118−121 followed by reductive amination afforded spirocyclic amino pyrazoles 122−125. In order to convert the bromide to carboxylic acid, compounds 122−125 were carbonylated using N-formylsaccharin as a source of carbon monoxide, which is slowly released in situ as described in the literature. 40 The acyl fluoride formed in the reaction as a result of the caesium fluoride base was quenched with H 2 O to form the final target compounds 31−34. For all the compounds discussed in this report, no alerts were found when they were processed through a PAINS filter.

■ CONCLUSIONS
Following on from the previous publication of a spirocycle series with potent inhibition of M. tuberculosis but limited by safety concerns, 30 the optimization of a related but novel pyrazole spirocyclic amine is described. This report identified a zwitterionic series, exemplified by lead 29 that when compared to the original series, 3 had improved selectivity over HepG2, as well as reduced hERG channel inhibition. As with the original series, 29 was a putative MmpL3 inhibitor as large shifts against MmpL3 mutant strains were observed. Although 29 has a better selectivity profile than the earlier molecules, this came at the expense of reduced potency; as such, while 3 had excellent activity in an acute in vivo model of TB infection, 29 was found not to be efficacious in the same model. The lack of efficacy of 29 was hypothesized to be as a result of the need for improved in vivo exposure to ensure maximum coverage above the MIC. Thus, despite the original authors highlighting the possibility of eliminating series liabilities while retaining the remarkable in vivo potency, 30 given the complexity of balancing metabolic stability and hERG channel inhibition with MIC activity for this series, no further work was planned to try and improve exposure.

■ EXPERIMENTAL SECTION
Determination of MIC. MICs were determined against M. tuberculosis H37Rv (ATCC 25618) and mutant strains grown in Middlebrook 7H9 medium containing 10% v/v OADC (oleic acid, albumin, dextrose, and catalase) supplement (Becton Dickinson) and 0.05% w/v Tween 80 (7H9-Tw-OADC) under aerobic conditions as previously described. Bacterial growth was measured after 5 days of incubation at 37°C . 41 MmpL3 Hypomorph. Wild-type H37Rv and P606-5C (in which the native copy of mmpL3 was replaced with kanR and a tetracycline-regulated copy of mmpL3 was inserted at the att-L5 site and also contains zeoR) were each cultured in 10 mL of Middlebrook 7H9 [with kanamycin and zeocin at 25 μg/mL and anhydrotetracycline (atc) at 500 ng/mL for the mutant strain] supplemented with 0.2% (v/v) glycerol, 0.05% (v/v) tyloxapol, and ADNaCl (0.5% [w/v] BSA, 0.2% [w/v] dextrose, and 0.85% [w/v] NaCl) in a 25 cm 2 tissue culture flask with a vented cap. After approximately 7 days at 37°C and 5% CO 2 in a humidified incubator, growing to mid-log to late-log phase, each of the cultures was washed with fresh 7H9 and suspended to an OD 580 of 0.05 in 30 mL of 7H9 (with selection antibiotics for the mutant but without atc to deplete the levels of MmpL3) in a 75 cm 2 tissue culture flask with a vented cap and was incubated for further 14 days with a passage to OD 580 = 0.05 in 30 mL of 7H9 at day 7. Bacteria were then washed once in fresh medium and single-cell suspensions were prepared to a final OD 580 of 0.01 in 7H9. Compounds were solubilized in DMSO and dispensed into 384-well plates using an HP D300e Digital Dispenser as 11-point, 4-fold dilution series in triplicate. DMSO at a final concentration of 1% was used as no-drug control. In all, 50 μL of single-cell suspension was pipetted to each well and cultures were incubated for 7−14 d at 37°C in the same conditions as mentioned above. Final OD 580 values were normalized to no-drug control.
M. tuberculosis Kill Kinetics. For replicating conditions, late-log phase bacteria were exposed to compounds in 5 mL medium under aerobic conditions in standing cultures over 21 days. For starvation conditions, bacteria were resuspended in phosphate-buffered saline plus 0.05% tyloxapol for 14 days before compound addition. Viable bacteria were measured by plating serial dilutions and counting cfus after 4 weeks.
Intracellular Activity. THP-1 cells were propagated in RPMI-1640 supplemented with 10% FBS, 2 mM glutagro, and 1 mM sodium pyruvate medium in a humidified atmosphere of 37°C with 5% CO 2 . Cells were differentiated with 80 nM PMA treatment overnight and infected with M. tuberculosis (H37Rv LuxG13) at a multiplicity of infection of 1. Infected cells were harvested with Accutase, 5 mM EDTA, washed twice with PBS, seeded into 96-well plates at 4 × 10 4 cells per well, and incubated for 24 h. Compounds were added as a 10-point threefold serial dilution (0.5% DMSO final concentration). Bacterial viability was measured after 72 h. Growth inhibition curves were fitted using the Levenberg−Marquardt algorithm. IC 50 and IC 90 were defined as the compound concentrations that inhibited 50 or 90% of the intracellular growth, respectively.
THP-1 Cytotoxicity. THP-1 cells were propagated, differentiated into macrophages, and harvested as described above and seeded into 96-well plates at 4 × 10 4 cells per well for 24 h. Compounds were added as a 10-point three-fold serial dilutions (0.5% DMSO final concentration). Viability was measured after 72 h using CellTiter-Glo. Growth inhibition curves were fitted using the Levenberg−Marquardt algorithm. IC 50 was defined as the compound concentration that reduced cell viability by 50%.
HepG2 Cytotoxicity. Compound dilution curves were plated directly using a Labcyte Echo 550 acoustic dispenser (125 nL) in 384-well white clear-bottomed plates (Greiner). HepG2 cells (ECACC 85011430) were cultured in minimum essential medium (supplemented with glutamax) with 10% FCS and plated (25 μL) using a WellMate dispenser (1 × 10 5 per well) and incubated for 72 h. Doxorubicin was used as a positive control drug. Resazurin was then added to each well at a final concentration of 45 μM, and fluorescence was measured using PHERAstar LS (BMG Labtech) after 4 h of further incubation (excitation of 528 nm and emission of 590 nm). Raw data were normalized to controls and expressed as % growth. IC 50 was defined as the compound concentration that resulted in 50% inhibition.
Intrinsic Clearance (Cli) Experiments. Test compound (0.5 μM) was incubated with female CD1 mouse liver microsomes (Xenotech LLC) or pooled human liver microsomes (Life Technologies) at a final concentration of 0.5 mg/ mL 50 mM potassium phosphate buffer, pH 7.4, and the reaction started with addition of excess NADPH (8 mg/mL 50 mM potassium phosphate buffer, pH 7.4). Immediately, at time zero, then at 3, 6, 9, 15, and 30 min, an aliquot (50 μL) of the incubation mixture was removed and mixed with acetonitrile (100 μL) to stop the reaction. Internal standard was added to all samples, the samples were centrifuged to sediment precipitated protein, and the plates were then sealed prior to ultra-performance liquid chromatography−mass spectrometry (UPLC/MS/MS) analysis using a Quattro Premier XE (Waters Corporation, USA). XLfit (IDBS, UK) was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard at each time point. The rate of intrinsic clearance (CLi) of each test compound was then calculated using the following calculation k V CLi (mL/min /g liver) microsomal protein yield = × × where V (mL/mg protein) is the incubation volume/mg protein added and microsomal protein yield is taken as 52.5 mg protein/g liver. Verapamil (0.5 μM) was used as a positive control to confirm acceptable assay performance.
Isoform-specific metabolism studies were performed as mentioned above except that mouse liver microsomes were replaced with incubation mixtures containing EasyCYP bactosomes (50 pmol/mL, 0.5 mg/mL Cypex).
Aqueous Solubility. The aqueous solubility of the test compounds was measured using laser nephelometry. Compounds were subject to serial dilution from 10 to 0.5 mM in DMSO. An aliquot was then mixed with Milli-Q water to obtain an aqueous dilution plate with a final concentration range of 250−12 μM with a final DMSO concentration of 2.5%. Triplicate aliquots were transferred to a flat-bottomed polystyrene plate, which was immediately read on the NEPHELOstar (BMG Lab Technologies). The amount of laser scatter caused by insoluble particulates (relative nephelometry units, RNU) was plotted against compound concentration using a segmental regression fit, with the point of inflection being quoted as the compound's aqueous solubility (μM).
Mouse Pharmacokinetics. The test compound was dosed as a bolus solution intravenously or dosed orally by gavage to female C57 black mice (n = 3/dose level). Blood samples were collected from each mouse tail vein at predetermined time points post-dose, mixed with two volumes of distilled water, and stored frozen until UPLC/MS/MS analysis. Pharmacokinetic parameters were derived from the blood concentration time curve using PK Solutions software v2.0 (Summit Research Services, USA). All regulated procedures, at the University of Dundee, on living animals were carried out under the authority of a project license issued by the Home Office under the hERG Assays. These were performed as previously described 42,43 in brief: thallium flux high-throughput testing, hERG functional activity was measured in an inducible hERG T-RExTM-CHO Cell line (Thermo Fisher #K1237) using thallium influx as a surrogate indicator of potassium ion channel activity. Thallium enhances the fluorescent signal of BTC-AM dye (Thermo Fisher #B6791). Cells were loaded with the dye for 90 min in a low potassium buffer, dye removed, and compound added to the cells in a high potassium buffer in a 6 pt. dose response. After 30 min of compound incubation, channel activity was recorded upon addition of thallium buffer using a Tetra plate reader. The slope of the kinetic read was used to calculate channel activity. For QPatch testing, HEK-293 cells stably transfected with the hERG channel were tested in either QPatch (Sophion) or PatchXpress (Molecular Devices), automated planar patch-clamp systems. The cells were added to each chamber, negative pressure was applied to obtain intracellular access, and cells were exposed to either three or four ascending concentrations of drug. hERG tail current was measured as the difference in amplitude between a −50 mV pre-pulse and the end of a five-second test pulse to −50 mV, preceded by a depolarizing pulse (+20 mV). The hERG IC 50 value was determined from tail current.
Murine Model of Acute TB Infection. All infections were performed at Colorado State University in a certified ABSL3 facility in accordance with the guidelines of the Colorado State University Institutional Animal Care and Use Committee. Six to eight week old female specific pathogen-free BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA). The mice were infected with M. tuberculosis H37Rv via a low-dose aerosol exposure in a Glas-Col aerosol generation device (Glas-Col Inc., Terre Haute, IN). 44 Each treatment group consisted of six mice. 29 was formulated in 1% CMC (Sigma). 3 was formulated in 1% MC (Sigma). Rifampin was prepared using a mortar and pestle in sterile water. The treatment was started 7 days post-aerosol and continued for 12 consecutive days. Drugs were administered by oral gavage in a 0.2 mL volume at 300, 100, and 20 mg/kg for 29, 3, and rifampicin, respectively. For endpoint analysis, mice were euthanized 3 days following the end of treatment and lungs were collected. The left lung lobe (1/3rd of the lung by weight) was homogenized for enumeration of cfu by plating dilutions of the organ homogenates on Middlebrook 7H11 medium supplemented with 10% (v/v) OADC, 0.01 mg/mL of cycloheximide, and 0.05 mg/mL of carbenicillin. The data were expressed as mean log 10 cfu ± the standard error of the mean for each group. Statistical analysis was done by one-way analysis of variance with Dunnet's post-test to control for multiple comparisons (SigmaPlot, San Jose, CA). Values were considered significant at the 95% confidence level.
General Chemistry Methods. Chemicals and solvents were purchased from commercial vendors and used as received unless otherwise stated. Dry solvents were purchased in Sure Seal bottles stored over molecular sieves. Unless otherwise stated herein, reactions have not been optimized. Analytical thin-layer chromatography (TLC) was performed on precoated TLC plates (Kieselgel 60 F254, BDH). Developed plates were air-dried and analyzed under a UV lamp (UV 254/365 nm) and/or KMnO 4 was used for visualization. Flash chromatography was performed using Combiflash Companion Rf (Teledyne ISCO) and prepacked silica gel columns purchased from Grace Davison Discovery Science or SiliCycle. Massdirected preparative HPLC separations were performed using a Waters HPLC (2545 binary gradient pumps, 515 HPLC makeup pump, and 2767 sample manager) connected to a Waters 2998 photodiode array and a Waters 3100 mass detector. Preparative HPLC separations were performed with a Gilson HPLC (321 pumps, 819 injection module, and 215 liquid handler/injector) connected to a Gilson 155 UV/vis detector. On both instruments, HPLC chromatographic separations were conducted using Waters XBridge C18 columns, 19 mm × 100 mm, 5 μm particle size, using 0.1% ammonia in water (solvent A) and acetonitrile (solvent B) as the mobile phase. 1 H NMR spectra were recorded on a Bruker ADVANCE II 500 or 400 spectrometer operating at 500 and 400 MHz (unless otherwise stated) using CDCl 3 , DMSO-d 6 , or CD 3 OD solutions. Chemical shifts (δ) are expressed in ppm recorded using the residual solvent as the internal reference in all cases. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), multiplet (m), broadened (br), or a combination thereof. Coupling constants (J) are quoted to the nearest 0.1 Hertz (Hz). Low-resolution electrospray mass spectra were recorded on a Bruker Daltonics MicrOTOF mass spectrometer run in the positive mode. High-resolution mass spectroscopy (HRMS) was performed using a Bruker Daltonics MicroTof mass spectrometer. LC−MS analysis and chromatographic separation were conducted with either a Bruker Daltonics MicroTOF mass spectrometer connected to an Agilent diode array detector or a Thermo Dionex Ultimate 3000 RSLC system with a diode array detector, the column used was a Waters XBridge column (50 mm × 2.1 mm, 3.5 μm particle size), and the compounds were eluted with a gradient of 5−95% acetonitrile/water + 0.1% ammonia or with an Advion Expression Mass Spectrometer connected to a Thermo Dionex Ultimate 3000 HPLC with a diode array detector, the column used was a Waters XBridge column (50 mm × 2.1 mm, 3.5 μm particle size), or a Waters X-select column (30 mm × 2.1 mm, 2.5 μm particle size) with a gradient of 5−90% acetonitrile/ water + 0.1% formic acid. All final compounds showed chemical purity of ≥95% as determined by the UV chromatogram (190− 450 nm) obtained by LC−MS analysis.
General Method 1. Respective spirocyclic amines (1 equiv) and aldehydes (1 equiv) were combined in DCM (0.1−0.2 M), one drop of acetic acid was added, and the mixtures were stirred at rt for 1 h. Sodium triacetoxyborohydride (1.5−2 equiv) was added and the mixtures were stirred at rt for 18 h. The mixtures were washed with NaHCO 3 (saturated solution) and the organics were concentrated in vacuo. The crude materials were purified by preparative HPLC (XBridge column, 0.1% NH 4 OH modifier) and the fractions were concentrated in vacuo to afford the titled compounds.
General Method 2. Respective pyrazoles (1 equiv), boronic acids (2 equiv), and copper acetate (1.5 equiv) were combined in DCM (0.1 M) and pyridine (20 equiv) and stirred at rt for 3−18 h. The solvents were removed in vacuo and the crude products were purified by either flash chromatography (0−100% EtOAc in heptane) or preparative HPLC (XBridge column, 0.1% NH 4 OH modifier), and the fractions were concentrated in vacuo to afford the titled compounds.
General Method 3a. Respective hydrazines (1 equiv) and 2,4-dioxo-butanoates (1 equiv) were dissolved in EtOH (0.15 M) and heated to reflux for 1−3 h. The solvent was removed in vacuo and H 2 O was added. The products were extracted with EtOAc and concentrated in vacuo, and the resulting crude ACS Omega http://pubs.acs.org/journal/acsodf Article products were purified by flash chromatography (5−50% EtOAc in heptane) to afford the titled compounds. General Method 3b. Respective hydrazines or hydrazine hydrochlorides (1 equiv) and 2,4-dioxo-butanoates (1 equiv) were dissolved in AcOH (0.7 M), and the reaction mixtures were heated to reflux for 2 h and then allowed to cool to rt. The solvent was removed in vacuo, and the residue was partitioned between DCM and 1 M NaOH and then passed through a hydrophobic frit. The crude materials were purified by flash chromatography (0−100% EtOAc in heptane) to afford the titled compounds.
General Method 4. Oxazolines (1 equiv) were dissolved in 3 M HCl (30 equiv) and heated to 100°C for 18 h and then allowed to cool to rt. The reaction mixtures were neutralized with NaHCO 3 (saturated solution) and extracted with DCM. The organics were separated, dried through a hydrophobic frit, and concentrated in vacuo to afford the titled compounds.