Identification of Morpholino Thiophenes as Novel Mycobacterium tuberculosis Inhibitors, Targeting QcrB

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis there is a pressing need for new oral drugs with novel mechanisms of action. Herein, we describe the identification of a novel morpholino–thiophenes (MOT) series following phenotypic screening of the Eli Lilly corporate library against M. tuberculosis strain H37Rv. The design, synthesis, and structure–activity relationships of a range of analogues around the confirmed actives are described. Optimized leads with potent whole cell activity against H37Rv, no cytotoxicity flags, and in vivo efficacy in an acute murine model of infection are described. Mode-of-action studies suggest that the novel scaffold targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase, part of the bc1-aa3-type cytochrome c oxidase complex that is responsible for driving oxygen-dependent respiration.


■ INTRODUCTION
Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB), an airborne transmitted disease that is fatal if not properly treated. TB disproportionately affects developing countries and now outranks HIV as the leading cause of death from an infection worldwide. In 2015, 1.8 million people died of TB (1.4 million HIV-negative and 0.4 million HIV positive). 1 The current treatment regimen is considered antiquated and inadequate, taking a minimum of 6 months to cure patients, which contributes to high default rates leading to increased transmission, drug resistance, and even death. 2−4 As a consequence, new TB drugs working through existing and novel mechanisms are urgently required. 5,6 Although there has been increased investment in TB drug discovery over the past 10 years this has remained a significant challenge, and high-quality novel chemical starting points are still urgently required. 7 Identification of TB drug discovery starting points through in vitro target-directed screens has been largely unsuccessful. 6 As a result, cell-based phenotypic screening has emerged as a preferred means to identify novel antitubercular scaffolds. In this study a cluster of novel morpholino−thiophenes (MOT) analogues was identified from the Lilly corporate screening deck using an aerobic whole cell phenotypic screen of M. tuberculosis strain H37Rv. The initial hit had good antitubercular activity with a minimum inhibitory concentration (MIC) of 0.72 ± 0.30 (n = 14). Resynthesis and profiling of hits from the primary screen confirmed antitubercular activity with good potency and selectivity over both VERO and HepG2 cytotoxicity counter screens (see Table 1 for an example). The representative confirmed hit 1 had good M. tuberculosis MIC-derived metrics such as lipophilic ligand efficiency, 8 where LLE M. tuberculosis = pMIC − c log P or log D and Property Forecast Index 9 where PFI = no. of aromatic rings + log P, suggesting that the series had potential to be optimized into a high-quality oral drug candidate. 10,11 Physiochemical properties such as MW, log P, log D, and TPSA were in an acceptable range for oral drugs. 11,12 Kinetic solubility suggested 1 was highly soluble, although later additional solubility profiling of analogues found thermodynamic solubility to be an issue. Compound 1 showed moderate CYP inhibition and no in vitro hERG liability. The perceived issue for 1 was metabolic stability as a result of high mouse microsomal clearance, albeit human microsomal clearance was moderate.
Related thiophene pharmacophores, where the morpholine is replaced by a benzimidazole group, have been investigated for other disease areas. Analogues with no ether chain and a benzimidazole functionality have been reported as inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase. 13 Analogues with a benzimidazole functionality and an ether side chain have been reported as polo-like kinase (PLK) and Never in Mitosis Gene A-Related Kinase 2 (Nek2) inhibitors. 14−17 The thiophene-2-carboxamide substructure has also been observed within antitubercular inhibitor agents ( Figure 1).
The structure of the thiophene 2 inhibitor of mycoyltransferase bound to M. tuberculosis FbpC has been solved. 18 Recently, TCA1 3 has been reported to target both decaprenylphosphoryl-β-D-ribose oxidase (DprE1) and MoeW and was found to have in vitro bactericidal activity against both replicating and nonreplicating M. tuberculosis as well as being efficacious in both acute and chronic mouse models of TB infection. 19 Herein, we report on the profiling of emerging leads from the MOT series that suggested that they likely target QcrB, a subunit of the menaquinol cytochrome c oxidoreductase (bc1 complex) that is part of the bc1-aa3-type cytochrome c oxidase complex, driving oxygen-dependent respiration. The imidazo-[1,2-a]pyridine-3-carboxamides have recently been shown to target QcrB and are currently undergoing clinical development. 20−24 The profile and novelty of representative confirmed hit 1 was considered promising, and a medicinal chemistry program was established to derive a better understanding of the pharmacophore based on structure−activity relationship (SAR) studies.
■ CHEMISTRY Synthetic Routes. Synthesis of the MOT scaffold was initially achieved using the route outlined in Scheme 1. Amide 5 was formed by reaction of acyl chloride 4 with morpholine and then converted to thioamide 6 following treatment with Lawesson's reagent. Thioamide 6 was reacted with 2aminoacetyl bromide to yield hydroxy-thiophene 7. Mitsunobu 25 or alkylation reactions yielded the desired thiophene 8. However, a major drawback of this route was that 2substituted-5-morpholino-3-oxo-2, 3-dihydrothiophene-2-carboxamide 9 was the major product, regardless of whether the Mitsunobu or alkylation reaction was employed to introduce the ether chain.
To circumvent the issues with formation of 9, an alternative route was developed that relied on the early introduction of the ether side chain followed by introduction of the morpholine (Scheme 2). Using methyl 3-hydroxythiophene-2-carboxylate 10 in the Mitsunobu ether formation afforded phenyl ethyl derivative 11. Regioselective iodination with LDA and iodine afforded the versatile iodo-intermediate 12.
The ester functional group of 12 was converted to the primary amide 13 by reaction with calcium chloride and ammonia in methanol. 26 Suzuki 27 or Buchwald 28 reactions afforded a range of derivatives in this position (for example, 32, 34, and 35). Hydroxamic acid derivative 16 was prepared from intermediate 12 using a Buchwald-type reaction to form 14, followed by reaction of the ester with a tetrahydropyran protected hydroxylamine to afford 15, which was treated with ptoluenesulfonic acid to afford 16. Treatment of intermediate 11 with LDA and DMF afforded aldehyde 17, which was reacted with a range of amines, such as morpholine to give 18.

■ RESULTS AND DISCUSSION
The initial SAR investigation focused on systematically probing the pharmacophore to understand the impact of each functional group on antibacterial activity. Deletion analogues and nearest neighbors were examined as well as functionality on the phenyl ring with an overall aim of identifying key structural modifications that could be made to improve microsomal stability.
Primary Amide SAR. Modifications to the primary amide provided scope to improve MIC potency and mouse microsomal stability ( Table 2). Removal of the primary amide 19 or replacement with an ester 14 resulted in a loss of activity, indicating it may be involved in the binding mode of the MOT scaffold with its target or uptake into M. tuberculosis. The methyl ketone 20 was found to be equipotent with 1 but had a negative impact on mouse microsomal clearance. The Nmethyl amide 21 showed similar activity but also had a

Journal of Medicinal Chemistry
Article negative impact on the mouse microsomal stability. The ethyl 22 and trifluoroethyl 23 derivatives both led to a drop in potency. There were significant improvements in microsomal stability for the N-cyclobutyl amide 24; however, it came with a loss in antitubercular activity. Encouragingly, both the Weinreb amide 25 and the hydroxamic acid 16 showed improved microsomal stability together with a moderate MIC. A mix and match approach between TCA1 compound 3 was also explored; however, 26 was found to be inactive. Overall, the SAR for the primary amide replacement suggested that the carbonyl is important for antitubercular activity, and microsomal stability can be improved (Table 2). Morpholine Side Chain SAR. Replacement of the morpholine 1 with hydrogen 27 or piperidine 28 resulted in a loss of activity. The morpholin-3-one 29 showed improved microsomal stability; however, there was a >10-fold drop in potency. Bridged morpholine 30 showed a >5-fold drop in activity, whereas the methyl-substituted morpholine 31 lost activity, significant improvements in microsomal stability were not observed. Increasing the flexibility by opening up the morpholine ring to give 32 resulted in loss of activity. In light of the similarity to reported PLK inhibitors, 14,15 heteroaromatic replacements were considered. The PLK-related benzimidazole derivative 33 was inactive; however, the triazine 34 and 4-pyridyl 35 displayed good potency. The microsomal stability was improved by extending the morpholine with a methylene linker 18; however, no antibacterial activity was observed for an array of amines (data not shown). Overall, the SAR for the morpholine substituent suggested that there was limited scope for improving potency and microsomal stability (Table 3).
Phenyl Ring and Ethyl Ether Linker SAR. The SAR around the phenyl ring and the ethyl linker was examined to see if the mouse microsomal stability could be improved by changing the substituents and physiochemical properties of the phenyl ethyl ether ( Table 4). The 4-methoxy 36 was equipotent with 1 and also had improved microsomal stability, presumably as a result of a blocking metabolism on the phenyl ring. The 4-trifluoromethoxyphenyl 37 also afforded improved antibacterial activity and microsomal stability. The o-, m-, and

Journal of Medicinal Chemistry
Article p-substitution patterns of fluoro (38, 39, and 40) and o-and m-methoxy (41 and 42) groups were tolerated but did not significantly improve the overall properties of the molecule, suggesting that p-substitution of the phenyl group is favored. The 4-pyridyl 43 and p-cyanophenyl 44 derivatives lost activity, suggesting that polarity was not tolerated in this region. Saturated polar heterocyclic alkyl analogues were prepared, 45, 46, and 47, while they significantly improved microsomal stability, they lost whole cell activity, supporting the notion that polarity was not tolerated in this region. The phenethyl linker was found to be important for potency; shortening the chain to a methylene linker 48 resulted in loss of activity. Replacement of the O-linker to afford an n-propyl 49 was tolerated but with a drop off in potency. Introduction of a gem-difluoro group 50 as well as hydroxylation 51 on the benzylic position of the ethyl chain, designed in an effort to block benzylic oxidation, resulted in a loss of activity. Constraining the ethyl linker to afford 52 and 53 gave improved microsomal stability but had a detrimental effect on potency. The SAR for the phenyl ring and ethyl linker suggested that there is scope in this region for improving microsomal stability (Table 4).
Core Replacement SAR. Phenyl was investigated as a bioisosteric thiophene replacement, employing two substitution patterns ( Table 5). The 4-morpholino 54 showed low whole cell activity, whereas the 5-morpholino 55 had moderate whole cell activity as well as improved microsomal stability. The m-fluorophenyl derivative 56 showed similar activity to 55. Additional SAR for the phenyl bioisosteric replacement was explored and is similar to the reported thiophene SAR (data not shown). The pyridyl core bioisoster 57 showed significantly improved microsomal stability with similar potency to 55. Overall, the SAR for the thiophene core and its bioisoster replacements suggested that there is scope in this region for improving microsomal stability while maintaining whole cell activity ( Table 5).
Summary of Pharmacokinetic (PK) Studies. Representative compounds 1, 55, 37, 46, and 25 were selected for mouse pharmacokinetic profiling to understand the correlation between in vitro microsomal turnover and in vivo clearance ( Table 6). Compounds 55, 37, and 46, with improved in vitro microsomal stability, showed improved C max and AUC oral exposures versus 1. However, there was an in vitro microsomal versus in vivo clearance disconnect for 1, 55, 37, and 46 where there is an overprediction of clearance. The phenyl bioisosteric replacement 55 showed improved in vitro clearance as well as in vivo exposure versus 1. However, it had a reduced MIC potency. Similarly, the saturated polar heterocycle 46 showed significant improved bioavailability but no measurable MIC activity. Overall 37 had the best activity and in vivo PK profile with a moderate volume of distribution and bioavailability. Weinreb amide 25, despite having excellent in vitro microsomal stability, showed similar poor C max and AUC compared to 1. To better understand the optimization parameters required to improve the oral exposures for compounds with improved in vitro microsomal stability, a rat hepatic portal vein (HPV) PK study was undertaken on lead 37.
Rat HPV PK Study for 37. The fraction of drug absorbed (Fa) from the gastrointestinal tract was low at 21% of the total administered drug, suggesting that the poor oral exposure was largely due to low solubility and/or metabolism in the gut wall during absorption since the permeability was observed to be rapid (see below). A 44% hepatic extraction ratio (ER), determined by measuring blood samples from the HPV (before first pass metabolism) versus the cardiac vein (systemic circulation), suggested that metabolic stability was also an issue, Table 7.
To better define the optimization parameters for improving oral exposure, the permeability and efflux of selected compounds were evaluated in a cell permeability assay. In addition, further solubility screens and metabolite identification studies were performed.
Madin Darby Canine Kidney (MDCK) Permeability Assay. Compounds 1, 37, 18, 46, and 56 were tested in an MDCK assay and found to show good permeability as well as no likely P-glycoprotein (P-gp) efflux issues (Table 8). This Minimum inhibitory concentration (MIC 90 ) is the minimum concentration required to inhibit the growth of M. tuberculosis in liquid culture. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). b Inhibitory concentration (IC 50 ) is the concentration required to inhibit growth of Vero cells by 50%. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). c Intrinsic clearance (Cli) using CD1 mouse liver microsomes

Journal of Medicinal Chemistry
Article was unsurprising considering the favorable MW, log P, low hydrogen bond donor (HBD) count, and TPSA observed for the examples in Tables 1−5. The good permeability data would suggest that solubility-limited absorption was likely to be an underlying optimization issue for the series, not reflected in the kinetic solubility screen. It is known that the electrondeficient bivalent sulfur atoms in thiophenes have low-lying σ* orbitals available for interaction with electron donors such as oxygen. As such an intramolecular interaction between the thiophene sulfur σ* orbital and the carbonyl is likely stabilizing a planar conformation. 29 Furthermore, primary amides, in particular aromatic ones, effectively generate two-dimensional intermolecular hydrogen-bonded networks 30 that can also have a dramatic effect on the thermodynamic solubility.
Solubility Screening. Considering the results of the HPV and permeability studies, we selected a number of compounds for additional solubility profiling. Compounds 37 showed poor solubility when tested in a high-throughput solubility assay (HTSA) at a range of pH values, pH 2/6/7.4. The ketone 20 showed improved solubility in the HTSA screens at different pH as well as in the FaSSIF and low pH HCl conditions. The improved solubility for the ketone versus the amide was likely to be as a result of the decreased potential for an intermolecular hydrogen-bonded network. While the methylene-homologated morpholine 18 was not active, it demonstrated improved solubility, in particular at low pH as a result of protonation of the morpholine. The phenyl bioisosteric replacement 55 also showed improved solubility versus 37, presumably as it is devoid of the intramolecular interaction between the thiophene sulfur and carbonyl, Table 9.
Metabolite Identification. In order to be able to address and modify the metabolically labile moieties in the compound series, the in vivo metabolites of 37 were determined. The main metabolites observed were the products of oxidation of the morpholine, whereas de-ethylation was observed only in small quantities (Supporting Information Table 3).
Efficacy Studies. Compound 37 was profiled in an acute 4 day screening model of TB infection to determine if it had in vivo efficacy (infected with H37Rv). 31 The compound was compared to moxifloxacin as the known control agent. The compounds were dosed orally at 100 mg/kg, and 37 showed a small but significant 0.8 log reduction of colony forming unit (CFU) counts in the lungs (Table 10). The in vivo efficacy, coupled with the understanding of the optimization parameters, led us to further investigate the mode of action for the series.
Mode of Action Studies. Since the SAR suggested that the phenethyl linker and the amide carbonyl were critical aspects of the minimum pharmacophore, it appeared that the series shared a similar pharmacophore to a number of reported scaffolds targeting QcrB. 24 QcrB is a subunit of the menaquinol cytochrome c oxidoreductase (bc1 complex) that is part of the bc1-aa3-type cytochrome c oxidase complex and is responsible for driving oxygen-dependent respiration. 21,22,32 A number of inhibitors of QcrB have been identified recently through phenotypic screens, 21,33−36 and Q203 is currently undergoing clinical development. Overlay of selected low local energy conformations of 37 with Q203 21 highlights the similarity in the pharmacophore ( Figure 2).
To test this hypothesis, a set of active compounds was tested against mutant strains of M. tuberculosis each with a single nucleotide polymorphism in QcrB; we used a QcrB T313I mutant since this was previously shown to confer resistance to multiple QcrB inhibitors including Q203 and the imidazopyridines, as well as QcrB M342T (Table 11). 21,24 A homology model for the structure of QcrB has been reported previously based on multiple cytochrome b homologues, 24 and the mutated residues map to the stigmatellin binding site within the bc 1 complex modifying the shape of the binding pocket. 24,37 The mutant strains were resistant to the panel of compounds chosen, with an increase in MIC of over 10-fold versus the wild-type (WT) strain. As for other QcrB inhibitors, 21,24,35 the resistant mutant data suggests that the MOT series targets QcrB directly acting like stigmatellin and disrupting electron transport.
To explore further the effect of the MOT compounds on QcrB, it was determined whether exposure resulted in a decrease in ATP levels, as would be expected for compounds which target QcrB. 21,34 ATP and growth were monitored in the same experiment, and it was clear that ATP levels were Table 3. Morpholine Side Chain SAR a Minimum inhibitory concentration (MIC 90 ) is the minimum concentration required to inhibit the growth of M. tuberculosis in liquid culture. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). b Inhibitory concentration (IC 50 ) is the concentration required to inhibit growth of Vero cells by 50%. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). c Intrinsic clearance (Cli) using CD1 mouse liver microsomes

Journal of Medicinal Chemistry
Article depleted in a dose-dependent manner ( Figure 3) as seen previously for compounds that also target QcrB. 21,34,35,38 The depletion of ATP occurred at lower concentrations than required to inhibit growth, as for Q203 (a known QcrB inhibitor), suggesting that this is the mode of action. In contrast, depletion of ATP by rifampicin which targets RNA polymerase correlated with growth. These data support the conclusion that the MOT series targets QcrB.

■ CONCLUSION
We describe the identification of a novel morpholino− thiophene series from whole cell screening against M. tuberculosis. The confirmed hit 1 was extensively profiled, and the profile suggested that microsomal stability was a key optimization parameter. Structure−activity relationships led to an understanding of the minimal pharmacophore as well as emerging leads such as 37 with improved whole cell activity Table 4. Phenyl Ring and Ethyl Linker SAR a Minimum inhibitory concentration (MIC 90 ) is the minimum concentration required to inhibit the growth of M. tuberculosis in liquid culture. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). b Inhibitory concentration (IC 50 ) is the concentration required to inhibit growth of Vero cells by 50%. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). c Intrinsic clearance (Cli) using CD1 mouse liver microsomes

Journal of Medicinal Chemistry
Article against M. tuberculosis and improved microsomal stability. Selected compounds were profiled in an acute in vivo efficacy model of TB, and 37 was found to have a small but significant effect. Mode of action profiling suggested the series targets QcrB, a subunit of the menaquinol cytochrome c oxidoreductase (bc1 complex) involved in bacterial respiration. In contrast to Q203, the novel lead series reported herein provides starting points with scope for optimization to reduce the high log P associated with Q203. Further lead optimization efforts will be reported elsewhere.

■ EXPERIMENTAL SECTION
Determination of Minimum Inhibitory Concentration (MIC 90 ). 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, catalase) supplement (Becton Dickinson) and 0.05% w/v Tween 80 (7H9-Tw-OADC) under aerobic conditions as previously described. 39 Bacterial growth was measured after 5 days of incubation at 37°C. MIC 90 was defined as the concentration of compound required to inhibit growth of M. tuberculosis by 90% and was determined by plotting growth and curve fitting using the Levenberg− Marquardt least-squares plot.
Measurement of Intrabacterial ATP Levels. M. tuberculosis H37Rv was exposed to compounds for 24 h. ATP levels were measured using the BacTiter-Glo assay kit (Promega) and expressed as RLU (relative luminescence units). Growth was monitored by OD 590 .
Intrinsic Clearance (Cli) Experiments. Test compound (0.5 μM) was incubated with female CD1 mouse liver microsomes (Xenotech LLC; 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, and then at 3, 6, 9, 15, and 30 min an aliquot (50 μL) of Table 5. Core Replacement SAR a Minimum inhibitory concentration (MIC 90 ) is the minimum concentration required to inhibit the growth of M. tuberculosis in liquid culture. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). b Inhibitory concentration (IC 50 ) is the concentration required to inhibit growth of Vero cells by 50%. Data are the average ± standard deviation of a minimum of two independent experiments; numbers of experiments is given (n). c Intrinsic clearance (Cli) using CD1 mouse liver microsomes  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. The human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents. 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 compounds aqueous solubility (μM).
Thermodynamic Solubility via High-Throughput Method, HTSA. Samples prepared in DMSO were dried for 12 h. The powder or film was redissolved in the solvent at various pH (2, 6, and 7.4) and DMSO control at 2 mM target concentration. Samples are stirred for 20 h and filtered through a 0.7 μm GF filter. The filtrate was analyzed by HPLC assay for concentration against DMSO standard curve. 40 Mouse Pharmacokinetics. Test compound was dosed as a bolus solution intravenously at 3 mg of free base/kilogram (dose volume 5 mL/kg; dose vehicle Saline or 10% DMSO; 40% PEG400; 50% saline) to female C57 Black (n = 3) or dosed orally by gavage as a solution at 10 mg of free base/kilogram (dose volume 10 mL/kg; dose vehicle 10% DMSO; 40% PEG400; 50% distilled water) to female C57 Black (n = 3/dose level). Blood samples were taken from each mouse tail vein at predetermined time points postdose, 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 v 2.0 (Summit Research Services, USA).
Therapeutic Efficacy of 37 against M. tuberculosis in an Acute Murine Model of Intratracheal Infection. Specific pathogen-free, 8−10 week old female C57BL/6 mice were purchased from Harlan Laboratories and allowed to acclimate for 1 week. Mice were intrathecal infected with 100.000 CFU/mouse (M. tuberculosis H37Rv strain). Compound 39 was administered for 4 consecutive days starting 5 days after the infection. Moxifloxacin was used as an interassay control and administered for 8 consecutive days starting 1 day after the infection. Lungs were harvested on day 9, 24 h after the last administration. All lung lobes were aseptically removed, homogenized, and frozen. Homogenates were plated in 10%    Table  10. The differences in the lung microorganism burden (log 10 CFUs/ lungs) obtained in the treated mice with respect to untreated controls (day 9 after infection) are shown in Table 10. No adverse clinical signs were observed in any animal. Moxifloxacin (30 mg/kg) reduced 2.8 logs the CFU lung number with respect to untreated mice. This quality control value is included in the accepted interval. CFU number in lungs of untreated mice: 6.8 log CFU. This value is inside the interval mean ±2 SD of the values of the last experiments. In these experimental conditions, 37 was able to inhibit the growth of the bacteria in the lungs of the mice compared to untreated mice. This difference was statistically significant. Rat Hepatic Portal Vein (HPV) Study. Test compound was dosed orally by gavage at 5 mg of free base/kilogram (dose volume 5 mL/kg; dose vehicle 1.0% carboxy methyl cellulose, CMC) to male Sprague−Dawley Rats (n = 1 per time point). At predetermined time points, blood samples (100 μL) were taken from the hepatic portal vein into a tube containing EDTA. Immediately afterward, the remaining blood was taken by cardiac puncture into a separate tube containing EDTA to determine the concentration of compound reaching the systemic circulation. The blood was 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 v 2.0 (Summit Research Services, USA).
General Chemistry Methods. Chemicals and solvents were purchased from commercial vendors and were 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, 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, 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 mobile phase. 1 H NMR spectra were recorded on a Bruker Advance II 500, 400, or 300 spectrometer operating at 500, 400, or 300 MHz (unless otherwise stated) using CDCl 3 or DMSO-d 6 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

Journal of Medicinal Chemistry
Article (s), doublet (d), triplet (t), multiplet (m), broadened (b), or a combination thereof. Coupling constants (J) are quoted to the nearest 0.1 Hz (Hz). Low-resolution electrospray (ES) mass spectra were recorded on a Bruker Daltonics MicrOTOF mass spectrometer run in 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 a Bruker Daltonics MicrOTOF mass spectrometer or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS where both instruments were connected to an Agilent 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. All final compounds showed chemical purity of ≥95% as determined by the UV chromatogram (190−450 nm) obtained by LC-MS analysis. High-resolution electrospray measurements were performed on a Bruker MicroTof mass spectrometer. Microwave-assisted chemistry was performed using a CEM microwave synthesizer. Methyl 3-morpholino-3-thioxopropanoate (6). 5 (28.1 g, 150 mmol) was dissolved in 1,4-dioxane (72 mL), and Lawesson's Reagent (34.0 g, 84 mmol) was added. The reaction was heated to 100°C for 2 h, stopped, and cooled to 0°C. The resulting precipitate was filtered washing with EtOAc, the filtrate was washed with NaHCO 3 (100 mL), and the organic layer was dried though a hydrophobic frit before being concentrated in vacuo to afford an orange oil. Trituration with Et 2 O afforded the desired product as a pale orange solid (22.5 g, 74%). 1  3-Hydroxy-5-morpholino-thiophene-2-carboxamide (7). Sodium hydride (1.2 g, 29.5 mmol) was dissolved in THF (18 mL) at rt, and 6 (6.0 g, 29.5 mmol) was added. The reaction was stirred at rt for 30 min and then cooled to 0°C. 2-Bromoacetamide (4.1 g, 29.5 mmol) was added. The reaction was maintained at 0°C for a further 1 h, then warmed to rt, and stirred for a further 2 h. A second equivalent of NaH (1.2 g, 29.5 mmol) was added, and the reaction was stirred at rt for 2 h. LCMS indicated that the desired product had formed. The reaction was quenched with water, and excess THF was removed in vacuo. The aqueous layer was acidified with 1 M HCl and partitioned with EtOAc. The desired product precipitated from the organic extracts (6.7 g, 65%). 1  General Procedure A: LDA. Thiophene derivative (1 equiv) was added to a flame-dried flask under N 2 and dry THF (4 mL/mmol) added. The solution was cooled to −78°C, and LDA (1.1 equiv) was added dropwise. The reaction mixture was stirred for 5 min, and iodine (1.1 equiv) was added. The reaction was further stirred for 10 min at −78°C, then warmed to rt for 3 h, quenched with water, and 1 N HCl (1 mL/mmol) added. The organic phase was separated and washed with an aqueous solution of sodium bisulphite and dried over MgSO 4 , and the solvent was removed in vacuo. The crude material was triturated with hexane to afford the desired product.
5-(Morpholinomethyl)-3-(2-phenylethoxy)thiophene-2-carboxamide (18). 3-Hydroxy-5-morpholinothiophene-2-carbonitrile (20a). NaOEt (10.4 g, 32 mmol) was dissolved in EtOH (90 mL), and 6 (5.00 g, 24.6 mmol) was added. The mixture was then cooled to 0°C, and 2bromoacetonitrile (2.95 mg, 24.6 mmol) was added. The reaction was stirred at 0°C for 1 h and then allowed to warm to rt for a further 1 h before addition of further NaOEt (2.18 g, 31.98 mmol). The reaction was stirred for 12 h at rt. The precipitate was filtered and dissolved in H 2 O before acidifying with 1 M HCl and extracting with DCM (3 × 50 mL). The combined organics were dried through a hydrophobic frit and concentrated in vacuo. The resulting solid was triturated with Et 2 O and filtered off to afford a beige solid (2.83 g, 55%). 1 (20). MeMgBr (53 mg, 0.45 mmol) was dissolved in toluene (5 mL) under N 2 , and 20b (70 mg, 0.22 mmol) was added. The mixture was heated to reflux for 3 h. The reaction mixture was allowed to cool to rt before acidifying with 2 M HCl to pH 2. The mixture was then heated to reflux for 30 min. The mixture was allowed to cool to rt and basified with a saturated aqueous NaHCO 3 solution and extracted with EtOAc (2 × 15 mL). The organics were dried through a phase separating frit and concentrated in vacuo to afford an orange oil which solidified on standing at rt. The crude material was columned eluting with 0−20% MeOH in DCM, pink solid (2 mg, 2% 3-Hydroxy-N-methyl-5-morpholinothiophene-2-carboxamide (21a). NaOEt (1.04 g, 3.20 mmol) was dissolved in EtOH (9 mL), and methyl 6 (500 mg, 2.5 mmol) was added. The mixture was then cooled to 0°C, and 2-bromo-N-methyl-acetamide (374 mg, 2.46 mmol) was added. The reaction was stirred at 0°C for 1 h and then allowed to warm to rt for a further 1 h before addition of further NaOEt (218 mg, 3.2 mmol). The reaction was then stirred at rt for 2 h. The reaction was quenched with H 2 O, and the volatiles were removed in vacuo. The mixture was acidified using 2 M HCl and extracted with DCM (2 × 20 mL). The combined organics were dried and concentrated in vacuo to afford an off-white solid which was purified by column chromatography eluting with 5% MeOH in DCM to afford the desired product as a white solid (127 mg, 21%). General Procedure C: AlCl 3 -Catalyzed Amide Formation. Acid (1 equiv) and trimethylaluminum in toluene (4 equiv) were stirred at rt, and ethanamine (1 equiv) was added. The reaction was heated at reflux for 3 h, stopped, and cooled to rt. Excess solvent was removed in vacuo; 1 M NaOH was added (20 mL/mmol), and the mixturewas extracted with EtOAc. The organic layer was dried in vacuo, and the crude residue was purified by column chromatography eluting with DCM to 10% DCM.