Encoded Library Technology as a Source of Hits ... - ACS Publications

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Encoded Library Technology as a Source of Hits for the Discovery and Lead Optimization of a Potent and Selective Class of Bactericidal Direct Inhibitors of Mycobacterium tuberculosis InhA Lourdes Encinas,‡ Heather O’Keefe,† Margarete Neu,§ Modesto J. Remuiñań ,‡ Amish M. Patel,† Ana Guardia,‡ Christopher P. Davie,† Natalia Pérez-Macías,∥ Hongfang Yang,† Maire A. Convery,§ Jeff A. Messer,† Esther Pérez-Herrán,‡ Paolo A. Centrella,⊥ Daniel Á lvarez-Gómez,‡ Matthew A. Clark,⊥ Sophie Huss,‡ Gary K. O’Donovan,† Fátima Ortega-Muro,‡ William McDowell,# Pablo Castañeda,‡ Christopher C. Arico-Muendel,† Stane Pajk,∞ Joaquín Rullás,‡ Iñigo Angulo-Barturen,‡ Emilio Á lvarez-Ruíz,× Alfonso Mendoza-Losana,‡ Lluís Ballell Pages,‡ Julia Castro-Pichel,*,‡ and Ghotas Evindar*,† †

ELT Boston, Platform Technology & Science, GlaxoSmithKline, Waltham, Massachusetts 02451, United States Diseases of the Developing World, Tres Cantos Medicines Development Campus, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain § Computational and Structural Chemistry, Platform Technology & Science, GlaxoSmithKline, Stevenage SG1 2NY, Hertfordshire, U.K. ∥ Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain ⊥ X-Chem Inc., 100 Beaver Street, Waltham, Massachusetts 02453, United States # Biological Reagent and Assay Development, Platform Technology & Science, GlaxoSmithKline, Stevenage SG1 2NY, Hertfordshire, U.K. ∞ Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia × Centro de Investigación Básica, GlaxoSmithKline, 28760 Tres Cantos, Madrid, Spain ‡

S Supporting Information *

ABSTRACT: Tuberculosis (TB) is one of the world’s oldest and deadliest diseases, killing a person every 20 s. InhA, the enoylACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure−activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.

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INTRODUCTION

compliance is monitored by healthcare workers and has been successful when appropriately implemented (cure rate of >90%). Despite this, there is an urgent need for the development of newer, safer, and effective antitubercular drugs with new targets and novel modes of action (MoA). InhA is an NADH-dependent 2-trans enoyl-acyl carrier protein (ACP) reductase of the type II fatty acid synthase

Despite the existence of treatments for tuberculosis (TB), 8.7 million people fell ill with TB and 1.4 million died from TB in 2011 (latest WHO report1). The increasing prevalence of multidrug resistant (MDR)2−7 and extremely drug resistant (XDR)8,9 TB strains represents a threat to public health worldwide. Resistance to TB drugs results primarily from nonadherence by patients, incorrect drug prescription, or poor quality drugs. The WHO sponsored implementation of DOTS10 (directly observed treatment short course) in which treatment © 2014 American Chemical Society

Received: September 4, 2013 Published: January 22, 2014 1276

dx.doi.org/10.1021/jm401326j | J. Med. Chem. 2014, 57, 1276−1288

Journal of Medicinal Chemistry

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(FASII) pathway in Mycobacterium tuberculosis (Mtb).11,12 There is a strong body of evidence indicating that it is the primary target of the frontline antitubercular drug isoniazid (INH).13,14 Clinical isolates and laboratory modified mycobacteria overexpressing InhA show resistance to INH.14−16 The drug inhibits InhA enzymatic activity, inducing an accumulation of saturated C24− C26 fatty acids and blocking the production of longer molecules, including mycolic acids. This inhibition correlates with mycobacterial cell death. The essentiality of InhA has also been demonstrated by the use of temperature sensitive mutants of InhA in Mycobacterium smegmatis, where a shift to the nonpermissive temperature results in rapid lysis and cell death.12 INH is a bactericidal drug and part of the first-line drug combination regimen for antitubercular therapy and is specifically active against M. tuberculosis. INH is activated within the mycobacterial cell by KatG. The activated form is thought to react with NADH within the InhA active site to form an inhibitory adduct. In vitro activated INH also forms adducts with NAD(P) cofactors that bind to and inhibit InhA and other enzymes like DHFR.17 The physiological relevance of these interactions in vivo is clear in the case of InhA, while the potential role of other possible targets of INH in the antitubercular activity of the drug remains to be solidly proved. Resistance to INH, which is one of the hallmarks of MDR strains, has been associated with at least five different genes (katG, inhA, ahpC, kasA, and ndh).18−20 While this finding still remains to be clearly explained, 60−70% of resistant isolates are directly linked to defects in the katG gene (often with compensatory mutations in other genes) and less commonly in the inhA structural gene and upstream promoter region. With this background, we and others21−26 were interested in the development of inhibitors targeted directly at InhA that do not require previous KatG activation. Specifically, it was our goal to develop a new agent that was able to interact with InhA differently from the known NAD-INH complex, enabling a new pharmacological profile when compared to INH that should result in activity against INH resistant clinical isolates. In search of potent and selective InhA inhibitors, we utilized encoded library technology (ELT), a proven novel and robust hit/lead identification platform27−29 wherein a large collection of chemotypically diverse DNA-encoded small molecule libraries are screened for affinity toward a desired protein target. The broad chemical diversity and modest target protein requirements are two of the major benefits of ELT for early drug discovery. Successful ELT hit identification campaigns against diverse targets have been previously reported.30−34 Herein we report the discovery of a novel chemotype identified through ELT affinity selection against M. tuberculosis InhA and its subsequent lead optimization.

Figure 1. Design of DNA-encoded library (DEL).

diamino acid scaffolds in cycle 1, followed by elaboration with over 800 amine-capping building blocks (carboxylic acids, aldehydes, sulfonyl chlorides, and isocyates) in cycles 2 and 3 to generate a 16.1 million compound library. The details of the library synthesis will be the subject of a different publication in the near future. A cubic scatter plot in which each axis represents a cycle of diversity in the library was used to analyze and visualize the enriched library members for the selected chemotype (Figure 2).35 Individual points, corresponding to discrete small molecule warheads in the library, are shown in pink and sized according to number of unique instances recorded by DNA sequencing. The display is set to a minimum of two unique copies per warhead, which should indicate significantly enriched binders under these selection conditions. In this analysis, the feature of interest is represented by a plane defined by the cycle 1 building block (BB1) (2S,4R)-4-aminopyrrolidine-2-carboxylic acid (1), which was attached to DNA through its carboxylic acid functional group. Within the selected plane, there are multiple lines due to preferred disynthon combinations of 1 with specific cycle 3 building blocks (BB3). The most prominent line is the BB3 3ethyl-1-methyl-1H-pyrazole-5-carboxylic acid (2) that is highlighted in Figure 2. The selected disynthon combination of this selected BB3 and the BB1 (2S,4R)-4-aminopyrrolidine-2carboxylic acid (1) provides the core structure of the InhA feature selected from this library as a single stereoisomer. There are a number of BB2s selected within the line each represented by a single point; therefore, the BB2 is a variable component of the selected scaffold that provides additional SAR in the form of possible moieties tolerated by the site of interaction on the protein. This “selection” SAR is a valuable tool in compound prioritization for off-DNA synthesis and can provide exquisite guidance in early chemotype lead optimization. In preparation for the off-DNA activity confirmation, three exemplars within the selected disynthon line were chosen to follow up. The synthetic strategy was designed to put together the pharmacophore earlier in the synthesis and add on the variable components later (Scheme 1). This allowed for testing of the intermediate in the activity assay and therefore additional SAR exploration. Synthesis was initiated with peptide coupling of amine 1 to acid 2 that afforded methyl ester 3. Saponification of the ester 3 followed by another amide formation with ethylamine gave the desired Boc-protected intermediate 4. TFA removal of the Boc protecting group followed by acylation of the free amine produced the desired final compounds (6, 7, and 8). In order to submit the corresponding ester and acids for testing, the desired compounds were prepared through Boc removal of precursor 3 followed by coupling with the appropriate carboxylic acid to give the corresponding methyl ester 9. After saponification of methyl ester 9 the corresponding acid 10 was accessible for testing as well. The prepared compounds were then assayed for their activity against M. tuberculosis InhA as reported in Table 1. The first three

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RESULTS AND DISCUSSION As described in earlier reports, affinity selection of InhA was performed by immobilizing biotinylated protein on a streptavidin matrix in PhyNexus tips.27 Selections were conducted in parallel against a panel of DNA-encoded libraries (DELs), representing a diversity of compounds, under conditions of protein alone, protein with NAD+, and protein with NADH. While enrichment of specific chemotypes was observed under all selection conditions, it was particularly evident for selections run in the presence of NADH cofactor. A putative hit from an aminoproline scaffold (Figure 1) was chosen for further follow-up. The DNAencoded library (Figure 1) utilized 22 orthogonally protected 1277



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Figure 2. (a) Spotfire cube data view of InhA selection feature in the presence of NADH. (b) Selected chemotype: BB1, cycle 1 building block; BB3, cycle 3 building block; RCO-, variable cycle 2 building block.

Taking into account the moderate intrinsic clearance found in the hepatocyte assay, the potential metabolites of 7 were examined after incubation with hepatocytes and metabolite follow-up by MS/MS. Results from N-dealkylation of the ethylamide and pyrazole unit in addition to the formation of oxidation products were the main detected metabolites (Figure S1 and Table S1). The complex crystal structure showed that compound 7 binds to InhA in the cleft normally occupied by substrate/product14,41 (Figure 3). The pyrazole 2-nitrogen makes a hydrogen bonded interaction to the 2′-hydroxyl group of NADH. In other InhA− inhibitor bound structures an interaction with the NADH 2′hydroxyl is also present (see Figure 3B for the triclosan complex structure14,21) typically as part of a hydrogen bond network involving Tyr158. In the current complex a network to Tyr158 is not formed in part because of Tyr158 maintaining the position it occupies in the apo-enzyme.14 The substrate binding loop is ordered in the structure with helix α6 (residues 197−206) closing the volume of the pocket. There are direct hydrogen bond interactions between main chain atoms of residues in the helix and atoms flanking the central proline of the ligand. In order to expand the explored chemical space beyond the building blocks contained within the ELT library and also with the idea of clarifying any possible bias introduced by the DNA tags, we decided to introduce novel modifications in the three positions of interest around the proline scaffold (Figure 4). The main objectives of this initial exploration were to increase both enzymatic activity and antitubercular potency. The 4-aminoproline central core was left unmodified, since we believed it provided a desirable scaffold for the appropriate disposition of the three different arms driving interactions within the InhA active site. Chirality at the proline core proved to be essential for activity (synthesis of 11’s enantiomer in Scheme S1). These

compounds (6, 7, and 8) synthesized off-DNA demonstrated inhibitory activity in the InhA assay. These data indicated some potential for further modification of the R1 moiety with the 3substitued benzofuran 7 giving an IC50 potency of 34 nM. Preliminary SAR exploration around the methylamide was initiated with synthesis and testing of the corresponding methyl ester and carboxylic acid. As demonstrated in Table 1, the inhibitor potency diminished to 215 nM for the methyl ester (9) and to 1290 nM for the corresponding carboxylic acid (10). In order to better understand the properties of compound 7, early activity and in vitro profiling of the hit structure were completed (Table 2) along with obtaining a complex crystal structure with InhA. As a key issue in any target based program, we were able to ascertain the relationship between enzymatic InhA inhibition and antitubercular MIC through the use of an InhAMTB overexpressor strain.36 The compound showed a selective antibacterial activity against M. tuberculosis and was found to be endowed with good potency against the target and modest, yet selective antitubercular activity. Furthermore, the compound exhibited an encouraging balance among solubility, lipophilicity, and permeability. The physicochemical properties of a compound are fundamental in the drug discovery process, mainly because of their influence on determining ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties and the overall suitability of drug candidates. For the oral route of administration the ‘ideal’ set of physicochemical properties is well established.37−40 The hit was metabolically stable when exposed to both human and mice microsomal fractions and showed moderate intrinsic clearence in hepatocytes. No measurable activity was found against the common cytochrome P450 (CYP450) isoforms (1A2, 2C9, 2D6, 3A4) or in the hERG dofetilide binding assay (pIC50 < 4.3). Cytotoxicity evaluation in HepG2 showed TOX50 > 50 μM. 1278



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Scheme 1. Off-DNA Synthesis of InhA Selected Hitsa

a Reagents and conditions: (i) N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethyene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU, 1.2 equiv), N,N-diisopropylethylamine (DIPEA, 3.0 equiv), N,N-dimethylformamide (DMF); (ii) lithium hydroxide (LiOH, 3.0 equiv); (iii) HATU (1.2 equiv), DIPEA (3.0 equiv); ethylamine hydrochloride (2.0 equiv); (iv) 20% trifluoroacetic acid in dichloromethane; (v) carboxylic acid (RCOOH, 1.2 equiv), HATU (1.2 equiv), DIPEA (3.0 equiv), DMF.

conclusions were supported by the evidence generated during the ELT selection process, where 22 orthogonally protected diamino acid scaffolds were deselected for InhA interaction. Preliminary SAR at P1, P2, and P3 Positions. The molecule was divided into three different regions, and one novel modification at a time was introduced in each position. This strategy allowed for the quick exploration and combination of the best chemical features in P1, P2, and P3 (Figure 4). We first turned our attention to the introduction of new diversity in P1. In order to confirm the importance of P1 in the pharmacophore, this position was suppressed. Compound 12 was still able to inhibit the enzyme with moderate activity (IC50 = 0.12 μM), but no whole cell activity was found (synthesis in Scheme S2). This evidence together with the realization that this was the DNA-anchoring site for the ELT studies prompted us to explore a wider chemical diversity at this site (Table 3). A number of modifications were attempted: truncation of the side chain to the primary amide, elongation and branching of the alkyl chain, aliphatic cycles, introduction of different heteroatoms and amino acids in addition to exploration of a number of aromatic substitutions as depicted in compounds 25−48 where ring changes, linker exploration, and variations of the substitution pattern in the aromatic rings were explored. Synthesis of the starting material 10 depicted in Scheme 1 allowed for a facile access of P1 modifications.

This early SAR investigation showed how the replacement of the amide bond by an amine group (compound 13) resulted in a complete loss of potency (synthesis in Scheme S3). N-alkylation of the amide (compound 15) was not tolerated. A number of replacements (Table 3) gave rise to compounds with excellent enzymatic potency and activities in cellular assay much less impressive or simply flat (see compounds 17, 26, 36, and 37). On the other hand, some compounds exhibited higher IC50 values that cannot be easily related to the potency of antitubercular activity (see compounds 32, 34, and 35). The fact that the context of the FAS II multienzyme complex is not truly represented in the enzymatic assay could be involved in this disconnection.42 Among the active compounds, 11 was 1 order of magnitude more potent in the whole cell assay than 7 and several times more potent than the rest and had the best physicochemical properties in terms of molecular weight, solubility, and lipophilicity (data not shown). This served as a basis for further optimization at other positions. Transformations in P2 required the synthesis of the special precursors 54 and 55 (Scheme 2). The effects of different heteroaromatic ring substitutions on the P2 2,4-pyrazole ring were examined. The SAR exploration was restricted to a small number of analogues of the pyrazole unit. Other P2 derivatives caused a significant or complete decrease in enzymatic activity (the SAR is summarized in Table S2). 1279



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Table 1. Activity of the First Compounds Made, Off-DNA

Figure 3. Compound 7 bound to the active site of InhA. In (A), compound 7 is shown in green ball-and-sticks, NADH is shown as thick lines, and hydrogen bonds are shown as dashed lines. Helix α6 and Tyr158 are also highlighted. In (B), the complex structure of triclosan (PDB code 1P45) is shown in gold superposed on the 7 complex structure. The different positions of helix α6 and Tyr 158 in the two complexes are visible.

Synthesis of the starting material 5 as depicted in Scheme 1 allowed for a facile access to P3 modifications. Among the heteroaromatic substituents investigated as possible P3 position replacements, only the benzofuran and benzothiophene moieties were associated with a significant in vitro antitubercular potency (Table S3). This tight SAR was confirmed with additional studies as shown further below.

Figure 4. Three positions of the target compound structure.

Table 2. Complete Profile of 7: In Vitro InhA Inhibition, Antitubercular Activity, Cytotoxicity, and Physicochemical Properties and in Vitro DMPK Profile

a

Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Drug Discovery Today 2011, 16, 822−830. bCLND solubility values that are within 85% of maximum possible concentration (as determined from DMSO stock concentration). 1280



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Table 3. P1 Modifications: Structures and Potency of Compounds 11−48

was fixed as the most suitable group in terms of potency. A selected group of mainly nonpolar aliphatic substitutions was evaluated around P2 (Table 4). The compounds bearing at position 4 cyclopropyl (58) and propyl (56) groups were found

Further Optimization of P1, P2, and P3 Positions. On the basis of the preliminary established SAR, a second round of in depth exploration of the P2 and P3 positions was initiated. Despite the obvious ester hydrolysis liabilities,43 the glycine at P1 1281



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Scheme 2. Synthesis of 54 and 55 Starting from 49a

a

Reagents and conditions: (i) ethylamine hydrochloride (1.2 equiv) or glycine methyl ester (1.1 equiv), HATU (1.2 equiv), DIPEA (3.0 equiv), DMF; (ii) 20% trifluoroacetic acid in dichloromethane; (iii) carboxylic acid (benzofuran-3-carboxylic acid, 1.2 equiv), HATU (1.2 equiv), DIPEA (3.0 equiv), DMF; (iv) piperidine/DMF 1:8 v/v, 0.6 M.

Table 4. SAR of the Pyrazole Derivatives: Structures and Potency of Compounds 56−62 Compared with 11

106). With the aim of generating isosteric substitution patterns more suitable for further development, different P1 analogues were prepared aimed at overcoming the blood instability issues associated with the ester group (Table S5).43 First, ester variations were attempted with the aim of altering the rate of hydrolysis (entries 107−113). This approach was unsuccessful as described in Table S5. Other alternatives, such as replacements with different amide combinations (primary, secondary or tertiary amides; entries 114, 115, 116) or oxazole (entry 45), were used, but all led to significant drops in whole cell potency. Intriguingly, while low enzymatic IC50 values could be achieved, no compounds with MIC < 1 μM could be identified. At this stage and given the improved activity of the diethylpyrazole P2 substitution, we chose to re-examine P1 (Table 5). The synthetic route to prepare the starting material 64 that afforded compounds 65−70 is outlined in Scheme S5. A selection of some of the best scaffolds in terms of potency, structural diversity, and physicochemical properties led to the synthesis of the new compounds 65−70. However, the desirable synergistic nature of the SAR of the diethylpyrazole and P1 substituents previously observed with the glycine derivative 59 was not transferable to the same extent to other P1 replacements. All the compounds 65−70 showed InhA inhibition at low concentrations combined with good activity against M. tuber-

to be equipotent to ethyl (11) in MIC. In contrast, compound 57, with the larger isobutyl in the 4-pyrazole position, was found to be inactive. With regard to possible pyrazole replacement patterns, the 2-ethyl substitution, compound 59, showed similar IC50 values but 1 order of magnitude improvement in terms of MIC. The three-carbon chain propyl (60) was still active but the polar 2-hydroxyethyl group (61) and the bulkier benzyl substitution (62) were not tolerated. From this moment onward, the diethylpyrazole was selected as the P2 substitution pattern of choice. With regard to P3, the SAR exploration is summarized in Table S4. The synthetic route to prepare the starting material 63 that afforded compounds 103−107 is outlined in Scheme S4. These studies showed how nonpolar group substitutions at the 2benzofuran position were tolerated. The fact that none of the replacements tested at P3 showed any significant improvement confirmed how the ELT work around this position was able to effectively cover a significant amount of chemical space. The benzofuran at P3 was hence fixed ahead of further P1 optimization. Unfortunately, the ester functionality of compound 11, which stood out with an MIC value of 0.5 μM, was deemed unsuitable because of the liabilities associated with enzymatic hydrolysis to generate the likely nonpermeable carboxylic acid product (entry 1282



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Table 5. SAR of Diethylpyrazole Derivatives: Structures and Potency of Compounds 65−70

a

Young, R. J.; Green, D. V. S.; Luscombe, C. N.; Hill, A. P. Getting physical in drug discovery II: the impact of chromatographic hydrophobicity measurements and aromaticity. Drug Discovery Today 2011, 16, 822−830. bLigand efficiency (LE): enzyme pIC50/heavy (non-hydrogen) atom count. cMortenson, P. N.; Murray, C. W. J. Comput.-Aided Mol. Des. 2011, 25, 663−667.

Table 6. Complete Profile of Compound 65: In Vitro InhA Inhibition, Antitubercular Activity, and Physicochemical Properties and Early in Vitro DMPK Profile

culosis with MIC values in the range of 0.2−1 μM. We have chosen to use the ligand lipophilicity efficiency index (LLE) designed by Leeson and Springthorpe. This useful index helps to rank compounds based on potency and lipophilicity along the lead optimization.44 We have also taken into account the LLEAT, which combines lipophilicity, size, and potency and has been designed to have the same target value of 0.3 kcal/mol and dynamic range as LE.45−48 As an indication of druglike properties, compound 65 showed the best ligand efficiency value of 0.37 kcal mol−1 atom−1, an LLEAT of 0.42. This was a significant improvement when compared to compound 7 (LE = 0.32, LLEAT = 0.36). The good potency values, combined with a reasonable lipophilicity and the lower molecular weight, led us to progress this lead molecule to further in vivo evaluation.

Profile of Compound 65. In order to complete its profile, compound 65 was tested for activity against intracellular bacteria, showing an MIC value of 0.24 μM. This significant intracellular activity (in THP-1 cells) was accompanied by a clean P450 profile (1A2, 2C9, 2C19, 2D6, and 3A4; IC50 > 50 μM in all cases) and good stability values in mouse and human microsomal fractions (Table 6). The bactericidal potential of compound 65 was assessed in killing rate experiments with linezolid and moxifloxacin as bacteriostatic and bactericidal controls, respectively (Figure S2). The results show that compound 65 was able to reduce >2 log cfu counts after 1 week of incubation very close to the moxifloxacine behavior. 1283


Journal of Medicinal Chemistry Given its interest, the pharmacokinetic profile of 65 in C57BL/6J mice following oral administration was evaluated at 50 mg/kg po (1% methylcellulose) (Figure 5 and Table 7).

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CONCLUSIONS

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EXPERIMENTAL SECTION

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A class of potent and selective M. tuberculosis direct InhA inhibitors was identified by means of the application of DNA encoded library technology. Compound 7 was considered a suitable starting point for further medicinal chemistry optimization. In order to systematically investigate the chemical space around the proline core, one modification at a time was introduced in the molecule around the three possible positions with the initial goal of improving potency. This approach was chemically flexible and was able to quickly provide SAR information. After combination of the best features in each position, P3 was found to be the least malleable part in terms of SAR manipulations. The nature of the group at this position was found to be key for potent enzymatic activity. The diethylpyrazole at position P2 emerged as the most suitable moiety for antitubercular potency optimizing. The SAR analysis around P1 provided the ability to improve the physicochemical and ADME properties. Feature combination resulted in the synthesis of the optimized lead compound 65. Despite its good balance between InhA inhibition, antitubercular potency, and pharmacokinetic profile, compound 65 was shown to be inactive in vivo against a murine TB acute infection model. Overall we presented here the optimization of a new antitubercular hit series showing good InhA enzymatic and antitubercular potencies and adequate physicochemical properties as reflected in attractive LE and LLE numbers. A clear disconnection was found between the antitubercular in vitro and in vivo profile of lead compound 65.

Figure 5. Peripheral blood levels of 65 after oral administration to C57BL/6J mice (n = 3) at a dose of 50 mg/kg as suspension in 1% methylcellulose. Individual values and mean values for each time point are represented in the plot.

Additionally, compound 65 was assayed by intravenous route at 4 mg/kg and showed a high in vivo clearance (CL = 85.5 mL min−1 kg−1) and a short half-life (t1/2 = 0.5 h) in mice (Table 7). The oral PK profile was characterized by a Cmax value of 1.75 μg/ mL at 0.83 h postdosing and an AUC(0−∞) of 3.59 μg·h/mL corresponding to 36% bioavailability (F). In view of its balanced pharmacological, physicochemical, and in vivo ADME profiles, compound 65 was progressed to both tolerability and acute efficacy studies in a murine model of M. tuberculosis infection.49 The aim of the preliminary safety study was to estimate the maximal tolerable single oral dose level of 65 in female C57BL/6J mice. The MNLD (maximal nonlethal dose) in mice treated with 65 (single oral dose formulated in 1% methylcellulose by gavage followed by observation for 7 days) was higher than 1000 mg/kg. No adverse clinical events were found. The mean total exposure (AUC), obtained at 24 h, associated with a 1000 mg/kg dose was 73.69 μg·h/mL, and the corresponding mean Cmax was 23 μg/mL. Subsequently, compound 65 was in vivo tested to determine the therapeutic efficacy against a murine acute M. tuberculosis infection model (Figure S3). No significant reduction of colony forming units (cfu) at any dose tested was observed in the lungs of infected mice. The positive control moxifloxacin (30 mg/kg body weight) reduced the cfu by 3.48 log with respect to untreated mice. Despite an acceptable in vivo pharmacokinetic profile and good antitubercular potency (0.5 μM), compound 65 was found to be inactive in an acute TB infection model. This result underlines how antitubercular compounds with good in vitro activity and aceptable PK properties can still be devoid of in vivo activity. Further studies regarding possible explanations for this disconnection would need to be done.

General Procedures. All commercially available reagents and solvents were used without further purification. (2S,4R)-1-tert-Butyl 2methyl 4-aminopyrrolidine-1,2-dicarboxylate hydrochloride 1 was purchased from Apollo, Advanced Tech, and Tyger. 3-Ethyl-1-methyl1H-pyrazole-5-carboxylic acid 2 was purchased from Chembridge. (2S,4R)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-1-(tertbutoxycarbonyl)pyrrolidine-2-carboxylic acid 49 was purchased from Aldrich and Cheminpex. Benzofuran-3-carboxylic acid 79 was purchased from Apollo, Bionet, and Maybridge. 1,3-Diethyl-1H-pyrazole-5carboxylic acid 84 was purchased from Sinova SL. HATU was purchased from Carbosynth. The rest of the reagents were obtained from Aldrich, Maybridge, Apollo, Panreac, Chembridge, Novabiochem, Enamine, Combiblocks, Artchem, and JW Pharmlab. Reactions were monitored by thin layer chromatography (TLC) using Merck 60 F254 silica gel glass backed plates. Elution was with suitable solvent mixtures, and visualization was by UV light. Reactions have also been followed by mass spectrometry and reversed phase chromatography using a high resolution spectrometer Waters ZMD 2000 coupled with LC Agilent 1100 with DAD detection. The products were purified by column chromatography or preparative HPLC. Automated flash chromatography was performed on a Flash Biotage Isolera SP4 system with peak detection at 254 nm, and preparative HPLC was performed in an Agilent 1100 and 1200 with peak detection at 254 nm. All products were obtained as amorphous solids, and melting

Table 7. Pharmacokinetic Parameters of 65 after Intravenous Administration of 4 mg/kg a Single Dose (20% Encapsin/%5 DMSO) and Oral Administration of 50 mg/kg b Single Dose (1% Methylcellulose) to C57BL/6J Mice (n = 3)c route iv po

Tmax b (h)

Cmax b (μg/mL)

AUC(0−∞) b (μg·h/mL)

t1/2 a (h)

Compound 65 0.47 (0.20) 0.83 (0.14)

1.75 (0.54)

3.59 (0.53)

Vd a (L/kg))

CL a (mL min−1 kg−1)

3.68 (2.08)

85.51 (15.93)

F (%)

35.65 (5.07)

a

t1/2, half-life; Vd, volume of distribution; CL, clearance. bTmax, time at maximum concentration; Cmax, maximum concentration; AUC(0−∞), area under the curve for total exposure to infinite time. cValues are included as the mean and standard deviation (SD) of individual values. 1284



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3-Ethyl-N-(5-(ethylcarbamoyl)pyrrolidin-3-yl)-1-methyl-1Hpyrazole-5-carboxamide 2,2,2-Trifluoroacetate (5). Compound 5 was synthesized from Boc-deprotection of (2S,4R)-tert-butyl 4-(3-ethyl1-methyl-1H-pyrazole-5-carboxamido)-2-(ethylcarbamoyl)pyrrolidine1-carboxylate (4) using the protocol described in general method C. The reaction mixture was concentrated to dryness (coevaporated with CH2Cl2 × 5, EtOAc × 1) to give the title compound (4.22 g, 128% yield) as an orange oil. The yield was >100% because the product contained EtOAc which could not be removed under reduced pressure. The crude product was carried on to next step. MS (ESI) m/z [M + H]+ = 294; 1H NMR (400 MHz, DMSO-d6) δ 9.46 (br s, 1H), 8.78 (br s, 1H), 8.56 (d, J = 6.3 Hz, 1H), 8.50−8.47 (m, 1H), 6.65 (s, 1H), 4.51−4.43 (m, 1H), 4.36−4.26 (m, 1H), 3.95 (s, 3H), 3.62−3.52 (m, 1H), 3.25−3.12 (m, 3H), 2.57−2.49 (m, 2H), 2.39−2.27 (m, 1H), 2.18−2.08 (m, 1H), 1.19−1.13 (m, 3H), 1.05 (t, J = 7.25, 3H). N-((3R,5S)-1-(Benzofuran-3-carbonyl)-5-(ethylcarbamoyl)pyrrolidin-3-yl)-3-ethyl-1-methyl-1H-pyrazole-5-carboxamide (7). Compound 7 was synthesized from Boc-deprotection of (2S,4R)tert-butyl 4-(3-ethyl-1-methyl-1H-pyrazole-5-carboxamido)-2(ethylcarbamoyl)pyrrolidine-1-carboxylate (4) using the protocol described in general method C followed by coupling with benzofuran3-carboxylic acid using the procedure described in general method A. The reaction mixture was concentrated in vacuo, and the residue was purified by preparative HPLC. The reaction afforded the title compound (40 mg, 29% yield). MS (ESI) m/z [M + H]+ = 437.9; 1H NMR (400 MHz, DMSO-d6) δ 8.49 (s br, 2H), 7.98 (m, 2H), 7.64 (d, J = 7.6 Hz, 1H), 7.34 (m, 2H), 6.61 (s, 1H), 4.59 (m, 2H), 4.11 (m, 1H), 3.99 (s, 3H), 3.67 (m, 3H), 3.08 (m, 2H), 2.24 (m, 1H), 2.08 (m, 1H), 1.12 (t, J = 7.2, 3H), 1.01 (t, J = 6.8, 3H); 13C NMR (100 MHz, DMSO-d6, 25 °C) δ ppm 13.7, 14.7, 20.6, 33.4, 34.6, 38.4, 48.6, 53.1, 59.3, 105.3, 111.4, 116.2, 122.3, 123.6, 125.2, 126.0, 135.3, 151.3, 153.9, 159.5, 161.8, 170.8. [α]23.2D −34.71 (c 0.68, EtOH). N-((3R,5S)-1-(Benzofuran-3-carbonyl)-5-carbamoylpyrrolidin-3-yl)-1,3-diethyl-1H-pyrazole-5-carboxamide (65). The title compound was synthesized from 64 using the protocol described in general method A. After preparative HPLC purification (XBridge-30, 35 mL/min, 10−100% acetonitrile in water, 10 nM NH4HCO3, basic, 20 min) the reaction afforded the title compound (423 mg, 77% yield). MS (ESI) m/z [M + H]+ = 424; 1H NMR (400 MHz, DMSO-d6, 80 °C) δ 8.34 (br s, 1H), 8.28 (d, J = 6.6 Hz, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.41−7.31 (m, 2H), 7.13−6.82 (br s, 2H), 6.60 (s, 1H), 4.73 (dd, J = 8.3, 4.9 Hz, 1H), 4−62−4.57 (m, 1H), 4.40−4.35 (m, 2H), 4.11−4.07 (m, 1H), 3.71 (dd, J = 10.8, 5.6 Hz, 1H), 2.58−2.53 (m, 2H), 2.34−2.22 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H), 1.19 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, DMSO-d6, 25 °C) δ ppm 13.7, 15.8, 20.7, 34.5, 45.4, 48.6, 53.1, 59.1, 105.4, 111.4, 116.2, 122.2, 123.6, 125.2, 126.0, 134.7, 151.4, 153.9, 159.5, 161.8, 173.2. [α]23.2D −46.53 (c 0.68, EtOH). Materials and Methods. The human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents. All animal studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and the GSK Policy on the Care, Welfare and Treatment of Animals. Affinity Selection. Selections were done using streptavidin matrix tips (PhyNexus) and biotinylated InhA protein (full length (270aa)). Three rounds of selections were performed. Each tip in each round of selection had 10 μg of protein loaded/attached to the matrix. The InhA protein was chemically biotinylated using NHS-chromogenic biotin. Biotinylated protein ran as a dimer (76 kDa) and retained activity. Before use, tips were prewashed in selection buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Tween 20) and 1.0 mg/mL sheared salmon sperm DNA (sssDNA, Ambion) and 10 mM β-mercaptoethanol (BME). For selection round 1, 10 μg of biotinylated InhA protein was immobilized on a previously prepared streptavidin matrix tip and the tip was washed four times with selection buffer to remove excess protein. The selection conditions included buffer with and without NADH. When the experimental conditions included NADH, it was added to all relevant buffers, with the exception of elution buffer, to a final concentration of 133μM. Then 5 nmol of pyrrolidine library was diluted in 60 mL of selection buffer. The appropriate library samples were

points were not measured. All NMR spectra were recorded on a Bruker DPX Avance 400 MHz instrument equipped with a QNP probe. Measurements were made at room temperature and at 80 °C and in an appropriate deuterated solvent using residual hydrogenated solvent as standard (CDCl3, δ = 7.26 ppm and DMSO-d6, δ = 2.50 ppm); conditions were indicated in each case. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants (J) are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), dd (double doublet), m (multiplet), br (broad). Analytical purity was ≥95% unless stated otherwise, as determined by 1H NMR and HPLC analyses. The purity of final compounds was checked using a Waters ZQ2000 coupled with LC Waters 2795 and Waters 2996 PDA detector. All mass spectra were performed by electrospray ionization (ESI). Representative procedures and physical properties and characterization for main compounds are described. Characterization data for the rest of the compounds are detailed in the Supporting Information. Synthesis of Compounds. General Method A. General Procedure for HATU-Mediated Coupling. To the desired carboxylic acid (1.0 equiv) and HATU (1.2 equiv) in N,N-dimethylformamide was added Hunig’s base (3.0 equiv). To the reaction mixture was then added the desired amine (1.0 equiv). The reaction mixture was allowed to stir at room temperature for 3−12 h. The reaction mixture was then diluted with EtOAc (100 mL), washed with 10% NH4Cl (2 × 100 mL), saturated NaHCO3 (1 × 100 mL), saturated NaCl (1 × 100 mL), dried (MgSO4), and evaporated to dryness. The crude product was assessed for purity and the desired mass by LCMS analysis. General Method B. General Procedure for the Deprotection of Methyl Esters. To the desired methyl ester starting material (1.0 equiv) in tetrahydrofuran (THF)/methanol (5:1) was added a solution of lithium hydroxide (3.0 equiv). The reaction mixture was stirred overnight at room temperature. The reaction was analyzed by LCMS. The reaction mixture was diluted with EtOAc, washed with 10% KH2SO4, saturated NaCl, dried over MgSO4, and evaporated to dryness. General Method C. General Procedure for Boc-Deprotection of Carbamates. To the desired Boc-protected starting material (1.0 equiv) in dichloromethane was added TFA (20% by volume).The reaction mixture was then stirred at room temperature for 2 h. Deprotection process was monitored by LCMS. The solvents were evaporated in vacuo, and the product was azeotroped with 2 × CH2Cl2. General Method D. Removal of Fmoc-Protection Group or Fmoc Deprotection. A solution of piperidine/dimethylformamide (1:8 v/v, 0.6 M) was added to the resin and shaken at room temperature for 1 h. The reaction was checked by LCMS and showed that the deprotection had been successful. The remaining piperidine and dimethylformamide were removed by rotary evaporator heating at 80 °C. This crude was dissolved in MeOH, and a white precipitated appeared. The solid was filtered off. The crude was added to a silica gel column and was eluted with CH2Cl2/MeOH 8%. (2S,4R)-1-tert-Butyl 2-Methyl 4-(3-ethyl-1-methyl-1H-pyrazole-5-carboxamido)pyrrolidine-1,2-dicarboxylate (3). Compound 3 was synthesized from the (2S,4R)-1-tert-butyl 2-methyl-4aminopyrrolidine-1,2-dicarboxylate (1) and 3-ethyl-1-methyl-1H-pyrazole-5-carboxylic acid (2) using the same procedure as described in general method A. The reaction produced a brown oil in 96% yield (1.57 g) with a purity of 95%. No further purification was needed. MS (ESI) m/z [M + H]+ = 380.8. (2S,4R)-tert-Butyl 4-(3-Ethyl-1-methyl-1H-pyrazole-5-carboxamido)-2-(ethylcarbamoyl)pyrrolidine-1-carboxylate (4). Compound 4 was synthesized from saponification of (2S,4R)-1-tert-butyl 2methyl 4-(3-ethyl-1-methyl-1H-pyrazole-5-carboxamido)pyrrolidine1,2-dicarboxylate (3) using the same procedure as described in general method B followed by coupling with ethylamine using the same procedure as described in general method A. The reaction produced a brown oil in 92% yield (1.38 g) with a purity of 95%. The crude product was carried on to next step as is. MS (ESI) m/z [M + H]+ = 393.8; 1H NMR (400 MHz, DMSO-d6) δ 8.42 (br s, 1H), 7.91 (br s, 1H), 6.66 (s, 1H), 4.41 (m, 1H), 4.11 (m, 1H), 3.93 (s, 3H), 3.63 (m, 2H), 3.31 (m, 1H), 3.26 (m, 2H), 3.09 (m, 2H), 2.14 (m, 1H), 2.01 (m, 1H), 1.32 (s, 9H), 1.14 (t, J = 7.2, 3H), 1.01 (t, J = 6.8, 3H). 1285



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they were incubated at 37 °C without shaking for 6 days. A resazurin solution was prepared by dissolving one tablet of resazurin (resazurin tablets for milk testing; ref 330884Y′ VWR International Ltd.) in 30 mL of sterile PBS (phosphate buffered saline). Of this solution, an amount of 25 μL was added to each well. Fluorescence was measured (Spectramax M5 Molecular Devices, excitation 530 nm, emission 590 nm) after 48 h to determine the MIC value. Pharmacokinetic Studies. For pharmacokinetic studies C57BL/6 female mice of 18−20 g weight were used (n = 3 mice). Experimental compounds were administered by intravenous (iv) bolus at 4 mg/kg at a volume of 10 mL/kg to n = 3 mice and by oral gavage (po) at 50 mg/kg single dose at a volume of 20 mL/kg to n = 3 mice. All mice received treatment in the fed state. Drugs were administered as solution for iv route in 20% encapsin and 5% DMSO and as suspension for po route in 1% methylcellulose. Peripheral total blood was the compartment chosen for the establishment of compound concentrations. Aliquots of 25 μL of blood were taken from the lateral tail vein for each mouse at the following time points: for iv route, 10, 20, and 30 min and 1, 2, 4, and 8 h; for po route, 15, 30, and 45 min and 1, 2, 4, and 8 h. LCMS was used as the analytical method of choice for the establishment of compound concentration in blood with a sensitivity of LLQ = 1−5 ng/mL in 25 μL of blood. The noncompartmental data analysis (NCA) was performed with WinNonlin Phoenix 6.3 (Pharsight, Certara L.P.), and supplementary analysis was performed with GraphPad Prism 5 (GraphPad Software, Inc.). In Vivo Efficacy Assessment. Specific pathogen-free, 8- to 10week-old female C57BL/6 mice were purchased from Harlan Laboratories and were allowed to acclimate for 1 week. The experimental design has been previously described.49 In brief, mice were intratracheally infected with 100 000 cfu/mouse (M. tuberculosis H37Rv strain). Products were administered for 8 consecutive days starting 1 day after infection. Lungs were harvested 24 h after the last administration. All lung lobes were aseptically removed, homogenized, and frozen. Homogenates were plated in 10% OADC-7H11 medium for 14 days at 37 °C.

passed over the streptavidin matrix tip for 1 h at room temperature. The tip was washed 8 times with selection buffer and 2 times with DNA free selection buffer. Binders were eluted by passing heated (80 °C) DNA free selection buffer over the tip for 12 min. The eluted material was passed over a fresh streptavidin matrix tip for 10 min at room temperature to remove any denatured protein (postclear). The postclear step was repeated a second time to the round 1 selection output (1 μL of the round 1 selection output was retained to be used for qPCR in order to monitor the output from this round of selection). The remaining round 1 selection output was brought to 60 μL by adding the necessary volume of sssDNA and selection buffer. For selection round 2, the above selection procedure was repeated with fresh protein and a previously prepared fresh streptavidin matrix tip. Then 5 μL of the round 2 selection output was retained to be used for qPCR in order to monitor the output from this round of selection. The remaining round 2 selection output was brought to 60 μL by adding the necessary volume of sssDNA and selection buffer. For selection round 3, the selection procedure was repeated with fresh protein, a previously prepared fresh tip, and the round 2 selection output. The postclear steps were not repeated at the end of round 3. Quantitative PCR was run with the outputs from each round of selection to assess selection yields for each step. The round 3 output was sequenced using Roche/454 technology. A no-target control selection was performed in the same fashion, using the same buffer but without the protein input. X-ray Crystallography. InhA was expressed in E. coli and purified in two steps by IEC and size exclusion chromatography. The protein (4.15 mgs/mL in 20 mM Tris, pH 8.0, 0.1 M NaCl, 2 mM DTT) was incubated with NADH and compound prior to crystallization. The complex crystallized in 10% PEG 8000 and 0.1 M Tris, pH 8.5, at 20 °C and was cryoprotected prior to data collection at ESRF. The structure was solved by molecular replacement. More details, statistics, and exemplars of the density are given in the Supporting Information. InhA Enzymatic Assay. Enzymatic activity was measured fluorimetrically by following NADH oxidation at λexc = 340 nm and λem= 480 nm, using 50 mM NADH and 50 mM 2-trans-dodecenoyl-CoA (DDCoA) as substrates. Dose−response experiments to determine IC50 were performed using 5 nM InhA. The percentage of remaining enzymatic activity (% AR) at different compound concentrations was calculated with the formula % AR = 100 × [(sample − control 2)/ (control 1 − control 2)] where sample is the enzymatic activity for each compound concentration, control 1 is enzyme activity in the absence of any compound, and control 2 is NADH oxidation in absence of enzyme. IC50 was calculated fitting % AR to a two parameter equation % AR = 100/[1 + (compound conc/IC50)s] where s is a slope factor. IC50 was calculated using GraFit 5.0.12 software (Eritacus Software Ltd.). All reactions were run in 30 mM PIPES buffer, pH 6.8, at 25 °C. Strain and Growth Conditions. M. tuberculosis H37Rv (ATC25618) wild type and H37Rv overexpressing InhA (HyrR) were grown in Middlebrook 7H9-ADC broth (Difco) supplemented with 0.05% Tween 80 and on 7H10-OADC or 7H11-OADC agar (Difco) at 37 °C. INH and Hyg were purchased from Sigma-Aldrich. Where required hygromycin (50 μg mL−1) was added to the culture medium. Mycobacterium tuberculosis Inhibition Assay. The measurement of the minimum inhibitory concentration (MIC) for each tested compound was performed in 96-well flat-bottom polystyrene microtiter plates. Ten 2-fold drug dilutions in neat DMSO were performed. An amount of 5 μL of these drug solutions was added to 95 μL of Middlebrook 7H9 medium (lines A−H, rows 1−10 of the plate layout). Isoniazid was used as a positive control. Eight 2-fold dilutions of isoniazid starting at 160 μg/mL were prepared, and 5 μL of this control curve was added to 95 μL of Middlebrook 7H9 medium (Difco catalogue ref 271310) (row 11, lines A−H). An amount of 5 μL of neat DMSO was added to row 12 (growth and blank controls). The inoculum was standardized to approximately 1 × 107 cfu/mL mL−1 and diluted 1 in 100 in Middlebrook 7H9 broth (10% ADC (Becton Dickinson catalog ref 211887) and 0.025% Tween 80) to produce the final inoculum. Then 100 μL of this inoculum was added to the entire plate except G-12 and H-12 wells (blank controls). All plates were placed in a sealed box to prevent drying out of the peripheral wells, and

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ASSOCIATED CONTENT

S Supporting Information *

Additional tables, schemes, figures, protocols and experimental procedures of some assays mentioned in the main text, synthetic procedures, and compound characterization. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*J.C.-P.: phone, +34-669-399879; fax, 34-918-070310; e-mail, [email protected]. *G.E.: phone, 1-781-795-4423; fax, 1-781-795-4496; e-mail, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Physicochemical Characterisation Group of Analytical Chemistry, GSK Tres Cantos, Mariá Teresa FraileGabaldón, and Rubén Gómez for carrying out chromlog D measurements for all the compounds and formulation studies, Angel Santos for protein binding measurements, and Jesús Gómez, Ana Á lvarez, and Raquel Gabarró for their help with LCMS and NMR analysis. Santiago Ferrer is thanked for his assistance with the pharmacology work. We acknowledge the ́ excellent technical expertise of Delia Blanco, Marı ́a Martinez, ́ Pedro A. Torres Gómez, and Mariá José Rubén González del Rio, Rebollo for biological evaluation. We gratefully acknowledge the Laboratory Animal Science (Pre-Clinical) Department for essential animal lab upkeep and maintenance. The research 1286



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(12) Vilcheze, C.; Morbidoni, H. R.; Weisbrod, T. R.; Iwamoto, H.; Kuo, M.; Sacchettini, J. C.; Jacobs, W. R., Jr. Inactivation of the inhAencoded fatty acid synthase II (FASII) enoyl-acyl carrier protein reductase induces accumulation of the FASI end products and cell lysis of Mycobacterium smegmatis. J. Bacteriol. 2000, 182, 4059−4067. (13) Banerjee, A.; Dubnau, E.; Quemard, A.; Balasubramanian, V.; Um, K. S.; Wilson, T.; Collins, D.; de Lisle, G.; Jacobs, W. R., Jr. InhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science 1994, 263, 227−230. (14) Dessen, A.; Quemard, A.; Blanchard, J. S.; Jacobs, W. R., Jr.; Sacchettini, J. C. Crystal structure and function of the isoniazid target of Mycobacterium tuberculosis. Science 1995, 267, 1638−1641. (15) Parikh, S. L.; Xiao, G.; Tonge, P. J. Inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis, by triclosan and isoniazid. Biochemistry 2000, 39, 7645−7650. (16) Scior, T.; Garces-Eisele, S. J. Isoniazid is not a lead compound for its pyridyl ring derivatives, isonicotinoyl amides, hydrazides, and hydrazones: a critical review. Curr. Med. Chem. 2006, 13, 2205−2219. (17) Argyrou, A.; Vetting, M. W.; Aladegbami, B.; Blanchard, J. S. Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid. Nat. Struct. Mol. Biol. 2006, 13, 408−413. (18) Schroeder, E. K.; de Souza, O. N.; Santos, D. S.; Blanchard, J. S.; Basso, L. A. Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tuberculosis. Curr. Pharm. Biotechnol. 2002, 3, 197−225. (19) Basso, L. A.; Santos, D. S. Drugs that inhibit mycolic acid biosynthesis in Mycobacterium tuberculosisan update. Med. Chem. Rev. 2005, 2, 393−413. (20) Hazbón, M. H.; Brimacombe, M.; Bobadilla Del Valle, M.; Cavatore, M.; Guerrero, M. I.; Varma-Basil, M.; Billman-Jacobe, H.; Lavender, C.; Fyfe, J.; Garcia-Garcia, L.; Leon, C. I.; Bose, M.; Chaves, F.; Murray, M.; Eisenach, K. D.; Sifuentes-Osornio, J.; Cave, M. D.; Ponce de Leon, A.; Alland, D. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2006, 50, 2640−2649. (21) Kuo, M. R.; Morbidoni, H. R.; Alland, D.; Sneddon, S. F.; Gourlie, B. B.; Staveski, M. M.; Leonard, M.; Gregory, J. S.; Janjigian, A. D.; Yee, C.; Musser, J. M.; Kreiswirth, B.; Iwamoto, H.; Perozzo, R.; Jacobs, W. R.; Sacchettini, J. C.; Fidock, D. A. Targeting tuberculosis and malaria through inhibition of enoyl reductase compound activity and structural data. J. Biol. Chem. 2003, 278, 20851−20859. (22) Lu, X. Y.; You, Q. D.; Chen, Y. D. Recent progress in the identification and development of InhA direct inhibitors of Mycobacterium tuberculosis. Mini-Rev. Med. Chem. 2010, 10, 182−193. (23) Ballell Pages, L.; Castro Pichel, J.; Fernandez Menendez, R.; Fernandez Velando, E. P.; Gonzalez del Valle S.; Leon Diaz M. L.; Mendoza Losana, A.; Wolfendale, M. J. (Pyrazol-3-yl)-1,3,4-thiadiazole2-amine and (Pyrazol-3-yl)-1,3,4-thiazol-2-amine Compounds. WO/ 2010/118852, 2010. (24) Pan, P.; Tonge, P. J. Targeting InhA, the FASII enoyl-ACP reductase: SAR studies on novel inhibitor scaffolds. Curr. Top. Med. Chem. 2012, 12, 672−693. (25) Castro Pichel, J.; Fernandez Menendez, R.; Fernandez Velando, E. P.; Gonzalez del Valle S.; Mallo-Rubio, A. 3-Amino-pyrazole Derivatives Useful against Tuberculosis. WO/2012/049161, 2012. (26) Horlacher, O. P.; Hartkoorn, R. C.; Cole, S. T.; Altmann, K.-H. Synthesis and antimycobacterial activity of 2,1′-dihydropyridomycins. ACS Med. Chem. Lett. 2013, 4, 264−268. (27) Clark, M. A.; Acharya, R. A.; Arico-Muendel, C. C.; Belyanskaya, S. L.; Benjamin, D. R.; Carlson, N. R.; Centrella, P. A.; Chiu, C. H.; Creaser, S. P.; Cuozzo, J. W.; Davie, C. P.; Ding, Y.; Franklin, G. J.; Franzen, K. D.; Gefter, M. L.; Hale, S. P.; Hansen, N. J. V.; Israel, D. I.; Jiang, J.; Kavarana, M. J.; Kelley, M. S.; Kollmann, C. S.; Li, F.; Lind, K.; Mataruse, S.; Medeiros, P. F.; Messer, J. A.; Myers, P.; O’Keefe, H.; Oliff, M. C.; Rise, C. E.; Satz, A. L.; Skinner, S. R.; Svendsen, J. L.; Tang, L.; Vloten, K. V.; Wagner, R. W.; Yao, G.; Zhao, B.; Morgan, B. A. Design, synthesis and selection of DNA-encoded small-molecule libraries. Nat. Chem. Biol. 2009, 5, 647−654. (28) Deng, H.; O’Keefe, H.; Davie, C. P.; Lind, K. E.; Acharya, R. A.; Franklin, G. J.; Larkin, J.; Matico, R.; Neeb, M.; Thompson, M. M.; Lohr,

leading to these results has received funding from the Global Alliance for TB Drug Development and from the European Union’s 7th Framework Program (FP7-2007-2013) under the Orchid Grant Agreement No. 261378. The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.

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ABBREVIATIONS USED ADMET, absorption, distribution, metabolism, excretion, and toxicity; AUC, area under the curve; BOC, tert-butyloxycarbonyl; CLND, chemiluminescent nitrogen detection; cfu, colony forming unit; DEL, DNA-encoded chemical libraries; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DMPK, drug metabolism and pharmacokinetics; DMSO, dimethyl sulfoxide; ELT, encoded library technology; HATU, N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate Noxide; INH, isoniazid; InhA, Mycobacterium tuberculosis InhA; LE, ligand efficiency; LLE, ligand lipophilicity efficiency; LLEAT, ligand lipophilicity efficiency Astex; MDR-TB, multidrugresistant tuberculosis; MIC, minimum inhibitory concentration; NMR, nuclear magnetic resonance; PFI, property forecast index, therapeutics; PK, pharmacokinetics; SAR, structure−activity relationship; TB, tuberculosis; TFA, trifluoroacetic acid; WHO, World Health Organization; XDR, extensively drug-resistant tuberculosis

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