4-Aminoquinolone Piperidine Amides: Noncovalent Inhibitors of

May 28, 2014 - ... Alderley Park, Macclesfield, Cheshire SK10 2NA, United Kingdom ..... Yong-Xia Zhu , Ying Xv , Wei-Qiong Zuo , Kai Ran , Hong-Xia De...
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4‑Aminoquinolone Piperidine Amides: Noncovalent Inhibitors of DprE1 with Long Residence Time and Potent Antimycobacterial Activity Maruti Naik,†,□ Vaishali Humnabadkar,‡,□ Subramanyam J. Tantry,†,□ Manoranjan Panda,†,□ Ashwini Narayan,‡ Supreeth Guptha,‡ Vijender Panduga,§ Praveena Manjrekar,† Lalit kumar Jena,† Krishna Koushik,† Gajanan Shanbhag,† Sandesh Jatheendranath,†,▲ M. R. Manjunatha,† Gopinath Gorai,† Chandramohan Bathula,† Suresh Rudrapatna,† Vijayashree Achar,† Sreevalli Sharma,‡ Anisha Ambady,‡ Naina Hegde,‡ Jyothi Mahadevaswamy,‡ Parvinder Kaur,‡ Vasan K. Sambandamurthy,‡ Disha Awasthy,‡ Chandan Narayan,‡,◆ Sudha Ravishankar,‡ Prashanti Madhavapeddi,‡ Jitendar Reddy,§ KR Prabhakar,§ Ramanatha Saralaya,§ Monalisa Chatterji,‡ James Whiteaker,⊥ Bob McLaughlin,∥ Laurent R. Chiarelli,∇ Giovanna Riccardi,∇ Maria Rosalia Pasca,∇ Claudia Binda,∇ Joaõ Neres,○ Neeraj Dhar,○ François Signorino-Gelo,○ John D. McKinney,○ Vasanthi Ramachandran,‡ Radha Shandil,§ Ruben Tommasi,∥ Pravin S. Iyer,† Shridhar Narayanan,‡ Vinayak Hosagrahara,§,¶ Stefan Kavanagh,# Neela Dinesh,*,‡ and Sandeep R. Ghorpade*,† †

Department of Medicinal Chemistry, ‡Department of Biosciences, and §DMPK (Drug Metabolism and Pharmacokinetics) and Animal Sciences, AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024, India ∥ Chemistry and ⊥Biosciences, Infection iMed, AstraZeneca, Waltham, Massachusetts 02451, United States # Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, Cheshire SK10 2NA, United Kingdom ∇ Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, 27100 Pavia, Italy ○ ́ Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland S Supporting Information *

ABSTRACT: 4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure−activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.



INTRODUCTION Among the few novel targets being investigated for developing new medicines for the treatment of tuberculosis (TB), decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1) offers promising opportunities for exploring various chemotypes with cidal activity against Mycobacterium tuberculosis (Mtb).1 DprE1, along with decaprenylphosphoryl-D-2-ketoerythropentose reductase (DprE2), is responsible for epimerization of decaprenylphosphorylribose (DPR) to decaprenylphosphorylarabinose (DPA), the sole precursor for synthesis of arabinan, which is an essential component of the mycobacterial cell wall.2 © 2014 American Chemical Society

DprE1 was identified as a primary target for a mechanism-based inhibitor containing a reactive nitro substituent, benzothiazinone (BTZ043; Figure 1), which acts by forming a covalent bond with Cys387 in the active site pocket of the enzyme.3,4 Currently, there are no marketed DprE1 inhibitors in use for the treatment of TB. Consequently, DprE1 is an attractive target for developing effective and safer medicines for the treatment of drug-sensitive as well as multi-drug-resistant Received: April 17, 2014 Published: May 28, 2014 5419

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Figure 1. Known DprE1 inhibitors.

Table 1. Properties of Compounds 1−3

property Mtb MIC (μM) Mtb MBC (μM) IC80 on hypoxic Mtb (μM) MMICa (μM) solubility at pH 7.4 (μM) logD human PPB (fraction unbound, fu) human microsomal CLint (μL/min/mg of protein) rat hepatocyte CLint (μL/min/106 cells) Caco-2 A−B/B−A (10−6 cm/s) hERG IC50 (μM) CYP inhibitionb (μM) secondary pharmacology hitsc (IC50, μM)

compd 1

compd 2

compd 3

6.3 6.3 >100 >200 255 1.6 0.3 100 >200 105 2.0 0.4 100 >100 26 2.2 0.4 12.5

1.5

5.6

2.4

0.5/14 >100 >30 active 1 of 24 targets (μ-opioid receptor, 21.9)

0.6/13 >100 >30 active 2 of 24 targets (μ-opioid receptor, −28.1; 5-HT2B receptor, −41.9)

0.2/11 >100 NDd active 1 of 24 targets (adenosine A1 receptor, −34)d

a

MIC on the A549 cell line used to determine cytotoxicity. bIsoforms tested include CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. Diverse set of binding, enzyme, and functional assays covering 24 distinct molecular targets; activity classified as a defined IC50/EC50. dNot determined. c



RESULTS AND DISCUSSION Whole-Cell-Based Screen. 4-Aminoquinolone piperidine amide 1 was obtained as a single active compound with moderate cidal activity on replicating Mtb (MIC = MBC = 6.25 μM) from a whole-cell-based screen of 320,000 compounds from the AstraZeneca corporate collection (Figure S1, Supporting Information). A limited number of available analogues of compound 1 either showed weak activity against Mtb or were inactive. These analogues were restricted to variation only in the amide portion of the molecule, thus demanding further systematic structure−activity relationship (SAR) exploration to demonstrate potential of the scaffold for hit to lead optimization. Compound 1, even though inactive against nonreplicating Mtb (hypoxia IC80 > 100 μM), had excellent leadlike properties, such as low molecular weight, logD < 2, and good solubility, and also had an attractive secondary pharmacological profile (Table 1). Exploration of the SAR on the quinolone ring resulted in more potent analogues 2 and 3, which retained leadlike properties (Table 1; refer to the section “SAR Optimization Strategy” for more details). Hence,

(MDR) and extensively-drug-resistant (XDR) TB as it does not pose a risk of pre-existing drug resistance in the clinic.5 Furthermore, DprE1 is specific to mycobacteria and actinomycetes and does not have a human homologue. PBTZ169 (Figure 1), a safer and more effective analogue of BTZ043, is poised to enter clinical trials.6 Recently, several noncovalent inhibitors of DprE1 have been reported which are shown to be effective in in vivo animal models of TB and have potential for further development as clinical candidates, e.g., TCA17 and azaindoles8 (Figure 1). Herein, we report 4-aminoquinolone piperidine amides (AQs) as a novel scaffold, identified from a whole cell screen, with potent cidality on replicating as well as intracellular Mtb. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1. Interestingly, AQs have slow on rates and long residence times of ∼100 min on the enzyme, which may have useful implications for the dosage regimen of these compounds. 5420

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Table 2. MIC Modulation against Various Overexpressed and Resistant Mutant Strainsa MIC (μM)

a

compd

Mtb H37Rv

DprE1 OE

InhA OE

TopA OE

PimA OE

BTZ043 (C387S) mutant

TMC207R- resistant mutant clone 8.1

MOX-resistant mutant clone 4.1

1 2 3 TMC 207 BTZ043 MOX rifampicin INH

6.3 1.6 0.4 0.78 0.003 0.15 0.01 0.45

>100 100 50 1.6 12.5 0.08 0.01 0.22

6.3 1.6 0.4 1.6 0.003 0.16 0.01 >7.25

3.1 0.8 0.4 0.8 0.012 0.31 0.03 0.45

3.1 1.6 0.4 0.8 0.003 0.15 0.004 0.22

100 25 3.12 0.4 25 0.08 0.004 0.45

3.1 1.6 0.2 50 0.002 0.15 0.01 0.23

1.6 1.6 0.2 1.6 0.003 10 0.02 0.45

OE = overexpressed strain, MOX = moxifloxacin, and INH = isoniazid.

target identification as well as SAR studies to further improve potency of the scaffold were initiated. Target Identification by MIC Modulation and Whole Genome Sequencing. In an attempt to identify the target for the AQ series, the compounds were tested against a panel of Mtb strains including overexpressed and resistant mutants of various target genes. The compounds from the AQ series showed a decrease in potency only against strains where DprE1 was mutated or overexpressed, but not when other targets were similarly modified (Table 2). This strongly indicated that DprE1 could be the target for this series. To further confirm that the target is DprE1, spontaneous mutants were raised against compound 2, which exhibited a 16−64-fold increase in MIC (the mutation frequency was 1.99 × 10−8). Sanger sequencing of the dprE1 gene from the mutant strains indicated a single nucleotide change, resulting in an amino acid substitution (Tyr → His) at position 314. Whole genome sequencing of multiple mutants confirmed this mutation and did not indicate any significant secondary target (data not shown). Time-Dependent Inhibition of DprE1 Enzyme Activity. To verify that AQs inhibit the catalytic activity of DprE1, representative compounds were tested against recombinant Mycobacterium smegmatis (Msm) DprE1 using the assay conditions as described in the Experimental Section. The sequence identity between the Mtb and Msm DprE1 is 84%, and the active site is fully conserved (sequence analysis in Figures S1 and S2, Supporting Information).1 Hence, the Msm DprE1 assay for which a thorough biochemical characterization was previously carried out2 was used for the purpose of this work. Potent IC50 values in the range of 10−100 nM were obtained, showing a good correlation with the MIC for the compounds tested. Mass-spectrometry-based analysis indicated that AQs are noncovalent inhibitors of DprE1 (Figure S7, Supporting Information). The reaction progress curve at different concentrations of compound 2 is shown in Figure 2. While the uninhibited reaction showed a linear increase in signal from 0 to 40 min, the reactions with several AQs showed a continuous decrease in the rate of reaction over time, indicating time-dependent inhibition. For routine screening, the IC50 of the compounds was measured by using the initial rate of reaction (0−6 min, IC50 early) and the rate of reaction after 30 min (30−36 min, IC50 late). A difference in IC50 early and IC50 late is indicative of a time-dependent inhibition (see Table S8, Supporting Information, for early and late IC50 values of the compounds). A good trend between IC50 late on Msm DprE1 enzyme and Mtb MIC was observed (Figure 3). In general, potent DprE1 inhibition

Figure 2. Kinetic analysis of Msm DprE1 enzyme inhibition by compound 2. Connecting lines are visual guides only.

Figure 3. Scatter plot of Msm DprE1 pIC50 late vs Mtb pMIC. pIC50 = −[log(IC50)] and pMIC = −[log(MIC)], wherein IC50 and MIC are in molar units. Most of the compounds made in the series are included in the plot even though not all are represented in the paper.

(IC50 ≤ 100 nM, pIC50 ≥ 7) was required to achieve potent Mtb MIC of ≤1 μM (pMIC > 6). 5421

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Representative compounds were tested in kinetic studies to measure the dissociation rate constant (koff) of the inhibitor from the enzyme by fitting the progress curve data to a singlestep reversible slow binding model as described in the Experimental Section. A typical data fit is shown in Figure 2 for compound 2. The koff values, residence time (1/koff), and dissociation half-life of the enzyme−inhibitor complex (0.693/ koff) for compounds 1−3 are shown in Table 3. The standard Table 3. koff and Residence Time of Representative Compounds compd

koff × 10−4 (s−1)

koff std error × 10−4 (s−1)

t1/2 (min)

residence time (min)

1 2 3

1.6 1.2 1.1

0.14 0.2 0.13

72 83 105

104 119 152

Figure 5. Cidality against intracellular Mtb by representative AQs 2 and 3. Error bars are standard deviations from duplicate experiments carried out in parallel.

error in the estimates of koff was less than 10%. The AQs showed a dissociation half-life (t1/2) of over 1 h. A longer residence time or longer t1/2 implies that the compound remains bound to the enzyme for a longer period of time, which may be advantageous under in vivo conditions where the compound may retain its inhibitory effect on the target even after the inhibitor is cleared from the plasma.9 Bactericidality of AQs. In vitro killing kinetic studies carried out with multiple compounds in the AQ series exhibited a maximum kill of ≥4 log by day 14. Interestingly, compounds with difluoro substitution at the 6- and 7-positions of the quinolone ring (e.g., compound 3) showed an improved kill of ∼3 log by day 3 and ≥4 log by day 7 (Figure 4; Table S6, Supporting Information). Compounds 2 and 3 were also profiled for their activity on intracellular Mtb. Both compounds were capable of killing intracellular Mtb, although no dose response was observed. Compound 3 showed a superior kill (2 log) compared to 2 (1 log) (Figure 5). This again suggests that analogues containing the 6,7-difluoro substitution on the quinolone ring have better killing properties. Single-Cell Analysis of the Effect of AQ Compounds on Mtb. Bactericidal activity of compound 2 was also evaluated at the single-cell level using microfluidics-based time lapse microscopy.10 Recombinant Mtb expressing GFP was cultured in customized microfluidic devices on the microscope. After several days of growth on the device, the bacteria were exposed to 30 μM of 2 for a period of 5−7 days (Figure 6). In response

to the compound, the bacteria decreased their growth rate rapidly; however, lysis was significantly delayed and only occurred after a lag period ranging from 40 to 80 h. A substantial fraction of cells lysed, with a few cells exhibiting strange morphologies and release of cytoplasmic material from the polar region. Lysis also continued to occur after drug washout, suggesting some postantibiotic killing. Some of the morphological changes observed are similar to the changes previously described in the case of exposure of Mtb to BTZ043.3 Activity on Nonmycobacterial Strains, Nonreplicating Phase (NRP) Mtb, and Drug-Resistant Strains of Mtb. Representative compounds from the AQ series were profiled for MIC against a panel of nonmycobacterial strains, including Candida albicans as well as Gram-negative and Gram-positive pathogens with no activity detected up to the maximum concentration tested (100 μM; refer to the Supporting Information for the nonmycobacterial strains used). This specific activity against Mtb is consistent with the fact that DprE1 is not present in other bacterial species. This could be advantageous in the treatment of chronic diseases such as tuberculosis, thereby avoiding unnecessary exposure of the commensal flora to the antibiotic. When tested in the hypoxia model of NRP Mtb, all AQ compounds were found to be inactive. This was expected as the target DprE1 is mainly involved in cell wall synthesis, which is down-regulated under NRP conditions. The compounds were

Figure 4. In vitro killing kinetics for AQ compounds (LOQ = limit of quantification). The lines connecting the data points are visual guides only. 5422

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Figure 6. Single-cell analysis of the effect of compound 2 on Mtb. Mtb expressing GFP was grown in a microfluidic device and imaged over 1 h intervals. Bacteria were exposed to a 30 μM concentration of compound 2 between t = 118 h and t = 284 h (days 5−12), after which the drug was washed out. Snapshot images of representative microcolonies are shown. GFP fluorescence is shown in green, and the phase channel is shown in red. Numbers (upper right) indicate days elapsed. Labels (upper left) indicate the presence or absence of AQ in the flow medium (7H9). The scale bars represent 2 μm.

Figure 7. Key interactions exhibited by (a) PBTZ169 (PDB ID 4NCR) in the DprE1 active site, (b) CT319 (PDB ID 4FDO), and (c) TCA1 (PDB ID 4KW5). (d) Overlaid picture of the ligands: CT319 (green), PBTZ169 (orange), TCA1 (violet). (e) The ordering of the loop constituting residues 316−322 to a small helix reported in the case of the CT319-bound structure is depicted as a green ribbon.

reported two loops, disordered to different extents, in the substrate binding domain (residues 269−283 and 314−322), leaving the active site open. Interestingly, one Mtb DprE1 structure reported by Batt et al.4 suggests the closure of loop residues 314−322 upon binding of the nitrobenzene derivative CT319 in a noncovalent manner (Figure 7b). However, the recently reported crystal structure of a non-nitro inhibitor TCA1 (Figure 7c) showed the disordered loop in the same region. This indicates that the ordering of the loop is likely driven by the interaction of residues 314−322 with the ligand and may behave differently with different scaffolds. Furthermore, given the expected association of DprE1 with the cell membrane of Mtb, the conformation of this flexible loop in the bacterium might be different from that observed in the crystal structures.1b,2 Key residues such as Cys387 (covalent binding), Asn385, and Ser228 are responsible for the interactions with the ligands. The backbone of residues 132−134 and the hydrophobic side chain of Lys367 and Phe369 form a cleft that binds to the CF3 group in the case of nitro compounds and the thiazole ring for TCA1. The ligand overlay of these three

equipotent on all single-drug-resistant and clinical Mtb strains tested, indicating that this scaffold has the potential to treat drug-resistant tuberculosis (Table S7, Supporting Information). Binding Mode of Aminoquinolones Proposed on the Basis of the DprE1 Active Site Structure. Repeated attempts to crystallize AQs with DprE1 protein were unsuccessful. Hence, docking studies of AQs using reported DprE1 crystal structures were undertaken. Several recent reports on the structural biology of Msm and Mtb DprE12,4,6a,7 led to insights into the active site and mode of binding of different chemical classes. The residue numbers referred to in this section are from Mtb DprE1 unless otherwise noted. The analyses of ligand-bound crystal structures of DprE1 from Mtb and Msm revealed several features that can be utilized for structure-based lead optimization. The covalent benzothiazinone inhibitors, e.g., PBTZ169, revealed the formation of a S−N covalent bond between Cys387 and the N atom of the activated nitro group (Figure 7a).4,6a The conformation of the flavin adenine dinucleotide (FAD) cofactor remains unchanged in all the structures. Most of the structures 5423

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Figure 8. Predicted Glide docking pose of compound 1 in Mtb DprE1.

Figure 9. SAR optimization strategy for 4-AQs.

different classes of inhibitors is shown in Figure 7d,e. The piperazine side chain in the case of PBTZ169, the terminal benzyl group in CT319, and the benzothiazole ring in TCA1 are all oriented toward the loop composed of residues 314− 322. Docking was performed against both open (PDB ID 4KW5) and closed (PDB ID 4FDO) conformations of the active site using Glide 6.1 (Schrodinger).11,12 The docking protocol reproduced the binding modes of TCA1 and CT319 with an RMSD of less than 1.5 Å from the crystallographic pose. Owing to the presence of multiple polar side chains and a partially exposed active site, the open structure gave multiple binding modes for AQ 1. On the other hand, the closed form predicted predominantly one mode of binding. The predicted pose and 2D interaction plot for compound 1 are shown in Figure 8. On the basis of the proposed binding mode, the AQ scaffold can be divided into site 1, a linker, and site 2. In site 1, the quinolone amide group makes polar contacts with Asn385. The NH group at C-4 of the quinolone forms a H-bond with one of the carbonyl oxygens of FAD. The amide group of site 2 makes a H-bond contact with Tyr60. Besides these polar contacts, compound 1 makes multiple favorable van der Waals contactsresidues 132−134 and Tyr314 with site 1 ring atoms, Leu317, Val365, and Trp230 with the linker alicyclic ring, and Gly321, Phe320, and Asp389 with the site 2

chloromethylpyrazole ring. This predicted pose was useful to explain a number of SAR trends exhibited by the series as described in the section “SAR Optimization Strategy”. Several attempts were made to obtain cocrystal structures of Mtb and Msm DprE1 with AQ analogues. For the Mtb protein, crystals were often obtained from cocrystallization experiments of soaking native protein crystals with the compounds, but these either diffracted poorly or presented no electron density to account for the inhibitor in the active site. A possible explanation for the difficulties in obtaining cocrystal structures with these compounds resides in the fact that they likely interact with Tyr314 at the edge of a flexible loop and might induce a conformation of this loop that is not compatible with the crystallization forms previously obtained for other inhibitors.2,4 SAR Optimization Strategy. Since compound 1 was obtained as a single active from the screen, very limited information was available for further expansion of the scaffold. Hence, modifications were made to the compound to understand key pharmacophoric features in the molecule (Figure 9). The major focus was the substitution on the quinolone ring to strengthen the van der Waals interaction as depicted by the docking studies followed by SAR exploration on the linker ring to improve potency. Limited SAR exploration on the site 2 pyrazole was planned as several analogues with 5424

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Table 5. Quinolone Ring Replacementa

amide variations were inactive in the screen as mentioned in the previous section. SAR at Site 1. Addition of F atoms at C-6, C-7, and C-8 (compounds 2−6, Table 4) improved the potency, which could Table 4. Substitution on the Quinolone Ring

a

compd

R

DprE1 IC50a (μM)

Mtb MIC (μM)

2 3 4 5 6 7 8 9 10

6-F 6,7-di-F 7-F 8-F 7,8-di-F 1-N-CH3 1-N-C2H5 4-O 4-N-CH3

0.005 0.006 0.007 0.011 0.009 0.14 >100 8 18

1.6 0.8 3.1 3.1 1.6 25 >100 50 >250

a

Unsubstituted piperidine ring as the linker and 5-chloro-1methylpyrazole as the site 2 ring. bIC50 at 30 min.

DprE1 IC50 of 3 μM. The docking analysis suggests that the phenyl group can be accommodated at the hydrophobic pocket but the position of the pyridone and piperidine rings are not optimal (Figure S6, Supporting Information). SAR at the Linker. Replacement of the piperidine ring with other alicyclic rings, such as pyrrolidine and azetidine, at the linker position led to weaker compounds 15 and 16 (Table 6). The van der Waals contact with piperidine methylene groups might be critical and was not optimal with pyrrolidine or aziridine ring systems. Further exploration of substituted piperidines and bicyclic systems containing a piperidine core

IC50 at 30 min.

be attributed to van der Waals contact with the backbone atoms of residues 132−134 and Phe369. The mutant residue Tyr314 is within van der Waals contact (3.7 Å) of the C-6 F atom of compound 2 (Figure S4, Supporting Information). The more potent MICs (0.8 μM) and killing properties observed with compound 3 having 6,7-difluoro substitution on the quinolone ring could be due to improved permeability across the Mtb cell wall or due to enhanced binding with the DprE1 protein. This is not reflected in the DprE1 IC50 as the IC50 values could not be measured accurately below 10 nM due to limitations on protein concentrations used for the assay. The N-methylquinolone derivative 7, while active and predicted to bind in the same orientation as compound 1, is 2−4-fold less potent than the corresponding nonmethylated derivative 1 (Table 4). This could be due to loss of H-bonding with the Asn385 amide. Additionally, modeling suggests that further extension in this direction may not be permitted, which is reflected in the result of N-ethyl derivative 8. Replacing NH linker at quinolone C-4 with “O” or “NCH3” led to much weaker compounds 9 and 10, respectively (Table 4), presumably due to loss of H-bonding with the FAD carbonyl. The quinolone ring was replaced with a few other bicyclic rings having donor−acceptor functionality to explore the scope of improving the potency and modulating the physicochemical properties. Masking of the CO−NH interactions in quinolone, as in methoxyquinoline 11 and cinnoline 12, led to loss of DprE1 inhibition as well as Mtb MIC (Table 5). Indazole derivative 13 with an identical linker and site 2 predicted to possess a binding mode similar to that of aminoquinolones (Figure S5, Supporting Information) showed submicromolar IC50 against DprE1 (Table 5). The design to convert quinolone into substituted pyridones was conceived to improve the physicochemical properties, but the compounds were weakly active. For example, 6-phenylpyridone analogue 14 showed a

Table 6. SAR at the Linker Piperidine Ringa

a

Unsusbtituted quinolone ring at site 1 and 5-chloro-1-methylpyrazole as the site 2 ring. bIC50 at 30 min.

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Figure 10. Stereoisomeric preference for branched piperidine derivatives in the linker moiety of AQs: (a) docking pose of the (1R,5S)azabicyclo[3,2,1]octanyl syn system 19 in the active site, (b) ligand overlay of the preferred stereoisomers for [3,2,1] bicyclic octane 19 and (2S,4R)2-methylpiperidine derivative 17.

Table 7. SAR at the Site 2 Aryl Ringa

revealed very interesting results with dramatic improvement in Mtb MICs (compounds 17−20, Table 6). A preference in stereochemistry was observed for the branched piperidine derivatives, such as 2-methylpiperidines 17 and 18 and azabicyclo[3,2,1]octanyl systems 19 and 20. This was well supported by the predicted binding mode for the series. The preferred stereoisomers in both the cases make hydrophobic contacts with Val365, Leu317, and Trp320 (Figure 10a), whereas the corresponding weakly active/inactive stereoisomer did not dock. Improved MICs observed with compound 19 could be due to enhanced permeation of this compound across the bacterial cell wall as there was no significant improvement in DprE1 IC50 compared to that of the piperidine analogue 2. SAR at the Site 2 Aryl Ring. Among several heteroaromatic rings explored at site 2, disubstituted five-membered heterocyclic rings such as pyrazole, isoxazole, and triazoles were the most preferred (compounds 22−25, Table 7). Substitution at the C-5 position of the pyrazole ring was found to be critical for potent DprE1 inhibition. Compound 21 with 1-methylpyrazole, lacking a C-5 substituent, was about 100-fold less potent than compounds 1 and 22 with disubstituted pyrazole. Extending the substitution beyond ethyl at the pyrazole N-1 position was detrimental as observed with 26 and 27. This is in agreement with the findings from the docking studies. The hydrophobic contacts (Cl−Me or Me−Me) from the disubstituted derivatives with residues such as Leu317, Phe320, and Trp322 may play a critical role. Additionally, this finding suggests that site 2 of AQs may trigger an induced fit for these residues that are otherwise part of a disordered loop (residue 316−322) in most of the reported crystal structures, hence possibly explaining the longer residence time observed for this scaffold. Potency Improvement. Several potent compounds (28− 36) could be made by combining fluorinated quinolone rings with piperidine and substituted piperidine rings (Table 8). Compound 28 with 6,7-difluoroquinolone at site 1, piperidine as the linker, and dimethylisoxazole as the aryl ring at site 2 showed better MIC compared to corresponding analogue 3 with 5-chloro-1-methylpyrazole at site 2. This was a critical observation for obtaining more potent compounds such as 30 with improved permeation properties as described in the section “DMPK and Safety Profile of the Compounds” (DMPK

a

Unsubstituted quinolone ring at site 1 and unsubstituted piperidine ring as the linker unless otherwise specified. bIC50 at 30 min. c6Fluoroquinolone derivatives.

= drug metabolism and pharmacokinetics). Compounds 32 and 33 with 6,7-difluoroquinolone at site 1 and an azabicyclo[3,2,1]octanyl ring as the linker showed the best MICs of 60 nM. Interestingly, N-methylation of the quinolone ring for compounds having an azabicyclo[3,2,1]octanyl ring at the linker position did not deteriorate the potency (compounds 34−36). This observation with a bicyclo[3,2,1]octanyl ring linker was exploited further by replacing the quinolone ring with indazole, phenylpyridone, and azaquinolones (compounds 37−40), which had previously led to weakly active compounds with an unsubstituted piperidine linker. Gratifyingly, all these compounds showed improved DprE1 inhibition and potent Mtb MICs. This is in agreement with the model as proposed in Figure 10 which suggests multiple favorable van der Waals 5426

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Table 8. Potent Compounds

a

IC50 at 30 min.

Table 9. IC50 and MIC Modulations against DprE1 Mutant Strains Mtb MIC (μM)

Msm DprE1 IC50 (μM)

compd

WT-Mtb H37Rv

DprE1 OE

BTZ043R (C387S)

2R (Y314H)

Y321H

WT

IC50 fold shift

1 2 3 30 19 31

6.25 1.56 0.78 0.78 0.39 0.39

>100 100 >100 100 6.25 12.5

100 50 12.5 6.25 1.56 6.25

100 25 50 25 10 9.47 >10 7.02 0.55 1.03

0.15 0.08 0.07 0.04 0.04 0.02

>63 111 >143 165 12 47

Interestingly, when the piperidine ring was substituted as in compounds 19 and 31, cross-resistance with the Y314H mutant was lost while that against the C387S mutant strain was retained. Furthermore, the shift in IC50 of the wild-type and mutant enzymes purified from M. smegmatis for these compounds is lower (12−47 fold) than that observed for compounds containing an unsubstituted piperidine (>100-fold, e.g., compound 1). The fact that the IC50 against the wild-type enzyme is retained suggests that these compounds target DprE1, but may interact differently compared to other AQs. It is also possible that a second target is engaged by these compounds, as reflected by the improved potency against the DprE1 overexpressing strains.

contacts between the ethylene bridge and residues such as Leu317, Trp230, and Val365, which may add a marked entropic advantage. This SAR observation may help for further scaffold morphing to generate diverse leads with different physicochemical and safety properties. MIC Modulation and Binding Kinetics of AQs with the Tyr314 Mutant. Since the resistant mutant mapping suggested that the AQs interact with Tyr314, IC50 values of a set of compounds were measured against Msm Y321H DprE1 (which corresponds to Y314H of Mtb DprE1). The IC50 values of the compounds were 10- to >100-fold higher on the tyrosine mutant enzyme compared to the wild type, confirming the importance of the tyrosine residue in the interaction with AQ analogues (Table 9). 5427

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solubilities of the compounds were low, ranging from 6 to 100 μM, and were improved 3−5-fold in FeSSIF media13 (FeSSIF = fed-state simulated intestinal fluid, pH 6.5). Compound 37, an indazole analogue, showed excellent aqueous solubility of more than 1 mM. In terms of in vitro plasma protein binding, NHquinolone analogues showed better unbound fraction (fu) in human plasma (e.g., fu ranged from 0.16 to 0.5 for compounds 1−3, Table 1) compared to N-methylquinolone analogues (fu ranged from 0.01 to 0.05 for compounds 7, 30, 36, 39, and 40). On the basis of in vitro metabolism studies with human microsomes and rat hepatocytes, the compounds were predicted to have low to moderate blood clearance (liver blood flow, LBF,14 ranged from 1% to 65% and from 5% to 37% when tested for rat hepatocyte CLint and human microsomal CLint, respectively). None of the compounds tested were found to inhibit the cytochrome P450 isoforms. Compounds with free NH-quinolone had poor Caco-2 permeability and a high efflux ratio. N-Methylquinolone analogues (e.g., 30, 36, and 40) and indazole analogue 37 showed high Caco-2 permeability and lower efflux ratios possibly due to fewer hydrogen bond donor−acceptor interactions. All the compounds tested were inactive on mammalian cells up to the highest concentration tested. Compounds with an unsubstituted piperidine ring, in general, had hERG IC50 > 33 μM, whereas a few compounds such as 36 which contained the bicyclo[3,2,1]octanyl ring showed hERG IC50 < 33 μM. This could be due to the higher logD of these compounds. However, less lipophilic analogues, such as 39 and 40 with azaquinolone rings and indazole analogue 37, were much cleaner in hERG channel assays (IC50 > 33 μM), demonstrating structural opportunities to allay cardiac risk with the bicyclo[3,2,1]octanyl ring system. Representative compounds from the AQ series were tested in a panel of secondary pharmacology assays designed to identify whether the series have intrinsic activity against targets with a clear linkage to toxicity (Table 10).15 Generally, the AQ compounds were shown to have good profiles in this panel for an early optimization program. Although isolated potential off-target activities were identified, these were considered optimizable through systemic medicinal chemistry exploration. One consistent activity (adenosine A1 receptor) was noted with several compounds where data were available. However, the observed Ki range of 8−34 μM suggests that an appropriate safety margin may be achievable.

The kinetics of inhibition for compounds that retained potency against the Y314H mutant enzyme (e.g., compound 19) indicates that binding equilibrium of the compounds to DprE1 Y321H is achieved rapidly (Figure 11b) in contrast to

Figure 11. Comparison of the binding kinetics of compound 19 on (a) wild-type Msm DprE1 and (b) the Msm DprE1 Y321H mutant. Connecting lines are visual guides only.

the slow binding of the compound to the wild-type enzyme (Figure 11a). This can be observed as a progressive decrease in the rate of reaction over time at inhibitory concentrations of the compound on the wild-type enzyme (Figure 11a, 41 and 123 nM concentrations of of compound), while the progress curves are linear at all concentrations with the Y321H mutant enzyme (Figure 11b). This suggests that Tyr314 may have a role in the slow binding kinetics of AQ compounds. DMPK and Safety Profile of the Compounds. Compounds were profiled for physicochemical properties and in vitro DMPK assays, including solubility, logD, human PPB (plasma protein binding), rat hepatocyte CLint, human microsomal CLint, and Caco-2 permeability. The aqueous

Table 10. In Vitro DMPK and Safety Properties of Representative Compounds property logD solubility at pH 7.4 (μM) FeSSIF solubility (μM) human PPB (fraction unbound, fu) human microsomal CLint (μL/min/mg of protein) (LBF, %)a rat hepatocyte CLint (μL/min/106 cells) (LBF, %)a Caco-2 A−B/B−A (10−6 cm/s) CYPb inhibition IC50 (μM) MMIC A549 IC50 (μM) hERG IC50 (μM) secondary pharmacology hitsd

compd 7

compd 30

compd 36

compd 37

2.0 2.3 2.8 1.9 78 28 6 >1000 NDc 91 NDc NDc 0.03 0.05 0.03 0.08 14 (6) 49 (20) 107 (25) 4 (6) 8 (7) 17 (19) 56 (31) 9 (19) 4/23 22/21 23/13 20/31 >30 >30 >30 >30 >100 >16 >50 >50 >33 NDc 16 >33 active on 1−3 targets; the adenosine A1 receptor was a common hit, Ki range 8−34 μM

compd 39

compd 40

1.8 20 100 0.01 37 (5) 3 (1) 1/36 >30 >50 >33 NDc

1.8 132 307 0.05 100 (28) 20 (19) 33/21 >30 >50 >33 NDc

a Liver blood flow (%) predicted on the basis of the well-stirred mode.14 bIsoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. cNot determined. dDiverse set of binding, enzyme, and functional assays covering 24 distinct molecular targets; activity classified as a defined Ki/IC50/ EC50.

5428

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Figure 12. Mean plasma concentration of compounds 3 and 7 (n = 2) following (a) an intravenous dose of 2 mg/kg and (b) an oral administration dose of 10 mg/kg. The lines connecting the data points are visual guides only.

Analogues of this novel series exhibit excellent cidal properties in vitro against replicating as well as intracellular Mtb. Interestingly, analysis of single cells carried out using microfluidics indicates that lysis of the bacteria continues to occur even after drug is washed out, suggesting a postantibiotic effect for this series. Further optimization based on the SAR observations will be useful for identifying compounds suitable to establish in vivo efficacy for the series. The impact of a long residence time on the dosing interval of these compounds, in principle, could be used to identify compounds requiring less frequent dosing, which is of paramount importance in TB treatment. This study reports 4-aminoquinolone piperidine amide as a potential lead series toward this purpose with adequate antitubercular properties and an attractive safety profile.

Pharmacokinetics studies on representative compounds were performed in male Wistar rats, following single intravenous and oral dose administration (Figure 12 and Table 11). Table 11. Pharmacokinetic (PK) Parameters of Compounds 3 and 7a intravenous (dose 2 mg/kg)

oral (dose 10 mg/kg)

PK param

compd 3

compd 7

compd 3

compd 7

C0 (iv) or oral Cmax (μM) AUC0−∞ (μM h) elimination half-life (h) CL (mL/min/kg) Vss (L/kg) bioavailability (F, %)

4.9 1.2 0.5 34.4 0.9

5.4 3.4 1.1 24.8 1.2

0.02 0.04 1.1

7 19 1.3

0.3

100



a

Key: iv, intravenous; C0, plasma concentration at 0 h following iv administration; Cmax, maximum (peak) plasma concentration following oral administration; AUC, area under the curve; CL, plasma clearance; Vss, volume distribution.

EXPERIMENTAL SECTION

Biochemistry: DprE1 Enzyme Assays and Residence Time Measurements. DprE1 assays were performed as described previously.2,8 Briefly, a 50 μL reaction was performed at 25 °C in 384-well black plates (catalog no. 3573, Corning Costar, Corning, NY) in buffer containing 50 mM glycylglycine, pH 8.5, 200 mM potassium glutamate, 10 μM FAD, and 0.002% Brij-35, 2% DMSO, and 75 nM purified DprE1 enzyme, either wild type or Y321H (using thioredoxin6His precision Msm Rv3790 protein). The enzyme was preincubated in the FAD-containing assay mix for 30 min prior to the start of the reaction. The reactions were started with mix containing the substrate 300 μM farnesylphosphoryl-β-D-ribofuranose, the coupling enzyme 0.01 mg/mL horseradish peroxidase (Sigma-Aldrich P-6782), and 50 μM amplex red (Invitrogen A-22177). The conversion of amplex red to resorufin was monitored on a Tecan Saffire II (excitation wavelength of 563 nm and emission wavelength of 585 nm) in the kinetic mode. This complete reaction was the positive control, and the rate of reaction in the presence of 10 μM BTZ043 was taken as the background. The background rate was subtracted from all reactions to get the background-subtracted rate of reaction. For IC50 measurements, the compounds were dissolved in DMSO and serially diluted to a concentration which was 50× the desired test concentration. A 1 μL volume of the stock was used in a 50 μL reaction volume. For koff measurements, the data were collected at varying inhibitor concentration. The single-step reversible slow binding model16 fitted well to the fluorescence vs time data. The following equations were used for the analysis:

Compounds with free quinolone NH (e.g., compound 3) had very poor oral exposures (F < 1%) in rats. As this compound showed a moderate plasma clearance (48% of rat LBF) following intravenous administration, the low oral exposure could be due to low permeability and high efflux associated with this compound (Table 1). However, N-methylquinolone analogues (e.g., compound 7) showed low plasma clearance (30% of rat LBF) following intravenous administration and an excellent bioavailability (F = 100%) following single oral dose administration. Thus, Caco-2 permeability along with other parameters such as clearance and solubility could be the key optimization parameters to achieve reasonable oral exposure with this class of compounds.



CONCLUSION 4-Aminoquinolones piperidine amides are established here as noncovalent inhibitors of DprE1, an essential enzyme involved in the cell wall synthesis of mycobacteria. The compounds showed potent, time-dependent inhibition of DprE1, which is unique among the noncovalent inhibitors of DprE1 reported so far. Compounds in this series could not be cocrystallized with DprE1 protein yet; however, on the basis of current structural knowledge of DprE1, a structural hypothesis was proposed to explain the SAR pattern observed in the series. Use of a bicyclo[3,2,1]octanyl ring at the linker position allows opportunities for scaffold morphing and modulation of the physicochemical and safety properties of the compounds.

[P] = vst +

vs − vi (1 − e−kobsdt ) kobsd

⎛ [I] ⎞ kobsd = koff ⎜1 + app ⎟ Ki ⎠ ⎝ 5429

(1)

(2)

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Scheme 1. Synthesis of AQsa

a

Reagents and conditions: (a) malonic acid, POCl3, reflux, 3 h; (b) concd HCl, dioxane, reflux; (c) NaH, DMF, alkyl halide, rt, 3 h; (d) 1-Boc-4aminopiperidine or corresponding Boc-protected alicyclic amine, Pd(OAc)2, BINAP, CsCO3, toluene, microwave, 130 °C, 45 min; (e) trifluoroacetic acid, CH2Cl2, rt, 2 h; (f) R2CO2H, HATU, N,N-diisopropylethylamine, DMF, rt; (g) NaOMe, toluene, reflux, 15 h; (h) NaNO2, concd HCl, water, −5 to 70 °C; (i) POCl3, reflux, 3 h; (j) iodine, KOH, DMF, 5 h; (k) di-tert-butyl dicarbonate, TEA, DCM, rt, 20 h; (l) phenylboronic acid, Pd(dppf)2Cl2, Cs2CO3, DME−water; (m) NaNO2, H2SO4, water; (n) EtOAc, NaH, 80 °C, 2 h; (o) diethyl malonate, NaOEt, ethanol, reflux, 15 h; (p) 15% NaOH, reflux, 4 h; (q) NaOAc, acetic acid, 120 °C, 20 h. intermediate 43 ready for Buchwald coupling.18 In the case of Nalkylated derivatives, intermediate 43 was reacted with the corresponding alkyl halide, such as CH3I, in the presence of NaH to give N-alkyl derivative 44. Reaction of 4-chloroquinolone derivative 43 or 44 with the corresponding Boc-protected piperidinamine led to intermediate 45. Intermediate 45 was then deprotected by treatment with trifluoroacetic acid to give free amine 46, which was then coupled with the corresponding aryl acid to furnish the final compounds 1−40. For the compounds with quinolone replaced with other bicyclic/ pyridone rings, the corresponding aryl halide was treated with piperidine derivatives under Buchwald conditions to furnish intermediates which were then treated in a similar way to furnish the required compounds. 2,4-Dichloroquinoline (42, R1 = H) when treated with sodium methoxide in toluene under reflux conditions led to 4-chloro-2methoxyquinoline (47) as a major product.19 Intermediate 47 was then processed as per the general protocol to yield compound 11. Cinnoline intermediate 50 was synthesized by reacting 2-acetylaniline (48) under diazotization conditions to give 4-hydroxycinnoline (49), which was then chlorinated by reaction with POCl3 under reflux.20 5Fluoroindazole (51) was iodinated by treatment with iodine under basic conditions to give intermediate 52.21 As this 3-iodoindazole derivative 52 did not react well under Buchwald conditions, the indazole NH was protected with a Boc group by treatment with di-tertbutyl dicarbonate to yield intermediate 53. Intermediate 53 could then be processed using the general protocol to furnish compounds 13 and

where t is time, [I] is the concentration of inhibitor, vi and vs are the initial and steady-state velocities of the reaction in the presence of the inhibitor, kobsd is the apparent first-order rate constant for the interconversion between vi and vs, Kiapp is the apparent Ki, which is related to Ki by different functions based on the modes of inhibition, and koff is the rate constant for dissociation of the enzyme−inhibitor complex. Equation 2 was then substituted into eq 1 to give [P] = vst +

vs − vi

(

koff 1 +

[I] K iapp

app

)

(1 − e−koff (1 + ([I]/ K i

))t

) (3)

Equation 3 was then fitted to the fluorescence vs time data at varying inhibitor concentration (see Figures 2 and 11), where vs and vi are the local parameters (one value each for every compound concentration) and koff and Kiapp are the global parameters (single value for the set of compound concentrations). Mass Spectrometry. Mass spectrometric determination of the binding mode of aminoquinolones with DprE1 enzyme was performed as described previously.17 Chemistry. All the compounds reported here were synthesized following the general protocol as shown in Scheme 1. The quinolone ring was constructed by reacting aniline or substituted aniline 41 with malonic acid and POCl3 under reflux conditions to give 2,4dichloroquinoline intermediate 42.18 Intermediate 42 underwent selective hydrolysis at 2-Cl after being refluxed in a mixture of concentrated HCl and 1,4-dioxane to furnish 4-chloroquinolone 5430

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4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-6,7-difluoroquinolin-2(1H)-one (3). Yield: 20%. 1H NMR (300 MHz, DMSO-d6): δ 1.47 (d, J = 11 Hz, 2H), 1.91−2.11 (m, 2H), 2.96 (br s, 1H), 3.17 (d, J = 4 Hz, 1H), 3.76 (m, 2H), 3.83 (s, 3H), 4.41 (br s, 1H), 5.36−5.58 (m, 1H), 6.53 (d, J = 7 Hz, 1H), 6.97−7.28 (m, 1H), 7.71 (s, 1H), 8.17 (dd, J = 12, 9 Hz, 1H), 10.89 (br s, 1H). HRMS: m/z (ES+) = 422.11183 (MH+) for C19H18ClF2N5O2. 4-((1-((5-chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-7-fluoroquinolin-2(1H)-one (4). Yield: 20%. 1H NMR (300 MHz, DMSO-d6): δ 1.50 (m, 2H), 1.89−2.12 (m, 2H), 2.91−3.06 (m, 2H), 3.73−3.80 (m, 2H), 3.83 (s, 3H), 4.41 (m, 1H), 5.42 (s, 1H), 6.59 (d, J = 8 Hz, 1H), 6.96 (d, J = 9 Hz, 2H), 7.71 (s, 1H), 7.98−8.13 (m, 1H), 10.84 (s, 1H). HRMS: m/z (ES+) = 404.12886 (MH+) for C19H19ClFN5O2. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-8-fluoroquinolin-2(1H)-one (5). Yield: 39%. 1H NMR (300 MHz, DMSO-d6): δ 1.51 (q, J = 11 Hz, 2H), 1.99 (d, J = 11 Hz, 2H), 2.76−3.09 (m, 1H), 3.17 (br s, 1H), 3.75 (d, J = 7 Hz, 1H), 3.68−3.81 (m, 1H), 3.83 (s, 3H), 4.43 (br s, 1H), 5.38−5.63 (m, 1H), 6.64 (d, J = 8 Hz, 1H), 6.97−7.19 (m, 1H), 7.27−7.46 (m, 1H), 7.71 (s, 1H), 7.84 (d, J = 8 Hz, 1H), 10.70 (br s, 1H). HRMS: m/z (ES+) = 404.12858 (MH+) for C19H19ClFN5O2. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-7,8-difluoroquinolin-2(1H)-one (6). Yield: 20%. 1H NMR (300 MHz, DMSO-d6): δ 1.49 (m, 2H), 1.88−2.09 (m, 2H), 2.94 (br s, 1H), 3.22−3.31 (m, 1H), 3.76 (br s, 2H), 3.83 (s, 3H), 4.41 (br s, 1H), 5.48 (s, 1H), 6.68 (d, J = 7 Hz, 1H), 7.00−7.32 (m, 1H), 7.71 (s, 1H), 7.87 (br s, 1H), 10.76−11.11 (m, 1H). HRMS: m/z (ES+) = 422.12033 (MH+) for C19H18ClF2N5O2. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-1-methylquinolin-2(1H)-one (7). Yield: 39%. 1H NMR (300 MHz, DMSO-d6): δ 1.35−1.64 (m, 2H), 2.00 (d, J = 10 Hz, 2H), 2.95 (br s, 1H), 3.34 (s, 4H), 3.49 (s, 3H), 3.76 (br s, 2H), 4.43 (br s, 1H), 5.63 (s, 1H), 6.58 (d, J = 8 Hz, 1H), 7.20 (t, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H), 7.58 (t, J = 8 Hz, 1H), 7.71 (s, 1H), 8.07 (d, J = 9 Hz, 1H). HRMS: m/z (ES+) = 400.15316 (MH+) for C20H22ClN5O2. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-1-ethylquinolin-2(1H)-one (8). Yield: 34%. 1H NMR (300 MHz, DMSO-d6): δ 1.13 (t, J = 7 Hz, 3H), 1.36−1.69 (m, 2H), 1.92− 2.12 (m, 2H), 2.98−3.08 (m, 2H), 3.60−3.81 (m, 2H), 3.83 (s, 3H), 4.18 (d, J = 7 Hz, 2H), 4.33−4.55 (m, 1H), 5.61 (s, 1H), 6.56 (d, J = 8 Hz, 1H), 7.18 (t, J = 7 Hz, 1H), 7.42−7.51 (m, 1H), 7.53−7.62 (m, 1H), 7.71 (s, 1H), 8.07 (d, J = 8 Hz, 1H). HRMS: m/z (ES+) = 414.16918 (MH+) for C21H24ClN5O2. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)oxy)quinolin-2(1H)-one (9). Yield: 26%. 1H NMR (300 MHz, DMSO-d6): δ 1.68−1.84 (m, 2H), 1.96−2.10 (m, 2H), 3.54 (br s, 2H), 3.64−3.79 (m, 1H), 3.79−3.91 (s, 4H), 4.92 (br s, 1H), 5.93− 6.11 (m, 1H), 7.16 (t, J = 7 Hz, 1H), 7.27 (d, J = 8 Hz, 1H), 7.46−7.56 (m, 1H), 7.75 (s, 1H), 7.84 (d, J = 8 Hz, 1H), 11.37 (s, 1H). HRMS: m/z (ES+) = 387.122 (MH+) for C19H19ClN4O3. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)methylamino)quinolin-2(1H)-one (10). Yield: 10%. 1H NMR (300 MHz, DMSO-d6): δ 1.78 (br s, 4H), 2.70 (s, 3H), 3.05 (br s, 1H), 3.16 (d, J = 5 Hz, 1H), 3.64 (br s, 2H), 3.82 (s, 3H), 4.48 (br s, 1H), 5.88 (s, 1H), 7.16 (t, J = 8 Hz, 1H), 7.28 (d, J = 8 Hz, 1H), 7.46 (t, J = 8 Hz, 1H), 7.64 (d, J = 8 Hz, 1H), 7.73 (s, 1H), 11.31 (br s, 1H) ;HRMS: m/z (ES+) = 400.1531 (MH+) for C20H22ClN5O2. (5-Chloro-1-methyl-1H-pyrazol-4-yl)(4-((2-methoxyquinolin-4yl)amino)piperidin-1-yl)methanone (11). Yield: 20%. 1H NMR (300 MHz, DMSO-d6): δ 1.59 (q, J = 11 Hz, 2H), 2.08 (d, J = 11 Hz, 2H), 3.42 (s, 3H), 3.90 (s, 4H), 3.95 (s, 3H), 4.50 (br s, 1H), 6.10 (s, 1H), 6.77 (d, J = 8 Hz, 1H), 7.25−7.41 (m, 1H), 7.55−7.69 (m, 2H), 7.78 (s, 1H), 8.20 (d, J = 8 Hz, 1H). HRMS: m/z (ES+) = 400.1535 (MH+) for C20H22ClN5O2. (5-Chloro-1-methyl-1H-pyrazol-4-yl)(4-(cinnolin-4-ylamino)piperidin-1-yl)methanone (12). Yield: 10%. 1H NMR (300 MHz, DMSO-d6): δ 1.40−1.50 (m, 2H), 1.95−2.05 (m, 2H), 3.01 (br s, 1H), 3.35−3.45(m, 1H), 3.83 (s, 4H), 4.08 (d, J = 7 Hz, 1H), 4.47 (br s, 1H), 7.18 (d, J = 8 Hz, 1H), 7.50−7.69 (m, 1H), 7.69−7.85 (m,

37. The 3-phenylpyridone intermediate 56 was synthesized from 2amino-4,6-dichloropyridine (54).22 Compound 54 was reacted with phenylboronic acid under Suzuki conditions to yield 55, which on diazotization followed by acid hydrolysis furnished intermediate 56. Compounds 14 and 38 were then synthesized from intermediate 56 following the general protocol. 1,8-Naphthyridine-2,4-diol (58) was constructed by condensation of ethyl acetate with ethyl 2-aminonicotinate (57) in the presence of NaH.23 Intermediate 58 was then converted to intermediate 59 through the sequence chlorination, hydrolysis, and N-methylation as described for other intermediates. Compound 39 was synthesized from intermediate 59 following the general protocol. Pyridopyrazine-6,8-diol (62) was synthesized by hydrolytic decarboxylation of ethyl ester 61, which in turn was synthesized by condensing methyl 3-aminopyrazine-2-carboxylate (60) with diethyl malonate in the presence of sodium ethoxide.24 Compound 40 was synthesized from intermediate 62 following the general protocol. All commercial reagents and solvents were used without further purification. (1R,3r,5S)-tert-butyl 3-amino-8-azabicyclo[3.2.1]octane-8carboxylate and (1R,3s,5S)-tert-butyl 3-amino-8-azabicyclo[3.2.1]octane-8-carboxylate were purchased from Wuxi APPTEC (Tianjin) Co., Ltd., China. Analytical thin-layer chromatography (TLC) was performed on SiO2 plates with aluminum backing. visualization was accomplished by UV irradiation at 254 and 220 nm. Flash column chromatography was performed using the Biotage Isolera flash purification system with SiO2 60 (particle size 0.040−0.055 mm, 230−400 mesh). The purity of all final derivatives for biological testing was confirmed to be >95% as determined using an Agilent 1100 series HPLC instrument at two wavelengths, 220 and 254 nm, using the following two conditions: (low-pH conditions) Waters Sunfire C18 column (50 mm × 4.6 mm, 5 μm particle size), 10 mM ammonium acetate (pH 4.0) (eluent A), acetonitrile (eluent B); (high-pH conditions) Waters XBridge C18 column (50 mm × 4.6 mm, 5 μm particle size), 0.2% (v/v) ammonia solution (25%) (pH 9.0) (eluent A), acetonitrile (eluent B). The structure of the intermediates and end products was confirmed by 1H NMR and mass spectroscopy. Proton magnetic resonance spectra were determined in DMSO-d6 unless otherwise stated using a Bruker DRX-300 or Bruker DRX-400 spectrometer operating at 300 or 400 MHz, respectively. LC−MS data were acquired using an Agilent LC−MS VL series instrument (source ES ionization) coupled with an Agilent 1100 series HPLC system and an Agilent 1100 series PDA instrument at the front end. HRMS data were acquired using an Agilent 6520 quadrupole time of flight tandem mass spectrometer (Q-TOF MS/MS) coupled with an Agilent 1200 series HPLC system. General Procedure for Coupling of Intermediate 46 with Aromatic Acids. The aromatic acid (2.45 mmol) was dissolved in DMF (10 mL). The intermediate 46 was added to the solution followed by the addition of N,N-diisopropylethylamine (1.3 mL, 7.36 mmol). The reaction mixture was stirred at rt for 3 h. After the completion of the reaction, the volatiles were evaporated under vacuum, and the residue obtained was purified by flash chromatography on a silica gel column using methanol−dichloromethane as the eluent. Pure fractions were combined and evaporated under vacuum to get the final compounds 1−40 as off-white solids in 10−60% yield. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)quinolin-2(1H)-one (1). Yield: 40%. 1H NMR (300 MHz, DMSO-d6): δ 1.45−1.60 (m, 2H), 1.88−1.95 (m, 2H), 1.99−2.10 (m, 2H), 2.82−3.03 (m, 1H), 3.75 (br s, 1H), 3.83 (s, 3H), 4.40 (br s, 1H), 5.45 (s, 1H), 6.53 (d, J = 7.35 Hz, 1H), 7.09 (t, J = 7 Hz, 1H), 7.21 (d, J = 8 Hz, 1H), 7.35−7.50 (m, 1H), 7.71 (s, 1H), 7.98 (d, J = 8 Hz, 1H), 10.75 (br s, 1H). HRMS: m/z (ES+) = 386.13818 (MH+) for C19H20ClN5O2. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-6-fluoroquinolin-2(1H)-one (2). Yield: 30%. 1H NMR (300 MHz, DMSO-d6): δ 1.48−158 (m, 2H), 1.97−2.10 (m, 2H), 2.85− 2.95 (m, 2H), 3.25−3.32 (m, 1H), 3.74 (br s, 1H), 3.83 (s, 3H), 4.41 (br s, 1H), 6.50 (d, J = 8 Hz, 1H), 5.49 (s, 1H), 7.16−7.27 (m, 1H), 7.29−7.41 (m, 1H), 7.71 (s, 1H), 7.92 (d, J = 11 Hz, 1H), 10.85 (br s, 1H). HRMS: m/z (ES+) = 404.1286 (MH+) for C19H19ClFN5O2. 5431

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Journal of Medicinal Chemistry

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4-((1-((1,5-Dimethyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-yl)amino)quinolin-2(1H)-one (22). Yield: 19%. 1H NMR (300 MHz, DMSO-d6): δ 1.50 (d, J = 11 Hz, 2H), 1.89−2.03 (m, 3H), 2.31 (s, 3H), 3.13−3.45 (m, 2H), 3.74 (s, 3H), 3.91−4.45 (m, 2H), 5.44 (s, 1H), 6.52 (d, J = 8 Hz, 1H), 7.08 (t, J = 7 Hz, 1H), 7.21 (d, J = 8 Hz, 1H), 7.39−7.47 (m, 2H), 7.85−8.05 (m, 1H), 10.75 (s, 1H). HRMS: m/z (ES+) = 366.19225 (MH+) for C20H23N5O2. 6-Fluoro-4-((1-((1-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)carbonyl)piperidin-4-yl)amino)quinolin-2(1H)-one (23). Yield: 38%. 1 H NMR (300 MHz, DMSO-d6): δ 1.29−1.60 (m, 2H), 1.80−2.12 (m, 2H), 2.96 (t, J = 12 Hz, 1H), 3.22 (t, J = 12 Hz, 1H), 3.57 (d, J = 13 Hz, 1H), 3.72 (br s, 1H), 4.00 (s, 3H), 4.48 (d, J = 13 Hz, 1H), 5.49 (s, 1H), 6.51 (d, J = 8 Hz, 1H), 7.15−7.29 (m, 1H), 7.30−7.40 (m, 1H), 7.73 (s, 1H), 7.93 (d, J = 11 Hz, 1H), 10.85 (br s, 1H). HRMS: m/z (ES+) = 438.15605 (MH+) for C20H19F4N5O2. 4-((1-((4,5-Dimethylisoxazol-3-yl)carbonyl)piperidin-4-yl)amino)6-fluoroquinolin-2(1H)-one (24). Yield: 33%. 1H NMR (300 MHz, DMSO-d6): δ 1.58 (m, 2H), 2.00 (s, 3H), 2.02−2.21 (m, 2H), 2.46 (s, 3H), 3.11 (m, 1H), 3.26−3.43 (m, 2H), 3.81 (m, 2H), 4.58 (m, 1H), 5.59 (s, 1H), 6.61 (d, J = 8 Hz, 1H), 7.25−7.37 (m, 1H), 7.37−7.48 (m, 1H), 7.99 (d, J = 11 Hz, 1H). HRMS: m/z (ES+) = 385.1681 (MH+) for C20H21FN4O3. 4-((1-((1,5-Dimethyl-1H-1,2,3-triazol-4-yl)carbonyl)piperidin-4yl)amino)-6-fluoroquinolin-2(1H)-one (25). Yield: 35%. 1H NMR (300 MHz, DMSO-d6): δ 1.49−1.70 (m, 2H), 2.15 (s, 3H), 2.35−2.42 (m, 2H), 2.46 (s, 3H), 2.98−3.05 (m, 1H), 3.76 (br s, 1H), 3.96 (m, 2H), 4.50 (d, J = 13 Hz, 1H), 5.51 (s, 1H), 6.39−6.67 (m, 1H), 7.15− 7.45 (m, 2H), 7.81−8.05 (m, 1H), 10.84 (br s, 1H). HRMS: m/z (ES+) = 385.17815 (MH+) for C19H21FN6O2. 4-((1-((5-Chloro-1-ethyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-yl)amino)-6-fluoroquinolin-2(1H)-one (26). Yield: 15%. 1H NMR (300 MHz, DMSO-d6): δ 1.35 (t, J = 7 Hz, 3H), 1.41−1.62 (m, 2H), 1.98 (br s, 2H), 2.95 (br s, 1H), 3.17 (br s, 1H), 3.75 (br s, 2H), 4.18 (q, J = 7 Hz, 2H), 4.42 (br s, 1H), 5.50 (s, 1H), 6.51 (d, J = 8 Hz, 1H), 7.14−7.28 (m, 1H), 7.29−7.42 (m, 1H), 7.74 (s, 1H), 7.92 (d, J = 11 Hz, 1H), 10.85 (br s, 1H). HRMS: m/z (ES+) = 418.13689 (MH+) for C20H21ClFN5O2. 4-((1-((5-Chloro-1-isopropyl-1H-pyrazol-4-yl)carbonyl)piperidin4-yl)amino)quinolin-2(1H)-one (27). Yield: 25%. 1H NMR (300 MHz, DMSO-d6): δ 1.41 (d, J = 7 Hz, 6H), 1.44−1.62 (m, 2H), 2.00 (d, J = 11 Hz, 2H), 2.95 (br s, 1H), 3.09−3.31 (m, 1H), 3.74 (d, J = 6 Hz, 2H), 4.43 (br s, 1H), 4.70 (qn, J = 7 Hz, 1H), 5.45 (s, 1H), 6.54 (d, J = 8 Hz, 1H), 7.09 (t, J = 8 Hz, 1H), 7.21 (d, J = 8 Hz, 1H), 7.36− 7.51 (m, 1H), 7.75 (s, 1H), 7.98 (d, J = 8 Hz, 1H), 10.76 (s, 1H). HRMS: m/z (ES+) = 414.16933 (MH+) for C21H24ClFN5O2. 4-((1-((4,5-Dimethyl-1,2-oxazol-3-yl)carbonyl)piperidin-4-yl)amino)-6,7-difluoroquinolin-2(1H)-one (28). Yield: 41%. 1H NMR (300 MHz, DMSO-d6): δ 1.41−1.67 (m, 2H), 1.99 (s, 4H), 2.45 (s, 3H), 2.98−3.19 (m, 1H), 3.34 (t, J = 12 Hz, 2H), 3.80 (d, J = 14 Hz, 2H), 4.56 (d, J = 14 Hz, 1H), 5.56 (s, 1H), 6.61 (d, J = 8 Hz, 1H), 7.22 (dd, J = 12, 8 Hz, 1H), 8.23 (dd, J = 13, 9 Hz, 1H), 10.9 (s, 1H). HRMS: m/z (ES+) = 403.15779 (MH+) for C20H20F2N4O3. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-6,7-difluoro-1-methylquinolin-2(1H)-one (29). Yield: 72%. 1 H NMR (300 MHz, DMSO-d6): δ 1.37−1.59 (m, 2H), 1.88−2.10 (m, 2H), 2.96 (br s, 1H), 3.31 (br s, 1H), 3.41−3.55 (m, 3H), 3.63− 3.96 (m, 5H), 4.41 (br s, 1H), 5.65 (s, 1H), 6.54 (d, J = 8 Hz, 1H), 7.54 (dd, J = 13, 7 Hz, 1H), 7.71 (s, 1H), 8.24 (dd, J = 13, 9 Hz, 1H). HRMS: m/z (ES+) = 436.13505 (MH+) for C20H20ClF2N5O2. 4-((1-((4,5-Dimethylisoxazol-3-yl)carbonyl)piperidin-4-yl)amino)6,7-difluoro-1-methylquinolin-2(1H)-one (30). Yield: 32%. 1H NMR (300 MHz, DMSO-d6): δ 1.37−1.61 (m, 2H), 1.92 (s, 3H), 1.95−2.14 (m, 2H), 2.38 (s, 3H), 2.92−3.11 (m, 1H), 3.20−3.29 (m, 1H), 3.46 (s, 3H), 3.64−3.89 (m, 2H), 4.38−4.60 (m, 1H), 5.66 (s, 1H), 6.46− 6.64 (m, 1H), 7.44−7.63 (m, 1H), 8.14−8.31 (m, 1H). HRMS: m/z (ES+) = 417.17333 (MH+) for C21H22F2N4O3. 4-(((2S,4R)-1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-2methylpiperidin-4-yl)amino)-6,7-difluoroquinolin-2(1H)-one (31). Yield: 23%. 1H NMR (300 MHz, DMSO-d6): δ 1.23−1.48 (m, 4H), 1.57−1.75 (m, 1H), 1.88 (s, 1H), 2.01 (br s, 1H), 3.17 (m, 1H), 3.83 (s, 3H), 3.91 (br s, 1H), 4.35 (br s, 1H), 4.91 (br s, 1H), 5.52 (s, 1H),

2H), 8.10 (d, J = 8 Hz, 1H), 8.32 (d, J = 8 Hz, 1H), 8.83 (s, 1H). HRMS: m/z (ES+) = 371.13831 (MH+) for C18H19ClN6O. (5-Chloro-1-methyl-1H-pyrazol-4-yl)(4-((5-fluoro-1H-indazol-3yl)amino)piperidin-1-yl)methanone (13). Yield: 34%. 1H NMR (300 MHz, DMSO): δ 1.37−1.53 (m, 2H), 2.09 (d, J = 9 Hz, 2H), 3.05 (br s, 1H), 3.19−3.30 (m, 1H), 3.58−3.80 (m, 2H), 3.82 (s, 3H), 4.28 (br s, 1H), 5.86 (d, J = 7 Hz, 1H), 7.12 (dt, J = 3, 9 Hz, 1H), 7.21−7.31 (m, 1H), 7.49 (dd, J = 3, 9 Hz, 1H), 7.70 (s, 1H), 11.51 (s, 1H). HRMS: m/z (ES+) = 377.12945 (MH+) for C17H18ClFN6O. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4yl)amino)-6-phenylpyridin-2(1H)-one (14). Yield: 33%. 1H NMR (300 MHz, DMSO-d6): δ 1.31−1.42 (m, 2H), 1.88−1.98 (m, 2H), 3.05 (br s, 1H), 3.30−3.33 (m, 1H), 3.53 (br s, 2H), 3.82 (s, 3H), 4.12−4.40 (m, 1H), 5.23 (s, 1H), 5.95 (s, 1H), 6.54 (d, J = 8 Hz, 1H), 7.42−7.49 (m, 3H), 7.56−7.64 (m, 2H), 7.71 (s, 1H), 10.60 (br s, 1H). HRMS: m/z (ES+) = 412.15279 (MH+) for C21H22ClN5O2. 4-((1-((5-chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)pyrrolidin-3yl)amino)quinolin-2(1H)-one (15). Yield: 24%. 1H NMR (300 MHz, DMSO-d6): δ 2.09 (br s, 1H), 2.18−2.30 (m, 1H), 3.51−3.71 (m, 3H), 3.79−3.86 (s, 3H), 3.91−4.07 (m, 1H), 4.19 (br s, 1H), 5.36 (d, J = 9 Hz, 1H), 6.79 (dd, J = 14, 6 Hz, 1H), 7.10 (t, J = 8 Hz, 1H), 7.21 (dd, J = 8, 4 Hz, 1H), 7.44 (t, J = 8 Hz, 1H), 7.88 (d, J = 8 Hz, 1H), 8.04 (dd, J = 12, 8 Hz, 1H), 10.85 (br s, 1H). HRMS: m/z (ES+) = 372.12198 (MH+) for C18H18ClN5O2. 4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)azetidin-3yl)amino)quinolin-2(1H)-one (16). Yield: 20%. 1H NMR (300 MHz, DMSO-d6): δ 3.82 (s, 3H), 4.10 (br s, 1H), 4.25 (d, J = 6 Hz, 1H), 4.38 (br s, 2H), 4.73 (d, J = 7 Hz, 1H), 5.15 (s, 1H), 7.13 (t, J = 8 Hz, 1H), 7.23 (d, J = 8 Hz, 1H), 7.30 (d, J = 4 Hz, 1H), 7.39−7.52 (m, 1H), 7.88 (s, 1H), 7.99 (d, J = 8 Hz, 1H), 10.90 (s, 1H). HRMS: m/z (ES+) = 358.10808 (MH+) for C17H16ClN5O2. 4-(((2S,4R)-1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-2methylpiperidin-4-yl)amino)quinolin-2(1H)-one (17). Yield: 27%. 1H NMR (300 MHz, DMSO-d6): δ 1.20−1.50 (m, 2H), 1.32 (d, J = 9 Hz, 3H), 1.64−1.75 (m, 1H), 1.81−1.94 (m, 1H), 1.97−2.08 (m, 1H), 3.37−3.72 (m, 1H), 3.83 (s, 3H), 3.87−3.92 (m, 1H), 4.11−4.47 (m, 1H), 5.48 (s, 1H), 6.48 (d, J = 8 Hz, 1H), 7.08 (t, J = 7 Hz, 1H), 7.20 (d, J = 8 Hz, 1H), 7.42 (t, J = 7 Hz, 1H), 7.69 (s, 1H), 7.97 (d, J = 8 Hz, 1H), 10.75 (br s, 1 H). HRMS: m/z (ES+) = 400.15462 (MH + 1) for C20H22ClN5O2. 4-(((2S,4S)-1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-2methylpiperidin-4-yl)amino)quinolin-2(1H)-one (18). Yield: 14%. 1H NMR (300 MHz, DMSO-d6): δ 0.76−0.89 (m, 2H), 1.22 (d, J = 7 Hz, 4H), 1.78−1.96 (m, 3H), 3.76 (br s, 1H), 3.83 (s, 3H), 4.25 (d, J = 7 Hz, 1H), 5.31 (s, 1H), 6.48 (d, J = 6 Hz, 1H),), 7.03−7.26 (m, 2H), 7.37−7.47 (m, 1H), 7.72 (s, 1H), 8.04 (d, J = 9 Hz, 1H),10.82 (s, 1H). HRMS: (ES+) m/z = 400.15391 (MH + 1) for C20H22ClN5O2. 4-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8azabicyclo[3.2.1]octan-3-yl)amino)-6-fluoroquinolin-2(1H)-one (19). Yield: 23%. 1H NMR (300 MHz, DMSO-d6): δ 1.60 (m, 1H), 1.83 (m, 1H), 1.87−2.14 (m, 6H), 3.83 (s, 3H), 3.96−4.10 (m, 1H), 4.23 (m, 1H), 4.60 (m, 1H), 5.50 (s, 1H), 6.45 (d, J = 8 Hz, 1H), 7.14−7.26 (m, 1H), 7.26−7.42 (m, 1H), 7.76 (s, 1H), 7.90 (dd, J = 11, 3 Hz, 1H), 10.82 (s, 1H). HRMS: m/z (ES+) = 430.14482 (MH+) for C21H21ClFN5O2. 4-(((1R,3s,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8azabicyclo[3.2.1]octan-3-yl)amino)-6-fluoroquinolin-2(1H)-one (20). Yield: 27%. 1H NMR (300 MHz, DMSO-d6): δ 1.21 (d, J = 7 Hz, 1H), 2.02−2.28 (m, 6H), 3.81 (br s, 1H), 3.87−3.94 (m, 4H), 4.22 (br s, 1H), 4.63 (br s, 1H), 5.29 (s, 1H), 6.46 (br s, 1H), 7.31 (dd, J = 9, 5 Hz, 1H), 7.43 (td, J = 9, 3 Hz, 1H), 7.82−7.94 (m, 2H), 11.00 (s, 1H). HRMS: m/z (ES+) = 430.14419 (MH+) for C21H21ClFN5O2. 4-((1-((1-Methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-yl)amino)quinolin-2(1H)-one (21). Yield: 11%. 1H NMR (300 MHz, DMSOd6): δ 1.50 (d, J = 12 Hz, 2H), 1.99 (d, J = 12 Hz, 2H), 3.14−3.19 (m, 2H), 3.6−3.75 (m, 2H), 3.82 (m, 1H), 3.86 (s, 3H), 5.45 (s, 1H), 6.54 (d, J = 8 Hz, 1H), 7.09 (t, J = 8 Hz, 1H), 7.21 (d, J = 8 Hz, 1H), 7.39− 7.47 (m, 1H), 7.66 (s, 1H), 7.98 (d, J = 8 Hz, 1H), 8.07 (s, 1H), 10.77 (s, 1H). HRMS: m/z (ES+) = 352.16966 (MH+) for C19H21N5O2. 5432

dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry

Article

6.47 (d, J = 8 Hz, 1H), 7.15 (dd, J = 12, 7 Hz, 1H), 7.69 (s, 1H), 8.16 (dd, J = 13, 9 Hz, 1H), 10.9 (br s, 1H). HRMS: m/z (ES+) = 436.13509 (MH+) for C20H20ClF2N5O2. 4-((3-endo-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8azabicyclo[3.2.1]oct-3-yl)amino)-6,7-difluoroquinolin-2(1H)-one (32). Yield: 74%. 1H NMR (300 MHz, DMSO-d6): δ 1.49−1.57 (m, 1H), 1.75−1.80 (m, 1H), 1.94−2.03 (m, 6H), 3.83 (s, 3H), 3.95−4.01 (m, 1H), 4.21−4.3 (m, 1H), 4.55−4.65 (m, 1H), 5.49 (br s, 1H), 6.46 (d, J = 8.10 Hz, 1H), 7.14 (dd, J = 12, 7 Hz, 1H), 7.76 (s, 1H), 8.15 (dd, J = 13, 9 Hz, 1H), 10.86 (s, 1H). HRMS: m/z (ES+) = 448.1352 (MH+) for C21H20ClF2N5O2. 4-(((1R,3r,5S)-8-((4,5-Dimethylisoxazol-3-yl)carbonyl)-8azabicyclo[3.2.1]octan-3-yl)amino)-6,7-difluoroquinolin-2(1H)-one (33). Yield: 25%. 1H NMR (300 MHz, DMSO-d6): δ 1.76 (m, 2H), 1.97 (s, 3H), 1.89−2.12 (m, 6H), 2.37 (s, 3H), 4.04 (m, 1H), 4.35 (br s, 1H), 4.71 (br s, 1H), 5.51 (s, 1H), 6.49 (d, J = 8 Hz, 1H), 7.14 (dd, J = 12, 7 Hz, 1H), 8.17 (dd, J = 13, 9 Hz, 1H), 10.87 (s, 1H). HRMS: m/z (ES+) = 429.17352 (MH+) for C22H22F2N4O3. 4-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8azabicyclo[3.2.1]octan-3-yl)amino)-6-fluoro-1-methylquinolin2(1H)-one (34). Yield: 30%. 1H NMR (300 MHz, DMSO-d6): δ 1.61 (br s, 1H), 1.90−2.16 (m, 6H), 3.49 (s, 5H), 3.83 (s, 3H), 3.93−4.17 (m, 1H), 4.60 (br s, 1H), 5.67 (s, 1H), 6.46 (d, J = 8 Hz, 1H), 7.44 (d, J = 6 Hz, 2H), 7.76 (s, 1H), 7.98 (d, J = 11 Hz, 1H). HRMS: m/z (ES+) = 444.16024 (MH+) for C22H23ClFN5O2. 4-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8azabicyclo[3.2.1]octan-3-yl)amino)-6,7-difluoro-1-methylquinolin2(1H)-one (35). Yield: 28%. 1H NMR (300 MHz, DMSO-d6): δ 1.58− 1.64 (m, 1H), 1.83−1.90 (m, 1H), 1.92−2.12 (m, 6H), 3.46 (s, 3H), 3.83 (s, 3H), 3.96−4.13 (m, 1H), 4.24 (m, 1H), 4.60 (m, 1H), 5.66 (s, 1H), 6.47 (d, J = 9 Hz, 1H), 7.54 (dd, J = 13, 7 Hz, 1H), 7.76 (s, 1H), 8.22 (dd, J = 13, 9 Hz, 1H). HRMS: m/z (ES+) = 462.14315 (MH+) for C22H22ClF2N5O2. 4-((3-endo-8-((4,5-Dimethyl-1,2-oxazol-3-yl)carbonyl)-8azabicyclo[3.2.1]oct-3-yl)amino)-6,7-difluoro-1-methylquinolin2(1H)-one (36). Yield: 30%. 1H NMR (300 MHz, DMSO-d6): δ 1.67− 1.86 (m, 2H), 1.97 (s, 6H), 2.03 (dd, J = 13, 6 Hz, 2H), 2.37 (s, 3H), 3.32 (br s, 1H), 3.46 (s, 3H), 4.06 (br s, 1H), 4.35 (br s, 1H), 4.71 (br s, 1H), 5.67 (s, 1H), 6.49 (d, J = 8 Hz, 1H), 7.52 (dd, J = 13, 7 Hz, 1H), 8.23 (dd, J = 13, 9 Hz, 1H). HRMS: m/z (ES+) = 443.18928 (MH+) for C23H24F2N4O3. (5-Chloro-1-methyl-1H-pyrazol-4-yl)(3-endo-3-((5-fluoro-1H-indazol-3-yl)amino)-8-azabicyclo[3.2.1]oct-8-yl)methanone (37). Yield: 18%. 1H NMR (300 MHz, DMSO-d6): δ 1.23 (s, 4H), 1.42 (br s, 1H), 1.55 (br s, 1H), 1.66 (br s, 1H), 1.99 (d, J = 16 Hz, 2H), 2.12 (br s, 2H), 4.13 (br s, 1H), 4.21 (br s, 1H), 4.59 (br s, 1H), 5.78 (d, J = 8 Hz, 1H), 7.10 (td, J = 9, 3 Hz, 1H), 7.24 (dd, J = 9, 4 Hz, 1H), 7.43 (dd, J = 9, 2 Hz, 1H), 7.76 (s, 1H), 11.47 (s, 1H). HRMS: m/z (ES+) = 403.14462 (MH+) for C19H20ClFN6O. 4-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8azabicyclo[3.2.1]octan-3-yl)amino)-6-phenylpyridin-2(1H)-one (38). Yield: 28%. 1H NMR (300 MHz, DMSO-d6): δ 1.35 (m, 1H), 1.60 (m, 1H), 1.91 (m, 3H), 1.99 (m, 3H), 3.83 (s, 3H), 3.9 (m,1H), 4.20 (m, 1H), 4.57 (m, 1H), 5.26 (d, J = 2 Hz, 1H), 5.89 (s, 1H), 6.44 (d, J = 8 Hz, 1H), 7.36−7.50 (m, 3H), 7.54−7.67 (m, 2H), 7.76 (s, 1H), 10.59 (br s, 1H) . HRMS: m/z (ES+) = 438.16935 (MH+) for C23H24ClN5O2. 4-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8azabicyclo[3.2.1]octan-3-yl)amino)-1-methyl-1,8-naphthyridin2(1H)-one (39). Yield: 32%. 1H NMR (300 MHz, DMSO-d6): δ 1.49− 1.69 (m, 1H), 1.74−1.89 (m, 1H), 1.92−2.14 (m, 6H), 3.55 (s, 3H), 3.83 (s, 3H), 3.97−4.12 (m, 1H), 4.17−4.34 (m, 1H), 4.49−4.70 (m, 1H), 5.68 (s, 1H), 6.55−6.75 (m, 1H), 7.14−7.35 (m, 1H), 7.76 (s, 1H), 8.39−8.52 (m, 1H), 8.53−8.65 (m, 1H). HRMS: m/z (ES+) = 427.16479 (MH+) for C21H23ClN6O2. 8-((3-endo-8-((4,5-Dimethyl-1,2-oxazol-3-yl)carbonyl)-8azabicyclo[3.2.1]oct-3-yl)amino)-5-methylpyrido[2,3-b]pyrazin6(5H)-one (40). Yield: 37%. 1H NMR (300 MHz, CDCl3): δ 1.82− 1.92 (m, 4H), 1.9 (s, 3H), 2.01- 2.09 (m, 4H), 2.29 (s, 3H), 3.62 (s, 3H), 3.85−4.10 (m, 1H), 4.65 (m, 1H), 4.89 (m, 1H), 5.84 (s, 1H),

6.02 (d, J = 8 Hz, 1H), 8.22 (d, J = 2 Hz, 1H), 8.47 (d, J = 2 Hz, 1H). HRMS: m/z (ES+) = 409.19847 (MH+) for C21H24N6O3.



ASSOCIATED CONTENT

S Supporting Information *

Sequence homology between Mtb DprE1 and Msm DprE1, synthetic procedures for the intermediates, MIC data for clinically resistant strains, microbiology experimental details, mass spectra of DprE1 with AQs, method for time lapse microscopy, bioanalytical methods, protocols for rat PK studies, physicochemical studies, and hERG assay, and CSV file listing the compounds and their molecular weights. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Authors

*Phone: +91 9535500085. E-mail: [email protected]. *Phone: +91 9880238194. E-mail: sandeepghorpade@hotmail. com. Present Addresses

◆ Pohang University of Science and Technology, Pohang, North Gyeongsang 790-784, Republic of Korea. ¶ DMPK, Infection iMed, AstraZeneca, Waltham, MA 02451. ▲ Syngenta India Ltd., Corlim, Goa 403110, India.

Author Contributions □

The first four authors contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The research leading to these results was funded by the European Community’s Seventh Framework Program (Grant 260872). We thank Dr. Saravana Kappuasamy, Syngene, and his team for profound chemistry support, Dr. Peter Warner and Suresh Solapure, AstraZeneca, for scientific discussions during the course of the work, Lyn Rosenbrier-Ribeiro and Duncan Armstrong, Discovery Safety, AstraZeneca, United Kingdom, for secondary pharmacology support, Dr. Matthew BridglandTaylor and Dr. Ann Woods, Discovery Sciences, AstraZeneca, United Kingdom, for hERG assay, Dr. Liz Flavell and Dr. Emma Cains, Discovery Sciences, AstraZeneca, for protein purifications, Sreenivasaiah Menasinakai for analytical and purification support, the compound management group at AstraZeneca for all compound-dispensing- and compounddispatch-related activities, and the members of the EU consortium for constructive discussions during the meetings.



ABBREVIATIONS USED TB, tuberculosis; Mtb, Mycobacterium tuberculosis; Msm, Mycobacterium smegmatis; MIC, minimum inhibitory concentration; NRP, nonreplicating phase; GFP, green fluorescent protein; SAR, structure−activity relationship; AUC, area under the curve; f , free fraction; qd, once a day; bid, twice a day; CFU, colony-forming units; rt, room temperature; DME, 1,2dimethoxyethane; THF, tetrahydrofuran; DMF, N,N-dimethylformamide



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