4-Aminoquinolone piperidine amides: Noncovalent inhibitors of DprE1 with long residence time and potent antimycobacterial activity

Article abstract of DOI:10.1021/jm5005978

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.

Full text of DOI:10.1021/jm5005978

Article
pubs.acs.org/jmc
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,
Chandramohan Bathula, Suresh Rudrapatna, Vijayashree Achar, Sreevalli Sharma, Anisha Ambady,
†
†
†,▲
†
†
‡
‡
§
M. R. Manjunatha, Gopinath Gorai,
†
†
†
‡
‡
‡
‡
‡
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, Joao
∇
∇
∇
○
○
̃
Neres, Neeraj Dhar,
○
○
‡
§
Franco
̧
is 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 Fed
́ ́
erale 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 decaprenylphos-
phoryl-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
DprE1 was identified as a primary target for a mechanism-based
inhibitor containing a reactive nitro substituent, benzothiazi-
none (BTZ043; Figure 1), which acts by forming a covalent
■
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
3,4
bond with Cys387 in the active site pocket of the enzyme.
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
1
cidal activity against Mycobacterium tuberculosis (Mtb). DprE1,
along with decaprenylphosphoryl-D-2-ketoerythropentose re-
ductase (DprE2), is responsible for epimerization of decap-
renylphosphorylribose (DPR) to decaprenylphosphorylarabi-
nose (DPA), the sole precursor for synthesis of arabinan, which2
is an essential component of the mycobacterial cell wall.
Received: April 17, 2014
Published: May 28, 2014
©
2014 American Chemical Society
5419
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
Figure 1. Known DprE1 inhibitors.
Table 1. Properties of Compounds 1−3
property
Mtb MIC (μM)
compd 1
compd 2
compd 3
6.3
1.6
0.8
Mtb MBC (μM)
6.3
3.1
0.8
IC80 on hypoxic Mtb (μM)
>100
>200
255
1.6
>100
>200
105
2.0
>100
>100
26
a
MMIC (μM)
solubility at pH 7.4 (μM)
logD
2.2
human PPB (fraction unbound, fu) 0.3
0.4
0.4
human microsomal CLint
μL/min/mg of protein)
<4
<4.8
12.5
(
rat hepatocyte CL
1.5
5.6
2.4
int
6
(
μL/min/10 cells)
−6
Caco-2 A−B/B−A (10 cm/s)
hERG IC50 (μM)
0.5/14
>100
>30
0.6/13
>100
>30
0.2/11
>100
b
d
CYP inhibition (μM)
ND
c
secondary pharmacology hits
active 1 of 24 targets
(μ-opioid receptor, 21.9)
active 2 of 24 targets (μ-opioid receptor, −28.1;
5-HT2B receptor, −41.9)
active 1 of 24 targets
(adenosine A1 receptor, −34)
d
(
IC , μM)
5
0
a
b
MIC on the A549 cell line used to determine cytotoxicity. Isoforms tested include CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2.
c
d
Diverse set of binding, enzyme, and functional assays covering 24 distinct molecular targets; activity classified as a defined IC /EC . Not
5
0
50
determined.
(
MDR) and extensively-drug-resistant (XDR) TB as it does not
RESULTS AND DISCUSSION
■
5
pose a risk of pre-existing drug resistance in the clinic.
Furthermore, DprE1 is specific to mycobacteria and actino-
mycetes and does not have a human homologue. PBTZ169
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
(
Figure 1), a safer and more effective analogue of BTZ043, is
6
poised to enter clinical trials. 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
7
further development as clinical candidates, e.g., TCA1 and
8
azaindoles (Figure 1). Herein, we report 4-aminoquinolone
(
SAR) exploration to demonstrate potential of the scaffold for
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.
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,
5
420
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
a
Table 2. MIC Modulation against Various Overexpressed and Resistant Mutant Strains
MIC (μM)
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
compd
1
2
3
6.3
>100
100
50
6.3
3.1
3.1
100
25
3.1
1.6
1.6
1.6
0.8
1.6
1.6
1.6
0.4
0.4
0.4
0.4
3.12
0.4
0.2
0.2
TMC 207
BTZ043
MOX
0.78
0.003
0.15
0.01
0.45
1.6
1.6
0.8
0.8
50
1.6
12.5
0.08
0.01
0.22
0.003
0.16
0.01
>7.25
0.012
0.31
0.03
0.45
0.003
0.15
0.004
0.22
25
0.002
0.15
0.01
0.23
0.003
10
0.08
0.004
0.45
rifampicin
INH
0.02
0.45
a
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
1
×
6−64-fold increase in MIC (the mutation frequency was 1.99
−8
10 ). 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
Figure 2. Kinetic analysis of Msm DprE1 enzyme inhibition by
compound 2. Connecting lines are visual guides only.
(
data not shown).
Time-Dependent Inhibition of DprE1 Enzyme Activ-
ity. 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
1
Figures S1 and S2, Supporting Information). Hence, the Msm
DprE1 assay for which a thorough biochemical characterization
2
was previously carried out 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.
Figure 3. Scatter plot of Msm DprE1 pIC50 late vs Mtb pMIC. pIC50
=
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, IC
−
[log(IC )] and pMIC = −[log(MIC)], wherein IC 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.
50
50
50
late). A difference in IC early and IC late is indicative of a
5
0
50
time-dependent inhibition (see Table S8, Supporting Informa-
tion, for early and late IC50 values of the compounds). A good
trend between IC late on Msm DprE1 enzyme and Mtb MIC
(IC50 ≤ 100 nM, pIC ≥ 7) was required to achieve potent
5
0
50
was observed (Figure 3). In general, potent DprE1 inhibition
Mtb MIC of ≤1 μM (pMIC > 6).
5
421
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
Representative compounds were tested in kinetic studies to
measure the dissociation rate constant (k ) of the inhibitor
off
from the enzyme by fitting the progress curve data to a single-
step 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/k ), and
off
dissociation half-life of the enzyme−inhibitor complex (0.693/
k ) for compounds 1−3 are shown in Table 3. The standard
off
Table 3. koff and Residence Time of Representative
Compounds
−
4
−4
koff × 10
koff std error × 10
t1/2
(min)
residence time
(min)
−
1
−1
compd
(s
)
(s
)
1
2
3
1.6
1.2
1.1
0.14
0.2
72
83
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.
0.13
105
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
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
9
after the inhibitor is cleared from the plasma.
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
BTZ043.
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.
∼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
1
0
microscopy. 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
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.
5
422
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
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.
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
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
4
structure reported by Batt et al. 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. Further-
more, 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
2
,4,6a,7
DprE1
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
1b,2
structures.
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
4,6a
N atom of the activated nitro group (Figure 7a).
The
conformation of the flavin adenine dinucleotide (FAD) cofactor
binds to the CF group in the case of nitro compounds and the
3
remains unchanged in all the structures. Most of the structures
thiazole ring for TCA1. The ligand overlay of these three
5
423
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
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−
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
3
22.
Docking was performed against both open (PDB ID 4KW5)
and closed (PDB ID 4FDO) conformations of the active site
1
1,12
using Glide 6.1 (Schrodinger).
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
2
,4
2D interaction plot for compound 1 are shown in Figure 8.
inhibitors.
On the basis of the proposed binding mode, the AQ scaffold
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
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
5
424
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
a
amide variations were inactive in the screen as mentioned in the
previous section.
Table 5. Quinolone Ring Replacement
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 IC50 (μM)
Mtb MIC (μM)
a
Unsubstituted piperidine ring as the linker and 5-chloro-1-
2
3
4
5
6
7
8
9
6-F
0.005
0.006
0.007
0.011
0.009
0.14
>100
8
1.6
b
methylpyrazole as the site 2 ring. IC50 at 30 min.
6,7-di-F
7-F
0.8
3.1
8-F
3.1
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
7,8-di-F
1.6
1-N-CH3
25
1-N-C H
>100
50
2
5
4-O
10
4-N-CH3
18
>250
a
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.
a
Table 6. SAR at the Linker Piperidine Ring
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 “NCH ” led to much
3
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
IC against DprE1 (Table 5). The design to convert quinolone
50
into substituted pyridones was conceived to improve the
physicochemical properties, but the compounds were weakly
active. For example, 6-phenylpyridone analogue 14 showed a
a
Unsusbtituted quinolone ring at site 1 and 5-chloro-1-methylpyrazole
b
as the site 2 ring. IC50 at 30 min.
5
425
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
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.
a
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 hetero-
cyclic 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
Table 7. SAR at the Site 2 Aryl Ring
a
Unsubstituted quinolone ring at site 1 and unsubstituted piperidine
ring as the linker unless otherwise specified. IC50 at 30 min. 6-
b
c
Fluoroquinolone derivatives.
=
3
[
drug metabolism and pharmacokinetics). Compounds 32 and
3 with 6,7-difluoroquinolone at site 1 and an azabicyclo-
3,2,1]octanyl ring as the linker showed the best MICs of 60
3
16−322) in most of the reported crystal structures, hence
possibly explaining the longer residence time observed for this
scaffold.
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
Potency Improvement. Several potent compounds (28−
6) could be made by combining fluorinated quinolone rings
3
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
5
426
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
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
6.25
1.56
0.78
0.78
0.39
0.39
>100
100
100
50
100
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
>100
100
12.5
6.25
1.56
6.25
50
3
1
3
0
9
1
25
6.25
12.5
<0.39
0.78
47
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 physicochem-
ical 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).
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 IC 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.
50
5
427
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
The kinetics of inhibition for compounds that retained
potency against the Y314H mutant enzyme (e.g., compound
solubilities of the compounds were low, ranging from 6 to 100
13
μM, and were improved 3−5-fold in FeSSIF media (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, NH-
quinolone analogues showed better unbound fraction (fu) in
human plasma (e.g., fu ranged from 0.16 to 0.5 for compounds
1
9) indicates that binding equilibrium of the compounds to
DprE1 Y321H is achieved rapidly (Figure 11b) in contrast to
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
14
blood flow, LBF, ranged from 1% to 65% and from 5% to
3
7% when tested for rat hepatocyte CLint and human
microsomal CL , respectively). None of the compounds
int
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
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
15
against targets with a clear linkage to toxicity (Table 10).
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
(
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 CL , human
available. However, the observed K range of 8−34 μM suggests
int
i
microsomal CL , and Caco-2 permeability. The aqueous
that an appropriate safety margin may be achievable.
int
Table 10. In Vitro DMPK and Safety Properties of Representative Compounds
property
compd 7
compd 30
compd 36
compd 37
compd 39
compd 40
logD
2.0
78
2.3
2.8
6
1.9
1.8
1.8
solubility at pH 7.4 (μM)
FeSSIF solubility (μM)
28
>1000
20
132
c
c
c
ND
91
ND
ND
100
307
human PPB (fraction unbound, fu)
0.03
0.05
0.03
0.08
0.01
37 (5)
3 (1)
1/36
>30
>50
>33
0.05
a
human microsomal CLint (μL/min/mg of protein) (LBF, %)
14 (6)
8 (7)
4/23
>30
49 (20)
17 (19)
22/21
>30
107 (25)
56 (31)
23/13
>30
4 (6)
9 (19)
20/31
>30
100 (28)
20 (19)
33/21
>30
6
a
rat hepatocyte CLint (μL/min/10 cells) (LBF, %)
−6
Caco-2 A−B/B−A (10 cm/s)
b
CYP inhibition IC50 (μM)
MMIC A549 IC50 (μM)
hERG IC50 (μM)
>100
>33
>16
>50
>50
>50
c
ND
16
>33
>33
d
c
c
secondary pharmacology hits
active on 1−3 targets; the adenosine A1 receptor was a
ND
ND
common hit, K range 8−34 μM
i
a
14
b
c
Liver blood flow (%) predicted on the basis of the well-stirred mode. Isoforms CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. Not
d
determined. Diverse set of binding, enzyme, and functional assays covering 24 distinct molecular targets; activity classified as a defined K/IC /
i
50
EC .
50
5
428
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
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.
Pharmacokinetics studies on representative compounds were
performed in male Wistar rats, following single intravenous and
oral dose administration (Figure 12 and Table 11).
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.
Table 11. Pharmacokinetic (PK) Parameters of Compounds
a
3
and 7
intravenous
dose 2 mg/kg)
(
oral (dose 10 mg/kg)
PK param
compd 3 compd 7 compd 3 compd 7
C0 (iv) or oral C
max
(μM)
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
AUC0
−∞
(μM h)
19
1.3
elimination half-life (h)
CL (mL/min/kg)
Vss (L/kg)
bioavailability (F, %)
0.3
100
a
Key: iv, intravenous; C , plasma concentration at 0 h following iv
0
EXPERIMENTAL SECTION
■
administration; Cmax, maximum (peak) plasma concentration following
oral administration; AUC, area under the curve; CL, plasma clearance;
Vss, volume distribution.
Biochemistry: DprE1 Enzyme Assays and Residence Time
Measurements. DprE1 assays were performed as described
2,8
previously. Briefly, a 50 μL reaction was performed at 25 °C in
3
84-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 thioredoxin-
6His 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
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
3
0
00 μM farnesylphosphoryl-β-D-ribofuranose, the coupling enzyme
.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.
(
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.
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.
CONCLUSION
-Aminoquinolones piperidine amides are established here as
■
4
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.
For koff measurements, the data were collected at varying inhibitor
16
concentration. The single-step reversible slow binding model fitted
well to the fluorescence vs time data. The following equations were
used for the analysis:
vs − v
i
obsdt
[
P] = vt +
(1 − e−k
)
s
kobsd
(1)
⎛
⎞
[
Ki
I]
kobsd = k ⎜1 +
⎟
⎠
off
app
⎝
(2)
5
429
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of AQs
a
Reagents and conditions: (a) malonic acid, POCl , reflux, 3 h; (b) concd HCl, dioxane, reflux; (c) NaH, DMF, alkyl halide, rt, 3 h; (d) 1-Boc-4-
3
aminopiperidine or corresponding Boc-protected alicyclic amine, Pd(OAc) , BINAP, CsCO , toluene, microwave, 130 °C, 45 min; (e) trifluoroacetic
2
3
acid, CH Cl , rt, 2 h; (f) R CO H, HATU, N,N-diisopropylethylamine, DMF, rt; (g) NaOMe, toluene, reflux, 15 h; (h) NaNO , concd HCl, water,
2
2
2
2
2
−
5 to 70 °C; (i) POCl , reflux, 3 h; (j) iodine, KOH, DMF, 5 h; (k) di-tert-butyl dicarbonate, TEA, DCM, rt, 20 h; (l) phenylboronic acid,
3
Pd(dppf) Cl , Cs CO , DME−water; (m) NaNO , H SO , water; (n) EtOAc, NaH, 80 °C, 2 h; (o) diethyl malonate, NaOEt, ethanol, reflux, 15 h;
2
2
2
3
2
2
4
(
p) 15% NaOH, reflux, 4 h; (q) NaOAc, acetic acid, 120 °C, 20 h.
1
8
where t is time, [I] is the concentration of inhibitor, v and v are the
initial and steady-state velocities of the reaction in the presence of the
intermediate 43 ready for Buchwald coupling. In the case of N-
alkylated derivatives, intermediate 43 was reacted with the
i
s
inhibitor, kobsd is the apparent first-order rate constant for the
corresponding alkyl halide, such as CH I, in the presence of NaH to
3
app
interconversion between v and v , K is the apparent K , which is
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.
i
s
i
i
related to K by different functions based on the modes of inhibition,
i
and koff is the rate constant for dissociation of the enzyme−inhibitor
complex. Equation 2 was then substituted into eq 1 to give
vs − vi
off(1+([I]/K ia pp
[
P] = vt +
(1 − e−k
))t)
s
[
I]
koff 1 + Kapp
(
)
i
(3)
Equation 3 was then fitted to the fluorescence vs time data at varying
inhibitor concentration (see Figures 2 and 11), where v and v are the
s
i
2
,4-Dichloroquinoline (42, R = H) when treated with sodium
local parameters (one value each for every compound concentration)
1
app
methoxide in toluene under reflux conditions led to 4-chloro-2-
and koff and Ki are the global parameters (single value for the set of
1
9
methoxyquinoline (47) as a major product. 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),
compound concentrations).
Mass Spectrometry. Mass spectrometric determination of the
binding mode of aminoquinolones with DprE1 enzyme was performed
17
as described previously.
20
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 2,4-
which was then chlorinated by reaction with POCl
3
under reflux. 5-
Fluoroindazole (51) was iodinated by treatment with iodine under
2
1
basic conditions to give intermediate 52. 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-tert-
butyl dicarbonate to yield intermediate 53. Intermediate 53 could then
be processed using the general protocol to furnish compounds 13 and
18
dichloroquinoline intermediate 42. 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
5
430
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
3
7. The 3-phenylpyridone intermediate 56 was synthesized from 2-
4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
22
1
amino-4,6-dichloropyridine (54). 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-amino-
nicotinate (57) in the presence of NaH. 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)
yl)amino)-6,7-difluoroquinolin-2(1H)-one (3). Yield: 20%. H NMR
(300 MHz, DMSO-d ): δ 1.47 (d, J = 11 Hz, 2H), 1.91−2.11 (m, 2H),
6
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 C19H18ClF N O .
2 5 2
23
4-((1-((5-chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
1
yl)amino)-7-fluoroquinolin-2(1H)-one (4). Yield: 20%. H NMR (300
MHz, DMSO-d ): δ 1.50 (m, 2H), 1.89−2.12 (m, 2H), 2.91−3.06 (m,
6
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
C
H19ClFN O .
5 2
19
2
4
with diethyl malonate in the presence of sodium ethoxide.
Compound 40 was synthesized from intermediate 62 following the
general protocol.
4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
1
yl)amino)-8-fluoroquinolin-2(1H)-one (5). Yield: 39%. H NMR (300
MHz, DMSO-d ): δ 1.51 (q, J = 11 Hz, 2H), 1.99 (d, J = 11 Hz, 2H),
6
All commercial reagents and solvents were used without further
purification. (1R,3r,5S)-tert-butyl 3-amino-8-azabicyclo[3.2.1]octane-8-
carboxylate 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
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 C19
H
19ClFN
O
2
.
5
performed on SiO plates with aluminum backing. visualization was
4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
2
1
yl)amino)-7,8-difluoroquinolin-2(1H)-one (6). Yield: 20%. H NMR
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,
(
300 MHz, DMSO-d ): δ 1.49 (m, 2H), 1.88−2.09 (m, 2H), 2.94 (br
6
s, 1H), 3.22−3.31 (m, 1H), 3.76 (br s, 2H), 3.83 (s, 3H), 4.41 (br s,
230−400 mesh). The purity of all final derivatives for biological testing
1H), 5.48 (s, 1H), 6.68 (d, J = 7 Hz, 1H), 7.00−7.32 (m, 1H), 7.71 (s,
+
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
1H), 7.87 (br s, 1H), 10.76−11.11 (m, 1H). HRMS: m/z (ES ) =
+
422.12033 (MH ) for C19
H
18ClF
N
O
5
.
2
2
4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
1
yl)amino)-1-methylquinolin-2(1H)-one (7). Yield: 39%. H NMR
(
2
1
300 MHz, DMSO-d ): δ 1.35−1.64 (m, 2H), 2.00 (d, J = 10 Hz, 2H),
6
.95 (br s, 1H), 3.34 (s, 4H), 3.49 (s, 3H), 3.76 (br s, 2H), 4.43 (br s,
H), 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
1
+
+
products was confirmed by H NMR and mass spectroscopy. Proton
Hz, 1H). HRMS: m/z (ES ) = 400.15316 (MH ) for C20H22ClN O .
5 2
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.
4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
1
yl)amino)-1-ethylquinolin-2(1H)-one (8). Yield: 34%. H NMR (300
MHz, DMSO-d ): δ 1.13 (t, J = 7 Hz, 3H), 1.36−1.69 (m, 2H), 1.92−
6
2
4
.12 (m, 2H), 2.98−3.08 (m, 2H), 3.60−3.81 (m, 2H), 3.83 (s, 3H),
.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,
+
1
H), 7.71 (s, 1H), 8.07 (d, J = 8 Hz, 1H). HRMS: m/z (ES ) =
+
4
14.16918 (MH ) for C H ClN O .
21 24
5
2
4
-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
1
yl)oxy)quinolin-2(1H)-one (9). Yield: 26%. H NMR (300 MHz,
DMSO-d
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 chromatog-
raphy 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.
): δ 1.68−1.84 (m, 2H), 1.96−2.10 (m, 2H), 3.54 (br s,
6
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 C19H19ClN O .
4 3
4-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
1
yl)methylamino)quinolin-2(1H)-one (10). Yield: 10%. H NMR (300
MHz, DMSO-d ): δ 1.78 (br s, 4H), 2.70 (s, 3H), 3.05 (br s, 1H), 3.16
6
(d, J = 5 Hz, 1H), 3.64 (br s, 2H), 3.82 (s, 3H), 4.48 (br s, 1H), 5.88
4
-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
(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)
1
yl)amino)quinolin-2(1H)-one (1). Yield: 40%. H NMR (300 MHz,
+
+
DMSO-d ): δ 1.45−1.60 (m, 2H), 1.88−1.95 (m, 2H), 1.99−2.10 (m,
;HRMS: m/z (ES ) = 400.1531 (MH ) for C H ClN O .
6
20 22 5 2
2
1
7
H), 2.82−3.03 (m, 1H), 3.75 (br s, 1H), 3.83 (s, 3H), 4.40 (br s,
H), 5.45 (s, 1H), 6.53 (d, J = 7.35 Hz, 1H), 7.09 (t, J = 7 Hz, 1H),
.21 (d, J = 8 Hz, 1H), 7.35−7.50 (m, 1H), 7.71 (s, 1H), 7.98 (d, J = 8
(5-Chloro-1-methyl-1H-pyrazol-4-yl)(4-((2-methoxyquinolin-4-
1
yl)amino)piperidin-1-yl)methanone (11). Yield: 20%. H NMR (300
MHz, DMSO-d ): δ 1.59 (q, J = 11 Hz, 2H), 2.08 (d, J = 11 Hz, 2H),
6
+
+
Hz, 1H), 10.75 (br s, 1H). HRMS: m/z (ES ) = 386.13818 (MH ) for
3.42 (s, 3H), 3.90 (s, 4H), 3.95 (s, 3H), 4.50 (br s, 1H), 6.10 (s, 1H),
C H ClN O .
6.77 (d, J = 8 Hz, 1H), 7.25−7.41 (m, 1H), 7.55−7.69 (m, 2H), 7.78
19
20
5
2
+
+
4
-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
(s, 1H), 8.20 (d, J = 8 Hz, 1H). HRMS: m/z (ES ) = 400.1535 (MH )
1
yl)amino)-6-fluoroquinolin-2(1H)-one (2). Yield: 30%. H NMR (300
for C H ClN O .
20
22
5
2
MHz, DMSO-d ): δ 1.48−158 (m, 2H), 1.97−2.10 (m, 2H), 2.85−
(5-Chloro-1-methyl-1H-pyrazol-4-yl)(4-(cinnolin-4-ylamino)-
6
1
2
.95 (m, 2H), 3.25−3.32 (m, 1H), 3.74 (br s, 1H), 3.83 (s, 3H), 4.41
piperidin-1-yl)methanone (12). Yield: 10%. H NMR (300 MHz,
(
7
1
br s, 1H), 6.50 (d, J = 8 Hz, 1H), 5.49 (s, 1H), 7.16−7.27 (m, 1H),
DMSO-d ): δ 1.40−1.50 (m, 2H), 1.95−2.05 (m, 2H), 3.01 (br s,
6
.29−7.41 (m, 1H), 7.71 (s, 1H), 7.92 (d, J = 11 Hz, 1H), 10.85 (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,
+
+
H). HRMS: m/z (ES ) = 404.1286 (MH ) for C H ClFN O .
19
19
5
2
5
431
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
2
H), 8.10 (d, J = 8 Hz, 1H), 8.32 (d, J = 8 Hz, 1H), 8.83 (s, 1H).
4-((1-((1,5-Dimethyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-yl)-
+
+
1
HRMS: m/z (ES ) = 371.13831 (MH ) for C H ClN O.
amino)quinolin-2(1H)-one (22). Yield: 19%. H NMR (300 MHz,
18
19
6
(
5-Chloro-1-methyl-1H-pyrazol-4-yl)(4-((5-fluoro-1H-indazol-3-
DMSO-d ): δ 1.50 (d, J = 11 Hz, 2H), 1.89−2.03 (m, 3H), 2.31 (s,
6
1
yl)amino)piperidin-1-yl)methanone (13). Yield: 34%. H 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
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 C20H N O .
23 5 2
(
m, 1H), 7.49 (dd, J = 3, 9 Hz, 1H), 7.70 (s, 1H), 11.51 (s, 1H).
6-Fluoro-4-((1-((1-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)-
carbonyl)piperidin-4-yl)amino)quinolin-2(1H)-one (23). Yield: 38%.
+
+
HRMS: m/z (ES ) = 377.12945 (MH ) for C H ClFN O.
17
18
6
1
H NMR (300 MHz, DMSO-d ): δ 1.29−1.60 (m, 2H), 1.80−2.12
4
-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
6
1
yl)amino)-6-phenylpyridin-2(1H)-one (14). Yield: 33%. H NMR
300 MHz, DMSO-d ): δ 1.31−1.42 (m, 2H), 1.88−1.98 (m, 2H),
(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).
(
6
3
4
7
1
.05 (br s, 1H), 3.30−3.33 (m, 1H), 3.53 (br s, 2H), 3.82 (s, 3H),
.12−4.40 (m, 1H), 5.23 (s, 1H), 5.95 (s, 1H), 6.54 (d, J = 8 Hz, 1H),
.42−7.49 (m, 3H), 7.56−7.64 (m, 2H), 7.71 (s, 1H), 10.60 (br s,
+
+
HRMS: m/z (ES ) = 438.15605 (MH ) for C H F N O .
20 19
4
5
2
+
+
4
-((1-((4,5-Dimethylisoxazol-3-yl)carbonyl)piperidin-4-yl)amino)-
H). HRMS: m/z (ES ) = 412.15279 (MH ) for C H ClN O .
21
22
5
2
1
6
-fluoroquinolin-2(1H)-one (24). Yield: 33%. H NMR (300 MHz,
4
-((1-((5-chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)pyrrolidin-3-
1
DMSO-d ): δ 1.58 (m, 2H), 2.00 (s, 3H), 2.02−2.21 (m, 2H), 2.46 (s,
yl)amino)quinolin-2(1H)-one (15). Yield: 24%. H NMR (300 MHz,
6
3
5
(
H), 3.11 (m, 1H), 3.26−3.43 (m, 2H), 3.81 (m, 2H), 4.58 (m, 1H),
DMSO-d ): δ 2.09 (br s, 1H), 2.18−2.30 (m, 1H), 3.51−3.71 (m,
6
.59 (s, 1H), 6.61 (d, J = 8 Hz, 1H), 7.25−7.37 (m, 1H), 7.37−7.48
3
=
H), 3.79−3.86 (s, 3H), 3.91−4.07 (m, 1H), 4.19 (br s, 1H), 5.36 (d, J
+
m, 1H), 7.99 (d, J = 11 Hz, 1H). HRMS: m/z (ES ) = 385.1681
9 Hz, 1H), 6.79 (dd, J = 14, 6 Hz, 1H), 7.10 (t, J = 8 Hz, 1H), 7.21
+
(
MH ) for C H FN O .
(
dd, J = 8, 4 Hz, 1H), 7.44 (t, J = 8 Hz, 1H), 7.88 (d, J = 8 Hz, 1H),
20 21
4
3
+
4-((1-((1,5-Dimethyl-1H-1,2,3-triazol-4-yl)carbonyl)piperidin-4-
8.04 (dd, J = 12, 8 Hz, 1H), 10.85 (br s, 1H). HRMS: m/z (ES ) =
1
+
yl)amino)-6-fluoroquinolin-2(1H)-one (25). Yield: 35%. H NMR
300 MHz, DMSO-d ): δ 1.49−1.70 (m, 2H), 2.15 (s, 3H), 2.35−2.42
3
72.12198 (MH ) for C H ClN O .
18
18
5
2
(
6
4
-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)azetidin-3-
1
(m, 2H), 2.46 (s, 3H), 2.98−3.05 (m, 1H), 3.76 (br s, 1H), 3.96 (m,
yl)amino)quinolin-2(1H)-one (16). Yield: 20%. H NMR (300 MHz,
DMSO-d ): δ 3.82 (s, 3H), 4.10 (br s, 1H), 4.25 (d, J = 6 Hz, 1H),
2
7
(
H), 4.50 (d, J = 13 Hz, 1H), 5.51 (s, 1H), 6.39−6.67 (m, 1H), 7.15−
.45 (m, 2H), 7.81−8.05 (m, 1H), 10.84 (br s, 1H). HRMS: m/z
6
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 ) = 385.17815 (MH ) for C H FN O .
19 21 6 2
4
-((1-((5-Chloro-1-ethyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-yl)-
1
amino)-6-fluoroquinolin-2(1H)-one (26). Yield: 15%. H NMR (300
+
+
(
ES ) = 358.10808 (MH ) for C H ClN O .
17 16 5 2
MHz, DMSO-d ): δ 1.35 (t, J = 7 Hz, 3H), 1.41−1.62 (m, 2H), 1.98
6
4
-(((2S,4R)-1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-2-
1
(br s, 2H), 2.95 (br s, 1H), 3.17 (br s, 1H), 3.75 (br s, 2H), 4.18 (q, J
methylpiperidin-4-yl)amino)quinolin-2(1H)-one (17). Yield: 27%. H
=
7
7 Hz, 2H), 4.42 (br s, 1H), 5.50 (s, 1H), 6.51 (d, J = 8 Hz, 1H),
NMR (300 MHz, DMSO-d ): δ 1.20−1.50 (m, 2H), 1.32 (d, J = 9 Hz,
6
.14−7.28 (m, 1H), 7.29−7.42 (m, 1H), 7.74 (s, 1H), 7.92 (d, J = 11
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
+
+
Hz, 1H), 10.85 (br s, 1H). HRMS: m/z (ES ) = 418.13689 (MH ) for
C H ClFN O .
2
0
21
5
2
4
-((1-((5-Chloro-1-isopropyl-1H-pyrazol-4-yl)carbonyl)piperidin-
(
d, J = 8 Hz, 1H), 7.42 (t, J = 7 Hz, 1H), 7.69 (s, 1H), 7.97 (d, J = 8
1
4
-yl)amino)quinolin-2(1H)-one (27). Yield: 25%. H NMR (300
+
Hz, 1H), 10.75 (br s, 1 H). HRMS: m/z (ES ) = 400.15462 (MH + 1)
for C H ClN O .
MHz, DMSO-d ): δ 1.41 (d, J = 7 Hz, 6H), 1.44−1.62 (m, 2H), 2.00
6
20
22
5
2
(d, J = 11 Hz, 2H), 2.95 (br s, 1H), 3.09−3.31 (m, 1H), 3.74 (d, J = 6
4
-(((2S,4S)-1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-2-
methylpiperidin-4-yl)amino)quinolin-2(1H)-one (18). Yield: 14%.
H NMR (300 MHz, DMSO-d ): δ 0.76−0.89 (m, 2H), 1.22 (d, J
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−
1
=
6
7.51 (m, 1H), 7.75 (s, 1H), 7.98 (d, J = 8 Hz, 1H), 10.76 (s, 1H).
7 Hz, 4H), 1.78−1.96 (m, 3H), 3.76 (br s, 1H), 3.83 (s, 3H), 4.25
+
+
HRMS: m/z (ES ) = 414.16933 (MH ) for C H ClFN O .
21
24
5
2
(
(
1
d, J = 7 Hz, 1H), 5.31 (s, 1H), 6.48 (d, J = 6 Hz, 1H),), 7.03−7.26
4
-((1-((4,5-Dimethyl-1,2-oxazol-3-yl)carbonyl)piperidin-4-yl)-
1
m, 2H), 7.37−7.47 (m, 1H), 7.72 (s, 1H), 8.04 (d, J = 9 Hz,
amino)-6,7-difluoroquinolin-2(1H)-one (28). Yield: 41%. H NMR
+
H),10.82 (s, 1H). HRMS: (ES ) m/z = 400.15391 (MH + 1) for
(300 MHz, DMSO-d ): δ 1.41−1.67 (m, 2H), 1.99 (s, 4H), 2.45 (s,
6
C H ClN O .
3H), 2.98−3.19 (m, 1H), 3.34 (t, J = 12 Hz, 2H), 3.80 (d, J = 14 Hz,
20
22
5
2
4
-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8-
2H), 4.56 (d, J = 14 Hz, 1H), 5.56 (s, 1H), 6.61 (d, J = 8 Hz, 1H),
azabicyclo[3.2.1]octan-3-yl)amino)-6-fluoroquinolin-2(1H)-one
7
.22 (dd, J = 12, 8 Hz, 1H), 8.23 (dd, J = 13, 9 Hz, 1H), 10.9 (s, 1H).
1
(19). Yield: 23%. H NMR (300 MHz, DMSO-d ): δ 1.60 (m, 1H),
+
+
6
HRMS: m/z (ES ) = 403.15779 (MH ) for C H F N O .
20 20 2 4 3
1
4
7
3
.83 (m, 1H), 1.87−2.14 (m, 6H), 3.83 (s, 3H), 3.96−4.10 (m, 1H),
.23 (m, 1H), 4.60 (m, 1H), 5.50 (s, 1H), 6.45 (d, J = 8 Hz, 1H),
.14−7.26 (m, 1H), 7.26−7.42 (m, 1H), 7.76 (s, 1H), 7.90 (dd, J = 11,
4
-((1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-
yl)amino)-6,7-difluoro-1-methylquinolin-2(1H)-one (29). Yield: 72%.
1
H NMR (300 MHz, DMSO-d ): δ 1.37−1.59 (m, 2H), 1.88−2.10
6
+
+
Hz, 1H), 10.82 (s, 1H). HRMS: m/z (ES ) = 430.14482 (MH ) for
(
m, 2H), 2.96 (br s, 1H), 3.31 (br s, 1H), 3.41−3.55 (m, 3H), 3.63−
C H ClFN O .
21
21
5
2
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).
4
-(((1R,3s,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8-
azabicyclo[3.2.1]octan-3-yl)amino)-6-fluoroquinolin-2(1H)-one
+
+
HRMS: m/z (ES ) = 436.13505 (MH ) for C H ClF N O .
20
20
2
5
2
1
(20). Yield: 27%. H NMR (300 MHz, DMSO-d ): δ 1.21 (d, J = 7 Hz,
6
4-((1-((4,5-Dimethylisoxazol-3-yl)carbonyl)piperidin-4-yl)amino)-
1
1
H), 2.02−2.28 (m, 6H), 3.81 (br s, 1H), 3.87−3.94 (m, 4H), 4.22 (br
6
(
,7-difluoro-1-methylquinolin-2(1H)-one (30). Yield: 32%. H NMR
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 C H ClFN O .
300 MHz, DMSO-d ): δ 1.37−1.61 (m, 2H), 1.92 (s, 3H), 1.95−2.14
6
(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
+
+
21
21
5
2
4
-((1-((1-Methyl-1H-pyrazol-4-yl)carbonyl)piperidin-4-yl)amino)-
1
+
+
quinolin-2(1H)-one (21). Yield: 11%. H NMR (300 MHz, DMSO-
d ): δ 1.50 (d, J = 12 Hz, 2H), 1.99 (d, J = 12 Hz, 2H), 3.14−3.19 (m,
(
ES ) = 417.17333 (MH ) for C H F N O .
21 22 2 4 3
6
4-(((2S,4R)-1-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-2-
2
(
7
H), 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−
.47 (m, 1H), 7.66 (s, 1H), 7.98 (d, J = 8 Hz, 1H), 8.07 (s, 1H), 10.77
methylpiperidin-4-yl)amino)-6,7-difluoroquinolin-2(1H)-one (31).
1
Yield: 23%. H NMR (300 MHz, DMSO-d ): δ 1.23−1.48 (m, 4H),
6
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),
+
+
(
s, 1H). HRMS: m/z (ES ) = 352.16966 (MH ) for C H N O .
19
21
5
2
5
432
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
6.02 (d, J = 8 Hz, 1H), 8.22 (d, J = 2 Hz, 1H), 8.47 (d, J = 2 Hz, 1H).
+
+
+
(
dd, J = 13, 9 Hz, 1H), 10.9 (br s, 1H). HRMS: m/z (ES ) =
HRMS: m/z (ES ) = 409.19847 (MH ) for C H N O .
21 24 6 3
+
436.13509 (MH ) for C H ClF N O .
20 20 2 5 2
4
-((3-endo-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8-
ASSOCIATED CONTENT
Supporting Information
■
azabicyclo[3.2.1]oct-3-yl)amino)-6,7-difluoroquinolin-2(1H)-one
1
*
S
(
32). Yield: 74%. H NMR (300 MHz, DMSO-d ): δ 1.49−1.57 (m,
6
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.
1
H), 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 C H ClF N O .
+
21
20
2
5
2
4
-(((1R,3r,5S)-8-((4,5-Dimethylisoxazol-3-yl)carbonyl)-8-
azabicyclo[3.2.1]octan-3-yl)amino)-6,7-difluoroquinolin-2(1H)-one
1
(
1
33). Yield: 25%. H NMR (300 MHz, DMSO-d ): δ 1.76 (m, 2H),
6
.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:
AUTHOR INFORMATION
Corresponding Authors
*Phone: +91 9535500085. E-mail: neeladin@gmail.com.
Phone: +91 9880238194. E-mail: sandeepghorpade@hotmail.
=
■
+
+
m/z (ES ) = 429.17352 (MH ) for C H F N O .
22
22
2
4
3
4
-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8-
azabicyclo[3.2.1]octan-3-yl)amino)-6-fluoro-1-methylquinolin-
*
1
2
(
(
(1H)-one (34). Yield: 30%. H NMR (300 MHz, DMSO-d ): δ 1.61
6
com.
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,
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.
◆
J = 6 Hz, 2H), 7.76 (s, 1H), 7.98 (d, J = 11 Hz, 1H). HRMS: m/z
+
+
(
ES ) = 444.16024 (MH ) for C H ClFN O .
22
23
5
2
¶
4
-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8-
▲
azabicyclo[3.2.1]octan-3-yl)amino)-6,7-difluoro-1-methylquinolin-
1
2
1
3
1
8
(1H)-one (35). Yield: 28%. H NMR (300 MHz, DMSO-d ): δ 1.58−
6
.64 (m, 1H), 1.83−1.90 (m, 1H), 1.92−2.12 (m, 6H), 3.46 (s, 3H),
.83 (s, 3H), 3.96−4.13 (m, 1H), 4.24 (m, 1H), 4.60 (m, 1H), 5.66 (s,
H), 6.47 (d, J = 9 Hz, 1H), 7.54 (dd, J = 13, 7 Hz, 1H), 7.76 (s, 1H),
□
Notes
+
+
.22 (dd, J = 13, 9 Hz, 1H). HRMS: m/z (ES ) = 462.14315 (MH )
for C H ClF N O .
The authors declare no competing financial interest.
22
22
2
5
2
4
-((3-endo-8-((4,5-Dimethyl-1,2-oxazol-3-yl)carbonyl)-8-
ACKNOWLEDGMENTS
■
azabicyclo[3.2.1]oct-3-yl)amino)-6,7-difluoro-1-methylquinolin-
1
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 Bridgland-
Taylor 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 compound-
dispatch-related activities, and the members of the EU
consortium for constructive discussions during the meetings.
2
1
3
(1H)-one (36). Yield: 30%. H NMR (300 MHz, DMSO-d ): δ 1.67−
.86 (m, 2H), 1.97 (s, 6H), 2.03 (dd, J = 13, 6 Hz, 2H), 2.37 (s, 3H),
.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,
H), 8.23 (dd, J = 13, 9 Hz, 1H). HRMS: m/z (ES ) = 443.18928
MH ) for C H F N O .
6
+
1
(
+
23 24 2 4 3
(
5-Chloro-1-methyl-1H-pyrazol-4-yl)(3-endo-3-((5-fluoro-1H-in-
dazol-3-yl)amino)-8-azabicyclo[3.2.1]oct-8-yl)methanone (37).
1
Yield: 18%. H NMR (300 MHz, DMSO-d ): δ 1.23 (s, 4H), 1.42
6
(
2
(
1
br s, 1H), 1.55 (br s, 1H), 1.66 (br s, 1H), 1.99 (d, J = 16 Hz, 2H),
.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,
H), 7.43 (dd, J = 9, 2 Hz, 1H), 7.76 (s, 1H), 11.47 (s, 1H). HRMS:
+
+
m/z (ES ) = 403.14462 (MH ) for C H ClFN O.
19
20
6
4
-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8-
azabicyclo[3.2.1]octan-3-yl)amino)-6-phenylpyridin-2(1H)-one
1
(
1
4
(
1
38). Yield: 28%. H NMR (300 MHz, DMSO-d ): δ 1.35 (m, 1H),
6
.60 (m, 1H), 1.91 (m, 3H), 1.99 (m, 3H), 3.83 (s, 3H), 3.9 (m,1H),
.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,
H), 10.59 (br s, 1H) . HRMS: m/z (ES ) = 438.16935 (MH ) for
ABBREVIATIONS USED
■
+
+
TB, tuberculosis; Mtb, Mycobacterium tuberculosis; Msm,
Mycobacterium smegmatis; MIC, minimum inhibitory concen-
tration; 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, 1,2-
dimethoxyethane; THF, tetrahydrofuran; DMF, N,N-dimethyl-
formamide
C H ClN O .
2
3
24
5
2
4
-(((1R,3r,5S)-8-((5-Chloro-1-methyl-1H-pyrazol-4-yl)carbonyl)-8-
azabicyclo[3.2.1]octan-3-yl)amino)-1-methyl-1,8-naphthyridin-
1
2
1
3
1
1
4
(1H)-one (39). Yield: 32%. H NMR (300 MHz, DMSO-d ): δ 1.49−
6
.69 (m, 1H), 1.74−1.89 (m, 1H), 1.92−2.14 (m, 6H), 3.55 (s, 3H),
.83 (s, 3H), 3.97−4.12 (m, 1H), 4.17−4.34 (m, 1H), 4.49−4.70 (m,
H), 5.68 (s, 1H), 6.55−6.75 (m, 1H), 7.14−7.35 (m, 1H), 7.76 (s,
H), 8.39−8.52 (m, 1H), 8.53−8.65 (m, 1H). HRMS: m/z (ES ) =
27.16479 (MH ) for C H ClN O .
+
+
21
23
6
2
REFERENCES
8
-((3-endo-8-((4,5-Dimethyl-1,2-oxazol-3-yl)carbonyl)-8-
■
azabicyclo[3.2.1]oct-3-yl)amino)-5-methylpyrido[2,3-b]pyrazin-
(1) (a) Riccardi, G.; Pasca, M. R.; Chiarelli, L. R.; Manina, G.;
Mattevi, A.; Binda, C. The DprE1 Enzyme, One of the Most
Vulnerable Targets of Mycobacterium tuberculosis. Appl. Microbiol.
1
6
1
3
(5H)-one (40). Yield: 37%. H NMR (300 MHz, CDCl ): δ 1.82−
3
.92 (m, 4H), 1.9 (s, 3H), 2.01- 2.09 (m, 4H), 2.29 (s, 3H), 3.62 (s,
H), 3.85−4.10 (m, 1H), 4.65 (m, 1H), 4.89 (m, 1H), 5.84 (s, 1H),
̌ ́
Biotechnol. 2013, 97, 8841−8848. (b) D. Mikusova, K.; Makarov, V.;
5
433
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434

Journal of Medicinal Chemistry
Article
Neres, J. DprE1From the Discovery to the Promising Tuberculosis
Drug Target. Curr. Pharm. Des. 2013, DOI: 10.2174/
Extra Precision Glide: Docking and Scoring Incorporating a Model of
Hydrophobic Enclosure for Protein−Ligand Complexes. J. Med. Chem.
2006, 49, 6177−6196.
(13) Clarysse, S.; Brouwers, J.; Tack, J.; Annaert, P.; Augustijns, P.
Intestinal Drug Solubility Estimation Based on Simulated Intestinal
Fluids: Comparison with Solubility in Human Intestinal Fluids. Eur. J.
Pharm. Sci. 2011, 43, 260.
1
(
381612819666131118163721.
2) Neres, J.; Pojer, F.; Molteni, E.; Chiarelli, L. R.; Dhar, N.; Boy-
Rottger, S.; Buroni, S.; Fullam, E.; Degiacomi, G.; Lucarelli, A. P.;
̈
Read, R. J.; Zanoni, G.; Edmondson, D. E.; De Rossi, E.; Pasca, M. R.;
McKinney, J. D.; Dyson, P. J.; Riccardi, G.; Mattevi, A.; Cole, S. T.;
Binda, C. Structural Basis for Benzothiazinone-Mediated Killing of
Mycobacterium tuberculosis. Sci. Transl. Med. 2012, 4, 150ra121.
(14) Sohlenius-Sternbeck, A.-K.; Jones, C.; Ferguson, D.; Middleton,
B. J.; Projean, D.; Floby, E.; Bylund, J.; Afzelius, L. Practical Use of the
Regression Offset Approach for the Prediction of in Vivo Intrinsic
Clearance from Hepatocytes. Xenobiotica 2012, 42, 841−853.
(15) The secondary pharmacology was conducted at CEREP, France.
(16) Copeland, R. A. Time-Dependent Inhibition. In Enzymes: A
Practical Introduction to Structure, Mechanism, and Data Analysis, 2nd
ed.; Copeland, R. A., Ed.; Wiley-VCH: New York, 2000; pp 318−349.
(
̈
3) Makarov, V.; Manina, G.; Mikusova, K.; Mollmann, U.; Ryabova,
O.; Saint-Joanis, B.; Dhar, N.; Pasca, M. R.; Buroni, S.; Lucarelli, A. P.;
Milano, A.; De Rossi, E.; Belanova, M.; Bobovska, A.; Dianiskova, P.;
Kordulakova, J.; Sala, C.; Fullam, E.; Schneider, P.; McKinney, J. D.;
Brodin, P.; Christophe, T.; Waddell, S.; Butcher, P.; Albrethsen, J.;
Rosenkrands, I.; Brosch, R.; Nandi, V.; Bharath, S.; Gaonkar, S.;
Shandil, R. K.; Balasubramanian, V.; Balganesh, T.; Tyagi, S.; Grosset,
J.; Riccardi, G.; Cole1, S. T. Benzothiazinones Kill Mycobacterium
tuberculosis by Blocking Arabinan Synthesis. Science 2009, 324, 801−
̌
́ ́
(17) Trefzer, C.; Skovierova, H.; Buroni, S.; Bobovska, A.; Nenci, S.;
Molteni, E.; Pojer, F.; Pasca, M. R.; Makarov, V. Benzothiazinones Are
Suicide Inhibitors of Mycobacterial Decaprenylphosphoryl-β-D-ribo-
furanose 2′-Oxidase DprE1. J. Am. Chem. Soc. 2012, 134, 912−915.
8
(
04.
4) Batt, S. M.; Jabeena, T.; Bhowrutha, V.; Quilla, L.; Lunda, P. A.;
Eggeling, L.; Alderwicka, L. J.; Futterera, K.; Besra, G. S. Structural
(18) Ramasamy, A. K.; Balasubramaniam, V.; Mohan, K. Synthesis
and Characterization of Substituted 4-Methoxy-1H-quinolin-2-ones. E-
̈
J. Chem. 2010, 7, 1066−1070.
Basis of Inhibition of Mycobacterium tuberculosis DprE1 by
Benzothiazinone Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 2012, 109,
(19) Osborne, A. G.; Miller, L. A. D. Regioselective Alkoxydehaloge-
nation of 2,4-Dihalogenoquinololines, a Reinvestigation of the
Bromination of 2-Methoxyquinoline. J. Chem. Soc., Perkin Trans. 1
1
(
1354−11359.
5) Pasca, M. R.; Degiacomi, G.; Ribeiro, A. L.; Zara, F.; De Mori, P.;
1
(
993, 181−184.
Heym, B.; Mirrione, M.; Brerra, R.; Pagani, L.; Pucillo, L.; Troupioti,
P.; Makarov, V.; Cole, S. T.; Riccardi, G. Clinical Isolates of
Mycobacterium tuberculosis in Four European Hospitals are Uniformly
Susceptible to Benzothiazinones. Antimicrob. Agents Chemother. 2010,
20) Arrington, M. P.; Liu, R.; Conticello, R. D.; Gauss, C. M.;
Hopper, A.; Nguyen, T. M.; Tehim, A. 4-Substituted 4,6-
Dialkoxycinnoline Derivatives as Phosphodiesterase 10 Inhibitors for
the Treatment of Psychatric or Neurological Syndromes. WO2006/
5
(
4, 1616−1618.
0
28957 A1.
6) (a) Makarov, V.; Lechartier, B.; Zhang, M.; Neres, J.; van der Sar,
(21) Bondy, S. S.; Cannizzaro, C. E.; Chou, C-h.; Halcomb, R. L.;
A. M.; Raadsen, S. A.; Hartkoorn, R. C.; Ryabova, O. B.; Vocat, A.;
Decosterd, L. A.; Widmer, N.; Buclin, T.; Bitter, W.; Andries, K.; Pojer,
F.; Dyson, P. J.; Cole, S. T. Towards a New Combination Therapy for
Tuberculosis with Next Generation Benzothiazinones. EMBO Mol.
Med. 2014, 6, 372−383. (b) Cole, S.; Makarov, V. 2-Piperazin-1-yl-4H-
Hu, Y. E.; Link, J. O.; Liu, Q.; Schroeder, S. D.; TSE, W. C.; Zhang, J.
R. Compounds for the Treatment of HIV. WO 2013/006738.
(22) Player, M. R.; Calvo, R.; Chen, J.; Meegalla, S.; Valley, G. Parks,
D.; Parsons, W.; Ballentine, S.; Branum, S. Substituted Aza-Bicyclic
Imidazole Derivatives Useful as TRPM8 Receptor Modulators. US
1,3-benzothiazin-4-one Derivatives and Their Use for The Treatment
2
(
011/0218197.
23) Cao, S.; Popovici-Muller, J.; Salituro, F. G.; Saunders, J.; Tan,
of Mammalian Infections. WO2012066518 A1, May 24, 2012. (c)
Stop TB Partnership. Drug PipelinePBTZ 169. http://www.
newtbdrugs.org/project.php?id=145554 (Accessed March 10, 2014).
X.; Travins, J.; Yan, S. Ye, Z. Therapeutically Active Compositions and
Their Methods of Use. WO 2012/171337.
(
7) Wang, F.; Sambandan, D.; Halder, R.; Wang, J.; Batt, S. M.;
(24) Carter, N. B.; Cordingley, M. R.; Crowely, P. J.; Turnbull, M. D.
Weinrick, B.; Ahmad, I.; Yang, P.; Zhang, Y.; Kim, J.; Hassani, M.;
Huszar, S.; Trefzer, C.; Ma, Z.; Kaneko, T.; Mdluli, K. E.; Franzblau,
Pyrido[2,3-b]pyrazine Derivatives Useful as Herbicidal Compounds.
WO 2008/009908.
̈
S.; Chatterjee, A. K.; Johnson, K.; Mikusova, K.; Besra, G. S.; Futterer,
K.; Jacobs, W. R., Jr.; Schultz, P. G. Identification of a Small Molecule
with Activity against Drug-Resistant and Persistent Tuberculosis. Proc.
Natl. Acad. Sci. U.S.A. 2013, 110, E2510−E2517.
(
8) Shirude, P. S.; Shandil, R.; Sadler, C.; Naik, M.; Hosagrahara, V.;
Hameed, S.; Shinde, V.; Bathula, C.; Humnabadkar, V.; Naveen
Kumar, C. N.; Reddy, J.; Panduga, V.; Sharma, S.; Ambady, A.; Hegde,
N.; Whiteaker, J.; McLaughlin, R. E.; Gardner, H.; Madhavapeddi, P.;
Ramachandran, V.; Kaur, P.; Narayan, A.; Guptha, S.; Awasthy, D.;
Narayan, C.; Mahadevaswamy, J.; Vishwas, K. G.; Ahuja, V.; Srivastava,
A.; Prabhakar, K. R.; Bharath, S.; Kale, R.; Ramaiah, M.; Roy
Choudhury, N.; Sambandamurthy, V. K.; Solapure, S.; Iyer, P. S.;
Narayanan, S.; Chatterji, M. Azaindoles: Noncovalent DprE1
Inhibitors from Scaffold Morphing Efforts, Kill Mycobacterium
tuberculosis and Are Efficacious in Vivo. J. Med. Chem. 2013, 56,
9
(
701−9708.
9) Copeland, R. A.; Pompliano, D. L.; Meek, T. D. Drug−Target
Residence Time and Its Implications for Lead Optimization. Nat. Rev.
Drug Discovery 2006, 5, 730−740.
(
10) Wakamoto, Y.; Dhar, N.; Chait, R.; Schneider, K.; Signorino-
Gelo, F.; Leibler, S.; McKinney, J. D. Dynamic Persistence of
Antibiotic-Stressed Mycobacteria. Science 2013, 39, 91−95.
(
11) Small-Molecule Drug Discovery Suite 2013-3: Glide, version 6.1;
Schro
12) Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.;
Greenwood, J. R.; Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T.
̈
dinger, LLC: New York, 2013.
(
5
434
dx.doi.org/10.1021/jm5005978 | J. Med. Chem. 2014, 57, 5419−5434