Novel N-linked aminopiperidine-based gyrase inhibitors with improved hERG and in vivo efficacy against mycobacterium tuberculosis

Article abstract of DOI:10.1021/jm500432n

DNA gyrase is a clinically validated target for developing drugs against Mycobacterium tuberculosis (Mtb). Despite the promise of fluoroquinolones (FQs) as anti-tuberculosis drugs, the prevalence of pre-existing resistance to FQs is likely to restrict their clinical value. We describe a novel class of N-linked aminopiperidinyl alkyl quinolones and naphthyridones that kills Mtb by inhibiting the DNA gyrase activity. The mechanism of inhibition of DNA gyrase was distinct from the fluoroquinolones, as shown by their ability to inhibit the growth of fluoroquinolone-resistant Mtb. Biochemical studies demonstrated this class to exert its action via single-strand cleavage rather than double-strand cleavage, as seen with fluoroquinolones. The compounds are highly bactericidal against extracellular as well as intracellular Mtb. Lead optimization resulted in the identification of potent compounds with improved oral bioavailability and reduced cardiac ion channel liability. Compounds from this series are efficacious in various murine models of tuberculosis.

Full text of DOI:10.1021/jm500432n

Article
pubs.acs.org/jmc
Novel N‑Linked Aminopiperidine-Based Gyrase Inhibitors with
Improved hERG and in Vivo Efficacy against Mycobacterium
tuberculosis
,
†
†
§
‡
†
‡
Shahul Hameed P,* Vikas Patil, Suresh Solapure, Umender Sharma, Prashanti Madhavapeddi,
†
†
†
Anandkumar Raichurkar, Murugan Chinnapattu, Praveena Manjrekar, Gajanan Shanbhag,
†
†
†
†
†
Jayashree Puttur, Vikas Shinde, Sreenivasaiah Menasinakai, Suresh Rudrapatana, Vijayashree Achar,
‡
‡
‡
‡
‡
Disha Awasthy, Radha Nandishaiah, Vaishali Humnabadkar, Anirban Ghosh, Chandan Narayan,
‡
‡
‡
#
#
§
V. K. Ramya, Parvinder Kaur, Sreevalli Sharma, Jim Werngren, Sven Hoffner, Vijender Panduga,
§
§
§
§
§
C. N. Naveen Kumar, Jitendar Reddy, Mahesh Kumar KN, Samit Ganguly, Sowmya Bharath,
†
‡
§
§
∥
Ugarkar Bheemarao, Kakoli Mukherjee, Uma Arora, Sheshagiri Gaonkar, Michelle Coulson,
⊥
‡
‡
David Waterson, Vasan K. Sambandamurthy, and Sunita M. de Sousa
†
‡
§
Department of Medicinal Chemistry; Department of Biosciences and DMPK and Animal Sciences, AstraZeneca India Pvt. Ltd.,
Bellary Road, Hebbal, Bangalore 560024, India
∥
Safety Assessment, AstraZeneca, Alderley Park, Macclesfield, U.K.
*
S Supporting Information
ABSTRACT: DNA gyrase is a clinically validated target for developing drugs against Mycobacterium tuberculosis (Mtb). Despite
the promise of fluoroquinolones (FQs) as anti-tuberculosis drugs, the prevalence of pre-existing resistance to FQs is likely to
restrict their clinical value. We describe a novel class of N-linked aminopiperidinyl alkyl quinolones and naphthyridones that kills
Mtb by inhibiting the DNA gyrase activity. The mechanism of inhibition of DNA gyrase was distinct from the fluoroquinolones,
as shown by their ability to inhibit the growth of fluoroquinolone-resistant Mtb. Biochemical studies demonstrated this class to
exert its action via single-strand cleavage rather than double-strand cleavage, as seen with fluoroquinolones. The compounds are
highly bactericidal against extracellular as well as intracellular Mtb. Lead optimization resulted in the identification of potent
compounds with improved oral bioavailability and reduced cardiac ion channel liability. Compounds from this series are
efficacious in various murine models of tuberculosis.
INTRODUCTION
Clinically validated targets of known antimycobacterial agents
offer a great promise in terms of designing novel inhibitors that
are likely to be bactericidal under the in vitro and in vivo
growth conditions. Among the extensively studied bacterial
targets, DNA gyrase, belonging to the type II topoisomerase
family, has been validated clinically by the fluoroquinolone
■
Tuberculosis (TB) continues to be a major global health
problem that claims ∼1.5 million lives each year and imposes a
huge economic burden on developing countries. This problem
is further aggravated by the emergence of drug-resistant strains
of Mtb that require up to 2 years of treatment with a
combination of six to eight drugs.
The availability of the genome sequence of Mtb has
propelled a huge effort toward target-based discovery of
1
5a,b
2
(
FQ) class of drugs.
Combination regimens containing
moxifloxacin have shown the potential to shorten the duration
5c
of TB treatment. However, pre-existing resistance to FQs is
3
antimycobacterial compounds. However, numerous efforts
aimed at the essential targets in Mtb have highlighted many4
Received: March 19, 2014
inherent challenges in identifying attractive chemical leads.
©
XXXX American Chemical Society
A
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Figure 1. Non-fluoroquinolone-based gyrase inhibitors reported in the literature.
Figure 2. Structures of initial leads (1a, 2b) and compounds with monocyclic RHS (4, 5).
1
2,13
likely to limit the clinical utility of this class of drugs for treating
TB in the long run.
FQs bind to the GyrA subunit of bacterial DNA gyrase and
model of TB.
Representative structures of the GyrA class,
5d
9−11
N-linked aminopiperidines (1a, 1b, 2a, and 2b) 1
from
from
2,13
AstraZeneca, and NBTIs (3a, 3b, 3c, 4a, and 4b)
trap the double strand cleaved DNA−gyrase complex, inducing
GSK are shown in Figure 1.
6
an SOS response that leads to bacterial cell death. A novel
The chemical structures of NBTIs including the N-linked
piperidines consist of three parts, namely, a bicyclic aromatic
left-hand side (LHS), a mono or bicyclic right-hand side
(RHS), and a linker region joining the RHS and LHS.
The mechanism of inhibition and the binding mode of
NBTIs for the antibacterial series have been reported. From
inhibitor of the DNA gyrase with a unique binding site is likely
to have activity against FQ-resistant TB. Numerous non-
fluoroquinolone-based bacterial type II topoisomerase inhib-
itors from both the GyrA and GyrB classes have been reported
7
8a
as antibacterial agents. Inhibitors of the DNA GyrB class
1
8a
18b
18c,e
include bisthiazoles, aminopyrazinamides,
and pyrrolamides,
aryl ureas,
the crystal structure studies of methoxyquinoline-3-carbonitrile
1
8d
8a
whereas inhibitors of the GyrA class
(GSK299423, 4a), one can infer that the LHS portion of the
include aminopiperidine-based novel bacterial type II topo-
isomerase inhibitors (NBTIs). AstraZeneca has reported novel
N-linked aminopiperidines as potent inhibitors of the bacterial
NBTIs binds to the DNA substrate, whereas the RHS portion
interacts with the protein dimer interface of the GyrA subunits.
The binding site of NBTIs is distinct from the FQ binding, and
they are reported to be active against FQ-resistant strains of
9
−11
type II topoisomerases with antibacterial activity.
Glax-
8a
oSmithKline (GSK) has reported antibacterial and anti-TB
Staphylococcus aureus. However, the mode of binding of
activity for NBTIs with in vivo efficacy in the acute mouse
NBTIs to the Mtb GyrA subunit is not known. As the active
B
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a
Scheme 1
a
Reagents: (a) Na CO , DMF, 70 °C; (b) 4 N HCl in dioxane, 55 °C; (c) NaBH CN, DCE or CHCl /EtOH, 65−80 °C; (d) NaOMe, MeOH, 75
2
3
3
3
°C; (e) 33, HATU, DIPEA, DMF, rt; (f) ZnCN , Zn(OAc) , Zn, Pd (dba) , dppf, DMF, 100 °C, 23%; (g) Pd (dba) , dppf, CuCN, DMF, 110 °C,
2 2 2 3 2 3
6
8%.
a
Scheme 2
a
Reagents: (a) MeI, Cs CO , DMSO, 60 °C, 83%; (b) 48% HBr, 80 °C, > 95%; (c) 2-bromoethanol, Cs CO , DMSO, 95 °C, >95%; (d) MsCl,
2
3
2
3
TEA, DCM, 0 °C, >95%; (e) 29e, Na CO , DMF, 70 °C, 98%.
2
3
site of DNA gyrases is highly conserved across the bacterial
species, we assumed that the binding mode could be similar to
what has been reported for S. aureus DNA gyrase.
aminopiperidines with a bicyclic RHS and an optimized linker
1
0,11
was shown to have lesser hERG liability.
Therefore, we
8b
screened a library of N-linked aminopiperidines against Mtb
and identified lead compounds that inhibited Mtb DNA gyrase
activity as well as the growth of Mtb in broth (Figure2). Herein,
we report medicinal chemistry efforts to reduce the hERG
liability and improve the Mtb MIC as well as the oral
bioavailability of these lead molecules. Additionally, we also
describe the mechanism of inhibition of Mtb gyrase, the
The NBTIs with a monocyclic RHS (3a−3c) display potent
minimum inhibitory concentration (MIC) against FQ-sensitive
and FQ-resistant strains of Mtb and were found to be
13a
efficacious in the acute mouse model of tuberculosis.
However, these compounds are also potent inhibitors of the
1
3b
cardiac hERG channel.
A different class of N-linked
C
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a
Scheme 3
a
Reagents: for R4 = CHO, (a) NaBH(OAc) , DMF, 70 °C; for R4 = −CH −OMs, (b) Na CO , DMF, 70 °C; (c) NaOMe, MeOH, 75 °C; (d) 4 N
3
2
2
3
HCl in dioxane, 55 °C or TFA/DCM, rt.
Table 1. Properties of the Initial Lead Compounds (1a, 2a, 2b) and the Novel Compounds with a Monocyclic RHS (4 and 5)
properties
Mtb MIC (μg/mL)
1a
2a
2b
0.13
4
5
2
0.25
0.150
>100
0.8
>16
0. 06
0.25
Mtb gyrase supercoiling IC50 (μM)
Msm GyrB ATPase IC50 (μM)
log D pH 7.4
1.05
>100
0.7
0.110
>100
0.4
>100
>100
1.1
>100
1.3
hERG IC50 (μM)
44
21
ND
5.3
26
−6
Caco-2: Papp A−B/B−A (1 × 10 cm/s)
1.9/38.3
1.1/25
2.0/12.6
17.1/9.1
13.6/7.4
characterization of spontaneous resistant mutants, and the
efficacy in the acute and chronic mouse model of TB.
Compound 8 was synthesized as described in Scheme 2. The
intermediate naphthyridone 24c was N-methylated with methyl
iodide in the presence of cesium carbonate to generate
intermediate 8-ii. O-Demethylation of 8-ii with HBr provided
the naphthyridine-2,6-dione 8-iii, which upon alkylation with 2-
bromoethanol gave both the N- and O-alkylated isomers. The
N-alkylated isomer 8-iv was converted to mesylate 8-v with
MsCl under basic conditions. The alkylation of 29e with
mesylate 8-v under basic conditions resulted in compound 8.
The synthesis of compounds with substituted amino-
piperidine linker is shown in Scheme 3. The RHS aldehyde
CHEMISTRY
■
The synthesis of compounds with unsubstituted amino-
piperidine and methoxy- or fluoro-substituted aminopiperidine
linker is described in Schemes 1,2, and 3, respectively. The
alkylation of the LHS (24a−h) with mesylate 25 in the
presence of a base resulted in the formation of N-alkylated
product (26a−i) as the major isomer along with a small
amount of O-alkylated product. Wherever possible, the minor
1
6
30 was synthesized as reported earlier. The reductive
amination of the modified aminopiperidine linkers (29a−d)
with the LHS aldehyde (28a) or alkylation with mesylates
(28b,c) in the presence of a base provided the final compounds
11
O-alkylated isomer was removed by column chromatography;
otherwise, the mixture was taken as such for N-Boc
deprotection. The free amines (27a−i) were obtained upon
N-Boc deprotection of the intermediates 26a−i in the presence
of an acid. Compounds 4−14 were synthesized from the
amines 27a−i either by reductive amination with RHS
aldehydes (31, 34), by amide coupling with RHS acid (33),
or by alkylation with mesylates (32, 35) (Scheme 1). The
mesylate 25 and LHS fragments 24a and 24f were synthesized
(
1
15−17, 19−21) in moderate to good yields. The compounds
8 and 23 were obtained by alkylation of 18b with mesylate 32
and reductive amination of 23b with aldehyde 30, respectively.
Synthesis of all the intermediates involved in Schemes 1−3 are
described in the Supporting Information.
1
0,11
as reported by Reck et al.,
whereas LHS fragments 24b and
RESULTS AND DISCUSSION
■
2
4c were synthesized using an optimized Heck protocol as
Identification of Lead Molecules. A focused library of N-
linked aminopiperidines, synthesized during the antibacterial
drug discovery program, was screened for MIC against
replicating Mtb in broth. This screening effort led to the
identification of compounds 1a, 2a, and 2b with submicromolar
MIC against Mtb. Properties of the representative lead
molecules are shown in Table 1.
1
4
described earlier. The synthesis of 24d as described in
Scheme 6 (Supporting Information). The LHS 24h was
synthesized from its fluoro precursor 24g using NaOMe.
15
Detailed synthesis of the precursor aldehydes 31 and 34, the
mesylates 32 and 35, and acid 33 are described in Schemes 7
and 8 (Supporting Information).
D
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As shown in Table 1, the compounds 1a, 2a, and 2b
displayed low micromolar or submicromolar Mtb MIC along
with potent IC50 in the Mtb DNA gyrase supercoiling (SC)
assay. The specific activity of the Mtb enzyme was too low to
set up a specific assay for ATPase activity of Mtb set up a
18b
specific assay for ATPase activity of Mtb GyrB. Therefore,
we have used Mycobacterium smegmatis (Msm) GyrB as a
18b
surrogate for the Mtb enzyme.
None of the initial leads
inhibited the ATPase activity of Msm GyrB, suggesting that the
holoenzyme (GyrA2B2 complex) or GyrA was inhibited. Initial
leads inhibited the hERG channel and had poor Caco2
permeability. Lead optimization efforts were primarily focused
on improving the Mtb MIC and Caco2 permeability and
reducing the hERG liability. During lead optimization, we
tracked the SAR based on Mtb MIC assuming that the
mechanism of gyrase inhibition was retained across newly
synthesized compounds in this series.
Lead Optimization and Hypothesis Generation. We
hypothesized that reducing the number of hydrogen-bonding
groups while keeping log D in the range of 1−2 would improve
the Caco-2 permeability and maintain MIC. Compounds 4 and
5
having disubstituted pyridines with hydrophilic and electron-
withdrawing cyano substitution were designed by opening the
bicyclic RHS ring. A shape-based overlay of the newly designed
analogues (4 and 5) with the initial leads (1a and 2a in Figure
1
9
2
) using the ROCS tool suggested that the overall shape of
molecules on the LHS or the linker region was similar (Figure
).
The RHS fragment with the 3-pyridyl ring in compound 5
3
fully occupies the shape of the bicyclic RHS (1a and 2b),
thereby suggesting that their binding mechanism could be
similar. In the case of compound 4, the RHS with 2-pyridyl ring
did not fully occupy the shape of the bicyclic RHS ring (1a and
2
b) of the initial leads. Hence, compound 4 may have different
behavior with respect to the enzyme potency. On the basis of
this hypothesis, compounds 4 and 5 were synthesized and
tested. Compound 5 maintained good enzyme potency and
MIC, whereas as compound 4 lost both of these activities.
Due to the nonavailability of a cocrystal structure of Mtb
gyrase, we docked compounds 4 and 5 into the published
crystal structure of S. aureus DNA bound GyrA subunit (PDB
8
a
ID 2XCS). Subsequently, a Mtb DNA gyrase subunit A (Mtb
GyrA) homology model was built on 2XCS as a template using
2
0
21
MOE. FRED docking tool was used understand the binding
interactions. Parts b,c and d of Figure 3 show the possible
binding modes of compounds 4 and 5 in the DNA-bound GyrA
subunit of S. aureus and in the Mtb GyrA model, respectively.
Docking analysis suggested that the LHS quinolone of
compounds 4 and 5 were positioned between the two base
pairs of DNA, as observed for NBTIs, whereas the RHS
pyridine binding showed a subtle difference with respect to the
methyl group orientation. The methyl substitution in the RHS
of compound 5 was oriented toward the hydrophobic region
occupied by Ala68, Val71, Gly72, and Met121 of the DNA-
bound GyrA subunit of S. aureus (Ala74, Val77, Ala78, and
Met127 in the Mtb GyrA, respectively) making several van der
Waals contacts. The methyl group also makes a stronger
interaction with the carbonyl of Ala74 and methyl of Ala78 in
Mtb. On the contrary, the methyl group of the RHS pyridine in
compound 4 was oriented away from the hydrophobic region
Figure 3. (a) Overlay of the initial leads with compounds containing a
novel monocyclic RHS; 4 (yellow) on 1a (green) and 2a (orange) (A)
and 5 (cyan) on 1a and 2a (B). (b) Docking pose of compound 4
(
yellow) and 5 (cyan) in the crystal structure of S. aureus DNA gyrase
8a
bound to 4a (green). (c) Docking pose of compound 4 (yellow) and
(cyan) on to the homology model of Mtb DNA gyrase subunit A.
d) Molecular surface of the active site depicting the RHS of
5
(
compound 4 and 5. The LHS part is not shown in part d for clarity
purposes.
(
Figure 3d) and the nitrile group was in close proximity to Ala
4. Additionally, the −NH group of the aminopiperidine ring in
compound 5 makes a key interaction with the carboxylate
7
group of Asp83 of the S. aureus DNA bound GyrA (Asp 89 in
Mtb GyrA). This observation was consistent with the binding
E
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8
a
mode of 4a. On the other hand, the −NH group’s hydrogen
in compound 4 was 4.1 Å away from the oxygen atom of the
carboxylate group in Asp83 (in the homology model of Mtb
GyrA, it was 4.7 Å away from Asp89), which is unlikely to make
a strong hydrogen-bonding interaction with the Asp83 residue.
Thus, the combination of loss of hydrophobic interaction and a
weak hydrogen-bonding contact with the Asp 83 may partially
explain the loss of potency for compound 4.
Table 2. SAR Modification around RHS and LHS Fragments
Compounds 4 and 5 also displayed improved Caco-2
permeability over the bicyclic RHS analogue (1a, 2a, and
2
1
2
5
b). Although, compounds 4 and 5 had similar log D (1.1−
.3), compound 5 showed moderate hERG inhibition (IC50
6 μM), whereas compound 4 was more potent (hERG IC =
=
50
.3 μM) against the hERG channel. The improved hERG
selectivity seen for compound 5 could be due to the
substitution pattern of CH and cyano groups on the RHS
3
ring. On the basis of the promising profile of compound 5, we
fixed 2-methyl-3-cyanopyridine as the RHS and started
exploring the SAR on the LHS. Replacement of the LHS
cyanoquinolone ring with 1,5-naphthyridone resulted in a 2−3-
fold improvement in the hERG liability, while maintaining the
MIC and Caco-2 permeability (compounds 6 and 7 in Table
2). Compound 7 showed a 4-fold improvement in MIC (0.06
μg/mL) as compared to the starting lead 2a. Moving the
methoxy group to the 6-position of the LHS 1,5-naphthyridone
weakened the MIC (compounds 7 and 7a shown as a match
pair in Table2), whereas converting compound 7a to a 1,5-
naphthyridinedione form (compound 8) completely abolished
the antimycobacterial activity, suggesting that the substitution
at the 7-position of the LHS ring was essential for retaining
MIC. The replacement of the 1,5-naphthyridone LHS ring with
a 5-cyano-7-methoxyquinolone (compound 9) improved the
Mtb MIC to 0.01 μg/mL at the cost of hERG selectivity. The
best MIC observed for compound 9 could be due to the
improved binding to enzyme (supercoiling assay IC50 = 0.065
μM) or better cell permeability driven by higher lipophilicity.
Changing the alkyl bridge of the RHS into an amide was
tolerated for both the Mtb MIC and hERG selectivity
(compound 7 vs 10 in Table 2). Introduction of additional
a
Not determined.
nitrogen in the RHS ring (pyridine to pyrazine) retained the
hERG IC50 but weakened the MIC by 15-fold (compound 7
versus 12 in Table 2).
monocyclic pyridine RHS with a bicyclic RHS along with a
fluoro-substituted linker also showed a similar trend in the
hERG selectivity (Table 3) without compromising the Mtb
MIC. It was also observed that a fluoro-substituted linker
modification with a combination of pyridoxazinone RHS and 6-
fluoro 1,5-naphthyridone LHS retained the Caco-2 perme-
ability (19 and 20 in Table 2). This could be due to the better
permeability of the fluoro-substituted linker and the LHS part.
Structure−Property Relationship for the in Vivo
Clearance and Oral Bioavailability in Rats. In general, a
majority of compounds from this series showed good solubility
(>1000 μM) at pH 7.4. The initial lead compound 2b with a
bicyclic RHS fragment showed 11% oral bioavailability in rats,
probably due to poor Caco-2 permeability and high clearance.
High clearance for compound 2b (∼2.5 times higher than rat
liver blood flow) suggested an extrahepatic route of elimination
in the rats (Table 4). Replacement of the bicyclic RHS
fragment with a monocyclic 2-methyl-3-cyanopyridine fragment
in compounds 5 and 9 improved the oral bioavailability.
Replacing the quinolone LHS ring with 1,5-naphthyridone and
introduction of a fluoro substitution in the linker region also
showed a similar trend (compounds 7, 16, and 17 in Table 4).
To further improve the hERG selectivity, we explored a
pyridopyrazinone ring on the LHS (compounds 13 and 14 in
Table 2). Compound 14 with a pyrido[2,3-b]pyrazin-2(1H)-
one improved the hERG inhibition (IC50 > 100 μM), and
compound 13 with a pyrido[2,3-b]pyrazin-3(4H)-one lost the
hERG selectivity (IC50 = 13 μM). The higher hERG potency
observed for compound 13 could be due to the high log D and
the enhanced π-stacking interaction with the hERG channel.
Overall, the reduction of hERG liability for this series tracked
well with reduced lipophilicity (lower log D) as reported earlier
1
0
for the antibacterial N-linked aminopiperidine series.
Modulation of the pK of aminopiperidine linker through a
a
substitution at the 3-position of piperidine moiety with fluoro
11
substitution lowered the hERG inhibition. Adopting a similar
strategy, a 2−3-fold improvement in hERG inhibition was
achieved by introducing a fluoro or methoxy group at the 3-
position of the aminopiperidine in cis configuration (com-
pounds 15−18 in Table 3). The compounds with trans
configuration were not synthesized due to synthetic complexity.
It was interesting to note that that the linker modification did
not affect Mtb MIC and the Caco-2 permeability. Replacing the
F
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Table 3. SAR around Linker Modification
a-vis the fluoroquinolones, few representative compounds were
tested in the Mtb DNA gyrase supercoiling assay (Table 5) and
Table 5. Aminopiperidines Inhibit the Mtb Gyrase
Supercoiling Activity
compd
Mtb gyrase supercoiling IC50 (μM) Mtb MIC (μg/mL)
2
5
7
9
1
1
2
2
b
0.11
0.25
0.14
0.065
0.25
0.35
0.24
0.17
0.13
0.06
0.06
0.01
0.06
0.13
0.13
0.1
7
9
0
2
moxifloxacin
12.5
0.06
the cleavable complex assay (Figure 4). Most of the compounds
with MIC against Mtb also inhibited the supercoiling activity of
Mtb gyrase, thus confirming their mode of inhibition.
a
Not determined.
Table 4. SPR for Oral Bioavailability
PSA Caco-2: Papp A−B/B−A rat clearance
rat F
(%)
compd log D (Å2)
(1 × 10 cm/s)
−6
(mL/min/kg)
2
5
9
7
1
1
1
2
1
b
0.4
105
82
91
83
74
83
92
97
97
2.0/12.6
13.6/7.4
10/4.7
187
41
10
32
30
42
54
63
87
11
1.31
1.71
1.07
0.92
1.29
0.99
0.47
0.47
>100
59
10.5/8.3
17.7/15.4
13.6/7.4
11.7/9.9
AB:8.1 BA:12.3
7.6/11.2
>100
>100
52
6
7
8
0
9
73
>100
87
Figure 4. Mtb DNA gyrase cleavable complex assay for (a) compound
7 and (b) moxifloxacin. A titration was performed with varying
concentrations of compound 7 and moxifloxacin. (c) Plot representing
the relative fractions of nicked, linear, and supercoiled DNA to the
total DNA in the sample is shown.
The methoxy substitution in the aminopiperidine linker with a
monocyclic RHS (compound 18) showed moderate clearance
with good bioavailability (73%). Modification of the bicyclic
pyrido-oxazinone RHS with 1,5-napthyridone and a fluoro
linker combination resulted in moderate to high in vivo
clearance with >50% oral bioavailability (compound 19 and 20
Table 4). Good bioavailability (>50%) of compounds 17−20
with moderate to high clearance could be due to the saturation
of clearance mechanism.
FQs trap the DNA gyrase bound to double-strand cleaved
DNA in a ternary complex, thereby resulting in the formation
of linearized DNA in the cleavable complex assay, as shown
with moxifloxacin (Figure 4b). In contrast, the titration of
compound 7 showed an increased formation of single-strand
cleaved (nicked) plasmid with increasing compound concen-
tration (Figure 4a,c). We propose that the chemical scaffold
described in our work arrests the reaction at the single-strand
cleavage (or nick) and, hence, is the primary mode of inhibition
Mechanism of DNA Gyrase Inhibition. In order to
understand the mechanism of inhibition at a molecular level vis-
G
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by trapping this as ∼70% of reaction product. A small fraction
of the enzyme−DNA complex may “proceed” through to the
double-stranded cleavage, thereby resulting in a linear product
as visualized on the gel. This level, one must note, is rather
small (20%), just above the basal level of 10% of intact DNA. A
much smaller increase in the formation of linear DNA was seen
primarily at lower concentrations, which saturated at 20% of the
total DNA in the reaction (basal level of intact DNA ∼ 10%).
This suggests that the primary mechanism of inhibition of the
enzyme−DNA complex by compound 7 appears to be via
trapping the transiently single-strand cleaved DNA as it
represents ∼70% of product formed. This is different from
the mechanism of inhibition of FQs which form in classical
displayed a ∼1.3 log kill as compared to the untreated control.
Moxifloxacin, used as a positive control, showed ∼2.3 log kill at
1 μg/mL. This data highlight the cidality of this compound
series against a physiologically relevant intracellular milieu
believed to be important in the pathogenesis of tuberculosis.
In order to measure the resistance frequency, spontaneously
resistant mutants of Mtb were selected on agar plates
containing varying concentrations of compounds 2b and 17
(Table 6). Mutants resistant to compound 2b arose at a
Table 6. Aminopiperidines Display a Low Frequency of
Spontaneous Resistance against Mtb
Mtb MIC
(μg/mL)
mutant selection concn
mutation
frequency
“cleavable complex”.
compd
(μg/mL)
Another phenomenon observed was that these inhibitors
2
2
1
1
b
b
7
7
0.13
0.13
0.06
0.06
0.5
0.5
1
2.9 × 10−
6
were equipotent in inhibition of the enzyme (IC ) and
50
−7
8.2 × 10
3.5 × 10
8.7 × 10
1.9 × 10
1.9 × 10
2.6 × 10
2.3 × 10
bacterial cell growth (MIC) as compared to the FQs (e.g.,
moxifloxacin), which exhibit MIC several orders of magnitude
lower than the enzyme inhibition. This phenomenon observed
with the FQs has been explained by the downstream effects of
double-stranded lesions in the chromosomal DNA, resulting in
an SOS response of the cell leading to a more rapid inhibition/
kill. This data suggests that the mechanism of action of the N-
linked aminopiperidinyl alkyl quinolones and naphthyridones
inhibitors could be different from that of the FQs.
Bactericidal Activity of Aminopiperidines against
Replicating Mtb in Broth and Macrophages. Detailed
time course kill studies were performed to understand the
influence of varying drug concentrations over a period of 10
days. As shown in Figure 5a, compounds 19 and 20 displayed a
−
−
−
−
−
−
8
9
7
7
8
8
0.5
1
ciprofloxacin
ciprofloxacin
moxifloxacin
moxifloxacin
4
0.5
8
0.06
0.06
0.25
0.5
frequency of 8.2 × 10−7 at 8× MIC as compared to 2 × 10−7
seen for ciprofloxacin at 8× MIC. In contrast, mutants resistant
−8
to compound 17 arose at a frequency of 3.5 × 10 at ∼10×
−9
MIC (8.7 × 10 at ∼20× MIC), which is similar to the
−8
observed frequency for moxifloxacin (2.3 × 10 ) at a similar
multiples of the MIC.
Compounds from this series were tested for their MIC
against resistant mutants selected using 2b, 17, and
moxifloxacin. As shown in Table 7, compounds 2b and 17, as
Table 7. MIC of Aminopiperidine against Mutants Resistant
to Compound 2b and 17
MIC (μg/mL)
Mtb
H37Rv
wild-type
compd 2bR
mutant
(A74V)
compd 17R
mutant
(D89N)
MoxiR
mutant
(G88N)
compd
2
1
5
2
b
7
0.13
0.06
0.06
0.13
0.06
0.25
0.5
2
>2
>1
>2
>2
1
0.03
0.03
0.06
0.06
4
1
>2
0
>2
moxifloxacin
ciprofloxacin
ofloxacin
0.06
0.25
1
4
8
4
16
isoniazid
0.06
0.015
0.06
0.008
0.03
0.015
0.06
0.008
rifampicin
Figure 5. (a) Compounds 19 and 20 display a concentration-
dependent as well as time-dependent killing of replicating Mtb grown
in broth. (b) In vitro resistance to aminopiperidines (2b, 17) maps to
the gyrA gene in Mtb.
well as other analogues, showed a ≥16-fold increase in MIC
compared to the wild-type H37Rv strain) to the 2b- or 17-
(
resistant mutant strains, while showing no change in their MIC
against the moxifloxacin-resistant isolate. Interestingly, moxi-
floxacin retained its wild-type MIC against the compound 2b-
resistant clone, but its MIC increased ∼16 times versus the
compound 17-resistant clone. Ciprofloxacin and ofloxacin
retained their MIC on the 2b-resistant clone, but their MIC
increased 16 and 8 times against the 17-resistant clone. The
aminopiperidines retained their MIC against moxifloxacin-
resistant mutants, whereas MIC for three FQs increased by
>16-fold (Table 7). Isoniazid and rifampicin retained their wild-
type MIC against all the resistant clones tested; indicating that
>
3-log reduction in the colony forming units (cfu) over a
period of 10 days. The extent of kill increased with both the
concentration and time, a property displayed by fluoroquino-
1
7
lones against Mtb. Furthermore, to probe the activity of this
series against slowly replicating Mtb residing inside human
macrophages, adherent THP-1 macrophages infected with Mtb
were exposed to different compounds at a concentration of 1
1
8b
μg/mL for 7 days at 37 °C, as described earlier.
The
representative compounds (17, 19, and 22) from this series
H
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Figure 6. Aminopiperidines (2b) retain their MIC against a diverse panel of clinical isolates of Mtb. Cumulative frequency is defined as the sum of all
previous frequencies up to the current point.
the resistant clones had a specific mutation in the gyrase gene.
The entire gyrA gene of six spontaneous resistant clones from
each compound were sequenced. The multiple sequence
alignment of the mutated and the wild-type genes confirmed
mutations in the gyrA gene. These mutations were clustered
around the N-terminal domain of the GyrA protein spanning
amino acid position 74−104. Mutants resistant to 2b had a
A74V amino acid change. Interestingly, the 17-resistant clone
lung cfu was observed at 40 or 80 mg/kg dose of compounds 5
and 7 (monocyclic RHS) as compared to 240 mg/kg dose of
compound 22 (bicyclic RHS). The percentage time of dosing
interval for free plasma concentration to be above the MIC was
>50 for all the compounds profiled (Table 8). However, the
free AUC/MIC ratio for compound 7 was nearly 8 times lower
than for compound 22. The efficacy for compounds 5 and 7 in
the chronic model improved significantly with an extended
duration of treatment (Figure 7C).
16.4 showed a different mutation: D89N in the vicinity of the
amino acid 88, which is critical for mediating resistance to
2
2
moxifloxacin. The mutation in the moxifloxacin resistant
clone was G88N, which is consistent with the earlier
observation that moxifloxacin resistance maps to G88N in the
CONCLUSION
■
In summary, screening of a focused antibacterial library
followed by a lead optimization effort led to the identification
of a novel class of N-linked aminopiperidine-based alkyl
quinolone and naphthyridone compounds with excellent Mtb
MIC, improved pharmacokinetic properties, and significantly
reduced hERG liability. These compounds inhibited the Mtb
DNA gyrase with a novel mode of inhibition that conferred the
ability to retain their MIC against FQ-resistant clinical isolates
of Mtb. Compound 7 with monocyclic RHS and compound 19
with bicyclic RHS displayed the best efficacy in the acute model
of TB. Compound 5 and 7 were also efficacious in the chronic
model of TB. Our lead optimization efforts led to a significant
improvement in the properties that are suitable for an oral
route of administration. Additionally, the medicinal chemistry
efforts have provided enough structural diversity to optimize
the safety and pharmacokinetic properties of this series. In
conclusion, the N-linked aminopiperidine lead series represents
an attractive class of gyrase inhibitors with the potential to treat
drug-susceptible as well as drug-resistant TB, including FQ-
resistant TB. Assessment of the in vivo safety is essential before
optimizing this class of compounds toward the development of
a novel anti-TB drug.
22
laboratory setting as well as in the clinic.
Furthermore, to prove that the novel mechanism of action of
aminopiperidines is relevant in the clinic, we tested compound
2
b against a panel 44 clinical isolates of Mtb using an agar plate
24
dilution method, as described earlier. This panel included 22
strains of Mtb that were fully susceptible to FQs and 22 strains
resistant to FQs. As shown in Figure 6, compound 2b retained
its wild-type MIC90 (<0.5 μg/mL) against all the 44 clinical
isolates, irrespective of their susceptibility pattern to FQs. On
the contrary, the MIC90 of ofloxacin shifted significantly from
0.25 to >8 μg/mL. This data clearly highlights the potential of
the aminopiperidines in the treatment of multidrug-resistant
2
TB, which is steadily increasing in numbers across the globe.
In Vivo Efficacy and Pharmacokinetic and Pharmaco-
dynamic (PK−PD) Relationship. Several compounds with
good MIC, cidality, and pharmacokinetic profile were selected
for establishing the in vivo proof of concept in a murine model
of TB and understanding the PK−PD relationship for this
series. The PK profile in healthy mice suggested that the
treatment with ABT before giving the first dose and twice daily
dosing were essential to maintain adequate plasma concen-
trations of the compound to drive efficacy. The data for five
compounds, representing both monocyclic and bicyclic RHS,
tested in the acute murine model of TB are shown in Figure 7A.
Compound 7 with a monocyclic RHS showed the best efficacy.
It reduced the bacterial burden in lungs to below the limit of
quantification at 20 mg/kg dose. Compounds with a bicyclic
RHS were relatively less efficacious (19, 20, and 22). The best
efficacy in this class was observed for compound 19: a 1.2 log
reduction in the bacterial cfu at 160 mg/kg. Similarly, in the
chronic model of mouse TB (Figure 7B), ∼0.5 log reduction in
EXPERIMENTAL SECTION
■
Computational Methods. Ligand Preparation. Throughout the
studies, CORINA (http://www.molecular-networks.com/products/
corina) was used for generating 3D structures from smiles, and
Omega 2.2.0 (OpenEye Scientific Software, Santa Fe, NM 87507) was
used for generating conformers. Default settings were retained for both
the tools.
Molecular Alignment of Compounds 4 and 5. ROCS, a shape-
based molecular alignment tool (Version 3.2, OpenEye Scientific
Software) was used for molecular alignment. Shape Tanimoto score
I
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Figure 7. Efficacy after 20 days of daily treatment with compound 5, 7, 19, 20, and 22 in the acute model (A) or chronic (B) model and when
treatment was given for 8 weeks (6 days/week) in the chronic model (C). Numbers in the bracket indicate the dose (mg/kg). The dotted line
indicates the lower limit of quantitation (LLOQ) for the lung cfu. The mean and standard deviation are shown for each dose group.
was used to rank the overlays. The omega-generated multiconformers
max number of output conformers per molecule = 30) of compound
1a and 2a were aligned to the crystal-bound conformation of 4a (PDB
ID 2XCS). The best scoring conformation of compounds 1a and 2a
(
J
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Journal of Medicinal Chemistry
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The docking of designed molecules was done in two steps. In the
first step the multiple conformers of the compound to be docked were
aligned using ROCS onto the best-aligned conformation of compound
2a onto the crystal ligand. A maximum of five conformations per
compound were opted as an output from ROCS. In the second step
the resultant conformations of designed compounds were docked
using the FRED protocol as described above. Consensus scoring and
visual inspection were used to select the pose. The complex of selected
pose and protein was subjected to constrained minimization using
Macromodel (Schrodinger) and the OPLS force field to remove
unfavorable contacts.
Table 8. Pharmacokinetic and Pharmacodynamic
Parameters in Mice
MIC
fraction unbound
dose
free
% free
compd (μg/mL) in mouse plasma (mg/kg) AUC/MIC T > MIC
5
7
9
0
2
0.062
0.062
0.125
0.125
0.062
0.43
0.23
0.32
0.44
0.48
20
20
159
54
58
58
70
58
83
1
2
2
80
129
175
434
80
160
Determining the Antimycobacterial Properties. Mtb H37Rv
ATCC 27294 was used for all the studies and was grown and tested as
was further utilized as reference to derive the best possible conformer
of compounds 4 and 5 from the pool of multiple conformers.
Homology Modeling. Mycobacterium tuberculosis gyrase subunit A
2
3
described earlier. The minimum inhibitory concentration (MIC) was
1
8b
determined as described earlier.
Cleavable Complex Assay with Mtb H37Rv Gyrase
Holoenzyme. The cleavable complex assay was performed in 96-
well PCR plates (Bio-Rad) in 30 μL volume. The assay mix contained
(
Mtb GyrA) was built by homology modeling using MOE (Molecular
Operating Environment, Version 2013.12, Chemical Computing
Group, Montreal, Canada). The published S. aureus DNA gyrase
subunit A (Sau GyrA) crystal structure was retrieved from Protein
Data Bank (PDB ID 2XCS 2.1 Å) and examined for (1) ligand
interaction and followed by (2) similarity with Mtb GyrA.
4
00 nM of −Mtb H37Rv holoenzyme, 105 ng of relaxed pBR322
DNA, 40 mM HEPES−KOH pH 7.5, 100 mM potassium glutamate,
5 mM KCl, 1 mM spermidine, 10 mM MgCl , 4 mM DTT, 6%
2
2
glycerol, and 0.35 mg/mL BSA. The assay was started with the
addition of DNA and carried out for 60 min at 37 °C. The reaction
was stopped by addition of 0.75 μL of proteinase K (20 mg/mL, 40
U/mg) and 3 μL of 2% SDS followed by incubation at 37 °C for 1 h.
This was followed by the addition of 4 μL of 10× DNA-loading dye to
samples followed by loading onto the gel. Nicked, linear, and
supercoiled forms of DNA were separated by gel electrophoresis for 16
h at 1 V/cm, on a 0.8% agarose in buffer containing 45 mM Tris−
borate, 1 mM EDTA, and 0.3 μg/mL ethidium bromide. An image of
the gel was captured using a Fujifilm FLA 5100 Phosphorimager and
used for quantitation of the DNA using Bio-Rad’s Quantity One
software.
The NBTI binds to a site adjacent to the FQ binding site (Figure 13
in Supporting Information) as evident in the published crystal
8a
structure. The NBTI binds midway between the two active sites,
with the quinoline-carbonitrile group sitting in between the two
central base pairs of the stretched DNA and the oxathiolopyridine
group occupying the noncatalytic pocket that opens up between the
8a
two GyrA subunits (refer to ref 8a for further details).
Overall primary sequence similarity of Mtb with Sau GyrA was
found to be 42%, whereas the active site similarity was >95% (Figure 8
in Supporting Information), and thus Sau GyrA served as template for
the Mtb GyrA model generation [GeneDoc Ver 2.6.002 (http://www.
nrbsc.org/gfx/genedoc/) with default options was used for multiple
alignments of sequences].
Model Generation Steps. Ten different models of Mtb GyrA were
generated with the default settings as implemented in MOE. The
bound ligand and DNA were used as the environment for induced fit.
Out of the 10 different models, the model with lowest RMSD (C-α) to
the average position of all of the intermediate models was selected.
Analysis of the Ramachandran plot of the selected model indicated
that >95% of the residues were in the allowed region. The coordinates
of DNA and the ligand from the 2XCS structure were merged into the
homology model of Mtb GyrA. The resultant complex of model was
Determination of IC for the Supercoiling Activity of Mtb
50
H37Rv Gyrase Holoenzyme. DNA supercoiling assay was
performed in 96-well PCR plates (Bio-Rad) in 30 μL volume. Assay
mix contained 12.5 nM Mtb H37Rv holoenzyme, 50 ng of relaxed
pBR322 DNA, 40 mM HEPES−KOH pH 7.5, 100 mM potassium
glutamate, 15 mM KCl, 1 mM spermidine, 10 mM MgCl , 4 mM
2
DTT, 8% glycerol, 0.36 mg/mL BSA, and 75 μM ATP. The assay was
started with the addition of DNA and ATP mix and continued for 90
min at 37 °C. The reaction was stopped by addition of 0.75 μL of
Proteinase K (20 mg/mL, 40 U/mg) and 3 μL of 2% SDS followed by
incubation at 37 °C for 1 h. This was followed by the addition of 4 μL
of 10× DNA-loading dye to samples followed by loading onto the gel.
Supercoiled and relaxed forms of DNA were separated by gel
electrophoresis for 16 h at 1 V/cm, on a 0.8% agarose in buffer
containing 45 mM Tris−borate and 1 mM EDTA. The gel was stained
for 10 min with a solution containing 0.7 μg/mL ethidium bromide in
water and destained in water for 30 min, followed by imaging and
quantitation of the gel bands.
̈
further analyzed and minimized in Maestro (version 8; Schrodinger,
LLC) using default parameters of the Protein Preparation Tool. The
RMSD (C-α) of the Mtb GyrA model and Sau GyrA was found be less
than 2 Å. No major structural differences were observed between the
Mtb GyrA model and Sau GyrA crystal structure and the overall
architecture remains the same (Figure 9 in Supporting Information).
The active site (5 Å radius from ligand, 2XCS) is very well conserved,
except that Gly72 of Sau GyrA is occupied by Ala78 in Mtb GyrA.
This difference is not disturbing the architecture of the active site and
provides additional hydrophobicity to the active site of Mtb GyrA
Time Kill Studies against Mtb in 7H9 Broth. This assay was
performed in a 200 μL volume using 96-well microplates with
Middlebrook 7H9 broth supplemented with 0.2% glycerol, 0.05%
Tween 80 (Sigma), and 10% albumin dextrose catalase (Difco
(
Figure 10 in Supporting Information). The energy-minimized model
was used for docking.
1
8b
Molecular Docking. Molecular dockings into the Sau GyrA and
Mtb GyrA model were performed using FRED (Version 2, OpenEye
Scientific Software). The Receptor Tool in FRED was used to prepare
the receptor input file for docking. The active site was defined by
selecting the ligand and a docking constrain involving Asp83/89 of Sau
and Mtb GyrA as a hydrogen bond acceptor was added. The Asp was
found to be involved in critical hydrogen bond interaction with the
Laboratories, Detroit, MI) as described earlier.
Serial 2-fold
dilutions of aminopiperidines spanning a wide concentration range
7
were added to each of the 96-wells containing approximately 3 × 10
cfu/mL of Mtb H37Rv. The plates were incubated at 37 °C. Bacterial
enumeration was performed on Middlebrook 7H11 agar plates at 0, 3,
7, and 10 days after drug exposure. The plates were incubated for up to
28 days at 37 °C, in 5% CO in a humidified atmosphere. The bacterial
2
8
a
ligand and mutation of it was found to affect the activity of NBTIs.
colonies were enumerated and data expressed as the log10 cfu for each
drug concentration.
Determination of Resistance Frequency. Spontaneous resistant
The docking protocol was validated by extracting the bound
conformation of the ligand and redocking into the receptor. The
best agreement was found between docked pose and crystal bound
conformation (RMSD < 0.8 Å in both the receptor). After this
validation, the receptor was used for docking. Apart from the default
Chemgauss3 scoring function, CGO scoring (based on the shape
overlay of ligands) was used.
mutants were raised against compound 2b, 17, ciprofloxacin, and
19
moxifloxacin using a single step selection method as described.
Briefly, a mid-logarithmic phase culture of Mtb H37Rv was centrifuged
1
0
and concentrated 100-fold to achieve a bacterial number of ∼10 cfu/
mL. Varying dilutions of the bacterial culture were plated onto
K
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Journal of Medicinal Chemistry
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compound-containing plates. Appropriate dilutions of the bacterial
culture were also plated on drug-free Middlebrook 7H11 agar to
enumerate the bacterial numbers in the culture. Plates were incubated
for 4 weeks at 37 °C and the cfu in drug-free plates were enumerated.
The drug-containing plates were incubated for up to 6−8 weeks at 37
compound-treated groups contained three mice each. An additional
three mice were sacrificed just after infection to serve as early controls.
Isoniazid was formulated in 0.5% (w/v) (hydroxypropyl)methyl
cellulose (HPMC), whereas test compounds were prepared in the
same formulation as used for the PK study. ABT was orally
administered before compound administration, as described above.
In the acute model, infected mice were treated with an oral dose of 20,
40, 80, or 160 mg/kg of compound 5, 7, 19, 20,22 given twice daily (8
h interval) for 20 days (7 days/week). In the first chronic model
efficacy study, infected mice were treated with an oral dose of 40, 80,
or 240 mg/kg of compound 5, 7, 19, 20, and 22 given twice daily (8 h
interval) for 20 days (7 days/week). In a second chronic model
efficacy study, infected mice were treated with an oral dose of 20, 40,
or 80 mg/kg of compound 5 or 7 twice daily (8 h interval) for 8 weeks
(6 days/week). Mice were sacrificed after completion of treatment and
suitable dilutions of lung homogenates were plated on 7H11 agar to
determine viable cfu per mouse lung tissue. One-way analysis of
variance (ANOVA) followed by the Bonferroni’s multiple comparison
test was used to analyze efficacy data.
°C to enumerate the number of resistant colonies.
The spontaneous rate of resistance was calculated by dividing the
number of colonies on drug-containing plates (at a given
concentration) divided by the total number of viable bacteria
estimated on drug-free plates. Resistant colonies were randomly
picked from the drug-containing plates and grown in complete 7H9
broth to determine their level of resistance against the parent
compound, close analogues, and TB drugs with different mechanisms
of action.
Genetic Mapping of Mutations Conferring Resistance to N-
Linked Aminopeperidinyl Alkyl Quinolones and Naphthyr-
idones. In order to map the genetic mutations arising following
compound exposure and mutant selection, genomic DNA from
microbiologically well characterized colonies was isolated. The entire
gyrA gene of resistant mutants and wild-type Mtb H37Rv were PCR
amplified using standard conditions from boiled supernatants using
specific Mtb primers to amplify the entire gyrA gene. PCR was
performed with cycling parameters of 94 °C for 30 s, 60 °C for 30 s,
and 72 °C for 2 min for 30 cycles in a DNA Engine Dyad cycler (Bio-
Rad). PCR products were purified from the gel (PCR purification kit,
Qiagen), quantitated, and sequenced (Microsynth). The sequences
from the resistant clones were aligned against the wild-type H37Rv
gyrA gene using Vector NTI software to detect mutations in the target
gene.
General Chemical Methods. All commercial reagents and
solvents were used without further purification. Analytical thin-layer
chromatography (TLC) was performed on SiO plates on alumina.
2
Visualization was accomplished by UV irradiation at 254 and 220 nm.
Flash column chromatography was performed using the Biotage
Isolera flash purification system with SiO 60 (particle size 0.040−
2
0
.055 mm, 230−400 mesh). The purity of all final derivatives for
biological testing was confirmed to be >95%, as determined using the
following conditions: a Shimadzu HPLC instrument with a Hamilton
reverse-phase column [HxSil, C18, 3 μm, 2.1 mm × 50 mm (H2)];
eluent A, 5% CH CN in H O; eluent B, 90% CH CN in H O; a flow
In Vivo Studies in Mice. All animal experimentation protocols
were approved by the Institutional Animal Ethics Committee
registered with the Government of India (Reg. No. 5/1999/
CPCSEA). Six to eight week old BALB/c mice, purchased from
RCC laboratories, Hyderabad, India, were used in the pharmacokinetic
3
2
3
2
rate of 0.2 mL/min was used with UV detection at 254 and 214 nm.
The structures of the intermediates and end products were confirmed
by 1H NMR and mass spectroscopy. Proton magnetic resonance
spectra were determined in DMSO-d , unless otherwise stated, using
6
(
PK) and pharmacodynamic (PD) studies. Animals randomly assigned
Bruker DRX-300 or Bruker DRX-400 spectrometers, operating at 300
or 400 MHz, respectively. Splitting patterns are indicated as follows: s,
singlet; d, doublet; t, triplet; m, multiplet; br, broad peak. LCMS data
were acquired using an Agilent LCMS VL series with ES ionization,
coupled with an Agilent 1100 series HPLC system and an Agilent
to cages were allowed a 2 week acclimatization period before initiating
the experiment. Feed and water were given ad libitum.
Pharmacokinetics. PK was analyzed in the infected mice during
the efficacy study. Blood samples from infected mice were collected on
day 18 or 19 of dosing and processed in a BSL3 laboratory.
Compounds 5, 7, 19, 20, and 22 were formulated in a solution
containing 5% dextrose, pH adjusted to 4−5 with 5 M acetic acid. Test
compounds, or the corresponding vehicle, were orally administered
through gavage at 10 mL/kg dose volume. Aminobenzotriazole
1
100 series PDA as the front end. HRMS data was acquired using an
Agilent 6520 quadrupole-time of flight tandem mass spectrometer (Q-
Tof MS/MS) coupled with an Agilent 1200 series HPLC system.
The synthesis of compound 1a, 2a, and 2b were previously reported
10
by Reck et al.
(
ABT), a known cytochrome P450 inhibitor, dissolved in water, was
1
-(2-(4-((5-Cyano-6-methylpyridin-2-yl)methylamino)-
orally administered at a dose of 100 mg/kg (10 mL/kg) 2 h before
dosing the compound to reduce the first pass metabolism and increase
plasma exposure. Blood samples were collected by puncturing the
saphenous vein at 5, 15, and 30 min and 1, 2, 4, and 6 h after dosing.
The same formulation as above was used for intravenous PK at 5 mg/
kg and per oral PK at 40 mg/kg dose in rats. Plasma samples from
mice/rats were mixed with chilled acetonitrile (1:10 v/v) to precipitate
proteins, vortexed, and centrifuged at 4000 rpm for 30 min at 10 °C.
The resulting supernatant was mixed with mobile phase containing
carbamazepine (internal standard, 10 ng/mL) and analyzed for drug
concentrations by liquid chromatography with tandem mass
spectrometry (LC−MS/MS). Chromatographic separation was
achieved on a Gemini C18 column (50 × 4.6 mm, particle size 5
μm, Phenomenex) by isocratic elution with 40% 10 mM ammonium
acetate buffer in acetonitrile at 500 μL/min flow rate over 4.5 min. An
AB Sciex API3000 triple quadrupole mass spectrometer (Applied
Biosystems) equipped with a turbo ionspray source was used for the
quantitation. The area under the concentration (AUC) vs time PK
profile was calculated by noncompartmental analysis (WinNonLin
piperidin-1-yl)ethyl)-2-oxo-1,2-dihydroquinoline-7-carboni-
1
1
trile (4). In a 100 mL round-bottomed flask, 27a (250 mg, 0.84
mmol) and 31 (185 mg, 1.27 mmol) were taken up in a DCE (8 mL)
and ethanol (2.000 mL) mixture. Acetic acid (0.050 mL, 0.84 mmol)
was added to the reaction mixture that was then heated at 80 °C for 1
h. The reaction mixture was then cooled to rt and then sodium
triacetoxyborohydride (536 mg, 2.53 mmol) was added to it
portionwise. The reaction mixture was then stirred at rt overnight.
The reaction was monitored by LCMS. The profile showed
formation of imine and only 20% product. Sodium cyanoborohydride
(106 mg, 1.69 mmol) was added to the reaction mixture and it was
stirred at rt for 4 h. The reaction mixture was then evaporated and the
residue was then neutralized with saturated NaHCO . It was then
3
partitioned between DCM (5%MeOH) and water. The organic layer
was then separated, dried (Na SO ), and evaporated to get a residue
2
4
that was then chromatographed with DCM/MeOH to get 4 (75 mg,
1
20.85%): H NMR (300 MHz, DMSO-d ) δ1.24 (d, J = 8.85 Hz, 2 H),
6
1.81 (d, J = 10.74 Hz, 2 H), 2.04 (t, J = 10.55 Hz, 2 H), 2.15 (br s, 1
H), 2.38 (br s, 1 H), 2.51 (s, 3 H), 2.55 (br s, 2 H), 2.92 (d, J = 11.11
Hz, 2 H), 3.76 (br s, 2 H), 4.37 (t, J = 6.50 Hz, 2 H), 6.79 (d, J = 9.42
Hz, 1 H), 7.66 (d, J = 8.10 Hz, 1 H), 7.84 (d, J = 7.72 Hz, 1 H), 7.93
(t, J = 8.29 Hz, 2 H), 8.01 (d, J = 9.61 Hz, 1 H), 8.09 (s, 1 H); HRMS
5
.2.1, Pharsight Inc.).
Pharmacodynamics. BALB/c mice were infected in an aerosol
18d
chamber as described earlier.
Infected mice were housed in
+
individually ventilated cages (Allentown Technologies) in a biosafety
level (BSL)-3 facility. Treatment was initiated after 3 days and 4 weeks
of infection in the acute and chronic model, respectively. Vehicle- or
m/z (ES+) 427.217 21 (MH ) for C H N O.
25
26
6
1-(2-(4-((5-Cyano-6-methylpyridin-3-yl)methylamino)-
piperidin-1-yl)ethyl)-2-oxo-1,2-dihydroquinoline-7-carboni-
L
dx.doi.org/10.1021/jm500432n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
trile (5). In a 500 mL round-bottomed flask, 27a (11 g, 37.12 mmol)
and sodium carbonate monohydrate (13.81 g, 111.35 mmol) were
taken up in DMF (100 mL). In another liquid addition funnel 32 (8.40
g, 37.12 mmol) was taken up in DMF (100 mL), and the solution was
then added slowly to the above reaction mixture. The reaction mixture
was then stirred at rt for 4 h. The solvent was evaporated to dryness
and the residue was then partitioned between DCM (5% MeOH) and
water. The organic layer was then separated, dried, and chromato-
graphed with DCM/MEOH to get a slightly crude product. The solid
was then triturated in succession with DCM/hexane and MeOH/ether
to get slightly impure 5 (9.30 g, 58.7%), which was again triturated
with water/MeOH to get 4.85 g of pure compound. The remaining
2.18−2.38 (m, 2 H), 2.64 (s, 3 H), 2.79−2.98 (m, 2 H), 3.73 (s, 2 H),
3.91 (s, 3 H), 4.31 (t, J = 6.78 Hz, 2 H), 6.79 (d, J = 9.80 Hz, 1 H),
7.12 (d, J = 9.23 Hz, 1 H), 7.80 (d, J = 9.80 Hz, 1 H), 8.00 (d, J = 9.23
Hz, 1 H), 8.15 (d, J = 1.88 Hz, 1 H), 8.66 (d, J = 1.88 Hz, 1 H);
+
HRMS m/z (ES+) 433.234 27 (MH ) for C H N O .
24
28
6
2
6-Methoxy-1-methyl-1,5-naphthyridin-2(1H)-one (8-ii). In a
100 mL round-bottomed flask, 24c (2.000 g, 11.35 mmol) and cesium
carbonate (4.07 g, 12.49 mmol) were taken up in DMSO (20 mL) to
give a tan suspension. Then to this was added methyl iodide (1.065
mL, 17.03 mmol), and the resulting reaction mixture was heated to 60
°C for 2 h. Water was added to the reaction mixture, which was
extracted using EtOAc (5 × 50 mL). The resulting organic layers were
washed with water, dried over sodium sulfate, and concentrated under
vacuum to obtain crude solid. The crude product was purified by silica
amount was later purified by preparative HPLC to get another 1.577 g
1
of product: H NMR (300 MHz, DMSO-d ) δ 1.20 (d, J = 12.43 Hz, 3
6
H), 1.76 (d, J = 11.87 Hz, 2 H), 1.92−2.11 (m, 2 H), 2.30 (d, J = 14.51
gel column using 0−9% methanol in DCM to yield 8-ii (1.800 g, 83%)
1
Hz, 2 H), 2.54 (br s, 1 H), 2.65 (s, 3 H), 2.90 (d, J = 11.68 Hz, 2 H),
as solid: H NMR (300 MHz, DMSO-d ) δ 3.61 (s, 3 H), 3.91 (s, 3
6
3
7
.74 (s, 2 H), 4.36 (t, J = 6.59 Hz, 2 H), 6.78 (d, J = 9.42 Hz, 1 H),
.65 (d, J = 7.91 Hz, 1 H), 7.91 (d, J = 8.10 Hz, 1 H), 8.00 (d, J = 9.61
H), 6.80 (d, J = 9.61 Hz, 1 H), 7.12 (d, J = 9.23 Hz, 1 H), 7.79 (d, J =
9.61 Hz, 1 H), 8.00 (d, J = 9.04 Hz, 1 H); HRMS m/z (ES+) 191.23
+
Hz, 1 H), 8.08 (s, 1 H), 8.15 (d, J = 1.88 Hz, 1 H), 8.66 (d, J = 1.88
(M + H) for C H N O .
10
10
2
2
+
Hz, 1 H); HRMS m/z (ES+) 427.225 70 (MH ) for C H N O.
1-Methyl-1,5-naphthyridine-2,6(1H,5H)-dione (8-iii). In a 250
mL round-bottomed flask, 8-ii (1.700 g, 8.94 mmol) was taken up in
25
26
6
5
-((1-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-
piperidin-4-ylamino)methyl)-2-methylnicotinonitrile (6). In a
4
8% HBr in water (15 mL, 276.23 mmol) to give a yellow solution.
1
5
00 mL round-bottomed flask, 27b (1.500 g, 5.17 mmol), 32 (1.286 g,
.68 mmol), and sodium carbonate (1.095 g, 10.33 mmol) were taken
The resulting reaction mass was stirred at 80 °C for 2 h. The reaction
mass was concentrated to dryness and the pH was adjusted to ∼7. The
yellow solid obtained was filtered, washed with hexane, and dried to
up in DMF (25 mL) to give a yellowish solution under nitrogen
atmosphere that was heated to 70 °C for 1 h. Solvent was evaporated
from the reaction mixture. Water was added to it and extracted using
1
yield 8-iii (1.800 g, 114%) as yellow solid: H NMR (300 MHz,
DMSO-d ) δ 3.49 (s, 3 H), 6.33 (d, J = 9.23 Hz, 1 H), 6.50 (d, J = 9.61
6
1
0% methanol in DCM (4 × 25 mL). The combined organic layers
Hz, 1 H), 7.43 (d, J = 9.42 Hz, 1 H), 7.36 (d, J = 9.61 Hz, 1 H);
were concentrated to dryness and purified by silica gel chromatog-
raphy using 0−18% methanol in DCM to yield 6 (1.400 g, 64.4%) as
brown solid; 200 mg of the product was purified by preparative HPLC
+
HRMS m/z (ES+) 177.19 (M + H) for C H N O .
9
8
2
2
1-(2-Hydroxyethyl)-5-methyl-1,5-naphthyridine-2,6(1H,5H)-
dione (8-iv). In a 100 mL round-bottomed flask, 8-iii (1.000 g, 5.68
mmol) and cesium carbonate (2.034 g, 6.24 mmol) were taken up in
DMSO (10 mL) under anitrogen atmosphere to give a yellowish
solution. To it was added 2-bromoethanol (0.561 mL, 7.95 mmol),
and the resulting reaction mass was heated to 95 °C overnight. Water
was added to the reaction mass and extracted using ethyl acetate (5 ×
1
to obtain product as the diacetate salt: H NMR (300 MHz, DMSO-
d ) δ1.15−1.29 (m, 2 H), 1.73−1.83 (m, 2 H), 1.91 (s, 6 H), 1.97−
6
2
2
6
1
.10 (m, 2 H), 2.31−2.43 (m, 1 H), 2.53−2.59 (m, 2 H), 2.65 (s, 3 H),
.90 (d, J = 11.68 Hz, 3 H), 3.77 (s, 2 H), 4.31 (t, J = 6.59 Hz, 2 H),
.83 (d, J = 9.80 Hz, 1 H), 7.95 (d, J = 9.80 Hz, 1 H), 8.01 (dd, J =
1.30, 2.07 Hz, 1 H), 8.16 (d, J = 1.88 Hz, 1 H), 8.56 (d, J = 2.45 Hz, 1
4
0 mL). The combined organic fractions were washed with water,
+
H), 8.67 (d, J = 1.88 Hz, 1 H); HRMS m/z (ES+) 421.21490 (MH )
dried with sodium sulfate, and concentrated to dryness to yield 8-iv
(1.300 g, 104%) as light yellow solid: HRMS m/z (ES+) 221.26 (M +
for C H FN O·2CH COOH.
23
25
6
3
+
5
-((1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-
piperidin-4-ylamino)methyl)-2-methylnicotinonitrile (7). In a
00 mL round-bottomed flask 6 (6.3 g, 14.98 mmol) was taken up in
H) for C H N O .
1
1
12
2
3
2
-(5-Methyl-2,6-dioxo-5,6-dihydro-1,5-naphthyridin-1(2H)-
5
yl)ethylmethanesulfonate (8-v). In 50 mL round-bottomed flask,
8-iv (400 mg, 1.82 mmol) and TEA (0.759 mL, 5.45 mmol) were
taken up in DCM (10 mL) under a nitrogen atmosphere to give a
yellowish solution. The resulting reaction mass was cooled to 0 °C and
Mesyl-Cl (0.184 mL, 2.36 mmol) was added to it very slowly. Then
the reaction mixture was stirred at 0 °C for 45 min. DCM was added
to the reaction mass and washed with water (2 × 20 mL). The
combined organic fractions were dried over sodium sulfate and
concentrated to dryness to yield 8-v (600 mg, 111%) as a yellow gel:
methanol (100 mL) under N . To this mixture was added sodium
2
methoxide (3.24 g, 59.93 mmol) with stirring. The resulting reaction
was stirred at 70 °C for 6 h. The reaction was cooled to rt, and solvent
was evaporated to dryness. The obtained solid was diluted with water
and extracted with 5% methanol in DCM. The organic layer was
separated, washed with brine, and dried to get a residue. The residue
was then purified on silica with DCM/MeOH to a slightly crude
compound, which on trituration with acetonitrile afforded the pure 7
1
(
4.01 g, 61.9%): H NMR (300 MHz, DMSO-d ) δ 1.11−1.32 (m, 2
+
6
HRMS m/z (ES+) 299.27 (M + H) for C H N O S.
12 14 2 5
H), 1.76 (d, J = 10.93 Hz, 2 H), 1.94−2.10 (m, 2 H), 2.28 (d, J = 18.46
Hz, 2 H), 2.54 (br s, 2 H), 2.64 (s, 3 H), 2.91 (d, J = 11.30 Hz, 2 H),
2
-Methyl-5-((1-(2-(5-methyl-2,6-dioxo-5,6-dihydro-1,5-
naphthyridin-1(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-
nicotinonitrile (8). In a 100 mL round-bottomed flask, 8-v (500 mg,
1.68 mmol), 29e (386 mg, 1.68 mmol), and sodium carbonate (355
mg, 3.35 mmol) were taken up in DMF (5 mL) to give a yellowish
solution under a nitrogen atmosphere that was heated to 70 °C for 1 h.
Solvent was evaporated from the reaction mixture. Water was added to
the residue and it was extracted using 10% methanol in DCM (3 × 25
mL). The combined organic layers were concentrated to dryness and
3
.34 (s, 2 H), 3.98 (s, 3 H), 4.35 (t, J = 6.78 Hz, 2 H), 6.66 (d, J = 9.80
Hz, 1 H), 7.41 (d, J = 2.26 Hz, 1 H), 7.86 (d, J = 9.61 Hz, 1 H), 8.15
d, J = 1.88 Hz, 1 H), 8.28 (d, J = 2.26 Hz, 1 H), 8.66 (d, J = 2.07 Hz,
(
1
+
H); HRMS m/z (ES+) 433.235 90 (MH ) for C H N O .
24 28 6 2
5
-((1-(2-(6-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-
piperidin-4-ylamino)methyl)-2-methylnicotinonitrile (7a). In a
1
2
00 mL round-bottomed flask, 27c (800 mg, 2.65 mmol), 32 (658 mg,
.91 mmol), and sodium carbonate (561 mg, 5.29 mmol) were taken
purified by preparative HPLC to yield 8 (160 mg, 22.07%) as a yellow
1
up in DMF (20 mL) to give a yellowish solution under nitrogen
atmosphere that was heated to 70 °C for 1 h. Solvent was evaporated
from the reaction mixture. Water was added to it and the mixture was
extracted using 10% methanol in DCM (3 × 50 mL). The combined
organic layers were concentrated to dryness and purified by silica gel
chromatography using 0−15% methanol in DCM to obtain an impure
solid: H NMR (400 MHz, DMSO-d ) δ 1.14−1.27 (m, 2 H), 1.70−
6
1
.80 (m, 2 H), 1.96−2.06 (m, 2 H), 2.25−2.36 (m, 2 H), 2.39−2.49
(
m, 2 H), 2.65 (s, 3 H), 2.84−2.89 (m, 2 H), 3.62 (s, 3 H), 3.73 (s, 2
H), 4.31 (t, J = 6.78 Hz, 2 H), 6.78 (s, 1 H), 6.76 (s, 1 H), 7.82−8.01
m, 2 H), 8.15 (d, J = 2.01 Hz, 1 H), 8.66 (d, J = 2.01 Hz, 1 H);
(
+
HRMS m/z (ES+) 433.235 18 (MH ) for C H N O .
24
28
6
2
product (270 mg) that was repurified by preparative HPLC to yield 7a
1
-(2-(4-((5-Cyano-6-methylpyridin-3-yl)methylamino)-
1
(
270 mg, 23.59%) as white solid: H NMR (300 MHz, DMSO-d ) δ
piperidin-1-yl)ethyl)-7-methoxy-2-oxo-1,2-dihydroquinoline-
6
1
.11−1.33 (m, 2 H), 1.67−1.84 (m, 2 H), 2.01 (t, J = 11.02 Hz, 2 H),
5-carbonitrile (9). In a 100 mL round-bottomed flask, 27i (500 mg,
M
dx.doi.org/10.1021/jm500432n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
3
.53 mmol), 32 (416 mg, 1.84 mmol), and sodium carbonate (325 mg,
.06 mmol) were taken up in DMF (10 mL) to give a yellowish
= 9.8 Hz, 1 H), 8.34 (s, 1 H), 9.05 (s, 1 H), 9.95 (br s, 2 H), 11.32 (br
+
s, 1 H); HRMS m/z (ES+) 434.230 91 (MH ) for C H N O ·2HCl.
23
27
7
2
solution under nitrogen atmosphere and heated to 70 °C for 1 h.
Solvent was evaporated from the reaction mixture. Water was added to
thr residue and it was extracted using 10% methanol in DCM (3 × 25
mL). The combined organic layers were concentrated to dryness and
purified by silica gel chromatography to obtain impure product (250
5-((1-(2-(6-Methoxy-3-oxopyrido[3,2-b]pyrazin-4(3H)-yl)-
ethyl)piperidin-4-ylamino)methyl)-2-methylnicotinonitrile
(
13). In a 100 mL round-bottomed flask, 4-(2-(4-aminopiperidin-1-
10
yl)ethyl)-6-methoxypyrido[3,2-b]pyrazin-3(4H)-one (27f) (1.5 g,
.94 mmol), 32 (1.342 g, 5.93 mmol), and sodium carbonate (0.545
4
mL, 9.89 mmol) were taken up in DMF (5 mL) to give a brown
solution. The resulting reaction mixture was heated to 74 °C for 1 h.
The reaction mixture was evaporated, and the residue was taken up in
water. The aqueous layer was extracted with DCM (three times). The
DCM layer was evaporated and the crude compound was purified by
mg) that was repurified by preparative HPLC to give 9 (60.0 mg,
1
8
.58%) as white solid: H NMR (400 MHz, DMSO-d ) δ 1.10−1.33
6
(
m, 2 H), 1.75−1.78 (m, 2 H), 2.02 (t, J = 10.54 Hz, 2 H), 2.15−2.39
(
m, 2 H), 2.55 (m, 2H) 2.65 (s, 3 H), 2.89−2.92 (m, 2 H), 3.74 (br s,
2
H), 3.96 (s, 3 H), 4.37 (t, J = 7.03 Hz, 2 H), 6.68 (d, J = 9.54 Hz, 1
preparative HPLC to give 13 (0.500 g, 20.49%) as a monoacetate salt:
H), 7.31 (d, J = 2.01 Hz, 1 H), 7.54 (d, J = 2.51 Hz, 1 H), 7.92 (d, J =
1
H NMR (300 MHz, DMSO-d ) δ 1.26−1.28 (m, 2 H), 1.56−1.57
1
0.04 Hz, 1 H), 8.15 (d, J = 2.01 Hz, 1 H), 8.66 (d, J = 2.01 Hz, 1 H);
6
+
(m, 2 H), 1.9 (s, 3 H), 2.0−2.1 (m, 2 H), 2.2−2.4 (m,1 H), 2.6−2.7
(
HRMS m/z (ES+) 457.234 16 (MH ) for C H N O .
26
28
6
2
m, 1 H), 2.8 (s, 3 H), 2.9−3.0 (m, 2 H), 3.7 (s, 2 H), 4.0 (s, 3 H),
5
-Cyano-N-(1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-
4
.2−4.5 (t, 2 H), 6.80 (d, 1 H), 8.0−8.2 (m, 3 H), 8.6 (s, 1 H); HRMS
yl)ethyl)piperidin-4-yl)-6-methylnicotinamide (10). In a 50 mL
round-bottomed flask 33 (0.161 g, 0.99 mmol) was dissolved in DMF
+
m/z (ES+) 434.230 02 (MH ) for C H N O ·CH COOH.
23 27
7
2
3
5
-((1-(2-(7-Methoxy-2-oxopyrido[3,2-b]pyrazin-1(2H)-yl)-
ethyl)piperidin-4-ylamino)methyl)-2-methylnicotinonitrile
14). In a 250 mL round-bottomed flask, 27h (2.000 g, 6.59 mmol),
2 (1.641 g, 7.25 mmol), DIPEA (3.45 mL, 19.78 mmol), and sodium
(
5 mL) under N . To this were added DIPEA (0.520 mL, 2.98 mmol)
2
and HATU (0.754 g, 1.98 mmol), and the mixture was stirred. After 5
min, 27e (0.300 g, 0.99 mmol) was added. The resulting reaction was
at rt for 3 h. DMF was evaporated to dryness, and the residue was
diluted with water, extracted with 10% methanol in DCM, and
concentrated. Purification was done by preparative HPLC to get pure
product 10 (0.200 g, 45.1%): H NMR (300 MHz, DMSO-d ) δ
1
2
3
(
3
carbonate (1.398 g, 13.19 mmol) were taken up in DMF (25 mL) to
give a yellowish solution under nitrogen atmosphere that was heated
to 70 °C for 1 h. Solvent was evaporated from the reaction mixture.
Water was added to the residue and the mixture was extracted using
1
6
.41−1.61 (m, 2 H), 1.80 (d, J = 9.98 Hz, 2 H), 2.05−2.22 (m, 2 H),
.59 (t, J = 6.59 Hz, 2 H), 2.73 (s, 3 H), 3.01 (d, J = 11.30 Hz, 2 H),
.77 (dd, J = 7.63, 3.67 Hz, 1 H), 4.00 (s, 3 H), 4.39 (t, J = 6.59 Hz, 2
1
0% methanol in DCM (3 × 50 mL). The combined organic layers
were concentrated to dryness and purified by silica gel chromatog-
raphy using 0−35% methanol in DCM and 0.1% ammonia to yield an
impure product (700 mg) that was repurified by preparative HPLC to
H), 6.67 (d, J = 9.61 Hz, 1 H), 7.44 (d, J = 2.07 Hz, 1 H), 7.88 (d, J =
9
.61 Hz, 1 H), 8.29 (d, J = 2.26 Hz, 1 H), 8.51 (d, J = 7.54 Hz, 1 H),
1
give 14 (0.100 g, 3.50%): H NMR (300 MHz, DMSO-d ) δ 1.07−
6
8
.59 (d, J = 2.07 Hz, 1 H), 9.08 (d, J = 2.26 Hz, 1 H); HRMS m/z (ES
1
.26 (m, 2 H), 1.72−1.75 (m, 2 H), 1.92−2.09 (m, 2 H), 2.22−2.36
+
+
) 447.213 98 (MH ) for C H N O .
24
26
6
3
(
m, 2H) 2.55 (t, J = 6.59 Hz, 2 H), 2.64 (s, 3 H), 2.82−2.96 (m, 2 H),
2
-Methoxy-5-((1-(2-(7-methoxy-2-oxo-1,5-naphthyridin-
3
.72 (s, 2 H), 4.00 (s, 3 H), 4.34 (t, J = 6.50 Hz, 2 H), 7.51 (d, J = 2.45
1
(2H)-yl)ethyl)piperidin-4-ylamino)methyl)nicotinonitrile (11).
Hz, 1 H), 8.14 (d, J = 1.70 Hz, 1 H), 8.25 (s, 1 H), 8.32 (d, J = 2.45
Hz, 1 H), 8.65 (d, J = 1.88 Hz, 1 H); HRMS m/z (ES+) 434.230 32
In a 50 mL round-bottomed flask, 27e (0.050 g, 0.17 mmol) and 34
0.027 g, 0.17 mmol) were taken in a mixture of ethanol (5 mL) and
(
(
MH+) for C H N O .
23 27 7 2
chloroform (5.00 mL) under N . To this was added acetic acid (0.095
2
1
-(2-((3S,4R)-4-((5-Cyano-6-methylpyridin-3-yl)-
μL, 1.65 μmol), and the reaction was stirred at 65 °C. A turbid
solution was observed. After 1 h, the reaction was cooled to rt and
sodium cyanoborohydride (0.021 g, 0.33 mmol) was added. The
resulting reaction was stirred at rt for another 6 h. Reaction solvents
methylamino)-3-fluoropiperidin-1-yl)ethyl)-2-oxo-1,2-dihy-
droquinoline-7-carbonitrile (15). In a 50 mL round-bottomed
flask, 29a (487 mg, 0.82 mmol) was dissolved in DMF (15 mL), and
DIPEA (0.576 mL, 3.30 mmol) was added. The mixture stirred for 5
were evaporated to dryness and diluted with sat. NaHCO solution.
11
3
min, and then 28a (175 mg, 0.82 mmol) was added. The reaction
The crude mixture was extracted with 5% methanol in DCM and
washed with water and brine. The organic layer was separated and
concentrated. Purification was done using methanol/DCM as a mobile
phase to get pure product 11 (0.045 g, 60.7%): H NMR (300 MHz,
DMSO-d ) δ 1.13−1.28 (m, 2 H), 1.77 (d, J = 11.30 Hz, 2 H), 2.04 (t,
J = 10.46 Hz, 2 H), 2.16 (br s, 1 H), 2.30 (d, J = 14.51 Hz, 1 H), 2.55
was heated to 70 °C for 2 h and then brought to rt. Sodium
triacetoxyborohydride (524 mg, 2.47 mmol) was added and the
mixture was stirred at rt for overnight. The reaction mixture was
filtered through a sintered funnel, washed with 20% MeOH in DCM,
and concentrated. The crude compound was basified with saturated
sodium bicarbonate solution and extracted with 15% MeOH/DCM.
The solvent was evaporated under vacuum to yield crude product that
1
6
(
(
m, 2H) 2.91 (d, J = 11.68 Hz, 2 H), 3.68 (s, 2 H), 3.98 (s, 6 H), 4.35
t, J = 6.88 Hz, 2 H), 6.66 (d, J = 9.61 Hz, 1 H), 7.41 (d, J = 2.07 Hz, 1
1
was purified by preparative HPLC to give 15 (200 mg, 54.6%): H
H), 7.87 (d, J = 9.61 Hz, 1 H), 8.19 (d, J = 2.26 Hz, 1 H), 8.28 (d, J =
NMR (400 MHz, DMSO-d ) δ 1.64 (m, 2 H), 2.13 (t, 1 H), 2.40−
6
2
2
.45 Hz, 1 H), 8.39 (d, J = 2.26 Hz, 1 H); HRMS m/z (ES+) 449.229
6 (MH ) for C H N O .
2
.20 (m, 1 H), 2.57 (t, 4 H), 2.87 (s, 3H) 3.14 (m,1H) 2.97 (m, 1 H),
+
24
28
6
3
3.80 (s, 2 H), 4.28−4.44 (m, 2 H), 4.65 and 4.78 (s, 1 H), 6.79 (d, J =
6
-((1-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-
piperidin-4-ylamino)methyl)-3-methylpyrazine-2-carbonitrile
12). In a 25 mL sealed vial, 12a (95 mg, 0.19 mmol), ZnCN (57.2
9
8
8
.54 Hz, 1 H), 7.66 (d, J = 8.03 Hz, 1 H), 7.91 (d, J = 8.03 Hz, 1 H),
.01 (d, J = 9.54 Hz, 1 H), 8.10 (s, 1 H), 8.17 (d, J = 2.01 Hz, 1 H),
.68 (d, J = 2.01 Hz, 1 H); HRMS m/z (ES+) 445.216 72 (MH ) for
(
+
2
mg, 0.49 mmol), Zn(OAc) (5.72 mg, 0.03 mmol), and Zn (12.75 mg,
2
C H FN O.
25 25 6
0
.19 mmol) were added in DMF (5 mL), and then Pd (dba) (14.28
2 3
5-(((3S,4R)-3-Fluoro-1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-
(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2-methylnicotino-
mg, 0.02 mmol) and dppf (17.29 mg, 0.03 mmol) were added. The
mixture was heated to 100 °C for 1 h, filtered through Celite, and
washed with 10% MeOH/DCM. The filtrate was concentrated under
vacuum, and the crude residue was purified by preparative HPLC to
get the required product, which was hygroscopic. The product was
dissolved in 1 mL of DCM and cooled to 0 °C, and 1 mL of HCl in
diethyl ether was added dropwise. The salt that precipitated was
1
nitrile (16). In a 100 mL round-bottomed flask, 29a (520 mg, 2.10
mmol), 28b (500 mg, 1.75 mmol), and sodium carbonate (370 mg,
3.49 mmol) were taken up in DMF (10 mL) to give a yellowish
solution under nitrogen atmosphere that was heated to 70 °C for 1 h.
Solvent was evaporated from the reaction mixture. Water was added to
the residue and it was extracted using 10% methanol in DCM (3 × 25
mL). The combined organic layers were concentrated to dryness and
filtered under nitrogen flow to get the 2HCl salt of 12 (23 mg,
1
2
2
3.30%): H NMR (300 MHz, DMSO-d ) δ 2.05−2.20 (m, 2 H),
purified by silica gel chromatography to yield 16 (450 mg, 58.8%) as
6
1
.35−2.45 (m, 2 H), 2.77 (s, 3 H), 3.05−3.20 (m, 2 H), 3.25−3.45
yellow gel: H NMR (400 MHz, DMSO-d ) δ 1.48−1.63 (m, 1 H),
6
(
4
m, 3 H), 3.80−3.88 (m, 2 H), 4.05 (s, 3 H), 4.50−4.55 (m, 2 H),
1.63−1.74 (m, 1 H), 2.10−2.23 (m, 1 H), 2.25−2.44 (m, 2 H), 2.46−
2.54 (m, 1 H), 2.63 (t, J = 6.53 Hz, 2 H), 2.70 (s, 3 H), 2.90−3.03 (m,
.70−4.75 (m, 2 H), 6.75 (d, J = 9.8 Hz, 1 H), 7.70 (s, 1 H), 7.90 (d, J
N
dx.doi.org/10.1021/jm500432n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
4
8
H), 3.11−3.23 (m, 1 H), 3.85 (s, 2 H), 4.26−4.45 (m, 2 H), 4.69−
.81 (2m, 1 H), 6.89 (d, J = 9.54 Hz, 1 H), 8.00 (d, J = 10.04 Hz, 1 H),
.09 (dd, J = 11.04, 2.01 Hz, 1 H), 8.21 (d, J = 1.51 Hz, 1 H), 8.61 (d,
H), 2.19−2.42 (m, 1 H), 2.57 (t, J = 6.69 Hz, 3 H), 2.90 (d, J = 11.30
Hz, 1 H), 3.02−3.22 (m, 1 H), 3.71 (d, J = 3.39 Hz, 2 H), 4.30 (tq, J =
13.10, 6.77 Hz, 2 H), 4.60 (s, 1 H), 4.64−4.79 (d, J = 45 Hz 1 H), 6.83
(d, J = 9.61 Hz, 1 H), 7.02 (d, J = 8.10 Hz, 1 H), 7.30 (d, J = 8.10 Hz,
1 H), 7.95 (d, J = 9.80 Hz, 1 H), 7.99−8.11 (m, 1 H), 8.56 (d, J = 2.07
J = 2.51 Hz, 1 H), 8.73 (d, J = 2.01 Hz, 1 H); HRMS m/z (ES+)
+
4
39.206 23 (MH ) for C H F N O.
23 24 2 6
+
5
-(((3S,4R)-3-Fluoro-1-(2-(7-methoxy-2-oxo-1,5-naphthyri-
Hz, 1 H), 11.19 (s, 1 H); HRMS m/z (ES+) 471.196 93 (MH ) for
din-1(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2-methylnico-
tinonitrile (17). In a 500 mL round-bottomed flask, 16 (8.50 g, 19.39
mmol) and sodium methoxide (4.19 g, 77.54 mmol) were taken up in
MeOH (150 mL) to give a yellow solution. The resulting reaction
mixture was heated to reflux for 2 h. Solvent was evaporated from the
reaction mixture, and water was added to the residue. This was
extracted using 10% methanol in DCM (3 × 200 mL), and the
combined organic layers were concentrated and purified by silica gel
chromatography using 0−10% methanol in DCM to yield 17 (5.5 g)
as off white solid, which was low-melting and hygroscopic. Therefore,
the free base was dissolved in DCM (60 mL), and HCl in ether (85
mL) was added to it. The precipitated solid was stirred at rt for 2 h and
filtered. The filtered solid was further triturated with methanol/
C H F N O .
23
24
2
6
3
6
-(((3S,4R)-3-Fluoro-1-(2-(7-methoxy-2-oxo-1,5-naphthyri-
din-1(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2H-pyrido[3,2-
b][1,4]oxazin-3(4H)-one (21). In a 50 mL round-bottomed flask,
28c (350 mg, 1.17 mmol), 29b (803 mg, 1.29 mmol), DIPEA (0.615
mL, 3.52 mmol), and sodium carbonate (249 mg, 2.35 mmol) were
taken up in DMF (5 mL) to give a yellowish solution under nitrogen
atmosphere that was heated to 70 °C for 1 h. Solvent was evaporated
from the reaction mixture. Water was added to the residue and
extracted using 10% methanol in DCM (3 × 25 mL). The combined
organic layers were concentrated to dryness and purified by
1
preparative HPLC to yield 21 (120 mg, 21.20%) as white solid: H
NMR (300 MHz, DMSO-d ) δ 1.46−1.74 (m, 2 H), 2.16 (t, J = 10.27
Hz, 1 H), 2.24−2.45 (m, 2 H), 2.59 (t, J = 6.88 Hz, 3 H), 2.86−2.98
(m, 1 H), 3.08−3.17 (m, 1 H), 3.73 (s, 2 H), 3.99 (s, 3 H), 4.27−4.44
(m, 2 H), 4.61 (s, 2 H), 4.65−4.82 (2m, 1 H), 6.66 (d, J = 9.61 Hz, 1
H), 7.02 (d, J = 8.10 Hz, 1 H), 7.30 (d, J = 7.91 Hz, 1 H), 7.42 (d, J =
2.26 Hz, 1 H), 7.87 (d, J = 9.61 Hz, 1 H), 8.28 (d, J = 2.26 Hz, 1 H),
6
acetonitrile, filtered, and dried to give the HCl salt of 17 (6.50 g,
1
6
2
4
4.1%) as pale yellow solid: H NMR (300 MHz, DMSO-d ) δ 2.18−
6
.47 (m, 2 H), 2.72 (s, 3 H), 3.23−3.75 (m, 7 H), 4.07 (s, 3 H), 4.26−
.46 (m, 2 H), 4.63−4.86 (m, 2 H), 5.50−5.66 (d, J = 45 Hz 1H), 6.73
d, J = 9.80 Hz, 1 H), 7.75 (s, 1 H), 7.95 (d, J = 9.80 Hz, 1 H), 8.34 (d,
(
+
J = 2.07 Hz, 1 H), 8.57 (d, J = 2.07 Hz, 1 H), 8.95 (d, J = 2.07 Hz, 1
11.15 (br s, 1 H); HRMS m/z (ES+) 483.215 93 (MH ) for
H), 10.22−10.33 (2 br s, 2 H), 11.25 (br s, 1 H); HRMS m/z (ES+)
C H FN O .
24
27
6
4
+
4
51.225 43 (MH ) for C H FN O ·2HCl.
6-(((3R,4S)-3-Fluoro-1-(2-(7-methoxy-2-oxo-1,5-naphthyri-
24
27
6
2
5-(((3S,4R)-3-Methoxy-1-(2-(7-methoxy-2-oxo-1,5-naphthyr-
din-1(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2H-pyrido[3,2-
b][1,4]oxazin-3(4H)-one (22). In a 100 mL round-bottomed flask,
22a (1.5 g, 3.19 mmol) was dissolved in MeOH (30 mL), and sodium
methoxide (0.689 g, 12.75 mmol) was added. The mixture was heated
to 80 °C overnight. The solvent was evaporated under vacuum, the
idin-1(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2-methylnico-
tinonitrile (18). In a 50 mL round-bottomed flask, 18b (150 mg, 0.45
mmol) was dissolved in DMF (5 mL), sodium carbonate (143 mg,
1
.35 mmol) was added, and the mixture was stirred for 2 min. Then 32
(
102 mg, 0.45 mmol) was added to the reaction and heated to 70 °C
residue was basified with saturated NaHCO solution and extracted
with 10% MEOH/DCM, and the solvent was evaporated to get the
3
for 2 h. The reaction mixture was concentrated under vacuum, and the
residue was basified with saturated NaHCO solution, concentrated
under vacuum, and purified by preparative HPLC to give 18 (30.0 mg,
crude compound, which was purified by preparative HPLC to give 22
3
1
(1.2 g, 78%): H NMR (300 MHz, DMSO-d ) δ 1.46−1.76 (m, 2 H),
6
1
1
2.42%) as a gum: H NMR (300 MHz, DMSO-d ) δ 1.35−1.52 (m, 1
1.96−2.22 (m, 2 H), 2.22−2.45 (m, 1 H), 2.57 (t, J = 6.78 Hz, 3 H),
2.93 (d, J = 11.30 Hz, 1 H), 3.15 (br s, 1 H), 3.72 (s, 2 H), 3.92−4.09
(m, 3 H), 4.35 (t, J = 6.22 Hz, 2 H), 4.60 (s, 2 H), 4.63−4.88 (d, J =
51 Hz, 1 H), 6.66 (d, J = 9.61 Hz, 1 H), 7.02 (d, J = 8.10 Hz, 1 H),
7.30 (d, J = 8.10 Hz, 1 H), 7.42 (s, 1 H), 7.87 (d, J = 9.61 Hz, 1 H),
6
H), 1.58−1.74 (m, 1 H), 1.97−2.11 (m, 1 H), 2.20−2.44 (m, 2 H),
2
(
.54−2.61 (m, 2 H), 2.61−2.85 (m, 6 H), 3.17 (s, 3 H), 3.25−3.32
m, 1 H), 3.74 (d, J = 4.33 Hz, 2 H), 3.98 (s, 3 H), 4.27−4.44 (m, 2
H), 6.66 (d, J = 9.61 Hz, 1 H), 7.43 (s, 1 H), 7.86 (d, J = 9.80 Hz, 1
H), 8.17 (s, 1 H), 8.27 (d, J = 2.07 Hz, 1 H), 8.67 (s, 1 H); HRMS m/
+
8.28 (s, 1 H), 11.19 (s, 1 H); HRMS m/z (ES+) 483.216 40 (MH )
+
z (ES+) 463.245 32 (MH ) for C H N O .
for C24H27FN O .
6 4
25
30
6
3
6
-(((3S,4R)-3-Fluoro-1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-
(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2H-pyrido[3,2-b]-
1,4]oxazin-3(4H)-one (19). In a 250 mL round-bottomed flask, 28b
7.86 g, 27.47 mmol), 29b (7.00 g, 24.97 mmol), and sodium
6-(((3S,4R)-3-Methoxy-1-(2-(7-methoxy-2-oxo-1,5-naphthyr-
idin-1(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2H-pyrido-
[3,2-b][1,4]oxazin-3(4H)-one (23). In a 250 mL round-bottomed
flask, 23c (2.300 g, 4.77 mmol) and sodium methoxide (1.288 g, 23.83
mmol) were taken up in MeOH (50 mL) to give a white solution
under nitrogen atmosphere and heated to 70 °C for 2 h. The solvent
was evaporated from the reaction mixture. Water was added to the
residue and extracted using 10% methanol in DCM (3 × 100 mL).
The combined organic layers were concentrated to dryness and
purified by silica gel chromatography using 0−10% methanol in DCM
1
[
(
carbonate (5.29 g, 49.95 mmol) were taken up in DMF (50 mL) to
give a yellowish solution under nitrogen atmosphere that was heated
to 70 °C for 1 h. Solvent was evaporated from the reaction mixture.
Water was added to the residue and it was extracted using 10%
methanol in DCM (5 × 75 mL). The combined organic layers were
concentrated to dryness and purified by silica gel column using 0−12%
1
1
methanol in DCM to yield 19 (5.40 g, 46.0%) as a white solid: H
and 0.1% ammonia to yield 23 (0.350 g, 14.85%) as off white solid: H
NMR (300 MHz, DMSO-d ) δ 1.44−1.68 (m, 2 H), 2.2−2.39 (m, 3
NMR (300 MHz, DMSO) δ 1.73 (br s, 2 H), 1.99−2.20 (m, 2 H),
2.51−2.71 (m, 2 H), 2.99 (br s, 1 H), 3.09−3.23 (m, 4 H), 3.62 (br s,
1H), 3.94 (s, 3 H), 3.95−4.10 (m, 3 H), 4.21−4.47 (m, 2 H), 4.63 (s,
2 H), 6.61 (d, J = 9.61 Hz, 1 H), 7.06 (d, J = 7.91 Hz, 1 H), 7.30−7.47
(m, 2 H), 7.83 (d, J = 9.61 Hz, 1 H), 8.24 (d, J = 2.07 Hz, 1H), 8.75
(br s, 1H), 11.30 (s, 1H); HRMS m/z (ES+) 495.230 61 (MH ) for
C H N O .
25 30 6 5
6
H), 2.57 (t, J = 6.59 Hz, 2 H), 2.84−2.97 (m, 1 H), 3.06−3.20 (m, 1
H), 3.60−3.82 (m, 2 H), 4.14−4.43 (m, 2 H), 4.61 (s, 3 H), 4.63−4.79
(
d, J = 45 Hz, 1 H), 6.83 (d, J = 9.80 Hz, 1 H), 7.02 (d, J = 8.10 Hz, 1
H), 7.30 (d, J = 7.91 Hz, 1 H), 7.95 (d, J = 9.80 Hz, 1 H), 8.04 (dd, J =
1.21, 2.17 Hz, 1 H), 8.56 (d, J = 2.26 Hz, 1 H), 11.20 (s, 1 H);
+
1
+
HRMS m/z (ES+) 471.196 74 (MH ) for C H F N O .
23
24
2
6
3
6
-(((3R,4S)-3-Fluoro-1-(2-(7-fluoro-2-oxo-1,5-naphthyridin-
(2H)-yl)ethyl)piperidin-4-ylamino)methyl)-2H-pyrido[3,2-b]-
1,4]oxazin-3(4H)-one (20). In a 250 mL round-bottomed flask, 29c
1
[
ASSOCIATED CONTENT
■
* Supporting Information
Sythetic schemes and procedures for intermediates; primary
sequence alignment of Mtb GyrA and Sau GyrA; Overlay of
secondary structures of Mtb GyrA model and Sau GyrA;
comparison of active site residues of Mtb GyrA model and Sau
GyrA crystal structure; docked poses of compounds 1a, 2a, and
(
(
(
3.50 g, 12.49 mmol) was dissolved in DMF (50 mL) and Na CO
3.97 g, 37.46 mmol) was added. The mixture stirred for 2 min, 28b
4.29 g, 14.98 mmol) was added, and the reaction was heated to 70 °C
2 3
S
for 2 h. The reaction mixture was concentrated under vacuum, and the
residue was basified with sat. NaHCO solution, concentrated under
3
vacuum, and purified by preparative HPLC to give 20 (2.90 g, 49.4%):
1
H NMR (300 MHz, DMSO) δ 1.41−1.70 (m, 2 H), 1.93−2.19 (m, 3
O
dx.doi.org/10.1021/jm500432n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
9; comparison of the binding mode of FQ and NBTIs in the
Sau DNA gyrase crystal structure; assay procedures for log D
and hERG measurement. This material is available free of
charge via the Internet at http://pubs.acs.org.
Article
1
phase 2 trial. Lancet 2009, 373, 1183−1189. (c) Diacon, A. H.;
Dawson, R.; von Groote-Bidlingmaler, F.; Symons, G.; Venter, A.;
Donald, P. R.; van Niekerk, C.; Everitt, D.; Winter, H.; Becker, P.;
Mendel, C. M.; Spigelman, M. K. 14 day bactericidal activity of PA-
8
24, bedaquiline, pyrazinamide, and moxifloxacin combinations: A
randomized trial. Lancet 2012, 380, 986−993. (d) Duong, D. A.;
Nguyen, T. H.; Nguyen, T. N.; Dai, V. H.; Dang, T. M.; Vo, S. K.; Do,
D. A.; Nguyen, V. V.; Nguyen, H. D.; Dinh, N. S.; Farrar, J.; Caws, M.
Beijing genotype of Mycobacterium tuberculosis is significantly
associated with high-level fluoroquinolone resistance in Vietnam.
Antimicrob. Agents Chemother. 2009, 53, 4835−4839.
AUTHOR INFORMATION
Corresponding Author
Phone: +91 80 23621212, Ext. 6292. Fax: +91 80 23621214.
E-mail: shahul.mehar@astrazeneca.com; shahulmehar@yahoo.
com.
■
*
(
6) Drlica, K.; Malik, M.; Kerns, R. J.; Zhao, X. Quinolone-mediated
bacterial death. Antimicrob. Agents Chemother. 2008, 52, 385−392.
7) (a) Bisacchi, G S.; Dumas, J. Recent advances in the inhibition of
Present Addresses
Medicines for Malaria Venture (MMV), Geneva, Switzerland.
Swedish Institute for Communicable Disease Control, S-171
2 Solna, Sweden.
⊥
(
#
bacterial type II topoisomerases. In Annual Reports in Medicinal
Chemistry; Macor, J. E., Ed.; Academic Press: Burlington, MA, 2009;
Vol. 44, pp 379−396. (b) Shirude, P. S.; Hameed, S. Non-
fluoroquinolone-based inhibitorsof mycobacterial type II topoisomer-
ase as potential therapeutic agentsfor TB. In Annual Reports in
Medicinal Chemistry; Desai, M. C., Ed.; Academic Press: Burlington,
MA, 2012; Vol. 47, pp 319−330.
8
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We sincerely thank CMG Bangalore, AZ Global DMPK, Safety,
Biosciences (AZ, Boston, MA), and Discovery Sciences (AZ,
Alderly Park, UK) for technical support in various assays. We
acknowledge Sandeep Ghorpade, Manoranjan Panda, and
Pravin Shirude for scientific discussions. We also thank V.
Balasubramanian, R. Tommasi, and Tanjore Balganesh for their
constant inspiration, support, and encouragement.
(8) (a) Bax, B. D.; Chan, P. F.; Eggleston, D. S.; Fosberry, A.; Gentry,
D. R.; Gorrec, F.; Giordano, I.; Hann, M. M.; Hennessy, A.; Hibbs, M.;
Huang, J.; Jones, E.; Jones, J.; Brown, K. K.; Lewis, C. J.; May, E. W.;
Saunders, M. R.; Singh, O.; Spitzfaden, C. E.; Shen, C.; Shillings, A.;
Theobald, A. J.; Wohlkonig, A.; Pearson, N. D.; Gwynn, M. N. Type
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ABBREVIATIONS USED
■
TB, tuberculosis; FQ, fluoroquinolone; Mtb, Mycobacterium
tuberculosis; Msm, Mycobacterium smegmatis; MIC, minimum
inhibitory concentration; HB, H-bond; SAR, structure−activity
relationship; SPR, structure−property relationship; AUC, area
under the curve; cfu, colony forming units; rt, room
temperature; DME, 1,2-dimethoxyethane; THF, tetrahydrofur-
an; DMF, N,N-dimethylformamide; ABT, aminobenzotriazole;
BSL-3, biosafety level 3 laboratory.
(9) (a) Breault, G.; Eyermann, C. J.; Geng, B.; Morningstar, M.;
Reck, F. Preparation of quinolinones and analogs for the treatment of
multi-drug resistant bacterial infections. WO2006134378, 2006.
(b) Janelle,C. P.; Mark, C.; Geng, B.; Aydon, G. A.; Reck, F.
Preparation of 2-quinolinone and 2-quinoxalinone derivatives as
antibacterial agents. WO2008071961, 2008. (c) Breault, G.; Vincent,
G.; Reck, F. Preparation of naphthyridines as bactericides for treating
bacterial infections. WO2008071964, 2008. (d) Geng, B.; Morningstar,
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infections. WO2008071981, 2008.
(10) Reck, F.; Alm, R.; Brassil, P.; Newman, J.; DeJonge, B.;
Eyermann, C. J.; Breault, G.; Breen, J.; Comita-Prevoir, J.; Cronin, M.;
Davis, H.; Ehmann, D.; Galullo, V.; Geng, B.; Grebe, T.; Morningstar,
M.; Walker, P.; Hayter, B.; Fisher, S. Novel N-linked aminopiperidine
inhibitors of bacterial topoisomerase type II: Broad-spectrum
antibacterial agents with reduced hERG activity. J. Med. Chem. 2011,
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