Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II: Broad-spectrum antibacterial agents with reduced hERG activity

Article abstract of DOI:10.1021/jm2008826

Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. Aminopiperidines that have a bicyclic aromatic moiety linked through a carbon to an ethyl bridge, such as 1, generally show potent broad-spectrum antibacterial activity, including quinolone-resistant isolates, but suffer from potent hERG inhibition (IC₅₀= 3 M for 1). We now disclose the finding that new analogues of 1 with an N-linked cyclic amide moiety attached to the ethyl bridge, such as 24m, retain the broad-spectrum antibacterial activity of 1 but show significantly less hERG inhibition (IC ₅₀= 31 M for 24m) and higher free fraction than 1. One optimized analogue, compound 24l, showed moderate clearance in the dog and promising efficacy against Staphylococcus aureus in a mouse thigh infection model.

Full text of DOI:10.1021/jm2008826

Article
pubs.acs.org/jmc
Novel N-Linked Aminopiperidine Inhibitors of Bacterial
Topoisomerase Type II: Broad-Spectrum Antibacterial Agents with
Reduced hERG Activity
Folkert Reck,*,† Richard Alm,† Patrick Brassil,† Joseph Newman,† Boudewijn DeJonge,†
Charles J. Eyermann,† Gloria Breault,† John Breen,† Janelle Comita-Prevoir,† Mark Cronin,†
Hajnalka Davis,† David Ehmann,† Vincent Galullo,† Bolin Geng,† Tyler Grebe,† Marshall Morningstar,†
Phil Walker,‡ Barry Hayter,‡ and Stewart Fisher†
†Infection Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
‡AstraZeneca Research Support Laboratory, Block 25, Mereside, Alderley Park Macclesfield, Cheshire, SK10 4TG, United Kingdom
ABSTRACT: Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases
(DNA gyrase and topoisomerase IV) are of interest for the development of new
antibacterial agents that are not impacted by target-mediated cross-resistance with
fluoroquinolones. Aminopiperidines that have a bicyclic aromatic moiety linked through
a carbon to an ethyl bridge, such as 1, generally show potent broad-spectrum anti-
bacterial activity, including quinolone-resistant isolates, but suffer from potent hERG
inhibition (IC50= 3 μM for 1). We now disclose the finding that new analogues of 1 with
an N-linked cyclic amide moiety attached to the ethyl bridge, such as 24m, retain the
broad-spectrum antibacterial activity of 1 but show significantly less hERG inhibition
(IC50= 31 μM for 24m) and higher free fraction than 1. One optimized analogue,
compound 24l, showed moderate clearance in the dog and promising efficacy against
Staphylococcus aureus in a mouse thigh infection model.
safety profile with this class of compounds. The chemical
structures of NBTIs are quite diverse but have an overall similar
topology: A bicyclic aromatic left-hand side (LHS) that stacks
between two base pairs of the DNA in the GyrA/DNA
complex, an aromatic right-hand side (RHS) that interacts with
the protein, and a linker between the LHS and RHS that is
mostly solvent exposed but does pick up an interaction with
Asp83 that has been shown to be important for binding.2
We were interested in the aminopiperidine subclass of
NBTIs exemplified by compounds 1 and 2 (Figure 1),
INTRODUCTION
■
The prevalence of drug-resistant pathogenic bacteria is
increasing globally1 and is necessitating research into develop-
ment of new antibacterial agents that lack cross-resistance
mediated by mutations in the bacterial targets. Bacterial type II
topoisomerases (DNA gyrase and topoisomerase IV) are
clinically proven antibacterial targets, with many important
agents of the fluoroquinolone class of drugs targeting the GyrA
and ParC subunits of DNA gyrase and topoisomerase IV,
respectively. Novel (non-fluoroquinolone) bacterial type II
topoisomerase inhibitors (NBTIs) have been described in the
literature2−4 and are not impacted by target mutations that
cause resistance to fluoroquinolones. The binding site of
NBTIs in the DNA-bound gyrase complex of Staphylococcus
aureus has been determined by crystallography2 and does not
overlap with the two fluoroquinolone binding sites. NBTIs are
thus of particular interest for the development of new
antibacterial agents because they act on a clinically proven
antibacterial target through a novel mechanism of inhibition
and retain activity against fluoroquinolone-resistant isolates.
NBTIs were first discovered in the laboratories of
GlaxoSmithKline and Aventis Pharma AG, and several
pharmaceutical companies are now filing patent applications
or publishing in this area.5−10 Viquidacin (NXL-101), an NBTI
discovered by Aventis and developed by Novexel, underwent
phase I trials but was discontinued as a result of QTc
prolongation observed in phase I trials,11 emphasizing the
intrinsic challenges for achieving an acceptable cardiovascular
Figure 1. C-Linked aminopiperidine leads from the patent literature
(GSK).6
Received: July 6, 2011
Published: October 14, 2011
© 2011 American Chemical Society
7834
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
Figure 2. Compounds from the literature6,38,39 used to build the pharmacophore model.
a
Scheme 1
a
Reagents: (a) Fe, AcOH, 90 °C, 93%.
Figure 3. Pharmacophore-based overlay of seven NBTI reference
compounds.
a
Scheme 2
a
Reagents: (a) NEt3, (CH3)3SiCHN2, CH2Cl2/MeOH/CH3CN,
room temp, 67%.
a
Scheme 3
Figure 4. (a) Active hit from the pharmacophore and shape virtual
screen. (b) N-Linked hit from the virtual screen. (c) Overlay of a N-linked
LHS hit (green carbons) onto the query molecule (cyan carbons) used to
define the pharmacophore search. (d) Orthogonal view of (c).
a
Reagents: (a) N-methylmorpholine, isobutyl chloroformate, THF,
room temp, then NaBH4, water, then Pd on C/H2, THF, 24%; (b)
NEt3, triphosgene, toluene, room temp, 36%.
identified by researchers at GSK,6 because 1 and 2 displayed
potent broad-spectrum antibacterial activity combined with
good physicochemical properties, such as sufficiently high
solubility for iv formulation. It soon became apparent to us
that 1 and 2 carried considerable cardiovascular safety risk
arising from potent hERG inhibition, with IC50 values of 3
and 4 μM, respectively. We embarked on a lead optimization
Figure 5. Overlay of a NBTI inhibitor from pharmacophore modeling
(cyan carbons) versus NBTI inhibitor conformation (gold carbons)
observed bound to GyrA from the reported crystal structure.2
7835
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
a
a
Scheme 4
Scheme 7
a
Reagents: (a) KOCN, AcOH, room temp, 64%.
a
Reagents: (a) ethyl glyoxylate, MeOH, room temp, 13% for 4m, 95%
for 4p.
a
Scheme 5
a
Scheme 8
a
Reagents: (a) DBU, N(iPr)2Et, 100−120 °C, 70%.
a
program with the goal of identifying novel analogues that
retain the potent antibacterial activity and good physico-
chemical properties of the leads but show significantly
reduced hERG liability.
Reagents: (a) NaOMe, MeOH, 70 °C, 71%.
a
Scheme 9
MOLECULAR MODELING
■
One well-established strategy for identifying new leads is virtual
screening of molecular databases for compounds matching a
pharmacophore model. Since the NBTI X-ray structure with
GyrA was not available at the outset, we employed a ligand-
based approach to building an NBTI pharmacophore model.
The known NBTI literature was examined. Compounds with
reported antibacterial activity were identified, and a diverse,
conformationally constrained subset of seven compounds was
selected (Figure 2). The TFIT pharmacophore modeling
software12 was used to identify low-energy conformations
that maximized the overlap of the LHS aromatic rings, the
basic center of the amino group, and the RHS aromatic rings
of the seven reference NBTIs. The array of tentative
“bioactive” conformations identified using this approach is
shown in Figure 3. A pharmacophore and shape search of a
multiconformer version of our corporate database yielded
several interesting hits that matched the LHS, RHS, and basic
center pharmacophore features of NBTIs. Some of the virtual
screening hits were selected for IC50 testing in a supercoiling
assay.13 While the virtual screening hit shown in Figure 4a was
active in the supercoiling assay (IC50 = 1.5uM), the most useful
aspect of the pharmacophore search output was in the
inspiration for new compound design. For example, our focus
quickly turned to considering the use of N-linked LHS
chemistry after seeing the overlays for an N-linked LHS hit
(Figure 4b−d). The recent publication of the X-ray structure of
an NBTI bound to a Staphylococcus aureus gyrase/DNA
complex provided experimental evidence for the bioactive
conformation of the aminopiperidine class of NBTI anti-
a
Reagents: (a) H2SO4, room temp, then NaNO2, 90%.
bacterials.2 The NBTI conformation observed in the X-ray
structure compared to the bioactive conformation from our
ligand-based approach (Figure 5) showed a high degree
of similarity, with only a slight rotation of the RHS in the
ligand-based model. This result is not surprising given that
the seven compounds used to build the pharmacophore
model contained more conformational constraints in the LHS
and linker and little information to constrain the
conformation of the RHS. Overall, the ligand-based
pharmacophore model has proven to be a powerful tool in
our search for improved NBTIs and has helped us design new
and potent NBTI chemotypes.
CHEMISTRY
■
Compounds were assembled through N-alkylation of hetero-
cycles 4, followed by reductive aminations with RHS aldehydes
as the key synthetic steps5 (Scheme 11).
The syntheses of LHS heterocycles 4 are outlined in
Schemes 1−9. Alkylations of 4 with mesylate 19 gave
aminopiperidine derivatives 20 (Scheme 11). Mesylate 19
was freshly prepared for immediate use from the alcohol 18
(Scheme 10). 19 is unstable under storage. Alkylations with 19
a
Scheme 6
a
Reagents: (a) ethyl bromoacetate, K2CO3, 150 °C; (b) H2, Pd/C, MeOH/AcOH, room temp, 64% (combined for steps a and b); (c) H2O2, water,
80 °C, then AcOH, room temp, 91%.
7836
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
a
Scheme 10
a
Reagents: (a) 2-bromoethanol, NEt3, CH3CN, 50 °C, 66%; (b) MsCl, NEt3, CH2Cl2, 0 °C.
a
Scheme 11
a
Reagents: (a) NaH, DMF, 0 °C to room temp; (b) TFA, CH2Cl2, 0 °C; (c) NaOMe, MeOH, 140 °C, 61%; (d) sodium dithionite, DMF/water,
100 °C, 27%; (e) 3 Å molecular sieves, CHCl3/MeOH, 70 °C, then NaBH(OAc)3, 0 °C to room temp; (f) NaOMe/MeOH, 78 °C, 38%.
generally afforded 20 together with smaller amounts of O-
alkylation products. O-Alkylated product generally had higher
Rf by TLC than N-alkylated product and was efficiently
removed by chromatography. N-Alkylation of 20 was
confirmed by NMR studies, with carbon chemical shifts for
the CH2N moiety observed in the characteristic range of 40−45
ppm vs 60−70 ppm for CH2O in the case of O-alkylation.
Further transformations on the LHS were carried out in some
cases on the Boc-protected intermediates (20q, 20o), rather
than unprotected interemediates 4, because of the higher
7837
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
solubility of 20 in most organic solvents. Cleavage of the Boc
group in 20 gave free amines 21, generally in quantitative
yields. Reductive aminations of aldehydes 2214 and 2315 with
amines 21 gave the best results when performed with the free
i
Table 1. SAR of Left-Hand Side (LHS)
7838
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
Table 1. continued
a
b
c
d
Methicillin-susceptible Staphylococcus aureus. Streptococcus pneumonia D39 (penicillin-susceptible). Pseudomonas aeruginosa PAO1. Escherichia
coli W3110. Escherichia coli TopoIV IC50. Partion coefficient at pH 7.4. Fraction unbound, human, % free. hERG Ionworks IC50.19 Minimum
e
f
g
h
i
inhibitory concentration (MIC, μg/mL): lowest drug concentration that reduced growth by 80% or more.
bases of 21a−t in the presence of freshly activated molecular
sieves. The mixtures were initially heated to drive imine
formation to completion, then cooled for the reduction with
sodium triacetoxyborohydride. When the reduction was carried
out at elevated temperatures or with more reactive reducing
agents, such as sodium borohydride, then over-reduction was
observed with quinoxalinones, for example, with 21l.
The methoxy substituent was replaced by a fluoro or a cyano
substituent to give compounds with similar potencies (25c,d).
4-Methyl substitution was tolerated (24i). A partially saturated
moiety in the LHS was also tolerated, as was apparent from the
potent IC50 values for benzoxazinones 24a,c,d and the cyclic
carbamate 25g. However, the thiazinone 24e and the
dihydroquinoline 25f lacked potent target affinity, which
indicated that the degree of puckering as a function of bond
length may be an important factor for recognition of the LHS.
Taking the 2-quinolone derivative 24h as a starting point, the
introduction of additional nitrogen atoms in the LHS was
tolerated. Derivatives containing 4- or 8-aza substituions (24l,
24m, and 24r) showed an increase in target potency, whereas
the introduction of a nitrogen in the 3- or 6-position resulted in
a reduction in potency (24j, 24n, 24p, 24s, and 25t).
The antibacterial activity (MIC) exhibited by N-linked
aminopiperidines was similar to that of the C-linked reference
compounds 1 and 2 and inhibited the growth of Gram-positive
(Staphylococcus aureus and Streptococcus pneumoniae) and
Gram-negative organisms (Escherichia coli and Pseudomonas
aeruginosa) (Table 1). Plasma protein binding for some N-
linked aminopiperidines (24h, 24l, 24m) was significantly
reduced compared to 1 and 2, likely due to lower log D values.
Aminopiperidines tended to be more potent against Gram-
positive organisms than against Gram-negatives. The situation
was reversed though for 25t, which had a relatively low log D of
−0.65 and retained Gram-negative activity but lacked potent
activity against Gram-positive organisms. This finding is in
agreement with the general observation that log D for agents
targeting Gram-positive organisms tend to be higher than for
agents that target Gram-negative organisms.17 This observation
is likely due to the structure of the outer membrane in Gram-
negatives, with hydrophilic porins presenting an important
access route.18
RESULTS AND DISCUSSION
■
A prototype for the N-linked aminopiperidine series was
identified as a hit in a virtual screen against the pharmacophore
model. We realized that linking the LHS through the nitrogen
of a cyclic amide moiety such as in 24m would enable us to
explore a novel molecular framework with the potential for
lower lipophilicity compared to earlier compounds from this
class. We rationalized that the carbonyl group of the LHS may
give good target potency by restraining conformational rotation
in the ethyl bridge, similar to the fluoro substituent in 1 and 2,6
albeit potentially with a lower log D. Reduction of log D was
seen as desirable to improve the safety profile of the
compounds, especially to reduce hERG inhibition.16 A series
of new compounds was prepared,5 combining novel bicyclic N-
linked LHSs with RHSs that were optimized for the
corresponding C-linked series.6 N-Linked aminopiperidines
24 and 25 and C-linked reference compounds 1 and 2 were
evaluated for inhibition of bacterial topoisomerase IV,
antibacterial activity, and inhibition of the hERG ion channel
(Table 1).
N-Linked aminopiperidines showed potent binding to
bacterial topoisomerase IV from E. coli with IC50 in the low
nanomolar range for the best compounds (25c, 25g, 24m, 24l).
The benzoxazinone RHS moiety gave slightly higher target
potency than the dioxinopyridine RHS (cf. 1 to 2). The
presence of a substituent in the LHS was important for potent
binding, which was evident by the 20-fold difference in IC50
between the unsubstituted 24b and the methoxy derivative 25a.
Aminopiperidines were evaluated for inhibition of the hERG
channel in a functional ionworks assay.19 IC50 correlated
7839
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
a
Table 2. Pharmacokinetics in Rat and Dog
compd
clearance (mL min−1 kg−1
)
volume (L/kg)
half-life (h)
AUC (iv) μg·h/mL
bioavailability (%)
64
rat
25c
24l
25c
24l
66
23
32
61
11
5.8
2.8
166
8.3
0.64
0.19
3.09
2.3
338
7.8
21
dog
a
Pharmacokinetic parameters following administration of 4 mg/kg (compound 24l, rat) or 3 mg/kg (compounds 25c and 24l, dog), iv bolus
injection. Oral bioavailability was assessed following a 10 mg/kg dose.
a
Table 3. Exposure and Efficacy of Compound 24l Compared to Levofloxacin in a Mouse Pneumonia Model
compound 24l
levofloxacin
log reduction in cfu/lung vs vehicle-
treated control
log reduction in cfu/lung vs vehicle-
treated control
AUC∞ (μg h−1 mL−1
)
AUC∞ (μg h−1 mL−1
)
12 (mg kg−1 day−1
24 (mg kg−1 day−1
36 (mg kg−1 day−1
48 (mg kg−1 day−1
)
)
)
)
0.47
0.76
1.17
1.79
0.05
1.17
1.50
ND
ND
b
b
2.50
1.46
ND
ND
ND
ND
2.79
a
b
In vivo activity against Staphylococcus aureus 516 (ARC516). 20 mg kg−1 day−1 dose.
roughly with log D,16 with the more polar N-linked compounds
like 24m showing a significantly improved hERG profile over
the C-linked reference compound 1 (31 μM vs 3 μM). The
structural feature of an N-linked LHS thus represents an
important advance for the design of NBTIs with reduced QT
liability. Still, further improvements of the hERG profile are
likely needed to eliminate QT liability in this series completely.
The pharmacokinetics of selected compounds were deter-
mined in the rat and dog (Table 2). N-Linked aminopiperidines
showed high volumes of distribution in both species, with high
clearance in the rat and moderate clearance in the dog.
Compound 25c showed good bioavailability in the dog (64%).
Compound 24l was active against Staphylococcus aureus in a
neutropenic mouse thigh infection model (Table 3), with a
1 log reduction in colony forming units (cfu) observed at a
dose of 24 mg kg−1 day−1. The response per dose was similar to
that of the comparator, levofloxacin, in this model.
according to the Clinical and Laboratory Standards Institute
guidelines.20 Compounds were dissolved in 100% DMSO and diluted
to 2% DMSO (v/v) in culture medium to 11 doubling dilutions from
64 to 0.06 μg/mL. Specific culture medium was as follows: For
Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli:
Mueller Hinton broth, Difco no. 275730. For Streptococcus pneumo-
niae: 2.2% (w/v) Mueller Hinton broth, 0.65% (v/v) lysed horse
blood (Hema Resource and Supply no. 15-14-0100-28). Inoculants
were incubated at 35 °C on blood agar plates (Remel no. 01202) for
18−24 h. Plates were read by spectrophotometry at 620 nm.
Topoisomerase IV Assay. The assay utilizes the ATPase activity
of the ParE subunit of reconstituted Escherichia coli ParC/ParE
tetramer protein. Inhibition of ATPase activity was monitored by
reduced production of inorganic phosphate, a product of the ATPase
reaction. Inorganic phosphate was quantified using the ammonium
molybdate/malachite green-based detection system. For determination
of IC50 values, assays were performed in 384-well microtiter plates.
Each well contained a dilution range of the compound dissolved in
DMSO. In addition, each well contained 20 mM Tris, pH 8.0, 50 mM
ammonium acetate, 0.16 mM ATP, 0.005% Brij-35, 8.0 mM
magnesium chloride, 0.5 mM EDTA, 2.5% v/v glycerol, 5 mM
dithiothreitol, 0.005 mg/mL sheared salmon sperm DNA, 0.5 nM E.
coli ParC protein, and 0.5 nM E. coli ParE protein. Final volume of
assays was 30 μL. Mixtures were incubated for 24 h at room
temperature, and reactions were quenched with the addition of 30 μL
of malachite green reagent21 via a bulk reagent dispenser. Plates were
incubated 3−5 min at room temperature, and then absorbance at
650 nM was measured using a Spectramax 384 plate reader. Values
reported are the mean from three replicate experiments.
Plasma Protein Binding. Plasma protein binding was deter-
mined using the Dianorm equilibrium dialysis chamber. Compound
(10 μM) was spiked in the plasma chamber (donor side); phosphate
buffer was placed in the receiver side. The unit was rotated at 37 °C
for 16 h. Drug concentration was determined for the plasma sample
that represents the bound fraction, and drug concentration was
determined for the buffer sample that represents the free fraction.
LC/MS−MS quantitative sample analysis was achieved using an Ace
C18 50 mm × 4.6 mm column (MacMod, PA) and electrospray
ionization MRM detection (PE Sciex API 4000 mass spectrometer,
Applied Biosystems CA). Plasma samples (50 μL) were treated with
methanol (150 μL) containing an internal standard to precipitate the
protein. Concentration determination was based on a standard curve
(10 nM to 10 μM). Data were processed by the Analyst, version 1.4.1,
software.
CONCLUSIONS
■
We report the use of a rationally conceived pharmacophore
model and a virtual screening exercise for idea generation in our
bacterial topoisomerase inhibitor program. This led to the
identification of novel aminopiperidine inhibitors with an
N-linked cyclic amide containing LHS instead of the usual
C-linked quinoline and naphthyridine LHS found in the patent
literature. We report on the synthesis and biological evaluation
of N-linked aminopiperidines and show that these analogues
display excellent broad-spectrum antibacterial activity with a
significantly improved hERG profile. Pharmacokinetic param-
eters for two optimized analogues (25c and 24l) were
determined in the rat and dog, showing high clearance in the
rat but moderate clearance in the dog. Compound 24l was
evaluated in a neutropenic mouse thigh infection model against
Staphylococcus aureus and found to be efficacious with a 1 log
reduction in cfu at a dose of 24 mg kg−1 day−1. N-Linked
aminopiperidines thus show promise for the development of
novel antibacterial agents.
EXPERIMENTAL SECTION
Minimum Inhibitory Concentration Testing. Minimum inhib-
itory concentrations were determined by broth microdilution
■
7840
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
log D Determination. The partition coefficient (log D) was
measured by shake flask method, using 10 mM phosphate buffer at
pH 7.4 and n-octanol. The samples were allowed to reach equilibrium
by shaking for 1 h at 1200 rpm, and sample analysis was done by LC/
UV, with MS for mass confirmation.
Animals. Wistar Han rats for pharmacokinetic studies were
obtained from Charles River Laboratories (Raleigh, NC). CD-1 mice
were obtained from Charles River Laboratories (Kingston, NY). All
animals were housed and acclimated in the animal facility on site
before each study. All experimental procedures were conducted in
accordance with protocols approved by the Institutional Animal Care
and Use Committee.
7-Methoxy-3,4-dihydroquinolin-2(1H)-one (4f). A mixture of
7-hydroxy-3,4-dihydroquinolin-2(1H)-one 524 (3 g, 17 mmol) and
triethylamine (3.14 mL, 22 mmol) in dichloromethane/methanol/
acetonitrile (10:1:10, 168 mL) was treated with (trimethylsilyl)-
diazomethane (2 M solution in hexanes, 10.25 mL, 20.5 mmol). The
mixture was stirred overnight at room temperature, and the solvent
was removed under reduced pressure. Chromatography on silica gel
with hexanes/acetone (1:1) gave 2.2 g (67%) of the product as a
1
colorless solid. MS (ESP) m/z 178.16 (MH+). H NMR (DMSO-d6)
δ: 2.38 (t, 2H); 2.81 (t, 2H); 3.68 (s, 3H); 6.68−6.78 (m, 3H); 9.90
(brs, 1H).
7-Methoxy-1,4-dihydro-2H-3,1-benzoxazin-2-one (4g). A
solution of (2-amino-4-methoxyphenyl)methanol 7 (744 mg, 4.85
mmol) in toluene (25 mL) was treated with triethylamine (1.36 mL,
9.7 mmol) and triphosgene (1.58 g, 5.34 mL). The reaction mixture
was stirred at room temperature for 1.5 h and then was heated to
110 °C for an additional 2 h. The reaction mixture was cooled to room
temperature, diluted with water (25 mL), and filtered. The aqueous
layer was extracted with toluene (3 × 30 mL) and ethyl acetate (2 ×
20 mL). The combined organic layers were washed with brine, dried
over sodium sulfate, and concentrated under reduced pressure.
Chromatography on silica gel with hexanes/ethyl acetate (3:2) gave
Pharmacokinetic Studies. Pharmacokinetic properties of se-
lected compounds were studied in the rat. Groups of three Wistar
Han rats were administered test compound at a dose of 3 mg/kg or
4 mg/kg by bolus injection into a cannulated jugular vein. Oral
bioavailability was determined following a 10 mg/kg dose given by oral
gavage. Serial 200 μL samples of whole blood were taken at time
intervals. Concentration of compound in plasma was determined by
LC−MS/MS, and pharmacokinetic parameters were estimated using a
noncompartmental model in WinNonLin (Pharsight). Exposure in
CD-1 mice was determined for analysis of the efficacy studies. At
timed intervals, groups of three mice were sacrificed and whole blood
samples collected by cardiac puncture. Plasma samples were prepared
and analyzed as described above. Similarly, plasma pharmacokinetics
were determined from 0 to 24 h in male Beagle dogs (n = 3) following
15 min iv infusions at 3 mg/kg or oral administration at 10 mg/kg.
Staphylococcus aureus Neutropenic Thigh Infection Model.
Compound 24l was studied in a neutropenic mouse thigh infection
model as described in the paper by Mills et al.22 Briefly, mice were
rendered neutropenic by injecting cyclophosphamide (Sigma-Aldrich,
St. Louis, MO) intraperitoneally for 4 days (150 mg/kg body weight)
and 1 day (100 mg/kg) before experimental infection. Mice were
infected with Methicillin-susceptible Staphylococcus aureus (ARC516,
AstraZeneca culture collection) to achieve a target inoculum of 5 × 105
cfu/thigh. Groups of five animals each received an intraperitoneal
injection of 24l at the doses specified in Table 3, prepared in 5%
dextrose with lactic acid, pH 5.0, on a q.d. regime starting 2 h after
infection. An additional group of five mice received vehicle alone.
Efficacy was determined 24 h after the start of treatment. Thigh tissue
was homogenized with an Omni TH homogenizer (Omni Interna-
tional, Warrenton, VA), plated onto tryptic soy agar plates, and
incubated at 37 °C overnight for cfu determination.
1
314 mg (36%) of the product as a colorless solid. H NMR (DMSO-
d6) δ: 3.78 (s, 3H); 5.26 (s, 2H); 6.38 (d, 1H); 6.59 (d, 1H); 6.98 (d,
1H); 8.36 (brs, 1H).
7-Methoxy-4-methylquinazolin-2(1H)-one (4j). To a solution
of commercially available 2-amino-4-methoxyacetophenone 8 (1.07 g)
in acetic acid (20 mL) was added potassium cyanate (1.19 g) at room
temperature. The reaction mixture was stirred overnight, then poured
into water and neutralized with solid sodium hydrogen carbonate. The
mixture was extracted with ethyl acetate and then with chloroform.
The combined organic phases were washed with brine, dried over
sodium sulfate, and concentrated under reduced pressure. Chromatog-
raphy on silica gel with 0−10% methanol in dichloromethane gave
0.79 g (64%) of the product as a colorless solid. MS (ESP) m/z 191
1
(MH+). H NMR (DMSO-d6) δ: 2.59 (s, 3H); 3.85 (s, 3H); 6.70 (d,
1H); 6.82 (dd, 1H); 7.87 (d, 1H).
7-Chloro-1,6-naphthyridin-2(1H)-one (4k). A solution of (E)-
ethyl 3-(4-amino-6-chloropyridin-3-yl)acrylate 925 (0.617 g, 2.73
mmol) in diisopropylethylamine (5 mL) was treated with DBU
(0.7 mL, 4.5 mmol). The reaction mixture was heated to 100 °C for
18 h. The temperature was increased to 120 °C for an additional 24 h.
The solvent was removed at reduced pressure. The residue was
purified by chromatography on silica gel, eluting with 0−6% methanol
in dichloromethane to give the product as a yellow solid (0.346 g,
General Chemical Methods. All commercially available solvents
and reagents were used without further purification. All moisture-
sensitive reactions were carried out under a nitrogen atmosphere in
commercially available anhydrous solvents. Column chromatography
was performed on 230−400 mesh silica gel 60. Aluminum-backed
sheets of silica gel 60 F254 (EM Science) were used for TLC. Melting
points were obtained with a Mel-Temp II melting point apparatus
1
70%). H NMR (methanol-d4) δ: 6.68 (d, J = 9.6 Hz, 1H); 7.30
(s, 1H); 8.03 (d, J = 9.6 Hz, 1H); 8.67 (s, 1H).
7-Methoxyquinoxalin-2(1H)-one (4l).26,27 To a solution of 8%
aqueous sodium hydroxide (1.32 L) was added 12 (100 g) followed by
a solution of 30 wt % hydrogen peroxide in water (1.17 L). The
reaction mixture was slowly heated to 80 °C and maintained at this
temperature for 4 h. The heating source was removed, and acetic acid
(150 mL) was added dropwise. The suspension was stirred overnight
at room temperature and the precipitated solid was collected by
filtration to afford the product as a tan solid (90 g, 91%). MS (ESP)
m/z 177 (MH+). 1H NMR (DMSO-d6) δ: 3.83 (s, 3H); 6.76 (d, 1H);
6.90 (dd, 1H); 7.67 (d, 1H); 7.97 (s, 1H); 12.32 (brs, 1H).
6-Methoxypyrido[2,3-b]pyrazin-3(4H)-one (4m). To a solu-
tion of commercially available 3,4-diamino-6-methoxypyridine 13
(1.11 g) in methanol (20 mL) was added ethyl glyoxalate (3.5 mL).
The reaction mixture was stirred overnight at room temperature, then
filtered and washed with methanol (the precipitate contained the
undesired regioisomer 6-methoxypyrido[2,3-b]pyrazin-2(1H)-one).
The filtrate was concentrated and suspended in diethyl ether to give
0.18 g product (13%) as a colorless solid. MS (ESI) m/z 178 (MH+).
1H NMR (DMSO-d6) δ: 6.77 (d, 1H); 8.01 (s, 1H); 8.07 (d, 1H);
12.83 (s, 1H).
1
from Laboratory Devices, Inc. and are uncorrected. H NMR spectra
were recorded at 300 or 400 MHz. Chemical shifts are reported in
ppm (δ) relative to solvent. The purity of tested compounds was
assessed by LC−MS. Reverse phase HPLC was carried out using YMC
Pack ODS-AQ (100 mm × 20 mm i.d., S-5μ particle size, 12 nm pore
size) on Agilent instruments. Mass spectrometry was performed using
a Micromass Quattro Micro mass spectrometer (for ESP) and an
Agilent 1100 MSD instrument (for APCI). All compounds tested
possessed a purity of at least 95%.
6-Methoxy-2H-1,4-benzothiazin-3(4H)-one (4e). To a solu-
tion of ethyl [(4-methoxy-2-nitrophenyl)thio]acetate23 (3 g, 11 mmol)
in acetic acid (30 mL) was added iron powder (1.7 g, 31 mmol). The
reaction mixture was heated at 90 °C for 3 h. It was cooled to room
temperature, diluted with ethyl acetate, filtered through Celite, and
concentrated to dryness under reduced pressure. Silica gel
chromatography with hexanes/ethyl acetate (3:2) gave 2 g (93%) of
the product as a colorless solid. MS (ESP) m/z 196 (MH+). 1H NMR
(CDCl3-d) δ: 3.39 (s, 2H); 3.78 (s, 3H); 6.44 (d, 1H); 6.59 (dd, 1H);
7.20 (d, 1H); 8.63 (brs, 1H).
2-Methoxy-8H-pyrido[2,3-d]pyrimidin-7-one (4n).28 A mix-
ture of 2-methanesulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one 1529
(740 mg, 3.5 mmol) in NaOMe solution (0.5 M in methanol,
7841
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
70 mL, 15 mmol) was heated at 70 °C for 2 h, then cooled to room
temperature. The mixture was quenched with glacial acetic acid,
filtered through Celite and the residue rinsed with methanol. The
combined filtrate and wash were concentrated, and the residue was
partitioned between water and dichloromethane. The layers were
separated, and the aqueous phase was back-extracted with chloroform/
isopropyl alcohol (3:1, 5 × 50 mL). The combined organic phases
were dried over sodium sulfate and concentrated under reduced
pressure. Chromatography on silica gel with dichloromethane/
methanol (3:1) gave the product as a colorless solid in 71% yield.
10.6 g of the crude product as a tan solid. This material was used
1
without further purification. MS (ESP) m/z 179 (MH+). H NMR
(DMSO-d6) δ: 3.61 (m, 5H); 5.57 (m, 1H); 6.35−6.40 (m, 2H); 6.60
(m, 1H); 10.13 (brs, 1H).
tert-Butyl [1-(2-Hydroxyethyl)piperidin-4-yl]carbamate
(18). A mixture of commercially available tert-butyl piperidin-4-
ylcarbamate 17 (5 g, 25 mmol), 2-bromoethanol (1.77 mL, 25
mmol), and triethylamine (3.86 mL, 27.5 mmol) in acetonitrile
(20 mL) was heated in a sealed tube at 50 °C for 16 h. The solvent
was removed under reduced pressure, and the residue was taken up in
ethyl acetate (300 mL) and washed with saturated aqueous sodium
hydrogen carbonate solution (100 mL). The aqueous phase was back-
extracted once with ethyl acetate (100 mL), and the combined organic
phases were dried over sodium sulfate and concentrated under
reduced pressure. Chromatography on silica gel with dichloro-
methane/methanol (4:1) gave 4.04 g (66%) of the product as a
1
MS (ESP) m/z 178 (MH+). H NMR (DMSO-d6) δ: 4.02 (s, 3H);
6.50 (d, J = 9.4 Hz, 1H); 7.97 (d, J = 9.4 Hz, 1H); 8.93 (s, 1H).
6-Methoxy-3-oxo-3,4-dihydrobenzo[e][1,2,4]triazine 1-
Oxide (4o).30 To a suspension of 3-amino-6-methoxybenzo[e]-
[1,2,4]triazine 1-oxide 1631 (1.17 g, 6.08 mmol) in water (15 mL)
was added concentrated sulfuric acid (3.5 mL), followed by sodium
nitrite (1.6 g, 23 mmol) in small portions. After 15 min the reaction
mixture was filtered and the residue was washed with water and dried
under reduced pressure at 105 °C to give the product as a colorless
1
colorless solid, mp 66 °C. MS (ESP) m/z 245 (MH+). H NMR
(DMSO-d6) δ: 1.33 (m, 2H); 1.36 (s, 9H); 1.62 (m, 2H); 1.92 (t,
2H); 2.32 (t, 2H); 2.77 (m, 2H); 3.17 (m, 1H); 3.43 (m, 2H); 4.34 (t,
1H); 6.73 (d, 1H).
1
solid in 90% yield, mp 238−240 °C. MS (ESP) m/z 194 (MH+). H
NMR (DMSO-d6) δ: 3.88 (s, 3H); 6.69 (s, 1H); 6.92 (d, J = 9.3 Hz,
1H); 8.03 (d, J = 9.3 Hz, 1H); 12.48 (s, 1H).
2-{4-[(tert-Butoxycarbonyl)amino]piperidin-1-yl}ethyl Meth-
anesulfonate (19). A mixture of 18 (1.7 g, 7 mmol) in dry
dichloromethane (20 mL) and triethylamine (1.4 mL, 9.8 mmol) was
treated at 0 °C with methanesulfonyl chloride (0.65 mL, 8.4 mmol).
After 45 min the reaction was complete by TLC (chloroform/
methanol 6:1, Rf = 0.54). Potassium phosphate buffer (pH 7, 1 M,
50 mL) was added. Dichloromethane was removed under reduced
pressure, and the residue was extracted with ice-cold ethyl acetate (2 ×
100 mL) and dried over sodium sulfate. The solvent was removed
under reduced pressure, and the crude preparation of the mesylate was
used without delay for the next step. MS (ESP) m/z 323.18 (MH+).
General Procedure for 20 by Alkylation of 4 with 19. A
solution of 4 (2 mmol) in dry dimethylformamide (10 mL) was
treated at 0 °C under stirring with sodium hydride (60% in oil,
2 mmol). The cooling bath was removed, and the mixture was stirred
for 30 min at room temperature. Freshly prepared 19 in a solution
with DMF (0.58 mmol/mL, 2 mmol) was added, and the resulting
mixture was stirred overnight at room temperature. DMF was removed
under reduced pressure, and the residue was taken up in ethyl acetate
(100 mL) and saturated aqueous sodium hydrogen carbonate solution
(30 mL). The aqueous phase was back-extracted once with ethyl
acetate (50 mL). The combined organic phases were dried over
sodium sulfate and concentrated under reduced pressure. Generally O-
alkylation was observed as a minor product and removed by
chromatography. Chromatography conditions are given below for
individual compounds.
2-Methoxypteridin-7(8H)-one (4p). To a solution of commer-
cially available 2-methoxy-4,5-pyrimidine diamine 14 (1.83 g) in
methanol (25 mL) was added ethyl glyoxalate (5 mL). The mixture
was stirred overnight at room temperature, then heated to reflux for
2 h. The reaction mixture was cooled with an ice bath and the resulting
precipitate was collected by filtration and washed with methanol to
yield the product as an off-white solid (2.20 g, 95%). MS (ESP) m/z
179 (MH+). 1H NMR (DMSO-d6) δ: 3.98 (s, 3H); 8.06 (s, 1H); 8.95
(s, 1H); 13.08 (s, 1H).
(2-Amino-4-methoxyphenyl)methanol (7).32 To a solution of
commercially available 4-methoxy-2-nitrobenzoic acid 6 (4 g, 20.66
mmol) in tetrahydrofuran (20 mL) at −15 °C under nitrogen was
added N-methylmorpholine (2.2 mL, 20 mmol) followed by isobutyl
chloroformate (2.6 mL, 20 mmol) dropwise. After 5 min, the reaction
mixture was filtered and the solid was rinsed with tetrahydrofuran
(20 mL). The filtrate was cooled to −15 °C, and a solution of sodium
borohydride in water (3 M, 10 mL) was added. The mixture was
stirred for 5 min, then partitioned between water and dichloro-
methane. The aqueous layer was extracted with dichloromethane (3 ×
100 mL), and the combined organic layers were washed with brine,
dried over magnesium sulfate, and concentrated under reduced
pressure. The residue was dissolved in tetrahydrofuran (35 mL).
Palladium (10% on carbon, 400 mg) was added, and the mixture was
stirred under hydrogen gas for 18 h. The reaction mixture was filtered
through Celite, and the filtrate was concentrated under reduced
pressure. Chromatography on silica gel with hexanes/ethyl acetate
tert-Butyl {1-[2-(6-Methoxy-3-oxo-2,3-dihydro-4H-1,4-ben-
zoxazin-4-yl)ethyl]piperidine-4-yl}carbamate (20a). 20a was
prepared from 4a33 and 19 according to the general procedure for
20. Chromatography on silica gel with hexanes/ethyl acetate (1:1)
1
(3:2) gave 744 mg (24%) of the product as a yellow solid. H NMR
(DMSO-d6) δ: 3.63 (s, 3H); 4.30 (s, 2H); 4.84−4.96 (m, 2H); 6.08
(dd, 1H); 6.20 (d, 1H); 6.90 (d, 1H).
1
afforded the product in 60% yield. MS (ESP) m/z 406 (MH+). H
Ethyl N-(4-Methoxy-2-nitrophenyl)glycinate (11). A mixture
of 4-methoxy-2-nitroaniline (25.0 g, 0.15 mol), ethyl bromoacetate
(200 mL, 1.8 mol), and potassium carbonate (31.1 g, 0.23 mol) was
heated at 150 °C for 4.5 h. The mixture was cooled to room
temperature, and aqueous sodium hydroxide solution (1M, 600 mL)
was added. This mixture was extracted with ethyl acetate (2 ×
500 mL). The combined organic phases were dried over magnesium
sulfate and concentrated under reduced pressure. Chromatography
was done on silica gel with 25−50% acetone in hexanes to give 22.1 g
NMR (CDCl3-d) δ: 1.34 (m, 2H); 1.36 (m, 2H); 1.62 (m, 2H); 1.98
(m, 2H); 2.43 (m, 2H); 2.84 (m, 2H); 3.20 (m, 1H); 3.73 (s, 3H);
3.96 (m, 2H); 4.53 (s, 2H); 6.56 (m, 1H); 6.76 (m, 1H); 6.92 (m,
1H).
tert-Butyl {1-[2-(3-Oxo-2,3-dihydro-4H-1,4-benzoxazin-4-yl)-
ethyl]piperidin-4-yl}carbamate (20b). 20b was prepared from
commercially available 4b and 19 according to the general procedure
for 20. Chromatography on silica gel with dichloromethane/methanol
(20:1) gave the product in 63% yield as a yellow gum. MS (ESP) m/z
376 (MH+).
1
of the crude product as a red solid. H NMR revealed the presence of
∼20% dialkylated product. This material was used without further
1
tert-Butyl {1-[2-(6-Cyano-3-oxo-2,3-dihydro-4H-1,4-benzox-
azin-4-yl)ethyl]piperidin-4-yl}carbamate (20c). 20c was pre-
pared from 4c34 and 19 according to the general procedure for 20.
Chromatography on silica gel with hexanes/ethyl acetate (1:3)
purification for the next step. H NMR (DMSO-d6) δ: 1.18−1.23 (t,
3H); 3.74 (s, 3H); 4.12−4.18 (q, 2H); 4.23−4.25 (d, 2H); 6.90 − 6.93
(d, 1H); 7.25−7.29 (dd, 1H); 7.51−7.52 (d, 1H); 8.23−8.27 (t, 1H).
7-Methoxy-3,4-dihydroquinoxalin-2(1H)-one (12).26,27 11
(15.8 g crude) was taken up in 200 mL of 1:1 methanol/acetic acid,
treated with 10% palladium on carbon (2 g), and stirred in an
atmosphere of hydrogen overnight. The reaction mixture was filtered
through Celite and the filtrate was concentrated to dryness to give
1
afforded the product in 76% yield. MS (ESP) m/z 401 (MH+). H
NMR (CDCl3-d) δ: 1.41 (m, 2H); 1.42 (s, 9H); 1.93 (m, 2H); 2.21
(m, 2H); 2.58 (m, 2H); 2.87 (m, 2H); 3.45 (m, 1H); 4.02 (m, 2H);
4.43 (m, 1H); 4.67 (s, 2H); 7.03 (d, 1H); 7.30 (m, 1H); 7.47 (s, 1H).
7842
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
tert-Butyl {1-[2-(6-Fluoro-3-oxo-2,3-dihydro-4H-1,4-benzox-
azin-4-yl)ethyl]piperidin-4-yl}carbamate (20d). 20d was pre-
pared from 4d35 and 19 according to the general procedure for 20.
Chromatography on silica gel with hexanes/ethyl acetate (1:1) gave
as a mixture with 4k. This mixture was carried on to the next step
without further purification. H NMR (methanol-d4) δ: 1.33 (s, 9H);
1
1.75 (d, J = 13.0 Hz, 2H); 1.99−2.22 (m, 2H); 2.47−2.68 (m, 2H);
2.71−2.98 (m, 2H); 3.48−3.61 (m, 1H); 4.30 (t, J = 7.1 Hz, 1H); 6.60
(dd, J = 18.3, 9.6 Hz, 1H); 7.57 (s, 1H); 7.90 (t, J = 9.8 Hz, 1H); 8.56
(d, J = 2.8 Hz, 1H).
tert-Butyl {1-[2-(7-Methoxy-2-oxoquinoxalin-1(2H)-yl)ethyl]-
piperidin-4-yl}carbamate (20l). 20l was prepared from 4l and 19
according to the general procedure for 20. Chromatography on silica
gel with 25% acetone in hexanes gave the product as a colorless solid,
29% yield. MS (ESP) m/z 403 (MH+). 1H NMR (DMSO-d6) δ:
1.26−1.40 (m, 11H); 1.57−1.72 (m, 2H); 1.97−2.11 (m, 2H); 2.51−
2.61 (m, 2H); 2.85−2.98 (m, 2H); 3.19 (s, 1H); 3.92 (s, 3H); 4.32 (t,
2H); 6.76 (d, 1H); 6.95−7.04 (m, 2H); 7.70−7.78 (m, 1H); 8.04 (s,
1H).
tert-Butyl {1-[2-(6-Methoxy-3-oxopyrido[2,3-b]pyrazin-
4(3H)-yl)ethyl]piperidin-4-yl}carbamate (20m). 20m was pre-
pared from 4m and 19 according to the general procedure for 20.
Chromatography on silica gel with 0−25% acetone in dichloro-
methane gave the product in 67% yield. MS (ESI) m/z 404 (MH+).
1H NMR (CDCl3) δ: 1.31−1.40 (m, 2H); 1.40−1.46 (m, 9H); 1.87−
1.95 (m, 2H); 2.15−2.27 (m, 2H); 2.69−2.75 (m, 2H); 2.93−3.02 (m,
2H); 3.40−3.51 (m, 1H); 4.02 (s, 3H); 4.35−4.46 (m, 1H); 4.51−
4.60 (m, 2H); 6.73 (d, 1H); 8.02 (d, 1H); 8.15 (s, 1H).
1
the product in 67% yield. MS (ESP) m/z 394.26 (MH+). H NMR
(CDCl3-d) δ: 1.39 (m, 2H); 1.43 (s, 9H); 1.92 (m, 2H); 2.20 (m,
2H); 2.58 (t, 2H); 2.88 (m, 2H); 3.45 (m, 1H); 3.98 (t, 2H); 4.41 (m,
1H); 4.55 (s, 2H); 6.68 (m, 1H); 6.85 (m, 1H); 6.91 (m, 1H).
tert-Butyl {1-[2-(6-Methoxy-3-oxo-2,3-dihydro-4H-1,4-ben-
zothiazin-4-yl)ethyl]piperidin-4-yl}carbamate (20e). 20e was
prepared from 4e and 19 according to the general procedure for 20.
Chromatography on silica gel with hexanes/ethyl acetate (1:3)
afforded the product in 85% yield. MS (ESP) m/z 422.24 (MH+).
1H NMR (CDCl3-d) δ: 1.40 (m, 2H); 1.45 (s, 9H); 1.92 (m, 2H);
2.22 (m, 2H); 2.62 (t, 2H); 2.88 (m, 2H); 3.35 (s, 2H); 3.49 (m, 1H);
3.82 (s, 3H); 4.08 (t, 2H); 4.43 (m, 1H); 6.61 (dd, 1H); 6.86 (d, 1H);
7.28 (s, 1H).
tert-Butyl {1-[2-(7-Methoxy-2-oxo-3,4-dihydroquinolin-
1(2H)-yl)ethyl]piperidin-4-yl}carbamate (20f). 20f was prepared
from 4f and 19 according to the general procedure for 20.
Chromatography on silica gel, eluting with ethyl acetate and then
acetone/dichloromethane (4:1), gave the product as a colorless oil,
(82%). MS (ESP) m/z 404 (MH+). 1H NMR (DMSO-d6) δ: 1.32 (m,
2H); 1.36 (s, 9H); 1.64 (m, 2H); 1.93−2.00 (m, 4H); 2.37 (m, 2H);
2.47 (m, 2H); 2.78 (m, 2H); 3.16 (m, 1H); 3.71 (s, 3H); 3.91(t, 2H);
6.72−6.82 (m, 3H); 7.05 (d, 1H).
tert-Butyl 1-(2-(2-Methoxy-7-oxopyrido[2,3-d]pyrimidin-
8(7H)-yl)ethyl)piperidin-4-ylcarbamate (20n). 20n was prepared
from 4n and 19 according to the general procedure for 20.
Chromatography on silica gel with 10% methanol in dichloromethane
tert-Butyl {1-[2-(7-Methoxy-2-oxo-2H-3,1-benzoxazin-1(4H)-
yl)ethyl]piperidin-4-yl}carbamate (20g). A solution of 4g (310 mg,
1.8 mmol) in dry N,N-dimethylformamide (5 mL) was treated at 0 °C
under stirring with lithium bis(trimethylsilyl)amide (1 M in
tetrahydrofuran, 1.9 mL, 1.9 mmol). The mixture was stirred for
30 min at room temperature and treated with 19 (2.03 mmol) as
described in the general procedure for 20. Chromatography on silica
gel with dichloromethane/methanol (95:5) gave 333 mg (47%) of the
1
gave the product in 59% yield. MS (ESP) m/z 404 (MH+). H NMR
(DMSO-d6) δ: 1.28 (m, 2H); 1.38 (s, 9H); 1.65 (m, 2H); 1.90−2.10
(m, 2H); 2.56 (m, 1H); 2.92 (m, 2H); 3.18 (m, 1H); 4.01 (s, 3H);
4.38 (t, J = 6.9 Hz, 2H); 6.50−6.59 (m, 1H); 6.76 (m, J = 7.5 Hz, 1H);
7.89−8.01 (m, 1H); 8.83−8.98 (m, 1H).
4-(2-(4-(tert-Butoxycarbonylamino)piperidin-1-yl)ethyl)-6-
methoxy-3-oxo-3,4-dihydrobenzo[e][1,2,4]triazine 1-Oxide
(20o). 20o was prepared from 4o and 19 according to the general
procedure for 20. The residue obtained after aqueous workup was
taken up in ether, and the precipitate was collected by filtration and
dried under reduced pressure to give the product in 39% yield. MS
1
product as a colorless solid. MS (ESP) m/z 406 (MH+). H NMR
(CDCl3) δ: 1.34−1.46 (m, 11H); 1.86 (d, 2H); 2.12−2.25 (m, 2H);
2.60−2.64 (m, 2H); 2.60−2.64 (m, 2H); 2.81−2.90 (m, 2H); 3.38−
3.45 (m, 1H); 3.76 (s, 3H); 3.97−3.91 (m, 2H); 4.32−4.42 (m, 1H);
5.05 (s, 2H); 6.49−6.56 (m, 2H); 6.94 (d, 1H).
tert-Butyl {1-[2-(7-Methoxy-2-oxoquinolin-1(2H)-yl)ethyl]-
piperidin-4-yl}carbamate (20h). 20h was prepared from 4h24
and 19 according to the general procedure for 20. Chromatography on
silica gel with hexanes/acetone (1:1) gave the product as a colorless
hard foam, 20% yield. MS (ESP) m/z 402 (MH+). 1H NMR (DMSO-
d6) δ: 1.30−1.42 (m, 2H); 1.36 (s, 9H); 1.66 (m, 2H); 2.04 (m, 2H);
2.45−2.53 (m, 2H); 2.92 (m, 2H); 3.15 (m, 1H); 3.88 (s, 3H); 4.31 (t,
2H); 6.39 (m, 1H); 6.76 (m, 1H); 6.88 (m, 1H); 6.93 (m, 1H); 7.62
(d, 1H); 7.80 (d, 1H).
tert-Butyl {1-[2-(7-Methoxy-4-methyl-2-oxoquinolin-1(2H)-
yl)ethyl]piperidin-4-yl}carbamate (20i). 20i was prepared from
4i36 and 19 according to the general procedure for 20.
Chromatography on silica gel, eluting with a gradient of 5−7%
methanol in methylene chloride, gave the product as a light brown oil,
74% yield. MS (ESP) m/z 411 (MH+).
1
(ESP) m/z 442 (MNa+). H NMR (CDCl3) δ: 1.22 (s, 1H); 1.26 (s,
1H); 1.30 (s, 1H); 1.35 (s, 9H); 1.64 (d, J = 10.93 Hz, 2H); 2.03 (t,
J = 10.83 Hz, 2H); 2.58 (t, J = 6.50 Hz, 2H); 2.85−2.95 (m, 2H); 3.17
(s, 1H); 3.98 (s, 3H); 4.28 (t, J = 6.5 Hz, 2H); 6.75 (d, J = 7.72 Hz,
1H); 6.99 (dd, J = 9.42, 2.26 Hz, 1H); 7.04 (d, J = 2.07 Hz, 1H); 8.14
(d, J = 9.42 Hz, 1H).
tert-Butyl {1-[2-(2-Methoxy-7-oxopteridin-8(7H)-yl)ethyl]-
piperidin-4-yl}carbamate (20p). 20p was prepared from 4p and
19 according to the general procedure for 20. Chromatography on
silica gel with a gradient of 0−5% methanol in dichloromethane gave
the product as a colorless hard foam in 83% yield. MS (ESP) m/z 405
(MH+). 1H NMR (CDCl3) δ: 1.24−1.35 (m, 2H); 1.43 (s, 9H); 1.84−
1.95 (m, 2H); 2.14−2.24 (m, 2H); 2.71 (t, 2H); 2.91−2.96 (m, 2H);
3.38−3.49 (m, 1H); 4.06−4.12 (m, 3H); 4.33−4.43 (m, 1H); 4.46 (t,
2H); 8.13 (s, 1H); 8.93 (s,1H).
tert-Butyl {1-[2-(7-Methoxy-4-methyl-2-oxoquinazolin-
1(2H)-yl)ethyl]piperidin-4-yl}carbamate (20j). 20j was prepared
from 4j and 19 according to the general procedure for 20. Reverse
phase HPLC chromatography on a 19 mm × 100 mm ODO AQ C18
column, eluting with a gradient of 0.1% TFA/water to 0.1% TFA/
acetonitrile, gave the product in 35% yield. MS (ESP) m/z 417
tert-Butyl-{1-[2-(7-chloro-2-oxo-1,8-naphthyridin-1(2H)-yl)-
ethyl]piperidin-4-yl}carbamate (20q). 20q was prepared from 7-
chloro-1,8-naphthyridin-2(1H)-one 4q37 and 19 according to the
general procedure for 20. Chromatography on silica gel with 20%
acetone in hexanes gave the product as a colorless solid in 67% yield.
1
MS (ESP) m/z 407 (MH+). H NMR (DMSO-d6) δ: 1.20 (s, 2H);
1
(MH+). H NMR (CDCl3) δ: 1.36−1.43 (m, 2H); 1.37−1.47 (m,
1.35 (s, 9H); 1.56−1.67 (m, 2H); 1.92−2.05 (m, 2H); 2.45−2.51 (m,
J = 3.20 Hz, 2H); 2.52 (s, 1H); 2.85−2.97 (m, 2H); 3.09−3.23 (m,
1H); 4.34−4.43 (m, 2H); 6.70 (d, J = 9.4 Hz, 1H); 7.39 (d, J = 8.1 Hz,
1H); 7.97 (d, J = 9.6 Hz, 1H); 8.21 (d, J = 8.1 Hz, 1H).
tert-Butyl {1-[2-(7-Methoxy-2-oxo-1,8-naphthyridin-1(2H)-
yl)ethyl]piperidine-4-yl}carbamate (20r). A solution of 20q
(0.68 g, 1.67 mmol) and sodium methoxide (2.55 mmol) in methanol
(5.0 mL) was heated in a sealed tube at 140 °C for 30 min using
microwave irradiation. The reaction mixture was concentrated under
reduced pressure and then quenched with water. The product was
extracted with ethyl acetate, and the organic phase was washed with
9H); 1.89−1.98 (m, 2H); 2.21−2.31 (m, 2H); 2.66−2.70 (m, 2H);
2.71 (s, 2H); 2.87−2.97 (m, 2H); 3.47 (s, 1H); 3.94 (s, 3H); 4.28−
4.37 (m, 2H); 4.43 (s, 1H); 6.78 (d, 1H); 6.82 (dd, 1H); 7.80 (d, 1H).
tert-Butyl 1-(2-(7-Chloro-2-oxo-1,6-naphthyridin-1(2H)-yl)-
ethyl)piperidin-4-ylcarbamate (20k). A suspension of 4k (0.348 g,
1.933 mmol) in DMF (7 mL) was treated with lithium bis-
(trimethylsilyl)amide (1 M, 2.0 mL). The mixture was stirred at
room temperature for 10 min, then cooled to 0 °C and reacted with 19
according to the general procedure for 20. Chromatography on silica
gel with 0−5% methanol in dichloromethane gave the crude product
7843
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
brine and then dried over sodium sulfate. Chromatography on silica gel
with 10% methanol in dichloromethane gave the product as a colorless
solid in 61% yield. MS (ESP) m/z 403 (MH+). 1H NMR (DMSO-d6) δ:
1.28 (s, 1H); 1.35 (s, 9H); 1.63−1.71 (m, 2H); 2.06 (t, 2H); 2.52 (m,
1H); 2.87−2.94 (m, 2H); 2.96 (m, 2H); 3.13−3.20 (m, 1H); 3.20 − 3.29
(m, 2H); 3.97 (s, 3H); 4.41−4.49 (m, 1H); 6.48 (d, J = 9.5 Hz, 1H); 6.70
(d, J = 8.1 Hz, 1H); 7.07 (d, J = 9.5 Hz, 1H); 8.04 (d, J = 8.1 Hz, 1H).
tert-Butyl 1-(2-(6-Methoxy-3-oxobenzo[e][1,2,4]triazin-
4(3H)-yl)ethyl)piperidin-4-ylcarbamate (20t). To a solution of
20o (0.440 g, 1.05 mmol) in DMF (7 mL) were added water (7 mL)
and sodium dithionite (900 mg). The mixture was heated in a
microwave reactor at 100 °C for 2 h. The reaction mixture was washed
with brine and extracted with ethyl acetate (40 mL). The organic layer
was dried over sodium sulfate and concentrated under reduced
pressure. Chromatography was done on silica gel with 0−10%
methanol in methylene chloride to give the product in 27% yield. MS
(ESP) m/z 404 (MH+). 1H NMR (DMSO-d6) δ: 1.17−1.33 (m, 2H);
1.37 (s, 9H); 1.64 (m, 2H); 1.90−2.14 (m, 2H); 2.61 (m, 2H); 3.17
(m, 1H); 4.01 (s, 3H); 4.27 (t, J = 6.3 Hz, 2H); 6.76 (d, 1H); 7.01 (m,
1H); 7.11 (d, 1H); 7.95 (m, 2H); 8.27 (d, 1H).
General Procedure for 21 by Deprotection of 20. A solution
of 20 (16.6 mmol) in dichloromethane (100 mL) was treated with
trifluoroacetic acid (40 mL) at 0 °C for 30 min. The solvent was
removed under reduced pressure and the residue codistilled once with
dichloromethane, then taken up in dichloromethane (200 mL) and
washed with saturated sodium hydrogen carbonate solution (50 mL,
pH adjusted to 10 with sodium hydroxide). The aqueous phase was
back-extracted with dichloromethane (3 × 100 mL) and dried over
sodium sulfate. The combined organic phases were concentrated
under reduced pressure to give the products.
procedure for 21a to give the trifluoroacetate salt of the product in
quantitative yield. MS (ESP) m/z 316 (MH+).
1-[2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxy-4-methylqui-
nazolin-2(1H)-one (21j). 21j was prepared from 20j according to
the procedure for 21a to give the trifluoroacetate salt of the product in
1
quantitative yield. MS (ESP) m/z 317 (MH+);. H NMR (CDCl3) δ:
1.36−1.46 (m, 2H); 1.80−1.88 (m, 2H); 2.21 (td, 2H); 2.67−2.76 (m,
6H); 2.98 (d, 2H); 3.95 (s, 3H); 4.30−4.38 (m, 2H); 6.80−6.85 (m,
2H); 7.77−7.83 (m, 1H).
1-(2-(4-Aminopiperidin-1-yl)ethyl)-7-chloro-1,6-naphthyri-
din-2(1H)-one (21k). A solution of 20k (0.449 g, 1.106 mmol) in
dichloromethane was treated with a solution of hydrochloric acid in
dioxane (4M, 8 mmol) The mixture was stirred at room temperature
overnight. The precipitate was collected by filtration to give the bis-
hydrochloride salt of the product as a colorless solid in quantitative
yield. MS (ESP) m/z 307 (MH+).
1-[2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxyquinoxalin-
2(1H)-one (21l). 21l was prepared from 20l according to the general
procedure for 21 to give the crude product in 77% yield as an oil. MS
(ESP) m/z 303 (MH+).
4-[2-(4-Aminopiperidin-1-yl)ethyl]-6-methoxypyrido[2,3-b]-
pyrazin-3(4H)-one (21m). 21m was prepared from 20m according
to the general procedure for 21 to give the crude product in 94% yield.
MS (ESP) m/z 304 (MH+).
8-(2-(4-Aminopiperidin-1-yl)ethyl)-2-methoxypyrido[2,3-d]-
pyrimidin-7(8H)-one (21n). 21n was prepared from 20n according
to the procedure for 21a to give the trifluoroacetate salt of the product
in quantitative yield as a brown hard foam. MS (ESP) m/z 304
1
(MH+). H NMR (DMSO-d6) δ: 1.73 (m, 2H); 1.94−2.24 (m, 2H);
3.14 (m, 2H); 3.30 (brs, 1H); 3.83 (m, 2H); 4.04 (s, 3H); 4.61 (m,
2H); 6.62 (m, 1H); 8.01 (d, J = 9.6 Hz, 1H); 8.15 (m, 3H); 8.97 (s,
1H); 9.66 (brs, 1H).
4-[2-(4-Aminopiperidin-1-yl)ethyl]-6-methoxy-2H-1,4-ben-
zoxazin 3(4H)-one (21a). 21a was prepared from 20a according to
the general procedure for 21, but aqueous workup was omitted, to give
the trifluoroacetate salt of the product in quantitative yield. MS (ESP)
m/z 306 (MH+).
4-[2-(4-Aminopiperidin-1-yl)ethyl]-2H-1,4-benzoxazin-
3(4H)-one (21b). 21b was prepared from 20b according to the
procedure for 21a to give the trifluoroacetate salt of the product in
quantitative yield. MS (ESP) m/z 276 (MH+).
8-[2-(4-Aminopiperidin-1-yl)ethyl]-2-methoxypteridin-
7(8H)-one (21p). 21p was prepared from 20p according to the
general procedure for 21 to give the crude product in quantitative
1
yield. MS (ESP) m/z 305 (MH+). H NMR (CDCl3) δ: 1.21−1.33
(m, 2H); 1.72−1.82 (m, 2H); 2.13 (td, 2H); 2.64 (s, 1H); 2.71 (t,
2H); 2.92−3.01 (m, 2H); 4.11 (s, 3H); 4.48 (t, 2H); 8.13 (s, 1H);
8.92 (s, 1H).
1-{2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxy-1,8-naph-
thyridin-2(1H)-one (21r). 21r was prepared from 20r according to
the general procedure for 21, except after evaporation of the reaction
mixture, the residue was diluted with water and the aqueous layer washed
with ethyl acetate. The aqueous layer was concentrated under vacuum
using acetonitrile as an azeotrope to afford the trifluoroacetic acid salt of
the product in 78% yield as an off-white solid. MS (ESP) m/z 303 (MH+).
4-(2-(4-Aminopiperidin-1-yl)ethyl)-6-methoxybenzo[e]-
[1,2,4]triazin-3(4H)-one (21t). 21t was prepared from 20t accord-
ing to the general procedure for 21 to give the product in 58% yield.
MS (ESP) m/z 304 (MH+).
General Procedure for 24 and 25 by Reductive Amination of
21. A solution of 21 (0.20 mmol) and 1 equiv of aldehyde, either
(2,3-dihydro[1,4]dioxino[2,3-c]pyridine-7-carbaldehyde 2214 for 24 or
3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6-carbaldehyde
2315for 25, in dry chloroform/methanol (5 mL, 1:1) was heated over
freshly activated 3 Å molecular sieves (pearled) at 70 °C for 3 h. The
reaction mixture was cooled to 0 °C, and sodium triacetoxyborohy-
dride (0.6 mmol) was added. The reaction mixture was stirred at room
temperature for 30 min, then filtered. The filtrate was concentrated to
dryness under reduced pressure. The residue was taken up in
dichloromethane (50 mL) and saturated aqueous sodium hydro-
gencarbonate solution (5 mL). The pH of the aqueous phase was
adjusted to a pH of 10 with 1 M aqueous sodium hydroxide solution.
The aqueous phase was back-extracted twice with dichloromethane (2 ×
20 mL), and the combined organic phases were dried over sodium sulfate
and concentrated under reduced pressure.
4-[2-(4-Aminopiperidin-1-yl)ethyl]-3-oxo-3,4-dihydro-2H-
1,4-benzoxazine-6-carbonitrile (21c). 21c was prepared from 20c
according to the procedure for 21a to give the trifluoroacetate salt of
the product in quantitative yield. MS (ESP) m/z 301 (MH+).
4-[2-(4-Aminopiperidin-1-yl)ethyl]-6-fluoro-2H-1,4-benzoxa-
zin-3(4H)-one (21d). 21d was prepared from 20d according to the
procedure for 21a to give the trifluoroacetate salt of the product in
quantitative yield. MS (ESP) m/z 294 (MH+).
4-[2-(4-Aminopiperidin-1-yl)ethyl]-6-methoxy-2H-1,4-ben-
zothiazin-3(4H)-one (21e). 21e was prepared from 20e according
to the procedure for 21a to give the trifluoroacetate salt of the product
in quantitative yield. MS (ESP) m/z 322 (MH+).
1-[2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxy-3,4-dihydro-
quinolin-2(1H)-one (21f). 21f was prepared from 20f according to
the general procedure for 21. The product was obtained in 82% yield
as a colorless oil. MS (ESP) m/z 304 (MH+). 1H NMR (DMSO-d6) δ:
1.19 (m, 2H); 1.62 (m, 2H); 1.96 (t, 2H); 2.36 (t, 2H); 2.46 (m, 2H);
2.73−2.80 (m, 5H); 3.71 (s, 3H); 3.91 (m, 2H); 6.77−6.84 (m, 2H);
7.04 (m, 1H).
1-[2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxy-1,4-dihydro-
2H-3,1-benzoxazin-2-one (21g). 21g was prepared from 20g
according to the procedure for 21a to give the trifluoroacetate salt of the
product in quantitative yield as a brown oil. MS (ESP) m/z 302 (MH+).
1-[2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxyquinolin-
2(1H)-one (21h). 21h was prepared from 20h according to the
general procedure for 21. The product was obtained in quantitative
yield as a colorless oil. MS (ESP) m/z 302 (MH+). 1H NMR (DMSO-
d6) δ: 1.21 (m, 2H); 1.65 (m, 2H); 2.04 (t, 2H); 2.40−2.52 (m, 2H);
2.89 (m, 2H); 3.69 (m, 1H); 3.88 (s, 3H); 4.31 (t, 2H); 6.40 (m, 1H);
6.88 (m, 1H); 6.94 (m, 1H); 7.63 (m, 1H); 7.80 (m, 1H).
4-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-6-methoxy-2H-1,4-benzoxazin-
3(4H)-one (24a). 24a was prepared from 21a, N,N-diisopropylethyl-
amine (15 equiv), and 2214 according to the general procedure for 24
except as reducing agent, sodium cyanoborohydride (0.3 mmol) was
1-[2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxy-4-methylqui-
nolin-2(1H)-one (21i). 21i was prepared from 20i according to the
7844
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
used. Reverse phase chromatography with water/acetonitrile and TFA
as modifier gave the trifluoroacetic acid salt of the product. The salt
was partitioned between chloroform and sodium hydrogen carbonate
solution. The aqueous phase was back-extracted with chloroform and
the combined organic phases were dried over magnesium sulfate and
concentrated under reduced pressure to give the free base of the
product in 32% yield as a gum. MS (ESP) m/z 455 (MH+). 1H NMR
(CDCl3-d) δ: 1.46 (m, 2H); 1.88 (m, 2H); 2.14 (m, 2H); 2.54 (m,
1H); 2.58 (t, J = 7.4 Hz, 2H); 2.95 (m, 2H); 3.78 (s, 3H); 3.79 (s,
2H); 4.01 (t, J = 7.4 Hz, 2H); 4.29 (m, 4H); 4.52 (s, 2H); 6.50 (dd,
J = 6.2, 2.7 Hz, 1H); 6.67 (d, J = 2.7 Hz, 1H); 6.81 (s, 1H); 6.89 (d,
J = 8.7 Hz, 1H); 8.08 (s, 1H).
4.70 (s, 2H); 6.80 (m, 1H); 6.86 (s, 1H); 7.23−7.28 (m, 2H); 7.45 (d,
1H); 9.70 (brs, 2H); 11.07 (brs, 1H); 11.37 (s, 1H).
6-[({1-[2-(7-Methoxy-2-oxo-2H-3,1-benzoxazin-1(4H)-yl)-
ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one (25g). 25g was prepared from 21g, N,N-
diisopropylethylamine (15 equiv), and 2315 according to the general
procedure for 25. Chromatography on silica gel with dichloro-
methane/methanol (9:1) gave the free base of the product as a
colorless foam. The free base was taken up in 1,4-dioxane (2 mL), and
HCl in 1,4-dioxane (4M, 2 mol equiv) was added dropwise under
rapid stirring. The precipitate was collected by filtration to give the bis-
hydrochloride salt of the product in 48% yield as a colorless solid. MS
(ESP) m/z 468 (MH+). 1H NMR (DMSO-d6) δ: 2.09 (m, 2H); 2.31−
2.44 (m, 2H); 3.00−3.15 (m, 2H); 3.66−3.78 (m, 2H); 3.82 (s, 3H);
4.13−4.22 (m, 2H); 4.22−4.35 (m, 2H); 4.69 (s, 2H); 5.25 (s, 2H);
6.70 (d, 1H); 6.85 (s, 1H); 7.20 (dd, 2H); 7.45 (d, 1H); 9.56−9.71
(m, 2H); 11.05−11.19 (m, 1H); 11.37 (s, 1H).
6-[({1-[2-(6-Methoxy-3-oxo-2,3-dihydro-4H-1,4-benzoxazin-
4-yl)ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one (25a). 25a was prepared from 21a, N,N-
diisopropylethylamine (15 equiv) and 2315 according to the procedure
for 24a (17% yield). MS (ESP) m/z 468 (MH+).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-7-methoxyquinolin-2(1H)-one
(24h). 24h was prepared from 21h and 2214 according to the general
procedure for 24. Chromatography on silica gel with dichloro-
methane/methanol (8:1 to 4:1) gave the free base of the title
compound as a colorless oil. The HCl salt was prepared according to
the protocol for 25f to give the bis-HCl salt of the product in 48%
yield as a colorless solid, mp 243 °C. MS (ESP) m/z 451 (MH+). 1H
NMR (DMSO-d6) δ: 2.00−3.80 (m, 11H); 3.96 (s, 3H); 4.20−4.45
(m, 6H); 4.68 (m, 2H); 6.45 (d, 1H); 6.93 (d, 1H); 7.18 (s, 1H); 7.30
(s, 1H); 7.68 (d, 1H); 7.88 (d, 1H); 8.25 (s, 1H); 9.74 (brs, 2H);
11.18 (brs, 1H).
4-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethy)-2H-1,4-benzoxazin-3(4H)-one
(24b). 24b was prepared from 21b, N,N-diisopropylethylamine (15
equiv), and 2214 according to the procedure for 24a (16% yield). MS
1
(ESP) m/z 425 (MH+). H NMR (DMSO-d6) δ: 2.2−3.8 (m, 14H);
3.87 (s, 1H); 4.42 (m, 4H); 4.75 (s, 2H); 7.11 (m, 3H); 7.40−7.60
(m, 2H); 8.29 (s, 1H); 10.01 (s, 2H); 10.01 (s, 2H); 11.35 (brs, 1H).
3-Oxo-4-[2-(4-{[(3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]-
oxazin-6-yl)methyl]amino}piperidin-1-yl)ethyl]-3,4-dihydro-
2H-1,4-benzoxazine-6-carbonitrile (25c). 25c was prepared from
21c, N,N-diisopropylethylamine (15 equiv), and 2315 according to the
procedure for 24a (19% yield). MS (ESP) m/z 463 (MH+). 1H NMR
(CDCl3-d) δ: 1.49 (m, 2H); 1.94 (m, 2H); 2.14 (m, 2H); 2.56 (m,
1H); 2.59 (t, J = 6.7 Hz, 2H); 2.94 (m, 2H); 3.84 (s, 2H); 4.03 (t, J =
6.7 Hz, 2H); 4.62 (s, 2H); 4.67 (s, 2H); 6.94 (d, J = 8.1 Hz, 1H); 7.03
(d, J = 8.5 Hz, 1H); 7.19 (d, J = 7.9 Hz, 1H); 7.30 (dd, J = 6.6, 1.7 Hz,
1H); 7.46 (d, J = 1.5 Hz, 1H).
6-[({1-[2-(6-Fluoro-3-oxo-2,3-dihydro-4H-1,4-benzoxazin-4-
yl)ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one (25d). 25d was prepared from 21d, N,N-
diisopropylethylamine (15 equiv), and 2315 according to the procedure
for 24a (16% yield). MS (ESP) m/z 456 (MH+). 1H NMR (CDCl3-d) δ:
1.52 (m, 2H); 1.94 (m, 2H); 2.15 (m, 2H); 2.57 (m, 1H); 2.59 (t, J =
align="left"7.1 Hz, 2H); 2.97 (m, 2H); 3.84 (s, 2H); 4.01 (t, J = 7.1 Hz,
2H); 4.54 (s, 2H); 4.62 (s, 2H); 6.68 (m, 1H); 6.86 (m, 1H); 6.89 (m,
1H); 6.95 (d, J = 8.1 Hz, 1H); 7.20 (d, J = 8.1 Hz, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-7-methoxy-4-methylquinolin-
2(1H)-one (24i). 24i was prepared from 21i, N,N-diisopropylethyl-
amine (15 equiv), and 2214 according to the procedure for 24a, except
the product was obtained as a TFA salt after reverse phase
1
chromatography (6% yield). MS (ESP) m/z 465 (MH+). H NMR
(DMSO-d6) δ: 2.35 (s, 3H); 3.79 (s, 3H); 6.20 (s, 1H); 6.80 (m, 2H);
7.58 (d, 1H); 11.44 (brs, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-7-methoxy-4-methylquinazolin-
2(1H)-one (24j). 24j was prepared from 21j and 2214 according to
the general procedure for 24. Purification of the crude product was
performed by reverse phase HPLC on a 19 mm × 100 mm ODO AQ
C18 column, eluting with a gradient of 0.1% TFA/water to 0.1% TFA/
acetonitrile. Fractions containing product were concentrated under
reduced pressure. The residue was taken up in methanol (10 mL), and
1 M HCl in ether (2 mol equiv) was added. The precipitate was
collected by filtration to give the bis-HCl salt of the product as a
colorless solid, 52% yield. MS (ESP) m/z 466 (MH+). 1H NMR
(DMSO-d6) δ: 2.03−2.14 (m, 2H); 2.31−2.42 (m, 2H); 2.82 (s, 3H);
3.07−3.14 (m, 2H); 3.32−3.44 (m, 2H); 3.77−3.86 (m, 2H); 4.07−
4.15 (m, 3H); 4.22−4.33 (m, 2H); 4.34−4.40 (m, 2H); 4.41−4.47 (m,
2H); 4.67−4.77 (m, 2H); 7.11 (d, 1H); 7.22 (s, 1H); 7.37 (s, 1H);
8.19 (d, 1H); 8.29 (s, 1H); 9.81 (s, 1H); 11.28 (s, 1H).
7-Chloro-1-(2-(4-((2,3-dihydro[1,4]dioxino[2,3-c]pyridin-7-
yl)methylamino)piperidin-1-yl)ethyl)-1,6-naphthyridin-2(1H)-
one (24k). 24k was prepared from 21k, N,N-diisopropylethylamine
(2.2 equiv), and 2214 according to the procedure for 24. The residue
obtained after aqueous workup was purified by silica gel chromatog-
raphy, eluting with 0−12% methanol in dichloromethane to give the
product as a colorless solid in 41% yield. 1H NMR (CDCl3-d) δ:
1.56−1.71 (m, 2H); 2.01 (d, J = 11.3 Hz, 2H); 2.19−2.36 (m, 1H);
2.69 (t, J = 7.1 Hz, 2H); 3.08 (t, J = 7.6 Hz, 2H); 3.60−3.76 (m, 2H);
3.90 (s, 2H); 4.23−4.42 (m, 5H); 6.72 (d, J = 9.6 Hz, 1H); 6.85 (s,
1H); 7.43 (s, 1H); 7.71 (d, J = 9.6 Hz, 1H); 8.11 (s, 1H); 8.55 (s, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-7-methoxyquinoxalin-2(1H)-one
(24l). 24l was prepared from 21l and 2214 according to the general
procedure for 24. Chromatography on silica gel with 5% methanol in
dichloromethane containing 0.25% ammonium hydroxide gave the
free base of the product as an oil. The free base was taken up in
isopropanol and treated with HCl in dioxane (4 M, 3 equiv). Solvent
4-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-6-methoxy-2H-1,4-benzothiazin-
3(4H)-one (24e). 24e was prepared from 21e, N,N-diisopropylethyl-
amine (15 equiv), and 2214 according to the procedure for 24a (13%
1
yield). MS (ESP) m/z 471 (MH+). H NMR (CDCl3-d) δ: 1.47 (m,
2H); 1.91 (m, 2H); 2.15 (m, 2H); 2.54 (m, 1H); 2.60 (t, J = 6.4 Hz,
2H); 2.94 (m, 2H); 3.33 (s, 2H); 3.47 (s, 2H); 3.80 (s, 5H); 4.08 (t,
J = 6.5 Hz, 2H); 4.29 (m, 4H); 6.58 (dd, J = 6.0, 2.6 Hz, 1H); 6.80 (s,
1H); 6.88 (d, J = 2.4 Hz, 1H); 7.23 (d, J = 8.4 Hz, 1H); 8.08 (s, 1H).
6-[({1-[2-(7-Methoxy-2-oxo-3,4-dihydroquinolin-1(2H)-yl)-
ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one (25f). 25f was prepared from 21f and 2315
according to the general procedure, but the aqueous workup was
omitted. Chromatography on a Phenomenex Synergy Polar-RP4um
column, eluting with 30−60% acetonitrile, containing 10 mM
ammonium acetate at pH 8, followed by chromatography on silica
gel with dichloromethane/methanol (7:1) gave the free base of the
product. The free base was dissolved in dichloromethane/ether (10
mL, 1:1), and HCl in ether (1 M, 3 mol equiv) was added under
vigorous stirring. The mixture was evaporated to dryness under
reduced pressure, and the residue was taken up as a suspension in
dichloromethane/hexanes (10 mL, 1:1). The solid was collected by
filtration and dried under reduced pressure to give the bis HCl salt of
the product as a colorless solid in 24% yield, mp >285 °C (dec). MS
1
(ESP) m/z 466 (MH+). H NMR (DMSO-d6) δ: 2.10 (m, 2H); 2.36
(m, 2H); 2.53 (t, 2H); 2.86 (t, 2H); 3.10 (m, 2H); 3.16 (m, 2H); 3.36
(m, 1H); 3.70 (m, 2H); 3.73 (s, 3H); 4.16 (m, 2H); 4.28 (m, 2H);
7845
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
was removed under reduced pressure to give the bis-hydrochloride salt
1H NMR (DMSO-d6) δ: 1.08−1.27 (m, 2H); 1.75 (m, 2H); 1.91 (m,
2H); 2.03 (m, 2H); 2.30−2.44 (m, 1 H); 2.61 (m, 2H); 2.89 (m, 2H);
3.94−4.05 (m, 3H); 4.28 (t, J = 6.5 Hz, 2H); 4.61 (s, 2H); 6.93−7.04 (m,
2H); 7.11 (dd, J = 9.0, 2.2 Hz, 1H); 7.29 (d, J = 8.1 Hz, 1H); 8.27 (d, J =
9.0 Hz, 1H); 11.16 (brs, 1H).
of the product in 27% yield as an off-white solid. MS (ESP) m/z 452
1
(MH+). H NMR (D2O) δ: 1.86−2.02 (m, 2H); 2.36−2.49 (m, 2H);
3.09−3.23 (m, 2H); 3.52 (t, 2H); 3.56−3.68 (m, 1H); 3.82−3.95 (m,
5H); 4.32−4.40 (m, 4H); 4.43−4.50 (m, 2H); 4.61 (t, 2H); 6.82 (d,
1H); 7.03 (dd, 1H); 7.29 (s, 1H); 7.71 (d, 1H); 7.97 (s, 1H); 8.22 (s,
1H).
AUTHOR INFORMATION
Corresponding Author
*Phone: 781-839-4617. Fax: 781-839-4630. E-mail: folkert.reck@
astrazeneca.com.
■
4-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-6-methoxypyrido[2,3-b]pyrazin-
3(4H)-one (24m). 24m was prepared from 21m and 2214 according
to the general procedure for 24. Chromatography on silica gel with 0−
2% methanol in dichloromethane gave the product in 64% yield. MS
1
(ESP) m/z 453 (MH+). H NMR (DMSO-d6) δ: 1.18 (q, 2H); 1.74
ACKNOWLEDGMENTS
■
(d, 2H); 2.02 (t, 2H); 2.30−2.40 (m, 1H); 2.60 (t, 2H); 2.89 (d, 2H);
3.65 (s, 2H); 3.98 (s, 3H); 4.26 (dd, 2H); 4.29−4.34 (m, 2H); 4.40 (t,
2H); 6.83 (d, 1H); 6.92 (s, 1H); 7.98 (s, 1H); 8.10 (s, 1H); 8.12 (d,
1H).
The authors thank Dr. Camil Joubran, Analytical Chemistry
Department of AstraZeneca R&D Boston, for performing NOE
and HMBC NMR experiments; Adam Shapiro, Bioscience
Department of AstraZeneca R&D Boston, for performing the
supercoiling assay; AstraZeneca R&D Susceptibility Testing
Group for MIC testing,; and Sussie Hopkins and Lena Grosser,
Pharmacology Department, AstraZeneca R&D Boston, for
efficacy studies.
8-(2-(4-((2,3-Dihydro-[1,4]dioxino[2,3-c]pyridin-7-yl)-
methylamino)piperidin-1-yl)ethyl)-2-methoxypyrido[2,3-d]-
pyrimidin-7(8H)-one (24n). 24n was prepared from 21n, N,N-
diisopropylethylamine (5 equiv), and 2214 according to the procedure
for 24a. Chromatography on silica gel with 15% methanol in
dichloromethane gave the product as a colorless solid in 44% yield. MS
1
(ESP) m/z 453 (MH+). H NMR (DMSO-d6) δ: 1.23 (m, 2H); 1.80
ABBREVIATIONS USED
(m, 2H); 2.07 (t, J = 10.8 Hz, 2H); 2.39 (brs, 2H); 2.95 (m, 2H); 3.71
(m, 2H); 4.06 (s, 3H); 4.34 (m, 2H); 4.36−4.48 (m, 4H); 6.61 (d, J =
9.5 Hz, 1H); 6.99 (s, 1H); 8.00 (d, J = 9.5 Hz, 1H); 8.06 (s, 1H); 8.97
(s, 1H).
■
NBTI, novel (non-fluoroquinolone) bacterial type II top-
oisomerase inhibitor; LHS, bicyclic aromatic left-hand side;
RHS, aromatic right-hand side; MIC, minimal inhibitory
concentration; cfu, colony forming units; ESP, electrospray
8-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-2-methoxypteridin-7(8H)-one
(24p). 24p was prepared from 21p and 2214 according to the general
procedure for 24. Chromatography on silica gel with 0−20% methanol
in dichloromethane gave the monoacetic acid salt of the product as a
REFERENCES
■
(1) Zhanel, G. G.; DeCorby, M.; Adam, H.; Mulvey, M. R.;
McCracken, M.; Lagace-Wiens, P.; Nichol, K. A.; Wierzbowski, A.;
Baudry, P. J.; Tailor, F.; Karlowsky, J. A.; Walkty, A.; Schweizer, F.;
Johnson, J.; Hoban, D. J. Prevalence of antimicrobial-resistant
pathogens in Canadian hospitals: results of the Canadian ward
surveillance study (CANWARD 2008). Antimicrob. Agents Chemother.
2010, 54, 4684−4693.
1
colorless solid in 43% yield. MS (ESP) m/z 454 (MH+). H NMR
(DMSO-d6) δ: 1.18−1.28 (m, 2H); 1.79 (d, 2H); 1.90 (s, 2H); 2.00
(t, 2H); 2.58 (t, 2H); 2.90 (d, 2H); 3.78 (s, 2H); 4.03 (s, 3H); 4.26−
4.35 (m, 6H); 6.96 (s, 1H); 8.04 (s, 1H); 8.15 (s, 1H); 8.99 (s, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidine-1-yl}ethyl)-7-methoxy-1,8-naphthyridin-
2(1H)-one (24r). 24r was prepared from 21r, N,N-diisopropylethyl-
amine (2 equiv), and 2214 according to the general procedure for 24.
Chromatography on silica gel with 5% methanol in dichloromethane,
containing 0.5% ammonium hydroxide, gave the product as an oil in
45% yield. MS (ESP) m/z 452 (MH+). 1H NMR (DMSO-d6) δ:
0.78−0.92 (m, 1H); 1.15−1.29 (m, 2H); 1.74 (dd, 2H); 2.02 (m, 2H);
2.26−2.40 (m, 1H); 2.49 (m, 2H); 2.51−2.61 (m, 2H); 2.91 (m, 2H);
3.66 (m, 2H); 3.96 (s, 3H); 4.23−4.29 (m, 2H); 4.29−4.35 (m, 2H);
4.38−4.50 (m, 2H); 6.48 (d, J = 9.4 Hz, 1H); 6.71 (d, J = 8.3 Hz, 1H);
6.92 (s, 1H); 7.84 (d, J = 9.4 Hz, 1H); 7.97−8.06 (m, 2H).
(2) 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
IIA topoisomerase inhibition by a new class of antibacterial agents.
Nature 2010, 466, 935−940.
(3) Wiener, J. J. M.; Gomez, L.; Venkatesan, H.; Santillan, A. Jr.;
Allison, B. D.; Schwarz, K. L.; Shinde, S.; Tang, L.; Hack, M. D.;
Morrow, B. J.; Motley, S. T.; Goldschmidt, R. M.; Shaw, K. J.; Jones, T.
K.; Grice, C. A. Tetrahydroindazole inhibitors of bacterial type II
topoisomerases. Part 2: SAR development and potency against
multidrug-resistant strains. Bioorg. Med. Chem. Lett. 2007, 17, 2718−
2722.
(4) Black, M. T.; Stachyra, T.; Platel, D.; Girard, A.; Claudon, M.;
Bruneau, J.; Miossec, C. Mechanism of action of the antibiotic
NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II
topoisomerases. Antimicrob. Agents Chemother. 2008, 52, 3339−3349.
(5) Breault, G.; Eyermann, C. J.; Geng, B.; Morningstar, M.; Reck, F.
WO2006134378, 2006.
(6) Axten, J. M.; Brooks, G.; Brown, P.; Davies, D.; Gallagher, T. F.;
Markwell, R. E.; Miller, W. H.; Pearson, N. D.; Seefeld, M.
WO004058144, 2004.
1-(2-(4-((2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-yl)-
methylamino)piperidin-1-yl)ethyl)-7-methoxy-1,6-naphthyri-
din-2(1H)-one (24s). To a solution of sodium methoxide in
methanol (0.5 M, 1.4 mL) was added 24k (0.103 g, 0.23 mmol).
The mixture was heated at 68 °C for 3 h. More sodium methoxide
solution (0.5 M, 1.4 mL) was added, and heating was continued for 18 h
at 78 °C. The mixture was cooled to room temperature and diluted
with ethyl acetate and water. The pH of the aqueous layer was adjusted
to pH 5 with 1 N HCl. The layers were separated, and the aqueous
layer was washed with ethyl acetate (2 × 15 mL). The combined
organic phases were dried over MgSO4 and concentrated under
1
reduced pressure to give the product as a tan solid in 38% yield. H
NMR (DMSO-d6) δ: 1.47 (m, 2H); 2.03 (m, 2H); 2.77 (brs, 1H);
3.00 (m, 2H); 3.88−4.06 (m, 5H); 4.17−4.47 (m, 7H); 6.47 (d, J =
9.6 Hz, 1H); 6.77 (s, 1H); 7.08 (s, 1H); 7.92 (d, J = 9.6 Hz, 1H); 8.12
(s, 1H); 8.56 (s, 1H); 9.13−10.02 (m, 2H).
6-((1-(2-(6-Methoxy-3-oxobenzo[e][1,2,4]triazin-4(3H)-yl)-
ethyl)piperidin-4-ylamino)methyl)-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one (25t). 25t was prepared from 21t, N,N-diisopro-
pylethylamine (15 equiv), and 2315 according to the general procedure for
25. Chromatography on silica gel with 0−20% methanol in dichloro-
methane gave the product in 30% yield. MS (ESP) m/z 466 (MH+).
(7) Kiyoto, T.; Tanaka, T.; Tsutsui, Y.; Ando, J.; Motono, M.;
Kawaguchi, Y.; Noguchi, T.; Ushiki, Y.; Ushiyama, F.; Urabe, H.
WO2006137485, 2006.
(8) Gauvin, J.; Hubschwerlen, C.; Surivet, J.; Zumbrunn Acklin, C.
WO2007105154, 2007.
(9) Inagaki, H.; Fujisawa, T.; Itoh, M.; Hayakawa, M.; Tsuda, T.;
Nagamochi, M.; Takahashi, H.; Takemura, M. WO2009125809, 2009.
(10) Gaucher, B.; Danel, F. H.; Roussel, P. WO2010084152, 2010.
7846
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847

Journal of Medicinal Chemistry
Article
(11) Black, M. T.; Coleman, K. New inhibitors of bacterial
topoisomerase GyrA/ParC subunits. Curr. Opin. Invest. Drugs 2009,
10, 804−810.
(12) McMartin, C.; Bohacek, R. S. Flexible matching of test ligands
to a 3D pharmacophore using a molecular superposition force field:
comparison of predicted and experimental conformations of inhibitors
of three enzymes. J. Comput.-Aided Mol. Des. 1995, 9, 237−250.
(13) Barrett, J. F.; Sutcliffe, J. A.; Gootz, T. D. In vitro assays used to
measure the activity of topoisomerases. Antimicrob. Agents Chemother.
1990, 34, 1−7.
(14) Barfoot, C. W.; Brown, P.; Dabbs, S.; Davies, D. T.; Hennessy,
A. J.; Miles, T. J.; Pearson, N. D. The design of efficient and selective
routes to pyridyl analogues of 2,3-dihydro-1,4-benzodioxin-6-carbalde-
hyde. Tetrahedron Lett. 2010, 51, 5038−5040.
(15) Brooks, G.; Dabbs, S.; Davies, D. T.; Hennessy, A. J.; Jones, G.
E.; Markwell, R. E.; Miles, T. J.; Owston, N. A.; Pearson, N. D.; Peng,
T. W. The design of efficient and selective routes to pyridyl analogues
of 3-oxo-3,4-dihydro-2H-1,4-(benzothiazine or benzoxazine)-6-carbal-
dehydes. Tetrahedron Lett. 2010, 51, 5035−5037.
(16) Waring, M. J.; Johnstone, C. A quantitative assessment of hERG
liability as a function of lipophilicity. Bioorg. Med. Chem. Lett. 2007, 17,
1759−1764.
(30) Schmidt, K. J.; Hammann, I. DE1809390, 1970.
(31) Hay, M. P.; Gamage, S. A.; Kovacs, M. S.; Pruijn, F. B.;
Anderson, R. F.; Patterson, A. V.; Wilson, W. R.; Brown, J. M.; Denny,
W. A. Structure−activity relationships of 1,2,4-benzotriazine 1,4-
dioxides as hypoxia-selective analogues of tirapazamine. J. Med. Chem.
2003, 46, 169−182.
(32) Joyeau, R.; Felk, A.; Guilaume, S.; Wakselman, M.; Vergely, I.;
Doucet, C.; Boggetto, N.; Reboud-Ravaux, M. Synthesis and inhibition
of human leukocyte elastase by functionalized N-aryl 2-azetidinones:
effect of different substituents on the aromatic ring. J. Pharm.
Pharmacol. 1996, 48, 1218−1230.
(33) Vermeulen, E. S.; Van Smeden, M.; Schmidt, A. W.; Sprouse, J.
S.; Wikstroem, H. V.; Grol, C. J. Novel 5-HT7 receptor inverse
agonists. Synthesis and molecular modeling of arylpiperazine- and
1,2,3,4-tetrahydroisoquinoline-based arylsulfonamides. J. Med. Chem.
2004, 47, 5451−5466.
(34) Caliendo, G.; Perissutti, E.; Santagada, V.; Fiorino, F.; Severino,
B.; d’Emmanuele di Villa Bianca, R.; Lippolis, L.; Pinto, A.; Sorrentino,
R. Synthesis and vasorelaxant activity of new 1,4-benzoxazine
derivatives potassium channel openers. Bioorg. Med. Chem. 2002, 10,
2663−2669.
(35) Reddy, P. S. N.; Reddy, P.; Reddy, G. J.; Rao, K. S. Synthesis of
pyrazolo[3′,4′:4,5]pyrimido[2,3-c][1,4]benzoxazines. A new hetero-
cyclic ring system. Heterocycl. Commun. 2003, 9, 503−506.
(36) Gogte, V. N.; Kulkarni, S. B.; Tilak, B. D. Synthesis of
heterocyclic compounds: part XX. 2,4-Disubstituted 1,2,3,4-tetrahy-
droquinolines. Indian J. Chem., Sect. B: Org. Chem. Incl. Med. Chem.
1977, 15B, 769−773.
(37) Turner, J. A. A general approach to the synthesis of 1,6-, 1,7-,
and 1,8-naphthyridines. J. Org. Chem. 1990, 55, 4744−4750.
(38) Davies, D. T.; Elder, J. S.; Forrest, A. K.; Jarvest, R. L.; Pearson,
N. D.; Sheppard, R. J. WO2004014361, 2004.
(17) O’Shea, R.; Moser, H. E. Physicochemical properties of
antibacterial compounds: implications for drug discovery. J. Med.
Chem. 2008, 51, 2871−2878.
(18) Nikaido, H. Molecular basis of bacterial outer membrane
permeability revisited. Microbiol. Mol. Biol. Rev. 2003, 67, 593−656.
(19) Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme,
A.; Henthorn, D. C.; Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C.
G.; Abi-Gerges, N.; Persson, F.; Jacobson, I.; Sullivan, M.; Albertson,
N.; Hammond, T. G.; Sullivan, E.; Valentin, J. P.; Pollard, C. E.
Optimisation and validation of a medium-throughput electrophysiol-
ogy-based hERG assay using IonWorks HT. J. Pharmacol. Toxicol.
Methods 2006, 54, 189−199.
(39) Axten, J. M.; Daines, R. A.; Davies, D. T.; Gallagher, T. F.;
Jones, G. E.; Miller, W. H.; Pearson, N. D.; Pendrak, I.
WO2004002992, 2004.
(20) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria
That Grow Aerobically: Approved Standard, 2nd ed.; National
Committee for Clinical Laboratory Standards: Villanova, PA, 1993.
(21) Lanzetta, P. A.; Alvarez, L. J.; Reinach, P. S.; Candia, O. A. An
improved assay for nanomole amounts of inorganic phosphate. Anal.
Biochem. 1979, 100, 95−97.
(22) Mills, S. D.; Eakin, A. E.; Buurman, E. T.; Newman, J. V.; Gao,
N.; Huynh, H.; Johnson, K. D.; Lahiri, S.; Shapiro, A. B.; Walkup, G.
K.; Yang, W.; Stokes, S. S. Novel bacterial NAD+-dependent DNA
ligase inhibitors with broad-spectrum activity and antibacterial efficacy
in vivo. Antimicrob. Agents Chemother. 2011, 55, 1088−1096.
(23) Teulon, J. M. EP162776, 1985.
(24) Sidhu, G. S.; Thyagarajan, G.; Ansari, S. Syntheses of
carbostyrils and dihydrocarbostyrils. Liebigs Ann. Chem. 1959, 627,
218−224.
(25) Clark, J. D.; Davis, J. M.; Favor, D.; Fay, L. K.; Franklin, L.;
Henegar, K. E.; Johnson, D. S.; Nichelson, B. J.; Ou, L.; Repine, J. T.;
Walters, M. A.; White, A. D.; Zhu, Z. Unpublished results, 2005.
(26) Chen, P.; Barrish, J. C.; Iwanowicz, E.; Lin, J.; Bednarz, M. S.;
Chen, B.-C. Reaction of quinoxalin-2-ones with TosMIC reagent:
synthesis of imidazo[1,5-a]quinoxalin-4-ones. Tetrahedron Lett. 2001,
42, 4293−4295.
(27) Curd, F. H. S.; Davey, D. G.; Stacey, G. J. Synthetic
antimalarials. XL. The effect of variation of substituents in 2-chloro-
3-(2-diethylaminoethylamino)quinoxaline. J. Chem. Soc. 1949, 1271−
1277.
(28) Kiyoto, T.; Ando, J.; Tanaka, T.; Tsutsui, Y.; Yokotani, M.;
Noguchi, T.; Ushiyama, F.; Urabe, H.; Horikiri, H. WO2007138974,
2007.
(29) Barvian, M.; Boschelli, D.; Cossrow, J.; Dobrusin, E.; Fattaey,
A.; Fritsch, A.; Fry, D.; Harvey, P.; Keller, P.; Garrett, M.; La, F.;
Leopold, W.; McNamara, D.; Quin, M.; Trumpp-Kallmeyer, S.;
Toogood, P.; Wu, Z.; Zhang, E. Pyrido[2,3-d]pyrimidin-7-one
inhibitors of cyclin-dependent kinases. J. Med. Chem. 2000, 43,
4606−4616.
7847
dx.doi.org/10.1021/jm2008826|J. Med. Chem. 2011, 54, 7834−7847