Antitubercular nitrofuran isoxazolines with improved pharmacokinetic properties

Article abstract of DOI:10.1016/j.bmc.2012.08.023

A series of tetracyclic nitrofuran isoxazoline anti-tuberculosis agents was designed and synthesized to improve the pharmacokinetic properties of an initial lead compound, which had potent anti-tuberculosis activity but suffered from poor solubility, high

Full text of DOI:10.1016/j.bmc.2012.08.023

Bioorganic & Medicinal Chemistry 20 (2012) 6063–6072
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antitubercular nitrofuran isoxazolines with improved
pharmacokinetic properties
Rakesh a, David Bruhn a, Dora B. Madhura b, Marcus Maddox a, Robin B. Lee a, Ashit Trivedi b, Lei Yang a,
Michael S. Scherman c, Janet C. Gilliland c, Veronica Gruppo c, Michael R. McNeil c, Anne J. Lenaerts c,
Bernd Meibohm b, Richard E. Lee a,
⇑
a Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN, USA
c Mycobacterial Research Laboratories, Department of Microbiology, Colorado State University, Fort Collins, CO, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tetracyclic nitrofuran isoxazoline anti-tuberculosis agents was designed and synthesized to
improve the pharmacokinetic properties of an initial lead compound, which had potent anti-tuberculosis
activity but suffered from poor solubility, high protein binding and rapid metabolism. In this study, struc-
tural modifications were carried on the outer phenyl and piperidine rings to introduce solubilizing and
metabolically blocking functional groups. The compounds generated were evaluated for their in vitro
antitubercular activity, bacterial spectrum of activity, solubility, permeability, microsomal stability and
protein binding. Pharmacokinetic profiles for the most promising candidates were then determined.
Compounds with phenyl morpholine and pyridyl morpholine outer rings were found to be the most
potent anti-tuberculosis agents in the series. These compounds retained a narrow antibacterial spectrum
of activity, with weak anti-Gram positive and no Gram negative activity, as well as good activity against
non-replicating Mycobacterium tuberculosis in a low oxygen model. Overall, the addition of solubilizing
and metabolically blocked outer rings did improve solubility and decrease protein binding as designed.
However, the metabolic stability for compounds in this series was generally lower than desired. The best
three compounds selected for in vivo pharmacokinetic testing all showed high oral bioavailability, with
one notable compound showing a significantly longer half-life and good tolerability supporting its further
advancement.
Received 16 May 2012
Revised 13 August 2012
Accepted 20 August 2012
Available online 28 August 2012
Keywords:
Tuberculosis
Antibiotic
Nitrofuran
Isoxazoline
Nitroaromatic
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
line functionality. Compounds in this series had excellent MIC
activity, but suffered from overall poor solubility, high protein
On a global scale Tuberculosis is one of the leading causes of
mortality from an infectious agent and a major AIDS-associated,
opportunistic infection.1 The standard six-month drug treatment
regime for tuberculosis is extremely long and often leads to patient
non-compliance and consequent development of resistance.2 The
long treatment time is believed to be caused by latent/slow growing
bacterial subpopulations that are poorly susceptible to front line
agents. These latent populations are thought to exist in part in an
acidic low oxygen/microaerophilic state within the granuloma.3
For this study, our efforts have focused on developing nitrofuran
based anti-tuberculosis therapeutics that are active in latent TB
models and thus have the potential to shorten tuberculosis therapy
regimen.4–9 Initial series were based around nitrofuranyl amides,
however the amide functionality proved to be a metabolic liability
and it was replaced with a more stable 3,5-disubstituted isoxazo-
binding and poor microsomal stability. Herein we describe the syn-
thesis of a new series of compounds designed to improve pharma-
cokinetic properties based on lead compound 1 (Fig. 1A). The
nitrofuran and isoxazoline A and B rings were kept intact to main-
tain antitubercular potency and the C and D rings were modified to
increase solubility and metabolic stability while decreasing protein
binding. Modification of the C and D rings employed a strategy
similar to that used for second generation oxazolidinones. These
antibacterial agents have a similar linear tetra cyclic structure with
the outer rings playing a significant role in modulating their
absorption, distribution and metabolic properties (Fig. 1B).10–12
2. Chemistry
The target compounds of the present study were synthesized
from their corresponding 4-bromo, 1-vinyl benzene C ring precur-
sors in two steps, using a modified Buchwald coupling of aryl ha-
lides with cyclic amines to add the substitute D rings followed
⇑
Corresponding author.
E-mail address: richard.lee@stjude.org (R.E. Lee).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.023

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Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
B
A
O
O
N
O
F
O
O
A
H
N
O
F
B
H
N
O2N
H3C
H3C
N
C
N
N
N
D
O
N
O
1
O
S
Linezolid
Sutezolid
MIC 0.006 ug/ml
Solubility 1.1 ug/ml
Protein binding 98.8%
Figure 1. (A) 1 Initial lead antitubercular nitrofuran. In this study targeted changes were made to the outer C and D ring systems to improve ADME properties. (B) Comparator
oxazolidinone antibacterial agents Linezolid and Sutezolid with similar linear structures.
Cl
O
O2N
N
O
OH
X
O
N
X
O2N
I
X
5
X
a
b
N
N
c
O
Br
X = N
Br
O
4a
X = CH
3b
3c X = C-F
2
6
7
X = CH
X = N
4b X = N
4c X = C-F
8 X = C-F
Scheme 1. Reagents and conditions: (a) Tributyl(vinyl)tin, PdCl2(PPh3)2, DMF, MW, 160 °C, 5 min; (b) morpholine, Pd(OAc)2, 2-(di-tert-butylphosphino)biphenyl, NaOtBu,
toluene, 80 °C, 12 h; (c) 5, Et3N, CHCl3, RT, 2 h.
O
N
O
N
O
O
O2N
O2N
c
b
a
N
N
N
S
S
O
Br
S
12
11
10
9
Scheme 2. Reagents and conditions: (a) Thiomorpholine, Pd(OAc)2, 2-(di-tert-butylphosphino)biphenyl, NaOtBu, toluene, 80 °C, 12 h; (b) 5, Et3N, CHCl3, RT, 2 h; (c)NalO4,
CH3CN, MeOH, H2O, RT, 12 h.
O
N
O
X
O
O
O
O2N
X
X
HN
a
b
+
N
N
Y
Br
Y
Y
15
X = CH; Y = N-CH3
16 X = N; Y = N-CH3
17
13
3b-c
14a-f
X = C-F; Y = N-CH3
18 X = CH; Y = O
19
X = N; Y = O
20 X = C-F; Y = O
Scheme 3. Reagents and conditions: (a) N,N0-Dimethyl ethylene diamine, CuI, K2CO3, toluene, reflux, 6 h; (b) 5, Et3N, CHCl3, RT, 2 h.
by 3+2 cyclo addition reaction to the vinyl functionality simulta-
neously adding the nitrofuran A ring and isoxazoline B ring as de-
scribed in Schemes 1–3. More specifically, compounds 6–8 were
synthesized starting from their corresponding 4-bromo aryl iodide
precursors 2 as depicted in Scheme 1. Iodobenzenes were con-
verted into their vinyl analogues 3b–c by treating with tribu-
tyl(vinyl)tin in presence of Pd catalyst in DMF under microwave
irradiation in 80–82% yields.13 The aryl amination reaction was
carried out using morpholine, palladium acetate and phosphorous
ligand in presence of sodium tert butoxide in toluene at 80 °C for
overnight to afford 4a–c in 69–83% yields.14 Finally, the isoxazoline
ring was constructed by treating the olefins with nitrofuranyl chlo-
roxime 5 in the presence of Et3N in CHCl3 at room temperature to
give compounds 6–8 in 75–80% yields.
The thiomorpholine compound 11 was synthesized from
4-bromo styrene 9 by treating with thiomorpholine, palladium ace-
tate and phosphorous ligand in presence of sodium tert butoxide in
toluene at 80 °C for 4 h to afford 4-(4-vinylphenyl) thiomorpholine
10 in 77% yield (Scheme 2). Then the isoxazoline ring construction
was carried out by treating 10 with nitrofuranyl chloroxime in the
presence of Et3N in CHCl3 at room temperature for 2 h to give 11
in 63% yield. The thiomorpholine compound was oxidized to
sulfoxide by treating with NaIO4 in methanol and water at room
temperature for overnight to yield 12 in 58% yield.15
4-Methylpiperazin-2-one and morpholin-3-one analogues 15–
20 were synthesized (Scheme 3) from appropriate aryl bromide
precursors 3b–c by treating with cyclic amides 13 (Y = NCH3 for
15–17, Y = O for 18–20) in presence of N,N0-dimethyl ethylene

Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
6065
Table 1
MIC and in vitro pharmacokinetic profile
Compound
MIC (mg/
mL)
Solubility pH 7.4
(mg/L)
Microsomal
stability t1/2 (h)
Caco-2
Protein binding
(%)
Mouse Human Papp A/B
(nm/s)
Papp B/A
(nm/s)
Efflux
ratio
Mouse
Human
N
O
O
O2N
O2N
0.006
0.006
0.013
1.35
0.7
98.8
ND
1.1
(0.2)
0.78
(0.03)
0.47
(0.02)
76.09
(38.6)
103
(5.19)
N
1
N
O
O
9.9
(0.2)
1.06
(0.07)
2.79
(0.18)
171.8
(75.9)
120.9
(19.8)
96.4
(0.82)
96.2
(1.06)
N
O
6
O
N
O
O2N
O2N
O2N
O2N
N
2.2
14.1
(0.3)
1.23
(0.07)
3.37
(0.26)
64.73
(26.7)
142.1
(98.5)
95.3
(2.32)
96.7
(0.63)
N
7
O
N
O
F
O
0.05
0.05
0.88
1.19
3.2
(0.2)
0.92
(0.06)
2.29
(0.10)
200.7
(76.6)
177
(54.6)
93.9
(0.43)
93.4
(0.75)
N
O
8
N
O
O
O
1.9
(0.1)
0.04
(0.00)
0.21
(0.00)
96.15
(22.4)
114.4
(12.6)
98.7
(0.25)
98.5
(0.21)
N
N
S
S
11
N
O
0.4
0.4
0.2
28.9
(0.2)
0.87
(0.06)
4.53
(0.17)
241.5
(48.7)
272.1
(3.27)
1.13
1.07
0.91
1.43
82.2
(3.47)
77.7
(0.69)
12
O
N
O
O
O
O
O
O
O2N
O2N
O2N
O2N
28.8
(0.3)
0.39
(0.01)
5.48
(0.48)
284
(30.5)
305.2
(40.2)
76.1
(2.77)
57.4
(8.49)
N
15
N
N
N
O
O
N
28.9
(0.6)
0.35
(0.01)
4.24
(0.24)
366
(37.9)
332.8
(28.9)
59.8
(10.2)
48.9
(8.8)
N
16
N
O
F
O
29.2
(0.3)
0.62
(0.03)
4.81
(0.31)
269.8
(34.3)
385.8
(36.2)
82.8
(6.38) (14.8)
60.9
0.4
0.4
N
17
N
N
O
O
25.88
(0.16)
1.4
(0.1)
10.03
(2.8)
126.4
(33.7)
565.4
(46.8)
66
(2.2)
64.3
(2.9)
4.47
5.31
2.75
N
18
O
N
O
O
O
O2N
O2N
O
N
21.94
(0.63)
1.01
(0.1)
1.52
(0.1)
86.05
(13.5)
457.3
(37.9)
58.9
(4.7)
60.2
(1.6)
0.2
0.8
N
F
19
O
N
O
O
12.58
(1.49)
1.38
(0.2)
8.31
(1.7)
154.1
(5.36)
424.1
(67.3)
79.7
(1.0)
70.8
(1.1)
N
20
O
Standard deviations are in parentheses; Papp: the apparent permeability coefficient.
diamine, CuI and K2CO3 in toluene at reflux temperature for 6 h.16
Then the isoxazoline ring was constructed using classical condi-
tions as discussed in Scheme 1 to obtain compounds 15–20 in
57–73% yields.
3. MIC and in vitro pharmacokinetics properties
Table 1 summarizes the anti-tuberculosis MIC activity and the
in vitro pharmacokinetic profiling results. Across the whole series

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Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
Table 2
Antibacterial spectrum of activity (
l
g/ml)
Compd
S. aureus
S. pyogenes
S. pneumonia
E. faecalis
B. subtilis
B. anthracis
E. coli K12
E. coli DtolC
1
6
7
12.5
6.25
12.5
25
12.5
6.25
6.25
12.5
6.25
6.25
12.5
6.25
100
25
25
200
>200
50
50
50
50
200
50
200
200
12.5
50
50
50
50
200
50
>200
>200
200
200
100
100
100
100
100
50
12.5
50
200
12.5
6.25
6.25
25
>200
12.5
25
12.5
12.5
12.5
12.5
25
25,
25
25
25
>200
>200
>200
>200
>200
100
50
50
200
25
50
6.25
6.25
6.25
12.5
6.25
12.5
6.25
25
8
11
12
15
16
17
18
19
20
25
100
100
100
>200
>200
200
6.25
12.5
12.5
6.25
12.5
12.5
25
>200
200
Organisms abbreviated above are as follows: S. aureus ATCC 29213; S. pyogenes ATCC 700294; S. pneumoniae DAW27; E. faecalis ATCC3186; B. subtilis ATCC 23857; B. anthracis
Sterne 34F2; E. coli K12; E. coli K12
DtolC.
Table 3
In vivo pharmacokinetic parameters in rats [mean (SD)]
Compound
Cmax (mg/L)
AUC0–1 (h mg/L)
t
(h)
CL (L/h/kg)
Vd (L/kg)
fe (%)
F (%)
Cytotoxicity (in vitro) IC50 lg/ml
½
1
2.28
2.75
2.79
3.77
10.9
0.00
29.4
2.5
(0.43)
0.83 (0.19)
(0.58)
1.63
(0.36)
2.39 (0.74)
(0.77)
6.50 (1.81)
(3.7)
35.0
(0.7)
2.9
6
0.00
100
(0.43)
3.36
(0.44)
0.96
(0.37)
15.5b
6.1–11.8b
(24.2)
2.86 (0.62)
(0.4)
3.2
(0.3)
20.3
(2.5)
—
7
2.73 (0.41)
1.19 (0.34)
0.72 (0.17)
0.61 (0.41)
3.02 (0.42)
0.02
(0.01)
0.68
(0.44)
73
47.9
58.3
15
11.61 (4.04)
3.1 (1.6)
Linezolida,21,22
Ofloxacina,23,24
15b
1.0
1.5–1.8
0.63
0.86–1.62
0.72
0.99
109
78
10.9b
46
—
a
Data shown taken from previously published reports as cited.
Based on a 10 mg/kg IV dose.
b
C ring substitutions did not have a major impact on measured
parameters. Substitution with a pyridyl group (7, 16 and 19) re-
sulted in a small decrease in protein binding overall. Compound
7 had slight improvements in solubility and microsomal stability
over 6, however the efflux ratio suggested active efflux transport.
As could be expected, fluorine substitution of the C ring (8, 17
and 20) did increase the lipophilicity and it decreased solubility
in combination with morpholine and morpholinone D rings.
More pronounced SAR was observed for the D ring substitu-
tions. Morpholines (6–8) retained good MIC, had improved solubil-
ity and microsomal stability, and a slight reduction in protein
binding. Thiomorpholine (11) retained good MIC activity, and its
S-oxide (12) has increased solubility and Caco-2 permeability,
and reduced protein binding. However, the metabolic stability
was very poor for 11, which is presumably rapidly S oxidized to
12 and then on to further metabolites. It was also noted that
S-oxide 12 has an 8-fold weaker MIC value than 11, which is unde-
sirable. The solubility and protein binding of the 4-methylpiperaz-
inones (15–17) was much improved and the Caco-2 permeability
was the highest observed in the series. However, the MIC increased
parison purposes due to its greater improvements in solubility and
protein binding as well as its availability at the time of testing. Thi-
omorpholines 11 and 12 were excluded from further testing due to
concerns over metabolic processing and observed toxicity. Mor-
pholinones 18–20 were also excluded primarily due to the high ef-
flux ratios as well as higher MIC values.
Previous compounds in the series of nitrofurans have demon-
strated a narrow range of activity with selectivity for Mycobacte-
rium tuberculosis and activity against non-replicating TB.17 To
ensure this series retained that narrow spectrum of activity, the
MIC of compounds was determined by microbroth dilution for a
representative panel of bacterial pathogens (Table 2). Weak to
moderate MIC activity was observed against some Gram positive
bacteria most notably Staphylococcus aureus, Bacillus subtilis, Bacil-
lus anthracis and an efflux pump deficient strain of Escherichia coli.
No activity was observed against any of the following Gram nega-
tive strains: Burkholderia cepacia, Proteus mirabilis, Proteus vulgaris,
Klebsiella pneumoniae, Acinetobacter baumannii, Strenotrophomonas
maltophilia, and Pseudomonas aeruginosa (data not shown). To en-
sure this series retained activity against non-replicating bacteria,
compound 7 was tested in an oxygen depletion model developed
by Voskuil and coworkers from the original Wayne model.18 It
significantly (0.4 lg/ml) for these compounds and they were rap-
idly metabolized, perhaps due the introduction of a new site of
metabolism via N-demethylation of the piperazine ring. The mor-
pholinones (18–20) had a similar profile to the 4-methylpiperazi-
nones with improved solubility and protein binding, but higher
MICs. Microsomal stability was much better for these compounds
and the best in the series. However, a comparatively high and
undesirable efflux ratio was observed in this assay.
killed 99.9% of bacteria at 50 lg/ml and 66.7% at 10 lg/ml demon-
strating 7 retained bactericidal activity against this important per-
sistent population of TB.
4. In vivo pharmacokinetics
Based on the retention of a good MIC and modest improvements
in solubility, microsomal stability and protein binding compounds
6 and 7 were advanced for further testing. Despite its higher MIC
and short half-life 4-methylpiperazinone 15 was selected for com-
For the prioritized set of compounds (1, 6, 7 and 15), the in vivo
pharmacokinetics were evaluated and the results are shown in Ta-
ble 3. After intravenous administration of 10 mg/kg in rats, com-
pounds 1, 6, 7 and 15 showed concentration–time profiles with

Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
6067
areas under the curve ranging from 0.96 to 3.36 mg h/L, and peak
plasma concentrations of 0.83–2.73 mg/L. These peak plasma con-
centrations when converted to unbound concentrations were 4.6
times the MIC for compound 1, 5.0 times the MIC for compound
6, 9.8 times the MIC for compound 7, and 0.71 times the MIC for
compound 15. Compounds 7 and 15 showed a similarly short elim-
ination half-life of 0.61–0.72 h, while compounds 1 and 6 exhibited
a longer half-life largely due to their larger volume of distribution.
Clearance was high in all four compounds close to or higher than
hepatic blood flow, indicating potential extrahepatic metabolism.
The fraction of dose excreted unchanged by the kidneys (fe) was
negligible for all compounds. Both of these observations indicate
rapid in vivo metabolic degradation for all four compounds as
the major elimination pathway, which is consistent with their lim-
ited microsomal stability (Table 1). Their relatively large volumes
of distribution and non-renal elimination are probably a reflection
of their high logP values. The oral bioavailability was high for all
compounds 29.4% for compound 1, nearly 100% for compound 6,
47.9% for 7 and 58.3% for 15. The lower bioavailability for com-
pound 1 may be due to its poor solubility and 7 may be the conse-
quence of active efflux counteracting absorption as indicated by
the Caco-2 efflux ratio of 2.2 (Table 1).
er than for the investigated nitrofuran isoxazolines, suggesting that
further stabilization against metabolic degradation may be a prom-
ising pathway to further optimize the current lead compound.
6. Experimental
6.1. Reagents and instrumentation
All anhydrous solvents and starting materials were purchased
from Aldrich Chemical Co. (Milwaukee, WI). All reagent grade sol-
vents used from chromatography were purchased from Fisher Sci-
entific (Suwanee, GA) and flash column chromatography silica
cartridges were obtained from Biotage Inc. (Lake Forest, VA). The
reactions were monitored by thin-layer chromatography (TLC) on
pre-coated Merch 60 F254 silica gel plates and visualized using
UV light (254 nm). A Biotage FLASH column chromatography sys-
tem was used to purify mixtures. All NMR spectra were recorded
on a Bruker-400 spectrometer. Chemical shifts (d) are reported in
parts per million relative to the residual solvent peak or internal
standard (tetramethylsilane), and coupling constants (J) are re-
ported in hertz (Hz). High resolution mass spectra were recorded
on a Waters Xevo G2 QTOF LC–MS using ESI. Purity of the products
was confirmed before testing by analytical RP-HPLC on a Shimadzu
HPLC system, and all final compounds had a purity of 95% or great-
er as determined by RP-HPLC. Gradient conditions M1: solvent A
(0.1% formic acid in water) and solvent B (0.1% formic acid in
MeOH): 0–1.00 min 95% A, 1.00–6.00 min 0–95% B (linear gradi-
ent), 6.00–9.50 min 100% B, 9.50–9.75 min 0–95% A, 9.75–
10.0 min 95% A, detection by UV at 254 nm and by ELSD. Gradient
conditions M2: same as M1 except solvent B is (0.1% formic acid in
acetonitrile).
In these in vivo pharmacokinetic studies, however, toxicity was
observed in rats after IV dosing with 15, leading to seizures and
death in one animal. No animals died after oral administration of
this compound, probably because of the lower peak plasma con-
centrations compared to the IV route. Compounds 1, 6 and 7 were
found to be non-toxic in both administration modes.
5. Conclusions
A novel series of tetra cyclic isoxazolines was synthesized to im-
prove the pharmacokinetic and pharmacodynamic properties over
an initial lead anti-tuberculosis agent. Introduction of solubilizing
and metabolically blocked outer rings was successful in improving
the overall solubility and decreasing protein binding. However, this
resulted in compounds with overall in vitro microsomal stability
that was lower than desired. When comparing ring substitutions
it was noted that introduction of more nonpolar-groups gave rise
to better MIC values but also generally worse solubility and meta-
bolic stabilities, a problem we have observed in other tuberculosis
drug development projects.19 This created a necessary tradeoff be-
tween these values when selecting lead candidates for advance-
ment into in vivo pharmacokinetic testing in rats. Thus,
compounds 6, 7 and 15 were put forward as they possessed the
best overall improvements in in vitro pharmacokinetic parameters
tested while retaining excellent to good MIC activity. Encourag-
ingly the pharmacokinetic profile of our compounds showed all
compounds to have high oral bioavailability with the exception
of initial compound 1 which was likely limited by poor solubility.
However, compounds 7 and 15 were rapidly cleared in vivo and
judged unsuitable for further advancement. Compound 6 demon-
strated a significantly longer half-life, higher volume of distribu-
tion and good tolerability. This compound has now advanced
forward into a in vivo efficacy testing phase that includes formula-
tion optimization studies to boost its solubility and absorption,
dose optimization using time kill experiments20 and efficacy test-
ing in a rapid mouse model of tuberculosis infection.
6.1.1. General procedure I, for preparation of 3b–c
4-Bromo aryl iodide (1 mmol), tributyl(vinyl)stannane
(1 mmol) and PdCl2(PPh3)2 (0.05 mmol) was disolved in anhydrous
DMF and heated to 160 °C in microwave for 5 min. The reaction
mass was cooled to room temperature, diethyl ether and water
were added to form a partition and the organic layer was washed
with water, brine, dried over anhydrous Na2SO4, concentrated un-
der reduced pressure and purified by flash chromatography to af-
ford 3b–c in 80–82% yields.
6.1.2. 2-Bromo-5-vinylpyridine (3b)
2-Bromo-5-iodopyridine (2 g, 7.04 mmol), tributyl(vinyl)stann-
ane (2.23 g, 7.04 mmol) and PdCl2(PPh3)2 (0.24 g, 0.35 mmol) was
disolved in anhydrous DMF (6 mL) and the reaction was carried
out as discribed in general procedure I to afford 3b (1.06 g) in
82% yield. 1H NMR (400 MHz, CDCl3): d 8.35 (d, J = 2.4 Hz, 1H),
7.60 (dd, J = 8.3, 2.6 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 6.65 (dd,
J = 17.7, 11.0 Hz, 1H), 5.83 (d, J = 17.6 Hz, 1H), 5.43 (d, J = 11.0 Hz,
1H); LC–MS: 186 (M++2).
6.1.3. 1-Bromo-2-fluoro-4-vinylbenzene (3c)
1-Bromo-2-fluoro-4-iodobenzene (2 g, 6.65 mmol), tribu-
tyl(vinyl)stannane (2.10 g, 6.65 mmol) and PdCl2(PPh3)2 (0.23 g,
0.33 mmol) was disolved in anhydrous DMF (6 mL) and the reac-
tion was carried out as discribed in general procedure I to afford
3c (1.06 g) in 80% yield. 1H NMR (400 MHz, CDCl3): d 7.46–7.55
(m, 1H), 7.16 (dd, J = 9.8, 2.0 Hz, 1H), 7.05 (dd, J = 8.2, 2.0 Hz, 1H),
6.63 (dd, J = 17.6, 10.9 Hz, 1H), 5.76 (d, J = 17.6 Hz, 1H), 5.34 (d,
J = 10.9 Hz, 1H); LC–MS: 203 (M++2).
To gain further insight the pharmacokinetic parameters of these
agents were compared to the reported values of synthetic antibac-
terial agents linezolid and ofloxacin both known for their excellent
bioavailability.21–24 As can be seen in Table 3 linezolid and ofloxa-
cin have a substantially lower clearance and smaller volume of dis-
tribution. Thus, despite of similarly short half-lives in rats, their
peak concentrations and systemic exposure are substantially high-
6.1.4. General procedure II, for preparation of 4a–c
A mixture of aryl bromide (1.0 mmol), morpholine (2.0 mmol),
diacetoxypalladium (0.2 mmol), sodium butan-1-olate (2.4 mmol)

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Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
and 2-(di-tert-butylphosphino)biphenyl (0.4 mmol) was dissolved
in anhydrous toluene and heated to 80 °C for 12 h, then the reac-
tion mass was concentrated under reduced pressure and purified
directly by flash chromatography to afford 4a–c in 69–83% yields.
6.1.10. 4-(5-(3-(5-Nitrofuran-2-yl)-4,5-dihydroisoxazol-5-
yl)pyridin-2-yl)morpholine (7)
To a solution of 4-(5-vinylpyridin-2-yl)morpholine 4b (1.00 g,
5.26 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl chloride
(1.20 g, 6.31 mmol) in anhydrous CHCl3 (15 mL) was added Et3N
(0.87 mL, 6.31 mmol) in CHCl3 (3 mL) and the reaction continued
as described in general procedure III to afford 7 (1.42 g) in 78.6%
yield. 1H NMR (400 MHz, CDCl3): d 8.18 (d, J = 2.4 Hz, 1H), 7.51
(dd, J = 8.9, 2.5 Hz, 1H), 7.40 (d, J = 3.9 Hz, 1H), 7.05 (d, J = 3.9 Hz,
1H), 6.66 (d, J = 8.8 Hz, 1H), 5.74 (dd, J = 11.0, 9.0 Hz, 1H), 3.85–
3.70 (m, 5H), 3.53 (t, 4H), 3.37 (dd, J = 17.2, 9.0 Hz, 1H); 13C NMR
6.1.5. 4-(4-Vinylphenyl)morpholine (4a)
1-Bromo-4-vinylbenzene
9 (0.2 g, 1.09 mmol), morpholine
(0.19 g, 2.18 mmol), diacetoxypalladium (0.05 g, 0.21 mmol), so-
dium butan-1-olate (0.25 g, 2.62 mmol) and 2-(di-tert-buty-
lphosphino)biphenyl (0.13 g, 0.43 mmol) was dissolved in
anhydrous toluene and the reaction was carried out as discribed
in general procedure II to afford 4a (0.16 g) in 77% yield. 1H NMR
(400 MHz, CDCl3): d 7.30–7.34 (m, 2H), 6.82–6.86 (m, 2H), 6.64
(dd, J = 17.6, 10.9 Hz, 1H), 5.59 (dd, J = 17.6, 1.0 Hz, 1H), 5.09 (dd,
J = 10.9, 1.0 Hz, 1H), 3.77–3.84 (m, 4H), 2.70–2.77 (m, 4H); LC–
MS: 190 (M++1).
(101 MHz, CDCl3)
d 159.79, 147.90, 147.23, 146.47, 135.50,
123.63, 113.04, 112.57, 106.95, 82.16, 66.66, 45.44, 40.86; HRMS
m/z [M+H]+ Calcd for C16H16N4O5: 345.119. Found: 345.119.
6.1.11. 4-(2-Fluoro-4-(3-(5-nitrofuran-2-yl)-4,5-
dihydroisoxazol-5-yl)phenyl)morpholine (8)
6.1.6. 4-(5-Vinylpyridin-2-yl)morpholine (4b)
To a solution of 4-(2-fluoro-4-vinylphenyl)morpholine 4c
2-Bromo-5-vinylpyridine 3b (0.6 g, 3.26 mmol), morpholine
(0.56 g, 6.52 mmol), diacetoxypalladium (0.14 g, 0.65 mmol), so-
dium butan-1-olate (0.75 g, 7.82 mmol) and 2-(di-tert-buty-
lphosphino)biphenyl (0.38 g, 1.30 mmol) was dissolved in
anhydrous toluene and the reaction was carried out as discribed
in general procedure II to afford 4b (0.51 g) in 83% yield. 1H NMR
(400 MHz, CDCl3): d 8.15–8.21 (m, 1H), 7.62 (dd, J = 8.9, 2.4 Hz,
1H), 6.55–6.67 (m, 2H), 5.58 (d, J = 17.6 Hz, 1H), 5.13 (d,
J = 11.0 Hz, 1H), 3.78–3.86 (m, 4H), 3.48–3.56 (m, 4H); LC–MS:
191 (M++1).
(0.38 g, 1.83 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl
chloride (0.41 g, 2.20 mmol) in anhydrous CHCl3 (6 mL) was added
Et3N (0.30 mL, 2.20 mmol) in CHCl3 (2 mL) and the reaction contin-
ued as described in general procedure III to afford 8 (0.5 g) in 75.3%
yield. 1H NMR (400 MHz, CDCl3): d 7.40 (d, J = 3.9 Hz, 1H), 7.06 (m,
3H), 6.94 (t, J = 8.6 Hz, 1H), 5.76 (dd, J = 11.2, 8.4 Hz, 1H), 3.87 (t,
J = 4.0 Hz 4H), 3.79 (dd, J = 17.2, 8.4 Hz, 1H) 3.38 (dd, J = 17.2,
8.3 Hz, 1H), 3.09 (t, J = 4.0 Hz 4H); 13C NMR (101 MHz, CDCl3) d
156.81, 154.35, 147.72, 147.13, 140.34, 133.69, 122.13, 118.84,
114.04, 113.82, 113.03, 112.66, 83.00, 66.90, 50.73, 41.53, 29.70;
HRMS m/z [M+H]+ Calcd for C17H16FN3O5: 362.114. Found:
362.113.
6.1.7. 4-(2-Fluoro-4-vinylphenyl)morpholine (4c)
1-Bromo-2-fluoro-4-vinylbenzene 3c (1.4 g, 6.96 mmol), mor-
pholine (1.21 g, 13.92 mmol), diacetoxypalladium (0.31 g,
1.39 mmol), sodium butan-1-olate (1.60 g, 16.71 mmol) and 2-
(di-tert-butylphosphino)biphenyl (0.83 g, 2.79 mmol) was dis-
solved in anhydrous toluene and the reaction was carried out as
discribed in general procedure II to afford 4c (1.0 g) in 69% yield.
6.1.12. 4-(4-Vinylphenyl)thiomorpholine (10)
A mixture of 1-bromo-4-vinylbenzene 9 (1.0 g, 5.46 mmol), thi-
omorpholine (1.127 g, 10.93 mmol), sodium butan-1-olate (1.26 g,
13.11 mmol), Pd(OAc)2 (0.245 g, 1.09 mmol) and 2-(di-tert-buty-
lphosphino)biphenyl (0.652 g, 2.18 mmol) in anhydrous toluene
was heated at 80 °C for 4 h. The reaction mass was then concen-
trated under reduced pressure and purified by flash chromatogra-
phy to afford 10 (0.86 g) in 76.6% yield. 1H NMR (400 MHz, CDCl3):
d 7.30–7.34 (m, 2H), 6.82–6.86 (m, 2H), 6.64 (dd, J = 17.6, 10.9 Hz,
1H), 5.59 (dd, J = 17.6, 1.0 Hz, 1H), 5.09 (dd, J = 10.9, 1.0 Hz, 1H),
3.53–3.62 (m, 4H), 2.70–2.77 (m, 4H); LC–MS: 206 (M++1).
1H NMR (400 MHz, CDCl3):
d 7.05–7.17 (m, 2H), 6.87 (t,
J = 8.5 Hz, 1H), 6.55–6.70 (m, 1H), 5.63 (d, J = 17.6 Hz, 1H), 5.19
(d, J = 10.8 Hz, 1H), 3.83–3.91 (m, 4H), 3.07–3.12 (m, 4H); LC–
MS: 208 (M++1).
6.1.8. General procedure III, for preparation of 6–8
At room temperature, with vigorous stirring, a solution of Et3N
(1.2 mmol) in anhydrous CHCl3 was slowly added to a solution of
olefin (1.0 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl chlo-
ride (1.2 mmol) in anhydrous CHCl3. The reaction mixture was stir-
red at room temperature for 2 h and then diluted with excess
CHCl3, washed with water, dried over anhydrous Na2SO4, concen-
trated under reduced pressure and purified by flash chromatogra-
phy to afford compounds 6–8 in 75.3–80% yields.
6.1.13. 3-(5-Nitrofuran-2-yl)-5-(4-thiomorpholinophenyl)-4,5-
dihydroisoxazole (11)
To a stirred solution of 4-(4-vinylphenyl)thiomorpholine 10
(0.65 g, 3.17 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl
chloride (0.72 g, 3.80 mmol) in anhydrous CHCl3 (10 mL), Et3N
(0.53 mL, 3.80 mmol) was added at room temperature and stirred
at the same temperature for an additional 2 h. The reaction mass
was then diluted with CHCl3 (20 mL), washed with water
(2 ꢀ 20 mL), dried over anhydrous Na2SO4, concentrated under re-
duced pressure and purified by flash chromatography to afford 11
(0.71 g) in 63% yield. 1H NMR (400 MHz, CDCl3): d 7.41 (d,
J = 3.9 Hz, 1H), 7.26 (d, J = 7.8 Hz, 2H), 7.05 (d, J = 3.9 Hz, 1H),
6.88 (d, J = 7.8 Hz , 2H), 5.76 (dd, J = 11.1, 8.9 Hz, 1H), 3.76 (dd,
J = 17.2, 11.1 Hz, 1H), 3.63–3.55 (m, 4H), 3.40 (dd, J = 17.2,
11.1 Hz, 1H), 2.77–2.70 (m, 4H); 13C NMR (101 MHz, CDCl3): d
151.31, 147.83, 147.51, 129.52, 127.33, 116.77, 113.08, 112.43,
84.01, 51.69, 41.16, 26.45; HRMS m/z [M+H]+ Calcd for
6.1.9. 4-(4-(3-(5-Nitrofuran-2-yl)-4,5-dihydroisoxazol-5-
yl)phenyl)morpholine (6)
To
a solution of 4-(4-vinylphenyl)morpholine 4a (0.2 g,
1.05 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl chloride
(0.24 g, 1.26 mmol) in anhydrous CHCl3 (5 mL) was added Et3N
(0.17 mL, 1.26 mmol) in CHCl3 (1 mL) and the reaction continued
as described in general procedure III to afford 0.29 g of 6 in 80%
yield. 1H NMR (500 MHz, CDCl3): d 7.39 (d, J = 3.9 Hz, 1H), 7.27
(d, J = 9.2 Hz, 2H), 7.03 (d, J = 3.9 Hz, 1H), 6.91 (d, J = 8.5 Hz, 2H),
5.75 (dd, J = 11.2, 9.03 Hz, 1H), 3.86 (t, J = 4.6 Hz, 4H), 3.75 (dd,
J = 17.0, 10.9 Hz, 1H), 3.39 (dd, J = 17.0, 8.7 Hz, 1H), 3.17 (t,
J = 4.8 Hz, 4H); 13C NMR (101 MHz, CDCl3) d 151.63, 147.81,
147.50, 129.97, 127.18, 115.61, 113.08, 112.43, 84.00, 66.78,
48.96, 41.20; HRMS m/z [M+H]+ Calcd for C17H17N3O5: 344.125.
Found: 344.124.
C17H17N3O4S: 360.101. Found: 360.101.
6.1.14. 4-(4-(3-(5-Nitrofuran-2-yl)-4,5-dihydroisoxazol-5-
yl)phenyl)thiomorpholine 1-oxide (12)
To a solution of 3-(5-nitrofuran-2-yl)-5-(4-thiomorpholinophe-
nyl)-4,5-dihydroisoxazole 11 (0.05 g, 0.139 mmol) in MeOH (2 mL)

Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
6069
and water (0.5 mL) was added NaIO4 (0.03 g, 0.15 mmol). The mix-
ture was allowed to stir at room temperature for 12 h and filtered.
The filtrate was concentrated under reduced pressure and purified
by flash chromatography to afford 12 (0.03 g) in 58% yield; 1H NMR
(400 MHz, CDCl3): d 7.40 (d, J = 3.8 Hz, 1H), 7.29 (d, J = 8.0 Hz , 2H),
7.05 (d, J = 3.9 Hz, 1H), 6.96 (d, J = 8.0 Hz, 2H), 5.77 (dd, J = 11.1,
8.8 Hz, 1H), 4.07–4.00 (m, 2H), 3.77 (dd, J = 17.2, 11.1 Hz, 1H),
3.67–3.57 (m, 2H), 3.40 (dd, J = 17.2, 8.8 Hz, 1H), 2.94–2.79 (m,
4H); HRMS m/z [M+H]+ Calcd for C17H17N3O5S: 376.095. Found:
376.095.
IV to afford 14d (0.14 g) in 64% yield. 1H NMR (400 MHz, CDCl3): d
7.44–7.47 (m, 2H), 7.28–7.32 (m, 2H), 6.71 (dd, J = 17.6, 10.9 Hz,
1H), 5.74 (dd, J = 17.6, 0.9 Hz, 1H), 5.27 (dd, J = 10.8, 0.9 Hz, 1H),
4.35 (s, 2H), 4.00–4.07 (m, 2H), 3.73–3.80 (m, 2H); LC–MS: 204
(M++1).
6.1.20. 4-(5-Vinylpyridin-2-yl)morpholin-3-one (14e)
A mixture of 2-bromo-5-vinylpyridine 3b (0.2 g, 1.08 mmol),
morpholin-3-one (0.22 g, 2.17 mmol), N,N0-dimethylethylene dia-
mine (0.01 g, 0.10 mmol), K2CO3 (0.30 g, 2.17 mmol) and CuI
(0.01 g, 0.05 mmol) in anhydrous toluene was heated to reflux
and the reaction was continued as described in general procedure
IV to afford 14e (0.15 g) in 67.5% yield. 1H NMR (400 MHz, CDCl3):
d 8.42 (d, J = 2.3 Hz, 1H), 8.11 (dd, J = 8.5, 0.8 Hz, 1H), 7.80 (dd,
J = 8.7, 2.4 Hz, 1H), 6.71 (dd, J = 17.7, 11.0 Hz, 1H), 5.81 (dd,
J = 17.7, 0.7 Hz, 1H), 5.38 (dd, J = 11.0, 0.7 Hz, 1H), 4.38 (s, 2H),
4.18–4.02 (m, 4H); LC–MS: 205 (M++1).
6.1.15. General procedure IV, for preparation of 14a–f
A mixture of aryl bromide (1.0 mmol), 4-methylpiperazin-2-one
(for compounds 15–17)/morpholin-3-one (for compounds 18–20)
(2.0 mmol), N,N0-dimethylethylene diamine (0.1 mmol), K2CO3
(2.0 mmol) and CuI (0.05 mmol) in anhydrous toluene was heated
to reflux with stirring for 6 h. Then the reaction mixture was cooled
to room temperature, poured into water, stirred vigorously and ex-
tracted thrice with ethyl acetate, dried over anhydrous Na2SO4,
concentrated under reduced pressure and purified by flash chro-
matography to afford compounds 14a–f in 54–68% yields.
6.1.21. 4-(2-Fluoro-4-vinylphenyl)morpholin-3-one (14f)
A
mixture of 1-bromo-2-fluoro-4-vinylbenzene 3c (0.2 g,
0.99 mmol), morpholin-3-one (0.20 g, 1.99 mmol), N,N0-dimethyl-
ethylene diamine (0.008 g, 0.09 mmol), K2CO3 (0.27 g, 1.99 mmol)
and CuI (0.01 g, 0.05 mmol) in anhydrous toluene was heated to re-
flux and the reaction was continued as described in general proce-
dure IV to afford 14f (0.11 g) in 53.6% yield. 1H NMR (400 MHz,
CDCl3): d 7.18–7.24 (m, 3H), 6.66 (dd, J = 17.5, 10.8 Hz, 1H), 5.74
(d, J = 17.5 Hz, 1H), 5.32 (d, J = 10.8 Hz, 1H), 4.39 (s, 2H), 4.14–
4.03 (m, 4H); LC–MS: 222 (M++1).
6.1.16. 4-Methyl-1-(4-vinylphenyl)piperazin-2-one (14a)
A mixture of 1-bromo-4-vinylbenzene 9 (1.0 g, 5.46 mmol), 4-
methylpiperazin-2-one (1.24 g, 10.93 mmol), N,N0-dimethylethyl-
ene diamine (0.04 g, 0.54 mmol), K2CO3 (1.51 g, 10.93 mmol) and
CuI (0.05 g, 0.27 mmol) in anhydrous toluene was heated to reflux
and the reaction was continued as described in general procedure
IV to afford 14a (0.7 g) in 60% yield. 1H NMR (400 MHz, CDCl3): d
7.39–7.48 (m, 2H), 7.24–7.27 (m, 2H), 6.70 (dd, J = 17.6, 10.9 Hz,
1H), 5.73 (dt, J = 17.6, 0.9 Hz, 1H), 5.26 (dt, J = 10.9, 0.9 Hz, 1H),
3.65–3.75 (m, 2H), 3.28 (s, 2H), 2.75–2.83 (m, 2H), 2.41 (s, 3H);
LC–MS: 217 (M++1).
6.1.22. 4-Methyl-1-(4-(3-(5-nitrofuran-2-yl)-4,5-
dihydroisoxazol-5-yl)phenyl)piperazin-2-one (15)
To a solution of 4-methyl-1-(4-vinylphenyl)piperazin-2-one
14a (0.82 g, 3.79 mmol) and N-hydroxy-5-nitrofuran-2-carbimi-
doyl chloride (0.86 g, 4.55 mmol) in anhydrous CHCl3 (10 mL)
was added Et3N (0.63 mL, 4.55 mmol) in CHCl3 (3 mL) and the reac-
tion was continued as described in general procedure III to afford
15 (0.94 g) in 67.2% yield. 1H NMR (400 MHz, CDCl3): d 7.44–7.29
(m, 5H), 7.03 (d, J = 3.8 Hz, 1H), 5.84 (dd, J = 11.1, 8.0 Hz, 1H),
3.82 (dd, J = 17.1, 11.2 Hz, 1H), 3.71 (dd, J = 6.2, 4.6 Hz, 2H), 3.40
(dd, J = 17.1, 8.1 Hz, 1H), 3.28 (s, 2H), 2.79 (t, J = 12.0 Hz, 2H),
2.41 (s, 3H); 13C NMR (101 MHz, CDCl3): d 166.88, 152.19,
147.66, 147.05, 142.22, 138.05, 126.67, 126.39, 112.87, 83.21,
59.77, 52.03, 50.03, 45.12, 41.76; HRMS m/z [M+H]+ Calcd for
6.1.17. 4-Methyl-1-(5-vinylpyridin-2-yl)piperazin-2-one (14b)
A mixture of 2-bromo-5-vinylpyridine 3b (0.15 g, 0.81 mmol),
4-methylpiperazin-2-one (0.18 g, 1.63 mmol), N,N0-dimethylethyl-
ene diamine (0.007 g, 0.08 mmol), K2CO3 (0.22 g, 1.63 mmol) and
CuI (0.007 g, 0.04 mmol) in anhydrous toluene was heated to reflux
and the reaction was continued as described in general procedure
IV to afford 14b (0.11 g) in 62.8% yield. 1H NMR (400 MHz, CDCl3):
d 8.40 (d, J = 2.3 Hz, 1H), 8.12 (dd, J = 8.6, 1.0 Hz, 1H), 7.80 (dd,
J = 8.7, 2.4 Hz, 1H), 6.72 (dd, J = 17.7, 11.0 Hz, 1H), 5.81 (dd,
J = 17.7, 0.7 Hz, 1H), 5.38 (dd, J = 11.0, 0.8 Hz, 1H), 3.66–3.76 (m,
2H), 3.28 (s, 2H), 2.76–2.82 (m, 2H), 2.41 (s, 3H); LC–MS: 218
(M++1).
C18H18N4O5: 371.134. Found: 371.134.
6.1.23. 4-Methyl-1-(5-(3-(5-nitrofuran-2-yl)-4,5-
dihydroisoxazol-5-yl)pyridin-2-yl)piperazin-2-one (16)
6.1.18. 1-(2-Fluoro-4-vinylphenyl)-4-methylpiperazin-2-one
(14c)
To a solution of 4-methyl-1-(5-vinylpyridin-2-yl)piperazin-2-
one 14b (0.06 g, 0.27 mmol) and N-hydroxy-5-nitrofuran-2-carb-
imidoyl chloride (0.06 g, 0.33 mmol) in anhydrous CHCl3 (3 mL)
was added Et3N (0.04 mL, 0.33 mmol) in CHCl3 (0.5 mL) and the
reaction was continued as described in general procedure III to af-
ford 16 (0.05 g) in 56.7% yield. 1H NMR (400 MHz, CDCl3): d 8.42 (d,
J = 2.4 Hz, 1H), 8.07 (d, J = 8.7 Hz, 1H), 7.69 (dd, J = 8.7, 2.5 Hz, 1H),
7.40 (d, J = 3.9 Hz, 1H), 7.06 (d, J = 3.8 Hz, 1H), 5.85 (dd, J = 11.2,
8.3 Hz, 1H), 4.14–3.95 (m, 2H), 3.86 (dd, J = 17.2, 11.1 Hz, 1H),
3.40 (dd, J = 17.2, 8.3 Hz, 1H), 3.31 (s, 2H), 2.80 (t, J = 5.5 Hz, 2H),
A
mixture of 1-bromo-2-fluoro-4-vinylbenzene 3c (0.2 g,
0.99 mmol), 4-methylpiperazin-2-one (0.22 g, 1.99 mmol), N,N0-
dimethylethylene diamine (0.008 g, 0.09 mmol), K2CO3 (0.27 g,
1.99 mmol) and CuI (0.009 g, 0.04 mmol) in anhydrous toluene
was heated to reflux and the reaction was continued as described
in general procedure IV to afford 14c (0.12 g) in 55% yield. 1H
NMR (400 MHz, CDCl3): d 7.18–7.24 (m, 3H), 6.66 (dd, J = 17.5,
10.8 Hz, 1H), 5.74 (d, J = 17.5 Hz, 1H), 5.32 (d, J = 10.8 Hz, 1H),
3.62–3.69 (m, 2H), 3.30 (s, 2H), 2.77–2.84 (m, 2H), 2.42 (s, 3H);
LC–MS: 235 (M++1).
2.40 (s, 3H); 13C NMR (101 MHz, CDCl3):
d 167.79, 153.72,
152.23, 147.73, 146.74, 145.40, 134.82, 131.54, 119.74, 112.99,
81.27, 60.32, 52.03, 46.38, 45.00, 41.53; HRMS m/z [M+H]+ Calcd
for C17H17N5O5: 372.130. Found: 372.129.
6.1.19. 4-(4-Vinylphenyl)morpholin-3-one (14d)
A mixture of 1-bromo-4-vinylbenzene 9 (0.2 g, 1.09 mmol),
morpholin-3-one (0.22 g, 2.18 mmol), N,N0-dimethylethylene dia-
mine (0.01 g, 0.10 mmol), K2CO3 (0.30 g, 2.18 mmol) and CuI
(0.01 g, 0.05 mmol) in anhydrous toluene was heated to reflux
and the reaction was continued as described in general procedure
6.1.24. 1-(2-Fluoro-4-(3-(5-nitrofuran-2-yl)-4,5-
dihydroisoxazol-5-yl)phenyl)-4-methylpiperazin-2-one (17)
To a solution of 1-(2-fluoro-4-vinylphenyl)-4-methylpiperazin-
2-one 14c (0.07 g, 0.29 mmol) and N-hydroxy-5-nitrofuran-2-

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Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
carbimidoyl chloride (0.06 g, 0.35 mmol) in anhydrous CHCl3
(3 mL) was added Et3N (0.05 mL, 0.35 mmol) in CHCl3 (0.5 mL)
and the reaction was continued as described in general procedure
III to afford 17 (0.07 g) in 61% yield. 1H NMR (400 MHz, CDCl3): d
7.39 (dd, J = 3.9, 0.6 Hz, 1H), 7.33–7.29 (m, 1H), 7.24–7.14 (m,
2H), 7.04 (d, J = 3.9 Hz, 1H), 5.83 (dd, J = 11.2, 7.6 Hz, 1H), 3.85
(dd, J = 17.2, 11.2 Hz, 1H), 3.65 (t, J = 12 Hz, 2H), 3.39 (dd, J = 17.1,
7.6 Hz, 1H), 3.29 (s, 2H), 2.81 (t, J = 12 Hz, 2H), 2.42 (s, 3H); 13C
NMR (101 MHz, CDCl3): d 166.95, 159.21, 156.70, 147.61, 146.73,
141.38, 129.75, 129.29, 121.88, 114.35, 114.13, 112.95, 82.34,
59.44, 51.84, 49.75, 45.11, 41.89; HRMS m/z [M+H]+ Calcd for
first prepared in 96-well round bottom microtiter plates (Nunc,
USA). An equivalent volume (100 lL) of bacterial broth inoculum
(containing approximately 106 bacterial CFU/mL for M. tuberculosis
and 105 cfu/mL for all other bacteria) was added to each well to
give final concentrations of drug starting at 200 lg/mL and the
plates were incubated aerobically at 37 °C. M. tuberculosis microti-
ter plates were incubated for 7 or 14 days and all other strains
were incubated overnight. The MIC was recorded as the lowest
concentration of drug which prevented visible growth.
6.3. Cytotoxicity
C18H17FN4O5: 389.125. Found: 389.125.
Vero cells (kidney epithelial cells; ATCC CCL-81) were cultured
in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented
with 10% fetal bovine serum (FBS) and maintained in a humidified
incubator (37 °C, 5% CO2). Monolayers were trypsinized, seeded at
ꢁ10% confluency in white-wall, clear-bottom 96-well microtiter
plates, and allowed to adhere overnight. The next day, media
was removed and replaced with fresh DMEM/FBS containing
two-fold serial dilutions of test compounds. Following additional
72 h of incubation, cell viability was evaluated using MTT (CellTit-
er96Ò, Promega) according to the manufacturer’s instructions, with
overnight solubilization. Absorbance at 570 nm was recorded and
IC50 values calculated from corresponding dose response curves.
Results reported are the average of at least two independent
experiments.
6.1.25. 4-(4-(3-(5-Nitrofuran-2-yl)-4,5-dihydroisoxazol-5-
yl)phenyl)morpholin-3-one (18)
To a solution of 4-(4-vinylphenyl)morpholin-3-one 14d (0.04 g,
0.19 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl chloride
(0.04 g, 0.23 mmol) in anhydrous CHCl3 (3 mL) was added Et3N
(0.03 mL, 0.23 mmol) in CHCl3 (0.5 mL) and the reaction was con-
tinued as described in general procedure III to afford 18 (0.05 g) in
72.5% yield. 1H NMR (400 MHz, CDCl3): d 7.46–7.33 (m, 5H), 7.04
(d, J = 3.9 Hz, 1H), 5.85 (dd, J = 11.2, 8.1 Hz, 1H), 4.35 (s, 2H),
4.08–4.01 (m, 2H), 3.89–3.73 (m, 3H), 3.40 (dd, J = 17.1, 8.1 Hz,
1H); 13C NMR (101 MHz, CDCl3):
d 166.74, 147.67, 147.01,
141.60, 138.25, 126.73, 125.88, 112.87, 83.13, 68.58, 64.10, 49.54,
41.80; HRMS m/z [M+H]+ Calcd for C17H15N3O6: 358.103. Found:
358.103.
6.4. Solubility
6.1.26. 4-(5-(3-(5-Nitrofuran-2-yl)-4,5-dihydroisoxazol-5-
yl)pyridin-2-yl)morpholin-3-one (19)
This assay is performed on Biomek FX ADME-TOX workstation
(Beckman Coulter, Inc.; Fullerton, CA). Thirty millilitre of 10 mM
compounds solution in DMSO was applied to each well in a stock
plate. In a reference plate, compounds were diluted 600-fold in
system solution buffer (SSB, pH 7.4; pION INC, Woburn, MA) and
iso-propanol (1:1, v/v). Concentrations were assessed by UV spec-
trometry (230–500 nm). In the sample plate, compounds were di-
luted 100-fold in system solution buffer, incubated at room
temperature for 18 h to allow the compounds to be fully stable,
and then filtered through a 96-well filter plate (pION Inc., Woburn,
MA). Fractions were collected from the filtered sample plate, di-
luted with iso-propanol by 1:1 (v/v), and determined concentra-
To a solution of 4-(5-vinylpyridin-2-yl)morpholin-3-one 14e
(0.08 g, 0.39 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl
chloride (0.09 g, 0.47 mmol) in anhydrous CHCl3 (4 mL) was added
Et3N (0.06 mL, 0.47 mmol) in CHCl3 (0.5 mL) and the reaction was
continued as described in general procedure III to afford 19 (0.09 g)
in 66.8% yield. 1H NMR (400 MHz, CDCl3): d 8.43 (d, J = 2.4 Hz, 1H),
8.20 (d, J = 8.7 Hz, 1H), 7.71 (dd, J = 8.6, 2.5 Hz, 1H), 7.41 (d,
J = 3.8 Hz, 1H), 7.07 (d, J = 3.9 Hz, 1H), 5.86 (dd, J = 11.2, 8.3 Hz,
1H), 4.36 (s, 2H), 4.19–4.01 (m, 4H), 3.87 (dd, J = 17.2, 11.2 Hz,
1H), 3.41 (dd, J = 17.2, 8.3 Hz, 1H); 13C NMR (101 MHz, CDCl3): d
167.86, 152.96, 147.74, 146.73, 145.36, 135.09, 131.72, 119.09,
112.95, 81.22, 68.63, 64.29, 45.63, 41.58; HRMS m/z [M+H]+ Calcd
for C16H14N4O6: 359.098. Found: 359.098.
tion via UV spectrometry. Calculation was carried out by
lSOL
Evolution software and all compounds were tested in triplicates.
6.1.27. 4-(2-Fluoro-4-(3-(5-nitrofuran-2-yl)-4,5-
6.5. Metabolic stability
dihydroisoxazol-5-yl)phenyl)morpholin-3-one (20)
To a solution of 4-(2-fluoro-4-vinylphenyl)morpholin-3-one 14f
(0.05 g, 0.22 mmol) and N-hydroxy-5-nitrofuran-2-carbimidoyl
chloride (0.05 g, 0.27 mmol) in anhydrous CHCl3 (3 mL) was added
Et3N (0.03 mL, 0.27 mmol) in CHCl3 (0.5 mL) and the reaction was
continued as described in general procedure III to afford 20 (0.05 g)
in 66.3% yield. 1H NMR (400 MHz, CDCl3): d 7.39 (d, J = 4.0 Hz, 1H),
7.33–7.37 (m, 1H), 7.19–7.24 (m, 2H), 7.05 (d, J = 3.9 Hz, 1H), 5.84
(dd, J = 11.2, 7.6 Hz, 1H), 4.36 (s, 2H), 4.06–4.01 (m, 2H), 3.86 (dd,
J = 17.2, 11.3 Hz, 1H), 3.75–3.68 (m, 2H), 3.40 (dd, J = 17.1, 7.6 Hz,
Sample preparation for microsomal stability was modified from
the published method of Di.26 DMSO stock solutions of test com-
pounds were prepared at 10 mM concentration. Mouse liver micro-
somal solution was prepared by adding 0.058 mL of concentrated
mouse liver microsomes (20 mg/mL protein concentration) to
1.756 mL of 0.1 M potassium phosphate buffer (pH 7.4) and 5 lL
of 0.5 M EDTA to make a 0.6381 mg/mL (protein) microsomal solu-
tion. NADPH regenerating agent contained 0.113 mL of NADPH A,
0.023 mL of NADPH B, and 0.315 mL of 0.1 M potassium phosphate
1H); 13C NMR (101 MHz, CDCl3):
d
166.75, 159.11, 156.60,
buffer (pH 7.4). 2.2
each added directly to 1.79 mL of liver microsomal solution. This
solution was mixed and 90 L was transferred to six time points
plates (each in triplicate wells). For the time 0 plate, 225 L of cold
acetonitrile with internal standard (4 g/ml warfarin) was added
to each well, followed by addition of NADPH regenerating agent
(22.5 L) and no incubation. For other five time points’ plate,
NADPH regenerating agent (22.5 L) was added to each well to ini-
tiate the reaction, the plate was incubated at 37 °C for required
time, followed by quenching of the reaction by adding 225 L of
cold acetonitrile with internal standard (4 g/ml warfarin) to each
lL of each test compound diluted solution was
147.61, 146.68, 141.72, 129.61, 128.64, 121.94, 114.44, 114.23,
112.96, 99.98, 82.25, 68.52, 64.04, 49.68, 41.93; HRMS m/z
[M+H]+ Calcd for C17H14FN3O6: 376.093. Found: 376.092.
l
l
l
6.2. MIC determination
l
MICs were determined using the microbroth dilution method
according to Clinical Laboratory Standards Institute (CLSI) stan-
dards25 and were read by visual inspection. Two-fold serial dilu-
l
l
tions of antibiotic in 100
lL of the appropriate broth media were
l

Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
6071
well. All of the plates were sealed and mixed well at 600 rpm for
10 min and were centrifuged at 4000 rpm for 20 min. The superna-
tants (120 lL) were transferred to analytical plates for analysis by
calculated on following equations: %free = (concentration buffer
chamber/concentration plasma chamber) ꢀ 100% and %bound =
100%–%free.
LC–MS. Conditions for Waters ACQUITY-UPLC–MS–UV system
were described separately. The metabolic stability is evaluated
via the half-life from least-squares fit of the multiple time points
based on first-order kinetics.
6.8. In vivo pharmacokinetics
Catheterized male Sprague-Dawley rats (jugular vein alone for
oral study and jugular vein and femoral vein for intravenous study)
weighing approximately 225 g were obtained from Harlan Biosci-
ence (Indianapolis, IN). Animals were kept on a 12 h light/dark cy-
cle with access to food and water ad libitum. Animal studies were
conducted according to the guideline of Animal Welfare Act and
the Public Health Service Policy on Humane Care and Use of Labo-
ratory Animals. The study protocol was approved by the institu-
tional animal care and use committee of the University of
Tennessee Health Science Center. Test compounds (1, 6, 7 and
15) were administered to a group of rats (n = 5) either intrave-
nously (IV) or per oral (PO) at a dose of 10 or 100 mg/kg respec-
tively. For oral administration, the animals were fasted overnight,
but had access to water ad libitum.
For the IV studies, compound 6 (10 mg/kg) was prepared by dis-
solving the drug in 2% ethanol and saline, whereas the formula-
tions of compounds 1, 7 and 15 (10 mg/kg) were prepared by
dissolving the drug in 30% DMSO, 30% propylene glycol, 20% PEG
3000 and 20% saline. The formulations were administered via fem-
oral vein catheter followed by flushing the catheter with heparin-
ized locking solution. For the PO studies, compound 6 (100 mg/kg)
was prepared by dissolving the drug in 2% methanol and sonicated
for 15 min, while the formulation for compounds 7 and 15
(100 mg/kg) were prepared in 60:40 PEG 3000 water and for com-
pound 1 in 10% vitamin E TPGS solution. The oral doses were
administered using oral gavage needle fitted to a 1.0 mL syringe.
6.6. Caco-2 permeability
High throughput Caco-2 permeability determinations were per-
formed in the 96-well Transwell system using a modified meth-
od.27 Caco-2 cells were maintained at 37 °C in a humidified
incubator with an atmosphere of 5% CO2. The cells were cultured
in eagle’s minimum essential medium containing 20% FBS in
75 cm2 flasks, 100 units/ml of penicillin, and 100
lg/ml of strepto-
mycin. The Caco-2 cells were seeded onto inserts of a 96-well plate
at a density of 0.165 ꢀ 105 cells/insert and cultured in the MEM
containing 10% FBS for 7 days. Each cultured monolayer on the
96-well plate was washed twice with HBSS/HEPES (10 mM, pH
7.4). The permeability assay was initiated by the addition of each
compound solution (50 lmol/L) into inserts (apical side, A) or
receivers (basolateral side, B). The Caco-2 cell monolayers were
incubated for 2 h at 37 °C. Fractions were collected from receivers
(if apical to basal permeability) or inserts (if basal to apical perme-
ability), and concentrations were assessed by UPLC/MS (Waters;
Milford, MA). All compounds were tested in triplicates. The A?B
(or B?A) apparent permeability coefficients (Papp, cm/s) of each
compound were calculated using the equation, Papp = (dQ/dt)/
(A ꢀ C0). The flux of drug across the monolayer is dQ/dt (
l
mol/s).
The initial drug concentration on the apical side is C0
The surface area of the monolayer is A (cm2).
(lmol/L).
Serial blood samples (approx. 250 lL) were collected at 0 (pre-
6.7. Plasma protein binding
dose), 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 12.0, 24.0, 36.0 and
48.0 h post-dose. Plasma was separated immediately by centrifu-
gation (3000ꢀg for 10 min at 4 °C) and stored at ꢂ80 °C until
analysis. Urine samples were collected at an interval of 0–6, 6–
12, 12–24, 24–36 and 36–48 h post-dose and stored at ꢂ80 °C until
analysis. Plasma and urine samples were analyzed for drug concen-
trations by LC–MS/MS assay.
Solutions were prepared at 10 mM in DMSO. Dulbecco’s phos-
phate buffered saline (DPBS; pH 7.4) was obtained from Invitrogen
(Carlsbad, CA). Single-Use RED (rapid equilibrium dialysis) device
was obtained from Thermo scientific (Rockford, IL). Mouse and hu-
man plasma was obtained Lampire Biological Laboratories (Pipers-
ville, PA). Sample preparation for plasma protein binding was
modified from the method of Waters.28 The Teflon base plate with
the RED inserts (MWCO 8K) were allowed to use without any pre-
condition the membrane inserts. Mouse plasma was thawed and
centrifuged at 1000 rpm for 2 min to remove any particulates. Each
6.9. LC–MS/MS assay for plasma and urine drug concentrations
Sample preparation involved a simple protein precipitation
method using acetonitrile. Plasma/urine proteins were precipitated
by the addition of three volumes of internal standard (IS) spiked
compound was prepared at 10
This was done by adding 1 L of drug solution (10 mM in DMSO)
to 1000 L of mouse or human plasma (0.1% DMSO). Spiked plasma
solutions (300 L) were placed into the sample chamber (indicated
by the red ring) and 500 L of DPBS into the adjacent chamber. The
lM in mouse and human plasma.
l
acetonitrile to 50 lL aliquots of plasma/urine test samples. The
l
samples were vortex mixed for 1 min and centrifuged at 3000ꢀg
for 10 min at 4 °C and the supernatants were collected for LC–
MS/MS assay.
l
l
plate was sealed and incubated at 37 °C on an orbital shaker
(100 rpm) for 4 h. After incubation, the seal was removed from
the RED plate and the volume of the insert confirmed—little to
Chromatographic separations were carried out using a Shima-
dzu liquid chromatograph (Shimadzu Corporation, USA) consisting
of two pumps, online degasser, system controller and a CTC Leap
auto sampler (Leap Technologies, Carrboro, NC). Mobile phase con-
sist of acetonitrile and 10 mM ammonium acetate buffer pH ꢁ3.8
was used at a flow rate of 0.3 mL/min in gradient mode. A Phenom-
no volume change occurred. Aliquots (50 lL) were removed from
each side of the insert and dispensed into a 96-well deep plate.
An equal volume of blank plasma or DPBS was added to the re-
quired wells to create analytically identical sample matrices (ma-
trix matching). To each sample 200 lL of ACN containing 4 lg/ml
warfarin internal standard was added. All of the plates were sealed
enexÒ C18(2), 3
CA) protected with a guard column was used for the separation.
The samples (20 L) were injected onto the column and the eluate
lm, 50 ꢀ 2.0 mm column (Phenomenex, Torrance,
l
and mixed well at 600 rpm for 10 min and were centrifuged at
4000 rpm for 20 min. The supernatants (120 lL) were transferred
to analytical plates for analysis by LC–MS. Conditions for Waters
ACQUITY-UPLC–MS–UV system were described separately. The
test compound concentrations were quantified in both buffer and
plasma chambers via peak areas relative to the internal standard.
The percentage of the test compound bound to plasma was
was led directly into a mass spectrometer. An API 3000 triple-
quadruple mass spectrometer (Applied Biosystems ABI/MDS-Sciex,
Foster City, CA) equipped with an electrospray ion source was
operated in the positive ion mode. The resulting multiple reaction
monitoring chromatograms were used for quantification using the
Analyst software version 1.4.2 (Applied Biosystems ABI/ MDS-
Sciex, Foster City, CA).
A calibration curve ranging from

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Rakesh et al. / Bioorg. Med. Chem. 20 (2012) 6063–6072
2. Yee, D.; Valiquette, C.; Pelletier, M.; Parisien, I.; Rocher, I.; Menzies, D. Am. J.
3.9–5000
the test compound into 50
l
g/L was constructed for each test compound by spiking
L of blank rat plasma or urine. A struc-
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l
3. Singh, R.; Manjunatha, U.; Boshoff, H. I.; Ha, Y. H.; Niyomrattanakit, P.;
Ledwidge, R.; Dowd, C. S.; Lee, I. Y.; Kim, P.; Zhang, L.; Kang, S.; Keller, T. H.;
Jiricek, J.; Barry, C. E., 3rd Science 2008, 322, 1392.
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6. Tangallapally, R. P.; Yendapally, R.; Lee, R. E.; Hevener, K.; Jones, V. C.; Lenaerts,
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turally similar analogue to the test compounds, Lee 1106,29 was
used as IS to all calibration standards and all plasma or urine spec-
imens. Linearity for calibration standards in triplicates was as-
sessed by subjecting the spiked concentrations and the
respective peak areas to least-square linear regression analysis
with and without intercepts, and a weighted least-square regres-
sion (1/x or 1/x2). A proper calibration model was chosen after
examination of residuals and coefficient of correlation in each case.
8. Moraski, G. C.; Thanassi, J. A.; Podos, S. D.; Pucci, M. J.; Miller, M. J. J. Antibiot.
(Tokyo) 2011, 64, 667.
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Hamel, J. C.; Schaadt, R. D.; Stapert, D.; Yagi, B. H.; Adams, W. J.; Friis, J. M.;
Slatter, J. G.; Sams, J. P.; Oien, N. L.; Zaya, M. J.; Wienkers, L. C.; Wynalda, M. A. J.
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15. Chaffee, S. C.; Albrecht, B. K.; Hodous, B. L.; Martin, M. W.; McGowan, D. C.;
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WO2006044823A2, 2006.
16. Iijima, T.; Yamamoto, Y.; Akatsuka, H.; Kawaguchi, T. WO2007089034A1, 2007.
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The pharmacokinetic profile of the test compounds was ana-
lyzed from plasma concentration-time data after IV and PO admin-
istration by non-compartmental analysis using Phoenix-
WinNonlin 6.2 (Pharsight Corporation, Mountain View, CA). The
terminal half-life (t1/2) was calculated as 0.693/kz, where kz is the
terminal phase rate constant. The peak plasma concentration
(Cmax) was obtained by visual inspection of the plasma concentra-
tion–time curves. The area under the plasma concentration–time
curve from time 0 to infinity (AUC0–1) was calculated by the trap-
ezoidal rule with extrapolation to time infinity. Volume of distribu-
tion (Vd) was calculated as ratio of the area under the first moment
curve (AUMC0–1) time dose divided by the square of AUC0–1. The
total body clearance (CL) was calculated using the equation
CL = Doseiv/AUC0–1,iv. Oral bioavailability (F) was calculated by
using F = (AUC0–1,oral ꢀ Doseiv)/(AUC0–1,iv ꢀ Doseoral), where, Do-
seoral, Doseiv, AUC0–1,iv, and AUC0–1,oral are the oral and IV dose
and the corresponding areas under the plasma concentration-time
curves from time 0 to infinity, respectively. The fraction (fe) of the
test compound excreted in urine was calculated as the cumulative
amount of dose excreted unchanged in urine divided by the admin-
istered dose of the test compound.
22. FDA Briefing Package: Anti-Infective Drugs Advisory Committee; Zyvox
Preclinical Summary, U.S. Food and Drug Administration 2000 http://
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Bacteria that Grow Aerobically, 7th ed.; Approved Standard M7–A7; CLSI:
Wayne, PA, USA, 2006.
Acknowledgments
This study was supported by the National Institutes of Health
Grant AI062415 and the American Lebanese Syrian Associated
Charities (ALSAC), St. Jude Children’s Research Hospital.
26. Di, L.; Kerns, E. H.; Ma, X. J.; Huang, Y.; Carter, G. T. Comb. Chem. High
Throughput Screen. 2008, 11, 469.
27. Uchida, M.; Fukazawa, T.; Yamazaki, Y.; Hashimoto, H.; Miyamoto, Y. J.
Pharmacol. Toxicol. Methods 2009, 59, 39.
28. Waters, N. J.; Jones, R.; Williams, G.; Sohal, B. J. Pharm. Sci. 2008, 97, 4586.
29. Hurdle, J. G.; O’Neill, A. J.; Chopra, I.; Lee, R. E. Nat. Rev. Microbiol. 2011, 9, 62.
References and notes
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