The effect of 5-substitution on the electrochemical behavior and antitubercular activity of PA-824

Article abstract of DOI:10.1016/j.bmcl.2010.11.093

Nitroimidazole PA-824 is part of an exciting new class of compounds currently undergoing clinical evaluation as novel TB therapeutics. The recently elucidated mechanism of action of PA-824 involves reduction of the nitroimidazole ring and subsequent nitri

Full text of DOI:10.1016/j.bmcl.2010.11.093

Bioorganic & Medicinal Chemistry Letters 21 (2011) 812–817
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The effect of 5-substitution on the electrochemical behavior
and antitubercular activity of PA-824
Soledad Bollo a, Luis J. Núñez-Vergara a, Sunhee Kang b, , Liang Zhang b,à, Helena I. Boshoff b,
b, ,§
Clifton E. Barry, III b, Juan A. Squella a, , Cynthia S. Dowd
⇑
⇑
a Bioelectrochemistry Laboratory, Chemical and Pharmaceutical Sciences Faculty, University of Chile, PO Box 233, Santiago 1, Chile
b Tuberculosis Research Section, LCID, National Institutes of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitroimidazole PA-824 is part of an exciting new class of compounds currently undergoing clinical
evaluation as novel TB therapeutics. The recently elucidated mechanism of action of PA-824 involves
reduction of the nitroimidazole ring and subsequent nitric oxide release. The importance of this
compound and its unique activity prompted us to explore how substitution of the nitroimidazole ring
would affect electrochemical reduction and antitubercular activity. We prepared analogs of PA-824 with
bromo, chloro, cyano, and amino substituents in the 5-position of the aromatic ring. We found that sub-
stitution of the imidazole ring greatly influences reduction and the stability of the corresponding nitro
radical anion. Further, the antitubercular activities of the bromo and chloro analogs may indicate that
an alternate nitroreductase pathway within Mycobacterium tuberculosis exists.
Received 23 September 2010
Revised 17 November 2010
Accepted 19 November 2010
Available online 25 November 2010
Keywords:
Nitro radical anion
Cyclic voltammetry
PA-824
Ó 2010 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis
Each year, tuberculosis, caused by Mycobacterium tuberculosis
(Mtb), kills over 2 million people and 8 million are newly infected.1
While there is a WHO-approved therapy for this disease, drug
resistance is widespread and the need for new drugs is pressing.2
In 2000, the structure and antitubercular activity of a unique nitro-
imidazole, PA-824 (1), was reported.3 Since then, many research
groups have investigated its potential as an addition to antituber-
cular therapy and it has entered Phase 2 clinical trials.4–9 Interest-
ingly, the mechanism of action of PA-824 remained elusive for
some time and is unique with respect to antibiotic therapies.7,10
The action of PA-824 relies on intracellular reduction of the
nitro-bearing imidazole by an Mtb-specific nitroreductase, Ddn.
of the corresponding nitro radical anion.11 In that work we con-
cluded that an aprotic medium was the best experimental condi-
tion to perform this study. Our work showed that PA-824 was
electrochemically reduced via formation of a relatively stable nitro
radical anion in a manner similar to that of the electrochemical
reduction of the well-known bactericidal drug metronidazole.
However, the PA-824-derived radical required more energy for for-
mation (about 200 mV) and was approximately 100 times less sta-
ble than the corresponding metronidazole radical anion.
In light of the importance of reduction in the bioactivity of
PA-824, we asked whether or not this reduction could be modu-
lated by substitution on the imidazole ring and, if so, what the
consequences would be on the antitubercular activity of such com-
pounds. Because of its proximity to the nitro group, substituents at
the 5-position of the imidazole ring might be expected to have
the most influence on the reduction potential of this series. We
therefore synthesized PA-824 analogs bearing either electron-
withdrawing (cyano, chloro, and bromo) or electron-donating
(amino) substituents in the 5-position to explore a breadth of elec-
tronic characteristics. We report here both the electrochemistry of
the nitro radical anion formation and its stability, as well as the
antitubercular activities of each of these compounds.
This 2eꢀ reduction is coupled to the oxidation/reduction of F420
,
an unusual deazaflavin cofactor that delivers a hydride atom in
the reaction.
In previous work we reported the cyclic voltammetric behavior
of PA-824 (1) with the aim of revealing the formation and stability
⇑
Corresponding authors. Address: Department of Chemistry, George Washington
University, 725 21st Street NW, Washington, DC 20052, USA. Tel.: +1 202 994 8405;
fax: +1 202 994 5873 (C.S.D.); tel.: +56 2 6782928; fax: +56 2 7371241 (J.A.S.).
E-mail addresses: asquella@ciq.uchile.cl (J.A. Squella), cdowd@gwu.edu (C.S.
Dowd).
The synthetic process used to prepare compounds 2–5 is shown
in the Scheme 1. PA-824 (1) was prepared as described.12,13
Bromination of PA-824 (1) under basic conditions proceeded in
high yield to give compound 2.14 Compound 3 was prepared in
moderate yield from compound 2 using potassium cyanide and
potassium iodide.15 Aromatic amination of compound 1 was
Present address: Division of Medicinal Chemistry, Institute Pasteur Korea,
Gyeonggi-do 463-400, South Korea.
à
Present address: Medicinal Chemistry, Walter Reed Army Institute of Research,
Silver Spring, MD 20910, USA.
§
Present address: Department of Chemistry, George Washington University,
Washington, DC 20052, USA.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.093

S. Bollo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 812–817
813
O2N
O2N
N
O2N
NC
N
N
a
b
Br
O
O
2
O
N
N
N
OCF3
OCF3
OCF3
O
O
O
3
1
c
d
O2N
H2N
O2N
Cl
N
N
O
O
5
N
N
OCF3
OCF3
O
4
O
Scheme 1. Synthesis of 5-position Analogs of PA-824. Reagents and conditions: (a) Br2, KHCO3, DMF, 0 °C, 89%; (b) KI, KCN, DMF, 100 °C, 65%; (c) KOtBu, (CH3)3NNH2I, DMSO,
rt, 29%; (d) NiCl2, DMF, mw, 100 W, rt to 170 °C, 5 min, 72%.
achieved using 1,1,1-trimethylhydrazinium iodide and potassium
tert-butoxide in low yield to give compound 4.16 Chloro analog 5
was prepared from bromo analog 2 by halogen exchange using
nickel(II) chloride in DMF under microwave conditions.17 This
reaction was also successful using standard conditions upon heat-
ing at 70 °C (not shown). Attempts to prepare the 5-fluoro com-
pound were unsuccessful.
The effect of different 5-position substituents on reduction of
the nitro group was evaluated using different electrochemical
techniques and working electrodes.18 Figure 1 shows differential
pulse voltammograms obtained in aprotic medium (100% DMF)
for each nitroimidazole. Each voltammogram shows a main peak
(I) due to reduction of the nitro group of the corresponding nitro-
imidazole derivative. This value is the peak potential value (EP), a
parameter that reflects the tendency of the molecule to be reduced.
As can be seen, substituents adjacent to the nitro group influence
the voltammograms significantly. Thus, compounds 2 (bromo), 3
(cyano), and 5 (chloro), with electron-withdrawing substituents,
induce a positive shift in the reduction potential while the amino
compound (4) produces a negative shift. In Table 1 the peak poten-
tial values of each nitro compound are shown. Comparing all of the
derivatives, the cyano compound (3) shows the greatest shift in
peak potential value, suggesting that the nitro radical anion result-
ing from this compound was the most thermodynamically favored.
As expected, the inclusion of an electron-donating amino substitu-
ent (4) produced a negative shift, hindering the reduction process.
This would impede formation of the nitro radical anion in biologi-
cal systems.
Cyclic voltammetry (CV), unlike differential pulse voltammetry,
not only gives information about the energy required for the reduc-
tion of the nitro group (Ep values) but also provides information
about the kinetics of the nitro group electron transfer reaction
(reversibility) and stability of the nitro radical anion formed (k2 val-
ues). The advantage of CV is that information about both the catho-
dic and anodic electrochemical reactions is uncovered. In this case,
the one-electron reduction of the nitro compound forms the corre-
sponding nitro radical anion. This reaction generates a cathodic peak
characterized by a cathodic peak potential (Epc) and a cathodic peak
current (Ipc). When the scan is reversed, the nitro radical anion is oxi-
dized back to the initial nitro derivative, generating an anodic peak
characterized by an anodic peak potential (Epa) and an anodic peak
current (Ipa). The current ratio, Ipa/Ipc, indicates the stability of the ni-
tro radical. Thus, a current ratio of one indicates a stable nitro radical
and the absence of further reaction. The ratio decreases if the radical
reacts subsequently.
I'
15 μA
I
Br-PA824
I
Cl-PA824
I
NH2-PA824
I
CN-PA824
I
H-PA824
Using CV, it was possible to evaluate the influence of each substi-
tuent on the reduction mechanism of the nitro group. Figure 2 shows
the cyclic voltammograms obtained using a mercury electrode. As
expected the cyano (3), bromo (2), and chloro (5) derivatives pro-
electrolyte
ꢁꢀ
0
-500
-1000 -1500 -2000 -2500
E / mV
duce a positive shift in the nitro reduction, but the ArNO2=ArNO2
couple is only clearly formed in the cyano derivative. Moreover,
the redox mechanism is clearly more complex in the case of the bro-
mo derivative, with two close signals (i.e., I and I0) that prevent the
exact determination of which peak corresponds to the nitro radical
Figure 1. Differential pulse voltammograms obtained for the reduction of 1 mM
5-position substituted PA-824 analogs using a glassy carbon electrode (GCE) in DMF
and 0.1 M tetrabutylammonium perchlorate (TBAP).

814
S. Bollo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 812–817
Table 1
Peak potentials (Epc) for reduction of 5-substituted PA-824 analogs
O2N
R
N
O
N
OCF3
O
Compound
R
DPV–GCEa
Epc (mV)
ꢀ1236
CV–Hgb
Epc (mV)
D
Ep (mV)
k2,dim (Mꢀ1 sꢀ1
)
1
2
3
4
5
H
Br
CN
NH2
Cl
ꢀ1312
72
—
71
—
1.9 ꢃ 105
ꢀ1107/ꢀ1236
ꢀ784/ꢀ1635
ꢀ1331
ꢀ1084/ꢀ1281
ꢀ852/ꢀ1808
ꢀ1528
—
6.7 ꢃ 102
—
—
ꢀ1126
ꢀ1217
276
a
Values obtained by differential pulse voltammetry (DPV).
Values obtained from cyclic voltammetric (CV) experiments.
b
- CN
5 μA
-800
-1000
-1200
-1400
-1600
-1800
I
I'
- Br (I')
- Cl
Br-PA824
I
- Br (I)
- H
- NH2
Cl-PA824
I
-0.9 -0.6 -0.3 0.0 0.3 0.6 0.9
NH2-PA824
Hammett constant, σp
I
Figure 3. Dependence of the peak potential (E) for the reductions of 5-position
substituted PA-824 analogs on the Hammett substituent constants (
p).27 Peak
r
CN-PA824
H-PA824
potentials were obtained from cyclic voltammetric measurements at 1 V/s. The two
points for bromine are from the two peaks found in the voltammogram.
I
cyano (3) derivative. For the chloro (5) derivative, an oxidation sig-
nal was observed but with a difference between cathodic and ano-
dic peak potentials,
DEp, that far exceeds the value expected for a
Electrolyte
reversible system suggesting a quasi-reversible behavior. Finally,
the bromo (2) and amino (4) derivatives did not show any oxida-
tion signal, meaning that the nitro reduction was completely irre-
0
-500 -1000 -1500 -2000 -2500
E / mV
versible. The calculated
DEp values are shown in Table 1.
Figure 2. Cyclic voltammograms obtained for the reduction of 1 mM 5-position
substituted PA-824 analogs using a drop mercury electrode (DME) in DMF and
0.1 M TBAP. Sweep rate = 1 V/s.
Furthermore, according to the different evolution with the sweep
rate of both peaks in the bromo derivative (I and I0, Fig. 4) and
knowing that adsorption peaks depend on sweep rate and diffusion
peaks depend on square root of the sweep rate, we conclude that
the product of the reduction is strongly adsorbed on the electrode
producing an adsorptive pre-peak I0 and a diffusion peak I.20
Kinetic analyses were possible only for the derivatives that
showed a reversible mechanism on the mercury electrode.21 The
anion. In fact the voltammetric behavior for the bromo derivative is
indicative of a strong adsorption of the reaction product on the elec-
trode surface as shown below.
The peak potential values obtained for each derivative using a
mercury electrode are presented in Table 1. As can be seen, the or-
der of reduction is similar to that obtained using a glassy carbon
electrode (GCE). Plotting reduction potential versus Hammett sub-
stituent constants often leads to a linear correlation.19 Figure 3
clearly shows a good correlation between the Hammett constant
and the peak potential value (correlation coefficient = 0.96).
The nitro radical anion stability was evaluated by CV measure-
ments varying the sweep rate during the experiment (Fig. 4). Var-
iation of the sweep rate in CV is equivalent to changing the time
schedule of the experiment thus permitting us to visualize free
radical species like the nitro radical anion. Under these conditions,
well-resolved, reversible cyclic voltammograms without interfer-
ing signals were obtained only for the parent PA-824 (1) and the
dependence of
D
Ep and the current ratio, Ipa/Ipc, on the sweep rate
plot are shown in Figure 5. From the Ep behav-
and the versus
x
s
D
ior, we can conclude that PA-824 (1) in DMF follows a quasi-
reversible mechanism at low scan rates, but over 1 V/s the value
is ꢂ70 mV indicating that the transference is reversible. For the
cyano derivative (3), the
DEp value was about 60–80 mV over all
sweep rates studied. On the other hand, the Ipa/Ipc values of both
compounds increase with increasing sweep rate up to 1, showing
the typical variation of an ECi mechanism,22 that is, with values
lower than 1 at low sweep rates and values ꢂ1 at higher sweep
rates. According to the literature,23 an electrochemically formed
nitro radical anion in this medium can undergo dimerization. Olm-
stead and Nicholson developed theoretical approaches to evaluate

S. Bollo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 812–817
815
CN - PA 824
H - PA 824
12
9
10
5
0
6
3
0
-5
-3
-800
-1000
-1200
-1400
-500 -600 -700 -800 -900
E / mV
E / mV
8
6
4
2
0
I'
8
Br - PA 824
Cl - PA 824
NH2 - PA 824
I
15
10
5
6
4
2
0
0
-5
-2
-800
-1000
-1200
-1200
-1400
E / mV
-1600
-300
-600
-900 -1200 -1500
E / mV
E / mV
Figure 4. Cyclic voltammograms of the isolated couple nitro/nitroradical anion from 1 mM PA-824 and its analogs in DMF and 0.1 M TBAP at different sweep rates.
24,25
the corresponding decay constants, k2,dim
.
Applying these the-
active against anaerobic (MAC) Mtb cultures at 2–4-fold higher
concentrations compared with PA-824.
oretical curves to our experimental data, we found a straight line in
the versus plot (Fig. 5). From this, we were able to determine
the decay constant values (Table 1). We conclude that the nitro
radical anion formed from the cyano derivative (3) is almost 300-
times more stable than the similar species formed from the parent
PA-824 (1).
x
s
The cyclic voltammetric results show that substitution in the
5-position of the nitroimidazole greatly influences the electrore-
duction of the adjacent nitro group. Thus, the bromo and chloro
derivatives produced a decrease in the redox potential but the
resulting nitro radical anion is not a stable species. On the other
hand, the amino derivative hindered the electroreduction of the ni-
tro group at about 200 mV (mercury electrode) and study of the
radical was not possible because it was not sufficiently stable in
the timescale of the cyclic voltammetric experiment. Finally, incor-
poration of a cyano group in the 5-position produced a dramatic
decrease in the energy required for nitro reduction (ꢂ460 mV)
and its nitro radical anion was shown to be ꢂ300 times more stable
than the parent PA-824. Interestingly, the bromo and chloro deriv-
atives were active against wild-type and F420 mutant strains of
Mtb, while the amino and cyano derivatives were devoid of antitu-
bercular activity.
Taken together, our results indicate that: (1) reduction of the
bromo and chloro derivatives may occur via an Mtb nitroreductase
that is distinct from Ddn and which may follow a different mech-
anism compared with PA-824; (2) electron-rich nitroimidazoles
may not be reducible within the Mtb cell; and (3) electron-defi-
cient nitroimidazoles are reduced slowly and result in a more sta-
ble nitro radical anion. One might expect these latter compounds
to have greater antitubercular activity due to an increased produc-
tion of NO. They did not display higher activity against wild-type
H37Rv. One reason for this may be that electrochemical reduction
is not perfectly mimicked within the cell, and steric constraints of
the relevant nitroreductase prevented facile nitroreduction. Addi-
tionally, NO production could be measured in a manner similar
The biological activity of compounds 2–5 is shown in Table 2.26
The data for PA-824 is given as a reference and has been reported
previously.5 Against wild-type Mtb, the bromo (2) and chloro (5)
derivatives retained antitubercular activity, albeit 30-fold lower
compared to the parent PA-824 (1). The cyano (3) and amino
(4) derivatives were inactive. Superficially, this data suggests
that while substitution at the 5-position is tolerated, strongly
electron-withdrawing or electron-donating substituents in this
position are not. Looking further, we see that compounds 2 and 5
are also active against Class B1 and C mutant Mtb strains where
PA-824 is inactive (B1 mutants are defective in production of coen-
zyme F420 while C mutants are defective in Ddn, the enzyme which
catalyzes reduction of this class of compounds using F420).7 These
data suggest that the antitubercular activity of compounds 2 and
5 is not reliant on the biosynthesis of cofactor F420 in the same
way that PA-824 is. The most likely explanation of these results is
that reduction of these compounds proceeds by a different pathway
than PA-824. Since the mechanism of action of PA-824 involves
reduction not of the aromatic nitro group per se but rather hydride
transfer to C-4 of the imidazole ring, one possible explanation is
that the 5-substituted compounds are instead reduced at the nitro
substituent. Due to the modest activity of these compounds, we
cannot rule out a non-specific mechanism within the cell or cell
membrane. It is interesting to note that the bromo derivative 2 is

816
S. Bollo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 812–817
H - PA 824
CN - PA 824
120
100
80
60
40
20
0
120
100
80
60
40
20
0
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0
log V
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0
log V
1.0
0.8
0.6
0.4
1.0
0.8
0.6
0.4
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0
log V
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0
log V
3
2
1
0
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.000
0.005
0.010
0.015
0.020
0.0
0.2
0.4
0.6
τ
τ
Figure 5.
DEp and Ipa/Ipc vs. log sweep rate plots and plot of the kinetic parameter, x, with the time constant, s, for PA-824 and its cyano analog according to the theoretical
approach of Nicholson and Shain for dimerization.21
Table 2
Biological activities of 5-substituted PA-824 analogs
O2N
R
N
O
N
OCF3
O
Compound
R
wtH37Rv MIC (
lM)
wtH37Rv MAC (
lM)
Class B1a MIC (
lM)
Class Cb MIC (
lM)
1
2
3
4
5
H
Br
CN
NH2
Cl
0.8
25
>100
>100
25
8–16
32.5
nd
>500
nd
>100
25
>100
>100
25
>100
25
>100
>100
50
nd = not done.
a
ꢀ
B1 mutant is F420
.
b
C mutant is Ddnꢀ.
to that for PA-82410 to show that a nitroreductive mechanism re-
mains at work. These experiments are beyond the scope of this
work.
The most intriguing result to come from this data is the possi-
bility of an alternate nitroreductase that reduces the bromo/chloro
derivatives. This possibility was raised in previous work by Hurdle

S. Bollo et al. / Bioorg. Med. Chem. Lett. 21 (2011) 812–817
817
et al.28 If such a protein exists, pursuing it and its substrate toward
a second-generation nitroimidazole series would seem worth-
while. These compounds would theoretically overcome resistance
to PA-824 and related clinical candidates.
16. Preparation of 4. 2-Nitro-6-(4-trifluoromethoxy-benzyloxy)-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazin-3-ylamine. KOtBu (0.44 g, 3.90 mmol) was added to
a stirred solution of 1 (0.70 g, 1.95 mmol) and 1,1,1-trimethylhydrazinium
iodide (0.43 g, 2.15 mmol) in DMSO (10 mL) at rt. The reaction mixture was
stirred further under an inert atmosphere overnight. Water (200 mL) was
added to the mixture, and the precipitate was filtered and washed with water
to give 4 (yellow solid, 0.21 g, 29%): 1H NMR (300 MHz, DMSO-d6) d 3.89–4.05
(m, 2H), 4.20–4.24 (m, 1H), 4.36 (d, J = 11.7 Hz, 1H), 4.59–4.71 (m, 3H), 7.33–
7.35 (m, 1H), 7.43–7.46 (m, 2H), 7.83 (br s, 2H); MS(ESI) m/e 375 [M+H]+; mp
235 °C (dec); HRMS m/e calcd for C14H14N4O5F3 375.0916, found 375.0918.
17. Preparation of 5. 3-Chloro-2-nitro-6-(4-trifluoromethoxy-benzyloxy)-6,7-
Acknowledgements
This study was funded by the Intramural Research Division
of the NIAID, NIH, and FONDECYT (Project No. 1090120).
dihydro-5H-imidazo[2,1-b][1,3]oxazine. Compound
2 (0.020 g, 0.045 mmol)
was placed in a glass tube with nickel(II) chloride (5.9 mg, 0.045 mmol), DMF
(0.5 mL) and stirring bar. The vessel was sealed with a septum and placed into
the microwave cavity. Microwave irradiation of 100 W was used, the
temperature being ramped from rt to 170 °C. Once this temperature was
reached, the reaction mixture was held at this temperature for 5 min. After the
reaction, the solvent was removed under reduced pressure. Water (5 mL) was
added to the residue and the mixture was extracted with ethyl acetate
(5 mL ꢃ 2). The organic layer was washed with brine (5 mL), dried (MgSO4) and
evaporated. The crude residue was purified by recrystallization (mixture of
ethyl acetate and hexanes) to give 5 (white solid, 0.013 g, 72%): 1H NMR
(300 MHz, CDCl3 + CD3OD) d 4.02–4.17 (m, 2H), 4.22–4.25 (m, 1H), 4.36 (dd,
J = 12.0 Hz, 1.5 Hz, 1H), 4.62 (d, J = 12.3 Hz, 1H), 4.66–4.71 (m, 1H), 4.72 (d,
J = 12.3 Hz, 1H), 7.21–7.23 (m, 2H), 7.35–7.39 (m, 2H); 13C NMR (75 MHz,
CDCl3) d 46.1, 66.3, 67.6, 70.4, 114.1, 118.9, 121.4, 122.3, 129.3, 135.4, 138.4,
145.4, 149.3; MS(ESI) m/e 394, 396 [M+H]+ (3:1 ratio of Cl isotope pattern); mp
141–142 °C; HRMS m/e calcd for C14H12N3O5Cl1F3 394.0418, found 394.0413;
References and notes
1. WHO International Fact Sheet No. 104. http://www.who.int/mediacentre/
factsheets/fs104/en/index.html, 2007.
2. Treatment of Tuberculosis: Guidelines for National Programmes, WHO/CDS/TB
2003.313; WHO: Geneva, 2003.
3. Stover, C. K.; Warrener, P.; VanDevanter, D. R.; Sherman, D. R.; Arain, T. M.;
Langhorne, M. H.; Anderson, S. W.; Towell, J. A.; Yuan, Y.; McMurray, D. N.;
Kreiswirth, B. N.; Barry, C. E.; Baker, W. R. Nature 2000, 405, 962.
4. Kim, P.; Kang, S.; Boshoff, H. I.; Jiricek, J.; Collins, M.; Singh, R.; Manjunatha, U.
H.; Niyomrattanakit, P.; Zhang, L.; Goodwin, M.; Dick, T.; Keller, T. H.; Dowd, C.
S., ; Barry, C. E., 3rd J. Med. Chem. 2009, 52, 1329.
5. Kim, P.; Zhang, L.; Manjunatha, U. H.; Singh, R.; Patel, S.; Jiricek, J.; Keller, T. H.;
Boshoff, H. I.; Barry, C. E., 3rd; Dowd, C. S. J. Med. Chem. 2009, 52, 1317.
6. Lenaerts, A. J.; Gruppo, V.; Marietta, K. S.; Johnson, C. M.; Driscoll, D. K.;
Tompkins, N. M.; Rose, J. D.; Reynolds, R. C.; Orme, I. M. Antimicrob. Agents
Chemother. 2005, 49, 2294.
7. Manjunatha, U. H.; Boshoff, H.; Dowd, C. S.; Zhang, L.; Albert, T. J.; Norton, J. E.;
Daniels, L.; Dick, T.; Pang, S. S.; Barry, C. E., 3rd Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 431.
8. http://new.tballiance.org/new/portfolio/html-portfolio.php.
9. Tyagi, S.; Nuermberger, E.; Yoshimatsu, T.; Williams, K.; Rosenthal, I.; Lounis,
N.; Bishai, W.; Grosset, J. Antimicrob. Agents Chemother. 2005, 49, 2289.
10. 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.
½ ꢄ ꢀ34.09 (c, 0.87, CHCl3).
a 2D0
18. Electrochemical experiments were performed using a totally automated BAS
CV-100 voltammetric analyzer. All experiments were carried out at a constant
temperature of 25 0.1 °C using a 10 mL thermostatic cell. A static mercury
drop electrode (SMDE) mode in a Controlling Growth Mercury Electrode stand
from BAS, with a drop area of 0.42 mm2, and a 3 mm diameter Glassy Carbon
Electrode (GCE) as working electrodes and a platinum wire as a counter
electrode were used. All potentials were measured against 3 M Ag/AgCl.
19. Zuman, P. Substituents Effect in Organic Polarography; Plenum Press: New York,
NY, 1973.
20. Wopschall, R. H.; Shain, I. Anal. Chem. 1967, 39, 1514.
21. For voltammetric studies, 1 mM of each drug in anhydrous dimethylformamide
(DMF) and 0.1 M of tetrabutylammonium perchlorate (TBAP) was used.18 In the
kinetic analysis carried out by cyclic voltammetry, the return-to-forward peak
11. Bollo, S.; Nunez-Vergara, L. J.; Squella, J. A. J. Electroanal. Chem. 2004, 562, 9.
12. Baker, W. R. S. C.; Keeler, E. L. U.S. Patent 5,668,127, 1997.
13. Baker, W. R. S. C.; Keeler, E. L. U.S. Patent 6,087,358, 2000.
14. Preparation of 2. 3-Bromo-2-nitro-6-(4-trifluoromethoxy-benzyloxy)-6,7-
ꢁꢀ
current ratio Ipa/Ipc for the reversible first-electron transfer (the ArNO2=ArNO2
couple) was measured from each cyclic voltammogram, varying the scan rate
from 0.1 to 10 V/s according to the procedure described by Nicholson.21 Using
the theoretical approach of Olmstead et al.,23,24 the Ipa/Ipc values measured
dihydro-5H-imidazo[2,1-b][1,3]oxazine. Bromine (62 lL, 0.50 mmol) was
added to a stirred suspension of PA-824 (1, 0.15 g, 0.42 mmol) and KHCO3
(0.063 g, 0.63 mmol) in anhydrous DMF (5 mL) at 0 °C. The resulting mixture
was stirred at room temperature for 2 h. The solvent was removed under
reduced pressure, and water (10 mL) was added to the residue, and the
mixture was extracted with ethyl acetate (10 mL ꢃ 2). The organic layer was
washed with brine (10 mL), dried (MgSO4) and evaporated. The crude residue
was purified by column chromatography (methylene chloride/methanol = 30:1
ratio) to give 2 (pale yellow solid, 0.16 g, 89%): 1H NMR (300 MHz, CDCl3)
d 3.98 (dd, J = 3.9, 13.2 Hz, 1H), 4.08–4.14 (m, 1H), 4.18–4.19 (m, 1H), 4.33–
4.37 (m, 1H), 4.61–4.68 (m, 2H), 4.71 (d, J = 12.0 Hz, 1H), 7.21–7.23 (m, 2H),
7.34–7.36 (m, 2H); 13C NMR (75 MHz, CDCl3) d 47.2, 66.5, 67.7, 70.5, 99.9,
118.9, 121.4, 122.3, 129.3, 135.3, 147.3, 149.4; MS(ESI) m/e 438, 440 [M+H]+
(1:1 ratio of Br isotope pattern); mp 124.8–126.1 °C; HRMS m/e calcd for
experimentally at each scan rate were inserted into
a working curve to
determine the parameter , which incorporates the effects of rate constant,
x
drug concentration and scan rate. A plot of
relationship described by the equation
x
versus
s
resulted in a linear
x
¼ k2 ꢃ Co
ꢃ s
ꢁꢀ
where k2 is the second-order rate constant for the chemical reaction of ArNO2
,
Co is the compound concentration and = (Ek ꢀ E )/
s
m. We obtained the second-
½
order rate constant for the decomposition of the nitro radical anion from the
slope of the straight line
of ArNO2 follows second-order kinetics is supported by the linear relation be-
x versus s. The assumption that the decomposition
ꢁꢀ
C
14H12N3O5BrF3 437.9912, found 437.9911.
15. Preparation of 3. 2-Nitro-6-(4-trifluoromethoxy-benzyloxy)-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine-3-carbonitrile. To solution of (0.20 g, 0.46
tween the kinetic parameter
22. Nicholson, R. S.; Shain, I. Anal. Chem. 1964, 36, 706.
23. Squella, J. A.; Bollo, S.; Nunez-Vergara, L. Curr. Org. Chem. 2005, 9, 565.
24. Olmstead, M. L.; Nicholson, R. S. Anal. Chem. 1969, 41, 862.
25. Olmstead, M. L.; Hamilton, R. G.; Nicholson, R. S. Anal. Chem. 1969, 41, 260.
26. The antitubercular activities of all compounds were measured as has been
previously described using the H37Rv laboratory strain of Mycobacterium
tuberculosis.5 Mutant strains B1 and C have been described previously.7
27. Hammett, L. P. Physical Organic Chemistry; McGraw-Hill: New York, NY, 1940.
28. Hurdle, J. G.; Lee, R. B.; Budha, N. R.; Carson, E. I.; Qi, J.; Scherman, M. S.; Cho, S.
H.; McNeil, M. R.; Lenaerts, A. J.; Franzblau, S. G.; Meibohm, B.; Lee, R. E. J.
Antimicrob. Chemother. 2008, 62, 1037.
x and the time constant s.
a
2
mmol) in DMF (10 mL) was added KI (0.015 g, 0.091 mmol) and KCN (0.059 g,
0.91 mmol). The reaction mixture was heated to 100 °C and stirred overnight.
The solvent was removed and water (40 mL) was added. The resulting mixture
was extracted with methylene chloride (40 mL ꢃ 2). The organic layer was
washed with brine (40 mL), dried (MgSO4) and evaporated. The crude residue
was purified by column chromatography (hexanes/ethyl acetate = 1:3 ratio) to
give 3 (white solid, 0.11 g, 65%): 1H NMR (300 MHz, CDCl3) d 4.19–4.32 (m, 3H),
4.39–4.44 (m, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.71–4.77 (m, 2H), 7.22–7.26 (m,
2H), 7.34–7.37 (m, 2H); MS(ESI) m/e 385 [M+H]+ ; mp 158–159 °C; HRMS m/e
calcd for C15H12N4O5F3 385.0760, found 385.0757; ½a D20
ꢀ34.1 (c, 0.51, CHCl3).
ꢄ