Structure-activity relationships of antitubercular nitroimidazoles. 1. structural features associated with aerobic and anaerobic activities of 4 - And 5-nitroimidazoles

Article abstract of DOI:10.1021/jm801246z

The 4-nitroimidazole PA-824 is active against aerobic and anaerobic Mycobacterium tuberculosis(Mtb)while 5-nitroimidazoles like metronidazole are active against only anaerobic Mtb. We have synthesized analogues of both 4 - and 5-nitroimidazoles and explor

Full text of DOI:10.1021/jm801246z

J. Med. Chem. 2009, 52, 1317–1328
1317
Structure-Activity Relationships of Antitubercular Nitroimidazoles. 1. Structural Features
Associated with Aerobic and Anaerobic Activities of 4- and 5-Nitroimidazoles
Pilho Kim,†,§ Liang Zhang,†,# Ujjini H. Manjunatha,†, Ramandeep Singh,† Sejal Patel,‡,| Jan Jiricek,‡ Thomas H. Keller,‡
Helena I. Boshoff,† Clifton E. Barry, III,† and Cynthia S. Dowd*,†,∞
Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20892, and NoVartis Institute for Tropical Diseases, 138670, Singapore
ReceiVed October 1, 2008
The 4-nitroimidazole PA-824 is active against aerobic and anaerobic Mycobacterium tuberculosis (Mtb)
while 5-nitroimidazoles like metronidazole are active against only anaerobic Mtb. We have synthesized
analogues of both 4- and 5-nitroimidazoles and explored their antitubercular activities. The nitro group is
required for both activities in all compounds. The key determinants of aerobic activity in the 4-nitroimidazoles
include the bicyclic oxazine, the lipophilic tail, and the 2-position oxygen. For the 5-nitroimidazoles, neither
the corresponding bicyclic analogue nor addition of a lipophilic tail conveyed aerobic activity. Incorporation
of a 2-position oxygen atom into a rigid 5-nitroimidazooxazine provided the first 5-nitroimidazole with
aerobic activity. Across both series, anaerobic and aerobic activities were not correlated and Mtb mutants
lacking the deazaflavin-dependent nitroreductase (Ddn) retained anaerobic sensitivity to some compounds.
Aerobic activity appears to be correlated with efficiency as a substrate for Ddn, suggesting a means of
structure-based optimization of improved nitroimidazoles.
Introduction
oxygen conditions Mtb cultures become sensitive to Mtz
treatment.7,8 However, Mtz has no activity against aerobic
populations of Mtb and is not used in the treatment of human
tuberculosis. 4-Nitroimidazoles based on Mtz were originally
synthesized as radiosensitizing agents9 and later found to have
significant activity against Mtb.10,11 From a large number of
2,3-dihydro-6-nitroimidazo[2,1-b]oxazole derivatives, compound
3 (2-ethyl-6-nitro-2,3-dihydroimidazo[2,1-b]oxazole, CGI-
17341) emerged as an interesting tool compound with potent
in vitro and in vivo activity against Mtb.10 Further work on
this class of compounds led to the discovery of PA-824, a
4-nitroimidazooxazine.5 PA-824 and its analogues have shown
potent activity against aerobic and anaerobic Mtb, no cross
resistance to current TB drugs, and efficacy in the mouse model
of TB infection.5,12-14 PA-824 is currently in clinical develop-
ment by the Global Alliance for TB Drug Development.15 More
recently, a structurally related 4-nitroimidazooxazole, compound
4 ((R)-2-methyl-6-nitro-2-((4-(4-(4-(trifluoromethoxy)phenoxy)-
piperidin-1-yl)phenoxy)methyl)-2,3-dihydroimidazo[2,1-b]ox-
azole, OPC-67683), has demonstrated significant antitubercular
activity and is also being evaluated in human clinical trials.16
A series of antitubercular and structurally similar nitrofurans
have also been reported.17 These compounds appear to be
reduced using FGD1 and F420, but through a nitroreductase
different from deazaflavin-dependent nitroreductase (Ddn)
(Rv3547) used by PA-824.18
Tuberculosis, caused by Mycobacterium tuberculosis (Mtba),
continues to be a major threat to global public health, killing
more than 1.5 million people in 2005.1 One major contributing
factor toward this mortality is the extended duration of
chemotherapy (6-9 months) required to completely cure
disease.2 Tuberculous lesions are hypoxic, with a pO2 less than
10 mmHg.3 Hypoxic Mtb does not actively replicate and is
significantly less sensitive to the standard antituberculosis drugs
that interfere with processes essential for rapid aerobic growth.4
Thus, hypoxia is thought to contribute to the extended duration
of therapy and new agents active against these metabolically
altered bacteria are urgently needed.
The nitroimidazoles, such as (S)-2-nitro-6-(4-(trifluoromethoxy)-
benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-
824, 1),5 are a promising new class of compounds that have
shown antitubercular activity under hypoxic conditions and are
currently in clinical evaluation. Metronidazole (Mtz, 2-(2-
methyl-5-nitro-1H-imidazol-1-yl)ethanol, 2), a 5-nitroimidazole,
is widely used for the treatment of diseases caused by anaerobic
bacteria.6 It has been known for almost 15 years that under low
* To whom correspondence should be addressed. Phone: 202-994-8405.
Fax: 202-994-5873. E-mail: cdowd@gwu.edu.
†
National Institute of Allergy and Infectious Diseases.
Present address: Korea Research Institute of Chemical Technology,
§
Daejeon, Korea.
#
Present address: Walter Reed Army Medical Hospital, Washington,
DC.
Present address: Novartis Institute for Tropical Diseases, Singapore.
Novartis Institute for Tropical Diseases.
Present address: Novartis Institute for Biomedical Research, Cambridge,
‡
|
MA.
∞
Present address: Department of Chemistry, George Washington
University, Washington, DC 20052.
a Abbreviations: FGD1, F420-dependent glucose-6-phosphate dehydro-
genase; MAC, minimum anaerobicidal concentration; MIC, minimum
inhibitory concentration; MBP, maltose-binding protein; Mtb, Mycobacte-
rium tuberculosis; Mtz, metronidazole; SAR, structure-activity relationship;
TB, tuberculosis; TBS or TBDMS, tert-butyldimethylsilyl; THP, tetrahydro-
2H-pyran; TBAF, tetrabutylammonium fluoride; TBAI, tetrabutylammonium
iodide; 2D NOESY, two-dimensional nuclear Overhauser effect spectroscopy.
10.1021/jm801246z CCC: $40.75
2009 American Chemical Society
Published on Web 02/11/2009

1318 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Scheme 1. Synthesis of 5-NO2-824 and des-NO2-824a
Kim et al.
a Reagents and conditions: (a) 6, K2CO3, EtOH, sealed tube, 70 °C, 20 h; (b) DHP, PPTS, CH2Cl2, room temp, 2 d; (c) TBAF, THF, room temp, 18 h;
(d) AcOH, THF, H2O, 60 °C, 18 h; (e) NaH, TBAI, 4-trifluoromethoxybenzyl bromide, DMF, -78 °C to room temp, 18 h. Compounds 10a and 12 were
prepared following the same scheme.
Our objective in the current study was to determine the
fundamental structure-activity relationship (SAR) of nitroimi-
dazoles as antitubercular compounds, with special emphasis on
the structural requirements for aerobic versus anaerobic activi-
ties. A better understanding of the SAR would help us to
determine if these diverse groups of nitroimidazole compounds
have the same cellular mode of action. In particular, we were
interested in the differences or similarities in SAR between the
4-nitroimidazooxazines (typified by PA-824) and the 5-nitroimi-
dazoles (such as Mtz) and whether the SAR for the aerobic
activity would mirror that of the anaerobic activity. To simplify
the discussion, we will use the numbering for the imidazole
ring throughout the paper. Thus, Mtz has a 5-nitro substituent,
while PA-824 and compound 3 are referred to as 4-nitro
derivatives.
Scheme 2. Synthesis of Analogues To Examine the Importance
of the Side Chain of Nitroimidazolesa
Chemistry
Simple comparison of the structures of Mtz and PA-824
revealed four key differences: (i) the location of the nitro group;
(ii) the presence or absence of the lipophilic side chain; (iii)
the electronic nature of the atom at the 2-position of the
nitroimidazole; (iv) a monocyclic versus a bicyclic ring structure.
We have synthesized a number of compounds that probe the
structural space defined by these parameters and then examined
their activity against Mtb.
Nitro isomers of PA-824 and Mtz. We initially prepared
the 5-nitro isomer of PA-824 (11 in Scheme 1). The original
synthesis of PA-824 and related compounds was published in
two U.S. patents several years ago.12,13 We have improved upon
this synthesis and have used this as the basis for much of our
synthetic work. The patented synthesis began with 2,4-dini-
troimidazole, an explosive. Instead, we used 2-chloro-4-ni-
a Reagents and conditions: (a) NaH, CH3I, DMF, -78 °C to room temp,
18 h; (b) NaH, TBAI, 4-trifluoromethoxybenzyl bromide, DMF, -78 °C to
room temp, 18 h; (c) 2-bromoethanol, K2CO3, TBAI, CH3CN, sealed tube, 75
°C, 2 h.

4- and 5-Nitroimidazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1319
Scheme 3. Synthesis of Ring-Opened Analogues of PA-824a
a Reagents and conditions: (a) BrCH2CH2OTBS, K2CO3, MeOH, sealed tube, 75 °C, 18 h; (b) NaOMe, MeOH, room temp, 24 h; (c) NaH, TBAI,
4-trifluoromethoxybenzyl bromide, DMF, -78 °C to room temp, 14 h.
Scheme 4. Synthesis of “Bicyclic” Mtza
a Reagents and conditions: (a) (i) 22, K2CO3, MeOH, sealed tube, 70 °C, 14 h; (ii) TBSCl, imidazole, CH2Cl2, room temp, 1 h; (b) tributyl(vinyl)stannane,
Pd(PPh3)4, LiCl, DMF, microwave, 120 °C, 10 min; (c) Hoveyda-Grubbs’ II catalyst, CH2Cl2, sealed tube, 60 °C, 2.5 h; (d) Wilkinson’s catalyst, H2(g),
t-BuOH, THF, room temp, 14 h; (e) TBAF, THF, room temp, 10 min. 27b was prepared following the same scheme using (S)-vinyloxirane.
Scheme 5. Synthesis of 2-Methoxy-Mtza
a Reagents and conditions: (a) BrCH2CH2OTBS, K2CO3, MeOH, sealed tube, 75 °C, 18 h; (b) NaOMe, MeOH, room temp, 15 h; (c) TBAF, THF, room
temp, 10 min.
troimidazole (5). Condensation of 5 with epoxide 6 gave an
isomeric mixture of products. After careful chromatographic
separation, alcohols 7a and 7b were isolated in a ratio of 3:1,
with a total yield of 86%.19 THP protection of 5-nitroimidazole
7b furnished protected diol 8 in 70% yield. Cyclization of 8 in
the presence of TBAF yielded bicyclic compound 9, which was
then deprotected with AcOH to give alcohol 10b. The 4-nitro
alcohol 10a was obtained following the same procedures from
7a (not shown). Alkylation of 10b using 4-trifluoromethoxy-
benzyl bromide with NaH and TBAI gave 5-nitro-824 (11).
Interestingly, movement of the nitro group from the 4-position
to the 5-position of the imidazole ring dramatically changes the
chemical reactivity of these compounds. The yields for each of
the reactions leading to compound 11 are significantly lower
than those using the corresponding 4-nitro isomers. This is
typified by the yield of the final alkylation reaction where PA-
824 was obtained in 70% yield,13 while compound 11 was
obtained in 13%. The desnitro analogue (12) was prepared using
the same methodology with 2-nitroimidazole as the starting
material (see Supporting Information).
To examine the importance of the location of the nitro group
in Mtz (2), we also prepared the 4-nitro isomer of this compound
(Scheme 2). 2-Methyl-4-nitroimidazole (13) was reacted with
2-bromoethanol and K2CO3 in the presence of TBAI to give
4-nitro-Mtz (14).20
Lipophilic Side Chain Analogues of PA-824 and Mtz. The
importance of the lipophilic side chain of PA-824 on aerobic
and anaerobic activity was addressed by the compounds shown
in Scheme 2. Methylation of alcohol 10a with CH3I and NaH
gave the analogue (15) with the 4-trifluoromethoxyphenyl side
chain removed in 57% yield.21 Simple alkylation of either Mtz
(2) or 4-nitro-Mtz (14)22 with 4-trifluoromethoxybenzyl bromide
in the presence of NaH and TBAI gave compounds 16 (33%)
and 17 (39%), respectively.
Monocyclic Analogue of PA-824 and Bicyclic Analogues
of Mtz. Compound 20, a ring-opened analogue of PA-824
(Scheme 3), was prepared as follows: 2-chloro-4-nitroimidazole
(5) was alkylated using TBS-protected bromoethanol to give
compound 18 (25%) in addition to the corresponding 5-nitro
isomer (28 (2.3%) in Scheme 5) as a minor product. Substitution

1320 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Scheme 6. Synthesis of 2-C-824a
Kim et al.
a Reagents and conditions: (a) 22, K2CO3, MeOH, sealed tube, 85 °C, 20 h; (b) NaH, TBAI, 4-trifluoromethoxybenzyl bromide, DMF, -78 °C to room
temp, 14 h; (c) tributyl(vinyl)stannane, Pd(PPh3)4, LiCl, DMF, microwave, 120 °C, 15 min; (d) Grubbs’ II catalyst, CH2Cl2, sealed tube, 55 °C, 15 h; (e)
Wilkinson’s catalyst, H2(g), t-BuOH, THF, room temp, 16 h.
Figure 1. Compounds examining the antitubercular SAR of 4-nitroimidazoles.
of the chlorine followed by TBS deprotection in the presence
of NaOMe afforded 19 in 61% yield.23 Alkylation of the
hydroxyl group in 19 gave rise to a ring-opened version of
PA-824 (20) in 58% yield. To give “bicyclic” analogues of
Mtz, we envisioned ring-closing metathesis as a key step
(Scheme 4).24 Considering the possible conformations of the
bicyclic product, both stereoisomers were prepared. Starting
from 2-bromo-4-nitroimidazole (21), an epoxide-opening
reaction using (R)-vinyloxirane (22) gave an allylic alcohol
which was TBS-protected to afford compound 23a (16% in
two steps). This procedure also produced the corresponding
4-isomer (e.g., 31 (47%) in Scheme 6). Stille coupling of
23a using tributyl(vinyl)stannane furnished the required diene
(24a, 67%).25 Ring-closing metathesis using the second
generation Hoveyda-Grubbs’ catalyst26 provided the unsat-
urated bicyclic compound 25a (72%). TBS protection was
required to avoid undesired elimination during the synthesis
of 25a. Selective reduction of 25a with Wilkinson’s catalyst
gave compound 26a in 65% yield.27 TBS deprotection of
26a with TBAF afforded bicyclic Mtz 27a (69%). The same

4- and 5-Nitroimidazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1321
Figure 2. Compounds examining the antitubercular SAR of 5-nitroimidazoles.
procedure was followed using (S)-vinyloxirane to obtain the
enantiomer 27b.
side chain on the activity of the 4-nitroimidazoles, we prepared
compounds 10a and 15. Removal of the side chain to give the
simple “4-nitro alcohol” (10a) resulted in a dramatic and
complete loss of both aerobic and anaerobic activities. To rule
out that this was simply the very different lipophilicities of an
ether compared to an alcohol, we also explored 15 (the simple
methyl ether). Compound 15 likewise showed a dramatic loss
of aerobic and anaerobic activities when compared to PA-824.
These results are in line with the results from Baker et al.12
who showed that 6-(benzyloxy)-2-nitro-6,7-dihydro-5H-imi-
dazo[2,1-b][1,3]oxazine (i.e., PA-824 without the OCF3) was
inactive against M. boVis BCG while compounds retaining the
lipophilic substituent in the para position of the aromatic ring
retained aerobic activity.
Compound 20 represents an open ring analogue of PA-824
with simple scission of the C-6/C-7 carbon bond of the oxazine
ring. As expected, this conservative change retained some of
the aerobic activity of the parent compound, PA-824, albeit at
a level reduced nearly 10-fold. The anaerobic activity was even
more significantly decreased. The response of both activities
suggests that the increase in rotational freedom accompanying
the loss of the oxazine ring results in a smaller population of
active conformers required for recognition by the activating
enzyme. Removal of the 2-position oxygen atom from 20 yields
compound 17. Interestingly, this change in the electron-donating
potential of the 2-substituent leads to a loss in aerobic activity
of more than 20-fold, arguing for a very important role of the
2-postion oxygen in determining aerobic activity of the 4-ni-
troimidazoles. The loss of anaerobic activity of 17 relative to
that of Mtz further supports that the 2-oxy substituent is a critical
determinant of both aerobic and anaerobic activities in the
4-nitroimidazoles but not 5-nitroimidazoles.
2-Position Substitutions in Each Series. To understand the
importance of the 2-position substituent on activity, both the
2-methoxy Mtz derivative (30) and the carba analogue (35) of
PA-824 were made. In Scheme 5, reaction of 2-chloro-4-
nitroimidazole (5) with TBS-protected bromoethanol gave the
4- and 5-nitro alkylation products in an 11:1 ratio. Interestingly,
preparation of the 5-nitro isomer required use of a protected
bromoethanol. The 4- and 5-nitro isomers were separated by
careful column chromatography, and their structures were
confirmed by 2D NOESY experiments and UV λmax differ-
ences.28 The 5-nitro isomer (28) was reacted with sodium
methoxide to give 29 (58%) in addition to a small amount of
30 (15%). Subsequent TBS deprotection of 29 with TBAF
afforded desired compound 30 in 69% yield. The synthesis of
analogue 35 is shown in Scheme 6 and follows the synthetic
methodology used to prepare compounds 27a and 27b. Alky-
lation of 2-bromo-4-nitroimidazole (21) furnished allylic alcohol
31. Compound 31 was then alkylated with 4-trifluoromethoxy-
benzyl bromide to afford 32 in 62% yield to install the side
chain of PA-824. 32 underwent Stille coupling, ring-closing
metathesis, and selective reduction reactions, furnishing com-
pounds 33, 34, and 35, respectively.
Results and Discussion
A Nitro Group is Essential for Both Aerobic and
Anaerobic Activities. To assess the importance of the presence
of the nitro group, we examined the aerobic MIC (minimal
inhibitory concentration, defined as the concentration that
prevents 99% of bacterial growth) as well as the anaerobic MAC
(minimum anaerobicidal concentration, defined as the drug
concentration that causes a 90% reduction in bacterial numbers
following 7 days of anaerobic drug exposure) of desnitro-824
(12). Desnitro-824 (12) retained neither the aerobic nor anaero-
bic activity of the parent compound, indicating that the nitro
group is indeed essential for the dual activities of PA-824 (Table
1).
Removal of the side chain from compound 17 yields a more
Mtz-like compound, 14. The sole difference between 14 and
Mtz lies in the position of the nitro group. Compound 14 did
not display either aerobic or anaerobic activity. This result
confirms the importance of the position of the nitro group in
Mtz’s anaerobic activity. The lack of activity of 14 compared
to 17 corroborates the observation that the side chain alone
cannot confer aerobic or anaerobic activity in the 4-nitro series.
To probe the importance of the 2-position of the imidazole
further, we returned to the bicyclic core structure of PA-824
SAR of the 4-Nitroimidazole Series Related to PA-824.
We approached the SAR of the 4-nitroimidazoles derived from
PA-824 systematically as shown in Figure 1. In order to
understand the importance of the trifluoromethoxybenzyl ether

1322 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Kim et al.
Table 1. Aerobic, Anaerobic, and Enzymatic Activities of Nitroimidazolesa
a NS ) not a substrate.
and replaced the oxazine oxygen with a methylene unit to give
compound 35. This modification restores the rotational con-
straints of the parent compound, PA-824, while eliminating the
electron-donating component at the 2-position. As expected, the
aerobic activity of compound 35 was reduced more than 30-
fold compared to PA-824 and this compound showed negligible
anaerobic activity. Interestingly, the olefinic precursor to 35,
compound 34, generated as an intermediate following the
metathesis reaction, showed comparably low aerobic activity.
While the aerobic activity was comparable, the anaerobic activity
of 34 was at least 4-fold better than its saturated analogue.
Together, these results indicate that the electronic nature of the
2-position substituent has profound importance for both activities
of the 4-nitroimidazoles. Combined with the increase in anaero-
bic activity seen with the unsaturated carba analogue of PA-
824 (34), these results suggest that the SAR associated with
aerobic activity and the SAR associated with anaerobic activity
are distinct within the 4-nitroimidazoles.

4- and 5-Nitroimidazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1323
SAR of the 5-Nitroimidazoles Related to Mtz. We em-
ployed a similar feature comparison in attempting to understand
the SAR of the antitubercular activities of the 5-nitroimidazoles
(Figure 2). Beginning with the lipophilic side chain, we found
that compound 16, containing the benzyl ether of PA-824
appended to the Mtz core structure, failed to display any aerobic
activity against Mtb and indeed showed a significant decrease
in anaerobic activity relative to that of Mtz. This observation
marks a clear distinction between the 4- and 5-nitroimidazole
series. The lipophilic side chain is required for both aerobic
and anaerobic activity of the 4-nitroimidazoles. Conversely, the
presence of the lipophilic side chain did not confer aerobic
activity and decreased anaerobic activity in the 5-nitro series.
Building structural elements of PA-824 into Mtz, compound
30 has a 2-position oxygen atom. While this feature is essential
for high aerobic and anaerobic activities in the 4-nitro series,
this modification alone is not sufficient to convey either activity
in the 5-nitro series. Indeed, 30 lost anaerobic activity relative
to the parent, Mtz.
the anaerobic activity of these two compounds was unrelated
to activation by this particular nitroreductase.
Kinetics of Activation of Nitroimidazoles by Ddn. In order
to better differentiate the activities of these compounds, we also
directly examined the efficiency of these compounds as sub-
strates of the F420-dependent nitroreductase Ddn (Rv3547). Table
1 shows the kcat/Km values of these compounds, including that
for PA-824. These data clearly show that PA-824 is the best
substrate for Ddn while Mtz is not a substrate at all. In general,
the activity of these compounds as substrates of Ddn is mostly
proportional to their observed aerobic killing activity. To a
limited extent, both 4- and 5-nitroimidazoles are substrates for
Ddn, although bicyclic 4-nitroimidazoles appear to be superior.
The SAR associated with aerobic activity in these nitroimida-
zoles is likely to reflect their interaction with Ddn, suggesting
that these compounds could be further optimized, informed by
the structure of their interaction with that receptor. Anaerobic
activity, however, appears to be more complicated than simple
efficiency as a substrate for Ddn.28
We were intrigued to note the biological activity data from
compound 10b, the “bicyclic” analogue of Mtz containing the
2-position oxygen atom. Compound 10b unexpectedly showed
aerobic activity and weak anaerobic activity. The rigid bicyclic
structure imparts both aerobic and anaerobic activity compared
with compound 30. Further, this result is contrary to that found
for the 4-nitro isomer 10a, which was inactive (Table 1).
The addition of the side chain from PA-824 to compound
10b yields compound 11. This change improves both the aerobic
and anaerobic activity of 10b. Compound 11 can also be viewed
as the 5-nitro isomer of PA-824 and is only slightly less active
compared to that parent.
To understand the influence of the 2-position oxygen atom
alone, we prepared compounds 27a/b, conformationally rigid,
bicyclic forms of Mtz. This change gave one of the most striking
results in this study. Comparing compounds 10b and 27a/b,
we find that replacing only the 2-position oxygen atom from
compound 10b with a methylene results in the loss of both
aerobic and anaerobic activities. This result indicates that for
5-nitroimidazooxazines the 2-position oxygen atom is an
essential requirement for both activities. As seen above, this
was not found to be the case with 4-nitroimidazooxazines.
Compound 35, with a 2-position methylene, retained both
aerobic and anaerobic activities.
Conclusion
This study suggests that, even though the 4-nitroimidazoles
represented by PA-824 are chemically related to 5-nitroimida-
zoles such as Mtz, the mechanism of cellular toxicity of these
molecules differs substantially. By exploring the common
elements of both series of molecules, we were able to both gain
aerobic activity (10b) in the 5-nitroimidazoles and lose aerobic
activity in the 4-nitro series (34 and 35). This work has
elucidated some key structural features that are important for
aerobic activity: the preference for the 4-nitroimidazole; the
nature of the substituent at the 2-position of the imidazole ring;
a hydrophobic substituent at the 6-position of the oxazine ring;
and the conformationally rigid bicyclic system. By correlating
these structural changes with whole cell activity against wild
type and defined mutants resistant to PA-824, we were able to
establish which structures made genuine contributions to
understanding SAR. By assessing the efficiency of these
compounds as substrates for Ddn, the nitroreductase that
activates these prodrugs, we were able to further delineate on-
target from off-target compounds. As antibacterial development
programs gradually shift toward more whole-cell based screen-
ing and lead optimization programs, these results illustrate the
ease with which multiple target effects can confuse and mislead
medicinal chemists. The improved understanding of the aerobic
SAR resulting from these studies should facilitate the rational
design and synthesis of even more potent analogues of PA-
824.
Relationship between Aerobic and Anaerobic SAR. Con-
sidering all the compounds together, we can deduce that the
structural requirements for aerobic and anaerobic activity for
the 4- and 5-nitroimidazoles are fundamentally different. To
explore this further we performed MIC and MAC determinations
against both wild type Mtb and defined Mtb mutants that were
selected for resistance to PA-824. PA-824 is activated by the
Experimental Section
Anhydrous solvents and reagents were purchased from Sigma-
Aldrich and used as received unless otherwise stated. Melting points
were obtained on an Electrothermal 9100 apparatus and are uncor-
rected. LC-MS analysis was conducted on an Agilent 1100 series
HPLC with attached Agilent quadrupole mass analyzer model G1946
D SL with electrospray ionization in positive ion mode. LC chroma-
tography used a Phenomenex Luna C18(2) column (2 mm × 50 mm,
3 µm) with a water/acetonitrile (each with 0.1% (v/v) formic acid)
gradient using a flow rate of 0.3 mL/min. UV detection was with an
Agilent diode array detector model G1315A spectrometer at 270 and
310 nm. Proton (1H) and carbon (13C) NMR spectra were recorded at
300 and 75.5 MHz, respectively, on a Varian Gemini spectrometer,
using TMS or the solvent peak as an internal standard. Column
chromatography was conducted using either silica gel (Geduran, 60,
mesh 40-63 µm) or prepacked RediSep columns (Teledyne Isco, Inc.,
Lincoln, NE) on an Isco CombiFlash Optix10 instrument. Elemental
analyses were performed by Atlantic Microlab, Inc. (Norcross, GA).
nitroreductase Ddn (Rv3547) at the expense of cofactor F420
.
Mutants resistant to this drug fall into three classes. Class A
mutants have lost the F420-dependent glucose 6-phosphate
dehydrogenase FGD1.29 Class B mutants have disruptions in
the biosynthetic genes for cofactor F420. Class C mutants have
lesions in Ddn (Rv3547), the protein that reductively activates
30
these prodrugs at the expense of reduced F420
.
Aerobically,
all classes of mutants show complete cross-resistance to all of
the compounds with aerobic activity described in this study,
suggesting that the aerobic SAR was the result of reduction of
these compounds only by Ddn (Table 1). Anaerobically,
however, compound 11 (5-nitro-824) and compound 34 (the
olefin precursor to the 2C analogue of PA-824) both showed
significant killing of class C mutants. This result suggests that

1324 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Kim et al.
HRMS analyses were performed at the W. M. Keck Foundation
Biotechnology Resource Laboratory (Yale University, New Haven,
CT) or at the National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK), NIH. Isoniazid, rifampicin, Mtz, and methylene
blue were obtained from Sigma-Aldrich. PA-824 was prepared as
described in U.S. Patent 5,668,127. All stocks were made in 20 mM
DMSO. Compound 5 was purchased from TimTec (Newark, DE).
6.6, 3.3 Hz, 1 H), 7.85 (s, 1 H); 13C (CD3OD) δ 46.2, 62.9, 89.8,
115.4; LC-MS m/z 185.9 [M + H+].
(S)-6-(4-(Trifluoromethoxy)benzyloxy)-6,7-dihydro-3-nitro-
5H-imidazo[2,1-b][1,3]oxazine (11). At -78 °C under argon
atmosphere, NaH (60% dispersion in mineral oil, 9.5 mg, 0.24
mmol) was added to a stirred solution of 10b (40.0 mg, 0.21 mmol),
4-trifluromethoxybenzyl bromide (66.0 mg, 0.26 mmol), and
tetrabutylammonium iodide (3.7 mg, 0.01 mmol) in anhydrous DMF
(5 mL). The mixture was then warmed to room temperature and
further stirred at room temperature for 18 h. The reaction was
quenched with methanol (0.5 mL). The solvent was removed under
reduced pressure. CH2Cl2 (10 mL) was added to the residue and
filtered to remove the inorganic salts. The CH2Cl2 solution was
then concentrated in vacuo. The yellow gum residue was purified
via column chromatography (0-2% MeOH in CH2Cl2) to give a
yellow solid, which was further purified via preparative TLC (eluted
with 5% MeOH in CH2Cl2) to give 11 as a yellow crystalline solid
(S)-1-(tert-Butyldimethylsilyloxy)-3-(2-chloro-4-nitro-1H-imi-
dazol-1-yl)propan-2-ol (7a) and (S)-1-(tert-Butyldimethylsily-
loxy)-3-(2-chloro-5-nitro-1H-imidazol-1-yl)propan-2-ol (7b). A
mixture of 5 (2.4 g, 16.2 mmol), 6 (4.6 g, 24.4 mmol), and
anhydrous K2CO3 (0.34 g, 2.4 mmol) in absolute EtOH (16 mL)
was heated in a sealed tube at 70 °C for 20 h. The solvent and
excess 6 were then removed under reduced pressure. CH2Cl2 (50
mL) was added to the residue, and filtered through a bed of Celite
(2 cm thick). The filtrate was then concentrated in vacuo to give a
brown oil, which was purified via column chromatography (silica
gel, 25-50% EtOAc in hexanes) to give 7a as a white crystalline
solid (3.9 g, 66%) and 7b as a colorless oil (1.1 g, 20%). 7a: Mp
1
(10 mg, 13%). [R]2D0 +7.4 (c 0.37, MeOH); H NMR (CDCl3) δ
3.78 (dd, J ) 11.1, 3.3 Hz, 1 H), 3.89 (dd, J ) 11.1, 3.9 Hz, 1 H),
4.27 (dd, J ) 9.9, 6.6 Hz, 1 H), 4.34 (dd, J ) 9.9, 8.7 Hz, 1 H),
4.60 (s, 2 H), 5.39-5.47 (m, 1 H), 7.18-7.31 (m, 1,4-disubstituted
pattern, 4 H), 7.54 (s, 1 H); 13C (CDCl3) δ 45.5, 69.4, 73.2, 85.3,
112.7, 121.3, 129.3, 135.8, 147.5, 149.2, 156.7; LC-MS m/z 360.1
[M + H+].
1
) 116.5-118.5 °C; H NMR (CDCl3) δ 0.10 (s, 6 H), 0.90 (s, 9
H), 2.74 (d, br, J ) 5.0 Hz, 1 H, alcohol-H), 3.61 (dd, J ) 13.5,
3.7 Hz, 1 H), 3.71 (dd, J ) 13.5, 4.2 Hz, 1 H); 13C NMR (CDCl3)
δ -5.7, 17.9, 25.6, 50.6, 63.9, 69.8, 122.6, 132.5, 144.8; LC-MS
m/z 336.4 [M + H+]. 7b: 1H NMR (CDCl3) δ -0.13 (s, 3 H), 0.04
(s, 3 H), 0.87 (s, 9 H), 3.60-3.64 (m, 2 H), 3.99-4.25 (m, 3 H),
7.80 (s, 1 H); 13C NMR (CDCl3) δ -4.9, 18.0, 25.8, 50.8, 63.8,
70.7, 122.4, 132.9, 145.6; LC-MS m/z 336.4 [M + H+].
2-(2-Methyl-4-nitro-1H-imidazol-1-yl)ethanol (14). A mixture
of 2-methyl-4-nitroimidazole (13, 254 mg, 2.0 mmol), 2-bromo-
ethanol (170 µL, 2.4 mmol), potassium carbonate (0.55 g, 4.0
mmol), tetrabutylammonium iodide (15 mg, 0.04 mmol), and
acetonitrile (2 mL) was stirred at 75 °C for 2 h. Silica gel (2 g)
was added, and the solvent was removed under reduced pressure.
The residue was purified twice via column chromatography (0-5%
MeOH in CH2Cl2) to give a white solid (76 mg, 22% yield). Mp )
1-((2S)-3-(tert-Butyldimethylsilyloxy)-2-(tetrahydro-2H-pyran-
2-yloxy)propyl)-2-chloro-5-nitro-1H-imidazole (8). A mixture of
7b (0.78 g, 2.3 mmol), DHP (0.43 mL, 4.6 mmol), and PPTS (0.88
g, 3.5 mmol) in CH2Cl2 (20 mL) was stirred under inert atmosphere
at room temperature for 2 days. Saturated NaHCO3 aqueous solution
(20 mL) was added to the reaction mixture. The CH2Cl2 layer was
separated, and the aqueous layer was extracted with CH2Cl2 (2 ×
15 mL). The organic extracts and the original CH2Cl2 layer were
combined, washed with brine (30 mL), and dried over MgSO4. The
solvent was then removed under reduced pressure to give a pale-
brown oil, which was purified via column chromatography (silica
gel, EtOAc/hexanes ) 3/1) to give 8 (a mixture of two diastere-
omers) as a colorless oil (0.68 g, 70%). 1H NMR (CDCl3) δ -0.12
(d, J ) 4.5 Hz, 3 H), -0.04 (d, J ) 1.8 Hz, 3 H), 0.85 (s, 4.5 H),
0.86 (s, 4.5 H), 1.50-1.83 (m), 3.25-3.31 (m, 1 H), 3.44 (dd, J )
12.3, 2.1 Hz, 1 H), 3.49-3.56 (m, 1 H), 3.63 (dd, J ) 10.0, 7.0
Hz, 1 H), 3.73-3.88 (m, 2 H), 3.99-4.15 (m, 3 H), 4.20-4.32
(m, 1 H), 4.54-4.59 (m, 1 H), 7.84 (s, 0.5 H), 7.90 (s, 0.5 H);
LC-MS m/z 419.5 [M + H+].
(S)-6,7-Dihydro-3-nitro-6-(tetrahydro-2H-pyran-2-yloxy)-5H-
imidazo[2,1-b][1,3]oxazine (9). TBAF (1.0 M in THF, 3.0 mL,
3.0 mmol) was added dropwise to a stirred solution of 8 (0.70 g,
1.6 mmol) in anhydrous THF (10 mL) at room temperature. The
reaction mixture was further stirred at room temperature under inert
atmosphere for 18 h. The solvent was removed under reduced
pressure. Aqueous NaHCO3 saturated solution (10 mL) was added
to the brown syrupy residue, and the mixture was extracted with
CH2Cl2 (3 × 10 mL). The organic extracts were combined, washed
with brine, dried over MgSO4, and the solvent was removed under
reduced pressure to give a pale-brown syrup, which was purified
via column chromatography (silica gel, EtOAc/hexanes ) 3:1 to
straight EtOAc) to give 9 as a white crystalline solid (0.10 g, 23%).
Mp ) 118.0-119.1 °C; 1H NMR (CDCl3) δ showed a mixture of
diastereomers; LC-MS m/z 270.0 [M + H+].
126.7-127.8 °C (lit.22 130-131 °C); H NMR (CD3OD) δ 2.44
1
(s, 3 H), 3.84 (t, J ) 5.1 Hz, 2 H), 4.12 (t, J ) 5.1 Hz, 2 H), 8.07
(s, 1 H).
(S)-6,7-Dihydro-6-methoxy-2-nitro-5H-imidazo[2,1-b][1,3]oxa-
zine (15). NaH (60% dispersion in mineral oil, 24 mg, 0.60 mmol)
was added to a stirred solution of 10a (93.0 mg, 0.50 mmol) and
CH3I (40.5 µL, 0.65 mmol) in anhydrous DMF (2 mL) at -78 °C.
The reaction mixture was then warmed to room temperature and
stirred under an inert atmosphere for 18 h. The reaction was
quenched with methanol (1 mL). Silica gel was added to the
mixture, and the solvent was removed under reduced pressure. The
residue was purified via column chromatography (silica gel, 2-5%
MeOH in CHCl3) to give 15 as a yellow solid (57 mg, 57%). Mp
1
) 80.0-80.9 °C; H NMR (CDCl3) δ 3.48 (s, 3 H), 3.91-3.96
(m, 1 H), 4.14 (dt, J ) 12.9, 2.4 Hz, 1 H), 4.21 (dd, J ) 12.9, 3.9
Hz, 1 H), 4.34 (d, br, J ) 12.0 Hz, 1 H), 4.63 (ddd, J ) 12.0, 3.6,
2.1 Hz, 1 H), 7.42 (s, 1 H); 13C NMR (CDCl3) δ 47.6, 56.7, 68.3,
69.7, 117.4, 148.3; LC-MS m/z 200.1 [M + H+].
1-(2-(4-(Trifluoromethoxy)benzyloxy)ethyl)-2-methyl-5-nitro-
1H-imidazole (16). NaH (60% dispersion in mineral oil, 44 mg,
1.1 mmol) was added to a stirred mixture of 1 (Mtz, 171 mg, 1.0
mmol), 4-trifluoromethoxybenzyl bromide (306 mg, 1.2 mmol), and
TBAI (18 mg, 0.05 mmol) in anhydrous DMF (5 mL) at -78 °C.
The reaction mixture was then warmed to room temperature and
stirred at room temperature under Ar for 18 h. The reaction was
quenched with MeOH (10 drops). The solvent was removed under
reduced pressure. H2O was added to the residue. The mixture was
extracted with CH2Cl2 (3 × 30 mL). The organic extracts were
combined, washed with H2O, and dried over MgSO4. The solvent
was then removed under reduced pressure to give a brown oil, which
was purified via preparative TLC (eluted with 5% MeOH in CH2Cl2)
to give 16 as a yellow solid (113 mg, 33%). Mp ) 64.5-65.5 °C;
1H NMR (CDCl3) δ 2.50 (s, 3H), 3.79 (t, J ) 4.8 Hz, 2H), 4.42 (s,
2H), 4.51 (t, J ) 4.8 Hz, 2H), 7.13-7.19 (m, 4H), 7.94 (s, 1H);
13C NMR (CDCl3) δ 14.8, 46.7, 68.9, 72.6, 121.2, 128.9, 133.3,
136.3, 149.0, 151.9; LC-MS m/z 346.1 [M + H+].
(S)-6,7-Dihydro-3-nitro-5H-imidazo[2,1-b][1,3]oxazin-6-ol (10b).
A mixture of 9 (0.22 g, 0.81 mmol), glacial acetic acid (4.0 mL,
69 mmol), tetrahydrofuran (2.0 mL), and H2O (1.0 mL) was heated
at 60 °C for 18 h. The solvent and extra reagents were removed
under reduced pressure. The residue was triturated with CH2Cl2 to
give 10b as a yellow crystalline solid (0.12 g, 75%). Mp )
174.0-175.2 °C; 1H NMR (CD3OD) δ 3.76 (dd, J ) 12.9, 3.6 Hz,
1 H), 3.98 (dd, J ) 12.9, 3.0 Hz, 1 H), 4.24 (dd, J ) 10.2, 6.6 Hz,
1 H), 4.40 (dd, J ) 10.2, 9.0 Hz, 1 H), 5.44 (dddd, J ) 12.6, 9.6,
1-(2-(4-(Trifluoromethoxy)benzyloxy)ethyl)-2-methyl-4-nitro-
1H-imidazole (17). NaH (60% dispersion in mineral oil, 40 mg,
1.0 mmol) was added to a stirred mixture of 14 (140 mg, 0.82

4- and 5-Nitroimidazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1325
mmol), 4-trifluoromethoxybenzyl bromide (255 mg, 1.0 mmol), and
TBAI (18 mg, 0.05 mmol) in anhydrous DMF (5 mL) at -78 °C.
The reaction mixture was then warmed to room temperature and
stirred under Ar for 18 h. The reaction was quenched with MeOH
(2 drops). The solvent was removed under reduced pressure. H2O
(30 mL) was added to the residue. The mixture was extracted with
CH2Cl2 (3 × 25 mL). The organic extracts were combined, washed
with H2O (2 × 100 mL), and dried over MgSO4. The solvent was
then removed under reduced pressure to give a brown solid, which
was purified via preparative TLC (eluted with EtOAc/hexanes )
3/1) to give 17 as a yellow solid (111 mg, 39%). Mp ) 86.9-88.5
with anhydrous MgSO4, filtered, and concentrated. To the crude
organic mixture (2.55 g) in CH2Cl2 (50 mL) was added TBSCl
(2.93 g, 19.5 mmol) and imidazole (1.33 g, 19.5 mol) at room
temperature. The reaction mixture was stirred at room temperature
for 1 h, quenched with H2O, and extracted with CH2Cl2 (2×). The
combined organic layers were dried with anhydrous MgSO4, filtered,
and concentrated. Chromatography on silica gel (EtOAc/hexanes
) 1/2 to 1/1) afforded (R)-23a (611 mg, 1.62 mmol, 16%) as a
slightly yellow oil in addition to the 4-NO2 desTBS product (31,
1.25 g, 4.76 mmol, 48%) as a viscous yellow oil. (R)-23a: [R]D20
1
-26.5 (c 1.89, CHCl3); H NMR (CDCl3) δ 0.01 (s, 6H), 0.83 (s,
1
°C; H NMR (CDCl3) δ 1.93 (s, 3 H), 3.24 (t, J ) 5.0 Hz, 2 H),
9H), 3.90-4.00 (m, 2H), 4.93-4.98 (m, 1H), 5.26 (dd, J ) 17.3,
1.4 Hz, 1H), 5.44 (dd, J ) 10.4, 1.4 Hz, 1H), 5.98 (ddd, J ) 17.3,
10.4, 6.0 Hz, 1H), 7.92 (s, 1H); 13C NMR (CHCl3) δ -5.75, -5.67,
18.0, 25.6, 62.5, 64.2, 119.5, 120.4, 120.7, 131.4, 147.3; HRMS
(ESMS) calcd for C13H23BrN3O3Si [M + H+] 376.0692, found
3.62 (t, J ) 5.0 Hz, 2 H), 4.00 (s, 2 H), 6.66-6.77 (m,
1,4-disubstituted benzene pattern); 13C NMR (CDCl3) δ 13.2, 47.2,
68.5, 72.5, 120.5, 121.1, 129.0, 136.0, 145.4, 146.4, 148.8; LC-MS
m/z 346.1 [M + H+].
1
376.0638. 31: [R]2D0 -6.86 (c 1.69, CHCl3); H NMR (CDCl3) δ
1-(2-(tert-Butyldimethylsilyloxy)ethyl)-2-chloro-4-nitro-1H-
imidazole (18) and 1-(2-(tert-Butyldimethylsilyloxy)ethyl)-2-
chloro-5-nitro-1H-imidazole (28). A solution of 5 (255 mg, 1.73
mmol), BrCH2CH2OTBS (605 mg, 2.53 mmol), and K2CO3 (99
mg, 0.72 mmol) in MeOH (5 mL) was heated in a sealed tube at
75 °C for 18 h. The reaction mixture was quenched with H2O and
extracted with EtOAc (2×). The combined organic layers were dried
with MgSO4, filtered, and concentrated. Chromatography on silica
gel (EtOAc/hexanes ) 1/1) afforded 18 as a white solid (130 mg,
0.424 mmol, 25%) and 28 as a white solid (19 mg, 0.061 mmol,
3.95-4.03 (m, 1H), 4.16 (dd, J ) 14.4, 3.3 Hz, 1H), 4.49-4.57
(m, 1H), 5.27 (d, J ) 10.5 Hz, 1H), 5.39 (d, J ) 17.1 Hz, 1H),
5.86 (ddd, J ) 16.8, 10.8, 6.0 Hz, 1H), 7.98 (s, 1H); 13C NMR
(CDCl3) δ 53.8, 71.2, 118.9, 120.7, 123.3, 135.9, 146.6; HRMS
(ESMS) calcd for C7H9N3O3Br [M + H+] 261.9827, found
261.9829.
(S)-2-Bromo-1-(2-(tert-butyldimethylsilyloxy)but-3-enyl)-5-ni-
tro-1H-imidazole (23b). This compound was prepared in the same
manner as 23a, using (S)-vinyloxirane. Yield: 23b (15%) and the
4-NO2-desTBS product (45%). 23b: [R]2D0 +23.7 (c 0.58, CHCl3);
1H NMR (CDCl3) δ -0.04 (s, 6H), 0.79 (s, 9H), 3.88-3.98 (m,
2H), 4.90-4.98 (m, 1H), 5.23 (d, J ) 17.3, 1H), 5.40 (d, J ) 10.4,
1H), 5.94 (ddd, J ) 17.3, 10.4, 6.0 Hz, 1H), 7.92 (s, 1H); 13C NMR
(CDCl3) δ -5.9, -5.9, 17.9, 25.5, 62.4, 64.1, 119.5, 120.4, 120.6,
131.3, 147.2; HRMS (ESMS) calcd for C13H22BrN3O3Si [M + H+]
376.0692, found 376.0704.
1
3.5%). 18: Mp ) 87.5-88.6 °C; H NMR (CDCl3) δ -0.07 (s,
6H), 0.78 (s, 9H), 3.86 (t, J ) 4.8 Hz, 2H), 4.13 (t, J ) 4.8 Hz,
2H), 7.83 (s, 1H); 13C NMR (CDCl3) δ -5.9, 17.9, 25.5, 50.0,
60.8, 121.9, 132.2, 145.2; HRMS (ESMS) calcd for C11H21ClN3O3Si
[M + H+] 306.1041, found 306.1035; presence of 2D-NOESY
correlationships, aromatic H with CH2, λmax ) 300 nm. 28: Mp )
1
47.0-47.8 °C; H NMR (CDCl3) δ -0.07 (s, 6H), 0.80 (s, 9H),
3.89 (t, J ) 5.3 Hz, 2H), 4.60 (t, J ) 5.3 Hz, 2H), 7.91 (s, 1H);
13C NMR (CDCl3) δ -5.8, 18.0, 25.6, 49.2, 61.1, 132.1, 138.8,
139.1; HRMS (ESMS) calcd for C11H21ClN3O3Si [M + H+]
306.1041, found 306.1035; absence of 2D-NOESY correlationships,
aromatic H with 2 CH2, λmax ) 310 nm.
2-(2-Methoxy-4-nitro-1H-imidazol-1-yl)ethanol (19). To a
solution of 18 (438 mg, 1.43 mmol) in MeOH (10 mL) was added
NaOMe (619 mg, 11.5 mmol) at room temperature. The reaction
mixture was stirred at room temperature for 24 h and concentrated.
Chromatography on silica gel (MeOH/CH2Cl2 ) 1/20) afforded 19
as a white solid (164 mg, 0.874 mmol, 61%). Mp ) 132-133 °C;
1H NMR (MeOH-d4) δ 3.79 (t, J ) 5.3 Hz, 2H), 3.97 (t, J ) 5.3
Hz, 2H), 4.07 (s, 3H), 7.85 (s, 1H); 13C NMR (MeOH-d4) δ 48.7,
58.6, 61.0, 120.0, 143.5, 152.9; HRMS (ESMS) calcd for
C6H10N3O4 [M + H+] 188.0671, found 188.0688.
(R)-1-(2-(tert-Butyldimethylsilyloxy)but-3-enyl)-5-nitro-2-vinyl-
1H-imidazole (24a). A mixture of (R)-23a (300 mg, 0.797 mmol),
tributylvinylstannane (0.255 mL, 0.874 mmol), Pd(PPh3)4 (37 mg,
0.032 mmol), and LiCl (33 mg, 0.80 mmol) in DMF (2 mL) was
heated under microwave at 120 °C for 10 min. The reaction mixture
was concentrated under vacuum. Chromatography on silica gel
(EtOAc/hexanes ) 1/2) afforded (R)-24a (144 mg, 0.446 mmol,
56%) as a slightly yellow oil. [R]2D0 -13.7 (c 0.37, CHCl3); 1H NMR
(CDCl3) δ -0.06 (s, 3H), -0.05 (s, 3H), 0.78 (s, 9H), 3.87 (dd, J
) 10.8, 6.6 Hz, 1H), 3.97 (dd, J ) 10.8, 4.1 H, 1H), 4.84-4.90
(m, 1H), 5.18 (dd, J ) 17.4, 1.5 Hz, 1H), 5.39 (dd, J ) 10.4, 1.5
Hz, 1H), 5.56 (dd, J ) 10.8, 1.5 Hz, 1H), 5.95 (ddd, J ) 17.4,
10.8, 5.7 Hz, 1H), 6.36 (dd, J ) 17.1, 1.5 Hz, 1H), 6.55 (dd, J )
17.1, 10.8 Hz, 1H), 7.85 (s, 1H); 13C NMR (CHCl3) δ -5.84,
-5.81, 17.9, 25.5, 60.3, 64.7, 118.6, 120.1, 121.3, 123.2, 132.0,
144.9, 147.1; HRMS (ESMS) calcd for C15H26N3O3Si [M + H+]
324.1743, found 324.1745.
1-(2-(4-(Trifluoromethoxy)benzyloxy)ethyl)-2-methoxy-4-ni-
tro-1H-imidazole (20). A mixture of 19 (19.6 mg, 0.105 mmol),
4-trifluoromethoxybenzyl bromide (40.1 mg, 0.157 mmol), and
n-Bu4NI (4.6 mg, 0.012 mmol) in DMF (1 mL) was prepared under
argon atmosphere. To this mixture 60% NaH (5.6 mg, 0.14 mmol)
was added at -78 °C. The reaction mixture was stirred at -78 °C
for 30 min and then at room temperature for 14 h, quenched with
H2O, and extracted with EtOAc (2×). The combined organic layers
were dried with MgSO4, filtered, and concentrated. Chromatography
on silica gel (EtOAc/hexanes ) 2/1) afforded 20 as a slightly yellow
(S)-1-(2-(tert-Butyldimethylsilyloxy)but-3-enyl)-5-nitro-2-vinyl-
1H-imidazole (24b). This compound was prepared in the same
manner as (R)-24a. Yield, (S)-24b (53%) as a slightly yellow oil;
1
[R]2D0 +14.6 (c 2.23, CHCl3); H NMR (CDCl3) δ -0.04 (s, 3H),
-0.03 (s, 3H), 0.80 (s, 9H), 3.85-3.91 (m, 2H), 4.82-4.92 (m,
1H), 5.20 (dd, J ) 17.4, 1.5 Hz, 1H), 5.41 (dd, J ) 10.5, 1.5 Hz,
1H), 5.59 (dd, J ) 10.5, 1.5 Hz, 1H), 5.91-6.02 (m, 1H), 6.39
(dd, J ) 17.1, 1.5 Hz, 1H), 6.52-6.61 (m, 1H), 7.86 (s, 1H); 13C
NMR (CHCl3) δ -5.79, -5.75, 18.0, 25.5, 60.3, 64.7, 118.6, 120.2,
121.3, 123.3, 132.0, 144.9, 147.2; HRMS (ESMS) calcd for
C15H26N3O3Si [M + H+] 324.1743, found 324.1749.
1
oil (22 mg, 0.061 mmol, 58%). H NMR (CDCl3) δ 3.67 (t, J )
5.0 Hz, 2H), 3.99 (t, J ) 5.0 Hz, 2H), 4.04 (s, 3H), 4.48 (s, 2H),
7.14 (d, J ) 8.4 Hz, 2H), 7.23 (d, J ) 8.4 Hz, 2H), 7.57 (s, 1H);
13C NMR (CDCl3) δ 44.8, 57.7, 67.5, 72.2, 117.9, 118.6, 120.9,
122.0, 128.8, 135.9, 142.5, 148.6, 150.7; HRMS (ESMS) calcd for
C14H15F3N3O5 [M + H+] 362.0964, found 362.0972.
(R)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6-dihydroimida-
zo[1,2-a]pyridine (25a). To a solution of (R)-24a (81 mg, 0.25
mmol) in CH2Cl2 (10 mL) was added Hoveyda-Grubbs second
generation catalyst (22 mg, 0.035 mmol). The reaction mixture was
stirred at 60 °C for 2.5 h. Chromatography on preparative TLC
(EtOAc/hexanes ) 1/1) afforded (R)-25a (54 mg, 0.18 mmol, 72%)
as a slightly brown solid. [R]2D0 -17.8 (c 0.48, CHCl3); mp )
(R)-2-Bromo-1-(2-(tert-butyldimethylsilyloxy)but-3-enyl)-5-ni-
tro-1H-imidazole (23a) and (R)-1-(2-Bromo-4-nitro-1H-imida-
zol-1-yl)but-3-en-2-ol (31). A mixture of 21 (2.0 g, 10.4 mmol),
(R)-vinyloxirane (22, 1.00 g, 13.5 mmol), and K2CO3 (560 mg,
4.05 mmol) in MeOH (10 mL) was heated in a sealed tube at 70
°C for 14 h. The reaction mixture was quenched with H2O and
extracted with EtOAc (3×). The combined organic layers were dried
1
106-108 °C; H NMR (CDCl3) δ 0.09 (d, J ) 0.9 Hz, 6H), 0.92
(s, 9H), 3.50 (dd, J ) 10.5, 9.0 Hz, 1H), 4.11 (dd, J ) 10.5, 4.2
Hz, 1H), 4.82-4.87 (m, 1H), 6.76 (s, 2H), 8.06 (s, 1H); 13C NMR

1326 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Kim et al.
(CHCl3) δ -5.3, -5.2, 18.4, 26.0, 63.9, 66.4, 118.0, 122.9, 132.3,
138.9, 153.7; HRMS (ESMS) calcd for C13H22N3O3Si [M + H+]
296.1430, found 296.1423.
61.1, 131.0, 135.3, 155.4; HRMS (ESMS) calcd for C12H24N3O4Si
[M + H+] 302.1536, found 302.1538.
2-(2-Methoxy-5-nitro-1H-imidazol-1-yl)ethanol (30). To a
solution of 29 (17 mg, 0.055 mmol) in THF (1 mL) was added 1.0
M TBAF in THF (0.070 mL, 0.070 mmol) at room temperature.
The reaction mixture was stirred at room temperature for 10 min
and concentrated. Chromatography with preparative TLC gave 30
as a white solid (7.1 mg, 0.038 mmol, 69%). Mp ) 99.0-99.8 °C;
1H NMR (CDCl3) δ 3.91 (t, J ) 5.6 Hz, 2H), 4.15 (s, 3H), 4.40 (t,
J ) 5.6 Hz, 2H), 7.78 (s, 1H); 13C NMR (CDCl3) δ 46.3, 57.9,
61.3, 131.5, 135.5, 155.4; LC-MS m/z 188.1 [M + H+].
(S)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6-dihydroimida-
zo[1,2-a]pyridine (25b). This compound was prepared in the same
manner as (R)-25a. Yield: (S)-25b (72%) as a slightly brown solid.
[R]2D0 +19.9 (c 1.36, CHCl3); mp ) 108-109 °C; 1H NMR (CDCl3)
δ 0.09 (d, J ) 0.9 Hz, 6H), 0.92 (s, 9H), 3.50 (dd, J ) 10.5, 9.0
Hz, 1H), 4.11 (dd, J ) 10.5, 4.2 Hz, 1H), 4.82-4.87 (m, 1H),
6.76 (s, 2H), 8.06 (s, 1H); 13C NMR (CHCl3) δ -5.6, -5.5, 18.1,
25.7, 63.3, 66.2, 117.8, 122.5, 138.8, 150.3, 153.7; HRMS (ESMS)
calcd for C13H22N3O3Si [M + H+] 296.1430, found 296.1429.
(R)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6,7,8-tetrahydroim-
idazo[1,2-a]pyridine (26a). A solution of (R)-25a(58 mg, 0.20
mmol) in a t-BuOH (1.5 mL) and THF (0.5 mL) was prepared and
degassed by freezing and thawing (3×). To the mixture was added
Wilkinson’s catalyst (17 mg, 0.018 mmol) and degassed by freezing
and thawing (1×). Under a hydrogen balloon atmosphere the
reaction mixture was stirred at room temperature for 14 h.
Chromatography on preparative TLC (EtOAc/hexanes ) 1/1)
afforded (R)-26a (34 mg, 0.11 mmol, 57%) as a slightly yellow
solid in addition to the starting material ((R)-25a, 22 mg, 0.074
mmol, 37%). [R]2D0 0.00 (c 0.31, CHCl3); mp ) 68.0-68.9 °C; 1H
NMR (CDCl3) δ 0.04 (s, 3H), 0.06 (s, 3H), 0.88 (s, 9H), 2.23-2.34
(m, 1H), 2.64-2.76 (m, 1H), 2.82-3.06 (m, 2H), 3.66 (dd, J )
10.8, 7.5 Hz, 1H), 3.89 (dd, J ) 10.8, 3.6 Hz, 1H), 4.39-4.47 (m,
1H), 7.81 (s, 1H); 13C NMR (CHCl3) δ -5.6, -5.5, 18.4, 23.1,
25.7, 27.9, 60.1, 65.4, 115.7, 128.6, 132.0, 152.9; HRMS (ESMS)
calcd for C13H24N3O3Si [M + H+] 298.1587, found 298.1594.
(S)-6-(tert-Butyldimethylsilyloxy)-3-nitro-5,6,7,8-tetrahydroim-
idazo[1,2-a]pyridine (26b). This compound was prepared in the
same manner as (R)-26a. Yield: (S)-26b (55%) as a slightly yellow
solid in addition to the starting material ((S)-25b, 12 mg, 0.039
mmol, 42%). [R]2D0 +0.01 (c 0.75, CHCl3); mp ) 67.2-68.0 °C;
1H NMR (CDCl3) δ 0.03 (s, 3H), 0.05 (s, 3H), 0.88 (s, 9H),
2.23-2.34 (m, 1H), 2.63-2.75 (m, 1H), 2.84-3.02 (m, 2H), 3.66
(dd, J ) 10.8, 7.5 Hz, 1H), 3.89 (dd, J ) 10.8, 3.6 Hz, 1H),
4.39-4.47 (m, 1H), 7.81 (s, 1H); 13C NMR (CHCl3) δ -5.61,
-5.56, 18.1, 23.0, 25.7, 27.9, 60.0, 65.4, 115.7, 150.5, 152.8;
HRMS (ESMS) calcd for C13H24N3O3Si [M + H+] 298.1587, found
298.1595.
1-((R)-2-(4-(Trifluoromethoxy)benzyloxy)but-3-enyl)-2-bromo-
4-nitro-1H-imidazole (32). A mixture of 31 (166 mg, 0.633 mmol),
4-trifluoromethoxybenzyl bromide (250 mg, 0.95 mmol), and
n-Bu4NI (23 mg, 0.063 mmol) in DMF (5 mL) was prepared under
argon atmosphere. To this mixture 60% NaH (30 mg, 0.76 mmol)
was added at -78 °C. The reaction mixture was stirred at 0 °C for
30 min and then at room temperature for 14 h, quenched with H2O,
and extracted with EtOAc (2×). The combined organic layers were
dried with MgSO4, filtered, and concentrated. Chromatography on
silica gel (EtOAc/hexanes ) 3/4) afforded 32 as a slightly yellow
1
oil (171 mg, 0.392 mmol, 62%). [R]2D0 -18.9 (c 1.95, CHCl3); H
NMR (CDCl3) δ 4.26-4.40 (m, 3H), 4.48 (d, J ) 12.0 Hz, 1H),
4.78 (d, J ) 12.0 Hz, 1H), 5.58-5.63 (m, 2H), 5.87-5.98 (m,
1H), 7.31 (d, J ) 8.7 Hz, 2H), 7.38 (d, J ) 8.7 Hz, 2H), 8.09 (s,
1H); 13C NMR (CDCl3) δ 52.3, 69.4, 77.6, 118.4, 120.4, 120.7,
121.1, 121.9, 122.9, 128.8, 133.0, 135.8, 146.6, 148.46, 148.48;
HRMS (ESMS) calcd for C15H14BrF3N3O4 [M + H+] 436.0120,
found 436.0132.
1-((R)-2-(4-(Trifluoromethoxy)benzyloxy)but-3-enyl)-4-nitro-
2-vinyl-1H-imidazole (33). A mixture of 32 (741 mg, 1.70 mmol),
tributyl(vinyl)stannane (0.55 mL, 1.9 mmol), Pd(PPh3)4 (79 mg,
0.068 mmol), and LiCl (72 mg, 1.70 mmol) in DMF (5 mL)
was prepared. The reaction mixture was heated under microwave
at 120 °C for 15 min. The reaction mixture was concentrated
under vacuum. Chromatography on silica gel (EtOAc/hexanes
) 1/3-1/1) afforded 33 as a slightly yellow oil (590 mg, 1.54
1
mmol, 91%). [R]2D0 -13.0 (c 0.59, CHCl3); H NMR (CDCl3) δ
3.96-4.13 (m, 3H), 4.25 (d, J ) 12.0 Hz, 1H), 4.57 (d, J )
12.3 Hz, 1H), 5.37-5.46 (m, 2H), 5.56 (d, J ) 10.8 Hz, 1H),
5.65-5.76 (d, J ) 10.8 Hz, 1H), 6.37 (dd, J ) 17.1, 1.5 Hz,
1H), 6.53 (dd, J ) 17.1, 11.1 Hz, 1H), 7.08-7.17 (m, 4H), 7.74
(s, 1H); 13C NMR (CDCl3) δ 50.8, 69.6, 78.5, 118.6, 120.9,
121.0, 121.2, 121.4, 122.0, 123.2, 129.0, 133.4, 135.7, 144.8,
146.9, 148.76, 148.78; HRMS (ESMS) calcd for C17H17F3N3O4
[M + H+] 384.1171, found 384.1167.
(R)-5,6,7,8-Tetrahydro-3-nitroimidazo[1,2-a]pyridin-6-ol (27a).
To a solution of (R)-26a (22 mg, 0.074 mmol) in THF (1 mL) was
added 1.0 M TBAF in THF (0.10 mL, 0.10 mmol) at room
temperature. The reaction mixture was stirred at room temperature
for 10 min. Chromatography on preparative TLC (MeOH/EtOAc
) 1/9) afforded (R)-27a (9.4 mg, 0.051 mmol, 69%) as a white
(R)-6-(4-(Trifluoromethoxy)benzyloxy)-5,6-dihydro-2-nitroim-
idazo[1,2-a]pyridine (34). A mixture of 33 (560 mg, 1.54 mmol)
and Grubbs’ second generation catalyst (130 mg, 0.15 mmol) in
CH2Cl2 (5 mL) was heated in a sealed tube at 55 °C for 15 h. The
reaction mixture was concentrated under vacuum. Chromatography
on silica gel (EtOAc/hexanes ) 1/1) afforded 34 as a gray-white
solid (320 mg, 0.901 mmol, 59%). [R]2D0 -189.6 (c 0.51, CHCl3);
1
solid. [R]2D3 -10.7 (c 0.43, MeOH); mp ) 163.5-164.1 °C; H
NMR (MeOH-d4) δ 2.34-2.45 (m, 1H), 2.66-2.78 (m, 1H),
2.83-3.02 (m, 2H), 3.66 (dd, J ) 12.0, 6.3 Hz, 1H), 3.92 (dd, J )
11.6, 3.5 Hz, 1H), 4.47-4.54 (m, 1H), 8.09 (s, 1H); 13C NMR
(MeOH-d4) δ 23.9, 29.1, 61.9, 64.6, 117.8, 151.2, 155.2; HRMS
(ESMS) calcd for C7H10N3O3 [M + H+] 184.0722, found 184.0724.
(S)-5,6,7,8-Tetrahydro-3-nitroimidazo[1,2-a]pyridin-6-ol (27b).
This compound was prepared in the same manner as 27a. Yield:
(S)-27b (91%) as a white solid. [R]2D0 +8.3 (c 0.29, MeOH); mp )
159.8-161.0 °C; 1H NMR (MeOH-d4) δ 2.34-2.46 (m, 1H),
2.63-2.80 (m, 1H), 2.82-3.02 (m, 2H), 3.66 (dd, J ) 12.0, 6.3
Hz, 1H), 3.92 (dd, J ) 11.7, 3.3 Hz, 1H), 4,47-4.53 (m, 1H),
8.09 (s, 1H); 13C NMR (MeOH-d4) δ 24.0, 29.1, 61.9, 64.9, 117.8,
151.2, 155.2; HRMS (ESMS) calcd for C7H10N3O3 [M + H+]
184.0722, found 184.0723.
1
mp ) 101.7-102.8 °C; H NMR (CDCl3) δ 4.14-4.22 (m, 1H),
4.34-4.42 (m, 2H), 4.59 (s, 2H), 6.43-6.48 (m, 1H), 6.65 (d, J )
10.2 Hz, 1H), 7.14 (d, J ) 8.4 Hz, 2H), 7.30 (d, J ) 8.7 Hz, 2H),
7.77 (s, 1H); 13C NMR (CDCl3) δ 48.3, 67.6, 69.7, 118.5, 120.2,
120.7, 120.9, 121.9, 129.0, 130.2, 135.8, 141.0, 147.3, 148.7; HRMS
(ESMS) calcd for C15H13F3N3O4 [M + H+] 356.0858, found
356.0851.
(R)-6-(4-(Trifluoromethoxy)benzyloxy)-5,6,7,8-tetrahydro-2-
nitroimidazo[1,2-a]pyridine (35). A solution of 34 (144 mg, 0.405
mmol) in THF/t-BuOH (2 mL:6 mL) was degassed by freezing
and thawing (3×). To this mixture was added Wilkinson’s catalyst
(72 mg, 0.078 mmol) at room temperature. The reaction mixture
was degassed by freezing and thawing (1×). The reaction mixture
was stirred at room temperature under a H2(g) balloon for 16 h
and concentrated under vacuum. Chromatography on silica gel
(EtOAc/hexanes ) 1/1) afforded 35 as a yellow oil (73 mg, 0.20
1-(2-(tert-Butyldimethylsilyloxy)ethyl)-2-methoxy-5-nitro-1H-
imidazole (29). To a solution of 28 (29 mg, 0.095 mmol) was added
NaOMe (50 mg, 0.93 mmol) at room temperature. The reaction
mixture was stirred at room temperature for 15 h and concentrated.
Chromatography on silica gel (EtOAc/hexanes ) 1/1) afforded 29
as a slightly yellow oil (17 mg, 0.055 mmol, 58%) in addition to
30 (2.7 mg, 0.014 mmol, 15%). 1H NMR (CDCl3) δ -0.07 (s, 6H),
0.80 (s, 9H), 3.89 (t, J ) 5.3 Hz, 2H), 4.60 (t, J ) 5.3 Hz, 2H),
7.91 (s, 1H); 13C NMR (CDCl3) δ -5.8, 18.0, 25.6, 46.2, 57.3,
1
mmol, 50%). [R]2D0 -13.2 (c 1.8, CHCl3); H NMR (CDCl3) δ
1.95-2.06 (m, 1H), 2.27-2.36 (m, 1H), 2.84-3.08 (m, 2H),

4- and 5-Nitroimidazoles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1327
4.07-4.19 (m, 3H), 4.55 (d, J ) 12.0 Hz, 1H), 4.63 (d, J ) 11.7
Hz, 1H), 7.16 (d, J ) 8.7 Hz, 2H), 7.31 (d, J ) 8.7 Hz, 2H), 7.61
(s, 1H); 13C NMR (CDCl3) δ 19.7, 24.2, 49.6, 69.6, 69.8, 118.6,
118.7, 121.0, 122.0, 128.8, 136.0, 144.1, 147.2, 148.7; HRMS
(ESMS) calcd for C16H15F3N3O6 [M + HCO2-] 402.0913, found
402.0929.
Mtb MIC Assay. A stock culture of Mtb H37Rv (ATCC 27294)
was grown to OD 0.5 in Middlebrook 7H9 broth (Difco) supple-
mented with 0.05% Tween-80, 0.2% glycerol, and albumin/NaCl/
glucose (ADC) complex. The culture was diluted 1:1000 in 7H9-
based medium before aliquoting 50 µL into each well of a 96-well
plate. The inhibitors were dissolved in DMSO to make stock
solutions of 50 µmol/mL. Inhibitors were added to the first row of
wells of the 96-well plate with 100 µL of 7H9-based medium. After
pipet mixing and use of a multichannel pipet, 50 µL was removed
from each well in the first row and added to the second row. 2-Fold
dilution in this manner was carried out to give eight dilutions of
each inhibitor (1000-7.8 µM). The plates were incubated for 2
weeks at 37 °C, and the MIC99 values were read macroscopically
using an inverted plate reader. Each measurement was made three
independent times.
mM-1 cm-1). Reactions were performed in phosphate buffer, pH
7.4, 50 µM F420, 0.6 µM Rv3457, and varying amounts of PA-824
and analogues. Apparent steady-state kinetic parameters, Km and
Vmax, were obtained by fitting initial rate data to the Michaelis-
Menten equation.
Acknowledgment. This work was funded (in part) by the
intramural research program of National Institute of Allergy and
Infectious Diseases, NIH, and (in part) by a grant from the Bill
and Melinda Gates Foundation and the Wellcome Trust through
the Grand Challenges in Global Health Initiative. We thank Dr.
Lacy Daniels (Texas A&M University Health Science Center)
for F420 and Michael Goodwin (TRS, NIAID, NIH), John Lloyd
(NIDDK, NIH), and Wesley White (NIDDK, NIH) with
assistance in obtaining analytical data.
Supporting Information Available: Experimental procedures
for the preparation of desnitro-824 (12) and purity information for
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Minimum Anaerobicidal Concentration Estimation Experi-
ment. For oxygen depletion assays, an early log phase Mtb culture
in glycerol free Dubos broth was diluted 100-fold and 20 mL was
transferred to tubes (Pyrex 16 mm × 125 mm culture tubes) to
maintain a head space ratio of 0.5 as described previously.31 The
tubes were sealed with paraplast and incubated for 20 days under
uniform stirring at 180 rpm using magnetic stirring bars. 1.5 µg/
mL of methylene blue was added to a reference tube to visualize
oxygen depletion. 100 µL of Mtb NRP-2 stage cells was exposed
to various concentrations of drugs in a 96-well microplate. 2-Fold
drug dilutions in DMSO (1.95-500 µM) and a DMSO control were
used. Handling of NRP-2 cells was done in a vinyl anaerobic
chamber (Coy Laboratories, MI) fitted with a Coy model 10 gas
analyzer and a vacuum air lock chamber. The anaerobic chamber
was maintained under 90% nitrogen and 10% hydrogen. 96-Well
plates were placed in a type A biobag anaerobic chamber (Becton
Dickinson, MD) along with an oxygen indicator strip and incubated
at 37 °C for 7 days. After drug exposure, the cells were washed
three times with fresh Dubos broth and serially diluted by 2-fold
dilutions. The minimal anaerobicidal concentration (MAC) against
Mtb was estimated by measuring the number of viable bacilli in
each dilution after drug treatment as follows: 5 µL of cell suspension
from each well was spotted in a single well of a 7H11 agar-
containing 96-well plate. After 3 weeks of incubation at 37 °C, the
MAC was read as the concentration of drug causing a 90%
reduction in visible growth.
Expression and Purification of Mtb-Rv3547 in E. coli. The
Mtb-Rv3547 genomic PCR fragment was amplified using XbaI-
FP (5′-cga ccc ttg gtc tct aga atc agc gtc atg ccg-3′) and D3-RP
(5′-tgg ctt cac gtt cgg caa gct tgg gaa cgg tca-3′). The PCR fragment
was digested with XbaI and HindIII and was cloned in a pMALc2x
vector digested with the same restriction enzymes for expression
of Mtb-Rv3547 with the N-terminal MBP tag. Rv3547 DNA
sequencing in pMAL-c2x-Rv3547 was performed using malE-FP
(NEB no. S12375) and M13 seq (NEB no. S12115) primers. MBP-
tagged Rv3547 was reproducibly expressed in E. coli turbo cells
(NEB). Protein induction at 37 °C with 300 µM IPTG yielded
Rv3547-MBP protein in the cytosol. The fusion protein was purified
to homogeneity using amylose resin affinity column chromatog-
raphy (NEB no. E8021) per the manufacturer’s instructions.
Nitroreductase Assay. F420 purified from Methanobacterium
thermoautotrophicum Marburg was a kind gift from Lacy Daniels,
Texas A&M University, Kingsville, TX. Reduced F420 (F420H2) was
prepared using a recombinant Mtb glucose 6-phosphate dehydro-
genase as described,29 followed by heat inactivation of FGD1 at
55 °C water bath for 15 min and ultrafiltration (Centricon 10 kDa).
Reduction of 1 or other nitroimidazoles by Rv3547 was monitored
by oxidation of F420H2 at 400 nm using a Cary UV spectropho-
tometer at 25 °C. Initial rates were calculated on the basis of the
gain of absorbance at 400 nm due to oxidation of F420 (ε400 ) 25.71
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