Synthesis and antitubercular activity of 7-(R)- and 7-(S)-methyl-2-nitro-6-(S)-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazines, analogues of PA-824

Article abstract of DOI:10.1016/j.bmcl.2008.03.011

Nitroimidazoles such as PA-824 and OPC-67683 are currently in clinical development as members of a promising new class of therapeutics for tuberculosis. While the antitubercular activity of these compounds is high, they both suffer from poor water solubility thus complicating development. We determined the single crystal X-ray structure of PA-824 and found a close packing of the nitroimidazoles facilitated by a pseudoaxial conformation of the p-trifluoromethoxybenzyl ether. To attempt to disrupt this tight packing by destabilizing the axial preference of this side chain, we prepared the two diastereomers of the 7-methyl-nitroimidazo-oxazine. Determination of the crystal structure of the 7-(S)-methyl derivative (5, cis) revealed that the benzylic side chain remained pseudoaxial while the 7-(R)-methyl derivative (6, trans) adopted the desired pseudoequatorial conformation. Both derivatives displayed similar activities against Mycobacterium tuberculosis, but neither showed improved aqueous solubility, suggesting that inherent lattice stability is not likely to be a major factor in limiting solubility. Conformational analysis revealed that all three compounds have similar energetically accessible conformations in solution. Additionally, these results suggest that the nitroreductase that initially recognizes PA-824 is somewhat insensitive to substitutions at the 7-position.

Full text of DOI:10.1016/j.bmcl.2008.03.011

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2256–2262
Synthesis and antitubercular activity of 7-(R)- and
7-(S)-methyl-2-nitro-6-(S)-(4-(trifluoromethoxy)benzyloxy)-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazines, analogues of PA-824
Xiaojin Li,a, Ujjini H. Manjunatha,a,à Michael B. Goodwin,a
John E. Knox,b,§ Christopher A. Lipinski,c Thomas H. Keller,b
Clifton E. Barry, IIIa and Cynthia S. Dowda,*,–
aTuberculosis Research Section, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA
bNovartis Institute for Tropical Diseases, 138670, Singapore
cMelior Discovery, Waterford, CT 06385, USA
Received 10 January 2008; revised 26 February 2008; accepted 4 March 2008
Available online 7 March 2008
Abstract—Nitroimidazoles such as PA-824 and OPC-67683 are currently in clinical development as members of a promising new
class of therapeutics for tuberculosis. While the antitubercular activity of these compounds is high, they both suffer from poor water
solubility thus complicating development. We determined the single crystal X-ray structure of PA-824 and found a close packing of
the nitroimidazoles facilitated by a pseudoaxial conformation of the p-trifluoromethoxybenzyl ether. To attempt to disrupt this tight
packing by destabilizing the axial preference of this side chain, we prepared the two diastereomers of the 7-methyl-nitroimidazo-oxa-
zine. Determination of the crystal structure of the 7-(S)-methyl derivative (5, cis) revealed that the benzylic side chain remained
pseudoaxial while the 7-(R)-methyl derivative (6, trans) adopted the desired pseudoequatorial conformation. Both derivatives dis-
played similar activities against Mycobacterium tuberculosis, but neither showed improved aqueous solubility, suggesting that inher-
ent lattice stability is not likely to be a major factor in limiting solubility. Conformational analysis revealed that all three compounds
have similar energetically accessible conformations in solution. Additionally, these results suggest that the nitroreductase that ini-
tially recognizes PA-824 is somewhat insensitive to substitutions at the 7-position.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Despite decades of research, tuberculosis continues to be
a major public health threat. The WHO estimates that,
in 2005, 1.6 million people died from tuberculosis (TB)
which is caused by the microorganism Mycobacterium
tuberculosis (Mtb).1 Current therapy for TB lasts for
several months and is called directly observed therapy
short-course (DOTS). This regimen calls for an intensive
phase of chemotherapy using four drugs for 2 months,
followed by a continuation phase using two drugs for
4–6 months.2 Rifampin is inarguably the most impor-
tant drug in the DOTS regimen as it allowed shortening
the course of therapy to 6 months from the standard 12
to 18 months of previous regimens. The potency of
rifampin is thought to be due in part to its action against
both aerobically growing and anaerobically adapted,
non-replicating mycobacteria.3
Keywords: Mycobacterium tuberculosis; PA-824; Solubility; Water
solubility.
*
Corresponding author. Tel.: +1 202 994 8405; fax: +1 202 994
5873; e-mail: cdowd@gwu.edu
Nitroimidazoles are a class of compounds widely used
clinically for the management of anaerobic bacterial
infections.4 Metronidazole (1) has activity only against
anaerobically adapted Mtb5 and is the subject of an
on-going clinical trial.6 Bicyclic nitroimidazoles have
also been developed that have both aerobic and anaero-
bic activity against Mtb (Fig. 1). CGI-17341 (2) and
OPC-67683 (3), both nitroimidazo-oxazoles, and PA-
824 (4), a nitroimidazo-oxazine, have activity against
Present address: Nalco Company, Naperville, IL 60563, USA.
Present address: Novartis Institute for Tropical Diseases, 138670,
Singapore.
à
§
Present address: Computational Chemistry, Neurology CEDD Cog-
nition & Neurodegeneration Centre, GlaxoSmithKline, 138667,
Singapore.
–
Present address: Department of Chemistry, George Washington
University, 725 21st St. NW, Corcoran Hall 107, Washington, DC
20052, USA.
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.011

X. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2256–2262
2257
2
2
2
2
Metronidazole (1)
CGI-17341 (2)
2
2
3
PA-824 (4)
7
7
6
6
3
OPC-67683 (3)
3
3
7-(
S
)-Methyl-PA-824 (5, cis) 7-(
R)-Methyl-PA-824 (6, trans)
Figure 1. Nitroimidazoles with antitubercular activity.
aerobic and anaerobic populations of Mtb.7–9 PA-824
has an aerobic MIC (minimum inhibitory concentra-
tion) of 0.4 lM and substantial anaerobic activity at
8–16 lM. Several groups have also confirmed the effi-
cacy of PA-824 in the mouse model of TB infection.10–13
PA-824, and more recently OPC-67683, have entered
clinical trials for the treatment of tuberculosis.14,15 Prior
work has shown that PA-824 acts as a prodrug requiring
bioreductive activation before exerting its biological ef-
fect.16 One serious issue with the clinical use of both
PA-824 and OPC-67683 lies in their low water solubility.
PA-824 is only soluble at 10.2 lg/mL17 (Table 1) and the
in vivo activity of OPC-67683 requires formulation in
5% gum arabic.15 Similarly, the low water solubility of
PA-824 necessitates the use of a complex formulation
in animals (CM-2) that may not be suitable for human
use and may require development of an expensive
alternate.
packing in the crystal lattice and that disruption of this
arrangement might result in improved water solubility.
We postulated that adding a substituent on the oxazine
ring adjacent to the trifluoromethoxybenzyl ether would
force this side chain into the pseudoequatorial position
disrupting the antiparallel stacking observed in the crys-
tal structure.
To this end, we targeted the 7-methyl analogues of PA-
824, 5 and 6 (Fig. 1). The synthesis of these compounds
is shown in Scheme 1. Briefly, crotonyl chloride (7)
underwent asymmetric dihydroxylation using AD-mix
a to give diol 8.20 Ring-closure under basic conditions
yielded epoxy alcohol 10.21 To prepare the opposite dia-
stereomer, racemic allylic alcohol 9 underwent Sharpless
epoxidation to give epoxy alcohol 11.22 Compounds 10
and 11 were protected to give 12 and 13,23 respectively.
Alkylation of 2,4-dinitroimidazole24 yielded the corre-
sponding secondary alcohols 14 and 15,25 which were
converted to the THP-protected derivatives 16 and
17.26 Desilylation and ring cyclization proceeded in
one step to give oxazines 18 and 19.27 Deprotection, giv-
ing free alcohols 20 and 21,28 followed by benzylation
with p-trifluoromethoxybenzyl bromide delivered de-
sired compounds 7-(S)-methyl-PA-824 (5) and 7-(R)-
methyl-PA-824 (6), respectively.29
Low solubility can often be related to thermodynamic
stability of a particular crystalline polymorph.18 There-
fore, in an effort to understand the reasons behind the
low water solubility of PA-824, we determined its crystal
structure (Fig. 2).19,31 This structure showed PA-824 ar-
ranged in antiparallel layers with stacked nitroimidaz-
oles alternating with stacked p-trifluoromethoxybenzyl
ethers. The oxazine ring is bent so that C-6, bearing
the trifluoromethoxybenzyl side chain, is out of the
plane of the imidazole ring. This forces the side chain
into a pseudoaxial orientation. We hypothesized that
the low water solubility of PA-824 could be due to tight
Compounds 5 and 6 displayed comparable antitubercu-
lar activities to PA-824 under aerobic conditions (Table
1). The MIC for 7-(S)-methyl-PA-824 (5) is 0.2–0.4 lM
and for 7-(R)-methyl-PA-824 (6) is 0.2 lM. Like PA-
Table 1. Biological activity and solubility of PA-824 and 7-methyl analogues
Compound
H37Rv MIC
(lM)
H37Rv MAC
(lM)
H37Rv-T3 MIC
(lM)
H37Rv-5A1
MIC (lM)
H37Rv-T2
MIC (lM)
Solubility
(lg/mL)
PA-824 (4)
7-(S)-Methyl-824 (5, cis)
7-(R)-Methyl-824 (6, trans)
0.4
0.2–0.4
0.2
8–16
16
>100
>100
>100
>100
>100
>100
>100
>100
>100
10.2 1.6
9.89 1.9
10.3 1.6
8–16
The phenotype and genetic changes in mutants H37Rv-T3, H37Rv-5A1 and H37Rv-T2 have been reported.16

2258
X. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2256–2262
Figure 2. Crystal structure of PA-824. Asymmetric unit of the PA-824 crystal shows a staggered configuration of individual molecules and four
molecules per unit cell (left). The space-filling diagram highlights this tight packing arrangement (right).
Scheme 1. Synthesis of 7-methyl analogues of PA-824. Reagents and conditions: (a) AD-mix a, CH3SO2NH2, NaHCO3, t-BuOH, H2O, 0 ꢁC; (b)
KOH, Et2O, 0 ꢁC; (c) D-(ꢀ)-DIPT, TBHP, Ti(OiPr)4, CH2Cl2, ꢀ20 ꢁC; (d) TBSCl, imidazole, CH2Cl2, 0 ꢁC ! rt; (e) 2,4-dinitroimidazole, EtOH,
70 ꢁC; (f) 3,4-dihydro-2H-pyran, PPTS, CH2Cl2, rt; (g) TBAF, THF, rt; (h) AcOH, THF, H2O, 45 ꢁC; (i) NaH, 4-trifluoromethoxybenzyl bromide,
DMF, ꢀ45 ꢁC ! rt.
824, the two methyl-oxazines both require reduced coen-
zyme F420. Mutants that either fail to reduce this cofac-
tor (H37Rv-T3) or mutants deficient in its biosynthesis
(H37Rv-5A1) are cross-resistant to these nitroimidaz-
oles. The reduction of these compounds is probably
mediated by the nitroreductase Rv3547 as mutants lack-
ing this enzyme (H37Rv-T2) are also cross-resistant.16
The MAC (minimum anaerobicidal concentration) val-
ues for compounds 4–6 were equivalent.30
determined the crystal structures of compounds 5 and
6 (Fig. 3).31 The crystal structure of compound 5 (cis)
shows the oxazine ring in the same conformation as in
that of PA-824. Thus, carbon-6 in 5 is out of the plane
of the imidazole ring, just as in the crystal structure of
PA-824. The side chain in 5 is similarly pseudoaxial.
As hoped, in compound 6, the trans isomer, the opposite
effect occurred. Carbon-6 in this compound is out of the
plane of the imidazole ring, but on the side opposite to
that found in PA-824 and compound 5. This conforma-
tion places the benzylic side chain in the desired pseud-
oequatorial conformation. The conformation of
compound 6 depicted in the crystal structure places
the substituents of both carbon-6 and carbon-7 in the
more favorable pseudoequatorial positions. Interest-
ingly, the packing diagrams for compounds 5 and 6 do
Unfortunately, when we determined the thermodynamic
solubility of compounds 5 and 6, they were not more
soluble than PA-824, each having a solubility limit of
approximately 10 lg/mL (Table 1).17 To understand
the consequence of the methyl substituent on the confor-
mation of these compounds in the crystalline form, we

X. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2256–2262
2259
Figure 3. Crystal structures of PA-824 (purple), 7-(S)-methyl-PA-824 (5, cis, green), and 7-(R)-methyl-PA-824 (6, trans, white). View on the left is
shown along the plane of the nitroimidazole to highlight the differing conformations of the oxazine rings in each structure. Arrows indicate the C-6
carbon atoms. Crystal packing diagrams of compounds 5 (right, top) and 6 (right, bottom) are distinct from that of PA-824.
not show the same arrangement of antiparallel mole-
cules as in the case of PA-824 (Fig. 3). Instead, the com-
minimized to yield the 100 lowest energy structures.
For PA-824 (4), all 100 structures agreed well with the
crystal structure (within 9 kJ/mol), the primary differ-
ences arising from rotation of the benzyl side chain
about the ether bond. For compound 5 (cis), 73 of the
100 lowest energy conformations agreed with the PA-
824 set. The remaining conformations agreed well with
compound 5 crystal structure, although these had mini-
mized energies of at least 12.2 kJ/mol above the global
minimum. For compound 6 (trans), 91 of 100 conforma-
tions were nearly the same as PA-824 (within 6 kJ/mol).
The remaining 9 solutions agreed with the crystal struc-
ture of 6, being at least 3.9 kJ/mol above the global min-
imum. These data indicate that the major conformation
in solution of both 7-methyl isomers will most likely be
very close to that of PA-824. The slightly higher activity
seen with compound 6 is intriguing in that it suggests
that the active conformer may, in fact, not be the mini-
mum energy conformer in solution. This result suggests
that the active conformer may be more abundant with
the trans substituent forcing an even more pronounced
pseudoequatorial orientation of the side chain.
pounds are arranged linearly in
a back-to-back
conformation with imidazole rings in a nearly perpen-
dicular arrangement between rows. The similarity be-
tween packing diagrams for compounds 5 and 6
indicates that the differences in single-molecule structure
do not impact the overall packing between molecules.
Despite changing the angle of the oxazine-trifluorometh-
oxybenzyl side chain to either more pseudoaxial (in
compound 5) or more pseudoequatorial (in compound
6) compared with PA-824, there was a negligible effect
on solubility. This result suggests that stability of this
crystalline form is not a major contributor to the overall
insolubility of this molecule. Further, the melting points
and crystal packing densities of these compounds are
very similar. The melting points are 149–150 ꢁC (PA-
824),32 146–148 ꢁC (compound 5), and 126.5–128.5 ꢁC
(compound 6). The crystal packing densities are 1.55–
1.59 g/cm3 for all three compounds. The similarities in
these numbers support the conclusion that the com-
pounds’ poor solubility is not related to the crystal
packing.
Thus, these results suggest that the low solubility of PA-
824 in aqueous media may not be the result of an intrin-
sically stable crystalline form. Future efforts will focus
on the introduction of solubilizing groups while preserv-
ing activity. Such studies would be greatly facilitated by
an in vitro nitroreductase assay from which detailed
SAR could be obtained. The activity of compounds 5
and 6 against wild-type H37Rv and all three classes of
mutants resistant to PA-824 suggests that these com-
pounds are acting through the same mechanism as
Nonetheless, the variation in angle between the imidazo-
oxazine and the trifluoromethoxybenzyl side chain
seemed incongruent with the minor variations in biolog-
ical activity, particularly since the R isomer of PA-824 is
nearly 50-fold less active than the S.10 To understand
this discrepancy, we performed a conformational analy-
sis on these compounds to identify the lowest energy
conformations in solution.33 Compounds 4–6 were

2260
X. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2256–2262
sion is shaken for 3 days. The sample is centrifuged at
10,000g for 5 min. The supernatant is diluted in water and
injected onto an LC/MS where the UV peak areas are
recorded at 310 nm. The concentration of the sample
solution is calculated using a previously determined
calibration curve, corrected for the dilution factor of the
sample.
PA-824 and are substrates of Rv3547. The activity of 5
and 6 demonstrates that the nitroreductase accepts at
least small substituents at the 7-position. These results
suggest that further derivatives at the 7-position of the
oxazine may be fruitfully explored to improve activity
and solubility of these important compounds.
18. Singhal, D.; Curatolo, W. Adv. Drug Deliv. Rev. 2004, 56,
335.
Acknowledgments
19. Crystal data for compound 4: C14H12N3O5F3,
MW = 359.26, monoclinic, P21; Z = 4, a = 14.7024(17),
˚
b = 5.7699(8), c = 17.702(3) A; b = 94.913(8)ꢁ; V =
We thank the Division of Intramural Research (NIAID,
NIH) for support of this work. Crystal structures were
determined by Lee Daniels at Rigaku/MSC, Inc. (PA-
824) and Kenneth Hardcastle of Emory University
(compounds 5 and 6).
3
3
˚
1496.2(3) A , F(000) = 736.00; Dx = 1.595 g/cm ; 2hmax
=
136.7ꢁ (Rigaku RAXIS RAPID detector with graphite
monochromated Cu–Ka radiation), R = 0.0430 (4733 data
with I > 2rI, 451 parameters).
20. Richardson, T. I.; Rychnovsky, S. D. J. Org. Chem. 1996,
61, 4219.
21. Vanhessche, K. P. M.; Wang, Z.-M.; Sharpless, K. B.
Tetrahedron Lett. 1994, 35, 3469.
References and notes
22. White, J. D.; Kang, M.-C.; Sheldon, B. G. Tetrahedron
Lett. 1983, 24, 4539.
23. Hindupur, R. M.; Panicker, B.; Valluri, M.; Avery, M.
Tetrahedron Lett. 2001, 42, 7341.
1. WHO International Fact Sheet No. 104. 2007. http://
www.who.int/mediacentre/factsheets/fs104/en/index.html.
2. Treatment of Tuberculosis: Guidelines for National Pro-
grammes; WHO/CDS/TB 2003.313; WHO: Geneva, 2003.
3. Duncan, K.; Barry, C. E., 3rd Curr. Opin. Microbiol. 2004,
7, 460.
4. Barry, C. E., 3rd.; Boshoff, H. I.; Dowd, C. S. Curr.
Pharm. Des. 2004, 10, 3239.
5. Wayne, L. G.; Sramek, H. A. Antimicrob. Agents Chemo-
ther. 1994, 38, 2054.
6. http://www.clinicaltrials.gov/ct2/show/NCT00425113?
term=metronidazole&cond=tuberculosis&rank=1.
7. Ashtekar, D. R.; Costa-Perira, R.; Nagrajan, K.; Vishva-
nathan, N.; Bhatt, A. D.; Rittel, W. Antimicrob. Agents
Chemother. 1993, 37, 183.
8. Matsumoto, M.; Hashizume, H.; Tomishige, T.; Kawasa-
ki, M.; Tsubouchi, H.; Sasaki, H.; Shimokawa, Y.;
Komatsu, M. PLoS Med. 2006, 3, 2131.
9. Stover, C. K.; Warrener, P.; VanDevanter, D. R.; Sher-
man, 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., 3rd; Baker, W. R. Nature
2000, 405, 962.
10. Baker, W. R.; Shaopei, C.; Keeler, E. L. U.S. Patent
5,668,127, 1997.
11. 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 Chemo-
ther. 2005, 49, 2294.
12. Manjunatha, U. H.; Dowd, C. S.; Zhang, L.; Barry, C. E.,
3rd. Unpublished work.
13. Tyagi, S.; Nuermberger, E.; Yoshimatsu, T.; Williams, K.;
Rosenthal, I.; Lounis, N.; Bishai, W.; Grosset, J. Anti-
microb. Agents Chemother. 2005, 49, 2289.
24. Sudarsanam, V. N. K.; George, T.; Shenoy, S. J.; Iyer, V. V.;
Kaulgud, A. P. Indian J. Chem., Sect. B 1982, 21B, 1022.
25. Preparation of 14: (2S,3S)-3-(TBDMS)-1-(2,4-dini-
troimidazol-1-yl)-butan-2-ol. A mixture of 2,4-dinitroim-
idazole (0.25 g, 1.58 mmol) and epoxide 12 (0.48 g,
2.37 mmol) in ethanol (0.53 mL) was heated at 70 ꢁC for
18 h in a sealed tube. After removal of the solvent, the
residue was separated by silica gel column chromatogra-
phy (10–20% EA in hexanes) to give 14 as an oil (0.22 g,
38%). 1H NMR (300 MHz, CDCl3) d 7.99 (s, 1H), 4.77
(dd, J = 13.8, 2.1 Hz, 1H), 4.29 (dd, J = 13.8, 9.9 Hz, 1H),
3.89 (m, 1H), 3.74 (m, 1H), 2.58 (d, J = 6.9 Hz, 1H), 1.31
(d, J = 6.3 Hz, 3H), 0.92 (s, 9H), 0.15 (s, 3H), 0.14 (s 3H).
13C NMR (75 MHz, CDCl3) d 143.14, 125.11, 74.10,
69.21, 60.05, 54.63, 25.86, 19.94, 18.10, ꢀ4.07, ꢀ4.81.
[a]D = +16.9 (c = 1.0 in CH2Cl2). HRMS calcd for
C13H23N3O4Si [MꢀNO2]+: 314.1536; found 314.1418.
Compound
15
(2S,3R)-3-(TBDMS)-1-(2,4-dini-
troimidazol-1-yl)-butan-2-ol was prepared in the same
way from epoxide 13 giving (0.193 g, 34%). 1H NMR
(300 MHz, CDCl3) d 8.03 (s, 1H), 4.92 (dd, J = 1.5,
13.6 Hz, 1H), 4.24 (dd, J = 9.9, 13.6 Hz, 1H), 4.00 (m,
1H), 3.86 (m, 1H), 2.62 (d, J = 6.0 Hz, 1H), 1.27 (d,
J = 6.3 Hz, 3H), 0.92 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H). 13
C
NMR (75 MHz, CDCl3) d 143.20, 131.30, 125.15, 74.20,
70.19, 53.26, 25.92, 19.12, 18.16, ꢀ4.11, ꢀ4.72. [a]D = +8.9
(c = 1.0 in CHCl3). HRMS calcd for C13H24N3O4Si
[MꢀNO2]+: 314.1536; found 314.1548.
26. Preparation of 16: (2S, 3S)1-[3-(TBDMS)-2-(tetrahydro-
pyran-2-yloxy)-butyl]-2,4-dinitro-1H-imidazole.
A mix-
ture of 14 (115 mg, 0.319 mmol), PPTS (16.7 mg,
0.0665 mmol), and 3,4-dihydro-2H-pyran (60.6 lL,
0.664 mmol) in methylene chloride (1.8 mL) was stirred
at rt for 20 h. The reaction was quenched with water
(1.8 mL). The organic layer was separated and the
aqueous layer was extracted with methylene chloride (3·
1.8 mL). The combined organic portions were washed
with brine and dried over Na2SO4. Solvent removal
yielded a pair of diastereomers (133 mg, 94%, ꢁ1:1) as a
white solid. The solid was used in the next reaction
without further purification. 1H NMR (300 MHz, CDCl3)
d 7.94 (s, 1H), 7.88 (s, 1H), 4.99 (dd, J = 2.6, 13.6 HZ, 1H),
4.86 (dd, J = 2.4, 13.8 Hz, 1H), 4.36 (dd, J = 10.2, 13.8 Hz,
1H), 4.26–4.08 (m, 4H), 3.91 (m, 2H), 3.75 (m, 2H), 3.36
(m, 2H), 3.09 (m, 1H), 1.65–1.09 (m, 18H), 0.85 (s, 18H),
14. Global Alliance for TB Drug Development. http://
www.tballiance.org/home/home.php.
15. Sasaki, H.; Haraguchi, Y.; Itotani, M.; Kuroda, H.;
Hashizume, H.; Tomishige, T.; Kawasaki, M.; Matsum-
oto, M.; Komatsu, M.; Tsubouchi, H. J. Med. Chem.
2006, 49, 7854.
16. 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.
17. The thermodynamic solubility determination was based
on the method of Avdeef and Testa (Avdeef, A.; Testa, B.
Cell Mol. Life Sci. 2002, 59, 1681). Briefly, 3–5 mg of
compound was exposed to 0.5 mL water and the suspen-

X. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2256–2262
2261
0.034 (m, 12H). 13C NMR (75 MHz, CDCl3) d 143.01,
142.58, 141.97, 141.60, 125.69, 124.90, 101.77, 100.25,
80.54, 76.93, 68.59, 68.22, 65.29, 63.83, 51.70, 50.56, 30.99,
30.81, 25.84, 25.80, 25.05, 24.88, 21.34, 20.07, 18.24, 18.09,
18.04, 17.01, ꢀ4.54, ꢀ4.84, ꢀ4.85, ꢀ4.95. HRMS calcd for
C18H33N4O7Si [M+H]+: 445.2119; found 445.2118. Com-
pound 17, (2S,3R)-3-[3-(TBDMS)-2-(tetrahydro-pyran-2-
yloxy)-butyl]-2,4-dinitro-1H-imidazole, was prepared in
the same way from 15 to give 0.174 g (94%). 1H NMR
(300 MHz, CDCl3) d 7.91 (s, 1H), 7.86 (s, 1H), 4.93 (d,
J = 14.1 Hz, 2H), 4.42 (dd, J = 9.0, 14.1 Hz, 1H), 4.30–
3.72 (m, 8H), 3.37–3.08 (m, 3H), 1.69–1.10 (m, 12H), 1.24
(d, J = 6.3 Hz, 3H), 1.23 (s, J = 6.3 Hz, 3H), 0.90 (s, 9H),
0.89 (s, 9H), 0.11 (s, 3H), 0.86 (s, 3H), 0.075 (s, 6H). 13C
NMR (75 MHz, CDCl3) d 143.07, 142.69, 142.02, 141.60,
125.77, 125.16, 101.55, 100.03, 80.62, 78.19, 70.55, 69.35,
65.48, 64.55, 52.26, 51.18, 31.12, 31.05, 25.95, 25.87, 25.16,
24.95, 21.53, 20.75, 20.63, 20.37, 18.23, 18.15, ꢀ4.32,
ꢀ4.36, ꢀ4.48, ꢀ4.67. HRMS calcd for C18H33N4O7Si
[M+H]+: 445.2119; found 445.2123.
(s, 1H), 4.55 (ddq, J = 0.9, 5.3, 6.6 Hz, 1H), 4.31 (dd,
J = 4.2, 12.7 Hz, 1H), 4.07 (ddd, J = 4.2, 5.3, 5.3 Hz, 1H),
3.97 (ddd, J = 0.9, 5.3, 12.7, 1H), 1.98 (br s, exchanged,
1H), 1.45 (d, J = 6.6 Hz, 3H). 13C NMR (75 MHz,
CD3OD) d 149.05, 144.25, 117.76, 80.02, 65.37, 48.37,
17.74. [a]D = 22.4 (c = 0.5 in CH3OH). HRMS calcd for
C7H10N3O4 [M+H]+: 200.0671; found 200.0683.
29. Preparation of 5. (6S,7S)-7-Methyl-2-nitro-6-(4-trifluoro-
methoxy-benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxa-
zine. To a solution of 20 (27 mg, 0.136 mmol) and
4-(trifluoromethoxy)-benzylbromide (26.3 lL, 0.163 mmol)
in anhydrous DMF (0.17 mL) was added NaH (3.9 mg,
0.163 mmol) at ꢀ45 ꢁC. After stirring for 1 h, the reaction
mixture was allowed to warm to rt where stirring
continued for 30 min. The mixture was separated by prep
TLC (2% MeOH in EA) to give the target molecule
(41.9 mg, 83%) as white needles. Mp: 146–148 ꢁC. 1H
NMR (300 MHz, CD3COCD3) d 7.70 (s, 1H), 7.50 (d,
J = 8.2 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 4.88 (d,
J = 12.2 Hz), 4.78 (m, 1H), 4.73 (d, J = 12.2), 4.61 (dd,
J = 13.8, 1.8 Hz, 1H), 4.34 (dd, J = 10.5, 3.2 Hz, 2H), 4.23
(m, 1H) 1.50 (d, J = 6.3 Hz, 3H). 13C NMR (75 MHz,
CD3COCD3) d 149.00, 149.50, 138.32, 130.43, 123.30,
121.91, 119.80, 117.30, 76.88, 71.32, 71.11, 46.84, 17.08.
[a]D = ꢀ11.6 (c = 0.5 in acetone). HRMS calcd for
C15H15F3N3O5 [M+H]+: 374.0964; found 374.0970. Com-
pound 6, (6S,7R)-7-methyl-2-nitro-6-(4-trifluoromethoxy-
benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine,
was prepared in the same way from 21 to give 37.1 mg
(62%) of the product. Mp: 126.5–128.5 ꢁC. 1H NMR
27. Preparation of 18. (6S,7S)-7-Methyl-2-nitro-6-(THP)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazine. To a solution of
16 (133 mg, 0.299 mmol) in THF (1.2 mL) was added
TBAF (0.90 mL, 0.9 mmol, 1 M in THF) at rt. After
stirring 0.5 h, the solvent was removed and the residue was
dissolved in chloroform (2.0 mL). The mixture was
washed with saturated NaHCO3 (1.5 mL), water
(1.5 mL), and was dried over MgSO4. After removing
the solvent, the residue was chromatographed (2.5–5%
MeOH in EA) to give the desired product (67.6 mg, 80%).
1H NMR (300 MHz, CDCl3) d 7.42 (s, 2H), 4.77 (m, 2H),
4.64 (m, 1H), 4.54 (m, 1H), 4.39 (dd, J = 2.7, 12.9 Hz, 1H),
4.28 (m, 1H), 4.21–4.04 (m, 4H), 3.86 (m, 1H), 3.68 (m,
1H), 3.53 (m, 2H), 1.59 (d, J = 6.6 Hz, 3H), 1.51 (d,
J = 6.3 Hz, 3H), 1.82–1.46 (m, 12). 13C NMR (75 MHz,
CDCl3) d 147.98, 147.92, 143.47, 115.70, 115.57, 102.05,
95.67, 76.40, 76.05, 70.26, 65.02, 63.37, 62.97, 48.93, 45.52,
30.59, 30.24, 25.11, 25.02, 19.59, 19.25, 16.80, 16.47.
HRMS calcd for C12H18N3O5 [M+H]+: 284.1246; found
284.1249. Compound 19, (6S,7R)-7-methyl-2-nitro-6-
(300 MHz, CD3COCD3)
d 7.77 (s, 1H), 7.50 (d,
J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 4.89 (dq,
J = 3.6, 6.9 Hz, 1H), 4.81 (s, 2H), 4.49 (dd, J = 3.8,
13.4 Hz, 1H), 4.39 (dd, J = 3.5, 13.4 Hz, 1H), 4.19 (ddd,
3.5, 3.6, 3.8, 1H), 1.47 (d, J = 6.9 Hz, 3H). 13C NMR
(75 MHz, CD3COCD3) d 149.72, 148.02, 138.66, 130.61,
123.49, 122.21, 120.10, 117.66, 76.83, 72.48, 71.13, 45.58,
18.08. [a]D = 6.4 (c = 0.5 in acetone). HRMS calcd for
C15H15F3N3O5 [M+H]+: 374.0964; found 374.0966.
30. Isoniazid, rifampicin, metronidazole, and methylene blue
were obtained from Sigma–Aldrich. All stocks were made
in 20 mM DMSO. The broth dilution method was used to
determine the MIC99 of all compounds against Mtb
(H37Rv, ATCC 27294) and all H37Rv mutants as
described previously in Domenech et al., Infect. Immunol.
2005, 73, 3492–3501. For oxygen depletion assays (MAC)
early log phase Mtb cultures in Dubos broth was diluted
100-fold and 20-mL was transferred to tubes (Pyrex
16 · 125 mm culture tubes) to maintain a head space ratio
of 0.5 as described previously [Wayne in Mycobacterium
tuberculosis Protocols. In Parish, T., Stoker, N. G., Eds.;
Humana Press: New Jersey, 2001; pp 247–270]. The tubes
were sealed with paraplast and incubated for 20 days
under uniform stirring at 180 rpm using magnetic stirring
bars. Methylene blue (1.5 lg/mL) was added to a reference
tube to visualize oxygen depletion. Mtb NRP-2 stage cells
(100 lL) were exposed to 1.95–500 lM of drug in a 96-well
microplate in 2-fold drug dilutions. Handling of NRP-2
cells was done in a vinyl anaerobic chamber (Coy
Laboratories, Michigan) fitted with a Coy Model 10 gas
analyzer and vacuum air lock chamber. The anaerobic
chamber was maintained under 90% nitrogen and 10%
hydrogen. The 96-well plates were placed in a Type A Bio-
bag anaerobic chamber (Becton and Dickinson, Mary-
land) 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. The MAC
against Mtb was estimated by measuring the number of
viable bacilli spotted on a 7H11 agar-containing 96-well
(THP)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine,
was
prepared in the same way from 17 (59.6 mg, 96%). 1H
NMR (300 MHz, CDCl3) d 7.46 (s, 1H), 7.43 (s, 1H), 4.76
(m, 3H), 4.56 (dq, J = 6.4, 6.4 Hz, 1H), 4.32 (dd, J = 4.6,
12.8 Hz, 1H), 4.26 (dd, J = 3.4, 12.4 Hz, 1H), 4.14–3.99
(m, 4H), 3.78 (m, 2H), 3.52 (m, 2H), 1.74–1.49 (m, 12H),
1.44 (d, J = 6.6 Hz, 3H), 1.44 (d, J = 7.2 Hz, 3H). 13C
NMR (75 MHz, CDCl3) d 147.16, 147.07, 143.87, 115.40,
115.23, 99.92, 97.46, 77.07, 75.73, 70.22, 67.91, 63.42,
62.79, 46.83, 44.74, 30.62, 30.50, 25.20, 25.14, 19.54, 19.02,
17.87, 17.55. HRMS calcd for C12H18N3O5 [M+H]+:
284.1246; found 284.1249.
28. Preparation of 20. (6S,7S)-7-Methyl-2-nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]oxazin-6-ol.
A
mixture of 18
(67.6 mg, 0.239 mmol) in acetic acid (1.0 mL), THF
(0.52 mL), and water (0.26 mL) was stirred for 11 h at
45 ꢁC. Prep TLC (5% MeOH in EA) of the reaction
mixture gave the desired compound (39.7 mg, 83%) as a
white solid, mp (dec.) >200 ꢁC. 1H NMR (300 MHz,
DMSO-d6) d 8.06 (s, 1H), 5.71 (br s, 1H), 4.59 (q,
J = 6.3 Hz, 1H), 4.18 (d, J = 13.0 Hz, 1H), 4.02 (s, 1H),
4.00 (d, J = 13.0 Hz, 1H), 1.36 (d, J = 6.3 Hz, 3H). 13C
NMR (75 MHz, DMSO-d6) d 147.81, 142.26, 118.04,
76.31, 61.69, 49.97, 16.52. [a]D = ꢀ26.4 (c = 1.0 in DMSO-
d6). HRMS calcd for C7H10N3O4 [M+H]+: 200.0671;
found 200.0690. Compound 21, (6S,7R)-7-methyl-2-nitro-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ol, was pre-
pared in the same way from 19 to give 33.1 mg (86%).
Mp: 182.1–182.6 ꢁC. 1H NMR (300 MHz, CD3OD) d 7.77

2262
X. Li et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2256–2262
plate. For spotting, 5 lL of a cell suspension from each
(2318 data with I > 2rI, 236 parameters). Crystallographic
data (excluding structure factors) for these compounds
have been deposited at the Cambridge Crystallographic
Data Centre [CCDC 669894 (for compound 4), 669895
(for compound 5), and 669896 (for compound 6)]. Copies
of the data can be obtained, free of charge, on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
[fax: +44(0) 1223 336033 or deposit@ccdc.cam.ac.uk].
32. Baker, W. R.; Shaopei, C.; Keeler, E. L. U.S. Patent
6,087,358, 2000.
33. All calculations were performed with Macromodel 9.1
(Schro¨dinger) using the conformational search module.
Automatic setup was used to describe the conformational
search parameters. OPLS2005 was the force field used
along with an implicit water solvent model. The torsional
sampling model (MCMM) was used with up to 10,000
steps saving the 100 lowest energy unique conformations.
well is spotted on a 7H11 agar-containing 96-well plate,
incubated for 3 weeks at 37 ꢁC, and the concentration of
the drug at which 90% reduction in visible growth is
recorded.
31. Crystal data for compound 5. C15H14F3N3O5,
MW = 373.29, orthorhombic, P212121; Z = 4, a =
˚
4.6223(2),
1596.45(11) A ,
b = 9.3690(4),
c = 36.8642(14) A;
Dx=1.553 Mg/m3;
V =
h
3
˚
F(000)=768;
range = 8.63–50.08ꢁ (Bruker CCD area detector with
graphite monochromated Cu–Ka radiation), R = 0.0197
(1648 data with I > 2rI, 248 parameters). For compound
6. C15H14F3N3O5, MW = 373.29, orthorhombic, P212121;
˚
Z = 4, a = 4.42660(10), b = 9.6295(3), c = 37.3504(10) A;
3
3
˚
V = 1592.10(7) A , F(000) = 768; Dx = 1.557 Mg/m ; h
range = 2.37–66.30ꢁ (Bruker CCD area detector with
graphite monochromated Cu–Ka radiation), R = 0.0264