Synthesis and structure-activity studies of biphenyl analogues of the tuberculosis drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro- 5H-imidazo[2,1-b][1,3]oxazine (PA-824)

Article abstract of DOI:10.1021/jm901207n

A series of biphenyl analogues of the new tuberculosis drug PA-824 was prepared, primarily by coupling the known (6S)-2-nitro-6,7-dihydro-5H-imidazo[2, 1-b][1,3]oxazin-6-ol with iodobenzyl halides, followed by Suzuki coupling of these iodides with appropr

Full text of DOI:10.1021/jm901207n

282 J. Med. Chem. 2010, 53, 282–294
DOI: 10.1021/jm901207n
Synthesis and Structure-Activity Studies of Biphenyl Analogues of the Tuberculosis Drug (6S)-
2-Nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824)
Brian D. Palmer,† Andrew M. Thompson,† Hamish S. Sutherland,† Adrian Blaser,† Iveta Kmentova,† Scott G. Franzblau,‡
Baojie Wan,‡ Yuehong Wang,‡ Zhenkun Ma,§ and William A. Denny*,†
†Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand, ‡Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612, and §Global Alliance for TB Drug Development, 40 Wall Street, New York, New York 10005
Received August 12, 2009
A series of biphenyl analogues of the new tuberculosis drug PA-824 was prepared, primarily by
coupling the known (6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ol with iodobenzyl
halides, followed by Suzuki coupling of these iodides with appropriate arylboronic acids or by assembly
of the complete biaryl side chain prior to coupling with the above alcohol. Antitubercular activity was
determined under both replicating (MABA) and nonreplicating (LORA) conditions. para-Linked
biaryls were the most active, followed by meta-linked and then ortho-linked analogues. A more detailed
study of a larger group of para-linked analogues showed a significant correlation between potency
(MABA) and both lipophilicity (CLOGP) and the electron-withdrawing properties of terminal ring
P
substituents ( σ). Selected compounds were evaluated for their efficacy in a mouse model of acute
Mycobacterium tuberculosis infection. In vivo activity correlated well with the stability of compounds to
microsomal metabolism. Three compounds bearing combinations of lipophilic, electron-withdrawing
groups achieved >200-fold higher efficacies than the parent drug.
Introduction
related 6-nitroimidazo[2,1-b][1,3]oxazoles, exemplified by 2
(OPC-67683).4,5 These drugs show activity against both active
and persistent M. tb in animal models, suggesting a novel
mechanism and the potential to shorten the long treatment
cycles that are needed with the current agents.6,7
Tuberculosis (TB),a caused by Mycobacterium tuberculosis
(M. tb), is of increasing concern due to the recent emergence
and spread of multidrug-resistant strains.1 While the majority
of cases are potentially curable, complex and lengthy multi-
drug combination treatment is required, due largely to the
presence of metabolically quiescent subpopulations of bacte-
ria which can survive long periods of treatment with the
conventional drugs. The current standard protocols employ
combinations of isoniazid, rifampin, pyrazinamide, and
ethambutol, given over a period of 6-9 months. This is a
major barrier to full patient compliance and has contributed
to the development of drug-resistant strains.2
A study of 1 in susceptible and resistant strains of M. tb
showed3 that it was converted to more polar metabolites only
bythe former strains, suggestinga bioreductiveactivationstep
(it has a low one-electron reduction potential of -534 mV).8
Genetic studies suggested the involvement of a protein similar
to the F420-dependent glucose-6-phosphate dehydrogenase
of Mycobacterium smegmatis.3 A later paper9 confirmed that
while resistance to 1 is caused by loss of either a bacterial
glucose-6-phosphate dehydrogenase (FGD1) or its deazafla-
vin cofactor (F420), some mutants wild type for both FGD1
and F420 were still resistant to 1 but not to the related
compound 3 (CGI-17341). Sequencing of these mutants
showed changes in an unknown gene product (Rv3547, a
putative nitroreductase) and that complementation with this
restored sensitivity to 1. Both 1 and 2 are currently in phase II
clinical trials for the treatment of drug-susceptible and drug-
resistant TB,10,11 following successful phase II early bacte-
ricidal activity (EBA) studies.6,10 A recent mouse study showed
that the combination of 1 at 100 mg/kg with pyrazinamide was
as effective as the standard first-line combination of rifampin,
isoniazid, and pyrazinamide, although the emergence of
strains resistant to 1 was observed.12
Recently, there has been an intensive effort seeking drugs
that hit new targets, or work in new ways, since these have
the potential to overcome multidrug resistance. An exciting
development has been the discovery of the 2-nitroimidazo-
[2,1-b][1,3]oxazines, exemplified by 1 (PA-824),3 and the
*Corresponding author. Phone: (þ649) 923 6144. Fax: (þ649) 373
7502. E-mail: b.denny@auckland.ac.nz.
a Abbreviations: TB, tuberculosis; M. tb, Mycobacterium tuberculo-
sis; FGD1, F420-dependent glucose-6-phosphate dehydrogenase; EBA,
early bactericidal activity; SAR, structure-activity relationship; MIC,
minimum inhibitory concentration; SD, standard deviation; HREIMS,
high-resolution electron impact mass spectrometry; HRFABMS, high-
resolution fast atom bombardment mass spectrometry; HRAPCIMS,
high-resolution atmospheric pressure chemical ionization mass spectro-
metry; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
THF, tetrahydrofuran; TBAF, tetra-n-butylammonium fluoride;
DMA, N,N-dimethylacetamide; THP, tetrahydropyranyl; SRM, se-
lected reaction monitoring; IS, internal standard; PAR, peak area ratio;
CFU, colony forming unit; CMC, carboxymethylcellulose; TBDMS,
tert-butyldimethylsilyl.
To date, structure-activity (SAR) studies in the 2-nitro-
imidazo[2,1-b][1,3]oxazine series have been reasonably lim-
ited. Early work with 1 and analogues showed that there was
a considerable difference between the enantiomers at C-6,
with the S enantiomer being much more active than the R.3,13
r
pubs.acs.org/jmc
Published on Web 11/24/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 283
Some data have been reported for a small number of alternate
linker analogues of 1, with urea, carbamate, and amide
variations all proving active in cultures of Mycobacterium
bovis.13 The synthesis and antitubercular activity of 7-methyl
derivatives of 1 have also been described.14 A recent broader
study of the key determinants of aerobic activity in the general
class of 2-nitroimidazo[2,1-b][1,3]oxazines showed that the
required components were the bicyclic oxazine, the lipophilic
tail, and an oxygen substituent in the 8-position.15 Extending
the linker between the 6-(S) position and the terminal aro-
matic ring also gave highly potent compounds for a more
soluble 6-amino series.16
There are conflicting reports about the effects of other
substituents at the 8-position. Two recent studies8,16 have
shown that replacing the 8-oxygen with sulfur (as thioether)
is acceptable but that replacement with oxidized versions of
the latter (sulfoxide, sulfone) or with carbon dramatically
reduces activity. However, one report suggests that replace-
ment of the 8-oxygen with nitrogen is also acceptable,16
while in a study of heterocyclic analogues of 1 having similar
one-electron reduction potentials we found the 2-nitroimidazo-
[1,2-a]pyrimidine 4 (-568 mV), the 2-nitropyrazolo[5,1-b]-
[1,3]oxazine 5 (-500 mV), and the 2-nitropyrazolo[1,5-
a]pyrimidine 6 (-517 mV) to all be inactive.8 A recent radio-
chemical study17 has shown that the reduction chemistry of 1 is
different to that of related nitroheterocycles, with imidazole
ring reduction occurring in preference to nitro group reduction
at the two-electron level. This is consistent with recent work18
suggesting that 1 kills anaerobic bacteria through the release of
reactive nitrogen species, including nitric oxide, with the level of
formation (percent) of the concomitant denitro metabolite
correlating with the amount of anaerobic killing of M. tb across
a series of eight analogues.
In considering the above SAR, we elected to retain the key
imidazooxazine chromophoreand probe the suggested hydro-
phobic pocket(s) by initially employing more conformation-
ally rigid biphenyl-type side chains. The desired final goal of
our studies was to develop a second generation analogue of 1
having an improved pharmacological profile over both 1 and
2 for potential advancement into clinical development for TB
therapy. While 1 itself shows excellent pharmacokinetics,
tolerability, and safety in current clinical trials,19 the initial
challenge for a second generation analogue, addressed in this
study, was to first improve potency against both replicating
and nonreplicating M. tb and to second demonstrate im-
proved efficacy in vivo, compared to 1. A more efficacious
compound may help to reduce the cost of therapy and
improve the therapeutic index, thereby delivering a safer,
more affordable drug.
In this paper we therefore present SAR for biphenyl
analogues of the nitroimidazooxazine 1, with the aim of using
this scaffold both to further delineate the nature of the
preferred pharmacophore and to provide compounds of
superior efficacy to 1.
Chemistry and Methods
The bulk of the compounds listed in Tables 1, 2, and 3 were
prepared from the known13 chiral alcohol 122 by NaH-induced
alkylations with iodobenzyl halides 123, 125, and 127 to give
the iodobenzyl ethers 124, 126, and 12813 which then under-
went Suzuki couplings with appropriate arylboronic acids
(Scheme 1). Compounds 36, 92, 109, 114, and 120 were syn-
thesized via the cyclic boronate esters 133 and 134, which were
conveniently prepared from the related bromobenzyl ethers
130 and 132 (Scheme 2). An alternative method, involving
assembly of the biaryl side chain prior to coupling with alco-
hol 122, was employed for compounds 98, 100, 106, 107, 111,
and 113 (Scheme 3). Novel iodobenzenes 137 and 138
were prepared from the commercial anilines 135 and 136
Table 1. MIC Data for ortho-Linked Biphenyl Analogues of 1
MIC (μM)a
compd
X
MABA
LORA
1
0.50 ( 0.30
0.64 ( 0.32
1.2 ( 0.4
0.80 ( 0.31
0.62 ( 0.17
1.9 ( 0.7
0.81 ( 0.07
17 ( 1
8.4 ( 1.4
1.2 ( 0.3
2.3 ( 0.4
1.3 ( 0.4
1.8 ( 0.3
1.6 ( 0.4
2.6 ( 1.4
2.5 ( 0.5
6.2 ( 0.3
8.2 ( 2.9
3.0 ( 0.6
6.3 ( 0.7
3.2 ( 0.3
36 ( 13
7
H
8
2-OCF3
3-CN
3-F
Regarding the benzyl ether side chain of 1, early studies had
shown the utility of various lipophilic substituents, particu-
larly at the 4-position on the benzyl ring (e.g., 4-nBu, 4-tBu,
4-CF3, 4-OBn, 4-OCF3, 4-OPh), to enhance potency against
cultures of M. bovis.13 To explain these and similar findings in
the related 6-amino series, a recently described QSAR model
predicted the presence of either two hydrophobic binding
areas (one relatively close and one more distant from the
imidazooxazine) or a single (proximal or more distant) hydro-
phobic pocket in the nitroreductase.16 However, it is impor-
tant tonote thatwhile a few analogues from some of the above
studies display better potency than 1 in vitro, to date none of
these have been shown to possess better efficacy than 1 in vivo.
9
10
11
12
13
14
15
16
17
18
19
3-OCF3
3-SMe
4-COMe
4-CN
19 ( 5
4-F
3.4 ( 0.2
4.3 ( 0.7
3.4 ( 0.2
3.8 ( 0.2
3.1 ( 0.8
4-OCF3
4-SMe
3-F, 4-OMe
3,4-benzo
a Minimum inhibitory concentration, determined under aerobic
(MABA)23 or anaerobic (LORA)22 conditions. Each value is the mean
ofatleasttwoindependentdeterminations(29 determinationsfor1) (SD.

284 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Table 2. MIC Data for meta-Linked Biphenyl Analogues of 1
Palmer et al.
via diazotization. Suzuki coupling of these and other com-
mercial halobenzenes (139-141) to 4-(hydroxymethyl)-
phenylboronic acid (142) gave the required hydroxymethylbi-
phenyls (143-147), which were quantitatively converted to the
bromides (148-152) by HBr/AcOH and coupled to 122 as
above. This method was preferred for the larger scale synthesis
of compounds when the required substituted phenylboronic
acids were not commercially available. Compound 113 was
similarly prepared via difluoromethylation20 of the hydroxybi-
phenyl ester 154 (obtained via Suzuki coupling of 5-bromo-2-
chlorophenol and 4-(methoxycarbonyl)phenylboronic acid 153),
followed by reduction to alcohol 156, bromination with
phosphorus tribromide, and coupling to 122. Finally, to eval-
uate the acceptability of increasing aqueous solubility via
alcohol and amino functionality linked to the terminal ring,
several alkoxy-linked amino analogues (28, 38, 66, and 91) were
prepared from the corresponding phenols (25, 34, 62, and 85,
respectively) by reaction with protected hydroxyalkyl bro-
mides, followed by elaboration of the deprotected alkoxy alco-
hols (27, 37, 64, and 90) via their iodo derivatives (Scheme 4).
ThecomparativelipophilicitiesofthecompoundsinTable3
wereestimated both by summing substituentπ values and also
by log P values calculated using ACD LogP/LogD prediction
software (v.8.0, Advanced Chemistry Development Inc.,
Toronto, Canada). In calculating the electronic properties of
multisubstituted compounds, 20- and 40-substituents were
assigned σp values, and 30- and 50-substituents were assigned
MIC (μM)a
compd
X
MABA
LORA
1
0.50 ( 0.30
0.095 ( 0.015
0.46 ( 0.15
0.34 ( 0.10
0.17 ( 0.01
0.18 ( 0.03
0.30 ( 0.16
0.40 ( 0.07
0.58 ( 0.33
1.4 ( 0.4
0.070 ( 0.037
0.30 ( 0.19
0.12 ( 0.01
0.23 ( 0.01
0.077 ( 0.034
0.54 ( 0.23
0.11 ( 0.05
0.06 ( 0.01
0.36 ( 0.12
0.69 ( 0.25
0.095 ( 0.025
0.19 ( 0.10
0.12 ( 0.01
2.6 ( 1.4
2.1 ( 0.8
3.3 ( 0.1
2.9 ( 1.0
3.7 ( 2.1
2.7 ( 0.8
3.7 ( 0.2
3.5 ( 0.1
2.5 ( 0.5
11 ( 7
1.4 ( 0.1
1.9 ( 0.4
2.9 ( 1.4
4.8 ( 1.1
3.8 ( 0.7
5.8 ( 0.1
2.2 ( 0.5
2.8 ( 1.0
1.5 ( 0.2
11 ( 5
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
H
2-OCF3
3-CF3
3-CN
3-F
3-OH
3-OCF3
3-O(CH2)3OH
3-O(CH2)3Nmorphb
3-SMe
4-COMe
4-CF3
4-CN
4-F
4-OH
4-OCF3
4-OCF2H
4-O(CH2)3OH
4-O(CH2)3Nmorphb
4-SMe
σ
m values, and the total was summed.
The in vitro activity of the compounds against M. tb (strain
H37Rv) was quantified by MICs (minimum inhibitory con-
centrations; the lowest compound concentration effecting a
growth inhibition of >90%). The values recorded represent
0.93 ( 0.08
2.2 ( 0.5
3.2 ( 0.4
3-F, 4-OMe
3,4-benzo
a As for Table 1. b N-Morpholinyl.
Table 3. MIC Data, Calculated log P, and Substituent Values for para-Linked Biphenyl Analogues of 1
MIC (μM)d
P
P
compd
X
πa
log Pb
σc
MABA
LORA
1
2.70
3.51
4.48
2.63
4.02
3.99
2.47
3.17
3.71
2.98
4.21
5.50
4.20
4.85
5.55
4.48
2.93
3.58
2.03
3.53
4.08
2.76
0.50 ( 0.30
0.045 ( 0.005
0.077 ( 0.031
0.08 ( 0.02
0.19 ( 0.01
0.15 ( 0.04
0.12 ( 0
0.065 ( 0.005
0.03 ( 0
0.34 ( 0.11
0.035 ( 0.005
0.06 ( 0
0.087 ( 0.025
0.14 ( 0.02
0.31 ( 0.09
0.067 ( 0.025
0.14 ( 0.02
0.12 ( 0.01
2.8 ( 0.9
0.045 ( 0.005
0.06 ( 0.01
0.14 ( 0.02
2.6 ( 1.4
3.9 ( 2.0
1.6 ( 0.4
0.78 ( 0.16
0.67 ( 0.12
1.5 ( 0.3
4.6 ( 1.1
1.4 ( 0.3
0.82 ( 0.19
2.7 ( 0.9
0.97 ( 0.08
1.8 ( 0.1
1.4 ( 0.3
4.2 ( 1.5
0.88 ( 0.02
1.3 ( 0.4
0.57 ( 0.01
1.3 ( 0.6
6.7 ( 1.9
2.2 ( 0.7
1.6 ( 0.5
3.1 ( 1.4
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
H
0
0
2-CF3
2-CHO
2-F
0.88
-0.65
0.14
0.54
0.42
0.06
0.23
2-Cl
0.71
2-OH
2-OMe
2-OEt
-0.67
-0.02
0.38
-0.37
-0.27
-0.24
-0.20
0.35
2-O(CH2)3OH
2-OCF3
2-OPh
2-SMe
3-iPr
-1.0
1.04
2.08
-0.03
0.0
0.61
1.53
-0.07
0.06
0.43
3-Ph
3-CF3
1.96
0.88
3-CHO
3-CN
-0.65
-0.57
-1.49
0.14
0.35
0.56
3-CONH2
3-F
3-Cl
0.28
0.34
0.71
-0.67
0.37
0.12
3-OH

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 285
Table 3. Continued
MIC (μM)d
P
P
compd
X
πa
log Pb
σc
MABA
LORA
63
64
65
66
67
68
3-OMe
-0.02
-1.47
-1.0
3.27
2.30
3.08
3.13
4.31
4.93
4.00
1.95
3.04
4.85
5.20
5.17
4.48
2.33
4.48
4.42
2.82
2.83
1.92
3.05
3.44
4.08
2.73
3.32
4.20
5.20
2.35
3.13
3.11
3.50
4.36
4.00
1.68
2.30
5.15
5.07
3.89
4.92
3.73
4.43
2.72
3.38
5.19
4.88
4.83
5.19
3.76
3.30
4.33
3.16
3.97
3.30
2.92
3.28
4.25
3.35
3.65
3.31
4.74
0.12
-0.02
-0.12
-0.12
0.38
0.27 ( 0.17
0.46 ( 0.01
0.18 ( 0.07
1.5 ( 0.5
1.9 ( 0
3-O(CH2)2OH
3-O(CH2)3OH
3-O(CH2)2NMe2
3-OCF3
5.4 ( 2.3
3.2 ( 1.2
18 ( 8
1.04
1.66
0.077 ( 0.026
0.12 ( 0.06
0.077 ( 0.026
0.12 ( 0
0.13 ( 0.06
0.10 ( 0.01
0.095 ( 0.015
0.09 ( 0
1.4 ( 0.6
0.88 ( 0
0.95 ( 0.04
1.4 ( 0.1
0.76 ( 0.18
>32
3-OCH2Ph
3-SMe
3-NH2
0.10
0.15
69
70
0.61
-1.23
-0.28
1.53
-0.16
0.71
71
72
3-NO2
4-iPr
-0.15
-0.20
-0.01
0.54
73
74
4-tBu
4-Ph
4-CF3
1.98
1.96
>32
>32
75
76
0.88
0.03 ( 0.01
0.54 ( 0.12
0.077 ( 0.012
0.060 ( 0.016
0.20 ( 0.07
0.025 ( 0.005
2.1 ( 1.1
0.04 ( 0.01
0.015 ( 0.005
0.015 ( 0.005
0.64 ( 0.23
0.065 ( 0.005
0.25 ( 0.03
0.04 ( 0.01
0.55 ( 0.31
0.60 ( 0.16
0.097 ( 0.026
0.05 ( 0.01
0.035 ( 0.015
0.075 ( 0.015
0.67 ( 0.20
0.49 ( 0.23
0.03 ( 0
1.4 ( 0.5
3.7 ( 1.7
3.1 ( 2.0
0.63 ( 0.30
1.0 ( 0.1
0.58 ( 0.33
15 ( 5
0.73 ( 0.22
1.4 ( 0.5
2.7 ( 0.6
4.3 ( 2.1
6.8 ( 3.2
3.8 ( 1.8
2.7 ( 1.2
3.3 ( 1.6
2.2 ( 1.3
1.0 ( 0
4-CH2OH
4-CH2OtBu
4-CH2NHPh
4-CHO
-1.03
1.55
1.0
0.0
77
78
-0.32
-0.15
0.42
79
80
-0.65
-0.57
-1.49
-0.55
0.14
4-CN
0.66
0.36
81
82
4-CONH2
4-COMe
4-F
0.50
0.06
83
84
4-Cl
4-OH
0.71
0.23
85
86
-0.67
-0.02
0.85
-0.37
-0.27
-0.45
-0.03
-0.16
-0.17
-0.17
0.18
4-OMe
4-OiPr
4-OPh
87
88
2.08
89
90
4-O(CH2)2OH
4-O(CH2)3OH
4-O(CH2)3Nmorphe
4-OCF2H
4-OCF3
-1.47
-1.0
-1.0
0.69
91
92
0.77 ( 0.24
1.3 ( 0.1
0.95 ( 0.01
14 ( 3
93
94
1.04
0.61
0.35
0.0
4-SMe
4-SO2Me
4-NH2
95
96
-1.63
-1.23
1.59
0.72
-0.66
0.77
0.58
1.9 ( 1.0
1.4 ( 0.1
0.78 ( 0.03
0.74 ( 0.04
0.72 ( 0.29
0.81 ( 0.24
0.93 ( 0.25
1.0 ( 0.1
1.2 ( 0.3
0.97 ( 0.44
0.90 ( 0.12
0.95 ( 0.15
1.9 ( 0.2
1.6 ( 0.1
1.9 ( 0.8
0.34 ( 0.16
1.5 ( 0.1
1.1 ( 0.4
2.1 ( 0.5
0.79 ( 0.24
0.86 ( 0.22
0.94 ( 0.44
0.76 ( 0.26
1.5 ( 0.6
1.3 ( 0.4
0.87 ( 0.13
97
98
2-Cl, 4-CF3
2-Cl, 4-OCF3
2-Cl, 6-OMe
2-F, 4-OCF3
2-F, 6-OMe
2,6-diMe
1.75
0.69
0.04 ( 0.01
0.055 ( 0.005
0.045 ( 0.015
0.13 ( 0.02
0.39 ( 0.06
0.31 ( 0.06
0.03 ( 0
99
-0.04
0.41
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
1.18
0.12
-0.21
-0.34
-0.54
0.40
1.12
2,6-diOMe
3,4-diF
-0.04
0.28
1.59
3-Cl, 4-CF3
3-Cl, 4-OCF3
3-OCF3, 4-Cl
3-CF3, 4-Cl
3-NO2, 4-OCF3
3-F, 4-OMe
3-F, 4-OCF3
3-OMe, 4-F
3-OCF2H, 4-Cl
3-OH, 4-Cl
3,5-diOMe
2-OMe, 3,5-diF
2,6-diMe, 4-OMe
3,4,5-triF
0.91
0.72
0.06 ( 0.03
0.03 ( 0
1.75
1.75
0.61
0.66
0.040 ( 0.014
0.035 ( 0.005
0.045 ( 0.015
0.040 ( 0.014
0.03 ( 0
1.59
0.76
1.06
0.07
0.12
1.18
0.69
0.18
0.12
1.40
0.045 ( 0.015
0.03 ( 0
0.54
0.35
0.04
0.20 ( 0.01
0.28 ( 0.06
0.04 ( 0.01
0.58 ( 0.07
0.04 ( 0.01
0.30 ( 0.08
0.045 ( 0.015
0.045 ( 0.025
-0.04
0.26
1.10
0.24
0.41
-0.61
0.74
0.42
0.45
3,5-diMe, 4-OH
3,5-diF, 4-OMe
3,4-benzo
-0.51
0.41
0.10
0.26
1.16
a Sum of substituent π values. b log P calculated with ACD LogP/LogD prediction software (v.8.0; Advanced Chemistry Development Inc., Toronto,
Canada). c Sum of substituent σ values. d Minimum inhibitory concentration, determined under aerobic (MABA)23 or anaerobic (LORA)22 conditions.
Each value is the mean of at least two independent determinations (29 determinations for 1) ( SD. e N-Morpholinyl.
the mean of at least two separate determinations ((SD).
Activity against replicating M. tb was measured using an
8 day microplate-based assay with Alamar blue readout
(added on day 7) for determination of bacterial growth
(MABA).21 Screening for activity against nonreplicating
M. tb employed an 11 day high-throughput, luminescence-based

286 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Palmer et al.
Scheme 1a
Scheme 3a
a Reagents and conditions: (i) NaH, DMF, 5-20 °C, 1-2 h;
(ii) ArB(OH)2, toluene, EtOH, 2 M K2CO3, Pd(dppf)Cl2 under N2,
reflux, 5-150 min.
Scheme 2a
a Reagents and conditions: (i) NaH, DMF, 20 °C, 2 h; (ii) bis-
(pinacolato)diboron, Pd(dppf)Cl2, KOAc, DMSO, 90 °C, 1 h; (iii)
4-(HF2CO)PhBr or (3-NO2, 4-OCF3)PhBr or (5-Br, 2-Cl)PhOH or
(3,5-diF, 4-OMe)PhBr, toluene, EtOH, 2 M K2CO3, Pd(dppf)Cl2 under
N2, reflux, 15-70 min.
a Reagents and conditions: (i) aqueous NaNO2, 25% H2SO4, 0 °C,
10-12 min, then aqueous KI, 20 °C, 10-15 min, then 51 °C, 2 h; (ii)
halobenzene (137, 138, 139, 140, or 141), toluene, EtOH, 2 M Na2CO3,
Pd(dppf)Cl2 under N2, reflux, 1-3.5 h; (iii) 33% HBr/AcOH, 20 °C,
6-11 h; (iv) 122, NaH, DMF, 0-20 °C, 3 h; (v) (5-Br, 2-Cl)PhOH,
toluene, EtOH, 2 M K2CO3, Pd(dppf)Cl2 under N2, reflux, 30 min; (vi)
NaOCOCClF2, K2CO3, DMF, 80 °C, 14 h; (vii) LiAlH4, Et2O, 0-20 °C,
2 h; (viii) PBr3, Et2O, 0-20 °C, 3 h; (ix) 122, NaH, DMF, 0-20 °C, 2 h.
low-oxygen-recovery assay (LORA), where the bacteria con-
tained a plasmid with an acetamidase promoter driving a
bacterial luciferase gene and were first adapted to low oxygen
conditions by extended culture.22 Mammalian cytotoxicity
was also assessed23 against VERO cells (CCL-81, American
Type Culture Collection) in a 72 h exposure, using a tetra-
zolium dye assay.
and MABA assays is much smaller for the ortho-linked
compounds as compared to their meta- and para-linked
analogues (2.6-fold, compared to 14.5- and 28.4-fold, res-
pectively), and there is little correlation between the two sets
of assay data. This may indicate that a different mechanism of
activation applies under aerobic and anaerobic conditions.
Across this particular data set (Table 4), no single substituent
gave consistently improved MIC results compared to the
parent (H) compounds (7, 20, and 42); nevertheless, slightly
improved potencies were observed in several para-linked
examples (e.g., 4-CN, 80; 4-F, 83; 4-OCF3, 93).
In general, addition of a second aryl ring to the benzyl ether
side chain of 1 led to analogues with lower aqueous solubili-
ties than 1, typically 0.5-5 μg/mL at pH = 7 for selected
compounds from Tables 1-3 (compared with 19 μg/mL for 1
and 0.6 μg/mL for 2; data not shown). The attachment of
potentially solubilizing alcohol and amino functionality to the
distal ring of meta- and para-linked biphenyls led to com-
pounds(27, 28, 37, 38, 50, 64, 65, 66, 76, 89, 90, 91) within vitro
activities generally comparable to 1, although amino ana-
logues 28, 38, and 66 were 4-7-fold less potent than 1 in the
LORAassay. Overall, the para-linked biphenyl analogues of 1
were significantly more potent than 1 itself in both the MABA
and LORA assays (an order of magnitude in MABA), as were
Results and Discussion
Tables 1-3 provide data for 115 biphenyl analogues of 1,
including their activities against both replicating and non-
replicating cultures of M. tb. All of the compounds were
relatively nontoxic to mammalian VERO cells, with IC50s
>1 2 5 μM (data not shown). General inspection of
Tables 1-3 shows that the para-linked biphenyl analogues
are the most active, followed by the meta-linked ones, with the
ortho-linked analoguesbeing the leastactive. This relationship
seems to hold regardless of the nature and positioning of
substituentsonthe terminal benzene ringand suggests that the
binding pocket is reasonably linear and restricted, at least
around the inner benzyl ether ring (moderate steric bulk was
well tolerated around the terminus of the second benzene ring,
as shown by 3,4-benzo-fused compounds 19, 41, and 121 and
by 3- and 4-Ph/OPh analogues, 55, 74, and 88). This is
demonstrated more quantitatively in Table 4, which shows a
comparison of all of the ortho-linked analogues of Table 1
with their corresponding meta- and para-linked analogues
bearing the same substituents on the terminal ring. The mean
MICs for this representative set of compounds are 3.0, 0.19,
and 0.05 μM in MABA and 7.9, 2.8, and 1.4 μM in LORA,
respectively. The difference between the MICs in the LORA

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 287
several of the meta-linked compounds. The set of para-linked
biphenyl analogues was therefore expanded to explore the
SAR in more detail.
a 4-substituent (compounds 72-121) there was a weak but
significant and useful relationship (eq 1) between aerobic
MIC (albeit having somewhat limited range) and the sum
of π values of the substituents, suggesting that lipophilic
compounds were more effective:
Previous reports have suggested that a lipophilic side chain
confers higher potency in both the nitroimidazooxazine and
-oxazole classes, as shown3 by the comparison of 1 (log P 2.70)
with the less effective 3 (log P 0.70) and by the much greater
inhibitory activity2,5 of the even more lipophilic 2 (log P 4.75).
In the present data set (Table 3) there were no clear relation-
ships between MABA MIC values and measures of global
lipophilicity (calculated log P values) for the (smaller) sets
of ortho- and meta-substituted derivatives (43-71). How-
ever, in the larger set of para-linked biphenyls containing
P
logðMICMABAÞ ¼ -0:29ð( 0:06Þ π - 0:91ð( 0:07Þ
(1Þ
n ¼ 50 R ¼ 0:56 F ¼ 22:3
P
The addition of a second parameter ( σ) measuring the
summed electronic properties of the ring B substituents gave
an equation with an improved fit, showing that this variable
was also significant across a broad range of values ( σ from
-0.66 to þ1.06):
P
Scheme 4a
P
logðMICMABAÞ ¼ -0:26ð( 0:05Þ π -0:52ð( 0:12Þ
X
σ -0:82ð( 0:06Þ
n ¼ 50 R ¼ 0:72 F ¼ 24:6
ðπ=σ cross-correlation coefficient 0:15Þ
(2Þ
Attempted fits using other electronic-related (F, R) and
lipophilicity-related (MR, PSA) parameters were examined
but were not as good. Given the close cross-correlation
P
between π for the ring B substituents and overall log P
for the compounds (R = 0.96), the related eq 3 using overall
log P values (which may be of more general utility for
compounds that incorporate additional changes) was very
similar:
logðMICMABAÞ ¼ -0:25ð(0:06Þ log P -0:52ð(0:13Þ
X
σ -0:014ð(0:22Þ
n ¼ 50 R ¼ 0:69 F ¼ 21:1
ðlog P=σ cross-correlation coefficient 0:15Þ
(3Þ
a Reagents and conditions: (i) Br(CH2)3OTBDMS, Cs2CO3, DMF,
90 °C, 1-2 h, then TBAF, THF, 20 °C, 2 h; (ii) MsCl, Et3N, THF, 0 °C,
1-2.5 h, then NaI, Me2CO, reflux, 1-3 h; (iii) morpholine, DMA, 20 °C,
16 h; (iv) Br(CH2)2OTHP, Cs2CO3, DMF, 90 °C, 2 h, then MsOH,
MeOH, 20 °C, 1 h; (v) aqueous Me2NH, DMA, 20 °C, 16 h.
Equations 2 and 3 suggest that, in addition to the impor-
tance of overall higher compound lipophilicity, possibly to
facilitate penetration of the exceptionally lipophilic cell wall
of M. tb,24 a relatively electron-deficient terminal ring is
Table 4. Comparison of MICs for 13 Sets of ortho/meta/para-Linked Biphenyl Analogues
ortho-link meta-link
MIC (μM) MIC (μM)
para-link
MIC (μM)
substituent
no.
MABA
LORA
no.
MABA
LORA
no.
MABA
LORA
H
7
0.64
1.2
2.5
6.2
8.2
3.0
6.3
3.2
36
20
21
23
24
26
29
30
32
33
35
39
40
41
0.095
0.46
0.17
0.18
0.40
0.07
0.30
0.23
0.077
0.11
0.095
0.19
0.12
2.1
3.3
3.7
2.7
3.5
1.4
1.9
4.8
3.8
2.2
0.93
2.2
3.2
42
51
58
60
67
69
82
80
83
93
94
110
121
0.045
0.035
0.12
3.9
2-OCF3
3-CN
3-F
8
0.97
1.3
9
0.80
0.62
1.9
10
11
12
13
14
15
16
17
18
19
0.045
0.077
0.077
0.04
2.2
1.4
3-OCF3
3-SMe
4-COMe
4-CN
0.81
17
8.4
0.95
0.73
0.58
1.4
19
0.025
0.015
0.035
0.075
0.04
4-F
1.2
2.3
3.4
4.3
3.4
3.8
3.1
4-OCF3
4-SMe
3-F, 4-OMe
3,4-benz
1.3
1.3
1.8
0.95
1.9
1.6
0.045
0.87
means
3.04
7.88
0.19
2.75
0.05
1.42

288 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Palmer et al.
separately beneficial. This suggests that charge-transfer bind-
ing may be important at the binding site of the nitroreductase
and is consistent with the empirical dominance of lipophilic,
electron-withdrawing groups such as CF3 and OCF3 in the
design of the more effective compounds. It also points to the
potential use of halogens as suitable substituents.
Table 5. Microsomal Stability and in Vivo Efficacy Data for Selected
Analogues
Examining SAR for the expanded para-linked biphenyl
class (Table 3) in more detail, there was only a slight potency
advantage for 4-substitution over 3- or 2-substitution (6/9
cases in MABA), with 4-CF3 (75) and 4-Cl (84) analogues
proving particularly effective, in addition to the 4-CN (80),
4-F (83), and 4-OCF3 (93) examples identified previously.
However, 4-SO2Me (95) and 3- or 4-CONH2 (59, 81) ana-
logues were notably poor. The combination of two or more
substituents (compounds 97-120) generally retained or en-
hanced MIC potency, particularly in LORA, with 111 (3-F,
4-OCF3: LORA MIC 0.34 μM) being the most potent of all
the compounds evaluated in this assay.
Selected compounds were evaluated for their efficacy in a
mouse model of acute M. tb infection, using a once daily oral
dose of 100 mg/kg for 5 days a week for 3 weeks, following
established protocols.23 The choice of compounds was pri-
marily based on their MIC potencies, with particular focus on
compounds with good dual activity against both replicating
and nonreplicating bacteria, but a variety of different types of
substituents were also studied. The clinical trial drug 1 was
employed as an internal standard, with activity recorded
(Table 5) as the ratio of the fold decrease in colony forming
units (CFUs) recovered from the lungs of compound-treated
mice compared to the corresponding fold CFU decrease
achieved by treatment with 1, to allow interexperiment com-
parisons (see Supporting Information for raw CFU data for
the compounds of Table 5). The data in Table 5 first illustrate
the apparent liability incurred by alkoxy groups on the
molecule, in that compounds 49, 50, and 91 (and, to a lesser
extent, 68) were markedly inferior to 1. Even when a methoxy
group was paired with a more electron-withdrawing group
(101, 110, and 116) the in vivo results were still very poor.
Similarly, anilino, ketone, andphenoxy-substituted analogues
(78, 82, and 88) displayed almost no efficacy at all in this
model. Conversely, compounds bearing lipophilic, electron-
withdrawing groups (75, 93, 98, 100, 106, 111, and 113),
particularly at the terminus (3- and 4-positions), were sub-
stantially more active than 1 (>10-fold). The three best
candidates were 93 (4-OCF3), 106 (3-Cl, 4-OCF3), and 111
(3-F, 4-OCF3), which were all >200-fold more efficacious
than 1 in this model.
The strong in vivo efficacy of some of the most lipophilic
analogues following oral dosing (as observed for 2)4,5 is
consistent with significant oral bioavailability for this class,
in addition to an improved binding to the nitroreductase.
Thus, in order to further understand the divergent in vivo
results above, the compounds were also evaluated for their
stability in a metabolism screen with human and mouse liver
microsome preparations. The compounds were incubated for
1 h at 37 °C, and the percentage of compound remaining was
determined by LC-MS-MS. The results (Table 5) allow some
SAR to be derived. The parent drug 1 showed good stability
toward both human and mouse microsomes (82% and 94%
remaining, respectively). Five analogues bearing O-alkyl
groups (49, 50, 91, 101, and 116) had poor stabilities, espe-
cially in mouse microsomes (68 and 110 also showed mar-
ginal stabilities in mouse microsomes). The remainder of the
compounds were relatively stable in the mouse assay (less than
microsomes (%
remaining at 1 h)
in vivo efficacyc
(ratio vs 1)
compd
X
Ha
Mb
1
49
82
50
59
64
60
12
95
36
75
22
11
94
97
92
91
88
91
90
95
92
80
93
83
72
94
0.2
21
50
85
52
58
70
55
48
14
77
96
93
86
89
0
1.00
0.15
<0.01
0.66
72
2-OEt
50
68
2-O(CH2)3OH
3-OCH2Ph
4-CF3
75
78
4-CH2NHPh
4-CN
4-COMe
0.03
3.6
80
82
<0.01
3.0
83
88
4-F
4-OPh
<0.01
0.05
8.5
91
92
4-O(CH2)3Nmorphd
4-OCF2H
4-OCF3
93
97
>205
7.2
23
2-Cl, 4-CF3
2-Cl, 4-OCF3
2-F, 4-OCF3
2-F, 6-OMe
3-Cl, 4-OCF3
3-OCF3, 4-Cl
3-CF3, 4-Cl
3-F, 4-OMe
3-F, 4-OCF3
3-OCF2H, 4-Cl
2-OMe, 3,5-diF
98
100
101
106
107
108
110
111
113
116
46
0.07
281
9.7
84
92
96
50
86
86
2
7.7
0.02
419
10
0.40
a Pooled human liver microsomes. b Pooled CD-1 mouse liver micro-
somes. c Fold reduction in lung CFU for compound compared with
the fold CFU reduction for 1 in a mouse model of acute TB infection
(see text). d N-Morpholinyl.
50% loss in 1 h), but a further three (78, 82, and 88) were
markedly unstable toward human microsomes. Thus the
lowest stability compounds were those bearing an alkoxy
or phenoxy group or having a ketone substituent or a free
NH function. Conversely, compounds that showed high
stabilities in both assays were those with halogen and/or
CF3/OCF3 substituents (75, 93, 97, 98, 100, 106, 107, 108,
and 111), reinforcing the apparent utility of these substi-
tuents. Compounds 92 and 113 also showed the usefulness
of the OCF2H group, although these compounds appeared
to be slightly less stable than their OCF3 counterparts
(93 and 107).
Across the whole data set, the microsomal stability results
appear, at least for this group of compounds, to be usefully
predictive for in vivo activity. If the microsomal assay data are
crudely quantified as the sum of the two “% compound
remaining” numbers, and the >,< modifiers are removed
from the fold reduction numbers, eq 4 shows that there is
a reasonable correlation between microsomal stability and
in vivo efficacy:
logðfold reduction in CFUÞ ¼ 4:84ð(0:99Þ
logðmicrosomal stabilityÞ -9:82
n ¼ 23 R ¼ 0:73 F ¼ 23:8
(4Þ

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 289
2 H), 4.67-4.60 (m, 2 H), 4.59 (d, J = 12.2 Hz, 1 H), 4.46 (d, J =
12.0 Hz, 1 H), 4.27-4.19 (m, 3 H). Anal. (C13H12IN3O4) C, H, N.
General Method for Suzuki Coupling. Iodides 124, 126, or 128
(1 equiv) and arylboronic acids (1.3 equiv) were suspended in
toluene/EtOH (5 mL/2 mL per 100 mg of iodide), and then
aqueous K2CO3 (1 mL of 2 M per 100 mg of iodide) was added.
The resulting mixture was purged with N2, treated with Pd-
(dppf)Cl2 (5 mol %), and heated under reflux in an N2 atmo-
sphere for 20 min (or longer if noted). The mixture was diluted
with water and extracted with EtOAc (3ꢀ). The dried (MgSO4)
organic layers were adsorbed onto silica gel and chromato-
graphed, eluting first with EtOAc/hexane (1:1) and then with
EtOAc to give the product. For more polar compounds, EtOAc/
MeOH (95:5) or CH2Cl2/MeOH (95:5) was used. Trituration of
the product in Et2O gave the pure compounds.
Conclusions
This study has demonstrated the potential utility of parti-
cularly para-linked biphenyl-based side chains to provide
highly potent analogues of 1, having significantly improved
activity in vitro against both replicating and nonreplicating
M. tb. Seven of the compounds showed substantially (>10-fold)
improved efficacies over 1 when evaluated in a mouse model
of acute M. tb infection, with three of these being >200-fold
more effective than 1. This is the first study to report analo-
gues of 1 with improved efficacy in vivo. Overall, there appears
to be predictive correlations between MABA MIC data and
the lipophilicity and electron-withdrawing properties of sub-
stituent groups on the distal ring of the side chain. The ability
of the compounds to resist degradation by liver microsome
preparations may also be a useful indicator of their potential
in vivo activity.
Details of the preparation of compounds for Tables 1-3 by
this method and their characterization (mp, 1H NMR, and
either combustion analysis or HPLC) are given in Supporting
Information.
Preparation of Biphenyl Analogues via Boronic Acid Pinacol
Esters and Aryl Halides (Scheme 2). (6S)-6-[(3-Bromobenzyl)-
oxy]-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (130).
Reaction of alcohol 122 with 3-bromobenzyl bromide (129) and
NaH inDMF for 2 h atroomtemperature (asdescribed above for
the preparation of 124) gave 130 (93%): mp (EtOAc/petroleum
ether) 59-62 °C; 1H NMR δ 8.01 (s, 1 H), 7.50-7.47 (m, 2 H),
7.33-7.30 (m, 2 H), 4.69-4.65 (m, 2 H), 4.63 (d, J = 12.3 Hz,
1 H), 4.47 (d, J = 12.0 Hz, 1 H), 4.28-4.20 (m, 3 H). Anal. (C13-
H12BrN3O4) C, H, N.
(6S)-6-[(4-Bromobenzyl)oxy]-2-nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazine (132). Similar reaction of alcohol 122 with
4-bromobenzyl bromide (131) and NaH in DMF for 2 h at room
temperature (as described for 124) gave 132 (88%): mp (Et2O)
188-190 °C; 1H NMR δ 8.01 (s, 1 H), 7.54 (dt, J = 8.4, 2.2 Hz,
2 H), 7.13 (dt, J = 8.5, 2.2 Hz, 2 H), 4.67-4.62 (m, 2 H), 4.61
(d, J = 12.2 Hz, 1 H), 4.46 (d, J = 12.0 Hz, 1 H), 4.28-4.19 (m,
3 H). Anal. (C13H12BrN3O4) C, H, N.
(6S)-2-Nitro-6-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzyloxy]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (133). A
mixture of the 3-bromide 130 (1.50 g, 4.24 mmol) and bis-
(pinacolato)diboron (1.195 g, 4.70 mmol) and KOAc (2.55 g,
26.0 mmol) in DMSO (40 mL) was purged with N2. Pd(dppf)Cl2
(0.15 g, 0.20 mmol) was added, and the mixture was then stirred
at 90 °C for 1 h while being continuously purged with N2 (gas
bubbled through the solution). The resulting cooled mixture was
partitioned between EtOAc and water, and the organic extract
was evaporated to dryness and then purified by chromato-
graphy on silica gel, eluting with EtOAc. The product was
triturated in Et2O to give 133 (1.033 g, 61%): mp 168-171 °C;
1H NMR δ 8.01 (s, 1 H), 7.62-7.58 (m, 2 H), 7.44 (dt, J = 7.8,
1.5 Hz, 1 H), 7.36 (t, J = 7.5 Hz, 1 H), 4.68-4.61 (m, 3 H), 4.47
(d, J = 11.9 Hz, 1 H), 4.30-4.20 (m, 3 H), 1.28 (s, 12 H). Anal.
(C19H24BN3O6) C, H, N.
(6S)-2-Nitro-6-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
benzyloxy]-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (134). Si-
milar reaction of the 4-bromide 132 with bis(pinacolato)-
diboron, as described above for the preparation of 133, followed
by chromatography on silica gel (eluting with EtOAc) and
trituration in Et2O, gave 134 (51%): mp 150-153 °C;
1H NMR δ 8.01 (s, 1 H), 7.65 (d, J = 8.0 Hz, 2 H), 7.32 (d,
J = 8.0 Hz, 2 H), 4.70 (d, J = 12.5 Hz, 1 H), 4.67-4.63 (m, 2 H),
4.46 (d, J = 11.9 Hz, 1 H), 4.29-4.20 (m, 3 H), 1.29 (s, 12 H).
Anal. (C19H24BN3O6) C, H, N.
Experimental Section
Analyses were performed by the Campbell Microanalytical
Laboratory, University of Otago, Dunedin, New Zealand. Melting
points were determined using an Electrothermal Model 9200
melting point apparatus and are as read. NMR spectra were
measured in (CD3)2SO (unless otherwise specified) on a Bruker
Avance 400 spectrometer at 400 MHz for 1H and are referenced to
Me4Si. Chemical shifts and coupling constants are recorded in
units of parts per million and hertz, respectively. High-resolution
electron impact (HREIMS) and fast atom bombardment
(HRFABMS) mass spectra were determined on a VG-70SE mass
spectrometer at nominal 5000 resolution. High-resolution atmo-
spheric pressure chemical ionization (HRAPCIMS) mass spectra
were determined on a Bruker micrOTOF-Q II mass spectrometer.
Thin-layer chromatography was carried out on aluminum-backed
silica gel plates (Merck 60 F254) with visualization of components
by UV light (254 nm) or exposure to I2. Column chromatography
was carried out on silica gel (Merck 230-400 mesh). Tested
compounds were g95% pure, as determined by combustion
analysis, or by HPLC conducted on an Agilent 1100 system, using
a reversed-phase C8 column with diode array detection.
Preparation of Biphenyl Analogues from Iodobenzyl Ethers
and Arylboronic Acids (Scheme 1). (6S)-6-[(2-Iodobenzyl)oxy]-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (124). A solu-
tion of (6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-ol13 (122) (3.188 g, 17.2 mmol) and 2-iodobenzyl chloride
(123) (5.00 g, 19.8 mmol) in anhydrous DMF (60 mL) was
treated at 5 °C with 60% NaH (0.893 g, 22.3 mmol). The mixture
was warmed to room temperature, stirred for 1 h, and then
quenched with water, and the precipitate was collected and
triturated in Et2O to give 124 (4.19 g, 61%): mp (Et2O)
1
114-117 °C; H NMR δ 8.04 (s, 1 H), 7.86 (d, J = 7.7 Hz,
1 H), 7.41-7.35 (m, 2 H), 7.10-7.04 (m, 1 H), 4.69-4.61 (m, 3
H), 4.51 (d, J = 11.6 Hz, 1 H), 4.35-4.23 (m, 3 H). Anal.
(C13H12IN3O4) C, H, N, I. HPLC purity: 98.7%.
(6S)-6-[(3-Iodobenzyl)oxy]-2-nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazine (126). Similar reaction of alcohol 122 with
3-iodobenzyl bromide (125) and NaH in DMF for 1 h at room
temperature (as described for 124) gave 126 (85%): mp (EtOAc/
petroleum ether) 144-146 °C; 1H NMR δ 8.01 (s, 1 H),
7.67-7.64 (m, 2 H), 7.33 (d, J = 7.6 Hz, 1 H), 7.16 (br t, J =
8.1 Hz, 1 H), 4.67-4.62 (m, 2 H), 4.60 (d, J = 12.3 Hz, 1 H), 4.47
(d, J = 12.0 Hz, 1 H), 4.28-4.20 (m, 3 H). Anal. (C13H12-
IN3O4 0.25EtOAc) C, H, N.
(6S)-6-{[40-(Difluoromethoxy)[1,10-biphenyl]-3-yl]methoxy}-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (36). Reac-
tion of 133 and 1-bromo-4-(difluoromethoxy)benzene under the
general Suzuki conditions described above for 15 min gave 36
(54%) as a white solid: mp 113-115 °C; 1H NMR δ 8.02 (s, 1 H),
7.67 (d, J = 8.8 Hz, 2 H), 7.60-7.53 (m, 2 H), 7.44 (t, J = 7.6 Hz,
1 H), 7.31 (br d, J = 7.6 Hz, 1 H), 7.27 (t, JH-F = 74.1 Hz, 1 H),
3
(6S)-6-[(4-Iodobenzyl)oxy]-2-nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazine (128). Similar reaction of alcohol 122 with
4-iodobenzyl bromide (127) and NaH in DMF for 2 h at room
temperature (as described for 124) gave 12813 (97%): mp
(EtOAc/petroleum ether) 210-212 °C; 1H NMR δ 8.01 (s,
1 H), 7.71 (dt, J = 8.3, 2.0 Hz, 2 H), 7.13 (br d, J = 8.3 Hz,

290 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Palmer et al.
1
(1.33 g, 87%) as a colorless oil (a white solid on freezing): H
7.25 (d, J = 8.8 Hz, 2 H), 4.75-4.63 (m, 3 H), 4.49 (d, J = 11.9
Hz, 1 H), 4.32-4.20 (m, 3 H). Anal. (C20H17F2N3O5) C, H, N.
(6S)-6-{[40-(Difluoromethoxy)[1,10-biphenyl]-4-yl]methoxy}-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (92). Reac-
tion of 134 and 1-bromo-4-(difluoromethoxy)benzene under
the general Suzuki conditions described above for 15 min gave
NMR (CDCl3) δ 7.64 (quintet, J = 1.5 Hz, 1 H), 7.57 (dd, J =
8.4, 1.9 Hz, 1 H), 7.19 (d, J = 8.4 Hz, 1 H); HRAPCIMS calcd
for C7H3ClF3IO m/z (Mþ) 323.8834, 321.8864, found 323.8847,
321.8873.
2-Chloro-4-iodo-1-(trifluoromethoxy)benzene (138). Similar
diazotization of 3-chloro-4-(trifluoromethoxy)aniline (136)
(1.00 g) in a cold mixture of 98% H2SO4 (0.75 mL) and water
(2.25 mL) (as described for 137) and chromatography of the
product on silica gel, eluting with pentane, gave 138 (1.24 g,
1
92 (77%) as a cream solid: mp 188-191 °C; H NMR δ 8.02
(s, 1 H), 7.71 (d, J = 8.8 Hz, 2 H), 7.63 (d, J = 8.3 Hz, 2 H), 7.40
(d, J = 8.3 Hz, 2 H), 7.26 (t, JH-F = 74.1 Hz, 1 H), 7.25 (d, J =
8.8 Hz, 2 H), 4.73-4.64 (m, 3 H), 4.48 (d, J = 11.9 Hz, 1 H),
4.32-4.21 (m, 3 H). Anal. (C20H17F2N3O5) C, H, N.
1
81%) as a colorless oil (a white solid on freezing): H NMR
(6S)-2-Nitro-6-{[30-nitro-40-(trifluoromethoxy)[1,10-biphenyl]-
4-yl]methoxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (109).
A stirred mixture of 134 (50.1 mg, 0.125 mmol) and Pd(dppf)Cl2
(9.2 mg, 0.0126 mmol) in toluene (2 mL) and EtOH (1 mL) was
degassed for 4 min (vacuum pump), and then N2 was added.
Aqueous Na2CO3 (0.35 mL of 2 M, 0.70 mmol) and 4-bromo-
2-nitro-1-(trifluoromethoxy)benzene (52 mg, 0.182 mmol) were
sequentially added by syringe, the stirred mixture was again
degassed for 4 min, and then N2 was added. The resulting
mixture was stirred at 90 °C for 70 min and then cooled, diluted
with aqueous NaHCO3 (50 mL), and extracted with CH2Cl2
(5 ꢀ 50 mL). The extracts were evaporated to dryness, and the
residue was chromatographed on silica gel. Elution with
0-0.5% EtOAc/CH2Cl2 gave foreruns, and then elution with
1.5-2.5% EtOAc/CH2Cl2 gave 109 (48 mg, 80%) as a light
yellow solid: mp (CH2Cl2/pentane) 165-168 °C; 1H NMR
(CDCl3) δ 8.16 (d, J = 2.3 Hz, 1 H), 7.83 (dd, J = 8.6, 2.3
Hz, 1 H), 7.58 (dt, J = 8.3, 1.8 Hz, 2 H), 7.52 (dq, J = 8.6, 1.4
Hz, 1 H), 7.44 (br d, J = 8.3 Hz, 2 H), 7.39 (s, 1 H), 4.80 (d, J =
12.1 Hz, 1 H), 4.69 (d, J = 12.1 Hz, 1 H), 4.65 (ddd, J = 12.2,
3.7, 2.1 Hz, 1 H), 4.38 (dd, J = 12.1, 1.3 Hz, 1 H), 4.25-4.13 (m,
3 H). Anal. (C20H15F3N4O7) C, H, N.
4-Chloro-40-({[(6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b]-
[1,3]oxazin-6-yl]oxy}methyl)[1,10-biphenyl]-3-ol (114). Reaction
of 134 and 5-bromo-2-chlorophenol under the general Suzuki
conditions described above for 1 h gave 114 (76%) as a light
yellow powder: mp 92-95 °C; 1H NMR δ 10.26 (br s, 1 H), 8.03
(s, 1 H), 7.55 (d, J = 8.3 Hz, 2 H), 7.42-7.37 (m, 3 H), 7.20
(d, J = 2.1 Hz, 1 H), 7.07 (dd, J = 8.3, 2.1 Hz, 1 H), 4.72-4.63
(m, 3 H), 4.48 (d, J = 11.9 Hz, 1 H), 4.32-4.21 (m, 3 H). Anal.
(C19H16ClN3O5) C, H, N.
(6S)-6-[(30,50-Difluoro-40-methoxy[1,10-biphenyl]-4-yl)methoxy]-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (120). Reac-
tion of 134 and 5-bromo-1,3-difluoro-2-methoxybenzene under
the general Suzuki conditions described above for 30 min gave
120 (70%) as a white solid: mp 168-171 °C; 1H NMR δ 8.02 (s,
1 H), 7.68 (d, J = 8.3 Hz, 2 H), 7.53-7.45 (m, 2 H), 7.39 (d, J =
8.3 Hz, 2 H), 4.70-4.64 (m, 3 H), 4.48 (d, J = 11.9 Hz, 1 H),
4.29-4.23 (m, 3 H), 3.95 (s, 3 H). Anal. (C20H17F2N3O5) C, H, N.
Preparation of Biphenyl Analogues via Hydroxymethylbiphe-
nyls (Scheme 3). 1-Chloro-4-iodo-2-(trifluoromethoxy)benzene
(137). An ice-cold mixture of 98% H2SO4 (1.0 mL) and water
(3.0 mL) was added to 4-chloro-3-(trifluoromethoxy)aniline
(135) (1.00 g, 4.73 mmol), and the resulting salt was crushed
(using a glass rod) and cooled in an ice bath. A solution of
NaNO2 (359 mg, 5.20 mmol) in cold water (0.75 mL, then
0.25 mL) was added dropwise (over 10 min with stirring), and
then the mixture was stirred at 0 °C for 10 min. A solution of
urea (43.5 mg, 0.724 mmol) in cold water (0.25 mL) was added,
and the mixture was stirred at 0 °C for 3 min. Finally, a solution
of KI (1.66 g, 10.0 mmol) in cold water (1.6 mL, then 0.2 mL)
was added slowly, and the mixture was stirred at room tem-
perature for 15 min and then at 51 °C for 2 h. The resulting
mixture was cooled in ice, diluted with ice-water (45 mL), and
extracted with pentane (4 ꢀ 50 mL). The extracts were sequen-
tially washed with an aqueous solution of Na2SO3 (30 mL of
0.5%) and then with water (40 mL) and finally concentrated
carefully under reduced pressure at 13 °C. The resulting oil was
chromatographed on silica gel, eluting with pentane, to give 137
(CDCl3) δ 7.82 (d, J = 2.1 Hz, 1 H), 7.61 (dd, J = 8.6, 2.1 Hz,
1 H), 7.05 (dq, J = 8.6, 2.0 Hz, 1 H); HRAPCIMS calcd for
C7H3ClF3IO m/z (Mþ) 323.8834, 321.8864, found 323.8834,
321.8861.
Procedure A. [20-Chloro-40-(trifluoromethoxy)[1,10-biphenyl]-
4-yl]methanol (143). A stirred mixture of 4-(hydroxymethyl)-
phenylboronic acid (142) (308 mg, 2.03 mmol) and Pd(dppf)Cl2
(191 mg, 0.261 mmol) in toluene (22 mL) and EtOH (11 mL) was
degassed for 8 min (vacuum pump), and then N2 was added.
Aqueous Na2CO3 (4.4 mL of 2 M, 8.8 mmol) was added by
syringe, the stirred mixture was again degassed for 8 min,
and then N2 was added. 2-Chloro-1-iodo-4-(trifluoromethoxy)-
benzene (139) (585 mg, 1.81 mmol) was added by syringe, and
the resulting mixture was stirred at 88 °C for 60 min. The cooled
mixture was then diluted with aqueous NaHCO3 (100 mL) and
extracted with CH2Cl2 (5 ꢀ 100 mL). The extracts were evapo-
rated to dryness, and the residue was chromatographed on silica
gel. Elution with 0-50% CH2Cl2/petroleum ether first gave
foreruns, and then further elution with 50% CH2Cl2/petroleum
ether gave 143 (537 mg, 98%) as a white solid: mp (pentane)
38-39 °C; 1H NMR (CDCl3) δ 7.46 (br d, J = 8.2 Hz, 2 H), 7.42
(dt, J = 8.3, 2.0 Hz, 2 H), 7.37 (br s, 1 H), 7.36 (d, J = 8.5 Hz, 1
H), 7.19 (m, 1 H), 4.77 (d, J = 5.9 Hz, 2 H), 1.70 (t, J = 5.9 Hz, 1
H); HREIMS calcd for C14H10ClF3O2 m/z (Mþ) 304.0292,
302.0321, found 304.0294, 302.0317.
[20-Fluoro-40-(trifluoromethoxy)[1,10-biphenyl]-4-yl]methanol
(144). Reaction of 142 and 1-bromo-2-fluoro-4-(trifluoro-
methoxy)benzene (140) under the Suzuki conditions described
in procedure A for 3.5 h, followed by chromatography of the
product on silica gel, eluting with 0-40% CH2Cl2/petroleum
ether (foreruns) and then 40% CH2Cl2/petroleum ether, gave
144 (73%) as a cream solid: mp (CH2Cl2/pentane) 72-74 °C; 1H
NMR (CDCl3) δ 7.52 (dq, J = 8.3, 1.8 Hz, 2 H), 7.49-7.42 (m,
3 H), 7.13-7.03 (m, 2 H), 4.76 (d, J = 5.9 Hz, 2 H), 1.69 (t, J =
5.9 Hz, 1 H); HREIMS calcd for C14H10F4O2 m/z (Mþ)
286.0617, found 286.0612.
[30-Chloro-40-(trifluoromethoxy)[1,10-biphenyl]-4-yl]methanol
(145). Reaction of 142 and 138 under the Suzuki conditions
described in procedure A for 95 min, followed by chromatogra-
phy of the product on silica gel, eluting with 0-40% CH2Cl2/
petroleum ether (foreruns) and then 40% CH2Cl2/petroleum
ether, gave 145 (84%) as a cream solid: mp (CH2Cl2/pentane)
65-66 °C; 1H NMR (CDCl3) δ 7.68 (d, J = 2.2 Hz, 1 H), 7.54 (dt,
J = 8.3, 1.9 Hz, 2 H), 7.48 (dd, J = 8.5, 2.2 Hz, 1 H), 7.46 (br d,
J = 8.4 Hz, 2 H), 7.38 (dq, J = 8.5, 1.4 Hz, 1 H), 4.76 (d, J = 5.9
Hz, 2 H), 1.70 (t, J = 5.9 Hz, 1 H); HREIMS calcd for C14H10-
ClF3O2 m/z (Mþ) 304.0292, 302.0321, found 304.0300, 302.0319.
[40-Chloro-30-(trifluoromethoxy)[1,10-biphenyl]-4-yl]methanol
(146). Reaction of 142 and 137 under the Suzuki conditions
described in procedure A for 120 min, followed by chromato-
graphy of the product on silica gel, eluting with CH2Cl2, gave
146 (77%) as a cream solid: mp (CH2Cl2/pentane) 70-71 °C;
1H NMR (CDCl3) δ 7.57-7.50 (m, 4 H), 7.49-7.43 (m, 3 H),
4.76 (d, J = 5.9 Hz, 2 H), 1.69 (t, J = 5.9 Hz, 1 H); HREIMS
calcd for C14H10ClF3O2 m/z (Mþ) 304.0292, 302.0321, found
304.0283, 302.0319.
[30-Fluoro-40-(trifluoromethoxy)[1,10-biphenyl]-4-yl]methanol
(147). Reaction of 142 and 4-bromo-2-fluoro-1-(trifluoro-
methoxy)benzene (141) under the Suzuki conditions described

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 291
in procedure A for 2.5 h, followed by chromatography of the
product on silica gel, eluting with 0-40% CH2Cl2/petroleum
ether (foreruns) and then 40% CH2Cl2/petroleum ether, gave
147 (73%) as a cream solid: mp (CH2Cl2/pentane) 70-71 °C; 1H
NMR (CDCl3) δ 7.54 (dt, J = 8.3, 1.8 Hz, 2 H), 7.46 (br d, J =
8.2 Hz, 2 H), 7.41 (br d, J = 11.2 Hz, 1 H), 7.40-7.32 (m, 2 H),
4.76 (d, J = 5.9 Hz, 2 H), 1.69 (t, J = 5.9 Hz, 1 H); HREIMS
calcd for C14H10F4O2 m/z (Mþ) 286.0617, found 286.0616.
Procedure B. 4-(Bromomethyl)-20-chloro-40-(trifluoromethoxy)-
1,10-biphenyl (148). HBr in AcOH (5 mL of 33% w/w) was added
to a solution of alcohol 143 (618 mg, 2.04 mmol) in glacial AcOH
(2.5 mL), and the mixture was stirred at room temperature for
11 h. The resulting orange solution was added slowly to ice-
water (50 mL) with stirring, and then the mixture was extracted
with pentane (6 ꢀ 50 mL). The extracts were washed with
ice-water (50 mL) and then evaporated to give 148 (743 mg,
100%) as an oil: 1H NMR (CDCl3) δ 7.47 (dt, J = 8.3, 1.9 Hz,
2 H), 7.39 (dt, J = 8.3, 1.9 Hz, 2 H), 7.37 (m, 1 H), 7.35 (d, J =
8.5 Hz, 1 H), 7.19 (m, 1 H), 4.55 (s, 2 H); HREIMS calcd for
C14H9BrClF3O m/z (Mþ) 367.9427, 365.9457, 363.9477, found
367.9428, 365.9453, 363.9485.
on silica gel, eluting with 0-1% EtOAc/CH2Cl2 (foreruns) and
then 1-2% EtOAc/CH2Cl2, gave 100 (79%) as a light yellow
solid: mp (CH2Cl2/hexane) 149-151 °C; 1H NMR (CDCl3)
δ 7.52 (dq, J = 8.3, 1.7 Hz, 2 H), 7.44 (t, J = 8.5 Hz, 1 H), 7.40
(br d, J = 8.4 Hz, 2 H), 7.38 (s, 1 H), 7.13-7.04 (m, 2 H), 4.79
(d, J = 12.1 Hz, 1 H), 4.67 (d, J = 12.1 Hz, 1 H), 4.64 (ddd, J =
12.1, 3.7, 2.1 Hz, 1 H), 4.37 (dd, J = 12.1, 1.5 Hz, 1 H),
4.23-4.11 (m, 3 H). Anal. (C20H15F4N3O5) C, H, N.
(6S)-6-{[30-Chloro-40-(trifluoromethoxy)[1,10-biphenyl]-4-yl]-
methoxy}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(106). Reaction of alcohol 122 with bromide 150 (0.99 equiv)
using procedure C, followed by chromatography of the product
on silica gel, eluting with CH2Cl2 (foreruns) and then 0-2%
EtOAc/CH2Cl2, gave 106 (70%) as a pale yellow solid: mp
(CH2Cl2/hexane) 150-151 °C; 1H NMR (CDCl3) δ 7.67 (d, J =
2.2 Hz, 1 H), 7.54 (dt, J = 8.2, 1.7 Hz, 2 H), 7.47 (dd, J = 8.5, 2.2
Hz, 1 H), 7.43-7.36 (m, 4 H), 4.78 (d, J = 12.0 Hz, 1 H), 4.67 (d,
J = 12.0 Hz, 1 H), 4.63 (ddd, J = 12.1, 3.6, 2.1 Hz, 1 H), 4.37
(dd, J = 12.1, 1.1 Hz, 1 H), 4.23-4.11 (m, 3 H). Anal. (C20H15-
ClF3N3O5) C, H, N.
(6S)-6-{[40-Chloro-30-(trifluoromethoxy)[1,10-biphenyl]-4-yl]-
methoxy}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(107). Reaction of alcohol 122 with bromide 151 (1.00 equiv)
using procedure C, followed by chromatography of the pro-
duct on silica gel, eluting with CH2Cl2 (foreruns) and then
2% EtOAc/CH2Cl2, gave 107 (78%) as a cream solid: mp
(CH2Cl2/pentane) 168-171 °C; 1H NMR (CDCl3) δ 7.57-
7.51 (m, 3 H), 7.50 (m, 1 H), 7.44 (dd, J = 8.3, 2.1 Hz, 1 H),
7.40 (br d, J = 8.2 Hz, 2 H), 7.38 (s, 1 H), 4.78 (d, J = 12.0 Hz,
1 H), 4.67 (d, J = 12.2 Hz, 1 H), 4.63 (ddd, J = 12.1, 3.7, 2.1 Hz,
1 H), 4.36 (br d, J = 12.1 Hz, 1 H), 4.23-4.11 (m, 3 H). Anal.
(C20H15ClF3N3O5) C, H, N.
4-(Bromomethyl)-20-fluoro-40-(trifluoromethoxy)-1,10-biphenyl
(149). Bromination of alcohol 144 using procedure B for 9 h gave
149 (100%) as a cream solid that was used directly in the next
step: 1H NMR (CDCl3) δ 7.52-7.42 (m, 5 H), 7.13-7.03
(m, 2 H), 4.54 (s, 2 H); HRAPCIMS calcd for C14H9F4O m/z
[M - Br]þ 269.0584, found 269.0574.
4-(Bromomethyl)-30-chloro-40-(trifluoromethoxy)-1,10-biphenyl
(150). Bromination of alcohol 145 using procedure B for 10 h
gave 150 (100%) as a cream solid that was used directly in the
next step: 1H NMR (CDCl3) δ 7.67 (d, J = 2.2 Hz, 1 H), 7.52 (dt,
J = 8.5, 2.2 Hz, 2 H), 7.48 (dt, J = 8.6, 2.3 Hz, 2 H), 7.48 (dd,
J = 8.6, 2.2 Hz, 1 H), 7.38 (dq, J = 8.5, 1.4 Hz, 1 H), 4.54
(s, 2 H); HRAPCIMS calcd for C14H9ClF3O m/z [M - Br]þ
287.0260, 285.0289, found 287.0264, 285.0291.
(6S)-6-{[30-Fluoro-40-(trifluoromethoxy)[1,10-biphenyl]-4-yl]-
methoxy}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(111). Reaction of alcohol 122 with bromide 152 (0.99 equiv)
using procedure C, followed by chromatography of the product
on silica gel, eluting with 0-2% EtOAc/CH2Cl2 (foreruns) and
then 2% EtOAc/CH2Cl2, gave 111 (76%) as a cream solid: mp
4-(Bromomethyl)-40-chloro-30-(trifluoromethoxy)-1,10-biphenyl
(151). Bromination of alcohol 146 using procedure B for 6 h gave
151 (100%) as a cream solid that was used directly in the next
step: 1H NMR (CDCl3) δ 7.55-7.46 (m, 6 H), 7.45 (dd, J = 8.3,
2.1 Hz, 1 H), 4.54 (s, 2 H); HRAPCIMS calcd for C14H9ClF3O
m/z [M - Br]þ 287.0260, 285.0289, found 287.0249, 285.0289.
4-(Bromomethyl)-30-fluoro-40-(trifluoromethoxy)-1,10-biphenyl
(152). Bromination of alcohol 147 using procedure B for 6 h gave
152 (100%) as a cream solid that was used directly in the next
step: 1H NMR (CDCl3) δ 7.52 (dt, J = 8.5, 2.2 Hz, 2 H), 7.48 (dt,
J = 8.5, 2.2 Hz, 2 H), 7.43-7.32 (m, 3 H), 4.54 (s, 2 H);
HRAPCIMS calcd for C14H9F4O m/z [M - Br]þ 269.0584,
found 269.0572.
1
(CH2Cl2/pentane) 169-171 °C; H NMR (CDCl3) δ 7.54 (dt,
J = 8.3, 1.8 Hz, 2 H), 7.43-7.32 (m, 6 H), 4.78 (d, J = 12.0 Hz,
1 H), 4.67 (d, J = 11.9 Hz, 1 H), 4.64 (ddd, J = 12.1, 3.7, 2.1 Hz,
1 H), 4.37 (dd, J = 12.1, 1.3 Hz, 1 H), 4.23-4.12 (m, 3 H). Anal.
(C20H15F4N3O5) C, H, N.
(6S)-6-{[40-Chloro-30-(difluoromethoxy)[1,10-biphenyl]-4-yl]-
methoxy}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(113). A mixture of 4-(methoxycarbonyl)phenylboronic acid
(153) (1.00 g, 5.56 mmol) and 5-bromo-2-chlorophenol (1.15
g, 5.54 mmol) in aqueous K2CO3 (5 mL of 2 M, 10 mmol), EtOH
(15 mL), and toluene (25 mL) was purged with N2. Pd(dppf)Cl2
(50 mg, 0.068 mmol) was added, and the mixture was refluxed
under N2 for 0.5 h and then partitioned between EtOAc and 0.1
M HCl. Column chromatography of the organic extract (eluting
with CH2Cl2) gave methyl 40-chloro-30-hydroxy[1,10-biphenyl]-
4-carboxylate (154) (1.177 g, 81%) as a white solid: mp 162-
164 °C; 1H NMR [(CD)3SO] δ 10.36 (br s, 1 H), 8.03 (d, J = 8.5
Hz, 2 H), 7.73 (d, J = 8.5 Hz, 2 H), 7.44 (d, J = 8.3 Hz, 1 H), 7.27
(d, J = 2.2 Hz, 1 H), 7.17 (dd, J = 8.3, 2.2 Hz, 1 H), 3.88 (s, 3 H).
APCI MS m/z 263, 265 [M þ H]þ.
Procedure C. (6S)-6-{[20-Chloro-40-(trifluoromethoxy)[1,10-
biphenyl]-4-yl]methoxy}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b]-
[1,3]oxazine (98). A stirred solution of (6S)-2-nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]oxazin-6-ol13 (122) (342 mg, 1.85 mmol)
and bromide 148 (741 mg, 2.03 mmol) in anhydrous DMF
(7 mL) under N2 at 0 °C was treated with 60% NaH (111 mg,
2.78 mmol) and then quickly degassed and resealed under N2.
After stirring at room temperature for 3 h, the reaction was
cooled (CO2/acetone), quenched with ice/aqueous NaHCO3
(20 mL), added to brine (80 mL), and extracted with CH2Cl2
(6ꢀ80 mL). The combined extracts were evaporated to dryness,
and the residue was chromatographed on silica gel, eluting with
CH2Cl2, to give 98 (694 mg, 80%) as a light yellow solid: mp
A mixture of 154 (1.119 g, 4.26 mmol), K2CO3 (0.735 g, 5.32
mmol), and sodium chlorodifluoroacetate (1.30 g, 8.52 mmol) in
anhydrous DMF (10 mL) was stirred at 80 °C under N2 for 14 h.
The mixture was partitioned between EtOAc and water, and the
organic fraction was dried and evaporated. Column chromato-
graphy (eluting with 1:1 hexanes:CH2Cl2) gave methyl 40-
chloro-30-(difluoromethoxy)[1,10-biphenyl]-4-carboxylate (155)
(0.493 g, 37%) as a white solid: mp 80-81 °C; 1H NMR (CDCl3)
δ 8.12 (d, J = 8.5 Hz, 2 H), 7.61 (d, J = 8.5 Hz, 2 H), 7.54 (d, J =
8.3 Hz, 1 H), 7.49-7.47 (m, 1 H), 7.43 (dd, J = 8.3, 2.1 Hz, 1 H),
6.60 (t, JH-F = 73.4 Hz, 1 H), 3.95 (s, 3 H). APCI MS m/z 313,
315 [M þ H]þ.
1
(CH2Cl2/pentane) 80-82 °C; H NMR (CDCl3) δ 7.45-7.35
(m, 6 H), 7.34 (d, J = 8.5 Hz, 1 H), 7.20 (m, 1 H), 4.79 (d, J =
12.0 Hz, 1 H), 4.68 (d, J = 12.1 Hz, 1 H), 4.65 (ddd, J = 12.1,
2.9, 2.5 Hz, 1 H), 4.37 (br d, J = 11.8 Hz, 1 H), 4.24-4.12 (m,
3 H). Anal. (C20H15ClF3N3O5) C, H, N.
(6S)-6-{[20-Fluoro-40-(trifluoromethoxy)[1,10-biphenyl]-4-yl]-
methoxy}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(100). Reaction of alcohol 122 with bromide 149 (0.99 equiv)
using procedure C, followed by chromatography of the product

292 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Palmer et al.
LiAlH4 (80 mg, 2.11 mmol) was added to a solution of 155
(0.314 g, 1.00 mmol) in Et2O (20 mL) at 0 °C. The mixture was
stirred at room temperature for 2 h, cooled to 0 °C, quenched
with ice, and then filtered through Celite. The organic frac-
tion was dried and evaporated, and then column chromato-
graphy (eluting with 9:1 CH2Cl2:Et2O) gave [40-chloro-
30-(difluoromethoxy)[1,10-biphenyl]-4-yl]methanol (156) (0.286
solution of alcohol 27 (0.436 g, 1.02 mmol) and Et3N (0.43 mL,
3.09 mmol) in THF (40 mL) at 0 °C. The mixture was stirred at
0 °C for 1 h, then diluted with EtOAc, washed with saturated
aqueous NaHCO3, and dried, and the solvent was removed to
give a crude mesylate. This mesylate was then refluxed with NaI
(1.54 g, 10.3 mmol) in Me2CO (100 mL) for 3 h. The solvent was
removed under reduced pressure, and the residue was parti-
tioned between EtOAc and water. The organic portion was
dried and evaporated, and the residue was chromatographed
(eluting with EtOAc) to give 158 (0.426 g, 78%) as a pale yellow
glass, which was used directly in the next step: 1H NMR δ 8.01
(s, 1 H), 7.61-7.57 (m, 2 H), 7.43 (t, J = 7.8 Hz, 1 H), 7.36 (t, J =
7.9 Hz, 1 H), 7.32 (d, J = 7.6 Hz, 1 H), 7.23-7.16 (m, 2 H), 6.96
(dd, J = 8.1, 4.9 Hz, 1 H), 4.76-4.65 (m, 3 H), 4.48 (d, J = 11.8
Hz, 1 H), 4.32-4.21 (m, 3 H), 4.10 (t, J = 6.0 Hz, 2 H), 3.42 (t,
J = 6.9 Hz, 2 H), 2.23 (tt, J = 6.9, 6.0 Hz, 2 H). APCI MS m/z
536 [M þ H]þ.
1
g, 100%) as a white solid: mp 50-51 °C; H NMR (CDCl3) δ
7.54 (d, J = 8.3 Hz, 2 H), 7.50 (d, J = 8.3 Hz, 1 H), 7.47-7.42
(m, 3 H), 7.39 (dd, J = 8.3, 2.1 Hz, 1 H), 6.58 (t, JH-F = 73.5 Hz,
1 H), 4.75 (d, J = 5.9 Hz, 2 H), 1.68 (t, J = 5.9 Hz, 1 H). APCI
MS m/z 285, 287 [M þ H]þ.
PBr3 (45 μL, 0.48 mmol) was added to a solution of 156 (0.271 g,
0.952 mmol) in Et2O (10 mL) at 0 °C. The mixture was
stirred at room temperature for 3 h, cooled to 0 °C, and
quenched with ice. The organic layer was washed with aqueous
NaHCO3, dried, and evaporated. Column chromatography
(eluting with 1:1 hexanes:CH2Cl2) gave 4-(bromomethyl)-
40-chloro-30-(difluoromethoxy)-1,10-biphenyl (157) (0.271 g,
82%) as white flakes, which was used directly in the next step:
Procedure F. (6S)-6-({30-[3-(4-Morpholinyl)propoxy][1,10-bi-
phenyl]-3-yl}methoxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b]-
[1,3]oxazine (28). A solution of iodide 158 (85 mg, 0.16 mmol)
and morpholine (70 μL, 0.80 mmol) in DMA (3 mL) was stirred
at room temperature for 16 h. The resulting mixture was
partitioned (EtOAc/water), the organic fraction was dried,
and then removal of the solvent gave a solid, which was
triturated in Et2O to give 28 (76 mg, 97%) as a pale yellow
1
mp (hexanes) 60-61 °C; H NMR (CDCl3) δ 7.54-7.43 (m,
6 H), 7.38 (dd, J = 8.3, 2.1 Hz, 1 H), 6.58 (t, JH-F = 73.5 Hz,
1 H), 4.54 (s, 2 H).
NaH (60%) (20 mg, 0.5 mmol) was added to a solution of 157
(0.120 g, 0.35 mmol) and 122 (0.064 g, 0.35 mmol) in anhydrous
DMF (7 mL) at 0 °C. The mixture was stirred at room
temperature for 2 h and then quenched with water and parti-
tioned between EtOAc and water. The organic fraction was
dried and evaporated, and then column chromatography using a
gradient (1:1 hexanes:EtOAc to EtOAc), followed by Et2O
trituration, gave 113 (0.117 g, 75%) as a white solid: mp
1
solid: mp 92-95 °C; H NMR δ 8.01 (s, 1 H), 7.60-7.55 (m,
2 H), 7.43 (br t, J = 7.7 Hz, 1 H), 7.37-7.29 (m, 2 H), 7.17 (br d,
J = 7.7 Hz, 1 H), 7.16 (t, J = 2.2 Hz, 1 H), 7.14 (ddd, J = 8.2,
2.5, 0.7 Hz, 1 H), 4.75-4.64 (m, 3 H), 4.48 (d, J = 11.8 Hz, 1 H),
4.32-4.20 (m, 3 H), 4.08 (t, J = 6.3 Hz, 2 H), 3.57 (t, J = 4.6 Hz,
4 H), 2.44 (t, J = 7.1 Hz, 2 H), 2.41-2.33 (m, 4 H), 1.89 (tt,
1
159-161 °C; H NMR δ 8.02 (s, 1 H), 7.71-7.64 (m, 3 H),
J = 7.1, 6.3 Hz, 2 H). Anal. (C26H30N4O6 0.5H2O) C, H, N.
3
7.62-7.55 (m, 2 H), 7.43 (d, J = 8.2 Hz, 2 H), 7.40 (t, JH-F
=
3-{[30-({[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl]oxy}methyl)[1,10-biphenyl]-4-yl]oxy}-1-propanol (37). Reac-
tion of phenol 34 (prepared by Suzuki coupling; see Supporting
Information) with (3-bromopropoxy)(tert-butyl)dimethylsilane
and Cs2CO3 in DMF, followed by desilylation with TBAF,
using procedure D, gave 37 (51%) as a white solid: mp 160-
162 °C; 1H NMR δ 8.02 (s, 1 H), 7.57-7.49 (m, 4 H), 7.40 (t, J =
7.6 Hz, 1 H), 7.25 (br d, J = 7.6 Hz, 1 H), 7.01 (d, J = 8.8 Hz,
2 H), 4.74-4.63 (m, 3 H), 4.53 (t, J = 5.2 Hz, 1 H), 4.48 (d, J =
11.7 Hz, 1 H), 4.32-4.19 (m, 3 H), 4.08 (t, J = 6.2 Hz, 2 H), 3.57
(td, J = 5.9, 5.2 Hz, 2 H), 1.88 (tt, J = 6.2, 5.9 Hz, 2 H). Anal.
(C22H23N3O6) C, H, N.
73.3 Hz, 1 H), 4.75-4.64 (m, 3 H), 4.48 (d, J = 11.9 Hz, 1 H),
4.32-4.21 (m, 3 H). Anal. (C20H16ClF2N3O5) C, H, N.
Preparation of Substituted Alkoxy Biphenyl Analogues
(Scheme 4). 30-({[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b]-
[1,3]oxazin-6-yl]oxy}methyl)[1,10-biphenyl]-3-ol (25). Reaction
of iodide 126 and 3-hydroxyphenylboronic acid under the
Suzuki conditions described above gave 25 (83%) as a tan solid:
mp 80-83 °C; 1H NMR δ 9.48 (s, 1 H), 8.02 (s, 1 H), 7.53-7.49
(m, 2 H), 7.42 (t, J = 7.5 Hz, 1 H), 7.29 (d, J = 7.5 Hz, 1 H), 7.24
(t, J = 7.8 Hz, 1 H), 7.04-6.98 (m, 2 H), 6.77 (ddd, J = 8.1, 2.4,
0.8 Hz, 1 H), 4.75-4.64 (m, 3 H), 4.48 (d, J = 11.9 Hz, 1 H),
4.32-4.21 (m, 3 H). Anal. (C19H17N3O5 0.25EtOAc) C, H, N.
(6S)-6-{[40-(3-Iodopropoxy)[1,10-biphenyl]-3-yl]methoxy}-2-
nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (159). Mesyla-
tion of alcohol 37, followed by reaction with NaI in Me2CO,
using procedure E, gave 159 (84%) as a glass, which was used
directly in the next step: 1H NMR δ 8.02 (s, 1 H), 7.58-7.50 (m, 4
H), 7.40 (t, J = 7.6 Hz, 1 H), 7.25 (br d, J = 7.6 Hz, 1 H), 7.02 (d,
J = 8.8 Hz, 2 H), 4.73-4.62 (m, 3 H), 4.48 (d, J = 11.7 Hz, 1 H),
4.33-4.20 (m, 3 H), 4.07 (t, J = 6.0 Hz, 2 H), 3.41 (t, J = 6.8 Hz,
2 H), 2.22 (tt, J = 6.8, 6.0 Hz, 2 H). APCI MS m/z 536 [M þ H]þ.
(6S)-6-({40-[3-(4-Morpholinyl)propoxy][1,10-biphenyl]-3-yl}-
methoxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (38).
Reactionof iodide 159 with morpholinein DMA usingprocedure
3
Procedure D. 3-{[30-({[(6S)-2-Nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazin-6-yl]oxy}methyl)[1,10-biphenyl]-3-yl]oxy}-1-
propanol (27). A mixture of phenol 25 (0.737 g, 2.01 mmol), (3-
bromopropoxy)(tert-butyl)dimethylsilane (0.740 g, 2.92 mmol),
and Cs2CO3 (3.2 g, 9.8 mmol) in anhydrous DMF (20 mL) was
stirred at 90 °C for 2 h. The resulting mixture was partitioned
between EtOAc and water, the organic fraction was dried, and
the solvent was removed under reduced pressure to give an oil,
which was dissolved in THF (100 mL) and treated with TBAF
(1 M in THF, 10 mL). The solution was stirred at room
temperature for 2 h, then the solvent was removed, and the
residue was partitioned between EtOAc and water. The organic
portion was washed with water, dried, and evaporated, and the
resulting oil was purified by column chromatography (eluting
with EtOAc) to give 27 (0.477 g, 56%) as a gum: 1H NMR δ 8.01
(s, 1 H), 7.60-7.55 (m, 2 H), 7.43 (t, J = 8.2 Hz, 1 H), 7.37-7.29
(m, 2 H), 7.20-7.13 (m, 2 H), 6.93 (ddd, J = 8.2, 2.5, 0.7 Hz,
1 H), 4.75-4.63 (m, 3 H), 4.52 (t, J = 5.2 Hz, 1 H), 4.47 (d, J =
11.7 Hz, 1 H), 4.33-4.20 (m, 3 H), 4.10 (t, J = 6.4 Hz, 2 H), 3.58
(td, J = 5.9, 5.2 Hz, 2 H), 1.89 (tt, J = 6.4, 5.9 Hz, 2 H);
HRFABMS calcd for C22H24N3O6 m/z [M þ H]þ 426.1665,
found 426.1673. HPLC purity: 93.2%.
1
F gave 38 (91%) as a white solid: mp 203-206 °C; H NMR
δ 8.02 (s, 1 H), 7.56-7.49 (m, 4 H), 7.40 (t, J = 7.6 Hz, 1 H), 7.24
(br d, J = 7.6 Hz, 1 H), 6.99 (d, J = 8.8 Hz, 2 H), 4.73-4.63 (m, 3
H), 4.48 (d, J = 11.8 Hz, 1 H), 4.32-4.20 (m, 3 H), 4.05 (t, J = 6.4
Hz, 2 H), 3.58 (t, J = 4.6 Hz, 4 H), 2.43 (t, J = 7.3 Hz, 2 H),
2.40-2.33 (m, 4 H), 1.89 (tt, J = 7.3, 6.4 Hz, 2 H). Anal. (C26H30-
N4O6) C, H, N.
3-{[40-({[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl]oxy}methyl)[1,10-biphenyl]-2-yl]oxy}-1-propanol (50). Reac-
tion of phenol 47 (prepared by Suzuki coupling; see Supporting
Information) with (3-bromopropoxy)(tert-butyl)dimethylsilane
and Cs2CO3 in DMF, followed by desilylation with TBAF (2.4
equiv), using procedure D, gave 50 (52%) as a gum: 1H NMR δ
8.03 (s, 1 H), 7.48 (d, J = 8.3 Hz, 2 H), 7.33 (d, J = 8.3 Hz, 2 H),
Procedure E. (6S)-6-{[30-(3-Iodopropoxy)[1,10-biphenyl]-3-yl]-
methoxy}-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(158). Mesyl chloride (0.16 mL, 2.04 mmol) was added to a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 293
7.31-7.25 (m, 2 H), 7.09 (br d, J = 7.6 Hz, 1 H), 7.00 (td, J =
7.4, 1.0 Hz, 1 H), 4.72-4.63 (m, 3 H), 4.49 (d, J = 11.9 Hz, 1 H),
4.45 (t, J = 5.2 Hz, 1 H), 4.33-4.21 (m, 3 H), 4.04 (t, J = 6.2 Hz,
2 H), 3.48 (td, J = 5.9, 5.2 Hz, 2 H), 1.78 (tt, J = 6.2, 5.9 Hz, 2
H); HRFABMS calcd for C22H24N3O6 m/z [M þ H]þ 426.1665,
found 426.1659. HPLC purity: 95.5%.
Information) (0.600 g, 1.20 mmol), 2-(2-bromoethoxy)tetra-
hydro-2H-pyran (0.68 g, 3.25 mmol), and Cs2CO3 (2.66 g,
8.16 mmol) in DMF (15 mL) was stirred at 90 °C for 2 h. The
resulting mixture was partitioned (EtOAc/water), the organic
fraction was evaporated, and the residue was chromatographed
(eluting with EtOAc) to give a crude THP ether (0.649 g, 80%).
This ether (0.573 g) was dissolved in MeOH (100 mL), treated
with MsOH (10 drops), and then stirred at room temperature for
1 h. The resulting solution was filtered through a plug of
NaHCO3, and the solvent was removed under reduced pressure.
Chromatography of the residue (eluting with EtOAc) gave 89
(0.410 g, 86%) as a white solid: mp 209-212 °C; 1H NMR δ 8.02
(s, 1 H), 7.61-7.55 (m, 4 H), 7.36 (d, J = 8.3 Hz, 2 H), 7.01 (d,
J = 8.8 Hz, 2 H), 4.85 (t, J = 5.5 Hz, 1 H), 4.71-4.62 (m, 3 H),
4.48 (d, J = 11.9 Hz, 1 H), 4.32-4.21 (m, 3 H), 4.02 (t, J =
4.9 Hz, 2 H), 3.73 (dt, J = 5.5, 4.9 Hz, 2 H). Anal. (C21H21N3O6)
C, H, N.
3-{[40-({[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl]oxy}methyl)[1,10-biphenyl]-4-yl]oxy}-1-propanol (90). Reac-
tion of phenol 85 with (3-bromopropoxy)(tert-butyl)dimethyl-
silane (2.0 equiv) and Cs2CO3 in DMF for 1 h, followed by
desilylation with TBAF (2.0 equiv), using procedure D, gave 90
(61%) as a white solid: mp 206-209 °C; 1H NMR δ 8.02 (s, 1 H),
7.61-7.55 (m, 4 H), 7.36 (d, J = 8.3 Hz, 2 H), 7.00 (d, J = 8.8
Hz, 2 H), 4.71-4.62 (m, 3 H), 4.52 (t, J = 5.2 Hz, 1 H), 4.47 (d,
J = 11.9 Hz, 1 H), 4.31-4.21 (m, 3 H), 4.07 (t, J = 6.4 Hz, 2 H),
3.56 (td, J = 5.9, 5.2 Hz, 2 H), 1.87 (tt, J = 6.4, 5.9 Hz, 2 H).
Anal. (C22H23N3O6) C, H, N.
2-{[40-({[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl]oxy}methyl)[1,10-biphenyl]-3-yl]oxy}ethanol (64). A mixture
of phenol 62 (prepared by Suzuki coupling; see Supporting
Information) (0.440 g, 1.20 mmol), 2-(2-bromoethoxy)tetra-
hydro-2H-pyran (0.500 g, 2.39 mmol), and Cs2CO3 (1.95 g,
5.98 mmol) in anhydrous DMF (10 mL) was stirred at 90 °C for
2 h. The resulting mixture was partitioned between EtOAc and
water, the organic fraction was evaporated, and the residue was
chromatographed (eluting with EtOAc) to give a crude THP
ether. This ether was dissolved in MeOH (100 mL), treated with
MsOH (5 drops), and then stirred at room temperature for 1 h.
The resulting solution was filtered through a plug of NaHCO3,
and the solvent was removed under reduced pressure. Chromato-
graphy of the residue (eluting with EtOAc) gave 64 (0.400 g,
67%) as a pale yellow glass: 1H NMR δ 8.03 (s, 1 H), 7.63 (d, J =
8.3 Hz, 2 H), 7.38 (d, J = 8.3 Hz, 2 H), 7.36 (t, J = 8.0 Hz, 1 H),
7.20 (br d, J = 7.7 Hz, 1 H), 7.17 (t, J = 2.2 Hz, 1 H), 6.93 (ddd,
J = 8.2, 2.5, 0.7 Hz, 1 H), 4.83 (t, J = 5.5 Hz, 1 H), 4.73-4.63 (m,
3 H), 4.48 (d, J = 11.9 Hz, 1 H), 4.33-4.21 (m, 3 H), 4.06 (t,
J = 4.9 Hz, 2 H), 3.74 (dt, J = 5.5, 4.9 Hz, 2 H); HRFABMS
calcd for C21H22N3O6 m/z [M þ H]þ 412.1509, found 412.1514.
HPLC purity: 96.8%.
3-{[40-({[(6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl]oxy}methyl)[1,10-biphenyl]-3-yl]oxy}-1-propanol (65). Reac-
tion of phenol 62 with (3-bromopropoxy)(tert-butyl)dimethyl-
silane (2.5 equiv) and Cs2CO3 in DMF for 1 h, followed by
desilylation with TBAF (2.1 equiv), using procedure D, and
chromatography of the product on silica gel, eluting with a
gradient of 1:1 hexanes:EtOAc to EtOAc, gave 65 (66%) as an
oil: 1H NMR δ 8.03 (s, 1 H), 7.64 (d, J = 8.3 Hz, 2 H), 7.39 (d,
J = 8.3 Hz, 2 H), 7.35 (dd, J = 8.2, 7.9 Hz, 1 H), 7.19 (br d, J =
7.9 Hz, 1 H), 7.16 (t, J = 2.2 Hz, 1 H), 6.92 (ddd, J = 8.2, 2.2, 0.7
Hz, 1 H), 4.73-4.64 (m, 3 H), 4.52 (t, J = 5.2 Hz, 1 H), 4.48 (d,
J = 11.9 Hz, 1 H), 4.32-4.21 (m, 3 H), 4.10 (t, J = 6.4 Hz, 2 H),
3.57 (td, J = 5.9, 5.2 Hz, 2 H), 1.88 (tt, J = 6.4, 5.9 Hz, 2 H);
HRFABMS calcd for C22H24N3O6 m/z [M þ H]þ 426.1665,
found 426.1659. HPLC purity: 95.7%.
(6S)-6-{[40-(3-Iodopropoxy)[1,10-biphenyl]-4-yl]methoxy}-2-
nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (161). Mesyla-
tion of alcohol 90 for 2.5 h, followed by reaction with NaI in
Me2CO for 1 h, using procedure E, gave 161 (75%) as a white
solid: mp 146-148 °C; 1H NMR δ 8.02 (s, 1 H), 7.61-7.55 (m,
4 H), 7.36 (d, J = 8.3 Hz, 2 H), 7.03 (d, J = 8.8 Hz, 2 H),
4.70-4.62 (m, 3 H), 4.47 (d, J = 11.9 Hz, 1 H), 4.30-4.20 (m,
3 H), 4.07 (t, J = 6.0 Hz, 2 H), 3.40 (t, J = 6.8 Hz, 2 H), 2.21 (tt,
J = 6.8, 6.0 Hz, 2 H). APCI MS m/z 536 [M þ H]þ.
(6S)-6-({40-[3-(4-Morpholinyl)propoxy][1,10-biphenyl]-4-yl}-
methoxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (91).
Reaction of iodide 161 with morpholine using procedure
1
F gave 91 (89%) as a white solid: mp 221-224 °C; H NMR
δ 8.02 (s, 1 H), 7.60-7.54 (m, 4 H), 7.36 (d, J = 8.3 Hz, 2 H),
7.00 (d, J = 8.8 Hz, 2 H), 4.70-4.62 (m, 3 H), 4.47 (d, J = 11.9
Hz, 1 H), 4.32-4.20 (m, 3 H), 4.05 (t, J = 6.4 Hz, 2 H), 3.57
(t, J = 4.6 Hz, 4 H), 2.42 (t, J = 7.3 Hz, 2 H), 2.40-2.33
(m, 4 H), 1.88 (tt, J = 7.3, 6.4 Hz, 2 H). Anal. (C26H30-
(6S)-6-{[30-(2-Iodoethoxy)[1,10-biphenyl]-4-yl]methoxy}-2-nitro-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (160). Mesylation of
alcohol 64, followed by reaction with NaI in Me2CO, using
procedure E, gave 160 (80%) as a foam, which was used directly
in the next step: 1H NMR δ 8.03 (s, 1 H), 7.65 (d, J = 8.3 Hz,
2 H), 7.42-7.35 (m, 3 H), 7.24 (br d, J = 8.3 Hz, 1 H), 7.18 (m,
1 H), 6.95 (ddd, J = 8.2, 2.5, 0.7 Hz, 1 H), 4.72-4.65 (m, 3 H),
4.48 (d, J = 11.9 Hz, 1 H), 4.34 (t, J = 6.1 Hz, 2 H), 4.32-4.21
(m, 3 H), 3.54 (t, J = 6.1 Hz, 2 H). APCI MS m/z 522 [M þ H]þ.
N,N-Dimethyl-2-{[40-({[(6S)-2-nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazin-6-yl]oxy}methyl)[1,10-biphenyl]-3-yl]oxy}ethana-
mine (66). A solution of iodide 160 (0.105 g, 0.202 mmol) and
dimethylamine (∼0.1 mL of a 40% aqueous solution) in DMA
(2 mL) was stirred at room temperature for 16 h in a sealed tube.
The resulting mixture was partitioned (EtOAc/water), the or-
ganic fraction was dried, and then removal of the solvent gave a
solid, which was triturated in Et2O to give 66 (58 mg, 65%) as a
white solid: mp 93-96 °C; 1H NMR δ 8.03 (s, 1 H), 7.64 (d, J =
8.3 Hz, 2 H), 7.39 (d, J = 8.3 Hz, 2 H), 7.35 (t, J = 8.0 Hz, 1 H),
7.19 (br d, J = 7.7 Hz, 1 H), 7.17 (t, J = 2.2 Hz, 1 H), 6.93 (ddd,
J = 8.2, 2.5, 0.7 Hz, 1 H), 4.73-4.64 (m, 3 H), 4.48 (d, J = 11.9
Hz, 1 H), 4.32-4.21 (m, 3 H), 4.11 (t, J = 5.8 Hz, 2 H), 2.64 (t,
N4O6 0.25H2O) C, H, N.
Biology. MABA and LORA Tuberculosis Assays. These were
3
carried out according to the published protocols.21-23
Microsomal Stability Assays. Compounds (1 μM) were in-
cubated with pooled human or CD-1 mouse liver microsome
preparations (0.5 mg/mL final protein concentration) and an
NADPH regenerating system (MgCl2, 3.3 mM; G6P, 3.3 mM;
G6PD, 0.4 unit/mL; NADPþ, 1.3 mM) in phosphate buffer
(75 mM, pH 7.4), with a final volume of 200 μL. Compounds
were dissolved in DMSO such that the final DMSO concentra-
tion was 0.5%. Positive controls (warfarin, propranolol, and
testosterone, incubated as a cocktail) were treated similarly.
Incubation was at 37 °C in a 96-well plate in a humidified
incubator, and reactions were stopped at 0 and 60 min by the
addition of MeCN (100 μL) containing metoprolol (0.2 μM) as
an internal standard. Samples were diluted 10ꢀ and centrifuged
prior to analysis by LC-MS/MS using electrospray ionization
and SRM monitoring using a gradient LC method (isocratic for
0.5 min, followed by a 5% to 91% MeCN linear gradient in
water containing 0.1% HCOOH over 1 min, with a 2.5 min hold
at 91% MeCN; total run time 6.5 min). An XDB-C18 2.1 ꢀ
50 mm column (Agilent) was employed, with an eluent flow rate
of 0.5 mL/min. LC peak areas were integrated and expressed as
analyte/IS peak area ratios (PAR), and a mean value for each
J = 5.8 Hz, 2 H), 2.22 (s, 6 H). Anal. (C23H26N4O5 0.5H2O) C,
3
H, N.
2-{[40-({[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl]oxy}methyl)[1,10-biphenyl]-4-yl]oxy}ethanol (89). A mixture
of phenol 85 (prepared by Suzuki coupling; see Supporting

294 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Palmer et al.
time point was calculated from the duplicates. The percent
remaining value was calculated as
(9) 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.
Identification of a nitroimidazo-oxazine-specific protein involved
in PA-824 resistance in Mycobacterium tuberculosis. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 431–436.
% remaining ¼ 100ðmean PART60=mean PART0
Þ
(10) Guillemont, J.; Lieby-Muller, F.; Lounis, N.; Balemans, W.; Koul,
A.; Andries, K. New anti-tuberculosis drugs in clinical develop-
ment: an overview. Curr. Bioactive Compd. 2009, 5, 137–154.
(11) Tam, C.-M.; Yew, W.-W.; Yuen, K.-Y. Treatment of multidrug-
resistant and extensively drug-resistant tuberculosis: current status
and future prospects. Exp. Rev. Clin. Pharmacol. 2009, 2, 405–421.
(12) Tasneen, R.; Tyagi, S.; Williams, K.; Grosset, J.; Nuermberger, E.
Enhanced bactericidal activity of rifampin and/or pyrazinamide
when combined with PA-824 in a murine model of tuberculosis.
Antimicrob. Agents Chemother. 2008, 52, 3664–3668.
(13) (a) Baker W. R.; Shaopei, C.; Keeler, E. L. Nitroimidazole anti-
bacterial compounds and methods of use thereof. U.S. Patent
5,668,127, 1997; (b) Nitro-[2,1-b]imidazopyran compounds and anti-
bacterial uses thereof. U.S. Patent 6,087,358, 2000.
(14) Li, X.; Manjunatha, U. H.; Goodwin, M. B.; Knox, J. E.; Lipinski,
C. A.; Keller, T. H.; Barry, C. E.; Dowd, C. S. 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. Bioorg. Med. Chem.
Lett. 2008, 18, 2256–2262.
The assays were conducted by MDS Pharma Services, 22011
30th Drive SE, Bothell, WA 98021.
In Vivo Acute TB Infection Assay. BALB/c mice were infected
via aerosol with a suspension of ∼2 ꢀ 106 colony forming units
(CFU) of M. tuberculosis Erdman/mL.23 Each compound was
given orally to a group of seven or eight mice at 100 mg/kg daily
for 5 days a week for 3 weeks, beginning on day 11 postinfection.
Compounds were administered as a suspension in 0.5% CMC/
0.08% Tween 80 in water. Mice were sacrificed on day 31, and
the numbers of CFU in the lungs were determined and com-
pared with the CFU for vehicle alone-treated mice at this time.23
The parent drug 1 was employed as a positive control in each
experiment, and the results are recorded as the ratio of the
average reduction in CFU in the compound-treated mice/the
average CFU reduction in the mice treated with 1. In this assay,
1 itself caused up to 2.5-3 log reductions in CFU.
(15) Kim, P.; Zhang, L.; Manjunatha, U. H.; Singh, R.; Patel, S.;
Jiricek, J.; Keller, T. H.; Boshoff, H. I.; Barry, C. E.; Dowd,
C. S. Structure-activity relationships of antitubercular nitroimida-
zoles. 1. Structural features associated with aerobic and anaerobic
activities of 4- and 5-nitroimidazoles. J. Med. Chem. 2009, 52,
1317–1328.
(16) 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. Structure-
activity relationships of antitubercular nitroimidazoles. 2. Deter-
minants of aerobic activity and quantitative structure-activity
relationships. J. Med. Chem. 2009, 52, 1329–1344.
Acknowledgment. The authors thank the Global Alliance
for Tuberculosis Drug Development for financial support
through a collaborative research agreement.
Supporting Information Available: Additional experimental
procedures and characterizations for compounds in Tables 1-3;
CFU data for compounds in Table 5; combustion analytical
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
(17) Anderson, R. F.; Shinde, S. S.; Maroz, A.; Boyd, M.; Palmer, B. D.;
Denny, W. A. Intermediates in the reductionof the antituberculosis
drug PA-824, (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine, in aqueous solution.
Org. Biomol. Chem. 2008, 6, 1973–1980.
(18) Singh, R.; Manjunatha, U.; Boshoff, H. I. M.; Ha, Y. H.; Niyom-
rattanakit, P.; Ledwidge, R.; Dowd, C. S.; Lee, I. Y.; Kim, P.;
Zhang, L.; Kang, S.; Keller, T. H.; Jiricek, J.; Barry, C. E. PA-824
kills nonreplicating Mycobacterium tuberculosis by intracellular
NO release. Science 2008, 322, 1392–1395.
(19) Ginsberg, A. M.; Laurenzi, M. W.; Rouse, D. J.; Whitney, K. D.;
Spigelman, M. K. Safety, tolerability, and pharmacokinetics of
PA-824 in healthy subjects. Antimicrob. Agents Chemother. 2009,
53, 3720–3725.
(20) O’Shea, P. D.; Chen, C.-Y.; Chen, W.; Dagneau, P.; Frey, L. F.;
Grabowski, E. J. J.; Marcantonio, K. M.; Reamer, R. A.; Tan, L.;
Tillyer, R. D.; Roy, A.; Wang, X.; Zhao, D. Practical asymmetric
synthesis of a potent PDE4 inhibitor via stereoselective enolate
alkylation of a chiral aryl-heteroaryl secondary tosylate. J. Org.
Chem. 2005, 70, 3021–3030.
(21) Collins, L. A.; Franzblau, S. G. Microplate Alamar blue assay
versus BACTEC 460 system for high-throughput screening of
compounds against Mycobacterium tuberculosis and Mycobacter-
ium avium. Antimicrob. Agents Chemother. 1997, 41, 1004–1009.
(22) Cho, S. H.; Warit, S.; Wan, B.; Hwang, C. H.; Pauli, G. F.;
Franzblau, S. G. Low-oxygen-recovery assay for high-throughput
screening of compounds against nonreplicating Mycobacterium
tuberculosis. Antimicrob. Agents Chemother. 2007, 51, 1380–1385.
(23) Falzari, K.; Zhu, Z.; Pan, D.; Liu, H.; Hongmanee, P.; Franzblau,
S. G. In vitro and in vivo activities of macrolide derivatives against
Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2005,
49, 1447–1454.
References
(1) World Health Organization (WHO) Tuberculosis fact sheet No.
104; revised March 2007.
(2) Laurenzi, M.; Ginsberg, A.; Spigelman, M. Challenges associated
with current and future TB treatment. Infect. Disord.;Drug
Targets 2007, 7, 105–119.
(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. A small-molecule nitroimidazopyran drug candidate
for the treatment of tuberculosis. Nature 2000, 405, 962–966.
(4) Sasaki, H.; Haraguchi, Y.; Itotani, M.; Kuroda, H.; Hashizume,
H.; Tomishige, T.; Kawasaki, M.; Matsumoto, M.; Komatsu,
M.; Tsubouchi, H. Synthesis and antituberculosis activity of
a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-
b]oxazoles. J. Med. Chem. 2006, 49, 7854–7860.
(5) Matsumoto, M.; Hashizume, H.; Tomishige, T.; Kawasaki, M.;
Tsubouchi, H.; Sasaki, H.; Shimokawa, Y.; Komatsu, M. OPC-
67683, a nitro-dihydro-imidazooxazole derivative with promising
action against tuberculosis in vitro and in mice. PLoS Med. 2006, 3,
2131–2143.
(6) Rivers, E. C.; Mancera, R. L. New anti-tuberculosis drugs with
novel mechanisms of action. Curr. Med. Chem. 2008, 15, 1956–
1967.
(7) Ma, Z.; Ginsberg, A. M.; Spigelman, M. Antimycobacterium
agents. In Comprehensive Medicinal Chemistry II; Taylor, J. B.;
Triggle, D. J., Eds.; Elsevier: Oxford, 2006; Vol. 7; pp 699-730.
(8) Thompson, A. M.; Blaser, A.; Anderson, R. F.; Shinde, S. S.;
Franzblau, S. G.; Ma, Z.; Denny, W. A.; Palmer, B. D. Synthesis,
reduction potentials, and antitubercular activity of ring A/B ana-
logues of the bioreductive drug (6S)-2-nitro-6-{[4-(trifluoro-
methoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(PA-824). J. Med. Chem. 2009, 52, 637–645.
(24) Barry, C. E.; Crick, D. C.; McNeil, M. R. Targeting the formation
of the cell wall core of M. tuberculosis. Infect. Disord.;Drug
Targets 2007, 7, 182–202.