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

Article abstract of DOI:10.1021/jm101288t

New heterocyclic analogues of the potent biphenyl class derived from antitubercular drug PA-824 were prepared, aiming to improve aqueous solubility but maintain high metabolic stability and efficacy. The strategy involved replacement of one or both phenyl

Full text of DOI:10.1021/jm101288t

J. Med. Chem. 2010, 53, 8421–8439 8421
DOI: 10.1021/jm101288t
Synthesis and Structure-Activity Relationships of Aza- and Diazabiphenyl Analogues of
the Antitubercular Drug (6S)-2-Nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,
7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824)
Iveta Kmentova,† Hamish S. Sutherland,† Brian D. Palmer,† Adrian Blaser,† Scott G. Franzblau,‡ Baojie Wan,‡
Yuehong Wang,‡ Zhenkun Ma,§ William A. Denny,† and Andrew M. Thompson*,†
†
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, United States, and Global Alliance for TB Drug Development, 40 Wall Street, New York, New York 10005, United States
§
Received October 6, 2010
New heterocyclic analogues of the potent biphenyl class derived from antitubercular drug PA-824 were
prepared, aiming to improve aqueous solubility but maintain high metabolic stability and efficacy. The
strategy involved replacement of one or both phenyl groups by pyridine, pyridazine, pyrazine, or pyrimi-
dine, in order to reduce lipophilicity. For para-linked biaryls, hydrophilicities (ClogP) correlated with
measured solubilities, but highly soluble bipyridine analogues displayed weak antitubercular activities. A
terminal pyridine or proximal heterocycle allowed retention of potency and provided solubility improve-
ments, particularly at low pH, with examples from the latter classes displaying the better in vivo efficacies,
high metabolic stabilities, and excellent pharmacokinetics. Five such compounds were >100-fold better
than the parent drug in a mouse model of acute Mycobacterium tuberculosis infection, and two orally
bioavailable pyridine analogues (3-4-fold more soluble than the parent at low pH) were superior to
antitubercular drug OPC-67683 in a chronic infection model.
Introduction
have TB, but treatment can be complicated by drug-drug
interactions and higher toxicities; poor treatment adherence
then leads todisease progression and drug resistance).1,3 More
effective, low-cost drugs are urgently required to shorten
treatment times and better meet the increasing challenges of
MDR-TB.1,3,4
The contagious airborne disease tuberculosis (TB)a has
reemerged as a serious health threat, fueled by the spread of
multidrug-resistant (MDR) strains and synergy with the HIV
pandemic, prompting the World Health Organization in 1993
to declare it a global emergency. Despite recent control efforts
based largely around DOTS (directly observed therapy short
course; using four drugs over 6-9 months), 9 million new TB
cases are reported annually (0.5 million MDR-TB), and many
die (mortality estimated at 1.8 million in 2008, including 0.5
million HIV-positive individuals).1,2 Furthermore, achieve-
ment of the Stop TB Partnership target of halving 1990 rates
of prevalence and mortality by 2015 has recently been de-
scribed as “appearing impossible in African countries” and
therefore “unlikely globally”, based on current trends.2 This is
largely a result of poverty, inadequate access to proper
therapy, and HIV coinfection (one-third of HIV sufferers also
The (6S)-2-nitroimidazo[2,1-b][1,3]oxazines, exemplified by
PA-824 (1),5 and the related 6-nitroimidazo[2,1-b][1,3]oxazoles,
exemplified by OPC-67683 (2),6 are highly efficacious against
both replicating and nonreplicating persistent forms of My-
cobacterium tuberculosis (M. tb, the causative agent of TB).
The latter activity reportedly arises from an unusual route of
reductive metabolism of the nitroimidazole ring,7 leading to
the release of nitric oxide as an active species.8 Compounds 1
and 2 and the even more lipophilic diarylquinoline TMC-207
(3)9 (Chart 1) are currently in phase II clinical trials for the
treatment of both drug-susceptible and drug-resistant TB.3,4
In a previous SAR study10 of biphenyl analogues of 1, we
showed that within this class there were positive correlations
between aerobic MIC potency and both overall compound
lipophilicity and the electron-withdrawing capability of
substituents on the terminal ring, with several analogues
(e.g., 4-6) showing markedly superior activity to 1, both in vitro
and in vivo. For example, 5 and 6 gave efficacy enhancements
over 1 of >205-fold and 419-fold in lung colony forming unit
(CFU) reduction, respectively, following oral dosing in a mouse
model of acute TB infection. The effectiveness of these highly
lipophilic agents (cf. 2, 3) may in part be related to their ability
to penetrate the exceptionally lipophilic cell wall of M. tb.11
Despite such encouraging results, we noted that the very
poor aqueous solubilities of the biphenyl analogues
*Corresponding author. Phone: (þ649) 923 6145. Fax: (þ649) 373
7502. E-mail: am.thompson@auckland.ac.nz.
a Abbreviations: TB, tuberculosis; MDR, multidrug-resistant; DOTS,
directly observed therapy short course; M. tb, Mycobacterium tuberculosis;
SAR, structure-activity relationship; MIC, minimum inhibitory concentra-
tion; CFU, colony forming unit; NBS, N-bromosuccinimide; DMF, N,N-
dimethylformamide; SD, standard deviation; AUC, area under the curve;
HREIMS, high-resolution electron impact mass spectrometry; HRFABMS,
high-resolution fast atom bombardment mass spectrometry; HRESIMS,
high-resolution electrospray ionization mass spectrometry; APCI MS, atmo-
spheric pressure chemical ionization mass spectrometry; THF, tetrahydro-
furan; AIBN, azobisisobutyronitrile; DME, dimethoxyethane; DMSO,
dimethyl sulfoxide; PBS, phosphate-buffered saline; PAR, peak area
ratio; IS, internal standard; TFAA, trifluoroacetic anhydride; NMM,
N-methylmorpholine.
r
2010 American Chemical Society
Published on Web 11/11/2010
pubs.acs.org/jmc

8422 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Chart 1. Structures of Antitubercular Agents
Kmentova et al.
Scheme 1a
(e.g., ,0.5 μg/mL for 5, .20-fold less than 112) would likely
limit their oral bioavailabilities at higher doses, reducing
their potential utility as TB drugs. Indeed, absorption issues
were noted during clinical trials of the similarly lipophilic 2 and
necessitated changes to its formulation.13 Furthermore, high
plasma binding (expected to be much greater for 5 and 6 than
the 94% found for 114), while providing the opportunity for
a longer half-life possibly suitable for intermittent adminis-
tration, may also be associated with lower than expected
activity in the clinical setting.15 Although dry powder aero-
sols show some promise for the alternative local drug delivery
of 1 (guinea pig model),16 such alternative routes of admin-
istration seem unlikely to overcome all of these challenges.
However, attempts to improve the aqueous solubility of the
biphenyl class simply with appended hydrophilic groups led
to a dramatic loss of potency (particularly in vivo).10
In an alternative solubilization strategy, we recently explored
the replacement of the proximal phenyl ring in the biaryl
side chain (closest to the ether linkage) with a variety of
more hydrophilic five-membered ring heterocycles.12 Several
of the resulting arylheterocyclic derivatives showed encourag-
ing solubility improvements over biphenyl analogues, and
four nitrogen heterocyclic subseries were found to provide
higher in vitro potencies than predicted (based on their lipo-
philicities). Two pyrazole-based examples (7, 8), displaying
improved solubilities over 5 (8 was twice as soluble as 1), were
also found to be significantly superior to 1 in the mouse model
above(efficacyenhancementsover1of12-foldand41-foldfor
7 and 8, respectively). However, pyrazoleanalogue 8 exhibited
a less ideal microsomal stability profile,12 a deficiency that we
hypothesized might be circumvented by using six-membered
ring heterocycles instead. We also expected that these new
heterocycles could provide analogues with higher efficacy by
providing a more linear geometry for the biaryl side chain,
which, based on our earlier findings,10 appeared particularly
favorable for binding to proposed17 hydrophobic regions in
the nitroreductase.
a Reagents and conditions: (i) ArB(OH)2 (or pinacol ester), toluene,
EtOH, 2 M K2CO3, Pd(dppf)Cl2 under N2, reflux, 10 min-3 h; (ii) sub-
stituted halopyridine, toluene, EtOH, 2 M K2CO3 (or Na2CO3), Pd-
(dppf)Cl2 under N2, reflux, 0.5-3 h; (iii) NaOCOCClF2, K2CO3, DMF,
80 °C, 20 h; (iv) NBS, PPh3, CH2Cl2, 20 °C, 3-4 h; (v) 138 or 139, NaH,
DMF, 0-20 °C, 2.2-2.5 h.
candidates having better aqueous solubilities, acceptable
pharmacokinetics, high oral bioavailabilities, and robust
efficacies in mouse models of advanced disease.
Chemistry
Compounds 9-42, having a substituted pyridine as the
terminal ring of the biaryl side chain, were generally prepared
from either the known10 iodobenzyl ethers 127-129 or the
known10 pinacol boronate esters 130 and 131 via Suzuki
couplings with the corresponding substituted pyridinylboronic
acids or halopyridines, respectively (Scheme 1A,B). Due to the
difficulty in obtaining the isomeric halo(trifluoromethoxy)-
pyridines,18 the corresponding difluoromethoxypyridine ana-
logues (16, 29, 37) were targeted instead. The OCF2H sub-
stituent has similar electronic properties to OCF3 (σm and σp
0.31 and 0.18, compared with 0.38 and 0.35, respectively),19 and
the required known20,21 bromopyridine derivatives 133 and 134
were readily accessed from 6-bromo-3-pyridinol (132) and
5-bromo-2(1H)-pyridinone, respectively, via difluoromethyla-
tions with sodium chlorodifluoroacetate.22 An alternative syn-
thetic route to the final compounds, involving initial assembly
of the biaryl side chain prior to coupling with the known23
chiral oxazine alcohol 140 (exemplified for 25 and 28), was
preferred for the larger scale synthesis of some analogues
obtained from boronate ester 131, due to better overall yields
from 140 and greater ease of purification (Scheme 1C). Thus
4-(hydroxymethyl)phenylboronic acid (135) readily underwent
Suzuki couplings with halopyridines to provide the required
biaryl alcohols (136, 137), which were then brominated with
NBS/PPh324 and coupled to alcohol 140.
Herein, we now describe the results of a systematic study of
aza- and diazabiphenyl analogues of 1, which represent alter-
native, less lipophilic, weakly basic (chargeable) derivatives of
the biphenyl class, seeking more stable second generation
Biaryl analogues containing a pyridine ring proximal to the
ether linkage (43-103) were predominantly synthesized via
Suzuki couplings on isomeric bromopicolyl ether derivatives

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8423
Scheme 3a
Scheme 2a
a Reagents and conditions: (i) SOCl2, CHCl3, 0-20 °C, 20 h, or reflux,
1 h; (ii) 140, NaH, DMF, -5 to 20 °C, 1-16 h; (iii) ArB(OH)2 (or pinacol
ester), toluene, EtOH, 2 M K2CO3, Pd(dppf)Cl2 under N2, reflux, 10 min
-6 h; (iv) SOBr2, CH2Cl2, 0-20 °C, 15 h; (v) TFAA, 20 °C, 30 min, then
reflux, 30 min, then aqueous NaHCO3, 20 °C, 16 h.
(143, 146, 148, 152, 156, 160), obtained by base-catalyzed alkyl-
ation of alcohol 140 with various picolyl halides (Schemes 2
and 3). For the meta-linked biaryls (Scheme 2), 2-bromo-
6-(chloromethyl)pyridine25 (142), 3-bromo-5-(chloromethyl)-
pyridine hydrochloride26 (147), and 4-bromo-2-(chloromethyl)-
pyridine (151) were prepared from precursor alcohols by
chlorination with thionyl chloride [in the case of 147, prior
conversion of (5-bromo-3-pyridinyl)methanol into its hydro-
chloride salt was necessary to avoid undesirable side reactions26].
(4-Bromo-2-pyridinyl)methanol27 (150) was more conveniently
obtained from 4-bromo-2-methylpyridine 1-oxide28 (149) by a
one-pot reaction with trifluoroacetic anhydride29 (rather than
Ac2O27), followed by a mild in situ base-catalyzed hydrolysis
of the resulting trifluoroacetate ester, with the product being
sufficiently pure to use directly in the following chlorination step
(to give 151). However, yields for the alkylation of alcohol 140
using these picolyl chlorides were only moderate (30-54%), and
attempts to acquire pure 146 by this methodology were largely
unsuccessful. Therefore, (2-bromo-4-pyridinyl)methanol (144)
was instead reacted with thionyl bromide to provide the picolyl
bromide 14530 (isolated as a crystalline HBr salt), from which
the desired ether 146 was obtained cleanly and in good yield
(65%, Scheme 2B), although Suzuki couplings on this substrate
were somewhat capricious.
In contrast to the above results for meta-linked biaryls,
para-halopicolyl ethers 156, 160, and 161 were synthesized in
high yield (>80%) from both picolyl chlorides and bromides
(Scheme 3A,B and Supporting Information). 5-Bromo-
2-(bromomethyl)pyridine31 (154) and 2-bromo-5-(bromomethyl)-
pyridine31,32 (158), usually prepared by free radical bromination
of the corresponding bromopicolines, were readily obtained in
much better yield and purity from the precursor alcohols (153,
157), by bromination with NBS/PPh3 (0.1-5 g scale), or by
mesylation and displacement with lithium bromide. 5-Bromo-
a Reagents and conditions: (i) NBS, PPh3, CH2Cl2, 20 °C, 3-3.5 h; or
MsCl, Et3N, THF, 0 °C, 1 h, then LiBr, Me2CO, reflux, 1 h; (ii) 140,
NaH, DMF, 0-20 °C, 2-3 h; (iii) ArB(OH)2 (or pinacol ester), toluene,
EtOH, 2 M K2CO3 (or Na2CO3), Pd(dppf)Cl2 under N2, reflux, 20 min
-21 h; (iv) nBuLi, B(OiPr)3, toluene, THF, -78 °C, 4 h, then -78 to
-20 °C, 2 h, then 2 N HCl, -20 to 20 °C, 40 min; (v) 2-Cl-5-CF3pyridine,
toluene, EtOH, 2 M Na2CO3, Pd(dppf)Cl2 under N2, 88 °C, 4 h.
2-(chloromethyl)pyridine29 (155) was also acquired from 153,
following treatment with thionyl chloride. However, whereas
the bromopicolyl ether derivatives were generally excellent
substrates for Suzuki coupling, a similar reaction employing
chloropicolyl ether 161 (derived from commercial chloro-
methylpyridine 159) was much slower and proved challenging
to complete. Rare halo(trifluoromethoxy)phenylboronic acids
167-17133-37 required for the synthesis of compounds 77-81
and 96-100 were readily obtained in high yield from (com-
mercial or known10) substituted iodo- or bromobenzenes (162-
166) via a lithium-halogen exchange and “in situ quench”
protocol (Scheme 3C).38 Following drying, some of these
arylboronic acids were found by NMR to contain varying
amounts of the corresponding arylboroxines39 (cyclic anhy-
dride form), as expected, but reacted without complication in
Suzuki coupling reactions to give the desired products cleanly
(except in the case of the 2-Cl analogue 96, where ∼10% de-
chlorination of the product was unexpectedly observed, ne-
cessitating repeated chromatographic purification cycles).
As noted above, construction of the side chain first, prior to
coupling with alcohol 140 (exemplified here for 80, Scheme 3D),
was alternatively preferred for the larger scale synthesis of
some analogues due to greater ease of final purification. Impor-
tantly, this approach also enabled synthesis of bipyridine 83

8424 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
Scheme 4a
iodide 181 (obtained by mesylation of alcohol 180 and iodide
displacement) after an attempted bromination (PBr3) of 180
resulted in partial replacement of the ring chlorine (leading to
potential side reactions in the alkylation step). Alcohol 18043
was itself derived44 from the commercial acid 179 via reduction
of the acid chloride. The 2-aryl-5-pyrimidinylmethyl analogues
(122-126) were initially accessed from a 2-methylthiopyrimi-
dine precursor, rather than a halopyrimidine derivative, in order
to avoid competing pyrimidinyl ether formation during base-
catalyzed coupling of the halomethylpyrimidine with alcohol
140. 2-(Methylthio)pyrimidine-5-carboxylic acid45 (186) (ob-
tained from its methyl ester46), was first reduced (with NaBH4,
via the in situ-formed mixed anhydride) to alcohol 187,47 which
was successively brominated (via mesylation and bromide dis-
placement) and reacted with 140 as usual (Scheme 4D).
Leibeskind-Srogl cross-coupling48 of the resulting
2-(methylthio)pyrimidine 189 with arylboronic acids then
furnished the required biaryl derivatives in moderate yield
(∼40%) on a small (50 mg) scale. However, since purification
of the final products from this reaction was not straightfor-
ward, an alternative route was developed for the larger scale
synthesis of 124, in which the biaryl side chain was assembled
first (Scheme4E). Thus, a selectiveSuzuki couplingon2-iodo-
5-bromopyrimidine (190) gave the known49 2-aryl derivative
191, from which pyrimidinecarboxaldehyde 192 was obtained
ingoodyield(74%) followinga sufficientlylow-temperature50
(-95 °C) metal-halogen exchange and rapid DMF quench.
Elaboration to bromide 194, via the mesylate of derived alco-
hol 193, followed by coupling to alcohol 140, then completed
an improved (0.5 g) synthesis of 124.
Results and Discussion
Tables 1-3 provide physicochemical and biological data
for 118 new analogues of 1, in which one or both of the phenyl
groups of the previously described10 biphenyl class was replaced
by pyridine, pyridazine, pyrazine, or pyrimidine. Compound
lipophilicities were estimated by CLogP values, calculated using
ACD LogP/LogD prediction software (version 8.0; Advanced
Chemistry Development Inc., Toronto, Canada). The compara-
tive effects of these heterocyclic replacements on ClogP values
were evaluated for representative compound subsets by calcu-
lating mean differences in ClogP values between compounds in
each subset and biphenyl analogues bearing the same substitu-
ents (Table 4). Broadly, these showed that replacement of one of
the phenyl groups by pyridine lowered ClogP values by ∼1.3
units, whereas replacement by a diaza heterocycle had a more
variable effect, with lipophilicity differences ranging from -1.41
(30,50-pyrimidine) to -2.84 (pyridazine). Additional replace-
ment of the second phenyl group by pyridine further reduced
ClogP values (by ∼0.6-1.1 units) providing particularly hydro-
philic analogues.
Solubilities in water at pH = 7 were measured for all of the
solid compounds with sufficient stock available (111 of the 118
analogues). In general, the compounds that displayed the
highest solubilities included monopyridine analogues lacking
substituents adjacent to the pyridine nitrogen (e.g., 9, 17, 31,
35, 59), bipyridine analogues (e.g., 56, 70, 82-89, 101-103),
and (to a lesser extent) the pyridylpyrazine and pyridylpyr-
imidine analogues (116, 121), consistent with their greater
hydrophilic character. The ortho- and meta-linked biaryls also
displayed higher solubilities than the para-linked biaryls over-
all. Although calculated log P values are only a broad
indicator of aqueous solubility, which is also influenced by
factors such as crystallinity and H-bond networks, eq 1 shows
a Reagents and conditions: (i) 140, NaH, DMF, -78 to 0 °C, 0.5-1 h
(-42 °C, 2 h for 178); (ii) ArB(OH)2, toluene, EtOH, 2 M K2CO3,
Pd(dppf)Cl2 under N2, reflux, 0.5 h; (iii) SOCl2, catalytic DMF, reflux,
1 h, then NaBH4, dioxane, H2O,10-20 °C, 1 h; (iv) MsCl, Et3N, THF,
0 °C, 0.5 h, then NaI, Me2CO, reflux, 1 h; (v) NBS, AIBN, CCl4, 60 °C,
3 h; (vi) iBuOCOCl, NMM, DME, 0 °C, 20 min, then NaBH4, H2O,
0 °C, 10 min; (vii) MsCl, Et3N, CH2Cl2 or THF, 0 °C, 0.5-1 h, then LiBr,
Me2CO, reflux, 1 h; (viii) ArB(OH)2, CuTC, P(2-furyl)3, Pd2(dba)3 CHCl3
3
under N2, THF,50°C, 18 h; (ix) 4-OCF3PhB(OH)2, toluene, Na2CO3,H2O,
Pd(PPh3)4 under N2, reflux, 17.5 h; (x) nBuLi, THF, -95 °C, 30 s, then
DMF, -90 °C, 20 min; (xi) NaBH4, MeOH, 0 °C, 1 h.
(Scheme 3E), which was challenging to prepare from 156 due
to facile protodeboronation of the highly electron deficient tri-
fluoromethyl-substituted 2-pyridyl boronate ester (a recently
reported copper(I)-facilitated Suzuki coupling reaction40 pro-
vides an alternative solution to this problem). Thus, 6-(hydro-
xymethyl)-3-pyridinylboronic acid (174) underwent a success-
ful Suzuki coupling with 2-chloro-5-(trifluoromethyl)pyridine
to give bipyridine alcohol 175 in low yield, which was elabo-
rated to 83 via bromination (NBS/PPh3) and alkylation
reactions, as above.
Further biaryl analogues containing a pyridazine, pyrazine,
or pyrimidine ring proximal to the ether linkage (104-126) were
predominantly generated via Suzuki couplings on chloro- or
bromoheterocyclic methyl ether derivatives of alcohol 140 (178,
182, and 185, Scheme 4A-C). Chloropyridazine 178 and bro-
mopyrimidine 185 were prepared from known bromomethyl
precursors, 17741 and 184,42 obtained via free radical bromina-
tion of methyl-substituted haloheterocycles. In contrast,
the synthesis of chloropyrazine 182 was achieved via unstable

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8425
Table 1. Physicochemical Properties and MIC Values for Heterobiaryl Analogues of 1 Having a Terminal Pyridine Ring
solubilityb (μg/mL)
MIC (μM)c
compd
link
R
CLogPa
pH = 7
pH = 1
MABA
LORA
1
2.70
4.48
4.36
2.19
2.19
2.69
4.35
2.12
2.16
3.54
2.59
2.19
3.57
2.19
2.69
4.35
2.12
2.16
3.54
4.46
1.82
2.25
2.09
2.59
2.41
2.19
3.57
1.86
2.19
2.21
2.16
2.87
2.69
4.35
2.12
2.62
2.21
19
0.43
1.2
116
35
0.50 ( 0.30
0.03 ( 0.01
0.035 ( 0.015
>8
2.6 ( 1.4
1.4 ( 0.5
1.3 ( 0.1
>32
4
5
1.1
9
ortho
ortho
ortho
ortho
ortho
meta
meta
meta
meta
meta
meta
meta
meta
meta
para
para
para
para
para
para
para
para
para
para
para
para
para
para
para
para
para
para
para
para
3-aza
3-aza, 4-F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
>8
5.2 ( 2.4
2.7 ( 1.2
>8
16 ( 1
3-aza, 4-OCH3
3-aza, 4-OCH2Ph
4-aza
48
8.3 ( 1.5
10 ( 2
>32
1.2
2-aza
1.4 ( 0.4
11 ( 4
2-aza, 4-CF3
2-aza, 4-OCF2H
3-aza
4.0
6.7
0.32 ( 0.08
0.21 ( 0.02
1.4 ( 0.3
0.47 ( 0.03
0.37 ( 0.12
0.40 ( 0
0.07 ( 0.01
1.4 ( 0.5
0.21 ( 0
0.06 ( 0.03
0.025 ( 0.005
0.08 ( 0.02
0.14 ( 0.03
0.035 ( 0.005
0.08 ( 0.03
0.13 ( 0.05
0.51 ( 0.04
0.03 ( 0
0.20 ( 0.05
0.095 ( 0.025
0.55 ( 0.25
2.6 ( 0.9
0.045 ( 0.005
0.045 ( 0.015
0.04 ( 0
0.50 ( 0.25
0.56 ( 0.29
0.23 ( 0.02
4.3 ( 2.3
6.4 ( 2.7
9.7 ( 2.6
8.4 ( 0.9
5.1 ( 1.6
1.7 ( 0.3
3.0 ( 0.5
>32
110
1.1
26
3-aza, 4-CF3
3-aza, 4-F
3-aza, 4-OCH3
3-aza, 4-OCH2Ph
4-aza
18
0.15
2-aza
7.3
0.20
3.3
1.7
4.3
67
1.7 ( 0.5
1.0 ( 0.2
1.0 ( 0.6
2.5 ( 0.8
1.1 ( 0.4
1.3 ( 0.3
1.5 ( 0.6
1.2 ( 0.5
5.7 ( 1.0
2.1 ( 0.2
2.4 ( 0.9
2.1 ( 0.2
2.8 ( 0.9
19 ( 7
2-aza, 4-CF3
2-aza, 4-CF3, 6-Cl
2-aza, 4-CN
2-aza, 3-F
5.1
2.0
2-aza, 4-F
275
23
2-aza, 4-OCF2H
2-aza, 4-OCH3
3-aza
2.9
2.5
29
3-aza, 4-CF3
3-aza, 4-CN
3-aza, 4-F
1.0
3.0
8.0
55
11
10
3-aza, 5-F
3-aza, 4-NH2
3-aza, 4-OCF2H
3-aza, 4-OCH3
3-aza, 4-OCH2Ph
4-aza
13
3.2
4.4
0.03
22
0.66 ( 0.30
0.61 ( 0.12
>32
9.2 ( 2.1
3.9 ( 2.1
1.4 ( 0.3
4-aza, 2-F
4-aza, 3-F
16
14
15
a CLogP values, calculated using the ACD LogP/LogD prediction software (version 8.0, Advanced Chemistry Development Inc., Toronto, Canada).
b Solubility in water at 20 °C, determined by HPLC (see Experimental Section, Method A). c Minimum inhibitory concentration, determined under
aerobic (MABA)53 or anaerobic (LORA)52 conditions. Each value is the mean of at least two independent determinations ( SD.
that these do provide a reasonable prediction of solubility
within the larger series of para-linked biaryls (across a solu-
bility range of ∼23000-fold):
119, 124; pyridazine 107, having a calculated pKa of 1.01,
showed a moderate 5-fold enhanced solubility at pH=1).
The compounds were screened in assays to quantify their
antitubercular activity under both aerobic and anaerobic con-
ditions (this permitted the elucidation of SAR for each, as
previously described10,12). Briefly, the 8 day microplate-based
assay using Alamar blue reagent (added on day 7) for deter-
mination of growth (MABA)51 gave an assessment of activity
against replicating M. tb, while the 11 day high-throughput,
luminescence-based low-oxygen-recovery assay (LORA)52
measured activity against bacteria in a nonreplicating state
that models clinical persistence. Minimum inhibitory concen-
trations (MICs) are defined as the lowest compound concen-
tration effecting >90% growth inhibition, and the values
recorded in Tables 1-3 represent the mean of at least two
independent determinations ((SD). The compounds were
additionally screened for mammalian cytotoxicity in a 72 h
log½solubility ðμg=mLÞꢀ ¼ - 0:46 ð ( 0:07Þ ClogP
þ 1:78 ð ( 0:16Þ
n ¼ 73 R ¼ 0:63 F ¼ 46:3
ð1Þ
Aqueous solubilities at pH = 1 were also measured for
selected compounds containing functionalities that may ex-
hibit weak base behavior. Encouragingly, most of the pyridine
analogues tested displayed markedly increased solubilities at
low pH (e.g., >100-fold for 59, 74, 92, 93, 97), consistent with
their calculated pKa values (ranging from 2.03 for 24 and 28 to
3.88 for 59 and 92). The exceptions to this (25, 34, 42) could be
accounted for by their negativecalculated pKa values, as could
the results for the pyrazine and pyrimidine analogues (114,

8426 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
Table 2. Physicochemical Properties and MIC Values for Heterobiaryl Analogues of 1 Having a Proximal Pyridine Ring
solubilityb (μg/mL)
MIC (μM)c
compd
link
aza
R
CLogPa
pH = 7
pH = 1
MABA
LORA
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
30 (meta)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
40 (para)
20
20
20
20
20
20
20
40
40
40
40
40
40
40
50
50
50
50
50
50
50
60
60
60
60
60
60
60
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
30
30
30
30
30
30
30
30
30
30
30
30
30
30
4-CF3
4-CN
4-F
3.14
1.48
2.10
3.01
2.16
1.95
1.35
3.14
1.48
2.10
3.01
2.16
1.95
1.35
3.17
1.51
2.13
3.04
2.19
1.98
1.38
3.10
1.44
2.06
2.97
2.12
1.91
1.31
3.17
1.51
2.13
3.04
2.19
1.98
3.75
3.56
3.60
3.02
3.51
0.93
2.22
2.25
0.87
0.89
1.38
1.30
0.89
3.14
1.48
2.10
3.01
2.16
1.95
3.72
3.53
3.57
2.99
3.48
2.22
0.84
1.35
5.0
24
0.29 ( 0.03
0.48 ( 0.24
0.30 ( 0.09
0.16 ( 0.10
0.17 ( 0.01
0.30 ( 0.06
0.71 ( 0.16
0.075 ( 0.015
0.38 ( 0.13
0.17 ( 0.06
0.035 ( 0.005
0.055 ( 0.005
0.34 ( 0.04
1.3 ( 0.5
0.15 ( 0.06
0.69 ( 0.21
0.31 ( 0.16
0.17 ( 0.05
0.53 ( 0.31
0.91 ( 0.01
2.8 ( 1.3
2.9 ( 1.0
10 ( 4
15
2.7 ( 0.8
1.4 ( 0.3
2.7 ( 0.8
4.3 ( 1.4
5.2 ( 1.8
4.4 ( 0.5
13 ( 1
1.2 ( 0.2
2.4 ( 0.6
2.7 ( 0.3
6.2 ( 0.5
12 ( 1
4-OCF3
3.0
9.6
10
4-OCF2H
3-F, 4-OCH3
3-aza, 4-OCH3
4-CF3
14
2.8
8.0
31
4-CN
4-F
4-OCF3
3.9
16
4-OCF2H
3-F, 4-OCH3
3-aza, 4-OCH3
4-CF3
7.2
93
2.8
29
3.5 ( 1.0
18 ( 5
4-CN
4-F
4-OCF3
45
41000
7.7 ( 1.1
2.4 ( 1.3
5.9 ( 2.9
9.0 ( 1.8
22 ( 2
9.5
9.2
24
4-OCF2H
3-F, 4-OCH3
3-aza, 4-OCH3
4-CF3
31
43
0.88 ( 0.04
2.1 ( 0.3
0.46 ( 0.25
0.76 ( 0.27
0.67 ( 0.20
2.3 ( 0.7
24 ( 11
>32
4-CN
4-F
4-OCF3
19
16
28 ( 1
18 ( 4
26 ( 6
16 ( 0
4-OCF2H
3-F, 4-OCH3
3-aza, 4-OCH3
4-CF3
86
25
96
3.8 ( 0.1
31 ( 8
2.3
45
0.04 ( 0.01
0.18 ( 0.08
0.13 ( 0.02
0.065 ( 0.038
0.02 ( 0
0.17 ( 0.05
0.14 ( 0.03
0.04 ( 0.01
0.06 ( 0.03
0.05 ( 0.02
0.06 ( 0
1.4 ( 0.5
0.83 ( 0.09
0.24 ( 0
0.49 ( 0
2.6 ( 0.8
0.36 ( 0.14
3.2 ( 0.8
1.7 ( 0.2
0.053 ( 0.005
0.18 ( 0.06
0.06 ( 0
0.05 ( 0.01
0.02 ( 0
0.09 ( 0.04
0.030 ( 0.014
0.017 ( 0.005
0.047 ( 0.017
0.025 ( 0.005
0.18 ( 0
0.19 ( 0.02
0.37 ( 0.12
0.24 ( 0.02
3.4 ( 0.1
7.5 ( 0.7
3.2 ( 1.5
3.7 ( 1.2
4.0 ( 0.9
3.4 ( 0.6
2.6 ( 0.9
2.4 ( 0.9
1.7 ( 0.8
1.3 ( 0.6
2.3 ( 0.7
20 ( 1
9.6 ( 3.0
16 ( 10
19 ( 9
21 ( 7
9.6 ( 3.4
20 ( 4
4-CN
4-F
4-OCF3
17
2.5
4.0
10
1360
4-OCF2H
3-F, 4-OCH3
2-Cl, 4-OCF3
3-Cl, 4-OCF3
2-F, 4-OCF3
3-F, 4-OCF3
3-OCF3, 4-Cl
2-aza, 3-F
2-aza, 4-CF3
3-aza, 4-CF3
3-aza, 4-F
3-aza, 5-F
3-aza, 4-OCH3
4-aza, 2-F
4-aza, 3-F
4-CF3
1.4
0.85
1.4
30
334
442
0.80
41
5.9
12
43
130
24
700
42
14 ( 4
1.2
2.9
3.8
2.3
1.6
2.1
8.8
0.18
2.2
3.0
0.22
7.0
51
0.53 ( 0.17
6.0 ( 0.6
2.9 ( 0.1
0.54 ( 0.24
3.4 ( 0.4
1.9 ( 0
1.6 ( 0.1
1.0 ( 0.1
1.9 ( 0.4
0.93 ( 0.29
2.4 ( 1.2
17 ( 9
4-CN
4-F
4-OCF3
1000
542
4-OCF2H
3-F, 4-OCH3
2-Cl, 4-OCF3
3-Cl, 4-OCF3
2-F, 4-OCF3
3-F, 4-OCF3
3-OCF3, 4-Cl
3-aza, 4-CF3
3-aza, 4-F
3-aza, 4-OCH3
39
71
13 ( 1
7.3 ( 0.5
51
a CLogP values, calculated using the ACD LogP/LogD prediction software (version 8.0, Advanced Chemistry Development Inc., Toronto, Canada).
b Solubility in water at 20 °C, determined by HPLC (see Experimental Section, Method A). c Minimum inhibitory concentration, determined under
aerobic (MABA)53 or anaerobic (LORA)52 conditions. Each value is the mean of at least two independent determinations ( SD.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8427
Table 3. Physicochemical Properties and MIC Values for Heterobiaryl Analogues of 1 Having a Proximal Pyridazine, Pyrazine, or Pyrimidine Ring
solubility (μg/mL)b
pH = 7 pH = 1
5.4
22
MIC (μM)c
compd
aza
R
CLogPa
MABA
LORA
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
20,30
20,30
20,30
20,30
20,30
20,30
20,30
20,30
20,50
20,50
20,50
20,50
20,50
20,60
20,60
20,60
20,60
20,60
30,50
30,50
30,50
30,50
30,50
4-CF3
4-CN
4-F
1.64
-0.01
0.60
1.52
0.67
0.46
0.73
-0.15
2.31
1.27
2.19
1.33
1.39
2.75
1.71
2.63
1.77
1.83
2.82
2.07
3.05
2.20
2.14
0.06 ( 0
2.8 ( 0.3
13 ( 3
0.36 ( 0.03
0.19 ( 0.02
8.3
6.1
1.3
6.8
5.6 ( 1.9
1.7 ( 0.1
7.5 ( 0.4
6.6 ( 1.2
14 ( 5
4-OCF3
31
0.075 ( 0.025
0.17 ( 0.05
0.25 ( 0.03
0.55 ( 0.22
0.72 ( 0.28
0.08 ( 0.01
0.085 ( 0.015
0.023 ( 0.005
0.20 ( 0.04
0.33 ( 0.10
0.06 ( 0
0.13 ( 0.01
0.11 ( 0.01
0.12 ( 0.04
0.28 ( 0.07
0.025 ( 0.005
0.11 ( 0.02
0.027 ( 0.009
0.13 ( 0.07
0.16 ( 0.07
4-OCF2H
3-F, 4-OCH3
3-aza, 4-CF3
3-aza, 4-OCH3
4-CF3
12
34
0.84
20 ( 8
0.97 ( 0.53
2.0 ( 0.8
1.0 ( 0.6
1.3 ( 0.6
3.7 ( 0.5
3.5 ( 1.4
12 ( 1
1.9 ( 0.5
7.4 ( 1.2
14 ( 2
9.4 ( 0.9
5.4 ( 0.3
1.8 ( 1.0
4.4 ( 1.7
12 ( 3
4-F
4-OCF3
8.9
2.6
5.0
2.8
5.1
3.0
4-OCF2H
3-aza, 4-CF3
4-CF3
27
2.7
50
4-F
4-OCF3
8.1
7.8
4-OCF2H
3-aza, 4-CF3
4-CF3
85
4-F
4-OCF3
2.1
1.1
4-OCF2H
3-aza, 4-CF3
a CLogP values, calculated using the ACD LogP/LogD prediction software (version 8.0, Advanced Chemistry Development Inc., Toronto, Canada).
b Solubility in water at 20 °C, determined by HPLC (see Experimental Section, Method A). c Minimum inhibitory concentration, determined under
aerobic (MABA)53 or anaerobic (LORA)52 conditions. Each value is the mean of at least two independent determinations ( SD.
tetrazolium dye assayusing VEROcells53 (CCL-81; American
Type Culture Collection); the compounds were all relatively
nontoxic in this assay, with IC50s >128 μM (data not shown).
InTable 1, wefirst exploredthe effect ofreplacingthe termi-
nal phenyl ring with a substituted pyridine by comparing ortho-
(compounds 9-13), meta- (compounds 14-22), and para-
(compounds 23-42) linked analogues. Each series was further
subdivided according to the position of the aza atom in the ring.
Substituents on this pyridine ring were selected to enable a broad
range of lipophilicities to be studied, but a greater emphasis was
placed on lipophilic, electron-withdrawing groups that have
previously provided particular utility10,12 (using OCF2H as a
substitute for the favored OCF3). The ortho-linked compounds
(9-13) were generally more soluble than related meta-andpara-
linked analogues but had poor activities, consistent with pre-
vious results for ortho-linked biphenyl analogues.10 The meta-
linked compounds (14-22) were more effective overall, parti-
cularly the lipophilic benzyloxy analogue 21 (MABA MIC
0.07 μM). Comparison of 15 (2-aza) with18 (3-aza) suggests that
the position of the aza atom has little effect, but compounds
lacking a ring substituent (14, 17, 22) had weaker activity
(>3-fold in MABA). As expected, the larger para-linked series
(23-42) provided the best activity, with compounds 24, 25,
28, 32, 37, and 38 standing out for their high potencies in both
assays. Compound 28 (2-aza, 4-F) was also particularly nota-
ble for its good aqueous solubility (67 μg/mL at pH = 7, 3.5-
fold better than 1), which was further improved at low pH
(to 275 μg/mL). Within this para-linked series, a closer
comparison of the 2-aza and 3-aza analogues bearing common
4-substituents (R = CF3, CN, F, OCF2H, OCH3) suggests
that again the position of the aza atom is not critical (mean
MABA MICs 0.077 and 0.083 μM, mean LORA MICs 1.50 and
1.57 μM, respectively). However, the 4-aza analogues (40-42),
amino compound 36, and other analogues lacking a substituent
adjacent to the pyridine nitrogen (31, 35) were markedly less
effective.
Table 4, part A, compares the mean MIC data for the various
subseries against the mean MICs for the corresponding sub-
stituted biphenyl analogues (where these were available), using
previously published data.10 In most cases where there was
more than a single compound in a subset, the pyridyl analogues
were 2-8-fold less potent than the corresponding biphenyls
in both the MABA and LORA assays (although compounds
23-30 displayed slightly higher mean potency in LORA than
the biphenyls). We have previously shown, for a large series of
para-linked biphenyl analogues,10 that MICs in the MABA
assay correlate positively with global compound lipophilicity
(eq 2).
X
logðMICMABAÞ ¼ - 0:25CLogP - 0:52
σ - 0:014 ð2Þ
According to this equation, the 2-5-fold reduced mean
MABA potency shown by the three subsets of para-linked
compounds in Table 1 could be explained (at least in part) by
their lower lipophilicities (average ΔCLogP of -0.9 to -1.4
units, leading to predicted potency values 1.4-3-fold higher
than observed, Table 4, part A). In summary, replacement of

8428 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
Table 4. Summary of Calculated Lipophilicity Differences, Mean Aqueous Solubilities, and Mean MICs for Compound Subsets
mean MABA MIC (μM)
mean LORA MIC (μM)
compd
link
aza
ΔCLogPa
av solubilityb
azac
deazad
ratioe
calcdf
azac
deazad
ratioe
(A) Compounds of Table 1
9-10
13
o
3
-1.29
-1.39
-1.07
-1.16
-1.39
-1.06
-0.90
-1.37
76
>8
>8
0.92
0.64
>8.7
>12
7.0
>24
>32
3.0
2.5
2.6
2.9
2.1
2.3
2.4
2.3
>8
>13
2.8
o
4
2
3
4
2
3
4
14-16
17-19
22
m
m
m
p
5.4
46
0.64
0.092
0.097
0.095
0.038
0.096
0.093
7.2
7.7
>32
0.75
1.4
7.7
2.7
15
>15
0.61
1.8
23-30
31-38
40-42
11
15
17
0.095
0.51
0.43
2.5
5.3
0.070
0.16
0.20
1.4
4.4
4.8
p
p
4.6
2.1
(B) Compounds of Table 2
43-48
49
m
m
m
m
m
m
m
m
p
20
20
40
40
50
50
60
60
20
20
20
30
30
30
-1.35
-1.97
-1.35
-1.97
-1.32
-1.94
-1.39
-2.01
-1.32
-1.32
-2.45
-1.35
-1.35
-2.28
11
14
0.28
0.71
0.18
1.3
0.13
0.40
2.2
1.8
1.4
3.3
3.5
7.0
9.2
9.5
3.0
1.9
6.7
2.3
1.6
4.7
4.0
5.2
5.0
3.1
1.7
3.1
1.7
3.1
1.7
3.1
1.7
1.2
0.74
1.9
1.2
0.74
1.6
1.3
3.1
1.6
7.1
2.5
13
50-55
56
11
93
0.13
0.40
12
7.8
22
57-62
63
20
31
0.46
2.8
0.13
0.40
64-69
70
38
96
1.2
3.8
0.13
0.40
>24
31
>7.7
18
71-76
77-81
82-89
90-95
96-100
101-103
13
0.10
0.070
1.4
0.033
0.037
0.21
0.071
0.079
0.86
4.2
2.1
16
3.5
2.8
8.4
2.1
2.2
7.5
p
6.9
125
2.3
2.9
36
p
p
0.076
0.060
0.27
0.033
0.037
0.057
0.072
0.080
0.21
2.5
1.6
p
p
12
(C) Compounds of Table 3
104-109
110
p
p
p
p
p
p
p
p
20,30
20,30
20,50
20,50
20,60
20,60
30,50
30,50
-2.84
-3.75
-2.17
-3.09
-1.73
-2.65
-1.41
-2.34
8.3
12
0.18
0.55
0.097
0.33
0.11
0.28
0.073
0.16
0.033
0.030
0.033
0.030
0.033
0.030
0.033
0.030
5.5
18
0.17
0.26
0.12
0.18
0.089
0.14
0.074
0.12
6.2
14
1.2
2.1
1.2
2.1
1.2
2.1
1.2
2.1
5.2
6.7
1.1
1.8
5.2
6.7
4.4
5.7
112-115
116
4.3
27
2.9
11
1.3
3.7
6.2
117-120
121
17
85
3.3
9.3
2.2
5.3
14
5.3
12
122-125
126
1.6
a Mean difference in CLogP values between the heterobiaryl subclass and the biphenyl compounds from ref 10 bearing the same substituents. b Average
solubility (μg/mL) in water at pH = 7. c Heterobiaryl compounds of this paper. d Comparison data for corresponding substituted biphenyl compounds from ref 10
(most subclasses) or for terminal pyridine ring analogues in Table 1 (for pyridylheterocycles of Tables 2 and 3). e Ratio of mean MICs (aza/deaza), as defined above.
f Predicted mean MABA MICs based on eq 2 (see text), using mean observed MABA MIC data for para-linked biphenyl analogues and ΔCLogP values for related
heterobiaryl subclasses.
the terminal phenyl ring by pyridine in the most promising
(4-substituted) para-linked series has provided several promis-
ing new analogues which combine improved aqueous solubil-
ities (particularly at low pH) with excellent potencies in both
antitubercular assays.
bipyridine 70) were ranked lowest by potency but provided
the highest aqueous solubilities of the meta-linked series (up to
0.1 mg/mL at pH=7 for 70). A similar comparison for the para-
linked series shows that the less soluble 30-aza subset (com-
pounds 90-95) was slightly more potent overall than the 20-aza
subset (compounds 71-76), particularly in LORA (mean
MICs 2.5 and 4.2 μM, respectively). Both 4-OCF2H ana-
logues (75, 94) had excellent MABA potencies (MICs 0.02 μM),
whereas the 4-CF3 and 4-OCF3 analogues with a 30-aza atom
(90, 93) had notably high LORA potencies (MICs ∼0.5 μM).
For the more potent para-linked series, we examined some
additional, more lipophilic phenyl ring substituent patterns
(2-Cl-4-OCF3, 3-Cl-4-OCF3, 2-F-4-OCF3, 3-F-4-OCF3, and
3-OCF3-4-Cl) that had provided improved LORA potencies
inthe biphenyl series.10 Both the 20-aza and30-aza subsets with
these substituents (compounds 77-81 and 96-100, respec-
tively) had higher mean potencies than the previous subsets
above (having the standard substituents), particularly in the
LORA assay (1.6-2-fold). While these were still somewhat less
(1.6-2.8-fold) than for related biphenyl analogues, the mean
measured MABA potencies were actually similar or slightly
better than predicted (Table 4, part B), based on their reduced
mean lipophilicities (ΔCLogP of -1.32 to -1.35 units). From
In Table 2, we next explored the effect of replacing the phenyl
ring proximal to the ether linkage with a substituted pyridine,
including examples of all six positional isomers of the more
favorable meta- and para-linked series. The use of a larger set of
common substituents in this work (4-CF3, 4-CN, 4-F, 4-OCF3,
4-OCF2H, 3-F-4-OCH3, and 3-aza-4-OCH3) enabled a more
definitive assessment of the subtle effects produced by this
variation, as well as the separate evaluation of a more hydro-
philic bipyridyl side chain (Table 4, part B). Thus, comparing
the four largest subsets of meta-linked compounds, an aza atom
at the 40-position (compounds 50-55) gave the highest mean
MABA potency (similar to biphenyl analogues), with com-
pound 53 (4-OCF3) the best overall (MABA MIC 0.035 μM).
An aza atom at the 20-position (compounds 43-48) was slightly
more advantageous for LORA potency across these subsets
(comparable to biphenyl analogues) and was preferred among
the more weakly active bipyridyl compounds (compound 49;
both assays). The two 60-aza subsets (compounds 64-69 and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8429
Table 5. Microsomal Stability and Acute in Vivo Efficacy Data for Selected Compounds
TD solubilitya
pH = 7 pH = 1
microsomes (% remaining at 1 h)
compd
link
aza
R
Hb
Mc
in vivo efficacyd (ratio vs 1)
1
11
<0.1
11
<0.1
82
97
93
86
83
98
86
88
95
40
93
54
71
97
97
88
83
87
100
94
98
92
96
94
96
90
80
61
80
91
79
75
61
86
31
39
97
86
80
87
77
90
92
91
87
87
1.00
>205
33
5
24
25
28
29
32
34
37
38
42
53
59
74
80
92
93
97
99
107
114
119
124
para
para
para
para
para
para
para
para
para
meta
meta
para
para
para
para
para
para
para
para
para
para
2
4-CF3
4-CF3, 6-Cl
4-F
0.42
0.84
2
5.1
2
0.65
3.3
2
4-OCF2H
4-CF3
4-F
3
0.17
0.38
15
3
0.66
1.9
3
4-OCF2H
4-OCH3
3-F
3
0.26
0.95
0.12
0.02
27
4
40
4-OCF3
4-F
50
20
4-OCF3
3-F, 4-OCF3
4-F
0.44
1.5
65
20
67
>93
45
233
30
5.9
<0.1
7.8
30
4-OCF3
3-Cl, 4-OCF3
3-F, 4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
>89
>933
>840
11
30
0.19
0.32
3.0
17
34
30
20,30
20,50
20,60
30,50
10
0.44
0.44
167
3.7
1.2
1.5
>1120
a Thermodynamic solubility (μg/mL) in aqueous buffer at pH = 7.4 or pH = 1.0 and 20 °C, determined by HPLC (see Experimental Section, Methods
B and C). b Pooled human liver microsomes. c Pooled CD-1 mouse liver microsomes. d Fold reduction in lung CFUs for compound compared with the
fold CFU reduction for 1 in a mouse model of acute TB infection (see text).
these subsets, compounds 97 (3-Cl-4-OCF3) and 99 (3-F-4-
OCF3) in particular stood out for their impressive overall po-
tency profiles, although 97 was significantly less soluble than
most at pH = 7.
The pyridazine class (compounds 104-109) was the most
hydrophilic of all the monoheterocyclic and bipyridyl deriva-
tives examined here (ΔCLogP value -2.84 units), although
the mean solubility value was only moderate (lower than for 20-
aza compounds 71-76 but better than for 30-aza compounds
90-95). Within this class, the more lipophilic examples, 104
(4-CF3) and107 (4-OCF3), showed fairly good potency profiles,
despite the overall activity of the set being about 5-fold less
than for related biphenyl analogues (and roughly 2-fold less
than for 20-aza or 30-aza analogues). Of greater interest was the
somewhat more lipophilic pyrazine class (compounds 112-
115) due to the very high LORA potencies determined (mean
MIC 1.3 μM; equivalent to biphenyl analogues) and good
overall MABA activity. Compound 114 (4-OCF3) had the best
activity of any diaza compound (MICs 0.023 and 1.0 μM in
MABA and LORA, respectively), comparable to some of the
best pyridine derivatives, although its aqueous solubility was
relatively low (2.6 μg/mL). 20,60-Pyrimidine derivatives (com-
pounds 117-120) had the highest mean aqueous solubility
of the four classes, despite their slightly higher lipophilicities
(ΔCLogP value -1.73 units), but their activity was moderate.
Conversely, the 30,50-pyrimidine isomers (compounds 122-125),
which were of very similar lipophilicities to the pyridine ana-
logues (Table 2), provided the highest mean MABA potency of
all (0.073 μM), with the more lipophilic examples, 122 (4-CF3)
and 124 (4-OCF3), standing out (MABA MICs 0.025 and
0.027 μM, respectively); however, this was the least soluble
class. More soluble pyridyl heterocylic derivatives (110, 116,
121, and 126) mirrored these trends but were generally about
2-fold less active in MABA than predicted and gave generally
A larger set of para-linked bipyridines, including more
favorable CF3 or F substituents, was also prepared and eval-
uated (compounds 82-89 and 101-103). These compounds
were considerably less lipophilic than the corresponding mono-
pyridines of Table 1 (mean CLogP values 1.13 and 0.93 units
lower, respectively), which translated into some dramatically
improved solubilities (up to 0.7 mg/mL at pH = 7 for 88).
However, unsurprisingly (based on the predicted MABA data),
their mean potencies were 5-8-fold less than for the related
monopyridine analogues (of Table 1) in both assays (some
4-24-fold less than for related biphenyl analogues), with the
particularly weak LORA results suggesting limited utility.
In an alternative exploration of the effect of diaza substitu-
tion, and based on previous success with pyrazole-, triazole-,
and tetrazole-based analogues,12 we finally investigated the
effect of replacing the phenyl ring proximal to the ether link-
age with a substituted pyridazine, pyrazine, or pyrimidine in the
most favored para-linked series (Table 3). All four possible
structural classes were studied, using a minimum of four term-
inal phenyl ring substituents (4-CF3, 4-F, 4-OCF3, 4-OCF2H;
4-CN and 3-F-4-OCH3 were also included in the first set for
comparison with the pyridine analogues of Table 2). The 3-
aza-4-CF3 substituent pattern, which was the most preferred
for the bipyridines above (and had previously proven quite
effective in a 5-aryl thiophene series12) was employed for the
study of related pyridyl heterocyclic compounds.

8430 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
Table 6. Pharmacokinetic Parameters for Selected Analogues in CD-1 Mice Following a Single Oral Dose of 40 mg/kg
plasma
Cmax (μg/mL)
lung
Cmax (μg/mL)
9.0
34
compd
AUC0-infa (μg h/mL)
t1/2 (h)
AUC0-infa (μg h/mL)
t1/2 (h)
AUC ratiob
3
3
5
198
20
7.4
1.1
14
218
67
13
10
1.1
3.4
24
32
74
80
92
93
97
99
107
114
124
8.8
c
-
5.4
8.4
2.7
24
217
418
137
61
14
26
7.6
5.3
12
414
427
155
136
81
373
35
9.1
8.2
7.9
2.8
2.2
14
1.9
1.0
9.1
1.1
2.2
11
18
10
12
296
88
0.27
3.2
2.9
7.0
3.6
9.6
1.2
22
14
284
251
94
175
52
12
1.4
1.8
2.9
7.2
23
7.0
23
4.0
3.4
>8
18
152
43
>324
131
>2.1
3.0
a Area under the curve, extrapolated to infinity. b Lung AUC/plasma AUC. c Not calculable.
weak LORA results, similar to bipyridine analogues, suggesting
little interest in further evaluating these compounds.
pyrazine 114 (ClogP 2.19; 167-fold better than 1) and less
hydrophilic 30,50-pyrimidine 124 (ClogP 3.05; >1120-fold
better than 1) also displayed outstanding efficacies. Even the
highly hydrophilic pyridazine 107 (ClogP 1.52) was 11-fold more
effective than 1, demonstrating that very high compound lipo-
philicities (e.g., ClogP > 4) were not mandatory to achieve
excellent in vivo efficacies.
A selection of the more interesting compounds was assayed
to determine relative metabolic stabilities using human and
mouse liver microsome preparations (Table 5; data for 1 and
the biphenyl analogue 5 are also provided for comparison).
All of the compounds evaluated (except for the 3-aza-4-OCH3
analogue 38) were acceptably stable toward human micro-
somes (>50% remaining after incubation at 37 °C for 1 h),
and all except meta-linked pyridyl analogues 53 and 59 were
similarly stable toward mouse microsomes. The very high
stabilities of many of the most potent para-linked heterocyclic
analogues (>80%, several comparable to 5) were particularly
encouraging, indicating that these heterocycles did not intro-
duce a significant metabolic liability.
The compounds selected above werealso evaluatedfortheir
antitubercular effects in a mouse model of acute M. tb infec-
tion using a once daily oral dose of 100 mg/kg for 5 days a week
for 3 weeks, following established protocols.53 In this assay, 1
was used as an internal reference standard, with the activity of
new analogues recorded as the ratio of the fold decrease in
CFUs recovered from the lungs of compound-treated mice
compared to the corresponding fold CFU decrease achieved
by treatment with 1, to allow interexperiment comparisons
(Table 5). Examining the series where the terminal phenyl ring
had been replaced by pyridine (24-42), the best compounds
were the isomeric trifluoromethyl-substituted analogues 24
and 32 (respectively 33-fold and 15-fold more effective than
1). Difluoromethoxy analogues 29 and 37 had disappointingly
low efficacies, suggesting that OCF2H was not an effective
substitute for the favored OCF3 group.
Of major interest were the compounds in which the prox-
imal phenyl ring had been replaced by pyridine (53-99) or
diaza heterocycles (107-124). While the low stability meta-
linked analogues 53 and 59 were ineffective (as anticipated),
all of the other five pyridine analogues containing a 4-OCF3
phenyl substitutent (with or without an adjacent halo atom)
were markedly superior to 1, with 80 (20-aza, 3-F-4-OCF3Ph;
233-fold), 93 (30-aza, 4-OCF3Ph; >89-fold), 97 (30-aza, 3-Cl-
4-OCF3Ph; >933-fold), and 99 (30-aza, 3-F-4-OCF3Ph;
>840-fold) clearly preeminent (comparable to the best biphe-
nyl analogues). A closer comparison of 93 and 99 with 74 and
80 indicated that 30-aza compounds were more efficacious
than 20-aza analogues and that 3-F-4-OCF3 (or 3-Cl-4-OCF3;
see 97) phenyl substitution may provide higher efficacy in this
model than 4-OCF3 substitution alone. Finally, among the
diaza heterocyclic derivatives, the slightly more hydrophilic
To further assess the merits of these leading candidates
for more advanced development, first, accurate (thermodyna-
mic) solubility measurements were performed at pH=7.4 and
pH=1 (Table 5; data for 1 and the biphenyl analogue 5 are
also provided for comparison). While all of the compounds
evaluated had inferior aqueous solubilities to 1 at pH=7.4 (the
best was 30-aza, 4-FPh analogue 92 at 5.9 μg/mL, which was
about 2-fold less than 1), several pyridine derivatives (74, 80,
92, 93, and 99) had significantly (3- to >8-fold) higher solu-
bilities than 1 at low pH, consistent with previous data. Mouse
pharmacokinetic data were also obtained for this compound
set, following single oral dosing at 40 mg/kg (Table 6; com-
parative data for 5 are also provided). Most compounds
(except perhaps 4-FPh-pyridine 92 and pyridazine 107) demon-
strated excellent plasma half-lives (5-24 h) and generally high
exposures in both lung tissue and plasma (AUCs >100 μg h/
3
mL, peak plasma levels >7 μg/mL), with some (notably 4-
CF3pyridine 24, 3-Cl-4-OCF3Ph-pyridine 97, pyrazine 114,
and 30,50-pyrimidine 124) exhibiting preferential accumula-
tion in lung tissue. Such high exposure levels (similar to 5)
were indicative of very good oral bioavailability, and this was
confirmed by similar pharmacokinetic assessments in the rat
(Table 7) for four compounds of particular interest (74, 93, 99,
and 114). Extended plasma half-lives were again found, and
the determined oral bioavailabilities ranged from 45% (for
pyrazine 114) to an impressive 93% (for 3-F-4-OCF3Ph-
pyridine 99).
Finally, a smaller selection of compounds was evaluated for
their antitubercular effects in a mouse model of chronic M. tb
infection, using a once daily oral dose of either 100 mg/kg for
5 days a week for 3 weeks or 30 mg/kg for 5 days a week for
8 weeks, starting∼50-70 days postinfection (Table 8). This was
a much more stringent assay, requiring the killing of bacteria in
a well-established, plateau phase of growth. In the initial 3 week
assay, both 1 and 2 were employed as internal reference stan-
dards for the two experiments, with the objective of identifying
lead compounds superior to both clinical trial drugs (2 was
about 10-fold more efficacious than 1 in this assay). Here,
pyridine analogues 92 (4-FPh) and 93 (4-OCF3Ph) respec-
tively showed 20-fold and 15-fold higher efficacies than 1, and

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8431
Table 7. Pharmacokinetic Parameters and Oral Bioavailability for Selected Analogues in Male Sprague-Dawley Rats Following a Single Dose
intravenous (5 mg/kg)a
oral (20 mg/kg)
compd
C0b (μg/mL)
Tmax (h)
t1/2 (h)
AUClastc(μg h/mL)
Cmax (μg/mL)
Tmax (h)
AUClastc(μg h/mL)
Fd (%)
3
3
74
93
8.7
0.083
0.083
0.083
0.167
>6
>6
33
35.9
12.1
6.97a
16.3
16
6
5
6
5
66.7
30.1
46
62
93
45
3.9
8.1
7.2
7.0
99
114
0.53a
3.9
129.4
29.4
24
a The intravenous dose for 99 was 1 mg/kg. b Maximum plasma concentration extrapolated to t = 0. c Area under the curve calculated to the last time
point; for 74, 93, and 114 this was 6 h but for 99 the last time point was 24 h. d Oral bioavailability, determined using dose normalized AUClast values.
Table 8. Chronic in Vivo Efficacy Data for Selected Analogues
was replaced by pyridine, pyridazine, pyrazine, or pyrimidine,
were generally more effective. Whereas two of the more hydro-
philic classes (20,60-pyrimidine and pyridazine) displayed better
solubilities at pH=7 but more modest potencies, the two para-
linked phenylpyridine classes in particular combined improved
solubilities (up to 1.4 mg/mL at low pH) with very high activ-
ities, both in vitro and in vivo. For these latter classes, addi-
tional lipophilic substitution on the terminal phenyl ring (e.g.,
3-F, 3-Cl) generally improved in vitro potency and efficacy in
the acute in vivo model.
Overall, ten compounds were substantially (>10-fold) more
efficacious than 1 in the mouse model of acute M. tb infection,
with five of these showing a >100-fold improvement over 1.
In several cases (93, 97, 99, 124), no CFUs could be detected
following the plating of undiluted lung homogenates, indicat-
ing complete sterilization. Pharmacokinetic assessments of
these compounds revealed lengthy half-lives and high expo-
sures in both plasma and lung tissue following oral dosing,
consistent with excellent oral bioavailabilities, and this was
established for four leading candidates. Two of these, pyr-
idines 93 and 99, were respectively 4-fold and 3-fold more
soluble than 1 at low pH and exhibited superior (3.7-6.2-fold
greater) efficacies compared to the clinical trial drug 2 in a
more stringent mouse model of chronic M. tb infection.
3 week study (100 mg/kg)
8 week study (30 mg/kg)
relative
fold CFU efficacy
relative
efficacy
relative
efficacy
fold CFU
compd reductiona (ratio vs 1) (ratio vs 2) reductiona (ratio vs 2)
1
157, 81b
1.0
0.07, 0.14b
2
2111, 593b 13, 7.3b 1.0
571, 1042b
1.0
24
32
74
92
93
99
114
271
1.7
4.6b
0.9
20
0.13
0.63b
0.07
1.5
371b
142
828
1.4
3156
1219b
15b
2.1b
3478, 6410b
3846b
1333
6.1, 6.2b
3.7b
2.3
a Fold reduction in lung CFUs in a mouse model of chronic TB
infection (see text). b Data from second experiment.
1.5-fold and 2.1-fold higher efficacies than 2, whereas the
trifluoromethyl pyridine analogues 24 and 32 and the 20-aza
isomer of 93 (74) had low activity. For the subsequent 8 week
study, the test compounds were evaluated against 2 only, also
in two separate experiments. Under these conditions, how-
ever, compound 74 demonstrated comparable activity to 2.
Pyridine analogue 93 (4-OCF3Ph) consistently showed the
best efficacy, about 6-fold greater than 2, while close analogue
99 (3-F-4-OCF3Ph) and pyrazine 114 also showed 3.7-fold
and 2.3-fold higher efficacies than 2, respectively.
Experimental Section
Combustion analyses were performed by the Campbell Micro-
analytical Laboratory, University of Otago, Dunedin, New
Zealand. Melting points were determined on an Electrothermal
IA9100 melting point apparatus and are as read. NMR spectra
were measured 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 electrospray ionization (HRESIMS) mass spec-
tra were determined on a Bruker micrOTOF-Q II mass spectro-
meter. Low-resolution atmospheric pressure chemical ionization
(APCI) mass spectra were measured for organic solutions on a
ThermoFinnigan Surveyor MSQ mass spectrometer, connected
to a Gilson autosampler. 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). Compounds of Tables 1-3 were isolated follow-
ing trituration in Et2O, unless otherwise indicated. Tested com-
pounds were g95% pure, as determined by combustion anal-
ysis, or by HPLC conducted on an Agilent 1100 system, using a
reversed-phase C8 column with diode array detection.
Conclusions
This study explored aza and diaza analogues of the biphe-
nyl class of 2-nitroimidazooxazines as a strategy for develop-
ing more solublecompoundsthatretained high antitubercular
activity, and efficient synthetic routes to a wide range of these
compounds were developed. The study was sparked by pre-
vious work describing replacement of one of the phenyl rings
by various five-membered heterocycles, which suggested that
potency and lipophilicity could be decoupled (to some extent).
The new aza analogues were appreciably less lipophilic than
the biphenyl class (ΔClogP values ranged from 0.9 to 3.75 units
lower), and there was a reasonable correlation between hydro-
philicity and measured aqueous solubility for the larger series
of para-linked biaryls. Bipyridine and pyridyl diazaheterocyc-
lic analogues provided the best solubility improvements (up
to 0.7 mg/mL at pH = 7) but displayed weak potencies in anti-
tubercular assays, as predicted by their very high hydrophili-
cities.
Several compounds in which the terminal phenyl ring was
replaced by pyridine retained high in vitro potencies against
both replicating and nonreplicating M. tb and showed im-
proved aqueous solubilities, particularly at low pH (e.g., 0.28
mg/mL for 28), although only two of these (trifluoromethyl
analogues 24 and 32) were significantly more efficacious than
1 in a mouse model of acute M. tb infection. Further analo-
gues, in which the phenyl ring proximal to the ether linkage
Compounds of Table 1. Procedure A. (6S)-2-Nitro-6-{[2-(3-
pyridinyl)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(9) (Scheme 1A). (6S)-6-[(2-Iodobenzyl)oxy]-2-nitro-6,7-dihy-
dro-5H-imidazo[2,1-b][1,3]oxazine10 (127) (0.100 g, 0.249 mmol)
and 3-pyridinylboronic acid (0.040 g, 0.325 mmol) were sus-
pended in toluene/EtOH (5 mL/2 mL), and then aqueous K2CO3

8432 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
(2 M, 1 mL) was added. The stirred mixture was purged with N2,
treated with Pd(dppf)Cl2 (9.5 mg, 0.013 mmol), and heated under
reflux in an N2 atmosphere for 30 min. The resulting mixture was
diluted with water (10 mL) and extracted with EtOAc (3 ꢁ 15 mL).
The dried (MgSO4) organic extracts were adsorbed onto silica gel
and chromatographed, eluting with EtOAc, to give 9 (0.085 g,
97%) as a light brown powder: mp 157-159 °C; 1H NMR
[(CD3)2SO] δ 8.59 (dd, J = 4.8, 1.6 Hz, 1 H), 8.55 (dd, J = 2.3,
0.8 Hz, 1 H), 7.98 (s, 1 H), 7.75 (ddd, J = 7.9, 2.2, 1.8 Hz, 1 H),
7.51-7.48 (m, 1 H), 7.45-7.40 (m, 3 H), 7.32-7.28 (m, 1 H),
4.57-4.48 (m, 3 H), 4.40(br d, J = 12.0 Hz, 1 H), 4.20-4.09 (m, 3
H). Anal. (C18H16N4O4) C, H, N.
evaporated to dryness, and the residue was chromatographed on
silica gel. Elution with 0-20% Et2O/petroleum ether first gave
foreruns, and then further elution with 20-50% Et2O/petroleum
ether gave the crude product, which was further chromatographed,
eluting with CH2Cl2 (foreruns) and then with 33% Et2O/CH2Cl2,
to give 136 (1.037 g, 64%) as a cream solid (following pentane
trituration): mp 70-71 °C; 1H NMR (CDCl3) δ 8.84 (br d, J = 1.0
Hz, 1 H), 8.04 (br d, J = 1.6 Hz, 1 H), 7.77 (dt, J = 8.3, 1.7 Hz, 2
H), 7.50 (br d, J=8.3 Hz, 2 H), 4.79 (d, J=5.9 Hz, 2 H), 1.74
(t, J = 6.0 Hz, 1 H); HRESIMS calcd for C13H10ClF3NO m/z
[M þ H]þ 290.0369, 288.0398, found 290.0372, 288.0400.
[4-(5-Fluoro-2-pyridinyl)phenyl]methanol (137). Reaction of
boronic acid 135 and 2-bromo-5-fluoropyridine (2.2 equiv) under
the Suzuki conditions described in procedure C (with 0.14 equiv
of Pd(dppf)Cl2 catalyst), followed by chromatography of the
product on silica gel, eluting with CH2Cl2 and 0-25% Et2O/
petroleum ether (foreruns) and then with 25-33% Et2O/petro-
leum ether, gave 137 (54%) as a cream solid (following pentane
trituration): mp 100-101 °C; 1H NMR (CDCl3) δ 8.54 (d, J =
2.9 Hz, 1 H), 7.94 (dt, J = 8.4, 1.9 Hz, 2 H), 7.72 (ddd, J = 8.8,
4.2, 0.5 Hz, 1 H), 7.50-7.43 (m, 3 H), 4.76 (d, J = 6.0 Hz, 2 H),
1.69 (t, J = 6.0 Hz, 1 H); HRESIMS calcd for C12H11FNO m/z
[M þ H]þ 204.0819, found 204.0824.
See Supporting Information for details of the syntheses of
related compounds of Table 1 from known10 iodobenzyl ethers
127, 128, or 129.
Procedure B. (6S)-2-Nitro-6-{[3-(2-pyridinyl)benzyl]oxy}-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazine (14) (Scheme 1B). A mix-
ture of (6S)-2-nitro-6-{[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine10 (130)
(0.101 g, 0.252 mmol) and 2-bromopyridine (0.055 g, 0.35 mmol) in
toluene (5 mL), EtOH (3 mL), and K2CO3 (2 M, 1 mL) was purged
with N2. Pd(dppf)Cl2 (8 mg, 0.011 mmol) was added, and the stirred
mixture was refluxed under N2 for 30 min and then partitioned
between EtOAc and water. The organic layer was dried and evap-
orated, and then column chromatography of the residue on
silica gel, eluting with a gradient of 1:1 hexanes:EtOAc to EtOAc,
gave 14 (74 mg, 83%) as a pale yellow gum: 1H NMR [(CD3)2SO]
δ 8.65 (ddd, J = 4.8, 1.8, 1.0 Hz, 1 H), 8.03-7.96 (m, 3 H), 7.92
(dt, J = 8.0, 1.1 Hz, 1 H), 7.87 (td, J = 8.0, 1.8 Hz, 1 H), 7.47 (t,
J = 7.6 Hz, 1 H), 7.40-7.33 (m, 2 H), 4.76 (d, J = 12.0 Hz, 1 H),
4.72 (d, J = 12.0 Hz, 1 H), 4.69 (dt, J = 11.9, 2.6Hz, 1 H), 4.49 (br
d, J = 11.9 Hz, 1 H), 4.33-4.20 (m, 3 H); HRFABMS calcd for
C18H17N4O4 m/z [M þ H]þ 353.1250, found 353.1247. HPLC
purity: 100%.
Procedure D. 2-[4-(Bromomethyl)phenyl]-3-chloro-5-(trifluoro-
methyl)pyridine (138). A solution of alcohol 136 (1.035 g, 3.60
mmol) and PPh3 (1.089 g, 4.15 mmol) in anhydrous CH2Cl2
(40 mL) was carefully treated with recrystallized N-bromosucci-
nimide (0.739 g, 4.15 mmol) (water bath cooling), and the mixture
was stirredatroom temperature for 4 h. The resulting solutionwas
added to excess petroleum ether at the top of a silica gel column
(30 g in petroleum ether), rinsing on with minimal extra CH2Cl2.
Elution with petroleum ether first gave foreruns, and then further
elution with 20% Et2O/petroleum ether gave 138 (1.219 g, 97%) as
1
a white solid that was used directly in the next step: H NMR
2-Bromo-5-(difluoromethoxy)pyridine (133). A mixture of
6-bromo-3-pyridinol (132) (2.40 g, 13.8 mmol), sodium chlorodi-
fluoroacetate (4.20 g, 27.5 mmol), and K2CO3 (2.40 g, 17.4 mmol)
in anhydrous DMF (20 mL) was stirred at 80 °C for 20 h, then
cooled, and partitioned between Et2O and water. The organic layer
was washed with water and brine, dried (MgSO4), and carefully
concentrated under reduced pressure to minimize evaporation of
the product. Column chromatography of the residue, eluting with
9:1 hexanes:Et2O, gave 13320 (1.98 g, 64%) as a colorless oil: 1H
NMR (CDCl3) δ 8.26 (d, J = 2.8 Hz, 1 H), 7.49 (d, J = 8.5 Hz, 1
H), 7.37 (dd, J = 8.5, 2.8 Hz, 1 H), 6.54 (t, JH-F = 72.2 Hz, 1 H).
APCI MS m/z 226, 224 [M þ H]þ.
(CDCl3) δ8.84 (br d, J= 1.0Hz, 1H), 8.04 (brd, J= 1.8 Hz, 1 H),
7.76 (dt, J = 8.4, 1.9 Hz, 2 H), 7.52 (dt, J = 8.3, 1.8 Hz, 2 H), 4.55
(s, 2 H); HRESIMS calcd for C13H9BrClF3N m/z [M þ H]þ
353.9504, 351.9532, 349.9554, found 353.9510, 351.9537, 349.9559.
2-[4-(Bromomethyl)phenyl]-5-fluoropyridine (139). Bromina-
tion of alcohol 137 using procedure D (with 1.2 equiv of PPh3
and NBS) for 3 h, followed by chromatography of the product
directly on silica gel, eluting with pentane (foreruns) and then with
20-50% Et2O/pentane, gave 139 (87%) as a white solid that was
used directly in the next step: 1H NMR (CDCl3) δ 8.54 (d, J = 2.9
Hz, 1 H), 7.92 (dt, J = 8.4, 1.9 Hz, 2 H), 7.72 (ddd, J = 8.7, 4.3, 0.4
Hz, 1 H), 7.52-7.43 (m, 3 H), 4.54 (s, 2 H); HRESIMS calcd for
C12H10BrFN m/z [M þ H]þ 267.9955, 265.9975, found 267.9959,
265.9979.
(6S)-6-({3-[5-(Difluoromethoxy)-2-pyridinyl]benzyl}oxy)-2-
nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (16). Reaction
of boronate ester 130 and bromide 133 under the Suzuki coupling
conditions described in procedure B gave 16 (76%) as a white
solid: mp 120-122 °C; 1H NMR [(CD3)2SO] δ 8.55 (d, J = 2.8
Hz, 1 H), 8.02-7.94 (m, 4 H), 7.74 (dd, J = 8.7, 2.8 Hz, 1 H), 7.48
Procedure E. (6S)-6-({4-[3-Chloro-5-(trifluoromethyl)-2-pyridinyl]-
benzyl}oxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (25). A
solution of (6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-ol23 (140) (0.644 g, 3.48 mmol) and bromide 138 (1.217 g, 3.47
mmol) in anhydrous DMF (13 mL) under N2 at 0 °C was treated
with 60% NaH (0.184 g, 4.60 mmol), then quickly degassed, and
resealed under N2. After being stirred at room temperature for 2.5 h,
the reaction was cooled (CO2/acetone), quenched with ice/aqueous
NaHCO3 (20 mL), added to brine (100 mL), and extracted with
CH2Cl2 (7 ꢁ 100 mL). The combined extracts were evaporated
to dryness, and the residue was chromatographed on silica gel.
Elution with 0-1% EtOAc/CH2Cl2 first gave foreruns, and then
further elution with 7-10% EtOAc/CH2Cl2 gave 25 (1.327 g,
(t, J = 7.6 Hz, 1 H), 7.39 (br d, J = 7.6 Hz, 1 H), 7.35 (t, JH-F
=
73.4 Hz, 1 H), 4.76 (d, J = 12.0 Hz, 1 H), 4.72 (d, J = 12.0 Hz, 1
H), 4.68 (dt, J = 12.0, 2.5 Hz, 1 H), 4.48 (br d, J = 11.9 Hz, 1 H),
4.32-4.21 (m, 3 H). Anal. (C19H16F2N4O5) C, H, N.
See Supporting Information for details of the syntheses of
related compounds of Table 1 from known10 boronic acidpinacol
esters 130 or 131.
Procedure C. {4-[3-Chloro-5-(trifluoromethyl)-2-pyridinyl]phenyl}-
methanol (136) (Scheme 1C). A stirred mixture of 4-(hydroxymethyl)-
phenylboronic acid (135) (0.850 g, 5.59 mmol) and Pd(dppf)Cl2
(0.826 g, 1.13 mmol) in toluene (56 mL) and EtOH (28 mL) was
degassed for 15 min (vacuum pump), and then N2 was added.
Aqueous Na2CO3 (14 mL of 2 M, 28 mmol) was added by syringe,
the stirredmixturewasagaindegassedfor15min, andthenN2 was
added. 2,3-Dichloro-5-(trifluoromethyl)pyridine (2.41 g, 11.2 mmol)
was added by syringe, and the resulting mixture was stirred at 88 °C
for 3 h. The cooled mixture was then diluted with aqueous NaHCO3
(100 mL) andextractedwithCH2Cl2 (5ꢁ 100 mL). The extracts were
1
84%) as a cream solid: mp (CH2Cl2/pentane) 159-160 °C; H
NMR (CDCl3) δ 8.84 (dd, J = 1.8, 0.7 Hz, 1 H), 8.05 (d, J = 1.6
Hz, 1 H), 7.78 (br d, J = 8.3 Hz, 2 H), 7.45 (br d, J = 8.3 Hz, 2 H),
7.39(s, 1 H), 4.82 (d, J = 12.4Hz, 1 H), 4.71 (d, J = 12.4 Hz, 1 H),
4.64 (ddd, J = 12.1, 3.6, 1.9 Hz, 1 H), 4.37 (dd, J = 12.1, 1.3 Hz, 1
H), 4.23-4.11 (m, 3 H). Anal. (C19H14ClF3N4O4) C, H, N.
(6S)-6-{[4-(5-Fluoro-2-pyridinyl)benzyl]oxy}-2-nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]oxazine (28). Reaction of alcohol 140 with
bromide 139 and NaH using procedure E for 135 min, followed by

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8433
chromatography of the product on silica gel, eluting with 0-6%
EtOAc/CH2Cl2 (foreruns) and then with 7-10% EtOAc/CH2Cl2,
gave crude material, which was further chromatographed, eluting
with 0-67% EtOAc/petroleum ether (foreruns) and then with
EtOAc and 30% EtOAc/CH2Cl2, to give 28 (0.357 g, 74%) as a
cream solid: mp (CH2Cl2/pentane) 180-181 °C; 1H NMR
(CDCl3) δ 8.54 (d, J = 2.9 Hz, 1 H), 7.95 (dt, J = 8.4, 1.9 Hz,
2 H), 7.72 (ddd, J = 8.8, 4.2, 0.4 Hz, 1 H), 7.48 (ddd, J = 8.7, 8.1,
2.9 Hz, 1 H), 7.41 (br d, J = 8.4 Hz, 2 H), 7.37 (s, 1 H), 4.79 (d, J =
12.2 Hz, 1 H), 4.68 (d, J = 12.2 Hz, 1 H), 4.61 (ddd, J = 12.1, 3.7,
1.9 Hz, 1 H), 4.35 (dd, J = 12.1, 1.5 Hz, 1 H), 4.20-4.09 (m, 3 H).
Anal. (C18H15FN4O4) C, H, N.
Compounds of Table 2. 2-Bromo-6-(chloromethyl)pyridine (142)
(Scheme 2A). A solution of (6-bromo-2-pyridinyl)methanol (141)
(3.74 g, 19.9 mmol) in CHCl3 (250 mL) at 0 °C was treated with
SOCl2 (26 mL, 356 mmol). The stirred mixture was refluxed for 1 h,
then cooled, poured into ice-water, and extracted into iPr2O. The
combined extracts were washed with brine, dried (MgSO4), and
evaporated to dryness to give 14225 (3.42 g, 83%) as a yellow oil: 1H
NMR (CDCl3) δ 7.59 (t, J = 7.7 Hz, 1 H), 7.47 (d, J = 7.4 Hz,
1 H), 7.44 (d, J = 7.9 Hz, 1 H), 4.63 (s, 2 H); HREIMS calcd for
C6H5BrClN m/z (Mþ) 208.9244, 206.9273, 204.9294, found
208.9233, 206.9273, 204.9294.
Procedure F. (6S)-6-[(6-Bromo-2-pyridinyl)methoxy]-2-nitro-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (143). A solution of
alcohol 140 (2.65 g, 14.3 mmol) and chloride 142 (3.22 g, 15.7
mmol) in anhydrous DMF (70 mL) at -5 °C was treated with 60%
NaH (0.74 g, 18.5 mmol), and the resulting mixture was stirred at
room temperature for 2 h. The reaction was quenched with water
(300 mL) and extracted with EtOAc, and the combined extracts
were dried (MgSO4) and evaporated to dryness. Column chroma-
tography of the residue, eluting with 5% MeOH/EtOAc, gave
crude 143 (2.56 g, 50%) as a pale brown solid (following Et2O/
EtOAc trituration): mp 163-165 °C; 1H NMR [(CD3)2SO] δ 8.02
(s, 1 H), 7.75 (t, J = 7.7 Hz, 1 H), 7.56 (d, J = 7.6 Hz, 1 H), 7.40 (d,
J =7.5 Hz, 1 H), 4.76-4.65 (m, 3 H), 4.49 (br d, J =12.0Hz, 1H),
4.35-4.21 (m, 3 H); HRFABMS calcd for C12H12BrN4O4 m/z
[M þ H]þ 357.0022, 355.0042, found 357.0013, 355.0034.
J=5.1, 1.3, 0.6Hz, 1 H), 4.75 (d, J=14.1 Hz, 1 H), 4.73-4.64(m,
2 H), 4.48(brd, J=11.9 Hz, 1 H), 4.32-4.20 (m, 3 H); HRESIMS
calcd for C12H12BrN4O4 m/z [M þ H]þ 357.0017, 355.0036, found
357.0017, 355.0038.
(6S)-2-Nitro-6-({2-[4-(trifluoromethyl)phenyl]-4-pyridinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (50). Reac-
tion of bromide 146 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described in procedure A
for 6 h, followed by chromatography of the product on silica gel,
eluting with EtOAc, gave 50 (42%) as a white solid: mp 199-
202 °C; 1H NMR [(CD3)2SO] δ 8.68 (dd, J = 5.0, 0.5 Hz, 1 H),
8.26 (br d, J = 8.1 Hz, 2 H), 8.03 (s, 1 H), 7.90 (br s, 1 H), 7.84 (br
d, J = 8.2 Hz, 2 H), 7.36 (br d, J = 5.0 Hz, 1 H), 4.83 (d, J = 13.8
Hz, 1 H), 4.79 (d, J = 13.8 Hz, 1 H), 4.72 (dt, J = 12.0, 2.6 Hz, 1
H), 4.51 (br d, J = 11.5 Hz, 1 H), 4.36-4.23 (m, 3 H). Anal.
(C19H15F3N4O4 0.25H2O) C, H, N. HPLC purity: 100%.
3
See Supporting Information for details of the syntheses of
related compounds 51-56 of Table 2 from bromide 146.
(6S)-6-[(5-Bromo-3-pyridinyl)methoxy]-2-nitro-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (148) (Scheme 2C). Reaction of alcohol
140 with 3-bromo-5-(chloromethyl)pyridine hydrochloride26 (147)
(1.0 equiv) and NaH (2.4 equiv) using procedure F for 16 h, fol-
lowed by chromatography of the product on silica gel, eluting with
2.5% MeOH/CH2Cl2, gave 148 (30%) as a light yellow solid: mp
169-171 °C; 1H NMR [(CD3)2SO] δ 8.64 (d, J = 2.3 Hz, 1 H),
8.52 (d, J = 1.7 Hz, 1 H), 8.03 (s, 1 H), 7.97 (br t, J = 2.0 Hz, 1 H),
4.73 (d, J = 12.6 Hz, 1 H), 4.70-4.64 (m, 2 H), 4.48 (br d, J = 12.0
Hz, 1 H), 4.32-4.20 (m, 3 H). Anal. (C12H11BrN4O4) C, H, N, Br.
(6S)-2-Nitro-6-({5-[4-(trifluoromethyl)phenyl]-3-pyridinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (57). Reac-
tion of bromide 148 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described inprocedure A for
15 min, followed by chromatography of the product on silica gel,
eluting with 0-5% MeOH/EtOAc, gave 57 (92%) as a light
1
brown solid: mp 227-229 °C; H NMR [(CD3)2SO] δ 8.89 (d,
J = 2.3 Hz, 1 H), 8.60 (d, J = 1.9 Hz, 1 H), 8.04 (t, J = 2.1 Hz, 1
H), 8.02 (s, 1 H), 7.94 (br d, J = 8.2 Hz, 2 H), 7.85 (br d, J = 8.3
Hz, 2 H), 4.81 (d, J = 12.4 Hz, 1 H), 4.77 (d, J = 12.4 Hz, 1 H),
4.70 (dt, J = 12.0, 2.5 Hz, 1 H), 4.50 (br d, J = 11.9 Hz, 1 H),
(6S)-2-Nitro-6-({6-[4-(trifluoromethyl)phenyl]-2-pyridinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (43). Reac-
tion of bromide 143 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described in procedure A,
followed by chromatography of the product on silica gel, eluting
with 5% MeOH/EtOAc, gave 43 (53%) as a light yellow powder:
mp 170-172 °C; 1H NMR [(CD3)2SO] δ 8.29 (br d, J = 8.1 Hz, 2
H), 8.03 (s, 1 H), 7.99 (dd, J = 7.9, 1.1 Hz, 1 H), 7.95 (t, J = 7.6
Hz, 1 H), 7.85 (br d, J = 8.2 Hz, 2 H), 7.42 (dd, J = 7.4, 1.0 Hz,
1 H), 4.86 (d, J = 13.4 Hz, 1 H), 4.83 (d, J = 13.4 Hz, 1 H), 4.74
(dt, J = 12.0, 2.6 Hz, 1 H), 4.52 (br d, J = 12.0 Hz, 1 H),
4.41-4.33 (m, 2 H), 4.27 (dd, J = 13.2, 2.9 Hz, 1 H). Anal.
4.35-4.22 (m, 3 H). Anal. (C19H15F3N4O4 0.25H2O) C, H, N, F.
3
HPLC purity: 99.8%.
See Supporting Information for details of the syntheses of
related compounds 58-63 of Table 2 from bromide 148.
(4-Bromo-2-pyridinyl)methanol (150) (Scheme 2D). Protected
from moisture, trifluoroacetic anhydride (17 mL, 122 mmol) was
carefully and slowly added to 4-bromo-2-methylpyridine 1-oxide28
(149) (4.33 g, 23.0 mmol). The orange mixture was stirred at room
temperature for 30 min, refluxed for 30 min, and then cooled.
Saturated aqueous NaHCO3 was added (to pH = 8), and the re-
sulting red solution was stirred at room temperature for 16 h. The
mixture was extracted with CH2Cl2, and the combined organic
layers were washed with brine and dried (MgSO4). Evaporation
of the solvent gave15027 (3.38g, 78%) as a dark orange-brown oil
that was used directly in the next step: 1H NMR (CDCl3) δ 8.38
(d, J = 5.3 Hz, 1 H), 7.48 (dd, J = 1.7, 0.7 Hz, 1 H), 7.38 (ddd,
J = 5.3, 1.2, 0.7 Hz, 1 H), 4.75 (s, 2 H), 3.32 (s, 1 H).
4-Bromo-2-(chloromethyl)pyridine (151). A solution of 150
(3.38 g, 18.0 mmol) in CH2Cl2 (210 mL) at 0 °C was carefully
treated with SOCl2 (21 mL, 288 mmol). The mixture was stirred
at room temperature for 20 h, and then saturated aqueous
NaHCO3 was added. The organic layer was washed with brine,
dried (MgSO4), and evaporated to dryness to give 151 (2.94 g,
79%) as a yellow oil: 1H NMR (CDCl3) δ 8.47 (d, J = 5.3 Hz, 1
H), 7.68 (d, J = 1.7 Hz, 1 H), 7.42 (dd, J = 5.3, 1.8 Hz, 1 H), 4.64
(s, 2 H); HRFABMS calcd for C6H6BrClN m/z [M þ H]þ
209.9322, 207.9352, 205.9372, found 209.9319, 207.9356,
205.9368.
(C19H15F3N4O4 0.25H2O) C, H, N. HPLC purity: 98.9%.
3
See Supporting Information for details of the syntheses of
related compounds 44-49 of Table 2 from bromide 143.
2-Bromo-4-(bromomethyl)pyridine Hydrobromide (145)
(Scheme 2B). A solution of (2-bromo-4-pyridinyl)methanol (144)
(2.00 g, 10.6 mmol) in anhydrous CH2Cl2 (10 mL) at 0 °C was
treated with SOBr2 (0.90 mL, 11.6 mmol). The resulting mixture
was stirred at room temperature for 15 h, then the solvent was
evaporated, and the residue was crystallized from MeOH/Et2O,
to give 14530 (2.77 g, 79%) as a white solid: mp 172-175 °C; 1H
NMR [(CD3)2SO] δ 8.39 (d, J = 5.0 Hz, 1 H), 7.74 (br d, J = 0.9
Hz, 1 H), 7.51 (dd, J = 5.0, 1.4 Hz, 1 H), 4.66 (s, 2 H). APCI MS
m/z 254, 252, 250 [M þ H]þ.
(6S)-6-[(2-Bromo-4-pyridinyl)methoxy]-2-nitro-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (146). Reaction of alcohol 140 with bro-
mide salt 145 (1.2 equiv) and NaH (2.4 equiv) using procedure F
for 1 h, followed by chromatography of the product on silica gel,
eluting with 2% MeOH/CH2Cl2, gave 146 (65%) as a light yellow
solid: mp 150-152 °C; 1H NMR [(CD3)2SO] δ 8.34 (dd, J = 5.1,
0.3 Hz, 1 H), 8.02 (s, 1 H), 7.51 (br d, J = 0.5 Hz, 1 H), 7.34 (ddd,
(6S)-6-[(4-Bromo-2-pyridinyl)methoxy]-2-nitro-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (152). Reaction of alcohol 140 with
chloride 151 (0.91 equiv) and NaH (1.1 equiv) using procedure F

8434 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
for 3 h, followed by chromatography of the product on silica gel,
eluting with 5% MeOH/EtOAc, gave 152 (54%) as a light yellow
solid: mp 155-158 °C; 1H NMR [(CD3)2SO] δ 8.41 (d, J = 5.3 Hz,
1 H), 8.01 (s, 1 H), 7.59 (dd, J = 5.3, 1.9 Hz, 1 H), 7.55 (d, J = 1.5
Hz, 1 H), 4.77 (d, J = 13.6 Hz, 1 H), 4.72 (d, J = 13.5 Hz, 1 H),
4.70 (dt, J = 12.0, 2.6 Hz, 1 H), 4.50 (br d, J = 12.0 Hz, 1 H),
-20 °C (over 2 h). HCl (2 N, 2.6 mL) was added, and the mixture
was stirred at room temperature for 40 min and then diluted with
water (40 mL) and extracted with EtOAc (5 ꢁ 50 mL). The extracts
were washed with brine (50 mL) and then evaporated to dryness.
The residue was triturated in hexane (∼5 mL), cooled to -20 °C,
and rapidly filtered cold (washing with pentane cooled to -20 °C)
to give 16733 (0.391 g, 64%) as a white solid: mp 150-152 °C (lit.33
mp 160-162); 1H NMR (CDCl3) δ 8.00 (d, J = 8.4 Hz, 1 H), 7.24
(m, 1 H), 7.18 (ddq, J = 8.4, 2.2, 1.1 Hz, 1 H), 5.25 (s, 2 H).
See Supporting Information for details of the syntheses of
related arylboronic acids 168-171 from halobenzenes 163-166.
Procedure H. (6S)-6-({5-[2-Chloro-4-(trifluoromethoxy)phenyl]-
2-pyridinyl}methoxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazine (77). A stirred mixture of bromide 156 (50.2 mg, 0.141
mmol), boronic acid 167 (63.5 mg, 0.264 mmol), and Pd(dppf)Cl2
(19.6 mg, 0.027 mmol) in toluene (2 mL) and EtOH (1 mL) was
degassed for 5 min (vacuum pump), and then N2 was added. An
aqueous solution of Na2CO3 (0.40 mL of 2 M, 0.80 mmol) was
added by syringe, the stirred mixture was again degassed for
5 min, and then N2 was added. The resulting mixture was stirred at
90 °C for 100 min and then cooled, diluted with aqueous NaHCO3
(50 mL), and extracted with CH2Cl2 (4ꢁ 50 mL). The extracts were
evaporated to dryness, and the residue was chromatographed on
silica gel. Elution with 0-0.75% MeOH/CH2Cl2 first gave fore-
runs, and then further elution with 0.75% MeOH/CH2Cl2 gave
77 (52 mg, 77%) as a cream solid: mp (CH2Cl2/pentane) 144-146
°C; 1H NMR (CDCl3) δ 8.61 (dd, J = 2.3, 0.7 Hz, 1 H), 7.80 (dd,
J = 8.0, 2.3 Hz, 1 H), 7.46 (d, J = 8.0 Hz, 1 H), 7.42 (s, 1 H), 7.41
(m, 1 H), 7.37(d, J = 8.5 Hz, 1 H), 7.25(m, 1 H), 4.89 (d, J = 13.0
Hz, 1 H), 4.82 (d, J = 13.0 Hz, 1 H), 4.73 (ddd, J = 12.2, 3.4, 2.0
Hz, 1 H), 4.41 (dd, J = 12.2, 1.2 Hz, 1 H), 4.35-4.22 (m, 3 H).
Anal. (C19H14ClF3N4O5) C, H, N.
4.37-4.21 (m, 3 H). Anal. (C12H11BrN4O4 0.25H2O) C, H, N, Br.
3
HPLC purity: 99.8%.
(6S)-2-Nitro-6-({4-[4-(trifluoromethyl)phenyl]-2-pyridinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (64). Reac-
tion of bromide 152 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described in procedure A
for 1 h, followed by chromatography of the product on silica gel,
eluting with 5% MeOH/EtOAc, gave 64 (87%) as a yellow solid:
mp 105-109 °C; 1H NMR [(CD3)2SO] δ 8.64 (dd, J = 5.1, 0.4 Hz,
1 H), 8.01 (s, 1 H), 7.95 (br d, J = 8.2 Hz, 2 H), 7.86 (br d, J = 8.3
Hz, 2 H), 7.69 (dd, J = 5.2, 1.9 Hz, 1 H), 7.64 (br s, 1 H), 4.85 (d,
J = 13.4 Hz, 1 H), 4.80 (d, J = 13.4 Hz, 1 H), 4.71 (dt, J = 11.8,
2.6 Hz, 1 H), 4.52 (br d, J = 12.0 Hz, 1 H), 4.40 (m, 1 H), 4.35 (dt,
J = 13.5, 2.1 Hz, 1 H), 4.26 (dd, J = 13.5, 3.2 Hz, 1 H). Anal.
(C19H15F3N4O4 H2O) C, H, N. HPLC purity: 97.2%.
3
See Supporting Information for details of the syntheses of
related compounds 65-70 of Table 2 from bromide 152.
5-Bromo-2-(bromomethyl)pyridine (154) (Scheme 3A). Bromi-
nation of (5-bromo-2-pyridinyl)methanol (153) using procedure
D (with 1.2 equiv of PPh3 and NBS) for 3.5 h, followed by chro-
matography of the product directly on silica gel, eluting with
petroleum ether (foreruns) and then with 10-30% Et2O/petro-
leum ether, gave 15431 (83%) as a lachramatory lilac oil that was
used directly in the next step: 1H NMR (CDCl3) δ 8.63 (d, J =
2.2 Hz, 1 H), 7.82 (dd, J = 8.3, 2.4 Hz, 1 H), 7.35 (d, J = 8.4 Hz,
1 H), 4.50 (s, 2 H).
See Supporting Information for details of alternative reaction
conditions to provide 154.
See Supporting Information for details of the syntheses of
related compounds 78-81 of Table 2 from bromide 156.
{5-[3-Fluoro-4-(trifluoromethoxy)phenyl]-2-pyridinyl}methanol
(172) (Scheme 3D). A stirred mixture of bromide 153 (0.753 g,
4.00 mmol), 3-fluoro-4-(trifluoromethoxy)phenylboronic acid
(170) (1.165 g, 5.20 mmol), and Pd(dppf)Cl2 (0.366 g, 0.50 mmol)
in toluene (40 mL) and EtOH (20 mL) was degassed for 15 min
(vacuum pump), and then N2 was added. An aqueous solution of
Na2CO3 (10 mL of 2 M, 20.0 mmol) was added by syringe, the
stirred mixture was again degassed for 15 min, and then N2 was
added. The resulting mixture was stirred at 89 °C for 2 h and then
cooled, diluted with aqueous NaHCO3 (120 mL), and extracted
with CH2Cl2 (6 ꢁ 100 mL). The extracts were evaporated to dry-
ness, and the residue was chromatographed on silica gel. Elution
with 50-75% CH2Cl2/petroleum ether first gave foreruns, and then
further elution with 75% CH2Cl2/petroleum ether and 0-0.5%
MeOH/CH2Cl2 gave 172 (0.687 g, 60%) as a light yellow-brown
solid: mp 51-53 °C; 1H NMR (CDCl3) δ 8.76 (br d, J = 1.9 Hz,
1 H), 7.84 (dd, J = 8.1, 2.3 Hz, 1 H), 7.46-7.34 (m, 4 H), 4.83 (d,
J = 5.1 Hz, 2 H), 3.47 (t, J = 5.2 Hz, 1 H); HRESIMS calcd for
C13H10F4NO2 m/z [M þ H]þ 288.0642, found 288.0641.
(6S)-6-[(5-Bromo-2-pyridinyl)methoxy]-2-nitro-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (156). Reaction of alcohol 140 with bro-
mide 154 and NaH (1.4 equiv) using procedure E for 3 h, followed
by chromatography of the product on silica gel, eluting with 0-
0.5% MeOH/CH2Cl2 (foreruns) and then with 0.5-1% MeOH/
CH2Cl2, gave 156 (81%) as a pale yellow solid: mp 171-172 °C;
1H NMR [(CD3)2SO] δ 8.64 (dd, J = 2.3, 0.4 Hz, 1 H), 8.04 (dd,
J = 8.4, 2.4 Hz, 1 H), 8.02 (s, 1 H), 7.35 (dd, J = 8.4, 0.4 Hz, 1 H),
4.76-4.65 (m, 3 H), 4.49 (br d, J = 12.0 Hz, 1 H), 4.35-4.28 (m, 2
H), 4.24 (dd, J=13.7, 3.5 Hz, 1 H). Anal. (C12H11BrN4O4) C,H,N.
See Supporting Information for details of alternative reaction
conditions to provide 156.
(6S)-2-Nitro-6-({5-[4-(trifluoromethyl)phenyl]-2-pyridinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (71). Reac-
tion of bromide 156 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described in procedure A,
followed by chromatography of the product on silica gel, eluting
with 5% MeOH/EtOAc, gave 71 (84%) as a light yellow powder:
mp 204-207 °C; 1H NMR [(CD3)2SO] δ 8.91 (d, J = 1.8 Hz, 1 H),
8.17 (dd, J = 8.1, 2.4 Hz, 1 H), 8.03 (s, 1 H), 7.96 (br d, J = 8.2 Hz,
2H), 7.85(brd, J=8.3Hz, 2H), 7.50 (d, J= 8.2Hz, 1H), 4.83(d,
J = 13.2 Hz, 1 H), 4.79 (d, J = 13.3 Hz, 1 H), 4.73 (dt, J = 12.0,
2.7 Hz, 1 H), 4.52 (br d, J = 12.0 Hz, 1 H), 4.39-4.24 (m, 3 H).
Anal. (C19H15F3N4O4) C, H, N, F.
2-(Bromomethyl)-5-[3-fluoro-4-(trifluoromethoxy)phenyl]-
pyridine (173). Bromination of alcohol 172 using procedure D
(with 1.2 equiv of PPh3 and NBS) for 3 h, followed by chroma-
tography of the product directly on silica gel, eluting with petro-
leum ether (foreruns) and then with 10-20% Et2O/pentane, gave
173 (75%) as a white solid that was used directly in the next step: 1H
NMR (CDCl3) δ 8.76 (dd, J = 2.4, 0.6 Hz, 1 H), 7.84 (dd, J = 8.1,
2.4 Hz, 1 H), 7.54 (dd, J = 8.0, 0.6 Hz, 1 H), 7.46-7.33 (m, 3 H),
4.60 (s, 2 H); HRESIMS calcd for C13H9BrF4NO m/z [M þ H]þ
351.9778, 349.9798, found 351.9778, 349.9798.
See Supporting Information for details of the syntheses of
related compounds 72-76, 82, and 84-89 of Table 2 from
bromide 156.
Procedure G. 2-Chloro-4-(trifluoromethoxy)phenylboronic Acid
(167) (Scheme 3C). Triisopropyl borate (0.72 mL, 3.12 mmol)
and 2-chloro-1-iodo-4-(trifluoromethoxy)benzene (162) (0.816 g,
2.53 mmol) were successively added via syringe to a mixture of
anhydrous toluene (4 mL) and anhydrous distilled THF (1 mL)
under N2, and the mixture was cooled to -78 °C. nBuLi (1.20 mL
of a 2.5 M solution in hexanes, 3.00 mmol) was added dropwise
over 30 min to the stirred solution (at -78 °C), and the mixture was
stirred at -78 °C for an additional 4 h and then slowly warmed to
(6S)-6-({5-[3-Fluoro-4-(trifluoromethoxy)phenyl]-2-pyridinyl}-
methoxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (80).
Reaction of alcohol 140 with bromide 173 (1.04 equiv) and NaH
using procedure E, followed by chromatography of the product
on silica gel, eluting with 0-0.75% MeOH/CH2Cl2 (foreruns)
and then with 0.75-1.5% MeOH/CH2Cl2, gave 80 (89%) as a
light yellow solid: mp (CH2Cl2/pentane) 182-184 °C; 1H NMR

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8435
(CDCl3) δ 8.74 (dd, J = 2.3, 0.7 Hz, 1 H), 7.86 (dd, J = 8.1, 2.4
Hz, 1 H), 7.48-7.38 (m, 4 H), 7.35 (ddd, J = 8.4, 2.2, 1.0 Hz, 1 H),
4.87 (d, J = 13.0 Hz, 1 H), 4.81 (d, J = 13.0 Hz, 1 H), 4.70 (ddd,
J = 12.2, 3.5, 1.5 Hz, 1 H), 4.40 (dd, J = 12.2, 1.4 Hz, 1 H),
4.33-4.20 (m, 3 H). Anal. (C19H14F4N4O5) C, H, N.
4.72-4.62 (m, 3 H), 4.47 (br d, J = 11.8 Hz, 1 H), 4.31-4.19 (m, 3
H). Anal. (C12H11BrN4O4) C, H, N.
See Supporting Information for details of the similar synthe-
sis of chloride 161.
(6S)-2-Nitro-6-({6-[4-(trifluoromethyl)phenyl]-3-pyridinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (90). Reac-
tion of bromide 160 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described in procedure A for
20 min, followed by chromatography of the product on silica gel,
eluting with EtOAc, gave 90 (51%) as a white solid: mp 267-
270 °C; 1H NMR [(CD3)2SO] δ 8.67 (d, J = 1.7 Hz, 1 H), 8.30 (br
d, J = 8.1 Hz, 2 H), 8.07 (br d, J = 8.1 Hz, 1 H), 8.03 (s, 1 H), 7.87
(dd, J = 8.3, 2.2 Hz, 1 H), 7.85 (br d, J = 8.7 Hz, 2 H), 4.79 (d, J =
12.4 Hz, 1 H), 4.75 (d, J = 12.4 Hz, 1 H), 4.71 (dt, J = 12.0, 2.5 Hz,
1 H), 4.50 (br d, J = 11.7 Hz, 1 H), 4.34-4.22 (m, 3 H). Anal.
[5-(Trifluoromethyl)[2,30-bipyridin]-60-yl]methanol (175)
(Scheme 3E). A stirred mixture of 2-chloro-5-(trifluoromethyl)-
pyridine (0.674 g, 3.71 mmol), 6-(hydroxymethyl)-3-pyridinyl-
boronic acid (174) (0.124 g, 0.814 mmol), and Pd(dppf)Cl2 (0.115
g, 0.157 mmol) in toluene (8 mL) and EtOH (4 mL) was degassed
for 8 min (vacuum pump), and then N2 was added. An aqueous
solution of Na2CO3 (2 mL of 2 M, 4 mmol) was added by syringe,
the stirred mixture was again degassed for 8 min, then N2 was
added, and the resulting mixture was stirred at 88 °C for 4 h. The
cooled mixture was then diluted withaqueous NaHCO3 (100 mL)
and extracted with CH2Cl2 (5 ꢁ 80 mL). The extracts were evap-
orated to dryness, and the residue was chromatographed on
silica gel. Elution with 0-30% EtOAc/CH2Cl2 first gave fore-
runs, and then further elution with 30-40% EtOAc/CH2Cl2 gave
the crude product, which was rechromatographed on silica gel,
eluting with Et2O, to give 175 (54 mg, 26%) as a cream solid: mp
(CH2Cl2/pentane) 99-100 °C; 1H NMR (CDCl3) δ 9.20 (d, J =
1.9 Hz, 1 H), 8.98 (dd, J = 1.3, 0.8 Hz, 1 H), 8.39 (dd, J = 8.2, 2.3
Hz, 1 H), 8.03 (dd, J = 8.4, 1.9 Hz, 1 H), 7.88 (d, J = 8.3 Hz, 1 H),
7.41 (dd, J=8.2, 0.6 Hz, 1 H), 4.86 (d, J=5.1 Hz, 2 H), 3.55
(t, J = 5.2 Hz, 1 H); HRESIMS calcd for C12H10F3N2O m/z
[M þ H]þ 255.0740, found 255.0735.
(C19H15F3N4O4 0.25H2O) C, H, N, F. HPLC purity: 98.9%.
3
See Supporting Information for details of the syntheses of
related compounds 91-103 of Table 2 from bromide 160 and the
alternative synthesis of 93 from chloride 161.
Compounds of Table 3. (6S)-6-[(6-Chloro-3-pyridazinyl)methoxy]-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (178) (Scheme 4A).
NaH (60%, 0.304 g, 7.60 mmol) was added to a solution of
alcohol 140 (0.893 g, 4.82 mmol) in anhydrous DMF (20 mL) at
0 °C. The mixture was cooled to -42 °C, and a solution of 3-
(bromomethyl)-6-chloropyridazine41 (177) (1.05 g, 5.06 mmol) in
anhydrous DMF (5 mL) was added. The resulting mixture was
stirred at -42 °C for 2 h and then quenched with ice and extracted
with EtOAc (200 mL). The organic layer was dried (MgSO4) and
evaporated, and then column chromatography of the residue on
silica gel, eluting with a gradient of 1:1 hexanes:EtOAc to EtOAc,
60-(Bromomethyl)-5-(trifluoromethyl)-2,30-bipyridine (176). Bro-
mination of alcohol 175 using procedure D (with 1.26 equiv of PPh3
and NBS) for 3 h, followed by chromatography of the product
directly on silica gel, eluting with 0-30% Et2O/petroleum ether
(foreruns) and then with 30% Et2O/petroleum ether, gave 176
(72%) as a lachrymatory white solid that was used directly in the
next step: 1H NMR (CDCl3) δ 9.20 (dd, J = 2.3, 0.6 Hz, 1 H),
8.98 (dd, J = 1.3, 0.9 Hz, 1 H), 8.39 (dd, J = 8.2, 2.3 Hz, 1 H),
8.04 (ddd, J = 8.4, 2.3, 0.4 Hz, 1 H), 7.88 (d, J = 8.3 Hz, 1 H),
7.59 (dd, J = 8.2, 0.7 Hz, 1 H), 4.62 (s, 2 H); HRESIMS calcd
for C12H9BrF3N2 m/z [M þ H]þ 318.9876, 316.9896, found
318.9885, 316.9903.
1
gave 178 (0.843 g, 56%) as a white solid: mp 180-184 °C; H
NMR [(CD3)2SO] δ 8.02 (s, 1 H), 7.93 (d, J = 8.8 Hz, 1 H), 7.74
(d, J = 8.9 Hz, 1 H), 4.97 (d, J = 13.2 Hz, 1 H), 4.94 (d, J = 13.2
Hz, 1 H), 4.69 (dt, J = 12.0, 2.6 Hz, 1 H), 4.50 (br d, J = 12.1 Hz,
1 H), 4.39-4.30 (m, 2 H), 4.25 (dd, J = 13.5, 3.3 Hz, 1 H). Anal.
(C11H10ClN5O4) C, H, N.
Procedure I. (6S)-2-Nitro-6-({6-[4-(trifluoromethyl)phenyl]-
3-pyridazinyl}methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(104). A mixture of chloride 178 (0.100 g, 0.32 mmol) and
4-(trifluoromethyl)phenylboronic acid (0.073 g, 0.38 mmol) in
toluene (5 mL), EtOH (3 mL), and aqueous K2CO3 (2M, 1 mL)
was purged with N2 for 5 min. Pd(dppf)Cl2 (5 mg, 6.83 μmol) was
added, and the mixture was refluxed under N2 for 0.5 h. The
resulting solution was partitioned between EtOAc and water, and
the organic layer was dried (MgSO4) and concentrated under
reduced pressure. Column chromatography of the residue on silica
gel, eluting with 1:1 hexanes:EtOAc and 0-5% MeOH/EtOAc,
gave 104 (92mg, 68%) as a whitesolid: mp 220-224 °C; 1H NMR
[(CD3)2SO] δ 8.39-8.33 (m, 3 H), 8.03 (s, 1 H), 7.92 (br d, J = 8.3
Hz, 2 H), 7.80 (d, J = 8.8 Hz, 1 H), 5.06 (d, J = 13.2 Hz, 1 H), 5.02
(d, J = 13.2 Hz, 1 H), 4.74 (dt, J = 12.0, 2.6 Hz, 1 H), 4.52 (br d,
J = 12.0 Hz, 1 H), 4.44-4.34 (m, 2 H), 4.28 (dd, J = 13.5, 3.2 Hz,
1 H). Anal. (C18H14F3N5O4) C, H, N.
See Supporting Information for details of the syntheses of
related compounds 105-111 of Table 3 from chloride 178.
(5-Chloro-2-pyrazinyl)methanol (180) (Scheme 4B). Asolution
of 5-oxo-4,5-dihydro-2-pyrazinecarboxylic acid (179) (6.70 g, 47.8
mmol) in SOCl2 (40 mL) and DMF (0.4 mL) was refluxed for 1 h.
The excess reagent was removed under reduced pressure to give a
black oil, which was extracted with refluxing hexanes (500 mL).
The hot filtrate was filtered three times through Celite (reheating
the filtrate each time) and evaporated to give the crude acid
chloride (6.03 g, 71%). A solution of the acid chloride (3.92 g,
22.1 mmol) in dioxane (20 mL) was added slowly to a solution of
NaBH4 (2.09 g, 55.2 mmol) in water (150 mL), keeping the
temperature of the solution at ∼10 °C. The solution was stirred
at room temperature for 1 h and then extracted with EtOAc. The
organic portion was dried and evaporated, and the residue was
chromatographed on silica gel, eluting with 1:1 CH2Cl2:EtOAc,
to give 18043 (2.18 g, 68%) as a white solid: mp 62-63 °C; 1H
(6S)-2-Nitro-6-{[5-(trifluoromethyl)[2,30-bipyridin]-60-yl]-
methoxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (83). Reac-
tion of alcohol 140 with bromide 176 and NaH (1.6 equiv) using
procedure E, followed by chromatography of the product on silica
gel, eluting with 0-1.25% MeOH/CH2Cl2 (foreruns) and then
with 1.5% MeOH/CH2Cl2, gave 83 (54%) as a pale yellow solid:
mp (MeOH/CH2Cl2/pentane) 218 °C dec; 1H NMR [(CD3)2SO] δ
9.28 (br d, J = 1.7 Hz, 1 H), 9.08 (dd, J = 1.3, 0.9 Hz, 1 H), 8.52
(dd, J = 8.2, 2.3 Hz, 1 H), 8.34 (dd, J = 8.5, 2.2 Hz, 1 H), 8.28 (d,
J = 8.4 Hz, 1 H), 8.04 (s, 1 H), 7.54 (d, J = 8.2 Hz, 1 H), 4.86 (d,
J = 13.6 Hz, 1 H), 4.82 (d, J = 13.6 Hz, 1 H), 4.73 (dt, J = 12.0,
2.6 Hz, 1 H), 4.52 (br d, J = 11.9 Hz, 1 H), 4.40-4.33 (m, 2 H),
4.27 (dd, J = 13.8, 3.6 Hz, 1 H). Anal. (C18H14F3N5O4) C, H, N.
2-Bromo-5-(bromomethyl)pyridine (158) (Scheme 3B). Bromi-
nation of (6-bromo-3-pyridinyl)methanol (157) using procedure
D for 3.5 h, followed by chromatography of the product directly on
silica gel, eluting with petroleum ether (foreruns) and then with
15-25% Et2O/pentane, gave 15832 (91%) as a lachrymatory white
1
solid that was used directly in the next step: H NMR (CDCl3)
δ 8.38 (d, J = 2.5 Hz, 1 H), 7.59 (dd, J = 8.2, 2.6 Hz, 1 H), 7.48 (d,
J = 8.2 Hz, 1 H), 4.42 (s, 2 H).
See Supporting Information for details of alternative reaction
conditions to provide 158.
(6S)-6-[(6-Bromo-3-pyridinyl)methoxy]-2-nitro-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (160). Reaction of alcohol 140 with bro-
mide 158 and NaH using procedure E, followed by chromatogra-
phy of the product on silica gel, eluting with 0-1% MeOH/CH2Cl2
(foreruns) and then with 1-1.5% MeOH/CH2Cl2, gave 160 (88%)
1
as a cream solid: mp (MeOH/CH2Cl2/hexane) 200-203 °C; H
NMR [(CD3)2SO] δ 8.35 (dd, J = 2.3, 0.4 Hz, 1 H), 8.02 (s, 1 H),
7.69 (dd, J = 8.2, 2.5 Hz, 1 H), 7.63 (dd, J = 8.1, 0.5 Hz, 1 H),

8436 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
1
NMR [(CD3)2SO] δ 8.71 (d, J = 1.3 Hz, 1 H), 8.53 (br d, J = 0.5
Hz, 1 H), 5.65 (t, J = 5.8 Hz, 1 H), 4.64 (d, J = 5.8 Hz, 2 H).
APCI MS m/z 147, 145 [M þ H]þ.
gave 117 (79%) as a white solid: mp 223-225 °C; H NMR
[(CD3)2SO] δ 9.20 (s, 2 H), 8.05 (s, 1 H), 8.04 (br d, J = 8.3 Hz, 2
H), 7.90 (br d, J = 8.3 Hz, 2 H), 4.93 (d, J = 14.5 Hz, 1 H), 4.89
(d, J = 14.5 Hz, 1 H), 4.73 (dt, J = 11.9, 2.6 Hz, 1 H), 4.52 (br d,
J = 11.9 Hz, 1 H), 4.46-4.43 (m, 1 H), 4.36 (dt, J = 13.5, 2.1 Hz,
1 H), 4.27 (dd, J = 13.4, 3.3 Hz, 1 H). Anal. (C18H14F3N5O4) C,
H, N.
2-Chloro-5-(iodomethyl)pyrazine (181). Mesyl chloride (1.57
mL, 20.3 mmol) was added to a solution of alcohol 180 (1.44 g,
9.98 mmol) and Et3N (4.17 mL, 29.9 mmol) in anhydrous THF
(20 mL) at 0 °C. The mixture was stirred at 0 °C for 0.5 h and
then partitioned between EtOAc and water. The organic fraction
was dried (MgSO4), and the solvent was removed under reduced
pressure to give a crude mesylate. The mesylate was dissolved in
acetone (40 mL), sodium iodide (7.5 g, 50 mmol) was added, and
the stirred mixture was refluxed for 1 h. The solvent was removed
under reduced pressure, and the residue was partitioned between
EtOAc and water. The organic portion was dried and evaporated,
and the residue was chromatographed on silica gel, eluting with
CH2Cl2, to give 181 (1.54 g, 61%) as a white solid, which was used
immediately due to its instability.
See Supporting Information for details of the syntheses of
related compounds 118-121 of Table 3 from bromide 185.
[2-(Methylsulfanyl)-5-pyrimidinyl]methanol (187) (Scheme 4D).
Isobutyl chloroformate (2.55 mL, 19.7 mmol) was added to a
solution of 2-(methylsulfanyl)-5-pyrimidinecarboxylic acid45 (186)
(3.33 g, 19.6 mmol) and N-methylmorpholine (2.16 mL, 19.6 mmol)
in anhydrous DME (100 mL) at 0 °C, and the mixture was stirred at
0 °C for 20 min. The resulting mixture was filtered through Celite,
and the filtrate was cooled to 0 °C and then treated with a solution
of NaBH4 (0.83 g, 21.9 mmol) in water (10 mL). The mixture was
stirred for 10 min, then diluted with water (100 mL), and extracted
with EtOAc (200 mL). The organic fraction was washed with brine,
dried (MgSO4), and concentrated under reduced pressure. Chro-
matography of the residue on silica gel, eluting with 1:1 EtOAc:
CH2Cl2, gave 187 (1.71 g, 56%) as a pale yellow solid: mp 59-61 °C
(lit.47 mp 63-64 °C); 1H NMR [(CD3)2SO] δ 8.57 (s, 2 H), 5.32 (t,
J = 5.6 Hz, 1 H), 4.47 (d, J = 5.6 Hz, 2 H), 2.50 (s, 3 H).
5-(Bromomethyl)-2-(methylsulfanyl)pyrimidine (188). Mesyl
chloride (1.38 mL, 17.6 mmol) was added to a solution of alco-
hol 187 (1.37 g, 8.77 mmol) and Et3N (3.67 mL, 26.3 mmol) in
anhydrous CH2Cl2 (25mL) at0 °C. The solution was stirred at 0 °C
for 0.5 h, then diluted with CH2Cl2 (50 mL), and washed with
water. The organic fraction was dried (MgSO4), and the solvent was
removed under reduced pressure to give a crude mesylate. The
mesylate was dissolved in acetone (50 mL), LiBr (7.60 g, 87.5 mmol)
was added, and the stirred mixture was refluxed for 1 h. The solvent
was evaporated, and the residue was partitioned between CH2Cl2
and water. The organic fraction was dried and evaporated, and then
column chromatography of the residue, eluting with 5% EtOAc/
CH2Cl2, gave 18855 (1.511 g, 79%) as a white solid: mp 74-76 °C;
1H NMR [(CD3)2SO] δ 8.73 (s, 2 H), 4.70 (s, 2 H), 2.52 (s, 3 H).
APCI MS m/z 221, 219 [M þ H]þ.
Procedure J. (6S)-6-[(5-Chloro-2-pyrazinyl)methoxy]-2-nitro-6,
7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (182). NaH (60%, 0.36 g,
9.0 mmol) was added to a solution of alcohol 140 (0.93 g, 5.02
mmol) and iodide 181 (1.54 g, 6.05 mmol) in anhydrous DMF
(10 mL) at -78 °C. The mixture was stirred at 0 °C for 1 h and then
quenched with ice and extracted with EtOAc (200 mL). The
organic layer was dried (MgSO4) and evaporated, and then column
chromatography of the residue on silica gel, eluting with 0-5%
MeOH/EtOAc, gave 182 (1.02 g, 65%) as a white solid: mp 181-
183 °C; 1H NMR [(CD3)2SO] δ 8.76 (d, J = 1.4 Hz, 1 H), 8.50 (d,
J = 1.4 Hz, 1 H), 8.02 (s, 1 H), 4.85 (d, J = 13.7 Hz, 1 H), 4.81 (d,
J = 13.7 Hz, 1 H), 4.70 (dt, J = 12.1, 2.6 Hz, 1 H), 4.49 (br d, J =
12.0 Hz, 1 H), 4.38-4.29 (m, 2 H), 4.25 (dd, J = 13.5, 3.3 Hz, 1 H).
Anal. (C11H10ClN5O4) C, H, N.
(6S)-2-Nitro-6-({5-[4-(trifluoromethyl)phenyl]-2-pyrazinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (112). Reac-
tion of chloride 182 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described in procedure I gave
112 (78%) as a white solid: mp 233-236 °C; 1H NMR [(CD3)2SO]
δ 9.29 (d, J = 1.4 Hz, 1 H), 8.76 (d, J = 1.4 Hz, 1 H), 8.34 (br d,
J = 8.2 Hz, 2 H), 8.04 (s, 1 H), 7.90 (br d, J = 8.2 Hz, 2 H), 4.91 (d,
J=13.4 Hz, 1 H), 4.88 (d, J=13.4 Hz, 1 H), 4.74 (dt, J=12.1, 2.6
Hz, 1 H), 4.52 (br d, J = 11.9 Hz, 1 H), 4.43-4.33 (m, 2 H), 4.27
(dd, J = 13.5, 3.3 Hz, 1 H). Anal. (C18H14F3N5O4) C, H, N.
See Supporting Information for details of the syntheses of
related compounds 113-116 of Table 3 from chloride 182.
5-Bromo-2-(bromomethyl)pyrimidine (184) (Scheme 4C). A
mixture of 5-bromo-2-methylpyrimidine54 (183) (1.34 g, 7.75
mmol), N-bromosuccinimide (1.40 g, 7.87 mmol), and AIBN
(0.13 g, 0.79 mmol) in CCl4 (15 mL) was stirred at 60 °C for 3 h.
The resulting mixture was filtered, the filter cake was washed
with Et2O (100 mL), and the combined filtrates were concen-
trated under reduced pressure. Column chromatography of the
residue, eluting with 2:1 CH2Cl2:hexanes, gave 18442 (0.214 g,
11%) as a white solid: mp 55-57 °C; 1H NMR (CDCl3) δ 8.79
(s, 2 H), 4.57 (s, 2 H). Anal. (C5H4Br2N2) C, H, N.
(6S)-6-{[2-(Methylsulfanyl)-5-pyrimidinyl]methoxy}-2-nitro-6,
7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (189). Reaction of alco-
hol 140 with bromide 188 and NaH (1.5 equiv), using procedure
J, gave 189 (87%) as a cream solid: mp 220-221 °C; 1H NMR
[(CD3)2SO] δ 8.60 (s, 2 H), 8.01 (s, 1 H), 4.68-4.59 (m, 3 H), 4.46
(br d, J = 11.6 Hz, 1 H), 4.28-4.19 (m, 3 H), 2.51 (s, 3 H). Anal.
(C12H13N5O4S) C, H, N.
Procedure K. (6S)-2-Nitro-6-({2-[4-(trifluoromethyl)phenyl]-
5-pyrimidinyl}methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine
(122). A mixture of methyl sulfide 189 (0.050 g, 0.16 mmol), tris(2-
furyl)phosphine (0.012 g, 52 μmol), copper benzothiophene-2-
carboxylate (0.097 g, 0.40 mmol), and 4-(trifluoromethyl)phenyl-
boronic acid (0.067 g, 0.35 mmol) in anhydrous THF (6 mL) was
purged with N2. Pd2(dba)3 CHCl3 (0.013 g, 13 μmol) was added,
3
Further elution of the column with 0-5% EtOAc/CH2Cl2
gave recovered 183 (0.676 g, 50%).
and the mixture was stirred at 50 °C for 18 h in a sealed tube under
N2. The resulting mixture was diluted with EtOAc (100 mL),
filtered through Celite, and washed with water. The organic fraction
was dried and evaporated, and then column chromatography of the
residue on silica gel, eluting with 0-5% MeOH/EtOAc, gave 122
(26 mg, 40%) as a pale yellow solid: mp (MeOH) 259-263 °C; 1H
NMR [(CD3)2SO] δ 8.92 (s, 2 H), 8.58 (br d, J = 8.1 Hz, 2 H), 8.03
(s, 1H), 7.90(brd, J=8.1 Hz, 2 H), 4.81 (d, J=12.8 Hz, 1 H), 4.78
(d, J = 12.8 Hz, 1 H), 4.71 (dt, J = 12.1, 2.5 Hz, 1 H), 4.50 (br d,
J = 12.0 Hz, 1 H), 4.36-4.23 (m, 3 H); HRESIMS calcd for
C18H15F3N5O4 m/z [M þ H]þ 422.1071, found 422.1059. HPLC
purity: 95.6%.
See Supporting Information for details of the syntheses of
related compounds 123-126 of Table 3 from methyl sulfide 189.
5-Bromo-2-[4-(trifluoromethoxy)phenyl]pyrimidine (191)
(Scheme 4E). A mixture of 5-bromo-2-iodopyrimidine (190)
(1.50 g, 5.27 mmol), 4-(trifluoromethoxy)phenylboronic acid
(6S)-6-[(5-Bromo-2-pyrimidinyl)methoxy]-2-nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]oxazine (185). Reaction of alcohol 140 with
bromide 184 (1.24 equiv) and NaH (1.5 equiv), using procedure
J for 0.5 h (but quenching with water, filtering the solid, washing
with water, then triturating with Et2O), gave 185 (51%) as a light
brown solid: mp >290 °C; 1H NMR [(CD3)2SO] δ 8.98 (s, 2 H),
8.04 (s, 1 H), 4.83 (d, J = 13.2 Hz, 1 H), 4.80 (d, J = 13.2 Hz, 1 H),
4.68 (dt, J = 12.0, 2.6 Hz, 1 H), 4.48 (br d, J = 11.9 Hz, 1 H),
4.40-4.36 (m, 1 H), 4.31 (dt, J = 13.5, 2.1 Hz, 1 H), 4.23 (dd, J =
13.5, 3.3 Hz, 1 H). Anal. (C11H10BrN5O4) C, H. N: calcd, 19.67;
found, 19.17.
(6S)-2-Nitro-6-({5-[4-(trifluoromethyl)phenyl]-2-pyrimidinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (117). Re-
action of bromide 185 and 4-(trifluoromethyl)phenylboronic acid
under the Suzuki coupling conditions described in procedure I

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23 8437
(1.185 g, 5.75 mmol), and Na2CO3 (1.11 g, 10.5 mmol) in toluene
(120 mL) and water (15 mL) was purged with N2. Pd(PPh3)4
(60 mg, 0.052 mmol) was added, and the stirred mixture was refluxed
under N2 for 17.5 h and then partitioned between EtOAc and water.
The organic fraction was dried and evaporated, and then column
chromatography of the residue on silica gel, eluting with 20%
CH2Cl2/hexanes, gave 19149 (1.26 g, 75%) as a white solid: mp
107-108 °C; 1H NMR (CDCl3) δ 8.83 (s, 2 H), 8.46 (d, J = 9.0 Hz,
2H),7.31(brd,J= 9.0 Hz, 2 H). APCI MS m/z321, 319 [M þ H]þ.
2-[4-(Trifluoromethoxy)phenyl]-5-pyrimidinecarbaldehyde (192).
nBuLi (1.88 mL of a 2.5 M solution in hexanes, 4.70 mmol) was
added to a stirred solution of bromide 191 (1.252 g, 3.92 mmol) in
anhydrous THF (40 mL) at -95 °C. The solution was stirred for
30 s, and then anhydrous DMF (5 mL) was added. The mixture
was stirred at -90 °C for 20 min, then quenched with aqueous
NH4Cl, and partitioned between EtOAc and water. The organic
fraction was dried and evaporated, and then column chromatog-
raphy of the residue on silica gel, eluting with 15% EtOAc/hexanes,
gave 192 (0.778 g, 74%) as a white solid: mp 114-115 °C; 1HNMR
(CDCl3) δ 10.16 (s, 1 H), 9.22 (s, 2 H), 8.62 (d, J = 9.0 Hz, 2 H),
7.36 (br d, J = 9.0 Hz, 2 H). APCI MS m/z 301 [M þ H þ
MeOH]þ, 269 [M þ H]þ.
600, Kalamazoo, MI 49008, using a published protocol (similar
to method C below).12 For pH 1 determinations, the pH 7.4
solution was replaced by pH 1 buffer.
Method C. The study of the remaining compounds (80, 97, 99,
107, and 124) was conducted by Cerep, 15318 NE 95th St.,
Redmond, WA 98052. according to the following procedure:
Aliquots of the compound DMSO stocks (10 mM) were trans-
ferred to pH 7.4 PBS buffer or simulated gastric fluid (target
concentrations were 200 μM in compound and 2% DMSO),
and the solutions were equilibrated for 24 h at room temperature.
The compound concentrations were determined by fast gradient
HPLC (using photodiode array detection) with reference to 200 μM
calibration standards. Reported values were the mean of two
determinations.
Minimum Inhibitory Concentration Assays (MABA and
LORA). These were carried out according to the published
protocols.52,53
Microsomal Stability Assays. These were conducted by MDS
Pharma Services, 22011 30th Drive SE, Bothell, WA 98021, using
a published protocol.10 The percentage of compound remaining
after 1 h incubation was calculated as
% remaining ¼ 100ðmean PART60=mean PART0
Þ
{2-[4-(Trifluoromethoxy)phenyl]-5-pyrimidinyl}methanol (193).
NaBH4 (0.22 g, 5.82 mmol) was added to a solution of aldehyde
192 (0.776 g, 2.89 mmol) in MeOH (100 mL) at 0 °C. The solution
was stirred at 0 °C for 1 h, then quenched with brine, and parti-
tioned between EtOAc and water. Column chromatography of
the organic portion on silica gel, eluting with 1:1 EtOAc/hexanes,
gave 193 (0.657 g, 84%) as a white solid: mp 84-85 °C; 1H NMR
[(CD3)2SO] δ 8.86 (s, 2 H), 8.50 (d, J = 8.9 Hz, 2 H), 7.51 (br d,
J = 8.9 Hz, 2 H), 5.46 (t, J = 5.5 Hz, 1 H), 4.60 (d, J = 5.5 Hz, 2
H). APCI MS m/z 271 [M þ H]þ.
where PAR = analyte/IS peak area ratio.
In Vivo Acute TB Infection Assay. Each compound was
administered orally to a group of seven M. tb-infected BALB/c
mice at a standard dose of 100 mg/kg, daily for 5 days a week for
3 weeks, beginning on day 11 postinfection, in accordance with
published protocols.10,53 The results are recorded as the ratio of
the average reduction in colony forming units (CFUs) in the
compound-treated mice/the average CFU reduction in the mice
treated with 1. In this assay, 1 caused up to 2.5-3 log reductions
in CFUs.
In Vivo Chronic TB Infection 3 Week Assay. Compounds were
administered orally as described for the acute assay but with
treatment beginning ∼70 days postinfection. In this assay, 1 caused
a ca. 2 log reduction in CFUs, whereas 2 caused a ca. 3 log reduc-
tion in CFUs.
In Vivo Chronic TB Infection 8 Week Assay. Each compound
was administered orally to a group of seven M. tb-infected
BALB/c mice at a dose of 30 mg/kg, daily for 5 days a week
for 8 weeks, with treatment beginning ∼50 days postinfection.
In this assay, 2 caused a ca. 3 log reduction in CFUs.
In Vivo Mouse Pharmacokinetics. Compounds were adminis-
tered orally to CD-1 mice at a standard dose of 40 mg/kg, as a
suspension in 0.5% carboxymethylcellulose/0.08% Tween 80 in
water. Samples derived from plasma and lungs were analyzed by
LC-MS/MS to generate the required pharmacokinetic para-
meters.
Studies of all compounds except 114 were conducted at
Cumbre Pharmaceuticals Inc., Dallas, TX, and UNT Health
Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX
76107-2699. The study of compound 114 (using the same
protocol) was conducted by MDS Pharma Services, 22002
26th Ave. SE, Suite 104, Bothell, WA 98021-4444.
5-(Bromomethyl)-2-[4-(trifluoromethoxy)phenyl]pyrimidine (194).
Mesyl chloride (0.55 mL, 7.0 mmol) was added to a solution of
alcohol 193 (0.951 g, 3.52 mmol) and Et3N (1.5 mL, 10.8mmol) in
anhydrous THF (40 mL) at 0 °C. The mixture was stirred at 0 °C
for 1 h and then partitioned between EtOAc and water. The orga-
nic fraction was dried, and the solvent was removed under reduced
pressure to give a crude mesylate. The mesylate was dissolved
in acetone (100 mL), LiBr (6.10 g, 70.2 mmol) was added, and the
stirred mixture was refluxed under N2 for 1 h. The resulting
mixture was filtered, the solvent was evaporated, and the residue
was partitioned between EtOAc and water. The organic fraction
was dried and evaporated, and then column chromatography of
the residue, eluting with CH2Cl2, gave 194 (1.10 g, 94%) as a white
solid:mp80-81 °C; 1H NMR(CDCl3) δ8.82 (s, 2 H), 8.50 (d, J =
9.0 Hz, 2 H), 7.32 (br d, J = 9.0 Hz, 2 H), 4.48 (s, 2 H). APCI MS
m/z 335, 333 [M þ H]þ.
(6S)-2-Nitro-6-({2-[4-(trifluoromethoxy)phenyl]-5-pyrimidinyl}-
methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (124). Reac-
tion of alcohol 140 with bromide 194 (1.05 equiv) and NaH (1.5
equiv), using procedure J, followed by chromatography of the
product on silica gel, eluting with 5% MeOH/EtOAc, gave 124
1
(0.512 g, 58%) as a white solid: mp (MeOH) 227-230 °C; H
NMR [(CD3)2SO] δ 8.88 (s, 2 H), 8.49 (br d, J = 8.9 Hz, 2 H), 8.03
(s, 1 H), 7.51 (br dd, J = 8.9, 0.8 Hz, 2 H), 4.79 (d, J = 12.6 Hz, 1
H), 4.76 (d, J = 12.6 Hz, 1 H), 4.70 (dt, J = 12.0, 2.5 Hz, 1 H), 4.49
(br d, J = 12.0 Hz, 1 H), 4.35-4.29 (m, 2 H), 4.25 (dd, J = 13.3,
3.1 Hz, 1 H). Anal. (C18H14F3N5O5) C, H, N.
In Vivo Rat Pharmacokinetics and Oral Bioavailability. Com-
pounds were administered to male Sprague-Dawley rats both
intravenously, at doses of 1 or 5 mg/kg, and orally, at a dose of
20 mg/kg, as 2 mg/mL solutions in 40% hydroxypropyl-β-
cyclodextrin/50 mM citrate buffer (pH = 3). Samples derived
from plasma were analyzed by LC-MS/MS to generate the
required pharmacokinetic parameters.
Solubility Determinations. Method A. The solid compound
sample was mixed with water (enough to make a 2 mM solution)
in an Eppendorf tube, and the suspension was sonicated for 15
min and then centrifuged at 13000 rpm for 6 min. An aliquot of
the clear supernatant was diluted 2-fold with water, and then
HPLC was conducted. The solubility was calculated by compar-
ing the peak area obtained with that from a standard solution of
the compound in DMSO (after allowing for varying dilution
factors and injection volumes).
The study was conducted by Absorption Systems, 436 Cream-
ery Way, Suite 600, Exton, PA 19341-2556.
Acknowledgment. The authors thank the Global Alliance
for Tuberculosis Drug Development for financial support
through a collaborative research agreement. Special thanks
belongto Dr. Annette Shadiack and Dr. Khisi Mdluli for their
efforts in designing and coordinating the ADME/PK studies.
Method B. The study of most compounds (1, 5, 24, 32, 74, 92,
93, 114) was conducted by Admetryx, 4717 Campus Drive, Suite

8438 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 23
Kmentova et al.
Supporting Information Available: Additional experimental
procedures and characterizations for compounds in Tables 1-3;
combustion analytical data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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