Structure-activity relationships of antitubercular nitroimidazoles. 3. Exploration of the linker and lipophilic tail of ((S)-2-nitro-6,7-dihydro-5 H -imidazo[2,1- b ][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (6-amino PA-824)

Article abstract of DOI:10.1021/jm1010644

The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2, 1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 μM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.

Full text of DOI:10.1021/jm1010644

ARTICLE
pubs.acs.org/jmc
StructureÀActivity Relationships of Antitubercular Nitroimidazoles. 3.
Exploration of the Linker and Lipophilic Tail of
((S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]
oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (6-Amino PA-824).
Joseph Cherian,§,# Inhee Choi,† Amit Nayyar,† Ujjini H. Manjunatha,§ Tathagata Mukherjee,† Yong Sok Lee,‡
||
Helena I. Boshoff,† Ramandeep Singh,†, Young Hwan Ha,†,^ Michael Goodwin,† Suresh B. Lakshminarayana,§
Pornwaratt Niyomrattanakit,§ Jan Jiricek,§ Sindhu Ravindran,§ Thomas Dick,§ Thomas H. Keller,§,#
Veronique Dartois,§ and Clifton E. Barry, III*,†
†Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892, United States
‡Center for Molecular Modeling, Division of Computational Bioscience, Center for Information Technology, National Institutes of
Health, Bethesda, Maryland 20892, United States
§Novartis Institute for Tropical Diseases, 10 Biopolis Road, Singapore, 138670
ABSTRACT: The (S)-2-nitro-6-(4-(trifluoromethoxy)ben-
zyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-
824 (1) has demonstrated antitubercular activity in vitro and in
animal models and is currently in clinical trials. We synthesized
derivatives at three positions of the 4-(trifluoromethoxy)-
benzylamino tail, and these were tested for whole-cell activity
against both replicating and nonreplicating Mycobacterium
tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase
(Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole
cell activity and 1.6 μM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule.
Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of
the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial
orientation of the linker and lipophilic tail.
’ INTRODUCTION
Recently, a significant amount of information on structureÀ
activity relations (SAR) within this series of molecules has begun
to emerge both in terms of whole cell activity and Ddn substrate
preferences. The key determinants of these molecules as sub-
strates include: an S configuration at C-6 of the oxazine ring, the
nitro group, an 8-oxy substituted bicyclic nitroimidazole, and a
lipophilic trifluoromethoxybenzyl tail.14 The lipophilic tail has
been the subject of considerable optimization chemistry since the
series was first reported in the patent literature.15,16 A wide
variety of substituted aromatics have been employed, in general,
with the most significant improvements in activity occurring with
para substituents. The coincidental finding that p-trifluoro-
methoxy substitution (which appears in both candidate drugs)
with linkers that vary in length by 4À5 Å consistently provided
the most active derivatives prompted us to explore that more
systematically with a homologous series of linkers increasing in
carbon length. This study showed an optimal spacing of four
carbons between the ether oxygen and the aromatic ring.14 These
findings and the extended lipophilic tail present in some
The nitroimidazo-oxazines, including PA-824 (1),1 and nitro-
imidazo-oxazoles, including OPC-676832,3 (Figure 1), are can-
didates for the treatment of tuberculosis (TB) that are currently
in phase 2 clinical trials.4 These compounds have shown good
activity in animal models of disease both as single agents and in
combination studies.5À8 This class has attracted attention be-
cause of its ability to not only kill rapidly dividing bacteria but also
to kill quiescent bacteria maintained under hypoxic conditions.1
It is hoped that this activity against nonreplicating organisms may
allow the shortening of the 6À8 month duration of TB che-
motherapy required to achieve a durable cure.9
These nitroimidazoles are pro-drugs and require bioreductive
activation to exert their bactericidal effect.10 In Mycobacterium
tuberculosis (Mtb) this reduction is mediated by a deazaflavin-
dependent nitroreductase, Ddn,11 and reduction occurs on the
imidazole ring, resulting in the elimination of bactericidal re-
duced nitrogen species such as nitric oxide.12 The anaerobic
activity of compounds in this series correlates with the extent of
production of reduced nitrogen species, and both aerobic and
anaerobic activities correlate roughly with the efficiency of
enzymatic reduction by Ddn.13
Received: August 16, 2010
Published: July 14, 2011
r
2011 American Chemical Society
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analogues inspired further exploration of various substituted
biphenyl analogues of this tail.17 This study reported a correla-
tion between lipophilicity of the side chain and identified
analogues with significantly improved in vivo activity in a
mouse model.
corresponding acyl chlorides in presence of NaH in DMF.
Formylation of 2 with formic acid in the presence of acetic
anhydride in THF yielded the N-formamide derivative (3) in
55% yield. The tertiary amine derivatives (5aÀc) were synthe-
sized by reductive amination of formaldehyde, propionaldehyde,
or acetone with amine 2 in moderate yield. Reaction of 2 with
triphosgene followed by ethylamine hydrochloride in the pre-
sence of triethylamine afforded the urea derivative 6 in 66% yield.
R2 Modifications. Oxazol-2-yl(4-(trifluoromethoxy)phenyl)-
methanone (9a) was prepared by copper mediated acylation18 of
oxazol-2-yl zinc chloride using 4-trifluoromethoxybenzoyl chlor-
ide and then subjected to reductive amination with amine (7) in
the presence of Ti(iPrO)4/AcOH/THF/NaCNBH3 to provide
17a as diastereomeric mixture (Scheme 2). Synthesis of the R2
methyl analogue 17b was accomplished by reductive amination
of the commercially available 4-trifluoromethoxyacetophenone
9b. Imidizolium ylide generated from N-benzylimidazole using
diisopropylcarbamyl chloride, and diisopropylethylamine was
treated with trifluoromethoxybenzaldehyde in refluxing CH3CN
to produce the carbamate, which upon hydrolysis afforded the
benzylic alcohol (11). N-Debenzylation using Pd/C followed by
mesylation of the benzylic alcohol and subsequent reaction with
amine 7 in THF in the presence of NaH at room temperature
produced the final product (17c) as a mixture of diastereomers.
The three alkyl derivatives (17dÀf) were all prepared in
moderate yield by alkylation of amine 7 using the corresponding
bromides 15aÀc, as illustrated in Scheme 2. Addition of ethyl
and n-propyl Grignard reagents to 4-trifluoromethoxybenzalde-
hyde (13) afforded the corresponding benzylic alcohols (14aÀb),
while 1-(4-(trifluoromethoxy)phenyl)butan-1-ol (14c) was pre-
pared by the addition of n-BuLi to trifluoromethoxybenzalde-
hyde. Alcohols 14aÀc were converted to their corresponding
bromides (15aÀc) using PBr3 in ether and treated with amine
(7) in DMF in the presence of K2CO3/KI at 90 ꢀC to provide the
required compounds 17dÀf as a mixture of diastereomers.
Synthesis of carboxamide derivative 17g was achieved by nu-
cleophilic addition of TMSCN to the imine formed between the
We have previously derived a 3D-quantitative structureÀ
activity relationship (QSAR) pharmacophore model that was
reasonably predictive for aerobic MIC among a series of 21
training and 22 test nitroimidazoles.14 Although the best model
suggested only one hydrophobic feature, other models suggested
the possibility of two distinct hydrophobes in the tail region of 1
and raised the prospect that additional analogues could be
synthesized, which would simultaneously engage both features.
In this paper, we describe our attempts to explore this hypothesis
with three series of analogues synthesized primarily from the
more soluble 6-S-amino series of compounds. The first (R1)
employs the amine as an attachment site, the second (R2) focuses
on the benzylic carbon, and the third (R3) explores additional
diversity on the aromatic nucleus of the trifluoromethoxybenzyl
moiety of the parent compound.
Chemistry. R1 Modifications. 2 was synthesized as previously
reported14 and utilized as a starting point for R1 (Scheme 1).
Amide derivatives 4aÀf were synthesized by reacting 2 with the
Figure 1
Scheme 1a
a Reaction conditions: (i) HCO2H, Ac2O, THF, 0 ꢀC, 1 h, 55%; (ii) R1COCl, NaH, DMF rt to 70 ꢀC; (iii) R1CHO, NaBH(OAc)3, MeOH/AcOH; (iv)
triphosgene, EtNH2 HCl, Et3N, THF, 0 ꢀC to rt, 66%.
3
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Scheme 2a
a Reaction conditions: (i) n-BuLi, ZnCl2, CuI, THF, À78 ꢀC to rt, 1.5 h, then 4-trifluoromethoxybenzoyl-chloride, rt, 1 h, 40%; (ii) diisopropylcarbamyl
chloride, DIPEA, 4-trifluoromethoxybenzaldehyde, CH3CN, reflux, 19 h, 73%; (iii) 50% TFA in water, THF, reflux, 15 h, 82%; (iv) H2, Pd/C, MeOH, 1
atm, 81%; (v) MsCl, Et3N, CH2Cl2, rt, 1 h; (vi) 7, NaH, THF, rt, 40 h, 15%; (vii) RMgBr, THF, 0 ꢀC to rt when R = Et and n-Pr; n-BuLi, THF, À78 ꢀC to
rt when R = n-Bu; (viii) PBr3, ether, 0 ꢀC to rt; (ix) 7, K2CO3, DMF, KI, 90 ꢀC; (x) 4-trifluoromethoxybenzaldehyde, neat, 100 ꢀC, 5 min then TMSCN,
100 ꢀC, 30 min, 50%; (xi) EtOH/HCl, À10 ꢀC, 38%; (xii) 7, NaCNBH3, AcOH, EtOH, 5%; (xiii) 4-methoxybenzylalcohol, KOtBu, 60 ꢀC, 2 h, 34%;
(xiv) PDC, CH2Cl2, rt, 24 h, 62%; (xv) TBDMSOTf, CH2Cl2, rt, 5 min, 86%; (xvi) ethylbromoacetate, Zn, CH2Cl2, rt, 3 h; (xvii) LiAlH4, THF, 0 ꢀC to
rt, 2 h, 25% over two steps; (xviii) MnO2, CH2Cl2, rt, 6 h, 50%; (xix) 7, Ti(iOPr)4, AcOH, NaBH3CN, 9%.
amine 7 and 4-trifluoromethoxybenzaldehyde (13) at 100 ꢀC
and subsequent hydrolysis in ethanolic HCl. Reaction of 4-
((trifluoromethoxy)phenyl)oxirane 18 with 4-trifluoromethoxy-
benzyl alcohol in presence of KOtBu at 60 ꢀC afforded alcohol
19. Further oxidation of the ring-opened product, followed by
removal of the p-methoxybenzyl group using TBDMSOTf,
afforded 2-hydroxy-1-(4-(trifluoromethoxy)phenyl)ethanone (20).
3-Hydroxy-1-(4-(trifluoromethoxy)-phenyl)propan-1-one (22)
was synthesized from 13 in three steps involving a Reformatsky
reaction with ethylbromoacetate in presence of Zn, LiAlH4
mediated reduction of the ester group to yield 21, and finally
oxidation of the benzylic alcohol using MnO2 to provide the
hydroxy ketone 22. Reductive amination of 20 and 22 with amine
7 afforded nitroimidazooxazines 17hÀi, respectively.
2-Cyclopropyloxy-4-trifluoromethoxy benzaldehyde (26) was
synthesized in five steps20 from ethyl-2-hydroxy-4-trifluoro-
methoxybenzoate (24). Alkylation of 24 with 1-bromo-2-chloro-
ethane using K2CO3 in DMF followed by saponification with
KOtBu in THF at 20 ꢀC resulted in the 4-(trifluoromethoxy)-
2-(vinyloxy)benzoic acid (25) in 63% yield. Cyclopropanation of
25 under SimmonsÀSmith conditions followed by reduction of
the carboxylic acid group using BH3 DMS and subsequent
3
oxidation of the benzylic alcohol using PCC provided
2-O-cyclopropyl-4-trifluoromethoxybenzaldehyde (26) in 40%
yield.
Reaction of ethyl-2-hydroxy-4-trifluoromethoxybenzoate (24)
with 4-fluoronitrobenzene in the presence of NaH in DMF
followed by Pd/C mediated reduction of the nitro group afforded
the amine (29). Removal of the amino group by diazotization
and subsequent reduction of the ester group using LiAlH4
provided the benzylic alcohol derivative (30) with 48% yield.
The required aldehyde 31 was then obtained by PCC mediated
oxidation of (30). Reductive amination of these aldehydes 26,
28aÀd, and 31 with amine 7 in presence of NaBH(OAc)3 in
DMFÀAcOH provided the nitroimidazooxazines 32aÀf. De-
protection of the O-methoxymethyl ether using 6N HCl in THF
provided 32g in 67% yield.
2-Fluoro and 2-chloro-4-trifluoromethoxybenzaldehyde (34aÀb)
were readily synthesized from commercially available 2-chloro
and 2-fluoro substituted 4-trifluoromethoxyiodobenzene (33aÀb)
by lithiation using n-BuLi at À78 ꢀC followed by quenching
with DMF in 72% and 57% yield, respectively (Scheme 4).
2-Bromo-4-trifluoromethoxybenzaldehyde 37 was synthe-
sized by oxidative cleavage of the product 36 of Pd(0)
R3 Modifications. R3 modified nitroimidazooxazines were
synthesized by the reductive amination of substituted trifluoro-
methoxybenzaldehydes with amine (7). Most of these alde-
hydes were not available commercially and were synthesized as
described in Schemes 3, 4, and 5. Thus 2-hydroxy-4-trifluoro-
methoxybenzoic acid (23)19 was used as a key intermediate in
the synthesis of 2-hydroxy bearing analogues and the corre-
sponding ether derivatives (32aÀc, Scheme 3). Esterification of
(23) with ethyl alcohol followed by alkylation with BnBr and
cyclopropylmethylbromide gave the respective ether derivatives
(27bÀc) in excellent yields. Protection of the phenolic ÀOH
group of 24 as its O-methoxymethyl ether was carried out using
MOMCl/DIPEA to afford 27a. LiAlH4 mediated reduction of
the ester group of 27aÀc and subsequent oxidation using
PCC in CH2Cl2 afforded the required aldehydes 28aÀc in
good yield.
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Scheme 3a
a Reaction conditions: (i) EtOH/H+, 80 ꢀC, 80%; (ii) 1-bromo-2-chloroethane, K2CO3, DMF, rt, 15 h, 80%; (iii) KOtBu, THF, 1 h, 20 ꢀC, 78% over two
steps; (iv) Et2Zn, CH2I2, ClCH2CH2Cl, toluene, rt, 17 h, 51%; (v) BH3-DMS, THF, reflux, 1.5 h; (vi) PCC, CH2Cl2, rt, 0.5 h, 80% over two steps; (vii)
RX, K2CO3, DMF, 70 ꢀC, 30 min to 2 h, when R = Bn, cyclopropylmethyl; MOMCl, DIPEA, CH2Cl2, rt, 3 h; (viii) LiAlH4, THF, 0 ꢀC to rt, 1 h; (ix)
4-fluoronitrobenzene, NaH, DMF, 100 ꢀC, 2 h, 67%; (x) H2, Pd/C, EtOAc, rt, 1.5 h, 87%; (xi) NaNO2, H3PO2, 6N HCl, 50 ꢀC, 1 h; (xii) NaBH(OAc)3,
AcOH, DMF, 20 h; (xiii) 6N HCl, THF, 1 h, rt.%.
Scheme 4a
a Reaction conditions: (i) styrene, Pd(OAc)2, Et3N, 95 ꢀC, 16 h, 64%; (ii) OsO4, NaIO4, acetoneÀwater, rt, 16 h, 16%; (iii) 7, NaCNBH3, AcOH, DMF,
20 h; (iv) ethylene glycol, p-TSA, benzene, 80 ꢀC, 8 h, 86%; (v) Pd(OAc)2, Cs2CO3, Xantphos, dioxane, amine, 90 ꢀC, 8 h; (vi) THF, 6 N HCl, 30 min,
room temperature; (vii) n-BuLi, THF, À78 ꢀC, DMF, 15 min.
mediated Heck coupling between 2-bromo-4-trifluoromethoxy-
iodobenzene (35) and styrene. Buchwald coupling of 36
with morpholine and piperidine followed by oxidative cleavage
of the olefin afforded aldehydes 40aÀb in moderate yields.
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Scheme 5a
a Reaction conditions: (i) TBSCl, imidazole, CH2Cl2, rt, 60%; (ii) s-BuLi, TMEDA, THF, À78 ꢀC, 1 h, then FB(OMe)2, À78 ꢀC, 30 min followed by alk,
H2O2, 30 min, 28%; (iii) K2CO3, MeI, DMF, 70 ꢀC; (iv) MOMCl, DIPEA, DMF, rt, 16 h, 82%; (v) TBAF, THF, rt, 1.5 h; (vi) PCC, CH2Cl2, rt, 1 h; (vii)
7, NaCNBH3, AcOH, DMF; (viii) 4-fluoronitrobenzene, NaH, DMF, 100 ꢀC, 2 h, 30%; (ix) Fe/NH4Cl, EtOAcÀwater, reflux, 1.5 h; (x) NaNO2,
H3PO2, 6N HCl, 50 ꢀC, 1 h, 52% over two steps; (xi) ethylene glycol, p-TSA, benzene, 80 ꢀC, 8 h, 72%; (xii) Pd(OAc)2, Cs2CO3, Xantphos, dioxane,
amine, 90 ꢀC, 8 h; (xiii) THF, 6 N HCl, 30 min, rt; (xiv) sec-BuLi, MeOCOCl, THF, À78 ꢀC to rt, 3 h.
2-(4-Methylpiperazin-1-yl)-4-(trifluoromethoxy)benzaldehyde 39
was synthesized by Buchwald coupling of N-methylpiperazine
with 2-[2-bromo-4-(trifluoromethoxy)phenyl]-1,3-dioxolane (38)
followed by deprotection of the acetal. The reductive amination
of aldehydes 34aÀb, 37, 39, and 40aÀb with amine 7 afforded
nitroimidazooxazines 41aÀf.
54 and subsequent deprotection of the acetal afforded the
required aldehydes 55aÀb in moderate yields. Methyl-5-for-
myl-2-(trifluoromethoxy)benzoate 48 was synthesized from 42
in three steps. Lithiation of 42 with sec-BuLi at À78 ꢀC followed
by the addition of methylchloroformate, subsequent deprotec-
tion of the TBS ether with TBAF, and PCC mediated oxidation
of the benzylic alcohol afforded 48.
Reductive amination of these aldehydes (47aÀb, 48, 49aÀb,
52, 55aÀb) with amine 7 was carried out in presence of
NaCNBH3 in DMF containing AcOH to provide the nitroimi-
dazole derivatives 56aÀh in 15À55% yields. Deprotection of the
O-methoxymethyl ether 56d was carried out as mentioned
previously to provide 56i. Syntheses of 1 derivatives 59aÀc with
R3 modification having 3-F, 3-OMe, and 3-OMOM substitutions
was achieved by alkylation of alcohol 57 with the corresponding
benzylic bromides 58aÀb (Scheme 6). Deprotection of the O-
methoxymethyl ether in 59a was carried out using 6N HCl in
THF to afford 59d.
4-Trifluoromethoxybenzyl alcohol (42) was protected as a
TBS ether (43) in 80% yield (Scheme 5), which was then reacted
with s-BuLi at À78 ꢀC in the presence of TMEDA and subse-
quently treated with FB(OMe)2 followed by alkaline H2O2
hydrolysis, affording the required phenol (44) in 30% yield.
Conversion of 44 to the corresponding O-methyl derivative (45)
and O-methoxymethyl ether (46) was achieved by using MeI/
K2CO3 and MOMCl/Et3N, respectively. Cleavage of the TBS
ether using TBAF followed by PCC-mediated oxidation pro-
duced the corresponding aldehydes 47aÀb. Synthesis of 3-phe-
noxy-4-trifluoromethoxybenzaldehyde 52 followed a similar
protocol used for the synthesis of 2-phenoxy derivative 31. Thus,
5-((tert-butyl(dimethyl)silyl)oxymethyl)-2-(trifluoromethoxy)-
phenol (44) was converted to the 3-(4-nitrophenoxy) derivative
50. Reduction to the corresponding aniline derivative followed
by diazotization gave 3-phenoxy-4-trifluoromethoxybenzylic al-
cohol 51 in 52% yield. Oxidation of 51 with PCC in CH2Cl2
produced the required aldehyde 52.
’ RESULTS AND DISCUSSION
R1 Modifications: SAR of Amides, Ureas, and Tertiary
Amines. Both benzyl ether and amine analogues of nitroimida-
zooxazines have been shown to be equipotent against M.
tuberculosis. The amenability of the benzylic amine in 2 allowed
us to explore further modifications in the series shown in Table 1.
N-Formylation (3) reduced both cellular activity and efficiency
as a substrate for Ddn by 2-fold compared to the parent
compound 2, whereas N-acetylation of the amino group was
3-Morpholino- and 3-(1-pipiridyl)-substituted 4-trifluoro-
methoxybenzaldehydes 55aÀb were synthesized following a
similar sequence of reaction used in the synthesis of 40aÀb.
Thus, Buchwald coupling of the morpholine and piperidine
with 2-(3-chloro-4-(trifluoromethoxy)phenyl)-[1,3]-dioxolane
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Scheme 6a
1, however, within this group there was in general only a weak
correlation between enzymatic activity and MIC. The same was
not true of the N-aroyl derivatives, some of which seem to be
significantly better substrates for the enzyme. However, this
improvement in substrate efficiency did not translate into
improvements in MIC. One potential explanation for this
phenomenon could be that the aroyl substituent, with its
similarity to the p-trifluoromethoxybenzyl amine substituent,
competes with this group and alters the binding mode of the
substrate, altering product formation and hence efficacy. Alter-
natively, these could be transported into the cell less efficiently
reducing the effective intracellular concentration.
The tertiary amine analogues (5aÀc) showed significantly
improved potency in comparison with the amide derivatives.
Higher alkyl derivatives such as N-n-Pr derivative 5b showed a
6-fold increase in the potency compared to 1 and was similar in
potency to the N-Me derivative (5a). Both 5a and 5b were 2-fold
more potent against anaerobic Mtb. However, the N-i-Pr deri-
vative 5c was less active against both replicating and nonreplicat-
ing Mtb when compared to N-n-Pr 5b, which indicates that
branched chains may not be suitable at this position. For 5a and
5b, improvements in MIC were paralleled by increases in kcat/KM,
suggesting that the binding mode of the substrate was maintained
to optimize aerobic activity. While these tertiary amines showed
improvements in the overall activity profile, the intrinsic clear-
ance of these compounds was unfortunately very high in mouse
liver microsomes and we discontinued exploration of this series
(Table 4).
a Reaction conditions: (i) NaH, DMF, À78 ꢀC to rt; (ii) 6N HCl, THF,
rt, 4 h.
Table 1. Minimum Inhibitory Concentration (MIC) and
Minimum Anaerobicidal Concentration (MAC) of R1
Modifications
R2 Modifications: Flat SAR and a Potential Site for Meta-
bolic Tailoring. Our efforts to systematically optimize the
benzylic position by incorporating lower alkyl groups (17b,
17dÀf) resulted in compounds with either comparable or very
slightly enhanced activity compared to 1 against both replicating
and nonreplicating Mtb (Table 2). Elongation of the alkyl chain
from methyl to n-butyl resulted in very slight enhancements in
potency (2-fold), suggesting the presence of a hydrophobic
pocket but the SAR here was notably flat, and the introduction
of a hydroxymethyl group at this position resulted in compound
(17h), which had activity profile similar to 2. The derivatives with
hydroxyethyl (17i) and carboxamide (17g) were 5- and 1.5-fold
more potent respectively against replicating Mtb although their
potency against anaerobic bacilli was compromised. Introduction
of heterocycles such as oxazole (17a) or imidazole (17c) led to
compounds with similar potency against both replicating and
nonreplicating Mtb. In general, the presence of more polar
groups resulted in the reduction of activity against nonreplicating
Mtb, whereas the potency against replicating Mtb remained in
the range of 0.15À0.4 μM. Although the stereochemistry of the
C-6 substituent is critical for 1 activity (the S isomer being 100
fold more potent than the R isomer), the introduction of another
chiral center at the benzylic position does not seem to affect the
activity as testing the individual diastereoisomers showed equiva-
lent activity in all assays. This flatness of the SAR is mirrored in
the kinetics of F420 reoxidation when they were tested as
substrates for Ddn. The kcat/KM was comparable to the parent
compound except for the compounds (17e, 17f) whose kcat/KM
was 0.31 and 0.4, respectively (Table 2). This 2-fold increase in
substrate efficiency translated into a slight improvement in MIC
only for compound 17f.
detrimental to both MIC and MAC and reduced the catalytic
efficiency of this as a substrate for Ddn by nearly 10-fold. The N-
propionyl derivative 4b was insoluble and could not be evaluated.
N-Aroyl amide derivatives behaved in a similar fashion with both
benzoyl (4c) and chlorobenzoyl (ortho, meta, and para) deriva-
tives (4dÀf) resulting in compounds that were significantly less
potent against replicating and nonreplicating Mtb. We also
explored one urea derivative (6) but did not elaborate on the
series after the observation that reducing the basicity of nitrogen
resulted in less potent compounds.
The catalytic activity of the simple amide substituted mol-
ecules as substrates for the Ddn enzyme, measured as kcat/KM for
reoxidation of reduced F420, was generally lower than that of
Benzylic positions in general are susceptible to metabolism
(Metasite predictions, for example, highlight the benzylic carbon
of 1 as the primary site of metabolic liability). Blocking of
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benzylic position would make this position metabolically more
stable. The flat SAR observed at this position suggests that this
does not form an important contact site for Ddn and therefore
Table 3. Continued
Table 2. Minimum Inhibitory Concentration (MIC) and
Minimum Anaerobicidal Concentration (MAC) of R2
Modifications
Mtb MIC99 μM Mtb MAC μM
Ddn
compd no.
R2
((SD)
((SD)
kcat/KM
1
ÀH
ÀH
0.63 ( 0.22
0.36 ( 0.05
0.40 ( 0.18
0.44 ( 0.16
0.52 ( 0.23
0.36 ( 0.18
0.27 ( 0.07
0.15 ( 0.00
0.41 ( 0.35
0.30 ( 0.22
0.13 ( 0.04
8.8 ( 3.4
14 ( 2
0.15
0.17
0.20
0.09
0.17
0.14
0.31
0.40
0.12
0.15
0.21
2
17a
17b
17c
17d
17e
17f
17g
17h
17i
2-oxazole
methyl
7.8 ( 3.1
5.6 ( 1.4
17 ( 7
2-Imidazole
Et
3.1 ( 2.2
3.8 ( 1.4
4.4 ( 1.7
30 ( 11
11 ( 3
n-Pr
n-Bu
CONH2
ÀCH2OH
ÀCH2CH2OH
23 ( 6
Table 3. Minimum Inhibitory Concentration (MIC) and
Minimum Anaerobicidal Concentration (MAC) of R3
Modifications
raises the possibility of further manipulation of this center to alter
metabolic stability or other metabolic properties without com-
promising activity.
R3 Modifications: Hydrophobic Groups Preferred. Signifi-
cant improvements in potency were realized upon substituting
at the 2-position of the aromatic ring of the benzyl ether
Table 3 (top). Substitution using halogens (41aÀc) as well as
hydroxyl (32g) groups improved MIC to lower than 100 nM.
Phenoxy (32d) and cyclopropyloxy (32f) produced the most
potent compounds in this series, showing MIC values down to
60 nM. The corresponding benzyloxy derivative (32b) was less
potent against both aerobic and anaerobic Mtb but still three
times as potent as 1. Similarly, cyclopropylmethoxy (32e) group
was tolerated less well in both cases, suggesting an optimal
spacing between a cyclic hydrophobe and the ether oxygen that
may play a critical role in binding to Ddn. Consistent with this
hypothesis, both cyclopropylmethoxy and benzyloxy substitu-
ents are poorer substrates for Ddn by comparison with their
correspondingly shorter analogues. In fact, phenoxy substituted
32d was the best substrate for Ddn seen in the present study with
a kcat/KM of 0.42. Compounds 41dÀe bearing a piperidine and
morpholine group also showed improved MIC, while N-methyl-
piperazine analogue 41f resulted in the least aerobically potent
compound among these amine-bearing substitutions. All three
compounds in this series resulted in disproportionate loss of
anaerobic activity and an inconsistent correlation with their
efficiency as substrates for Ddn.
The 3-position of the trifluoromethoxyphenyl ring likewise
tolerated most of the substituents tested, resulting in compounds
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Table 4. Stability in Mouse Liver Microsomes and Solubility
mouse liver microsomes
CL μL.minÀ1mgÀ1
compd no.
structure
ClogP
solubility pH 6.8 μg/mL
t1/2 min
predicted hepatic extraction ratio21 (%)
5a
N-Me
N-nPr
N-iPr
2-OPh
3.2
4.2
4.1
4.7
3.7
3.3
2.1
2.9
3.4
2.7
3.2
1.9
4.3
10
<2
<2
3
124.9
11
85
95
95
90
97
87
81
81
75
72
82
73
87
5b
407
3.4
3.2
5c
433
32d
32e
32f
32g
41a
41b
56a
56b
56i
56e
216.6
866.3
150.7
96.9
95.6
69.7
58.7
103.4
61.9
149
6.4
2-O-cyclopropylmethyl
6
1.6
2-O-cyclopropyl
2-OH
2-F
11
25
72
30
43
77
56
<2
9.2
14.3
14.5
19.9
23.6
13.4
22.4
9.3
2-Cl
3-F
3-Cl
3-OH
3-OPh
with superior potency to 1. Halogen substitutions (56aÀb, 59b)
at the 3-position of the phenyl ring exhibited improved potency
similar to that seen in the case of the corresponding 2-halo
substituted analogues Table 3 (bottom). Compounds 56i and
59d both bear 3-OH moieties and differ in being the analogous
benzyl ether and benzyl amines and behave identically in all assays,
showing comparable aerobic and anaerobic potency to 1. Com-
pounds 56c and 59c are the corresponding methyl ethers and
unremarkable except in the distinction that the amino analogue
was slightly superior to the ether analogue. This compound 56c,
however, does not show a significant increase in kcat/KM,
suggesting that the effect may be complex. Surprisingly, the
3-phenoxy substituted compound 56e showed relatively similar
activities to the hydroxy and methoxy substituents. The methyl
carboxylate (56f), morpholine (56g), and piperazine (56h) all
showed comparable cellular activity as well as enzymatic activity,
resulting in a very flat picture of the SAR at the 3-position.
In general, ortho and meta positions of the trifluoromethox-
yphenyl ring exhibited similar activity profile for the groups
tested, except for OMe and N-methylpiperazine groups, for
which a preference for meta position was observed. The loss of
potency against nonreplicating Mtb was more significant when
amine substituents were used in the R3 modification. The activity
of these analogues was not dependent on the nature of the
substituents tested. Although benzylic ether analogues and their
amine derivatives exhibited similar profile in their activities
against both replicating and nonreplicating Mtb, the amino series
exhibited a better solubility profile and a 2-fold improvement in
potency.
Solubility, Microsomal Stability and in Vivo Clearance
Rates in Mice. Solubility and stability in mouse liver microsomes
were determined for selected potent compounds (Table 4).
Hydroxyl group and halogen substitution on the aryl ring
resulted in compounds with reasonable solubility, while N-alkyl
derivatives such as 5aÀc and ether derivatives such as 32dÀf and
56e had significantly reduced solubility. This is probably due to
the higher lipophilicity of these derivatives as reflected in their
ClogP values. Metabolic stability studies in mouse liver micro-
somes revealed high clearance rates for the more lipophilic
compounds. Although the 3-Cl derivative 56b and 2-F derivative
41a were the most soluble compounds, their intrinsic clearance
was comparatively higher than that of 56a and 56i (3-F and
3-OH derivatives respectively) that exhibited the most attractive
metabolic stability. We used these intrinsic clearance rates to
calculate a predicted hepatic extraction ratio.21 On the basis of
these values, we attempted to verify whether low in vitro
clearance would be translated in vivo. We therefore performed
in vivo pharmacokinetic studies with compounds 56a, 56b, and
56i. Following intravenous injection at 5 mg/kg, compounds 56a
and 56b showed low to moderate clearance with good systemic
exposure and elimination half-lives of 1.3 and 4.3 h, respectively.
Compound 56i, on the other hand, was cleared more rapidly,
with a half-life of 0.4 h resulting in low systemic exposure. This
relative disconnect between in vitro and in vivo clearance for 56i
is suspected to be due to glucuronidation, which is not captured
in microsomal stability studies that only measure phase I
metabolism.
Ddn May Prefer Substrates in a Pseudoequatorial Con-
formation. We have previously shown that in this bicyclic system
the lipophilic tail can adopt a pseudoaxial or pseudoequatorial
conformation at C-6 and that the preferred form in crystalline 1
was pseudoaxial. In addition, we found that 7R-methylated 1
crystallized in a pseudoequatorial conformation.22 To investigate
the energetics of 1 in solution, we have calculated the Gibbs free
energy of both conformers of 1 using density functional theory at
the level of B3LYP/6-31G*23 in a solvent reaction field of
cyclohexane. These calculations reveal that the pseudoaxial form
is 0.9 kcal/mol more stable than the pseudoequatorial form,
indicating that only about 18% of 1 would be in the pseudoe-
quatorial form in solution. Furthermore, the calculated energy
barrier between the two conformers is less than 6 kcal/mol,
suggesting that both conformers exist in solution at room
temperature with rapid interconversion on the nanosecond time
scale. Interestingly, the ortho substituted R3 derivatives yielded
the largest improvements in potency, with two candidates in the
60 nM range with substantial improvements in their activity as
substrates for Ddn. Figure 2A (showing the pseudoequatorial
conformer) and Figure 2B (showing the pseudoaxial conformer)
show an overlay of the geometry optimized ortho substituted
analogues including two promising molecules (2-phenoxy-
(32d) and cyclopropyloxy- (32f)) as well as the methoxy-
(32c), chloro- (41b), and N-methylpiperazino- (41f) analogues.
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Figure 2. Superpositions of PA-824 (1) and some R3 derivatives that were geometry optimized at the level of B3LYP/6-31G*. In each, the head, tail, and
linker region of 1 is shown with yellow carbons, in (A), the tail is shown in the pseudoequatorial conformation, while in (B), the tail is shown in the
pseudoaxial conformation. (A) and (B) show R3 analogues 32c, 32d, 32f, 41b, and 41f. The inset table shows the difference in enthalpy (ΔH) and Gibbs
free energy (ΔG) at 298.15 K calculated for the pseudoequatorial conformer compared with the pseudoaxial conformer for each compound with a
solvent reaction field of cyclohexane. Atoms represented by colors are as follows: green, carbon; dark green, fluorine or chlorine; blue, nitrogen; red,
oxygen. Hydrogen atoms are not shown.
Parts A and B of Figure 2 depict an overlay of the head portion
of each ortho analogue with 1 to illustrate the conformational
deviation of the tail portion of each derivative. In general, the
tail group of the pseudoequatorial conformers better overlap
with 1 than the tail groups of the pseudoaxial conformers. In
terms of energetics, relative to 1, each ortho substitution
stabilizes the equatorial form from 0.5 kcal/mol (32c) to 1.6
kcal/mol (41f) as seen in the values of ΔH. Additional
stabilization of the equatorial conformation from 0.1 kcal/mol
(32d) to 0.9 kcal/mol (32C) arises from the larger vibrational
entropy as seen in the calculated ΔG values; the equatorial
conformation, which is more extended in molecular shape than
the axial, is more flexible and thus tends to have larger
vibrational entropy. This stabilization energy directly translates
into an increase in the concentration of the pseudoequatorial
conformer at equilibrium; for example, about 70% of com-
pound 41b would be in the pseudoequatorial form in cyclo-
hexane. Accordingly, it can provide a rationale at a qualitative
level for the 10- to 20-fold enhanced potency of the ortho-
substituted compounds listed in Table 3A provided that Ddn
preferentially recognizes the pseudoequatorial conformation.
With larger and more polar substituents such as 41f there might
be other factors (e.g., steric repulsion) at the binding site of Ddn
contributing to its relative inactivity. Besides the increase in the
concentration of the pseudoequatorial conformer, the im-
proved catalytic efficiency of several of these compounds,
including 2-phenoxy (32d), cyclopropoxy (32f), Cl (41b),
and Br (41c), suggests that these groups might well increase
either the polarizability of the tail group by donating π-electron
density (e.g., cyclopropoxy) and/or the separation of the
electrostatic charge on the tail group by an electron with-
drawing group (e.g., Cl). This increased polarizability and/or
charge separation might be responsible for enhancing potential
π-stacking interactions24,25 with an aromatic residue at the
binding site, resulting in improved catalytic efficiency of 32d,
32f, 41b, and 41c.
’ CONCLUSIONS
We have explored three positions of diversity in the
4-(trifluoromethoxy)benzylamino tail region of 1 using our pre-
viously described 3D-QSAR pharmacophore model for predict-
ing MICs of compounds in this series. All of these analogues were
predicted by our previously described QSAR model to be highly
active (MIC <1 μM), and in general this was the case. In the case
of the N-acylated and N-alkylated derivative in Table 1, however,
there were some notable exceptions. Two of the three positions
(R1 and R3) provided substantial SAR and improved lead
compounds in terms of both potency and solubility, while one
site provided little SAR (R2). This is nonetheless a useful
observation because this position could be potentially manipu-
lated to modulate the pharmacological properties of additional
derivatives. A recent report on a series of biphenyl analogues of
these compounds highlighted the importance of microsomal
stability in determining in vivo activity of compounds in this
series.17 In this study, we have confirmed good in vitro metabolic
stability with in vivo pharmacokinetic studies. On the basis of
our results, a subset of these compounds are both stable and
soluble enough to merit further testing in animals. In general,
the QSAR model was successful in predicting active com-
pounds with the exception of compounds substituted at the
benzylic nitrogen that are likely to affect the conformation of
the oxazine ring. The present study provides an additional
insight into the preferential binding mode of 1 to Ddn based
on the conformational energetics, and this should facilitate
further optimization of this exciting class of molecules for the
treatment of TB.
’ EXPERIMENTAL SECTION
General Methods. Reagents and solvents were purchased from
Aldrich, Acros, or other commercial sources and used without further
purification. Thin layer chromatography (TLC) was carried out on
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precoated Silica Gel 60 F254 plates from Merck. Compounds were
visualized under UV light or phosphomolybdic acid (PMA) stain. NMR
spectra were obtained on a Varian 400 MHz Oxford NMR. Preparative
HPLC separation was performed using Agilent reverse phase HPLC
with Atlantis dC18 column, 19 mm  250 mm, 10 μm. Unless otherwise
noted, purity of compounds was established to be >95% by LC/MS
(Aquity UPLC with PDA detector and ELSD using Waters Quattro
Micro-API Micromass with multimode ionization as detector and
Acquity BEH C18, 50 mm  2.1 mm, 1.7 μm column) and Waters
Aquity UPLC with PDA detector, using Acquity BEH C18, 100 mm Â
2.1 mm, 1.7 μm column. Column chromatography was carried out on
silica gel (100À200 mesh). MIC, MAC, and enzyme kinetic assays were
carried out as previously described.14 MIC, MAC, and enzyme assays
were carried out multiple times in two independent laboratories, and
discrepancies were repeated until concordant results were obtained.
Values reported are averages of multiple measurements, and reported
errors are standard deviations.
1H NMR (CDCl3): δ 4.08 (dd, 1H, J = 4.8, 12.0 Hz), 4.43À4.46 (m, 2H),
4.60À4.75 (m, 3H), 4.95 (m, 1H), 7.21 (m, 5H), 7.45 (m, 5H). HRMS
calcd for C21H17F3N4O5 [M + H+] 463.1229, found 463.1232.
(S)-2-Chloro-N-(2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl)-N-(4-trifluoromethoxybenzyl)-benzamide (4d).
Yield 51%. 1H NMR (DMSO-d6): δ 4.10À4.20 (m, 1H), 4.20À4.70 (m,
4H), 4.70À5.05 (m, 2H), 7.00À7.70 (m, 8H), 7.91 (s, 1H). HRMS
calcd for C21H16ClF3N4O5 [M + H+] 497.0840, found 497.0847. HPLC
purity: 88%.
(S)-3-Chloro-N-(2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl)-N-(4-trifluoromethoxybenzyl)-benzamide (4e).
Yield 55% yield. 1H NMR (CDCl3): δ 4.09 (dd, 1H, J = 4.8, 12.0 Hz),
4.43À4.61 (m, 2H), 4.65À4.73 (m, 3H), 4.93 (m, 1H), 7.18À7.26 (m,
6H), 7.32 (d, 1H, J = 7.2 Hz), 7.83 (t, 1H, J = 7.6 Hz), 7.45 (br s, 1H).
HRMS calcd for C21H16ClF3N4O5 [M + H+] 497.0840, found
497.0829.
(S)-4-Chloro-N-(2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl)-N-(4-trifluoromethoxybenzyl)-benzamide (4f).
(S)-N-(2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl)-N-(4-trifluoromethoxybenzyl)-formamide (3). Acetic an-
hydride (26 μL, 0.28 mmol) was added to formic acid (0.11 mL, 2.78
mmol) at 0 ꢀC. After 30 min at 0 ꢀC, a solution of ((S)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)-
amine 2 (50 mg, 0.14 mmol) in THF (1 mL) was added to the mixture
and stirred for 1 h. The solvent was removed in vacuo and the residue
partitioned between EtOAc and water. The organic layer was washed
with water and brine and dried (anhyd Na2SO4) and concentrated under
reduced pressure. Chromatographic purification of the residue on silica
gel eluting with 1% MeOH in CH2Cl2 gave 3 (30 mg, 55%). 1H NMR
(CDCl3): δ 4.03 (dd, 1H, J = 5.6, 12.0 Hz), 4.30À4.40 (m, 2H),
4.52À4.54 (m, 1H), 4.55 (s, 2H), 4.79 (dd, 1H, J = 6.8, 12.0 Hz), 7.17 (s,
1H), 7.23À7.24 (s, 4H), 8.37 (s, 1H). 13C NMR (CDCl3): δ 44.8, 46.2,
50.9, 67.3, 114.8, 121.7, 121.9, 128.6, 129.1, 134.4, 147.0, 149.7, 164.0.
HRMS calcd for C15H13F3N4O5 [M + H+] 387.0916, found 387.0926.
(S)-N-(2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl)-N-(4-trifluoromethoxybenzyl)-acetamide (4a). To a so-
lution of 2 (50 mg, 0.14 mmol) in dry DMF (0.5 mL) was added NaH
(60% dispersion in mineral oil, 18 mg, 0.42 mmol) at 0 ꢀC. After 0.5 h,
acetyl chloride (29 μL, 0.42 mmol) was added, and stirring continued at
room temperature for 2 h. The reaction mixture was diluted with water
(5 mL) and extracted with EtOAc (3 Â 10 mL). The combined organic
layer was washed with 1N aqueous HCl (2 Â 5 mL), water (10 mL), and
brine (10 mL), dried (anhyd Na2SO4), and concentrated under reduced
pressure. Chromatographic purification of the residue on silica gel
1
Yield 53%. H NMR (CDCl3): δ 4.11 (m, 1H), 4.42À4.73 (m, 5H),
4.94 (m, 1H), 7.21À7.41 (m, 9H). 13C NMR (CDCl3): δ 44.8, 49.5,
67.6, 115.1, 122.0, 124.9, 127.3, 128.3, 130.7, 131.2, 134.9, 136.7. HRMS
calcd for C21H16ClF3N4O5 [M + H+] 497.0840, found 497.0827. HPLC
purity: 91.3%.
(S)-N-Methyl-2-nitro-N-4-trifluoromethoxybenzyl-6,7-di-
hydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (5a). To solu-
tion of 2 (70 mg. 0.196 mmol) in a mixture of methanol (1 mL) and
AcOH (0.2 mL) was added aqueous formaldehyde (5.9 μL, 0.196
mmol) followed by NaBH(OAc)3 (24.6 mg, 0.392 mmol). After stirring
at room temperature for 3 h, the solvent was removed under reduced
pressure and the residue partitioned between water and ethyl acetate.
The organic layer was washed with water and brine, dried (anhyd
Na2SO4), and concentrated under reduced pressure. The residue was
purified by column chromatography over silica gel using 10À20%
gradient mixture of ethyl acetate/chloroform as eluent to give 5a (20
mg, 27%). 1H NMR (CDCl3): δ 2.35 (s, 3H), 3.34 (m, 1H), 3.74 (ABq,
2H, J = 14.0, 19.0 Hz), 4.15 (m, 2H), 4.53 (m, 2H), 7.18 (d, 2H, J = 8.0
Hz), 7.29 (d, 2H, J = 8.0 Hz), 7.40 (s, 1H). 13C NMR (CDCl3): δ 38.6,
45.3, 53.5, 58.4, 68.0, 115.0, 121.4, 129.7, 136.8, 147.8, 148.8. HRMS
calcd for C15H15F3N4O4 [M + H+] 373.1124, found 373.1110. HPLC
purity: 93.5%.
(S)-2-Nitro-N-propyl-N-(4-trifluoromethoxybenzyl)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (5b). To a
stirred solution of 2 (75 mg, 0.3 mmol) in THF (2 mL) containing
AcOH (0.5 mL) was added propionaldehyde (53 μL, 0.73 mmol)) and
Ti(i-OPr)4 (0.27 mL, 0.9 mmol) at room temperature. After 30 min,
NaBH(OAc)3 (190 mg, 0.9 mmol) was added and the reaction was
stirred for 15 h. The reaction mixture was neutralized with satd aq
NaHCO3 and extracted with EtOAc (3 Â 20 mL). The combined
organic layer was washed with water (10 mL) and brine (10 mL), dried
(anhyd Na2SO4), and concentrated under reduced pressure. The
residue was purified by column chromatography over silica gel using
gradient mixture of 0.5À1% MeOH in CHCl3 to afford 75 mg of 5b
(64%). 1H NMR (DMSO-d6): 0.77 (t, 3H, J = 7.2 Hz), 1.38 (m, 2H),
2.50 (m, 2H), 3.44 (m, 1H), 3.77 (ABq, 2H, J = 14.8, 20.4 Hz), 4.13 (m,
2H), 4.52 (d, 2H, J = 6.0 Hz), 7.29 (d, 2H, J = 8.0 Hz), 7.39 (d, 2H, J = 8.0
Hz), 7.98 (s, 1H). HRMS calcd for C17H19F3N4O4 [M + H+] 401.1437,
found 401.1434.
1
eluting with 1% MeOH in CH2Cl2 gave 4a (28 mg, 50%). H NMR
(CDCl3): δ 2.24 (s, 3H), 4.08 (dd, 1H, J = 5.6, 12.0 Hz), 4.30À4.40 (m,
2H), 4.52À4.54 (m, 1H), 4.55 (s, 2H), 4.79 (dd, 1H, J = 6.8, 12.0 Hz),
7.19 (d, 2H, J = 8.0 Hz), 7.23 (d, 2H, J = 8.0 Hz), 7.24 (s, 1H).
(S)-N-(2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl)-N-(4-trifluoromethoxybenzyl)-propionamide (4b). To a
solution of 2 (50 mg, 0.14 mmol) in dry THF (1 mL) was added NaH
(18 mg, 0.42 mmol) at 0 ꢀC. After 10 min, propionyl chloride (36 μL,
0.42 mmol) was added and the reaction mixture heated at 70 ꢀC for 3 h.
The reaction mixture was diluted with water (5 mL) and extracted with
EtOAc (3 Â 10 mL). The combined organic layer was washed with water
(10 mL) and brine (10 mL), dried (anhyd Na2SO4), and concentrated
under reduced pressure. Chromatographic purification of the residue on
silica gel eluting with 1% MeOH in CH2Cl2 gave 4b (30 mg, 52%).
1HNMR (CDCl3): δ 1.19 (t, 3H, J = 7.2 Hz), 2.45 (q, 2H, J = 7.2, 14.4
Hz), 4.08 (dd, 1H, J = 6.0, 12.5 Hz), 4.40 (dd, 2H, J = 7.2, 12.0 Hz), 4.61
(m, 3H), 4.77 (m, 1H), 7.17 (d, 2H, J = 8.0 Hz), 7.24À7.27 (m, 3H).
Using the same procedure, the following compounds were
synthesized:
(S)-N-Isopropyl-2-nitro-N-(4-trifluoromethoxybenzyl)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (5c). To a stir-
red solution of 2 (100 mg, 0.279 mmol) and acetone (0.2 mL, 1.39
mmol) in methanol (1.5 mL) containing AcOH (1.5 mL) was added
titanium isopropoxide (0.5 mL, 1.67 mmol) at room temperature. After
stirring for 2 h, NaBH(OAc)3 (235 mg, 1.1 mmol) was added and the
reaction was stirred for 3 days. Saturated aq NaHCO3 was added, and the
(S)-N-(2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yl)-N-(4-trifluoromethoxybenzyl)benzamide (4c). Yield 48%.
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reaction mixture extracted with ethyl acetate (2 Â 75 mL). The organic
layer was washed with water and brine, dried (anhyd Na2SO4), and then
concentrated under reduced pressure. The residue was purified over
silica gel column chromatography using 1% MeOH in CHCl3 as eluent
to give 5c (30 mg, 29%). 1H NMR (CDCl3): δ 1.13, 1.14 (2 d, 6H, J =
6.8 Hz), 3.15 (m, 1H), 3.55 (m, 1H), 3.73 (d, 1H, J = 15.2, Hz), 3.86 (d,
1H, J = 15.2 Hz), 3.99 (m, 2H), 4.31 (dd, 1H, J = 8.8, 11.2 Hz), 4.47 (dd,
1H, J = 2.4, 11.2 Hz), 7.16 (d, 2H, J = 8.0 Hz), 7.25 (s, 1H), 7.29 (d, 2H,
J = 8.0 Hz). 13C NMR (CDCl3): δ 19.3, 20.8, 46.2, 49.1, 49.2, 68.7,
115.0, 119.0, 121.0, 128.6, 138.7, 143.6, 147.5, 148.3. HRMS calcd for
C17H19F3N4O4 [M + H+] 401.1437, found 401.1451.
(S)-3-Ethyl-1-(2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl)-1-(4-trifluoromethoxybenzyl)-urea (6). To a stir-
red solution of ethylamine hydrochloride (42 mg, 0.52 mmol) and
triphosgene (312 mg, 1.05 mmol) in dry THF (4 mL) at 0 ꢀC was added
Et3N (0.5 mL, 2.1 mmol). After 15 min, a solution of 2 (75 mg, 0.2
mmol) in THF (2 mL) was added and the resulting mixture was stirred
at room temperature for 2 h. The reaction mixture was diluted with water
(10 mL) and extracted with EtOAc (3 Â 20 mL). The combined organic
layer was washed with water (10 mL) and brine (10 mL), dried (anhyd
Na2SO4), and then concentrated under reduced pressure. The residue
thus obtained (50 mg) was redissolved in DMF (3 mL) and treated with
Et3N (83 μL, 0.6 mmol), followed by ethylamine hydrochloride (49 mg,
0.6 mmol). After stirring for 3 h at room temperature, the reaction
mixture was diluted with water (10 mL) and extracted with EtOAc (3 Â
20 mL). The combined organic layer was washed with brine (10 mL),
dried (anhyd Na2SO4), and then concentrated under reduced pressure.
The crude compound was purified by column chromatography over
silica gel using 1À2% MeOH in CHCl3 as eluent to afford 35 mg (66%)
of 6. 1H NMR (DMSO-d6): 0.99 (t, 3H, J = 7.2 Hz), 3.08 (m, 2H), 4.15
(d, 2H, J = 7.2 Hz), 4.36 (dd, 1H, J = 3.2, 10.4 Hz), 4.46À4.68 (m, 4H),
6.78 (t, 1H, J = 4.8 Hz), 7.26 (d, 2H, J = 8.8 Hz), 7.30 (d, 2H, J = 8.8 Hz),
7.98 (s, 1H). 13C NMR (CDCl3): δ 15.4, 36.1, 45.8, 48.4, 49.1, 68.5,
115.2, 122.1, 127.3, 135.2, 143.9, 157.6.
Oxazol-2-yl-(4-trifluoromethoxyphenyl)methanone (9a).
To a solution of oxazole (600 mg, 8.69 mmol) in dry THF (10 mL)
at À78 ꢀC was added n-BuLi (2.9 M in hexane, 3.3 mL, 9.57 mmol).
After 30 min at À78 ꢀC, ZnCl2 (0.5 M in THF, 13.7 mL, 17.39 mmol)
was added and the mixture warmed to room temperature. After 45 min,
CuI (1.65 g, 8.69 mmol) was added and stirring was continued for 10
min. A solution of 4-trifluoromethoxybenzoylchloride (obtained by
refluxing 4-trifluoromethoxybenzoic acid (2.14 g, 9.57 mmol) in dry
THF (15 mL) containing oxalyl chloride (4.16 mL) and cat. pyridine for
3 h followed by evaporation) in THF (15 mL) was added at room
temperature, and the mixture was stirred for 1 h. The reaction mixture
was diluted with water (5 mL) and extracted with EtOAc (3 Â 10 mL).
The combined organic layer was washed with water (10 mL) and brine
(10 mL), dried (anhyd Na2SO4), and concentrated under reduced
pressure. The crude compound was purified by column chromatography
over silica gel using a gradient of 1À2% MeOH in CHCl3 to afford 9a
(900 mg, 40%). 1H NMR (CDCl3): δ 7.35 (d, 2H, J = 8.0 Hz), 7.44 (s,
1H), 7.93 (s, 1H), 8.60 (d, 2H, J = 8.0 Hz). ESI MS: m/z 258.0 (M + H).
(6S)-2-Nitro-N-(oxazol-2-yl(4-trifluoromethoxyphenyl)-
methyl)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine
(17a). To a stirred solution of oxazol-2-yl-(4-(trifluoromethoxy)-
phenyl)methanone (280 mg, 1.09 mmol) and amine 7 (200 mg,
1.09 mmol) in dry THF (4 mL) containing AcOH (2 mL) was added
Ti(i-OPr)4 (0.98 mL, 3.27 mmol) and stirred at room temperature. After
15 h, NaBH3CN (203 mg, 3.27 mmol) was added and stirring continued
for 2 h. The reaction mixture was quenched with water, neutralized to
pH 7 (satd aq NaHCO3), and extracted with EtOAc (3 Â 20 mL). The
combined organic layer was washed with water (10 mL) and brine
(10 mL) dried (anhyd Na2SO4), and concentrated under reduced
pressure. The residue was purified using preparative HPLC to afford
17a (40 mg, 9%) as a mixture of diastereomers. 1H NMR (DMSO-d6): δ
3.20 (m, 1H), 3.51À3.57 (m, 1H), 4.06À4.14 (m, 2H), 4.37À4.46 (m,
2H), 5.39 (d, 1H, J = 9.0 Hz), 7.19 (s, 1H), 7.34À7.37 (m, 2H),
7.55À7.587 (m, 2H), 7.97 and 8.00 (2s, 1H), 8.07 (s, 1H). HRMS calcd
for C17H14F3N5O5 [M + H+] 426.1025, found 426.1025.
(6S)-2-Nitro-N-(1-(4-trifluoromethoxyphenyl)ethyl)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (17b). Com-
pound 17b was prepared in a similar fashion to 17a as a mixture of
diastereomers from 9b and amine 7 in 28% yield. 1H NMR (DMSO-d6):
δ 1.26 (d, 3H, J = 6.4 Hz), 2.77 (m, 1H), 3.01 (br s, 1H), 3.78 (m, 1H),
3.92À4.07 (m, 2H), 4.19À4.35 (m, 2H), 7.31 (t, 2H, J = 8.0 Hz), 7.49 (t,
2H, J = 8.0 Hz), 7.94 and 8.01 (2s, 1H). FT-ICR HRMS calcd for
C15H15F3N4O4 [M + H+] 373.1118, found 373.1120.
(1-Benzyl-1H-imidazol-2-yl)(4-trifluoromethoxyphenyl)-
methanol (11). To a stirred solution of N-benzylimidazole (1.0 g, 6.32
mmol) in acetonitrile (15 mL) was added diisopropylcarbamoyl chlor-
ide (1.24 g, 7.59 mmol), 4-trifluoromethoxybenzaldehyde (1.4 mL, 9.49
mmol), and DIPEA (3.4 mL, 19.61 mmol) at 0 ꢀC under nitrogen atmo-
sphere. The reaction mixture was heated at reflux for 19 h, quenched
with water (30 mL), and extracted with EtOAc (20 mL Â 2). The
combined organic layer was washed with water (20 mL) and brine
(10 mL), dried (anhyd Na2SO4), and concentrated under reduced
pressure. The crude compound was purified by column chromatography
over silica gel using a gradient mixture of 0À15% of EtOAc in hexane
as eluent to afford 2.2 g (73%) of (1-benzyl-1H-imidazol-2-yl)(4-
(trifluoromethoxy)phenyl)-methyldiisopropylcarbamate. 1H NMR
(DMSO-d6): δ 1.00À1.15 (m, 12H), 3.81 (br s, 2H), 5.24À5.40 (m,
2H), 6.92 (d, 2H, J = 8.0 Hz), 7.06 (d, 2H, J = 8.0 Hz), 7.21 (s, 1H),
7.22À7.35 (m, 5H), 7.41 (d, 2H, J = 8.0 Hz). ESI MS: m/z 476.1
(M + H). To a stirred solution of the above product (2.2 g, 4.6 mmol) in THF
(20 mL) was added TFA (2 mL) and water (2 mL). After heating at
reflux for 15 h, the mixture was quenched with aqueous NaHCO3
(30 mL) and extracted with EtOAc (2 Â 20 mL). The combined organic
layer was washed with water (20 mL) and brine solution (10 mL), dried
(anhyd Na2SO4), and concentrated under reduced pressure to afford
1.32 g (82%) of 11. 1H NMR (DMSO-d6): δ 5.21 (s, 2H), 5.92 (d, 1H,
J = 4.0 Hz), 6.38 (d, 1H, J = 4.0 Hz), 6.83 (br s, 1H), 6.95À7.10 (m,
3H), 7.23À7.25 (m, 5H), 7.39 (d, 2H, J = 8.0 Hz). ESI MS: m/z 349.1
(M + H).
(1H-Imidazol-2-yl)(4-trifluoromethoxyphenyl)methanol
(12). To a stirred solution of 11 (500 mg, 1.43 mmol) in MeOH
(10 mL) was added 5% Pd/C (100 mg) and two drops of AcOH and
stirred under hydrogen (balloon) atmosphere at room temperature for
2.5 h. The reaction mixture was filtered through a Celite pad and washed
with EtOAc (50 mL). The filtrate was washed with aqueous NaHCO3
solution (20 mL), water (10 mL), and brine solution (10 mL). The
organic phase was dried (anhyd Na2SO4) and concentrated under
reduced pressure to afford 12 (300 mg, 81%). 1H NMR (DMSO-d6):
δ 5.76 (s, 1H), 6.26 (s, 1H), 6.89 (s, 2H), 7.30 (d, 2H, J = 8.4 Hz), 7.51
(d, 2H, J = 8.4 Hz), 11.94 (br s, 1H). ESI MS: m/z 259.0 (M + H).
(6S)-N-((1H-Imidazol-2-yl)(4-trifluoromethoxyphenyl)-
methyl)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-amine (17c). To a stirred solution of 12 (300 mg, 1.20 mmol) in
CH2Cl2(10 mL) was added methanesulfonyl chloride (0.11 mL, 1.45
mmol) and triethylamine (0.25 mL, 1.81 mmol) at 0 ꢀC under nitrogen
atmosphere and stirred at room temperature for 1 h. The reaction
mixture was diluted with CH2Cl2 (20 mL) and washed with water
(10 mL) and brine solution (10 mL). The organic phase was dried
(anhyd Na2SO4) and concentrated under reduced pressure to afford 400
mg of mesylate, which was redissolved in dry THF (3 mL) and added to
a previously stirred solution of (2-nitro-6,7-dihydro-5H-imidazo[2,1-
b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (0.19 mg, 1.03
mmol) in THF (10 mL) containing NaH (85 mg, 60% dispersion in
mineral oil, 2.06 mmol) at room temperature under the nitrogen
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ARTICLE
atmosphere. After 40 h, the reaction was quenched with aqueous solution
of NaHCO3 (20 mL) and extracted with EtOAc (20 mL Â 2). The
combined organic layer was washed with water (20 mL) and
brine (10 mL) and dried (anhyd Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromatography
over silica gel using a gradient mixture of 0À10% of MeOH in EtOAc as
stirred solution of amine 7 (200 mg, 1.086 mmol) and 1-(1-
bromopropyl)-4-(trifluoromethoxy)benzene (500 mg, 1.63 mmol) in
acetonitrile was added K2CO3 (449 mg, 3.26 mmol) and KI (16 mg,
0.108 mmol). After heating at reflux for 48 h, the solvent was evaporated
and the residue partitioned between ethyl acetate and water. The organic
layer was washed with brine (150 mL), dried (anhyd Na2SO4), and
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel (100À200 mesh) using a solvent
gradient mixture of 0À10% MeOH in CHCl3 to afford 17d (100 mg,
25%) as a mixture of diastereomers. 1H NMR (DMSO-d6): δ 0.67, 0.72
(2 t, 3H, J = 7.6 Hz), 1.40À1.70 (m, 2H), 2.70À2.80 (m, 1H),
2.90À3.00 (m, 1H), 3.60À4.10 (m, 3H), 4.10À4.46 (m, 2H), 7.33 (d,
2H, J = 8.0 Hz), 7.47 (d, 2H, J = 8.0 Hz), 7.94 and 8.01 (2s, 1H). HRMS
calcd for C16H17F3N4O4 [M + H+] 387.1280, found 387.1267.
1
eluent to afford 17c (65 mg, 15%) as a mixture of diastereomers. H
NMR (DMSO-d6): δ 3.10À3.30 (m, 2H), 3.90À4.10 (m, 1H), 4.13 (dd,
1H, J = 4.0, 12.0 Hz), 4.30À4.45 (m, 2H), 5.17 (d, 1H, J = 7.6 Hz),
6.60À7.10 (br s, 2H), 7.25À7.35 (m, 2H), 7.51 (dd, 2H, J = 2.4, 8.8 Hz),
7.98 (d, 1H, J = 2.4 Hz), 11.8 (br s, 1H). HRMS calcd for
C17H15F3N6O4 [M + H+] 425.1185, found 425.1187.
1-(4-Trifluoromethoxyphenyl)propan-1-ol (14a). To a stir-
red solution of 4-trifluoromethoxybenzaldehyde (5 g, 26.31 mmol) in
THF was added ethylmagnesium bromide (2 M solution, 32.8 mL, 65.7
mmol) at À15 ꢀC and warmed to room temperature over 30 min. After 3
h, the reaction mixture was quenched with aqueous NH4Cl and
extracted with ethyl acetate. The organic layer was washed with water
(2 Â 200 mL and brine (200 mL), dried over (anhyd Na2SO4), and
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel using a solvent gradient mixture
of 0À5% EtOAc in hexane as eluent to afford 14a (3.5 g, 62%). 1H NMR
(DMSO-d6): δ 0.81 (t, 3H, J = 7.6 Hz), 1.50À1.70 (m, 2H), 4.48
(m, 1H), 5.24 (d, 1H, J = 4.4 Hz), 7.29 (d, 2H, J = 8.4 Hz), 7.42 (d, 2H,
J = 8.4 Hz).
In a similar manner, the following compounds were synthesized:
(6S)-2-Nitro-N-(1-(4-trifluoromethoxyphenyl)butyl)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (17e). Yield
1
10%. H NMR (DMSO-d6): δ 0.70À0.85 (m, 3H), 1.00À1.25 (m,
2H), 1.35À1.70 (m, 2H), 2.72À2.79 (m, 1H), 2.95 (s, 1H), 3.74À3.83
(m, 1H), 4.01À4.05 (m, 2H), 4.15À4.36 (m, 2H), 7.32 (m, 2H), 7.46À
7.49 (m, 2H), 7.96 and 8.02 (2 s, 1H). HRMS calcd for C17H19F3N4O4
[M + H+] 401.1437, found 401.1431.
(6S)-2-Nitro-N-(1-(4-trifluoromethoxyphenyl)pentyl)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (17f). Yield
10%. 1H NMR (DMSO-d6): δ 0.75À0.79 (2 t, 3H, J = 5.2 Hz),
1.00À1.23 (m, 4H), 1.42À1.63 (m, 2H), 2.71À2.76 (m, 1H), 2.95
(br s, 1H), 3.67À3.80 (m, 2H), 4.01À4.05 (m, 1H), 4.16À4.34 (m,
2H), 7.30À7.43 (m, 2H), 7.46, 7.48 (2 dd, 2H, J = 8.0 Hz), 7.95 and 8.01
(2s, 1H). HRMS calcd for C18H21F3N4O4 [M + H+] 415.1593, found
415.1600.
In a similar fashion, the following compounds were synthesized:
1-(4-Trifluoromethoxyphenyl)butan-1-ol (14b). Yield 65%.
1H NMR (DMSO-d6): δ 0.88 (t, 3H, J = 7.2 Hz), 1.22À1.36 (m, 2H),
1.34À1.61 (m, 2H), 4.53À4.58 (m, 1H), 5.20 (d, 1H, J = 4.0 Hz), 7.29
(d, 2H, J = 8.0 Hz), 7.43 (d, 2H, J = 8.0 Hz).
1-(4-Trifluoromethoxyphenyl)pentan-1-ol (14c). To solu-
tion of 4-trifluoromethoxy benzaldehyde (3 g, 15.78 mmol) in THF
at À78 ꢀC was added n-BuLi (1.6 M in hexane, 11 mL, 17.36 mmol) and
warmed to room temperature over 30 min. After 2 h, the reaction
mixture was quenched with ice and extracted with diethyl ether (2 Â
60 mL). The combined organic layer was washed with water and brine
solution, dried over (anhyd Na2SO4), and concentrated under reduced
pressure. The crude compound was purified by column chromatography
over silica gel using a solvent gradient mixture of 0À2% EtOAc in hexane
as eluent to afford 14c (3.1 g, 79%). 1H NMR (DMSO-d6): δ 0.82À0.89
(m, 3H), 1.15À1.41 (m, 4H), 1.50À1.63 (m, 2H), 4.51À4.57 (m, 1H),
5.21À5.24 (m, 1H), 7.26À7.32 (m, 2H), 7.40À7.45 (m, 2H).
1-(1-Bromopropyl)-4-trifluoromethoxybenzene (15a). To
a stirred solution of 14a (3.5 g, 15.98 mmol, 1 equiv) in ether was added
PBr3 (0.5 g, 0.75 mL, 7.99 mmol) at 0 ꢀC. After stirring at 0 ꢀC for 1 h,
the reaction mixture was diluted with cold water and extracted into ether
(800 mL). The organic layer was washed with water (150 mL) and brine
(150 mL), dried over (anhyd Na2SO4), and concentrated under reduced
pressure. The residue was purified by column chromatography over
silica gel (100À200mesh) using a 5À10% gradient mixture of EtOAc in
hexane as eluent to afford 15a (2.5 g, 51%). 1H NMR (CDCl3): δ 1.00
(t, 3H, J = 6.8 Hz), 2.10À2.30 (m, 2H), 4.80À4.96 (m, 1H), 7.18 (d, 2H,
J = 8.4 Hz), 7.41 (d, 2H, J = 8.4 Hz).
2-((S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-ylamino)-2-(4-trifluoromethoxyphenyl)-acetamide (17g).
A mixture of amine 7 (100 mg, 0.543 mmol) and trifluoromethoxyben-
zaldehyde (206 mg, 1.086 mmol) were heated at 100 ꢀC for 5 min. The
reaction mixture was brought to room temperature and added TMSCN
(216 mg, 2.172 mmol) to the reaction mixture. After heating for 30 min,
the reaction mixture was cooled to room temperature and diluted with
ethyl acetate (50 mL). Organic layer was washed with water (2 Â
10 mL) and brine, dried (anhyd Na2SO4), and concentrated under
reduced pressure. The residue was purified by column chromatography
over silica gel (100À200 mesh) using a solvent gradient of 10À60%
EtOAcÀhexane as eluent to afford 105 mg (50%) of 2-((S)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ylamino)-2-(4-trifluorometh-
1
oxyphenyl)acetonitrile (16). H NMR (DMSO-d6): δ 3.46À3.52 (m,
1H), 3.80À3.95 (m, 1H), 4.00À4.10 (m, 1H), 4.17À4.20 (m, 1H),
4.42À4.59 (m, 2H), 5.33À5.42 (m, 1H), 7.42À7.47 (m, 2H), 7.55À
7.61 (m, 2H), 8.00 and 8.08 (2s, 1H). ESI MS: m/z 384.0 (M + H). A
solution of 16 (100 mg, 0.257 mmol) in ethanol (5 mL) was purged with
HCl gas for 1 h at À10 ꢀC. After 15 min, reaction mixture was
neutralized with saturated NaHCO3 solution and extracted with ethyl
acetate (2 Â 30 mL). The organic layer was washed with water and
brine, dried over (anhyd Na2SO4), and concentrated under reduced
pressure. The crude compound was purified by column chromatography
over silica gel using a solvent gradient of 15% MeOH in CHCl3 as eluent
to afford 40 mg (38%) of 17g as a mixture of diastereomers. 1H NMR
(DMSO-d6): δ 3.11À3.16 (m, 2H), 3.95À4.20 (m, 2H), 4.38À4.46 (m,
3H), 7.19 (s, 1H), 7.31À7.35 (m, 2H), 7.52À7.56 (m, 3H), 7.97 and
8.05 (2s, 1H). FT-ICR MS calcd for C15H14F3N5O5 [M + H+]
402.1019, found 402.1023. HPLC purity 95.6%.
In a similar fashion, the following compounds were synthesized:
1-(1-Bromobutyl)-4-trifluoromethoxybenzene (15b). Yield
50%. 1H NMR (CDCl3): δ 0.86 (t, 3H, J = 7.6 Hz), 1.26À1.57 (m, 2H),
2.03À2.12 (m, 1H), 2.18À2.29 (m, 1H), 4.94 (t, 1H, J = 7.6 Hz), 7.18
(d, 2H, J = 8.0 Hz), 7.42 (d, 2H, J = 8.0 Hz).
1-(1-Bromopentyl)-4-trifluoromethoxybenzene (15c). Yield
1
52%. H NMR (CDCl3): δ 0.87À0.98 (m, 3H), 1.25À1.49 (m, 4H),
2-(4-Methoxybenzyloxy)-1-(4-trifluoromethoxyphenyl)-
ethanol (19). To a mixture of 2-(4-trifluoromethoxyphenyl)oxirane
(2 g, 9.80 mmol) and p-methoxybenzyl alcohol (2.19 g, 19.60 mmol)
was added KOtBu (2.19 g, 19.6 mmol) and heated at 60 ꢀC for 2 h. The
reaction mixture was diluted with water (100 mL) and extracted with
2.00À2.40 (m, 2H), 4.90À5.00 (m, 1H), 7.16À7.20 (m, 2H), 7.39À7.44
(m, 2H).
(6S)-2-Nitro-N-(1-(4-trifluoromethoxyphenyl)propyl)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (17d). To a
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Journal of Medicinal Chemistry
ARTICLE
ethyl acetate (150 mL). The organic layer was washed with water
(2 Â 50 mL) and brine (1 Â 50 mL), dried over (anhyd Na2SO4), and
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel (100À200 mesh) using a
solvent gradient of 0À5% EtOAc in hexane as eluent to afford 19
3-Hydroxy-1-(4-trifluoromethoxyphenyl)propan-1-one
(22). A solution of compound 21 (1.2 g, 5.08 mmol) in THF (20 mL)
was treated with MnO2 (4.2 g, 50.84 mmol) at room temperature. After
6 h, the reaction mixture was filtered through Celite, washed with excess
CH2Cl2 (100 mL), and concentrated. The residue was purified by
column chromatography over silica gel using a solvent gradient of
1
(1.3 g, 34%). H NMR (DMSO-d6): δ 3.35À3.55 (m, 2H), 3.73 (s,
1
3H), 4.42 (s, 2H), 4.74À4.77 (m, 1H), 5.52 (d, 1H, J = 4.8 Hz), 6.87 (d,
2H, J = 8.8 Hz), 7.19 (d, 2H, J = 8.0 Hz), 7.30 (d, 2H, J = 8.0 Hz), 7.47
(d, 2H, J = 8.8 Hz).
0À30% EtOAc in hexane as eluent to afford 22 (450 mg, 50%). H
NMR (DMSO-d6): δ 3.14À3.18 (t, 2H, J = 6.0 Hz), 3.78 (m, 2H), 4.650
(t, 1H, J = 4.8 Hz), 7.51(d, 2H, J = 8.2 Hz), 8.10 (d, 2H, J = 8.2 Hz).
2-((S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-ylamino)-2-(4-trifluoromethoxyphenyl)-ethanol (17i). A
mixture of amine 7 (300 mg, 1.630 mmol) and 22 (457 mg, 1.456
mmol) in ethanol (5 mL) was heated at 70 ꢀC for 36 h. After cooling the
reaction mixture to room temperature, AcOH (1 drop) and NaCNBH3
(202 mg, 3.26 mmol) were added and stirring continued for 4 h. The
solvent was removed and the residue partitioned between ethyl acetate
and water. The organic layer was washed with satd aq NaHCO3 solution
(2 Â 50 mL), water (50 mL), and brine (1 Â 50 mL), dried over (anhyd
Na2SO4), and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography eluting with 2% MeOH in
CHCl3 to afford 30 mg (5%) of 17i as a mixture of diastereomers. 1H
NMR (DMSO-d6): δ 1.69À1.70 (br s, 2H), 2.23 (s, 1H), 2.64 (br s,
2H), 3.24 (s, 1H), 3.92 (d, 1H, J = 12.0 Hz), 4.15 (d, 1H, J = 12.0 Hz),
4.29À4.33 (m, 1H), 4.39À4.45 (m, 1H), 4.66 (s, 1H), 5.39 (m, 1H),
7.28À7.31 (m, 2H), 7.40À7.44 (m, 2H), 8.06 (s, 1H). HRMS calcd for
C16H17F3N4O5 [M + H+] 403.1229, found 403.1218.
2-Hydroxy-1-(4-trifluoromethoxyphenyl)ethanone (20).
To a solution of 19 (1.3 g, 3.82 mmol) in CH2Cl2 (20 mL) was added
pyridinium dichromate (2.6 g, 68.8 mmol) and 4 Å molecular sieves
powder and stirred at room temperature for 24 h. The reaction mixture
was filtered through Celite and filtrate concentrated under reduced
pressure. The crude compound was purified by column chromatography
over silica gel using a solvent gradient of 0À30% EtOAc in hexane as
eluent to afford 800 mg (62%) of 2-(4-methoxybenzyloxy)-1-(4-
(trifluoromethoxy)phenyl)ethanone. This compound (800 mg, 2.36
mmol) was dissolved in CH2Cl2 (10 mL) and added TBDMSOTf
(1.08 mL, 4.73 mmol) at 0 ꢀC. After stirring for 15 min at room
temperature, the reaction mixture was quenched with satd NaHCO3
solution and extracted with ethyl acetate (75 mL). The organic layer was
washed with water (2 Â 50 mL) and brine (50 mL) and dried (over
anhyd Na2SO4) and concentrated under reduced pressure. The crude
compound was purified by column chromatography over silica gel using
a solvent gradient of 0À30% EtOAc in hexane as eluent to afford 20 (450
mg, 86%). 1H NMR (DMSO-d6): δ 4.80 (d, 2H, J = 5.6 Hz), 5.19 (t, 1H,
J = 6.0 Hz), 7.51 (d, 2H, J = 8.0 Hz), 8.06 (d, 2H, J = 8.0 Hz).
2-((S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-ylamino)-2-(4-trifluoromethoxyphenyl)-ethanol (17h). A
mixture of amine 7 (300 mg, 1.63 mmol) and 2-hydroxy-1-(4-trifluor-
omethoxyphenyl)ethanone (430 mg, 1.95 mmol) in ethanol (2 mL) was
heated at 70 ꢀC for 1 h. After cooling, the reaction mixture to room
temperature, AcOH (1 drop) and NaCNBH3 (101.08 mg, 1.63 mmol)
were added, and stirring continued for 16 h. The solvent was removed
and the residue partitioned between ethyl acetate and water. The organic
layer was washed with satd aq NaHCO3 solution (2 Â 50 mL), water
(50 mL), and brine (1 Â 50 mL), dried over (anhyd Na2SO4), and
concentrated under reduced pressure. The residue was purified by silica
gel column chromatogrpahy eluting with 2% MeOH in CHCl3 to afford
30 mg (5%) of 17h as a mixture of diasteromers. 1H NMR (DMSO-d6):
δ 2.69 (br s, 1H), 3.09 (s, 1H), 3.30À3.43 (m, 2H), 3.70À4.60 (m, 5H),
4.80À5.00 (m, 1H), 7.20À7.60 (m, 4H), 7.91 and 8.04 (2s, 1H). HRMS
calcd for C15H15F3N4O5 [M + H+] 389.1073, found 389.1079.
1-(4-Trifluoromethoxyphenyl)propane-1,3-diol (21). To a
stirred solution of 4-trifluoromethoxybenzaldehyde (6.0 g, 31.57 mmol)
in THF (40 mL) and methyl bromoacetate (11.5 mL, 126.31 mmol), Zn
powder (16.5 g, 252.6 mmol) was added portionwise at room tempera-
ture and sonicated for 3 h. The reaction mixture was filtered through
Celite and washed with CH2Cl2 (500 mL). The filtrate was concentrated
under reduced pressure and the crude residue purified by column
chromatography over silica gel using a solvent gradient of 0À5% EtOAc
in hexane as eluent to afford 3 g of ethyl 3-hydroxy-3-(4-trifluoro-
methoxyphenyl)propanoate. This compound (3 g, 10.8 mmol) was
dissolved in anhydrous THF (30 mL) and treated with LiAlH4 (514 mg,
15.15 mmol) at 0 ꢀC. After 2 h, the reaction mixture was quenched at
0 ꢀC with satd aq Na2SO4 solution and filtered. The filtrate was washed
with water (2 Â 75 mL) and brine (75 mL), dried over (anhyd Na2SO4),
and concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel using a solvent gradient of
0À40% EtOAc in hexane as eluent to afford 21 (1.8 g, 25%). 1H
NMR (CDCl3): δ 1.80À2.10 (m, 2H), 2.31 (s, 1H), 3.31 (s, 1H), 3.90
(s, 2H), 4.80À5.05 (m, 1H), 7.10À7.30 (m, 2H), 7.35À7.45 (m, 2H).
ESI MS: m/z 237.0 (M + H).
Ethyl-2-benzyloxy-4-trifluoromethoxybenzoate (27b). To
a stirred solution of ethyl-2-hydroxy-4-trifluoromethoxybenzoate 24
(300 mg, 1.20 mmol) in DMF (4 mL) was added benzyl bromide
(0.17 mL, 1.44 mmol) and K2CO3 (331 mg, 2.4 mmol) and heated at
70 ꢀC for 30 min. It was diluted with EtOAc (50 mL) and washed with
water (30 mL Â 2) and brine solution (20 mL). The organic phase was
separated, dried (anhyd Na2SO4), and concentrated under reduced
pressure to afford 400 mg (quant) of 27b. 1H NMR (CDCl3): δ 1.37
(t, 3H, J = 7.2 Hz), 4.36 (q, 2H, J = 3.2, 14.4 Hz), 5.15 (s, 2H), 6.84 (m,
2H), 6.29À7.49 (m, 5H), 7.87 (d, 1H, J = 8.4 Hz). ESI MS: m/z 341.1
(M + H).
Ethyl 2-Methoxymethoxy-4-trifluoromethoxybenzoate
(27a). To a solution of ethyl-2-hydroxy-4-trifluoromethoxybenzoate 24
(500 mg, 2.00 mmol) in CH2Cl2 (5 mL) was added diisopropylethy-
lamine (1.09 mL, 6.72 mmol) followed by methoxymethylchloride
(0.53 mL, 6.72 mmol) at room temperature. After 3 h, the reaction
mixture was poured into water and extracted with CH2Cl2 (10 mL). The
organic layer was washed with water and brine, dried (Na2SO4), and
concentrated under reduced pressure to give crude product, which upon
purification by silica gel column chromatography using 2% ethyl acetate
in hexane as eluent gave 480 mg (81%) 27a. 1H NMR (CDCl3): δ 1.38
(t, 3H, J = 7.2 Hz), 3.52 (s, 3H), 4.36 (q, 2H, J = 3.2, 14.4 Hz), 5.25 (s,
2H), 6.89 (d, 1H, J = 8.4 Hz), 7.05 (s, 1H), 7.82 (d, 1H, J = 8.4 Hz). ESI
MS: m/z 295.0 (M + H).
Ethyl (2-Cyclopropylmethoxy)-(4-trifluoromethoxy)benz-
oate (27c). To a solution of 24 (300 mg, 1.20 mmol) in DMF (4 mL)
was added bromomethylcyclopropane (0.17 mL, 1.44 mmol) and
K2CO3 (331 mg, 2.4 mmol). After heating at 100 ꢀC for 3 h, the
reaction mixture was poured into water and extracted with EtOAc
(50 mL). The organic layer was washed with water (30 mL Â 2) and
brine solution (20 mL), dried (anhyd Na2SO4), and concentrated under
reduced pressure. The crude compound was purified by column
chromatography over silica gel using a gradient mixture of 0À3% of
EtOAcÀhexane as eluent to afford 27c (300 mg, 82%). 1H NMR
(CDCl3): δ 0.39À0.43 (m, 2H), 0.63À0.67 (m, 2H), 1.22À1.29 (m,
1H), 1.37 (t, 3H, J = 7.2 Hz), 3.89 (d, 2H, J = 6.8 Hz), 4.37 (q, 2H, J = 3.2,
14.4 Hz), 6.75 (s, 1H), 6.82 (d, 1H, J = 8.4 Hz), 7.82 (d, 1H, J = 8.4 Hz).
ESI MS: m/z 305.0 (M + H).
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2-Benzyloxy-4-trifluoromethoxybenzaldehyde (28b). To a
stirred solution of 27b (400 mg, 1.17 mmol) in THF (10 mL) was added
LiAlH4 (66 mg, 1.76 mmol) at 0 ꢀC under nitrogen atmosphere. After
stirring 0 ꢀC for 1 h, the reaction mixture was quenched with water
(30 mL) and then extracted with EtOAc (20 mL Â 2). The organic layer
was washed with water (20 mL) and brine solution (10 mL), dried
(anhyd Na2SO4), and concentrated under reduced pressure to afford
320 mg of (2-benzyloxy-4-trifluoromethoxyphenyl)methanol which was
dissolved in CH2Cl2 (10 mL) and treated with PCC (300 mg) at room
temperature under nitrogen atmosphere. After 1 h, the reaction mixture
was filtered through Celite pad and the filtrate concentrated under
reduced pressure. The residue was passed through a short pad of silica
gel eluting with EtOAcÀhexane (1:5) to afford 220 mg (63%) of 28b.
1H NMR (DMSO-d6): δ 5.34 (s, 2H), 7.08 (d, 1H, J = 8.4 Hz), 7.34 (s,
1H), 7.35 À7.44 (m, 4H), 7.51 (d, 1H, J = 7.2 Hz), 7.84 (d, 1H, J = 8.4
Hz), 10.35 (s, 1H). ESI MS: m/z 297.0 (M + H).
2-(Methoxymethoxy)-4-(trifluoromethoxy)benzaldehyde
(28a). To a stirred solution of 27a (480 mg, 1.63 mmol) in THF (5 mL)
at (0 ꢀC) was added LiAlH4 (74.2 mg, 1.96 mmol) and stirred at room
temperature for 30 min. The reaction mixture was quenched with moist
ethyl acetate. The organic layer was washed with water and brine, dried
(anhyd Na2SO4), and concentrated under reduced pressure. The crude
product was filtered through short silica plug using 10% ethyl acetate in
hexane as eluent to get 380 mg of (2-methoxymethoxy-4-trifluoro-
methoxyphenyl)-methanol, which was dissolved (380 mg, 1.29 mmol)
in CH2Cl2 (5 mL) and treated with pyridinium chlorochromate (760 mg)
at room temperature for 1 h. The reaction mixture was filtered through
Celite and the filtrate concentrated under reduced pressure. The residue
was purified by column chromatography over silica gel using gradient
mixture of 0À2% ethyl acetate in hexanes as eluent to give 28a (350 mg,
62%). 1H NMR (CDCl3): δ 3.54 (s, 3H), 5.31 (s, 2H), 6.94 (d, 1H, J =
8.4 Hz), 7.08 (s, 1H), 7.89 (d, 1H, J = 8.4 Hz), 10.43 (s, 1H).
2-Cyclopropylmethoxy-4-trifluoromethoxybenzaldehyde
(28c). To a stirred solution of 27c (280 mg, 0.92 mmol) in THF
(10 mL) was added LiAlH4 (52 mg, 1.38 mmol) at 0 ꢀC under nitrogen
atmosphere. After stirring 0 ꢀC for 1 h, the reaction mixture was
quenched with water (30 mL) and extracted with EtOAc (20 mL Â 2).
The organic layer was washed with water (20 mL) and brine solution
(10 mL), dried (anhyd Na2SO4), and concentrated under reduced
pressure to afford 190 mg of (2-cyclopropylmethoxy-4-trifluoromethox-
yphenyl)methanol. This compound was dissolved in CH2Cl2 (10 mL)
and added PCC (400 mg) at room temperature under nitrogen atmo-
sphere. After 2 h, the reaction mixture was filtered through Celite pad
and the filtrate concentrated under reduced pressure. The residue was
purified by column chromatography over silica gel using a gradient
mixture of 0À2% EtOAc in hexane as eluent to afford 28c (180 mg,
75%). 1H NMR (CDCl3): δ 0.38À0.42 (m, 2H), 0.67À0.72 (m, 2H),
1.25À1.33 (m, 1H), 3.93 (d, 2H, J = 6.8 Hz), 6.75 (s, 1H), 6.86 (d, 1H,
J = 8.4 Hz), 7.88 (d, 1H, J = 8.4 Hz), 10.48 (s, 1H). ESI MS: m/z 261.0
(M + H).
stirred solution of ethyl-[2-(2-chloro-ethoxy)-4-trifluoromethoxy]-
benzoate (800 mg, 2.56 mmol) in THF (10 mL) was added KO-t-Bu
(575 mg, 5.12 mmol) at 0 ꢀC under nitrogen atmosphere. After stirring
at 20 ꢀC for 1 h, saturated NH4Cl solution (20 mL) was added and the
reaction mixture extracted with EtOAc (15 mL Â 2). The combined
organic layer was washed with water (10 mL) and brine. The organic
phase was dried (anhyd Na2SO4) and concentrated under reduced
1
pressure to afford 25 (500 mg, 78%). H NMR (CDCl3): δ 4.86 (m,
1H), 5.09 (dd, 1H, J = 2.0, 13.2 Hz), 6.60 (dd, 1H, J = 5.6, 13.2 Hz), 6.94
(s, 1H), 7.07 (d, 1H, J = 8.0 Hz), 8.19 (d, 1H, J = 8.0 Hz). ESI MS: m/z
249.0 (M + H).
2-Cyclopropoxy-4-trifluoromethoxybenzaldehyde (26).
To a solution of Et2Zn (4 mL, 4.01 mmol) in dichloroethane (5 mL)
at À20 ꢀC was added CH2I2 (0.32 mL, 4.01 mmol) under nitrogen
atmosphere and stirred for 10 min at À20 ꢀC. A cold (0 ꢀC) solution of
4-trifluoromethoxy-2-vinyloxybenzoic acid (500 mg, 2.01 mmol, solu-
tion in 30% tolueneÀdichloroethane) was added at À20 ꢀC, and the
resulting mixture stirred at room temperature for 17 h. It was quenched
with 2N aqueous HCl (20 mL) and then extracted with EtOAc
(10 mL Â 2). The combined organic layer was washed with water
(10 mL) and brine (10 mL), dried (Na2SO4), and concentrated under
reduced pressure. The residue was purified by column chromatography
over silica gel using a gradient mixture of 0À20% EtOAc in hexane as
eluent to afford 270 mg (51%) of 2-cyclopropoxy-4-trifluoromethox-
1
ybenzoic acid. H NMR (DMSO-d6): δ 0.69 (m, 2H), 0.83 (m, 2H),
3.98 (m, 1H), 7.00 (d, 1H, J = 8.0 Hz), 7.35 (s, 1H), 7.76 (d, 1H, J = 8.0
Hz), 12.84 (s, 1H). ESI MS: m/z 261.0 (M-H). To a cold stirred solution
of 2-cyclopropoxy-4-trifluoromethoxybenzoic acid (270 mg, 1.03
mmol) in THF (10 mL) at 0 ꢀC was added BH3-DMS (0.19 mL, 2.06
mmol) under nitrogen atmosphere. After refluxing for 1.5 h at room
temperature, the reaction mixture was cooled to 0 ꢀC and quenched with
saturated aqueous NH4Cl solution (20 mL). The crude product was
extracted with EtOAc (10 mL Â 2), organic layer washed with water
(10 mL) and brine, dried (anhyd Na2SO4), and evaporated to afford 230
mg of (2-cyclopropoxy-4-trifluoromethoxyphenyl)methanol as color-
less oil. This compound (230 mg, 0.927 mmol) was dissolved in CH2Cl2
(10 mL) and treated with PCC (500 mg) under nitrogen atmosphere at
room temperature. After 0.5 h, the mixture was filtered through Celite
pad and the filtrate concentrated under reduced pressure. The residue
was purified by column chromatography over silica gel using a gradient
mixture of 0À5% EtOAc in hexane as eluent to afford 26 (200 mg, 80%).
1H NMR (CDCl3): δ 0.89 (m, 4H), 3.84 (m, 1H), 6.89 (d, 1H, J = 8.0
Hz), 7.18 (s, 1H), 7.86 (d, 1H, J = 8.0 Hz), 10.32 (s, 1H). ESI MS: m/z
247.0 (M + H).
Ethyl 2-(4-Aminophenoxy)-4-(trifluoromethoxy)benzoate
(29). To a stirred solution of 24 (250 mg, 1.0 mmol) in DMF
(5 mL) was added NaH (45 mg, 1.20 mmol) at 0 ꢀC under nitrogen
atmosphere and stirred at 0 ꢀC for 10 min. 1-Fluoro-4-nitrobenzene
(0.3 mL, 3.0 mmol) was added and the mixture heated at 100 ꢀC. After 2
h, the reaction mixture was cooled to 0 ꢀC, diluted with water (50 mL),
and then extracted with EtOAc (20 mL Â 2). The combined organic
layer was washed with water (50 mL) and brine, dried (anhyd Na2SO4),
and concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel using a gradient mixture of
0À4% EtOAc in hexane as eluent to afford 250 mg (67%) of ethyl-2-(4-
nitrophenoxy)-4-trifluoromethoxybenzoate. 1H NMR (CDCl3): δ 1.81
(t, 3H, J = 7.2 Hz), 4.21 (q, 2H, J = 7.2, 14.0 Hz), 6.94À6.99 (m, 3H),
7.22 (d, 1H, J = 8.0 Hz), 8.10 (d, 1H, J = 8.0 Hz), 8.21À8.24 (m, 2H).
ESI MS: m/z 370.0 (M À H). To a stirred solution of ethyl-2-(4-
nitrophenoxy-4-trifluoromethoxybenzoate (250 mg, 0.65 mmol) in
EtOAc (15 mL) was added 5% Pd/C (20 mg) and stirred under the
hydrogen atmosphere. After 1.5 h, the reaction mixture was filtered
through Celite pad and the filtrate concentrated under reduced pressure
to afford 29 (200 mg, 87%). 1H NMR (DMSO-d6): δ 1.23 (t, 3H, J = 7.2
4-(Trifluoromethoxy)-2-(vinyloxy)benzoic Acid (25). To a
stirred solution of 24 (800 mg, 3.20 mmol) in DMF (5 mL) was added
K2CO3 (1.32 g, 9.60 mmol) and 1-bromo-2-chloroethane (1.32 mL,
16.00 mmol) under nitrogen atmosphere and stirred at room tempera-
ture for 15 h. It was diluted with cold water (20 mL) and extracted with
EtOAc (15 mL Â 2). The combined organic layer was washed with water
(10 mL) and brine. The organic phase was dried (anhyd Na2SO4) and
then concentrated under reduced pressure to give crude compound. The
residue was purified by column chromatography over silica gel using a
gradient mixture of 0À5% EtOAcÀhexane as eluent to afford 800 mg
1
(80%) of ethyl-[2-(2-chloro-ethoxy)-4-trifluoromethoxy]benzoate. H
NMR (CDCl3): δ 1.39 (t, 3H, J = 6.8 Hz), 3.87 (t, 2H, J = 6.0 Hz), 4.28
(t, 2H, J = 6.0 Hz), 4.36 (q, 2H, J = 6.8 Hz), 6.77 (s, 1H), 6.89 (d, 1H, J =
8.4 Hz), 7.86 (d, 1H, J = 8.4 Hz). ESI MS: m/z 313.0 (M + H). To a
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Journal of Medicinal Chemistry
ARTICLE
Hz), 4.25 (q, 2H, J = 7.2, 14.0 Hz), 5.10 (br s, 2H), 6.59À6.63 (m, 3H),
6.78 (d, 2H, J = 8.2 Hz), 7.10 (d, 1H, J = 8.2 Hz), 7.86 (d, 1H, J = 8.2 Hz).
ESI MS: m/z 342.0 (M + H).
4.32À4.44 (m, 2H), 5.20 (s, 2H), 6.86 (d, 1H, J = 8.4 Hz), 7.00 (s, 1H),
7.27 (m, 1H), 7.38 (s, 1H). ESI MS: m/z 419.2 (M + H).
(S)-N-(2-Cyclopropylmethoxy-4-trifluoromethoxybenzyl)-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine
(32e). Yield 11%. 1H NMR (DMSO-d6): δ 0.32À0.35 (m, 2H),
0.55À0.59 (m, 2H), 1.19À1.25 (m, 1H), 3.27 (m, 1H), 3.78 (d, 2H,
J = 6.8 Hz), 3.87 (d, 2H, J = 6.8 Hz), 4.02 (dd, 1H, J = 3.2, 13.0 Hz), 4.17
(dd, 1H, J = 4.0, 13.0 Hz), 4.41 (m, 2H), 6.87 (d, 1H, J = 8.4 Hz), 6.91 (s,
1H), 7.39 (d, 1H, J = 8.4 Hz), 8.02 (s, 1H). HRMS calcd for
C18H19F3N4O5 [M + H+] 429.1386, found 429.1391.
(S)-N-(2-Cyclopropoxy-4-trifluoromethoxybenzyl)-2-nitro-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (32f). Yield
8%. 1H NMR (DMSO-d6): δ 0.69 (m, 2H), 0.81 (m, 2H), 3.21 (br s, 1H),
3.68 (d, 2H, J = 6.8 Hz), 3.92À4.00 (m, 2H), 4.14 (dd, 1H, J = 4.0, 12.0
Hz), 4.37À4.43 (m, 2H), 6.92 (d, 1H, J = 8.0 Hz), 7.19 (s, 1H), 7.39 (d,
1H, J = 8.0 Hz), 8.02 (s, 1H). 13C NMR (CDCl3): δ 6.3, 45.5, 47.5, 47.7,
51.4, 69.4, 106.2, 112.8, 115.2, 125.4, 130.1, 143.5, 147.3, 149.3, 157.6.
HRMS calcd for C17H17F3N4O5 [M + H+] 415.1229, found 415.1231.
HPLC purity: 93.88%.
(2-Phenoxy-4-(trifluoromethoxy)phenyl)methanol (30). To
a solution of 29 (190 mg, 0.55 mmol) in aqueous 6N HCl (5 mL) was
added aqueous NaNO2 solution (50 mg, 0.72 mmol in 1 mL of water) at
0 ꢀC. After stirring at 0 ꢀC for 30 min, a cold solution of 50% H3PO2
(0.3 mL, 5.57 mol) was added and stirred at 50 ꢀC for 1 h. The reaction
mixture was dilutedwithwater (15mL) and extractedwith EtOAc(10mL
 2). The combined organic layer was washed with water (10 mL) and
brine (10 mL), dried (anhyd Na2SO4), and concentratedto afford 130 mg
of ethyl-2-phenoxy-4-trifluoromethoxybenzoate (0.39 mmol), which was
dissolvedinTHF (10mL) and treatedwith LiAlH4 (22mg, 0.59 mmol) at
0 ꢀC under nitrogenatmosphere. After stirringatroomtemperature for 30
min, the reaction mixture was quenched with ice and filtered. The filtrate
was diluted with EtOAc (20 mL), washed with water (20 mL) and brine
solution (10mL), and dried (anhyd Na2SO4). Evaporationunder reduced
pressure and purification of the residue by column chromatography over
silica gel using a gradient mixture of 0À10% EtOAc in hexane as eluent
afforded 30 (55 mg, 48%). 1H NMR (CDCl3): δ 4.77 (d, 2H, J = 5.6 Hz),
6.67 (s, 1H), 6.97À6.99 (m, 2H), 7.03 (d, 1H, J = 8.0 Hz), 7.18 (t, 1H, J =
7.2 Hz), 7.38 (t, 2H, J = 7.2 Hz), 7.48 (d, 1H, J = 8.0 Hz). ESI MS: m/z
283.1 (M À H).
(S)-2-Nitro-N-(2-phenoxy-4-trifluoromethoxybenzyl)-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (32d). Yield
1
12%. H NMR (DMSO-d6): δ 3.28 (br s, 1H), 3.83 (d, 2H, J = 6.0
Hz), 4.00 (br d, 1H, J = 12.0 Hz), 4.14 (dd, 1H, J = 3.2, 12.0 Hz),
4.35À4.43 (m, 2H), 6.73 (s, 1H), 7.02 (d, 2H, J = 8.0 Hz), 7.14À7.20
(m, 2H), 7.41 (t, 2H, J = 7.2 Hz), 7.60 (d, 2H, J = 8.0 Hz), 8.00 (s, 1H).
HRMS calcd for C20H17F3N4O5 [M + H+] 451.1270, found 451.1251.
(S)-2-((2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-ylamino)methyl)-5-trifluoromethoxyphenol (32g). To a so-
lution of 32a (200 mg, 0.47 mmol) in THF (2 mL) was added 6N
aqueous HCl (5 mL) and stirred at room temperature for 4 h. The
reaction mixture was neutralized using aq NaHCO3 solution and
extracted with ethyl acetate (2 Â 50 mL). The organic layer was washed
with water and brine, dried (anhyd Na2SO4), and concentrated under
reduced pressure. The residue was purified by column chromatography
over silica gel using 0À5% MeOH in CHCl3 as eluent to give 32g (120
mg, 67%). 1H NMR (DMSO-d6): δ 3.25 (m, 1H), 3.76 (s, 2H), 4.01 (d,
1H, J = 12.0 Hz), 4.15 (d, 1H, J = 12.0 Hz), 4.41 (br s, 2H), 6.69À6.71
(m, 2H), 7.26 (d, 1H, J = 7.6 Hz), 8.01 (s, 1H), 10.21 (br s, 1H). 13C
NMR (CD3OD): δ 47.3, 48.0, 48.7, 70.0, 109.1, 112.5, 117.8,123.1,
125.0, 131.6, 143.9, 149.2, 150.6, 158.8. HRMS calcd for C14H13F3N4O5
[M + H+] 375.0916, found 375.0916.
2-Chloro-4-trifluoromethoxybenzaldehyde (34b). To a so-
lution of 2-chloro-4-triflouromethoxyiodobenzene (100 mg, 0.310
mmol) in THF (4 mL) at 78 ꢀC was added n-butyl lithium (1 M in
hexane, 0.62 mL, 0.021 mmol) and stirred at À78 ꢀC for 30 min. The
pale-yellow suspension was treated with a solution of DMF (0.048 mL,
0.621 mmol) in diethyl ether (1 mL) at À78 ꢀC. The reaction mixture
was stirred for 15 min and quenched with dilute aqueous H2SO4. The
organic layer was separated. The aqueous layer was extracted with
diethylether, and the combined organic layer washed with water and
brine, dried (anhyd Na2SO4), and evaporated under reduced pressure.
The residue was purified by column chromatography over silica gel by
using mixture of 0À3% ethyl acetate in hexane to get 34b (50 mg, 72%).
1H NMR (CDCl3): δ 7.28À7.33(m, 1H), 7.45 (d, 1H, J = 8.0 Hz), 7.53
(t, 1H, J = 8.0 Hz), 10.45 (s, 1H).
2-Phenoxy-4-trifluoromethoxybenzaldehyde (31). To a
stirred solution of 30 (55 mg, 0.19 mmol) in CH2Cl2 (10 mL) was
added PCC (120 mg) at room temperature under nitrogen atmosphere.
After 30 min, the reaction mixture was filtered through Celite and the
filtrate concentrated under reduced pressure. The residue was purified
by silica gel column chromatography, eluting with 10% EtOAc in hexane
to afford 31 (50 mg, quant). 1H NMR (CDCl3): δ 6.65 (s, 1H), 7.00 (d,
1H, J = 8.0 Hz), 7.11 (d, 2H, J = 8.2 Hz), 7.24À7.28 (m, 1H), 7.45 (t, 2H,
J = 8.0 Hz), 7.98 (d, 1H, J = 8.2 Hz), 10.45 (s, 1H). ESI MS: m/z 283
(M + H).
(S)-N-(2-Benzyloxy-4-trifluoromethoxybenzyl)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (32b). To a
stirred solution of 28b (100 mg, 0.33 mmol) in DMFÀAcOH (5 mL,
1:1) was amine 7 (68 mg, 0.37 mmol) at 0 ꢀC under the nitrogen
atmosphere. After stirring at room temperature for 1 h, NaBH(OAc)3
(214 mg, 1.01 mmol) was added and stirring continued for 24 h. The
reaction mixture was diluted with EtOAc (50 mL), washed with
saturated aqueous NaHCO3 solution (20 mL) and water (20 mL),
and brine solution. The organic layer was dried (anhyd Na2SO4),
evaporated under reduced pressure, and the residue purified by column
chromatography over silica gel using a gradient mixture of 0À1% MeOH
in CHCl3 as eluent to afford 32b (35 mg, 24%). 1H NMR (DMSO-d6):
3.78 (br s, 2H), 3.94À3.97 (m, 1H), 4.11À4.13 (m, 1H), 4.34À4.40 (m,
3H), 5.15 (s, 2H), 6.88 (d, 1H, J = 8.0 Hz), 7.04 (s, 1H), 7.30À7.40 (m,
5H), 7.44 (d, 1H, J = 7.8 Hz), 7.94 (s, 1H), 13C NMR (acetone-d6): δ
45.7, 48.3, 49.0, 70.3, 71.2, 106.6, 113.4, 117.4, 128.6, 129.1, 129.6, 131.2,
137.7, 149.8, 158.5. HRMS calcd for C21H19F3N4O5 [M + H+]
465.1386, found 465.1387.
The following compounds were synthesized in a similar manner:
(S)-N-(2-Methoxy-4-trifluoromethoxybenzyl)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (32c). Yield
1
23%. H NMR (DMSO-d6): δ 3.20À3.30 (m, 1H), 3.75 (d, 3H, J =
9.2 Hz), 3.81 (s, 3H), 3.90À4.10 (dd, 1H, J = 5.6, 16.0 Hz), 4.16 (dd, 1H,
J = 5.6, 16.0 Hz), 4.30À4.50 (m, 2H), 6.89 (d, 1H, J = 10.2 Hz), 6.95
(s, 1H), 7.39 (d, 1H, J = 10.2 Hz), 8.01 (s, 1H). HRMS calcd for
C15H15F3N4O5 [M + H+] 389.1059, found 389.1061. HPLC
purity: 94%.
2-Fluoro-4-trifluoromethoxybenzaldehyde (34a). To a so-
lution of 2-fluoro-4-triflouromethoxyiodobenzene (1.0 g, 3.86 mmol) in
THF (20 mL) at À78 ꢀC was added n-butyl lithium (1 M solution,
7.7 mL, 7.72 mmol) and stirred at À78 ꢀC for 30 min. The pale-yellow
suspension was treated with a solution of DMF (0.59 mL, 7.72 mmol) in
diethyl ether (5 mL) at À78 ꢀC. The reaction mixture was stirred for 15
min and quenched with dilute aqueous H2SO4. The organic layer was
separated. The aqueous layer was extracted with diethyl ether, and the
combined organic layer was washed with water and brine, dried
(S)-N-(2-Methoxymethoxy-4-trifluoromethoxybenzyl)-2-
nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (32a).
Yield 35%. 1H NMR (CDCl3): δ 3.37À3.39 (m, 1H), 3.46 (s, 3H), 3.90
(s, 2H), 3.94 (dd, 1H, J = 4, 12.4 Hz), 4.15 (dd, 1H, J = 4, 12.4 Hz),
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(Na2SO4), and evaporated under reduced pressure. The residue was
purified by column chromatography over silica gel by using mixture of
0À3% ethyl acetate in hexane to get 460 mg (57%) of 34a. 1H NMR
(CDCl3): δ 7.06 (d, 1H, J = 10.0 Hz), 7.13 (d, 1H, J = 8.0 Hz), 7.95 (t,
1H, J = 8.0 Hz), 10.32 (s, 1H).
washed with water (20 mL) and brine, dried over (anhyd Na2SO4), and
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel using a gradient of 0À7% EtOAc
in hexane as eluent to afford 900 mg (59%) of (E)-4-(2-styryl-5-(tri-
fluoromethoxy)phenyl)morpholine. 1H NMR (CDCl3): δ 2.99 (t, 4H,
J = 4.8 Hz)), 3.89 (t, 4H, J = 4.8 Hz), 6.85 (br s, 1H), 6.94 (d, 1H, J = 8.8
Hz), 7.03 (d, 1H, J = 16.0 Hz), 7.29 (d, 1H, J = 7.2 Hz), 7.35 (d, 1H, J =
8.4 Hz), 7.39 (t, 2H, J = 7.2 Hz), 7.52 (d, 2H, J = 7.2 Hz), 7.58 (d, 1H, J =
8.8 Hz). ESI MS: m/z 350.1 (M + H). To a solution of (E)-4-(2-styryl-
5-(trifluoromethoxy)phenyl)morpholine (900 mg, 2.57 mmol) in acet-
one (15 mL), water (30 mL) at 0 ꢀC was added NaIO4 (823 mg, 3.86
mmol) followed by OsO4 (1 M solution in t-BuOH, 0.05 mL, 0.02
mmol). After stirring at room temperature for 19 h, the reaction mixture
was quenched with aq sodium metabisulfite solution (20 mL) and
extracted with EtOAc (2 Â 15 mL). The combined organic layer was
washed with water (10 mL) and brine, dried (anhyd Na2SO4), and
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel using a gradient of 0À5% EtOAc
in hexane as eluent to afford 40b (350 mg, 49%). 1H NMR (CDCl3): δ
3.10 (t, 4H, J = 4.8 Hz), 3.91 (t, 4H, J = 4.8 Hz), 6.88 (s, 1H), 6.97 (d, 1H,
J = 8.4 Hz), 7.85 (d, 1H, J = 8.4 Hz), 10.23 (s, 1H).
(E)-2-Bromo-1-styryl-4-(trifluoromethoxy)benzene (36). To
a solution of 2-bromo-1-iodo-4-trifluoromethoxybenzene (200 mg, 0.544
mmol) in acetonitrile (5 mL) was added styrene (0.08 mL, 0.708 mmol)
and triethylamine (0.1 mL, 0.71 mmol). After purging with argon for 30
min, Pd(OAc)2 (10 mg, 0.04 mmol) was added and the reaction mixture
was stirred at 95 ꢀC. After 16 h, the reaction mixture was cooled to room
temperature and poured into 10% aq HCl (10 mL). The product was
extracted using diethyl ether. The combined organic layer was washed
with water and brine, dried (anhyd Na2SO4), and concentrated under
reduced pressure. The residue was purified by column chromatography
over silica gel by using a solvent gradient mixture of 0À1% EtOAc in
hexane to afford 36 (120 mg, 64%). 1H NMR (CDCl3): δ 7.02 (d, 1H, J =
16.0 Hz), 7.20 (m, 1H), 7.29À7.32 (m, 1H), 7.37À7.42 (m, 3H), 7.47
(m, 1H), 7.54 (d, 2H, J = 8.0 Hz), 7.68 (d, 1H, J = 8.0 Hz).
2-Bromo-4-(trifluoromethoxy)benzaldehyde (37). To a so-
lution of 36 (1.0 g, 2.91 mmol) in acetone (20 mL) and water (5 mL) at
0 ꢀC was added OsO4 (0.4 M in t-BuOH, 0.072 mL, 0.029 mmol) and
NaIO4 (931 mg, 4.37 mmol). After 16 h at room temperature, the
reaction mixture was diluted with aq sodium metabisulphite solution and
extracted with EtOAc (2 Â 50 mL). The combined organic layer was
washed with water and brine solution, dried (Na2SO4), and concen-
trated under reduced pressure. The residue upon purification by column
chromatography over silica gel using a gradient mixture of 0À2% EtOAc
in hexane afforded 37 (130 mg, 16%). 1H NMR (CDCl3): δ 7.27À7.30
(m, 1H), 7.52 (dd, 1H, J = 1.2, 2.4 Hz), 7.98 (d, 1H, J = 8.5 Hz), 10.32
(s, 1H).
2-(Piperidin-1-yl)-4-(trifluoromethoxy)benzaldehyde (40a).
To a stirred solution of 36 (1.0 g, 2.91 mmol), piperidine (0.34 mL,
3.49 mmol), and Cs2CO3 (2.83 g, 8.73 mmol) in dioxane (15 mL) was
added Xantphos (804 mg, 0.873 mmol) under argon. After degassing,
the reaction mixture with argon for 30 min, Pd(OAc)2 (65 mg, 0.291
mmol) was added and continued the argon purging for another 10 min.
The resulting reaction mixture was stirred at 80 ꢀC for 16 h. After cooling
to room temperature, the reaction mixture was diluted with water
(20 mL) and extracted with diethyl ether. The organic layer was washed
with water and brine, dried over (anhyd Na2SO4), and concentrated
under reduced pressure. The residue was purified by column chroma-
tography over silica gel by using a solvent gradient of 0À10% EtOAc in
hexane as eluent to afford 800 mg of (E)-1-(2-styryl-5-(trifluoromethox-
yphenyl)piperidine. This compound (0.8 g, 2.30 mmol) was dissolved in
acetone (30 mL), water (6 mL) was added OsO4 (1 M solution in
t-BuOH, 72 μL, 0.028 mmol), and NaIO4 (920 mg, 4.32 mmol) was
added at 0 ꢀC and stirred at room temperature for 16 h. The reaction
mixture was quenched with aq metabisulphite solution and extracted
with ethyl acetate. The combined ethyl acetate layer was washed with aq
Na2S2O3, water and brine and dried over (anhyd Na2SO4), and
concentrated under reduced pressure. The crude compound was
purified by column chromatography over silica gel (100À200 mesh)
by using a solvent gradient mixture of 0À15% EtOAc in hexane as eluent
to afford 40a (250 mg, 31%). 1H NMR (CDCl3): δ 1.58À1.77 (m, 6H),
3.10 (t, 4H, J = 5.2 Hz), 6.86À6.89 (m, 2H), 7.82 (d, 1H, J = 8.4 Hz),
10.18 (s, 1H).
2-(2-Bromo-4-(trifluoromethoxy)phenyl)-1,3-dioxolane
(38). To a stirred solution of 2-bromo-4-trifluoromethoxybenzaldehyde
(1.5 g, 5.57 mmol) in benzene (30 mL) was added p-TSA (15 mg) and
ethylene glycol (0.94 mL, 16.72 mmol). After heating at reflux using
DeanÀStark apparatus for 8 h, the reaction mixture was cooled to room
temperature. The organic layer was diluted with ethyl acetate and
washed with satd aq NaHCO3 (50 mL), water, and brine. The organic
layer was dried (anhyd Na2SO4), evaporated under reduced pressure,
and the residue purified by column chromatography over neutral
alumina using a solvent gradient of 0À1% EtOAc in hexane as eluent
to afford 38 (1.5 g, 86%). 1H NMR (CDCl3): δ 4.06À4.17 (m, 4H), 6.06
(s, 1H), 7.21 (d, 1H, J = 8.0 Hz), 7.45 (br s, 1H), 7.64 (d, 1H, J = 8.0 Hz).
In a similar manner, 2-(3-chloro-4-(trifluoromethoxy)phenyl)-1,3-
dioxolane (54) was prepared in 72% yield from 3-chloro-4-trifluoro-
methoxybenzaldehyde. 1H NMR (CDCl3): δ 4.00À4.15 (m, 4H), 5.80 (s,
1H), 7.32 (d, 1H, J = 8.4 Hz), 7.39À7.42 (m, 1H), 7.61 (d, 1H, J = 1.6 Hz).
2-(4-Methylpiperazin-1-yl)-4-(trifluoromethoxy)benzal-
dehyde (39). To a stirred solution of 38 (200 mg, 0.63 mmol), N-
methylpiperzine (0.085 mL, 0.766 mmol) and Cs2CO3 (622 mg, 1.914
mmol) in dioxane (3 mL) was added Xantphos (110 mg, 0.191 mmol)
and degassed with argon for 20 min. Pd(OAc)2 (14 mg, 0.063 mmol)
was added and continued the argon purging for another 10 min. After
stirring at 90 ꢀC for 8 h, the reaction mixture was cooled to room
temperature, diluted with ethyl acetate (10 mL), and washed with water
and brine and dried (anhyd Na2SO4). Evaporation of the organic solvent
under reduced pressure and purification of the residue by column
chromatography over neutral alumina using a solvent gradient of
0À1% MeOH in CHCl3 as eluent to afford 1-(2-(1,3-dioxolan-2-yl)-
5-(trifluoromethoxyphenyl)-4-methylpiperazine (120 mg). 1H NMR
(DMSO-d6): δ 2.22 (s, 3H), 2.48 (m, 4H), 2.96 (m, 4H), 3.95À4.11
(m, 4H), 5.95 (s, 1H), 7.01 (br s, 1H), 7.06 (d, 1H, J = 8.0 Hz), 7.56 (d,
1H, J = 6.4 Hz). ESI MS: 333.0 (M + H). This compound (120 mg, 0.361
mmol) was dissolved in THF (5 mL) and treated with 6 N HCl (10 mL)
at room temperature for 30 min. The reaction mixture was neutralized
with solid NaHCO3 and extracted with ethyl acetate. The organic layer
was washed with water and brine, dried (anhyd Na2SO4), and concen-
1
trated under reduced pressure to afford 39 (100 mg, 55%). H NMR
2-Morpholino-4-(trifluoromethoxy)benzaldehyde (40b).
To a stirred solution of 36 (1.5 g, 4.37 mmol) in 1,4-dioxane (20 mL)
was added morpholine (0.42 mL, 5.24 mmol) and Cs2CO3 (4.2 g, 13.11
mmol). After degassing the reaction mixture with argon, Pd(OAc)2 (98
mg, 0.43 mmol) was added and stirred at 100 ꢀC. After 18 h, the reaction
mixture was filtered through Celite. The filtrate was diluted with water
(40 mL) and extracted with EtOAc (2 Â 30 mL). The organic layer was
(DMSO-d6): δ 2.24 (s, 3H), 2.52 (m, 4H), 3.10 (m, 4H), 7.08 (m, 2H),
7.81 (d, 1H, J = 9.6 Hz), 10.09 (s, 1H). ESI MS: 289.0 (M + H).
Adopting a similar procedure, the following compounds were
synthesized:
3-Morpholino-4-(trifluoromethoxy)benzaldehyde (55a).
Yield 21%. 1H NMR (CDCl3): δ 3.12 (t, 4H, J = 5.2 Hz), 3.87 (t, 4H,
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ARTICLE
J = 5.2 Hz), 7.38 (dd, 1H, J = 2.0, 8.0 Hz), 7.50À7.60 (m, 2H), 9.96
(s, 1H).
3-(4-Methylpiperazin-1-yl)-4-(trifluoromethoxy)benzald-
0.56 mmol). After stirring at room temperature for 16 h, the reaction
mixture was diluted with CH2Cl2, washed with water and brine, dried
(anhyd Na2SO4), and concentrated under reduced pressure. The
residue upon purification by column chromatography over silica gel
using a solvent gradient of 10% EtOAc in hexane as eluent afforded 46
(140 mg, 82%). 1H NMR (DMSO-d6): δ 0.00 (s, 6H), 0.82 (s, 9H), 3.30
(s, 3H), 4.63 (s, 2H), 5.17 (s, 2H), 6.92 (d, 1H, J = 8.00 Hz), 7.19 (s,
1H), 7.25 (d, 1H, J = 8.0 Hz).
1
ehyde (55b). Yield 15%. H NMR (CDCl3): δ 2.36 (s, 3H), 2.59
(t, 4H, J = 4.4 Hz), 3.16 (t, 4H, J = 4.4 Hz), 7.35 (dd, 1H, J = 1.2, 8.0 Hz),
7.50 (dd, 1H, J = 2.0, 8.0 Hz), 7.53 (d, 1H, J = 1.6 Hz), 9.95 (s, 1H).
tert-Butyldimethyl-(4-(trifluoromethoxy)benzyloxy)silane
(43). To a solution of (4-(trifluoromethoxy)phenyl)methanol (1.49 g,
7.76 mmol) in CH2Cl2 (20 mL) at 0 ꢀC was added imidazole (686 mg,
10.08 mmol) followed by TBDMSCl (1.40 g, 9.31 mmol). After stirring
for 16 h at room temperature, the reaction mixture was diluted with
CH2Cl2, washed with water and brine and dried (anhyd Na2SO4) and
concentrated under reduced pressure. The residue was purified by
column chromatography over silica gel using 10% ethyl acetate in
hexane as eluent to give 43 (1.70 g, 60%). 1H NMR (CDCl3): δ 0.10
(s, 6H), 0.94 (s, 9H), 4.73 (s, 2H), 7.17 (d, 2H, J = 8.0 Hz), 7.34 (d, 2H,
J = 8.0 Hz).
3-(Methoxymethoxy)-4-(trifluoromethoxy)benzaldehyde
(47b). To a solution of 46 (280 mg, 0.82 mmol) in THF (2 mL) was
added tetra-n-butyl ammonium fluoride trihydrate (250 mg, 0.92
mmol). After stirring at room temperature for 30 min, it was diluted
with water and extracted with CH2Cl2. The organic layer was washed
with brine, dried (anhyd Na2SO4), and concentrated to give
(3-(methoxymethoxy)-4-(trifluoromethoxyphenyl)methanol (160 mg,
0.65 mol), which was dissolved in CH2Cl2 (4 mL) and treated with
pyridinium chlorochromate (240 mg, 1.1 mmol). After 1 h at room
temperature, the reaction mixture was diluted with ethyl acetate. The
organic layer was washed with water and brine, dried (anhyd Na2SO4),
and evaporated under reduced pressure. The residue was purified over
silica gel eluting with 10% EtOAc in hexane to give 47b (140 mg, 73%).
1H NMR (CDCl3): δ 3.51 (s, 3H), 5.30 (s, 2H), 7.41 (d, 1H, J = 8.0 Hz),
7.55 (dd, 1H, J = 1.2, 8.0 Hz), 7.75 (d, 1H, J = 1.2 Hz), 9.96 (s, 1H).
Methyl 5-Formyl-2-(trifluoromethoxy)benzoate (48). To a
solution of 43 (3.0 g, 9.8 mmol) in dry THF (45 mL) at À78 ꢀC was
added sec-BuLi (1.4 M in cyclohexane, 14.7 mL, 20.56 mmol). After 1 h
at À78 ꢀC, a solution of methyl chloroformate (1.13 mL, 14.75 mmol) in
dry THF (5 mL) was added and the reaction mixture allowed to warm to
room temperature over 1 h. After 3 h at room temperature, the reaction
was quenched with satd aq NH4Cl solution and extracted with diethyl
ether (2 Â 30 mL). The organic layer was washed with brine (50 mL),
dried (anhyd Na2SO4), and concentrated under reduced pressure to give
4 g of crude product, which was dissolved in dry THF (40 mL) and
5-((tert-Butyldimethylsilyloxy)methyl)-2-(trifluoromethoxy)-
phenol (44). To a solution of 43 (500 mg, 1.63 mmol) in THF
(10 mL) at À78 ꢀC was added TMEDA (0.245 mL, 1.63 mmol),
followed by sec-BuLi (1.4 M in cyclohexane, 1.16 mL, 1.63 mmol). After
stirring at À78 ꢀC for 1 h, fluorodimethoxyboraneÀdimethyl ether
complex (prepared by adding BF3 OEt2 (0.17 mL, 1.40 mmol) and
3
trimethylborate (0.311 mL, 2.81 mmol) in a separate flask followed by
diethyl ether (0.27 mL, 2.81 mmol) was added at À78 ꢀC. After warming
the reaction mixture to room temperature over 30 min, alkaline H2O2
was added and stirred for 20 min. The reaction mixture was poured into
1N HCl and extracted with ether. The organic layer was washed with
water and brine, dried (anhyd Na2SO4), and concentrated under
reduced pressure. The crude compound was purified by column
chromatography over silica gel using 10% ethyl acetate in hexane as
eluent to give 44 (150 mg, 28%). 1H NMR (DMSO-d6): δ 0.08 (s, 6H),
0.91 (s, 9H), 4.63 (s, 2H), 6.76 (d, 1H, J = 8.0 Hz), 6.99 (s, 1H), 7.18 (d,
1H, J = 8.0 Hz), 10.07 (s, 1H).
treated with TBAF 3H2O (4.16 g, 13.1 mmol) for 1 h. The reaction
3
mixture was diluted with water (50 mL), extracted with diethyl ether
(2 Â 50 mL), washed with water (3 Â 50 mL) and brine (50 mL), dried
(anhyd Na2SO4), and concentrated under reduced pressure. The crude
product (3 g, 12.0 mmol) was dissolved in dry CH2Cl2 (30 mL) and
treated with PCC (3.0 g, 13.9 mmol). After 30 min, the reaction mixture
was diluted with diethyl ether (50 mL), filtered through Celite, and
concentrated. The residue was purified by column chromatography over
silica gel by using a solvent gradient mixture of 2À3% EtOAc in hexane
as eluent to afford 48 (500 mg, 21%). 1H NMR (CDCl3): δ 3.98 (s, 3H),
7.51 (d, 1H, J = 8.4 Hz), 8.10 (dd, 1H, J = 1.6, 8.4 Hz), 8.47 (d, 1H, J =
1.6 Hz), 10.06 (s, 1H).
tert-Butyldimethyl(3-(4-nitrophenoxy)-4-(trifluoromethoxy)-
benzyloxy)silane (50). To a stirred solution of 44 (250 mg, 0.776
mmol) in DMF (3 mL) at 0 ꢀC was added NaH (60% in oil, 40 mg, 0.931
mmol) and followed by 4-nitro-1-fluorobenzene (0.25 mL, 2.33 mmol)
and stirred at 100 ꢀC for 1 h. The reaction mixture was diluted with water
and extracted with ethyl acetate (2 Â 20 mL). The organic layer was
washed with water and brine, dried (Na2SO4), and concentrated under
reduced pressure. The crude compound was purified by column
chromatography over silica gel (100À200 mesh) by using gradient
mixture of 0À10% ethyl acetate in hexane as eluent to afford 50 (100 mg,
30%). 1H NMR (CDCl3): δ 0.09 (s, 6H), 0.91 (s, 9H), 4.73 (s, 2H), 7.02
(d, 2H, J = 9.0 Hz), 7.15 (br s, 1H), 7.22 (dd, 1H, J = 1.2, 8.8 Hz), 7.35
(d, 1H, J = 8.4 Hz), 8.22 (d, 2H, J = 8.8 Hz).
3-Methoxy-4-(trifluoromethoxy)benzaldehyde (47a). To
the solution of 44 (3 g, 9.31 mmol) in dry DMF (10 mL) was added
K2CO3 (2.57 g, 18.63 mmol) followed by MeI (1.16 mL, 18.63 mmol)
and stirred for 3 h at room temperature. Reaction mixture was diluted
with ether (50 mL) and washed with water (2 Â 10 mL) and brine, dried
(anhyd Na2SO4), and concentrated under reduced pressure to afford 3 g
(8.928 mmol) of product which was dissolved in dry THF (12 mL) and
treated with TBAF 3H2O (2.812 g, 8.928 mmol) at room temperature.
3
After stirring the reaction mixture for 1.5 h, it was diluted with ether
(30 mL). The organic layer was washed with water (2 Â 5 mL) and brine
solution, dried (anhyd Na2SO4), and concentrated under reduced
pressure. The residue upon purification by column chromatography
over silica gel (100À200 mesh) using a solvent gradient of 20À23%
EtOAc in hexane as eluent afforded 3-methoxy-4-(trifluoromethoxy)-
benzyl alcohol (1.0 g, 52%). 1H NMR (DMSO-d6): δ 3.84 (s, 3H), 4.50
(d, 2H, J = 5.6 Hz), 5.28 (t, 1H, J = 5.6 Hz), 6.95 (d, 1H, J = 8.4 Hz), 7.17
(s, 1H), 7.27 (d, 1H, J = 8.4 Hz). A solution of the above benzylic alcohol
(900 mg, 4.484 mmol) in CH2Cl2 (10 mL) was added PCC (966 mg,
4.484 mmol) at room temperature. After stirring the reaction mixture for
1 h, it was diluted with CH2Cl2 (25 mL), washed with water (2 Â 5 mL)
and brine, and concentrated under reduced pressure. The crude
compound was purified by column chromatography over silica gel using
a solvent gradient of 10À15% EtOAcÀpetroleum ether as eluent to
afford 47a (700 mg, 91%) of 3-methoxy-4-(trifluoromethoxy)ben-
1
zaldehyde. H NMR (DMSO-d6): δ 3.95 (s, 3H), 7.62 (s, 2H), 7.71
(3-(4-Phenoxy)-4-(trifluoromethoxy)phenyl)methanol (51).
To stirred solution of 50 (300 mg, 0.677 mmol) in ethyl acetate (5 mL)
was added iron powder (380 mg, 6.77 mmol), NH4Cl (109 mg, 2.031
mmol), and H2O (1.5 mL) at room temperature. The reaction mixture
was heated at reflux for 16 h. Reaction mixture was filtered through
Celite and the filtrate diluted with ethyl acetate. The organic layer was
(s, 1H), 10.01 (s, 1H).
tert-Butyl-(3-(methoxymethoxy)-4-(trifluoromethoxyben-
zyloxy))dimethylsilane (46). To a solution of 44 (150 mg, 0.46
mmol) in CH2Cl2 (2 mL) at 0 ꢀC was added diisopropylethylamine
(0.09 mL, 0.56 mmol) followed by methoxymethylchloride (0.04 mL,
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Journal of Medicinal Chemistry
ARTICLE
washed with water and brine, dried (Na2SO4), and concentrated under
reduced pressure to afford 200 mg (0.48 mmol) of 4-(5-((tert-
butyldimethylsilyloxy)methyl)-2-(trifluoromethoxy)phenoxy)aniline,
which was dissolved in 6N aqueous HCl (3 mL) and treated with
aqueous NaNO2 (43 mg, 0.62 mmol). After stirring at 0 ꢀC for 30 min, a
cold solution of 50% H3PO2 (50%, 0.290 mL, 2.672 mmol) was added
and stirred at 50 ꢀC for 1 h. The reaction mixture was diluted with water
(5 mL) and extracted with ethyl acetate (2 Â 10 mL). The combined
organic layer was washed with aq NaHCO3, water and brine, and dried
(Na2SO4) and concentrated under reduced pressure to afford 51 (50
mg, 52%). 1H NMR (CDCl3): δ 4.63 (s, 2H), 7.00 (m, 3H), 7.10À7.14
(m, 2H), 7.31À7.37 (m, 3H).
1H NMR (DMSO-d6):.δ 2.67 (br s, 1H), 2.80À2.95 (m, 4H), 3.26À3.37
(m, 1H), 3.70À3.82 (m, 6H), 4.01 (dd, 1H, J = 3.6, 12.4 Hz), 4.18 (dd, 1H,
J = 3.6, 12.4 Hz), 4.44 (d, 2H, J = 2.4 Hz), 6.97 (s, 1H), 7.03 (d, 1H, J = 8.0
Hz), 7.49 (d, 1H, J = 8.0 Hz), 8.05 (s, 1H). 13C NMR (CDCl3): δ 46.17,
47.9, 52.9, 67.1, 69.1, 112.9, 115.3, 116.2, 130.7, 132.0, 143.4, 147.2, 149.1,
152.5. HRMS calcd for C18H20F3N5O5 [M + H+] 444.1495, found
444.1490. HPLC purity: 95.4%.
(S)-N-(2-(4-Methylpiperazin-1-yl)-4-(trifluoromethoxy)-
benzyl)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
1
6-amine (41f). Yield 32%. H NMR (DMSO-d6): δ 2.24 (s, 3H),
2.40À2.60 (m, 4H), 2.67 (br s, 1H), 2.80À3.00 (m, 4H), 3.20À3.40 (br
s, 1H), 3.77 (br s, 2H), 3.98À4.02 (m, 1H), 4.16À4.20 (m, 1H), 4.43 (br
s, 2H), 6.94 (s, 1H), 7.00 (d, 1H, J = 8.4 Hz), 7.48 (d, 1H, J = 8.4 Hz),
8.04 (s, 1H). FT-ICR MS calcd for C19H23F3N6O4 [M + H+] 457.1805,
found 457.1804.
(S)-N-(3-Morpholino-4-(trifluoromethoxybenzyl)-2-nitro-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (56g). Yield
31%. 1H NMR (DMSO-d6): δ 2.80À2.90 (m, 1H), 2.90À3.00 (m, 4H),
3.20À3.30 (m, 1H), 3.65À3.85 (m, 6H), 4.00 (dd, 1H, J = 2.8, 12.8 Hz),
4.15 (dd, 1H, J = 4.0, 12.8 Hz), 4.35À4.45 (m, 2H), 7.00À7.10 (m, 2H),
7.21 (d, 1H, J = 8.4 Hz), 8.01 (s, 1H). 13C NMR (DMSO-d6): δ 38.6, 39.8,
41.5, 42.9 (2C), 58.6 (2C), 61.2, 108.3, 111.2, 113.2, 113.7, 132.0, 133.0,
134.4, 136.9, 139.9. HRMS calcd for C18H20F3N5O5 [M + H+] 444.1495,
found 444.1506. HPLC purity: 95.3%.
3-Phenoxy-4-(trifluoromethoxy)benzaldehyde (52). To a
stirred solution of 51 (25 mg, 0.088 mmol) in CH2Cl2 (2 mL) was added
PCC (19 mg, 0.088 mmol). After stirring for 1 h at room temperature,
the reaction mixture was filtered through Celite. The filtrate was
evaporated under reduced pressure and the residue purified by column
chromatography over silica gel using a gradient mixture of 0À5% EtOAc
1
in hexane as eluent to afford 52 (15 mg, 62%). H NMR (CDCl3): δ
7.04 (d, 2H, J = 8.4 Hz), 7.21 (t, 1H, J = 7.2 Hz), 7.42 (t, 2H, J = 7.2 Hz),
7.45 (s, 1H), 7.50 (d, 1H,J =8.4 Hz), 7.63 (d, 1H,J =8.4 Hz), 9.89 (s, 1H).
(S)-N-(2-Chloro-4-trifluoromethoxybenzyl)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (41b). A mix-
ture of 34b (483 mg, 2.16 mmol) and amine 7 (200 mg 1.08, mmol) in
DMF (5 mL) containing AcOH (1 mL) was stirred at room temperature
for 1 h. NaBH3CN (165 mg, 2.52 mmol) was added to the reaction
mixture, and stirring continued for 24 h. The reaction mixture was
diluted with water and extracted with EtOAc. The combined organic
layer was washed with water and brine solution, dried (Na2SO4), and
evaporated under reduced pressure. The residue was purified by column
chromatography over silica gel using a gradient mixture of 0À5%
(S)-N-(3-(4-Methylpiperazin-1-yl)-4-(trifluoromethoxy)-
benzyl)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-
1
amine (56h). Yield 20% yield. H NMR (CDCl3): δ 2.39 (s, 3H),
2.60À2.70 (t, 4H, J = 4.0 Hz), 3.05À3.15 (t, 4H, J = 4.0 Hz), 3.35À3.45
(m, 1H), 3.88 (d, 2H, J = 10.0 Hz), 3.94 (dd, 1H, J = 4.4, 12.0 Hz), 4.16
(dd, 1H, J = 4.8, 12.0 Hz), 4.35À4.45 (m, 2H), 6.92 (dd, 1H, J = 2.0, 8.0
Hz), 6.96 (d, 1H, J = 2.0 Hz), 7.13 (dd, 1H, J = 1.2, 8.0 Hz), 7.38 (s, 1H).
HRMS calcd for C19H23F3N6O4 [M + H+] 457.1811, found 457.1798.
HPLC purity: 93.4%.
(S)-Methyl 5-((2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-ylamino)methyl)-2-(trifluoromethoxy)benzoate
(56f). Yield 55%. 1H NMR (DMSO-d6): δ 2.93À2.94 (m, 1H),
3.26À3.29 (m, 1H), 3.84À3.87 (m, 5H), 3.97À4.00 (m, 1H), 4.15
(dd, 1H, J = 3.6, 12.4 Hz), 4.37À4.45 (m, 2H), 7.46 (d, 1H, J = 8.4 Hz),
7.67 (d, 1H, J = 8.4 Hz), 7.88 (s, 1H), 7.99 (s, 1H). ESI MS: m/z 417.1
(M + H). HRMS calcd for C16H15F3N4O6 [M + H+] 417.1022, found
417.1014. HPLC purity: 95.3%.
1
MeOH-CHCl3 to afford 41b (110 mg, 26%). H NMR (DMSO-d6):
δ 2.81À2.83 (m, 1H), 3.88 (d, 2H, J = 6.8 Hz), 4.01À4.04 (br d, 1H, J =
12.0 Hz), 4.118 (dd, 1H, J = 4.0, 12.0 Hz), 4.39À4.48 (m, 2H), 7.36 (d,
1H, J = 8.0 Hz), 7.52À7.53 (m, 1H), 7.61 (d, 1H, J = 8.0 Hz), 8.03 (s,
1H). HRMS calcd for C14H12ClF3N4O4 [M + H+] 393.0577, found
393.0572.
Adopting similar procedure, the following compounds were
synthesized:
(S)-N-(2-Fluoro-4-(trifluoromethoxy)benzyl)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (41a). Yield
1
15%. H NMR (CDCl3): δ 3.34 (br s, 1H), 3.97 (m, 3H), 4.19 (dd,
(S)-N-(3-Methoxy-4-(trifluoromethoxy)benzyl)-2-nitro-6,
7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (56c). Yield
1H, J = 4.0, 12.0 Hz), 4.39À4.43 (m, 2H), 6.97À6.98 (m, 1H), 7.02 (s,
1H, J = 8.0 Hz), 7.38 (d, 1H, J = 8.0 Hz), 7.40 (s, 1H). HRMS calcd for
C14H12F4N4O4 [M + H+] 377.0873, found 377.0878. HPLC purity:
95.86%.
1
13%. H NMR (DMSO-d6): δ 2.80À2.90 (m, 1H), 3.24 (br s, 1H),
3.65À3.88 (m, 5H), 3.90À4.00 (m, 1H), 4.10À4.20 (m, 1H),
4.38À4.45 (m, 2H), 6.95 (d, 1H, J = 11.0 Hz), 7.19 (s, 1H), 7.25 (d,
1H, J = 11.0 Hz), 8.00 (s, 1H). FT-ICR HRMS calcd for C15H14F3N4O5
[M + H+] 389.1067, found 389.1069.
(S)-N-(2-Bromo-4-(trifluoromethoxy)benzyl)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (41c). Yield
1
46%. H NMR (DMSO-d6): δ 2.81À2.85 (m, 1H), 3.32À3.34 (m,
(S)-N-(3-(Methoxymethoxy)-4-(trifluoromethoxy)benzyl)-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine
(56d). Yield 12%. 1H NMR (CDCl3): δ 3.41 (m, 1H), 3.45 (s, 3H), 3.90
(d, 2H, J = 10 Hz), 3.94 (m, 1H), 4.15 (dd, 1H, J = 4.0, 12.0 Hz),
4.35À4.45 (m, 2H), 5.21 (s, 2H), 6.95 (d, 1H, J = 8.4 Hz), 7.19À7.21
(m, 2H), 7.38 (s, 1H). ESI MS: m/z 419.2 (M + H).
(S)-N-(3-Fluoro-4-trifluoromethoxybenzyl)-2-nitro-6,7-di-
hydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (56a). Yield
35%. 1H NMR (CDCl3): δ 3.40 (br s, 1H), 3.88À3.97 (m, 4H), 4.20
(dd, 1H, J = 4.0, 12.0 Hz), 4.38À4.46 (m, 2H), 7.11 (d, 1H, J = 8.0 Hz),
7.22 (dd, 1H, J = 1.6, 10.0 Hz), 7.28 (d, 1H, J = 8.0 Hz), 7.39 (s, 1H). 13C
NMR (CDCl3): δ 47.5, 47.8, 49.7, 69.3, 115.5, 116.4, 116.6, 119.1,
121.6, 123.7, 135.4, 140.3, 143.3, 147.3, 153.2, 155.7. HRMS calcd for
C14H12F4N4O5 [M + H+] 378.0742, found 378.0729.
1H), 3.85 (d, 2H, J = 7.0 Hz), 4.03 (dd, 1H, J = 2.4, 120 Hz), 4.20 (dd,
1H, J = 4.0, 12.0 Hz), 4.38À4.48 (m, 2H), 7.40 (dd, 1H, J = 1.2, 7.5 Hz),
7.0 (d, 1H, J = 7.5 Hz), 7.66 (d, 1H, J = 1.6 Hz), 8.04 (s, 1H). 13C NMR
(CDCl3): δ 47.5, 47.9, 49.9, 69.4, 115.6, 120.1, 121.5, 123.5, 125.3,
130.7, 136.9, 143.3, 147.2, 148.4. HRMS calcd for C14H12BrF3N4O4
[M + H+] 437.0072, found 437.0064. HPLC purity: 94.5%.
(S)-2-Nitro-N-(2-(piperidin-1-yl)-4-(trifluoromethoxy)benzyl)-
6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (41d). Yield
13%. 1H NMR (DMSO-d6): δ 1.50À1.65 (m, 6H), 2.69À2.84 (m, 5H),
3.24 (m, 1H), 3.77 (d, 2H, J = 7.2 Hz), 4.00 (dd, 1H, J = 3.2, 13.0 Hz), 4.17
(dd, 1H, J = 3.2, 13.0 Hz), 4.42À4.45 (m, 2H), 6.93 (br s, 1H), 6.98 (d, 1H,
J = 8.4 Hz), 7.48 (d, 1H, J = 8.4 Hz), 8.03 (s, 1H). FT-ICR MS calcd for
C19H22F3N5O4 [M + H+] 442.1696, found 442.1698.
(S)-N-(2-Morpholino-4-(trifluoromethoxy)benzyl)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (41e). Yield 24%.
(S)-N-(3-Chloro-4-trifluoromethoxybenzyl)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (56b). Yield
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Journal of Medicinal Chemistry
ARTICLE
37%. 1H NMR (CDCl3): δ 3.40 (br s, 1H), 3.87À3.97 (m, 4H), 4.19 (dd,
1H, J = 4.4, 12.0 Hz), 4.18À4.46 (m, 2H), 7.26À7.30 (m, 2H), 7.40 (s,
1H), 7.46 (s, 1H). 13C NMR (CDCl3): δ 47.5, 47.9, 49.6, 69.3, 115.4,
119.1, 121.7, 122.6, 127.2, 127.5, 130.1, 139.6, 143.4, 144.2, 147.3. HRMS
calcd for C14H12ClF3N4O4 [M + H+] 393.0577, found 393.0574.
(S)-2-Nitro-N-(3-phenoxy-4-(trifluoromethoxy)benzyl)-6,
7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine (56e). Yield
In
a
similar manner, (S)-6-(3-methoxy-4-(trifluoromethoxy)-
benzyloxy)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (59c)
was synthesized in 30% yield. H NMR (DMSO-d6): δ 3.80 (s, 3H),
1
4.20À4.30 (m, 3H), 4.48 (m, 1H), 4.60À4.70 (m, 3H), 6.96 (d, 1H, J =
8.4 Hz), 7.14 (s, 1H), 7.31 (d, 1H, J = 8.4 Hz), 8.03 (s, 1H). FT-ICR
HRMS calcd for C15H14F3N3O6 [M + H+] 390.0907, found 390.0909.
(S)-6-(3-Fluoro-4-(trifluoromethoxy)benzyloxy)-2-nitro-6,
7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (59b). To a stirred
solution of 49b (7.0 g, 33.65 mmol) in methanol (50 mL) was added
NaBH4 (1.9 g, 50.48 mmol) at room temperature under the N2
atmosphere. After 1 h, ice water (100 mL) was added and the reaction
mixture extracted with EtOAc (3 Â 60 mL). The combined organic layer
was washed with water (50 mL) and brine, dried over (anhyd Na2SO4),
and concentrated under reduced pressure to afford 3-fluoro-4-trifluoro-
methoxybenzyl alcohol (7 g, quant.). 1H NMR (DMSO-d6): δ 4.53 (d,
2H, J = 5.6 Hz), 5.44 (t, 1H, J = 5.6 Hz), 7.26 (d, 1H, J = 8.4 Hz), 7.40 (d,
1H, J = 11.2 Hz), 7.48À7.54 (t, 1H, J = 8.4 Hz). ESI MS: m/z 211.0 (M +
H). To a solution of 3-fluoro-4-trifluoromethoxybenzyl alcohol (7 g,
33.33 mmol) in CH2Cl2 (70 mL) at 0 ꢀC was added triphenylphosphene
(13.0 g, 49.99 mmol), followed by NBS (8.9 g, 49.99 mmol) in
portionwise at room temperature under the N2 atmosphere. After 1 h,
water (100 mL) was added and the reaction mixture extracted with
EtOAc (3 Â 40 mL). The combined organic layer was washed with water
(30 mL) and brine, dried over (anhyd Na2SO4), and concentrated under
reduced pressure. The crude compound was purified by column
chromatography over silica gel using a gradient of 0À5% EtOAcÀ
hexane as eluent to afford 4-(bromomethyl)-2-fluoro-1-(trifluorom-
1
11%. H NMR (CDCl3): δ 3.35 (br s, 1H), 3.80À3.90 (m, 3H), 4.14
(dd, 1H, J = 4.0, 12.0 Hz), 4.30À4.41 (m, 2H), 6.95 (d, 1H, J = 1.6 Hz),
6.98 (d, 2H, J = 8.0 Hz), 7.07 (dd, 1H, J = 1.6, 8.0 Hz), 7.16 (t, 1H, J = 8.0
Hz), 7.30 (d, 1H, J = 8.4 Hz), 7.34À7.38 (m, 3H). 13C NMR (CDCl3): δ
47.6, 47.7, 50.0, 69.2, 115.2, 118.5, 119.5, 122.9, 123.5, 123.8, 129.8, 139.4,
143.4, 147.2, 149.5, 156.4. HRMS calcd for C20H17F3N4O5 [M + H+]
451.1229, found 451.1228.
(S)-5-((2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-ylamino)methyl)-2-(trifluoromethoxy)-phenol (56i). To a
solution of 56d (20 mg, 0.047 mmol) in THF (1 mL) was added 6N HCl
(1 mL). After 1 h, the reaction mixture was neutralized with aq NaHCO3
solution and extracted with ethyl acetate. The organic layer was washed
with water and brine and dried (anhyd Na2SO4) and concentrated under
reduced pressure. The residue was triturated with ether/pentane to
obtain 56i (8.0 mg 47%). 1H NMR (CDCl3): δ 3.40 (m, 1H), 3.91 (d,
2H, J = 10 Hz), 3.95 (m, 1H), 4.16 (dd, 1H, J = 4.0, 12.0 Hz), 4.39À4.44
(m, 2H), 5.50 (br s, 1H), 6.86 (d, 1H, J = 8.4 Hz), 7.02 (br s, 1H), 7.19
(d, 1H, J = 8.4 Hz), 7.35 (s, 1H). HRMS calcd for C14H13F3N4O5 [M +
H+] 375.0916, found 375.0929. HPLC purity: 95.7%.
4-(Bromomethyl)-2-(methoxymethoxy)-1-(trifluoromethoxy)-
benzene (58a). To a solution of 46 (1.1 g, 3.0 mmol) in THF (10 mL)
was added TBAF (1.0 g, 3.6 mmol). After stirring at room temperature
for 1 h, the reaction mixture was diluted with water and extracted with
CH2Cl2. The organic layer was washed with brine, dried (anhyd
Na2SO4), and concentrated to give (3-(methoxymethoxy)-4-(trifluoro-
methoxyphenyl)methanol (600 mg, 1.039 mmol), which was dissolved
in CH2Cl2 (20 mL) and treated with triphenylphosphine (644 mg, 2.46
mmol) and NBS (437 mg, 2.458 mmol) at 0 ꢀC. After stirring for 30 min
at room temperature, the reaction mixture was poured into water (5 mL)
and extracted with diethyl ether (2 Â 15 mL). The organic layer was
washed with water, NaHCO3 solution, and brine and dried (Na2SO4).
The solvent was evaporated under reduced pressure and the residue
passed through a short silica pad to afford 58a (500 mg, 66%), which was
contaminated with 15% triphenylphosphine oxide. This material was
1
ethoxy)benzene (58b, 6.0 g, 66%). H NMR (DMSO-d6): 4.73 (s,
2H), 7.42 (d, 1H, J = 8.4 Hz), 7.55À7.71 (m, 2H). Alkylation of 57 using
58b was carried out as described previously during the synthesis of 59a
to afford 59b (32% yield). 1H NMR (DMSO-d6): δ 4.20À4.35 (m, 3H),
4.47 (d, 1H, J = 11.6 Hz), 4.65À4.75 (m, 3H), 7.26 (d, 1H, J = 8.0 Hz),
7.44 (d, 1H, J = 10.8 Hz), 7.55 (t, 1H, J = 8.0 Hz), 8.04 (s, 1H). 13C NMR
(CD3OD): δ 58.3, 68.6, 69.2, 70.1, 117.1, 117.3, 117.7, 120.6, 123.1,
124.8, 124.9, 136.8, 141.0, 143.9, 149.1, 154.4, 156.9. HRMS calcd for
C14H11F4N3O5 [M + H+] 378.0742, found 378.0729.
(S)-5-((2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-
6-yloxy)methyl)-2-(trifluoromethoxy) phenol (59d). To a so-
lution of 59a (150 mg, 0.357 mmol) in THF (5 mL) was added 6N HCl
(10 mL) and stirred for 1 h at room temperature. The reaction mixture
was neutralized with satd NaHCO3 solution and extracted with EtOAc
(2 Â 20 mL). The organic layer was washed with water and brine
solution, dried (Na2SO4), and evaporated under reduced pressure. The
residue was triturated with diethyl ether/pentane (1:1) mixture to afford
59d (90 mg, 67%). 1H NMR (DMSO-d6): δ 4.21À4.27 (m, 3H), 4.47
(d, 1H, J = 12.0 Hz), 4.55À4.67 (m, 3H), 6.78 (dd, 1H, J = 1.6, 8.4 Hz),
6.94 (d, 1H, J = 1.2 Hz), 7.20 (d, 1H, J = 8.0 Hz), 8.03 (s, 1H), 10.16 (s,
1H). 13C NMR (DMSO-d6): δ 46.7, 66.5, 67.8, 68.9, 116.3, 118.0, 119.0,
121.0, 122.8, 135.3, 138.3, 142.1, 147.0, 149.7. HRMS calcd for
C14H12F3N3O6 [M + H+] 376.0756, found 376.0750.
In Vivo Pharmacokinetic Studies. The Novartis Institute for
Tropical Disease’s Animal Care and Use Committee approved all animal
experimentalprotocols and animalsuse. The compoundswere formulated
in 10% w/v hydroxypropyl-β-cyclodextrin (Acros) and 10% w/v lecithin
(Acros/Organics, New Jersey, USA). The formulation was centrifuged,
and the supernatant was injected into animals via the intravenous route at
5 mg/kg. Blood samples were collected at various time points between 1
min and 24 h postdose. Plasmasampleswereextracted and analyzedusing
optimum LC/MS/MS conditions. Pharmacokinetic parameters were
determined using WinNonLin Professional, version 5.0.1 (Pharsight,
CA, USA), by noncompartmental analysis. The extraction ratio (ER)
was calculated using the formula as previously described.21
1
used as such for next reaction, without further purification. H NMR
(DMSO-d6): δ 3.40 (s, 3H), 4.69 (s, 2H), 5.29 (s, 2H), 7.17 (dd, 1H, J =
2.0, 8.4 Hz), 7.37 (d, 1H, J = 8.4 Hz), 7.41 (d, 1H, J = 2.0 Hz).
In a similar manner, 4-(bromomethyl)-2-methoxy-1-(trifluoromet-
hoxy)benzene (58c) was prepared from (3-methoxy-4-(trifluorome-
1
thoxy)phenyl)methanol in 46% yield. H NMR (CDCl3): δ 3.89 (s,
3H), 4.46 (s, 2H), 6.96 (dd, 1H, J = 2.8, 10.8 Hz), 7.02 (d, 1H, J = 2.8
Hz), 7.18 (dd, 1H, J = 1.2, 10.8 Hz).
(S)-6-(3-(Methoxymethoxy)-4-(trifluoromethoxy)benzyloxy)-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (59a). To
a solution of 4-(bromomethyl)-2-(methoxymethoxy)-1-(trifluorometh-
oxy)benzene (0.3 g, 0.9 mmol) (S)-2-nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazin-6-ol (57, 0.15 g, 0.81 mmol) in DMF (10 mL) was
added NaH (60% in mineral oil, 36 mg, 0.9 mmol) at À78 ꢀC and
warmed to room temperature over 1 h. Water (5 mL) was added and the
reaction mixture extracted with EtOAc (2 Â 30 mL). The organic layer
was washed with water and brine, dried (Na2SO4), and evaporated
under reduced pressure. The residue was purified by column chroma-
tography over silica gel using a solvent gradient of 0À50% EtOAc in
petroleum ether to afford 59a (115 mg, 34%). 1H NMR (DMSO-d6): δ
3.37 (s, 3H), 4.25 (m, 3H), 4.46 (d, 1H, J = 12.4 Hz), 4.60À4.70 (m,
3H), 5.26 (s, 2H), 7.02 (d, 1H, J = 8.4 Hz), 7.22 (s, 1H), 7.34 (d, 1H, J =
8.4 Hz), 8.03 (s, 1H). ESI MS: m/z 420.3 (M + H).
Quantum Chemistry and Superposition Study. In our
modeling studies, both pseudoequatorial and pseudoaxial forms of 1
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Journal of Medicinal Chemistry
ARTICLE
were used as reference structures.22 The pseudoequatorial conformer of
1 was built by replacing the methyl of 7-(R)-methyl-1 (X-ray structure)
with a hydrogen atom while the X-ray structure of 1 was used for the
pseudoaxial conformer without further modification. The geometries of
these two conformers of 1 were then energy minimized with the density
functional theory at the level of B3LYP/6-31G*.23 The two resulting
conformers were subsequently modified to build structures of the
corresponding conformers of 32c, 32d, 32f, 41b, and 41f. These
compounds (each with two conformations) were then energy mini-
mized at the level of B3LYP/6-31G* in cyclohexane utilizing the
polarized continuum solvent model with the UAKS parameters set.
The resulting structures were taken as the assumed geometries of these
compounds. Alternative conformers were considered by varying the
hydrogen position of the linker group (NH), which can H-bond with the
corresponding ortho-substituent. This was done by first changing the
dihedral angle of HÀNÀC6ÀC5, and then after another cycle of full
geometry optimization, the respective low energy conformers were
chosen for comparison (see Figure 2). For the superposition study,
the optimized structures of 32c, 32d, 32f, 41b, and 41f were overlaid
onto the geometry optimized 1 with the rigid fit of Quanta 2008
(Accelrys) using the nine heavy atoms of the imidazo-oxazine as a
common alignment site. The root-mean-square deviations of such fit for
each compound were all less than 0.01 Å and 0.03 Å for the pseudoe-
quatorial and the pseudoaxial conformers, respectively. The energy
barrier for the conversion of pseudoaxial 1 to the pseudoequatorial
form was calculated by varying the dihedral angle of O8ÀC7ÀC6ÀO
and then further optimizing the geometry with the highest enegy on the
potential energy surface with the keyword, opt=(ts,calcfs,noeigentest) as
implemented in Gausssian 09 software.23
MsCl, methanesulfonyl chloride; Mtb, Mycobacterium tuber-
culosis; Mtz, metronidazole; NBS, N-bromosuccinimide; SAR,
structureÀactivity relationship; TB, tuberculosis; TBAF, tetra-
n-butylammonium fluoride; TBS or TBDMS, tert-butyldimethyl-
silyl; Tf, triflate; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TMEDA, tetramethlyethylenediamine; TMSCN, trimethylsilyl
cyanide
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’ AUTHOR INFORMATION
Corresponding Author
*Phone: 301-435-7509. Fax: 301-480-5712, cbarry@mail.nih.
gov. CEB3, Room 2W20D Building 33, 9000 Rockville Pike,
Bethesda, Maryland 20892.
Present Addresses
Translational Health Science and Technology Institute, Vaccine
and Infectious Disease Research Centre, Haryana 122016, India.
^Curachem, Inc., Gyeonggi-do, South Korea.
#Experimental Therapeutics Center, 31 Biopolis Way, Singapore
138669.
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’ ACKNOWLEDGMENT
This work was funded, in part, by the intramural research
program of NIH, NIAID, by NITD, and by a grant from the Bill
and Melinda Gates Foundation and the Wellcome Trust through
the Grand Challenges in Global Health Initiative. We thank Anne
Goh and the bioanalytical team of NITD for the determination of
compound concentrations in microsomal and plasma samples
and Dr. Noel Whittaker, NIDDK, for HRMS of some of the
compounds. The quantum chemical study utilized PC/LINUX
clusters at the Center for Molecular Modeling of the NIH
(http://cit.nih.gov), and this research was also supported in part
by the NIH Intramural Research Program through the Center for
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’ ABBREVIATIONS USED
AcOH, acetic acid; DIPEA, N,N-diiospropylethylamine; DMF,
dimethylformamide; ER extraction ratioMAC, minimum anaero-
bicidal concentration; MIC, minimum inhibitory concentration;
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