Structure-activity relationships for amide-, carbamate-, and urea-linked analogues of the tuberculosis drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl] oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824)

Article abstract of DOI:10.1021/jm2012276

Analogues of clinical tuberculosis drug (6S)-2-nitro-6-{[4- (trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824), in which the OCH₂ linkage was replaced with amide, carbamate, and urea functionality, were investigate

Full text of DOI:10.1021/jm2012276

Article
pubs.acs.org/jmc
Structure−Activity Relationships for Amide-, Carbamate-,
And Urea-Linked Analogues of the Tuberculosis Drug
(6S)-2-Nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (PA-824)
Adrian Blaser,† Brian D. Palmer,† Hamish S. Sutherland,† Iveta Kmentova,† Scott G. Franzblau,‡
Baojie Wan,‡ Yuehong Wang,‡ Zhenkun Ma,§ Andrew M. Thompson,*,† and William A. Denny†
†Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland
1142, New Zealand
‡Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois
60612, United States
§Global Alliance for TB Drug Development, 40 Wall Street, New York 10005, United States
S
* Supporting Information
ABSTRACT: Analogues of clinical tuberculosis drug (6S)-
2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-
imidazo[2,1-b][1,3]oxazine (PA-824), in which the OCH2
linkage was replaced with amide, carbamate, and urea func-
tionality, were investigated as an alternative approach to
address oxidative metabolism, reduce lipophilicity, and improve
aqueous solubility. Several soluble monoaryl examples dis-
played moderately improved (∼2- to 4-fold) potencies against
replicating Mycobacterium tuberculosis but were generally
inferior inhibitors under anaerobic (nonreplicating) conditions.
More lipophilic biaryl derivatives mostly displayed similar or reduced potencies to these in contrast to the parent biaryl series.
The leading biaryl carbamate demonstrated exceptional metabolic stability and a 5-fold better efficacy than the parent drug in
a mouse model of acute M. tuberculosis infection but was poorly soluble. Bioisosteric replacement of this biaryl moiety by
arylpiperazine resulted in a soluble, orally bioavailable carbamate analogue providing identical activity in the acute model,
comparable efficacy to OPC-67683 in a chronic infection model, favorable pharmacokinetic profiles across several species, and
enhanced safety.
respiratory poisoning (via nitric oxide release) under aerobic
and anaerobic conditions, respectively.6,8,9 Recent clinical
data for 1, indicating excellent early bactericidal activity at
200 mg/day,10 has been shown to correlate well with the time-
dependent activity observed in vivo, where percentage time that
the free drug concentration exceeds the minimum inhibitory
concentration (MIC) was found to be the crucial parameter for
dose optimization.11
In considering the challenges for further antituberculosis
drug development,1 it is useful to reiterate here some key de-
sirable attributes for a new drug in terms of pharmacological
profile and safety. These include good stability under various
conditions, high oral bioavailability, sufficient lung penetration,
a lengthy elimination half-life (suitable for once-daily or less
frequent dosing), acceptable protein binding, freedom from
genotoxicity/mutagenicity, satisfactory cardiac safety (minimal
hERG inhibition), and avoidance of other toxicities.4 An absence
INTRODUCTION
■
With levels of tuberculosis (TB) incidence at their highest ever,
there is an urgent need for new drugs with greater efficacy,
safety, and affordability that can reduce the high pill burden and
shorten lengthy treatment times (a minimum of 6 months for
drug-susceptible TB with current therapy).1,2 This is partic-
ularly true in cases of multi- and extensively drug resistant
tuberculosis (MDR-TB and XDR-TB), and in persistent forms
of the disease, where the existing drugs are less effective.3 After
more than four decades of minimal effort in this regard, intense
recent efforts spearheaded by the Global Alliance for
Tuberculosis Drug Development (TB Alliance, founded
February 2000) have led to more than 10 agents in current
clinical trials.4,5 The bicyclic nitroimidazole analogues, PA-824
(1)6 and OPC-67683 (2),7 currently in phase II clinical trials,
are of particular interest because these drugs are active against
both replicating and nonreplicating persistent Mycobacterium
tuberculosis (M. tb) infections in animal models and are useful
against MDR-TB.4,5 Compound 1 reportedly acts via a
bioreductive mechanism to cause both cell wall inhibition and
Received: September 14, 2011
Published: December 8, 2011
© 2011 American Chemical Society
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Article
of drug−drug interactions is particularly critical with combina-
tion treatments but also because HIV coinfection is a significant
problem (estimated at up to 15% of the global TB burden;
∼2 million people), especially in Africa.2,12 The first-line TB drug
rifampicin, for example, is a potent inducer of cytochrome
P450 (CYP) enzymes (especially CYP3A4) that can metabolize
comedications such as antiretroviral agents (mostly HIV
protease inhibitors), leading to subtherapeutic concentrations;
another first-line TB drug, isoniazid, is a CYP inhibitor, which
can cause toxicities due to higher than expected levels of co-
administered drugs.1,4 Beyond these properties, the new agent
should further display good bactericidal activity and good
sterilizing activity (against persistent bacilli), with no cross-
resistance to current drugs, and must be affordable.4 These are
characteristics that we have been pursuing for second-generation
analogues of 1. Finally, regarding challenges encountered in
early stage drug development, the ability of M. tb to exist in a
range of metabolic states (such that subpopulations of bacteria
in different microenvironments in vivo are able to exhibit dif-
ferential drug susceptibilities) clearly adds additional complex-
ity both to the discovery of structure−activity relationships
(SARs) and to the possible correlation of in vitro potency with
in vivo efficacy.3,4,12
toxicity risks associated with molecular fragmentation. How-
ever, α-methyl substitution, described19 as a possible means to
suppress oxidative metabolism of benzyl ethers, proved an in-
effective stabilization strategy in our hands. In contrast, removal
of the benzylic methylene provided one biaryl analogue (5) that
exhibited reasonable efficacy in the acute model (8-fold better
than 1) and negligible fragmentation to alcohol metabolite 6 in
liver microsomes. Unfortunately, both this compound (5) and
the other leading candidate that arose from this ether mod-
ification study (7: 89-fold better than 1 in the same in vivo
model) were poorly soluble (<0.5 μg/mL in water at pH = 7)
and pyridine analogues of these were less effective. Therefore,
additional linker options were considered to better enable us to
address this metabolic stability issue.
More than a decade ago, Baker et al. (PathoGenesis Corporation)
disclosed the high in vitro potencies of a few amide, urea, and
carbamate-linked analogues of 1 against Mycobacterium bovis
(similar data against M. tb were not provided).20 Very recently,
more soluble amine-linked congeners of 1 were also exam-
ined21 (by Novartis), but no reports have further investigated
the former linker classes. In this current paper, we therefore
describe replacement of the OCH2 linkage of 1 (and its iso-
mers, selected biaryl analogues, and their bioisosteres) with
various amide, carbamate, and urea functionality and evaluate
this as a possible alternative strategy to circumvent oxidative
metabolism, reduce compound lipophilicity, and improve aqueous
solubility (while maintaining reasonable in vivo efficacy) in
order to provide a superior TB drug candidate with enhanced
safety.
Following on from our initial investigations13−15 of (hetero)-
biaryl side chain analogues of 1 (including early biphenyl lead 3)
that culminated in the more soluble, orally bioavailable pyridine
derivative 4 (having markedly superior efficacies to 1 in mouse
models of both acute and chronic M. tb infection), we recently
described16 an SAR study of alternative side chain ether link-
ages (to OCH2), seeking new candidates with both high ef-
ficacy and enhanced metabolic stability (reduced toxicity potential)
(Figure 1). The latter issue was related to the possible muta-
CHEMISTRY
■
The reference benzyl ethers 822 and 9, together with acetamide
3622 (previously reported by amide formation on an acid
precursor), were prepared by NaH-assisted alkylation of the
known20 oxazine alcohol 6 and the requisite halides (Scheme 1).
a
Scheme 1
Figure 1. Structures of antitubercular agents.
a
Reagents and conditions: (i) 2-/3-OCF3 BnBr or
4-OCF3PhNHCOCH2Cl, NaH, DMF, 0−20 °C, 2.5−6 h; (ii) RPhNCO
or RBnNCO, CuCl, DMF, 20 °C, 2−4 h; (iii) MeI, NaH, DMF, 20
°C, 3 h; (iv) ArB(OH)2 (or pinacol ester), toluene, EtOH, 2 M
K2CO3, Pd(dppf)Cl2 under N2, reflux, 1−3 h.
genicity of smaller metabolites formed by oxidative cleavage of
the benzyl ether (or picolyl ether) side chain based upon re-
ported Ames data for 6-nitroimidazooxazoles.17 While com-
pounds such as 1 and 2 are not mutagenic (both in vitro and in
vivo),6,7 and 1 has displayed excellent safety in clinical trials,18
we deemed that removal of, or substitution on, the benzylic
methylene moiety should be evaluated as first options to
enhance compound stability and further minimize any possible
The known20 6-O-carbamates 33 and 35, as well as halide
analogues 86−90 (needed for the synthesis of biaryl derivatives
67−81 using Suzuki coupling), were also derived from 6 via
Cu(I)-catalyzed condensation with aryl isocyanates; N-methylation
313
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Journal of Medicinal Chemistry
Article
a
of 33 then yielded 34. The required 4-(trifluoromethoxy)benzyl
isocyanate23 was made by Curtius rearrangement of the acyl
azide (as reported).
Scheme 3
Synthesis of the remaining linker variants began with the
known20,21 chiral amine 91 (stored as its hydrochloride salt for
improved stability). Acylation or sulfonylation reactions of 91
using the relevant acyl or sulfonyl chlorides (sourced from the
available acids via standard procedures where necessary)
yielded the desired amides (10−12, 22, 23, 92, and 93) and
sulfonamides (16−18), which were subsequently N-methylated
in a few cases (Scheme 2A−C). Halides 92 and 93 were also
a
Scheme 2
a
Reagents and conditions: (i) ArOCOCl, DIPEA, CH2Cl2, 20 °C,
1.5−3 h; (ii) ArB(OH)2 (or pinacol ester), toluene, EtOH, 2 M
K2CO3, Pd(dppf)Cl2 under N2, reflux, 1−3 h; (iii) CO(OCCl3)2,
DIPEA, CH2Cl2, 0−20 °C, 2−4 h; (iv) ArNCO or 4-OCF3PhNCS,
NMM, Bu2Sn(OAc)2, DMF, 20 °C, 2−5 h; (v) MeI, NaH, DMF,
0−20 °C, 1 h.
a
Reagents and conditions: (i) ArCOCl or ArSO2Cl or
a
Scheme 4
RPhOCH2COCl, DIPEA, DMF, or THF or dioxane, 20 °C, 1−4 h;
(ii) MeI, NaH or K2CO3, DMF, 0 or 20 °C, 1.5−3 h; (iii) ArB(OH)2,
toluene, EtOH, 2 M K2CO3, Pd(dppf)Cl2 under N2, reflux, 1−3 h;
(iv) 4-OCF3PhCHO, NaBH3CN, AcOH, DMF, 20 °C, 2.5 h;
(v) 4-OCF3PhCOCH2Br or 4-OCF3PhNHCOCH2Cl, DIPEA, DMF,
20−65 °C, 2.5−4.5 h.
elaborated to biaryl derivatives 37−46 by Suzuki coupling.
The 6-amino congener of 1 (24)21 was prepared via reduc-
tive alkylation of 91 (as reported), whereas extended linker
analogues (25 and 26) were made in poor yields from un-
optimized reactions employing the commercial halides
(Scheme 2D).
Aryl chloroformates, required for the assembly of N-
carbamates 27, 28,20 and 94, were generated by base-catalyzed
reaction of triphosgene with the appropriately substituted phenol
or benzyl alcohol24,25 and reacted in situ with 91 (Scheme 3A).
Alternatively, treatment of 91 with aryl isocyanates (using
NMM and catalytic dibutyltin diacetate) gave ureas 29 and
32 and their halide analogues 95−99; use of the related aryl
isothiocyanate also led to thiourea 31 (Scheme 3B,C). N-
Methylation on both nitrogen atoms of urea 29 provided 30,
while Suzuki couplings on halides 94−99 produced compounds
47−66.
Finally, the O-carbamates 82−85 were formed by chlorofor-
mylation of alcohol 6, followed by one-pot coupling with the
cyclic amine derivatives 103, 107, 112, or 113 (Scheme 4).
The required intermediates 103 and 107 were prepared via
Mitsunobu reactions on the N-Boc hydroxy-amines 100 and
104, followed by Boc deprotection. Synthesis of 112 was
achieved via successive Schiff base formation between amine
a
Reagents and conditions: (i) DEAD, PPh3, THF, reflux, 5 h; (ii)
TFA, CH2Cl2, 20 °C, 1−4 h; (iii) MeOH, reflux, 4 h, then
NaBH4, 20 °C, 30 min; (iv) MeI, K2CO3, Me2CO, 20 °C, 18 h; (v)
CO(OCCl3)2, Et3N, THF, 5−20 °C, 2 h, then 103, 107, 112, or 113,
THF, 20 °C, 2 h.
108 and aldehyde 109, in situ reduction with NaBH4 (to give
110), N-methylation (111) and deprotection, while 11326 was
obtained using Buchwald coupling on N-Boc piperazine.
314
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Article
treatment times.4,29 Because 1 displays an alternative mechanism
of action under these conditions,8,9 it is unsurprising that
different SAR trends can result from testing analogues of 1 in
these two assays.13−16,21 Mammalian cytotoxicity was also
evaluated30 against VERO cells (CCL-81, American Type
Culture Collection) in a 72 h exposure, using a tetrazolium dye
assay. Here, the compounds generally showed very low toxicity,
with IC50 values of >128 μM (the highest concentration tested;
data not shown) for all except five analogues, none of which
showed notable activities.
In addition to these biological assays, the lipophilicities of the
new analogues were studied using CLogP estimations obtained
from the latest ACD LogP/LogD software (version 12.0;
Advanced Chemistry Development Inc., Toronto, Canada); in
most cases the compounds in this study were significantly more
hydrophilic than their OCH2-linked counterparts (by up to 1.8
log units). Aqueous solubility data (at pH = 7) were also
measured for almost all of the 4-OCF3 substituted examples;
about half of the monoaryl analogues (particularly those con-
taining amine, amide, or urea functionality, Table 1) demon-
strated superior results in comparison to 1 itself. For biaryl
RESULTS AND DISCUSSION
■
A total of 68 new and 10 known analogues of 1 in which the
OCH2 linkage was modified or replaced with amine, amide,
carbamate, and urea functionality were initially screened in two
in vitro assays for determination of their aerobic and anaerobic
activities (minimum inhibitory concentrations, MICs) against
M. tb strain H37Rv (Tables 1, 3 and 6). The MABA (aerobic)
assay27 assessed growth inhibition of replicating M. tb over an
8-day exposure, with the MIC being the lowest drug
concentration to effect an inhibition of >90% (recorded values
being the mean of 2−5 independent determinations
SD).
The luminescence-based low-oxygen-recovery (LORA, anaero-
bic) assay28 further examined compound effects against bacteria
in the nonreplicating state that models clinical persistence,
using an 11-day exposure to M. tb that had first been adapted to
low oxygen conditions by extended culture. Screening for
antitubercular activity under oxygen depletion conditions (or
related stresses) has been recommended as a reasonable
starting point for the identification of agents that are better at
removing persistent bacteria in vivo to potentially shorten
Table 1. Physicochemical Properties and MIC Values for Amide, Urea, And Carbamate-Linked Analogues of 1 (Monoaryls)
c
MIC (μM)
a
b
compd
X
R
CLogP
solubility
MABA
LORA
d
8
OCH2
OCH2
OCH2
2-OCF3
3-OCF3
4-OCF3
2-OCF3
3-OCF3
4-OCF3
2-OCF3
3-OCF3
4-OCF3
2-OCF3
3-OCF3
4-OCF3
2-OCF3
3-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
4-OCF3
3.49
3.29
3.11
2.99
2.65
2.48
2.26
2.41
1.98
1.95
2.24
1.71
3.05
3.40
2.98
2.56
2.40
2.95
1.97
2.54
2.58
2.86
2.48
1.96
2.17
2.03
2.62
1.75
2.38
2.67
1.8
0
8.6 1.7
2.1 0.3
2.6 1.4
>64
9
0.36 0.06
0.50 0.30
e
1
19
109
17
10
11
NHCO
27
7
NHCO
4.9 1.2
1.8 0.1
45 18
>64
e
12
NHCO
19
>64
>64
>64
>64
>64
>64
45
2
13
14
15
16
17
18
19
20
21
N(Me)CO
N(Me)CO
N(Me)CO
NHSO2
24
22
8
4
>64
>64
NHSO2
NHSO2
6.1
9.7 2.2
N(Me)SO2
N(Me)SO2
N(Me)SO2
NHCOCH2
NHCOCH2O
NHCH2
20
12
6
4
4
6
1
6
4
64
15
64
0.53 0.04
0.27 0.03
0.16 0.05
0.22 0.02
7.6 0.6
22
e
22
21
f
23
43
10
f
24
131
21
5.3 1.3
>64
25
26
27
NHCH2CO
NHCH2CONH
NHCO2
0.70 0.22
46 16
g
g
>64
>64
e
28
NHCO2CH2
NHCONH
NMeCONMe
NHCSNH
NHCONHCH2
OCONH
0.15 0.04
0.23 0.01
11
3
e
29
93
38
12
32
5.4
18
10
68
4.2 0.3
>64
30
31
32
12
2
8.0 0.2
0.27 0.07
0.12 0.01
0.21 0.03
0.25 0.02
>64
4.1 0.4
2.7 0.8
5.4 0.8
e
33
34
OCON(Me)
OCONHCH2
OCH2CONH
e
35
17
4
d
36
0.24
0
7.8 0.9
a
CLogP values, calculated using the ACD LogP/LogD prediction software (version 12.0, Advanced Chemistry Development Inc., Toronto,
b
c
Canada). Solubility (μg/mL) in water at pH = 7 and 20 °C, determined by HPLC (see Experimental Section). Minimum inhibitory concentration,
determined under aerobic (MABA)30 or anaerobic (LORA)28 conditions. Each value is the mean of at least two independent determinations SD.
d
e
f
g
Ref 22. Ref 20. Ref 21. Unstable under assay conditions.
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Journal of Medicinal Chemistry
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compounds (Table 3), replacement of the terminal phenyl ring
by pyridine and meta linkage of the biaryl moiety gave
improved solubility values, as found previously.15,16
identical potency to 1 in the LORA assay and was 4-fold more
effective in the MABA assay, albeit this compound had inferior
aqueous solubility. Unexpectedly, N-methylation of 33 (34)
lowered the CLogP value and improved solubility compared to
33 (∼3-fold), although the activity of 34 was concomitantly
reduced (∼2-fold in both assays). Finally, both homologation
of 33 (35) and extension of the 6-OCH2 linker with an amide
moiety (36) gave inferior results to 33 (notably under non-
replicating conditions), although the latter compound did
provide enhanced solubility.
The activities of a few of the compounds in Table 1 against
M. bovis have been reported previously.20 Against M. tb, these
compounds were found to be on average about 3-fold less
potent but showed a similar rank order of aerobic activity
(Table 2). Overall then, the results suggested several possible
An initial examination of direct (monoaryl) analogues of 1
(Table 1) began with a small study investigating the effect of
varying the position of the trifluoromethoxy substituent for
some relatively rigid amide and sulfonamide linkers (NHCO,
NMeCO, NHSO2, NMeSO2; note that we elected to consider
only this particular substituent based on previous studies13−16
indicating broad utility and superior in vivo efficacy). In the
parent OCH2-linked series, the 2-OCF3 analogue 8 was the
least effective in both replicating and nonreplicating M. tb
assays, compared with the 3- and 4-OCF3 analogues 9 and 1.
This pattern also held for the amide and sulfonamide linkers
(10−21), where 4-OCF3 was preferred; therefore, the latter
positioning was retained for all of the remaining linker ana-
logues, enabling a direct comparison with 1. However, com-
pounds 10−21 were generally much less active than their cor-
responding OCH2-linked congeners, likely due to the increased
rigidity imposed, which could project the phenyl ring at an
inappropriate orientation in the binding site of the activating
enzyme. A similar reduction in potency was recently observed
for a phenyl ether variant of 1 (X = O),16 while previous
studies13,15 have also shown consistent and significant differ-
ences in the mean MIC potencies of ortho-, meta-, and para-
linked biaryl analogues, consistent with an elongated but
constricted side chain binding site. N-Methylation was
disfavored in the amide series (13−15) but was unexpectedly
activating in the less impressive sulfonamide series, with the
4-OCF3 analogue 21 showing equivalent aerobic potency to the
similarly lipophilic 1 (but 8.5-fold lower potency under an-
aerobic assay conditions). Homologation of the more soluble
amide 12 (to the less constrained 22) significantly improved
the MABA MIC value (∼7-fold; 0.27 μM) although the
anaerobic activity remained weak (21 μM). Further known
linker extension (23)21 provided a more modest (∼2-fold) ad-
ditional potency gain in both MIC assays and still permitted
good aqueous solubility (43 μg/mL). Analogous improvements
in aerobic MIC potency with longer flexible linkers have been
noted in earlier reports.16,21
Table 2. Comparative Activity of Selected Compounds of
Table 1 against M. bovis and M. tb
MIC (μM)
a
b
compd
X
M. bovis
M. tb
1
OCH2
0.167
1.34
0.50
1.8
12
28
29
33
NHCO
NHCO2CH2
NHCONH
OCONH
0.037
0.039
0.039
0.075
0.15
0.23
0.12
0.25
35
OCONHCH2
a
b
Ref 20. This work.
alternatives to OCH2 as linking groups for additional analogues
of 1. The seven best of these (NHCOCH2, NHCOCH2O,
NHCO2CH2, NHCONH, NHCONHCH2, OCONH, and
OCONHCH2) were therefore selected for the syntheses of
small series of related compounds bearing predominantly para-
linked biaryl side chains (Table 3). We have shown pre-
viously13,15 that for compounds containing the OCH2 linker,
para-linked biaryl analogues were substantially more effective
than 1 against both replicating and nonreplicating M. tb, with
meta-linked congeners generally providing slightly inferior
results but ortho-linked compounds being poor, consistent
with a long, linear binding site. However, in the current study,
we elected to investigate both para- and meta-linkage for two
cases (X = NHCONH, OCONH) where there was greater
conformational restriction to allow for the possibility that a
meta-linked geometry might be more favorable in such
instances. On the basis of our earlier findings in the para-
linked biphenyl series,13 showing a significant correlation of
aerobic M. tb activity with both overall lipophilicity, and with
the electron-withdrawing ability of substituents, we selected
three such substituents (CF3, F, OCF3) for the terminal phenyl
ring in addition to pyridine comparators (e.g., 3-aza-4-CF3) that
might enhance aqueous solubility.15,16
The previously described 6-amino analogue of 1 (24)21
provided greater solubility than 1 and had better activity against
replicating M. tb (∼2-fold), as reported. However, this com-
pound seemed slightly less effective than 1 under nonrepli-
cating conditions, and ketone- or amide-containing extended
linker derivatives of this (25, 26), designed to introduce some
conformational restriction, were much worse. Alternatively, the
novel N-carbamate 27, while quite stable in nonpolar solvents,
was surprisingly not sufficiently stable in DMSO (the solvent
employed to dissolve test compounds for these in vitro assays),
resulting in very poor MIC results. In marked contrast to this,
the known20 homologue of 27 (28), an isomer of the more
hydrophilic extended amide 23 above, gave very good MABA
data (0.15 μM), being equipotent to 23 in both assays.
Furthermore, both the urea analogue of 27 (29) and its
homologue (32) demonstrated a comparable MIC profile to
amine 24, but N-methylation (30) or thiation (31) significantly
diminished potency. Encouragingly, urea 29 also showed a
5-fold higher aqueous solubility than 1 (93 μg/mL).
To provide a broader assessment of the utility of the selected
linkers in this new context, Table 4 compares the activities of
the different subseries of biaryl compounds of Table 3 with
their corresponding OCH2-linked analogues by examining the
mean MIC values for each subseries having the four common
aryl substituents indicated above. This analysis suggests that, for
the 4-linked biaryls, all of the new linkers resulted in considerably
Noting that all of the 6-N-linked compounds above had
anaerobic activities that were inferior to the 6-OCH2-linked
trial drug 1, we finally examined a few 6-O-linked congeners of
the compounds above. Pleasingly, O-carbamate 33 displayed an
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Journal of Medicinal Chemistry
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Table 3. Physicochemical Properties and MIC Values for Amide, Urea, and Carbamate-Linked Analogues of 1 (Biaryls)
c
MIC (μM)
a
b
compd
X
link
R
CLogP
solubility
1.2
MABA
LORA
d
3
OCH2
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
3′
3′
3′
3′
3′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
3′
3′
3′
3′
3′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4′
4-OCF3
4-CF3
4.95
3.45
3.48
4.40
1.69
2.31
3.12
3.14
4.07
1.35
1.97
3.75
3.78
4.70
1.99
2.61
2.18
2.20
3.13
0.42
1.03
3.45
3.48
4.40
1.69
2.31
2.21
2.92
2.94
3.87
1.16
3.26
3.28
4.21
1.50
2.11
3.92
3.94
4.86
2.15
2.77
2.67
3.27
3.30
4.22
1.51
0.035 0.015
0.44 0.15
0.33 0.11
0.22 0.03
0.46 0.01
0.68 0.28
0.25 0.01
0.17 0.05
0.18 0.07
0.69 0.15
0.69 0.25
0.08 0.02
0.085 0.025
0.11 0.01
0.23 0.01
0.34 0.10
0.035 0.005
1.3 0.1
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
NHCOCH2
NHCOCH2
NHCOCH2
NHCOCH2
NHCOCH2
NHCOCH2O
NHCOCH2O
NHCOCH2O
NHCOCH2O
NHCOCH2O
NHCO2CH2
NHCO2CH2
NHCO2CH2
NHCO2CH2
NHCO2CH2
NHCONH
NHCONH
NHCONH
NHCONH
NHCONH
NHCONH
NHCONH
NHCONH
NHCONH
NHCONH
NHCONH
NHCONHCH2
NHCONHCH2
NHCONHCH2
NHCONHCH2
OCONH
23
11
4
2
4-F
4-OCF3
3-aza, 4-CF3
3-aza, 4-F
4-CF3
1.8
5.0 0.1
>32
>32
>32
4-F
26
25
30
31
1
4
2
1
4-OCF3
3-aza, 4-CF3
3-aza, 4-F
4-CF3
0.96
0.67
3.0
4.5 2.0
4.2 0.9
5.0 2.1
4.7 1.3
4.3 0.6
3.6 0.6
3.8 1.2
5.8 3.4
4-F
4-OCF3
3-aza, 4-CF3
3-aza, 4-F
4-CF3
4-F
0.03
0
4-OCF3
3-aza, 4-CF3
3-aza, 4-F
4-CF3
0.25 0.10
0.22 0.01
0.23 0.01
0.19 0.03
1.3 0.5
13
15
1
1
>32
4-F
9.1 3.4
4-OCF3
3-aza, 4-CF3
3-aza, 4-F
4-aza, 3-CF3
4-CF3
0.17
0.06
0
18
17
5
3
2
0
0.17 0.07
0.24 0.01
0.36 0.14
0.32 0.13
0.11 0.02
1.0 0.2
11
13
>32
26
4-F
3
4-OCF3
3-aza, 4-CF3
4-CF3
0.94
32 16
>32
0.40 0.21
0.04 0.0
1.0 0.4
1.5 0.4
0.85 0.25
2.9 0.8
5.8 2.0
>32
OCONH
4-F
0.027 0.005
OCONH
4-OCF3
3-aza, 4-CF3
3-aza, 4-F
4-CF3
0.07
5.8
0.06
0
OCONH
0.11 0.01
0.24 0.01
0.055 0.005
1.3 0.4
OCONH
OCONH
OCONH
4-F
18
4
OCONH
4-OCF3
3-aza, 4-CF3
3-aza, 4-F
4-aza, 3-CF3
4-CF3
0.06
0.05 0.01
>32
OCONH
0.25
0
4.1 0.4
4.3 1.7
6.2 2.7
>32
OCONH
0.13 0.04
0.75 0.11
0.24 0.01
0.11 0.01
0.52 0.08
0.69 0.19
OCONH
OCONHCH2
OCONHCH2
OCONHCH2
OCONHCH2
4-F
4.5 0.8
2.4 0.8
4-OCF3
3-aza, 4-CF3
0.34
11
6
a
CLogP values, calculated using the ACD LogP/LogD prediction software (version 12.0, Advanced Chemistry Development Inc., Toronto,
b
c
Canada). Solubility (μg/mL) in water at pH = 7 and 20 °C, determined by HPLC (see Experimental Section). Minimum inhibitory concentration,
determined under aerobic (MABA)30 or anaerobic (LORA)28 conditions. Each value is the mean of at least two independent determinations SD.
d
Ref 13.
lower activity than the parent OCH2 linker in both the aerobic
and anaerobic assays (X/OCH2 ratios mostly >10), with only
the N-linked carbamate analogues (47−50; the most lipophilic
of the 6-acylamino derivatives) showing reasonable overall
potencies. In contrast, in the two less lipophilic 3-linked biaryl
subseries investigated, the alternative linkers (X = NHCONH
and OCONH) were slightly more effective than OCH2 in pro-
viding activity against replicating M. tb (mean MABA MIC values
1.5- to 3.2-fold better) and gave comparable (X = NHCONH)
or improved potencies (X = OCONH) in the LORA assay
317
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Journal of Medicinal Chemistry
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Table 4. Influence of the Linker X on the Mean Inhibitory Potencies of Biaryl Analogues
b
mean MICs (μM)
ratio (X/OCH2)
a
compds
link
X
ΔCLogP
MABA
LORA
MABA LORA
37−40
42−45
47−50
52−55
57−60
63−66
67−70
72−75
78−81
4′
4′
4′
3′
4′
4′
3′
4′
4′
NHCOCH2
NHCOCH2O
NHCO2CH2
NHCONH
NHCONH
NHCONHCH2
OCONH
−0.55
−0.89
−0.25
−1.83
−0.55
−1.09
−0.75
−0.09
−0.73
0.36
0.32
0.13
0.13
0.43
0.46
0.059
0.41
0.39
>18
>28
4.6
13
11
11
18
4.6
0.68
15
16
0.31
2.9
1.5
>12
>19
0.37
>14
>7.5
6.6
>19
>31
1.6
OCONH
>22
>12
15
14
OCONHCH2
a
Mean difference in CLogP values between compounds in the linker subclass X and the OCH2-linked analogues having the same biaryl geometry
(4′ or 3′ link) and bearing the same substituents. Ratio of mean MIC values (X/OCH2) for the various linker subclasses; data for the parent series
b
(para: MABA 0.028, LORA 1.6 μM; meta: MABA 0.19, LORA 4.3 μM) were derived from refs 13 and 15 (see text).
Table 5. Comparative Activities of Mono- and Biphenyl Analogues: Influence of Overall Lipophilicity
MIC (μM)
b
compds
MABA
LORA
MIC ratio (I/II)
a
I
II
X
ΔCLogP
I
II
I
II
1.3
5.0
25
5.0
18
MABA
14
1.2
LORA
1
3
OCH2
1.84
1.84
1.67
1.84
1.92
1.84
2.24
1.84
0.50
0.27
0.16
0.15
0.23
0.27
0.12
0.25
0.035
0.22
0.18
0.11
0.06
1.0
2.6
2.0
4.2
22
23
28
29
32
33
35
39
44
49
59
65
74
80
NHCOCH2
NHCOCH2O
NHCO2CH2
NHCONH
21
10
11
0.89
1.4
0.40
2.2
4.2
3.8
0.23
0.13
<0.084
7.1
NHCONHCH2
OCONH
4.1
2.7
32
0.27
2.4
0.05
0.52
>32
OCONHCH2
17
2.4
0.48
a
b
Difference in CLogP values between compounds of forms I and II in each linker class. Ratio of MIC values (I/II) for mono- vs biphenyl
compounds in each linker class.
as well. These results are illustrated graphically in Figure S1
(see Supporting Information). Thus, the O-carbamates 67−69
showed the best MIC profiles of all of the biaryl derivatives,
comparable to 3. However, a closer examination of the MIC
results for the para-linked biaryl analogues having the latter two
linkers (X = NHCONH and OCONH; compounds 57−60 and
72−75) indicated that there was an unusually high variability in
the MABA data across these sets (Table 3), with the very
poorly active 4-F compounds (58 and 73, each tested 3−5
times) severely distorting the averages. Exclusion of these
outliers led to mean MABA MIC values of 0.14 and 0.12 μM,
respectively, essentially the same as for the N-linked carbamates
(47−50), and with the 4-OCF3 analogues (59 and 74) having
useful aerobic potencies (0.05−0.06 μM) although very poor
anaerobic activities.
Table 5 compares the MIC data for the 4-OCF3 biphenyl
compounds with those of the corresponding monophenyl
analogues bearing the same linker groups. Despite an increase
of 1.7−2.2 units in CLogP values for the biphenyl compounds,
which in the OCH2-linked series resulted in a 14-fold increase
in MABA potency,13 in the present work the MIC values of the
biphenyl series against replicating M. tb were scarcely improved
over those of their monoaryl congeners (and were poorer in
some cases; see Figure S2 in Supporting Information). Poten-
cies in the LORA assay were also generally similar or worse (see
Figure S3 in Supporting Information). These results are re-
miniscent of those found for the previous study16 of extended
ether linkers and are again consistent with a rather constrained
hydrophobic binding domain in the activating nitroreductase
that requires a very specific disposition of the larger biphenyl
side chain for optimal interactions, which the alternative linkers
may not allow.
Because the biaryl compounds of Table 3 generally showed
similar MIC potencies to the monoaryl compounds of Table 1
but were much less soluble, they demonstrate little apparent
therapeutic advantage (at least in vitro). In a further attempt to
utilize the favorable properties of the O-carbamate linker, a
small set of aryl-terminating cyclic amine-based analogues was
prepared and evaluated (Table 6). Similar side chains have pre-
viously been reported to be effective replacements for a biaryl
substituent, with arylpiperazine being recognized as a bio-
isostere of the biaryl moiety.31−33 The obvious major potential
benefit of such substitution is significantly improved aqueous
solubility, and this was demonstrated here, with the 4-OCF3
318
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Journal of Medicinal Chemistry
Article
phenyl analogues (82, 83, and 85) showing comparable
solubility values to 1 (at pH = 7), and the more hydrophilic
pyridine derivative 84 being 5-fold better. Arylpiperazine 82
also displayed an excellent MIC profile (MABA 0.033 μM,
LORA 1.1 μM), similar to that of the much more lipophilic and
poorly soluble biphenyl analogue 3, and aryloxypyrrolidine 83
was only slightly less impressive. In contrast, compounds 84
and 85 provided significantly reduced potencies, especially in
the LORA (nonreplicating) assay.
Representative compounds bearing a variety of different
linkers and with good MABA activity (IC50 values ranging from
0.03 to 0.25 μM) were subsequently evaluated for metabolic
stability in human (HLM) and mouse liver microsomes
(MLM) as an indicator of suitability for in vivo evaluation
(Table 7; comparative data for 1 and 3 are also provided).13 All
of the compounds except N-methyl carbamate 34 showed
notably high stabilities toward both HLM and MLM (>80%
parent remaining after 1 h). However, examination of the
metabolite profile of 34 showed only a single compound, the
desmethyl carbamate 33, which displayed excellent metabolic
stability (superior to 1 in HLM). The biphenyl derivative of 33,
74, similarly demonstrated higher stability than the OCH2-linked
analogue 3, with negligible metabolism by either HLM or MLM
occurring over the 1 h incubation period. Importantly, the more
soluble arylpiperazine carbamate 82 (a bioisostere of 74) was also
acceptably stable (similar to 1 in HLM, though inferior to 74).
Table 6. Physicochemical Properties and MIC Values for Cyclic Amine Carbamate-Linked Analogues of 1
a
CLogP values, calculated using the ACD LogP/LogD prediction software (version 12.0, Advanced Chemistry Development Inc., Toronto,
b
c
Canada). Solubility (μg/mL) in water at pH = 7 and 20 °C, determined by HPLC (see Experimental Section). Minimum inhibitory concentration,
determined under aerobic (MABA)30 or anaerobic (LORA)28 conditions. Each value is the mean of at least two independent determinations SD.
Table 7. Microsomal Stability and in Vivo Efficacy Data for Selected Compounds
a
b
Solubility (μg/mL) in water at pH = 7 and 20 °C, determined by HPLC. Pooled human (H) or CD-1 mouse (M) liver microsomes.
c
Fold reduction in lung CFUs for compound compared with the fold CFU reduction for 1 in a mouse model of acute TB infection (see text).
d
Fold reduction in lung CFUs for compound compared with the fold CFU reduction for 2 in a mouse model of chronic TB infection (see text).
e
f
Rapid metabolism to the desmethyl carbamate 33 was observed. Eight week study, dosing at 30 mg/kg.
319
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Table 8. Pharmacokinetic Parameters for Selected Analogues in CD-1 Mice Following a Single Oral Dose of 40 mg/kg
plasma
Cmax (μg/mL)
7.4
12
lung
Cmax (μg/mL)
9.0
18
a
a
b
compd
AUC0−inf (μg·h/mL)
t1/2 (h)
AUC0−inf (μg·h/mL)
t1/2 (h)
AUC ratio
3
198
296
28.5
6.61
44.9
53.4
14.4
24
218
81
12.8
2.2
4.1
c
1.1
0.27
1.5
4
33
59
74
82
1.4
9.8
43.3
>60
>136
3.8
4.7
0.48
0.52
6.3
>9.0
>3.0
3.2
62.3
10.5
6.9
c
2.5
169
11.7
10.5
a
b
c
Area under the curve, extrapolated to infinity. Lung AUC/plasma AUC. Not calculable.
Table 9. Pharmacokinetic Parameters and Oral Bioavailability Data for 82 in Rats and Dogs
a
intravenous
oral (20 mg/kg)
b
c
d
food type
normal
C0 (μg/mL)
Tmax (h)
t1/2 (h)
AUClast (μg·h/mL)
Cmax (μg/mL)
Tmax (h)
t1/2 (h)
AUClast (μg·h/mL)
F
(%)
57
84
Rats (Sprague−Dawley)
5.93
0.70
1.7
0.083
0.5
8.3
28
4.4
5.0
e
67.4
232
Dogs (Beagle)
normal
high fat
27.6
5.8
5.2
8.0
12
29
30
182
a
b
c
The intravenous dose was 1 mg/kg for rats and 2 mg/kg for dogs. Maximum plasma concentration extrapolated to t = 0. Area under the curve
d
calculated to the last time point; for rats the last time point was 24 h, while for dogs it was 72 h. Oral bioavailability, determined using dose
normalized AUClast values. Not calculable.
e
Further confirmation of the metabolic stability of these urea
and carbamate linkers was obtained from mouse pharma-
cokinetic studies on selected examples (Table 8). Here, the four
compounds examined each showed good plasma half-lives (>6 h)
and satisfactory exposure levels in lung tissue following oral
dosing, but 82 possessed the best profile overall.
demonstrated equivalent in vivo efficacy to its bioisostere 74
(5.2-fold better than 1) and showed a 283-fold higher aqueous
solubility than biaryl 74 at pH = 7 (4083-fold higher at pH = 1,
consistent with its calculated pKa value of 2.44).
These interesting results prompted a further appraisal of the
antitubercular activities of O-carbamates 33, 74, and 82 in a
more stringent chronic disease mouse model (Table 7), where
the infection was first established for ∼50−70 days (resulting in
a growth plateau) prior to daily oral dosing (for 5 days per
week over 3−8 weeks).15 Compared to the clinical trial drug 2
(an order of magnitude more efficacious than 1 in this assay),
monophenyl carbamate 33 was almost completely ineffective,
while its biaryl derivative 74 was weakly active (0.35-fold, but
∼3-fold superior to 1). Intriguingly, despite its fairly modest
activity in the acute in vivo assay, the arylpiperazine carbamate
82 displayed essentially equivalent efficacy to both 2 and 3 in
this chronic model and was much more soluble than both
compounds (>1000-fold at pH = 7.4 in a thermodynamic
solubility assay); therefore 82 was selected for advanced
studies.
Additional pharmacokinetic data for 82 in the rat (see Table 9)
may be compared to previously reported data for pyridine 4.15
While 4 gave a higher maximum plasma concentration fol-
lowing oral dosing (8.1 versus 4.4 μg/mL), both compounds
provided similar oral bioavailabilities (4, 62%; 82, 57%). More-
over, a re-examination of the mouse pharmacokinetic data
(Table 8) revealed that 82 afforded a more prolonged exposure
than 4 in the lung (the primary infection site). A further study
of 82 was then conducted in dogs to ascertain the effect of diet
on the pharmacokinetic profile observed. Knowledge about the
influence of food is important in the treatment of tuberculosis,
where the drugs are taken once daily or less; among the first
line agents, isoniazid shows reduced bioavailability with food.34
Dietary fat commonly increases the absorption of less bioavailable
lipophilic drugs by improving their dissolution, but hydrophilic
drugs are not significantly affected.35 The results (Table 9) show
that 82 has excellent oral bioavailability in dogs (84%), providing
The compounds above were further assessed in an acute
infection mouse tuberculosis model, measuring the fold
reduction in colony-forming units (CFUs) in the lung following
oral dosing at 100 mg/kg daily for 5 days per week over 3
weeks13,30 (Table 7). The fold reductions in CFUs were
compared to that achieved with the OCH2-linked compound 1,
which was employed as an internal reference standard in each
of the experiments. In the monoaryl series, N-methyl carba-
mate 34 was unexpectedly found to be moderately better than
1 (5-fold), but its des-methyl metabolite 33 was less effec-
tive than 1 (consistent with a previous study of 3320). The
greater efficacy for the N-methyl analogue (despite its poor
stability) may in part be related to its 3-fold higher aqueous
solubility (consistent with its lower CLogP value), which might
allow better absorption and distribution. In contrast, the highly
soluble ether-extended amide analogue 36 was no better than
33. Figure S4 (in Supporting Information) illustrates these
various solubility and in vivo efficacy effects. Examining the
more lipophilic biaryl series, poorly soluble O-carbamates 69
and 74 (respectively 3.4- and 5-fold better than 1) were sup-
erior to the corresponding urea analogues, 54 and 59, with the
para-linked examples having better efficacies than their meta-
linked counterparts, in agreement with previous studies.15,16
While the biaryl carbamate 74 was found to be 13-fold more
efficacious than the monoaryl analogue 33, it is worth noting
that this is significantly less than the more than 205-fold im-
provement seen for OCH2-linked biphenyl analogue 3 over 1,
confirming the superiority of the OCH2-linker for high in vivo
efficacy, as previously observed.13−16 An attempt to improve
aqueous solubility using trifluoromethylpyridine as a replacement
for the terminal aryl ring14−16 (70) concomitantly abolished the
in vivo activity. In contrast, the arylpiperazine carbamate 82
320
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Journal of Medicinal Chemistry
Article
high exposures and a long half-life (29 h) that are not markedly
altered by a high fat diet.
(nonextended) O-carbamate analogue of 1 (33) proved to be
the best and displayed high microsomal stability.
With the attrition of new drug candidates in clinical trials
estimated at 95%, it has been recommended that chemists pay
greater attention to fundamental compound properties that can
help to predict fitness for survival as potential drugs, rather than
focusing too much on potency alone.36 Whereas issues of
pharmacokinetics and bioavailability were historically the major
reasons for drug failure, other factors, such as toxicology and
clinical safety, as well as efficacy, are now more predominant.36
There is increasing recognition that high molecular weight and
high lipophilicity are associated not only with poor oral drug-
like properties (particularly low solubility) but also with a
higher risk of observing toxicity or unwanted side effects.37
Thus CLogP values of between 2 and 3 are often considered
optimal for oral drugs, and although better antitubercular
activity has been observed in several studies for somewhat more
lipophilic compounds (CLogP ∼4), the majority of current TB
drugs still have LogP values ranging from 2.5 to 4.38 With this
in mind, we obtained further physical and safety data for 82 and
compared these with results for the parent clinical trial agent 1
(Table 10). While 82 has a higher molecular weight than 1
These encouraging findings prompted combination of some
of the linkers together with particular biaryl side chains that had
been proven to greatly enhance both in vitro and (notably) in
vivo activity in the parent (OCH2 linked) series. However, the
resulting biaryl compounds were generally not greater in
potency than their monoaryl counterparts. Furthermore, only
for meta-linked examples were the urea and carbamate linkers
superior to OCH2, reinforcing previous conclusions regarding
structural limitations for preferred binding to the activating
nitroreductase.16,21 Nevertheless, upon further evaluation in a
mouse model of acute M. tb infection, the highly stable para-
linked biaryl (O-)carbamate 74 was found to be superior to
both the leading monoaryl analogue 33 (13-fold) and the
parent drug 1 (5-fold), although it was very poorly soluble. A
consideration of possible biaryl replacements in this most pro-
mising (O-carbamate) series led to the potent, more hydrophilic
arylpiperazine analogue 82, a bioisostere of biaryl carbamate 74.
This compound provided equivalent in vivo efficacy to the
latter in the acute in vivo model but was as soluble as 1 at
neutral pH (and 5.6-fold more soluble than 1 at low pH).
Carbamate 82 additionally provided a similar level of in vivo
efficacy to the clinical trial drug 2 in a more stringent chronic
infection model (with greatly superior aqueous solubility over 2)
and compared well to the parent drug 1 in other respects
(e.g., reduced binding to human plasma proteins). Pharmacokinetic
assessments of 82 across several species also revealed favorable
profiles, including high oral bioavailabilities and minimal food
effects, suggesting that this compound may be worthy of further
study.
Table 10. Additional Comparative Data for 1 and 82
property
1
82
molecular weight (Da)
log Pexp
359.3
457.4
2.52 0.02
2.82 0.03
thermodynamic solubility (μM):
pH = 7.4
30
30
33
169
91
pH = 1
a
human plasma protein binding (%)
94
b
mutagenic effect (Ames test)
no
no
EXPERIMENTAL SECTION
■
a
b
Reference 39. Not mutagenic up to precipitating concentrations
Combustion analyses were performed by the Campbell Micro-
analytical Laboratory, University of Otago, Dunedin, New Zealand.
Melting points were determined using an Electrothermal IA9100
melting point apparatus and are as read. NMR spectra were measured
(>75 μg/well) in strains TA98 and TA100, both in the presence and
absence of metabolic activation (S9 fraction).
(457 versus 359), the measured LogP results for these com-
pounds were comparable (2.82 versus 2.52), as were their
thermodynamic solubility values at pH = 7.4 (30−33 μM);
compound 82 was also about 6-fold more soluble than 1 at low
pH (0.17 mM). Moreover, 82 displayed a reduced binding to
human plasma proteins (91% versus 94% for 139), which
together with its very low inhibition of hERG (IC50 >30 μM)
suggested an improved potential for safety.40 Finally, we con-
firmed that (like 1) carbamate 82 did not show any mutagenic
effect (Ames test, Salmonella tester strains TA98 and TA100,
conducted with and without metabolic activation).
1
on a Bruker Avance 400 spectrometer at 400 MHz for H and are
referenced to Me4Si. Chemical shifts and coupling constants are re-
corded in units of ppm and hertz, respectively. High-resolution electron
impact (HREIMS) and fast atom bombardment (HRFABMS) mass
spectra were determined on a VG-70SE mass spectrometer at nominal
5000 resolution. High-resolution electrospray ionization (HRESIMS)
mass spectra were determined on a Bruker micrOTOF-Q II mass
spectrometer. Low-resolution atmospheric pressure chemical ioniza-
tion (APCI) mass spectra were measured for organic solutions on a
ThermoFinnigan Surveyor MSQ mass spectrometer, connected to a
Gilson autosampler. Thin-layer chromatography was carried out on
aluminum-backed silica gel plates (Merck 60 F254), with visualization
of components by UV light (254 nm) or exposure to I2. Column
chromatography was carried out on silica gel (Merck 230−400 mesh).
Compounds of Tables 1, 3, and 6 were isolated following trituration in
Et2O (or CH2Cl2/Et2O) unless otherwise indicated. HPLC determi-
nations of purity were conducted on an Agilent 1100 system using a
reversed phase C8 column with diode array detection.
Compounds of Table 1. Procedure A: (6S)-2-Nitro-6-{[2-(tri-
fluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazine (8) (Scheme 1A). 1-(Bromomethyl)-2-(trifluoromethoxy)-
benzene (105 μL, 0.65 mmol) and NaH (30 mg of 60% dispersion in
mineral oil, 0.75 mmol) were successively added to a solution of (6S)-
2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-ol20 (6) (100 mg,
0.54 mmol) in anhydrous DMF (3 mL). The mixture was stirred at
20 °C for 2.5 h and then diluted with EtOAc (150 mL) and washed
with water. The aqueous layer was re-extracted with EtOAc, and the
combined organic layers were washed with brine, dried (Na2SO4), and
concentrated under reduced pressure. The residue was chromatographed
CONCLUSIONS
■
The current investigation set out to evaluate various amide,
carbamate, and urea linkers as possible replacements for the
OCH2 linkage of 1 in order to address oxidative metabolism,
minimize compound lipophilicity, and enhance aqueous
solubility, with the overall aim of improved safety. The new
compounds were generally more hydrophilic than the OCH2
linked analogues, as assessed by CLogP values, and many of the
monoaryl analogues tested (particularly those having amide or
urea linkers) offered solubility increases, compared to 1 itself.
Extended amide, N- or O-carbamate monoaryl derivatives
provided moderately improved aerobic potencies (but reduced
anaerobic activity), urea analogues a more balanced MIC
profile (similar to the 6-amino analogue of 1), while the
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Article
on silica gel, eluting with a gradient of 0−4% MeOH/CH2Cl2, to give 822
(135 mg, 70%) as a white solid: mp 108−110 °C. 1H NMR [(CD3)2SO]
δ 8.02 (s, 1 H), 7.51 (dd, J = 7.4, 1.8 Hz, 1 H), 7.45 (dd, J = 7.8, 1.9 Hz,
1 H), 7.41−7.33 (m, 2 H), 4.72 (s, 2 H), 4.66 (dt, J = 12.1, 2.4 Hz, 1 H),
4.49 (d, J = 11.7 Hz, 1 H), 4.32−4.21 (m, 3 H). Anal. (C14H12F3N3O5)
C, H, N.
light-yellow solid: mp 120−124 °C. H NMR (CDCl3) δ 8.01−7.97
(m, 2 H), 7.42 (s, 1 H), 7.33 (br d, J = 8.0 Hz, 2 H), 4.48−4.36 (m, 2 H),
4.28−4.17 (m, 3 H), 3.97 (ddd, J = 12.3, 5.2, 1.2 Hz, 1 H), 3.49−3.43
(m, 1 H), 2.30 (br s, 1 H). HRFABMS calcd for C15H14F3N4O5 m/z
[M + H]+ 387.0916, found 387.0913. HPLC purity: 93.2%.
1
Procedure F: 4-(Trifluoromethoxy)phenyl [(6S)-2-nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]oxazin-6-yl]carbamate (27) (Scheme 3A).
4-(Trifluoromethoxy)phenol (65 μL, 0.50 mmol) and DIPEA
(90 μL, 0.52 mmol) were successively added to a solution of tri-
phosgene (77 mg, 0.25 mmol) in anhydrous CH2Cl2 (2 mL) at 0 °C.
The mixture was stirred at 0−20 °C for 4 h to give a solution of the
crude aryl chloroformate. A solution of amine salt 91 (86 mg, 0.39
mmol) and DIPEA (175 μL, 1.00 mmol) in anhydrous CH2Cl2
(3 mL) was then added, and the mixture was stirred at 20 °C for a
further 90 min. Following dilution with EtOAc (200 mL), the solution
was washed with saturated aqueous NaHCO3 and brine. The aqueous
layer was re-extracted with EtOAc and the combined organic layers
were dried (Na2SO4) and concentrated under reduced pressure. The
residue was chromatographed on silica gel, eluting with a gradient of
0−4% MeOH/CH2Cl2 to give 27 (60 mg, 40%) as an off-white solid:
Procedure B: N-[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl]-2-(trifluoromethoxy)benzamide (10) (Scheme 2A).
2-(Trifluoromethoxy)benzoyl chloride (140 mg, 0.623 mmol) and
DIPEA (200 μL, 1.15 mmol) were successively added to a solution
of (6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-amine
hydrochloride20 (91) (101 mg, 0.458 mmol) in anhydrous DMF (3 mL).
The mixture was stirred at 20 °C for 1 h and then diluted with EtOAc
(200 mL) and washed with saturated aqueous NaHCO3 and brine.
The aqueous layer was re-extracted with EtOAc, and the com-
bined organic layers were dried (Na2SO4) and concentrated under
reduced pressure. The residue was chromatographed on silica gel,
eluting with a gradient of 0−4% MeOH/CH2Cl2, to give 10 (140 mg,
82%) as a white solid: mp 172−174 °C. 1H NMR [(CD3)2SO] δ 9.08
(d, J = 6.5 Hz, 1 H), 8.09 (s, 1 H), 7.62−7.54 (m, 2 H), 7.49−7.40
(m, 2 H), 4.64−4.58 (m, 1 H), 4.56 (dd, J = 11.2, 2.1 Hz, 1 H), 4.45
(ddd, J = 11.2, 3.8, 1.8 Hz, 1 H), 4.39 (dd, J = 13.0, 4.8 Hz, 1 H), 4.09
(ddd, J = 13.0, 2.9, 1.8 Hz, 1 H). Anal. (C14H11F3N4O5·0.5H2O) C, H, N.
Procedure C: N-Methyl-N-[(6S)-2-nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazin-6-yl]-2-(trifluoromethoxy)benzamide (13). A sol-
ution of amide 10 (48 mg, 0.13 mmol) in anhydrous DMF (1 mL) at
0 °C was successively treated with NaH (12 mg of 60%, 0.30 mmol) and
MeI (12 μL, 0.19 mmol). The mixture was stirred at 0 °C for 90 min
and then water (100 mL) was added and the product was extracted
with EtOAc (3 × 100 mL). The combined extracts were washed with
brine, dried (Na2SO4), and concentrated under reduced pressure. The
residue was chromatographed on silica gel, eluting with a gradient
of 0−4% MeOH/CH2Cl2, to give 13 (39 mg, 78%) as a white solid:
1
mp 177−179 °C. H NMR [(CD3)2SO] δ 8.58 (d, J = 6.2 Hz, 1 H),
8.08 (s, 1 H), 7.40 (d, J = 8.6 Hz, 2 H), 7.28 (d, J = 9.0 Hz, 2 H),
4.56−4.42 (m, 2 H), 4.36 (dd, J = 12.8, 4.7 Hz, 1 H), 4.32−4.24 (m,
1 H), 4.15−4.08 (m, 1 H). Anal. (C14H11F3N4O6) C, H, N.
1-[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl]-
3-[4-(trifluoromethoxy)phenyl]urea (29) (Scheme 3B). A mixture of
the free base of amine salt 91 (50 mg, 0.27 mmol) and 1-isocyanato-
4-(trifluoromethoxy)benzene (50 μL, 0.33 mmol) in anhydrous DMF
(2 mL) was stirred at 20 °C for 5 h. Brine (100 mL) was then added,
and the product was extracted with EtOAc (3 × 100 mL). The
combined extracts were washed with brine, dried (Na2SO4), and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel, eluting with a gradient of 0−2% MeOH/CH2Cl2,
to give 2920 (14 mg, 13%) as a light-yellow solid: mp 166 °C (dec). 1H
NMR [(CD3)2SO] δ 8.61 (s, 1 H), 8.09 (s, 1 H), 7.49−7.44 (m, 2 H),
7.23 (br d, J = 8.4 Hz, 2 H), 6.91 (d, J = 6.8 Hz, 1 H), 4.56 (dd, J =
11.2, 1.9 Hz, 1 H), 4.45 (ddd, J = 11.2, 3.3, 2.0 Hz, 1 H), 4.41−4.35
(m, 1 H), 4.30 (dd, J = 12.8, 4.1 Hz, 1 H), 4.10 (dt, J = 12.8, 2.2 Hz, 1
H). HRFABMS calcd for C14H13F3N5O5 m/z [M + H]+ 388.0869,
found 388.0862. HPLC purity: 95.1%.
Procedure G: 1-[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl]-3-[4-(trifluoromethoxy)phenyl]thiourea (31). A solution
of amine salt 91 (101 mg, 0.458 mmol) and NMM (100 μL, 0.91
mmol) in anhydrous DMF (2 mL) was successively treated with
1-isothiocyanato-4-(trifluoromethoxy)benzene (110 μL, 0.68 mmol)
and dibutyltin diacetate (3 drops). The mixture was stirred at 20 °C
for 2 h, then diluted with EtOAc (150 mL) and washed with brine.
The aqueous layer was re-extracted with EtOAc, and the combined
organic layers were washed with brine, dried (Na2SO4), and
concentrated under reduced pressure. The residue was chromato-
graphed on silica gel, eluting with a gradient of 0−2% MeOH/CH2Cl2,
to give 31 (32 mg, 17%) as a cream solid: mp (CH2Cl2/hexane) 178 °C
1
mp 187−190 °C. H NMR [(CD3)2SO] δ 8.13 (s, 1 H), 7.64−7.45
(m, 4 H), 5.10−5.01 (m, 1 H), 4.75−4.60 (m, 2 H), 4.47−4.26 (m, 2 H),
2.73 (s, 3 H). Anal. (C15H13F3N4O5) C, H, N.
N-[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl]-
2-(trifluoromethoxy)benzenesulfonamide (16). Reaction of amine salt
91 with 2-(trifluoromethoxy)benzenesulfonyl chloride (1.25 equiv)
and DIPEA, using procedure B, gave 16 (83%) as a white solid: mp
214−219 °C. 1H NMR [(CD3)2SO] δ 8.83 (br s, 1 H), 7.99 (s, 1 H),
7.98−7.93 (m, 1 H), 7.83−7.78 (m, 1 H), 7.62−7.56 (m, 2 H), 4.43
(dd, J = 11.3, 2.1 Hz, 1 H), 4.30 (ddd, J = 11.3, 4.5, 1.7 Hz, 1 H), 4.25
(dd, J = 12.9, 4.6 Hz, 1 H), 4.08 (br s, 1 H), 3.98 (ddd, J = 12.9, 3.7,
1.7 Hz, 1 H). Anal. (C13H11F3N4O6S) C, H, N.
Procedure D: N-Methyl-N-[(6S)-2-nitro-6,7-dihydro-5H-imidazo-
[2,1-b][1,3]oxazin-6-yl]-2-(trifluoromethoxy)benzenesulfonamide
(19). A solution of sulfonamide 16 (75 mg, 0.18 mmol) in anhydrous
DMF (2 mL) was successively treated with K2CO3 (77 mg, 0.56
mmol) and MeI (23 μL, 0.37 mmol). The mixture was stirred at 20 °C
for 2 h and then diluted with EtOAc (200 mL) and washed with
saturated aqueous NaHCO3 and brine. The aqueous layer was re-
extracted with EtOAc, and the combined organic layers were dried
(Na2SO4) and concentrated under reduced pressure. The residue was
chromatographed on silica gel, eluting with a gradient of 0−4%
MeOH/CH2Cl2, to give 19 (70 mg, 90%) as a white solid: mp 160−
162 °C. 1H NMR [(CD3)2SO] δ 8.03 (dd, J = 7.8, 1.7 Hz, 1 H), 8.03
(s, 1 H), 7.89−7.84 (m, 1 H), 7.68−7.64 (m, 1 H), 7.62 (dt, J = 7.7,
1.0 Hz, 1 H), 4.59−4.51 (m, 2 H), 4.47−4.40 (m, 1 H), 4.29−4.18
(m, 2 H), 2.83 (s, 3 H). Anal. (C14H13F3N4O6S) C, H, N.
Procedure E: 2-{[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl]amino}-1-[4-(trifluoromethoxy)phenyl]ethanone (25)
(Scheme 2D). 2-Bromo-1-[4-(trifluoromethoxy)phenyl]ethanone
(157 mg, 0.555 mmol) and DIPEA (200 μL, 1.15 mmol) were
successively added to a solution of amine salt 91 (103 mg, 0.467
mmol) in anhydrous DMF (2 mL). The mixture was stirred at 20 °C
for 2.5 h, and then brine (100 mL) was added and the product was
extracted with EtOAc (3 × 150 mL). The combined extracts were
washed with brine, dried (Na2SO4), and concentrated under reduced
pressure. The residue was chromatographed on silica gel, eluting with
a gradient of 0−2% MeOH/CH2Cl2, to give 25 (11 mg, 6%) as a
1
(dec). H NMR (CDCl3) δ 8.43 (d, J = 8.6 Hz, 1 H), 8.36 (s, 1 H),
7.44−7.39 (m, 2 H), 7.21 (s, 1 H), 7.13 (d, J = 8.3 Hz, 2 H), 5.65−5.59
(m, 1 H), 4.87 (dt, J = 11.7, 2.0 Hz, 1 H), 4.45 (dd, J = 11.6, 1.5 Hz, 1 H),
4.32−4.20 (m, 2 H). Anal. (C14H12F3N5O4S·0.25EtOAc) C, H, N.
Procedure H: (6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl [4-(trifluoromethoxy)phenyl]carbamate (33) (Scheme
1A). 1-Isocyanato-4-(trifluoromethoxy)benzene (1.50 mL, 9.94 mmol)
and CuCl (95 mg, 0.96 mmol) were successively added to a solution of
alcohol 6 (1.50 g, 8.10 mmol) in anhydrous DMF (20 mL). The mix-
ture was stirred at 20 °C for 3 h and then diluted with EtOAc (150 mL)
and washed with 0.2 N HCl, water, and brine. The aqueous layer was
re-extracted with EtOAc and the combined extracts were dried
(Na2SO4) and concentrated under reduced pressure. The residue was
chromatographed on silica gel, eluting with a gradient of 0−2%
MeOH/CH2Cl2, to give 3320 (2.50 g, 80%) as a white solid: mp 211−
213 °C [lit.20 220 °C (dec)]. 1H NMR [(CD3)2SO] δ 10.08 (s, 1 H),
8.08 (s, 1 H), 7.56 (d, J = 8.8 Hz, 2 H), 7.30 (d, J = 8.4 Hz, 2 H),
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5.44 (br s, 1 H), 4.64 (br s, 2 H), 4.45−4.40 (m, 1 H), 4.35−4.30
(m, 1 H). Anal. (C14H11F3N4O6·0.25Et2O) C, H, N. HPLC purity: 100%.
2-{[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl]-
oxy}-N-[4-(trifluoromethoxy)phenyl]acetamide (36). Reaction of
alcohol 6 with 2-chloro-N-[4-(trifluoromethoxy)phenyl]acetamide and
NaH (1.55 equiv), using procedure A (but at 0−20 °C for 6 h), gave 3622
(19%) as a white solid: mp (CH2Cl2/petroleum ether) 73−76 °C.
1H NMR (CDCl3) δ 8.10 (s, 1 H), 7.58−7.54 (m, 2 H), 7.46 (s, 1 H),
7.19 (d, J = 8.4 Hz, 2 H), 4.74−4.68 (m, 1 H), 4.42 (d, J = 12.5 Hz,
1 H), 4.36−4.25 (m, 5 H). Anal. (C15H13F3N4O6·0.25EtOAc) C, H, N.
HPLC purity: 96.8%.
Compounds of Table 3. Procedure I. General Method for
Suzuki Couplings. A stirred mixture of aryl-bromide or aryl-iodide
(0.19 mmol) and arylboronic acid (or pinacol ester derivative) (0.25
mmol) in toluene (5 mL), EtOH (3 mL) and 2 M K2CO3 (1 mL) was
purged with N2 for 5 min. Pd(dppf)Cl2 (9 μmol) was added, and the
stirred mixture was heated under reflux in an N2 atmosphere for 1 h
(or until the reaction was complete). After cooling to 20 °C, the
mixture was diluted with EtOAc (150 mL) and washed with water.
The aqueous layer was re-extracted with EtOAc, and the combined or-
ganic layers were washed with brine, dried (Na2SO4), and con-
centrated under reduced pressure. The residue was chromatographed
on silica gel, eluting with a gradient of 0−4% MeOH/CH2Cl2, to give
the required products.
1-[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl]-3-
[4′-(trifluoromethyl)biphenyl-3-yl]urea (52). Reaction of bromide 95
and 4-(trifluoromethyl)phenylboronic acid, using procedure I, gave 52
(72%) as a brown solid: mp (EtOAc/petroleum ether) 156 °C (dec).
1H NMR [(CD3)2SO] δ 8.57 (s, 1 H), 8.10 (s, 1 H), 7.81 (s, 5 H),
7.39−7.35 (m, 2 H), 7.31−7.26 (m, 1 H), 6.93 (d, J = 6.8 Hz, 1 H),
4.58 (dd, J = 11.2, 1.8 Hz, 1 H), 4.47 (ddd, J = 11.2, 3.3, 1.9 Hz, 1 H),
4.42−4.35 (m, 1 H), 4.31 (dd, J = 12.7, 4.1 Hz, 1 H), 4.13 (dt, J =
12.7, 2.2 Hz, 1 H). Anal. (C20H16F3N5O4·0.5H2O) C, H, N.
(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl
(3-bromophenyl)carbamate (86) (Scheme 1A). Reaction of alcohol 6
with 1-bromo-3-isocyanatobenzene and CuCl, using procedure H for
1
2.5 h, gave 86 (80%) as a white solid: mp 226 °C (dec). H NMR
[(CD3)2SO] δ 10.08 (s, 1 H), 8.07 (s, 1 H), 7.75 (br s, 1 H), 7.41 (br
d, J = 7.8 Hz, 1 H), 7.25 (t, J = 7.9 Hz, 1 H), 7.20 (dt, J = 7.9, 1.5 Hz,
1 H), 5.44 (q, J = 1.6 Hz, 1 H), 4.65 (s, 2 H), 4.43 (dd, J = 14.0,
3.4 Hz, 1 H), 4.34 (dd, J = 14.0, 1.3 Hz, 1 H). Anal. (C13H11BrN4O5)
C, H, N.
(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl [4′-
(trifluoromethyl)biphenyl-3-yl]carbamate (67). Reaction of bromide
86 and 4-(trifluoromethyl)phenylboronic acid, using procedure I, gave
67 (58%) as a cream solid: mp 125−128 °C. 1H NMR [(CD3)2SO] δ
10.03 (s, 1 H), 8.09 (s, 1 H), 7.89−7.76 (m, 5 H), 7.54−7.47 (m, 1
H), 7.43 (t, J = 7.7 Hz, 1 H), 7.38 (dt, J = 7.6, 1.4 Hz, 1 H), 5.46 (br d,
J = 1.4 Hz, 1 H), 4.66 (s, 2 H), 4.45 (dd, J = 13.9, 3.4 Hz, 1 H), 4.35
(d, J = 13.9 Hz, 1 H). Anal. (C20H15F3N4O5) C, H, N.
2-(4-Bromophenyl)-N-[(6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b]-
[1,3]oxazin-6-yl]acetamide (92) (Scheme 2B). Reaction of amine salt
91 with (4-bromophenyl)acetyl chloride (1.5 equiv) and DIPEA, using
procedure B for 3 h, gave 92 (83%) as a light-yellow solid: mp 199 °C
(dec). 1H NMR [(CD3)2SO] δ 8.70 (d, J = 6.4 Hz, 1 H), 8.08 (s, 1 H),
7.49−7.44 (m, 2 H), 7.21−7.16 (m, 2 H), 4.50−4.45 (m, 1 H), 4.41−
4.32 (m, 2 H), 4.29 (dd, J = 12.9, 4.6 Hz, 1 H), 4.00 (dt, J = 12.9, 2.4
Hz, 1 H), 3.44 (s, 2 H). Anal. (C14H13BrN4O4) C, H, N.
Compounds of Table 6. Procedure J: (6S)-2-Nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]oxazin-6-yl 4-[4-(trifluoromethoxy)phenyl]-
piperazine-1-carboxylate (82) (Scheme 4C). Triphosgene (3.05 g,
10.3 mmol) was added in portions over 20 min to a stirred suspension
of alcohol 6 (3.81 g, 20.6 mmol) and Et3N (8.60 mL, 61.7 mmol) in
anhydrous THF (120 mL) at 5 °C, such that the temperature of the
mixture remained below 10 °C. After 10 min, the cooling bath was
removed and the mixture was stirred at 20 °C for 90 min. A solution of
1-[4-(trifluoromethoxy)phenyl]piperazine26 (113) (5.32 g, 21.6 mmol)
in THF (20 mL) was added, and stirring was continued for another
2 h. Water was added, and the mixture was extracted with EtOAc.
Evaporation of this extract gave an oily solid, which was chromato-
graphed on silica gel. Elution with CH2Cl2 gave foreruns, and then
further elution with 50% EtOAc/CH2Cl2 gave 82 (5.68 g, 62%) as a
N-[(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl]-
2-[4′-(trifluoromethyl)biphenyl-4-yl]acetamide (37). Reaction of bro-
mide 92 and 4-(trifluoromethyl)phenylboronic acid, using procedure I,
1
gave 37 (62%) as a light-brown solid: mp 178−182 °C. H NMR
[(CD3)2SO] δ 8.74 (d, J = 6.3 Hz, 1 H), 8.09 (s, 1 H), 7.87 (d, J = 8.3
Hz, 2 H), 7.80 (d, J = 8.3 Hz, 2 H), 7.66 (d, J = 8.3 Hz, 2 H), 7.37 (d,
J = 8.3 Hz, 2 H), 4.51 (br d, J = 9.2 Hz, 1 H), 4.45−4.35 (m, 2 H),
4.30 (dd, J = 12.9, 4.4 Hz, 1 H), 4.03 (dt, J = 12.9, 2.3 Hz, 1 H), 3.53
(s, 2 H). Anal. (C21H17F3N4O4·0.25H2O) C, H, N.
1
light-yellow solid: mp 166−168 °C. H NMR [(CD3)2SO] δ 8.06 (s,
1 H), 7.19 (d, J = 9.0 Hz, 2 H), 6.99 (d, J = 9.0 Hz, 2 H), 5.32 (br s,
1 H), 4.62−4.55 (m, 2 H), 4.39 (dd, J = 13.9, 3.5 Hz, 1 H), 4.27 (br d,
J = 13.9 Hz, 1 H), 3.45 (m, 4 H), 3.13 (m, 4 H). Anal. (C18H18F3N5O6)
C, H, N.
4-Iodobenzyl [(6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]-
oxazin-6-yl]carbamate (94) (Scheme 3A). Reaction of triphosgene,
4-iodobenzyl alcohol, and DIPEA for 2 h, followed by reaction of the
crude aryl chloroformate with amine salt 91 (1.0 equiv) and DIPEA
for 3 h, using procedure F, gave 94 (43%) as an off-white solid: mp
(3S)-3-[4-(Trifluoromethoxy)phenoxy]pyrrolidine (103)
(Scheme 4A). Diethyl azodicarboxylate (6.57 mL, 38.7 mmol) was
added dropwise to a refluxing solution of tert-butyl (3R)-3-hydroxypyrroli-
dine-1-carboxylate (100) (4.84 g, 25.9 mmol), 4-(trifluoromethoxy)-
phenol (101) (3.34 mL, 25.8 mmol), and triphenylphosphine (10.2 g,
38.9 mmol) in anhydrous THF (100 mL), and the solution was
refluxed under N2 for 5 h. The cooled solution was concentrated to
dryness directly onto silica gel and chromatographed on silica gel.
Elution with 3% EtOAc/petroleum ether gave foreruns, and then
further elution with 17% EtOAc/petroleum ether gave the crude ether
102 as a colorless oil, which was used directly in the next step. A
solution of this crude 102 in 1:1 CH2Cl2/TFA (120 mL) was stirred at
20 °C for 4 h. After concentration to dryness, the residue was parti-
tioned between 1 N NaOH solution and CH2Cl2. The organic portion
was acidified with 4 N HCl in dioxane and concentrated to dryness.
The residue was triturated with petroleum ether and then dissolved in
CH2Cl2. The solution was washed with 1 N NaOH, dried (Na2SO4),
and concentrated under reduced pressure, to give 103 (3.93 g, 62%
1
204−208 °C. H NMR [(CD3)2SO] δ 8.04 (s, 1 H), 8.01 (br d, J =
5.7 Hz, 1 H), 7.75−7.71 (m, 2 H), 7.19−7.14 (m, 2 H), 5.01 (s, 2 H),
4.46 (dd, J = 11.2, 2.1 Hz, 1 H), 4.37 (dd, J = 11.2, 2.8 Hz, 1 H), 4.29
(dd, J = 12.8, 4.7 Hz, 1 H), 4.23−4.16 (m, 1 H), 4.05−3.97 (m, 1 H).
Anal. (C14H13IN4O5) C, H, N.
[4′-(Trifluoromethyl)biphenyl-4-yl]methyl [(6S)-2-nitro-6,7-dihydro-
5H-imidazo[2,1-b][1,3]oxazin-6-yl]carbamate (47). Reaction of
iodide 94 and 4-(trifluoromethyl)phenylboronic acid, using procedure
1
I, gave 47 (77%) as a light-brown solid: mp 189−191 °C. H NMR
[(CD3)2SO] δ 8.05 (s, 1 H), 8.03 (d, J = 4.9 Hz, 1 H), 7.90 (br d,
J = 8.2 Hz, 2 H), 7.81 (br d, J = 8.3 Hz, 2 H), 7.74 (d, J = 8.3 Hz,
2 H), 7.49 (br d, J = 8.0 Hz, 2 H), 5.13 (s, 2 H), 4.48 (dd, J = 11.2, 2.1 Hz,
1 H), 4.39 (dd, J = 11.2, 2.9 Hz, 1 H), 4.31 (dd, J = 12.7, 4.7 Hz,
1 H), 4.26−4.18 (m, 1 H), 4.07−4.00 (m, 1 H). Anal. (C21H17F3N4O5)
C, H, N.
1-(3-Bromophenyl)-3-[(6S)-2-nitro-6,7-dihydro-5H-imidazo[2,1-
b][1,3]oxazin-6-yl]urea (95) (Scheme 3B). Reaction of amine salt 91
and NMM with 1-bromo-3-isocyanatobenzene and dibutyltin dia-
cetate, using procedure G for 4 h, gave 95 (92%) as a white solid: mp
1
overall) as a pale-yellow oil. H NMR (CDCl3) δ 7.13 (d, J = 8.9 Hz,
2 H), 6.84 (d, J = 8.9 Hz, 2 H), 4.82−4.77 (m, 1 H), 3.21−3.13 (m,
2 H), 3.03 (dd, J = 12.6, 4.8 Hz, 1 H), 2.95−2.89 (m, 1 H), 2.13−2.04
(m, 1 H), 1.99−1.91 (m, 1 H). APCI MS m/z 248 [M + H]+.
(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl (3S)-
3-[4-(trifluoromethoxy)phenoxy]pyrrolidine-1-carboxylate (83)
(Scheme 4C). Reaction of alcohol 6 with triphosgene and Et3N,
followed by reaction of the crude chloroformate with 103, using
1
160−165 °C. H NMR [(CD3)2SO] δ 8.59 (s, 1 H), 8.08 (s, 1 H),
7.80−7.78 (m, 1 H), 7.22−7.15 (m, 2 H), 7.12−7.07 (m, 1 H), 6.94
(d, J = 6.7 Hz, 1 H), 4.56 (dd, J = 11.2, 2.0 Hz, 1 H), 4.45 (ddd, J =
11.2, 3.4, 2.0 Hz, 1 H), 4.39−4.33 (m, 1 H), 4.30 (dd, J = 12.7, 4.1 Hz,
1 H), 4.11 (dt, J = 12.7, 2.3 Hz, 1 H). Anal. (C13H12BrN5O4) C, H, N.
323
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procedure J (except that the product was eluted from the silica gel
column using EtOAc), gave 83 (39%) as a pale-yellow foam: mp 81−
84 °C. 1H NMR [(CD3)2SO] δ (1:1 mixture of amide rotamers) 8.06,
8.02 (2 s, 1 H), 7.30−7.21 (2 d, J = 8.5 Hz, 2 H), 7.07−6.97 (2 d, J =
8.5 Hz, 2 H), 5.27 (br s, 1 H), 5.01 (br s, 1 H), 4.60−4.52 (m, 2 H),
4.41−4.32 (m, 1 H), 4.28−4.21 (m, 1 H), 3.62−3.25 (m, 4 H), 2.18−
1.99 (m, 2 H). HRFABMS calcd for C18H18F3N4O7 m/z [M + H]+
459.1128, found 459.1124. HPLC purity: 96.9%.
(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl 4-
{methyl[4-(trifluoromethoxy)benzyl]amino}piperidine-1-carboxyl-
ate (85) (Scheme 4C). Reaction of alcohol 6 with triphosgene and
Et3N, followed by reaction of the crude chloroformate with 112, using
procedure J (except that the product was eluted from the silica gel
column using 5% MeOH/EtOAc), gave 85 (39%) as a light-yellow
solid: mp (Et2O/petroleum ether) 55−58 °C. 1H NMR [(CD3)2SO] δ
8.06 (s, 1 H), 7.40 (d, J = 8.5 Hz, 2 H), 7.27 (d, J = 8.5 Hz, 2 H), 5.26
(br s, 1 H), 4.60−4.51 (m, 2 H), 4.36 (dd, J = 13.8, 3.5 Hz, 1 H), 4.24
(br d, J = 13.8 Hz, 1 H), 4.01 (br m, 1 H), 3.79 (br m, 1 H), 3.53 (s, 2 H),
2.76 (br t, J = 12.4 Hz, 2 H), 2.60−2.51 (m, 1 H), 2.06 (s, 3 H),
1.79−1.63 (m, 2 H), 1.44−1.31 (m, 2 H). HREIMS calcd for
C21H24F3N5O6 m/z (M+) 499.1679, found 499.1677. HPLC purity:
97.3%.
Minimum Inhibitory Concentration Assays (MABA and
LORA). These were carried out according to the published proto-
cols.28,30
Partition Coefficients. The octanol−water partition coefficients
were measured using the shake-flask method, according to a published
protocol.41
Solubility Determinations. Method A. Kinetic solubility assess-
ments were made on powdered solids using a published protocol.16
Method B. The thermodynamic solubility of compound 82 (at
pH = 1 and 7.4) was assessed by Cerep, 15318 NE 95th Street,
Redmond, WA 98052, according to a published protocol (method C).15
Plasma Protein Binding Assay. The study of compound 82 was
conducted by Cerep, 15318 NE 95th Street, Redmond, WA 98052,
using an equilibrium dialysis technique with overnight incubation of
the compound (at 10 μM, containing 1% DMSO), according to a
published protocol.42
hERG Assay. The study of the effect of compound 82 on cloned
hERG potassium channels expressed in human embryonic kidney cells
(HEK293) was conducted by ChanTest Corporation, 14656 Neo
Parkway, Cleveland, OH 44128 (FASTPatch IC50 screen).
Ames Test. Compound 82 (at concentrations of 0.25−250 μg/
well) was evaluated in the microAmes reverse mutation screen
conducted by Midwest BioResearch, LLC, 8025 Lamon Avenue,
Skokie, IL 60077, testing against Salmonella strains TA98 and TA100
(preincubated both with and without rat liver S9).
tert-Butyl 4-{[6-(trifluoromethyl)pyridin-3-yl]oxy}piperidine-
1-carboxylate (106) (Scheme 4A). Diethyl azodicarboxylate (7.16 mL,
45.5 mmol) was added dropwise to a refluxing solution of tert-butyl
4-hydroxypiperidine-1-carboxylate (104) (6.10 g, 30.3 mmol), 6-(trifluo-
romethyl)pyridin-3-ol (105) (4.95 g, 30.3 mmol), and triphenylphos-
phine (11.9 g, 45.4 mmol) in anhydrous THF (80 mL), and the solution
was refluxed for 5 h. The cooled solution was concentrated to dryness
directly onto silica gel, and chromatographed on silica gel. Elution with
5% EtOAc/petroleum ether gave 106 (5.78 g, 55%) as a pale-yellow
solid: mp 81−82 °C. 1H NMR (CDCl3) δ 8.37 (d, J = 2.7 Hz, 1 H), 7.61
(d, J = 8.7 Hz, 1 H), 7.29 (dd, J = 8.7, 2.7 Hz, 1 H), 4.61−4.57 (m, 1 H),
3.74−3.67 (m, 2 H), 3.41−3.34 (m, 2 H), 2.01−1.91 (m, 2 H), 1.83−
1.73 (m, 2 H), 1.47 (s, 9 H). Anal. (C16H21F3N2O3) C, H, N.
5-(Piperidin-4-yloxy)-2-(trifluoromethyl)pyridine (107). A solution
of 106 (4.08 g, 11.8 mmol) in 1:1 CH2Cl2/TFA (50 mL) was stirred
at 20 °C for 4 h. After concentration to dryness, the residue was
partitioned between saturated aqueous NaHCO3 solution and CH2Cl2.
The aqueous portion was basified to pH = 14 with 1 N KOH, and the
resulting mixture was extracted with CH2Cl2. Evaporation of this
extract gave 107 (2.68 g, 92%) as a low melting solid that was used
1
directly in the next step. H NMR [(CD3)2SO] δ 8.42 (d, J = 2.8 Hz,
1 H), 7.79 (d, J = 8.7 Hz, 1 H), 7.63 (dd, J = 8.7, 2.8 Hz, 1 H), 4.66−
4.59 (m, 1 H), 3.28 (br s, 1 H), 2.98−2.90 (m, 2 H), 2.62−2.55 (m,
2 H), 1.98−1.92 (m, 2 H), 1.52−1.42 (m, 2 H). HRFABMS calcd for
C11H14F3N2O m/z [M + H]+ 247.1058, found 247.1059.
(6S)-2-Nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl 4-
{[6-(trifluoromethyl)pyridin-3-yl]oxy}piperidine-1-carboxylate (84)
(Scheme 4C). Reaction of alcohol 6 with triphosgene and Et3N,
followed by reaction of the crude chloroformate with 107, using
procedure J (except that the product was eluted from the silica gel
column using 5% MeOH/EtOAc), gave 84 (33%) as a cream solid:
Microsomal Stability Assays. These were conducted by MDS
Pharma Services, 22011 30th Drive SE, Bothell, WA 98021−4444,
using a published protocol.13 The percentage of compound remaining
after a 1 h incubation was calculated as:
1
mp 105−106 °C. H NMR [(CD3)2SO] δ 8.44 (d, J = 2.6 Hz, 1 H),
8.06 (s, 1 H), 7.82 (d, J = 8.7 Hz, 1 H), 7.65 (dd, J = 8.7, 2.6 Hz, 1 H),
5.31−5.26 (m, 1 H), 4.79 (m, 1 H), 4.60−4.54 (m, 2 H), 4.39 (dd, J =
13.9, 3.5 Hz, 1 H), 4.26 (d, J = 13.9 Hz, 1 H), 3.80−3.48 (br m, 2 H),
3.33−3.18 (br m, 2 H), 2.03−1.83 (br m, 2 H), 1.67−1.47 (br m, 2 H).
Anal. (C18H18F3N5O6) C, H, N.
% remaining = 100 × (mean PAR /mean PAR
)
T60
T0
N-Methyl-N-[4-(trifluoromethoxy)benzyl]piperidin-4-amine (112)
(Scheme 4B). A solution of tert-butyl 4-aminopiperidine-1-carboxylate
(108) (3.00 g, 15.0 mmol) and 4-(trifluoromethoxy)benzaldehyde
(109) (2.14 mL, 15.0 mmol) in MeOH (60 mL) was refluxed for 4 h.
After cooling to 20 °C, NaBH4 (1.13 g, 29.9 mmol) was added in
portions and the mixture was stirred for a further 30 min. Water was
added, the mixture was extracted with EtOAc, and the extract was
evaporated to give the crude amine 110 (5.75 g), which was used
directly in the next step. A mixture of this crude 110 (5.75 g, 15.0 mmol),
MeI (0.95 mL, 15.3 mmol), and K2CO3 (4.56 g, 33.0 mmol) in
anhydrous acetone (150 mL) was stirred at 20 °C for 18 h. After
removal of the solvent under reduced pressure, the residue was
partitioned between CH2Cl2 and water, and the organic layer was
evaporated to give tert-butyl 4-{methyl[4-(trifluoromethoxy)benzyl]-
amino}piperidine-1-carboxylate (111) (4.77 g, 82% overall) as a crude
solid: mp 35−40 °C, which was used directly in the next step.
A solution of crude 111 (4.77 g, 12.3 mmol) in 1:1 CH2Cl2/TFA (150 mL)
was stirred at 20 °C for 1 h. After concentration to dryness under
reduced pressure, the residue was partitioned between 2 N NaOH and
CH2Cl2, and the organic layer was evaporated to give 112 (3.16 g, 73%
overall) as an oil. 1H NMR (CDCl3) δ 7.34 (d, J = 8.5 Hz, 2 H), 7.14
(d, J = 8.5 Hz, 2 H), 3.57 (s, 2 H), 3.22−3.16 (m, 2 H), 2.66−2.49
(m, 3 H), 2.19 (s, 3 H), 2.13 (br m, 1 H), 1.87−1.80 (m, 2 H), 1.59−1.48
(m, 2 H). HREIMS calcd for C14H19F3N2O m/z (M+) 288.1449, found
288.1448.
where PAR = analyte/IS peak area ratio
In Vivo Acute TB Infection Assay. Each compound was admin-
istered orally to a group of 7 M. tb-infected BALB/c mice at a standard
dose of 100 mg/kg, daily for 5 days a week for 3 weeks, beginning on
day 11 postinfection, in accordance with published protocols.13,30 The
results are recorded as the ratio of the average reduction in colony
forming units (CFUs) in the compound-treated mice/the average
CFU reduction in the mice treated with 1. In this assay, 1 caused up to
2.5−3 log reductions in CFUs.
In Vivo Chronic TB Infection 3 Week Assay. Compounds were
administered orally as described for the acute assay but with treatment
beginning ∼70 days postinfection. In this assay, 1 caused a ca. 2 log
reduction in CFUs, whereas 2 caused a ca. 3 log reduction in CFUs.
In Vivo Chronic TB Infection 8 Week Assay. Each compound
was administered orally to a group of 7 M. tb-infected BALB/c mice at
a dose of 30 mg/kg, daily for 5 days a week for 8 weeks, with treatment
beginning ∼50 days post infection. In this assay, 2 caused a ca. 3 log
reduction in CFUs.
In Vivo Mouse Pharmacokinetics. Compounds were adminis-
tered orally to CD-1 mice at a standard dose of 40 mg/kg, as a
suspension in 0.5% carboxymethylcellulose/0.08% Tween 80 in water.
Samples derived from plasma and lungs were analyzed by LC-MS/MS
to generate the required pharmacokinetic parameters.
Studies of compounds 3, 33, and 82 were conducted at Cumbre
Pharmaceuticals Inc., Dallas, Texas, and UNT Health Science Center,
324
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Journal of Medicinal Chemistry
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3500 Camp Bowie Blvd., Fort Worth TX 76107−2699. The study of
compounds 59 and 74 (using the same protocol) was conducted by
MDS Pharma Services, 22002 26th Avenue SE, Suite 104, Bothell,
WA 98021−4444.
In Vivo Rat Pharmacokinetics and Oral Bioavailability.
Compound 82 was administered to groups of 3 male Sprague−
Dawley rats, both intravenously, at a single dose of 1 mg/kg, and
orally, at a single dose of 20 mg/kg, as 1 or 2 mg/mL solutions in 40%
hydroxypropyl-β-cyclodextrin/50 mM citrate buffer (pH = 3). Samples
derived from plasma were analyzed by LC-MS/MS to generate the
required pharmacokinetic parameters.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional experimental procedures and characterizations for com-
pounds; graphs of selected tabular data; combustion analytical
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (+649) 923 6145. Fax: (+649) 373 7502. E-mail:
am.thompson@auckland.ac.nz.
■
ACKNOWLEDGMENTS
■
We thank the Global Alliance for Tuberculosis Drug Develop-
ment for financial support through a collaborative research
agreement. Special thanks belong to Drs. Anna Upton, Annette
Shadiack, and Khisi Mdluli for their efforts in designing and
coordinating the ADME/pharmacokinetic studies. The authors
also thank Sisira Kumara and H. D. Sarath Liyanage,
respectively, for the solubility and log P measurements, and
Dr Kevin Hicks for his assistance with processing some
pharmacokinetic data.
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ABBREVIATIONS USED
■
TB, tuberculosis; MDR, multidrug-resistant; XDR, extensively
drug resistant; M. tb, Mycobacterium tuberculosis; MIC,
minimum inhibitory concentration; HIV, human immunodefi-
ciency virus; CYP, cytochrome P450; SAR, structure−activity
relationship; NMM, N-methylmorpholine; SD, standard
deviation; DMSO, dimethyl sulfoxide; HLM, human liver
microsomes; MLM, mouse liver microsomes; CFU, colony
forming unit; HREIMS, high resolution electron impact mass
spectrometry; HRFABMS, high resolution fast atom bombard-
ment mass spectrometry; HRESIMS, high resolution electro-
spray ionization mass spectrometry; DMF, N,N-dimethylform-
amide; DIPEA, diisopropylethylamine; THF, tetrahydrofuran;
TFA, trifluoroacetic acid; PAR, peak area ratio; IS, internal
standard; AUC, area under the curve; DEAD, diethyl
azodicarboxylate
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