Rational design of 5-phenyl-3-isoxazolecarboxylic acid ethyl esters as growth inhibitors of Mycobacterium tuberculosis. A potent and selective series for further drug development

Article abstract of DOI:10.1021/jm901273n

New antituberculosis (anti-TB) drugs are urgently needed to shorten the 6-12month treatment regimen and especially to battle drug-resistant Mycobacterium tuberculosis (Mtb) strains. In this study, we have continued our efforts to develop isoxazole-based a

Full text of DOI:10.1021/jm901273n

678 J. Med. Chem. 2010, 53, 678–688
DOI: 10.1021/jm901273n
Rational Design of 5-Phenyl-3-isoxazolecarboxylic Acid Ethyl Esters as Growth Inhibitors of
Mycobacterium tuberculosis. A Potent and Selective Series for Further Drug Development
Annamaria Lilienkampf,† Marco Pieroni,† Baojie Wan,‡ Yuehong Wang,‡ Scott G. Franzblau,‡ and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago,
833 S. Wood Street, Chicago, Illinois 60612 and ‡Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago,
833 S. Wood Street, Chicago, Illinois 60612
Received August 25, 2009
New antituberculosis (anti-TB) drugs are urgently needed to shorten the 6-12 month treatment regimen
and especially to battle drug-resistant Mycobacterium tuberculosis (Mtb) strains. In this study, we have
continued our efforts to develop isoxazole-based anti-TB compounds by applying rational drug design
approach. The biological activity and the structure-activity relationships (SAR) for a designed series of
5-phenyl-3-isoxazolecarboxylic acid ethyl ester derived anti-TB compounds were investigated. Several
compounds were found to exhibit nanomolar activity against the replicating bacteria (R-TB) and low
micromolar activity against the nonreplicating bacteria (NRP-TB). The series showed excellent
selectivity toward Mtb, and in general, no cytotoxicity was observed in Vero cells (IC50 >128 μM).
Notably, selected compounds also retained their activity against isoniazid (INH), rifampin (RMP), and
streptomycin (SM) resistant Mtb strains. Hence, benzyloxy, benzylamino, and phenoxy derivatives of
5-phenyl-3-isoxazolecarboxylic acid ethyl esters represent a highly potent, selective, and versatile series
of anti-TB compounds and as such present attractive lead compounds for further TB drug development.
Introduction
second-line drugs, i.e., kanamycin, capreomycin, or amikacin.
According to the WHO, over 400000 people develop MDR-
TB each year, and cases have been reported across all regions
of the world. Failed MDR-TB treatment regimen may result
in XDR-TB, which in turn can be spread from one individual
to another. Thus, in 2006, XDR-TB was named as a global
threat to public health.4 All the foregoing facts emphasize the
urgent demand for new anti-TB drugs with novel mechanisms
of action. However, TB is a neglected disease and new anti-TB
drugs have not been introduced for the past 4 decades.5 While
more researchhasbeen placedon early stagedrug discoveryas
a consequence of funding from the Bill and Melinda Gates
Foundation and others, only a few compounds have been
advanced to the clinic. In this connection we note that
compounds such as arylquinoline (RS,βR)-6-bromo-R-[2-
(dimethylamino)ethyl]-2-methoxy-R-1-naphthalenyl-β-phen-
yl-3-quinolineethanol (1) (TMC207)6 and nitroimidazoles
(6S)-6,7-dihydro-2-nitro-6-[[4-(trifluoromethoxy)phenyl]meth-
oxy]-5H-imidazo[2,1-b][1,3]oxazine (2) (PA-824)7 and (2R)-
2,3-dihydro-2-methyl-6-nitro-2-[[4-[4-[4-(trifluoromethoxy)phen-
oxy]-1-piperidinyl]phenoxy]methyl]-imidazo[2,1-b]oxazole
(3) (OPC-67683)8 have entered clinical trials as new anti-TB
agents.9 1 targets the c subunit of ATP synthase,10 whereas
2 and 3 have been suggested to disrupt the synthesis of vital
cell wall mycolic acids.7,8 In addition, 2 kills NRP-TB by
intracellular nitric oxide (NO) release.11
Mycobacterium tuberculosis (Mtba) is the predominant
cause of tuberculosis (TB), a life-threatening chronic infection
primarily affecting the lungs. The World Health Organization
(WHO)hasestimatedthatone-thirdofthe world’s population
is infected with Mtb, resulting in 1.7 million deaths from TB in
2006.1 As a frequent coinfection, TB is aggravated by the
spread of HIV and is a major cause of death among HIV/
AIDS patients. Current TB treatment regimen DOTS
(directly observed therapy short-course) requires patients to
take a combination of three or four drugs, namely, isoniazid
(INH), rifampin (RMP), pyrazinamide (PZA), and ethambu-
tol (EMB) (or alternatively streptomycin (SM)), throughout a
6-12 month period. The long treatment regimen, which is
necessary due to the presence of a nonreplicating persistent
Mtb phenotype (NRP-TB),2 results in poor patient compli-
ance, causes undesired side effects, and makes a significant
contribution to the emergence of drug resistant Mtb strains.3
Multidrug-resistant TB (MDR-TB) is resistant to the most
common first-line drugs, i.e., INH and RMP, whereas exten-
sively drug-resistant TB (XDR-TB) is also resistant to
the fluoroquinolones and at least one of the intravenous
*To whom correspondence should be addressed. Phone: þ1-312-996-
7577. Fax: þ1-312-996-7107. E-mail: kozikowa@uic.edu.
a Abbreviations: DOTS, directly observed therapy short-course;
INH, isoniazid; LORA, low oxygen recovery assay; MABA, microplate
Alamar blue assay; MIC, minimum inhibitory concentration; MDR-
TB, multidrug-resistant tuberculosis; Mtb, Mycobacterium tuberculosis;
NRP-TB, nonreplicating persistent tuberculosis; R-TB, replicating tu-
berculosis; RMP, rifampin; SDR-TB, single drug resistant tuberculosis;
SM, streptomycin; TB, tuberculosis; XDR-TB, extensively drug-resis-
tant tuberculosis.
We have previously reported certain isoxazole-based com-
pounds, created by combining phenotypic screening and
rational drug design, to exhibit good activity against Mtb.
When a 3-isoxazolecarboxylic acid ethyl ester moiety was
linked to the quinoline core of the antimalarial drug meflo-
quine (Mtb MIC = 13 μM),12 the original lead compound 4
r
pubs.acs.org/jmc
Published on Web 12/11/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 679
(MIC = 0.9 μM) was obtained (Figure 1).13 Equal activity
was observed with compound 5 (MIC = 0.95 μM) in which
the oxymethylene linker was replaced with a phenoxy linker.14
In further optimization studies, the activity, as well as meta-
bolic stability, was improved when the oxymethylene linker
was replaced with a trans-ethenyl linker15 and the quinoline
core was replaced with a phenyl ring to obtain the structurally
simplified derivative 6 (MIC = 0.73 μM).16 Our previous
work has also shown that the isoxazole14 and the C3 ester
moiety13,14 are crucial for the anti-TB activity. However, the
ester may act as a prodrug for the corresponding carboxylic
acid that is formed within the bacteria possibly by esterase
activity. The carboxylic acid derivatives are inactive in vitro
which may be due to insufficient penetration through the Mtb
cell wall.13,16 In a related study, Lee and co-workers have
recently reported certain derivatives of 5-phenyl-3-isoxazoli-
necarboxylic acid esters to have low micromolar activity
against R-TB.17
Here, we describe the design, synthesis, and biological
evaluation of a series of anti-TB agents based on a 5-phenyl-
3-isoxazolecarboxylic acid ethyl ester core. Compounds 4, 5,
and 6 were used as lead compounds, and a rational approach
was employed to design compound 7a that showed excellent
activity against Mtb with an MIC of 0.6 μM (Figure 1).
Encouraged by this finding, we engaged in a more in-depth
study of the structure-activity relationships (SAR) for this
isoxazole-based anti-TB compound series. The modifications
were focused on the substitution pattern of the side-chain aryl
moiety, on the oxymethylene linker, and in one example on the
isoxazole ring. The target compounds were evaluated for their
activity against R-TB and NRP-TB. The 5-phenyl-3-isoxazo-
lecarboxylic acid ethyl ester derivatives were found, in several
cases, to exhibit nanomolar activity against R-TB and also to
have low micromolar activity against NRP-TB. These 5-
phenyl-3-isoxazolecarboxylic acid ethyl esters were also found
to have activity against single drug-resistant Mtb strains
(SDR-TB), as described herein.
Chemistry
Two different strategies were employed in the synthesis of
target compounds 7a-s and 8a-k. Compounds 7a, 7b, 7h,
7m, and 7q-r were synthesized in two steps starting from 3-
hydroxyphenylacetylene (9) (Scheme 1, method A). Alkyla-
tion of 9 with a suitably substituted benzyl bromide produced
the acetylene intermediates 10a-e in excellent yields (81-
91%). Next, a dipolar cycloaddition of the nitrile oxide
Figure 1. Evolution of isoxazole-based anti-TB agents.
Scheme 1a
a Reagents and conditions: (a) ArCH2Br or ArCH2Cl, K2CO3, KI, acetone, reflux; (b) ethyl 2-chloro-2-(hydroxyimino)acetate, Et3N, ether or THF,
room temp; (c) TBDPSCl, imidazole, CH2Cl2; (d) TBAF, THF, room temp.

680 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Lilienkampf et al.
Scheme 2a
Scheme 3a
a Reagents and conditions: (a) 3-methoxyphenylmagnesium bromide,
THF, 0 °C to room temp; (b) ethyl 2-chloro-2-(hydroxyimino)acetate,
Et3N, THF, room temp.
a Reagents and conditions: (a) (1-bromoethyl)benzene, K2CO3, KI,
acetone, reflux; (b) 3-(trifluoromethyl)phenylboronic acid, Cu(OAc)2,
Et3N, CH2Cl2, room temp; (c) 4-(chloroacetyl)morpholine, K2CO3, KI,
acetone, reflux.
acid ethyl ester (27) gave 28 in 65% yield (Scheme 4). The
aniline intermediate 27 was in turn synthesized from 3-ethy-
nylaniline (22) via Boc-protected intermediate 26 according to
a published procedure.18 Similarly compound 30, bearing an
aminomethylene linker at the para position, was synthesized
in two steps via reductive amination and the dipolar cycload-
dition. The meta amide linked derivative 31 was also synthe-
sized from 27 in 79% yield by an amide coupling reaction with
3-(trifluoromethyl)benzoic acid. The ortho amide 33 was
obtained via the acetylene intermediate 32. The pyrazole
derivative 34 was synthesized from the corresponding acet-
ylene intermediate 10f and ethyl diazoacetate under micro-
wave irradiation (Scheme 5). According to 1H NMR, 34 is a
mixture of 1-H and 2-H tautomers in DMSO-d6.
derived from ethyl 2-chloro-2-(hydroxyimino)acetate with the
acetylene intermediates furnished the desired 5-phenyl-3-isoxa-
zolecarboxylic acid ethyl esters 7a, 7b, 7h, 7m, and 7q-r in
43-77% yield. In an alternative route, we chose to synthesize
5-(3-hydroxyphenyl)-3-isoxazolecarboxylic acid ethyl ester (13)
as an intermediate, which could efficiently be employed in the
focused library synthesis of the monosubstituted derivatives
7c-g, 7j-l, 7n-p, and 7s and the di- and trisubstituted com-
pounds 8a-k (Scheme 1, method B). The protection of 9 with
TBDPSCl produced the intermediate 11, which in turn gave the
isoxazole intermediate 12 via the dipolar cycloaddition reaction.
Deprotection of 12 with TBAF in THF gave the key intermedi-
ate 13 in 67% yield. The final compounds 7c-g, 7j-l, 7n-p, 7s,
and 8a-k were synthesized from 13 in 63-99% yields by
straightforward Williamson reaction with various benzyl ha-
lides, as described above. All the benzyl halides employed
were commercially available with the exception of [3-(chloro-
methyl)phenyl]-4-morpholinylmethanone, which was synthe-
sized from morpholine and 3-(chloromethyl)benzoylchloride.
The 3-amino derivative 7i was synthesized by reducing the
corresponding 3-nitro derivative 7h with SnCl2. The acetylmor-
pholine derivative 14 was synthesized from phenol 13 and
4-(chloroacetyl)morpholine (Scheme 2).
Next, we focused our efforts on the modifications of
the oxymethylene linker moiety. The R-methyl derivative
15 was synthesized from the phenol 13 and (1-bromo-
ethyl)benzene by employing the same strategy as described
above (Scheme 2). The diphenyl ether derivative 16, bearing
the phenoxy moiety atthe meta position, was obtained in 67%
yield by Cu(OAc)2-catalyzed coupling of 13 with 3-(trifluo-
romethyl)phenylboronic acid (Scheme 2), whereas the para
substituted derivative 19 was synthesized from the commer-
cially available 1-ethynyl-4-phenoxybenzene (17) as outlined
above (Scheme 3). Grignard reaction of 3-methoxyphenyl-
magnesium bromide with 4-ethynylbenzaldehyde (18) gave
the acetylene intermediate 20 which in turn gave 21, bearing a
hydroxymethylene linker at the para position.
Results and Discussion
A total of 42 compounds were synthesized and evaluated
first for their activity against the Mtb strain H37Rv in a
microplate Alamar blue assay (MABA).19 In addition, the
compounds were further evaluated for their potency in a low
oxygen recovery assay (LORA),20 which is a luminescence-
based high-throughput assay suggested for assessment of
activity against NRP-TB in oxygen-deprived conditions. Sev-
eralcompounds werefound toeffectively inhibitthe growth of
R-TB in MABA with nanomolar MICs. In addition, several
compounds exhibited low micromolar activity. First, we
investigated the effect of the aromatic substitution pattern
of the benzyloxy moiety on the anti-TB activity (Table 1),
which was shown to affect compound potency and from
which some whole cell SAR could be derived. In all modifica-
tions, the oxymethylene linker was kept atthe meta position in
relation to the isoxazole moiety. The designed lead compound
7a, which had an excellent MIC of 0.6 μM, carries a -CF3
group at the meta position (R2) of the benzene ring. Removal
of this substituent (7b, MIC = 1.2 μM) led to 2-fold reduced
activity relative to 7a, indicating that substitution of the ring
plays some role in the anti-TB activity. At the meta position,
various substituents were investigated. All the tested halogen
substituents (-F, -Cl, and -Br), as well as -OCF3, -CN,
and -NO2 (compounds 7c-h), yielded activity comparable to
thatof the lead 7a with MICs ranging between 0.6 and 1.0μM.
Keeping the linker in the original meta position, NaB-
(OAc)3H mediated reductive amination of 3-(trifluoromethyl)-
benzaldehyde with 5-(3-aminophenyl)-3-isoxazolecarboxylic

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 681
Scheme 4a
a Reagents and conditions: (a) 3-(trifluoromethyl)benzaldehyde, NaB(OAc)3H, AcOH, 1,2-dichloroethane, room temp; (b) ethyl 2-chloro-
2-(hydroxyimino)acetate, Et3N, ether or THF, room temp; (c) benzoylchloride, Et3N, DMAP, CH2Cl2, room temp; (d) Boc2O, THF, reflux, 16 h;
(e) TFA, CH2Cl2, room temp; (f) 3-(trifluoromethyl)benzoic acid, DMAP, EDCI, CH2Cl2, 0 °C to room temp.
Scheme 5a
(compound 7q) led to a slight improvement in the activity
relativetothe meta substituted lead 7a (MIC of 0.4vs 0.6μM).
Comparable activity was obtained with 7r (MIC = 0.4 μM),
bearing a -CO2Et moiety at the para position, while -F (7s,
MIC=1.8 μM) substitution was somewhat detrimental. In general,
the preferred monosubstitution position seemed to be para g
meta > ortho. A range of substituents were tolerated at the
meta position where the electronic character of the substitu-
tion seemed to influence the activity. However, at the ortho
position, small substituents seemed to be favored, whereas at
the para position larger groups yielded the best activity.
Next, various di- and trisubstitution patterns were explored
with halogens and the -CF3 group (8a-k, Table 1). Fluorine
was chosen as the preferred substituent because of its
small size and previous success (good overall potency) in
the monosubstituted series. Several difluorosubstitution pat-
terns were investigated. With the exception of 8e (R2,R4 = F,
MIC = 4.4 μM), all the difluorinated compounds (8a, 8c, and
8f-h, MIC = 0.6-1.6 μM) exhibited very good activity
against Mtb, in particular 8c (R2,R3 = F) with an MIC of
0.6 μM. However, the corresponding dichloro substitution
pattern (R2,R3 = Cl) led to decreased activity (8d, MIC=
2.7 μM). Compound 8b, bearing both an R1 = F and an R2 =
CF3 group in the ring, also exhibited good activity, as did 8i
(R1 = Cl, R4 = F), withMICs of0.6 and 0.9 μM, respectively.
Trifluorosubstitution patterns, namely, R1, R2, and R3 (8j,
MIC = 0.9 μM) or R1, R4, and R5 (8k, MIC = 1.0 μM),
were also well tolerated. In addition, the benzyloxy side
chain in question was shown to be as important as the
intermediate 5-(3-hydroxyphenyl)-3-isoxazolecarboxylic acid
ethyl ester (13, MIC = 7.3 μM), as well as the acetylmorpho-
line derivative 14 (MIC = 13.8 μM), exhibited reduced anti-
TB activity.
a Reagents and conditions: (a) N2CHCO2Et, benzene, microwave,
140 °C.
Althoughstill active, -CH3, -OCH3, and -NH2 substitution
led to slightly decreased potency (compounds 7i-k, MIC =
1.2-2.1 μM). However, the activity was completely lost with
7l, bearing a 4-morpholinecarbonyl moiety, probably because
of increased steric hindrance at the target site. The electronic
character of the substituent apparently contributes to the anti-
TB activity as can be seen when comparing -CH3 and -CF3
(7k, MIC = 1.2 μM vs 7a, MIC = 0.6 μM) or -OCH3 and
-OCF3 (7j, MIC = 2.1 μM vs 7f, MIC = 0.7 μM). Introduc-
tion of -CF3 group, as well -NO2, to the ortho position (R1)
decreased the potency 2- to 3-fold, although 7m (MIC =
1.5 μM) and 7p (MIC = 1.5 μM) still exhibited good anti-TB
activity. With halogens as ortho substituents, i.e., -Cl and
-F, the activity was retained as compared to the correspond-
ing meta substituted compounds 7n and 7o having an MIC
of 0.9 and 1.1 μM, respectively. The introduction of a -CF3
group to the para position (R3) of the benzene ring

682 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Lilienkampf et al.
Table 1. The effect of the benzyl side chain substitution on the anti-TB activity
R1
R2
R3
R4
R5
MABAa MIC (μM)
LORAa MIC (μM)
Vero cells IC50 (μM)
7a
7b
7c
7d
7e
7f
H
H
-CF3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-H
-F
-F
H
H
-F
H
-F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-F
H
H
H
-F
0.6
1.2
0.8
1.0
0.8
0.7
0.6
0.6
1.5
2.1
1.2
>128
1.5
0.9
1.1
1.5
0.4
0.4
1.8
1.6
0.6
0.6
2.7
4.4
0.9
1.2
1.0
0.9
0.9
1.0
7.3
13.8
0.1
0.5
0.3
8.0
41.5
18.9
10.8
12.7
13.1
26.5
8.7
>128
>128
>128
>128
>128
101
H
-F
H
H
H
-Cl
-Br
H
H
H
H
-OCF3
-CꢀN
H
7g
7h
7i
H
H
>128
>128
>128
>128
>128
>128
>128
>128
ndb
H
-NO2
-NH2
H
H
H
25.8
26.7
14.9
>128
34.8
10.1
19.1
25.8
11.8
32.7
24.0
26.8
7.0
7j
H
-OCH3
-CH3
-CON(CH2CH2)2O
H
7k
7l
H
H
H
H
7m
7n
7o
7p
7q
7r
-CF3
-Cl
-F
-NO2
H
H
H
H
H
H
H
H
H
>128
>128
>128
nd
H
-CF3
-CO2Et
-F
H
H
H
7s
H
H
8a
8b
8c
8d
8e
8f
-F
-F
H
-F
-CF3
-F
-Cl
-F
H
>128
>128
>128
nd
H
-F
-Cl
H
19.2
10.6
16.6
23.7
12.8
15.7
15.1
12.6
10.9
113.9
48.9
1.9
H
H
>128
>128
>128
>128
nd
-F
-F
-F
-Cl
-F
-F
H
8g
8h
8i
H
H
H
-F
H
H
8j
-F
H
-F
H
>128
nd
nd
8k
13
14
RMP
INH
2
nd
127-144
>128
nd
>128
3.8
a Mtb strain H37Rv; b not determined.
Finally, we investigated the effect of the oxymethylene
linker moiety on the anti-TB activity (Table 2). The introduc-
tion of an R-methyl group to the linker (compound 15) led to
complete loss of the activity. Elimination of the methylene
moiety, to give the phenoxy derivative 16 (MIC = 1.0 μM),
did not significantly alter the potency. Insertion of a nonsub-
stituted phenoxy moiety to the para position resulted in
similar activity (compound 19, MIC = 0.9 μM). A hydro-
xymethylene linker at the para position was also tolerated
(compound 21, MIC = 2.8 μM), which is interesting because
the related compound 15 was found to be inactive. This
suggests that hydrophilic, but not hydrophobic, branching
at the linker may be acceptable. The original oxymethylene
linker could also be successfully replaced with an amino-
methylene linker, as in compounds 28 (MIC = 1.1 μM) and
30 (MIC = 0.6 μM). In fact, 30, bearing the aminomethylene
linker at the para position, was 2-fold more active than the
corresponding meta derivative 28, which in turn was 2-fold
lessactivethanthecorrespondingoxymethylenederivative7a.
The planar amide linker at the meta position (31,MIC=0.9μM)
yielded activity comparable to that of the corresponding
aminomethylene linker derivative, 28, and slightly decreased
activity as compared to the lead 7a. However, an amide linker
at the ortho position (compound 33), which significantly
alters the 3D geometry of the molecule, was not tolerated
and the activity was lost. Surprisingly, the Boc-protected
intermediate 26, with a carbamate linker and a bulky tert-
butyl moiety, also exhibited good activity against Mtb.
Although modifications of the isoxazole moiety in the past
have been unsuccessful, we decided to synthesize the pyrazole
derivative 34. The pyrazole ring was chosen, since our earlier
SAR studies on the isoxazole moiety had suggested that the
position of the ring nitrogen at this moiety may play an
important role in the anti-TB activity.14 However, the pyr-
azole derivative 34 had significantly reduced activity with an
MIC of 32.9 μM, thus further confirming the crucial role of
the isoxazole moiety for the anti-TB activity.
The compounds were also tested for potency in LORA, a
plausible model for NRP-TB. Notably, these compounds also
exhibited micromolar activity in LORA (Tables 1 and 2). In
general, the compounds that were the most active in MABA
also showed good activity in LORA. In particular, 8b and 30

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 683
Table 2. The effect of the linker moiety and its position on the anti-TB activity
Linker position
Linker
R
MABA MIC (μM)
LORA MIC (μM)
Vero cells IC50 (μM)
15
16
meta
meta
para
para
meta
para
meta
ortho
-CH(CH3)O-
-O-
-O-
H
-CF3
H
>128
1.0
0.9
>128
10.5
13.8
23.9
15.3
6.3
nd
>128
>128
nd
19
21
-CH(OH)-
-CH2NH-
-CH2NH-
-CONH-
-CONH-
-OCH3
-CF3
-CF3
-CF3
H
2.8
1.1
28
30
>128
>128
>128
nd
0.6
1.4
31
33
28.3
>128
12.6
>128
1.9
>128
1.1
26
>128
nd
34c
RMP
32.0
0.1
127-144
a Mtb strain H37Rv; b not determined; c A mixture of 1-H and 2-H tautomers.
Table 3. Activity against drug-resistant strains of Mtb
MABA MIC (μM)
retained their activity relatively well against the oxygen
starved bacteria with a LORA MIC of 7.0 and 6.3 μM,
respectively. In addition, several other compounds, namely,
7a, 7d-f, 7h, 7n, 7q, 8d, 8g, 8j-k, 16, and 26, had LORA MIC
values of e13 μM. This can be considered as an encouraging
finding, since RMP has a LORA MIC of ∼2 μM and is thus
20-fold less active under low oxygen conditions. Among the
current TB drugs, only RMP, SM, and PZA have been
reported to show good activity against this phenotype and it
has been suggested that the key to shortening the long TB
treatment is to target NRP-TB.
r-RMPa
r-INHb
r-SMc
7a
1.0
1.0
1.0
1.0
1.0
1.0
1.0
>32
0.4
0.2
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.2
1.1
0.1
0.5
>32
7e
7n
1.0
1.0
8a
8b
0.9
1.0
8f
8g
1.1
0.1
RMP
INH
SM
>128
0.4
To eliminate the possibility that the anti-TB activity arises
from general toxicity, Vero cells were used for an in vitro
cytotoxicity evaluation. These compounds are highly selective
toward Mtb, and with the exception of 7f (IC50 = 101 μM,
selectivity index of 144), all the compounds had IC50 > 128 μM.
In addition, these compounds are not broad-spectrum
antibiotics as was shown by the fact that 7a did not exhibit
activity against M. smegmatis, E. coli, S. aureus, or fungus
C. albicans (MIC > 100 μM). In addition, no activity was
found against parasite T. gondii. Finally, selected compounds
were evaluated against Mtb strains that are resistant to three
first-line TB drugs (Table 3). All the tested compounds,
namely, 7a, 7e, 7n, 8a, 8b, 8f, and 8g, retain their activity
against RMP, INH, and SM resistant strains, as do 4 and 5,14
suggesting a different mode of action and indicating that this
compound class also holds promise as lead structures for
drug-resistant TB.
The discovery of 5-phenyl-3-isoxazolecarboxylic acid esters
as potent anti-TB compounds, although driven by rational
design, originates from phenotypicscreening, thus proving the
power of the approach of bringing lead discovery to the level
ofthe organism. Phenotypic screening iswell justifiedforMtb,
since one of the major challenges in TB drug discovery is the
compound penetration through the thick and waxy Myco-
bacterium cell wall. However, the major disadvantage of
phenotypic screening is that often the molecular target of a
compound remains unknown and lead optimization depends
solely on ligand-based design and the chemists’ intuition.
Although SAR can be derived for this anti-TB compound
class, it should be noted that the whole cell SAR not only
includes the binding affinity to a plausible molecular target
but also is affected by Mtb cell permeability and intracellular
a RMP resistant strain. b INH resistant strain. c SM resistant strain.
metabolism. The SAR obtained from this series, including the
particular structural requirements for the activity, i.e., the
isoxazole moiety and the carboxy functionality at its C3
position, suggests that a specific, yet to be identified, molec-
ular target may exist for these anti-TB compounds. Although
not much can be said regarding the target at this point, the
additional activity against NRP-TB indicates that the target is
not likely to lie somewhere along the Mtb cell wall synthesis
pathway. The fact that a variety of substituents and substitu-
tion patterns on the side chain benzene ring, as well as various
linker moieties, are tolerated suggests that while the planar 5-
phenyl-3-isoxazolecarboxylic moiety may reside in a specific
binding cavity, the flexible benzylic side chain of the molecule
may, at least partly, extend to the surface area. Alternatively,
the side chain may occupy a relatively large and flexible
hydrophobic cavity. The lack of activity of ortho amide 33
can be explained by the difference in the spatial orientation of
side chain.
Conclusions
Benzyloxy, benzylamino, and phenoxy derivatives of 5-
phenyl-3-isoxazolecarboxylic acid ethyl esters are highly po-
tent anti-TB agents with several compounds exhibiting nano-
molar activity against R-TB phenotype and thus comparable
activity to the current first-line anti-TB drugs. In addition,
although being somewhat weaker, these anti-TB compounds
show activity against the NRP-TB phenotype in low oxygen
conditions. Various mono-, di-, and trisubstitution patterns

684 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Lilienkampf et al.
on the benzyloxy moiety were explored, for which a range
of substituents yield good anti-TB activity. The preferred
monosubstitution position seems to be para g meta>ortho,
with a variety of substituents being tolerated at the meta
position. At the ortho position, small substituents are pre-
ferred, while at the para position larger groups furnish the
best activity. Also, di- and trisubstituted derivatives, mostly
fluorinated compounds, yield good anti-TB activity. The
nature and the position of the linker moiety affect the anti-
TB activity. The original oxymethylene linker at the meta
position can be replaced with an oxy, aminomethylene, or
amide linker, of which the oxy linker yields comparable
activity, with respect to the original system. Insertion of
the aminomethylene linker to the para position is beneficial
for the activity.
Tested compounds, namely, certain benzyloxy derivatives,
retain their activity against Mtb strains that are resistant to
first-line TB drugs INH and RMP or to second-line drug SM.
The compound class shows high selectivity toward Mtb and,
in general, does not exhibit cytotoxity toward Vero cells up to
128 μM. Overall, the high selectivity and potency against drug
susceptible and drug-resistant R-TB, as well as the excellent
activity against NRP-TB, establish these 5-phenyl-3-isoxazo-
lecarboxylic acid ethyl esters derivatives as a promising anti-
TB chemotype.
EtOAc-hexane. The reactions were typically performed in
50-100 mg quantities.
5-[3-[[(3-Trifluoromethyl)phenyl]methoxy]phenyl]-3-isoxazole-
carboxylic AcidEthyl Ester (7a). Procedure A (in ether) wasused.
Yield 64% (white powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J =
7.1 Hz), 4.48 (2H, q, J = 7.1 Hz), 5.18 (2H, s), 6.92 (1H, s), 7.09
(1H, m), 7.42 (3H, m), 7.53 (1H, m), 7.63 (2H, m), 7.74 (1H, br s).
13C NMR (CDCl3) δ 14.3, 62.4, 69.5, 100.4, 112.2, 117.5, 119.2,
124.2 (q, J = 272 Hz), 124.3 (q, J = 4 Hz), 125.1 (q, J = 4 Hz),
128.1, 129.3, 130.6, 130.8, 131.2 (q, J = 32 Hz), 137.6, 157.2,
159.0, 160.1, 171.5. HRMS (ESI) calculated for C20H16F3NO4
[M þ H]þ 392.1104, found 392.1111.
5-[3-[(Phenyl)methoxy]phenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (7b). Procedure A (in ether) was used. Purificiation
was by preparative HPLC. Yield 68% (white powder). 1H NMR
(CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q, J = 7.1 Hz), 5.14
(2H, s), 6.91 (1H, s), 7.08 (1H, m), 7.33-7.47 (8H, m). HRMS
(ESI) calculated for C19H17NO4 [M þ H]þ 324.1230, found
324.1247.
5-[3-[(3-Fluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7c). Procedure B was used. Yield 97% (white
powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H,
q, J = 7.1 Hz), 5.13 (2H, s), 6.91 (1H, s), 7.01-7.09 (2H, m),
7.17-7.23 (2H, m), 7.34-7.42 (4H, m). HRMS (ESI) calculated
for C19H16FNO4 [M þ H]þ 342.1136, found 342.1151.
5-[3-[(3-Chlorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7d). Procedure B was used. Yield 93% (white
solid). 1H NMR (CDCl3) δ 1.44 (3H, t, J = 7.1 Hz), 4.47 (2H, q,
J = 7.1 Hz), 5.10 (2H, s), 6.91 (1H, s), 7.07 (1H, m), 7.32 (3H, m),
7.38-7.41 (3H, m), 7.46 (1H, br s). HRMS (ESI) calculated for
C19H16ClNO4 [M þ H]þ 358.0841, found 358.0853.
Experimental Section
Chemistry. 1H NMR and 13C NMR spectra were recorded on
Bruker spectrometer at 400 and 100 MHz or 300 and 75 MHz,
respectively, with TMS as an internal standard. 19F NMR
spectra were recorded on Bruker spectrometer at 376 MHz with
TFA as an external standard. HRMS experiments were per-
formed on Q-TOF-2TM (Micromass). TLC was performed with
Merck 60 F254 silica gel plates and column chromatography by
using CombiFlash Rf system with RediSep columns. Prepara-
tive HPLC was carried out on a Shimadzu SCL-10A VP
instrument with an ACE 5-AQ (21.2 mm ꢁ 150 mm) column.
The purity of the target compounds was determined to be g95%
by analytical HPLC using Agilent 1100 HPLC system with a
Synergi 4 μm Hydro-RP 80A column, with detection at 254 nm
on a variable wavelength detector G1314A; flow rate = 1.4 mL/
min; gradient elution over 20 min, from 30% MeOH-H2O to
100% MeOH with 0.05% TFA or alternatively from 10%
MeOH-H2O to 100% MeOH with 0.05% TFA.
General Procedure A for the Synthesis of 7a, 7b, 7h, 7m, 7q, 7r,
12, 19, 21, 30, and 33. The appropriate acetylene intermediate
(1 equiv) and Et3N (3 equiv) were dissolved into anhydrous THF
or ether (15 mL/mmol). Subsequently, ethyl 2-chloro-2-(hydroxy-
imino)acetate (3 equiv) in anhydrous THF or ether (2 mL/mmol)
was added to the solution via syringe pump over 8 h, and the
reaction mixture was stirred overnight at room temperature.
The reaction mixture was filtered and washed with THF, and
the filtrate was evaporated in vacuo. The crude material
was purified by flash chromatography using gradient elution
from hexane to 30-40% EtOAc-hexane to give the product.
The reactions were typically performed in 100-300 mg quan-
tities.
5-[3-[(3-Bromophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7e). Procedure B was used. Yield 99% (white
powder). 1H NMR (CDCl3) 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H,
q, J = 7.1 Hz), 5.10 (2H, s), 6.92 (1H, s), 7.07 (1H, m), 7.28 (1H,
m), 7.37-7.42 (4H, m), 7.48 (1H, m), 7.46 (1H, br s). HRMS
(ESI) calculated for C19H16BrNO4 [M þ H]þ 402.0336 found
402.0355.
5-[3-[[(3-Trifluoromethoxy)phenyl]methoxy]phenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (7f). Procedure B was used. Yield 71%
(colorless oil). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48
(2H, q, J = 7.1 Hz), 5.15 (2H, s), 6.92 (1H, s), 7.08 (1H, m), 7.24
(1H, m), 7.33 (1H, br s), 7.38-7.44 (5H, m). HRMS (ESI)
calculated for C20H16F3NO5 [M þ H]þ 408.1053, found 408.1036.
5-[3-[(3-Cyanophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7g). Procedure B was used. Yield 95% (white
solid). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H,
q, J = 7.1 Hz), 5.17 (2H, s), 6.93 (1H, s), 7.08 (1H, m), 7.43
(3H, m), 7.53 (1H, m), 7.67 (2H, m), 7.78 (1H, br s). HRMS
(ESI) calculated for C20H16N2O4 [M þ H]þ 349.1183, found
349.1200.
5-[3-[(3-Nitrophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7h). Procedure A (in ether) was used. Yield
77% (white solid). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz),
4.49 (2H, q, J = 7.1), 5.24 (2H, s), 6.94 (1H, s), 7.11 (1H, m), 7.45
(3H, m), 7.61 (1H, apparent t, J = 7.9 Hz), 7.81 (1H, d, J = 7.5),
7.23 (1H, d, J = 7.9), 8.37 (1H, s). HRMS (ESI) calculated for
C19H16N2O6 [M þ H]þ 369.1081, found 369.1097.
5-[3-[(3-Methoxyphenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7j). Procedure B was used. Yield 96% (white
solid). 1H NMR (CDCl3) δ 1.46 (3H, t, J = 7.1 Hz), 3.84 (3H, s),
4.49 (2H, q, J = 7.1 Hz), 5.12 (2H, s), 6.90 (2H, m), 7.02-7.11
(3H, m), 7.33 (1H, apparent t, J = 7.8 Hz), 7.42 (3H, m). HRMS
(ESI) calculated for C20H19NO5 [M þ H]þ 354.1336, found
354.1354.
General Procedure B for the Synthesis of 7c-g, 7j-l, 7n-p, 7s,
8a-k, 14, and 15. Anhydrous K2CO3 (6 equiv) and 13 (1 equiv)
in acetone (12 mL/mmol, HPLC grade) were refluxed for 15 min.
Subsequently, KI (0.5 equiv) and an appropriate benzyl halide
or alkyl halide (1.05 equiv) were added and the reaction mixture
was refluxed for 0.5-3 h until disappearance of the starting
material on TLC (1:4 EtOAc-hexane as an eluent). The reaction
mixture was cooled and filtered, and the filtrate was evaporated
in vacuo. The crude product was purified by flash chromato-
graphy using gradient elution from hexane to 30-90%
5-[3-[(3-Methylphenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7k). Procedure B was used. Yield 97% (white
solid). 1H NMR (CDCl3) δ 1.44 (3H, t, J = 7.1 Hz), 2.38 (3H, s),
4.48 (2H, q, J = 7.1 Hz), 5.09 (2H, s), 6.90 (1H, s), 7.08 (1H, m),
7.16 (1H, m), 7.24-7.31 (3H, m), 7.39-7.43 (3H, m). HRMS

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 685
(ESI) calculated for C20H19NO4 [M þ H]þ 338.1387 found
388.1399.
5-[3-[(3,4-Difluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (8c). Procedure B was used. Yield 99% (white
solid). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q,
J =7.1 Hz), 5.08(2H, s), 6.92 (1H, s), 7.07(1H, m), 7.17-7.32 (3H,
m), 7.42 (3H, m). HRMS (ESI) calculated for C19H15F2NO4 [M þ
H]þ 360.1042, found 360.1061.
5-[3-[[3-(4-Morpholinylcarbonyl)phenyl]methoxy]phenyl]-3-iso-
xazolecarboxylic Acid Ethyl Ester (7l). Procedure B was used.
Yield 92% (white powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J =
7.1 Hz), 3.35-3.90(8H, m), 4.48 (2H, q, J = 7.1 Hz), 5.17 (2H, s),
6.92 (1H, s), 7.08 (1H, m), 7.37-7.53 (7H, m). HRMS (ESI)
calculated for C24H24N2O6 [M þ H]þ 437.1707, found 437.1728.
5-[3-[[(2-Trifluoromethyl)phenyl]methoxy]phenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (7m). Procedure A (in ether) was
used. Purification was by preparative HPLC. Yield 43% (white
powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H,
q, J = 7.1 Hz), 5.33 (2H, s), 6.91 (1H, s), 7.07 (1H, m), 7.43 (4H,
m), 7.59 (1H, m), 7.74 (2H, m). HRMS (ESI) calculated for
C20H16F3NO4 [M þ H]þ 392.1104, found 392.1114.
5-[3-[(3,4-Dichlorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (8d). Procedure B was used. Yield 99% (white
powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H,
q, J = 7.1 Hz), 5.09 (2H, s), 6.92 (1H, s), 7.06 (1H, m), 7.29 (1H,
dd, J = 1.7 Hz, J = 8.3 Hz), 7.42 (3H, m), 7.48 (1H, d, J =
8.2 Hz), 7.57 (1H, d, J =1.6 Hz). HRMS (ESI) calculated for
C19H15Cl2NO4 [M þ H]þ 392.0451, found 392.0462.
5-[3-[(3,5-Difluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (8e). Procedure B was used. Yield 89% (white
solid). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q,
J = 7.1 Hz), 5.12 (2H, s), 6.78 (1H, m), 6.92 (1H, s), 6.99 (2H, m),
7.05 (1H, m), 7.41-7.43 (3H, m). HRMS (ESI) calculated for
C19H15F2NO4 [M þ H]þ 360.1042, found 360.1060.
5-[3-[(2-Chlorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7n). Procedure B was used. Yield 88% (white
powder). 1H NMR (CDCl3) δ 1.44 (3H, t, J = 7.1 Hz), 4.47 (2H,
q, J = 7.1 Hz), 5.22 (2H, s), 6.92 (1H, s), 7.09 (1H, m), 7.29 (2H,
m), 7.42 (4H, m), 7.57 (1H, m). HRMS (ESI) calculated for
C19H16ClNO4 [M þ H]þ 358.0841, found 358.0857.
5-[3-[(2,5-Difluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (8f). Procedure B was used. Yield 91% (white
powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q,
J =7.1 Hz), 5.18(2H, s), 6.93 (1H, s), 6.98-7.11 (3H, m), 7.25 (1H,
m), 7.42 (3H, m). HRMS (ESI) calculated for C19H15F2NO4 [M þ
H]þ 360.1042, found 360.1061.
5-[3-[(2-Fluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7o). Procedure B was used. Yield 93% (white
powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H,
q, J = 7.1 Hz), 5.20 (2H, s), 6.92 (1H, s), 7.08-7.21 (3H, m),
7.32-7.44 (4H, m), 7.53 (1H, m). HRMS (ESI) calculated for
C19H16FNO4 [M þ H]þ 342.1136, found 342.1149.
5-[3-[(2,6-Difluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (8g). Procedure B was used. Yield 90% (white
powder). 1H NMR (CDCl3) δ 1.44 (3H, t, J = 7.1 Hz), 4.47 (2H, q,
J = 7.1 Hz), 5.20 (2H, s), 6.95 (3H, m), 7.11 (1H, m), 7.32-7.45
(4H, m). HRMS (ESI) calculated for C19H15F2NO4 [M þ H]þ
360.1042, found 360.1053.
5-[3-[(2-Nitrorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7p). Procedure B was used. Yield 63% (white
solid). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q,
J = 7.1 Hz), 5.56 (2H, s), 6.94 (1H, s), 7.10 (1H, m), 7.42 (3H, m),
7.53 (1H, apparent t, J = 7.7 Hz), 7.72 (1H, apparent t, J = 7.6
Hz), 7.91 (1H, d, J = 7.9 Hz), 8.20 (1H, d, J = 7.9 Hz). HRMS
(ESI) calculated for C19H16N2O6 [M þ H]þ 369.1081, found
369.1101.
5-[3-[(2,4-Difluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (8h). Procedure B was used. Yield 77% (white
solid). 1H NMR (CDCl3) δ 1.46 (3H, t, J = 7.1 Hz), 4.48 (2H, q,
J = 7.1 Hz), 5.14 (2H, s), 6.84-6.94 (3H, m), 7.08 (1H, m), 7.41
(3H, m), 7.49 (1H, m). HRMS (ESI) calculated for C19H15F2NO4
[M þ H]þ 360.1042, found 360.1055.
5-[3-[[(4-Trifluoromethyl)phenyl]methoxy]phenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (7q). Procedure A (in ether) was
used. Purification was by preparative HPLC. Yield 57% (white
powder). 1H NMR (CDCl3) δ 1.46 (3H, t, J = 7.1 Hz), 4.48 (2H,
q, J = 7.1 Hz), 5.20 (2H, s), 6.92 (1H, s), 7.07 (1H, m), 7.42 (3H,
m), 7.58 (2H, d, J = 8.2 Hz), 7.67 (2H, d, J = 8.2 Hz). HRMS
(ESI) calculated for C20H16F3NO4 [M þ H]þ 392.1104, found
392.1101.
5-[3-[(4-Ethoxycarbonylphenyl)methoxy]phenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (7r). Procedure A (in ether) was
used. Yield 77% (white powder). 1H NMR (CDCl3) δ 1.40 (3H,
t, J = 7.1 Hz), 1.45 (3H, t, J = 7.1 Hz), 4.39 (2H, q, J = 7.1 Hz),
4.48 (2H, q, J = 7.1 Hz), 5.20 (2H, s), 6.91 (1H, s), 7.07 (1H, m),
7.41 (3H, m), 7.53 (2H, d, J = 8.2 Hz), 8.08 (2H, d, J = 8.3 Hz).
HRMS (ESI) calculated for C22H21NO6 [M þ H]þ 396.1442,
found 396.1460.
5-[3-[(4-Fluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7s). Procedure B was used. Yield 99% (white
solid). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q,
J = 7.1 Hz), 5.10 (2H, s), 6.91 (1H, s), 7.08 (3H, m), 7.43 (5H,
m). HRMS (ESI) calculated for C19H16FNO4 [M þ H]þ
342.1136, found 342.1138.
5-[3-[(2,3-Difluorophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (8a). Procedure B was used. Yield 99% (white
powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q,
J= 7.1 Hz), 5.22 (2H, s), 6.93 (1H, s), 7.08-7.20 (3H, m), 7.29 (1H,
m), 7.42 (3H, m). HRMS (ESI) calculated for C19H15F2NO4 [M þ
H]þ 360.1042, found 360.1056.
5-[3-[(2-Chloro-5-fluorophenyl)methoxy]phenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (8i). Procedure B was used. Yield
95% (white powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1
Hz), 4.48 (2H, q, J = 7.1 Hz), 5.20 (2H, s), 6.93 (1H, s), 7.00
(1H, dt, J = 2.9, Hz, J = 8.2 Hz), 7.09 (1H, m), 7.34 (1H, dd,
J = 2.9 Hz, J = 9.1 Hz), 7.38 (1H, m), 7.44 (3H, m). HRMS
(ESI) calculated for C19H15ClFNO4 [M þ H]þ 376.0746, found
376.0758.
5-[3-[(2,3,4-Trifluorophenyl)methoxy]phenyl]-3-isoxazolecar-
boxylic Acid Ethyl Ester (8j). Procedure B was used. Yield 88%
(white powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz),
4.49 (2H, q, J = 7.1 Hz), 5.16 (2H, s), 6.93 (1H, s), 7.01-7.09
(2H, m), 7.24 (1H, m), 7.43 (3H, m). HRMS (ESI) calculated for
C19H14F3NO4 [M þ H]þ 378.0948, found 378.0957.
5-[3-[(2,3,6-Trifluorophenyl)methoxy]phenyl]-3-isoxazolecar-
boxylic Acid Ethyl Ester (8k). Procedure B was used. Yield 75%
(white powder). 1H NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz),
4.48 (2H, q, J = 7.1 Hz), 5.21 (2H, s), 6.91 (2H, m), 7.11 (1H,
m), 7.19 (1H, m), 7.43 (3H, m). HRMS (ESI) calculated for
C19H14F3NO4 [M þ H]þ 378.0948, found 378.0960.
5-[3-[(3-Aminophenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (7i). SnCl2 2H2O (1.2 g, 6.2 mmol) was added
portionwise to compound 7h (1.23 g, 0.62 mmol) in 50%
3
EtOH-EtOAc (50 mL), and the reaction mixture was stirred
3
overnight at room temperature. Approximately
/ of the
4
solvent was evaporated under reduced pressure, and 1 M NaOH
(15 mL) was added to the residue. The mixture was extracted
with EtOAc (3 ꢁ 25 mL), and combined organic layers were
washed with brine (20 mL) and dried with Na2SO4. After
filtration, the solvent was evaporated and the residue was
purified by flash chromatography using gradient elution from
10% EtOAc-hexane to 50% EtOAc-hexane to give 7i as a
pale-yellow powder in 61% yield. 1H NMR (CDCl3) δ 1.45 (3H,
5-[3-[[2-Fluoro-(3-trifluoromethyl)phenyl]methoxy]phenyl]-3-
isoxazolecarboxylic Acid Ethyl Ester (8b). Procedure B was
used. Yield 91% (white powder). 1H NMR (CDCl3) δ 1.45 (3H,
t, J = 7.1 Hz), 4.48 (2H, q, J = 7.1 Hz), 5.24 (2H, s), 6.93 (1H,
m), 7.10 (1H, m), 7.30 (1H, m), 7.44 (3H, m), 7.62 (1H, m), 7.77
(1H, m). HRMS (ESI) calculated for C20H15F4NO4 [M þ H]þ
410.1010, found 410.1028.

686 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Lilienkampf et al.
t, J = 7.1 Hz), 3.71 (2H, br s), 4.48 (2H, q, J = 7.1 Hz), 5.05 (2H,
s), 6.66 (1H, m), 6.78 (1H, m), 6.82 (1H, d, J = 7.6 Hz), 6.90 (1H,
s), 7.07 (1H, m), 7.18 (1H, t, J = 7.7 Hz), 7.39 (2H, m), 7.42 (1H,
m). HRMS (ESI) calculated for C19H18N2O4 [M þ H]þ
339.1339, found 339.1351.
as a white solid in 67% yield. 1H NMR δ 1.46 (3H, t, J = 7.1 Hz),
4.49 (2H, q, J = 7.1 Hz), 6.94 (1H, s), 7.15 (1H, m), 7.22-7.31
(2H, m), 7.42-7.54 (4H, m), 7.63 (1H, m). 13C NMR (CDCl3) δ
14.4, 62.5, 100.8, 116.0 (q, J = 4 Hz), 116.7, 120.7, (q, J = 4 Hz),
121.6, 121.7, 122.2, 123.8 (q, J = 273 Hz), 128.7, 130.8, 131.2,
132.8 (q, J = 33 Hz), 157.25, 157.26, 157.30, 160.1, 170.9. HRMS
(ESI) calculated for C19H14F3NO4 [M þ H]þ 378.0948, found
378.0950.
5-[(4-Phenoxy)phenyl]-3-isoxazolecarboxylic Acid Ethyl Ester
(19). 19 was synthesized as described in general method A (in
ether) by using 1-ethynyl-4-phenoxybenzene (17) as a starting
material and purified by preparative HPLC. Yield 63% (white
powder). 1H NMR δ 1.45 (3H, t, J = 7.1 Hz), 4.48 (2H, q, J =
7.1 Hz), 6.85 (1H, s), 7.08 (4H, d, J = 8.1), 7.20 (1H, m), 7.40
(2H, m), 7.77 (2H, d, J = 8.5). 13C NMR (CDCl3) δ 14.4, 62.4,
99.3, 118.7, 120.1, 121.5, 124.6, 127.9, 130.2, 156.0, 157.2, 160.1,
160.3, 171.5. HRMS (ESI) calculated for C18H15NO4 [M þ H]þ
310.1074, found 310.1088.
3-[(tert-Butyldiphenyl)silanyloxy]phenylacetylene (11). To 3-
hydroxyphenylacetylene (0.85 g, 7.2 mmol) in anhydrous
CH2Cl2 (20 mL), imidazole (0.65 g, 9.4 mmol) was added
followed by tert-butyldiphenylchlorosilane (2.4 mL, 9.4 mmol),
and the reaction mixture was stirred for 1 h at room tempera-
ture. Upon completion, the reaction mixture was filtered and the
filtrate was evaporated in vacuo. The residue was purified by
flash chromatography using gradient elution from hexane to
10% EtOAc-hexane to give 11 as a pale-yellow oil in 90% yield.
1H NMR (CDCl3) δ 1.09 (9H, s), 2.97 (1H, s), 6.67 (1H, m), 6.99
(3H, m), 7.35-7.43 (6H, m), 7.70 (4H, m). 13C NMR (CDCl3) δ
19.5, 26.5, 76.8, 83.4, 120.6, 122.9, 123.4, 125.1, 127.8, 129.1,
130.0, 132.5, 135.5, 155.3.
5-[[(tert-Butyldiphenyl)silanyloxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (12). 12 was synthesized as described above in
general method A (in THF) and purified by flash chromatography
from hexane to 30% EtOAc-hexane. Yield 83% (colorless oil).
1H NMR (CDCl3) δ 1.13 (9H, s), 1.43 (3H, t, J = 7.1 Hz), 4.45
(2H, q, J = 7.1 Hz), 6.8 (1H, s), 6.80 (1H, m), 7.17 (2H, m), 7.30
(1H, m), 7.36-7.45 (6H, m), 7.70 (4H, m). 13C NMR (CDCl3) δ
14.2, 19.5, 26.5, 62.2, 100.0, 117.2, 118.7, 122.2, 127.6, 127.9, 130.0,
130.2, 132.4, 135.5, 156.1, 156.8, 160.0, 171.5.
5-(3-Hydroxyphenyl)-3-isoxazolecarboxylic Acid Ethyl Ester
(13). To compound 12 (1.8 g, 3.9 mmol) in anhydrous THF
(20 mL), 1 M TBAF in THF (5.8 mL, 5.8 mmol) was added
dropwise, and the reaction mixture was stirred at room tem-
perature for 3 h. The solvent was evaporated in vacuo, saturated
NH4Cl (aq, 50 mL) was added to the residue, and the mixture
was stirred for 30 min followed by extraction with CH2Cl2 (3 ꢁ
20 mL). The combined organic layers were washed with brine
(20 mL), dried with Na2SO4, and evaporated. The residue was
purified by flash chromatography using gradient elution from
25% EtOAc-hexane to 100% EtOAc to give 13 as a white
powder in 67% yield. 1H NMR (DMSO-d6) δ 1.34 (3H, t, J =
7.1 Hz), 4.39 (2H, q, J = 7.1 Hz), 6.94 (1H, s), 7.29-7.41 (4H,
m), 9.90 (1H, s). 13C NMR (DMSO-d6) δ 14.0, 61.9, 100.7, 112.2,
116.7, 118.1, 127.1, 130.6, 156.8, 157.9, 159.4, 171.2. HRMS
(ESI) calculated for C12H11NO4 [M þ H]þ 234.0761, found
234.0771.
5-[3-[(1-Methyl-1-phenyl)methoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (15). 15 was synthesized as described in the general
method B by using (1-bromoethyl)benzene as a benzyl halide. Yield
71% (white solid). 1H NMR (CDCl3) δ 1.44 (3H, t, J = 7.1 Hz),
1.67 (3H, d, J = 6.4 Hz), 4.46 (2H, q, J = 7.1 Hz), 5.38 (1H, q, J =
6.4 Hz), 6.83 (1H, s), 6.95 (1H, m), 7.24-7.40 (8H, m). HRMS
(ESI) calculated for C20H19NO4 [M þ H]þ 338.1387, found
338.1394.
5-[2-(4-Morpholinyl)-2-oxoethoxy]phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (15). 15 was synthesized as described in the general
method B by using 4-(chloroacetyl)morpholine as an alkyl halide.
Yield 88% (white powder). 1H NMR (CDCl3) δ 1.45 (3H, t,
J = 7.1 Hz), 3.61-3.69 (8H, m), 4.48 (2H, q, J = 7.1 Hz), 4.78
(2H, s), 6.93 (1H, s), 7.09 (1H, m), 7.38-7.44 (3H, m). HRMS
(ESI) calculated for C18H20N2O6 [M þ H]þ 361.1394, found
361.1404.
5-[4-[(3-Methoxyphenyl)hydroxymethyl]phenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (21). 21 was synthesized as de-
scribed in general method A (in ether) by using intermediate 20
1
as a starting material. Yield 50% (white powder). H NMR
(CDCl3) δ 1.44 (3H, t, J = 7.1 Hz), 2.35 (1H, br s), 3.79 (3H, s),
4.47 (2H, m, J = 7.1 Hz), 5.86 (1H, s), 6.83 (1H, m), 6.90 (1H,
s), 6.95 (2H, m), 7.27 (1H, m), 7.52 (2H, d, J = 8.2 Hz), 7.77
(2H, d, J = 8.2 Hz). 13C NMR (CDCl3) δ 14.2, 55.3, 62.4, 75.8,
99.9, 112.2, 113.3, 118.9, 125.8, 126.1, 127.1, 129.8, 144.9,
146.4, 157.0, 159.9, 160.0, 171.5. HRMS (ESI) calculated for
C20H19NO5 [M þ H]þ 354.1336, found 354.1338.
5-[3-[[[(3-Trifluoromethyl)phenyl]methyl]amino]phenyl]-3-iso-
xazolecarboxylic Acid Ethyl Ester (28). To a solution of
3-(trifluomethyl)benzaldehyde (75 mg, 0.43 mmol) in 1,2-di-
chloroethane (8 mL) were added aniline 27 (100 mg, 0.43 mmol),
NaB(OAc)3H (0.11 g, 0.52 mmol), and AcOH (0.03 mg, 0.027
mL, 0.47 mmol). The mixture was stirred at room temperature
for 3 h. The reaction mixture was poured into water (30 mL) and
extracted with CH2Cl2 (3 ꢁ 15 mL). The combined organic
layers were washed with saturated NaHCO3 (20 mL) and brine
(20 mL) and dried with MgSO4. After filtration, the solvent was
evaporated and the crude product was purified first by flash
chromatography (elution from hexane to 40% EtOAc-hexane)
followed by purification by preparative HPLC. Yield 65%
(beige powder). 1H NMR (CDCl3) δ 1.44 (3H, t, J = 7.1 Hz),
4.47 (2H, m, J = 7.1 Hz), 6.71 (2H, d, J = 8.8 Hz), 6.85 (1H, s),
7.08 (1H, br s), 7.16 (1H, m), 7.28 (2H, m), 7.49 (1H, m), 7.58
(2H, m), 7.66 (1H, br s). 13C NMR (CDCl3) δ 14.4, 48.0, 62.4,
100.1, 110.0, 115.4, 115.9, 124.25 (q, J = 273 Hz), 124.33 (q, J =
4 Hz), 124.6 (q, J = 4 Hz), 127.8, 129.5, 130.4, 130.9, 131.4 (q,
J = 33 Hz), 140.1, 148.4, 157.1, 160.3, 172.3. HRMS (ESI)
calculated for C20H17F3N2O3 [M þ H]þ 391.1264, found
391.1274.
5-[4-[[[(3-Trifluoromethyl)phenyl]methyl]amino]phenyl]-3-iso-
xazolecarboxylic Acid Ethyl Ester (30). 30 was synthesized as
described in general method A (in THF) by using intermediate
29 as a starting material. Yield 54% (pale-yellow powder). 1H
NMR (DMSO-d6) δ 1.32 (3H, t, J = 7.1 Hz), 4.36 (2H, q, J =
7.1 Hz), 4.46 (2H, d, J = 6.0 Hz), 6.69 (2H, d, J = 8.8 Hz),
7.07 (2H, m), 7.57-7.71 (6H, m). 13C NMR (DMSO-d6)
δ 14.0, 45.3, 61.8, 97.1, 112.3, 113.8, 123.6 (two over lapping
quartets), 124.3 (q, J = 273 Hz), 127.3, 129.2 (q, J = 31 Hz),
129.5, 131.3, 141.3, 150.6, 156.6, 159.7, 172.2. HRMS (ESI)
calculated for C20H17F3N2O3 [M þ H]þ 391.1264, found
391.1273.
5-[3-[[3-(Trifluoromethyl)benzoyl]amino]phenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (31). A mixture of the aniline 27
(80 mg, 0.34 mmol), DMAP (34 mg, 0.28 mmol), and EDCI (80 mg,
0.52 mmol) in CH2Cl2 (20 mL) was cooled to 0 °C. (3-Trifluoro-
methyl)benzoic acid (65 mg, 0.34 mmol) was added, and the
reaction mixture was stirred at 0 °C for 4 h followed by
overnight at room temperature. Upon completion, CH2Cl2
5-[(3-(3-Trifluoromethyl)phenoxy)phenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (16). The phenol 13 (55 mg, 0.24 mmol),
3-(trifluoromethyl)phenylboronic acid (90 mg, 0.47 mmol), Et3N
˚
(0.3 mL, 2.1 mmol), and powdered 4 A molecular sieves (500 mg)
were mixed in anhydrous CH2Cl2 (10 mL). Cu(OAc)2 (64 mg, 0.35
mmol) was added, and the reaction mixture was stirred at room
temperature for 3 h. The mixture was diluted with CH2Cl2 (10 mL)
and filtered, and the filtrate was evaporated in vacuo. The crude
product was purified by flash chromatography using gradient
elution from hexane to 50% EtOAc-hexane to give the product

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 687
(40 mL) was added and the mixture was washed with 5% HCl
(2 ꢁ 20 mL), 1 M NaOH (2 ꢁ 20 mL), and brine (20 mL). The
organic layer was dried with Na2SO4, filtered, and evaporated.
The crude product was purified by flash chromatography using
gradient elution from hexane to 70% EtOAc-hexane. Yield
79% (white powder). 1H NMR (CDCl3) δ 1.43 (3H, t, J = 7.1
Hz), 4.46 (2H, q, J = 7.1 Hz), 6.98 (1H, s), 7.51 (1H, m), 7.64
(2H, m), 7.82 (2H, m), 8.14 (4H, m). 13C NMR (CDCl3) δ 14.3,
62.5, 100.6, 118.0, 122.4, 123.1, 123.8 (q, J = 273 Hz), 124.4 (q,
J = 4 Hz), 127.5, 128.8 (q, J = 4 Hz), 129.6, 130.2, 130.8, 131.5
(q, J = 33 Hz), 135.5, 138.7, 157.2, 160.1, 165.0, 171.4. HRMS
(ESI) calculated for C20H15F3N2O4 [M þ H]þ 405.1057, found
405.1073.
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recrystallizations from EtOAc. Yield 41% (white powder). H
NMR (CDCl3) δ 1.45 (3H, t, J = 7.1 Hz), 4.49 (2H, q, J = 7.1
Hz), 6.96 (1H, s), 7.27 (1H, m), 7.51-7.60 (4H, m), 7.66 (1H, dd,
J = 1.4 Hz, J = 7.8), 7.96 (2H, m), 8.58 (1H, d, J = 8.3), 9.28
(1H, br s). 13C NMR (CDCl3) δ 14.4, 62.8, 102.9, 117.0, 123.6,
125.0, 127.3, 129.2, 129.3, 132.3, 132.5, 134.5, 136.1, 157.2,
159.8, 165.8, 171.8. HRMS (ESI) calculated for C19H16N2O4
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5-[3-[(3,4-Difluorophenyl)methoxy]phenyl]-1H-pyrazole-3-car-
boxylic Acid Ethyl Ester (34). Ethyl diazoacetate (0.53 g, 3.9
mmol, 0.48 mL) was added to acetylene intermediate 10f (0.80 g,
3.3 mmol) in benzene (3 mL). The reaction mixture was heated to
140 °C for 1.5 h in a sealed vessel under microwave irradiation
(Biotage Initiator). After the mixture was cooled, the solvent was
evaporated and the residue was purified by preparative HPLC to
give the title compound as a light-yellow powder in 80% yield.
According to 1H NMR, the product is a mixture of 1-H and 2-H
tautomers in DMSO-d6. 1H NMR (DMSO-d6) δ 1.32 (3H, m),
4.31 (2H, m), 7.00 (1H, m), 7.25-7.59 (7H, m), 13.92 (N-H),
14.05 (N-H). HRMS (ESI) calculated for C19H16F2N2O3 [M þ
H]þ 359.1202, found 359.1194.
Biology. The MICs were determined using Mtb H37Rv ATCC
27294 in MABA19 and LORA20 assays according to published
procedures. The reported MICs are average values from two to
three individual experiments. Similarly, cytotoxicities were de-
termined on Vero cells according to a published procedure.19b
For a brief description of the biological assays see the Support-
ing Information.
Acknowledgment. A.L. thanks The Academy of Finland
(Grant 120441) and The Finnish Cultural Foundation for
fellowships. Dr. S. H. Cho and K. Sidell are acknowledged for
the bacterial and fungal selectivity assays. Dr. R. McLeod is
acknowledged for the T. gondii assay, and Global Alliance for
TB Drug Development is acknowledged for financial support.
Supporting Information Available: Synthesis of acetylene
intermediates 10a-f, 20, 29, and 32, synthesis of 3-[-
(chloromethyl)phenyl]-4-morpholinylmethanone, and a brief
description of the biological assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
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