Synthesis, biological evaluation, and structure-activity relationships for 5-[(E)-2-arylethenyl]-3-isoxazolecarboxylic acid alkyl ester derivatives as valuable antitubercular chemotypes

Article abstract of DOI:10.1021/jm900513a

Tuberculosis (TB), mostly caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death from infectious disease worldwide. Its coinfection with HIV and the emergence of multidrugresistant TB(MDR-TB) and extensively drug-resistant TB (X

Full text of DOI:10.1021/jm900513a

J. Med. Chem. 2009, 52, 6287–6296 6287
DOI: 10.1021/jm900513a
Synthesis, Biological Evaluation, and Structure-Activity Relationships for 5-[(E)-2-Arylethenyl]-
3-isoxazolecarboxylic Acid Alkyl Ester Derivatives as Valuable Antitubercular Chemotypes
Marco Pieroni,† Annamaria Lilienkampf,† Baojie Wan,‡ Yuehong Wang,‡ Scott G. Franzblau,‡ and Alan P. Kozikowski*,†
†Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,
833 South Wood Street, Chicago, Illinois 60612, and ‡Institute for Tuberculosis Research, College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612
Received April 22, 2009
Tuberculosis (TB), mostly caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of
death from infectious disease worldwide. Its coinfection with HIV and the emergence of multidrug-
resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains have further worsened the
TB pandemic. Despite its global impact, TB is considered a neglected disease and no new anti-TB
therapeutics have been introduced over the last four decades. The nonreplicating persistent form of TB
(NRP-TB) is responsible for the length of the treatment and is the putative cause of treatment failure.
Therefore, new anti-TB agents, which are active against both the replicating form of Mtb (R-TB) and
NRP-TB, are urgently needed. Herein, we report the synthesis and structure-activity relationships
(SAR) of a series of 5-[(E)-2-arylethenyl]-3-isoxazolecarboxylic acid alkyl esters as potent anti-TB
agents. Several compounds had submicromolar minimum inhibitory concentrations (MIC) against
R-TB and were active against NRP-TB in the low micromolar range, thus representing attractive lead
compounds for the possible development of new anti-TB agents.
Introduction
form (NRP-TB) while withstanding chemotherapy.9
Although the DOTS strategy has significantly improved the
treatment outcome, the duration of the 6-12 month treat-
ment, the high cost, and the undesired side effects often lead to
a poor patient compliance, which, in turn, contributes to the
emergence of multidrug resistant TB (MDR-TB) and exten-
sively drug resistant TB (XDR-TB).10,11 MDR-TB is usually
resistant to at least two of the first-line drugs, principally INH
and RMP, whereas XDR-TB additionally fails to respond to
any fluoroquinolone and at least one of three injectable
second line drugs (amikacin, capreomycin, or kanamycin),11
thereby being extremely difficult to cure. The treatment of
resistant strains requires a prolongation of the therapy,
employs more toxic drugs, and increases the financial burden,
thus making TB a vicious cycle.
The foregoing facts highlight the crucial need for novel anti-
TB agents, which are synthetically feasible, lack side effects,
and have physicochemical properties allowing oral adminis-
tration. Moreover, to shorten the duration of the treatment,
they should target NRP-TB and have a novel mechanism of
action.12-15 As part of our efforts in developing new che-
motherapeutics for TB,16,17 we report herein the synthesis and
an in-depth SAR study of new 5-[(E)-2-arylethenyl]-3-isoxa-
zolecarboxylic acid alkyl ester derivatives (Chart 1) with
excellent activity against both replicating Mtb (R-TB) and
NRP-TB and devoid of apparent toxicity to Vero cells. We
have previously reported the design and study of anti-TB
mefloquine-isoxazole hybrid compounds, like compound
1,16b followed by their optimization (compounds 2 and 3)18
in order to improve their potency and metabolic profile
(Figure 1). The good activity shown by 2 and 3 encouraged
us to search for a simplified structure that could be efficiently
Among the infectious diseases, tuberculosis (TBa), a lung
infectioncaused mainlyby Mycobacteriumtuberculosis(Mtb),
is considered one of the most threatening for public health.
This concern is justified by recent reports from the World
Health Organization (WHO), according to which there were
9.2 million new cases of TB and 1.7 million deaths from TB in
2006. Moreover, one-third of the world’s population is esti-
matedtobe latently infectedbyMtb.1 The new cases, as well as
the deaths, occur mostly in developing countries and the
number of HIV-positive patients coinfected with Mtb is
constantly rising.2 In spite of the research efforts being made,
no new drugs have actually been brought to the market for
decades.3-5 In fact, among the first-line anti-TB drugs,
namely isoniazide (INH), pyrazinamide (PZA), ethambutol
(EMB), and rifampin (RMP), the latter represents the most
recent one, although discovered in 1966.6 The current recom-
mended therapeutic strategy Directly Observed Therapy,
Short-course (DOTS)7 is based on the coadministration of
the four above-mentioned drugs for the first two months,
followed by a prolonged treatment with INH and RMP for an
additional 4-7 months.7,8 The cause of this long therapy is the
peculiar ability of Mtb to survive in a nonreplicating persistent
*To whom correspondence should be addressed. Phone: þ1-312-996-
7577. Fax: þ1-312-996-7107. E-mail: kozikowa@uic.edu.
a Abbreviations: EMB, ethambutol ; INH, isoniazid; LORA, low
oxygen recovery assay; MABA, microplate Alamar Blue assay; MIC,
minimum inhibitory concentration; MDR-TB, multidrug-resistant tu-
berculosis; MOX, moxifloxacin; Mtb, Mycobacterium tuberculosis;
NRP-TB, nonreplicating persistent tuberculosis; PZA, pyrazinamide,
R-TB, replicating tuberculosis; RMP, rifampin; TB, tuberculosis; XDR-
TB, extensively drug-resistant tuberculosis.
r
2009 American Chemical Society
Published on Web 09/16/2009
pubs.acs.org/jmc

6288 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Pieroni et al.
described previously. A dipolar cycloaddition reaction of the
nitrile oxide derived from ethyl 2-chloro-2-(hydroxyimino)-
acetate with propargyl bromide gave the 5-bromomethylisox-
azole derivative 4,21 which was subsequently reacted with
triphenylphosphine in THF to give the salt 5. Compound 51
was synthesized starting from 5-methylisoxazole, which was
first brominated with NBS to give 50 and then reacted with
triphenylphosphine to produce the desired intermediate. The
Wittig reaction of 5 and 51 with an appropriate aldehyde in
anhydrous THF using sodium bistrimethylsilylamide as a base
produced the target compounds 6-10, 12-26, 28-36, and 47.
For compounds 11 and 37, NaH was employed as the base in
anhydrous CH2Cl2 because the above conditions failed to
afford the desired products. In most of the reactions carried
out, the alkenes obtained largely possessed trans stereochem-
istry with yields ranging from 15% to 73%. In cases where some
1
cis-isomer was formed (5-10% according to H NMR), the
pure trans-isomer was isolated either by additional column
chromatography or by crystallization from EtOH. In the few
cases where an approximately one to one mixture of both iso-
mers was formed, as found in the case of 19a, 19b, 24a, 24b, 34a,
and 34b, the isomers were separated by preparative HPLC.
Ester hydrolysis of 6 and 9 using LiOH produced the corre-
sponding acids 38 and 39 in excellent yields. Treatment of
alkenes 9 and 22 with H2/Pd in MeOH gave the alkyl deriva-
tives 48 and 49 in satisfactory yields. The selective reduction of
nitro derivative 24b to the aniline 27 was accomplished by using
SnCl2 in EtOH. Finally, esters 40-46 were obtained in good
yields by a Ti(EtO)4 catalyzed transesterification between the
corresponding ethyl ester and an excess of the appropriate
alcohol.
Figure 1. Examples of mefloquine-isoxazole hybrids and their
simplified derivatives. For R and R1, see Chart 1.
modified in order to investigate the SAR and to thereby
elucidate the anti-TB potential of these isoxazole-based com-
pounds. Hence, keeping intact the 5-ethenyl-3-isoxazolecar-
boxylic acid ester core, which is suggested to act as the
pharmacophore,16-18 we first substituted the quinoline ring
with a naphthalene group (compound 6) in order to assess
whether the quinoline ring nitrogen was essential for the
activity. Next, we investigated whether an appropriately sub-
stituted single aromatic ring, such as benzene or pyridine,
could lead to good anti-TB activity. Inour earlier studies,16-18
halogenated and trifluoromethyl substituted compounds
showed the best activity and thus we chose to further inves-
tigate the effect of these substituents in the present compound
class. Substituents such as amino, hydroxyl, and methoxy
were chosen to lower the ClogP in order to improve the drug-
likeness of these compounds. More hindered groups were
chosen as substituents to probe the presence and the size of
any lipophilic pockets in the binding site. The majority of the
new molecules synthesized herein were found to show excellent
activity against Mtb while not exhibiting toxicity to Vero cells.
For several compounds, the MICs were found to be in the
submicromolar range against R-TB and in the low micromolar
against NRP-TB. To some extent, the activity appeared to be
influenced by the degree of lipophilicity, and thus for selected
derivatives the ethyl ester was replaced with other more
hindered esters, resulting in a slight improvement in their
anti-TB activity as described below.
Results and Discussion
All of the target compounds were tested for their ability to
inhibit the growth of R-TB strain H37Rv in a microplate
Alamar Blue assay (MABA)22 and subsequently tested for
their activity against NRP-TB in a low oxygen recovery assay
(LORA).23
The majority of the compounds were found to be highly
active in inhibiting R-TB, some of them being active at
submicromolar concentrations (Table 1). The naphthyl deri-
vative 6 retained good activity against R-TB (MIC 1.6 μM)
although less active than the corresponding quinoline deriva-
tive 3 (MIC 0.2 μM). When the structure was simplified by
replacing the naphthyl core with simple aromatic rings, i.e.,
pyridine and benzene (compound 7 MIC 15.5 μM and com-
pound 8 MIC 11.7 μM), the activity decreased significantly as
compared to 6. The introduction of various substituents to the
pyridine and benzene rings led to a variable range of activities
and allowed us to construct a plausible SAR as will be
described below. The MICs were, in almost all the cases, lower
than those of the unsubstituted species. Introduction of a
lipophilic trifluoromethyl group in the meta position of the
aromatic ring led to a sharp increase in potency, with com-
pound 9 being very active against R-TB with an MIC of
0.73 μM. The meta position was found to be favored because
moving the trifluoromethyl group around the benzene ring, as
with derivatives 10 (MIC 3.9 μM) and 11 (MIC 3.9 μM), led to
a decrease in anti-TB potency. The replacement of the triflu-
oromethyl group with a chlorine atom (compound 18) led to a
slight decrease in activity (MIC 2.3 μM), with 18 being 3-fold
less active than 9. The appendage of an additional chlorine
atom in the benzene ring led to derivatives 21 and 35 (MIC 5.4
Chemistry
The target compounds 6-26, 28-37, and 47 were synthe-
sized by employing straightforward and efficient Wittig pro-
tocols (Scheme 1). (3-Ethoxycarbonyl-5-isoxazolylmethyl)tri-
phenylphosphonium bromide19 (5) and (5-isoxazolylmethyl)-
triphenylphosphonium bromide20 (51) were prepared as

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6289
Chart 1. Structures of the Anti-TB Derivatives Synthesized in This Study
a cis-isomer.
and 2.5 μM, respectively), both of which showed comparable
activity to that of 18. The dichloro substitution in the ortho
position of the pyridine ring produced the most potent
derivative in the series against R-TB (compound 30, MIC
0.59 μM). The replacement of the chlorine atoms with fluorine
at the benzene ring led to derivatives with the same range of
activities asthe dichlorosubstituted analogues, compounds12
and 19b having an MIC of 5.3 and 3.7 μM, respectively.
Introduction of small alkyl groups to the benzene ring was
generally well tolerated (14 and 15), with the ortho-ethyl
group producing the best activity (14, MIC 0.98 μM).
Conversely, the meta-substituted toluene derivative 17
(MIC 6.5 μM) was found to be one of the least potent
compounds in the series. Derivative 13, bearing a chlorine,
a fluorine, and a methyl group at the benzene ring, had an
MIC of 3.5 μM, with a difficult assessment of its colloca-
tion within the SAR. With methoxy substitution, in both
the benzene and in the pyridine ring, a good activity was
still maintained. The 6-methoxypyridine derivative 29
(MIC 1.4 μM) was 3-fold more potent than the correspond-
ing regioisomer 34b (MIC 3.8 μM). Monomethoxy sub-
stitution at the meta position of the benzene ring was well
tolerated (26, MIC 3.7 μM), and the introduction of an
additional methoxy group at the other meta position of the
benzene ring (16, MIC 2.0 μM) did not significantly alter
the potency. Derivative 20 (MIC 0.96 μM), bearing an
ortho-methylthio group, also showed good potency. Next,
bulkier substituents were introduced in order to investigate
the presence of lipophilic pockets in the binding site. When
a second aromatic ring was attached to the phenyl group,
either directly (compound 33, MIC 2.0 μM) or via an
oxymethylene linker (compound 25, MIC 1.7 μM), the
activity was retained. However, the dibenzyloxy substi-
tuted derivative 22 (MIC 6.3 μM) was found to be 4-fold
less active than the corresponding monosubstituted com-
pound. In addition to the pyridine and benzene rings, five-
membered heterocycles were investigated. The thiophene
derivative 31 and the furan derivative 32 both showed good
activity. In particular, 31 (MIC 1.6 μM) was found to be
only 3-fold less active than the most potent compound 30.
As expected, more polar compounds, such as the amino-
derivative 27, the phenol 28, and much more markedly, the
N-methylpiperazine derivative 37, showed lower activities
with MICs of 7.5, 7.8, and 15.2 μM, respectively.

6290 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Pieroni et al.
Scheme 1a,b
a Reagents and conditions: (a) Ethyl 2-chloro-2-(hydroxyimino)acetate, triethylamine, anhydrous Et2O, 10 h through syringe pump, rt (58%);
(b) NBS, benzoylperoxide (cat.), anhydrous CH2Cl2, 3 h, 80 °C (68%); (c) Ph3P, MeCN, reflux, 8 h (95%); (d) R1CHO, Na[(CH3)3Si]2N, anhydrous
THF, -78 °C to rt, overnight (15-73%) or R1CHO, NaH, anhydrous CH2Cl2, 0 °C to rt, overnight, (49-61%); (e) LiOH, THF-MeOH-H2O 3:1:1, rt,
1 h; (f) SnCl2, EtOH, reflux, 3 h (51%); (g) Pd/C 5%, H2, EtOH, rt, 1 h (76%); (h) Ti(EtO)4 (cat), R2OH, neat, 80 °C, 3 h (59-89%).
b For complete structures, see Chart 1.
Table 1. Anti-TB Activity of Compounds 6-49 against M. tuberculosis Strain H37Rv
MABA
MIC (μM)
LORA
MIC (μM)
Vero cells
IC50 (μM)
MABA
MIC (μM)
LORA
MIC (μM)
Vero cells
IC50 (μM)
compd
ClogPa
compd
ClogPa
6
1.6
15.5
11.7
0.73
3.9
4.0
52.4
18.0
21.1
6.8
>128
>128
ndb
4.8
2.1
3.6
4.5
4.5
4.5
3.9
4.9
4.6
5.0
3.6
4.1
4.3
3.9
3.9
4.1
5.0
7.1
5.3
3.3
3.3
5.3
3.5
2.4
2.3
2.9
30
31
0.59
1.6
5.1
26.9
50.2
12.3
45.9
16.0
3.5
>128
>128
>128
113
nd
3.1
3.2
2.8
4.2
2.9
2.9
5.0
3.8
2.7
4.2
3.9
4.6
4.0
5.0
6.8
6.6
6.3
4.8
2.8
4.0
1.8
7
8
32
33
7.9
2.0
9
>128
nd
nd
10
11
12
13
14
15
16
17
18
19a
19b
20
21
22
23
24a
24b
25
26
27
28
29
34a
34b
35
27.5
3.8
3.9
5.3
12.1
13.8
7.0
nd
nd
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
2.5
3.8
3.5
36
37
10.7
37.4
>128
>128
7.6
nd
nd
0.98
3.1
2.0
8.3
15.2
>128
>128
1.8
38
39
nd
nd
2.0
6.5
3.6
7.4
40
41
nd
nd
2.3
6.7
2.0
7.9
6.7
8.9
29.2
3.7
12.3
8.9
42
43
>128
>128
>128
nd
10.9
8.3
9.2
0.96
5.4
7.8
5.9
44
45
24.2
9.3
7.3
1.1
6.3
4.7
3.6
46
47
1.8
>128
nd
nd
17.3
5.4
>128
14.2
>128
0.1
>128
15.6
>128
1.9
10.9
5.1
48
49
1.2
1.7
nd
9.9
8.8
RMP
INH
MOX
127
128
3.7
7.5
0.84
0.46
>128
>128
20.7
28.7
86.6
7.8
1.4
a Calculated with ChemDraw Ultra 7.0, CambridgeSoft. b Not determined
The above results suggest that the overall lipophilicity, more
than the position of the substituents at the ring, may be the
main variable responsible for the achievement of satisfactory
anti-TB activity. The cis-isomers were found to be less potent
than the corresponding trans-isomers, the difference in activity
ranging from 4-fold (24a vs 24b) to 8-fold (19a vs 19b). The
reduction of the planar ethenyl moiety to an ethylene had
a detrimental effect on the potency. In fact, compound 48
(MIC 7.5 μM) was more than 20-fold less active that the
corresponding unsaturated derivative 9. The dihydroxy deri-
vative 49 did not show any anti-TB activity, however, the loss
of activity may be due to its more polar character that may
prevent penetration through the thick and greasy Mtb cell wall.
Finally, more hindered esters, which could exhibit improved
cellular penetration due to increased lipophilicity, were inves-
tigated. The n-butyl ester 40 (MIC 1.8 μM) was found to be

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6291
slightly more active than the parent compound 26, whereas 41
(MIC 2.0 μM) had a potency comparable to the corresponding
ethyl ester 29. Compound 46, derived from the ethyl ester 2, was
less active than the parent compound. Further elongation of the
ester alkyl chain, as for compounds 42-45, led to a pronounced
decrease in potency along with an unfavorable increase in
ClogP. The acid derivatives 38 and 39, and the derivative 47,
which lack the carboxylic acid moiety on the isoxazole ring,
showed no anti-TB activity. It is possible that the acid, resulting
from the hydrolysis of the ester, may be the species responsible
for the anti-TB activity;24 however, the marked polar character
of the acid may prevent its penetration through the cell wall. On
the other hand, the lack of activity of 47 may be due to the
absence of a carboxylic acid precursor, demonstrating that this
moeity may represent a crucial element for anti-TB activity.
With the exceptions of derivatives 23 and 43, the MICs in
LORA were found to be higher than those in MABA,
although in the low micromolar range (<10 μM) for almost
half of the compounds. With few exceptions, the overall SAR
outlined for MABA activity may, to a certain extent, be
applied to activity against NRP-TB. In fact, the most active
compounds in LORA, namely 46 (LORA MIC 1.8 μM), 14
(LORA MIC 2.0 μM), 35 (LORA MIC 3.5 μM), and 16
(LORA MIC 3.6 μM), were also found to be among the most
potent compounds in MABA. The dichloro-substituted pyr-
idine 30, which in the MABA assay had the best MIC, also
maintained satisfactory activity in LORA. Compounds 9 and
29, which showed good activity in MABA, failed to maintain
good activity in LORA with MICs that were 27-fold and
62-fold higher than those exhibited against R-TB, respec-
tively. In contrast to the general trend, some of the more
hindered esters, namely 42, 43, and 45, exhibited very similar
activities both in MABA and in LORA. Surprisingly, 23 was
5-fold more potent against the NRP-TB than the R-TB.
Next, the selectivity of the two most promising compounds
against Mtb was evaluated by testing their activity against
three other organisms. Compounds 9 and 30 failed to inhibit
the growth of a Gram positive microorganism (Staphylococcus
aureus), a Gram negative microorganism (Escherichia coli), a
fungus (Candida albicans), and another mycobacterium
(Mycobacterium smegmatis), thus indicating selectivity toward
Mtb (Table 2, Supporting Information). Compound 30 was
also evaluated for its ability to induce mutations in Mtb at a
concentration 4-fold higher than its MIC. The frequency of
mutation to resistance was found to be 1.6 ꢀ 10-7, similar to
that shown by RMP and superior to that of INH. With the
exception of 33 (IC50 113 μM), the compounds synthesized in
this study did not show toxicity to Vero cells up to 128 μM test
concentration (Table 1).
active against both R-TB and NRP-TB. Most of the synthe-
sized derivatives exhibited good activity, with several com-
pounds being active in the submicromolar range against
R-TB and in the low micromolar range against NRP-TB.
5-[(E)-2-(3,5-Dichloro-4-pyridinyl)ethenyl]-3-isoxazolecarboxylic
acid ethyl ester (30) was the most active compound in the series
against R-TB (MABA MIC 0.59 μM), whereas 5-[(E)-2-(6-
methoxy-4-quinolinyl)ethenyl]-3-isoxazolecarboxylic acid butyl
ester (46) showed the best activity against NRP-TB (LORA
MIC 1.8 μM). These MIC values are slightly higher than those
of RMP, a first line anti-TB drug, and comparable or better
than those of other anti-TB agents currently marketed or in
clinical trials. Moreover, 30 and 46, as almost all of the
derivatives synthesized in this study, are devoid of toxicity
to Vero cells and possess overall satisfactory drug-like proper-
ties. The SAR from the series provided interesting insights
and, in general, all the compounds endowed with a higher
ClogP value, a feature likely topromoteMtb cellpermeability,
were found to show good anti-TB activity. Therefore halo-
gens, small alkyl appendages, bulkier aromatic and hetero-
aromatic rings, and methoxy groups were all found to be
suitable substituents in order to obtain good anti-TB proper-
ties. The more hydrophilic substituents, i.e., hydroxyl, amino,
and N-methylpiperazine, had a detrimental effect on the
activity. The stereochemistry of the alkene linker played an
important role, with a 4-8-fold difference in potency between
the more active trans- and the less active cis-isomers. More-
over, the reduction of the double bond led to a marked
decrease of potency. The n-butyl ester derivatives were found
to have good anti-TB potency, especially against NRP-TB,
while bulkier alkyl esters had an unfavorable impact on the
activity. Finally, hydrolysis of the ester or the removal of the
carboxylic function led to a loss of activity. These findings
provide significant information about the 5-[(E)-2-aryl-
ethenyl]-3-isoxazolecarboxylic acid alkyl esters as a valuable
lead scaffold for the development of new anti-TB drugs active
against both R-TB and NRP-TB. Further studies on this
promising anti-TB phenotype are currently underway in our
laboratory.
Experimental Section
Biology. The MICs were determined using Mtb H37Rv ATCC
27294 in MABA21 and LORA22 assays according to published
procedures. Reported MICs are an average of three individual
measurements. For a description of the biological, assays see
Supporting Information.
Chemistry. 1H NMR and 13C NMR spectra were recorded on
a Bruker spectrometer at 400 and 100 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. Standard abbreviation indicating multiplicity was
used as follows: s = singlet, d = doublet, dd = doublet of
doublets, t = triplet, q = quadruplet, m = multiplet, and br =
broad. HRMS experiments were performed on Q-TOF-2TM
(Micromass). TLC was performed with Merck 60 F254 silica gel
plates. Column chromatography was performed using Combi-
Flash Rf system with RediSep columns or alternatively using
Merck silica gel (40-60 mesh). Preparative HPLC was carried
out on a Shimadzu SCL-10A VP instrument with an ACE 5-AQ
(21.2 mm ꢀ 150 mm) column. Analytical HPLC was carried out
on Agilent 1100 HPLC system with a Synergi 4 μ Hydro-RP 80A
column, on a variable wavelength detector G1314A. Method 1:
flow rate = 1.4 mL/min; gradient eluation over 20 min, from
30% CH3CN-H2O to 100% CH3CN with 0.05% TFA. Meth-
od 2: flow rate = 1.4 mL/min; gradient eluation over 20 min,
from 10% CH3CN-H2O to 100% CH3CN with 0.05% TFA.
Our previous studies have indicated that the 3-isoxazole-
carboxylic moiety is crucial for the anti-TB activity. Inhibition
of the Mtb virulence factor protein tyrosine phosphatase
(MptpB) by isoxazole-3-carboxylic acid derivatives has been
reported.25 Thus, if indeed the ester moiety acts a prodrug for
the corresponding acid, our compounds may be ligands for
MptpB. However, MptpB is not essential for the survival of
Mtb in vitro. Hence, the mechanism by which these the 5-[(E)-
2-arylethenyl]-3-isoxazolecarboxylic acid alkyl esters exhibit
their activity against R-TB and NRP-TB remains unknown.
Conclusions
Utilizing simple and flexible synthetic chemistry protocols,
we have built a small library of 5-[(E)-2-arylethenyl]-3-iso-
xazolecarboxylic acid alkyl esters as anti-TB compounds

6292 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Pieroni et al.
Method 3: flow rate = 1.4 mL/min; gradient eluation over
20 min, from 50% CH3CN-H2O to 70% CH3CN with 0.05%
TFA. The purity of the target compounds was determined to be
>95% by analytical HPLC.
(q, J = 7.2 Hz, 2H), 6.71 (s, 1H), 6.85-6.98 (m, 2H), 7.04 (d, J =
16.8 Hz, 1H), 7.45 (d, J = 16.8 Hz, 1H), 7.50-7.62 (m, 1H). 19
F
NMR (CDCl3) δ -111.9 (d, J = 8.0 Hz), -108.1 (d, J = 8.0 Hz).
HRMS (ESI) calculated for C14H11F2NO3 [M þ H]þ 280.0779,
found 280.0793.
General Procedure for the Synthesis of 6-26, 28-37, and 47.
To a stirred mixture of (3-ethoxycarbonyl-5-isoxazolylmethyl)tri-
phenylphosphonium bromide19 (5) (1 equiv) or (5-bromomethy-
lisoxazole)triphenylphosphonium bromide20 (51) (1 equiv) in
anhydrous THF at -78 °C, sodiumbistrimethylsilylamide (1.05
equiv) was added dropwise. After 0.5 h, the appropriate aldehyde
(1 equiv) in anhydrous THF was added dropwise and the reaction
mixture was stirred at rt until consumption of the starting material
according to TLC. The mixture was cooled to 0 °C and sat. aq
NH4Cl (10 mL) was added, followed by removal of the solvent
under reduced pressure. H2O (20 mL) was added and the mixture
was extracted with chloroform (3 ꢀ 20 mL), and then the organic
layers were separated, washed with brine, dried over anhydrous
Na2SO4, filtered, and concentrated under reduced pressure. The
residue was purified by flash chromatography to obtain the
product. Analytical data for compounds 8,26 18,27 and 3228
matched the data previously published.
5-[(E)-2-(6-Chloro-2-fluoro-3-methylphenyl)ethenyl]-3-isoxa-
zolecarboxylic Acid Ethyl Ester (13). Purified by column chro-
matography (EtOAc-hexane 1:4) followed by recrystallization
1
with EtOH. Yield 47% (white powder). H NMR (CDCl3) δ
1.45 (t, J = 7.2 Hz, 3H), 2.28 (s, 3H), 4.47 (q, J = 7.2 Hz, 2H),
6.74 (s, 1H), 7.05-7.18 (m, 2H), 7.31 (d, J = 16.8 Hz, 1H), 7.58
(d, J = 16.8 Hz, 1H). 19F NMR (CDCl3) δ -113.4. HRMS (ESI)
calculated for C15H13ClFNO3 [M þ H]þ 310.0640, found
310.0653.
5-[(E)-2-(2-Ethylphenyl)ethenyl]-3-isoxazolecarboxylic Acid Ethyl
Ester (14). Purified by column chromatography (EtOAc-hexane
1:9). Yield 46% (white powder). 1H NMR (CDCl3) δ 1.26 (t, J =
7.6 Hz, 3H), 1.45 (t, J = 7.2 Hz, 3H), 2.82 (q, J = 7.6 Hz, 2H), 4.81
(q, J = 7.2 Hz, 2H), 6.68 (s, 1H), 6.91 (d, J = 16.2 Hz, 1H),
7.22-7.36 (m, 3H), 7.59 (d, J = 7.2 Hz, 1H), 7.65 (d, J = 16.2 Hz,
1H). 13C NMR (CDCl3) δ 13.8, 15.3, 26.0, 61.8, 101.1, 113.1, 125.4,
126.0, 128.9, 129.2, 133.0, 133.2, 142.8, 156.4, 159.7, 170.1. HRMS
(ESI) calculated for C16H17NO3 [M þ H]þ 272.1281, found
272.1285.
5-[(E)-2-(1-Naphthyl)ethenyl]-3-isoxazolecarboxylicAcidEthyl
Ester (6). Purified by column chromatography (EtOAc-hexane
1:4). Yield 51% (white powder). 1H NMR (CDCl3) δ 1.47 (t, J =
7.2 Hz, 3H), 4.95 (q, J = 7.2 Hz, 2H), 6.76 (s, 1H), 7.07 (d, J =
16.4 Hz, 1H), 7.50-7.65 (m, 3H), 7.99 (d, J = 7.2 Hz, 1H), 7.90
(d, J = 8.0 Hz, 2H), 8.20-8.27 (m, 2H). 13C NMR (CDCl3) δ
13.8, 61.8, 101.5, 114.5, 122.9, 123.8, 125.1, 125.9, 126.4, 128.4,
129.5, 130.9, 132.2, 132.7, 133.3, 156.4, 159.6, 169.9. HRMS(ESI)
calculated for C18H15NO3 [M þ Na]þ 316.0944, found 316.0955.
5-[(E)-2-(4-Pyridinyl)ethenyl]-3-isoxazolecarboxylic Acid Ethyl
Ester (7). Purified by column chromatography (EtOAc-hexane
3:7). Yield 49% (white powder). 1H NMR (CDCl3) δ 1.43 (t, J =
7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H), 6.77 (s, 1H), 7.16 (d, J =
5-[(E)-2-[3-(1-Methylethyl)phenyl]ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (15). Purified by column chromatography
(EtOAc-hexane 1:9). Yield 39% (white powder). 1H NMR
(CDCl3) δ 1.29 (d, J = 6.8 Hz, 6H), 1.45 (t, J = 7.2 Hz, 3H),
2.88-2.99 (m, 1H), 4.47 (q, J = 7.2 Hz, 2H), 6.68 (s, 1H), 6.99 (d,
J = 16.4 Hz, 1H), 7.22-7.28 (m, 1H), 7.30-7.45 (m, 4H). 13C
NMR (CDCl3) δ 13.8, 23.5, 33.7, 61.8, 101.0, 111.7, 124.4, 125.1,
127.5, 128.5, 134.7, 136.1, 149.2, 156.3, 159.7, 170.1. HRMS (ESI)
calculated for C17H19NO3 [M þ H]þ 286.1437, found 286.1442.
5-[(E)-2-(3,5-Dimethoxyphenyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (16). Purified by column chromatography
(EtOAc-hexane 1:4) followed by recrystallization with EtOH.
16.4 Hz, 1H), 7.30-7.40 (m, 3H), 8.66 (d, J = 6.0 Hz, 2H). 13
C
NMR (CDCl3) δ 13.8, 61.9, 102.9, 116.0, 120.8, 132.9, 141.8,
150.2, 156.5, 159.3, 168.5. HRMS (ESI) calculated for
C13H12N2O3 [M þ H]þ 245.0920, found 245.0922.
1
Yield 33% (white powder). H NMR (CDCl3) δ 1.45 (t, J =
7.2 Hz, 3H), 3.85 (s, 6H), 4.47 (q, J = 7.2 Hz, 2H), 6.48-6.50 (m,
1H), 6.69 (bds, 3H), 6.90 (d, J = 16.4 Hz, 1H), 7.34 (d, J =
16.4 Hz, 1H). 13C NMR (CDCl3) δ 13.8, 55.0, 61.8, 101.3, 101.4,
104.9, 112.4, 135.7, 136.7, 156.3, 159.6, 160.4, 171.4. HRMS
(ESI) calculated for C16H17NO5 [M þ H]þ 304.1179, found
304.1188.
5-[(E)-2-Phenylethenyl]-3-isoxazolecarboxylic Acid Ethyl Ester26
(8). Purified by column chromatography (EtOAc-hexane 1:4).
1
Yield 50% (white powder). H NMR (CDCl3) δ 1.45 (t, J =
7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H), 6.69 (s, 1H), 6.99 (d, J =
16.4 Hz, 1H), 7.33-7.45 (m, 4H), 7.55 (d, J = 7.2 Hz, 2H). 13
C
5-[(E)-2-(3-Methylphenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (17). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 45% (white powder). 1H NMR
(CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 2.40 (s, 3H), 4.47 (q, J =
7.2 Hz, 2H), 6.67 (s, 1H), 6.97 (d, J = 16.4 Hz, 1H), 7.19 (d, J =
7.2 Hz, 1H), 7.27-7.41 (m, 4H). 13C NMR (CDCl3) δ 13.8, 21.0,
61.8, 101.0, 111.7, 124.1, 127.6, 128.4, 130.0, 134.6, 135.9, 138.2,
156.3, 159.6, 170.0. HRMS (ESI) calculated for C15H15NO3
[M þ H]þ 258.1124, found 258.1135.
NMR (CDCl3) δ13.8, 61.8, 101.2, 111.9, 126.9, 128.6, 129.2, 134.7,
135.7, 156.3, 159.6, 169.9. HRMS (ESI) calculated for C14H14NO3
[M þ H]þ 244.0968, found 244.0974.
5-[(E)-2-(3-Trifluoromethylphenyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (9). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 46% (white crystals). 1H NMR
(CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H),
6.73 (s, 1H), 7.06 (d, J = 16.8 Hz, 1H), 7.44 (d, J = 16.8 Hz, 1H),
7.55 (t, J = 8.0 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.71 (d, J =
8.0 Hz, 1H), 7.79 (s, 1H). 13C NMR (CDCl3) δ 13.8, 61.9, 102.0,
113.6, 123.4 (q, J = 4 Hz), 123.5 (q, J = 271 Hz), 125.5 (q, J =
4 Hz), 129.1, 129.9, 131.1 (q, J = 32 Hz), 133.9, 135.5, 156.4, 159.5,
169.1. HRMS (ESI) calculated for C15H12F3NO3 [M þ H]þ
312.0842, found 312.0854.
5-[(E)-2-(3-Chlorophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (18).27 Purifiedbycolumnchromatography (EtOAc-
hexane 1:4) followed by recrystallization with EtOH. Yield 63%
(white powder). 1H NMR (CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H),
4.47 (q, J = 7.2 Hz, 2H), 6.70 (s, 1H), 6.99 (d, J = 16.4 Hz, 1H),
7.30-7.42 (m, 4H), 7.54 (bds, 1H). 13C NMR (CDCl3) δ 13.8,
61.9, 101.8, 113.2, 125.1, 126.6, 129.0, 129.8, 134.1, 134.6, 136.5,
156.4, 159.5, 169.2. HRMS (ESI) calculated for C14H12ClNO3
[M þ H]þ 278.0578, found 278.0588.
5-[(E)-2-(4-Trifluoromethylphenyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (10). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 39% (white powder). 1H NMR
(CDCl3) δ 1.45 (t, J = 6.8 Hz, 3H), 4.48 (q, J = 6.8 Hz, 2H),
6.75 (s, 1H), 7.06 (d, J = 16.8, 1H), 7.44 (d, J = 16.8 Hz, 1H),
7.65-7.69 (m, 4H). 13C NMR (CDCl3) δ 13.8, 61.9, 102.2, 114.2,
123,5 (q, J = 270 Hz), 125.6 (q, J = 4 Hz), 127.0, 130.8 (q, J =
40 Hz), 133.9, 138.1, 156.4, 159.5, 169.0. HRMS (ESI) calculated
for C15H12F3NO3 [M þ H]þ 312.0842, found 312.0853.
5-[(Z)-2-(3,5-Difluorophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (19a). Purified by preparative HPLC. Yield 21% (white
powder). 1H NMR(CDCl3) δ1.41(t, J = 7.2 Hz, 3H), 4.43 (q, J =
7.2 Hz, 2H), 6.48 (s, 1H), 6.59 (d, J = 12.4 Hz, 1H), 6.78-6.93 (m,
4H). 19F NMR (CDCl3) δ -109.9. HRMS (ESI) calculated for
C14H11F2NO3 [M þ H]þ 280.0779, found 280.0794.
5-[(E)-2-(2,4-Difluorophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (12). Purified by column chromatography (EtOAc-
hexane 1:4) and recrystallized by EtOH. Yield 39% (white
5-[(E)-2-(3,5-Difluorophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (19b). Purified by preparative HPLC. Yield 29% (white
powder). 1HNMR(CDCl3) δ1.45 (t, J= 7.2 Hz, 3H), 4.47 (q, J=
1
powder). H NMR (CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.47

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6293
7.2 Hz, 2H), 6.73 (s, 1H), 6.78-6.84 (m, 1H), 6.98 (d, J = 16.4 Hz,
1H), 7.03-7.08 (m, 2H), 7.32 (d, J = 16.4 Hz, 1H). 19F NMR
(CDCl3) δ -110.0. HRMS (ESI) calculated for C14H11F2NO3
[M þ H]þ 280.0779, found 280.0793.
5-[(E)-2-(3-Methoxyphenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (26). Purified by column chromatography (EtOAc-
hexane 1:4) followed by recrystallization with EtOH. Yield 51%
(whitepowder). 1H NMR (CDCl3) δ 1.45 (t, J = 7.2Hz, 3H), 3.87
(s, 3H), 4.47 (q, J = 7.2 Hz, 2H), 6.69 (s, 1H), 6.93 (dd, J1 = 2 Hz,
J2 = 8.0 Hz, 1H), 6.97 (d, J = 16.4 Hz, 1H), 7.06 (bds, 1H), 7.14
(d, J = 7.2 Hz, 1H), 7.30-7.41 (m, 2H). 13C NMR (CDCl3) δ
13.8, 61.8, 101.6, 104.3, 111.8, 114.0, 119.2, 127.5, 129.0, 156.4,
159.4, 159.6, 161.8, 164.1, 169.8. HRMS (ESI) calculated for
C15H15NO4 [M þ H]þ 274.1073, found 274.1084.
5-[(E)-2-(2-Methylthiophenyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (20). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 45% (pale-yellow powder). 1H
NMR (CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 2.50 (s, 3H), 4.46
(q, J = 7.2 Hz, 2H), 6.73 (s, 1H), 6.93 (d, J = 15.6 Hz, 1H),
7.16-7.34 (m, 3H), 7.57 (d, J = 1.8 Hz, 1H), 7.87 (d, J = 15.6
Hz, 1H). 13C NMR (CDCl3) δ 13.8, 16.3, 61.8, 101.2, 113.6,
125.4, 125.9, 127.2, 129.3, 132.8, 134.0, 138.2, 156.3, 159.6,
170.1. HRMS (ESI) calculated for C15H15NO3S [M þ H]þ
290.0845, found 290.0859.
5-[(E)-2-(3-Hydroxyphenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (28). Purified by column chromatography (EtOAc-
1
hexane 1:4). Yield 39% (white powder). H NMR (CD3OD) δ
1.41 (t, J = 7.2 Hz, 3H), 4.43 (q, J = 7.2 Hz, 2H), 6.77-6.87 (m,
2H), 7.00-7.17 (m, 3H), 7.22 (t, J = 8.0 Hz, 1H), 7.39 (d, J =
16.4 Hz, 1H). 13C NMR (CD3OD) δ 12.6, 61.4, 100.6, 111.5,
112.8, 116.0, 118.2, 129.2, 135.8, 136.2, 156.3, 157.3, 159.4, 170.4.
HRMS (ESI) calculated for C14H13NO4 [M þ H]þ 260.0917,
found 260.0929.
5-[(E)-2-(3,5-Dichlorophenyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (21). Purified by column chromatography
1
(EtOAc-hexane 1:4). Yield 73% (white powder). H NMR
(CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.48 (q, J = 7.2 Hz, 2H),
6.72 (s, 1H), 6.99 (d, J = 16.0 Hz, 1H), 7.24-7.42 (m, 4H). 13C
NMR (CDCl3) δ 13.8, 61.9, 102.4, 114.4, 125.1, 128.7, 132.7,
135.2, 137.7, 156.5, 159.4, 168.7. HRMS (ESI) calculated for
5-[(E)-2-(6-Methoxy-3-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid] Ethyl Ester (29). Purified by column chromatography
(EtOAc-hexane 1:4) followed by recrystallization with EtOH.
C
14H11NO3Cl2 [M þ H]þ 312.0188, found 312.0197.
1
Yield 46% (white powder). H NMR (CDCl3) δ 1.44 (t, J =
5-[(E)-2-[3,5-Bis(phenylmethoxy)phenyl]ethenyl]-3-isoxazolecar-
7.2 Hz, 3H), 3.99 (s, 3H), 4.47 (q, J = 7.2 Hz, 2H), 6.66 (s, 1H),
6.80 (d, J = 8.8 Hz, 1H), 6.87 (d, J = 16.4 Hz, 1H), 7.35 (d, J =
16.4 Hz, 1H), 7.80 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H) 8.28 (d, J =
2.4 Hz, 1H). 13C NMR (CDCl3) δ 13.8, 53.4, 61.8, 101.0, 111.0,
111.2, 124.1, 132.0, 135.2, 147.0, 156.4, 159.6, 164.4, 169.7. HRMS
(ESI) calculated for C14H14N2O4 [M þ H]þ 275.1026, found
275.1037.
boxylic Acid Ethyl Ester (22). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 29% (white powder). 1H NMR
(CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H),
5.00 (s, 4H), 6.64-6.70 (m, 2H), 6.78 (d, J = 1.8 Hz, 1H), 6.93 (d,
J = 16.0 Hz, 1H), 7.28-7.48 (m, 11H). 13C NMR (CDCl3) δ 13.8,
61.8, 69.8, 101.4, 103.0, 106.1, 112.5, 127.1, 127.7, 128.3, 135.6,
136.2, 136.7, 156.5, 159.6, 159.8, 169.7. HRMS (ESI) calculated for
C28H25NO3 [M þ H]þ 456.1805, found 456.1810.
5-[(E)-2-(3,5-Dichloro-4-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (30). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 50% (white powder). 1H NMR
(CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.48 (q, J = 7.2 Hz, 2H),
6.85 (s, 1H), 7.40 (d, J = 17.2 Hz, 1H), 7.47 (d, J = 17.2 Hz, 1H),
8.56 (s, 2H). 13C NMR (CDCl3) δ 13.8, 62.0, 103.8, 122.6, 126.4,
130.8, 138.5, 148.0, 156.6, 159.2, 168.0. HRMS (ESI) calculated for
C13H10Cl2N2O3 [M þ H]þ 313.0141, found 313.0152.
5-[(E)-2-(4-Methyl-1-naphthyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (23). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 37% (white powder). 1H NMR
(CDCl3) δ 1.47 (t, J = 7.2 Hz, 3H), 2.75 (s, 3H), 4.49 (q, J =
7.2 Hz, 2H), 6.73 (s, 1H), 7.03 (d, J = 16.0 Hz, 1H), 7.37 (d, J =
7.2 Hz, H) 7.50-7.74 (m, 3H), 8.01-8.08 (m, 1H), 8.21-8.25 (m,
2H). 13C NMR (CDCl3) δ 13.8, 19.2, 61.6, 102.4, 116.3, 124.4,
125.4, 125.7, 125.8, 125.9, 126.0, 130.5, 130.9, 132.4, 135.4,
135.8, 155.8, 159.6, 169.0. HRMS (ESI) calculated for
C19H17NO3 [M þ H]þ 308.1281, found 308.1296.
5-[(E)-2-(3-Thiophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (31). Purified by column chromatography
(EtOAc-hexane 1:4) followed by recrystallization with
1
EtOH. Yield 59% (white powder). H NMR (CDCl3) δ 1.44
5-[(Z)-2-(3-Nitrophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (24a). Purified by preparative HPLC. Yield 15%
(white powder). 1H NMR (CDCl3) δ 1.41 (t, J = 7.2 Hz, 3H),
4.46 (q, J = 7.2 Hz, 2H), 6.48 (s, 1H), 6.67 (d, J = 12.4 Hz, 1H),
6.97 (d, J = 12.4 Hz, 1H), 7.56 (t, J = 7.6 Hz, 1H), 7.73 (d, J =
7.6 Hz, 1H), 8.22-8.24 (m, 2H). 13C NMR (CDCl3) δ 13.7, 61.9,
99.6, 103.9, 116.2, 123.1, 123.2, 129.2, 133.7, 133.9, 146.2, 156.0,
159.8, 168.8. HRMS (ESI) calculated for C14H12N2O5 [M þ H]þ
289.0819, found 289.0824.
5-[(E)-2-(3-Nitrophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (24b). Purified by preparative HPLC. Yield 29%
(white powder). 1H NMR (CDCl3) δ 1.46 (t, J = 7.2 Hz, 3H),
4.48 (q, J = 7.2 Hz, 2H), 6.77 (s, 1H), 7.13 (d, J = 16.4 Hz,
1H), 7.47 (d, J = 16.4 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.84
(d, J = 8.0 Hz, 1H), 8.21-8.23 (m, 1H), 8.42 (brs, 1H). 13C
NMR (CDCl3) δ 14.4, 62.4, 103.0, 115.1, 121.5, 123.8, 130.0,
133.0, 133.2, 136.9, 148.0, 156.9, 159.8, 169.0. HRMS (ESI)
calculated for C14H12N2O5 [M þ H]þ 289.0819, found
289.0829.
(t, J = 7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H), 6.64 (s, 1H), 6.81
(d, J = 16.4 Hz, 1H) 7.33-7.46 (m, 4H). 13C NMR (CDCl3) δ
13.8, 61.8, 100.9, 111.8, 124.2, 125.8, 126.6, 129.6, 137.8,
156.3, 159.6, 170.0. HRMS (ESI) calculated for C14H14N2O4
[M þ H]þ 275.1026, found 275.1037.
5-[(E)-2-(2-Furanyl)ethenyl]-3-isoxazolecarboxylic Acid Ethyl
Ester28 (32). Purified by column chromatography (EtOAc-
hexane 1:4) followed by recrystallization with EtOH. Yield
1
49% (pale-yellow powder). H NMR (CDCl3) δ 1.44 (t, J =
6.8 Hz, 3H), 4.46 (q, J = 6.8 Hz, 2H), 6.49 (dd, J1 = 2 Hz, J2 =
3.2 Hz, 1H), 6.63 (s, 1H), 6.87 (d, J = 16.0 Hz, 1H), 7.17 (d, J =
16.0 Hz, 1H), 7.49 (s, 1H). 13C NMR (CDCl3) δ 13.8, 61.8, 101.3,
109.8, 111.9, 112.6, 122.4, 143.6, 150.9, 156.4, 159.6, 169.6.
HRMS (ESI) calculated for C12H11NO4 [M þ H]þ 234.0760,
found 234.0769.
5-[(E)-2-[4-(2-Pyridinyl)phenyl]ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (33). Purified by column chromatography
(EtOAc-hexane 1:4). Yield 48% (white powder). 1H NMR
(CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H),
6.70 (s, 1H), 7.05 (d, J = 16.4 Hz, 1H), 7.20-7.30 (m, 1H), 7.45
(d, J = 16.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.76-7.80 (m,
2H), 8.06 (d, J = 8.4 Hz, 2H), 8.72 (d, J = 4.4 Hz, 1H). 13C
NMR (CDCl3) δ 13.8, 61.8, 101.3, 112.3, 120.1, 122.1, 127.0,
127.3, 135.1, 135.2, 136.4, 139.9, 149.4, 156.0, 156.4, 159.6,
169.8. HRMS (ESI) calculated for C19H16N2O3 [M þ H]þ
321.1233, found 321.1242.
5-[(E)-2-[(3-Phenylmethoxy)phenyl]ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (25). Purified by column chromatography
(EtOAc-hexane 1:4) followed by recrystallization with EtOH.
1
Yield 41% (white powder). H NMR (CDCl3) δ 1.45 (t, J =
7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H), 5.12 (s, 2H), 6.68 (s, 1H),
C
6.92-7.01 (m, 2H), 7.14-7.16 (m, 2H), 7.28-7.48 (m, 7H). 13
NMR (CDCl3) δ 13.8, 61.8, 69.7, 101.2, 112.3, 113.0, 115.7, 119.8,
127.1, 127.7, 128.3, 129.6, 135.6, 136.1, 136.3, 156.3, 158.8, 159.6,
169.8. HRMS (ESI) calculated for C21H19NO4 [M þ H]þ
350.1386, found 350.1390.
5-[(Z)-2-(6-Methoxy-2-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (34a). Purified by preparative HPLC. Yield 19%

6294 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Pieroni et al.
(white powder). 1H NMR (CDCl3) δ 1.42 (t, J = 7.2 Hz, 3H), 3.91
(s, 3H), 4.42 (q, J = 7.2 Hz, 2H), 6.59 (d, J = 12.8 Hz, 1H),
6.71-6.78 (m, 2H), 6.97 (d, J = 7.2 Hz, 1H), 7.37 (s, 1H),
7.55-7.62 (m, 1H). 13C NMR (CDCl3) δ 13.7, 53.5, 61.7, 105.4,
111.2, 115.1, 118.6, 134.3, 138.4, 151.0, 155.9, 159.7, 163.3, 169.0.
HRMS (ESI) calculated for C14H14N2O4 [M þ H]þ 275.1026,
found 275.1039.
5-[(E)-2-(1-Naphthyl)ethenyl]-3-isoxazolecarboxylic Acid (38).
Toa stirred solutionof6 (20mg, 0.31 mmol) ina mixture ofTHF-
H2O-MeOH 3:1:1 (4 mL), LiOH H2O (8.9 mg, 1.23 mmol) was
3
added portionwise. After 30 min at rt, the mixture was concen-
trated under reduced pressure to evaporate the organic layer and
then 0.1N HCl was cautiously added dropwise until a precipitate
appeared. The precipitate was collected to give the title com-
pound 38 (Yield 93%) as a white solid without further purifica-
tion. 1H NMR(CD3OD) δ 6.95 (s, 1H), 7.25 (d, J =16.2Hz, 1H),
5-[(E)-2-(6-Methoxy-2-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (34b). Purified by preparative HPLC. Yield 29%
(white powder). 1H NMR (CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.02
(s, 3H), 4.47 (q, J = 7.2 Hz, 2H), 6.70-6.75 (m, 2H), 6.94 (d, J =
7.51-7.63 (m, 3H), 7.85-7.93 (m, 3H), 8.25-8.34 (m, 2H). 13
C
NMR (CD3OD) δ 102.2, 116.2, 123.5, 124.7, 125.2, 125.6, 125.8,
127.9, 128.2, 130.3, 133.0, 133.4, 135.4, 156.3, 160.6, 169.0.
HRMS (ESI) calculated for C16H11NO3 [M þ H]þ 266.0811,
found 266.0820.
6.8 Hz, 1H), 7.33 (d, J = 16.4 Hz, 1H), 7.55-7.62 (m, 1H). 13
C
NMR (CDCl3) δ 13.8, 52.9, 61.8, 102.3, 111.6, 115.0, 117.3, 134.1,
138.7, 150.2, 156.4, 159.6, 163.3, 169.7. HRMS (ESI) calculated
for C14H14N2O4 [M þ H]þ 275.1026, found 275.1039.
5-[(E)-2-(3-Trifluoromethylphenyl)ethenyl]-3-isoxazolecarboxylic
Acid (39). Synthesized as 38, except 9 was used as a starting
material. Yield 86% (white solid). 1H NMR (CD3OD) δ 6.89 (s,
1H), 7.34 (d, J = 16.4 Hz, 1H), 7.53 (d, J = 16.8 Hz, 1H),
7.59-7.66 (m, 2H), 7.88-7.94 (m, 2H). 13C NMR (CD3OD) δ
102.2, 116.2, 123.5, 124.7, 125.2, 125.6, 125.8, 127.9, 128.2, 130.3,
133.0, 133.4, 135.4, 156.3, 160.6, 169.0. HRMS (ESI) calculated for
C13H8F3NO3 [M þ H]þ 284.0529, found 284.0525.
5-[(E)-2-(2,6-Dichlorophenyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (35). Purified by column chromatography
1
(EtOAc-hexane 1:4). Yield 41% (white powder). H NMR
(CDCl3) δ 1.45 (t, J = 7.2 Hz, 3H), 4.47 (q, J = 7.2 Hz, 2H),
6.77 (s, 1H), 7.16-7.22 (m, 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.51
(d, J = 16.8 Hz, 1H). 13C NMR (CDCl3) δ 13.8, 61.9, 102.3,
120.3, 128.5, 129.1, 129.3, 131.9, 134.4, 156.4, 159.5, 169.0.
HRMS (ESI) calculated for C14H11Cl2NO3 [M þ H]þ
312.0188, found 312.0199.
General Procedure for the Synthesis of Esters 40-46. Under
nitrogen atmosphere, the ethyl ester derivative (1 equiv), the
appropriate alcohol (10 equiv) and Ti(EtO)4 (10% mol) were
stirred at 80 °C until consumption of the limiting reagent. After
the addition of H2O, the mixture was extracted with CHCl3 and
then the organic layer was separated, washed with brine, dried
over Na2SO4 anhydrous, filtered, and concentrated under re-
duced pressure. The residue was purified by flash chromatog-
raphy (EtOAc-hexane 1:4) to give the product.
5-[(E)-2-(2-Fluoro-4-methoxyphenyl)ethenyl]-3-isoxazolecar-
boxylic Acid Ethyl Ester (36). Purified by column chromatog-
raphy (EtOAc-hexane 0.5:4.5). Yield 25% (white powder). 1H
NMR (CDCl3) δ 1.44 (t, J = 7.2 Hz, 3H), 3.84 (s, 3H), 4.47 (q,
J = 7.2 Hz, 2H), 6.66-6.70 (m, 2H), 6.75 (dd, J1 = 2 Hz, J2 =
8.4 Hz, 1H) 6.97 (d, J = 16.8 Hz, 1H), 7.42-7.49 (m, 2H). 13
C
NMR (CDCl3) δ 13.8, 55.3, 61.8, 100.8, 101.4, 110.4, 112.0,
115.3, 128.4, 128.7, 156.3, 159.9, 161.5, 162.8, 170.2. HRMS
(ESI) calculated for C15H14FNO4 [M þ H]þ 292.0979, found
292.0985.
5-[(E)-2-(3-Methoxyphenyl)ethenyl]-3-isoxazolecarboxylic Acid
Butyl Ester (40). Synthesized from 26. Yield 83% (white powder).
1H NMR (CDCl3) δ 0.99 (t, J = 7.2 Hz, 3H), 1.43-1.54 (m, 2H),
1.76-1.83 (m, 2H), 3.86 (s, 3H), 4.44 (t, J = 7.2 Hz, 2H), 6.68 (s,
1H), 6.93 (dd, J1 =2.0Hz, J2 = 8.0Hz, 1H), 6.97 (d, J = 16.4 Hz,
1H), 7.06 ( br s, 1H), 7.14 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 8.0 Hz,
1H), 7.38 (d, J = 16.4 Hz, 1H). 13C NMR (CDCl3) 13.4, 18.7,
30.1, 54.9, 65.6, 101.2, 112.0, 112.2, 114.9, 119.5, 129.6, 135.6,
136.1, 156.3, 159.6, 159.7, 169.8. HRMS (ESI) calculated for
C17H19NO4 [M þ H]þ 302.1386, found 302.1402.
5-[(E)-2-(3,5-Dichloro-4-pyridinyl)ethenyl]-isoxazole (47). Pur-
ified by column chromatography (EtOAc-hexane 3:7). Yield
38% (orange powder). 1H NMR (CDCl3) 6.53 (s, 1H), 7.39-7.47
(m, 2H), 8.31 (s, 1H), 8.55 (bds, 2H). 13C NMR (CDCl3) δ 103.1,
123.0, 125.3, 130.8, 139.0, 147.8, 150.4, 165.6. HRMS (ESI)
calculated for C10H6Cl2N2O [M þ H]þ 240.9929, found
240.9936.
5-[(E)-2-[4-(4-Methyl-1-piperazinyl)phenyl]ethenyl]-3-isoxazole-
carboxylic Acid Ethyl Ester (37). To a stirred solution of 3-ethyl-
isoxazolecarboxylate-5-methyltriphenylphosphonium bromide
5 (267 mg, 0.54 mmol) in anhydrous CH2Cl2 (12 mL), NaH
(82 mg, 3.43 mmol) was added at 0 °C. After 20 min, 4-(4-
methylpiperazin-1-yl)benzaldehyde (100 mg, 0.49 mmol) in
CH2Cl2 (2 mL) was added dropwise and the reaction mixture
was stirred at rt overnight. The mixture was quenched with H2O,
and the organic layer was separated, washed with brine, dried
over anhydrous Na2SO4, filtered, and concentrated. Purification
by flash chromatography (CH2Cl2-MeOH 9:1) gave the title
compound as a yellow powder. Yield 61%. 1H NMR (CDCl3) δ
1.44 (t, J = 7.2 Hz, 3H), 2.38 (s, 3H), 2.58-2.60 (t, 4H),
3.29-3.33 (t, 4H), 4.46 (q, J = 7.2 Hz, 2H), 6.60 (s, 1H), 6.81
(d, J = 16.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 16.4
Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H). 13C NMR (CDCl3) δ 13.8,
45.8, 47.6, 54.5, 61.7, 99.6, 99.5, 108.6, 114.8, 125.4, 128.2, 135.6,
151.5, 159.8, 170.7. HRMS (ESI) calculated for C19H23N3O3
[M þ H]þ 342.1812, found 342.1811.
5-[(E)-2-(6-Methoxy-3-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid Butyl Ester (41). Synthesized from 29. Yield 89% (white
powder). 1H NMR (CDCl3) δ 0.95-1.00 (m, 3H), 1.43-1.52 (m,
2H), 1.75-1.82, (m, 2H), 3.98 (s, 3H), 4.42 (t, J = 6.8 Hz, 2H),
6.65 (s, 1H), 6.80 (d, J = 4.4 Hz, 1H), 6.87 (d, J = 16.4 Hz, 1H),
7.34 (d, J = 16.4 Hz, 1H), 7.78-7.81 (m, 1H), 8.28 (br s, 1H). 13
C
NMR (CDCl3) 13.3, 18.7, 30.1, 53.4, 65.6, 101.0, 111.0, 111.2,
124.1, 132.0, 135.2, 147.0, 156.3, 159.7, 164.4, 169.7. HRMS (ESI)
calculated for C16H18N2O4 [M þ H]þ 303.1339, found 303.1351.
5-[(E)-2-(3,5-Dichloro-4-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid 3-Pentyl Ester (42). Synthesized from 30. Yield 59% (white
powder). 1H NMR (CDCl3) δ 0.98 (t, J = 7.2 Hz 6H), 1.74-1.79
(m, 4H), 5.09-5.12(m, 1H), 6.84(s, 1H), 7.39(d,J=16.8Hz, 1H),
7.47 (d, J = 16.8 Hz, 1H), 8.56 (s, 2H). 13C NMR (CDCl3) 9.24,
25.9, 37.6, 44.0, 79.2, 103.0, 122.6, 126.4, 130.7, 138.0, 147.2,
157.3, 159.2, 168.0. HRMS (ESI) calculated for C16H16Cl2N2O3
[M þ H]þ 355.0610, found 355.0620.
5-[(E)-2-(3,5-Dichloro-4-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid Octyl Ester (43). Synthesized from 30. Yield 81% (white
powder). 1H NMR (CDCl3) δ 0.89-0.91 (m, 3H), 1.28-1.50 (m,
10H), 1.78-1.83, (m, 2H), 4.42 (t, J = 6.8 Hz, 2H,), 6.84 (s, 1H),
7.39 (d, J = 16.8 Hz, 1H), 7.47 (d, J = 16.8 Hz, 1H), 8.56 (s, 2H).
13C NMR (CDCl3) 13.7, 22.2, 25.4, 28.1, 28.8, 28.9, 31.4, 66.1,
103.8, 122.6, 126.4, 130.8, 138.5, 148.0, 156.2, 159.3, 167.9. HRMS
(ESI) calculated for C19H22Cl2N2O3 [M þ H]þ 397.1080, found
397.1086.
5-[(E)-2-(2-Trifluoromethylphenyl)ethenyl]-3-isoxazolecarboxylic
Acid Ethyl Ester (11). Synthesized as 37, except 2-(trifluoromethyl)-
benzaldehyde was used as a starting material. Purified by prepara-
tive HPLC. Yield 49% (white powder). 1HNMR(CDCl3) δ1.45 (t,
J = 7.2 Hz, 3H), 4.48 (q, J = 7.2 Hz, 2H), 6.8 (s, 1H), 6.98 (d, J =
16.0 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H),
7.70-7.80 (m, 3H). 13C NMR (CDCl3) δ 13.8, 61.9, 102.0, 115.9,
123.3 (q, J= 270 Hz), 125.8 (q, J= 6 Hz), 126.8, 128.2, 128.6, 130.4
(q, J = 32 Hz), 131.7, 135.5, 156.4, 159.5, 169.1. HRMS (ESI)
calculated for C15H12F3NO3 [M þ H]þ 312.0842, found 312.0846.
5-[(E)-2-(3,5-Dichloro-4-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid 2-Octyl Ester (44). Synthesized from 30. Yield 64% (white
powder). 1H NMR (CDCl3) δ 0.86-0.92 (m, 3H), 1.28-1.42 (m,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6295
11H), 1.63-1.82, (m, 2H), 5.24-5.28 (m, 1H), 6.84 (s, 1H), 7.39 (d,
J=16.8Hz, 1H),7.47(d, J= 16.8 Hz, 1H), 8.56 (s, 2H). 13CNMR
(CDCl3) 13.7, 19.5, 22.2, 25.0, 28.6, 31.3, 35.4, 73.5, 103.8, 122.6,
126.3, 130.8, 138.6, 148.0, 156.8, 158.9, 167.8. HRMS (ESI)
calculated for C19H22Cl2N2O3 [M þ H]þ 397.1080, found
397.1087.
fellowships. Dr. S. C. Cho and Dr. K. Sidell are acknowledged
for the selectivity and the frequency of mutations assays.
Supporting Information Available: HPLC purity determina-
tions for the target compounds 6-49 and a brief description of
the biological assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
5-[(E)-2-(3,5-Dichloro-4-pyridinyl)ethenyl]-3-isoxazolecarboxylic
Acid 3-Cyclopentanepropyl Ester (45). Synthesized from 30. Yield
1
69% (white powder). H NMR (CDCl3) δ 1.02-1.12 (m, 2H),
References
1.41-1.89 (m, 9H), 4.41 (d, J = 6.8 Hz, 2H), 6.84 (s, 1H), 7.40 (d,
J=16.8Hz, 1H),7.47(d, J= 16.8 Hz, 1H), 8.56 (s, 2H). 13CNMR
(CDCl3) δ 24.8, 27.4, 31.7, 32.2, 39.3, 66.3, 103.8, 122.6, 126.4,
130.8, 138.5, 148.0, 156.6, 159.3, 167.9. HRMS (ESI) calculated for
C19H20Cl2N2O3 [M þ H]þ 395.0923, found 395.0930.
(1) World Health Organization. Global Tuberculosis Control;Surveil-
lance, Planning, Financing; World Health Organization: Geneva,
2008; http://www.who.int/tb/publications/global_report/en/index.html
(accessed February 2009).
(2) Brooks, J. T.; Kaplan, J. E.; Holmes, K. K.; Benson, C.; Pau, A.;
Masur, H. HIV-associated opportunistic infections;going, going,
but not gone: the continued need for prevention and treatment
guidelines. Clin. Infect. Dis. 2009, 48, 609–611.
(3) Zhang, Y.; Post-Martens, K.; Denkin, S. New drug candidates and
therapeutic targets for tuberculosis therapy. Drug Discovery Today
2006, 11, 21–27.
(4) Rivers, E. C.; Mancera, R. L. New anti-tuberculosis drugs in
clinical trials with novel mechanism of action. Drug Discovery
Today 2008, 13, 23–24.
(5) Chaisson, R. E. Tuberculosis chemotherapy. Still a double-edged
sword. Am. J. Respir. Crit. Care Med. 2003, 167, 1461–1466.
(6) Maggi, N.; Pasqualucci, C. R.; Ballotta, R.; Sensi, P. Rifampicin, a
new orally active rifamycin. Farmaco Sci. 1966, 21, 68–75.
(7) The Five Elements of DOTS; World Health Organization: Geneva,
2009; http://www.who.int/tb/dots/whatisdots/en/index.html (accessed
February 2009).
(8) Blumberg, H. M.; Burman, W. J.; Chaisson, R. E.; Daley, C. L.;
Etkind, S. C.; Friedman, L. N.; Fujiwara, P.; Grzemska, M.;
Hopewell, P. C.; Iseman, M. D.; Jasmer, R. M.; Koppaka, V.;
Menzies, R. I.; O’Brien, R. J.; Reves, R. R.; Reichman, L. B.;
Simone, P. M.; Starke, J. R.; Vernon, A. A. American Thoracic
Society/Centers for Disease Control and Prevention/Infectious
Diseases Society of America: Treatment of Tuberculosis. Am. J.
Respir. Crit. Care Med. 2003, 15, 603–662.
(9) (a) McKinney, J. D.; Jacobs, W. R. J.; Bloom, B. R. Persisting
problems in Tuberculosis. In Emerging Infections; Krause, R. M., Ed.;
Academic Press: New York, 1998; pp 51-146. (b) Honer zu Bentrup, K.;
Russell, D. G. Mycobacterial persistence: adaptation to a changing
environment. Trends Microbiol. 2001, 9, 597–605. (c) Boshoff,
H. I. M.; Barry, C. E. Tuberculosis;metabolism and respiration in the
absence of growth. Nat. Rev. Microbiol. 2005, 3, 70–80.
(10) Johnson, R.; Streicher, E. M.; Louw, G. E.; Warren, R. M.;
Van Helden, P. D.; Victor, T. C. Drug resistance Mycobacterium
tuberculosis. Curr. Issues Mol. Biol. 2006, 8, 97–112.
(11) (a) Dorman, S. E.; Chaisson, R. E. From magic bullets back to the
magic mountain: the rise of extensively drug-resistant tuberculosis.
Nat. Med 2007, 3, 295–298. (b) CNC. Extensively drug-resistant
tuberculosis;United States, 1993-2006. MMWR. Morb. Mortal.
Wkly Rep. 2007, 56, 250–253.
(12) Global alliance for tuberculosis drug development. Executive
summary of the scientific blueprint for TB drug development.
Tuberculosis 2001, 81, (Suppl.), 1-52.
(13) O’Brien, R. J.; Nunn, P. P. The need for new drugs against
tuberculosis. Obstacles, opportunities, and next steps. Am. J.
Respir. Crit. Care Med. 2001, 163, 1055–1058.
(14) Dunkan, K.; Sacchettini, J. D. Approaches to tuberculosis drug
development. In: Hatfull, J. F., Jacobs, W. R. J., Eds.; Molecular
Genetics of Mycobacteria; ASM Press: Washington, DC; 2000,
297-307.
(15) Zhang, Y. The magic bullets and tuberculosis drug targets. Annu.
Rev. Pharmacol. Toxicol. 2005, 45, 529–564.
(16) (a) Mao, J.; Wan, B.; Wang, Y.; Franzblau, S. G.; Kozikowski, A.
P. HTS chemical hybridization, and drug design identify a chemi-
cally unique antituberculosis agents coupling serendipity and ra-
tional approaches to drug discovery. ChemMedChem 2007, 2, 811–
813. (b) Mao, J.; Wang, Y.; Wan, B.; Kozikowski, A. P.; Franzblau, S. G.
Design, synthesis, and pharmacological evaluation of mefloquine-
based ligands as novel antituberculosis agents. ChemMedChem
2007, 2, 1624–1630.
(17) Lilienkampf, A.; Mao, J.; Wan, B.; Wang, Y.; Franzblau, S. G.;
Kozikowski, A. P. Structure-Activity Relationships for a series of
quinoline-based compounds active against replicating and nonreplicat-
ing Mycobacterium tuberculosis. J. Med. Chem. 2009, 52, 2109–2118.
(18) Mao, J.; Yuan, H.; Wang, Y.; Pieroni, M.; Kim, K. H.; Huang, Q.;
Wan, B.; van Breemen, R. B.; Kozikowski, A. P.; Franzblau, S. G.
Unpublished results.
5-[(E)-2-(6-Methoxy-4-quinolinyl)ethenyl]-3-isoxazolecarboxylic
Acid Butyl Ester (46). Synthesized from 2. Yield 69% (yellow
powder). 1H NMR (CDCl3) δ 1.00 (t, J = 7.2 Hz, 3H), 1.43-1.54
(m, 2H), 1.78-1.85 (m, 2H), 4.01 (s, 3H), 4.44 (t, J = 6.8 Hz, 2H),
6.81 (s, 1H), 7.19 (d, J = 16.4 Hz, 1H), 7.35 (d, J = 2.8 Hz, 1H),
7.44 (dd, J1 = 2.8 Hz, J2 = 9.2 Hz, 1H), 7.55 (d, J = 4.4 Hz, 1H),
8.05-8.09 (m, 2H), 8.80 (d, J = 4.4 Hz, 1H). 13C NMR (CDCl3) δ
13.3, 18.7, 30.1, 55.4, 65.8, 99.6, 100.7, 103.0, 117.3, 122.0, 126.7,
130.2, 131.4, 138.7, 144.6, 147.1, 156.6, 158.0, 159.5, 168.7. HRMS
(ESI) calculated for C20H20N2O4 [M þ H]þ 353.1495, found
353.1503.
5-[2-(3-Trifluoromethylphenyl)ethyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (48). 5% Pd/C (100 mg) was added to a solution of 9
(76 mg, 0.24 mmol) in anhydrous EtOH (10 mL) and the flask was
saturated with H2. After 10 min, Pd/C was filtered over celite and
the filtrate evaporated under reduced pressure. The crude material
was purified by flash chromatography (EtOAc-hexane 1:4) to
1
give the title compound as a white solid (yield 75%). H NMR
(CDCl3) δ 1.41 (t, J = 7.2 Hz, 3H), 3.09-3.18 (m, 4H), 4.44 (q,
J = 7.2 Hz, 2H), 6.38 (s, 1H), 7.37-7.51 (m, 4H). 13C NMR
(CDCl3) δ 13.7, 27.8, 32.8, 61.7, 101.7, 123.2 (q, J = 4 Hz), 123.6
(q, J = 271 Hz), 124.6 (q, J = 4 Hz), 128.7, 130.6 (q, J = 32 Hz),
131.3, 140.0, 156.0, 159.6, 173.2. HRMS (ESI) calculated for
C15H14F3NO3 [M þ H]þ 314.0998, found 314.1006.
5-[2-(3,5-Dihydroxyphenyl)ethyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (49). Synthesized as 48 by using 22 as a starting
material. Purified by column chromatography (EtOAc-hexane
3:7). Yield 87% (white powder). 1H NMR (CD3OD) δ 1.38 (t,
J = 7.2 Hz, 3H), 2.88 (t, J = 7.2 Hz, 3H), 3.11 (t, J = 7.2 Hz,
3H), 4.39 (q, J = 7.2 Hz, 2H), 6.10-6.16 (m, 3H), 6.47 (s, 1H).
13C NMR (CD3OD) δ 12.5, 27.3, 32.6, 61.3, 99.9, 101.0, 105.9,
141.6, 155.8, 157.8, 159.5, 174.8. HRMS (ESI) calculated for
C14H15NO5 [M þ H]þ 278.1023, found 278.1035.
5-[(E)-2-(3-Aminophenyl)ethenyl]-3-isoxazolecarboxylic Acid
Ethyl Ester (27). To a stirred solution of 24b (35 mg, 0.12 mmol)
in anhydrous EtOH (5 mL), SnCl2 H2O (270 mg, 1.20 mmol)
3
was added portionwise and the mixture was refluxed until
consumption of the starting material on TLC. The solvent was
evaporated under reduced pressure, and an aqueous solution of
1N NaOH (1 mL) was added. The mixture was extracted several
times with EtOAc and the combined organic layers were washed
with brine, dried over anhydrous Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was purified by
preparative TLC (EtOAc-hexane 3:7), to give the title com-
pound as a pale-yellow solid (yield 51%). 1H NMR (CDCl3) δ
1.44 (t, J = 7.2 Hz, 3H), 3.76 (s, 2H), 4.47 (q, J = 7.2 Hz, 2H),
6.66 (s, 1H), 6.70 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 6.85 ( br s,
1H), 6.90-6.99 (m, 2H), 7.16-7.22 (m, 1H), 7.32 (d, J = 16.4
Hz, 1H). 13C NMR (CDCl3) δ 13.8, 61.8, 101.4, 112.1, 113.4,
116.4, 117.9, 129.8, 135.4, 136.4, 146.8, 156.4, 159.7, 170.1.
HRMS (ESI) calculated for C14H14N2O3 [M þ H]þ 259.1077,
found 259.1082.
Acknowledgment. TB Alliance is acknowledged for finan-
cial support. A. Lilienkampf thanks The Academy of Finland
(grant 120441) and The Finnish Cultural Foundation for

6296 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Pieroni et al.
(19) Bravo, P.; Ricca, A.; Ticozzi, C.; Vajna de Pava, O. New r-oxo-
γ-(enaminoalkyl)triphenylphosphonium salts and ylides from iso-
xazole derivatives: synthesis of r,γ-dioxox olefins. Gazz. Chim.
Ital. 1976, 106, 743–759.
(20) DeShong, P.; Cipollina, J. A.; Lowmaster, N. K. A general method
for the synthesis of tetramic acid derivatives. J. Org. Chem. 1988,
53, 1356–1364.
(21) Micetich, R. G.; Shaw, C. C.; Hall, T. W.; Spevak, P.; Fortier, R.
A.; Wolfert, P.; Foster, B. C.; Bains, B. K. The 5-alkoxymethyl-,
5-alkylthiomethyl-, and 5-dialkylaminomethylisoxazoles. Hetero-
cycles 1985, 23, 571–583.
screening of compounds against nonreplicating Mycobacterium
tuberculosis. Antimicrob. Agents Chemother. 2007, 51, 1380–
1385.
(24) Mao, J.; Yuan, H.; Wang, Y.; Wan, B.; Pak, D.; Lilienkampf, A.;
Pieroni, M.; Franzblau, S. G.; Kozikowski, A. P. Unpublished
results.
(25) Soellner, M. B.; Rawls, K. A.; Grundner, C.; Alber, T.; Ellman,
J. A. Fragment-Based Substrate Activity Screening Method for
the Identification of Potent Inhibitors of the Mycobacterium
tuberculosis Phosphatase PtpB. J. Am. Chem. Soc. 2007, 129,
9613–9615.
(22) Franzblau, S. G.; Witzig, R. S.; Mclaughlin, J. C.; Torres, P.;
Madico, G.; Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer,
V. K.; Ferguson, R. M.; Gilman, R. H. Rapid, low-technology
MIC determination with clinical Mycobacterium tuberculosis iso-
lates by using the microplate Alamar Blue assay. J. Clin. Microbiol.
1998, 36, 362–366.
(26) Omar, F. A.; Abdel-Hafez, A. A.; Ahmed, M. S. Design and
synthesis of some p-substituted styrylisoxazole carboxylic acid
derivatives as anti-inflammatory agents. Bull. Pharm. Sci. Assiut
Univ. 2004, 27, 171–186.
(27) Mokhtar, H. M. Synthesis of nitrogenous compounds. Part II.
Pak. J. Sci. Ind. Res. 1990, 33, 30–36.
(28) Soliman, G.; Rateb, L. Synthesis of heterocyclic compounds from
δ-unsaturated 1,3-dioxo esters. J. Chem. Soc. 1956, 3663–3668.
(23) Cho, S. H.; Warit, S.; Wan, B.; Hwang, C. H.; Pauli, G. F.;
Franzblau, S. G. Low-Oxygen-Recovery Assay for high-throughput