Synthesis and evaluation as antitubercular agents of 5-arylethenyl and 5-(hetero)aryl-3-isoxazolecarboxylate

Article abstract of DOI:10.1002/ddr.21057

Preclinical Research A new series of 5-aryl and 5-arylethenylisoxazole carboxylate derivatives was synthesized and evaluated for in vitro activity against Mycobacterium tuberculosis H₃₇R_v. Several compounds exhibited minimum inhibitory concentrations in the low micromolar range (2.3-11.4 μM). A variety of substituents introduced around the isoxazole ring allowed the delineation of preliminary SARs for this new series of compounds.

Full text of DOI:10.1002/ddr.21057

DRUG DEVELOPMENT RESEARCH •• : ••–•• (2012)
Research Article
Synthesis and Evaluation as Antitubercular Agents of
5-Arylethenyl and 5-(Hetero)aryl-3-Isoxazolecarboxylate
Claudia Vergelli,1 Agostino Cilibrizzi,1 Letizia Crocetti,1 Alessia Graziano,1
Vittorio Dal Piaz,1 Baojie Wan,2 Yuehong Wang,2 Scott Franzblau,2 and
Maria Paola Giovannoni1*
1Dipartimento di Scienze Farmaceutiche, University of Florence, via Ugo Schiff 6, 50019 Sesto
Fiorentino, Florence, Italy
2Institute for Tuberculosis Research, University of Illinois at Chicago, Chicago, IL 60612, USA
Strategy, Management and Health Policy
Enabling
Preclinical Development Clinical Development
Technology,
Genomics,
Proteomics
Preclinical
Research
Toxicology, Formulation
Drug Delivery,
Pharmacokinetics
Phases I-III
Regulatory, Quality,
Manufacturing
Postmarketing
Phase IV
ABSTRACT
A new series of 5-aryl and 5-arylethenylisoxazole carboxylate derivatives was synthesized
and evaluated for in vitro activity against Mycobacterium tuberculosis H37Rv. Several compounds exhibited
minimum inhibitory concentrations in the low micromolar range (2.3–11.4 mM). A variety of substituents
introduced around the isoxazole ring allowed the delineation of preliminary SARs for this new series of
compounds. Drug Dev Res •• : ••–••, 2012.
© 2012 Wiley Periodicals, Inc.
Key words: Mycobacterium tuberculosis; antituberculosis agents; 3-isoxazolecarboxylic acid alkyl esters
INTRODUCTION
For many years, TB was considered a poverty-
related disease, but it now occurs in the developed
world. This is due to several reasons, the most impor-
tant being the development of new virulent strains,
resistant to antitubercular drugs, as well as the stream
of immigrants from countries where TB is endemic.
In addition, the recurrence of TB is directly connected
to the explosive spread of HIV as the number of HIV-
positive patients coinfected with Mtb is constantly rising
[Brooks et al., 2009]. WHO reports that one third of
HIV-infected people are coinfected with TB [World
Tuberculosis (TB) is a chronic and often deadly
infectious disease caused by various species of Mycobac-
terium, mainly Mycobacterium tuberculosis (Mtb)
[Russell et al., 2010]. The World Health Organization
(WHO) has estimated that one-third of the world’s
population is infected with Mtb, resulting in 9.4 million
new TB cases and 1.7 million deaths from TB in 2009, for
the most part in developing countries [World Health
Organization, 2010; World Health Organization, 2010/
2011].
Despite the severe worldwide impact of TB, it
remains a neglected disease as demonstrated by the
evidence that no new anti-TB drugs have been intro-
*Correspondence
to:
Maria
Paola
Giovannoni,
duced in therapy over the past 40 years [Janin, 2007; Dipartimento di Scienze Farmaceutiche, University of Florence,
Via Ugo Schiff 6, Sesto Fiorentino 50019, Florence, Italy.
Spigelman, 2007] with rifampin (RMP), discovered in
E-mail: mariapaola.giovannoni@unifi.it
1966, the most recent drug in this field [Maggi et al.,
Received 24 October 2012; Accepted 1 December 2012
1966]. At present, only a few drug candidates are in
Phase II clinical trials, e.g., PA-824 [Stover et al., 2000]
and TMC-207 [Andries et al., 2005] (Fig. 1).
Published online in Wiley Online Library (wileyonlinelibrary.
com). DOI: 10.1002/ddr.21057
© 2012 Wiley Periodicals, Inc.

2
VERGELLI ET AL.
N
O
O
F3C
HO
O
N
N
O
PA-824
NO2
N
Br
NO2
O
TMC-207
EtOOC
N
O
N
N
N
O
CH2Ph
N
N
CH2Ph
B
MIC = 1.56 μg/mL
A
MIC = 0.00005 μg/mL
EtOOC
CF3
EtOOC
N
O
N
O
O
N
D
MIC MABA = 0.60 μM
C
MIC MABA = 0.21 μM
R2
ROOC
R1
N
O
E
Fig. 1. Examples of anti-TB agents.
Health Organization, 2002], and 90% of these die because of the presence of a nonreplicating persistent
within a few months of the appearance of clinical symp- Mtb phenotype (NRP-TB) [Wayne and Sohaskey,
toms. This situation has prompted the WHO to declare 2001] is often associated with poor patient compliances
TB as a global emergency [Janin, 2007].
that contribute to the development of multidrug
The current standard therapy, Directly Observed resistance. There are two types of drug resistance:
Therapy Short-course [World Health Organization, multidrug resistant TB (MDR-TB) and extensively
2009] is based on a combination of isoniazide (INH), drug resistant TB (XDR-TB) [Johnson et al., 2006;
Rifampin (RMP), pyrazinamide, and ethambutol (or Dorman and Chaisson, 2007]; the former defined as
streptomycin) for 2 months followed by further treat- resistance to at least INI and RMP (MDR-TB) and
ment with INH and RMP for an additional 4–7 the latter including concomitant resistance to any
months. This long treatment regimen is necessary fluoroquinolone and at least one second-line drug, e.g.
Drug Dev. Res.

ANTITUBERCULAR AGENTS
3
O
O
X
X
EtOOC
EtOOC
A
N
N
CH3
CH=CH-Ar
O
O
1a,b
2a-f
for 2a and 2f
B
2
Ar
X
1
X
O
a
b
Ph
H
a
b
c
d
Ph
4-Cl-Ph
3-Cl-Ph
H
H
H
H
HOOC
N
CH=CH-Ar
3a,b
O
3
Ar
Ph
e
f
Cl
H
a
b
C
O
ROOC
4
Ar
R
N
CH=CH-Ar
O
a
b
c
Ph
Ph
CH3
iC3H7
iC3H7
4a-c
Fig. 2. Reagent and conditions: (A) ArCHO, EtONa, EtOH, 0°C, 2–20 h; (B) NaOH, EtOH, r.t., 1 h; (C) ROH, H2SO4, 80°C, 30–90 min.
MATERIALS AND METHODS
Chemistry
kanamycin, amikacin, or capreomycin (XDR-TB).
These data highlight the need for new, safer, and more
potent anti-TB agents with novel mechanisms of
action.
All the final compounds were synthesized as
Many classes of organic compounds have been reported in Figures 2 and 3. Figure 2 shows the syn-
synthesized and tested as anti-TB agents, and in par- thetic procedure leading to the 4-benzoyl-5-
ticular nitrogen heterocycles derivatives such as isox- styrylisoxazoles 2a–f and 4a–c. The precursors were
azolines or isoxazoles have been described as good in represented by previously described isoxazoles 1a,b
vitro antitubercular agents [Johar et al., 2005; Janin, [Renzi et al., 1968; Dal Piaz et al., 1991], which were
2007; Eswarana et al., 2010]. Some representative com- condensed with the appropriate arylaldehydes to give
pounds with this scaffold are shown in Figure 1. The the corresponding vinyl derivatives 2a–f. Hydrolysis of
isoxazolines A and B [Tangalapally et al., 2007] and the 2a and 2f gives intermediate carboxylic acids 3a,b
isoxazole derivatives C [Pieroni et al., 2009] and D [Lil- which, in turn, were transformed into the ester deriva-
ienkampf et al., 2010] had interesting levels of activity tives 4a–c using the appropriate alcohol and conc.
against Mtb with minimum inhibitory concentration H2SO4 at 80°C. Figure 2 shows the synthesis of the
(MIC) values in the low or submicromolar range.
4-unsubstituted derivatives 8a–g and 10 a–e. The key
In this article, we describe the synthesis and intermediates 7a–g (7a [Fatutta and Balestra, 1958];
the biological activity of a new series of anti-TB 7b [Thormann et al., 2008]; 7c [Deavegowda et al.,
compounds with an isoxazole scaffold (Fig. 1, struc- 2010]; 7f [Janculev and Podolesov, 1961]; 7g [Patil
ture E), structurally related to compounds C and D. et al., 2007] were obtained by condensation of commer-
Various substituents were introduced around the isox- cially available 5a–g and diethyl oxalate 6 [Fatutta and
azole core in order to find the best substitution Balestra, 1958] and transformed into the final isoxazoles
pattern and to define the importance of different 8a–g by treatment with hydroxylamine hydrochloride
substituents.
in ethanol and H2SO4. Finally, for compounds 8a–e, a
Drug Dev. Res.

4
VERGELLI ET AL.
A
R
CH2
COOEt
R
CH3
COOEt
COOEt
6
+
O
O
O
7a-g
5a-g
B
COOEt
5, 7-10
R
N
O
a
R
8a-g
C
b
c
S
COOH
S
N
d
e
R
O
OCH3
O
9a-e
D
H3CO
N
COOC3H7i
OCH3
f
N
O
R
10a-e
g
Fig. 3. Reagent and conditions: (A) Na, Et2O, r.t-40°C; (B) NH2OH.HCl, H2SO4, EtOH, reflux; (C) 1 N NaOH, EtOH, rt-60°C; (D) iC3H7OH,
H2SO4, reflux.
further transformation in the corresponding isopropyl
esters (10a–e) was performed following the same pro-
cedure described for compounds 4a–c.
General Procedures for 2a–f
To a cooled (0°C) and stirred mixture of 1a,b
[Renzi et al., 1968; Dal Piaz et al., 1991] (3.86 mmol)
and the appropriate arylaldehyde (3.86 mmol) in
4–6 ml of anhydrous EtOH, a solution of sodium ethox-
ide, obtained from sodium (7.72 mmol) and anhydrous
EtOH (5 ml), was slowly added. The mixture was
stirred at 0°C for 2–20 h, then it was acidified with 6 N
HCl and diluted with 30 ml of cold water. The final
compounds were recovered by suction and purified by
crystallization.
Experimental
All melting points were determined on a Büchi
1
apparatus and are uncorrected. H-NMR spectra were
recorded with an Avance 400 instrument (Bruker
Biospin Version 002 with SGU, Bruker AXS Inc.,
Madison, WI USA). Chemical shifts are reported in
ppm, using the solvent as internal standard. Extracts
were dried over Na2SO4, and the solvents were removed
under reduced pressure. Merck F-254 commercial 4-Benzoyl-5-Styryl-Isoxazole-3-Carboxylic Acid
plates (Merck-Gruppe, Darmstadt, Germany) were Ethyl Ester, 2a
used for analytical thin layer chromatography (TLC)
Yield = 67%; mp = 56°C (EtOH); 1H-NMR
to follow the reaction course. Silica gel 60 (Merck (DMSO-d6) d 0.97 (t, 3H, CH3CH2, J = 7.2 Hz), 4.07
70–230 mesh) was used for column chromatography. (q, 2H, CH3CH2, J = 7.2 Hz), 7.13 (d, 1H, CH = CH,
Microanalyses were performed with a Perkin-Elmer 260 J = 16.8 Hz), 7.45–7.49 (m, 3H, Ar), 7.58 (m, 2H, Ar),
elemental analyzer (Perkin-Elmer, Waltham, MA USA) 7.62–7.78 (m, 4H: 3H, Ar; 1H, CH = CH), 7.82 (m,
for C, H, and N. Results were within Ϯ0.4% of the 2H, Ar). MS m/z 348 [M+]. Anal. Calcd for C21H17NO4:
theoretical values unless otherwise stated. Reagents and C, 72.61; H, 4.93; N, 4.03. Found C, 7.40; H, 4.92;
starting materials were commercially available.
N, 4.04.
Drug Dev. Res.

ANTITUBERCULAR AGENTS
5
4-Benzoyl-5-[2-(4-Chlorophenyl)-Vinyl]-Isoxazole-
3-Carboxylic Acid Ethyl Ester, 2b
7.60–7.65 (m, 6H, Ar), 7.70 (d, 1H, CH = CH, J =
16.4 Hz), 7.85 (m, 2H, Ar). MS m/z 424 [M+]. Anal.
Yield = 75%; mp = 75–77°C (Et2O); 1H-NMR Calcd for C27H21NO4: C, 76.58; H, 5.00; N, 3.31. Found
(DMSO-d6) d 0.96 (t, 3H, CH3CH2, J = 7.2 Hz), 4.07 C, 76.77; H, 4.99; N, 3.32.
(q, 2H, CH3CH2, J = 7.2 Hz), 7.15 (d, 1H, CH = CH,
J = 16.8 Hz), 7.48 (m, 2H, Ar), 7.58 (m, 2H, Ar), 7.70–
4-Benzoyl-5-(2-Biphenyl-4-yl-Vinyl)-Isoxazole-3-
Carboxylic Acid, 3b
7.78 (m, 4H: 3H, Ar; 1H, CH = CH), 7.82 (m, 2H, Ar).
MS m/z 382 [M+]. Anal. Calcd for C21H16ClNO4: C,
66.06; H, 4.22; N, 3.67. Found C, 66.22; H, 4.23; N, 3.68.
To a suspension of 2f (0.28 mmol) in 2.5 ml of
EtOH, 1 ml of 1N NaOH was added, and the mixture
was stirred at room temperature for 1 h. After cooling,
the mixture was acidified with 2N HCl and extracted
with ethyl acetate (3 x 10 ml). Evaporation of the
solvent under vacuum afforded the desired final com-
pound. Yield = 73%; mp = 187–190°C (EtOH); 1H-
NMR (CDCl3) d 6.70 (d, 1H, CH = CH, J = 16.4 Hz),
7.36–7.68 (m, 11H: 10H, Ar; 1H, CH = CH), 7.70–7.87
(m, 3H, Ar), 8.05 (m, 2H, Ar). MS m/z 396 [M+]. Anal.
Calcd for C25H17NO4: C, 75.94; H, 4.33; N, 3.54. Found
C, 75.73; H, 4.34; N, 3.54.
4-Benzoyl-5-[2-(3-Chlorophenyl)-Vinyl]-Isoxazole-
3-Carboxylic Acid Ethyl Ester, 2c
Yield = 67%; mp = 68–70°C (Et2O); 1H-NMR
(CDCl3) d 1.08 (t, 3H, CH3CH2, J = 7.2 Hz), 4.13 (q,
2H, CH3CH2, J = 7.2 Hz), 7.05 (d, 1H, CH = CH, J =
16.4 Hz), 7.30–7.40 (m, 3H, Ar), 7.50–7.60 (m, 4H: 3H,
Ar; 1H, CH = CH), 7.65 (m, 1H, Ar), 7.82 (m, 2H,
Ar). MS m/z 382 [M+]. Anal. Calcd for C21H16ClNO4:
C, 66.06; H, 4.22; N, 3.67. Found C, 66.20; H, 4.21;
N, 3.67.
General Procedure for Compounds 4a–c
4-Benzoyl-5-(2-Naphthalen-1-yl-Vinyl)-Isoxazole-3-
Carboxylic Acid Ethyl Ester, 2d
To a mixture of 3a [Renzi et al., 1968] and 3b
(0.313 mmol) in 1.5–2 ml of the appropriate alcohol
(methanol or isopropyl alcohol), 0.5 ml of conc. H2SO4
was added. The suspension was refluxed for 30–
120 min, and after cooling, cold water was added, and
the mixture was extracted with CH2Cl2 (3 ¥ 15 ml). The
solvent was evaporated in vacuum affording final com-
pounds 4a and 4b. For compound 4c, after dilution
with water, the precipitate was recovered by suction
and recrystallized with ethanol.
1
Yield = 48%; mp = 137–139°C (Et2O); H-NMR
(DMSO-d6) d 1.00 (t, 3H, CH3CH2, J = 7.2 Hz), 4.10
(q, 2H, CH3CH2, J = 7.2 Hz), 7.24 (d, 1H, CH = CH,
J = 16.4), 7.50–7.65 (m, 5H, Ar), 7.73 (m, 1H, Ar), 7.90
(m, 2H, Ar), 7.95 (m, 1H, Ar), 7.98–8.04 (m, 2H, Ar),
8.20 (m, 1H, Ar), 8.40 (d, 1H, CH = CH, J = 16.4 H).
MS m/z 398 [M+]. Anal. Calcd for C25H19NO4: C, 75.55;
H, 4.82; N, 3.52. Found C, 75.31; H, 4.81; N, 3.51.
4-(4-Chlorobenzoyl)-5-(2-Naphthalen-1-yl-Vinyl)-
Isoxazole-3-Carboxylic Acid Ethyl Ester, 2e
4-Benzoyl-5-Styryl-Isoxazole-3-Carboxylic Acid
Methyl Ester, 4a
Yield = 28%; mp = 125–126°C (EtOH); 1H-NMR
(CDCl3) d 1.20 (t, 3H, CH3CH2, J = 7.2 Hz), 4.24 (q,
2H, CH3CH2, J = 7.2 Hz), 7.12 (d, 1H, CH = CH,
J = 16.4 Hz), 7.50 (d, 2H, Ar, J = 8.4 Hz), 7.57–7.64 (m,
3H, Ar), 7.77 (m, 1H, Ar), 7.80–7.90 (m, 2H, Ar), 7.92
(d, 2H, Ar, J = 8.4 Hz), 8.17 (m, 1H, Ar), 8.48 (d, 1H,
CH = CH, J = 16.4 Hz). MS m/z 432 [M+]. Anal. Calcd
for C25H18ClNO4: C, 69.53; H, 4.20; N, 3.24. Found C,
69.69; H, 4.21; N, 3.23.
Yield = 94%; oil; 1H-NMR (CDCl3) d 3.70 (s, 3H,
CH3), 7.03 (d, 1H, CH = CH, J = 16.4 Hz), 7.36–7.40
(m, 3H, Ar), 7.45–7.55 (m, 4H, Ar), 7.60–7.70 (m, 2H:
1H, Ar; 1H, CH = CH), 7.82 (d, 2H, Ar). MS m/z 334
[M+]. Anal. Calcd for C20H15NO4: C, 72.06; H, 4.54; N,
4.20. Found C, 72.27; H, 4.55; N, 4.21.
4-Benzoyl-5-Styryl-Isoxazole-3-Carboxylic Acid
Isopropyl Ester, 4b
Yield = 92%; oil; 1H-NMR (CDCl3) d 1.06 (d, 6H,
CH(CH3)2, J = 6.4 Hz), 5.03 (m, 1H, CH(CH3)2), 7.05
(d, 1H, CH = CH, J = 16.4 Hz), 7.35–7.40 (m, 3H, Ar),
4-Benzoyl-5-(2-Biphenyl-4-yl-Vinyl)-Isoxazole-3-
Carboxylic Acid Ethyl Ester, 2f
Yield = 47%; mp = 128–129°C (EtOH); 1H-NMR 7.42–7.55 (m, 4H, Ar), 7.60–7.70 (m, 2H: 1H, Ar; 1H,
(CDCl3) d 1.10 (t, 3H, CH3CH2, J = 7.2 Hz), 4.15 (q, CH = CH), 7.85 (d, 2H, Ar). MS m/z 362 [M+]. Anal.
2H, CH3CH2, J = 7.2 Hz), 7.10 (d, 1H, CH = CH, Calcd for C20H15NO4: C, 73.12; H, 5.30; N, 3.88. Found
J = 16.4 Hz), 7.40 (m, 1H, Ar), 7.46–7.55 (m, 5H, Ar), C, 72.91; H, 5.29; N, 3.89.
Drug Dev. Res.

6
VERGELLI ET AL.
4-Benzoyl-5-(2-Biphenyl-4-yl-Vinyl)-Isoxazole-3-
Carboxylic Acid Isopropyl Ester, 4c
15–45 min. After cooling, the solvent was evaporated
under vacuum to afford a residue which, after treat-
Yield = 65%; mp = 123–125°C (EtOH); 1H-NMR ment with cold water (15 ml), gave rise to a crude
(CDCl3) d 1.07 (d, 6H, CH(CH3)2, J = 6.4 Hz), 5.03 precipitate that was recovered by suction. The final
(m, 1H, CH(CH3)2), 7.10 (d, 1H, CH = CH, compounds 8b, 8e, and 8g were purified by crystalli-
J = 16.4 Hz), 7.35–7.56 (m, 6H, Ar), 7.60–7.77 (m, zation, whereas compounds 8d and 8f were purified by
6H: 5H, Ar, 1H, CH = CH), 7.82 (m, 1H, Ar), 7.85 (d, column chromatography using toluene/ethyl acetate
2H, Ar). MS m/z 438 [M+]. Anal. Calcd for C28H23NO4: 9.5:0.5 as eluent.
C, 76.87; H, 5.30; N, 3.20. Found C, 76.85; H, 5.29; N,
3.21.
5-Benzo[b]thiophen-2-yl-Isoxazole-3-Carboxylic
Acid Ethyl Ester, 8b
Yield = 69%; mp = 130–131°C, dec. (EtOH);
General Procedure for Compounds 7d and 7e
1H-NMR (CDCl3) d 1.47 (t, 3H, CH3CH2, J = 7.2 Hz),
To a stirred mixture of the commercially available
5d or 5e (5,26 mmol) and diethyl oxalate (5–15 mmol)
4.51 (q, 2H, CH3CH2, J = 7.2 Hz), 6.92 (s, 1H, isox-
azole), 7.45 (m, 2H, Ar), 7.85 (s, 1H, Ar), 7.90 (m, 2H,
Ar). MS m/z 274 [M+]. Anal. Calcd for C14H11NO3S: C,
61.52; H, 4.06; N, 5.12. Found C, 61.64; H, 4.07; N,
5.11.
in anhydrous ether (50 ml), 5.25–7.40 mmol of Na
was added in a dropwise manner. The suspension was
stirred at room temperature for 4 h and then heated at
40°C for 4–7 h. After cooling, the precipitate was fil-
tered off and washed with anhydrous ether. The solid
was dissolved in water and acidified with 6N HCl to
afford a crude precipitate that was collected by suction
and purified by crystallization from ethanol.
5-(7-Methoxybenzofuran-2-yl)-Isoxazole-3-
Carboxylic Acid Ethyl Ester, 8d
Yield = 40%; mp = 119–121°C; purified by
column chromatography (toluene/ethyl acetate 9.5:0.5);
1H-NMR (CDCl3) d 1.45 (t, 3H, CH3CH2, J = 7.2 Hz),
4.07 (s, 3H,OCH3), 4.50 (q, 2H, CH3CH2, J = 7.2 Hz),
6.93 (m, 1H, Ar), 7.14 (s, 1H, isoxazole), 7.24–7.30 (m,
4-Hydroxy-4-(7-Methoxybenzofuran-2-yl)-2-oxo-
but-3-Enoic Acid Ethyl Ester, 7d
Yield = 85%; mp = 108–111°C (EtOH); 1H-NMR 2H, Ar), 7.36 (s, 1H, Ar). MS m/z 288 [M+]. Anal. Calcd
(CDCl3) d 1.45 (t, 3H, CH3CH2, J = 7.2 Hz), 4.07 (s, for C15H13NO5: C, 62.72; H, 4.56; N, 4.88. Found C,
3H, OCH3), 4.42 (q, 2H, CH3CH2, J = 7.2 Hz), 7.00 (m, 62.94; H, 4.57; N, 4.87.
1H, Ar), 7.20 (s, 1H, Ar), 7.25–7.33 (m, 2H, Ar), 7.65
(s, 1H, Ar). MS m/z 291 [M+]. Anal. Calcd for C15H14O6:
C, 62.07; H, 4.86. Found C, 62.22; H, 4.85.
5-(2,6-Dimethoxypyridin-3-yl)-Isoxazole-3-
Carboxylic Acid Ethyl Ester, 8e
Yield = 82%; mp = 131–133°C, (Ether); 1H-NMR
(CDCl3) d 1.47 (t, 3H, CH3CH2, J = 7.2 Hz), 4.01 (s,
3H, OCH3), 4.13 (s, 3H, OCH3), 4.50 (q, 2H, CH3CH2,
J = 7.2 Hz), 6.48 (d, 1H, Ar, J = 8.4 Hz), 7.05 (s, 1H,
isoxazole), 8.19 (d, 1H, Ar, J = 8.4 Hz). MS m/z 279
[M+]. Anal. Calcd for C13H14N2O5: C, 56,11; H, 5,07; N,
10,07. Found C, 56.24; H, 5.06; N, 10.04.
4-(2,6-Dimethoxypyridin-3-yl)-4-Hydroxy-2-oxo-
but-3-Enoic Acid Ethyl Ester, 7e
Yield = 84%; mp = 86–89°C (EtOH); 1H-NMR
(CDCl3) d 1.43 (t, 3H, CH3CH2, J = 7.2 Hz), 4.03 (s,
3H, OCH3), 4.12 (s, 3H, OCH3), 4.40 (q, 2H, CH3CH2,
J = 7.2 Hz), 6.45 (d, 1H, Ar, J = 8.8 Hz), 7.41 (s, 1H,
Ar), 8.28 (d, 1H, Ar, J = 8.8 Hz). MS m/z 282 [M+].
Anal. Calcd for C13H15NO6: C, 55.51; H, 5.38; N, 4.98.
Found C, 55.68; H, 5.37; N, 4.99
5-(9H-Fluoren-2-yl)-Isoxazole-3-Carboxylic Acid
Ethyl Ester, 8f
Yield = 68%; mp = 158–161°C, dec.; purified by
column chromatography (toluene/ethyl acetate 9.5:0.5);
1H-NMR (CDCl3) d 1.46 (t, 3H, CH3CH2, J = 7.2 Hz),
General Procedure for Compounds 8b, 8d-g
To a suspension of 7b [Thormann et al., 2008] or 4.02 (s, 2H, PhCH2Ph), 4.51 (q, 2H, CH3CH2,
7d-g [Janculev and Podolesov, 1961; Patil et al., 2007; J = 7.2 Hz), 6.98 (s, 1H, isoxazole), 7.38–7.47 (m, 2H,
Deavegowda et al., 2010] (0.51 mmol) and hydroxy- Ar), 7.61 (m, 1H, Ar), 7.85–7.92 (m, 3H, Ar), 8.03 (s,
lamine hydrochloride (2.5–3.02 mmol, dissolved in 1H, Ar). MS m/z 306 [M+]. Anal. Calcd for C19H15NO3:
0.5 ml of water) in 2–6 ml of EtOH, 0.3–0.4 ml of conc. C, 74.74; H, 4.95; N, 4.59. Found C, 74.56; H, 4.96;
H2SO4 was added. The mixture was heated at 80°C for N, 4.58.
Drug Dev. Res.

ANTITUBERCULAR AGENTS
7
5-Anthracen-9-yl-Isoxazole-3-Carboxylic Acid Ethyl 9c [Schneider et al., 2008]) following the general
Ester, 8g
procedure described for compounds 4a–c. For com-
Yield = 74%; mp = 139–141°C, (EtOH); 1H-NMR pounds 10b–d, after dilution with cold water, the
(CDCl3) d 1.53 (t, 3H, CH3CH2, J = 7.2 Hz), 4.60 (q, 2H, precipitate was recovered by suction and purified by
CH3CH2, J = 7.2 Hz), 7.09 (s, 1H, isoxazole), 7.51–7.57 crystallization, whereas compound 10e was purified
(m, 4H, Ar), 7.80 (m, 2H, Ar), 8.10 (m, 2H, Ar), 8.67 (s, by column chromatography using cyclohexane/ethyl
1H, Ar). MS m/z 318 [M+]. Anal. Calcd for C20H15NO3: acetate 1/1 as eluent.
C, 75.70; H, 4.76; N, 4.41. Found C, 75.88; H, 4.76;
N, 4.42.
5-Biphenyl-4-yl-Isoxazole-3-Carboxylic Acid
Isopropyl Ester, 10a
Yield = 55%; mp = 101–103°C; (EtOH); 1H-NMR
General Procedure for 9b and 9d,e
(CDCl3) d 1.46 (d, 6H, CH(CH3)2, J = 6.4 Hz), 5.32–
A mixture of 8b or 8d,e (0.91 mmol) and 1N
5.42 (m, 1H, CH(CH3)2), 6.97 (s, 1H, isoxazole), 7.43 (m,
NaOH (1–1.5 ml) in 3–4 ml of EtOH was stirred at
1H, Ar), 7.50 (m, 2H, Ar), 7.67 (m, 2H, Ar), 7.75 (d, 2H,
room temperature for 20–30 min. For compound 9b,
Ar, J = 8.4 Hz), 7.92 (d, 2H, Ar, J = 8.4 Hz). MS m/z 308
the suspension was heated at 50°C for 30 min. After
[M+]. Anal. Calcd for C19H17NO3: C, 74.25; H, 5.58; N,
evaporation of the solvent under vacuum, water was
4.56. Found C, 74.50; H, 5.56; N, 4.57.
added, and the suspension was acidified with 6N HCl
(1–2 ml). The crude precipitate was recovered by
suction (compounds 9b and 9d), whereas compound
9e was extracted with ethyl acetate (3 ¥ 20 ml).
5-Benzo[b]thiophen-2-yl-Isoxazole-3-Carboxylic
Acid Isopropyl Ester, 10b
1
Yield = 85%; mp = 101–103°C, (cicloexane); H-
NMR (CDCl3) d 1.46 (d, 6H, CH(CH3)2, J = 6.4 Hz),
5.32–5.41 (m, 1H, CH(CH3)2), 6.91 (s, 1H, isoxazole),
7.45 (m, 2H, Ar), 7.85 (s, 1H, Ar), 7.89 (m, 2H, Ar). MS
m/z 288 [M+]. Anal. Calcd for C15H13NO3S: C, 62.70; H,
4.56; N, 4.87. Found C, 62.87; H, 4.57; N, 4.86.
5-Benzo[b]thiophen-2-yl-Isoxazole-3-Carboxylic
Acid, 9b
Yield = 47%; mp = 192–195°C, dec.; (EtOH); 1H-
NMR (DMSO-d6) d 7.41 (s, 1H, isoxazole), 7.49 (m, 2H,
Ar), 7.99 (m, 1H, Ar), 8.10 (m, 1H, Ar), 8.18 (s, 1H, Ar).
MS m/z 246 [M+]. Anal. Calcd for C12H7NO3S: C, 58.77;
H, 2.88; N, 5.71. Found C, 58.94; H, 2.87; N, 5.73.
5-Thiophen-2-yl-Isoxazole-3-Carboxylic Acid
Isopropyl Ester, 10c
1
Yield = 59%; mp = 53–56°C, (EtOH); H-NMR
5-(7-Methoxybenzofuran-2-yl)-Isoxazole-3-
Carboxylic Acid, 9d
(CDCl3) d 1.44 (d, 6H, CH(CH3)2, J = 6.4 Hz), 5.35 (m,
1H, CH(CH3)2), 6.79 (s, 1H, isoxazole), 7.17 (m, 1H,
Ar), 7.52 (m, 1H, Ar), 7.59 (m, 1H, Ar). MS m/z 238
[M+]. Anal. Calcd for C11H11NO3S: C, 55.68; H, 4.67; N,
5.90. Found C, 55.89; H, 4.67; N, 5.88.
Yield = 76%; mp = 179–182°C, dec.; (EtOH);
1H-NMR (DMSO-d6) d 3.99 (s, 3H, OCH3), 7.09 (m,
1H, Ar), 7.27–7.35 (m, 3H: 2H, Ar; 1H, isoxazole),
7.72 (s, 1H, Ar). MS m/z 260 [M+]. Anal. Calcd for
C13H9NO5: C, 60.24; H, 3.50; N, 5.40 Found C, 60.36;
H, 3.51; N, 5.41.
5-(7-Methoxybenzofuran-2-yl)-Isoxazole-3-
Carboxylic Acid Isopropyl Ester, 10d
Yield = 82%; mp = 96–100°C; purified by column
chromatography (cicloexane/ethyl acetate 1:1); 1H-
NMR (CDCl3) d 1.45 (d, 6H, CH(CH3)2, J = 6.4 Hz),
4.07 (s, 3H, OCH3), 5.35 (m, 1H, CH(CH3)2), 6.93
(m, 1H, Ar), 7.13 (s, 1H, isoxazole), 7.26 (m, 2H, Ar),
7.36 (s, 1H, Ar). MS m/z 302 [M+]. Anal. Calcd for
C16H15NO5: C, 63.78; H, 5.02; N, 4.65. Found C, 63.59;
H, 5.04; N, 4.66.
5-(2,6-Dimethoxypyridin-3-yl)-Isoxazole-3-
Carboxylic Acid, 9e
Yield = 56%; mp = 200–203°C, dec.; (EtOH);
1H-NMR (DMSO-d6) d 3.96 (s, 3H, OCH3), 4.08 (s, 3H,
OCH3), 6.59 (d, 2H, Ar, J = 8.4 Hz), 6.97 (s, 1H, isox-
azole), 8.21 (d, 2H, Ar, J = 8.4 Hz). MS m/z 251 [M+].
Anal. Calcd for C11H10N2O5: C, 52.80; H, 4.03; N, 11.20.
Found C, 52.94; H, 4.02; N, 11.17.
5-(2,6-Dimethoxypyridin-3-yl)-Isoxazole-3-
Carboxylic Acid Isopropyl Ester, 10e
General Procedure for 10a–e
Compounds 10a–e were obtained starting from
Yield = 86%; mp = 119–121°C, (EtOH); 1H-NMR
9a–e (compounds 9a [Fatutta and Balestra, 1958] and (CDCl3) d 1.45 (d, 6H, CH(CH3)2, J = 6.4 Hz), 4.00
Drug Dev. Res.

8
VERGELLI ET AL.
(s, 3H, OCH3), 4.13 (s, 3H, OCH3), 5.35 (m, 1H, and INH as positive controls. Compound stock solu-
CH(CH3)2), 6.48 (d, 1H, Ar, J = 8.4 Hz), 7.04 (s, 1H, tions were prepared in DMSO at concentration of
isoxazole), 8.19 (d, 1H, Ar, J = 8.4 Hz). MS m/z 293 [M+]. 12.8 mM, and the final test concentrations ranged from
Anal. Calcd for C14H16N2O5: C, 57.53; H, 5.52; N, 9.58. 128 mM to 0.5 mM. Twofold dilutions of compounds
Found C, 57.82; H, 5.53; N, 9.61.
were prepared in Middlebrook 7H12 medium (7H9
broth containing 0.1% w/v casitone, 5.6 mg/ml palmitic
acid, 5 mg/ml bovine serum albumin, 4 mg/ml catalase,
filter-sterilizes) in a volume of 100 ml in 96-well micro-
plates (BD OptiluxTM, 96-well Microplates, black/clear
flat bottom; Becton,-Dickenson, Franklin Lakes, NJ
USA). Mtb cultures (100 ml inoculum of 2 ¥ 105 cfu/ml)
were added, yielding a final test volume of 200 ml. The
Biological Assays
Microplate Almar blue assay (MABA)
[Franzblau et al., 1998]
Test compound MICs against Mtb H37RV
(ATCC27294) were assessed by the MABA using RMP
TABLE 1. Anti-TB Activity of Compounds 2a–f and 4a–c
O
X
ROOC
N
O
CH=CH-Ar
2a-f, 4a-c
Compound
R
Ar
X
MABAa MIC (mM)
LORAa MIC (mM)
Vero cell IC50 (mM)
2a
C2H5
H
H
58.2
96.6
95.17
Cl
Cl
2b
2c
C2H5
C2H5
35.5
81.6
109.1
62.3
>128
H
H
108.4
2d
2e
C2H5
C2H5
26.9
54.7
>128
Cl
>128
>128
NDb
2f
C2H5
CH3
H
H
H
H
114.6
26.8
61.2
64.0
101.3
54.1
111.5
88.07
100.6
56.74
4a
4b
4c
iC3H7
iC3H7
>128
82.3
INH
RMP
PA-824
0.4
0.1
0.4
>128
1.9
>128
110
>128
3.0
aMtb strain H37Rv.
bNot determined.
INH, isoniazide; LORA, low oxygen recovery assay; MABA, microplate Alamar blue assay; MIC, minimum inhibitory concentration;
RMP, rifampin.
Drug Dev. Res.

ANTITUBERCULAR AGENTS
9
plates were incubated at 37°C. On the seventh day of fluorescence of Ն 90% relative to the mean of replicate
incubation, 12.5 ml of 20% Tween 80 and 20 ml of bacteria-only controls.
Alamar Blue (Invitrogen BioSourceTM; Life Technolo-
gies, Grand Island, NY USA) were added to the wells. Low-Oxygen-Recovery Assay (LORA)
After incubation at 37°C for 16–24 h, well fluorescence [Cho et al., 2007]
was measured (ex 530, em 590 nm). MICs were defined
A low-oxygen adapted culture of recombinant
as the lowest concentration effecting a reduction in H37Rv (pFCA-luxAB), expressing a Vibrio harveyii
TABLE 2. Anti-TB Activity of Compounds 8a–g and 10a–e
ROOC
N
Ar
O
8a-g, 10a-e
Compound
R
Ar
MABAa MIC (mM)
LORAa MIC (mM)
Vero cell IC50 (mM)
8a
8b
C2H5
C2H5
3.3
2.7
30.1
25.3
102
>128
S
8c
8d
C2H5
C2H5
3.5
2.2
58.9
13.1
>128
>128
S
O
O
NDb
NDb
O
8e
8f
C2H5
C2H5
>128
>128
>128
>128
N
O
8g
10a
10b
C2H5
iC3H7
iC3H7
>128
2.3
>128
63.5
26.6
NDb
>128
>128
11.4
S
10c
10d
iC3H7
iC3H7
2.4
5.6
14.0
7.3
>128
>128
S
N
O
O
O
10e
iC3H7
>128
>128
NDb
O
INH
RMP
PA-824
0.4
0.1
0.4
>128
1.9
>128
>128
>128
3.0
aMtb strain H37Rv.
bNot determined.
INH, isoniazide; LORA, low oxygen recovery assay; MABA, microplate Alamar blue assay; MIC, minimum inhibitory concentration;
RMP, rifampin.
Drug Dev. Res.

10
VERGELLI ET AL.
luciferase gene with an acetamidase promoter, was differ only by a chlorine in the para–position of benzoyl
grown in a BiostatQ fermentor (Sartorius Group, Got- fragment. Compound 2d was one of the more active in
tingen, Germany). Cells were collected on ice, washed this series (MIC = 26.9 mM) with 2e being inactive
in PBS, and stored at -80°C. Approximately 105 cfu/ml (MIC > 128 mM), suggesting that the chlorine is detri-
of thawed NRP cells were exposed to twofold serial mental to activity. For most compounds, there is a cor-
dilutions of test compound in 7H9 broth in 96-well respondence between MABA and LORA values and
plates, incubated for 10 days anaerobically at 37°C. with the exception of compounds 2b and 2d (Vero cell
Luminescence readings were obtained following a 28-h IC50 > 128 mM), they also showed little in vitro cytotox-
recovery in an aerobic environment (5% CO2). The data icity (Vero cell 56.74 < IC50 < 128 mM)
were analyzed graphically, and the lowest concentration
Data on the series of 5-(hetero)aryl-4-
of test compound preventing metabolic recovery (90% unsubstituted isoxazoles 8a–g and 10a–e are shown in
reduction relative to untreated cultures) was deter- Table 2. With the exception of compounds 8e, 8f, 8g,
mined as described previously.
and 10e improved activity was observed as compared
with the previous compounds. The residue R of the
ester function can be changed as the 3-ethyl and the
3-isopropyl ester derivatives show comparable activity.
However, there is a crucial role for the substituent
at position 5. Compounds with a diphenyl, thiophene,
benzothiophene, or a methoxybenzofurane had MIC
values in the low micromolar range (MIC = 2.2–
5.6 mM), whereas the introduction of aromatic portions
endowed with major bulk (fluorene and anthracene
nucleus) resulted to inactive compounds (8f and 8g,
MIC > 128 mM). The same inactivity was observed for
the two 5-(2,6-dimethoxypiridine) derivatives, 8e and
10e, that contain a strongly basic nitrogen. An analo-
gous activity trend occurred in the LORA, whereas
MIC values were approximately an order of magnitude
higher than in MABA. Moreover, with the exception of
8a, all active compounds had no toxicity in Vero cell
with IC50 values > 128 mM. These initial results suggest
that position 4 of the isoxazole ring must be unsubsti-
tuted and the ethylenic spacer must be deleted from
position 5 and the aromatic portion, with an appropriate
steric hindrance directly linked to C-5 isoxazole.
Further studies are in progress to better define the
requirements in the isoxazole scaffold to improve anti-
tubercular activity.
Cytotoxicity Assay [Falzari et al., 2005]
Cytotoxicity was determined by exposing different
concentrations of samples to Vero cells. Briefly, samples
were dissolved at 12.8 mM in DMSO. Six threefold
dilutions were performed in growth medium MEM
(Gibco, Grand Island, NY, USA), containing 10% fetal
bovine serum (HyClone, Logan, UT, USA), 25 mM N-
(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid
(HEPES, Gibco), 0.2% NaHCO3 (Gibco), and 2 mM
glutamine (Irvine Scientific, Santa Ana, CA, USA).
Final DMSO concentrations did not exceed 1% v/v.
Compound dilutions were assayed in duplicate in 96-
well tissue culture plates (Becton Dickinson Labware,
Lincoln Park, NJ, USA) in a volume of 50 ml per well.
An equal volume containing 5 ¥ 105 log phase Vero cells
(CCL-81; ATTC, Rockville, MD, USA) was added to
each well, and cultures were incubated at 37°C in 5%
CO2 atmosphere. After 72 h, cell viability was measured
using the CellTiter 96 aqueous nonradioactive cell pro-
liferation assay (Promega Corp. Madison, WI, USA)
according to the manufacturer’s instructions. Absor-
bance at 490 nm was read in a Victor2 multilabel reader
(Perkin Elmer). The IC50 values were determined using
a curve-fitting program.
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