Synthesis, optimization and structure-activity relationships of 3,5-disubstituted isoxazolines as new anti-tuberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2008.04.007

In the course of the development of a potent series of nitrofuranylamide anti-tuberculosis agents, we investigated if the exceptional activity resulted in part from the isoxazoline core and if it possessed any intrinsic anti-tuberculosis activity. This led to the discovery of an isoxazoline ester with appreciable anti-tuberculosis activity. In this study we explored the anti-tuberculosis structure-activity relationship of the isoxazoline ester compound through systematic modification of the 3,5-di-substituted isoxazoline core. Two approaches were used: (i) modification of the potentially metabolically labile ester functionality at the 3 position with acids, amines, amides, reverse amides, alcohols, hydrazides, and 1,3,4-oxadiazoles; (ii) substitution of the distal benzyl piperazine ring in the 5 position of the isoxazoline ring with piperazyl-ureas, piperazyl-carbamates, biaryl systems, piperidines and morpholine. Attempts to replace the ester group at C-3 position of isoxazoline with a variety of bioisosteric head groups led to significant loss of the tuberculosis inhibition indicating that an ester is required for anti-tuberculosis activity. Optimization of the isoxazoline C-5 position produced compounds with improved anti-tuberculosis activity, most notably the piperazyl-urea and piperazyl-carbamate analogs.

Full text of DOI:10.1016/j.ejmech.2008.04.007

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 460e472
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, optimization and structureeactivity relationships of 3,
5-disubstituted isoxazolines as new anti-tuberculosis agents
*
Rakesh, Dianqing Sun, Robin B. Lee, Rajendra P. Tangallapally, Richard E. Lee
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center,
847 Monroe Ave Rm327, Memphis, TN 38163, United States
Received 29 December 2007; received in revised form 4 April 2008; accepted 15 April 2008
Available online 29 April 2008
Abstract
In the course of the development of a potent series of nitrofuranylamide anti-tuberculosis agents, we investigated if the exceptional activity
resulted in part from the isoxazoline core and if it possessed any intrinsic anti-tuberculosis activity. This led to the discovery of an isoxazoline
ester with appreciable anti-tuberculosis activity. In this study we explored the anti-tuberculosis structureeactivity relationship of the isoxazoline
ester compound through systematic modification of the 3,5-di-substituted isoxazoline core. Two approaches were used: (i) modification of the
potentially metabolically labile ester functionality at the 3 position with acids, amines, amides, reverse amides, alcohols, hydrazides, and 1,3,4-
oxadiazoles; (ii) substitution of the distal benzyl piperazine ring in the 5 position of the isoxazoline ring with piperazyl-ureas, piperazyl-
carbamates, biaryl systems, piperidines and morpholine. Attempts to replace the ester group at C-3 position of isoxazoline with a variety of
bioisosteric head groups led to significant loss of the tuberculosis inhibition indicating that an ester is required for anti-tuberculosis activity.
Optimization of the isoxazoline C-5 position produced compounds with improved anti-tuberculosis activity, most notably the piperazyl-urea
and piperazyl-carbamate analogs.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Anti-tuberculosis agents; Antibacterial; Nitrofurans; Isoxazolines; Lead optimization
1. Introduction
the drugs in the current tuberculosis regime result from
research performed over 50 years ago [5]. At that time with
the successful introduction of those agents it was widely
believed that tuberculosis could be eliminated and this led to
significant underinvestment in the development of new thera-
peutics to treat tuberculosis for many decades [6]. Today
more people die from tuberculosis than ever before, and this
has catalyzed a renewed effort to develop improved therapies
[7]. Hence, there is an urgent need to develop potent and fast
acting anti-tuberculosis drugs with new modes of action to
overcome the cross-resistance with current drugs and low tox-
icity profiles that can be tolerated for long treatment periods
required for tuberculosis chemotherapy.
Tuberculosis is caused by Mycobacterium tuberculosis,
a deadly obligate bacterial pathogen. The global effect of
tuberculosis is immense [1,2]. According to the World Health
Organization, currently one-third of world’s population is
infected with latent tuberculosis [3]. Based on the trend over
the past few years, a total of 225 million new cases and 79 mil-
lion deaths are expected from tuberculosis between 1998 and
2030. The major concerns for current tuberculosis treatment
are its latency, co-infection with HIV, poor patient compliance,
and drug resistance issues caused by the emergence of multi-
drug resistant tuberculosis (MDR-TB) and the recent advent of
extensively drug resistant tuberculosis (XDR-TB) [4]. Most of
In an ongoing effort to develop novel anti-tuberculosis
therapeutics, previously, we reported a series of nitrofuranyl
compounds with potent inhibitory activity against M. tubercu-
losis [8e11]. The outstanding in vitro activity of Lee-878 from
this series led us to explore if the isoxazoline core was itself
* Corresponding author. Tel.: þ1 901 448 6018; fax: þ1 901 448 6828.
E-mail address: relee@utmem.edu (R.E. Lee).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.04.007

Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
461
privileged and if it had any intrinsic anti-tuberculosis activity.
A series of non-nitrofuran isoxazolines were subsequently
synthesized in which the nitrofuran head group was systemat-
ically replaced. This led to the discovery of isoxazoline ester
1, which showed appreciable anti-tuberculosis activity (MIC:
1.56 mg/mL) (Fig. 1) [12]. Excitingly, this represents a new
synthetically tractable anti-tuberculosis chemotype that has
not been previously reported. The study described herein
concerns our efforts to optimize this hit to increase its meta-
bolic stability and to explore the structural similarity with
the oxazolidinone class of antibiotics including linezolid
[13e15]. The outline of the approach is displayed in Fig. 1,
and features systematic modification and optimization of the
isoxazoline ring to replace the 3 position with bioisosteric
head groups and the 5 position with modified piperazyl
C-rings.
10aed were synthesized from amine 7 by reaction with alkyl
halides in moderate yields (48e58%).
For the second series of analogs, the piperazine C-ring was
completely replaced with a series aryl rings (Scheme 2, I). The
required biaryl systems were synthesized using the Suzuki
chemistry. 4-Bromo styrene 2 was treated with a variety of
aryl boronic acids in the presence of Pd(PPh3)4 using 1 M
aqueous K2CO3 solution in DME at reflux to afford corre-
sponding biaryl compounds 14aed in 61e73% yields. These
substituted olefins were then reacted to form their correspond-
ing isoxazoline derivatives 15aed using identical synthetic
conditions described for the synthesis of compound 6.
For the third series of analogs, the piperazine C-ring was
substituted with a piperidine system (Scheme 2, II). These
analogs were synthesized using the same aryl amination reac-
tion conditions as described earlier for the synthesis of piper-
azine analog 4. Compounds 16a and 16b were prepared by
nucleophilic aromatic amination of 2 with 4-(4-trifluorome-
thoxy-phenoxy)-piperidine and piperidine in 78% and 71%
yields, respectively. The isoxazoline ring was then constructed
on these cores using standard chemistries to afford the target
compounds 17a and 17b in 74% and 77% yields, respectively.
For the fourth series of analogs, the piperazine C-ring was
replaced with heteroaryl oxadiazole rings (Scheme 2, III). The
synthetic approach to this series utilized the synthetic utility
of phenyl substituted cyano group as a synthon for introduction
of heteroaryl rings [18]. Accordingly, 4-cyano styrene was
treated with hydroxylamine hydrochloride in ethanol in the
presence of Et3N to produce amidine intermediate 18. Amidine
18 was treated with triethyl orthoformate and a catalytic amount
of boron trifluoride diethyl ether at 80 ꢀC to give 3-phenyl 1,2,4-
oxadiazole derivative 19a in 59% yield. Amidine 18 was treated
with benzoyl chloride in pyridine at reflux temperature to afford
3,5-disubstituted 1,2,4-oxadiazole derivative 19b in 48% yield.
The olefin moiety in both 19a and 19b was used for the synthesis
of isoxazoline derivatives 20a and 20b, respectively, using stan-
dard procedures in 59% and 60% yields.
2. Chemistry
2.1. Modifications of the C-5 side chain of the
isoxazoline ring
Four series of C-5 analogs were synthesized as described in
Schemes 1 and 2. Our first target was the synthesis of a set of
compounds possessing varying substitutions to the piperazine
C-ring of lead compound 1. N-Boc protected phenyl pipera-
zine 4 was synthesized from 4-bromo styrene 2 and Boc-piper-
azine 3 using standard aromatic amination reaction conditions
in 58% yield (Scheme 1) [16]. Olefin 4 was treated with
commercially available oxime 5 in the presence of Et3N in
dichloromethane at room temperature to afford isoxazoline 6
following a [3 þ 2] regioselective cycloaddition mechanism
in 71% yield [17]. The deprotection of Boc protected
compound 6 was achieved by treating with trifluoroacetic
acid in THF at room temperature to give 7 in quantitative
yield. Compound 7 was used as a key intermediate for the
synthesis of several derivatives. Disubstituted urea derivatives
8aee were synthesized by treating free amine 7 with a variety
of alkyl and aryl isocyanates in greater than 80% yields. A set
of carbamate derivatives 9aee were then synthesized from
amine 7 by reaction with the corresponding alkyl chlorofor-
mates also in 70e89% yields. N-Alkylated derivatives
2.2. Modifications on C-3 side chain of isoxazoline ring
Synthetic modification to the C-3 side chain ester of the
isoxazoline ring in 1 was approached by the synthesis of three
O
N
O
1
5
N
O
A
N
O
O
O
3
B
N
N
N
O
N
C
N
Lee-878
1
COOH
CONHR
O
N
CONHR
COOR
R
X
O
H
F
CONHNH2
COAr
CH2OH
CH2N3
N
N
N
O
O
O
N
Linezolid
X
CH2NH2
CH2NHCOR
X
Fig. 1.

462
Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
H
N
O
N
a
O
b
+
N
N
O
N
N
N
O
O
O
N
O
N
Br
O
O
O
OH
6
O
4
2
3
5
Cl
O
O
c
N
O
NH
7
d
e
f
O
N
N O
O
N
O
O
O
N
O
N
O
O
N
N
O
O
N
N
R
8a R = iPr
HN
9a R = Me
9b R = Et
9c R = Allyl
9d R = iBu
9e R = nBu
R
10a R = Allyl
O
8b R = nPr
8c R = nHex
8d R = Ph
R
10b R = Cyclopropylmethyl
10c R = 4-NO2Bn
10d R = Bn; (methyl ester at 3 position)
8e R = PhCH2CH2
g
g
g
N O
O
N
HO
O
N
HO
HO
O
N
O
N
O
O
O
N
N
N
N
11a R = nHex
11b R = PhCH2CH2
12a R = iBu
12b R = nBu
O
HN
R
R
13 from 10d
Scheme 1. Reagents and conditions: (a) PdCl2[P(o-Tol)3]2, NaOtBu, toluene, 100 ꢀC; (b) Et3N, CH2Cl2; (c) TFA, THF, rt; (d) Et3N, RNCO, CH2Cl2, rt; (e) Et3N,
ROCOCl, CH2Cl2, rt; (f) K2CO3, RBr, DMF; (g) LiOH, THF/H2O, rt.
series of compounds with acid, amine, amide, reverse amide,
alcohol, hydrazide, and 1,3,4-oxadiazole substitutions at the
3 position. The rationale for these substitutions was to use a bi-
oisosteric approach and synthesize derivatives with increased
metabolic stability over labile ester functionality of 1 while
retaining a similar pattern of heteroatoms. For the first series,
free acids with a variety of 5 position substitutions were gen-
erated by hydrolysis of the esters 8c, 8e, 9d, 9e and 10d, using
I:
N
O
O
a
b
Ar
14a Ar = Ph
14b Ar = 4-CHO Ph
14c Ar = 3-F, 4-OMe Ph
14d Ar = 2-furyl
O
Br
Ar
2
15a Ar = Ph
15b Ar = 4-CHO Ph
15c Ar = 3-F, 4-OMe Ph
15d Ar = 2-furyl
II:
N
O
O
c
b
O
N
N
Br
X
X
2
16a x = O(4-OCF3)Ph
16b x = H
17a x = O(4-OCF3)Ph
17b x = H
III:
O
N
O
d
e
b
N
H
N
N
O
O
OH
O
CN
N
N
NH
X
X
20a x = H
20b x = Ph
19a x = H
19b x = Ph
18
Scheme 2. Reagents and conditions: (a) aryl boronic acid, Pd(Ph3P)4, 1 M aqueous K2CO3, DME, reflux; (b) ethyl chlorooximido acetate, Et3N, CH2Cl2, rt; (c)
4-(4-trifluoromethoxy-phenoxy)-piperidine or piperidine, NaOtBu, PdCl2[(o-Tol)3P]2, toluene, reflux; (d) NH2OH$HCl, Et3N, EtOH, reflux; (e) CH(OEt)3,
BF3$OEt2, reflux or PhCOCl, Py, reflux.

Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
463
LiOH, in aqueous THF at room temperature to afford the cor-
3. Anti-tuberculosis activity
responding free acids 11a, 11b, 12a, 12b and 13 in 62e93%
yields (Scheme 1). For the second series, modifications to
the C-3 side chain were carried out keeping the isoxazoline
5 position unaltered as found in compound 1. The ester func-
tionality in 1 was converted into amide 21 in 66% yield by
treating with ammonium hydroxide in 1,4-dioxane at room
temperature (Scheme 3). Propyl amide 23 was synthesized
by hydrolysis of 1, followed by activation of the resulting
free acid in situ as acid chloride and coupling to n-propyl
amine in THF in 59% overall yield. The hydrazide derivative
24 was synthesized by treating the ester with hydrazine
hydrate in ethanol at reflux temperature in 52% yield. Then
24 was used to construct a 1,3,4-oxadiazole ring at C-3 posi-
tion of isoxazoline ring. Accordingly, diacyl hydrazine 25
was synthesized from 24 by treatment with acetyl chloride
in dichloromethane using Et3N as a base in 61% yield. Subse-
quent treatment of 25 with p-TsCl, Et3N in dichoromethane
and 40% aqueous K2CO3 sequentially afforded 2,5-disubsti-
tuted 1,3,4-oxadiazole analog 26 in 58% yield.
For the third series, free amines and reverse amides were
substituted on the C-3 side chain of the isoxazoline ring
producing compounds with structural resemblance to the
oxazolidinone class of antibiotics. The esters in isoxazoline
derivatives 1 and 28, with C-5 N-benzyl piperazine and
morpholine moieties, respectively, were reduced into the
corresponding primary alcohols 29a and 29b by DIBAL-H
in THF at 0 ꢀC in 56% and 60% yields. The primary alcohols
29a and 29b were then converted into their azides 30a and 30b
by treating with CBr4, Ph3P and NaN3 in DMF at room
temperature in 59% and 61% yields, followed by reduction
with Ph3P in 1,4-dioxane to yield primary amines 31a and
31b in 67% and 60% yields. Amines 31a and 31b were finally
treated with acetyl chloride using Et3N in CH2Cl2 to afford
acetamides 32a and 32b in 78% and 71% yields (Scheme 4).
All compounds synthesized with the complete isoxazoline
moiety were tested against M. tuberculosis (H37Rv) using
microbroth dilution and MIC values were determined by vi-
sual inspection [19]. The anti-tuberculosis activities of the
new isoxazolines esters bearing varying substitutions to the
phenyl B in the C-5 position are shown in Table 1. Compounds
with a piperazyl-urea substitution 8aee displayed varying
anti-tuberculosis activity with MICs ranging from 0.4 to
25 mg/mL. Those with simple substitutions of i-propyl 8a
(12.5 mg/mL) and n-propyl 8b (25 mg/mL) were least active
with anti-tuberculosis activity inferior to the original hit com-
pound 1 (1.56 mg/mL). Most active in this series were n-hexyl
8c (0.4 mg/mL) and phenethyl 8e (0.8 mg/mL), which had bet-
ter MIC values than that of 1. Compounds in the piperazyl-car-
bamate series 6 and 9aee all demonstrated equal or 1-fold
more potent anti-tuberculosis activity than the lead compound
1. In a comparative analysis it was notable that the pharmaco-
logically more desirable and smaller side chains were much
better tolerated in the carbamate series than the urea series
(e.g., 9c and 9d over 8a and 8b). In the alkylated piperazine
series 10aed and 1, larger lipophilic substituents were clearly
favored by comparing 1, 10c and 10d over 10a and 10b. Ethyl
ester lead compound 1 and it’s methyl ester 10d had the same
MIC value (1.56 mg/mL). For the biphenyl systems 15aec,
functionalization of the outer phenyl ring was required for
appreciable anti-tuberculosis activity and in general activity
of this series was lower than that of the previous carbamate
series. The biheteroaryl series 15d, 20a, and 20b showed
that a furan substitution 15d was significantly favored over
the isosteric oxadiazole ring 20a and phenyl substitution of
the oxadizole ring 20b restored similar activity to that of furan
15d. para-Substitution of the C-5 phenyl with nitrogen-
containing alkyl rings produced piperazine compound 7 with
N
O
N
O
O
H2N
a
O
N
O
N
N
N
1
21
b
N
O
H
N
N
Li+-O
O
c
O
N
O
N
N
N
22
23
O
N
O
H
N
O
H2NHN
N
d
e
N
H
1
O
N
O
N
N
25
N
24
N
O
O
N
f
H3C
N
N
N
26
Scheme 3. Reagents and conditions: (a) NH4OH, 1,4-dioxane, 12 h, rt; (b) LiOH, H2O/THF; (c) (i) (COCl)2, DCM, cat. DMF; (ii) n-PrNH2, THF; (d)
NH2NH2$H2O, EtOH, reflux; (e) CH3COCl, Et3N, CH2Cl2, rt; (f) (i) p-TsCl2, Et3N, CH2Cl2, reflux; (ii) 40% K2CO3.

464
Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
N
O
O
a
c
b
O
N
N
Br
X
X
1 x = N-Bn
28 x = O
27a x = N-Bn
27b x = O
N
O
N
O
N
O
H2N
N3
HO
e
d
N
N
N
X
X
X
31a x = N-Bn
31b x = O
29a x = N-Bn
29b x = O
30a x = N-Bn
30b x = O
H
N
N
O
f
O
N
X
32a x = N-Bn
32b x = O
Scheme 4. Reagents and conditions: (a) morpholine or 1-benzyl piperazine, NaOtBu, PdCl2[(o-Tol)3P]2, toluene, reflux; (b) ethyl chlorooximido acetate, Et3N,
CH2Cl2, rt; (c) DIBAL-H, THF, 0 ꢀC; (d) CBr4, Ph3P, NaN3, DMF, rt; (e) Ph3P, 1,4-dioxane; (f) CH3COCl, Et3N,CH2Cl2, rt.
limited activity and morpholine compound 28 with no activity.
In this series, the simple piperidine derivative 17b was the
most active and it was notably more active than the more com-
plex and lipophilic 4-(4-trifluoromethoxy-phenoxy)-piperidine
substitution 17a that resembles the side chain found in the new
anti-tuberculosis agent OPC-68638 [20].
the serum stability for these esters is extremely short,
precluding the advancement of this class for testing of their
efficacy in animal models of tuberculosis. Presumably, the ac-
tive compounds are taken up as esters by the tubercle bacilli
in vitro and then hydrolyzed to active free acids once the
compounds are internalized in the bacteria. The free acids
of the corresponding active esters are presumably inactive
in vitro as they cannot penetrate the complex M. tuberculosis
cell wall, perhaps in part due to their residual negative
charge. The structural resemblance of this series to the oxazo-
lidinone class of antibiotics was also explored through the
synthesis of reverse amides 32b with high structural similar-
ity to linezolid (Fig. 1). However, 32b was inactive in con-
trast to linezolid, which has good anti-tuberculosis activity
[21]. This suggests that this series of compounds works
through a different mechanism of action, a hypothesis that is
further supported by the lack of antimicrobial activity of these
compounds against other bacteria including Staphylococcus
aureus, Streptococcus pneumoniae and Escherichia coli.
The anti-tuberculosis activity for the optimization of C-3
isoxazoline ester position is shown in Table 2. Hydrolysis of
the esters to free acids 11a, 11b, 12a, 12b, 13, and 22 led
to a complete loss in activity when compared to their cor-
responding esters 8c, 8e, 9d, 9e, 10d, and 1 regardless of
C-5 substitution. The loss of activity is likely attributed to
poor cell membrane penetration. Ester bioisosteric replace-
ments such as primary amide 21, n-propyl amide 23, hydra-
zide 24 and 1,3,4-oxadiazole 26 were also largely inactive.
Potential metabolite alcohols 29a and 29b were inactive
when compared to the corresponding esters. Finally, azides
30a, 30b, primary amines 31a, 31b and reverse amides
32a, 32b that mimic linezolid functionalization also had
negligible anti-tuberculosis activity. These findings strongly
suggest that an ester functionality at the C-3 position of iso-
xazoline ring is absolutely required for anti-tuberculosis ac-
tivity in this series.
5. Experimental
All the anhydrous solvents and starting materials were
purchased from Aldrich Chemical Co. (Milwaukee, WI).
All reagent grade solvents used for chromatography were
purchased from Fisher Scientific (Suwanee, GA) and flash
column chromatography silica cartridges were obtained
from Biotage Inc. (Lake Forest, VA). The reactions were
monitored by thin-layer chromatography (TLC) on pre-
coated Merck 60 F254 silica gel plates and visualized using
UV light (254 nm). A Biotage FLASH column chromatogra-
4. Conclusions
There is a clear need to develop novel anti-tuberculosis
agents that are chemically tractable and possess drug-like
properties. In this study a new interesting chemotype with
anti-tuberculosis activity was examined. Optimization of the
isoxazoline C-5 position produced compounds with improved
anti-tuberculosis activity, including most notably urea analog
(8c) and carbamates (9cee). Attempts to replace the metabol-
ically labile ester group at the C-3 position of isoxazoline
with a variety of bioisosteric head groups led to complete
loss of the tuberculosis inhibition. This is unfortunate because
1
phy system was used to purify mixtures. All H NMR spectra
were recorded on a Varian INOVA-500 spectrometer. Chem-
ical shifts (d) are reported in parts per million relative to the
residual solvent peak or internal standard (tetramethylsilane),
and coupling constants (J ) are reported in hertz (Hz). Mass

Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
465
Table 1
In vitro anti-tuberculosis activity of C-5 modified isoxazoline ester analogs
Compd Structure MIC (mg/mL) Compd Structure
MIC (mg/mL)
O
O
N
N
O
O
O
1
1.56
0.8
10a
6.25
N
N
N
N
O
O
O
O
O
O
N
N
N
O
O
O
6
10b
10c
10d
12.5
0.8
N
N
N
N
N
N
O
O
O
O
O
NO2
N
N
O
O
7
50
N
N
NH
N
O
O
O
N
H
N
O
8a
12.5
25
1.56
N
N
O
O
O
O
O
N
N
N
H
N
O
N
8b
15a
100
N
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
H
H
N
O
O
O
O
O
O
O
O
O
O
8c
8d
8e
9a
9b
9c
9d
9e
0.4
15b
15c
15d
17a
17b
20a
20b
28
6.25
3.125
3.25
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
F
O
O
N
N
H
N
6.25
O
O
O
O
H
N
O
O
0.8
F
N O
F
O
1.56
1.56
0.8
25
O
O
O
O
O
F
N
O
O
O
O
N
O
O
N
6.25
O
O
N
N
O
50
N
N
N
O
O
N
0.8
3.125
O
O
O
O
O
N
0.8
N
>200
O
spectra were recorded on a Bruker Esquire LCMS using ESI.
Purity of the products was confirmed before testing by
analytical RP-HPLC on Shimadzu HPLC system. Gradient
conditions. solvent A (0.1% TFA in water) and solvent B
(acetonitrile): 0e2.00 min 100% A, 2.00e7.00 min
0e100% B (linear gradient), 7.00e8.00 min 100% B, UV
detection at 254 nm.
5.1. General procedure for aryl amination reactions
A mixture of 4-bromo styrene (1 mmol), amine (1.3 mmol),
NaOtBu (1.5 mmol) and PdCl2[P(o-Tol)3]2 (0.03 mmol) in tol-
uene was heated to reflux for 4 h. The solvent was evaporated
under reduced pressure and the crude residue was purified by
flash chromatography.

466
Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
Table 2
In vitro anti-tuberculosis activity of C-3 modified isoxazoline analogs
Compd
Structure
MIC (mg/mL)
Compd
Structure
MIC (mg/mL)
N
O
O
N
O
HO
H
N
N
11a
>200
26
200
N
H3C
N
N
N
O
N
O
O
O
O
N
O
N
HO
H
N
N
N
N
11b
12a
12b
13
>200
>200
>200
>200
200
29a
29b
30a
30b
31a
31b
200
>200
50
N
N
N
HO
N
N
N
O
O
O
O
O
N
O
N
HO
HO
O
O
O
N
O
O
N
HO
N3
N
N
O
O
N
N
HO
200
N3
N
N
N
O
O
O
N
O
N
H2N
21
200
H2N
H2N
N
N
N
N
O
O
N
O
N
Li+-O
22
100
>200
N
N
O
N
N
N
O
O
N
O
N
H
N
H
23
24
200
50
32a
32b
>200
>200
N
N
N
O
O
O
O
N
N
H
N
H3C
H2NHN
N
N
O
N
O
O
5.2. Representative aryl amination
was added in portions and stirred at room temperature over-
night. The reaction mixture was washed with water, brine,
dried (anhyd. Na2SO4), concentrated under reduced pressure
and crude products were purified by flash chromatography.
5.2.1. 4-(4-Vinyl-phenyl)-piperazine-1-carboxylic
acid tert-butyl ester (4)
A mixture of 4-bromo styrene (1.0 g, 5.46 mmol), 1-Boc-
piperazine (1.32 g, 7.10 mmol), sodium tert-butoxide (0.78 g,
8.19 mmol) and PdCl2[P(o-Tol)3]2 (0.12 g, 0.16 mmol) was
heated in anhydrous toluene (20 mL) at 100 ꢀC under argon
for 3 h. The reaction mixture was then concentrated under
reduced pressure and subjected to flash column chromatogra-
phy to give 4 (0.91 g) in 58% yield. 1H NMR (500 MHz,
CDCl3): d 1.51 (9H, s), 3.18 (4H, s), 3.61 (4H, s), 5.13 (1H,
d, J ¼ 10.7 Hz), 5.63 (1H, d, J ¼ 17.5 Hz), 6.67 (1H, dd,
J ¼ 10.9, 17.5 Hz), 6.88e6.95 (2H, br d), 7.36 (2H, d,
J ¼ 8.7 Hz); ESI MS: 311.8 (M þ 23).
5.3.1. 4-[4-(3-Ethoxycarbonyl-4,5-dihydro-isoxazol-
5-yl)-phenyl]-piperazine-1-carboxylic acid
tert-butyl ester (6)
To a stirred solution of olefin 4 (0.8 g, 2.77 mmol) in
CH2Cl2 (10 mL), Et3N (0.77 mL, 5.55 mmol) and ethyl chlor-
ooximido acetate (0.631 g, 4.16 mmol) were added at 0 ꢀC and
stirred at room temperature overnight. The reaction mixture
was diluted with excess CH2Cl2 (20 mL) and washed with
water (20 mL), dried (anhyd. Na2SO4), concentrated under
reduced pressure and the crude residue was purified by flash
chromatography to afford 6 (0.788 g) in 71% yield. 1H
NMR (300 MHz, CDCl3): d 1.39 (3H, t, J ¼ 7.1 Hz), 1.49e
1.51 (9H, s), 3.13e3.27 (5H, m), 3.52e3.65 (5H, m), 4.38
(2H, q, J ¼ 7.1 Hz), 5.72 (1H, dd, J ¼ 9.3, 11.4 Hz), 6.92
(2H, d, J ¼ 8.7 Hz), 7.24 (2H, d, J ¼ 8.7 Hz). ESI MS: 426.3
(M þ Na); HPLC purity: 97%, tR ¼ 6.6 min.
5.3. General procedure for the synthesis
of isoxazoline derivatives
To a cooled (0 ꢀC) solution of olefin (1 mmol) and Et3N
(2 mmol) in CH2Cl2, ethyl chlorooximido acetate (1.5 mmol)

Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
467
5.3.2. 5-(4-Piperazin-1-yl-phenyl)-4,5-dihydro-
isoxazole-3-carboxylic acid ethyl ester (7)
150.66, 150.78, 157.17, 160.16; ESI MS: 453.3 (M þ Na);
HPLC purity: 98%, tR ¼ 6.4 min.
A solution of 6 (0.8 g, 1.98 mmol) in THF/TFA (1:1,
10 mL) was stirred at room temperature for 1 h. The reaction
mixture was concentrated under reduced pressure to give 7
5.4.4. 5-[4-(4-Phenylcarbamoyl-piperazin-1-yl)-
phenyl]-4,5-dihydro-isoxazole-3-carboxylic
acid ethyl ester (8d)
1
(0.552 g) in 92% yield. H NMR (500 MHz, CDCl3): d 1.41
(3H, t, J ¼ 7.1 Hz), 3.22 (1H, dd, J ¼ 9.0, 17.8 Hz), 3.34e
3.42 (4H, br s), 3.43e3.52 (4H, br s), 3.62 (1H, dd,
J ¼ 11.5, 17.8 Hz), 4.39 (2H, q, J ¼ 7.1 Hz), 5.75 (1H, dd,
J ¼ 9.3, 11.5 Hz), 6.95 (2H, d, J ¼ 8.8 Hz), 7.29 (2H, d,
J ¼ 8.5 Hz), 9.62 (2H, br s); 13C NMR (500 MHz, CDCl3):
d 14.15, 41.08, 43.41, 46.71, 62.24, 84.83, 117.31, 127.45,
132.28, 150.23, 151.26, 160.65; ESI MS: 304.2 (M þ 1);
HPLC purity: 98%, tR ¼ 4.7 min.
1
Yield: 95.5%; H NMR (500 MHz, CDCl3): d 1.37 (3H, t,
J ¼ 7.1 Hz), 3.17e3.26 (5H, m), 3.54e3.66 (5H, m), 4.36
(2H, q, J ¼ 7.1 Hz), 5.71 (1H, dd, J ¼ 9.3, 11.5 Hz), 6.67
(1H, s), 6.89 (2H, d, J ¼ 8.5 Hz), 7.04 (1H, t, J ¼ 7.3 Hz),
7.24 (2H, t, J ¼ 8.8 Hz), 7.28 (2H, m), 7.37 (2H, d,
J ¼ 7.6 Hz); 13C NMR (300 MHz, CDCl3): d 13.59, 40.36,
43.36, 48.16, 61.57, 84.57, 115.68, 119.63, 122.73, 126.78,
128.35, 129.87, 138.41, 150.65, 150.71, 154.54, 160.15; ESI
MS: 445.2 (M þ Na); HPLC purity: 96%, tR ¼ 6.0 min.
5.4. General procedure for the synthesis of urea
and carbamate derivatives
5.4.5. 5-[4-(4-Phenethylcarbamoyl-piperazin-1-yl)-
phenyl]-4,5-dihydro-isoxazole-3-carboxylic
acid ethyl ester (8e)
Piperazine TFA salt 7 (1 mmol) was dissolved in anhydrous
dichloromethane, and then triethylamine (4 mmol) was added,
followed by isocyanate or chloroformate (3 mmol). The reaction
mixture was stirred at room temperature overnight and evapo-
rated. The residue was purified by column chromatography.
1
Yield: 82.6%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
J ¼ 7.1 Hz), 2.85 (2H, t, J ¼ 6.8 Hz), 3.17 (4H, t,
J ¼ 5.1 Hz), 3.21 (1H, dd, J ¼ 9.3, 17.8 Hz), 3.47 (2H, t,
J ¼ 4.9 Hz), 3.52 (2H, q, J ¼ 6.6 Hz), 3.57 (1H, dd, J ¼ 11.5,
17.8 Hz), 4.37 (2H, q, J ¼ 7.3 Hz), 4.48 (1H, t, J ¼ 5.4 Hz),
5.71 (1H, dd, J ¼ 9.3, 11.2 Hz), 6.89 (2H, d, J ¼ 8.8 Hz),
7.19e7.25 (5H, m), 7.32 (2H, t, J ¼ 7.3 Hz); 13C NMR
(500 MHz, CDCl3): d 13.59, 35.75, 40.36, 41.47, 43.01,
48.12, 61.53, 84.55, 115.65, 125.91, 126.74, 128.08, 128.30,
129.82, 138.76, 150.64, 150.74, 156.90, 160.18; ESI MS:
473.2 (M þ Na); HPLC purity: 98%, tR ¼ 6.1 min.
5.4.1. 5-[4-(4-Isopropylcarbamoyl-piperazin-1-yl)-phenyl]-
4,5-dihydro-isoxazole-3-carboxylic acid ethyl ester (8a)
1
Yield: 91.1%; H NMR (500 MHz, CDCl3): d 1.20 (6H, d,
J ¼ 6.6 Hz), 1.41 (3H, t, J ¼ 7.1 Hz), 3.21e3.26 (5H, m),
3.53e3.63 (5H, m), 4.02 (1H, m), 4.29 (1H, d, J ¼ 6.6 Hz),
4.39 (2H, q, J ¼ 7.1 Hz), 5.74 (1H, dd, J ¼ 9.0, 11.2 Hz),
6.95 (2H, d, J ¼ 8.3 Hz), 7.27 (2H, d, J ¼ 8.5 Hz); 13C NMR
(500 MHz, CDCl3): d 14.17, 23.49, 40.96, 42.71, 43.44,
48.92, 62.17, 85.07, 116.45, 127.36, 151.23, 156.97, 160.73;
ESI MS: 411.3 (M þ Na); HPLC purity: 99%, tR ¼ 5.6 min.
5.4.6. 4-[4-(3-Ethoxycarbonyl-4,5-dihydro-isoxazol-5-yl)-
phenyl]-piperazine-1-carboxylic acid methyl ester (9a)
1
Yield: 76.3%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
5.4.2. 5-[4-(4-Propylcarbamoyl-piperazin-1-yl)-
phenyl]-4,5-dihydro-isoxazole-3-carboxylic
acid ethyl ester (8b)
J ¼ 7.1 Hz), 3.10e3.25 (5H, m), 3.53e3.67 (5H, m), 3.73
(3H, s), 4.37 (2H, q, J ¼ 7.1 Hz), 5.71 (1H, dd, J ¼ 9.8,
11.0 Hz), 6.91 (2H, d, J ¼ 8.5 Hz), 7.24 (2H, d, J ¼ 8.3 Hz);
13C NMR (500 MHz, CDCl3): d 14.17, 40.93, 43.59, 49.01,
52.76, 62.15, 85.12, 116.54, 127.32, 130.54, 151.22, 151.48,
155.87, 160.74; ESI MS: 384.1 (M þ Na); HPLC purity:
98%, tR ¼ 5.8 min.
1
Yield: 97.8%; H NMR (500 MHz, CDCl3): d 0.93 (3H, t,
J ¼ 7.1 Hz), 1.38 (3H, t, J ¼ 7.1 Hz), 1.49e1.60 (2H, m),
3.10e3.28 (7H, m), 3.46e3.64 (5H, m), 4.37 (2H, q,
J ¼ 7.1 Hz), 4.55 (1H, br s), 5.71 (1H, t, J ¼ 10.0 Hz), 6.90
(2H, d, J ¼ 8.1 Hz), 7.23 (2H, d, J ¼ 8.1 Hz); 13C NMR
(500 MHz, CDCl3): d 11.42, 14.17, 23.46, 40.92, 42.71,
43.60, 48.71, 62.15, 85.16, 116.22, 127.34, 130.31, 151.23,
151.33, 157.71, 160.74; ESI MS: 411.3 (M þ Na); HPLC pu-
rity: 100%, tR ¼ 5.6 min.
5.4.7. 4-[4-(3-Ethoxycarbonyl-4,5-dihydro-isoxazol-5-yl)-
phenyl]-piperazine-1-carboxylic acid ethyl ester (9b)
Yield: 89.1%; 1H NMR (500 MHz, CDCl3): d 1.27 (3H, t),
1.38 (3H, t), 3.16 (4H, t, J ¼ 4.9 Hz), 3.21 (1H, dd, J ¼ 9.3,
17.8 Hz), 3.57 (1H, dd, J ¼ 11.5, 17.6 Hz), 3.63 (4H, t,
J ¼ 4.9 Hz), 4.17 (2H, q, J ¼ 7.1 Hz), 4.37 (2H, q,
J ¼ 7.1 Hz), 5.71 (1H, dd, J ¼ 9.3, 11.2 Hz), 6.91 (2H, d,
J ¼ 8.8 Hz), 7.24 (2H, d, J ¼ 8.8 Hz); 13C NMR (500 MHz,
CDCl3): d 14.17, 14.70, 40.93, 43.51, 49.03, 61.56, 62.15,
85.14, 116.52, 127.32, 130.49, 151.22, 151.52, 155.49,
160.75; ESI MS: 398.3 (M þ Na); HPLC purity: 96%,
tR ¼ 6.1 min.
5.4.3. 5-[4-(4-Hexylcarbamoyl-piperazin-1-yl)-phenyl]-4,
5-dihydro-isoxazole-3-carboxylic acid ethyl ester (8c)
1
Yield: 100%; H NMR (500 MHz, CDCl3): d 0.91e0.97
(3H, t), 1.22e1.41 (9H, m), 1.40e1.58 (2H, m), 3.16e3.27
(7H, m), 3.48e3.61 (5H, m), 4.36 (2H, q, J ¼ 7.1 Hz), 4.58
(1H, t), 5.71 (1H, dd, J ¼ 9.5, 11.0 Hz), 6.90 (2H, d,
J ¼ 8.5 Hz), 7.23 (2H, d, J ¼ 8.8 Hz); 13C NMR (300 MHz,
CDCl3): d 13.45, 13.58, 22.01, 26.08, 29.69, 31.01, 40.35,
40.50, 43.07, 48.15, 61.52, 84.57, 115.63, 126.74, 129.74,

468
Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
5.4.8. 4-[4-(3-Ethoxycarbonyl-4,5-dihydro-isoxazol-5-yl)-
115.26, 117.64, 126.68, 129.05, 134.26, 150.65, 151.10,
160.22; ESI MS: 366.3 (M þ Na); HPLC purity: 98%,
tR ¼ 4.9 min.
phenyl]-piperazine-1-carboxylic acid allyl ester (9c)
1
Yield: 69.6%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
J ¼ 7.1 Hz), 3.12e3.28 (5H, m), 3.57 (1H, dd, J ¼ 11.5,
17.8 Hz), 3.65 (4H, t, J ¼ 4.9 Hz), 4.37 (2H, q, J ¼ 7.1 Hz),
4.62 (2H, d, J ¼ 5.4 Hz), 5.23 (1H, d, J ¼ 10.5 Hz), 5.32
(1H, d, J ¼ 17.1 Hz), 5.71 (1H, dd, J ¼ 9.5, 11.0 Hz), 5.96
(1H, m), 6.92 (2H, d, J ¼ 8.5 Hz), 7.24 (2H, d, J ¼ 8.3 Hz);
13C NMR (500 MHz, CDCl3): d 14.15, 40.91, 43.60, 49.01,
62.13, 66.19, 85.10, 116.52, 117.60, 127.30, 130.54, 132.93,
151.20, 151.45, 155.04, 160.72; ESI MS: 410.3 (M þ Na);
HPLC purity: 98%, tR ¼ 6.3 min.
5.5.2. 5-[4-(4-Cyclopropylmethyl-piperazin-1-yl)-
phenyl]-4,5-dihydro-isoxazole-3-carboxylic
acid ethyl ester (10b)
1
Yield: 57.8%; H NMR (500 MHz, CDCl3): d 0.14 (2H, q,
J ¼ 4.6 Hz), 0.53e0.57 (2H, m), 0.86e0.94 (1H, m), 1.38 (3H,
t, J ¼ 7.1 Hz), 2.31 (2H, d, J ¼ 6.4 Hz), 2.68 (4H, t,
J ¼ 5.1 Hz), 3.18e3.28 (5H, m), 3.56 (1H, dd, J ¼ 11.5,
17.8 Hz), 4.36 (2H, q, J ¼ 7.1 Hz), 5.70 (1H, dd, J ¼ 9.5,
11.5 Hz), 6.92 (2H, d, J ¼ 8.8 Hz), 7.22 (2H, d, J ¼ 8.8 Hz);
13C NMR (500 MHz, CDCl3): d 4.19, 8.59, 14.41, 41.06,
48.96, 53.38, 62.34, 64.02, 85.55, 116.05, 127.51, 129.79,
151.46, 151.96, 161.03; ESI MS: 380.2 (M þ Na); HPLC pu-
rity: 96%, tR ¼ 5.0 min.
5.4.9. 4-[4-(3-Ethoxycarbonyl-4,5-dihydro-isoxazol-
5-yl)-phenyl]-piperazine-1-carboxylic
acid isobutyl ester (9d)
1
Yield: 82.0%; H NMR (500 MHz, CDCl3): d 0.95 (6H, d,
J ¼ 6.6 Hz), 1.38 (3H, t, J ¼ 6.8 Hz), 1.96 (1H, m), 3.12e3.27
(5H, m), 3.53e3.68 (5H, m), 3.90 (2H, d, J ¼ 6.6 Hz), 4.37
(2H, q, J ¼ 6.8 Hz), 5.71 (1H, dd, J ¼ 9.8, 10.7 Hz), 6.92
(2H, d, J ¼ 8.1 Hz), 7.24 (2H, d, J ¼ 8.3 Hz); 13C NMR
(500 MHz, CDCl3): d 14.17, 19.14, 28.03, 40.93, 43.51,
49.02, 62.14, 71.71, 85.13, 116.51, 127.33, 130.50, 151.22,
151.50, 155.54, 160.74; ESI MS: 426.3 (M þ Na); HPLC pu-
rity: 89%, tR ¼ 6.6 min.
5.5.3. 5-{4-[4-(4-Nitro-benzyl)-piperazin-1-yl]-
phenyl}-4,5-dihydro-isoxazole-3-carboxylic
acid ethyl ester (10c)
1
Yield: 57.9%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
J ¼ 7.1 Hz), 2.62 (4H, t, J ¼ 4.4 Hz), 3.18e3.28 (5H, m),
3.56 (1H, dd, J ¼ 11.7, 17.8 Hz), 3.65 (2H, s), 4.37 (2H, q,
J ¼ 7.1 Hz), 5.71 (1H, dd, J ¼ 9.8, 10.7 Hz), 6.90 (2H, d,
J ¼ 8.5 Hz), 7.22 (2H, d, J ¼ 8.5 Hz), 7.55 (2H, d,
J ¼ 8.3 Hz), 8.20 (2H, d, J ¼ 8.5 Hz); 13C NMR (500 MHz,
CDCl3): d 14.41, 41.09, 49.03, 53.32, 62.37, 85.49, 116.17,
123.86, 127.54, 129.76, 130.07, 146.34, 151.47, 151.78,
161.02; ESI MS: 461.1 (M þ Na); HPLC purity: 99%,
tR ¼ 5.3 min.
5.4.10. 4-[4-(3-Ethoxycarbonyl-4,5-dihydro-isoxazol-5-yl)-
phenyl]-piperazine-1-carboxylic acid butyl ester (9e)
1
Yield: 84.9%; H NMR (500 MHz, CDCl3): d 0.95 (3H, t,
J ¼ 6.8 Hz), 1.34e1.45 (5H, m), 1.60e1.68 (2H, m), 3.10e
3.26 (5H, m), 3.53e3.68 (5H, m), 4.12 (2H, t, J ¼ 5.9 Hz),
4.37 (2H, q, J ¼ 6.8 Hz), 5.71 (1H, dd, J ¼ 10.0, 10.3 Hz),
6.92 (2H, d, J ¼ 7.6 Hz), 7.24 (2H, d, J ¼ 8.1 Hz); 13C NMR
(500 MHz, CDCl3): d 13.81, 14.17, 19.22, 31.09, 40.93,
43.52, 49.02, 62.14, 65.50, 85.13, 116.51, 127.32, 130.49,
151.22, 151.51, 155.57, 160.74; ESI MS: 426.3 (M þ Na);
HPLC purity: 91%, tR ¼ 6.7 min.
5.5.4. 5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-dihydro-
isoxazole-3-carboxylic acid methyl ester (10d)
Complete ester transformation occurred during the post-
1
workup evaporation process in methanol. Yield: 61.2%; H
NMR (500 MHz, CDCl3): d 2.60 (4H, t, J ¼ 4.2 Hz), 3.18e
3.26 (5H, m), 3.51e3.60 (3H, m), 3.90 (3H, s), 5.70 (1H,
dd, J ¼ 10.0, 10.7 Hz), 6.89 (2H, d, J ¼ 8.5 Hz), 7.20 (2H, d,
J ¼ 8.5 Hz), 7.24e7.38 (5H, m); 13C NMR (500 MHz,
CDCl3): d 40.71, 48.75, 52.83, 52.97, 63.08, 85.45, 115.81,
127.20, 127.29, 128.32, 129.22, 129.39, 137.96, 150.98,
151.75, 161.21; ESI MS: 402.2 (M þ Na); HPLC purity:
98%, tR ¼ 5.0 min.
5.5. General procedure for the synthesis
of alkylated derivatives
Piperazine TFA salt 7 (1 mmol) was dissolved in anhydrous
dimethylformamide, and then K2CO3 (3 mmol) and alkyl bro-
mide (1.2 mmol) were added. The reaction mixture was stirred
at room temperature overnight and evaporated, and the residue
was purified by column chromatography on silica gel.
5.6. General procedures for the synthesis of free acids
5.5.1. 5-[4-(4-Allyl-piperazin-1-yl)-phenyl]-4,5-dihydro-
isoxazole-3-carboxylic acid ethyl ester (10a)
Ethyl ester (1 mmol) was dissolved in tetrahydrofuran/wa-
ter (8 mL) and then lithium hydroxide hydrate (1 mmol) was
added. The reaction mixture was stirred at room temperature
for 3 h, the solvent was evaporated in vacuo at ambient tem-
perature, and then H2O (10 mL) was added and extracted
with ethyl acetate (40 mL). The organic phase was washed
with H2O (3 ꢁ 10 mL), dried (anhyd. Na2SO4) and evaporated
to give free acid. Acid 13 was obtained by the evaporation of
the combined aqueous phases, analytical sample was obtained
1
Yield: 48.3%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
J ¼ 7.3 Hz), 2.60 (4H, t, J ¼ 5.1 Hz), 3.06 (2H, d,
J ¼ 6.6 Hz), 3.18e3.25 (5H, m), 3.56 (1H, dd, J ¼ 11.5,
17.6 Hz), 4.36 (2H, q, J ¼ 7.3 Hz), 5.19 (1H, dd, J ¼ 0.7,
10.0 Hz), 5.23 (1H, dd, J ¼ 1.7, 17.3 Hz), 5.70 (1H, dd,
J ¼ 9.5, 11.5 Hz), 5.85e5.92 (1H, m), 6.91 (2H, d,
J ¼ 8.8 Hz), 7.22 (2H, d, J ¼ 8.8 Hz); 13C NMR (300 MHz,
CDCl3): d 13.59, 40.28, 48.19, 52.38, 61.18, 61.49, 84.71,

Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
469
as white powder after the solid residue was washed with min-
imum amount of ice-H2O, filtered, and dried.
60.36, 84.23, 115.38, 127.48, 128.13, 128.42, 129.86,
130.04, 150.31, 152.46, 161.59; ESI MS: 366.3 (M þ H);
HPLC purity: 100%, tR ¼ 4.8 min.
5.6.1. 5-[4-(4-Hexylcarbamoyl-piperazin-1-yl)-phenyl]-
4,5-dihydro-isoxazole-3-carboxylic acid (11a)
5.7. General procedure for the synthesis of biaryl
compounds 14aed and isoxazoline compounds 15aed
1
Yield: 62.7%; H NMR (500 MHz, CDCl3): d 0.87 (3H, t,
J ¼ 6.0 Hz), 1.22e1.36 (6H, m), 1.46e1.54 (2H, m), 3.10e
3.30 (7H, m), 3.48e3.64 (5H, m), 5.69 (1H, dd, J ¼ 9.5,
9.8 Hz), 6.90 (2H, d, J ¼ 7.6 Hz), 7.21 (2H, d, J ¼ 7.3 Hz);
13C NMR (500 MHz, CDCl3): d 14.09, 22.60, 26.62, 30.10,
30.34, 31.56, 34.25, 40.92, 41.22, 43.49, 48.96, 84.57,
116.57, 125.54, 127.37, 131.14, 135.79, 150.74, 158.18,
162.18; ESI MS: 425.3 (M þ Na); HPLC purity: 98%,
tR ¼ 5.7 min.
A mixture of 4-bromo styrene (1 mmol), aryl boronic acid
(1.1 mmol), Pd(Ph3P)4 (0.03 mmol) and 1 M K2CO3 solution
in dimethoxy ethane was degassed, and purged with argon
twice and heated to reflux overnight. The solvent was evapo-
rated and ethyl acetate was added, the organic phase was
washed with water, brine, dried (anhyd. Na2SO4) and concen-
trated under reduced pressure. The crude residue was purified
by flash chromatography to afford biaryl compounds (14aed)
in 50e70% yields. These olefins were converted into the cor-
responding isoxazolines (15aed) following the general proce-
dure discussed earlier.
5.6.2. 5-[4-(4-Phenethylcarbamoyl-piperazin-1-yl)-phenyl]-
4,5-dihydro-isoxazole-3-carboxylic acid (11b)
Yield: 86.0%; 1H NMR (500 MHz, DMSO-d6): d 2.74 (2H,
t, J ¼ 7.5 Hz), 3.08e3.15 (5H, m), 3.23e3.28 (2H, m), 3.43
(2H, t, J ¼ 5.0 Hz), 3.57 (1H, dd, J ¼ 11.5, 17.8 Hz), 5.68
(1H, dd, J ¼ 10.0, 11.0 Hz), 6.73 (1H, t, J ¼ 5.4 Hz), 6.78
(2H, d, J ¼ 8.8 Hz); 7.18e7.32 (7H, m); 13C NMR
(500 MHz, DMSO-d6): d 36.49, 40.58, 40.84, 42.40, 43.67,
48.45, 84.87, 116.00, 126.43, 128.04, 128.76, 129.13,
130.14, 140.30, 151.59, 153.02, 157.77, 162.17; ESI MS:
445.1 (M þ Na); HPLC purity: 98%, tR ¼ 5.4 min.
5.7.1. 5-Biphenyl-4-yl-4,5-dihydro-isoxazole-3-
carboxylic acid ethyl ester (15a)
1
Yield: 72.6%; H NMR (500 MHz, CDCl3): d 1.39 (3H, t,
J ¼ 7.0 Hz), 3.27 (1H, dd, J ¼ 8.7, 17.5 Hz), 3.66 (1H, dd,
J ¼ 8.7, 17.5 Hz), 4.37 (2H, q, J ¼ 7.0 Hz), 5.83 (1H, dd, J ¼
8.7, 11.4 Hz), 7.34e7.46 (5H, m), 7.57e7.62 (4H, m); ESI
MS: 296 (M þ 1); HPLC purity: 98.6%, tR ¼ 6.8 min.
5.6.3. 5-[4-(4-Isobutoxycarbonyl-piperazin-1-yl)-phenyl]-
5.7.2. 5-(40-Formyl-biphenyl-4-yl)-4,5-dihydro-isoxazole-3-
4,5-dihydro-isoxazole-3-carboxylic acid (12a)
carboxylic acid ethyl ester (15b)
1
Yield: 92.6%; H NMR (500 MHz, CDCl3): d 0.95 (6H, d,
1
Yield: 61%; H NMR (500 MHz, CDCl3): d 1.39 (3H, t,
J ¼ 6.6 Hz), 1.91e2.00 (1H, m), 3.10e3.26 (5H, m), 3.50e
3.72 (5H, m), 3.91 (2H, d, J ¼ 6.6 Hz), 5.74 (1H, t,
J ¼ 9.8 Hz), 6.97 (2H, d, J ¼ 7.6 Hz), 7.24 (2H, d,
J ¼ 6.8 Hz); 13C NMR (500 MHz, CDCl3): d 19.12, 27.99,
30.35, 43.38, 49.47, 72.05, 85.80, 117.07, 125.54, 127.44,
131.36, 150.88, 155.82, 162.66; ESI MS: 376.3 (M þ H);
HPLC purity: 98%, tR ¼ 5.8 min.
J ¼ 7.1 Hz), 3.28 (1H, dd, J ¼ 8.8, 17.5 Hz), 3.66 (1H, dd,
J ¼ 8.7, 17.5 Hz), 4.36 (2H, q, J ¼ 7.2 Hz), 5.83 (1H, dd,
J ¼ 8.7, 11.4 Hz), 7.42e7.45 (2H, m), 7.59e7.67 (4H, m),
7.74 (1H, d, J ¼ 8.0 Hz), 7.96 (1H, d, J ¼ 8.0 Hz), 10.06
(1H, s); ESI MS: 346 (M þ Na); HPLC purity: 99.2%,
tR ¼ 6.8 min.
5.7.3. 5-(30-Fluoro-40-methoxy-biphenyl-4-yl)-4,5-dihydro-
5.6.4. 5-[4-(4-Butoxycarbonyl-piperazin-1-yl)-phenyl]-
isoxazole-3-carboxylic acid ethyl ester (15c)
4,5-dihydro-isoxazole-3-carboxylic acid (12b)
1
Yield: 70%; H NMR (500 MHz, CDCl3): d 1.39 (3H, t,
1
Yield: 83.2%; H NMR (500 MHz, CDCl3): d 0.94 (3H, t,
J ¼ 7.3 Hz), 3.25 (1H, dd, J ¼ 8.7, 17.5 Hz), 3.66 (1H, dd,
J ¼ 11.4, 17.8 Hz), 3.93 (3H, s), 4.37 (2H, q, J ¼ 7.0 Hz),
5.81 (1H, dd, J ¼ 8.7, 11.4 Hz), 7.02 (1H, t, J ¼ 8.3 Hz),
7.29e7.33 (2H, m), 7.38 (2H, d, J ¼ 8.3 Hz), 7.54 (2H, d,
J ¼ 8.3 Hz); ESI MS: 366.1 (M þ Na); HPLC purity: 99.0%,
tR ¼ 6.8 min.
J ¼ 7.3 Hz), 1.35e1.45 (2H, m), 1.60e1.68 (2H, m), 3.12e
3.26 (5H, m), 3.51e3.72 (5H, m), 4.13 (2H, t, J ¼ 6.6 Hz),
5.72 (1H, dd, J ¼ 9.3, 9.5 Hz), 6.97 (2H, d, J ¼ 7.8 Hz), 7.24
(2H, d, J ¼ 7.3 Hz); 13C NMR (500 MHz, CDCl3): d 13.79,
19.19, 30.35, 31.01, 40.70, 43.34, 49.49, 65.90, 85.55,
117.09, 127.42, 131.44, 150.82, 155.86, 162.64; ESI MS:
376.3 (M þ H); HPLC purity: 99%, tR ¼ 5.9 min.
5.7.4. 5-(4-Furan-2-yl-phenyl)-4,5-dihydro-isoxazole-3-
5.6.5. 5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-
dihydro-isoxazole-3-carboxylic acid (13)
carboxylic acid ethyl ester (15d)
1
Yield: 58%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
Yield: 61.9%; 1H NMR (500 MHz, DMSO-d6): d 2.83 (4H,
br s), 3.30 (4H, overlapped with H2O peak), 3.13 (1H, dd,
J ¼ 9.8, 17.8 Hz), 3.59 (1H, dd, J ¼ 11.2, 17.8 Hz), 3.90
(2H, s), 5.71 (1H, dd, J ¼ 10.0, 10.7 Hz), 6.99 (2H, d,
J ¼ 8.5 Hz), 7.27 (2H, d, J ¼ 8.5 Hz), 7.36e7.52 (5H, m);
13C NMR (300 MHz, DMSO-d6): d 40.36, 46.53, 51.29,
J ¼ 7.0 Hz), 3.22 (1H, dd, J ¼ 9.0, 17.8 Hz), 3.64 (1H, dd, J ¼
11.7, 17.8 Hz), 4.36 (2H, q, J ¼ 6.3 Hz), 5.78 (1H, dd, J ¼ 9.0,
11.4 Hz), 6.47e6.48 (1H, m), 6.66 (1H, d, J ¼ 3.4 Hz), 7.33
(2H, d, J ¼ 8.0 Hz), 7.47 (1H, d, J ¼ 0.7 Hz), 7.67 (2H, d,
J ¼ 8.3 Hz); ESI MS: 286.2 (M þ 1); HPLC purity: 99.1%,
tR ¼ 6.6 min.

470
Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
5.7.5. 5-{4-[4-(4-Trifluoromethoxy-phenoxy)-piperidin-1-
yl]-phenyl}-4,5-dihydro-isoxazole-3-carboxylic acid ethyl
ester (17a)
5.8.3. 5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-dihydro-
isoxazole-3-carboxylic acid amide (21)
To a stirred solution of ester 1 in 1,4-dioxane, ammonium
hydroxide (2 eq) was added and stirred at room temperature
overnight. The reaction mass was concentrated under reduced
1
Yield: 74.3%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
J ¼ 7.3 Hz), 1.89e1.95 (2H, m), 2.06e2.11 (2H, m), 3.12e
3.16 (2H, m), 3.21 (1H, dd, J ¼ 9.2, 17.5 Hz), 3.49e3.59
(3H, m), 4.36 (2H, q, J ¼ 7.0 Hz), 4.43e4.46 (1H, m), 5.70
(1H, dd, J ¼ 9.2, 11.2 Hz), 6.90e6.94 (4H, m), 7.14 (2H, d,
J ¼ 8.7 Hz), 7.22 (2H, d, J ¼ 8.7 Hz); ESI MS: 501.1
(M þ Na); HPLC purity: 98.8%, tR ¼ 6.4 min.
1
pressure to give amide 21 in 66% yield. H NMR (500 MHz,
CDCl3): d 2.61e2.69 (4H, m), 3.2e3.3 (5H, m), 3.53e3.63
(3H, m), 5.49e5.56 (1H, br s), 5.69 (1H, dd, J ¼ 9.5,
11.2 Hz), 6.56e6.63 (1H, br s), 6.9 (2H, d, J ¼ 8.7 Hz), 7.21
(2H, d, J ¼ 8.7 Hz), 7.27e7.3 (1H, m), 7.32e7.38 (4H, m);
ESI MS: 387.2 (M þ Na); HPLC purity: 99%, tR ¼ 4.6 min.
5.7.6. 5-(4-Piperidin-1-yl-phenyl)-4,5-dihydro-isoxazole-3-
5.8.4. Lithium; 5-[4-(4-benzyl-piperazin-1-yl)-phenyl]-4,5-
dihydro-isoxazole-3-carboxylate (22)
carboxylic acid ethyl ester (17b)
1
Yield: 77%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
To a stirred solution of ester 1 in THF/H2O, LiOH (5 eq)
was added and stirred at room temperature for 2 h. The reac-
tion mass was concentrated under reduced pressure to give
lithium salt 22 in 90% yield. 1H NMR (500 MHz, D2O):
d 2.54e2.6 (4H, m), 3.03e3.13 (5H, m), 3.46e3.54 (3H,
m), 5.6 (1H, dd, J ¼ 8.5, 10.9 Hz), 7.0 (2H, d, J ¼ 9.0 Hz),
7.24 (2H, d, J ¼ 9.0 Hz), 7.26e7.34 (5H, m); HPLC purity:
100%, tR ¼ 4.7 min.
J ¼ 7.0 Hz), 1.55e1.62 (2H, m), 1.66e1.72 (4H, m), 3.17
(4H, t, J ¼ 5.6 Hz), 3.21 (1H, dd, J ¼ 10.7, 19.0 Hz), 3.55
(1H, dd, J ¼ 11.4, 17.5 Hz), 4.36 (2H, q, J ¼ 7.0 Hz), 5.68
(1H, dd, J ¼ 9.2, 11.2 Hz), 6.90 (2H, d, J ¼ 8.7 Hz), 7.19
(2H, d, J ¼ 8.7 Hz); ESI MS: 303.2 (M þ 1); HPLC purity:
99.0%, tR ¼ 4.7 min.
5.8. General procedure for the synthesis of 20a and 20b
5.8.5. 5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-dihydro-
isoxazole-3-carboxylic acid propyl amide (23)
A solution of 4-cyano styrene (1 mmol), NH2OH$HCl
(2 mmol) and Et3N (1.5 mmol) in EtOH was refluxed for
4 h. The reaction mixture was concentrated under reduced
pressure and the crude amidine 18 was used as such for the
next reaction without further purification.
To a solution of amidine 18 (1 mmol) in triethyl orthofor-
mate (5 mL), BF3$OEt2 (cat)/pyridine (5 mL) or benzoyl chlo-
ride (1.2 mmol) was added and the reaction mixtures were
heated at 80 ꢀC for 2 h, concentrated under reduced pressure
and purified by flash chromatography to afford 19a and 19b
in 51% and 45% yields, respectively.
To a stirred solution of 22 (1 eq) in THF, oxaloyl chloride
(2 eq) and DMF (catalytic) were added and stirred at room
temperature for 4 h. Then added n-propyl amine (6 eq) to
the acid chloride and again stirred at room temperature for an-
other 2 h. The reaction mixture was diluted with excess ethyl
acetate and washed with 1 N NaOH solution, water, brine,
dried (anhyd. Na2SO4), concentrated under reduced pressure
and purified by flash chromatography to give 23 in 59% yield.
1H NMR (500 MHz, CDCl3): d 0.93 (3H, t, J ¼ 7.3 Hz), 1.62
(2H, m), 2.6e2.7 (4H, br s), 3.21e3.32 (5H, m), 3.35 (2H, dq,
J ¼ 1.9, 7.0 Hz), 3.56e3.66 (3H, m), 5.68 (1H, dd, J ¼ 9.5,
11.2 Hz), 6.68e6.74 (1H, m), 6.92 (2H, d, J ¼ 8.7 Hz), 7.23
(2H, d, J ¼ 8.7 Hz), 7.29e7.33 (1H, m), 7.35e7.42 (4H, m);
ESI MS: 429.3 (M þ Na); HPLC purity: 100%, tR ¼ 5.1 min.
Compounds 19a and 19b were converted into the corre-
sponding isoxazolines 20a and 20b following the synthetic
procedure discussed earlier.
5.8.1. 5-(4-[1,2,4]Oxadiazol-3-yl-phenyl)-4,5-dihydro-
isoxazole-3-carboxylic acid ethyl ester (20a)
1
Yield: 59%; H NMR (500 MHz, CDCl3): d 1.38 (3H, t,
5.8.6. 5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-dihydro-
isoxazole-3-carboxylic acid hydrazide (24)
J ¼ 7.0 Hz), 3.24 (1H, dd, J ¼ 8.5, 17.8 Hz), 3.70 (1H,
dd, J ¼ 11.7, 17.8 Hz), 4.37 (2H, q, J ¼ 7.0 Hz), 5.85 (1H,
dd, J ¼ 8.5, 11.7 Hz), 7.47 (2H, d, J ¼ 8.3 Hz), 8.14 (2H, d,
J ¼ 8.0 Hz), 8.77 (1H, s); ESI MS: 288.1 (M þ 1).
To a stirred solution of ethyl ester 1 (0.2 g, 0.508 mmol) in
ethanol (10 mL), hydrazine hydrate (0.05 mL, 1.017 mmol)
was added and the mixture was refluxed overnight. The reac-
tion mixture was concentrated under reduced pressure and the
crude residue was purified by flash chromatography to afford
17 (0.1 g) in 52% yield. 1H NMR (500 MHz, CDCl3):
d 2.60 (4H, t, J ¼ 5.1 Hz), 3.18e3.30 (5H, m), 3.54e3.62
(3H, m), 3.96 (2H, d), 6.89 (2H, d, J ¼ 8.7 Hz), 7.19 (2H, d,
J ¼ 8.7 Hz), 7.24e7.29 (3H, m), 7.31e7.38 (4H, m), 7.78
(1H, br s); 13C NMR (500 MHz, CDCl3): d 40.60, 48.66,
52.89, 63.05, 84.63, 115.87, 127.26, 128.31, 129.36, 151.62,
152.42; ESI MS: 380.4 (M þ 1); HPLC purity: 99.3%,
tR ¼ 5.4 min.
5.8.2. 5-[4-(5-Phenyl-[1,2,4]oxadiazol-3-yl)-phenyl]-4,5-
dihydro-isoxazole-3-carboxylic acid ethyl ester (20b)
1
Yield: 60%; H NMR (500 MHz, CDCl3): d 1.39 (3H, t,
J ¼ 7.0 Hz), 3.25 (1H, dd, J ¼ 8.5, 17.5 Hz), 3.70 (1H, dd,
J ¼ 11.7, 17.8 Hz), 4.37 (2H, q, J ¼ 7.3 Hz), 5.86 (1H, dd,
J ¼ 8.7, 11.7 Hz), 7.48 (2H, d, J ¼ 8.0 Hz), 7.54e7.64 (3H,
m), 8.21 (4H, t, J ¼ 8.5 Hz); ESI MS: 362.3 (M ꢂ 1); HPLC
purity: 98.4%, tR ¼ 7.1 min.

Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
471
5.8.7. 1-Benzyl-4-{4-[3-(5-methyl-[1,3,4]oxadiazol-2-yl)-
4,5-dihydro-isoxazol-5-yl]-phenyl}-piperazine (26)
5.9.2. [5-(4-Morpholin-4-yl-phenyl)-4,5-dihydro-isoxazol-3-
yl]-methanol (29b)
1
To a solution of 24 (0.85 g, 2.24 mmol) in CH2Cl2 (15 mL),
Et3N (0.93 mL, 6.728 mmol) and acetyl chloride (0.23 mL,
3.36 mmol) were added at 0 ꢀC and stirred at room tempera-
ture for 2 h. The reaction mixture was concentrated under re-
duced pressure and purified by flash chromatography to afford
5-[4-(4-benzyl-piperazin-1-yl)-phenyl]-4,5-dihydro-isoxazole-
3-carboxylic acid N0-acetyl-hydrazide (25) (0.58 g) in 61%
yield. Compound 25 (0.47 g, 1.11 mmol) was dissolved in
CH2Cl2 (20 mL), and Et3N (0.31 mL, 2.23 mmol) and p-
TsCl (0.424 g, 2.23 mmol) were added at 0 ꢀC and allowed
to reflux gently for 4 h. The reaction mixture was cooled to
room temperature and washed vigorously with 40% K2CO3
solution, dried (anhyd. Na2SO4), concentrated under reduced
pressure and purified by flash chromatography to afford 26
Yield: 60%; H NMR (500 MHz, CDCl3): d 3.02 (1H, dd,
J ¼ 8.7, 17.0 Hz), 3.16 (4H, t, J ¼ 4.6 Hz), 3.41 (1H,
dd, J ¼ 10.7, 17.0 Hz), 3.86 (4H, t, J ¼ 4.8 Hz), 4.46 (2H, d,
J ¼ 5.8 Hz), 5.57 (1H, dd, J ¼ 8.7, 10.5 Hz), 6.90 (2H, d,
J ¼ 8.7 Hz), 7.25 (2H, d, J ¼ 6.3 Hz); ESI MS: 263.1
(M þ 1); HPLC purity: 98.7%, tR ¼ 4.2 min.
5.10. General procedure for the synthesis of 30a and 30b
To a stirred solution of alcohol 29a or 29b (1 mmol) in an-
hydrous DMF, CBr4 (1 mmol), Ph3P (1 mmol) and NaN3
(5 mmol) were added sequentially and the resulting mixture
was stirred at room temperature for 2 h. The reaction mixtures
were quenched with MeOH, filtered and concentrated. The
crude residues were purified by flash chromatography.
1
(0.26 g) in 58% yield. H NMR (500 MHz, CDCl3): d 2.54
(3H, s), 2.60 (4H, t, J ¼ 5.1 Hz), 3.18e3.30 (5H, m), 3.54e
3.62 (3H, m), 5.52 (1H, dd, J ¼ 9.5, 11.2 Hz), 6.84 (2H, d,
J ¼ 8.7 Hz), 7.04 (2H, d, J ¼ 8.7 Hz), 7.26e7.35 (3H, m),
7.93 (2H, d, J ¼ 8.6 Hz); ESI MS: 426.4 (M þ Na); HPLC pu-
rity: 98.5%, tR ¼ 5.8 min.
5.10.1. 1-[4-(3-Azidomethyl-4,5-dihydro-isoxazol-5-yl)-
phenyl]-4-benzyl-piperazine (30a)
1
Yield: 59%; H NMR (500 MHz, CDCl3): d 2.60 (4H, t,
J ¼ 4.8 Hz), 3.01 (1H, q, J ¼ 7.8 Hz), 3.20 (4H, t,
J ¼ 5.1 Hz), 3.37 (1H, q, J ¼ 7.8 Hz), 3.56 (2H, s), 4.13 (2H,
s), 5.57 (1H, t, J ¼ 5.1 Hz), 6.89 (2H, d, J ¼ 8.7 Hz), 7.21
(2H, d, J ¼ 8.5 Hz), 7.24e7.29 (1H, m), 7.30e7.37 (4H, m);
13C NMR (500 MHz, CDCl3): d 42.66, 42.71, 47.49, 48.85,
48.89, 52.99, 63.06, 82.91, 115.90, 126.99, 127.06, 128.36,
129.28, 130.45, 151.52, 153.79; ESI MS: 377.4 (M þ 1);
HPLC purity: 98.5%, tR ¼ 5.1 min.
5.8.8. 5-(4-Morpholin-4-yl-phenyl)-4,5-dihydro-isoxazole-
3-carboxylic acid ethyl ester (28)
Compound 28 was synthesized following the general proce-
dure described earlier in 78% yield. 1H NMR (500 MHz,
CDCl3): d 1.38 (3H, t, J ¼ 7.0 Hz), 3.17 (4H, t, J ¼ 4.8 Hz),
3.21 (1H, dd, J ¼ 9.7, 18.3 Hz), 3.56 (1H, dd, J ¼ 11.4,
17.8 Hz), 3.85 (4H, t, J ¼ 4.8 Hz), 4.36 (2H, q, J ¼ 7.0 Hz),
5.71 (1H, dd, J ¼ 9.2, 11.4 Hz), 6.90 (2H, d, J ¼ 8.5 Hz),
7.23 (2H, d, J ¼ 8.7 Hz); ESI MS: 327.1 (M þ Na); HPLC pu-
rity: 99.7%, tR ¼ 5.6 min.
5.10.2. 4-[4-(3-Azidomethyl-4,5-dihydro-isoxazol-5-yl)-
phenyl]-morpholine (30b)
1
Yield: 61%; H NMR (500 MHz, CDCl3): d 3.02 (1H, dd,
J ¼ 8.7, 17.0 Hz), 3.16 (4H, t, J ¼ 4.6 Hz), 3.40 (1H, dd,
J ¼ 10.9, 17.3 Hz), 3.85 (4H, t, J ¼ 4.8 Hz), 4.15 (2H, s),
5.59 (1H, dd, J ¼ 9.0, 10.5 Hz), 6.90 (2H, d, J ¼ 8.5 Hz),
7.24 (2H, d, J ¼ 8.5 Hz); ESI MS: 288.3 (M þ 1); HPLC pu-
rity: 98.8%, tR ¼ 5.3 min.
5.9. General procedure for the synthesis of 29a and 29b
To a stirred solution of ester 1 or 28 (1 mmol) in anhydrous
THF, DIBAL-H (2 mmol) was added dropwise at 0 ꢀC and
stirred at the same temperature for 4 h. The reaction mass
was quenched with saturated solution of potassium sodium tar-
trate and the product was extracted with EtOAc, washed with
water, brine, dried (anhyd. Na2SO4) and concentrated. The
crude residue was purified by flash chromatography to give
pure compounds.
5.11. General procedure for the synthesis of 31a and 31b
To a stirred solution of azide 30a or 30b (1 mmol) in 1,4-
dioxane (5 mL), Ph3P (1.6 mmol) was added and stirred at
room temperature overnight. The reaction mixtures were con-
centrated under reduced pressure and the crude residues were
purified by flash chromatography to afford 31a or 31b.
5.9.1. {5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-dihydro-
isoxazol-3-yl}-methanol (29a)
1
Yield: 56%; H NMR (500 MHz, CDCl3): d 2.60 (4H, t,
5.11.1. C-{5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-
dihydro-isoxazol-3-yl}-methylamine (31a)
J ¼ 5.1 Hz), 3.01 (1H, q, J ¼ 8.7 Hz), 3.19 (4H, t,
J ¼ 5.1 Hz), 3.37 (1H, q, J ¼ 8.7 Hz), 3.56 (2H, s), 4.43
(2H, s), 5.53 (1H, t, J ¼ 9.2 Hz), 6.88 (2H, d, J ¼ 8.7 Hz),
7.21 (2H, d, J ¼ 8.7 Hz), 7.24e7.29 (1H, m), 7.30e7.39
(4H, m); 13C NMR (500 MHz, CDCl3): d 42.59, 48.89,
52.96, 58.00, 63.06, 82.34, 115.95, 127.10, 127.27, 128.34,
129.32, 130.94, 137.71, 151.34, 158.61; ESI MS: 374.4
(M þ 23); HPLC purity: 99%, tR ¼ 4.6 min.
Yield: 67%; 1H NMR (500 MHz, CDCl3): d 1.67 (2H, br s),
2.59 (4H, t, J ¼ 4.8 Hz), 2.95 (1H, q, J ¼ 8.5 Hz), 3.19 (4H, t,
J ¼ 5.1 Hz), 3.32 (1H, q, J ¼ 8.5 Hz), 3.56 (2H, s), 3.61 (2H,
s), 5.51 (1H, t, J ¼ 8.7 Hz), 6.88 (2H, d, J ¼ 8.5 Hz), 7.21
(2H, d, J ¼ 8.5 Hz), 7.27e7.28 (1H, m), 7.30e7.36 (4H, m);
13C NMR (500 MHz, CDCl3): d 43.33, 48.90, 53.00, 63.06,
63.15, 82.13, 115.89, 127.02, 128.33, 129.23, 131.08,

472
Rakesh et al. / European Journal of Medicinal Chemistry 44 (2009) 460e472
137.90, 151.33; ESI MS: 373.1 (M þ 23); HPLC purity:
98.8%, tR ¼ 4.3 min.
Appendix. Supplementary data
Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.ejmech.2008.04.
007.
5.11.2. C-[5-(4-Morpholin-4-yl-phenyl)-4,5-dihydro-
isoxazol-3-yl]-methylamine (31b)
1
Yield: 60%; H NMR (500 MHz, CDCl3): d 2.97 (1H, dd,
J ¼ 8.7, 17.0 Hz), 3.15 (4H, t, J ¼ 4.8 Hz), 3.35 (1H, dd,
J ¼ 6.3, 17.0 Hz), 3.63 (2H, s), 3.85 (4H, t, J ¼ 4.8 Hz), 5.53
(1H, dd, J ¼ 9.0, 10.5 Hz), 6.89 (2H, d, J ¼ 8.5 Hz), 7.24
(2H, d, J ¼ 8.5 Hz); ESI MS: 284.2 (M þ 23); HPLC purity:
100%, tR ¼ 4.0 min.
References
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5.12. General procedure for the synthesis of 32a and 32b
[3] ‘‘ Tuberculosis’’ WHO Fact Sheet No 104, Health Communications,
WHO, Geneva, 2006. http://www.who.int/mediacentre/factsheets/fs104/
en/index.html.
To a stirred solutions of amines 31a and 31b (1 mmol) in
CH2Cl2 (5 mL), Et3N (3 mmol) and acetyl chloride
(1.5 mmol) were added at 0 ꢀC and allowed to stir at room
temperature for 1 h. The reaction mixtures were diluted with
excess CH2Cl2 (15 mL) and washed with aqueous NaHCO3
(10 mL), dried (anhyd. Na2SO4), concentrated under reduced
pressure and the crude residues were purified by flash
chromatography.
[4] B.R. Bloom (Ed.), Tuberculosis: Pathogenesis, Protection, and Control,
ASM Press, Washington, DC, 1994.
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5.12.1. N-{5-[4-(4-Benzyl-piperazin-1-yl)-phenyl]-4,5-
dihydro-isoxazol-3-ylmethyl}-acetamide (32a)
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1
Yield: 78%; H NMR (500 MHz, CDCl3): d 2.00 (3H, s),
2.59 (4H, t, J ¼ 4.8 Hz), 2.96 (1H, q, J ¼ 8.5 Hz), 3.19 (4H,
t, J ¼ 5.1 Hz), 3.30 (1H, q, J ¼ 8.5 Hz), 3.55 (2H, s), 4.18
(2H, t, J ¼ 4.8 Hz), 5.51 (1H, t, J ¼ 5.1 Hz), 6.30 (1H, br s),
6.87 (2H, d, J ¼ 8.5 Hz), 7.18 (2H, d, J ¼ 8.5 Hz), 7.24e
7.28 (1H, m), 7.30e7.36 (4H, m); 13C NMR (500 MHz,
CDCl3): d 22.91, 22.96, 36.95, 37.00, 43.35, 48.85, 48.87,
52.97, 53.03, 62.95, 63.05, 82.41, 82.44, 115.85, 127.03,
127.08, 127.20, 128.31, 128.33, 129.24, 130.52, 137.91,
151.44, 156.15, 170.53; ESI MS: 415 (M þ 23); HPLC purity:
98.7%, tR ¼ 4.6 min.
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5.12.2. N-[5-(4-Morpholin-4-yl-phenyl)-4,5-dihydro-
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1984.
isoxazol-3-ylmethyl]-acetamide (32b)
1
Yield: 71%; H NMR (500 MHz, CDCl3): d 2.97 (1H, dd,
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J ¼ 9.0, 17.3 Hz), 3.15 (4H, t, J ¼ 4.8 Hz), 3.34 (1H, dd,
J ¼ 10.7, 17.0 Hz), 3.85 (4H, t, J ¼ 4.8 Hz), 4.20 (2H, q,
J ¼ 3.4 Hz), 5.54 (1H, dd, J ¼ 9.2, 10.2 Hz), 6.19 (1H, br s),
6.89 (2H, d, J ¼ 8.5 Hz), 7.22 (2H, d, J ¼ 8.5 Hz); ESI MS:
326.1 (M þ Na); HPLC purity: 99.1%, tR ¼ 4.2 min.
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Chemother. 56 (2005) 180e185.
Acknowledgments
We thank National Institutes of Health grant AI062415 for
financial support.