Design, synthesis and antimycobacterial activity of various 3-(4-(substitutedsulfonyl)piperazin-1-yl)benzo[ d[isoxazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.09.043

In this communication, we synthesized a series of twenty four novel 3-(4-(substitutedsulfonyl)piperazin-1-yl)benzo[d]isoxazole analogues, characterized using various spectroscopic techniques and evaluated for their in vitro anti-tubercular activity against Mycobacterium tuberculosis (MTB) H₃₇Rv strain. The titled compounds exhibited Minimum inhibitory concentration (MIC) between 3.125 and >50 μg/mL. Among the tested compounds, 5c, 6a, 6j and 6p exhibited moderate activity (MIC Combining double low line 12.5 μg/mL), while 5a and 6i exhibited good activity (MIC Combining double low line 6.25 μg/mL) and 6b (MIC Combining double low line 3.125 μg/mL) exhibited very good anti-tubercular activity. In addition, the analogues 5a, 5c, 6a, 6b, 6i, 6j and 6p were subjected to toxicity studies against mouse macrophage (RAW 264.7) cell lines to analyse the selectivity profile of the newly synthesized compounds and selectivity index of the most active compound was found to be >130 indicating suitability of the compound for further drug development. Structure of 6b was further substantiated through single crystal XRD.

Full text of DOI:10.1016/j.ejmech.2014.09.043

European Journal of Medicinal Chemistry 87 (2014) 71e78
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and antimycobacterial activity of various 3-(4-
(substitutedsulfonyl)piperazin-1-yl)benzo[d]isoxazole derivatives
Kalaga Mahalakshmi Naidu a, Amaroju Suresh a, Jayanty Subbalakshmi a,
Dharmarajan Sriram b, Perumal Yogeeswari b, Pallepogu Raghavaiah c,
,
*
Kondapalli Venkata Gowri Chandra Sekhar a
a Department of Chemistry, Birla Institute of Technology & Science-Pilani, Hyderabad Campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad 500 078,
Telangana, India
b Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad 500 078,
Telangana, India
c Department of Chemistry, University of Cape Town, Rondebosch 7707, Cape Town, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
In this communication, we synthesized a series of twenty four novel 3-(4-(substitutedsulfonyl)piperazin-
1-yl)benzo[d]isoxazole analogues, characterized using various spectroscopic techniques and evaluated
for their in vitro anti-tubercular activity against Mycobacterium tuberculosis (MTB) H37Rv strain. The titled
Received 14 April 2014
Received in revised form
10 September 2014
Accepted 12 September 2014
Available online 16 September 2014
compounds exhibited Minimum inhibitory concentration (MIC) between 3.125 and >50
the tested compounds, 5c, 6a, 6j and 6p exhibited moderate activity (MIC ¼ 12.5 g/mL), while 5a and 6i
exhibited good activity (MIC ¼ 6.25 g/mL) and 6b (MIC ¼ 3.125 g/mL) exhibited very good anti-
mg/mL. Among
m
m
m
tubercular activity. In addition, the analogues 5a, 5c, 6a, 6b, 6i, 6j and 6p were subjected to toxicity
studies against mouse macrophage (RAW 264.7) cell lines to analyse the selectivity profile of the newly
synthesized compounds and selectivity index of the most active compound was found to be >130
indicating suitability of the compound for further drug development. Structure of 6b was further sub-
stantiated through single crystal XRD.
Keywords:
Benzo[d]isoxazole
Piperazine sulphonamide
Antimycobacterial activity
Mycobacterium tuberculosis
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
(0.9 millione1.1 million), South Africa (0.4 millione0.6 million),
Indonesia
(0.4
millione0.5
million)
and
Pakistan
Tuberculosis (TB), an airborne infectious disease is caused by
MTB. The MTB commonly attacks the lungs (pulmonary TB) and it
can also attack any part of the human body such as kidney, spine
and brain (extrapulmonary TB). TB represents one of the prime
public health concerns worldwide after the human immunodefi-
ciency virus (HIV). TB is one of the major causes of death in HIV
patients. According to the World Health Organization (WHO),
about one-third of the world's population is latently infected with
MTB. In 2012, there were an estimated 8.6e9.4 million cases and
1.2e1.5 million deaths (including deaths from TB among HIV-
positive people). The vast majority of TB deaths are in the devel-
oping world and more than 50% of all deaths occur in Asia alone.
The five countries with the largest number of incident cases in
(0.3 millione0.5 million). India and China solitarily accounted for
26% and 12% of global cases, respectively. If TB is not treated
properly, it is serious and can be a cause of death [1]. Multidrug
resistant TB (MDR-TB) is a form of TB which occurs when MTB
strain turns resistant to the most effective anti-TB drugs i.e.
isoniazid and rifampin. In 2012, worldwide 0.45 million people
developed MDR-TB and there were 0.17 million deaths resulting
from MDR-TB [2]. Extensively drug resistant TB (XDR-TB) occurs
when MTB strain is resistant to at least isoniazid and rifampin in
addition to being resistant to one of the fluoroquinolones, as well
as resistant to at least one of the second line injectable drugs i.e.
amikacin, kanamycin or capreomycin [3]. By the end of 2012, XDR-
TB was found worldwide in 92 countries. Approximately, 9.6% of
MDR-TB cases have XDR-TB. It is associated with a much higher
mortality rate than MDR-TB [2]. Totally drug resistant TB (TDR-TB)
or extremely drug resistant TB (XXDR-TB) is not been clearly
defined by WHO. It occurs when MTB strain becomes resistant to
all first and second line anti-TB drugs. It is very uncontrollable
2012
were
India
(2.0
millione2.4
million),
China
* Corresponding author.
E-mail addresses:
(K.V.G. Chandra Sekhar).
kvgc@hyderabad.bits-pilani.ac.in,
kvgcs@yahoo.com
http://dx.doi.org/10.1016/j.ejmech.2014.09.043
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

72
K.M. Naidu et al. / European Journal of Medicinal Chemistry 87 (2014) 71e78
Fig. 1. General synthetic procedure for title compounds.
although not always difficult to treat [3e5]. Prevalence of XDR-TB
and TDR-TB has increased worldwide [5e7]. Except Bedaquiline
(TMC-207) there were no new drugs approved for treatment of TB
in past few decades, recently it is specifically approved to treat
MDR-TB [8].
Heterocyclic compounds have excellent biological importance.
Mainly, benzisoxazoles and its derivatives have broad spectrum of
biological activities such as antimicrobial [9,10] anti-cancer [11],
anti-HIV [12], anti-diabetic [13], antipsychotic [14,15], anticonvul-
sant [16]. Also, in particular Subash et al., reported 5-tert-Butyl-N-
pyrazol-4-yl-4,5,6,7-tetrahydrobenzo[d]isoxazole-3-carboxamide
derivatives with excellent anti-TB activity [17]. Till date, there are
no reports of benzisoxazole derivatives as anti-TB agents.
Impressed by their broad range of biological activities and in
continuation of our ongoing research on novel anti-TB agents
[18,19] we made an attempt to explore this heterocycle for anti-TB
activity.
On other hand piperazine sulfonamide derivatives are known to
exhibit wide range of pharmacological activities like antimicrobial
[20], antiproliferative [21], anti-HIV [22], antifungal [23], anti-
protozoal [24], anticonvulsant [25], anti-diabetic [26], sigma re-
ceptor ligands [27] and in precise 7-[4-(5-amino-[1,3,4]thiadiazole-
2-sulfonyl)-piperazin-1-yl]-1-cyclopropyl and ethyl-6-fluoro-4-
oxo-1,4-dihydro-quinoline-3-carboxylic acid as antibacterial and
anti-TB agents [28].
2. Chemistry
In this report, we synthesized 3-(4-substitutedsulfonylpiperazin-
1-yl)benz[d]isoxazole derivatives as depicted in Fig.1. Substituted 2-
chlorobenzaldehyde (1aed) on treatment with NH2OH.HCl and
CH3COONa in EtOH and H2O at 0 ꢀC e rt for 1 hyielded substituted 2-
chlorobenzaldehyde oxime derivatives (2aed) [31]. Further chlori-
nation with N-chlorosuccinimide in CCl4 stirred at 0 ꢀC e rt for
45 min afforded substituted 2-chloro-N-hydroxybenzimidoyl chlo-
ride derivatives (3aed) [32]. Substituted (2-chlorophenyl) (piper-
azin-1-yl)methanone oxime (4aed) [33] was synthesized by
reacting 3aed with anhydrous piperazine and N(C2H5)3 as base in
CH2Cl2 at rt for 2 h. Literature survey reveals employing expensive
and toxic catalysts for the effective cyclization of 4aed to afford
5aed [34e36]. We herein optimised this cyclization by employing
Table 1
Optimisation of reaction conditions of 5a.a
Entry Solvent
Base Heating
source
Temperature
(ꢀC)
Time Yield
(%)b
1
2
3
MeCN
Dioxane
DMF
K2CO3 Conventional
75
12 h
12 h
12 h
12 h
12 h
12 h
12 h
NRc
NRc
NRc
NRc
38
DIPEA Conventional 100
K2CO3 Conventional 100
TEA Conventional 100
KOH Conventional 100
KOH Conventional 100
4
DMF
5
DMF
6
7
8
9
10
11
12
13
14
15
Dioxane
42
54
Benzo[d]isoxazole derivatives target pantothenate synthetase
(PS) enzyme which catalyzes amide bond formation of pantothe-
nate from D-pantoate and b-alanine accompanied by hydrolysis of
Dioxane:H2Od KOH Conventional 100
Dioxane:H2Od KOH Microwave
Dioxane:H2Od KOH Microwave
Dioxane:H2Od KOH Microwave
120
120
120
15 min 55
30 min 64
45 min 62
Mg-adenosine 50-triphosphate (ATP) into adenosine 50-mono-
phosphate (AMP) and Mg-pyrophosphate (PPi). PS is recognized as
a suitable target for discovery of new therapeutics to treat TB
[17,29,30].
Dioxane:H2Od KOH Conventionale 120
Dioxane:H2Od KOH Conventionale 120
Dioxane:H2Od KOH Conventionale 120
Dioxane:H2Od KOH Conventionale 120
Dioxane:H2Od KOH Conventionale 120
1 h
2 h
3 h
4 h
5 h
52
58
63
82
80
Inspired by the anti-mycobacterial activity of benzisoxazoles
targeting the PS and piperazine sulfonamide skeleton, we chalked
out a synthetic path to incorporate these two pharmacophores into
a single framework. We synthesized new 3-(4-substituted sulfo-
nylpiperazin-1-yl)benzo[d]isoxazole derivatives and evaluated for
their antimycobacterial activity.
a
All the reactions were carried out with 4a (1.0 equiv.) and base (3.0 equiv.) in
solvent (4 mL).
b
c
Isolated yield after column chromatography.
NR ¼ no reaction.
d
e
Dioxane:H2O (3:1).
Reactions were carried out in sealed tube.

K.M. Naidu et al. / European Journal of Medicinal Chemistry 87 (2014) 71e78
73
Table 2
inexpensive base (Table 1). As a model reaction, 4a was treated with
Antimycobacterial activities of compound 5ae5d and 6ae6x against MTB H37Rv.
potassium carbonate (K2CO3) in acetonitrile (MeCN) at 75 ꢀC for 12 h.
The reaction did not proceed to yield 5a. With this failure, we
scrutinized optimal reaction condition with variety of bases and
solvents as summarised in Table 1. Employing KOH and DMF at
100 ꢀC under conventional heating source for 12 h the desired
product was obtained in about 38%. Under similar reaction condi-
tions, an increase in the yield was noticed (entry 6 and 7) when we
changed solvent to dioxane and dioxane: water (3:1 ratio). Accen-
tuation on microwave assisted organic synthesis; reaction was car-
ried out at 120 ꢀC in Biotage initiator with a pre-stirring of 30 s and
stirring rate at 600 rpm. The reaction was carried out by irradiating
microwaves at various intervals of time (entry 8e10) to afford the
desired compound in moderate yield. Having better results under
closed vessel condition, we further focussed to carry out the reaction
in sealed tube under conventional heating source at various intervals
of time (entry 11e15). These findings noticeably increased the yield
and diminished the reaction time to 4 h. Having the optimised re-
action condition handy various 5aed derivatives were synthesized
in good yield. Further, title compounds 6aex were synthesized by
reacting substituted-3-(piperazin-1-yl)benzo[d]isoxazole (5aed)
with various sulfonyl chlorides, using N(C2H5)3 as base and CH2Cl2 as
solvent at 0 ꢀC to rt. All the title compounds were purified by column
chromatography using 45e80% of ethyl acetate in hexane. The 1H
NMR showed multiplets in the range 3.22e3.68 ppm due to piper-
azine (eCH2e) protons, aromatic protons of substituted sulfonyl
group and benzoisoxazole appeared at 6.98e8.42 ppm; 13C NMR
showed aromatic carbons in the range of 110e166 ppm and piper-
azine carbons 45e47 ppm. IR spectroscopy revealed the absence of
peak at ~3230 cm-1 due to NH stretch in the title compounds 6aex,
clearly confirming the formation of products. All the synthesized
compounds were confirmed by 1H NMR, 13C NMR, IR and HRMS. In
addition single crystal structurewas determined for one of the active
compounds, 6b.
Entry
R
X
aMP (ꢀC) MIC(
37Rv
mg/mL) against MTB
H
5a
5b
e
e
e
75e76
6.25
4- 101e102 25
Cl
5c
5d
6a
e
e
6-
Cl
7-
Cl
H
81e82
88e89
12.5
25
168e169 12.5
6b
6c
6d
6e
6f
H
H
H
H
H
174e175
3.12
162e163 50
181e182 >50
230e231 >50
202e203 50
3. Results and discussion
3.1. Antimycobacterial activity
All the synthesized compounds were tested for their ability to
inhibit the growth of MTB H37RV (ATCC 27294) strain by Microplate
Alamar Blue Assay (MABA) with compound concentration ranges
from 50 to 0.78 mg/mL. Isoniazid and Rifampin were used as the
positive drug standards. The in vitro antimycobacterial results of
6g
6h
4- 169e170 >50
Cl
4- 164e165 25
Cl
title compounds are tabulated in Table 2 as MIC and the activity
range between 3.125 and >50
and 6p displayed moderate activity (MIC ¼ 12.5
and 6i showed good activity (MIC 6.25
(MIC ¼ 3.125 g/mL) exhibited very good anti-tubercular activity. In
addition, compounds 5a, 5c, 6a, 6b, 6i, 6j and 6p with MIC
g/mL were further subjected to toxicity studies on normal
m
g/mL. In these derivatives 5c, 6a, 6j
g/mL), while 5a
g/mL) and 6b
m
m
¼
6i
6j
6k
6l
4- 187e188
6.25
m
Cl
ꢁ12.5
m
cell lines to analyse the selectivity profile. In these analogous,
compound 6b displayed selectivity index >130 indicating suit-
ability of the compound in an endeavour to attain lead molecule for
further drug development.
4- 116e118 12.5
Cl
Amongst the series, compound 6i inhibits 99% growth of MTB
H37Rv (ATCC 27294) strain at a concentration 6.25 mg/mL. Com-
4- 216e217 50
Cl
pound 6b emerged as the most promising candidate by inhibiting
99% growth of MTB H37Rv (ATCC 27294) strain at a concentration
3.125 mg/mL.
Among the synthesized compounds, electronic effects of sub-
stituent play a crucial role in displaying anti-TB activity. Consid-
ering chloro substituent at 4th, 6th and 7th position of
benzisoxazole moiety (6gex), moderate activity was displayed by
6-chloro derivatives bearing electron withdrawing substituent. In
4- 195e196 50
Cl
(continued on next page)

74
K.M. Naidu et al. / European Journal of Medicinal Chemistry 87 (2014) 71e78
Table 2 (continued )
activity by 4 fold (MIC ¼ 12.5
mg/mL). While varying electron
releasing group, methyl substituent exhibited 8 fold increase in
Entry
R
X
aMP (ꢀC) MIC(
37Rv
mg/mL) against MTB
activity (6i, MIC ¼ 6.25
mg/mL) compared to bromo substituent (6f,
MIC ¼ 50 g/mL). In general, unsubstituted benzene and methyl-
H
m
sulfonyl core of chloro substituted benzisoxazole moiety did not
have much impact on the activity spectrum. Eventually, when
considering unsubstituted benzisoxazole core, both electron with-
drawing and releasing groups on benzenesulfonyl moiety were
unsuccessful in influencing the activity profile. Nevertheless,
replacing methylsulfonyl group exhibited 4 fold increase in activity
6m
6n
6- 194e195 >50
Cl
6- 202e203 25
Cl
(6a, MIC ¼ 12.5
mg/mL) and influx of unsubstituted benzenesulfonyl
moiety (6b) markedly amplified with 16 fold increase in activity.
Thus, 6b emerged as most promising candidate by inhibiting 99%
6o
6p
6q
6r
6- 185e186 >50
Cl
growth of MTB H37Rv strain at a concentration 3.125 mg/mL. Over
and above to promote and validate the necessity of arylsulfonyl
core, we subjected the ability of intermediate 3-(piperazin-1-yl)
benzo[d]isoxazole substituents (5aed) to inhibit the growth of MTB
H
37RV strain. From the activity profile it is apparent that, 6-chloro
substituent 5c exhibited fold increase in activity
(MIC ¼ 12.5 g/mL) and indeed the unsubstituted molecule 5a
exhibited 4 fold increase in activity (MIC ¼ 6.25 g/mL) compared
to 5b and 5d (MIC ¼ 25 g/mL). Focussing on slight manipulation of
6- 176e177 12.5
Cl
2
m
m
m
6- 232e233 25
Cl
electronic environment by introducing various lipophilic substitu-
ent on benzenesulfonyl and benz[d]isoxazole core might lead to
more potent molecules. Further, compared to the earlier reported
terahydrobenzo[d]isoxazole-3-carboxamide derivatives [17] the
present reported compounds exhibit better activity towards MTB in
MABA.
6- 212e213 >50
Cl
6s
6t
7- 172e173 25
Cl
3.2. IC50 assay
The active compounds 5a, 5c, 6a, 6b, 6i, 6j and 6p were sub-
jected to Promega Cell Titer 96 non-radioactive cell proliferation
assay to analyse the selectivity profile against mouse macrophage
(RAW264.7) cell lines. The IC50 values [37] and selectivity index (SI)
values are tabulated in Table 3 and the results imply suitability of
the compounds in drug development for tuberculosis.
7- 175e176 25
Cl
6u
6v
7- 159e160 >50
Cl
3.3. X-ray crystallographic study of compound 6b
7- 170e171 >50
Cl
3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole
(6b)
crystals were grown from the slow evaporation of a 1:2:7 ratio of
methanol/dichloromethane/hexane solvent mixture at rt, to get a
white flake crystal.
A
suitable crystal (Crystal size/mm3
6w
7- 242e243 50
0.39 ꢂ 0.34 ꢂ 0.22), was selected and mounted on an Xcalibur, Eos,
Gemini diffractometer. The crystal was kept at 293.15 K during data
collection. Using Olex2 [38] the structure was solved with the
Unknown structure solution program using Unknown and refined
with the olex2.refine [39] refinement package using Gauss-Newton
minimization. The basic crystallographic data and structure
refinement are shown in Table 4 and molecular structure is given as
an ORTEP diagram in Fig. 2. Crystallographic data of the compound
6b was deposited with the Cambridge Crystallographic Data Centre
and the deposition number is CCDC 968913.
Cl
6x
7- 187e188 25
Cl
Isoniazid
Rifampicin e
e
e
e
e
e
0.36
1.0
a
Uncorrected.
particular 6q, with nitro group at para position of benzenesulfonyl
core found to be fairly active (MIC ¼ 25 g/mL), while tri-
fluoromethoxy group increased the activity by fold (6p,
g/mL). However, complete loss of activity was noticed
for electron releasing groups. In case of 7-chloro derivatives both
electron withdrawing and releasing groups were found to be futile.
Among 4-chloro derivatives electron withdrawing nitro substituent
(6k) was ineffective, and trifluoromethoxy group increased the
4. Conclusion
m
2
Our preliminary anti-tubercular screening results drive us to
contrive the chemical structure of benzo[d]isoxazole derivative to
generate essential pharmacophoric features that could lead to the
synthesis of promising candidate in developing anti-tubercular
agents. These findings unfold the possibility of employing various
lipophilic groups on these derivatives.
MIC ¼ 12.5
m

K.M. Naidu et al. / European Journal of Medicinal Chemistry 87 (2014) 71e78
75
Table 3
IC50 g/mL) and selectivity index (SI) values of active compounds.
129.26, 127.76, 122.61, 121.65, 115.68, 110.57, 47.75, 45.27. HRMS:
(ESI m/z) for C17H18N3O3S calculated: 344.1069, found: 344.1074
(MþH)þ. Anal. Calcd for C17H17N3O3S: (%) C 59.46, H 4.99, N 12.24.
Found: C 59.61, H 5.11, N 12.37.
(
m
Entry MIC ( g/mL) in MTB % Cell inhibition at IC50
m
aSI value
H
37Rv 50 g/mL approximation IC50/MIC
m
5a
5c
6a
6b
6i
6.25
12.5
12.5
3.125
6.25
12.5
12.5
36.84
24.12
28.42
5.82
21.60
42.16
18.10
67.86
103.64
87.96
429.55
115.74
59.29
10.85
8.29
7.03
137.45
18.51
4.74
5.2.3. 3-(4-Tosylpiperazin-1-yl)benzo[d]isoxazole (6c)
Pale yellow solid (85%); 1H NMR (400 MHz, CDCl3)
d 7.85 (d,
J ¼ 7.2 Hz, 1H), 7.81 (dd, J ¼ 9.2 Hz & J ¼ 5.6 Hz, 1H), 7.68 (m, 1H),
7.52 (d, J ¼ 7.2 Hz, 4H), 7.34 (d, J ¼ 7.4 Hz, 1H), 3.52 (t, J ¼ 5.2 Hz &
6j
6p
J ¼ 4.8 Hz, 4H), 3.25 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H), 2.96 (s, 3H). 13
C
138.12
11.04
a
NMR (100 MHz, CDCl3)
d 164.48, 160.12, 135.14, 133.28,129.81,
Selectivity index.
129.26, 127.76, 123.42, 122.61, 121.65, 110.64, 47.75, 45.27, 28.45.
HRMS: (ESI m/z) for C18H20N3O3S calculated: 358.1225, found:
358.1231 (MþH) þ. Anal. Calcd for C17H19N3O3S: (%) C 60.49, H 5.36,
N 11.76. Found: C 60.64, H 5.18, N 11.93.
5. Experimental section
5.1. Materials and methods
5.2.4. 3-(4-(4-(Trifluoromethoxy)phenylsulfonyl)piperazin-1-yl)
benzo[d]isoxazole (6d)
Chemicals and solvents were procured from commercial sources
and are analytically pure. Thin-layer chromatography (TLC) was
carried out on aluminium-supported silica gel plates (Merck 60
F254) with visualization of components by UV light (254 nm).
Column chromatography was carried out on silica gel (Merck
100e200 mesh). 1H NMR spectra and 13C NMR spectra were
recorded at 400 MHz using a Bruker AV 400 spectrometer (Bruker
CO., Switzerland) in CDCl3 and DMSO-d6 solution with tetrame-
White solid (80%); 1H NMR (400 MHz, CDCl3)
d 8.04 (d,
J ¼ 7.4 Hz, 1H), 7.98 (dd, J ¼ 9.2 Hz & J ¼ 5.6 Hz, 1H), 7.81 (dd,
J ¼ 7.8 Hz & J ¼ 5.6 Hz,1H), 7.52 (d, J ¼ 7.2 Hz, 2H), 7.34 (d, J ¼ 7.4 Hz,
1H), 7.08 (d, J ¼ 7.6 Hz, 2H), 3.68 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.24
(t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 164.29,
160.23, 154.62, 135.14, 132.28, 129.32, 128.72, 127.76,123.42, 122.61,
116.76, 110.64, 47.75, 45.27. HRMS: (ESI m/z) for C18H17F3N3O4S
calculated: 428.0892, found: 428.0898 (MþH)þ. Anal. Calcd for
thylsilane as the internal standard, and chemical shift values (d)
were given in ppm. Melting points were determined on an electro
thermal melting point apparatus (Stuart-SMP30) in open capillary
tubes and are uncorrected. IR spectra were recorded with an FT-IR
spectrophotometer (Jasco FTIR-4200). Elemental analyses were
analysed by Elementar Analysensysteme GmbH vario MICRO cube
CHNS/O Analyzer. All tested compounds purity were greater than
95%. High-resolution mass spectra (HRMS) were recorded on
QSTAR XL Hybrid MS/MS mass spectrometer.
C18H16F3N3O4S: (%) C 50.58, H 3.77, N 9.83. Found: C 50.68, H 3.54, N
9.97.
5.2.5. 3-(4-(4-Nitrophenylsulfonyl)piperazin-1-yl)benzo[d]
isoxazole (6e)
Pale yellow solid (82%); 1H NMR (400 MHz, CDCl3)
d 8.28 (d,
J ¼ 8.4 Hz, 1H), 8.24 (d, J ¼ 8.0 Hz, 2H), 8.14 (d, J ¼ 7.6 Hz, 2H), 8.04
(dd, J ¼ 7.2 Hz & J ¼ 5.6 Hz, 1H), 7.78 (dd, J ¼ 7.8 Hz & J ¼ 5.6 Hz, 1H),
7.38 (d, J ¼ 7.4 Hz, 1H), 3.56 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.26 (t,
J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 164.32,
5.2. Synthesis of title compounds (6aex)
160.41, 159.62, 134.64, 133.28, 129.54, 129.22, 127.76, 123.42, 122.61,
110.64, 47.75, 45.27. HRMS: (ESI m/z) for C17H17N4O5S calculated:
389.0920, found: 389.0916 (MþH)þ. Anal. Calcd for C17H16N4O5S:
(%) C 52.57, H 4.15, N 14.43. Found: C 52.73, H 4.32, N 14.57.
To a stirred solution of 3-(piperazin-1-yl)benzo[d]isoxazole
derivatives (5aed) (1.0 equiv.), triethylamine (2.0 equiv.) was added
in dichloromethane and cooled to 0 ꢀC; to this mixture substituted
sulfonylchlorides (1.2 equiv.) were added and allowed to rt, stirred
for 1 h. After completion of the reaction, as indicated by TLC, the
reaction was quenched with ice cold water and extracted with
CH2Cl2. The organic layers were collected, washed with saturated
brine solution, dried over anhydrous Na2SO4 and concentrated in
vacuo. The resultant crude products were purified by column
chromatography [ethyl acetate/hexane (45e80%)] to get the title
compounds in yields ranging from 80 to 98%.
5.2.6. 3-(4-(4-Bromophenylsulfonyl)piperazin-1-yl)benzo[d]
isoxazole (6f)
White solid (85%); 1H NMR (400 MHz, CDCl3)
d 8.21 (d,
J ¼ 7.6 Hz, 1H), 8.08 (dd, J ¼ 9.2 Hz & J ¼ 5.6 Hz, 1H), 7.94 (d,
J ¼ 7.6 Hz, 2H), 7.86 (d, J ¼ 7.2 Hz, 2H), 7.54 (dd, J ¼ 7.8 Hz &
J ¼ 5.6 Hz,1H), 7.32 (d, J ¼ 7.6 Hz, 1H), 3.68 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz,
4H), 3.24 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d
164.89,161.08,139.12,135.14,133.28,129.81,129.42,127.76,123.42,
5.2.1. 3-(4-(Methylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6a)
122.61, 110.64, 47.64, 45.25. HRMS: (ESI m/z) for C17H17BrN3O3S
calculated: 422.0174, found: 422.0181 (MþH)þ. Anal. Calcd for
White solid (94%); 1H NMR (400 MHz, CDCl3)
d 7.67 (d,
J ¼ 8.0 Hz, 1H), 7.53e7.46 (m, 2H), 7.27 (d, J ¼ 12.0 Hz, 1H), 3.70 (t,
C17H16BrN3O3S: (%) C 48.35, H 3.82, N 9.95. Found: C 48.47, H 4.03, N
J ¼ 4.0 Hz, 4H), 3.44 (t, J ¼ 4.0 Hz, 4H), 2.83 (s, 3H). 13C NMR
10.13.
(100 MHz, CDCl3) d164.06, 160.66, 129.90, 122.71, 121.72, 115.74,
110.62, 48.00, 44.93, 34.65. HRMS: (ESI m/z) for C12H16N3O3S
5.2.7. 4-Chloro-3-(4-(methylsulfonyl)piperazin-1-yl)benzo[d]
calculated: 282.0912, found: 282.0916 (MþH)þ. Anal. Calcd for
isoxazole (6g)
White solid (95%); 1H NMR (400 MHz, CDCl3)
d
7.80 (d, J ¼ 7.6 Hz,
C
12H15N3O3S: (%) C 51.23, H 5.37, N 14.94. Found: C 51.39, H 5.44, N
15.11.
1H), 7.58 (m, 1H), 7.21 (d, J ¼ 8.0 Hz, 1H), 3.58 (t, J ¼ 5.2 Hz &
J ¼ 4.8 Hz, 4H), 3.25 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H), 2.98 (s, 3H). 13
C
5.2.2. 3-(4-(Phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6b)
NMR (100 MHz, CDCl3) d 164.54, 160.59, 133.18, 123.11, 122.61,
White solid (88%); 1H NMR (400 MHz, CDCl3)
d
7.80 (d, J ¼ 7.2 Hz,
121.65, 115.65, 47.75, 45.27, 38.18. HRMS: (ESI m/z) for
2H), 7.65e7.48 (m, 4H), 7.45 (d, J ¼ 7.6 Hz, 2H), 7.19 (m, 1H), 3.68 (t,
C
12H15ClN3O3S calculated: 316.0523, found: 316.0529 (MþH)þ.
J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.24 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13
C
Anal. Calcd for C12H14ClN3O3S: (%) C 45.64, H 4.47, N 13.31. Found: C
45.47, H 4.63, N 13.55.
NMR (100 MHz, CDCl3)
d 164.99, 160.59, 135.44, 133.18, 129.81,

76
Table 4
K.M. Naidu et al. / European Journal of Medicinal Chemistry 87 (2014) 71e78
5.2.10. 4-Chloro-3-(4-(4-(trifluoromethoxy)phenylsulfonyl)
Crystal data and structure refinement for 6b.
piperazin-1-yl)benzo[d]isoxazole (6j)
White solid (82%); 1H NMR (400 MHz, CDCl3)
d 7.88 (d,
Compound
6b
J ¼ 8.8 Hz, 1H), 7.45 (d, J ¼ 8.8 Hz, 2H), 7.41 (d, J ¼ 5.2 Hz, 1H), 7.39
(m, 1H), 7.25 (d, J ¼ 8.4 Hz, 2H), 3.53 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H),
3.25 (t, J ¼ 4.8 Hz & J ¼ 4.4 Hz, 4H), 2.86 (s, 3H). 13C NMR (100 MHz,
Formula
C17H17N3O3S
343.39
293.15
0.7107
monoclinic
P21/c
8.2112(6)
19.5119(9)
10.7941(8)
90
109.787(7)
90
Formula weight
Temperature/K
Wavelength (Å)
Crystal system
Space group
a (Å)
CDCl3)
d 164.45, 161.12, 138.22, 133.28, 129.81, 129.26, 127.76,
123.42, 122.61, 121.23, 121.02, 110.64, 47.75, 45.27. HRMS: (ESI m/z)
for
C18H16ClF3N3O4S calculated: 462.0502, found: 462.0506
(MþH)þ. Anal. Calcd for C18H15ClF3N3O4S: (%) C 46.81, H 3.27, N
b (Å)
c (Å)
9.10. Found: C 47.02, H 3.38, N 9.31.
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
V (Å3)
1627.3(2)
4
5.2.11. 4-Chloro-3-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)benzo
Z
[d]isoxazole (6k)
F(000)
720.0
1.402
0.220
5.67e52.744ꢀ
0.0284
Dcalc (g/mm3)
Pale yellow solid (85%); 1H NMR (400 MHz, CDCl3)
d 8.28 (d,
m
2
(mmꢃ1
Q
)
J ¼ 7.6 Hz, 2H), 8.14 (d, J ¼ 8.4 Hz, 2H), 7.88 (d, J ¼ 7.6 Hz,1H), 7.54 (d,
(ꢀ)
J ¼ 7.2 Hz, 1H), 7.38 (m, 1H), 3.56 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.26
Rint
(t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 164.92,
Crystal size/mm3
0.39 ꢂ 0.34 ꢂ 0.22
Goodness-of-fit on F2
R1 indices [I > 2sigma(I)]
wR2 (all data)
1.039
160.41, 156.62, 138.64, 133.28, 129.80, 125.22, 124.76, 122.42, 121.61,
110.64, 47.75, 45.27. HRMS: (ESI m/z) for C17H16ClN4O5S calculated:
423.0530, found: 423.0534 (MþH)þ. Anal. Calcd for C17H15ClN4O5S:
(%) C 48.29, H 3.58, N 13.25. Found: C 48.47, H 3.72, N 13.48.
0.0501
0.1148
0.18/ꢃ0.0.27
Largest diff. peak/hole (e Åꢃ3
)
Fig. 2. ORTEP plot for the compound 6b. All the non-hydrogen atoms are presented by their 30% probability thermal ellipsoids.
5.2.8. 4-Chloro-3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]
5.2.12. 3-(4-(4-Bromophenylsulfonyl)piperazin-1-yl)-4-
chlorobenzo[d]isoxazole (6l)
isoxazole (6h)
White solid (84%); 1H NMR (400 MHz, CDCl3)
d
7.92 (d,
White solid (85%); 1H NMR (400 MHz, CDCl3)
d 8.21 (d,
J ¼ 8.4 Hz, 2H), 7.74 (d, J ¼ 7.2 Hz, 1H), 7.71e7.64 (m, 3H), 7.52 (dd,
J ¼ 7.2 Hz & J ¼ 4.8 Hz, 1H), 7.38 (d, J ¼ 8.2 Hz, 1H), 3.68 (t, J ¼ 5.2 Hz
& J ¼ 4.8 Hz, 4H), 3.24 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR
J ¼ 7.6 Hz, 1H), 8.08 (dd, J ¼ 9.2 Hz & J ¼ 5.6 Hz, 1H), 7.94 (d,
J ¼ 7.6 Hz, 2H), 7.86 (d, J ¼ 7.2 Hz, 2H), 7.54 (dd, J ¼ 7.8 Hz &
J ¼ 5.6 Hz,1H), 7.32 (d, J ¼ 7.6 Hz,1H), 3.68 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz,
4H), 3.24 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
(100 MHz, CDCl3)
d 164.99, 160.59, 135.44, 133.18, 129.81, 129.26,
127.76, 122.61, 121.65, 115.68, 110.57, 47.75, 45.27. HRMS: (ESI m/z)
for C17H17ClN3O3S calculated: 378.0679, found: 378.0684 (MþH)þ.
Anal. Calcd for C17H16ClN3O3S: (%) C 54.04, H 4.27, N 11.12. Found: C
54.27, H 4.45, N 11.37.
d 163.89, 161.08, 139.12, 135.14, 133.28, 129.81, 129.24, 124.76,
123.42, 121.61, 110.64, 47.64, 45.25. HRMS: (ESI m/z) for
C
17H16BrClN3O3S calculated: 455.9784, found: 455.9788 (MþH)þ.
Anal. Calcd for C17H15BrClN3O3S: (%) C 44.70, H 3.31, N 9.20. Found:
C 44.89, H 3.54, N 9.63.
5.2.9. 4-Chloro-3-(4-tosylpiperazin-1-yl)benzo[d]isoxazole (6i)
White solid (95%); 1H NMR (400 MHz, CDCl3)
d 7.70 (d,
J ¼ 8.4 Hz, 2H), 7.44 (d, J ¼ 8.4 Hz, 1H), 7.39 (d, J ¼ 8.0 Hz, 2H), 7.37
(m, 1H), 7.23 (d, J ¼ 8.4 Hz, 1H), 3.53 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H),
3.25 (t, J ¼ 4.8 Hz & J ¼ 4.4 Hz, 4H), 2.86 (s, 3H). 13C NMR (100 MHz,
5.2.13. 6-Chloro-3-(4-(methylsulfonyl)piperazin-1-yl)benzo[d]
isoxazole (6m)
White solid (92%); 1H NMR (400 MHz, CDCl3)
d
7.58 (d, J ¼ 7.6 Hz,
CDCl3)
d
164.98, 160.42, 138.14, 133.28, 129.22, 129.04, 123.76,
1H), 7.52 (d, J ¼ 8.0 Hz, 1H), 7.36 (s, 1H), 3.58 (t, J ¼ 5.2 Hz &
123.42, 122.61, 121.65, 110.64, 47.75, 45.27, 28.14. HRMS: (ESI m/z)
for C18H19ClN3O3S calculated: 392.0836, found: 392.0840 (MþH)þ.
Anal. Calcd for C18H18ClN3O3S: (%) C 55.17, H 4.63, N 10.72 Found: C
55.34, H 4.84, N 10.91.
J ¼ 4.8 Hz, 4H), 3.25 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H), 2.98 (s, 3H). 13
C
NMR (100 MHz, CDCl3)
d 164.12, 152.32, 133.18, 123.11, 122.61,
121.65, 115.65, 47.75, 45.27, 38.18. HRMS: (ESI m/z) for
C
12H15ClN3O3S calculated: 316.0523, found: 316.0529 (MþH)þ.

K.M. Naidu et al. / European Journal of Medicinal Chemistry 87 (2014) 71e78
77
Anal. Calcd for C12H14ClN3O3S: (%) C 45.64, H 4.47, N 13.31. Found: C
45.82, H 4.61, N 13.59.
& J ¼ 4.8 Hz, 4H), 3.28 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H), 2.98 (s, 3H). 13
C
NMR (100 MHz, CDCl3)
d
164.14, 152.54, 132.18, 124.26, 122.61,
121.65, 115.65, 47.75, 45.27, 38.18. HRMS: (ESI m/z) for
5.2.14. 6-Chloro-3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]
C
12H15ClN3O3S calculated: 316.0523, found: 316.0529 (MþH)þ.
isoxazole (6n)
Anal. Calcd for C12H14ClN3O3S: (%) C 45.64, H 4.47, N 13.31. Found: C
45.78, H 4.62, N 13.51.
White solid (98%); 1H NMR (400 MHz, CDCl3)
d 8.02 (d,
J ¼ 8.4 Hz, 2H), 7.78e7.67 (m, 3H), 7.58 (d, J ¼ 7.6 Hz, 1H), 7.52 (d,
J ¼ 8.0 Hz, 1H), 7.36 (s, 1H), 3.68 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.24
5.2.20. 7-Chloro-3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]
(t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 165.57,
isoxazole (6t)
156.64, 136.44, 134.18, 129.81, 128.24, 127.76, 122.61, 121.65, 115.68,
110.57, 47.75, 45.27. HRMS: (ESI m/z) for C17H17ClN3O3S calculated:
378.0679, found: 378.0684 (MþH)þ. Anal. Calcd for C17H16ClN3O3S:
(%) C 54.04, H 4.27, N 11.12. Found: C 54.24, H 4.41, N 11.39.
White solid (95%); 1H NMR (400 MHz, CDCl3)
d 7.92 (d,
J ¼ 8.4 Hz, 2H), 7.74e7.66 (m, 3H), 7.52 (d, J ¼ 7.2 Hz, 1H), 7.48 (d,
J ¼ 8.2 Hz,1H), 7.38 (m, 1H), 3.68 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.29
(t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 163.44,
154.12, 136.44, 133.18, 129.81, 129.26, 127.76, 122.61, 121.65, 116.68,
110.57, 47.75, 45.27. HRMS: (ESI m/z) for C17H17ClN3O3S calculated:
378.0679, found: 378.0684 (MþH)þ. Anal. Calcd for C17H16ClN3O3S:
(%) C 54.04, H 4.27, N 11.12. Found: C 54.28, H 4.42, N 11.33.
5.2.15. 6-Chloro-3-(4-tosylpiperazin-1-yl)benzo[d]isoxazole (6o)
White solid (85%); 1H NMR (400 MHz, CDCl3)
d 7.89 (d,
J ¼ 8.4 Hz, 2H), 7.62 (d, J ¼ 8.0 Hz, 1H), 7.54 (d, J ¼ 7.6 Hz, 1H), 7.42
(d, J ¼ 8.0 Hz, 2H), 7.34 (s, 1H), 3.56 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H),
3.29 (t, J ¼ 4.8 Hz & J ¼ 4.4 Hz, 4H), 2.76 (s, 3H). 13C NMR (100 MHz,
5.2.21. 7-Chloro-3-(4-tosylpiperazin-1-yl)benzo[d]isoxazole (6u)
CDCl3)
d 165.98, 152.42, 137.14, 133.28, 129.81, 128.24, 127.76,
White solid (85%); 1H NMR (400 MHz, CDCl3)
d 7.82 (d,
123.42, 122.61, 121.65, 110.64, 47.75, 45.27, 28.14. HRMS: (ESI m/z)
for C18H19ClN3O3S calculated: 392.0836, found: 392.0840 (MþH)þ.
Anal. Calcd for C18H18ClN3O3S: (%) C 55.17, H 4.63, N 10.72 Found: C
55.38, H 4.88, N 10.97.
J ¼ 8.4 Hz, 2H), 7.64 (d, J ¼ 8.0 Hz, 1H), 7.52 (d, J ¼ 7.6 Hz, 1H), 7.44
(d, J ¼ 8.0 Hz, 2H), 7.38 (m, 1H), 3.54 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H),
3.29 (t, J ¼ 4.8 Hz & J ¼ 4.4 Hz, 4H), 2.86 (s, 3H). 13C NMR (100 MHz,
CDCl3)
d 164.18, 159.42, 136.22, 132.34, 129.81, 128.14, 124.76,
123.42, 122.61, 121.65, 110.64, 47.75, 45.27, 28.14. HRMS: (ESI m/z)
for C18H19ClN3O3S calculated: 392.0836, found: 392.0840 (MþH)þ.
Anal. Calcd for C18H18ClN3O3S: (%) C 55.17, H 4.63, N 10.72 Found: C
55.41, H 4.82, N 10.93.
5.2.16. 6-Chloro-3-(4-(4-(trifluoromethoxy)phenylsulfonyl)
piperazin-1-yl)benzo[d]isoxazole (6p)
White solid (80%); 1H NMR (400 MHz, CDCl3)
d 7.87 (d,
J ¼ 8.0 Hz, 2H), 7.52 (d, J ¼ 8.0 Hz,1H), 7.46 (s,1H), 7.41 (d, J ¼ 8.0 Hz,
2H), 7.22 (d, J ¼ 8.0 Hz, 1H), 3.68 (t, J ¼ 8.0 Hz & J ¼ 4.0 Hz, 4H), 3.27
(t, J ¼ 4.0 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 164.32, 160.19,
5.2.22. 7-Chloro-3-(4-(4-(trifluoromethoxy)phenylsulfonyl)
piperazin-1-yl)benzo[d]isoxazole (6v)
152.55, 136.46, 133.94, 129.90, 123.63, 121.15, 121.05, 118.91, 114.46,
110.98, 47.72, 45.12. HRMS: (ESI m/z) for C18H16ClF3N3O4S calcu-
lated: 462.0502, found: 462.0506 (MþH)þ. Anal. Calcd for
White solid (84%); 1H NMR (400 MHz, CDCl3)
d 7.86 (d,
J ¼ 8.4 Hz, 2H), 7.56 (d, J ¼ 8.0 Hz, 1H), 7.48 (d, J ¼ 7.6 Hz, 1H), 7.38
(m, 1H), 7.18 (d, J ¼ 7.2 Hz, 2H), 3.56 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H),
3.27 (t, J ¼ 4.8 Hz & J ¼ 4.4 Hz, 4H). 13C NMR (100 MHz, CDCl3)
C
18H15ClF3N3O4S: (%) C 46.81, H 3.27, N 9.10. Found: C 46.98, H 3.42,
N 9.35.
d
164.14, 160.12, 148.22, 136.28, 132.81, 129.26, 124.76, 123.42,
5.2.17. 6-Chloro-3-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)benzo
122.61, 120.23, 114.14, 110.64, 47.75, 45.27. HRMS: (ESI m/z) for
18H16ClF3N3O4S calculated: 462.0502, found: 462.0506 (MþH)þ.
[d]isoxazole (6q)
C
Yellow solid (85%); 1H NMR (400 MHz, CDCl3)
d 8.28 (d,
Anal. Calcd for C18H15ClF3N3O4S: (%) C 46.81, H 3.27, N 9.10. Found:
C 47.04, H 3.46, N 9.29.
J ¼ 8.0 Hz, 2H), 8.14 (d, J ¼ 8.4 Hz, 2H), 7.58 (d, J ¼ 7.6 Hz, 1H), 7.48
(d, J ¼ 7.6 Hz, 1H), 7.38 (s, 1H), 3.56 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H),
3.29 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
5.2.23. 7-Chloro-3-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)benzo
d
164.96, 154.41, 152.62, 138.64, 133.28, 129.81, 125.26, 124.76,
[d]isoxazole (6w)
122.42, 121.61, 110.64, 47.75, 45.27. HRMS: (ESI m/z) for
Yellow solid (85%); 1H NMR (400 MHz, CDCl3)
d 8.28 (d,
C
17H16ClN4O5S calculated: 423.0530, found: 423.0534 (MþH)þ.
J ¼ 7.6 Hz, 2H), 8.14 (d, J ¼ 8.4 Hz, 2H), 7.58 (d, J ¼ 7.6 Hz,1H), 7.46 (d,
Anal. Calcd for C17H15ClN4O5S: (%) C 48.29, H 3.58, N 13.25. Found: C
48.44, H 3.69, N 13.07.
J ¼ 7.2 Hz, 1H), 7.41 (m, 1H), 3.56 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.29
(t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d 164.92,
160.41, 156.62, 137.64, 132.28, 128.82, 125.26, 122.76, 122.42, 120.24,
116.64, 47.75, 45.27. HRMS: (ESI m/z) for C17H16ClN4O5S calculated:
423.0530, found: 423.0534 (MþH)þ. Anal. Calcd for C17H15ClN4O5S:
(%) C 48.29, H 3.58, N 13.25. Found: C 48.49, H 3.71, N 13.44.
5.2.18. 3-(4-(4-Bromophenylsulfonyl)piperazin-1-yl)-6-
chlorobenzo[d]isoxazole (6r)
White solid (94%); 1H NMR (400 MHz, CDCl3)
d
8.12 (d, J ¼ 7.6 Hz,
2H), 8.08 (d, J ¼ 7.4 Hz, 2H), 7.56 (d, J ¼ 7.6 Hz,1H), 7.42 (d, J ¼ 7.2 Hz,
1H), 7.32 (s, 1H), 3.58 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H), 3.29 (t,
J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
d
164.88,
5.2.24. 3-(4-(4-Bromophenylsulfonyl)piperazin-1-yl)-7-
152.08, 139.12, 135.14, 133.28, 129.81, 129.04, 124.76,123.42, 122.61,
110.64, 47.64, 45.25. HRMS: (ESI m/z) for C17H16BrClN3O3S calcu-
lated: 455.9784, found: 455.9788 (MþH)þ. Anal. Calcd for
chlorobenzo[d]isoxazole (6x)
White solid (85%); 1H NMR (400 MHz, CDCl3)
d 8.08 (d,
J ¼ 8.0 Hz, 2H), 8.02 (d, J ¼ 7.6 Hz, 2H), 7.56 (d, J ¼ 7.2 Hz, 1H), 7.46
(d, J ¼ 7.8 Hz, 1H), 7.36 (m, 1H), 3.58 (t, J ¼ 5.2 Hz & J ¼ 4.8 Hz, 4H),
3.26 (t, J ¼ 4.8 Hz & J ¼ 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3)
C
17H15BrClN3O3S: (%) C 44.70, H 3.31, N 9.20. Found: C 44.91, H 3.50,
N 9.57.
d
163.74, 160.08, 139.12, 133.14, 132.28, 129.81, 124.84, 123.76,
5.2.19. 7-Chloro-3-(4-(methylsulfonyl)piperazin-1-yl)benzo[d]
122.42, 121.61, 111.12, 47.64, 45.25. HRMS: (ESI m/z) for
C
Anal. Calcd for C17H15BrClN3O3S: (%) C 44.70, H 3.31, N 9.20. Found:
C 44.94, H 3.58, N 9.59.
isoxazole (6s)
17H16BrClN3O3S calculated: 455.9784, found: 455.9788 (MþH)þ.
White solid (94%); 1H NMR (400 MHz, CDCl3)
d
7.67 (d,
J ¼ 8.0 Hz,1H), 7.54 (m,1H), 7.46 (d, J ¼ 8.0 Hz,1H), 3.56 (t, J ¼ 5.2 Hz

78
K.M. Naidu et al. / European Journal of Medicinal Chemistry 87 (2014) 71e78
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5.3. Microplate Alamar Blue Assay (MABA)
The antimycobacterial activities of title compounds 5aed and
6ae6x were evaluated against MTB H37Rv (ATCC 27294) strain by
the MABA [40,41]. Isoniazid and rifampin are used as positive
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mg/mL, and the final test concentrations ranged
6085e6094.
from 50 to 0.78 g/mL. Two fold serial dilutions of compounds were
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prepared in Middlebrook 7H9GC medium in a volume of 100 mL in
96-well microplates. TB culture (100
m
L inoculum of 2 ꢂ 105 CFU/
mL) was added, yielding a final testing volume of 200 mL. The plates
were incubated at 37 ꢀC. On the seventh day of incubation 25
10% Tween 80 and 25 L of Alamar Blue (Accumed International,
mL of
m
Westlake, Ohio) were added to the wells of test plate. After incu-
bation at 37 ꢀC for 24 h, colours of all wells were recorded. A blue
colour in the well was interpreted as no growth, and a pink colour
was scored as growth. The MICs were defined as the lowest con-
centration which prevented a colour change from blue to pink.
5.4. IC50 assay
The most active compounds (5a, 5c, 6a, 6b, 6i, 6j and 6p) were
further examined for selectivity profile in RAW 264.7 cell line at the
concentration of 50 mg/mL. After 72 h of exposure, viability was
assessed on the basis of cellular conversion of MTT into a formazan
product using the Promega Cell Titer 96 non-radioactive cell pro-
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