Accepted Manuscript

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

Kalaga Mahalakshmi Naidu, Amaroju Suresh, Jayanty Subbalakshmi, DharmarajanSriram, Perumal Yogeeswari, Pallepogu Raghavaiah, Kondapalli Venkata GowriChandra Sekhar

PII: S0223-5234(14)00861-7

DOI: 10.1016/j.ejmech.2014.09.043

Reference: EJMECH 7349

To appear in: European Journal of Medicinal Chemistry

Received Date: 14 April 2014

Revised Date: 10 September 2014

Accepted Date: 12 September 2014

Please cite this article as: K.M. Naidu, A. Suresh, J. Subbalakshmi, D. Sriram, P. Yogeeswari, P.Raghavaiah, K.V. Gowri Chandra Sekhar, Design, synthesis and antimycobacterial activity of various3-(4-(substitutedsulfonyl)piperazin-1-yl)benzo[d]isoxazole derivatives, European Journal of MedicinalChemistry (2014), doi: 10.1016/j.ejmech.2014.09.043.

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6b, MIC = 3.125 µg/mL, SI = 137.45 Single crystal structure of 6b

Design, synthesis and antimycobacterial activity of various 3-(4-(substitutedsulfonyl)piperazin-1-yl)benzo[d]isoxazole derivatives Kalaga Mahalakshmi Naidua, Amaroju Suresh,a Jayanty Subbalakshmia, Dharmarajan Sriramb, Perumal Yogeeswarib, Pallepogu Raghavaiah,c Kondapalli Venkata Gowri Chandra Sekhar a*

aDepartment of Chemistry Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad-500 078, Andhra Pradesh, India.

bDepartment of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad-500 078, Andhra Pradesh, India.

cDepartment of Chemistry, University of Cape Town, Rondebosch-7707, Cape Town, South Africa.

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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 Sekhara∗

aDepartment of Chemistry, Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad-500 078, Andhra Pradesh, India.

bDepartment of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad-500 078, Andhra Pradesh, India.

cDepartment of Chemistry, University of Cape Town, Rondebosch-7707, Cape Town, South Africa.

Abstract: 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 - >50 µg/mL. Among the tested compounds,

5c, 6a, 6j and 6p exhibited moderate activity (MIC = 12.5 µg/mL), while 5a and 6i exhibited

good activity (MIC = 6.25 µg/mL) and 6b (MIC = 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.

Keywords:

Benzo[d]isoxazole; Piperazine sulphonamide; Antimycobacterial activity; Mycobacterium

tuberculosis;

1. Introduction

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 immunodeficiency virus (HIV). TB is one of the major

causes of death in HIV patients. According to the World Health Organization (WHO), about ∗ Corresponding author

Tel: +91- 40-66303527, E-mail: kvgc@hyderabad.bits-pilani.ac.in; kvgcs@yahoo.com

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one-third of the world’s population is latently infected with MTB. In 2012, there were an

estimated 8.6–9.4 million cases and 1.2–1.5 million deaths (including deaths from TB among

HIV-positive people). The vast majority of TB deaths are in the developing world and more

than 50 % of all deaths occur in Asia alone. The five countries with the largest number of

incident cases in 2012 were India (2.0 million–2.4 million), China (0.9 million–1.1 million),

South Africa (0.4 million–0.6 million), Indonesia (0.4 million–0.5 million) and Pakistan (0.3

million–0.5 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 although

not always difficult to treat [3-5]. Prevalence of XDR-TB and TDR-TB has increased

worldwide [5-7]. 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], anticonvulsant [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], antiprotozoal [24], anticonvulsant [25], anti-diabetic [26], sigma receptor

ligands [27] and in precise 7-[4-(5-amino-[1,3,4]thiadiazole-2-sulfonyl)-piperazin-1-yl]-1-

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cyclopropyl and ethyl-6-fluoro-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid as

antibacterial and anti-TB agents [28].

Benzo[d]isoxazole derivatives target Pantothenate synthetase (PS) enzyme which catalyzes

amide bond formation of pantothenate from D-pantoate and β-alanine accompanied by

hydrolysis of Mg-adenosine 5′-triphosphate (ATP) into adenosine 5′-monophosphate (AMP)

and Mg-pyrophosphate (PPi). PS is recognized as a suitable target for discovery of new

therapeutics to treat TB [17,29,30].

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

sulfonylpiperazin-1-yl)benzo[d]isoxazole derivatives and evaluated for their

antimycobacterial activity.

2. Chemistry

In this report, we synthesized 3-(4-substituted sulfonylpiperazin-1-yl)benz[d]isoxazole

derivatives as depicted in Figure 1. Substituted 2-chlorobenzaldehyde (1a-d) on treatment

with NH2OH.HCl and CH3COONa in EtOH and H2O at 0 ºC - rt for 1h yielded substituted 2-

chlorobenzaldehyde oxime derivatives (2a-d) [31]. Further chlorination with N-

chlorosuccinimide in CCl4 stirred at 0 ºC – rt for 45 min afforded substituted 2-chloro-N-

hydroxybenzimidoyl chloride derivatives (3a-d) [32]. Substituted (2-chlorophenyl)

(piperazin-1-yl)methanone oxime (4a-d) [33] was synthesized by reacting 3a-d with

anhydrous piperazine and N(C2H5)3 as base in CH2Cl2 at rt for 2h. Literature survey reveals

employing expensive and toxic catalysts for the effective cyclization of 4a-d to afford 5a-d

[34-36]. We herein optimised this cyclization by employing inexpensive base (Table 1). As a

model reaction, 4a was treated with potassium carbonate (K2CO3) in acetonitrile (MeCN) at

75 oC for 12h. 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 oC under conventional heating source for 12h the desired

product was obtained in about 38%. Under similar reaction conditions, an increase in the

yield was noticed (entry 6 and 7) when we changed solvent to dioxane and dioxane : water

(3:1 ratio). Accentuation on microwave assisted organic synthesis; reaction was carried out at

120 oC 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 8-10) to

afford the desired compound in moderate yield. Having better results under closed vessel

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condition, we further focussed to carry out the reaction in sealed tube under conventional

heating source at various intervals of time (entry 11-15). These findings noticeably increased

the yield and diminished the reaction time to 4h. Having the optimised reaction condition

handy various 5a-d derivatives were synthesized in good yield. Further, title compounds 6a-x

were synthesized by reacting substituted-3-(piperazin-1-yl)benzo[d]isoxazole (5a-d) with

various sulfonyl chlorides, using N(C2H5)3 as base and CH2Cl2 as solvent at 0 ºC to rt. All the

title compounds were purified by column chromatography using 45-80% of ethyl acetate in

hexane. The 1H NMR showed multiplets in the range 3.22-3.68 ppm due to piperazine (-CH2-

) protons, aromatic protons of substituted sulfonyl group and benzoisoxazole appeared at

6.98-8.42 ppm; 13C NMR showed aromatic carbons in the range of 110-166 ppm and

piperazine carbons 45-47 ppm. IR spectroscopy revealed the absence of peak at ∼ 3230 cm-1

due to NH stretch in the title compounds 6a-x, clearly confirming the formation of products.

All the synthesized compounds were confirmed by 1H NMR, 13C NMR, IR and HRMS. In

addition single crystal structure was determined for one of the active compounds, 6b.

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 µg/mL. Isoniazid and Rifampin were used as the

positive drug standards. The in vitro antimycobacterial results of title compounds are

tabulated in Table 2 as MIC and the activity range between 3.125 - >50 µg/mL. In these

derivatives 5c, 6a, 6j and 6p displayed moderate activity (MIC = 12.5 µg/mL), while 5a and

6i showed good activity (MIC = 6.25 µg/mL) and 6b (MIC = 3.125 µg/mL) exhibited very

good anti-tubercular activity. In addition, compounds 5a, 5c, 6a, 6b, 6i, 6j and 6p with MIC

≤ 12.5 µg/mL were further subjected to toxicity studies on normal cell lines to analyse the

selectivity profile. In these analogous, compound 6b displayed selectivity index > 130

indicating suitability of the compound in an endeavour to attain lead molecule for further

drug development.

Amongst the series, compound 6i inhibits 99% growth of MTB H37Rv (ATCC 27294) strain

at a concentration 6.25 µg/mL. Compound 6b emerged as the most promising candidate by

inhibiting 99% growth of MTB H37Rv (ATCC 27294) strain at a concentration 3.125 µg/mL.

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Among the synthesized compounds, electronic effects of substituent play a crucial role in

displaying anti-TB activity. Considering chloro substituent at 4th, 6th and 7th position of

benzisoxazole moiety (6g-x), moderate activity was displayed by 6-chloro derivatives bearing

electron withdrawing substituent. In particular 6q, with nitro group at para position of

benzenesulfonyl core found to be fairly active (MIC = 25 µg/mL), while trifluoromethoxy

group increased the activity by 2 fold (6p, MIC = 12.5 µg/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 activity by 4 fold (MIC = 12.5 µg/mL). While varying electron releasing

group, methyl substituent exhibited 8 fold increase in activity (6i, MIC = 6.25 µg/mL)

compared to bromo substituent (6f, MIC = 50 µg/mL). In general, unsubstituted benzene and

methylsulfonyl core of chloro substituted benzisoxazole moiety did not have much impact on

the activity spectrum. Eventually, when considering unsubstituted benzisoxazole core, both

electron withdrawing and releasing groups on benzenesulfonyl moiety were unsuccessful in

influencing the activity profile. Nevertheless, replacing methylsulfonyl group exhibited 4 fold

increase in activity (6a, MIC = 12.5 µg/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% growth of MTB H37Rv strain at a concentration 3.125

µg/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 (5a-d)

to inhibit the growth of MTB H37RV strain. From the activity profile it is apparent that, 6-

chloro substituent 5c exhibited 2 fold increase in activity (MIC = 12.5 µg/mL) and indeed the

unsubstituted molecule 5a exhibited 4 fold increase in activity (MIC = 6.25 µg/mL)

compared to 5b and 5d (MIC = 25 µg/mL). Focussing on slight manipulation of electronic

environment by introducing various lipophilic substituent 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.

3.2 IC50 assay

The active compounds 5a, 5c, 6a, 6b, 6i, 6j and 6p were subjected 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

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tabulated in Table 3 and the results imply suitability of the compounds in drug development

for tuberculosis.

3.3. X-ray crystallographic study of compound 6b

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 0.39 × 0.34 × 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 Figure 2. Crystallographic data of the compound 6b was deposited with the

Cambridge Crystallographic Data Centre and the deposition number is CCDC 968913.

4. Conclusion

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.

5. Experimental Section

5.1. Materials and methods

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 100-200 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 tetramethylsilane as the internal

standard, and chemical shift values (δ) 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 analyzed 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.

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5.2 Synthesis of title compounds (6a-x)

To a stirred solution of 3-(piperazin-1-yl)benzo[d]isoxazole derivatives (5a-d ) (1.0 equiv.),

triethylamine (2.0 equiv.) was added in dichloromethane and cooled to 0 ºC; to this mixture

substituted sulfonylchlorides (1.2 equiv.) were added and allowed to rt, stirred for 1h. 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 (45 - 80 %)] to get

the title compounds in yields ranging from 80-98%.

5.2.1. 3-(4-(methylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6a)

White solid (94%); 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 8.0 Hz, 1H), 7.53 – 7.46 (m,

2H), 7.27 (d, J = 12.0 Hz, 1H), 3.70 (t, J = 4.0 Hz, 4H), 3.44 (t, J = 4.0 Hz, 4H), 2.83 (s, 3H). 13C NMR (100 MHz, CDCl3) δ164.06, 160.66, 129.90, 122.71, 121.72, 115.74, 110.62,

48.00, 44.93, 34.65. HRMS: (ESI m/z) for C12H16N3O3S calculated: 282.0912, found:

282.0916 (M+H) +. Anal. Calcd for C12H15N3O3S: (%) C 51.23, H 5.37, N 14.94. Found: C

51.39, H 5.44, N 15.11.

5.2.2. 3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6b)

White solid (88%); 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 7.2 Hz, 2H), 7.65 – 7.48 (m,

4H), 7.45 (d, J = 7.6 Hz, 2H), 7.19 (m, 1H), 3.68 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.24 (t, J

= 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 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

C17H18N3O3S calculated: 344.1069, found: 344.1074 (M+H) +. Anal. Calcd for C17H17N3O3S:

(%) C 59.46, H 4.99, N 12.24. Found: C 59.61, H 5.11, N 12.37.

5.2.3. 3-(4-tosylpiperazin-1-yl)benzo[d]isoxazole (6c)

Pale yellow solid (85%); 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 7.2 Hz, 1H), 7.81 (dd, J

= 9.2 Hz & J = 5.6 Hz, 1H), 7.68 (m, 1H), 7.52 (d, J = 7.2 Hz, 4H), 7.34 (d, J = 7.4 Hz, 1H),

3.52 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.25 (t, J = 4.8 Hz & J = 5.2 Hz, 4H), 2.96 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 164.48, 160.12, 135.14, 133.28,129.81, 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 (M+H) +. Anal. Calcd for C17H19N3O3S: (%) C 60.49,

H 5.36, N 11.76. Found: C 60.64, H 5.18, N 11.93.

5.2.4. 3-(4-(4-(trifluoromethoxy)phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6d)

White solid (80%); 1H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 7.4 Hz, 1H), 7.98 (dd, J = 9.2

Hz & J = 5.6 Hz, 1H), 7.81 (dd, J = 7.8 Hz & J = 5.6 Hz, 1H), 7.52 (d, J = 7.2 Hz, 2H), 7.34

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(d, J = 7.4 Hz, 1H), 7.08 (d, J = 7.6 Hz, 2H), 3.68 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.24 (t, J

= 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 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 (M+H) +. Anal. Calcd for

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) δ 8.28 (d, J = 8.4 Hz, 1H), 8.24 (d, J =

8.0 Hz, 2H), 8.14 (d, J = 7.6 Hz, 2H), 8.04 (dd, J = 7.2 Hz & J = 5.6 Hz, 1H), 7.78 (dd, J =

7.8 Hz & J = 5.6 Hz, 1H), 7.38 (d, J = 7.4 Hz, 1H), 3.56 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H),

3.26 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 164.32, 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 (M+H) +. Anal. Calcd for

C17H16N4O5S: (%) C 52.57, H 4.15, N 14.43. Found: C 52.73, H 4.32, N 14.57.

5.2.6. 3-(4-(4-bromophenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6f)

White solid (85%); 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 7.6 Hz, 1H), 8.08 (dd, J = 9.2

Hz & J = 5.6 Hz, 1H), 7.94 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 7.2 Hz, 2H), 7.54 (dd, J = 7.8

Hz & J = 5.6 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 3.68 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.24

(t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 164.89, 161.08, 139.12,

135.14, 133.28, 129.81, 129.42, 127.76,123.42, 122.61, 110.64, 47.64, 45.25. HRMS: (ESI

m/z) for C17H17BrN3O3S calculated: 422.0174, found: 422.0181 (M+H) +. Anal. Calcd for

C17H16BrN3O3S: (%) C 48.35, H 3.82, N 9.95. Found: C 48.47, H 4.03, N 10.13.

5.2.7. 4-chloro-3-(4-(methylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6g)

White solid (95%); 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 7.6 Hz, 1H), 7.58 (m, 1H),

7.21 (d, J = 8.0 Hz, 1H), 3.58 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.25 (t, J = 4.8 Hz & J = 5.2

Hz, 4H), 2.98 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 164.54, 160.59, 133.18, 123.11,

122.61, 121.65, 115.65, 47.75, 45.27, 38.18. HRMS: (ESI m/z) for C12H15ClN3O3S

calculated: 316.0523, found: 316.0529 (M+H) +. Anal. Calcd for C12H14ClN3O3S: (%) C

45.64, H 4.47, N 13.31. Found: C 45.47, H 4.63, N 13.55.

5.2.8. 4-chloro-3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6h)

White solid (84%); 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.2

Hz, 1H), 7.71 – 7.64 (m, 3H), 7.52 (dd, J = 7.2 Hz & J = 4.8 Hz, 1H), 7.38 (d, J = 8.2 Hz,

1H), 3.68 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.24 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR

(100 MHz, CDCl3) δ 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:

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378.0679, found: 378.0684 (M+H) +. Anal. Calcd for C17H16ClN3O3S: (%) C 54.04, H 4.27,

N 11.12. Found: C 54.27, H 4.45, N 11.37.

5.2.9. 4-chloro-3-(4-tosylpiperazin-1-yl)benzo[d]isoxazole (6i)

White solid (95%); 1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4

Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.37 (m, 1H), 7.23 (d, J = 8.4 Hz, 1H), 3.53 (t, J = 5.2 Hz

& J = 4.8 Hz,, 4H), 3.25 (t, J = 4.8 Hz & J = 4.4 Hz, 4H), 2.86 (s, 3H). 13C NMR (100 MHz,

CDCl3) δ 164.98, 160.42, 138.14, 133.28, 129.22, 129.04, 123.76, 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 (M+H) +. Anal. Calcd for C18H18ClN3O3S: (%) C 55.17, H 4.63, N 10.72

Found: C 55.34, H 4.84, N 10.91.

5.2.10. 4-chloro-3-(4-(4-(trifluoromethoxy)phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole

(6j)

White solid (82%); 1H NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.8

Hz, 2H), 7.41 (d, J = 5.2 Hz, 1H), 7.39 (m, 1H), 7.25 (d, J = 8.4 Hz, 2H), 3.53 (t, J = 5.2 Hz

& J = 4.8 Hz, 4H), 3.25 (t, J = 4.8 Hz & J = 4.4 Hz, 4H), 2.86 (s, 3H). 13C NMR (100 MHz,

CDCl3) δ 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 (M+H) +. Anal. Calcd for C18H15ClF3N3O4S: (%) C 46.81, H 3.27, N 9.10.

Found: C 47.02, H 3.38, N 9.31.

5.2.11. 4-chloro-3-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6k)

Pale yellow solid (85%); 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 7.6 Hz, 2H), 8.14 (d, J =

8.4 Hz, 2H), 7.88 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 7.2 Hz, 1H), 7.38 (m, 1H), 3.56 (t, J = 5.2

Hz & J = 4.8 Hz,, 4H), 3.26 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ

164.92, 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

(M+H) +. Anal. Calcd for C17H15ClN4O5S: (%) C 48.29, H 3.58, N 13.25. Found: C 48.47, H

3.72, N 13.48.

5.2.12. 3-(4-(4-bromophenylsulfonyl)piperazin-1-yl)-4-chlorobenzo[d]isoxazole (6l)

White solid (85%); 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 7.6 Hz, 1H), 8.08 (dd, J = 9.2

Hz & J = 5.6 Hz, 1H), 7.94 (d, J = 7.6 Hz, 2H), 7.86 (d, J = 7.2 Hz, 2H), 7.54 (dd, J = 7.8

Hz & J = 5.6 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 3.68 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.24

(t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 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

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m/z) for C17H16BrClN3O3S calculated: 455.9784, found: 455.9788 (M+H) +. Anal. Calcd for

C17H15BrClN3O3S: (%) C 44.70, H 3.31, N 9.20. Found: C 44.89, H 3.54, N 9.63.

5.2.13. 6-chloro-3-(4-(methylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6m)

White solid (92%); 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.6 Hz, 1H), 7.52 (d, J = 8.0

Hz, 1H), 7.36 (s, 1H), 3.58 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.25 (t, J = 4.8 Hz & J = 5.2

Hz, 4H), 2.98 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 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 C12H15ClN3O3S

calculated: 316.0523, found: 316.0529 (M+H) +. Anal. Calcd for C12H14ClN3O3S: (%) C

45.64, H 4.47, N 13.31. Found: C 45.82, H 4.61, N 13.59.

5.2.14. 6-chloro-3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6n)

White solid (98%); 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 8.4 Hz, 2H), 7.78 – 7.67 (m,

3H), 7.58 (d, J = 7.6 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.36 (s, 1H), 3.68 (t, J = 5.2 Hz & J =

4.8 Hz,, 4H), 3.24 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 165.57,

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 (M+H) +.

Anal. Calcd for C17H16ClN3O3S: (%) C 54.04, H 4.27, N 11.12. Found: C 54.24, H 4.41, N

11.39.

5.2.15. 6-chloro-3-(4-tosylpiperazin-1-yl)benzo[d]isoxazole (6o)

White solid (85%); 1H NMR (400 MHz, CDCl3) δ 7.89 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.0

Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.34 (s, 1H), 3.56 (t, J = 5.2 Hz

& J = 4.8 Hz,, 4H), 3.29 (t, J = 4.8 Hz & J = 4.4 Hz, 4H), 2.76 (s, 3H). 13C NMR (100 MHz,

CDCl3) δ 165.98, 152.42, 137.14, 133.28, 129.81, 128.24, 127.76, 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 (M+H) +. Anal. Calcd for C18H18ClN3O3S: (%) C 55.17, H 4.63, N 10.72

Found: C 55.38, H 4.88, N 10.97.

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) δ 7.87 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0

Hz, 1H), 7.46 (s, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 1H), 3.68 (t, J = 8.0 Hz

& J = 4.0 Hz,, 4H), 3.27 (t, J = 4.0 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 164.32, 160.19,

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 calculated: 462.0502, found: 462.0506

(M+H) +. Anal. Calcd for C18H15ClF3N3O4S: (%) C 46.81, H 3.27, N 9.10. Found: C 46.98, H

3.42, N 9.35.

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5.2.17. 6-chloro-3-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6q)

Yellow solid (85%); 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 8.0 Hz, 2H), 8.14 (d, J = 8.4

Hz, 2H), 7.58 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.38 (s, 1H), 3.56 (t, J = 5.2 Hz

& J = 4.8 Hz,, 4H), 3.29 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ

164.96, 154.41, 152.62, 138.64, 133.28, 129.81, 125.26, 124.76, 122.42, 121.61, 110.64,

47.75, 45.27. HRMS: (ESI m/z) for C17H16ClN4O5S calculated: 423.0530, found: 423.0534

(M+H) +. Anal. Calcd for C17H15ClN4O5S: (%) C 48.29, H 3.58, N 13.25. Found: C 48.44, H

3.69, N 13.07.

5.2.18. 3-(4-(4-bromophenylsulfonyl)piperazin-1-yl)-6-chlorobenzo[d]isoxazole (6r)

White solid (94%); 1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 7.6 Hz, 2H), 8.08 (d, J = 7.4

Hz, 2H), 7.56 (d, J = 7.6 Hz, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.32 (s, 1H), 3.58 (t, J = 5.2 Hz

& J = 4.8 Hz,, 4H), 3.29 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ

164.88, 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 calculated: 455.9784, found: 455.9788

(M+H) +. Anal. Calcd for C17H15BrClN3O3S: (%) C 44.70, H 3.31, N 9.20. Found: C 44.91, H

3.50, N 9.57.

5.2.19. 7-chloro-3-(4-(methylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6s)

White solid (94%); 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 8.0 Hz, 1H), 7.54 (m, 1H),

7.46 (d, J = 8.0 Hz, 1H), 3.56 (t, J = 5.2 Hz & J = 4.8 Hz,, 4H), 3.28 (t, J = 4.8 Hz & J = 5.2

Hz, 4H), 2.98 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 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 C12H15ClN3O3S

calculated: 316.0523, found: 316.0529 (M+H) +. Anal. Calcd for C12H14ClN3O3S: (%) C

45.64, H 4.47, N 13.31. Found: C 45.78, H 4.62, N 13.51.

5.2.20. 7-chloro-3-(4-(phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6t)

White solid (95%); 1H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 8.4 Hz, 2H), 7.74 – 7.66 (m,

3H), 7.52 (d, J = 7.2 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.38 (m, 1H), 3.68 (t, J = 5.2 Hz & J

= 4.8 Hz,, 4H), 3.29 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ 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 (M+H) +.

Anal. Calcd for C17H16ClN3O3S: (%) C 54.04, H 4.27, N 11.12. Found: C 54.28, H 4.42, N

11.33.

5.2.21. 7-chloro-3-(4-tosylpiperazin-1-yl)benzo[d]isoxazole (6u)

White solid (85%); 1H NMR (400MHz, CDCl3) δ 7.82 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.0

Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.38 (m, 1H), 3.54 (t, J = 5.2 Hz

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& J = 4.8 Hz,, 4H), 3.29 (t, J = 4.8 Hz & J = 4.4 Hz, 4H), 2.86 (s, 3H). 13C NMR (100 MHz,

CDCl3) δ 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 (M+H) +. Anal. Calcd for C18H18ClN3O3S: (%) C 55.17, H 4.63, N 10.72

Found: C 55.41, H 4.82, N 10.93.

5.2.22. 7-chloro-3-(4-(4-(trifluoromethoxy)phenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole

(6v)

White solid (84%); 1H NMR (400 MHz, CDCl3) δ 7.86 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 8.0

Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.38 (m, 1H), 7.18 (d, J = 7.2 Hz, 2H), 3.56 (t, J = 5.2 Hz

& J = 4.8 Hz,, 4H), 3.27 (t, J = 4.8 Hz & J = 4.4 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ

164.14, 160.12, 148.22, 136.28, 132.81, 129.26, 124.76, 123.42, 122.61, 120.23, 114.14,

110.64, 47.75, 45.27. HRMS: (ESI m/z) for C18H16ClF3N3O4S calculated: 462.0502, found:

462.0506 (M+H) +. Anal. Calcd for C18H15ClF3N3O4S: (%) C 46.81, H 3.27, N 9.10. Found:

C 47.04, H 3.46, N 9.29.

5.2.23. 7-chloro-3-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)benzo[d]isoxazole (6w)

Yellow solid (85%); 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 7.6 Hz, 2H), 8.14 (d, J = 8.4

Hz, 2H), 7.58 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 7.2 Hz, 1H), 7.41 (m, 1H), 3.56 (t, J = 5.2 Hz

& J = 4.8 Hz,, 4H), 3.29 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ

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

(M+H) +. Anal. Calcd for C17H15ClN4O5S: (%) C 48.29, H 3.58, N 13.25. Found: C 48.49, H

3.71, N 13.44.

5.2.24. 3-(4-(4-bromophenylsulfonyl)piperazin-1-yl)-7-chlorobenzo[d]isoxazole (6x)

White solid (85%); 1H NMR (400 MHz, CDCl3) δ 8.08 (d, J = 8.0 Hz, 2H), 8.02 (d, J = 7.6

Hz, 2H), 7.56 (d, J = 7.2 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.36 (m, 1H), 3.58 (t, J = 5.2 Hz

& J = 4.8 Hz,, 4H), 3.26 (t, J = 4.8 Hz & J = 5.2 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ

163.74, 160.08, 139.12, 133.14, 132.28, 129.81, 124.84, 123.76, 122.42, 121.61, 111.12,

47.64, 45.25. HRMS: (ESI m/z) for C17H16BrClN3O3S calculated: 455.9784, found: 455.9788

(M+H) +. Anal. Calcd for C17H15BrClN3O3S: (%) C 44.70, H 3.31, N 9.20. Found: C 44.94, H

3.58, N 9.59.

5.3. Microplate Alamar Blue Assay (MABA)

The antimycobacterial activities of title compounds 5a-d and 6a-6x were evaluated against

MTB H37Rv (ATCC 27294) strain by the MABA [40,41]. Isoniazid and rifampin are used as

positive controls. Compound stock solutions were prepared in DMSO at a concentration 100

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µg/mL, and the final test concentrations ranged from 50 to 0.78 µg/mL. Two fold serial

dilutions of compounds were prepared in Middlebrook 7H9GC medium in a volume of 100

µL in 96-well microplates. TB culture (100 µL inoculum of 2 × 105 CFU/mL) was added,

yielding a final testing volume of 200 µL. The plates were incubated at 37 °C. On the seventh

day of incubation 25 µL of 10% Tween 80 and 25 µL of Alamar Blue (Accumed

International, Westlake, Ohio) were added to the wells of test plate. After incubation at 37 °C

for 24 h, colors of all wells were recorded. A blue color in the well was interpreted as no

growth, and a pink color was scored as growth. The MICs were defined as the lowest

concentration which prevented a color change from blue to pink. The MIC was defined as the

lowest drug concentration which prevented a color 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 µg/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 proliferation assay [42].

Acknowledgements

We acknowledge the financial assistance provided by DST (F.No.SB/S1/OC-60/2013), New

Delhi, India.

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Figure 1: General synthetic procedure for title compounds

Figure 2: ORTEP plot for the compound 6b. All the non-hydrogen atoms are presented by

their 30% probability thermal ellipsoids.

Table 1: Optimisation of reaction conditions of 5aa

Table 2: Antimycobacterial activities of compound 5a-5d and 6a – 6x against MTB H37Rv

Table 3: IC50 (µg/mL) and selectivity index (SI) values of active compounds

Table 4: Crystal data and structure refinement for 6b

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� 24 novel benz[d]isoxazoles were synthesized & evaluated for anti-TB activity.

� 3 compounds 5a, 6b and 6i exhibited good anti-TB activity against MTB H37RV

strain.

� In vitro toxicity studies were carried out for most active compounds.

� SI value (>130) indicates, suitability of 6b for further development to treat TB.

� Most active compound, 6b was substantiated by single crystal X-ray studies.

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Table 1: Optimisation of reaction conditions of 5aa

Entry Solvent Base Heating source Temperature ( oC) Time Yield (%)b

1 MeCN K2CO3 Conventional 75 12 h NRc

2 Dioxane DIPEA Conventional 100 12 h NRc

3 DMF K2CO3 Conventional 100 12 h NRc

4 DMF TEA Conventional 100 12 h NRc

5 DMF KOH Conventional 100 12 h 38

6 Dioxane KOH Conventional 100 12 h 42

7 Dioxane:H2Od KOH Conventional 100 12 h 54

8 Dioxane:H2Od KOH Microwave 120 15 min 55

9 Dioxane:H2Od KOH Microwave 120 30 min 64

10 Dioxane:H2Od KOH Microwave 120 45 min 62

11 Dioxane:H2Od KOH Conventionale 120 1 h 52

12 Dioxane:H2Od KOH Conventionale 120 2 h 58

13 Dioxane:H2Od KOH Conventionale 120 3 h 63

14 Dioxane:H2Od KOH Conventionale 120 4 h 82

15 Dioxane:H2Od KOH Conventionale 120 5 h 80

aAll the reactions were carried out with 4a (1.0 equiv.) and base (3.0 equiv.) in solvent (4 mL), bIsolated yield after column chromatography, cNR = no reaction, dDioxane:H2O (3:1), ereactions were carried out in sealed tube.

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Table 2: Antimycobacterial activities of compound 5a – 5d and 6a – 6x against MTB H37Rv

Entry R

X

aMP (ºC)

MIC(µg/mL) against MTB

H37Rv

5a - -

75-76

6.25

5b -

4-Cl

101-102 25

5c -

6-Cl

81-82 12.5

5d -

7-Cl

88-89 25

6a

H

168-169 12.5

6b

H

174-175 3.12

6c

H

162-163 50

6d

H

181-182 >50

6e

H

230-231 >50

6f

H

202-203 50

6g

4-Cl

169-170 >50

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6h

4-Cl

164-165 25

6i

4-Cl

187-188 6.25

6j

4-Cl

116-118 12.5

6k

4-Cl

216-217 50

6l

4-Cl

195-196 50

6m

6-Cl

194-195 >50

6n

6-Cl

202-203 25

6o

6-Cl

185-186 >50

6p

6-Cl

176-177 12.5

6q

6-Cl

232-233 25

6r

6-Cl

212-213 >50

6s

7-Cl

172-173 25

6t

7-Cl

175-176 25

6u

7-Cl

159-160 >50

6v

7-Cl

170-171 >50

6w

7-Cl

242-243 50

6x

7-Cl

187-188 25

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Isoniazid -

-

- 0.36

Rifampicin -

-

- 1.0

aUncorrected

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Table 3: IC50 (µg/mL) and selectivity index (SI) values of active compounds

Entry MIC (µg/mL) in

MTB H37Rv

% cell inhibition

at 50 µg/mL

IC50

approximation

aSI value

IC50/MIC

5a 6.25 36.84 67.86 10.85

5c 12.5 24.12 103.64 8.29

6a 12.5 28.42 87.96 7.03

6b 3.125 5.82 429.55 137.45

6i 6.25 21.60 115.74 18.51

6j 12.5 42.16 59.29 4.74

6p 12.5 18.10 138.12 11.04 a Selectivity index

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Table 4: Crystal data and structure refinement for 6b

Compound 6b

Formula C17H17N3O3S

Formula weight 343.39

Temperature/K 293.15

Wavelength (Å) 0.7107

Crystal system monoclinic

Space group P21/c

a (Å) 8.2112(6)

b (Å) 19.5119(9)

c (Å) 10.7941(8)

α (°) 90

β (°) 109.787(7)

γ (°) 90

V (Å3) 1627.3(2)

Z 4

F(000) 720.0

Dcalc (g/mm3) 1.402

µ (mm-1) 0.220

2Θ (°) 5.67 to 52.744°

Rint 0.0284

Crystal size/mm3 0.39 × 0.34 × 0.22

Goodness-of-fit on F2 1.039

R1 indices [I>2sigma(I)] 0.0501

wR2 (all data) 0.1148

Largest diff. peak/hole (e Å-3) 0.18/-0. 0.27

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Figure 1:

Reagents and conditions: (i) NH2OH.HCl (1.2 equiv.), CH3COONa(2.0 equiv.), EtOH, H2O, 0 ºC- rt, 1h, (80-98%); (ii) N-Chlorosuccinimide (1.2 equiv.), CCl4, 0 ºC - rt, 45 min, (75-95%); (iii) Piperazine (8.0 equiv.), N(C2H5)3 (2.0 equiv.), CH2Cl2, rt, 2h, (63-92%); (iv) KOH (3.0 equiv.), Dioxane:H2O (3:1), 120 ºC, 4h, (65-90%); (v) RSO2Cl (1.2 equiv.), N(C2H5)3

(2.0 equiv.), CH2Cl2, 0 ºC - rt, 1h, (80-98%);

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Figure 2:

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Supplementary Data

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 Sekhara∗

aDepartment of Chemistry, Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar Nagar,Shamirpet Mandal, Hyderabad-500 078, Andhra Pradesh, India.

bDepartment of Pharmacy,Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad-500 078, Andhra Pradesh, India.

cDepartment of Chemistry, University of Cape Town, Rondebosch-7707, Cape Town, South Africa.

*Corresponding author:

K.V.G. Chandra Sekhar, Department of Chemistry,

Birla Institute of Technology and Science-Pilani, Hyderabad, INDIA, PIN-500 078;

Phone: +91-40-66303527; E-mail address: kvgc@hyderabad.bits-pilani.ac.in, kvgcs@yahoo.com

∗ Corresponding author

Tel: +9140-66303527, E-mail: kvgc@hyderabad.bits-pilani.ac.in; kvgcs@yahoo.com

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Contents: Page

1. Spectral Data and spectra’s S3

2. Tables 1S-5S: Full list of atomic coordinates thermal parameters, bond distances and

angles of 6b crystal structure S10

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1. Spectral Data

1.1. 1H and 13C NMR spectra’s of represented final compounds

6a 1H NMR

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6a 13C NMR

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6b 1H NMR

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6b 13C NMR

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6i 1H NMR

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6j 1H NMR

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6p 1H NMR

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6p 13C NMR

2. Tables 1S-5S: Full list of atomic coordinates thermal parameters, bond

distances and angles of 6b crystal structure

Table 1S: Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2×103) for 6b. Ueq is defined as 1/3 of of the trace of the orthogonalised UIJ tensor. Atom x y z U(eq) C1 6045(3) 2959(1) 10178(2) 44.4(5)

C2 6791(3) 3327.4(11) 11324(2) 54.4(6)

C3 8566(3) 3364.2(13) 11864(2) 64.9(7)

C4 9589(3) 3042.3(13) 11271(3) 66.5(7)

C5 8858(3) 2676.4(12) 10141(3) 63.9(7)

C6 7080(3) 2625.1(11) 9588(2) 55.0(6)

C7 3701(3) 3359.3(10) 7053(2) 55.5(6)

C8 2709(3) 3836.6(10) 5966(2) 61.3(7)

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C9 2927(3) 4728.2(11) 7598(2) 57.3(6)

C10 3856(3) 4234.7(10) 8670(2) 53.8(6)

C11 2654(3) 5045.1(10) 5398(2) 50.8(6)

C12 2464(3) 5005.2(11) 4021(2) 48.2(5)

C13 2620(3) 4522.1(12) 3124(2) 61.9(7)

C14 2223(4) 4715.3(15) 1830(3) 77.5(8)

C15 1691(4) 5375.6(17) 1418(3) 79.4(8)

C16 1560(4) 5863.5(15) 2277(3) 74.1(8)

C17 1965(3) 5659.7(12) 3569(3) 60.8(6)

N1 3328(2) 3529.2(8) 8251.6(17) 48.7(5)

N2 3175(2) 4549.9(8) 6357.7(17) 50.0(5)

N3 2302(3) 5657.8(10) 5719(2) 77.9(7)

O1 1885(3) 6066.8(8) 4557(2) 85.7(6)

O2 3277(2) 2308.6(8) 8804.4(17) 73.1(5)

O3 3050(2) 3198.5(9) 10364.8(17) 72.2(5)

S1 3788.5(8) 2956.3(3) 9423.4(6) 54.9(2)

Table 2S: Anisotropic Displacement Parameters (Å2×103) for 6b. The Anisotropic displacement factor exponent takes the form: -2π2[h2a*2U11+...+2hka×b×U12] Atom U11 U22 U33 U23 U13 U12 C1 56.6(13) 36.0(11) 40.9(12) 3.8(9) 16.8(10) -3.0(9)

C2 66.4(16) 51.2(13) 48.8(13) -0.6(11) 23.7(12) -2.0(11)

C3 66.9(18) 68.5(16) 50.4(14) -4.4(12) 8.1(13) -7.2(13)

C4 55.1(16) 67.1(16) 69.4(17) 10.9(14) 10.7(13) 6.5(13)

C5 67.7(17) 56.0(14) 73.9(18) 9.8(14) 31.6(14) 17.3(13)

C6 71.9(17) 41.9(12) 51.8(13) 0.1(11) 21.7(12) 3.0(11)

C7 76.2(17) 36.7(11) 50.4(13) -2.2(11) 17.1(12) -0.5(11)

C8 86.4(19) 37.0(12) 51.1(14) -2.7(11) 11.0(13) -2.1(12)

C9 77.1(17) 41.1(12) 56.6(14) -5.9(11) 26.5(13) -1.6(11)

C10 69.3(16) 44.6(12) 49.1(13) -4.0(11) 22.1(12) -5.7(11)

C11 58.2(14) 36.4(12) 55.8(14) -0.8(11) 16.6(11) -1.7(10)

C12 47.6(13) 41.4(12) 55.3(14) 4.6(11) 17.3(11) -4.4(10)

C13 77.4(18) 52.4(14) 60.1(15) 1.7(12) 28.9(13) -7.1(12)

C14 101(2) 79.9(19) 60.1(17) 1.6(15) 37.8(16) -13.5(16)

C15 78(2) 97(2) 62.6(18) 21.0(18) 23.8(15) -17.3(17)

C16 69.2(18) 69.6(18) 83(2) 30.5(17) 24.6(15) 0.6(14)

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C17 63.3(16) 51.5(14) 67.3(17) 10.7(13) 21.8(13) 1.0(12)

N1 54.1(11) 39.6(9) 50.4(11) 1.7(8) 15.1(9) -7.7(8)

N2 68.7(13) 32.7(9) 47.3(11) -1.5(8) 17.8(9) -1.2(8)

N3 124(2) 42.7(12) 69.5(15) 6.1(11) 36.0(14) 17.4(12)

O1 129.3(18) 43.4(10) 85.4(14) 15.7(10) 37.7(12) 21.7(10)

O2 81.1(13) 48.7(9) 75.5(12) 7.7(9) 8(1) -26.5(9)

O3 68.5(12) 86.2(12) 76.4(12) 16.9(10) 43.3(10) -2.1(9)

S1 55.4(4) 50.4(4) 58.6(4) 7.9(3) 19.0(3) -11.6(3)

Table 3S: Bond Lengths for 6b. Atom Atom Length/Å Atom Atom Length/Å C1 C2 1.381(3) C11 N2 1.374(3)

C1 C6 1.385(3) C11 N3 1.304(3)

C1 S1 1.754(2) C12 C13 1.388(3)

C2 C3 1.376(3) C12 C17 1.379(3)

C3 C4 1.369(3) C13 C14 1.375(3)

C4 C5 1.365(3) C14 C15 1.385(4)

C5 C6 1.381(3) C15 C16 1.358(4)

C7 C8 1.504(3) C16 C17 1.378(3)

C7 N1 1.465(3) C17 O1 1.348(3)

C8 N2 1.467(3) N1 S1 1.6336(18)

C9 C10 1.500(3) N3 O1 1.428(3)

C9 N2 1.463(3) O2 S1 1.4248(16)

C10 N1 1.468(3) O3 S1 1.4284(18)

C11 C12 1.444(3)

Table 4S: Bond Angles for 6b. Atom Atom Atom Angle/˚ Atom Atom Atom Angle/˚ C2 C1 C6 120.1(2) C16 C15 C14 121.5(3)

C2 C1 S1 119.75(17) C15 C16 C17 116.1(3)

C6 C1 S1 120.06(17) C16 C17 C12 124.5(3)

C3 C2 C1 119.5(2) O1 C17 C12 110.8(2)

C4 C3 C2 120.5(2) O1 C17 C16 124.8(2)

C5 C4 C3 120.3(2) C7 N1 C10 110.85(17)

C4 C5 C6 120.3(2) C7 N1 S1 117.78(14)

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C5 C6 C1 119.4(2) C10 N1 S1 115.89(14)

N1 C7 C8 109.21(19) C9 N2 C8 112.70(18)

N2 C8 C7 110.08(18) C11 N2 C8 117.75(17)

N2 C9 C10 111.19(18) C11 N2 C9 114.72(17)

N1 C10 C9 110.14(18) C11 N3 O1 106.72(19)

N2 C11 C12 129.46(19) C17 O1 N3 107.71(17)

N3 C11 C12 111.5(2) N1 S1 C1 106.79(9)

N3 C11 N2 119.0(2) O2 S1 C1 108.55(11)

C13 C12 C11 138.8(2) O2 S1 N1 106.84(10)

C17 C12 C11 103.3(2) O2 S1 O3 119.88(11)

C17 C12 C13 118.0(2) O3 S1 C1 107.73(11)

C14 C13 C12 118.4(2) O3 S1 N1 106.36(10)

C13 C14 C15 121.5(3)

Table 5S: Hydrogen Atom Coordinates (Å×104) and Isotropic Displacement Parameters (Å2×103) for 6b. Atom x y z U(eq) H2 6100 3549 11726 65

H3 9074 3609 12638 78

H4 10788 3073 11640 80

H5 9560 2461 9741 77

H6 6582 2369 8827 66

H7A 3367 2889 6800 67

H7B 4932 3404 7213 67

H8A 2971 3734 5175 74

H8B 1477 3772 5775 74

H9A 1701 4723 7475 69

H9B 3357 5188 7855 69

H10A 5096 4281 8875 65

H10B 3590 4339 9459 65

H13 2984 4078 3392 74

H14 2314 4395 1219 93

H15 1417 5488 534 95

H16 1216 6309 2009 89