Synthesis and biological evaluation of novel isoxazolines linked via piperazine to 2- benzoisothiazoles as potent apoptotic agents

Article abstract of DOI:10.1016/j.ejmech.2016.09.094

Synthesis of 3-(4-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)piperazin-1-yl) benzoisothiazole derivatives (5a-i), which constitute a new class of isoxazolines, has been accomplished in regio-selective manner. These derivatives have been prepared by employing the reaction between substituted aldoximes (4a-i) and 3-(4-Allylpiperazin-1-yl) benzoisothiazole in presence of chloramine-T which afforded in good yields. These compounds were screened for cytotoxic activity on tumor cells. Four among the nine synthesized compounds were found to exhibit potent cytotoxic and antineoplastic activities in comparison to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) protein in mammalian cancer cells. The rest of the derivatives showed moderate activity.

Full text of DOI:10.1016/j.ejmech.2016.09.094

Accepted Manuscript
Synthesis and biological evaluation of novel isoxazolines linked via piperazine to 2-
benzoisothiazoles as potent apoptotic agents
Sathish Byrappa, M. Harsha Raj, Tenzin Kungyal, Narayana U. Kudva N, Bharathi P.
Salimath, K.M. Lokanatha Rai
PII:
S0223-5234(16)30857-1
10.1016/j.ejmech.2016.09.094
EJMECH 8960
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 May 2016
Revised Date: 9 July 2016
Accepted Date: 28 September 2016
Please cite this article as: S. Byrappa, M. Harsha Raj, T. Kungyal, N.U. Kudva N, B.P. Salimath, K.M.
Lokanatha Rai, Synthesis and biological evaluation of novel isoxazolines linked via piperazine to 2-
benzoisothiazoles as potent apoptotic agents, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.09.094.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of novel isoxazolines
linked via piperazine to 2- benzoisothiazoles as potent
apoptotic agents
Sathish Byrappaa, M. Harsha Rajb, Tenzin Kungyalb, Narayana U.
Kudva Na, Bharathi P Salimathb, K. M. Lokanatha Rai a,*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri,
Mysore 570 006, India
b Department of Studies in Biotechnology, University of Mysore, Manasagangotri,
Mysore 570 006, India
*Corresponding author. Tel: +0821 2419667.E-mail addersses: kmlrai@yahoo.com,
† Electronic supplementary information (ESI) available, 1H NMR, 13C NMR and
LCMS spectra for all compounds
Abstract
S
ynthesis of 3-(4-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)piperazin-1-yl)
benzoisothiazole derivatives (5a-i), which constitute a new class of isoxazolines,
has been accomplished in regio-selective manner. These derivatives have been
prepared by employing the reaction between substituted aldoximes (4a-i) and
3-(4-Allylpiperazin-1-yl) benzoisothiazole in presence of chloramine-T which
afforded in good yields. These compounds were screened for cytotoxic activity on
tumor cells. Four among the nine synthesized compounds were found to exhibit
potent cytotoxic and antineoplastic activities in comparison to tumour necrosis
factor-related apoptosis inducing ligand (TRAIL) protein in mammalian cancer cells.
The rest of the derivatives showed moderate activity.
Key words: Benzoisothiazoles, Piperazine, Isoxazole and Cytotoxic activity.
1. Introduction
Increasing cases of neoplastic diseases like cancer is associated with high
mortality rate worldwide, accounting for almost 8.2 million deaths [1]. This has
stimulated an unprecedented level of research directed towards the discovery of new
structure leads that might be of use in designing novel chemotherapeutic drugs.
Despite breakthroughs in anticancer therapies, chemotherapy still remains a reliable
approach for cancer therapy. A typical attribute of neoplastic cells is their ability to
evade apoptosis consequently due to accumulation of mutations that block apoptosis
pathways [2]. Many studies have indicated the induction of apoptosis upon
efficacious cancer treatment [3]. Whilst chemotherapeutic drugs are known to induce
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autophagy, cell cycle arrest and cell senescence [4, 5], it is imperative that there is
induction of apoptosis and permanent removal of neoplastic cells for effective cancer
chemotherapy. Recently, benzoisothiazole, Isoxazole and piperazine rings have been
shown to exhibit varied biological activities including anticancer properties [6–11].
5-((4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)methyl)-3-phenyl-4,5-dihydro
isoxazole is a class of heterocyclic compounds having benzisothiazole, piperazine
and isoxazoline rings in the same system.
Derivatives of 2-Benzo/benzisothiazolimino-5-aryliden-4-thiazolidinones [12]
act as balanced inhibitors of cyclooxygenase/lipoxygenase enzymes. Amidino
benzisothiazole derivatives [13] showed remarkable analgesic activity and
antiphlogistic activity.
3-benzisothiazolyl derivatives [14] exhibit potent affinity
for the 5-Hydroxytryptamine-2A (5-HT2A) receptor combined with moderate to low
5-HT1A and D2 affinities. 2-(Benzylidineamino)-benzo[d]isothiazol-3-ones [15]
were found to be possessing good antimicrobial activity with the parent compound,
2-amino-benzo[d]isothiazol-3-one being the most effective.
Introduction of the methyl sulfonamide group at the 7-position of the
Benzo[d]isothiazole-1,1-dioxides [16] provided an excellent boost in potency (IC50 =
0.005 µM) but showed poor replicon inhibition Effective Concentration (EC50) =
4.924 µM). 3-Aminobenzo[d]isothiazole derivatives [17] show a local anaesthetic
activity in the rat sciatic nerve model. Benzoisothiazole derivatives [18] linked to
N,N-Dimethylformimidamide group was found to significantly inhibit the
5-HT-induced Ca2+ increases making them potent 5-HT6 receptor antagonists.
Isoxazolines are heterocycles that belong to the azole family which have gained
much importance in the field of medicinal chemistry as anticancer agents [8–9].
3,5-Diaryl isoxazoline /isoxazole linked to 2, 3-dihydroquinazolinone [19] exhibits
significant anticancer activity against 18 human cancer cell lines with growth
inhibition (GI50) values less than 1 mM. Isoxazole linked to pyrrolobenzodiazepine
[20] showed promising anticancer activity with GI50 values in the range of
<0.1–3.6 µM. Isoxazole analogues of retinoids and its derivatives [21] have shown
good antiproliferative activity and a capability to induce differentiation of tumor
cells. Isoxazolo[4,5-d]pyrimidine derivatives [22] significantly inhibited the
humoral immune response in vivo to sheep erythrocytes at a dose of 100 mg.
Methylenebis-isoxazolo[4,5-b]azepines bearing furan and thiophene rings [23] were
shown to be most toxic towards cancer cell lines in comparison with that of standard
drug Cisplatin.
5-(3-Alkylquinolin-2-yl)-3-aryl isoxazole derivatives [24] showed excellent
potent cytotoxicity against human cancer cell lines (A549, COLO 205, MDA-MB
231 and PC-3) having IC50 values of <12 µM. Isoxazole derivatives are also known
to show good antiproliferative against Hela cells [25]. Derivatives of isoxazole
containing 6-hydroxycoumarin [26] showed exceptional cytotoxic activity with IC50
of 8.2 and 13.6µM against PC-3 cancer cell line. Isoxazole derivatives [27] showed
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potent antibacterial activity against all the tested microorganisms at a concentration
of 100µg/mL similar to that of the standard antibiotic ciprofloxacin. Ether-linked
bis(isoxazoline) derivatives [28] were tested for antimicrobial activity by disc
diffusion and micro dilution methods, showing moderate to potent inhibition.
Piperazine-based compounds [29] were tested for high-throughput screening (HTS)
approaches and exhibited submicromolar inhibition of Mycobacterium tuberculosis
InhA (MtInhA) enzyme activity. Sulfonyl(piperazin-1-yl)benzo[d]isoxazole
analogues [30] were screened for in vitro anti-tubercular activity against
Mycobacterium tuberculosis (MTB) H37Rv strain. The titled compounds exhibited
Minimum inhibitory concentration (MIC) between 3.125 and >50 mg/mL.
Piperazine based derivatives as
a
new class of S1 ligands.
4-benzyl-1-(3-iodobenzylsulfonyl)piperidine [31] sigma ligand was identified with
96-fold σ1/σ2 selectivity ratio (Kiσ1 = 0.96 ± 0.05 nM and Kiσ2 = 91.8 ± 8.1 nM).
1-(4-Substitutedbenzoyl)-4-(4-chlorobenzhydryl)piperazine derivatives [32] showed
significant cell growth inhibitory activity on the selected cancer cell lines,
especially had lower GI50 values when compared to 5-fluorouracil (5-FU).
Arylpiperazine containing 2-fluorophenyl derivatives [33] exhibited strong
cytotoxic activities against LNCaP cells (IC50 < 5 µM).
In this study, based on the survey of recent literature studies on
benzo[d]isothiazole, isoxazole and piperazine and in our effort to discover potent
anticancer agents, we synthesized benzo[d]isothiazole derivatives containing
piperazine and isoxazoline rings and screened them for their antineoplastic efficacy
using the human breast cancer cell line MDA-MB-231 and the Ehrlich ascites tumor
(EAT), murine mammary carcinoma model. As a positive control, we have used the
soluble form of recombinant TNF-related apoptosis-inducing ligand (rTRAIL)
which has been shown to induce apoptosis selectively in cancer cells like MDA MB
231 [34].
2. Results and discussion
2.1. Chemistry
Synthesis of novel heterocyclic ring systems by using nitrone-alkene
cycloaddition reaction has gained prominence over the last few decades [35]. In
synthetic perspective, nitrone-alkene cycloaddition is used to connect multiple
pharmacore moieties with a single step [36]. As a result, the cyclo adducts emanated
due to nitrone-alkene cycloaddition reaction serves an excellent precursors in
organic as well as in medicinal chemistry [37].
Based on the above facts, it would thus be interesting to synthesize molecules
possessing benzisothiazole, piperazine and isoxazoline rings in the same system and
evaluate their biological properties. So an attempt to design, synthesize and
evaluation of in vitro cytotoxic activity of novel isoxazolines linked via piperazine
to 2- benzoisothiazoles is made. Therefore, we describe herein utilization of
nitrone-alkene cyclo addition as a synthetic method for the preparation of
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isoxazolines linked via piperazine to 2- benzoisothiazoles.
Thus the compounds (3-(4-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)
piperazine-1-yl)benzo[d]isothiazole) 5(a-i) were synthesized from starting material
Benzo[d]isothiazol-3(2H)-one (1), which was treated with phosphoryl chloride
(
POCl3) in dichloromethane to get an intermediate 3-chlorobenzoisothiazole which
were intercepted in situ by piperazine to afford compound 3-(Piperazin-1-yl)benzo
[d]isothiazole (2). The compound 2 was subjected to allylation with excess of allyl
bromide in the presence of tri ethylamine in Dimethyl Formamide (DMF) to get
compound 3-(4-Allylpiperazin-1-yl)benzoisothiazole (3). With the compound 3 in
hand, we turned our attention to find suitable dipole precursors and in turn
substituted aldoximes (4a-i) are found to be excellent precursors. These aldoximes
were prepared from the corresponding aldehydes from the procedure outlined in
already reported method [38]. The obtained substituted aldoximes were subjected to
nitrone-alkene cycloaddition reaction by treatment with 3-(4-Allylpiperazin-1-yl)
benzoisothiazole (3) and chloramine-T in ethanol at reflux condition for 1 hour. The
oximes will undergo oxidative dehydrogenation to yield nitrile oxides. These
nitriloxides further react with olefinic group of compound 3 and undergo
cycloaddition to get desired 3-(4-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)
piperazin-1-yl)benzo[d]isothiazole
derivatives
(5a-i),
which
contain
Benzo[d]isothiazole, piperazine and isoxazoline rings. The numbering of the
skeleton is shown in Fig. 1.
3. Materials and methods
3.1. Cell lines and culture media
MDA-MB-231, human triple negative breast cancer cells were procured from
National Centre for Cell Science, Pune, India and cultured in Dulbecco’s modified
Eagle’s medium (DMEM), containing 10% fetal bovine serum (FBS), (Gibco, USA)
and 100 mg/mL penicillin streptomycin (Invitrogen). The Ehrlich ascites tumor
cells/mouse mammary carcinoma cells were maintained in Swiss albino mice (6-8
weeks old) obtained from the animal house facility, Department of Studies in
Zoology, University of Mysore, Karnataka, India. The animal experiments were
approved by the institutional animal care and use committee, University of Mysore,
Karnataka, India. The culture medium used throughout these experiments was
DMEM. Recombinant human soluble TRAIL was purified from pGEX-2TK
plasmid containing TRAIL-GST fusion construct, a kind gift from Dr. Stanley
Lipkowitz, National Cancer Institute, Bethesda. Other chemicals and reagents used
were of analytical grade.
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3.2. Test compound preparation
All the test compounds were dissolved in Dimethyl sulfoxide
(
DMSO) (0.1%)
solvent and filter sterilized using a 0.22µm syringe filter for the in vitro cytotoxicity
studies. In control samples, equimolar concentration of DMSO was added as vehicle
to rule out its cytotoxic effect. As a standard anti-cancer, apoptosis inducing drug,
soluble recombinant rTRAIL (25 pM) was used.
3.3. MTT assay to study the effect of compounds on proliferation and the metabolic
activity of cancer cells
Human Triple negative breast cancer, MDA-MB-231 cells (1x104) were seeded
into 96 well tissue culture plate (Nunc MicroWell™) in complete DMEM and
incubated overnight at 37°C, 5% CO2. Media was replaced with fresh media
containing different concentrations (2, 5, 10, 20, 40, 80, 100 and 200 µM/ml) of the
compounds (5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h and 5i) in triplicates with appropriate
vehicle (0.1% DMSO) treated controls and media blanks. After 16hrs of treatment,
10µl MTT (5mg/ml) was added to each well and incubated for 4 hours at 37°C, 5%
CO2. The resultant purple formazan crystals were solubilized in 100µl of DMSO. The
optical density of the coloured solution was measured at a test wavelength of 570nm
and a reference wavelength of 630 nm. (Varioskan™ Flash Multimode Reader,
Thermo Scientific). The percentage cytotoxicity and IC50 values were calculated with
respect to control wells.
3.4. In vivo cytotoxicity of compounds assessed by Trypan blue dye exclusion assay
Mouse mammary carcinoma, EAT cells growing in exponential phase, were
seeded in 6 well culture plates at 3 x 104 cells per well in 2ml complete DMEM
medium. After 16h, the media was replaced with complete medium containing
50µM of compounds (5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h and 5i) to the respective wells.
Positive control cells were treated with rTRAIL (25 pM) and the negative control
cells were vehicle (0.1% DMSO) treated and incubated for 16 h at 37°C with 5%
CO2 in humidified incubator. The semi adherent cells were harvested by
centrifugation at 2500 x g for 5 min. The cell pellet was then re-suspended in 100 µl
of PBS; 10 µl of the cell suspension was stained with an equal volume of 0.4%
trypan blue in 980 µl of PBS and incubated for 2 min at 37°C. The total number of
viable cells was estimated by counting using a Neubauer Chamber.
3.5. Apoptosis detection by morphological changes in cells by nuclear staining
Analysis of apoptosis by morphological changes in EAT cells upon treatment
with benzoisothiazole derivatives by Acridine orange and Ethidium bromide
(AO/EtBr) dual staining was performed. AO is a cell permeable dye that stains both
live and dead cells, whereas EtBr only stains nucleus of cells that have lost
membrane integrity. Necrotic cells also stain orange, but resemble viable cells.
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Briefly, 5x104 cells per well were seeded into 6 well plate in complete DMEM
media and treated with compounds (5a, 5b, 5c, 5d, 5e, 5f, 5g, 5h and 5i) (50µM),
positive control cells were treated with rTRAIL (25 pM) and the negative control
cells were vehicle (0.1% DMSO) treated and incubated for 16h. The cells were
harvested and washed with PBS. 10µl of 1mg/ml AO and EtBr mixture was added
to the cell pellet in PBS and 20 µl of cells were placed on a microscopic slide with a
coverslip and immediately analyzed under a fluorescence microsope 40X objective
(Zeiss AxioVert). Nuclei were visualized; apoptotic cells in five random fields were
counted and photographed.
3.6. Statistics
All experiments were performed in triplicates. Wherever appropriate, the data
are expressed as the mean ± SD and means were compared using one-way analysis
of variance. Statistical significance of differences between control, rTRAIL and
compound treated cells were determined by Dunnet’s test. For all tests, P<0.05 was
considered statistically significant.
4. Results
1
The H NMR spectrum of compound (5a) 3-(4-((3-(4-Methoxyphenyl)-4,5-
dihydroisoxazol-5-yl)methyl)piperazin-1-yl)benzo [d] isothiazole. (5a) (R = OCH3),
exhibited a doublet at δ 8.07 (J = 9 Hz) due to the C1 and C5 - protons. A multiplet
was observed in the region between δ 7.64 - 7.54 due to C17, C19 and C20 protons.
Triplet was observed at δ 7.46 - 7.41 (J = 6 Hz) due to presence of C18 - proton. A
doublet in the region δ 7.02 (J = 9 Hz) due to presence of C2 and C4 - protons. A
multiplet was observed at δ 4.93 - 4.83 due to C9 - proton. A singlet in the region δ
3.8 due to presence of C22 - proton. A multiplet was observed at the range between δ
3.56 - 3.45 due to C12, C14 and C8 - protons. A multiplet was observed at the range
between δ 3.22 - 3.14 due to C8 - proton. A multiplet was observed at the range
between δ 2.78 - 2.63 due to the presence of C10, C11, and C13 - protons. The mass
1
spectrum (LCMS) of the compound (5a) showed a [M + 1] peak at 409.1. The H,
13C NMR and Mass spectral (LCMS) data of all compounds are in complete
agreement with assigned structure and details are included in the experimental
section.
4.1. Cytotoxicity of compounds on MDA-MB-231 cells
The dose response and IC50 values of all the nine compounds were determined
using the breast cancer cell line MDA-MB-231. These cells were treated with
increasing concentrations (2, 5, 10, 20, 40, 80, 100 and 200 µM/ml) of each
compound for 16 hours. The results revealed that all the test compounds induced
cytotoxicity in the tumor cell line, while compounds 5c, 5g and 5e exhibited high
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cytoxicity with IC50 values of 30, 75 and 95µM/ml respectively. Compounds 5b, 5d
and 5a showed intermediate activity and compounds 5f, 5i and 5h were weakly
cytotoxic.
4.2. Benzoisothiazole derivatives induced time dependent cell death
The cytotoxic effects of the benzoisothiazole derivatives was determined in
vitro using EAT cells. The number of viable cells present in the treated cell
suspension was counted by differentiating the live cells from the dead ones. A time
dependent decrease in viable cells was observed in treated cells. The percentage
viability in cells treated with compounds 5c, 5f, 5b and 5e showed higher
cytotoxicity of 53%, 50%, 45.6% and 44.6% compared to the others, while the
rTRAIL treated positive control showed 43% cell death in comparison with the
vehicle (0.1% DMSO) treated negative control cells as shown in fig 3.
4.3. Benzoisothiazole derivatives induce apoptosis in EAT cells
In order to ascertain the compound induced cytotoxity was due to apoptosis,
EAT cells were subjected to morphological examination by staining the treated and
control cells with AO/EtBr. Cells with intact nuclei and cytoplasm fluoresced green.
But positive control and compound treated cells showed early apoptotic morphology
with membrane blebbing, condensed nuclei and appeared bright green. The late
apoptotic cells fluoresced orange-red with condensed half moon shaped or
fragmented nuclei. From the data it is clear that out of the nine benzoisothiazole
derivatives, 5c, 5g, 5b and 5e induced apoptosis as early as 16h by 67.3%, 61.3%,
59% and 50% respectively whereas the positive and negative control samples
showed 63.6% and 5% apoptosis respectively. From the data shown in Fig. 4A and
B, it is clear that 50µM of these compounds could induce apoptosis in EAT cells
which corroborate with the previous results.
4.4. Discussion
Antineoplastic chemotherapeutics drugs kill tumor cells by inducing apoptosis
either by intrinsic or extrinsic pathways. Increase in apoptosis induction in cancer
cells is due to the sensitivity of the cancer cells towards the chemotherapeutic drug
[39]. Thus novel bioactive drugs like ours need to be tested for drug sensitivity with
respect to induction of apoptosis.
In in vitro conditions, cytotoxic compounds impede with cellular attachment,
resulting in morphological changes, contrarily affecting their proliferation rate, or
causing cell death [40]. Hence, the effect of compounds on cell proliferation was
studied using the MTT assay which is a well-characterized colorimetric assay, based
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on the enzymatic reduction of the tetrazolium salt MTT in living, metabolically
active cells, but not in dead cells. We determined the median inhibitory
concentration (IC50) of the three highly cytotoxic compounds. Since all the eight
compounds were cytotoxic in general, they were subjected to further evaluation of
their cytotoxic activity on a murine mammary carcinoma (EAT), with the objective
of determining the mode of cell death along with a known apoptosis inducing
ligand, TRAIL.
Methods like Hoechst staining, PI staining, annexin staining, acidic denaturation,
and thermal denaturation, in situ nick translation assays have all been developed to
study apoptosis induction by monitoring cytological-morphological changes, but
have their own shortcomings in processing steps [41]. While MTT assay, a
metabolic profiling assay is also generally used as a drug sensitivity assay, but this
assay cannot differentiate between apoptotic and necrotic cells. Hence, a qualitative
and quantitative assay like the AO/EtBr dual staining is a well suited method to
assess apoptotic cell death [42].
In our present study, four out of nine compounds showed potent pro-apoptotic
activity in the EAT tumor model, which was assessed by both trypan blue dye
exclusion assay and AO/EtBr dual staining, which was on par or better than the
rTRAIL induced apoptosis. In terms of the identification of potent and selective
activity in MDA MB 231 breast cancer cell lines, the compounds 5c, 5g and 5e
exhibited high cytoxicity. Compounds 5b, 5d and 5a showed intermediate activity
and compounds 5f, 5i and 5h were weakly cytotoxic. From the experimental results
it is evident that compound 5f and 5i show negligible cytotoxic activity and
compounds 5c, 5b, 5e and 5g are cytotoxic and induce apoptosis and this may be
due to the substitution of benzyloxy group at the para position as in 5b, which
shows good apoptotic activity and by inclusion of methoxy group at meta position
with benzyloxy group as in 5c, results in increased rate of apoptosis compared to
5b. When the benzyloxy group is replaced to meta position (5e), we observed a
decrease in the activity compared to 5b and 5c. But in the absence of benzyloxy
groups compounds (5a, 5f, 5d and 5h) lose their apoptosis inducing ability and the
presence of fluorine group (5i) at para position decreases the activity drastically.
5. Experimental section.
The melting points were determined by open capillary method using electric
melting point apparatus and are uncorrected. The IR spectra were recorded on
1
Agilent Fourier transform infrared (FT-IR) Spectrophotometer. H NMR spectra
13
were recorded on Bruker 400 MHz spectrometer. C NMR spectra were recorded
on Bruker 100 MHz spectrometer by using DMSO-d6 as a solvent and TMS as an
internal standard. The chemical shifts are expressed in δ ppm. The LC-MS spectra
were recorded using Agilent-Single Quartz. The purity of the compounds was
checked by TLC.
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5.1. General procedure for the preparation of compounds 5a–i
An equimolar mixture of 3-(4-Allylpiperazin-1-yl)benzoisothiazole (1 g, 3.86
mmoles) and aldoximes (4a-i)(3.86 mmoles) in ethanol (15 ml) was stirred for 10
minutes at room temperature. To the above reaction mass, chloramine-T (3.86
mmoles) was charged slowly over a period of 10 min. Then the reaction mixture
o
was heated to 60 to 65 C and maintained for 2hour. The reaction progress was
monitored by TLC. Upon completion of the reaction (disappearance of starting
material on TLC), Reaction mass was cooled to room temperature and distilled to a
minimum volume and dilute with ethyl acetate (5 mL). The above solution was
washed with 2% aqueous sodium hydroxide solution (25 mL x 2) followed by with
5% aqueous sodium hydroxide solutions (25 mL x 2). The organic layer was again
washed with water (25 mL x 2). The organic layer was concentrated to dryness to
afford a crude derivative. The crude derivative was diluted with methanol (40 mL)
to precipitate a solid. The solid was filtered off and wash with 10 ml methanol. The
solid was dried and further recrystallized from 1:4 ethylacetate and heptane to
afford a pure derivative as an off white solid.
5.1.1. 3-(4-((3-(4-Methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)piperazine-1-yl)
benzo[d]isothiazole (5a).
o
1
White powder, yield (75%), m.p.: 109−111 C. H NMR (400 MHz,
DMSO-d6) δ: 8.07−8.04 (d, J = 9 Hz, 2H), 7.64−7.54 (m, 3H), 7.46−7.41 (t, J = 6
Hz, 1H), 7.02−6.99 (d, J = 9Hz, 2H), 4.93 - 4.83 (m, 1H), 3.80 (s, 3H), 3.56 - 3.45
(m, 5H), 3.22−3.14 (m, 1H), 2.78−2.63 (m, 6H) ppm. 13C NMR (100 MHz,
DMSO-d6) δ: 163.48, 163.53, 156.05, 151.95, 128.08, 127.81, 127.32, 124.36,
124.12, 121.95, 121.01, 114.18, 78.80, 61.33, 55.25, 54.86, 52.98, 49.65, 38.68 ppm.
LC MS (m/z): [M+H] 409.1.Anal. calcd. for C22H24N4O2S (408.16).
5.1.2.
3-(4-((3-(4-(Benzyloxy)phenyl)-4,5-dihydroisoxazol-5-yl)methyl)piperazin
-1-yl)benzo[d]isothiazole (5b).
Off white solid. yield (78%), m.p.: 130-134oC; 1H NMR (400 MHz, DMSO) δ:
8.07 - 8.04 (d, 2H, J = 9Hz); 7.64 - 7.53 (m, 3H); 7.47 - 7.31 (m, 6H); 7.10 - 7.07 (d,
2H, J = 9Hz); 5.16 (s, 2H); 4.93 - 4.83 (m, 1H); 3.53 - 3.45 (m, 5H); 3.21 - 3.05 (m,
13
1H); 2.77 - 2.58 (m, 6H). C NMR (100 MHz, DMSO) δ: 163.48, 159.59, 156.03,
151.96, 136.72, 128.42, 128.08, 127.87, 127.81, 127.66, 127.32, 124.36, 124.13,
122.17, 121.01, 115.05, 78.82, 69.28, 61.32, 52.98, 49.65,38.69. LC MS (m/z):
485( M+H). Anal. calcd. for C28H28N4O2S (484.19).
5.1.3. 3-(4-((3-(4-(Benzyloxy)-3-methoxyphenyl)-4,5-dihydroisoxazol-5-yl) methyl)
piperazin-1-yl)benzo[d]isothiazole (5c).
Off white solid. yield (73%), m.p.: 146-148oC; 1H NMR (400 MHz, DMSO) δ:
8.07 - 8.04 (d, 2H, J = 9Hz); 7.59 - 7.53 (t, 1H, J = 9Hz); 7.46 - 7.29 (m, 7H); 7.17 -
7.08 (m, 2H); 5.14 (s, 2H); 4.94 - 4.84 (m, 1H); 3.81 (s, 3H); 3.57 - 3.43 (m, 5H);
3.22 - 3.14 (m, 1H); 2.79 - 2.67 (m, 4H); 2.65 - 2.57 (m, 2H). 13C NMR (100 MHz,
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DMSO) δ: 163.48, 156.27, 151.95, 149.35, 149.06, 136.76, 128.40, 127.88, 127.81,
127.77, 127.32, 124.36, 124.13, 122.40, 121.02, 120.04, 113.14, 109.32, 78.91,
69.80, 61.34, 55.56, 52.99, 49.64, 38.68. LC MS(m/z): 515.1( M+H). Anal. calcd.
for C29H30N4O3S (514.2).
5.1.4.
3-(4-((3-(3,4-Dimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)piperazin
-1-yl)benzo[d]isothiazole (5d).
Off white solid. yield (67%), m.p.: 152-154oC; 1H NMR (400 MHz, DMSO) δ:
8.07 - 8.04 (d, 2H, J = 3Hz); 7.59 - 7.54 (t, 1H, J = 9Hz); 7.46 - 7.41 (t, 1H, J = 9Hz);
7.27 (s, 1H); 7.19 - 7.16 (m, 1H); 7.03 - 7.00 (d, 1H, J = 9Hz); 4.94 - 4.76 (m, 1H);
3.80 (s, 6H); 3.64 - 3.44 (m, 5H); 3.23 - 3.14 (m, 1H); 2.79 - 2.68 (m, 4H); 2.65 -
2.58(m, 2H). 13C NMR (100 MHz, DMSO) δ: 163.48, 156.29, 151.95, 150.41,
148.72, 127.81, 127.32, 124.36, 124.12, 122.05, 121.01, 120.15, 111.45, 109.03,
78.89, 61.35, 55.54, 55.47,52.99, 49.64,38.68. LC MS(m/z): 438.9( M+H). Anal.
calcd. for C23H26N4O3S (438.17).
5.1.5. 3-(4-((3-(3-(Benzyloxy)-4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl) methyl)
piperazin-1-yl)benzo[d]isothiazole (5e).
Off white solid. yield (83%), m.p.: 138 - 140oC; 1H NMR (400 MHz, DMSO) δ:
8.07 - 8.04 (d, 2H, J = 9Hz); 7.59 - 7.54 (t, 1H, J = 9Hz); 7.47 - 7.30 (m, 7H); 7.21 -
7.18 (m, 1H); 7.06 - 7.03 (d, 1H, J = 9Hz); 5.13 (s, 2H); 4.93 - 4.83 (m, 1H); 3.81 (s,
3H); 3.52 - 3.43 (m, 5H); 3.21 - 3.13 (m, 1H); 2.77 - 2.70 (m, 4H); 2.68 - 2.58 (m,
2H). 13C NMR (100 MHz, DMSO) δ: 163.48, 156.21, 151.96, 150.77, 147.70,
136.90, 128.38, 127.83, 127.75, 127.32, 124.36, 124.13, 122.00, 121.02, 120.50,
111.78, 110.96, 78.89, 69.92, 61.32, 55.64, 53.00, 49.64, 38.69. EI MS(m/z):
515.1 ( M+H). Anal. calcd. for C29H30N4O3S (514.2).
5.1.6.
isothiazole (5f).
Off white solid. yield (79%), m.p.: 118-126oC; 1H NMR (400 MHz, DMSO) δ:
3-(4-((3-Phenyl-4,5-dihydroisoxazol-5-yl)methyl)piperazin-1-yl)benzo[d]
8.08 - 8.05(d, 2H, J = 9Hz); 7.71 - 7.68(m, 2H); 7.59-7.41(m, 5H); 4.98 - 4.88 (m,
1H); 3.57 - 3.52 (t, 1H, J = 9Hz); 3.48 - 3.44 (m, 4H); 3.26 - 3.18 (m, 1H); 2.80 -
2.60 (m, 6H); 13C NMR (100 MHz, DMSO) δ:163.48, 156.58, 151.96, 129.93,
129.50, 128.75, 127.81, 127.32, 126.51, 124.36, 124.12, 121.01, 79.16, 61.28, 52.98,
49.65, 38.39. LC MS (m/z): 379 (M+H). Anal. calcd. for C21H22N4OS (378.15).
5.1.7.
3-(4-((3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)methyl)piperazin-1-yl)
benzo[d]isothiazole (5g).
Off white solid. yield (82%), m.p.: 132-134oC; 1H NMR (400 MHz, DMSO) δ:
8.07 - 8.04 (d, 2H, J = 9Hz); 7.72 - 7.69 (t, 2H, J = 6Hz); 7.59 - 7.51 (m, 3H); 7.46 -
7.41 (t, 1H, J = 6Hz); 5.0 - 4.90 (m, 1H); 3.57 - 3.43 (m, 5H); 3.25 - 3.17 (m, 1H);
2.82 - 2.57 (m, 6H). 13C NMR (100 MHz, DMSO) δ: 163.47, 155.86, 151.96,
134.49, 128.84, 128.40, 128.26, 127.81, 127.32, 124.36, 124.12, 121.01, 79.52,
61.22, 52.97, 49.65, 38.24. LC MS (m/z): 413.0(M+H). Anal. calcd. for
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C21H21ClN4OS (412.11).
5.1.8. 3-(4-((3-(3,4,5-Trimethoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)
piperazin-1-yl)benzo[d]isothiazole (5h).
Off white solid. yield (63%), m.p.: 128-130oC; 1H NMR (400 MHz, DMSO) δ:
8.08 - 8.05 (d, 2H, J = 9Hz); 7.59 - 7.54 (t, 1H, J = 6Hz); 7.47 - 7.41 (t, 1H, J = 9Hz);
6.97 (s, 2H); 4.93 - 4.92 (m, 1H) 3.83 (s, 6H); 3.70(s, 3H); 3.58 - 3.45 (m, 5H); 3.27
- 3.18 (m, 1H); 2.79 - 2.63 (m, 6H). 13C NMR (100 MHz, DMSO) δ: 163.48, 156.59,
152.96, 151.95, 139.05, 127.82, 127.31, 124.96, 124.37, 124.12, 121.01, 104.07,
79.20, 61.33, 60.09, 56.00, 52.99, 49.62, 38.65. EI MS(m/z): 469.0(M+H). Anal.
calcd. for C24H28N4O4S (468.18).
5.1.9.
3-(4-((3-(4-Fluorophenyl)-4,5-dihydroisoxazol-5-yl)methyl)piperazin-1-yl)
benzo[d]isothiazole (5i).
Off white solid. yield (77%), m.p.: 106-118oC; 1H NMR (400 MHz, DMSO) δ:
8.08 - 8.05 (d, 2H, J = 9Hz); 7.78 - 7.70 (m, 2H); 7.59 - 7.54 (m, 1H); 7.46 - 7.41 (t,
1H, J = 6Hz); 7.35 - 7.26 (m, 2H); 4.97 - 4.88 (m, 1H); 3.57 - 3.34 (m, 5H); 3.26 -
13
3.18(m, 1H); 2.80 - 2.62 (m, 6H). C NMR (100 MHz, DMSO) δ: 164.58, 163.47,
161.30, 155.75, 151.96, 129.25, 128.87, 128.76, 127.80, 127.32, 126.14, 126.10,
125.58, 124.35, 124.12, 121.01, 115.94, 115.65, 79.30, 61.24, 52.97, 49.64, 38.49.
EI MS(m/z): 397(M+H). Anal. calcd. for C21H21FN4OS (396.14).
6. Conclusion
In conclusion we have synthesized novel 5-((4-(Benzo[d]isothiazol-3-yl)
piperazin-1-yl)methyl)-3-phenyl-4,5-dihydroisoxazole
derivatives.
These
derivatives were synthesized in moderate to good yields. They were characterized
1
by H, 13C NMR and Mass spectroscopy. Our results demonstrate a promising
evidence in treatment of cancer using these benzoisothiazole derivatives and further
studies on pro-apoptotic, anti-angiogenic, anti-metastatic signal transduction
pathways need to be ascertained to know the primary mode of action.
7. Acknowledgement
We acknowledge funding by University with Potential for Excellence
f.No.14/4/2012(NS/PE), University Grants Commission-Government of India No. f
4-1/2013(SAP II) and DOE, Central Tibetan Administration, Dharamasala.
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Table and Figure Legends
Scheme 1. General synthetic route
Table 1. List of is compounds
Fig. 1. Numbering of the compound (5a)
Fig. 2. Cytotoxicity of compounds on MDA-MB-231 cells as assessed by MTT
assay: Cells (3 x 104) were treated with test compounds at different concentrations
(2, 5, 10, 20, 40, 80, 100 and 200µM/ml) for 16 h, incubated with MTT solution for
4 h and the formazan crystals were dissolved in DMSO and read at 570nm. Data are
presented as the mean ±SEM of three independent experiments; *** P <0.001,
**P<0.01 and *P<0.05.
Fig. 3. Cytotoxicity of EAT cells as assessed by Trypan blue dye exclusion assay:
Cells (3 x 104) were treated with benzoisothiazole derivatives (50µM) along with
respective positive and negative controls for 45 mins, 1.5h, 3h and 6h intervals. The
cells were harvested, washed with PBS and counted after adding trypan blue dye.
The viable cells were counted and the percentage viability was calculated. Data are
presented as the mean ± SEM of three independent experiments; **P<0.01.
Fig. 4. (A and B): Treatment of EAT cells with benzoisothiazole derivatives
induced apoptosis, as assessed by nuclear staining and morphological changes using
AO/EtBr dual staining. EAT cells 5x104 were seeded in 6-well plates, treated with
compounds (50µM) or rTRAIL (25 pM) for positive control and vehicle (0.1%
DMSO) treated negative control. The cells were incubated for 16 hours and then
harvested, stained, photographed and counted. Data are presented as the mean ±
SEM of three independent experiments; **P<0.01.
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Table 1. List of is compounds
Compounds
5a
5b
5c
5d
5e
5f
5g
5h
5i
R
R1
R2
H
H
OCH3
OCH3 OCH2Ph
H
H
H
H
Cl
H
OCH3
OCH3
OCH3
H
F
H
OCH3 OCH2Ph OCH2Ph OCH3 OCH3
H
H
H
H
H

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Highlights:
1. Synthesis of new benzoisothiazole derivatives.
2. They comprise a new class of isoxazolines synthesized in a regioselective manner.
3. All compounds were screened for anti-cancer and pro-apoptotic activity.
4. Four out of nine compounds were potent apoptosis inducers in cancer cells.