Synthesis and antioxidant activity of quinolinobenzothiazinones

Article abstract of DOI:10.1016/j.ejmech.2010.07.006

A new series of structurally diverse quinolinobenzothiazinones has been synthesized with the annulation of heterocyclic structural pharmacophores. The synthesized quinolinobenzothiazinones have been evaluated for their antioxidant (LPO & GSH) and radical scavenging activities (DPPH and ABTS assays).

Full text of DOI:10.1016/j.ejmech.2010.07.006

European Journal of Medicinal Chemistry 45 (2010) 4467e4472
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antioxidant activity of quinolinobenzothiazinones
,
a
b
b
M. Kumar a , Kshitija Sharma , R.M. Samarth , A. Kumar
*
a Department of Chemistry, University of Rajasthan, JLN Marg, Jaipur 302055, Rajasthan, India
b Department of Zoology, University of Rajasthan, Jaipur 302055, Rajasthan, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of structurally diverse quinolinobenzothiazinones has been synthesized with the annula-
tion of heterocyclic structural pharmacophores. The synthesized quinolinobenzothiazinones have been
evaluated for their antioxidant (LPO & GSH) and radical scavenging activities (DPPH and ABTS assays).
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 24 January 2010
Received in revised form
29 June 2010
Accepted 2 July 2010
Available online 5 August 2010
Keywords:
Quinolinobenzothiazinones
DPPH (2,2-diphenyl-1-picrylhydrazyl)
ABTS [2,2-azinobis(3-ethylbenzothiazoline-
6-sulphonic acid)]
GSH (glutathione)
LPO (lipid peroxidation)
Quinolino[3,4-b][1,4] benzothiazin-6(5H)-
ones
MDA (malondialdehyde)
1. Introduction
approaches in rational drug design in generating new structurally
diverse drugs (with fused heterocycles) as structural diversity is
The synthesis of heterocyclic systems is of continuing interest in
the field of organic chemistry because most of the compounds with
biological activity are derived from heterocyclic structures. 1,4-
Benzothiazines constitute an interesting class of privileged
heterocycles with promising biological and therapeutically activity
obviously directly related to the compounds potentiality endowed
with pharmacological activities. The annulation of quinolone ring
system with other heterosystems, such as with pyrane, results in
pyranoquinolone structural scaffold and its presence in the natural
products exhibits diverse biological activities such as antibacterial
[16e18], antifungal and antialgal [19], anti-inflammatory [20] and
antimalarial [21] as well as inhibition of calcium signaling [22],
platelet aggregation [23] and nitric oxide production [24]. The
alkaloids with such fused structural motif have also been reported
to exhibit cancer cell growth inhibitory activity and are investigated
as potential anticancer agents [25,26]. In addition, xanthosimuline
with pyranoquinolone structural motif is active against multi drug
resistant kB-VI cancer cells, while huajiasimuline exhibits a -
selective cytotoxicity profile showing greatest activity with
estrogen-positive ZR-75-I breast cancer cells [27]. Similarly ben-
zopyranobenzothiazines incorporating benzopyrane fused with
1,4-benzothiazine have shown interesting cytoprotective effects
against t-BHP-induced cytotoxicity [28].
and have been reported as calcium channel blockers [1], KATP
-
channel openers [2], phosphodiesterase 7 inhibitors [3], 5-HT3
antagonists [4], anticataract agents [5], dopamine D4 [6], Naþ/Hþ
exchange inhibitors [7], coagulation factor Xa inhibitors [8] and
matrix metalloproteinase inhibitors [9]. 1,4-Benzothiazines have
also been reported as new antiallergic [10] and antirheumatic [11]
agents. Similarly, quinolone ring system is also a privileged heter-
osystem and appears in several natural and synthetic compounds
of significant pharmacological properties with their use as HIV
protease inhibitors [12], antimalarials [13], drugs for treatment of
asthma [14], broad spectrum antibacterial agents [15], etc.
The annulation or incorporation of two or more heterocyclic
systems (pharmacophoric structures) has been one of the best
Encouraged by the promising biological activities of the struc-
turally diverse heterocycles with fused heterocyclic systems and
our continuing research programme on the synthesis of thera-
peutically interesting heterocycles [29e32], we have designed and
* Corresponding author. Tel.: þ91 141 2702720.
E-mail address: mahendrakpathak@gmail.com (M. Kumar).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.006

4468
M. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4467e4472
R2
R2
R2
NH4SCN
S
N
Br2
C
NH2.HBr
H2O
CHCl3
NH
C
S
NH2
NH2
R1
R1
1
R1
liq. NH3
2
3
R2
R2
SH
1a; R1 = CH3, R2 = H
1b; R1 = CF3, R2 = H
1c; R1 = OCH3, R2 = H
1d; R1 = H, R2 = Br
S
C
N
(i) KOH
(ii) gl. acetic acid
NH2
NH2
R1
R1
5
4
5a; R1 = CH3, R2 = H
5b; R1 = CF3, R2 = H
5c; R1 = OCH3, R2 = H
5d; R1 = H, R2 = Br
Scheme 1. Reaction protocol for the synthesis of 2-aminobenzenethiols.
synthesized quinolinobenzothiazinones, based on two privileged
biodynamic heterocyclic scaffolds; quinolone and 1,4-benzothia-
zine, with an aim of developing new structural motif which will
provide the multi-target drugs with promising bioactivity. The
synthesized quinolinobenzothiazinones have been screened for
their antioxidant activity by DPPH radical scavenging and ABTSþ
radical cation decolourization assays. The synthesized compounds
have also been evaluated for their antioxidant activity in Swiss
albino mice.
3. Results and discussion
In the present work, a series of six new compounds were
synthesized and all the synthesized quinolinobenzothiazinones
9aef were screened for their antioxidant activity by the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and 2,2-azi-
nobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTSꢀþ) radical
cation decolorizaton assays.
The results of DPPHꢀ and (ABTSꢀþ) inhibition by compounds
9aef are summarized in Table 1 and Fig. 1. In the present
investigation, the compounds 9c, 9d and 9f showed excellent
2. Chemistry
percent inhibition of DPPHꢀ activity (54.29
ꢁ
0.05%,
53.82 ꢁ 1.2%, 22.45 ꢁ 0.06% respectively) and were the most
effective DPPH radical scavengers. The percentages can be
considered as a full absorbance inhibition of DPPHꢀ, as after
completing the reaction, the final solution always possessed
some yellowish colour and, therefore, its absorbance inhibition
compared to the colourless methanol solution could not reach
100% [34,35].
2-Aminobenzenethiols were synthesized by alkaline hydrolysis
of 2-aminobenzothiazoles which, in turn, were prepared by cycli-
zation of phenylthioureas obtained by the reaction of substituted
anilines with ammonium thiocyanate (Scheme 1) [30,31].
4-Hydroxyquinolin-2-ones were synthesized by the reaction of
substituted aniline with malonic ester in the presence of poly-
phosphoric acid (Scheme 2) [33].
Another antioxidant activity screening method, i.e., ABTS radical
cation decolorization assay, showed results similar to those
obtained by DPPHꢀ assay. The compounds 9c, 9d and 9f were the
most active as they nearly fully scavenged (ABTSꢀþ). The absorbance
after 6 min were 0.052, 0.095 and 0.318 respectively. It was
observed that the reaction with (ABTSꢀþ) was fast in almost all the
cases and completed within 1 min. During the remainder of the
reaction time, the changes in absorbance were negligible. Thus,
compounds 9c, 9d and 9f showed the highest radical scavenging
activities (Fig. 1).
Quinolino[3,4-b][1,4]benzothiazin-6(5H)-ones were synthe-
sized in quantitative yields in a single step involving the reaction of
2-aminobenzenethiols with 4-hydroxyquinolin-2-ones in the
presence of dimethyl sulphoxide. Under the reaction conditions,
dimethyl sulphoxide acts as a solvent as well as oxidizing agent and
the reaction proceeds regioselectively involving in situ formation of
enaminoketone intermediate 8 which undergoes intramolecular
cyclization with the cleavage of SeS bond (Scheme 3).
The synthesized compounds were crystallized from methanol
and their structures were assigned by their analytical and spectral
data.
OC2H5
CH2
OH
O
R4
C
R4
R4
PPA
(I) CH2(COOC2H5)2
(II) Na2CO3
C
O
N
H
NH2
N
H
O
R3
R3
R3
7
6
6a; R3 = CH3, R4 = H
6b; R3 = OCH3, R4 = H
6c; R3 = H, R4 = CH3
7a; R3 = CH3, R4 = H
7b; R3 = OCH3, R4 = H
7c; R3 = H, R4 = CH3
Scheme 2. Synthesis of 4-hydroxyquinolin-2-ones.

M. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4467e4472
4469
R4
R4
R1
R1
NH2
N
R3
HO
+
R3
NH
R2
R2
S
S
SH
NH
R2
5
O
O
7
DMSO
NH2
R4
R1
R1
R4
H
N
R3
R1
H
NH
R2
S
S
N
R3
+
O
NH
R2
S
H
O
R2
NH2
R4
–H+
R1
(8)
R1
H
N
R3
NH
R2
S
9
O
9a; R1 = CH3, R2 = H, R3 = CH3, R4 = H
9b; R1 = CH3, R2 = H, R3 = H, R4 = CH3
9c; R1 = OCH3, R2 = H, R3 = OCH3, R4 = H
9d; R1 = H, R2 = Br, R3 = OCH3, R4 = H
9e; R1 = H, R2 = Br, R3 = H, R4 = CH3
9f; R1 = CF3, R2 = H, R3 = H, R4 = CH3
Scheme 3. Reaction sequence for the synthesis of quinolinobenzothiazinones.
A
significant increase in liver reduced glutathione (GSH)
showed highly significant increase in GSH contents (P < 0.005).
Treatment with compounds 9a, 9c and 9f showed comparatively
less significant increase (P < 0.05) in GSH content over the
normal (Fig. 2). Also, significant (P < 0.05) decrease in lipid
peroxidation (LPO) level was observed in animals treated with
the synthesized compounds (Fig. 3).
content along with decrease in lipid peroxidation (LPO) level
was observed in animals treated with compounds 9aef
(Table 2). However, treatment with compound 9d and 9e
Table 1
Antioxidant activity of synthesized quinolinobenzothiazinones (DPPHꢀ & ABTSꢀþ
assays).
0.8
a
b
c
d
e
f
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
S. No.
DPPHꢀ % inhibition
of 1 mg/ml of
the compound
ABTSꢀþ activity at different time
intervals (minutes)
0 min
1 min
2 min
4 min
6 min
9a
9b
9c
9d
9e
9f
2.707 ꢁ 0.03
8.33 ꢁ 0.02
54.29 ꢁ 0.05
53.82 ꢁ 1.2
13.69 ꢁ 0.09
22.45 ꢁ 0.06
0.722
0.721
0.726
0.725
0.724
0.722
0.620
0.560
0.055
0.141
0.412
0.328
0.610
0.520
0.052
0.098
0.406
0.318
0.610
0.520
0.052
0.098
0.406
0.318
0.610
0.520
0.052
0.095
0.406
0.318
0 min.
1 min.
2 min.
4 min.
6 min.
Time (minutes)
Fig. 1. ABTS activity at different time intervals by quinolinobenzothiazinones.

4470
M. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4467e4472
Table 2
4. Conclusion
Antioxidant properties of quinolinobenzothiazinones in the liver in Swiss Albino
Mice (LPO & GSH).
In the present communication, quinolinobenzothiazinones have
been synthesized bya simple andconvenient method keeping inview
multi-target drug design strategy with the annulation of heterocyclic
structural pharmacophores. The synthesized compounds have been
evaluated for their antioxidant activity by DPPHꢀ and ABTSꢀþ assays.
The synthesized compounds 9c, 9d and 9f have shown significant
þ
antioxidant activity as interpreted by the results of DPPHꢀ and ABTSꢀ
assays. The synthesized compounds have also shown interesting
antioxidant activity as measured by estimating reduced glutathione
(GSH) and lipid peroxidation (LPO) in liver of Swiss albino mice. The
antioxidant activities of these compounds are attributed to the pres-
ence of quinolinobenzothiazine heterosystem (tetracyclic fused
heterocycles with NeH bonds). Along with, we observed that the
presenceofsubstituentssuchasCF3, OCH3, CH3 andBronthearomatic
ring also play an important role in deciding antioxidant activity.
S. No.
LPO (n mole/mg tissue)
GSH (n mole/mg tissue)
9a
9b
9c
9d
9e
9f
6.72 ꢁ 0.17, P < 0.05
6.69 ꢁ 0.16
4.51 ꢁ 0.14, P < 0.05
4.50 ꢁ 0.11
6.81 ꢁ 0.06, P < 0.05
6.51 ꢁ 0.16, P < 0.05
6.42 ꢁ 0.08, P < 0.5
6.53 ꢁ 0.17, P < 0.5
4.80 ꢁ 0.13, P < 0.05
5.01 ꢁ 0.09, P < 0.005
5.0 ꢁ 0.15, P < 0.005
4.90 ꢁ 0.25, P < 0.05
5. Experimental protocols
5.1. Chemistry
5.4
The melting points of all the synthesized compounds were deter-
mined on the electrothermal melting point apparatus (PT-122) in
open capillary tubes and are uncorrected. Substituted anilines were
purchased from commercial sources and used without purification.
The purity of all the compounds was checked by TLC. IR spectra were
recorded on a Shimadzu 8400S FTIR spectrometer. 1H NMR and 13C
NMR spectra were recorded on JEOL 300 MHz spectrometer using
tetramethylsilane (TMS) as an internal standard. The mass spectra of
some of the synthesized compounds were provided by RSIC-CDRI
(Lucknow). Analyticalandspectraldataofthesynthesizedcompounds
are included in the experimental section and are in agreement with
the proposed structures of the synthesized compounds.
5.2
5
4.8
4.6
4.4
4.2
4
3.8
a
b
c
d
e
f
Treatment Compound
5.1.1. General procedure for the synthesis of quinolino[3,4-b][1,4]
benzothiazin-6(5H)-ones
To the stirred suspension of 4-hydroxyquinolin-2-one (0.01 mol)
in DMSO (6 ml) was added substituted 2-aminobenzenethiol
(0.01 mol) and the resulting mixture was refluxed for 1 h. The
Fig. 2. Antioxidant influence of quinolinobenzothiazinones on GSH level in liver of
Swiss albino mice.
7
6.9
6.8
6.7
6.6
6.5
6.4
6.3
6.2
6.1
6
a
b
c
d
e
f
Treatment Compound
Fig. 3. Antioxidant influence of quinolinobenzothiazinones on LPO content in liver of Swiss albino mice.

M. Kumar et al. / European Journal of Medicinal Chemistry 45 (2010) 4467e4472
4471
mixture was cooled down to room temperature and the product
separated was filtered and crystallized from methanol.
30 min of incubation in dark at room temperature, the absorbance
was observed against a blank at 517 nm.
The assay was carried out in triplicate and the percentage of
inhibition was calculated using the following formula.
5.1.2. 4,11-Dimethyl-6,12-dihydroquinolino[3,4-b][1,4]
benzothiazin-6(5H)-one (9a)
ðAB ꢃ AAÞ
Yield: 65%, m.p.: 286e288 ꢂC, IR (KBr) (cmꢃ1): 3410, 3330, 2900,
% Inhibition ¼
ꢄ 100
AA
1682, 1624, 1494, 818. 1H NMR (DMSO-d6)
d
(ppm): 2.36 (3H, s,
CH3), 2.54 (3H, s, CH3), 6.90e7.89 (6H, m, Ar-H), 8.35 (NH), 10.80
(CONH). 13C NMR (DMSO-d6)
(ppm): 17.41, 18.09, 115.92, 116.54,
where, AB ¼ absorption of blank and AA ¼ absorption of test
d
5.2.2. ABTS radical cation decolorization assay
119.95, 121.29, 122.19, 122.46, 122.82, 127.55, 132.20, 133.28, 135.02,
147.57, 170.40. MS (m/z): 294 [Mþ]; Anal. calcd. for C17H14N2OS: C,
69.36; H, 4.79; N, 9.52. Found: C 69.38; H, 4.76; N, 9.50.
The 2,2-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)
radical cation (ABTSꢀþ) decolorization test [37] was also used to
evaluate the antioxidant activity of compounds 9aef. (ABTSꢀþ) was
generated by oxidation of ABTS with potassium persulphate. For
this purpose, ABTS was dissolved in deionized water to 7 mM
concentration, and potassium persulphate was added to a concen-
tration of 2.45 mM. The reaction mixture was left at room
temperature overnight (12e16 h) in the dark before its use. The
(ABTSꢀþ) solution was then diluted with ethanol to an absorbance
of 0.700 ꢁ 0.020 at 734 nm. After addition of 1 ml of the diluted
5.1.3. 2,11-Dimethyl-6,12-dihydroquinolino[3,4-b][1,4]
benzothiazin-6(5H)-one (9b)
Yield: 68%, m.p.: 284e287 ꢂC, IR (KBr) (cmꢃ1): 3410, 3340, 2890,
1685,1622,1492, 816. 1H NMR (DMSO-d6)
d
(ppm): 2.31 (3H, s, CH3),
2.50 (3H, s, CH3), 7.02e7.53 (6H, m, Ar-H), 8.41 (1H, s, NH),10.91 (1H,
s, CONH). 13C NMR (DMSO-d6)
(ppm): 17.42, 18.09, 115.92, 116.55,
d
119.95, 121.29, 122.19, 122.45, 122.82, 127.55, 132.20, 133.29, 135.01,
147.57, 170.41. MS (m/z): 294 [Mþ], Anal. calcd. for C17H14N2OS; C,
69.36; H, 4.79; N, 9.52, Found: C, 69.39; H, 4.78; N, 9.54.
(ABTSꢀþ) solution (A 734 nm ¼ 0.700 ꢁ 0.020) to 10
ml of the
compound, the absorbance readings were taken at 30 ꢂC at inter-
vals of exactly 1e6 min after mixing later. All determinations were
carried out in triplicate.
5.1.4. 4,11-Dimethoxy-6,12-dihydroquinolino[3,4-b][1,4]
benzothiazin-6(5H)-one (9c)
Yield: 70%, m.p.: 292e294 ꢂC, IR (KBr) (cmꢃ1): 3390, 3325, 2880,
5.2.3. In vivo studies in Swiss albino mice
1680, 1610, 1490, 1250, 810. 1H NMR (DMSO-d6)
d
(ppm): 3.84 (3H, s,
OCH3), 4.12 (3H, s, OCH3), 6.89e7.91 (6H, m, Ar-H), 8.53 (1H, s, NH),
11.73 (1H, s, CONH). 13C NMR (DMSO-d6)
(ppm): 54.6, 55.7, 110.60,
The compounds were further treated for evaluation of anti-
oxidative properties in Swiss albino mice. Results showed that
there was significant decrease in lipid peroxidation (LPO) level and
elevation in reduced glutathione (GSH) in Swiss albino mice.
d
115.86, 119.96, 120.24, 122.32, 122.42, 127.56, 133.24, 135.23, 121.24,
147.54, 156.0, 158.26, 169.60. MS (m/z): 326 [Mþ]; Anal. calcd. for
C17H14N2O3S; C, 62.56; H, 4.32; N, 8.58, Found: C, 62.57; H, 4.29; N, 8.60.
5.2.3.1. Material and methods
5.2.3.1.1. Animals. Swiss albino mice were obtained from Jawa-
harlal Nehru University, New Delhi, India. Rendom-bred, Males
Swiss albino mice weighing 24 ꢁ 2 g were used for experiments.
These animals were maintained in the animal house at temperature
of 24ꢂꢁ3 ꢂC.
5.1.5. 9-Bromo-4-methoxy-6,12-dihydroquinolino [3,4-b][1,4]
benzothiazin-6(5H)-one (9d)
Yield: 75%, m.p.: 298e300 ꢂC, IR (KBr) (cme1): 3455, 3330, 2925,
1682, 1645, 1530, 1252, 835, 550. 1H NMR (DMSO-d6)
d (ppm): 3.89
(3H, s, OCH3), 6.95e7.69 (6H, m, Ar-H), 8.89 (1H, s, NH), 11.70 (1H, s,
CONH). Anal. calcd. for C16H11BrN2O2S; C 51.21, H 2.95, N 7.47;
Found: C 51.33, H 2.96, N 7.48.
The mice were obtained in two groups. Group-I animals were
fed orally with 0.1 ml of double-distilled water once a day for 7 days
before radiation and served as the control group, while animal of
Group-II received compound in 0.1 ml of double-distilled water in
a similar fashion.
5.1.6. 9-Bromo-2-methyl-6,12-dihydroquinolino [3,4-b][1,4]
benzothiazin-6(5H)-one (9e)
Animals were sacrificed by cervical dislocation and liver was
perfused in situ immediately with cold 0.9% NaCl and thereafter
carefully removed and rinsed in chilled 0.15 ml tris KCl buffer (pH 7.4)
toyielda10%(w/v)homogenate.Aliquots(0.5ml)ofthishomogenate
were used for assaying reduced glutathione and lipid peroxidation.
Yield: 72%, m.p.: 296e298 ꢂC, IR (KBr) (cmꢃ1): 3460, 3340, 2940,
1688, 1650, 1550, 845, 560. 1H NMR (DMSO-d6)
d (ppm): 2.37 (3H, s,
CH3), 6.98e7.75 (6H, m, Ar-H), 8.55 (1H, s, NH), 10.68 (1H, s, CONH).
Anal. calcd. for C16H11BrN2OS; C, 53.49, H, 3.09, N, 7.80; Found: C,
53.63, H, 3.12, N, 7.82.
5.2.3.1.2. Chemicals. Synthesized
quinolinobenzothiazinones
DPPH, (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2-azinobis) 3-eth-
ylbenzothiozoline-6-sulfonic acid, potassium persulphate, tri-
chloroacetic acid (TCA), 5-dithiobis-2-nitrobenzoic acid (DTNB),
thiobarbituric acid (TBA), etc.
5.1.7. 2-Methyl-11-trifluoromethyl-6,12-dihydroquinolino [3,4-b]
[1,4] benzothiazin-6 (5H)-one (9f)
Yield: 60%, m.p.: 294e296 ꢂC, IR (KBr) (cmꢃ1): 3450, 3350, 2930,
1686, 1640, 1535, 1130, 840. 1H NMR (DMSO-d6)
d (ppm): 2.39 (3H,
5.2.3.2. Biochemical studies
s, CH3), 7.0e7.86 (6H, m, Ar-H), 8.59 (1H, s, NH),10.89 (1H, s, CONH).
Anal. calcd. for C17H11F3N2OS; C, 58.62, H, 3.18, N, 8.04, Found: C,
58.66; H, 3.20, N, 8.10.
5.2.3.2.1. Lipid peroxidation. Lipid peroxidation level in liver
was estimated spectrophotometrically by thiobarbituric acid e
reactive substances (TBARS) method of Ohkhawa [38] and
expressed in terms of malondialdehyde formed per mg protein. In
brief, 0.4 ml of microsomal sample was mixed with 1.6 ml of 0.15 M
tris KCl buffer to which 0.5 ml of 30% TCA was added. Then 0.5 ml of
52 mM TBA was added and mixture placed in a water bath for
25 min at 80 ꢂC, cooled in ice and centrifuged at room temperature
for 10 min at 3000 rpm. The absorbance of the clear supernatant
was measured against a reference blank of distilled water at
531.8 nm.
5.2. Pharmacology
5.2.1. DPPH radical scavenging assay
Radical scavenging activity of the compounds 9aef was deter-
mined against stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
spectrophotometrically [36]. A stock solution (1 mg/ml) of the
compound was prepared in methanol. 50
ml of the compounds were
added to 5 ml of a 0.004% methanol solution of DPPH radical. After

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Acknowledgement
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