Antioxidant and DNA damage inhibition activities of 4-Aryl-N-(4-aryl-thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides

Article abstract of DOI:10.1007/s12039-014-0638-4

A series of 4-aryl-N-(4-pheny-thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides were synthesized by condensing 4-aryl-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxylic acid with 2-amino-4-aryl-thiazole derivatives. The newly synthesized molecules were characterized by spectral analysis and subjected to antioxidant and DNA damage inhibition studies.

Full text of DOI:10.1007/s12039-014-0638-4

c
J. Chem. Sci. Vol. 126, No. 6, November 2014, pp. 1913–1921. ꢀ Indian Academy of Sciences.
Antioxidant and DNA damage inhibition activities of 4-Aryl-N-(4-aryl-
thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides
K SHUBAKARAa, K B UMESHAa,∗, N SRIKANTAMURTHYa and J CHETHANb
aDepartment of Chemistry, Yuvaraja’s College, University of Mysore, Mysore 570 005, India
bDepartment of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, India
e-mail: kbu68umesha@rediffmail.com
MS received 17 September 2013; revised 13 March 2014; accepted 14 March 2014
Abstract. A series of 4-aryl-N-(4-pheny-thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides
were synthesized by condensing 4-aryl-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxylic acid with 2-amino-
4-aryl-thiazole derivatives. The newly synthesized molecules were characterized by spectral analysis and
subjected to antioxidant and DNA damage inhibition studies.
Keywords. HOBt; EDC; HCl; 1,3,4-oxadiazine carboxamides; antioxidant.
1. Introduction
DNA damage by the reactive oxygen species and chem-
ical carcinogens can lead to mutations and is suspected
to be a major cause of cancer.12 In order to protect
the genome from deleterious effects of ROS, cells have
antioxidants, ROS-eliminating enzymes and efficient
DNA repair pathways. Among DNA damage causing
cancer development, approximately 80% of the damage
is caused by ROS such as hydrogen peroxide (H2O2),
singlet oxygen (1O2) and hydroxyl radical (•OH). There-
fore, protection of oxidative DNA damage induced by
ROS is very important for cancer prevention.13
Benzoxadiazines derived from embelin (herbs
extract) is reported to exhibit antioxidant and free radi-
cal scavenging activities.14 Various oxadiazines linked
with other heterocyclic moieties show enhanced bio-
logical activities.15–17 In addition, oxadiazines bearing
thiazole moiety exhibit antifungal activities.18
In order to achieve promising antioxidants, we have
synthesized a new series of 4-aryl-N-(4-aryl-thiazol-2-
yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides
(7a–l) by condensing 4-aryl-5,6-dihydro-4H-1,3,4-
oxadiazine-2-carboxylic acid (6a–d) with 2-amino-
4-aryl-thiazole derivatives (2a–d) as illustrated in
schemes 1 and 2.
Oxadiazines are interesting and promising six-
membered heterocyclic compounds with oxygen and
nitrogen atoms. 1,3,4-oxadiazine derivatives exhibit
biological activities such as cardiovascular, antibacte-
rial, plant growth regulating, miticidal and nematocidal,
acricidal, insecticidal and anticonvulsive activities.1
In addition, oxadiazine derivatives are also used as
drugs for the treatment of anaemia2 and viral diseases.3
Thiazoles are important class of natural and synthetic
compounds and display a wide range of biological
activities such as cardiotonic,4 sedative,5 anaesthetic,6
bactericidal7 and anti-inflammatory.8
The main characteristic of an antioxidant is its ability
to trap free radicals. Highly reactive free radicals or
reactive oxygen species (ROS) are present in biologi-
cal systems from a wide variety of sources.9 These free
radicals can cause a number of chain reactions and may
oxidize nucleic acids, proteins, lipids or DNA and can
initiate several diseases.10 Antioxidants terminate these
chain reactions by removing free radical intermediates
and inhibit other oxidation reactions, thus inhibiting
the oxidative mechanism that leads to degenerative
diseases. They do this by oxidizing themselves, so
antioxidants are often reducing agents. Antioxidants are
widely used as dietary supplements and are being inves-
tigated for the prevention of diseases such as cancer,
coronary heart disease and even altitude sickness.11
DNA damage can lead to mutation if replication pro-
ceeds without proper repair. Oxidative stress-induced
2. Experimental
The melting points were measured with micro mel-
ting point apparatus and are uncorrected. All chemi-
cals/reagents used were purchased from Merck Chem-
icals (India), Fluka Chemicals (India) and IR spec-
tra were recorded in KBr pellets on Shimadzu 8300
∗For correspondence
1913

1914
K Shubakara et al.
Scheme 1. Synthesis of 2-amino-4-aryl-thiazole.
Scheme 2. Synthesis of 4-aryl-5,6-dihydro-4H-1,3,4- 51 oxadiazine-2-carboxylic acid.
Scheme 3. Synthesis of 4-aryl-N-(4-aryl-thiazol-2-yl)-5,6-dihydro-4h-1,3,4-oxadiazine-2-carboxamides.
spectrometer. The 1H NMR spectra were recorded
on a Bruker supercon 400 MHz spectrophotometer
using DMSO-d6 as solvent and Tetra methyl silane
(TMS) as an internal standard. The chemical shifts
were expressed in ppm. Mass spectra were obtained
on LC-MSD-Trap-XCT-Plus spectrophotometer. Thin
layer chromatography (TLC) was performed on
pre-coated Silica Gel sheet (HF 254, Sd-fine) and
visualization of the spots was done in iodine vapour
and UV light. Chromatographic separations were car-
ried out on silica gel (60-120) mesh using petroleum
ether: acetone (9:1) as eluent (scheme 3).
2.1 General procedure for the synthesis
of 2-amino-4-aryl-thiazole 2a-d
2-amino-4-aryl thiazole derivatives were prepared19–21
using different substituted phenacyl bromides by

Antioxidant activities of oxadiazines
1915
refluxing with excess thiourea in presence of conc. HCl (C-6), 63.0 (C-5), 116.2 (C-9 and C-13), 126.4 (C-11),
for about 20 min. After the completion of reaction, the 129.9 (C-10 and C-12), 141.9 (C-8), 150.9 (C-2), 168.1
reaction mixture was cooled to room temperature; the (C-7); LC-MS: 242.6 (M+H)+. Anal. % Calcd for
pale yellow solid thus separated was filtered, washed C10H9ClN2O3: C: 50.02, H: 4.89, N: 11.73. Found: C:
with chloroform, dried and recrystallized from ethanol 49.91, H: 4.72, N: 11.64.
to obtain 2-amino-4-aryl thiazole derivatives.
2.2b 4-(4-Methoxyphenyl)-5,6-dihydro-4H-1,3,4-
oxadiazine-2-carboxylic acid (6c): Obtained from 5c
2.2 Typical procedure for the synthesis of 4-phenyl-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxylic acid:
(2.64 g, 10.00 mmol), yield 81% (1.91 g); IR (KBr
cm−1): ν 2845 (-OCH3), 1683 (>C=O), 2735 (-OH),
(6a)
1242 (C-O), 3061 (Ar-CH), 1132 (C-O-C), 1580
1
(Ar-C=C); H NMR (DMSO-d6): δ 3.82 (t, 2H, J =
5.2 Hz), 3.90 (s, 3H, -OCH3) 4.43 (t, 2H, J = 5.6 Hz),
6.64–7.11 (m, 4H), 11.8 (s, 1H); 13C NMR (DMSO-
d6): δ 54.2 (C-6), 55.9 (-OCH3), 63.1 (C-5), 114.8
(C-9 and C-13), 115.3 (C-10 and C-12), 136.4 (C-8),
152.4 (C-11), 150.8 (C-2), 168.1 (C-7); LC-MS: 237.3
(M+H)+; Anal. % Calcd for C11H12N2O4: C: 55.95,
H: 5.12, N: 11.86. Found: C: 55.91, H: 5.05, N: 11.74.
2.2c 4-(4-Nitrophenyl)-5,6-dihydro-4H-1,3,4-
oxadiazine-2-carboxylic acid (6d): Obtained from 5d
(2.80 g, 10.00 mmol), yield 85% (2.14 g); IR (KBr
cm−1): ν 1545 and 1350 (-NO2), 1695 (>C=O), 2742
(-OH), 1248 (C-O), 3071 (Ar-CH), 1142 (C-O-C),
An oven-dried two-neck round bottom flask was
charged with a solution of 4-phenyl-5,6-dihydro-
4H-1,3,4-oxadiazine-2-carboxylate22 –24 (5a, 2.35 g,
10.00 mmol) in 25 mL of methanol. A volume of 10 mL
of 5% NaOH solution was added dropwise to the reac-
tion mixture and allowed to stir at room temperature for
24 h. After completion of reaction (monitored by TLC),
methanol was evaporated under reduced pressure and
the residue was acidified with dil. HCl. The white solid
thus separated was filtered, dried and recrystallized
from 20 mL ethanol to obtain 4-phenyl-5,6-dihydro-
4H-1,3,4-oxadiazine-2-carboxylic acid (6a, 1.64 g)
80% yield; IR (KBr cm−1): ν 3061 (Ar-CH), 1133
(C-O-C), 1680 (>C=O), 2737 (-OH), 1256 (C-O),
1579 (Ar-C=C); 1H NMR (DMSO-d6): δ 3.81 (t, 2H,
J = 5.2 Hz), 4.43 (t, 2H, J = 5.6 Hz), 6.95–7.42
(m, 5H), 11.82 (s, 1H); 13C NMR (DMSO-d6): δ 54.2
(C-6), 63.1 (C-5), 113.8 (C-9 and C-13), 121.3
(C-11), 129.8 (C-10 and C-12), 144.1 (C-8), 150.9 (C-
2), 168.1 (C-7). LC-MS: 207.0 (M+H)+. Anal. %
Calcd for C10H10N2O3: C: 58.51, H: 4.97, N: 13.36;
Found; C: 58.25, H: 4.89, N: 13.45. Same procedure
was followed for all the derivatives.
1
1582 (Ar-C=C); H NMR (DMSO-d6): δ 3.82 (t, 2H,
J = 5.2 Hz), 4.42 (t, 2H, J = 5.6 Hz), 7.32–8.24 (m,
4H), 11.8 (s, 1H); 13C NMR (DMSO-d6): δ 54.3 (C-6),
63.1 (C-5), 114.7 (C-9 and C-13), 124.9 (C-10 and
C-12), 141.3 (C-11), 150.1 (C-8), 150.9 (C-2), 168.1
(C-7); LC-MS: 252.3 (M+H)+; Anal. % Calcd for
C10H9N3O5: C: 47.77, H: 3.57, N: 16.78. Found: C:
47.88, H: 3.61, N: 16. 63.
2.3 Typical procedure for the synthesis
of 4-phenyl-N-(4-phenyl-thiazol-2-yl)-5,6-dihydro-4H-
1,3,4-oxadiazine-2-carboxamides (7a)
2.2a 4-(4-Chlorophenyl)-5,6-dihydro-4H-1,3,4-
oxadiazine-2-carboxylic acid (6b): Obtained from
5b (2.70 g, 10.00 mmol), yield 82% (1.98 g); IR
(KBr cm−1): ν 2350 (Ar-Cl), 3063 (Ar-CH), 1130
(C-O-C), 1685 (>C=O), 2734 (-OH), 1242 (C-O),
1
1578 (Ar-C=C); H NMR (DMSO-d6): δ 3.81 (t, 2H,
4H), 11.78 (s, 1H); 13C NMR (DMSO-d6): δ 54.2 xylic acid (6a, 2.30 g, 11.00 mmol) was taken in round
J = 5.2 Hz), 4.43 (t, 2H, J = 5.6 Hz), 7.16–7.44 (m,
4-phenyl-5,6-dihydro-4H-1,3,4-oxadiazine-2-carbo-

1916
K Shubakara et al.
bottomed flask and dissolved in 30 mL of CHCl3, after Obtained from 6a (2.30 g, 11.00 mmol) and 2c (1.92 g,
10 min, HOBt (2.00 g, 15.00 mmol) and 1-ethyl-3-(3- 10.00 mmol), yield 56% (2.13 g); IR (KBr cm−1): ν
dimethylaminopropyl) carbodimide (EDC.HCl) (2.81 g, 3113 (-NH), 1684 (-C=O), 3058 (Ar-CH), 1135 (C-
1
15.00 mmol) were added. The reaction mixture was O-C), 1576 (Ar-C=C), 722 (C-S), 3650 (Ar-OH); H
stirred at room temperature for 15 min, then 2-amino-4- NMR (DMSO-d6): δ 3.81 (t, 2H, J = 5.2 Hz), 4.43
phenyl thiazole (2a, 1.76 g, 10.00 mmol) and 1.15 mL (t, 2H, J = 5.6 Hz), 6.91–7.44 (m, 5H), 6.94–7.62 (m,
(11.00 mmol) of triethylamine were added and stirred 4H), 5.42 (s, 1H), 7.64 (s, 1H), 12.26 (s, 1H); 13C NMR
at room temperature for about 5–6 h. After completion (DMSO-d6): δ 53.9 (C-6), 62.8 (C-5), 105.8 (C-5ꢁ),
of reaction (monitored by TLC), the reaction mixture 113.8 (C-9 and (C-13), 121.3 (C-11), 129.8 (C-10 and
was extracted with CHCl3 (3 × 20 mL). The combined C-12), 144.2 (C-8), 129.1 (C-8ꢁ and C-12ꢁ), 117.8 (C-9ꢁ
organic layer was washed with 5% NaHCO3 solution and C-11ꢁ), 155.8 (C-10ꢁ), 126.3 (C-7ꢁ), 150.5 (C-4ꢁ),
followed by 5% HCl solution and finally with distilled 150.9 (C-2), 161.2 (C-7), 164.6 (C-2ꢁ). LC-MS: 381.4
water and then dried over anhydrous Na2SO4. The (M+H)+; Anal. % Calcd for C19H16N4O3S: C: 60.12,
CHCl3 layer was evaporated under reduced pressure; H: 4.24, N: 14.73; Found: 59.99, H: 4.20, N: 14.75.
the crude solid thus obtained was purified by column
chromatography using petroleum ether: acetone (8:2) as
2.3c 4-Phenyl-N-(4-p-tolyl)-thiazol-2-yl)-5,6-dihydro-
eluent, which afforded 4-phenyl-N-(4-phenyl-thiazol-
H-1,3,4-oxadiazine-2-carboxamide
(7d): Obtained
2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide
(7a, 2.05 g) as pale yellow solid in 55% yield, mp
126–128◦C. IR (KBr cm−1): ν 3114.70 (-NH), 1680
(>C=O), 3059.1 (Ar-CH), 1133 (C–O–C), 1579 (Ar-
C=C), 719 (C–S); 1H NMR (DMSO-d6): δ 3.81 (t, 2H,
J = 5.2 Hz), 4.42 (t, 2H, J = 5.6 Hz), 6.95–7.42 (m,
5H), 7.52–7.95 (m, 5H), 7.68 (s, 1H) 12.26 (s, 1H); 13C
NMR (DMSO-d6): δ 53.9 (C-6), 62.9 (C-5), 105.8 (C-5ꢁ),
113.7 (C-13 and C-9), 121.3 (C-11), 129.7 (C-12 and
C-10), 144.1 (C-8), 127.8 (C-8ꢁ and C-12ꢁ), 128.9 (C-
10ꢁ), 129.5 (C-9ꢁ and C-11ꢁ), 133.3 (C-7ꢁ), 150.4 (C-4ꢁ),
150.9 (C-2), 161.2 (C-7), 164.5 (C-2ꢁ); LC-MS: 365.2
(M+H)+. Anal. % Calcd for C19H16N4O2S: C: 62.66,
H: 4.43, N: 15.45. Found: C: 62.60, H: 4.39, N: 15.37.
Same procedure was followed for all the derivatives.
from 6a (2.30 g, 11.00 mmol) and 2d (1.90 g
10.00 mmol) yield 57% (2.16 g); IR (KBr cm−1): ν
3115 (-NH), 1682 (-C=O), 3061 (Ar-CH), 1131 (C-O-
C), 1581 (Ar-C=C), 716 (C-S); 1H NMR (DMSO-d6):
δ 2.33 (s, 3H), 3.80 (t, 2H, J = 5.2 Hz), 4.43 (t, 2H,
J = 5.6 Hz), 6.95–7.42 (m, 5H), 7.40–7.82 (m, 4H),
7.60 (s, 1H) 12.26 (s, 1H); 13C NMR (DMSO-d6):
δ 21.5 (-CH3), 53.9 (C-6), 62.8 (C-5), 105.8 (C-5’),
113.9 (C-9 and C-13), 121.3 (C-11), 129.8 (C-10 and
C-12), 144.2 (C-8), 127.6 (C-8ꢁ and C-12ꢁ), 129.9 (C-9ꢁ
and C-11ꢁ), 137.8 (C-10ꢁ), 130.4 (C-7ꢁ), 150.4 (C-4ꢁ),
150.9 (C-2), 161.2 (C-7), 164.5 (C-2ꢁ); LC-MS: 379.1
(M+H)+; Anal. % Calcd for C20H18N4O2S: C: 63.51,
H: 4.79, N: 14.83; Found: C: 63.47, H: 4.76, N: 14.80.
2.3d 4-(4-Chlorophenyl)-N-(4-(4-chlorophenyl)-thiazol-
2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide
(7e): Obtained from 6b (2.62 g, 11.00 mmol) and 2b
(2.10 g, 10.00 mmol) yield 56.5% (2.45 g); IR (KBr
cm−1): ν 3112 (-NH), 1690 (-C=O), 3055 (Ar-CH),
1135 (C-O-C), 1575 (Ar- C=C), 715 (C-S), 2350 (Ar-
2.3a 4-Phenyl-N-(4-(4-chlorophenyl)-thiazol-2-yl)-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide (7b):
Obtained from 6a (2.30 g, 11.00 mmol) and 2b (2.10 g,
10.00 mmol), yield 58% (2.32 g); IR (KBr cm−1): ν
3112 (-NH), 1686 (-C=O), 3061 (Ar-CH), 1130 (C-
1
O-C), 1582 (Ar-C=C), 720 (C-S), 2350 (Ar-Cl); H
1
Cl); H NMR (DMSO-d6): δ 3.81(t, 2H, J = 5.2 Hz),
NMR (DMSO-d6): δ 3.81 (t, 2H, J = 5.2 Hz), 4.42
(t, 2H, J = 5.6 Hz), 6.93–7.41 (m, 5H), 7.66–8.14 (m,
4H), 7.66 (s, 1H) 12.26 (s, 1H); 13C NMR (DMSO-d6):
δ 53.9 (C-6), 62.8 (C-5), 105.8 (C-5ꢁ), 113.9 (C-9 and
C-13), 121.3 (C-11), 129.8 (C-10 and C-12), 144.2
(C-8), 129.0 (C-8ꢁ and C-12ꢁ), 129.9 (C-9ꢁ and C-11ꢁ),
135.1 (C-10ꢁ), 131.32 (C-7ꢁ), 150.5 (C-4ꢁ), 150.9 (C-2),
161.2 (C-7), 164.5 (C-2ꢁ); LC-MS: 400.1 (M+H)+.
Anal. % Calcd for C19H15N4ClO2S: C: 57.21, H: 3.94,
N: 14.15; Found: C: 57.17, H: 3.81, N: 14.04.
4.43 (t, 2H, J = 5.6 Hz), 6.55–7.40 (m, 4H), 7.66–8.14
(m, 4H), 7.67 (s, 1H) 12.26 (s, 1H); 13C NMR (DMSO-
d6): δ 53.9 (C-6), 62.9 (C-5), 105.8 (C-5ꢁ), 116.2 (C-9
and C-13), 126.4 (C-11), 129.9 (C-10 and C-12), 141.9
(C-8), 129.0 (C-8ꢁ and C-12ꢁ), 129.9 (C-9ꢁ and C-11ꢁ),
135.1 (C-10ꢁ), 131.3 (C-7ꢁ), 150.5 (C-4ꢁ), 150.9 (C-2),
161.2 (C-7), 164.5 (C-2ꢁ). LC-MS: 434.3 (M+H)+;
Anal. % Calc for C19H14Cl2N4O2S: C: 52.67, H: 3.26,
N: 12.93; Found: C: 52.59, H: 3.20, N: 12.89.
2.3b 4-Phenyl-N-(4-(4-hydroxyphenyl)-thiazol-2-yl)- 2.3e 4-(4-Chlorophenyl)-N-(4-(4-hydroxyphenyl)-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide (7c): thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-car-

Antioxidant activities of oxadiazines
1917
boxamide (7f): Obtained from 6b (2.62 g, 2.3h 4-(4-Methoxyphenyl)-N-(4-(4-hydroxyphenyl)-
11.00 mmol) and 2c (1.92 g, 10.00 mmol), yield 58% thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-car-
(2.40 g); IR (KBr cm−1): ν 3114 (-NH), 1690 (-C=O), boxamide (7i): Obtained from 6c (2.68 g,
3058 (Ar-CH), 1136 (C-O-C), 1578 (Ar-C=C), 722 11.00 mmol) and 2c (1.92 g, 10.00 mmol), yield 59.5%
(C-S); H NMR (DMSO-d6): δ 3.81 (t, 2H, J = (2.44 g) IR (KBr cm−1): ν 3113 (-NH), 1686 (-C=O),
1
5.2 Hz), 4.42 (t, 2H, J = 5.6 Hz), 6.55–7.39 (m, 4H), 3063 (Ar-CH), 1132 (C-O-C), 1584 (Ar-C=C), 723
1
6.97–7.65 (m, 4H), 5.43 (s, 1H), 7.66 (s, 1H) 12.26 (s, (C-S), 3650 (Ar-OH); H NMR (DMSO-d6): δ 3.81
1H); 13C NMR (DMSO-d6): δ 54.0 (C-6), 62.9 (C-5), (t, 2H, J = 5.2 Hz), 3.88 (s, 3H, -OCH3), 4.43 (t, 2H,
105.8 (C-5ꢁ), 116.3 (C-9 and C-13), 126.4 (C-11), J = 5.6 Hz), 6.64–7.10 (m, 4H), 6.97–7.65 (m, 4H),
129.9 (C-10 and C-12), 142.0 (C-8), 129.1 (C-8ꢁ and 5.47 (s, 1H), 7.67 (s, 1H) 12.26 (s, 1H); 13C NMR
C-12ꢁ), 117.8 (C-9ꢁ and C-11ꢁ), 155.8 (C-10ꢁ), 126.3 (DMSO-d6): δ 54.1 (C-6), 55.9 (-OCH3), 63.1(C-5),
(C-7ꢁ), 150.5 (C-4ꢁ), 150.9 (C-2), 161.2 (C-7), 164.5 105.8 (C-5ꢁ), 114.8 (C-9 and C-13), 115.3 (C-10 and
(C-2ꢁ); LC-MS: 415.1 (M+H)+; Anal. % Calc for C-12), 117.8 (C-9ꢁ and C-11ꢁ), 129.1 (C-8ꢁ and C-
C19H15ClN4O3S: C: 55.10, H: 3.64, N: 13.55; Found: 12ꢁ), 155.8 (C-10ꢁ), 126.3 (C-7ꢁ), 136.4 (C-8), 150.5
C: 55.01, H: 3.56, N: 13.50.
(C-4ꢁ), 150.9 (C-2), 152.4 (C-11), 161.2 (C-7), 164.5
(C-2ꢁ); LC-MS: 411.1 (M+H)+; Anal. % Calc for
C20H18N4O4S: C: 58.58, H: 4.42, N: 13.65; Found: C:
58.53, H: 4.38, N: 13.61.
2.3f 4-(4-Chlorophenyl)-N-(4-(p-tolyl)-thiazol-2-yl)-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide (7g):
Obtained from 6b (2.62 g, 11.00 mmol) and 2d (1.90 g
10.00 mmol), yield 60% (2.48 g); IR (KBr cm−1): ν
3112 (-NH), 1688 (-C=O), 3061 (Ar-CH), 1136 (C-
O-C), 1583 (Ar- C=C), 722 (C-S), 2230 (Ar-C), 2350
2.3i 4-(4-Methoxyphenyl)-N-(4-(p-tolyl)-thiazol-2-
yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide
(7j): Obtained from 6c (2.68 g, 11.00 mmol) and 2d
(1.90 g 10.00 mmol), yield 59% (2.40 g); IR (KBr
cm−1): ν 3116 (-NH), 1695 (-C=O), 3062 (Ar-CH),
1137 (C-O-C), 1582 (Ar-C=C), 722 (C-S), 3654 (Ar-
1
(Ar-Cl); H NMR (DMSO-d6): δ 2.34 (s, 3H), 3.80
(t, 2H, J = 5.2 Hz), 4.42 (t, 2H, J = 5.6 Hz), 6.55–
7.39 (m, 4H), 7.40–7.82 (m, 4H), 7.68 (s,1H), 12.26
(s, 1H); 13C NMR (DMSO-d6): δ 21.5 (-CH3), 53.9
(C-6), 62.9 (C-5), 105.8 (C-5ꢁ), 116.2 (C-9 and C-13),
126.4 (C-11), 129.9 (C-10 and C-12), 141.9 (C-8),
127.6 (C-8ꢁ and C-12ꢁ), 129.9 (C-9ꢁ and C-11ꢁ), 137.8
(C-10ꢁ), 130.4 (C-7ꢁ), 150.4 (C-4ꢁ), 150.9 (C-2), 161.3
(C-7), 164.5 (C-2ꢁ); LC-MS: 413.1 (M+H)+; Anal. %
Calc for C20H17ClN4O2S: C: 55.10, H: 3.64, N: 13.55.
Found: C: 55.01, H: 3.56, N: 13.50.
1
C); H NMR (DMSO-d6) : δ 2.35 (s, 3H, CH3), 3.81
(t, 2H, J = 5.2 Hz), 3.93 (s, 3H, -OCH3), 4.42 (t, 2H,
J = 5.6 Hz), 6.64–7.14 (m, 4H), 7.40–7.84 (m, 4H),
7.69 (s, 1H) 12.26 (s, 1H); 13C NMR (DMSO-d6): δ
21.5 (-CH3), 54.1 (C-6), 55.9 (-OCH3), 63.1 (C-5),
105.9 (C-5ꢁ), 114.8 (C-9 and C-13), 115.3 (C-10 and
C-12), 127.6 (C-8ꢁ and C-12ꢁ), 129.9 (C-9ꢁ and C-
11ꢁ), 137.8 (C-10ꢁ), 130.4 (C-7ꢁ), 136.4 (C-8), 150.5
(C-4ꢁ), 150.9 (C-2), 152.4 (C-11), 161.6 (C-7), 164.5
(C-2ꢁ); LC-MS: 409.2 (M+H)+; Anal. % Calc for
C21H20N4O3S: C: 61.75, H: 4.94, N: 13.79; Found: C:
61.69, H: 4.89, N: 13.72.
2.3g 4-(4-Methoxyphenyl)-N-(4-(4-chlorophenyl)-
thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-car-
boxamide (7h): Obtained from 6c (2.68 g,
11.00 mmol) and 2b (2.10 g, 10.00 mmol) yield 59% 2.3j 4-(4-Nitrophenyl)-N-(4-(4-chlorophenyl)-thiazol-
(2.52 g); IR (KBr cm−1): ν 3114 (-NH), 1680 (-C=O), 2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide
3059 (Ar-CH), 1133 (C-O-C), 1579 (Ar-C=C), 719 (7k): Obtained from 6d (2.72 g, 11.00 mmol) and
1
(C-S), 2350 (Ar-Cl); H NMR (DMSO-d6): δ 3.81 2b (2.10 g, 10.00 mmol) yield 57% (2.41 g); IR (KBr
(t, 2H, J = 5.2 Hz), 3.90 (s, 3H, -OCH3), 4.43 (t, 2H, cm−1): ν 3119 (-NH), 1698 (-C=O), 3062 (Ar-CH),
J = 5.6 Hz), 6.55–7.39 (m, 4H), 7.16–7.71 (m, 4H), 1135 (C-O-C), 1589 (Ar-C=C), 724 (C-S), 2350 (Ar-
1
7.65 (s, 1H) 12.26 (s, 1H); 13C NMR (DMSO-d6): δ Cl), 1545 and 1350 (NO2); H NMR (DMSO-d6):
54.1 (C-6), 55.9 (-OCH3), 63.1 (C-5), 105.9 (C-5ꢁ), δ 3.82 (t, 2H, J = 5.2 Hz), 4.43 (t, 2H, J = 5.6 Hz),
114.8 (C-9 and C-13), 115.3 (C-10 and C-12), 129.1 7.32–8.21 (m, 4H), 7.16–7.72 (m, 4H), 7.66 (s, 1H)
(C-8ꢁ and C-12ꢁ), 130.0 (C-9ꢁ and C-11ꢁ), 131.3 (C-7ꢁ), 12.26 (s, 1H); 13C NMR (DMSO-d6): δ 54.2 (C-6),
135.1 (C-10ꢁ), 136.4 (C-8), 150.5 (C-4ꢁ), 150.9 (C-2), 63.2 (C-5), 105.9 (C-5ꢁ), 114.7 (C-9 and C-13), 124.9
152.4 (C-11), 161.6 (C-7), 165.3 (C-2ꢁ); LC-MS: 428.6 (C-10 and C-12), 129.1 (C-8ꢁ and C-12ꢁ), 130.0 (C-9ꢁ
(M+H)+; Anal. % Calc for C20H17ClN4O3S: C: 56.11, and C-11ꢁ), 135.1 (C-10ꢁ), 131.3 (C-7ꢁ), 141.3 (C-11),
H: 4.00, N: 13.16; Found: C: 56.01, H: 3.87, N: 13.06.
150.1 (C-8), 150.5 (C-4ꢁ), 150.9 (C-2), 166.3 (C-7),

1918
K Shubakara et al.
164.6 (C-2ꢁ); LC-MS: 424.4 (M+H)+; Anal. % Calc
for C19H14ClN5O4S: C: 51.48, H: 3.18, N: 15.78;
Found: C: 51.41, H: 3.15, N: 15.70.
was determined according to the method of Yen and
Chen.26 The samples 7a-l (10, 50 and 100 μg/mL) were
mixed with equal volumes of 0.2 M phosphate buffer
(pH 6.6) and 1% potassium ferricyanide. The mixture
was incubated at 50◦C for 20 min. Then, an equal vol-
ume of 10% trichloroacetic acid was added to the mix-
ture and then centrifuged at 5000 rpm for 10 min.
The upper layer of solution was mixed with distilled
water and 0.1% ferric chloride at a ratio of 1:1:2 and
the absorbance were measured at 700 nm. Increased
absorbance of the reaction mixture indicated increased
reducing power.
2.3k 4-(4-Nitrophenyl)-N-(4-(p-tolyl)-thiazol-2-yl)-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamide (7l):
Obtained from 6d (2.72 g 11.00 mmol) and 2d (1.90 g
10.00 mmol), yield 58% (2.45 g); IR (KBr cm−1):
ν 3114 (-NH), 1683 (-C=O), 3062 (Ar-CH), 1135
(C-O-C), 1581 (Ar-C=C), 721 (C-S), 2232 (Ar-C),
1
1545 and 1350 (-NO2); H NMR (DMSO-d6): δ 2.36
(s, 3H, CH3), 3.81 (t, 2H, J = 5.2 Hz), 4.43 (t, 2H, J =
5.6 Hz), 7.32–8.21 (m, 4H), 7.40–7.84 (m, 4H), 7.68
(s, 1H), 12.26 (s, 1H); 13C NMR (DMSO-d6): δ 21.5
(-CH3), 54.3 (C-6), 63.1 (C-5), 105.8 (C-5ꢁ), 114.7
(C-9 and C-13), 124.9 (C-10 and C-12), 141.3 (C-11),
150.1 (C-8), 127.6 (C-8ꢁ and C-12ꢁ), 130.0 (C-9ꢁ and
C-11ꢁ), 137.8 (C-10ꢁ), 130.44 (C-7ꢁ), 150.4 (C-4ꢁ),
150.9 (C-2), 161.2 (C-7), 164.5 (C-2ꢁ); LC-MS: 424.5
(M+H)+; Anal. % Calc for C20H17N5O4S: C: 56.76,
H: 4.06, N: 16.54; Found: C: 56.70, H: 4.02, N: 16.47.
2.4d DNA damage inhibition assay: DNA dam-
age inhibition ability of synthesized 4-aryl-N-(4-
aryl-thiazol- 2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-
carboxamides 7a-l was performed using lambda phage
DNA.27,28 Briefly, lambda phage DNA (0.6 μg) was
subjected to oxidation using Fenton’s reagent (0.3 mM
hydrogen peroxide, 0.5 μM ascorbic acid and 0.8 μM
ferric chloride) in presence and absence of the sam-
ple (0.2 mg) for 2 h at 37◦C. The samples 7a-l were
subjected to electrophoresis (Bio-rad Gel electrophore-
sis unit) on 1% agarose for 2 h at 50 volts DC. Gels
were stained with ethidium bromide (0.5 μg/mL) and
documented (Bio-rad gel documentation unit).
2.4 Biological studies of 4-Aryl-N-(4-aryl-thiazol-
2-yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-
carboxamides 7a–l
2.4a Antioxidant studies:
2.4e Statistical analysis: All the experiments were
carried out in triplicates (n = 3) and the results
2.4b DPPH radical scavenging assay: Antioxidant
activity of the synthesized 4-aryl-N-(4-aryl-thiazol-2-
yl)-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides
7a-l in comparison to standard antioxidant butylated
hydroxyl toluene (BHT) on DPPH radical was esti-
mated according to the method of Lai et al.25 Samples
7a-l (10, 50 and 100 µg/mL) and BHT (0–5 µg/mL)
were taken in methanol in 200 µL aliquot and mixed
with 100 mM Tris-HCl buffer (800 µL, pH 7.4) and
then 1 mL of 500 µM DPPH was added in ethanol
(final concentration of 250 µM). The mixture was
shaken vigorously and left to settle for 20 min at room
temperature in the dark. Absorbance of the result-
ing solution was measured spectrophotometrically at
517 nm. The capability to scavenge DPPH radical was
calculated using eq. (1).
were expressed as mean
standard deviation
(SD) as shown in table 1. Graphical representa-
tion of DPPH radical scavenging activity of the
synthesized 4-aryl-N-(4-aryl-thiazol-2-yl)-5,6-dihydro-
4H-1,3,4-oxadiazine-2-carboxamides 7a-l is as shown
in figures 1–3. Pictorial representation of DNA damage
inhibition activity is shown in figure 4.
3. Results and Discussion
Derivatives of 4-aryl-5,6-dihydro-4H-1,3,4-oxadiazine-
2-carboxylic acid 6a-d were obtained from 4-aryl-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxylates 5a-d
on treating with 5% NaOH solution using methanol
as a solvent for about 24 h. The obtained com-
pounds 6a-d were soluble in 5% NaHCO3 and re-
precipitated on acidification, which indicated the for-
mation of 4-phenyl-5,6-dihydro-4H-1,3,4-oxadiazine-
2-carboxylic acid. Structural proof for the compounds
6a-d was confirmed by spectral studies. For instance,
DPPH Scavenging activity (%)
ꢀꢁ
ꢂ
ꢃ
=
Acontrol − Asample /Acontrol × 100 (1)
2.4c Measurement of reducing power: The reducing in IR spectra, signal at 1725–1736 cm−1 of >C=O
power of synthesized 4-aryl-N-(4-aryl-thiazol-2-yl)- is shifted to 1680–1695 cm−1 and a signal appeared
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides 7a-l at 2733–2745 cm−1 due to -OH group. In H NMR
1

Antioxidant activities of oxadiazines
1919
Table 1. DPPH radical scavenging activity and reducing power of samples 7a–l compared with standard antioxidant BHT.
Compounds
% DPPH radical scavenging activity*
% Reducing power determination*
10 (μg/mL)
50 (μg/mL)
100 (μg/mL)
10 (μg/mL)
50 (μg/mL)
100 (μg/mL)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
16 0.022
18 0.092
24 0.016
23 0.022
29 0.013
27 0.191
23 0.226
29 0.10
25 0.012
18 0.076
15 0.067
17 0.421
32 0.018
28 0.091
33 0.013
41 0.086
42 0.012
39 0.032
56 0.111
40 0.183
53 0.099
42 0.082
28 0.036
29 0.036
27 0.136
57 0.083
51 0.099
55 0.066
76 0.055
67 0.079
69 0.086
76 0.093
68 0.082
82 0.093
44 0.016
39 0.032
41 0.034
38 0.133
94 0.012
06 0.091
07 0.019
11 0.036
08 0.086
12 0.083
13 0.019
10 0.086
14 0.016
07 0.016
08 0.019
06 0.024
05 0.213
16 0.183
12 0.082
22 0.027
34 0.011
22 0.092
28 0.016
29 0.016
26 0.092
32 0.082
27 0.082
16 0.016
15 0.026
17 0.417
32 112
56 0.088
62 0.082
79 0.045
65 0.012
68 0.023
69 0.061
62 0.012
82 0.082
64 0.082
58 0.061
57 0.053
56 0.396
86 0.163
(BHT) Standard
* Values are expressed as mean standard deviation (n = 3)
frequency in the region 3112.4–3120.9 cm−1 and
-C=O frequency region 1670.2–1690.5 cm−1. 1H NMR
spectra shows, the absence of carboxylic acid peak at
δ 11.82–11.85 ppm and the appearance of single small
peak due to -CONH- protons in the region δ 12.24–
12.31 ppm. The peaks of aromatic protons due to
substituted thiazole moiety appear at δ 7.4–8.12 ppm,
and in all the derivatives, one singlet peak of thiazole -
CH- proton appeared at δ 7.65–7.69 ppm. In 13C NMR,
in addition to aromatic carbon peaks at δ 113–154 ppm,
a single peak at δ 105.4–105.9 ppm appeared in all
the derivatives. All the 4-aryl-N-(4-aryl-thiazol-2-yl)-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxamides 7a-l
showed (M+H)+ peak in the mass spectra. The above
spectral evidence confirms the formation of products.
spectra, the absence of quartet in the region δ 4.12–
4.31 ppm, (2H for -OCH2 group) and triplet in the
region δ 1.18–1.30 ppm, (3H for -OCH2-CH3 group)
and the presence of carboxylic acid peak at δ 11.82–
11.85 ppm confirmed the formation of product 6a-
d. In 13C NMR, peak due to -CH3 at δ 13.7–
13.9 ppm and -OCH2 at δ 60.9–61.4 ppm disap-
peared and the peak, due to -C=O shifted to δ
168.1–168.2 ppm from δ 158.4–158.6 ppm. All the
synthesized 4-aryl-5,6-dihydro-4H-1,3,4-oxadiazine-2-
carboxylic acids showed (M+H)+ peak which was con-
sistent with the assigned structure in the mass spectra.
The obtained compounds 6a-d were condensed with
2-amino-4-aryl-thiazole derivatives 2a-d to give 4-
aryl-N-(4-aryl-thiazol-2-yl)-5,6-dihydro-4H-1,3,4-oxa-
diazine-2-carboxamides 7a-l. The obtained samples
1
7a-l were confirmed by IR, H NMR, 13C NMR and
3.1 Antioxidant activity of samples 7a–l
MS studies. The IR spectra shows the amide -NH
3.1a DPPH radical scavenging assay: The free radi-
cal scavenging ability of samples 7a-l was evaluated
Figure 2. Bar graph representation of DDPH radical sca-
venging assay of 7a-l.
Figure 1. DDPH radical scavenging assay of 7a-l.

1920
K Shubakara et al.
Figure 3. Graphical representation of reducing power of
7a-l.
Figure 4. Electrophoretic mobility of DNA in treated
samples.
by DPPH scavenging model system using eq. (1). All
the samples 7a-l showed free radical scavenging abil-
ity. The data from table 1 reveals that compounds 7a-
l of synthesized derivatives 7a-l exhibit good DPPH
free radical scavenging activity compared to standard
antioxidant (BHT). These results indicate the potential
electron donating ability of the samples. The DDPH
radical scavenging assays of 7a-l are shown graphically
in figures 1 and 2.
inhibition. Electrophoretic mobility of DNA is shown
in figure 4.
4. Conclusions
We have synthesized a new series of compounds 7a–
l and evaluated them for their DNA damage inhibi-
tion and antioxidant activities. Compounds 7e, 7h, 7c
and 7f having electron donating groups at para position
of aryl moiety demonstrated potent antioxidant activ-
ity and DNA damage inhibition compared to the stan-
dard. These compounds might be blocking the process
of oxidation by neutralizing free radicals via interaction
of the lone pair of electrons (on -Cl, -OCH3 and -OH)
with DPPH radical.
3.1b Reducing ability study of 7a-l: Reducing power
of samples 7a–l was also evaluated (table 1) for
their ability to reduce ferric chloride and potassium
ferricyanide complex. At the initial concentrations
(10 μg/mL), no significant differences in the activ-
ity were observed. However, as the concentration
was increased (50–100 μg/mL), reducing power also
increased. Increased absorbance at 700 nm indicated
the reducing ability of the synthesized compounds. The
Acknowledgements
enhanced reducing power of 7h and 7c may be due to The authors thank the Principal, Yuvaraja’s College,
the presence of electron donating groups at para posi- University of Mysore, Mysore, for providing laboratory
tion of the phenyl ring. Graphical representation of facilities to carry out research work.
reducing power is shown in figure 3.
References
3.1c DNA damage inhibition studies: DNA damage
1. Pfeiffer W D 2008 Review on oxadiazines and thiadi-
inhibition ability of samples 7a-l was evaluated on
azines. (Greifswald: University of Greifswald)
lambda phage DNA oxidation. The hydroxyl radical
2. Stoltefuss Braunlich G, Logers M, Schmeck C,
generated by Fenton’s reagent caused DNA fragmen-
Fugmann B, Nielsch U, Bechem M, Gerdes C, Sperzel
tation with increase in its electrophoresis mobility and
thereby the fragments had run out of the gel (lane
2). Treatment of samples 7a-l prior to oxidation min-
imized the extent of DNA damage. As evidenced by
gel documentation analysis, compared to negative DNA
(figure 4), out of all the samples compounds having
electron donating groups at para-position of the aryl
group, 7e, 7h, 7c and 7f, showed better DNA damage
M, Lustig K and Sturmer W 2001 PCT Int. Appl. WO
2001000601 Chem. Abstr. 134 86284
3. Stefanova Z, Nikolova N, Dimov T, Michailov Y and
Neychev H 1997 Pharmazie 409
4. Suri K A, Suri O P and Atal C K 1986 Indian Drug. 23
207
5. Louis L 1998 EurPat,263,020 Chem. Abstr. 109128995q
6. Geronikaki A and Theophilidis G 1992 Eur. J.
Med. Chem. 27 709

Antioxidant activities of oxadiazines
1921
7. Mean R G N and Mocoelo C R O 1994 Chem. Abstr. 120 19. Kidwai M, Venkataramanan R and Dave B 2002
8. Giridhar T, Reddy R B, Prasanna B and Chandra Mouli
G V P 2001 Indian J. Chem. 40B 1279
9. Apel K and Hirt H 2004 Annu. Rev. Plant Biol. 55 373
10. Jain P K and Agrawal R K 2008 Asian J. Exp. Sci. 22
213
11. Re R, Pellegrini N, Proteggente A, Pannala A, Yang
M and Riceevans C 1999 Free Radic. Biol. Med. 26
1231
12. Jeong J B et al. 2009 Phytomedicine 16 85
13. Ghosal D, Omelchenko Gaidamakova M V and Fems E
K 2005 Microbiol. Rev. 29 361
14. Joshi R, Kamat J P and Mukerjee T 2007 Chem. Biol.
Interact. 167(2) 125
J. Heterocycl. Chem. 39 1045
20. Venkata Rao C et al. 2013 Der Pharm. Chem. 5 181
21. Prajapati A K and Modi Vishal P 2010 J. Chil. Chem.
Soc. 55
22. Enders, E 1967 Methoden Der Organischen Chemie,
Muller E (ed.), (Stuttgart: George Thieme Verlag) Vol.
X/2, p. 180
23. Boger D L and Weinreb (eds.) 1987 Hetero Diels–
Alder methodology in organic synthesis (San Diego:
Academic Press) p. 270
24. Stevenson, Thomas, Martin and Newark 2001 EP
0737188 B1
25. Lai L S, Chou S T and Chao W W 2001 J. Agric. Food
15. Koraiem A I M, El-Hand A and Shindy H A 1995 Chem.
Pap. 49 192
Chem. 49 963
26. Yen G C and Chen H Y 1995 J. Agric. Food Chem. 43
27
27. Kumar G S, Nayaka Harish, Shylaja M D and Salimath
P V 2006 J. Food C. Anal. 19 446
28. Umesha K B, Rai K M L and Harish Nayaka M A 2009
Int. J. Biomed. Sci. 5 359
16. Dekeyser M A and McDonald P T 1998 US Patent 5804
579
17. Batenko N G, Karlivans G A and Valters R E 2005
Heterocycles 65 1569
18. Patel K H and Mehta A G 2012 Der Chem. Sin. 3 1410