Synthesis and biological evaluation of coumarin clubbed thiazines scaffolds as antimicrobial and antioxidant

Article abstract of DOI:10.1007/s00044-018-2222-9

A new series of 4-methyl-6-nitro-2-oxo-2H-chroman-7yl-2-(4-(4-fluorophenyl)-6-phenyl-2H-1,3-thiazin-2-yl-amino)acetates 5a–j were synthesized from 6-nitro-4-methyl coumarinyl chloroacetate (5) and 2-amino thiazines (IIIa–j). The structure of the final compounds was adequately confirmed via spectroscopic techniques (IR, ¹H NMR, ¹³C NMR, Mass) and characterization of physical properties. Final compounds were screened for their antimicrobial, antitubercular, and antioxidant activities. Compounds 5c and 5h found to have antibacterial potency against E. coli with MIC values 50 μg/mL compared to standard drugs. Compound 5d demonstrated better antifungal potency (MIC = 200 μg/mL) against C. albicans when compared with griseofulvin. Compounds 5b and 5h found to be encouraging antitubercular (MIC = 62.5 μg/mL with 98–99% inhibition) against M. tuberculosis H₃₇Rv. The newly synthesized 5h and 5b were appeared to have high radical scavenging efficacies as 33.99 ± 0.301 and 35.35 ± 0.470 μg/mL ± SD of IC₅₀ values, respectively, in DPPH and ABTS bioassay.

Full text of DOI:10.1007/s00044-018-2222-9

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2222-9
ORIGINAL RESEARCH
Synthesis and biological evaluation of coumarin clubbed thiazines
scaffolds as antimicrobial and antioxidant
Nilesh B. Chauhan1 Navin B. Patel Vatsal M. Patel Bhupendra M. Mistry3
1 ●
2 ●
●
Received: 19 April 2018 / Accepted: 9 July 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A new series of 4-methyl-6-nitro-2-oxo-2H-chroman-7yl-2-(4-(4-fluorophenyl)-6-phenyl-2H-1,3-thiazin-2-yl-amino)acet-
ates 5a–j were synthesized from 6-nitro-4-methyl coumarinyl chloroacetate (5) and 2-amino thiazines (IIIa–j). The structure
1
13
of the final compounds was adequately confirmed via spectroscopic techniques (IR, H NMR, C NMR, Mass) and
characterization of physical properties. Final compounds were screened for their antimicrobial, antitubercular, and
antioxidant activities. Compounds 5c and 5h found to have antibacterial potency against E. coli with MIC values 50 µg/mL
compared to standard drugs. Compound 5d demonstrated better antifungal potency (MIC = 200 µg/mL) against C. albicans
when compared with griseofulvin. Compounds 5b and 5h found to be encouraging antitubercular (MIC = 62.5 µg/mL with
9
8–99% inhibition) against M. tuberculosis H Rv. The newly synthesized 5h and 5b were appeared to have high radical
37
scavenging efficacies as 33.99 ± 0.301 and 35.35 ± 0.470 μg/mL ± SD of IC50 values, respectively, in DPPH and ABTS
bioassay.
●
●
●
Keywords 1,3-thiazine-2-amines Coumarin Antibacterial Antioxidant and antitubercular
Introduction
2016), anticancer (Sashidhara et al. 2010; Thakur et al.
015), antiplatelet (Roma et al. 2003), antithrombotic
2
The natural compound such as coumarin has served as
valuable leads for the development of newer biological
potent analogs (Kostova et al. 2011; Patel et al. 2017).
Coumarins form an elite class of compounds, which exhibit
a variety of therapeutic activities including antibacterial
(Kontogiorgis et al. 2015), analgesic (Keri et al. 2010),
antifungal (Al-Amiery et al. 2012; Rehman et al. 2005),
antiviral (Hassan et al. 2016), anticoagulant (Rost et al.
2005), anti-inflammatory (Bansal et al. 2013), and anti-
tumor (Chen et al. 2013). On the other hand, the nitrogen
and sulfur heterocyclic ring families are very interesting due
to their physicochemical properties, especially in the sense
of design of new drugs and new materials. The core moiety
of 1,3-thiazines has N-C-S linkage have been used as
antimicrobial activity (Koketsu et al. 2002), antitumor
(Wang et al. 2012), antituberculosis (Tiwari et al. 2016),
analgesic and anti-inflammatory (Jupudi et al. 2013), and
antioxidant (Jeleń et al. 2015).
(
Bhat et al. 2009; Muratovic et al. 2013), antimalarial (Patel
et al. 2012), antioxidant (Kostova 2006; Nagamallu et al.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2222-9) contains supplementary
material, which is available to authorized users.
*
Navin B. Patel
The development of coumarins as antioxidant agents has
attracted much attention in recent years. Coumarins afford an
opportunity for the discovery of new antioxidants with truly
novel mechanisms of action. The present article deals with the
rational design of coumarin clubbed thiazine motif with an
understanding of the mechanisms of existing synthetic and
natural coumarins. Antimicrobial activity of 4- and 7-hydroxy
and nitrocoumarins has been reviewed extensively (Debeljak
et al. 2007; Dekić et al. 2011) and it has been observed that
when it has been nitrated, its antimicrobial activities is
drnavinbpatel@gmail.com
1
2
Organic Research Laboratory, Department of Chemistry, Veer
Narmad South Gujarat University, Surat 395007 Gujarat, India
Department of Chemistry, Jamanaben Narottambhai Motiram
Patel Science College, Bharthana (Vesu), Surat 395017 Gujarat,
India
3
Department of Food Science and Biotechnology, College of Life
and Biotechnology, Dongguk University, Biomedical Campus, 32
Dongguk-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, Republic of
Korea

Medicinal Chemistry Research
1
enhanced. Additionally, a recent Quantitative Structure
Activity Relationship (QSAR) study of the antimicrobial
activity of some 3-nitrocoumarins has put forward some new
arguments in this direction. With this consideration and
continuation of our ongoing interest in the synthesis of the
thiazine clubbed coumarin derivatives, we have been
prompted to synthesize newer, possibly more potent, phar-
macologically active compounds. We have condensed chlor-
oacetate of 4-methyl-7-hydroxy-6-nitrocoumarin with
substituted amino derivatives of thiazines obtained via chal-
cone derivatives. The synthesized compounds were assigned
1148 (–C–O–C). H NMR (DMSO-d ) δ (ppm): 10.35 (s,
6
1H, phenolic -OH), 7.48 (d, 1H, aromatic) 6.76 (d,1H,aro-
matic), 6.69 (s, 1H,aromatic), 2.37 (s, 3H, –CH ) (Furniss
3
et al. 1989).
7-Hydroxy-4-methyl-6-nitro-2H-chromen-2-one (4) has
been prepared as described in literature
−
1
Yield: 72%, mp: 193–195 °C (as reported). IR (KBr, cm ):
3490 (–OH), 3094, 2827 (–CH , asym, sym), 1745 (>C=O
str), 1190 (–C–O–C), 1536, 1357 (–NO2). H NMR
(DMSO-d ) δ (ppm):13.26 (s, 1H, Phenolic –OH), 8.44 (s,
1H, aromatic), 7.09 (s, 1H, aromatic), 6.35 (s, 1H, aro-
matic), 2.37 (s, 3H, –CH ) (Ganguly et al. 2001).
3
1
1
13
on the basis of IR, H NMR, C NMR and mass spectral
data. The in vitro evaluation of these derivatives viz., anti-
microbial activity on four different bacteria (Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus, and
Streptococcus pyogenes) and three different fungi (Candida
albicans, Aspergillus niger, and Aspergillus clavatus), anti-
tubercular activity on Mycobacterium tuberculosis (H Rv)
6
3
4-Methyl-6-nitro-2-oxo-2H-chromen-7-yl 2-chloroacetate (5)
has been prepared as described in literature
3
7
−
1
virulent strain is presented. Antioxidant acticity of these
derivatives have been evaluated by DPPH (2,2’-diphenyl-1-
picrylhydrazyl) and ABTS (2,2’-Azino-bis-3-ethylbenzthia-
zoline-6-sulfonic acid) radical scavenging assay.
Yield: 69%, mp: 109–112 °C. IR (KBr, cm ): 3003, 2799
(–CH , asym, sym), 1751, 1657 (>C=O str), 1190
(–C–O–C), 1571, 1346 (–NO ), 831 (–C–Cl). H NMR
(DMSO-d ) δ (ppm): 8.38 (s, 1H, aromatic), 7.27 (s, 1H,
3
1
2
6
aromatic), 6.37 (s, 1H, aromatic), 4.47 (s, 2H, –COCH2),
2
.48 (s, 3H, –CH ) (Qandil and Fakhouri 2012).
3
Materials and methods
General method for the synthesis of (E)-1-(4-fluorophenyl)-
All solvents, chemicals, and reagents were purchased from
Sigma-Aldrich with the highest purity and used without
further purification. Melting points were determined with an
open capillary method on ‘‘Equiptronics’’ digital melting
point apparatus, model no. EQ-730 and are uncorrected. IR
spectra were recorded on a Perkin Elmer spectrophotometer
3-phenylprop-2-en-1-one (IIa)
p-Fluoro acetophenone 6 (0.01 mol) and benzaldehyde Ia
(0.01 mol) were dissolved in 15 mL ethanol. NaOH solution
(0.02 mol) in ethanol was added slowly and the mixture was
stirred at 20 °C for 2 h until the entire mixture becomes very
thick. The progress of the reaction was monitored by TLC
(toluene: acetone, 80:20). Then the reaction mixture was
poured slowly onto 400 mL of water with stirring and kept
in refrigerator for 24 h. The precipitate obtained was fil-
tered, washed, and recrystallized from ethanol. The other
compounds IIb–j were prepared by the same method using
substituted benzaldehydes Ib–j.
1
13
(
KBr pellets) instrument. H and C NMR spectra were
recorded on Bruker Avance II 400 MHz NMR Spectrometer
using DMSO-d as solvent and TMS as an internal standard.
6
All chemical shifts were reported as δ values (ppm). Mass
spectra were recorded using Expression CMS from Advion,
USA using ESI as ion source (mobile phase 0.1% formic
acid in 80:20, Methanol: Water). Analytical thin-layer
chromatography (TLC) was performed with Merck silica
gel plates and visualized with ultraviolet (UV) irradiation
General method for the synthesis of 4-(4-Fluorophenyl)-6-
phenyl-2H-1,3-thiazin-2-amine (IIIa)
(
254 nm) or iodine.
A mixture of (E)-1-(4-fluorophenyl)-3-phenylprop-2-en-1-
one IIa (0.01 mol), thiourea (0.01 mol) was dissolved in
ethanolic sodium hydroxide (10 mL) was refluxed about
2–3 h. The progress of the reaction was monitored by TLC
Experimental procedure
Chemistry
(ethylacetate: n-hexane (1:3)). The reacting mixture was
4
-Methyl-7-hydroxy-coumarin (3) has been prepared as
poured onto 400 mL of cold water and stirred for an hour
and then kept in refrigerator for 24 h. The precipitate of 4-
described in literature
(
4-fluorophenyl)-6-phenyl-2H-1,3-thiazin-2-amine
IIIa
−
1
Yield: 85%, mp: 183 to 185 °C. IR (KBr, cm ): 3493
–OH), 3098, 2817 (–CH , asym, sym), 1735 (>C=O str),
obtained was filtered, washed, and recrystallized with
ethanol. The other compounds IIIb–j were prepared by the
(
3

Medicinal Chemistry Research
same method using substituted chalcones IIb–j. Compound
19.41 (C-12), 168.17 (C-14), 40.96 (C-16), 111.13 (C-
18),157.37 (C-20), 74.16 (C-21), 160.14 (C-22), 134.13 (C-
24), 130.16 (C-25, C-29), 115.52 (C-26, C-28), 165.12 (C-
27), 131.51 (C-32), 133.10 (C-33), 131.56 (C-37), 127.80
(C-34), 131.25 (C-36), 128.35 (C-35),; m/z: 563.09 (M ),
565.09(M+2).
−
1
IIIh: IR (KBr, cm ): 3182 (–NH ), 2959 (–CH ), 1599
2
3
1
(
(
–C=N), 1170 (C–F); H NMR (DMSO-d ) δ (ppm): 8.76
6
s, 2H, –NH ), 6.89–7.61 (m, 8H, aromatic), 6.45 (s, 1H,
2
+
aromatic), 5.28 (s, 1H, aromatic), 2.97 (t, 2H, –
CH CH CH ), 1.65 (m, 2H, –CH CH CH ), 0.94 (t, 2H,
2
2
3
2
2
3
–
CH CH CH ).
2 2 3
4
-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
nyl)-6-4-chlorophenyl-2H-1,3-thiazin-2-yl-amino)acetates
5c) Pale yellow solid, yield: 65%, mp: 163–165 °C, M.F.:
General method for the synthesis of (5a–j)
(
−
1
All the reactions were carried out under nitrogen atmo-
sphere. In a round bottom containing compound 5 (0.1 mol)
and IIIa–j (0.1 mol), DMF and K CO (2.4 equivalent)
C H ClFN O S, IR (KBr) ν cm : 3238 (–NH), 1740,
2
8
19
3
6
1669 (–C=O), 1599 (–C=N–), 3067, 2840 (–CH ), 1538,
3
1358 (–NO ), 1258, 1057 (–C–O–C–), 1170 (C–F), 758
2
3
2
1
were added under constant stirring. Reaction Mixture was
(C–Cl); H NMR (400 MHz, DMSO-d6, TMS) δ: 8.54 (s,
refluxed for 8–9 h. After the completion of the reaction
1H, –CH), 6.30–7.77 (m, 11H, aromatic), 5.34 (s, 1H,
–CH), 3.70 (s, 2H, –CH NH), 3.23 (s, 1H, –CH NH), 2.41
(
TLC monitored), reaction mixture was poured over cru-
2
2
1
3
shed ice, solids that are separated out was filtered, washed
(s, 3H, –CH3); C NMR (100 MHz, DMSO-d TMS) δ:
6
with saturated solution of NaHCO and dried. The crude
160.12 (C-2),113.94 (C-3), 152.51 (C-4), 120.72 (C-5),
139.44 (C-6), 135.64 (C-7), 115.77 (C-8), 159.12 (C-9),
118.67 (C-10), 19.41 (C-12), 168.17 (C-14), 40.96 (C-16),
111.13 (C-18),157.37 (C-20), 74.16 (C-21), 160.14 (C-22),
134.13 (C-24), 130.16 (C-25, C-29), 115.52 (C-26, C-28),
3
product was purified by column chromatography using
silica gel 100–200 mesh and gradient (0–80%) ethylacetate
in hexane as eluent. The precipitate obtained was filtered,
washed, and recrystallized.
165.12 (C-27), 131.51 (C-32), 133.10 (C-33), 131.56 (C-
4
-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
37), 127.80 (C-34), 131.25 (C-36), 128.35 (C-35); m/z:
563.09 (M ), 565.09(M+2).
+
nyl)-6-phenyl-2H-1,3-thiazin-2-yl-amino) acetate (5a) Yel-
low solid, yield: 59 %, m.p.: 129–131 °C, M.F.:
C H N FO S.
4-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
nyl)-6-2-hydroxyphenyl-2H-1,3-thiazin-2-yl-amino) acetate
(5d) Pale yellow solid, yield: 61 %, m.p.: 174–176 °C,
M.F.: C H FN O S.
2
8
19
3
6
−
1
IR (KBr) ν cm : 3192 (–NH), 1715, 1671 (–C=O), 1601
C=N), 2961, 2840 (–CH ), 1508, 1364 (–NO ), 1177
C–F); H NMR (400 MHz, DMSO-d , TMS) δ ppm: 8.55
(
(
(
3
2
1
6
28 20
3
7
−
1
s, 1H, –CH), 6.32–7.78 (m, 12H, aromatic), 5.23 (s, 1H,
IR (KBr) ν cm : 3484 (–OH), 3190 (–NH), 1758, 1664
–
CH), 3.71 (s, 2H, –CH NH), 3.23 (s, 1H, –CH NH), 2.40
(–C=O), 1622 (C=N), 2965, 2842 (–CH ), 1523, 1351
2
2
3
1
3
1
(
s, 3H, –CH3); C NMR (100 MHz, DMSO-d TMS) δ
(–NO ), 1174 (C–F); H NMR (400 MHz, DMSO-d , TMS):
6
2
6
ppm: 160.11 (C-2),113.88 (C-3), 152.54 (C-4), 120.68 (C-
δ ppm: 10.11 (s, 1H, –OH), 8.58 (s, 1H, –CH), 6.37–7.77
(m, 11H, aromatic), 5.22 (s, 1H, –CH), 3.71 (s, 2H, –
5
1
8
1
1
), 139.40 (C-6), 135.67 (C-7), 115.79 (C-8), 159.14 (C-9),
17.65 (C-10), 19.40 (C-12), 168.16 (C-14), 43.98 (C-16),
1.13 (C-18),157.35 (C-20), 95.50 (C-21), 153.11 (C-22),
34.18 (C-24), 130.12 (C-25, C-29), 115.57 (C-26, C-28),
13
CH NH), 3.23 (s, 1H, –CH NH), 2.44 (s, 3H, –CH );
C
2
2
3
NMR (100 MHz, DMSO-d , TMS) δ ppm: 160.12 (C-
6
2),113.92 (C-3), 152.50 (C-4), 120.68 (C-5), 139.46 (C-6),
135.67 (C-7), 115.72 (C-8), 159.10 (C-9), 117.64 (C-10),
19.44(C-12), 168.15 (C-14), 43.93 (C-16), 81.12 (C-
65.09 (C-27), 136.32 (C-32), 128.55 (C-33, C-37), 129.04
+
(
C-34, C-36), 126.32 (C-35); m/z: 545.54 (M ).
18),157.35 (C-20), 95.51 (C-21), 153.12 (C-22), 134.11
4
-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
nyl)-6-2-chlorophenyl-2H-1,3-thiazin-2-yl-amino)acetates
5b) Cream yellow solid, yield: 59%, mp: 156–158 °C, M.
(C-24), 130.12 (C-25, C-29), 115.59 (C-26, C-28), 165.15
(C-27), 122.32 (C-32), 158.54 (C-33), 117.52 (C-34),
128.35 (C-35), 121,44 (C-36), 125.02 (C-37); m/z: 561.54
(
−
1
+
F.: C H ClFN O S, IR (KBr) ν cm : 3185 (–NH), 1720,
(M ).
2
8
19
3
6
1
667 (–C=O), 1591 (–C=N-), 2956, 2835 (–CH ), 1514,
3
1
1
354 (–NO ), 1172 (C–F), 749 (C–Cl); H NMR
4-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
nyl)-6-4-hydroxyphenyl-2H-1,3-thiazin-2-yl-amino) acetate
(5e) Pale yellow solid, yield: 69 %, m.p.: 184–186 °C,
M.F.: C H N FO S.
2
(
6
400 MHz, DMSO-d6, TMS) δ: 8.54 (s, 1H, –CH),
.31–7.78 (m, 11H, aromatic), 5.39 (s, 1H, –CH), 3.69 (s,
H, –CH NH), 3.30 (s, 1H, –CH NH), 2.39 (s, 3H, –CH );
2
2
2
3
28 20
3
7
−1
1
3
C NMR (100 MHz, DMSO-d6 TMS) δ: 160.12 (C-
),113.94 (C-3), 152.51 (C-4), 120.72 (C-5), 139.44 (C-6),
35.64 (C-7), 115.77 (C-8), 159.12 (C-9), 118.67 (C-10),
IR (KBr) ν cm : 3478 (–OH), 3186 (–NH), 1765, 1671
2
1
(>C=O), 1627 (C=N), 2959, 2851 (–CH ), 1530, 1356
3
1
(–NO ), 1180 (C–F); H NMR (400 MHz, DMSO-d , TMS):
2
6

Medicinal Chemistry Research
δ ppm: 10.20 (s, 1H, –OH), 8.51 (s, 1H, –CH), 6.32–7.74
(5h) Dark yellow solid, yield: 68%, m.p.: 140–142 °C, M.
(
m, 11H, aromatic), 5.27 (s, 1H, –CH), 3.69 (s, 2H, –
F.: C H F N O S.
IR (KBr) ν cm : 3192 (–NH), 1715, 1671 (>C=O), 1601
(–C=N), 2961, 2840 (–CH ), 1509, 1364 (–NO ), 1233,
1055 (–C–O–C), 1177 (C–F); H NMR (400 MHz, DMSO-
d6, TMS): δ ppm: 8.57 (s, 1H, –CH), 6.34–7.76 (m, 11H,
3
1
26
3
6
−1
1
3
CH NH), 3.30 (s, 1H, –CH NH), 2.51 (s, 3H, –CH );
NMR (100 MHz, DMSO-d , TMS) δ ppm: 160.09 (C-
2
1
1
1
C
2
2
3
6
3
2
1
),113.88 (C-3), 152.46 (C-4), 120.74 (C-5), 139.41 (C-6),
35.61 (C-7), 115.72 (C-8), 159.10 (C-9), 117.64 (C-10),
9.44(C-12), 168.15 (C-14), 43.93 (C-16), 81.12 (C-
8),157.35 (C-20), 95.51 (C-21), 153.12 (C-22), 134.11
aromatic), 5.23 (s, 1H, –CH), 3.74 (s, 2H, –CH NH), 3.22
2
(s, 1H, –CH NH), 2.42 (s, 3H, –CH ), 2.99 (t, 2H, –
2
3
(
(
C-24), 130.12 (C-25, C-29), 115.59 (C-26, C-28), 165.15
C-27), 122.32 (C-32), 158.54 (C-33), 117.52 (C-34),
CH CH CH ), 1.67 (m, 2H, –CH CH CH ), 0.96 (t, 2H,
2 2 3 2 2 3
1
3
–CH CH CH ); C NMR (100 MHz, DMSO-d TMS) δ
2
2
3
6
1
28.35 (C-35), 121,44 (C-36), 125.02 (C-37); m/z: 561.45
ppm: 160.10 (C-2),113.90 (C-3), 152.50 (C-4), 120.70 (C-
5), 139.44 (C-6), 135.64 (C-7), 115.77 (C-8), 159.13 (C-9),
+
(
M ).
117.67 (C-10), 19.41(C-12), 168.18 (C-14), 43.94 (C-16),
4
-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
nyl)-6-4-fluorophenyl-2H-1,3-thiazin-2-yl-amino) acetate
5f) Light yellow solid, yield: 64%, m.p.: 158–160 °C,
M.F.: C H N F O S.
81.10 (C-18), 157.34 (C-20), 95.50 (C-21), 153.10 (C-22),
134.14 (C-24), 130.14 (C-25, C-29), 115.56 (C-26, C-28),
165.10 (C-27), 133.35 (C-32), 126.52 (C-33, C-37), 128.00
(C-34, C-36), 140.34 (C-35), 34.41 (C-38), 24.01 (C-39),
(
2
8
19
3
2
6
−
1
+
IR (KBr) ν cm : 3219 (–NH), 1745, 1669 (–C=O), 1619
13.31 (C-40); m/z: 587.15 (M ).
(
(
(
C=N), 2954, 2851 (–CH ), 1168 (aryl-F), 1525, 1358
3
1
–NO ); H NMR (400 MHz, DMSO-d , TMS) δ ppm: 8.53
4-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
nyl)-6-3-bromophenyl-2H-1,3-thiazin-2-yl-amino) acetate
(5i) Pale yellow solid, yield: 62%, m.p.: 125–127 °C., M.
F.: C H BrFN O S.
2
6
s, 1H, –CH), 6.33–7.79 (m, 11H, aromatic), 5.25 (s, 1H,
CH), 3.71 (s, 2H, –CH NH), 3.22 (s, 1H, –CH NH), 2.43
–
2
2
s, 3H, –CH3); 1 C NMR (100 MHz, DMSO-d , TMS) δ
3
(
6
28 19
3
6
−
1
ppm: 160.11 (C-2),113.88 (C-3), 152.48 (C-4), 120.69 (C-
IR (KBr) ν cm : 3326 (–NH), 1749 (–C=O), 1623
1
5
1
8
1
1
), 139.42 (C-6), 135.62 (C-7), 115.76 (C-8), 159.11 (C-9),
17.65 (C-10), 19.40 (C-12), 168.15 (C-14), 43.90 (C-16),
1.12 (C-18),157.33 (C-20), 95.50 (C-21), 153.10 (C-22),
34.14 (C-24), 130.12 (C-25, C-29), 115.52 (C-26, C-28),
(C=N), 2895, 2958 (–CH ), 1514, 1331 (–NO ); H NMR
3
2
(400 MHz, DMSO-d , TMS) δ: 8.57 (s, 1H, –CH),
6
6.38–7.77(m, 11H, aromatic), 5.25 (s, 1H, –CH), 3.73 (s,
2H, –CH NH), 3.22 (s, 1H, –CH NH), 2.42 (s, 3H, –CH );
2
2
3
1
3
65.16 (C-27), 131.33 (C-32), 128.51 (C-33, C-37), 115.06
C NMR (100 MHz, DMSO-d , TMS) δ ppm: 160.12 (C-
6
+
(
C-34, C-36), 161.34 (C-35); m/z: 563.53 (M ).
2),113.91 (C-3), 152.50 (C-4), 120.89 (C-5), 139.43 (C-6),
35.61 (C-7), 115.72 (C-8), 159.10 (C-9), 117.66 (C-10),
1
4
-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
19.40 (C-12), 168.16 (C-14), 43.95 (C-16), 81.12 (C-
18),157.33 (C-20), 95.51 (C-21), 153.11 (C-22), 134.14 (C-
24), 130.15 (C-25, C-29), 115.55 (C-26, C-28), 165.12 (C-
27), 138.37 (C-32), 128.59 (C-33), 127.50 (C-37), 123.06
nyl)-6-p-tolyl-2H-1,3-thiazin-2-yl-amino) acetate (5g) Yel-
low solid, yield: 62%, m.p.: 165–167 °C, M.F.:
C H FN O S.
2
9
22
3
6
−
1
+
IR (KBr) ν cm : 3211(–NH), 1741, 1664 (–C=O), 1629
(C-34), 130.09 (C-36),129.35 (C-35); m/z: 623.32 (M ),
(
(
(
C=N), 2941, 2861 (–CH ), 1171 (aryl-F), 1517, 1353
625.18 (M+2), 627.15 (M+4).
3
1
–NO ); H NMR (400 MHz, DMSO-d , TMS) δ ppm: 8.56
2
6
s, 1H, –CH), 6.38–7.79(m, 11H, aromatic), 5.24 (s, 1H,
CH), 3.70 (s, 2H, –CH NH), 3.21 (s, 1H, –CH NH), 2.41
4-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
nyl)-6-3-phenoxy-2H-1,3-thiazin-2-yl-amino) acetate (5j)
Dark yellow solid, yield: 62%, m.p.: 132–134 °C., M.F.:
C H FN O S.
–
2
2
13
(
s, 3H, –CH ), 2.38 (s, 3H, –CH ); C NMR (100 MHz,
3 3
DMSO-d TMS) δ ppm: 160.09 (C-2),113.91 (C-3), 152.54
6
34 24
3
7
−
1
(
C-4), 120.66 (C-5), 139.46 (C-6), 135.67 (C-7), 115.79 (C-
IR (KBr) ν cm : 3318 (–NH), 1749 (–C=O), 1623
(C=N), 2895, 2958 (–CH ), 1514, 1331 (–NO ) 1249, 1050
(–C–O–C–), 1161 (C–F); H NMR (400 MHz, DMSO-d ,
8
1
2
1
), 159.15 (C-9), 117.65 (C-10), 19.47 (C-12), 168.18 (C-
4), 43.95 (C-16), 81.11 (C-18),157.32 (C-20), 95.51 (C-
1), 153.09 (C-22), 134.16 (C-24), 130.14 (C-25, C-29),
15.55 (C-26, C-28), 165.13 (C-27), 133.34 (C-32), 125.51
3
2
1
6
TMS) δ: 8.57 (s, 1H, –CH), 6.38–7.77(m, 11H, aromatic),
5.25 (s, 1H, –CH), 3.73 (s, 2H, –CH NH), 3.22 (s, 1H,
–CH NH), 2.42 (s, 3H, –CH ); C NMR (100 MHz,
2 3
DMSO-d , TMS) δ ppm: 160.23 (C-2),113.88 (C-3), 152.65
6
2
1
3
(
(
C-33, C-37), 129.04 (C-34, C-36), 136.35 (C-35), 22.37
C-38); m/z: 559.56 (M ).
+
(C-4), 120.69 (C-5), 139.51 (C-6), 135.69 (C-7), 115.66 (C-
4
-Methyl-6-nitro-2-oxo-2H-chromen-7yl-2-(4-(4-fluorophe-
8), 159.54 (C-9), 118.56 (C-10), 19.42 (C-12), 168.21 (C-
14), 40.89 (C-16), 111.56 (C-18), 157.24 (C-20), 74.19 (C-
nyl)-6-4-propylphenyl-2H-1,3-thiazin-2-yl-amino)acetate
21), 160.12 (C-22), 134.18 (C-24), 130.16 (C-25, C-29),

Medicinal Chemistry Research
Antioxidant activity
F
O
1
50
00
H
N
N
1
O
5
0
S
NO2
0
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
A A
DPPH IC50 μg/mL 40.02 35.35 37.8 64.21 69.17 58.9 51.97 33.99 69.58 49.15 36.22
ABTS IC50 μg/mL 90.16 104.8 83.67 69.3 91.93 53.92 52 83.03 61.27 123.9 22.64
O
R
Fig. 2 The plot of antioxidant assay result of 5a–j
O
drugs to test upon standard bacterial strains. Each synthe-
sized compound was diluted obtaining 2000 μg/mL con-
centration, as a stock solution and then many dilutions were
made shown as in antimicrobial activities (Fig. 1).
Fig. 1 General structure of 4-methyl-6-nitro-2-oxo-2H-chromen-7yl-2-
4-(4-fluorophenyl)-6-substituted phenyl-2H-1,3-thiazin-2-yl -amino)
acetate 5a–j
(
1
15.62 (C-26, C-28), 165.20 (C-27), 130.66 (C-32), 127.69
(
C-33), 142.14 (C-34), 127.39 (C-35), 127.09 (C-36),
Antioxidant evaluation
1
1
26.19 (C-37), 160.61 (C-39), 118.13 (C-40, C-44),
30.11 (C-41, C-43) 122.79 (C-42); m/z: 637.13 (M ).
+
DPPH method Reduction of 2,2-diphenyl-1-picrylhydrazyl
(free radical) is the base of the DPPH antioxidant bioassay.
Biology
It has an odd electron that shows a maximum absorption
band of 517 nm (deep violet color) in ethanol. The DPPH
bioassay is the widely used and acceptable method for
evaluating the free radical scavenging action of the tested
compounds. Such substances donate a hydrogen atom when
mixed with the DPPH, thereby introducing its reduced
congener, diphenylpicrylhydrazine (non-radical) with the
loss of violet color. In the present study, DPPH bioassay
was adopted to screen the berberine-based compounds for
their in vitro antioxidant profiles. The results of this
bioassay investigation were introduced in the form of the
percentage of radical scavenging antioxidant activity (RSA
%) of each substance. The investigation of the DPPH
radical scavenging activity was operated according to the
methodology described by (Brand-Williams et al. 1995)
with some modifications (Mistry et al. 2016). A stable free
radical, DPPH, was allowed to react with test compounds in
methanol as 20 μg/mL (100, 10, 1, and 0.1) quantities of
title compounds were mixed up with 180 μg/mL of DPPH in
methanol. Titled compounds donated hydrogen during the
mixing thereby introduced the reduction of DPPH and
hence a change in the color was observed from deep violet
to light yellow at 517 nm after 25 min of reaction in a
UV–Visible spectrophotometer (Perkin Elmer). The blank
reading was also performed using the mixture of methanol
(20 μg/mL) and sample (180 μg/mL of DPPH). Ascorbic
acid served as a control drug in this assay, and its solution
was prepared by mixing methanol (20 μg/mL) and DPPH
radical solution (180 μg/mL). The results of this bioassay,
RSA % was determined according to (Mensor et al. 2001)
as described in the below equation.
In vitro antimicrobial assay
The broth microdilution method has been employed to
determine the MICs of synthesized compounds as described
in the literature (Patel et al. 2010). Dimethylsulfoxide
(
DMSO) was used as diluent to get desired concentration of
drugs to test upon standard bacterial strains. Prepare a sol-
vent control of a 1:10 dilution of the DMSO used to dis-
solve the antimicrobial agent being tested. This
1
0
:10 solution is prepared by adding 0.1 ml of solvent to
.9 ml of the appropriate diluent. The highest dilution
showing at least 99% inhibition is taken as MIC. To eval-
uate the antimicrobial potency of the final derivatives, they
were screened against different strains viz., two Gram-
positive bacteria S. aureus (MTCC–96) and S. pyogenes
(
(
MTCC–442), two Gram-negative bacteria E. coli
MTCC–443) and P. aeruginosa (MTCC–1688), and fungi,
C. albicans (MTCC–227), A. niger (MTCC–282), and A.
clavatus (MTCC– 1323), and compared with standard
drugs, chloramphenicol, ciprofloxacin, and griseofulvin.
In vitro antitubercular assay
Tubercle bacilli are aerobes, grow in specially enriched
media containing egg, asparagines, potatoes, serum, and
meat extracts. Colonies appear in 2–6 weeks. The drug
susceptibility test to determine MIC by LJ Slope method
has been employed (Muralidhar and Srivastava 2004). M.
tuberculosis H Rv [acid fast bacilli] (MTCC–200) was
3
7
used for screening of antitubercular activity. DMSO was
used as diluents/vehicle to get desired concentration of
Absorbance of blankÀAbsorbance of test
%
Scavenging ¼
 100
Absorbance of blank

Medicinal Chemistry Research
Scheme 1 a Cooled (5–10 °C), conc. H2SO4; b CAN, 30% H2O2 +
2–3 h refluxed;
f
4-methyl-6-nitro-2-oxo-2H-chromen-7-yl 2-
5
mL H2O, stirred; c α-chloroacetyl chloride, CH2Cl2, triethyl amine
chloroacetate (5), DMF, K2CO3, 8-9 h refluxed. Where, R = -H, 2-
Cl, 4-Cl, 2-OH, 4-OH, 4-F, 4-CH3, 4-C3H7, 3-Br, 3-OPh
1
h stirred; d NaOH, EtOH, 2 -3 h stirred; e thiourea, ethanolic NaOH,
A plot of concentration (Fig. 2) of test compounds and %
Results and discussion
scavenging introduced IC50 in the presence of an ascorbic
acid as standard.
Chemistry
ABTS method The ABTS• + radical cation scavenging
efficacies of the test compounds was determined according
to the method described earlier with some modifications
The synthetic protocol for the lead molecule 4-methyl-6-
nitro-2-oxo-2H-chromen-7-yl 2-chloroacetate (5) and final
compounds (5a–j) is depicted in Scheme 1. According to
the scheme, 4-methyl-7-hydroxy-coumarin (2) has been
synthesized by pechmann condentation, which is followed
by nitration with nitric acid and acetic acid and then reacting
with chloroacetyl chloride to get 4-methyl-3-nitro-2-oxo-
2H-chromen-7-yl 2-chloroacetate (5). For the synthesis of
final compounds, key intermediate (5) was condensed with
amino thiazines derivatives (IIIa–j) obtained from
cycloaddition reaction between substituted chalcones (IIa–
j) and thiourea. The reaction protocol is illustrated in the
following scheme.
(
Mistry et al. 2016). Mixing of an equal amount of 7.0 mM
ABTS• + stock solution with 2.45 mM potassium persulfate
stock solution produces the ABTS• + cation. The mixture
was kept in a dark place at 0 °C temperature for 12 h and
ABTS solution was diluted with MeOH so that it gives UV
absorption value of 0.700 ( ± 0.200) at the 734 nm. The
1
000 μL stock solutions of titled compounds 5a–j were
dissolved in methanol and further dilutions of 100, 10, 1,
and 0.1 μg/mL taken for test samples. In all 180 μg/mL
solutions of compounds to be evaluated and 20 μg/mL of
the ABTS solution were mixed in 96-well plates in a dark
place and then incubated for 10 min to measure UV
absorption at 734 nm. The solution of 180 μg/mL ABTS in
Characterizations of intermediate and final compounds
were confirmed by their spectral analysis. The characteristic
band of 3192 cm− of –NH, while two bands for –C=O
1
appeared at 1715 and 1671 cm− and 1523, 1351 cm for
1
−1
2
0 μg/mL methanol was used as a control determination,
while ascorbic acid was used as a reference drug. The UV
absorption data represented the radical scavenging rates that
give the corresponding IC50s for the test compounds.
The scavenging capability of ABTS• + radical was
calculated using the following equation:
–NO in IR spectrum confirmed the structure of the final
compounds 5a–j. H NMR spectrum of final compounds
2
1
showed singlet at 8.55 of –CH confirmed the neighboring
–NO group while –CH NH showed two singlet at 3.71 and
2
2
1
3
3.23, respectively. C NMR spectrum of 5a–j showed
peaks nearer at 160.10 and 168.20 for two different C=O
Absorbance of blankÀAbsorbance of test
%
Scavenging ¼
 100
Absorbance of blank
and one peak at 19.40 for –CH of coumarin. Peaks at
3

Medicinal Chemistry Research
4
3.90, 81.10, and 111.30 were obtained for –CHNH, –CH
–CH CH CH at position 4 on benzaldehyde demonstrated
2 2 3
of thiazine (C-18) ring, which confirming the structure of
final compounds.
remarkable activity (MIC = 50 µg/mL) against E. coli,
comparable to chloramphenicol and ciprofloxacin. While
other compounds showed poor activity against S. aureus
and S. pyogenes. Compounds 5a and 5b, having substituent
Biology
–
H, 2-Cl, respectively, exhibited significant activity with
Antimicrobial activity
MIC value 250 µg/mL, while compound 5d showed
encouraging potency (MIC = 200 µg/mL) against C. albi-
cans compared with griseofulvin. Other compounds with
very high MIC values and seem to be poor to moderately
active (Table 2).
The minimum inhibitory concentrations (MIC) for the
antimicrobial potency of 5a–j were screened against four
different bacterial strains and three different fungal strains.
The susceptibility of the organisms was determined by the
broth microdilution method (Rattan 2000) and compared
with standard drugs; chloramphenicol, ciprofloxacin, and
griseofulvin. The results of this activity are described in
Table 1. Compound 5c with a –Cl group and 5 h with
Table 3 Antitubercular data of compounds 5a–j
Minimum inhibitory concentration
Compound no.
MIC (μg/ml)
% Inhibition
Table 1 Spectral characterization of the compounds
5
5
a
100
99
98
98
99
98
97
99
99
99
98
99
99
Comp. no.
R
Molecular Formula
M.P. °C
Yield %
b
62.5
500
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
–C6H5
2-Cl
C28H20FN3O6S
C28H19FN3O6SCl
C28H19N3O6SCl
C28H20FN3O7S
C28H20FN3O7S
C28H19F2N3O6S
C29H22FN3O6S
C31H26FN3O6S
C H BrFN O S
129–131
156–158
163–165
174–176
184–186
158–160
165–167
140–142
125–127
132–134
56
59
65
61
69
64
62
68
62
69
5c
5d
1000
1000
>1000
500
4-Cl
5e
2-OH
4-OH
4-F
5f
5g
5h
62.5
500
g
h
i
4-CH3
4-C3H7
3-Br
5i
5j
>1000
40
Rifampicin
Isoniazid
28
19
3
6
j
3-OPh
C34H24FN3O7S
0.2
Table 2 Antibacterial and
antifungal data of compounds
Compound no.
MIC (μg/mL)
Antibacterial activity
S. S.
5
a–j
Antifungal activity
E. coli P.
C.
A. niger
A.
aureus pyogenes MTCC aeruginosa albicans
MTCC282 clavatusMTCC
MTCC MTCC
443
MTCC
1688
MTCC227
1323
9
6
442
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
250
100
500
500
250
200
250
500
500
250
500
100
500
250
250
200
250
500
500
125
50
100
200
50
200
200
100
250
250
250
250
100
100
200
50
250
250
500
200
1000
1000
1000
>1000
500
500
–
1000
200
1000
500
1000
500
>1000
250
100
250
125
250
50
1000
>1000
500
1000
>1000
>1000
500
g
h
i
1000
1000
1000
–
100
200
50
1000
>1000
–
j
Chloramphenicol 50
Ciprofloxacin
25
25
50
50
–
–
–
Griseofulvin
–
–
–
–
500
100
100

Medicinal Chemistry Research
Table 4 Screening results of DPPH and ABTS radical scavenging
activity of 5a–j
DPPH and ABTS bioassays, respectively. Also, a MIC of
5a–j using broth microdilution method towards bacterial
Compd. no.
DPPH
ABTS
and fungal strains was studied and the derivatives 5c and 5h
displayed remarkable potency against E. coli with MIC
values 50 µg/mL compare to standard drugs. It has been
observed that the potent antibacterial and antitubercular
candidate proved to possess significant antioxidant activity.
The present of propyl group on phenyl ring plays an
important role for the potency in above mentioned biolo-
gical assay.
IC50 μg/mL ± SD
IC50 μg/mL ± SD
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
40.02 ± 0.199
35.35 ± 0.470
37.80 ± 0.246
64.21 ± 0.895
69.17 ± 0.866
58.90 ± 1.069
51.97 ± 1.098
33.99 ± 0.301
69.58 ± 0.330
49.15 ± 0.140
36.22 ± 0.469
90.16206 ± 1.091
104.78 ± 0.744
83.67 ± 0.553
69.30 ± 0.893
91.93 ± 1.020
53.92 ± 0.777
52.00 ± 0.109
83.03 ± 0.713
61.27 ± 2.330
123.86 ± 1.951
22.64 ± 0.260
g
h
i
Acknowledgements We are thankful to Veer Narmad South Gujarat
University for providing necessary facilities, D. Rajani, Microcare
Laboratory, Surat, for antimicrobial and antitubercular activity. Nilesh
Chauhan is highly obliged to University Grants Commission, New
Delhi for awarding Teacher Fellowship Award under Faculty
Improvement Program. Vatsal M. Patel is grateful to University Grant
Commission for providing BSR-SAP fellowship (F.7-233/2009
j
Ascorbic acid
(BSR)).
Antitubercular activity
Compliance with ethical standards
The MIC of the titled compounds were tested for anti-
tuberculer activities (Andrews 2001). The results are men-
tioned in Table 3. Antituberculer activity results showed
that, compound 5b and 5h having 2-Cl and 4-propyl group
demonstrated better activity 62.5 µg/mL with 98–99 %
inhibition against M. tuberculosis H Rv (Table 4).
Conflict of interest The authors declare that they have no conflict of
interest.
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