Synthesis, carbonic anhydrase inhibitory activity and antioxidant activity of some 1,3-oxazine derivatives

Article abstract of DOI:10.1002/ddr.21464

(Table presented.). A series of 1-(6-methyl-2-substituted phenyl-4-thioxo-4H-1,3-oxazin-5-yl)ethanones (3a-n) were synthesized by the reaction of benzoyl isothiocyanates with active methylene compound acetylacetone in the presence of triethyl amine in a one-pot process. The structures of the products were elucidated by elemental analyses, FT-IR, ¹H NMR, ¹³C NMR, and mass spectroscopy. These new 1,3-oxazine derivatives were evaluated for their inhibitory activity against carbonic anhydrase II. Results for in vitro assay revealed that compound 3b having 4-methoxy phenyl moiety was the most potent inhibitor with IC₅₀ value of 0.144 ± 0.008 μM. It exhibited higher enzyme inhibitory activity as compared to the standard acetazolamide (IC₅₀ = 0.997 ± 0.061 μM). The compounds 3c, 3h, and 3n also displayed superior inhibitory activities compared to the rest of the synthesized oxazine derivatives. The radical scavenging activity of oxazine derivatives was also performed and it was found that compounds showed moderate antioxidant activity. Lipinski rule confirmed the therapeutic potential of the synthesized compounds. Molecular docking studies were also performed to further understand the binding affinity of these compounds with PDBID 1V9E which confirmed that the synthesized derivatives bind in the active binding site of the target protein. Based upon our results, it is proposed that compound 3b may serve as a lead structure to design more potent carbonic anhydrase inhibitors.

Full text of DOI:10.1002/ddr.21464

Received: 20 April 2018
DOI: 10.1002/ddr.21464
Revised: 10 August 2018
Accepted: 14 August 2018
R E S E A R C H A R T I C L E
Synthesis, carbonic anhydrase inhibitory activity and
antioxidant activity of some 1,3-oxazine derivatives
Rabia Qamar1 | Aamer Saeed1 | Maria Saeed1 | Zaman Ashraf2 | Qamar Abbas3 |
Mubashir Hassan4 | Fernando Albericio5
Enabling Technologies
Strategy, Management & Health Policy
Hit, Lead &
Candidate Discovery
Preclinical Research
& Development
Clinical
Research
Post-Market
Research
1Department of Chemistry, Quaid-i-Azam
Abstract
University, Islamabad, Pakistan
2Department of Chemistry, Allama Iqbal Open
University, Islamabad, Pakistan
A series of 1-(6-methyl-2-substituted phenyl-4-thioxo-4H-1,3-oxazin-5-yl)ethanones (3a-n) were
synthesized by the reaction of benzoyl isothiocyanates with active methylene compound acetyla-
cetone in the presence of triethyl amine in a one-pot process. The structures of the products were
elucidated by elemental analyses, FT-IR, 1H NMR, 13C NMR, and mass spectroscopy. These new
1,3-oxazine derivatives were evaluated for their inhibitory activity against carbonic anhydrase
II. Results for in vitro assay revealed that compound 3b having 4-methoxy phenyl moiety was the
most potent inhibitor with IC50 value of 0.144 ꢀ 0.008 μM. It exhibited higher enzyme inhibitory
activity as compared to the standard acetazolamide (IC50 = 0.997 ꢀ 0.061 μM). The compounds
3c, 3h, and 3n also displayed superior inhibitory activities compared to the rest of the synthesized
oxazine derivatives. The radical scavenging activity of oxazine derivatives was also performed and
it was found that compounds showed moderate antioxidant activity. Lipinski rule confirmed the
therapeutic potential of the synthesized compounds. Molecular docking studies were also per-
formed to further understand the binding affinity of these compounds with PDBID 1V9E which
confirmed that the synthesized derivatives bind in the active binding site of the target protein.
Based upon our results, it is proposed that compound 3b may serve as a lead structure to design
more potent carbonic anhydrase inhibitors.
3Department of Physiology, University of
Sindh, Jamshoro, Pakistan
4Department of Biology, College of Natural
Sciences, Kongju National University, Gongju,
Republic of Korea
5School of Chemistry and Physics, University
of KwaZulu-Natal, Durban, South Africa
Correspondence
Aamer Saeed, Department of Chemistry,
Quaid-i-Azam University, 45320 Islamabad,
Pakistan.
Emails: asaeed@qau.edu.pk;
aamersaeed@yahoo.com
and
Zaman Ashraf, Department of Chemistry,
Allama Iqbal Open University, Islamabad,
Pakistan.
Email: mzchem@yahoo.com
KEYWORDS
1,3-oxazine, acyl isothiocyanates, antioxidant activity, carbonic anhydrase inhibition,
molecular docking
other biosynthetic reactions (e.g., gluconeogenesis, lipogenesis, and
ureagenesis; Alterio et al., 2006; Ebbesen et al., 2009; Hilvo et al.,
1
| INTRODUCTION
Carbonic anhydrase zinc metallo-enzyme catalyzes the reversible
hydration of carbon dioxide (CO2) into a proton (H+) and bicarbonate
ðHCO3− ; Mincione, Scozzafava, & Supuran, 2009; Supuran, 2008;
Temperini, Innocenti, Scozzafava, Parkkila, & Supuran, 2009). The bio-
sequestration of CO2 using carbonic anhydrase in situ encapsulated
inside electrospun hollow fibers has been described by Cui et al. (2014). It
has widely been documented that carbonic anhydrases contribute in vital
physiological processes associated with homeostasis, CO2=HCO3− , elec-
trolyte secretion, tumorigenicity, respiration, calcification, and many
2008; Thiry, Dogne, Masereel, & Supuran, 2006). Acetazolamide is a
well-known example of clinically recognized CA inhibitor used for the
treatment of epilepsy, glaucoma, and altitude sickness (Avvaru et al.,
2009). Moreover, it has been revealed that CAIs could be employed
as active agents for the cure of hypoxic tumor and obesity (Boztas
et al., 2014; Sethi et al., 2014; Simone, Fiore, & Supuran, 2008). How-
ever, there is a need to enhance the selectivity and inhibition activity
of CA inhibitors. So, the design and synthesis of safe and effective
CAIs as useful clinical agents is an active area of research in medicinal
Drug Dev Res. 2018;1–10.
wileyonlinelibrary.com/journal/ddr
© 2018 Wiley Periodicals, Inc.
1

2
QAMAR ET AL.
ꢀ
chemistry (Gulçin & Taslimi, 2018; Yigit et al., 2018). Ren et al. have
reported the CO2 sequestration by immobilized carbonic anhydrase on
mesoporous cruciate flower-like metal organic framework (Ren et al., 2018).
Oxazines display useful biological activities and constitute an
important class of natural and nonnatural products, making these
compounds of special interest (Turgut, Pelit, & Köycü, 2007). Most of
them are relevant to the biochemistry of enzymes (Lanni et al., 2007)
and cytotoxic drugs (Ouberai et al., 2006). Not only a potential candi-
date for the synthesis of various types of drug sources (Tomasulo,
Sortino, & Raymo, 2005), 1,3-oxazine ring system is also useful for
thermal ring closing and photo induced ring opening (Sawant,
Mhaske, & Wadekar, 2012). A wide range of biological applications
such as anticoagulant, antitubercular, antitumor, antimicrobial, as non-
steroidal PR agonist and as neuroprotective drugs (Beena & Akelesh,
2013; Bhat & Pawar, 2008; Fensome et al., 2005; Narita et al., 2009;
Sukhorukov et al., 2014) are known to exhibited by 1,3-oxazine con-
taining heterocycles as this 1,3-oxazine motif is found to be unique
and versatile in its bio-active functions (Mayekar et al., 2011).
2.1.1
| Synthesis of acyl isothiocyanates (2a-n), general
procedure
The suitably substituted aromatic acids (2 mmol) in slight excess of
thionyl chloride (1.2 equiv.) were heated under reflux for 2 hr to yield
corresponding acid chloride derivatives. To these freshly prepared
acid chlorides, solution of potassium thiocyanate (2 mmol) in dry ace-
tone was added and then stirred for 1.5 hr at room temperature to
afford acyl isothiocyanates that were further used in situ.
2.1.2
| Synthesis of 1-(6-methyl-2-substituted phenyl-
4-thioxo-4H-1,3-oxazin-5-yl)ethanones (3a-n), general
procedure
To a solution of freshly prepared benzoyl isothiocyanate (2 mmol) in
dry acetone, acetylacetone (2 mmol), and triethyl amine (2 mL) were
added and heated under reflux for 8 hr. The progress of the reaction
was monitored by thin layer chromatography, upon reaction com-
pletion the reaction mixture was poured onto crushed ice. The
precipitated solid was filtered off, washed well with distilled
water, dried, and then recrystallized from n-hexane: ethyl acetate
4:1 to afford pure products (3a-n).
So far, sulfonamides as CA inhibitors have extensively been
reported. We want to report a new class of potent carbonic anhydrase
inhibitors, and to the best of our knowledge 1,3-oxazine scaffold was
not explored as CA inhibitor. By taking into account the biological sig-
nificance of 1,3-oxazine derivatives, this is an exciting structure to work
with for the development of new chemical entities to combat various
diseases. Based on the known facts and possible future applications of
CA-II inhibitors in various diseases, the identification of new com-
pounds that suppress the CA activity is the current objective of several
research groups. In the present study, we reported the expedient syn-
thesis of a library of 1,3-oxazines and evaluated their inhibition profile
against therapeutically relevant carbonic anhydrase II. The antioxidant
activity of the synthesized compounds was also evaluated as some of
the researchers reported the antioxidant potential of the carbonic anhy-
drase inhibitors (Aksu et al., 2016; Bulut et al., 2018; Öztaskın, Taslimi,
Maras¸, Gülcin, & Göksu, 2017; Taslimi et al., 2018). The potent inhibitors
were also assessed for binding to CA through molecular docking studies.
1-(2-[4-Methylphenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3a)
Off white solid; yield: 83%; Rf: 0.58 (n-hexane: ethyl acetate, 1:1); m.
p.: 123–124 ꢁC; IR (pure, cm−1): 2,968, 2,879 Csp3 -H , 1,672 (C O),
À
Á
1,610 (C N), 1,564, 1,455 (Ar
C
C), 1,303 (C S); 1H NMR
(300 MHz, [CD3]2CO): δ (ppm) 7.92–7.86 (m, 2H, Ar H), 7.35–7.31
(m, 2H, Ar H), 2.57 (s, 3H, COCH3), 2.41 (s, 3H, Ar CH3), 2.32 (s, 3H,
CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ (ppm) 191.0 (COCH3), 189.1
(C S), 166.7 (C N), 164.0, 150.3, 144.0, 131.1, 129.7, 128.8, 24.3
(COCH3), 21.1 (Ar CH3), 17.2 (CH3); GC–MS: m/z (%) = 259.07 (100)
[M+]; anal. calcd. for C14H13NO2S: C, 64.84; H, 5.05; N, 5.40; S,
12.36; found: C, 64.73; H, 5.09; N, 5.57; S, 12.22.
1-(2-[4-Methoxyphenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5-yl)
ethanone (3b)
White crystalline solid; yield: 80%; Rf: 0.50 (n-hexane: ethyl acetate,
À
Á
2
| MATERIALS AND METHODS
1:1); m.p.: 110–111ꢁC; IR (pure, cm−1): 2,932, 2,840 Csp3 -H , 1,660
(C O), 1,640 (C N), 1,526, 1,430 (Ar
C
C), 1,305 (C S), 1,237,
1,050 (C O); 1H NMR (300 MHz, [CD3]2CO): δ (ppm) 7.99–7.94 (m,
2H, Ar H), 7.06–7.00 (m, 2H, Ar H), 4.10 (s, 3H, OCH3), 2.46 (s, 3H,
COCH3), 2.31 (s, 3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ (ppm)
190.9 (COCH3), 186.3 (C S), 166.0 (C N), 163.5, 159.2, 155.5,
131.2, 122.5, 113.7, 55.0 (Ar OCH3), 23.6 (COCH3), 19.1 (CH3); GC–
MS: m/z (%) = 275.06 (100) [M+]; anal. calcd. for C14H13NO3S: C,
61.07; H, 4.76; N, 5.09; S, 11.65; found: C, 61.22; H, 4.70; N,
5.12; S, 11.53.
2.1 | Chemistry
All chemicals, reagents, and solvents were of best quality, purchased
from Sigma–Aldrich Chemical Co. and Merck (Darmstadt, Germany),
and were used without further purification. Rf values were determined
using aluminum precoated silica gel plates Kiesel 60 F254 from Merck
(Darmstadt, Germany). Melting points of the compounds were mea-
sured in open capillaries using Stuart melting point apparatus (SMP3)
and are uncorrected. The IR spectra were recorded on FTS 3000 MX,
Bio-Rad Merlin (Excalibur Model) spectrophotometer as pure com-
pounds. 1H NMR and 13C NMR spectra were run on a Bruker
300 MHz and 75.5 MHz NMR spectrometer in [CD3]2CO using TMS
as an internal standard. Mass spectra were recorded on Agilent tech-
nologies 6890N gas chromatograph and an inert mass selective
detector 5973 mass spectrometer and the elemental analyses were
conducted using a LECO-183 CHNS analyzer.
1-(2-[2,6-Dimethoxyphenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-
5yl)ethanone (3c)
Off white solid; yield: 78%; Rf: 0.48 (n-hexane: ethyl acetate, 1:1); m.
À
Á
p.: 115–116 ꢁC; IR (pure, cm−1): 2,935, 2,852 Csp3 -H , 1,645 (C O),
1,595 (C N), 1,522, 1,435 (Ar
C
C), 1,302 (C S), 1,232, 1,045
(C O); 1H NMR (300 MHz, [CD3]2CO): δ (ppm) 7.29–7.23 (m, 1H,

QAMAR ET AL.
3
SCHEME 1 Synthetic route to 1-(6-methyl-2-substituted phenyl-4-thioxo-4H-1,3-oxazin-5-yl)ethanones 3a-n
Ar H), 6.84–6.79 (m, 2H, Ar H), 3.89 (s, 6H, OCH3), 2.29 (s, 3H,
COCH3), 2.22 (s, 3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ (ppm)
194.3 (COCH3), 189.5 (C S), 164.1 (C N), 163.4, 162.7, 152.3,
133.1, 128.4, 115.2, 59.2 (Ar OCH3), 22.3 (COCH3), 18.7 (CH3); GC–
MS: m/z (%) = 305.07 (100) [M+]; anal. calcd. for C15H15NO4S: C,
59.00; H, 4.95; N, 4.59; S, 10.50; found: C, 59.07; H, 4.67; N,
4.83; S, 10.42.
1H NMR (300 MHz, [CD3]2CO): δ (ppm) 7.56 (d, 2H, J = 7.5 Hz,
Ar H), 7.30 (d, 2H, J = 7.5 Hz, Ar H), 2.25 (s, 3H, COCH3), 2.11 (s,
3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ (ppm) 195.0 (COCH3),
188.3 (C S), 164.2 (C N), 163.7, 162.4, 136.6, 130.6, 129.0, 128.5,
23.1 (COCH3), 18.0 (CH3); GC–MS: m/z (%) = 279.01 (100) [M+]; anal.
calcd. for C13H10ClNO2S: C, 55.82; H, 3.60; N, 5.01; S, 11.46;
found: C, 55.88; H, 3.45; N, 5.09; S, 11.62.
1-(2-[2-Chlorophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5-yl)
ethanone (3d)
1-(2-[2,4-Dichlorophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-
5yl)ethanone (3g)
Off white powder; yield: 85%; Rf: 0.55 (n-hexane: ethyl acetate, 1:1);
Brown sticky oil; yield: 55%; Rf: 0.49 (n-hexane: ethyl acetate, 1:1); IR
À
Á
À
Á
m.p.: 184–185 ꢁC; IR (pure, cm−1): 2,931, 2,849 Csp3 -H , 1,659
(pure, cm−1): 2,948, 2,861 Csp3 -H , 1,658 (C O), 1,599 (C N),
(C O), 1,593 (C N), 1,530, 1,455 (Ar
C
C), 1,309 (C S), 778 (C Cl);
1,554, 1,447 (Ar
C
C), 1,295 (C S), 780 (C Cl); 1H NMR (300 MHz,
1H NMR (300 MHz, [CD3]2CO): δ (ppm) 7.70 (d, 1H, J = 6.9 Hz,
Ar H), 7.66 (d, 1H, J = 7.3 Hz, Ar H), 7.59–7.53 (m, 2H, Ar H), 2.33
(s, 3H, COCH3), 2.21 (s, 3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ
(ppm) 193.5 (COCH3), 190.5 (C S), 163.5 (C N), 162.7, 162.5, 154.7,
134.0, 132.5, 130.6, 129.0, 127.3, 24.1 (COCH3), 17.5 (CH3); GC–MS:
m/z (%) = 279.01 (100) [M+]; anal. calcd. for C13H10ClNO2S: C,
55.82; H, 3.60; N, 5.01; S, 11.46; found: C, 55.88; H, 3.45; N,
5.09; S, 11.62.
[CD3]2CO): δ (ppm) 7.55–7.48 (m, 2H, Ar H), 7.39 (s, 1H, Ar H), 2.40
(s, 3H, COCH3), 2.23 (s, 3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ
(ppm) 194.3 (COCH3), 189.5 (C S), 165.4 (C N), 163.0, 163.2, 138.5,
135.4, 132.0, 130.5, 129.7, 127.1, 24.3 (COCH3), 19.5 (CH3); GC–MS:
m/z (%) = 312.97 (100) [M+]; anal. calcd. for C13H9Cl2NO2S: C,
49.70; H, 2.89; N, 4.46; S, 10.21; found: C, 49.67; H, 2.94; N,
4.61; S, 10.18.
1-(2-[2-Bromophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3h)
1-(2-[3-Chlorophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5-yl)
ethanone (3e)
Light brown powder; yield: 74%; Rf: 0.50 (n-hexane: ethyl acetate,
À
Á
1:1); m.p.: 142–143 ꢁC; IR (pure, cm−1): 2,955, 2,869 Csp3 -H , 1,669
Off white powder; yield: 83%; Rf: 0.53 (n-hexane: ethyl acetate, 1:1);
À
Á
m.p.: 180–181 ꢁC; IR (pure, cm−1): 2,938, 2,845 Csp3 -H , 1,655
(C O), 1,609 (C N), 1,557, 1,452 (Ar
C
C), 1,280 (C S), 662 (C Br);
(C O), 1,597 (C N), 1,532, 1,454 (Ar
C
C), 1,299 (C S), 775 (C Cl);
1H NMR (300 MHz, [CD3]2CO): δ (ppm) 7.72 (d, 1H, J = 6.3 Hz,
Ar H), 7.58–7.55 (m, 1H, Ar H), 7.46–7.41 (m, 2H, Ar H), 2.29 (s,
3H, COCH3), 2.23 (s, 3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ
(ppm) 193.5 (COCH3), 190.2 (C S), 165.6 (C N), 164.3, 163.0, 155.3,
134.5, 133.3, 132.8, 131.5, 127.9, 22.4 (COCH3), 17.4 (CH3); GC–MS:
m/z (%) = 324.96 (100) [M+]; anal. calcd. for C13H10 BrNO2S: C,
48.16; H, 3.11; N, 4.32; S, 9.89; found: C, 48.35; H, 3.22; N,
4.39; S, 9.82.
1H NMR (300 MHz, [CD3]2CO): δ (ppm) 7.91–7.87 (m, 1H, Ar H),
7.82–7.79 (m, 1H, Ar H), 7.55–7.51 (m, 1H, Ar H), 7.46–7.40 (m,
1H, Ar H), 2.25 (s, 3H, COCH3), 2.12 (s, 3H, CH3); 13C NMR
(75.5 MHz, [CD3]2CO): δ (ppm) 194.2 (COCH3), 187.3 (C S), 166.5
(C N), 163.3, 163.7, 135.2, 134.4, 132.4, 131.2, 130.3, 129.5, 23.6
(COCH3), 19.3 (CH3); GC–MS: m/z (%) = 279.01 (100) [M+]; anal.
calcd. for C13H10ClNO2S: C, 55.82; H, 3.60; N, 5.01; S, 11.46;
found: C, 55.88; H, 3.45; N, 5.09; S, 11.62.
1-(2-[2-Flourophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3i)
1-(2-[4-Chlorophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5-yl)
ethanone (3f )
Brown sticky; yield: 53%; Rf: 0.53 (n-hexane: ethyl acetate, 1:1); IR
À
Á
(pure, cm−1): 2,962, 2,874 Csp3 -H , 1,661 (C O), 1,622 (C N),
Off white powder; yield: 84%; Rf: 0.52 (n-hexane: ethyl acetate, 1:1);
À
Á
m.p.: 186–187 ꢁC; IR (pure, cm−1): 2,945, 2,852 Csp3 -H , 1,650
1,560, 1,455 (Ar
C
C), 1,264 (C S); 1H NMR (300 MHz, [CD3]2CO):
(C O), 1,604 (C N), 1,548, 1,445 (Ar
C
C), 1,289 (C S), 772 (C Cl);
δ (ppm) 7.81 (d, 1H, J = 8.8 Hz, Ar H), 7.50–7.46 (m, 1H, Ar H), 7.38

4
QAMAR ET AL.
TABLE 1 Carbonic anhydrase II inhibitory activities (IC50 in μM) of
synthesized 1,3-oxazine derivatives (3a-n) and standard
acetazolmamide
Ar H), 8.21–8.03 (m, 3H, Ar H), 2.31 (s, 3H, COCH3), 2.20 (s, 3H,
CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ (ppm) 195.0 (COCH3), 190.2
(C S), 166.2 (C N), 165.0, 162.5, 140.5, 135.7, 132.4, 129.8, 126.3,
124.3, 22.7 (COCH3), 17.3 (CH3); GC–MS: m/z (%) = 290.04 (100)
[M+]; anal. calcd. for C13H10N2O4S: C, 53.79; H, 3.47; N, 9.65; S,
11.05; found: C, 53.90; H, 3.51; N, 9.33; S, 11.22.
R
O
CH3
N
COCH3
S
3a-n
1-(2-[4-Nitrophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3l)
Compound
R
IC50 ꢀ SEM (μM)
1.679 ꢀ 0.099
0.144 ꢀ 0.008
0.328 ꢀ 0.018
2.196 ꢀ 0.125
16.161 ꢀ 0.592
8.235 ꢀ 0.386
1.349 ꢀ 0.067
0.301 ꢀ 0.012
3.453 ꢀ 0.214
27.113 ꢀ 1.612
32.474 ꢀ 2.134
2.973 ꢀ 0.175
17.210 ꢀ 1.125
0.317 ꢀ 0.014
0.997 ꢀ 0.061
Off white powder; yield: 81%; Rf: 0.43 (n-hexane: Ethyl acetate, 1:1);
3a
4-CH3
À
Á
m.p.: 164–165 ꢁC; IR (pure, cm−1): 2,953, 2,862 Csp3 -H , 1,665
3b
4-OCH3
2,6-OCH3
2-Cl
(C O), 1,594 (C N), 1,556, 1,444 (Ar
C
C), 1,542, 1,348 (N O),
3c
1,310 (C S); 1H NMR (300 MHz, [CD3]2CO): δ (ppm) 8.40–8.37 (m,
2H, Ar H), 8.32–8.25 (m, 2H, Ar H), 2.56 (s, 3H, COCH3), 2.40 (s,
3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ (ppm) 193.5 (COCH3),
186.6 (C S), 165.1 (C N), 164.8, 150.6, 148.9, 136.0, 135.5, 130.9,
123.6, 24.3 (COCH3), 18.9 (CH3); GC–MS: m/z (%) = 290.04 (100)
[M+]; anal. calcd. for C13H10N2O4S: C, 53.79; H, 3.47; N, 9.65; S,
11.05; found: C, 53.90; H, 3.51; N, 9.33; S, 11.22.
3d
3e
3-Cl
3f
4-Cl
3g
2,4-Cl
2-Br
3h
3i
2-F
3j
4-F
3k
3-NO2
4-NO2
3,5-NO2
1-Naph
3l
1-(2-[3,5-dinitrophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3m)
3m
3n
Acetazolamide
Light yellow solid; yield: 87%; Rf: 0.40 (n-hexane: ethyl acetate, 1:1);
À
Á
m.p.: 178–179 ꢁC; IR (pure, cm−1): 2,957, 2,865 Csp3 -H , 1,680
(d, 1H, J = 7.3 Hz, Ar H), 7.29–7.24 (m, 1H, Ar H), 2.30 (s, 3H,
COCH3), 2.19 (s, 3H, CH3); 13C NMR (75.5 MHz, [CD3]2CO): δ (ppm)
195.2 (COCH3), 186.4 (C S), 166.2 (C N), 163.7, 161.8, 159.7,
132.7, 130.9, 124.5, 118.5, 115.6, 24.2 (COCH3), 19.2 (CH3); GC–MS:
m/z (%) = 263.04 (100) [M+]; anal. calcd. for C13H10FNO2S: C,
59.30; H, 3.83; N, 5.32; S, 12.18; found: C, 59.34; H, 3.79; N,
5.37; S, 12.23.
(C O), 1,601 (C N), 1,553, 1,448 (Ar
C
C), 1,545, 1,336 (N O),
1,290 (C S); 1H NMR (300 MHz, CD3OD: δ (ppm) 9.21–9.18 (m, 1H,
Ar H), 9.11 (d, 2H, J = 3.0 Hz, Ar H), 2.18 (s, 3H, COCH3), 2.05 (s,
3H, CH3); 13C NMR (75.5 MHz, CD3OD: δ (ppm) 194.3 (COCH3),
189.6 (C S), 166.4 (C N), 164.5, 163.2, 148.7, 145.4, 133.3, 128.7,
121.9, 25.0 (COCH3), 19.8 (CH3); GC–MS: m/z (%) = 335.02 (100)
[M+]; anal. calcd. for C13H9N3O6S: C, 46.57; H, 2.71; N, 12.53; S,
9.56; found: C, 46.69; H, 2.53; N, 12.71; S, 9.80.
1-(2-[4-Flourophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3j)
TABLE 2 Antioxidant activities (radical scavenging %) of synthesized
1,3-oxazine derivatives (3a-n) and standard ascorbic acid
Light yellow crystalline; yield: 82%; Rf: 0.55 (n-hexane: ethyl acetate,
À
Á
Radical scavenging %
(100 μg/mL)
1:1); m.p.: 162–163 ꢁC; IR (pure, cm−1): 2,965, 2,876 Csp3 -H , 1,663
Compound
R
(C O), 1,590 (C N), 1,567, 1,458 (Ar
C
C), 1,264 (C S); 1H NMR
3a
4-CH3
4-OCH3
2,6-OCH3
2-Cl
2.942 ꢀ 0.107
28.030 ꢀ 0.691
1.421 ꢀ 0.0472
8.261 ꢀ 0.285
27.380 ꢀ 0.947
21.194 ꢀ 0.733
28.928 ꢀ 1.56
0.571 ꢀ 0.097
36.560 ꢀ 2.265
12.177 ꢀ 0.621
1.738 ꢀ 0.040
0.981 ꢀ0.039
21.635 ꢀ0.986
1.421 ꢀ 0.071
96.464 ꢀ 2.164
(300 MHz, [CD3]2CO): δ (ppm) 8.08–8.03 (m, 2H, Ar H), 7.33–7.27
(m, 2H, Ar H), 2.72 (s, 3H, COCH3), 2.34 (s, 3H, CH3); 13C NMR
(75.5 MHz, [CD3]2CO): δ (ppm) 190.4 (COCH3), 185.2 (C S), 167.0
(C N), 163.7, 162.8, 153.4, 131.1, 129.9, 115.6, 31.6 (COCH3), 21.4
(CH3); GC–MS: m/z (%) = 263.04 (100) [M+]; anal. calcd. for
3b
3c
3d
3e
3-Cl
3f
4-Cl
C
13H10FNO2S: C, 59.30; H, 3.83; N, 5.32; S, 12.18; found: C,
3g
2,4-Cl
2-Br
59.34; H, 3.79; N, 5.37; S, 12.23.
3h
3i
2-F
1-(2-[3-Nitrophenyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3k)
3j
4-F
3k
3-NO2
4-NO2
3,5-NO2
1-Naph
3l
Light yellow solid; yield: 79%; Rf: 0.45 (n-hexane: ethyl acetate, 1:1);
À
Á
m.p.: 192–193 ꢁC; IR (pure, cm−1): 2,950, 2,865 Csp3 -H , 1,662
3m
3n
(C O), 1,591 (C N), 1,550, 1,442 (Ar
C
C), 1,547, 1,342 (N O),
Vitamin C
1,292 (C S); 1H NMR (300 MHz, [CD3]2CO): δ (ppm) 8.60 (s, 1H,

QAMAR ET AL.
5
FIGURE 1 Crystal structure of bovine anhydrase II along with hydrophobicity and Ramachandran graphs which shows most of protein residues
(93.8%) lies in favored region [Color figure can be viewed at wieyonlinelibrary.com]
2011). The method is based on the principle that p-nitrophenyl ace-
tate is hydrolyzed by carbonic anhydrase to form yellow colored p-
nitrophenol which was measured spectrophotometrically. Briefly,
reaction mixture contained 120 μL of 50 mM tris–sulfate buffer
(pH 7.6 containing 0.1 mM ZnCl2), 20 μL of inhibitor and 20 μL (50 U)
bovine enzyme per well. Contents were well mixed and preincubated
at 25 ꢁC for 10 min. Substrate p-nitrophenyl acetate was prepared
(6 mM stock using <5% acetonitrile in buffer and used fresh every
time) and 40 μL was added per well to achieve 0.6 mM concentration
per well. Total reaction volume was made to 200 μL. After 30 min
incubation at 25 ꢁC, contents were mixed and absorbance was mea-
sured at 348 nm using a microplate reader. Acetazolamide was used
as a reference inhibitor and tris–sulfate buffer was used as negative
control. Each concentration was analyzed in three independent exper-
iments. The IC50 values were determined by the data analysis and
graphing software Origin 8.6, 64-bit. The percent of inhibition of car-
bonic anhydrase was calculated as following.
1-(2-[1-Naphthoyl]-6-methyl-4-thioxo-4H-1,3-oxazin-5yl)
ethanone (3n)
Dark brown sticky; yield: 49%; Rf: 0.44 (n-hexane: ethyl acetate, 1:1);
À
Á
IR (pure, cm−1): 2,962, 2,874 Csp3 -H , 1,752 (C O), 1,602 (C N),
1,559, 1,463 (Ar
C
C), 1,310 (C S); 1H NMR (300 MHz, [CD3]2CO):
δ
(ppm) 8.39–8.35 (m, 1H, Ar H), 8.33–8.08 (m, 2H, Ar H),
7.79–7.60 (m, 1H, Ar H), 7.93–7.55 (m, 3H, Ar H), 2.27 (s, 3H,
COCH3), 2.20 (s, 3H, CH3); 13C NMR (75.5 MHz, (CD3)2CO): δ (ppm)
196.2 (COCH3), 181.3 (C S), 163.7, 159.4 (C N), 142.4, 134.2, 133.8,
130.7, 128.3, 127.5, 126.7, 125.9, 29.1 (COCH3), 20.2 (CH3); GC–MS:
m/z (%) = 295.07 (100) [M+]; anal. calcd. for C17H13NO2S: C, 69.13; H,
4.44; N, 4.74; S, 10.86; found: C, 69.20; H, 4.61; N, 4.55; S, 10.79.
2.2 | Carbonic anhydrase assay
Carbonic anhydrase II inhibition was measured as described previously
with some modifications (Al-Rashida, Ashraf, Hussain, Nagra, & Abbas,
TABLE 3 Chemo-informatics properties of synthesized 1,3-oxazine derivatives (3a-n)
Mol. LogP
PSA
Mol. vol
(cm3)
Ligands
Mol. wt (g/mol)
259.07
275.06
305.07
279.01
279.01
279.01
312.97
322.96
263.04
263.04
290.04
290.04
335.02
295.07
No. HBA
No. HBD
(mg/L)
(A2)
Drug score
−1.39
−1.43
−1.49
−0.99
−1.33
−1.06
−0.36
−1.35
−1.23
−1.28
−1.12
−1.14
−1.05
−1.43
3a
4
5
6
4
4
4
4
4
4
4
6
6
8
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2.07
1.76
1.61
2.26
2.38
2.38
2.98
2.40
1.82
1.94
1.39
1.39
1.12
3.00
31.01
38.56
46.28
31.01
31.01
31.01
31.01
31.01
31.01
31.01
69.28
69.28
107.54
30.74
284.25
295.15
327.80
278.63
280.58
280.50
295.90
284.32
268.19
269.22
289.01
288.94
314.80
312.78
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
Abbrevations (HBA = no. of hydrogen bond acceptor; HBD = no. of hydrogen bond donor; LogP = lippophilicity of partition coefficient; PSA = polar sur-
face area).

6
QAMAR ET AL.
TABLE 4 Docking results of synthesized 1,3-oxazine derivatives (3a-n) using glide showing binding affinity with target protein PDBID 1V9E
Docking complexes
Docking score
−4.22
−4.22
−3.20
−3.47
−3.61
−3.73
−3.54
−2.99
−3.55
−4.49
−4.03
−4.57
−3.69
−3.74
Glide ligand efficacy
−0.235
Glide score
−4.449
−4.449
−3.414
−3.591
−3.804
−3.871
−3.662
−3.152
−3.764
−4.51
Glide energy
−26.45
−26.45
−22.33
−21.61
−25.08
−23.24
−19.87
−18.50
−17.89
−31.89
−25.99
−31.86
−27.96
−23.95
3a
3b
3c
3d
3e
3f
−0.235
−0.153
−0.193
−0.201
−0.207
3g
3h
3i
−0.187
−0.166
−0.198
3j
−0.225
3k
3l
−0.202
−4.055
−4.589
−3.719
−3.956
−0.229
3m
3n
−0.161
−0.374
rates were compared and the percent inhibition because of the
presence of tested inhibitors was calculated. Each concentration
was analyzed in three independent experiments.
Inhibitionð%Þ ¼ ½ðB−SÞ=Bꢂ × 100:
ð1Þ
Here, the B and S are the absorbances for the blank and samples.
2.3 | Free radical scavenging assay
2.4 | Computational methodology
Radical scavenging activity was determined by modifying method
by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Blois, 1958; Ashraf
et al., 2015). The assay solution consisted of 100 μL of (150 μ)
2,2-diphenyl-1 picrylhydrazyl (DPPH), 20 μL of increasing concen-
tration of test compounds and the volume was adjusted to 200 μL
in each. This reaction mixture was then incubated for 30 min at
room temperature. Ascorbic acid (vitamin C) was used as a refer-
ence inhibitor. The measurements were carried out by using a
micro plate reader (OPTIMax, tunable) at 517 nm. The reaction
2.4.1
| Assessment of protein structure from PDB
The three dimensional (3D) crystal structure of carbonic anhydrase II
was retrieved form the Protein Data Bank (PDB) having PDBID
1V9E (www.rcsb.org). Energy minimization of target structure was
carried out by using conjugate gradient algorithm and Amber force
field in UCSF Chimera 1.10.1 (Pettersen et al., 2004). The stereo-
chemical properties, Ramachandran graph and values (Lovell et al.,
2003) of carbonic anhydrase II structure were assessed by
FIGURE 2 Graph depiction between glide energy values and glide score showing ligands efficacy for the target protein PDBID 1V9E [Color
figure can be viewed at wieyonlinelibrary.com]

QAMAR ET AL.
7
FIGURE 3 Docking interactions of compound 3b with receptor protein showing carbonyl oxygen of ligand 3b interacts with Lys168 of target
protein, the 2D binding interactions also depicted [Color figure can be viewed at wieyonlinelibrary.com]
Molprobity server (Chen et al., 2010), while the hydrophobicity
3
|
RESULTS AND DISCUSSION
graph was generated by Discovery Studio 4.1 Client (Studio, 2008).
The protein architecture and statistical percentage values of helices,
beta-sheets, coils and turns were accessed by using online tool
VADAR 1.8 (Willard et al., 2003).
3.1 | Chemistry
The reaction sequence employed for the synthesis of title compounds
is shown in Scheme 1. The interaction of equimolar quantities of reac-
tive key substrate benzoyl isothiocyanate (2a-n) and methylene active
compound acetylacetone in the presence of base triethyl amine gave
target products (3a-n) in good yields. Acyl isothiocyanates (2a-n) were
prepared by heating various substituted aromatic acids in slight excess
of thionyl chloride. Acyl halides as prepared above gave the corre-
sponding acyl isothiocyanates directly on reaction with metal thiocya-
nate. The commercially available acetylacetone was employed as
carbon nucleophile to react with corresponding aroyl isothiocyanate
at its C N to afford an adduct that underwent subsequent cyclization
to yield a variety of desired 1,3-oxazine derivatives in a one-pot
process.
1H NMR and 13C NMR data of 1,3-oxazines (3a-n) demonstrated
in the experimental section is in close agreement with their antici-
pated structures. 1H NMR revealed the presence of two methyl pro-
ton singlets, CH3 next to the ketone carbonyl appeared at 2.29 ppm
while the other CH3 resonated at a slightly shielded value of
2.22 ppm. 13C NMR displayed three distinct peaks for carbonyl, thio-
carbonyl and C N around 193.2, 190.7 and 165.4 ppm, respectively
while the peaks corresponding to two methyl carbons were observed
in aliphatic region at δ 23.5 and 19.2. FT-IR spectra exhibited charac-
teristic asymmetric and symmetric stretching absorptions for sp3C-H
at 2,968, 2,879 cm−1. The intense absorption bands for C N at
1,610 cm−1 and for C O at 1,663 cm−1, in addition to the stretching
frequencies of C C at 1,680 cm−1 and C S around 1,303 cm−1 all
correspond to oxazine ring structure.
2.4.2
| Ligands preparation
The synthesized compounds (3a-n) were sketched in drawing ACD/-
ChemSketch tool (http://www.acdlabs.com/products/draw_nom/
draw/chemsketch/). The designed ligands were further visualized and
minimized by UCSF Chimera 1.10.1. The Molinspiration (http://www.
molinspiration.com/) and Molsoft (http://www.molsoft.com/) online
computational tools were used to predict the drug-likeness and basic
biological properties of these synthesized compounds. Moreover,
Lipinski’s rule of five was analyzed using Molsoft and Molinspiraion
tools. The number of rotatable bonds, H-bond acceptors (HBA), and
H-bond donors (HBD) were also confirmed by PubChem (https://
pubchem.ncbi.nlm.nih.gov/).
2.4.3
| Grid generation and molecular docking
Prior to molecular docking, the optimized carbonic anhydrase struc-
ture was prepared using the “Protein Preparation Wizard” workflow
in Schrödinger Suite. Bond orders were assigned and hydrogen
atoms were added to the protein. The structure was then mini-
mized to reach the converged root mean square deviation (RMSD)
of 0.30 Å with the OPLS_2005 force field. The active site of the
enzyme is defined from the co-crystallized ligands from PDB and
literature data (Fattah et al., 2018). Furthermore, docking experi-
ment was performed against all synthesized ligands and target pro-
tein by using glide docking protocol (Friesner et al., 2006). The
predicted binding energies (docking scores) and conformational
positions of ligands within active region of protein were also per-
formed using glide experiment. Throughout the docking simula-
tions, both partial flexibility and full flexibility around the active site
residues are performed by Glide/SP/XP and induced fit docking
(IFD) approaches (Farid, Day, Friesner, & Pearlstein, 2006).
3.2 | Carbonic anhydrase inhibition
The heterocyclic compounds have been synthesized as carbonic anhy-
drase inhibitors as a different heterocyclic core have been extensively
used in the design of more potent carbonic anhydrase inhibitors

8
QAMAR ET AL.
coils, respectively. The overall the reliability and efficacy of CA-II was
confirmed by Ramachandran graph and values. It has been observed
that 93.8% of all residues were present in favored regions and only six
poor rotamers lies in unfavored regions (Figure 1).
3.5 | Chemo-informatic properties and Lipinski rule
(RO5) evaluation of ligands
The synthesized oxazine derivatives were investigated computationally
to predict their chemical and bio-molecular properties. The basic
prophesied chemoinformatic properties such as molecular weight
(MW, g/mol), LogP, HBD, HBA, molar volume, polar surface area (PSA),
and drug likeness values of all synthesized chemical scaffolds are tabu-
lated in Table 3. Prior research displayed the standard values range for
MW and PSA are (160–480 g/mol) and (<89 Å2), respectively (Ghose,
Herbertz, Hudkins, Dorsey, & Mallamo, 2012; Kadam & Roy, 2007).
The predicted results of synthesized ligands showed that compounds
3a-n possessed good MW and PSA values which were comparable
with standard values. The RO5 analysis also confirmed the therapeutic
potential of all the synthesized ligands and all compounds obeyed the
RO5 rule. The hydrogen bonding has been recognized as significant
parameter for drug permeability. It has been observed that exceeded
numbers of HBA (>10) and HBD (>5) results in poor permeation
(Bakht, Yar, Abdel-Hamid, Al Qasoumi, & Samad, 2010). Our predicted
results showed that all the synthesized ligands possessed <10 HBA and
<5 HBD, respectively and their LogP values were also comparable with
standard value (5). However, multiple examples are available for RO5
violation among the existing drugs (Tian et al., 2015).
FIGURE 4 Docking interactions of acetazolamide with receptor
protein showing sulfonamide oxygen interacts with Asn10 having
binding distance 2.44 Å (binding affinity is 6.00 kcal/mol) [Color
figure can be viewed at wieyonlinelibrary.com]
(Krasavin et al., 2018). All synthesized compounds (3a-n) were tested
for their ability to act as carbonic anhydrase II inhibitors. The inhibi-
tory activities of these compounds were examined using standard clin-
ically employed inhibitor acetazolamide. As listed in Table 1, the
studied compounds showed different enzyme inhibition profiles.
Compound 3b having 4-methoxy phenyl moiety was the best inhibitor
with IC50 value 0.144 ꢀ 0.008 μM. Among the series, compounds 3c,
3h, and 3n also proved to be the potent inhibitors of carbonic anhy-
drase. Halogen on phenyl ring sharply decreased the inhibitory activity
of derivatives 3e, 3f, and 3j (16.161, 8.235, 27.113 μM, respectively).
While, the analogues with strong electron withdrawing nitro groups
that is, 3k (32.474 ꢀ 2.134 μM) and 3m (17.210 ꢀ 1.125 μM) also
displayed a considerable loss in inhibitory potency.
3.6 | Docking energy and binding analysis of
synthesized compounds
The docked complexes of all oxazines (3a-n) against carbonic anhy-
drase II were analyzed separately and evaluated on the basis of
docking score and ligand interactions pattern. Results disclosed that
all analogues (3a-n) showed good docking scores in the active region
of target protein. Docking analysis also justified their good glide
ligand efficacy, glide score, and glide energy values as mentioned in
Table 4. Although, the basic nucleus of all the synthesized com-
pounds was similar, therefore most of ligands possess good efficient
energy values and no big energy difference was observed in all dock-
ing complexes. The glide score and ligand efficacy correlation is men-
tioned in Figure 2.
3.3 | Free radical scavenging
The antioxidant potential of all synthesized compounds (3a-n) has
been determined using vitamin C as reference to compare the antioxi-
dant activity of the title compounds. Table 2 presented the values
obtained from DPPH assay and it was found that the synthesized
compounds exhibited low to moderate antioxidant activity. The com-
pound 3b exhibited 28% radical scavenging activity while 3i which
possess 2-fouloro substitution at phenyl ring showed 36% radical
scavenging potential. None of the synthesized compound showed
better antioxidant activity than standard ascorbic acid. It is concluded
that the synthesized compounds showed good enzyme inhibitory
activity compared to antioxidant activity.
The ligands–protein binding analyses showed that 3b is confined
in the active binding pocket of target protein as mentioned in
Figure 3. The carbonyl group in 3b interacts with Lys168 and form
hydrogen bonding interactions. The other residues which are present
around 3b are Ser1, His2, His3, Trp4, Gly5, His9, Asn10, Gly63,
His63, Phe229, and Leu237. Recent literature data also favor our
docking results (Fattah et al., 2018). The 3c-receptor docked complex
revealed the good conformational state with good interaction pattern
within the receptor binding pocket. The docking result of 3c-receptor
docked complex showed that π–π and hydrophobic bonds were
observed at His3 and Lys17 residues, respectively. The residues which
are partially involved in surrounding area of ligand are His9, Asn10,
3.4 | Structural assessment of carbonic anhydrase II
Carbonic anhydrase II (CA-II) is a zinc containing protein which con-
tains 259 amino acids (Behçet et al., 2018). The structural architecture
CA-II showed that it consists of 9% helices, 45% β-sheets, and 45%

QAMAR ET AL.
9
Al-Rashida, M., Ashraf, M., Hussain, B., Nagra, S. A., & Abbas, G. (2011).
Discovery of new chromone containing sulfonamides as potent inhibi-
tors of bovine cytosolic carbonic anhydrase. Bioorganic & Medicinal
Chemistry, 19, 3367–3371.
Alterio, V., Vitale, R. M., Monti, S. M., Pedone, C., Scozzafava, A.,
Cecchi, A., … Supuran, C. T. (2006). Carbonic anhydrase inhibitors:
X-ray and molecular modeling study for the interaction of a fluorescent
antitumor sulfonamide with isozyme II and IX. Journal of American
Chemical Society, 128(25), 8329–8335.
Ashraf, Z., Rafiq, M., Seo, S. -Y., Babar, M. M., & Zaidi, N. -U. -S. S. (2015).
Design, synthesis and bioevaluation of novel umbelliferone analogues
as potential mushroom tyrosinase inhibitors. Journal of Enzyme Inhibi-
tion and Medicinal Chemistry, 30, 874–883.
Avvaru, B. S., Busby, S. A., Chalmers, M. J., Griffin, P. R.,
Venkatakrishnan, B., Agbandje-McKenna, M., … McKenna, R. (2009).
Apo-human carbonic anhydrase II revisited: Implications of the loss of
a metal in protein structure, stability, and solvent network. Biochemis-
try, 48(31), 7365–7372.
His14, and Asp18. The ligand benzene shows good contact with His3
and form π–π stacking interaction (Supporting Information Figure S2).
The 3D and 2D conformations of all docking complexes are men-
tioned in supplementary data (Supporting Information Figures S1–13).
Acetazolamide docking was performed to check the accuracy of
synthetic compounds docking results. Figure 4 showed that Acetazol-
amide binds against target protein having similar pattern and common
residues were seen which binds with drug molecule. A good docking
energy value was observed in acetazolamide-carbonic anhydrase
docking complex. Some common residues such as His3, Trp4, Gly5,
Asn10, Gly62, His63, Lys168, Phe229, and Glu234 were seen around
the drug structure. Acetazolamide forms an active hydrogen bond
against Asn10 at a distance 2.44 Å. Comparative analyses showed
that our designed ligands structure and acetazolamide binding pattern
are common in interactions which strengthen our docking results.
Bakht, M. A., Yar, M. S., Abdel-Hamid, S. G., Al Qasoumi, S. I., & Samad, A.
(2010). Molecular properties prediction, synthesis and antimicrobial
activity of some newer oxadiazole derivatives. European Journal of
Medicinal Chemistry, 45(12), 5862–5869.
4
| CONCLUSIONS
Beena, K., & Akelesh, T. (2013). Design, synthesis, characterization and
evaluation of some 1,3-oxazine derivatives as potent antimicrobial
agents. Scholars Research Library, 5(4), 257–260.
Herein, we reported
a simple and straightforward synthesis of
Behçet, A., Celepci, D. B., Aktas¸, A., Taslimi, P., Gök, Y., Aygün, M., &
Gülçin, İ. (2018). Synthesis, characterization and crystal structure of
2-(4-hydroxy-phenyl)-ethyl and 2-(4-nitro-phenyl)-ethyl substituted
benzimidazole salts: Their inhibitory properties against carbonic anhy-
drase and acetylcholinesterase. Journal of Molecular Structure, 1170,
160–169.
Bhat, A., & Pawar, P. (2008). Synthesis and biological evaluation of some
[1,4]thiazin-2-one and [1,4]oxazin-2-one derivatives. Indian Drugs,
45(12), 962–965.
1-(6-methyl-2-substituted phenyl-4-thioxo-4H-1,3-oxazin-5-yl)etha-
nones (3a-n) by employing the chemistry of aroyl isothiocyanates.
These new compounds possess CA-II inhibition activity and prelimi-
nary SAR studies revealed that substituents on phenyl ring play an
essential role towards inhibition profile. Docking studies were carried
out to predict the binding affinity of synthesized compounds with tar-
get protein which assured that most of the synthesized compounds
bind at active binding site. It was further analyzed that 3b-receptor
docked complex revealed good interactions with the receptor binding
pocket and showed hydrogen bonding interaction with Lys168 resi-
dues. Pharmacological investigations confirmed that all ligands pos-
sess therapeutic potential so it can be inferred that these new
1,3-oxazine analogues could be further manipulated in drug discovery.
Based upon our results it is proposed that compound 3b may serve as
a lead structure to design more potent carbonic anhydrase inhibitors.
Blois, M. S. (1958). Antioxidant determinations by the use of a stable free
radical. Nature, 181(4617), 1199–1200.
Boztas, M., Cetinkaya, Y., Topal, M., Gülçin, I. l., Menzek, A., Sahin, E., …
Supuran, C. T. (2014). Synthesis and carbonic anhydrase isoenzymes I,
II, IX, and XII inhibitory effects of dimethoxybromophenol derivatives
incorporating cyclopropane moieties. Journal of Medicinal Chemistry,
58(2), 640–650.
Bulut, N., Kocyigit, U. M., Gecibesler, I. H., Dastan, T., Karci, H.,
Taslimi, P., & Cetin, A. (2018). Synthesis of some novel pyridine com-
pounds containing bis-1,2,4-triazole/thiosemicarbazide moiety and
investigation of their antioxidant properties, carbonic anhydrase, and
acetylcholinesterase enzymes inhibition profiles. Journal of Biochemical
and Molecular Toxicology, 32(1), e22006.
Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M.,
Kapral, G. J., … Richardson, D. C. (2010). MolProbity: All-atom struc-
ture validation for macromolecular crystallography. Acta Crystallogra-
phica Section D: Biological Crystallography, 66(1), 12–21.
Cui, J., Li, Y., Ji, X., Bian, H., Zhang, Y., Su, Z., … Zhang, S. (2014).
Bio-sequestration of CO2 using carbonic anhydrase in situ encapsu-
lated inside electrospun hollow fibers. Chemical Journal of Chinese Uni-
versities, 35, 1999–2006.
ACKNOWLEDGMENTS
We are thankful to Department of Chemistry, Quaid-i-Azam Univer-
sity, Islamabad, Pakistan for providing encouraging environment and
facilities for research work.
Ebbesen, P., Pettersen, E. O., Gorr, T. A., Jobst, G., Williams, K.,
Kieninger, J., … Lambin, P. (2009). Taking advantage of tumor cell adap-
tations to hypoxia for developing new tumor markers and treatment
strategies. Journal of Enzyme Inhibition and Medicinal Chemistry, 24-
(Suppl 1), 1–39.
Farid, R., Day, T., Friesner, R. A., & Pearlstein, R. A. (2006). New insights
about HERG blockade obtained from protein modeling, potential
energy mapping, and docking studies. Bioorganic & Medicinal Chemistry,
14(9), 3160–3173.
CONFLICT OF INTEREST
Authors declare no conflict of interest.
ORCID
Aamer Saeed
Zaman Ashraf
http://orcid.org/0000-0002-7112-9296
http://orcid.org/0000-0002-6620-9080
Fattah, T. A., Saeed, A., Channar, P. A., Larik, F. A., Hassan, M., Raza, H., …
Seo, S. -Y. (2018). Synthesis and molecular docking studies of (E)-4--
(substituted-benzylideneamino)-2H-chromen-2-one derivatives: Entry
to new carbonic anhydrase class of inhibitors. Drug Research, 68,
378–386. https://doi.org/10.1055/s-0043-123998
REFERENCES
Aksu, K., Özgeris¸, B., Taslimi, P., Naderi, A., Gülçin, İ., & Göksu, S. (2016).
Antioxidant activity, acetylcholinesterase, and carbonic anhydrase
inhibitory properties of novel ureas derived from phenethylamines.
Archiv der Pharmazie, 349(12), 944–954.
Fensome, A., Bender, R., Chopra, R., Cohen, J., Collins, M. A., Hudak, V., …
Svenson, K. (2005). Synthesis and structure-activity relationship of

10
QAMAR ET AL.
novel 6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2-thiones as progester-
one receptor modulators leading to the potent and selective nonsteroi-
dal progesterone receptor agonist tanaproget. Journal of Medicinal
Chemistry, 48(16), 5092–5095.
Sawant, R. L., Mhaske, M. S., & Wadekar, J. B. (2012). Anticoagulant
potential of schiff bases of 1,3-oxazines. International Journal of Phar-
macy and Pharmaceutical Sciences, 4(4), 320–323.
Sethi, K. K., Verma, S. M., Tanç, M., Purper, G., Calafato, G., Carta, F., &
Supuran, C. T. (2014). Carbonic anhydrase inhibitors: Synthesis and
inhibition of the human carbonic anhydrase isoforms I, II, IX and XII
with benzene sulfonamides incorporating 4- and 3-nitrophthalimide
moieties. Bioorganic & Medicinal Chemistry, 22(5), 1586–1595.
Simone, G. D., Fiore, A. D., & Supuran, C. T. (2008). Are carbonic anhy-
drase inhibitors suitable for obtaining antiobesity drugs? Current Phar-
maceutical Design, 14(7), 655–660.
Studio, D. (2008). Discovery, version 2.1. San Diego, CA: Accelrys.
Sukhorukov, A. Y., Nirvanappa, A. C., Swamy, J., Ioffe, S. L.,
Swamy, S. N., & Rangappa, K. S. (2014). Synthesis and characterization
of novel 1,2-oxazine based small molecules that targets acetylcholines-
terase. Bioorganic & Medicinal Chemistry Letters, 24(15), 3618–3621.
Supuran, C. T. (2008). Carbonic anhydrases: Novel therapeutic applications
for inhibitors and activators. Nature Reviews Drug Discovery, 7(2),
168–181.
Taslimi, P., Sujayev, A., Turkan, F., Garibov, E., Huyut, Z., Farzaliyev, V., &
Gulçin, İ. (2018). Synthesis and investigation of the conversion reactions
of pyrimidine-thiones with nucleophilic reagent and evaluation of their
acetylcholinesterase, carbonic anhydrase inhibition, and antioxidant
activities. Journal of Biochemical and Molecular Toxicology, 32(2), e22019.
Temperini, C., Innocenti, A., Scozzafava, A., Parkkila, S., & Supuran, C. T.
(2009). The coumarin-binding site in carbonic anhydrase accommo-
dates structurally diverse inhibitors: The antiepileptic lacosamide as an
example and lead molecule for novel classes of carbonic anhydrase
inhibitors. Journal of Medicinal Chemistry, 53(2), 850–854.
Friesner, R. A., Murphy, R. B., Repasky, M. P., Frye, L. L., Greenwood, J. R.,
Halgren, T. A., … Mainz, D. T. (2006). Extra precision glide: Docking
and scoring incorporating a model of hydrophobic enclosure for pro-
tein−ligand complexes. Journal of Medicinal Chemistry, 49(21),
6177–6196.
Ghose, A. K., Herbertz, T., Hudkins, R. L., Dorsey, B. D., & Mallamo, J. P.
(2012). Knowledge-based, central nervous system (CNS) lead selection
and lead optimization for CNS drug discovery. ACS Chemical Neurosci-
ence, 3(1), 50–68.
Gulçin, İ., & Taslimi, P. (2018). Sulfonamide inhibitors: A patent review
2013–present. Expert Opinion on Therapeutic Patents, 28(7), 541–549.
Hilvo, M., Salzano, A. M., Innocenti, A., Kulomaa, M. S., Scozzafava, A.,
Scaloni, A., … Supuran, C. T. (2008). Cloning, expression, post-translational
modifications and inhibition studies on the latest mammalian carbonic
anhydrase isoform, CA XV. Journal of Medicinal Chemistry, 52(3),
646–654.
Kadam, R., & Roy, N. (2007). Recent trends in drug-likeness prediction: A
comprehensive review of in silico methods. Indian Journal of Pharma-
ceutical Sciences, 69(5), 609–615.
Krasavin, M., Shetnev, A., Sharonova, T., Baykov, S., Tuccinardi, T.,
Kalinin, S., … Supuran, C. T. (2018). Heterocyclic periphery in the
design of carbonic anhydrase inhibitors: 1,2,4-Oxadiazol-5-yl benzene-
sulfonamides as potent and selective inhibitors of cytosolic hCA II and
membrane-bound hCA IX isoforms. Bioorganic Chemistry, 76, 88–97.
Lanni, T. B., Greene, K. L., Kolz, C. N., Para, K. S., Visnick, M., Mobley, J. L.,
… Liimatta, M. B. (2007). Design and synthesis of phenethyl benzo[1,4]
oxazine-3-ones as potent inhibitors of PI3Kinaseγ. Bioorganic & Medici-
nal Chemistry Letters, 17(3), 756–760.
Thiry, A., Dogne, J. -M., Masereel, B., & Supuran, C. T. (2006). Targeting
tumor-associated carbonic anhydrase IX in cancer therapy. Trends in
Pharmacological Sciences, 27(11), 566–573.
Lovell, S. C., Davis, I. W., Arendall, W. B., De Bakker, P. I., Word, J. M.,
Prisant, M. G., … Richardson, D. C. (2003). Structure validation by Cα
geometry: φ, ψ and Cβ deviation. Proteins: Structure, Function, and Bio-
informatics, 50(3), 437–450.
Mayekar, A. N., Yathirajan, H. S., Narayana, B., Sarojini, B. K.,
Kumari, N. S., & Harrison, W. T. (2011). Synthesis and antimicrobial
study of new 8-bromo-1,3-diaryl-2,3-dihydro-1H-naphtho[1,2e][1,3]
oxazines. International Journal of Chemistry, 3(1), 74–86.
Mincione, F., Scozzafava, A., & Supuran, C. T. (2009). Antiglaucoma carbonic
anhydrase inhibitors as ophthalomologic drugs. Hoboken, NJ: Wiley.
Narita, T., Suga, A., Kobayashi, M., Hashimoto, K., Sakagami, H.,
Motohashi, N., … Wakabayashi, H. (2009). Tumor-specific cytotoxicity
and type of cell death induced by benzo[b]cyclohept[e][1,4]oxazine and
2-aminotropone derivatives. Anticancer Research, 29(4), 1123–1130.
Ouberai, M., Asche, C., Carrez, D., Croisy, A., Dumy, P., & Demeunynck, M.
(2006). 3,4-Dihydro-1H-[1,3]oxazino[4,5-c]acridines as a new family of
Tian, S., Wang, J., Li, Y., Li, D., Xu, L., & Hou, T. (2015). The application of
in silico drug-likeness predictions in pharmaceutical research. Advanced
Drug Delivery Reviews, 86, 2–10.
Tomasulo, M., Sortino, S., & Raymo, F. M. (2005). A fast and stable photo-
chromic switch based on the opening and closing of an oxazine ring.
Organic Letters, 7(6), 1109–1112.
Turgut, Z., Pelit, E.,
&
Köycü, A. (2007). Synthesis of new
1,3-disubstituted-2,3-dihydro-1H-naphth [1,2e][1,3]oxazines. Mole-
cules, 12(3), 345–352.
Willard, L., Ranjan, A., Zhang, H., Monzavi, H., Boyko, R. F., Sykes, B. D., &
Wishart, D. S. (2003). VADAR: A web server for quantitative evaluation
of protein structure quality. Nucleic Acids Research, 31(13), 3316–3319.
ꢀ
ꢀ
Yigit, B., Yigit, M., Barut Celepci, D., Gök, Y., Aktas¸, A., Aygün, M., & Gülçin, İ.
(2018). Novel benzylic substituted imidazolinium, tetrahydropyrimidinium
and tetrahydrodiazepinium salts: Potent carbonic anhydrase and acetyl-
cholinesterase inhibitors. ChemistrySelect, 3(27), 7976–7982.
cytotoxic drugs. Bioorganic
4641–4643.
Öztaskın, N., Taslimi, P., Maras¸, A., Gülcin, İ., & Göksu, S. (2017). Novel
antioxidant bromophenols with acetylcholinesterase, butyrylcholines-
terase and carbonic anhydrase inhibitory actions. Bioorganic Chemistry,
74, 104–114.
& Medicinal Chemistry Letters, 16(17),
SUPPORTING INFORMATION
Additional supporting information may be found online in the Sup-
porting Information section at the end of the article.
Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S.,
Greenblatt, D. M., Meng, E. C., & Ferrin, T. E. (2004). UCSF chimera—A
visualization system for exploratory research and analysis. Journal of
Computational Chemistry, 25(13), 1605–1612.
Ren, S., Feng, Y., Wen, H., Li, C., Cui, J., & Jia, S. (2018). Immobilized car-
bonic anhydrase on mesoporous cruciate flower-like metal organic
framework for promoting CO2 sequestration. International Journal of
Biological Macromolecules, 117, 189–198.
How to cite this article: Qamar R, Saeed A, Saeed M, et al.
Synthesis, carbonic anhydrase inhibitory activity and antioxi-
dant activity of some 1,3-oxazine derivatives. Drug Dev Res.
2018;1–10. https://doi.org/10.1002/ddr.21464