Identification of 3-hydroxy-4[3,4-dihydro-3-oxo-2H-1,4-benzoxazin-4-yl]-2,2-dimethyldihydro-2H-benzopyran derivatives as potassium channel activators and anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-015-1344-6

The present study described the design, synthesis and identification of 3-hydroxy-4[3,4-dihydro-3-oxo-2H-1,4-benzoxazin-4-yl]-2,2-dimethyldihydro-2H-benzopyran derivatives. Their biological activity was tested for K_ATP channel opener as antihypertensives, COX-1 and COX-2 activity. The results were compared with the activity of cromakalim, ibuprofen and celecoxib. The study aimed at exploring the influence of introduction of a benzoxazine substituent at position 6 of various derivatives of benzopyrans in order to improve biological activity. Several compounds were found to be equipotent or even more potent than cromakalim. Out of these nitro-substituted benzopyrans, nitro substitution at benzoxazino group possessed potent antihypertensive activity in the R/S isomers. With amino derivatives, activity remains constant when compared with standard cromakalim. Similarly, compounds 17b, 17c, 17e and 17h have exhibited around 40 % inhibition of COX-1 as compared to the inhibition of COX-2. Only two compounds 17g and 17i exhibited effective inhibition more than 50 % of COX-2 compared with the inhibition of COX-1 at a concentration of 0.3 mg/ml.

Full text of DOI:10.1007/s00044-015-1344-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1344-6
ORIGINAL RESEARCH
Identification of 3-hydroxy-4[3,4-dihydro-3-oxo-2H-1,4-
benzoxazin-4-yl]-2,2-dimethyldihydro-2H-benzopyran derivatives
as potassium channel activators and anti-inflammatory agents
Mohsina Bano
Shailesh V. Jain
•
Kuldipsinh P. Barot
Manjunath D. Ghate
•
•
Received: 18 June 2014 / Accepted: 18 February 2015
Ó Springer Science+Business Media New York 2015
Abstract The present study described the design, syn-
thesis and identification of 3-hydroxy-4[3,4-dihydro-3-oxo-
Graphical Abstract
2H-1,4-benzoxazin-4-yl]-2,2-dimethyldihydro-2H-benzopy-
ran derivatives. Their biological activity was tested for KATP
channel opener as antihypertensives, COX-1 and COX-2
activity. The results were compared with the activity of
cromakalim, ibuprofen and celecoxib. The study aimed at
exploring the influence of introduction of a benzoxazine
substituent at position 6 of various derivatives of benzopy-
rans in order to improve biological activity. Several com-
pounds were found to be equipotent or even more potent
than cromakalim. Out of these nitro-substituted benzopy-
rans, nitro substitution at benzoxazino group possessed po-
tent antihypertensive activity in the R/S isomers. With amino
derivatives, activity remains constant when compared with
standard cromakalim. Similarly, compounds 17b, 17c, 17e
and 17h have exhibited around 40 % inhibition of COX-1 as
compared to the inhibition of COX-2. Only two compounds
Keywords Benzopyran ꢀ Benzoxazine ꢀ
K channel opener ꢀ COX-1 ꢀ COX-2 ꢀ Anti-inflammatory
Introduction
1
7g and 17i exhibited effective inhibition more than 50 % of
COX-2 compared with the inhibition of COX-1 at a con-
centration of 0.3 mg/ml.
Benzopyran-based cromakalim is KATP channel opener,
which is reported as potential class of therapeutic agents
possessing wide variety of cardioprotective activities such
as antihypertensive, antiischaemic, myorelaxant, antiar-
rhythmic, skeletal muscle relaxants, COX-2 inhibitors,
antimicrobials, anti-inflammatory, urinary incontinence,
antiasthmatics, anticonvulsants and antiangiogenic (Flor-
ence et al., 2014). Out of all the numerous activities related
to KATP channels, antihypertensive activity is the most
widely studied, used and well established (Sanfilippo et al.,
M. Bano
Department of Pharmaceutical Chemistry, Krupanidhi College
of Pharmacy, Carmelaram Post, Chikkabellandur,
Bangalore 560035, India
e-mail: 11ftphdp9@nirmauni.ac.in
K. P. Barot ꢀ S. V. Jain ꢀ M. D. Ghate (&)
Department of Pharmaceutical Chemistry, Institute of Pharmacy,
Nirma University, Ahmedabad 382481, Gujarat, India
e-mail: manjunath.ghate@nirmauni.ac.in
1993). These KATP channel openers acts mainly by the
opening of potassium channels. Mechanism of action of
cromakalim suggests that its antihypertensive effect is as-
sociated with cell membrane hyperpolarization and their
affinity towards potassium channels. They express pre- and
K. P. Barot
e-mail: barotkuldip@gmail.com
S. V. Jain
e-mail: shal.jain2007@gmail.com
123

Med Chem Res
post-synaptically in many brain regions; they open and
close in response to changes in intra-cellular ATP ratios.
Also slight reduction in potency was also observed with the
elimination of the pyran oxygen as in the case of indanes
(Buckle et al., 1991a, b). However, replacements of the pyran
ring with oxazine systems have shown potent vasodilators
(Yamada et al., 1993).
?
Low ATP levels open these channels, allowing K efflux
and cell hyperpolarization (Burrell et al., 1990). KATP
channel openers such as benzopyrans have been intensely
investigated because of their wide distribution, key role in
linking cellular metabolism to membrane potential and
therapeutic values. They have attracted considerable at-
tention because of the evidence of their potential value in
the treatment of those disorders in which smooth muscle
contraction is involved (Caliendo et al., 2002).
Hence, we planned the synthesis of the benzoxazine ben-
zopyrans without altering the nucleus of the basic cro-
makalim.Wehaveattemptedtosynthesizevarioussubstituted
benzopyrans and carry out the biological studies. To test
whether the synthesized molecules are biologically active and
do they possess activity, they were tested for antihypertensive
and COX-2 activity, and the results were compared with the
activity of existing cromakalim. Antihypertensive activity of
the synthesized compounds was carried out by direct method
intheanaesthetized rats(Sanfilippoetal., 1993). Benzopyran-
based cromakalim, pinacidil, aprikalim and diazoxide are
ATP-sensitive potassium channel openers which are potent
antihypertensive agents acting via peripheral vasodilation;
because of the ability of these agents to open potassium
channels in several tissue types, their usage is somewhat
limited over the existing antihypertensive agents. Tissue-
selective KATP openers are clearly required to explore the
potential of these agents in disease states. Hence, various
derivatives have been synthesized in correlation with these
benzopyrans with tissue selectivity. The objective was
achieved, and antihypertensive activity was carried out by the
direct method using anaesthetized albino rats.
Benzopyran nucleus has been modified in both the aro-
matic ring and in the pyran moiety. Structure–activity studies
have shown that the different positions of the benzopyran ring
have been variously substituted permitting the optimal ac-
tivity to be correlated with a specific set of structural charac-
teristics and stereo chemical features in the molecule (Lang
and Wenk, 1988). When cromakalim aromatic ring was re-
placed by pyridine and thiophene systems, potent pyran[3,2-
c]pyridines and thieno[3,2-b]pyrans were identified (Sanfil-
ippo et al., 1992; Press et al., 1993). The replacement of the
pyran nucleus with different heterocyclic and non-hetero-
cyclic systems often produced low or moderately active
compounds when compared with the benzopyran series
(
Ashwood et al., 1991). Similarly, there are several benzox-
azine derivatives depending on the position of the oxygen and
nitrogen in the ring, possessing large number of pharmaco-
logically active molecules such as calcium channel an-
tagonists, central nervous system drugs, analgesic and others
Number of uses of benzopyran-based cromakalim are as
follows: widely used as non-steroidal anti-inflammatory
drugs by the inhibition of COX-2 inhibitory activity without
influencing much on the normal physiological functions of
COX-1, expressed virtually in all tissues and involved in the
regulation of physiological functions and in maintaining
platelet aggregation and homeostasis of the GI tract and the
kidney; mainly, 2,3-diarylbenzopyran derivatives have been
extensively used as COX-2 inhibitors (Matralis et al., 2011).
Based on the above information, newly synthesized mole-
cules were evaluated for their ability to inhibit COX-2 and
COX-1 enzymes by in vitro colorimetric COX (ovine) in-
hibitor assay method which utilizes the peroxidase compo-
nent of the enzyme cyclooxygenase. Docking studies were
also performed to find out in silicon interaction of benzox-
azine benzopyrans derivatives with COX-1 and COX-2 tar-
get protein (Zhang et al., 2002).
(
Verma et al., 2011). Therefore, benzoxazine derivatives have
been reported to show considerable pharmacological actions
such as antimicrobial, antimycobacterial, antidiabetic, anti-
hypolipidemic, and antidepressant (Mac ´ı as et al., 2005). The
literature review showed that Hiroshi K and co-workers have
synthesized novel benzoxazine derivatives (1) and claimed
?
that these compounds possess K channel opening activity
and can be used as antiasthmatic, antiepileptic and antihy-
pertensive agent (Koga et al., 1999). Russell et al. (Russell
et al., 1993) have synthesized highly potent fluoroalkyl ben-
zoxazine pyridine-N-oxide derivatives (2) and (3) as potassi-
um channel openers. Kusumoto et al. (1994) have studied the
antihypertensive and cardiovascular properties of a new
potassium channel opener, TCV-295 (4) in rats and dogs.
Cecchetti et al. (2003) have synthesized highly potent 1,4-
benzothiazine derivatives (5) as KATP openers. Koini et al.
(2009) have synthesized and studied 5,7,8-trimethyl-ben-
zopyran (6) and 5,7,8-trimethyl-1,4-benzoxazine amino
amide derivatives (7) as novel antiarrhythmic agents against
ischaemia–reperfusion injury. Structure–activity studies
indicated thatthe replacementof the pyran oxygenwithNH or
Results and discussion
Chemistry
CH to produce tetrahydroquinolines and tetrahydronaph-
2
6-Substituted-2,2-dimethyl-2,7b-dihydro-1aH-oxireno[2,3-c]
chromene (epoxide) derivatives were synthesized using R/R-
and S/S Jacobsen’s catalyst for the synthesis of targeted
thalenes, as well as the ring expansion to benzoxazepines, had
detrimental effect on the potency (Buckle et al., 1991a, b).
1
23

Med Chem Res
3
-hydroxy-4[3,4-dihydro-3-oxo-2H-1,4-benzoxazin-4-yl]-2,
-dimethyldihydro-2H-benzopyrans. The synthesis of 6-sub-
water and drying over anhydrous sodium sulphate, anhy-
2
drous K CO was added and heated at 80 °C with stirring for
2
3
stituted-2,2-dimethyl-2,7b-dihydro-1aH-oxireno[2,3-c]chro-
mene (epoxide) using R/R- and S/S Jacobsen’s catalyst
provided optimum yield. Compounds 7a–e and 8a–e were
confirmed by spectral and elemental methods of analysis.
Synthetic route for the targeted compounds are summarized in
Scheme 1.
3 h. After washing with water and extracted several times
with CHCl , combined extracts were dried over anhydrous
3
Na SO and evaporated in vacuum to get white solid product
2
4
-
16a–c. 3470.25 cm (N–H), 1214.56 cm (C–O–C) and
1
-1
-
1
2964.42 cm (CH–Ali) indicated the synthesis of above
intermediates. The target molecule 3-hydroxy-4[3,4-dihy-
dro-3-oxo-2H-1,4-benzoxazin-4-yl]-2,2-dimethyldihydro-
2H-benzopyrans [17a–i and 18a–i] was synthesized using
Synthesis of substituted benzoxazino benzopyrans was
achieved in several steps involving the synthesis of inter-
mediate benzoxazino moiety and synthesis of the above
epoxides using various derivatives of benzopyrans. 6-Sub-
stituted 3,4-dihydro-3-oxo-2H-1,4-benzoxazine 16a–c were
synthesized using bromoacetyl bromide in ice-cold bath
solution of 4-substituted 2-amino phenols in saturated solu-
various R/S epoxy benzopyran derivatives with COCl in
2
acetonitrile solution. After washing with aqueous NaHCO3
and extracting with ethyl acetate, the organic layers are dried
over NaSO ; the residue was further recrystallized to get the
4
above product (Scheme 2). Synthesis of targeted molecules
was confirmed using elemental analysis; spectrophotometric
evaluations and optical rotations were confirmed by
tion of sodium carbonate in CHCl at room temperature for
3
3
h to obtain crude oil. After washing several times with
Scheme 1 Scheme for the
preparation of 6-substituted-2,2-
dimethyl-2,7b-dihydro-1aH-
oxireno[2,3c]chromene
(
epoxidation using R/R- or S/S
Jacobsen’s catalyst) [7a–e] and
8a–e]
[
123

Med Chem Res
Scheme 2 Scheme for the
synthesis of 3,4-dihydro-3-
hydroxy 2,2-dimethyl-4-[N(3,4-
dihydro-3-oxo-2H-1,4
benzoxazine)]-2H-benzopyran
polarimetric studies. FTIR spectra showed that C–N stretch
moderate action. Amino derivative substituted with bromo-
oxazino group possess minimal activity. Surprisingly, in the
benzoxazino derivative where R = NO and R = NO ,
-
1
-1
at 1263.56 cm and Ar–Cl at 710.72 cm confirms the
identification of the functional groups. Mass spectrum shows
the presence of molecular ion peak at 394.21 m/z, whereas
the calculated mass of the compound was 395.0 m/z.
2
1
2
both the R/S isomers have shown good activity, whereas the
same nitro group with a bromo substituent at benzoxazino
position has shown no activity. But when nitro derivative of
benzopyran substituted with a bromo benzoxazino com-
pound, there was no activity observed. Even R isomer con-
taining cyano substitution at position 6 and bromo group at
the benzoxazino group did not show activity. Amino sub-
stitution at position 6 in both R and S has shown moderate
activity (Table 2).
Biological evolution and structure–activity relationship
(
SAR)
When benzoxazino benzopyrans were subjected to anti-
hypertensive activity, we have observed that the nitro
group-substituted benzopyrans with the nitro substitution at
benzoxazino group possessed good antihypertensive ac-
tivity in the R/S isomers, even so with the amino deriva-
tives the activity remains when compared with the standard
cromakalim. The cyano derivative as usual was also active
with R/S isomer; this was not the case with the other
derivatives that are summarized in Table 1.
When synthesized compounds 17b, 17c, 17e, 17g, 17h
and 17i were subjected to in vitro colorimetric COX
(ovine) inhibitor assay for evaluation of their ability to
inhibit COX-2 and COX-1, the peroxidase component of
cyclooxygenase was utilized. The peroxidase activity is
assayed colorimetrically by monitoring the appearance of
0
0
From the above observation, it is clear that when substi-
oxidized N,N,N ,N -tetramethyl-p-phenylenediamine (TMPD)
tuted with cyano group at position 6 and R with bromine
1
during the reduction of PGG to PGH , at 590 nm. The re-
2
2
group, the compound has shown good antihypertensive ac-
tivity and R isomer of the same substitutions has also shown
sults of in vitro COX enzyme inhibition assay studies are
summarized in Table 3. The results showed that the
1
23

Med Chem Res
Table 1 Physical and analytical data of compounds 17a–i and 18a–i
1
7a-i
18a-i
Comp. No.
R
R
1
Mol. formula
Mol. weight
Melting point
% Yield
Rf value
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
7a
7b
7c
7d
7e
7f
Cl
Cl
C
19
C
19
C
19
C
20
C
19
C
19
C
19
C
19
C
19
C
19
C
19
C
19
C
20
C
19
C
19
C
19
C
19
C
19
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
17Cl
2
NO
4
394.25
449.25
449.25
429.26
374.82
419.27
385.37
404.8
240
210
184
189
213
178
195
176
149
231
201
182
176
208
172
179
182
153
40
44
60
45
36
35
49
37
67
36
41
53
42
38
35
45
40
43
0.7
0.15
0.11
0.2
0.7
0.9
0.3
0.4
0.12
0.1
0.2
0.12
0.2
0.7
0.7
0.3
0.5
0.4
Br
NO
Br
2
17BrN
17BrN
17BrN
2
2
2
O
6
6
4
NO
CN
NH
NH
NH
NO
NO
Cl
2
O
O
Br
2
2
2
2
2
Cl
19ClN
2 4
O
Br
19BrN
2
O
4
6
4
7g
7h
7i
NO
Cl
2
2
2
19 3 6
N O
2
17ClN O
NO
Cl
N
17 3
O
8
415.35
394.25
449.25
449.25
429.26
374.82
419.27
385.37
404.8
8a
8b
8c
8d
8e
8f
2
17Cl NO
Br
NO
Br
17BrN
17BrN
17BrN
2
2
2
O
6
6
4
NO
CN
NH
NH
NH
NO
NO
2
O
O
Br
2
2
2
2
2
Cl
19ClN
2 4
O
Br
19BrN
2
O
4
8g
8h
8i
NO
Cl
2
2
19 3 6
N O
2
17ClN O
6
NO
N
17 3
O
8
415.35
compounds 17b, 17c, 17e and 17h have exhibited around
0 % inhibition of COX-1 (39.07, 42.89, 39.21 and 51.68 %),
constriction must expand for any substrates, inhibitors or
products to access or depart the active site. The cat-
4
˚
alytically essential Tyr-385 is located approximately 13 A
as compared to the inhibition of COX-2 (9.09, 21.08, 14.06
and 16.93 %) at a concentration of 0.3 mg/ml. Only two
compounds 17g and 17i exhibited effective inhibition of
COX-2 (51.04 and 54.88 %), compared with the inhibition of
COX-1 (17.63 and 26.7 %) at a concentration of 0.3 mg/ml.
The blank has 38.09 % COX-1 inhibition and 8.69 % COX-2
inhibition activity. The standard celecoxib has 86.95 % in-
hibition of COX-2 and 33.33 % COX-1 inhibition, whereas
ibuprofen has 80.95 % inhibition of COX-1 and 13.04 %
COX-2 inhibition (Fig. 1).
above Arg-120 and across the active site from Ser-530.
Ser-530 is not essential for COX. Most of the residues in
the COX active sites are identical between the two iso-
forms, but there are notable exceptions in a region across
the active site from Arg-120 and bordered by hydrophobic
residues at position 523 (Val in COX-2, Ile in COX-1)
(Marnett, 2002). The difference in steric bulk at position
523 provides differential access to space in a side pocket
(accessible in COX-2, not accessible in COX-1). Con-
served differences also exist at the base of the side pocket
(Arg-513 in COX-2, His-513 in COX-1) (Schneider et al.,
Docking studies
2001). Evaluation was done with docking score, and single
All molecules have shown similar docking mode in the
active site of the enzyme. The active site is separated from
the initial substrate binding site by a constriction comprised
of three residues: Arg-120, Tyr-355 and Glu-524. This
best pose is generated as the output for particular ligand.
The docked conformation of the highest docking score
molecule 17i is shown in Fig. 2. 17i compound shows the
strong H-bonding interaction with Thr-206, His-207 of
123

Med Chem Res
Table 2 Antihypertensive activity of compounds 17a–i and 18a–i
1
7a-i
18a-i
Comp. No
Parameter
Baseline
With Adrenaline
With test alone
With test ?Adrenaline
Inference
1
7b
R = Br
= NO
2
SBP
114.8 (± 0.256)
66.0 (± 1.250)
91.7 (± 0.773)
316.1 (± 4.077)
129.7 (± 1.980)
86.1 (± 1.489)
114.8 (± 2.058)
397.2 (± 1.258)
124.5 (± 3.150)
90.7 (± 1.054)
116.7 (± 0.117)
308.7 (± 0.114)
130.8 (± 2.112)
86.7 (± 1.220)
109.6 (± 2.365)
311.1 (± 5.708)
112.7 (± 0.228)
72.7 (± 1.007)
95.7 (± 4.228)
326.1 (± 1.056)
117.9 (± 3.146)
67.8 (± 1.057)
90.2 (± 1.007)
304.0 (± 4.127)
105.1 (± 0.404)
60.7 (± 1.056)
78.9 (± 1.008)
268.1 (± 1.470)
122.7 (± 2.022)
74.8 (± 2.228)
100.4 (± 2.085)
309.7 (± 1.228)
123.6 (± 2.050)
83.9 (± 3.691)
107.9 (± 5.180)
290.7 (± 0.170)
159.3 (± 1.250)
92.8 (± 2.930)
129.7 (± 2.145)
390.8 (± 1.225)
188.4 (± 2.158)
118.4 (± 1.012)
136.7 (± 2.008)
433.8 (± 3.113)
167.9 (± 0.225)
123.0 (± 0.704)
154.7 (± 0.140)
359.6 (± 0.488)
172.9 (± 1.088)
120.9 (± 3.500)
148.2 (± 2.238)
342.8 (± 0.117)
168.9 (± 1.580)
100.1 (± 2.080)
126.0 (± 2.710)
364.7 (± 2.117)
159.8 (± 2.118)
98.7 (± 1.228)
119.7 (± 3.189)
346.8 (± 0.221)
144.7 (± 0.786)
91.5 (± 0.258)
115.0 (± 3.489)
317.9 (± 3.118)
168.0 (± 1.058)
102.6 (± 0.019)
141.8 (± 1.007)
341.0 (± 4.057)
168.2 (± 1.204)
120.6 (± 1.227)
140.8 (± 1.239)
326.9 (± 3.229)
149.6 (± 1.022)
86.5 (± 1.110)
119.0 (± 2.410)
376.0 (± 3.008)
132.0 (± 0.057)
85.7 (± 1.550)
116.0 (± 1.546)
409.2 (± 0.018)
156.0 (± 1.087)
112.8 (± 1.883)
138.4 (± 1.478)
327.9 (± 1.048)
139.6 (± 1.159)
89.8 (± 2.004)
119.0 (± 2.112)
328.9 (± 2.007)
126.0 (± 1.050)
79.8 (± 0.502)
102.7 (± 1.007)
334.0 (± 1.443)
156.7 (± 1.084)
97.3 (± 1.008)
120.6 (± 2.107)
349.7 (± 2.397)
116.9 (± 1.228)
68.7 (± 2.115)
80.8 (± 2.007)
278.0 (± 0.220)
159.7 (± 2.580)
89.7 (± 3.007)
126.7 (± 0.227)
322.7 (± 5.227)
132.0 (± 0.229)
89.6 (± 0.580)
118.0 (± 1.500)
302.7 (± 2.007)
152.1 (± 0.228)
88.2 (± 1.027)
122.6 (± 3.009)
380.4 (± 3.505)
141.8 (± 2.880)
90.2 (± 1.709)
126.9 (± 1.148)
420.7 (± 2.220)
159.7 (± 1.009)
115.4 (± 1.007)
142.0 (± 1.888)
322.0 (± 4.258)
144.0 (± 1.029)
92.0 (± 1.780)
126.8 (± 1.155)
331.0 (± 0.770)
129.7 (± 2.550)
82.0 (± 3.008)
108.1 (± 0.013)
339.7 (± 0.227)
157.6 (± 1.228)
100.2 (± 3.096)
120.8 (± 1.528)
350.4 (± 1.886)
120.0 (± 2.505)
76.1 (± 0.156)
91.5 (± 0.117)
289.7 (± 1.551)
160.8 (± 2.008)
93.9 (± 1.739)
129.8 (± 0.728)
336.0 (± 1.270)
149.2 (± 3.237)
100.9 (± 4.336)
122.9 (± 4.179)
315.8 (± 2.775)
Minimal activity
Antihypertensive
Moderately active
Antihypertensive
Antihypertensive
No Activity
DBP
MABP
HR
R
1
1
7d
R = CN
= Br
SBP
DBP
MABP
HR
R
1
1
7f
R = NH
= Br
SBP
2
DBP
MABP
HR
R
1
1
7g R = NH
= Br
2
SBP
R
1
DBP
MABP
HR
1
7i
R = NO
= NO
2
SBP
2
DBP
MABP
HR
R
1
1
8b
R = Br
= NO
2
SBP
DBP
MABP
HR
R
1
1
8d
R = CN
= Br
SBP
Antihypertensive
Moderately active
Antihypertensive
DBP
MABP
HR
R
1
1
8g
R = NH
= Br
SBP
2
DBP
MABP
HR
R
1
1
8i
SBP
R = NO
2
DBP
MABP
HR
R
1
= NO
2
1
23

Med Chem Res
Table 3 In vitro COX-1 and COX-2 enzyme inhibition activity data for standard drugs’ blank and synthesized compounds 17a–i
1
7a-i
a,b
Compounds
% Inhibition
COX-1 (0.3 mg/ml)
COX-2 (0.3 mg/ml)
Celecoxib
Ibuprofen
Blank
33.33
80.95
38.09
39.07
86.95
13.04
8.69
1
7b
R = Br, R
7c
R = NO
7e
R = NH
7g
R = NH
7h
R = NO
7i
R = NO
9.09
1
= NO
2
1
42.89
39.21
17.63
51.68
26.70
21.08
14.06
51.07
16.93
54.88
2
, R
2
, R
2
, R
2
, R
2
, R
1
1
1
1
1
= Br
1
= Cl
1
= NO
= Cl
2
1
1
= NO
2
Test concentration used for evaluation is 0.3 mg/ml, dissolved in Methanol
a
Values are acquired using in vitro ovine COX-1/COX-2 assay kit (Catalog No. 760 111, Cayman Chemicals Inc., Ann Arbor, MI)
Experiments were carried out in duplicate and have less than 10 % error
b
Cox-1 target protein (Fig. 2a) and Thr-221, Asp-294, Phe-
925 of Cox-2 target protein (Fig. 2c). Aromatic rings
Synthesis of 6-substituted-2,2-dimethyl-2,7b-dihydro-1aH-
oxireno[2,3-c]chromene (epoxide) using R/R- and S/
S Jacobsen’s catalyst [7a–d and 8a–d]
3
provide necessary lipophilic characters to allocate in lipo-
philic cleft of protein (Fig. 2b, d). All other molecules
showed similar docking mode in the active site of the en-
zyme (Table 4).
Substituted benzopyran epoxide was synthesized by several
steps utilizing trial-and-error method starting from phenols.
Various substituted phenols were successfully acetylated
using acetyl chloride and triethylamine under dichlor-
omethane. These acetylated phenols underwent Fries re-
arrangement in the presence of aluminium trichloride under
controlled temperature of 120–130 °C. The compounds
were further cyclized using pyrrolidine under Dean Stack
set-up using acetone to get cyclized nucleus, which were
further treated with p-toluenesulphonic acid (PTSA) to get
the nucleus of various substituted benzopyrans. The dou-
ble-bonded compound was further converted to chiral
derivative by treating with a chiral catalyst, Jacobsen’s
catalyst; the respective R/S catalyst resulted in the chiral
epoxides which are highly reactive.
Experimental work
Chemistry
Progress of all the reactions were monitored by TLC
studies using pre-coated alumina plate using various sol-
vent systems for elution. Formation of the products was
carried by the determination of their melting points.
Structure of all the compounds was authenticated by the
1
detailed spectral analysis using FTIR and HNMR.
123

Med Chem Res
Fig. 1 First-generation KATP
channels openers as potent
antihypertensive agents
N
O
N
N
O
O
NC
OH
CH3
O SPh
N
O N
2
N
O
2
CH3
CF3
CF3
CF3
O
CH3
O
(
2)
(3)
Cromakalim
Synthesis of 6-substituted 3,4-dihydro-3-oxo-2H-1,4-
benzoxazine intermediate [16a–c]
sulphate and concentrated in vacuum to provide a crude oil.
To the solution of crude oil product, anhydrous K CO
2
3
(0.1 mol) in 250 ml of DMF was added and heated at
Bromoacetyl bromide (3.5 ml, 0.15 mol) was added drop-
wise to an ice-cold bath solution of 4-substituted 2-amino
phenol. To this solution, 0.1 mmol saturated solution of
80 °C and stirred for 3 h. After cooling, the reaction
mixture was poured into water and extracted several times
with CHCl . Combined organic extracts were dried over
3
sodium carbonate in approximately 600 ml of CHCl was
3
anhydrous Na SO and evaporated in vacuum to get white
2
4
added. The reaction mixture was stirred at room tem-
perature for 3 h and monitored by TLC continuously;
layers were separated; and the organic phase was washed
several times with water and dried over anhydrous sodium
solid product 16a–c. Completion of the reaction was con-
firmed by the help of TLC, using diethyl ether: n-hexane
1:1 as solvent system; percentage yield of the compound
was found to 68 %.
1
23

Med Chem Res
Fig. 2 Docking poses of
synthesized compound.
a Binding interaction of 17i
compound in the binding pocket
of Cox-1 target protein (PDB:
3
1
N8X). b Hydrophobic fitting of
7i compound in binding pocket
of COX-1 target protein.
c Binding interaction of 17i
compound in the binding pocket
of Cox-2 target protein (PDB:
4COX). d Hydrophobic fitting
of 17i compound in binding
pocket of COX-2 target protein
Table 4 Docking result of synthesized molecules in Cox-1 and Cox-2 target
Molecule
Cox-1
Cox-2
Docking score
Crash
Docking score
Crash
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
7a
7b
7c
7d
7e
7f
4.2361
4.6229
3.1293
2.676
-2.9798
-0.6516
-1.1717
-1.0507
-1.0989
-2.9823
-1.0446
-1.4756
-1.2149
-1.3396
-1.3362
-0.2928
-1.4703
-1.0013
-1.3137
-0.9563
-1.6318
-0.6875
-1.4525
-0.5562
3.8123
3.0801
1.2349
3.5041
2.2297
2.685
-0.9143
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
-1.7626
2.8071
2.6483
3.7211
3.3094
4.8597
2.9451
2.7012
4.2698
3.9045
3.766
7g
7h
7i
5.244
3.9415
5.9193
4.4946
5.0748
5.366
8a
8b
8c
8d
8e
8f
3.1873
3.4567
3.3603
5.0594
4.1827
3.8871
6.2437
6.4778
3.4929
3.4131
4.796
8g
8h
8i
4.1976
6.0995
5.3483
Ibuprofen
Celecoxib
123

Med Chem Res
Synthesis of 3,4-dihydro-3-hydroxy 2,2-dimethyl-4-[N(3,4-
dihydro-3-oxo-2H-1,4-benzoxazine)]-2H-benzopyran
(C–Br) 92.3 (CH–OH) 70.2 (CH ) 21.2 (CH ) MS (ESI, m/
2
3
z): 450.0 (M ? 1, 96 %), 448.2 (M - 1, 65 %). Anal.
Calcd. for C19H17BrN2O6: C, 50.80; H, 3.81; N, 6.24.
Found: C, 50.84; H, 3.79; N, 6.12.
[
17a–i and 18a–i]
Various R/S epoxy benzopyran derivatives (1 g, 4.97 m-
mol) and 1.03 g of the above intermediate and 0.64 g
6-Bromo-4-((3S, 4R)-3-hydroxy-2,2-dimethyl-6-
nitrochroman-4-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one
(17c)
COCl were added to 4 ml of acetonitrile solution and
2
heated to 60 °C, stirring continuously for 50–60 min. After
1
h, TLC was carried out to check the completion of the
reaction. The reaction mixture was washed with aqueous
Yield 60.53 %, mp 184–186 °C. [a]25D ?43.12 (c 0.006,
0.5 N HCl) IR (KBr) vmax: 3648, 3092, 2966, 1651, 1600,
-1 1
1520, 1340, 1214 545 cm . H NMR (DMSO 400 MHz),
NaHCO and extracted with ethyl acetate; organic layers
3
are dried over MgSO ; and the residue obtained was further
4
recrystallized by hexane–ethyl acetate and hexane–chlo-
roform to get pure 3,4-dihydro-3-hydroxy 2,2-dimethyl-4-
d ppm 1.577 (s, 6H, CH ), 4.608 (s, 1H, OH), 8.709–8.716
(d, 2H, CH), 8.343 (d, 1H, J = 2.8, CH), 6.935–6.941 (d,
3
[
N(3,4-dihydro-3-oxo-2H-1,4-benzoxazine)]-2H-benzopy-
1H, J = 2.4 CH), 7.107 (s, 1H, CH), 7.084 (s, 1H, CH),
ran derivatives 17a–i and 18a–i. Formation of the above
product was confirmed by TLC, using hexane: ethyl acetate
2.749 (t, 2H, CH ), 8.366 (d, 1H, J = 2.8, CH), 6.847 (d,
2
1H, CH d = 166.3 (C=O) 151.4, 150.8 (C–O) 129.3, 127.2
(CH, benzene) 138.5 (C–NO ) 115.4 (C–Br) 92.8 (CH–
5
:1 as solvent system, and further the structure of the
2
1
synthesized compound was confirmed using FTIR and H
NMR studies. Optical rotations were confirmed by polari-
metric studies.
OH) 70.2 (CH ) 21.2 (CH ) MS (ESI, m/z): 430.2 (M ? 1,
2
3
94 %), 428.0 (M - 1, 55 %). Anal. Calcd. for
C19H17BrN2O6: C, 50.80; H, 3.81; N, 6.24. Found: C,
50.74; H, 3.85; N, 6.32.
6
-Chloro-4-((3S,4R)-6-chloro-3-hydroxy-2,2-dimethyl-3,4-
dihydro-2H-chromen-4-yl)-2H-benzo[b][1,4] oxazin-
(4H)-one (17a)
(3S, 4R)-4-(6-Bromo-3-oxo-2,3-
3
dihydrobenzo[b][1,4]oxazin-4-yl)-3-hydroxy-2,2-
dimethylchroman carbonitrile (17d)
Yield 40.38 %, mp 240–242 °C; [a]25D ?38.00 (c 0.01,
Water) IR (KBr) vmax: 1266, 3459, 2929.08, 2977, 1637,
Yield 45.27 %, mp 189–191 °C. [a]25D ?23.5 (c 0.01,
Water) IR (KBr) vmax: 3574, 3045, 2914, 2315, 1661,
-1 1
1610, 1510, 1218, 545 cm . H NMR (DMSO, 400 MHz)
-
1
1
1
589, 1363 710 cm
.
H NMR (DMSO, 400 MHz),
d = 1.40 (6H, s, CH ), 4.58 (1H, s, OH), 7.032 (2H, d,
3
J = 2.4, CH), 6.928–6.934 (2H, d, J = 2.4, CH), 7.24 (1H,
s, CH), 7.173 (1H, s, CH), 4.093–4.146 (2H, t, J = 8.2,
CH ), 2.038 (2H, t, J = 3.2 CH ), 5.619 (1H, d, J = 12,
d = 1.457 (6H, s, CH ), 4.609 (1H, s, OH), 7.239 (d, 2H,
3
J = 2.0 CH), 7.362–7.367 (1H, d, J = 2.0, CH), 7.383
(1H, d, J = 2.0, CH), 6.783 (1H, s, CH), 6.295 (1H, s, CH),
6.818 (1H, d, J = 2.0, CH), 6.927 (1H, d, J = 2.4, CH).
13C NMR (DMSO, 400 MHz d = 166.3 (C=O) 151.2,
150.8 (C–O) 129.2, 127.1 (CH, benzene) 125.4 (C–Br)
105.4 (C–CN) 92.2 (CH–OH) 70.1 (CH ) 21.2 (CH ) MS
2
2
CH), 6.220 (1H, d, J = 9.6, CH). 13C NMR (DMSO,
4
1
7
9
00 MHz): d = 166.3 (C=O) 151.1, 150.3 (C–O) 129.3,
27.2 (CH, benzene) 125.4, 126.1 (C–Cl) 92.2 (CH–OH)
0.3 (CH ) 21.2 (CH ) MS (ESI, m/z): 395.2 (M ? 1,
2
3
2
3
8 %), 393.0 (M - 1, 52 %). Anal. Calcd. for
(ESI, m/z): 430.0 (M ? 1, 97 %), 428.2 (M - 1, 62 %).
Anal. Calcd. for C19H17BrN2O6: C, 50.80; H, 3.81; N,
6.24. Found: C, 50.83; H, 3.86; N, 6.22.
C19H17Cl2NO4: C, 57.88; H, 4.35; N, 3.55. Found: C,
5
7.94; H, 4.41; N, 3.27.
4
2
-((3S, 4R)-6-Bromo-3-hydroxy-2,2-dimethyl-3,4-dihydro-
H-chromen-4-yl)-6-nitro-2H-benzo[b][1,4] oxazin-3(4H)-
(3S, 4R)-4-(6-Bromo-3-oxo-2,3-
dihydrobenzo[b][1,4]oxazin-4-yl)-3-hydroxy-2,2-
dimethylchroman-6-carbonitrile (17e)
one (17b)
Yield 44.25 %, mp 210–212 °C. [a]25D ?21.40 (c 0.009,
Water) IR (KBr) vmax: 3044, 2927, 2976, 1638, 1597,
Yield 66.53 %, mp 213–215 °C. [a]25D ?0.12 (c 0.01,
Water) IR (KBr) vmax: 3709, 2924, 1649, 1475, 1290,
-1 1
-
1 1
361, 1266, 522, 561, cm . H NMR (DMSO, 400 MHz),
1
1203 452 cm . H NMR (400 MHz, DMSO) d ppm 1.564
(6H, s, CH ), 7.210 (1H, s, Ar–H), 7.198 (1H, d, J = 4.0
d = 1.407 (6H, s, CH ), 5.608 (1H, s, OH), 7.067 (2H, d,
3
3
J = 2.4, CH), 7.149 (1H, d, J = 2.4, CH), 7.171 (d, 1H,
J = 2.4, CH), 7.241 (1H, s, CH), 6.649 (1H, s, CH). 13C
NMR (DMSO, 400 MHz): d = 166.1 (C=O) 151.2, 150.5
Ar–H), 7.266–7.269 (d, 1H, Ar–H), 7.345–7.348 (1H d,
J = 1.2, CH), 7.90 (d, 1H, J = 4.0, Ar–H), 4.024 (2H, s,
NH), 7.90 (2H, d, J = 4.0 Ar–H), 7.084 (s, OH, 1H), 7.24
(1H, s CH). 13C NMR (DMSO, 400 MHz) d = 166.3
(
C–O) 142.4(C-NO ) 129.5, 127.2 (CH, benzene) 116.1
2
1
23

Med Chem Res
(
C=O) 151.4, 150.2 (C–O) 129.3, 127.2 (CH, benzene)
7.385 (1H, d, J = 2.4 Ar–H), 7.323 (1H, d, J = 2.4 CH),
8.024 (1H, d, J = 2.4, Ar–H), 7.205 (2H, d, J = 2.4, Ar–
H), 8.051 (1H, d, J = 2.4, Ar–H), 7.310 (2H, d, J = 2.4,
Ar–H), 7.090 (OH, s, 1H), 5.041 (2H, d, J = 2.4, CH),
4.599 (t, CH, 1H). 13C NMR (DMSO, 400 MHz): 13C
NMR (DMSO, 400 MHz): d = 166.3 (C=O) 151.1, 150.3
1
41.4, (C–NH ), 128.4 (C–Cl) 92.2 (CH–OH) 70.3 (CH )
2 2
2
1.8 (CH ). MS (ESI, m/z): 375.1 (M ? 1, 95 %), 373.0
3
(
M - 1, 69 %). Anal. Calcd. for C19H19ClN2O4: C,
6
0.88; H, 5.11; N, 7.47. Found: C, 60.81; H, 5.18; N, 7.42.
4
2
3
-((3S, 4R)-6-Amino-3-hydroxy-2,2-dimethyl-3,4-dihydro-
H-chromen-4-yl)-6-bromo-2H-benzo[b][1,4] oxazin-
(4H)-one (17f)
(C–O) 129.3, 127.2 (CH, benzene) 139.4 (C–NO ) 126.1
2
(C–Cl) 92.2 (CH–OH) 70.3 (CH ) 21.4 (CH ). MS (ESI, m/
2
3
z): 405.1 (M ? 1, 96 %), 403.4 (M - 1, 65 %). Anal.
Calcd. for C19H17ClN2O6: C, 56.37; H, 4.23; N, 6.92.
Found: C, 56.34; H, 4.29; N, 6.84.
Yield 61.27 %, mp 178–180 °C. [a]25D ?46.00 (c 0.01,
Water) IR (KBr) vmax: 3399, 2980, 1615, 1463, 1295,
-
1 1
1268, 617.83, cm . H NMR (400 MHz, DMSO) d ppm
1.561 (6H, s, CH ), 7.215 (1H, s, Ar–H), 7.187 (1H, d,
4-((3S,4R)-3-Hydroxy-2,2-dimethyl-6-nitrochroman-4-yl)-
6-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (17i)
3
J = 4.0 Ar–H), 7.266–7.269 (d, 1H, Ar–H), 7.345–7.348
(
1H d, J = 1.2, CH), 7.92 (d, 1H, J = 4.0, Ar–H), 4.021
2H, s, NH), 7.95 (2H, d, J = 4.0 Ar–H), 7.089 (s, OH,
Yield 56.46 %, mp 181–183 °C. [a]25D ?0.22 (c 0.005, N
HCl) IR (KBr) vmax: 3453, 2934, 1634, 1525, 1434,
(
-
1
1
1
H), 7.24 (1H, s CH). 13C NMR (DMSO, 400 MHz)
12,385, 446 cm
.
H
NMR (DMSO, 400 MHz),
d = 166.3 (C=O) 151.1, 150.3 (C–O) 129.3, 127.2 (CH,
benzene) 141.4 (C–NH ), 116.1 (C–Br) 92.2 (CH–OH)
d = 1.305 (6H, s, CH ), 7.066 (1H, s, Ar–H), 6.806 (1H, d,
3
J = 2.4, Ar–H), 7.384 (1H, d, J = 2.4 Ar–H), 7.325 (1H,
d, J = 2.4 CH), 8.024 (1H, d, J = 2.4, Ar–H), 7.203 (2H,
d, J = 2.4, Ar–H), 8.050 (1H, d, J = 2.4, Ar–H), 7.311
(2H, d, J = 2.4, Ar–H), 7.093 (OH, s, 1H), 5.041 (2H, d,
J = 2.4, CH), 4.599 (t, CH, 1H). 13C NMR (DMSO,
2
7
9
0.3 (CH ) 21.3 (CH ). MS (ESI, m/z): 420.3 (M ? 1,
2 3
3 %), 418.0 (M - 1, 64 %). Anal. Calcd. for
C19H19BrN2O4: C, 54.43; H, 4.57; N, 6.68. Found: C,
5
4.48; H, 4.59; N, 6.74.
400 MHz): 13C NMR (DMSO, 400 MHz): d = 166.3
4
2
-((3S, 4R)-6-Amino-3-hydroxy-2,2-dimethyl-3,4-dihydro-
H-chromen-4-yl)-6-nitro-2H-benzo[b][1,4] oxazin-3(4H)-
(C=O) 151.1, 150.3 (C–O) 129.3, 127.2 (CH, benzene)
139.6 (C–NO ) 92.2 (CH–OH) 70.5 (CH ) 21.5 (CH ). MS
2
2
3
one (17g)
(ESI, m/z): 416.1 (M ? 1, 95 %), 413.4 (M - 1, 66 %).
Anal. Calcd. for C19H17N3O8: C, 54.94; H, 4.13; N,
10.12. Found: C, 54.99; H, 4.19; N, 10.14.
Yield 69.52 %, mp 195–197 °C. [a]25D ?25.0 (c 0.01,
Water) IR (KBr) vmax: 3449, 2822, 1601, 1297, 1352,
-
1
1
H
1
252, 1214 cm
.
NMR (DMSO, 400 MHz),
Docking studies
d = 1.302 (6H, s, CH ), 7.069 (1H, s, Ar–H), 6.800 (1H, d,
3
J = 2.4, Ar–H), 7.382 (1H, d, J = 2.4 Ar–H), 7.328 (1H,
d, J = 2.4 CH), 8.028 (1H, d, J = 2.4, Ar–H), 7.204 (2H,
d, J = 2.4, Ar–H), 8.050 (1H, d, J = 2.4, Ar–H), 4.599 (s,
NH, 2H), 7.310 (2H, d, J = 2.4, Ar–H), 7.090 (OH, s, 1H),
Molecular docking is a key tool in structural molecular
biology and computer-assisted drug design. The goal of
ligand–protein docking is to predict the predominant
binding mode of a ligand with a protein of known 3D
structure. Docking can be used to perform virtual screening
on large libraries of compounds, rank of results and pro-
pose structural hypotheses of how the ligand inhibits the
target, which is valuable in lead optimization. To investi-
gate the detailed intermolecular interactions, docking
studies were carried out between the synthesized deriva-
tives COX-1 and COX-2 target protein. The 3D structure of
COX-1 and COX-2 enzyme is well known. For our studies,
X-ray crystal structure of COX-1 (3N8X) and COX-2
(4COX) was taken from PDB having resolution of more
5
.041 (2H, d, J = 2.4, CH), 4.599 (t, CH, 1H). 13C NMR
DMSO, 400 MHz): d = 166.3 (C=O) 151.1, 150.3 (C–O)
29.3, 127.2 (CH, benzene) 141.3, (C–NO ) 140.1 (C–
(
1
2
NH ) 92.2 (CH–OH) 70.3 (CH ) 21.3 (CH ). MS (ESI, m/
2
2
3
z): 386.2 (M ? 1, 95 %), 384.1 (M - 1, 52 %). Anal.
Calcd. for C19H19N3O6: C, 59.22; H, 4.97; N, 10.90.
Found: C, 59.30; H, 4.99; N, 10.96.
4
2
-((3S, 4R)-6-Amino-3-hydroxy-2,2-dimethyl-3,4-dihydro-
H-chromen-4-yl)-6-nitro-2H-benzo[b][1,4] oxazin-3(4H)-
˚
than 2.0 A. The molecular docking of different synthesized
one (17h)
benzopyran derivatives into COX-1 and COX-2 target
protein was performed using Surflex-Dock 2.1 module of
SYBYL X 1.2 software. The protein–ligand interactions
responsible for the observed activity to identify the binding
orientations and processing of the protein included the
Yield 47.26 %, mp 176–178 °C. [a]25D ?0.21 (c 0.005, N
HCl) IR (KBr) vmax: 3484, 2978, 1651, 1519, 1471, 1297,
-
1 1
440 cm . H NMR (DMSO, 400 MHz), d = 1.308 (6H, s,
CH ), 7.061 (1H, s, Ar–H), 6.801 (1H, d, J = 2.4, Ar–H),
3
123

Med Chem Res
deletion of the ligand and the solvent molecules as well as
the addition of hydrogen atoms. After ensuring chemical
correctness, water molecules in the crystal structures were
deleted and hydrogens were added where hydrogen atoms
were missing and bond order for crystal ligand and protein
administration was surgically cannulated, and left carotid
artery by dissection for blood-pressure recording was iso-
lated and exposed, using PE-50 tubing. By means of a
three-way plastic stop cock and a stainless steel needle at
the end of the PE tubing, the arterial cannula to a blood-
pressure transducer and a venous cannula to a syringe were
attached, and both cannulae with heparinized saline before
cannulation were fluid filled. Afterthought, the arterial
cannula via the BSL pressure transducer (SS13L) to the
BIOPAC Systems, Inc was connected. Criterion for anti-
hypertensive activity will be reduction in systolic arterial
pressure by about 10–20 mmHg. Sympathetic system ac-
tivation to induce hypertension is done by administering
adrenaline, 5 lg/kg i.v., flushing the venous cannula with
0.2 ml of normal saline, and then allowing returning to pre-
injection level. Effect of antihypertensive blood-pressure
compounds on induced hypertension is measured by in-
jecting the test compound 20 mg/kg solution intravenously
and allowing to equilibration in the system, and then,
adrenaline, 5 lg/kg i.v., as described previously was re-
peated and blood-pressure response to each procedure was
observed and recorded.
˚
were adjusted and minimized up to 0.30 A RMSD.
Antihypertensive activity
BIOPAC System, Santa Barbara, with calibration was used
to record the BP. Adrenaline was used to induce hyper-
tension, left carotid artery was exposed, and the arterial
cannula to a blood-pressure transducer and a venous can-
nula to a syringe were attached. Arterial cannula was
connected via the BSL pressure transducer (SS13L) to the
BIOPAC Systems.
Toxicity studies to fix up LD50
In order to fix up the dose to carry out the antihypertensive
activity, toxicity studies were carried out according to the
OECD Guidelines No. 420 and 421. Wister albino rats
weighing 200–250 g were chosen; route of administration
of the drug was chosen as oral route. Six groups of animals
each containing three animals were initially selected. At
first, as per the Guidelines No. 420 and 421, given a dose of
SBP—systolic blood pressure
DBP—diastolic blood pressure
MABP—mean arterial blood pressure
HR—heart rate
7
0 mg/kg body weight monitored the animal for the toxic
Values are expressed in mean ± SEM. No of readings: 03
symptoms as well as mortality; the animals showed high
toxicity symptoms such as increased intestinal motility,
diarrhoea, tail erection and irritation to nose; and after 3 h,
all the animals were dead. Hence, we decreased the dose to
Colorimetric COX (ovine) inhibitor screening assay
5
0 mg/kg body weight and administered it to the next
There are several assay kits available for measuring COX
inhibition. Cayman’s colorimetric COX (ovine) inhibitor
screening assay measures the peroxidase component of
COXs. The peroxidase activity is assayed colorimetrically
group of animals and monitored for toxic symptoms and
mortality. In this dose, animals were safe but showed fewer
toxic symptoms and only few were mortal, and toxicity
symptoms were diarrhoea, tail erection and irritation to
nose. Once again, we decreased the dose, and it was fixed
to a dose of 20 mg/kg body weight to the next set of ani-
mals and observed for the toxic symptoms and mortality.
At this dose, all the animals were safe and no toxic
symptoms were seen. Hence, it was concluded that 20 mg/
kg body weight dose was safe and recommended dose for
further studies (antihypertensive activity).
0
0
by monitoring the appearance of oxidized N,N,N ,N -te-
tramethyl-p-phenylenediamine (TMPD) at 590 nm. Inhi-
bition of COX activity, by a variety of selective and non-
selective inhibitors, showed potencies similar to those ob-
served with in vitro methods. Test concentration used for
evaluation is 0.3 mg/ml which is dissolved in Methanol.
X = Values are acquired using in vitro ovine COX-1/
COX-2 assay kit (Catalog No. 760 111, Cayman
Chemicals Inc., Ann Arbor, MI).
Direct antihypertensive activity
Y = Experiments were carried out in duplicate and have
\
10 % error.
Direct antihypertensive activity was carried out using the
instrument BIOPAC System (MP-36, Santa Barbara,
California) for recording BP response. Male albino rats,
weighing 200–250 g were used, the rats were anaesthetized
with urethane hydrochloride, 1.25 g/kg i.p, and then, the
rats were prepared by shaving the neck and inguinal region
with animal hair clippers. The jugular vein for drug
Conclusion
Benzoxazine scaffold with different substituents have also
been used in the development phase as potential new drugs.
1
23

Med Chem Res
The versatility of the benzoxazine skeleton, in addition to
its relative chemical simplicity and accessibility, makes
these chemicals amongst the most promising sources of
bioactive compounds. This has led to the discovery of a
wide variety of compounds that are of high interest from
the point of view of its antimicrobial, antimycobacterial,
antidiabetic and antidepressant effects amongst the others.
Hence, an attempt has been made to combine benzopyran
nucleus along with various benzoxazines. The combination
of these two heteroaromatic nucleus is expected to show a
great deal of pharmacologic responses. In this series of
benzoxazino benzopyrans, we have observed details and
can conclude that the Nitro group-substituted benzopyrans
with the a nitro substitution at benzoxazino group pos-
sessed good antihypertensive activity in the R/S isomers,
even so with the Amino derivatives the activity remains
when compared with the standard cromakalim.
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macy, Nirma University for providing necessary facilities for ex-
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to Department of Science and Technology (DST) and Council of
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