In silico Investigation and Biological Evaluation of Synthesized Sulfamethoxazole Derivatives

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January-February 2020 Indian Journal of Pharmaceutical Sciences 123
Research Paper
*Address for correspondence
E-mail: sairampaidesetty@gmail.com
Accepted 02 December 2019
Revised 18 September 2019
Received 08 Juy 2019
Indian J Pharm Sci 2020;82(1):123-130
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In silico Investigation and Biological Evaluation of
Synthesized Sulfamethoxazole Derivatives
JYOTIRMAYA SAHOO1, PRIYAMBADA KSHIRODA, NANDINI SARANGI2, S. K. ROUT3, AND S. K. PAIDESETTY3*
School of Pharmacy, ARKA Jain University, Gamharia, Seraikela, Kharsawan, Jameshedpur, Jharkand-832 108,
1Department of Pharmaceutics, 2Department of Pharmaceutical Chemistry, Sri Jayadev College of Pharmaceutical Sciences,
Bhubaneswar-752 101, 3Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Siksha ‘O’ Anusand-
han (Deemed to be University) Bhubaneswar-751 030, India
Sahoo et al.: In silico investigation and biological evaluation of Sulfamethoxazole derivatives
A series of 4-[4-(substitutedaryl/heteroaryldiazenyl]-N-(5-methylisoxazol-3-yl)benzene sulphonamide
derivatives (4a-4f) were designed and synthesized coupling a mixture of diazotized sulfamethoxazole
with six different phenolic and enolic compounds in an in situ reaction. The structural environment of
synthesis of each molecule was conrmed by Fourier-transform infrared spectroscopy, proton nuclear
magnetic resonance and elemental analysis. These derivatives were further screened in various biological
assays in vivo for analgesic and antiinammatory activities and in vitro for antioxidant and antimicrobial
activities. When tested for analgesic activity at a dose of 50 mg/kg, compounds 4-((2-hydroxynaphthalen-
1-yl)diazenyl)-N-(5-methylisoxazol-3-yl)benzene sulphonamide (4d) and 4-((4-hydroxy-5-isopropyl-2-
methylphenyl)diazenyl)-N-(5-methylisoxazol-3- yl)benzene sulphonamide (4f) showed 58.33 and 57.76 %
of pain inhibition, respectively. These two molecules also exhibited signicant antioxidant activity at
10 and 50 µg/µl. The compound 4-[(4-hydroxy-2-oxo-2H-chromen-3-yl)diazenyl]-N-(5-methylisoxazol-
3-yl)benzene sulphonamide (4a) exhibited antibacterial activity against Staphylococcus aureus resistance,
Candida albicans and DescriptionCryptococcus neoformans at a concentration of 31.25 µg/ml. The analgesic
action of these synthesised analogues was predicted in molecular docking experiments with a specic
target protein, cyclooxgenase-2 of Mus musculus and results indicated all tested compounds to exhibit
good binding interaction with the active site amino acid of the target enzyme.
Key words: Sulfanilamide, thymol, 4-hydroxy coumarin, antioxidant, antimicrobial, antiinammatory
Sulfanilamides were established molecules in the
eld of medicine. Though these are rarely prescribed
these days, but in medicinal chemistry the importance
of sulfanilamide entity is well-recognised as it
continues to offer several therapeutic benets for drug
development[1]. Some 70-80 y ago, a red dye Prontosil,
an azo-linked sulfonamide pro-drug was widely used to
treat streptococcal infections[2]. For antibacterial drug
development identication of correct lead candidates
is a major challenge. Thus, sulfanilamide analogues
continue to offer insights for the development of newer
antimicrobials.
Isoxazole is a heterocyclic azole moiety with
oxygen and nitrogen atoms in cyclopentadiene ring.
Compounds bearing the isoxazole ring serve as an
important source for developing useful drug candidates,
for treating several infectious diseases. Although,
majority of isoxazole derivatives have exhibited
immunosuppressant and antiinammatory activities,
sulfonamide containing isoxazole analogues were
found to display potent analgesic and antiinammatory
action. The commercially available isoxazole bearing
drugs are COX-2 inhibitor, valdecoxib, leunomide,
dihydrofolate synthetase inhibitor, sulfamethoxazole
(SMZ), sulsofurazole and β-lactamase-resistant
antibiotics, such as cloxacillin and dicloxacillin to
mention some.
Moreover, literature revealed that molecules with a
diazenyl (–N=N-) group showed versatile biological
properties[3] and also that nitrogen bearing heterocyclic

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Indian Journal of Pharmaceutical Sciences
124
compounds possess good biological action[4].
Microorganisms cause infectious disease and due to
irrational use of antibiotics, some of the pathogenic
organisms have developed resistance against these
antimicrobial drugs. Thus, Research efforts are to be
focused more towards developing drugs that act against
the resistant pathogenic organisms with minimal side
effects to create socio-economic benet. Cellular
oxidative stress in biological system due to generation
of reactive oxygen species (ROS) leads to alterations
in genes through oxidation of nucleic acid, impairment
of muscle function by protein denaturation, while lipid
peroxidation in cells cause perturbation of homeostasis
resulting in cell death. ROS are produced during
tissue injury and antioxidants play a vital role during
wound healing. Although, inammation is a defensive
mechanism in response to foreign bodies but leads to
cellular damage due to producing oxidative stress[5].
Under these circumstances, synthetic and natural
antioxidants have a major role to protect cells against
oxidative stress[6] . Hence, it is obligatory to develop
new drugs at minimum cost, which are effective against
resistant organisms and also aid in quick healing of
infected wounds through free radical scavenging[7].
Previous studies from our laboratory explored the
synthesis and characterisation of phenolic/enolic
substituted diazenyl SMZ derivatives[8-14]. To continue
these efforts, biological activities of these analogues
needs to be evaluated using different models. The present
work is conceptualized on the basis of literature to
design some hybrid molecules containing sulfonamide,
5-methyl-isoxazole moiety and diazenyl group all
together with different phenolic/enolic systems with
in these structures. In this drug design attempt it was
planned to couple 6 bioactive neutral nucleophiles such
as, 4-hydroxy coumarin (4-HC), 8-hydroxy quinoline
(8-HQ), salicylic acid, β-napthol, salicylaldehyde
and thymol with diazonium salt of SMZ to produce
the desired molecules 4a-4f to evaluate the biological
actions possessed by these molecules. In addition,
molecular docking study was carried out to predict
the potential of the molecules to exhibit analgesic
and antiinammatory activities. Thus, all designed
molecules were subjected to molecular docking against
the cyclooxygenase (COX-2) enzyme, the structure of
which was retrieved from the Protein Data Bank.
MATERIALS AND METHODS
All chemicals (Merck Specialties Ltd., and Hi-Media
Laboratories Pvt. Ltd., Mumbai, India) used were of
synthetic and analytical grade. Melting points (°) were
determined using the open capillary method (Elico)
and were uncorrected. The IR spectra, molecular mass,
1HNMR spectra and elemental analysis were carried
out on Jasco FT/IR 4100, Japan, Shimadzu, Bruker 1H
NMR, 400 MHz and Perkin Elmer 2400, respectively.
The UV absorption (λmax) maximum was measured on a
Jasco V-630 spectrophotometer
In silico investigation:
COX-2 of Mus musculus with PDB ID: 1CX2 was
retrieved from Protein Data Bank (PDB) and docked
against the proposed molecules with Arugus Lab
4.0 individually. The protein-ligand interaction was
carried out by Discovery Studio Visualizer 3.1 software.
Synthesis of the proposed molecules (4a-4f):
The synthetic procedures of compounds (4a-4f) have
been previously reported (g. 1)[8-12]. A cold solution of
sodium nitrite was added dropwise to an aqueous solution
of the desired SMZ with concentrated hydrochloric acid;
the temperature of the reaction mixture was maintained
at 0-5°. When addition was completed, the solution
was to stand a few minutes with occasional stirring to
complete diazotization. Then it was poured into an ice
cold solution of individual phenolic/enolic compounds
(2a-2f) in 20 % sodium hydroxide the reaction mixture
was stirred and kept overnight in a refrigerator. The
nal precipitate obtained was ltered and recrystallized
from hot ethanol.
Spectral characterization:
The spectral data of individual analogues 4a-4f
was previously reported. 4-[(4-hydroxy-2-oxo-2H-
chromen-3-yl)diazenyl]-N-(5-methylisoxazol-3-yl)
benzene sulphonamide (4a)- red colour; yield, 72 %; Rf;
0.8, mp (°); 225-228; UV/Vis (λmax, nm, CH3OH): 403;
IR (KBr, γ, cm-1): 3179 (O-H str.), 1726 (C=O str.), 1617
(-C=C- str.), 2925 (CH2 str.), 1506 (-N=N-), 1390, 1157
(SO2 str.); 1HNMR (CDCl3 δ ppm, 400 MHZ): 2.43 (s,
3H, CH3), 7.93 (d, coumarin H-5), 7.654 (m, coumarin
H-6), 7.65 (m, coumarin H-7), 7.42 (d, coumarin H-8),
5.62 (s, 1H, isoxazol-4-yl H); LC-MS (% area); 100;
m/z; 427.18 (M+1); analysis for C19H13N4O6S: Calcd %
C, 53.52; H, 3.31; N, 13.14;S,7.52; found %: C, 53.47;
H, 3.36; N, 13.21;S,7.49.
4-((8-hydroxyquinolin-5-yl)diazenyl)-N-(5-
methylisoxazol-3-yl)benzene sulphonamide (4b)-
orange colour; yield 92 %; Rf; 0.8, mp (°); 220-223;
UV/Vis (λmax, nm, CH3OH): 403; IR (KBr, γ, cm-1):
3297 (O-H str.), 1616 (-C=C- str.), 2847 (CH2 str.),

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Indian Journal of Pharmaceutical Sciences 125
January-February 2020
1573 (C=N str.), 1506 (-N=N-), 1341 1170 (SO2str.);
1HNMR (DMSO-d6, δ ppm, 400 MHZ): 2.83
(s,3H,CH3), 8.01-8.05 (m,4H,ArH), 9.28 (d, quinolinyl
H-2), 7.63 (d, quinolinyl H-3), 8.03 (d, quinolinyl H-4),
8.07 (d, quinolinyl H-6), 7.26 (d, quinolinyl H-7), 6.28
(s,1H,isoxazol-4-yl H); LC-MS (% area); 100; m/z;
410.30 (M+1); Analysis for C19H15N5O4S: Calcd % C,
55.74; H, 3.69; N, 17.11 S, 7.67; found %: C, 55.72; H,
3.73; N, 17.13; S,7.85.
2-hydroxy-5-((4-(N-(5-methylisoxazol-3-yl)
sulfamoyl)phenyl)diazenyl)benzoic acid (4c)- Grey
colour powder; yield 73 %; Rf; 0.8, mp (°); 227-230;
UV/Vis (λmax, nm, C2H5OH): 370; IR (KBr, γ, cm-1):
3461 (N-H str.), 1668 (C=O str.), 1614 (-C=C- str.),
2922 (CH2 str.), 1573, (C=N str.), 1510 (-N=N-), 1315,
1170 (SO2str.); 1HNMR (DMSO-d6, δ ppm, 400 MHZ):
7.25-8.01 (m,7H,ArH), 2.43 (s, 3H, CH3), 11.67 (s, IH,
OH), 12,11 (sb, 1H,COOH), 6.17 (s, 1H, isoxazol-4-yl
H); LC-MS (% area); 100; m/z; 403.18 (M+1); analysis
for C17H14N4O6S: Calcd % C, 50.75; H, 3.51; N, 13.92;
S,7.97; found %: C, 50.47; H, 3.55; N, 13.96; S,7.89.
4-((2-hydroxynaphthalen-1-yl)diazenyl)-N-(5-
methylisoxazol-3-yl)benzene sulfonamide (4d)- Orange
red colour powder; yield 90 %; Rf; 0.8, mp (°); 170-
178; UV/Vis (λmax, nm, C2H5OH): 470; IR (KBr, γ,
cm-1): 3251 (O-H/NH str.), 1511, 1607 (C=C str.),1460
(-N=N-), 1385 (O-H bend.), 1316, 1159 (SO2 str.),1212
(C-O str.), 956 (S-N str.); 1H NMR (CDCl3, δ ppm, 400
MHZ): 17.49 (s, 1H, SO2NH), 16.00 (1H, OH), 7.85-
8.34 (m, 4H, ArH), 7.14-7.85 (6H, naphthyl H), 6.26
(s, 1H. isoxazol-4yl H), 2.37 (s, 3H, CH3); LC-MS (%
area); 97; m/z; 409.68 (M+1); analysis for C20H16N4O4S:
Calcd % C, 58.84; H, 3.97; N, 13.71; S,7.86; found %:
C, 50.81; H, 3.95; N, 13.72; S,7.85.
4-((3-formyl-4-hydroxyphenyl) diazenyl)-N-(5-
methylisoxazol-3-yl) benzene sulfonamide (4e)- Grey
colour powder; yield 90 %; Rf; 0.8, mp (°); 170-175;
UV/Vis (λmax, nm, C2H5OH): 450; IR (KBr, γ, cm-1):
3442 (O-H str.), 2915 (CH str.), 1660 (C=O str.), 1615
(C=C str.), 1478 (-N=N-), 1305, 1175 (SO2 str.), 1137
(C-O str.), 901 (S-N str.); 1HNMR (DMSO-d6, δ ppm,
400 MHZ): 11.42 (s, 1H, OH), 11.02 (s, 1H, SO2NH),
Fig. 1: Synthesis of sulfamethoxazole analogues 4a-4f

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Indian Journal of Pharmaceutical Sciences
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10.03 (s, 1H, CHO), 8.18-8.23 (m, 4H, Ar H), 8.01
(d, 1H, salicylaldehyde H-6), 7.98 (s, 1H, salicylaldehyde
H-2), 7.53 (d, 1H, salicylaldehyde H-5), 6.24 (s, 1H,
isoxazole H-4), 2.38 (s, 3H, =C-CH3); LC-MS (% area);
100; m/z; 386.22 (M+1); analysis for C17H14N4O5S:
Calcd % C, 52.83; H, 3.67; N, 14.51;S,8.29; found %:
C, 50.85; H, 3.68; N, 14.52; S, 8.33.
4-((4-hydroxy-5-isopropyl-2-methylphenyl)diazenyl)-
N-(5-methylisoxazol-3-yl)benzene sulphonamide
(4f)- Brick red colour powder; yield 84 %; Rf; 0.8,
mp (°); 304-310; UV/Vis (λmax, nm, 1,4-dioxane):
409; IR (KBr, γ, cm-1): 3489, 3163 (O-H str.), 2961
(CH2 str.), 1619 (C=C str.), 1466 (-N=N-), 1341, 1129
(SO2 str.), 899 (S-N str.). 1H NMR (DMSO-d6, δ ppm,
400 MHZ): 9.75 (s, 1H, SO2NH), 9.56 (s, 1H, OH),
7.95-8.50 (m, 4H, Ar H), 7.85 (s, 1H, thymol H-6), 6.95
(s, 1H, thymol H-3), 6.80 (s, 1H, isoxazolyl H), 2.97
(m, 1H, CH (CH3)2), 2.38 (s, 3H, methyl), 1.25 (d, 6H,
CH (CH3)2; LC-MS (% area); 100; m/z; 415.18 (M+1);
analysis for C20H22N4O4S: Calcd % C, 57.85; H, 5.29;
N, 13.45; S,7.68; found %: C, 57.96; H, 5.55; N, 13.52;
S,7.74 (Table 1, g. 2)
Pharmacological evaluation:
Acute oral toxicity of the synthesised molecules was
determined using recommended procedure as prior
OECD guideline No. 420. The animals were orally
administered with the test molecules at the doses of 5,
50, 300 and 2000 mg/kg at an interval of 24 h between
of each dose. The entire study was carried out as per
CPCSEA and IAEC guidelines (registration number
1171/C/08/CPCSEA and Ref. No. 60/SPS/IAEC/
SOAU).
In vivo acetic acid-induced writhing method was
carried out with a little modication to evaluate the
analgesic activity[15]. The group I was treated as a
negative control and the group II was as positive
control and was administered orally 100 mg/kg
acetylsalicylic acid (ASA). Animals from groups III-
XIV were orally administered with test molecules (4a-
4f) at the dose level of 25 and 50 mg/kg. After 1 h of the
administrations, all the groups were treated with 0.6 %
v/v acetic acid solution (1 ml/100 g) intra peritoneally
and the onset and number of writhings was noted.
Finally percent of analgesic activity was calculated
as follows, % analgesic activity=mean writhing
count (control−treated group)/mean writhing count of
control group×100. The reaction time was expressed as
mean±SEM. The statistical analysis was done by one
way-ANOVA followed by Dunnett’s t-test.
Eddy’s hot plate (Techno) was used to induce pain by
thermal stimuli and to measure the response latencies
as per method rst described by Eddy and Leimback[13]
with a slight modication and the instrument was
adjusted to 55±0.5° and the basal reaction time of each
animal were recorded before administering either the
test or standard compounds. The reaction time for all
the groups was measured at an interval of 60 min for
3 h after administration of test molecules and nally
the percent analgesic activity (reaction time) was
determined. Carrageenan-induced rat hind paw oedema
method was followed with a slight deviation using the
Ugo Basile plethysmometer (7150). All treatments were
given orally I h before the injection of carrageenan.
Percent inhibition of inammation was calculated using
the formula, % inhibition = 100 (1-Vt/Vc), where Vc
represents oedema volume in control and Vt oedema
volume in group treated with test molecules.
In vitro radical scavenging capacity:
The antioxidant activity of the prepared isoxazole
derivatives were measured using the 2,2-diphenyl-
Comps. Heteroaryl M. formula m/z Rf mp (°) Color Yield
(%)
4a 4-[(4-hydroxy-2-oxo-2H-chromen-3-yl)diazenyl]-N-
(5-methylisoxazol-3-yl)benzene sulphonamide C19H14N4O6S 427.00 0.8 260-270 Bright
yellow 75
4b 4-((8-hydroxyquinolin-5-yl) diazenyl) -N- (5-methyl
isoxazol-3-yl) benzene Sulphonamide C19H15N5O4S 410.30 0.8 220-223 Brick
red 92
4c
2-hydroxy-5-
((4-(N-(5-methylisoxazol-3-yl)
sulfamoyl) phenyl) diazenyl)
benzoic acid
C17H14N4O6S 403. 00 0.8 227-230 Grey 73
4d 4-((2-hydroxynaphthalen-1-yl)diazenyl)-N-(5-
methylisoxazol-3-yl) benzene sulphonamide C20H16N4O4S 409.30 0.8 170-174 Orange
red 90
4e 4-((3-formyl-4-hydroxyphenyl) diazenyl)-N-(5-
methylisoxazol-3-yl) benzene sulfonamide C17H14N4O5S 386.22 0.8 170-172 Grey 90
4f 4-((4-hydroxy-5-isopropyl-2-methylphenyl)diazenyl)-
N-(5-methylisoxazol-3-yl)benzenesulphonamide C20H22N4O4S 414.14 0.8 304-310 Brick
red 84
TABLE 1: PHYSICAL CHARACTERISTIC DATA OF SULFAMETHOXAZOLE ANALGUES 4A-4F

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uconazole was used as reference standards against
bacterial and fungal strains, respectively. The lowest
concentration of the test compounds inhibiting visible
growth for bacteria and fungi was termed as minimum
inhibitory concentration (MIC). Test solutions of
synthesized molecules were prepared suing the two-
fold dilution method at a concentration level ranging
from 500-31.25 µg/ml using DMF to evaluate the MIC.
Statistical analysis:
Dunnett’s post hoc test and Dunnett’s t-test were used
and the observed data subjected to one way analysis of
variance (ANOVA).
RESULTS AND DISCUSSION
A series of phenolic/enolic substituted
azosulfamethoxazoles 4a-4f were synthesized by the
in situ reactions between diazotised SMZ and 6 different
individual phenolic and enolic compound under mild
conditions. All the proposed isoxazole derivatives
were synthesized, physical properties and spectral
interpretation were made (Table 1), which were similar
to those reported earlier[8-12].
The research work was aimed to theoretically validate
the binding of the synthesized SMZ derivatives
(4a-4f) using molecular docking against COX-2
protein of Mus musculus as depicted in Table 2. The
docking score and molecule interactions (g. 3) were
obtained in least binding energy of the compound 4d
and 4f at value -12.980 and -12.386 kcal/mol with
1-picrylhydrazyl (DPPH) assay procedure with some
modications[6] at several concentrations, the absorbance
of the test molecules and the standard butylated
hydroxy toluene (BHT) was measured at 517 nm on a
UV/Vis spectrophotometer and the antioxidant activity
was calculated. A mixture of DPPH and methanol was
considered as control. All experiments were carried out
in triplicate. Followed by Dunnett’s post hoc test, the
IC50 values were expressed as mean±SD. Inhibition (%)
= [(Acont- Atest)/Atest]×100, Acont= absorbance of control,
Atest= absorbance of the test and standard samples, The
IC50 value was graphically determined.
Microbiological evaluation:
The antimicrobial activity of the synthesized molecules
was investigated according to the agar well diffusion
method using the nutrient agar and Sabouraud dextrose
agar medium (HI-Media) for bacteria and fungi,
respectively. The antimicrobial diffusion test was
performed using a cell suspension of about 1.5×106
CFU/ml employing a McFarland turbidity standard
No. 0.5[14]. The microbial strains K. pneumonia
(MTCC 109) Candida albicans (MTCC 3017) and
C. neoformans (MTCC 3019) were procured from
the Institute of Microbial Technology and Gene bank
(IMTECH), Chandigarh, India. Escherichia coli
and Staphylococcus aureus resistant to noroxacin,
ooxacin, and ampicillin were isolated from the
urine sample collected from UTI patients at the IMS,
SUM Hospital, Bhubaneswar, India. Amoxicillin and
Fig. 2: 1HNMR of 4-((8-hydroxyquinolin-5-yl)diazenyl)-N-(5-methylisoxazol-3-yl)benzene sulfonamide (4b)

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Indian Journal of Pharmaceutical Sciences
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Comps. Chemical name 3D- structure
Docking
score (kcal/
mol)
Interaction with amino acid of
target enzyme during docking
4a
4-[(4-hydroxy-2-oxo-2H-
chromen-3-yl)diazenyl]-N-
(5-methylisoxazol-3-yl)
benzene sulphonamide
-10.958 LYS83, PRO8, VAL89, LEU93,
ILE112, TYR115, TYR122, PHE35
4b
4-((8-hydroxyquinolin-5-
yl) diazenyl) -N- (5-methyl
isoxazol-3-yl) benzene
Sulphonamide
-10.652 PHE200, VAL295, LEU391,
PHE395, LEU408, TYR409, VAL444
4c
2-hydroxy-5-
((4-(N-(5-methylisoxazol-3-yl)
sulfamoyl) phenyl) diazenyl)
benzoic acid
-11.386
MET196, PHE200, GLN203,
VAL291, PHE292, LEU294,
VAL295, LEU298, LEU391,
PHE395, LEU408
4d
4-((2-hydroxynaphthalen-
1-yl) diazenyl)-N-(5-
methylisoxazol-3-yl) benzene
sulfonamide
-12.980
LYS83, VAL89, LEU93, ILE112,
TYR115, VAL116, SER119,
ARG120, TYR122
4e
4-((3-formyl-4-hydroxyphenyl)
diazenyl)-N-(5-
methylisoxazol-3-yl) benzene
sulfonamide
-11.159
MET196, PHE200, GLN203,
VAL291, LEU294, VAL295, LEU298,
HIS388, LEU391, PHE395, LEU408
4f
4-((4-hydroxy-5-isopropyl-
2-methylphenyl)diazenyl)-
N-(5-methylisoxazol-3-yl)
benzenesulphonamide
-12.386
PHE200, GLN208, VAL295,
LEU391, PHE395, PHE404,
LYS405, PHE407, LEU408, TYR409,
VAL444, VAL447
Acetyl
salicylic
acid
2-acetoxybenzoic acid -10.256 PHE210, ASN375, ILE377, ALA378,
PHE381
TABLE 2: CONVERTED 3D- STRUCTURES AND DOCKING SCORES OF INDIVIDUAL SMZ ANALGUES 4A-
4F AGAINST TARGET ENZYME CYCLOOXYGENASE-2
Fig. 3: Docking images captured by the software discovery studio visualizer
Docking images captured during interaction of 1CX2 with SMZ derived molecules A. 4c, B. 4d, C. 4f and D. standard acetylsalicylic
acid, respectively
highest binding afnity to COX-2 and compared
to standard those of ASA at –10.256 kcal/mol.
Compound 4-((2-hydroxynaphthalen-1-yl)diazenyl)-
N-(5-methylisoxazol-3-yl)benzene sulphonamide (4d)
binds to several amino acids, LYS83, VAL89, LEU93,
ILE112, TYR115, VAL116, SER119, ARG120 and
TYR122 of the active site of COX-2.
The absorption spectra of the molecule 4-((4-hydroxy-2-
oxo-2H-chromen-3-yl)diazenyl)-N-(5-methylisoxazol-
3-yl)benzene sulphonamide (4a) gave the largest

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bathochromic shift 425 nm with isopropanol in
comparison to other solvents. Molecule 4a and 4c
showed the maximum wave length (λmax) with ethanol
at 420 and 370 nm, respectively; the molecule 4b,
4d and 4e showed at 403.8, 480 and 360 nm with
methanol, respectively, while the molecule 4f showed λ
max at 409 nm with1 4-dioxane. The molecule 4d showed
maximum bathochromic shift with methanol, ethanol,
DMSO and THF in comparison to other molecules.
Results of acute oral toxicity study indicated that the
synthesized isoxazole derivatives were safe up to 2000
mg/kg with no mortality, no toxic symptoms and no
gross behavioural changes observed in Wistar rats. In
the acetic acid-induced model in the control group,
acetic acid produced 52.8±5.3 writhes in 10 min of
observation period and the standard ASA (100 mg/
kg) inhibited pain by 63.63 % and compounds (4a-4f)
produced 16.09, 13.25, 44.88, 58.33, 36.553 and 57.76
% pain inhibition at 50 mg/kg dose level. Among which
the 2-naphthol and thymol coupled diazotized isoxazole
molecules 4d and 4f reported with signicant percent of
pain inhibition 58.33 and 57.76 % respectively at a dose
of 50 mg/kg body weight. In radiant heat model, the
negative control group of animal showed 4.03±0.18 of
reaction time by the end of 60 min, while the compound
4d showed signicant analgesic activity by the end of
120 and 180 min for a reaction time of 6.7±0.59 and
6.3±0.5. The compound 4f noticed with reaction time
of 5.8±0.52 and 5.7±0.5 by the end of 120 and 180 min,
respectively. The maximum percent inhibition of pain
produced by the test compounds at a dose of 50 mg/
kg were for compound 4d (80), 4f (62.85), 4e (51.42)
and 4a (48.57) and for the standard (77.14). However,
in both acetic acid-induced writhing and radiant heat
models molecules 4d and 4f exerted signicant analgesic
activity. The molecules 4d and 4f signicantly (p<0.05)
inhibited the inammatory oedema when compared to
negative control by the end 120 min of carrageenan
injection.
The antioxidant activity of the isoxazole derivatives was
investigated using the DPPH assay and the results were
compared with those of standard BHT. The IC50 values
of 4-[(4-hydroxy-2-oxo-2H-chromen-3-yl)diazenyl]-
N-(5-methylisoxazol-3-yl)benzene sulphonamide
(4a), 4-((8-hydroxyquinolin-5-yl)diazenyl)-N-
(5-methylisoxazol-3-yl)benzene sulphonamide
(4b), 2-hydroxy-5-((4-(N-(5-methylisoxazol-3-
yl)sulfamoyl)phenyl)diazenyl)benzoic acid (4c),
4-((2-hydroxynaphthalen-1-yl)diazenyl)-N-(5-
methylisoxazol-3-yl)benzene sulphonamide (4d),
4-((3-formyl-4-hydroxyphenyl)diazenyl)-N-(5-
methylisoxazol-3-yl)benzene sulphonamide (4e),
4-((4-hydroxy-5-isopropyl-2-methylphenyl)diazenyl)-
N-(5-methylisoxazol-3-yl)benzene sulphonamide
(4f) and the standard BHT were, 30±0.78, 52±1.13,
59.7±0.03, 38±1.4, 48±0.89, 40±0.59 and 31±0.70 µg/
µl, respectively. However, compound (4a) exhibited
IC50 at the lowest concentration level of 30±0.78 µg/µl
in comparison to other compounds tested. The molecule
4d and 4f showed signicant antioxidant activity at 10
and 50 µg/µl, with % inhibitions of 36.53±0.02 and
61.41±0.04 when compared to the standard.
Compounds 4a and 4e showed signicant antimicrobial
activity against S. aureus(res) and C. neoformans
in comparison to amoxicillin and uconazole,
respectively, whereas compounds 4b-4d showed
signicant antibacterial activity against E. coli(res)
in comparison to standard (Table 3). The inhibitory
property of the isoxazole derivatives was determined
in the concentration range of 500-31.25 µg/ml to nd
out the MIC values in µg/ml. The compound 4a have
been exhibited more potential antibacterial activity by
inhibiting the growth of S. aureus(res), C. Albicans and
C. neoformans at a concentration level of 31.25
µg/ml. The reported MIC results against different
microbial strains by the compound 4b would be
considered as highly effective antimicrobial compound
Comps. E. coli(res) K. pneumonia S. aureus(res) C. albicans C. neoformans
4a - - 24.00 ± 0.63* 24.17 ± 0.75 31.50 ± 0.84*
4b 20±2* 15.83±1.17 25.33±1.97*8.83±1.33 17.5±2.17
4c 22±1.55* 25±1.1* - 21±2.83 21.17±0.98
4d 15.67±1.21* 13±2.19 15.17±2.23 - 14.17±0.75
4e - - 28.17±1.47* - 18.17±2.32*
4f 9.83±1.84 9.57 9.41 - 7.83±1.17
RA/RA112.67±1.51 15.33±1.97 13±1.67 19.33±4.68 24.17±1.94
TABLE 3: ANTIMICROBIAL ACTIVITY OF SMZ ANALGUES 4A-4F AGAINST DIFFERENT MICROBIAL
STRAINS
Results expressed in mean±SD of zone of inhibitions in mm (n=6), data were analyzed using One Way ANOVA followed by Dunnett’s Post
Hoc test, statistical signicance at *p<0.05 in comparison to the reference antibiotic (RA), - no zone of inhibition, E. coli(res)- Escherichia
coli (resistant), K. pneumonia- Klebsiella pneumonia, S. aureus(res)- Staphylococcus aureus (resistant), C. albicans- Candida albicans ,C.
neoformans- Cryptococcus neoformans

www.ijpsonline.com
January-February 2020
Indian Journal of Pharmaceutical Sciences
130
when compared to other synthesized isoxazole
derivatives. However, all the synthesized molecules
showed potential fungal inhibitory property against
C. neoformans.
The resultant docking score of the molecules suggested
that the molecules 4d and 4f would be potent COX-
2 inhibitors. It was also found that from the in vivo
evaluations that the molecule 4d and 4f showed highest
analgesic and antiinammatory activity among all
compounds tested; its action could be due to linking
5-methylisoxazolyl moiety to 2-naphthol and thymol,
respectively. Literature supports that isoxazole derived
molecules have shown to possess the inhibitory property
of COX-2 and also revealed that the nitrogen heterocyclic
molecules bearing –N=N- are responsible for inhibition
of COX[14]. In present study, the synthesized molecules
contained diazenyl function group along with isoxazole
nucleus and sulphanilamide together in their structures.
Overall structural activity relationships (SAR) study
of all analogues had suggested that the presence of
phenolic/enolic hydroxyl, diazenyl group and nitrogen
containing heterocyclic rings in their structures could be
responsible for exhibiting the antioxidant, antimicrobial
and antioxidant property.
A series of isoxazolyl derivatives were prepared by
azo-coupling reactions and evaluated to investigate
their various biological actions. The result suggested
that the analogue 4d and 4f exhibited signicant
analgesic, antioxidant and antimicrobial activities
in comparison to standard drugs. Furthermore, the
plausible binding sites of these synthesized derivatives
could be designated through molecular docking. Thus,
SARs of synthesized derivatives suggested that the
presence of 5-methylisoxazolyl moiety and a phenolic
system, could yield potential leads for developing new
therapeutic agents.
Acknowledgements:
The authors thank the Dean, School of Pharmaceutical
Sciences, Siksha ‘O’ Anusandhan University and
Deputy Director RIPAES, Bhubaneswar, India.
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