Synthesis, biological evaluation, molecular docking, and ADMET studies of some isoxazole-based amides

Article abstract of DOI:10.1007/s00044-017-2070-z

Some isoxazole-based amides were synthesized by the reaction of 3-(2-chlorophenyl)-5-methylisoxazole-4-carbonyl chloride with various aliphatic, aromatic and heterocyclic amines; characterized by analysis of spectroscopic data and evaluated for in vivo an

Full text of DOI:10.1007/s00044-017-2070-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2070-z
ORIGINAL RESEARCH
Synthesis, biological evaluation, molecular docking, and ADMET
studies of some isoxazole-based amides
Sushama Kauthale1 Sunil Tekale1 Manoj Damale2 Jaiprakash Sangshetti3
Rajendra Pawar1
●
●
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Received: 17 June 2017 / Accepted: 9 September 2017
© Springer Science+Business Media, LLC 2017
Abstract Some isoxazole-based amides were synthesized
by the reaction of 3-(2-chlorophenyl)-5-methylisoxazole-4-
carbonyl chloride with various aliphatic, aromatic and het-
erocyclic amines; characterized by analysis of spectroscopic
data and evaluated for in vivo anti-inflammatory, ulcero-
genic, and antimicrobial activity. Compounds A1, A7, and
A10 were identified as the potent anti-inflammatory agents
in carrageenan-induced albino rat paw edema assay exhi-
biting 92.85–93.57% edema inhibition after 5 h with lower
ulcer index (2.5) than the standard diclofinac sodium (6.15).
Antibacterial activity against the bacteria Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus, and
Bacillus subtilis was found to be good in comparison with
the standard ampicillin in terms of minimum inhibitory
concentration values. Anti-inflammatory activity results
were supported by molecular docking—possible binding
modes, interactions, and docking scores of titled com-
pounds with the active site of cyclooxygenase-2 enzyme.
In silico absorption, distribution, metabolism, and
excretion–toxicity study was also performed to predict the
preliminary pharmacological, pharmacokinetic, and toxicity
profile of the synthesized anti-inflammatory agents sug-
gesting that these derivatives have good oral drug like
behavior and non-toxic nature.
●
●
Keywords Molecular docking ADMET
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Anti-inflammatory Ulcerogenic Antibacterial.
Introduction
Most of the commercially marketed drugs are small mole-
cules; contributing about 90% of the therapeutics among
pharmaceuticals. Small size of drug molecules assists in
enhancing possibility of rapid diffusion through cell mem-
branes; so that the drug molecule can reach intracellular
sites of receptor. Thus, from pharmacology and molecular
microbiology point of view discovery of therapeutically and
medicinally active small molecules is highly desirable
(Scott et al. 2016).
Anti-inflammatory drugs are the compounds used to
alleviate inflammation or swelling. These drugs inhibit one
or more isoforms of cyclooxygenase (COX) enzyme which
catalyzes arachidonic acid present in cell membranes into
inflammatory mediators such as prostaglandins (PGs),
prostacyclins, and thromboxanes. Unfortunately some of
the classical non-steroidal anti-inflammatory drugs induce
their action with side effects of ulcer generation, gastric
bleeding, and cardiovascular effects as a consequence of
inhibition of COX-2 enzyme. Few years back anti-
inflammatory drugs like valdecoxib and rofecoxib were
withdrawn from pharmaceutical market due to such side
effects. Consequently design, synthesis, and discovery of
non-steroidal anti-inflammatory agents with minimum
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2070-z) contains supplementary
material, which is available to authorized users.
* Rajendra Pawar
rppawar@yahoo.com
1
Department of Chemistry, Deogiri College, Station Road,
Aurangabad, MS 431 005, India
2
Department of Pharmaceutical Chemistry, Shri. Bhagwan College
of Pharmacy, Aurangabad, MS 431003, India
3
Y. B. Chavan College of Pharmacy, Dr. Rafiq Zakaria Campus,
Aurangabad, MS 431001, India

Med Chem Res
Fig. 1 Some isoxazole and
amide-based commercial drugs
F3C
N
O
O
N
O
H
N
N
NH
N
H
O
H2N
O
S
O
O
(1)
(2)
(3)
N
O
O
N
S
O
HN
N
NH2
N
R
N
Z
(4)
(5)
(6)
ulcerogenic properties becomes a challenging goal in
medicinal chemistry. An antibacterial agent is a compound
which kills or inhibits the growth of bacteria. Search for the
discovery of new antibacterial agents is essential due to
resistance shown by bacteria to existing antibacterial drugs
on account of their genetic modifications.
a
Cl
O
Cl
O
Base, solvent
r. t. , 1 h
RNH2
R
N
O
Cl
N
O
N
H
(Scheme 1)
(A1-14)
Isoxazole ring is an imperative skeleton of many phar-
maceutically active ingredients such as valdecoxib (1),
isocarboxazid (2), leflunomide (3) etc. (Fig. 1). Valdecoxib
is a nonsteroidal anti-inflammatory drug. Isocarboxazid is
an antidepressant drug that acts irreversible monoamine
oxidase inhibitor and mainly used to treat mood, anxiety,
Parkinson’s disease, and dementia-related disorders. Leflu-
nomide; an immunosuppressive antirheumatic drug is used
in active moderate-to-severer heumatoid arthritis and psor-
iatic arthritis (Krensky et al. 2006). Simple amide molecules
including pyrazinamide (bacteriostatic antitubercular agent)
(4), mepivacaine (5) (local anesthetic) etc. also serve as
drugs for the welfare of mankind (Zimhony et al. 2000;
Porto et al. 2007). Furthermore studies reveal that benzo[d]
thiazol-2-yl-(piperazin-1-yl)methanones (6) act as potent
anti-mycobacterial chemo types (Pancholia et al. 2016).
Biological potential of isoxazole and amide functionality
bearing molecules is reviewed by various researchers
(Rajput et al. 2015; Kaur et al. 2014). Recently some novel
furancarboxamide derivatives were shown to be potent
antifungal targets against Rhizoctonia solani, Botrytis cir-
erea, Valsa mali, and Sphaceloma ampelimum (Wen et al.
2016).
b
Cl
Cl
O
O
Reflux, 100 oC, 1h
NH3
N
O
(A14)
NH2
N
O
Cl
(Scheme 2)
Fig. 2 a Synthesis of isoxazole-based amides A1-13. b Synthesis of
isoxazole amides 3-(2-chlorophenyl)-5-methylisoxazole-4-carbox-
amide (A14)
Molecular docking has given brief insight into strength of
molecular complexes, suggesting that the synthesized deri-
vatives have a strong potential to inhibit the prostaglandin
precursor. The experimentally observed results of anti-
inflammatory activity were supported by theoretical pre-
dictions of possible interactions and binding modes of these
“drug like” molecules with COX-2 enzyme from molecular
docking studies. In silico physicochemical and pharmaco-
logical properties of the synthesized compounds were pre-
liminarily studied using absorption, diffusion, metabolism,
excretion toxicity (ADMET).
Considering therapeutic potential of isoxazoles, sig-
nificant biological activities of amide derivatives and
mechanism of action of non-steroidal anti-inflammatory
drugs, in the present studies we report synthesis of some
isoxazole-based amides (Fig. 2a, b). Furthermore, screening
of the synthesized isoxazole amides was aimed to explore
their potential as non-steroidal anti-inflammatory agents
with minimum ulcerogenecity and improved safety profile
as an attempt to search for “small drug like” molecules.
Materials and methods
Chemicals used were SD fine or Sigma Aldrich made and
used without further purification. Reaction progress was
monitored using thin layer chromatography (TLC) (30%
ethyl acetate: n-hexane) on silica gel precoated aluminum
plates. Structures of products were confirmed by analysis of
spectroscopic data (1H nuclear magnetic resonance (NMR),

Med Chem Res
1
13C NMR, infrared (IR), and mass spectra). H NMR (400
MHz) and 13C NMR (100.6 MHz) spectra of the products
were scanned on Brucker Vector 400 MHz spectro-
photometer in CDCl3 solvent using tetramethyl silane
(TMS) as an internal standard. IR spectra were recorded on
a Shimadzu (Prestige IR21) spectrophotometer. Diclofenac
sodium (Themis Pharmaceuticals, Mumbai), carrageenin
(Sigma Chemical Co, St Louis, MO, USA) were used for
screening the biological activity of the synthesized amides.
as monitored by TLC, the contents were cooled to room
temperature and the precipitated solid was filtered off under
vacuum as the crude product which was washed with ice-
cold water (3 × 5 mL), dried and further recrystallized from
ethanol.
All the synthesized compounds were characterized by
analysis of spectroscopic data—1H NMR, 13C NMR, IR,
and mass.
Acute toxicity study
Synthesis of isoxazole amides
Preliminary experiments were carried out on rats (n = 6).
Compounds A1–A14 were administered orally in different
doses to find out the range of doses which cause zero and
100% mortality of animals. Acute oral toxicity was con-
ducted according to the method of Organization for Eco-
nomic Co-operation and Development (OECD) (OECD
2001). Animals were kept fasting providing only water.
Compounds A1–A14 were given p.o. in doses of 100, 200,
300, and 400 mg/kg/p.o. administered orally for 4 days of
six groups of rats (n = 6) and the animals were kept under
observation for mortality as well as any behavioral changes
for evaluation of a possible anti-inflammatory effect. The
minimum lethal dose for our present study was found to be
>400 mg/kg per body weight.
The isoxazole based amides were synthesized by the reac-
tion of various aliphatic, aromatic and heterocyclic amines
with
3-(2-chlorophenyl)-5-methylisoxazole-4-carbonyl
chloride in presence of triethyl amine or sodium bicarbonate
base at 0 °C-room temperature or refluxing with ammonia
within 1 h (Fig. 2a, b).
Procedure for the synthesis of A1
A solution of thiosemicarbazide (5 mmol) in acetone (5 mL)
was reacted with portion wise addition of 3-(2-chlor-
ophenyl)-5-methylisoxazole-4-carbonyl chloride (5 mmol)
in the presence of sodium bicarbonate (7.5 mmol) with
continuous stirring under ice cold condition. After com-
pletion of reaction (monitored by TLC); acetone was eva-
porated under reduced pressure, poured onto ice-cold water,
resulting solid precipitated was filtered, washed (3 × 5 mL)
with water and finally recrystallized from ethanol.
Anti-inflammatory activity of isoxazole based amides
(A1–A14)
In vivo anti-inflammatory activity of the synthesized com-
pounds A1–A14 was evaluated using carrageenan induced
paw edema bioassay in rats and results were compared with
diclofinac sodium as the reference drug. Animal studies
were approved by Institutional Animal Ethics Committee
(IAEC), constituted for purpose of control and supervision
of experimental animals by the Ministry of Environment
and Forests, Government of India, New Delhi, India.
Wistar rats (150–200 g) were group housed (n = 6) under
a standard 12 h light/dark cycle at controlled conditions of
temperature and humidity (25 2 °C, 55–65%). Rats
received standard rodent chow and water ad libitum. Rats
were acclimatized to laboratory conditions for 7 days before
carrying the experiments. All experiments were carried out
in a noise-free room between 08.00 and 15.00 h. Separate
group (n = 6) of rats was used for each set of experiments.
Animals were kept fasting providing only water. Com-
pounds A1–A14 were given doses of 10 and 20 mg/kg/p.o.
and administered orally for 4 days of six groups of rats (n =
6) and the animals were kept under observation for mor-
tality as well as for any behavioral changes for evaluation of
a possible anti-inflammatory effect (Antarkar et al. 1994;
Winter et al. 1962; Kasahara et al. 1985; El-Sayed et al.
2010; Abbas et al. 2010).
General procedure for the synthesis of amides A2–A13
Triethyl amine (6 mmol) was added to an ice cold solution
of amine (5 mmol) in dichloromethane (5 mL) followed by
portion wise addition of 3-(2-chlorophenyl)-5-methylisox-
azole-4-carbonyl chloride (5 mmol). The contents were
slowly warmed to room temperature and stirred for 1 h.
After completion of reaction as monitored by TLC, DCM
was evaporated under vacuum on rotary evaporator; the
reaction mixture was basified with an ice cold aqueous
saturated sodium bicarbonate and extracted with DCM (3 ×
10 mL). The combined organic layers was dried over
anhydrous sodium sulfate and concentrated to get the crude
product which was further recrystallized by triturating with
DCM and n-hexane.
Procedure for the synthesis of 3-(2-chlorophenyl)-5-
methylisoxazole-4-carboxamide (A14)
3-(2-Chlorophenyl)-5-methylisoxazole-4-carbonyl chloride
(5 mmol) in concentrated ammonia (10 mL) was refluxed at
100 °C for 1 h in a sealed tube. After completion of reaction

Med Chem Res
Table 1 Effect of different isoxazole based amides (A1–A14) on paw
edema induced by carrageenan in rats
Carrageenan induced hind paw edema
Treatment
Dose
(mg/kg)
Mean differences
in paw volume
(mL)
Percentage
of inhibition
(%)
The animals were divided into five groups of six animals
each and were fasted for a period of 24 h prior to study.
Group 1 was treated as control (0.1 mL of 1% (w/v) of
Carrageenin subcutaneously), Group 2 was received diclo-
fenac sodium 30 mg/kg, p.o. Group 3 were treated with the
compounds A1–A14 (10 and 20 mg/kg, p.o.). Edema was
induced by injecting 0.1 mL of 1% solution of carrageenan
in saline into sub plantar region of right hind paw of the
rats. Volumes of edema of injected and contra lateral paws
were measured at time interval after induction of inflam-
mation using a plethysomgraph to calculate the percentage
of paw edema inhibition using Eq. (1) (Table 1).
Normal
0.1 mL of
1% (w/v)
1.40 0.05
–
Diclofenac sodium
30
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
0.60 0.07*
0.95 0.05
0.65 0.07*
0.97 0.05
0.80 0.06*
0.95 0.05
0.69 0.05*
0.99 0.04
0.82 0.06*
0.98 0.07
0.71 0.05*
0.97 0.06
0.72 0.05*
0.95 0.06
0.66 0.05*
0.97 0.05
0.71 0.06*
0.98 0.05
0.72 0.07*
0.94 0.05
0.66 0.05*
0.96 0.06
0.73 0.07*
0.96 0.05
0.78 0.05*
0.95 0.05
0.76 0.05*
0.95 0.04
0.78 0.06*
97.14
72.14
93.57
70.71
82.85
72.14
90.71
69.28
81.42
70.00
89.28
70.71
88.57
72.14
92.85
70.71
89.28
70.00
88.57
72.85
92.85
71.42
87.85
71.42
84.28
72.14
85.71
72.14
84.28
A1
A2
A3
A4
Vc À Vt
Percentage inhibition ¼
 100
ð1Þ
Vc
A5
where Vc is the Edema volume of control group, Vt the
Edema volume of test group
A6
Ulcerogenic activity study
A7
Rats were fasted 20 h before drug administration. Com-
pounds A1–A14 and diclofenac sodium were given orally
in different doses respectively suspended in 1% Tween;
while one group received vehicle (1% Tween). Rats were
fasted for 2 h, allowed to feed for 2 h then fasted for another
20 h. Rats were given another two doses on the second and
third days. On fourth day, rats were sacrificed, stomach
removed, opened along with the greater curvature and
rinsed with 0.9% saline. The number of mucosal damage
(red spots) was counted and their severity (ulcerogenic
severity) was graded from 0 to 4 (Table 2) according to the
following score assignment using Eqs. (2) and (3):
A8
A9
A10
A11
A12
A13
A14
% Incidence=10 ¼½number of rats showing ulcer of any grade
divided by total number of rats in the group  100Š=10:
ð2Þ
Each data suggests mean SEM (n = 6). One-way ANOVA using
Dunnett’s test was applied for statistical analysis, treatment groups
compared with control group. Significant at *p < 0.01, compared to
control group
Total ulcerscore
ð3Þ
Ulcer index ðUIÞ ¼
Number of animals ulcerated
Antimicrobial activity
antibacterial activity. Dimethyl sulfoxide was used as the
control.
Antibacterial activity was evaluated against four different
gram-positive and gram-negative bacterial strains such as
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus
aureus, and Bacillus subtilis. Minimum inhibitory con-
centration (MIC) values were determined using standard
agar method (Cruickshank et al. 1975; Collins 1976; Khan
1997; Duraiswamy et al. 2006; Saundane et al. 1989).
Ampicillin was used as the standard for comparison of
Molecular docking
To explore binding affinity, binding mode and possible
molecular interactions of the synthesized isoxazole based
amides; compounds were docked against active site of
COX-2, an isoform of cycloxygenase or prostaglandin
endoperoxidase synthease (PGHS) using Surflex-Dock

Med Chem Res
Table 2 Effect of isoxazole based amides (A1–A14) on diclofenac
sodium induced ulcer in rats
which is used to describe disposition of a pharmaceutical
compound within an organism. In the present study,
ADMET properties of the synthesized isoxazole amides
were carried using ADMET predictor FAF Drugs2 which
runs on Linux OS. This tool is freely available and used for
in silico ADMET filtering.
Treatment Dose
(mg/kg)
No. of
animals
%
Ulcer index
Incidence
with ulcer /10
Normal
0.1 ml of 1% (w/v)
0
0.0
0
Diclofenac 30
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
6.15 1.10
3.50 0.10
2.70 0.10
3.80 0.12
2.95 0.10
3.90 0.13
3.00 0.10
3.90 0.15
3.00 0.10
3.80 0.12
2.90 0.10
3.80 0.15
2.90 0.15
3.60 0.10
2.70 0.10
3.90 0.12
3.00 0.10
3.80 0.10
2.90 0.15
3.50 0.10
2.70 0.15
3.80 0.10
2.90 0.10
3.80 0.12
2.90 0.15
3.70 0.10
2.80 0.15
3.90 0.10
3.00 0.17
A1
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
Results and discussion
A2
In case of amides A1–A13, addition of 3-(2-chlorophenyl)-
5-methylisoxazole-4-carbonyl chloride to thiosemicarbazide
or amine was carried out in the presence of appropriate base
(NaHCO3 or triethyl amine) under ice-cold condition and
then the contents were stirred at room temperature for 1 h.
For synthesis of 3-(2-chlorophenyl)-5-methylisoxazole-4-
carboxamide (A14), starting material 3-(2-chlorophenyl)-5-
methylisoxazole-4-carbonyl chloride was refluxed with
ammonia at 100 °C for 1 h in a sealed tube. Almost all the
amines reacted smoothly with the acid chloride to afford
corresponding amide derivatives in good to excellent yields.
Yields of the products were comparatively higher in case of
amines possessing electron donating groups (Entries 5, 12,
14; Table 4). Obtained products were purified by crystal-
lization with ethanol (A1, A14) or by triturating with DCM
and n-hexane (A2–A13). Thus no chromatographic techni-
que was required to purify the obtained products. All the
compounds were fully characterized by analysis of spec-
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
1
troscopic data H, 13C NMR, IR, and mass. A broad signal
1
around 12.5 δ ppm in H NMR spectra indicated the for-
mation of amide. In 13C NMR, carbonyl carbon of amide
group appeared in the range of 168–175 whereas in IR
spectra N–H stretch of amide appeared as an absorption
band around between 3200 and 3400 cm−1 confirming the
formation of amide. Products were confirmed by presence
of molecular ion peaks at appropriate values in the mass
spectra.
Each data suggests mean SEM (n = 5). One-way ANOVA using
Dunnett’s test was applied for statistical analysis, treatment groups
compared with control group; significant at *p < 0.01, compared to
control group
Anti-inflammatory activity
All analysis was performed using graph pad prism for
Windows. Statistical analysis was expressed as mean
standard error of the mean (SEM). Data was analyzed by
one way ANOVA, where applicable p < 0.05 was con-
sidered statistically significant, compared with vehicle fol-
lowed by Dunnett’s test. Results of experiments for 10 and
20 mg/kg concentrations are summarized in Table 1; Fig. 3.
Satisfactory results were observed for 20 mg/kg
concentration.
module of Sybyl 2.1.1 package. Post docking analysis was
performed in order to validate docking accuracy of the
software and ability of docking software for reproducing
docking results by analyzing and comparing RMSD values
(Table 3).
ADMET study
Anti-inflammatory activity of synthesized compounds
was carried out by carrageenan-induced rat paw edema
model using diclofenac sodium as the reference drug. Mean
changes in paw edema thickness of animals treated with the
In silico pharmacokinetic and pharmacological studies were
performed on the basis of absorption, distribution, meta-
bolism and excretion toxicity abbreviated as (ADMET)

Med Chem Res
RMSD Å
Table 3 Effect of different
compounds A1–14 on paw
edema induced by carrageenan
in rats and molecular docking
details
Compounds
Inhibition (%)
Molecular docking score
Total score (-log Ki)
Crash score
Polar score
A-1
93.57
82.85
90.71
89.28
81.42
88.57
92.85
89.28
88.57
92.85
87.85
84.28
85.71
84.28
97.14
4.3283
3.35
−3.2819
−2.458
1.6202
1.4119
0.8769
0.3364
0
0.045
0.056
0.049
0.057
0.046
0.053
0.052
0.05
A-2
A-3
3.4348
2.7991
2.8845
2.8713
3.2007
3.1285
2.6974
3.6011
2.7763
2.4909
2.4111
2.4224
3.4402
−2.9451
−0.6882
−1.0032
−1.4905
−1.4473
−0.4659
−0.9204
−0.5275
−1.7416
−0.7506
−0.546
A-4
A-5
A-6
0.0037
1.3894
0.8413
0.065
A-7
A-8
A-9
0.056
0.05
A-10
A-11
A-12
A-13
A-14
Diclofenac
0.8224
0.519
0.04
1.5604
0.6554
1.0196
1.4606
0.044
0.052
0.052
0.065
−0.9423
−1.8105
tested compounds from induction of inflammation was
measured, together with inhibition percent of edema and
results of anti-inflammatory activity are summarized in
Table 1 which reveals good results obtained at 20 mg/kg,
p.o. dose; comparable with the standard drug. Anti-
inflammatory activity of the compounds was found in fol-
lowing order:
Acute ulcerogenecity study
All analysis was performed using graph pad prism for
Windows and expressed as mean SEM. Data was ana-
lyzed by one way ANOVA, wherever applicable p < 0.05
was considered statistically significant, compared with
vehicle followed by Dunnett’s test (Finney 1964; Gierse
et al. 1999).
A1 > A7 > A10 > A3 > A5 > A8 > A6 > A9 > A11 >
A13 > A12 > A14 > A2 > A4
Thus, the compounds A1, A3, A5, A7, A8, and A10
were found to be more effective than rest of the derivatives.
From Table 1, compound A1 with thiosemicarbazide
moiety at 20 mg/kg concentration was the most promising
anti-inflammatory (93.57% inhibition) agent. In addition,
the compound A7 with 2-aminothiazole and A10 with
piperidine ring was found to be equipotent in terms of %
paw edema inhibition (92.85% inhibition). Compound A3
with 4-fluoroaniline had 90.85% paw edema inhibition had
good anti-inflammatory activity as compared with A2
having Cl-atom at the same position which could be prob-
ably due to small size of fluorine atom. Similar results were
observed in case of A7 and A8 with 2-aminothiazole and 2-
aminobenzothiazole moieties respectively. However sur-
prisingly, compound A4 with 3-Cl, 4-F substituent had the
least anti-inflammatory activity (81.42% inhibition) with
higher ulcer index. Further increase in ring size of amine
part of the tested compounds had no significant effect on
anti-inflammatory activity as in the case of A11 having
naphthyl ring. Compounds A7 and A10, A5 and A8, A6
and A9, A12 and A14 were observed to be equipotent anti-
inflammatory agents in terms of % edema inhibition.
Acute ulcerogenicity study
Acute ulcerogenicity is usually expressed as a side effect
and consequence of anti-inflammatory action. Compounds
A1-A14 with best overall outline in animal efficacy model
were evaluated for gastric ulcerogenic potential in rats
(Table 2; Figs. 4 and 5). Ulcerogenic effect was compared
to a classical NSAID, diclofenac sodium (U.I. = 6.15).
Interestingly all the synthesized compounds showed mild
ulcerogenic potential as they have lower ulcer indices
(2.7–3.8) at 20 mg/kg p.o. dose than the standard diclofinac
sodium (U. I. = 6.15). Tested compounds showed ulcero-
genic activity in the following order:
(A1 = A7) < A10 < A13 < (A5 = A11)<(A6 = A9 =
A12) < A2 < (A3 = A4 = A8) < A14
Results revealed that compound A14 has the highest UI
(3.90) and lowest UI was observed in case of A1 and A7
(2.7). Thus the compounds A1, A7, A10, and A13 con-
taining Cl, F and—NO2 substituents were found to be safer
with lower ulcerogenic index and A14 was found to be the
least safe anti-inflammatory agent.

Med Chem Res
Table 4 Yield and melting points of isoxazole-based amides
Table 4 continued
Yield@
M. P.
(°C)
Entry
Compound
Structure
Yield@
84
M. P.
(°C)
Entry
Compound
Structure
1
A-1
87
175–177
9
A-9
124–126
116–118
179–181
119–121
122–124
138–140
Cl
O
Cl
O
H
N
S
N
O
N
H
N
O
N
NH2
O
2
3
4
5
6
A-2
A-3
A-4
A-5
A-6
92
90
86
90
93
171–173
10
11
12
13
14
A-10
A-11
A-12
A-13
A-14
87
90
92
81
93
Cl
Cl
O
O
O
Cl
O
N
N
N
O
N
H
O
158–160
151–153
148–150
111–113
F
Cl
Cl
N
O
N
O
N
H
N
H
O
F
Cl
Me
Cl
O
N
N
H
Cl
N
O
N
O
H
OCF3
Cl
O
Cl
O
N
O
N
N
O
N
NO2
H
H
Cl
O
Cl
O
N
O
N
N
O
NH2
H
F
7
A-7
82
172–174
@Yields isolated for reaction of amine (5 mmol) with 3-(2-
chlorophenyl)-5-methylisoxazole-4-carbonyl chloride (5 mmol) in the
presence of triethyl amine or NaHCO3
Cl
O
N
S
N
O
N
H
Antibacterial activity
8
A-8
80
161–163
Results of antibacterial activity of the isoxazole-based
amides against various bacteria E. coli, P. aeruginosa, S.
aureus and B. subtilis were found to be good in terms of
MIC values and were compared with the standard ampicilin
as summarized in Table 5. The isoxazole amides A2, A4,
and A13 were found to be highly active against all the tested
Cl
O
N
S
N
O
N
H

Med Chem Res
Fig. 3 Effect of isoxazole based
amides on paw edema induced
by carrageenan in rats
Fig. 4 Determination of ulcer index in excise stomach of rats
Fig. 5 Antiulcerogenic effect of
isoxazole based amides against
diclofenac sodium in rats
bacterial strains. Compound A6 possessing 4-fluoro-
benzylamine moiety had noticeable antibacterial activity
against P. aeruginosa, S. aureus, and B. subtilis having
MIC value (75–125 µg/mL) in comparison with the stan-
dard ampicillin having MIC (100–250 µg/mL). In general
MIC values of isoxazole based amides in case of S. aureus
and B. subtilis were comparable with the standard ampicilin
and the compounds were less potent antibacterial agents in
case of E. coli and P. aeruginosa.

Med Chem Res
Table 5 MIC values for the isoxazole-based amides
MIC values (µg/mL)a
Compounds
E. coli
P. aeruginosa
S. aureus
B. subtilis
A-1
200 0.16
250 .24
225 0.56
287.5 0.31
250 0.23
*
212.1 .54
275 1.13
187.5 0.32
267.5 0.6
225 0.65
125 0.19
*
*
A-2
250 0.13
275 0.24
*
A-3
250 0.02
A-4
175 0.32
225 0.25
200 0.45
125 0.58
250 0.34
125 0.37
*
A-5
275 0.24
A-6
*
75 0.84
275 0.08
*
A-7
*
A-8
250 0.24
200 0.28
225 0.53
*
A-9
*
267.5 0.44
*
A-10
A-11
A-12
A-13
A-14
Ampicillin
175 0.95
250 0.12
*
*
150 0.57
*
250 0.18
112 0.30
262.5 0.19
*
*
167.5.5 0.99
212.5 0.24
225 0.51
250 0.88
262.5 0.70
*
225 0.64
225 0.90
100 2.14
100 1.24
250 2.99
* No activity was observed up to 400 μg/mL
a
Values are the average of three readings standard deviation
Molecular docking
are separated by rotatable bonds of compounds and (c)
Polar score giving an idea about contribution of polar non-
hydrogen bonding interactions to the total score are shown
in Table 3.
COX-1 is the first isoform of PGHS mostly responsible for
maintaining gastric functions such as it provides integrity
and vascular homeostasis in case of gastric protective layer
i.e., gastric mucosa. COX-2 is unique in its function that it
only expresses inflammatory response so it acts as strong
target for non steroidal anti-inflammatory agents (Ranga-
hanumaiah et al. 2016).
Analysis of binding affinity (−logki) values and mole-
cular interactions of isoxazole-based amides such as A-1
(4.3283), A-10 (3.6011), and A-3 (3.4348) suggests that
they are the most active among all the synthesized deriva-
tives. When the most active isoxazole based amide A-1 was
compared with the reference co-crystallized ligand diclo-
fenac, it was found to exhibit a strong potential to inhibit
COX-2. Compounds A-2, A-4, A-5, A-6, A-8, A-9, A-11,
A-12, A-13, and A-14 had a potential to act as anti-
inflammatory agents which is indicated by intermediate
total docking score (3.3–2.4) given in Table 3. The highly
active isoxazole amide derivatives such as A-1 (4.3283), A-
10 (3.6011), and A-3 (3.4348) showed an efficient binding
mode and penetrating active site cavity by forming hydro-
gen bond and π interactions with the active site residues of
COX-2.
The most active derivative A-1 (4.3283) interacts with
active site amino acid residue SER 530, forms conventional
hydrogen bond interactions with oxazole oxygen at a dis-
tance 2.56 Å and TYR355 forms both conventional hydro-
gen bond interaction and pi-sulfur bond interactions with
thiourea (=S) and terminal nitrogen atoms of thiourea at a
distance of 5.59 and 2.08 Å respectively. Aliphatic amino
acid at active site GLY526 forms halogen bond interactions
with (–Cl) of phenyl ring at the distance of 3.24 Å. The
active site amino acid residues such as LEU352, VAL523,
VAL349, LEU531, and ALA527 interact with phenyl,
isoxazole ring and methyl substituent of isoxazole rings and
To identify the possible identical binding modes of
synthesized amides, they were docked into active site of
protein and further ranked based on total docking score and
possible interactions using three dimensional X-ray crystal
structure of COX-2 (PDB ID: 1PXX Resolution 2.90 Å)
complex with diclofenac (Kurumbail et al. 1996). The
complex 1PXX is advantageous over other COX-2 com-
plexes as COX-2 protein present in the complex diclofenac
showed a number of activities such as antimicrobial,
ulcerogenic, analgesic, anti-inflammatory, lipid peroxida-
tion, antitumor, and inhibitor formation of transthyretin
amyloid so it will provide important clue in understanding
details of inhibition at molecular level (El-Henawy 2013).
The number of compounds showing high inhibition per-
centage of edema calculated in carrageenan-induced rat paw
edema model were shown to posses very high total docking
score, polar score, and low crash score indicating the pre-
sence of non-covalent interactions such as hydrogen bond
interaction and π interactions (Table 3) (Huang et al. 2004).
To represent the details of docking score following terms
were used (a) Total score as total docking score (b) Crash
score as degree of inappropriate penetration by ligand into
the protein and interpenetration between ligand atoms that

Med Chem Res
Fig. 6 a Binding pose and molecular interactions of A-1 into the active site of COX-2. b Binding pose and molecular interactions of A-10 into the
active site of COX-2

Med Chem Res
sulfur atom of terminal thiourea to form various π interac-
tions such as pi-sigma, alkyl, pi-alkyl, and pi-sulfur shown
in Fig. 6a.
The second most active isoxazole amide A-10 (3.6011)
forms hydrogen bond interactions with amino acids
SER530 and conventional hydrogen bond interactions with
isoxazole oxygen of distance of 1.97 Å. The amino acids
TYR348, VAL349, LEU352, LEU359, LEU531, AL527,
PHE518, MET522, and VAL523 form weak kind of pi
interactions such as alkyl and pi-alkyl interactions with
phenyl ring Cl atom, phenyl ring, methyl substituent of
oxazole rings, piperidine ring and nitrogen atom of iso-
xazole ring (Fig. 6b).
ADMET study
In silico ADMET study was performed to evaluate phar-
macokinetic and safety potential of the synthesized
isoxazole-based amides A1–A14 and standard diclofenac
using ADMET predictor FAFDrugs2 which runs on Linux
OS. This tool is freely available and used for in silico
ADMET filtering (Lagorce et al. 2008; Sangshetti et al.
2014). Compliance of the synthesized compounds to
Lipinski’s rule of five was studied and the results are
summarized in Table 6 (Lipinski et al. 2001). This approach
has been widely used as a filter for lead molecules that
would likely be further developed for drug design programs.
Numerous non-cardiovascular drugs have been banned and
withdrawn from the drug market due to their severe cardiac
side effect such as heart arrhythmia and person may leads to
death. So it is very important to find out affinity of potential
lead compounds in drug discovery process. In order to find
cardiac activity or not various experimental techniques are
employed where they focus on testing binding affinity of
leads towards the hERG K+ channel. The hERG K+
channel in heart controls the conductions of impulses
through cardiac cycle and blockage of hERG K+ channel
may lead to server shock of arrhythmic attack. In order to
find binding affinity towards the hERG K+ channel insilico
tool Pred-hERG were used (http://labmol.farmacia.ufg.br/
predherg/) (Ertl et al. 2000). All the compounds in the study
has been Insilico tested to find out their binding affinity
towards the hERG K+ channel and all were found to be
non-blockers of hERG K+ channel (Table 6).
In order to find out role of probable metabolic cyto-
chrome (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and
CYP3A4) in metabolism of potential leads in the study,
compounds were tested for CYP450 inhibition by
employing online tool Swiss ADME (http://www.swissa
dme.ch/index.php) (Vincent et al. 2017). On the basis of
CYP450 inhibition it was found that these compounds are
potential inhibitors of CYP450 such as CYP1A2 and
CYP2C19 mentioned in Table 6.

Med Chem Res
We assessed parameters like molecular percent absorp-
tion (%ABS > 100), weight (MW > 500), partition coeffi-
cient (logP > 5), number of rotatable bonds (>10) and ratio
of H/C(>1). All the above mentioned parameters signify
oral bio-availability and good intestinal absorption. Topo-
logical polar surface area (TPSA) i.e., surface belonging to
polar atoms and molecular weight are the descriptors which
correlate with passive molecular transport through mem-
branes that allows prediction of route of transport of drugs
through the barrier membranes, intestine and blood-brain
barrier (BBB). The percentage of absorption (% ABS) was
calculated using TPSA from the formula %ABS = 109
−(0.345 × TPSA) (Winter et al. 1962).
Prediction of in silico pharmacokinetic parameters suggest
that the synthesized compounds have potential of high oral
drug bio-availability indicating that isoxazole-based amides
have new opportunities for the possible modification and
future discovery of drug candidates in medicinal chemistry.
Acknowledgements Authors are thankful to the Director, Sophisti-
cated Analytical and Instrumentation Facility (SAIF), Panjab Uni-
versity Chandigarh (India) for providing spectral data and to the
Director, Scan research laboratory, Bhopal for providing the results of
biological activity.
Compliance with ethical standards
All the synthesized compounds exhibited a very good %
ABS ranging from 62.93 to 78.64%. Values of partition
coefficient (logP > 5), number of rotatable bonds (>10),
number of rigid bonds (>25) and ratio of H/C (>1) deter-
mine the absorption performance through the lipophilic
phospholipid membranes and toxicity. Furthermore, none of
the isoxazole amides A1–14 violated Lipinski’s rule of five
indicating that these isoxazole derivatives have a potential
to be developed as orally active small anti-inflammatory
drug candidates with minimum ulcerogenic properties and
may be potentially active against COX-2.
Conflict of interest The authors declare that they have no competing
interests.
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