Structural studies of 2,5-disubstituted 1,3,4-thiadiazole derivatives from dithioesters under the mild condition: Studies on antioxidant, antimicrobial activities, and molecular docking

Article abstract of DOI:10.1080/00397911.2020.1745843

A series of 2,5-disubstituted 1,3,4-thiadiazole was synthesized and evaluated for their antioxidant and molecular docking studies. These molecules were efficiently synthesized under mild and inexpensive starting material. Construction of these molecules developed using substituted aldehydes and substituted dithioesters in presence of NCS (N-Chorosuccinimide). The compounds 4a, 4b, 4c, 4f, and 4k showed good antibacterial and antioxidant activity among which, 4k possess excellent antibacterial and antioxidant activity. The results of antioxidant activity studies revealed that compound 4k manifested profound antioxidant potential. The molecular docking study was performed with respective newly synthesized compounds to ascertain the binding mode of 4k with respect to the critical proteins involved in biosynthesis and metabolic pathways.

Full text of DOI:10.1080/00397911.2020.1745843

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Structural studies of 2,5-disubstituted 1,3,4-
thiadiazole derivatives from dithioesters under
the mild condition: Studies on antioxidant,
antimicrobial activities, and molecular docking
Kemparaje Gowda, Hassan A. Swarup, Sandhya C. Nagarakere, Shobith
Rangappa, Rangappa S. Kanchugarkoppal & Mantelingu Kempegowda
To cite this article: Kemparaje Gowda, Hassan A. Swarup, Sandhya C. Nagarakere, Shobith
Rangappa, Rangappa S. Kanchugarkoppal & Mantelingu Kempegowda (2020): Structural studies
of 2,5-disubstituted 1,3,4-thiadiazole derivatives from dithioesters under the mild condition: Studies
on antioxidant, antimicrobial activities, and molecular docking, Synthetic Communications, DOI:
10.1080/00397911.2020.1745843
To link to this article: https://doi.org/10.1080/00397911.2020.1745843
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https://doi.org/10.1080/00397911.2020.1745843
Structural studies of 2,5-disubstituted 1,3,4-thiadiazole
derivatives from dithioesters under the mild condition: Studies
on antioxidant, antimicrobial activities, and molecular docking
Kemparaje Gowdaa, Hassan A. Swarupa, Sandhya C. Nagarakereb, Shobith
Rangappac, Rangappa S. Kanchugarkoppalb , and Mantelingu Kempegowdaa
b
aDepartment of Studies in Chemistry, University of Mysore, Mysuru, India; Department of Chemistry,
c
St. Philomena’s College, Mysuru, India; Adichunchanagiri Institute for Molecular Medicine,
Mandya, India
ABSTRACT
ARTICLE HISTORY
Received 18 November 2019
A series of 2,5-disubstituted 1,3,4-thiadiazole was synthesized and eval-
uated for their antioxidant and molecular docking studies. These mole-
cules were efficiently synthesized under mild and inexpensive starting
material. Construction of these molecules developed using substituted
aldehydes and substituted dithioesters in presence of NCS (N-
Chorosuccinimide). The compounds 4a, 4b, 4c, 4f, and 4k showed
good antibacterial and antioxidant activity among which, 4k possess
excellent antibacterial and antioxidant activity. The results of antioxi-
dant activity studies revealed that compound 4k manifested profound
antioxidant potential. The molecular docking study was performed
with respective newly synthesized compounds to ascertain the binding
mode of 4k with respect to the critical proteins involved in biosyn-
thesis and metabolic pathways.
KEYWORDS
Aldehyde; antibacterial;
docking; NCS; thiadiazole
GRAPHICAL ABSTRACT
CONTACT Mantelingu Kempegowda
kmlingu@gmail.com; Rangappa Kanchugarakoppal S
rangappaks@gmail.com
Synthetic Laboratory, DOS in Chemistry, University of Mysore, Mysuru, Karnataka, 570006, India
Supplemental data for this article can be accessed on the publisher’s website.
ß 2020 Taylor & Francis Group, LLC

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K. GOWDA ET AL.
Introduction
Among the heterocycles 1,3,4-thiadiazole having wide applications in the field of bio-
logical systems.[1,2] These 1, 3, 4-thiadiazole ring has been found to be vital in
pharmacological activities because of their pharmaceutical interest.[3,4] It shows a
broad spectrum of biological activities like anticancer,[5–7] antioxidant,[8] antimicro-
bial,[9] antihypertensive activity,[10] anti-inflammatory,[11] anticonvulsants[12] and anti-
fungal activity.[13,14] Some of the important drugs containing 1,3,4-thiadiazole ring
are methazolamide, acetazolamide, sulfamethizole, and furidiazine.
Apart from pharmaceutical interests, 1,3,4-thiadiazole plays an important role in the field
of material science like it is used in photoconductivity,[15] corrosion inhibitor[16] and also in
liquid crystals.[17] Distinct methods have been reported for the synthesis of 2,5-disubstituted
1,3,4-thiadiazole in conventional methods like the use of metal catalysts under harsh reaction
conditions. Eycken and group[18] reported the synthesis of 2,5-disubstituted 1,3,4-thiadiazole
using N-tosylhydrazones and elemental sulfur in the presence of CuI and Cs2CO3. In add-
ition to this reaction between an aryl halide and tert-butyl isocyanide via isocyanide cycliza-
tion using PdCl2 and DPPP were also reported by Zhu and coworkers.[19]

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The use of the microwave irradiation method was also developed by Varma and
group[20] for the synthesis of 2,5-disubstituted 1,3,4-thiadiazole through a condensation
reaction of triethyl orthoalkanates and acid hydrazide in presence of Nafion NR50 and
phosphorus pentasulfide with alumina. The drawbacks with these protocols are, it requires
the use of transition-metal catalysts which are toxic in nature under harsh reaction condi-
tions like microwave radiation followed by tedious work-up procedure and poor func-
tional-group tolerance, have a narrow scope. Dithioesters occupy a foremost position in
organic synthesis and these are adaptable starting materials in the preparation of many
heterocyclic since their impartially stable and methods for the preparation of dithioesters
are easy. In continuation of our interest in the synthesis of a heterocyclic ring, cycloadd-
ition reaction compounds[21–26] and also based on our experiences on thioesters,[22,27–32]
we explored a method to construct 2,5-disubstituted 1,3,4-thiadiazole which were synthe-
sized from simple and inexpensive starting materials and reagents like substituted alde-
hydes and substituted dithioesters using Chlorosuccinimide.
Result and discussion
Before optimizing the method for the synthesis of 2,5-disubstituted 1,3,4-thiadiazole, we
synthesized benzohydrazonoyl chloride 2a by using hydrazine hydrate and NCS. We
took up 2a and 3a as the pilot substrate for optimizing the method. During optimiza-
tion, primarily we choose DMF as solvent and TEA as a base, the suspension was
stirred at 90 ꢀC for 2 days (Table 1, entry 1), fortunately, we observed a new spot
between 2a and 3a while monitoring the reaction. After the spectral analysis, we came
to know that the generated spot was our desired compound 4a. With our interest to
determine suitable solvent for cyclization, we have screened the solvents like DMF,
Ethanol, DMSO, THF, methanol, CH3CN, Toluene, and water (Table 1, entries 1, 5 and
13–18) and we found that there was no reaction taking place when the solvent system
became acetonitrile/toluene/water, in DMF, DMSO, THF, MeOH yields were low and
in ethanol, the reaction went smoothly to give a good yield. Further, We want to opti-
mize the base by using different bases like Na2CO3, K2CO3, NaOH, TEA, N,N-
Diisopropylethylamine (DIPEA), KOH and LiOH (Table 1, entries 2–5, 10–12). We
observed that TEA was a suitable base for the cyclization and formation of the product

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K. GOWDA ET AL.
was isolated in 80% yield (Table 1, entry 5). In order to know the stoichiometric effect of
TEA on the yield of product which we performed the reaction by changing the equivalence
of TEA (Table 1, entries 5–7), and maximum yield of the product 4a was observed at 1 Eq.
of TEA. Finally, the effect of temperature was also tested by varying the temperature, at
lower temperature yields less comparing to reflux conditions (Table 1, entries, 5, 6 and 8).
Eventually, we found that reaction of 2a (1 Eq.) and dithioesters (1 Eq.) with base TEA
(1 Eq.) by using Ethanol (10 vol) as a solvent at reflux condition for 48 h was ideal (Table
1, entry 6). With the aforementioned optimized condition we want to check the substrate
scope of the method by varying the substitution at the para position of the aryl group of
dithioesters 3, in that OMe, Me, F, Cl, Et give corresponding products 4(b–f) with the
yield of 89–95%.
Similarly, we checked the substrate scope by varying different substitution at aryl
group of benzohydrazonoyl chloride 2, such as OMe, Me, F, Cl and 3,4 dimethoxy
groups which give corresponding products 4(f–o) with the yield of 88–95% (Table 2).
Biology
Antioxidant activity
Antibacterial and antioxidant activity are closely related.[33] Hence, we further aimed to
access the antioxidant activity for 4(a–o). Radical scavenging potency of all the com-
pounds 4(a–o) was assessed in vitro by various antioxidant activities like superoxide
radical (Fig. 1a), hydroxyl radical (Fig. 1b), lipid peroxidation (Fig. 1c), and DPPH
Table 1. Optimization of reaction conditions.
Entry
Base
Equivalence (Eq.)
Solvent
Temp (ꢀC)
Time (hr)
Yield (%)
1
2
3
4
5
6
7
8
Et3N
Na2CO3
K2CO3
NaOH
Et3N
Et3N
Et3N
Et3N
Et3N
DIPEA
KOH
LiOH
Et3N
2.5
2.5
2.5
2.5
2.5
1.0
0.5
1.0
0.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
DMF
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
Ethanol
DMSO
90
90
90
90
90
Reflux
50
40
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
48
48
48
48
48
48
48
30
20
20
48
48
10
48
48
48
48
48
10
20
23
22
80
95
85
70
60
21
46
48
10
20
20
0
9
10
11
12
13
14
15
16
17
18
Et3N
Et3N
Et3N
Et3N
THF
MeOH
MeCN
Toluene
0
0
Et3N
H2O
Bold values represent the best optimized condition obtained for the synthesis of compounds.

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Table 2. The substrate scope of reactions.
(Fig. 1d).[34] The experiments were performed in triplicate at five different concentra-
tions. The results were taken as a mean standard deviation (SD). In complex metabolic
system drives the most common superoxide anions to generate in a variety of biological
systems which result in oxidative stress. Hence, an NBT assay gives a whether the syn-
thesized compounds are capable to scavenge superoxide anions. Results deliberate that
the 4k shows potent superoxide anion scavenge activity compared to 4(a–c), and 4f
(Fig. 1a). Another devastating free radical is hydroxyl radicals which are known to initi-
ate cell damage, break DNA and cause strand breakage which, in turn, result in carcino-
genesis, mutagenesis, and cytotoxicity in vivo. Thus we attempt to explore the effect of
4(a–o) on hydroxyl radicals generated by Fe3þ ions were measured by the extent of
deoxyribose degradation, as an indicator of TBA–MDA adducts formation, among the
series 4(a–o). Among the studied, compounds 4b and 4f exhibited a remarkable cap-
acity for scavenging hydroxyl radical, significantly higher than that of the standard
BHA, whereas compound 4a showed moderate scavenging activity. 4k exerts more
inhibition of hydroxyl radicals among the series (Fig. 1b). To determine the production
of malondialdehyde and related lipid peroxides in the RBC membrane, the lipid peroxi-
dation assay was carried out. The reactive system of TBA generates lipid peroxides as a

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K. GOWDA ET AL.
Figure 1. Comparison of radical scavenging activities like superoxide radical (a), hydroxy radical (b),
lipid peroxidation (c), and DPPH (d) of compounds 4a, 4b, 4c, 4f, and 4k.
byproduct induced by Ferrous sulfate: Ascorbate system. 4k showed the highest inhib-
ition compared with the 4a, 4b, 4c, and 4f of ghost lipid peroxidation, about 36 3% at
a lower concentration (75 mg) (Fig. 1c). The freshly prepared DPPH solution shows a
deep purple color with an absorption maximum at 517 nm. Based on the experimental
results, compound 4f and 4k having Methyl and methoxy substituent showed stronger
DPPH scavenging activity than others (Fig. 1d). Compounds 4b and 4c having a
methoxy substitution showed poor antioxidant properties, whereas the rest of com-
pounds showed less activity compared to the standard BHA and tocopherol
Antimicrobial activities
The results of the antimicrobial activities of the synthesized compounds 4(a–o) are
summarized in Table 3. Results confirm that the newly synthesized compounds 4(a–o)
exert a good antibacterial activity ranging from 12.5–100 mg/mL against bacterial spe-
cies. Among the series 4(a–o), compounds 4(a–c), 4f, and 4k, poses the highest antibac-
terial activity against Escherichia coli, S. pyogenes, and S. aureus (Table 3). Substitution
at R1 and R2 with Methyl (4k) and Methoxy (4f) at para position (Table 3) possess
good antimicrobial activity against the test bacterial species, Whereas the increase of
hydrophobic at R1, and R2 results in a decrease in the activity which is seen in com-
pound 4k at para and meta position, Hence the introducing methyl or methoxy at meta

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ꢁ
Table 3. Minimum inhibitory concentrations (MIC) (in mg/mL ) of the synthesized compounds
4(a–o) against bacterial species.
Compound
S. aureus
S. pyogenes
E. coli
4a
4b
4c
4d
75.0 0.16
50.0 0.21
12.5 1.05
25.0 0.26
25.0 0.17
50.0 0.82
25.0 0.44
50.0 0.87
50.0 0.41
4e
4f
4g
4h
4i
4j
4k
50.0 0.15
50.0 0.44
12.5 1.05
12.5 0.71
25.0 0.16
50.0 0.03
25.0 0.75
25.0 0.27
50.0 0.22
50.0 0.13
50.0 1.42
50.0 0.96
25.0 0.23
50.0 0.15
12.5 0.16
12.5 0.32
75.0 0.12
4l
4m
50.0 0.42
4n
4o
75.0 1.05
75.0 0.44
12.5 1.04
100.0 0.27
50.0 1.23
75.0 0.23
25.0 1.16
Ciprofloxacin
ꢁ
Values are mean SD of three replicates. Ciprofloxacin was used as a positive control against bacteria species.
position has resulted in low activity. When the same organism was tested on the agar-
well diffusion test, resulting in the same activity as seen in the serial dilution method of
determining antimicrobial activity is (Fig. 2a–c). Methyl or methoxy group at R2 at para
position alone will bear little activity (4b and 4c). These validate that the methyl/
Methoxy group very much essential for antibacterial activity. The poorer inhibitory
effect of these compounds might be attributed to the presence of fluoro, chloro, and
combination of methyl or methoxy group also in poor activity.
Molecular docking
In order to understand possible mechanisms of 4(a–o) synthesized compounds which
exerted antioxidant and antibacterial activity, we have performed molecular docking
studies to ascertain the probable bind of a potent molecule at the active site. The
molecular docking studies showed an interacting map of AmpC b-lactamase and cata-
lase. b-Lactamases are the most culprit that cause resistance to b-lactam containing
antibiotics. Several b-Lactamases inhibitors along with antibiotics are an attractive target
for drug discovery and have been determined, in making this enzyme an attractive tar-
get for a computational biologist. Hence we attempted to check the mode of binding
with b-lactamase (Fig. 3), with a hydrogen bonding with Ala307, Ser257, and Ala79.
Whereas ascertain the binding mode with catalase in exerting antioxidant activity, we
had, dock 4k into the active site of catalase. Ligand 4k interacted catalase with Leu134
and Ser196 with hydrogen bonding and with Phe140 it interacted via p–p stacking with
benzene ring of Phe140 with 1,3,4-thiadiazole ring (Fig. 3). Based on docking score, lig-
and 4j and 4k possess higher bioactivity with promising scoring function, when com-
pared to other structurally related compounds as tabulated in Table 4.
Conclusion
In the present study highlights the new synthesis route of 2,5-disubstituted 1,3,4-thiadiazole
from dithioesters in mild condition. The method is a simple and reliable approach toward
the synthesis of 2,5-disubstituted 1,3,4-thiadiazole, and paves the way for further

8
K. GOWDA ET AL.
Figure 2. Inhibition zone of newly synthesized molecules against E. coli (a), S. pyogenes (b), and
S. aureus (c) bacterial strains.
development of thiadiazole and dithioesterschemistry. The presence of 1,3,4-thiadiazole and
methyl/methoxy substitutions in an aromatic ring is posited to be the key feature for the bio-
logical potency of the synthesized compounds. Preliminary studies show that the compounds
4(a–d), and 4k possess excellent antimicrobial and antioxidant activities.
General experimental conditions
Materials
All work relating to analytical thin-layer chromatography was performed with Merck
silica gel 60 F254 aluminum plates and were visualized with UV light. The following
mobile phases were employed for TLC: chloroform, methanol, hexane, and ethyl acetate
in different ratios. The instrumental techniques employed for the characterization of the
1
1
newly synthesized compounds include H and 13C NMR and mass spectroscopy. H

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Figure 3. Molecular docking interactive map of compounds 4j (a) and 4k (b) into the AmpCb-lacta-
mase (PDB id 1KE4), binding deep inside the active site, depicting the best docking pose.
and 13C NMR spectra were recorded on a Bruker-AV (500, 400 and 126, 101 MHz,
respectively) and Agilent WM (400 and 100 MHz) Fourier transforms spectrophotom-
eter in CDCl3 or DMSO-d6 solution using tetramethylsilane (TMS) as an internal stand-
ard. Chemical shifts were recorded in ppm relative to TMS. Mass and purity were
recorded on an LC–MSD-Trap-XCT (Agilent Technologies, Inc., Santa Clara, CA). All
the reagents and chemicals used were from Sigma Aldrich chemicals.
General procedure for the preparation of product
Step 1: General procedure for the synthesis of benzylidene hydrazine using aldehyde
and hydrazine hydrate
To the solution of aryl aldehyde 1 (5.0 mmol, 1 Eq.) in Ethanol (10 vol), Hydrazine
hydrate (5.0 mmol, 1 Eq.) was added and stirred at room temperature for 6 h. Then the
reaction was monitored by TLC, after the completion of the reaction, the reaction mix-
ture was allowed to cool at room temperature, which was diluted with water and the
aqueous layer was extracted with ethyl acetate (25 mL ꢂ 3), dried over anhydrous
sodium sulfate, the solvent was removed under reduced pressure to get benzylidene
hydrazine which was then taken to the next step without any purification.
Step 2: General procedures for the synthesis of benzohydrozonychloride using chlor-
osuccinamide andbenzylidene hydrazine
To the solution of benzylidenehydrazine(4.8 mmol, 1 Eq.) in DCM (10 vol), N-
Chlorosuccinamide (NCS) was added (4.8 mmol, 1 Eq.) and stirred the reaction mixture at
room temperature for 6 h. Then the reaction was monitored by TLC, after the completion of
the reaction, the reaction mixture was allowed to cool at room temperature, which was then
diluted with water, the aqueous layer was extracted with ethyl acetate (25 mL ꢂ 3), dried
over anhydrous sodium sulfate, the solvent was removed under reduced pressure to get ben-
zohydrozonychloride, which was then taken to the next step without any purification.
Step 3: General procedure for the synthesis of 2,5-disubstituted 1,3,4-thiadiazoles
using benzohydrozonychloride and dithioesters
To the solution of benzohydrozonylchloride (1.2 mmol, 1 Eq.) in Ethanol, dithioester
(1.2 mmol, 1 Eq.) and triethylamine (1.2 mmol, 1 Eq.) were added and refluxed for 48h.
Then the reaction was monitored by TLC. After the completion of the reaction, the

10
K. GOWDA ET AL.

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reaction mixture was allowed to cool at room temperature, which was then diluted with
water, the aqueous layer was extracted with ethyl acetate (25mL ꢂ 3), dried over anhyd-
rous sodium sulfate, the solvent was removed with reduced pressure and obtained residue
was purified by silica gel chromatography to get 2,5-disubstituted 1,3,4-Thiadiazole.
Mechanism
Lone pair of electrons from Amine group in the benzohydrazonoyl chloride attacks
electrophilic carbon of dithioester to give intermediate A which eliminates upon MeSH
to give intermediate B. Then TEA abstract proton form B and in situ cyclization taking
place to generate final product 2,5-disubstituted 1,3,4-thiadiazole with the elimination of
Hydrochloric acid.
Antimicrobialactivity
Minimum inhibitory concentrations (MICs) were determined by serial dilution tech-
nique.[35] The nutrient broth, which contains the logarithmic serially two-fold diluted
amount of test compound 4(a–o) and controls were inoculated with approximately 1 to
2 ꢂ 105cells/mL of actively dividing the bacteria cells. The bacterial culture was
incubated for 24 h at 37 ꢀC, the growth was monitored visually. The lowest concentra-
tion required to arrest the growth of microorganisms which regarded as minimumin
hibitory concentration. The synthesized compounds were screened for their antibacterial
activity against E. coli, S. pyogenes, and S. aureus. The antibiotics Ciprofloxacin was
used as a positive control against bacterial species. Dimethyl sulfoxide was used as solv-
ent control. The experiments were carried out in triplicate; the results were taken as a
mean standard deviation (SD).
Agar-well diffusion testing
The agar well diffusion method was followed according to Marasini, et al.[36]
Antibacterial activities of the 4(a–o) were investigated by the agar well diffusion
method. Staphylococcus aureus and E. coli test cultures were first spread into the surface
of sterilized Mueller Hinton Agar plates using sterile cotton swabs. Five wells were
made on each plate with a sterile cork borer instrument. The potent among the serially

12
K. GOWDA ET AL.
diluted test compounds were chosen for agar–well diffusion. 15 lg in DMSO (<5%) of
each of 4(a–c), 4f, and 4k were added in one well of each plate. The antibiotics
Ciprofloxacin was used as a positive control against bacterial species. Diffusion of com-
pound, antibiotics, and DMSO were allowed at room temperature for 1 h. All of the
plates were then covered with lids and incubated at 37 ꢀC for 24 h. After incubation,
plates were observed for the zone of bacterial growth inhibition. The size of inhibition
zones was measured and antimicrobial activity of the 4(a–c), 4f, and 4k was expressed
in terms of an average diameter of inhibition zone in millimeters.
Superoxide scavenging activity
The Superoxide radical (O2ꢄꢃ) scavenging activity of 4(a–o) was measured according to
the method of Lee et al.[37] The reaction mixture containing 100 lL of 30 mM EDTA
(pH 7.4), 10 lL of 30 mM hypoxanthine in 50 mM NaOH, and 200 lL of 1.42 mM nitro
blue tetrazolium with or without 4(a–o) and SOD serving as a positive control in vari-
ous concentrations ranging from 0–50lg. After a pre-incubated solution at ambient
temperature for 3 min, 100 lL of xanthine oxidase solution (0.5 U/mL) was added to the
mixture and incubated for 1 h at 37 ꢀC and the volume were made up to 3 mL with
20 mM phosphate buffer (pH 7.4). The solution was incubated at room temperature for
20 min, absorbance was measured at 560 nm. Appropriate controls were included ruling
out the artifacts induced reaction. The control was without any inhibitor. Inhibitory
effect of 4(a–o) on superoxide radicals was calculated as follows:
Superoxide radical scavenging activityð%Þ
Absorbance of control ꢃ Absorbance of test sample
¼
ꢂ100
Absorbance of control
Hydroxyl radical scavenging activity of 2,5-disubstituted 1,3,4-thiadiazole
The hydroxyl radical scavenging activity of 4(a–o) was according to the method of
Halliwell and Gutteridge.[38] The reaction mixture containing FeCl3 (100 lM), EDTA
(104 lM), H2O2 (1 mM) and 2-deoxy- D-ribose (2.8 mM) were mixed 4(a–o) (0–50 mM)
in 1 mL final reaction volume made of potassium phosphate buffer (20 mM pH 7.4) and
incubated for one hour at 37 ꢀC. a-tocopherol and BHA were used as a positive control.
The mixture was heated at 95 ꢀC in a water bath for 15 min followed by the addition of
1 mL each of TCA (2.8%) and TBA (0.5% TBA in 0.025 M NaOH containing 0.02%
BHA). eventually, the reaction mixture was cooled on ice and centrifuged at 5000 rpm
till 15 min. The absorbance of the supernatant was measured at 532 nm using the nega-
tive control without any antioxidant considering 100% oxidation. The percentage
hydroxyl radical scavenging activity of 4(a–o) was determined.
Lipid peroxidation inhibition assay
The evaluation of the antioxidant activity of 4(a–o) based on the inhibition of peroxida-
tion in RBC ghost was done according to Shimasaki et al.[39] The evaluation of oxida-
tion was done measure by the TBA reactive substances.[40] The human erythrocyte
ghost was isolated according to the method of Dodge et al. 100 lL of ghost suspension
(300 lg membrane protein equivalent) was subjected to peroxidation by ferrous sulfate

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and ascorbic acid (10:100 lmol) in a final volume of 1 mL of Tris-buffered saline
(20 mM, pH 7.4,150 mM NaCl). The reaction mixture was treated with or without
4(a–o) (0–75 mM), a-tocopherol and BHA (400 lM) were used as the positive control.
The contents were incubated for 1 h at 37 ꢀC. The reaction was terminated by the add-
ition of 10 lL of 5% phenol and 1 mL of 1% TCA. For each system 1 mL of 1% TBA
was added, the contents were kept in a boiling water bath for 15 min, cooled and centri-
fuged at 6000 rpm for 10 min. The absorbance of supernatants was measured colorimet-
rically at 535 nm. Appropriate blanks were included for each measurement. The
negative control sample was considered as 100% peroxidation. Without any test. The %
inhibition of lipid peroxidation was determined accordingly by comparison of the
absorbance of the test samples with a negative control.
DPPH radical scavenging activity
DPPH radical scavenging activity was assessed according to the method of Shimada
et al.[41] The 4(a–o) at concentrations ranging from 0 to 75 mg were mixed in 1 mL of
freshly prepared 0.5 mM DPPH ethanolic solution and 2 mL of 0.1 M acetate buffer pH
5.5. The resulting solution was then incubated at 37 ꢀC for 30 min and measured colori-
metrically at 517 nm. a-tocopherol and BHA (0–75 mg) was used as positive control
under the same assay conditions. Negative control was without any inhibitor or 4(a–o).
Lower absorbance at 517 nm represented higher DPPH scavenging activity. The %
DPPH radical scavenging activity of 4(a–o) was calculated from the decrease in absorb-
ance at 517 nm in comparison with the negative control.
Molecular docking studies
Molecular docking protocol was followed according to the reported method by Vivek
et al.[42] Briefly, The structural drawing and geometry cleaning of novel potentials of
4(a–o) subjected to other parameters, viz., energy minimization by using OPLS 2005
force field, the addition of hydrogen atoms, neutralization of charged groups, generation
of ionization states and set pH 7.5 using Epik. Generation of tautomers and stereoisom-
ers of 32 per ligand and low-energy ring conformations and optimize the geometries
followed by generating low energy ring conformation per ligand were computed, opti-
mized by LigPrep and used for molecular docking.
The crystal structure of AmpC beta-lactamase from E. coli, and Catalasewere obtained
from the Brookhaven Protein Data Bank, whose PDB id, 1KE4 (AmpC b-lactamase),[43]
and 2CAG (Catalase).[44] The crystal structure was imported and refined by a multistep
process through the protein preparation wizard of workspace, which includes energy
minimization using OPLS-2005 force field, correct bond orders were assigned, hydrogen
atoms were added and the water molecules were removed from 5 Å hetero atom, formal
charges, amide groups of Asn and Gln were optimized. All amino acid flips were
assigned to correct geometry and hydrogen bonds were optimized. Using PROPKA, pH
was fixed and optimized to 7.5. Non-hydrogen atoms were minimized by restrained
minimization to default RMSD to 0.3 Å. Using Extra-precision (XP) docking and scor-
ing each compound docking into the receptor grid of radii 20 Å ꢂ 20 Å ꢂ 20 Å and the
docking calculation was judge based on the score.

14
K. GOWDA ET AL.
Acknowledgments
All authors thank the University of Mysore, Mysuru for NMR, spectral analysis, and
Mass analysis.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Rangappa S. Kanchugarkoppal
http://orcid.org/0000-0003-0572-6305
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