Synthesis, biological evaluation and molecular dynamics studies of oxadiazine derivatives as potential anti-hepatotoxic agents

Article abstract of DOI:10.1080/07391102.2021.1938233

Generally, herbal medicines having remarkable popularity for treating liver ailments, but they are still unacceptable because of the deprivation of herbal drug standardization. Therefore, there is a need for promising synthetic drugs to overcome the criti

Full text of DOI:10.1080/07391102.2021.1938233

Journal of Biomolecular Structure and Dynamics
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Synthesis, biological evaluation and molecular
dynamics studies of oxadiazine derivatives as
potential anti-hepatotoxic agents
Saleem Akbar, Subham Das, Ashif Iqubal & Bahar Ahmed
To cite this article: Saleem Akbar, Subham Das, Ashif Iqubal & Bahar Ahmed (2021):
Synthesis, biological evaluation and molecular dynamics studies of oxadiazine derivatives
as potential anti-hepatotoxic agents, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2021.1938233
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2021.1938233
Synthesis, biological evaluation and molecular dynamics studies of oxadiazine
derivatives as potential anti-hepatotoxic agents
a
a
, Ashif Iqubalb and Bahar Ahmeda
ꢀ
ꢀ
Saleem Akbar
, Subham Das
aDepartment of Pharmaceutical Chemistry, School of Pharmaceutical Education & Research, Anti-hepatotoxic Research laboratory, New
Delhi, India; Department of Pharmacology, School of Pharmaceutical Education & Research, New Delhi, India
b
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 23 April 2021
Accepted 28 May 2021
Generally, herbal medicines having remarkable popularity for treating liver ailments, but they are
still unacceptable because of the deprivation of herbal drug standardization. Therefore, there is a
need for promising synthetic drugs to overcome the critical liver problem. We introduce 1, 3, 4-
oxadiazine ring in this study to identify better anti-hepatotoxic agents via a suitable synthetic
route. These oxadiazine-based derivatives were structurally confirmed by analytical and spectral
data and evaluated for their anti-hepatotoxic potential. Further, in vitro hepatotoxicity studies have
been done to check the toxicity level in the synthesized compound. Compounds 5a, 5b, 5c and
9d were selected for further biological evaluation according to in vitro results. After that, CCl4-
induced animal model was used to evaluate in vivo anti-hepatotoxicity activity. Compound 5a with
52.99%, 59.3%, 79.34% and 5b with 52.16%, 57.65%, 75.10% revealed to be most promising for
reduction in level of SGPT, SGOT and ALKP, respectively. Moreover, it was also observed that the
compound 5a with 411.01%, 53.39% and 5b with 378.63%, 48.9% level of albumin and total pro-
tein were respectively. The induced-fit docking results of the compounds 5a and 5b reveal some
essential binding information and exhibited desirable ADME properties, and obeyed Lipinski’s rule
of five. In addition, molecular dynamics studies for 100ns further confirm the protein-ligand com-
plex’s stability, supporting the in vitro and in vivo data, and help in establishing the SAR of synthe-
sized compounds. Two compounds, 5a and 5 b, exhibited higher anti-hepatotoxic activity than the
standard drug silymarin.
KEYWORDS
Liver; hepatotoxicity; anti-
hepatotoxic agents; hepatic
disorder; oxadiazine;
benzoxadiazine; induced-fit
docking; molecu-
lar dynamics
Abbreviations: ADMET: absorption, distribution, metabolism, excretion, and toxicity; ALB: albumin;
ALKP: alkaline phosphatase; RMSD: root-mean-square deviation; RMSF: root-means-square fluctuation;
SAR: structure–activity relationship; SGOT: serum glutamate oxaloacetate transaminase; SGPT: serum
glutamate pyruvate transaminase; TP: total protein
CONTACT Bahar Ahmed
bakhan_ph@jamiahamdard.ac.in
Department of Pharmaceutical Chemistry, School of Pharmaceutical Education and Research,
Anti-hepatotoxic Research Laboratory, Jamia Hamdard, Hamdard Nagar, New Delhi, 110062, India
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2021.1938233.
ß 2021 Informa UK Limited, trading as Taylor & Francis Group

2
S. AKBAR ET AL.
they do not show anti-hepatotoxic activity. Previously,
Ahmed et al. (2003) reported that compounds having a 1,4-
dioxane heterocyclic ring exhibit a crucial component in
showing anti-hepatotoxic potential (Khalilullah et al., 2011).
The design and activity of 1,4-Benzodioxane-based deriva-
tives with promising anti-hepatotoxic potential were previ-
ously reported from the same research laboratory (Ahmed et
al., 2003; Bahar Ahmed et al, 2011; Khan et al., 2006). Based
on a literature survey of the anti-hepatotoxic agents, we
hypothesized the importance of oxadiazine skeleton and also
found that there is no evidence that the oxadiazine ring has an
anti-hepatotoxic activity, so it is interesting to investigate the
activity of oxadiazine-based derivatives. An induced-fit docking
(IFD) approach and molecular dynamics (MD) simulation techni-
ques are also used to better understand the molecular architec-
ture of the synthesized compounds (Gaur et al., 2015; Yadav,
Dhawan, et al., 2014; Yadav, Kalani, et al., 2013; Yadav, Kumar,
et al., 2017; Yadav & Khan, 2013). According to the IFD and MD
simulation studies, these oxadiazine-containing derivatives
mimic the necessary binding interaction of silybin with the
amino acid residue of the active site of the receptor.
Compounds 5a and 5b also show some interesting H-bonds
with ARG100 and SER431, which could be the reason for the
higher activity when compared with the standard.
1. Introduction
The liver is reflected to be the foremost vital organ having
an exocrine, endocrine and regulatory activity (Goldfischer et
al., 1981). Various etiological factors, such as nutrition, infec-
tion, toxic and environmental factors, are the primary cause
of various liver diseases. Multiple pathogenic mechanisms
contribut to different liver diseases (Joshi et al., 2018). The
liver is one of the most important organs that work with vari-
ous metabolic functions such as metabolism of lipids, protein,
and carbohydrates and remove toxic metabolites. The liver is
also involved in detoxification and the elimination of foreign
substances. Worldwide, liver disorders affect millions of people.
Autoimmune liver disorder, drug-induced liver disorder, non-
alcoholic fatty hepatic disorder and hepatitis C are the most
common reasons behind hepatopathy. Most of the hepatic dis-
orders can be prevented or cured; otherwise, they may lead to
the cause of chronic hepatic disease. Herbal medicine is used in
India since traditional times.
Generally, herbal medicines have remarkable popularity
for treating liver ailments, but they are still unacceptable
because of deprivation of identification of active pharma-
ceutical ingredients, deprivation of herbal drug standardiza-
tion, deprivation of randomized control trial and deprivation
of evaluation of toxins (Khalilullah et al., 2011). Six mem-
bered heterocyclic rings having three heteroatoms are very
less in nature, but they possess various biological applica-
tions. Oxadiazine is such a new scaffold having promising
therapeutic potential.
Various biological application is related to oxadiazine con-
taining heteroatoms at 1,2,4 or 1,3,4 places. Derivatives of
1,3,4-oxadiazine exhibited various biological applications
such as plant growth regulator, anticonvulsive activity, anti-
bacterial, nematocidal, cardiovascular, matricidal, anti-HIV,
antitumor, COX-1 inhibitors, anemic disease, viral disease,
anti-allergic, FTase inhibitors, analgesics, neuroleptics, anti-
estrogens and acaricidal activity. Oxadiazine derivatives are
used as an intermediate to synthesize some of the b-lactam
antibiotics or prodrugs, mainly penems and carbapenems
(Gurunanjappa & Kariyappa, 2017; Mohareb & Schatz, 2011;
Shet et al., 2010; Shubakara et al., 2014).
This study was undertaken to prepare and evaluate its
anti-hepatotoxic potential of some heterocyclic compounds
containing oxadiazine ring system against liver toxicity intoxi-
cated with (CCl4) in Wistar rats. Some of the compounds,
namely compounds 5a and 5b, revealed as potent anti-hep-
atotoxic agents, whereas other exhibited only a moderate
activity compared with the standard drug silymarin.
2. Materials and method
2.1. General
The open capillary tube method is used for determining the
melting point and is uncorrected. Thin-layer chromatography
(TLC) was used to validate the homogeneity of the synthe-
sized molecule using Merck silica gel 60 ASTM (70–230
mesh) opting toluene: ethyl acetate: formic acid (TEF) in the
ratio of (5:4:1) as a solvent system. The visualization of TLC
spots was done in UV light. IRAffinity-1S Fourier Transform
Infrared Spectrophotometer was used to record all FTIR
(Shimadzu Corporation, Kyoto, Japan) spectra in KBr pellets.
CDCl3 or dimethyl sulfoxide (DMSO) was used as solvents,
and tetramethylsilane as an internal standard, using Bruker
300 MHz spectrometer, was used for recording 1H NMR
(Bruker Corporation, Massachusetts, United States) spectra.
Chemical shifts were expressed in (ppm, d) parts per million,
and the signals of NMR were narrated as m (multiplets), q
(quartet), t (triplet), d (doublet) and s (singlet). The mass
spectrum was recorded on the triple quadrupole mass spec-
trometer system using argon/helium/nitrogen as FAB gas in
Waters LCMS/MS (Waters Corporation, Massachusetts, United
States). Mass spectra of some compounds were found on the
LC-MSD-Trap-XCT-Plus spectrophotometer. Elemental analysis
Besides, derivatives of oxadiazine also exhibited antifungal
ꢀꢁ
ꢀ
(Mehta, 2013), anti-apoptotic (Voracova et al., 2017), anti-
leishmanial (Mohareb & Schatz, 2011), antioxidant, DNA dam-
age inhibitory activity (Shubakara et al., 2014),
antiproliferative activity (Bakavoli et al., 2010; Jin et al., 2018),
insecticidal activity (Xu et al., 2018), antimicrobial and cyto-
toxic activity (Jin et al., 2018), and MAO-A inhibitory activity
(Khan et al., 2002). Indoxacarb has oxadiazine scaffolds origi-
nated by the E.I. DuPont Company (Wilmington, Delaware,
USA) and used as broad-spectrum insecticides (Xu et
al., 2018).
Seeds of Silybum marianum contain silymarin, also called
‘milk thistle’, which has been revealed to be the most effect-
ive anti-hepatotoxic in response to various toxicants. The iso-
meric mixture of silymarin having silybin (a), silychristin (b)
and silydianin (c) (Figure 1). Silybin is one of the most potent
components bearing 1,4-dioxane moiety, whereas there is no
1,4-dioxane ring component in the silychristin and silydianin; (PerkinElmer, Inc., Waltham, Massachusetts, United States) (C,

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Figure 1. Chemical structure of isomers of silymarin: silybin (a), silychristin (b) and silydianin (c).
H and N) data were within 0.4% of theoretical values using done for obtaining the solid, evaporated to dryness and
a Perkin–Elmer 2400 analyzer (USA).
recrystallized using ethanol (20 mL) to give 4-substituted
phenyl-5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxylic
acid 5(a–e).
2.2. Chemistry
2.2.1.4. 4-Phenyl-5,6-dihydro-4H-1,3,4-oxadiazine-2-carbox-
ylic acid (5a). Yield 82%; white Solid; m.p. 114–123 ꢂC; IR
(KBr cmꢃ1): 2713 (–COOH), 1650 (>C ¼ O), 1166 (>C–O),
2.2.1. Plan of synthesis 1
2.2.1.1. Standard method of preparation of ethyl-2-oxo-
2(2-substituted aryl hydrazinyl acetates derivatives 3(a–e).
Diethyl oxalate (0.01 moles) was added into a solvent of
ethanol with a dropwise addition of excess derivatives of
phenylhydrazine for 5–6 h at room temperature to be found
as yellow solid, which was separated by a routine workup.
1
1076 (>C–O–C<); H NMR (DMSO-d6, 300 MHz, d, ppm): 3.82
(t, 2H, >N–CH2–), 4.44 (t, 2H,–O– CH2–), 6.93–7.30 (m, 5H,
Ar–H), 11.61 (s, 1H, –COOH); 13C NMR (DMSO-d6, 300 MHz, d,
ppm): 54.3, 63.2, 113.9, 121.4, 129.8, 144.2, 150.9, 168.2; LC
MS/MS m/z: 207.2 (Mþ); Anal. calcd for C10H10N2O3: C, 58.25;
H, 4.89; N, 13.59. Found: C, 58.21; H, 4.88; N, 13.60.
2.2.1.2. Standard method of preparation of 4-substituted
phenyl-5,6-dihydro-4H-dihydro-1,3,4-oxadiazine-2-carboxyl-
ate 4(a–e). In a setup of one necked RBF with a magnetic
stirrer, 10 mL of dried dimethylformamide (DMF), 0.01moles
of ethyl-2-oxo-2(2-substituted phenyl hydrazinyl) acetate
derivatives 3(a–e) and 0.01 moles of dibromo ethane
refluxed in the presence of anhydrous potassium carbonate
for 4 h. Diethyl ether (3 ꢁ 25 mL) is used as extracting solvent
for extraction of the reaction mixture after completing the
reaction, dried with anhydrous sodium sulfate, and evapor-
ation of ether to obtain red residue of 4-substituted phenyl-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxylate 4(a–e) and
recrystallized using ethyl alcohol to get reddish-brown solid.
2.2.1.5. 4-(4-Fluorophenyl)-5,6-dihydro-4H-1,3,4-oxadiazine-
2-carboxylic acid (5 b). Yield 53%; light maroon solid; m.p.
288 ꢂC; IR (KBr cmꢃ1): 2717 (–COOH), 2364 (Ar–F), 1645
1
(>C ¼ O), 1224 (>C–O), 1072 (>C–O–C<); H NMR (DMSO-d6,
300 MHz, d, ppm): 3.80 (t, 2H, >N–CH2–), 4.43 (t, 2H, –O–
CH2–), 7.31–8.26 (m, 4H, Ar–H), 11.59 (s, 1H, –COOH); 13C
NMR (DMSO-d6, 300 MHz, d, ppm): 55.4, 67.1, 116.0, 117.3,
140.1, 151.1, 158.5, 169.2; LC MS/MS m/z: 225.01 (Mþ); Anal.
calcd for C10H9FN2O3: C, 53.57; H, 4.05; N, 12.50. Found: C,
53.60; H, 4.08; N, 13.46.
2.2.1.6. 4-(4-Bromophenyl-5,6-dihydro-4H-1,3,4-oxadiazine-2-
carboxylic acid (5c). Yield 63%; soily solid; m.p.
270 ꢂC–280 ꢂC; FTIR (KBr cmꢃ1): 2854 (–COOH), 2368 (Ar–Br),
1676 (>C ¼ O), 1296 (>C–O), 1103 (>C–O–C<); 1H NMR
(DMSO-d6, 300 MHz, d, ppm): 3.55 (t, 2H,>N–CH2–), 4.61 (t,
2H,–O– CH2–), 6.98-7.33 (m, 5H, Ar–H), 11.53 (s, 1H, –COOH);
2.2.1.3. Procedure for preparation of 4-substituted phenyl-
5,6-dihydro-4H-1,3,4-oxadiazine-2-carboxylic acid 5(a–e).
To a solution of 4-substituted phenyl-5,6-dihydro-4H-1,3,4-
oxadiazine-2-carboxylate 4(a–e) with a dropwise addition of
2% NaOH solution, 11 mmol in 25 mL of ethanol as well as
stirred well at room temperature 22 ꢂC–25 ꢂC for about 4–5 h. 13C NMR (DMSO-d6, 300 MHz, d, ppm): 54.1, 64.3, 115.9,
TLC was used for the progression of the reaction. After com- 116.1, 133.0, 143.8, 152.7, 168.3; LC MS/MS m/z: 285.83 (Mþ);
pleting the reaction, ethanol was evaporated under reduced Anal. calcd for C10H9BrN2O3: C, 42.13; H, 3.18; N, 9.83. Found:
pressure, and dilute HCl is used to acidify it. Filtration was C, 42.16; H, 3.20; N, 9.80.

4
S. AKBAR ET AL.
thiosemicarbazide (0.01 mol) in DMSO 10 mL added. At that
point, this reaction proceeded under normal conditions by
2.2.1.7. 4-(2,4-Dichlorophenyl-5,6-dihydro-4H-1,3,4-oxadia-
zine-2-carboxylic acid (5d). Yield 55%; brown solid; m.p.
290 ꢂC; FTIR (KBr cmꢃ1): 2854 (–OH), 2364 (Ar–Cl), 1676 stirring for 7–8 h, and monitoring of completion of the reac-
1
(>C ¼ O), 1269 (>C–O), 1103 (>C–O–C<); H NMR (DMSO-d6, tion is done by TLC. After completion, cold water was
300 MHz, d, ppm): 3.72 (t, 2H,>N–CH2–), 4.51 (t,
2H,–O–CH2–),7.23–7.46 (m, 3H, Ar-H), 11.54 (s, 1H, –COOH);
13C NMR (DMSO-d6, 300 MHz, d, ppm): 54.1, 63.3, 118.7,
124.9, 128.6, 133.8, 143.1, 153.0, 169.2; LC MS/MS m/z: 275.39
(Mþ); Anal. calcd for C10H8Cl2N2O3: C, 43.66; H, 2.93; N, 10.18.
Found: C, 43.64; H, 2.96; N, 10.14.
poured and solid precipitate filtered off and dried to obtain
hydrazonate (8e). Then recrystallized it.
Reaction assembly composed of single naked RBF, oil
heating system and magnetic stirrer. 5 gm of 2-chlorophenyl
N-carbamothioyl benzene carbohydrazonate (8e), 25 mL
dimethyl acetamide (DMAC) solvent and 0.7 gm of potassium
carbonate used as an acid binder were taken. The reaction
mixture was refluxed up to 115–120 ꢂC and kept over 8 h.
DMAC solvent was distilled out under vacuum. Then, the fil-
tered off solid, rinsed with water and evaporated to dryness,
and recrystallized it using 20 mL methanol.
2.2.1.8. 4-(2,4-Dinitrophenyl)-5,6-dihydro-4H-1,3,4-oxadia-
zine-2-carboxylic acid (5e). Yield 68%; Dark brown solid;
m.p. 292 ꢂC–300 ꢂC; FTIR (KBr cmꢃ1): 2858 (–OH), 1695 (>C═
O), 1545 and 1350 (–NO2), 1228 (>C–O), 1112 (>C–O–C<);
1H NMR (DMSO-d6, 300 MHz, d, ppm): 3.72 (t, 2H, >N–CH2–),
4.47 (t, 2H, –O–CH2–), 7.29–8.07 (3H, m, Ar-H) 11.63 (s, 1H,
–COOH); 13C NMR (DMSO-d6, 300 MHz, d, ppm): 56.7, 63.4,
122.3, 125.5, 132.3, 138.9, 143.1, 150.3 169.0; LC MS/MS m/z:
297.19 (Mþ); Anal. calcd for C10H8N4O7: C, 40.55; H, 2.72; N,
18.92. Found: C, 40.52; H, 2.70; N, 18.93.
2.2.2.4. 3-Phenyl-1H-4,1,2-benzoxadiazine (9a). Yield 65%;
white solid; m.p. 130 ꢂC; FTIR (KBr) cmꢃ1: 3396.64 (–NH),
3007.02 (–C ¼ C, aromatic), 1445.75 (–C ¼ N), 1070.49 (–C–O);
1H NMR (DMSO-d6, 300 MHz, d, ppm): 3.27 (1H, S, –NH),
7.52–7.65 (br S, 5H, Ar-H, B-ring), 7.85–8.35 (br S, 4H, Ar-H, A-
ring); 13C NMR (DMSO-d6, 300 MHz, d, ppm): 112.9, 115.6,
120.1, 129.2, 130.4, 143.1, 148.1; LC-MS/MS m/z: 223 (Mþ),
Adduct peak; Anal. calcd for C13H10N2O: C, 74.27; H, 4.79; N,
13.33. Found: C, 74.25; H, 4.83; N, 13.32.
2.2.2. Plan of synthesis 2
2.2.2.1. Common method of preparation of 2-chloro-
phenyl 2-substituted benzoate (6). To make a solution by
dissolving 5 gm of phenol in 50 mL of water and 5 gm of
benzoyl chloride or substituted benzoyl chloride added into
it. Until the whole mixture became alkaline, add a 10%
sodium hydroxide solution slowly. The mixture is heated and
stirred well until the odor of benzoyl chloride or 2-chloro-
benzoyl chloride has gone off. The phenyl benzoate was col-
lected by filtration. The solid residue was rinsed with cold
water, evaporated to dryness and recrystallized from
dilute alcohol.
2.2.2.5. 3-(2-Chlorophenyl)-1H-4,1,2-benzoxadiazine (9 b).
Yield 74%; white solid; m.p. 65 ꢂC; FTIR (KBr) cmꢃ1: 3396.64
(–NH), 2902.87 (¼C-H, aromatic), 1538.56 (–C ¼ C), 1442.75
(–C ¼ N), 1029 (–C–O), 680.87 (–Cl); 1H NMR (DMSO-d6,
300 MHz, d, ppm): –NH (D2O Unexchangable), 7.52–7.65 (br S,
5H, Ar-H, B-ring), 7.85–8.35 (br S, 4H, Ar-H, A-ring); 13C NMR
(DMSO–d6, 300 MHz, d, ppm): 111.2, 116.3, 120.1, 127.1,
130.4, 132.5, 142.1, 147.6; LC-MS/MS m/z: 244 (Mþ); Anal.
calcd for C13H9ClN2O: C, 63.82; H, 3.71; N, 11.45. Found: C,
63.85; H, 3.73; N, 11.40.
2.2.2.2. Procedure for the synthesis of aryl N-substituted
benzene carbohydrazonate 9(a–d). Six equivalent hydrazine
hydrochlorides (7a–d), (0.06 mol) were added into a solution
of 2-chloro phenyl benzoate in DMF (4 mL). This reaction pro-
ceeded for 24 h at 25 ꢂC. For monitoring the reaction, TLC
was used. After completion, ice water was poured into the
reaction mixture, and filtered off solid precipitate and dried
and recrystallized to obtain hydrazonate (8a–d).
Reaction assembly consisted of a single round bottom
flask, magnetic stirrer, air condenser and oil heating system.
3 gm of hydrazonate (8a–d) is dissolved in 15 mL of dimethy-
lacetamide, and 0.4 gm of potassium carbonate added into
it, used as an acid binder, was taken. Then, the reaction mix-
ture was refluxed up to 110 ꢂC–120 ꢂC, and TLC did the pro-
gress of the reaction. Cold water was added to the reaction
mixture after the completion of the reaction, and filtered off
and evaporated to dry it and finally recrystallized with
methanol to obtain oxadiazine derivatives (9a–d).
2.2.2.6. 1,3-Diphenyl-1H-4,1,2-benzoxadiazine (9c). Yield
48%; yellowish brown; m.p. 120 ꢂC; FTIR (KBr) cmꢃ1: 3292.49
(¼C-H, aromatic), 1691.57 (–C ¼ N), 1290.38 (–C–O), 935
(–C ¼ C);1H NMR (DMSO-d6, 300 MHz, d, ppm): 7.21–7.48 (m,
5H, Ar-H, B-ring), 7.46–7.62 (m, 5H, Ar-H, D-ring), 7.64–8.23
(m, 4H, Ar-H, A-ring); 13C NMR (DMSO-d6, 300 MHz, d, ppm):
112.1, 119.3, 120.5, 122.6, 128.3, 131.0, 143.9, 146.7, 148.3; LC
MS/MS m/z: 286.11 (Mþ); Anal. calcd for C19H14N2O: C, 79.70;
H, 4.93; N, 9.78. Found: C, 79.66; H, 4.94; N, 9.76.
2.2.2.7. 1-(2,4-Dinitrophenyl)-3-phenyl-1H-4,1,2-benzoxa-
diazine (9d). Yield 55%; reddish brown; m.p. 115 ꢂC; FTIR
(KBr) cmꢃ1: 3246.20 (Ar-C ¼ C), 1531.48 (N-O,asymmetric),
1344.38 (N-O, symmetric), 1128.36 (–C–O);1H NMR (DMSO-d6,
300 MHz, d, ppm): 6.90–7.27 (br S, 5H, Ar-H, B-ring), 7.45–7.54
(m, 4H, Ar-H, A-ring), 8.07 (br S, 1H, Ar-H, D-ring), 8.32 (dd,
1H, Ar-H, D-ring), 8.35 (dd, 1H, Ar-H, D-ring); 13C NMR
(DMSO-d6, 300 MHz, d, ppm): 115.9, 118.9, 123.5, 125.9, 128.5,
129.0, 129.6, 129.8, 130.0, 130.4, 132.1, 137.0, 138.3, 145.7,
2.2.2.3. Method for the synthesis of 3-phenyl-1H-4,1,2-
benzoxadiazine-1-carbothioamide (9e). To a solution of 2-
chloro phenyl benzoate in DMSO (10 mL), a solution of 148.4, 148.9, 169.5); LC MS/MS m/z: 376 (Mþ); Anal. calcd for

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
C19H12N4O5: C, 60.64; H, 3.21; N, 14.89. Found: C, 60.60; H, four rats from every group were taken for their biochem-
3.24; N, 14.85. ical evaluation.
2.2.2.8.
3-Phenyl-1H-4,1,2-benzoxadiazine-1-carbothioa- 2.3.2.1. Treatment schedule. Normal saline was given to
group I, which is used as normal control. Group II (toxic con-
mide (9e). Yield 48%; yellow; m.p. 120 ꢂC; FTIR (KBr) cmꢃ1
:
3402.43 (–N–H, primary amine), 3051.39 (Ar-C ¼ C), 1445 trol) used as carbon tetrachloride-intoxicated control and
1
(–C ¼ N), 1024 (–C ¼ S); H NMR (DMSO-d6, 300 MHz, d, ppm): liquid paraffin assorted with carbon tetrachloride in the ratio
3.80 (S, 2H, –NH2), 7.42–7.52 (m, 5H, Ar-H, B-ring), 7.55–8.06 of 1:1 and dose (1.5 mL/kg) were given only on day one.
(br S, 4H, Ar-H, A-ring; 13C NMR (DMSO-d6, 300 MHz, d, ppm): Group III served as standard control, and CCl4 blended with
110.4, 126.1, 128.9, 132.4, 137.3, 148.1, 153.0, 178.1; LC-MS/ liquid paraffin on day one and subsequently, silymarin
MS m/z: 269.06 (Mþ); Anal. calcd for C14H11N3OS: C, 62.44; H, (Silybon-70) is administered daily dose (10 mg/kg) for seven
4.12; N, 15.60. Found: C, 62.47; H, 4.10; N, 15.62.
days. On the first day, a single dose of carbon tetrachloride
was administered to groups IV, V, VI and VII, followed by a
daily dose (10 mg/kg) of the synthesized compounds (5a,
5b, 5c and 9d) orally for seven days.
2.3. Biological evaluations
2.3.1. In vitro hepatotoxicity assay
2.3.2.2. Analysis of the liver functions. Biochemical meas-
ures such as SGOT, ALKP and SGPT by treatment using syn-
thesized compounds (10 mg/kg) were considered for
evaluating their potency of anti-hepatotoxic potential. Using
the standard method, we have also evaluated other parame-
ters such as total protein (TP) and total albumin (Doumas et
al., 1971; Wootton, 1964).
MTT assay was used to determine the safety profile of the
synthesized compounds on human liver cell lines (HepG2) to
verify in vitro hepatotoxicity, as mentioned by Verma et al.
(2019). The reduction of MTT to formazan by cellular
enzymes is the basis for this assay. The test samples were
diluted with DMEM and then transferred to the 96-well
plates in different concentrations (300, 250, 200, 150, 100
and 50 mg/mL). The concentration of DMSO was kept at ꢄ
0.1%. In a 96-well plate, a volume of 100 mL of sample was
transferred to each well containing HepG2 cells (104 cells/
well), along with a positive (inoculum) and negative (media)
control. After 72 h of incubation at 37 ꢂC, 50 mL of MTT
reagent was applied to each well in the dark and incubated
for 4 h at 37 ꢂC. The formazan crystals produced by the cells
were dissolved in 50 mL of sterile DMSO after 4 h. The optical
density (OD) of cells was calculated at 540 nm using an ELISA
plate reader. The CC50 values were calculated using a curve
fitting program (Ajay et al., 2010; Guo et al., 2010).
2.3.2.3. Statistical analysis. Analysis of the found data was
carried out using one-way ANOVA and further processes
were by Dennett’s test. The data obtained from biochemical
evaluation were expressed as mean S.E. Determination of
significance was measured by Student’s t-tests and Dennett’s
test (Dunnets, 1964; Woolson, & Clarke, 2002).
2.3.2.4. Histopathology of liver. Previously reported method
was used for determining the histopathological studies
(Luna, 1968). The animals were sacrificed after 24 h of the
last dose using thiopentone Sodium, and the liver was
removed out and washed using normal saline. The liver was
serially sectioned and examined under an electron micro-
scope after staining with eosin and hematoxylin.
For this assay, 5ꢁ 103 cells/well of HepG2 cells were used,
incubated for 24 h, and MTT was applied. Using the below-given
equations, the percentage Cell Viability (CV) and Percentage Cell
Inhibition (CI) were determined (Verma et al., 2019).
%CV ¼ ðODofTestðsynthesizedcompoundsÞ
=ODofControlÞ=100
2.4. Molecular simulation studies
%CI ¼ 100ꢃ%CV
Molecular docking simulation studies mostly comprise a selec-
tion of protein followed by the preparation of the protein, grid
generation, and development of the ligand, docking, free
energy calculation (MM-GBSA), and in silico absorption, distribu-
tion, metabolism and excretion (ADME) predictions using
QikProp. Schrodinger software was used for molecular docking
2.3.2. In vivo studies
Khalilullah et al. (2011) reported that Wistar rats of either sex
were used for anti-hepatotoxic studies. Neat and clean cages
are used for housing Wistar rats, and standard pellet diet
and water ad libitum fed them. The Wistar rats were kept at
(23 2 ꢂC) with 12-h day and night cycle and 60%–70% RH
€
and MD studies (Maestro j Schrodinger, 2018).
before their use in the experiment. The Institutional Animal 2.4.1. Selection of target
The carbon tetrachloride-induced liver toxicity mediated by
CCl4, a toxic metabolite of carbon tetrachloride, is formed by
cytochrome P-450 2E1 (CYPE2E1) in hepatocytes. These free
Ethics committee, founded by Jamia Hamdard, approved this
proposal for this proposed work. All the experimental sets
contain five Wistar rats, every seven groups of either sex.
Toxic control is composed of a mixture of 1:1 ratio of car- radicals react with lipids, DNA and protein to produce tri-
bon tetrachloride and liquid paraffin. The test drugs were chloromethyl peroxyl radicals, which destroys the functional
given in the form of aqueous suspension for seven days after integrity of cell organelles. The size of cytochrome P-450 2E1
carbon tetrachloride administration. On the 8th day of study, is in the range of 70-dalton molecular weight xenobiotic to

6
S. AKBAR ET AL.
Scheme 1. Synthetic route for the preparation of the compounds 5(a–e).
much larger fatty acid signaling molecule. The structure of
2.4.3. Generation of grid
CYP2E1 contains 2.2 angstroms for an Indazole complex and Grid is prepared using minimized protein, which further
2.6 angstroms for a 4-methyl pyrazole complex. Both com- implicates the selection of ligands as a reference. It also indi-
plexes bind to their active site and show the interaction of cates drugs having binding sites related to the specific tar-
heme iron and hydrogen bonds to Thr 303. The active site of get. A prepared grid is used in further processing in the
CYP2E1 contains two adjacent neighboring voids. One advanced docking process. The grid box was generated
encircles the l-helix, and the second one forms a channel to according to the active site of the protein where the center
the protein surface.
was X: 39.98, Y: 29.95, Z: 9.93 (coordinates). With no restric-
tions, the Van der Waals radius scaling factor was set to 1.0.
Finally, the grid was created with a partial charge cutoff of
around 0.25. The Receptor Grid Generation tool in Maestro
was used to generate the grid.
2.4.2. Selection and preparation of the protein
Protein (PDB ID: 3E4E) with a resolution of 2.6 Å was carefully
chosen and downloaded by Protein Data Bank, which is
processed for simulation study of molecular docking. After
selection, the protein was imported and refine, optimized,
and minimized after eliminating the undesirable water mole-
cules and faults reports with the help of the Protein
Preparation Wizard module in Maestro (Schrodinger 2018-3).
The Prime tool was used to fill in the missing side chains
and residues. The essential water molecules were reserved,
and lastly, protein structure having the lowest energy was
obtained, which is used in the further study of docking. In
the protein structure, the active site and catalytically essen-
tial residues were preserved. Beyond 5 Å, non-protein water
2.4.4. Ligand preparation
LigPrep module in Maestro was in use for the refinement of
the drawn structure. For the preparation of ligands, the OPLS3e
force field was used. Ligand preparation process further
includes generation of tautomers, ionization state at pH
7.0 1.0 using Epik, charged group neutralization, and the add-
ition of hydrogen bond and ligand geometry was optimized.
2.4.5. Docking studies of the designed compounds
molecules were removed. Energy minimization was per- Using Glide, molecular docking studies were performed by
formed using the optimized potential for liquid stimulation the Extra precision (XP) method, which also provided favor-
(OPLS3e) force field to produce low-energy state protein able ligand poses for further examining active sites for the lig-
with a default root-mean-square deviation (RMSD) value of and binding. The docking results gave the best poses along
0.30 Å, which was then used for molecular docking. For with the dock score and glide score. The outcomes of the dock-
molecular docking, the nature of the protein was rigid.
ing studies are mentioned concisely in Table 2 and Figure 5.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Scheme 2. Synthetic route for the preparation of the compounds 9(a–e).
ligand. Additional prime side chain prediction and mini-
2.4.6. Calculation-free binding energy (MM-GBSA)
Using XP docking, we get a decent correlation between mization were performed (refinement of all residues within
good poses and a good score. However, for the whole idea
about the potential biological response of the free binding
energy of the ligands binding to the protein active site in
the docked complex in XP mode, it was further subjected to
the binding energy calculation (MM-GBSA) using Prime in
Maestro (Lyne et al., 2006).
5 Å), which helps the ligand structure and conformation
manage reorienting side chains close by (Sankhe et
al., 2021).
2.4.9. MD simulation
MD simulation was widely used to understand the function-
ality and dynamics of protein and protein-ligand systems.
Molecular docking does not represent the real biological
processes in which the protein and ligand are solvated in
water. Therefore, MD simulation was performed to tackle this
problem. The docked protein-ligand complex was further
used for the MD simulation studies. To better understand
the protein’s dynamic behavior in the presence of ligand,
MD simulation was performed using Desmond integrated
2.4.7. In silico prediction of ADME properties
After completing the docking studies, it was exciting to perform
the ADME properties prediction of the compounds. ADME pre-
diction helps us in whether the designed compounds showed
drug-like action or not. ADME prediction was made using the
QikProp module of Maestro, Schrodinger software.
€
with Maestro (Desmond j Schrodinger, n.d.). MD simulation is
2.4.8. IFD studies
a three-step process in which the first step is system builder,
second minimization and finally, the dynamics. For the simu-
lation, the OPLS3e force field was used. In the system
builder, the solvent model was selected as simple point
IFD module from the Maestro was used to perform IFD-XP.
Synthesized compounds 5a and 5b and standard were
chosen for IFD-XP docking based on the XP docking score
ranking, ADME analysis and MMGBSA free binding energy
score. For IFD, a maximum of 20 poses were set for each charge (SPC) model, which was put in an orthorhombic box,

8
S. AKBAR ET AL.
Table 1. Evaluation of anti-hepatotoxicity of synthesized compounds on SGOT, SGPT, albumin, alkaline phosphatase, total protein in Wistar rats intoxicated with
carbon tetrachloride.
Group
N 5 5
Treatment
Dosea
SGOT (IU/mL)
30.63 0.04
SGPT (IU/mL)
41.73 0.53
ALKP (KSA units)
24.58 0.16
Albumin (g/dl)
2.30 0.10
Total Protein (g/dl)
7.06 0.28
1
2
3
4
5
Normal
Toxic
Standard
Test 1 (5a)
Test 2 (5b)
Normal saline only
1.5 ml/kg (p.o.)
10 mg/kg (p.o.)
10 mg/kg (p.o.)
10 mg/kg (p.o.)
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀ
42.65 0.03
34.68 0.51
36.28 0.02
36.38 0.02
66.48 0.06
43.46 0.03
26.82 0.09
28.48 0.36
29.28 0.42
1.70 0.04
3.47 0.02
4.16 0.04
4.49 0.42
5.67 0.13
5.86 0.25
5.75 0.24
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
48.23 1.0
51.78 0.20
52.21 0.53
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀ
3.97
ꢀꢀꢀꢀ
0.05
3.63 0.04
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀ
ꢀꢀꢀ
6
7
Test 3 (9d)
Test 4 (5c)
10 mg/kg (p.o.)
10 mg/kg (p.o.)
39.89 0.02
37.1 0.03
59.88 0.04
56.11 0.06
36.33 0.13
35.26 0.07
4.80 0.16
5.32 0.04
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀ
ꢀꢀꢀ
3.87 0.04
Values are expressed in mean S.E.M (n ¼ 5). SGOT, Serum glutamic acid transaminase; SGPT, Serum glutamic pyruvic transaminase; ALKP, Alkaline Phosphatase;
TP, Total protein; p.o., peroral.
aSingle dose of carbon tetrachloride on day one, and the daily dose of synthesized compounds were given for seven days.
ꢀ
ꢀꢀ
ꢀꢀꢀ
p < 0.001,
ꢀꢀꢀꢀ
p < 0.0001, vs CCl4.
p > 0.05 ns; p < 0.1, p < 0.01,
One-way ANOVA is used, followed by Dennett’s test.
Figure 2. Different biochemical parameters.
and the method of measurement of box size was Buffer.
Sodium and chloride ions were used to neutralize the
charges. Then, the energy minimization and equilibration of
the prepared system was done. Temperature and pressure
were set, respectively, at 300 K and 1.01325 bar via NPT
ensemble. The recording interval of trajectory is 20 ps. The
simulation study was analyzed using the Simulation
Interaction Diagram of Desmond. A MD simulation study for
20 ns was carried out for the best docking score show-
ing compounds.
Table 2. Docking score and prime MMGBSA score of the synthe-
sized compounds.
Docking
MMGBSA DG binding
Sl. No.
Compound
score (XP)
score (Kcal/mol)
1.
2.
3.
4.
5.
6.
7.
8.
5a
5b
5c
5d
5e
9a
9b
9c
–12.51
–12.12
–8.57
–8.63
–7.73
–5.33
–6.44
–3.31
–3.05
–8.17
–11.48
–47.96
–48.48
–37.63
–43.64
–28.92
–36.42
–45.63
–45.91
–30.31
–38.07
–55.89
9.
10.
11.
9d
9e
Silybin (Std.)
3. Results and discussion
3.1. Chemistry
Phenyl hydrazine derivatives 2(a–e) on condensation with
diethyl oxalate (1) yielded ethyl-2-oxo-2(2-substituted aryl
hydrazinyl) acetates 3(a–e). The compounds 4-aryl ꢃ5,6-dihy-
3.1.1. Plan of synthetic scheme 1
Shubakara et al. (2014)1 designed the synthetic scheme to dro-4H-1,3,4-oxadiazine-2-carboxylates (4a–e) were prepared
synthesize derivatives of 1,3,4-oxadiazine-2-carboxylic acid. by condensation and cyclizing the obtained compounds 2-

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Figure 3. Histopathological analysis showed marked and significant damage to hepatic tissue when exposed to CCl4. CCl4-treated group showed congestion in a
central vein, degenerated cellular architecture, pyknotic nucleus, vacuolization and the sign of fatty changes, as compared with control, and exhibited the hepato-
toxic effect. Treatment with synthesized derivatives of oxadiazine showed varied anti-hepatotoxic effects where we observed reduced congestion, reduced vacuoli-
zation, pyknosis and improvement in cellular architecture, thereby exhibiting the anti-hepatotoxic effect. Additionally, we did not find any signs of steatosis in the
treatment group. These results showed that anti-hepatotoxic activity determined by biochemical studies are in accordance with and supportive to each other.
substituted aryl hydrazinyl acetates 3(a–e) with dibromo- 11.61 ppm. In 13C NMR, the absence of ester peak due to
ethane. The obtained compounds 2-substituted aryl hydra-
–CH3 at d 13.7 ppm, –OCH2 at d 60.9 ppm and > C¼O peak at
zinyl acetates 4(a–e) on hydrolysis to give derivatives of
d 158.4 ppm and also the presence of carboxylic acid > C¼O
1,3,4-oxadiazine-2-carboxylic acid 5(a–e).
peak at d 168.1 ppm confirmed the formation of product
(Scheme 1).
Prepared molecule structures were confirmed using vari-
ous spectral techniques such as IR, mass and NMR spectros-
copy. In the case of IR spectra of 4(a–e), the ester > C¼O
group at 1725 cmꢃ1 was replaced by the acid > C¼O group
at 1680 cmꢃ1 and also the acid –OH group was shown at
2737 cmꢃ1. In 1H NMR spectra, ester group as a triplet in
the region d 1.20 ppm, (3H for – OCH2–CH3 group), and
quartet in the region d 4.15 ppm, (2H for –OCH2 group) dis-
3.1.2. Plan of synthetic route 2
A new chemical series of 1-aryl-3-substituted phenyl-1H-
4,1,2-benzoxadiazine 9(a–e) were synthesized by condensa-
tion and dehydrohalogenation of compounds. Reactions
involved in the cyclo-condensation of phenyl hydrazonte
appeared. It also showed the presence of –COOH peak at d 8(a–e) were catalyzed by a mild amount of potassium

10
S. AKBAR ET AL.
Figure 4. The influence of CCl4 and synthesized compared with standard drug on hepatic tissue. CCl4 treatment damaged hepatic tissue as evident from conges-
tion in the central vein (long black arrow), degenerated cellular architecture (small yellow arrow), pyknotic nucleus (long yellow arrow), vacuolization (little black
arrow) and a sign of fatty changes (blue arrow), whereas treatment with derivatives of oxadiazine showed the varied anti-hepatotoxic effect.
Figure 5. 2D interaction diagrams of the synthesized compounds and standard drug.
carbonate to produce 1,3,4-oxadiazine in good yields. The the IR spectra of compound 9a and 9 b, and –Cl group
arrangement of atoms in the compound was confirmed by appears at 657 cmꢃ1 disappeared.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Figure 6. 3D IFD ligand interactions of the compounds 5a and 5b along with standard.
The primary amine of hydrazonate is converted into a sec- trichloromethyl peroxyl radicals, which destroys the func-
ondary amine, which is looked at 3396 cmꢃ1. >C¼ N group is
tional integrity of cell organelles. Administration of toxic
dose of carbon tetrachloride and hepatic glutathione was
visible at 1445 cmꢃ1. –NH2 peak of hydrazonate visible at d
depleted by 80% of the Wistar rats, and that 80% depleted
glutathione in the liver cells is essential for producing necro-
sis. The mechanism involved in glutathione depletion may
be expected to reduce peroxide by Fe2þ ions and form
highly reactive hydroxyl radicals, contributing to lipid peroxi-
dation and oxidation of DNA and proteins. The necrosis was
mainly seen in the centrilobular areas. Upraise levels of SGOT
and SGPT are an indicator of cellular discharge and disrupt
the mobility of the cell due to CCl4-induced toxicity.
Upraising the levels of SGOT and SGPT is an indicator of cel-
lular leakage and disrupts the integrity of cells due to CCl4-
induced toxicity. SGOT and SGPT could be considered as
standard parameters of hepatic damage. It promotes the
deposition of fatty acid in the liver and shows a vital part in
the depletion of glutathione boost lipid oxidation, dysregula-
tion in protein synthesis and fall of enzyme activity. It has
exposed that anti-hepatotoxic agents exhibited their action
counter to carbon tetrachloride mediated lipid peroxidation
by decreasing the production of free radical species by giv-
ing the anti-oxidative activity of the anti-hepatotoxic com-
pounds fundamentally.
4.23 ppm was replaced by the –NH peak of the oxadiazine ring,
which is looked at d 3.27 ppm in H NMR spectra. The IR spec-
1
tra of compounds 9c, 9d and 9e, the formation of the oxadia-
zine ring, are confirmed by the disappearance of the secondary
amine of hydrazonate, which appeared in the region at
3350 cmꢃ1. The –Cl group of hydrazonate looked at 640 cmꢃ1
was replaced by the oxadiazine ring containing –NH group at
3292 cmꢃ1. In 1H NMR, –NH peak of a secondary amine of
hydrazonate as a singlet in the region d 3.81 ppm disappeared,
which confirmed the structure of final products (Scheme 2).
3.2. Biological evaluation
3.2.1. In vitro hepatotoxicity assay
The MTT assay was used to assess the in vitro hepatotoxicity of
the synthesized compounds against HepG2 cell lines. The 50%
cytotoxic concentration (CC50) value of few synthesized com-
pounds was found to be >200 lM in HepG2 cell lines, indicat-
ing that they were safe for testing in vivo and also support the
molecular modeling studies (Supplementary Table S1).
The actions of hepatic enzymes such as alkaline phosphat-
ase (ALKP), serum glutamate oxaloacetate transaminase
(SGOT), serum glutamate pyruvate transaminase (SGPT) sig-
nificantly hiked, although the level of TP declined by the
liver damage intoxicated with carbon tetrachloride in Wistar
3.2.2. In vivo studies
The carbon tetrachloride-induced liver toxicity mediated by
CCl3, a toxic metabolite of carbon tetrachloride, was formed
by cytochrome P-450 2E1 (CYPE2E1) in hepatocytes. These
free radicals react with lipids, DNA and protein to produce rats in response to average values, as displayed in Table 1.

12
S. AKBAR ET AL.
Figure 7. 3D Superimposed structure of compounds 5a and 5 b with the standard at the binding site.
Table 3. ADMET properties analysis of the synthesized compounds alongside standard drug-using QikProp.
Sl. No.
Compound
aLog Po/w
bPSA
cLog S
H donor
H acceptor
dRule of five
% Human oral adsorption
1.
2.
3.
4.
5.
6.
7.
8.
5a
5b
5c
5d
5e
9a
9b
9c
1.39
1.61
1.90
2.26
0.11
2.92
3.34
1.57
3.77
3.07
1.76
62.13
62.13
62.13
62.13
148.41
33.62
33.62
41.90
111.11
50.85
155.14
–2.43
–2.73
–3.52
–3.93
–3.84
–3.78
–4.53
–3.36
–6.64
–4.73
–4.37
1
1
1
1
1
1
1
0
0
5
5
5
5
7
2.5
2.5
5
4
3.5
9.65
0
0
0
0
1
0
0
0
0
0
0
74.90
76.13
76.31
81.07
25.32
100
100
95.59
89.88
100
9.
10.
11.
9d
9e
1.8
4
Silybin (Std.)
63.41
aPartition coefficient of octanol/water (<5).
bPolar surface area (range 7–200).
cSolubility (range –6.5 to 0.5).
dLipinski’s Rule of five violations (ꢄ1).
The increased enzyme concentration of SGOT, SGPT and enzymatic concentration of total albumin from 3.63 to
ALKP was found after treated with carbon tetrachloride to
42.65, 66.48 and 43.46 units/mL in comparison with average
values of 30.63, 41.73 and 24.58, respectively. The level of
enzymes decreased after the administration of compounds
under investigation in the range of 39.89 to 34.63 units/mL
for SGOT, 59.88 to 48.23 units/mL for SGPT and 36.33 to
26.82 units/mL for ALKP, which were obtained to be compar-
able results with standard drug 34.68, 48.23 and 26.82 units/
mL, respectively. It has been found that compounds 5a and
5b exhibited an anti-hepatotoxic activity in comparison with
standard (Silybon-70) and were found to be 36.28, 51.78 and
28.48 units/mL, respectively, and compounds 36.38, 52.21
and 29.28 units/mL, respectively. The obtained results
showed that derivatives of oxadiazine exhibited comparable
results with those of the standard drug (silymarin), which has a
structural analog of the 1,4-dioxane ring system. Thus, oxadia-
zine moiety also shows a significant role in the anti-hepato-
toxic activity.
4.16 g/dl, in response to a standard (silymarin) 5.67 and
3.47 g/dl, respectively (Figure 2).
Treatment of Wistar rats with compounds 5a and 5 b
(10 mg/kg, p.o.) resulted in 52.99%, 59.3%, 79.34% and
52.16%, 57.65%, 75.10% reduction of SGPT, SGOT and ALKP
as compared with CCl4, respectively. Promotion of ALB and
TP for compounds 5a and 5b led to 411.01%, 53.39% and
378.63%, 48.9% as compared with toxic control group,
respectively. Percentage reduction of SGPT, SGOT and ALKP
as compared with CCl4 for compounds 9d and 5c was
22.96%, 26.67%, 37.76% and 46.17%, 41.89%, 43.43%
obtained, respectively. In addition, promotion of ALB and TP
for compounds 9d and 5c was 322.03%, 11.76% and
362.60%, 32.34% obtained, respectively.
3.3. Histopathological study of liver tissues
Histopathological analysis showed marked and significant
damage to hepatic tissue when exposed to CCl4. CCl4-treated
group showed congestion in a central vein, degenerated cel-
lular architecture, pyknotic nucleus, vacuolization and the
After administering toxic agents, carbon tetrachloride, the
enzymatic concentration of TP 4.49 g/dl and albumin 1.70 g/
dl was decreased, respectively. Treatment with the synthe-
sized compounds upraised the decreased enzymatic concen-
tration of TP from 4.80 to 5.86 g/dl and increased its reduced sign of fatty changes, as compared with control, and

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
Figure 8. RMSD and RMSF plot of ligand-protein complexes (a) RMSD and RMSF plot of compound 5a with the active site of the protein, (b) RMSD and RMSF plot
of compound 5b with the active site of the protein and (c) RMSD and RMSF plot of standard with the active site of the protein.
exhibited the hepatotoxic effect. Treatment with the synthe- be the reason for the better activity when it is compared
sized derivatives of oxadiazine showed varied anti-hepato- with the standard (Figures 5–7). Docking of standard along-
toxic effects where we observed reduced congestion, side the generated grid exhibited alike docking mode and H-
reduced vacuolization, pyknosis and improvement in cellular
architecture, thereby exhibiting the anti-hepatotoxic effect
(Figure 3).
bonds with an RMSD value of 0.76 and, therefore, validated
the generated grid. First, a molecular docking simulation was
done for the synthesized compounds and displayed good
MMGBSA DG binding energies in the range of ꢃ55.89 to
ꢃ28.92 kcal/mol (Table 2). All the units presented a respect-
able glide score. Nevertheless, the best encouraging mole-
cules were 5a, 5b, with an excellent glide score of 12.5, 12.1
separately. However, the glide score of the standard was
11.4 kcal/mol (Table 2).
Prediction of ADME properties of the synthesized com-
pounds 5(a–e) and 9(a–e) for the desirable pharmacokinetic
value was found within the range using the QikProp tool in
Schrodinger software (Table 3). For lead optimization, synthe-
sized compounds should follow the Lipinski rule of five for a
better activity profile. During ADMET prediction, synthesized
compounds obeyed the rule.
Additionally, we did not find any signs of steatosis in the
treatment group. These results showed that anti-hepatotoxic
activity determined by biochemical studies is under and sup-
portive of each other, showing the influence of CCl4 and syn-
thesized compounds compared with standard drug on
hepatic tissue. CCl4 treatment damaged hepatic tissue as evi-
dent from congestion in a central vein (long black arrow),
degenerated cellular architecture (small yellow arrow),
pyknotic nucleus (long yellow arrow), vacuolization (little
black arrow) and a sign of fatty changes (blue arrow),
whereas treatment with derivatives of oxadiazine showed
the varied anti-hepatotoxic effect (Figure 4).
Given a rigid macromolecule state, re-docking refers to
the capacity to repeat the associated ligand’s co-crystallized
binding geometry and orientation (Gaur et al., 2015; Yadav,
Dhawan, et al., 2014; Yadav, Kalani, et al., 2013; Yadav,
Kumar, et al., 2017; Yadav & Khan, 2013). Following docking
analysis validation, compounds 5a and 5b with good scores,
ligand interaction and binding energy were chosen for IFD.
The interactions in the docking mainly take place between
the rigid protein’s binding site and the flexible ligand.
However, in actual ligand-protein interactions in the body,
the target protein undergoes backbone or side chain move-
3.4. Molecular docking
The above-synthesized compounds 5(a–e) and 9(a–e) were
docked for their molecular docking simulation studies
against the particular position. The study of molecular dock-
ing delivers an understanding of molecular binding modes
of the molecules within the large pocket of receptors.
Schrodinger software was used for the molecular docking
process of the synthesized compounds. Standard (Silybin)
shows hydrogen bonding with THR 307, GLN 358, LEU 363
and ARG 435, in which ARG 435 was considered an essential
one for the anti-hepatotoxic property. In the above-synthe- ments after binding with ligands, resulting in changes in the
sized compounds, 5a and 5b displayed H-bonding with ARG protein’s binding sites. IFD-XP was designed to solve the
435 residue of the protein, which was essential for better flaws in rigid docking protocols. The primary goal of IFD is
activity, additional binding with ARG 100, and SER 431 may to produce a most accurate active complex structure of a

14
S. AKBAR ET AL.
Figure 9. Graph exhibiting interaction fraction of different Protein-ligand contacts between synthesized ligands and standard with different amino acids of protein;
(a) protein-ligand contacts of compound 5a with respective amino acids of the protein, (b) protein-ligand contacts of compound 5b with respective amino acids of
the protein, (c) protein-ligand contacts of standard with respective amino acids of the protein.
Figure 10. Timeline representation of Protein-ligand interaction after 100-ns molecular dynamics; (a) protein-ligand interaction of compound 5a with respective
amino acids of the protein, (b) protein-ligand interaction of compound 5b with respective amino acids of the protein, (c) protein-ligand interaction of standard
with respective amino acids of the protein. (Darker color indicates intense binding).
ligand, which is impossible to achieve using XP docking with single conformation of the ligand. But in the case of IFD,
a rigid protein structure. IFD prevents false-negative XP dock- each compound was given 20 confirmers. As a result, IFD-XP
ing output. Compound docking in XP was achieved with a was used to generate a maximum of 20 conformers for each

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
15
Figure 11. Post-docking 2D protein-ligand interaction diagram generated after 100-ns molecular dynamics; (a) ligand interaction plot representing the interactions
that occur between compound 5a with respective amino acids of the protein, (b) ligand interaction plot representing the interactions that occur between com-
pound 5b with respective amino acids of the protein, (c) ligand interaction plot representing the interactions that occur between of standard with respective
amino acids of the protein.
ligand based on molecular docking and binding energy for and standard were used for the MD simulation study. MD
simulation studies were carried out to study the various
aspects such as protein-ligand complex stability, binding
mode prediction, type of interactions with the binding site
of the selected protein (PDB: 3E4E) and synthesized com-
pounds 5a, 5b and standard. A frame was captured every
20 ps during the simulation and saved onto a trajectory.
Overall, the simulation exercise produced about 1000 frames.
the synthesized compounds 5a and 5b, and standard.
Through IFD-XP, mainly interactions were checked.
Compounds 5a and 5 b showed similar interactions as pre-
dicted in XP docking. No major change was found between
XP docking and IFD-XP docking. The standard drug also
demonstrates similar bindings when compared with the out-
put of XP docking and IFD-XP docking.
3.4.1.1. RMSD. The stability of the docking poses was deter-
mined by the values of RMSD. RMSD provides information
about the structural deviation and protein stability. Figure 8
shows the RMSD of synthesized compounds 5a and 5 b and
standard with selected protein, and it was observed that the
3.4.1. MD
Desmond (Schrodinger Release 2018-3) was used to investi-
gate the dynamic interactions of docked complexes. The
simulation interaction of the ligands at the binding site was
tested for 100 ns. The synthesized compounds 5a and 5b complex was stable for the simulation period. A slight drift

16
S. AKBAR ET AL.
Figure 12. SAR of most potent synthesized compound 5a.
was observed, but it was well stable throughout the com- some protein residues may make several interactions of the
plete simulation process. No major changes were found in same subtype with the ligand, values above 1.0 are possible.
the simulation trajectory as opposed to the protein in the An analysis report was generated for the potential protein-lig-
presence of synthesized compounds 5a and 5 b. Both of our
synthesized compounds (5a and 5 b) complexes were within
the range, as variations of the order of 1–3Å are perfectly
acceptable for globular proteins. The RMSD value of the syn-
thesized compounds 5a and 5 b complex was lower than
that of the standard compound. The RMSD of the protein-lig-
and complex was stable throughout the simulation period
of 100 ns.
and contacts (Figure 9). Compound 5a has an interaction frac-
tion of 2.5 with amino acid ARG100 and close to 1 with HIS370
and SER431, indicating that it has 250 percent contact with
ARG100 and 100 percent contact with HIS370 and SER431.
Compound 5b, on the other hand, has a close to three inter-
action fraction values with ARG100 and a close to one inter-
action fraction value with HIS370 and SER431. Various other
interactions were also observed between synthesized com-
pounds and amino acid residue of the protein.
A timeline depicts the interactions between the synthe-
sized compound, the standard and the protein. The darker
color indicates more powerful contacts (Figure 10).
Compounds 5a and 5 b exhibit dark color interaction with
ARG100, HIS370, SER431 and ARG435. The standard com-
3.4.1.2. Root-mean-square fluctuation. Root-mean-square
fluctuations (RMSF) were defined to find the protein’s resi-
due-wise fluctuations over the simulation time. The RMSF
study focuses on residue fluctuations, demonstrating their
importance in the flexibility of the functionally significant res-
idues that would affect the protein. Figure 8 illustrates, in pound shows similar interactions like ARG100 and ARG435
the presence of synthesized compounds 5a and 5 b and while also demonstrating some different interactions with
standard, the overall fluctuations of the protein residue. PHE298, ALA299, THR303, GLN358, HIS370 VAL436 and
Initially, compounds 5a and 5b show some minor fluctu- CYS437. Figure 11 represents interactions between various
ation; after that it was well stable throughout the simulation amino acids and synthesized compounds along with the
period. Some minor fluctuation was also seen in the later standard that occurs during the simulation period of 100 ns.
stages for compound 5a, but no major changes were seen
during the simulation. At first, the standard compound fluc-
tuated as well, but no significant changes were observed
later. Throughout the 100-ns simulation period, the RMSF of
the protein-ligand complex remained stable.
Compound 5a exhibited strong hydrogen bond interactions
with different amino acids such as ARG100, HIS370, SER431,
THR432 and ARG435, while compound 5b shows ARG100,
HIS370, SER431, THR432 and ARG435, which were consistent
throughout the simulation. Standard demonstrates H-bond
with ARG100, PHE298, ALA299, THR303, GLN358, HIS370,
ARG435, VAL436 and CYS437 during the simulation period
(Figure 11). Both compounds 5a and 5 b illustrate similar
contacts with the respective amino acids of the protein.
In contrast, standard compounds show similar interaction
with ARG100 and ARG435, essential for the particular activity.
It was assumed that the synthesized compounds had better
activity because their interaction patterns were different
from the standard. The MD simulation study results demon-
3.4.1.3. Protein-ligand contacts. The consistency of protein-
ligand interactions was also studied during the simulation pro-
cess to better understand the protein-ligand complex.
Throughout the 100-ns MD simulation, interactions between
protein with synthesized compounds and standard were
observed. As shown in Figure 9, these interactions can be div-
ided and summarized into four types of protein-ligand interac-
tions or contacts: hydrophobic, hydrogen bonds, ionic and
water bridges. The stacked bar charts are normalized through- strated that the protein-ligand complex is well stable
out the trajectory: a value of 0.5 indicates that the specific con- throughout 100 ns and shows strong binding interactions
tact is maintained for 50% of the simulation duration. Because with different amino acids.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
17
Ahmed, B., Habibullah, & Khan, S. (2011). Synthesis and antihepatotoxic
activity of 2-(substituted-phenyl)-5-(2,3- dihydro-1,4-benzodioxane-2-
yl)-1,3,4-oxadiazole derivatives. Journal of Enzyme Inhibition and
Medicinal Chemistry, 26(2), 216–221. https://doi.org/10.3109/14756366.
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Ajay, A., Singh, V., Singh, S., Pandey, S., Gunjan, S., Dubey, D., Sinha,
S. K., Singh, B. N., Chaturvedi, V., Tripathi, R., Ramchandran, R., &
Tripathi, R. P. (2010). Synthesis and bio-evaluation of alkylaminoaryl
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3.5. Structure–activity relationship
The structure–activity relationship (SAR) of the prepared
compounds is carried out on the support of the central oxa-
diazine ring attached to the aryl ring with different substitu-
tions. The compound having a phenyl ring attached with the
central oxadiazine core ring (5a) displayed the most potent
activity (Figure 12). A compound containing a substitution of
the terminal phenyl ring at the para-position also revealed
promising anti-hepatotoxic activity. Substitution with elec-
tron-donating groups (EDG) shows a decline in the activity.
4. Conclusion
€
Desmond j Schrodinger. (n.d.). Retrieved August 2, 2020, from https://
We have synthesized two series of oxadiazine derivatives and
evaluated their anti-hepatotoxic activity in Wistar rats (CCl4-
induced model). In Scheme 1, two synthesized compounds (5a
and 5 b) showed the most potent anti-hepatotoxic activity
than the standard drug silymarin. However, compound 5a is
more potent than compound 5 b. Synthesized compound 5c
showed moderate activity. In Scheme 2, Compound 9d exhib-
ited mild anti-hepatotoxic activity compared with standard
drug (silymarin). The biochemical and histopathological results
of the synthesized compounds (5a, 5b, 5c and 9d) reveal that
they have some anti-hepatotoxic properties to prevent hep-
atotoxicity in the liver. Molecular docking simulation data fur-
ther support the results, which correlate with the outcomes
too. Hence, we conclude that the synthesized compounds
have promising activity to prevent necrosis in the liver.
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Acknowledgments
The authors acknowledge with thanks to AICTE for providing GPAT
scholarship & thanks to Jamia Hamdard, New Delhi, for providing facili-
ties for this work. The study is exempted and approved by the
Institutional Animal Ethics Committee of Jamia Hamdard based on the
basis that this type of study is animal subject research.
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Disclosure statement
No potential conflict of interest was reported by the authors.
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Note
1. The synthethic scheme was used in this process was reported by
Shubakara et al.
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CO;2-A
ORCID
Saleem Akbar
Subham Das
Bahar Ahmed
http://orcid.org/0000-0001-5661-371X
http://orcid.org/0000-0001-7327-7631
http://orcid.org/0000-0002-2945-2794
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