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Synthesis, Structural Elucidation, In Silico and In Vitro Studies of
New Class of Methylenedioxyphenyl-Based Amide Derivatives as
Potential Myeloperoxidase Inhibitors for Cardiovascular Protection
Reshma Rajan, Sambantham Karthikeyan, and Rajagopal Desikan*
Cite This: https://doi.org/10.1021/acsomega.3c07555
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sı Supporting Information
ABSTRACT: Novel methylenedioxyphenyl-based amides, espe-
cially N-(4-methoxybenzyl)-6-nitrobenzo-[1,3]-dioxole-5-carboxa-
mide (MDC) and N-(3-acetylphenyl)-6-nitrobenzo-[1,3]-dioxole-
5-carboxamide (ADC), potential cardiovascular preventive agents,
are successfully synthesized, and their chemical structures are
verified by 1H and 13C NMR, Fourier transform infrared (FT-IR),
high-resolution mass spectrometry (HRMS), and single-crystal X-
ray diraction (SC-XRD) analyses. Data obtained from SC-XRD
reveal that MDC and ADC are both monoclinic molecules with Z=
2 and 4, respectively. From density functional theory (DFT)
calculations, 3.54 and 3.96 eV are the energy gaps of the optimized
MDC and ADC structures, respectively. MDC and ADC exhibit an
electrophilicity index value of more than 1.5 eV, suggesting that they can act as an electrophile, facilitating bond formation with
biomolecules. Hirshfeld surface analysis demonstrates that more than 25% of atomic interactions in both MDC and ADC are from
H···H interactions. Based on pharmacokinetic predictions, MDC and ADC exhibit drug-like properties, and molecular docking
simulations revealed favorable interactions with active site pockets. Both MDC and ADC achieved higher docking scores of −7.74
and −7.79 kcal/mol, respectively, with myeloperoxidase (MPO) protein. From docking results, MPO was found to be most
favorable followed by dipeptidyl peptidase-4 (DPP-4) and α-glucosidase (α-GD). Antioxidant, anti-inflammatory, and in vitro
enzymatic studies of MDC and ADC indicate that MDC is more selective toward MPO and more potent than ADC. The application
of MDC to inhibit myeloperoxidase could be ascertained to reduce the cardiovascular risk factor. This can be supported from the
results of computational docking (based on hydrogen bonding and docking score), in vitro antioxidant and anti-inflammatory
properties, and MPO enzymatic inhibition (based on the percentage of inhibition and IC50 values).
1. INTRODUCTION
A dicult undertaking is the creation of a new drug. Drugs are
produced either entirely from natural sources or by semi-
synthetic methods. Therapeutic drugs for the treatment of
cardiovascular disease (CVD) can be obtained from natural
products and their structural equivalents. All social groups are
aected by the serious global health problem of CVD. A class
of bioactive scaolds known as substituted methylenediox-
yphenyl (MDP) derivatives have been shown to possess
antioxidant, antibacterial, antiobesity, antidiabetic, and anti-
inflammatory properties.
1−4
The MDP pharmacophore
remains of great interest to many research teams as it routinely
provides encouraging results for the treatment of CVD.
5−9
A
naturally occurring phytochemical derived from Piper nigrum
(spice bell pepper), piperonylic acid, has MDP pharmaco-
phore. The piperine in P. nigrum has been shown to increase
the bioavailability of drugs such as resveratrol, quercetin, and
nevirapine.
10−12
Potent bioactive drugs in the MDP group that
are on the market are shown in Figure 1. Parkinson’s disease,
diabetes, epilepsy, and urinary tract infections are treated with
piribedil, berberine, stiripentol, and cinoxacin, respec-
tively.
13−16
From the reported structure−activity relationship
studies, chemical linking of the MDP group to the parent
structure appeared to enhance the bioactivity. This is probably
due to nucleophilic oxygen in the diether group which readily
donates electrons to the electrophilic center for interacting
with protein residues within the active site to facilitate
biological interactions. Also, the fifth and sixth positions of
the MDP group are shown to influence the bioactivity of the
molecule.
5,17−19
From the literature, MDP with nitro
substitution at the sixth position is shown to oer better
cardioprotective eects.
6
Therefore, the nitro group at the sixth
Received: September 29, 2023
Revised: December 18, 2023
Accepted: December 21, 2023
Article
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position is kept intact and further modification was done at the
fifth position to get better bioactive derivatives.
Methoxy is an essential functional group found in
phytochemicals such as curcumin, guaiacol, and methoxy-
flavone.
20−22
Likewise, the acetophenone group plays a crucial
role in the action of numerous medications, including
pyrovalerone, amfepramone, and bupropion.
23−25
For the
preparation of medications such as zolpidem, oxiconazole, and
cinacalcet, basic acetophenone derivatives are used as raw
materials.
26
It is therefore anticipated that the combination of
4-methoxybenzylamine, 3-aminoacetophenone, and 6-nitro-
piperonal (carrying the MDP group) will exhibit significant
biological activity. In the present study, a new class of
carboxamide derivatives is synthesized with the intention of
preserving the pharmacophoric potency of the parent structure
and improving the overall bioactivity after creating the amide
bond when coupled with piperonylic acid and amines. We first
investigated their drug-likeness, molecular docking, and
pharmacokinetic properties. The successful results encouraged
us to further explore their biological activities, such as
antioxidant and anti-inflammatory properties and enzyme
inhibitions. α-Glucosidase (α-GD), dipeptidyl peptidase-4
(DPP-4), and myeloperoxidase (MPO) proteins are used for
molecular docking. The development of CVD, including
atherosclerosis (MPO)
27
and type 2 diabetes (DPP-4
28
and
α-GD
29
), is related to these three proteins (MPO, DPP-4, and
α-GD). When a macrophage cell dies due to neutrophil
extracellular trap (NETosis) activation, chromatin is released
into the extracellular environment to trap and eliminate
microorganisms. One of the direct links between diabetes and
atherosclerosis that can be demonstrated in the inflammatory
pathways is NETosis.
30,31
Clinical studies have shown that the
link between inflammation, diabetes, and atherosclerosis
increases the risk of CVD.
32,33
Therefore, it is essential to
look into the anti-inflammatory characteristics of the molecules
in relation to CVD. 1H, 13C NMR, Fourier transform infrared
(FT-IR), and High-resolution mass spectrometry (HRMS) are
used to characterize the synthesized structures. Furthermore,
single-crystal X-ray diraction (SC-XRD) data unequivocally
corroborated the molecules’ entire chemical structure. To
assess the quantum chemical parameters and to expose the
crystals’ theoretical and experimental bond angles and bond
lengths, the Hirshfeld surface analysis is carried out with SC-
XRD CIF data. From the Hirshfeld surface analysis, the
molecules’ volume, asphericity, inner area, di,de,dnorm, and
globularity are determined, and their global hardness, global
electrophilicity index, dipole moment, global softness, and
electron negativity are determined from density functional
theory (DFT).
The recently synthesized small molecule drug for MPO
inhibition in clinical trials, Verdiperstat, is produced by
AstraZeneca and Biohaven Pharmaceuticals. Mild adverse
eects including nausea, headache, and insomnia have been
linked to verdiperstat.
34,35
Therefore, the appending of a
brand-new medication for MPO inhibition that has fewer
negative eects is urgently needed. The present study focuses
on the synthesis and application of phytochemical-based
organic scaolds targeting cardiovascular disease risk factors,
namely, diabetes and atherosclerosis. Sci-Finder search showed
limited scientific reports in the literature for nitro-substituted
methylenedioxyphenyl amide-based molecules.
36,37
Imidazole,
isoindoline, pyridine, triazole, quinoline, and thiosemicarba-
zone-based heterocyclic derivatives targeting diabetes and
atherosclerosis have been reported.
38−43
Relatively, a few
phytochemical-based scaolds are used for treating diabetes
and atherosclerosis.
44−46
Chemically linking phytochemical
derivatives to organic molecules improves the ecacy and
reduces the toxicity of the drug. Thus, minimal toxicity and
maximal eectiveness are anticipated when piperine derivatives
are chemically linked to an amine via an amide bond.
Introducing a novel class of small compounds for MPO
inhibition that are inspired by nature is the goal of this
endeavor.
2. MATERIALS AND METHODS
Starting materials, deuterated solvents, and reagents are bought
from TCI Chemicals, Avra, and Sigma-Aldrich. Solvents are
bought from SD Fine Chemicals. ProTox-II predicts molecule
toxicity, while SwissADME and pkCSM predict ADME
characteristics. Molecular docking is performed using Auto-
DockTools 1.5.1, and visualizations are performed using
Discovery Studio Visualizer v21.1.0.20298. Myeloperoxidase,
dipeptidyl peptidase-4, and α-glucosidase are purchased from
Sigma-Aldrich.
2.1. Pharmacokinetic Studies. The pharmacokinetic
(body action on the drug) features of MDC and ADC are
calculated using the free online programs SwissADME,
pkCSM, and ProTox-II.
47−49
Toxicological qualities of drugs
are predicted using ProTox-II, and SwissADME and pkCSM
are used to identify ADME (absorption, distribution,
metabolism, and excretion) properties. Clinical trial HITS
rejection rates are significantly lowered by ADMET inves-
tigations, which have grown to be an essential component in
the development of new drugs. Drug-likeness, oral bioavail-
ability, and physicochemical characteristics are all clarified by
ADMET.
2.2. Molecular Docking. The binding interactions of hit
compounds with targeted proteins are determined using an in
silico molecular docking (MD) simulation. To ascertain
whether ligands and proteins interact well, this method is
employed as a reference tool. Three key proteins
myeloperoxidase (MPO), dipeptidyl peptidase-4 (DPP-4),
and α-glucosidase (α-GD)were chosen for MD simulations
to examine the binding capacity of MDC and ADC for various
disease targets, including atherosclerosis and antidiabetic
eects. You can download the crystal structures of the proteins
Figure 1. Commercial drugs with methylenedioxyphenyl pharmaco-
phore.
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B
MPO (PDB code 5FIW), DPP-4 (PDB code 6B1E), and α-
GD (PDB code 3A4A) from the RCSB Protein Data
Bank.
50−52
Salicylhydroxamic acid, sitagliptin, and acarbose
are used as positive controls for MPO, DPP-4, and α-GD,
respectively. In preparation for docking, the proteins and
ligands are created using discovery studio. For docking,
AutoDockTool 1.5.6 makes use of the Lamarckian genetic
algorithm. Active protein sites are chosen, and a grid box is
created to frame all of the active sites before docking.
2.3. Synthesis and Crystallization. The syntheses of
MDC and ADC are achieved through two steps. Initially 6-
nitropiperonal is oxidized to 6-nitrobenzo[1,3]dioxole-5-
carboxylic acid
53
and coupled with 4-methoxybenzylamine/3-
aminoacetophenone to form 6-nitrobenzo[1,3]dioxole-5-car-
boxamide derivatives (Scheme 1). Similar syntheses with
dierent reaction conditions are reported in the literature.
54,55
The method used in this research is well known, simple, and
highly useful for researchers to accomplish the synthesis of
amide derivatives from aldehydes by using a simple synthetic
procedure. The complete methodology is provided in the
Supporting Information. The reaction is optimized by
changing various experimental parameters like reagents,
solvents, and time to achieve a maximum yield with highest
purity. When the reaction was conducted in methanol at 32 °C
for 4 h with KMnO4and Na2HPO4as reagents, the maximum
yield was achieved (Table 1). Similarly, for step 2, coupling
agents, bases, and solvents were varied. The maximum yield
was obtained when dimethylaminopyridine was used as the
base and (3-dimethylamino-propyl)-ethyl-carbodiimide hydro-
chloride as the coupling agent. The reaction was carried out
under an inert atmosphere for 12 h at 32 °C in dichloro-
methane (Table 2). Dimethyl sulfoxide and hexane (1:1)
solvents were used to crystallize MDC and ADC. The crystals
were separated, dried, and analyzed for single-crystal X-ray
diraction.
2.4. Single-Crystal X-Ray Diraction Studies. Table 3
summarizes the crystal and structure refinement data for MDC
and ADC. The X-ray crystallographic investigation is
performed on MDC and ADC with approximate dimensions
of 0.098 mm ×0.170 mm ×0.200 mm and 0.120 mm ×0.145
mm ×0.186 mm, respectively. X-ray diraction intensities are
measured on a Bruker D8 Quest with i-mu-s microfocus
molybdenum source Mo Kα(λ= 0.71073 Å) radiation. A
Bruker SHELXL-2019/1 is used to solve and refine the crystal
structure of molecules. The CCDC deposition numbers for
single crystals of MDC and ADC are 2239892 and 2282346,
respectively.
2.5. Computational Analysis. Quantum chemical com-
putational analysis has advanced significantly in recent years
and is now a highly eective method for confirming
experimental results. The B3LYP/6-311G** level of theory
in the Gaussian-16 program is used to study the quantum
chemical parameters. Using DFT, energy calculations of the
optimized structures are examined. The structure’s chemical
potential is examined along with its molecular orbital energies,
ELUMO and EHOMO, three-dimensional (3D) surface image,
molecular electrostatic potential (MEP), and dipole mo-
ment.We conducted the Hirshfeld surface analysis using
CrystalExplorer version 3.1 to determine the intermolecular
interactions between each atom.
2.6. In Vitro and Ex Vivo Biological Studies. The
established method from scientific literature was followed for
in vitro antioxidant, ex vivo anti-inflammatory, and in vitro α-
GD, DPP-4, and MPO enzymatic inhibition studies.
56−60
Ascorbic acid and aceclofenac were used as positive controls
for antioxidant and anti-inflammatory studies, respectively. For
α-GD, DPP-4, and MPO enzymatic inhibition studies,
acarbose, sitagliptin, and salicylhydroxamic acid were used as
positive controls, respectively. The complete procedures for
the biological studies are given in the SI.
Scheme 1. Synthesis of MDC and ADC.
Table 1. Oxidation of Aldehyde under Dierent Reaction
Conditions
a
s.
no. oxidizing
agent reagent solvent reaction
condition yield
(%)
1 MnO2NaHSO3ethanol 12 h, 32 °C 60
2 KMnO4NaHSO3methanol 30 min, 32 °C 55
3 KMnO4Na2HPO4methanol 6 h, 32 °C 75
4 KMnO4Na2HPO4,
NH4Cl methanol 4 h, 32 °C 81
a
All reactions were carried out on a 100 mg scale. Aldehyde (1.0
equiv), oxidizing agent (1.0 equiv), reagent (1.0 equiv), and solvent
(5 mL). MnO2: manganese dioxide, NaHSO3: sodium bisulfite,
KMnO4: potassium permanganate, Na2HPO4: disodium hydrogen
phosphate, NH4Cl: ammonium chloride, hhour.
Table 2. Acid Amine Coupling under Dierent Reaction
Conditions
a
s. no base reagents solvent reaction condition yield (%)
1 TEA EDCI·HCl DCM 32 °C, 12 h 65
2 DMAP DCC DMF 32 °C, 12 h 77
3 DMAP EDCI·HCl CHCl332 °C, 12 h, N276
4 DMAP EDCI·HCl DCM 32 °C, 12 h, N285
5 TEA DCC Benzene 32 °C, 12 h, N269
a
All reactions were carried out on a 100 mg scale. Acid (1.0 equiv),
amine (1.0 equiv), base (2.0 equiv), reagent (2.0 equiv), and solvent
(10 mL). TEA: triethylamine, EDCI·HCl: (3-dimethylamino-propyl)-
ethyl-carbodiimide hydrochloride, DMAP: 4 dimethylaminopyridine,
DCC: N,N′-dicyclohexylcarbodiimide, h: hour.
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3. RESULTS AND DISCUSSION
3.1. Pharmacokinetic Studies. 3.1.1. Drug-Likeness. The
Lipinski’s rule of five is used to assess a molecule’s medicinal
similarity. According to Lipinski, any oral medicine should
have the following properties: a molecular weight (MW) of
≤500, a partition coecient (log P) of ≤5, a number of
rotatable bonds of ≤10, and a number of hydrogen-bond
donors and acceptors of ≤5 and ≤10, respectively.
61−63
Molecules must meet all five criteria for use in medication
delivery. Any molecule that satisfies Ghose’s law must have the
following properties: a molecular weight between 160 and 480,
a log Pvalue between −0.4 and 5.6, a molar refractive range
between 40 and 130, and an overall atom count between 20
and 70.
64
Veber’s rule states that a standard drug to have good
oral bioavailability should meet the following criteria: rotatable
bonds ≤10 and TPSA ≤140 Å2.
65
According to Egan, good
oral bioavailability standards are TPSA ≤130 Å2and log P
values within the limit of −1.0 ≤log P≤5.8.
66
To distinguish
between compounds that are similar to drugs and those that
are not, Muegge’s rule modified the characteristics range and
added more factors. According to Muegge’s rule, the ideal MW
and log Pranges are 200−600 and −2 to 5, respectively.
Additionally, there should be more than four total carbon
atoms, at least one heteroatom, and seven or fewer rings.
According to Muegge, the hydrogen-bond acceptors and
donors must be ≤5 and ≤10, respectively, and the rotatable
bond must be ≤15.
67
Using the web resources SwissADME
and ChemDraw Professional 16.0, the drug-likeness rules are
anticipated. Table 4 shows that the MDC and ADC molecules
adhere to all five rules without any deviation. Results from
MW, HBA, and HBD suggest that the compounds have
significant levels of penetration and absorption in the body.
The total polar surface areas (TPSAs) of the MDC and ADC
molecules are, respectively, 102.612 and 110.45 Å2. TPSA is
described as the overall surface sum of polar molecules or
atoms (oxygen and nitrogen) and their hydrogen attachments.
TPSA predicts cellular absorption, and the ideal range for
absorption is 60−140 Å2.
68
Table 5 shows that MDC and
ADC each have a bioavailability score of 55%. Bioavailability is
the rate at which a molecule enters the bloodstream and
reaches the site of action. Any moiety with a bioavailability
score of ≥0.55 is considered ideal and absorbed very well by
the body.
69
A bioavailability score of 0.55 and a TPSA score
between 60 and 140 Å2indicate optimal absorption for both
MDC and ADC. Owing to their high bioavailability scores and
flexibility, MDC and ADC can quickly enter the body’s
systemic circulation, go to the site of action, and engage the
binding pocket of biomolecules.
3.1.2. In Silico ADMET Prediction. In silico ADMET
prediction is a crucial drug profiling step in the process of
discovering new medicines. Table 6 summarizes the in silico
ADMET parameters for MDC and ADC ligands, using online
free tools SwissADME, pkCSM, and ProTox-II. The ADMET
predictions for the well-known methylenedioxyphenyl-based
drugs, piribedil (PB), berberine (BB), stiripentol (SP), and
cinoxacin (CX), are calculated for a comparative study. Any
Table 3. X-ray Details of MDC and ADC
a
MDC ADC
chemical formula C16H14N2O6C16H12N2O6
formula weight 330.29 g/mol 328.28 g/mol
temperature 298(2) K 300(2) K
wavelength 0.71073 Å 0.71073 Å
crystal size 0.098 mm ×0.170 mm ×
0.200 mm 0.120 mm ×0.145 mm ×
0.186 mm
crystal system monoclinic monoclinic
space group P1211P121/c1
unit cell
dimensions a= 4.9569(5) Å a= 16.823(3) Å
b= 8.3653(8) Å b= 7.2581(12) Å
c= 18.1827(18) Å c= 11.904(2) Å
α= 90°α= 90°
β= 95.723(3)°β= 97.046(5)°
γ= 90°γ= 90°
volume 750.20(13) Å31442.5(4) Å3
Z2 4
F(000) 344 680
density
(calculated) 1.462 g/cm31.511 g/cm3
absorption
coecient 0.114 mm−10.118 mm−1
theta range for
data collection 2.25−28.30°2.44−28.29°
index ranges −6≤h≤6, −11 ≤k≤11
,−24 ≤l≤24
−22 ≤h≤22, −9≤k≤9
,−15 ≤l≤15
reflections
collected 21,073 32,344
independent
reflections 3701 [R(int) = 0.0420] 3569 [R(int) = 0.0561]
refinement
method full-matrix least-squares on
F2full-matrix least-squares on
F2
data/restraints/
parameters 3701/1/221 3569/0/218
goodness-of-fit on
F21.076 1.067
final Rindices
[I> 2σ(I)] R1= 0.0389 R1= 0.0483
wR2= 0.0824 wR2= 0.1108
Rindices [all data] R1= 0.0623 R1= 0.1015
wR2= 0.0963 wR2= 0.1463
largest di. peak
and hole 0.139 and −0.147 e Å−30.230 and −0.171 e Å−3
a
Computer programs: Bruker APEX4/SAINT, SHELXL2019/1
(Sheldrick, 2019), and Bruker SHELXTL.
Table 4. Lipinski’s Rule of Five for the Proposed Molecules
descriptors
compound MW (molecular weight
in g/mol) log P(partition
coecient) RB (rotatable
bond) HBA (hydrogen-bond
acceptor) HBD (hydrogen-
bond donor) TPSA (topological polar surface
area in Å2)
MDC 330.29 1.56 6 6 1 102.61
ADC 328.28 1.22 5 6 1 110.45
Table 5. Drug-Likeness Prediction for the Proposed
Molecules
compound Lipinski Ghose Veber Egan Muegge bioavailability
score
MDC yes yes yes yes yes 0.55
ADC yes yes yes yes yes 0.55
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drug molecule administered to the body needs to be first
absorbed by the body and then be able to be removed from the
body with the fewest negative eects possible. Every molecule
must be water soluble to be a medicine molecule because the
human body is composed of about 60% water. The Cancer
coli-2 (Caco-2) cell line, which is made up of human epithelial
colorectal adenocarcinoma cells, eectively absorbs anything
with a Caco-2 permeability score of more than 0.90. The
majority of medications taken by mouth must be absorbed in
the intestines, and any molecule that absorbs more than 30% is
regarded as having exceptional absorption. MDC and ADC
have HIA values greater than 90%, indicating adequate
intestine absorption. The boiled egg diagram between TPSA
and partition coecient (log P) can predict the molecule’s
ability to traverse the blood−brain barrier (BBB) or human
intestinal absorption (HIA).
70
The egg yolk represents the
BBB, whereas the white albumin of the egg stands in for
HIA.
71
Figure 2 depicts the penetration of PB, SP, and BB
through BBB whereas CX, MDC, and ADC through HIA. The
log Kpvalue greater than 2.5 denotes low skin permeability for
PB, BB, CX, MDC, and ADC. The bioavailability radar plot is
depicted in Figure 3 for the drugs PB, BB, SP, CX, MDC, and
ADC. The steady-state volume of distribution (VDss) refers to
the amount of medication needed for an even distribution to
produce a blood plasma-like concentration. The higher VDss
value denotes that the tissue has a more extensive drug
Table 6. In Silico ADMET Parameters for Proposed Molecules
a
absorption
Com Ws (log mol/L) Caco2-P (log in 10−6cm/s) HIA (% A) SP (log Kp) P-gs P-GI i P-GII i
PB −2.55 1.30 96.15 −2.81 no no no
BB −3.20 1.62 100 −2.67 no no yes
SP −3.09 1.89 92.85 −2.28 no no no
CX −3.20 1.26 78.86 −2.73 no no no
MDC −3.71 0.37 90.84 −2.74 yes no no
ADC −3.60 0.33 96.29 −2.73 yes no no
distribution
HVDss (log L/kg) Hf (Fu) BBB permeability (log BB) CNS permeability (log PS)
PB 0.24 0.35 −0.025 −2.515
BB 0.81 0.35 0.633 −1.675
SP 0.23 0.13 0.267 −1.677
CX −0.66 0.33 −0.653 −3.043
MDC −0.44 0 −0.603 −2.533
ADC −0.49 0.05 −0.552 −2.365
metabolism
PC-4 CYP2D6 Sub CYP3A4 Sub CYP1A2 In CYP2C19 In CYP2C9 In CYP2D6 In CYP3A4 In
PB no yes yes no no no no
BB no yes yes no no yes no
SP no yes yes yes no no no
CX no no no no no no no
MDC no yes yes yes yes no yes
ADC no yes no yes yes no yes
excretion
total clearance (log mL/min/kg) renal OCT2 Sub
PB 0.855 yes
BB 1.272 yes
SP −0.008 no
CX 0.643 no
MDC 0.171 no
ADC 0.039 no
toxicity
AMES HTD (log
mg/kg/day) h I
inh h II
inh OAT (LD50)
mol/kg OCT (LOAEL) (log
mg/kg/D) HT SS TP-T
(log μg/L) MT
(log mM)
PB no −0.54 no yes 2.929 1.384 yes no 0.386 0.032
BB no −0.132 no no 3.313 1.275 no no 0.288 −0.869
SP no 0.219 no no 1.959 2.156 no yes 2.211 0.811
CX yes 0.87 no no 1.956 1.004 yes no 0.284 2.66
MDC yes 0.154 no no 2.122 1.548 yes no 0.34 −0.061
ADC yes 0.18 no no 2.117 1.476 yes no 0.327 0.603
a
PC, positive control; Com, compound; Ws, water solubility; Caco2-P, Caco2-permeability; HIA, human intestinal absorption; SP, skin
permeability; P gs, P-glycoprotein substrate; P gIi, P-glycoprotein I inhibitor; P-gIIi, P-glycoprotein II inhibitor; HVDss, steady-state volume of
distribution; Hf, fraction unbound (human); BBB, blood−brain barrier; CNS, central nervous system; CYP, cytochrome P; Sub, substrate; In,
inhibitor; OCT2, organic cation transport 2; HTD, maximum tolerated dose (human); OAT, oral rat acute toxicity (LD50); OCT, oral rat chronic
toxicity (LOAEL); HT, hepatotoxicity; SS, skin sensitization; TP-T, Tetrahymena pyriformis toxicity; MT, Minnow toxicity.
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dispersion than plasma. Log VDss value is optimum within the
range −0.15 ≥log VDss ≥0.45. The VDss values for MDC
and ADC are both more than −0.15, indicating a good volume
of dispersion. Having log PS values of −2.5 and −2.3,
respectively, which are greater than −2, MDC and ADC are
able to quickly enter the central nervous system (CNS). The
Cytochrome P450 (CYP) family of enzymes, which metabo-
lizes 50% of medications by itself, is essential for the
metabolism of substances. The liver’s detoxifying enzyme
Cytochrome P450 oxidizes xenobiotics to encourage excretion.
The drug molecule must be processed by one of two CYP450
isoforms, CYP2D6 or CYP3A4, which are linked to drug
metabolism. Except for CX, all substances can be metabolized
by CYP3A4 substrates. The sum of renal clearance and hepatic
clearance determines the total clearance, which is correlated
with bioavailability. All of the compounds have extremely low
Figure 2. Boiled egg diagram for piribedil (PB), berberine (BB), stiripentol (SP), cinoxacin (CX), MDC, and ADC.
Figure 3. Bioavailability radar for piribedil (PB), berberine (BB), stiripentol (SP), cinoxacin (CX), MDC, and ADC.
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clearance indices, which indicates greater drug persistence in
the body. Lethal dose 50 (LD50), AMES toxicity (Salmonella
typhimurium reverse mutation assay), and hepatotoxicity are
the three basic toxicological parameters used to interpret the
toxicity of the compounds. AMES can determine which
molecules are more hazardous and which molecules are
mutagenic based on their LD50 values. In general, a drug’s
maximum tolerated dose (HTD) should not be more than
0.47. The estimated hazardous dosage of chemicals in humans
is minimal because the HTD values for MDC and ADC are
0.15 and 0.18, respectively, which are both less than 0.47.
According to the toxicity prediction, the compounds are either
nontoxic or only tolerably poisonous to a certain degree.
72−74
The proposed MDC and ADC compounds exhibit drug
similarity based on in silico predictions and are suitable for the
drug development process.
3.2. Molecular Docking. A docking investigation is
conducted to determine the binding interaction between
MDC and ADC and the residues in the active sites of MPO,
DPP-4, and α-GD.
75
Table 7 provides examples of the ligand−
protein interactions and binding energies of MDC and ADC to
the proteins. Salicylhydroxamic acid, sitagliptin, and acarbose,
recognized for inhibiting MPO, DPP-4, and α-GD, are used as
positive controls. Figure 4 shows images of positive controls,
MDC, and ADC docked with proteins in two dimensions. The
binding energy value assesses both the stability of the protein−
ligand complex and the intensity of the interaction between a
protein and a ligand. Lower binding energy encourages a
stronger association between the protein and the ligand and
helps the targeted protein form stable complexes that are well
suited for biological function.
Compared with the positive control (−5.88 kcal/mol),
MDC and ADC appear to have higher binding energies,
according to docking scores of MPO (−7.79 and −7.74 kcal/
mol, respectively). The oxygen in the nitro group and
ARG:333 and ARG:424 form a hydrogen bond in both
molecules. To maintain the stability and specificity of the
protein−ligand complex, many intermolecular hydrogen bonds
are extremely important. MDC and ADC achieved docking
scores of −4.30 and −4.20 kcal/mol for DPP-4, respectively.
Positive controls have a higher binding score (−7.66 kcal/mol)
than do MDC and ADC.
MDC interacts with amino acids ARG:125, HIS:126,
SER:209, SER:630, and TYR:631 via intermolecular hydrogen
bonding. Three groups in the ligand, the methylenedioxy,
nitro, and methoxy groups, are primarily where hydrogen
bonds are formed. Through hydrogen bonds, amino acids
GLN:553 and LYS:554 bind to the protein in ADC. MDC and
ADC achieved docking scores of −3.83 and −4.67 kcal/mol
with α-GD, respectively. GLN:279, HIS:280, and ARG:315 for
MDC and LYS:156 and ASN:415 for ADC are the hydrogen-
bond interactions. Methylenedioxy bridge and nitro group
both produce hydrogen bonds. When docked with MPO,
DPP-4, and α-GD, the MDC and ADC molecules fit well into
active site pockets and exhibit positive interactions with
proteins.
In conclusion, MDC exhibits more hydrogen bonds and
binding energy than ADC, and it exhibits potential ligand−
protein interactions with excellent docking stability. In brief,
we may deduce from the docking studies that the MDC ligand
exhibits the best binding and that, in comparison with other
proteins, it binds to MPO specifically.
3.3. Structure−Activity Relationship (SAR). Based on
the in silico results and structure−activity relationship, we plan
to synthesize MDC and ADC and proceed with further
screening. Both MDC and ADC have key functional groups,
nitro, diether, and amide, in common (Figure 5). Nitro
substitution in the parent aromatic ring has a positive
electrostatic potential which can interact with the proteins -
carbonyls, sulfurs, and water via “π−hole interaction”.
76
Molecular electrostatic potential studies conducted for MDC
and ADC substantiate this result. The presence of amide bonds
is another salient feature for a better bioactivity. The carbonyl
group and the amine group, which may function as a
hydrogen-bond acceptor (HBA) and a hydrogen-bond donor
(HBD), respectively, are two dierent types of hydrogen-
bonding sites that are often present in an amide.
77
This feature
helps amides form hydrogen-bonding interactions with
biomolecules. The presence of the methylenedioxyphenyl
pharmacophore in the molecules further enhances the
bioactivity. Nucleophilic oxygen in the diether functional
group readily donates electrons to interact with protein
residues for facilitating biological interactions. Upon compar-
ing MDC and ADC, MDC has an electron-donating methoxy
group that readily donates electrons with biomolecules. The
role of the methoxy group in many phytochemicals like
curcumin
20
and resveratrol is significantly explored and proven
in many scientific studies.
78,79
The acetophenone group is an
electron-withdrawing group, and it can accept electrons from
biomolecules to form an electrovalent bond. The conducted in
vitro study results also suggest that MDC has better inhibition
capacity than ADC. This might be due to the presence of
electron-donating methoxy substitution which readily interacts
with the residues in the active site pocket of the protein.
3.4. Characterization. 3.4.1. NMR and IR. 6-Nitrobenzo-
[1,3]dioxole-5-carboxylic acid: The purified product was
analyzed for 1H and 13C NMR using dimethyl sulfoxide
(DMSO) as solvent in 0 delta TMS calibration method. In
Table 7. Binding Energy and Interaction of Proteins with Positive Controls, MDC, and ADC
protein ligand binding energy
(kcal/mol) binding interaction
α-GD acarbose −4.61 ASP:69, TYR:72, HIS:112, TYR:158, ASP:215, VAL:216, LEU:219, ARG:315, ARG:446
MDC −3.83 TYR:158, ASP:215, ASP:216, GLN:279, HIS:280, SER:311, PRO:312, PHE:314, ARG:315,
ASP:352
ADC −2.67 LYS:156, TYR:158, LEU:313, ARG:315, GLU:411, ASN:415
DPP-4 sitagliptin −5.66 GLU:205, GLU:206, PHE:357, TYR:547, TRP:629, SER:630, HIS:740
MDC −5.30 ARG:125, HIS:126, GLU:206, SER:209, SER:630, TYR:631, VAL:656, TRP:659, TYR:666
ADC −4.20 TYR:553, GLN:553, LYS:554, TRP:629
MPO salicylhydroxamic acid −7.88 GLY:90, TYR:296, MET:87, PHE:332
MDC −7.79 GLU:242, ARG:333, LEU:406, LEU:417, LEU:420, ARG:424
ADC −7.74 GLU:102, ARG:333, LEU:217, LEU:420, ARG:424
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proton NMR, −CH2−in the methylenedioxy bridge appeared
as a singlet at 6.21 ppm, and in carbon NMR, the characteristic
acid carbonyl peak appeared at 166.1 ppm. Detailed analysis
data are available in the SI.
N-(4-Methoxybenzyl)-6-nitrobenzo[1,3]dioxole-5-carboxa-
mide (MDC): In proton NMR, the characteristic amide −
NH−was observed as a triplet at 9.00 ppm, and the −CH2−
proton of the methylenedioxy bridge and the methoxy proton
appeared as singlets at 6.27 and 3.74 ppm, respectively. In
carbon NMR, the characteristic amide carbonyl and methoxy
carbon appeared at 165.5 and 55.5 ppm, respectively. The
functional groups in MDC are confirmed using FT-IR analysis.
The characteristic amide stretch (N−H) is observed at
3278.99 cm−1, the carbonyl group (−CO−) stretch at
Figure 4. Two-dimensional (2D) molecular docking binding interaction images of salicylhydroxamic acid, sitagliptin, acarbose, MDC, and ADC
with myeloperoxidase (MPO), dipeptidyl peptidase-4 (DPP-4), and α-glucosidase (α-GD) proteins.
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1643.16 cm−1, and the NO2stretch at 1501 cm−1(asym-
metrical) and 1345.97 cm−1(symmetrical).
N-(3-Acetylphenyl)-6-nitrobenzo[1,3]dioxole-5-carboxa-
mide (ADC): In proton NMR, the amide −NH−is identified
as a singlet at 10.73 ppm. The −CH2−proton of the
methylenedioxy bridge and the methyl proton of acetophenone
appeared as singlets at 6.32 and 2.58 ppm, respectively. In
carbon NMR, acetophenone carbonyl and amide carbonyl are
observed at 198.0 and 164.4 ppm, respectively. Acetophenone
methyl carbon appeared at 27.2 ppm. FT-IR analysis validates
the functional groups in ADC. The characteristic amide
stretching (N−H) is detected at 3311.45 cm−1, the carbonyl
group stretching (−CO−) at 1666.50 cm−1, and the NO2
stretching at 1602.8 cm−1(asymmetric) and 1338.60 cm−1
(symmetric).
Figure 5. Structure−activity relationship of MDC and ADC.
Figure 6. Mass fragmentation pattern of MDC.
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3.4.2. Mass Spectral Data. The exact mass of N-(4-
methoxybenzyl)-6-nitrobenzo-[1,3]-dioxole-5-carboxamide
(MDC) is 330.0852, and 331.0932 is the measured mass in
positive mode [M + 1]. We examined the pattern of mass
spectral fragmentation to further support the predicted
structure. The potential predicted MDC fragments are
C8H4NO5•and C8H9O•, with masses 194.0085 and
121.0653, respectively. A possible dimer is C32H28N4O12,
which has a calculated mass of 661.1704. According to
experimental data (Figure 6), the masses of the dimer
Figure 7. Mass fragmentation pattern of ADC.
Figure 8. ORTEP diagram for the crystal structure.
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C32H28N4O12 and the fragments C8H4NO5•and C8H9O•are
661.1777 and 194.0085 and 121.0647, respectively. The
theoretical masses of the fragments and the dimer agree well
with the experimental data. Apart from these fragments, there
are possibilities of sodium adduct formation,
80
which is shown
in Figure 6.
The exact mass of N-(3-acetylphenyl)-6-nitrobenzo[1,3]-
dioxole-5-carboxamide referred to as ADC is 328.0695, and
329.0766 is the mass obtained in positive mode [M + 1]. Here,
only one fragment is observed; the expected mass for that
fragment (C8H4NO5•) is 194.0089, and the experimental mass
is 194.0078. The theoretical mass agrees well with the
experimental data. As in MDC above, we can see the
formation of three sodium adducts, as shown in Figure 7.
The original mass spectral file images of MDC and ADC are
available in the SI.
3.5. Single-Crystal X-ray Diraction Studies. Unques-
tionably, the crystallization process aids in determining the
final architectures of MDC and ADC. The final crystals are
seen in a 1:1 mixture of dimethyl sulfoxide and hexane after
experiments with various techniques and solvent systems. At
room temperature, a method of gradual solvent evaporation
was used. SC-XRD analysis is performed on the produced
crystals and the resultant ORTEP plot is shown in Figure 8.
Bond angles and bond lengths from DFT (theoretical) and SC-
XRD (calculated) are shown in S3.1.
For MDC, XRD data reveal that the methylenedioxyphenyl
bridge, O3−C3, and O1−C3 show bond lengths of 1.43 and
1.42 Å, respectively with a bond angle of 105.4°for the O3−
C3−O1 bond. The linking amide bond length of nitrogen and
the adjacent methyl group (N1−C9) is 1.46 Å and that of
nitrogen with the carbonyl group (N1−C8) is 1.32 Å. The
corresponding bond angle for C8−N1−C9 is 121.7°and that
for N1−C8−C5 is 115.9°. The amide carbonyl (C8−O5)
bond length is 1.23 Å, and the bond angle for N1−C8−O5 is
123°. The bond length is 1.41 Å for O6−C17 in the methoxy
carbon with a bond angle of 117.6°for C13−O6−C17. For the
nitro group, the bond length of O2−N16 was slightly higher
than that of O4−N6 with a bond angle of 124.4 for the O5−
N1−O2 bond. With cell dimensions a= 4.9569(5) Å, b=
8.3653(8) Å, and c= 18.1827(18) Å, a single crystal is
monoclinic with space group P1211 and Z= 2.
For ADC, XRD data reveal that the methylenedioxyphenyl
bridge, O3−C2, and O1−C2 show a bond length of 1.43 and
1.44 Å, respectively, with a bond angle of 108.29°for the O3−
C2−O1 bond. The linking amide bond length of nitrogen and
the benzene ring (N2−C9) is 1.40 Å and that of nitrogen with
the carbonyl group (N2−C8) is 1.35 Å. The corresponding
bond angle for C8−N2−C9 is 127.36°and that for N2−C8−
C6 is 114.20°. The amide carbonyl (C8−O4) bond length is
1.20 Å, and the bond angle for N2−C8−O4 is 124.03°. The
acetophenone carbonyl (C15−O6) bond length and bond
angle (C16−C15−O6) are 1.22 Å and 120.4°, respectively.
For the nitro group, the bond length of O5−N1 showed
slightly higher bond length than O2−N1. The bond angle for
the O5−N1−O2 bond was found to be 118.3°. When
compared to amide carbonyl, acetophenone carbonyl has
slightly greater values for bond length and bond angle. With
cell dimensions a= 16.823(3) Å, b= 7.2581(12) Å, and c=
11.904(2) Å, a single crystal is monoclinic with space group
P121/c1 and Z= 4.
These bond angles and lengths are in good agreement with
theoretical calculations performed by DFT (Table S1).
Comparable bond lengths exist between the methylenedioxy
bridges of MDC and ADC, but ADC has a larger bond angle;
similar to how the bond length of the amide group is longer in
MDC and the bond angle is greater in ADC. The larger bond
angle in ADC reveals that the atoms are more open and widely
spaced away than those in MDC. Greater flexibility and
rotation are made possible by larger bond angles. The atoms
are tightly spaced, and the molecule is smaller in MDC. The
link between two atoms weakens, becomes less stable, and is
more susceptible to cleavage as the bond length increases.
Figure 9. HOMO and LUMO electron distribution level.
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3.6. Computational Studies. 3.6.1. Frontier Molecular
Orbital Analysis (FMO). To determine a molecule’s chemical
reactivity, the molecular orbital theory is frequently used.
Gaussian-16 software is used to conduct quantum chemistry
research at the B3LYP/6-311G** level of theory. The frontier
electron theory’s crucial component focuses on the highest
occupied molecular orbital (HOMO) and lowest unoccupied
molecular orbital (LUMO) energy orbitals. The HOMO and
LUMO surfaces for MDC and ADC are shown in Figure 9.
The electron-rich nature of HOMO tends to donate electrons,
and the electron-deficient nature of LUMO accepts electrons.
The nucleophilic heteroatoms like nitrogen in MDC and ADC
readily donate electrons, facilitating bond formation. In MDC,
the HOMO surface is located near the amide group and the
methoxy-substituted aromatic ring. The nucleophilic-methoxy
group in MDC can help in bond formation with biomolecules
via electron sharing. Hydrogen-bonding sites of the amide
group facilitate hydrogen-bond formation with the active site
pocket of protein. In ADC, HOMO surfaces are occupied in
the methylenedioxyphenyl ring, amide, and nitro group. The
nucleophilic nature of the diether and the presence of
heteroatoms like nitrogen and oxygen help in bond formation.
For both MDC and ADC, the LUMO surface is located at the
methylenedioxyphenyl ring. Deficiency of electrons is seen
across the region, and it facilitates bond formation with
electron-donating pockets of biomolecules by readily accepting
electrons. The heteroatoms in the ADC−MDC that contribute
to a negative charge are shown in red. Similarly, the atoms like
carbon and hydrogen that contribute to a positive charge are
indicated by green-colored surfaces.
81
The HOMO and
LUMO energies for MDC and ADC are −6.30 and −2.76
eV and −6.65 and −2.68 eV, with energy gaps (ΔE) of 3.54
and 3.96 eV, respectively. Higher energy gap molecules are
typically more stable and less polarizable, resulting in the
hardness of the entity. On the other hand, due to the
molecules’ strong polarizability, and narrow energy gap,
molecules tend to exhibit chemical softness.
82
Pearson
described chemical hardness as the energy gap between two
FMOs.
83
The energy gap for ADC is higher when compared to
MDC, suggesting that ADC shows chemical hardness and is
more stable. The presence of an electron-withdrawing carbonyl
group in acetophenone substitution increased the hardness of
ADC when compared to MDC.
Global chemical reactivity parametersglobal hardness,
global softness, chemical potential, electron negativity, and
global electrophilicity indexare calculated using Koopman’s
theorem.
84,85
Table 8 depicts the global chemical reactivity
parameters. Electronegativity is a term used to describe an
atom’s or functional groups’ ability to pull inbound electrons to
itself.
86
The electronegative groups nitro and carbonyl in MDC
and ADC have a tendency to accept the electrons facilitating
bond formation with biomolecules. The global electrophilicity
index represents the stabilization energy of the molecule. A
smaller global electrophilicity index implies a strong
nucleophilic molecule, while a higher value denotes a strong
electrophilic molecule. Molecules are classified as potent
electrophiles if values are greater than 1.5 eV, moderate
electrophiles if values range in between 0.8 and 1.5 eV, and
minor electrophiles if the values are less than 0.8 eV.
87,88
The
electrophilicity index value for MDC and ADC is larger than
1.5 eV, indicating that the molecules have a potent
electrophilic nature that facilitates the formation of bonds
with biomolecules. Molecules having greater HOMO−LUMO
energy gap show lesser tendency for interaction and bond
formation, while molecules with less HOMO−LUMO energy
gap are more interactive and are more willing to participate in
chemical events involving bond breaking or bond formation.
As can be seen from the DFT values, MDC has low HOMO−
LUMO values, and it is anticipated to participate in the bond
formations like covalent, hydrogen, and ionic bonds.
3.6.2. Hirshfeld Surface Calculations. To get a qualitative
understanding of the role of the principal intermolecular
interaction within the crystal, Hirshfeld surface (HFS) analysis,
a graphical visualization technique, was performed.
89
Based on
the crystallographic information file (CIF), the computational
tool CrystalExplorer 3.1 is used to assess the molecular
Hirshfeld surface analysis and its accompanying two-dimen-
sional fingerprint plots. With the use of the fundamental energy
model B3LYP/6-31G(d,p), the HFS analysis is used to
determine the overall interaction energy of the crystal
structure. With the help of dnorm, it is possible to identify
molecular sites crucial for intermolecular interaction. Equation
1is utilized to pinpoint the crystal compounds’ surface areas
that are especially important for intermolecular interactions.
The normalized contact distance dnorm can be correlated with
the radii of the atoms (vdW),
90,91
the distance to the closest
nucleus interior to the surface (di), and the distance to the
closest nucleus exterior to the surface (de).
dd r
r
d r
r
( )
( )
( )
norm
i i
vdW
i
vdW
e
e
vdW
e
= +
(1)
Spackman and Jayatilaka’s
89
approach oers insightful
information regarding the strength of intermolecular inter-
actions inside crystal structures. Intermolecular hydrogen
bonds are crucial for the interaction of drugs with macro-
molecules. Figures 10 and 11 show the Hirshfeld surface with
dnorm visualization graphs of MDC and ADC and two-
dimensional (2D) fingerprint plots of intermolecular inter-
action surfaces, respectively. The nearest contacts are shown in
red in dnorm, the farthest contacts in blue, and the contacts
equivalent to the sum of the van der Waals radii
92
are shown in
white regions. On Hirshfeld surfaces that are mapped using the
shape index, red and blue triangular pairings may be seen
interacting. This is additional proof that stacking interactions
occur. Table 9 provides information about the molecule’s
surface characteristics.
From the Hirshfeld surface analysis, the corresponding
volume (368.22 Å3), asphericity (0.274), inner area (344.05
Å2), and globularity (0.722) of MDC are calculated. According
to the 2D fingerprint diagram, the divs deof the crystal and
Table 8. DFT Molecular Orbital Energy Calculations
energy
DFT analysis MDC ADC
dipole moment 6.24 D 8.46 D
E(HOMO) −6.30 eV −6.65 eV
E(LUMO) −2.76 eV −2.68 eV
ΔE(HOMO−LUMO) 3.54 eV 3.96 eV
electronegativity (χ) 4.53 4.67
chemical potential (μ)−4.53 −4.67
global hardness (η) 1.7 1.9
softness (σ) 0.28 0.50
global electrophilicity index (ω) 5.79 5.50
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important contacts for crystal packing are found to be H···H
(31.1%), O···H (20.5%), C···H (10.6%), O···O (3.4%), O···C
(2.9%), C−C(1.1%), O···N (0.5%), H···N (0.5%), and N···C
(0.1%). Similarly, for ADC, the corresponding volume,
asphericity, inner area, and globularity are found to be
353.85 Å3, 0.313, 334.08 Å2, and 0.724, respectively. From
the 2D fingerprint diagram, important contacts for crystal
packing are H···H (26.5%), O···H (20.4%), C···H (8.0%), O···
O (4.9%), O···C (4.1%), C···C(5.4%), O···N (0.6%), H···N
(0.9%), and N···C (0.0%).
In 2D fingerprint diagrams, more than 25% of interactions in
both MDC and ADC are for the H···H interaction. Both ADC
and MDC have 20% of O···H interactions. The O···H bonding
interactions are shown by the bright red point in the dnorm
Figure 10. Hirshfeld surface maps for MDC and ADC.
Figure 11. Full and disaggregated 2D fingerprint plot for the specific contacts.
Table 9. Surface Property Information
MDC ADC
name minimum maximum minimum maximum
di0.743 2.569 0.818 2.527
de0.743 2.522 0.818 2.409
dnorm −0.601 1.194 −0.487 1.665
shape index −0.999 0.999 −0.992 0.998
curvedness −3.853 0.259 −3.851 0.356
Figure 12. Molecular electrostatic potential surfaces for MDC and ADC.
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(Figure 11) diagram. Thus, MDC and ADC may easily form
bonds and interact with biomolecules via hydrogen bonding.
3.6.3. Molecular Electrostatic Potential (MEP). DFT at the
B3LYP/6-311G** level is used to calculate the theoretical
MEP. Figure 12 shows the MEP surfaces of MDC and ADC.
When evaluating how molecular interactions are directed, the
MEP is used to forecast the nucleophilic and electrophilic
centers in the molecule. The red area has a value of 6.784 ×
10−2, which indicates high electron density (nucleophilic);
whereas, the blue zone has a value of −6.784 ×10−2, which
indicates low electron density (electrophilic) with a negative
sign. White is a symbol of a neutral atom. MEP focuses in
particular on the electrostatic environment, charge distribution,
and potential for molecular interaction.
In MDC, the electron-rich nucleophilic center is located in
the amide carbonyl and nitro groups. The electrophilic center
is seen in amide nitrogen and methylene groups in the
methylenedioxyphenyl ring. Similarly, for ADC, the nucleo-
philic center is in amide carbonyl, acetophenone carbonyl, and
nitro groups. Electron deficiency is seen across the amide
nitrogen and methylene groups in the methylenedioxyphenyl
ring. We can foresee the groups that will readily interact with
the macromolecules from the MEP results. Here, the
macromolecules can bind with MDC and ADC via electron
sharing through carbonyl and nitro groups. Amide nitrogen
and methylene groups in the methylenedioxyphenyl ring can
accept electrons from macromolecules and form bonds. Both
MDC and ADC have the potential to engage with any target
protein’s active site. They are acceptable for use in medicinal
chemistry.
93
3.7. In Vitro and Ex Vivo Biological Studies. 3.7.1. In
Vitro Antioxidant Assay. In healthy human cells, unstable
radicals have a propensity to form stable pairs with biological
macromolecules like proteins. This leads to the damage to cells
that are crucial in the development of cancer and heart
diseases. The result of an oxidation process that takes place
within the human body is the resulting free radicals.
Antioxidants help in stopping cell deterioration by scavenging
the free radicals that are created. The antioxidant activity of
MDC and ADC is quantified by this study, and the inhibition
percentage graph is plotted in Figure 13. The initial color of
DPPH in ethanol is purple or violet, and we can see that the
color fades to various shades of yellow when MDC and ADC
are added. The amount of electrons increases and the
discoloration turns yellow when an antioxidant adds a
hydrogen atom to DPPH to form DPPH-H.
94
By using a
similar method of action to acquire electrons from the
antioxidant, the addition of antioxidant causes the color of
the ABTS•+solution to change from deep blue to colorless.
95
Ascorbic acid is used as the positive control. From DPPH and
ABTS•+scavenging assay results, the corresponding IC50 values
for MDC and ADC are 25.28 ±0.12, 55.05 ±0.3 and 41.04 ±
0.25, 66.13 ±0.34 μM, respectively; whereas ascorbic acid, a
positive control, has IC50 values of 11.61 ±0.31 and 11.26 ±
0.55 μM, respectively. The results of the antioxidant
investigation demonstrate that MDC and ADC both eectively
scavenge the free radicals present in the solution and exhibit a
promising antioxidant activity when compared to the positive
control. The DPPH and ABTS•+acquire stability by taking
electrons from MDC and ADC. From scientific literature,
96,97
the presence of a methoxy group, a +M substituted group in
the para position, and a −M substitution in the meta position
favor the antioxidant activity. The methoxy group, an electron
donor with beneficial mesomeric action, is present in the core
moiety of MDC in the para position. Both MDC and ADC
exhibit good antioxidant properties, although MDC is a more
powerful antioxidant when comparing IC50 values and SAR
observations.
3.7.2. Ex Vivo Anti-inflammatory Activity. On investigating
the hemolytic activity of MDC and ADC, the anti-
inflammatory medicine aceclofenac exhibits an increase in
activity with concentration, as shown in Figure 14. When red
blood cells (RBCs) are added to the hypotonic solution, the
hemoglobin begins to oxidize, which causes the RBC
membrane to burst. Free radical production in ruptured cells
is then sparked by lipid peroxidation. At a concentration of 20
mM, MDC and ADC demonstrated 55 and 51% anti-
inflammatory action, respectively, and as the concentration
increases, the activity likewise increases to 90 and 88%. The
positive control has an IC50 value of 3.87 ±0.28 μM, while
MDC and ADC have values of 21.05 ±0.10 and 27.54 ±0.22
μM, respectively. The anti-inflammatory activity of both MDC
and ADC is extremely good and promising, although ADC
outperformed MDC in terms of IC50 values and the percentage
of anti-inflammatory activity when compared with the industry
standard.
3.7.3. In Vitro α-GD Inhibition Assay. Newly synthesized
MDC and ADC are investigated for their ability to inhibit α-
GD protein, and the results are compared with the positive
control acarbose. The enzyme α-glucosidase will catalyze the
Figure 13. DPPH and ABTS antioxidant activities of compounds.
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transformation of the substrate 4-nitrophenyl-α-D-glucopyrano-
side into α-D-glucopyranoside and p-nitrophenol under the
given experimental conditions (pH = 6.8; T= 37 °C). The
latter product’s yellow hue is spectrophotometrically evaluated
at 405 nm, and the inhibition percentage graph is plotted in
Figure 15. At a concentration of 10 mM, ADC and MDC
demonstrated 35 and 39% α-GD inhibition, respectively, and
as the concentration increased, the activity was found to
increase by 81 and 83%. The positive control has an IC50 value
of 14.27 ±0.21 μM, while ADC and MDC have values of
46.05 ±0.10 and 41.11 ±0.52 μM, respectively. Both MDC
and ADC have the potential to inhibit α-GD; while
considering IC50 values and comparison with the positive
control, MDC is a better α-GD inhibitor.
3.7.4. In Vitro DPP-4 Inhibition Assay. The potential of
MDC and ADC to inhibit DPP-4 is tested via fluorometric
assay with sitagliptin as the positive control. The investigation
involved monitoring the release of 7-amino-4-methylcoumarin
(AMC), a fluorescent compound that is produced when DPP-
4 cleaves H-Gly-Pro-7-amido-4-methylcoumarin hydrobro-
mide (GP-AMC). The study is performed with 50, 100, 150,
and 200 μM concentrations of MDC and ADC for 5−60 min
after adding the inhibitor. The results are plotted in a line
graph of time vs concentration, and the slope values for each
concentration are calculated. Simultaneously, slope values are
plotted for sitagliptin and the blank solution (without
inhibitor). The slope values calculated from the line graph
are utilized to calculate the percentage of inhibition, as shown
Figure 16. The IC50 value was calculated by plotting the
percentage of inhibition versus dierent concentrations of
MDC and ADC. A similar process was employed for the
positive control. The IC50 value for sitagliptin is 3.88 ±0.23
μM and that for ADC and MDC is 31.02 ±0.17 and 25.34 ±
0.11 μM, respectively. MDC and ADC moderately inhibit
DPP-4, and MDC with less IC50 value upon comparing ADC
exhibits better inhibition.
3.7.5. In Vitro MPO Inhibition Assay. MPO inhibition is
carried out in a cell-free system by utilizing 3,5,3′,5′-
tetramethylbenzidine (TMB) as an enzyme substrate and
salicylhydroxamic acid as a positive control. Percentage of
inhibition vs absorbance is plotted with varying concentrations
of MDC and ADC, and the IC50 values are then calculated.
The positive control has an IC50 value of 2.9 ±0.13 μM, while
MDC and ADC have values of 4.5 ±0.19 and 10 ±0.27 μM,
respectively. At low concentrations (5 μM), MDC exhibits
more than 50% inhibition. Even though we achieved 50%
inhibition at low concentration, we still wanted to evaluate the
maximum inhibition. At 25 μM, it exhibits a maximal
inhibition that is more similar to the positive control. From
the percentage inhibition graph in Figure 17 and the IC50 value
in Table 10, MDC exhibited good inhibition at very low
concentrations.
Upon comparison of IC50 values of MDC and ADC with α-
GD, DPP-4, and MPO (Table 10), MDC exhibited selective
MPO inhibition with an IC50 value closer to the positive
control. Docking results put forward that MDC has good
interaction with the MPO protein’s active site pockets. This
makes it easier for MDC to dock with a protein and stop it
from acting. Results from theoretical molecular docking and
experimental in vitro studies of MDC are in good agreement
with one another. In light of the performed studies, we strongly
Figure 14. Anti-inflammatory activity of MDC and ADC.
Figure 15. α-Glucosidase inhibition by MDC and ADC.
Figure 16. Dipeptidyl peptidase-4 inhibition by MDC and ADC.
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recommend MDC for MPO inhibition after performing in vivo
studies.
4. CONCLUSIONS
The findings of the current study suggest that theoretical and
experimental biological studies using synthesized derivatives of
6-nitrobenzo-[1,3]-dioxole-5-carboxamide yield promising re-
sults. Molecular docking, ADMET, and Lipinski rules opine
that the compounds are similar to drugs and the ligands exhibit
strong protein-binding interactions. The ligands exhibit good
binding energy to MPO and are selective for the oxidative
protein. MDC exhibited more activity than ADC in anti-
inflammatory and antioxidant studies. SC-XRD analysis
revealed the precise structural characteristics of the com-
pounds. The experimental (SC-XRD) and theoretical (DFT)
measurements of MDC and ADC bond lengths are consistent.
Molecules’ chemical behavior is predicted using DFT
calculations, and a smaller energy gap from FMO revealed
that these molecules are very interactive. Methylenediox-
yphenyl ring and the amide group in both FMO and MEP
molecules are capable of easy interaction and binding with the
active sites of proteins or other biomolecules. The in vitro α-
GD, DPP-4, and MPO inhibition by both MDC and ADC
appeared to be promising. MDC outperformed ADC in terms
of IC50 values and the percentage of inhibition activity when
compared with the positive control. MDC’s overall bioactivity
is favored by the inclusion of an electron-donating group,
methoxy group, in the para position, providing appropriate
geometrical properties to the molecules. This oers avenues for
MDC to fit into the target protein intact. In view of this proper
geometrical orientation, MDC exhibits selective myeloperox-
idase inhibition in in vitro enzymatic studies. Therefore, for the
development of myeloperoxidase-targeted medicines for
cardiovascular disease, derivatives of 6-nitrobenzo[1,3]-
dioxole-5-carboxamide, which have not yet been identified in
the literature, are ideal.
■ASSOCIATED CONTENT
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsomega.3c07555.
Synthesis and crystallization; characterization data for
synthesized derivatives and starting material; crystal
structure report for MDC and ADC; procedures for in
vitro and ex vivo biological studies (PDF)
■AUTHOR INFORMATION
Corresponding Author
Rajagopal Desikan −Department of Chemistry, School of
Advanced Sciences, Vellore Institute of Technology, Vellore
632014 Tamilnadu, India; orcid.org/0000-0002-6118-
8823; Email: desikanrajagopal@gmail.com
Authors
Reshma Rajan −Department of Chemistry, School of
Advanced Sciences, Vellore Institute of Technology, Vellore
632014 Tamilnadu, India
Sambantham Karthikeyan −Department of Chemistry,
School of Advanced Sciences, Vellore Institute of Technology,
Vellore 632014 Tamilnadu, India; orcid.org/0000-
0001-7807-151X
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsomega.3c07555
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
The authors are grateful to VIT-RGEMS for the financial
support. They also thank DST-VIT-FIST for NMR and single-
crystal XRD, VIT-SIF for FT-IR, and VIT for HRMS and other
instrumentation facilities.
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