Identification of 2-benzoxazolinone derivatives as lead against molecular targets of diabetic complications

Article abstract of DOI:10.1111/cbdd.13369

Diabetic complications follow multiple pathophysiological pathways involving aldose reductase (ALR2)-mediated polyol pathway, advanced glycation end products (AGEs) and reactive oxygen species formation. Literature suggests ALR2 inhibitors such as epalres

Full text of DOI:10.1111/cbdd.13369

DR OM SILAKARI (Orcid ID : 0000-0002-1174-1125)
Article type : Research Article
Identification of 2-benzoxazolinone derivatives as lead against molecular targets of diabetic
complications
Bhawna Vyasa, Shalki Choudharyb, Pankaj Kumar Singhb, Baldev Singha, Renu Bahadurc, Ashok Kumar
Malika, Om Silakarib*
aDepartment of Chemistry, Punjabi University, Patiala, Punjab, India, 147002
bMolecular Modeling Lab, Department of Pharmaceutical Sciences and Drug Research, Punjabi
University, Patiala, Punjab, India, 147002.
cIndian Council of Medical Research, New Delhi, 110029.
*E-mail: omsilakari@gmail.com
Telephone: +919501542696
Abstract
Diabetic complications follow multiple pathophysiological pathways involving aldose reductase
(ALR2) mediated polyol pathway, advanced glycation end products (AGEs) and reactive oxygen
species formation. Literature suggests ALR2 inhibitors such as Epalrestat to possess significant
potential in retinopathy and neuropathy. Thus in this study multiple pathophysiology directed
molecules targeting ALR2,AGEs and free radicals formation were designed using in-silico techniques.
Initially, database was screened via in-silico tools to obtain hitswith affinity for the catalytic domain
of ALR2. Additional focus was laid on the presence of structural attributes responsible for AGE’s
inhibitory and anti-oxidant potential. Out of obtained hits, 2-benzoxazolinone scaffold was selected
and ten derivatives were synthesized accordingly. Finally, the synthesized molecules were evaluated
for their ALR2 and AGEs inhibitory activities along with free radical scavenging potency.
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13369
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Keywords: neuropathy; retinopathy; 2-benzoxazolinone; in-silico analysis
1. Introduction
Chronic diabetes is associated with persistent hyperglycaemia, which further leads to secondary
complications. These diabetic complications (DC) are multi-factorial disorders postulated to cause
various irreversible pathological conditions viz. macrovascular and microvascular complications (1).
The microvascular complications such as retinopathy, neuropathy and nephropathy progress
through different pathways such as polyol pathway, advanced glycation end product (AGEs)
pathway, protein kinase c pathway (PKC) and hexosamine pathway, which are less active under
normal condition (2, 3). Additionally in diabetic complications, oxidative stress increases the
production of reactive oxygen species through mitochondrial electron transport chain and triggers
all the above mentioned pathways through unifying mechanism(4).
In the last couple of decades, the management of diabetic complications has been tried via
development of aldose reductase inhibitors (i.e. epalrestat, tolrestat), protein kinase Cβ inhibitors
(i.e. ruboxistaurin) but none of them has received approval by US-FDA. The major road block in the
management of diabetic complications is the identification of new chemical entities to target
multiple pathological processes underpinning the pathogenesis of diabetic complications. In this
article, we report molecular modelling based designing of some 2-benzoxazolinone derivatives as
inhibitors of multiple molecular targets of diabetic complications. Based on the in-silico results, the
designed molecules were synthesized and evaluated against molecular targetssuch as ALR2, AGEs
and oxidative stress using in-vitro assays.
2. Experimental
2.1. Pharmacophore based virtual screening
Initially a previously published ligand based pharmacophore model was utilized to screen an in-
house heterocycle database (5). Fit value of least “fit” active molecule was considered as a cut-off
value to screen hits. Further obtained hits were filtered to obtain molecules having structural
requirements for anti-AGE and anti-oxidant potential.The basic features required for AGEs inhibition
include a hetero-aromatic nucleus with hydrogen bonding capability, one or more hydrophobic
region and a protonable nitrogen atom which is connected to central core with alkyl linker (6).
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2.2. Molecular docking and Dynamics Simulations
To perform molecular docking analysis, X-ray crystallographic structures of ALR2 (PDB ID: 1US0) was
downloaded and prepared using automated Preparation Wizard in Schrodinger. The ligands were
sketched using drawing tools and were optimized using Ligprep tool (pH 6.5-7.5). Docking analysis
was carried out to identify the putative binding pose of the molecules within the active site of the
proteins. Glide was used to perform the docking simulations, while Desmond software working on
OPLS_2005 forcefield was used for molecular dynamics simulations. The protein complex was
neutralized by adding Na+ ions and the system was then solvated in orthorhombic box of TIP3P
water molecules having a salt concentration of 0.15M. A temperature of 310K was maintained using
Nose-Hoover Chain thermostat and a pressure of 1.01 bar using Martyn-Tobias-Klein barostat(7, 8).
2.3. MMGBSA and ADME calculation
The ligand binding energies (kcal/mol) of final hits complexed with respective proteins were
calculated using Prime MM/GBSA module employing an equation:
MMGBSA ΔGbind = ER: EL – EL – ER
Where ER: EL, EL and ER are the prime energies of the optimized complex, free ligand and free
receptor, respectively (9).
Additionally, the ADME properties of all synthesized compounds were also calculated using QikProp
module of Schrödinger software.
2.4. Chemical synthesis
All the organic/inorganic chemicals, solvents and reagents used for the synthesis were of analytical
grade and purchased from Merck, Himedia, SD fine and Sigma Aldrich with >99% purity as certified
by the manufacturer. Progress of the reaction was monitored at each step with thin layer
chromatography using pet ether:ethyl acetate (8:2) as a solvent system. Meltingpoint of all
compounds was recorded on Labtronics digital automatic melting point apparatus and was
uncorrected. Finally purified compounds were characterized using IR, NMR and mass techniques. IR
spectra were recorded using Bruker (Alpha E) FTIR spectrometer. Proton and carbon NMR spectra
were recorded at 400MHz and 100MHz respectively on Bruker advance II NMR spectrometer using
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CDCl3 as a solvent and tetramethylsilane (TMS) as an internal standard. Mass spectra were recorded
on waters Q-TOF micro MS spectrometer at ESI+mode as m/z ratio.
2.4.1. General procedure for the synthesis of BV (1-10)
Protocol followed for the synthesis of all compounds is displayed in scheme 1. In first step, 4mmol of
different benzyl chlorides (2) and 10 mmol of K2CO3 were added to a solution of 2 mmol of 2-
benzoxazolinone (1) in acetone at room temperature.After completion of reaction, as monitored by
TLC, the reaction mixture was filtered and concentrated to obtain crude products. Resulting
products were then purified by column chromatography using pet ether:ethyl acetate (8:2) as
solvent system.
2.5 In vitro antioxidant activity (DPPH assay)
1,1-diphenyl-2-picryl hydrazyl (DPPH) radical was used for the determination of free radical-
scavenging activity of the synthesized compounds. DPPH free radical is reduced to the corresponding
hydrazine by abstracting a hydrogen atom from antioxidant (10). Like other free radicals, it do not
dimerize and give rise to deep violet color with an absorption in ethanol solution at around 520 nm.
When mixed with antioxidants, DPPH gives rise to the reduced form (yellow color) by accepting
hydrogen atom from the antioxidant (11).
The 0.1 mM solution of DPPH in methanol (39.4 mg in 1000 ml) was freshly prepared and
covered with black paper. Different concentrations of test compounds were prepared in
methanol and added with an equal volume to the methanolic solution of DPPH. After 30 min
the absorbance was recorded at 517 nm. Ascorbic acid was used as a standard or positive
control in the assay. EC50 values were calculated for standard and all test compounds by
plotting concentration versus % inhibition graph. EC50 value is the concentration of
antioxidants required to scavenge 50% of DPPH free radicals (12).
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2.6. In vitro ALR2 inhibitory activity
The goat eyes were obtained from a local slaughter house just after slaughtering and stored in PBS
pH 7 for transportation. Followed by extraction of enzyme aldose reductase from goat eye lens. The
eye lenses from the freshly slaughtered goats were removedand washed with saline. 2.5 g of eye
lenses were pooled and homogenized with 7.5 mL of 2-mercaptoethanol in cold water (1:3 W/V).
Any insoluble material was removed from the homogenate by centrifugation at 10,000 rpm for 30
min (4°C) and obtained supernatant liquid was further saturated with 40% solution of ammonium
sulfate to remove inert proteins from the mixture. The saturatedsolution was filtered and
centrifuged again for 30 min at 4°C toobtain pure ALR2 enzyme, which was further used in the assay
(13, 14).
2.6.1. Determination of enzymatic activity- Absorbance of a cuvette containing assay reaction
mixture i.e. 1 mL of freshly extracted ALR2 enzyme, 1 mL of different concentrations of inhibitors in
1% DMSO and 1 mL of 0.067 M phosphate buffer (0.104mM NADPH, 10 mM glyceraldehydes and 0.4
M LiSO4) with final pH 6.2 was recorded againstnegative controlgroup containing all the components
but no inhibitors using UV spectrophotometer at 340 nm for 3 min. The enzyme reaction is started
upon the addition of a substrate (glyceraldehydes). The enzyme inhibitory activity was calculated
using following formula:
Activity U/ml =
ΔA test per min × total volume of assay mixture
(6.2 µM, extinction coefficient of NADPH at 340nm) x volume of enzyme taken
Where, ΔA test/min is the rate of change of absorbance per minute of test compounds.
The percentage inhibition of all test compounds was calculated by considering 100% activity in
negative control groupagainst ALR2. The 50% inhibitory concentration (IC50) of each test compound
was determined by plotting log concentration of test compounds versus percentage inhibition (15).
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2.7. In vitroALR1 inhibitory activity
Extraction and purification of ALR1 enzyme was performed by following standard procedure as
reported in the literature (16). Freshly obtained rat kidney was kept in PBS solution, which was
further homogenized by taking 3:1 ratio of 10mM sodium phosphate buffer (pH 7.2) having 2.5mM
2-mercaptoethanol 2.0mM EDTA, 0.25 M sucrose and rat kidney (1). The resulting homogenate was
further centrifuged at 10,000 rpm for 20 min and supernatant solution was saturated with
ammonium sulfate solution. Obtained precipitates were then filtered and filtrate was again
centrifuged at 10,000 rpm for 20 minutes. Finally obtained supernatant was used as a source of ALR1
enzyme for the assay.
2.7.1. Determination of enzymatic activity
Absorbance of assay mixture (1ml of 50mM sodium phosphate buffer pH 7.2 containing 10 mM DL-
glyceraldehyde, 0.1 mM NADPH, 0.2 M ammonium sulfate and 5mM β-mercaptoethanol + 1ml of
enzyme supernatant + 1ml of different concentrations of inhibitors) was recorded
spectrophotometrically at λmax 340nm and 37°C temperatureat an interval of 1 minute for a period of
3 minutes. Reaction starts after the addition of substrate DL-glyceraldehyde and the ALR1 activity
was monitored as a function of consumption of NADPH due to its oxidation via ALR1. Enzyme
inhibitory activity and IC50 values werecalculated using the same formula as used in case of ALR2.
Selectivity index of all compounds was calculated by taking the ratio of IC50 values of ALR1 and ALR2.
2.8. In vitro AGEs inhibitory activity
Glycation reaction between free amino group of biomolecules and carbonyl group of glucose leads
to the production of advanced glycation end products (AGEs), which are implicated in diabetic
complications(17). Amadori products obtained as a result of glycated protein were measured using
bovine serum albumin (BSA)-glucose assay(18). In this assay, different concentrations of test
compounds were prepared by dissolving in 1% DMSO. 100 µl of each test compound was incubated
with 500 µl of Bovine serum albumin (BSA, 1mg/ml) and 400 µl of 500 mM glucose at 60°C for 24h.
After the incubation period, 10 µl of 100% (w/v) trichloroacetic acid (TCA) was added to the reaction
mixture in order to stop the reaction.Themixture was then keptat 4°C for 10 min and then
centrifuged at 15,000 rpm at 4°C. Precipitates of glycated protein were redissolved in 1ml PBS
(pH10) and immediately quantified in triplicate for glycated BSA on the basis of fluorescence
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intensity measured on spectrofluorometer at 370 nm (excitation) and 440nm (emission).
Fluorometer cuvette containing BSA, Glucose and different concentration of test compounds was
read against negative control (BSA and glucose alone without any inhibitor) and aminoguanidine was
used as a positive control (19). The percentage inhibition was calculated by using formula
Intensity of control - Intensity of test
Intensity of control
× 100
% Inhibition =
IC50 of all test compounds and standard were calculated by plotting % inhibition versus log
concentration curve.
3. Results and discussion
3.1. In-silico analysis
Previously reported Ligand-based pharmacophore models for ALR2(20), generated using Phase
module of Schrödinger was employed to screen an in-house small molecule database. This screening
yielded 2-benzoxazolinone derivatives as hits, which were then subjected to docking analysis using
co-crystallized 3-D structure of ALR2 followed by molecular dynamic simulations and calculation of
MM-GBSA score (binding energies). Molecular docking analysis in ALR2 showed that the carbonyl
group in the core nucleus acts as an acceptor group and interacts via hydrogen bond with His110 in
many of the designed compounds while the fused aromatic ring of the scaffold formed π-π
interactions with various hinge region residues such as Trp111. All the compounds showed good
docking scores (Glide XP G-score) ranging from -1.74 to -1.49 within the ALR2. Docking was followed
with molecular dynamic simulations of the designed molecules in complex with ALR2, for a period of
10 ns. Simulations disclosed that almost all the molecules were maintaining key H-bond interactions
with catalytic domain amino acids. The RMSD values of the protein and ligands in the complex with
top compound (BV-4) were calculated to observe the overall stability of the complexfor the given
period of time and the graphs for the same are represented in fig.1. The 3D interaction diagram of
the designed molecules BV-4 in ALR2 is shown in Fig. 2. Finally, binding energy scores (MM-GBSA
scores) of the compounds lied in the range of -72.83 to -37.54 in ALR2 (Supplementary table S2).
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Finally, ADME properties of synthesized compounds were calculated. Pharmacokinetic prediction
resulted in the selection of ligands which are more likely to exhibit optimum ADME properties.
Values of ADME properties for BV1-BV10 fall in the acceptable limits according to lipinski’s rule of
five (21) (Supplementary table S3).
3.2. Chemistry
For the synthesis of molecules pertaining to scheme 1 given in supplementary, in the step one,
benzylation of 2-benzoxazolinone was performed by treating it with variedly substituted benzyl
chlorides in the presence K2CO3 using acetone as solvent to yield different benzylated2-
benzoxazolinone. The obtained compounds were purified via recrystallization. All the compounds
1
13
were characterized by IR, H-NMR and C-NMR. In IR spectrum, the 2-benzoxazolinone derivatives
showed the presence of strong absorption bands of C=O ~1750 and C=N from ~1610 to ~1620 cm-1.
1
The synthesis of final compounds was confirmed in H-NMR. Almost each spectrum showed
presence of singlet of two –CH2 proton at ~5.0 ppm. Rest of the aromatic protons were observed in
13
similar pattern from ~6.8 to ~ 7.8 ppm. C spectrum exhibited characteristic peak at ~154 ppm for
carbonyl and aromatic carbons were observed from 110 to 134 ppm with a characteristic peak at 55
ppm for –CH2 carbon. Similarly, mass spectrometry also showed quasi ion peaks at expected m/z
values.
3.3. In vitro antioxidant activity (DPPH assay)
Oxidative stress is the unifying mechanism which triggers all the pathways of diabetic complications.
All the synthesized compounds BV(1-10) were screened for free radical scavenging activity using
DPPH assay. Results in the form of EC50 values are summarized in Table 1. All compounds have
shown activity in range of 180-620 nM in term of EC50.The anti-radical power (ARP) of the standard
ascorbate was found to be 0.126, the stoichiometry was found to be 0.398 and number of molecules
of DPPH quenched was found to be 2.512.
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3.4. In vitroALR2 inhibitory activity:
The synthesized compounds were evaluated for their ALR2 inhibitory activity by considering negative
control group (with no inhibitor) with 100% ALR2 activity. All experiments were performed in
triplicate and results are summarized in Table 1 in terms of IC50 values with standard error mean
(SEM) at 0.05 level of significance.All compounds have shown promising ALR2 inhibitory activity
which is in range of 1.53-9.77 µM. Out of all, compound BV-6 with fluoro substitution at 4th position
of benzyl ring and BV-4 with chloro substitution at 2nd position of benzyl ring have shown best ALR2
inhibitory activity with IC50 value of 1.53±0.98 µM and 1.89±0.90 µM respectively.
3.5. In vitro ALR1 inhibitory activity:
ALR1 inhibitory activity was performed on freshly removed rat kidney.Valproic acid was used as a
standard drug and was used as a positive control value (16, 22). The IC50 value and selectivity index
of BV1-BV10 are displayed in Table 1. Our results clearly indicate that the compounds with nitro,
chloro and methyl substitutions (BV2, BV4 and BV7) are less active on ALR1 when compared to their
activity on ALR2. Furthermore, BV4 was found to be most selective ALR2 inhibitor with selectivity
index 10.73.
3.6. In vitro advanced glycation end-product (AGEs) formation inhibitory activity:
In diabetic complications, AGEs formation leads to various pathological conditions. All the
synthesized compounds BV (1-10) were evaluated for in vitro AGEs inhibitory activity and their
corresponding results are shown in Table 1. Activity was found in range of 1.82-21.31 µM for all
screened molecules. Compound BV-1 and BV-8 displayed best activity with IC50 values 2.157±1.44
and 1.82±1.14 respectively.
4. Conclusion
Present study is simply an in-silico guided effort to identify lead molecules having ALR2 inhibitory
potential with additional ability to inhibit formation of AGEs and process of free radicals generation,
which may be beneficial for diabetic complications. On the basis of this study it can be concluded
that ten 2-benzoxazolinone derivatives, effective against ALR2, AGEs and oxidative stress were
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identified. This scaffold can be further explored to develop molecules with better activity profile for
the treatment of diabetic complications.
Acknowledgement
BV would like to thank ICMR for Research Associate Fellowship.
Conflict of interest
Declared none
Legends
Fig. 1 RMSD plot of BV-4 in complex with ALR-2 for the time period of 10 ns.
Fig. 2 Compound BV-4 in the catalytic domain of ALR2 after the time period of 10 ns, i) 3D-view, ii)
2D-view.
Table 1 The Aldose reductase inhibitory, advanced glycation end products (AGEs) formation
inhibitory and radical scavenging activities of synthesized compounds (BV-1 to BV-10).
Legends for supporting information
Scheme 1 General synthetic scheme for the synthesis of BV (1-10)
Table S1 All the synthesized molecules along with their physicochemical data
Table S2 MMGBSA score, predicted activity and fit value of BV1-BV10
Table S3 ADME properties of all synthesized compounds using Qikprop module of schrödinger
Fig. S1 HNMR spectra of BV-1
Fig. S2 C13 NMR spectra of BV-1
Fig. S3 Mass spectra of BV-1
Fig. S4 HNMR spectra of BV-2
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Fig. S5 C13NMR spectra of BV-2
Fig. S6 Mass spectra of BV-2
Fig. S7 HNMR spectra of BV-3
Fig. S8 C13NMR spectra of BV-3
Fig. S9 Mass spectra of BV-3
Fig. S10 HNMR spectra of BV-4
Fig. S11 C13NMR spectra of BV-4
Fig. S12 Mass spectra of BV-4
Fig. S13 HNMR spectra of BV-5
Fig. S14 C13NMR spectra of BV-5
Fig. S15 Mass spectra of BV-5
Fig. S16 HNMR spectra of BV-6
Fig. S17 C13NMR spectra of BV-6
Fig. S18 Mass spectra of BV-6
Fig. S19 HNMR spectra of BV-7
Fig. S20 Mass spectra of BV-7
Fig. S21 HNMR spectra of BV-8
Fig. S22 Mass spectra of BV-8
Fig. S23 HNMR spectra of BV-9
Fig. S24 Mass spectra of BV-9
Fig. S25 HNMR spectra of BV-10
Fig. S26 Mass spectra of BV-10
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Table 1: The Aldose reductase inhibitory, advanced glycation end products (AGEs) formation
inhibitory and radical scavenging activities ofsynthesizedcompounds (BV-1 to BV-10).
ALR2 inhibitory
activity
ALR1 inhibitory
activity
AGEs inhibitory
activity
Radical scavenging
activity
Substituent
(R)
Selectivity
indexb
C. No.
(IC50a±SDa, µM)
2.46±0.67
3.16±0.71
5.84±0.64
1.89±0.90
6.73±1.49
1.53±0.98
7.44±1.07
7.45±0.24
9.77±0.53
7.15±1.15
0.8±1.49
-
(IC50a±SDa, µM)
(IC50c± SDa, µM)
2.157±1.44
11.76±3.28
5.06±1.52
8.64±1.20
21.31±1.89
12.24±1.51
2.5±1.21
1.82±1.14
8.544±1.75
3.299±0.97
-
(EC50d± SDa, nM)
254.49±0.60
180.93±1.17
246.08±1.13
213.38±2.73
387.63±1.30
453.09±6.18
458.68±14.63
267.61±6.67
409.95±5.09
620.62±10.60
-
BV-1
4-NO2
3-NO2
2-NO2
2-Cl
9.58±1.27
3.89
5.01
2.42
10.73
2.18
5.99
2.12
1.21
1.09
1.62
BV-2
15.86±0.77
14.19±1.12
20.28±5.4
BV-3
BV-4
BV-5
3-Cl
14.69±1.59
9.17±2.46
BV-6
4-F
BV-7
4-CH3
4-(CH3)2CH
4-(CH3)3C
3-CF3
-
15.82±1.67
9.04±0.95
BV-8
BV-9
10.67±1.92
11.64±2.95
BV-10
Epalrestat
Valproic acid
-
56.1±2.7
-
-
-
-
-
Aminoguanidi
ne
-
-
-
-
-
-
0.45±1.71
-
-
Ascorbic acid
199.4±0.466
a SD: Data are expressed as mean ± SD (n=3). Data were statistically analyzed by one way ANOVA; *P<0.05 vs Std.
bDefined as IC50 [ALR1]:IC50 [ALR2].
c IC50 was defined as the concentration resulting in 50% inhibition of AGEs product formation.
d EC50 was defined as the effective concentration resulting in 50% scavenging activity.
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Scheme 1 General synthetic scheme for the synthesis of BV (1-10)
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