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Synthesis and Antidiabetic Evaluation of NAo-Benzylidenebenzohydrazide Derivatives by In Silico Studies Yusuf Syaril Alam1.
Pratiwi Pudjiastuti2*.
Saipul Malulana3.
Nur Rahmayanti Affifah1.
Fahimah Martak1.
Arif Fadlan1.
Tutik Sri Wahyuni3, and Syukri Arief4 Department of Chemistry.
Faculty of Science and Data Analytics.
Institut Teknologi Sepuluh Nopember.
Jl.
Arif Rahman Hakim.
Surabaya 60115.
Indonesia Department of Chemistry.
Faculty of Science and Technology.
Airlangga University.
Surabaya 60115.
Indonesia Department of Pharmaceutical Science.
Faculty of Pharmacy.
Airlangga University.
Surabaya 60115.
Indonesia Department of Chemistry.
Faculty of Mathematics and Natural Sciences.
Andalas University.
Limau Manis Campus.
Padang 25163.
Indonesia * Corresponding author:
email: pratiwi-p@fst.
Received: February 6, 2023 Accepted: June 7, 2023 DOI: 10.
22146/ijc.
Abstract: Three new NAo-benzylidenebenzohydrazide (NBB) derivatives were successfully synthesized and yielded 50Ae58%.
FTIR.
ESI-MS, 1H- and 13C-NMR were used to investigate the characteristic of NBB derivates.
The structure and relationship of NBB derivatives into -glucosidase and -amylase as good targets for diabetes treatment were evaluated using in silico screening.
Molecular mechanics-Poisson Boltzmann/generalized born surface area (MM-PB/GBSA) was used to calculate the free binding energy .
Gbind (MM-GBSA)) of NBB to -glucosidase and -amylase receptors showed that the results of Oe0.
45 and Oe20.
79 kcal/mol, respectively.
In the ortho position.
NBB derivatives exhibited electron donating groups (EDG like -OCH3, -OH and -Cl with binding free energies of Oe21.
Oe6.
71, and 21.
94, respectively, and acarbose, a native ligand energy of Oe32.
kcal/mol.
In addition, the binding free energy of N-2-(-OCH3, -OH and -C.
-NBB to the -amylase receptor showed the number of Oe39.
Oe43.
Oe42.
81, respectively and Oe46.
51 kcal/mol in comparing with a native ligand.
As a result, it was found that all the NBB derivatives were able to interact with several amino acids in the -glucosidase cavity as well as the native ones, including Ala281.
Asp282, and Asp616.
NBB and native ligand showed similar interaction between -amylase with Gly110 amino acid residue.
Keywords: NAo-benzylidenebenzohydrazide.
-amylase.
n INTRODUCTION Diabetes, in general, is a chronic metabolic disease characterized by elevated levels of blood glucose, which is divided into several types.
Specifically, type-2 diabetes is a hyperglycemia and either complete or partial deficiencies of insulin secretion .
Over the past few decades, there has been a rise in the prevalence of type-2 diabetes in many countries of the world from a wide range of income An alternative therapeutic approach for controlling Yusuf Syaril Alam et al.
hyperglycemia associated with type-2 diabetes is to target -glucosidase and -amylase enzymes that catalyze starch hydrolysis in the intestine.
Inhibition of -glucosidase -amylase hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) and retard the absorption of glucose .
Acarbose, miglitol, and voglibose are the three glucosidase inhibitors that have been approved for use in clinical trials at this time .
Voglibose comes from a microbial origin, whereas miglitol is synthetically derived Indones.
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Fig 1.
Synthesis of NBB and derivatives from 1Aadeoxynojirimycin .
The -amylase is a calcium metalloenzyme that helps in the digestion of polysaccharide molecules into small saccharides.
As same as -glucosidase, the -amylase enzyme causes postprandial hyperglycaemia and increased blood glucose With these characteristics, -amylase is a wellknown therapeutic target for the treatment and maintenance of elevated postprandial blood glucose .
NAo-benzylidenebenzohydrazide (NBB) and its derivatives are important to the development of a significant class of new drugs .
Taha et al.
recently reported benzothiazole NBB derivatives containing benzohydrazide as -glucosidase inhibitor with a wide range of IC50 values.
Ullah et al.
also reported that benzohydrazide-based imine and thiazolidine-4-one inhibit -glucosidase and -amylase enzymes.
According to Fan et al.
chromone-based NBB derivatives may have the potential to perform as -glucosidase inhibitors.
The core of benzohydrazide is crucial to the inhibition of their enzymes.
Therefore, the research aims to synthesize and evaluate NBB-derived with electron-donating groups (-OCH3, -OH and -C.
(Fig.
as antidiabetic to target -glucosidase and -amylase enzymes by using molecular docking.
n EXPERIMENTAL SECTION Materials Analytical grades of benzohydrazide, benzaldehyde, o-anisaldehyde, ochlorobenzaldehyde were purchased from Sigma Aldrich.
Analytical grades of hexane, dimethylformamide, dimethyl sulfoxide, dichloromethane, sodium acetate and glacial acid were purchased from Merck.
The pure solvents of ethanol 99.
9%, methanol 99.
8%, chloroform, and ethyl acetate were prepared from Fulltime.
Instrumentation NMR measurement was used TMS as an internal reference.
NMR spectra were recorded on a JEOL Yusuf Syaril Alam et al.
Resonance 400 MHz spectrometer, and the chemical shifts were reported in .
TLC was performed using silica gel 60 F254 aluminium sheet, while ESI-MS data were obtained using water mass spectrometer QTOF XEVO.
At last, functional groups were analyzed using FTIR Bruker Opus with KBr pellet preparation.
Software for docking analysis: the computing study in this research was performed under a Dell WorkStation Personal Computer.
Linux Ubuntu 20.
3 LTS OS,
IntelA Xeon(R) W-2223 CPU @3.
60 GHz octa-core.
RAM 16 GB and GPU NVIDIA Quadro P2200.
Meanwhile, molecular docking was conducted with Maestro Schrydinger 2022-1 software (Schrydinger.
New York.
NY.
USA).
Procedure Synthesis of NBB derivatives NBB synthesis was conducted by using the reflux process and several modified procedures from Jubie et .
The ligands were prepared by adding 6 mmol .
of benzohydrazide in 30 mL of ethanol.
Then, 6 mmol of the o-benzaldehyde derivative and 30 mL of ethanol were added to the flask with a small amount of acetic acid.
The mixture was refluxed for 2 h at 70 AC After that, the product was cooled overnight at 4 AC and separated by using a funnel.
As the last step, an aluminium sheet with TLC gel 60 F254 was used for product tracing.
This procedure was repeated for the synthesis of the other four derivates, such as N-2(-Cl, -OH, and -OM.
NAo-.
-chlorobenzyliden.
-benzohydrazide 1.
Yield:
FTIR (KBr, cmOe.
: 3181 (-Csp2H aromati.
(-C=O).
1555 (-N=N-).
1H-NMR (DMSO-d.
: 12.
, 1H, -NH).
, 1H, -CH=N).
, 1H).
, 2H).
, 1H).
d, 3H).
, 2H).
13C-NMR
(DMSO-d.
: 163.
8 (-C=O).
2 (-CH=N).
ESIMS: 259.
%) [L H] .
%) [L N.
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NAo-.
-hydroxybenzyliden.
-benzohydrazide 2.
Yield:
FTIR (KBr, cmOe.
: 3268 (-NH).
3268 (-OH).
(-Csp2H aromati.
1672 (-C=O).
1538 (-N=N).
1271 (-CO).
1H-NMR (DMSO-d.
: 12.
, 1H, -NH).
, 1H, -CH=N).
, 1H).
, 2H).
, 1H).
, 3H).
d, 1H).
, 2H).
13C-NMR (DMSOd.
: 163.
4 (-C=O).
0 (-C-OH).
9 (-CH=N)-.
ESIMS: 241.
%) [L H] .
NAo-.
-methoxybenzyliden.
-benzohydrazide Yield: 57.
FTIR (KBr, cmOe.
3184 (-Csp2H aromati.
2988 (-Csp3H of methy.
1640 (C=O).
1556 (-N=N).
(-C-O).
1H-NMR (DMSO-d.
: 11.
, 1H, -NH).
, 1H, -CH=N).
, 2H).
d, 1H).
d, 1H).
, 2H), 7.
, 1H).
, 1H).
, 1H).
, 3H, -OCH.
13C-NMR (DMSO-d.
: 163.
5 (-C=O).
3 (-C=OCH.
8 (-CH=N).
2 (-OCH.
ESIMS: 255.
%) [L H] .
Ligands and receptor preparation ChemDraw was used to generate the ligand structures, which were then converted into a 3D model using LigPrep .
module in Schrodinger 2022-1 as well as protonated at pH 7.
4 with Epik .
and OPLS4 forcefield .
These processes aim to restore improper or missing bonds, assign protonation, possible ionization, and tautomeric states .
Moreover, -glucosidase (PDB ID: 5NN.
protein was prepared by removing the residual solvent, optimizing the hydrogen bond, and protonating using ProtAssign .
and PROPKA .
the same time, in the Auprotein preparation wizardAy that is incorporated into Maestro Schrodinger 2022-1 .
, the partial charge was also added using OPLS4 forcefield.
Molecular docking The docking study was performed using Glide .
Maestro Schrodinger's 2022-1 to predict the binding affinities and molecular interactions of synthesized compounds against two receptor targets: -glucosidase (PDB ID: 5NN.
and -amylase (PDB ID: 6GXV).
Acarbose, an inhibitor that acts as the native ligand, was used for comparing to compounds.
A grid box was placed at the center of acarbose position with similar dimensions Yusuf Syaril Alam et al.
for both receptors .
y 20 y 20 yI) for setting the docking region.
The docking grid box for -glucosidase and -amylase were assigned based on the coordinates of the native ligand at .
= Oe13.
92, y = Oe38.
29, z = 95.
= 44.
03, y = 22.
72, and z = Oe11.
, respectively.
Redocking the native ligand was conducted to validate docking protocols and calculated their root mean square deviation (RMSD) with 100 conformations limit The docking protocol was deemed valid if the RMSD value was < 2 yI .
The docking process was carried out with Glide in extra precision (XP) mode under rigid receptors and flexible ligand conditions.
The molecular mechanics-generalized Born surface area (MM-GBSA) was calculated to assess the docking pose of the ligands and determine the potency of each compound .
The Auligand interactions panelAy on Maestro Schrodinger was used to visualize the molecular The results provide valuable insights into the binding modes and mechanisms of these receptors.
It could help to guide the design of new compounds with improved efficacy and specificity.
n RESULTS AND DISCUSSION Computer-Aided Drug Discovery Computer-aided drug discovery is the most important tool to predict drug activity through computational structure-based drug discovery.
The relationship between sites of protein action and compounds acting as ligands can be explained using a variety of software.
In point of fact, a physics-based equation is used to determine the binding free energy .
The -glucosidase is an essential enzyme that is found on the luminal surface of enterocytes that functions to regulate blood glucose by converting complex carbohydrates into absorbable glucose, which is required for energy metabolism .
The removal of the anomeric carbon from the glucosyl group and the glycosidic oxygen (C1AeO) is the first step in the hydrolysis reactions carried out by -glucosidases.
Then, the glucosyl group is replaced by a proton from water resulting in the process of hydrolysis and transglycosylation exchange process between glucosyl residues and the protons .
Acarbose (Fig.
inhibits Indones.
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Fig 2.
Acarbose intestinal -glucosidases, the enzymes responsible for the metabolism of complex carbohydrates into absorbable monosaccharide units in a reversible manner.
This mechanism might be used to identify and develop new diabetic medications that are useful for showing diabetes progression .
Molecular Docking Molecular docking is an important and helpful tool to predict the binding affinity of molecules to proteins .
In this study, the docking protocol was validated by internal validation after redocking the native ligand in its original positions resulting in RMSD values of 1.
812 and 165 yI for -glucosidase and -amylase receptors.
Based on these findings, it is possible to determine the test compound's activity against -glucosidase as well as amylase receptors using either docking protocol .
Prior to docking the molecule.
RMSD was used to determine the native acarbose ligandAos docking position, as depicted in Fig.
Compounds NBB 1, 2, and 3 with aromatic parts in their structures were evaluated for the protein-ligand complexes in the structure-activity relationship.
It was discovered that a more negative score of binding affinity indicated a stronger binding.
This ligand-protein binding process is correlated with this score which is also known as the change of the free energy.
The measurement of how strong the interaction between the ligand and the protein is often directly related to the potential for ligand activity .
NBB without substituents resulted in an MMGBSA score of Oe0.
45 on the binding affinity of complexes ligand-receptors.
The ortho positions of the NBB derivatives with EDG (-Cl, -OH and -OM.
were Oe18.
Oe6.
71, and Oe21.
94 kcal/mol.
As can be seen in Table 1 and Fig.
4, the results of the ligand-receptor binding indicated that these compounds were bound to the same residues (Ala284.
Asp282 and Asp.
in the entry area of the glucosidase active site.
The native acarbose inhibitor was validated by a redocking process on -amylase in its original positions, which resulted in an RMSD value of 1.
165 yI.
In Table 2, the docking result between NBB derivatives and the amylase receptors showed that the value of MMGBSA as a binding affinity score for the ligand-receptors complex had a nearly equal range between Oe43.
09 to Oe39.
42, with native ligand acarbose Oe46.
51 kcal/mol.
Similar to glucosidase, the NBB grid score.
Oe20.
78 kcal/mol, was found to be greater than the NBB derivatives.
Through the use of a hydrogen bond.
NBB derivatives were able to interact with the same amino acids in the cavity of amylase Gly110 in a strong A-A interaction .
It should Fig 3.
The acarbose native ligand on .
-glucosidase and .
-amylase receptors (Blue = original position and green = native ligand after redockin.
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Fig 4.
The binding of NBB derivatives and -glucosidase Yusuf Syaril Alam et al.
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Table 1.
Molecular docking result against -glucosidase receptor No.
Compound N'-benzylidenebenzohydrazide N'-.
-chlorobenzyliden.
benzohydrazide, 1 N'-.
-hydroxybenzyliden.
benzohydrazide, 2 N'-.
-methoxybenzyliden.
benzohydrazide, 3 Acarbose (Native ligan.
MMGBSA .
cal/mo.
Oe0.
Oe17.
Oe6.
Oe21.
Oe32.
Amino acid interaction Asp282.
Arg600 Ala284.
Trp481.
Asp616 Asp518.
Asp616.
Phe649 Asp282.
Trp481 Asp 282.
Ala284.
Asp404.
Asp616.
His674 Table 2.
Molecular docking results against -amylase receptor No.
Compound Acarbose (Native ligan.
N'-benzylidenebenzohydrazide N'-.
-chlorobenzyliden.
benzohydrazide, 1 N'-.
-hydroxybenzyliden.
benzohydrazide, 2 N'-.
-methoxybenzyliden.
benzohydrazide, 3 be noted that NBB was not involved in binding interactions with Gly110 and other amino acid residues that include the native ligand (Table .
Through a variety of hydrophobic and hydrogen bonds, the active site of residue -amylase receptor with NBB and its derivates can be followed in Fig.
Yusuf Syaril Alam et al.
MMGBSA
cal/mo.
Oe46.
Oe20.
Oe42.
Oe43.
Oe39.
Amino acids interaction Gly110.
Asp166.
Asp234.
Arg232.
Asp332 Tyr58.
Tyr196 Gly110.
Ala111 Gly110.
Gln51 Gly110.
Ala111 The compounds synthesized exhibit affinity energy values that are suboptimal compared to native ligands.
However, some of the synthesized compounds show interactions with the catalytic site of the -glucosidase receptor, specifically N'-.
-chlorobenzyliden.
benzo hydrazide.
N'-.
-hydroxybenzyliden.
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Fig 5.
The binding of NBB and derivatives toward -amylase receptor and N'-.
-nitrobenzyliden.
benzohydrazide on amino acids Asp518 and Asp616.
Although these interactions can inhibit the activity of the -glucosidase enzyme, they do not perform as well as the native ligand due to the lack of other molecular interactions that could enhance the affinity of the interaction.
Despite this limitation, these three compounds hold the potential for further development as -glucosidase inhibitor agents .
n CONCLUSION In drug design, the molecular docking technique has been used extensively to predict the ligands-receptor Substituents on the benzene ring play a role in antidiabetic activity.
NBB derivatives ligand with Yusuf Syaril Alam et al.
electron-donating groups at the ortho position has the potential to increase the activity of antidiabetic target receptors for both -glucosidase and -amylase.
Numerous functional groups and para positions will be performed in the subsequent experiment.
n ACKNOWLEDGMENTS The authors would like to thank Research.
Innovation and Community Development and LPPM.
Airlangga University for funding this research number 1076/UN3.
15/PT/2022 through the "Riset Kolaborasi Indonesia" Research Grant organized by Ministry of Education.
Culture.
Research and Technology.
Republic Indonesia in FY 2022.
n Indones.
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AUTHOR CONTRIBUTIONS
Yusuf Syarif Alam.
Nur Rahmayanti Afifah.
Arif Fadlan conducted the synthesis experiment.
Tutik Sri Wahyuni and Saipul Maulana performed docking Pratiwi Pudjiastuti.
Fahimah Martak and Arif Syukri wrote and revised the manuscript.
All authors agreed to the final version of this manuscript.
n REFERENCES