Antidiabetic, antioxidant and cytotoxicity activities of ortho - and para -substituted Schi ﬀ bases derived from metformin hydrochloride: Validation by molecular docking and in silico ADME studies

: This work evaluates the in vitro antioxidant and antidiabetic activities of two metformin hydrochloride-based Schi ﬀ bases. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to examine the in vitro cytotoxic e ﬀ ects of HL1 and HL2 on the A549 lung cancer cell line. The two Schi ﬀ bases that have been previously synthesized by using two e ﬀ ective, green techniques, namely stirring and microwave-assisted, are N , N -dimethyl- N ′ -[( Z )-(2-nitrophenyl) methylidene] imi-dodicarbonimidic diamide and N , N -dimethyl- N ′ -[( Z )-(4-nitro-phenyl) methylidene] imidodicarbonimidic diamide, indicated by HL1 and HL2, respectively. Studies of antidiabetic e ﬃ - cacy using alpha-amylase revealed that HL2 has a higher inhibition than HL1, but the results on sucrase enzyme showed that HL1 had the highest inhibitory action, whereas the outcome of the antioxidant test with the 2,2-diphenyl-1-picrylhydrazyl assay demonstrated that HL2 was the most e ﬀ ective antioxidant, followed by ascorbic acid and HL1. In the MTT assay, HL1 had the best result, with an IC 50 value of 57.13 µg/mL compared to HL2 with an IC 50 value of 76.83 µg/mL. It was observed that HL1 was the most e ﬀ ective against the human lung cancer cell line A459. The ﬁ ndings were supported by computational and pharmacokinetic studies (SwissADME). Based on empirical and computational studies, we suggest that HL1 and HL2 are promising candidates as antioxidants and antidiabetics after being examined in vivo .


Introduction
Important organic compounds known as "Schiff bases" have a carbon-nitrogen double bond, with the nitrogen atom attached to either an aryl or an alkyl group.They form when an amine condenses with a carbonyl substance, generally an aldehyde or a ketone.
They have a variety of biological effects, such as antidiabetic, antioxidant, and antitumor abilities, and they are desirable candidates for drug development and additional pharmacological and medicinal chemistry research due to their adaptability and prospective therapeutic applications [1][2][3][4][5].
Hyperglycemia is caused by abnormalities in insulin secretion, action, or both that characterize diabetes mellitus (DM).According to the World Health Organization, there are two types of DM: type (I) insulin-dependent diabetes mellitus and type (II) noninsulin-dependent diabetes mellitus (NIDDM).A complete lack of insulin secretion is the root cause of type (I), while a combination of insulin resistance and an insufficient compensatory insulin-secretory response causes type (II) diabetes [6][7][8].Diabetes affects 371 million people worldwide, with 90% of people developing type (II) diabetes as a result of their lifestyle, genetic tampering in (agriculture and dietary products) and a lack of physical exercise.The most prominent symptoms are increased thirst, hunger, polyuria, blurred vision, and weight loss.Over time, DM can lead to cardiovascular disease (five million people died in 2011), sexual dysfunction, neuropathy, nephropathy (which can lead to renal failure), cancer, and stroke [9][10][11][12][13][14].
Even though metformin is the most extensively used oral antidiabetic medication and has significant advantages over other treatments, 20-30% of the patients experience gastrointestinal side effects, and 5% cannot tolerate it.Metformin, which is derived from the medicinally named plant Galega officinalis and was historically used to treat diabetes in medieval Europe, is still the only ethical medicine licensed for the treatment of NIDDM patients [22,25,26].
Furthermore, the emergence of diabetes problems has been linked to oxidative stress and free radical overproduction.Atom or molecule fragments with one or more unpaired electrons in atomic or molecular orbitals are called free radicals, which are often unstable, so they assault the nearest stable molecule as soon as possible to collect the electron that it requires to gain stability [27].The two types of free radicals that are most frequently produced in the human body are reactive oxygen species (ROS) and reactive nitrogen species.Both endogenous and exogenous sources produce free radicals, exogenous sources such as pollution, alcohol, tobacco smoke, heavy metals, transition metals, industrial solvents, pesticides, certain drugs like halothane, paracetamol, and radiation, as well as endogenous sources such as mitochondria, peroxisomes, endoplasmic reticulum, phagocytic cells, and so on [28][29][30][31][32].
Oxidative stress is caused by high concentrations of reactive free radical species, which can damage nucleic acids, lipids, proteins, and cell structure.It can also lead to many diseases, such as lung disease, liver disease, cirrhosis, heart disease, atherosclerosis, diabetes, rheumatoid arthritis, and Parkinson's disease.So, free radical production and antioxidant defenses need to be in balance for an organism to function properly [29][30][31][32][33][34][35][36][37].
Antioxidants are substances found in small amounts in the body compared to free radicals.Enzymatic and nonenzymatic antioxidants are two types of antioxidants that scavenge unpaired electrons, prevent the generation of free radicals, stop chain reactions, and dampen the energy of executed molecules [27,31].
Natural and synthetic antioxidants are two types of antioxidants that stabilize free radicals and suppress them.Natural antioxidants are composed of minerals, vitamins, and phytochemicals.Minerals are essential for enzymatic processes, and vitamins are essential for redox reaction regulation.Phytochemicals suppress free radical reactivity by stabilizing free radicals.Synthetic antioxidants are phenolic compounds that interact with free radicals to suppress free radical chain reactions.Plant and fungi extracts contain antioxidant compounds such as flavones, isoflavones, flavonoids, anthocyanins, coumarins, lignans, catechins, and isocatechins [27,31,38].
It is also fascinating to note that metformin has been linked to a variety of biological processes, including weight loss, anti-aging, and anticancer action [39,40].
Since 10 years ago, increasing evidence has emerged suggesting that biguanide medications like metformin may be helpful in the prevention and treatment of a variety of cancers, such as prostate cancer [41].Individuals with DM in particular have a higher chance of developing cancer, and metformin has been discovered to significantly inhibit a number of malignancies [42,43].
Previously, two novel Schiff bases HL1 and HL2, were successfully synthesized via an eco-friendly methodology and characterized using elemental analysis, differential scanning calorimetry, fourier-transform infrared spectroscopy, UV-Vis spectroscopy, 1 H NMR spectroscopy, 13 C NMR spectroscopy, and mass spectroscopy.The antibacterial activity of both metformin derivatives was evaluated using the agar well diffusion method against a range of grampositive and gram-negative pathogens [6].
Our current work focuses on evaluating the in vitro cytotoxic, antidiabetic, and antioxidant tests of metformin derivatives (HL1 and HL2).The inhibition of α-amylase and sucrase enzymes by Schiff bases HL1 and HL2 was used to measure the level of antidiabetic activity.In contrast, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was used to measure the antioxidant activity of metformin derivatives while assessing their cytotoxic effects on the A549 lung cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test.Molecular docking studies were carried out to investigate the possible binding mechanisms of HL1 and HL2 with their target proteins in order to support their antioxidant and antidiabetic effects.Additionally, absorption, distribution, metabolism, and excretion (ADME) is used for predicting the medicinal chemistry and drug-like characteristics of metformin derivatives.

Synthesis of metformin Schiff bases HL1 and HL2
Earlier, our study was done on the synthesis of the metformin-based Schiff bases HL1 and HL2, utilizing both conventional and green techniques.In the traditional procedure, equimolar quantities of metformin-HCl and (ortho) para-substituted benzaldehyde were refluxed in methanolic basic medium.In the environmentally friendly procedures, equimolar solutions of metformin and nitro-substituted benzaldehydes were dissolved in a basic aqueous medium and either refluxed under microwave irradiation (Method I) or stirred at room temperature using a magnetic stirrer (Method II) [6].

Biological tests 2.2.1 Antioxidant
The antioxidant efficacy of HL1 and HL2 was measured using the DPPH test.

Procedure
In vitro, antioxidant efficiency was assessed by the DPPH assay with few adjustments [32].About 0.05 mL of HL1 and HL2 dissolved in methanol were diluted to 1.0 mL using ethanol to attain concentrations of 1-200 μg/mL and were added with DPPH (final concentration: 200 μM, in 95% ethanol).The control contains dimethyl sulfoxide (DMSO) and ethanol in similar amounts.Triplicate aliquots were used for each test.The reference standard (positive control) applied was ascorbic acid.A decrease in the absorbance of the test compounds was detected at 515 nm after 20 min using UV-vis spectroscopy (Shimadzu UV-1800, Tokyo, Japan), and the following formula was used for calculating the % inhibition: The sample absorbance is the measurement of the DPPH solution with compounds, whereas the control absorbance is the measurement of the DPPH solution without compounds.

Antidiabetic
In vitro, the antidiabetic activity of HL1 and HL2 was estimated by how well they blocked the actions of the enzymes alpha-amylase and sucrase.

Procedure for the alpha-amylase inhibition assay
The alpha-amylase inhibition assay was carried out according to the protocol outlined by Puneeth and Sharada [44].The alpha-amylase enzyme (1 unit/mL) from Bacillus species was dissolved using 0.1 M phosphate-buffered saline (pH 6.9).The enzyme solution was pre-incubated with the various concentrations of HL1 and HL2 (50, 100, and 200 μg) for 10 min at 37°C.The enzymatic reaction was allowed to occur for 30 min at 37°C after the starch solution (0.1%) was added to the incubation medium to start the reaction.By adding 3,5-dinitrosalicylic acid (DNS reagent) to the reaction mixture, the reaction was stopped.The tubes were then placed in a boiling water bath for 10 min.By adding a 40% sodium potassium tartrate solution and allowing it to cool to room temperature, the color was stabilized.At 540 nm, the optical density was determined.A positive control was used, which was metformin.The following formula was used to determine the compounds' % activity: Sample absorption Control absorption 100.

Procedure for the sucrase inhibition assay
The inhibitory studies of HL1 and HL2 on sucrase enzyme were performed based on the procedure given by Sharath Chandra et al. [45] with a few changes.In brief, HL1 and HL2 were pre-incubated with enzyme solution at various concentrations (50, 100, and 200 μM) in maleate buffer (0.1 M, pH 6) for 10 min at 37°C.Sucrose solution (60 mM) was added to start the reaction and incubated for 30 min at 37°C.After incubation, the reaction was stopped by placing the reaction mixture in a water bath for 10 min.The amount of glucose liberated in the reaction mixture was estimated using the glucose oxidase-peroxidase method (GOD-POD strategy).

Estimation of glucose by the GOD-POD method
The glucose generated in the mixture of reaction was calculated by the GOD-POD test kit instructions [45].In short, 50 μL of the incubated medium was added to a 96-well ELISA plate.Each well received 200 μL of the GOD-POD color reagent and then will be incubated for 30 min at 37°C in the dark for the development of color.At 505 nm the optical density was determined.Metformin was used as a positive control.The following formula was used to calculate each compound's % activity: Sample absorption Control absorption 100.

Cytotoxicity assay
Ligands HL1 and HL2 were examined for in vitro cytotoxicity against the A549 lung cancer cell line obtained from the DSMZ Leibniz Institute (German Collection of Microorganisms and Cell Cultures Braunschweig, Germany).The cell viability assay was performed by the MTT method, which was provided by Invitrogen, Waltham, MA, USA.The solvent used for preparation (DMSO, acidified isopropanol) obtained from Sigma, St. Louis, MO, USA, UV-vis analysis Nuaire, Plymouth, MN, USA.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was used to measure cell viability as already mentioned [46][47][48].
The cytotoxic activity of the synthesized compounds (HL1 and HL2) was evaluated in vitro using the MTT assay.In brief, the A549 lung cancer cell line was grown for 24 h at 37°C after being plated at a density of 5 × 10 4 cells per well in a 24-well plate.Following that, the cells were subjected to various concentrations (100, 50, 25, and 12.5 μg/mL) of each substance.About 100 µL of MTT (5 mg/mL in phosphate-buffered saline) was added to each well after 48 h of incubation.After a further 4 h at 37°C incubation of the plates, the formazan crystals were dissolved in 1 mL of acidified isopropanol.The cell viability was calculated at 570 nm using the following formula:

= × % Cell viability Mean absorbance of treated cells Mean absorbance control 100
The dose-response curve was used to get the inhibitory concentration (IC 50 ) value.

Statistical analysis
Mean ± standard error is used to display experimental data.Statistical analyses were carried out by one-way ANOVA using SPSS ver.20.0 software.

Software program 2.3.1 Molecular docking using molecular operating environment (MOE)
By using a protein data bank (http://www.rcsb.org/pdb,accessed on 30 August 2022), the X-ray crystal structures of water-forming NAD (P) H oxidase (PDB ID: 2CDU, 1.80 Å resolution) and human pancreatic α-amylase (PDB ID: 1B2Y with a resolution of 3.20 Å) were retrieved [49,50].For docking, the MOE was utilized for preparing the protein structures.First, the Sequence Editor window of MOE was used to remove undesirable atoms, molecules, chains, and ligands (2CDU: H 2 O molecules, co-crystallized ligands ADP and FAD were removed [51]; 1B2Y: H 2 O molecules, ion metals, and the PCA ligand were removed).Second, polar hydrogen atoms were added.Third, energy was minimized using Amber12.EHT with an RMS of 0.1 kcal/mol/A 2 .Fourth, missing atoms were corrected and typed (2CDU: 6,943 atoms; 1B2Y: 7,763 atoms).Fifth, the site finder module of MOE was utilized to identify protein's active site (2CDU: the largest pocket contains 229 amino acids, IB2Y: the ligand pocket contains 129 amino acids).ChemDraw was used to draw the ligands' 2D structures, while MOE software (protonation, partial charges, and energy minimization) was utilized to prepare 3D structures.At last, the database file of ligands was created and saved for further docking [52,53].
In order to locate and analyze the interaction between ligand and protein, docking was conducted with certain settings (Rescoring function1 and rescoring function 2: London dG, placement: Triangle matcher, retain:2 and Refinement: force field).After that, the compound that had the best overall characteristics in terms of energy, Hbond count, and location was selected as the one to be visualized [54,55].

SwissADME
SwissADME is a free web application on a website (http:// www.swissadme.ch/,accessed on 20 July 2022).ADME supports the development of new drugs by precisely predicting the medicinal chemistry, drug-like properties, solubility, lipophilicity (LIPO), physicochemical properties, and pharmacokinetic parameters of small molecules [56].
SwissADME also exhibited the structure and bioavailability radar using canonical SMILES.The first part showed the chemical structure in two dimensions.The second part is the bioavailability radar, which allows for a rapid peek at how much the target compounds resemble medicines.For each feature anticipated to be orally accessible, the optimum physicochemical environment is illustrated by the pink area as LIPO: −0.7 < XLOGP3 < +5.0, SIZE (Molecular weight (MW)) 150 g/mol < MW < 500 g/mol, POLAR (Polarity) 20Å 2 < Molecular polar surface area (TPSA) < 130 Å 2 , INSOLU (Insolubility) −6 < Log S(ESOL) < 0, INSATU (Insaturation) 0.25 < FractionCsp3 < 1 and FLEX (Flexibility) 0 < RP (Number of rotatable bonds) < 9 are the six physicochemical qualities that are taken into consideration [57].

Results and discussion
HL1 and HL2 in Scheme 1 represent the Schiff bases derived from metformin hydrochloride and substituted (ortho)para-nitrobenzaldehyde, and they have been effectively synthesized utilizing conventional and microwaveassisted techniques earlier published [6].
The DPPH test was used to examine the antioxidant activity of HL1 and HL2.Besides that, the alpha-amylase and sucrase enzymes were inhibited to assess the antidiabetic activity of metformin derivatives, and the result is supported by a docking study and SwissADME prediction.

In vitro study 3.1.1 Antioxidant
The scavenging activity of the free radical DPPH was investigated with minor modifications [58] by the spectrophotometric method.
The scavenging mechanism of free radical DPPH is the reduction process, as depicted in Figure 1.The stable free radical DPPH has the color purple, but when either HL1 or HL2 is added, DPPH gets a proton and turns yellow [59].
Table 1 and Figure 2 include the results of HL1 and HL2's antioxidant activity.Both exhibited significant antioxidant activity.HL2 had the strongest antioxidant action and then ascorbic acid and HL1, with IC 50 values of 4.89, 5.24, and 5.35 µg/mL, respectively.

Alpha-amylase inhibition activity
The alpha-amylase inhibitory activity results and IC 50 are presented in Figure 3 and Table 2 for HL1, HL2, and standard metformin.HL1 and HL2 inhibited α-amylase most effectively when compared to regular metformin.HL2 has the greatest level of inhibitory activity with an IC 50 of 76.1 µg/mL, followed by HL1 with an IC 50 of 99.3 µg/mL.Metformin had very little potency against α-amylase with an IC 50 of 272.6 µg/mL.

Sucrase inhibition activity
Table 3 and Figure 4 show that both HL1 and HL2 and standard metformin can stop sucrase enzymes from working.According to the observations, HL1 showed the maximum inhibition activity with an IC 50 of 89.3 µg/mL, followed by HL2 and metformin with an IC 50 of 93.3 and 492.4 µg/mL, respectively.Standard metformin displayed a relatively low level of sucrase enzyme inhibitory action.

MTT assay
Through the use of the MTT assay, cytotoxicity was identified.This assay based on the reduction of the calorimetrically measurable yellow tetrazolium salt 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) into the purple formazan.This mechanism occurs in the mitochondria of metabolically active cells and indirectly measures cellular metabolic activity.
The cytotoxicity activity of HL1 and HL2 against the A549 lung cancer cell line is represented in Table 4 and Figure 5.To assess the cytotoxicity of Schiff bases, the lung cancer cell line A549 has been used.The MTT assay was performed to check each compound's cytotoxicity after 48 h of treatment with various concentrations.Results obtained indicated that at concentrations of 12.5, 25, 50, and 100 μg/mL, HL1 showed inhibition activity against the A549 lung cancer cell line at 93.42, 68.24, 56.23, and 13.49%, respectively.IC 50 of HL1 was 57.13 μg/mL, but HL2 has inhibition activities of 87.59, 76.19, 65.47, and 37.38%,     respectively.Actually, compound HL2 demonstrated a less potent cytotoxic effect on cell line A549 lung cancer (IC 50 = 76.83)[60,61].

Theoretical study 3.2.1 Docking study
Molecular docking studies are routinely utilized in the pharmaceutical industry to assess the efficacy of new drug candidates.The binding mode in which bioactive compounds (also known as ligands) interact with their target proteins provides a useful way to categorize these compounds.

Antioxidant
The findings of our experiments indicate that our compounds have a significant potential for exhibiting antioxidant activity.To corroborate these findings, we carried out a molecular docking analysis to gain a deeper comprehension of one of the potential mechanisms underlying these findings.Then, due to its essential function in the production of ROS, we chose the NADPH enzyme (PDB code: 2CDU).According to the research that has been done, this enzyme is a significant contributor to the production of superoxide anion (O 2 -), which is the precursor to the vast majority of other ROS.Because of this, chemicals that are capable of inhibiting the NADPH oxidase (NOX) contribute significantly to the overall equilibrium of oxidative stress.The result indicates that HL2 was the highest effective antioxidant.With a docking score of −6.49 kcal/mol, it outperformed both ascorbic acid (−5.04 kcal/mol) and HL1 (−4.9 kcal/mol), as shown in Table 5 and Figures 6-8.At the protein's active site, it made two hydrogen bonds with amino acid residues ASP282 and CSX42 at distances of 2.94 and 4.23 Å, respectively (Figure 7).Furthermore, it formed two ionic interactions with ASP282 at 3.33 and 2.94 Å, respectively.HL1 formed one hydrogen bond with the amino acid residue ASP282 at a distance of 3.30 and two ionic bond contacts with ASP282 at distances of 3.46 and     3.30 Å.However, it does not interact with the important amino acid CSX42, as shown in Figure 6.

Antidiabetic activity
The examined compounds demonstrated a more significant association between in vitro activity and in silico study results, according to the docking results of these compounds against human pancreatic alpha-amylase (PDB ID: 1B2Y).The findings of the docking analysis showed that the metformin derivatives (Hl1 and HL2) have higher negative docking scores (−5.41 and −5.47 kcal/mol) than metformin (−4.95 kcal/mol), which suggests that these two derivatives have strong affinities and excellent interactions (hydrogen bonds) within the active binding site of 1B2Y.
Three hydrogen bond interactions (red dashes) were formed between HL1 and amino acid residues SER145, ASN105, and ASP147 at distances of 3.17, 3.36, and 2.85 Å respectively, as shown in the 2D and 3D representations of HL1 in Figure 9.It also interacted with the amino acid residue ASP147 via an ionic interaction at a distance of 3.34 Å. HL2, on the other hand, formed two hydrogen bonds and two ionic interactions (Figure 10, Table 6).It formed two hydrogen bond interactions with amino acid residues SER145 and ASN105 at distances of 3.33 and 3.05 Å, respectively, and two ionic bond interactions with amino acid residue ASP147 at distances of 3.33 and 3.90 Å.The standard drug (metformin) formed only one hydrogen bond with amino acid LEU162 at a distance of 3.28 Å as shown in Figure 11.Ortho and para-substituted Schiff bases derived from metformin hydrochloride  9

SwissADME
According to the SwissADME program's predictions, the novel metformin derivatives can be taken orally after being tested in clinical settings for their antidiabetic and antioxidant abilities, where a high level of antidiabetic and antioxidant activity is shown by the in vitro findings.
The five criteria laid out by Lipinski, upon which the SwissADME program is based are MWs in the range (150-500) g/mol, hydrogen bond acceptors (HBA) must be less than 10, hydrogen bond donors (HBD) should be less than 5, TPSA which is between 20 and 130 Å 2 , and lastly, high LIPO needed of XLOGP3 g(LogP < 5).In contrast, the proposed compounds are considered unsuitable for druglikeness if they violate more than two of Lipinski's five rules.Table 7 displays ADME predictions for HL1 and HL2, as well as metformin and ascorbic acid, and demonstrates that HL1 and HL2, which adhered to Lipinski's five criteria, also possess the chemical and physical qualities necessary for oral bioavailability [62].
HL1 and HL2 have molecular weights of 262.27 g/mol, which are within the permitted limit; the number of rotatable bonds of the derivatives of metformin is equal to 6, indicating good structural flexibility; there are 5 (HBA) and 3 (HBD), both acceptors and donors falling inside the required range, TPSA has gained increased importance in medicinal chemistry.When the TPSA value is greater than 60 Å 2 and less than 130 Å 2 , it can be used to accurately predict intestinal absorption; when it is less than 60 Å 2 , it shows excellent blood-brain barrier permeability.Metformin derivatives have TPSA values of 121.15 Å 2 , which means they will be more readily absorbed in the gastrointestinal tract, which has a 68.48% absorption rate, but the absorbance of the standard drugs is higher.In order for a new synthetic molecule to meet Lipinski's rules, it must have adequate oral and intestinal absorption, which is necessary for it to be drug-like, and the value of Log p must be less than 5.The values of XLOGP3 for the Schiff bases fall within the ideal range for LIPO and the examined HL1 and HL2 have drug-like properties that can be easily determined using bioavailability radars; these parameters are indicated by the pink region through the ideal range for each feature (LIPO, size, solubility, polarity, flexibility, and saturation) as well as standard drugs as displayed in Figures 12-15.Moreover, the range of synthetic accessibility was 2.80 to 3.04; consequently, there is a good chance of synthesizing all of them.
Oral medication must be able to penetrate either the intestinal or brain barrier; thus, both of them are the most crucial ADMET features, assigned by BBB and GI in Table 7, and illustrated by the BOILED-Egg.According to the ADME prediction, molecules in the white region have a higher chance of being absorbed by the GI tract, but those in the yellow zone are more likely to penetrate the brain.Metformin derivatives were not expected to permeate the brain and had a high intestinal absorption rate, as evidenced by their placement inside the white region in Figures 12 and 13, and the basic function of P-glycoprotein (P-gp) is to protect the body from toxic substances by maintaining the BBB and removing medications from the kidneys and liver into urine and bile, since metformin derivatives have no effect on the permeability of gp, in addition, the data in Table 7 reveal that the metformin derivative has no effect on the excretion or functionality of the five CYP enzymes confirming that the new drug will be properly metabolized and will not accumulate.Also, derivatives of metformin showed skin permeability values of −7.14 cm/s, which are within the acceptable range of Log Kp for drug candidates [56,57,63].

Conclusions
Experiments done in vitro on the alpha-amylase enzyme, the sucrase enzyme, and the DPPH scavenging radical for antidiabetic and antioxidant purposes showed excellent   results that were in line with theoretical studies.HL2 displayed superior inhibitory activity on the alpha-amylase enzyme, while HL1 showed a more powerful effect on the sucrase enzyme.By contrast, the DPPH assay revealed that maximum antioxidant activity was shown by HL2, followed by ascorbic acid and HL1, using the DPPH assay.The A549 lung cancer cell line was the subject of our investigation, which demonstrated that both HL1 and HL2 have anticancer potential.However, HL1 had superior activity.The anticancer effect against other cancer cell lines will need to be investigated further, though.Furthermore, the molecular docking study demonstrated that HL1 and HL2 interacted strongly at the active sites of the targeted proteins 2CDU and 1B2Y, with HL2 being the most potent α-amylase enzyme inhibitor and the best antioxidant.In contrast, the ADME predictions of metformin derivatives suggest that these compounds have good bioavailability and adhere to Lipinski's five principles.As was previously stated and is evident from all outcomes, metformin derivatives are a strong option for therapeutic usage, but additional studies are required to support these findings, first on rats and then subsequently on human patients.

Figure 5 :
Figure 5: Cytotoxic activity of HL1 and HL2 against A549 lung cancer cell line.

Figure 8 :
Figure 8: 2D and 3D of ascorbic acid at the active site of NAD (P) H oxidase (PDB ID: 2CDU).

Figure 15 :
Figure 15: Structure, bioavailability radar, and the Boiled-Egg graph of ascorbic acid based on SwissADME.

Figure 13 :
Figure 13: Structure, bioavailability radar, and the Boiled-Egg graph of HL2 based on SwissADME.

Figure 14 :
Figure 14: Structure, bioavailability radar, and the Boiled-Egg graph of metformin based on SwissADME.

Table 1 :
Antioxidant activity of HL1, HL2 and ascorbic acid