Exploring chalcone-sulfonyl piperazine hybrids as anti-diabetes candidates: Design, synthesis, biological evaluation, and molecular docking study

To combat the rising rates of diabetes mellitus over the world, novel compounds are required. The demand for more affordable and efficient methods of managing diabetes is increasing due to the unavoidable side effects associated with the existing antidiabetic medications. In order to develop inhibitors against alpha-glucosidase and alpha-amylase, various chalcone-sulfonyl piperazine hybrid compounds ( 5a – k ) were designed and synthesized in this present research. In addition, several spectroscopic methods, including FT-IR, 1 H-NMR, 13 C-NMR, and HRMS, were used to confirm the exact structures of the synthesized derivatives. All synthetic compounds were evaluated for their ability to inhibit alpha-glucosidase and alpha-amylase in vitro using acarbose as the reference standard and they showed excellent to good inhibitory potentials. Compound 5k exhibited excellent inhibitory activity against alpha-glucosidase (IC 50 = 0.31 ± 0.01 μM) and alpha-amylase (IC 50 = 4.51 ± 1.15 μM), which is 27-fold more active against alpha-glucosidase and 7-fold more active against alpha-amylase compared to acarbose, which had IC 50 values of 8.62 ± 1.66 μM for alpha-glucosidase and 30.9 7 ± 2.91 μM for alpha-amylase. It was discovered from the Lineweaver-Burk plot that 5k exhibited competitive inhibition against alpha-glucosidase. Furthermore, cytotoxicity screening assay results against human fibroblast HT1080 cells showed that all compounds had a good level of safety profile. To explore the binding interactions of the most active compound ( 5k ) with the active site of enzymes, molecular docking research was also conducted, and the results obtained supported the experimental data.


Introduction
Diabetes mellitus (DM) is a major global health issue characterized by abnormalities in insulin action (type 2 diabetes), insulin secretion (type 1 diabetes), or both, resulting in a range of metabolic disorders, such as hypertension and coronary heart disease, which pose a significant risk to individuals' overall health [1][2][3].
According to the World Health Organization (WHO) reports, the prevalence of diabetes has surpassed 420 million individuals, and it is projected to increase to over 642 million by the year 2040 [4].The prevailing type, which accounts for approximately 80-90% of all diabetic cases, is type-2 diabetes, leading to high blood glucose levels (hyperglycemia) [5], insulin resistance, and the inadequate production of insulin [6].Reducing blood glucose levels is a highly effective approach to manage diabetes and prevent its associated complications, such as eye, kidney, heart, and vascular diseases [7].
Disaccharides or polysaccharides of carbohydrates are broken down by various intestinal enzymes, such as alphaglucosidase and alpha-amylase [8].Alpha-amylase hydrolyzes dietary starch to maltose and dextrin, which are then transformed into glucose by alpha-glucosidase, leading to an increase in blood glucose levels [9].Therefore, inhibition of both enzymes is a useful strategy for lowering postprandial hyperglycemia [10].In fact, these two enzymes may be the primary targets for the development of lead medications for the treatment of diabetes.
Acarbose, voglibose, and miglitol are some examples of alpha-glucosidase inhibitors that effectively delay the absorption of sugars from the gut and are used therapeutically to treat diabetes.However, their less frequent utilization can be attributed to their relatively high cost and significant side effects, such as stomachache, diarrhea, and flatulence [11,12].Therefore, creating novel small hybrid molecules that act as inhibitors of both alphaamylase and alpha-glucosidase is a crucial emphasis in medicinal chemistry.
Chalcones are open chain flavonoids and predominantly biosynthesized by plants, but they can also be obtained through numerous synthetic methodologies.Chalcone consists of two aromatic rings that are connected by a threecarbon system, known as an alpha, beta unsaturated carbonyl system [13].Their simple chemistry, ease of synthesis, and diversity of substituents make them a subject of great fascination among researchers in the 21 st   Century.They also display a wide range of therapeutic activities, including anti-inflammatory [14], anticancer [15], antioxidants [16], antihypertensive [17], antiviral [18], and antimicrobial [19].Additionally, chalcones have been identified as potent antidiabetic agents [20,21].Fig. 1 displays some chalcone compounds (A-D) exhibiting alphaamylase and alpha-glucosidase inhibitory activity [22,23].
Heterocycles are an important class with biological and pharmacological significance that contain at least one ring and an element other than carbon [24].Heterocycles incorporating nitrogen are extremely important and are the focus of a lot of research [25].Among these, piperazine, which is a significant six-membered cyclic molecule containing two nitrogen atoms in positions 1 and 4, holds particular importance in the field of medicinal chemistry.This moiety is present in numerous well-known medications with a wide range of therapeutic applications, including antipsychotics [26,27], antihistamines [28,29], antianginals [30,31], antidepressants [32,33], cancer treatments [34,35], antivirals [36,37], and cardio protectors [38,39].It is also important to note that the oral DPP-4 inhibitor Teneligliptin, which contains piperazine, has been licensed for the treatment of type-II diabetes [40] (Fig. 1).To enhance the design and synthesis of hybrid analogs with improved biological potency, molecular hybridization is an invaluable and potent tool [41].The main goal of this method is to integrate two or more different pharmacophore moieties into a single molecule with a common scaffold, which may have more advantages over standard drugs.Our research team is persistently engaged in the design and synthesis of diverse heterocyclic scaffolds to explore potent therapeutic and inhibitors options [42][43][44][45][46]. Therefore, in our present research, we are combining the three biologically significant moieties of chalcone, sulfonyl, and piperazine utilizing molecular hybridization to create new hybrid compounds that are effective alpha-amylase and alpha-glucosidase inhibitors.
Moreover, we conducted kinetic mechanism studies and evaluated cytotoxicity activity, and the outcomes of all these investigations are correlated with the results obtained from molecular docking studies.

Alpha-glucosidase and alpha-amylase inhibitory activity and structure-activity relationship (SAR)
All the compounds (5a-k) were subjected to in vitro assessment to evaluate their inhibitory potential against alpha-glucosidase and alpha-amylase enzymes.The results demonstrated that these molecules displayed excellent to good inhibitory activity, as evidenced by their IC50 values (Table 1).Among these series, compound 5k exhibited a significant inhibitory potential, as indicated by its IC50 value of 0.31 ± 0.01 μM against alphaglucosidase and 4.51 ± 1.15 μM against alpha-amylase.Notably, these values were superior to those of the standard reference acarbose, which displayed IC50 values of 8.62 ± 1.66 μM and 30.97 ± 2.91 μM against alphaglucosidase and alpha-amylase, respectively.
Structure-activity relationship (SAR) studies were conducted to examine the impact of different moieties (R) on the activities of alpha-glucosidase and alpha-amylase.Compound 5a, which contains an unsubstituted phenyl ring, exhibited an IC50 value of 14.66 ± 0.81 µM for alpha-glucosidase and an IC50 value of 26.06 ± 2.81 µM for alpha-amylase.Comparatively, it demonstrated better activity than acarbose (IC50 = 30.97± 2.91 µM) in inhibiting alpha-amylase.However, it did not demonstrate superior activity compared to acarbose (IC50 = 8.62 ± 1.66 µM) in inhibiting alpha-glucosidase.This suggests that the phenyl group may have a stronger affinity for the active site of alpha-amylase when compared to acarbose.Compound 5a, was also explored for its anti-inflammatory properties (with swelling degree of 3.7 ± 0.9 mg) by Li et al [47].However, it did not exhibit significant activity when compared to celecoxib (swelling degree = 1.6 ± 0.7 mg).Compounds 5b-5d, which possess electron-withdrawing groups at the para position of the phenyl ring, exhibited better inhibition activity against both alpha-glucosidase and alpha-amylase compared to 5a (unsubstituted phenyl ring) and even outperform the standard acarbose.Among these compounds, 5d (IC50 = 0.63 ± 0.01 µM) with a fluoro group demonstrated better activity against alpha-glucosidase compared to 5b (IC50 = 2.61 ± 0.21 µM) with a chloro group and 5c (IC50 = 5.03 ± 0.79 µM) with a bromo group.This improved activity can be attributed to the specific electronic and steric properties of the fluorine substituent.The smaller size of the fluorine substituent enables a better fit within the active site of alpha-glucosidase, enhancing the interactions with the active site residues.Furthermore, fluorine, with its high electronegativity, affects the electron density distribution in the molecule.This electronic effect may further strengthen the interactions between the 4-fluoro compound and the active site residues of alpha-glucosidase, resulting in enhanced inhibitory activity.
This can be attributed to the larger size of the bromo group, which allowed for a more optimal fit within the active site of alpha-amylase.The increased size of the bromo substituent potentially facilitates stronger interactions with the active site residues, resulting in enhanced binding and inhibition of the enzyme's activity.
Compounds 5e-5g displayed enhanced inhibitory activity against both alpha-glucosidase and alpha-amylase compared to the standard acarbose, owing to the presence of electron-donating groups at the para position of the phenyl ring.Among these compounds, compound 5e (IC50 = 0.36 ± 0.01 µM), which contains a methyl group, exhibited superior activity against alpha-glucosidase compared to compound 5f (IC50 = 2.98 ± 0.19 µM) with a methoxy group and compound 5g (IC50 = 47.84 ± 1.03 µM) with an ethoxy group.It can be attributed to the relatively small size of methyl group, allowing for better fitting and accommodation within the active site of alphaglucosidase.The weak activity of the ethoxy group in compound 5g compared to the methyl and the methoxy groups against alpha-glucosidase can be attributed to two main factors.Firstly, the ethoxy group is larger in size, which may lead to steric hindrance and less favorable interactions with the active site residues of alphaglucosidase.Secondly, the ethoxy group is less electron-donating than the methyl and methoxy groups, leading to a lower distribution of electron density within the molecule.This reduced electron density distribution can weaken the interactions between compound 5g and the active site residues of alpha-glucosidase.Compound 5f (IC50 = 7.58 ± 0.83 µM), containing the methoxy group, exhibited better activity against alpha-amylase compared to compound 5e (IC50 = 9.66 ± 0.79 µM) with the methyl group and compound 5g (IC50 = 43.11± 1.56 µM) with the ethoxy group.This observation can be attributed to the electronic properties of the methoxy group, which is a strong electron-donating group.The electron-donating nature of the methoxy group likely contributes to its improved activity by facilitating stronger interactions with the active site of alpha-amylase.
The moderate activity observed for compound 5h (IC50 = 13.42 ± 1.66 µM for alpha-glucosidase and IC50 = 32.60 ± 4.84 µM for alpha-amylase), which combines benzyloxy and methoxy groups on the phenyl ring, against both alpha-glucosidase and alpha-amylase can be attributed to the presence of bulkier substituents.The larger size of the benzyloxy and methoxy groups introduces steric hindrance, which can influence the optimal fit of the compound within the active sites of both enzymes.
Compound 5i (IC50 = 7.32 ± 0.92 µM for alpha-glucosidase and IC50 = 11.13 ± 0.43 µM for alpha-amylase), which contains a thiophene moiety, demonstrated superior activity compared to compound 5j (IC50 = 30.79± 6.28 µM for alpha-glucosidase and IC50 = 31.75± 6.23 µM for alpha-amylase), which contains a furan moiety, against both alpha-glucosidase and alpha-amylase.This can be attributed to two main factors.Firstly, the sulfur atom possesses a higher electronegativity than the oxygen atom and this higher electronegativity of sulfur facilitates stronger interactions between thiophene and the active site residues of the enzymes.Secondly, the greater aromatic character of thiophene can enhance its stability and increase its affinity for the active sites of alpha-glucosidase and alpha-amylase.The antibacterial properties of compound 5i were evaluated by Li et al [48].against Bacillus subtilis, Escherichia coli, and Staphylococcus aureus Rosenbach.In comparison to reference standards including Ofloxacin, Levofloxacin, and Moxifloxacin, it displayed a moderate level of activity.
Compound 5k (IC50 = 0.31 ± 0.01 µM for alpha-glucosidase and IC50 = 4.51 ± 1.15 µM for alpha-amylase), which possesses a pyridine scaffold, exhibited the most significant activity against both alpha-glucosidase and alphaamylase compared to other compounds and even standard acarbose.The literature demonstrates that nitrogen atoms often participate in hydrogen bonding interactions at enzyme binding sites, which helps ligands fit properly within the cavity [5].There were two reasons for pyridine potent activity.First, the pyridine scaffold offered a unique chemical structure with specific functional groups that can effectively interact with the active sites of the enzymes.The nitrogen atom in the pyridine ring possesses a lone pair of electrons, which can participate in hydrogen bonding and other favorable interactions with the active site residues of alpha-glucosidase and alphaamylase.Secondly, the aromatic nature of the pyridine ring enhanced its stability and affinity for the active sites of alpha-glucosidase and alpha-amylase.The conjugated π-electron system in the pyridine scaffold promoted favorable π-π interactions with aromatic residues in the active sites, further strengthening the binding and inhibitory activity.Compound 5k exhibited remarkable potential, both in vitro and in silico analyses, suggesting that this compound could play a pivotal role in affecting the targets.In the context of alpha-glucosidase, it formed a hydrogen bond with LYS480 through its nitrogen unit in the pyridine ring, hydrophobic interactions with VAL451 and established a π-anion interaction with ASP452 via its pyridine ring.On the other hand, in the case of alpha-amylase, it established hydrophobic interaction with TRP59 via piperazine ring.Hydrogen bonds are formed with ARG195 and GLN63 through the sulfonyl and carbonyl units, respectively.Furthermore, a π-anion interaction occurred with ASP300 through the pyridine ring, and a π-π stacked interaction established with HIS299 and TRP59, involving the pyridine and bromophenyl rings.

Kinetic analysis
To gain insight into the inhibitory mechanism of the synthesized compounds on alpha-glucosidase, an inhibition kinetics study was conducted.This study aimed to determine the inhibition type and the inhibition constant of the most potent compound 5k, as identified through our IC50 results.During our analysis of the enzyme kinetics, a set of straight lines was observed in the Lineweaver-Burk plot of 1/V (inverse of reaction velocity) versus 1/[S] (inverse of substrate concentration) at various inhibitor concentrations.In the Lineweaver-Burk plot of compound 5k, it was observed that the slopes of the lines were not significantly affected, indicating that Vmax (maximum reaction velocity) remained constant.However, with increasing concentration, Km (Michaelis constant) showed an increase while Vmax remained relatively unchanged.The observed pattern suggests that compound 5k acts as a competitive inhibitor of alpha-glucosidase (Fig. 2A).Additionally,

Cell viability
The impact of the synthesized compounds (5a-k) on the viability of human fibroblast HT1080 cells was assessed using an MTT assay.The cells were incubated in 96-well plates until reaching 70% density for 24 hours.
Subsequently, the cells were treated with the compounds (5a-k) as well as acarbose, employed as a positive control for comparative purposes.Concentrations of 10, 20, 30, 40, 50, 60, and 100 μM were utilized for both the synthesized compounds and acarbose.The effects on cell viability were assessed, and the corresponding results are outlined in Table 2. Based on these findings, it can be inferred that all our synthesized compounds displayed a relatively safe profile.The control group, which was treated with acarbose, exhibited a cell viability of approximately 79% at the highest concentration of 100 μM.Some compounds demonstrated an improved safety profile in comparison to acarbose, while others (5a, 5b, 5e, 5f, and 5i) exhibited a similar safety profile to that of acarbose.Additionally, compounds 5c (89%), 5g (93%), 5h (85%), 5j (87%), and 5k (82%) were identified as the safest, even at the highest concentration (100 μM), when compared to acarbose (79%).Hence, these compounds show great potential as prospective drug candidates in future pharmaceutical development.* Indicates no toxicity on the cell viability.

Molecular docking
The refined models of receptors and ligands were used for molecular docking studies.The binding site specified for alpha-glucosidases was MIG, which is attached to these residues (ASP327, ASP443, MET444, ASP542, ASP452), and for alpha-amylase was 3L9 (TYR62, VAL98, HIS101, THR163, LEU168, ASP197, ALA198, LEU162, ARG195, HIS201, TYR151, GLU240, LYS200, ILE235, GLU233, HIS299, and ASP300).Acarbose was used as a standard to compare the docking results with the given ligands.Among these synthesized compounds, compound 5k showed interaction with both receptors by establishing hydrogen bonds with the specified active site residues, while the other did not establish any notable interactions, which were ignored from further analysis.These results were further analyzed with standard; it was observed that compound 5k interacted with alpha-glucosidase by forming multiple hydrophobic bonds (Pi-sulfur, Pi-Anion, Pi-Alkyl, and Pi-Pi) and established three hydrogen bonds with LYS480, ARG526, and ARG598 by utilizing the pyridine, sulfonyl, and bromide groups of the compound 5k.On the other hand, acarbose established multiple hydrogen bonds by exploiting the hydroxyl group in its structure, interacting with ASP203, ASP327, GLU404, ARG526, and ASP542, resulting in a binding energy of 118.1 kJ/mol.In the case of alpha-amylase, the carbonyl and sulfonyl groups of the compound 5k participated in establishing hydrogen bonds with ARG195 and GLN63, resulting in a binding energy of -101.4 kJ/mol.Here, acarbose formed nine (9) hydrogen bonds and a single hydrophobic interaction with the residues of alpha-amylase, such as TYR151, ARG195, ASP197, LYS200, GLU233, ILE235, ASP300, HIS305, ASP356, and TRP59.These interactions were facilitated by both the hydroxyl and hydrogen groups present in acarbose.By comparing the results of compound 5k with acarbose, it can be concluded that the activity of 5k possesses adequate affinity to inhibit the protein activity.This is attributed to the ability of compound 5k to establish hydrogen bonds with the target residues, which can potentially lead to protein inhibition.The results are illustrated in Figs. 3 and 4.

ADMET Prediction Results
The ADMET properties of both the standard drug and compound 5k were comparatively analyzed (Table 3).It was observed that the water solubility value of compound 5k is -5.58 log mol/L, whereas acarbose has a water solubility value of -1.482 log mol/L.The CaCo-2 permeability and intestinal absorption of compound 5k were found to be higher than that of acarbose.However, both compounds exhibited similar skin permeability.
Additionally, it was observed that acarbose acts as a substrate for P-glycoprotein, whereas compound 5k inhibits the activity of both P-glycoprotein I and II.The volume of distribution at a steady rate of 5k was -0.316 log L/kg with no fraction unbound and acarbose was -0.836 log L/kg with a fraction unbound value of 0.505 Fu.Both compounds exhibited minimal BBB and CNS permeability.Acarbose does not affect the activity of CYP variants, whereas compound 5k acts as a substrate for CYP3A4 and inhibits CYP2C19, CYP2C9, and CYP3A4.The total clearance value for compound 5k was -0.205 log ml/min/kg, while for acarbose, the value was 0.428.Both compounds are non-toxic; however, they interact with the hERG II protein by inhibiting its function.

Conclusion
In conclusion, a multi-step synthesis of chalcone-sulfonyl piperazine derivatives was effectively carried out via molecular hybridization, and all compounds were subjected to in vitro inhibitory potentials against alphaglucosidase and alpha-amylase as compared to standard reference acarbose.The synthesized analogues revealed excellent to good inhibitory potentials, with IC50 values for alpha-glucosidase ranging from 0.31 ± 0.01 to 47.84 ± 1.03 μM and for alpha-amylase from 4.51 ± 1.15 to 43.11 ± 1.56 μM.The MTT assay method was employed to demonstrate the cytotoxicity of all compounds on human fibroblast HT1080 cells.The results clearly indicated that all the synthesized compounds exhibit non-toxicity even at high concentrations.A molecular docking study was conducted to investigate the binding interactions between the most active analogue (5k) and the active sites of both enzymes.The results displayed that 5k forms several essential interactions with the active sites of the enzymes.Considering the comprehensive experimental data, these compounds have the potential to emerge as effective medications for the treatment of type-2 diabetes.

Chemistry
All commercially available chemicals and solvents were utilized without any additional purification.The melting points were determined in open capillary tubes using an uncorrected MPA 160 apparatus from Fisher Scientific (USA). 1 H NMR signals were recorded at 400 MHz and 13 C NMR at 100 MHz in DMSO-d6 employing Bruker Avance III (Germany).FT-IR spectra were obtained using a Frontier IR spectrophotometer from Perkin Elmer, USA.High-resolution mass spectra (HRMS) spectra were acquired on UHPLC/High Resolution Mass Spectrometer, Model: 1290 infinity II/Triple TOF 5600 plus (USA).The progression of the reactions was monitored using thin-layer chromatography (TLC) analysis.
Upon completion, the mixture was poured into crushed ice and neutralized by HCl (if no precipitate was formed, HCl was added to acidify the solution).The resulting mixture was stirred for an additional 30 minutes and then.

Synthesis of 1-(4-{4-[(4-bromophenyl)sulfonyl]piperazin-1-yl}phenyl)-3-phenylprop-2-en-1-one derivatives (5a-k)
Compounds 5a-k were synthesized using a previously reported method from the literature [45,49,50].To a solution containing 1 mmol of compound 3, pure EtOH was added, and the mixture was stirred for 5 minutes at 0°C.Subsequently, 1.1 mmol of NaOH was added portion wise, followed by the addition of 1 mmol of various aldehydes (4a-k).The reaction mixture was then refluxed for a duration of 24 hours.Once the reaction was completed, the mixture was poured into crushed ice and neutralized using HCl.The resulting precipitate was stirred at room temperature for 30 minutes and subsequently filtered.The obtained product was recrystallized from EtOH, resulting in the desired chalcones (5a-k).The physical and spectral data for compounds 5a-k, are listed below.

Alpha-glucosidase inhibition assay
The inhibitory activities of the synthesized compounds against α-glucosidase were evaluated using a methodology described in the literature [11].Acarbose was employed as a standard reference drug.

Alpha-amylase inhibition assay
The α-amylase inhibitory activity of the synthesized compounds was assessed using a method described in a previously published study [11].

Alpha-glucosidase kinetic mechanism
Among the inhibitors tested, compound 5k demonstrated the highest potency (IC50 value).Consequently, compound 5k was selected for kinetic analysis.Several experiments were conducted to calculate the inhibition kinetics of compound 5k [51].Various concentrations of 5k (0.00, 0.16, 0.31, and 0.62) were tested.The concentration of the substrate, p-nitrophenyl-α-D-glucopyranoside, ranged from 0.3125 to 10 mM in all the kinetics experiments.The pre-incubation and measurement periods matched those specified in the procedure for the α-glucosidase inhibition assay.The maximal initial velocity was determined from the initial linear portion of absorbance in the first five minutes following the addition of the enzyme at 30 s intervals.To determine the type of enzyme inhibition, Lineweaver-Burk plots were constructed by plotting the inverse of the velocities (1/V) against the inverse of the substrate concentration (1/[S]) in units of mM -1 .The dissociation constant (Ki) for the enzyme-inhibitor (EI) complex was determined by generating a secondary plot of 1/V versus the inhibitor concentration.

Cell culture and treatment
Human fibroblast HT1080 cells were procured from the Korean Cell Line Bank in Seoul, Korea, and utilized for evaluating the cytotoxicity of the synthesized compounds (5a-k).The cultivation and treatment procedures were performed according to the methods described in the literature [52].

Cell viability
To determine the cell viability of the compounds, we employed the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay.HT1080 cells were seeded onto 96-well plates at a density of 0.4 × 10 5 cells/well with three replicate wells per group and incubated in media for 24 hours.The compounds (5a-k) were treated at different concentrations (10,20,30,40,50,60, and 100 µM), while acarbose was employed as a control group at the same concentrations.After 24 hours of incubation, the media was discarded, and 100 µl of 0.5 mg/ml MTT solution, diluted in DMEM, was added to each well for 4 hours.The resulting formazan crystals were solubilized by adding 100 µl of DMSO, and the absorbance of the 96-well plate was measured at 570 nm using a microplate reader (Thermo Fisher, MA, USA).

Chemical Structure Preparation
The structures of the synthesized compounds (5a-k) were prepared for molecular docking studies by converting the Chemdraw format files into mol2 files using Discovery Studio [53] and OpenBabel [54].The structure of acarbose was retrieved from PubChem and converted to mol2 format using Discovery studio.These structures were hydrogenated polarly and minimized using the PRODRG server [55].

Receptor Structure Preparation
The structures of α-glucosidase and α-amylase were obtained from the Protein Data Bank database, with IDs 3L4W and 4W93, respectively.These structures were prepared by removing water ions and in-bound ligands.Subsequently, they were submitted to the Modrefiner database [56] for structural minimization.

Fig. 1 .
Fig. 1.Design strategy of novel chalcone-sulfonyl piperazine derivatives as new antidiabetic agents based on molecular hybridization of pharmacophoric units of potent chalcone reported alpha-glucosidase and alphaamylase inhibitors A-D and marketed antidiabetic agent.
Fig. 2B illustrates the relationship between the slope and the concentration of 5k, enabling the determination of the EI dissociation constant.By analyzing the concentration of 5k in relation to the slope, a Ki value of 0.4 µM was calculated.

Fig. 2 .
Fig. 2. Lineweaver-Burk plots for inhibition of alpha-glucosidase in the presence of compound 5k.(A) Concentrations of 5k were 0.00, 0.16, 0.31 and 0.62 mM.(B).The insets display the plot of the slope as a function

Table 2 .
Cell survived (%) after exposure with compounds for in vitro HT1080 fibroblast cell lines.

Table 3 .
Chemoinformatic profile of compound 5k compared with acarbose.