## Introduction

Diabetes mellitus is a chronic disease that presents as both fasting and postprandial hyperglycemia, with disruptions in the metabolizing of carbohydrates and proteins. Chronic hyperglycemia can cause impairment to all organs of the human body by decreasing blood vessels' elasticity and narrowing them, impeding blood flow. This can cause a reduction in blood and oxygen supplies to all organs, increase the risk of hypertension and cause multiple damages to large and small blood vessels. Thus, it can be implicated in many macrovascular and microvascular illnesses, including heart attack, stroke, peripheral arterial disease, retinopathy, neuropathy, and nephropathy1,2. In recent years, there has been a rise in the global incidence of diabetes. According to the International Diabetes Federation, the estimated number of individuals with diabetes reached 30 million in 1985 and is expected to reach 380 million by 20253.

Antioxidant agents are free radical scavenging molecules that may prevent or delay several forms of cell damage by neutralizing the harmful free radicals in the living cells and thereby preventing or delaying various diseases4,5,6,7.

According to the World Health Organization, obesity is one of the most prevalent health concerns nowadays, and it is believed to be one of the major causes of cancer and a variety of other ailments8. In general, obesity is often caused by a number of variables, beginning with a high caloric food intake and inadequate physical activity, and it may be influenced by genetic susceptibility9,10.

Many structures containing hydroxyl functional groups are considered pharmacologically active toward different biological targets, such as combretastatin A-4 (CA-4), which has potent anticancer activity11, trolox has a powerful antioxidant effect, and acarbose has antidiabetic property (Fig. 1).

Regarding these structures, the current investigation aims to evaluate new molecules containing a hydroxyl functional group prepared from the esterification of thio-acid with benzoic acid derivatives, including benzoic acid (2a), 2-hydroxybenzoic acid (2b), 3-hydroxybenzoic acid (2c), and 4-hydroxybenzoic acid (2d) with 2-thiophenylethanol as antidiabetic, anti-obesity, and antioxidant agents by the inhibition of core metabolic enzymes implicated in hyperglycemia (α-amylase and α-glucosidase), lipids metabolism and oxidative stress.

## Materials and methods

### Chemicals and instruments

The following chemicals and biological materials were purchased from Frutarom (UK): β-carotene, DPPH, Na2HPO4/NaH2PO4, NaCl, gallic acid, trolox, methanol, starch, porcine pancreatic amylase, lipase, glucosidase, dinitrosalicylic acid (DNSA), Na2CO3, dimethyl sulfoxide (DMSO), and acarbose. The evaluated compounds 2a2d were synthesized previously by our team, as outlined in Fig. 212.

### α-Amylase inhibitory activity

The α-amylase inhibitory evaluation was established following the Wickramaratne et al. procedure13 with minor changes. Working solutions with concentrations of 10, 50, 100, 500, and 1000 µg/ml were produced from our synthesized compounds, diluting them with a buffer of Na2HPO4/NaH2PO4 (0.02 M), NaCl (0.006 M) at pH 6.9 and then brined up to 10 ml using a 10 ml volumetric flask. The acarbose was considered a positive control and was established following the same previous steps utilized for the synthesized compounds. As percent inhibition, the enzyme inhibitory potential was expressed, and the following equation was utilized to estimate the IC50 dose for the samples14.

$$\%a-Amylase\ Inhibiton=\frac{Abs100\%\ control-Abs\ Sample}{Abs\ 100\%\ Control}\times 100$$

### α-Glucosidase activity

The inhibition of α-glucosidase activity by the synthesized molecules was measured. The α-glucosidase inhibitory activity is expressed as percentages of inhibition, which was calculated using the following formula:

$${\text{Inhibitory}}\,{\text{effect }}\% \, = \, \left( {A_{b} - \, A_{s} } \right)/ \, A_{b} *100 \, \%$$

where Ab and As are the absorbance values of the blank (containing PBS, α-glucosidase, and PNPG, the colored substrate of glucosidase) and the tested samples containing PBS, samples, α-glycosidase, and PNPG, respectively.

The α-glucosidase activity was measured using 10 and 20 mg/ml of the synthesized molecules. Each concentration was recorded by using 1, 3, 6, 9, and 12 mm PNPG. The inhibitory pattern was assessed using a Lineweaver–Burk plot. The constant Ki of enzyme inhibitory effect was determined15.

### Antioxidant activity

#### DPPH method

Four synthetic compounds were tested for the efficiency of scavenging free radicals matched with trolox and gallic acid as a basic. From the compounds, 1 mg/ml concentration solutions in methanol were prepared, and the solutions were used to prepare concentrations of 5, 10, 20, 30, 40, and 50 μg/ml. The DPPH reagent (0.002% w/v) was dissolved in methanol before mixed with working concentrations in ratios of 1:1:1 (compound:DPPH:methanol). The methanol solution was adopted as a blank. All the solutions were incubated for 30 min at room temperature in the dark. When the antioxidant compound reacts with DPPH, which can donate hydrogen, it is reduced the color changes from deep violet to light. Absorbance values were estimated by using a UV–Vis spectrophotometer at a wavelength of 517 nm. The DPPH inhibition percentage of all tested compound, trolox and gallic acid, was estimated according to the formula:

$${\text{Inhibition}}\,{\text{ percentage }} = \, \left( {A_{b} - A_{s} } \right)/A_{b} \times 100\% ,$$

where Ab is blank absorbance, and AS is sample absorbance. The IC50 values for tested synthetic compounds, trolox and gallic acid were determined from the curves16.

#### β-Carotene method

The activity of the synthesized compounds was assessed via performing a modified assay of Gazzani and Miller17. The method was based on the coupled oxidation of β-carotene and linoleic acid emulsion. The bleaching mechanism of β-carotene is produced from the hydro-peroxides created from linoleic acid18. During the oxidation process, the characteristic orange color of β-carotene and the chromophore will be lost. The presence of antioxidants can hinder the extent of the β-carotene bleaching. Briefly, 1 mg of β-carotene was dissolved in 2 ml of chloroform, 20 mg of linoleic acid, and 200 mg of Tween 20. A rotary evaporator entirely vaporized chloroform at a temperature of less than 30 °C under reduced pressure. Then 200 ml of distilled water saturated with oxygen was added to the flask, which was shaken vigorously for 30 min. A sample (5 ml) of the prepared emulsion was transferred to a series of tubes containing 0.1 ml of the synthesized compounds or tocopherol (2 mg/ml).

The control sample was prepared in the same way but without added antioxidants. Each sample type was prepared in triplicate. The samples were inserted in a water bath at 50 °C for 2 h. The absorbance of the samples was measured using a spectrophotometer at a wavelength of 470 nm, immediately after the preparation of the samples, and at 15-min intervals until the end (t = 120 min) of the experiment.

### Anti-lipase activity

The lipase inhibitory activity was conducted by following the method in the references with minor modifications19,20,21. The prepared synthesized compounds solutions (1 mg/ml) were diluted with 10% DMSO to produce five different dilutions (0.2, 0.4, 0.6, 0.8, and 1 mg/ml). Orlistat anti-obesity drug was considered a positive control in this protocol and was then tested following the same steps used previously. A Tris–HCl buffer was used to zero UV–Vis spectrophotometer at 405 nm. The % inhibition of the synthesized compounds and orlistat was calculated by using the following equation:

$${\text{Inhibitory}}\,{\text{lipase}}\,{\text{percentage}}\,\left( \% \right) = \left[ {\left( {A_{b} - \, A_{s} } \right)/A_{b} } \right]*100$$

where Ab is blank absorbance, and AS is sample absorbance. The IC50 values for tested synthetic compounds and orlistat were determined from the constructed curves.

### Molecular docking

The docking studies on the α-amylase enzyme were based on the human pancreatic amylase (the X-ray crystallographic structure (PDB code 4W93) that was obtained from the RCSB repository. This 3D protein structure was chosen based on its good resolution of crystallography (X-ray diffraction 1.352 Ao). Additionally, this work is considered a continuation of our previous work on the α-amylase enzyme that utilized the ID: 4W93 α-amylase crystal structure22.

Preparing this crude obtained crystal structure for docking, using the PyMOL program (v1.8), started with removing the native ligand, non-protein atoms, and the present crystallographic waters. This step was followed by protonating imidazole and amide side chains via an H++ server. Then the polar hydrogen atoms were added and the structure was brought up to a physiological pH. The open-source program rDOCK (rdock.sourceforge.net), a development of RiboDock, was applied to perform the docking studies23. Considering the cavity occupied by the native ligand (a flavonol glycoside called montbretin A) of the utilized protein crystal structure (code 4W93), the binding site for docking studies was defined within 6Ao around it. The default docking procedure was followed that includes 3 stages of Genetic Algorithm search (GA-3, GA-2, and GA-1). This was followed by low-temperature Monte Carlo (MC) and Simplex minimization (MIN) stages22.

In the current study, the most potent compound against α-amylase was the 2a compound. Applying the empirical score function of Rdock, the 2a molecule was chosen to be docked in the binding pocket of the prepared crystal structure, keeping twenty docking solutions for each inhibitor to be sorted by their binding scores and later visually investigated for the connections between the inhibitors and the pocket’s residues.

A set of ADME-T related properties were calculated by using the QiKProp module (schrödinger 10.9, LLC, NY) running in normal mode. QikProp generates relevant descriptors and uses them to perform ADMET predictions24,25.

### Statistical analysis

The antidiabetic, antioxidant, and antilipase experimental works were conducted in triplicates and the results were expressed as means ± SD standard deviation while the result was considered significant when the p value was < 0.05.

## Results and discussion

### Antidiabetic activity

Hyperglycemia can be controlled by several therapeutic protocols, one of which is the inhibition of α-amylase and α-glucosidase metabolic enzymes6,26. In fact, α-amylase is a responsible enzyme for the hydrolysis of starch into simple monosaccharides27,28,29. While α-glucosidase enzyme can be found on the border of the small intestine and act upon hydrolysis of α-(1,4) bonds between monosaccharide units30,31,32.

The antidiabetic potential of the synthesized compounds was estimated by assessing their α-amylase and α-glycosidase inhibitory effects. Acarbose, which is a commercial antidiabetic drug, was used as a positive control. An evaluation of the plot of % α-amylase and α-glycosidase inhibition activities of each compound is shown in Figs. 3 and 4, respectively. The IC50 values were calculated (Table 1). All synthesized compounds show potential activity against the α-amylase enzyme.

Compound 2a showed potent α-amylase inhibitory activity with an IC50 value of 3.57 ± 1.08 µg/ml in comparison with positive control acarbose (IC50 = 6.47 ± 1.01 µg/ml), as for compound 2a, it demonstrated potent α-glycosidase inhibitory activity with an IC50 dose of 10.09 ± 0.70 µg/ml in comparison with acarbose (IC50 = 44.79 µg/ml). These results are reasonable and justified since the synthesized compounds are modified acid esters and have the same functional groups as phenolic compounds, such as trans-p-coumaric acid.

### Antioxidant activity

Antioxidants are valuable organic substances that can neutralize the harmful free radicals in living cells. There are two types of these radicals; natural pathways create the first type in our bodies due to the aging of cells and other factors; the second type is entered into our bodies from external sources like smoking, sun exposure, and other dangerous pollution sources. However, the body needs exogenous sources of synthetic antioxidants or dietary antioxidants from fruits and vegetables to scavenge these harmful radicals6,33,34.

### DPPH inhibitory activity

1,1-Diphenyl-2-picryl-hydroxyl (DPPH) is one of the most widely used methods for measuring the antioxidant activity of different compounds such as phenols and phenolic acid esters. The decrease in absorbance at 517 nm induced by antioxidants determines the reduction ability of the DPPH. When antioxidants donate hydrogen atoms to the radicals, they lose their purple color. This, in turn, leads to decreased absorption. The decrease in absorption is taken as a measure of the extent of radical scavenging. All the compounds showed free radical scavenging activity near gallic acid at a concentration of 30 µg/ml (Fig. 5). Compound 2a showed potential antioxidant activity with an IC50 value of 47.01 ± 1.07 µg/ml compared with the positive controls, gallic acid and trolox, which have IC50 doses of 31.72 ± 1.22 and 4.28 ± 0.47 µg/ml, respectively (Table 2).

On the other hand, there is not much difference between percent inhibition values for the compounds. This means that the position of functional groups as ortho-, para- or meta- has a slight effect on the antioxidant activity. Our results are in agreement with literature done on polyphenolic compounds, which showed that the structure did not affect the antioxidant activity35.

### β-Carotene linoleic acid activity

The same compounds were tested for their antioxidant activity using the emulsion system of β-carotene linoleic acid, depending on the fact that β-carotene loses its color in the absence of antioxidants. The synthetic compounds 2a and 2b showed higher antioxidant efficiency in concentration 0.2 mg/ml compared with water (control) and α-tocopherol and the other synthetic compounds antioxidant, which gave the lowest antioxidant efficiency. Figure 6 showed the antioxidant activities of the synthetic acid esters and positive reference standard with the coupled oxidation of β-carotene in a concentration of 0.2 mg/ml.

The antioxidant activity of all the compounds gradually decreases with the increase of time. Water showed the highest β-carotene bleaching activity, followed by α-tocopherol after 1 h and (2a) compound, which has the best antioxidant activity with the absorbance of 1.04 after 75 min compared with the other synthetic tested compounds. The second one is a 2b compound which showed absorbance 0.84 after 75 min.

### Porcine pancreatic lipase enzyme activity

The hydrolysis of p-nitrophenyl butyrate to p-nitrophenyl was used to measure the influence of the synthetic acid esters on the porcine pancreatic lipase enzyme. The assay measures the inhibition percentages of the synthesized compounds and the obtained results compared with orlistat, a pharmaceutical anti-obesity drug, and used in the current study as a positive control. The results of inhibitory percentages for the compounds and orlistat are shown in Fig. 7.

The anti-lipase activity IC50 values of the synthesized compounds were with the range of 107.25–341.06 µg/ml in comparison with orlistat (IC50 = 25.01 ± 0.78 µg/ml). The best inhibition activity was observed in compounds 2a and 2d with IC50 values of 107.95 ± 1.88 and 119.05 ± 2.04 µg/ml, respectively. Compound 2c has the lowest inhibitory activity with an IC50 value of 341.06 ± 2.40 µg/ml.

### Docking study

The 2a compound, which exhibited a super anti-diabetic and anti-lipase activity besides super antioxidant activity (considering the β-Carotene method) over the other designed compounds, was docked to the binding pocket of the α-amylase protein structure ID: 4W93). This docking revealed the formation of hydrogen bonds with the residue E233 and charge–π interactions with H201; stabilized by interactions with other surrounding residues like π–π stacking with Y62. Our docking studies suggest the potent inhibition of α-amylase is derived from strong interactions with the catalytic residue E233 and the neighbor residue H201, besides other stabilizing interactions with surrounding residues (Fig. 8) as well as the molecular Docking scores and α-amylase inhibtory activities are presented in Table 3.