In silico design, synthesis and in-vitro evaluation of thiazolidine derivatives

 Dr. Sundhararajan, Silpa N^*, Jeevitha J, Jayashree KR, Jameerul Hasan S

Mohamed Sathak A.J.College of Pharmacy,Sholinganallur,Chennai-119

*Correspondence: slpvasuraj@gmail.com

DOI: https://doi.org/10.71431/IJRPAS.2025.4307

INTRODUCTION

In the hopes of improving the condition's prognosis and curing it, novel anti-diabetic medications are still needed even if there are several antidiabetic medications on the market. To expedite the drug development process, insilico laboratory researchers compare virtual libraries (databases) to virtual receptors (targets) using computer techniques. The process of building the in silico design begins with the identification of a group of high affinity ligands that share the same binding mechanism. The set ought to be chosen based on the low flexibility and structural diversity criteria.

The functional groups deemed necessary for biological activity are then determined. This study was aimed to design and arrive for potentially active scaffolds of thiazolidine (TD) heterocyclic derivatives as anti-diabetic agents, to synthesize the selected thiazolidine derivatives based on the synthetic feasibility, to characterize synthesised compounds , and  finally evaluating the in-vitro activity of the synthesized compounds for antidiabetic activity by alpha amylase inhibitory potential. Thiazolidine Derivatives are mentioned as TD1, TD2, TD3, TD4, TD5 for convenience.

MATERIALS AND METHODS

Docking studies

Software used

In this research work, we used various bio-informatics tools to carry out for the in-silico work. In current work we used following offline software’s like Marvin sketch for sketching molecules, PyRx for performing the molecular docking studies and some online software such as PDB, PubChem database, SPDBV, and Protein-ligand interaction profile (PLIP) [1].

Preparation of protein

We retrieved the targeted protein human MAO-A (PDB ID: 2Z5X) from the online program PDB website and the protein preparation were started from  the removal of water molecules, and followed this we added the missing H-atoms, ionization and energy minimization of proteins. The energy minimization was done by applying force filed through SPDBV software and it was validated by Ramachandran plot [2].

Identification of active sites

After preparation of protein, it was subjected to identify the active amino acid present in its structure by Protein-ligand interaction profile. By using PLIP we found the active amino acid residue present in the protein [3].

Preparation of Ligands

The 3D and 2D structure of designed derivatives were sketched by using Marvin sketch software. The sketched molecules are optimized and save as .pdb format for further processing.

Molecular Docking

The PyRx software was used for the docking process. The docking process was performed using molecular docking engine of PyRx using grid resolution. During the docking process the default setting was used for the calculation [4]. Substituted thiazolidines are screened and named as TD1, TD2 for convenience in this study. Thaiazolidine as scaffolds, substituted or derivatives were chosen with side chain feasible for synthesis.

Figure 1: In-silico inhibition

Table 1. Docking score

Table 2. Alpha amylase inhibition

Figure 2. 2D and 3D view of docking fit of compound TD1

Figure 3. 2D and 3D view of docking fit of compound TD2

Schematic representation of the synthesis

                                                            Table 3 synthesised thiazolidines                                                    

Experimental method of synthesis

The availability of ingredients in our lab and the viability of the synthetic strategy were taken into consideration.

Step 1: Synthesis of (E)-N-substituted benzylidenebenzohydrazide (A)

Equimolar quantities of benzohydrazide (1 mmol) and different aromatic aldehydes (2 mmol) was refluxed in alcohol for 4 h in the presence of few drops of glacial acetic acid. The reaction mixture on cooling was poured into cold water, filtered and dried. The crude solid was recrystallized in DMF–water mixture to give the products.

Step 2: title compound (TD1) N-(4-oxo-2-(p-tolyl)thiazolidin-3-yl)benzamide

A mixture of (E)-N-substituted benzylidenebenzohydrazide (1 mmol) and thioglycolic acid (mercapto acetic acid) (2 mmol) was refluxed in dry benzene (25 ml) for 8–10 h. After completion of reaction excess benzene was evaporated in vacuum. The resulting residue was neutralized with saturated NaHCO₃ solution until CO₂ evolution ceased. The solid product was washed with water, dried and recrystallized from DMF–water mixture.

Chemical Formula: C17H16N2O2S,Exact Mass: 312.09,Molecular Weight: 312.39

m/z: 312.09 (100.0%), 313.10 (18.6%), 314.09 (4.7%), 314.10 (2.2%), 313.09 (1.5%)

Elemental Analysis: C, 65.36; H, 5.16; N, 8.97; O, 10.24; S, 10.26

¹H NMR (500 MHz, DMSO) δ 9.60 (s, 1H), 7.89 – 7.80 (m, 3H), 7.47 (s, 1H), 7.46 – 7.39 (m, 2H), 7.29 – 7.15 (m, 3H), 7.14 (s, 1H), 6.82 (s, 1H), 3.64 (d, J = 15.0 Hz, 2H), 2.34 – 2.30 (m, 3H).

¹³C NMR (125 MHz, DMSO) δ 172.33, 160.65, 138.50, 137.55, 132.71, 132.03, 129.56, 128.47, 128.28, 125.70, 67.56, 33.25, 21.12.

TD2

N-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)benzamide,Chemical Formula: C17H16N2O3S

Exact Mass: 328.09 Molecular Weight: 328.39 m/z: 328.09 (100.0%), 329.09 (20.2%), 330.08 (4.5%), 330.09 (2.5%) Elemental Analysis: C, 62.18; H, 4.91; N, 8.53; O, 14.62; S, 9.76

¹H NMR (500 MHz, DMSO) δ 9.70 (s, 1H), 7.90 – 7.78 (m, 2H), 7.47 (s, 1H), 7.45 – 7.39 (m, 2H), 7.35 – 7.21 (m, 2H), 7.11 (s, 7429H), 6.98 – 6.83 (m, 1H), 6.02 (s, 2H), 3.82 – 3.78 (m, 3H), 3.64 (d, J = 15.0 Hz, 2H).

¹³C NMR (125 MHz, DMSO) δ 172.33, 161.84, 160.65, 135.53, 132.71, 132.03, 128.47, 128.28, 126.67, 114.04, 67.56, 56.03, 33.25.

In vitro antidiabetic study

Inhibition of α-amylase enzyme

0.1g of starch was dissolved in 100 mL of sodium acetate buffer (pH = 4.8, 16 mM) to create starch solution (0.1%). Dissolve 27.5 mg of α-amylase in 100 mL of deionized water to make an enzyme solution. In order to create a colorimetric reagent, 1 g of 3,5-dinitro salicylic acid was dissolved in 20 mL of deionized water, 0.16 g of sodium hydroxide was added gradually to 10 mL of deionized water, and 4 g of sodium potassium tartrate was added all at once. After the mixture was well combined, 100 milliliters of deionized water were added.  After mixing 100 μL of each of the control and sulfonylurea derivatives with 100 μL of the starch solution, they were left to react with the α-amylase solution for 30 minutes at 25°C in an alkaline environment. Five minutes later, the action was caught. Through the reduction of 3,5-dinitro salicylic acid to 3-amino-5-nitrosalicylic acid, the quantitative determination of liberated maltose was carried out. At 540 nm, the response was noticed.

RESULT AND DISCUSSION

Out of the proposed substituted thiazolidine compounds, the one with the best match and configuration was chosen (Figure 1 and Figure 2).

In order to compare the insilico and invitro outcomes, compounds TD1 and TD2 (table 1) were chosen based on their score and synthetic feasibility.

The IR, 1H NMR, 13C NMR, and mass spectrometric data of compounds TD1 and TD2 were taken into consideration when studying their structures. The 1H NMR spectrum of TD1 reveals a singlet for methyl protons at 3.32 ppm. The carbonyl group signal was detected at 165.7 ppm in the TD1 13C NMR spectra. The molecular ion peak at m/z = 319 was present in the mass spectra of TD1

Compounds TD1 and TD2 demonstrated synthetic feasibility, however compounds TD4 and TD5 had a higher docking score.

 According to the docking score, TD2 should have demonstrated stronger inhibitory activity than TD1as; nevertheless, TD1's invitro inhibition potential was significantly higher than TD2's (table 2) which recommends conducting in vitro research and synthesizing further compounds, This article gives an insight of comparing insilico and biological evaluation,. Research on a larger number of compounds and an extension to invivo evaluation will provide a more comprehensive picture of the uniqueness of particular substituted thiazolidine derivatives.

CONCLUSION

Thiazolidine Derivatives were screened for their in silico inhibitory potential, synthesized, characterized and invitro evaluation taken care as planned. Despite the fact that smaller number of  compounds are used, a thorough assessment of these compounds has been conducted using insilico design, synthesis, and invitro activity. More molecules in the substituted thiazolidine basket will result from this study's screening of additional thiazolidine compounds, the development of a simplified synthesis pathway, and their assessment. There are plans to synthesize and test more substituted thiazolidine compounds, which will be published later.

ACKNOWLEDGEMENT

The Faculty team of Mohamed Sathak A.J. College of Pharmacy is acknowledged by the authors for their assistance in this study.

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