Virtual Screening, Synthesis, and Biological Evaluation of Some Carbohydrazide Derivatives as Potential DPP-IV Inhibitors

Dipeptidyl peptidase-4 (DPP-IV) inhibitors are known as safe and well-tolerated antidiabetic medicine. Therefore, the aim of the present work was to synthesize some carbohydrazide derivatives (1a–5d) as DPP-IV inhibitors. In addition, this work involves simulations using molecular docking, ADMET analysis, and Lipinski and Veber’s guidelines. Wet-lab synthesis was used to make derivatives that met all requirements, and then FTIR, NMR, and mass spectrometry were used to confirm the structures and perform biological assays. In this context, in vitro enzymatic and in vivo antidiabetic activity evaluations were carried out. None of the molecules had broken the majority of the drug-likeness rules. Furthermore, these molecules were put through additional screening using molecular docking. In molecular docking experiments (PDB ID: 2P8S), many molecules displayed more potent interactions than native ligands, exhibiting more hydrogen bonds, especially those with chloro- or fluoro substitutions. Our findings indicated that compounds 5b and 4c have IC50 values of 28.13 and 34.94 µM, respectively, under in vitro enzymatic assays. On the 21st day of administration to animals, compound 5b exhibited a significant reduction in serum blood glucose level (157.33 ± 5.75 mg/dL) compared with the diabetic control (Sitagliptin), which showed 280.00 ± 13.29 mg/dL. The antihyperglycemic activity showed that the synthesized compounds have good hypoglycemic potential in fasting blood glucose in the type 2 diabetes animal model (T2DM). Taken all together, our findings indicate that the synthesized compounds exhibit excellent hypoglycemic potential and could be used as leads in developing novel antidiabetic agents.


Introduction
Hyperglycemia and many other alterations of carbohydrate and protein metabolisms are symptoms of diabetes mellitus, often known as "DM", a chronic metabolic disorder. The It was observed that nitrogen ring system and amide core was common in most of the approved drugs Amide core Nitrogen ring system as pyrimidine core Structures of some DPP-IV approved drugs    Pharmacokinetic characteristics are essential for drug development because they enable researchers to assess the biological components of effective medicines. Lipinski's rule of five and Veber's criterion was used to determine if the compounds were appropriate for oral bioavailability ( Table 2). All proposed compounds were studied for their ADME features to get a deeper comprehension of their pharmacokinetic profiles and the drug-like qualities each of them (Table 3). Oral acute toxicity, LD 50 (in mg/kg), toxicity class, and other toxicity parameters have all been anticipated (Table 4).
Where: NL, Native ligand; GI abs., gastrointestinal absorption; BBB pen., blood-brain barrier penetration; P-gp sub., p-glycoprotein substrate; H, High; Y, Yes; N, No. In developing new medications, the goal is to convert a therapeutic chemical into a dosage form suitable for administering to patients. The pharmacological effects of a medicine, which must occur at the site of action and then disappear after a specific amount of time, are preferable to the once-daily administration of a drug. By analyzing a new drug's absorption, distribution, metabolism, and excretion (ADME) characteristics, one can make risk-based assessments of the medication's potential for adverse effects [12][13][14]. This contributes to understanding the course of the pharmacokinetic processes and helps in their investigation. We have synthesized a few 6-methyl-2-oxo-4-substituted-N'-[(E)substitutedmethylidene]-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide derivatives by using a rational drug design technique, that can block the activity of DPP-IV. The ADMET study was carried out on each of the newly generated compounds.
All the molecules' log P values fell within the range of 1.61 to 3.29, which suggests that an optimal amount of lipophilicity was achieved. The Log P value evaluates the permeability of a drug in the body enabling it to reach the target tissues [15]. All of the investigated compounds had molecular weights of less than 500 Da, suggesting that they can easily move across biological membranes [16,17]. To our relief, none of the compounds negatively impacted the validity of the Lipinski Rule of Five. It has been discovered that the total polar surface area (TPSA) and the number of rotatable bonds can differentiate between compounds that are active and that are not when taken orally. Veber's rule dictates that the TPSA should be less than 140 Å 2 , and the number of rotatable bonds should be less than 10. None of the synthesized molecules broke Veber's law in any way. It is an indication that these compounds can be converted into dosage forms that can be taken orally. Despite the fact that many drugs available on the market violate Rule 5, they are still approved by the FDA for oral use. Care must therefore be taken in screening molecules according to the Rule of Five, and a detailed report can be found at https://www.nature.com/articles/nrd.2018.197 (accessed on 16 November 2022).
The pharmacokinetics and drug-likeness features of each molecule were analyzed and estimated so that additional improvements might be made to these compounds. There was, for instance, no evidence that any of these chemicals could cross the blood-brain barrier (BBB). The values of log Kp (skin penetration, cm/s) and bioavailability of all the compounds were well within the scope of acceptable ranges. It was observed that all investigated compounds exhibit a high level of gastrointestinal (GI) absorption. Except for the native ligand, none of the compounds broke any of the norms set out by the Ghose, Egan, and Muegge rule. Numerous compounds have been discovered to be inhibitors of cytochrome enzymes, which suggests that these chemicals will likely interfere with the metabolism of other medications. Even more fascinating is that each of the synthesized compounds was a P-gp substrate inhibitor, which suggests that they can prevent drug efflux from the cell. Moreover, acute toxicity predictions showed that all of these compounds belong to class IV of toxicity, which indicates that they are harmful if swallowed (300 < LD50 ≤ 2000) [18].

Molecular Docking
From the very first screening to the application of the Lipinski and Veber rules, the ADMET calculations, and the drug-likeness attributes, none of the molecules had broken the majority of those rules. As a result, these molecules underwent additional screening through molecular docking. Many compounds have demonstrated more powerful interactions and higher binding energies with the target than the native ligand. Table 5 presents details of the interactions and docking scores of the most potent molecules with the DPP-IV enzyme. Furthermore, Table 6 describes the 2D and 3D docking postures of the most potent compounds (docking interactions, docking scores, and binding poses of resting molecules are depicted in the Supplementary File in Tables S1 and S2).   Table 6. The 2D-and 3D-binding postures of the most potent molecules.  Table 6. The 2D-and 3D-binding postures of the most potent molecules.  Table 6. The 2D-and 3D-binding postures of the most potent molecules. Table 6. Cont.

3D-Docking Poses 2D-Docking Poses
Compound 4a Molecules 2023, 28, 149 9 of 23 Compound 5b Molecular docking on the human DPP-IV enzyme was used to get further insight into the structure-activity connection of our new derivatives through the molecules' binding mode. The lowest energy conformers of all the ligands were docked onto the active site grid of the enzyme. The native ligand had a binding free energy of −9.1 kcal/mol and established just one conventional hydrogen bond with TYR662 through the N-H of the amide group. It formed electrostatic bonds with ARG125 and ARG358, and hydrophobic bonds with ARG358 and PHE357.
All investigated compounds demonstrated good binding affinity ranging from −8.9 to −8.0 kcal/mol. From series 1a-1d, molecule 1b showed the most potent interactions with a docking score of −9.4 kcal/mol. It formed five conventional hydrogen bonds and one carbon-hydrogen bond with SER349, SER376, THR351, and GLU378. It additionally displayed two electrostatic (attractive charge) bonds with GLU378. It formed two Pi-donor hydrogen bonds with THR350 and SER376 and exhibited hydrophobic interactions with PHE396, CYS394, and VAL354. From the series 2a-2d, molecule 2b exhibited the most potent binding affinity with a docking score of −9.3 kcal/mol and formed two conventional hydrogen bonds with ARG560 and ASN562. It also formed numerous hydrophobic interactions (Pi-sigma, Pi-Pi T-shaped, alkyl, and Pi-alkyl) with LYS512, ILE529, ARG560, PHE559, PRO475, ALA564, and PHE559.
In the series 3a-3d, compound 3b exhibited potent interactions and showed −9.3 kcal/mol binding affinity. It formed only one conventional hydrogen bond with HIS740 and two electrostatic (attractive charge) bonds with GLU205. It displayed numerous hydrophobic interactions (Pi-Pi stacked, Pi-Pi T-shaped, amide Pi-stacked, alkyl, and Pi-alkyl) with PHE357, TYR662, TYR547, TYR666, SER630; TYR631, LYS554, VAL656, and TYR547. In the series 4a-4d, compound 4a showed the highest binding affinity with the Compound 5b Molecular docking on the human DPP-IV enzyme was used to get further insight into the structure-activity connection of our new derivatives through the molecules' binding mode. The lowest energy conformers of all the ligands were docked onto the active site grid of the enzyme. The native ligand had a binding free energy of −9.1 kcal/mol and established just one conventional hydrogen bond with TYR662 through the N-H of the amide group. It formed electrostatic bonds with ARG125 and ARG358, and hydrophobic bonds with ARG358 and PHE357.
All investigated compounds demonstrated good binding affinity ranging from −8.9 to −8.0 kcal/mol. From series 1a-1d, molecule 1b showed the most potent interactions with a docking score of −9.4 kcal/mol. It formed five conventional hydrogen bonds and one carbon-hydrogen bond with SER349, SER376, THR351, and GLU378. It additionally displayed two electrostatic (attractive charge) bonds with GLU378. It formed two Pi-donor hydrogen bonds with THR350 and SER376 and exhibited hydrophobic interactions with PHE396, CYS394, and VAL354. From the series 2a-2d, molecule 2b exhibited the most potent binding affinity with a docking score of −9.3 kcal/mol and formed two conventional hydrogen bonds with ARG560 and ASN562. It also formed numerous hydrophobic interactions (Pi-sigma, Pi-Pi T-shaped, alkyl, and Pi-alkyl) with LYS512, ILE529, ARG560, PHE559, PRO475, ALA564, and PHE559.
In the series 3a-3d, compound 3b exhibited potent interactions and showed −9.3 kcal/mol binding affinity. It formed only one conventional hydrogen bond with HIS740 and two electrostatic (attractive charge) bonds with GLU205. It displayed numerous hydrophobic interactions (Pi-Pi stacked, Pi-Pi T-shaped, amide Pi-stacked, alkyl, and Pi-alkyl) with PHE357, TYR662, TYR547, TYR666, SER630; TYR631, LYS554, VAL656, and TYR547. In the series 4a-4d, compound 4a showed the highest binding affinity with the Compound 5b Molecular docking on the human DPP-IV enzyme was used to get further insight into the structure-activity connection of our new derivatives through the molecules' binding mode. The lowest energy conformers of all the ligands were docked onto the active site grid of the enzyme. The native ligand had a binding free energy of −9.1 kcal/mol and established just one conventional hydrogen bond with TYR662 through the N-H of the amide group. It formed electrostatic bonds with ARG125 and ARG358, and hydrophobic bonds with ARG358 and PHE357.
All investigated compounds demonstrated good binding affinity ranging from −8.9 to −8.0 kcal/mol. From series 1a-1d, molecule 1b showed the most potent interactions with a docking score of −9.4 kcal/mol. It formed five conventional hydrogen bonds and one carbon-hydrogen bond with SER349, SER376, THR351, and GLU378. It additionally displayed two electrostatic (attractive charge) bonds with GLU378. It formed two Pi-donor hydrogen bonds with THR350 and SER376 and exhibited hydrophobic interactions with PHE396, CYS394, and VAL354. From the series 2a-2d, molecule 2b exhibited the most potent binding affinity with a docking score of −9.3 kcal/mol and formed two conventional hydrogen bonds with ARG560 and ASN562. It also formed numerous hydrophobic interactions (Pi-sigma, Pi-Pi T-shaped, alkyl, and Pi-alkyl) with LYS512, ILE529, ARG560, PHE559, PRO475, ALA564, and PHE559.
In the series 3a-3d, compound 3b exhibited potent interactions and showed −9.3 kcal/mol binding affinity. It formed only one conventional hydrogen bond with HIS740 and two electrostatic (attractive charge) bonds with GLU205. It displayed numerous hydrophobic interactions (Pi-Pi stacked, Pi-Pi T-shaped, amide Pi-stacked, alkyl, and Pi-alkyl) with PHE357, TYR662, TYR547, TYR666, SER630; TYR631, LYS554, VAL656, and TYR547. In the series 4a-4d, compound 4a showed the highest binding affinity with the Compound 5b Molecular docking on the human DPP-IV enzyme was used to get further insight into the structure-activity connection of our new derivatives through the molecules' binding mode. The lowest energy conformers of all the ligands were docked onto the active site grid of the enzyme. The native ligand had a binding free energy of −9.1 kcal/mol and established just one conventional hydrogen bond with TYR662 through the N-H of the amide group. It formed electrostatic bonds with ARG125 and ARG358, and hydrophobic bonds with ARG358 and PHE357.
All investigated compounds demonstrated good binding affinity ranging from −8.9 to −8.0 kcal/mol. From series 1a-1d, molecule 1b showed the most potent interactions with a docking score of −9.4 kcal/mol. It formed five conventional hydrogen bonds and one carbon-hydrogen bond with SER349, SER376, THR351, and GLU378. It additionally displayed two electrostatic (attractive charge) bonds with GLU378. It formed two Pi-donor hydrogen bonds with THR350 and SER376 and exhibited hydrophobic interactions with PHE396, CYS394, and VAL354. From the series 2a-2d, molecule 2b exhibited the most potent binding affinity with a docking score of −9.3 kcal/mol and formed two conventional hydrogen bonds with ARG560 and ASN562. It also formed numerous hydrophobic interactions (Pi-sigma, Pi-Pi T-shaped, alkyl, and Pi-alkyl) with LYS512, ILE529, ARG560, PHE559, PRO475, ALA564, and PHE559.
In the series 3a-3d, compound 3b exhibited potent interactions and showed −9.3 kcal/mol binding affinity. It formed only one conventional hydrogen bond with HIS740 and two electrostatic (attractive charge) bonds with GLU205. It displayed numerous hydrophobic interactions (Pi-Pi stacked, Pi-Pi T-shaped, amide Pi-stacked, alkyl, and Pi-alkyl) with PHE357, TYR662, TYR547, TYR666, SER630; TYR631, LYS554, VAL656, and TYR547. In the series 4a-4d, compound 4a showed the highest binding affinity with the Molecular docking on the human DPP-IV enzyme was used to get further insight into the structure-activity connection of our new derivatives through the molecules' binding mode. The lowest energy conformers of all the ligands were docked onto the active site grid of the enzyme. The native ligand had a binding free energy of −9.1 kcal/mol and established just one conventional hydrogen bond with TYR662 through the N-H of the amide group. It formed electrostatic bonds with ARG125 and ARG358, and hydrophobic bonds with ARG358 and PHE357.
All investigated compounds demonstrated good binding affinity ranging from −8.9 to −8.0 kcal/mol. From series 1a-1d, molecule 1b showed the most potent interactions with a docking score of −9.4 kcal/mol. It formed five conventional hydrogen bonds and one carbon-hydrogen bond with SER349, SER376, THR351, and GLU378. It additionally displayed two electrostatic (attractive charge) bonds with GLU378. It formed two Pi-donor hydrogen bonds with THR350 and SER376 and exhibited hydrophobic interactions with PHE396, CYS394, and VAL354. From the series 2a-2d, molecule 2b exhibited the most potent binding affinity with a docking score of −9.3 kcal/mol and formed two conventional hydrogen bonds with ARG560 and ASN562. It also formed numerous hydrophobic interactions (Pi-sigma, Pi-Pi T-shaped, alkyl, and Pi-alkyl) with LYS512, ILE529, ARG560, PHE559, PRO475, ALA564, and PHE559.
In the series 3a-3d, compound 3b exhibited potent interactions and showed −9.3 kcal/mol binding affinity. It formed only one conventional hydrogen bond with HIS740 and two electrostatic (attractive charge) bonds with GLU205. It displayed numerous hydrophobic interactions (Pi-Pi stacked, Pi-Pi T-shaped, amide Pi-stacked, alkyl, and Pi-alkyl) with PHE357, TYR662, TYR547, TYR666, SER630; TYR631, LYS554, VAL656, and TYR547. In the series 4a-4d, compound 4a showed the highest binding affinity with the target. It exhibited -9.5 kcal/mol binding affinity and formed only two carbon-hydrogen (halogen, F) bonds with PHE559 and VAL558. It formed hydrophobic interactions with LEU514, PRO510, LYS512, and ILE529. On the other hand, compound 5b from the series 5a-5d exhibited the highest interactions and displayed -9.6 kcal/mol binding affinity. It formed three conventional hydrogen bonds with ILE102, HIS100, and ILE102. It also formed one fluorine halogen bond with ASN74 and hydrophobic interactions with LYS71, TYR105, ILE76, and ILE102. All the aromatic substitutions displayed hydrophobic interactions with the enzyme cavity. The NH of the imidazole ring system has an important function in binding the derivatives to the enzyme cavity. Although most synthesized molecules displayed more potent interactions than native ligands, many displayed more hydrogen bonds, especially molecules with chloro-or fluoro-substitutions. All the designed derivatives exhibited potent interactions and optimized binding affinity with the target. Therefore, all the designed derivatives were subjected to wet lab synthesis and biological evaluation.

In vitro DPP-IV Enzyme Assay
All the synthesized compounds were subjected to in vitro enzyme assays and the results are shown in Table 7. Results revealed that all of these compounds exhibit excellent in vitro inhibitory potential. Of all the synthesized molecules, 4c (which possesses a 2-fluoro phenyl substitution as -Ar position and a -2-methoxy phenyl substitution as -Ar') showed 60.40 ± 0.08% inhibition at 50 µM and a 34.94 µM IC 50 value, while compound 5b (which possesses a -2,4-difluoro phenyl substitution as -Ar position whereas -2-hydroxy phenyl substitution as -Ar') displayed 70.70 ± 0.37% inhibition at 50 µM and an IC 50 value of 28.13 µM. Compound 4c has a fluoro group that withdraws electrons; this makes it more likely to bind to the active site of the enzyme DPP-IV. Due to the presence of an electron-donating methoxy group at Ar' of compound 4c, electron density increases on the pyrimidine 2-one ring, which increases the lipophilicity and activity of the compound. Similarly, compound 5b possesses an electron-withdrawing fluoro group and an electron-donating hydroxyl group. The NH of the carbohydrazones 4c and 5b forms hydrogen bonds with the amino acids of the enzyme DPP-IV's active site. We incorporated various steric, electronic, and hydrophobic groups in the basic nucleus. The synthesized compounds thus showed promising in vitro DPP-IV inhibitory activity.

In Vivo Antidiabetic Activity
Compounds 1b, 4c, and 5b were chosen for further in vivo antidiabetic research based on virtual screening, in vitro enzymatic assay results, and ultimately to better understand structure-activity relationships. Results obtained from in vivo evaluations of the antihyperglycemic activity of the synthesized compounds are displayed in Table 8. These results show the effect of the selected compounds on fasting blood glucose levels compared to sitagliptin. The graph of the in vivo antidiabetic activity is given in Figure 2. results show the effect of the selected compounds on fasting blood glucose levels compared to sitagliptin. The graph of the in vivo antidiabetic activity is given in Figure 2.  The DPP-IV inhibitor sitagliptin is a fast-acting, strongly bound, reversible, and competitive inhibitor. Long-term inhibition of >70% after 8 h was demonstrated in ob/ob mice at 10 mg/kg (p.o.). In the same model, duration was correlated with long-lasting effectiveness (35% glucose excursion at 8 h) [19]. Therefore, in the present investigation, a 10 mg/kg dose of sitagliptin was employed. The compounds showed significant hypoglycemic potential in fasting blood glucose in an animal model. After the trial, blood glucose levels The DPP-IV inhibitor sitagliptin is a fast-acting, strongly bound, reversible, and competitive inhibitor. Long-term inhibition of >70% after 8 h was demonstrated in ob/ob mice at 10 mg/kg (p.o.). In the same model, duration was correlated with long-lasting effectiveness (35% glucose excursion at 8 h) [19]. Therefore, in the present investigation, a 10 mg/kg dose of sitagliptin was employed. The compounds showed significant hypoglycemic potential in fasting blood glucose in an animal model. After the trial, blood glucose levels were considerably higher in diabetes-control animals than in normal control ones. This difference was dramatically reduced in the diabetic treatment groups (compound 5b at 50 mg/kg and standard treatment with sitagliptin at 10 mg/kg). The serum blood glucose level recovered with compound 5b was significantly lower on day 21 (157.33 ± 5.75 mg/dL) than that of the diabetic control group (280.00 ± 13.29 mg/dL), whereas that of regular sitagliptin was higher (133.50 ± 11.80 mg/dL). As far as in vitro enzyme assay results are concerned, compound 5b exhibited an IC 50 value that is >1400 times higher than that of sitagliptin, though, in animals, 5b displayed a very potent activity that was significantly comparable with the sitagliptin. This may be linked to the fact that 5b can inhibit other rate-limiting enzymes contributing to diabetes. This point can be further investigated by performing computational analysis and other in vitro enzyme assays. We are thus aiming to develop more anti-diabetic derivatives considering 5b as a leading nucleus.
The SAR analysis of all the derivatives revealed important insights into essential structural requirements for effective DPP-IV inhibition. Phenyl ring substitutions are necessary for binding at two sites of the DPP IV enzyme. Removal of the 2-hydroxy substitution of the phenyl ring decreases the inhibitory activity of the derivatives. Hence, the introduction of a polar substituent is essential for the activity. In addition, a significant activity increase was observed due to the structural features of the synthesized compounds. The different structural characteristics, such as the electron-withdrawing groups -Cl and -F, make it easier for the synthesized compounds to connect to the active sites of DPP-IV. In contrast, electron-donating groups such as -OH, -CH 3 , and -OCH 3 increase electron density on the derivatives and increase lipophilicity, thus enhancing the activity of the synthesized compounds.

In Silico ADMET Analysis
The Lipinski rule of five and the pharmacokinetic properties of the designed molecules were calculated with the help of the molinspiration and SwissADME servers [20,21]. ProTox-II is a web server that is freely accessible to the public and used to carry out an in silico toxicity prediction of the proposed derivatives (http://tox.charite.de/protox_II accessed on 20 October 2022) [18,22,23].

Molecular Docking
The molecular docking was performed using Autodock Vina 1.1.2 in PyRx 0.8 [24]. The process of ligands and protein purification is described in our previously published paper [25]. The Universal Force Field (UFF) has been assigned to carry out energy reduction and optimization [26]. Figure 3 depicts the structure of the DPP-IV enzyme (PDB ID: 2P8S) acquired from the PDB site. The enzyme structures were refined, purified, and prepared for molecular docking using Discovery Studio Visualizer 2019 [27]. The whole molecular docking approach, including identifying cavities and active amino acid residues, was carried out as reported by Khan et al. [15,[28][29][30][31][32].

Chemistry
All chemicals and reagents used throughout this work were purchased from Lab Trading Laboratories in Aurangabad, Maharashtra, India, and employed as received. Thin Layer Chromatography (TLC) was used to monitor the reaction, and spots were observed under UV light and exposed to iodine vapors. Melting points (uncorrected) were determined manually using the open capillary technique. 1 H and 13 C NMR spectra were obtained with a 500 MHz spectrometer (JEOL) with CDCl3 as a solvent and tetramethyl silane (TMS) as an internal standard. Chemical shifts are expressed in δ units or parts per million (ppm), together with the coupling frequencies as singlet (s), double (d), triplet (t), and multiplet (m); 1 H-1 H coupling constants are given in Hertz. Mass (m/z) spectra of the synthesized compounds were acquired with a Shimadzu LC-MS system. IR spectra of the synthesized compounds were recorded with an Agilent Technologies' Microlab IR Spectrophotometer.

Synthesis of ethyl 6-methyl-2-oxo-4--1,2,3,4-tetrahydropyrimidine-5-carboxylate
A mixture of 1 mmol of an aromatic aldehyde, ethyl acetoacetate, and urea, and catalytic amounts of CuCl2 were ground together for 5-10 min. The solid mass was left standing overnight and later washed with cold water. The product was dried and recrystallized from ethyl alcohol to obtain the pure product [15,29,33].

Chemistry
All chemicals and reagents used throughout this work were purchased from Lab Trading Laboratories in Aurangabad, Maharashtra, India, and employed as received. Thin Layer Chromatography (TLC) was used to monitor the reaction, and spots were observed under UV light and exposed to iodine vapors. Melting points (uncorrected) were determined manually using the open capillary technique. 1 H and 13 C NMR spectra were obtained with a 500 MHz spectrometer (JEOL) with CDCl 3 as a solvent and tetramethyl silane (TMS) as an internal standard. Chemical shifts are expressed in δ units or parts per million (ppm), together with the coupling frequencies as singlet (s), double (d), triplet (t), and multiplet (m); 1 H-1 H coupling constants are given in Hertz. Mass (m/z) spectra of the synthesized compounds were acquired with a Shimadzu LC-MS system. IR spectra of the synthesized compounds were recorded with an Agilent Technologies' Microlab IR Spectrophotometer.

Synthesis of 6-Methyl-2-oxo-4-substituted-N'-((E) Substituted-methylidene)-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide Derivatives
The product obtained from step II (6-methyl-2-oxo-4-substituted-1,2,3,4 tetrahydropyrimidine-5-carbohydrazide) (10 mmol) was reacted for 2 h under reflux with 10 mmol of an aromatic aldehyde and acetic acid in 5 mL of ethanol. The mixture was then allowed to cool. The obtained Schiff bases were collected by filtration and recrystallized from ethanol as solid products. Purification of the synthesized compounds was performed using column chromatography [25,35]. Pre-prepared TLC plates were employed to test the purity of the synthesized compounds and reaction completeness using n-hexane: ethyl acetate (7:3) as a mobile phase and iodine vapor as a visualization agent. Depicted in Figure 4 is the suggested reaction pathway for synthesizing all the designed derivatives. The different substitutions with compound codes are listed in Table 1 already. The structures of all the synthesized derivatives are depicted in Figure 5. The product obtained from step II (6-methyl-2-oxo-4-substituted-1,2,3,4 tetrahydropyrimidine-5-carbohydrazide) (10 mmol) was reacted for 2 h under reflux with 10 mmol of an aromatic aldehyde and acetic acid in 5 mL of ethanol. The mixture was then allowed to cool. The obtained Schiff bases were collected by filtration and recrystallized from ethanol as solid products. Purification of the synthesized compounds was performed using column chromatography [25,35]. Pre-prepared TLC plates were employed to test the purity of the synthesized compounds and reaction completeness using n-hexane: ethyl acetate (7:3) as a mobile phase and iodine vapor as a visualization agent. Depicted in Figure  4 is the suggested reaction pathway for synthesizing all the designed derivatives. The different substitutions with compound codes are listed in Table 1 already. The structures of all the synthesized derivatives are depicted in Figure 5.  Characterization data (E)-N-Benzylidene-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine -5-carbohydrazide (1a).  Characterization data (E)-N-Benzylidene-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide (1a).

Animals and Ethical Approvals
The antihyperglycemic potential of certain compounds was investigated in Wistar rats. STZ-induced diabetic rats were employed for the antihyperglycemic testing. The animal experiments were approved by the Institutional Animal Ethics Committee (IAEC) in New Delhi, India (Protocol Number: IAEC/MCP/009/2020). During the research period, the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and IAEC criteria for animal care were followed.

Acute Toxicity Studies
Acute toxicity studies were performed as per the OECD guideline 423. For compounds 1b, 4c, and 5b, three groups of six rats weighing 80-120 g were starved overnight and given the test compounds orally at doses of 200, 500, 1000, and 2000 mg/kg body weight. The animals were monitored for 72 h, for symptoms of acute toxicity, such as increased or reduced motor activity, tremors, convulsions, and drowsiness, among others. It was observed that at a dosage of 500 mg/kg body weight, more than half of the animals died. As a result, the OECD-recommended dosage for assessing the antidiabetic action was set at 50 mg/kg (i.e., one-tenth of the 2000 mg/kg body weight) [37].

STZ-Induced T2DM Model
Rats were given a high-fat diet after one week of acclimation. Diabetes was induced in the animals as per the method each described in [25]. Rats were then divided into six groups with six animals. Group I served as normal control with normal rats receiving the vehicle only. Group II served as diabetes control in which rats received the vehicle only. Group III served as standard and received sitagliptin at a dose of 10 mg/kg body weight (b.w.). Groups IV, V, and VI served as test groups and received compounds 1b, 5b, and 4c, respectively, at doses of 50 mg/kg b.w. The diabetic rats were given the tested compound suspended in 0.5% w/v CMC every day for 21 days. On days 1, 7, 14, and 21, blood samples were taken from the tail veins of each animal that had fasted overnight. A glucometer was used to measure blood glucose levels [38,39].

Conclusions
In summary, we have designed and developed a few compounds that can inhibit DPP-IV. The 6-methyl-2-oxo-4-substituted-N'-((E)substituted-methylidene)-1,2,3,4-tetrahydropyrimidine-5-carbohydrazide derivatives (1a-5d) were synthesized and characterized by spectral analysis. From the very first screening to the application of the Lipinski rule, the Veber rule, the ADMET calculations, and the drug-likeness attributes, none of these molecules had broken the majority of the drug-likeness rules. All the designed derivatives exhibited potent interactions and optimized binding affinity with the target enzyme. Therefore, wet lab synthesis and biological assessment were performed on all the proposed derivatives. In vitro DPP-IV inhibitory effects of the synthesized compounds were promising. The antihyperglycemic activity of the developed compounds in fasting blood glucose in animal models demonstrates that they have excellent hypoglycemic potential. As the developed molecules demonstrated potent in vitro and in vivo activities in the present study, we have concluded that this nucleus can be explored further and treated as a lead nucleus for developing novel antidiabetic agents. SAR analysis of all the derivatives revealed important insights into the essential structural requirements for effective DPP-IV inhibition. Phenyl ring substitutions are necessary for the binding at the two sites of the DPP IV enzyme. Hence, the introduction of a polar substituent is essential for the activity. In addition, a significant increase in activity was observed due to certain structural characteristics of the synthesized compounds. Different structural features, such as electron-withdrawing groups, for example -Cl and -F, facilitate the binding of the synthesized compounds to the active sites of DPP-IV. In contrast, electron-donating groups, such as -OH, -CH 3 , and -OCH 3 , increase the electron density of the derivatives and increase their lipophilicity, thus enhancing the activity of the synthesized compounds. By studying the structural requirements necessary for their activity, researchers can thus develop more carbohydrazide derivatives as potential DPP-IV inhibitors.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28010149/s1, Table S1: The binding interactions of the molecules with the target enzyme; Table S2: