Green synthesis, density functional theory (DFT), molecular docking, molecular dynamics simulation and biological activity of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives as PLA2 and proteinase K inhibitors

2.1.1 General procedure for the synthesis of tetrahydropyrimidine-5-carbonitrile derivatives (4a-h)

In a 50-mL beaker, a mixture of aldehydes (1a-h) (1 mmol), ethylcynoacetate (2) (1 mmol), urea/thiourea (3) (1.2 mmol), and 20 mg of TiO₂ was stirred after adding absolute ethanol (10 mL) (Yasmin et al., 2021). We added ethanol (98 %) (10 mL) to the reaction mixture and exposed it to a visible light source (100 mW cm-2). Thin-layer chromatography (TLC), using toluene, ethyl acetate, and formic acid in a 5:4:1 ratio, continuously monitored the reaction. At the end of the process, we added ethyl acetate and 20 % aqueous NaOH, and used centrifugation to extract TiO₂ (Yasmin et al., 2021). A solid product was obtained after being evaporated and purified by recrystallization with ethanol. The synthesized compounds were further characterized by FT-IR, ¹H NMR, and ¹³C NMR and are presented below.

2.1.2 2,4-dioxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydro-5-pyrimidinecarbonitrile(4a)

White solid; Yield: 91 %; M.P 236–238 ⁰C; FT-IR (cm⁻¹, ATR); 3445, 3329 (2 N-H), 2202 (CN), 1694 (C = O), 1449 (C = C); ¹H NMR (DMSO–d6, 400 MHz): 8.33 (s, 1H, NH), 7.51 (s, 2H, Ar-H), 5.40 (s, 1H, NH), 3.83–3.34(s, 9H, 3x OCH₃); ¹³C NMR (DMSO–d6, 100 MHz): 162.51, 160.00, 155.43, 153.33, 142.50, 126.93, 116.53, 109.30, 101.24, 62.74, 60.81, 56.51.

2.1.3 1,2,3,4-tetrahydro-2,4-dioxo-6-p-tolylprimidine-5-carbonitrile (4b)

White solid; Yield: 89 %; M.P 223–224 ⁰C FT-IR (cm⁻¹, ATR); 3431, 3280 (2 N-H), 2227(CN), 1668 (C = O), 1449 (C = C); ¹H NMR (DMSO–d6, 400 MHz): 8.4 (s, 2H, 2xNH), 8.04–6.75 (m, 4H, Ar −H), 2.48(s, 3H, CH₃); ¹³C NMR (DMSO–d6, 100 MHz): 160.21, 155.54, 144.99, 131.53, 130.53, 126.41, 116.37, 101.70, 62.83, 21.91.

2.1.4 6-(furan-2-yl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4c)

Light brown solid; Yield: 88 %; M.P 196–198 ⁰C FT-IR (cm⁻¹, ATR); 3369, 3166(2 N-H), 2218(CN), 1710(C = O), 1410(C = C); ¹H NMR (DMSO–d6, 400 MHz): 8.21(s, 1H, NH), 8.13(s, 1H, Ar –H), 7.53–7.52 (d, 1H, J = 3.6, Ar −H), 6.87–6.86(t, 1H, J = 3.4, Ar −H), 5.42(s, 1H, NH); ¹³C NMR (DMSO–d6, 100 MHz): 162.68, 160.07, 150.57, 148.64, 139.59, 125.15, 114.80, 97.39, 97.36, 62.61.

2.1.5 6-[5-(hydroxylmethyl)furan-2-yl]-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4d)

White solid; 85 %; M.P 204–206 ⁰C FT-IR (cm⁻¹, ATR); 3424 (OH), 3290 (2 N-H), 2226(CN), 1651 (C = O), 1443 (C = C); ¹H NMR (DMSO–d6, 400 MHz): 8.08 (s, 1H, NH), 7.49 (s, 1H, Ar-H), 6.71(s, 1H, Ar-H), 5.41(s, 1H, NH) 4.53(s, 1H, CH₂OH); ¹³C NMR (DMSO–d6, 100 MHz): 178.45, 163.86, 160.05, 152.19, 147.84, 139.44, 111.79, 96.21, 62.54, 56.57.

2.1.6 1,2,3,4-tetrahydro-6-(3,4,5-trimethoxyphenyl)-4-oxo-2-thioxopyrimidine-5-carbonitrile (4e)

Yellow solid; 87 %; 242–243 ⁰C FT-IR (cm⁻¹, ATR); 3351, 3140 (2 N-H), 2220 (CN),1719 (C = O), 1401 (C = C); ¹H NMR (DMSO–d6, 400 MHz): 9.88(s, 1H, NH), 7.25 (s, 2H, Ar-H), 4.31(s, 1H, NH) 3.86–3.36(s, 9H, 3x OCH₃); ¹³C NMR (DMSO–d6, 100 MHz): 192.36, 184.30,162.52, 153.79, 143.28, 132.11, 107.19, 101.27, 62.75, 60.68, 56.52.

2.1.7 1,2,3,4-tetrahydro-4-oxo-2-thioxo-6-p-tolylpyrimidine-5-carbonitrile (4f)

Brown solid; 84 %; 228–229 ⁰C; FT-IR (cm⁻¹, ATR); 3363, 3260 (2 N-H), 2211 (CN), 1719 (C = O), 1401 (C = C); ¹H NMR (DMSO–d6, 400 MHz): 8.33(s, 1H, NH), 7.96–7.94 (d, 1H, J = 8.12, Ar −H), 7.41–7.39(d, 1H, J = 8.04, Ar −H),7.18(s, 1H, Ar −H), 7.07–7.05(d, 1H, J = 8.24, Ar −H), 4.29(s, 1H, NH), 2.39(s, 3H, CH₃); ¹³C NMR (DMSO–d6, 100 MHz): 169.02, 162.92, 149.66, 146.66, 138.29, 132.11, 128.09, 115.31, 74.18. 21.10.

2.1.8 6-(furan-2-yl)-1,2,3,4-tetrahydro-4-oxo-2-thioxopyrimidine-5-carbonitrile (4g)

Yellow solid; 80 %; M.P 212–214 ⁰C; FT-IR (cm⁻¹, ATR); 3130, 3041 (2 N-H), 2217 (CN), 1709 (C = O), 1529 (C = C); ¹H NMR (DMSO–d6, 400 MHz): 8.21(s, 1H, NH), 8.13 (s, 1H, Ar-H), 7.52–7.51 (d, 1H, J = 3.3, Ar −H), 6.87 (s, 1H, Ar −H), 4.29(s, 1H, NH); ¹³C NMR (DMSO–d6, 100 MHz): 162.68, 150.57, 148.65, 139.59, 125.13, 115.79, 114.79, 97.37, 62.60.

2.1.9 1,2,3,4-tetrahydro-6-(5-(hydroxymethyl)furan-2-yl)-4-oxo-2-thioxopyrimidine-5-carbonitrile (4h)

Yellow solid; 79 %; M.P 221–223 ⁰C FT-IR (cm⁻¹, ATR); 3410 (OH), 3319, 3215 (2 N-H), 2211(CN), 1611 (C = O), 1665 (C = C); ¹H NMR (DMSO–d6, 400 MHz): 8.21 (s, 1H, NH), 6.83 (s, 1H, Ar-H), 6.46–6.38 (d, 1H, J = 29.9, Ar −H),5.44 (s, 1H, NH), 4.47(s, 1H, CH₂OH); ¹³C NMR (DMSO–d6, 100 MHz): 165.71, 158.11, 150.48, 149.32, 132.74, 128.74, 116.85, 115.11, 109.65, 56.21.

2.2.1 Density functional theory computations

The Orca 5.0.3 program was used to compute density functional theory (DFT) (Neese, 2012; Neese, 2018). Compounds 4a–4 h were optimized using the Lee-Yang-Parr correlation functional (B3LYP) (Becke, 1988; Lee et al., 1988). Chemcraft, a graphical software for quantum chemistry computations (https://www.chemcraftprog.com), visualized the output file of the DFT-optimized structure. We utilized the Avogadro program (Hanwell et al., 2012) to illustrate the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). Additionally, we calculated global reactivity characteristics like electronegativity using the previously reported HOMO and LUMO energies (Azam et al., 2018).

2.2.2 Molecular docking

2.3.1 Phospholipase A2 (PLA2) inhibitory activity

The PLA2 assay was carried out as previously discussed by De Araujo and Radvany (de Araújo and Radvanyi, 1987). Phospholipase A2 is on the open market, purchased from Sigma Aldrich in the United States (P6534). The substrate contained 100 mM NaCl, 10 mM CaCl₂, red phenol as a colorimetric indicator of 0.055 mM, 3.5 mM lecithin, 3 mM NaTDC, and 100 mM NaCl. Phosphate buffer assisted in raising the pH of the reaction to 7.6. In 10 % acetonitrile, the sPLA2 protein was solubilized at concentrations ranging from 0.01 to 0.08 g/L. These PLA2 solutions were incubated at room temperature for 20 min in 10 L holding 10 g of each chemical. After adding 1 mL of PLA2 substrate, researchers observed the hydrolysis kinetics for 5 min by measuring the optical density at 558 nm. Comparing the outcomes to a control experiment (one without the compound) allowed researchers to assess the proportion of inhibition.

The PLA2 activity assay was conducted in accordance with De Arajo and Radvany bm's earlier discussion (de Araújo and Radvanyi, 1987). Acquired phospholipase A2 from Sigma Aldrich in the US on the open market (P6534). The substrate was made up of 100 mM NaCl, 10 mM CaCl₂, 3.5 mM lecithin, 3 mM NaTDC, and red phenol as a colorimetric indicator of 0.055 mM. Phosphate buffer was used to bring the reaction mixture's pH up to 7.6. The sPLA2 protein was soluble in 10 % acetonitrile at a concentration of 0.01 to 0.08 g/L. After adding 1 mL of PLA2 substrate, the optical density at 558 nm was monitored for 5 min to determine the hydrolysis kinetics. Researchers were able to determine the inhibition percentage by comparing the results to a control experiment (one in which the chemical was not used).

2.3.2 Proteinase K inhibitory activity

The proteinase K utilized in this investigation was purchased commercially (P2308, Sigma Aldrich, USA). Hammerstein casein was used as the substrate for proteinase K testing utilizing the Kunitz caseinolytic method (Kunitz, 1946). The same procedures were used to evaluate the activities of proteinase K inhibitors, with the addition of the inhibitor (0.1 mg/mL) to the reaction mixture and a 10-minute pre-incubation at 37 °C. 2 mL of 1 % casein was added after the remaining enzyme activity test, and the mixture was left to stand for 30 min at 37 °C. The reaction was halted by adding 2.5 mL of a 5 % TCA solution. The reaction mixture's absorbance was assessed at 280 nm following centrifugation (12,000 rpm, 15 min). A proteinase K inhibitor unit is the volume of proteinase K inhibitor that reduces one unit of the related enzyme activity. The assay also contained the proper blanks for the enzyme, inhibitor, and substrate.

In this investigation, proteinase K was procured from commercially accessible sources (P2308, Sigma-Aldrich, Saint Louis, MO, USA). In short, the Kunitz caseinolytic approach was used to perform proteinase K assays using Hammerstein casein as the substrate (Kunitz, 1946). The same procedures were used to assess the proteinase K inhibitory activities: the reaction mixture was pre-incubated for 10 min at 37 °C, and the inhibitor (0.1 mg/mL) was added. After the analysis for remaining enzyme activity, 2 ml of 1 % casein were added, and the mixture was left to stand at 37 ◦C for 30 min. The reaction was halted by adding 2.5 mL of a 5 % TCA solution. After centrifuging the reaction mixture for 15 min at 12,000 rpm, the absorbance was measured at 280 nm. A proteinase K inhibitor unit is the quantity of proteinase K inhibitor that inhibits one unit of related enzyme activity. Appropriate blanks for the substrate, inhibitor, and enzyme were run in parallel with the test. As a positive control, a proteinase K inhibitor cocktail from Sigma was employed. Data are expressed as mean ± standard error of the mean (n = number of experiments in triplicate). P < 0.05 is considered to be statistically significant.

Some of the recently reported anti-phospholipase A2 and anti-proteinase K compounds are presented as in Fig. 1.

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Fig. 1

Compounds that have been reported as examples (I-IV) compared with compounds 4d and 4f as lowest and highest antiphospholipase A2, proteinase k activities in present studies.

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3.1.1 Density functional theory (DFT)

The HOMO-LUMO gap and global reactivity descriptors are important parts of understanding how chemicals react and what molecules are made of (Ramírez-Martínez et al., 2023). Density Functional Theory (DFT) gives us a way to study them. The DFT-optimized structures are shown in Fig. 2. The HOMO-LUMO gap is the energy difference between the highest occupied molecular orbital (HOMO) and the lowest occupied molecular orbital (LUMO) in a molecule. The HOMO-LUMO gap closely relates to the electronic properties of molecules, reflecting the energy required for electron excitation or transfer. A smaller gap indicates easier electron transfer and greater reactivity. The synthesized compounds' HOMO-LUMO gap fell within the range of 3.82–4.61 eV. Interestingly, thione derivatives (4e–4 h) exhibited a lower energy gap in comparison to keto derivatives. Regarding electrophilicity, the index value was at its lowest for the compound 4a, with 4b, 4d, 4e, 4f, 4c, 4 h, and 4 g following in increasing order. However, minimum chemical potential was noted with 4 g, while 4 a displayed the maximum value. Similarly, other DFT-based HOMO and LUMO were also used to compute global reactivity characteristics such as hardness, softness, and electronegativity, as presented in Table 1. The disparities in these values can be attributed to an alteration in chemical structures through diverse substitutions. Red is considered statistically significant.

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Fig. 2

Optimized geometries of the compounds 4a-4 h.

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Fig. 3 depicts the visualization of calculated HOMO/LUMO, which indicates the delocalization of electrons between HOMO and LUMO, which is regarded as critical for determining the possibility of molecular reactivity. Consequently, they are thought to be the basic orbitals that help determine how a molecule interacts with other species, such as the interaction between a drug and its macromolecular target (Azam et al., 2018; Ahmad et al., 2023). The HOMO and LUMO orbitals on the molecules are staggered with respect to one another, where cyan represents negative surfaces and purple signifies positive surfaces (Fig. 3). The thione group of pyrimidines was mostly covered by HOMO in compounds 4e–4 h, whereas HOMO engulfed the cyano group in compounds bearing the keto group (4a–4d). Compounds 4f and 4 g were dominated by LUMO, sparing the thione group.

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Fig. 3

Compounds 4a-4 h displaying HOMO and LUMO.

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3.1.2 ADMET analysis of the ligands

As evident from supplementary table 1, all the compounds are following Lipinsky’s rule of five and show high GI absorption with moderate to high solubility. None of them showed potential to cross the blood–brain barrier and bind as a Pgp substrate (except compound 4f). These properties make them suitable for lead-like candidates. The toxicity prediction from supplementary table 2 shows that all the compounds are non-mutagenic and non-carcinogenic. Furthermore, they showed low honey bee toxicity, with the exception of compound 4e.

3.1.3 Structural similarity of phospholipase A2 and proteinase K

The electrostatic analysis of target proteins Proteinase K and Phospholipase A2 was done using Adaptive Poisson-Boltzmann Solver (APBS) plugin in PyMol. The blue color in the color bar shows a positive charge, and the red color indicates a negative charge. As evident from supplementary Fig. 2, the ligand-binding cavities in both proteins were found to be negatively charged. In the overall structural alignment (supplementary Fig. 3) of phospholipase A2 on proteinase K, a significant amount of good alignment was found in terms of lower RMSD, as marked by the blue color, while the region in red shows a higher RMSD in alignment, and the region in grey shows the residues not used during alignment. As evident from the above data, both target proteins share negatively charged ligand binding pockets and align well over the binding pocket residues. This might be the probable reason for both target proteins to bind similar ligands.

3.1.4 Molecular docking

Before moving on to the docking, the structural integrity of the target proteins was assessed using the Ramachandran plot, where both target proteins were shown to have 100 % residues in the allowed region with no outliers, as shown in supplementary Fig. 1. The primary objective of this study was to elucidate the binding mode and energetics of compounds 4a–4 h with the target proteins phospholipase A2 (PDB ID: 1DB4) and proteinase K (PDB ID: 2PWB) using molecular docking simulations. First of all, the native co-crystallized ligands 8-indole ([3-(1-benzyl-3-carbamoylmethyl-2-methyl-1H-indol-5-yloxy)-propyl-]-phosphonic acid) and coumarin were redocked to their respective proteins in order to validate the docking protocol. The crystal structures of the docked conformations were well aligned.

The docking scores in kcal/mol are shown in Supplementary table 3. Our results revealed that ligands 4a–4 h interact with the specific binding sites on both proteins, phospholipase A2 and proteinase K, through a network of hydrogen bonds and hydrophobic interactions. The intermolecular interactions of the selected compounds have been presented in Figs. 4a and 4b. In general, the interactions are in good agreement with known binding modes of native ligands, bolstering the reliability of docking predictions. In the phospholipase A2 binding site, Gly29, Gly31, His47, Asp48, and Asp91 were the most important residues for polar interactions. However, residues such as Leu2, Phe5, Ala18, Tyr21, and Gly22 were contributors to hydrophobic contacts. In proteinase K, hydrogen bonds were formed with His69, Leu133, Gly134, Ala158, and Ser224, whereas hydrophobic interactions were afforded by Leu96, Gly100, Ala158, Asn161, and Tyr169. However, His69, Leu133, and Gly134 were involved in the polar and non-polar interactions. Overall, these results give us useful information about the structure of the ligand–protein complex. They help us understand how the molecules interact and what their possible functional effects might be.

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Fig. 4a

Molecular docking results showing 3D docked pose and two-dimensional binding interactions of phospholipase A2 (PDBID: 1DB4) with compound 4a (A), compound 4b (B), and 8-indole (C).

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Fig. 4b

Molecular docking results showing 3D docked pose and two-dimensional binding interactions of proteinase K (PDBID: 2PWB) with compound 4d (D), compound 4e (E), and coumarin (F).

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3.1.5 Molecular dynamics (MD) simulation

Since MD simulation mimics the actual biological phenomenon of protein–ligand interaction in a biological environment, this study is thought to be more relevant than docking (Durrant and McCammon, 2011; Singh et al., 2023). In order to provide information regarding the dynamic and thermodynamic properties and structure of molecules, MD simulations are now regarded as an integral part of the modern drug development process (Karplus and McCammon, 2002; Parveen et al., 2023; Hospital et al., 2015). In all-atom MD simulation, the modeled system comprises interacting particles placed in a giant simulation box, and Newton’s law of motion solves the movements. The simulation trajectories have been analyzed for structure stability using carbon-α atoms. To assess this, the root-mean-square deviation (RMSD) analysis was conducted, which measures the average distance between the superimposed protein atoms (Maiorov and Crippen, 1994). A high RMSD value throughout the simulation suggests larger structure deviations, while a low RMSD value indicates fewer structural changes. Protein-ligand complexes with reduced RMSD values show strong intermolecular contacts and stable docked conformation since large carbon alpha fluctuations in this context lead to the dissociation of ligand-bound receptors (Fusani et al., 2020). The RMSD values for the phospholipase A2-bound complexes of compounds 4a, 4b, and 8-indole (as a positive control) were 2.02 Å, 1.90 Å, and 2.04 Å, in that order (Fig. 5A). It seems that the compounds were effectively attached to the phospholipase A2 binding site, and according to the VMD analysis, there was no noticeable change in the binding mode. The minor RMSD jumps in the systems were due to the flexible loops in the enzyme that are naturally present to aid in enzyme catalytic mechanisms. The proteinase K-bound systems, including compounds 4d, 4e, and coumarin, displayed average RMSDs of 1.31 Å, 1.41 Å, and 1.25 Å, respectively (Fig. 5B).

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Fig. 5

The molecular dynamics simulation trajectories have been analyzed to investigate the stability of the structure showing root-mean square deviation (RMSD) of compounds 4a, 4b and 8-indole in complex with phospholipase A2 (A). RMSD plot of the compounds 4d, 4e and coumarin (B).

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Root mean square fluctuation (RMSF) on carbon-α atoms recognized the local residue level fluctuations. The mean RMSF of the compounds 4a, 4b, and 8-indole, in complex with phospholipase A2, was noted as 1.30 Å, 1.08 Å, and 1.23 Å, respectively (Fig. 6A). It can be see that the figure depicts a high stability of protein residues in the presence of the compound, with only a few noticeable exceptions where there are some jumps in the upper region. Likewise, an excellent RMSF profile was shown by the docked compounds in complex with proteinase K. Compounds 4d, 4e and coumarin demonstrated average RMSF of 0.71 Å, 0.72 Å, and 0.71 Å, respectively (Fig. 6B).

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Fig. 6

The root-mean square fluctuation (RMSF) of compounds 4a, 4b and 8-indole in complex with phospholipase A2 (A). RMSF plot of the compounds 4d, 4e and coumarin (B).

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The radius of gyration (RoG) analysis is an effective method for describing the compactness and relaxed nature of protein 3D structure during simulation time. (Lobanov et al., 2008). The compact nature of the protein is necessary for solid compounds to bind to phospholipase A2 and proteinase K where getting relax confers loose ligand binding. The mean RoG values of compounds 4a (14.64 Å), 4b (14.40 Å), 8-indole (14.59 Å) in complex with phospholipase A2 has been presented in Fig. 7A. A slight expansion in the Phospholipase A structure was observed in complex compound 4a as compared to compound 4b and 8-indole. Fig. 7B displays the RoG plot of compounds 4d (16.96 Å), 4e (16.95 Å), and coumarin (16.97 Å) with Proteinase K where a similar trend in of RoG values were observed in each complex. The low RoG value of complexes determines stable secondary element positioning of the protein in the compound’s presence, exerting less pressure on the compound to dissociate from the binding sites.

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Fig. 7

Radius of gyration analysis of complexes done on carbon-α atoms.

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The number of hydrogen bonds formed between the compounds 4a, 4b and 8-indole with phospholipase A2 and compounds 4d, 4e and coumarin with proteinase K were calculated as shown in Fig. 8A and 8B respectively. Compound 4a and 8-indole maintained only one hydrogen bond while compound 4b formed 1–2 hydrogen bonds with phospholipase A2 over the trajectory. In case of proteinase K, coumarin formed only one hydrogen bond intermittently while compound 4d and 4e formed 2–4 hydrogen bonds over the simulation trajectory indicating more stability with proteinase K as compared to the coumarin (positive control).

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Fig. 8

The number of hydrogen bonds formed between compounds 4a, 4b and 8-indole in complex with phospholipase A2 (A). The number of hydrogen bonds formed between compounds 4d, 4e and coumarin with Proteinase K (B).

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3.1.6 Principal component analysis (PCA) with free energy landscape (FEL)

The MD simulation trajectories were subjected to principal component analysis (PCA) for 4a, 4b, and 8-indole compounds bound to the Phospholipase A2 to analyze the conformational subspace of the complexes in order to comprehend the stable dynamic behavior. Compared to the positive control (8-indole), higher conformational subspace was covered by 4a and 4b over the PC2. Similarly, the trajectories of compounds 4d, 4e and coumarin in complex with proteinase K were also studied. The plot shown in Fig. 9 displays the displacement of atomic fluctuations and movements in the structures as a function of eigenvectors 1 and 2 (PC1 and PC2). The positive values are indicative of corelated movements while the negative values are indicative of anti-corelated movements. Compared to the positive control (coumarin), complexes of proteinase K with compounds 4d and 4e showed the protein minimum subspaces are covered by the clusters, which are well characterized.

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Fig. 9

Principle component analysis (PCA) was performed on 10,000 frames from MD simulations of all complexes. (A) 4a-phospholipase A2 (B) 4b-phospholipase A2, (C) 8-indole-phospholipase A2, (D) 4d-proteinase K, (E) 4e-proteinase K, and (F) coumarin-proteinase K complexes.

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Gibbs free energy landscape was created using the RMSD and RG values obtained from the MD trajectory. Fig. 10 depicts that the FEL was calculated for several compounds, such as 4a, 4b, and 8-indole, which were found to be bound to phospholipase A2 and 4d, 4e, and coumarin, which were found to be bound to proteinase K. The corresponding Gibbs free energies (kJ/mol) of different conformational states were depicted using a color bar, with blue representing the lowest energy state and dark red representing the highest energy state. The FEL analysis revealed that each compound acquired the least energy, corresponding to the most stable conformations. Additionally, enriched energy minima, indicated by the blue color with ample spacing, were detected in all.

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Fig. 10

The Gibbs free energy landscape (FEL) was plotted and color-coded using Root Mean Square Deviation (RMSD) and Radius of Gyration (RoG) values.

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The color bar in the diagram represents the Gibbs free energies (kJ/mol) for the different conformational states. The blue color represents the state with the lowest energy, while the red color represents the state with the highest energy. 4a, 4b and 8-indole were bound to phospholipase A2 while 4d, 4e and coumarin were bound to proteinase K.

3.2.1 Characterization of synthesized compounds

Synthesis of tetrahydropyrimidine-5-carbonitrile derivatives using TiO2 as photocatalyst in alcoholic medium is represented as scheme1.

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Scheme1

Synthesis of tetrahydropyrimidine-5-carbonitrile derivatives under the effect of TiO₂ as photocatalyst in ethanolic medium.

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FT-IR analysis revealed a peak at around 2210 cm⁻¹ corresponding to −C≡N, while stretching vibrations of –NH were detected at 3200, 3300, and 3400 cm⁻¹. Most compounds show distinctive peaks in 1H NMR spectroscopy for two NH groups, one at δ 8 ppm (downfield) and the other at 5 ppm (upfield). However, compound 4b displays both NH singlets at δ 8.4 ppm. Each compound justifies the total number of aromatic protons by their representation as a singlet, doublet, or triplet. Reading the 13CNMR readily reveals additional features. The C = C bond in the pyrimidine ring exhibits two significant peaks: one above δ 160 ppm in the downfield region and the other at δ 62–78 ppm in the most upfield region. Compounds 4a–d have 13CNMR peaks between µ 150–160 ppm because they contain a cyclic C = O group. Compounds 4a–h, on the other hand, have C = S peaks at higher chemical shifts, reaching as high as 192.36 ppm (4e) and 162.68 ppm (4 g). In ¹³CNMR, thiourea compounds show peaks that are farther downfield for the C = C pyrimidine ring. Compounds 4e–h also exhibit C = S peaks that extend further downfield. Furthermore, additional peaks were detected in their original positions, slightly upfield or downfield based on each compound substitution.

3.2.2 Green synthesis of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives

Harvesting of solar light using a lab solar simulator has been used to convert solar energy to chemical energy to synthesize 1, 2, 3, 4-tetrahydropyrimidine-5-carbonitrile derivatives of TiO₂ nanoparticles as photocatalysts. The results confirmed that using TiO₂ (20 mg) as a photocatalyst exhibits excellent performance in terms of yield and reaction time as compared to the conventional thermal method (Alharthi et al., 2023). The yield of all the synthesized compounds is as high as 91 % (compound 4a) in just 60 min. Our previous experiments yielded similar results (Alharthi et al., 2023).

3.3.1 Inhibitory activity of phospholipase A2 (PLA2)

As presented in Table 2, the 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives 4a-h have been found to exhibit phospholipase inhibitory action at concentrations ranging from 0.01 to 0.08 g/L. For compounds 4a-h, PLA2 activity was seen to increase in proportion to concentration increases. Compounds 4a to 4 h showed 37.98 %±2.66 %, 40 ± 2.54 %, 36.70 ± 1.82 %, 36.49 ± 2.67 %, 38.41 ± 3.11 %, 41.54 ± 2.22 %, 32.42 ± 2.39 %, and 38.60 %±2.78 % at the minimum concentration of 0.01 g/l, while the highest activity was shown at 92.67 %±2.98 %, 95.80 %±3.29, 90.45 %±2.27, 90.46 %±3.33, 92.40 %±3.02, 96.55 %±2.89, 88.67 %±3.66, and 92.45 %±2.74 at concentration of 0.05 g/l, as depicted Table 2.

The results are somewhat in agreement with our previous work with the same enzyme, and the PLA2 inhibitory efficiencies of the current molecules are found to be even better than those of other established moieties (El-Sayed et al., 2016; Bakht et al., 1944). Oleonolic acid, as a reference molecule, demonstrated comparatively lower potency (75.58 %±2.48 %) in comparison to compounds at maximum concentration. Given the high levels of PLA2 in many inflammatory disorders (Alafeefy et al., 2015), we will investigate these chemicals for their clinical value in these conditions.

Compounds 4a-h exhibit moderate to high PLA2 inhibitory activities, with IC50 values ranging from 0.023 to 0.031 g/L. Compounds 4b, 4d, 4e, 4f, and 4 h have the highest significant activity; their respective IC50 values are 0.031 g/L (Table 3). We will investigate these compounds in inflammatory conditions because many inflammatory conditions have elevated PLA2 levels (El-Sayed et al., 2016), potentially explaining their clinical significance.

3.3.2 Antiproteinase K activity

Table 4 shows that we screened the synthesized 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives (4-h) against the proteinase K enzyme at concentrations of 0.1 to 0.8 mg/mL. The activity of the proteinase K inhibitor was demonstrated in a dose-dependent fashion. The compounds 4c and 4d showed greater activity than the other compounds (4a, 4b, 4e, 4f, 4 g, and 4 h) at all concentrations. These poor compounds had the least proteinase K inhibitory activity at 0.1 mg/mL, with only 13 % ± 2.53 % for compound 4f and 54 % ± 1.96 % for compound 4e at 0.7 mg/mL. Compounds 4c and 4d demonstrated the highest inhibition (63 % ± 4.66 %) and (63 % ± 5.12 %) at a concentration of 0.7 mg/mL, respectively.

Compound 4d showed maximal proteinase K inhibition (%) at concentrations ranging from 0.2 to 0.8 mg/mL. Compounds 4 h and 4 g reached maximum action at concentrations of 0.1 and 0.4 mg/mL, similar to compound 4d. These results are in comparison to a few prior studies that achieved proteinase K inhibition at concentrations as high as 75 % (Bakht et al., 1944).

A proteinase K inhibitor cocktail (Sigma) was used as the positive control, and the inhibitory potential was found to be close to maximum inhibition (80 ± 2.48). IC50 results for compounds 4a-h for antiproteinase K activities demonstrated a range of 0.042–0.048 mg/L.

The most promising candidates in terms of antiproteinase K activity were reported with IC50 values of 0.047 and 0.048 mg/L, respectively, for compounds 4b and 4c.

3.3.3 Structure activity relationship (SAR) of studied compounds (4a-h)

We synthesized a series of 1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives (4-h) through a one-pot multicomponent reaction, which involved condensation of substituted aldehydes (1a–d), cynoacetate (2), and urea/thiourea (3), using TiO2 as a photocatalyst in ethanolic medium. All the compounds demonstrated better PLA2 (%) activity than proteinase K (%) activity. After establishing the structure–activity relationship (SAR) of all studied compounds, we found that compounds (4b, 4f) with electron-donating substituents exhibit good PLA2 Inhibitory (%) activity compared to those with electron withdrawing substituents (4a, 4e) or those with heterocyclic moieties (4c, 4d, 4 g, 4 h). On the other hand, compounds (4c, 4d) showed comparatively better Proteinase K inhibitory (%) activity with heterocyclic rings as compared to other electron-donating or substituent-withdrawing compounds. In general, all the compounds tested against PLA2 (%) and proteinase K (%) act in a dose-dependent manner.