Cytotoxic and Infection-Controlled Investigations of Novel Dihydropyridine Hybrids: An Efficient Synthesis and Molecular-Docking Studies

A sequence of novel 1,4-dihydropyridines (DHP) and their hybrids was developed using a multicomponent strategy under environmentally benign conditions. In addition, computational studies were performed, and the ligand–protein interactions calculated in different bacteria and two fungal strains. Para-hydroxy-linked DHP (5f) showed the best binding energies of 3.591, 3.916, 8.499 and 6.895 kcal/mol against various pathogens used and other substances received a good docking score. The pathogen resistance potential of the synthesized targets against four bacteria and two fungi showed that whole DHP substances exhibit different levels of resistance to each microorganism. Gram-positive bacteria, which are highly sensitive to all molecules, and the MTCC-1884-encoded fungus strongly rejected the studied compounds compared to comparator drugs. In particular, the 5f candidate showed remarkable antimicrobial activity, followed by the substances 5a, 5b, 5j, 5k and 5l. Furthermore, MIC and MBC/MFC properties showed that 5f had a minimum bacterial concentration of 12.5 μg/mL against E. coli and against two fungal pathogens, with its killing activity being effective even at low concentrations. On the other hand, whole motifs were tested for their cytotoxic activity, revealing that the methoxy and hydroxy-linked compounds (5h) showed greater cytotoxic potency, followed by the two hydroxy linked compounds (5d and 5f). Overall, this synthetic approach used represents a prototype for future nature-favored synthesis methods and these biological results serve as a guide for future therapeutic drug research. However, the computer results play an important role in the further development of biological experiments.


Introduction
The selection of a key chemical compound for synthesis is important because these substances must have shown therapeutic efficacy in previous studies. Numerous investigators have reported and proven that DHP-core-owning compounds exhibit diverse biological characteristics. Also, it is used in medicines such as amlodipine to treat blood pressure and Scheme 1. Single-step synthesis of dihydropyridine analogues. Table 1. Different strategies and yields of compound (5a-c).

Molecular-Docking Studies
The interaction at the molecular level between the ligands and protein receptors was determined using molecular docking. The selected ligands were docked to the binding region of the germs Bacillus subtilis, Staphylococcus aureus, ATCC 8739 code bacteria, Proteus vulgaris, MTCC 1881 and Aspergillus flavus using Glide (Standard Precision Mode) Scheme 1. Single-step synthesis of dihydropyridine analogues. Table 1. Different strategies and yields of compound (5a-c).
In the same solvent medium, we changed the catalyst and continued the reactions, resulting in a trace yield of compounds with values of 11% (5a), 19% (5b), and 15% (5c) in the case of indium chloride as a catalyst (Entry 3, Table 1), while the product 5a has a 32 percent yield in tin chloride promoter media, 5b has a 25% yield, and the percentage of 5c is about 21 (Entry 4, Table 1). Apart from a variety of acidic montmorillonite K-10 acid clay was considered a promoters for the production of 5a-c units using a mixed-solvent concept. The clay initiator, the 2:1 miscible ethanol-water solvent combination and all of the reactants of 5a were heated in an autoclave under argon conditions to yield the target 5a in an amount of 86%. When the reactions of 5b and 5c were successfully carried out using the same strategy, the yields of these targets were 83 percent and 88 percent, showing a high productivity (Entry 5, Table 1). Thus, with this environmentally benign reaction strategy, the products (5(d-l)) were prepared in impressive yields. The interaction at the molecular level between the ligands and protein receptors was determined using molecular docking. The selected ligands were docked to the binding region of the germs Bacillus subtilis, Staphylococcus aureus, ATCC 8739 code bacteria, Proteus vulgaris, MTCC 1881 and Aspergillus flavus using Glide (Standard Precision Mode) in the Glide module [27]. The exact coordinates of the active sites of all protein receptors were determined using the Schrödinger grid generation method. To allow more accurate Pharmaceuticals 2023, 16, 1159 4 of 29 predictions for the ligand positions, using several sets of specifications, the receptors characteristics and shape were taken into consideration in a grid. The essential zone of the lattice, which designates the residue as the lattice center, should be covered by it [28][29][30][31][32]. Additional default settings were used. The optimal docking conformation of each ligand with the lowest docking score was considered.

Binding Poses and Interaction Profiling
The best docking scores for bond conformations were determined after molecular docking and compared. Furthermore, the default settings of the academic A Schrödinger-Maestro v11.4 suite were used to study molecular interactions at a distance of 4 Ao around the docked ligand (Release 2018-3 from Schrödinger: Maestro, Schrödinger, LLC, 2018, New York, NY, USA). For docked ligands and receptors, intermolecular interactions like cation, hydrophobic and hydrogen-bonding interactions that are not covalent have been identified in discovered negative contacts, positive contacts, glycine interactions and the formation of salt bridges.

Bacillus subtilis
Binding Mode of Best Hit Ligands 5a and 5f with Bacillus subtilis The best projected binding energy for ligand 5a is −3.593 kcal/mol. In the active site of Bacillus subtilis, this molecule and essential amino acids have created numerous, robust connections ( Figure 1). For example, the amino acids Arg205 and Asp212 can interact three times with the ligand 5a (Table 2). This ligand promotes additional receptor contacts (hydrophobic interactions, polar interactions) by building new connections. With −3.591 kcal/mol, ligand 5f has the second-best binding energy. Ligand 5f was reported to have developed some strong interactions with key amino acids in the Bacillus active site ( Figure 2). For example, ligand 5f may interact three times with Ser103, Arg205, Gln270, and Hip271 (Table 2). New interactions can arise as a result of increased contacts between the ligand and receptor (hydrophobic interactions, polar interactions). in the Glide module [27]. The exact coordinates of the active sites of all protein receptors were determined using the Schrödinger grid generation method. To allow more accurate predictions for the ligand positions, using several sets of specifications, the receptors characteristics and shape were taken into consideration in a grid. The essential zone of the lattice, which designates the residue as the lattice center, should be covered by it. [28][29][30][31][32] Additional default settings were used. The optimal docking conformation of each ligand with the lowest docking score was considered.

Binding Poses and Interaction Profiling
The best docking scores for bond conformations were determined after molecular docking and compared. Furthermore, the default settings of the academic A Schrödinger-Maestro v11.4 suite were used to study molecular interactions at a distance of 4 Ao around the docked ligand (Release 2018-3 from Schrödinger: Maestro, Schrödinger, LLC, 2018, New York, NY, USA). For docked ligands and receptors, intermolecular interactions like cation, hydrophobic and hydrogen-bonding interactions that are not covalent have been identified in discovered negative contacts, positive contacts, glycine interactions and the formation of salt bridges.

Bacillus subtilis
Binding Mode of Best Hit Ligands 5a and 5f with Bacillus subtilis The best projected binding energy for ligand 5a is −3.593 kcal/mol. In the active site of Bacillus subtilis, this molecule and essential amino acids have created numerous, robust connections ( Figure 1). For example, the amino acids Arg205 and Asp212 can interact three times with the ligand 5a (Table 2). This ligand promotes additional receptor contacts (hydrophobic interactions, polar interactions) by building new connections. With −3.591 kcal/mol, ligand 5f has the second-best binding energy. Ligand 5f was reported to have developed some strong interactions with key amino acids in the Bacillus active site ( Figure  2). For example, ligand 5f may interact three times with Ser103, Arg205, Gln270, and Hip271 (Table 2). New interactions can arise as a result of increased contacts between the ligand and receptor (hydrophobic interactions, polar interactions).

Staphylococcus aureus
Binding Mode of Best Ligands 5f and 5j with Staphylococcus aureus One of the best ligands, 5f, has a binding energy of −3.916 kcal/mol. The Staphylococcus aureus active site has evolved numerous strong connections between this molecule and important amino acids ( Figure 3). For example, ligand 5f may interact three times with amino acids Ser445, Asn468, and Lys499 (Table 3). This ligand creates novel connections that allow stronger hydrophobic and polar interactions with the receptor. Ligand 5j has a binding energy of −3.919 kcal/mol. This ligand has formed several, strong interactions with key amino acids at the active site of Staphylococcus aureus ( Figure 4). For example, ligand 5j shows a threefold interaction with the amino acid Lys499 ( One of the best ligands, 5f, has a binding energy of −3.916 kcal/mol. The Staphylococcus aureus active site has evolved numerous strong connections between this molecule and important amino acids ( Figure 3). For example, ligand 5f may interact three times with amino acids Ser445, Asn468, and Lys499 (Table 3). This ligand creates novel connections that allow stronger hydrophobic and polar interactions with the receptor. Ligand 5j has a binding energy of −3.919 kcal/mol. This ligand has formed several, strong interactions with key amino acids at the active site of Staphylococcus aureus ( Figure 4). For example, ligand 5j shows a threefold interaction with the amino acid Lys499 (Table 3). By forming new connections, this ligand allows more contacts (hydrophobic interactions, polar interactions) between the receptor Staphylococcus aureus.

Escherichia coli
Binding Mode of Best Ligand 5k and 5l with Escherichia coli With a calculated binding energy of −5.414 kcal/mol, ligand 5k gave the best results by fully occupying the binding site. Some cluster residues, such as Asn247, show strong hydrogen bonding ( Figure 5). The protein-ligand has also become more stable due to additional hydrophobic and polar interactions, as can be seen in Table 4. By fully occupying the binding site, 5l achieved the best result with an estimated binding energy of −5900 kcal/mol. Some residues in clusters, such as Asn210, feature two hydrogen bonds ( Figure 6). Furthermore, as shown in Table 4, additional hydrophobic interactions and polar interactions increased the stability of the protein-ligand complex. With a calculated binding energy of −5.414 kcal/mol, ligand 5k gave the best results by fully occupying the binding site. Some cluster residues, such as Asn247, show strong hydrogen bonding ( Figure 5). The protein-ligand has also become more stable due to additional hydrophobic and polar interactions, as can be seen in Table 4. By fully occupying the binding site, 5l achieved the best result with an estimated binding energy of −5900 kcal/mol. Some residues in clusters, such as Asn210, feature two hydrogen bonds ( Figure  6). Furthermore, as shown in Table 4, additional hydrophobic interactions and polar interactions increased the stability of the protein-ligand complex.   Thr37, Thr153, Thr206, Asn210, Asn247 Thr28, Thr37, Asn210, Asn247 Arg36, Trp32 Asp27 Arg36, Arg151, Arg249 Gly152, Gly209, Trp32, Ile155, Ile156, Met207, Ala208, Phe213, Ala246, Ile250 Thr28, Ser29, Thr37, Asn210, Asn247 Thr37, Thr153, Thr206, Asn210, Asn247 Arg36, --Asp150 Arg36, Arg249 Gly152, Gly209, Figure 5. The molecular interactions and binding modes of best-hit ligand 5k (ball-stick structure) at the binding site of Escherichia coli protein (grey cartoon structure).

Proteus vulgaris
Binding Mode of Best Ligand 5f and 5k with Proteus vulgaris The best ligand 5f has a binding energy of −8.499 kcal/mol. This ligand has formed several tight interactions with critical amino acids at the active site of Proteus vulgaris (Figure 7). For example, three hydrogen bonds were generated with amino acids Gln92, Gly98, and Gln99 by ligand 5f (Table 5). Additional interactions (hydrophobic contacts, polar interactions, etc.) between the receptors are made possible by the shapes of these ligands. The second-best ligand 5k has a binding energy of −8.322 kcal/mol. This molecule has formed several tight interactions with critical amino acids at the Proteus vulgaris active site ( Figure 8). For instance, ligand 5k can interact three times with amino acids Gln92, Tyr97, Gly98, and Gln99 through strong hydrogen bonding (Table 5). This ligand creates new interactions that allow the Proteus vulgaris receptors to make more contacts (hydrophobic interactions and polar interactions). The best ligand 5f has a binding energy of −8.499 kcal/mol. This ligand has formed several tight interactions with critical amino acids at the active site of Proteus vulgaris (Figure 7). For example, three hydrogen bonds were generated with amino acids Gln92, Gly98, and Gln99 by ligand 5f (Table 5). Additional interactions (hydrophobic contacts, polar interactions, etc.) between the receptors are made possible by the shapes of these ligands. The second-best ligand 5k has a binding energy of −8.322 kcal/mol. This molecule has formed several tight interactions with critical amino acids at the Proteus vulgaris active site ( Figure 8). For instance, ligand 5k can interact three times with amino acids Gln92, Tyr97, Gly98, and Gln99 through strong hydrogen bonding (Table 5). This ligand creates new interactions that allow the Proteus vulgaris receptors to make more contacts (hydrophobic interactions and polar interactions).

Aspergillus niger
Binding Mode of Best Ligand 5b and 5f with Aspergillus niger With a binding energy of −6.828 kcal/mol, 5b is the best ligand. At the active site of Aspergillus niger, this ligand has developed several significant interactions with important amino acids (Figure 9). Indeed, ligand 5b can interact with amino acids, such as Asn134 and Asn341 through hydrogen bonding (Table 6). This ligand creates new connections that allow strong interactions with the receptors. The binding energy of the second-best ligand 5f is −6.895 kcal/mol. At the Aspergillus niger active site, this molecule has developed numerous close contacts with crucial amino acids ( Figure 10). Also, ligand 5f can participate in hydrogen bonding with amino acids such as Asn134 and Asn341 (Table 6). With a binding energy of −6.828 kcal/mol, 5b is the best ligand. At the active site of Aspergillus niger, this ligand has developed several significant interactions with important amino acids ( Figure 9). Indeed, ligand 5b can interact with amino acids, such as Asn134 and Asn341 through hydrogen bonding (Table 6). This ligand creates new connections that allow strong interactions with the receptors. The binding energy of the second-best ligand 5f is −6.895 kcal/mol. At the Aspergillus niger active site, this molecule has developed numerous close contacts with crucial amino acids ( Figure 10). Also, ligand 5f can participate in hydrogen bonding with amino acids such as Asn134 and Asn341 (Table 6).   --------

Aspergillus flavus
Binding Mode of Best Ligand 5f and 5h with Aspergillus flavus The binding energy of the best ligand 5f is −5.991 kcal/mol. At the Aspergillus flavus active site, this molecule, which contains an important amino acid, has created some powerful connections (Figure 11). For example, ligand 5f can form three hydrogen bonds with amino acids Arg197, Gly227, and Asp296 (Table 7). This ligand allows additional contacts (polar and hydrophobic) between the receptors. The ideal ligand is 5h, with a binding energy of −5.980 kcal/mol. In the active site of Aspergillus flavus, there are several strong interactions between ligand and critical amino acids. (Figure 12). For example, the amino acids Gly227 and Asp296 could form three separate hydrogen bonds with the ligand 5h ( Table 7). The unique connections this ligand makes allow for stronger hydrophobic and polar interactions with the receptor. The binding energy of the best ligand 5f is −5.991 kcal/mol. At the Aspergillus flavus active site, this molecule, which contains an important amino acid, has created some powerful connections (Figure 11). For example, ligand 5f can form three hydrogen bonds with amino acids Arg197, Gly227, and Asp296 (Table 7). This ligand allows additional contacts (polar and hydrophobic) between the receptors. The ideal ligand is 5h, with a binding energy of −5.980 kcal/mol. In the active site of Aspergillus flavus, there are several strong interactions between ligand and critical amino acids. (Figure 12). For example, the amino acids Gly227 and Asp296 could form three separate hydrogen bonds with the ligand 5h ( Table 7). The unique connections this ligand makes allow for stronger hydrophobic and polar interactions with the receptor. Figure 11. The molecular interactions and binding modes of best-hit ligand 5f (ball-stick structure) at the binding site of Aspergillus flavus protein (red cartoon structure). Figure 11. The molecular interactions and binding modes of best-hit ligand 5f (ball-stick structure) at the binding site of Aspergillus flavus protein (red cartoon structure). Arg197, Arg291 Gly227, Gly228, Gly229 Arg197, Arg291, Arg295 Gly227, Gly228, Gly229 Figure 11. The molecular interactions and binding modes of best-hit ligand 5f (ball-stick structure) at the binding site of Aspergillus flavus protein (red cartoon structure).

Overall Docking Results and Discussion
The computational molecular-docking analysis of the targets revealed that the entire substances showed numerous interactions in binding energy regions of all pathogens (Figures 13-15). Overall, 5f targets show strong protein-ligand interactions in all docking studies except the machine-related Escherichia coli studies. The binding potential of this substance was 3.591 kcal/mol for Bacillus subtilis, and against Staphylococcus aureus, the value was 3.916 kcal/mol, 8.499 kcal/mol for Proteus vulgaris and 6.895 kcal/mol in the case against Aspergillus niger (Figure 13 and 14). Fascinatingly, in computer experiments with the pathogen Escherichia coli, the docking score had the highest value of all at 5.900 kcal/mol, which corresponds to 5l ( Figure 15). Furthermore, synthesized compound 5k showed a possible protein-ligand interaction with a binding energy level of 5.414 kcal/mol, and since it has the second-best dock value, it is of importance. In fact, 5f noted the most favorable binding energy value. Additionally, the remaining compounds showed a range of protein-ligand docking scores in the bacterial and fungal strains tested.

Overall Docking Results and Discussion
The computational molecular-docking analysis of the targets revealed that the entire substances showed numerous interactions in binding energy regions of all pathogens (Figures 13-15). Overall, 5f targets show strong protein-ligand interactions in all docking studies except the machine-related Escherichia coli studies. The binding potential of this substance was 3.591 kcal/mol for Bacillus subtilis, and against Staphylococcus aureus, the value was 3.916 kcal/mol, 8.499 kcal/mol for Proteus vulgaris and 6.895 kcal/mol in the case against Aspergillus niger (Figure 13 and 14). Fascinatingly, in computer experiments with the pathogen Escherichia coli, the docking score had the highest value of all at 5.900 kcal/mol, which corresponds to 5l ( Figure 15). Furthermore, synthesized compound 5k showed a possible protein-ligand interaction with a binding energy level of 5.414 kcal/mol, and since it has the second-best dock value, it is of importance. In fact, 5f noted the most favorable binding energy value. Additionally, the remaining compounds showed a range of protein-ligand docking scores in the bacterial and fungal strains tested.

Antimicrobial Discussion
The compounds and their antimicrobial effectiveness against various bacterial and fungal strains were measured using established methods. Ciprofloxacin is a drug used as a standard for bacterial resistance properties and the antifungal potential of the target molecules was compared to Ketoconazole drugs. To evaluate the biological activity of compounds, the bacteria ATCC 6633 (Bacillus subtilis), ATCC 8739 (Escherichia coli), ATCC 19,433 (Staphylococcus aureus), ATCC 29,213 (Proteus vulgaris) and the fungal MTCC 1881 (Aspergillus niger), MTCC-1884 (Aspergillus flavus) were used in this study. The results of the tested substances are listed in Table 8. From the data, dihydropyridine 5f exposed higher levels of resistance to all four bacterial and two fungal strains than other compounds compared to the reference medicine. Its potential value is 40.12 mm in diameter at 200 µg/mL against B. subtilis., for the ATCC 19,433 pathogen, a resistance area of 42.14 mm, 36.19 mm against A. niger and an inhibition zone of 40.14 mm in the case of the A. flavus fungus. Moreover, compounds 5a, 5b, 5j, 5k and 5l then showed good activity against all pathogens. Although the whole tested motifs showed different resistance energies to all microbes used, the bacterial strains ATCC 6633 and ATCC 19,433 are more strongly attacked by entire tested substances than the other two bacteria. Also, in between

Antimicrobial Discussion
The compounds and their antimicrobial effectiveness against various bacterial and fungal strains were measured using established methods. Ciprofloxacin is a drug used as a standard for bacterial resistance properties and the antifungal potential of the target molecules was compared to Ketoconazole drugs. To evaluate the biological activity of compounds, the bacteria ATCC 6633 (Bacillus subtilis), ATCC 8739 (Escherichia coli), ATCC 19,433 (Staphylococcus aureus), ATCC 29,213 (Proteus vulgaris) and the fungal MTCC 1881 (Aspergillus niger), MTCC-1884 (Aspergillus flavus) were used in this study. The results of the tested substances are listed in Table 8. From the data, dihydropyridine 5f exposed higher levels of resistance to all four bacterial and two fungal strains than other compounds compared to the reference medicine. Its potential value is 40.12 mm in diameter at 200 µg/mL against B. subtilis., for the ATCC 19,433 pathogen, a resistance area of 42.14 mm, 36.19 mm against A. niger and an inhibition zone of 40.14 mm in the case of the A. flavus fungus. Moreover, compounds 5a, 5b, 5j, 5k and 5l then showed good activity against all pathogens. Although the whole tested motifs showed different resistance energies to all microbes used, the bacterial strains ATCC 6633 and ATCC 19,433 are more strongly attacked by entire tested substances than the other two bacteria. Also, in between fungal pathogens, the MTCC-1884 code strain shows stronger resistance to compounds than the A. flavus strain, which means that the screened compounds showed highly antifungal characteristics against A. flavus. On the other hand, 5c, 5e, and 5h showed moderate potency in resistance to all pathogens, while the other compounds were showed mild potency in killing fungal and bacterial pathogens (Figures 16 and 17). Table 8. The in vitro pathogenic potential of targets 5(a-l).

Compound
No.

Bacterial and Its Resistance Fungal and Its Resistance Gram-Positive
Gram-Negative  Table 9 shows the test results of the smallest inhibitory dose (MIC) and smallest bacteria/fungal dose (MBC/MFC) of the five best compounds, which already showed a more pronounced zone of inhibition than other compounds against all pathogens. Among these, compound 5a has since shown the lowest bacterial concentration (MBC) of 50 µg/mL, which is twice the MI concentration in B. subtilies. In fact, composite 5b gave the lowest bacterial volume (MBC) (100 µg/mL) versus E. coli bacteria. This value corresponds to twice the MIC (50 µg/mL). Substances 5k and 5l had the lowest bacterial concentration characteristics against E. coli (25 and 50 µg/mL), which is twice the MI concentration. Further, it was found that the MB concentrations of 5f and 5l were twice the MI concentration using the S. aureus pathogen. Moreover, 5f had the lowest fungal volume (MFC) of 50 and 100 µg/mL against all fungal pathogens, which was twice the MI concentration. The 100 µg/mL value of 5a and the 25 µg/mL value of 5k were found using the fungus A. flavus. Both of these results are double their MIC. In addition, the MF concentration in 5b in the case of the pathogen A. niger is 2XMIC. ------ Figure 16. Targets' resistance potential towards screened bacteria. Figure 16. Targets' resistance potential towards screened bacteria.  Table 9 shows the test results of the smallest inhibitory dose (MIC) and smallest bacteria/fungal dose (MBC/MFC) of the five best compounds, which already showed a more pronounced zone of inhibition than other compounds against all pathogens. Among these, compound 5a has since shown the lowest bacterial concentration (MBC) of 50 µg/mL, which is twice the MI concentration in B. subtilies. In fact, composite 5b gave the lowest bacterial volume (MBC) (100 µg/mL) versus E. coli bacteria. This value corresponds to twice the MIC (50 µg/mL). Substances 5k and 5l had the lowest bacterial concentration characteristics against E. coli (25 and 50 µg/mL), which is twice the MI concentration. Further, it was found that the MB concentrations of 5f and 5l were twice the MI concentration using the S. aureus pathogen. Moreover, 5f had the lowest fungal volume (MFC) of 50 and 100 µg/mL against all fungal pathogens, which was twice the MI concentration. The 100

The In Vitro Cytotoxic Assay
The cytotoxic potency of the substances 5(a-l) was tested using the MTT assay against the cell lines HepG2, U937 and SKOV3. Etoposide was used as a reference and Table 10 discloses the cytotoxicity results showing that the compounds showed promising cytotoxic activity against the HepG2 cancer cell line than the other two cell lines tested. Among all the test results of substances against the three cell lines, 5h exhibited the most significant efficacy value of 29 µg/mL (HepG2), 36 (U937) and 51 µg/mL (SKOV3) compared to the standard cell line. Also, amalgams 5d, 5f and 5k showed good effectiveness at IC50s; 57, 74, 88 µg/mL, 42, 60, 75 µg/mL and 59, 83, 95 µg/mL against three cell lines. While the ortho-methyl-substituted dihydropyridine (5a), 5g (para-chloro attached DHP) and paracyano linked DHP compound (5l) gave moderate biological potency compared to other motifs. In fact, in the case of the U937 and SKOV3 cell lines, the substance 5c displayed a very weak therapeutic activity with more than 200 µg/mL, whereas the compounds 5e and 5i gave similarly lower activity towards SKOV3 at more than 200 µg/mL. Fortunately, motif 5c resulted in an identifiable biological potency against HepG2 than the other two cell lines. Moreover, the other evaluated motifs showed some cytotoxicity related to the referee motif. The values of 86, 123, 160 µg/mL belong to 5b and for 5j to the IC 50 values 84, 117, 129, respectively. The structure-activity relationships are crucial for the relationship between the biologically active results and the targets tested. In this contradiction, most of the potency values of 5h (Table 10) associated with other motifs could be due to the presence of a methoxy group as a substituted compound at the fourth position of benzene ( Figure 18). Also, the amalgams 5d and 5f displayed the most cytotoxic results next to compound 5h. The existence of a hydroxy group on the ring of benzene may be the cause of this. Moreover, the para-hydroxy benzene ring bearing the compound 5f shows the best potency compared to motif 5d bound to the ortho-hydroxybenzene ring. From the above statements, it can be concluded that the compounds linked with methoxy and hydroxy groups (5h) showed a greater therapeutic efficacy than the compounds linked with two hydroxy groups (5d and 5f).

Way of Preparation for the Target Compounds 5(a-l)
Malononitrile (4, 1 mmol) and methylbenzaldehyde (3a, 1 mmol) were added to an autoclave glassware that already had 7 mL of an ethanol-water solvent, and the mixture was stirred under environmental conditions for 30 min. After adding the aminomethoxyphenol (1 1mmol), dimethyl acetylenedicarboxylate (2, 1 mmol), and catalyst K-10, the process was conducted for 5 h at 70 °C. The target was obtained by means of crude filtration and extraction of crude with dichloromethane solvent followed by concentration. Utilizing 2-propanol, the color solid 5a was recrystallized. This technique achieved 5(b-l) targets.

Way of Preparation for the Target Compounds 5(a-l)
Malononitrile (4, 1 mmol) and methylbenzaldehyde (3a, 1 mmol) were added to an autoclave glassware that already had 7 mL of an ethanol-water solvent, and the mixture was stirred under environmental conditions for 30 min. After adding the aminomethoxyphenol (1 1mmol), dimethyl acetylenedicarboxylate (2, 1 mmol), and catalyst K-10, the process was conducted for 5 h at 70 • C. The target was obtained by means of crude filtration and extraction of crude with dichloromethane solvent followed by concentration. Utilizing 2-propanol, the color solid 5a was recrystallized. This technique achieved 5(b-l) targets.

Antibacterial Screening Test
Two different groups of Gram-positive and Gram-negative pathogens mentioned in Section 2.3.1 became accustomed to screening the assay and used the method using a disc of filter paper [44]. The fundamental medium for evaluating bacteria was nutrient agar (NA). Only DMSO reagent was used for the disc controller. The agar Petri dishes were seeded with 0.5 mL of previously organized microbial suspension at 1 × 10 7 cells mL -1 . The dishes were incubated for 12 h at 37 • C for microbes and 24 h at 5 • C for diffusion time. The diameter of the visible area of inhibition was measured on a millimeter scale as a measure of the inhibition effect.

Antifungal Screening
The test was performed using the poisoned diet procedure [45] with few adjustments and estimated for two plant pathogens and fungi, namely MTCC 1884 and MTCC 1881. The fungi were tested on a base medium called potato Dextrose agar (PDA). The further procedure followed the method cited in [26].

MIC and MBC/MFC Effectiveness
The broth-dilution method was used for MIC screening [46]. In this method, freshly prepared food broth was used as the diluent. The earlier microorganism and fungal cultures (one day old) were mixed 100-fold in dietary broth (100 µL cultures of bacterial in 10 mL of NB). The experimental tubes containing the provided bacterial and fungal cultures received increasing amounts of the test samples (40,20,10,5,205, and 1.25 µL of stock solution holds 100, 50, 25, 12.5 and 6.25 µg of the test motifs). In addition, these portions underwent cultivation for fungus and germs, respectively, at 28 • C for 48 h and 37 • C for 1 day. The solvent control and discernible turbidity of each part were checked using an NB regulator, and no test-sample controls were tested simultaneously. The MIC was measured as the minimum concentration that prevented visible progression of the organisms tested, using each test tube set in the MIC determination to screen for MBC [47] and MFC [48]. From the tubes showing no growth, a loop of the broth was removed and inoculated by streaking with sterile nutrient broth against microbes and PDA against fungi. The portions hatched at 37 • C for one day after being injected with bacteria and inoculated with fungi, followed by culturing at 28 • C for 48 h. The MBC or MFC has been restricted as the lowest concentration of the test samples intended to kill microbes or fungi.

Cell Lines and Cell Culture
The human histiocytic carcinoma U937, SKOV3 (human ovarian carcinoma), HepG2 (human hepatocellular carcinoma) and B16F10 (mouse melanoma) through the NCCS (National Centre for Cellular Sciences), India-Pune, cell lines were gathered. The appropriate selection media were used to cultivate the cells (U937 (1640-RPMI), MEM (HepG2), and DMEM (SKOV3, B16F10)), and 10% FBS (fetal bovine serum) that has been heat inactivated was added. A total of 100 Units/mL of penicillin, 2 mM of glutamine, 1 mM of sodium bicarbonate, and 100 g/mL of streptomycin were all used. At 37 degrees Celsius, in a humidified atmosphere with 5% CO 2 , all cell lines were cultivated.

Concentrations Tested
Before the commencement of every study, fresh stock solutions of each chemical and a positive control were made. Each chemical was produced as a stock solution at an 8 mg/mL concentration in 100% DMSO. By successive dilutions with the suitable medium for cultivation, functioning solutions of every experiment chemical were created.
All substances underwent testing at the necessary concentrations ranging from 10 to 200 µg/mL.

Cytotoxicity
Following the Mossman protocol [49], the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] was used to quantify the cytotoxicity. In 96-well plates, cells (2x104) were cultivated into every well with 100 µL of medium. An exact 100 µL of medium with various experimental doses (10 g to 200 µg/mL) was injected to the cell culture after incubation for an overnight period (at 37 • C and in an atmosphere that was humidified with 5% carbon dioxide). This equates to between 2 and 40 µg per 200 µL test quantity. After one day, the viability of cells was determined via incorporating 10 µL of MTT (5 mg/mL), each well and continuing the incubation for an additional 3 h at 37 • C. The formazan blue that had developed in the tissues disappeared in 100 µL of DMSO after the medium had been removed. At 570 nm, a spectrophotometer (MAX Plus Spectra; SOFTmax PRO-5.4 supported molecular device) was used to measure the brightness of color production. The proportion of viable cells that were inhibited was calculated in comparison to control (no test substance) readings. Mean values were evaluated using regression analysis to find the finest straight-line agreement after collecting data from three different experiments. Using appropriate regression algorithms, IC50 concentrations (resulting in a 50% reduction in cell count compared to control measurements) were determined.

Conclusions
An environmental favor approach was established for the synthesis of novel 1,4dihydropyridine substances. There was good yield in the formation of whole compounds. Also, docking studies were performed for targets with four bacterial and two fungal pathogens, in which compound 5f showed a higher binding energy versus four microbes. In fact, other dihydropyridines and their docking scores against each pathogen have been widely discussed. Besides, the inhibitory activity of the compound against microbes was examined. Among other things, compound 5f was responsible for highly antimicrobial activity. Moreover, MIC and MBC/MFC data showed that 5f had a minimal bacterial concentration versus the E. coli bacterium. Also, whole motifs were tested for their cytotoxic activity. It was found that the methoxy-and hydroxy-linked compounds (5h) showed greater cytotoxic potency, followed by the two hydroxy-linked compounds (5d and 5f). A description of the biological investigation results are as follows: DHP analogs serve as fertile ground for the next generation of innovative antibacterial/cytotoxic drugs.