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INTRODUCTION
Azo compounds, characterized by the -N=N- (azo) linkage, are classified in IUPAC nomenclature as diazenes and have played a key role in organic chemistry since P. Griess's discovery in 1858. These compounds commonly serve as bridges between two or more molecular groups and may even contain multiple azo linkages. Today, synthetic azo compounds are widely used across many industries, including pharmaceuticals, cosmetics, food, dyes, paints, plastics, and more. Aromatic azo compounds are especially significant due to their diverse practical applications: they are utilized as dyes, pigments, indicators, and food additives, and are important in textiles, pharmaceuticals, and cosmetics for therapeutic as well as diagnostic purposes. Additionally, modern research explores their roles as anticancer, antibacterial, antifungal, antioxidant, anti-inflammatory, and antiviral agents, as well as their use as corrosion inhibitors and drug carriers. One notable feature is their good thermal stability and the tunability of their optical properties by changing substituents, which further broadens their utility in science and industry (Razali et al.,, 2023; Kyei et al., 2020).
The rise of resistant pathogens is a major global health threat, reducing the effectiveness of existing antimicrobial drugs. This has sparked urgent research to develop new treatments, as bacterial resistance is inevitable through evolution. Without new drugs, there's a risk of returning to a pre-antibiotic era. Additionally, helminth-related neglected tropical diseases cause significant illness and death worldwide. Limited effective antimicrobials and anthelminthics push the need for ongoing drug discovery. Nitrogen-containing heterocyclic compounds, particularly pyrazole derivatives, are vital in drug development due to their broad biological activity and potential for creating new medicines to combat resistance and neglected diseases .It has been noticed that significant development has taken in the research of Schiff ‘s bases that have shown variety of pharmacological activities, still a threat from various factors including microbes to these organic compounds led to the development of new coordinate compounds containing schiff’s bases with different metals (Joseyphus et al., 2022; Dinnimath et al., 2024). This chelation of Schiff bases with metals has resulted in the development of enhanced potency in their pharmacological activities, mainly anticancer activity and hence this has gained momentum now (Ibrahim et al., 2024; Mishra et al., 2024).
Azo-Schiff bases combine azo groups (-N=N-) with Schiff bases (-CH=N-) to form versatile compounds. These photochromic azo dyes can undergo reversible trans-cis isomerization under light or heat, making them useful in catalysis, antimicrobial applications, photo pharmacology, food, and textiles. Typically, azo groups attach to benzene or naphthalene rings but may also bind to aromatic heterocycles or enolizable aliphatic groups. Schiff bases form through amines reacting with carbonyl compounds, creating imine linkages. Recent syntheses of azo-Schiff base ligands via coupling diazonium salts with imidazole derivatives have expanded their chemical and biological applications, especially enhancing photostability and antimicrobial activity, making them promising for medicinal and industrial uses (Di Martino et al., 2022; Tahir et al., 2021; Abd El Salam et al., 2023).
MATERIALS AND METHODS
Materials
All the chemicals used were of LR grade purchased from Himedia. Initially the compounds were identified preliminarily by TLC by conventional method.
FTIR has been used to identify the functional groups in final products, using Shimadzu IR Affinity using DRS 8000 spectrometer. To predict the hypothetical binding interactions of ligand with the receptor. Molecular docking studies were performed by using glide and Qikprop module of schrodinger software.
Methods
The proposed Azo-Schiff’s base derivatives were synthesized in three steps (Moaeaa et al., 2022: Naeimi et al., 2015; Razali et al., 2023).
Step 1: Synthesis of diazotised Schiff’s bases.
Step 2: Synthesis of Diazotised Schiff base derivatives.
Step 3: Coupling of Diazotised Schiff’s bases with beta naphthol.
Step 1: Synthesis of Schiff’s base
Equimolar concentration of substituted aromatic aldehyde and Orthophenelene diamine dissolved in 20 mL abs. Ethanol and refluxed for 6-7 hours using AlCl3 as a catalyst. This has yielded a coloured product, which was filtered, washed and recrystallized from ethanol and water (1:1).
Step 2: Diazotisation of Schiff’s bases
In an RBF weighed quantities of (1 g) of Schiff’s base was beaker 2 mL of distilled water with 4 mL of Conc.Hcl is taken and added to the RBF and is kept on a magnetic stirrer with temperature between (0-5°C). In a separate beaker 3.2 g of sodium Nitrite is dissolved in 15 mL of distilled water and the temperature of the solution is maintained (0-5°C). Add solution 2 in 1 slowly with vigorous stirring (0-5°C).
Step 3: Coupling of beta naphthol with Diazotised Schiff’s base
In a separate beaker dissolved weighed quantity (6.24 g) of β-naphthol in 40 mL NaOH solution with temperature (0-5°C). Now slowly added solution of Diazotised Schiff’s base to it and refluxed the whole mixture for 30-40 min on the magnetic stirrer and then filtered the product by vacuum filtration and dried. The reaction is monitored by TLC in (3:7) ethyl acetate: Hexane and melting point.
Details of the synthesized compounds (C1 to C7)
RESULTS
Molecular docking (in silico study) studies
Molecular docking is a computational technique used in drug discovery to predict the preferred orientation of one molecule (the ligand) when bound to another molecule (the receptor) to form a stable complex. It’s essential for understanding how potential drug interact with their target proteins at the molecular level. Molecular docking study was performed using the Schrodinger’s glide software. Compounds were subjected to docking with enzymes of the bacteria downloaded from pdb (Kakkassery et al., 2021; Hissam et al., 2024).
Antimicrobial (in vitro method) study results (Fernandez et al., 2021)
Antibacterial analysis
Gram +ve bacterium (S. aureus)
The MBCs were recorded Instead of MIC for the samples against S. aureus at a concentration of 10 mg/mL where as the standard taken was amoxicillin with 1 mg/mL of working solution.
Gram -ve bacteria (E. coli)
Initially, MIC and MBC were recorded for samples C2, C3, C4, and C6 at a concentration of 1 mg/mL. Due to these negative results, a second round of testing was performed using a higher concentration of 10 mg/mL. The standard drug was amoxicillin with 1 mg/mL of working solution.
While the MIC results cannot be presented due to the compounds' distinct colors affecting the visual analysis, hence the MBC results are as follows:
Antifungal analysis (C. albicans)
Since the compounds were coloured, MFCs were recorded instead of MFC against Candida albicans for the study and the standard Fluconazole with 1 mg/mL as working solution was used.
DISCUSSION
Privileged Schiff bases were synthesized in one step, which then diazotized and coupled with beta-naphthol to yield new azo dye compounds. These new compounds were preliminarily identified using IR analysis, the confirmation of Schiff base formation by characteristic functional group vibrations such as phenolic-OH, imine (-CH=N), C=C, aromatic CH, and aldehyde (-CHO). As mentioned in Table 1, the prominent group of Schiff’s bases imine group (-CH=N-) was clearly notable in the range of 1500 cm-1 to 1650 cm-1. Another important functional group which is characteristic of diazotisation was N=N noticed in the range of 1030-1630 cm-1 for all the compounds. Figure 1 illustrates that the IR spectra of diazotized and coupled schiff’s base derivatives confirmed the attachment between the diazotized Schiff base and beta-naphthol. Table 2 reveals the Molecular ion values of peaks in LCMS spectra were in the range of 400 to 625 for all the compounds almost close to their molecular ion values which indicates the formation of the compounds and hence confirmed the compounds' structures as observed in Figure 2.
| Name of Compound | Functional Group | Vibration in cm-1 |
|---|---|---|
| C1 | OH C=NC=CN=N | 3201.01 - 3322.531514.1916021631 |
| C2 | OH C-H C=CC=NN=N | 3221-3357 cm-12902 cm-11602 cm-11631 cm-11514 cm-1 |
| C3 | C=NC=CC-H | 1515 cm-11631 cm-1 & 1602 cm-12897 cm-1 |
| C4 | C=O C=CN=NC=N | 1707 cm-11487 cm-11600 cm-11500 cm-1 |
| C5 | OH C=N C=CN=NC-H | 3221-3303 cm-11514 cm-11602 cm-11631 cm-12922 cm-1 |
| C6 | OH C=NC=CN=N | 3209-3246 cm-11550 cm-11602 cm-1 &1631 cm-1 |
| C7 | C=C-OH C=NN=N | 1602 cm-13217-3444 cm-11514 cm-11631 cm-1 |
| Name (code) of the compound | Molecular weight | Observed peak value |
|---|---|---|
| C1 | 418 | 415 |
| C2 | 463 | 463 |
| C3 | 418 | 392.81 |
| C4 | 465 | 460 |
| C5 | 403 | 401.79 |
| C6 | 624 | 623.51 |
| C7 | 450 | 442 |
The synthesized compounds were evaluated for their antibacterial potentials using Schrödinger Glide software, in which docking with bacterial and fungal proteins was carried out and compared with amoxicillin as a standard. As evident in Table 3, among all, compound C7 showed superior docking scores and binding energies while interacting with key amino acids such as THR, VAL, PHE, and LEU, suggesting probable good antibacterial potential and scope for structural optimization. As illustrated in Figures 3 and 4, the docking images clearly reveal the potential interactions of the synthesized compounds with amino acids, wherein the molecules clearly inserted into the pockets of the targeted proteins. The compounds C4 and C5 have shown glide energy -37.096 and -37.194 respectively which is lower than the glide energy of std, drug -38.057, which indicates the significance of the interactions of these compounds with the target proteins. The glide energy given by C5 and C6 were -44.25 and -40.036 respectively which is lesser that that of the std. drug against proteins. However, the glide energy of the compounds against the proteins of fungus C. albicans, is more than the energy generated by the std. drug which was38.039 (Fernandez et al., 2021; Joseyphus et al., 2022; Hissam et al., 2024).
| Drug | Scores | Glide energy (K cal/mol) |
|---|---|---|
| C4 | -4.779 | -37.096 |
| C6 | -4.97 | -40.077 |
| C5 | -4.213 | -37.194 |
| C7 | -5.111 | -42.507 |
| Amoxicillin (std drug) | -4.363 | -38.057 |
| Microorganism & Protein | E. coli(1 Q81) | --- |
| C4 | -8.589 | -60.335 |
| C6 | -5.649 | -40.036 |
| C5 | -5.718 | -44.25 |
| C7 | -5.743 | -45.805 |
| Amoxicillin (std) | -4.396 | -44.589 |
| Microorganism & Protein | S. aereus(5 XDT) | ---- |
| C2 | -5.066 | -42.76 |
| C4 | -8.084 | -60.137 |
| C6 | -5.116 | -42.583 |
| C5 | -5.368 | -42.436 |
| C7 | -6.334 | -48.58 |
| Fluconazole (std) | -6.302 | -38.039 |
| Microorganism & Protein | C. albicancs(4 HOE) | ---- |
In vitro tests assessed antibacterial activity against S. aureus and E. coli, and antifungal activity against C. albicans over range of concentrations from 10 to 0.019 mg/mL. Since compounds were coloured, MBC and MFC were measured instead of MIC, using amoxicillin and fluconazole as standards, ensuring consistent, comparable results.
As mentioned in Table 4, among all the compounds tested against Gram-positive bacterium S. aureus, C2 showed the highest MBC of 2.5 mg/dL, compared to C4 and C6 had the lowest MBCs of 0.625 mg/dL, indicating strong antibacterial effects likely due to functional groups such as OH, NO2, and alkyl amine. C2 contained OH, NO2, and alkyl amine; C4 possess -CHO, -NO2, and -OH; and C6 contains -NO2 and -OH groups as seen in Figure 5. Similarly, against Gram-negative E. coli, Figure 6 illustrates that C7 exhibited the highest MBC of 2.5 mg/mL, while C3 showed the lowest at 0.0195 mg/mL. These functional groups probable contributions to antibacterial activity which may be due to their electronegative property sufficient to damage the cell wall and it require further study in understanding their mode of action; however, these compounds showed promising, dose-dependent activity compared to the standard drug (Kaur et al., 2020; Kasare et al., 2022; Kyei et al., 2020).
| Compound name | Concentration of the compounds (mg/mL) 10 mg/mL to 0.019 mg/mL | Microorganism | MBC(MIC) value (mg/mL) |
|---|---|---|---|
| C2 | S. aureus | 2.5 | |
| C3 | S. aureus | 1.25 | |
| C4 | S. aureus | 0.625 | |
| C6 | S. aureus | 0.625 | |
| Standard drug (Amoxycillin) | 1 mg/mL | S. aureus | 0.0625 |
| C2 | E. coli | 1.25 | |
| C3 | E. coli | 0.0195 | |
| C4 | E. coli | 0.625 | |
| C6 | E. coli | 0.625 | |
| C7 | E. coli | 2.5 | |
| Standard drug (Amoxycillin) | 1 mg/mL | E. coli | 0.125 |
| C2 | C. albicans | 0.625 | |
| C3 | C. albicans | 0.625 | |
| C4 | C. albicans | 2.5 | |
| C6 | C. albicans | 0.3125 | |
| C7 | C. albicans | 1.25 | |
| Standard drug (Fluconazole) | 1 mg/mL | C. albicans | 0.0625 |
Table also reveals regarding the antifungal activity study of the compounds in which compound C2 exhibited the highest MBC of 2.5 mg/mL, while C4 and C6 exhibited lower MBC values of 0.625 mg/mL. This indicates that basic structure with electronegative groups like -NO2 and -OH likely to contribute to their activity. It is observed from Figure and Figure 7, that compound C7 demonstrated superior activity against both Gram-negative bacteria and fungi, possibly may be due to dimethoxy and hydroxyl groups because of their electronegative property which may damage the cell or inhibit the growth of the cell. Hydrophilic groups, especially hydroxyls, enhance the penetration of Schiff bases through microbial cell walls, improving their efficacy. Among tested compounds, it is evident from Figure that C6 demonstrated the strongest antifungal effect against C. albicans, followed by C2 and C3. It is also evident that these effects were dose-dependent compared to the standard drug. However, further study is needed to justify this claim (Morsy et al., 2023; Ibrahim et al., 2024).
In nutshell, the synthesized compounds were proven efficacious as antimicrobial agents in this study and open new opportunities for their utility. These compounds deserve an opportunity to be explored commercially, as antimicrobial and industrial dyes on various products which elicit new opportunities for the Azo dyes due to their antimicrobial applications.
CONCLUSION
A series of Schiff base derivatives coupled with beta naphthol via diazo link were synthesized and characterised by spectral analysis. Later, these compounds were screened for their antibacterial and antifungal activity by in silico followed by in vitro methods which have justified their antimicrobial potentials. As these compounds are coloured in nature, and possess antimicrobial potentials, these new Azo dyes would possibly find commercial application as dyes for pharmaceutical and industrial utility along with healthcare benefits in future.