Abstract

Fifteen symmetrical 1,2-phenylenediamine Schiff’s base derivatives were designed as DNA intercalators. Adsorption, distribution, metabolism, elimination, and toxicity (ADMET) properties and drug-likeliness of the designed compounds were predicted by Swiss-ADME software, and the molecular docking study was performed in the PyRx tool. Four Compounds NHM01, NHM04, NHM06 and NMH11 were synthesized and the structure of each compound was analyzed using Fourier transform infrared spectroscopy (FTIR), 1H NMR and 13C NMR. Moreover, the four compounds were in vitro biologically screened for their interactions with the genomic DNA. The binding properties of these compounds to genomic DNA (G-DNA) were investigated by UV-visible absorption and fluorescence spectroscopy. The molecular docking studies of compounds revealed that all the compounds are bioactive, however, compound NHM01 shows significant DNA binding affinity over standard drugs. The biological results indicate that the four synthesized compounds can interact with G-DNA by intercalation binding. Compound NHM01 showed the highest key selection vector (KSV) value, followed by NHM04, which suggests that compound NHM01 binds most tightly to G-DNA. Our results demonstrate that NHM01 and NHM04 may serve as novel lead compounds for the discovery of more 1,2-phenylenediamine Schiff’s base derivatives with improved anticancer potency and selectivity.

1. Introduction

Deoxyribonucleic acid (DNA) is the storage unit of genetic information and considered as major biological target for numerous anticancer agents. The anticancer agents that target DNA generally interact with DNA through reversible binding by three main modes: static electronic interactions, groove binding, or intercalation. The static electronic interactions (external surface binding) could be related to the cationic molecules that bind non-specifically with the anionic sugar phosphate backbone of DNA through outer edge stacking (Serec et al., 2016). The second mode of interaction is groove binding which comprises the insertion of the molecule into the base edges of the minor or major groove (usually G•C sites in the major groove, A•T sites in the minor groove). The major groove has multiple sites of interaction, its dimensions are 8.5Å in depth and 11.6Å in width, where bulky molecules can easily access the major groove. Conversely, the minor groove, which has a depth of 8.2 Å, is narrower and has fewer binding sites; however, binding to the minor groove is the most common. The third mode of interaction is intercalation which is closely related to most anticancer drugs which are currently in clinical use. In essence, small molecules can interact with DNA involving a single or mixed binding mode (Bhaduri et al., 2018). In this paper, our focus will be on DNA intercalation.

2. DNA Intercalators

DNA intercalators are a class of compounds that can reversibly bind to DNA through insertion between adjacent base pairs perpendicularly to the axis of the helix. These interactions can lead to conformational changes involving an increase in the vertical separation between base pairs, unwinding and lengthening of the DNA helix. It may also interfere with the detection and function of associated proteins or enzymes leading to the failure of DNA repair systems, transcription processes or replication of the DNA (Biebricher et al., 2015). Numerous clinical intercalating agents are available as anticancer agents, for example, daunomycin, doxorubicin, mitoxantrone or dactinomycin. Ethidium bromide is a phenanthridine monomer dye frequently used as a fluorescent marker (nucleic acid stain) in molecular biology for many procedures such as visualization of nucleic acid. Ethidium bromide intercalates between adjacent base pairs in the double-stranded DNA molecule and leads to deformation of the DNA. The deformation of DNA will possibly affect DNA biological processes, such as DNA transcription or replication (Binaschi et al., 2001).

3. Structural Features of DNA Intercalators

DNA intercalators have three main structural features: i) Chromophores, which are planar polyaromatic rings that bind to DNA, ii) cationic moieties, such as protonated amino groups which may interact with the phosphate backbone, and iii) side chains that can interact with DNA minor grooves. In general, DNA intercalators are classified into two major classes: monofunctional and bifunctional (El-Adl et al., 2020). Monofunctional intercalators, also referred to as simple intercalators, consist of one intercalating unit, often carrying a positive charge on their ring system (Wadler et al., 1994). Secondly, bifunctional intercalators, also called bis-intercalators or threading intercalation, have two typically cationic intercalating units separated by flexible linker groups long enough to permit double intercalation. Since bis-intercalators have two different intercalating moieties, they can cause stronger binding with DNA than simple mono-intercalators (Wadler et al., 1994).

4. Schiff Bases

Schiff bases are ranked among the most privileged synthetic organic intermediates extensively employed in industrial, pharmaceutical, and medical concerns. These compounds are also known as imines or azomethines with a general formula R-CH=NR’, where R and R’ are an alkyl or aryl group (Peng et al., 2014). Schiff bases have been reported to exhibit a wide spectrum of biological properties and can also interact with other ligands like ortho-phenylenediamines which leads to diverse and multipurpose applications (Koll, 2003). Bis-Schiff bases are usually synthesized from the condensation reaction of diamines with two equivalents of aldehyde or ketone in a 1:2 molar ratio. The bases can be either symmetric or asymmetric structures, depending on the reacting aldehydes or ketones (Vernekar & Sawant, 2023).

5. Literature Review

Literature review revealed a great interest in ortho-phenylenediamines Schiff complexes and their applications. In contrast, less attention has been focused on the uncoordinated Schiff bases of ortho-phenylenediamines. In this regard, the search for other uncoordinated anticancer agents with improved safety profiles has become an attractive area of study for cancer management (Lucaciu et al., 2022). The main aim of this work is to design and synthesize some symmetrical Schiff’s base derivatives of ortho-phenylenediamine and study their DNA intercalating ability using an ethidium bromide competition fluorescent assay and UV-visible spectroscopic method. In addition, there is increasing interest to explore how different structural features of 1,2-Phenylenediamine Schiff’s base derivatives can affect the DNA binding capability using molecular modeling tools, which may serve as a basis for understanding the molecular mechanism of action of the 1,2-Phenylenediamine Schiff’s base derivatives and can help to design new chemotherapeutic molecules.

2. Material and Methods

2.1. Molecular Docking

Molecular docking is an in silico method for recognizing the correct binding pose of a DNA-ligand complex and evaluating its strength using various scoring functions for selecting the best pose generated by each molecule to rank-order. The molecular docking studies of all compounds with DNA were performed using PyRx software to generate grid, calculate dock scores, and evaluate conformers. The steps of molecular docking include DNA structure optimization, in silico preparation for standard and designed compounds, and the process of docking calculations (Adelusi et al., 2022).

2.1.1. Optimization of DNA Molecule

The crystal structures of B-DNA fragments (PDB ID: 1BNA, 1D60, 1ZEW, 2DES, and 1DCO) were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb) and analyzed for its active site by Biovia Discovery Studio Visualizer (http://accelrys.com). Auto Dock 4.2 software was used to correct the imported DNA coordinates (the corrections involved deleting unwanted water molecules, adding hydrogen atoms (polar only), followed by adding Kollman charge, and saving as pdpqt format used as input in PyRx software).

2.1.2. Designing and In Silico Preparation of NHM Compounds

The in silico procedure has become a very important tool in drug identification and screening. Ligand absorption, distribution, metabolism, and excretion (ADME) pharmacokinetic properties of the designed Schiff’s base derivatives of o-phenylenediamine are evaluated to determine their activity within the human body, and prediction of these properties in advance will save the cost of drug discovery substantially. To verify drug-likeliness, canonical SMILES format of the designed NHM compounds were used in swissADME (http://www.swissadme.ch/index.php). The main bioavailability parameters were molecular weight (g/mol), lipophilicity (XlogP3), solubility (log S), polarity (Topological Polar Surface Area - TPSA in Ų), saturation (fraction of carbon atoms in Sp³ hybridization – Csp³), and flexibility (No of rotatable bonds). Consequently, absorption parameters such as Human Intestinal Absorption, Blood Brain Barrier, P-glycoprotein interaction (substrate or inhibitor), and metabolism (inhibitor or substrate) of the bio-active molecules with different cytochrome P450 enzymes were also estimated using admetSAR, which is based on QSAR data for prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET). Drug-like molecules considering Lipinski rules and good ADMET properties were chosen as ligands in the consequent molecular docking procedure.

2.1.3. Docking Process and Analysis

B-DNA crystal structures (PDB ID: 1BNA, 1D60, 1ZEW, 2DES, and 1DCO) were loaded in PyRx virtual screening tools as pdbqt format. Standard drug and designed compounds pdb files were loaded and automatically converted to pdbqt format. Docking was performed using Autodock Vina incorporated in PyRx software. The centers of the grid box were assigned automatically and the dimensions of the box were set to 25 × 25 × 25 Å. After docking, the complexes with the lowest binding energy (kcal/mol) were selected and their interaction modes were visualized by Discovery Studio.

2.2. Prediction of ADMET Properties and Drug-Likeliness

The in silico procedure has become a very important tool in drug identification and screening. Ligand absorption, distribution, metabolism, and excretion (ADME) pharmacokinetic properties of the designed Schiff’s base derivatives of o-phenylenediamine are evaluated to determine their activity within the human body, and prediction of these properties in advance will save the cost of drug discovery substantially. To verify drug-likeliness, canonical SMILES format of the designed NHM compounds were used in swissADME (http://www.swissadme.ch/index.php). The main bioavailability parameters were molecular weight (g/mol), lipophilicity (XlogP3), solubility (log S), polarity (Topological Polar Surface Area - TPSA in Ų), saturation (fraction of carbon atoms in Sp³ hybridization – Csp³), and flexibility (No of rotatable bonds). Consequently, absorption parameters such as Human Intestinal Absorption, Blood Brain Barrier, P-glycoprotein interaction (substrate or inhibitor), and metabolism (inhibitor or substrate) of the bio-active molecules with different cytochrome P450 enzymes were also estimated using admetSAR, which is based on QSAR data for prediction of absorption, distribution, metabolism, excretion, and toxicity (ADMET). Drug-like molecules considering Lipinski rules and good ADMET properties were chosen as ligands in the consequent molecular docking procedure.

2.2.1. Swiss ADME

Swiss ADME is a free web tool developed by the Swiss Institute of Bioinformatics (SIB); it was employed to evaluate pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of small molecules and is freely available at www.swissadme.ch. It calculates six physicochemical properties: lipophilicity, size, polarity, solubility, flexibility, and saturation. In addition, it promotes the assessment of ADME parameters (absorption, distribution, metabolism, and excretion) of drug candidates and molecules and provides information to determine Lipinski’s rule of five for drug likeness of oral bioavailability (Daina et al., 2017). The drug-likeness assessment is based on the following factors: molar mass smaller than 500 g/mol, log P smaller than five, number of hydrogen bond acceptors smaller than 10 (accounted for in the molecule function of N or O atoms), and number of hydrogen bond donors smaller than 5 (accounted for in the molecule function of NH or OH groups). Hence, molecules outside this range will be unlikely to become orally bioavailable as a drug (Lipinski et al., 2001).

2.3. Chemistry

2.3.1. General Procedure for Synthesis of Schiff Base (NHM Compounds)

Schiff’s bases were prepared by reacting one mole of phenylenediamine and two moles of substituted aromatic aldehydes or ketones in 100 ml ethanol. The solution was refluxed for 7 hours under constant stirring. The condensation reaction was carried out using glacial acetic acid as a catalyst and monitored by thin layer chromatography (TLC). The mixture was left for cooling and the solid product (crude) was gathered by filtration and washed four times with ethanol, then dried utilizing vacuum. The gained product was re-dissolved in ethanol for recrystallization and then dried to give a clean pure product (Scheme 1). The crystalline products obtained were characterized by Fourier Transform Infrared (FTIR) spectra (Cary 600 FTIR, USA), which was operated in a wavenumber range of 4000–400 cm−1, and 1H and 13C nuclear magnetic resonance (NMR) spectroscopies were performed with dimethyl disulfide (DMDS) used as solvent. Tetramethylsilane (TMS) was the standard reference, and the probe temperature was 25°C (Hamil et al., 2009).

2. Materials and Methods

2.1. Molecular Docking

Molecular docking is an in silico method used to identify the correct binding pose of a DNA-ligand complex and evaluate its strength through various scoring functions. The molecular docking studies of all compounds with DNA were performed using PyRx software, which generates grids, calculates dock scores, and evaluates conformers.

DNA Structure Optimization

Crystal structures of B-DNA fragments (PDB IDs: 1BNA, 1D60, 1ZEW, 2DES, and 1DCO) were downloaded from the Protein Data Bank (http://www.rcsb.org/pdb) and analyzed for their active sites using Biovia Discovery Studio Visualizer.

In Silico Preparation of NHM Compounds

In silico methodologies are essential for drug identification and screening. The absorption, distribution, metabolism, and excretion (ADME) properties of designed Schiff's base derivatives of o-phenylenediamine were evaluated to predict their activity in the human body.

Docking Process and Analysis

The B-DNA crystal structures (PDB IDs: 1BNA, 1D60, 1ZEW, 2DES, and 1DCO) were loaded into PyRx as pdbqt format. The docking was performed using AutoDock Vina, and grid box dimensions were set to 25 × 25 × 25 Å. Complexes with the lowest binding energy were selected for further analysis.

2.2. Prediction of ADMET Properties and Drug-Likeness

The in silico evaluation of ADME pharmacokinetic properties for the designed Schiff’s base derivatives was performed using swissADME. Key bioavailability parameters were assessed, including molecular weight, lipophilicity, polarity, saturation, and flexibility.

2.3. Chemistry

2.3.1. General Procedure for Synthesis of Schiff Bases (NHM Compounds)

Schiff bases were synthesized by reacting one mole of phenylenediamine with two moles of substituted aromatic aldehydes or ketones in 100 ml ethanol. The condensation reaction was catalyzed by glacial acetic acid, monitored by thin-layer chromatography (TLC). The solid product was collected via filtration, washed with ethanol, dried, and recrystallized.

2.4. DNA Binding Studies

To investigate the interaction of synthesized compounds with G-DNA, fluorescence emission spectra were used. All experiments utilized Tris buffer (0.01M Tris, 0.1M NaCl, pH 7.4). The pH of solutions was measured with a calibrated Jenway pH-meter.

Absorbance Spectra

Absorbance spectra were recorded on a Jenway UV-visible spectrophotometer within the 200-500 nm range. The concentration of genomic DNA used was 75 μg/ml.

Fluorescence Spectra and DNA-Binding Studies

Fluorescence emission spectra were measured using a Jasco FP-6200 spectrofluorometer. The ethidium bromide (EB) displacement experiment involved sequential addition of Tris buffer, EB, G-DNA, and NHM compounds.

3. Results and Discussion

3.1. Design Strategy of Ortho-Phenylenediamine Schiff’s Base Derivatives

Given the significant role of phenylenediamine Schiff's base derivatives as DNA intercalators, ortho-phenylenediamine was chosen as the key scaffold to develop a series of aromatic imino analogues.

Table 1: The chemical structures of designed compounds.

3. Results and Discussion

3.1. Design Strategy of NHM Compounds

Fifteen compounds named with NHM symbol were designed, the strategy which applied to design these compounds was based on the symmetrical distribution of the amino groups of ortho-phenylenediamine with different carbonyl compounds including naphthaldehyde, naphthquinone and anthraquinone derivatives, where the aldehydes or ketones differ in their lipophilicity, hydrogen bond ability as donor or acceptors, and ionizability. The applied strategy yielded the scaffold which contains three parts:

1. A planar polycyclic pattern to intercalate between DNA base-pairs and to provide π-π interactions.
2. Some polar functions providing hydrogen bond interactions with nucleic acids.
3. A variety of substituents to improve the interactions and promote the physicochemical properties.

To the best of our knowledge, there have been no literature reports regarding Schiff’s base of ortho-phenylenediamine with naphthoquinones and anthraquinone derivatives as DNA intercalating agents so far. The designed compounds were investigated for their docking energies. In addition, the effect of replacing the naphthyl ring with a three-ring system (anthracene) or aromatic rings with flexible linkage was also investigated. The chemical structures are listed in Table 1.

3.2. Molecular Docking Analysis

In this part of the current study, the molecular dockings of NHM compounds with five B-DNA fragments were performed using AutoDock Vina in PyRx Virtual Screening Tool to understand the possible binding mode. The molecular docking study was employed to identify the binding interactions and estimate the binding affinity of the most active derivatives to the DNA duplex by combining and optimizing variables such as hydrophobic, steric, and electrostatic complementation.

Two well-known biologically active DNA intercalating agents were used as references, namely doxorubicin and daunorubicin. Considering the values of free energies of binding mentioned in Table 2, it could be deduced that the compounds were properly accommodated between the DNA base pairs. The molecular docking analysis revealed that the compounds contributed to hydrophobic and π-π interactions, and hydrogen bonding with the DNA base pairs.

(1BNA): D(CGCGAATTCGCG)
(1DC0): D(CATGGGCCCATG)
(2DES): D(CGTACG)
(1D60): D(CCAACNTTGG)
(1ZEW): D(CCTCTAGAGG)

ΔG_a is the binding free energy change in the binding process. The more negative the numerical values for the binding affinity, the better is the predicted binding between a ligand and a macromolecule. In this particular case of screening five DNA fragments (1BNA, 1ZEW, 2DES, 1D60, 1BNA and 1DCO) with designed compounds, Table 2 shows that all the designed compounds have good binding energies when compared to standard drugs. Moreover, NMH01 and NMH11 are both predicted to have the best binding affinity.

The DNA fragment (1ZEW) was used to discuss and compare the intercalating activity of all compounds. It appears worth noting that compounds which exhibit high activity against (1ZEW) also showed high activity against the other DNA fragments. Simultaneously, any modification which might decrease the activity against (1ZEW) will produce the same effect against all other DNA fragments. Molecular docking results were identified based on the strongest ligand binding poses, where identified using the low binding energy and the number of H-bonding and hydrophobic interactions. From the docking studies, the molecular docking analysis revealed that all the docked molecules were inserted between the adjacent nucleotides at the active site. These compounds were stabilized at the active site through different interactions. The planar system was involved in hydrophobic stacking with the base pairs of different nucleotides, and the hydrophilic moieties formed several hydrogen bonds with different nucleotides.

It was observed that the introduction of hydrophilic substituents to the aromatic ring increases the intercalating activity (decrease of the docking energy) as shown with NMH01, NMH09, and NMH13. While the introduction of hydrophobic substituents (R) to the same aromatic ring leads to a decrease of the intercalating activity (increase of the docking energy) as shown for NMH04 and NMH08. A substantial observation was that the highest activity was predicted to be with the introduction of 2-hydroxynaphthalene-1,4-dione at NMH01. The importance of hydroxyl substituents at meta-positions of the aromatic ring are attributed to the formation of seven hydrogen bonds at intercalating sites, keeping the planarity which is needed for optimum localization at the intercalating site. Whereas, hydroxyl substituent at position five builds three H-bonds as shown in NMH02. H-bonding characteristics of compounds rendered significantly high binding affinity with DNA.

Lipophilic substituents (Cl or Br) at position 3 of the naphthyl side chain had little effect on the intercalating activity when compared with the non-substituted derivative (NMH03, NMH07). Furthermore, the introduction of a hydrogen bond acceptor substituent such as a carbonyl group on the aromatic ring leads to an increase of the intercalating activity, as could be seen from the comparison of 2-hydroxynaphthalene-1,4-dione derivatives with the corresponding non-substituted 2-hydroxynaphthalene derivatives as shown with NMH01, NMH02, and NMH09. In the case of compounds NMH04, NMH08, NMH05, and NMH10, replacement of the methyl moiety on aromatic rings with the isopropyl moiety leads to a decrease of the intercalating activity, which may be caused by steric hindrance of branched groups. Furthermore, as observed in the docking study, compound NMH14 was located at the groove pocket interacting with DNA by π-anion interaction with DGB:17 and DGA:7. Compound NMH15 was located at the groove pocket and interacted with DNA by six hydrogen bonds with DGA:10, DGA:9, DTB:15, and DAB:16. Nevertheless, it was evident that NMH14 and NMH15 had the lowest binding energy scores, which may be due to the lack of the planarity pattern of the polyaromatic systems.

Figure 1: Localization of NHM01 (yellow) at the intercalating site of DNA.

(ii) Compound NMH01 interacting with DNA strands with seven intermolecular hydrogen bonds.

(iii) 2-dimensional structure of NMH01 at the active site.

It can be clearly seen that the interaction mode of most compounds consists of a classical intercalation mode except the binding of NMH03, NMH05, NMH08, and NMH10 with DNA, which involves partial intercalation and groove binding. Moreover, compounds NMH14 and NMH15 bind to DNA via groove binding. It is worth mentioning that compounds NMH01, NMH04, NMH06, and NMH11 were the most active compounds for each DNA fragment (Table 2). For this reason, they have been selected for further synthesis and analysis.

3.3. ADMET properties

3.3.1. Analysis of Physicochemical Properties:

General characteristics of designed Schiff’s base derivatives of o-phenylenediamine revealed that all these compounds have a molecular weight less than 500 g/mol, except NMH03, NMH07, and NMH13 with molecular weights of 546.21, 542.26, and 552.53 g/mol, respectively. This appears a prime property to be called as drug likeness of the small molecules. It is evident from Table 3 that topological polar surface area (TPSA) values of all compounds were found to be within the limit of ≤ 140 Å. The prediction of TPSA parameters helps to understand the passive molecular transport of drug molecules. TPSA is an important tool in drug discovery and development. By analyzing a drug candidate's TPSA, we can predict its potential for oral bioavailability and ability to reach target sites within the body. This prediction hinges on a drug's ability to permeate biological barriers. The Molar refractivity (MR) of all compounds is within the ranges of 121.50–158.01; their number of rotatable bonds (nRotb), number of H-bond donors (nHBD), and the number of hydrogen bond acceptors (NHBAs) of all o-phenylenediamine derivatives are within the recognized limits of ≤10, ≤5, and ≤10, respectively.

SwissADME predictions suggest that symmetrical phenylenediamine Schiff’s base derivatives have optimum parameters for anticancer activity and can be considered as lead molecules for further modifications.

Molecular weight: MW, Number of heavy atom: nHA, Number of rotatable bonds: nRB, Number of H-bond acceptors: nHBA, Number of H-bond donors: nHBD, Molar refractivity: MR, topological polar surface area: TPSA.

3.3.2. Lipophilicity and Water Solubility

Log Po/w values of all derivatives range from 2.85 to 8.00 reflecting its partition preferably into the lipid compartment (Table 4). This implies that these compounds will be well absorbed through the membranes into the systemic circulation to achieve high bioavailability. On the other hand, solubility, Log S is aqueous solubility, exhibit a defined range of -4.4 to -8.72 as shown in Table 5. The compounds NMH01, NMH02, NMH03, NMH15 predicted as moderately soluble (with optimal lipophilicity and moderate water solubility) can achieve good bioavailability when administered orally. All other derivatives are poorly water soluble.

3.3.3. Pharmacokinetic Profile:

Predictions of gastrointestinal absorption were found to be high for compounds NHM01, NHM02, NHM04, and NHM15. In contrast, all other compounds were shown to exhibit low gastrointestinal absorption potential (Table 5). None of the selected compounds penetrate the blood-brain barrier which can diminish the chance of side effects in the CNS. Interestingly, some of the derivatives act as inhibitors of various CYP isoforms, indicating that they may present significant drug interactions.

3.3.4. Drug Likeness:

The drug likeness was determined using the rule of five, which suggests that NMH01 passes all drug likeness parameters while all other derivatives violate at least one of these. Compounds violating at least one of the drug-likeness criteria must be further studied before development. Lipinski’s Rule of Five indicated that most of the designed derivatives obeyed the rules of MW < 500, Log P < 5, and HBD < 5. Exceptionally, compound NMH07 is excluded from being a drug candidate due to the violation of weight and logP limits.

3.3.5. Chemical Synthesis:

The four most promising compounds (NHM01, NHM04, NHM06, and NHM11) were synthesized successfully via a previously established method with a good yield (73–82%) in two steps. The synthesized compounds were found to be soluble in organic solvents like DMSO and DMF but insoluble in water. Characterization of the synthesized compounds was achieved through various spectroscopic analyses.

Overall, the synthesized compounds (especially NHM01, NHM04, NHM06, and NHM11) show considerable potential for further development as anticancer agents.

3.5. Analysis of spectra

3.5.1. Proton 1H and Carbon 13C NMR spectra:

NMR analysis is very important to confirm the structure and functional groups. Experimental H-NMR chemical shifts were expressed in ppm, dispersed through the spectrum starting from tetramethylsilane as the internal standard and recorded in DMSO-d. Importantly, the spectra lacked the signal of the amino group (NH) characteristic of the starting material.

The ¹H-NMR spectrum of NHM01 displayed a singlet at 15 ppm and a multiplet at 7.84-8.29 ppm as a signal to the O-H proton and the protons of aromatic rings, respectively. Moreover, a peak appears at 2.09 ppm attributed to the aliphatic–CH (6H) compound contained in NHM04, and multiplet signals at 7.30-8.05 ppm were ascribed to aromatic protons. Besides, the signal due to –CH=N appears at 9.1 ppm for the compound NHM06 corresponding to the azomethine group, which confirms the formation of Schiff bases and the multiple peaks attributed to the aromatic protons between 7.32 and 8.05 ppm.

Additionally, the ¹H-NMR spectrum of compound NMH11 exhibited a singlet signal at 3.81 ppm corresponding to the CH2 group. The multiplet signals at 7.30-7.72 ppm were assigned to the 20 H aromatic protons. Consistently, 13C-NMR spectra showed a chemical shift at 189.10, 184.81, 156.40, and 185.01 ppm assigned to the imine group, revealing the formation of Schiff's base derivatives of o-phenylenediamine (NHM01, NHM04, NHM06, and NMH11), respectively. In addition, the peak observed at 16.50 ppm was attributed to a methyl carbon (C20) integrated at the aromatic group of compound NHM04. The peaks observed at 157.04 ppm and 183.00 ppm correspond to carbonyl groups of the compounds NMH01 and NMH04, respectively. The peak at 50.01 ppm attributes to the methylene group of compound NMH11. The peaks at 103.11-148.62 ppm range are ascribed to aromatic carbons.

3.5.2. Fourier-Transformed Infrared

In order to continue the characterization of NHM compounds, the FT-IR spectrum was carried out. The FT-IR spectra of NHM compounds showed the disappearance of both carbonyl groups at the aldehyde, ketone, or amino groups at the amine, whereas NH is vanished or hidden underneath the broad bands at 3450-3300 cm^-1 in Schiff’s base. Additionally, the presence of the azomethine CH=N stretching at about 1529−1655 cm^-1 depending on the nature of R side chain substituents, indicates the formation of the Schiff base compounds.

The FTIR spectrum of the compound NMH01 showed a stretched band around 3200 − 3570 (O-H) cm^-1, which is attributed to the intramolecular hydrogen bond of the hydroxyl group. Furthermore, compound NHM01 exhibits absorption bands at 1675-1619 cm^-1 corresponding to the C=C and C=O, respectively. Additionally, the band at 1446−1450-1577 cm^-1 corresponds to C=C of compounds NHM06, NHM04, and NMH11, respectively.

3.6. DNA binding studies

3.6.1. UV Absorbance Spectra:

UV-spectroscopy, being an important qualitative and quantitative analysis technique, has been exploited to investigate the interaction of ligands with DNA. The electronic absorption spectrum of DNA exhibits a broad band in the range of 200-350 nm and shows maximum absorption at ~260 nm. This maximum is a consequence of chromophoric groups in purine and pyrimidine moieties responsible for the electronic transitions (González-ruiz et al., 2011). Therefore, the changes in the absorption spectra of the compound alone and compound–DNA mixture indicate DNA–compound interaction, and the mode and strength of binding can also be determined from the shift.

The absorbance spectra of DNA could show hypochromism (decreased absorbance intensity) and hyperchromism (increased absorbance intensity) upon titration with the ligand. In general, the intercalative mode of binding usually results in hypochromism and a red-shift because of the strong stacking interaction between an aromatic chromophore and the nitrogen bases of DNA. This results in structural changes of the DNA that entail the local unwinding of the double helix. Whereas for weak interactions such as hydrogen bonding, groove binding, and electrostatic interaction, no significant shift of the absorption maxima occurs (Banerjee et al., 2016).

The absorption spectra resulting from titrations are depicted in Figure 2. As observed from spectral curves, the complexes showed pronounced hypochromism with a slight red shift of 2 nm. The results suggest that these compounds bind to DNA through an intercalation mode. The resultant hypochromic effect was found to be 0.244 nm for NMH01, 0.201 nm for NMH04, 1.53 nm for NMH06, and 1.42 nm for NMH11, followed by a red shift of magnitude 2.01 nm, 1.89 nm, 1.73 nm, and 1.88 nm for NMH01, NMH04, NMH06, and NMH11, respectively.

Figure 2: UV–Vis absorption spectra of DNA in the absence and presence of NMH01 (A) the G-DNA-NMH01 complex, (B) the 30 µM NMH01, (C) G-DNA-NMH01 complex subtracted from the G-DNA absorbance to show the hypochromic shift (D) G-DNA alone. The experiment was conducted in Tris buffer solution (0.01M Tris, 0.1M NaCl, at pH 7.4). Genomic DNA was used in a concentration of 75 μg/ml and NMH01 at 30 µM.

3.6.2. Ethidium bromide (EB) Competition Assay

A competitive displacement assay was conducted to obtain further evidence of the binding pattern of the tested compounds and DNA. Ethidium bromide (EB) is a strong fluorescent dye, known to bind to DNA through intercalation with maximum emission at 661 nm upon binding to double-stranded DNA. Free Ethidium bromide exhibits very low fluorescence. However, with the addition of DNA, due to its intercalation, the fluorescence intensity magnifies remarkably (Zhao et al., 2013).

The gradual addition of compounds 1, 4, 6, and 11 to the EB–DNA complex caused a decrease of the emission (quenching effect) (Figure 3). The hypochromic shift appears as a result of the formation of a complex between the compound and DNA, and this reduction of the fluorescence intensity is a characteristic sign of intercalation. The fluorescence spectra of the EB-DNA complex in the absence and presence of NMH01 is shown in Fig. 3. The figure shows that the fluorescence intensity of EB-DNA can be quenched significantly by the gradual addition of NMH01. This result indicates that NMH01 was able to replace EB in the DNA helix, indicating an intercalative NMH01 DNA binding mode.

The fluorescence quenching data were analyzed by the Stern–Volmer equation and the quenching constants (K_sv) were calculated in which the synthesized compounds were the quenchers:

I0/I = 1 + KSV [Q] (1)

I₀ and I represent the fluorescence intensities in the absence and presence of quencher, respectively; K_SV is the linear Stern-Volmer quenching constant; Q is the concentration of quencher. The K_SV values were given by the ratio of the slope to intercept. The K_SV value suggests a strong affinity of the compounds to EB-bound G-DNA and that it can competitively displace EB from DNA via an intercalative mode of binding. As shown in Table 8, compound NMH01 had the highest K_SV value, suggesting that compound NMH01 bound most strongly to G-DNA even more than daunorubicin. The K_SV values for the tested compounds are listed in Table 8.

Based upon the variation in absorbance and the fluorescence intensities, the binding constant for the compounds to determine the strength of the interaction of compounds with DNA at 298 K was determined from fluorescence titration data using the Benesi–Hildebrand (BH) equation:

1/ΔA = 1/(KA·ΔA0) [DNA] + 1/(ΔA0) ······ (2)

Figure 3: Fluorescence changes of the NMH01 system that contains (A) the G-DNA-ethidium bromide complex, (B) the G-DNA-ethidium bromide complex with 30 µM NMH01, and (C) ethidium bromide alone. The experiment was conducted in Tris buffer solution (0.01M Tris, 0.1M NaCl, at pH 7.4), λ_ex = 480 nm. Inset: plot of F₀/F versus different concentrations of NMH01 (µM) for the titration of NMH01 to G-DNA-EB complex. Genomic DNA was used in a concentration of 75 μg/ml and ethidium bromide at 72 µM.