Bioactivity of a radical scavenger bis(pyrazolium p-toluenesulphonate) on ctDNA and certain microbes: a combined experimental and theoretical analysis

Free radical scavenging capability of BPPTS was proved by biological experiments, docking analysis and theoretical calculations.

Abstract

A small organic molecule, bis(pyrazolium p-toluenesulphonate) (BPPTS), was crystallized, characterized and used to scavenge free radicals in biological systems. SXRD and spectroscopic analyses were used to confirm the structure of BPPTS. Methanolic and ethanolic solutions of BPPTS were used to assess the stability of the proposed drug using the UV-vis spectrophotometric technique. Optimization of the molecular structure was carried out by DFT with B3LYP/6-311++G(d,p) level of basis set. MEP and Fukui functions that elaborate theoretically the predominant electrophilic, nucleophilic and radical sites in BPPTS were correlated with experimental biological screening. BPPTS exhibits strong activity against Bacillus subtilis and Escherichia coli, comparable with all other analyzed pathogens. The free radical scavenging activity of BPPTS was assessed by both experimental studies and theoretical calculations. The binding sites of DPPH, which can bind to BPPTS, were also predicted by Fukui functions. DNA binding of BPPTS in UV-vis studies revealed the groove mode of binding due to the occurrence of hyperchromism. The phenomenon of hyperchromism was established by the Hirshfeld surface analysis of BPPTS, which confirmed the presence of π···π interactions (2.4%). Molecular docking established a positive correlation between experimental bio-screening reports and simulated data. ADMET properties were also calculated.

Introduction

The electron transfer process plays a vital role in chemical, physical and biological processes.1 Understanding the electron transfer processes in biological systems has been the subject of intensive research for a long time.2,3 Free radicals are chemical motifs possessing an unpaired electron with high reactivity and low stability. It can be formed either by loss or gain of a single electron or a homolytic cleavage of a covalent bond. Oxidative stress is the excessive production of reactive oxygenated/nitrogenated (ROS/RNS) species4 that cannot be defended by naturally occurring antioxidants in our body. The production of ROS/RNS overpowers the antioxidant defences and results in the oxidative stress of cellular membranes and serious tissue damage.5 Poor nutrition, pollution, waste water sources, stress and UV radiation are a few causes that damage tissues in living organisms and are responsible for cancer, diabetes, ageing, cardiovascular disease and kidney malfunction.6,7 ROS such as superoxide and hydrogen peroxide8,9 radicals affect the metabolism of proteins, DNA and lipids. This unbalanced state between oxidant species and antioxidants in turn triggers damage to cells.10,11 The DNA damage can have profound biological consequences if the resulting injury is not efficiently repaired. Synthetic antioxidants support and substitute the natural oxidants in healing the DNA impairment by capturing free radicals.12

Heterocyclic based azo derivatives13–15 are composed of a large number of natural products including antibiotics, alkaloids, flavanoids, vitamins and proteins.16 Pyrazole based heterocyclics are active antioxidants, anti-inflammatory agents,17 and antibacterial, anticancer and anticonvulsant agents.18 In the present investigation, the free radical scavenging activity of an antioxidant, bis(pyrazolium p-toluenesulphonate) (BPPTS), was analysed by both experimental and theoretical calculations against standard diphenyl picryl hydrazyl (DPPH) radicals. In order to validate the findings of the study, molecular geometrical optimization and Fukui function calculations were theoretically carried out to explore the sites for radical attack. Experimental analyses and theoretical findings are presented together in each section of this article to correlate both.

Results and discussion

Clinical biological evaluation and its bioinformatics correlation of BPPTS

Antimicrobial evaluation (in vitro and in silico)

The antibiotic activity of BPPTS on seven pathogens, namely Staphylococcus aureus, Bacillus subtilis, Salmonella paratyphi, Escherchia coli, Candida albicans, Aspergillus niger and Aspergillus fumigatus were clinically assessed using the Petri dish culturing method. The first four pathogens belong to the bacterial strains and the last three belong to fungal organisms. The former organisms were tested against standard ciprofloxacin and the latter were assessed using standard clotrimazole. Zone of inhibition (ZoI) and minimum inhibitory concentration (MIC, Table S1†) measurements were also performed.19 Experimentally evaluated results were validated with the aid of iGEMdock software in order to understand various intra and intermolecular interactions (electrostatic N–H···O, C–H···O, and O–H···O interactions). The antibacterial activity was evaluated using a single dose of 10 μg mL^–1 of standard ciprofloxacin and 100 μg mL^–1 of BPPTS. It exhibits strong to moderate inhibition activities of 63% against Bacillus subtilis (G^+ve) and 47% inhibition against Escherichia coli (G^–ve). Lower inhibitions were also observed on Salmonella aureus (25%) and Salmonella paratyphi (23%). The results show that BPPTS is the number one inhibitor against Bacillus subtilis (G^+ve) among the assessed pathogens. This was validated by molecular docking through various intermolecular interactions through minimum binding energy values. In Bacillus subtilis (PDB: ; 4OZ5), there are two strong intermolecular interactions observed between HD21 (N–H site) of ASN70 and ‘O’ atom of the p-toluenesulphonate moiety (N–H···O) at a distance of 2.36 Å, and between the phenolic ‘O’ atom of TYR134 and the N–H of pyrazolium moiety (O···H–N) at a distance of 2.76 Å. The total binding energy of the system was calculated to be –96.96 kcal mol^–1, which is a combination of three interactions, namely, van der Waals interaction (VDW –70.50 kcal mol^–1), H-bonding (–22.01 kcal mol^–1) and electrostatic interactions (–4.45 kcal mol^–1). On the other hand, Escherichia coli was inhibited to a lower extent by BPPTS, which was evidenced by both experimental and in silico methods. The binding energy of Escherichia coli with BPPTS is –83.92 kcal mol^–1, which is composed of –55.37 kcal mol^–1 (VDW), –22.60 kcal mol^–1 (H-Bond) and –5.94 kcal mol^–1 (electrostatic interaction). Therefore, it was concluded that the clinically tested ZoI results excellently match theoretically calculated binding energy values. Bacillus subtilis shows an experimental ZoI of about 63% with a greater binding energy while Escherichia coli has an experimental inhibition of 47% with a lower binding energy. Molecular docking studies clearly revealed that N–H···O and O–H···O interactions are responsible for drug–receptor binding and the results were further ascertained by the Hirshfeld surface analysis discussed earlier. Experimental and simulated results are depicted in Fig. S1.†

Fungal strains were also effectively inhibited by BPPTS but not to the extent of bacterial strains. The inhibition efficacy of BPPTS on Aspergillus fumigatus and Aspergillus niger are 30 and 21%, respectively. There is no inhibition against Candida albicans. These results show that BPPTS is a favorable inhibitor against bacterial strains rather than fungal strains.

The fundamental reason for the different sensitivities of BPPTS against bacteria and fungi is because of structural differences between them.20 The cell wall of bacteria is made up of peptidoglycon, which is nothing but proteins (amino acids), while in the fungal pathogens, the cell wall is made up of chitin, a polysaccharide. The cell wall of fungi is harder and stabler than that of bacteria. The greater percentage inhibition of BPPTS against Bacillus subtilis is due to the easy penetration of the drug into the cell.21 In the case of Candida albicans, BPPTS was not able to damage the cell wall, which is stronger than the cell wall of bacteria.22

Antioxidant activity (in vitro and in silico)

To find out the action of antibiotic analysis in detail, the electron pairing mechanism between the drug and the target has to be found out. In order to predict the electron scavenging mechanism, antioxidant analysis is introduced. The free radical scavenging activity of BPPTS was determined by elimination of radicals from the cell damaged system by means of the decolouration observed in UV-vis spectrophotometric studies.23 During combative interactions with BPPTS, the λ_max (517 nm) of standard DPPH˙ radicals shifted towards the red region, confirming the free radical scavenging capability by pairing of electrons between BPPTS (antioxidant) and DPPH˙ (oxidant). The excessive attack by reactive oxygen species (ROS) on DNA and proteins promotes the replication of cancerous tissues in the human system. Designed chemicals were used to denature the radicals to prevent their replication. The BPPTS molecule donates a hydrogen free radical to DPPH˙ to reduce it into a yellow coloured non-radical (DPPH-H), which leads to a decrease in the absorbance at 517 nm. Three different concentrations were used to assess the scavenging activity of BPPTS, namely 100, 200 and 300 μL from a stock solution of 0.01 mg per 2 mL. The ligand scavenged the DPPH˙ at a 300 μL concentration. This is an appreciable scavenging activity compared to the standard ascorbic acid. The experimental scavenging action was further reestablished by quantum chemical calculations. For predicting the active radical sites, DPPH has also been optimized separately by employing the DFT/B3LYP/6-311++G(d,p) level of basis set and the optimized molecular geometry is depicted in Fig. 1. Natural bond orbital (NBO) analysis from the optimized geometry is also used to find out the respective radical pairs that would couple. Fukui function calculation24,25 is one of the elements in the Gaussian 09 suite to predict radical sites of the scavenger that can join with free radicals. For calculating Fukui functions, three different charges and multiplicities were employed, namely 0 1, –1 2 and 1 2 for neutral, anion and cation species, respectively. Fukui functions of BPPTS and DPPH are presented in Tables S2 and S3.† In general, the atomic centres with maximal values of Fukui indices are found to be the preferred sites for electrophilic and nucleophilic attacks.26 Unfortunately, the radical site selectivity has not been analyzed for a large number of reactions involving radicals. However, those atoms with a high value of softness (S⁰) are susceptible to radical attack.27 Additionally, the redistribution of electron density with the change in the number of electrons is not clearly predictable. So the relative electrophilicity (f_k⁺/f_k^–) and relative nucleophilicity (f_k^–/f_k⁺) of BPPTS and DPPH were calculated.28 The site with the maximal value of f_k⁺/f_k^– is the most expected site to be attacked by nucleophilic attack and those with the highest value of f_k^–/f_k⁺ have the greatest chances of electrophilic attack.

Fig. 1. Optimized molecular geometry of DPPH.

The global electrophilicity index (ω) is a measure of energy stabilization when the system acquires an additional electronic charge from the surroundings. The indexes (ω) of BPPTS and DPPH were found to be 2.816 eV and 0.062 eV, respectively. The local electrophilicity indexes ω_fK⁺ (nucleophilic sites), ω_fK^– (electrophilic sites) and ω_fK⁰ (radical sites) were calculated for both BPPTS and DPPH. In BPPTS, the electrophilicity index at ω_fK⁺ exhibits a maximal value of 0.154 eV on atom H18, which confirms the chances of an electrophilic attack on this atom. On the other hand, the ω_fK⁺ of DPPH is found to be 0.014 eV on atom O39. The atoms H17 of BPPTS and N20 of DPPH are likely to be attacked by nucleophiles with values of 0.087 and 0.047 eV (ω_fK^–). It is interesting to note that for the radical site attack (ω_fK⁰), atoms S35 of BPPTS and N20 of DPPH are readily available to interact with each other with electrophilicity indices (ω_fK⁰) of 3.284 eV and 0.382 eV, respectively. In the structure of DPPH˙, the free radical site is found at the hydrazyl nitrogen attached to the picryl moiety. Theoretical calculations strongly endorse the same free radical site at the same nitrogen.

DNA binding studies (in vitro and in silico)

Antioxidant analysis reveals the greater scavenging activity against DPPH radicals and this affirms the capability of the proposed drug to act as a predictable DNA binder. The binding affinity between the drug and DNA was predicted using the UV-vis spectroscopic technique. Upon a steady increase in the concentration of ctDNA with a fixed concentration of BPPTS, there was a hyperchromic shift observed at 265 nm without any blue or red shift. The minor isosbestic point at 285 nm reveals the formation of new species during the combative reaction. The groove mode of interaction between the aromatic chromophore of base pairs (CG/AT) and BPPTS was confirmed by the hyperchromic shift at 265 nm. The interaction of BPPTS with ctDNA is evident from the UV-vis spectra (Fig. 2). The UV-visible spectroscopy studies confirmed that there is a DNA–ligand binding interaction but were not able to pinpoint the exact type of interaction. Therefore, in silico experiments were carried out to identify the exact type of interaction. During docking of BPPTS with ; 1BNA, assessed from Protein Data Bank (PDB), there is a strong C–H···O_guanine interaction observed between DG10 (O4) of ; 1BNA and the C–H site of the pyrazolium moiety of BPPTS at a distance of 2.44 Å. Similarly, DC9 (O2) of ; 1BNA interacted through a weak C–H···O_cytosine intermolecular interaction with the C–H site of the pyrazolium moiety of the drug at a distance of 3.08 Å. There is also a weak N–H···O intermolecular interaction between the N2 atom of guanine (DG16) and the oxygen atom of the p-toluenesulphonate moiety of BPPTS observed at a distance of 3.08 Å. A very weak T-shaped π···π interaction between DC11 and the centroid of the p-toluenesulphonate moiety is also observed (5.97 Å). The low % of C···C contacts (2.4) in the Hirshfeld surface analysis described earlier is now validated through docking.

Fig. 2. DNA binding: (a) in silico binding of ctDNA (; 1BNA) and BPPTS; (b) UV-vis spectra of ctDNA–BPPTS binding.

ADMET properties of BPPTS

Apart from the antibiotic, antioxidant and DNA binding studies, it is essential to find out the drug-likeness and toxic properties of the proposed drug alone. So the toxicological and ADMET properties were calculated theoretically with the help of the online server PreADMET. Parameters which are executed by PreADMET are human intestinal absorption (HIA), permeability to Madin-Darby Canine Kidney cells (P_MDCK), carcinogenicity, drug penetration of the blood–brain barrier (P_brain–P_blood), permeability of skin (P_skin), mutagenicity and carcinogenicity. Evaluating the ADMET properties is the pre requisite for the use of a ligand in a clinical screening.

The cell permeability values in intestinal epithelium cells in PreADMET are as follows. If the value is less than 4 nm s^–1, then it is low permeability, if the value is between 4 and 70 nm s^–1, then the permeability is medium and if it is above 70 nm s^–1, then the permeability is high. BPPTS exhibits 0.38 nm s^–1 in the intestinal epithelium and this indicates the existence of low permeability. Similarly, for the human intestinal absorption (HIA), the BPPTS ligand exhibits 76.5% and this is very high absorption in the human intestine, which is based on the general ranges: 0–20% – poor, 20–70% – moderate and 70–100% – high. For P_MDCK, BPPTS shows low permeability with 1.09 nm s^–1 since this value is less than 25 nm s^–1. The blood–brain barrier (P_BBB) was used to measure the penetration of the ligand into the central nervous system (CNS). The penetration of BPPTS into the CNS was found to be in the category middle with a value of 0.151 brain/blood. The general ranges are: if the BBB value lies below 0.1 b/b, then it is low absorption, if the value falls between 0.1 and 2.0 b/b, it is middle and if it exceeds 2.0 b/b, it is considered to be high absorption in the CNS.

Plasma protein binding (PPB) of any ligand influences not only the ligand's action and but also disturbs the ligand's activity and efficacy. For drug targeting properties, the drug should not bind to the plasma protein when it is injected into our body to facilitate diffusion and transport across the cell membrane and finally reach the target section. The normal values of PPB are as follows: if the value is above 90%, it indicates a strong plasma protein binding with the ligand and if the value lies below 90%, then it reveals a weak binding. The ligand BPPTS has a weak binding with a value of 34.92%.

The Ames test (a DNA mutation test in bacteria) interprets the mutagenicity of compounds and this test is used to determine whether the synthesis of histidine in DNA is enhanced or inhibited by the new ligand and also calculates the growth inhibition of the mutagenic agent.29,30 The test using BPPTS showed a positive mutagenic prediction.

Spectral analyses

NMR spectral analysis confirms the different proton environment (¹H) and carbon (¹³C) skeleton of the BPPTS. ¹H and ¹³C NMR spectra are shown in Fig. S2a and S2b,† and the atom labeling is given in parentheses. ¹H NMR signal assignments are as follows: solvent CDCl₃ (δ 7.29 ppm), H(1) & H(1′) (δ 13.6 & 13.3, respectively), H(2) (δ 7.83 ppm), H(3) (δ 6.59 ppm), H(4) (δ 2.38 ppm), H(5) (δ 7.21 ppm) and H(6) (δ 8.07 ppm). ¹³C NMR signals exhibited by BPPTS are: solvent CDCl₃ (δ 77 ppm), C(1) (δ 125 ppm), C(2) (δ 129 ppm), C(3) (δ 107 ppm), C(4) (δ 21 ppm), C(5) (δ 140 ppm) and C(6) (δ 133 ppm). Fourier transform infrared and Raman spectra of BPPTS are shown in Fig. S3a and S3b.† FT-IR spectroscopic analysis shows that there exists a stretching band at 2362 cm^–1 confirming the protonated N⁺–H tertiary amine cation.31 The bands at ν 1259 cm^–1 (asymmetric) and ν 1031 cm^–1 (symmetric) of BPPTS confirm the deprotonation in the –SO₃H moiety. The other significant bands are: 3163 cm^–1 (ν N–H 2° amine), 3057 cm^–1 (ν C–H), 2983 cm^–1 (ν asy CH₃), 2880 cm^–1 (ν sy CH₃), 1355 cm^–1 (ν C–N aryl 2° amine), 862 cm^–1 (ν C–S), 1540 cm^–1 (δ N–H) and 1473 cm^–1 (skeletal vibration of the aromatic ring). For Raman spectroscopic analysis, the significant bands are: 3130 cm^–1 (ν N–H 2° amine), 3064 cm^–1 (ν C–H), 2923 cm^–1 (ν –CH₃), 1381 (ν C N), 1211 cm^–1 (ν C–N), 1171 (ν asy –CH₃), 1032 cm^–1 (ν sy –CH₃) and 802 cm^–1 (δ N–H). The potential energy distribution (PED) of BPPTS was calculated using DFT with B3LYP/6-311++G(d,p) level of basis set to validate the experimental findings. The PED analysis of BPPTS is presented in Table S4.†

Stability analysis

Although BPPTS is stable in methanol as the solvent, its stability in various solvents were checked in order to check its stability to be used as a proposed drug.32,33 The UV-visible absorption spectra of BPPTS in methanol and ethanol are shown in Fig. 3(a and b).

Fig. 3. UV-vis absorbance spectra of BPPTS in (a) methanol and (b) ethanol.

The strong absorption band at 236 nm is due to the π–π* transition in ethanol. Similarly, in methanol, the bands at 234 and 261 nm are due to the n–π* and π–π* transitions, respectively. The absorbance was measured in a ∼3 hour interval for 50 h. It was noticed that there was no blue or red shift throughout the analysis. There was also only insignificant changes in absorbance values, supporting the stability of the proposed drug. BPPTS's solubility and stability in a biocompatible solvent like methanol is an additional advantage in pharmaceutical applications. The stability of BPPTS was further determined by NMR spectroscopy at different time intervals ranging from 0 to 120 minutes using DMSO-d₆ as the solvent. NMR spectra of BPPTS are depicted in Fig. S4.†

Theoretical calculations

Optimized molecular geometry

Optimized molecular geometry was obtained theoretically to understand the stability of BPPTS through various inter- and intramolecular hydrogen bonding interactions such as N–H···O, N⁺–H···O^– and C–H···O. The DFT method with the B3LYP34–36 functional at the 6311++G(d,p) level of basis set was employed37 and the optimized structure is shown in Fig. 4. Intermolecular hydrogen bonding interactions in BPPTS, N19⁺–H24···O9^– (2.643 Å), N20–H25···O38 (2.618 Å), N47⁺–H52···O9^– (2.651 Å) and N48–H53···O37 (2.614 Å), stabilise the molecular system. The N20–H25···O38 and N48–H53···O37 interactions are much stronger than the other two N⁺–H···O^– interactions due to the steric hindrance of O36 on O37 and O38 atoms. A weak C6–H18···O36 intermolecular interaction also supports the stability of the system with the bond distance of 3.514 Å. In the p-toluenesulphonate moiety, the bond distance of S7–O9 (1.544 Å) is shorter than the normal bond distance of S–O (1.652 Å), which confirms the loss of protons from the sulphonic acid group. This is evident from the optimization of free p-toluenesulphonic acid and free pyrazole under the same level of basis set [B3LYP/6311++G(d,p)*], which isshown in Fig. S5a and S5b.† The bond distance of C1–S7 (1.803 Å) in BPPTS is longer than the normal bond distance of C–S (1.787 Å) and this elongation is due to the influence of stronger double bond character of S7–O9. Similarly, in the pyrazolium moiety, the bond distances of N47⁺–H52 and N48–H53 are 1.060 and 1.066 Å, respectively, which are longer than the normal bond distance of N–H (1.007 Å) due to the higher electronegative nature of the oxygen atom in N–H···O interactions. The bond angle of C1–S7–O9 (S–O) is 104.1° (p-toluenesulphonate), which is higher than the value of 102.6° found in free p-toluenesulphonic acid. The C1–S7–O8 (S O) and C1–S7–O10 (S O) bond angle values are 107.6° and 107.5° (p-toluenesulphonate), respectively, which are shorter than the normal angle (109.0°) in free p-toluenesulphonic acid. These alterations in the calculated bond angles established the deprotonation of p-toluenesulphonic acid. It is interesting to observe that the stability of the system is further reinforced by the hydrogen bonded supramolecular ring graph set motif.38 The optimized geometrical parameters for an isolated molecule match quite well the observed structural parameters in SXRD and these results were discussed in the experimental SXRD section of this article. Optimization of BPPTS was exclusively carried out to derive stabilized interaction profiles between the drug and the target in molecular docking studies.

Fig. 4. Optimized molecular geometry of BPPTS.

Molecular electrostatic potential analysis

The molecular electrostatic potential (MEP) is a widely used analytical tool for interpreting the relationship between the molecular structure and reactivity through three dimensional visualisation of charge distribution and theoretical calculations.39–42 Politzer et al.43 exploited MEP to describe a variety of chemical phenomena such as bonding, inductive effect, chemical reactivity, resonance, etc. The regions conducive to the electrophilic interactions are governed by substantial negative values (the surfaces of the atoms are red in colour) of MEP. The presence of positive value in MEP explains the electron deficient regions (the surfaces of the atoms are blue in colour). The decreasing order of positive potential in the atoms is blue > green > yellow > orange > red. It is readily seen that the negative potential is largely localized at oxygen atoms of the p-toluenesulphonate moiety such as S7–O8, S7–O10 and S35–O36 and these nucleophilic atoms can easily interact with the electrophilic sites of proteins and nucleic acids. Similarly, the electron deficient centres of the pyrazolium moiety such as C22–H27, C50–H55 and the remaining C–H sites of pyrazolium are exclusively expected to interact with the electron rich sites of pathogens and DNA. Aromatic C–H and centroid sites available in BPPTS are also electrophilic (surfaces of the atoms are green in colour) in nature and they would interact with the nucleophilic centroids of amino and nucleic acids of proteins and DNA, respectively. The mode of interaction between the proposed drug (BPPTS) and targets is through a strong intermolecular N–H···O, C–H···O, O–H···O hydrogen bonding and C–H···π, π···π stacking interactions.44 This is further evidenced by the experimental biological screening and molecular docking studies discussed elsewhere in this paper. Fig. 5 depicts the MEP showing the active nucleophilic and electrophilic sites of BPPTS.

Fig. 5. Molecular electrostatic potential of BPPTS.

HOMO–LUMO energy gap analysis

Calculation of the HOMO–LUMO energy gap from frontier molecular orbital theory aids in determining the chemical reactivity and kinetic stability.45 A molecule with a small energy gap is essential for the large stabilization of LUMO.46 In order to predict the band gap of BPPTS, the DFT method with B3LYP/6-311++G(d,p) level of basis set was employed. The transition of the electronic cluster from the HOMO to the LUMO explains the existence of charges around the atoms in their excited state. The distribution of charges is represented by means of red and green colours for positive and negative phases, respectively.

The entire electron density of the p-toluenesulphonate moiety is located in the HOMO and it is moved to the pyrazole centre (LUMO) during the transition in BPPTS. The HOMO–LUMO energy gap is predicted to be 5.439 eV, which is responsible for the eventual charge transfer between p-toluenesulphonate and pyrazolium moieties enhancing the chemical reactivity. This band gap energy is lower than that of both the reactant molecules, with energy values of 5.969 for p-toluenesulphonic acid and 6.721 eV for pyrazole. The HOMO and LUMO of all three molecules are shown in Fig. 6. The HOMO–LUMO energy gap of BPPTS shows a great correlation with the experimentally observed biological activity of BPPTS on microbes and DNA.47

Fig. 6. HOMO–LUMO energy gap analyses of (a) BPPTS, (b) p-toluene sulphonic acid and (c) pyrazole.

Hirshfeld surface analysis

Hirshfeld surface analysis is an excellent tool to explore the partitioning of the crystal electron density into molecular fragments.48,49 To illustrate the connectivity of individual molecules in BPPTS crystals and to study the atom–atom interactions, the computational software CrystalExplorer v3.150 was employed. The first and foremost step of the Hirshfeld surface analysis is to map the d_e (distances of an atom external) and d_i (distances of an atom internal). The fingerprint plots (Fig. 7) generated from the combination of both d_e and d_i represent the intensity change of the atom–atom interaction from blue to green to red.51 The percentages of contacts that contribute to the total Hirshfeld surface area for various contacts such as O···H/H···O, N···H/H···N, N···C/C···N, C···H/H···C, C···C and H···H are 43.1, 2.6, 0.8, 14.2, 2.4 and 36.1, respectively.

Fig. 7. 2D Fingerprint plots of BPPTS and its various intermolecular contacts.

Five red spots observed in the d_norm of BPPTS (Fig. 8) vividly expose the strong interaction sites. The strength of contacts is further ascertained by the relative size of the spots on d_norm. The size of the red spots is larger at the C–H and N–H surfaces of pyrazolium followed by p-toluenesulphonate oxygens, and by N⁺–H of the pyrazolium moiety.52 In addition, some countable numbers of faint red spots are observed in BPPTS which are due to C–H···O/C–H···N interactions. The highest proportion of H···H contacts of BPPTS makes up 36.1%, which is due to the interaction between the C–H hydrogen of p-toluenesulphonate and the C–H hydrogen of the adjacent p-toluenesulphonate (C–H···H–C). The H of BPPTS has chances of interacting with the other H atoms of proteins and DNA at a weaker level of electrostatic interaction. It is interesting to observe that the hydrogens present in methyl groups are not involved in H···H contacts, whereas the ring hydrogens make possible contacts and the same trend was observed in the SXRD packing diagram shown in Fig. S6.† The flat red regions denoted as 1, 2, 3 & 4 in d_e (Fig. 9a) arise from the hydrogen bond acceptors O4 and O3 of p-toluenesulphonate involved in N2–H18···O4 (2.479 Å) and N4–H23···O3 (2.799 Å) hydrogen bonding interactions and the same interactions were observed in SXRD experiments also. The sharp spikes represented in the 2D fingerprint plot (Fig. 7) confirm the O···H/H···O counter contacts with a percentage contribution of 43.1. The nitrogen atom (N3) of the pyrazolium moiety does not involve in intermolecular N···H contacts due to its positive charge and the small contribution of N···H (2.6%) is due to the involvement of uncharged nitrogen (N4) with nearby hydrogens. The contribution of C···C contacts is 2.4% only due to the ring carbon. It is concrete proof to validate the DNA binding of BPPTS through the groove mode of interaction.53 It is noted that there is a hyperchromism observed in UV-vis absorbance spectroscopy and this has been confirmed by molecular docking studies discussed elsewhere in this manuscript. The shape index confirms the presence of weaker π···π interactions in the system with inverted blue and red coloured triangles. 3D plots also reinforce the weaker π···π interactions (Fig. 9b).

Fig. 8. d _norm of BPPTS.

Fig. 9. Hirshfeld surface maps of BPPTS.

Experimental and theoretical methods

Synthesis of BPPTS

Equimolar proportions of 1H-pyrazole and p-toluenesulphonic acid were used to synthesize the proton transfer compound bis(pyrazolium-p-toluenesulphonate) (BPPTS). Both the reactants were purchased from Sigma–Aldrich and they were used without any further purification. A methanol–water mixture was used as a solvent for dissolution. The resultant solution was stirred at 30 °C for 8 hours and the solution was filtered off to get rid of impurities and kept aside for slow evaporation under a dust free atmosphere at 30 °C. The pale pinkish coloured crystals were harvested after 15 days and were recrystallized several times for better purity. The chemical reaction for the synthesis of BPPTS is shown below.

Physico-chemical characterization and structural analysis

Various functional groups present in BPPTS were confirmed using Fourier transform infrared (FT-IR) and Fourier transform Raman (FT-Raman) spectra recorded in the ranges of 4000–400 cm^–1 and 4000–50 cm^–1, respectively. The FT-IR spectrum was recorded using a PerkinElmer RX1 spectrophotometer using the KBr pellet technique. The FT-Raman spectrum was recorded using a Bruker RFS 27 stand-alone FT-Raman spectrophotometer with a 1064 Nd:YAG laser of 100 mW power. UV-visible analysis was carried out using a JASCO spectrometer (V-570) between 200 and 800 nm. ¹H and ¹³C nuclear magnetic resonance spectra of BPPTS were recorded using a BRUKER-DRK 50–400 MHz instrument with tetramethylsilane (TMS) as an internal standard and CDCl₃ as a solvent. Crystal data of BPPTS were collected at 293 K on an Oxford diffractometer (CrysAlis^Pro CCD) using a Cu-X-Ray source equipped with a graphite monochromator. Data reduction was carried out using CrysAlis^PRO and the structure was solved and refined using SHELXS9754 and SHELXTL97,55 respectively. All non-hydrogen atoms were refined anisotropically. PLATON was employed to acquire the ORTEP of BPPTS.

Methodology for theoretical calculations

Quantum chemical calculations (QCC) data were computed using the Gaussian 09, Revision E.01 suite.56 Geometry optimization, molecular energies and molecular properties were calculated by DFT with B3LYP/6-311++G(d,p) level of basis set. Molecular electrostatic potential analysis (MEP),57 and HOMO–LUMO band gap and Fukui function calculations of BPPTS were carried out using the same level of the basis set. The data on various inter- and intramolecular contacts of BPPTS were obtained from Hirshfeld surface analysis using CrystalExplorer 3.1 software package.

Biological screening procedures

Antimicrobial screening

The inoculums were prepared in fresh nutrient broth from well-maintained slant culture. McFarland standard58 was used as a reference for maintaining the turbidity of the inoculums by the addition of sterile saline. The prepared inoculums were dried at room temperature. Ciprofloxacin (10 μg) was used as a standard drug for antibacterial evaluation. For an effective distribution, the Petri dishes were placed in the refrigerator at 4 °C for 60 minutes followed by an incubation process at 37 °C for 24 hours. The clotrimazole (10 μg) drug was used as a standard for antifungal screening and it was preserved in Sabouraud's dextrose broth. Various zones of inhibition of BPPTS were observed and measured. The technique of Perez et al.59 was employed to screen the antimicrobial activity of BPPTS. Seven pathogens commonly encountered in the literature, Escherichia coli, Staphylococcus aureus, Salmonella paratyphi and Bacillus subtilis were used and the fungal strains Candida albicans, Aspergillus niger and Aspergillus fumigatus were used for biological screening.

DPPH assay

The free radical scavenging activities of BPPTS and the standard solution (ascorbic acid) were investigated using the DPPH˙ (1,1-diphenyl-2-picrylhydrazyl radical) scavenging method as reported in the literature.60 The assay mixture contained 2 mL of a 1.0 mmol L^–1 DPPH radical solution prepared in methanol and 1 mL of a methanolic drug solution at different concentrations (10–500 μg mL^–1). The solution was rapidly mixed and incubated in the dark at 37 °C for 20 minutes. The DPPH˙ solution was stable at 517 nm and the same wavelength was used as the initial wavelength. The percentage of radical scavenging activity was calculated using the following formula:where Ac = absorbance of the control at 517 nm and As = absorbance of the sample.

DNA-binding studies

Preparation of stock solution

50 mM of NaCl is used to dissolve 1 g of ctDNA to obtain a concentration of 2.27 × 10^–4 mol L^–1, which was calculated using a molar extinction coefficient of 6600 M^–1 cm^–1 at 260 nm. The test solution was prepared using pH maintained Tris-HCl with pH 7.4 adjusted by adding hydrochloric acid. The purity of ctDNA was checked by recording the absorbance spectrum using a UV-visible spectrophotometer and it was noticed that ctDNA was free from protein. Test solutions were prepared by appropriate dilution of the stock solutions with doubly distilled water. The concentration of the BPPTS stock was 2.40 × 10^–4 mol L^–1 and methanol was used as a solvent. Doubly distilled water was used to prepare the DNA stock solution and was kept at 4 °C and the experiment was carried out at 25 °C and was used within four days from the day of preparation. The absorbance spectra were recorded after making a homogeneous mixture of all additives against the blank solution.

UV-visible absorption analysis

A DNA binding study through electronic absorption titration experiments for BPPTS was performed at 25 °C with a fixed concentration of the compound and varying concentrations of DNA (0–40 μM).

Molecular docking programs

Docking validation for antimicrobial and DNA-binding studies

Molecular docking validation of drug–pathogen and drug–DNA binding interaction studies was carried out using iGEMdock61,62 and Patchdock63,64 programs, respectively, to generate the protein–ligand and nucleic acid–ligand interaction profiles. During ligand–protein binding, 120 runs were implemented to find out the binding energy profile. Out of 120 runs, the ligand–protein pair with the highest binding affinity was selected and the interaction profiles were visualized using PyMol,65 open source Schrodinger software. PatchDock was used to study the drug–DNA interactions through an automated online server for molecular docking. Both protein binding and nucleic acid binding were validated using experimental results and findings.

PreADMET analysis

Absorption, distribution, metabolism, excretion and toxic (ADMET) properties were calculated with the help of the online server PreADMET.66 For BPPTS, the ADMET properties were calculated.

Conclusion

Bis(pyrazolium p-toluenesulphonate) (BPPTS) acts as an active scavenger on DPPH radicals, which was confirmed by the Fukui function calculations. It was interesting to note that for a radical site attack (ω_fK⁰), atoms S35 of BPPTS and N20 of DPPH are readily available for sharing their electrons mutually with electrophilicity indexes of 3.284 eV and 0.382 eV, respectively. The formation of bonds by mutual sharing of electrons allows the possibility of preventing active radicals in cellular organisms. The effectiveness of the laboratory synthesized BPPTS was studied through various in silico methodologies such as HOMO–LUMO analysis, molecular electrostatic potential analysis, Hirshfeld surface analysis and molecular docking analysis. In all theoretical calculations, the experimental results matched well the simulated data. In addition, it was observed that BPPTS interacted with the DNA model (; 1BNA) through various non-covalent and electrostatic interactions. The active amino acids that participated in the interactions are guanine and cytosine. Strong C–H···O_guanine and weak C–H···O_cytosine intermolecular interactions act as a bridge to prevent the repercussion of DNA damage. Antimicrobial screening also reveals a greater inhibition efficacy of BPPTS against Bacillus subtilis. It was clear that the cell wall of both bacterial and fungal strains influenced the inhibition. From the above observation, BPPTS can easily destroy the cell wall of Bacillus subtilis because of the greater interaction on peptidoglycon of bacteria than chitin of fungal pathogens. The experimentally assessed antimicrobial screening was corroborated well with in silico docking analysis. The ADMET properties of BPPTS show a greater human intestinal absorption (HIA) of about 76.5% and this was very high absorption in the human intestine.

Conflicts of interest

There are no conflicts of interest to declare.