1,2,3-Triazoles of 8-Hydroxyquinoline and HBT: Synthesis and Studies (DNA Binding, Antimicrobial, Molecular Docking, ADME, and DFT)

Abstract

A new series of 1,2,3-triazole hybrids containing either 2- or 4-hydroxyphenyl benzothiazole (2- or 4-HBT) and naphthalen-1-ol or 8-hydroxyquinoline (8-HQ) was synthesized in high yields and fully characterized. In vitro DNA binding studies with herring fish sperm DNA (hs-DNA) showed that quinoline- and 2-HBT-linked 1,2,3-triazoles of shorter alkyl linkers such as 6a are better with a high binding affinity (3.90 × 10⁵ L mol^–1) with hs-DNA as compared to naphthol- and 4-HBT-linked 1,2,3-triazoles bound to longer alkyl linkers. Molecular docking of most active 1,2,3-triazoles 6a–f showed high binding energy of 6a (−8.7 kcal mol^–1). Also, compound 6a displayed considerable antibacterial activity and superior antifungal activity with reference to ciprofloxacin and fluconazole, respectively. The docking results of the fungal enzyme lanosterol 14-α-demethylase showed high binding energy for 6a (−9.7 kcal mol^–1) involving dominating H-bonds, electrostatic interaction, and hydrophobic interaction. The absorption, distribution, metabolism, and excretion (ADME) parameter, Molinspiration bioactivity score, and the PreADMET properties revealed that most of the synthesized 1,2,3-triazole molecules possess desirable physicochemical properties for drug-likeness and may be considered as orally active potential drugs. The electrophilicity index and chemical hardness properties were also studied by density functional theory (DFT) using the B3LYP/6-311G(d,p) level/basis set.

Introduction

Five-membered 1,2,3-triazoles are considered to be an important class of biologically active units.¹ 1,2,3-Triazoles possess significant chemical properties such as aromatic stability, resistance to acid–base hydrolysis, high dipole moment, and the ability to form H-bonds.^2,3 Triazoles have been used as precursors in the synthesis of various pharmaceutical drugs of medicinal use, and they also play important roles in organic synthetic chemistry. 1,2,3-Triazoles exhibit peculiar biological properties such as antibacterial,⁴⁻⁶ antitubercular,⁴ anti-inflammatory,⁴ antifungal,^7,8 antiallergic,⁹ and anticancer properties.^2,4,10−12 The triazole moiety readily associates with biological targets such as DNA via H-bonds and other noncovalent interactions, improving solubility and metabolic stability.¹³ In the past few decades, various metal-catalyzed synthetic methods have been developed for triazole synthesis. However, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is the most promising and exclusively used method to obtain products in high selectivity and yield.¹⁴⁻¹⁷

Thiazoles, an important class of heterocycles, consist of one N- and one S-atom in five-member rings. From a biological point of view, thiazole units are diversified as a core structure in many biologically important compounds and drugs.^18,19 For example, a group of xenobiotic compounds having the benzene ring fused with the thiazole unit is used worldwide for numerous therapeutic applications.¹⁹ Among them, hydroxyphenyl benzothiazoles (2-HBT and 4-HBT) and their analogues have attracted attention due to their wide pharmacological activities.^17,20−22 From a chemistry point of view, 2-HBT and 4-HBT are easily synthesizable, highly stable, and exhibit a fluorescence property.^17,23 For example, 2-HBT is considered one of the most common molecules that show the ESIPT (excited-state intramolecular proton transfer) mechanism, and our group recently reported it for cysteine sensing.²⁴

Quinolines are heterocyclic compounds with two fused six-member rings and a N-atom. Nowadays, 8-hydroxyquinoline (8-HQ) has gained significant attention due to various applications in organic and inorganic chemistry for biological activity.²⁵ The synthetic versatility of 8-HQ helps to synthesize a variety of multifunctional molecules such as sensors, metal-binding units, drug designing, anti-COVID-19, etc.²⁶

DNA, a key molecule, contains genetic instructions to control the structure and functions of cells. DNA is a principal target in drug design and development strategies to produce novel therapeutics for various diseases.²⁷ Therefore, studying various possible interactions of small molecules or drugs with DNA is important to understand the mechanism of small molecules or their actions to design specific DNA-targeting drugs. The action of small molecules or drugs with DNA is reported to involve either electrostatic or groove binding.^27,28

Because of the biological significance and the DNA-binding propensity of both 1,2,3-triazole and 8-HQ, it was of significant interest to build a single molecular framework with these biologically important units. Therefore, we aimed at synthesizing a series of 8-HQ-linked triazole hybrids of O-alkylated 2-HBT or 4-HBT (Figure 1). The 8-HQ-derived 1,2,3-triazole linked with O-alkylated 2-HBT or 4-HBT has not been reported in the literature so far. However, propargylated 8-HQ-derived 1,2,3-triazole hybrids have been reported for various pharmacological applications in recent years.^18,29−32 In our present study, we have investigated the effect of 2-HBT or 4-HBT and the quinoline ring present in 1,2,3-triazole derivatives on DNA binding and biological studies. Considering our research interest in click chemistry, herewith we present the synthesis and a biological study of triazole hybrid molecules containing 8-HQ or 1-naphthol and 2-HBT or 4-HBT molecular units.

Figure 1.

Designed 1,2,3-triazole hybrid molecules containing biologically active units.

Results and Discussion

For the one-pot synthesis of 1,2,3-triazoles (6a–l), we adopted the widely known CuAAC reaction by reacting alkyne with in situ generated organic azides as shown in Scheme 1.¹⁴ The free −OH groups of 8-HQ and 1-naphthol were derivatized with the propargyl group as alkyne (5a–b). Organic azides were in situ generated by the reaction of the bromoalkoxy derivatives of 2-HBT (3a–c) and 4-HBT (4a–c), which in turn were prepared using the reported procedure.³³

Scheme 1. Synthesis of Compounds Containing 8-Hydroxyquinoline/2-Naphthol and 2-HBT/4-HBT, 6a–l.

1,2,3-Triazole hybrid molecules were then synthesized using the CuAAC reaction of as formed organic azide and alkyne precursors.⁷ The resulting 1,2,3-triazole molecules were purified using silica gel column chromatography in good-to-high yields. Detailed experimental procedures and various characterization data of 6a–l are given in a Supporting Information (SI) file. The complete details on reaction conditions and product yields are compiled in Table 1. The synthesized compounds were fully characterized using various spectroscopic techniques. For instance, the ¹H NMR spectrum of compound 6a displayed peaks of hydrogens of the alkyl at δ 4.70 and 4.96 ppm as triplet each and a singlet due to −CH₂ of the propargyl group at δ 5.36 ppm in the aliphatic region.

Table 1. Synthesis of Compounds Containing 2-HBT or 4-HBT and Quinoline or Naphthalene Derivatives^a.

^aReaction conditions (6a–l): 3a–c or 4a–c (1 equiv), 5a or 5b (1 equiv), 1.2 equiv NaN₃, 10 mol % CuSO₄.5H₂O and 20 mol % Na-ascorbate, 8:2; v/v tetrahydrofuran (THF)/H₂O, 60 °C, and 6–8 h.

^bYield refers to the compound obtained by column chromatography with an increased polarity gradient of ethyl acetate:n-hexane as a mobile phase.

Quinoline and naphthol were located in the aromatic region protons of 2-HBT. A singlet at δ 8.32 ppm due to the triazolyl proton confirmed the cycloaddition reaction. ¹³C NMR spectrum showed three characteristic signals at δ 49.58, 62.61, and 67.65 ppm due to the ethyl linker chain having two aliphatic C-atoms and one carbon of the −OCH₂ group from the propargyl unit. The infrared spectrum showed characteristic absorption peaks at 1448, 1240, and 3142 cm^–1 corresponding to N=N aromatic, cyclic N–N=N, and C–H proton, indicating formation of the 1,2,3-triazole ring.^8,34 In the UV–vis spectrum, compound 6a showed a characteristic broad spectrum with maxima at around 330 nm, which could be attributed to the π–π* transitions of the 2-HBT skeleton. Finally, the structure of 6a was supported and assigned at m/z 480.14 (molecular ion) peak for C₂₇H₂₁N₅O₂S in electrospray ionization mass spectrometry (ESI-MS). For other synthesized compounds 6b–l, similar spectral patterns were obtained, and detailed characterization is provided in the Experimental Section and a SI file.

DNA Binding Studies

Synthesized 1,2,3-triazoles are supposed to form an adduct with DNA, which is stabilized by the hydrophobic and/or H-bonding interactions.³⁵ Electronic absorption technology is the most commonly used technique for in vitro DNA binding studies. The changes in the absorption spectra provided information about interactions between molecules and DNA.^28,35 Systematic absorption titrations were carried out between 1,2,3-triazoles (6a–l) and the herring fish sperm DNA (hs-DNA) for quantitative evaluation of binding interactions. The synthesized 1,2,3-triazoles showed a characteristic absorbance peak in the region of 320–340 nm due to π–π* transition of 2- or 4-HBT-linked 1,2,3-triazoles. The absorption spectroscopic results showed that increasing the DNA concentration decreases the absorbance peak at 330 nm for different 1,2,3-triazole hybrid molecules, and the recorded change is shown as a linear plot for calculation of the binding constant, as shown in Figure 2 insets.³⁶ The UV–vis titration spectra of the remaining compounds 6g–l with DNA and the binding constant calculation using the respective linear plot method are given in the Supporting Information (Figure SI-53). The calculated hypochromism% (H%) and binding constant (K_b) of synthesized compounds 6a–l are shown in Table 2.

Figure 2.

Absorption titration spectra of 6 (a–f); the insets show the linear binding plot and the arrow shows the observed change in absorbance with increasing concentration of hs-DNA (0–2.6 × 10^–6 M).

Table 2. Calculated Hypochromism% (H%) and Binding Constant (K_b) of Synthesized 1,2,3-Triazoles 6a–l with hs-DNA.

^aH% or hypochromism %: [(A_f – A_b)/A_f] × 100, where A_f is the absorbance of free compounds and A_b is the absorbance of bound compounds.

^bIntrinsic binding constant is represented by K_b.

On studying the calculated binding constant values of the triazoles, it was found that 2-HBT-derived triazole derivatives linked to 8-hydroxyquinoline 6a–c showed a higher binding affinity to hs-DNA as compared to 4-HBT-derived triazoles linked to 8- hydroxyquinoline 6g–i. A similar trend was observed for 2-HBT- and 4-HBT-derived naphthol-linked triazoles 6d–f and 6j–l, respectively, i.e., (6d–f) > (6j–l). This makes us conclude that 2-HBT-derived triazoles possess better binding affinity than 4-HBT-derived triazoles. The quinoline-linked triazoles have a comparatively higher binding affinity than naphthol-linked triazoles, i.e., 6a–c > 6d–f. Also, triazoles with shorter alkyl linkers (n = 2) showed better binding affinity than triazoles with longer chain lengths, i.e., 6a > 6b > 6c and 6d > 6e > 6f. The binding constant of the triazoles was found to be in the range of (0.09–3.9) × 10⁵ L mol^–1. To understand the mechanism and mode of DNA binding, a molecular docking study was performed, and the results correlate well with in vitro DNA-binding analysis results.

Molecular Docking Studies

Molecular docking was performed on 6a–f compounds with the DNA dodecamer duplex sequenced (CGCGAATTCGCG)₂ (PDB ID: 1BNA) using AutoDock Vina³⁷ (open source). For a better insight into the mechanism of interaction, we performed molecular docking studies. For this, we found that the model with PBD ID: 1BNA has been extensively used in the literature³⁸⁻⁴² to predict the mode of binding with DNA. Inspired by this, we performed molecular docking on 6a–f compounds with the DNA dodecamer duplex using AutoDock Vina (open source).

In this study, the structures of compounds 6a–f were made flexible to obtain different conformations to predict the binding mode with DNA. The docked structure with the energetically most favorable conformation was analyzed. The interactions of compound 6a with the DNA residue are shown in Figure 3 and the remaining in Figures SI-54–SI-58. The best docking poses are shown in Figures SI-59 and SI-60.

Figure 3.

(a) Three-dimensional (3D) and (b) two-dimensional (2D) diagrams showing binding interactions of compound 6a with the DNA residue.

All of the six compounds bind to DNA preferably through the groove mode in the CG-rich region. The docking result of 6a showed that the O- and N-atoms of 8-HQ formed conventional H-bonds with DG2, and the N-atom of the triazole showed H-bonding with DG4. The C-atom of the alkyl linker next to the O-atom and the C-atom of the triazole ring reflected C–H-bond formation with DC23. The π-orbitals of 6a were involved in π-donor H-bonds, and the triazole ring was involved in a π–π T-shaped hydrophobic interaction with DG22. The calculated binding energy of 6a was the highest (−8.7 kcal mol^–1) among all compared compounds 6a–f, and the binding energies are listed in Table SI-1.

As depicted in Figures SI-54–SI-58 for compounds 6b–f, the O-atom of 6b linked to 8-HQ formed conventional H-bonds with DG2, similar to 6a. The O- and N-atoms of 8-HQ are involved in H-bond formation. The π-orbitals of 8-HQ and triazole are also involved in π-donor H-bond formation with DG24. Compound 6c showed that the middle N-atom of the triazole ring was involved in H-bond formation with DG4, while N- and O-atoms of 8-HQ formed H-bonds with DG2. The π-orbital of the triazole ring formed π-donor H-bonds with DG22. One carbon atom each from the alkyl linker and the triazole ring showed C–H bond formation with the DC23 residue of DNA. The N-atom of the 6d triazole ring formed H-bonds with DG22, and π-orbitals of the triazole ring showed π-donor H-bond formation with DG4. The N-atom of 2-HBT acts as a H-acceptor by forming C–H bonds with DA6. One of the N-atoms of 6e triazole was involved in H-bond formation with DG22, whereas the C-atom of the −CH₂ group toward the naphthol ring also showed C–H bond formation with DC23. The O-atom of 6f 1-naphthol and one of the N-atoms of the triazole ring formed H-bonds and π-anion H-bonds with DG22, respectively. The triazole ring was also involved in π-donor H-bond formation with the DG4 residue of DNA. We also docked positive compounds 8-HQ, 2-HBT, and 4-HBT and calculated the binding energy corresponding to the energetically most favorable conformation. It was found that 6a has a large negative value of binding energy compared to positive compounds (8-HQ, 2-HBT, and 4-HBT), which is indicative of the higher binding potential of 6a to DNA.

The dominant forces of the binding process are hydrogen bonding and hydrophobic interactions, followed by electrostatic forces. The compounds bound themselves in the area rich in CG base pairs, and compound 6a was found to be the most promising candidate. The shorter bond lengths and the large negative value of binding energy are indicative of higher binding potential. Computational simulation showed that all of the six compounds 6a–f bind DNA preferably through the groove mode in the CG-rich region. Hence, theoretical results are in good agreement with experimental DNA-binding analysis results.

Evaluation of Pharmacological Properties

Antibacterial Activity

Synthesized 1,2,3-triazoles 6a–l were screened against two Gram-positive strains (Staphylococcus aureus and Bacillus subtilis) and two Gram-negative strains (Escherichia coli and Pseudomonas aeruginosa) using the agar-well diffusion method⁴³ as shown in Table 3. To determine the activity of the sample, 50 μL of the compound test sample was added and incubated overnight at 37 °C; then, the diameter of the sample was recorded in millimeters as diameter of inhibition zone (DIZ). Further, ciprofloxacin and dimethyl sulfoxide (DMSO) were used as the reference and the negative control, respectively. The synthesized 1,2,3-triazole hybrid molecules displayed considerable in vitro antibacterial activity against the tested organisms. Compound 6a displayed a comparatively better result among all with the zone of inhibition in the range of 15.5–17.6 mm.

Table 3. Antibacterial Activity of Synthesized Triazoles 6a–l.

	DIZ (diameter of inhibition zone in mm)
	Gram-positive strain		Gram-negative strain
compound	S. aureus	B. subtilis	E. coli	P. aeruginosa
6a	17.6 ± 0.2	15.5 ± 0.1	17.2 ± 0.4	15.8 ± 0.2
6b	11.4 ± 0.1	10.8 ± 0.2	12.3 ± 0.3	11.9 ± 0.1
6c	9.6 ± 0.1	9.4 ± 0.4	9.2 ± 0.1	8.8 ± 0.3
6d	15.4 ± 0.3	14.1 ± 0.1	14.5 ± 0.2	13.8 ± 0.2
6e	10.9 ± 0.1	10.3 ± 0.3	11.6 ± 0.4	11.2 ± 0.1
6f	9.2 ± 0.1	9.6 ± 0.4	10.0 ± 0.1	9.8 ± 0.2
6g	13.2 ± 0.2	12.7 ± 0.3	12.1 ± 0.3	11.3 ± 0.2
6h	9.9 ± 0.3	9.4 ± 0.2	10.6 ± 1.1	10.7 ± 0.1
6i	9.5 ± 0.4	9.3 ± 0.1	9.5 ± 0.2	9.0 ± 0.2
6j	8.7 ± 0.3	9.2 ± 0.3	8.5 ± 0.1	8.6 ± 0.2
6k	9.4 ± 0.4	9.1 ± 0.3	9.3 ± 0.3	9.9 ± 0.1
6l	8.5 ± 0.1	8.8 ± 0.2	8.2 ± 0.2	8.7 ± 0.1
DMSO	0	0	0	0
ciprofloxacin	24.1	23.4	22.3	22.8

Antifungal Activity

The synthesized triazole hybrids 6a–l were also screened for antifungal activity against two fungal strains (Candida tropicalis and Aspergillus terreus) with DMSO as the negative control. The DIZ was measured under similar conditions as above and is detailed in Table 4. Synthesized triazoles were found to give good-to-better results against both fungal strains, and compound 6a showed the best results. The antifungal activity experiments revealed that the synthesized compounds exhibited a notable inhibition of fungal strains. The antifungal agent azole primarily shows efficacy by suppressing the cytochrome 450-dependent transformation of lanosterol to ergosterol.

Table 4. Antifungal Activity of Synthesized Triazoles 6a–l.

However, triazoles also act as inhibitors to 14α-demethylase and thereby suppress the synthesis of ergosterol. To investigate all of the possible interactions, molecular docking studies were performed, in which the synthesized triazoles were docked into the active site of the fungal enzyme lanosterol 14-α-demethylase (PDB ID: 1EA1)^44,45 as shown in Table SI-2. The 3D diagram showing the active site of the enzyme and the docked pose of 6a in the active site of the lanosterol 14-α-demethylase enzyme is shown in Figure 4. The 2D and 3D interacting modes of compound 6a in the active region of lanosterol 14-α-demethylase are presented in Figure 5. The 2D diagrams representing the possible interaction of compounds 6a–l with the lanosterol 14-α-demethylase enzyme are shown in Figures SI-61–SI-66.

Figure 4.

(a) 3D diagram showing the active site of the enzyme (yellow sphere) and (b) the docked pose of 6a with lanosterol 14-α-demethylase (active site). Heme molecule is shown in pink.

Figure 5.

(a) 2D and (b) 3D diagrams showing the interaction of 6a docked in the active site of lanosterol 14-α-demethylase. Heme molecule is shown in orange.

The O- and N-atoms of 8-HQ showed H-bond interaction with ARG96 having bond lengths of 2.29 and 2.42 Å, respectively. The π-orbitals of the triazole ring were involved in an electrostatic interaction with NH₂ of ARG96 and MET79, and the π-orbitals of the 2-HBT-phenyl ring with HEM460. Also, π-orbitals of the triazole ring are engaged in a π–σ interaction, and a π–sulfur interaction is present between HEM460 and the S-atom of 2-HBT. The π–π stacked interaction is seen between π-orbitals of 2-HBT and HEM460. The overall H-bond interaction, electrostatic interaction (π–cation), and hydrophobic interaction (π–σ) were found to be dominating nonbonding interactions. The binding energy of 6a was found to be −9.7 kcal mol^–1. Similar to our in vitro antifungal activity, 6a showed better interaction with the fungal enzyme among all of the triazoles.

Absorption, Distribution, Metabolism, and Excretion (ADME) Prediction

The oral bioavailability of any chemical compound is predicted by the calculation of certain physicochemical properties, i.e., log P, TPSA, molecular weight, the number of H-bond donors and acceptors, etc. For this, Lipinski’s rule of five is widely used, which states that an orally active drug needs to meet the criteria of (a) MW ≤ 500, (b) MLogP ≤ 4.15, (c) HBD ≤ 5, (d) HBA ≤ 10, and (e) the number of violations ≤1.⁴⁶ These parameters were calculated by the online available swissADME web tool (http://www.swissadme.ch/) and are presented in Table SI-3. The more positive the value of the drug-likeness score, the more likely the chemical compound to be a potential drug. The molecular parameters indicate that most of the synthesized 1,2,3-triazole hybrid molecules meet the criteria of Lipinski’s rule of five, except 6f and 6l. The MLogP (octanol/water partition coefficient) was also calculated, which predicts the lipophilic efficiency of the compound. The hydrophobicity of the molecule can be predicted by the log P value and plays an important role in the distribution of drug after absorption in the body. TPSA gives information about the polarity of the compound. It is a useful physicochemical parameter to analyze the transport property of the molecule, and it is in the acceptable range of <160 A². The more the number of rotatable H-bonds, the more the flexibility of the molecule to achieve different conformations. For almost all compounds, it was found to be <10. The topological parameter and the number of rotatable bonds are considered to be good descriptors of the oral bioavailability of drugs.⁴⁷

The drug under study is supposed to bind with the biological target. The biological target can be any common protein such as ion channels, enzymes, and receptors. The biological target is also known as the drug target. The larger the bioactivity score, the higher the probability of the chemical compound to be biologically active. For the G protein-coupled receptor (GPCR) ligand, the bioactivity score was found to be in the inactive range for most of the triazoles, which indicated that triazole produces physiological action by interacting with the GPCR ligand (Table SI-4). The bioactivity score for an ion channel modulator is in the −0.32 to −0.42 range, which suggests moderate interaction with the target. The bioactivity scores for kinase and enzyme inhibitors are in the active range for all of the synthesized triazoles. According to the literature, if the bioactivity score is greater than 0, the compound is active biologically, if it is between 0 and −0.5, moderately active, and if much lesser, inactive.⁴⁸ Therefore, it could be concluded that most of the triazole derivatives are biologically significant and exhibit desirable physicochemical properties for drug-likeness.

Results of our study displayed that most of the compounds are bound strongly to the plasma protein. The highly selective barrier of the brain and the remaining organs of the body are maintained by the blood–brain barrier (BBB); thus, all drugs that target the CNS (central nervous system) should show better BBB penetration. However, drugs that target peripheral organs must show low BBB penetration so as to minimize all associated side effects to CNS (Table SI-5). The compounds that show BBB less than 2.0 reflect high absorption to CNS, BBB between 2.0 and 0.1 shows intermediate absorption to CNS, and low absorption to CNS is observed if the BB value is less than 0.1. Compounds 6a–I showed intermediate-to-low absorption to CNS. Also, all compounds showed good human intestinal absorption (HIA).

Density Functional Theory (DFT) Study

Frontier molecular orbitals (abbreviated as FMOs) have extensively been studied with the help of DFT, an important property to calculate molecular reactivity parameters and design important drugs efficiently from the available database or a newly explored library of various chemical compounds of interest.^8,14,49 Molecular geometry optimization of all of the synthesized compounds containing 8-hydroxyquinoline/naphthalene and o,p-hydroxyphenyl benzothiazoles 6a–l was performed using the Gaussian 09 program at B3LYP/6-311G(d,p).⁵⁰ The synthesized compounds 6a–l were successfully optimized and verified at local minima. The lowest unoccupied molecular orbitals (LUMOs) of appropriate acceptor molecules accommodate electrons from higher-value highest occupied molecular orbitals (HOMOs). Koopman’s relation was applied for the LUMO–HOMO energy gap (ΔE_LUMO–HOMO) and other frontier orbital energy parameters such as the chemical potential of the tested molecules (μ) and the chemical hardness (η).⁵¹ However, Robert G. Parr approximation was applied for determining the electrophilicity index (ω) of the molecules.⁵² With the help of these three relations as detailed in our recent report,^7,8 all chemical reactivity parameters were computed and are summarized in Table SI-6.

The chemical hardness (η), which reflects the reactivity of any molecule, can be easily determined by the HOMO–LUMO energy gap. The higher the value of the HOMO–LUMO energy gap, the higher the hardness of the molecule. However, a lower value of the HOMO–LUMO energy gap implies a higher softness of the molecule. As we know, molecules with a higher energy gap or band gap require high excitation energy to overcome the energy barrier and thus are less reactive with respect to molecules possessing a lower energy gap. The FMO distribution pattern of all of the 1,2,3-triazole hybrid molecules 6a–l is shown in Figure SI-69. However, the FMOs of compound 6a, which showed high DNA-binding affinity and was found to be superior among all derivatives, are shown in Figure 6. The stabilizing energy of the compound is represented by ω and shows the augmentation of the electronic charge from the outer environment. The greater and lower value of ω and η, respectively, determine the reactivity of the compounds. Among all synthesized compounds 6a–l, compound 6d showed the least value of chemical hardness (1.82 eV) and a high value of the electrophilicity index (4.15 eV). However, 6a showed 2.19 and 3.42 eV for chemical hardness and the electrophilicity index, respectively.

Figure 6.

FMO distributions at the ground state of the 6a molecule.

Conclusions

A new series of 2-HBT and 4-HBT-derived triazoles linked with 8-hydroxyquinoline and 1-naphthol was efficiently synthesized via click chemistry, and the compounds were efficiently characterized using various spectroscopic techniques. The in vitro DNA binding studies of the synthesized 1,2,3-triazoles with herring fish sperm DNA (hs-DNA) revealed that the quinoline- and 2-HBT-linked 1,2,3-triazoles containing shorter alkyl linkers (n = 2) showed a better binding affinity with hs-DNA compared to naphthol- and 4-HBT-linked 1,2,3-triazoles bound to longer alkyl linkers (n = 3 or 4). The order of DNA-binding affinity is 6a (3.90 × 10⁵ L mol^–1) > 6b > 6d > 6g > 6e > 6h > 6c ≈ 6f > 6i > 6j > 6k > 6l (0.09 × 10⁵ L mol^–1). Molecular docking on screened 1,2,3-triazoles 6a–f also showed the high binding energy of 6a (−8.7 kcal mol^–1) wherein O- and N-atoms of 8-hydroxyquinoline were involved in H-bond formation with DG2, the N-atom of the triazole ring in H-bond formation with DG4, the C-atom of the alkyl linker in C–H bond formation with DC23, and the triazole ring showed a π–π T-shaped hydrophobic interaction with DG22. Compound 6a displayed considerable antibacterial activity (zone of inhibition 15.5–17.6 mm) with reference to ciprofloxacin and superior antifungal activity (zone of inhibition 33.7 and 30.8 mm) among all derivatives 6a–l with reference to fluconazole. Molecular docking with lanosterol 14-α-demethylase (fungal enzyme; PDB ID: 1EA1) revealed that the H-bond, electrostatic, and hydrophobic interactions are dominating nonbonding interactions. The binding energy of 6a was found to be high (−9.7 kcal mol^–1), and it showed better interaction with the fungal enzyme among all of the triazoles. The ADME parameter of all compounds met the criteria of Lipinski’s rule of five, except 6f and 6l. The Molinspiration bioactivity score for the drug targets revealed that most of the synthesized 1,2,3-triazole molecules possess desirable physicochemical properties for drug-likeness. Also, the PreADMET properties revealed that all of the compounds have good human intestinal absorption.

Experimental Section

General Information

All chemicals used were purchased from commercial suppliers. ¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectra were recorded in CDCl₃ or DMSO-d₆ [tetramethylsilane (TMS), internal standard] on a Bruker Avance 500 MHz spectrometer, and chemical shifts (δ) are quoted in ppm. ESI-mass spectrometry and FT-IR (using anhydrous KBr pellets) analysis were performed on a WATERS XEVO G2-XS QTOF spectrometer and a Shimadzu IR-Instrument, respectively. All of the solvents used for purification in column chromatography were of analytical grade, and silica gel of 100–200 mesh was used. The melting point was determined using the open tube capillary method and was uncorrected.

DNA Binding Studies

For DNA binding studies, free acid-degraded hs-DNA was purchased from SRL Pvt. Limited. A Thermo Fisher Scientific Evolution 300 UV–vis spectrophotometer was used to perform all UV–vis titrations (spectral range = 240–450 nm) in the presence of hs-DNA (0–2.6 × 10^–6 M). Using a 5 mM Tris-HCl/50 mM NaCl buffer medium, the hs-DNA stock solution was prepared, which gives a ratio of 1.8–1.9 of absorbance at 260 and 280 nm showing the protein-free nature of DNA.

The stock solution (1 mg mL^–1) of hs-DNA was prepared in the 5 mM Tris-HCl/50 mM NaCl pH 7.0 buffered medium, and the stock solutions (2 mM) of 1,2,3-triazoles 6a–l were prepared in DMSO. The systematic addition of DNA (0–2.6 × 10^–6 M) into a known and fixed concentration of the synthesized triazole (2.25 × 10^–5 M) solution helped in the analysis of DNA binding. The intrinsic binding constant (K_b) was calculated using the equation

where A₀ is the initial absorbance, A is the final absorbance, C_DNA is the DNA concentration, and k is a binding constant.

Molecular Docking Study

The crystal structures of both fragments, i.e., the DNA dodecamer [PDB ID: 1BNA; sequenced (CGCGAATTCGCG)₂] and the target enzyme of Mycobacterium tuberculosis termed cytochrome P450 14α-sterol demethylase (CYP51) complexed with fluconazole (PDB ID: 1EA1), were downloaded from the RCSB protein data bank (www.rcsb.org) in PDB format. CHEMSKETCH (www.acdlabs.com) was used for 3D structures, and UCSF Chimera^6,53 was used for energy minimization of the triazoles. Ligand and receptor preparation by adding Gasteiger charges followed by the removal of water molecules and addition of polar hydrogens were performed using AutoDockTools-1.5.6 software. Calculations and simulations were performed in the AutoDock Vina program, and result visualization was performed using the Discovery studio visualizer.^7,54

Pharmacological Study

The method used for the antifungal activity against C. tropicalis and A. terreus fungal strains was agar-well diffusion. DMSO was used to dissolve the synthesized compounds (4 mg mL^–1) and also used as the negative control. Fluconazole was used as the reference compound. To determine the activity, 50 μL of the compound test sample was added and incubated overnight at 37 °C; then, the DIZ (diameter of inhibition zone in millimeter) was measured for each sample. To investigate the antifungal properties through docking analysis, the X-ray crystal structure of lanosterol 14-α-demethylase from Mycobacterium tuberculosis in complex with fluconazole (PDB ID: 1EA1) was downloaded from the RSCB site.

DMSO and ciprofloxacin were used as the negative control and the reference, respectively, for determining the antibacterial activity against S. aureus and B. subtilis (Gram-positive strains) and E. coli and P. aeruginosa (Gram-negative strains) and the fluconazole reference for the antifungal activity against C. tropicalis and A. terreus using agar-well diffusion method. Stock solutions of triazoles 6a–l were dissolved in DMSO (4 mg mL^–1). The diameter of inhibition zone (DIZ) around each well was measured in millimeters.

ADME Prediction

The drug-likeness score was computed using Molsoft software (freely available on http://www.molsoft.com/mprop), and the score predicts whether a particular chemical compound is similar to known drugs or not. Drug-likeness helps to describe the pharmacokinetic properties of drug compounds for estimation of important parameters such as absorption, distribution, metabolism, and excretion (ADME). The bioactivity score for the drug targets (GPCR ligand, a kinase inhibitor, ion channel modulator) was calculated using Molinspiration online software (https://www. molinspiration.com/cgi-bin/properties) for all of the synthesized 1,2,3-triazole molecules, as shown in Table SI-3.

PreADMET, a web-based program, was used to rapidly predict absorption properties such as human intestinal absorption, blood–brain barrier penetration, and plasma protein binding as shown in Table SI-5. In the in vivo study, drugs may bind reversibly to proteins and lipids in plasma protein to monitor the drug concentration in clinical trials and predict the therapeutic dose of the drug. The strongly bound compounds have a score of >90 and weakly bound compounds, a score of <90%. Prediction of human intestinal absorption (HIA) plays an important role in the design, optimization, and selection of oral drugs. The poorly absorbed compounds have HIA between 0 and 20%, moderately absorbed compounds between 20 and 70%, and well-absorbed compounds between 70 and 100%.

DFT Study

In the present study, we have used density functional theory to explain the chemical reactivity and the kinetic stability of the synthesized molecules, which derive properties based on the electron density. The frontier molecular orbital (FMO) theory involving the highest occupied molecular orbitals (HOMOs) and the lowest unoccupied molecular orbitals (LUMOs) is one of the best theories to explain the chemical reactivity and the kinetic stability of the molecule.^{8−12,55−59} Frontier orbital theory for molecular orbital-based reaction analysis was developed by Fukui and co-workers and has led to the development of conceptual density functional theory (DFT) for qualitative reaction analyses.^{8,55,13−16,60−63} This theory intuitively assumed that the molecular orbitals contributing to chemical reactions were the HOMOs and LUMOs. HOMO–LUMO are frontier orbitals as they lie at the outermost boundaries of the electrons of the molecules, and their analysis has been carried out to explain charge transfer within the molecule. The negative magnitude of E_HOMO and E_LUMO establishes the stability of compounds.^17,18,64,65 The energy gap is (E_HOMO–E_LUMO) the chemical reactivity and kinetic stability of a molecules;^{19−22,66−69} a molecule with a large HOMO–LUMO gap is described as a hard molecule and is much less polarizable.

The HOMO and LUMO energies represent the ability to donate and gain an electron, respectively. The high HOMO energy corresponds to the more reactive molecule in reaction with electrophiles while the low LUMO energy is for molecular reactions with nucleophiles.^23,70 The reactive sites for electrophilic reactions are regions around atoms providing high HOMO electron densities of the ground state, while the reactive sites for nucleophilic reactions are regions around atoms providing high LUMO electron densities of the ground state. The reaction occurs with the highly possible interaction between the HOMO of one moiety and the LUMO of another. Using HOMO and LUMO energy values for a molecule, the global chemical reactivity descriptors of molecules such as chemical hardness, chemical potential, softness, electronegativity, and electrophilicity index have been defined.^{24−31,71−78}

The DFT/B3LYP method is a widely used method for theoretical calculations including geometry optimization, stability, reactivity, and molecular orbital descriptors HOMO and LUMO.^{32−36,79−83} The geometry-optimized parameters with the B3LYP method are close to single XRD data in many reported studies. High levels of theory are available in Gaussian such as CCSD(T), G3, G4, etc., but they are computationally more expensive. Hence, we have used DFT/B3LYP in this work, which gave satisfactory results.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c03668.

• ¹H NMR, ¹³C NMR, ESI-MS, and characterization data for all synthesized compounds; absorption titration spectra; 3D and 2D diagrams of binding interactions; binding energies and interactions of docking results; cartoon image showing the docked pose; surface image showing the docked pose; absorption titration; antibacterial and antifungal activity results; ADME prediction; Molinspiration bioactivity; and chemical reactivity parameters by DFT (PDF)

The authors declare no competing financial interest.