Exploration of the Hybrids of Imidazole-Thione and Benzotriazole Scaffolds: Chemistry, Bioactivity, and Drug Design Perspectives

Research and interest in creating new pharmaceutical agents are constantly expanding. A class of chemical compounds known as heterocycles that contain atleast one atom different from have long been recognized as repeating scaffolds in rings with higher carbon content than medicinal chemistry for their significance in the creation and identification of novel pharmaceuticals. They are appealing building blocks, because of their structural diversity and adaptability utilized in the creation of medication such as antioxidants, antitumorals, antivirals, antifungals and antibiotics, nephroprotective substance etc opening the door for novel treatments and enhanced result for patients. Highlighting some of the most recent developments in these fascinating field is the goal of this special issue, by compiling the most recent findings from studies on bioactive heterocyclic compounds. 3-diaza-2,4-cyclopentadiene or imidazole, is a five-3C and 2N atoms in 1 and 3 positions make up this member ring system. It has been said that structural frameworks are privileged structures, and specifically N-containing polycyclic structures have been linked to a variety of different biological activity. Imidazole nuclei are found in the field of five memberedred heterocycle structures displays a variety of properties. The imida-related medications' excellent therapeutic qualities have inspired medicinal chemists to create numerous innovative chemotherapeutic agents, antifungal, antibacterial, anti-inflammatory and antiviral agents etc. When modified to incorporate a thione group (-C=S ), imidazole derivatives often exhibit improved pharmacological profiles, including enhanced binding interaction with biological targets via hydrogen bonding and sulfur coordination. The bicyclic heterocyclic compound benzotriazole is made up of a fused benzene ring and three nitrogen atoms, exhibits a broad spectrum of pharmacological and biological activities. Given the speed at which microorganisms are changing genetically and becoming resistant to numerous antibiotics and treatment agents for a range of illnesses faster than the production of new medications accessible, making the fight against infectious diseases an ongoing endeavour. Bess, in the previous the triazole class has attracted a lot of attention in recent decades because of its extensive industrial use and agriculture. Medicinal chemistry greatly benefits from benzotriazole and its derivatives. One crucial synthetic approach in drug discovery is the incorporation of benzotriazole nuclei. The associated medications' strong therapeutic qualities have prompted medicinal chemists to create the numerous new chemotherapeutic substances. Molecular hybridization, the process of covalently linking two or more pharmacophores with known bioactivities to form a single hybrid molecule, has emerged as a powerful tool in drug discovery. Hybrid molecules can have synergistic or dual-target activity, improved physicochemical properties, and a lower resistance potential.

The fusion of imidazole-thione and benzotriazole motifs creates a novel hybrid framework capable of interacting with a wide range of biological targets. Several studies over the last decade have focused on developing such hybrids in order to investigate their therapeutic potential. This review seeks to:

• Describe the chemistry and synthesis of these hybrids.
• Analyze their biological activities.
• Highlight the relevant SAR insights.
• Provide a vision for future research into these promising molecular architectures.

CHEMISTRY^[7]

2-Imidazolidinethione is an organosulfur compound that has the formula C2H2(NH)2C=S. It is a cyclic, unsaturated thiourea with a short C=S bond length of 169 pm.The compound is frequently referred to as 2-mercaptoimidazole, a tautomer that is not observed. The compound forms a number of metal complexes. 2-imidazolidinethione and mercaptobenzimidazole have similar bonding and reactivity properties.

BTA (benzotriazole) is a heterocyclic compound with the chemical formula C6H4N3H. It can be thought of as the fusion of benzene and triazole rings. It is a white solid, but impure samples may appear tan. It acts as a corrosion inhibitor for copper.

Figure 1 -Chemical Background & Hybridization Strategy

Figure 2 – Bioactivity Spectrum & Drug Design Rationale

CHEMICAL BACKGROUND AND SIGNIFICANCE OF THE TWO SCAFFOLDS:^[8,9]

The pharmacological potential of heterocyclic compounds has long been recognized, with imidazole-thione and benzotriazole scaffolds standing out for their structural variety and reactivity. The hybridization of these two heterocycles is driven by their respective bioactivities and complementary chemical properties, allowing for the development of multifunctional drug candidates.

Imidazole-Thione core: Structural and biological Overview

Imidazoles are well-known heterocyclic compounds that are common and play important roles in a variety of medicinal agents. Imidazole is a five-membered planar ring that dissolves in water and other polar solvents. It exists in two equivalent tautomeric forms because the hydrogen atom can be located on either of the two nitrogen atoms. It is a highly polar compound, with a calculated dipole of 3.61D, and is completely soluble in water. The presence of a sextet of n-electrons, consisting of two electrons from the protonated nitrogen atom and one from each of the ring's remaining four atoms, qualifies the compound as aromatic. Imidazole is amphoteric, meaning it can act as both an acid and a base. Substitution of a thione group (-C=S) at the 2- or 4/5-position produces imidazole-thione derivatives, which have gained popularity due to their increased biological activity.

Key Properties:

• The thione moiety increases nucleophilicity and hydrogen bonding capacity.
• Functions as a metal-binding domain, which is useful for inhibiting metalloproteins.
• Shows tautomeric equilibrium with imidazole-thiol forms, which can influence biological interactions.

Biological Activities:

• Imidazole-thione derivatives are said to exhibit:
• Antimicrobial (for both Gram-positive and Gram-negative bacteria)
• Antifungal (especially against Candida spp).
• Anticancer (by inhibiting kinases and inducing apoptosis).
• Antioxidant, anti-inflammatory, and enzyme inhibiting effects

Benzotriazole Scaffold: Structural and Medicinal Relevance

Benzotriazole is an important scaffold in the development of new pharmaceutically active compounds. Its diverse biological activities, as well as its structural modification potential, make it a promising candidate for future drug development. Benzotriazole is a bicyclic compound consisting of a benzene ring fused with a 1,2,3-triazole ring. The presence of three nitrogen atoms contributes to its electron-donating and -withdrawing nature, allowing fine-tuning of physicochemical properties through substitution.

Key Points:

• Enhanced chemical stability, aromaticity, and electron density.
• Ability to form hydrogen bonds and coordinate with metal ions.
• Functions as a bioisostere for amides or carboxylic acids in drug design.

Biological Activities:

• Antimicrobial (broad-spectrum antibacterial, antifungal)
• Antiviral (primarily anti-HIV and anti-hepatitis C)
• Anti-inflammatory and anticancer properties
• Antitubercular, enzyme-inhibitory, and neuroprotective properties

Hybridization Rationale: Imidazole-Thione + Benzotriazole

The rationale for combining these two privileged scaffolds lies in their complementary pharmacophore.

Table:1

Combining both scaffolds enables flexible pharmacophore mapping, where:

• The imidazole-thione core may bind to the biological target.
• The benzotriazole ring interacts with the hydrophobic and polar regions.
• Linkers can be optimized for 3D conformational fit, hydrogen bonding, and π-π stacking interactions.

Figure:3

The design and synthesis of hybrid molecules containing both imidazole-thione and benzotriazole cores necessitates careful consideration of linkers, reaction conditions, and substitution sites. The synthetic approaches are generally classified as traditional solution-phase synthesis, microwave-assisted techniques, and green chemistry-based protocols. This section describes the most common methods using representative schemes.

General Synthetic Strategy

Most synthetic routes have three major steps:

1. Formation of the imidazole-thione core.
2. Synthesis of a functionalized benzotriazole derivative
3. Linking using nucleophilic substitution, amide/ester bond formation, or alkylation.

Common Linkers Used:

• Alkyl chains (-CH2- and -(CH2)n-)
• Amide and ester bridges
• Sulfonamide or thioether linkages

Stepwise Synthesis Approaches

1. Synthesis of Benzotriazole^[18,19,20]

Benzotriazole is synthesized by the diazotization and intramolecular cyclization of o-phenylenediamine with nitrous acid, which is generated in situ from sodium nitrite and an acid like acetic acid. The reaction is typically performed in an aqueous or aqueous-acetic acid solution, where the o-phenylenediamine is cooled, and the sodium nitrite is added slowly while stirring to form the benzotriazole product. 

Figure:4(Scheme-1)

2. Synthesis of substituted phenyl thioura derivatives^[21,22,23]

Substituted phenyl thioureas are commonly synthesized by reacting a substituted aniline with an isothiocyanate in a suitable solvent, or by the reaction of a substituted phenylammonium salt (derived from the aniline) with an ammonium thiocyanate. Another method involves the use of carbon disulfide and amines in an aqueous medium for the synthesis of substituted thioureas. 

Figure:5(Scheme-2)

• BI1 R=H
• BI2 R = 4- CH₃
• BI3 R = 4- CH₂CH₃
• BI4 R= 4-OCH₃
• BI5 R = 4 - OH
• B16 R = 4 - CI
• B17 R = 2 - Cl
• B18 R = 3 - Cl
• B19 R = 2 ,4-(Cl) 2
• BI10 R = 2 - F
• BI11 R = 4-Br
• BI12 R = 4 - (SO₂NH₂)

3. Synthesis of 1-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-chloroethanone^[24]

1-(1H-Benzo[d][1,2,3]triazol-1-yl)-2-chloroethanone (0.01 mole), anhydrous sodium acetate and substituted thiourea  (0.01 mole) were dissolved in ethanol, and the mixture was refluxed for 6 h. The mixture was poured into cold water and the solid formed was recrystallized using ethanol to afford the final compounds.

Figure:6 (Scheme-3)

A series of heterocyclic derivatives containing imidazole-thione linked benzotriazole were synthesised(scheme-3) and evaluated for anticancer activity. The synthesised derivatives are:

• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-Phenyl-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(4-Methylphenyl)-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(4-Ethylphenyl)-1H-Imidazole-2(3H)-Thione ,
• -(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(4-Methoxyphenyl)-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(4-Hydroxyphenyl)-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(4-Chlorophenyl)-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(2-Chlorophenyl)-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(3-Chlorophenyl)-1H-Imidazole-2(3H)-Thione ,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(2,4-Dichlorophenyl)-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(2-Florophenyl)-1H-Imidazole-2(3H)-Thione,
• 4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-1-(4-Bromophenyl)-1H-Imidazole-2(3H)-Thione,
• -(4-(1H-Benzo[d][1,2,3]Triazol-1-yl)-2-Thioxo-2,3-Dihydro-1H-Imidazol-1-yl)Benzenesulfonamide 

Microwave-Assisted Synthesis^{[25,26,27,28,29,30]}

Microwave irradiation is frequently used to:

• Enhance reaction rates
• Improve yields
• Reduce solvent usage

Example:

Microwave-assisted one-pot synthesis of imidazole-thione-benzotriazole hybrids using DMF at 120 °C for 10-15 minutes leads to higher yields than conventional reflux.

Green Chemistry Approaches^{[31,32,33,34,35]}

• Use of ethanol or water as green solvents
• Use of ionic liquids or deep eutectic solvents for eco-friendly catalysis
• Solvent-free grinding under ambient conditions (where possible)

Biological Activities and Structure-Activity Relationship (SAR)^{[36.37;38;39;40,41]}

Hybrid molecules incorporating imidazole-thione and benzotriazole moieties have demonstrated diverse biological activities due to their multi-pharmacophoric nature. These hybrids often act on multiple biological targets, offering potential for the development of broad-spectrum or selective therapeutic agents. This section reviews the key pharmacological properties, categorized by activity type, and discusses relevant structure-activity relationships (SAR).

1. Antimicrobial activity

The majority of imidazole-thione-benzotriazole hybrids have been studied for their antibacterial and antifungal properties.

Reported observations:

• Compounds with electron-drawing groups (CI, NO?) on the benzotriazole ring show increased antibacterial activity.
• The presence of alkyl or aryl groups on the imidazole ring improves both lipophilicity and cell membrane penetration.
• Thioether linkages demonstrated higher activity than amide linkers.

Target Pathogens:

• Gram-positive: Staphylococcus aureus, Bacillus subtilis.
• E. coli and Pseudomonas aeruginosa are both Gram-negative bacteria.
• Fungi: Candida albicar, Spergillus niger.

2. Anticancer activity

Some hybrids have been tested against human cancer cell lines, with promising cytotoxicity results.

Reported observations:

• Compounds containing halogenated benzotriazole rings exhibit high activity against MCF-7 (breast cancer) and HeLa (cervical cancer) cells.
• Electron-donating groups on the imidazole ring inhibit activity, most likely due to altered binding with DNA or enzymes.
• SAR suggests that using short alkyl chains as linkers promotes cell uptake and nuclear localization.

3. Antioxidant Activity.

• The addition of hydroxyl or methoxy substituents to the phenyl ring of either scaffold improves radical scavenging ability.
• In DPPH and ABTS assays, active derivatives had IC50 values ranging from 10 to 40 μM.

Anti-inflammatory and enzymatic inhibition

• Certain hybrids inhibit COX-2 and lipoxygenase, indicating anti-inflammatory activity.
• Molecular docking revealed hydrogen bonding between COX active site residues, particularly through the C=S and N-H groups.

Summary Table of Bioactivities

Here is a representative table that summarizes the biological activities of selected hybrid molecules.

Table:2 (BTA= Benzotriazole, Imz= Imidazole-thione)

Figure:6

The pharmacological profile of imidazole-thione-benzotriazole hybrids clearly demonstrates their multifunctionality. Rational substitutions and linker modifications significantly influence activity. Further SAR-based optimization are in vivo validation are needed to identify potent lead candidates.

Pharmacophore Modeling ^{[42,43,44,45,46]}

Benzotriazole is a privileged heteroaromatic nucleus widely recognized for its diverse biological activities, including anticancer, anti-inflammatory, antimicrobial, and kinase inhibitory effects. On the other hand, imidazole derivatives, particularly thione-substituted analogues, exhibit significant pharmacological importance due to their ability to act as hydrogen bond donors/acceptors, metal chelators, and enzyme inhibitors. The strategic hybridization of these two scaffolds results in the imidazole-thione linked benzotriazole  framework (Figure 7), which unites multiple pharmacophoric features in a single molecular entity.

Structurally, this hybrid offers:

• Aromatic π-systems from the fused imidazole-thione and benzotriazole rings, favoring π–π stacking interactions with aromatic residues of proteins.
• Hydrogen bond donor/acceptor groups (NH linker, heteroaromatic nitrogens, thione sulfur), enabling versatile interaction with enzymatic active sites.
• An ionizable center at the terminal substituted nitrogen, enhancing potential salt-bridge or water-mediated hydrogen bonding.
• Lipophilic patches provided by the benzotriazole ring, contributing to hydrophobic stabilization in protein pockets.

Preliminary reports on related scaffolds suggest that the introduction of a thione moiety enhances antimicrobial, antioxidant, and anticancer properties, attributed to its ability to participate in redox balance and metal chelation. Furthermore, the benzotriazole nucleus contributes to kinase inhibition and anti-inflammatory effects, making the hybrid scaffold suitable for multi-target drug design.

From a pharmacophore perspective, the benzotriazole–imidazole-thione hybrid can be considered a multifunctional template, displaying the essential features commonly identified in bioactive compounds:

• Hydrogen bond donor (HBD) – NH linker between the two heterocycles.
• Hydrogen bond acceptors (HBA) – heteroatoms of benzotriazole and imidazole-thione rings.
• Aromatic features (AR) – Benzotriazole  ring and imidazole moiety.
• Positive ionizable centre (PI) – terminal nitrogen substituent.
• Hydrophobic/aromatic surface – benzene portion of benzotriazole.

These characteristics justify the growing attention towards benzotriazole–imidazole-thione hybrids in drug discovery. Although experimental studies are still limited, the scaffold holds promise as a starting point for the design of novel anticancer, antimicrobial, and enzyme inhibitory agents. Future investigations should emphasize systematic structure–activity relationship (SAR) exploration, docking/pharmacophore validation, and in vivo evaluations to unlock its full therapeutic potential.

Computational Insights in Drug Discovery: Role of ADME and Docking Studies ^{[47,48,49,50,51]}

In recent years, computational approaches have become an indispensable part of modern drug discovery and development. Among these, ADME (Absorption, Distribution, Metabolism, and Excretion) predictions and molecular docking studies play a central role in evaluating drug-likeness and optimizing lead compounds before costly experimental procedures.

ADME Predictions:

Computational tools such as SwissADME, pkCSM, ADMETlab, and QikProp allow rapid screening of pharmacokinetic properties. Parameters including oral bioavailability, gastrointestinal absorption, blood–brain barrier penetration, metabolic stability, and toxicity risks can be predicted at an early stage. Such in silico profiling not only minimizes late-stage failures but also accelerates the lead optimization process.

Molecular Docking:

Docking is widely applied to study the binding interactions of small molecules with target proteins. Programs like AutoDock, Glide, GOLD, and MOE generate possible ligand conformations and provide scoring functions to estimate binding affinity. Several studies have demonstrated the usefulness of docking in rationalizing SAR (structure–activity relationships), identifying key binding residues, and guiding structural modifications of lead compounds.

Integrated Approach:

The combination of ADME prediction and docking provides a holistic computational framework to identify promising lead molecules. Docking ensures strong interaction with the biological target, while ADME screening validates the pharmacokinetic feasibility of the compound. Despite their predictive power, these approaches should be considered as complementary to experimental studies, since computational models may not fully replicate biological complexity.

Commonly used computational tools for ADME prediction and docking studies