Introduction

Tuberculosis (TB), being a major health concern at the global level, has seen a rise in the number of TB infections, deaths, and drug-resistant strains of TB during the period from 2020 to 2022 due to the COVID-19 pandemic [1,2]. The appearance of drug-resistant strains of TB has increased the urgency of the search for new drugs that can increase the number of existing drug options against TB. Existing TB drugs are effective against only a limited number of non-replicating bacterial populations because they target processes essential for the growth and replication of Mycobacterium tuberculosis. Therefore, the duration of treatment regimens of drug-susceptible TB (DS-TB) and drug-resistant TB (DR-TB) is long, taking months to cure the disease. In addition, the existing regimens of DR-TB are associated with low cure rates and high toxicity, making it imperative to look for effective and safe alternatives against DR-TB [3,4]. Mycobacterium tuberculosis (Mtb), Mycobacterium caprae, Mycobacterium canettii, Mycobacterium bovis, Mycobacterium pinnipedii, Mycobacterium africanum, and Mycobacterium microti are among the Mycobacterium species that cause tuberculosis. Mtb is the most prevalent of all Mycobacterium species and is responsible for causing pulmonary infections, but Mtb can also infect other vital organs [5-7].

Despite the identification of a number of natural and synthetic compounds with potent activity against resistant strains of Mtb, many of them have failed in early clinical trials and thus far, there are only a handful of drugs that have been registered for the therapy of TB during the last 50 years. The long duration of therapy and the associated toxic effects of existing antimycobacterial agents highlight the urgent need for the discovery of newer, safer chemotherapeutic agents [8,9].

Significant improvements have been made in understanding the molecular and cellular biology of TB bacteria. This has led to the identification of several molecular targets involved in the development and progression of TB. This has also led to the development of novel, rationally designed molecularly targeted agents that have been and are being applied in clinical practice. Among such targets is the enzyme dihydrofolate reductase (DHFR), a crucial enzyme in the folate metabolic pathway. This enzyme is responsible for reducing dihydrofolate (DHF) to its active form, tetrahydrofolate (THF). THF is a crucial cofactor required for the biosynthesis of purines, thymidylate, and several amino acids. THF is also required for the synthesis of DNA, RNA, and proteins. In microbial cells, disruption of THF regeneration rapidly inhibits cell growth and ultimately leads to cell death [10,11].

The structural features of mycobacterial dihydrofolate reductase (Mtb-DHFR) include an adenosine-binding subdomain and a flexible loop subdomain comprising the L1, L4, and L5 loops. The conformational changes in the loops play a crucial role in regulating the enzyme during the reduction of DHF to THF. Crystallographic studies have also revealed a small hydrophilic pocket adjacent to the folate-binding site in Mtb-DHFR, but not in human DHFR. This structural feature of the enzyme provides a promising avenue for the design of selective inhibitors for the treatment of tuberculosis.

Recent studies have also shown that alternative reductases such as RV2671 can partially compensate for DHFR activity, highlighting the central importance of THF regeneration for mycobacterial survival. Taken together, the essential role of DHFR in folate metabolism, its structural divergence from the human counterpart, and the availability of high-resolution crystal structures strongly support Mtb-DHFR as a rational and promising target for antitubercular drug discovery [12]. On this basis, the present study was undertaken to design and synthesize novel 5,6-diphenyl-1,2,4-triazine-piperazine derivatives and to evaluate their antitubercular potential as DHFR-targeted inhibitors.

The development of novel compounds based on medicinally relevant heterocycles is one of the most commonly employed methods in contemporary drug discovery. Among these is the class of nitrogen heterocycles, which have attracted researchers' interest due to their biological activities and potential.

In particular, heterocycles with bridgehead nitrogen atoms, such as (1,2,4,5)-tetrazine, (1,2,4)-triazole, and (1,2,4)-triazine derivatives, have attracted interest from drug researchers due to their potential for various biological and pharmaceutical applications. Among these is the 1,2,4-triazine nucleus, which is of interest for its potential in various biological and pharmaceutical applications, owing to its structural properties and stability in pharmacological interactions [13-15].

The derivatives of the 1,2,4-triazine ring system have been found to possess considerable biological activity. For example, lamotrigine, an anticonvulsant drug, and tirapazamine, an antitumor drug, are all derived from the triazine structure. In addition, fused 1,2,4-triazine derivatives have been found to possess potent antimicrobial, antiviral, and antimycobacterial activity. This unique structure of the 1,2,4-triazine ring system and its ability to interact with various biological pathways have led to further research into the development of drug molecules [16,17].

Piperazine, another nitrogen-heterocyclic compound, has also emerged as an important compound in medicinal chemistry due to its wide variety of pharmacological activities, such as antimycobacterial [18], antibacterial [19,20], antiviral [21], antifungal [22], antitumor [23], analgesic [24], anti-Alzheimer [25] and anticonvulsant activities [26]. The piperazine ring is recognized as a potential pharmacophore for the treatment of mycobacterial infections, offering enhanced hydrogen-bonding potential, dipole moment, stability, and rigidity in vivo [27]. These properties have been correlated to the enhanced pharmacological activities of the piperazine derivatives. This has also been attributed to the enhanced pharmacological activity of piperazine derivatives, which have been proposed as promising candidates for developing new anti-TB agents [28]. In addition, the presence of piperazine in the specific DprE1 inhibitor benzothiazinone (PBTZ169) has established piperazine as a promising compound [29,30].

In the last few years, the term ‘molecular hybridization’ (MH) has been recognized as a potent tool in medicinal chemistry. This method of drug design involves combining different pharmacophore groups of compounds to generate a single hybrid with the desired pharmacological activity [31,32]. The hybrids have been found to possess superior therapeutic activity and physicochemical characteristics, making them of considerable interest as potential drug candidates against DHFR inhibitors Figure 1 [33-35].

Figure 1.: Designing strategy for 5,6-diphenyl-1,2,4-triazine-piperazine derivatives as potential anti-TB leads

In view of the urgent need to develop novel and potent anti-tuberculosis agents, the search for novel molecular hybrids of 1,2,4-triazine and piperazine moieties presents an exciting research opportunity. Through the systematic synthesis of these compounds, it is possible to fully exploit their unique characteristics to develop potent antimycobacterial agents. This approach not only helps to resolve the challenges associated with the treatment of tuberculosis but also offers a pathway to the development of novel second-generation chemotherapeutic agents with a wide range of activity and reduced toxicity. As a part of our ongoing research towards the synthesis of novel biologically active 1,2,4-triazine derivatives, here we report the synthesis and pharmacological activity of novel 1-(4-benzoyl-piperazin-1-yl)-2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one.