Vision on Synthetic and Medicinal Facets of 1,2,4-Triazolo[3,4-b][1,3,4]thiadiazine Scaffold

The present review article strives to compile the latest synthetic approaches for the synthesis of triazolothiadiazine and its derivatives, along with their diverse pharmacological activities, viz. anticancer, antimicrobial, analgesic and anti-inflammatory, antioxidant, antiviral, enzyme inhibitors (carbonic anhydrase inhibitors, cholinesterase inhibitors, alkaline phosphatase inhibitors, anti-lipase activity, and aromatase inhibitors) and antitubercular agents. The review focuses particularly on the structure–activity relationship of biologically important 1,2,4-triazolo[3,4-b][1,3,4]thiadiazines, which have profound importance in drug design, discovery and development. In silico pharmacokinetic and molecular modeling studies have also been summarized. It is hoped that this review article will be of help to researchers engaged in the development of new biologically active entities for the rational design and development of new target-oriented 1,2,4-triazolo[3,4-b][1,3,4]thiadiazine-based drugs for the treatment of multifunctional diseases. Graphical Abstract


Multicomponent Reaction Approach
Recently, multicomponent reactions (MCRs) due to their high efficiency, atom economy, and reduced time are being explored to access functionalized triazolothiadiazines in a one-pot one-step fashion. In general, construction of triazolothiadiazine scaffold using MCR involves condensation of two components, namely, α-halocarbonyl compounds and 4-amino-5-hydrazinyl-3-mercapto-1,2,4-triazole and the third component is being employed for preparing derivatives or variations at hydrazinyl group.
Puthiyapurayil et al. [77] synthesized several 6-arylsubstituted-3-      (113) and studied their anticancer potential against full NCI-60 cancer cell lines. Compound 113h was found to show the highest percent inhibition over the 60 cell lines, with mean growth inhibition percentage of 45.44%. SAR studies revealed that compounds with p-cholorophenyl substitution at C-6 postion of triazolothiadiazine nucleus had better activity than analogs with an unsubstituted phenyl ring (Fig. 27). Mechanistic   having cyclopropyl substituted fluoroquinolones displayed better activity against L1210 cell lines, with an IC 50 value of 0.14 µmol/L (Fig. 28).
Ghosh et al. [92] introduced a series of triazolothiadiazines (121) bearing substituted phenylquinolin-2-one moiety and evaluated them for their antibacterial   pyridin-5-one moiety activity against Pseudomonas aeruginosa, E. coli, S. aureus, and antifungal activity against A. fumigatus, A. niger and C. albicans. SAR study revealed that triazolothiadiazine derivatives bearing phenyl quinoline moiety possessed moderate to good antimicrobial activities and halogen substituted compounds 121d, 121g and 121h showed the promising antifunfal activity against C. albicans with zones of inhibition of 20 mm, 23 mm and 19 mm, respectively (Fig. 35).
Indole clubbed derivatives of triazolothiadiazine (136) were evaluated for their antibacterial potential against B. subtilis, B. cereus, S. aureus, M. luteus, E. coli, P. aeruginosa, S. typhimurium and P. vulgaris by Sim et al. [104]. Compound 136d was found to show significant inhibition against B. subtilis, B. cereus and S. aureus with MIC = 3.91 μg ml −1 . SAR studies revealed the importance of chlorine on indole moiety and p-methylphenyl group at C-6 position of triazolothiadiazine ring (Fig. 49).

Rational Design Based on SAR of Triazolothiadiazine Derivatives
The present review captures the diverse biological activities exhibited by triazolothiadiazines reported in the literature. After careful analysis, general structural features have been identified that can be used in the design and development of novel triazolothiadiazine deivatives having clinical efficiency. Unfused 5-membered triazole ring systems were found to be biologically inactive; however, fusion of the 5-membered triazole ring with 6-membered thiadiazine ring is important for bioactivity [59,84]. Unsaturation in the 6-membered thiadiazine ring fueled bioactivity, in comparison with saturated derivatives [84]. At position-3 of the triazole ring, aromatic substitution connected through a spacer (0-3 atoms) exhibited better activity. Aromatic substituents at position-6 of the triazolothiadiazine ring displayed improved potency over the aliphatic analogs. Halogen-substituted aryl groups, specifically chloro-substituted aryl results in better activity than electron-donating groups, as aliphatic substitution bring down the activity. Substitution at position-7 of the triazolothiadiazine ring can be important for activity, but is not essential. Figure 72 summarizes the structural modifications in terms of different substituents at position-3, 6 and 7 (R 1 , R 2 and R 3 ) with respect to various biological activities, namely anticancer, antiviral, antimicrobial, antioxidant, antitubercular, enzyme inhibitiors, analgesic and anti-inflammatory activities.

Pharmacokinetic Profile
Pharmacokinetics is the study of processes by which drugs are absorbed, distributed, metabolized and eliminated from the body. Performing standard experimental procedures for absorption, distribution, metabolism and excretion (ADME) profiling for a vast number of compounds would have been time-consuming and economically unfeasible. Most drug molecules fail to enter the market due to their adverse pharmacokinetic properties. Recently, computer simulation platforms are gradually becoming popular as they can accelerate the pharmacokinetic development of the drug molecule [124,125]. In the primary phase of drug discovery, in silico approaches for the prediction of pharmacokinetics of drug candidates play an essential role in terms of cost effectiveness, time factors and avoidance of the ethical problems that arise from a large number of animals required for experiments. A successful lead should obey Lipinski's rule of five, which states that the drug should be easily absorbed, distributed to its target site, not easily metabolized, and excreted easily.
The drug-likeness of a compound was assessed according to Jorgensen's rule of three [126] and Lipinski's rule of five [127], which indicates that a chemical substance can be orally active in humans by estimating structural or physiochemical properties (descriptors) which are important for drug pharmacokinetics. Jorgensen's rule of three states that Caco-2 cell permeability (P Caco ) > 22 nm/s, predicted aqueous solubility (PlogS) > − 5.7 and number of primary metabolites < 7. Lipinski's rule of five states that orally active drug has no more than one violation of the following criteria: molecular weight < 500 Da; hydrogen bond donors ≤ 5; hydrogen bond acceptors ≤ 10; and octanol/water partition coefficient (log P) ≤ 5 (or MlogP not greater than 4.15) [128,129]. Topological polar surface area (TPSA) ≤ 140 Å 2 and the number of rotatable bonds ≤ 10 are also considered additional criteria used to determine oral bioavailability [130].
Triazolo [3,4-b] [1,3,4]thiadiazine derivatives have been identified as compounds with broad-spectrum activity, high safety, and excellent pharmacokinetic properties in a variety of studies. Therefore, analysis of arrangement of substituents around triazolothiadiazine scaffold (Fig. 73) might provide useful information to construct novel triazolothiadiazine derivatives with better biological proficiency.
Çevik et al. [76] calculated ADME parameters of benzimidazole-triazolothiadiazine derivatives 105 by QikProp 4.8 software for comparing with those of 95% of known drugs. All compounds 105 were found in accordance with the Jorgensen's rule of three and Lipinski's rule of five for drug-likeness properties by causing no more than one violation. According to predictions of ADME properties for compounds 105e and 105l, including molecular weight 433.489 and 467.934, respectively, the number of rotatable bond values (1), only one hydrogen bond donor group, hydrogen bond acceptors 6 and logP 4.468 and 4.96, respectively, it can be suggested that the active compounds 105e and 105l have desirable pharmacokinetic properties.
Sathyanarayana et al. [113] reported that novel [1,2,4]triazolo [3,4- 72 Structural modifications on triazolothiadiazine scaffold and their effect on bioactivities ADME parameters. Most of these compounds showed TPSA < 80 Å 2 , indicating their good intestinal absorption. The most potent antioxidant 146c showed excellent percentage human oral absorption (100%). Instead of having molecular weight ≥ 500 Da, these compounds have high percentage of human oral absorption and a violation of Lipinski's rule of five will not affect oral absorption [131]. QikProp predicted the number of possible metabolic reactions of these compounds in the range 2-5 (recommended range 1-8), indicating the compounds can easily reach the target site. Moreover, the LogKp values for compounds 146 range from −1.35 to −3.26 (acceptable range −8.0 to −10.0), which indicates that compounds 146 can be administrated dermally.
In silico ADME properties of the most selective triazolothiadiazine derivatives 160a and 160b as a new oral anti-inflammatory agent were accessed using the Swis-sADME web tool by Abdellatif et al. [132], which revealed that both compounds violated Lipinski's rule of five by two represented by molecular weight (589.94 and 663.61 Da, respectively) and MlogP values (6.46 and 4.55, respectively). Consequently, these triazolothiadiazine derivatives have lower bioavailability scores (0.17) compared with that of the reference drug celecoxib (0.55).
Alsaedi et al. [133] synthesized triazolo [3,4-b] [1,3,4]thiadiazine derivative 161 as potent VEGFR2 inhibitor with anti-hepatocellular carcinoma activity and predicted pharmacokinetic properties using SwissADME tool. Compound 161 showed good gastrointestinal (GI) absorption, high oral bioavailability, and fitted well in the Lipiniski's rule. ADME prediction revealed that compound 161 has a desirable pharmacokinetic profile and can be considered as a hopeful drug candidate.
Sever et al. [134] predicted pharmacokinetic profiles of triazolothiadiazine derivatives using QikProp program of Schrodinger's Maestro molecular modelling package. Compounds exhibited human oral absorption percentages ranging from 91.010% to 100.000%. All compounds comply with Lipinski's rule of five and Jorgensen's rule of three. On the basis of these findings, they are expected to possess favorable oral bioavailability and drug-like properties. According to in silico ADME studies for triazolothiadiazine 162, the total solvent accessible surface area (SASA) was 622.356 Å 2 , the predicted octanol/water partition coefficient (logPo/w) was 5.224, logarithm of predicted binding constant to human serum albumin, logK HSA value was 0.957 and the predicted apparent Caco-2 cell permeability P Caco-2 was 1763.718. These results were in agreement with recommended range, thus indicating compound 162 to possess a favorable ADME profile.

Molecular Modeling
In receptor-based drug design, the 3D structure of the binding site is modelled on the target with the aid of molecular simulations. Docking experiments are carried out to identify the binding sites through computationally model ligand-receptor interactions as well as lead candidates from the binding energy scores [135]. A literature survey reflected the prominence of the 1,2,4-triazolo [3,4-b] [1,3,4]thiadiazine structural motif in the drug designing aimed at diverse pharmacological activities by its robust interactions with various receptors. Extensive research has focused on structural attributes of 1,2,4-triazolo [3,4-b] [1,3,4]thiadiazines responsible for its bioactivity on particular targets via computational studies (docking and molecular dynamics). Structural variations on triazolothiadiazine scaffold and their interactions with different biological receptors are shown in Fig. 74. 1,2,4-Triazolo[3,4-b] [1,3,4]thiadiazine derivative 111j exhibited promising anticancer potential, which is supported by several interactions of its core nucleus with amino acids Lys 352 , Ala 250 , Lys 254 , Leu 248 , Asn 258 and the H-bond between oxygen atom of its trimethoxyphenyl ring and the thiol group of Cys 241 in the colchicine binding site of tubulin polymerase [82]. The methylpropylphenyl moiety of triazolothiadiazines 88b-d inserted into a hydrophobic basin formed by Val 349 , Leu 352 , Phe 518 and Ile 523 amino acids of the COX-1 protein to form a hydrophobic interaction [59]. In compound 113h, the N2 of the triazole ring and NH at position-3 interacted with Val 135 via two H-bonds inside the ATP binding pocket of GSK-3β, one of the targets of anticancer activity of this compound [84]. Compound 104a displayed H-bond interactions with Asp 479 and Lys 456 present at ATP catalytic binding domain of Topo-Iiβ [75]. Docking studies displayed that compound 102a formed strong H-bond interactions with Ser 93 , His 321 and His 154 in active site of tissue-nonspecific alkaline phosphatase (h-TNAP), while compound 102d formed important interactions with the active site of intestinal alkaline phosphatase (h-IAP), which included two H-bonds between two nitrogen atoms of triazole ring and Arg 166 and metal interaction of carbonyl group of coumarin core with Zn 2+ [73]. Docking studies indicated that compound 105e could inhibit aromatase enzyme through an H-bond interaction of CN group at the paraposition of the phenyl ring with Ser 314 . Besides, benzimidazole ring formed two π-π interactions with Arg 115 and Phe 134 and the triazolothiadiazine ring could interact by a salt bridge with the Hem 600 molecule and Cys 437 [76].
Further modifications and changes in substitution patterns at the C-3 and C-6 positions could result in higher affinity towards different receptors with additional interactions. This wide statistics will impart directions to researchers in innovating target selective, optimized and diverse 1,2,4-triazolo [3,4-b] [1,3,4]thiadiazine analogs for the treatment of multifactorial diseases.

Conclusion
In conclusion, the present review article deliberated the various synthetic protocols engaged for synthesis of diverse array of triazolothiadiazine analogs via multicomponent reactions (MCRs), conventional heating and visible-light-mediated organic reactions. Moreover, the triazolothiadiazine scaffold is an important structural motif in biologically active compounds, exhibiting more potency than standard marketed drugs. This review presented an in-depth analysis of medicinal aspects of 1,2,4-triazolo [3,4-b] [1,3,4]thiadiazines for different biological activities such as anticancer, antimicrobial, analgesic and anti-inflammatory, antioxidant, antiviral, enzyme inhibitors and antitubercular activities. The activity profile of this scaffold particularly offers structural modifications at the C3 and C6 positions of the triazolothiadiazine ring to achieve new analogues. This study also revealed the structural diversity, biological importance and SAR examination of heterocyclic compounds containing a triazolothiadiazine nucleus, which will help the practitioners in further exploration of biological efficacy.