Synthetic Methods and Pharmacological Potentials of Triazolothiadiazines: A Review

This review article examines the synthetic pathways for triazolothiadiazine derivatives, such as triazolo[3,4-b]thiadiazines, triazolo[5,1-b]thiadiazines, and triazolo[4,3-c]thiadiazines, originating from triazole derivatives, thiadiazine derivatives, or thiocarbohydrazide. The triazolothiadiazine derivatives exhibit several biological actions, including antibacterial, anticancer, antiviral, antiproliferative, analgesic, anti-inflammatory, and antioxidant properties. The review article aims to assist researchers in creating new biologically active compounds for designing target-oriented triazolothiadiazine-based medicines to treat multifunctional disorders.


Introduction
In recent years, nitrogen-and sulfur-containing scaffolds have become prominent in health, agriculture, and industry due to their extensive applications [1].One nitrogenand sulfur-containing heterocycle is triazolothiadiazine.This is a bicyclic molecule of nine members, consisting of four carbon atoms, four nitrogen atoms, and one sulfur atom.This heterocycle is generated by the combination of two active components, a five-membered triazole and a six-membered thiadiazine.As shown in Figure 1, triazolothiadiazine has nine types of isomers.

Scheme 9. Synthesis of triazolothiadiazines 23a-l.
Synthesis of triazolothiadiazine 23 involved a chemoselective sequential double addition reaction.This included an InCl 3 -catalyzed dehydrative nucleophilic addition of an amino group to an aromatic aldehyde, followed by the addition of the resulting Schiff's base 25 to cyclohexyl isocyanide.Adduct 26 undergoes intramolecular cyclization to produce the required molecules [29] (Scheme 10).Scheme 9. Synthesis of triazolothiadiazines 23a-l.
Synthesis of triazolothiadiazine 23 involved a chemoselective sequential double addition reaction.This included an InCl3-catalyzed dehydrative nucleophilic addition of an amino group to an aromatic aldehyde, followed by the addition of the resulting Schiff's base 25 to cyclohexyl isocyanide.Adduct 26 undergoes intramolecular cyclization to produce the required molecules [29] (Scheme 10).

Scheme 10. Mechanism of formation of triazolothiadiazines 23a-l.
Triazolo [3,4-b][1,3,4]thiadiazines 31 were produced with high efficiency by a catalystfree one-pot reaction between compound 1 and dibenzoylacetylene 28.Single crystal Xray diffraction was used to determine the structures of the generated molecules [30] (Scheme 11).Triazolo [3,4-b][1,3,4]thiadiazines 31 were produced with high efficiency by a catalystfree one-pot reaction between compound 1 and dibenzoylacetylene 28.Single crystal X-ray diffraction was used to determine the structures of the generated molecules [30] (Scheme 11).3-Phenylpropiolaldehyde 32 was reacted with 1 in i-PrOH containing p-TsOH to give 4-((3-phenylprop-2-yn-1-ylidene)amino)-4H-1,2,4-triazole-3-thiol 33 then hydrogenation using sodium borohydride in methanol to afford 34.Intramolecular cyclization of the latter compound in DMF with KOH as a catalyst at 100 °C afforded 3-substituted (Z)-7benzylidene-6,7-dihydro-5H- [1,2,4]triazolo [3,4-b][1,3,4]thiadiazines 35.Oxidation of 35 in acetonitrile using MnO2 gave [1,2,4]triazolo [3,4-b][1,3,4]thiadiazines 36.However, hydrogenation 36 with sodium borohydride afforded 35 [11,31] (Scheme 12).Scheme 12. Synthesis of [1,2,4]triazolo [3,4- A one-pot reaction is performed utilizing visible-light photoredox catalysis to react 1,3-diketones 37 with NBS and compound 1.The reaction can be catalyzed by either p-TsOH or CFL (27 W) in a mixture of ethanol and water (4:1).The synthesis of 7-aroyl-6-methyl- [1,2,4]triazolo [3,4-b][1,3,4]thiadiazines 38 was achieved [32] (Scheme 13).The plausible mechanism for the regioselective production of 38 was detailed in Scheme 14.The C-Br bond of α-bromodiketone 37A and the S-H bond of compound 1 underwent homolytic fission when exposed to visible light.This resulted in the formation of free radicals 37AA and 39.The radicals were combined to create an S-alkylated open chain intermediate 40.After the N-H bond is broken homolytically with the help of a Br radical and the carbonyl carbon is cleaved with the assistance of a hydrogen free radical, there is a promotion of intramolecular free radical combination between the amine and the carbonyl carbon near the methyl group.Removing water led to the successful production of the desired product [32] (Scheme 14).The plausible mechanism for the regioselective production of 38 was detailed in Scheme 14.The C-Br bond of α-bromodiketone 37A and the S-H bond of compound 1 underwent homolytic fission when exposed to visible light.This resulted in the formation of free radicals 37AA and 39.The radicals were combined to create an S-alkylated open chain intermediate 40.After the N-H bond is broken homolytically with the help of a Br radical and the carbonyl carbon is cleaved with the assistance of a hydrogen free radical, there is a promotion of intramolecular free radical combination between the amine and the carbonyl carbon near the methyl group.Removing water led to the successful production of the desired product [32] (Scheme 14).
underwent homolytic fission when exposed to visible light.This resulted in the formation of free radicals 37AA and 39.The radicals were combined to create an S-alkylated open chain intermediate 40.After the N-H bond is broken homolytically with the help of a Br radical and the carbonyl carbon is cleaved with the assistance of a hydrogen free radical, there is a promotion of intramolecular free radical combination between the amine and the carbonyl carbon near the methyl group.Removing water led to the successful production of the desired product [32] (Scheme 14).

Scheme 14. Mechanism of formation of triazolothiadiazines 38.
A very effective synthesis of triazolothiadiazinols 44 was produced using a catalystfree method.The synthesis involves the reaction of compound 1 with substituted nitroepoxide 43 in methanol at an ambient temperature, resulting in excellent regio-and Scheme 14. Mechanism of formation of triazolothiadiazines 38.
A very effective synthesis of triazolothiadiazinols 44 was produced using a catalystfree method.The synthesis involves the reaction of compound 1 with substituted nitroepoxide 43 in methanol at an ambient temperature, resulting in excellent regio-and diastereoselectivity.The dehydration of compound 44 using p-TsOH in EtOH at 70 Compound 45′s synthesis process is depicted in Scheme 16.The thiol group opens the nitroepoxide ring, leading to the creation of intermediate 46 by eliminating nitric acid.The amino group then reacts via a nucleophilic attack on the carbonyl, leading to the creation of triazolothiadiazinol 44.The stereochemistry of the compounds is explained using two Newman projections (46a and 46b).The reaction proceeded through conformer 46a due to the unfavorable contact between the methyl and aryl groups in 46b, resulting in the products with the observed stereochemistry [33].Compound 45 s synthesis process is depicted in Scheme 16.The thiol group opens the nitroepoxide ring, leading to the creation of intermediate 46 by eliminating nitric acid.The amino group then reacts via a nucleophilic attack on the carbonyl, leading to the creation of triazolothiadiazinol 44.The stereochemistry of the compounds is explained using two Newman projections (46a and 46b).The reaction proceeded through conformer 46a due to the unfavorable contact between the methyl and aryl groups in 46b, resulting in the products with the observed stereochemistry [33].
Compound 45′s synthesis process is depicted in Scheme 16.The thiol group opens the nitroepoxide ring, leading to the creation of intermediate 46 by eliminating nitric acid.The amino group then reacts via a nucleophilic attack on the carbonyl, leading to the creation of triazolothiadiazinol 44.The stereochemistry of the compounds is explained using two Newman projections (46a and 46b).The reaction proceeded through conformer 46a due to the unfavorable contact between the methyl and aryl groups in 46b, resulting in the products with the observed stereochemistry [33].The molecule 1 was alkylated with 2-bromo-1-(3,4,5-trimethoxyphenyl)ethan-1-one 47 in dry acetone with K 2 CO 3 to produce S-phenacyl derivatives 48.When the subsequent reaction was carried out in refluxing ethanol, it produced the cyclized analogs 49.In addition, the annulation of 48 was achieved by subjecting it to reflux in absolute ethanol, resulting in the formation of 49 (Scheme 17).The produced compounds exhibited remarkable selectivity towards cancer cells compared to normal cells, since they did not demonstrate any notable cytotoxicity against L929 cells.Furthermore, compound 49a has the ability to hinder the process of tubulin polymerization at micro-molar concentrations [12].Similarly, triazolothiadiazines 50 were achieved by condensing 1 with phenacyl bromides in refluxing ethanol, as reported [34].Triazolothiadiazines 51 were produced by reacting 1 with α-bromoacetophenone in the presence of K2CO3 and p-TsOH [35] (Scheme 18).The synthesized compounds were assessed as very effective and specific inhibitors of electric eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) using Ellman's method, with neostigmine and donepezil serving as standard inhibitors.

Scheme 17. Synthesis of triazolothiadiazines 49a-g.
Similarly, triazolothiadiazines 50 were achieved by condensing 1 with phenacyl bromides in refluxing ethanol, as reported [34].Triazolothiadiazines 51 were produced by reacting 1 with α-bromoacetophenone in the presence of K 2 CO 3 and p-TsOH [35] (Scheme 18).The synthesized compounds were assessed as very effective and specific inhibitors of electric eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) using Ellman's method, with neostigmine and donepezil serving as standard inhibitors.
Similarly, triazolothiadiazines 50 were achieved by condensing 1 with phenacyl bromides in refluxing ethanol, as reported [34].Triazolothiadiazines 51 were produced by reacting 1 with α-bromoacetophenone in the presence of K2CO3 and p-TsOH [35] (Scheme 18).The synthesized compounds were assessed as very effective and specific inhibitors of electric eel acetylcholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) using Ellman's method, with neostigmine and donepezil serving as standard inhibitors.
In the same manner, substituted phenacyl bromide was reacted with compound 1 to give benzimidazole-triazolothiadiazine derivatives 52 (Scheme 19).The synthesized compounds exhibited anticancer efficacy against the MCF-7 human breast cancer cell lines.Compounds 52c, 52e, 52k, and 52m, which showed the highest activity on the MCF-7 cell line, were selected for additional in vitro experiments to investigate the potential mechanisms responsible for their activity.These assays focused on determining their ability to inhibit the aromatase enzyme [13].In the same manner, substituted phenacyl bromide was reacted with compound 1 to give benzimidazole-triazolothiadiazine derivatives 52 (Scheme 19).The synthesized compounds exhibited anticancer efficacy against the MCF-7 human breast cancer cell lines.Compounds 52c, 52e, 52k, and 52m, which showed the highest activity on the MCF-7 cell line, were selected for additional in vitro experiments to investigate the potential mechanisms responsible for their activity.These assays focused on determining their ability to inhibit the aromatase enzyme [13].In addition, 4-amino-3-[1-[4-(substituted phenyl]ethyl]-1,2,4-triazole-5-thiones 1I-3I were condensed with phenacyl halides in reflux ethanol or under microwave irradiation (350 Watt) to give 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines 53a-j (Scheme 20).The produced compounds were screened for their bioactivity against epithelial cancer cells and this demonstrated that those derivatives are promising drug candidates for epithelial cancers, especially liver cancer [36].
Molecules 2024, 29, x FOR PEER REVIEW 17 of 38 were investigated, with the aim of establishing a connection between their alkaline phosphatase inhibitory properties and potential anti-proliferative and pro-apoptotic effects.The examined compounds had significant inhibitory effects on h-TNAP and h-IAP enzymes, surpassing the inhibitory activity of conventional medicines [40].Additionally, their antibacterial activity against four human pathogenic bacteria were evaluated: 6 3,4]thiadiazine demonstrates significant efficacy against all bacteria [41].The synthesized compounds were assessed for their in vitro antiproliferative properties against MCF-7 and MDA-MB-231 breast cancer cell lines using the MTT test [47].Furthermore, the synthesized compounds were assessed for their in vitro antibacterial activity.The majority of compounds showed a satisfactory level of antibacterial activity and are notably more potent than ampicillin [49].Long chain alkenyl-6-phenyl-7H-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines 55a-d, were synthesized from the reaction of compound 1 and phenacyl bromide via a ring closure reaction (Scheme 22).The prepared compounds are promising anticancer agents [50].

Scheme 22. Synthesis of long chain triazolothiadiazines 55a-d.
In the same fashion, triazolothiadiazine derivatives 56 were synthesized through the reaction of 1 with phenacyl halides in EtOH at reflux conditions [14,15,51] (Scheme 23).The synthesized compounds were tested for their ability to relieve pain and reduce inflammation.Also, all compounds were tested for their ability to harm the digestive system and their antioxidant activity.Most of the substances worked well in both the carrageenan-induced oedema and acetic acid-induced writhing tests, with only minor effects on the digestive system.Most successful results were seen with molecules that added a chlorine or fluorine atom to the sixth position of the fused phenyl ring [15].The effects of triazolothiadiazines on hepatocellular carcinoma (HCC) cells were described.Compound 56t had the highest efficacy in halting the cell cycle specifically in the G2/M phase and inducing cell death in HCC cells.Oxidative stress induced activation of the JNK protein, resulting in a reconfiguration of the roles of ASK1, MKK7, and c-Jun proteins.In addition, nude mice treated with 56t had reduced growth tissue and experienced an extended lifespan without succumbing to illness.Additionally, 56t hindered the movement of HCC cells and the accumulation of liver cancer stem cells (LCSCs), both individually and when used in conjunction with sorafenib.Compound 56t has the potential to impact the proliferation, stemness, and migration of HCC cells, making it a possible treatment option for this increasingly prevalent disease, which is seeing an annual growth rate of approximately 3% [51].

Scheme 22. Synthesis of long chain triazolothiadiazines 55a-d.
In the same fashion, triazolothiadiazine derivatives 56 were synthesized through the reaction of 1 with phenacyl halides in EtOH at reflux conditions [14,15,51] (Scheme 23).The synthesized compounds were tested for their ability to relieve pain and reduce inflammation.Also, all compounds were tested for their ability to harm the digestive system and their antioxidant activity.Most of the substances worked well in both the carrageenan-induced oedema and acetic acid-induced writhing tests, with only minor effects on the digestive system.Most successful results were seen with molecules that added a chlorine or fluorine atom to the sixth position of the fused phenyl ring [15].The effects of triazolothiadiazines on hepatocellular carcinoma (HCC) cells were described.Compound 56t had the highest efficacy in halting the cell cycle specifically in the G2/M phase and inducing cell death in HCC cells.Oxidative stress induced activation of the JNK protein, resulting in a reconfiguration of the roles of ASK1, MKK7, and c-Jun proteins.In addition, nude mice treated with 56t had reduced growth tissue and experienced an extended lifespan without succumbing to illness.Additionally, 56t hindered the movement of HCC cells and the accumulation of liver cancer stem cells (LCSCs), both individually and when used in conjunction with sorafenib.Compound 56t has the potential to impact the proliferation, stemness, and migration of HCC cells, making it a possible treatment option for this increasingly prevalent disease, which is seeing an annual growth rate of approximately 3% [51].
When 1 was treated with 1-phenylbutane-1,3-dione and aromatic ketones in glacial acetic acid with drops of conc.sulfuric acid as a catalyst, it produced 7-benzoyl-3,6dimethyl-s-triazolo[3,4-b]1,3,4-thiadiazine 60 and 3-methyl-7H-s-triazolo[3,4-b]-1,3,4-thiadia zines 61, respectively (Scheme 26).The synthesized compounds show significant potential for eliminating heavy metal ions and inorganic anions from water-based systems.The metal ion removal efficiency achieved a high level of 76.29%, whereas the removal ef-ficiency for inorganic anions reached a perfect 100%.These findings indicate that the synthesized compounds possess significant potential as adsorbents for the purpose of water filtration [53].acetic acid with drops of conc.sulfuric acid as a catalyst, it produced 7-benzoyl-3,6dimethyl-s-triazolo [3,4-b]1,3,4-thiadiazine 60 and 3-methyl-7H-s-triazolo [3,4-b]-1,3,4thiadiazines 61, respectively (Scheme 26).The synthesized compounds show significant potential for eliminating heavy metal ions and inorganic anions from water-based systems.The metal ion removal efficiency achieved a high level of 76.29%, whereas the removal efficiency for inorganic anions reached a perfect 100%.These findings indicate that the synthesized compounds possess significant potential as adsorbents for the purpose of water filtration [53].(E)-4-(Arylideneamino)-5-methyl-4H-1,2,4-triazole-3-thiol 62 was treated with appropriate substituted ω-bromoacetophenone in EtOH containing Et 3 N at room temperature to give (3-methyl-6-aryl-6,7-dihydro-5H- [1,2,4]triazolo [3,4-b][1,3,4]thiadiazin-7-yl)(aryl)meth anones 64 via intermediate 63 (Scheme 28).The synthesized compounds were assessed for their antiproliferative activity against HepG2 cell lines in vitro.Additionally, their effects on plant growth regulation were evaluated on wheat and radish.The results revealed that all of the synthesized compounds showed a remarkably low antiproliferative activity against HepG2 cell lines in vitro, contrary to expectations.However, they demonstrated significant plant growth-regulating effects on both wheat and radish [54].3,4]thiadiazin-7-yl)(aryl)methanones 64 via intermediate 63 (Scheme 28).The synthesized compounds were assessed for their antiproliferative activity against HepG2 cell lines in vitro.Additionally, their effects on plant growth regulation were evaluated on wheat and radish.The results revealed that all of the synthesized compounds showed a remarkably low antiproliferative activity against HepG2 cell lines in vitro, contrary to expectations.However, they demonstrated significant plant growth-regulating effects on both wheat and radish [54].Aminotriazolethione 1 was reacted with thiophene-2-carbaldehyde to afford the corresponding Schiff's base 65.The latter compound was then treated with phenacyl bromides leading to fused heterocycle triazolothiadiazines 66.Moreover, compounds 67 were obtained directly by the reaction of 1 with phenacyl bromides [55] (Scheme 29).b] [1,3,4]thiadiazin-7-yl)(aryl)methanones 64 via intermediate 63 (Scheme 28).The synthesized compounds were assessed for their antiproliferative activity against HepG2 cell lines in vitro.Additionally, their effects on plant growth regulation were evaluated on wheat and radish.The results revealed that all of the synthesized compounds showed a remarkably low antiproliferative activity against HepG2 cell lines in vitro, contrary to expectations.However, they demonstrated significant plant growth-regulating effects on both wheat and radish [54].Aminotriazolethione 1 was reacted with thiophene-2-carbaldehyde to afford the corresponding Schiff's base 65.The latter compound was then treated with phenacyl bromides leading to fused heterocycle triazolothiadiazines 66.Moreover, compounds 67 were obtained directly by the reaction of 1 with phenacyl bromides [55] (Scheme 29).On the other hand, a series of triazolothiadiazine hydrazone derivatives 76 was synthesized via a one-pot, three-components reaction of Purpald 68, phenacyl bromides, and various aromatic aldehydes in EtOH at a reflex temperature [60,61] (Scheme 33).On the other hand, a series of triazolothiadiazine hydrazone derivatives 76 was synthesized via a one-pot, three-components reaction of Purpald 68, phenacyl bromides, and various aromatic aldehydes in EtOH at a reflex temperature [60,61]  On the other hand, a series of triazolothiadiazine hydrazone derivatives 76 was synthesized via a one-pot, three-components reaction of Purpald 68, phenacyl bromides, and various aromatic aldehydes in EtOH at a reflex temperature [60,61] (Scheme 33).Similarly, triazolothiadiazine hydrazone derivatives 77 were produced via a one-pot four component approach involving the condensation of 68, aromatic aldehydes and various phenacyl bromides in absolute EtOH containing Et3N [62] (Scheme 34).The mechanism of producing compound 77 is described in the Scheme.Firstly, dianils 78 were formed; then a nucleophilic substitution reaction between the latter dianils and phanacyl bromide was followed by an annulation reaction via intermediates 79 and 80 [62]  The mechanism of producing compound 77 is described in the Scheme.Firstly, dianils 78 were formed; then a nucleophilic substitution reaction between the latter dianils and phanacyl bromide was followed by an annulation reaction via intermediates 79 and 80 [62] (Scheme 35).The mechanism of producing compound 77 is described in the Scheme.Firstly, dianils 78 were formed; then a nucleophilic substitution reaction between the latter dianils and phanacyl bromide was followed by an annulation reaction via intermediates 79 and 80 [62]  (±)-3-(1H-Pyrazol-1-yl)-6,7-dihydro-5H- [1,2,4]triazolo [3,4-b][1,3,4]thiadiazines 82 were developed using a two-step, one pot four-components reaction of 68, acetylacetone, aldehyde in EtOH with drops of conc.HCl at reflux to give 4-(arylideneamino)-5-(3,5-dimethyl-1H-pyrazol-1-yl)-4H-1,2,4-triazole-3-thiol 81, not isolated, then substituted phenacyl bromide and Et 3 N were added.Then the mixture was refluxed to produce the target compounds (Scheme 36).The prepared compounds were tried to see how well they worked against viruses and tumors.It was shown that small changes in the structure of the phenyl group could change the biological qualities to make them more effective against viruses or tumors.These compounds have the ability to fight tumors due to stopping tubulin from polymerizing [63].

Cpd
Molecules 2024, 29, x FOR PEER REVIEW 27 of 38 phenacyl bromide and Et3N were added.Then the mixture was refluxed to produce the target compounds (Scheme 36).The prepared compounds were tried to see how well they worked against viruses and tumors.It was shown that small changes in the structure of the phenyl group could change the biological qualities to make them more effective against viruses or tumors.compounds have the ability to fight due to stopping tubulin from polymerizing [63].

Scheme 26 .
Scheme 26.Synthesis of triazolothiadiazines 60 and 61.The proposed mechanisms for the formation of compounds 60 and 61 are summarized in Scheme 27.The s-triazole is initially oxidized to form the disulfide intermediate A, which is followed by the nucleophilic attack of the enolate form of the ketone to give the S-alkylation intermediate B. This formed intermediate then undergoes intramolecular cyclization to yield the desired products.
[53]s-triazolo[3,4-b]1,3,4-thiadiazine 60 and 3-methyl-7H-s-triazolo[3,4-b]-1,3,4thiadiazines 61, respectively (Scheme 26).The synthesized compounds show significant potential for eliminating heavy metal ions and inorganic anions from water-based systems.The metal ion removal efficiency achieved a high level of 76.29%, whereas the removal efficiency for inorganic anions reached a perfect 100%.These findings indicate that the synthesized compounds possess significant potential as adsorbents for the purpose of water filtration[53].The proposed mechanisms for the formation of compounds 60 and 61 are summarized in Scheme 27.The s-triazole is initially oxidized to form the disulfide intermediate A, which is followed by the nucleophilic attack of the enolate form of the ketone to give the S-alkylation intermediate B. This formed intermediate then undergoes intramolecular cyclization to yield the desired products.