Syntheses and Applications of 1,2,3-Triazole-Fused Pyrazines and Pyridazines

Pyrazines and pyridazines fused to 1,2,3-triazoles comprise a set of heterocycles obtained through a variety of synthetic routes. Two typical modes of constructing these heterocyclic ring systems are cyclizing a heterocyclic diamine with a nitrite or reacting hydrazine hydrate with dicarbonyl 1,2,3-triazoles. Several unique methods are known, particularly for the synthesis of 1,2,3-triazolo[1,5-a]pyrazines and their benzo-fused quinoxaline and quinoxalinone-containing analogs. Recent applications detail the use of these heterocycles in medicinal chemistry (c-Met inhibition or GABAA modulating activity) as fluorescent probes and as structural units of polymers.


Synthetic Approaches
This overview of synthetic methods is organized according to the type of heterocycle. In the case of 1H-1,2,3-triazolo [1,5-a]pyrazines, methods are subdivided into pyrazines and benzopyrazines. Reaction times are included along with solvents, catalysts, and other reagents in most examples. Commercial availability of precursors is emphasized where applicable.

Synthetic Approaches
This overview of synthetic methods is organized according to the type of heterocycle. In the case of 1H-1,2,3-triazolo [1,5-a]pyrazines, methods are subdivided into pyrazines and benzopyrazines. Reaction times are included along with solvents, catalysts, and other reagents in most examples. Commercial availability of precursors is emphasized where applicable.
Molecules 2022, 27, x FOR PEER REVIEW 11 Synthesizing compounds of the same type, Bertelli, and coworkers [48] first for a triazole diester on a ring ortho to a nitro group, 65, which was intramolecularly cycl to form ethyl 4,5-dihydro-4-oxo-[1,2,3]triazolo[1,5-a]quinoxaline-3-carboxylate, (Scheme 16). This reaction was conducted by hydrogenation with a 10% Pd/C cataly by reaction with FeCl3 and Fe powder. Biagi and coworkers [49] cyclized the tria diester into 1,2,3-triazoloquinoxalinone 66 with 10% Pd/C in ethanol in an excellent yield. Shen and coworkers further modified the ester group of 66 to prepare a deriva suitable for biological testing [50].   Saha and coworkers [52] used the intramolecular cyclization of ortho-substituted anilines with tethered 1,2,3-triazoles, 72, a Pictet-Spengler reaction, to form 1,2,3-triazoloquinoxalines 73 in yields in the range 61-70% (Scheme 18). This sequence offers two-point diversity: one from 72, and the other from an aryl aldehyde 73. The prerequisite triazole 72 was conveniently prepared from readily available starting materials, including o-fluoronitrobenzene 70, phenylacetylene 71, and sodium azide.  Using photoredox catalysis, He and coworkers [54] used [fac-Ir(ppy)3] as a photoca lyst to afford the corresponding 1,2,3-triazoloquinoxaline 78 from isonitrile 77 in 60% yi (Scheme 20). Due to poor solubility of the catalyst, ACN resulted in decreased yields co pared to DMF. This work is a rare example of free-radical generation of 1,2,3-triaz fused ring systems, as cyclohexyl radicals are proposed to have formed from pheny dine(III)dicarboxylate. The radicals yield isonitrile carbon radicals, followed by react with carbon 5 of the triazole. Various fused rings were synthesized in addition to 1, triazoles including tetrazoles, pyrazoles, and imidazoles in yields as high as 80%.  Using photoredox catalysis, He and coworkers [54] used [fac-Ir(ppy) 3 ] as a photocatalyst to afford the corresponding 1,2,3-triazoloquinoxaline 78 from isonitrile 77 in 60% yield (Scheme 20). Due to poor solubility of the catalyst, ACN resulted in decreased yields compared to DMF. This work is a rare example of free-radical generation of 1,2,3triazole-fused ring systems, as cyclohexyl radicals are proposed to have formed from phenyliodine(III)dicarboxylate. The radicals yield isonitrile carbon radicals, followed by reaction with carbon 5 of the triazole. Various fused rings were synthesized in addition to 1,2,3-triazoles including tetrazoles, pyrazoles, and imidazoles in yields as high as 80%.
forming 84, or phenylboronic acid, forming 85. Derivatives of 84 were prepared in yields in the range 26-78%, and one synthesis of 85 yielded 86%.

Syntheses of 1,2,3-Triazolo[1,5-b]pyridazines
Despite being reported as early as 1949 by Schofield and coworkers [85] in their study of cinnolines, 1,2,3-triazolo [1,5-b]pyridazines remain rare in the literature, in part owing to few methods available for their synthesis. While synthesizing azepinones, Evans and coworkers [86] instead serendipitously obtained 3,6-diphenyl-1,2,3-triazolo[1,5-b]pyridazine 108. This was obtained from the intramolecular cyclization of diketo-oxime 107 (Scheme 29) after refluxing in HCl. This gave up to 22% of a pyrazinylhydrazone byproduct. A similar method in the same report used HOAc, but this resulted in poor yields (about 15%) and up to three products. A number of methods exist for the preparation of molecules containing the 1,2,3triazolo [4,5-d]pyridazine core, the majority of which involve the treatment of 1,2,3-triazole dicarbonyl species with hydrazine hydrate followed by acid or heat-promoted cyclization, or the cyclization of a diaminopyridazine with nitrite.

Syntheses of 1,2,3-Triazolo[1,5-b]pyridazines
Despite being reported as early as 1949 by Schofield and coworkers [85] in their study of cinnolines, 1,2,3-triazolo [1,5-b]pyridazines remain rare in the literature, in part owing to few methods available for their synthesis. While synthesizing azepinones, Evans and coworkers [86] instead serendipitously obtained 3,6-diphenyl-1,2,3-triazolo[1,5b]pyridazine 108. This was obtained from the intramolecular cyclization of diketo-oxime 107 (Scheme 29) after refluxing in HCl. This gave up to 22% of a pyrazinylhydrazone byproduct. A similar method in the same report used HOAc, but this resulted in poor yields (about 15%) and up to three products. A fluoroborate salt was prepared by Riedl and coworkers [87] in a manner similar to that of Beres and coworkers [36]. The acyl-substituted pyridazine, 111, after treatment with p-bromophenyl hydrazine hydrochloride 112 gave the hydrazone 113. Tribromophenol bromine (TBP) in DCM afforded the desired ring-closed product 114 in 67% yield (Scheme 30). The initial bromide salt was converted to the fluoroborate salt with 40% fluoroboric acid in ACN. Ketone 111 was prepared by the same group via reaction of a commercially available 3-cyanopyridizine 109 with p-chlorophenylmagnesium bromide 110, also synthesized from commercially available p-chlorobromobenzene and Mg. This was followed by acidic workup to afford the desired ketone. Compounds of this type were also prepared by Vasko and coworkers [88] using a similar method, which gave a 27% yield. A third method for the synthesis of 1,2,3-triazolo [ A fluoroborate salt was prepared by Riedl and coworkers [87] in a manner similar to that of Beres and coworkers [36]. The acyl-substituted pyridazine, 111, after treatment with p-bromophenyl hydrazine hydrochloride 112 gave the hydrazone 113. Tribromophenol bromine (TBP) in DCM afforded the desired ring-closed product 114 in 67% yield (Scheme 30). The initial bromide salt was converted to the fluoroborate salt with 40% fluoroboric acid in ACN. Ketone 111 was prepared by the same group via reaction of a commercially available 3-cyanopyridizine 109 with p-chlorophenylmagnesium bromide 110, also synthesized from commercially available p-chlorobromobenzene and Mg. This was followed by acidic workup to afford the desired ketone. Compounds of this type were also prepared by Vasko and coworkers [88] using a similar method, which gave a 27% yield. A third method for the synthesis of 1,2,3-triazolo[1,5-b]pyridazines consisted of intramolecular oxidative ring closure of a hydrazone derived from 111 to afford the neutral 1,2,3-triazolo [1,5-b]pyridazine 115 [89]. Kvaskoff and coworkers employed MnO 2 as an oxidant using a similar procedure [35,89,90], where purification by sublimation afforded the desired product 115 (where R 1 = R 2 = H) in 71% yield.
A fluoroborate salt was prepared by Riedl and coworkers [87] in a manner similar to that of Beres and coworkers [36]. The acyl-substituted pyridazine, 111, after treatment with p-bromophenyl hydrazine hydrochloride 112 gave the hydrazone 113. Tribromophenol bromine (TBP) in DCM afforded the desired ring-closed product 114 in 67% yield (Scheme 30). The initial bromide salt was converted to the fluoroborate salt with 40% fluoroboric acid in ACN. Ketone 111 was prepared by the same group via reaction of a commercially available 3-cyanopyridizine 109 with p-chlorophenylmagnesium bromide 110, also synthesized from commercially available p-chlorobromobenzene and Mg. This was followed by acidic workup to afford the desired ketone. Compounds of this type were also prepared by Vasko and coworkers [88] using a similar method, which gave a 27% yield. A third method for the synthesis of 1,2,3-triazolo [1,5-b]pyridazines consisted of intramolecular oxidative ring closure of a hydrazone derived from 111 to afford the neutral 1,2,3-triazolo [1,5-b]pyridazine 115 [89]. Kvaskoff and coworkers employed MnO2 as an oxidant using a similar procedure [35,89,90], where purification by sublimation afforded the desired product 115 (where R1 = R2 = H) in 71% yield.

Applications
Recent applications of the aforementioned heterocyclic systems, covering both medicinal and non-medicinal topics, are discussed in the following section.
Later, using PF-04217903 as a reference, Jia, and coworkers [1] reported the discovery of a compound now known as Savolitinib (Figure 3). This compound, also an exquisite c-Met inhibitor with an equal IC 50 of 0.005 µM, demonstrated favorable pharmacokinetic properties in mice [1]. Savolitinib possessed equal potency. Having recently passed phase II clinical trials for the treatment of metastatic non-small cell lung cancer, papillary and clear cell renal cell carcinoma, gastric cancer, and colorectal cancer, Savolitinib has been granted conditional approval for use in China at the time of this review [97]. A review of c-Met inhibitors in non-small cell lung cancer has recently appeared [98]. tent (IC50 = 0.005 µM) and selective inhibition of over 200 c-Met kinases [2]. This heterocyclic scaffold in general gave rise to derivatives (altering substituents at the 2 and 6 ring positions) with potent inhibition, of which PF-04217903 was the best. This compound was selected as a preclinical candidate for the treatment of cancer [96].
Later, using PF-04217903 as a reference, Jia, and coworkers [1] reported the discovery of a compound now known as Savolitinib (Figure 3). This compound, also an exquisite c-Met inhibitor with an equal IC50 of 0.005 µM, demonstrated favorable pharmacokinetic properties in mice [1]. Savolitinib possessed equal potency. Having recently passed phase II clinical trials for the treatment of metastatic non-small cell lung cancer, papillary and clear cell renal cell carcinoma, gastric cancer, and colorectal cancer, Savolitinib has been granted conditional approval for use in China at the time of this review [97]. A review of c-Met inhibitors in non-small cell lung cancer has recently appeared [98]. Sirbu and coworkers [20] recently reported a novel class of small molecules containing the 1,2,3-triazolo [4,5-b]pyrazine scaffold with excellent properties for use as versatile fluorescent probes in optical imaging ( Figure 4). Specifically, a phenyl ester derivative was used to dye HeLa cells in epifluorescence microscopy. Compared to commercially available LysoTracker Green DND-26, the tested triazolopyrazine derivative demonstrated comparable properties. In addition, it showed low cytotoxicity when evaluated in Alamar Blue assay (>95% cell viability up to 170 µM) and showed high solubility with a variety of desirable characteristics. A phenyl ester derivative, when evaluated as a dye in HeLa cells, showed high photostability and low cytotoxicity [20].  Sirbu and coworkers [20] recently reported a novel class of small molecules containing the 1,2,3-triazolo [4,5-b]pyrazine scaffold with excellent properties for use as versatile fluorescent probes in optical imaging ( Figure 4). Specifically, a phenyl ester derivative was used to dye HeLa cells in epifluorescence microscopy. Compared to commercially available LysoTracker Green DND-26, the tested triazolopyrazine derivative demonstrated comparable properties. In addition, it showed low cytotoxicity when evaluated in Alamar Blue assay (>95% cell viability up to 170 µM) and showed high solubility with a variety of desirable characteristics. A phenyl ester derivative, when evaluated as a dye in HeLa cells, showed high photostability and low cytotoxicity [20].  Intriguingly, another application lay in the monitoring of hypoxic regions w mor cells. This was explored by Janczy-Cempa and coworkers [23], who looke fluorescent products produced after reduction of nitrotriazolopyrazine probes by ductases (enzymes often overexpressed in tumor regions). Both probes studied (   [20] for use in optical and/or cellular imaging. Intriguingly, another application lay in the monitoring of hypoxic regions within tumor cells. This was explored by Janczy-Cempa and coworkers [23], who looked at the fluorescent products produced after reduction of nitrotriazolopyrazine probes by nitroreductases (enzymes often overexpressed in tumor regions). Both probes studied ( Figure 5) had very weak fluorescence in normoxic regions, but their reduction by nitroreductases led to a 15-fold increase in intensity in hypoxic regions. This was evaluated using the human melanoma cell line A2058. In contrast to the fluorescence probes developed by Sirbu and coworkers [20], probes in this study had substitutions on the pyrazine ring as opposed to the triazole-fused pyrazole. While additional work is still to be done, this report demonstrates the potential for these highly conjugated compounds to be useful in biomedical monitoring. Legentil and coworkers [99] obtained compounds similar to the structure on the right in Figure 5 in yields as high as 79%, which were used to develop a luminescence layered double-hydroxide filter. This material was dispersed into a polymer for use as a dye.  [20] for use in optical and/or cellular imaging.
Intriguingly, another application lay in the monitoring of hypoxic regions within tumor cells. This was explored by Janczy-Cempa and coworkers [23], who looked at the fluorescent products produced after reduction of nitrotriazolopyrazine probes by nitroreductases (enzymes often overexpressed in tumor regions). Both probes studied ( Figure 5) had very weak fluorescence in normoxic regions, but their reduction by nitroreductases led to a 15-fold increase in intensity in hypoxic regions. This was evaluated using the human melanoma cell line A2058. In contrast to the fluorescence probes developed by Sirbu and coworkers [20], probes in this study had substitutions on the pyrazine ring as opposed to the triazole-fused pyrazole. While additional work is still to be done, this report demonstrates the potential for these highly conjugated compounds to be useful in biomedical monitoring. Legentil and coworkers [99] obtained compounds similar to the structure on the right in Figure 5 in yields as high as 79%, which were used to develop a luminescence layered double-hydroxide filter. This material was dispersed into a polymer for use as a dye. Overall, applications of compounds containing 1,2,3-triazolo [4,5-b]pyrazines in the current literature are focused on c-Met inhibition (i.e., the treatment of distinct types of cancers), and optical and/or cellular imaging, with triazapentalene-type molecules demonstrating a wide range of favorable characteristics as fluorescent probes.
Other recent patents have been filed regarding fused pyridazines with herbicidal activity, of which 1,2,3-triazolo [4,5-c]pyridazine is included [102]. In another recent patent, compounds of this type were implicated in controlling unwanted plant growth [103].
Reports of compounds containing the 1,2,3-triazolo[4,5-c]pyridazine scaffold are uncommon in the current literature beyond synthetic reports and patents. Undoubtedly, there is still work to be done in exploring the potential applications of this unique heterocyclic system.

Applications of 1H-1,2,3-Triazolo[4,5-d]pyridazines
In a recent development, Li, and coworkers [4] outlined a series of triazole-based structures for the construction of conjugated polymers for solar cells. In addition to demonstrating desirable properties as units incorporated into polymers (Figure 6), their reported synthetic route uses affordable, commercially available starting materials and produces units compatible with other monomers. Structures containing 1,2,3-triazolo[4,5-d]pyridazine components offer a privileged, conjugated unit for the construction of polymers owing in part to the convenient para substitution of the pyridazine ring and perpendicular N2 substitution of the triazole ring.
Reports of compounds containing the 1,2,3-triazolo [4,5-c]pyridazine scaffold are uncommon in the current literature beyond synthetic reports and patents. Undoubtedly, there is still work to be done in exploring the potential applications of this unique heterocyclic system.

Applications of 1H-1,2,3-triazolo[4,5-d]pyridazines
In a recent development, Li, and coworkers [4] outlined a series of triazole-based structures for the construction of conjugated polymers for solar cells. In addition to demonstrating desirable properties as units incorporated into polymers (Figure 6), their reported synthetic route uses affordable, commercially available starting materials and produces units compatible with other monomers. Structures containing 1,2,3-triazolo [4,5d]pyridazine components offer a privileged, conjugated unit for the construction of polymers owing in part to the convenient para substitution of the pyridazine ring and perpendicular N2 substitution of the triazole ring. Another notable outcome of the study of 1,2,3-triazolo [4,5-d]pyridazines was that from Biagi and coworkers [104], who reported compounds of this type with high selectivity for the A1 receptor subtype in radioligand binding assays at bovine brain adenosine A1 and A2A receptors. The most potent compound contained a 4-amino-substituted 7-hydroxy-1,2,3-triazolo [4,5-d]pyridazine, and after substitution of the hydroxyl group for a chlorine, affinity decreased and suggested a hydrogen-bond donating substituent at position 7 was critical for binding affinity.

Applications of 1,2,3-Triazolo[1,5-a]pyrazines
Among applications of compounds containing the 1,2,3-triazolo[1,5-a]pyrazine unit are those of benzo-fused 1,2,3-triazoloquinoxalines and saturated 1,2,3-triazole-fused piperidines. In a recent report by Pérez Morales and coworkers [105], a 1,2,3-triazoloquinoxalinone (Structure A, Figure 7) was identified via high-throughput screening as inducing expression of Rgg2/3-regulated genes in the presence of short hydrophobic pheromones at low concentrations. This work stemmed from interest in the Rgg2/3 quorum sensing circuit of the pathogen Streptococcus pyogenes, with the objective of manipulating and inhibiting the bacteria. After analyzing its mode of action, it was determined this compound directly uncompetitively inhibited recombinant PepO in vitro, and induced quorum sensing signaling by stabilizing short hydrophobic pheromones. triazoloquinoxalinone (Structure A, Figure 7) was identified via high-throughput screening as inducing expression of Rgg2/3-regulated genes in the presence of short hydrophobic pheromones at low concentrations. This work stemmed from interest in the Rgg2/3 quorum sensing circuit of the pathogen Streptococcus pyogenes, with the objective of manipulating and inhibiting the bacteria. After analyzing its mode of action, it was determined this compound directly uncompetitively inhibited recombinant PepO in vitro, and induced quorum sensing signaling by stabilizing short hydrophobic pheromones. Figure 7. Compounds containing congeners of the 1,2,3-triazolo[1,5-a]pyrazine core with a diverse set of biological activities: (A) an inducer of Rgg2/3-related genes of the human pathogen Streptococcus pyogenes [105], (B) a potent DPP-IV inhibitor evaluated for the treatment of type II diabetes [7], and (C), an identified BACE-1 inhibitor [6].
These reports, while not exhaustive, demonstrate recent applications of compounds containing the 1,2,3-triazolo[1,5-a]pyrazine scaffold or congeners thereof. Particularly prominent in the literature are benzo-fused and piperazine-containing analogs.

Applications of 1,2,3-Triazolo[1,5-b]pyridazines
There are no reported applications of compounds containing the 1,2,3-triazolo[1,5b]pyridazine ring system, and little regarding its physiological and/or pharmacological effects are known. Aside from one recent patent [106] regarding immunoregulatory functions, additional applications remain scarce at the time of this review.
The potential for new synthetic contributions is considerable for the triazole-fused pyrazines and pyridazines. Given the diversity of synthetic methods summarized in this review, new contributions that could be most beneficial are new routes to some of the precursors of the fused systems. In many of the reports cited, the starting materials are either not available commercially or are very expensive. For example, some diamino pyrazines are available as unsubstituted compounds or as halogenated derivatives, but all are USD 500-1000 per gram. Future studies of methods employing additional intramolecular cycloadditions leading to 1,2,3-triazolo[1,5-a]pyrazine derivatives would appear to have potential. Work on synthesis of the 1H-1,2,3-triazolo [4,5-c]pyridazines and the 1,2,3triazolo [1,5-b]pyridazines would be welcome for these less frequently studied areas.
Overall, diverse methods exist for the preparation of 1,2,3-triazole-fused diazines, spanning the last seven decades with numerous reports in the last five years. Currently, drugs containing these ring systems remain scarce with only a handful of exceptions, particularly containing either the 1,2,3-triazolo[4,5-b]pyrazine or 1,2,3-triazolo[4,5-c]pyridazine scaffold. Applications of the aforementioned types of compounds span from medicinal chemistry into the development of dyes, probes, and inhibitors of enzymes implicated in various diseases. Despite this, there lies underrealized and exciting potential for employing triazolopyrazines and triazolopyridazines as diverse substrates in the generation of novel molecules with a wide array of applications.