Synthesis of Benzo[4,5]thiazolo[2,3-c][1,2,4]triazole Derivatives via C-H Bond Functionalization of Disulfide Intermediates

Many nitrogen- and sulfur-containing heterocyclic compounds exhibit biological activity. Among these heterocycles are benzo[4,5]thiazolo[2,3-c][1,2,4]triazoles for which two main synthetic approaches exist. Here we report a new synthetic protocol that allows the preparation of these tricyclic compounds via the oxidation of a mercaptophenyl moiety to its corresponding disulfide. Subsequent C-H bond functionalization is thought to enable an intramolecular ring closure, thus forming the desired benzo[4,5]thiazolo[2,3-c][1,2,4]triazole. This method combines a high functional group tolerance with short reaction times and good to excellent yields.

An alternative approach for the synthesis of benzo [4,5]thiazolo [2,3-c] [1,2,4]triazoles starts with a 3-mercaptotriazole derivative that is fused with the benzene ring, thus forming the thiazole ring in the last step (Scheme 1C,F). An example of this synthetic route is shown in Scheme 1C, where 1-chloro-2-isothiocyanatobenzene, N, is reacted with a hydrazide derivative to give compound O [40]. In the presence of a strong base, such as sodium hydride in boiling DMF, the tricyclic product, Q, can be obtained directly from O. Insight into the mechanism of this reaction was gained by the use of a weaker base, which allowed the isolation of the reaction intermediate, P, upon acidification. Subsequent treatment of this intermediate, P, with sodium hydride in DMF under reflux affords the tricyclic compound, Q. As shown in Scheme 1D, cyclization of this intermediate, P , can also be achieved photochemically by subjecting it to 254 nm irradiation to yield compound R in a moderate yield [41]. Scheme 1E highlights a different route to the triazolo species (U) via two sequential oxidation steps. In the first step, a sodium salt (S) is converted to its disulfide (T), which, in the presence of bromine or iodine, undergoes an oxidative cyclization to the target molecule, U [42]. Unfortunately, a very narrow substrate scope, in combination with carbon tetrachloride being used as a solvent, restricts the application of this procedure significantly [43,44]. While the copper-catalyzed sequential diarylation of 2-mercaptotriazole, W, with 1-bromo-2-iodobenzene, V, in Scheme 1F was also shown to be possible, this reaction gave heterocycle, M, in only 14% yield [45].
A third approach leading to the benzo [4,5]thiazolo [2,3-c] [1,2,4]triazole ring system is based on forming the thiazole ring in the last reaction step by a bond formation between the sulfur of the 2-mercaptophenyl substituent of a triazole and the unfunctionalized triazole carbon (Scheme 1G,H). The Straub group has observed two of their thiol-containing triazolium salts (X) undergo an oxidative cyclization in DMSO (Scheme 1G), thereby forming charged N-substituted heteroaromatic compounds, Y [46,47]. As opposed to the acidic triazolium starting materials of the Straub group, we demonstrate in this report that a similar bond formation can be employed to obtain neutral benzo [4,5]thiazolo [2,3-c] [1,2,4]triazoles containing a wide variety of functional groups on the benzene ring from non-acidic triazoles (Scheme 1H). We were aiming to include both electron-donating and electron-withdrawing groups, halides, amines, alcohols, carboxylic acids, and other synthetically valuable substituents. As shown in Scheme 1H, we developed a two-step process for the conversion of a 4-(2-((4-methoxybenzyl)thio)phenyl)-4H-1,2,4-triazole species (Z) into the target tricyclic compound (AB).After the selective removal of the p-methoxybenzyl protecting group in the first step, the resulting free thiol (AA) is subsequently oxidized with DMSO to a disulfide intermediate (not shown in this scheme), which, upon deprotonation of the triazole carbon, undergoes an intramolecular ring closure. Herein, we present both the preparation of the starting triazoles (Z) from commercially available precursors, as well as their conversion to the benzo [4,5]thiazolo [2,3-c] [1,2,4]triazole derivative, AB.
A third approach leading to the benzo [4,5]thiazolo [2,3-c] [1,2,4]triazole ring system based on forming the thiazole ring in the last reaction step by a bond formation betwe the sulfur of the 2-mercaptophenyl substituent of a triazole and the unfunctionalized t azole carbon (Scheme 1G,H). The Straub group has observed two of their thiol-containi we present both the preparation of the starting triazoles (Z) from commercially available precursors, as well as their conversion to the benzo [4,5]thiazolo [2,3-c] [1,2,4]triazole derivative, AB.

Preparation of Triazole Precursors
Triazole, 3a, was prepared according to the literature procedure shown in Scheme 2 [46,48]. While the p-methoxybenzyl-protected aniline, 2a, could be obtained in this case by protecting 2-mercaptoaniline, 1a, under argon atmosphere, the lack of commercial availability of substituted 2-mercaptoanilines required the development of a different synthesis route. An obvious method for introducing a sulfur substituent on an aromatic ring would be a classical nucleophilic aromatic substitution using a 2-halonitrobenzene derivative starting material [49][50][51]. Subsequent reduction of the nitro group would afford the corresponding aniline derivative. This alternative route to the p-methoxybenzyl-protected 2-mercaptoanilines, 2b-2r, starting from 1-fluoro-2-nitrobenzene derivatives, is shown in Scheme 3. By using (4methoxyphenyl)methanethiol as a reagent in the nucleophilic aromatic substitution, we were able to obtain the p-methoxybenzyl-protected 2-mercaptonitrobenzene derivatives, 1b-1r, in excellent yields (77-99%) in one step. An exception was 1h, which was prepared from tert-butyl(4-fluoro-3-nitrophenoxy)dimethylsilane in 27% yield. The silyl-protecting group was required to prevent self-condensation of the starting material under basic conditions and is cleaved during the nucleophilic aromatic substitution due to the generation of fluoride, thus forming the free phenol 1h. The ester species 1m′ was obtained quantitatively from the carboxylic acid 1m. Reduction of the nitrobenzene derivatives 1b-1r in the presence of excess iron powder and ammonium chloride gave the corresponding amines, 2b-2r, in excellent yields (79-99%).

Scheme 2. Synthesis of triazole, 3a.
This alternative route to the p-methoxybenzyl-protected 2-mercaptoanilines, 2b-2r, starting from 1-fluoro-2-nitrobenzene derivatives, is shown in Scheme 3. By using (4methoxyphenyl)methanethiol as a reagent in the nucleophilic aromatic substitution, we were able to obtain the p-methoxybenzyl-protected 2-mercaptonitrobenzene derivatives, 1b-1r, in excellent yields (77-99%) in one step. An exception was 1h, which was prepared from tert-butyl(4-fluoro-3-nitrophenoxy)dimethylsilane in 27% yield. The silyl-protecting group was required to prevent self-condensation of the starting material under basic conditions and is cleaved during the nucleophilic aromatic substitution due to the generation of fluoride, thus forming the free phenol 1h. The ester species 1m was obtained quantitatively from the carboxylic acid 1m. Reduction of the nitrobenzene derivatives 1b-1r in the presence of excess iron powder and ammonium chloride gave the corresponding amines, 2b-2r, in excellent yields (79-99%).
Compound 2s was not available via a nucleophilic substitution followed by a reduction due to the reactivity of the second fluorine substituent. Instead, we prepared 2s in two steps from 2,2 -disulfanediylbis(4-fluoroaniline) by first reducing it with NaBH 4 to free the thiol that was subsequently protected with the p-methoxybenzyl group (Scheme 4). The synthesis of 2,2 -disulfanediylbis(4-fluoroaniline) can be found in the experimental procedures (Section 3.6.1).
In analogy to 3a, which was prepared in 69% yield following a literature procedure [46,49], triazoles 3b-3s were obtained by heating the aniline derivatives 2b-2s in the presence of N,N-dimethylformamide azine dihydrochloride at 150 • C for 16 h (Scheme 5). While the carboxylic acid 3m can be formed using the procedure described above, we were unable to separate it from the salts and eventually synthesized it in 81% yield by hydrolysis of the ester 3m . The yields of the triazoles obtained using this method were extremely variable (10-72%), as can be seen in Scheme 4, and certainly could use improvement. The low yields depended on the exact structure of the aniline. In many cases, the starting amine could be isolated back. However, leaving the reaction for a longer duration did not increase the amount of product isolated, which suggests that the N,N-dimethylformamide azine dihydrochloride may degrade over time. In some cases, the starting amine was not detected at the end of a low-yielding reaction, and we believe that it may undergo decomposition due to the high reaction temperature. Loss of the p-methoxybenzyl protecting group was observed in all reactions, generally resulting in the formation of trace amounts of the target benzo [4,5]thiazolo [2,3-c] [1,2,4]triazoles as byproducts of the triazole formation. However, this reaction was found to be the most dominant transformation in the preparation of 3d, which was obtained in only 17% yield. In addition, dehalogenation further lowered the yields of the bromo-and chloro-derivatives 3n-3q, whereas deamination was the major cause for the low isolated yields of the pyridine derivatives 3i and 3j. While we are currently investigating higher yielding routes to triazoles 3a-3s, we were able to isolate sufficient material to carry out a thorough investigation of the new oxidative cyclization reaction leading to the benzo [4,5]thiazolo [2,3-c] [1,2,4]triazole derivatives.
Compound 2s was not available via a nucleophilic substitution followed by a reduction due to the reactivity of the second fluorine substituent. Instead, we prepared 2s in two steps from 2,2′-disulfanediylbis(4-fluoroaniline) by first reducing it with NaBH4 to free the thiol that was subsequently protected with the p-methoxybenzyl group (Scheme 4). The synthesis of 2,2′-disulfanediylbis(4-fluoroaniline) can be found in the experimental procedures (Section 3.6.1).
Compound 2s was not available via a nucleophilic substitution followed by a reduction due to the reactivity of the second fluorine substituent. Instead, we prepared 2s in two steps from 2,2′-disulfanediylbis(4-fluoroaniline) by first reducing it with NaBH4 to free the thiol that was subsequently protected with the p-methoxybenzyl group (Scheme 4). The synthesis of 2,2′-disulfanediylbis(4-fluoroaniline) can be found in the experimental procedures (Section 3.6.1).

Scheme 4. Synthesis of 2s.
In analogy to 3a, which was prepared in 69% yield following a literature procedure [46,49], triazoles 3b-3s were obtained by heating the aniline derivatives 2b-2s in the presence of N,N-dimethylformamide azine dihydrochloride at 150 °C for 16 h (Scheme 5). While the carboxylic acid 3m can be formed using the procedure described above, we were unable to separate it from the salts and eventually synthesized it in 81% yield by hydrolysis of the ester 3m′. The yields of the triazoles obtained using this method were extremely variable (10-72%), as can be seen in Scheme 4, and certainly could use improvement. The low yields depended on the exact structure of the aniline. In many cases, the starting amine could be isolated back. However, leaving the reaction for a longer duration did not increase the amount of product isolated, which suggests that the N,N-dimethylformamide azine dihydrochloride may degrade over time. In some cases, the starting

Reaction Optimization
Complete deprotection of the thiol group of triazole 3a was achieved within 1 h by treatment with triflic acid and anisole in TFA under an argon atmosphere at 0 • C, thus forming the free thiol 4a (Scheme 6). mation in the preparation of 3d, which was obtained in only 17% yield. In addition, dehalogenation further lowered the yields of the bromo-and chloro-derivatives 3n-3q, whereas deamination was the major cause for the low isolated yields of the pyridine derivatives 3i and 3j. While we are currently investigating higher yielding routes to triazoles 3a-3s, we were able to isolate sufficient material to carry out a thorough investigation of the new oxidative cyclization reaction leading to the benzo [4,5]thiazolo [2,3-c] [1,2,4]triazole derivatives. Scheme 5. Preparation of triazole precursors 3a-3s. a All yields shown here are isolated yields. b Obtained as a by-product from the synthesis of 3f′. c Synthesized from 3m′.

Reaction Optimization
Complete deprotection of the thiol group of triazole 3a was achieved within 1 h by treatment with triflic acid and anisole in TFA under an argon atmosphere at 0 °C, thus forming the free thiol 4a (Scheme 6). Compound 4a served as our model substrate for optimization of the reaction conditions leading to our tricyclic heteroaromatic molecule, 6a (Table 1). Based on previous studies in our group, we rationalized that an oxidative cyclization should be possible via a symmetrical disulfide intermediate, 5a. Therefore, standard oxidation conditions for the formation of a disulfide bond were explored for this transformation, and the reaction was followed using LCMS. Scheme 5. Preparation of triazole precursors 3a-3s. a All yields shown here are isolated yields. b Obtained as a by-product from the synthesis of 3f . c Synthesized from 3m .

Scheme 5.
Preparation of triazole precursors 3a-3s. a All yields shown here are isolated yi Obtained as a by-product from the synthesis of 3f′. c Synthesized from 3m′.

Reaction Optimization
Complete deprotection of the thiol group of triazole 3a was achieved withi treatment with triflic acid and anisole in TFA under an argon atmosphere at 0 forming the free thiol 4a (Scheme 6). Compound 4a served as our model substrate for optimization of the reactio tions leading to our tricyclic heteroaromatic molecule, 6a (Table 1). Based on p studies in our group, we rationalized that an oxidative cyclization should be pos a symmetrical disulfide intermediate, 5a. Therefore, standard oxidation condition formation of a disulfide bond were explored for this transformation, and the reac followed using LCMS. Compound 4a served as our model substrate for optimization of the reaction conditions leading to our tricyclic heteroaromatic molecule, 6a (Table 1). Based on previous studies in our group, we rationalized that an oxidative cyclization should be possible via a symmetrical disulfide intermediate, 5a. Therefore, standard oxidation conditions for the formation of a disulfide bond were explored for this transformation, and the reaction was followed using LCMS.
The use of stoichiometric hydrogen peroxide in aqueous ethanol, a widely used oxidant for disulfide coupling, led to the disulfide 5a at room temperature (Entry 1). Increasing the temperature to 80 • C under otherwise identical conditions resulted in complete conversion to the desired product, 6a, within 16 h, which could be isolated in 78% yield (Entry 2). In an attempt to reduce the reaction time, we used an excess of hydrogen peroxide in ethanol; however, neither the reaction time nor the isolated yield showed any improvements (Entry 3). In addition, hydrogen peroxide was used in 10% aqueous NaOH. In this case, the desired product was not obtained at all since the reaction stopped at the disulfide stage (Entry 4). We decided to change our solvent to DMSO. Using DMSO as both the oxidant and solvent at room temperature, gave only the disulfide 5a, even after 16 h (Entry 5), and no cyclized product was observed. After increasing the temperature to 100 • C, we observed full conversion to compound 6a in 4 h and were able to isolate the heterocycle in 83% yield (Entry 6). In an attempt to reduce the reaction temperature, one equivalent of iodine was added to the DMSO solution, but this did not change the outcome of the reaction; at room temperature, the disulfide 5a was obtained as the sole product (Entry 7), while, at 100 • C, the reaction time for complete conversion to 6a was still 4 h, with a comparable isolated yield of 82% (Entry 8). With these results in hand, we decided that carrying out the reaction in DMSO at 100 • C (Entry 6) without an additional oxidant were the best reaction conditions to convert thiol 4a to the tricyclic heteroaromatic species 6a. The use of stoichiometric hydrogen peroxide in aqueous ethanol, a widely used oxidant for disulfide coupling, led to the disulfide 5a at room temperature (Entry 1). Increasing the temperature to 80 °C under otherwise identical conditions resulted in complete conversion to the desired product, 6a, within 16 h, which could be isolated in 78% yield (Entry 2). In an attempt to reduce the reaction time, we used an excess of hydrogen peroxide in ethanol; however, neither the reaction time nor the isolated yield showed any improvements (Entry 3). In addition, hydrogen peroxide was used in 10% aqueous NaOH. In this case, the desired product was not obtained at all since the reaction stopped at the disulfide stage (Entry 4). We decided to change our solvent to DMSO. Using DMSO as both the oxidant and solvent at room temperature, gave only the disulfide 5a, even after 16 h (Entry 5), and no cyclized product was observed. After increasing the temperature to 100 °C, we observed full conversion to compound 6a in 4 h and were able to isolate the heterocycle in 83% yield (Entry 6). In an attempt to reduce the reaction temperature, one equivalent of iodine was added to the DMSO solution, but this did not change the outcome of the reaction; at room temperature, the disulfide 5a was obtained as the sole product (Entry 7), while, at 100 °C, the reaction time for complete conversion to 6a was still 4 h, with a comparable isolated yield of 82% (Entry 8). With these results in hand, we decided that carrying out the reaction in DMSO at 100 °C (Entry 6) without an additional oxidant were the best reaction conditions to convert thiol 4a to the tricyclic heteroaromatic species 6a. [4,5]thiazolo [2,3-c] [1,2,4]

triazole Derivatives
We found that, by using the optimized conditions developed above, 3a could be converted to the tricyclic heteroaromatic compound 6a in one step without isolating the free thiol 4a. However, we generally carried out a brief aqueous extraction after the deprotection in order to remove residual acid and characterized the free thiol by LCMS. As shown in Scheme 7, triazoles 3a-3s were subjected to the deprotection conditions to give the corresponding free thiols 4a-4s, which were used without purification in the next step after removal of the solvent under high vacuum, followed by an aqueous extraction. During the oxidative cyclization step carried out in DMSO, we observed the free thiols 4a-4s to rapidly undergo oxidative disulfide coupling, leading to disulfides 5a-5s. At elevated  We found that, by using the optimized conditions developed above, 3a could be converted to the tricyclic heteroaromatic compound 6a in one step without isolating the free thiol 4a. However, we generally carried out a brief aqueous extraction after the deprotection in order to remove residual acid and characterized the free thiol by LCMS. As shown in Scheme 7, triazoles 3a-3s were subjected to the deprotection conditions to give the corresponding free thiols 4a-4s, which were used without purification in the next step after removal of the solvent under high vacuum, followed by an aqueous extraction. During the oxidative cyclization step carried out in DMSO, we observed the free thiols 4a-4s to rapidly undergo oxidative disulfide coupling, leading to disulfides 5a-5s. At elevated temperatures, the disulfide intermediates were found to undergo ring closure to give the desired compounds 6a-6s in 100% conversions by LCMS within less than 4 h. Regardless of whether the substituents on the aryl ring were electron-donating or electron-withdrawing, the tricyclic heteroaromatic compounds 6a-6s were obtained in good to excellent yields. For example, weakly electron-donating groups, such as methyl (compounds 6b and 6c) as well as the naphthalene derivative (6d) were obtained in 82-89% yields. Various nitrogen substituents were tolerated (compounds 6e, 6f, and 6f ): While the acetyl-protected amine 6e was isolated in good yield (82%), a lower yield was observed for the unprotected primary amine 6f (60%). Compound 6f , with its triazole substituent, was, on the other hand, isolated in excellent yield (94%). Since complete conversions were observed by LCMS in all cases and no other byproducts were detected, the lower yield of 6f is thought to be a result of its water solubility. Both 6e and 6f are not only insoluble in water but also showed poor solubility in organic solvents, such as methylene chloride, methanol, and even DMSO.
The two examples of oxygen-containing substituents at position 6 (6g and 6h) also gave good yields (81% and 85%, respectively). Pyridine derivatives (6i and 6j) were found to tolerate our reaction conditions; however, the products were obtained in lower yields (75% and 70% respectively). Strongly electron-withdrawing groups, such as a trifluoromethyl and a carbonitrile substituent in the 6-position, gave the tricyclic compounds 6k and 6l in good yields (86% and 80%, respectively). Similarly, the carboxylic acid 6m and ester 6m derivatives were synthesized in 85% and 86% yields, respectively. Additionally, all halogen-containing derivatives 6n-6s were isolated in excellent yields, ranging from 88% to 98%. It is noteworthy that the reaction time for both compounds with halogens at position 7 (6o and 6s) was 30 min shorter than the time required for the cyclization of any of the other compounds under identical conditions. is thought to be a result of its water solubility. Both 6e and 6f′ are not only insoluble in water but also showed poor solubility in organic solvents, such as methylene chloride, methanol, and even DMSO. The two examples of oxygen-containing substituents at position 6 (6g and 6h) also gave good yields (81% and 85%, respectively). Pyridine derivatives (6i and 6j) were found to tolerate our reaction conditions; however, the products were obtained in lower yields (75% and 70% respectively). Strongly electron-withdrawing groups, such as a trifluoromethyl and a carbonitrile substituent in the 6-position, gave the tricyclic compounds 6k and 6l in good yields (86% and 80%, respectively). Similarly, the carboxylic acid 6m and ester 6m′ derivatives were synthesized in 85% and 86% yields, respectively. Additionally, all halogen-containing derivatives 6n-6s were isolated in excellent yields, ranging from 88% to 98%. It is noteworthy that the reaction time for both compounds with halogens at position 7 (6o and 6s) was 30 min shorter than the time required for the cyclization of any of the other compounds under identical conditions. While most of the reactions in Scheme 7 were carried out on a 0.5 mmol or 1 mmol scale, smaller-scale reactions of 0.25 mmol (3g, 3i, and 3j) still allowed the isolation of the products 6g, 6i, and 6j in acceptable yields. However, as demonstrated with 3a and 3o, increasing the reaction scale to 5 mmol and above generally afforded the products 6a and 6o in excellent yields of over 90%. On a 5 mmol scale, heterocycle 6a was obtained in 90% Scheme 7. Formation of benzo [4,5] While most of the reactions in Scheme 7 were carried out on a 0.5 mmol or 1 mmol scale, smaller-scale reactions of 0.25 mmol (3g, 3i, and 3j) still allowed the isolation of the products 6g, 6i, and 6j in acceptable yields. However, as demonstrated with 3a and 3o, increasing the reaction scale to 5 mmol and above generally afforded the products 6a and 6o in excellent yields of over 90%. On a 5 mmol scale, heterocycle 6a was obtained in 90% yield compared to 85% on a 1 mmol scale. Likewise, increasing the scale from 1 mmol to 10 mmol for the preparation of 6o led to a small increase in yield from 90% to 92%.

Basic Mechanistic Investigations
We were interested in following the conversion of thiol 4a to compound 6a by NMR to confirm the disulfide intermediate 5a detected previously by LCMS. Therefore, compound 4a was dissolved in DMSO-d 6 and an 1 H NMR was recorded immediately after the addition of the solvent. This NMR showed the presence of a singlet at 8.78 ppm (H a ), which was assigned to the triazole C-H of the free thiol 4a ( Figure 2, 5 min RT). The NMR reaction tube was then heated to 100 • C, and a second 1 H NMR was measured after 1 h, which showed the presence of two new singlets at 9.64 (H c ) and 8.70 ppm (H b ). The singlet at 9.64 ppm corresponded to H c of heterocycle 6a, while the singlet at 8.70 ppm was assigned to H b of the disulfide intermediate 5a. LCMS of the NMR solution confirmed the presence of these two molecules, while no free thiol was detected. After 30 h, the disulfide 5a was consumed completely, and only the compound 6a was observed by both NMR and LCMS.
Interestingly, the reaction carried out in dried deuterated DMSO took longer to reach completion than in reagent grade DMSO, which suggested that water might play a role in the reaction mechanism. While the conversion of each thiol to the heterocycle is expected to generate one equivalent of dimethyl sulfide and one equivalent of water if carried out in DMSO, we were particularly interested to see how the rate of the cyclization step was affected by the amount of water present. To study this effect of water, the disulfide 5a was isolated and heated at 100 • C in DMSO containing known amounts of water ( Figure 3). Samples taken at regular intervals were immediately analyzed by LCMS, which, after calibration, allowed the quantification of the species present. Indeed, as expected, we observed that increases in the water content corresponded to increases in the rate of conversion of the disulfide 5a to product 6a. A possible explanation for the rate-enhancing effect of water could be that it is involved in the C-H bond functionalization of the triazole C-H bond. Although it cannot be excluded that some residual acid is present from the deprotection of the thiol despite the aqueous extraction, we hypothesize that the role of water might be twofold. On the one hand, reversible protonation of a triazole nitrogen is expected to significantly increase the acidity of the C-H bond in the resulting triazolium species. However, water is also the strongest base present and is likely to be involved in the deprotonation of this triazolium species, thereby leading to a nucleophilic carbene intermediate, which could attack one of the sulfur atoms in the disulfide bond to afford our heterocyclic product 6a and a thiolate. This thiolate leaving group would immediately undergo oxidative disulfide coupling, thus generating the next active disulfide intermediate.

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yield compared to 85% on a 1 mmol scale. Likewise, increasing the scale from 1 mmol to 10 mmol for the preparation of 6o led to a small increase in yield from 90% to 92%.

Basic Mechanistic Investigations
We were interested in following the conversion of thiol 4a to compound 6a by NMR to confirm the disulfide intermediate 5a detected previously by LCMS. Therefore, compound 4a was dissolved in DMSO-d6 and an 1 H NMR was recorded immediately after the addition of the solvent. This NMR showed the presence of a singlet at 8.78 ppm (Ha), which was assigned to the triazole C-H of the free thiol 4a (Figure 2, 5 min RT). The NMR reaction tube was then heated to 100 °C, and a second 1 H NMR was measured after 1 h, which showed the presence of two new singlets at 9.64 (Hc) and 8.70 ppm (Hb). The singlet at 9.64 ppm corresponded to Hc of heterocycle 6a, while the singlet at 8.70 ppm was assigned to Hb of the disulfide intermediate 5a. LCMS of the NMR solution confirmed the presence of these two molecules, while no free thiol was detected. After 30 h, the disulfide 5a was consumed completely, and only the compound 6a was observed by both NMR and LCMS. Interestingly, the reaction carried out in dried deuterated DMSO took longer to reach completion than in reagent grade DMSO, which suggested that water might play a role in the reaction mechanism. While the conversion of each thiol to the heterocycle is expected to generate one equivalent of dimethyl sulfide and one equivalent of water if carried out in DMSO, we were particularly interested to see how the rate of the cyclization step was affected by the amount of water present. To study this effect of water, the disulfide 5a was isolated and heated at 100 °C in DMSO containing known amounts of water ( Figure 3). Samples taken at regular intervals were immediately analyzed by LCMS, which, after calibration, allowed the quantification of the species present. Indeed, as expected, we observed that increases in the water content corresponded to increases in the rate of conversion of the disulfide 5a to product 6a. A possible explanation for the rate-enhancing effect of water could be that it is involved in the C-H bond functionalization of the triazole C-H bond. Although it cannot be excluded that some residual acid is present from the depro- Preliminary NMR studies into substituent effects on the reaction rate show that a very electron-withdrawing group, such as trifluoromethyl, in the para-position to the disulfide bond significantly increases the rate of conversion of this disulfide (5k) to the tricyclic heteroaromatic compound 6k (Figure 4). This effect is in agreement with the aforementioned mechanism, as the sulfur atom in the disulfide would become significantly more electrophilic while simultaneously the hydrogen of the C-H bond of the triazole would increase in acidity. Meanwhile, disulfide 5g (containing the electron-donating methoxy group) displayed a similar initial reaction rate as the unsubstituted disulfide 5a. Full conversion to both the unsubstituted heterocycle 6a and the methoxy derivative 6g was only observed after 30 h.
Although our hypothetical mechanism provides both a potential explanation for the role of water and some of the substituent effects observed, we cannot exclude other mechanisms for the formation of the heterocycle in the absence of a more rigorous kinetic investigation.
Molecules 2022, 27, x 10 of 32 expected to significantly increase the acidity of the C-H bond in the resulting triazolium species. However, water is also the strongest base present and is likely to be involved in the deprotonation of this triazolium species, thereby leading to a nucleophilic carbene intermediate, which could attack one of the sulfur atoms in the disulfide bond to afford our heterocyclic product 6a and a thiolate. This thiolate leaving group would immediately undergo oxidative disulfide coupling, thus generating the next active disulfide intermediate. Preliminary NMR studies into substituent effects on the reaction rate show that a very electron-withdrawing group, such as trifluoromethyl, in the para-position to the disulfide bond significantly increases the rate of conversion of this disulfide (5k) to the tricyclic heteroaromatic compound 6k ( Figure 4). This effect is in agreement with the aforementioned mechanism, as the sulfur atom in the disulfide would become significantly more electrophilic while simultaneously the hydrogen of the C-H bond of the triazole would increase in acidity. Meanwhile, disulfide 5g (containing the electron-donating methoxy group) displayed a similar initial reaction rate as the unsubstituted disulfide 5a. Full conversion to both the unsubstituted heterocycle 6a and the methoxy derivative 6g was only observed after 30 h.
Although our hypothetical mechanism provides both a potential explanation for the role of water and some of the substituent effects observed, we cannot exclude other mechanisms for the formation of the heterocycle in the absence of a more rigorous kinetic investigation.

General Information
Most reagents and solvents were purchased from Fisher Scientific (Waltham, MA, USA), Oakwood Chemical (Estill, SC, USA), TCI America (Portland, OR, USA), and Avan-

General Information
Most reagents and solvents were purchased from Fisher Scientific (Waltham, MA, USA), Oakwood Chemical (Estill, SC, USA), TCI America (Portland, OR, USA), and Avantor (Radnor, PA, USA) and were used as supplied unless otherwise noted. Thermo Scien-tific™ silica gel (for column chromatography, 0.035-0.070 mm, 60Å) from Fisher Scientific (Waltham, MA, USA) was used for chromatographic separations. DMSO-d 6 was dried over molecular sieves.

Analyses
1 H NMR, 13 C{ 1 H} NMR spectra, and 19 F{ 1 H} NMR spectra were all recorded using a 400 MHz Bruker Avance III spectrometer with a 5 mm liquid-state Smart Probe. Chemical shifts (δ H and δ C ) are expressed in parts per million (ppm) and reported relative to the resonance of the residual protons of the DMSO-d 6 (δ H = 2.50 ppm), CD 3 CN (δ H = 1.94 ppm), or CDCl 3 (δ H = 7.26 ppm) or in 13 C{ 1 H} NMR spectra relative to the resonance of the deuterated solvent DMSO-d 6 (δ C = 39.52 ppm), CD 3 CN (δ C = 1.32 ppm), or CDCl 3 (δ C = 77.16 ppm). Chemical shifts in the 19 F{ 1 H} NMR spectra are reported relative to the internal standard fluorobenzene (δ F = −113.15). The coupling constants (J) are given in Hz. All measurements were carried out at 298 K. The abbreviations used in the description of the NMR data are as follows: s, singlet; d, duplet; t, triplet; m, multiplet; and sept., septet. Copies of the 1 H NMR and 13 C NMR spectra for compounds 1b-1r, 2b-2s, 3b-3s, and 6a-6s are provided in the Supporting Information. High-resolution mass spectrometry (HRMS) data were obtained on an LTQ Orbitrap Fusion in FT orbitrap mode at a resolution of 240,000.

General Procedure 1 for the Synthesis of (4-Methoxybenzyl)(2-Nitrophenyl)sulfanes (1b-1r)
The following description is for a 30 mmol scale reaction. The solvent quantities and flask size were adjusted accordingly for smaller-scale reactions.
A 500 mL round-bottomed flask equipped with a stir bar was loaded with the 1fluoro-2-nitrobenzene derivative (1 equiv.) and 200 mL of ethanol and placed under an atmosphere of argon. (4-methoxyphenyl)methanethiol (1 equiv.) was added with a syringe, followed by a dropwise addition of NaOH (1 equiv.) dissolved in 10 mL of H 2 O. The reaction mixture was stirred at room temperature until TLC indicated the completion of the reaction (typically within 2 h). After removing the solvent under reduced pressure, the residue was diluted with 150 mL of H 2 O and extracted twice with dichloromethane. The organic phases were combined, dried over MgSO 4 , filtered, and concentrated. The resulting crude product was purified by recrystallization or column chromatography as described below.

Ethyl 4-((4-Methoxybenzyl)thio)-3-Nitrobenzoate (1m )
A 250 mL round-bottomed flask was charged with compound 1m (23 mmol, 1 equiv.), oxalyl chloride (3 equiv.), and dichloromethane (50 mL). After adding DMF (3 drops), the reaction mixture was stirred for 1 h at 0 • C in an ice bath. EtOH (1.3 equiv.), triethylamine (1.3 equiv), and dichloromethane (50 mL) were added at 0 • C, the ice bath was removed, and the reaction flask was fitted with a reflux condenser. The reaction mixture was stirred at 40 • C for 2 h, then cooled to room temperature. After washing the dichloromethane solution with water, the organic phase was dried over MgSO 4 , filtered, and evaporated. Recrystallization of the crude product from diethyl ether/hexanes (1:2) afforded the product as a bright yellow solid in 99.99% The following description is for a 20-25 mmol scale reaction. The solvent quantities and flask size were adjusted accordingly for smaller-scale reactions.
A 250 mL round-bottomed flask equipped with a stir bar was loaded with the (4methoxybenzyl)(2-nitrophenyl)sulfane derivative (1b-1r, 1 equiv.), iron power (5.0 equiv.), NH 4 Cl (5.0 equiv.), and 150 mL of EtOH/H 2 O (4:1). The reaction flask was placed under inert atmosphere (argon), fitted with a reflux condenser, and stirred at 80 • C until TLC indicated a complete reduction (typically between 1 and 4 h). After cooling to room temperature, the reaction mixture was filtered through celite and concentrated under reduced pressure. The residue was then basified with 1M NaOH and extracted twice with dichloromethane, dried over MgSO 4 , and evaporated. The crystalline solid was washed with hexanes or diethyl ether as described below.

2-((4-Methoxybenzyl)thio)-3-Methylaniline (2b)
The title compound was prepared according to general procedure 2 on a 20.0 mmol scale. The crystalline light brown solid was washed with hexanes, which gave the desired product in 94%

2,2 -Disulfanediylbis(4-Fluoroaniline)
A 500 mL round-bottomed flask equipped with a stir bar was charged with 16.802 g (100 mml) of 2-amino-6-fluorobenzothiazole, 100 mL of 10 M NaOH, and 100 mL of 2methoxyethanol. The flask was fitted with a reflux condenser and the reaction mixture was stirred at 100 • C for 4 days. After cooling to room temperature, the reaction mixture was acidified with HCl to pH 3 and extracted twice with diethyl ether. The combined organic phases were washed with brine, dried over MgSO 4, filtered, and evaporated. The product obtained from the extraction was sufficiently pure to be used without further purification. Yield: 69% (9.815 g, 34.52 mmol). 1  While stirring in an ice bath, NaBH 4 (5.22 g, 138 mmol, 4 equiv.) was added in small portions over a period of 15 min under a counterflow of argon. The ice bath was then removed, and the reaction mixture was stirred at room temperature for 1h. The reaction mixture was concentrated, water was added, and the product was extracted with diethyl ether. After drying the organic phase over MgSO 4 , filtration, and removal of the solvent, the crude 2-amino-5-fluorobenzenethiol was used immediately in the next step. Protection of 2-amino-5-fluorobenzenethiol: The crude 2-amino-5-fluorobenzenethiol (max. 69 mmol, 1 equiv.) was dissolved in 100 mL of EtOH and transferred to a 250 mL round-bottomed flask equipped with a stir bar, and 10.41 g (75 mmol, 1.08 equiv.) of 1-(chloromethyl)-4methoxybenzene was added. After setting the reaction under argon, 3.25 g (81.3 mmol, 1.18 equiv.) of NaOH dissolved in 10 mL of water was added dropwise under a counterflow of argon. Then, the reaction flask was fitted with a reflux condenser and stirred under an argon atmosphere at 50 • C for 4 h. After cooling to room temperature, the reaction mixture was concentrated. Basification with 1 M NaOH, followed by extraction with diethyl ether and drying over MgSO 4 gave the crude product as an oil upon filtration and solvent removal. Recrystallization from hot diethyl ether gave the product as a pale yellow powder in 9.747 g (37.01 mmol, 54%) yield. 1

General Procedure 3 for the Synthesis of Substituted Triazoles (3b-3s)
A 50 mL round-bottomed flask equipped with a stir bar was loaded the 2-((4-methoxybenzyl)thio)aniline derivative (2b-2s, 1 equiv.) and N,N-dimethylformamide azine dihydrochloride (1.1 equiv). After mixing the two compounds thoroughly, the reaction flask was placed under an inert atmosphere (argon) and stirred in an oil bath at 150 • C. The two solid compounds were observed to melt within an hour and turn dark red. After 16 h, the reaction flask was cooled to room temperature. The resulting dark solid was basified with 1 M NaOH and extracted with dichloromethane, dried over MgSO 4 , filtered, and concen- The title compound was obtained as a by-product of the synthesis of compound 3f , which was prepared according to general procedure 3 on a 3.64 mg (14. A 50 mL round-bottomed flask was charged with compound 3m (1.50 mmol, 1 equiv.), KOH (2 equiv.), and MeOH, and then heated to 35 • C for 1 h. After removal of the solvent under reduced pressure, the crude product was purified by column chromatography (silica gel, 1. dichloromethane, and 2. dichloromethane/methanol 10:1, R f = 0.19), which gave the product as a white powder in 87% (413 mg, 1.21 mmol) yield; m.p. > 360 • C. 1

Synthesis and Isolation of 2-(4H-1,2,4-Triazol-4-Yl)benzenethiol (4a) for Optimization Reactions and NMR Studies
A 25 mL round-bottomed flask equipped with a stir bar was loaded with 594.8 mg of triazole 3a (2 mmol, 1 equiv.) and 7.65 mL of trifluoroacetic acid (100 mmol, 50 equiv.). After flushing with argon, 1.09 mL of anisole (10 mmol, 5 equiv.) and 0.99 mL of trifluoromethanesulfonic acid (10 mmol, 5 equiv.) were added to this solution, and the reaction mixture was stirred at 0 • C for 1 h. The solution was then concentrated under high vacuum. The resulting dark red oil was triturated with hexanes and then diluted with 20 mL of water and extracted twice with dichloromethane (50 mL). The combined organic phases were dried over MgSO 4 , filtered, and concentrated. The target compound was obtained as a yellow oil (304.8 mg, 86%) and was used without any further purification. 1  3.9. General Procedure 4 for the Synthesis of benzo [4,5]thiazolo [2,3-c] [1,2,4]triazoles (6a-6s) A 25 mL round-bottomed flask equipped with a stir bar was loaded with the triazole (3a-3s, 1 equiv.) and trifluoroacetic acid (50 equiv.). After flushing with argon, anisole (5 equiv.) and trifluoromethanesulfonic acid (5 equiv.) were added to this solution, and the reaction mixture was stirred at 0 • C for 1 h. The solution was then concentrated under high vacuum. The resulting dark red oil was diluted with 20 mL of water and extracted twice with dichloromethane (50 mL). Following concentration of the combined organic phases, the crude thiol was dissolved in 2 mL of DMSO and heated to 100 C. After completion of the cyclization reaction, the reaction mixture was diluted with 20 mL of sat. NaHCO 3 and extracted twice with dichloromethane (50 mL). The combined organic phases were dried over MgSO 4 , filtered, and concentrated. The resulting crude product was purified by column chromatography or recrystallization as described below.