1. Introduction

2. Results and Discussion

2.2. Inhibitory Activity of Thienobenzo-Triazoles 14–21 and triazolium Salts 22–31 Toward Enzymes Cholinesterases

Newly prepared thienobenzo-triazoles 14–21 and triazolium salts 22–31 were tested for inhibitory activity toward AChE and BChE in various concentrations, depending on solubility. Spectrophotometric Ellman assay was applied [22], and the results obtained were compared to those of commercially available Galantamine as the reference standard. The IC₅₀ values of triazole derivatives 14–31 for AChE and BChE inhibition are presented in Table 1.

The tested compounds were divided into two main groups: thienobenzo-triazoles, presented on the left side of Table 1, and triazolium salts, presented on the right. Analogs can be found between these two groups that differ only in charge, while the type and position of the substituents are the same. According to structural features, the following structure-activity relationship can be noted. The presence of a charge on the triazole ring favored more potent inhibition toward both enzymes, AChE and BChE. Leading compounds were 22, 23, 26, 27, and 30, which inhibited both enzymes in the excellent to very good range of concentrations compared to the standard (Table 1, Figure 3 and Figure 4). The strongest inhibitor of both enzymes was derivative 23, with a value for BChE in the nanomolar range that was better than the standard (Table 1) and a slightly higher but still excellent value for AChE. The exception among the salts was 25, whose IC₅₀ values were not in such a good range of concentrations. The presence of chlorine on the benzyl ring supports the activity since three salts with excellent results, 23, 26, and 30, possess this substituent. Introduction of methyl substituent on thiophene ring, from 22 to 25, significantly reduced activity toward enzymes. The same structural change from 23 to 26 resulted similarly. Derivatives 24 and 27 were an exception, but only toward AChE, because the methylated charged derivative 27 was a better inhibitor of this enzyme than the unmethylated 24.

Uncharged thienobenzo-triazoles 14–21 prefer inhibition of BChE over AChE. This observation is consistent with the conclusions of our previous research, which indicated that neutral photoproducts tend to be weaker inhibitors and show selectivity toward BChE [20]. It is supported additionally by the fact that the uncharged analog of 30 analyzed in the previous research [20] showed weaker inhibition of both enzymes. For herein tested thienobenzo-triazoles, conversion to salt significantly increased inhibition of AChE and, to a good extent, also of BChE. Exceptions were 16 and 17, which achieved better IC₅₀ values for BChE than the corresponding salts 24 and 25.

It can be concluded that the most important structural feature for thienobenzo-triazoles is the presence/absence of charge. Triazolium salts were shown to be dual inihibitors whose potency of inhibition varies depending on the substituent of thiophene and triazole heterocycles.

Table 1. Calculated IC₅₀ values for the inhibition of AChE and BChE by compounds 14–31.

Table 1. Calculated IC₅₀ values for the inhibition of AChE and BChE by compounds 14–31.

Compound	IC50 (AChE/BChE) /μM	Compound	IC50 (AChE/BChE)/μM
	117.2 / 24.3		39.2 / 1.00
	77.0 / 34.6		4.4 / 0.47
	77.1 / 2.9		55.1 / 7.0
	> 250 / 57.6		211.1 / 102.3
	> 100 / 59.0		8.1 / 13.4
	> 250 / 70.3		8.3 / 9.7
	185.8 / 90.2		54.8 / 10.6
	464.7 / 171.9		12.2 / 2.7
Galantamine	0.15 / 7.9		157.0 / 16.1

Figure 3. Dose-response curve for the inhibition of AChE (a) and BChE (b) by 22.

Figure 3. Dose-response curve for the inhibition of AChE (a) and BChE (b) by 22.

Figure 4. Dose-response curve for the inhibition of AChE (a) and BChE (b) by 23.

Figure 4. Dose-response curve for the inhibition of AChE (a) and BChE (b) by 23.

2.3. ADMET Properties of Biologically Active Thienobenzo-Triazoles and Triazolium Salts

Absorption, distribution, metabolism, excretion, and toxicity (ADME(T)) studies are critical in drug development. They give insights into whether the compound exhibits drug-like pharmacokinetic properties and whether it has properties that will cause safety concerns in people [23]. ADMET properties are investigated in silico, in vitro, and in vivo. In early drug discovery, in silico tools are indispensable. For this study, free in silico tools [24,25] were employed to screen the candidate compounds.

Table 2. In silico study for more indicators about the absorption, distribution, metabolism, excretion, and toxicity (ADME(T)) in the human body of some pairs of thienobenzo-triazoles 14, 15, and 17 and their biologically active charged triazolium salt 22, 23, 25 and 30.

Table 2. In silico study for more indicators about the absorption, distribution, metabolism, excretion, and toxicity (ADME(T)) in the human body of some pairs of thienobenzo-triazoles 14, 15, and 17 and their biologically active charged triazolium salt 22, 23, 25 and 30.

Property Model Name					Compound				Unit
		14	22	15	23	17	25	30
Absorption	Water solubility	-6,53	-4,782	-6,30	-5,18	-6,2	-4,62	-4,69	log mol/L
Absorption	Caco2 permeability	0.82	0.852	0,892	1.209	0,794	0,78	0,761	log Papp in 10-6 cm/s
Absorption	Intestinal absorption (human)	87,71	95,798	76,09	95,903	76,1	76,2	76,1	% Absorbed
Distribution	BBB permeability	0,6378	0,484	0,563	0,457	0,63	0,453	0,396	log BB
Metabolism	CYP2D6 substrate	No	No	No	No	No	No	No
Metabolism	CYP3A4 substrate	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Metabolism	CYP2C9 inhibitior	No	No	No	No	No	No	No
Metabolism	CYP2D6 inhibitior	Yes	Yes	Yes	No	No	No	No
Metabolism	CYP3A4 inhibitior	No	No	No	No	No	No	No
Excretion	Total Clearance	0,96	0,874	0,901	0,855	0,873	0,882	0,925	log ml/min/kg
Excretion	Renal OCT2 substrate	No	No	No	No	No	No	No
Toxicity	AMES toxicity	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Toxicity	hERG I inhibitor	No	No	No	No	No	No	No
Toxicity	hERG II inhibitor	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Toxicity	Oral Rat Acute Toxicity (LD50)	2,31	2,251	2,47	2,322	2,25	2,25	2,24	mol/kg
Toxicity	Oral Rat Chronic Toxicity (LOAEL)	-	0.286	-	1.444	-	-	-	log mg/kg_bw/day
Toxicity	Hepatotoxicity	No	No	No	Yes	Yes	Yes	Yes
Toxicity	Skin Sensitisation	No	No	No	No	No	No	No

As the compounds in the form of salts (22–31) are developed mainly to enhance the ADmet part of the properties, it is important to compare the neutral molecule against its salt counterpart. It can be seen (Table 2) that the water solubility, although higher, remains very low, and all other properties are alike. To determine whether the in silico models could differentiate between the neutral molecule and the salt, multiple software programs were utilized, revealing variations in the absorption estimates. As other parts of the adMET are concerned, the results are similar. The CNS bioavailability is somewhat reduced, as can be expected, as this is a delicate balance between water solubility and lipophilicity.

2.4. Molecular Docking of Biologically Active Thienobenzo-Triazoles and Triazolium Salts into Cholinesterases

To reveal the structure of non-covalent complexes formed between the tested compounds and enzymes and identify the interactions responsible for their stabilization, molecular docking of two thienobenzo-triazoles and their triazolium counterparts into the active sites of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) was performed.

The most promising inhibitory activity was observed for triazolium salts 22 and 23. Their corresponding neutral thienobenzo-triazoles 14 and 15, which contain the same substituents, also performed very well (Table 1). Figure 5 presents the structures of the complexes resulting from the docking of compounds 14 (left panel) and 15 (right panel) into the active site of AChE, while the results of docking of cations 22 and 23 into the AChE are shown in Figure 6.

Both compounds occupy a similar position within the peripheral anionic subsite (PAS), allowing for π-π stacking between the thienobenzo moiety and Trp286 and similar interactions with Tyr341. The phenyl substituent in both cases is favorably oriented towards Phe338, also engaging in π-π stacking. In compound 14, the methyl group at the para position of the phenyl substituent participates in an alkyl-π interaction with Phe338. In the protein-ligand complex with 15, the chlorine atom is situated 4.2 Å from the centroid of Phe338, suggesting the presence of halogen-π dispersive attraction. The attractive interactions between the chlorine of the ligand and the π-system of the aromatic protein residues can occur in two distinct geometries, depending on the positioning of the Cl atom relative to the aromatic ring: “edge-on” and “face-on” [26]. When the difference between the distance from the chlorine to the center of the aromatic ring and the distance from Cl to the closest carbon of the aromatic ring exceeds 0.3 Å, the “edge-on” geometry is observed. In this case, the distances are 4.2 Å and 3.8 Å, indicating that the interaction can be classified as “edge-on.”

The structures of the complexes formed between acetylcholinesterase and triazolium cations 22 and 23 (Figure 6) reveal that, in both cases, the quaternary nitrogen of the triazole engages in electrostatic interaction with the anionic residues of the active site, specifically aspartate in the peripheral anionic subdomain (Asp74). The distance between the quaternary nitrogen and the oxygen of Asp74 is 3.9 Å, forming a salt bridge that significantly contributes to the stability of the complex. The conformations of the docked cations 22 and 23 are nearly identical (Figure 6), facilitating alkyl-π interactions between the methyl group on the triazole and Tyr332, as well as parallel π-π stacking of the triazolo-benzo fragment with Tyr341 and the thiophene core with Phe338. For cation 23, the preferred conformation of the ligand is unfavorable for establishing a chlorine-π interaction, as the Cl atom is positioned at distances exceeding 4.5 Å from the closest aromatic residues.

Figure 6. The AChE’s active site structure docked with triazolium salts 22 (left) and 23 (right panel). Distances in angstroms; hydrogen atoms of the residues omitted for clarity.

Figure 6. The AChE’s active site structure docked with triazolium salts 22 (left) and 23 (right panel). Distances in angstroms; hydrogen atoms of the residues omitted for clarity.

The docking of compounds 14 and 15 into butyrylcholinesterase (BChE) resulted in the structures shown in Figure 7. In both cases, the ligands adopt similar orientations within the active site. The thienobenzo moiety engages in π-π stacking interactions with Trp82, which belongs to the anionic subdomain of the enzyme’s active site. Simultaneously, the triazole ring interacts similarly with the histidine residue in the esteratic site. The phenyl substituent is oriented to facilitate π-π stacking with residue Phe329.

In compound 14, the methyl group on the phenyl substituent is conveniently positioned within the acyl pocket of BChE, interacting with residues Leu286 and Val288 to achieve alkyl-alkyl dispersive interaction. In molecule 15, the chlorine atom behaves similarly, establishing hydrophobic contacts with the aliphatic carbons of the residues in the acyl pocket.

Finally, the structures of the complexes between 22 and 23 and BChE, shown in Figure 8, again reveal the presence of the electrostatic attraction between the positively charged ligands’ region and the anionic residue of the enzyme.

As noted previously, the anion involved in AChE is aspartate from the peripheral anionic subsite (PAS); however, in BChE, the anionic residue engaged in this charge-charge interaction is Glu197, which is part of the active site’s anionic subdomain. Similar to the structures of the complexes with ligands 14 and 15, the thienobenzo moiety establishes parallel π-π stacking with the tryptophan in the anionic subsite, Trp82. In both cations, the phenyl substituent participates in stacking with Phe329. The methyl group on the phenyl of 22 is engaged in alkyl-alkyl dispersion with the acyl pocket. The chlorine atom in 23 is positioned too far from the nearest aromatic residue to form halogen-π dispersion effectively; however, it achieves hydrophobic interaction with residues of the acyl pocket.

2.5. Potential Genotoxicity of Thienobenzo-Triazoles 14–21 and Triazolium Salts 22–31

The pharmaceutical development process necessitates the evaluation of all involved compounds (including drug substances, impurities, intermediates, etc.) for their potential mutagenic or carcinogenic effects. The ICH M7 Guideline governs the regulation of such compounds, requiring that levels present in the drug substance or drug product be calculated based on their acceptable daily intake (ADI) and the drug’s maximum daily dose (MDD). Experimental data for new drug substances and products are often unavailable in the literature databases. The quantitative structure-activity relationship (QSAR) approach is essential in these cases. QSAR models predict biological activity based on structural components [27]. This approach is particularly useful during the early stages of identifying potentially active drug substances, as it helps eliminate compounds that exhibit biological activity but may also have mutagenic potential. The most commonly utilized tool for this purpose is Lhasa software (Nexus v.2.5.2 (Build 5, Jul 2022), Derek Nexus v.6.2.1, Meteor Nexus v.3.1.0, Sarah Nexus v.3.2.1, Vitic Link v.2.4.2), which incorporates two complementary models—one rule-based and the other statistical. Predictions from these models are subsequently reviewed by an expert to ensure accuracy.

In the case of compounds 14–31 that are investigated for their potential biological activity with the emphasis on the inhibition of enzymes cholinesterases, the software, as it only recognizes special functionalities, cannot determine the potential for salts, so the results are inconclusive. It does not treat them as their neutral counterparts, as the Derek Nexus will give them a negative, but with less assurance. As for the neutral molecules 14–21, the best candidate is compound 18. Here the Sarah prediction is inconclusive, but as Derek is strongly negative, the overall prediction comes out negative. For the rest of the neutral compounds, Sarah prediction has a positive result, and if they are to be tested further, it would be wise to do an experimental mutagenicity AMES test.

Table 3. The mutagenic potential of thienobenzo-triazoles 14–21 and triazolium salts 22–31 by Lhasa M7 evaluation (green square – negative, red square – positive, white square – no data available); grey highlight – negative, orange highlight – positive, white – strongly negative).

Table 3. The mutagenic potential of thienobenzo-triazoles 14–21 and triazolium salts 22–31 by Lhasa M7 evaluation (green square – negative, red square – positive, white square – no data available); grey highlight – negative, orange highlight – positive, white – strongly negative).

Structure	ICH M7 Class	DerekPrediction	Sarah Prediction	Overall In Silico
14	Class 3			Positive
15	Class 3			Positive
16	Class 3			Positive
17	Class 3			Positive
18	Inconclusive			Negative
19	Class 3			Positive
20	Class 3			Positive
21	Class 3			Positive
22	Inconclusive			Inconclusive
23	Inconclusive			Inconclusive
24	Inconclusive			Inconclusive
25	Inconclusive			Inconclusive
26	Inconclusive			Inconclusive
27	Inconclusive			Inconclusive
28	Inconclusive			Inconclusive
29	Inconclusive			Inconclusive
30	Inconclusive			Inconclusive
31	Inconclusive			Inconclusive

3. Materials and Methods

3.2. Synthesis of Phosphonium Salts 1’ and 2’

In a 250 mL three-necked flask, 7.9 g (0.063 mol) of 2-thiophene methanol was dissolved in 72.3 mL of dry solvent E and added to the flask. Then, 5.71 g (0.022 mol) of phosphorus tribromide, PBr₃ was dissolved in 7.23 mL of dry solvent E and added dropwise to the reaction flask over 40 minutes. The reaction mixture was stirred at room temperature on a magnetic stirrer for 1 hour. Subsequently, 15 mL of methanol and 100 mL of distilled water were added, and the mixture was extracted with a total of 300 mL of solvent E. The ether layer was dried overnight with anhydrous magnesium sulfate, MgSO₄, filtered, and evaporated to dryness using a rotary evaporator, leaving 2-thiophene bromide as a yellow oil in the flask. This oil was then dissolved in 20 mL of toluene, while 16.52 g (0.037 mol) of triphenylphosphine, PPh₃ was dissolved in 50 mL of toluene, and both solutions were added to the reaction flask. The mixture was stirred on a magnetic stirrer for three days and then filtered through a Büchner funnel under reduced pressure. The resulting light-yellow salt was dried in a desiccator for 12 hours. The dry thiophene-phosphonium salt 1’ obtained was used in all subsequent experiments for synthesizing compounds 6–8.

In a 250 mL round flask, 4.925 g (0.0440 mol) of dimethylthiophene was dissolved in 133 mL of carbon tetrachloride, CCl₄. Then, 7.83 g (1 equivalent) of N-bromosuccinimide, NBS, was added to the mixture. The chemicals were stirred in an oil bath at a reflux temperature (150 ºC). Once reflux started, a small amount of α,α-azobisisobutyronitrile, AIBN catalyst was added. The oil bath temperature was then reduced to approximately 100 ºC due to excessive reflux. With reflux established, the reaction mixture was illuminated with a lamp to initiate the reaction. After 1.5 hours, bromide formation began, accompanied by the appearance of white, fluffy succinimide crystals on the surface. After another hour, a second dose of AIBN was added, and the mixture was refluxed for an additional 3 hours. The succinimide was filtered out using pleated filter paper, and the clear orange liquid was evaporated using a rotary evaporator, leaving a brown, oily precipitate of thiophene bromide in the flask. In the second step, the thiophene bromide was dissolved in 10 mL of benzene with added PPh₃. The mixture was heated to reflux (80 ºC, with the mixer set to 110 ºC) and stirred at that temperature for 1 hour. The heating was then turned off, and the mixture was stirred at room temperature for the next 24 hours. The resulting salt was filtered using a Büchner funnel, and the crystallizer was weighed before and after adding the salt. The 2-methylthiophene salt 2’ was dried in a desiccator under vacuum for 4-5 hours and then used for synthesizing compounds 9–13.

3.3. Synthesis of Triazole Aldehydes 1–5

Triazole aldehydes 1–5 were synthesized in small glass vials from 200 mg of 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde [21] dissolved in 2 mL of dry 1,4-dioxane. The 1.2 eq of appropriate benzylamine was then added to the reaction mixture, and the mixture was briefly purged with argon. Depending on the applied benzylamine, the reaction took place for a certain period of time (24 – 48 h). The course of the reaction was monitored by thin-layer chromatography. At the end of the reaction, the solvent was removed by evaporation on a rotary vacuum evaporator. The solid product was purified by column chromatography using an appropriate solvent system, mostly DCM/EtOAc.

1-(4-methylbenzyl)-1H-1,2,3-triazole-4-carbaldehyde (1): 46 mg (isolated 23%), yellow oil; Rf (DCM) = 0.15; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 10.11 (s, 1H), 7.95 (s, 1H), 7.22 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.7 Hz, 2H), 5.54 (s, 2H), 2.37 (s, 3H).

1-(4-chlorobenzyl)-1H-1,2,3-triazole-4-carbaldehyde (2): 131 mg (isolated 65%), yellow oil; Rf (DCM) = 0.40; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 10.13 (s, 1H), 8.01 (s, 1H), 7.39 (d, J = 7.4 Hz, 2H), 7.25 (d, J = 8.9 Hz, 2H), 5.57 (s, 2H).

1-(3-methylbenzyl)-1H-1,2,3-triazole-4-carbaldehyde (3): 130 mg (isolated 65%), yellow oil; Rf (DCM) = 0.22; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 10.12 (s, 1H), 7.98 (s, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.21 (d, J = 8.1 Hz, 1H), 7.10 (d, J = 6.5 Hz, 2H), 5.54 (s, 2H), 2.36 (s, 3H).

1-(3-methoxybenzyl)-1H-1,2,3-triazole-4-carbaldehyde (4): 121 mg (isolated 61%), yellow oil; Rf (DCM) = 0.30; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 10.13 (s, 1H), 7.98 (s, 1H), 7.32 (t, J = 7.8 Hz, 1H), 6.93 (dd, J = 8.7, 2.6 Hz, 1H), 6.88 (d, J = 7.8 Hz, 1H), 6.82 (s, 1H), 5.55 (s, 2H), 3.80 (s, 3H).

1-(3-fluorobenzyl)-1H-1,2,3-triazole-4-carbaldehyde (5): 163 mg (isolated 82%), yellow oil; Rf (DCM) = 0.42; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 10.14 (s, 1H), 8.03 (s, 1H), 7.41 – 7.37 (m, 1H), 7.11 (dd, J = 8.0, 2.4 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 5.59 (s, 2H).

3.4. Synthesis of Triazolostilbenes 6–13 by Wittig Reaction

The apparatus, in a three-necked flask, a dropping funnel, a chlorine-calcium tube, and a balloon filled with nitrogen, was blown with argon for 5 min. A magnet was placed in the flask, the addition funnel was closed, and 30 mL of absolute ethanol was poured in (depending on the amount of starting reactants). A portion of absolute ethanol (10 mL) was poured into the flask, and the required amount of triphenylphosphonium salt was added. Sodium previously weighed in PE on an analytical balance with a precision of 0.0001 g was added to the remaining amount of absolute ethanol. After all the sodium had reacted in the ethanol with the evolution of hydrogen, a little portion of NaOEt was added to the flask. The aldehyde was dissolved in ethanol and transferred to a flask, then the rest of the NaOEt from the funnel was added dropwise. The flask was closed with a glass stopper, and the reaction mixture was left to stir in a magnetic stirrer at room temperature (6 – 108 h, 7 – 72 h, 8 – 48 h, 9 – 96 h, 10 – 96 h, 11 – 96 h, 12 – 168 h and 13 – 120 h). Compounds 6–13 were synthesized as mixtures of isomers and their pure isomers were not isolated due to the next reaction step.

1-(4-methylbenzyl)-4-(2-(thiophen-2-yl)vinyl)-1H-1,2,3-triazole (6): 37 mg (mixture of isomers, cis : trans = 1 : 1, 80%), white oil; Rf (PE/E = 80%) = 0.48.

1-(4-chlorobenzyl)-4-(2-(thiophen-2-yl)vinyl)-1H-1,2,3-triazole (7): 145 mg (mixture of isomers, cis : trans = 1 : 0.6, 90%), white oil; Rf (PE/E = 60%) = 0.25. MS (ESI) m/z (%, fragment): 302 (100); HRMS (m/z) for C₁₅H₁₂ClN₃S: [M + H]⁺_calcd = 301.0441, and [M + H]⁺_measured = 301.0440.

1-(3-methylbenzyl)-4-(2-(thiophen-2-yl)vinyl)-1H-1,2,3-triazole (8): 108 mg (mixture of isomers, cis : trans = 1 : 0.8, 91%), white oil; Rf (PE/E = 80%) = 0.67.

1-(4-methylbenzyl)-4-(2-(5-methylthiophen-2-yl)vinyl)-1H-1,2,3-triazole (9): 13 mg (mixture of isomers, cis : trans = 1 : 0.9, 13%), white oil; Rf (PE/E = 50%) = 0.60. MS (ESI) m/z (%, fragment): 296 (100); HRMS (m/z) for C₁₇H₁₇N₃S: [M + H]⁺_calcd = 295.1143, and [M + H]⁺_measured = 295.1147.

1-(4-chlorobenzyl)-4-(2-(5-methylthiophen-2-yl)vinyl)-1H-1,2,3-triazole (10): 50 mg (mixture of isomers, cis : trans = 1 : 0.8, 32%), white oil; Rf (PE/E = 50%) = 0.50. MS (ESI) m/z (%, fragment): 316 (100); HRMS (m/z) for C₁₆H₁₄ClN₃S: [M + H]⁺_calcd = 315.0597, and [M + H]⁺_measured = 315.0599.

1-(3-methylbenzyl)-4-(2-(5-methylthiophen-2-yl)vinyl)-1H-1,2,3-triazole (11): 12 mg (mixture of isomers, cis : trans = 1 : 0.7, 10%), white oil; Rf (PE/E = 90%) = 0.58. MS (ESI) m/z (%, fragment): 296 (100); HRMS (m/z) for C₁₇H₁₇N₃S: [M + H]⁺_calcd = 295.1143, and [M + H]⁺_measured = 295.1139.

1-(3-methoxybenzyl)-4-(2-(5-methylthiophen-2-yl)vinyl)-1H-1,2,3-triazole (12): 35 mg (mixture of isomers, cis : trans = 1 : 0.7, 24%), white oil; Rf (PE/E = 50 %) = 0.50. MS (ESI) m/z (%, fragment): 312 (100); HRMS (m/z) for C₁₇H₁₇N₃OS: [M + H]⁺_calcd = 311.1092, and [M + H]⁺_measured = 311.1098.

1-(3-fluorobenzyl)-4-(2-(5-methylthiophen-2-yl)vinyl)-1H-1,2,3-triazole (13): 16 mg (mixture of isomers, cis : trans = 1 : 0.7, 23%), white oil; Rf (PE/E = 50%) = 0.54.

3.5. Synthesis of Photoproducts 14–21

The corresponding 1,2,3-triazolostilbenes 6–13, as mixtures of isomers, were dissolved in 50 mL of toluene. These mixtures were then transferred to quartz tubes, which allowed light to pass through. A small amount of iodine was added as an oxidizing agent. The solutions, with a concentration of approximately 10⁻³ mol L⁻¹, were exposed to light for 1–4 hours using ten lamps emitting at a wavelength of about 313 nm in a Rayonet photoreactor. The resulting products, 14–21, were purified through column chromatography. Spectroscopic data for photoproducts 14–21 are provided below.

1-(4-methylbenzyl)-1H-thieno [3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (14): 27 mg (isolated 73%), yellow oil; Rf (PE/E (40 %)) = 0.34; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 7.99 (d, J = 9.0 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.55 (d, J = 6.0 Hz, 1H), 7.51 (d, J = 5.6 Hz, 1H), 7.12 (d, J = 8.2 Hz, 2H), 7.09 (d, J = 8.2 Hz, 2H), 6.08 (s, 2H), 2.29 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 144.7, 140.0, 138.1, 132.1, 129.8, 128.7, 127.7, 126.5, 122.7, 120.3, 119.2, 116.1, 52.9, 21.1; MS (ESI) m/z (%, fragment): 280 (100); HRMS (m/z) for C₁₆H₁₃N₃S: [M + H]⁺_calcd = 279.0830, and [M + H]⁺_measured = 279.0832.

1-(4-chlorobenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (15): 36 mg (isolated 25%), yellow oil; Rf (PE/E (80%)) = 0.25; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 8.00 (d, J = 8.6 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.58 (d, J = 5.4 Hz, 1H), 7.46 (d, J = 5.4 Hz, 1H), 7.29 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 9.2 Hz, 2H), 6.09 (s, 2H); ¹³C NMR (CDCl₃, 150 MHz) δ/ppm: 144.7, 140.2, 134.3, 133.6, 129.4, 128.6, 128.0, 127.9, 122.5, 119.9, 119.4, 116.1, 52.4; MS (ESI) m/z (%, fragment): 300 (100); HRMS (m/z) for C₁₅H₁₀ClN₃S: [M + H]⁺_calcd = 299.0284, and [M + H]⁺_measured = 299.0283.

1-(3-methylbenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (16): 26 mg (isolated 24%), yellow oil; Rf (PE/E (40%)) = 0.35; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 8.00 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 9.1 Hz, 1H), 7.55 (d, J = 5.9 Hz, 1H), 7.51 (d, J = 5.7 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 8.4 Hz, 2H), 6.09 (s, 2H), 2.27 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 144.7, 140.0, 139.0, 135.1, 129.1, 128.9, 127.7, 127.2, 123.6, 122.8, 120.3, 119.2, 116.1, 53.1, 21.4; MS (ESI) m/z (%, fragment): 280 (100); HRMS (m/z) for C₁₆H₁₃N₃S: [M + H]⁺_calcd = 279.0830, and [M + H]⁺_measured = 279.0828.

7-methyl-1-(4-methylbenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (17): 12 mg (isolated 15%), yellow oil; R_f (PE/E (30%)) = 0.46; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 7.91 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.18 (s, 1H), 7.12 (d, J = 8.3 Hz, 2H), 7.06 (d, J = 8.3 Hz, 2H), 6.04 (s, 2H), 2.60 (s, 3H), 2.30 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 142.6, 138.0, 132.3, 129.7, 126.6, 123.0, 118.9, 118.1, 115.0, 52.8, 21.1, 16.1; MS (ESI) m/z (%, fragment): 294 (100); HRMS (m/z) for C₁₇H₁₅N₃S: [M + H]⁺_calcd = 293.0987, and [M + H]⁺_measured = 293.0988.

1-(4-chlorobenzyl)-7-methyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (18): 14 mg (isolated 28%), colourless oil; R_f (PE/E = 50%) = 0.51; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 7.91 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 7.15 – 7.09 (m, 3H), 6.05 (s, 2H), 2.61 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 144.7, 143.0, 139.5, 134.3, 133.9, 129.3, 128.0, 122.8, 119.1, 117.7, 115.1, 52.3, 16.2; MS (ESI) m/z (%, fragment): 314 (100); HRMS (m/z) for C₁₆H₁₂ClN₃S: [M + H]⁺_calcd = 313.0440, and [M + H]⁺_measured = 313.0441.

7-methyl-1-(3-methylbenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (19): 13 mg (isolated 29%), yellow oil; R_f (PE/E (40%)) = 0.46; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 7.92 (d, J = 8.9 Hz, 1H), 7.65 (d, J = 8.8 Hz, 1H), 7.23 – 7.18 (m, 2H), 7.09 (d, J = 7.5 Hz, 1H), 7.03 – 6.96 (m, 2H), 6.04 (s, 2H), 2.60 (s, 3H), 2.28 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 142.6, 139.3, 138.9, 135.3, 129.1, 128.9, 127.3, 123.6, 123.0, 118.9, 118.1, 115.1, 52.9, 21.4, 16.2; MS (ESI) m/z (%, fragment): 294 (100); HRMS (m/z) for C₁₇H₁₅N₃S: [M + H]⁺_calcd = 293.0987, and [M + H]⁺_measured = 293.0981.

1-(3-methoxybenzyl)-7-methyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (20): 14 mg (isolated 35%), yellow oil; R_f (PE/E (40%)) = 0.38; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 7.91 (d, J = 8.9 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 7.17 (s, 1H), 6.85 – 6.75 (m, 2H), 6.70 (s, 1H), 6.05 (s, 2H), 3.71 (s, 3H), 2.60 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 160.1, 144.7, 142.6, 139.3, 136.9, 130.2, 128.3, 123.0, 118.9, 118.8, 118.1, 115.0, 113.6, 112.3, 55.2, 52.8, 16.2; MS (ESI) m/z (%, fragment): 310 (100); HRMS (m/z) for C₁₇H₁₅N₃OS: [M + H]⁺_calcd = 309.0936, and [M + H]⁺_measured = 309.0939.

1-(3-fluorobenzyl)-7-methyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazole (21): 8 mg (isolated 16%), colourless oil; R_f (PE/E (40%)) = 0.44; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 7.85 (d, J = 8.9 Hz, 1H), 7.63 (d, J = 8.9 Hz, 1H), 7.27 – 7.19 (m, 1H), 7.05 (s, 1H), 6.96 – 6.87 (m, 2H), 6.80 (d, J = 9.5 Hz, 1H), 6.01 (s, 2H), 2.51 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 161.9 (d, J_CF = 247.8 Hz), 143.7, 141.9, 138.5, 136.8 (d, J_CF = 7.4 Hz), 129.8 (d, J_CF = 8.5 Hz), 127.1, 121.8, 121.1 (d, J_CF = 3.1 Hz), 118.1, 116.7, 114.3 (d, J_CF = 20.9 Hz), 114.1, 112.7 (d, J_CF = 22.4 Hz), 51.4, 15.3; MS (ESI) m/z (%, fragment): 298 (100); HRMS (m/z) for C₁₆H₁₂FN₃S: [M + H]⁺_calcd = 297.0736, and [M + H]⁺_measured = 297.0734.

3.6. Synthesis of Triazolium Salts 22–31

In a glass reaction tube with a magnetic stirring bar, the corresponding thienobenzo-triazoles 14–21 (0.2 mmol, 1 equivalent), dry DCM (0.4 mL), and iodomethane (2 mmol, 10 equivalents) were added. The reaction mixtures were purged with argon and then stirred at 60 °C for 24 hours. After this period, the mixtures were cooled to 0 °C and diluted with 5 mL of solvent E. Following precipitation, the tubes were centrifuged three times for 10 minutes at 3000 rpm. The solvent was decanted, and the precipitate was washed three to five times with solvent E. The crude powders were then dried under high vacuum, yielding the pure triazolium salts 22–29. The solubility of triazolium salts 22–29 is low in CDCl₃, which is reflected in the signals, especially in the ¹³C NMR spectra.

3-methyl-1-(4-methylbenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (22): 8 mg (isolated 30%), yellow powder; R_f (DCM (100%)) = 0.19; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 7.99 (d, J = 9.1 Hz, 1H), 7.80 (dd, J = 9.1, 0.7 Hz, 1H), 7.55 (d, J = 5.5 Hz, 1H), 7.51 (dd, J = 5.5, 0.7 Hz, 1H), 7.10 (q, J = 17.3, 8.7 Hz, 4H), 2.29 (s, 3H), 1.59 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 143.1, 139.3, 135.1, 132.8, 130.9, 130.4, 127.8, 127.5, 127.2, 126.5, 120.6, 109.0, 57.2, 40.2, 21.2; MS (ESI) m/z (%, fragment): 294 (100); HRMS (m/z) for C₁₇H₁₆N₃S⁺: [M + H]⁺_calcd = 293.0987, and [M + H]⁺_measured = 293.0985.

1-(4-chlorobenzyl)-3-methyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (23): 15 mg (isolated 42%), yellow powder; R_f (DCM (100%)) = 0.15; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 8.34 (d, J = 8.6 Hz, 1H), 8.10 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 4.9 Hz, 1H), 7.85 (d, J = 5.7 Hz, 1H), 7.43 – 7.40 (m, 4H), 6.45 (s, 2H), 4.86 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 150.9, 130.9, 130.1, 129.5, 129.4, 128.9, 122.6, 121.3, 57.3, 34.6 (signals for 4 quaternary carbons are missing); MS (ESI) m/z (%, fragment): 314 (100); HRMS (m/z) for C₁₆H₁₃ClN₃S⁺: [M + H]⁺_calcd = 313.0440, and [M + H]⁺_measured = 313.0436.

3-methyl-1-(3-methylbenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (24): 4 mg (isolated 15%), yellow powder; R_f (DCM (100%)) = 0.20; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 8.34 (d, J = 11.1 Hz, 1H), 8.25 (d, J = 11.1 Hz, 1H), 7.94 (d, J = 6.3 Hz, 1H), 7.78 (d, J = 6.3 Hz, 1H), 7.31 (t, J = 7.4 Hz, 1H), 7.22 (d, J = 7.4 Hz, 1H), 7.18 – 7.16 (m, 2H), 6.34 (s, 2H), 4.92 (s, 3H), 2.35 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 142.7, 132.8, 130.7, 127.6, 127.1, 124.5, 122.7, 120.5, 116.0, 53.4, 30.9, 21.5 (signals for 5 quaternary carbons are missing); MS (ESI) m/z (%, fragment): 294 (100); HRMS (m/z) for C₁₇H₁₆N₃S⁺: [M + H]⁺_calcd = 293.0987, and [M + H]⁺_measured = 293.0991.

3,7-dimethyl-1-(4-methylbenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (25): 6 mg (isolated 27%), white powder; R_f (DCM (100%)) = 0.22; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 8.21 (d, J = 6.6 Hz, 1H), 8.07 – 8.04 (m, 1H), 7.52 (s, 1H), 7.30 – 7.28 (m, 1H), 7.28 – 7.27 (m, 1H), 7.24 – 7.23 (m, 1H), 7.23 – 7.21 (m, 1H), 6.31 (s, 2H), 4.84 (s, 3H), 2.74 (s, 3H), 2.36 (s, 3H); MS (ESI) m/z (%, fragment): 308 (100); HRMS (m/z) for C₁₈H₁₈N₃S⁺: [M + H]⁺_calcd = 307.1216, and [M + H]⁺_measured = 307.1218.

1-(4-chlorobenzyl)-3,7-dimethyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (26): 5 mg, (isolated 22%), white powder; Ry_f (DCM (100%)) = 0.21; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 8.20 (d, J = 6.2 Hz, 1H), 7.98 – 7.90 (m, 1H), 7.58 (s, 1H), 7.44 – 7.38 (m, 4H), 6.41 (s, 2H), 4.79 (s, 3H), 2.75 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ/ppm: 156.2, 149.1, 136.0, 129.9, 129.4, 127.3, 126.9, 123.4, 118.4, 65.9, 16.6, 15.3 (signals for 3 quaternary carbons are missing); MS (ESI) m/z (%, fragment): 328 (100); HRMS (m/z) for C₁₇H₁₅ClN₃S⁺: [M + H]⁺_calcd = 327.0122, and [M + H]⁺_measured = 327.0120.

3,7-dimethyl-1-(3-methylbenzyl)-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (27): 2 mg (isolated 15%), white powder; R_f (DCM (100%)) = 0.24; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 8.21 (d, J = 9.3 Hz), 8.11 – 8.09 (m, 1H), 7.49 (s, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.23 (d, J = 7.1 Hz, 1H), 7.19 – 7.16 (m, 2H), 6.31 (s, 2H), 4.86 (s, 3H), 2.73 (s, 3H), 2.36 (s, 3H); MS (ESI) m/z (%, fragment): MS (ESI) m/z (%, fragment): 308 (100); HRMS (m/z) for C₁₈H₁₈N₃S⁺: [M + H]⁺_calcd = 307.1216, and [M + H]⁺_measured = 307.1218.

1-(3-methoxybenzyl)-3,7-dimethyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (28): 4 mg, (isolated 26%), yellow powder; R_f (DCM (100%)) = 0.18; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 8.19 (br s, 1H), 8.00 (br s, 1H), 7.53 (br s, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.13 – 7.10 (m, 1H), 6.95 – 6.91 (m, 2H), 6.35 (br s, 2H), 4.81 (br s, 3H), 3.81 (s, 3H), 2.73 (s, 3H); MS (ESI) m/z (%, fragment): 324 (100).

1-(3-fluorobenzyl)-3,7-dimethyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (29): obtained only in traces according to NMR analysis, not isolated and evaluated. Its synthesis should be optimized in the following investigations.

Two additional triazolium salts, 30 and 31, were also prepared from the previously obtained and characterized neutral thienobenzo-triazole [20] for comparison of the non-charged and charged analogues.

1-(3-chlorobenzyl)-3-methyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (30): 10 mg (isolated 43%), yellow powder; R_f (DCM (100%)) = 0.20; ¹H NMR (CDCl₃, 300 MHz) δ/ppm: 8.39 – 8.31 (m, 2H), 8.13 – 8.07 (m, 2H), 8.02 – 7.96 (m, 2H), 7.86 – 7.82 (m, 2H), 6.47 (s, 2H), 4.87 (s, 3H); MS (ESI) m/z (%, fragment): 314 (100); HRMS (m/z) for C₁₆H₁₃ClN₃S⁺: [M + H]⁺_calcd = 313.0440, and [M + H]⁺_measured = 313.0436.

1-(furan-2-ylmethyl)-3-methyl-1H-thieno[3’,2’:3,4]benzo[1,2-d][1,2,3]triazol-3-ium (31): 6 mg (isolated 29%), yellow powder; R_f (DCM (100%)) = 0.17; ¹H NMR (CDCl₃, 600 MHz) δ/ppm: 8.34 (d, J = 9.0 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.99 (d, J = 5.8 Hz, 1H), 7.86 (d, J = 5.8 Hz, 1H), 7.50 – 7.47 (m, 2H), 7.13 (d, J = 3.8 Hz, 2H), 6.43 (s, 2H), 4.82 (s, 3H); MS (ESI) m/z (%, fragment): 270 (100); HRMS (m/z) for C₁₄H₁₂N₃OS⁺: [M + H]⁺_calcd = 269.0623, and [M + H]⁺_measured = 269.0627.

4. Conclusions

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