New Charged Cholinesterase Inhibitors: Design, Synthesis, and Characterization

Triazoles and triazolium salts are very common subunits in the structures of various drugs. Medicaments with a characteristic 1,2,3-triazole core are also being developed to treat neurodegenerative disorders associated with cholinesterase enzyme activity. Several naphtho- and thienobenzo-triazoles from our previous research emerged as being particularly promising in that sense. For this reason, in this research, new naphtho- and thienobenzo-triazoles 23–34, as well as 1,2,3-triazolium salts 44–51, were synthesized and tested. Triazolium salts 44–46 showed excellent activity while salts 47 and 49 showed very good inhibition toward both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) enzymes. In contrast, neutral photoproducts were shown to be selective towards BChE but with very good inhibition potential as molecules 24–27. The representative of newly prepared compounds, 45 and 50, were stable in aqueous solution and revealed intriguing fluorimetric properties, characterized by a strong Stokes shift of >160 nm. Despite their condensed polycyclic structure shaped similarly to well-known DNA-intercalator ethidium bromide, the studied compounds did not show any interaction with ds-DNA, likely due to the unfavorable steric hindrance of substituents. However, the studied dyes bind proteins, particularly showing very diverse inhibition properties toward AChE and BChE. In contrast, neutral photoproducts were shown to be selective towards a certain enzyme but with moderate inhibition potential. The molecular docking of the best-performing candidates to cholinesterases’ active sites identified cation–π interactions as the most responsible for the stability of the enzyme–ligand complexes. As genotoxicity studies are crucial when developing new active substances and finished drug forms, in silico studies for all the compounds synthesized have been performed.


Introduction
DNA, RNA, and proteins are biomolecules that govern living organisms, are included in various biological processes, and are often the targets of novel drugs.Binding small molecules to these biomolecules can result in changes in biological properties, resulting in various biological activities [1].Triazoles constitute an important class of heterocyclic molecules that exhibit a wide range of pharmacological activities [2].A wide variety of drugs containing 1,2,3-triazole as a central heterocyclic structural component prove its pharmacological importance, such as anticonvulsants [3], antimalarials [4], antimicrobials [5,6], antivirals [7], antiproliferative drugs [8], antitumor drugs [9], analgesics [10], and antidiabetics [11].This diversity of the triazole core has stimulated scientists' interest in developing new triazole molecules with promising biological activities [12].Drugs with a characteristic 1,2,3-triazole core are also being developed for the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) [13].
In the last few years, a broad spectrum of naphtho-and thienobenzo-triazoles has been prepared mostly photochemically (Figure 1, structures A and B) to analyze their potential inhibitory activity towards enzymes acetyl-(AChE) and butyrylcholinesterase (BChE).AChE has an important physiological role in the body because it controls the transmission of nerve impulses in cholinergic synapses of the nervous system by hydrolysis of the positively charged neurotransmitter acetylcholine [25].BChE serves as a co-regulator of cholinergic neurotransmission and is able to catalyze the hydrolysis of acetylcholine, and high levels of BChE are associated with the neuropathological hallmarks of AD [26].Several compounds from previous studies emerged as being particularly promising [27,28], and some displayed selectivity toward BChE.Based on these results, and taking into account the potential of 1,2,3-triazolium salts, a new series of naphthotriazoles (Figure 1, structure C) was synthesized in this work to examine the influence of the halogen as a substituent on the inhibitory potential towards cholinesterases.Additionally, for the first time, a series of charged thienobenzo-triazolium salts (Figure 1, structure D) was also prepared to see the effect of charge compared to their uncharged analogs (aromatic and non-aromatic) [29].It was also challenging to see the influence of the position of the substituent on the aromatic ring on triazole, the influence of the nature of the substituent (OCH 3 group in comparison to Cl), and the influence of the new dimethylamino group.
In the last few years, a broad spectrum of naphtho-and thienobenzo-triazoles has been prepared mostly photochemically (Figure 1, structures A and B) to analyze their potential inhibitory activity towards enzymes acetyl-(AChE) and butyrylcholinesterase (BChE).AChE has an important physiological role in the body because it controls the transmission of nerve impulses in cholinergic synapses of the nervous system by hydrolysis of the positively charged neurotransmitter acetylcholine [25].BChE serves as a co-regulator of cholinergic neurotransmission and is able to catalyze the hydrolysis of acetylcholine, and high levels of BChE are associated with the neuropathological hallmarks of AD [26].Several compounds from previous studies emerged as being particularly promising [27,28], and some displayed selectivity toward BChE.Based on these results, and taking into account the potential of 1,2,3-triazolium salts, a new series of naphthotriazoles (Figure 1, structure C) was synthesized in this work to examine the influence of the halogen as a substituent on the inhibitory potential towards cholinesterases.Additionally, for the first time, a series of charged thienobenzo-triazolium salts (Figure 1, structure D) was also prepared to see the effect of charge compared to their uncharged analogs (aromatic and non-aromatic) [29].It was also challenging to see the influence of the position of the substituent on the aromatic ring on triazole, the influence of the nature of the substituent (OCH3 group in comparison to Cl), and the influence of the new dimethylamino group.
For the synthesis of 1,2,3-triazolostilbene 10-22, it was necessary to prepare the correspond- The Wittig reaction produced two configurational isomers, each of 1,2,3-triazolo stilbenes 10-22, with different ratios of cis-and trans-isomers depending on th substituents.The obtained mixtures of isomers were purified by extraction and column chromatography, where the petroleum ether/ether (PE/E) solvent system of variabl proportions was used.In the resulting mixture of isomers, the cis-isomer eluted first, and the trans-isomer last at almost the same rate as the traces of the starting aldehyde.Th residual p-nitroaniline was successfully separated if it was introduced into the reaction mixture with aldehyde during the synthesis, which is also where it is formed.For th synthesis of triazolostilbenes 10-16, (thiophen-2-ylmethyl)triphenylphosphonium sal was used, while in the case of 17-22, the (4-methoxybenzyl)-or (4 chlorobenzyl)triphenylphosphonium, salt, along the addition of base sodium ethoxide was used.The Wittig reaction produced two configurational isomers, each of 1,2,3-triazolostilbenes 10-22, with different ratios of cisand trans-isomers depending on the substituents.The obtained mixtures of isomers were purified by extraction and column chromatography, where the petroleum ether/ether (PE/E) solvent system of variable proportions was used.In the resulting mixture of isomers, the cis-isomer eluted first, and the trans-isomer last at almost the same rate as the traces of the starting aldehyde.The residual p-nitroaniline was successfully separated if it was introduced into the reaction mixture with aldehyde during the synthesis, which is also where it is formed.For the synthesis of triazolostilbenes 10-16, (thiophen-2-ylmethyl)triphenylphosphonium salt was used, while in the case of 17-22, the (4-methoxybenzyl)-or (4-chlorobenzyl)triphenylphosphonium, salt, along the addition of base sodium ethoxide, was used.

Photocyclization of 1,2,3-Triazolo-Stilbenes 10-22 to the Targeted Final Photoproducts 23-35
Thienobenzo-and naphthotriazoles 23-34 were synthesized by photochemical reactions of triazolostilbenes 10-21 (Scheme 2).Given that the wavelength ranges in which thienobenzo-and naphtho-triazoles absorb are known from previous research, a wavelength of 313 nm was used for photochemical cyclization reactions.The corresponding triazolostilbenes 10-22 were dissolved in toluene and transferred to a light-transmitting quartz tube.Iodine, which serves as an oxidizing agent, was added to the mixture.The course of the reaction was monitored using thin-layer chromatography in the PE/E solvent system.In the case of triazolo-stilbene 20, naphthotriazole 35 was not formed.Thienobenzo-and naphthotriazoles 23-34 were synthesized by photochemica reactions of triazolostilbenes 10-21 (Scheme 2).Given that the wavelength ranges in which thienobenzo-and naphtho-triazoles absorb are known from previous research, wavelength of 313 nm was used for photochemical cyclization reactions.Th corresponding triazolostilbenes 10-22 were dissolved in toluene and transferred to a light transmitting quartz tube.Iodine, which serves as an oxidizing agent, was added to th mixture.The course of the reaction was monitored using thin-layer chromatography in the PE/E solvent system.In the case of triazolo-stilbene 20, naphthotriazole 35 was no formed.

Synthesis of the Triazolium Salts 44-51
Thienobenzo-triazolium salts 44-51 were synthesized in dry dichloromethane using iodomethane from the previously published triazoles 36-43 [29] over a period of 24 h a 60 °C (Scheme 3).After the workup, the crude powder was dried under a high vacuum t afford the pure triazolium salts 44-51.

Synthesis of the Triazolium Salts 44-51
Thienobenzo-triazolium salts 44-51 were synthesized in dry dichloromethane using iodomethane from the previously published triazoles 36-43 [29] over a period of 24 h at 60 • C (Scheme 3).After the workup, the crude powder was dried under a high vacuum to afford the pure triazolium salts 44-51.

Spectroscopic Characterization in Biorelevant Medium and DNA-Binding Ability of Triazolium Salts
Prior to any biological studies, novel compounds should be evaluated for their stability in biorelevant conditions, whereby spectrophotometric characterization is the most common approach.Also, such characterization would often reveal beneficial properties of compounds, such as fluorescence, which could eventually be applied to studying interactions with biological targets.
The stock solutions of the compounds were prepared in DMSO at 1 mM concentration and further diluted in aqueous solutions prior to the experiment.The stock solutions were kept at +4 • C, and working aliquots were kept at +25 • C. No visible precipitation or change in the UV spectra of working solutions was noticed, pointing to the high stability of compounds.Experiments were further conducted in a biologically relevant aqueous medium at pH = 7.0 (sodium cacodylate buffer, I = 0.05 M).Both compounds show adherence to the Beer-Lambert law to up to 10 µM concentrations (Figures S110 and S111), excluding aggregation in water.The difference in the UV/Vis spectra (Figure 2, Table 1) agrees well with the structures of the compounds.Namely, the absorption maximum of condensed aromatic compound 45 is bathochromically shifted with respect to compound 50; the latter shows separate absorption maxima that could be related to two moieties, the thieno-benzo core and the CF 3 -substituted benzyl moiety.Both studied compounds exhibit fluorescence, the condensed aromatic analog 45 showing a bathochromically shifted emission maximum with respect to 50, but 50 has a higher fluorescence intensity (Figure 2, Figures S112 and S113, Table 1).The emission intensity of 45 and 50 is proportional to the concentration at up to 2 µM concentrations (Figures S112 and S113).The excitation maxima correlate with the absorption maxima.Emission was stable upon heating and cooling the samples (Figure S114).Both compounds exhibit a large Stokes shift (Table 1, Figure S115), which is generally favorable for fluorescent dyes, excluding self-absorption.
Another feasible spectroscopic property is solvatochromism, which can be employed in water-exclusion sensing [31,32] or lipid-membrane studies [33].UV/Vis and fluorescence spectra were also taken in 1-octanol as a referent nonpolar solvent to evaluate potential solvatochromic effects.For both 45 and 50, emission in 1-octanol was hypsochromically shifted for 20 nm (Figures S116-S118); such a minor change is not applicable for fine water-content sensing.
Compounds 44-51 are cationic polycyclic structures, and particularly 44-49 are also condensed heteroaromatics rich in nitrogen atoms, with a crescent shape very similar to ethidium bromide, thus pointing toward possible interactions with ds-DNA [34].Therefore, we have chosen two triazolium salts, condensed aromatic 45 and its noncondensed analog 50, to study their DNA-binding ability.
To preliminarily probe the binding ability of 45 and 50 to ds-DNA, we used calf thymus ct-DNA as a representative of the B-helical secondary structure and containing equal amounts of AT-and GC-base pairs.The additions of ct-DNA to aqueous solutions of 45 and 50 at up to 50-fold excess did not yield any change in the UV/Vis spectra of compound 45, as well as yielding no change in the emission spectra of both compounds (Figures S119-S121), thus indicating no bio-relevant affinity of the dyes towards ds-DNA.Further, employing the fluorescence of the compounds, we performed fluorimetric titrations of the compounds with either bovine serum albumin (BSA) or human serum albumin (HSA) as common carrier proteins included in the transfer of numerous small molecules [35].The results showed an indication of binding; however, at given  Emission was stable upon heating and cooling the samples (Figure S114).Both compounds exhibit a large Stokes shift (Table 1, Figure S115), which is generally favorable for fluorescent dyes, excluding self-absorption.
Another feasible spectroscopic property is solvatochromism, which can be employed in water-exclusion sensing [31,32] or lipid-membrane studies [33].UV/Vis and fluorescence spectra were also taken in 1-octanol as a referent nonpolar solvent to evaluate potential solvatochromic effects.For both 45 and 50, emission in 1-octanol was hypsochromically shifted for 20 nm (Figures S116-S118); such a minor change is not applicable for fine water-content sensing.
Compounds 44-51 are cationic polycyclic structures, and particularly 44-49 are also condensed heteroaromatics rich in nitrogen atoms, with a crescent shape very similar to ethidium bromide, thus pointing toward possible interactions with ds-DNA [34].Therefore, we have chosen two triazolium salts, condensed aromatic 45 and its non-condensed analog 50, to study their DNA-binding ability.
To preliminarily probe the binding ability of 45 and 50 to ds-DNA, we used calf thymus ct-DNA as a representative of the B-helical secondary structure and containing equal amounts of AT-and GC-base pairs.The additions of ct-DNA to aqueous solutions of 45 and 50 at up to 50-fold excess did not yield any change in the UV/Vis spectra of compound 45, as well as yielding no change in the emission spectra of both compounds (Figures S119-S121), thus indicating no bio-relevant affinity of the dyes towards ds-DNA.Further, employing the fluorescence of the compounds, we performed fluorimetric titrations of the compounds with either bovine serum albumin (BSA) or human serum albumin (HSA) as common carrier proteins included in the transfer of numerous small molecules [35].The results showed an indication of binding; however, at given experimental conditions, the autofluorescence of the proteins hampered the accurate determination of binding constants.
The studied compounds did not show biorelevant activity toward ds-DNA, thus avoiding possible genotoxicity; however, they did show binding to serum albumin protein, which strongly supported further studies with relevant protein targets.As noted in the Introduction, similar analogs showed intriguing activity against enzymes AChE and BChE; thus, we studied this interaction in all compounds in detail.

ChEs Inhibition
Triazoles 23-34 and 44-51 were tested for their ability to inhibit acetylcholinesterase and butyrylcholinesterase, applying Ellman's assay [36] with slight modifications, using galantamine as a standard.From the results shown in Table 2 and considering our previous findings on structurally similar compounds, the following structure-activity relationship can be noted.Newly synthesized thienobenzo-triazole derivatives 23-34 effectively inhibited BChE with very good to moderate IC 50 values (Table 2).Very good inhibitory activity was observed for 24, 25, 26, and 27, with IC 50 values three−four times lower than the IC 50 of standard galantamine.These thienobenzo-triazoles possess the following substituents at the triazole unit: 3-fluorobenzyl, 3-methoxybenzyl, 3-chlorobenzyl, and 4-dimethylaminobenzyl.Derivative 24, with a fluorine substituent in the meta position, was the only one that reached the IC 50 value for AChE.In the previously tested series of thienobenzo-triazoles [28], the derivative with a fluorine substituent in the para position reached IC 50 values for both enzymes with the same concentration value.The derivative 25 with the methoxy group in the meta position also has a pair of analogs in the previous series [28] with the para position of the substituent.Both derivatives, 24 and 25, inhibit BChE in a similar concentration value, and in comparison with their analogs from [28], this leads to the conclusion that the position of the substituent on the aromatic ring does not significantly change the inhibitory activity.The introduction of a new substituent, 4-dimethylamino benzyl present in 27, led to a very good inhibition of BChE.A comparison of the derivatives with and without a methyl substituent at the thiophene ring is possible, too.The methyl-substituted thiophene analog of 23 was tested earlier [26], while 27 and 29 tested here represent such analogs too.The introduction of a methyl group at the thiophene reduced affinity toward enzyme BChE.
Another group of triazole derivatives, naphtotriazoles 30-34, showed weak potency toward the inhibition of the cholinesterases.None of the tested compounds within this group achieved significant results.Only derivative 32 with a buthenyl substituent at the triazole ring inhibited both enzymes, with a weak IC 50 value for AChE, lower by three orders of magnitude than for the standard galantamine, and a good value for BChE, an order of magnitude below the value measured for the standard.Derivatives 31-34 with the chlorine substituent on the naphtho moiety have methoxy-substituted analogs with the same substituent at the triazole ring (compound 30 and the compound previously tested in [28]).The replacement of the methoxy group with the chlorine decreased affinity towards enzymes.
Both AChE and BChE were effectively inhibited by the last studied group, the 1,2,3triazolium salts 44-51 (Table 2, Figures 3 and 4).The most potent inhibitory effects were observed for 44, 45, 46, 47, and 49, with IC 50 values better or similar to the standard galantamine for BChE and somewhat weaker for AChE.These derivatives can be compared to the uncharged analogs studied earlier [26]-the analog of the most effective salt 44 was highly selective, but an excellent inhibitor of BChE with an IC 50 value and order of magnitude better than for the standard galantamine.A similar observation also applies to the uncharged analog of 45: transformation to the salt form retains successful inhibition toward BChE, but activity toward AChE is drastically increased.Salts 46 and 49 increased the activity toward both enzymes compared to their uncharged analogs studied earlier.
Regarding the type of the central ring, whether aromatic or non-aromatic, 46 had better IC 50 values than the non-aromatized analog 50, while 47 and its non-aromatized analog 51 have almost the same affinities for both enzymes.Somewhat weaker inhibition values were achieved with 48, so of all the substituents, 4-hydroxybutyl proved to be the one that reduces activity most.Therefore, the type of substituent and charge at the nitrogen of the triazole ring are critical structural features that govern inhibitory potency, albeit with the loss of selectivity.IC50 values than the non-aromatized analog 50, while 47 and its non-aromatized analog 51 have almost the same affinities for both enzymes.Somewhat weaker inhibition values were achieved with 48, so of all the substituents, 4-hydroxybutyl proved to be the one that reduces activity most.Therefore, the type of substituent and charge at the nitrogen of the triazole ring are critical structural features that govern inhibitory potency, albeit with the loss of selectivity.The obtained results were summarized in a structure-activity relationship overview (Figure 5).The key determinants in a structure-activity relationship are the type of (hetero)cyclic ring, the molecule's geometry, the type of substituent, and the charge on the triazole ring.The obtained results were summarized in a structure-activity relationship overview (Figure 5).The key determinants in a structure-activity relationship are the type of (hetero)cyclic ring, the molecule's geometry, the type of substituent, and the charge on the triazole ring.The obtained results were summarized in a structure-activity relationship overview (Figure 5).The key determinants in a structure-activity relationship are the type of (hetero)cyclic ring, the molecule's geometry, the type of substituent, and the charge on the triazole ring.

Molecular Docking into Cholinesterases
According to experimental results, two charged triazole derivatives, compounds 44 and 45, showed excellent inhibitory potential toward both cholinesterases.Molecular docking was performed to obtain insight into the binding modes of these ligands within the active site and identify interactions responsible for the stability of the enzyme-ligand complex.Figure 6 shows the structures of the most stable complexes formed between triazolium ions 44 and 45, respectively, and the active site of AChE obtained by docking.
Both ligands have similar orientations, with the main molecular framework comprising thiophene, phenyl, and triazole rings positioned within a 5 Å distance from the esteratic active site (Ser203, Glu343, and His438).The sulfur from thiophene is positioned in proximity to the serine hydroxyl group, forming a weak hydrogen bond, and there is a π-π stacking between thiophene and His438.In both complexes, an energetically significant cation-π interaction is observed between the methyl-substituted triazole nitrogen and residue Trp86 of the anionic subsite.

Molecular Docking into Cholinesterases
According to experimental results, two charged triazole derivatives, compounds 44 and 45, showed excellent inhibitory potential toward both cholinesterases.Molecular docking was performed to obtain insight into the binding modes of these ligands within the active site and identify interactions responsible for the stability of the enzyme-ligand complex.Figure 6 shows the structures of the most stable complexes formed between triazolium ions 44 and 45, respectively, and the active site of AChE obtained by docking.The calculated electrostatic potential maps, illustrating the charge distribution within the cations of compounds 44 and 45 (Figure 7), indicate that the triazole ring is the most positively charged region of the scaffold; therefore, the triazole involvement in cation-π interactions is expected.In the AChE complex with 44 (Figure 6a), a cation-π interaction is also observed between the triazolium nitrogen and residue Trp341 belonging to the peripheral anionic site (PAS).However, in the complex involving compound 45 (Figure 6b), the Tyr341 participates in the alkyl-π interaction with the ethyl chain that connects triazole and the p-methoxyphenyl, while the positive triazolium nitrogen establishes an electrostatic attraction with Asp74, which is also part of the PAS.Positioning the pmethoxyphenyl substituent of compound 44 within the complex enables the alkyl-π interaction with residue Trp286, similar to the situation observed in the complex with 45, where the terminal ethyl group of the substituent interacts with Trp286.Both ligands have similar orientations, with the main molecular framework comprising thiophene, phenyl, and triazole rings positioned within a 5 Å distance from the esteratic active site (Ser203, Glu343, and His438).The sulfur from thiophene is positioned in proximity to the serine hydroxyl group, forming a weak hydrogen bond, and there is a π-π stacking between thiophene and His438.In both complexes, an energetically significant cation-π interaction is observed between the methyl-substituted triazole nitrogen and residue Trp86 of the anionic subsite.
The calculated electrostatic potential maps, illustrating the charge distribution within the cations of compounds 44 and 45 (Figure 7), indicate that the triazole ring is the most positively charged region of the scaffold; therefore, the triazole involvement in cation-π interactions is expected.In the AChE complex with 44 (Figure 6a), a cation-π interaction is also observed between the triazolium nitrogen and residue Trp341 belonging to the peripheral anionic site (PAS).However, in the complex involving compound 45 (Figure 6b), the Tyr341 participates in the alkyl-π interaction with the ethyl chain that connects triazole and the p-methoxyphenyl, while the positive triazolium nitrogen establishes an electrostatic attraction with Asp74, which is also part of the PAS.Positioning the p-methoxyphenyl substituent of compound 44 within the complex enables the alkyl-π interaction with residue Trp286, similar to the situation observed in the complex with 45, where the terminal ethyl group of the substituent interacts with Trp286.The calculated electrostatic potential maps, illustrating the charge distribution within the cations of compounds 44 and 45 (Figure 7), indicate that the triazole ring is the most positively charged region of the scaffold; therefore, the triazole involvement in cation-π interactions is expected.In the AChE complex with 44 (Figure 6a), a cation-π interaction is also observed between the triazolium nitrogen and residue Trp341 belonging to the peripheral anionic site (PAS).However, in the complex involving compound 45 (Figure 6b), the Tyr341 participates in the alkyl-π interaction with the ethyl chain that connects triazole and the p-methoxyphenyl, while the positive triazolium nitrogen establishes an electrostatic attraction with Asp74, which is also part of the PAS.Positioning the pmethoxyphenyl substituent of compound 44 within the complex enables the alkyl-π interaction with residue Trp286, similar to the situation observed in the complex with 45, where the terminal ethyl group of the substituent interacts with Trp286.The docking of cations 44 and 45 into the active site of BChE yielded structures depicted in Figure 8. Notably, the main scaffolds did not adopt mutually similar orientations.In the complex formed between 44 and BChE, a cation-π interaction is The docking of cations 44 and 45 into the active site of BChE yielded structures depicted in Figure 8. Notably, the main scaffolds did not adopt mutually similar orientations.In the complex formed between 44 and BChE, a cation-π interaction is evident between the triazolium nitrogen and Trp82 of the anionic site.Simultaneously, there is an electrostatic attraction between the methyl group at triazolium and Glu197, a residue also belonging to the anionic sub-domain (Figure 8a).While the scaffold of 45 is situated between Trp82 and the esteratic site, akin to the complex with 44, its orientation allows for the electrostatic attraction between the most positively charged part of the triazolium and residue Asp70, belonging to the PAS (Figure 8b).Additional stabilizing factors within these complexes involve the parallel π-π stacking between the thiophene and Trp82 in the complex with 44, whereas the complex with 45 features an alkyl-π interaction between the terminal ethyl group of the substituent and residue Tyr332.evident between the triazolium nitrogen and Trp82 of the anionic site.Simultaneously, there is an electrostatic attraction between the methyl group at triazolium and Glu197, a residue also belonging to the anionic sub-domain (Figure 8a).While the scaffold of 45 is situated between Trp82 and the esteratic site, akin to the complex with 44, its orientation allows for the electrostatic attraction between the most positively charged part of the triazolium and residue Asp70, belonging to the PAS (Figure 8b).Additional stabilizing factors within these complexes involve the parallel π-π stacking between the thiophene and Trp82 in the complex with 44, whereas the complex with 45 features an alkyl-π interaction between the terminal ethyl group of the substituent and residue Tyr332.

Genotoxicity
Impurities that can be present in the active pharmaceutical ingredient (API) as well as the related finished drug product cannot be completely avoided and an investigation into these compounds is a pivotal part of drug product development and is of critical importance for drug safety.Impurities that can be present in the active substance, as well as in each intermediate during the manufacturing process, have to be evaluated in regard to their genotoxic potential properties.Compounds with proven or potential genotoxic properties will be more strictly regulated and have to be controlled at very low levels,

Genotoxicity
Impurities that can be present in the active pharmaceutical ingredient (API) as well as the related finished drug product cannot be completely avoided and an investigation into these compounds is a pivotal part of drug product development and is of critical importance for drug safety.Impurities that can be present in the active substance, as well as in each intermediate during the manufacturing process, have to be evaluated in regard to their genotoxic potential properties.Compounds with proven or potential genotoxic properties will be more strictly regulated and have to be controlled at very low levels, significantly lower than other impurities (ICH M7 Guideline).These levels that are then allowed to be present in the drug substance/product will be calculated based on their experimental acceptable daily intake (AI) and the maximum daily dose (MDD) of the drug.With new compounds, the AI is usually not yet determined by toxicological studies on animals, and then the most conservative approach has to be taken with the strictest presumed AI as described in the guideline itself.Evaluations are primarily performed consistently by the use of in silico Q(SAR) tools.When developing new active substances and finished drug forms, these impurities will be new compounds, and usually no experimental data will be available.In these cases, the Q(SAR) approach is of vital importance.(Q)SAR models make predictions of biological activity based on structural components [37].This approach is vital during the early stages of searching for potentially active drug substances.Eliminating all compounds with mutagenic potential saves a lot of money and time.The Lhasa software package (Nexus v.2.5.2 (Build 5, Jul 2022), Derek Nexus v.6.2.1 and Sarah Nexus v.3.2.1) is the most commonly used software because it has two complementary models; it is also very important that all predictions are again reviewed an toxicology expert.
In the case of compounds 44-51, no structural alerts were found by Derek Nexus (Table 3).For Sarah, these compounds are out of scope, as will be the case often with very new synthetic compounds that are only starting to be investigated for their biological activity.This is an excellent example of how a complimentary model is a must, especially in this early development.With compound 10, Sarah Nexus has found a similar training set, and this compound can be negative.These compounds would be considered the safest ones for the continuation of the early stages of development.As for the compounds 27, 31, 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 and 24-26 substituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis provided by Sarah cannot be overruled by the negative by Derek, so they would probably be investigated further with utmost scrutiny and only if there were no other better active ingredients as new leads.These compounds would require an experimental AMES test to be conducted to determine their genotoxic potential.

Similarity to API Overall In Silico 23 Inconclusive
Molecules 2024, 29,1622 and Sarah Nexus v.3.2.1) is the most commonly used software because mentary models; it is also very important that all predictions are again cology expert.
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In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because mentary models; it is also very important that all predictions are again cology expert.
In the case of compounds 44-51, no structural alerts were found (Table 3).For Sarah, these compounds are out of scope, as will be the ca new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their b tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because mentary models; it is also very important that all predictions are again cology expert.
In the case of compounds 44-51, no structural alerts were found (Table 3).For Sarah, these compounds are out of scope, as will be the ca new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their b tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because mentary models; it is also very important that all predictions are again cology expert.
In the case of compounds 44-51, no structural alerts were found (Table 3).For Sarah, these compounds are out of scope, as will be the ca new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their b tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because mentary models; it is also very important that all predictions are again cology expert.
In the case of compounds 44-51, no structural alerts were found (Table 3).For Sarah, these compounds are out of scope, as will be the ca new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their b tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because mentary models; it is also very important that all predictions are again cology expert.
In the case of compounds 44-51, no structural alerts were found (Table 3).For Sarah, these compounds are out of scope, as will be the ca new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their b tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.(Table 3).For Sarah, these compounds are out of scope, as will be the ca new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.new synthetic compounds that are only starting to be investigated for their b tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.cology expert.
In the case of compounds 44-51, no structural alerts were found (Table 3).For Sarah, these compounds are out of scope, as will be the ca new synthetic compounds that are only starting to be investigated for tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their b tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.tivity.This is an excellent example of how a complimentary model is a this early development.With compound 10, Sarah Nexus has found a s and this compound can be negative.These compounds would be con ones for the continuation of the early stages of development.As for the 34, and 30 that have the dimethyl-amino-aryl moiety and compounds stituted halogen-aryl and methoxy-aryl moieties, the positive hypothes rah cannot be overruled by the negative by Derek, so they would probab further with utmost scrutiny and only if there were no other better ac new leads.These compounds would require an experimental AMES te to determine their genotoxic potential.and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.
In the case of compounds 44-51, no structural alerts were found by D (Table 3).For Sarah, these compounds are out of scope, as will be the case oft new synthetic compounds that are only starting to be investigated for their tivity.This is an excellent example of how a complimentary model is a must, this early development.With compound 10, Sarah Nexus has found a similar and this compound can be negative.These compounds would be consider ones for the continuation of the early stages of development.As for the comp 34, and 30 that have the dimethyl-amino-aryl moiety and compounds 33 an stituted halogen-aryl and methoxy-aryl moieties, the positive hypothesis pro rah cannot be overruled by the negative by Derek, so they would probably be further with utmost scrutiny and only if there were no other better active in new leads.These compounds would require an experimental AMES test to b to determine their genotoxic potential.[38].The concentration of ct-DNA was determined as the concentration of phosphates (corresponding to c(nucleobase)) spectroscopically at λ max = 260 nm [39].Bovine serum albumin (BSA, Sigma Aldrich) and human serum albumin (HSA, Sigma Aldrich) were dissolved in MilliQ water at c = 1 mM.

Synthesis of Triazole Aldehydes 1-9
Triazole aldehydes 1-9 were synthesized in small glass vials from 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde dissolved in dry 1,4-dioxane.The appropriate amine was then added to the reaction mixture, and the mixture was briefly purged with argon.Depending on the applied amine, the reaction took place for a certain period of time (most often around 24 h).Care must be taken in the reaction time to avoid amine polymerization.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 with an appropriate solvent system.

Synthesis of Triazolostilbenes 10-22 by Wittig Reaction
The apparatus, consisting of a three-necked flask, a dropping funnel, a chlorinecalcium tube, and a balloon filled with nitrogen, was blown with nitrogen for 15 min.A magnet was placed in the flask, the addition funnel was closed, and 30 or 40 mL of absolute ethanol was poured in (depending on the amount of starting reactants).A portion of absolute ethanol (10 or 20 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 for the next 72 h at room temperature.

Synthesis of Triazolostilbenes 10-22 by Wittig Reaction
The apparatus, consisting of a three-necked flask, a dropping fu calcium tube, and a balloon filled with nitrogen, was blown with nitro magnet was placed in the flask, the addition funnel was closed, and 30 or ethanol was poured in (depending on the amount of starting reactan absolute ethanol (10 or 20 mL) was poured into the flask, and the re triphenylphosphonium salt was added.Sodium previously weigh analytical balance with a precision of 0.0001 g was added to the rem absolute ethanol.After all the sodium had reacted in the ethanol with hydrogen, a little portion of NaOEt was added to the flask.The aldehy in ethanol and transferred to a flask, then the rest of the NaOEt from the dropwise.The flask was closed with a glass stopper, and the reaction m stir in a magnetic stirrer for the next 72 h at room temperature.mmol) with sodium in 10% excess (14.2 mg) and 40 mL of NaOEt.The obtained product was purified by column chromatography (ϕ = 3 cm; h = 17 cm; PE/E system (50%)).67.4 mg, 68% of the isolated mixture of isomers; yellow oil; Rf (PE/E (20%)) = 0.51.with sodium in 10% excess (7.6 mg) and 40 mL of NaOEt.The obtained product was purified by column chromatography (φ = 1.5 cm; h = 10 cm; system PE/E (70%)).46.0 mg, 33% of the isolated mixture of isomers; yellow oil; R f (PE/E (70%)) = 0.12.was purified by column chromatography (φ = 2 cm; h = 10 cm; PE/E system (80%)).62.6 mg, 64% of the isolated mixture of isomers; yellow oil; R f (PE/E (90%)) = 0.40.

Photochemical Synthesis of Naphthotriazoles 23-34
Corresponding 1,2,3-triazoles as mixtures of isomers (except pure isolated cis-18 and cis-19) were dissolved in 1 to 3 mL of toluene.The mixtures were then transferred to quartz tubes (they transmit light), and the rest of the toluene and a little iodine on the tip of a

Photochemical Synthesis of Naphthotriazoles 23-34
Corresponding 1,2,3-triazoles as mixtures of isomers (except pure isolated cis-18 and cis-19) were dissolved in 1 to 3 mL of toluene.The mixtures were then transferred to quartz tubes (they transmit light), and the rest of the toluene and a little iodine on the tip of a spatula (oxidizing agent) were added.The solutions (~10 −3 mol L −1 ) were illuminated for 1-3 h using 10 lamps with a wavelength of 313 nm in a Rayonet photoreactor.The obtained products 23-34 were purified by column chromatography.Only compound 20 did not react to give the corresponding photoproduct 35.The spectroscopic data for the photoproducts 23-34 are given below.

General Methylation Procedure for the Synthesis of 44-51
In a glass reaction tube equipped with a magnetic stirring bar, the corresponding thienobenzo-triazoles 36-43 (0.2 mmol, 1equiv), dry dichloromethane (p.a., 0.4 mL), and iodomethane (2 mmol, 10 equiv.)were added.Firstly, the reaction mixtures were purged with nitrogen, N 2 , and stirred at 60 • C for 24 h.After 24 h, the reaction mixtures were cooled to 0 • C and diluted with 5 mL of diethyl ether.After precipitation, the reaction tubes were centrifuged three times for 10 min at 3000 rpm.The solvent was decanted, and the precipitate was washed with diethyl ether five times.The crude powders were dried under a high vacuum to afford the pure triazolium salts 44-51.

General Methylation Procedure for the Synthesis of 44-51
In a glass reaction tube equipped with a magnetic stirring bar, the corresponding thienobenzo-triazoles 36-43 (0.2 mmol, 1equiv), dry dichloromethane (p.a., 0.4 mL), and iodomethane (2 mmol, 10 equiv.)were added.Firstly, the reaction mixtures were purged with nitrogen, N2, and stirred at 60 °C for 24 h.After 24 h, the reaction mixtures were cooled to 0 °C and diluted with 5 mL of diethyl ether.After precipitation, the reaction tubes were centrifuged three times for 10 min at 3000 rpm.The solvent was decanted, and the precipitate was washed with diethyl ether five times.The crude powders were dried under a high vacuum to afford the pure triazolium salts 44-51.

Figure 1 .
Figure 1.The basic skeleton of some previously synthesized thienobenzo-and naphtho-triazoles (structures A and B) and newly prepared uncharged and charged analogs in this work (structures C and D).

Figure 1 .
Figure 1.The basic skeleton of some previously synthesized thienobenzo-and naphtho-triazoles (structures A and B) and newly prepared uncharged and charged analogs in this work (structures C and D).

Figure 4 .
Figure 4. Dose-response curve for the inhibition AChE (a) and BChE (b) by via 46.

Figure 4 .
Figure 4. Dose-response curve for the inhibition AChE (a) and BChE (b) by via 46.

Figure 4 .
Figure 4. Dose-response curve for the inhibition AChE (a) and BChE (b) by via 46.

Figure 5 .
Figure 5. SAR of triazole fragment-based library concerning their cholinesterase-inhibition activity.

Figure 5 .
Figure 5. SAR of triazole fragment-based library concerning their cholinesterase-inhibition activity.

Figure 6 .
Figure 6.(a) The structure of the AChE's active site docked with 44 and (b) docked with 45.Distances given in angstroms; hydrogen atoms of the residues omitted for clarity.

Figure 6 .
Figure 6.(a) The structure of the AChE's active site docked with 44 and (b) docked with 45.Distances given in angstroms; hydrogen atoms of the residues omitted for clarity.

Figure 6 .
Figure 6.(a) The structure of the AChE's active site docked with 44 and (b) docked with 45.Distances given in angstroms; hydrogen atoms of the residues omitted for clarity.

Figure 7 .
Figure 7.The electrostatic potential maps of triazolium cations of compounds 44 and 45.Electrostatic surface potentials are colored blue and red for positive and negative charges, respectively, and green represents neutral regions.

Figure 7 .
Figure 7.The electrostatic potential maps of triazolium cations of compounds 44 and 45.Electrostatic surface potentials are colored blue and red for positive and negative charges, respectively, and green represents neutral regions.

Figure 8 .
Figure 8.(a) The structure of the BChE's active site docked with 44 and (b) docked with 45.Distances given in angstroms; hydrogen atoms of the residues omitted for clarity.

Figure 8 .
Figure 8.(a) The structure of the BChE's active site docked with 44 and (b) docked with 45.Distances given in angstroms; hydrogen atoms of the residues omitted for clarity.

Table 1 .
Spectrophotometric properties of studied compounds.

Table 1 .
Spectrophotometric properties of studied compounds.
1excitation at the longest absorption wavelength.
* Numbers given in parentheses represent maximal concentrations tested in µM.

Table 2 .
Inhibition of AChE and BChE and calculated IC50 values by compounds

Structure ICH M7 Class Derek Prediction Sarah Prediction Similarity to API
No Derek Alerts found P Class 3 No Derek Alerts found P Class 3 No Derek Alerts found P Class 5 No Derek Alerts found N Molecules 2024, 29, 1622

Structure ICH M7 Class Derek Prediction Sarah Prediction Similarity to API
No Derek Alerts found P Class 3 No Derek Alerts found P Class 3 No Derek Alerts found P Class 5 No Derek Alerts found N Molecules 2024, 29, 1622

Structure ICH M7 Class Derek Prediction Sarah Prediction Similarity to API O 23
Inconclusive No Derek Alerts found 24 Class 3 No Derek Alerts found 25 Class 3 No Derek Alerts found 26 Class 3 No Derek Alerts found 27 Class 3 No Derek Alerts found 28 Class 5 No Derek Alerts found 29 Class 3 No Derek Alerts found 30 Class 3 No Derek Alerts found Molecules 2024, 29, 1622

Structure ICH M7 Class Derek Prediction Sarah Prediction Similarity to API O 23
Inconclusive No Derek Alerts found 24 Class 3 No Derek Alerts found 25 Class 3 No Derek Alerts found 26 Class 3 No Derek Alerts found 27 Class 3 No Derek Alerts found 28 Class 5 No Derek Alerts found 29 Class 3 No Derek Alerts found Molecules 2024, 29, 1622

Structure ICH M7 Class Derek Prediction Sarah Prediction Similarity to API O 23
Inconclusive No Derek Alerts found 24 Class 3 No Derek Alerts found 25 Class 3 No Derek Alerts found 26 Class 3 No Derek Alerts found 27 Class 3 No Derek Alerts found 28 Class 5 No Derek Alerts found Molecules 2024, 29, 1622

ICH M7 Class Derek Prediction Sarah Prediction Similarity to API O 23
Inconclusive No Derek Alerts found 24 Class 3 No Derek Alerts found 25 Class 3 No Derek Alerts found 26 Class 3 No Derek Alerts found 27 Class 3 No Derek Alerts found Molecules 2024, 29, 1622

Structure ICH M7 Class Derek Prediction Sarah Prediction Similarity to API Overa
available); grey highlight-negative, orange highlight-positive, white-strongStructure ICH

M7 Class Derek Prediction Sarah Prediction Similarity to API O
and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.

M7 Class Derek Prediction Sarah Prediction Similarity to API O
and Sarah Nexus v.3.2.1) is the most commonly used software because it has mentary models; it is also very important that all predictions are again revie cology expert.