A Sustainable Synthetic Approach to Tacrine and Cholinesterase Inhibitors in Deep Eutectic Solvents under Aerobic Conditions

An enhanced, sustainable, and efficient method for synthesizing tacrine, achieving a 98% yield, has been developed by replacing volatile organic compounds with more eco-friendly solvents such as deep eutectic solvent (DESs). The optimized protocol scales easily to 3 g of substrate without yield loss and extends successfully to tacrine derivatives with reduced hepatotoxicity. Particularly notable is the synthesis of novel triazole-based derivatives, yielding 90–95%, by integrating an in situ preparation of aryl azides in DESs with N-propargyl-substituted tacrine derivatives. Quantitative metrics validate the green aspects of the reported drug development processes.


Introduction
In a world with dwindling petroleum resources, researchers are continuously nurturing the development of safer methodologies and waste prevention as a response to the growing demand for sustainable and environmentally friendly processes with a low carbon footprint.In particular, owing to the heavy impact of solvents on pollution and the organic waste produced (over 80%), green and sustainable solvents are progressively replacing volatile organic compounds (VOCs) in several chemical and extraction processes both in academia and industry [1,2].Deep eutectic solvents (DESs) are among the latest breakthroughs in the realm of green solvents due to their low volatility, nonflammability, and tunability properties.Additionally, the remarkably low toxicity of some of them (particularly those derived from bio-based components) aligns well with their suitability for use in the pharmaceutical field [3][4][5][6].They are usually obtained by the combination of safe, inexpensive, and nature-inspired components (Brønsted or Lewis acids and bases, anionic and/or cationic species) that, when mixed in a proper molar ratio and heated, give rise to eutectic mixtures with a eutectic point temperature lower to that of an ideal liquid mixture [7].
Nitrogen-containing heterocyclic compounds are a valuable source of therapeutic agents in medicinal chemistry.Thanks to their broad chemical structure, they can play the role of "spacers" and/or bioisosteres of various functional groups, often interacting effectively with receptors, enzymes, and biological targets [8].DESs have been widely used in organic synthesis [9], in particular for the preparation of N-containing heterocyclic scaffolds because of their dual solvent-catalyst role [10][11][12].Over the last decade, valuable sustainable methodologies have been introduced in the literature, both by our group and others, for the synthesis of pharmacologically active heterocycles with central nervous system activity or anti-inflammatory or antiproliferative properties (e.g., functionalized triazoles, pyrimidines, imidazoles, pyrazones, benzoxazines, tetrahydrofuran, and tetrahydropyran derivatives) [13][14][15][16][17][18] and of Active Pharmaceutical Ingredients (APIs) like Molecules 2024, 29, 1399 2 of 10 COX-1 inhibitors [19] and antihistamine drugs (e.g., thenfadyl and some analogs, such as dimethindene) [20,21].
Tacrine is a cholinesterase inhibitor that increases the synaptic levels of acetylcholine in the treatment of neurological disorders.Moreover, it was also found to act as an anticancer inhibitor of topoisomerases and DNA transcription [22].The preparation of tacrine derivatives with reduced toxicity (e.g., by modification of the heterocyclic ring or the chemical structure or by increasing or decreasing the ring size with new functional groups) is currently an active research area.Conventional approaches to synthesize tacrine (3a) often rely on intermolecular cyclodehydration reactions between anthranilonitrile (1a) (Scheme 1a) [23][24][25] or anthranilic acid [26] (1b) (Scheme 1b) [via 9-chlorotetrahydroacridine (3b)] and cyclohexanone (2a); however, these often use energy-intensive conditions with long reactions times (16-48 h), toxic VOCs (e.g., POCl 3 , toluene), and laborious work-up procedures based on VOCs (e.g., CHCl 3 , Et 2 O, MeOH), with 3a being often purified by column chromatography.The reaction between 1a and 2a has also been documented to occur in xylene with the aid of p-toluensulfonic acid (PTSA), yielding 48% under reflux for 3 h [27], or utilizing a solid catalyst combination of PTSA/silica gel under microwave irradiation, achieving a 70% yield [28].
in the treatment of neurological disorders.Moreover, it was also found to act as an anticancer inhibitor of topoisomerases and DNA transcription [22].The preparation of tacrine derivatives with reduced toxicity (e.g., by modification of the heterocyclic ring or the chemical structure or by increasing or decreasing the ring size with new functional groups) is currently an active research area.Conventional approaches to synthesize tacrine (3a) often rely on intermolecular cyclodehydration reactions between anthranilonitrile (1a) (Scheme 1a) [23][24][25] or anthranilic acid [26] (1b) (Scheme 1b) [via 9chlorotetrahydroacridine (3b)] and cyclohexanone (2a); however, these often use energyintensive conditions with long reactions times (16-48 h), toxic VOCs (e.g., POCl3, toluene), and laborious work-up procedures based on VOCs (e.g., CHCl3, Et2O, MeOH), with 3a being often purified by column chromatography.The reaction between 1a and 2a has also been documented to occur in xylene with the aid of p-toluensulfonic acid (PTSA), yielding 48% under reflux for 3 h [27], or utilizing a solid catalyst combination of PTSA/silica gel under microwave irradiation, achieving a 70% yield [28].
Herein, we report that the Friedländer annulation en route to quinoline skeletons of the type of 3a,c can straightforwardly be realized by reacting 2a with 2-aminobenzonitrile derivatives 1a,c, using DESs as privileged reactions media, with tacrine derivatives 3a,c being isolated in 95-98% yield, after short reaction times (3 h) at 120 °C, with no further purification required.In addition, the practicality of the proposed protocol was demonstrated by setting up a sustainable preparation of pharmacologically relevant Nsubstituted derivatives 4 (Scheme 1c) [29][30][31][32][33].Typical metrics applied at First and Second Pass, according to the Chem21 Metrics Toolkit [34], have also been calculated for the synthesis of 3a to demonstrate a significant advance in sustainability with respect to the state of the art.Herein, we report that the Friedländer annulation en route to quinoline skeletons of the type of 3a,c can straightforwardly be realized by reacting 2a with 2-aminobenzonitrile derivatives 1a,c, using DESs as privileged reactions media, with tacrine derivatives 3a,c being isolated in 95-98% yield, after short reaction times (3 h) at 120 • C, with no further purification required.In addition, the practicality of the proposed protocol was demonstrated by setting up a sustainable preparation of pharmacologically relevant N-substituted derivatives 4 (Scheme 1c) [29][30][31][32][33].Typical metrics applied at First and Second Pass, according to the Chem21 Metrics Toolkit [34], have also been calculated for the synthesis of 3a to demonstrate a significant advance in sustainability with respect to the state of the art.
In order to prove the applicability of the method, we also carried out the synthesis of 3a on a 3 g scale.The condensation reaction between 1a and 2a (25.4 mmol, 3 g; 25.4 mmol, 2.6 mL) in a ZnCl2/ChCl LADES (15 g) proceeded to uneventfully provide 3a in 98% yield (4.9 g) as a yellow solid.The product was isolated by filtration after the addition of a 10% solution of NaOH, with no requirement for chromatography (Table 1, entry 4) (see details in the Materials and Methods Section).Similarly, the condensation between 1c and 2a (1 mmol each), working either in ZnCl2/ChCl (1:1) or in FeCl3•6H2O/urea (2:1) (1 g) LADESs, furnished tacrine derivative 3c in a 98% yield after 3 h at 120 °C (Scheme 2).We next focused on primary amine functionalization, as this has been proven to lead to derivatives with fewer side effects, especially in the liver district, while opening up the way for the inclusion of pharmacophores to identify a suitable molecular platform for a multitargeting approach on muscarinic agonists and antagonists.N-propargyl derivatives, in particular, are potent acetylcholinesterase (AChE) and In order to prove the applicability of the method, we also carried out the synthesis of 3a on a 3 g scale.The condensation reaction between 1a and 2a (25.4 mmol, 3 g; 25.4 mmol, 2.6 mL) in a ZnCl 2 /ChCl LADES (15 g) proceeded to uneventfully provide 3a in 98% yield (4.9 g) as a yellow solid.The product was isolated by filtration after the addition of a 10% solution of NaOH, with no requirement for chromatography (Table 1, entry 4) (see details in the Materials and Methods Section).Similarly, the condensation between 1c and 2a (1 mmol each), working either in ZnCl 2 /ChCl (1:1) or in FeCl 3 •6H 2 O/urea (2:1) (1 g) LADESs, furnished tacrine derivative 3c in a 98% yield after 3 h at 120 • C (Scheme 2).

Entry
Molecules 2024, 29, x FOR PEER REVIEW 3 of 10
In order to prove the applicability of the method, we also carried out the synthesis of 3a on a 3 g scale.The condensation reaction between 1a and 2a (25.4 mmol, 3 g; 25.4 mmol, 2.6 mL) in a ZnCl2/ChCl LADES (15 g) proceeded to uneventfully provide 3a in 98% yield (4.9 g) as a yellow solid.The product was isolated by filtration after the addition of a 10% solution of NaOH, with no requirement for chromatography (Table 1, entry 4) (see details in the Materials and Methods Section).Similarly, the condensation between 1c and 2a (1 mmol each), working either in ZnCl2/ChCl (1:1) or in FeCl3•6H2O/urea (2:1) (1 g) LADESs, furnished tacrine derivative 3c in a 98% yield after 3 h at 120 °C (Scheme 2).We next focused on primary amine functionalization, as this has been proven to lead to derivatives with fewer side effects, especially in the liver district, while opening up the way for the inclusion of pharmacophores to identify a suitable molecular platform for a multitargeting approach on muscarinic agonists and antagonists.N-propargyl derivatives, in particular, are potent acetylcholinesterase (AChE) and We next focused on primary amine functionalization, as this has been proven to lead to derivatives with fewer side effects, especially in the liver district, while opening up the way for the inclusion of pharmacophores to identify a suitable molecular platform for a multitargeting approach on muscarinic agonists and antagonists.N-propargyl derivatives, in particular, are potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors [IC 50 values: 11.2-51.3(electric eel) and 77.6-83.5 (equine serum) nM, respectively) [37] with lower cytotoxicity and hepatotoxicity in vitro than tacrine itself [38].After screening several DES mixtures [e.g., ChCl/Gly (1:2), ChCl/urea (1:2), ChOAc/Gly (1:2)], other environmentally friendly solvents [e.g., 2-MeTHF and ciclopentyl methyl ether (CPME)] (see ESI), and various alkaline bases [e.g., KOH, K 2 CO 3 , Cs 2 CO 3 , NaH, LiOH) (see ESI), we found that CPME [39] allowed for the isolation of derivative 4a,c in 50-60% yield when treating 3a,c (1 mmol) with propargyl bromide (1.5 equiv.) in the presence of KOH (2 equiv.)as the base, and when stirring the resulting mixture at room temperature (RT) for 12 h (Scheme 3).Building on our recent achievements in the synthesis of functionalized triazoles by Cu-catalyzed cycloaddition reactions in DESs [40], we envisaged a truly green methodology to target pharmacologically relevant tacrine derivatives 7a-c, which are effective for the treatment of neuro-degenerative diseases.The inhibitory activity of chloro derivative 7c, in particular, is noteworthy, with IC50 values of 0.52 and 0.05 μM against AChE and BChE, respectively.Notably, while the removal of chloride resulted in a fourfold reduction in AChE inhibitory activity, compound 7b exhibited a striking increase in BChE inhibitory activity, surpassing that of 7c by 37-fold [41].To this end, benzyl azides 6a,b were first synthesized by reacting the corresponding benzyl bromides 5a,b (1 mmol) with NaN3 (2.3 equiv.) in ChCl/Gly (1:2) or ChCl/urea (1:2) (1 g) (98% yield by 1 H NMR analysis after 12 h at RT).The latter compound, without isolation, was then straightforwardly subjected to a cycloaddition reaction with N-propargyl-substituted tacrine derivatives 4a,c (0.5 mmol) in the presence of CuI (7 mol%), thereby smoothly providing derivatives 7a-c in 90-95% yield while working under aerobic conditions and vigorous stirring at RT for 24 h (Scheme 4).To better quantify the green credentials of the synthetic pathways developed for the synthesis of tacrine and its analogs, we have calculated the Sheldon's E-factor [42] and also have made use of some of the First and Second Pass CHEM21 Metrics Toolkit Building on our recent achievements in the synthesis of functionalized triazoles by Cu-catalyzed cycloaddition reactions in DESs [40], we envisaged a truly green methodology to target pharmacologically relevant tacrine derivatives 7a-c, which are effective for the treatment of neuro-degenerative diseases.The inhibitory activity of chloro derivative 7c, in particular, is noteworthy, with IC 50 values of 0.52 and 0.05 µM against AChE and BChE, respectively.Notably, while the removal of chloride resulted in a fourfold reduction in AChE inhibitory activity, compound 7b exhibited a striking increase in BChE inhibitory activity, surpassing that of 7c by 37-fold [41].To this end, benzyl azides 6a,b were first synthesized by reacting the corresponding benzyl bromides 5a,b (1 mmol) with NaN 3 (2.3 equiv.) in ChCl/Gly (1:2) or ChCl/urea (1:2) (1 g) (98% yield by 1 H NMR analysis after 12 h at RT).The latter compound, without isolation, was then straightforwardly subjected to a cycloaddition reaction with N-propargyl-substituted tacrine derivatives 4a,c (0.5 mmol) in the presence of CuI (7 mol%), thereby smoothly providing derivatives 7a-c in 90-95% yield while working under aerobic conditions and vigorous stirring at RT for 24 h (Scheme 4).Building on our recent achievements in the synthesis of functionalized triazoles by Cu-catalyzed cycloaddition reactions in DESs [40], we envisaged a truly green methodology to target pharmacologically relevant tacrine derivatives 7a-c, which are effective for the treatment of neuro-degenerative diseases.The inhibitory activity of chloro derivative 7c, in particular, is noteworthy, with IC50 values of 0.52 and 0.05 μM against AChE and BChE, respectively.Notably, while the removal of chloride resulted in a fourfold reduction in AChE inhibitory activity, compound 7b exhibited a striking increase in BChE inhibitory activity, surpassing that of 7c by 37-fold [41].To this end, benzyl azides 6a,b were first synthesized by reacting the corresponding benzyl bromides 5a,b (1 mmol) with NaN3 (2.3 equiv.) in ChCl/Gly (1:2) or ChCl/urea (1:2) (1 g) (98% yield by 1 H NMR analysis after 12 h at RT).The latter compound, without isolation, was then straightforwardly subjected to a cycloaddition reaction with N-propargyl-substituted tacrine derivatives 4a,c (0.5 mmol) in the presence of CuI (7 mol%), thereby smoothly providing derivatives 7a-c in 90-95% yield while working under aerobic conditions and vigorous stirring at RT for 24 h (Scheme 4).To better quantify the green credentials of the synthetic pathways developed for the synthesis of tacrine and its analogs, we have calculated the Sheldon's E-factor [42] and also have made use of some of the First and Second Pass CHEM21 Metrics Toolkit To better quantify the green credentials of the synthetic pathways developed for the synthesis of tacrine and its analogs, we have calculated the Sheldon's E-factor [42] and also have made use of some of the First and Second Pass CHEM21 Metrics Toolkit developed by Clark et al. [34], calculating atom economy (AE), reaction mass efficiency (RME), effective mass yield (EM), optimum efficiency (OE), renewable intensity (RI), renewable percentage (RP), and process mass intensity (PMI) metrics, with a breakdown of the latter for "chemicals" (reactants, reagents, and catalyst) (PMI rxn ) and work-up and reaction solvents (PMI WU ), and these values were compared with the corresponding ones related to the last available synthetic procedure developed in VOCs [24] (Table 2).The classical Friedländer condensation method for the synthesis of 3a yields an E-factor of 20 [24], whereas utilizing a ChCl-based DES reduces this value significantly to 7.An in-depth examination of the derived parameters reveals the markedly lower environmental impact of the DES-based synthetic route outlined in this study.This is particularly evident when comparing the following metrics: RME (89% in DES vs. 15% in VOCs), OE (97% in DES vs. 16% in VOCs), (36.3% in DES vs. 8.3% in VOCs), and RP (66.5% in DES vs. 25.2% in VOCs).It is also noteworthy that the sustainable synthetic pathway devised for tacrine synthesis eschews the use of VOCs during the isolation/purification step, which is characteristic of the traditional method employed by Dallanoce et al. [24].This is particularly evident when comparing the PMI WU values, which stand at 48.3 in VOCs versus 20.0 in DES.
3.4.Scale-Up Synthesis of Tacrine 3a in ZnCl 2 /ChCl (1:1 mol mol −1 ) LADES 2-Aminobenzonitrile (1a) (25.4 mmol, 3 g) and cyclohexanone (2a) (25.4 mmol, 2.6 mL) were added to ZnCl 2 /ChCl (1:1 mol mol −1 ) LADES (15 g).The reaction was kept at 120 • C for 3 h, then cooled to RT, and the volatiles evaporated under reduced pressure.A 10% solution of NaOH (7 mL) was added to the residue, and the mixture was stirred for an additional 3 h.After filtration, the cake was washed with water and then kept under stirring for 1 h with 25 mL of iPrOH.The solid was filtered and the solvent was evaporated under reduced pressure to give tacrine 3a as a yellow solid in 98% yield (4.9 g).

Representative Procedure for the Synthesis of N-Propargyl-Substituted Tacrine Derivatives 4a,c in CPME: Synthesis of 4a
To a solution of tacrine 3a (1 mmol, 198 mg), in CPME (2 mL), KOH (2 equiv., 112 mg) was added at 0 • C, and the mixture was stirred for 10 min.Then, propargyl bromide (1.5 eq., 114 µL) was added at RT, and the resulting mixture was stirred for 12 h.After the addition of 2 mL of water, the reaction mixture was extracted with CPME (3 × 1 mL).The collected organic phases were dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure.The crude was purified by column chromatography on silica gel (EtOAc as the eluent) to afford N-propargyl-substituted tacrine derivative 4a as a brown solid in 50% yield (118 mg).

Table 1 .
Synthesis of tacrine 3a in eutectic mixtures a .

Table 1 .
Synthesis of tacrine 3a in eutectic mixtures a .

Table 1 .
Synthesis of tacrine 3a in eutectic mixtures a .

Table 2 .
[24]titative metrics calculated for both classical and DES-based approaches for the synthesis of tacrine 3a a .Each synthesis of 3a was run on 25 mmol substrates.bClassicalsynthesis of 3a, as outlined in ref.[24]; Cy: cyclohexanone.c Process mass intensity (PMI) RXN : chemicals and reaction solvents.d Process mass intensity (PMI) WU : chemicals and reaction solvents, solvents, and reagents in workup.
a e Renewable sources: water, DES.f This value does not consider the amount of water solution used for work-up.g ZnCl 2 /ChCl (1:1 mol mol −1 ).