Deep Eutectic Solvents as Effective Reaction Media for the Synthesis of 2-Hydroxyphenylbenzimidazole-Based Scaffolds en Route to Donepezil-Like Compounds

An unsubstituted 2-hydroxyphenylbenzimidazole has recently been included as a scaffold in a series of hybrids (including the hit compound PZ1) based on the framework of the acetylcholinesterase (AChE) inhibitor Donepezil, which is a new promising multi-target ligand in Alzheimer’s disease (AD) treatment. Building upon these findings, we have now designed and completed the whole synthesis of PZ1 in the so-called deep eutectic solvents (DESs), which have emerged as an unconventional class of bio-renewable reaction media in green synthesis. Under optimized reaction conditions, the preparation of a series of 2-hydroxyphenylbenzimidazole-based nuclei has also been perfected in DESs, and comparison with other routes which employ toxic and volatile organic solvents (VOCs) provided. The functionalization of the aromatic ring can have implications on some important biological properties of the described derivatives and will be the subject of future studies of structure-activity relationships (SARs).


Introduction
Alzheimer's disease (AD) is recognized as a social and economic problem with an annual incidence of 34/1000 persons over 60 years old [1][2][3][4]. It is estimated that, in the absence of effective therapies, the number of people with dementia will reach more than 130 million worldwide by 2050 [5]. Although numerous clinical trials have been projected and realized [5][6][7], only five symptomatic drugs have been approved to date for the use as anti-AD agents at international level. Tacrine, which represented the first breakthrough in the AD therapy, has been discontinued from the market in several countries because of its severe adverse events [8].
Considering the specific pathogenesis of AD, a new generation of ligands has recently been explored as multifunctional molecules aimed at simultaneously acting on two or more disease features (multi-target directed ligands) so as to achieve synergistic or at least complementary therapeutic effects. The model based on the "one molecule/multiple targets" concept has led to the design of novel molecules frequently inspired by natural products or bio-active synthetic molecules [2]. The most explored strategy for multi-target anti-AD drugs is based on the "cholinergic hypothesis".

Results and Discussion
The role played by Zn and Cu cations in the Aβ deposition and stabilization has been a matter of debate in the literature in the last decade as well as the possibility that metal chelating agents can lead to the dissolution of Aβ and/or aggregation by preventing the interaction between the metal and the protein [2,11,17,21,[65][66][67][68][69]. According to the metal hypothesis of AD, it is important the inclusion of chelating moieties in the design of therapeutic/diagnostic agents [69]. Our groups recently focused on the synthesis and the biological evaluation of new Donepezil-like conjugates, and especially interesting were the results obtained from biological assays on PZ1 [19][20][21]. The synthesis of this new hit compound was designed using VOCs ( [19], Figure 1, red). Particularly disappointing from an environmental viewpoint were Steps 2, 4 and 5 in which it was made use of toxic and anhydrous CH 3 CN (step 2), N,N-dimethylacetamide (DMA) (Step 4), N,N-dimethylformamide (DMF) (Step 5), and of a carcinogenic reactant like hydrazine hydrate in Step 3. In addition, Step 4 also required up to Molecules 2020, 25, 574 3 of 14 12 h reaction time for completion. Thus, we decided to reshape the whole synthesis of PZ1 using DESs as environmentally responsible reaction media (Figure 1, blue).
The selective protection of the primary amine moiety of 1, performed by reacting 1 with phthalic acid anhydride at 180 • C for 5 h under solventless conditions, delivered adduct 2 in quantitative yield (>98% yield) [19,70] (Figure 1, Step 1). The benzylation of the secondary amine of the piperazine moiety of 2 en route to adduct 3 ( Figure 1, Step 2) was originally carried out in acetonitrile and in the presence of a couple of bases, used in excess (64% yield) ( Table 1, entry 1). An extensive screening of bases [KOH, t-BuOK, K 2 CO 3 , triethylamine (TEA)] in different hydrophilic [28,71,72] and hydrophobic [73] eutectic mixtures as solvents, at a temperature of 50 or 100 • C ( Table 1), revealed that TEA (2 equiv) either in ChCl/propylene glycol (PG) (1:3 mol mol −1 ) or in Bu 4 NCl/gly (1:4 mol mol −1 ) [72] were the best combinations as they delivered 3 in 64-68% yield (Table 1, entries 15,19).  The deprotection of the phthalimido moiety of 3 ( Figure 1, Step 3) was realized using MeNH2 (40% aq. solution) in place of a carcinogenic reactant such as hydrazine hydrate [19,74]. In this way, the N-benzylated adduct 4 was isolated in 95% yield. A similar yield (95%) was observed by reacting 2 with MeNH2 in a ChCl/gly (1:2) eutectic mixture with 40 w% water. On the other hand, by alternatively using the eutectic mixture ChCl/PG (1:3) + 40 w%, adduct 4 was obtained in 45% yield only. The synthesis of 2-hydroxyphenylbenzimidazole 7a, via a cyclodehydration reaction between 3,4-diaminobenzoic acid (5) and salicylaldehyde (6a) (Figure 1, Step 4), was also optimized in DESs using Na2S2O5 as the oxidant (Table 2). We screened three prototypical ChCl-based eutectic mixtures whose hydrogen bond component was basic (ChCl/urea; 1:2 mol mol -1 ), neutral (ChCl/gly; 1:2 mol mol −1 ) or acidic [(ChCl-L-lactic acid (LA); 1:2 mol mol -1 )]. All reactions were monitored through TLC analysis and stopped after complete consumption of the starting materials. As shown in Table 2, very good yields (up to 80%) were obtained in each case in short reaction times (30 min) at 100 °C ( Table  2, entries 1-3). As for the ChCl/gly eutectic mixture, the percentage yield of 7a could be increased to up to 84% running the reaction at 50 °C (Table 2, entry 4), whereas a temperature as low as 25 °C was detrimental even after 24 h reaction time (36% yield) (Table 2, entry 5). By changing the oxidant from Na2S2O5 to the commercially available urea-hydrogen peroxide or by alternatively running the cyclodehydration reaction under air in the absence of any additional oxidant reagent, the yield of 7a dropped down to 17% and 29% ( 1 H NMR analysis), respectively, the remaining being a complex   The deprotection of the phthalimido moiety of 3 ( Figure 1, Step 3) was realized using MeNH 2 (40% aq. solution) in place of a carcinogenic reactant such as hydrazine hydrate [19,74]. In this way, the N-benzylated adduct 4 was isolated in 95% yield. A similar yield (95%) was observed by reacting 2 with MeNH 2 in a ChCl/gly (1:2) eutectic mixture with 40 w% water. On the other hand, by alternatively using the eutectic mixture ChCl/PG (1:3) + 40 w%, adduct 4 was obtained in 45% yield only. The synthesis of 2-hydroxyphenylbenzimidazole 7a, via a cyclodehydration reaction between 3,4-diaminobenzoic acid (5) and salicylaldehyde (6a) (Figure 1, Step 4), was also optimized in DESs using Na 2 S 2 O 5 as the oxidant (Table 2). We screened three prototypical ChCl-based eutectic mixtures whose hydrogen bond component was basic (ChCl/urea; 1:2 mol mol −1 ), neutral (ChCl/gly; 1:2 mol mol −1 ) or acidic [(ChCl-l-lactic acid (LA); 1:2 mol mol −1 )]. All reactions were monitored through TLC analysis and stopped after complete consumption of the starting materials. As shown in Table 2, very good yields (up to 80%) were obtained in each case in short reaction times (30 min) at 100 • C ( Table 2, entries 1-3). As for the ChCl/gly eutectic mixture, the percentage yield of 7a could be increased to up to 84% running the reaction at 50 • C ( Table 2, entry 4), whereas a temperature as low as 25 • C was detrimental even after 24 h reaction time (36% yield) ( Table 2, entry 5). By changing the oxidant from Na 2 S 2 O 5 to the commercially available urea-hydrogen peroxide or by alternatively running the cyclodehydration reaction under air in the absence of any additional oxidant reagent, the yield of 7a dropped down to 17% and 29% ( 1 H NMR analysis), respectively, the remaining being a complex mixture of unidentified products (Table 2, entries 6,7). Compound 7a was found to precipitate directly from the above ChCl/gly mixture after dilution with water. Thus, it was isolated by simple filtration on a Büchner funnel and washing with a few drops of CH 2 Cl 2 . The same reaction, run in DMA at 100 • C, in the presence of Na 2 S 2 O 5 , provided 7a in 67% yield only after 12 h reaction time ( Table 2, entry 8). mixture of unidentified products (Table 2, entries 6,7). Compound 7a was found to precipitate directly from the above ChCl/gly mixture after dilution with water. Thus, it was isolated by simple filtration on a Büchner funnel and washing with a few drops of CH2Cl2. The same reaction, run in DMA at 100 °C, in the presence of Na2S2O5, provided 7a in 67% yield only after 12 h reaction time ( Table 2, entry 8). Possibly looking forward to developing and testing novel donepezil-hybrids, we decided to broaden already at this stage the scope of the aforementioned cyclodehydration reaction using ChCl/gly as a privileged reaction medium. The functionalization of the phenolic acid component may indeed contribute to modify the biological properties of the corresponding adducts, thereby tuning their chelating properties for the treatment of AD. In line with this strategy, Liang et al. recently synthesized a series of novel halogenated 8-hydroxyquinolines as derivatives of Clioquinol (CQ), which is a well-known prototypical metal-chelating drug [68]. CQ was studied up to phase II clinical trial with promising results, but it was later discontinued because of issues encountered during the development of the industrial production process [2,75]. In particular, it was noticed that the introduction in the CQ structure of powerful electron-withdrawing groups led to an improved metalchelating activity [68]. Another research group recently explored also the potentiality of these substituted nuclei as antioxidant agents [76]. To our delight, by reacting a variety of salicylaldehyde derivatives 6b-h, decorated with electron-donating and electron-withdrawing substituents, with 5, the desired 2-hydroxyphenylbenzimidazole derivatives 7b-h could be smoothly synthesized in 72-97% yield within 30 min reaction tim at 50 °C ( Figure 2). Of note, using DMA as the reaction medium, the above adducts have been reported to be prepared in 51-74% yield after 12 h reaction time at 100 °C [77]. The synthesis of 1,2-disubstituted or 2-substituted benzimidazoles has also been recently achieved using o-phenylenediamine (o-PDA) both as a component of the eutectic mixture ChCl/o-PDA (1:1 mol mol -1 ) and as a reagent in combination with different aldehydes [78]. Possibly looking forward to developing and testing novel donepezil-hybrids, we decided to broaden already at this stage the scope of the aforementioned cyclodehydration reaction using ChCl/gly as a privileged reaction medium. The functionalization of the phenolic acid component may indeed contribute to modify the biological properties of the corresponding adducts, thereby tuning their chelating properties for the treatment of AD. In line with this strategy, Liang et al. recently synthesized a series of novel halogenated 8-hydroxyquinolines as derivatives of Clioquinol (CQ), which is a well-known prototypical metal-chelating drug [68]. CQ was studied up to phase II clinical trial with promising results, but it was later discontinued because of issues encountered during the development of the industrial production process [2,75]. In particular, it was noticed that the introduction in the CQ structure of powerful electron-withdrawing groups led to an improved metal-chelating activity [68]. Another research group recently explored also the potentiality of these substituted nuclei as antioxidant agents [76]. To our delight, by reacting a variety of salicylaldehyde derivatives 6b-h, decorated with electron-donating and electron-withdrawing substituents, with 5, the desired 2-hydroxyphenylbenzimidazole derivatives 7b-h could be smoothly synthesized in 72-97% yield within 30 min reaction tim at 50 • C ( Figure 2). Of note, using DMA as the reaction medium, the above adducts have been reported to be prepared in 51-74% yield after 12 h reaction time at 100 • C [77]. The synthesis of 1,2-disubstituted or 2-substituted benzimidazoles has also been recently achieved using o-phenylenediamine (o-PDA) both as a component of the eutectic mixture ChCl/o-PDA (1:1 mol mol −1 ) and as a reagent in combination with different aldehydes [78].  Table 3). The preparation of PZ1 from 4 and 7a has only been performed to date in DMF, working at 25 °C for 60 h, with the product isolated in 21% yield (Table 3, entry 1). The use of ChCl/PG (1:3) as the solvent proved to be better with respect to other hydrophilic and hydrophobic eutectic mixtures as it provided PZ1 in an overall 30% yield at 60 °C after 60 h reaction time (Table 3, entries 2-8). The presence of both N,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) was also essential for the in situ formation of the amide moiety of 8 (Table 3, entries 9,10).   Table 3). The preparation of PZ1 from 4 and 7a has only been performed to date in DMF, working at 25 • C for 60 h, with the product isolated in 21% yield (Table 3, entry 1). The use of ChCl/PG (1:3) as the solvent proved to be better with respect to other hydrophilic and hydrophobic eutectic mixtures as it provided PZ1 in an overall 30% yield at 60 • C after 60 h reaction time (Table 3, entries 2-8). The presence of both N,N -dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) was also essential for the in situ formation of the amide moiety of 8 (Table 3, entries 9,10).   Table 3). The preparation of PZ1 from 4 and 7a has only been performed to date in DMF, working at 25 °C for 60 h, with the product isolated in 21% yield (Table 3, entry 1). The use of ChCl/PG (1:3) as the solvent proved to be better with respect to other hydrophilic and hydrophobic eutectic mixtures as it provided PZ1 in an overall 30% yield at 60 °C after 60 h reaction time (Table 3, entries 2-8). The presence of both N,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) was also essential for the in situ formation of the amide moiety of 8 (Table 3, entries 9,10).

H NMR and 13 C NMR spectra were recorded on a Bruker 600 MHz spectrometer and chemical
shifts are reported in parts per million (δ). FT-IR spectra were recorded on a Perkin-Elmer 681 spectrometer. GC analyses were performed on a HP 6890 model, Series II by using a HP1 column (methyl siloxane; 30 m, 0.32 mm, 0.25 µm film thickness). Analytical thin-layer chromatography (TLC) was carried out on pre-coated 0.25 mm thick plates of Kieselgel 60 F 254 ; visualization was accomplished by UV light (254 nm) or by spraying a solution of 5 % (w/v) ammonium molybdate and 0.2 % (w/v) cerium(III) sulfate in 100 mL 17.6 % (w/v) aq. sulfuric acid and heating to 473 K until blue spots appeared. Chromatography was conducted by using silica gel 60 with a particle size distribution 40-63 µm and 230-400 ASTM. GC-MS analyses were performed on HP 5995C model. High-resolution mass spectrometry (HRMS) analyses were performed using a Bruker microTOF QII mass spectrometer equipped with an electrospray ion source (ESI). Reagents and solvents, unless otherwise specified, were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and used without any further purification.

Synthesis and Characterization Data of 2-(4-benzyl-1-piperazinyl)ethanamine (4)
Compound 3 (1.0 mmol) was dissolved in an aq. solution of MeNH 2 (40% w/w, 3.0 mL) under magnetic stirring for 24 h at room temperature. After this time, an aq. solution of NaOH (20% w/w, 3.0 mL) was added. After 2 h, NaCl (0.3 g) was added, and the resulting solution was extracted with AcOEt (3 × 10 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to give 4 as a yellow oil (95% yield). 1 Table 2, entry 8: to a solution of salicylaldehyde (0.5 mmol) in N,N-dimethylacetamide (2 mL), 3,4-diaminobenzoic acid (0.5 mmol) and Na 2 S 2 O 5 (0.7 mmol) were progressively added under magnetic stirring. The resulting mixture was heated at 100 • C for 12 h, and then cooled to room temperature. The mixture was finally diluted with AcOEt (10 mL), washed with brine (3 × 10 mL), dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to give 7a as a pale brown solid (67% yield). Table 2, entry 4: 3,4-diaminobenzoic acid (0.5 mmol), salicylaldehyde (0.5 mmol) and Na 2 S 2 O 5 (0.7 mmol) were progressively dissolved in a ChCl/gly eutectic mixture (1.0 g), and the resulting mixture was warmed at 50 • C for 30 min. After this time, the reaction mixture was cooled to room temperature and 10 mL of H 2 O were added. This caused the precipitation of 7a as a pale brown solid, which was isolated by filtration on a Büchner funnel and washing with a few drops of CH 2 Cl 2 (84% yield).

Synthesis and Characterization Data of 2-(2-hydroxyphenyl)-1H-benzo[d]imidazole-5-carboxylic Acid Derivatives (7b-h)
Compounds 7b-h were synthesized in the ChCl/gly eutectic mixture according to the procedure described for 7a in Section 3.5.    Table 3, entry 1: a mixture of 4 (0.5 mmol), 7a (0.5 mmol), N-hydroxysuccinimide (0.5 mmol) and N,N -dicyclohexylcarbodiimide (0.5 mmol) in anhydrous DMF (3 mL) was stirred at room temperature for 60 h under a nitrogen atmosphere. After this time, the resulting mixture was filtered on a Büchner funnel, diluted with AcOEt (10 mL), and washed with brine (10 mL). The organic phase was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was purified by flash-chromatography (silica gel; eluent: CH 2 Cl 2 /MeOH 98:2:) to give 8 as a yellow solid (21% yield). Table 3, entry 3: compounds 4 (0.5 mmol), 7a (0.5 mmol), N-hydroxysuccinimide (0.5 mmol) and N,N -dicyclohexylcarbodiimide (0.5 mmol) were progressively dissolved in a ChCl/PG eutectic mixture (1.0 g) under magnetic stirring, and the resulting mixture was then heated at 60 • C for 60 h. After this time, the reaction mixture was cooled to room temperature and 5 mL of H 2 O were added. The resulting aqueous suspension was then extracted with AcOEt (3 × 10 mL). The combined organic phases were washed with brine (10 mL), dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. The crude product was purified by flash-chromatography (silica gel; eluent: AcOEt/MeOH 98:2) to give 8 as a yellow solid (30% yield).

Conclusions
In this paper, we have described an efficient condensation-mediated synthesis of 2-hydroxyphenylbenzimidazole derivatives and the whole synthesis of the hit, Donepezil-like compound PZ1, in selected DESs as environmentally responsible, safe, and nonconventional reaction media. Compared to VOCs, the synthesis of 2-hydroxyphenylbenzimidazoles in ChCl/gly takes place in better yields, shorter reaction time (30 min vs. 12 h) and under milder conditions (50 vs. 100 • C), and provides a way of easy functionalization of the phenolic moiety which may have implication on some important biological properties of these nuclei, such as the effective chelation of heavy metals. Moreover, these adducts were found to precipitate from the eutectic mixture after adding water during the work-up procedure, and thus they could easily be isolated by simple filtration. Further investigation into the application of these ligands in the therapy of Alzheimer's disease are underway in our laboratory and results will be reported in due course.