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

Article abstract of DOI:10.3390/molecules25030574

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).

Full text of DOI:10.3390/molecules25030574

Article
Deep Eutectic Solvents as Effective Reaction Media
for the Synthesis of 2‐Hydroxyphenylbenzimidazole‐
based Scaffolds en Route to Donepezil‐Like
Compounds
1,
1
1
1
Luca Piemontese *, Roberta Sergio , Federica Rinaldo , Leonardo Brunetti , Filippo M. Perna
1,2
3
1,2,
, M. Amélia Santos , Vito Capriati
*
1
Dipartimento di Farmacia–Scienze del Farmaco, Università degli Studi di Bari «Aldo Moro», Via E.
Orabona 4, I‐70125 Bari, Italy; r.sergio2@studenti.uniba.it (R.S.); f.rinaldo@studenti.uniba.it (F.R.);
l.brunetti2@studenti.uniba.it (L.B.); filippo.perna@uniba.it (F.M.P.)
Consorzio C.I.N.M.P.I.S., Via E. Orabona 4, I‐70125 Bari, Italy
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1,
1049‐001 Lisboa, Portugal; masantos@ist.utl.pt
2
3
* Correspondence: luca.piemontese@uniba.it (L.P.); vito.capriati@uniba.it (V.C.); Tel.: +39 080 5442174 (V.C.)
Academic editor: Paula Sério Branco
Received: 19 December 2019; Accepted: 27 January 2020; Published: 28 January 2020
Abstract: 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).
Keywords: deep eutectic solvents; 2‐hydroxyphenylbenzimidazole; Alzheimer’s disease
1. 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 [1–4]. 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 [5–7], 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
Molecules 2020, 25, 574; doi:10.3390/molecules25030574
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[2]. The most explored strategy for multi‐target anti‐AD drugs is based on the “cholinergic
hypothesis”. This involves the repositioning of drugs already used in therapy such as tacrine,
donepezil, memantine, rivastigmine [2,9–11] as starting molecules to hit other AD targets [12].
According to the so‐called “metal hypothesis”, several metal‐chelating moieties have been
incorporated in these structures [5,7,8]. Indeed, the biometal dyshomeostasis (copper and zinc cations
but also iron and aluminum) in AD is known to be involved in Aβ aggregation [13–15]. Moreover,
the redox activity of these ions may lead to the formation of reactive oxygen species (ROS), which are
known to play an important role in chronic inflammation eventually responsible for the oxidative
stress of neuronal cells [2,6,16–18].
In the last years, our research groups have synthesized and assayed new multi‐functional
2+
2+
ligands with chelating abilities towards Cu and Zn , potentially useful in the AD treatment based
on the framework of the acetylcholinesterase (AChE) inhibitor Donepezil and 2‐
hydroxyphenylbenzimidazole [19–21]. Benzimidazole‐based nuclei have been the subject of intense
investigation in recent years as they proved to be important scaffolds for the preparation of other
bioactive compounds [22–25]. For example, they have been included in several drugs and drug‐like
candidates such as the antihypertensive drug Telmisartan and the antiviral drug Maribavir, in several
anti‐inflammatory and antiulcer agents like the blockbuster drug Omeprazole [22,23], and in various
β‐secretase (BACE1) inhibitors and other AD targets [25–27].
Because of stringent environmental legislation to address the climate crisis, urgent actions are
needed to be taken in the chemical production, in particular, to progressively replace extensively
used conventional and often hazardous volatile organic compounds (VOCs), which are known to
contribute to over 80% of the organic waste produced, in favor of safer and more environmentally
responsible solvents. In this context, the so‐called deep eutectic solvents (DESs) have emerged as a
novel promising class of green solvents as they are non‐flammable, highly thermally stable, with
practically no vapor pressure, and thus low volatility [28]. They are combination of two or three safe
and cheap components (Lewis or Bronsted acids and bases, which can contain a variety of anionic
and/or cationic species) that form, through hydrogen bond formation, a eutectic liquid mixture at a
temperature far below than that of either of the individual components. Typical DES components
come from renewable sources [e.g., choline chloride (ChCl), glycerol (gly), urea, natural carboxylic
acids, amino acids, polyalcohols]. Therefore, their biodegradability is high and their toxicity is non‐
existent or very low. In addition, they can be easily prepared and exhibit tunable physicochemical
properties [28]. Because of their ability to act also as catalysts and reagents [29,30], DESs have been
primarily investigated in extraction and separation processes [31–34], in material sciences [35], for
metal electrodeposition [36], and for the synthesis of heterocycles [37]. Emerging and hot fields of
applications are represented by organometallics [38–40], metal‐ [41–48], bio‐ [49–53], and organo‐
catalysis [54–57], electrochemistry [58], photosynthesis [59] and energy technology [60,61]. Building
upon our interests in the synthesis of drugs and heterocycles using eco‐friendly reaction media like
DESs [43–47,55,62,63] and water [64], herein we report the sustainable preparation of several 2‐
hydroxyphenylbenzimidazole derivatives and the whole synthesis of PZ1 [19] in selected eutectic
mixtures.
2. 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 [2,11,17,21,65–69]. 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 [19–21]. 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
CH3CN (step 2), N,N‐dimethylacetamide (DMA) (Step 4), N,N‐dimethylformamide (DMF) (Step 5),

Molecules 2020, 25, 574
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and of a carcinogenic reactant like hydrazine hydrate in Step 3. In addition, Step 4 also required up
to 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).
Figure 1. Classical [red, 19] and new green (blue) procedure for the synthesis of PZ1.
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 , triethylamine (TEA)] in different hydrophilic [28,71,72] and
3
hydrophobic [73] eutectic mixtures as solvents, at a temperature of 50 or 100 °C (Table 1), revealed
−1
that TEA (2 equiv) either in ChCl/propylene glycol (PG) (1:3 mol mol ) or in Bu
4
NCl/gly (1:4 mol
−1
mol ) [72] were the best combinations as they delivered 3 in 64–68% yield (Table 1, entries 15,19).

Molecules 2020, 25, 574
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Table 1. Optimization of the benzylation reaction for the synthesis of 3.a
Br
O
O
N
NH
Base-1 and/or Base-2
T, t, solvent
N
N
N
N
O
2
3
O
b
Entry
Base‐1 (Equiv)
TEA (1.4)
KOH (1.1)
Base‐2 (Equiv)
K2CO3 (6.6)
BnBr (Equiv)
T (°C)
50
50
50
50
T (h)
3
24
24
24
24
24
Solvent
CH3CN
3, Yield (%)
c
1
2
3
4
5
6
2.3
1.0
1.0
1.0
1.0
1.0
64
NR
NR
24
d
‐
‐
‐
‐
‐
ChCl/gly
ChCl/urea
ChCl/gly
ChCl/urea
ChCl/PG
D‐
fructose/ChCl
D‐fructose
/urea
d
KOH (1.1)
t‐BuOK (1.1)
t‐BuOK (1.1)
t‐BuOK (1.1)
50
50
10
20
7
8
t‐BuOK (1.1)
t‐BuOK (1.1)
‐
‐
1.0
1.0
100
100
24
24
8
8
9
t‐BuOK (1.1)
t‐BuOK (1.1)
TEA (1.4)
K2CO3 (2.0)
TEA (2.0)
TEA (2.0)
TEA (2.0)
‐
‐
1.0
1.0
2.3
1.0
1.0
1.0
2.0
2.0
50
50
50
50
50
50
50
50
24
24
24
24
24
24
24
24
ChCl/gly
ChCl/urea
ChCl/gly
ChCl/gly
ChCl/gly
ChCl/PG
ChCl/PG
ChCl/gly
DL‐
24
10
NR
NR
26
10
11
12
13
14
15
16
d
d
K2CO3 (6.6)
‐
‐
‐
‐
‐
44
c
64
61
c
TEA (2.0)
d
17
TEA (2.0)
‐
1.0
50
24
NR
menthol/LA
Bu4NBr/gly
Bu4NCl/gly
c
18
19
a
TEA (2.0)
TEA (2.0)
‐
‐
2.0
2.0
50
50
24
24
44
68
c
Reaction conditions in deep eutectic solvent (DES): 1.0 g DES per 0.5 mmol of 2; DES:
ChCl/propylene glycol (PG) (1:3, mol mol−1); ChCl/gly (1:2 mol mol−1); ChCl/urea (1:2 mol mol−1); D‐
fructose/ChCl (2:1 mol mol−1); D‐fructose/urea (3:2 w/w); DL‐menthol/L‐lactic acid (LA) (1:2 mol mol−1);
Bu4NBr/gly (1:4 mol mol−1); Bu4NCl/gly (1:4 mol mol−1). b Calculated via 1H‐NMR analysis of the crude
reaction mixture using an internal standard technique (NMR internal standard: dibromomethane). c
The yields reported are for isolated products. d NR = no reaction.
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
–1
whose hydrogen bond component was basic (ChCl/urea; 1:2 mol mol ), neutral (ChCl/gly; 1:2 mol
−
1
–1
mol ) or acidic [(ChCl‐L‐lactic acid (LA); 1:2 mol mol )]. 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
1
dropped down to 17% and 29% ( H NMR analysis), respectively, the remaining being a complex

Molecules 2020, 25, 574
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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
(Table 2, entry 8).
2
S2
O
5
, provided 7a in 67% yield only after 12 h reaction time
Table 2. Optimization of the synthesis of 2‐hydroxyphenylbenzimidazole 7a. a
b
Entry T (°C) T (h) Oxidant
Solvent
ChCl/urea
ChCl/gly
ChCl/LA
ChCl/gly
ChCl/gly
ChCl/gly
ChCl/gly
DMA
7a, Yield (%)
1
2
3
4
5
6
7
8
100
100
100
50
25
50
0.5
0.5
0.5
0.5
24
0.5
0.5
12
Na
Na
Na
Na
Na
2
2
2
2
2
S
S
S
S
S
2
2
2
2
2
O
O
O
O
O
5
66
80
76
84
36
5
5
5
5
c
urea‐H
2
O
2
17
d
c
50
100
‐
Na
29
2S
2
O
5
67
a Reaction conditions in DES: 1.0 g DES per 0.5 mmol of 5, 0.5 mmol of 6a and 0.7 mmol of oxidant;
DES: ChCl/gly (1:2 mol mol−1); ChCl/urea (1:2 mol mol−1); ChCl/LA (1:2 mol mol−1); The yields
reported are for isolated products. Calculated via H‐NMR analysis of the crude reaction mixture
using an internal standard technique (NMR internal standard: dibromomethane). d Under air.
b
c
1
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‐
–1
PDA (1:1 mol mol ) and as a reagent in combination with different aldehydes [78].

Molecules 2020, 25, 574
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H
N
NH2
NH2
Na2S2O5
O
R
N
HOOC
HOOC
ChCl/Gly (1:2)
30 min, 50 °C
HO
R
HO
7a–h
5
6a–h
F
Br
H
N
H
H
N
N
N
HOOC
N
N
HOOC
HOOC
HO
HO
OCH3
HO
7a: 84%
7b: 72%
7c: 97%
Cl
NO2
H
N
H
N
H
N
N
N
N
HOOC
HOOC
HOOC
HO
HO
HO
7d: 72%
7e: 78%
7f: 67%
H
H
N
N
OCH3
OH
N
N
HOOC
HOOC
HO
HO
7g: 79%
7h: 94%
Figure 2. Scope of the cyclodehydration reaction for the synthesis of 2‐hydroxyphenylbenzimidazole
derivatives 7. Yields refer to isolated products.
Finally, the one‐pot two‐step synthesis of PZ1 (8) in DESs (Figure 1, Step 5) was investigated by
reacting the N‐benzylated adduct 4 (Figure 1, Step 3) with 2‐hydroxyphenylbenzimidazole derivative
7a (Figure 1, Step 4) (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. Optimization of the synthesis of PZ1 (8).a
Reagent 1
Reagent 2
H
N
H
N
N
N
+
N
N
solvent
time, 60 h
N
N
NH2
N
H
HOOC
HO
HO
PZ1 (8)
O
4
7a
b
Entry Reagent 1 (Equiv) Reagent 2 (Equiv) T (°C)
Solvent
DMF
ChCl/gly
ChCl/PG
ChCl/urea
menthol/LA
D‐fructose/urea
Bu4NBr/gly
Bu4NCl/gly
ChCl/PG
8, Yield (%)
1
2
NHS (1)
NHS (1)
NHS (1)
NHS (1)
NHS (1)
NHS (1)
NHS (1)
NHS (1)
NHS (1)
‐
DCC (1)
DCC (1)
DCC (1)
DCC (1)
DCC (1)
DCC (1)
DCC (1)
DCC (1)
‐
25
60
60
60
60
60
60
60
60
60
21
16
30
13
NR
NR
3
4
5
6
7
8
c
c
d
7
19
<5
d
d
9
10
c
DCC (1)
ChCl/PG
NR
a Reaction conditions in DES: 1.0 g DES per 0.5 mmol of 4; DES: ChCl/gly (1:2 mol mol−1); ChCl/urea
(1:2 mol mol−1); ChCl/PG (1:3 mol mol−1); D‐fructose/urea (3:2 w/w); DL‐menthol/LA (1:2 mol mol−1);
Bu4NBr/gly (1:4 mol mol−1); Bu4NCl/gly (1:4 mol mol−1). b The yields reported are for isolated products.
c
d
1
No reaction. Calculated via H‐NMR analysis of the crude reaction mixture using an internal
standard technique (NMR internal standard: dibromomethane).

Molecules 2020, 25, 574
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3. Materials and Methods
3.1. General Methods
1
13
H NMR and 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 F254; 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.
3.2. Preparation of Deep Eutectic Solvents (DESs)
−1
DESs [choline chloride (ChCl)/propylene glycol (PG) (1:3 mol mol ); ChCl/L‐lactic acid (LA) (1:2
−1
−1
−1
mol mol ); D‐fructose/urea (3:2 w/w); D‐fructose/ChCl (2:1 mol mol ); ChCl/urea (1:2 mol mol );
−1
−1
−1
ChCl/glycerol (gly) (1:2 mol mol ; DL‐menthol/LA (1:2 mol mol ); Bu4NBr/gly (1:4 mol mol );
Bu4NCl/gly (1:4 mol mol )] were prepared by heating under stirring at 60–80 °C for 10–30 min the
–1
corresponding individual components until a clear solution was obtained.
3.3. Synthesis and Characterization Data of 2‐[2‐(4‐benzylpiperazin‐1‐yl)ethyl]isoindoline‐1,3‐dione (2)
Phthalic acid anhydride (0.5 mmol) and 1‐(2‐aminoethyl)piperazine 1 (0.5 mmol) were heated at
180 °C for 5 h to give 2‐[2‐(piperazin‐1‐yl)ethyl]isoindoline‐1,3‐dione (2) as a dark brown solid in
1
>98% yield ( H NMR analysis) [19,70].
Table 1, entry 1: the resulting dark brown solid (2), (0.5 mmol) was mixed with K2CO3 (3.3 mmol),
triethylamine (TEA) (1.0 mmol) and benzyl bromide (1.15 mmol) and refluxed in acetonitrile (2 mL)
for 3 h at 50 °C. Then, the mixture was cooled to room temperature and 5 mL of H2O were added.
The resulting aqueous suspension was then extracted with AcOEt (3 × 10 mL). The combined organic
phase were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude
product was purified by flash‐chromatography (silica gel; eluent: CH2Cl2/MeOH/TEA 98:2:0.1) to
give 3 as a yellow solid (64% yield).
Table 1, entry 19: the resulting dark brown solid (2), (0.5 mmol), TEA (1.0 mmol) and benzyl
bromide (1.0 mmol) were dissolved in the Bu4NCl/gly eutectic mixture (1.0 g) under magnetic
stirring, and the resulting mixture was then heated at 50 °C for 24 h. After this time, the reaction
mixture was cooled to room temperature and 5 mL of H2O 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 Na2SO4 and concentrated under reduced pressure. The
crude product was purified by flash‐chromatography (silica gel; eluent: CH2Cl2/MeOH/TEA 98:2:0.1)
to give 3 as a yellow solid (68% yield).
1
2‐[2‐(4‐benzylpiperazin‐1‐yl)ethyl]isoindoline‐1,3‐dione (3). H NMR (400 MHz, CDCl3), δ (ppm):
2.39–2.52 (m, 8 H, piperazine), 2.60 (t, 2 H, J = 6.7 Hz, phthalimide‐CH2CH2N), 3.44 (s, 2 H, NCH2Ph),
3.78 (t, 2 H, J = 6.7 Hz, phthalimide–CH2CH2N), 7.19–7.27 (m, 5 H, aromatics, Ph–CH2), 7.67–7.69 and
13
7.80–7.82 (m, 4 H, phthalimide). C NMR (150 MHz, CDCl3) δ (ppm): 35.3, 53.0, 53.1, 55.7, 63.0, 123.2,
+
127.0, 128.2, 129.2, 132.2, 133.8, 138.1, 168.3. GC‐MS (m/z): 349 (M , 7), 189 (100), 91 (47). HRMS [M +
+
H] : calculated 350.1863; found 350.1858.

Molecules 2020, 25, 574
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3.4. Synthesis and Characterization Data of 2‐(4‐benzyl‐1‐piperazinyl)ethanamine (4)
Compound 3 (1.0 mmol) was dissolved in an aq. solution of MeNH2 (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 Na2SO4 and concentrated under reduced pressure to give 4 as a yellow oil (95% yield).
1
H NMR (400 MHz, CDCl3), δ (ppm): 2.35–2.43 (m, 10 H, piperazine, NH2–CH2CH2N), 2.73 (t, 2
13
H, J = 6.1 Hz, NH2–CH2CH2N), 3.46 (s, 2 H, NCH2Ph), 7.18–7.27 (m, 5 H, aromatics, Ph–CH2). C NMR
(150 MHz, CDCl3) δ (ppm): 38.7, 53.1, 53.2, 61.1, 63.1, 127.0, 128.2, 129.2, 138.0. GC‐MS (m/z): 219 (M ,
1), 189 (100), 91 (94). HRMS [M + H] : calculated 220.1808; found 220.1809.
+
+
3.5. Synthesis and Characterization Data of 2‐(2‐hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic Acid
(7a)
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 Na2S2O5 (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 Na2SO4 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 Na2S2O5
(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 H2O 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 CH2Cl2 (84%
yield).
1
2‐(2‐Hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7a) H NMR (400 MHz, DMSO‐d6) δ
(ppm): 7.02–7.08 (m, 2 H), 7.41 (t, J = 7.4 Hz, 1 H), 7.73 (d, J = 7.7 Hz, 1 H), 7.91 (d, J = 7.9 Hz, 1 H), 8.11
13
(d, J = 8.1 Hz, 1 H), 8.24 (s, 1 H). C NMR (100 MHz, DMSO‐d6) δ (ppm): 168.1, 158.5, 132.7, 127.2,
−
125.7, 124.55, 119.7, 117.7, 112.9. ESI‐MS (m/z): 253 (M − 1), 255 (M + 1). HRMS [(M − H] : calculated
253.0619; found 253.0637.
3.6. 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.
2‐(5‐Bromo‐2‐hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7b). White solid, m.p. > 300
1
°C, 72% yield. H NMR (600 MHz, DMSO‐d6) δ (ppm): 7.05 (d, J = 8.7 Hz, 1 H, BIM‐H‐4), 7.55 (d, J =
8.7 Hz, 1 H, BIM‐H‐5), 7.74 (d, J = 8.3 Hz, 1 H, BIM‐H‐2), 7.91 (d, J = 8.3 Hz, 1 H, BIM‐H‐3), 8.24 (s, 1
13
H, BIM‐H‐7), 8.30 (s, 1 H, BIM‐H‐1), 12.95 (broad s, 1 H, COOH). C NMR (150 MHz, DMSO‐d6) δ
(ppm): 110.8, 114.9, 115.0, 120.0, 124.8, 124.9, 126.0, 129.5, 135.1, 152.4, 152.5, 157.5, 168.0. HRMS [M −
−
H] : calculated 330.9724; found 330.9722.
2‐(5‐Fluoro‐2‐hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7c). White solid, m.p. 250 °C,
1
97% yield. H NMR (600 MHz, DMSO‐d6) δ (ppm): 7.04–7.06 (m, 1 H), 7.22–7.25 (m, 1 H), 7.70–7.72
13
(m, 1 H), 7.88–7.90 (m, 2 H), 7.98–8.01 (m, 1 H), 8.25 (broad s, 1 H), 12.78 (broad s, 1 H). C NMR (150
2
3
3
MHz, DMSO‐d6) δ (ppm): 112.8 (d, JC − F = 25.0 Hz), 113.3 (d, JC − F = 8.1 Hz), 119.0 (d, JC − F = 7.8 Hz),
2
2
119.4 ( JC − F = 23.3 Hz), 124.6, 124.7, 125.9, 153.0, 153.2, 154.6, 155.5 (d, JC − F = 234.5 Hz), 168.1. HRMS
[M − H] : calculated 271.0524; found 271.0525.
−
2‐(5‐Chloro‐2‐hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7d). White solid, m.p. > 300
1
°C, 72% yield. H NMR (300 MHz, DMSO‐d6), δ (ppm): 7.10 (d, 1 H, J = 6.0 Hz), 7.44 (d, 1 H, J = 6.0

Molecules 2020, 25, 574
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Hz), 7.73–7.75 (m, 1 H), 7.90 (d, 1 H, J = 6.0 Hz), 8.19 (s, 1 H), 8.24 (broad s, 1 H), 12.86 (broad s, 1 H,
13
COOH). C NMR (150 MHz, DMSO‐d6) δ (ppm): 119.6, 123.4, 124.8, 125.9, 126.5, 132.2, 152.7, 157.1,
−
168.0. HRMS [M − H] : calculated 287.0229; found 287.0228.
2‐(5‐Nitro‐2‐hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7e). White solid, m.p. > 300 °C,
1
78% yield. H NMR (600 MHz, DMSO‐d6) δ (ppm): 7.06–7.11 (m, 1 H),7.25–7.32 (m, 1 H), 7.73–7.76 (m,
13
1 H), 7.89–7.96 (m, 2 H), 8.24 (broad s, 1 H), 12.78 (broad s, 1 H). C NMR (150 MHz, DMSO‐d6) δ
−
(ppm): 113.3, 114.4, 118.9, 122.1, 123.4, 126.9, 127.5, 128.1, 129.1, 129.5, 152.7, 169.2. HRMS [M − H] :
calculated 298.0469; found 298.0468.
2‐(4‐Methoxy‐2‐hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7f). White solid, m.p. > 300
1
°C, 67% yield. H NMR (600 MHz, DMSO‐d6), δ (ppm): 3.79 (s, 3 H), 6.59–6.63 (m, 2 H) 7.66 (d, J = 9.0
13
Hz, 1 H), 7.86 (d, J = 9.0 Hz, 1 H), 8.10 (d, J = 9.0 Hz, 1 H), 8.18 (s, 1 H), 12.96 (broad s, 1 H). C NMR
(150 MHz, DMSO‐d6) δ (ppm): 55.9, 101.9, 105.8, 107.2, 124.4, 125.3, 128.6, 154.4, 160.4, 163.1, 168.1.
−
HRMS [M − H] : calculated 283.0724; found 283.0728.
2‐(5‐Methoxy‐2‐hydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7g). White solid, m.p. > 300
1
°C, 79% yield. H NMR (600 MHz, DMSO‐d6), δ (ppm): 3.33 (s, 3 H), 6.64 (d, J = 9.0 Hz, 1 H), 6.74 (d, J
13
= 9.0 Hz, 1 H), 7.19 (s, 1 H), 7.90–7.92 (m, 1 H), 8.09–8.11 (m, 1 H), 8.27 (s, 1 H), 10.2 (broad s, 1 H).
C
NMR (150 MHz, DMSO‐d6) δ (ppm): 56.2, 110.7, 112.7, 118.6, 119.8, 124.3, 125.7, 150.2, 152.5, 154.1,
168.1. HRMS [M − H]−: calculated 283.0724; found 283.0725.
2‐(2,4‐Dihydroxyphenyl)‐1H‐benzo[d]imidazole‐5‐carboxylic acid (7h). White solid, m.p. > 300 °C,
1
94% yield. H NMR (300 MHz, DMSO‐d6), δ (ppm): 6.43 (s, 1 H) 6.47 (d, J = 6.0 Hz, 1 H), 7.65 (d, J = 6.0
13
Hz, 1 H), 7.86 (d, J = 9.0 Hz, 1 H), 7.89 (d, J = 9.0 Hz, 1 H), 8.15 (s, 1 H), 12.96 (broad s, 1 H). C NMR
(150 MHz, DMSO‐d6) δ (ppm): 103.5, 104.7, 108.3, 113.1, 113.2, 115.8, 120.8, 125.2, 128.4, 140.5, 155.9,
160.4, 161.7, 168.5. HRMS [M − H]−: calculated 269.0568; found 269.0564.
3.7. Synthesis and Characterization Data of N‐(2‐(4‐benzylpiperazin‐1‐yl)ethyl)‐2‐(2‐hydroxy‐phenyl)‐1H‐
benzo[d]imidazole‐5‐carboxamide (PZ1) (8).
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 Na2SO4 and concentrated under reduced pressure. The crude product was
purified by flash‐chromatography (silica gel; eluent: CH2Cl2/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 H2O 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 Na2SO4 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).
N‐(2‐(4‐Benzylpiperazin‐1‐yl)ethyl)‐2‐(2‐hydroxy‐phenyl)‐1H‐benzo[d]imidazole‐5‐carboxamide (PZ1)
1
(8). Yellow solid, m.p.= 249–252 °C. H NMR (400 MHz, DMSO‐d6), δ (ppm): 2.34–2.47 (m, 10 H, 8
piperazine and 2 NH2–CH2CH2N), 3.36–3.41 (m, 4 H, 2 NCH2 Ph and 2 NH2–CH2CH2N), 6.99–7.09 (m,
2 H), 7.19–7.34 (m, 4 H), 7.39–7.44 (m, 1 H), 7.65–7.72 (m, 1 H), 7.76–7.83 (m, 1 H), 8.05–7.11 (m, 1 H),
13
8.13–8.15 (m, 1 H), 8.41 (t, J = 5.3 Hz, 1 H). C NMR (100.5 MHz, DMSO‐d6), δ (ppm): 37.0, 52.7, 52.9,
57.1, 62.2, 112.5, 117.3, 119.3, 122.4, 126.6, 126.9, 128.2, 128.6, 129.4, 132.2, 138.3, 153.3, 158.1, 166.4.
+
HRMS [M + H] : calculated 456.2394; found 456.2375.

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4. 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.
Author Contributions: L.P. conceived and designed the experiments and wrote the first draft of the paper; R.S.,
F.R., L.B. and F.M.P. carried out the experimental work; V.C., and M.A.S. provided reagents/materials/analysis
tools; L.P., V.C., and F.M.P. analyzed the data and participated in the discussion of the obtained results; V.C.
wrote the last draft of the paper and all authors participated in revising it. All authors have read and agreed to
the published version of the manuscript. All authors have read and agreed to the published version of the
manuscript.
Funding: This work was carried out under the framework of the national PRIN project “Unlocking Sustainable
Technologies Through Nature‐inspired Solvents (NATUREChem) (grant number: 2017A5HXFC_002)
financially supported by the University of Bari “Aldo Moro”, the Interuniversity Consortium C.I.N.M.P.I.S., and
the Ministero dell’Università e della Ricerca (MIUR‐PRIN).
Acknowledgments: L.P. thanks Fondo di Sviluppo e Coesione 2007–2013–APQ Ricerca Regione Puglia
“Programma regionale a sostegno della specializzazione intelligente e della sostenibilità sociale ed ambientale ‐
Future In Research”, Project ID: I2PCTF6. L.P. and F.R. thank the Erasmus+ EU programme.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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