Synthesis and in vitro screening of novel heterocyclic β-D-gluco- And β-D-galactoconjugates as butyrylcholinesterase inhibitors

Article abstract of DOI:10.3390/molecules24152833

The development of selective butyrylcholinesterase (BChE) inhibitors may improve the treatment of Alzheimer’s disease by increasing lower synaptic levels of the neurotransmitter acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and improved cognitive functions. A series of 14 novel heterocyclic β-d-gluco- and β-d-galactoconjugates were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and 1-benzylbenzimidazolium substituted β-d-gluco- and β-d-galacto-derivatives in a 10–50 micromolar range. The analysis performed by molecular modelling indicated key residues of the BChE active site, which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor should enable better blood–brain barrier permeability, and thus increase bioavailability in the central nervous system of these compounds.

Full text of DOI:10.3390/molecules24152833

molecules
Article
Synthesis and In Vitro Screening of Novel
Heterocyclic β-d-Gluco- and β-d-Galactoconjugates
as Butyrylcholinesterase Inhibitors
1
1
Krešimir Baumann 1, Lorena Kordic´ , Marko Mocˇibob 1, Goran Šinko 2,
*
and Srđanka Tomic´
1
Department of Chemistry, Faculty of Science, University of Zagreb, HR-10001 Zagreb, Croatia
Biochemistry and Organic Analytical Chemistry Unit, Institute for Medical Research and Occupational
Health, P.O. Box 291, HR-10001 Zagreb, Croatia
2
*
Correspondence: gsinko@imi.hr; Tel.: +385-1-4682-500
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 26 June 2019; Accepted: 3 August 2019; Published: 4 August 2019
Abstract: The development of selective butyrylcholinesterase (BChE) inhibitors may improve
the treatment of Alzheimer’s disease by increasing lower synaptic levels of the neurotransmitter
acetylcholine, which is hydrolysed by acetylcholinesterase, as well as by overexpressed BChE. An
increase in the synaptic levels of acetylcholine leads to normal cholinergic neurotransmission and
improved cognitive functions. A series of 14 novel heterocyclic β-d-gluco- and β-d-galactoconjugates
were designed and screened for inhibitory activity against BChE. In the kinetic studies, 4 out
of 14 compounds showed an inhibitory effect towards BChE, with benzimidazolium and
1-benzylbenzimidazolium substituted β-d-gluco- and β-d-galacto-derivatives in a 10–50 micromolar
range. The analysis performed by molecular modelling indicated key residues of the BChE active site,
which contributed to a higher affinity toward the selected compounds. Sugar moiety in the inhibitor
should enable better blood–brain barrier permeability, and thus increase bioavailability in the central
nervous system of these compounds.
Keywords: anticholinesterase; butyrylcholinesterase; glucose; galactose; heterocycles
1. Introduction
Alzheimer’s disease (AD) is a chronic and progressive age-related neurological disorder, and
the most common form of dementia in the industrialised part of the world. AD is a sum of genetic,
physiological, and environmental factors, which affect the risk of cognitive and neurodegenerative
manifestations in patients [
the aggregation of neurofibrillary tangles (NFTs), senile, tau and
exposure to aluminium and brain inflammation [ ]. Genetic factors that play a significant role in AD
progression are associated with the brain-derived neurotrophic factor (BDNF) and butyrylcholinesterase
gene (BCHE) [ ]. AD is characterized by low levels of the neurotransmitter acetylcholine (ACh), due
to the decreased rate of its biosynthesis [ ]. It has been reported that cholinesterases, particularly
butyrylcholinesterase (BChE, EC 3.1.1.8.), are associated with the pathogenesis and progression of
AD [ ]. BChE transforms the normal brain amyloid precursor protein to the -amyloid protein,
which then deposits and forms -amyloid plaques. Neurofibrillary tangles and -amyloid plaques
accumulate in brain cells, resulting in the loss of cholinergic neurons [ ]. Thus, BChE inhibitors
1]. The development of AD is related to several abnormal elements, such as
β-amyloid plaques, and, additionally,
2
3
4
5
,6
β
β
β
1,7
may have a greater role in the treatment of AD in the future. Though a cure has not yet been found,
cholinesterase inhibitors (ChEI) have been used as an effective palliative treatment for AD symptoms.
The currently used ChEI therapeutics for the treatment of mild to moderate AD are neostigmine
(Prostigmin®, Roche), rivastigmine (Exelon®, Novartis), donepezil (Aricept®, Pfizer), and galantamine
(Razadyne® or Reminyl®, Janssen), all of which act as ChEI (Figure 1) [8].
Molecules 2019, 24, 2833; doi:10.3390/molecules24152833
www.mdpi.com/journal/molecules

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Figure 1. Structures of cholinesterase inhibitors used as therapeutics in Alzheimer’s disease.
Studies of anti-ChEs show different strategies in the development of new molecules as potential
drugs for the treatment of Alzheimer’s disease. Different scaffolds were recently developed or
isolated and tested for anticholinesterase activity, including quinoxaline tacrine derivate [9], piperazine,
piperidine, morpholinecarbodithioate derivatives [10], and the natural compound glucosinalbin,
4-hydroxybenzyl glucosinolate, isolated from Bunias erucago [11].
In a recent study, a structure–activity relationship analysis for a large number of AChE inhibitors
was performed, quantifying several in silico parameters and inhibition potency [12]. Ligands with a
higher number of sp2 hybridised atoms showed greater AChE inhibition potency. Some of these ligands
are also active toward BChE [13–16]. Furthermore, compounds with potentially favourable motif sugar
moiety or charged species (quaternary ammonium group) have shown increased bioavailability and
are easily transported across cell membranes (e.g., through the blood–brain barrier) or by facilitative
sugar transporters [6,17].
Considering these facts, 14 novel heterocyclic derivatives of
β-d-glucose and β-d-galactose
have been synthesized and evaluated for their inhibitory activity against BChE (Scheme 1). The
heterocyclic moieties used in the design of these novel compounds were pyridinium, imidazolium, and
benzimidazolium derivatives, containing sp2 hybridised atoms, found in many natural compounds
and therapeutics with diverse biological activities. The idea of introducing a C2 spacer between the
glycon moiety and heterocyclic moiety was to reduce the steric hindrance effect, allowing the molecules
more flexibility (increased number of conformations) and better accommodation within the BChE
active site.
Scheme 1. Synthetic pathway to target glucose/galactose heterocyclic derivatives. (a) (CH3CO)2O,
I2, 5–10 min, 25 ◦C; (b) HOCH2CH2Br, BF3 Et2O, dry dichloromethane, N2, 0 ◦C; (c) dry
·
dimethylformamide, 24 h, 100 ◦C; (d) NaOCH3/CH3OH, 25 ◦C, 60 min.

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2. Results and Discussion
2.1. Preparation of Compounds
The preparation of the target compounds was conducted in four steps. Taking into account all
synthetic steps and the desired stereochemistry of the target compounds, we chose the ester protecting
group due to its key role as a neighbouring participating group in the preparation of glycosides. There
are several procedures described in the literature for the acetylation of the sugar moiety, differing in
the use of catalysts, solvents, and activators [18,19]. The most commonly used are pyridine, sodium
acetate, ZnCl2, or HClO4. Herein, we used a fast and more environmentally friendly method of sugar
acetylation, employing a catalytic amount of iodine as an activator of acetic anhydride [20].
Several glycosylation procedures have been reported and new, improved methods are constantly
developed [21]. The three main reactions of introducing aglycon to a sugar moiety are the direct,
Koenigs–Knorr, and trichloroacetimidate methods [21]. The introduction of a C2 spacer was conducted
in the second step, by attaching 2-bromoethanol to the anomeric C-atom of peracetylated d-pyranose,
using the direct method of O-glycosidation, in the presence of BF3 Et2O as an activator. A neighbouring
·
equatorial acetyl group at the sugar’s C-2 atom leads to 1,2-trans glycoside, since the nucleophilic
attack of alcohol at the anomeric C-atom is controlled by the steric hindrance of the acyloxonium ion
intermediate [21]. The heterocyclic compounds (pyridine, imidazole, benzimidazole, and their 1-methyl
and 1-benzyl derivatives) were attached to the previously prepared β-d-glucoside or β-d-galactoside.
Keeping in mind the pKa values of the used heterocyclic compounds and the solubility of the reactants,
reactions with heterocycles were carried out in polar aprotic dimethylformamide (DMF) as a solvent.
Finally, three deprotection methods were tested. First, by using dibutyltin oxide (DBTO) in dry
methanol, second by using dry methanol with type 4Å molecular sieves, and lastly, the Zempl
én
method, by adding sodium methoxide in dry methanol [22 25]. The Zempl n method showed to be
–
é
fast and efficient even with a catalytic amount of sodium methoxide and had fewer byproducts despite
the fact that methoxide is a hard nucleophile capable of attacking and destroying a previously formed
glycoside bond.
2.2. Kinetic Measurements-Inhibition
The ability of the studied heterocyclic glycoconjugates to inhibit BChE is summarised in Table 1.
Out of the 14 tested heterocyclic glycoconjugates, four compounds showed inhibition potency in the
micromolar range towards equine BChE (Figure 2). Specifically, they can be structurally related to their
glycon moiety as two glucoconjugates (
moiety as two benzimidazolium tertiary amines (
salts ( 14). Compounds and 12, and and 14 can be treated as stereoisomers with the inversion of
5,
7
) and two galactoconjugates (12
, 14), or on their aglycon
5
,
12) and two N-benzylbenzimidazolium quaternary
7,
5
7
the chiral centre configuration at the sugar C-4 atom.
The results of the kinetic measurements revealed that the tested compounds act as mixed inhibitors
of BChE, showing both competitive and noncompetitive inhibition with moderate inhibition potency.
According to these results, the tested compounds can bind to free enzymes, as well to the Michaelis
complex formed between the enzyme and substrate (ATCh). Parameter
the BChE Michaelis complex affinity towards compounds, due to the presence of a substrate within
the BChE active site. An increase in the value of a parameter confirms indirectly which residues of
α > 1 indicates a reduction in
α
the active site stabilise the binding of an inhibitor in a complex with a free enzyme also involved in
interactions with the substrate molecule. Usually, those were residues of the choline binding site Trp82
and Tyr332 [26].

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Table 1. Prepared heterocyclic
serum BChE.
β-d-glycoconjugates 1–14 and inhibition potency toward horse
Substitution
Position on
Aglycon
Concentration
Ki ± SEM
(µM) 1
Compound
Sugar/Glycon
Heterocycle/Aglycon
α ± SEM 1
(µM)
n.d.2
n.d.2
n.d.2
n.d.2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Pyridine (Py)
Imidazole (Im)
1-methylimidazole (MeIm)
1-benzylimidazole (BnIm)
Benzimidazole (BIm)
1-methylbenzimidazole (MeBIm)
1-benzylbenzimidazole (BnBIm)
Pyridine (Py)
N
10–140
10–150
10–150
10–150
20–240
10–120
10–110
10–110
15–200
10–160
15–160
15–170
10–140
15–160
N1
N3
N3
N1
N3
N3
N
N1
N3
N3
N1
N3
N3
n.d.2
n.d.2
n.d.2
n.d.2
D-GLUCOSE
16 ± 1.6
3.3 ± 0.7
n.d.2
n.d.2
52 ± 3.0
n.d.2
5.1 ± 0.8
n.d.2
n.d.2
n.d.2
Imidazole (Im)
n.d.2
n.d.2
1-methylimidazole (MeIm)
1-benzylimidazole (BnIm)
Benzimidazole (BIm)
1-methylbenzimidazole (MeBIm)
1-benzylbenzimidazole (BnBIm)
n.d.2
n.d.2
D-GALACTOSE
782 ± 306
2.1 ± 1.7
n.d.2
n.d.2
26 ± 2.7
5.2 ± 1.4
1
Inhibition parameters were obtained by nonlinear regression using Equation (1). Values are the mean
±
standard
2
error of the mean (SEM) of three independent measurements. Not determined.
Figure 2. Concentration-dependent inhibition of horse serum butyrylcholinesterase (BChE) by
glucoconjugate and galactoconjugate 14. The graphs represent the inhibition of BChE activity
5
at different concentrations of acetylthiocholine as a substrate. Nonlinear regression analysis was used
to determine the inhibition constant (Ki) and parameter α by Equation (1).

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Compounds
7
and 14 had an
α
of about 5, indicating a 5-fold lower affinity of BChE Michaelis
complex compared to free enzymes. Interestingly, compound 12 had the lowest
α value, about 2, and
also the highest constant of inhibition (Ki). This indicates that 12 is more likely to bind outside of the
substrate binding site, and therefore the presence of a substrate in the active site does not greatly affect
the affinity of the BChE Michaelis complex.
Other types of natural compounds with multiple hydroxyl groups are flavonoids, and their
effect on BChE was reported in the literature [27]. The flavonoids galangin, kaempferol, quercetin,
myricetin, fisetin, apigenin, luteolin, and rutin, reversibly inhibited BChE. The inhibition potency of
the flavonoids was attributed to their chemical structure, i.e., the number of OH groups and their
position on the phenyl ring. Galangin was the most potent inhibitor, with a Ki = (6.9
rutin was the weakest one, with Ki = (501 105) M [27]. Interestingly, in the structure of rutin,
a derivate of galangin, one can find two fused pyranosyl sugars via a 1–6 bond. It was shown that
the sugar moiety constitutes a steric constraint for the accommodation of rutin to the BChE active
site, resulting in a lower inhibition potency. In this study, compound was the most potent one,
±
2.2) µM, while
±
µ
β
5
with a 30-fold higher affinity of BChE than that for rutin. This confirms that the proper selection
of a sugar aromatic substituent positively increases the inhibition potency of derivatised pyranosyl
sugars toward BChE. Gobec and co-workers showed in a recent study that tryptophan-derived BChE
inhibitors have potential as lead compounds for symptomatic therapy against Alzheimer’s disease,
with passive diffusion of the blood–brain barrier [28]. The tryptophan indol ring is similar to the
benzimidazole moiety used in compounds 5 and 12, moreover, glycon moiety should enable active
transport over the blood–brain barrier. Their tryptophan-derived inhibitors showed more potency
if an additional amine was present in the inhibitor molecule. Kucˇa and co-workers used a different
approach, creating heterodimers of tacrine and tryptophan as multi-target agents with the potential
to treat Alzheimer’s disease [29]. These compounds were also predicted to cross the blood–brain
barrier via passive diffusion. Taking this into account, a modification in aromatic moiety that would be
complementary with the BChE aromatic residues that align the active site gorge would increase the
inhibition potency of aromatic pyranosyl sugars toward BChE.
2.3. Molecular Modelling
For a better understanding of BChE interactions with pyranosyl sugars, we performed molecular
modelling. The results of molecular modelling for compound
5
are shown in Figure 3A. Compound
, and 14 were
5
was the most potent inhibitor in this set, although the difference in Ki values for 5, 7
not significant enough to be rationalized with molecular docking with respect to a specific interaction
between the ligand and active site residues.
Compound 5, with its hydroxyl groups, forms multiple hydrogen bonds with active site gorge
residues: Glu197, Ser198, and His438. These residues constitute a BChE catalytic site and this is the
reason why parameter
α is increased for the binding of 5 to the Michaelis complex—the presence of
substrate (ATCh) resulted in a reduction in its affinity.
The benzimidazole radical is analogue with a tryptophan indole ring where one nitrogen atom is
replaced with a carbon atom. In the crystal structure of BChE, tryptophans are very often involved in
an interaction with other aromatic residues. Interestingly, BChE has a network of aromatic residues in
the active site. This network, rich in π-electrons, includes Trp82, the residue involved in the mechanism
of substrate hydrolysis, and the surrounding four additional residues Tyr128, Tyr332, Trp430, and
Tyr440 [30]. Close to this network are two also relevant aromatic residues: Trp231 and Phe329. The
same residues can be found in the active site of AChE: Trp86, Tyr133, Tyr341, Trp439, Tyr449, Trp236,
and Phe338. The main site of tacrine (see Figure 1) interaction with BChE is Trp82 [26] and with AChE
Trp86 [31], a conserved residue that forms a choline binding site. All these aromatic residues may
contribute to the stabilisation of the benzimidazole or benzylbenzimidazole part of tested compounds.
Figure 3A depicts the interaction of benzimidazole with Trp82, Tyr332, and a close proximity to Phe329

Molecules 2019, 24, 2833
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is visible. There is also a
peripheral binding site.
π
-anion interaction of benzimidazole with Asp70, a residue of BChE substrate
Figure 3. Docked conformation of tested compounds in the active site of the BChE (PDB: 2PM8).
Top-ranked conformation of compound ). Top-ranked conformation of compound 14 ).
Non-covalent interactions are shown as green or light green dashed lines (H-bonds) and orange
dashed lines ( interactions). Hydrogen atoms of residues are omitted for better visibility, oxygen is
5
(
A
(B
π
red, nitrogen is blue, and carbon is grey or yellow.
The binding mode of compound 14 is shown in Figure 3B. The major difference between
5
and 14 was the replacement of a hydrogen atom with a benzyl substituent at the benzimidazole
ring. This change enlarged the aromatic radical attached via C2-spacer to the sugar. Additionally,
a positive charge occurred at the N3 nitrogen. Compound 14, like compound 5, formed multiple
hydrogen bonds with active site gorge residues: Glu197, Ser198, and His438. The sugar moiety was
positioned close to Trp82. There was a T-shaped orientation between the benzyl group and Trp332, and
a carbon–hydrogen bond with Thr120. The C2-spacer was in close contact with Phe329, the distance

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being 4.23 Å. Benzimidazole was located in the pocket formed by Gly117 from an oxyanion hole, and
Val288 from an acyl binding site.
Since the central nervous system (CNS) active drugs show specific physicochemical properties,
we compared a set of properties to those that exist for known AD drugs (see Figure 1). As can be seen,
the properties for compounds
as well as donepezil (Ki, AChE = 5.7 nM), galantamine (Ki, AChE = 0.5
AChE = 0.7 µM) 12]. Compounds showed a higher number of hydrogen bonds donors due to
the presence of hydroxyl groups in the sugar moiety. For the same reason, the polar surface area
was also larger than that for CNS active drugs. Compounds 14 were stereoisomers with and
1–
7
were within the recommended values for the CNS active drugs,
µM), and rivastigmine (Ki,
[
1–7
8
–
1–7
were not included in the analysis, since they shared their physicochemical properties. Table 2 lists
six physicochemical properties in relation to the recommended values of CNS active drugs, which
generally have lower molecular weights (MW < 450), have moderate hydrophobicity (logP < 5), have
fewer hydrogen bonds donors and acceptors (HBD < 3 and HBA < 7), fewer rotatable bonds (RB < 8),
and are less polar (polar surface area PSA < 70 Å2) than drugs that are not active in the CNS [32].
Table 2. Comparison of physicochemical properties for compounds 1–7 with recommended values for
the central nervous system (CNS) active drugs. Known anti-AD (Alzheimer’s disease) drugs donepezil,
galantamine, and rivastigmine are given for comparison.
Physicochemical Properties
Compound
MW 1
logP 2
HBD 3
HBA 4
RB 5
PSA 6
1
2
3
4
5
6
7
286
275
289
365
325
339
415
379
287
250
–0.85
–1.23
–1.02
0.56
0.26
0.47
2.05
4.57
1.44
2.60
4
5
4
4
5
4
4
0
1
0
2
2
2
2
2
2
2
3
2
2
5
5
5
7
5
5
7
6
1
5
107
122
112
112
122
112
112
39
Donepezil
Galantamine
Rivastigmine
The recommended values for
CNS drugs [32]
42
33
450
5
3
7
8
70
1
2
3
MW—molecular weight; logP—lipophilicity coefficient; HBD—the number of hydrogen bonds donors;
4
HBA—the number of hydrogen bonds acceptors; 5 RB—rotatable bonds; 6 PSA—the polar surface area.
3. Materials and Methods
3.1. Chemicals
All reagents and solvents were used as purchased from commercial suppliers (Kemika, Zagreb,
Croatia; Sigma Chemical Co., St. Louis, MO, USA) without further purification. d-glucose, d-galactose,
1,2,3,4,6-penta-O-acetyl-β-d-glucose, pyridine, imidazole, and benzimidazole were commercially
available (Sigma Chemical Co., St. Louis, MO, USA). The procedure for the preparation of 1-methyl
and 1-benzyl imidazole/benzimidazole derivatives was given in our previously published paper [33].
The reactions were monitored by thin-layer chromatography (TLC) plates, Silica Gel 60 F254 plates
(Merck, Darmstadt, Germany). TLC plates were visualized by ultraviolet irradiation (254 nm) or by
iodine fumes. Iodine promoted solvent free sugar acetylation was conducted, employing an already
known procedure [19,20], as well as the introduction of an ethyl spacer in the second step by the direct
method of O-glycosidation [21], resulting in pure 2-bromoethyl
β
-d-glycosides. Target molecules
were prepared according to the general procedure described below. H- and 13C-nuclear magnetic
resonance (NMR) spectra were recorded at 22 ◦C with a Bruker Avance III HD 400 MHz/54 mm Ascend
spectrometer (Bruker Optics Inc., Billerica, MA, USA). Chemical shifts were given in ppm downfield
1
from tetramethylsilane (TMS) as an internal standard (δ = 0.00 ppm) and coupling constants (J) in

Molecules 2019, 24, 2833
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Hz. Splitting patterns were designated as s (singlet), d (doublet), dd (doublet of doublets), t (triplet),
q (quartet), or m (multiplet). Atoms of glycon moiety were numerated 1–6, while atoms in aglycon
moiety linking glycon with heterocycle were marked with (‘) and numbers 1 or 2, i.e., (H’-1, C’-1).
Atoms in heterocycle were marked with the corresponding abbreviation (Py, Im, BIm) and numerated
(HIm-1, CIm-4). Mass spectra were obtained using an Agilent 1200 series high-performance liquid
chromatography (HPLC) and an Agilent 6410 Triple Quadrupole mass spectrometer equipped with an
electrospray ionization source (Agilent Technologies, Lexington, MA, USA). Infrared (IR) spectra were
recorded on a Fourier-transform infrared spectroscopy (FTIR) PerkinElmer Spectrum Two spectrometer
(PerkinElmer, Inc., Waltham, MA, USA). The purity of the final products, tested with Agilent 1200
series HPLC, was more than 94%.
General procedure: A stirred solution of 2-bromoethyl-peracetylated-D-glycoside (0.1 mol) and
an appropriate heterocyclic compound (0.15 mol) in dry dimethylformamide (0.5 mL) was heated
approximately for 24 h at 100 ◦C under an inert argon atmosphere. The completion of the reaction
was monitored by TLC (CHCl3:CH3OH = 9:1). The reaction mixture was concentrated on the rotary
evaporator, and the resulting residue was purified by column chromatography on silica gel using
gradient eluation (CHCl3:CH3OH = 9:1 to 100% CH3OH), yielding an oily syrup. To the obtained
syrup dissolved in dry methanol, 25% sodium methoxide in methanol was added (~50 µL). The
completion of the reaction was monitored by TLC or IR. After no reactant material or no absorption
band for C=O (1750–1735 cm−1) was observed, the reaction mixture was concentrated under reduced
pressure and the residue was purified by column chromatography on silica gel using gradient eluation
(CHCl3:CH3OH = 9:1 to 100% CH3OH).
N-[2-(
Yield 89%, [α]2D5
1H-NMR (400 MHz, CD3OD)
4.89 (m, 2H), 4.3 (d, 1H, HGlu-1), 4.17–4.09 (m, 3H), 4.0 (m, 1H), 3.78 (m, 2H), 3.68–3.63 (m, 2H), 3.6–3.46
β
-d-glucopyranozyloxy)ethyl]pyridinium bromide (
48◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3392, 2955–2856 (C-H), 1636 (C=N), 1076–1041;
/ppm: 8.89 (d, 2H, HPy-2,6), 8.74 (t, 2H, HPy-3,5), 8.22 (t, 1H, HPy-4),
1) Colourless syrup, quaternary salt, (0.1159 g,
=
−
ν
δ
(m, 3H); 13C-NMR (101 MHz, CD3OD)
δ/ppm: 146.1 (CPy-2,4,6), 128.4 (CPy-3,5), 111.8 (CGlu-1),
76.8, 73.8, 71.5, 68.8 (CGlu-2,3,4,5), 66.4 (CGlu-6), 62.2 (C0-2, CH2-O), 59.1 (C0-1, CH2-N+); ESI-MS
(C13H20NO6+) m/z: found 286.1 (calculated 286.1).
N1-[2-(
[α]2D5
1H-NMR (400 MHz, CD3OD)
(s, 2H), 4.32-4.25 (m, 3H), 4.16–4.13 (m, 2H), 3.9–3.87 (m, 2H), 3.69–3.67 (m, 3H), 3.36–3.21 (m, 3H);
β
-d-glucopyranozyloxy)ethyl]imidazole (
40◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3401 (O-H), 2942–2863 (C-H), 1632 (C=N), 1444, 1075;
/ppm: 7.75 (s, 1H, HIm-2), 7.23 (s, 1H, HIm-4), 6.95 (s, 1H, HIm-5), 4.82
2) Yellowish syrup, tertiary amine, (0.098 g, Yield 73%,
=
−
ν
δ
13C-NMR (101 MHz, CD3OD)
δ/ppm: 137.6 (CIm-2), 127.2 (CIm-4), 119.8 (CIm-5) 103.1 (CGlu-1), 76.6
(CGlu-5), 73.6 (CGlu-3), 70.1 (CGlu-2), 68.6 (CGlu-4), 68.6 (CGlu-6), 61.3 (C0-2, CH2-O), 46.8 (C0-1,
CH2-N+); ESI-MS (C11H18N2O6-H+) m/z: found 275.1 (calculated 274.1).
N1-methyl-N3-[2-(
(0.075 g, Yield 63%, [α]D25
(C=N), 1447, 1085; 1H-NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6)
β
-d-glucopyranozyloxy)ethyl]imidazolium bromide (
36◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3392 (O-H), 2942–2855 (C-H), 1631
/ppm: 9.07 (s, 1H, HIm-2),
3) Yellowish syrup, quaternary salt,
=
−
ν
δ
7.77 (d, 1H, J = 2 Hz, HIm-4), 7.69 (d, 1H, J = 2.1 Hz, HIm-5), 5.15 (d, 1H, J = 1.59 Hz, HGlu-1),
5.02–4.96 (m, 2H, CH2-N+), 4.56 (t, 1H), 4.37 (t, 1H), 4.21–4.19 (d, 1H), 3.9 (t, 1H), 3.85 (s, 3H, CH3-N),
3.68 (m, 1H), 3.42 (m, 1H), 3.12 (m, 2H), 2.99 (m, 2H); 13C-NMR (101 MHz, DMSO-d6)
δ/ppm: 137.6
(CIm-2), 123.7 (CIm-5), 123.1 (CIm-4), 103.2 (CGlu-1), 77.4, 76.9, 73.7, 70.4 (CGal-2-5), 67.4 (CGal-6),
61.5 (C0-1, CH2-N+), 49.5 (CH3-N), 36.2 (C0-2, CH2-O); ESI-MS (C12H21N2O6+) m/z: found 289.1
(calculated 289.1).
N1-benzyl-N3-[2-(
(0.056 g, Yield 58%, [α]D25
(C=N), 1447, 1077; 1H-NMR (400 MHz, DMSO-d6)
7.44–7.42 (m, 5H, HBn-2-6), 5.47 (s, 2H), 5.17 (d, 1H), 5.04 (d, 1H), 4.97 (d, 1H), 4.61 (t, 2H), 4.42 (t,
β
-d-glucopyranozyloxy)ethyl]imidazolium bromide (
39◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3381 (O-H), 2950–2835 (C-H), 1628
/ppm: 9.36 (s, 1H, HIm-2), 7.84 (d, 2H, HIm-4,5),
4) Yellowish syrup, quaternary salt,
=
−
ν
δ

Molecules 2019, 24, 2833
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1H), 4.24–4.22 (d, 1H), 4.03 (m, 1H), 3.93 (m, 1H), 3.68 (m, 1H), 3.4 (m, 1H), 3.36 (s, 1H), 3.15 (m, 1H),
3.03–2.96 (m, 2H); 13C-NMR (101 MHz, DMSO-d6)
/ppm: 137.4 (CIm-2), 135.3 (CBn-1), 129.4 (CBn-3,5),
δ
129.1 (CBn-4), 128.7 (CBn-2,6), 123.7 (CIm-5), 122.5 (CIm-4), 103.2 (CGlu-1), 77.3 (CGlu-5), 76.9 (CGlu-3),
73.7 (CGlu-2), 70.4 (CGlu-4), 67.3 (CGlu-6), 61.4 (C0-2, CH2-O), 52.2 (C0-1, CH2-N+), 49.7 (Ph-CH2-N);
ESI-MS (C18H25N2O6+) m/z: found 365.1 (calculated 365.1).
N1-[2-(
63%, [α]2D5
1H-NMR (400 MHz, DMSO-d6)
1H), 4.57 (s, 2H), 4.46 (s, 1H), 4.25 (d, 1H), 4.05 (m, 1H), 3.91 (m, 2H), 3.75 (d, 1H), 3.41 (s, 1H), 3.18–3.11
(m, 1H), 3.07–3.02 (m, 2H); 13C-NMR (101 MHz, DMSO-d6)
/ppm: 145.2 (CBIm-2), 142.7 (CBIm-9),
β
-d-glucopyranozyloxy)ethyl]benzimidazole (
42◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3387 (O-H), 2936–2853 (C-H), 1645 (C=N), 1474, 1073;
/ppm: 8.27 (s, 1H), 7.67–7.64 (m, 3H), 7.27–7.18 (m, 2H), 5.12–5.04 (d,
5) Colourless syrup, tertiary amine, (0.063 g, Yield
=
−
ν
δ
δ
136.4 (CBIm-8), 123 (CBIm-5), 122.1 (CBIm-6), 119.5 (CBIm-4), 111.1 (CBIm-7), 102.4 (CGlu-1), 78.1, 76.3,
72.9, 71.2, 66.9 (CGlu-2,3,4,5,6), 61.3 (C0-2, CH2-O), 48.8 (C0-1, CH2-N); ESI-MS (C15H20N2O6-H+) m/z:
found 325.0 (calculated 324.1). See Figures S1–S3.
N1-methyl-N3-[2-(
salt, (0.089 g, Yield 44%, [α]D25
1651 (C=N), 1569, 1364, 1071; 1H-NMR (400 MHz, DMSO-d6)
HBIm-4,5), 7.76 (m, 2H, HBIm-6,7), 4.83 (t, 2H, CH2-N+), 4.39 (s, 3H, CH3-N), 5.02 (m, 1H, HGlu-1),
3.86–3.7 (m, 4H), 3.6-3.49 (m, 8H); 13C-NMR (101 MHz, DMSO-d6)
/ppm: 141.2 (CBIm-2), 129.6
β
-d-glucopyranozyloxy)ethyl]benzimidazolium bromide (
58◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3393 (O-H), 2940–2837 (C-H),
/ppm: 8.86 (s, 1H, HBIm-2), 8.4 (m, 2H,
6) Yellowish syrup, quaternary
=
−
ν
δ
δ
(CBIm-9), 128.2 (CBIm-8), 127.3, 126.7 (CBIm-5,6), 116.7, 111.9 (CBIm-4,7), 103.7 (CGlu-1), 75.2, 73.5,
+
71.7, 67.4 (CGlu-6), 61.1 (C0-1, CH2-N+), 48.5 (CH3-N), 42.8 (C0-2, CH2-O); ESI-MS (C16H23N2O6
m/z: found 339.2 (calculated 339.1).
)
N1-benzyl-N3-[2-(
β
-d-glucopyranozyloxy)ethyl]benzimidazolium bromide (
7
) Yellowish syrup, quaternary
/cm−1: 3416 (O-H), 2954–2851 (C-H),
δ/ppm: 8.7 (s, 1H, HBIm-2), 8.5 (m, 2H,
salt, (0.117 g, Yield 39%, [α]D25
= ν
52◦ (c 0.1, MeOH)); IR (KBr)
−
1
1639 (C=N), 1473, 1077; H-NMR (400 MHz, DMSO-d6)
HBIm-6,7), 7.7 (m, 2H, HBIm-4,5), 7.32–7.25 (m, 5H, HBn-2,3,4,5,6), 5.4 (s, 2H, Ph-CH2-N), 4.9 (m, 3H),
4.4 (m, 1H), 4.3 (m, 1H), 3.89 (d, 1H), 3.9–3.62 (m, 5H), 3.4 (m, 4H); 13C-NMR (101 MHz, DMSO-d6)
δ
/ppm: 145.9 (CBIm-2), 137.4 (CBn-1), 133.7, 131.13 (CBIm-8,9), 129.06, 128.7, 128.08, 126.8, 126.7
(CBn-2,3,4,5,6), 125.4 (CBIm-5,6), 113.5 (CBIm-4,7), 106.7 (CGlu-1), 76.2, 73.6, 71.4, 68.3 (CGlu-2,3,4,5),
64.4 (CGlu-6), 60.8 (C0-2, CH2-O), 52.6 (C0-1, CH2-N+), 49.7 (N-CH2-Ph); ESI-MS (C22H27N2O6+) m/z:
found 415.2 (calculated 415.1). See Figures S4–S6.
N-[2-(
Yield 69%, [α]2D5
1488, 1383, 1088; 1H-NMR (400 MHz, CD3OD)
β
-d-galactopyranozyloxy)ethyl]pyridinium bromide (
30◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3401 (O-H), 2950–2919 (C-H), 1629 (C=N),
/ppm: 9.11 (d, 2H, J = 5.79 Hz, HPy-2,6), 8.65 (t, 2H,
8) Colourless syrup, quaternary salt, (0.0849 g,
=
−
ν
δ
J1 = 7.73 Hz, J2 = 7.73 Hz, HPy-3,5), 8.15 (t, 2H, J1 = 7.24 Hz, J2 = 7.24 Hz, HPy-4), 4.97–4.88 (m, 2H)
4.38–4.32 (m, 2H), 4.26–4.21 (m,1H), 4.14–4.08 (m, 1H), 3.94–3.88 (m, 3H), 3.85 (d, 1H, J = 1,88 Hz,
HGal-1), 3.7–3.69 (d, 2H), 3.6–3.46 (m, 3H); 13C-NMR (101 MHz, CD3OD)
δ/ppm: 145.7 (CPy-2,4,6),
127.85 (CPy-3,5), 103.8 (CGal-1), 75.4, 73.3, 70.8, 68.8 (CGal-2,3,4,5), 67.9 (CGal-6), 61.6 (C0-2, CH2-O),
61.1 (C0-1, CH2-N+); ESI-MS (C13H20NO6+) m/z: found 286.1 (calculated 286.1).
N1-[2-(
[α]2D5
1H-NMR (400 MHz, CD3OD)
Hz, HIm-4,5), 6.95 (t, 1H), 4.28–4.24 (m, 3H), 4.17–4.13 (m, 1H), 3.9-3.84 (m, 2H), 3.79–3.71 (m, 3H),
β
-d-galactopyranozyloxy)ethyl]imidazole (
60◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3353 (O-H), 2938–2870 (C-H), 1631 (C=N), 1463, 1080;
/ppm: 7.75 (s, 1H, HIm-2), 7.7 (s, 1H), 7.23 (t, 1H), 7.07 (d, 2H, J = 0.96
9) Yellowish syrup, tertiary amine, (0.1258 g, Yield 98%,
=
−
ν
δ
3.58–3.47 (m, 3H); 13C-NMR (101 MHz, CD3OD)
δ/ppm: 137.6 (CIm-2) 127.2 (CIm-4), 119.9 (CIm-5)
103.8 (CGal-1), 75.4 (CGal-5), 73.5 (CGal-3), 71.1 (CGal-2), 68.9 (CGal-4), 68.6 (CGal-6), 61.4 (C0-2,
CH2-O), 46.9 (C0-1, CH2-N+); ESI-MS (C11H18N2O6-H+) m/z: found 275.1 (calculated 274.1).
N1-methyl-N3-[2-(
salt, (0.0779 g, Yield 19%, [α]D25
1628 (C=N), 1575, 1474, 1454, 1383, 1087, 1033; 1H-NMR (400 MHz, CDCl3)
β
-d-galactopyranozyloxy)ethyl]imidazolium bromide (10) Yellowish syrup, quaternary
30◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3368 (O-H), 2948–2833 (C-H),
/ppm: 8.02 (s, 1H, HIm-2),
=
−
ν
δ

Molecules 2019, 24, 2833
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7.73 (d, 1H, J = 1.97 Hz, HIm-4), 7.64 (d, 1H, J = 1.9 Hz, HIm-5), 4.85 (d, 1H, J = 1.59 Hz, HGal-1),
4.55–4.52 (m, 2H, H0-2, CH2-N+), 4.13–4.07 (m, 1H), 4.01 (s, 3H, CH3-N), 3.90–3.86 (m, 2H), 3.81–3.69
(m, 3H), 3.66–3.62 (t, 2H, J1 = 9.5 Hz, J2 = 9.57 Hz, H0-1), 3.36 (s, 1H), 3.24–3.2 (m, 3H); 13C-NMR
(101 MHz, CDCl3) δ/ppm: 137.5 (CIm-2), 127.2 (CIm-5), 119.8 (CIm-4), 103.8 (CGal-1), 75.4, 73.5,
71.0, 68.9 (CGal-2-5), 68.6 (CGal-6), 61.1 (C0-1, CH2-N+), 48.4 (CH3-N), 46.9 (C0-2, CH2-O); ESI-MS
(C12H21N2O6+) m/z: found 353.3 (289.1 + 2CH3OH) (calculated 289.1).
N1-benzyl-N3-[2-(
β-d-galactopyranozyloxy)ethyl]imidazolium bromide (11) Yellowish syrup, quaternary
salt, (0.0117 g, Yield 25%, [α]D25
=
−
30◦ (c 0.1, MeOH)); IR (KBr)
ν
/cm-1: 3391 (O-H), 2928–2890 (C-H),
/ppm: 7.76 (d, 1H), 7.62 (d, 1H),
1
1638 (C=N), 1562, 1455, 1155, 1072; H-NMR (400 MHz, CDCl3)
δ
7.52–7.4 (m, 6H), 5.49 (s, 2H), 4.58–4.48 (m,3H), 4.37–4.35 (d, 1H), 4.23–4.18 (m, 1H), 4.08–4.03 (m, 1H),
3.89 (d, 1H), 3.86–3.82 (m, 1H), 3.76–3.68 (m, 2H), 3.62–3.55 (m, 3H); 13C-NMR (101 MHz, CDCl3)
δ
/ppm: 136.8 (CIm-2), 134.0 (CBn-1), 129.1 (CBn-3,5), 128.9 (CBn-4), 128.4 (CBn-2,6), 123.3 (CIm-5),
121.8 (CIm-4), 103.7 (CGal-1), 75.4 (CGal-5), 73.4 (CGal-3), 70.9 (CGal-2), 68.9 (CGal-4), 67.4 (CGal-6),
61.2 (C0-2, CH2-O), 52.7 (C0-1, CH2-N+), 49.88 (Ph-CH2-N); ESI-MS (C18H25N2O6+) m/z: found 365.2
(calculated 365.1).
N1-[2-(β-d-galactopyranozyloxy)ethyl]benzimidazole (12) Yellowish syrup, tertiary amine, (0.0641g, Yield
18%, [α]2D5 = −33◦ (c = 0.1, MeOH)); IR (KBr) ν/cm−1: 3380 (O-H), 2926 (C-H), 1655 (C=N), 1499, 1292,
1075; 1H-NMR (400 MHz, CDCl3)
δ/ppm: 7.75 (s, 1H, HBim-2), 7.69 (m, 2H), 7.27 (m, 2H), 4.90 (m, 2H,
C0-1, CH2-N), 4.28–4.26 (m, 3H), 4.17–4.12 (m, 3H), 3.91–3.83 (m, 2H), 3.80–3.70 (m, 2H), 3.57–3.45 (m,
1H); 13C-NMR (101 MHz, CDCl3)
δ/ppm: 137.57 (CBim-2), 134.93 (CBim-8,9), 127.73 (CBim-5,6), 119.86
(CBim-4,7), 103.8 (CGal-1), 75.4, 73.5, 71.0, 68.9, (CGal-2,3,4,5), 68.6 (CGal-6), 61.2 (C0-2, CH2-O), 48.4
(C0-1, CH2-N); ESI-MS (C15H20N2O6-H+) m/z: found 325.2 (calculated 324.1). See Figures S7–S9.
N1-methyl-N3-[2-(
salt, (0.1051 g, Yield 20%, [α]D25
1642 (C=N), 1572, 1384, 1073; 1H-NMR (400 MHz, CDCl3)
1H), 8.0–7.99 (m, 1H), 7.77–7.74 (m, 1H), 7.3–6.69 (m, 1H), 4.86 (t, 2H, H0-1, CH2-N+), 4.23 (s, 3H,
CH3-N), 4.20 (m, H0-2), 4.01-3.96 (m, 4H), 3.83-3.56 (m, 5H); 13C-NMR (101 MHz, CDCl3)
/ppm:
β
-d-galactopyranozyloxy)ethyl]benzimidazolium bromide (13) Yellowish syrup, quaternary
78◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3413 (O-H), 2926–2860 (C-H),
/ppm: 8.33 (s, 1H, HBim-2), 8.1–8.07 (m,
=
−
ν
δ
δ
144.7 (CBIm-2), 129.6 (CBIm-9), 128.2 (CBIm-8), 127.3, 126.7 (CBIm-5,6), 116.7, 111.9 (CBIm-4,7), 103.7
(CGal-1), 75.2 (CBIm-5), 73.5 (CBIm-3), 71.7 (CBIm-2), 96.0 (CBIm-4), 67.4 (CBIm-6), 61.1 (C0-1, CH2-N+),
48.5 (CH3-N), 42.8 (C0-2, CH2-O); ESI-MS (C16H23N2O6+) m/z: found 339.2 (calculated 339.1).
N1-benzyl-N3-[2-(
salt, (0.1726 g, Yield 31%, [α]D25
1629 (C=N), 1563, 1478, 1456, 1384, 1077; 1H-NMR (400 MHz, CDCl3)
β
-d-galactopyranozyloxy)ethyl]benzimidazolium bromide (14) Yellowish syrup, quaternary
57◦ (c 0.1, MeOH)); IR (KBr) /cm−1: 3416 (O-H), 3054–2951 (C-H),
/ppm: 8.1–8.08 (d, 2H, J = 7.83 Hz,
=
−
ν
δ
HBIm-6,7), 7.87–7.84 (d, 2H, J = 8.2 Hz, HBim-4,5), 7.38–7.7 (m, 7H, 5 HBn), 5.83 (s, 2H, Ph-CH2-N),
4.39 (d, 1H), 4.31 (m, 1H), 4.20 (m, 1H), 3.89 (d, 1H, J = 1.93 Hz, HGal-1), 3.83–3.55 (m, 6H), 3.36 (m,
4H); 13C-NMR (101 MHz, CDCl3)
δ/ppm: 133.37 (CBim-2, CBn-1), 131.7, 131.13 (CBim-8,9), 129.06,
128.7, 128.08, 126.8, 126.7 (CBn-2,3,4,5,6), 113.16, 113.5 (CBIm-4,7), 103.7 (CGal-1), 75.2, 73.3, 70.9,
68.8 (CGal-2,3,4,5), 66.4 (CGal-6), 61.0 (C0-2, CH2-O), 50.6 (C0-1, CH2-N+), 47.7 (N-CH2-Ph); ESI-MS
(C22H27N2O6+) m/z: found 415.2 (calculated 415.2). See Figures S10–S12.
3.2. Inhibition of BChE by Heterocyclic β-d-Glycoconjugates
The stock solutions were prepared in water or 0.1 M phosphate buffer (pH 7.4), calibrated prior to
the measurements. If necessary, DMSO was added to aid the dissolution of the test compounds; the final
DMSO concentration was kept under 0.5% to eliminate its influence on enzyme activity [34]. Lyophilised
BChE from horse serum, acetylthiocholine iodide (ATCh), and thiol reagent 5,50-dihiobis(2-nitrobenzoic
acid) (DTNB) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Lyophilised BChE was
chosen over AChE from human red blood cells and BChE from human serum due to the competitive
reactions with other enzymes/proteins (primary proteases and albumin) that could interfere with

Molecules 2019, 24, 2833
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experimental parameters, such as enzyme kinetics, during the biochemical assay. Horse serum BChE
was also chosen due to a very high sequence homology with human plasma BChE [35]. Moreover, the
activity of BChE, unlike AChE, was not affected by the addition of 1–5% DMSO concentration used
for compound dissolution [34]. The activity of BChE (0.08 U/mL final concentration) was assayed by
the Ellman spectrophotometric method [36], using ATCh-iodide as a substrate (concentration range
0.04–1 mM), and the thiol reagent DTNB (0.3 mM, ε
= 14,250 dm3 mol−1 cm−1; [37]) in the presence of
compounds 1–14 (10–240 µM final concentration range).
The increase in the absorbance of the TNB anion was measured in a 0.1 M sodium phosphate buffer
(pH 7.4) at 25 ◦C and 412 nm up to 90 s in 2 mL total volume quartz cuvettes on an AnalytikJena Specord
200 spectrophotometer (Analitik, Jena, Germany). Compound-induced ATCh-iodide nonenzymatic
hydrolysis was not observed. Enzyme activity was measured at six substrate concentrations for five
compound concentrations in triplicate. The experimentally obtained initial reaction rates at different
substrate concentrations (ATCh) were fitted to Michaelis–Menten kinetics, wherefrom Km and Vm
were obtained, employing nonlinear regression using GraphPad Prism6 software (GraphPad, San
Diego, CA, USA). The enzyme-inhibitor dissociation constant, Ki, and binding affinity parameter
α
were evaluated from the four-parameter model of mixed inhibition related to Equation (1), using a
global optimization algorithm [38]:
[I]
Ki
Vm0 [S]
Km0 + [S]
1 +
1 +
1
vi =
Vm0 = Vm
Km0 = Km
.
(1)
[I]
αKi
[I]
αKi
1 +
3.3. Molecular Modelling
The compounds to be docked in the active site of human BChE were modelled and later minimized
with the MMFF94 force field using ChemBio3D Ultra 12.0 (PerkinElmer, Inc., Waltham, MA, USA).
The Discovery Studio 2017 R2 (Accelrys, San Diego, CA, USA) Dock Ligands protocol (CDOCKER)
with a CHARMm force field was used for the docking study [39,40]. The model of human BChE was
the crystal structure of BChE (PDB: 2PM8) and it was used as the rigid receptor [41]. The binding site
within the BChE was defined by a sphere (r = 13 Å) [42]. Details about docking procedure using the
CDOCKER protocol and scoring of generated ligand poses by a CHARMm energy were described
previously [42].
3.4. In Silico Prediction of Blood–Brain Barrier Penetration
The potential of novel compounds to cross the blood–brain barrier via passive diffusion was
evaluated by molecular descriptors: the polar surface area (PSA), the molecular weight (MW), the
calculated logarithm of the octanol/water partition coefficient (logP), the number of hydrogen bond
acceptors (HBA), the number of hydrogen bond donors (HBD), and the number of rotatable bonds
(RB). Molecular descriptors were determined using the Discovery Studio 2017 R2 (BioVia/Accelrys,
San Diego, CA, USA). The in silico determined results were compared to the recommendations of
physicochemical properties for known central nervous system drugs [32].
4. Conclusions
In conclusion, a series of multifunctional heterocyclic-based sugar analogues were designed
by merging two known biologically active structural moieties that can show favourable effects for
counteracting the progression and pathogenesis of Alzheimer’s disease. In this study, we presented the
inhibition of lyophilised equine BChE by 14 novel heterocyclic β-d-gluco- and β-d-galactoconjugates.
Compounds were prepared in four synthetic steps, in good or moderate yields. The direct measurement
of inhibition showed that 4 out of 14 tested compounds acted as mixed inhibitors of BChE. Compounds
5
µ
and 14 showed good inhibition potency in the micromolar range towards BChE with Ki = (16
±
1.6)
M and (26.2 2.7) M, respectively. Benzimidazole derivatives of pyranosyl sugars represent the
±
µ

Molecules 2019, 24, 2833
12 of 14
structural scaffold that can serve as a lead in the development of anti-AD drugs. The introduction of a
modification in the aromatic moiety that would be complementary with BChE aromatic residues that
align the active site gorge would increase the inhibition potency of aromatic pyranosyl sugars toward
BChE. Additionally, the introduction of another type of tertiary amines may also have positive effects
on inhibition potency, as Gobec and co-workers showed [28]. A comparison of the physicochemical
properties of the tested compounds in relation to the recommended values of CNS active drugs
confirmed their potential in the treatment of AD.
Supplementary Materials: The following are available online, ESI-MS, 1H and 13C NMR spectra of active
compounds (5, 7, 12 and 14; Figures S1–S12).
Author Contributions: K.B., L.K. and S.T. designed and synthesized the compounds. K.B., L.K., M.M., G.Š. and
S.T. conceived and designed the experiments. K.B., L.K. and M.M. performed the experiments and analysed the
data. K.B., L.K., M.M., G.Š. and S.T. contributed to writing the paper.
Funding: This research was funded by Hrvatska Zaklada za Znanost, grant number IP-2014-09-7899.
Acknowledgments: We thank Makso Herman for language editing.
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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