Synthesis, crystal structure, and biological evaluation of fused thiazolo[3,2-a]pyrimidines as new acetylcholinesterase inhibitors

Article abstract of DOI:10.3390/molecules24122306

A new series of thiazolo[3,2-a]pyrimidine bromide salt derivatives 7a-d were synthesized from 3,4-dihydropyrimidinethione precursors. The target compounds were fully characterized by 1D- and 2D-NMR, high resolution ESI-MS/MS and single crystal X-ray diffraction analysis, which confirmed a regioselective 5H cyclization of the dihydropyrimidinethiones. All target compounds were evaluated in vitro as human acetylcholinesterase (hAChE) inhibitors via an Ellman-based colorimetric assay and showed good inhibition activities (better than 70% at 10 μM and IC₅₀ values in the 1 μM range). Molecular docking simulations for all target products into hAChE were performed and confirmed strong binding to the enzyme. These results provide a promising and new starting point to improve acetylcholinesterase inhibitors and explore novel treatment options against Alzheimer's disease.

Full text of DOI:10.3390/molecules24122306

molecules
Article
Synthesis, Crystal Structure, and Biological
Evaluation of Fused Thiazolo[3,2-a]Pyrimidines as
New Acetylcholinesterase Inhibitors
Mohamed Y. Mahgoub 1
,2
, Awatef M. Elmaghraby * , Abd-Elfttah A. Harb ,
1,
1
João L. Ferreira da Silva 2 , Gonçalo C. Justino 2 and M. Matilde Marques 2
1
Chemistry Department, Faculty of Science, South Valley University, Qena 83523, Egypt;
m.mahgoub2013@gmail.com (M.Y.M.); alifaragharb@gmail.com (A.-E.A.H.)
Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal;
2
joao.luis@tecnico.ulisboa.pt (J.L.F.d.S.); goncalo.justino@tecnico.ulisboa.pt (G.C.J.);
matilde.marques@tecnico.ulisboa.pt (M.M.M.)
*
Correspondence: awatef_elmaghraby@yahoo.com
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: David Díez and María Ángeles Castro
Received: 26 May 2019; Accepted: 19 June 2019; Published: 21 June 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: A new series of thiazolo[3,2-a]pyrimidine bromide salt derivatives 7a–d were synthesized
from 3,4-dihydropyrimidinethione precursors. The target compounds were fully characterized
by 1D- and 2D-NMR, high resolution ESI-MS/MS and single crystal X-ray diffraction analysis,
which confirmed a regioselective 5H cyclization of the dihydropyrimidinethiones. All target
compounds were evaluated in vitro as human acetylcholinesterase (hAChE) inhibitors via an
Ellman-based colorimetric assay and showed good inhibition activities (better than 70% at 10
µM and
IC50 values in the 1 M range). Molecular docking simulations for all target products into hAChE
µ
were performed and confirmed strong binding to the enzyme. These results provide a promising and
new starting point to improve acetylcholinesterase inhibitors and explore novel treatment options
against Alzheimer’s disease.
Keywords: thiazolopyrimidines; acetylcholinesterase inhibition; molecular docking; crystal structures
1
. Introduction
Alzheimer’s disease (AD) is a chronic and progressive neurodegenerative disorder that is the
leading cause of dementia in elderly people, accounting for 60–80% of all cases. According to the World
Health Organization (WHO), AD affected about 50 million people worldwide in 2018 [ ]. AD destroys
1
brain cells and nerves, disrupting the neurotransmitters that carry messages in the brain, especially
those responsible for storing memories. Acetylcholinesterase (AChE) is the enzyme that catalyzes
the hydrolysis of the neurotransmitter acetylcholine (ACh) into acetic acid and choline (Scheme 1).
According to the cholinergic hypothesis, depleted levels of ACh are associated with AD [2,3].
As a result, AChE inhibitors, such as tacrine, donepezil, galantamine, and rivastigmine (Figure 1),
represent the major class of drugs approved and prescribed for AD. These drugs promote memory
function and delay the cognitive decline, although they do not change the underlying pathology [4].
Thiazolopyrimidine derivatives have been the focus of intense research in recent years. Among the
various members of this class, thiazolo[3,2-a]pyrimidines are particularly interesting. They have been
reported to inhibit specific enzymes, such as AChE [
casein kinase 2 [ ], YycG histidine kinase [ ], 2-methylerythritol 2,4-cyclodiphosphate synthase
IspF) proteins from Mycobacterium tuberculosis, Plasmodium falciparum, and Arabidopsis thaliana [ ],
and xanthine oxidase [10]. While the precise biological targets remain to be elucidated in some cases,
5], cell division cycle (Cdc) 25B phosphatase [6],
7
8
(
9
Molecules 2019, 24, 2306; doi:10.3390/molecules24122306
www.mdpi.com/journal/molecules

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thiazolo[3,2-a]pyrimidines have widely been reported to possess anti-cancer [11], anti-inflammatory
and analgesic [12 15], anti-microbial, antibiofilm, antioxidant, DNA cleavage [ 16 20], antiviral [21 22],
anti-Parkinsonian [23], antidiabetic [24], antihypertensive, and inotropic [25] activities. Other possible
applications include their use as antimalarials and inhibitors of HIV reverse transcriptase [ 26],
as well as antagonists of the 5-HT2 serotonin receptor, with potential interest for the treatment of
depression [27 28]. Other non-therapeutic uses for thiazolo[3,2-a]pyrimidines include inhibition of mild
–
8,
–
,
9,
,
steel corrosion [29], and dimeric thiazolo[3,2-a]pyrimidinones have been proposed for the development
of pigments, sensitizers in solar cells, and molecular switches [30]. As part of a program aiming to
obtain new potent AD modifying agents, we report herein a new small series of AChE inhibitors
based on a fused thiazolopyrimidine core. The plausible binding interactions of two representative
compounds at the active site of human AChE are also discussed.
Scheme 1. The hydrolysis of acetylcholine (ACh) by acetylcholinesterase (AChE) into acetic acid
and choline.
Figure 1. Structures of approved AChE inhibitors.
2
. Results and Discussion
2
.1. Synthesis
Herein, a new series of thiazolo[3,2-a]pyrimidines 7a–d, were designed and synthesized. Initially,
3
,4-dihydropyrimidinethiones (DHPMs) 4a
,
b
1
were prepared by a multicomponent Biginelli-type
, ethyl benzoylacetate and thiourea , as previously
described [31] (Scheme 2). Then, a series of fused thiazolo[3,2-a]pyrimidines 7a were synthesized
in good yields, in a regioselective manner, through the intermediate by cyclization of DHPMs 4a
reaction based on condensation of aldehydes
2
3
–d
6
,b

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with phenacyl bromides
5
under refluxing conditions in ethanol for 12–15 h (Scheme 3). The bromide
salts were used for biological activity measurements. For comparative characterization purposes,
compound 7b was also neutralized to the corresponding product 9.
Scheme 2. Synthesis of 3,4-dihydropyrimidinethiones (DHPMs) 4a,b by a multicomponent
Biginelli-type reaction.
Scheme 3. Synthesis of fused thiazolo[3,2-a]pyrimidines 7a–d and 9.
2
.2. Structural Characterization
The DHPMs 4a and the thiazolo[3,2-a]pyrimidine salts 7a
and 2D-NMR, high resolution mass spectrometry (HRMS), and single crystal X-ray diffraction analysis.
,b
–d were fully characterized by 1D-
2
.2.1. NMR Data
The NMR spectra were fully consistent with the expected structures. The most characteristic
features in the NMR spectra of DHPMs 4a,b were the methine proton and carbon shifts, observed at δ

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5.27 and 54.08 ppm, respectively, in 4a and at
δ
5.22 and 53.52 ppm, respectively, in 4b. Additionally,
the two exchangeable NHs were observed at downfield shifts around
δ 9.7 ppm for N3-H and
δ
10.4 ppm for N1- . Using a HMBC pulse sequence, 2- and 3-bond correlations were observed
H
between the methine proton and nearby carbons (C=S, C=O, C5, and C6), thus confirming the
structural characterization.
Theoretically, the intramolecular cyclization of DHPMs with phenacyl bromide derivatives
might afford two possible isomeric products: 5H-thiazolo[3,2-a]pyrimidines 7a
H-thiazolo[3,2-a]pyrimidines 8a . However, a single product was obtained in all instances,
presumably due to the higher conjugation (and thereby increased contribution) of the tautomer
shown in Scheme 3 for the non-fused precursor , compared to the other possible tautomeric form (not
shown). Cyclization was confirmed by the absence of thioamide NH resonances in the NMR spectra of
, along with the presence of an aromatic proton singlet (C2 H) at around 7.15–7.30 ppm and
the corresponding C2 resonance at 113.00–114.60 ppm. Moreover, compared with 4a , the further
downfield shifts observed for the methine proton and carbon resonances upon cyclization (Table 1) were
consistent with formation of the 5H isomers 7a . This was further corroborated by single crystal X-ray
diffraction characterization (cf. paragraph below). Neutralization of 7b yielded compound , which
had characteristic proton (C2-H) and carbon (C2) resonances at 6.20 and 103.04 ppm, respectively,
slightly upfield from those of 7b, as expected (cf. experimental section and Supplementary data).
–d and
7
–d
6
7
a
–
d
-
δ
δ
,b
–
d
9
δ
Table 1. 1H- and 13C-NMR chemical shifts (
δ, in ppm) for the methine proton (C5-H) and carbon (C5)
for all compounds discussed in the paper.
Comp.
1H-NMR
13C-NMR
4a
4b
7a
7b
7c
7d
9
5.27
54.08
5.22
53.52
6.44
6.41
59.63
6.36
59.93
6.73
59.41
6.27
58.36
59.87
For comparative purposes, the relevant carbon chemical shifts of compounds 7b and
9 are listed
in Table 2. The most notable changes were observed for C2, C7, and C9, which is consistent with
protonation of N8 in compound 7b. This assumption was further confirmed by single crystal X-ray
diffraction characterization (cf. Figure 2).
Table 2. Comparison of the relevant carbon chemical shifts (δ, in ppm) for the protonated compound
7
b and the non-protonated compound 9.
Comp.
C2
C5
C6
C7
C9
C=O
113.00
103.04
58.36
100.57
156.26
166.53
167.04
59.63
103.10
144.24
160.70
164.74

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Figure 2. Diagrams of the molecular structures of compounds 4a, 7b, and 7d showing the atomic
labelling scheme.
2
.2.2. X-Ray Diffraction Studies
Single crystal X-ray diffraction analyses allowed us to confirm the structures of compounds 4a
,
7
b, 7d without ambiguity (Figure 2). Some of the relevant crystal parameters are reported in Table S1,
in the Supplementary data section, while selected geometrical parameters are presented in Tables 3
and 4. Structurally-comparable bond lengths and angles were similar in all molecules and well within
the expected range, as judged from extensive analysis of the values included in the CSD [32,33] (see also
Tables S2 and S3, in the Supplementary data section, for full geometric parameters). All compounds
had phenyl groups bonded to a quasi-planar dihydropyrimidine-containing central ring (Figure 2
and Tables 3 and 4) displaying very similar angles between the planes. Another fragment present in
◦ ◦ ◦
all compounds (4a, 7b, and 7d) was the ester group, with torsion angle values 11.5 , 21.9 , and 32.7 ,
respectively (Tables 3 and 4). Despite these slight differences, they all presented similar packing index
values; in the absence of any stereochemical constraint it is possible to attribute the main geometrical
features in these compounds to an attempt of achieving the most compact structures possible [34].
Concerning compound 4a it was clear that S(2)-C(2) has all the characteristics of a double bond: angles
◦
around C(2) in the 116.4–121.9 range and a bond length of 1.680 Å; C=S double bonds usually display
values between 1.630 and 1.720 Å, while single bonds are longer, as expected (1.749–1.856 Å) [32].
In compounds 7b and 7d there was a Z C(2)-C(3) double bond, with bond lengths of 1.335 and
1.339 Å, respectively. Moreover, the bond length values for S(1)-C(2) and S(1)-C(9) in both compounds
suggest an extended delocalization spanning the sulfur atom.

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Table 3. Selected geometrical parameters for compound 4a.
Bond
Bond Length [Å] Angle
Angle [°]
123.1(2)
116.4(2)
121.9(2)
121.70(18)
126.8(2)
N(1)-C(2)
1.360(3)
1.387(3)
1.680(3)
1.327(3)
1.469(3)
1.508(3)
1.353(3)
C(2)-N(1)-C(6)
N(1)-C(6)
S(2)-C(2)
N(3)-C(2)
N(3)-C(4)
C(4)-C(5)
C(5)-C(6)
N(3)-C(2)-N(1)
N(3)-C(2)-S(2)
N(1)-C(2)-S(2)
C(2)-N(3)-C(4)
N(3)-C(4)-C(5)
108.95(18)
N(3)-C(4)-C(20) 110.4(2)
Torsion Angle (R-molecule) Angle [°]
C(5)-C(4)-C(20) 113.3(2)
O(7)-C(7)-C(5)-C(4)
−11.5(4)
C(6)-C(5)-C(7)
C(6)-C(5)-C(4)
C(7)-C(5)-C(4)
125.1(2)
120.6(2)
114.2(2)
C(5)-C(6)-C(30) 126.6(2)
N(1)-C(6)-C(30) 113.9(2)
C(5)-C(6)-N(1)
119.5(2)
Angles between Planes
N1-C2-N3- C4-C5-C6
N1-C2-N3- C4-C5-C6
Angles [°]
85.68(13)
66.15(17)
C20-…-C25
C30-…-C35
Table 4. Selected geometrical parameters for compounds 7b and 7d.
Molecule
7b
Bond Length [Å]
7d
S(1)-C(2)
S(1)-C(9)
C(3)-C(2)
N(4)-C(3)
N(4)-C(5)
C(5)-C(6)
C(6)-C(7)
N(8)-C(7)
N(8)-C(9)
N(4)-C(9)
1.731(4)
1.712(3)
1.335(5)
1.425(4)
1.503(4)
1.520(5)
1.339(5)
1.417(4)
1.340(4)
1.337(4)
1.717(4)
1.706(4)
1.339(6)
1.410(5)
1.488(5)
1.514(5)
1.340(5)
1.394(5)
1.335(5)
1.332(4)
◦
Angles [ ]
N(4)-C(3)-C(ring)
C(2)-C(3)-C(ring)
C(3)-N(4)-C(5)
C(9)-N(4)-C(3)
C(9)-N(4)-C(5)
N(4)-C(5)-C(6)
N(4)-C(5)-C(ring)
C(6)-C(5)-C(ring)
C(7)-C(6)-C(10)
C(7)-C(6)-C(5)
C(10)-C(6)-C(5)
C(6)-C(7)-N(8)
122.3(3)
126.8(3)
123.5(3)
113.2(3)
121.7(3)
108.7(3)
108.7(3)
114.0(3)
126.0(3)
123.4(3)
110.6(3)
119.1(3)
122.7(4)
126.2(4)
123.9(3)
112.6(3)
123.5(3)
109.3(3)
111.2(3)
110.6(3)
122.2(3)
124.0(3)
113.7(3)
119.6(3)

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Table 4. Cont.
Molecule
C(6)-C(7)-C(ring)
7b
7d
◦
Angles [ ]
129.7(3)
111.1(3)
120.5(3)
122.3(3)
125.0(3)
112.7(3)
127.1(3)
113.3(3)
120.8(3)
122.7(3)
123.9(3)
113.4(3)
N(8)-C(7)-C(ring)
C(9)-N(8)-C(7)
N(8)-C(9)-N(4)
N(8)-C(9)-S(1)
N(4)-C(9)-S(1)
◦
Torsion Angles [ ]
O(10)-C(10)-C(6)-C(5)
21.9(6)
−32.7(6)
◦
Angles between planes [ ]
Center ring –C5 phenyl ring b
a
89.49(12)
127.57(12)
55.68(12)
83.33(11)
74.09(14)
56.66(12)
a
c
Center ring –C7 phenyl ring
Center ring –C3 phenyl ring d
a
a—S1, C2, C3, N4, C5, C6, C7, N8, C9. b—Phenyl ring bonded to C5. c—Phenyl ring bonded to C7. d—Phenyl ring
bonded to C3.
2
.3. Supramolecular Interactions
2
.3.1. Hirshfeld Surface Analysis
The 3D normalized contact distance (dnorm) Hirshfeld surface map for compounds 4a, 7b, and 7d
was generated using the Crystal Explorer 3.1 software [35] to investigate the intermolecular interactions
in the crystal lattices. This parameter is defined as
dnorm = (d − rivdw)/rivdw + (de − revdw)/revdw
i
where rivdw and r
vdw
e
e
are the van der Waals radii of the atoms involved, d represents the distance from
the Hirshfeld surface to the nearest atom external to the surface, and d represents the distance from
i
the Hirshfeld surface to the nearest atom internal to the surface [36
or negative when intermolecular contacts are longer or shorter than r
,
37]. The value of dnorm is positive
vdw
, respectively. The Hirshfeld
surface map uses a coloring code to represent the proximity of intermolecular interactions around the
molecule within a network: white for d around the van der Waals distance, blue for d longer than
van der Waals distance, and red for d shorter than the van der Waals distance [38]. The 3D dnorm
Hirshfeld surface plots of the selected compounds 4a, 7b, and 7d included in this study are shown
in Figure 3a. The plots show the main different intermolecular interactions present in each molecule.
The large and deep red spots on the 3D Hirshfeld surfaces indicate the mainly strong close-contact
hydrogen-bonding interactions. Additionally, the presence of weak hydrogen bonds is predicted in all
molecules, involving C-H . X (X = O, N, S, Br) interactions. Other possible intermolecular interactions
. .
e
i
appear as lighter red regions (Figure 3a). The combination of d and d in the form of two-dimensional
(2D) fingerprint plots provides a summary of the intermolecular contacts in the crystal [37]. Thus,
the histograms in Figure 3b present the summary of the percentage contributions of the various types of
potential interactions. For all compounds, the major contribution was related to the proximity-induced
. .
. . .
(
H . H) (48–54%) and (C H) interactions (15.4–19.9%) (Figure 3b). The major contacts expected in
compound 4a were N-H . S (13.8%) and N-H O (7.8%). Noteworthy, in the case of compounds
and 7d, was the prediction of strong Br
respectively), together with weak C-H O hydrogen bonds (10.7 and 8.7%, respectively) (Figure 3b).
. .
. . .
-
. . .
7
b
H8-N8 hydrogen bond contributions (11.4 and 9.8%,
.
. .

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Figure 3. (a) 3D Hirshfeld surfaces mapped with dnorm; red colored regions show shorter contacts; (b)
histograms showing the percentage contributions of the various types of interactions predicted for
compounds 4a, 7b, and 7d.

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2
.3.2. Crystal Packings
Analysis of the crystal packing for these compounds confirmed that they are all racemic mixtures,
as expected from the lack of stereoselectivity of the reaction conditions involved in their synthesis.
The interactions detected on each compound using Platon and visualized using Mercury were consistent
with the Hirshfeld analysis results.
Compound 4a was found to have four molecules in the unit cell, two of isomer R and two
of isomer S. The molecule has a good hydrogen donor system that formed classical hydrogen
.
..
2
bonds: N(3)-H(3N) O(7) between molecules inside the unit cell and an R (8) synthon involving
2
...
N(1)-H(1N) S(2) interactions with molecules outside the unit cell (Figure 4 and Table 5).
Figure 4. Hydrogen bonds in compound 4a: Inside the unit cell (left) and with other molecules outside
the unit cell (right).
Figure 5. Main supramolecular interactions in compounds 7b and 7d (classical hydrogen bonds are
represented in orange, weak hydrogen bonds in blue).
-
Due to the presence of Br ions, the crystal structure of compound 7d was dominated by the
strong anion . H-N hydrogen bond with the protonated N8, but it also included weak C-H
. .
. . .
O
.
..
2
hydrogen bonds, one of them (C21-H21C O20) forming an R (6) synthon between molecules of the
two stereoisomers. A similarly strong Br- H8-N8 hydrogen bond was formed in compound 7b
2
. . .
,
in which case the anion was also involved in a weak hydrogen bond, acting as a bridge between two
2
. . .
cations of the same enantiomer. R (8) synthons were formed by C-H O hydrogen bonds between R-
2
and S- molecules (Figure 5 and Table 5).

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Table 5. Interaction parameters for compounds 4a, 7b, and 7d.
D-H . . .
A
H . . . A [Å]
D . . . A [Å]
D-H . . . A [ ]
◦
Symmetry Operation
Compound 4a
N(3)-H(3N) O(7)
.
..
1
2.02
2.52
2.872(2)
3.337(2)
159
173
− x,1/2 + y,1/2 − z
2
.
..
N(1)-H(1N) S(2)
−x,1 − y,1 − z
Compound 7b
.
..
a
N8-H8 Br1
2.48
2.81
2.49
2.60
3.199(3)
3.742(4)
3.397(6)
3.507(6)
141
175
166
166
x,y,z
.
..
b
C32-H32 Br1
x, −1 + y, z
1 − x, 1/2 + y, 1/2 − z
1 − x, 1 − y, 1 − z
.
..
C18-H18 O10
.
..
C24-H24 O26
Compound 7d
.
..
a
N8-H8 Br1
2.28
2.51
2.66
3.167(3)
3.372(5)
3.456(6)
165
153
140
x, y, z
.
..
C19-H19 O10
x, −1 + y, z
2 − x, −y, 1 − z
.
..
C21-H21C O20
a—classical hydrogen bond. b—for reasons of clarity these contacts are not represented in Figure 5.
2
.4. Molecular Docking Studies to Human Acetylcholinesterase
In order to investigate the affinities of the fused thiazolo[3,2-a]pyrimidines 7a–d toward human
acetylcholinesterase (hAChE), we started by performing molecular docking simulations of the target
compounds into the active site gorge of hAChE (PDB ID: 4 m0f, chain A), using Autodock Vina
software [39]. Docking simulations were performed concomitantly with the known hAChE inhibitors,
donepezil, tacrine, and rivastigmine, used as reference drugs for comparative purposes. The results for
the binding free energies of all compounds investigated are summarized in Figure 6.
Binding Affinity (kcal/mol)
0
.0
-10.0
-20.0
-30.0
-40.0
-50.0
-60.0
Figure 6. Comparison of the binding free energies of compounds 7a–d and the reference hAChE
inhibitors donepezil, tacrine, and rivastigmine to the active site gorge of hAChE.
Molecular docking indicated good binding to hAChE, with all compounds 7a
–d binding equally
or more strongly than any of the positive controls, which suggests that the compounds are potentially
powerful AChE inhibitors. We further probed the structural features responsible for the strong binding
to the enzyme. As representative examples, Figure 7A,B displays the docking poses for compounds 7b
and 7d (cf. next paragraph).

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(
A)
(
B)
Figure 7. (A) and (B) A close view of 7b and 7d docked into the active site gorge of human AChE.
Catalytic residues are shown in blue and the peripheral anionic site residues are shown in grey;
the ligands are shown in light blue.
A number of electrostatic interactions were found between 7b and the side chain O atoms of
Ser125 (at 4.0 Å) and of Asp74 (at 4.0 Å), and between the keto O atom of the ligand and the side chain
N atom of Trp286 (at 3.0 Å). An H bond is also possible between the latter atom pair (with a H-acceptor
distance of 2.1 Å and a donor-acceptor distance of 3.0 Å), albeit at a donor-H-acceptor angle on the
◦
lower limit of the allowed region (135 ). Aromatic interactions are also quite evident between the

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ligand and nearby amino acid residues, in particular a
π
-π
stacking involving the planes of the Trp286
side-chain and of one of the ligand aromatic rings (at a 3.9 Å distance, with an inter-plane angle of
◦
9
.3 ). Another
π
-π
stacking between the aromatic plane of Tyr341 and an aromatic plane of the ligand
◦
is also possible (at a 3.7 Å distance, with an inter-plane angle of 2.1 ).
Concerning ligand 7d, aromatic interactions are also evident between the ligand aromatic planes
and the aromatic planes of Tyr341 (at a 4.3 Å) and Trp86 (at 4.3 Å); an anion-π interaction is also likely to
occur between the carbonyl O atom of the ligand and the imidazole ring of His 447. Various electrostatic
interactions are also possible between the ligand and protein amino acid residues, noticeably from the
ligand S atom to the phenolic O atom of Tyr337 (at a 4.0 Å distance).
Although the poses differ between the compounds, the two panels in Figure 7 show both
hydrophobic and hydrogen bond interactions with amino acids located in the peripheral anionic site
(PAS) at the mouth of the gorge. PAS is a well-known substrate-binding site in AChE and binding
of ligands at this location has been amply reported to affect the catalytic activity of the enzyme,
by blocking access to the catalytic site and/or inducing an allosteric alteration of the catalytic triad
conformation and efficiency [40–42].
2
.5. In Vitro hAChE Inhibition
In view of the encouraging results obtained in the in silico study, compounds 7a–d were evaluated
for their hAChE inhibitory activity at up to 50 µM concentrations using a standard Ellman-based
colorimetric protocol [43]. For comparative purposes, the powerful hAChE inhibitor methyl paraoxon
was used as positive control.
As shown in Figure 8, all compounds demostrated hAChE inhibitory activities at the micromolar
(
µ
M) level, ranging from 73–77.5% at 10
control inhibitor yielded approximately 100% inhibition. IC50 values for hAChE inhibition, obtained
for compounds 7a , were approximately 1 M (7a, 1.59 M; 7b, 1.00 M; 7c, 1.17 M), as compared to
.9 nM for the positive control, methyl paraoxon, under the conditions of the assay. Likewise, hAChE
µM to 59–74% at 5 µM; at the same concentrations, the positive
–c
µ
µ
µ
µ
4
inhibitors such as donepezil, rivastigmine, and tacrine are reported to have IC50 values in the low
nanomolar range, under optimal assay conditions [44].
%
of hAChE inhibition
1
20
at 5 μM
at 10 μM
100
80
60
40
20
0
Figure 8. Experimental in vitro hAChE inhibition activities of the fused thiazolo[3,2-a]pyrimidines
and the positive control, methyl paraoxon, at 5 and 10 M. The data are expressed as average
standard deviation of two independent experiments.
7
a
–d
µ
±

Molecules 2019, 24, 2306
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3
. Materials and Methods
All reagents were purchased from commercial sources and used without further purification,
unless stated otherwise. Whenever necessary, solvents were dried by standard methods [45]. Melting
temperatures were measured with a Leica Galen III hot stage apparatus and are uncorrected.
1
H-NMR spectra were recorded on a Bruker Avance III 400 spectrometer, operating at 400 MHz.
13
C-NMR spectra were recorded on the same instrument, operating at 100.62 MHz. Chemical shifts
are reported in ppm downfield from tetramethylsilane and coupling constants (J) are reported in
Hz. Resonance and structural assignments (indicated using the IUPAC nomenclature system) were
based on the analysis of coupling patterns, including the 13C- H coupling profiles obtained from
heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC)
experiments, performed with standard pulse programs. The abbreviations Ph and Ar represent phenyl
and aryl groups, respectively.
1
Low resolution mass spectra (MS) were recorded on an LCQ Fleet ion trap mass spectrometer
equipped with an electrospray (ESI) ion source (Thermo Scientific). The mass spectrometer was
operated in the ESI positive ion mode, with the following optimized parameters: Ion spray voltage,
+
4.5 kV; capillary voltage, 16 V; tube lens offset,
−
63 V; sheath gas (N ), 80 arbitrary units; auxiliary
2
◦
gas (N ), five arbitrary units; capillary temperature, 300 C. Spectra typically correspond to the average
2
of 20
(
−
35 scans and were recorded in the range between 100 and 1500 Da. Tandem mass spectra
collision-induced dissociation experiments) were obtained with an isolation window of 4 9 Da,
30% relative collision energy, and an activation energy of 30 ms. Data acquisition and processing
−
a 20
−
were performed using the Xcalibur software.
High-resolution mass spectra (HRMS) were obtained on a Bruker Impact II quadrupole
time-of-flight mass spectrometer (Bruker Daltoniks). Aliquots (10
µL) of the samples dissolved
in methanol were analyzed by flow injection analysis (FIA) using an isocratic gradient (50 A:50 B) of
−
1
0
1
.1% formic acid in water (A) and 0.1% of formic acid in acetonitrile (B), at a flow rate of 10
5 min. The mass spectrometer was operated in the ESI positive ion mode and in the high-resolution
mode. The following optimized parameters were used: Capillary voltage: +4.5 kV; end plate offset:
µLmin over
−1 ◦
–
500 V; nebulizer: 40 psi; dry gas: 4.0 L
·
min ; dry heater: 200 C; m/z 100–2000; acquisition mode: full
scan acquisition with a scan rate of 1.0 Hz. Prior to analysis, the mass scale was calibrated by infusion
µL min flow rate. Recalibration of acquired data was
performed via lock mass internal calibration (1221.9906) located within the source. Data processing
was performed using the Data Analysis 4.1 software.
−1
of ESI low concentration tune mix at a 15
·
3
.1. X-Ray Crystallographic Analysis
X-ray crystallographic data for compounds 4a, 7b, and 7d were collected from single crystals using
an area detector diffractometer (Bruker AXS-KAPPA APEX II, Madison, WI, USA) at room temperature
and graphite-monochromated Mo K = 0.71073 Å) radiation. Cell parameters were retrieved using
α (λ
Bruker SMART software and refined with Bruker SAINT [46] on all observed reflections. Absorption
corrections were applied using SADABS [47]. The structures were solved by direct methods using
2
SHELXT [48] and refined with full-matrix least-squares refinement against F using SHELXL [49].
All the programs are included in the WINGX package (version 2014.01) [50]. All non-hydrogen atoms
were refined anisotropically, and the hydrogen atoms were inserted in idealized positions, riding on the
parent C atom, except for those connected to nitrogen atoms, placed according to the electron density
maps. Drawings were made using Mercury CSD 3.9 (Build RC1) [51]. Geometrical parameters such as
angles between planes and torsion angles were calculated using Olex2
−
1.2.9 [52], while intermolecular
interactions were determined using Platon 30715 [53] and visualized using Mercury. Despite several
attempts, good quality crystals of compound 7d could not be obtained, a circumstance that is reflected
in the refinement of the crystal structure. Relevant details of the X-ray data analysis are displayed in
Table 6 (cf. X-ray diffraction studies section).

Molecules 2019, 24, 2306
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Table 6. Structure refinement data of compounds 4a, 7b, and 7d.
Compound
Absorption coefficient (mm 1)
4a
0.194
7b
1.694
7d
−
1.680
1128
F(000)
712
1128
Crystal size (mm)
0.30 × 0.15 × 0.15
2.776 to 26.497
–16 ≤ h ≤16,
0.200 × 0.100 × 0.040
2.808 to 26.358
0.150 × 0.060 × 0.040
◦
θ range ( )
2.873 to 26.440
Index ranges
−18 ≤ h ≤18,
−10 ≤ k ≤10,
−19 ≤ l ≤ 26
−16 ≤ h ≤ 16,
−8 ≤ k ≤ 8,
–
10 ≤ k ≤ 10,
−
20 ≤ l ≤ 20
49,517
−35 ≤ l ≤ 35
Reflections collected
Independent reflections
Completeness to Θ max (%)
Data/restraints/parameters
Goodness-of-fit on F2
14,223
5233 [R(int) = 0.0695]
99.6
99,174
5395 [R(int) = 0.2244]
99.9
3618 [R(int) = 0.0299]
98.4
3618/1/219
1.051
5233/0/316
0.955
5395/1/320
1.054
Final R indices [I > 2sigma(I)]
R1 = 0.0711.
wR2 = 0.1922
R1 = 0.0491,
wR2 = 0.1000
R1 = 0.0491,
wR2 = 0.0873
R indices (all data)
R1 = 0.0842,
wR2 = 0.2063
R1 = 0.1095,
wR2 = 0.1210
R1 = 0.1299,
wR2 = 0.1163
Extinction coefficient
n/a
n/a
n/a
Largest diff. peak/hole (e. Å 3)
−
0.872/−0.992
0.300 and −0.501
0.610 and −0.503
Crystallographic data for compounds 4a, 7b, and 7d were deposited with the Cambridge
Crystallographic Data Centre (CCDC 1496341, 1911742, and 1911743) and can be obtained free
of charge from: CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: 44-1223-226033; e-mail:
deposit@ccdc.cam.ac.uk) or http://www.ccdc.cam.ac.uk/deposit.
3
.2. General Procedure for the Preparation of DHPMs 4a,b
The DHPMs 4a were prepared as previously described [31]. Briefly, a mixture of aldehyde
1 mmol), ethyl benzoylacetate (1 mmol), thiourea (1 mmol), and quartz (0.5 g) in ethanol (15 mL) was
,
b
(
refluxed for 3 h. After completion of the reaction, as monitored by thin layer chromatography (tlc),
the catalyst was filtered off and the solution was kept overnight. The precipitate formed on setting was
filtered under suction and then recrystallized from ethanol to afford the pure product 4a,b.
3
.2.1. Ethyl 4,6-diphenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4a)
◦
◦
1
Yield 85%; R 0.54 (tlc, silica, 2:1 hexane:ethyl acetate); mp 193–195 C [lit [54], 192 C]; H-NMR
f
(400 MHz, DMSO-d6)
δ
0.73 (t, 3H, J 7.0, CH3CH O), 3.74 (q, 2H, J 7.1, CH CH2O), 5.27 (d, 1H, J 3.4,
2
3
C4-
H
), 7.30–7.41 (m, 10 H, Ph-
H
), 9.75 (s, 1H, exchangeable, N3-
H
), 10.47 (s, 1H, exchangeable, N1-H);
13
C-NMR (400 MHz, DMSO-d6)
δ
13.31 (
H), 133.98, 143.00 (Ph-Cquat), 145.79 (C6), 164.87 (
C=S); ESI-MS m/z 339 [MH] ; ESI-MS/MS (339) m/z 322, 280, 276, 263, 234, 219; HRMS (ESI-TOF) Calcd
CH ), 54.08 (C4), 59.44 (CH ), 101.82 (C5), 126.38, 127.68,
3 2
1
(
27.78, 128.63, 128.66, 129.08 (Ph-
C
C=O), 174.51
+
+
for C H N O S, m/z 339.1162 ([MH] ), Found 339.1154 (∆ = –2.4 ppm).
1
9
19
2
2
3
.2.2. Ethyl 4-(4-methoxyphenyl)-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4b)
◦
1
Yield 68%; R 0.43 (tlc, silica, 2:1 hexane:ethyl acetate); mp 130–132 C; H-NMR (400 MHz,
f
DMSO-d6)
δ
0.74 (t, 3H, J 7.0, CH3CH O), 3.73 (q, 2H, J 7.0, CH CH2O), 3.75 (s, 3H, OCH3), 5.22
2
3
(
s, 1H, C4-
H
), 6.96 (d, 2H, Jortho 7.8, 4-OCH Ph-C3,C5-
H
), 7.30 (m, 5H, Ph-
H
), 7.40 (d, 2H, Jortho
3
7
.8, 4-OCH Ph-C2,C6-
H
), 9.67 (s, 1H, exchangeable, N3-
C-NMR (400 MHz, DMSO-d6) 13.30 ( H ), 53.52 (C4), 55.08 (O
14.0 (4-OCH Ph-C3 C5), 127.60 (4-OCH Ph-C2
H
), 10.40 (s, 1H, exchangeable, N1-
H
);
3
1
3
δ
C
C
H ), 59.44 (
3
C
H ), 102.08 (C5),
3
2
1
,
,
C6), 127.63, 128.60, 129.08 (Ph-
CH), 134.04 (Ph-C1),
3
3

Molecules 2019, 24, 2306
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1
3
35.16 (4-OCH Ph-C1), 145.46 (C6), 158.82 (4-OCH Ph-C4), 164.85 (
C
=O), 174.30 (
C
=S); ESI-MS m/z
3
3
+
69 [MH] ; ESI-MS/MS (369) m/z 352, 310, 293, 264, 249, 217; HRMS (ESI-TOF) Calcd for C H N O S,
20
21
2
3
+
m/z 369.1273 ([MH] ), Found 369.1267 (∆ = −1.6 ppm).
.2.3. Synthesis of the Thiazolo[3,2-a]pyrimidine Bromides 7a–d and the Neutralized Compound 9
A mixture of each DHPM 4a (1 mmol) and the appropriate phenacyl bromide derivative 5
3
,b
(1 mmol) in ethanol was heated under reflux for 12 h. The reaction mixture was monitored by tlc.
The solvent was removed under reduced pressure. The residue was washed several times with
a mixture of n-hexane:ethyl acetate 2:1. After washing, the final precipitate was dried to give the
bromide salt of thiazolo[3,2-a]pyrimidines 7a
–d with good yields. Compound 7b was neutralized to
the corresponding product 9 using a saturated sodium carbonate solution.
3
.2.4. 6-(Ethoxycarbonyl)-3,5,7-triphenyl-5H-thiazolo[3,2-a]pyrimidin-8-ium bromide (7a)
◦
1
Yield 80%; R 0.44 (tlc, silica, 2:1 hexane:ethyl acetate); mp 205–207 C; H-NMR (400 MHz, CDCl )
f
3
δ
0.78 (t, 3H, J 7.0, CH3CH O), 3.85 (q, 2H, J 7.0, CH CH2O), 6.44 (s, 1H, C5-
H
), 6.80 (d, 2H, J 7.5,
2
3
+
13
Ph-C
H
), 7.14–7.55 (m, 14H, Ph-C
H
and C2-
H
), 13.40 (br, 1H, N 8-
CH CH O), 59.81 (C5), 61.10 (CH3CH O), 103.33 (C6), 113.72 (C2), 126.70 (Ph-CH), 127.00
3 2 2
H); C-NMR (400 MHz, CDCl3)
δ
13.43 (
Ph-Cquat.), 128.35, 129.15, 129.40, 129.62, 129.91, 130.90, 130.95 (Ph-
39.80 (C3), 144.12 (C7), 160.93 (C9), 164.70 (O- =O); ESI-MS/MS (439) m/z 393, 315, 263, 219; HRMS
(
1
CH), 131.75, 138.92 (Ph-Cquat.),
C
+
+
(ESI-TOF) Calcd for C H N O S , 439.1480 [M] , Found 439.1480 (∆ = 0.0 ppm).
27 23 2 2
3
.2.5. 6-(Ethoxycarbonyl)-3-(4-methoxyphenyl)-5,7-diphenyl-5H-thiazolo[3,2-a]pyrimidin-8-ium
bromide (7b)
◦
1
Yield 83%; R 0.55 (tlc, silica, 2:1 hexane:ethyl acetate); mp 236–238 C; H-NMR (400 MHz, CDCl )
f
3
δ
0.78 (t, 3H, J 7.1, CH3CH O), 3.85 (q, 2H, J 7.1, CH CH2O), 3.87 (s, 3H, OCH3), 6.41 (s, 1H, C5-
H
), 6.85
2
3
(
d, 2H, J 7.5, Ph-C
H
), 6.92 (d, 2H, J 8.5, 4-OCH Ph-C3
,
5
), 7.10 (d, 2H, J 8.5, 4-OCH Ph-C2
,
6
), 7.15 (s,
); C-NMR (400
CH O), 103.10 (C6), 113.00
2
3
3
+
13
1
H, C2-H
), 7.17–7.44 (m, 6H, Ph-C
13.44 ( H CH O), 55.63 (O
C2), 114.55 (Ph- H), 118.91 (4-OCH Ph-C1), 126.64, 128.35, 129.17, 129.38, 129.60, 130.91, 131.38
H
), 7.53 (d, 2H, J 7.7, Ph-C
H), 13.34 (br, 1H, N 8-H
MHz, CDCl3)
δ
C
C
H ), 59.63 (C5), 61.10 (CH3
3
2
3
(
(
(
C
3
Ph-
O-
C
H), 131.71, 139.00 (Ph-Cquat.), 139.84 (C3), 144.24 (C7), 160.70 (C9), 161.56 (
C
-OCH ), 164.74
3
+
C
=O); ESI-MS m/z 469 [M] , ESI-MS/MS (469) m/z 423, 345, 263, 219, 191; HRMS (ESI-TOF) Calcd
+
+
for C H N O S , 469.1586 [M] , Found 469.1598 (∆ = 2.6 ppm).
28
25
2
3
3
.2.6. 6-(Ethoxycarbonyl)-3-(4-fluorophenyl)-5,7-diphenyl-5H-thiazolo[3,2-a]pyrimidin-8-ium
bromide (7c)
◦
1
Yield 74%; R 0.52 (tlc, silica, 2:1 hexane:ethyl acetate); mp 208–210 C; H-NMR (400 MHz, CDCl )
f
3
δ
2
7
0.78 (t, 3H, J 7.1, CH3CH O), 3.85 (m, 2H, CH CH2O), 3.87 (s, 3H, OCH3), 6.36 (s, 1H, C5-
H
), 6.83 (d,
2
3
H, J 7.5, Ph-C
.1, Ph-C
H O), 103.36 (C6), 114.60 (C2), 116.30, 116.50 (4-FPh-C
H
), 7.10–7.27 (m, 7H, Ph-C
H
), 7.30 (s, 1H, C2-
H
), 7.37-7.43 (m, 3H, Ph-C
13.43 ( H CH O), 59.93 (C5), 61.13
H), 123.10 (4-FPh-C1), 126.66, 128.34,
H), 131.75 (Ph-Cquat.), 132.13, 132.22 (4-FPh- H), 138.51 (C3),
4); ESI-MS m/z
H), 7.52 (d, 2H, J
+
13
H
), 13.30 (br, 1H, N 8-
H
); C-NMR (400 MHz, CDCl3)
δ
C
3
2
(CH3
C
2
1
29.23, 129.32, 129.70, 130.91, 130.85 (Ph-
C
C
C
138.95 (Ph-Cquat.), 143.83 (C7), 160.82 (C9), 164.60 (O-
C
=O), 162.86, 165.40 (4-FPh-
+
+
4
4
57 [M] , ESI-MS/MS (457) m/z 411, 333, 263, 219, 191; HRMS (ESI-TOF) Calcd for C H FN O S ,
27
22
2
2
+
57.1386 [M] , Found 457.1396 (∆ = 2.2ppm).
3
.2.7. 6-(Ethoxycarbonyl)-5-(4-methoxyphenyl)-3,7-diphenyl-5H-thiazolo[3,2-a]pyrimidin-8-ium
bromide (7d)
o
1
Yield 70%; R 0.40 (tlc, silica, 2:1 hexane:ethyl acetate); mp 193-195 C; H-NMR (400 MHz,
f
CDCl3)
δ
0.78 (t, 3H, J 7.1, CH3CH O), 3.74 (s, 3H, OCH3), 3.85 (q, 2H, J 7.1, CH CH2O), 6.73 (s, 1H,
2
3
C5- ), 6.65 (d, 2H, J 8.6, Ph-C
H
H
), 6.71 (d, 2H, J 8.6, 4-OCH Ph-C3
,5
), 7.17 (d, 3H, Ph-CH and C2-H),
3

Molecules 2019, 24, 2306
16 of 20
H), 13.37 (br, 1H, N 8-H
); 13C-NMR (400 MHz,
+
7
.41–7.47 (m, 5H, Ph-C
CDCl3) 13.43 ( H CH O), 55.42 (O
14.41 (Ph- H), 127.15 (Ph-Cquat.), 128.24, 128.37, 129.18, 129.36, 129.91, 130.87, 130.96 (Ph-
H
), 7.54 (d, 2H, J 7.0, Ph-C
H ), 59.41 (C5), 61.06 (CH3CH O), 103.45 (C6), 113.26 (C2),
2
δ
C
C
3
2
3
1
1
C
CH), 131.71,
31.77 (Ph-Cquat.), 139.88 (C3), 143.80 (C7), 160.50 (
C
-OCH ), 160.65 (C9), 164.74 (O-
69 [M] , ESI-MS/MS (469) m/z 423, 315, 293, 263, 249; HRMS (ESI-TOF) Calcd for C H N O S ,
C
=O); ESI-MS m/z
3
+
+
4
4
28
25
2
3
+
69.1586 [M] , Found 469.1595 (∆ = 1.9 ppm).
3
.2.8. Ethyl 3-(4-methoxyphenyl)-5,7-diphenyl-5H-thiazolo[3,2-a]pyrimidine-6-carboxylate (9)
o
1
Yield 70%; R 0.52 (tlc, silica, 2:1 hexane:ethyl acetate); mp 205-207 C; H-NMR (400 MHz, CDCl )
f
3
δ
0.78 (t, 3H, J 7.1, CH3CH O), 3.82 (q, 2H, J 7.1, CH CH2O), 3.86 (s, 3H, OCH3), 6.18 (s, 1H, C2-
H
), 6.27
2
3
(
(
(
1
(
s, 1H, C5-
m, 8H, Ar-C
H
), 6.91 (d, 2H, J 8.4, Ar-C
H
), 6.96 (d, 2H, J 7.0, Ar-C
H
), 7.08 (d, 2H, J 8.4, Ar-C
CH CH O), 55.53 (OCH ), 58.36 (C5), 59.75
3 2 3
H), 7.174–7.40
13
H
); C-NMR (400 MHz, CDCl3)
δ
13.62 (
H), 121.82 (C3), 126.56, 127.70, 128.26, 128.40, 128.50,
H), 139.70, 141.04, 142.40 (Ar-Cquat.), 156.26 (C7), 160.75 ( -OCH ), 166.53 (C9), 167.04
CH3
30.60 (Ph-
O-C=O).
CH O), 100.57 (C6), 103.04 (C2), 114.21 (Ar-C
2
C
C
3
3
.3. Molecular Docking Studies
The protein structure was prepared starting from the crystallographic complex of human AChE
in complex with the AChE inhibitor Territrem B (PDB ID: 4m0f, chain A). Territrem B and other
non-protein molecules were deleted and only the enzyme chain was retained. Hydrogen atoms and
missing heavy atoms were added. Zero occupancy side chains, polar hydrogen atoms, and the positions
of asparagine and glutamine side chain amidic groups were optimized and assigned the lowest energy
conformation. The Autodock Vina software [39] was used for the molecular docking process using a
flexible protein-flexible ligand approach. The protein flexible regions were defined as spherical regions
with a radius of 11 Å centered at the geometrical center of the Territrem B binding site. The docking
3
search space was defined as a cubic 30
×
45
×
30 Å box co-centrical with the flexible region. Docking
was performed with an exhaustiveness of 32 and the top 100 binding modes within 10 kcal/mol of the
top binding mode were recorded and analyzed. Protein-ligand analyses were performed with UCSF
Chimera [55].
3
.4. Enzyme Inhibition Activity
3
.4.1. Enzyme and Assay Reagents
Recombinant Human Acetylcholinesterase (rhAChE, catalog number 7574-CE, expressed in
0
Chinese hamster ovary cells) was purchased from R&D Systems. 5,5 -Dithiobis(2-nitrobenzoic acid)
(
DTNB, Ellman’s reagent), acetylthiocholine iodide (ACTI), and the methyl paraoxon used as reference
AChE inhibitor were purchased from Sigma-Aldrich (Schnelldorf, Germany).
3
.4.2. In Vitro Acetylcholinesterase Activity Assay
The inhibitory activities of the synthesized compounds on hAChE were evaluated using
a colorimetric Ellman-based method, with methyl paraoxon as the reference compound [43].
The inhibitors were dissolved in DMSO, acetylthiocholine iodide (ACTI, 20 mM) was dissolved
in distilled water, and the dithiobis(2-nitrobenzoic acid) (DTNB, 10 mM) and methyl paraoxon (20 mM)
were dissolved in assay buffer. Prior to use, all refrigerated solutions were allowed to reach room
temperature. Enzyme (100
µL, 0.2 µg/mL) and inhibitor solutions (20 µL) were added into a cuvette
containing 4.0 mL assay buffer (100 mM sodium phosphate, pH 7.4/1 mM EDTA). After 10 min incubation
at room temperature, required aliquots of the DTNB (20
µ
L) and the ACTI (20
µ
L) solutions were
◦
added. After gently mixing, the total mixture was incubated at 37 C for 35 min. The absorption was
measured at 405 nm on a Shimadzu 3101PC UV-vis spectrophotometer and corrected for background.
The specific inhibition extents were calculated in reference to measurements performed under the same

Molecules 2019, 24, 2306
17 of 20
conditions in the absence of inhibitor and expressed as percentage inhibition. For comparative purposes,
the standard inhibitor, methyl paraoxon, was tested concomitantly, giving >95–100% inhibition in all
instances. The enzyme activity was determined in the presence of concentrations up to 50 µM for any
given inhibitor. The same concentrations were used for methyl paraoxon. The measurements were
conducted immediately after sample preparation. Inhibition curves were produced to calculate the
IC50 values.
4
. Conclusions
In summary, we synthesized a series of fused thiazolo[3,2-a]pyrimidines in good yields from
3
2
,4-dihydropyrimidinethione precursors. All compounds were fully characterized by 1D- and
D-NMR, high resolution ESI-MS/MS, and single crystal X-ray diffraction analysis, which confirmed a
regioselective 5H cyclization of the DHPMs. All target compounds were screened in vitro as hAChE
inhibitors via an Ellman-based colorimetric assay and showed good inhibition activities (better than
7
0%) at 10 µM, with IC s in the 1 µM range. Molecular docking simulations for all target products
50
into hAChE were performed and confirmed strong binding to the peripheral anionic site of the enzyme,
at the mouth of the catalytic gorge, which funnels access to the catalytic site. These preliminary data
set the ground for further improvement of the core structure and provide a new and promising starting
point to develop new acetylcholinesterase inhibitors and explore novel treatment options against
Alzheimer’s disease.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: A.M.E., A.-E.A.H and M.M.M. designed the study. M.Y.M. was responsible for the
synthesis, NMR characterization, Hirshfeld surface analysis, and in vitro hAChE inhibition studies. He wrote the
preliminary draft of the manuscript. J.L.F.d.S was responsible for the X-ray characterization studies and wrote the
corresponding sections. G.C.J. performed the docking studies and wrote the corresponding section. A.M.E. and
M.M.M. critically reviewed and edited the whole manuscript, with input from all the authors.
Funding: This work was supported in part by Fundação para a Ciência e a Tecnologia (FCT, Portugal),
through grants UID/QUI/00100/2013, UID/QUI/100/2019, SAICTPAC/0019/2015, and PTDC/QUI-QAN/32242/2017,
funded by national funds through FCT/MEC and when appropriate co-financed by FEDER under the PT2020
Partnership Agreement. MYM also thanks the Egyptian Higher Education Ministry, Cultural Affairs, and Missions
sector for financial support. Thanks are also due to the Portuguese Mass Spectrometry Network (Grant
LISBOA-01-0145-FEDER-022125).
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.
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Sample Availability: Samples of the compounds 4a–b and 7a–d are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(