Novel N-benzylpiperidine carboxamide derivatives as potential cholinesterase inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.06.088

A series of fifteen acetylcholinesterase inhibitors were designed and synthesised based upon the previously identified lead compound 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl 1-benzylpiperidine-4-carboxylate (5) which showed good inhibitory activity (IC₅₀ 0.03 ± 0.07 μM) against acetylcholinesterase. A series of compounds were prepared wherein the ester linker in the original lead compound was exchanged for a more metabolically stable amide linker and the indanone moiety was exchanged for a range of aryl and aromatic heterocycles. The two most active analogues 1-benzyl-N-(5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-yl)piperidine-4-carboxamide (28) and 1-benzyl-N-(1-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) piperidine-4-carboxamide (20) afforded in vitro IC₅₀ values of 0.41 ± 1.25 and 5.94 ± 1.08 μM, respectively. In silico screening predicts that 20 will be a blood brain-barrier permeant, and molecular dynamic simulations are indicative of a close correlation between the binding of 20 and the Food and Drug Administration-approved cholinesterase inhibitor donepezil (1).

Full text of DOI:10.1016/j.ejmech.2019.06.088

Accepted Manuscript
Novel N-benzylpiperidine carboxamide derivatives as potential cholinesterase
inhibitors for the treatment of Alzheimer's disease
Divan G. van Greunen, C. Johan van der Westhuizen, Werner Cordier, Margo Nell,
Andre Stander, Vanessa Steenkamp, Jenny-Lee Panayides, Darren L. Riley
PII:
S0223-5234(19)30615-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.088
EJMECH 11491
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 May 2019
Revised Date: 28 June 2019
Accepted Date: 28 June 2019
Please cite this article as: D.G. van Greunen, C. Johan van der Westhuizen, W. Cordier, M. Nell, A.
Stander, V. Steenkamp, J.-L. Panayides, D.L. Riley, Novel N-benzylpiperidine carboxamide derivatives
as potential cholinesterase inhibitors for the treatment of Alzheimer's disease, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.06.088.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel N-benzylpiperidine carboxamide derivatives as potential
cholinesterase inhibitors for the treatment of Alzheimer’s disease
Divan G. van Greunen,a C. Johan van der Westhuizen,a Werner Cordier,b Margo Nell,b Andre Standerc
Vanessa Steenkamp,b Jenny-Lee Panayides,d* and Darren L. Riley.a*
aDepartment of Chemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria,
Lynnwood Road, Pretoria, South Africa
bDepartment of Pharmacology, Faculty of Health Sciences, University of Pretoria, Bophelo Road,
Pretoria, South Africa
cDepartment of Physiology, Faculty of Health Sciences, University of Pretoria, Lynnwood Road,
Pretoria, South Africa
dPioneering Health Sciences, CSIR Biosciences, Meiring Naudé Road, Pretoria, South Africa
* Corresponding authors: darren.riley@up.ac.za (D.L. Riley) jpanayides@csir.co.za (J.-L. Panayides)
ABSTRACT
A series of fifteen acetylcholinesterase inhibitors were designed and synthesised based upon the
previously identified lead compound 5,6-dimethoxy-1-oxo-2,3-dihydro-1H-inden-2-yl 1-
benzylpiperidine-4-carboxylate (5) which showed good inhibitory activity (IC50 0.03 ± 0.07 µM)
against acetylcholinesterase. A series of compounds were prepared wherein the ester linker in the
original lead compound was exchanged for a more metabolically stable amide linker and the
indanone moiety was exchanged for a range of aryl and aromatic heterocycles. The two most active
analogues 1-benzyl-N-(5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-yl)piperidine-4-carboxamide (28)
and 1-benzyl-N-(1-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) piperidine-4-carboxamide
(20) afforded in vitro IC50 values of 0.41 ± 1.25 and 5.94 ± 1.08 µM, respectively. In silico screening
predicts that 20 will be a blood brain-barrier permeant, and molecular dynamic simulations are
indicative of a close correlation between the binding of 20 and the Food and Drug Association-
approved cholinesterase inhibitor donepezil (1).
Key Words: Acetylcholinesterase, Alzheimer’s disease, benzylpiperidine, butyrylcholinesterase,
carboxamide, cytotoxicity, donepezil.
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1. Introduction
Alzheimer’s disease (AD) is the most common form of dementia accounting for an estimated 60 –
80% of all cases in the United States of America (USA) [1]. In 2017, it was estimated that 5.3 million
Americans aged 65 and older were living with AD, which is predicted to increase to 13.8 million by
2050 [1].
Alzheimer’s disease is neurodegenerative in nature and characterised by a gradual onset and
progression of deficits in more than one area of cognition, including disruption in behavioural,
language and memory skills [2, 3]. To date, consensus on the cause of AD has not been reached,
however, several hypotheses have been put forward based upon the various pathophysiological
factors observed. One of the most widely investigated being the cholinergic hypothesis [3, 4], which,
is linked to the observation that AD is characterised by a deficiency of the neurotransmitter,
acetylcholine. The deficiency arises either as a result of a decreased production of acetylcholine, or
through the amplification of the enzymatic degradation thereof by acetylcholinesterase (AChE), and
to a lesser extent, butyrylcholinesterase (BuChE) [5, 6]. Furthermore, AChE has also been shown to
bind to amyloid-ß (Aβ) and play a role in the deposition of Aβ plaques, another hallmark of AD [7]. As
a result, the inhibition of AChE is regarded as an important neuroprotective target in AD drug
discovery, acting by increasing relative acetylcholine concentrations in the brain and reducing the
deposition of toxic Aβ plaques [7].
Currently, three AChE inhibitors, donepezil 1, rivastigmine 2 and galantamine 3 (Figure 1), are
approved by the US Food and Drug Administration (FDA) for the treatment of the symptoms
associated with mild to advanced AD, [8] with a fourth, tacrine 4, having previously been
discontinued due to concerns linked to hepatotoxicity [9].
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Figure 1. Current and former FDA approved AChE inhibitors for the treatment of AD [8].
The AChE enzyme consists of two domains, the active catalytic anionic site (CAS) at the bottom of
the pocket and a peripheral anionic site (PAS) at the entrance to the binding pocket [10]. During the
development of donepezil 1, Sugimoto and co-workers focused largely on the optimisation of
binding in the CAS, showing that an N-benzylpiperidine moiety was critical for activity affording
strong binding interactions with the Trp84, Trp279, Phe330 and Phe331 residues in the active site. In
addition, the dimethoxy indanone moiety has indicated binding interactions with Phe288 and Trp279
in the PAS site [10].
Subsequent structure activity relationship (SAR) studies have been undertaken involving the
exploration of chemical space surrounding donepezil 1. In several examples, the indanone moiety
has been replaced with different heterocycles including coumarin, isoindoline-1,3-dione and
phthalimide [11-14] and in some cases have shown that concomitant inhibition of Aβ aggregation is
linked to systems displaying binding to the PAS site [12, 13].
In previous work reported by our group, the N-benzylpiperidine ring system of donepezil 1 was
replaced with a range of different saturated N-benzylated ring systems and the methyl bridge
between the indanone and the N-benzylpiperidine ring system with various different linkers (Figure
2) [15]. The SAR conducted identified compound 5 as the most active ligand with a half-maximal
inhibitory concentration (IC50) of 0.03 ± 0.07 µM with no observable cytotoxicity in the SH-SY5Y
neuroblastoma cell line (IC50 of >100 µM). In the present study, compound 5 was modified by
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replacing the ester linkage (Part B) with that of an amide linker to reduce the metabolic liability
associated with the ester. Furthermore, to explore the effect of binding at the PAS site, the indanone
moiety (Part A) was replaced with several aromatic and aryl heterocycles.
Figure 2. Progression from previously reported work involving the incorporation of an amide linker
and the replacement of the indanone moiety with different heterocyclic systems [15].
In this study, the synthesis of fifteen novel analogues of donepezil 1 are described, and in vitro
inhibition against AChE and BuChE as well as cytotoxicity are reported. In addition, in silico
assessment of compound-binding using molecular docking and molecular dynamic (MD) simulations,
is described.
2. Chemistry
The preparation of the desired carboxamide derivatives was envisaged using standard amide
coupling chemistry to allow the coupling of imidazole 6 with various commercial and synthetic
heterocyclic amines. The N-benzylpiperidine imidazole derivative 6 was prepared in three steps from
ethyl isonipecotate 7, [16, 17] initial treatment with benzyl chloride afforded the N-benzylated ester
8, thereafter, hydrolysis in the presence of methanolic sodium hydroxide afforded acid 9 (Scheme 1).
Subsequent treatment with carbonyl diimidazole afforded the imidazole derivative 6 in a 71% yield
over three steps.
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Scheme 1: (i) 1.05 eq. BnCl, 3 eq. Et3N, CH3CN, reflux, 3 h, 88%, (ii) 2 eq. NaOH, MeOH, reflux, 24 h,
98%, (iii) 1.2 eq. carbonyl diimidazole, CH2Cl2, 0°C, 2 h, 82%.
Initially, it was envisaged to use this approach to access the amide analogue 10 of previously
identified compound 5. To access the required amine 11, 5,6-dimethoxy-1-indanone 12 was treated
with isopentyl nitrite in an acidic methanolic solution affording the oximino compound 13 as a
precipitate in 81% yield (Scheme 2) [18]. Thereafter, catalytic reduction under acidic conditions
afforded the amine as an ammonium salt 11 in 95% yield [19]. Unfortunately, the subsequent
coupling attempts proved unsuccessful.
Scheme 2: (i) 1 eq. isopentyl nitrite, conc. HCl, MeOH, 40 °C, 30 min, 81%, (ii) H2, Pd/C, 1.5 eq. conc. HCl,
EtOH, rt, 1 h, 95%, (iii) 6 eq. NaH, DMSO, rt, 3 h, 0%.
Interestingly, the difficulties noted with the preparation of 10 did not appear to be general, and a
range of carboxamide derivatives 14 - 28 were readily prepared by coupling 6 with commercial and
synthetic amines under mild conditions in the presence of catalytic 4-(dimethylamino)pyridine (4-
DMAP) (Scheme 3, Table 1) in low to good yields [20].
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Scheme 3: (i) 1 eq. R-NH2, 0.6 eq. 4-DMAP, 1 eq. Et3N, CH2Cl2, rt – reflux, 12 h.
In the case of carboxamide 28, the required substituted aminothiazole heterocycle 29 was prepared
by bromination of 5,6-dimethoxy-1-indanone 12, [15, 21] affording the α-halogenated indanone 30
followed by treatment with thiourea to afford 29 in a 70% yield across two steps (Scheme 4).
Thereafter, the coupling required the initial deprotonation of amine 29 with sodium hydride prior to
reaction with 6 affording carboxamide 28 in 12% yield.
Scheme 4: (i) 1.05 eq. Br2, MeOH, rt, 30 min, 84%, (ii) 1 eq. thiourea, 5 eq. 3M aq. NH3, H2O, rt-reflux,
12h, 83%.
3. Structure activity relationship study
The inhibitory activity of all compounds was assessed against Electrophorus electricus AChE (EeAChE)
and BuChE from equine serum (eqBuChE) using Ellman’s spectrophotometric method [22] with some
minor modifications, [23] using galantamine as the positive control. The anticholinesterase activities
for the synthesised compounds expressed as IC50 values are provided in Table 1. In addition, all
compounds were assessed for cytotoxicity in the SH-SY5Y neuroblastoma cell line using the
sulforhodamine B (SRB) staining assay as described by Vichai and Kirtikara, [24] although the SH-SY5Y
cell line is cancerous in nature, it does present as a model of a neurological cellular environment [24].
Table 1. In vitro AChE, BuChE inhibitory activity and cytotoxicity of compounds 14 - 28.
EeAChE
IC50 ± SEM
(µM)
eqBuChE
IC50 ± SEM
(µM)
Cytotoxicity
IC50 ± SEM
(µM)
Compound
R
Yield (%)
91
14
25.60 ± 1.10
>100
40.48 ± 1.21
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15
16
90
85
13.98 ± 1.16
>100
>100
13.57 ± 1.15
73.42 ± 1.35
>100
17
18
19
99
59
93
44.39 ± 1.17
51.10 ± 1.12
>100
>100
>100
>100
50.90 ± 1.58
76.62 ± 1.11
>100
20
34
5.94 ± 1.08
>100
>100
21
22
77
59
30.88 ± 1.19
58.89 ± 1.15
>100
>100
>100
>100
23
24
77
97
58.18. ± 1.14
>100
>100
>100
3.37 ± 1.19
>100
25
26
53
38
>100
>100
>100
>100
99.32 ± 1.32
>100
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27
28
59
12
82.10 ± 1.14
0.41 ± 1.25
>100
>100
>100
31.31 ± 1.32
-
-
Donepezil 1
0.08 ± 0.01
3.58 ± 1.24
5.20 ± 0.01 95.13 ± 1.30
Galantamine 3
-
-
Two compounds were found to have EeAChE IC50 values of less than 10 µM, namely 20 (IC50 = 5.94
µM) and 28 (IC50 = 0.41 µM), with the latter having 5-fold less activity than donepezil. In both
instances, 20 and 28 have a proton acceptor in a similar position to the indanone carbonyl in 1 and 5,
suggesting, that although the PAS is not the primary binding site, it is still crucial for the
development of efficient AChE inhibitors. It was further noted, that the position of the h-bond
acceptor binding to the PAS site appears to play a role in inhibition. When the nitrogen was changed
from an ortho position to that of meta or para position in 21 – 23, an approximate 50% decrease in
activity was noted. Interestingly, thiazole 18 exhibited an IC50 of 51.10 µM, however, when
converted to the analogous tricyclic 28 there was a 125-fold increase in activity to 0.41 µM. This
trend suggests that binding in the PAS is highly susceptible to subtle changes in the electronic nature
of the proton acceptor and/or that the additional aromatic ring systems are improving binding,
through π-π stacking. In all cases, activity was selective for AChE with no observable inhibition of
BuChE below 100 µM, furthermore, compounds 20 and 28 did not display appreciable cytotoxicity
with IC50 values of 31.31 ± 1.32 µM (selectivity index of 76) for 28 and >100 µM for 20.
4. In silico screening
4.1. Molecular docking
Preliminary estimation of the binding affinity of the compounds was calculated using the binding
energy according to the combined Molecular Mechanics-Generalized Born Surface Area (MM-GBSA)
method. Molecular docking was performed using the crystal structure of Torpedo eel AChE (PDB:
1EVE) [25] using Glide XP and Prime MM-GBSA from the Schrödinger program suite [26, 27]. The
binding pocket comprises of the CAS containing Trp84 and Phe330, while the PAS includes the Tyr70,
Asp72, Try 121, Tyr334 and Trp279 residues.
The crystal structure contains donepezil 1 bound into the 20 Å deep gorge which is highly solvated,
with the R-configuration of the alpha carbon of the indanone carbonyl being noted. Donepezil 1 was
docked back into the crystal structure and it was noted that several of the water molecules needed
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to be included during the docking process to obtain the correct binding pose (RMSD of redocked
pose: 0.918 Å). This resulted in water-bridged interactions between the ligand and the residues
within the binding pocket. Thus, the water bridge network was kept as all the synthesised ligands
have an N-benzylpiperidine moiety in common with donepezil 1 around which several water
molecules are located.
The common scaffold, N-benzylpiperidine found in donepezil 1 and the synthesised compounds
produced similar poses and interactions, with the exception of compounds 16, 21 and 27, which
formed binding poses in the reverse orientation. These poses had N-benzylpiperidine interactions
with the PAS residues and the analogue structures deep within the binding pocket at the CAS. When
all the structural waters within the binding pocket were included, docking and MM-GBSA
calculations provided similar energies for the ligands entering the pocket from different orientations.
Thus, poses were visually inspected to select the correct conformation. The synthesised ligands with
the correct binding pose had comparable binding energy when compared with that of donepezil 1.
For example, the binding energy of 28 was -66.4 kcal/mol whereas donepezil 1 was -63.4 kcal/mol.
An overlay of the best pose for donepezil 1, 20 and 28 (which displayed the highest inhibitory
activity against AChE) shows that the N-benzylpiperidine moiety, which is common to all three,
overlaps almost perfectly (Figures 3 and 4) with analogous π-π stacking interactions between the
benzyl ring and Trp84. The charged nitrogen on the piperidine interacts with Trp84, Tyr334 and
Phe330 via π-cation interactions and hydrogen bonding through a water bridge is noted for the
protonated nitrogen to Try121. While donepezil 1 forms a second water bridge to Phe288 and
Phe331, 28 has a second hydrogen bond to Tyr121. The primary amide of 28 lies perpendicular
between the two cyclic structures (piperidine and tricyclic indenothiazole ring systems), which allows
the nitrogen to form a hydrogen bond with the hydroxyl on Tyr121 at 2.18 Å. Further π-π stacking is
noted between compounds 1 and 28 with the PAS residue Trp279. In the case of 28 a second π-π
stacking interaction with Tyr121 is observed which is not possible with ligand 1, as the
cyclopentanone substructure within indanone ring of 1 is not aromatic, suggesting that the
aforementioned differences between compounds 18 and 28 (Table 1) are arising due to additional π-
π stacking interactions present in 28. In contrast, compound 20 and donepezil 1 share mostly similar
interactions with Trp84, Tyr334, Phe330, Trp84, Phe288, Phe331 and Trp279 (Figures 3 and 4).
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Figure 3. Overlaid docked poses of donepezil 1 (green) and compound 28 (white) in torpedo eel
AChE. In both instances’ compounds have π-π stacking interactions with Trp279 and Trp84 shown in
blue, and the π-cation interactions with Tyr334, Phe330 and Trp84 shown in green. HOH1159 acts as
a water bridge between Tyr121 and the protonated nitrogen on the piperidine ring of both
compounds shown in yellow. Compound 1 forms an additional water bridge with HOH1254 to
Phe288 and Phe331, while 28 has a hydrogen bond between the secondary amine and Trp121 as
well as has π-π stacking between the heterocycle and Tyr121. Only selected residues and water
molecules are shown within the binding pocket for clarity.
Figure 4. Overlaid docked pose of donepezil 1 (green) and compound 20 (white) in torpedo eel AChE.
Both compounds have π-π stacking interactions with Trp84 shown in blue, while donepezil has an
additional stacking interaction with Trp279. π-Cation interactions with Try334, Phe330 and Trp84 for
both complexes are shown in green. Furthermore, both compounds share water bridged
interactions with HOH1254 to Phe288 and Phe331. Only selected residues and water molecules are
shown within the binding pocket for clarity.
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4.2. Molecular dynamics simulations
In order to assess how ligand-enzyme interactions vary over time, 40 ns molecular dynamic
simulations were performed at 310 °K for AChE complexed with 1, 28 and 20. Analysis of the
interactions between the compound and the protein during the simulation were done using the
simulation interaction diagrams tool of Schrödinger, as depicted in Figure 5 (a-c), for 1, 28 and 20
respectively (refer to Figure 6 for a schematic representation of the various interactions). Compound
28 is shown to have an increased frequency of interactions with various residues (Try70, Asp72, Tyr
121, Ser286, Phe330, Phe331 and Tyr334) but a decrease in interaction frequency was noted with
Trp279 and Phe288 when compared to that of donepezil 1. This loss in interaction frequency for 28
is ascribed to the tricyclic indenothiazole ring system which spins/rotates during the simulation and
does not show any preference to stay in a fixed planar orientation. Interestingly, compound 20 is
shown to have increased frequency of interactions with Tyr121, Phe288, Arg289, Phe330, Phe331
and Tyr334 when compared with donepezil 1, and unlike 28 there are no major losses in
interactions.
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Figure 5. Protein-ligand contacts monitored during a 40 ns MD simulation at 310 K for the (a) AChE-1
complex, (b) AChE-28 complex and (c) AChE-20 complex. The contacts are categorised into four
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types: hydrogen bonds (green), hydrophobic (purple), ionic (red) and water bridges (blue), with the
frequency of the contacts which the ligand makes residues in the binding pocket shown. The stacked
bar charts of the contacts are normalised, with values over 1.0 representing protein residues that
make multiple contacts with the ligand.
Figure 6. Schematic representation showing interactions which the ligands (a) donepezil 1, (b) 28
and (c) 20 makes with the surrounding residues of AChE during a 40 ns MD simulation at 310 K.
Interactions that occur more than 14% of the simulation time are shown.
The variation of the dihedral angle between the PAS binding heterocycle (Part A, figure 2) and the
CAS binding benzyl piperidine group or 1, 28 and 20 (Figure 6) affords a more in-depth
understanding of the ligand-enzyme interactions. It is apparent that 1 prefers a torsion of
approximately -90° due to the water bridged assisted hydrogen bond between the ketone of the
indanone and Phe288, preventing the indanone from rotating during the simulation. A similar affect
is noted for 20 where the torsion is also kept at approximately -90° due to the water bridges
between the cyclic amide carbonyl and Arg289 and Phe288. In contrast, in the case of 28, the
carbonyl present is located closer to the piperidine ring than in the case of 1, preventing interactions
with Phe288, instead forming infrequent interactions with Tyr70 and Asp72. Furthermore, although
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the nitrogen and sulphur atoms within the tricyclic ring system can act as h-bond acceptors they do
not show a clear interaction preference, resulting in the indeno-thiazole tricyclic system rotating by
~ 310˚ during the simulation for 28.
Figure 6. Schematic representation showing selected torsion of (a) donepezil 1, (b) 27 and (c) 20
during the 40 ns MD simulation at 310 K bound to AChE. Each colour represents a separate torsion
for the ligands.
4.3. Pharmacokinetics, ADME parameters and drug-like nature
The library of compounds was further assessed in terms of physiochemical descriptors, predictive
absorption, distribution, metabolism and excretion (ADME) parameters and pharmacokinetic
properties using the Swiss Institute of Bioinformatics SwissADME web tool [28]. All compounds
prepared showed good druglikeness in terms of the Lipinski guidelines and no pan assay interference
structures (PAINS) were noted [29]. Ligands 14 - 27 were computed to have total polar surface areas
(TSPA) in the range of 32 to 73 Å and a WLogP in the range of 0.43 to 3.56, suggesting that they will
act as blood brain barrier (BBB) permeants (Figure 7). In the case of the most active ligand identified
28 (IC50 0.41 µM), a TPSA of 91.93 Å is indicative of a non-BBB permeant (requires a TPSA < 80 Å).
However, as is the case with the other ligands prepared, gastrointestinal absorption is predicted to
be high. All ligands are predicted to be P-glycoprotein substrates except for compound 19 and
previously reported compound 5, suggesting that absorption from the gastrointestinal tract and
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across the BBB may be compromised as compounds may be effluxed, thus decreasing bioavailability.
The physiochemical descriptors for the two most active ligands 20 and 28, compound 5 and
donepezil 1 are summarised in Table 2. Ligand 20 (IC50 5.94 µM) which indicated 15-fold less activity
than 28 is arguably more attractive as a lead-like compound with a TPSA and WLogP which places
the ligand well within the range for BBB-permeants and with a molecular mass that is 45.07 g.mol-1
less than that of 28.
Table 2. Physiochemical descriptors of selected ligands
Compound
MW
Num.
H-bond
H-bond
donors
TPSA (Å)
WLogP
(g/mol)
rotatable
acceptors
bonds
5
20
409.47
404.50
449.57
379.49
7
6
7
6
6
3
5
4
0
1
1
0
65.07
59.27
91.93
38.77
2.73
2.61
3.86
3.83
28
Donepezil 1
Figure 7. Boiled egg plot of WLogP vs. TPSA, yellow indicates BBB-permeant, white indicates
gastrointestinal permeant, blue indicates P-glycoprotein +, red indicates P-glycoprotein -.
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5. Conclusion
A small library of fifteen potential AChE inhibitors was designed and prepared based upon the
molecular skeleton of a potent lead compound 5 (IC50 0.03 µM) which we previously reported.
Analogues were prepared by changing the ester linker present in 5 to that of an amide linker to
reduce the metabolic liability associated with the ester functional group. Introduction of the amide
linker appears to reduce activity, although it should be noted that the direct amide analogue 10 of
compound 5 could not be accessed synthetically. That been said, two potential lead compounds
were identified with AChE inhibitory IC50 activities below 10 µM, namely 20 (5.94 µM) and 28 (0.41
µM). The SAR and molecular modelling simulations suggest that although the use of the benzyl
piperidine group affords distinctive binding to the CAS, concomitant binding to the PAS is critical for
the development of an efficient inhibitor. This is evidenced as activities ranged from 0.41 µM to >
100 µM (deemed not active), even though all compounds shared a common benzyl piperidine group
with simple variations to the group at the PAS site. In the case of the most active compound 28, in
silico assessments revealed that 28 and donepezil 1 share similar binding interactions. MD
simulations, however, revealed that there are subtle differences in interactions, most notably, the
indanone moiety in donepezil 1 is rigidly bound within the PAS site, whereas the tricyclic
indenothiazole ring system of 28 appears to be able rotate by ~ 310˚. The observed rotation arises as
there is no clear interaction preference between the nitrogen and sulfur atoms of the thiazole ring.
In contrast, compound 20 approximates the binding of donepezil 1 more closely, showing increased
frequency of interactions with Tyr121, Phe288, Arg289, Phe330, Phe331 and Tyr334 and no major
losses in frequency of interactions with other residues. In silico ADME predictions suggest that
compound 20, although less active, is potentially more attractive as a lead compound with WLogP
and TPSA values which indicative of being a potential BBB permeant. In addition, 20 has a molecular
weight which is 45.07 g.mol-1 less than that of 28 and has less H-bond acceptors suggesting that it is
more attractive as a lead compound.
6. Experimental
6.1. Chemistry
6.1.1. General methods
All solvents, chemicals, and reagents were obtained commercially and used without further
purification. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were recorded on Bruker AVANCE-III-
300 instrument using CDCl3 and DMSO-d6. CDCl3 contained tetramethylsilane as an internal
standard. Chemical shifts, δ, are reported in parts per million (ppm), and splitting patterns are given
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as singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m). Coupling constants, J, are
expressed in hertz (Hz). Mass spectra were recorded in ESI mode on a Waters Synapt G2 Mass
Spectrometer at 70 eV and 200 mA. Samples were prepared in acetonitrile (containing 0.1% formic
acid) to an approximate concentration of 10 μg/mL. Infrared spectra were run on a Bruker ALPHA
Platinum ATR spectrometer. The absorptions are reported on the wavenumber (cm-1) scale, in the
range 400 - 4000 cm-1. Melting points were measured on a Stuart Melting Point SMP10 microscope
and are uncorrected. The retention factor (Rf) values quoted are for thin layer chromatography (TLC)
on aluminium-backed Macherey-Nagel ALUGRAM Sil G/UV254 plates pre-coated with 0.25 mm silica
gel 60, spots were visualised with UV light and basic KMnO4 spray reagent. Chromatographic
separations were performed on Macherey-Nagel Silica gel 60 (particle size 0.063 – 0.200 mm). Yields
refer to isolated pure products unless stated otherwise.
6.1.2. Ethyl 1-benzylpiperidine-4-carboxylate 8
A mixture of ethyl isonipecotate 7 (10.00 g, 63.61 mmol, 1 eq.), benzyl chloride (7.69 mL, 66.79
mmol, 1.05 eq.) and triethylamine (26.45 mL, 190.8 mmol, 3 eq.) in anhydrous acetonitrile (100 mL)
were refluxed for 3 h after which time the reaction mixture was left to cool to room temperature
and the solvent was evaporated in vacuo. The obtained oil was diluted with ethyl acetate (100 mL)
and washed with a 10% aqueous solution of sodium hydroxide (3 x 50 mL) and water (50 mL). The
organic layer was dried (Na2SO4), filtered and evaporated in vacuo to afford ethyl 1-benzylpiperidine-
4-carboxylate 8 as an orange oil (88%) which was used as is without any further purification; Rf 0.42
(1:3 ethyl acetate: hexane); νmax (neat)/cm-1 2944, 2801, 2760, 1728, 1448, 1168, 1046, 736, 698; 1H
NMR (300 MHz, CDCl3); δ 7.41 – 7.15 (m, 5H, ArH’s), 4.13 (t, 2H, J = 7.1 Hz, OCH2CH3), 3.51 (s, 2H,
CH2Ph), 2.87 (dt, 2H, J = 11.4, 3.1 Hz, CH2N), 2.30 (tt, 1H, J = 10.9, 4.2 Hz, CHCO), 2.05 (td, 2H, J =
11.3, 2.7 Hz, CH2N), 1.95 – 1.71 (m, 4H, CH2), 1.26 (t, J = 7.1 Hz, OCH2CH3); 13C NMR (75 MHz, CDCl3);
δ 175.33, 138.49, 129.16, 128.28, 127.07, 63.35, 60.33, 53.02, 41.32, 28.39, 14.32; HRMS m/z (ESI)
248.1682 ([M + H]+ requires 248.1645).
6.1.3. 1-Benzylpiperidine-4-carboxylic acid 9
To a stirring solution of sodium hydroxide (3.44 g, 86.02 mmol, 2 eq.) in methanol (100 mL) at room
temperature was added ethyl 1-benzylpiperidine-4-carboxylate 8 (10.01 g, 41.82 mmol, 1 eq.) in one
portion. The resulting mixture was heated to reflux temperature and left to stir overnight. The
resulting solution was cooled to room temperature and the solvent was evaporated in vacuo to
afford a yellow solid. The solid was re-dissolved in distilled water (100 mL) and the pH adjusted to
5.5. The solvent was removed in vacuo to afford a yellow solid. A pre-mixed solution of ethanol:
chloroform (1:1, 200 mL) was added and the mixture was left to stir for 1 h after which time the
solids were collected by filtration and washed with chloroform (10 mL). The filtrate was collected,
17

ACCEPTED MANUSCRIPT
and the solvent was evaporated in vacuo to afford 1-benzylpiperidine-4-carboxylic acid 9. As an off-
white solid (98%) which was used as is without any further purification. (Yield 98%); Off-white solid;
Rf 0.07 (4:1 dichloromethane: methanol); mp 168 °C; νmax (neat)/cm-1 2969, 2864, 1602, 1448, 1382,
921, 756, 702, 659; 1H NMR (300 MHz, CDCl3); δ 10.71 (s, 1H, OH), 7.38 – 7.27 (m, 5H, ArH’s), 3.89 (s,
2H, CH2Ph), 3.14 (d, 2H, J = 11.2 Hz, CH2N), 2.46 (t, 2H, J = 8.9 Hz, CH2N), 2.34 – 2.21 (m, 1H, CHCO),
13
2.07 – 1.82 (m, 4H, CH2); C NMR (75 MHz, CDCl3); δ 178.88, 132.25, 130.75, 128.74, 60.81, 51.50,
40.68, 26.72; HRMS m/z (ESI) 220.1353 ([M + H]+ requires 220.1332).
6.1.4. (1-Benzylpiperidin-4-yl)(1H-imidazol-1-yl)methanone 6
1-Benzylpiperidine-4-carboxylic acid 9 (6.24 g, 28.48 mmol, 1 eq.) in dry dichloromethane (100 mL)
was cooled to 0 °C, and carbonyl diimidazole (5.57 g, 34.36 mmol, 1.2 eq.) was added portion-wise
over 5 min. After 2 h, the mixture was diluted with dichloromethane (10 mL), and water (100 mL)
was added carefully. The organic layer was washed with water (2 x 40 mL) and brine (30 mL), dried
(Na2SO4) and evaporated in vacuo to afford (1-benzylpiperidin-4-yl)(1H-imidazol-1-yl)methanone 6
as an off-white solid (82%) which was used as is without any further purification; mp 119-121 °C;
1
νmax (neat)/cm-1 2932, 1604, 1447, 1383, 1065, 922, 825, 753; H NMR (300 MHz, CDCl3); δ 8.16 (s,
1H, imidazole NH), 7.46 (s, 1H, imidazole CH), 7.37 – 7.23 (m, 5H, ArH’s), 7.09 (s, 1H, imidazole CH),
3.58 (s, 2H, CH2Ph), 3.00 (d, 2H, J = 11.7 Hz, CH2N), 2.99 – 2.84 (m, 1H, CHCO), 2.27 – 2.09 (m, 2H,
CH2N), 2.08 – 1.91 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); δ 171.91, 136.18, 131.25, 129.28, 128.46,
127.46, 116.15, 77.16, 63.03, 52.38, 41.65, 28.54; HRMS m/z (ESI) 270.1707 ([M + H]+ requires
270.1601).
6.1.5. 2-Oximino-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one 13
To a solution of 5,6-dimethoxy-1-indanone 12 (1.50 g, 7.82 mmol, 1.0 eq.) in methanol (25 mL) at
40°C was added 96% isopentyl nitrite (1.31 mL, 9.38 mmol, 1.2 eq.) followed by concentrated
aqueous hydrochloric acid (0.78 mL, 9.38 mmol, 1.2 eq.). The solution was stirred for 30 min during
which time a precipitate formed. The precipitate was collected and dried to yield 2-oximino-5,6-
dimethoxy-2,3-dihydro-1H-inden-1-one 13 as a yellow solid (81%) which was used as is without any
further purification; Rf 0.45 (3:1 ethyl acetate: hexane); mp 240 °C (literature [18] 227-228 °C); νmax
(neat)/cm-1 3189, 1694, 1579, 1498, 1449, 1304, 1233, 1119, 1029, 978, 915, 801, 746; 1H NMR (300
MHz, DMSO); δ 12.41 (s, 1H, NOH), 7.17 (s, 1H, ArH), 7.16 (s, 1H, ArH), 3.89 (s, 3H, OCH3), 3.82 (s,
13
3H, OCH3), 3.64 (s, 2H, CH2); C NMR (75 MHz, DMSO); δ 187.57, 155.98, 154.71, 149.32, 142.48,
130.54, 108.57, 104.51, 56.15, 56.13, 55.72, 55.69, 27.85; HRMS m/z (ESI) 292.1558 ([M + H]+
requires 292.0761).
18

ACCEPTED MANUSCRIPT
6.1.6. 2-Amino-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one hydrochloride 11
A suspension of 2-oximino-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one 13 (0.21 g, 0.93
mmol, 1.0 eq.), concentrated aqueous hydrochloric acid (0.12 mL, 1.40 mmol, 1.5 eq.) and
10% palladium on carbon (0.05 g, 50 mg.mmol-1) in ethanol (15 mL) was hydrogenated at
atmospheric pressure and room temperature until the incorporation of the hydrogen was
complete. The catalyst was then filtered off and washed with ethanol (3 x 25 mL). The
solvent of the filtrate was then removed in vacuo to afford 2-amino-5,6-dimethoxy-2,3-
dihydro-1H-inden-1-one hydrochloride 11 as a light yellow powder (95%) which was used as is
without any further purification; Rf 0.54 (3:1 ethyl acetate: hexane); mp 240 °C decomposed
(literature [19] 240 °C decomposed); νmax (neat)/cm-1 2915, 1708, 1590, 1496, 1318, 1270, 1128,
1087, 1014, 801; 1H NMR (300 MHz, DMSO); δ 8.90 (s, 3H, NH3 ), 7.20 (s, 1H, ArH), 7.14 (s, 1H, ArH),
+
+
4.17 (s, 1H, COCH), 3.88 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.46 (d, 1H, J = 7.9 Hz, CH2aCHNH3 ), 3.07
+
13
(dd, 1H, J = 16.8, 4.6 Hz, CH2bCHNH3 ); C NMR (75 MHz, DMSO); δ 198.45, 156.30, 149.66, 147.21,
126.38, 108.40, 104.30, 56.24, 56.21, 55.84, 55.81, 53.32, 31.04; HRMS m/z (ESI) 208.1000 ([M + H]+
requires 208.0968).
6.1.7. 2-Bromo-5,6-dimethoxy-1-indanone 30
To a stirring solution of 5,6-dimethoxy-1-indanone 12 (10.00 g, 52.03 mmol, 1 eq.) in methanol (100
mL), was added bromine (2.82 mL, 54.63 mmol, 1.05 eq.) drop-wise. After addition of bromine the
product precipitated out of solution. The mixture was left to stir for 30 min after which time the
precipitate was filtered off, washed with ice cold methanol (50 mL), and dried in vacuo to afford 2-
bromo-5,6-dimethoxy-1-indanone 30 as a light-yellow powder (84%) which was used as is without
any further purification; Rf 0.72 (3:1 ethyl acetate: hexane); mp 162-163 °C [15]; νmax (neat)/cm-1
2959, 2688, 1687, 1583, 1497, 1309, 1261, 1218, 1107, 1015, 727; 1H NMR (300 MHz, CDCl3); δ 7.19
(s, 1H, ArH), 6.82 (s, 1H, ArH), 4.61 (dd, 1H, J = 2.9 & 7.3 Hz, CHBr), 3.95 (s, 3H, OCH3), 3.88 (s, 3H,
13
OCH3), 3.72 (dd, 1H, J = 7.2 & 17.9 Hz, CH2aCHBr), 3.30 (dd, 1H, J = 2.98 & 17.9 Hz, CH2bCHBr); C
NMR (75 MHz, CDCl3); δ 198.17, 156.75, 150.19, 146.73, 126.44, 107.27, 105.24, 56.49, 56.46, 56.28,
56.25, 44.70, 37.86.
6.1.8. 5,6-Dimethoxy-8H-indeno[1,2-d]thiazol-2-amine 29
To a suspension of thiourea (1.97 g, 25.89 mmol, 1 eq.) in distilled water (100 mL) was added 2-
bromo-5,6-dimethoxy-1-indanone 30 (7.00 g, 25.82 mmol, 1 eq.) in three portions over 30 min at
room temperature. The resulting mixture was refluxed for 12 h. The solution was then cooled down
to room temperature and a 3 M ammonium hydroxide solution (64.55 mL, 129.10 mmol, 5 eq.) was
added and which resulted in the precipitation of the product. The resulting mixture was left to stir
19

ACCEPTED MANUSCRIPT
for 30 min. and the solids collected by filtration, followed by washing with distilled water (100 mL)
after which time the solid was dried in vacuo, affording 5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-
amine 29 as a yellow solid (83%) which was used as is without further purification; Rf 0.27 (3:1 ethyl
acetate: hexane); mp 204 °C (decomp.); νmax (neat)/cm-1 3376, 3111, 2933, 1524, 1456, 1377, 1271,
1
1203, 1148, 1097, 750; H NMR (300 MHz, DMSO-d6); δ 7.16 (s, 1H, ArH), 7.06 (s, 2H, NH2), 6.98 (s,
13
1H, ArH), 3.79 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 3.58 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6); δ
172.75, 156.01, 148.05, 146.37, 137.51, 130.80, 120.70, 110.11, 102.13, 55.96, 55.67, 31.98; HRMS
m/z (ESI) 249.0697 ([M + H]+ requires 249.0692).
6.1.9. General method for amide couplings
A mixture of amine (1.00 mmol, 1 eq.), (1-benzylpiperidin-4-yl)(1H-imidazol-1-yl) methanone 6 (1.11
mmol, 1.1 eq.), 4-dimethylaminopyridine (0.60 mmol, 0.6 eq.) and triethylamine (1.00 mmol, 1 eq.)
in anhydrous dichloromethane (15 mL) was left to stir overnight at room temperature. The mixture
was then refluxed for 2 h after which time the mixture was left to cool down to room temperature. A
solution of 3 M aqueous sodium hydroxide (50 mL) was added to the reaction mixture. The organic
layer was separated and the aqueous layer was washed with dichloromethane (2 x 30 mL). The
organic layers were combined and washed with distilled water (30 mL). The organic layer was dried
(Na2SO4), filtered and evaporated in vacuo. The product was purified by column chromatography.
6.1.9.1. N-[2-(1H-indole-3-yl)ethyl]-1-benzylpiperidine-4-carboxamide 14
(Yield, 91%); Off-white solid; Rf 0.42 (9:1 dichloromethane: methanol); mp 144 - 145 °C; νmax
(neat)/cm-1 3309, 2929, 2816, 1630, 1532, 1445, 1348, 1294, 1207, 1105, 1031, 990, 922, 741, 702;
1H NMR (300 MHz, CDCl3); δ 8.45 (s, 1H, indole NH), 8.45 (s, 1H, NHCO), 7.59 (d, 1H, J = 7.8 Hz,
ArH’s), 7.39 – 7.24 (m, 6H, ArH’s), 7.20 (t, 1H, J = 7.4 Hz, ArH), 7.11 (t, 1H, J = 7.2 Hz, ArH), 6.98 (d,
1H, J = 1.9 Hz, ArH), 5.66 (t, 1H, J = 4.5 Hz), 3.58 (q, 2H, J = 6.5 Hz, CH2NHCO), 3.51 (s, 2H, CH2Ph),
13
3.01 – 2.86 (m, 4H, CH2), 2.08 – 1.92 (m, 3H, CHCO & CH2), 1.82 – 1.62 (m, 4H, CH2); C NMR (75
MHz, CDCl3); δ 175.02, 136.54, 129.38, 128.40, 127.42, 127.37, 122.26, 122.24, 119.49, 118.79,
112.91, 111.47, 63.08, 52.94, 43.06, 39.64, 28.71, 25.38; HRMS m/z (ESI) 362.2209 ([M + H]+
requires 362.2227).
6.1.9.2. 1-Benzyl-N-[2-(naphthalen-1-ylamino)ethyl] piperidine-4-carboxamide 15
(Yield, 90%); Green solid; Rf 0.61 (9:1 dichloromethane: methanol); mp 120 - 121 °C; νmax (neat)/cm-1
1
3402, 3314, 2929, 1628, 1582, 1538, 1412, 1292, 1135, 954, 753, 689; H NMR (300 MHz, CDCl3); δ
7.89 – 7.82 (m, 1H, ArH), 7.81 – 7.74 (m, 1H, ArH), 7.47 – 7.40 (m, 2H, ArH’s), 7.36 – 7.24 (m, 6H,
ArH’s), 7.22 (d, J = 8.2 Hz, 1H, ArH), 6.51 (d, J = 7.4 Hz, 1H, ArH), 6.15 (s, 1H, NHCO), 5.20 (s, 1H, NH),
3.64 (q, J = 5.8 Hz, 2H,CH2NHCO), 3.47 (s, 2H, CH2Ph), 3.36 (t, J = 5.4 Hz, 2H, CH2NH), 2.89 (d, J = 11.5
20

ACCEPTED MANUSCRIPT
Hz, 2H, CH2N), 2.17 – 1.89 (m, 3H, CHCO & CH2N), 1.84 – 1.72 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3);
δ 176.78, 143.66, 134.39, 129.30, 128.59, 128.37, 127.33, 126.67, 125.92, 124.94, 123.36, 120.46,
117.19, 103.46, 63.02, 52.84, 45.39, 42.93, 42.89, 38.87, 28.72; HRMS m/z (ESI) 388.2424 ([M + H]+
requires 388.2383).
6.1.9.3. 1-Benzyl-N-(2-morpholinoethyl) piperidine-4-carboxamide 16
(Yield, 85%); Yellow solid; Rf 0.30 (9:1 dichloromethane: methanol); mp 89 - 90 °C; νmax (neat)/cm-1
1
3266, 2928, 1638, 1540, 1441, 1227, 1114, 1002, 913, 863, 732, 698; H NMR (300 MHz, CDCl3); δ
7.35 – 7.19 (m, 5H, ArH’s), 6.15 (s, 1H, NHCO), 3.68 (m, 4H, CH2O), 3.52 (s, 2H, CH2Ph), 3.32 (dd, 2H, J
= 11.2, 5.7 Hz, CH2NHCO), 2.94 (d, 2H, J = 11.6 Hz, CH2N), 2.49 – 2.38 (m, 6H, (CH2)2NCH2, & CH2N),
13
2.18 – 1.98 (m, 3H, CHCO & CH2N), 1.89 – 1.67 (m, 4H, CH2); C NMR (75 MHz, CDCl3); δ 175.05,
137.65, 129.33, 128.34, 127.29, 66.98, 63.14, 57.07, 53.37, 52.99, 43.01, 35.51, 28.78; HRMS m/z
(ESI) 332.2341 ([M + H]+ requires 332.2332).
6.1.9.4. 1-Benzyl-N-(2-methoxybenzyl) piperidine-4-carboxamide 17
(Yield, 99%); Yellow crystalline solid; Rf 0.50 (9:1 dichloromethane: methanol); mp 120 °C; νmax
1
(neat)/cm-1 3274, 2932, 1642, 1546, 1489, 1445, 1236, 1114, 1025, 985, 736, 696; H NMR (300
MHz, CDCl3); δ 7.34 – 7.20 (m, 7H, ArH’s), 6.89 (dd, 2H, J = 15.1, 7.7 Hz, ArH’s), 6.06 (s, 1H, NHCO),
4.43 (d, 2H, J = 5.7 Hz, CH2NHCO), 3.84 (s, 3H, CH3O), 3.50 (s, 2H, CH2Ph), 2.93 (d, 2H, J = 11.7 Hz,
13
CH2N), 2.18 – 1.93 (m, 3H, CHCO & CH2N), 1.90 – 1.67 (m, 4H, CH2); C NMR (75 MHz, CDCl3); δ
174.61, 157.61, 138.04, 129.78, 129.25, 128.90, 128.31, 127.18, 126.40, 120.78, 110.38, 63.19,
55.44, 55.38, 53.10, 43.27, 39.35, 28.86; HRMS m/z (ESI) 339.2069 ([M + H]+ requires 339.2067).
6.1.9.5. 1-Benzyl-N-(thiazol-2-yl) piperidine-4-carboxamide 18
(Yield, 59%); Off-white solid; Rf 0.50 (9:1 dichloromethane: methanol); mp 179 - 181 °C; νmax
(neat)/cm-1 3156, 2928, 1679, 1560, 1494, 1442, 1368, 1329, 1290, 1267, 1186, 1167, 1141, 1108,
1075, 1010, 964, 799, 743, 726, 700; 1H NMR (300 MHz, CDCl3); δ 12.53 (s, 1H, NHCO), 7.50 (d, 1H, J
= 3.6 Hz, CHN), 7.38 – 7.23 (m, 5H, ArH’s), 7.02 (d, 1H, J = 3.6 Hz, CHS), 3.55 (s, 2H, CH2Ph), 3.01 (d,
13
2H, J = 11.0 Hz, CH2N), 2.59 – 2.46 (m, 1H, CHCO), 2.16 – 1.84 (m, 6H, CH2N & CH2); C NMR (75
MHz, CDCl3); δ 173.68, 160.50, 138.30, 136.24, 129.15, 128.38, 127.23, 113.74, 63.27, 53.08, 43.09,
28.54; HRMS m/z (ESI) 302.1362 ([M + H]+ requires 302.1322).
6.1.9.6. 1-Benzyl-N-(3,4,5-trimethoxyphenethyl)piperidine-4-carboxamide 19
(Yield, 93%); Light yellow solid; Rf 0.56 (9:1 dichloromethane: methanol); mp 119 - 121 °C; νmax
(neat)/cm-1 3327, 2926, 1635, 1589, 1547, 1509, 1454, 1435, 1324, 1245, 1128, 999, 817, 714, 676;
1H NMR (300 MHz, CDCl3); δ 7.32 – 7.20 (m, 5H, ArH’s), 6.38 (s, 2H, ArH’s), 5.69 (s, 1H, NHCO), 3.82
(s, 6H, CH3O), 3.81 (s, 3H, CH3O), 3.53 – 3.44 (m, 4H, CH2Ph & CH2NHCO), 2.91 (d, 2H, J = 11.6 Hz,
21

ACCEPTED MANUSCRIPT
CH2N), 2.73 (t, 2H, J = 7.0 Hz, PhCH2CH2NH), 2.15 – 1.91 (m, 3H, CHCO & CH2N), 1.85 – 1.63 (m, 4H,
13
CH2); C NMR (75 MHz, CDCl3); δ 175.04, 153.34, 136.58, 134.69, 129.23, 128.33, 127.26, 105.66,
105.64, 63.09, 60.88, 56.18, 52.93, 43.07, 40.47, 36.13, 28.83; HRMS m/z (ESI) 413.2458 ([M + H]+
requires 413.2453).
6.1.9.7. 1-Benzyl-N-(1-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) piperidine-4-carboxamide
20
(Yield, 34%); Off-white crystalline solid; Rf 0.33 (9:1 dichloromethane: methanol); mp 235 - 236 °C;
νmax (neat)/cm-1 3228, 3185, 2925, 1684, 1640, 1612, 1587, 1451, 1296, 1195, 1109, 957, 746, 692;
1H NMR (300 MHz, CDCl3); δ 8.33 (s, 1H, NHCO), 7.49 – 7.19 (m, 10H, ArH’s), 3.51 (s, 2H, CH2Ph), 3.04
(d, 3H, J = 0.8 Hz, NCH3), 2.90 (d, 2H, J = 11.0 Hz, CH2N), 2.33 (dt, 1H, J = 14.4, 7.3 Hz, CHCO), 2.19 (s,
13
3H, CCH3), 2.09 – 1.92 (m, 2H, CH2N), 1.90 – 1.77 (m, 4H, CH2); C NMR (75 MHz, CDCl3); δ 174.65,
161.93, 149.69, 134.70, 129.37, 128.34, 127.22, 127.06, 124.34, 109.04, 63.05, 52.84, 42.56, 36.32,
28.77, 12.66; HRMS m/z (ESI) 405.2292 ([M + H]+ requires 405.2285).
6.1.9.8. 1-Benzyl-N-(pyridine-2-ylmethyl) piperidine-4-carboxamide 21
(Yield, 77%); Yellow viscous oil; Rf 0.33 (9:1 dichloromethane: methanol); νmax (neat)/cm-1 3343,
1
2940, 2792, 1639, 1592, 1537, 1429, 1295, 1214, 1133, 993, 732; H NMR (300 MHz, CDCl3); δ 8.50
(d, 1H, J = 4.7 Hz, ArH), 7.63 (td, 1H, J = 7.7, 1.6 Hz, ArH), 7.36 – 7.13 (m, 7H, ArH’s), 6.97 (s, 1H,
NHCO), 4.53 (d, 2H, J = 4.8 Hz, CH2NHCO), 3.53 (s, 2H, CH2Ph), 2.96 (d, 2H, J = 11.6 Hz, CH2N), 2.30 –
2.16 (m, 1H, CHCO), 2.07 (m, 2H, CH2N), 1.96 – 1.74 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); δ
175.11, 156.45, 149.02, 137.68, 136.86, 129.34, 128.35, 127.29, 122.43, 122.14, 63.11, 53.00, 44.31,
42.99, 28.74; HRMS m/z (ESI) 310.1939 ([M + H]+ requires 310.1914).
6.1.9.9. 1-Benzyl-N-(pyridine-3-ylmethyl) piperidine-4-carboxamide 22
(Yield, 59%); Yellow viscous oil; Rf 0.23 (9:1 dichloromethane: methanol); νmax (neat)/cm-1 3312,
1
3029, 2947, 2921, 2793, 2750, 1635, 1533, 1430, 1296, 1218, 1132, 1020, 978, 704, 657; H NMR
(300 MHz, CDCl3); δ 8.45 (d, 1H, J = 4.7 Hz, ArH), 8.45 (s, 1H, ArH), 7.57 (d, 1H, J = 7.8 Hz, ArH), 7.32 –
7.18 (m, 6H, ArH’s), 6.45 (s, 1H, NHCO), 4.40 (d, 2H, J = 5.9 Hz, CH2NHCO), 3.49 (s, 2H, CH2Ph), 2.92
(d, 2H, J = 11.7 Hz, CH2N), 2.22 – 2.09 (m, 1H, CHCO), 2.00 (td, 2H, J = 11.1, 3.5 Hz, CH2N), 1.87 – 1.69
13
(m, 4H, CH2); C NMR (75 MHz, CDCl3); δ 175.30, 149.05, 148.80, 137.87, 135.59, 134.35, 129.21,
128.33, 127.24, 123.68, 63.10, 52.96, 43.05, 40.83, 28.83; HRMS m/z (ESI) 310.1939 ([M + H]+
requires 310.1914).
6.1.9.10.
1-Benzyl-N-(pyridine-4-ylmethyl) piperidine-4-carboxamide 23
(Yield, 77%); Yellow viscous oil; Rf 0.33 (9:1 dichloromethane: methanol); νmax (neat)/cm-1 3290,
2917, 2800, 1638, 1542, 1428, 1374, 1302, 1073, 990, 795, 736, 693, 638; 1H NMR (300 MHz, CDCl3);
22

ACCEPTED MANUSCRIPT
δ 8.48 (d, 2H, J = 4.8 Hz, pyridine CH’s), 7.35 – 7.20 (m, 5H, ArH’s), 7.12 (d, 2H, J = 5.0 Hz, pyridine
CH’s), 6.48 (s, 1H, NHCO), 4.41 (d, 2H, J = 5.9 Hz, CH2NHCO), 3.52 (s, 2H, CH2Ph), 2.95 (d, 2H, J = 11.2
Hz, CH2N), 2.21 (ddd, 1H, J = 15.2, 10.1, 4.8 Hz, CHCO), 2.04 (td, 2H, J = 10.8, 2.6 Hz, CH2N), 1.90 –
1.73 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3); δ 175.40, 150.00, 147.82, 137.70, 129.26, 128.37,
127.32, 122.26, 63.07, 52.91, 42.99, 42.15, 28.83; HRMS m/z (ESI) 310.1939 ([M + H]+ requires
310.1914).
6.1.9.11.
1-Benzyl-N-(furan-2-ylmethyl) piperidine-4-carboxamide 24
(Yield, 97%); Off-white crystalline solid; Rf 0.46 (9:1 dichloromethane: methanol); mp 110 °C; νmax
(neat)/cm-1 3308, 2939, 2922, 2815, 2767, 1639, 1535, 1450, 1256, 1143, 1109, 1076, 1011, 981,
911, 822, 749, 697, 643; 1H NMR (300 MHz, CDCl3); δ 7.36 – 7.20 (m, 6H, ArH’s), 6.30 (dd, 1H, J = 3.1,
1.9 Hz, CHCHCH), 6.19 (d, 1H, J = 2.9 Hz, CHCHC), 6.01 (s, 1H, NHCO), 4.41 (d, 2H, J = 5.4 Hz, CH2),
3.51 (s, 2H, CH2Ph), 2.94 (d, 2H, J = 11.7 Hz, CH2N), 2.20 – 1.95 (m, 3H, CHCO & CH2N), 1.89 – 1.68 (m,
4H, CH2); 13C NMR (75 MHz, CDCl3); δ 174.83, 151.43, 142.23, 137.87, 129.26, 128.34, 127.25,
110.53, 107.47, 63.12, 52.97, 43.03, 36.49, 28.75; HRMS m/z (ESI) 299.1740 ([M + H]+ requires
299.1754).
6.1.9.12.
1-Benzyl-N-(1,2,3,4-tetrahydro-naphthalen-1-yl) piperidine-4-carboxamide 25
(Yield, 53%); Pink crystalline solid; Rf 0.56 (9:1 dichloromethane: methanol); mp 135 - 136 °C; νmax
1
(neat)/cm-1 3256, 2929, 1637, 1539, 1446, 1223, 1119, 963, 737, 695; H NMR (300 MHz, CDCl3); δ
7.37 – 7.04 (m, 9H, ArH’s), 5.80 (d, 1H, J = 8.3 Hz, NHCO), 5.22 – 5.12 (m, CHN), 3.52 (s, 2H, CH2Ph),
2.95 (d, 2H, J = 11.6 Hz, CH2N), 2.77 (q, 2H, J = 5.7 Hz, PhCH2CH2), 2.19 – 1.96 (m, 3H, CHCO & CH2N),
1.92 – 1.71 (m, 8H, CH2); 13C NMR (75 MHz, CDCl3); δ 174.25, 137.66, 136.86, 129.27, 128.61, 128.35,
127.33, 127.24, 126.36, 63.15, 53.03, 47.19, 43.30, 30.29, 29.33, 28.91, 20.09; HRMS m/z (ESI)
349.2260 ([M + H]+ requires 349.2274).
6.1.9.13.
N-[2-(1H-imidazol-4-yl)ethyl]-1-benzylpiperidine-4-carboxamide 26
(Yield, 38%); Yellow viscous oil; Rf 0.13 (9:1 dichloromethane: methanol); νmax (neat)/cm-1 3246,
1
3084, 2938, 2796, 2764, 1632, 1555, 1442, 1336, 1226, 1104, 987, 931, 801, 728, 698, 659; H NMR
(300 MHz, CDCl3); δ 9.74 (s, 1H, imidazole NH), 7.56 (s, 1H, imidazole CH), 7.32 – 7.18 (m, 5H, ArH’s),
6.95 (t, 1H, J = 5.4 Hz, NHCO), 6.77 (s, 1H, imidazole CH), 3.53 – 3.44 (m, 4H, CH2Ph & CH2NHCO),
2.90 (d, 2H, J = 11.4 Hz, CH2N), 2.78 (t, 2H, J = 6.5 Hz, CH2CH2NH), 2.17 – 2.04 (m, 1H, CHCO), 1.97 (td,
13
2H, J = 11.1, 3.6 Hz, CH2N), 1.81 – 1.64 (m, 4H, CH2); C NMR (75 MHz, CDCl3); δ 175.71, 137.95,
135.76, 134.94, 129.29, 128.34, 127.24, 116.35, 63.22, 53.09, 43.24, 39.36, 28.86, 26.80; HRMS m/z
(ESI) 313.2023 ([M + H]+ requires 313.2023).
23

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6.1.9.14.
1-Benzyl-N-(2-(piperidin-1-yl)ethyl)piperidine-4-carboxamide 27
(Yield, 59%); Yellow viscous oil; Rf 0.24 (9:1 dichloromethane: methanol); νmax (neat)/cm-1 3294,
2933, 2770, 1637, 1551, 1443, 1300, 1126, 1038, 785, 726, 687; 1H NMR (300 MHz, CDCl3); δ 7.33 –
7.21 (m, 5H, ArH’s), 6.46 (s, 1H, NHCO), 3.50 (s, 2H, CH2Ph), 3.35 (dd, 2H, J = 11.2, 5.6 Hz, CH2NHCO),
2.93 (d, 2H, J = 11.6 Hz, CH2N), 2.48 (dd, 6H, J = 13.3, 7.3 Hz, (CH2)2NCH2), 2.12 (m, 1H, CHCO), 2.00
(td, 2H, J = 11.4, 2.8 Hz, CH2N), 1.88 – 1.70 (m, 4H, CH2), 1.61 (dt, 4H, J = 10.7, 5.5 Hz, CH2), 1.51 –
1.41 (m, 2H, CH2); 13C NMR (75 MHz, CDCl3); δ 175.25, 138.41, 129.21, 128.26, 127.06, 63.32, 57.16,
54.28, 53.25, 43.36, 35.68, 29.00, 25.69, 24.17; HRMS m/z (ESI) 330.2541 ([M + H]+ requires
330.2540).
6.1.9.15.
28
1-Benzyl-N-(5,6-dimethoxy-8H-indeno[1,2-d]thiazol-2-yl)piperidine-4-carboxamide
A mixture of 5,6-dimethoxy-8H-indeno[1,2-d]thiazole-2-amine 11 (0.25 g, 1.00 mmol, 1 eq.), (1-
benzylpiperidin-4-yl)(1H-imidazol-1-yl) methanone 9 (0.30 g, 1.11 mmol, 1.1 eq.) and sodium hydride
(0.08 g, 2.03 mmol, 2 eq., 60% in oil) in anhydrous tetrahydrofuran (15 mL) was refluxed overnight
after which time the reaction mixture was left to cool down to room temperature. The reaction was
quenched with the slow addition of a saturated aqueous solution of sodium sulfate (1 mL). A
solution of 3M aqueous sodium hydroxide was added and left to stir for 5 min. The organic layer was
separated and the aqueous layer was washed with dichloromethane (2 x 30 mL). The organic layers
were combined, washed with distilled water (30 mL), dried (Na2SO4), filtered and evaporated in
vacuo. The product was purified by column chromatography. (Yield, 12%); Brown solid; Rf 0.55 (9:1
dichloromethane: methanol); mp 218 °C (decomp.); νmax (neat)/cm-1 2932, 1687, 1536, 1452, 1383,
1272, 1208, 1142, 1070, 989, 846, 738, 697; 1H NMR (300 MHz, CDCl3); δ 10.29 (s, 1H, NHCO), 7.33 –
7.19 (m, 5H, ArH’s), 7.20 (s, 1H, ArH), 7.13 (s, 1H, ArH), 3.95 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.74 (s,
2H, indene CH2), 3.35 (s, 2H, CH2Ph), 2.79 (d, 2H, J = 11.3 Hz, CH2N), 2.34 – 2.20 (m, 1H, CHCO), 1.89
13
– 1.56 (m, 6H, CH2N & CH2); C NMR (75 MHz, CDCl3); δ 172.89, 162.30, 155.29, 148.84, 147.72,
138.28, 137.98, 129.90, 129.18, 129.08, 128.35, 127.23, 109.48, 102.35, 63.11, 56.45, 56.37, 52.47,
42.88, 32.50, 28.52; HRMS m/z (ESI) 450.1859 ([M + H]+ requires 450.1846).
6.2. Bioactivity screening
Prior to screening, all compounds were dissolved in dimethyl sulfoxide (DMSO) due to the
compounds being insoluble in water to a stock concentration of 10 mM. The final concentration of
DMSO in reaction was <1%.
24

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6.2.1. Cholinesterase inhibitory activity
Cholinesterase inhibitory activity for EeAChE and eqBuChE were determined using the 5,5-dithiobis-
2-nitrobenzoic acid (DTNB) assay as described by Ellman [22] and modified by Eldeen and co-workers
[23]. Three buffers were prepared: Buffer A – 50 mM Tris-hydrochloride (pH 8); Buffer B - 50 mM
Tris-hydrochloride (pH 8), containing 0.1% bovine serum albumin; Buffer C - 50 mM Tris-
hydrochloride (pH 8), fortified with 0.1 M sodium chloride and 0.02 M magnesium chloride. Into 96-
well plates were pipetted: 25 µL acetylthiocholine iodide (15 mM in distilled water), 125 µL DTNB (3
mM in buffer C), 50 µL buffer B and either 25 µL buffer A (negative control), galantamine (positive
control at 10 µM) or compounds 1, 14 – 28 (0.1 - 1 mM). Absorbance was measured at 405 nm (four
times) to account for baseline noise. An aliquot of 25 mL EeAChE (0.2 U/mL in buffer A) was pipetted
into the plates and the absorbance measured every 45 s for fifteen cycles. The EeAChE inhibition (%
relative to negative control) was determined through the rate of the reaction (correcting for
spontaneous colour changes) relative to the negative control. In the case of the BuChE inhibitory
activity assay, EeAChE was replaced with eqBuChE (0.2 U/mL in buffer A) as well as acetylcholine
iodide with butyrylcholine iodide (15 mM in distilled water).
6.2.2. Cytotoxicity screening
Cytotoxicity was assessed using the SRB staining assay on the SH-SY5Y neuroblastoma cell line as
described by Vichai and Kirtikara with minor modifications [24]. Although the SH-SY5Y cell line is
cancerous in nature, it does present as a model of a neurological cellular environment. The SH-SY5Y
cell line was cultured in DMEM/Ham's F12 nutrient medium (1:1) supplemented with 10% foetal calf
serum (FCS) in 75 mL flasks at 37 °C and 5% CO2 in a humidified incubator. Culture flasks with
confluent cells were rinsed with phosphate buffered saline and harvested using TrypLe™Express to
detach the cells. Detached cells were centrifuged (200 x g, 5 min), counted using the trypan blue
exclusion assay (0.1%), and diluted to 1 x 105 cells/µL in 10% FCS-fortified medium. The cell
suspension (100 µL) was seeded into sterile, clear 96-well plates, and incubated overnight to allow
the cells to attach. Blank wells contained 200 µL FCS (5%)-fortified media without cells to account for
background noise and sterility. Attached cells were exposed to 100 µL medium (negative control),
compounds 1, 14 - 28 (0.01 - 100 µM) or saponin (1%; positive control) prepared in FCS negative
medium for 72 h at 37 °C and 5% CO2 in a humidified incubator. Cells were fixed in the wells by
adding 50 µL trichloroacetic acid (50%) and left overnight at 4 °C. Plates containing the fixed cells
were washed three times with tap water and stained using 100 µL SRB solution (0.057% in 1% acetic
acid) for 30 min at room temperature in the dark. Stained cells were washed four times with 150 µL
acetic acid (1%) and air-dried. The bound dye was eluted using 200 µL Tris-buffer (10 mM, pH 10.5)
and the absorbance measured at 510 nm (reference 630 nm) using a ELx 800 microplate plate reader
25

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(Bio-Tek Instruments, Inc.). The blank value was subtracted from all the other values and the cell
density was expressed relative to the negative control as a percentage.
6.2.3. Statistics
Assays were performed with three intra- and inter-replicates. Statistical analyses were performed
using Graph-Pad Prism 5.0 (GraphPad). The IC50 values were determined using non-linear regression
analysis (variable slope).
6.3. Molecular modelling
Ligand structures were prepared using ligprep, with protonation states being predicted using epic
with a target pH of 7.0 ± 2.0. The protein structure was prepared using protein preparation wizard
from Schrödinger [30, 31] in which protonation states were assigned followed by an energy
minimisation to relieve unfavourable constraints. Molecular docking was done using Glide extra
precision (XP) scoring function and ligand structures were treated as flexible, which allowed for
sampling of ring conformations and nitrogen inversion, to obtain 5 poses for each ligand. The free
binding energy was calculated by submitting each docked pose complex to the Prime MM-GBSA tool
with the VSGB solvation model and OPLS3 force field [26, 27, 32].
The complexes with the highest binding pose score for donepezil 1, 20 and 28 were submitted for
MD simulations using Desmond found within the Schrödinger Suite and the OPLS3 force field [33-
35]. Solvent molecules within the binding pocket from the docking procedure were kept and the
system was further solvated using the Simple point-charge (SPC) water model for a simulation box
with boundaries 10 Å away from the protein, with an orthorhombic box containing approximately
52,000 atoms. The minimisation of the system was done using steepest descent, followed by default
relaxation protocol (see SI) and then MD simulation was done for 40 ns with NPT conditions using
the Berendsen thermostat (310 K, 1.103 bar) and particle mesh Ewald (PME) electrostatics with a
cut-off of 9 Å. Frames were extracted every 200 ps. Analysis of the MD simulations was done using
the simulation interaction diagram tool within the Schrödinger Suite [36]. Using Prime MM-GBSA the
average binding free energy was calculated for each frame of each simulation – see SI.
Acknowledgements
This work was supported by the National Research Foundation (NRF) of South Africa (Thuthuka grant
number 106959), the University of Pretoria (University, Science Faculty Research Councils and
Research and Development Program) and the Council for Scientific and Industrial Research (CSIR),
South Africa. Opinions expressed in this publication and the conclusions arrived at, are those of the
authors, and are not necessarily attributed to the NRF. The authors would like to gratefully
26

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acknowledge Ms Madelien Wooding (University of Pretoria) for LC-MS services; and the Centre for
High Performance Computing (CHPC) for access to the Schrödinger Suite and computational
resources used in this work.
Appendix A. Supplementary data
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