Design, synthesis and evaluation of cholinesterase hybrid inhibitors using a natural steroidal alkaloid as precursor

Article abstract of DOI:10.1016/j.bioorg.2021.104893

To date, Alzheimer's disease is the most alarming neurodegenerative disorder worldwide. This illness is multifactorial in nature and cholinesterase inhibitors have been the ones used in clinical treatments. In this context, many of these drugs selectively inhibit the acetylcholinesterase enzyme interacting in both the active site and the peripheric anionic site. Besides, some agents have exhibited extensive benefits being able to co-inhibit butyrylcholinesterase. In this contribution, a strategy previously explored by numerous authors is reported; the synthesis of hybrid cholinesterase inhibitors. This strategy uses a molecule of recognized high inhibitory activity (tacrine) together with a steroidal alkaloid of natural origin using different connectors. The biological assays demonstrated the improvement in the inhibitory activity compared to the alkaloidal precursor, together with the reinforcement of the interactions in multiple sites of the enzymatic cavity. This strategy should be explored and exploited in this area. Docking and molecular dynamic studies were performed to explain enzyme-ligand interactions, assisting a structure–activity relationship analysis.

Full text of DOI:10.1016/j.bioorg.2021.104893

Bioorganic Chemistry 111 (2021) 104893
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and evaluation of cholinesterase hybrid inhibitors using a
natural steroidal alkaloid as precursor
b
,
*
c
c
b
b
´
˜´˜
Jose L. Borioni , Valeria Cavallaro , Ana P. Murray , Alicia B. Penenory , Marcelo Puiatti ,
Manuela E. Garcíaa
,
*
a
´
´
´
IMBIV-CONICET, Departamento de Química Organica, Facultad de Ciencias Químicas, Universidad Nacional de Cordoba, Ciudad Universitaria, X5000HUA Cordoba,
Argentina
b
´
´
INFIQC-CONICET, Departamento de Química Organica, Facultad de Ciencias Químicas, Universidad Nacional de Cordoba, Argentina
c INQUISUR-CONICET, Departamento de Química, Universidad Nacional del Sur, B8000CPB, Bahía Blanca, Argentina
A R T I C L E I N F O
A B S T R A C T
To date, Alzheimer’s disease is the most alarming neurodegenerative disorder worldwide. This illness is multi-
factorial in nature and cholinesterase inhibitors have been the ones used in clinical treatments. In this context,
many of these drugs selectively inhibit the acetylcholinesterase enzyme interacting in both the active site and the
peripheric anionic site. Besides, some agents have exhibited extensive benefits being able to co-inhibit
butyrylcholinesterase.
In memory of Adriana B. Pierini (1953–2016).
Keywords:
Acetylcholinesterase inhibitors
Butyrylcholinesterase inhibitors
Molecular dynamic simulations
Structure activity relationships
Steroidal alkaloids
In this contribution, a strategy previously explored by numerous authors is reported; the synthesis of hybrid
cholinesterase inhibitors. This strategy uses a molecule of recognized high inhibitory activity (tacrine) together
with a steroidal alkaloid of natural origin using different connectors. The biological assays demonstrated the
improvement in the inhibitory activity compared to the alkaloidal precursor, together with the reinforcement of
the interactions in multiple sites of the enzymatic cavity. This strategy should be explored and exploited in this
area.
Solanocapsine-tacrine hybrids
Docking and molecular dynamic studies were performed to explain enzyme-ligand interactions, assisting a
structure–activity relationship analysis.
1. Introduction
peptide aggregation and tau protein phosphorylation [4]. Among these,
the most explored and effective strategy to date (as symptomatic drug
Alzheimer’s disease (AD) is one of the most common forms of de-
mentia with an alarming worldwide incidence, largely due to population
aging [1]. This is a chronic and progressive syndrome that affects the
central nervous system, causing neurodegeneration that results in
cognitive and language problems [2]. For this reason, countless efforts
are invested in the search for a treatment that can alleviate or reverse the
symptoms of the condition.
therapy) is enhancing cholinergic neurotransmission in the brain by an
increase in the level of acetylcholine (ACh) [5].
According to the first postulated hypothesis, the reduction in Ach
neurotransmitter synthesis is one of the main causes of AD. Conse-
quently, one of the therapeutic strategies is to increase the cholinergic
levels in the brain by inhibiting the biological activity of acetylcholin-
esterase (AChE), the enzyme responsible for the hydrolysis of ACh. Very
few AChE inhibitors (AChEI) including tacrine, donepezil, galantamine
and rivastigmine have been approved by the FDA. Tacrine was the first
AChEI drug approved to treat AD (chemically, 1,2,3,4-tetrahydroami-
noacridine, for abbreviation purposes, THA), but is no longer recom-
mended for use due to its hepatotoxicity (the structure of this inhibitor is
shown in Fig. 2) [6].
Currently, very few drugs are available for AD treatment, and their
effectiveness is limited by their poor long-term therapeutic efficacy (due
to low bioavailability) and their toxicity, which results in side effects.
These include naturally derived inhibitors, synthetic analogs and hy-
brids that, at the moment, have not shown the ability to inhibit the
progress of the disease [3].
The pathogenesis of AD involves several pathways including prin-
cipally, deficiency in cholinergic neurotransmission, beta-amyloid (Aβ)
In addition to AChE, there is another enzyme in the mammalian’s
brain that participates in these processes: Butyrylcholinesterase
* Corresponding authors.
E-mail address: manuelagarcia@fcq.unc.edu.ar (M.E. García).
https://doi.org/10.1016/j.bioorg.2021.104893
Received 16 September 2020; Received in revised form 1 April 2021; Accepted 3 April 2021
Available online 8 April 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
(BuChE), which catalyzes the hydrolysis of ACh and of other choline
esters neurotransmitters, disrupting neurotransmission. Thus, the inhi-
bition of both cholinesterases enhances the concentration of ACh in the
synaptic space and consequently increases cholinergic transmission [7].
Although they have a 65% homology in their amino acid sequence and a
very similar tertiary structure, they differ in the affinity for substrates,
catalytic activity and distribution. In most species, AChE is the pre-
dominant hydrolyzing enzyme in the nervous system (nearly 80% of
ACh is hydrolyzed) and in erythrocytes. Meanwhile, BuChE is present at
high concentrations in the blood plasma [8].
associated with the main door. In BuChE, six of these residues are
exchanged for smaller ones, increasing the entrance radius of the cata-
lytic cavity [17,18].The altered residues belong to the PAS and acyl
binding subsite of CAS. The main differences between both catalytic
cavities are illustrated in Fig. 1.
Since AD is a multifactorial disease, the urgent development of multi-
target inhibitors is required. Furthermore, and even though it is a
difficult task and a challenge of design and synthesis, future approaches
should focus on the development of a molecule capable of targeting
multiple factors involved in AD [19]. Currently, this task is supported by
computational approaches, including docking and molecular dynamics
(MD). These methods provide extremely useful information on the
design and mode of binding of new inhibitors, reducing development
time and costs [20].
Even though AChE is the primary cholinesterase(ChE) responsible
for the hydrolysis of ACh, BuChE has been found as a co-regulator in AD
brains with depleted AChE levels as a compensatory mechanism [9].
Indeed, an increased BuChE activity also plays an important role in Aβ-
aggregation during the early stages of senile plaque formation [10].
Consequently, inhibition of both AChE and BuChE enzymes may
serve as a good strategy for exploration. For example, rivastigmine,
which works as an inhibitor of both AChE and BuChE, displays a more
potent effect than donepezil and was recognized as one promising
candidate to positively improve the course of AD [11].
To date, it is well-known that alkaloidal compounds of natural origin
generally have a good ChE inhibitory potential [21]. Simply, the addi-
tional advantage added to the structural complexity lies in the presence
of protonable nitrogen atoms, capable of interacting with the different
subsites of the enzymatic cavity [22]. In this sense and continuing with
the previously reported development of new cholinesterase inhibitors
from the alkaloid solanocapsine (see structure in Fig. 2) and its de-
rivatives, [23] the strategy of hybrid formation from two bioactive
molecules with a different complexity linker was explored [24]. In these
previous reports, some of the hybrid compounds synthesized showed
better inhibition values concerning the alkaloidal precursor. Regarding
the binding mode of the new compounds, multiple interactions in
different sites of the enzymatic cavity were added as a result of
hybridization.
The AChE has an ellipsoidal shape and approximate size of 45 × 60
× 65 Å. The substrate accesses the active site through a deep groove
(gorge) on the surface of the protein, which is approximately 20 Å deep
[12]. This enzyme contains two main regions: the catalytic active site
(CAS), located at the bottom of the cavity, and the peripheral anionic
site (PAS), near the entrance of the gorge. In particular, different sub-
sites in the CAS could be identified: the anionic, oxyanionic, acyl
binding, and catalytic triad [13].
Indeed, several studies have shown that AChE is not merely the
enzyme that hydrolyzes ACh but also plays an essential role in the Aβ-
peptide aggregation. It has been suggested that the PAS is the enzymatic
region responsible for this role[14]. For this reason, it has been proposed
that inhibitors capable of interacting with CAS and PAS of the enzyme,
would perform a double function (called dual inhibitors), increasing the
levels of the neurotransmitter ACh and preventing the formation of
aggregates of Aβ [15].
Through the years, some of the synthesized inhibitors have in their
structure the well-known tacrine moiety (see its structure in Fig. 2). It
has been used as a starting molecule to develop novel multifunctional
agents with improved pharmacological profiles [25]. To date, it is one of
the most studied molecules related to this pathology. Its structure was
derivatized, substituted in almost all positions, simplified and it was
employed in the first attempts of dual inhibition (bis(7)-tacrine) [26].
Thus, tacrine homo- and heterodimers exhibit high potency even higher
than the tacrine monomer [27,28,29].Studies changing the nature and
length of the linker were also carried out with very interesting results
[30,31,32].
On the other hand, the BuChE gorge is considerably more volumi-
nous (approximately 200 Å3 larger), making it less selective than AChE
[16]. The main difference between the two enzymes is around the main
entrance of the catalytic cavity area. AChE has eight aromatic residues
According to these previous results and continuing our studies on
Fig. 1. Schematic comparison of the catalytic cavities of Homo sapiens BuChE (left) and Torpedo californica AChE (right) enzymes which PDB codes are 4BDS and
1EVE respectively. In schematic mode view are highlighted the reported residues from the different regions and subsites of each enzyme.
2

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
Fig. 2. Molecular structure of natural alkaloid solanocapsine (S) and tacrine (THA) building blocks. The primary amine groups were transformed to connect both
moieties with the different selected linkers for hybrid synthesis.
cholinesterase inhibitors, in the current report we present the synthesis
of novel dual hybrid inhibitors employing tacrine and solanocapsine as
scaffolds connected by linkers of different types. Also, determinations of
in-vitro inhibition on AChE and BuChE enzymes were performed to
evaluate the affinity and selectivity. Finally, structure–activity re-
lationships were performed employing the predicted binding modes
according to docking and molecular dynamic simulations of the com-
plexes between hybrid compounds and the enzymes. The simulations
made it possible to understand the binding mode of each inhibitor at an
atomistic level, being able to rationalize the experimental activities
presented. We propose that this integrated approach could serve as a
basis for more comprehensive developments in the future.
capabilities [39]. On the other hand, in an attempt to assess the impact
of increased rigidity or the possibility to increase non-polar interactions,
we introduced an aromatic ring into derivative 4 (see structure in
scheme 1).
Finally, it is known that one of the improvements to the tetrahy-
droacridine nucleus is the introduction of a chlorine atom at position 6
or 7, that in general terms produce an improved activity, causing
different selectivity and potency between cholinesterases [40,41,42].
Therefore, to evaluate the impact of this substitution, we synthesized the
compound 5, analog of compound 1.
2.2. Chemistry
2. Results and discussion
The synthesis of compounds hybrids 1–5 was accomplished as shown
in scheme 1. Complete detail of the procedures performed to obtain
them, and their structural characterization is found in the Materials and
methods section.
2.1. Synthetic planning and design
To fulfill the main synthetic goal of the present contribution, the
primary amine groups of solanocapsine and tacrine were modified to
connect both scaffolds with linkers of different nature and flexibility (see
Fig. 2). In our previous work, binding energies of the complexes formed
by AChE and hybrid compounds between solanocapsine and tacrine
with linkers of two, four, and five methylene units were computed [23].
The values were obtained using classical molecular mechanics methods.
Based on these results, we were able to conclude that the optimal length
of the flexible spacer bridge was five carbon atoms. This length allows
the inhibitor to fix in a better way with the catalytic cavity of the enzyme
maximizing the favorable interactions and resulting in better binding
energy. Also, the most favorable interactions were achieved through the
amine (non-amide) bond close to tacrine. Furthermore, a derivative with
a structural simplification of tacrine was assayed but did not lead to
better inhibition results. According to those findings, compound 1 was
designed and synthesized (see scheme 1).
For the synthesis of compounds 1–5, simple (widely reported)
sequential reactions were used. In all cases, just the final product of the
reaction sequence was purified. The course of the reactions was moni-
tored by TLC, presuming the presence of synthetic intermediates by the
finding of new spots compared with starting materials.
As shown in scheme 1, compounds 1 and 5 share the reaction
sequence, but starting from different precursors: tacrine in the case of 1
and 7-chlorotacrine for the synthesis of 5. In the first place a nucleo-
philic substitution between the tacrine derivative and 1,5-dibromopen-
tane in basic medium [43]. For the union with solanocapsine, bromine
was exchanged by iodine including a better leaving group in the syn-
thetic intermediate. Hence the last step is a final substitution by the
primary amine nucleophilic group of solanocapsine also in basic me-
dium. In the case of compound 5, a previous step is needed: the chlo-
rination of tacrine with N-chlorosuccinimide [44].
For the synthesis of compound 2 a convergent procedure was
employed. In the first place by parallel reactions of tacrine and sol-
anocapsine with iodoethanol in basic medium followed by a tosylation
activation step of the resulting alcohols yielding in two synthetic in-
termediates (in brackets in scheme 1) [45]. Finally, both intermediates
reacted with piperazine by disubstitution.
It has been observed that the insertion of at least one protonable
atom at physiological pH in the linker, capable of being located in the
neck of the enzyme, would reinforce the polar interactions. For this
reason and inspired by hybrids containing pharmacophores or natural
biologically active organic compounds we synthesized the hybrids 2 and
3, by adding nitrogen-containing heterocycles as piperazine and tri-
azole, respectively. Both nuclei possess versatile binding properties,
being identified as responsible for beneficial interactions in multiple
biological targets [33,34]. These rings are widespread structural motifs
in drug discovery, with a high number of positive hits encountered in
biological applications and thus are considered as privileged structures
in the medicinal chemistry fields [35,36,37,38].
In the case of compound 3 containing a triazole ring, a click reaction
was carried out to build the ring. In order to do so, the propargyl was
added to the tacrine scaffold and the corresponding azide were syn-
thesized. After the click reaction [46] an intermediary with tacrine and
the triazole was obtained with a free OH group. This group was activated
as a leaving group by a tosylation and in this way, by a nucleophilic
attack of the amine of solanocapsine in basic media the fusion final
product was obtained.
Numerous reports have shown that the triazole functionality results
in an effective bioisosteric replacement of a trans-amide bond because of
their similar size, dipole moment, planarity, and hydrogen bond
Finally, for the synthesis of hybrid 4, in the first stages the linker was
prepared, starting from terephthalic acid as a precursor of the methyl
3

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
Scheme 1. Summarized synthetic procedures followed to obtain the hybrid compounds from solanocapsine (S) and tacrine (THA) building blocks.
ester. Terephthalic alcohol was prepared in two steps after methylation
and reduction of the diester with LiAlH4. Once again, both hydroxyl
groups were activated and then after subsequent fusion nucleophilic
reaction with tacrine and solanocapsine, compound 4 was formed.
are shown in table 1. The graphs of the enzyme inhibition calculation for
both enzymes are presented in figure S1 of supporting information. In-
hibition percentages for each tested concentration are presented in table
S1 of supporting information. Samples were evaluated in concentration
ranging from 5 nM to 20
BuChE.
μM for AChE and from 5 nM to 50 μM for
2.3. Biological activity
All tested hybrid compounds, with exception of compound 2, showed
remarkable inhibitory activity in both enzymes, within the nanomolar
range close to the inhibitory value of tacrine (IC50 AChE = 0.029 nM and
IC50 BuChE = 0.004 nM). In all these cases, the hybrids present lower
biological activity than tacrine but an improvement with respect to the
The obtained hybrids were evaluated as cholinesterase inhibitors by
the modified Ellman’s method. The natural compound solanocapsine
[23] and tacrine were used as reference AChE and BuChE inhibitors. The
results obtained, along with their standard deviations, for the enzymes
4

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
Table 1
In-vitro Electric eel AChE and Horse serum BuChE inhibition activity of hybrids and their tacrine (THA) and solanocapsine (S) scaffolds obtained by the modified Ellman’s
method. Results are expressed as IC50 values (µM) ± SD. Each value is the mean of three replications. aSelectivity index = IC50 AChE/IC50 BuChE.
Comp.
Structure
AChE-IC50 ± SD(
μM)
BuChE-IC50 ± SD(
μM)
Selectivity indexa
4.92
1
0.064 ± 0.001
0.013 ± 0.001
2
3
5.210 ± 0.120
0.170 ± 0.020
35.500 ± 1.270
0.030 ± 0.005
0.15
5.67
4
0.290 ± 0.061
0.028 ± 0.002
10.36
5
S
0.026 ± 0.003
0.009 ± 0.001
2.89
3.220 ± 0.100
0.029 ± 0.003
2.220 ± 0.043
0.004 ± 0.001
1.45
7.25
THA
solanocapsine scaffold.
logically, it is expected some limitations in some medicinal chemistry
descriptors related to bioavailability properties. Nevertheless, this
research constitutes a proof of concept for subsequent designs of com-
pounds of this nature, which possess improved properties as suggested
by experimental findings.
All the inhibitors, except compound 2, present a slight selectivity for
BuChE over AChE. Compound 4 is the most selective, even more than
solanocapsine and tacrine, being approximately 10-fold more active
against BuChE. The origin of this selectivity may be due to the possibility
of a better accommodation of the bulky hybrids in the bigger BuChE
catalytic cavity.
The biological results are noteworthy not only for the IC50 value per
se, but also for the possible interactions at the enzymatic level that these
compounds could establish acting as dual inhibitors. This potential dual
nature represents an acquired characteristic that the inhibitor tacrine
does not exhibit. Molecular modelling studies could help to support this
hypothesis.
As shown in table 1, the activity results obtained are very promising,
indicating the potential strength of the employed strategy. Even though
there are still many issues to improve. Among these issues it should be
mentioned: the limited amount of natural starting material, the low
yields obtained for some of the products, either due to problems in the
purification of complex reaction mixtures, or to the degradation of some
of the products.
2.4. Docking and molecular dynamics analysis
To date, there are numerous compounds reported as inhibitors of
both cholinesterases, some even with outstanding activity of the nano-
molar order, for example huperzine, [47] donepezil-tacrine [48] hybrids
among others. Beyond this, although some exert dual inhibition, none of
those reported are structurally related to steroidal compounds, so this
constitutes the first finding of this nature to date.
To date, in addition to our preliminary attempt, there are no steroid-
tacrine hybrids reported as cholinesterase inhibitors. For this reason, it is
necessary to know the molecular determinant aspects of these com-
pounds for their biological activity in each enzyme specifically. To
answer this question and to give more validity to the structure–activity
relationships that may arise, computational studies were performed to
explore the binding interactions of these hybrids against both AChE and
The hybrid molecules have a high volume and molecular weight and
5

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
BuChE enzymes.
Although no specific interactions with the chlorine atom are observed,
its presence causes a slight variation of the position of the THA nucleus.
Thus, the hybrid adopted a relaxed geometry and most stable position
(Fig. 3a).
The coordinates of AChE were taken from the experimental structure
of Torpedo californica (TcAChE). The structure of the complex with the
bis-tacrine dimer as a ligand with a linker with five CH2 (PDB code:
2CMF) was used. This receptor presents the residue Phe330 (belonging
to the bottleneck region) closing the gate of the catalytic cavity to
At this point, it is valid to inquire whether the addition of hetero-
atoms or rigidity in the linker will enhance the present interactions. In
the case of compound 2, where a piperazine ring containing two nitro-
gen atoms was added, it is important to highlight that at pH 7.5 two
possible protonation states with a marked balance in the species distri-
bution + 3 (44.6%) and + 4 (51.1%) could be present, according to the
model employed for an estimation of the pKa values. This is the only
hybrid where this behavior exists, which could further explain the
decrease in inhibitory activity, due to the distinctive component of
solvation energy of one of the predominant species (this aspect is
referred to later). The pKa values of the protonable groups of this hybrid
are shown in figure S2in the supporting information.
facilitate
π-π interaction with one of the tacrine fragments. In our
experience, we consider that this interaction is crucial for the complexes
between the enzyme and voluminous ligands. For this reason, the
mentioned structure of the AChE was selected to perform the compu-
tational assays. In the case of BuChE (hBuChE), a structure from Homo
sapiens in a complex with tacrine was used (PDB code: 4BDS).
Before starting the docking simulation, the pKa values of the hybrid
compounds (1–5) were evaluated to determine the ionization state of
each one, [49] (see figure S2 in the supporting information). Compounds
1, 3–5 presented a charge of + 3. It should be mentioned that, due to the
estimated pKa of one of the amino groups of the piperazine ring of
compound 2, two possible states with + 3 and + 4 charges could be
formed, both were analyzed.
In both modeled species of compound 2, it is observed that the THA
part is almost superimposable with respect to 1, with the same favorable
interactions in PAS and the anionic subsite. In this subsite, for the spe-
cies with net charge + 4 an enhancement in the interaction with Trp279
is observed. On the other hand, with the other protonation state + 3, the
biggest difference lies in an unfavorable interaction (the only one
described in this analysis) within the piperazine ring and Asp72 residue
of the PAS that indicate some steric repulsion; this could probably be the
reason of its lower AChE inhibition value.
After the docking, 10 complexes of each molecule were obtained.
The complexes were minimized and their binding energy was recom-
puted by the MM-GBSA approximation. A control of this docking plus
refinement protocol was performed with the complexes of TcAChE with
bis-tacrine dimmer [50] and hBuChE with tacrine [51] these were
compared to the experimental ones. The geometries obtained, shown in
the figure S3 of the supporting information, are similar to those of the
experimental ones with low RMSD values, of 2.07 Å and 1.07 Å for
tacrine (4BDS) and bis-5-tacrine (2CMF) respectively, validating the
used protocol.
When the spacer or linker contains the triazole functionality (3),
there are no obvious favorable interactions with this nitrogenated ring
other than a modest van der Waals type with Ser122, in PAS (see
Fig. 4c). Instead of an important interaction between the linker and the
protein, it was observed a slight twist of solanocapsine through its
extension due to the position of the linker. Thus, in the entire cavity new
interactions appear, in addition to those already present in 1 such as
Asp72 and Glu74 of remarkable value, although the biological activity
does not show improvement. This could be due to the position of the
ligand (compared to 1) leading to a loss of important interactions such as
that with Glu199 of the anionic subsite, see table S2 in supporting in-
formation for total energy interactions values.
In the next stage, the most stable complex obtained after the
refinement, was selected to carry out MD simulations. Then, the binding
energy was calculated from a set of configurations, obtained from the
equilibrated part of the MD simulations, by employing the MM-GBSA
method. A list with the binding energies obtained with the refinement
docking and MD for both enzymes is shown in the table S2 in the sup-
porting information.
With the aim of identifying the residues that stabilize the formed
complex, a decomposition of the total binding energy per-residue was
performed with both cholinesterases (only the interactions with values
lower than ꢀ 1 kcal/mol were considered as relevant). For a better un-
derstanding, the results for each enzyme are presented and then, a
comparative and global analysis was performed. Indeed, in tables S3 and
S4 in the supporting information are summarized the residues with the
most relevant binding interactions of the complexes.
If the fragment added to the linker is an aromatic ring derived from
benzene (4), it is observed that the entire molecule takes a different
position, especially THA moiety (with respect to 1). This generates new
interactions in CAS through Ser200 and His440 with the tacrine scaffold
(hydrogen bond and van der Waals, respectively). Indeed, amine of
solanocapsine ring A interacts by a hydrogen bond with Ser81 added to
salt bridge with Asp72 (PAS), but they would not generate an
improvement in the inhibition value.
As seen in our previous report, for AChE enzyme, the solanocapsine
nucleus showed a good fit with the shape of the gorge (CAS, PAS, and
acyl binding subsite) with pronounced global stabilization (total binding
energy = -40.5 kcal/mol). An important detail to point out is that the
entry of solanocapsine into the AChE cavity is carried out through the F
ring of the molecule (see Fig. 2). On the contrary, for the tacrine
molecule, the most stabilizing interactions are limited to the bottleneck
region and the anionic subsite (total binding energy = -31.9 kcal/mol).
For more details, these interactions are shown in the Fig. 4a, b.
Regarding the hybrid ligands, more stabilizing interactions were
observed. In general, the tacrine moiety acts directing the entry of the
hybrid inhibitors towards the CAS. For compound 1, which for us rep-
resents the “zero point” of structural variation, outstanding interactions
are observed between tacrine fragment and residues of the anionic
subsite (Trp84 and Glu199, van der Waals and salt bridge, respectively).
In addition, interaction of van der Waals type between the rings A and B
of solanocapsine moiety with the PAS is observed (Tyr334 and Trp279).
If a comparative analysis is made with compound 5 that has a
chlorine atom on the THA unit, there is a reinforcement of interactions
of the solanocapsine moiety, with the PAS and anionic subsite in addi-
tion to an extra stabilization that already had compound 1 (for example
a salt bridge into amine of solanocapsine of ring A with Asp72 in PAS).
Similarly, the analysis of the interactions of the hybrid ligands, sol-
anocapsine and tacrine scaffolds with the BuChE enzyme was carried
out. By possessing a substantially bulkier cavity, this enzyme allows
substrates to accommodate maximizing favorable interactions and
enhancing inhibition, as was demonstrated in our experimental mea-
surement (added to the selectivity index of the compounds, slightly
higher for this enzyme).
Concerning the building blocks and their performance as BuChE
inhibitors, two highly stabilizing interactions are observed for tacrine
(with Trp82 at the cation-π site, and a salt bridge with Glu197). As for
solanocapsine, there is already a global interaction behavior, with
different sites of the protein, including PAS (Phe329 and Tyr332). Thus,
there are remarkable interactions between the primary nitrogen in the
alkaloid nucleus and Trp82, added to a salt bridge with Glu197 and
hydrogen bonds with Gly439. Secondary nitrogen has a strong hydrogen
bond with Ser287. Furthermore, there is a marked van der Waals
interaction with Gln119. These interactions are illustrated in the Fig. 4
d, e.
In general terms, for all hybrids it is observed that the tacrine scaffold
maintains strong interactions with Trp82 (cation-π site). Indeed, the
rigidity of the linker could be a determining factor for the binding mode
6

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
Fig. 3. Ligand-receptor 3D representation obtained after clustering over MD trajectory against AChE and BuChE enzymes. For a more comprehensive interpretation
of the adopted position in the catalytic cavities, compound 5 in AChE (a) and BuChE (b) enzymes was selected. Comparison and superimposition of compounds with
generic different linkers; 1 (flexible, cyan) and 4 (rigid, red) into AChE (c) and BuChE (d) enzymes. To clarify, only the most important interactions are highlighted.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
in BuChE, since some inhibitors show the ability to place part of the
solanocapsine skeleton “lying down” outside the catalytic cavity due to
the flexible carbon bridge (see figure S4 in supporting information, for
details).
(Gly116 and Thr120). A strong van der Waals interaction with Tyr440
and the THA fragment is highlighted. It is important to note that unlike
compound 5, due to these interactions of the new rigid bridge, all the
molecule changes its global orientation (fundamentally in the external
part of the enzyme cavity, Fig. 4f).
In specific terms, compound 1 exhibits van der Waals interactions
with oxyanionic subsite residues (Gly116 and Gly117) through its car-
bon bridge. A highly stabilizing hydrogen bond is observed between the
solanocapsine amino group bound to the linker and the Gln119, Pro285,
and Ser287 residues. About the solanocapsine skeleton, van der Waals
interactions with PAS (Phe329) and hydrogen binding with Gln71 are
found, likewise, the tacrine nucleus reinforces the interactions through
its aromatic nitrogen and Glu197.
For compounds 2 (with two protonation states, +3 and + 4 species)
and 4, a substantial loss in the number of stabilizing interactions was
found. The hydrophobic interactions between the PAS and the
solanocapsine-linker moieties are conserved, while the others are lost.
For all inhibitors evaluated, in both enzymes, tacrine moiety binds in
the active site region. One of the interactions observed in all complexes
is with Tpr84 in AChE (anionic subsite) and Trp82 in BuChE (cation-
π
Hybrid 5 shares quite a few interactions with 1, only some hydrogen
bonds are lost, although other notable ones are added between the ar-
omatic nitrogen of the scaffold tacrine with the CAS (His438), in addi-
tion to hydrophobic interactions between the Leu286 residue and the
linker in the acyl binding subsite. The chlorine atom introduced into the
tacrine moiety seems to serve as a positioner for the compound globally,
it does not present relevant interactions by itself as in AChE (see Fig. 3b),
although here there is a marked increase in inhibitory activity. This
greater selectivity for BuChE is in agreement with that reported by other
authors and may be due to the small differences in the catalytic sites of
both cholinesterases [52,53].
site). This hydrophobic interaction is relevant for the binding of hybrid
compounds to both enzymes. Furthermore, inhibitors with good IC50
values showed van der Waals type interactions with PAS residues for
both enzymes. These observations highlight the potential of these
compounds as dual inhibitors with very good inhibitory activity.
Specifically, in AChE the two more active compounds (compounds 1
and 5) interact with Glu199 (anionic subsite) through salt bridge
interaction. This interaction added to the presence of a flexible linker
that enables a better accommodation within the catalytic cavity may be
the origin of the remarkable inhibition values shown in both compounds
in AChE.
Regarding hybrids with a modified linker, some interactions are
conserved for compound 3. If compared with compound 5, stabilizing
interactions are added through the triazole ring with the oxyanionic hole
In the BuChE enzyme, the hybrid compounds tested, with the
exception of compound 2, have comparable biological activity values
and a general improvement in the inhibitory activity on the enzyme
7

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
Fig. 4. This figure illustrates the most relevant interactions in both enzymes, on the left AChE represented in orange and BuChE on the right, in green. The building
blocks tacrine (a,d), solanocapsine (b-e) and hybrid 3 (c-f) are represented. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
BuChE. This is given by the greater volume of its cavity which supposes a
greater degree of freedom when establishing interactions for molecules
of considerable size.
3. Conclusions
This is a contribution to the creation of hybrids of bulky molecules as
cholinesterase inhibitors, insufficiently explored at the moment. In this
sense, the results highlight the excellent performance of hybrids in
BuChE inhibition, ascribed to the larger size of the enzyme cavity, ac-
cording to docking and MD calculations.
In general, the protonation state reached by hybrids in cholinester-
ases at pH = 7.5 deserves special attention. In the case of compound 2,
the possibility of two protonation states is observed at this pH value. It
can be seen that in both proteins the compound with a net charge state
+ 4 presents a weaker binding. This can be explained since it presents a
difference in solvation energy for the most unfavorable complex, which
is also reflected in the total binding energy. For more details about the
binding energy components of each complex, see table S2 the supple-
mentary information.
Likewise, regarding the nature of the linkers the results allow to
conclude that different global positions were adopted by the inhibitors
yielding in most of the cases better inhibitory activity than sol-
anocapsine. This could be ascribed to an enhancement of the in-
teractions with the enzymes. Besides, it was observed that rotations and
twists at the linker occur as a “domino effect” causing the loss of certain
favorable interactions, which could be the origin of the decrease in the
activity.
Since the hybrids generated are large molecules, they occupy the
active site as well as the PAS, establishing more interactions with the
enzymes with respect to the isolated counterparts (THA and sol-
anocapsine moieties), see Fig. 4 for more details. According to these
results of the modelling studies, the hybrids could potentially act as dual
inhibitors, unlike THA.
Although the analysis carried out turns out to be special in each type
of system to be addressed, the logic seems to indicate the premise that
the use of N-containing steroidal nuclei (not necessarily alkaloidal) with
a suitable spacer and tacrine (to direct the entrance to the cavity) may be
a good strategy to be explored in this field of research.
8

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
́
́
Finally, it is important to highlight the relevance of linking properly
(1H, H-3), 3.11 m (2H, H2-4), 3.04 m (1H, H2-26a), 2.69 m (2H, H2-1),
2.16 m (1H, H2-26b), 2.00 m (1H, H-22), 1.93 m (1H, H-25), 1.91 m (4H,
scaffolds of reported bioactivity, which results in
a substantial
́
́
improvement in the interactions that lead to a very good inhibition of
both enzymes in general terms and their potential use as dual inhibitors,
due to the interactions achieved.
H2-2,3), 1.82 m (2H, H2-2), 1.82 m (1H, H2-4a), 1.82 m (1H, H2-24a),
́
́
́
́
1.81 m (2H, H2-2), 1.80 m (1H, H-20), 1.73 m (2H, H2-4), 1.68 m (2H,
́
́
H2-1), 1.58 m (2H, H2-3), 1.57 m (2H, H2-15), 1.38 m (1H, H-5), 1.32 m
(1H, H-8), 1.23 m (1H, H2-4b), 1.21 m (1H, H2-24b), 1.18 m (1H, H-9),
4. Materials and methods
1.08 m (1H, H-14), 0.96 d (3H, J = 6.7 Hz, H3-21), 0.86 d (3H, J = 6.7
Hz, H3-27), 0.80 s (3H, H3-19), 0.75 s (3H, H3-18), 0.72 m (1H, H-17).
13
́
́
C NMR (CDCl3 100.03 MHz): 157.1 (C, C-4a), 151.6 (C, C-9), 145.5 (C,
4.1. General
́
́
́
́
C-10a), 129.1 (CH, C-6), 127.0 (CH, C-5), 123.9 (CH, C-8), 122.9 (CH, C-
́
́
́
Optical rotation indexes were measured on JASCO P-1010 polarim-
eter. IR spectra were obtained in a Nicolet iZ10 ThermoScientifics
spectrophotometer (each compound was dissolved in a minimum
amount of solvent and a drop of solution was added to the AgCl IR
plates). NMR experiments were performed on Bruker AVANCE II 400
MHz instrument. Multiplicity determinations (HSQC-DEPT) and 2D
spectra (COSY, HSQC,and HMBC) were obtained using standard Bruker
software. Chemical shifts are expressed in ppm (δ) units using tetra-
methylsilane as the standard. Exact mass spectra were obtained on a
Bruker microTOF-Q II mass spectrometer, equipped with an ESI source
operating in positive mode. Chromatographic separations were per-
formed by column chromatography on silica gel 60 (0.063–0.200 mm),
and preparative TLC on silica gel 60 F254 (0.2 mm thick) plates. The
presence of alkaloids was revealed by Dragendorff’s reagent. Acetyl-
cholinesterase from Electric eel (type VI-S), 5,50-dithiobis (2-nitro-
7), 119.3 (C, C-8a), 115.2 (C, C-9a), 98.5C, C-23), 74.4 (CH, C-16), 68.7
(CH, C-22), 60.4 (CH, C-14), 54.8 (CH2, C-26), 54.7 (CH, C-5), 54.5 (CH,
́
́
́
́
C-17), 48.9 (CH2, C-5), 46.6 (CH, C-3), 45.4 (CH, C-9), 44.5 (CH2, C-1),
46.2 (CH2, C-24), 34.9 (CH, C-8), 39.2 (CH2, C-4), 35.6 (C, C-10), 32.9
́
(CH, C-20), 32.8 (CH2, C-4), 39.0 (C, C-13), 37.2 (CH2, C-1), 31.8 (CH2,
́
́
́
́
C-2), 30.9 (CH2, C-4), 29.9 (CH, C-25), 32.1 (CH2, C-2), 28.3 (CH2, C-
́
́
́
́
́
15), 22.8 (CH2, C-2/C-3), 24.5 (CH2, C-1), 24.1 (CH2, C-3), 18.6 (CH3, C-
27), 13.6 (CH3, C-18), 15.0 (CH3, C-21), 12.2 (CH3, C-19). HRESIMS m/z
[M + H]+697.54806 (calcd for C45H69N4O+2 , 697.54150).
4.3.2. (22R,23S,25R)-3β-N-[2-(4-(2-aminoethyl)piperazin-1-yl)ethyl]-
1,2,3,4-tetrahydroacridin-9-yl)amine-22,26-imino-16β,23-epoxy-5α-
cholestan-23β-ol (2)
Step 1:
80 mg (0.4 mmol) of tetrahydroacridine hydrochloride were placed
in a reaction flask and dissolved in 3 mL of dry DMF. 32 µL of iodoe-
thanol (0.4 mmol) were added and then 0.64 mmol (36 mg) of KOH. The
reaction mixture was vigorously stirred for 20 h (at room temperature),
and then 5 mL of H2O were added and partitioned with diethyl ether (5
× 10 mL). The organic layers were combined, dried with anhydrous
Na2SO4 and evaporated under reduced pressure. The resulting solid (67
mg) was used as recovered in Step 3.
benzoic
acid)
(DTNB),
acetylthiocholine
iodide
(ATCI),
butyrylthiocholine iodide (BTCI) and tacrine (99%) were purchased
from Sigma. BuChE (Horse serum) was purchased from MP Biomedicals.
4.2. Solanocapsine isolation
The natural compound solanocapsine was extracted from Solanum
pseudocapsicum using the procedures outlined in the previous report
[23].
Step 2:
Furthermore, 100 mg of solanocapsine (0.23 mmol) were dissolved
in 3 mL of DMF, 16 mg of KOH (0.28 mmol) and 18 µL of iodoethanol
(0.23 mmol) were added under stirring at 50 ◦C for 24 h. Then 2 mL of
H2O were added and extracted with diethyl ether (5 × 3 mL). The
organic phases were combined, dried with anhydrous Na2SO4 and
evaporated under reduced pressure, recovering 87 mg of impure
elongated-solanocapsine ready for activation in Step 3.
4.3. Preparation of solanocapsine derivatives
A schematic and simplified description of the reactions carried out
for the synthesis of compounds 1 to 5 is presented in the supporting
information, scheme S1.
Step 3:
The products of steps 1 and 2 (reaction crudes taken as pure) were
tosylated in DMF (3 mL) under stirring at room temperature for 12 h. For
this purpose, 13.2 and 20.5 mg (0.108, 0.168 mmol) of dimethylami-
nopiridine, 41.2 and 64.1 mg (0.22 and 0.34 mmol) of p-toluenesulfonic
acid, 25 and 39 µL (0.18 and 0.28 mmol) of triethylamine were added to
the tetrahydroacridine (step 1) and solanocapsine (step 2) derivatives,
respectively. The reaction mixtures were extracted with diethyl ether
(5x 5 mL) and H2O (5 mL). The organic phases were combined, dried
with anhydrous Na2SO4 and evaporated under reduced pressure. 25.6
mg and 8.3 mg of elongated-tosylated solanocapsine and tacrine crude
were recovered, respectively.
4.3.1. (22R, 23S, 25R)-3β-N-(1,2,3,4-tetrahydroacridin-9-yl)pentane-
1,5-diamine-22,26-imino-16β,23-epoxy-5α-cholestan-23β-ol (1)
Step 1:
80 mg of tetrahydroacridine (0.4 mmol) were dissolved in 2 mL of
dry DMSO and 42 mg of KOH (0.64 mmol) were added with stirring.
Then, 60 µL (0.43 mmol) of 1,5-dibromopentane were added, and the
reaction mixture was stirred at room temperature for 24 h. After that,
the reaction mixture was diluted with H2O (10 mL) and extracted with
AcOEt (4 × 8 mL). The combined organic layers were washed with H2O
(3 × 15 mL), dried with anhydrous Na2SO4 and evaporated under
reduced pressure.
Step 4:
Step 2:
The two mixtures obtained after Step 3 were assumed pure, and a
mass equivalent to 0.02 mmol of each was taken, that is, 13 mg and 8.3
mg elongated-tosylated solanocapsine and tacrine crudes, respectively.
They were dissolved in 1 mL of DMF and added to a reaction flask that
previously contained 2 µL of piperazine with 8 µL of triethylamine (0.09
mmol) dissolved in 1 mL of DMF. The mixture was kept under stirring at
50 ◦C for 48 h. 5 mL of H2O were added and the reaction mixture was
partitioned with diethyl ether (5 × 10 mL). The organic phases were
combined, dried with anhydrous Na2SO4 and evaporated under reduced
pressure. The reaction mixture (21 mg) was purified by preparative TLC
using a sequential elution of DCM: MeOH (15%). 4.1 mg of the product 2
were obtained (2.3%) as a yellow amorphous solid.
The organic crude (55 mg) was dissolved in 5 mL of DCM and 0.2
mmol (33 mg) of KI were added stirred for 30 min. After that, sol-
anocapsine (0.09 mmol, 40 mg) and triethylamine (40 µL) were added.
The reaction mixture was stirred at room temperature for another 20 h.
Later, it was concentrated to dryness and purified by preparative TLC
using DCM: MeOH (20%, sequential elution) to obtain 6.8 mg of com-
pound 1 (10.5%) as a yellow amorphous solid.
Chemical characterization:
25
[
α]
D: ꢀ 4.5 (c 0.06, MeOH), IR (film) νmax: 3342.2, 2931.4, 2859.1,
1667.2, 1581.4, 1410.7, 1383.7, 1121.6, 1034.7, 1008.6, 762.7 cmꢀ 1
.
1
́
H NMR (CDCl3, 400.13 MHz): 8.04 d (1H, J = 8.3, H-5), 7.89 d (1H, J =
́
́
8.2, H-8), 7.71 td (1H, J = 8.2, 1.1 Hz, H-7), 7.58 td (1H, J = 8.3, 1.1 Hz,
́
́
́
́
́
Chemical characterization:
H-6), 4.47 m (1H, H-16), 3.61 m (2H, H2-5), 3.55 m (2H, H2-1),3.14 m
9

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
25
[
α]
D: ꢀ 43.0 (c 0.02, MeOH), IR (film) νmax: 3391.4, 2928.5,
added, and the reaction mixture is partitioned with diethyl ether (5 ×
10 mL). The organic phases were combined, dried with anhydrous
Na2SO4 and evaporated under reduced pressure. The crude (18 mg) was
purified by preparative TLC using a sequential elution of DCM: MeOH
(10%). 3.2 mg of the product 3 were obtained (9.4%) as a yellowish
brown amorphous solid.
2854.3, 1598.8, 1448.3, 1381.8, 1353.8, 1160.0, 871.1 cmꢀ 1.1H NMR
́
(CDCl3, 400.13 MHz): 7.74 dd (1H, J = 8.3, H-5), 7.68 brd (1H, J = 8.2,
́
́
́
1.1 Hz, H-7), 7.28 m (1H, J = 8.2, H-8), 7.28 m (1H, J = 8.3, 1.1 Hz, H-6)
́
́
, 4.28 m (1H, H-16), 4.05 m (2H, H2-2) , 3.80 m (1H, H2-26a), 3.64 m
́
́
́
́
́
́
́
́
́
́
(2H, H2-7), 3.22 m (2H, H2-1), 3.08 m (1H, H-3), 2.98 m (8H, H2-3, 4, 5,
́
́
́
́
6), 2.96 m (1H, H2-26b) , 2.46 m (2H, H2-2) , 2.40 m (2H, H2-3) , 2.31 m
Chemical characterization:
́
́
25
(2H, H2-8), 2.13 m (1H, H2-4a) , 1.82 m (1H, H-22) , 1.68 m (1H, H2-1a) ,
[α
]
D: +15.3 (c 0.15, MeOH), IR (film) νmax: 3383.6, 3201.4,
1.61 m (2H, H2-2) , 1.61 m (2H, H2-15), 1.60 m (1H, H2-7a) , 1.60 m
3133.9, 2932.4, 2858.1, 2697.1, 1658.6, 1589.1, 1466.1, 1376.9,
(1H, H2-1a) , 1.44 m (1H, H2-4b) , 1.44 m (1H, H2-4a) , 1.36 m (1H, H-5)
1036.6, 767.9 cmꢀ 1. 1H NMR (CDCl3, 400.13 MHz): 8.11 brd (1H, J =
́
́
́
́
́
, 1.32 m (1H, H-8) , 1.27 m (2H, H2-6) , 1.26 m (1H, H2-7b) , 1.23 m (1H,
8.2 Hz, H-7), 7.91 d (1H, J = 8.2 Hz, H-5), 7.69 brt (1H, J = 7.5 Hz, H-6),
́
́
́
́
́
́
́
H-14) ,1.19 m (1H, H2-1b), 1.14 m (1H, H2-4b), 1.12 d (3H, J = 6.7 Hz,
H3-21), 1.05 m (1H, H-9), 0.92 d (3H, J = 6.7 Hz, H3-27), 0.90 m (1H,
7.43 d (2H, J = 7.8 Hz, H-4, H-6), 7.42 m (1H, H-8), 7.36 d (2H, J = 7.8
́
́
́
́
́
́
Hz, H-3, H-5), 4.48 m (1H, H-16), 3.72 m (2H, H2-8), 3.49 m (1H, H-3),
H2-1b), 0.88 s (3H, H3-19), 0.73 s (3H, H3-18),0.60 m (1H, H-17). 13
C
3.47 m (2H, H2-1), 3.29 m (2H, H2-4),2.58 m (2H, H2-1), 2.00 m (1H, H-
́
́
́
́
́
́
́
́
NMR (CDCl3 100.03 MHz): 146.0 (C, C-4a), 142.8 (C, C-9), 143.1 (C, C-
22), 1.95 m (4H, H2-2,3), 0.98 d (3H, J = 6.8 Hz, H3-21), 0.87 d (3H, J =
10a), 138.4 (C, C-8a), 129.7 (CH, C-8), 129.7 (CH, C-6), 127.0 (CH, C-7),
6.7 Hz, H3-27), 0.80 s (3H, H3-19), 0.75 s (3H, H3-18). 13C NMR (CDCl3
́
́
́
́
́
́
́
́
́
́
́
100.03 MHz): 154.3 (C, C-9), 148.5 (C, C-4a), 141.8 (C, C-7), 141.7 (C,
126.9 (CH, C-5), 110.0 (C, C-9a), 95.8 (C, C-23), 74.9 (CH, C-16), 68.8
́
́
́
́
́
́
́
́
́
́ ́
C-2), 140.7 (C, C-10a), 137.6 (C, C-8a), 131.7 (CH, C-6), 127.8 (CH, C-4,
(CH, C-22), 70.0 (CH2, C-7), 61.5 (CH, C-14), 64.2 (CH2, C-2), 58.2 (CH2,
́
́
́
́
́
́
́
́
́
́
́
́
́
́
́
C-26), 58.2 (CH2, C-3, 4,5,6), 55.2 (CH, C-5), 54.2 (CH, C-17), 53.2 (CH,
C-6), 127.7 (CH, C-3, C-5), 126.1 (CH, C-5), 125.2 (CH, C-8), 123.5 (CH,
́
́
́
́
C-7), 116.8 (C, C-9a), 99.3 (C, C-23), 74.2 (CH, C-16), 68.5 (CH, C-22),
C-3), 45.5 (CH, C-9), 42.5 (C, C-13), 44.0 (CH2, C-1), 34.8 (CH, C-8),
́
́
́
́
́
́
́
33.9 (CH2, C-8), 31.9 (CH2, C-7), 36.3 (CH2, C-4), 37.1 (CH2, C-1), 36.1
(CH2, C-4), 35.2 (C, C-10), 29.6 (CH2, C-2), 29.6 (CH2, C-15), 28.3 (CH2,
65.5 (CH2, C-1), 60.4 (CH, C-14), 58.4 (CH2, C-8), 54.8 (CH, C-17), 54.6
(CH, C-5), 54.5 (CH2, C-26), 50.6 (CH, C-3), 45.8 (CH2, C-24), 45.4 (CH,
C-9), 38.7 (CH2, C-1), 32.8 (CH, C-20), 39.0 (C, C-13), 35.4 (CH, C-8),
́
́
́
C-1), 22.6 (CH2, C-6), 21.5 (CH2, C-2), 18.7 (CH3, C-27), 21.4 (CH2, C-3),
14.1 (CH3, C-19), 14.0 (CH3, C-21), 12.1 (CH3, C-18). HRESIMS m/z [M
+ H]+767.59552 (calcd for C48H75N6O2+, 767.59460).
́
́
́
́
29.6 (CH, C-25), 29.6 (CH2, C-4), 23.7 (CH2, C-1), 22.1 (CH2, C-2, C-3),
18.5 (CH3, C-27), 14.9 (CH3, C-21), 13.5 (CH3, C-18), 12.2 (CH3, C-19).
HRESIMS m/z[M + H]+731.52756 (calcd for C48H67N4O+2 , 731.52585).
4.3.3. (22R, 23S, 25R)- 3β-N-[(1-(2-aminoethyl)-1H-1,2,3-triazol-4-yl)
methyl)]-1,2,3,4-tetrahydroacridin-9-yl)amine-22,26-imino-16β,23-epoxy-
4.3.4. (22R, 23S, 25R)- 3β-N-[4-(aminomethyl)benzyl]-(1,2,3,4-
5
α
-cholestan-23β-ol (3)
tetrahydroacridin-9-yl)amine-22,26-imino-16β,23-epoxy-5α-cholestan-
Step 1:
23β-ol (4)
A solution of 54 mg (0.83 mmol) of NaN3 and 50 µL of iodoethanol
Step 1:
(0.64 mmol) dissolved in 1 mL DMF were stirred at room temperature
for 2 h. Then 2 mL of H2O were added and extracted with diethyl ether
(5x 3 mL). The organic layers were combined, dried with anhydrous
Na2SO4 and evaporated under reduced pressure, recovering 67 mg of a
clear liquid, which is assumed to be azidoethanol pure.
Step 2:
300 mg of terephthalic acid (1.8 mmol) were dissolved in 3 mL of
MeOH in a flask and 100 µL of concentrated H2SO4 were added. The
mixture was refluxed for 3 h, then neutralized with 1 M NaOH solution.
After that, 7 mL of H2O were added and extracted with AcOEt (4 × 8
mL). The combined organic layers were dried with anhydrous Na2SO4
and evaporated under reduced pressure. 265 mg of esterified product
(1.4 mmol) were recovered.
Moreover, 145 mg (0.73 mmol) of tetrahydroacridine hydrochloride
were placed in a reaction flask and dissolved in 3 mL of dry DMF, 140 µL
of tosylated propargyl alcohol (0.8 mmol) were added and then 1.36
mmol (76 mg) of KOH. The reaction mixture was vigorously stirred for 3
h, and then 5 mL of H2O were added and partitioned with diethyl ether
(5 × 10 mL). The organic layers were combined, dried with anhydrous
Na2SO4 and evaporated under reduced pressure. 86 mg of impure
tacrine derivative were recovered.
Step 2:
The product of the reaction carried out in step 1 was dissolved in 4
mL of dry THF and then 4 mmol of LiAlH4 (155 mg) were added and the
mixture was vigorously stirred for 15 h at room temperature under an
inert atmosphere of N2. Then 5 mL of AcOEt and 5 mL of an aqueous
solution of H2SO4 (10%) were added. The aqueous phase was extracted
with successive portions of AcOEt (8 × 5 mL). The organic phases were
combined, dried with anhydrous Na2SO4 and evaporated under reduced
pressure. 160 mg of the corresponding diol (that was assumed as 1.16
mmol, 1,4-phenylenedimethanol) were recovered.
A solution of 21 mg of azidoethanol (0.24 mmol), obtained in step 1
in a MeOH:H2O (1:5) mixture were added to the solid obtained in step 2
and was stirred at room temperature. After that, 66 µL of triethylamine
(0.48 mmol) and 6 mg of CuI (0.03 mmol) were added. It was allowed to
react for 20 h. The reaction mixture was extracted with AcOEt (5 × 5
mL). The organic layers were combined, dried with anhydrous Na2SO4
and evaporated under reduced pressure. 119 mg of an impure product
were recovered.
Step 3:
The product of step 2 was then placed in a reaction flask and dis-
solved in 5 mL of DCM and 1 mL of dry DMF. 170 mg (1.4 mmol) of
dimethylaminopyridine, 534 mg (2.8 mmol) of p-toluenesulfonic acid
and 320 µL (2.3 mmol) of triethylamine were added. The mixture was
stirred at room temperature for 2 h. After that reaction time, the reaction
crude was concentrated to dryness.
Step 3:
The reaction mixture of the click reaction was then placed in a flask
and dissolved in 3 mL of DCM. 28 mg (0.23 mmol) of dimethylamino-
pyridine, 87 mg (0.46 mmol) of p-toluenesulfonic acid and 53 µL (0.38
mmol) of triethylamine were added to this mixture and stirred at room
temperature for 1 h. The crude was extracted with diethyl ether (5x 5
mL). The organic phases were combined, dried with anhydrous Na2SO4
and evaporated under reduced pressure. 21.5 mg of crude was
recovered.
Step 4:
80 mg of the activated linker were dissolved in 3 mL of dry DMF,
with 12 mg of KOH. Then, 18 mg of tetrahydroacridine hydrochloride
(0.09 mmol) were added and placed under stirring for 5 h at room
temperature. Then an aqueous solution of saturated Na2CO3 (5 mL) was
added and extracted with diethyl ether (5x 10 mL). The organic phases
were combined, dried with anhydrous Na2SO4 and evaporated under
reduced pressure. After that, 307 mg of reaction mixture were recovered
and subjected to flash chromatography using hexane and DCM as sol-
vents to remove tosylate-excess and a solution of DCM-MeOH (20%) for
the elution of the compound of interest. This was subjected to
Step 4:
The crude of the reaction carried out in step 3 was redissolved in 2
mL of DMF, then 4 mg of KOH and 20 mg of solanocapsine (0.046 mmol)
were added, under stirring for 48 h at 50 ◦C. Then, 5 mL of H2O were
10

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
purification by preparative TLC, and 9.5 mg of product were recovered
(that was assumed as the coupling product by its polarity in TLC).
Step 5:
DCM: acetonitrile (1: 1), 0.09 mmol (15 mg) of KI were added, stirred for
30 min and then solanocapsine (0.07 mmol, 30 mg) and triethylamine
(40 µL) were added. The mixture was allowed to react for 24 h, then was
concentrated to dryness and purified by preparative TLC using DCM:
MeOH (10%, sequential elution) to obtain 3.5 mg of compound 5(7%) as
a brown amorphous solid.
The tacrine derivative obtained in step 4 was dissolved in 2 mL of dry
DMF, then 2 mg of KOH and 10 mg of solanocapsine (0.026 mmol) were
added, and the reaction was stirred at 50 ◦C for 24 h. After that, 5 mL of
H2O was added, and the reaction mixture was partitioned with ethyl
ether (5 × 10 mL). The organic layers were combined, dried with
anhydrous Na2SO4 and evaporated under reduced pressure. The mixture
was purified by preparative TLC using a sequential elution of DCM:
MeOH (20%). Finally, 2.8 mg of the product 4 were obtained as a yellow
amorphous solid (16.5%).
Chemical characterization:
25
[α
]
D: +44.0 (c 0.08, MeOH), IR (film) νmax: 3338.3, 2930.4,
2858.1, 1729.9, 1668.2, 1636.4, 1589.1, 1521.6, 1459.9, 1176.4,
1082.9, 1033.7, 755.0 cmꢀ 1. 1H NMR (CDCl3, 400.13 MHz): 7.89 d (1H,
́
́
́
J = 8.9, H-6), 7.78 brs (1H, H-8), 7.48 dd (1H, J = 8.9, 1.7 Hz, H-5), 4.44
́
́
́
́
m (1H, H-16), 3.87 m (2H, H2-5), 3.51 m (2H, H2-1), 3.17 m (1H, H-3),
́
́
3.04 m (1H, H2-26a), 3.02 m (2H, H2-2a), 2.57 m (2H, H-1), 2.18 m (1H,
Chemical characterization:
25
́
́
[
α]
D: +10.9 (c 0.10, MeOH), IR (film) νmax: 3347.0, 2926.6,
H2-26b), 2.02 m (1H, H-22), 1.95 m (1H, H-25), 1.93 m (4H, H2-2, 3),
́
́
2853.3, 1670.1, 1591.1, 1457.0, 1411.7, 1381.8, 1350.9, 1211.1,
1.84 m (2H, H2-2), 1.82 m (1H, H2-4a), 1.82 m (1H, H2-4a), 1.81 m (1H,
1173.5, 1034.7, 1013.5, 759.9 cmꢀ 1
.
1H NMR (CDCl3, 400.13 MHz):
H-20), 1.69 m (1H, H2-1a), 1.63 m (1H, H2-2a), 1.57 m (2H, H2-15),
́
́
́
́
8.09 m (1H, J = 8.3, H-5), 8.00 m (1H, J = 8.2, H-8), 7.59 dd (1H, J =
1.50 m (1H, H2-11a), 1.50 m (1H, H2-3a), 1.37 m (1H, H-5), 1.35 m (1H,
́
́
́
́
́
́
7.0, 0.9 Hz, H-6), 7.39 dd (1H, J = 7.6, 1.2 Hz, H-7), 6.53 d (1H, J = 1.6
H-8), 1.34 m (2H, H2-4), 1.33 m (1H, H2-11b), 1.31 m (1H, H2-3b), 1.27
m (2H, H2-6), 1.23 m (1H, H2-4b), 1.21 m (1H, H2-24b), 1.18 m (1H, H-
9), 1.06 m (1H, H-14), 1.02 m (1H, H2-1b), 0.96 d (3H, J = 6.7 Hz, H3-
21), 0.87 m (1H, H2-2b), 0.85 d (3H, J = 6.6 Hz, H3-27), 0.79 s (3H, H3-
19), 0.73 s (3H, H3-18), 0.72 m (1H, H-17). 13C NMR (CDCl3 100.03
́
́
́
́
́
́
Hz, H-3), 4.88 brs (2H, H2-4), 4.49 brd (2H, J = 4.7 Hz, H2-2), 4.46 m
́
́
́
(1H, H-16), 4.04 brd (2H, J = 4.7 Hz, H2-1), 3.11 m (2H, H2-1), 3.11 m
́
́
(2H, H2-4), 3.04 m (1H, H2-26a), 2.77 m (1H, H-3), 2.70 m (2H, H2-3),
́
2.48 m (1H, H2-2a), 2.18 m (1H, H2-26b), 2.01 m (1H, H-22), 1.87 m
́
́
́
́ ́
MHz): 157.3 (C, C-4a), 147.4 (C, C-10a), 142.5 (C, C-9), 129.9 (CH, C-5),
(1H, H2-2b), 1.81 m (1H, H-20), 1.69 m (1H, H2-1a), 1.63 m (1H, H2-2a),
́
́
́
́
1.57 m (2H, H2-15), 1.49 m (1H, H2-11a), 1.38 m (1H, H-5), 1.34 m (1H,
H-8),1.28 m (1H, H2-11b), 1.26 m (2H, H2-6), 1.13 m (1H, H-9), 1.09 m
(1H, H-14), 0.98 m (1H, H2-1b), 0.96 d (3H, J = 6.7 Hz, H3-21), 0.87 m
(1H, H2-2b), 0.86 d (3H, J = 6.8 Hz, H3-27), 0.80 s (3H, H3-18), 0.75 s
(3H, H3-19), 0.71 m (1H, H-17). 13C NMR (CDCl3 100.03 MHz): 152.3
128.9 (CH, C-6), 129.7 (C, C-7), 119.5 (CH, C-8), 116.9 (C, C-8a), 110.5
́
(C, C-9a), 95.9 (C, C-23),74.4 (CH, C-16), 68.9 (CH, C-22), 60.3 (CH, C-
14), 54.9 (CH2, C-26), 54.8 (CH, C-5), 54.6 (CH, C-17), 46.4 (CH, C-3),
́
́
́
́
46.2 (CH2, C-24), 46.0 (CH2, C-5), 45.4 (CH, C-9), 41.8 (CH2, C-1), 34.8
(CH, C-8), 35.6 (C, C-10), 39.1 (C, C-13), 39.0 (CH2, C-4), 32.7 (CH2, C-
́
́
́
́
́
́
́
(C, C-4a), 144.3 (C, C-9), 143.3 (C, C-10a), 130.0 (CH, C-6), 124.8 (CH,
4), 37.1 (CH2, C-1), 31.7 (CH2, C-2), 29.9 (CH, C-25), 29.8 (CH2, C-2),
́
́
́
́
́
́
́
́
C-7), 123.6 (C, C-4), 123.4 (CH, C-5), 121.3 (CH, C-8), 118.4 (C, C-8a),
28.8 (CH2, C-4), 33.0 (CH, C-20), 28.4 (CH2, C-6), 28.4 (CH2, C-15), 23.3
́
́
́
́
́
́
115.4 (C, C-9a), 106.5 (CH, C-3), 95.7 (C, C-23), 74.5 (CH, C-16), 68.8
(CH2, C-1), 18.6 (CH3, C-27), 20.3 (CH2, C-11),20.3 (CH2, C-3), 22.1
́
́
́
́
(CH, C-22), 61.0 (CH2, C-1), 60.5 (CH, C-14), 55.0 (CH, C-5), 54.9 (CH2,
(CH2, C-2, C-3), 13.5 (CH3, C-18), 12.3 (CH3, C-19), 15.0 (CH3, C-21).
C-26), 54.6 (CH, C-17), 52.6 (CH2, C-2), 51.0 (CH, C-3), 45.4 (C, C-13),
HRESIMS m/z[M
+
H]+731.50621(calcd for C45H68ClN4O+2 ,
́
́
́
́
45.3 (CH, C-9), 44.4 (CH2, C-5), 41.6 (C, C-10), 34.8 (CH, C-8), 39.0
731.50253).
(CH2, C-4), 37.1 (CH2, C-1), 32.9 (CH, C-20), 31.6 (CH2, C-2), 31.6 (CH2,
́
́
C-4), 30.1 (CH2, C-1), 29.9 (CH, C-25), 28.5 (CH2, C-6), 28.2 (CH2, C-
4.4. Cholinesterases inhibition assay
́
́
15), 24.3 (CH2, C-3), 22.7 (CH2, C-2), 20.5 (CH2, C-11), 18.7 (CH3, C-
27), 15.1 (CH3, C-21), 13.6 (CH3, C-19), 12.4 (CH3, C-18). HRESIMS m/z
[M + H]+736.52899 (calcd for C45H66N7O+2 , 736.52725).
Electric eel AChE and horse serum BuChE were used as a source of both
the cholinesterases. AChE and BuChE inhibiting activities were
measured in-vitro by the spectrophotometric method developed by Ell-
man with slight modification [54]. The lyophilized enzyme, 500U AChE
/300U BuChE was prepared in buffer A (8 mM K2HPO4, 2.3 mM
NaH2PO4) to obtain 5/3 U/mL stock solution. Further enzyme dilution
was carried out with buffer B (8 mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M
NaCl, 0.05% Tween 20, pH = 7.6) to produce 0.126/0.06 U/mL enzyme
solution. Samples were dissolved in buffer B with 2.5% of MeOH as
4.3.5. (22R, 23S, 25R)-3β-N-(7-chloro-1,2,3,4-tetrahydroacridin-9-yl)
pentane-1,5-diamine-22,26-imino-16β,23-epoxy-5α-cholestan-23β-ol (5)
Step 1:
In a first step, 125 mg of tetrahydroacridine hydrochloride (0.63
mmol) were placed in a reaction flask. Then a solution of 86 mg of N-
chlorosuccinimide (0.63 mmol) in a mixture of DCM (2 mL) and MeOH
(0.5 mL) was added to this first flask, and the reaction was kept under
stirring at room temperature. After 1 h of reaction, 67 mg of Na2CO3
(0.63 mmol) were added. The mixture was concentrated to dryness until
evaporation of the organic solvent, redissolved with H2O (10 mL) and
extracted with DCM (3x 10 mL). The organic layers were combined,
dried with anhydrous Na2SO4. The reaction mixture (138 mg) was pu-
rified by column chromatography, using a sequential elution of DCM:
MeOH (20%). The fractions corresponding to the elution with DCM:
MeOH (97: 3%) were combined and 63 mg (0.27 mmol), assumed as
chloro-tetrahydroacridine were obtained.
cosolvent in concentrations ranging from 5 nM to 20
from 5 nM to 50 M for BuChE. Enzyme solution (300
solution (300 L) were mixed in a test tube and incubated for 60/120
min at room temperature. The reaction was started by adding 600 L of
μ
M for AChE and
μ
μ
L) and sample
μ
μ
the substrate solution (0.5 mM DTNB, 0.6 mM ATCI/BTCI, 0.1 M
Na2HPO4, pH 7.5). The absorbance was read at 405 nm for 180 s at
27 ◦C. Enzyme activity was calculated by comparing reaction rates for
the sample to the blank. All the reactions were performed in triplicate.
IC50 values were determined with GraphPad Prism 5. Tacrine was used
as a reference AChE/BuChE inhibitor. For more details, see table S1 in
supporting information.
Step 2:
Chlorated precursor was dissolved in 1 mL of DMSO, 0.68 mmol of
KOH (38.2 mg) and then 0.28 mmol of 1,5-dibromopentane (40 µL) were
added. The reaction mixture was vigorously stirred at room temperature
for 15 h. Then, this mixture was diluted with H2O (10 mL) and extracted
with AcOEt (4x 8 mL). The combined organic extracts were washed with
H2O (3 × 15 mL), dried with anhydrous Na2SO4 and evaporated under
reduced pressure.
5. Computational methods
5.1. Molecular docking studies
The complexes between the ligands and the enzymes were obtained
by molecular docking employing the coordinates from experimental X-
ray structures for each receptor. The AChE enzyme was taken from
Torpedo californica, TcAChE, and BuChE from Homo sapiens, hBuChE,
Step 3:
Elongated chloro-tacrine crude (38 mg) was taken up in a mixture of
11

J.L. Borioni et al.
Bioorganic Chemistry 111 (2021) 104893
(PDB code TcAChE: 2CMF and PDB code hBuChE: 4BDS) [50,51]. The
procedure for preparing the protein consisted of a treatment of the
titratable residues within the publicly accessible H++ server [55,56]
and removal of glycosides, the ligand and crystallographic waters.
Finally, the sulfide bonds were made between corresponding cysteines.
The receptors were prepared using Make Receptor software from OpenEye
package in combination with a shape-based site detection algorithm and
the position of a well-known bound ligand.
equilibrated part, of each MD run, were considered.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Before starting the docking simulations, the pKa values were esti-
mated in silico with the pka calculator plugin available in MarvinSketch
v20.12.0 software package, assuming a pH of 7.4 as physiological value
[49]. The ligand conformer libraries were obtained with Omega2
v3.0.1.2 software, with conformers generated at an energy threshold of
10 kcal/mol [57]. The docking runs were performed with Fred v3.0.1
software and ranked using the Chemmgauss4 scoring functions [58,59].
The visualization of the docking results were performed with VMD
v1.9.3 [60].
Acknowledgements
We would like to thank to Ph.D. Fabricio Bisogno for helpful com-
ments and discussions about synthetic aspects and Federico Brigante for
the English revision. NMR assistance by Ph.D. Gloria Bonetto is grate-
fully acknowledged. This work has been financed by Consejo Nacional
´
de Investigaciones Científicas y Tecnologicas (CONICET), Agencia
´
´
Nacional de Promocion Científica y Tecnologica (ANPCyT, Argentina),
´
Secretaría de Ciencia y Tecnica (SECyT- Universidad Nacional de
The structure of each complex obtained after the docking procedure,
was minimized, and the binding energy recalculated according to the
following procedure. The ff99SB force field was selected to model the
protein. The ligand units were built with the Antechamber module, using
GAFF force field and AM1-BCC fitted charges [61,62].The input files for
the simulations were made with the Xleap package included in Amber-
tools package. Then, two minimizations, of 5000 steps each, of the
complexes were performed employing Sander software. The first one
keeping the protein heavy atoms retrained at their initial positions and
the second one with the whole system free. Finally, after the minimi-
zation steps, the binding free energies were calculated with the molec-
ular mechanics-Generalized Born surface area (MM-GBSA) approximation
[63,64]. After this refinement, the most stable complex with each in-
hibitor was selected for MD simulations.
´
Cordoba).
INFIQC and IMBIV are jointly sponsored by CONICET and the Uni-
´
versidad Nacional de Cordoba. All calculations were performed with
´
computational resources from the Centro de Computacion de Alto
˜
´
Desempeno - Universidad Nacional de Cordoba (http://ccad.unc.edu.
ar/), in particular the Mendieta Cluster that belongs to the Facultad de
´
Matematica, Astronomía y Física, that is also part of SNCAD, República
Argentina.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104893.
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