Tuning the activity of iminosugars: novel N-alkylated deoxynojirimycin derivatives as strong BuChE inhibitors

Article abstract of DOI:10.1080/14756366.2020.1847101

We have designed unprecedented cholinesterase inhibitors based on 1-deoxynojirimycin as potential anti-Alzheimer’s agents. Compounds are comprised of three key structural motifs: the iminosugar, for interaction with cholinesterase catalytic anionic site (

Full text of DOI:10.1080/14756366.2020.1847101

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Tuning the activity of iminosugars: novel N-
alkylated deoxynojirimycin derivatives as strong
BuChE inhibitors
Ana I. Ahuja-Casarín , Penélope Merino-Montiel , José Luis Vega-Baez , Sara
Montiel-Smith , Miguel X. Fernandes , Irene Lagunes , Inés Maya , José M.
Padrón , Óscar López & José G. Fernández-Bolaños
To cite this article: Ana I. Ahuja-Casarín , Penélope Merino-Montiel , José Luis Vega-Baez ,
Sara Montiel-Smith , Miguel X. Fernandes , Irene Lagunes , Inés Maya , José M. Padrón , Óscar
López & José G. Fernández-Bolaños (2021) Tuning the activity of iminosugars: novel N-alkylated
deoxynojirimycin derivatives as strong BuChE inhibitors, Journal of Enzyme Inhibition and
Medicinal Chemistry, 36:1, 138-146, DOI: 10.1080/14756366.2020.1847101
To link to this article: https://doi.org/10.1080/14756366.2020.1847101
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 138–146
https://doi.org/10.1080/14756366.2020.1847101
BRIEF REPORT
Tuning the activity of iminosugars: novel N-alkylated deoxynojirimycin derivatives
as strong BuChE inhibitors
a
a
a
a
b
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Ana I. Ahuja-Casarın , Penelope Merino-Montiel , Jose Luis Vega-Baez , Sara Montiel-Smith , Miguel X. Fernandes ,
b
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b
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Irene Lagunes , Ines Maya , Jose M. Padron , Oscar Lopez and Jose G. Fernandez-Bolanos
a
b
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Facultad de Ciencias Quımicas, Ciudad Universitaria, Benemerita Universidad Autonoma de Puebla, Puebla, Mexico; BioLab, Instituto
c
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Universitario de Bio-Organica “Antonio Gonzalez” (IUBO-AG), Universidad de La Laguna, La Laguna, Spain; Departamento de Quımica Organica,
Facultad de Quımica, Universidad de Sevilla, Seville, Spain
ꢀ
ABSTRACT
ARTICLE HISTORY
Received 21 September 2020
Revised 18 October 2020
Accepted 23 October 2020
We have designed unprecedented cholinesterase inhibitors based on 1-deoxynojirimycin as potential anti-
Alzheimer’s agents. Compounds are comprised of three key structural motifs: the iminosugar, for inter-
action with cholinesterase catalytic anionic site (CAS); a hydrocarbon tether with variable lengths, and a
fragment derived from 2-phenylethanol for promoting interactions with peripheral anionic site (PAS). Title
compounds exhibited good selectivity towards BuChE, strongly depending on the substitution pattern
and the length of the tether. The lead compounds were found to be strong mixed inhibitors of BuChE
(IC50 ¼ 1.8 and 1.9 mM). The presumptive binding mode of the lead compound was analysed using
molecular docking simulations, revealing H-bond interactions with the catalytic subsite (His438) and CAS
(Trp82 and Glu197) and van der Waals interactions with PAS (Thr284, Pro285, Asn289). They also lacked
significant antiproliferative activity against tumour and non-tumour cells at 100mM, making them promis-
ing new agents for tackling Alzheimer’s disease through the cholinergic approach.
KEYWORDS
Iminosugars; 1-DNJ;
cholinesterase inhibitors;
anti-Alzheimer’s agents;
docking simulations
GRAPHICAL ABSTRACT
Introduction
damage, particularly to the respiratory system. Iminosugars can
act as correctors of defective cystic fibrosis transmembrane con-
ductance regulator (CFTR)14,15, an ABC transporter-class protein
that controls the content of ion and water in epithelial cells, or
alternatively they can reduce the inflammatory response of patho-
gens, like P. aeruginosa16. More recently, conjugation of iminosu-
gars with sulphonamides resulted in the inhibition of relevant
human carbonic anhydrases17. Among the vast number of imino-
sugars and related structures reported so far, 1-deoxynojirimycin
(1-DNJ), a natural iminosugar isolated from mulberry leaves18, is
the basis for the so-far two marketed drugs derived from iminosu-
Imino- and azasugars, that is, carbohydrate mimetics where the
endocyclic oxygen or a carbon atom, respectively, has been
replaced by a nitrogen atom that has attracted great attention
within Medicinal Chemistry since several decades ago1. The key
structural motif of such glycomimetics is a protonable nitrogen
atom, which might allow them to mimic transition states (in terms
of charge and geometry)2 of the glycosidase-mediated hydrolysis
of glycosidic bonds. Because of this, many imino- and azasugars
behave as potent inhibitors of ubiquitous glycosidases and glyco-
syltransferases1, key enzymes in a plethora of biological processes
where carbohydrates are involved, including metabolic pathways,
cell wall formation, and recognition events. Numerous imino- and
azasugars have shown relevant pharmacological activities3, such
as antidiabetic4, anticancer5, antifungal6, antiprotozoal7 or antiviral
agents8, among others. In this sense, Sirona Biochem Corp. has
just announced9 the launching of an intense programme search-
ing for new antivirals for combating the pandemic caused by
COVID-19, in which iminosugars are one of the three categories in
the study. This choice is strongly supported by the World
Laureates Association Shanghai Centre. Some iminosugars have
also been found to act as chaperones10 against lysosomal storage
disorders, like Gaucher’s11 and Fabry’s12 diseases. Iminosugars
have also been found to be effective against cystic fibrosis13, an
R
R
gars: MiglitolV (N-hydroxyethyl-1-deoxynojirimycin)19 and ZavescaV
(N-butyl-1-deoxynojirimycin)20, used for tackling non-insulin-
dependent diabetes, and Gaucher’s disease, respectively.
Nevertheless, a field that still remains relatively unexplored con-
cerning iminosugars is Alzheimer’s disease. Alzheimer’s is a devas-
tating neurodegenerative disease, considered nowadays the most
prevalent form of dementia, counting for roughly 70% of total
cases21. It causes severe impairment of cognitive functionality, neu-
rodegeneration, and even Parkinsonian symptoms, leading the
patient to a complete dependence even for accomplishing daily
tasks22. According to the World Alzheimer Report 2019, roughly 50
million people have been diagnosed worldwide, and this number is
expected to increase almost exponentially to 152 million people by
autosomal recessive disease-causing severe multi-system organ 205023. Another worrying aspect is that, although Alzheimer’s
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Facultad de Ciencias Quımicas, Ciudad Universitaria, Benemerita Universidad
CONTACT Penelope Merino-Montiel
penelope.merino@correo.buap.mx
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BioLab, Instituto Universitario de Bio-Organica “Antonio Gonzalez” (IUBO-AG),
Autonoma de Puebla, Puebla 72570, Mexico; Jose M. Padron
jmpadron@ull.es
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ꢀ
ꢀ
ꢀ
ꢀ
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Departamento de Quımica Organica,
Universidad de La Laguna, c/Astrofısico Francisco Sanchez 2, La Laguna E-38206, Spain; Oscar Lopez
osc-lopez@us.es
ꢀ
Facultad de Quımica, Universidad de Sevilla, Apartado 1203, Seville E-41071, Spain
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
139
disease has been classically associated with elderly people, the case to give compounds
7 and 8 as colourless oils (See
average age of patients has decreased significantly24.
Supplementary Material).
From a pathophysiological point of view, Alzheimer’s disease is
one of the most complex diseases nowadays, whose multifactorial
aetiology is not even completely understood. This hampers enor-
mously finding a treatment. The most recognised hallmarks are25
the deposit of toxic amyloid plaques, neurofibrillary tangles (via
hyperphosphorylation of tau proteins), and low levels of the
neurotransmitter acetylcholine, which is associated with the cogni-
tive decline.
General procedure for the preparation of compounds 9 and 10
To a solution of per-O-benzylated 1-deoxynojirimycin 6 (250 mg,
0.48 mmol) in DMF (5 ml), K2CO3 (199 mg, 1.44 mmol, 3.0 equiv.)
and O-alkylated derivatives 7 or 8 (1.19 mmol, 2.5 equiv.) were
added. The corresponding mixture was kept stirring at 85 ꢀC for
15 h. Then water was added, and products were portioned
between brine (50 ml) and EtOAc (3 ꢁ 50 ml). The combined
organic fractions were dried over Na2SO4, filtered and the filtrate
was concentrated to dryness under reduced pressure. The residue
was purified by column chromatography using the eluents indi-
cated in each case, to give compounds 9 and 10 as colourless syr-
ups (See Supplementary Material).
Currently, there are four marketed drugs for ameliorating the
symptoms of Alzheimer’s disease26 aiming at two therapeutic tar-
gets: the inhibitors of cholinesterases (acetylcholinesterase, AChE;
and butyrylcholinesterase, BuChE) donepezil, rivastigmine and gal-
antamine, which increase the levels of acetylcholine (cholinergic
hypothesis), and the antagonist of N-methyl-D-aspartic receptors
(NMDA) memantine, for regulating glutamate levels in brain cells.
R
In connection with iminosugars, ZavescaV was recently found
General procedure for the preparation of compounds 11 and 12
To a solution of benzylated derivative 9 or 10 (0.20 mmol) in a 1:1
CH2Cl2–MeOH mixture (6 ml), AcOH (0.4 ml) and Pd(OH)2/C
(150 mg) were added, and the corresponding mixture was hydro-
to reduce the amyloid plaque production in a cell model27.
Compain and co-workers analysed the cholinesterase inhibition of
a series of iminosugars with different substituents motifs and
stereochemistry28. In our research group, we recently combined 1-
DNJ with a selenoureido appendage to achieve strong b-glucosi-
dase and AChE inhibitors29. To the best of our knowledge, these
are the only examples of iminosugars exhibiting anti-Alzheimer’s
properties.
R
genated for 24 h. After that, it was filtered over a CeliteV pad and
washed with MeOH. The filtrate was concentrated to dryness and
purified by column chromatography (190:10:1
!
140:60:1
CH2Cl2–MeOH–Et3N) in the case of 11. For compounds 12, no fur-
ther purification was accomplished after filtration. Derivatives 11
and 12 were obtained as colourless syrups (See Supplementary
Material).
Our main target herein has been the design of new potential
AChE and/or BuChE inhibitors against Alzheimer’s disease using 1-
DNJ as the core template. Rationalisation of their biological prop-
erties by analysing interactions with key enzymatic domains is
also covered herein.
Enzymatic assays
For both families of enzymes used herein (glycosidases and choli-
nesterases), stock solutions of the inhibitors were prepared in
DMSO; DMSO content was kept at 5% (V/V) for glycosidases and
1.25% (V/V) for cholinesterases. Enzymes were properly dissolved
in water in a concentration in which the rate of the reaction when
using [S] ¼ 4 ꢁ KM is in the range 0.12–0.15 Abs/min. In both
kinds of enzymes, an initial screening at 100 mM inhibitor concen-
tration was performed, using the following substrate concentra-
tions: [S] ¼ 0.25 mM for a-glucosidase (Saccharomyces cerevisiae),
4.0 mM for b-glucosidase (almonds), 0.60 mM for a-galactosidase
(green coffee beans), 0.51 mM for b-galactosidase (Escherichia coli),
1.5 mM for b-galactosidase (Aspergillus Oryzae), 0.12 mM for AChE
(Electrophorus electricus), 0.11 mM for BuChE (equine serum). When
the percentage of inhibition was higher than 50%, the kinetic
parameters and the inhibition constants were calculated using five
Materials and methods
General procedures
1H (300.1 MHz) and 13C (75.5 MHz) NMR spectra were recorded at
25 ꢀC on a Bruker Avance 300 spectrometer using the deuterated
solvent indicated in each case. H and 13C assignments were con-
1
firmed by 2D COSY and HSQC experiments. Mass spectra (ESI)
were recorded on a Q Exactive mass spectrometer. TLCs were per-
formed on aluminium pre-coated sheets (E. Merck Silica gel 60
F254); spots were visualised by UV light, and by charring with 10%
vanillin in EtOH containing 1% of H2SO4, or with 10% H2SO4 in
EtOH. Column chromatography was performed using E. Merck
Silica Gel 60 (40–63 lm), using the eluent indicated in each case.
1
different substrate concentrations ranging from = K to 4 ꢁ KM,
4
M
Chemistry
and 2–4 different inhibitor concentrations (affording roughly
20–70% inhibition). Each assay was run in duplicate.
General procedure for the preparation of compounds 7 and 8
To a solution of the corresponding 2-arylethanol derivatives
14a–g (2.19 mmol) in DMF (5 ml), 60% NaH (525 mg, 13.14 mmol,
6.0 equiv.) was added and the resulting mixture was kept stirring
at rt under an inert atmosphere for 30 min. Then, 1,6-dibromohex-
ane or 1,5-dibromopentane was added (17.52 mmol, 8.0 equiv.),
and stirring was kept for a further 5 h in the case of 7, and 4 h in
the case of 8. The reaction was quenched by the careful addition
of water at 0 ꢀC. After that, the crude reaction mixture was diluted
with brine (50 ml) and extracted with ethyl acetate (3 ꢁ 50 ml). The
Glycosidase assays were accomplished using the methodology
reported by Bols and co-workers30. Each set of experiments is pre-
pared in PS cuvettes using 0.1 M phosphate buffer (pH 6.8) and
the corresponding o- or p-nitrophenyl glycopyranosides as sub-
strates. Reactions were monitored at 25 ꢀC by following the forma-
tion of the corresponding nitrophenolates at 400 nm (glucosidases
and a-galactosidase) or 420 nm (b-galactosidases) over a time
of 125 s.
For cholinesterases, minor modifications on Ellman’s assay31
combined organic fractions were dried over Na2SO4, filtered and were performed. The activity was measured in PS cuvettes con-
the filtrate was concentrated to dryness. The residue was purified taining 0.1 mM phosphate buffer (pH 8.0), 0.88 mM DTNB, and
by column chromatography, using the eluent indicated in each substrate (acetyl- and butyryl-thiocholine iodides for AChE and

140
A. I. AHUJA-CASARÍN
BuChE, respectively), The formation of the chromophore was side-effects. In particular, we envisioned the possibility of using 1-
monitored at 405 nm over a time of 125 s.
DNJ as the key core.
For strong inhibitors, the mode of inhibition was determined
using the Cornish–Bowden method, which involves the use of two
different plots: 1/v vs. [I] (Dixon plot) and [S]/v vs. [I]. For the cal-
culation of kinetic parameters (KM, Vmax) a nonlinear regression
analysis (least squares fit, GraphPad Prism 8.01) was used. Data
are expressed as the mean SD.
On the one hand, N-alkyl-DNJ derivatives could be partially
protonated at physiological pH, thus mimicking the ammonium
cation of acetylcholine, and enabling favourable interactions
within the CAS. Although it has been reported that the presence
of aromatic substituents connected to iminosugars through a
short alkyl tether (2–3 carbon atoms) leads to pKa of 6.037 or
below 6.038, because of the electron-withdrawing effect of the aryl
moiety, compounds prepared herein, with longer alkyl fragments
are expected to tentatively exhibit higher pKa values, probably in
between for those reported for N-nonyl-DNJ and N-butyl-DNJ (6.7
and 7.1, respectively)39. On the other hand, as aforementioned, 1-
DNJ has positive effects against amyloidogenesis. Moreover, 1-DNJ
has recently been found40 to ameliorate stable angina pectoris in
patients with coronary heart disease by reducing inflammatory
responses and by increasing the antioxidant machinery. Taking
into consideration that profound oxidative stress and neuroinflam-
mation are common features of Alzheimer’s disease41, the use of
1-DNJ as the pharmacophore might also provide activity against
other targets of this multifactorial disease. Furthermore, different
substitution patterns on the aromatic appendage might modulate
the interactions within the PAS region.
For accessing compounds 11 and 12, depicted in Scheme 1,
the key intermediate is per-O-benzylated 1-DNJ 6, which was
accessed in a 5-step linear pathway (36% overall yield), following
the procedure reported in the literature42, starting from commer-
cially-available methyl a-D-glucopyranoside 1 (Scheme 1). Such
procedure involves fully O-protection, acidic hydrolysis of the
glycoside moiety, reduction of the masked aldehyde of 3, Swern
oxidation43, and fast reductive amination of transient unstable
dicarbonyl compound 5; the last step consists of a double reduc-
tive amination (DRA), a cascade reaction that provides a straight-
Docking simulations
Interactions of cholinesterases with the compounds were analysed
by computational docking using MOE software (Chemical
Computing Group). Crystallographic structures of human AChE
and human BuChE was obtained from Protein Data Bank (PDB
code 4EY6 and 4AQD, respectively). Protein structures was ener-
getically minimised using Amber10 force field with EHT parame-
ters for small molecules, R-field solvation model, dielectric
constant of 1 for the protein interior and 80 for the exterior.
Ligand structure was drawn in MOE software, and its energy was
minimised with the above parameters using as stop criterion an
RMS gradient lower than 0.01 kcal/mol/Å. For the docking calcula-
tions: in the placement stage we used the Triangle Matcher algo-
rithm with the London dG scoring scheme. In the refinement
stage, we kept the receptor rigid and used the GBVI/WSA dG scor-
ing scheme. 2D diagrams were obtained from MOE software and
3D illustrations were obtained using Pymol software.
Antiproliferative assays
We selected the human neuroblastoma cell line SH-SY5Y and the
fibroblast cell line BJ-hTERT to evaluate the antiproliferative activ-
ity of the compounds. The tests were performed in 96-well plates
using the SRB assay32 with the following specifications. Cell seed-
ing densities were 5000 cells/well for SH-SY5Y and 7000 cells/well
for BJ-hTERT. Drug incubation times were 48 h. The optical density
of each well was measured at 530 (primary) and 620 (secondary)
nm. The antiproliferative activity expressed as 50% growth inhib-
ition (GI50), was calculated according to NCI formulas33.
forward
methodology
for
accessing
polyhydroxylated
piperidines44. Base-promoted N-alkylation with bromoderivatives 7
and 8, and deprotection by hydrogenolysis, furnished compounds
11 and 12, respectively, with the substitution pattern depicted in
Scheme 1. We hypothesised that the presence of one or two
methoxy groups could resemble the dimethoxy arene-based frag-
ment of the indanone moiety in donepezil, and thus, might
enable interaction with the PAS region of cholinesterases.
Moreover, the use of a o-dihydroxyphenyl ring (catechol moiety)
could afford relevant antioxidant properties45, as well as the cap-
acity for chelating metal ions (Zn, Cu, Fe are present at toxic levels
in the brain of Alzheimer’s patients)46.
Results and discussion
Chemistry
In terms of inhibition, AChE contains two key regions34: the cata-
lytic anionic site (CAS), which enables cation–p interactions with
the quaternary ammonium fragment of acetylcholine, the natural
substrate of the enzyme, and the peripheral anionic site (PAS),
enriched with aromatic amino acid residues, and located roughly
15 Å from CAS through a narrow gorge, capable of establishing
interactions with planar and aromatic residues. It is important to
mention that PAS is also involved in the initial step of b-amyloid
aggregation35. Accordingly, dual inhibitors establishing favourable
interactions with both, CAS and PAS, can be not only strong
inhibitors, but they can also contribute to ameliorate the forma-
tion of neurotoxic amyloid plaques.
To avoid side-reactions, those starting materials bearing free
phenolic hydroxyl groups (natural tyrosol 13b, hydroxytyrosol 13d
and homovanillic alcohol 13f) were protected using a chemoselec-
tive benzylation under mild basic conditions (See Supporting
Material, compounds 14b, d, and f). Moreover, for the scaffold
incorporating a methylidene motif, commercially-available 2–(30,40-
R1
OH
O
n
N
R2
Inspired by the structure of AChE, we propose herein the prep-
aration of a series of iminosugars-based cholinesterase inhibitors.
The general structure for such derivatives is depicted in Figure 1,
with three key structural motifs: the iminosugar residue, an alkoxy
tether, and an aromatic motif with different substitution patterns.
Oxygenated N-alkyl iminosugars have been found to reduce cyto-
HO
PAS interaction
OH
HO
CAS interaction
Figure 1. General structure of 1-DNJ derivatives as potential cholinesterase inhib-
toxicity when evaluated as antiviral agents36, thus diminishing itors designed on this work.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
141
Scheme 1. General procedure for the preparation of N-alkylated derivatives 11 and 12.
methylenedioxyphenyl)acetic acid 15 was reduced (See under such conditions, the kinetic parameters (KM,app and Vmax,app
)
were obtained, that were used for calculating the inhibition con-
stants (Ki’s). The mode of inhibition was also established for the
most potent compounds, using the Cornish–Bowden method51.
Supporting Material) under mild conditions, using a combination
of NaBH4 and I247. It has been reported48 that such conditions
lead to the in situ generations of borane, which reduces efficiently
the carboxyl group to a primary alcohol. Such treatment furnished
14g in a quantitative yield after chromatographic purification.
Mono-alkylation of 2-arylethanol derivatives 14a–g was accom-
plished using an excess of 1,6-dibromohexane under basic condi-
tions (NaH) in general from excellent to quantitative yields
(Scheme 1) to give 7, 8. Alkylation on the endocyclic nitrogen
atom of 6 (Scheme 1) was more complicated than previously
anticipated. Nucleophilic displacement of 6 on the terminal brom-
ine atom of 7a–g accomplished derivatives 9a–g in moderate
yields (46–68%). Final deprotection using standard hydrogenolysis
conditions furnished derivatives 11a–g in moderate to quantita-
tive yields.
To analyse the influence of the linkage length on the biological
properties, homologous 12a, c, and d were also prepared as rep-
resentative examples (Scheme 1). Noteworthy, extraordinary differ-
ences in activity towards cholinesterases were found by our group
upon assessment of tacrine-derived homo- and heterodimers as
anti-Alzheimer’s agents by small variations in the tether length49.
Again, the limiting step in terms of yields was the N-alkylation of
O-protected 1-DNJ 6. Analogously, previous attempts50 to alkylate
the same position on azafagomine, a cyclic hydrazine analogue of
1-DNJ via nucleophilic substitutions failed. This was attributed to
the high sterical hindrance exerted by the bulky exocyclic benzy-
loxymethyl moiety, in its major gt conformation, that occluded the
entry of the reagents.
Glycosidase and cholinesterase inhibition studies
As aforementioned, glycosidases play a pivotal role in controlling
not only the metabolic pathways where carbohydrates are present
but also many other significant biochemical events, like cell-to-cell
communication or recognition processes. Herein, a panel of five
commercially-available glycosidases have been tested, as models
of
carbohydrate-mediated
diseases:
a-glucosidase
(from
Saccharomyces cerevisiae), b-glucosidase (from almonds), a-galacto-
sidase (from green coffee beans) and b-galactosidase (from A. ory-
zae and from E. coli). Deficiencies in such enzymes are associated
with diabetes, Gaucher’s disease, Fabry’s disease, and gangliosido-
sis, respectively3.
Assessment of the inhibitory capacity against glycosidases was
accomplished using the methodology reported by Bols and co-
workers30. p-Nitrophenyl glycosides (o- for b-galactosidases) were
used as model substrates, and the release of the corresponding p-
nitrophenolates at pH 6.8 was monitored spectrophotometrically
(kmax ¼ 400 or 420 nm for p- or o-nitrophenolates, respectively).
Selected results are shown in Table 1, where the IC50 and
inhibition constants values, together with the mode of inhibition
are depicted. 1-DNJ is a good inhibitor of a-galactosidase and
moderate of a-glucosidase (IC50 ¼ 16 and 35 mM, respectively),
and a weak inhibitor of b-glucosidase (IC50 ¼ 71 mM). However,
compounds 11 and 12 lacked significant activity against a-glucosi-
dase and a/b-galactosidases (Table 1 and Table S1), and regarding
b-glucosidase, compounds 11 ranged from good (11b, d, and e,
IC50: 12–15 mM) to strong competitive inhibitors (11a, c, f, and g,
IC50: 4.6–8.4 mM). This means a complete reversal of selectivity
towards b-glucosidase, with a 15-fold increased activity compared
to natural 1-DNJ, suggesting that the more lipophilic substituents
favour interaction with b-glucosidase. The same behaviour was
Biological assays
The ten 1-DNJ derivatives prepared herein (11a–g, 12a, c, and d)
were subjected to inhibition assays on two families of enzymes:
glycosidases and cholinesterases. In both cases, an initial screen-
ing was performed at 100 mM inhibitor concentration. For those
compounds exhibiting a percentage of inhibition higher than 50% found upon alkylation on N-2 position of azafagomine50.

142
A. I. AHUJA-CASARÍN
Table 1. Enzyme inhibition for selected compounds 11a–g (IC50, Ki in mM).
Enzyme
Compound
a-Glucosidase
b-Glucosidase
AChE
BuChE
11a
IC50 ¼ 126
Kia ¼ 93 32
(Competitive)
IC50 >100
IC50 5 4.6
Kia 5 6.5 0.1
(Competitive)
IC50 ¼ 12
IC50 >100
IC50 >100
R1¼R2¼H
11b
IC50 >100
IC50 >100
IC50 >100
IC50 >100
IC50 >100
IC50¼ 76
R1¼OH, R2¼H
Kia ¼ 17
5
(Competitive)
IC50 5 6.0
11c
IC50 >100
IC50 >100
IC50 >100
R1¼OMe, R2¼H
Kia 5 4.5 0.7
(Competitive)
IC50 ¼ 15
11d
R1¼R2¼OH
Kia ¼ 11
4
(Competitive)
11e
IC50 ¼ 14
IC505 5.8
Kia 5 9.3 1.2
Kib 5 5.2 0.5
(Mixed)
IC50 5 1.9
Kia 5 1.4 0.4
Kib 5 4.5 1.4
(Mixed)
R1¼R2¼OMe
Kia ¼ 15
3
(Competitive)
11f
IC50 >100
IC50 >100
IC50 ¼ 35
IC50 5 8.2
Kia 5 9.6 0.8
(Competitive)
IC505 7.3
IC50 5 1.8
R1¼OH, R2¼OMe
Kia ¼ Kib ¼ 17
5
Kia 5 1.5 0.2
Kib 5 5.0 2.0
(Mixed)
IC50 5 7.3
Kia5 Kib 59.2 1.9
(Non-competitive)
(Non-competitive)
11g
IC505 8.4
Kia 5 9.0 3.0
(Competitive)
IC50 ¼ 48
Kia¼ 56 10
Kib¼ 43 14
(Mixed)
R1,R2¼OCH2O
1-DNJ
IC50 ¼ 71
IC50 >100
IC50 ¼ 10
Kia ¼ Kib ¼ 16
3
(Non-competitive)
Interestingly, a decrease in the tether length (compounds 12) was cholinesterase) but also for retarding or ameliorating neurotoxicity
found to be detrimental for the inhibition of b-glucosidase (e.g. associated with amyloidogenesis in advanced stages of
4–6-fold for 12a, c), showing the great influence of such struc-
tural motif.
the disease.
The absence of substitution on the aromatic scaffold, mono-
Derivatives 11 and 12 were tested also against AChE
(Electrophorus electricus) and BuChE (equine serum), as recognised
models for human cholinesterases due to their close structural
resemblance52. For that purpose, Ellman’s colorimetric assay was
used; this is an indirect test in which acetyl and butyryl thiocho-
line iodides are used as model substrates, which upon interaction
with the enzyme release thiocholine, which in turn reacts with the
chromogen reagent 5,50-dithiobis(2-nitrobenzoic acid (DTNB) to
give 2-nitro-5-mercaptobenzoate (at pH 8.0). The monitoring of
the latter at 405 nm furnishes the kinetic parameters.
Interesting structure-activity relationships can be extracted also
from activity data (Table 1). Regarding AChE, clearly, the presence
of a dimethoxyphenyl moiety furnishes the most potent com-
pound (11e, IC50, and Ki’s in the low micromolar range, mixed
inhibitor), as was initially hypothesised.
However, what is more, interesting about the anticholinergic
activity of these compounds is that they show a clear preference
towards BuChE. Thus, relatively lipophilic disubstitution (11e–g)
affords the strongest compounds in the series, within the low
micromolar range (IC50 1.8–7.3 mM) (di-OMeꢂOMe/OH > OCH2O).
An issue that must be addressed is that BuChE has a more
prominent role than AChE in elderly people and in more
advanced stages of Alzheimer’s disease53. Moreover, BuChE has
also been reported to participate in amyloid plaques and neuro-
fibrillary tangles-mediated neurodegeneration54. This means that
substitution, or the presence of a polar catechol moiety com-
pletely abolished activity.
Compounds 11e–g turned out to be either mixed or non-com-
petitive inhibitors, as evidenced by the Cornish–Bowden plots.
This means that the title compound can bind either the free
enzyme (Kia) or the complex enzyme-substrate (Kib). In order to
illustrate this issue, Cornish–Bowden plots for derivative 11e
against BuChE (mixed inhibition) are depicted in Figure 2.
Docking studies
Docking calculations predict that compound 11e forms a complex
with AChE (Figure 3). The iminosugar moiety establishes H-bond
interactions with Trp86 and Glu202 at the cationic anionic subsite
(CAS). Additionally, the aromatic ring moiety is involved in p stack-
ing interactions with Trp286 at the peripheral anionic subsite
(PAS). The aromatic ring also participates in van der Waals interac-
tions with several residues at the peripheral anionic subsite.
Docking calculations also predict that compound 11e forms a
complex with BuChE (Figure 4). The iminosugar moiety establishes
H-bond interactions with Trp82 and Glu197 at the cationic active
subsite (CAS) and with His438 at the catalytic subsite. Additionally,
the aromatic ring moiety is involved in van der Waals interactions
with residues Thr284, Pro285, Asn289 at the peripheral anionic
subsite (PAS). Taken together, these calculations predict that 11e
compounds 11e–g could be useful not only for re-establishing the interacts in similar modes with both enzymes (as seen in the 3D
cognitive functionality of the patients (by increasing the levels of figures) except for the interaction with His438 in BuChE which is

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
143
missing in AChE. The p stacking interactions with Trp286 seen in and one non-tumour cell line (BJ-hTERT human fibroblasts) were
used. Data show no disruption of cell growth for most of the
compounds tested. Only catechol-containing 11d, exhibited a
moderate antiproliferative effect against the non-tumour cell line
(GI50 ¼ 39 5 mM). Interestingly, the rest of the compounds, and
particularly the lead compounds (11e–g) lacked significant growth
inhibition effects when tested as a concentration as high as
AChE are not present in BuChE because there is no equivalent
residue at the BuChE peripheral anionic subsite.
Antiproliferative activity
To analyse the potential cytotoxicity of title compounds, their
antiproliferative activity was measured. For that purpose, one solid 100 mM (GI50 > 100 mM).
tumour cell line (SH-SY5Y, glioblastoma) as a model of brain cells,
Figure 2. Cornish–Bowden plots (BuChE) for compound 11e.
Figure 3. (a, b) Docking simulations for the interactions 11e-AChE.
Figure 4. (a, b) Docking simulations for the interactions 11e-BuChE.

144
A. I. AHUJA-CASARÍN
4. Ferhati X, Matassini C, Fabbrini MG, et al. Dual targeting of
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Conclusions
We have accomplished the straightforward preparation of hitherto
unknown cholinesterases inhibitors based on the use of the imino-
sugar 1-DNJ as the key pharmacophore for struggling with the
low levels of the neurotransmitter acetylcholine in Alzheimer’s dis-
ease. Appendage of an ether-connected aromatic motif with dif-
ferent substitution patterns allowed the modulation of the activity
towards cholinesterases. Compounds bearing relatively lipophilic
disubstituted phenyl moieties (3,4-diOMe, 3-OH/4-OMe, 3,4-
OCH2O) turned out to be strong inhibitors of AChE, and particu-
larly of BuChE, with IC50 and Ki values within the low micromolar
range. The presumptive binding mode of the lead compound to
cholinesterases was analysed using molecular docking simulations,
revealing interactions with two key regions of the enzyme, CAS,
and PAS. Moreover, as well as the strong anticholinergic activity,
the lack of remarkable growth inhibition effects at a concentration
as high as 100 mM suggests a good profile for the development of
future anti-Alzheimer’s drugs based on iminosugars.
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Acknowledgement
The authors would like to thank the Servicio de Resonancia
ꢀ
ꢀ
ꢀ
ꢀ
10. Sanchez-Fernandez EM, Garcıa Fernandez JM, Ortiz Mellet C.
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ꢀ
Magnetica Nuclear, CITIUS (University of Seville) for the perform-
ance of NMR experiments.
Disclosure statement
11. Clemente F, Matassini C, Faggi C, et al. Glucocerebrosidase
(GCase) activity modulation by 2-alkyl trihydroxypiperidines:
inhibition and pharmacological chaperoning. Bioorg Chem
2020;98:103740.
No potential conflict of interest was reported by the author(s).
Funding
ꢀ
ꢀ
12. Martınez-Bailen M, Carmona AT, Cardona F, et al. Synthesis
of multimeric pyrrolidine iminosugar inhibitors of human b
glucocerebrosidase and a-galactosidase A: first example of a
multivalent enzyme activity enhancer for Fabry disease.
Bioorg Chem 2020;192:112173.
ꢀ
The present work was financially supported by Direccion General
ꢀ
de Investigacion of Spain [CTQ2016-78703-P], the Junta de
ꢀ
Andalucıa [FQM134], the European Regional Development Fund
(FEDER) and the Mexican CONACYT [CB-2015/257465]. M.X.F. and
J.M.P. were supported by Spanish Government through project
PGC2018-094503-B-C22 (MCIU/AEI/FEDER, UE). A.I.A.-C. and I.L.
thank the CONACYT (Mexico) for support through a fellowship
13. Esposito A, D’Alonzo D, De Fenza M, et al. Synthesis and
therapeutic applications of iminosugars in cystic fibrosis. Int
J Mol Sci 2020;21:3353.
14. Jenkinson SF, Best D, Saville AW, et al. C-Branched iminosu-
gars: a-glucosidase inhibition by enantiomers of isoDMDP,
isoDGDP, and isoDAB-l-isoDMDP compared to miglitol and
miglustat. J Org Chem 2013;78:7380–97.
ꢀ
award. M.X.F. was supported by a Cabildo de Tenerife “Agustın de
Betancourt” contract.
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