Benzoic acid-derived nitrones: A new class of potential acetylcholinesterase inhibitors and neuroprotective agents

Article abstract of DOI:10.1016/j.ejmech.2019.04.026

The discovery of new chemical entities endowed with potent and selective acetylcholinesterase (AChE)and/or butyrylcholinesterase (BChE)inhibitory activity is still a relevant subject for Alzheimer's disease therapy. Therefore, a small library of benzoic b

Full text of DOI:10.1016/j.ejmech.2019.04.026

Accepted Manuscript
Benzoic acid-derived nitrones: A new class of potential acetylcholinesterase inhibitors
and neuroprotective agents
Catarina Oliveira, Donatella Bagetta, Fernando Cagide, José Teixeira, Ricardo
Amorim, Tiago Silva, Jorge Garrido, Fernando Remião, Eugenio Uriarte, Paulo J.
Oliveira, Stefano Alcaro, Francesco Ortuso, Fernanda Borges
PII:
S0223-5234(19)30332-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.026
EJMECH 11261
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2019
Revised Date: 10 April 2019
Accepted Date: 10 April 2019
Please cite this article as: C. Oliveira, D. Bagetta, F. Cagide, José. Teixeira, R. Amorim, T. Silva, J.
Garrido, F. Remião, E. Uriarte, P.J. Oliveira, S. Alcaro, F. Ortuso, F. Borges, Benzoic acid-derived
nitrones: A new class of potential acetylcholinesterase inhibitors and neuroprotective agents, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.026.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Benzoic acid-derived nitrones: a new class of potential
acetylcholinesterase inhibitors and neuroprotective agents
Catarina Oliveira1¥, Donatella Bagetta2,3¥, Fernando Cagide1, José Teixeira1,4, Ricardo
Amorim4, Tiago Silva1, Jorge Garrido1,5, Fernando Remião6, Eugenio Uriarte7, Paulo J.
Oliveira4, Stefano Alcaro2,3, Francesco Ortuso2,3*, Fernanda Borges1*
1CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of
Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal;
2Department of "Scienze della Salute", University "Magna Græcia" of Catanzaro,
Campus Universitario "S.Venuta", Catanzaro, Italy;
3Net4Science Academic Spin-Off, University "Magna Græcia" of Catanzaro, Campus
Universitario "S.Venuta", Catanzaro, Italy;
4CNC – Center for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech,
Biocant Park, Cantanhede 3060-197, Portugal;
5Department of Chemical Engineering, Superior Institute of Engineering of Porto
(ISEP), IPP, Rua Dr. António Bernardino de Almeida, 431, 4200-072, Porto, Portugal;
6UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,
Faculty of Pharmacy, University of Porto, Porto, Portugal;
7Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago
Compostela, Santiago de Compostela, Spain.
¥ These authors contributed equally to the work.
* Corresponding Authors:
Fernanda Borges, CIQUP/Department of Chemistry and Biochemistry, Faculty of
Sciences, University of Porto, Porto 4169-007, Portugal. E-mail: fborges@fc.up.pt
Francesco Ortuso, Department of "Scienze della Salute", University "Magna Græcia" of
Catanzaro, Campus Universitario "S.Venuta", Catanzaro, Italy. E-mail: ortuso@unicz.it

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ABSTRACT
The discovery of new chemical entities endowed with potent and selective
acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE) inhibitory activity is
still a relevant subject for Alzheimer’s disease therapy. Therefore, a small library of
benzoic based amide nitrones (compounds 24 to 42) was synthesized and screened
toward cholinesterase enzymes. SAR studies showed that the tert-butyl moiety is the
most favourable nitrone pattern. In general, tert-butyl derivatives effectively inhibited
AChE, being compound 33 the most potent (IC50 = 8.3 ± 0.3 µM; Ki 5.2 µM ). The data
pointed to a non-competitive inhibition mechanism of action, which was also observed
for the standard donepezil. None of compounds showed BChE inhibitory activity.
Molecular modelling studies provided insights into the enzyme-inhibitor interactions
and rationalised the experimental data, confirming that the binding mode of nitrones 33
and 38 towards AChE has the most favourable binding free energy.
The tert-butylnitrones 33 and 38 were not cytotoxic on different cell lines (SH-SY5Y
and HepG2). Moreover, compound 33 was able to prevent t-BHP-induced oxidative
stress in SH-SY5Y differentiated cells.
Due to its AChE selectivity and promising cytoprotective properties, as well as its
appropriate drug-like profile pointing toward blood-brain barrier permeability,
compound 33 is proposed as a valid lead for a further optimization step.
Keywords: Benzoic acid; Nitrones; Spin traps; Alzheimer’s disease; Cholinesterase
inhibitors; Acetylcholinesterase; Oxidative Stress.

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1. Introduction
Alzheimer’s disease (AD) is the most common type of dementia [1], accounting for up
to 70 % of cases worldwide [2, 3], and being characterized as a multifactorial disease
[4-7]. AD is associated with a decrease of cholinergic activity and is also related with
increased oxidative stress (OS) [8, 9]. Cholinesterases (ChE), a family of enzymes that
mainly catalyse the hydrolysis of the neurotransmitter acetylcholine (ACh), are involved
in the restoration of the cholinergic pathway at the end of the nerve transmission [9, 10].
There are two main types of ChEs identified so far, namely acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE): AChE hydrolyses ACh and BChE hydrolyses
butyrylcholine (BCh) [10, 11]. While AChE prevails in the healthy brain, BChE has a
negligible starring role in the regulation of synaptic ACh levels [9, 12]. Accordingly,
the use of selective AChE inhibitors (AChEI) is an important therapeutic approach for
AD. Cholinesterase inhibitors (ChEIs) have shown several benefits including reduced
degradation of synaptic ACh, improvement of brain ACh levels in a dose-dependent
manner resulting in an enhanced cholinergic transmission in patients with AD and other
dementias. However, the drugs currently approved by Food and Drug Administration
(FDA) [8, 14], namely donepezil and galantamine (selective and reversible AChEIs)
and rivastigmine (a dual cholinesterase inhibitor, which can inhibit both AChE and
BChE) are unable to modify disease progression.
Oxidative stress-related events are also relevant for AD progression. For instance, OS
and mitochondrial damage have been associated with AD associated events [4], as the
OS redox changes in specific cellular components cause a more oxidized state, bringing
about to an augmented production of reactive oxygen species (ROS) and/or less
effective intrinsic antioxidant activity [15].
Over the past decade, there have been substantial efforts to design multi-target drugs
(MTDs) as a therapeutic solution for AD, an approach that is moved by the increase
knowledge that AD is a complex and multifactorial disease affecting many interlinked
pathological pathways. In this context, the development of new chemical entities able to
prevent and/or minimize OS-related events with remarkable capacities to inhibit ChE
activity is still a relevant issue.
Nitrones, a class of compounds known as spin traps, were described as having the
ability to stabilize or trap free radicals and reduce the damage associated with

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unbalanced production of radical reactive species [16, 17]. These compounds comprise
the general structure R1-CH=NO-R2 (Figure 1a) and the underlying mechanism behind
their free radical trapping action is related to their ability to interact with highly reactive
oxygen- and carbon-centred radicals (X•) yielding nitroxide products (Figure 1b), which
are then stabilized by resonance [18, 19].
X
X
X
R2
R2
R2
R1
N
O
R1
R1
N
O
N
O
(a)
(b)
Figure 1. Free radical trapping mechanism of nitrones.
Following a MTD strategy in the present work we report the design and synthesis of
new hybrid nitrones (benzoic based amide nitrones) as potential ChEIs endowed with
neuroprotective properties. Structural modifications were performed on the romatic
pattern, spacer length and type of nitrone covalently bound to the carbon flexible
aliphatic chain (Figure 2). All derivatives were evaluated for ChEs inhibition, kinetics
and mechanism of enzymatic inhibition, cytotoxicity, antioxidant profile in cell-based
systems and drug-like properties. In addition, in order to rationalize our results, docking
experiments were performed using models built based on the crystal structures of
human AChE and BChE.
Figure 2. General structure of novel ChEIs new amide nitrones based on benzoic acid (BA).

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2. Results and Discussion
2.1. Chemistry
The synthesis of novel hybrid nitrones structurally based on benzoic acid was
performed following the synthetic strategy depicted in Scheme 1. The compounds were
obtained in three synthetic steps using benzoic acid (1), 3-methoxybenzoic acid (2), 4-
methoxybenzoic acid (3), 3,4-dimethoxybenzoic acid (4) and 3,4,5-trimethoxybenzoic
acid (5) as starting materials. The first synthetic step consisted of an amidation reaction
of benzoic acids 1-5 using dissimilar coupling agents, and different length of the linker
spacers. Therefore, ethyl chloroformate (step a) was used for the introduction of the 6-
aminohexan-1-ol spacer, yielding compounds 6-9 and 12. Alternatively, phosphorus
oxychloride (step b) was used with the 8-aminooctan-1-ol and 10-aminodecan-1-ol
spacers, yielding derivatives 10-11 and 13-14. The following reaction (step c) was the
oxidation of the alcohol group of compounds 6-14 to the corresponding aldehydes 15-
23 with pyridinium chlorochromate. Nitrones 24-42 were then obtained via a
microwave-assisted reaction (step d) using three different hydroxylamines (N-tert-
butylhydroxylamine hydrochloride, N-benzylhydroxylamine hydrochloride and N-
cyclohexylhydroxylamine hydrochloride). Following this strategy, we successfully
synthesized a series of derivatives with different aromatic substitution patterns, aliphatic
chain length spacers and nitrone moieties.

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Scheme 1. Synthetic strategy followed to obtain nitrone derivatives 24-42 from benzoic acids 1-5. Reagents and
conditions: a. Et3N, ClCOOC2H5, NH2(CH2)5CH2OH, r.t., 10 h; b. POCl3, NH2(CH2)7CH2OH or NH2(CH2)9CH2OH,
DIPEA, 1-2 h; c. PCC, CH2Cl2, 2 h; d. (CH3)3CNHOH·HCl, C6H5CH2NHOH·HCl or C6H11NHOH·HCl, NaHCO3,
MW, 90 ºC, 10-15 min.
2.2. Assessment of acetyl and butyrylcholinesterase inhibition
AChE and BChE inhibitory activity of nitrones 24-42 was evaluated following the
Ellman’s method [20, 21], with AChE from Electrophorus electricus (electric eel,
eelAChE) and BChE from equine serum (eqBChE). Acetylthiocholine iodide (ATCI) or
butyrylthiocholine iodide (BTCI) were used as substrates for AChE or BChE,
respectively, releasing thiocholine and acetate or butyrate. Then, thiocholine reacts with
5,5'-dithiobis-(2-nitrobenzoate) (DTNB) ion to produce 5-thio-2-nitrobenzoate (TNB2-)
anion, which was determined by UV/Vis spectroscopy [20, 21], enabling the screening
for ChE inhibition after incubation with the test compounds. Donepezil used as standard
ChE inhibitor [10] showed a higher potency for AChE than BChE (IC50 = 25 ±1 nM and
2.2 ± 0.2 µM, respectively). The results of the inhibitory potency (IC50 values) of
compounds under study and standard inhibitor (donepezil) are shown in Table 1.
Compounds 27, 30, 33, 36-38, 41 and 42 operated as effective and selective AChEIs.
The aromatic ring substituents as well as the spacer length and the nitrone moiety had a
significant influence on AChE inhibitory activity. Firstly, the type of substituent of the

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nitrone group (24-42) markedly influenced the activity, as only the derivatives bearing a
tert-butyl group (27, 30, 33, 36-38, 41 and 42) were active toward AChE (IC50 = 8.3 –
27.2 µM). Curiously, the introduction of benzyl and cyclohexyl nitrone moieties did not
lead to the same outcome.
Table 1. AChE and BChE inhibitory activity (IC50) of nitrone derivatives 24-42 and donepezil.
IC50 (µM ± SD)
Compound
24
Structure
eelAChE
eqBChE
*
*
*
*
*
*
*
*
*
*
*
*
25
26
27
28
29
30
31
32
33
*
27.2 ± 2.9
*
*
26.1 ± 2.7
*
*
8.3 ± 0.3

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*
*
*
*
*
*
*
*
*
*
34
35
36
37
38
39
40
41
42
*
12.2 ± 0.4
12.6 ± 0.6
17.2 ± 1.3
*
*
16.9 ± 1.3
11.8 ± 0.8
0.025 ± 0.001
2.2 ± 0.2
Donepezil
* Inactive at 50 µM (highest concentration tested).
The aromatic ring substituents also had a significant effect on AChE inhibition. Indeed,
while benzoic acid derivatives (24-26) lacked inhibitory activity toward AChE, the
introduction of methoxy groups led to an enhancement of the inhibitory effect.
Although the introduction of m- or p-OCH3 substituents (compounds 27 and 30) led to
similar inhibitory potencies (IC50 = 27.2 ± 2.9 and 26.1 ± 2.7 µM, respectively), a
significant improvement was observed for the 3,4,5-trissubstituted derivative 38 (IC50 =

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17.2 ± 1.3 µM) and, in particular, for the 3,4-dimethoxy derivative 33 (IC50 = 8.3 ± 0.3
µM).
Then, we studied the effect of the length of the alkyl linker for the derivatives with
optimal aromatic patterns (3,4-dimethoxy and 3,4,5-trimethoxy) and nitrone moiety
(tert-butylnitrone). Accordingly, the spacer was replaced by an eight- and ten-carbon
chain. It was observed that the increase of the spacer for the 3,4-OCH3 derivatives
(compounds 36 and 37) did not progress the inhibitory potency but for 3,4,5-OCH3
derivatives (compounds 41 and 42) although a slight improvement of inhibitory activity
was noticed, reaching a mild 1.5-fold increase for nitrone 42 (IC50 = 11.8 ± 0.8 µM),
which had a ten-carbon spacer.
As none of the precursors (1-5) were active against AChE at the highest concentration
tested (50 µM) it can be concluded that the presence of a positively charged terminal
nitrogen ((tert-butylnitrone)) and an alkyl spacer is required for activity. Moreover,
nitrones are selective for AChE as none of the compounds (nitrones and precursors)
showed inhibitory activity for BChE at the highest concentration tested (50 µM).
2.3. Assessment of drug-like properties
The drug-like properties were determined for all the nitrone derivatives (24-42),
donepezil and precursors 1-5 (see SI). The calculated parameters encompassed:
molecular weight (MW), partition coefficient (clog P), topological polar surface area
(tPSA in Å2), number of hydrogen acceptors (HBA), number of hydrogen donors
(HBD), number of rotatable bonds (nrotb) and blood (plasma)-brain partitioning
(logBB) (Table 2).
For nitrones with tert-butyl moiety (27, 30, 33, 36-38, 41 and 42, Table 2), we observed
that the values of HBA and HBD were in agreement with the drug-likeness
requirements of the Linpinski’s “Rule of 5” (with HBA ˂ 10 and HBD ˂ 5) [22]. In
general, all compounds exhibited a clogP value lower than 5, with the clogP values
ranging from 2.59 to 5.05, which is within the optimal range for orally administered and
central nervous system (CNS) drugs [22, 23]. However, comparing with CNS+ drug
parameters, compounds 38, 41 and 42 displayed a value of HBA = 7, which is out of the
proposed range.
The prediction of blood-brain barrier (BBB) permeability, determined by the logBB (the
ratio of the steady-state concentrations of the drug in the brain and in the blood) was
also assessed. Compounds with logBB below −1 are poorly distributed to the brain and

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are improbable to operate as effective CNS drugs [24]. All nitrones depicted on Table 2
displayed logBB > −1, pointing towards potential BBB permeability.
Table 2. Drug-like properties of nitrones derivatives with tert-butyl moiety (27, 30, 33, 36-38, 41 and 42)
Compound
MWa
clog Pa tPSA (Å2)a HBAa
HBDa
nrotba log BBa
320.4
320.4
350.5
378.5
406.6
380.5
408.5
436.6
˂ 450
2.99
2.99
2.80
4.15
5.05
2.59
3.94
4.85
˂ 5
67.08
67.08
76.31
76.31
76.31
85.54
85.54
85.54
˂ 60-70
5
5
1
1
10
10
11
13
15
12
14
16
˂ 8
0.232
0.232
0.086
0.109
0.115
0.004
0.028
0.035
≥ -1
27
30
6
1
33
6
1
36
6
1
37
7
1
38
7
1
41
7
1
42
CNS+ drugs7, 25-28
˂ 7
˂ 3
MW: molecular weight; clog P: logarithm of the octanol-water partition coefficient; tPSA: topological polar surface
area; HBA: number of hydrogen acceptors; HBD: number of hydrogen donors; nrotb: number of rotatable bonds; log
BB: logarithm of the ratio of the concentration of a drug in the brain and in the blood. a Properties calculated using
StarDrop software.
2.4. Modelling studies
To study the influence of the nitrone substituents on hChEs molecular recognition,
compounds 27-35 and 38-40 were submitted to molecular docking simulations and the
resulting
theoretical
complexes
were
scored
using
the
Molecular
Mechanics/Generalized Born Surface Area (MM-GBSA) binding free energy estimation
[29]. Differently from which was observed for hBChE, all evaluated compounds in
hAChE assumed an extended conformation. The reason of this different behaviour
could be related to the known structural differences between the two isoenzymes active
sites [30, 31] (Figure S1). Indeed, overlapping hBChE poses into the hAChE pocket, it
was observed that the aromatic residues Phe295, Phe297 and Tyr337 prevented the
ligand folded conformation. In fact, these aminoacids, in hBChE, are replaced by
Leu286, Val288 and Ala328, respectively, resulting in less steric hindrance. Although
the studied compounds were able to bind the active site of both isoforms their binding
free energies suggested for the tert-butyl derivatives 27, 30, 33, 38 a hAChE preference
over hBChE (Table 3), with nitrones 33 and 38 as the most energetically favourite
hAChE ligands, which are in a qualitative agreement with the experimental data.

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Table 3. Ligands-target theoretical binding free energy (in kcal/mol).
Compound hAChE hBChE
-49.78 -36.43
-42.71 -39.04
-35.63 -38.07
-44.57 -25.81
-39.26 -28.15
-38.11 -13.07
-50.66 -16.72
-39.98 -34.92
-35.62 -36.00
-52.90 -33.84
-25.37 -31.06
-33.25 -36.91
-69.65 -44.45
27
28
29
30
31
32
33
34
35
38
39
40
(R)-Donepezil
-72.81 -39.22
(S)-Donepezil
In particular, into the hAChE active site compounds 33, 27, 30, and 38 (Figures 3, S2-
S4, respectively) shared both the orientation of the tert-butyl group towards the inner
side of the gorge and established hydrogen bond to Phe295 backbone by means of the
amide oxygen. Into the hAChE, stacking interactions with the external Trp286 further
stabilized 27, 33 and 38 complexes.

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Figure 3. Best docking pose of compound 33 into hAChE active site displayed as light blue mesh. The most relevant
interacting residues and the ligand are respectively depicted in light blue and yellow tubes. Stacking interactions and
hydrogen bonds are respectively represented in green and purple.
The corresponding benzyl derivatives 28, 31, 34 (Figure S5-S7) equally interacted with
Phe295 and Trp286 and maintained the same orientation. On the contrary, 3,4,5-
trimethoxy phenyl ring of the derivative 39 was positioned near the internal Trp86.
Nevertheless, this compound performed hydrogen bond to Phe295 backbone by the
nitrone oxygen, and its benzyl ring was oriented towards the Trp286 (Figure S8). Thus,
both the number and position of the methoxy group(s) at the aromatic ring and the tert-
butyl/benzyl substituents seemed to have the same influence on the hAChE interactions.
Regarding the cyclohexyl ring substituted derivatives 29, 32, 35 and 40 (Figure S9-
S12), it was observed that 29 and 35, conversely to their tert-butyl analogues,
respectively directed the 3-methoxy and 3,4-dimethoxy phenyl ring towards to the
internal Trp86 establishing stacking interactions, while the hydrogen bond with Phe295
was established by the nitro group oxygen, similarly to compound 39. Contrariwise, the
binding modes of 32 and 40 were similar with those observed for 30 and 38.
Therefore, docking findings indicated that all studied compounds were able to bind to
hAChE active site mainly interacting with Trp286 and Phe295, belonging to the
peripheral anion site (PAS) and to the acyl pocket, respectively, which play a key role in
ligand binding and specificity [32, 33].

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However, according to the biological data attained with the nitrone group substituted by
benzyl (28, 31, 34, 39) and cyclohexyl moieties (29, 32, 35, 40) were endowed with a
worst hAChE binding free energy compared to the corresponding tert-butyl analogous.
Analysing each MM-GBSA term contributing to the binding free energy definition it
was observed that the solvation free energy (Generalized Born electrostatic solvation
energy) mostly penalized the benzyl and cyclohexyl derivatives (Table S2).
Focusing on the hBChE complexes, as previously reported, these inhibitors showed a
folded conformation not dependent from the substituent at the phenyl and nitro
moieties. Specifically, the tert-butyl derivatives 27, 30 and 38 (Figures S13-S15)
respectively oriented the 3-methoxy, 4-methoxy and 3,4,5-trimethoxyphenyl ring
towards the Phe329 performing stacking contacts, while the 3,4-dimethoxyphenyl ring
of 33 interacted to Tyr332 (Figure S16).
Concerning the benzyl derivatives, the docking poses of 28 (Figure S17) and 34 (Figure
S18) were quite similar to the 31 (Figure S19) and 39 (Figure S20) ones. In particular,
28 and 31 directed the methoxyphenyl ring towards the Tyr332 and the benzyl one
towards the Trp231; in the docking geometries of 34 and 39 such moiety was arranged
in an opposite manner.
Similar configuration of 34 and 39 was observed for the cyclohexyl derivatives 29, 35
and 40 (Figures S21-S23). Instead, regarding 32 both the 4-methoxyphenyl and
cyclohexyl ring were located near to Tyr231 and no productive interactions were
observed (Figure S24). Finally, 27, 30, 31, 39 and 40 poses highlighted steric hindrance
penalties with the residues of the catalytic triad, which could disfavour the hBChE
recognition. Any issue related with Pan Assay INterference compoundS (PAINS) was
found for the compounds under study.
2.5. Assessment of enzyme-inhibition mechanism
To evaluate the inhibition mechanism of the most promising AChEIs (compounds 33
and 38) kinetic experiments were performed. For this purpose, the enzyme inhibition
kinetics was evaluated using different substrate concentrations (ATCI), in absence or
presence of compounds 33, 38 and donepezil at different concentrations. The results are
shown in Figure 4. Graphical analyses of the reciprocal Lineweaver-Burk plots were
used to determine Michaelis-Menten reaction kinetic parameters (Michaelis constant,
Km and maximum velocity, Vmax).

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Figure 4. Kinetic studies on the mechanism of AChE inhibition by (A) compounds 33 and (B) 38, and (C) donepezil.
Details in reference39
Concerning compound 33, it was found that the Vmax decreased while Km appears to
remain unchanged (Figure 4A), displaying a series of converging lines on the same
point of the x-axis (1/[S]). The data pointed to a non-competitive inhibition mechanism
of action, which was also observed for the standard donepezil (Figure 4C), as expected
[34, 35]. The Lineweaver-Burk plots obtained for compound 38 (Figure 4B) presented a
series of converging lines displaying a behaviour corresponding to a mixed inhibition,
which is characterized by the decrease of Vmax and Km. Actually, a mixed inhibitor can
hinder the binding of substrate and decrease the turnover number of the enzyme [36].
From the Dixon plots, obtained from the replots of the slopes of the Lineweaver-Burk
plots vs. inhibitor concentrations (Figure 4, upper right corners), the AChE inhibition
binding affinities, determined as inhibition constants (Ki), were calculated. Compounds
33 (Figure 4A) and 38 (Figure 4B) displayed Ki values of 5.2 and 10.4 µM,
respectively. The Ki values of compounds 33 (IC50 = 8.3 µM) and 38 (IC50 = 17.2 µM)
correlated well with their experimental IC50, displaying IC50 and Ki values slightly
equal. Donepezil showed a similar behaviour (Ki = 16.4 nM and IC50 = 24.6 nM, Figure
4C).

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2.6. Assessment of cytotoxicity
The cytotoxic profile of the compounds 24-42 (see SI and Figure 5) was determined by
measuring the cellular viability, in human differentiated neuroblastoma (SH-SY5Y cell
line) and hepatocarcinoma cells (HepG2), after a 24 h incubation period at three
different concentrations (1, 10 and 50 µM). Both cell lines are often used in the
preclinical safety assessment of CNS drug candidates.37 Cellular viabilities were
estimated through the capability of living cells to metabolically reduce MTT and
resazurin to formazan and resorufin, respectively, providing an indirect measure of
metabolic function [38]. The results obtained are shown in Figure 5.
In general, the most promising compounds 33 (Figure 5A) and 38 (Figure 5B) with tert-
butyl nitrone moiety did not exhibit a cytotoxicity toward SH-SY5Y and HepG2 cells
for all tested concentrations. Interestingly, these compounds slightly increased cell
viability (106.8 – 122.1 %) for all tested concentrations in differentiated SH-SY5Y
cells, an effect that was not observed in HepG2 cells.
In brief, the data showed that the nitrone derivatives under study did not display
significant toxicity effects neither in human SH-SY5Y nor HepG2 cells at
concentrations in which they exhibited AChE inhibitory activities, revealing a
satisfactory safety window.
Figure 5. Cellular viability of human neurablastoma SH-SY5Y and human hepatocarcinoma HepG2 cells after a 24 h
treatment with three different concentrations (1, 10 and 50 µM) of nitrone compounds (A) 33 and (B) 38. Cellular
viability was evaluated through variations in cell metabolic activity using two methods: MTT and resazurin reduction
assays in differentiated SH-SY5Y and HepG2 cells, respectively. Untreated cells were used as control. Results are
expressed as mean % of untreated controls ± SEM. (n = 4).
2.7. Assessment of OS-induced cell death prevention
The antioxidant properties of the most promising nitrone compounds (33 and 38)
against OS-induced cell damage were evaluated in SH-SY5Y differentiated cells, at 10,

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50, 100 µM. Two different strategies were used: a) the tested compounds were pre-
incubated for 24 h at non-cytotoxic concentrations, and then pro-oxidant agents were
added to the cell culture; and b) the pro-oxidant agents were first added to the cell
culture and then the tested compounds were incubated for 24 h, at non-cytotoxic
concentrations (Figure 6A).
In the present study, classical pro-oxidant agents were used: hydrogen peroxide (H2O2),
tert-butyl hydroperoxide (t-BHP), the mitochondrial inhibitors rotenone and antimycin
A (ROT/AA), and the anti-cancer agent doxorubicin (DOX). The selected oxidative
stressors induced oxidative events by different mechanisms: H2O2 is a product of
enzymatic activity and dopamine oxidation and can be converted into hydroxyl radicals
via Fenton-like reactions [15]; t-BHP is an organic peroxide that causes lipid
peroxidation, opening of mitochondria nonspecific Ca2+-dependent pore, and cell death
[39]; ROT/AA are inhibitors of the mitochondrial electron transport chain, resulting in a
burst of superoxide anion production and induction of a ROS-dependent cell damage
cascade events; and DOX is a chemotherapeutic drug which generates a redox cycle at
different dehydrogenases, including mitochondrial complex I, leading to superoxide
anion production, and consequently to mitochondrial dysfunction. Cells treated with
H2O2 (1 mM, Figure 6B), t-BHP (250 µM, Figure 6B), ROT/AA (1 µM, Figure 6B), and
DOX (1 µM, Figure 6B) caused a significant reduction, of about 50.3 ± 1.1 %, 44.3 ±
6.4 %, 20.7 ± 2.1 % and 32.9 ± 6.7 %, respectively, in cell metabolic activity when
compared with nontreated cells.
In general, none of the promising AChEIs showed remarkable antioxidant effects
(Figure 6B and 6C). However, compound 33 was able to prevent the t-BHP-induced cell
damage in a dose-dependent manner (Figure 6B), a property that can be enhanced after
a subsequent optimization step.

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Figure 6. Antioxidant cytoprotective effects of tert-butyl nitrones, 33 and 38. (A) Schematic representation of
strategies used to evaluate nitrones’ antioxidant properties. Antioxidant activity of compounds (B) 33 and (C) 38,
were evaluated in human neuroblastoma SH-SY5Y cells against H2O2-, t-BHP-, ROT/AA-, and DOX-induced
decrease in cell metabolic activity. The comparisons were performed by using one-way ANOVA between the control
(oxidative stressors) vs. nitrones under study when were incubated. Data are means ± SEM of four independent
experiments and the results are expressed as percentage of control (control = 100 %), which represents the cells
without any treatment in the respective time point. Significance was accepted with * P < 0.05, ** P < 0.01, **** P <
0.0001.
3. Conclusions

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The development of new benzoic based amide nitrones (compounds 24-42), with
different nitrone substituents (tert-butyl, benzyl and cyclohexyl), was successfully
achieved. The compounds were screened toward cholinesterase enzymes and SAR
studies showed that the tert-butyl moiety is the most favourable nitrone pattern. Only
compounds with the tert-butyl moiety (27, 30, 33, 36, 38, 41 and 42) displayed
significant AChE inhibitory activity. Moreover, the presence and number of methoxy
substituents, as well as the spacer length, were found to be important contributors for
AChEI modulation potency. Compound 33, with two methoxy functions and a six-
carbon aliphatic chain, presented the best inhibitory activity toward AChE (IC50 = 8.3 ±
0.3 µM; Ki 5.2 µM). The data pointed to a non-competitive inhibition mechanism of
action, which was also observed for the standard donepezil None of compounds showed
BChE inhibitory activity.
Molecular modelling studies provided insights into enzyme-inhibitor interactions and a
rationale for the selectivity and potency observed, confirming that nitrones 33 and 38
resulted in the most energetically favourable hAChE ligands.
The most promising tert-butylnitrones 33 and 38 slightly increased the cell viability
(106.8 – 122.1 %) for all tested concentrations in differentiated SH-SY5Y cells and did
not have a significant effect on the cellular viability in HepG2 cells. Nitrone derivatives
33 and 38 revealed a satisfactory safety window as they did not display toxic effects in
both cell lines. Furthermore, compound 33 was able to prevent the t-BHP-induced cell
damage in a dose-dependent manner in SH-SY5Y differentiated cells, a property that
can be enhanced after a subsequent optimization step.
Due to its AChE selectivity and promising cytoprotection properties, as well as its
appropriate drug-like properties, pointing towards BBB permeability compound 33 is
proposed as a valid lead for further optimization step.
4. Experimental section
4.1. Chemistry
4.1.1. Synthesis of benzoic acid-derived nitrones
4.1.1.1. General procedures to obtain benzamide derivatives (6-14)

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A1) The appropriate benzoic acid (benzoic acid (1), 3-methoxybenzoic acid (2), 4-
methoxybenzoic acid (3), 3,4-dimethoxybenzoic acid (4) or 3,4,5-trimethoxybenzoic
acid (5), 1 mmol), was dissolved in dichloromethane (40 mL) and triethylamine (2
mmol) was added. Then, ethyl chloroformate (2 mmol) was added dropwise to the
stirred solution kept in an ice bath. After stirring 2 h at room temperature, the mixture
was cooled again and the 6-aminohexan-1-ol (2 mmol) was added. The purification
conditions are described in literature [15, 39].
A2) The appropriate benzoic acid (3,4-dimethoxybenzoic acid (4) or 3,4,5-
trimethoxybenzoic acid (5), 1 mmol) was dissolved in dichloromethane (15 mL) and
POCl3 (1 mmol) was added at room temperature. After 30 min, the reactional mixture
was cooled (ice bath) and 8-aminooctan-1-ol or 10-aminodecan-1-ol (1.2 mmol) and
DIPEA (4 mmol) were added. The reaction was stirred for 1-2 h at room temperature.
The purification conditions are described in literature [15].
1
N-(6-Hydroxyhexyl)benzamide (6). Procedure A1. η = 81 %. H RMN (CDCl3): δ =
1.40 – 1.42 (4H, m, N(CH2)2(CH2)2), 1.54 – 1.66 (4H, m, NCH2CH2(CH2)2CH2), 1.77
(1H, s, OH), 3.42 – 3.47 (2H, m, NCH2), 3.63 (2H, t, J = 6.5 Hz, CH2O), 6.28 (1H, s,
NH), 7.39 – 7.43 (2H, m, H(3) and H(5)), 7.46 – 7.50 (1H, m, H(4)), 7.74 – 7.77 (2H, m,
13
H(2) and H(6)). C RMN (CDCl3): δ = 25.4 (N(CH2)3CH2), 26.7 (N(CH2)2CH2), 29.8
(NCH2CH2), 32.7 (N(CH2)4CH2), 40.0 (NCH2), 62.8 (CH2O), 127.0 (C(2) and C(6)),
128.7 (C(3) and C(5)), 131.5 (C(4)), 134.9 (C(1)), 167.8 (CO). ESI/MS m/z (%): 222
(M++H, 100), 204 (41).
1
N-(6-Hydroxyhexyl)-3-methoxybenzamide (7). Procedure A1. η = 88 %. H RMN
(CDCl3): δ = 1.32 – 1.38 (4H, m, N(CH2)2(CH2)2), 1.48 – 1.62 (4H, m,
NCH2CH2(CH2)2CH2), 2.85 (1H, s, OH), 3.36 – 3.41 (2H, m, NCH2), 3.58 (2H, t, J =
6.5 Hz, CH2O), 3.79 (3H, s, OCH3), 6.89 (1H, t, J = 5.4 Hz, NH), 6.99 (1H, ddd, J =
1.4, 2.4, 7.7 Hz, H(4)), 7.27 (1H, dd, J = 7.6, 7.7 Hz, H(5)), 7.31 (1H, ddd, J = 1.4, 1.6,
7.6 Hz, H(6)), 7.36 (1H, dd, J = 1.6, 2.4 Hz, H(2)). 13C RMN (CDCl3): δ = 25.3
(N(CH2)3CH2), 26.6 (N(CH2)2CH2), 29.5 (NCH2CH2), 32.5 (N(CH2)4CH2), 40.0
(NCH2), 55.4 (OCH3), 62.4 (CH2O), 112.5 (C(4)), 117.4 (C(6)), 118.9 (C(2)), 129.5
(C(5)), 136.2 (C(1)), 159.7 (C(3)), 167.7 (CO). ESI/MS m/z (%): 274 (M++Na, 53), 252
(M++H, 18), 135 (100).

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1
N-(6-Hydroxyhexyl)-4-methoxybenzamide (8). Procedure A1. η = 69 %. H RMN
(CDCl3): δ = 1.36 – 1.46 (4H, m, N(CH2)2(CH2)2), 1.52 – 1.65 (4H, m,
NCH2CH2(CH2)2CH2), 1.76 (1H, s, OH), 3.38 – 3.49 (2H, m, NCH2), 3.63 (2H, t, J =
6.4 Hz, CH2O), 3.83 (3H, s, OCH3), 6.16 (1H, s, NH), 6.82 – 6.99 (2H, m, H(3) and
H(5)), 7.66 – 7.81 (2H, m, H(2) and H(6)). 13C RMN (CDCl3): δ = 25.4 (N(CH2)3CH2),
26.7 (N(CH2)2CH2), 29.9 (NCH2CH2), 32.7 (N(CH2)4CH2), 39.9 (NCH2), 55.5 (OCH3),
62.8 (CH2O), 113.9 (C(3) and C(5)), 127.2 (C(1)), 128.8 (C(2) and C(6)), 162.2 (C(4)),
167.3 (CO). ESI/MS m/z (%): 274 (M++Na, 48), 252 (M++H, 14), 135 (100).
N-(6-Hydroxyhexyl)-3,4-dimethoxybenzamide (9) and N-(6-Hydroxyhexyl)-3,4,5-
trimethoxybenzamide (12). Procedure A1. Structural analysis described in literature
[39].
N-(8-Hydroxyoctyl)-3,4-dimethoxybenzamide (10). Procedure A2. Structural analysis
described in literature [15].
N-(10-Hydroxydecyl)-3,4-dimethoxybenzamide (11), N-(8-Hydroxyoctyl)-3,4,5-
trimethoxybenzamide (13) and N-(10-Hydroxydecyl)-3,4,5-trimethoxybenzamide
(14). Procedure A2. Structural analysis described in literature [15].
4.1.1.2. General procedure to obtain aldehyde derivatives (15-23)
Pyridinium chlorochromate (1.5 mmol) and dichloromethane (20 mL) were added and
kept under stirring for 5-7 min. Benzoic acid amide derivative (6-14) was added and
stirred for 2 h. Thereafter diethyl ether (15 mL) was added and the solid was filtrated
using a celite pad. The solvent was evaporated and the compound purified by silica gel
flash chromatography using ethyl acetate as eluting system. The control reaction was
performed by TLC (silica gel, ethyl acetate). The procedure was adapted from the
literature [40].
N-(6-Oxohexyl)benzamide (15). η = 54 %. 1H RMN (CDCl3): δ = 1.28 – 1.40 (2H, m,
N(CH2)2CH2), 1.51 – 1.66 (4H, m, NCH2CH2CH2CH2), 2.39 (2H, td, J = 1.7, 7.2 Hz,
CH2CHO), 3.31 – 3.42 (2H, m, NCH2), 6.74 (1H, s, NH), 7.30 – 7.38 (2H, m, H(3) and
H(5)), 7.38 – 7.46 (1H, m, H(4)), 7.64 – 7.88 (2H, m, H(2) and H(6)), 9.69 (1H, t, J =

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13
1.7 Hz, CHO). C RMN (CDCl3): δ = 21.6 (N(CH2)2CH2), 26.4 (N(CH2)3CH2), 29.3
(NCH2CH2), 39.8 (NCH2), 43.7 (CH2CHO), 127.0 (C(2) and C(6)), 128.5 (C(3) and
C(5)), 131.4 (C(4)), 134.6 (C(1)), 167.8 (CONH), 202.7 (CHO). ESI/MS m/z (%): 220
(M++H, 7), 105 (100).
1
3-Methoxy-N-(6-oxohexyl)benzamide (16). η = 56 %. H RMN (CDCl3): δ = 1.28 –
1.38 (2H, m, N(CH2)2CH2), 1.51 – 1.64 (4H, m, NCH2CH2CH2CH2), 2.38 (2H, td, J =
1.6, 7.2 Hz, CH2CHO), 3.33 – 3.41 (2H, m, NCH2), 3.76 (3H, s, OCH3), 6.83 (1H, s,
NH), 6.96 (1H, ddd, J = 1.4, 2.6, 7.7 Hz, H(4)), 7.24 (1H, dd, J = 7.6, 7.7 Hz, H(5)),
7.28 (1H, ddd, J = 1.4, 1.6, 7.6 Hz, H(6)), 7.33 (1H, dd, J = 1.6, 2.3 Hz, H(2)), 9.69
(1H, t, J = 1.6 Hz, CHO). 13C RMN (CDCl3): δ = 21.5 (N(CH2)2CH2), 26.3
(N(CH2)3CH2), 29.3 (NCH2CH2), 39.7 (NCH2), 43.6 (CH2CHO), 55.3 (OCH3), 112.3
(C(4)), 117.4 (C(6)), 118.8 (C(2)), 129.4 (C(5)), 136.1 (C(1)), 159.7 (C(3)), 167.5
(CONH), 202.7 (CHO). ESI/MS m/z (%): 250 (M++H, 35), 135 (100).
1
4-Methoxy-N-(6-oxohexyl)benzamide (17). η = 43 %. H RMN (CDCl3): δ = 1.35 –
1.45 (2H, m, N(CH2)2CH2), 1.57 – 1.70 (4H, m, NCH2CH2CH2CH2), 2.45 (2H, td, J =
1.6, 7.2 Hz, CH2CHO), 3.35 – 3.48 (2H, m, NCH2), 3.83 (3H, s, OCH3), 6.19 (1H, s,
NH), 6.82 – 6.99 (2H, m, H(3) and H(5)), 7.66 – 7.81 (2H, m, H(2) and H(6)), 9.76 (1H,
13
t, J = 1.6 Hz, CHO). C RMN (CDCl3): δ = 21.7 (N(CH2)2CH2), 26.5 (N(CH2)3CH2),
29.6 (NCH2CH2), 39.8 (NCH2), 43.8 (CH2CHO), 55.5 (OCH3), 113.8 (C(3) and C(5)),
127.1 (C(1)), 128.8 (C(2) and C(6)), 162.2 (C(4)), 167.2 (CONH), 202.6 (CHO).
ESI/MS m/z (%): 250 (M++H, 12), 135 (100).
3,4-Dimethoxy-N-(6-oxohexyl)benzamide (18). η = 45 %. 1H RMN (CDCl3): δ = 1.34
– 1.46 (2H, m, N(CH2)2CH2), 1.54 – 1.73 (4H, m, NCH2CH2CH2CH2), 2.44 (2H, td, J =
1.6, 7.2 Hz, CH2CHO), 3.38 – 3.48 (2H, m, NCH2), 3.90 (6H, s, 2 × OCH3), 6.29 (1H, s,
NH), 6.83 (1H, d, J = 8.4 Hz, H(5)), 7.27 (1H, dd, J = 2.0, 8.4 Hz, H(6)), 7.41 (1H, d, J
= 2.0 Hz, H(2)), 9.75 (1H, t, J = 1.6 Hz, CHO). 13C RMN (CDCl3): δ = 21.6
(N(CH2)2CH2), 26.5 (N(CH2)3CH2), 29.5 (NCH2CH2), 39.8 (NCH2), 43.8 (CH2CHO),
56.1 (2 × OCH3), 110.4 (C(5)), 110.7 (C(2)), 119.3 (C(6)), 127.5 (C(1)), 149.1 (C(3)),
151.8 (C(4)), 167.2 (CONH), 202.6 (CHO). EI/MS m/z (%): 279 (M+, 41), 251 (87),
250 (26), 236 (72), 222 (28), 195 (58), 194 (40), 182 (22), 181 (90), 166 (82), 165
(100), 137 (32), 122 (26), 92 (20), 79 (35), 77 (42).

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1
3,4-Dimethoxy-N-(8-oxooctyl)benzamide (19). η = 66 %. H RMN (CDCl3): δ = 1.22
– 1.44 (6H, m, N(CH2)2(CH2)3), 1.54 – 1.68 (4H, m, NCH2CH2(CH2)3CH2), 2.41 (2H,
td, J = 1.8, 7.3 Hz, CH2CHO), 3.34 – 3.46 (2H, m, NCH2), 3.90 (6H, s, 2 × OCH3), 6.17
(1H, s, NH), 6.84 (1H, d, J = 8.4 Hz, H(5)), 7.25 (1H, dd, J = 2.0, 8.4 Hz, H(6)), 7.41
13
(1H, d, J = 2.0 Hz, H(2)), 9.75 (1H, t, J = 1.8 Hz, CHO). C RMN (CDCl3): δ = 22.0
(N(CH2)2CH2), 26.9 (N(CH2)5CH2), 29.1 (N(CH2)3(CH2)2), 29.8 (NCH2CH2), 40.1
(NCH2), 43.9 (CH2CHO), 56.1 (2 × OCH3), 110.4 (C(5)), 110.8 (C(2)), 119.2 (C(6)),
127.6 (C(1)), 149.1 (C(3)), 151.7 (C(4)), 167.2 (CONH), 202.9 (CHO). ESI/MS m/z
(%): 308 (M++H, 100), 165 (60), 124 (23).
1
3,4-Dimethoxy-N-(10-oxodecyl)benzamide (20). η = 71 %. H RMN (CDCl3): δ =
1.23 – 1.37 (10H, m, N(CH2)2(CH2)5), 1.51 – 1.64 (4H, m, NCH2CH2(CH2)5CH2), 2.40
(2H, td, J = 1.8, 7.3 Hz, CH2CHO), 3.37 – 3.44 (2H, m, NCH2), 3.90 (6H, s, 2 × OCH3),
6.20 (1H, s, NH), 6.83 (1H, d, J = 8.4 Hz, H(5)), 7.25 (1H, dd, J = 2.0, 8.4 Hz, H(6)),
13
7.41 (1H, d, J = 2.0 Hz, H(2)), 9.74 (1H, t, J = 1.8 Hz, CHO). C RMN (CDCl3): δ =
22.1 (N(CH2)2CH2), 27.0 (N(CH2)7CH2), 29.2 (N(CH2)3CH2), 29.3 (N(CH2)6CH2), 29.4
(N(CH2)4(CH2)2), 29.8 (NCH2CH2), 40.2 (NCH2), 44.0 (CH2CHO), 56.1 (2 × OCH3),
110.4 (C(5)), 110.8 (C(2)), 119.2 (C(6)), 127.6 (C(1)), 149.1 (C(3)), 151.7 (C(4)), 167.2
(CONH), 203.0 (CHO).
1
3,4,5-Trimethoxy-N-(6-oxohexyl)benzamide (21). η = 50 %. H RMN (CDCl3): δ =
1.35 – 1.45 (2H, m, N(CH2)2CH2), 1.57 – 1.72 (4H, m, NCH2CH2CH2CH2), 2.46 (2H,
td, J = 1.6, 7.1 Hz, CH2CHO), 3.41 – 3,49 (2H, m, NCH2), 3.86 (3H, s, OCH3), 3.88
(6H, s, 2 × OCH3), 6.32 (1H, s, NH), 7.00 (2H, s, H(2) and H(6)), 9.76 (1H, t, J = 1.6
Hz, CHO). 13C RMN (CDCl3): δ = 21.5 (N(CH2)2CH2), 26.4 (N(CH2)3CH2), 29.4
(NCH2CH2), 39.9 (NCH2), 43.8 (CH2CHO), 56.4 (2 × OCH3), 61.0 (OCH3), 104.5 (C(2)
and C(6)), 130.2 (C(1)), 141.0 (C(4)), 153.3 (C(3) and C(5)), 167.4 (CONH), 202.6
(CHO). EI/MS m/z (%): 309 (M+, 92), 281 (35), 280 (21), 266 (59), 225 (37), 224 (27),
211 (89), 196 (96), 195 (100), 154 (20), 152 (29), 137 (26), 109 (20), 81 (25).
1
3,4,5-Trimethoxy-N-(8-oxooctyl)benzamide (22). η = 63 %. H RMN (CDCl3): δ =
1.27 – 1.45 (6H, m, N(CH2)2(CH2)3), 1.54 – 1.71 (4H, m, NCH2CH2(CH2)3CH2), 2.43
(2H, t, J = 6.8 Hz, CH2CHO), 3.36 – 3.48 (2H, m, NCH2), 3.87 (3H, s, OCH3), 3.90

ACCEPTED MANUSCRIPT
(6H, s, 2 × OCH3), 6.08 (1H, s, CONH), 6.98 (2H, s, H(2) and H(6)), 9.76 (1H, s,
CHO). 13C RMN (CDCl3): δ = 22.0 (N(CH2)2CH2), 26.9 (N(CH2)5CH2), 29.1
(N(CH2)3(CH2)2), 29.8 (NCH2CH2), 40.3 (NCH2), 44.0 (CH2CHO), 56.5 (2 × OCH3),
61.0 (OCH3), 104.5 (C(2) and C(6)), 130.5 (C(1)), 141.0 (C(4)), 153.3 (C(3) and C(5)),
167.4 (CONH), 202.9 (CHO). ESI/MS m/z (%): 360 (M++Na, 20), 338 (M++H, 100),
195 (88), 169 (24), 154 (78).
1
3,4,5-Trimethoxy-N-(10-oxodecyl)benzamide (23). η = 77 %. H RMN (CDCl3): δ =
1.23 – 1.43 (10H, m, N(CH2)2(CH2)5), 1.52 – 1.71 (4H, m, NCH2CH2(CH2)5CH2), 2.46
(2H, t, J = 7.2 Hz, CH2CHO), 3.38 – 3.47 (2H, m, NCH2), 3.87 (3H, s, OCH3), 3.90
(6H, s, 2 × OCH3), 6.09 (1H, s, NH), 6.98 (2H, s, H(2) and H(6)), 9.75 (1H, s, CHO).
13C RMN (CDCl3): δ = 22.2 (N(CH2)2CH2), 27.1 (N(CH2)7CH2), 29.2 (N(CH2)3CH2),
29.3 (N(CH2)6CH2), 29.4 (N(CH2)4(CH2)2), 29.9 (NCH2CH2), 40.4 (NCH2), 44.0
(CH2CHO), 56.5 (2 × OCH3), 61.0 (OCH3), 104.5 (C(2) and C(6)), 130.5 (C(1)), 141.0
(C(4)), 153.3 (C(3) and C(5)), 167.3 (CONH), 203.0 (CHO). ESI/MS m/z (%): 366
(M++H, 100), 195 (33), 154 (35).
4.1.1.3. General procedure to obtain nitrone derivatives (24-42)
In a microwave vial the aldehyde derivative (15-23, 1 mmol), hydroxylamine
hydrochloride (N-tert-butyl, N-benzyl or N-cyclohexyl, 1.5 mmol) and NaHCO3 (1.5
mmol) were added in 3-5 mL of tetrahydrofuran at 90 ºC for 10 min with 10 sec of pre-
stirring. Dichloromethane (20 mL) was added and extracted with water (2 × 10 mL).
The organic phases were combined, the solvent was evaporated and the compound
purified by silica gel flash chromatography using ethyl acetate:methanol (9:1) as eluting
system. The control reaction was performed by TLC (silica gel, ethyl acetate).
1
α-5-Benzamidopentyl-N-tert-butyl nitrone (24). η = 51 %. H RMN (CD3OD): δ =
1.38 – 1.53 (11H, m, C(CH3)3 and N(CH2)3CH2), 1.57 – 1.73 (4H, m,
N(CH2)2CH2CH2CH2), 2.39 – 2.58 (2H, m, NCH2CH2), 3.40 (2H, t, J = 7.0, NCH2),
7.25 (1H, t, J = 5.7 Hz, CH=N+), 7.41 – 7.48 (2H, m, H(3) and H(5)), 7.49 – 7.55 (1H,
m, H(4)), 7.77 – 7.85 (2H, m, H(2) and H(6)). 13C RMN (CD3OD): δ = 26.1
(N(CH2)2CH2), 27.9 (N(CH2)3CH2), 28.0 (C(CH3)3), 28.2 (NCH2CH2), 30.1
(N(CH2)4CH2), 40.7 (NCH2), 70.5 (C(CH3)3), 128.2 (C(2) and C(6)), 129.5 (C(3) and
C(5)), 132.5 (C(4)), 135.8 (C(1)), 142.2 (CH=N+), 170.2 (CO). ESI/MS m/z (%): 313

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(M++Na, 26), 291 (M++H, 7), 290 (M+, 5), 105 (100). ESI/HRMS calcd for C17H26N2O2
(M+): 290.1994, found 290.1966.
1
α-5-Benzamidopentyl-N-benzyl nitrone (25). η = 31 %. H RMN (CD3OD): δ = 1.38
– 1.53 (2H, m, N(CH2)3CH2), 1.57 – 1.72 (4H, m, N(CH2)2CH2CH2CH2), 2.44 – 2.53
(2H, m, NCH2CH2), 3.38 (2H, t, J = 7.0 Hz, NCH2), 4.93 (2H, s, N+CH2), 7.31 – 7.48
(8H, m, H(2’–6’), H(3) and H(5) and CH=N+), 7.49 – 7.56 (1H, m, H(4)), 7.78 – 7.84
(2H, m, H(2) and H(6)). 13C RMN (CD3OD): δ = 26.0 (N(CH2)2CH2), 27.8
(N(CH2)3(CH2)2), 30.1 (NCH2CH2), 40.7 (NCH2), 69.4 (N+CH2), 128.2 (C(2) and C(6)),
129.5 (C(3) and C(5)), 129.8 (C(2’) and C(6’)), 130.0 (C(4’)), 130.1 (C(3’) and C(5’)),
132.6 (C(4)), 134.7 (C(1’)), 135.9 (C(1)), 146.3 (CH=N+), 170.2 (CO). ESI/MS m/z
(%): 348 (M++Na+H, 22), 347 (M++Na, 87), 325 (M++H, 100), 105 (60), 91 (22).
ESI/HRMS calcd for C20H25N2O2 (M++H): 325.1911, found 325.1906.
1
α-5-Benzamidopentyl-N-cyclohexyl nitrone (26). η = 58 %. H RMN (CD3OD): δ =
1.20 – 1.94 (16H, m, N(CH2)2(CH2)3 and N+CH(CH2)5), 2.41 – 2.52 (2H, m,
NCH2CH2), 3.39 (2H, t, J = 7.0 Hz, NCH2), 3.74 – 3.86 (1H, m, N+CH), 7.21 (1H, t, J =
5.9 Hz, CH=N+), 7.41 – 7.48 (2H, m, H(3) and H(5)), 7.49 – 7.56 (1H, m, H(4)), 7.76 –
13
7.85 (2H, m, H(2) and H(6)). C RMN (CD3OD): δ = 25.9 (N+CHCH2CH2CH2CH2),
26.1 (N+CH(CH2)2CH2 and N(CH2)2CH2), 27.5 (N(CH2)3CH2), 27.7 (NCH2CH2), 30.1
(N(CH2)4CH2), 31.9 (N+CHCH2(CH2)3CH2), 40.7 (NCH2), 74.5 (N+CH), 128.2 (C(2)
and C(6)), 129.5 (C(3) and C(5)), 132.5 (C(4)), 135.9 (C(1)), 144.2 (CH=N+), 170.2
(CO). ESI/MS m/z (%): 339 (M++Na, 94), 317 (M++H, 100). ESI/HRMS calcd for
C19H29N2O2 (M++H): 317.2224, found 317.2222.
1
α-5-(3-Methoxybenzamido)pentyl-N-tert-butyl nitrone (27). η = 83 %. H RMN
(CD3OD): δ = 1.43 – 1.49 (11H, m, C(CH3)3 and N(CH2)3CH2), 1.58 – 1.71 (4H, m,
N(CH2)2CH2CH2CH2), 2.45 – 2.52 (2H, m, NCH2CH2), 3.39 (2H, t, J = 7.0 Hz, NCH2),
3.84 (3H, s, OCH3), 7.06 – 7.10 (1H, m, H(2)), 7.25 (1H, t, J = 5.7 Hz, CH=N+), 7.32 –
7.40 (3H, m, H(4–6)). 13C RMN (CD3OD): δ = 26.1 (N(CH2)2CH2), 27.9 (N(CH2)3CH2),
28.0 (C(CH3)3), 28.2 (NCH2CH2), 30.1 (N(CH2)4CH2), 40.7 (NCH2), 55.9 (OCH3), 70.5
(C(CH3)3), 113.6 (C(4)), 118.3 (C(6)), 120.3 (C(2)), 130.6 (C(5)), 137.2 (C(1)), 142.2
(CH=N+), 161.3 (C(3)), 170.0 (CO). ESI/MS m/z (%): 321 (M++H, 8), 135 (100).
ESI/HRMS calcd for C19H29N2O3 (M++H): 321.2173, found 321.2163.

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α-5-(3-Methoxybenzamido)pentyl-N-benzyl nitrone (28). η = 38 %. H RMN
(CD3OD): δ = 1.39 – 1.49 (2H, m, N(CH2)3CH2), 1.56 – 1.69 (4H, m,
N(CH2)2CH2CH2CH2), 2.43 – 2.52 (2H, m, NCH2CH2), 3.37 (2H, t, J = 7.0 Hz, NCH2),
3.83 (3H, s, OCH3), 4.92 (2H, s, N+CH2), 7.05 – 7.10 (1H, m, H(2)), 7.31 – 7.45 (9H, m,
H(2’–6’), H(4–6) and CH=N+). 13C RMN (100 MHz, CD3OD): δ = 26.0 (N(CH2)2CH2),
27.7 (N(CH2)3CH2), 27.8 (N(CH2)4CH2), 30.1 (NCH2CH2), 40.7 (NCH2), 55.9 (OCH3),
69.4 (N+CH2), 113.6 (C(4)), 118.3 (C(6)), 120.3 (C(2)), 129.8 (C(2’) and C(6’)), 129.9
(C(4’)), 130.1 (C(3’) and C(5’)), 130.6 (C(5)), 134.7 (C(1’)), 137.2 (C(1)), 146.2
(CH=N+), 161.3 (C(3)), 170.0 (CO). ESI/MS m/z (%): 377 (M++Na, 58), 355 (M++H,
100), 135 (84), 91 (25). ESI/HRMS calcd for C21H27N2O3 (M++H): 355.2016, found
355.2046.
1
α-5-(3-Methoxybenzamido)pentyl-N-cyclohexyl nitrone (29). η = 78 %. H RMN
(CD3OD): δ = 1.20 – 1.93 (16H, m, N(CH2)2(CH2)3 and N+CH(CH2)5), 2.42 – 2.52 (2H,
m, NCH2CH2), 3.39 (2H, t, J = 7.0 Hz, NCH2), 3.74 – 3.83 (1H, m, N+CH), 3.84 (3H, s,
OCH3), 7.06 – 7.10 (1H, m, H(2)), 7.20 (1H, t, J = 5.9 Hz, CH=N+), 7.32 – 7.40 (3H, m,
H(4–6)). 13C RMN (CD3OD):
δ
=
25.9 (N+CHCH2CH2CH2CH2), 26.1
(N+CH(CH2)2CH2 and N(CH2)2CH2), 27.5 (N(CH2)3CH2), 27.7 (NCH2CH2), 30.1
(N(CH2)4CH2), 31.9 (N+CHCH2(CH2)3CH2), 40.7 (NCH2), 55.9 (OCH3), 74.6 (N+CH),
113.7 (C(4)), 118.3 (C(6)), 120.3 (C(2)), 130.6 (C(5)), 137.2 (C(1)), 144.2 (CH=N+),
161.3 (C(3)), 170.0 (CO). ESI/MS m/z (%): 369 (M++Na, 62), 347 (M++H, 48), 135
(100). ESI/HRMS calcd for C20H31N2O3 (M++H): 347.2329, found 347.2324.
1
α-5-(4-Methoxybenzamido)pentyl-N-tert-butyl nitrone (30). η = 62 %. H RMN
(CD3OD): δ = 1.39 – 1.51 (11H, m, C(CH3)3 and N(CH2)3CH2), 1.56 – 1.71 (4H, m,
N(CH2)2CH2CH2CH2), 2.42 – 2.53 (2H, m, NCH2CH2), 3.38 (2H, t, J = 7.0 Hz, NCH2),
3.84 (3H, s, OCH3), 6.93 – 7.01 (2H, m, H(3) and H(5)), 7.25 (1H, t, J = 5.7 Hz,
CH=N+), 7.76 – 7.81 (2H, m, H(2) and H(6)). 13C RMN (CD3OD): δ = 26.1
(N(CH2)2CH2), 27.9 (N(CH2)3CH2), 28.0 (C(CH3)3), 28.2 (NCH2CH2), 30.2
(N(CH2)4CH2), 40.6 (NCH2), 55.9 (OCH3), 70.5 (C(CH3)3), 114.7 (C(3) and C(5)),
127.9 (C(1)), 130.1 (C(2) and C(6)), 142.2 (CH=N+), 163.8 (C(4)), 169.8 (CO). ESI/MS
m/z (%): 343 (M++Na, 16), 321 (M++H, 4), 135 (100). ESI/HRMS calcd for C18H29N2O3
(M++H): 321.2173, found 321.2172.

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α-5-(4-Methoxybenzamido)pentyl-N-benzyl nitrone (31). η = 31 %. H RMN
(CD3OD): δ = 1.37 – 1.49 (2H, m, N(CH2)3CH2), 1.56 – 1.69 (4H, m,
N(CH2)2CH2CH2CH2), 2.43 – 2.53 (2H, m, NCH2CH2), 3.36 (2H, t, J = 7.0 Hz, NCH2),
3.83 (3H, s, OCH3), 4.93 (2H, s, N+CH2), 6.92 – 7.03 (2H, m, H(3) and H(5)), 7.30 –
13
7.46 (6H, m, H(2’–6’) and CH=N+), 7.73 – 7.83 (2H, m, H(2) and H(6)). C RMN
(CD3OD): δ = 26.0 (N(CH2)2CH2), 27.8 (N(CH2)3(CH2)2), 30.2 (NCH2CH2), 40.6
(NCH2), 55.9 (OCH3), 69.4 (N+CH2), 114.7 (C(3) and C(5)), 127.9 (C(1)), 129.8 (C(2’)
and C(6’)), 129.9 (C(4’)), 130.1 (C(2), C(6), C(3’) and C(5’)), 134.7 (C(1’)), 146.2
(CH=N+), 163.9 (C(4)), 169.8 (CO). ESI/MS m/z (%): 378 (M++Na+H, 24), 377
(M++Na, 100), 286 (26), 228 (32). ESI/HRMS calcd for C21H26N2O3Na (M++Na):
377.1836, found 377.1840.
1
α-5-(4-Methoxybenzamido)pentyl-N-cyclohexyl nitrone (32). η = 39 %. H RMN
(CD3OD): δ = 1.19 – 1.92 (16H, m, N(CH2)2(CH2)3 and N+CH(CH2)5), 2.41 – 2.51 (2H,
m, NCH2CH2), 3.37 (2H, t, J = 7.0 Hz, NCH2), 3.74 – 3.83 (1H, m, N+CH), 3.84 (3H, s,
OCH3), 6.93 – 7.00 (2H, m, H(3) and H(5)), 7.20 (1H, t, J = 5.9 Hz, CH=N+), 7.75 –
13
7.82 (2H, m, H(2) and H(6)). C RMN (CD3OD): δ = 25.9 (N+CHCH2CH2CH2CH2),
26.1 (N+CH(CH2)2CH2 and N(CH2)2CH2), 27.5 (N(CH2)3CH2), 27.7 (NCH2CH2), 30.2
(N(CH2)4CH2), 31.9 (N+CHCH2(CH2)3CH2), 40.6 (NCH2), 55.9 (OCH3), 74.5 (N+CH),
114.7 (C(3) and C(5)), 127.9 (C(1)), 130.1 (C(2) and C(6)), 144.2 (CH=N+), 163.8
(C(4)), 169.8 (CO). ESI/MS m/z (%): 369 (M++Na, 17), 135 (100). ESI/HRMS calcd for
C20H30N2O3Na (M++Na): 369.2149, found 369.2146.
1
α-5-(3,4-Dimethoxybenzamido)pentyl-N-tert-butyl nitrone (33). η = 90 %. H RMN
(CD3OD): δ = 1.40 – 1.49 (11H, m, C(CH3)3 and N(CH2)3CH2), 1.58 – 1.71 (4H, m,
N(CH2)2CH2CH2CH2), 2.43 – 2.52 (2H, m, NCH2CH2), 3.38 (2H, t, J = 7.0 Hz, NCH2),
3.88 (6H, s, 2 × OCH3), 7.00 (1H, d, J = 8.4 Hz, H(5)), 7.25 (1H, t, J = 5.7 Hz, CH=N+),
7.42 – 7.49 (2H, m, H(2) and H(6)). 13C RMN (CD3OD): δ = 26.1 (N(CH2)2CH2), 27.9
(N(CH2)3CH2), 28.0 (C(CH3)3), 28.2 (NCH2CH2), 30.2 (N(CH2)4CH2), 40.7 (NCH2),
56.5 (2 × OCH3), 70.5 (C(CH3)3), 112.0 (C(5) and C(2)), 121.8 (C(6)), 128.2 (C(1)),
142.2 (CH=N+), 150.3 (C(3)), 153.4 (C(4)), 169.7 (CO). ESI/MS m/z (%): 373 (M++Na,
16), 373 (M++H, 8), 165 (100). ESI/HRMS calcd for C19H31N2O4 (M++H): 351.2278,
found 351.2260.

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α-5-(3,4-Dimethoxybenzamido)pentyl-N-benzyl nitrone (34). η = 43 %. H RMN
(CD3OD): δ = 1.39 – 1.49 (2H, m, N(CH2)3CH2), 1.55 – 1.69 (4H, m,
N(CH2)2CH2CH2CH2), 2.43 – 2.53 (2H, m, NCH2CH2), 3.37 (2H, t, J = 7.0 Hz, NCH2),
3.87 (6H, s, 2 × OCH3), 4.93 (2H, s, N+CH2), 7.00 (1H, d, J = 8.2 Hz, H(5)), 7.31 – 7.48
(8H, m, H(2’–6’), CH=N+, H(2) and H(6)). 13C RMN (CD3OD): δ = 26.0
(N(CH2)2CH2), 27.8 (N(CH2)3(CH2)2), 30.2 (NCH2CH2), 40.7 (NCH2), 56.5 (2 ×
OCH3), 69.4 (N+CH2), 112.0 (C(5) and C(2)), 121.8 (C(6)), 128.2 (C(1)), 129.8 (C(2’)
and C(6’)), 129.9 (C(4’)), 130.1 (C(3’) and C(5’)), 134.7 (C(1’)), 146.2 (CH=N+), 150.3
(C(3)), 153.4 (C(4)), 169.7 (CO). ESI/MS m/z (%): 407 (M++Na, 28), 385 (M++H, 95),
165 (100). ESI/HRMS calcd for C22H29N2O4 (M++H): 385.2122, found 385.2121.
α-5-(3,4-Dimethoxybenzamido)pentyl-N-cyclohexyl nitrone (35). η = 57 %. 1H RMN
(CD3OD): δ = 1.19 – 1.91 (16H, m, N(CH2)2(CH2)3 and N+CH(CH2)5), 2.40 – 2.59 (2H,
m, NCH2CH2), 3.38 (2H, t, J = 7.0 Hz, NCH2), 3.72 – 3.84 (1H, m, N+CH), 3.88 (6H, s,
2 × OCH3), 7.00 (1H, d, J = 8.3 Hz, H(5)), 7.19 (1H, t, J = 5.7 Hz, CH=N+), 7.40 – 7.49
13
(2H, m, H(2) and H(6)). C RMN (CD3OD): δ = 25.9 (N+CHCH2CH2CH2CH2), 26.1
(N+CH(CH2)2CH2 and N(CH2)2CH2), 27.5 (N(CH2)3CH2), 27.7 (NCH2CH2), 30.2
(N(CH2)4CH2), 31.9 (N+CHCH2(CH2)3CH2), 40.7 (NCH2), 56.5 (2 × OCH3), 74.6
(N+CH), 112.0 (C(5) and C(2)), 121.8 (C(6)), 128.2 (C(1)), 144.2 (CH=N+), 150.3
(C(3)), 153.4 (C(4)), 169.7 (CO). ESI/MS m/z (%): 399 (M++Na, 40), 377 (M++H, 24),
165 (100). ESI/HRMS calcd for C21H33N2O4 (M++H): 377.2435, found 377.2426.
1
α-7-(3,4-Dimethoxybenzamido)heptyl-N-tert-butyl nitrone (36). η = 71 %. H RMN
(CD3OD): δ = 1.37 – 1.43 (6H, m, N(CH2)2(CH2)2CH2CH2), 1.47 (9H, s, C(CH3)3), 1.53
– 1.65 (4H, m, N(CH2)4CH2CH2CH2), 2.40 – 2.49 (2H, m, NCH2CH2), 3.36 (2H, t, J =
7.2 Hz, NCH2), 3.87 (6H, s, 2 × OCH3), 7.00 (1H, d, J = 8.5 Hz, H(5)), 7.24 (1H, t, J =
13
5.7 Hz, CH=N+), 7.42 – 7.47 (2H, m, H(2) and H(6)). C RMN (CD3OD): δ = 26.3
(N(CH2)2CH2), 27.9 (N(CH2)5CH2), 28.0 (C(CH3)3), 28.3 (N(CH2)3CH2), 30.1
(N(CH2)4CH2), 30.5 (NCH2CH2), 30.6 (N(CH2)6CH2), 41.0 (NCH2), 56.5 (2 × OCH3),
70.5 (C(CH3)3), 112.0 (C(5) and C(2)), 121.7 (C(6)), 128.2 (C(1)), 142.4 (CH=N+),
150.2 (C(3)), 153.4 (C(4)), 169.7 (CO). ESI/MS m/z (%): 401 (M++Na, 3), 379 (M++H,
1), 308 (58), 165 (100), 124 (28). ESI/HRMS calcd for C21H35N2O4 (M++H): 379.2591,
found 379.2577.

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α-9-(3,4-Dimethoxybenzamido)nonyl-N-tert-butyl nitrone (37). η = 82 %. H RMN
(CD3OD): δ = 1.31 – 1.42 (10H, m, N(CH2)2(CH2)4CH2CH2), 1.47 (9H, s, C(CH3)3),
1.53 – 1.64 (4H, m, N(CH2)6CH2CH2CH2), 2.40 – 2.49 (2H, m, NCH2CH2), 3.35 (2H, t,
J = 7.2 Hz, NCH2), 3.87 (6H, s, 2 × OCH3), 7.00 (1H, d, J = 8.6 Hz, H(5)), 7.24 (1H, t,
J = 5.7 Hz, CH=N+), 7.41 – 7.47 (2H, m, H(2) and H(6)). 13C RMN (CD3OD): δ = 26.4
(N(CH2)2CH2), 28.0 (C(CH3)3), 28.1 (N(CH2)7CH2), 28.3 (N(CH2)3CH2), 30.3
(N(CH2)6CH2), 30.4 (N(CH2)4CH2), 30.5 (N(CH2)5CH2), 30.6 (NCH2CH2(CH2)6CH2),
41.0 (NCH2), 56.5 (2 × OCH3), 70.5 (C(CH3)3), 111.9 (C(5)), 112.0 (C(2)), 121.7
(C(6)), 128.3 (C(1)), 142.4 (CH=N+), 150.2 (C(3)), 153.4 (C(4)), 169.7 (CO). ESI/MS
m/z (%): 406 (M++Na, 3), 165 (100), 139 (27), 124 (47). ESI/HRMS calcd for
C23H38N2O4Na (M++Na): 429.2724, found 429.2716.
1
α-5-(3,4,5-Trimethoxybenzamido)pentyl-N-tert-butyl nitrone (38). η = 94 %. H
RMN (CD3OD): δ = 1.40 – 1.50 (11H, m, C(CH3)3 and N(CH2)3CH2), 1.59 – 1.71 (4H,
m, N(CH2)2CH2CH2CH2), 2.44 – 2.52 (2H, m, NCH2CH2), 3.39 (2H, t, J = 7.0 Hz,
NCH2), 3.80 (3H, s, OCH3), 3.89 (6H, s, 2 × OCH3), 7.17 (2H, s, H(2) and H(6)), 7.25
(1H, t, J = 5.7 Hz, CH=N+). 13C RMN (CD3OD): δ = 26.1 (N(CH2)2CH2), 27.9
(N(CH2)3CH2), 28.0 (C(CH3)3), 28.2 (NCH2CH2), 30.2 (N(CH2)4CH2), 40.8 (NCH2),
56.7 (2 × OCH3), 61.1 (OCH3), 70.5 (C(CH3)3), 106.0 (C(2) and C(6)), 131.1 (C(1)),
142.1 (C(4)), 142.2 (CH=N+), 154.4 (C(3) and C(5)), 169.5 (CO). EI/MS m/z (%): 380
(M+, 37), 307 (20), 266 (31), 212 (45), 196 (51), 195 (100), 96 (34).
1
α-5-(3,4,5-Trimethoxybenzamido)pentyl-N-benzyl nitrone (39). η = 41 %. H RMN
(CD3OD): δ = 1.39 – 1.48 (2H, m, N(CH2)3CH2), 1.58 – 1.69 (4H, m,
N(CH2)2CH2CH2CH2), 2.45 – 2.53 (2H, m, NCH2CH2), 3.37 (2H, t, J = 7.0 Hz, NCH2),
3.80 (3H, s, OCH3), 3.88 (6H, s, 2 × OCH3), 4.92 (2H, s, N+CH2), 7.17 (2H, s, H(2) and
H(6)), 7.32 – 7.44 (6H, m, H(2’–6’) and CH=N+). 13C RMN (CD3OD): δ = 26.0
(N(CH2)2CH2), 27.7 (N(CH2)3CH2), 27.8 (N(CH2)4CH2), 30.1 (NCH2CH2), 40.8
(NCH2), 56.7 (2 × OCH3), 61.1 (OCH3), 69.4 (N+CH2), 106.0 (C(2) and C(6)), 129.8
(C(2’) and C(6’)), 129.9 (C(4’)), 130.1 (C(3’) and C(5’)), 131.1 (C(1)), 134.7 (C(1’)),
142.1 (C(4)), 146.2 (CH=N+),154.4 (C(3) and C(5)), 169.5 (CO). ESI/MS m/z (%): 415
(M++H, 100). ESI/HRMS calcd for C23H31N2O5 (M++H): 415.2227, found 415.2229.