Bifunctional phenolic-choline conjugates as anti-oxidants and acetylcholinesterase inhibitors

Article abstract of DOI:10.3109/14756366.2010.529806

Because of the complex cascade of molecular events that can occur in the brain of an Alzheimer's disease (AD) patient, the therapy of this neurodegenerative disease seems more likely to be achieved by multifunctional drugs. Herein, a new series of dual-targeting ligands have been developed and in vitro bioevaluated. Their architecture is based on conjugating the acetylcholinesterase inhibition and anti-oxidant properties in one molecular entity. Specifically, a series of naturally occurring phenolic acids with recognized anti-oxidant properties (derivatives of caffeic acid, rosmarinic acid, and trolox) have been conjugated with choline to account for the recognition by acetylcholinesterase (AChE). The synthesized hybrid compounds evidenced AChE inhibitory capacity of micromolar range (rationalized by molecular modeling studies) and good antioxidant properties. Their effects on human neuroblastoma cells, previously treated with beta-amyloid peptides and 1-methyl-4-phenylpyridinium ion neurotoxins (to simulate AD and Parkinson's disease, respectively), also demonstrated a considerable capacity for protection against the cytotoxicity of these stressors.

Full text of DOI:10.3109/14756366.2010.529806

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(4): 485–497
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.529806
ORIGINAL ARTICLE
Bifunctional phenolic-choline conjugates as anti-oxidants and
acetylcholinesterase inhibitors
Jaroslav Šebestík1, Sérgio M. Marques1, Pedro L. Falé2,3, Susana Santos2, Daniela M. Arduíno4,
Sandra M. Cardoso4,5, Catarina R. Oliveira4,6, M. Luísa M. Serralheiro2, and M. Amélia Santos1
1Centro de Química Estrutural, Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001 Lisboa, Portugal, 2Centro de
Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal,
3Centro de Biotecnologia Vegetal (IBB), Faculdade de Ciências da Universidade de Lisboa, Campo Grande 1749-016,
Lisboa, Portugal, 4Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3030 Coimbra, Portugal,
5Instituto de Biologia, Faculdade de Medicina, Universidade de Coimbra, 3030 Coimbra, Portugal, and 6Instituto de
Bioquímica, Faculdade de Medicina, Universidade de Coimbra, 3030 Coimbra, Portugal
Abstract
Because of the complex cascade of molecular events that can occur in the brain of an Alzheimer’s disease (AD) patient,
the therapy of this neurodegenerative disease seems more likely to be achieved by multifunctional drugs. Herein,
a new series of dual-targeting ligands have been developed and in vitro bioevaluated. Their architecture is based
on conjugating the acetylcholinesterase inhibition and anti-oxidant properties in one molecular entity. Specifically,
a series of naturally occurring phenolic acids with recognized anti-oxidant properties (derivatives of caffeic acid,
rosmarinic acid, and trolox) have been conjugated with choline to account for the recognition by acetylcholinesterase
(AChE). The synthesized hybrid compounds evidenced AChE inhibitory capacity of micromolar range (rationalized
by molecular modeling studies) and good antioxidant properties. Their effects on human neuroblastoma cells,
previously treated with beta-amyloid peptides and 1-methyl-4-phenylpyridinium ion neurotoxins (to simulate AD and
Parkinson’s disease, respectively), also demonstrated a considerable capacity for protection against the cytotoxicity
of these stressors.
Keywords: Acetylcholinesterase inhibitors, antioxidants, hybrid ligands, anti-neurodegeneratives, Alzheimer’s
disease
Introduction
plaque formation,3 up to date the only Food and Drug
Administration (FDA)-approved AD drugs consist of
acetylcholinesterase inhibitors (AChEI; Figure 1) and a
N-methyl D-aspartate (NMDA) receptor antagonist.4 e
AChEI-based therapies lead to the recovery of the cho-
linergic system to enhance synapses, but they may treat
only soft to moderate severity cases, and do not reverse
or heal the disease.5,6
Alzheimer’s disease (AD) is the most common form of
age-related neurodegenerative disorders, accounting
for 50–70% of all cases.1,2 AD individuals exhibit progres-
sive memory loss and subsequent deficits in cognitive
abilities. AD is typified by pathological depositions of
β-amyloid (Abeta) plaques and neurofibrillary tangles
within specific regions of the brain.
Complementary coadjutant therapeutics have also
been proposed for AD treatments,7 due to the brain
vulnerability to oxidative damage. In fact, as a result of
several mechanisms, radical reactive species can pro-
mote the formation of neurotoxic aggregates, such as the
e complexity of the mechanisms underlying the
progression of AD presents a current challenge for the
development of new therapeutic agents. Despite the
huge amount of research focused on tackling upstream
or downstream pathways of the disease onset and
Address for Correspondence: M. Amélia Santos, Centro de Química Estrutural, Instituto Superior Técnico, Av. Rovisco Pais 1, 1049-001
Lisboa, Portugal. Tel: +351-21-8419273; fax: +351-21-8464455. E-mail: masantos@ist.utl.pt
(Received 28 April 2010; revised 17 September 2010; accepted 04 October 2010)
485

486 J. Šebestík et al.
Abbreviations
MPP+
Abeta1–42
DPPH
BHT
ROS
NMDA
1-methyl-4-phenylpyridinium ion
42 residues-long β-amyloid peptides
2,2-diphenyl-1-picrylhydrazyl radical
3,5-di-tert-butyl-4-hydroxytoluene
reactive oxygen species
AD
Alzheimer’s disease
AChE
AChEI
PD
acetylcholinesterase
acetylcholinesterase inhibitors or inhibition
Parkinson’s disease
Abeta
β-amyloid plaques
N-methyl D-aspartate
has proven antiinflammatory activity,18 and caffeic acid
has anti-(5-lipoxygenage) activity.19
O
NH2
N
O
Concerning the molecular fragment for the AChE
inhibition, we aimed at getting some emulation of done-
pezil (Dnp), one of the most potent AChEIs (IC50 value of
33 nM) still in use.20 However, our main design strategy
was to take profit of hybrid drugs, even with individual
moderate effects, instead of very potent mono-targeting
drug with eventual severe side effects (e.g., tacrine).6
In the past, derivatives of cinnamoylcholine have been
described as quite potent inhibitors of AChE, with
inhibitory constants (KI) between sub-micromolar
and sub-millimolar range.21 On the other hand, α,β-
dehydrophenylalanine choline esters have recently been
found to inhibit that enzyme at micromolar level con-
centration.22 us, we have decided to study the choline
esters of these phenolic carboxylic acids, to conjugate, in
each molecular entity, their antioxidant properties with
the AChE inhibitory activity.
O
N
Tacrine
Donepezil
OH
N
O
O
O
N
N
O
Rivastigmine
Galantamine
Figure 1. Inhibitors of AChE currently approved by FDA as drugs
against AD.
Herein, we describe the preparation of the bifunc-
tional choline esters of three caffeic acid derivatives,
rosmarinic acid, trolox (Figure 3), as well as the resulting
biological (neuroprotective) properties. ese include
their AChE inhibitory and antioxidant activities, and the
neuroprotective efficacy in 1-methyl-4-phenylpyridin-
ium ion (MPP+) and 42 residues-long β-amyloid peptides
(Abeta1–42) induced cell death, as mimics of Parkinson’s
disease (PD) and AD, respectively.16
Abeta plaques. It is also known that AD brains display
evidence of metal dyshomeostasis (namely of calcium,
iron and copper), which in some cases may be related
to an increase in the oxidative stress.3,8 Anti-oxidant
compounds and metal chelators are able, respectively,
to scavenge free radical species and modulate the metal
excess. us, oxidative stress, protein aggregation, and
redox active metal ions are all considered to be promis-
ing pharmacological targets.9–11
Methods
However, the complexity of combined therapy proto-
cols leads to the recent search for alternative therapeutic
strategies based on multifunctional compounds.12–16
us, following the same strategy, we have decided to
develop new bifunctional compounds as drug candi-
dates for anti-AD therapy, by conjugating, in the same
compound, molecular fragments for both neurorestor-
ative and neuroprotective roles, such as the inhibition of
acetylcholinesterase (AChE) and the antioxidation.
Concerningtheantioxidantapproach,wehaveselected
molecular fragments containing a phenol (or catechol)
group with an α,β-unsaturated carboxylic acid chain,
namely caffeic and rosmarinic acids, as well as trolox
(Figure 2). ese are well-known naturally occurring
potent antioxidants with high radical scavenging capac-
ity and also some metal chelating ability.1,17 Noteworthy
is the fact that, besides having antioxidant properties,
trolox is also an analogue of vitamin E, rosmarinic acid
Chemicals and equipment
Analytical grade reagents from Aldrich, Fluka, and
Acros, were purchased from commercial suppliers
(in Lisbon), and they were used as supplied. AChE
type VI-S, from electric eel 349 U/mg solid, 411 U/mg
protein, 5,5′-dithiobis(2-nitrobenzoic) acid] (DTNB),
tris(hydroxymethyl)-aminomethane
(Tris
buffer),
acetylthiocholine iodide (AChI), caffeic acid, and ros-
marinic acid were obtained from Sigma. Solvents were
dried according to standard methods.23 e chemical
reactions were monitored by thin-layer chromatogra-
phy (TLC) using aluminium plates coated with silica
gel 60 F254 (Merck). TLC detection methods were used:
UV 254 nm, staining with I2 (general), ninhydrin (for
amino groups), and dragendorff (for quaternary ammo-
nium salts). e common mobile phases were used: C1
(nBuOH–HCOOH–H2O 75:14:12); C2 (CHCl3–MeOH
Journal of Enzyme Inhibition and Medicinal Chemistry

Bifunctional phenolic-choline conjugates 487
O
HO
HO
OH
O
O
OH
O
HO
HO
Caffeic acid
OH
OH
Rosmarinic acid
HO
O
OH
O
Trolox
Figure 2. Caffeic acid, rosmarinic acid, and trolox, all nontoxic natural antioxidants.
O
O
N+
N+
CH3O
CH3O
O
O
2
1
3
O
O
O
O
N+
HO
HO
N+
O
HO
HO
O
OH
OH
4
HO
O
N+
O
O
5
Figure 3. e synthesized choline ester derivatives of: 3,4-dimethoxycinnamic acid (1), cinnamic acid (2), caffeic acid (3), rosmarinic acid
(4), and trolox (5).
9:1); C3 (acetonitrile (ACN)-H2O 7:1 0.1% trifluo-
roacetic acid (TFA)); and C4 (ACN-H2O 5:1). Column
flash chromatography separations were performed on
silica gel Merck 230–400 mesh. e reverse-phase high-
performance liquid chromatography (RP-HPLC) analy-
sis was carried out in a Liquid Chromatograph Finnigan
Surveyor Plus Modular LC System equipped with a
Purospher STAR RP-18 column, from Merck and Xcalibur
software. e compounds were analyzed by injecting a
0.5 mg/ml solution (25 μL) and using a gradient com-
posed of solution A (0.05% trifluoroacetic acid), solution
B (ACN), and solution C (methanol) as follows: 0 min 90%
A, 10% B and 40 min 22% A, 66% B, 12% C. For choline
caffeate, a better resolution was obtained when using a
gradient composed of solution A (0.05% trifluoroacetic
acid) and solution B (methanol) as follows: 0 min 70% A,
30% B; 20 min 10% A, 90% B; and 25 min 10% A, 90% B.
e detection was carried out at wavelength between 200
and 600 nm with a diode array detector. To further purify
the compounds, the HPLC system referred previously
was also used, the peaks were collected separately, and
this process was repeated several times. Melting points
were measured with a Leica Galen III hot stage apparatus
and are uncorrected. Compensating anions were deter-
mined by Ionex HPLC using Dionex ICS-1500 instrument
equipped with Dionex IonPac AS14A 7 μm (4 × 250 mm)
anion exchange column, anion self-regenerating sup-
pressor (ASRS Ultra II), Dionex DS6 conductivity detec-
tor, and a 25 μL inject loop. Isocratic elution was carried
out with 1.1 ml/min flow of carbonate buffer (16 mM
© 2011 Informa UK, Ltd.

488 J. Šebestík et al.
Na2CO3, 1 mM NaHCO3) for 15 min. RT (Cl−) 3.7 0.3 min.
RT (CF3COO−) 9.7 0.9 min. Chromatograms were col-
lected by PC with Chromeleon software. e mass
spectra of the compounds were normally performed in
a Varian 500-MS LC Ion Trap instrument equipped with
an electrospray ionisation (ESI) ion source. e high-
resolution mass spectra (HRMS) were performed in a
Bruker MicrOTOF ESI–TOF apparatus. e NMR spectra
3,4-Dimethoxycinnamic acid (8)
Procedure was adopted according to Bisogno et al.26
Compound 7 (235 mg, 1.1 mmol) was dissolved in diox-
ane (8 ml) and water (5 ml) mixture. e pH was set up
to 12 with 2 M NaOH. After 2 h stirring, pH was again
raised to 12 with several drops of 2M NaOH. After the
next 2 h, dioxane was evaporated and reaction mixture
was remained at pH 12–14. Reaction mixture was washed
with ether (2 × 6 ml). e pH was set up to 1–2, and the
compound was taken to EtOAc (3× 6 ml). It was dried
over Na2SO4, filtered off, and the solvent was evaporated
to dryness. Yield 186 mg (0.9 mmol, 81%) of compound
1
(for H, 13C, attached proton test (APT), and 2D experi-
ments) were measured on Bruker AVANCE III 300 MHz
and Bruker AVANCE III 400 MHz spectrometers. e
structural assignment of the hydrogen and carbon
chemical shifts was achieved using homonuclear two-
1
8. m.p. 182–190°C. TLC Rf 0.53 in C2 mixture. H-NMR
1
1
dimensional H–1H correlation spectroscopy, 2D H–13C
heteronuclear multiple quantum coherence, and 1H–13C
heteronuclear multibond correlation experiments. e
UV measurements were carried out in a Camspec M 350
Double beam UV–vis spectrophotometer.
(300 MHz, CDCl3), δ: 7.74 (d, 1H, 16 Hz, Ph–CH=), 7.14
(dd, 1H, 8 Hz, 2 Hz, HPh-6), 7.08 (d, 1H, 2 Hz, HPh-2), 6.88 (d,
1H, 8 Hz, HPh-5), 6.32 (d, 1H, 16 Hz, =CH–CO), 3.93 (s, 6H,
2 × CH3O–Ph). 13C-APT-NMR (400 MHz, CDCl3), δ: 172.62
(COO), 151.64 (CPh-4), 149.38 (CPh-3), 147.09 (Ph–CH=),
127.16 (CPh-1), 123.26 (CPh-6), 115.02 (=CH–CO),
111.15 (CPh-5), 109.89 (CPh-2), 56.11 (Ph–OCH3), 56.03
(Ph–OCH3).
Synthesis of the compounds
Chlorocholine chloride (6)
Choline chloride (2.89 g, 20.1 mmol) was dried in high
vacuum at 200°C for 2 h. Freshly distilled SOCl2 (20 ml)
was added to the white solid.24 e solution was heated
under reflux for 1 h. After cooling, solvent was evaporated
to dryness. Solid was redissolved in MeOH (3 × 20 ml),
and re-evaporated to dryness. White crystalline solid was
dried in high vacuum at 80°C until constant mass. Yield
2.58 g (16.3 mmol, 81%) of white extremely hygroscopic
solid 3. 1H-NMR (300 MHz, D2O), δ: 4.04 (m, 2H, Cl–CH2),
3.80 (t, 2H, 7 Hz, N–CH2), 3.24 (s, 9H, 3 × CH3). 13C-APT-
NMR (400 MHz, D2O), δ: 67.29 (CH2N), 67.26 (CH2N),
67.23 (CH2N), 54.59 (CH3), 54.55 (CH3), 54.51 (CH3), 36.59
(CH2–Cl).
3,4-Dimethoxycinnamoylcholine chloride (1)
e procedure was modified according to the method of
Alemany et al.21 for the syntheses of choline esters of vari-
ous cinnamates. To a solution of compound 8 (186 mg,
0.89 mmol) in a dioxane–water (3:2) mixture (5 ml), a
solution of K2CO3 (61.7 mg, 0.45 mmol) in water (1 ml)
was added. After 10 min stirring, it was evaporated to
dryness. To the remaining solid, compound 6 (167 mg,
1.05 mmol) was added. e combined solids were dried
in high vacuum at 70°C for 1 h. Dry DMSO was added,
the mixture was heated up to 60°C, and it was left stirring
at that temperature for 44 h. DMSO was distilled off and
the residue was dried under vacuum for 1 h. It was redis-
solved in CHCl3 and loaded on 1-mm TLC plates from
Merck. Chromatographic separation was performed
using an ACN–H2O mixture (3:1) as eluent. Yield 107 mg
(0.32 mmol, 36%) of white hygroscopic solid 1. TLC Rf
0.26 in C1 mixture. RP-HPLC RT 15.4 min (84%). 1H-NMR
(300 MHz, CDCl3), δ: 7.64 (d, 1H, 16 Hz, Ph–CH=), 7.36
(d, 1H, 2 Hz, HPh-2), 7.27 (dd, 1H, 8 Hz, 2 Hz, HPh-6), 7.00
(d, 1H, 8Hz, HPh-5), 6.58 (d, 1H, 16 Hz, =CH–CO), 4.58
(m, 2H, COO–CH2), 3.81 (s, 3H, 3-CH3O–Ph), 3.80 (s, 3H,
4-CH3O–Ph), 3.73 (m, 2H, N–CH2), 3.18 (s, 9H, 3 × CH3).
13C-APT-NMR(400 MHz,DMSO-d6),δ:165.8(COO),151.2
(CPh-4), 149.0 (CPh-3), 145.5 (Ph–CH=), 126.6 (CPh-1), 123.1
(CPh-6), 114.7 (=CH–CO), 111.5 (CPh-5), 110.5 (CPh-2), 63.9
(N–CH2), 57.7 (COO–CH2), 55.7 (O–CH3), 55.6 (O–CH3),
Methyl 3,4-dimethoxycinnamate (7)
is compound was prepared according to the pro-
cedure of Jung et al.25 To a solution of caffeic acid (1 g,
5.6 mmol) and K2CO3 (2.49 g, 18 mmol) in THF (20 ml),
dimethylsulfate (1.58 ml, 16.6 mmol) was added drop-
wise. e mixture was heated under reflux over night.
Because the reaction was not completed, new portions
of dimethylsulfate (0.5 ml) and K2CO3 (1 g) were added.
After one more day of reflux, there was no progress.
Reaction mixture was evaporated to dryness and sepa-
rated by flash column chromatography (using CH2Cl2 as
eluent). After solvent evaporation, the residue was dried
on vacuum line, affording compound 7 as a white solid
(0.291 g, 24%); m.p. 66–70°C. TLC Rf 0.93 in C2 mixture.
TLC Rf 0.52 in CH2Cl2. 1H-NMR (300 MHz, CDCl3), δ: 7.64
(d, 1H, 16 Hz, Ph–CH=), 7.11 (dd, 1H, 8 Hz, 2 Hz, HPh-6),
7.05 (d, 1H, 2 Hz, HPh-2), 6.86 (d, 1H, 8 Hz, HPh-5), 6.32 (d,
1H, 16 Hz, =CH–CO), 3.91 (s, 6H, 2× CH3O–Ph), 3.79 (s,
3H, COOCH3). 13C-APT-NMR (400 MHz, CDCl3), δ: 167.82
(COO), 151.29 (CPh-4), 149.36 (CPh-3), 144.94 (Ph–CH=),
127.52 (CPh-1), 122.75 (CPh-6), 115.64 (=CH–CO),
111.18 (CPh-5), 109.77 (CPh-2), 56.12 (Ph–OCH3), 56.04
(Ph–OCH3), 51.77 (COOCH3).
+
52.9 (N–CH3). HRMS (ESI–TOF), for C16H24NO4 : calcu-
lated 294.1705; found 294.1707.
Cinnamoylcholine chloride (2)
It was prepared from cinnamic acid and the chloride
6, following the same procedure used for compound 1.
Yield 167 mg (0.62 mmol, 45%) of white hygroscopic solid
2. TLC Rf 0.12 in C1 mixture. TLC Rf 0.36 in C4 mixture.
RP-HPLC RT 18.5 min (97%). 1H-NMR (300 MHz, DMSO-
Journal of Enzyme Inhibition and Medicinal Chemistry

Bifunctional phenolic-choline conjugates 489
d6), δ: 7.73 (m, 3H, Ph–CH=, HPh-2), 7.45 (m, 3H, HPh-3,
HPh-4), 6.69 (d, 1H, 16 Hz, =CH–CO), 4.60 (m, 2H, COO–
CH2), 3.75 (m, 2H, N–CH2), 3.18 (s, 9H, 3 × CH3). 13C-NMR
(400 MHz, DMSO-d6), δ:165.6 (COO), 145.4 (Ph–CH=),
133.9 (CPh-1), 130.8 (CPh-4), 129.0 (CPh-3), 128.5 (CPh-2),
117.6 (=CH–CO), 63.9 (N–CH2), 58.0 (COO–CH2), 53.0
4.47 (m, 2H, COO–CH2), 3.63 (m, 2H, N–CH2), 3.01 (s,
9H, 3 × CH3), 2.58 (m, 1H, H-4), 2.45 (m, 1H, H-4), 2.32
(m, 1H, H-3), 2.06 (s, 3H, 7-CH3), 2.03 (s, 3H, 8-CH3), 1.99
(s, 3H, 5-CH3), 1.82 (m, 1H, H-3), 1.54 (s, 3H, 2-CH3). 13C-
APT-NMR (300 MHz, DMSO-d6), δ: 172.510 (COO), 145.9
(C-6), 144.5 (C-8a), 122.8 (C-8), 120.8 (C-7), 120.3 (C-5),
116.4 (C-4a), 76.4 (C-2), 63.7 (N–CH2), 58.8 (COO–CH2),
52.7 (N–CH3), 30.2 (C-3), 25.0 (2-CH3), 20.3 (C-4), 12.8
(7-CH3), 11.8 (5-CH3, 8-CH3). HRMS (ESI–TOF), for
+
(N–CH3). HRMS (ESI–TOF), for C14H20NO2 : calculated
234.1494; found 234.1487.
+
Caffeoylcholine trifluoroacetate (3)
C19H30NO4 : calculated 336.2175; found 336.2172.
Prepared as compound 1, but starting from caffeic acid
and compound 6. Separation was performed by column
chromatography (RP-18 silica gel, gradient from 1 to 88%
ACN (with 0.1% TFA)). Yield 100 mg (0.26 mmol, 19%)
of white hygroscopic solid 3. TLC Rf 0.19 in C1 mixture.
TLC Rf 0.33 in C4 mixture. RP-HPLC RT 11.8 min (97%).
1H-NMR (300 MHz, D2O), δ: 7.64 (d, 1H, 16 Hz, Ph–CH=),
7.19 (d, 1H, 2 Hz, HPh-2), 7.12 (dd, 1H, 8 Hz, 2 Hz, HPh-6),
6.94 (d, 1H, 8 Hz, HPh-5), 6.40 (d, 1H, 16 Hz, =CH–CO),
4.67 (m, 2H, COO–CH2), 3.80 (m, 2H, N–CH2), 3.25 (s, 9H,
3 × CH3). 13C-APT-NMR (300 MHz, D2O), δ: 168.1 (COO),
147.2 (CPh-4), 146.5 (Ph–CH=), 144.2 (CPh-3), 126.7
(CPh-1), 122.8 (CPh-6), 116.0 (CPh-5), 115.0 (CPh-2), 113.3
(=CH–CO), 64.6 (N–CH2), 58.1 (COO–CH2), 53.7 (CH3).
Bioassays of AChE inhibition
AChE enzymatic activity was measured using an adapta-
tion of the method previously described24; 98 μl of 50 mM
Tris–HCl buffer (pH 8), 30 μl of a solution sample of the
inhibitor, at different concentrations in methanol, and 7.5
μl of AChE solution containing 0.26 U/ml were mixed in a
microplate and left to incubate for 15 min. Subsequently,
22.5 μl of 0.023 mg/ml AChI and 142 μl of 3 mM DTNB
were added. e initial rate of the enzymatic reaction
was followed by reading the absorbance at 405 nm dur-
ing the first 5 min of reaction. Samples were prepared
in a range of concentrations of the compounds in water
(choline caffeate, choline trolox, choline cinnamate) or
in an aqueous solution of 50% methanol (choline 3,4-
dimethoxicinnamate, choline rosmarinate). A control
reaction was carried out using the sample solvent instead
of sample and it was considered 100% activity.
+
HRMS (ESI–TOF), for C14H20NO4 : calculated 266.1392;
found 266.1395.
Rosmarinylcholine trifluoroacetate (4)
Prepared as compound 1, but starting from rosmarinic
acid and compound 6. Yield 254 mg (0.45 mmol, 45%) of
yellow hygroscopic oil 4. TLC Rf 0.33 in C1 mixture. TLC
Asample
I%=100−
×100
Acontrol
1
Rf 0.37 in C4 mixture. RP-HPLC RT 18.7 min (84%). H-
where Asample is the absorbance of the compound con-
taining reaction, and Acontrol is the absorbance of the con-
trol reaction, which were recorded on a Camspec M350
double-beam scanning UV-vis spectrophotometer. Tests
were carried out in triplicate, and a blank with Tris–HCl
buffer instead of the enzyme solution was used.
NMR (300 MHz, DMSO-d6), δ: 9.79 (s, 1H, cin. Ph–OH),
9.23 (s, 1H, cin. Ph–OH), 8.92 (s, 1H, lact. Ph–OH), 8.89
(s, 1H, lact. Ph–OH), 7.49 (d, 1H, 16 Hz, Ph–CH=), 7.08 (d,
1H, 2 Hz, cin. HPh-2), 7.02 (dd, 1H, 8 Hz, 2 Hz, cin. HPh-6),
6.78 (d, 1H, 8 Hz, cin. HPh-5), 6.68 (m, 2H, lact. HPh-2, lact.
HPh-5), 6.55 (dd, 8 Hz, 2 Hz, lact. HPh-6), 6.28 (d, 1H, 16
Hz, =CH–CO), 5.18 (t, 1H, 7 Hz, lact. α-CH), 4.50 (m, 2H,
COO–CH2), 3.61 (m, 2H, N–CH2), 3.05 (m, 11H, 3 × N–CH3,
lact. β-CH2). 13C-APT-NMR (400 MHz, DMSO-d6), δ: 169.1
(lact. COO), 166.0 (cin. COO), 148.9 (cin. CPh–OH), 146.7
(Ph–CH=), 145.7 (cin. CPh–OH), 145.2 (lact. CPh–OH),
144.4 (lact. CPh–OH), 126.5 (lact. ipso-CPh), 125.3 (cin.
ipso-CPh), 121.8 (lact. CPh-2), 120.2 (cin. CPh-6), 116.8 (lact.
CPh-6), 115.8 (lact. CPh-5), 115.6 (cin. CPh-5), 115.0 (cin.
CPh-2), 112.7 (=CH–CO), 72.7 (lact. δ-CH), 63.6 (N–CH2),
58.6(COO–CH2–CH2–N), 52.9(N–CH3), 36.1(lact. δ-CH2).
Antioxidant activity
Antioxidant activity was measured by the 2,2-diphenyl-
1-picrylhydrazyl radical (DPPH) method, as described
by Tepe et al.27 To a 2.5 ml solution of DPPH (0.002% in
methanol), 25 μl of compound solution was added. e
mixture was incubated for 30 min at room temperature.
e absorbance was measured at 517 nm against the cor-
responding blank. e antioxidant activity was calculated
as:
+
HRMS (ESI–TOF), for C23H28NO8 : calculated 446.1805;
found 446.1815.
ADPPH − Asample
AA%=−
×100
ADPPH
2-(6-Hydroxy-2,5,7,8-tetramethylchroman-2-carbonyloxy)-
N,N,N-trimethylethanaminium chloride (5)
where AA is the antioxidant activity, ADPPH is the absorp-
tion of the DPPH solution against the blank, Asample is the
absorption of the sample compound against the blank.
e tests were carried out in triplicate and the compound
concentration providing 50% of antioxidant activity (IC50)
was obtained by plotting the antioxidant activity against
the compound concentration.
It was prepared as compound 1, starting from trolox and
compound 6. Yield 43 mg (0.11 mmol, 11%) of hygro-
scopic oil 5. TLC Rf 0.15 in C1 mixture. TLC Rf 0.41 in C4
mixture. TLC Rf 0.43 in C3. RP-HPLC RT 19.2 min (83%).
1H-NMR (300 MHz, DMSO-d6), δ: 7.57 (br, 1H, OH), δ
© 2011 Informa UK, Ltd.

490 J. Šebestík et al.
of some relevant properties in drug delivery, namely,
octanol/water partition coefficient (log P); brain/blood
partition coefficient (log BB); polar surface activity (PSA);
% human oral absorption, and the central nervous sys-
tem activity.
e compounds were built and minimized as previ-
ously mentioned for the docking calculations but, in
this case, a chloride ion was used as counter-ion for all
the compounds (except for the neutral Dnp). e final
structures were then submitted to the calculation of
some relevant pharmacokinetic properties and descrip-
tors, using the QikProp, version 2.5.32 ese predictions
are for orally delivered drugs and assume nonactive
transport.33
Docking of ligands
All X-ray structures of AChE complexed with different
inhibitors were taken from the RCSB Protein Data Bank
(PDB).28 To define the best docking procedure, the crystal
structures with PDB entry codes 1EVE (containing Dnp)
and 1ACL [containing trimethyl-(10-trimethylazanium-
yldecyl) azanium, a bi-quaternary ligand; see Figure S1,
Supplementary Material] were used. Solvent, co-
crystallization molecules, and counterions were removed
from the original structure whenever they were present,
and hydrogen atoms were added by means of Maestro
7.5.29 e ligands were extracted from the complexes and
were submitted to a conformational search (CS) of 1000
cycles, using a water environment model (generalized-
Born/surface-area model) by means of Macromodel.30
e algorithm used was based on the Monte-Carlo
method with the molecular Merck force field (MMFFs)
and a distance-dependent dielectric constant of 1.0.
Each minimized ligand was then docked to the respective
AChE structure, by means of the Gold program,31 where
the region of interest was defined to contain the residues
within 15 Å from the original position of the ligand. e
Gold default parameters were used, and the ligands were
submitted to 100 genetic algorithm runs, applying the
three fitness functions available with Gold (GoldScore,
ChemScore, and astex statistical potential (ASP)), at a
first stage without any other constraints. e best docked
conformation for each scoring function was then com-
pared with the experimental conformation of the ligands
in the crystal structure, and the root-mean-square devia-
tion (rmsd) between the positions of the heavy atoms was
calculated; this parameter being considered as a measure
of the docking accuracy. e rmsd values for Dnp and the
quaternary ligand were 1.199 and 1.101 Å for ASP, 1.211
and 2.828 Å for ChemScore, and 10.165 and 2.456 Å for
GoldScore fitness function, respectively, resulting in ASP
as the best scoring function to dock these ligands into
their original AChE structures with program Gold.
Cell culture and treatments
Human neuroblastoma SH-SY5Y cells (ATCC number
CRL-2266) were grown in Dulbecco’s modified Eagle’s
medium with nutrient mixture F-12 (DEMEM/F12)
with 10% heat-inactivated fetal calf serum, containing
50 U/ml penicillin and 50 µg/ml streptomycin, under
a humidified atmosphere of 95% air–5% CO2 at 37°C.
Cells were plated at 0.15 × 106 cells/ml for cell viability
assays.
Medium was changed after 24 h and immediately
before treatments. MPP+ was prepared as 200 mM
stocks (in H2O) immediately before use and added to
the medium to 1 mM final concentration. Abeta1–42 (1
μM) peptides were added from a 221.5 μM stock phos-
phate buffered saline (PBS) solution. Previously, the
peptides were dissolved in sterile distilled water at a
concentration of 6 mg/ml and diluted to 1 mg/ml (221.5
μM) with PBS and then incubated for 5–7 days to induce
fibril formation. e tested phenolic-choline conjugates
(1−3) were freshly prepared on each experiment day.
First, they were dissolved in H2O at a concentration
of 1 mM and then added to the medium to 5 μM final
concentration.
For combinatorial treatments, both simultaneous
and sequential treatment approaches were tested and
the selected compound’s concentrations shown to exert
the maximal protective action and had no toxic effects to
cells.
For all conditions tested, control experiments were
performed in which the compounds were tested or the
stress agents was not added.
e choline conjugates were built using program
Maestro 7.5 and underwent CS and structure optimi-
zation with Macromodel, using the same procedures
described earlier. To perform the docking calculations
of these ligands into AChE, the protein structure of 1ACL
complex was used as receptor, with the same docking
procedure mentioned earlier, and ASP as scoring func-
tion. Because of some difficulties in correctly placing
the trimethylammonium moiety in the active site of the
gorge, a scaffold match constraint was added. Here, the
N+(CH3)3 moiety of the quaternary ligand (from structure
1ACL, see Figure S1 on Supplementary Material), in its
original position, was saved and used by Gold as a scaf-
fold be matched; the scaffold match constraint weight,
a parameter determining how closely the ligand atoms
should fit onto the scaffold, was set to 5.0.
Cell reduction ability assay
Cell reduction ability as a surrogate of cell viability was
measured by using a quantitative colorimetric assay with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), according to the method of Mosmann.34
In brief, cells were incubated with MTT solution (in
Krebs medium) for 3 h at 37°C. e medium was then
discarded, the stained cells were dissolved with isopro-
panol/HCl, and thereafter the absorbance at 570 nm was
measured using a Spectramax Plus 384 spectrophotom-
eter (Molecular Devices). MTT reduction ability was
Prediction of pharmacokinetic properties
For the compounds in study, a brief in silico calcula-
tion was performed to enable a comparative prediction
Journal of Enzyme Inhibition and Medicinal Chemistry

Bifunctional phenolic-choline conjugates 491
i
N+
N+
Cl−
HO
Cl
Cl−
6
O
O
O
O
O
O
ii
iii
OH
O
HO
HO
OH
O
8
7
iv,v
O
O
O
N+
O
1
Figure 4. Synthesis of compound 1. Reagents and conditions: (i) SOCl2, reflux; (ii) (MeO)2SO2, K2CO3, THF, reflux; (iii) NaOH, dioxane/H2O,
pH 12–14, r.t.; (iv) K2CO3, dioxane/H2O, r.t.; and (v) DMSO, chlorocholine chloride (6), 60°C.
expressed as a percentage of the control value obtained
for untreated cells.
Table 1. Results of AChE inhibition and antioxidant activity
(DPPH assay) measured for the compounds in study, some
parent compounds and standard AChE inhibitors.
AChE
inhibition
Antioxidant
activity
Results and discussion
IC50 (μM)a EC50 (μM)b
Compound
Chemistry
Parent compounds (carboxylic acids)
3,4-Dimethoxycinnamic acid
Cinnamic acid
For the preparation of the new compounds, the first step
consisted of the conversion of choline into chlorocholine
(6) with thionyl chloride (Figure 4). e synthesis of the
cinnamoylcholine esters was based on a reported method
for alkylation of the respective silver carboxylates with
chlorocholine,21 but the silver salts were substituted with
cheaper potassium salts. e preparation of 2,6-dime-
thoxycinnamoylcholine analogue, 1, was slightly more
complex, because the caffeic acid was used as the starting
material, which was methylated using dimethylsulfate,
in the presence of K2CO3, to afford 7 (Figure 4). e ester
7 was then hydrolyzed to the corresponding carboxylic
acid, 8, under aqueous basic conditions (NaOH).
e carboxylic acid (8, cinnamic, caffeic, rosmarinic,
or trolox) was converted to the corresponding potas-
sium salt, using K2CO3. It was then alkylated with the
chlorocholine derivative, 6, to afford the respective target
compounds (1–5, respectively). Although compounds 1,
2, and 5 were obtained as the chloride salts, compounds
3 and 4 were obtained as trifluoroacetates, due to the use
of TFA in ion-pairing reverse-phase chromatography for
purification purpose.
7204c
9900 700
5551c
480
675d
Caffeic acid
24.8 0.2
6.4 0.1
13.2 0.4
Rosmarinic acid
Trolox
1220 80
3995
6
Choline esters
3,4-Dimethoxycinnamoylcholine
chloride, 1
7.3 0.8
303d
371d
Cinnamoylcholine chloride, 2
Caffeoylcholine trifluoroacetate, 3
Rosmarinylcholine trifluoroacetate, 4
Troloxcholine trifluoroacetate, 5
Standards
50
91
7
6
30
6
300 30
4.3 0.2
1193 93
38.4 0.9
Tacrine
0.2e
0.03f
1.07g
1.5h; 48g
–
–
–
104c
Donepezil
Galantamine
Rivastigmine
BHTi
–
15.7j
aAChEI (IC50) in Tris buffer (50mM, pH 8); bDPPH assay;
cmaximal dose in the assay leading to a value inferior than 25%
inhibition; dmaximal dose in the assay leading to a value inferior
than 5% inhibition; eref. 52; fref. 20; gref. 35; href. 37; iBHT=3,5-di-
tert-butyl-4-hydroxytoluene; jref. 53.
e stability of the new compounds in aqueous solu-
1
1
tion was evaluated by H-NMR in neutral D2O. e H-
NMR spectra of compounds 1 and 3 were recorded for a
long time, and no changes were observed in their spectra
in a 48-h timeframe, thus meaning that the compounds
are stable in aqueous medium, at least along that time
window.
Inhibition of AChE
Bioassays
e AChE inhibitory activities of the new phenolic-
choline esters, and their parent compounds (Figures 2
and 3), were evaluated and the results are summarized
© 2011 Informa UK, Ltd.

492 J. Šebestík et al.
in Table 1. All the carboxylic homologues displayed low
AChE inhibitory activities, but the insertion of the cho-
line moiety resulted, for all cases, in a drastic increase
in those inhibitory properties. e choline-conjugate
compounds proved to inhibit AChE, with IC50 values in
micromolar range. Although, the new compounds are in
general weaker AChEI than the standard AChEI drugs,
some of them present activities with the same order of
magnitude (low micromolar range). However, direct
comparison between the new and the reference inhibi-
tors renders difficult, because the corresponding inhibi-
tor–enzyme interactions are quite different, except Dnp,
which presents some structural analogies with these
compounds, and is the strongest standard inhibitor (IC50
value of 30 nM). Among these new compounds, 3,4-di-
methoxycinnamic choline ester (compound 1) appeared
as the strongest inhibitor, with IC50 values (7.3 μM) close
to those reported for some reference inhibitors, such as
galantamine (1.07 μM),35,36 rivastigmine (1.5 μM),37 or a
choline ester derivative of α,β-dehydrophenylalanine
(37 μM).22 Within this series of caffeic analogues (1–3),
compound 3 displayed the lowest activity, with IC50
value of 91 μM. e activity decreases from compounds
with 3,4-dimethoxy-substituted aromatic ring (1) to the
non-substituted (2), and then to the 3,4-dihydroxy-sub-
stituted (3) aromatic system; thus, it is suggested that the
methoxy and hydroxyl groups can establish favorable and
unfavorable interactions, respectively, with the active
site of AChE, with concomitant effects on the stability of
the corresponding protein-inhibitor adduct. is trend
also suggests that those moieties should interact with
the hydrophobic residues at the active site of AChE.
Regarding the other studied compounds, the ros-
marinyl- and trolox-choline derivatives (4 and 5, respec-
tively), and their AChE inhibitory activities (IC50 300
and 1190 μM) are much lower than those found for the
caffeic analogues. is activity decrease may be due to
the bulkiness of these compounds, which may hamper
their fitting into the enzyme-active cavity and/or to their
hydroxyl groups that may form particularly unfavor-
able interactions with the protein, as was observed for
compound 3. is shall be further clarified below by the
docking studies.
cationic–π interactions with the positively charged cho-
line, and help the stabilization of the transition state. e
catalytic triad (Ser200, His440, and Glu327; Figure 5 and
Figure S1 of Supplementary Material) binds covalently to
the substrate (through Ser200) and, aided by a water mol-
ecule, leads to the hydrolysis of the acetylcholine, gener-
ating choline and acetic acid. ere is also a peripheral
anionic site (PAS), located at the entrance of the gorge
and formed by Trp279, Tyr70, and Asp72, which may
act as a “trap” for binding substrate molecules, before
directing them into the active site gorge. e structure
of this enzyme seems to be highly optimized, in terms of
electrostatic field orientation and hydrophobic interac-
tions, to rapidly guide the substrate into the bottom of
the active site.
Aimed at getting some rationalization of the inhibitory
activities of our new ligands toward AChE, a protein–
ligand docking study was carried out using the program
Gold, version 4.0.31 To define the best docking procedure,
the crystal structures of two complexes of TcAChE were
taken from the RCSB PDB,28 containing one of the stron-
gest AChEI, the FDA-approved drug Dnp,41 and another
one with decamethonium ion, a biquaternary ligand42
(PDB entries 1EVE and 1ACL, respectively) (see Figure
S2, Supplementary Material). ese two complexes were
chosen because of some similarity between the corre-
sponding inhibitors and our ligands. In the first case, Dnp
has a dimethoxybenzene moiety, as compound 1; in the
second case, the inhibitor is a quaternary ligand, inserted
in the catalytic site but positioned along the gorge, rather
than being close to the catalytic Ser200, as we expect
to happen with our inhibitors, for sterical reasons. To
validate the method, the ligands were redocked into the
respective proteins, using the three scoring functions
available with Gold (ASP, ChemScore and GoldScore).
e rmsd of the best scored conformations was calcu-
lated for each case, with respect to the experimental,
and ASP was found to give the best prediction for both
ligands (rmsd values of 1.20 and 1.10 Å, for Dnp and the
quaternary inhibitor, respectively), so it was used on the
docking calculations with our inhibitors. Furthermore,
1EVE and 1ACL structures are very similar in their 3D
conformations (the overall rmsd for the heteroatoms was
calculated as 0.41 Å), but 1ACL structure was chosen for
docking, for containing a quaternary ligand.
Globally, this new type of bifunctional antioxidant
compounds revealed interesting properties as AChEI,
with activities of the same order of magnitude as some
reference inhibitors, but further efforts on structure
optimization may enable the improvement of the AChE
inhibitory capacity.
e first results were not always reliable. Either the
choline moiety was pointing outward the gorge, unlike
what is known for some ligands of this type, or it was in
variable positions within the active site, which made no
sense. Hence, we decided to apply a scaffold match con-
straint, allowing to define (constrain) the position that a
certain fragment should occupy along the docking calcu-
lations. In this case, the N+(CH3)3 fragment from the origi-
nal inhibitor was used as “scaffold” to dock the ligands
into the active site of 1ACL structure, and the constraint
weight was set to the value of 5.0.
Molecular modeling
e AChE protein is a very efficient enzyme with quite
interesting structural features.38–40 e active site is bur-
ied in a narrow deep gorge, which is mostly surrounded
by aromatic residues, and it includes two main subsites.
e catalytic anionic subsite (CAS) is formed by Trp84,
Phe330, and Glu199 (sequence numbering of Torpedo
e docking results for our compounds (1–5) with
TcAChE showed the ligands well inserted into its active
californica AChE, TcAChE)38–40
, and can establish
Journal of Enzyme Inhibition and Medicinal Chemistry

Bifunctional phenolic-choline conjugates 493
Entrance to
the Gorge
Trp279
Tyr70
Tyr334
Asp72
Ile287
Tyr121
Phe330
Phe331
1
Trp84
His440
Glu327
Ser200
Glu199
Figure 5. Docking of compound 1 (blue) into the active site of TcAChE (PDB entry 1ACL). e catalytic triad is formed by Ser200, His440,
and Glu327, the CAS is formed by Trp84, Phe330, and Glu199, and the PAS located at the entrance of the gorge is formed by Trp279, Tyr70,
and Asp72.
gorge, blocking the entrance of the substrate and water
molecules, and therefore hindering the catalytic activ-
ity of this enzyme. For the most active compound (1,
Figure 5), the quaternary ammonium moiety could
establish van der Waals contact with the indole ring
of Trp84, and electrostatic interactions with Glu199.
e spacer chain seems well accommodated along the
hydrophobic cavity, and the aromatic system (the cin-
namic conjugated system) forms a π–π contact with
Tyr334. One of the methoxy groups is pointing toward
the PAS, forming van der Waals interactions with the
Trp279 and Tyr70 aromatic systems, although the other
methoxy group is pointing to the opposite side of the
cavity, forming weak interactions with the Ile287 and
Phe331 side chains. Superimposition of the experimen-
tal conformations found for Dnp (on crystal structure
1EVE) and that calculated for compound 1 evidenced
that the area of contact with the enzyme is larger for
Dnp than for 1 (see Figures S1 and S3, Supplementary
Material), thus explaining its concomitant higher
inhibitory potency (IC50 values of 0.03 and 7.3 μM,
respectively). In fact, although Dnp forms strong π–π
and van der Waals contacts with the gorge wall residues
(namely benzyl-Trp84 and piperidine-Phe330) and
the peripheral site (dimethoxyphenyl-Trp279), in the
1-AChE complex these interactions are weaker. is can
be due to differences on the spacer (the linker between
the quaternary system and the phenolic aromatic ring),
namely on the length and on the surface, which are
smaller in compound 1 than in Dnp, thus accounting
for weaker hydrophobic interactions with the gorge
residues (Figure S3b).
Analyzing the docking of the cinnamic and caffeic
derivatives (2 and 3) with AChE, in comparison with
that found for compound 1, their binding conformations
are very similar, namely with the spacers and the aro-
matic rings almost perfectly superimposed (Figure S4,
Supplementary Material). Hence, most of the interactions
observed are maintained. e only differences between
these compounds are due to the substituent groups at
the 3 and 4 positions of the benzene rings, and they are
responsible for the differences in their inhibitory activi-
ties (which decreased 6.8- and 12.5-fold for compounds
2 and 3, with respect to that of compound 1). Concerning
compound 2, the absence of the methoxy groups, with
their favorable interactions with the enzyme (as found
for compound 1), accounts for a lower stability of the
respective ligand–enzyme complex, compared with that
of compound 1. Regarding compound 3, the presence of
two hydrophilic hydroxyl substituent groups hampered
the hydrophobic interactions established with the meth-
oxyl groups of compound 1. Instead, only a weak H bond
may be established between the 3-hydroxy and the car-
bonyl oxygen of Tyr334, but, apparently, it is not enough
to compensate the loss in hydrophobic interactions, thus
resulting in a decrease in inhibitory activity.
© 2011 Informa UK, Ltd.

494 J. Šebestík et al.
Concerning the rosmarinic (4) and trolox (5) deriva-
tives, comparison between the interactions of these
compounds with the enzyme resulted a bit more diffi-
cult, because they have different main backbones and
side chains. For compound 4 (Figure S5, Supplementary
Material), the docking results indicated that one of its
aromatic rings (the caffeic part) is able to establish a
π–π contact with the aromatic residues on the periph-
eral site (with Trp279 and Tyr70), although the other
aromatic ring forms weak van der Waals interactions
with Phe331. e spacer between the choline and the
aromatic moieties of compound 4 seems to favor a posi-
tional accommodation very similar to that of compound
1, although compound 4 seems to be able to establish
more aromatic interactions with the enzyme than com-
pound 1. erefore, the decrease in its inhibitory activ-
ity with respect to compound 1 (41-fold decrease) may
be explained mainly by the hydrophilic–hydrophobic
repulsion caused by the hydroxyl substituent groups of
the aromatic rings, which decreases the overall stability
of the 4-AChE complex. Regarding compound 5, its aro-
matic rings seem to be well accommodated at the center
of the gorge channel, apparently disabling the formation
of any strong π–π stacking with the enzyme, but only van
der Waals contacts (namely through the heterocyclic ring
with Tyr334, and the methyl groups with the peripheral
site aromatic residues). Furthermore, the hydroxyl group
must contribute unfavorably for the binding interactions
of this compound within the AChE active gorge, result-
ing in the lowest inhibitory activity among these new
compounds.
in the reactive oxygen species (ROS)-scavenging action
of polyphenols (through high constant rates of the
H-atom abstraction reactions).43,44 It is known that the
presence of these phenolic hydroxyl groups increases
the ROS-scavenging activity, but reduces the lipophi-
licity of the compounds. is fact may decrease their
ability to penetrate the blood–brain barrier (BBB) and
the cell membranes, and, as discussed before, also the
anti-AChE activity. Hence, these two factors must be
well balanced in the development of this type of drugs,
to make them valuable as potential anti-neurodegen-
erative drugs.
In vitro neuroprotective activity
Preliminary assays were performed to evaluate the
potential therapeutic action of a set of bifunctional
phenolic-choline conjugates (compounds 1–3, which
presented the best AChEI) on neuronal cells (SH-SY5Y)
pretreated with the stressors MPP+ and Abeta1–42. SH-SY5Y
cell line was selected because it has been shown to exhibit
a neuronal phenotype and have several neurochemical
markers.45 On the other hand, Abeta1–42 and MPP+ treat-
ments in SH-SY5Y cells constitute a reliable model for
screening potential neuroprotective compounds, since it
mimics some aspects of neurodegeneration underlying
the AD and PD, respectively.46,47 Both stressors induce
mitochondrial deficits, increase in ROS production, and
125
§§
§
Antioxidant activity
100
75
50
25
0
##
#
e choline-ester conjugates and the corresponding
carboxylic analogues were screened for their antioxidant
activities based on their interaction with the DPPH stable
free radical, and the results are shown in Table 1.
#
***
***
Among the carboxylic analogues, the rosmarinic
acid was the best antioxidant, with EC50 value of 6.4
μM, which was very close to trolox activity (13.2 μM),
whereas cinnamic acid was the weakest one (675 μM).
As expected, the antioxidant activities of these com-
pounds were not much affected by the introduction
of a choline moiety, but actually some increases were
observed, such as for the rosmarinylcholine, 4, which
appeared as the strongest antioxidant (EC50 of 4.3 μM).
is value is even lower than those observed for the
standard antioxidants 3,5-di-tert-butyl-4-hydroxytolu-
ene (BHT; an antioxidant food and cosmetic additive)
and trolox (a vitamin E-like antioxidant, quite used in
biochemical systems for oxidative stress protection).
is may be due to the extra catechol group in the ros-
marinic structure when compared with others. On the
other hand, among the compounds in this study, the
cinnamic derivatives (both for the carboxylic acid and
the choline ester 2) displayed the lowest antioxidant
activities. is is probably due to the absence of any
phenol hydroxyl groups, which have an important role
Figure 6. Effect of bifunctional phenolic-choline conjugates (1, 2,
3) on Abeta1–42 and MPP+ toxicity in SH-SY5Y cells. SH-SY5Y cells
were treated with Abeta1–42 (1 μM) and MPP+ (1mM) for 24h, in the
absence or the presence of 5 μM of compounds 1, 2, and 3. Results
are expressed as the percentage of SH-SY5Y untreated cells, with
themean S.E.M. derivedfromsixdifferentexperiments. P<0.001,
significantly different when compared with SH-SY5Y untreated
cells; #P<0.05; ##P<0.01, significantly different when compared
with Abeta1–42 treated cells; §P<0.05; §§P<0.01, significantly
different when compared with MPP+ treated cells.
Journal of Enzyme Inhibition and Medicinal Chemistry

Bifunctional phenolic-choline conjugates 495
apoptotic cell death48,49; so all three compounds, due to
their chemical properties, may act as strong antioxidants,
and thus prevent ROS cellular damage.
Conclusions
A small set of new bifunctional compounds has been
developed and studied on their potential antineurode-
generative roles. e design strategy was based on con-
jugating known naturally occurring antioxidant phenolic
acids (three caffeic acid derivatives, rosmarinic acid, and
trolox) with choline through ester formation, to enable
the AChEI.
e results depicted in Figure 6 evidenced a significant
decrease in the cell viability after a 24-h treatment with
1 μM Abeta1–42 (ca. 30%) and 1mM MPP+ (ca. 20%). e
bifunctional phenolic-choline conjugates, by themselves,
did not significantly affect the cell viability at the same
end point (a maximum of 10% increase was achieved
with compound 2). On the other hand, all the tested
compounds provided some protection against the cell
death induced by Abeta1–42 and MPP+ on cultured human
SH-SY5Y cells. e Abeta1–42-treated cells showed the
highest viability increments, approximately 19–22%
increases relative to the absence of ligands. Compound
1 seemed to be slightly less effective than compounds
2 and 3 in preventing the Abeta1–42-mediated toxicity.
However, the differences are not significant enough to
establish good correlation with the previously cited bio-
logical properties, since compound 2 displayed the worst
balance in terms of anti-AChE and antioxidant activities.
us, other factors may contribute to protecting role of
these compounds against cell Abeta1–42 injury, namely
their ability for cell membrane crossing, which depend on
several molecular parameters, such as the lipophilicity;
also the caspase cascade stress-induced apoptosis, which
was not evaluated herein.50,51
Concerning the effect of the compounds on MPP+-
treated cells, the viability improvement was only of
approximately 5–12%, compared with the absence of
the compounds, and compound 3 presents the lowest
protective activity. Differences between the cell perme-
ability properties of this set of compounds may also,
somehow, account for differences on their cell protec-
tive roles. Among this set of cell tested compounds,
the calculated values32 of log P and log BB (Table S1,
Supplementary Material) was 5.82 and −0.057 for com-
pounds 1, 5.18 and 0.47) for 2, and 3.84 and −1.07 for 3,
suggesting the lowest lipophilicity and BBB permeabil-
ity for compound 3. erefore, this compound should
present some extra difficulties to enter the cell, which
may explain the lowest protective effects on MPP+-
treated cells.
ese conjugates demonstrated hybrid properties,
namely AChE inhibitory and antioxidant activities in
micromolar range, two drug properties of relevance
for combating the symptoms and the progression of
multifactorial neurodegenerative diseases, namely
AD. Concerning the AChEI activity, 3,4-dimethoxycin-
namoylcholine was identified as the most potent inhibi-
tor, from our set, with IC50 of 7.3 μM. Interestingly, it
contains a catechol group protected with methyl groups,
and in that moiety it is similar to the FDA-approved drug
Dnp. Among these compounds, the rosmarinic and
trolox derivatives, presented the lowest AChEI activi-
ties, although they are among the strongest antioxidants
(EC50 values of 4.3 and 38.4 μM). eir different activi-
ties are rationalized on the basis of their structures and
docking simulations. e most active AChE inhibitors
(the caffeic acid derivatives) were also assessed for pro-
tective effects on the death of neuroblastoma SH-SY5Y
cells treated with Abeta1–42 and MPP+. ese compounds
proved to reduce the cell viability loss induced by each
of the stressors, thus evidencing neuroprotective roles.
Further studies are envisaged to optimize the structure
of the bifunctional analogous compounds as potential
anti-neurodegeneratives.
Acknowledgements
e authors thank Portuguese NMR Network (IST-UTL
Center) for providing access to the NMR facility. e mass
spectra were obtained at the IST Node, which is part of the
National Mass Spectrometry Network (RNEM) created by
the Portuguese Foundation for Science and Technology
(FCT). e authors thank Leonel Gil Nogueira for helping
with Ionex HPLC.
In summary, our preliminary results on ex vivo cells
indicate that the new phenolic-choline hybrids 1–3 exert
some protective effects on the death of SH-SY5Y cells
induced by Abeta1–42 or MPP+. However, the mechanism
of their cell-protecting roles still needs to be clarified,
namely in relation with their biological activity as AChE
inhibitors and antioxidative agents. e neuroprotective
properties are also likely to be improved with the lipophi-
licity of the design compounds, which can be predicted
by computational tools. us, from this work, a new type
of bifunctional derivatives of naturally occurring com-
pounds appeared with proved neuroprotective roles, and
they represent a challenge to pursuit with further struc-
tural optimizations to improve both target activities and
their membrane-cross abilities.
Declaration of interest
e authors are grateful to the Portuguese Foundation
for Science and Technology (FCT) (Postdoc grant SFRH/
BPD/42511/2007, PhD grant SFRH/BD/37547/2007,
PhD grant SFRH/BD/32470/2006, and PhD grant SFRH/
BD/38743/2007) for financial support. e authors are
also thankful to Centro de Neurociências e Biologia
Celular da Universidade de Coimbra (Portugal) for
financial support. All molecular modeling figures
were produced using the UCSF Chimera package from
the Resource for Biocomputing, Visualization, and
Informatics at the University of California, San Francisco
(supported by NIH Grant P41 RR-01081).
© 2011 Informa UK, Ltd.

496 J. Šebestík et al.
inhibicion de colinesterasa por esteres de colina. Anales Real Soc
Espan Fis Quim 1962;B58:531–538.
References
1. Casetta I, Govoni V, Granieri E. Oxidative stress, antioxidants and
neurodegenerative diseases. Curr Pharm Des 2005;11:2033–2052.
2. Lau I-F, Brodney MA. erapeutic approaches for the treatment
of Alzheimer disease: an Overview. In: Lau I-F, Brodney MA (eds),
Alzheimer’s Disease. Berlin: Springer-Verlag, 2008, pp. 1–25.
3. Jakob-Roetne R, Jacobsen H. Alzheimer’s disease: from
pathology to therapeutic approaches. Angew Chem Int Ed Engl
2009;48:3030–3059.
22. Grigoryan HA, Hambardzumyan AA, Mkrtchyan MV, Topuzyan
VO, Halebyan GP, Asatryan RS. alpha,beta-Dehydrophenylalanine
choline esters, a new class of reversible inhibitors of human
acetylcholinesterase and butyrylcholinesterase. Chem Biol
Interact 2008;171:108–116.
23. Armarego WLF, Perrin DD. Purification of Laboratory Chemicals,
4th edn. Oxford: Butterworth-Heinemann, 1996.
24. Ingkaninan K, Temkitthawon P, Chuenchom K, Yuyaem T,
ongnoi W. Screening for acetylcholinesterase inhibitory activity
in plants used in ai traditional rejuvenating and neurotonic
remedies. J Ethnopharmacol 2003;89:261–264.
4. Mimica N, Presecki P. Current treatment options for people
with Alzheimer’s disease in Croatia. Chem Biol Interact
2010;187:409–410.
5. Tumiatti V, Bolognesi ML, Minarini A, Rosini M, Milelli A, Matera
R, Melchiorre C. Progress in acetylcholinesterase inhibitors
for Alzheimer’s disease: an update. Expert Opin er Patents
2008;18:387–401.
6. Kao J, Grossberg G. Cholinesterase inhibitors. In: Lau I-F, Brodney
MA (eds), Alzheimer’s Disease. Berlin: Springer-Verlag, 2008, pp.
26–51.
25. Jung YH, Kim JD. Mechanism studies on the CSI reaction with
allyl ethers by varying p-substituent. Arch Pharm Res 2003;26:
667–678.
26. Bisogno F, Mascoti L, Sanchez C, Garibotto F, Giannini F,
Kurina-Sanz M et al. Structure-antifungal activity relationship
of cinnamic acid derivatives. J Agric Food Chem 2007;55:10635–
10640.
7. Cornelli U. Treatment of Alzheimer’s disease with a cholinesterase
inhibitor combined with antioxidants. Neurodegener Dis
2010;7:193–202.
8. Mattson MP. Pathways towards and away from Alzheimer’s disease.
Nature 2004;430:631–639.
27. Tepe B, Daferera D, Sokmen A, Sokmen M, Polissiou M.
Antimicrobial and antioxidant activities of the essential oil and
various extracts of Salvia tomentosa Miller (Lamiaceae). Food
Chem 2005;90:333–340.
28. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig
H et al. e Protein Data Bank. Nucleic Acids Res 2000;28:
235–242.
9. Sayre LM, Perry G, Smith MA. Oxidative stress and neurotoxicity.
Chem Res Toxicol 2008;21:172–188.
10. Gaeta A, Hider RC. e crucial role of metal ions in
neurodegeneration: the basis for a promising therapeutic strategy.
Br J Pharmacol 2005;146:1041–1059.
11. Biran Y, Masters CL, Barnham KJ, Bush AI, Adlard PA.
Pharmacotherapeutic targets in Alzheimer’s disease. J Cell Mol
Med 2009;13:61–86.
12. Rosini M, Andrisano V, Bartolini M, Bolognesi ML, Hrelia P,
Minarini A et al. Rational approach to discover multipotent anti-
Alzheimer drugs. J Med Chem 2005;48:360–363.
13. Bolognesi ML, Cavalli A, Valgimigli L, Bartolini M, Rosini M,
Andrisano V et al. Multi-target-directed drug design strategy:
29. Maestro, version 7.5; Schrödinger Inc.: Portland, OR, 2005.
30. Macromodel, version 8.5; Schrödinger Inc.: Portland, OR, 1999.
31. Jones G, Willett P, Glen RC, Leach AR, Taylor R. Development and
validation of a genetic algorithm for flexible docking. J Mol Biol
1997;267:727–748.
32. QikProp, version 2.5, Schrödinger, LLC, New York, NY, 2005.
33. Di L, Kerns EH, Carter GT. Strategies to assess blood–brain barrier
penetration. Expert Opin Drug Discov 2008;3:677–687.
34. Mosmann T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 1983;65:55–63.
from
a dual binding site acetylcholinesterase inhibitor to a
35. López S, Bastida J, Viladomat F, Codina C. Acetylcholinesterase
inhibitory activity of some Amaryllidaceae alkaloids and Narcissus
extracts. Life Sci 2002;71:2521–2529.
trifunctional compound against Alzheimer’s disease. J Med Chem
2007;50:6446–6449.
14. Bolognesi ML, Matera R, Minarini A, Rosini M, Melchiorre C.
Alzheimer’s disease: new approaches to drug discovery. Curr Opin
Chem Biol 2009;13:303–308.
36. Giacobini E. Drugs that target cholinesterase. In: Buccafusco J
(ed), Cognitive Enhancing Drugs. Switzerland: Birkhauser Verlag,
2004, pp. 11–37.
15. Zheng H, Youdim MB, Fridkin M. Site-activated multifunctional
chelator with acetylcholinesterase and neuroprotective-
neurorestorative moieties for Alzheimer’s therapy. J Med Chem
2009;52:4095–4098.
37. Bolognesi ML, Bartolini M, Cavalli A, Andrisano V, Rosini M,
Minarini A et al. Design, synthesis, and biological evaluation of
conformationally restricted rivastigmine analogues. J Med Chem
2004;47:5945–5952.
16. Arduino D, Silva D, Cardoso SM, Chaves S, Oliveira CR, Santos
MA. New hydroxypyridinone iron-chelators as potential
anti-neurodegenerative drugs. Front Biosci 2008;13:6763–
6774.
38. Wlodek ST, Antosiewicz J, Briggs JM. On the Mechanism of
acetylcholinesterase action: the electrostatically induced
acceleration of the catalytic acylation step. J Am Chem Soc
1997;119:8159–8165.
17. Koufaki M, eodorou E, Galaris D, Nousis L, Katsanou ES, Alexis
MN. Chroman/catechol hybrids: synthesis and evaluation of their
activity against oxidative stress induced cellular damage. J Med
Chem 2006;49:300–306.
18. Sanbongi C, Takano H, Osakabe N, Sasa N, Natsume M,
Yanagisawa R et al. Rosmarinic acid inhibits lung injury induced
by diesel exhaust particles. Free Radic Biol Med 2003;34:1060–
1069.
19. Yang JQ, Zhou QX, Liu BZ, He BC. Protection of mouse brain from
aluminum-induced damage by caffeic acid. CNS Neurosci er
2008;14:10–16.
20. Snape MF, Misra A, Murray TK, De Souza RJ, Williams JL,
Cross AJ et al. A comparative study in rats of the in vitro and
in vivo pharmacology of the acetylcholinesterase inhibitors
tacrine, donepezil and NXX-066. Neuropharmacology 1999;38:
181–193.
39. Tõugu V. Acetylcholinesterase: mechanism of catalysis and
inhibition. Curr Med Chem CNS Agents 2001;1:155–170.
40. Colletier J-P, Fournier D, Greenblatt HM, Stojan J, Sussman
JL, Zaccai G, Silman I, Weik M. Structural insights into
substrate traffic and inhibition in acetylcholinesterase. EMBO J
2006;25:2746–2756.
41. Kryger G, Silman I, Sussman JL. Structure of acetylcholinesterase
complexed with E2020 (Aricept): implications for the design of
new anti-Alzheimer drugs. Structure 1999;7:297–307.
42. Harel M, Schalk I, Ehret-Sabatier L, Bouet F, Goeldner M, Hirth
C et al. Quaternary ligand binding to aromatic residues in the
active-site gorge of acetylcholinesterase. Proc Natl Acad Sci USA
1993;90:9031–9035.
43. Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M. Free radicals,
metals and antioxidants in oxidative stress-induced cancer. Chem
Biol Interact 2006;160:1–40.
21. Alemany A, Baluja G, Corral C, Leon JLF. Inhibidores de
colinesterasa. 1. Influencia de la estructura del grupo acilo en la
44. Denisov ET, Afanas’ev IB. Oxidation and Antioxidants in Organic
Chemistry and Biology. Boca Raton, FL: CRC Press, 2005:835–894.
Journal of Enzyme Inhibition and Medicinal Chemistry

Bifunctional phenolic-choline conjugates 497
45. Youdim MB, Amit T, Bar-Am O, Weinstock M, Yogev-Falach
M. Amyloid processing and signal transduction properties of
antiparkinson-antialzheimer neuroprotective drugs rasagiline
and TV3326. Ann N Y Acad Sci 2003;993:378–86; discussion 387.
46. Cardoso SM, Moreira PI, Agostinho P, Pereira C, Oliveira CR.
Neurodegenerative pathways in Parkinson’s disease: therapeutic
strategies. Curr Drug Targets CNS Neurol Disord 2005;4:
405–419.
47. Pereira C, Agostinho P, Moreira PI, Cardoso SM, Oliveira CR.
Alzheimer’s disease-associated neurotoxic mechanisms and
neuroprotective strategies. Curr Drug Targets CNS Neurol Disord
2005;4:383–403.
49. Cardoso SM, Santos S, Swerdlow RH, Oliveira CR. Functional
mitochondria are required for amyloid beta-mediated
neurotoxicity. Faseb J 2001;15:1439–1441.
50. Troy CM, Shelanski ML. Caspase-2 redux. Cell Death Differ
2003;10:101–107.
51. Hitomi J, Katayama T, Eguchi Y, Kudo T, Taniguchi M, Koyama
Y et al. Involvement of caspase-4 in endoplasmic reticulum
stress-induced apoptosis and Abeta-induced cell death. J Cell
Biol 2004;165:347–356.
52. Otoguro K, Kuno F, Omura S. Arisugacins, selective
acetylcholinesterase inhibitors of microbial origin. Pharmacol
er 1997;76:45–54.
48. Esteves AR, Domingues AF, Ferreira IL, Januário C, Swerdlow RH,
Oliveira CR et al. Mitochondrial function in Parkinson’s disease
cybrids containing an nt2 neuron-like nuclear background.
Mitochondrion 2008;8:219–228.
53. Mata T, Proença C, Ferreira AR, Serralheiro MLM, Nogueira
JMF, Araújo MEM. Antioxidant and antiacetylcholinesterase
activities of five plants used as Portuguese food spices. Food Chem
2007;103:778–786.
© 2011 Informa UK, Ltd.