New cinnamic - N-benzylpiperidine and cinnamic - N,N-dibenzyl(N-methyl)amine hybrids as Alzheimer-directed multitarget drugs with antioxidant, cholinergic, neuroprotective and neurogenic properties

Article abstract of DOI:10.1016/j.ejmech.2016.05.055

Here we describe new families of multi-target directed ligands obtained by linking antioxidant cinnamic-related structures with N-benzylpiperidine (NBP) or N,N-dibenzyl(N-methyl)amine (DBMA) fragments. Resulting hybrids, in addition to their antioxidant a

Full text of DOI:10.1016/j.ejmech.2016.05.055

Accepted Manuscript
New cinnamic – N-benzylpiperidine and cinnamic – N,N-dibenzyl(N-methyl)amine
hybrids as Alzheimer-directed multitarget drugs with antioxidant, cholinergic,
neuroprotective and neurogenic properties
Martín Estrada, Clara Herrera-Arozamena, Concepción Pérez, Dolores Viña,
Alejandro Romero, José A. Morales-García, Ana Pérez-Castillo, María Isabel
Rodríguez-Franco
PII:
S0223-5234(16)30453-6
10.1016/j.ejmech.2016.05.055
EJMECH 8647
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 February 2016
Revised Date: 25 April 2016
Accepted Date: 23 May 2016
Please cite this article as: M. Estrada, C. Herrera-Arozamena, C. Pérez, D. Viña, A. Romero, J.A.
Morales-García, A. Pérez-Castillo, M.I. Rodríguez-Franco, New cinnamic – N-benzylpiperidine
and cinnamic – N,N-dibenzyl(N-methyl)amine hybrids as Alzheimer-directed multitarget drugs with
antioxidant, cholinergic, neuroprotective and neurogenic properties, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.05.055.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New cinnamic – N-benzylpiperidine and cinnamic – N,N-
dibenzyl(N-methyl)amine hybrids as Alzheimer-directed
multitarget drugs with antioxidant, cholinergic,
neuroprotective and neurogenic properties
a
a
a
b
Martín Estrada, Clara Herrera-Arozamena, Concepción Pérez, Dolores Viña,
c
d,e
d,e
Alejandro Romero, José A. Morales-García, Ana Pérez-Castillo, and María Isabel
a,
Rodríguez-Franco *
a
Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (IQM-
CSIC), C/ Juan de la Cierva 3, 28006-Madrid (Spain)
b
Centro de Investigación en Medicina Molecular y Enfermedades Crónicas (CIMUS),
Universidad de Santiago de Compostela, 15782 Santiago de Compostela (Spain)
c
Departamento de Toxicología y Farmacología, Facultad de Veterinaria, Universidad
Complutense de Madrid, 28040-Madrid, Spain.
d
Instituto de Investigaciones Biomédicas “Alberto Sols”, (IIB-CSIC), C/Arturo
Duperier 4, 28029-Madrid, Spain.
e
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas
(CIBERNED), C/ Valderrebollo 5, 28031-Madrid, Spain.
Corresponding author:
María Isabel Rodríguez-Franco, PhD
Tel.: +34 915622900; fax: +34 915644853
E-mail: isabelrguez@iqm.csic.es
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Abstract
Here we describe new families of multi-target directed ligands obtained by linking
antioxidant cinnamic-related structures with N-benzylpiperidine (NBP) or N,N-
dibenzyl(N-methyl)amine (DBMA) fragments. Resulting hybrids, in addition to their
antioxidant and neuroprotective properties against mitochondrial oxidative stress, are
active at relevant molecular targets in Alzheimer’s disease, such as cholinesterases
(hAChE and hBuChE) and monoamine oxidases (hMAO-A and hMAO-B). Hybrids
derived from umbellic – NBP (8), caffeic – NBP (9), and ferulic – DBMA (12)
displayed balanced biological profiles, with IC s in the low-micromolar and
5
0
submicromolar range for hChEs and hMAOs, and an antioxidant potency comparable to
vitamin E. Moreover, the caffeic – NBP hybrid 9 is able to improve the differentiation
of adult SGZ-derived neural stem cells into a neuronal phenotype in vitro.
2

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Keywords: cinnamic-based hybrids; antioxidants; neuroprotectants; neurogenic agents;
human cholinesterases; human monoamine oxidases
Abbreviations: AChE, acetylcholinesterase; AD, Alzheimer’s disease; βA, beta-
amyloid peptide; BACE-1, beta-site amyloid precursor protein cleaving enzyme 1;
BuChE, butyrylcholinesterase; DBMA, N,N-dibenzyl(N-methyl)amine; HMBC,
heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum
correlation; MAOs, Monoamine oxidases; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; NBP, N-benzylpiperidine; NMDA, N-methyl-D-aspartate
receptor; ORAC, oxygen radical absorbance capacity; ROS, reactive oxygen species;
SGZ, subgranular zone; SH-SY5Y, a human neuroblastoma cell line; SVZ,
subventricular zone.
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1
. Introduction
The current therapeutic arsenal against Alzheimer’s disease (AD) is composed by
three acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine, and galantamine)
and one antagonist of the N-methyl- -aspartate (NMDA) receptor, named memantine
1]. These marketed drugs modestly alleviate some symptoms, like memory
D
[
impairment, but they do not stop neuronal degeneration or repair brain damage [2].
Nowadays it is widely accepted that neuronal loss in AD and other
neurodegenerative diseases is the outcome of a wide variety of factors highly
interconnected. In the core of such complex scenario, oxidative stress emerges as an
important actor that could trigger many pathological cascades [3]. The oxidative stress
hypothesis states that toxicity exerted by free radical species is in the basis of many
abnormal signalling pathways that finally lead to neuronal death. Reactive entities are
able to interact with lipids in cellular membranes, altering their composition, function
and permeability. By-products from peroxidation of unsaturated fatty acids include
highly reactive and toxic species such as 4-hydroxynonenal, malondialdehyde and
acrolein, which seems to be especially increased in AD. Moreover, other vital
biomolecules are affected by free radicals. Arachidonic acid and docosahexaenoic acid,
two important molecules whose concentrations reach the highest levels in brain, are
susceptible to be oxidized under oxidative conditions [4-6].
Despite of the effective system of the human body to counteract the harmful effects
of oxidative metabolism (e.g., superoxide dismutase, aldehyde dehydrogenase, and
glutathione peroxidase), during aging the brain becomes particularly sensitive to
oxidative damage due to its great requirement of oxygen, its high levels of unsaturated
fatty acids and its relatively low levels of antioxidant enzymes [7].
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Several investigations have demonstrated a correlation between the severity of AD
and the levels of free radicals. Moreover, the AD-hallmark beta-amyloid peptide (βA)
and subsequent amyloid plaques deposition has been proven to induce the production of
hydrogen peroxide by reduction of metals such as iron and copper [8]. Simultaneously,
βA is able to stimulate glial cells, enhancing the production of an oxidative environment
and pro-inflammatory molecules [9-11]. Pathologic Aβs are the consequence of an
abnormal cleavage of the amyloid precursor protein by beta-site amyloid precursor
protein cleaving enzyme 1 (BACE-1) and several clinical studies have demonstrated a
correlation between low levels of BACE-1 and an effective protection against AD and
cognitive decline in the elderly [12]. Thus, in the last years BACE-1 inhibitors have
been developed as potential drugs for AD [13].
Monoamine oxidases (MAO) are metabolic enzymes whose levels are increased in
neurodegenerative diseases, such as AD and Parkinson disease. These high levels of
MAOs correlate with the exacerbated production of reactive oxygen species (ROS),
which are responsible of the toxic environment characteristic of neurodegeneration [14].
Consequently, MAO inhibitors have been explored as a complementary alternative in
the search for new drugs for AD treatment, both to reduce ROS in the case of MAO-B,
and to treat concomitant depression of AD patients, in the case of MAO-A [15].
Recently, neurogenic agents have emerged as innovative arms to combat AD and
other neurological diseases [16]. The adult brain preserves a vestigial neurogenic
activity in two stem-cells niches: the subventricular zone (SVZ) of the lateral ventricle
and the subgranular zone (SGZ) in the hippocampus. Neurogenesis has been related to
auto-repair processes in the brain and different molecular targets and signalling
pathways involved in such processes have been identified. As a consequence, different
drugs have been tested in neuronal plasticity [17]. For instance, it has been
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demonstrated that several antioxidants promote the transition of pluripotent stem-cells
to a fully reprogrammed phenotype and increase the number of connections between
neurons [18,19]
According to the oxidative stress hypothesis, antioxidants could play an important
role in prevention and therapy of AD. Among the most investigated natural
antioxidants, there are some derivatives of the cinnamic acid (e.g., ferulic, caffeic, p-
coumaric and umbellic acids) and natural molecules containing its structure, such as
curcumin derivatives [20-25] (Figure 1).
Figure 1. Structures of natural antioxidants containing the cinnamic acid structure (p-
coumaric, caffeic, umbellic, and ferulic acids, curcumin), donepezil, AP2238, and new
cinnamic – N-benzylpiperidine (NBP) (1-9) and cinnamic – N,N-dibenzyl(N-
methyl)amine (DBMA) hybrids (10-17).
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However, antioxidant molecules per se might not be sufficient to treat such highly
complex pathologies like AD, as shown by the ambiguous results obtained from several
clinical trials with this kind of compounds [26]. In this sense, multi-target directed
ligands (MTLs) that combine antioxidant properties and other activities in additional
targets of interest for AD (e.g, AChE, BACE, MAO) may greatly improve the treatment
of the disease. In the last years, several MTLs have been developed [27,28], such as
ladostigil
[(R)-3-(prop-2-yn-1-ylamino)-2,3-dihydro-1H-inden-5-yl
ethyl(methyl)carbamate] [29]. This dual AChE-MAO inhibitor enhances the expression
of neurotrophic factors and reverses neuronal damage by promoting endogenous
neurogenic processes.
Continuing with our interest in AD-directed drugs, which some of them displayed
interesting neurogenic properties [30-35], in this work we planned to obtain new
cinnamic-based antioxidants endowed with other fragments with AChE and MAO
inhibition. For this purpose, we selected N-benzylpiperidine (NBP) and N,N-
dibenzyl(N-methyl)amine (DBMA) fragments, which are present in the well-known
AChE inhibitors donepezil and 3-(4-((benzyl(methyl)amino)methyl)phenyl)-6,7-
dimethoxy-2H-chromen-2-one (AP2238), respectively [36-38] (Figure 1).
In this work, we describe the synthesis of new cinnamic – N-benzylpiperidine (1-9)
and cinnamic – N,N-dibenzyl(N-methyl)amine (10-17) hybrids and their biological
evaluation, which includes inhibition of human cholinesterases (hChEs) and
monoamino oxidases (MAOs), a study of their oxygen radical absorbance capacity
(ORAC) and their neuroprotective effects against death provoked by mitochondrial
oxidative stress in the human neuroblastoma cell line SH-SY5Y. Finally, we explored
the neurogenic effects of a selected hybrid using primary SGZ stem-cells from adult
mice.
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2
. Results and discussion
Synthesis of hybrids 1-17. Scheme 1 illustrates the synthetic routes to obtain cinnamic-
NBP (1-9) and cinnamic-DBMA hybrids (10-17) from commercially available
cinnamic-derived acids and amines carrying the NBP or the DBMA fragments. 2-(1-
Benzylpiperidin-4-yl)ethan-1-amine is commercially available, whereas 3- and 4-
((benzyl(methyl)amino)methyl)aniline were obtained as previously described (see
Supplementary Information for further details). In a microwave oven operating at 120
ºC, activation of the corresponding acid was performed with 1,1'-carbonyldiimidazole
(CDI) in THF during 7 minutes and the subsequent reaction with the proper amine
during 10 or 40 minutes depending on its character, aliphatic or aromatic. Derivatives 1-
5
and 10-15 were isolated in yields comprised between 60 and 95% (Scheme 1).
Deprotection of methoxy groups was carried out under mild conditions by
overnight treatment with boron tribromide (BBr ) at room temperature, giving hybrids
3
6
-9,16,17 in moderate to excellent yields (60-97%). It is worth mentioning that
increased yields were obtained when we used 1 equiv of BBr per ether group to be
3
cleavage, plus an additional equiv per each heteroatom included in the molecule, due to
the well-known complexation tendency of boron with free electron pairs, which
decreases the effective amount of reactive BBr [39,40].
3
All cinnamic-based hybrids (1-17) were purified using an automatic
chromatographic apparatus (IsoleraOne, Biotage) in silica gel cartridges and were
1
13
characterized by their analytical (HPLC, HRMS) and spectroscopic data ( H NMR, C
NMR). Complete NMR assignment of their hydrogen and carbon atoms (see
1
13
Experimental Part for details) were made by H – C two-dimensional diagrams,
mainly HSQC (heteronuclear single quantum correlation) and HMBC (heteronuclear
multiple bond correlation).
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O
OH
R
N
N
H N
2
H N
2
CH3
a, b
a, c
o
4
'
i
O
o
m
p
O
N
5
i
1'
m
p
5
N
6
7
3'
4
2'
1
N
6
7
4
1
N
4'
CH3
R
H
3'
R
H
1
2
3
4
, R = 6,7-diOMe (87%)
, R = 6-OMe (81%)
, R = 7-OMe (77%)
, R = 5,7-diOMe (88%)
10, R = 5,7-diOMe (60%)
11, R = 6,7-diOMe (60%)
12, R = 6-OMe,7-OH (3'-subst) (89%)
13, R = 6-OMe,7-OH (86%)
14, R = 6-OMe (79%)
d
5, R = 6-OMe,7-OH (95%)
6
7
8
9
, R = 6-OH (97%)
, R = 7-OH (87%)
, R = 5,7-diOH (60%)
, R = 6,7-diOH (65%)
15, R = 7-OMe (70%)
16, R = 6-OH (72%)
17, R = 7-OH (60%)
d
(All DBMA derivatives are 4'-substituted, except 12)
Scheme 1. Reagents and conditions: (a) CDI, THF, MW, 7 min, 120 °C; (b) 2-(1-
benzylpiperidin-4-yl)ethan-1-amine, MW, 10 min, 120 °C; (c) 3- or 4-
(
(benzyl(methyl)amino)methyl)aniline, MW, 40 min, 120 °C; (d) BBr , THF, rt,
3
overnight.
Biological Evaluation. The preliminary biological evaluation of new cinnamic-based
hybrids comprised: (i) Inhibition of recombinant human cholinesterases (hAChE and
hBuChE), using donepezil as reference and following the spectrophotometric method
described by Ellman et al. [41]. (ii) Inhibition of recombinant human monoamine
oxidases (hMAO-A and hMAO-B), expressed in baculovirus infected BTI insect cells,
using three known MAO inhibitors as references (selegiline, iproniazid, and
moclobemide) and following a described method [42]. (iii) Assessment of the radical
scavenger ability, using trolox (an analogue of vitamin E) as internal standard and
9

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following the oxygen radical absorbance capacity method (ORAC), as previously
described [43,44]. Results are gathered in Table 1.
Regarding hAChE and hBuChE, the NBP series displayed a moderate inhibition of
both isoforms, with IC in the micro- and sub-micromolar range, and in general, with a
5
0
slight preference for hAChE. Methoxy derivatives showed just a little better inhibition
than their hydroxy counterparts. The best hChEs inhibitor of the NBP series is 5 that
unexpectedly exhibited the inversed preference than the rest of its congeners, with an
IC = 76 nM towards hBuChE. Replacement of the NBP part with a DBMA fragment
5
0
maintained the hAChE inhibition in the low-micromolar range, but reduced drastically
the potency towards hBuChE. Thus, the cinnamic – DBMA hybrids showed a better
hAChE-selectivity than the cinnamic – NBP series.
In relation to hMAO-A and hMAO-B inhibition, both series displayed almost the
same pattern. Methoxy derivatives were found to be inactive and only hybrids bearing a
p-hydroxy group in the cinnamic fragment showed a meaningful inhibitory activity in
the micromolar range, with a slight preference for the hMAO-A isoform. In contrast,
when the hydroxy group was located at position meta- the resulting derivative was
almost inactive, as it can see in the couple 17 (7-OH, active) and 16 (6-OH, not active).
Maintaining the p-hydroxy group, introduction of a second hydroxy functionality in
position ortho- or meta- increased the inhibitory potency towards both isoforms. In fact,
8
(5,7-diOH) and 9 (6,7-diOH) were the most potent inhibitors of hMAO-A and hMAO-
B described in this work, displaying one-digit-micromolar IC s values for both
5
0
enzymes. These inhibitory activities are comparable with iproniazide, a drug that has
been clinically used for treating depression in humans [45] and that was employed in
our MAO experiments for comparative purposes (Table 1).
1
0

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Concerning ORAC assay, methoxy derivatives were almost inactive. On the
contrary, hydroxy substituted hybrids exhibited good antioxidant capacity in both series
with ORAC values between 1.0 and 3.2 trolox equivalents. This means they were up to
3
.2-fold more potent than trolox, the aromatic part of vitamin E and the responsible for
the radical scavenger capacity. Thus, cinnamic-based hybrids bearing one or two
hydroxy groups could be considered as good antioxidant agents. In contrast with
hMAOs in which a p-methoxy group is essential to find inhibition, for the radical
capture activity, cinnamic hybrids bearing one m-hydroxy group were superior. Hybrids
6
and 16 (6-OH) exhibited the best results with ORAC values of 2.2 and 3.2 in the NBP
and DBMA series, respectively. Simultaneously, they inhibited both hChEs in the low-
micromolar range, but were unable to inhibit any isoform of hMAO. Finally, hybrids
bearing one hydroxy group in the p-cinnamic position or two in o,p- or m,p-positions
showed also good antioxidant activities, ranging from the same than trolox to 1.8-fold
more potent (Table 1).
1
1

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Table 1. Inhibition of human cholinesterases (hAChE and hBuChE) and monoamine
oxidases (hMAO-A and hMAO-B) (IC , µM); oxygen radical absorbance capacity
5
0
a
(ORAC, trolox equiv.)
Compd.
hAChE
hBuChE
MAO-A
MAO-B
ORAC
b
1
2
3
4
5
6
7
8
9
0.26±0.04
0.75±0.10
0.49±0.06
0.63±0.08
0.39±0.05
1.12±0.10
1.01±0.08
0.99±0.10
1.75±0.12
8.73±0.92
3.98±0.29
5.99±0.49
0.69±0.10
0.98±0.12
0.76±0.20
4.39±0.86
0.076±0.01
1.02±0.21
0.93±0.16
0.26±0.08
0.69±0.12
>10
>100
45-55%
0.4±0.04
n.a.
b
45-55%
>100
b
b
45-55%
45-55%
n.a.
>100
>100
13.1±0.8
>100
n.a.
11.4±0.7
1.8±0.2
2.2±0.1
1.7±0.1
1.3±0.6
1.0±0.1
0.5±0.1
0.4±0.08
1.4±0.11
1.5±0.18
0.6±0.06
n.a.
b
45-55%
57.5±3.8
5.5±0.3
3.5±0.2
>100
>100
8.3±0.5
6.0±0.4
>100
10
11
12
13
14
15
16
17
>10
n.d.
n.d.
5.89±0.48
4.5±0.3
n.d.
7.7± 0.5
n.d.
c
c
33%
45%
c
4.67±0.31
5.76±0.40
3.49±0.31
35%
>100
>100
c
25%
>100
>100
c
c
c
46%
45-55%
45-55%
3.2±0.2
1.8±0.14
n.d.
c
c
46%
30%
29.7 ± 1.9
n.d.
29.0 ±1.9
n.d.
Donepezil
AP2238
0.010±0.002 2.50±0.07
d
d
0.044
n.d.
48.9
n.d.
n.d.
n.d.
R-(-)-deprenyl
Iproniazide
Moclobemide
Trolox
n.d.
n.d.
n.d.
n.d.
68.7 ± 4.2 0.017 ± 0.0019
n.d.
n.d.
6.6 ± 0.8
361 ± 19
n.d.
7.5 ± 0.4
>1000
n.d.
n.d.
n.d.
n.d.
n.d.
1.0
a
b
Results are expressed as mean ± SEM (n =5). Inhibition percentage at 100 µM
c
d
(
highest concentration tested). Inhibition percentage at 10 µM. Taken from ref.
38] n.a.: not active at 10 µM. n.d.: not determined
[
1
2

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A joint analysis of the above results points out that three hybrids belonging to the
NBP series showed interesting multitarget profiles. The ferulic-based compound 5
showed a potent antioxidant capacity of 1.8 trolox equivalents, nanomolar and sub-
micromolar inhibition of hBuChE and hAChE (76 nM and 0.39 µM, respectively), and
moderate non-selective inhibitory activity towards hMAO-A and hMAO-B in the
micromolar range (11 ꢀM and 13 ꢀM, respectively). Interestingly, dihydroxy substituted
hybrids derived from umbellic acid 8 (5,7-diOH) and caffeic acid 9 (6,7-diOH)
exhibited more balanced profiles, with IC s in the one-digit-micromolar and sub-
5
0
micromolar range for enzymatic targets (hChEs and hMAOs) and an antioxidant
potency comparable to vitamin E. Similar balanced multifunctional profile was found in
the ferulic – DBMA hybrid 12, which showed one-digit-micromolar IC ’s in all tested
5
0
targets (hAChE: 6.0 ꢀM; hBuChE: 5.9 ꢀM; hMAO-A: 4.5 ꢀM; hMAO-B: 7.7 ꢀM) and
a radical scavenger activity 1.5-fold more potent than vitamin E.
Moreover, cinnamic – DBP and cinnamic – DBMA hybrids were tested as
inhibitors of the human recombinant beta-secretase (hBACE-1), using a fluorescence
resonance energy transfer (FRET)-based assay as previously described [40,46].
Cinnamic – DBMA hybrids 12-15 were capable to inhibit poorly this enzyme with
percentages around 23-33% at 10 ꢀM (data not shown).
To evaluate the behaviour of new hybrids at the cellular level, their neuroprotective
capacity against mitochondrial oxidative stress were tested, using the human
neuroblastoma cell line SH-SY5Y and a toxic insult composed by the mixture of
rotenone and oligomycin A at concentrations of 30 and 10 ꢀM, respectively. The
combination of such toxics blocks complexes I and V of the mitochondrial electronic
chain, generating a great amount of free radicals that finally provoke mitochondrial
dysfunction and cell death [47,48]. Therefore, this assay is considered a good model of
1
3

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mitochondrial oxidative stress. Compounds were co-incubated at four concentrations
(0.1, 0.3, 1, and 3 ꢀM) with a mixture of rotenone and oligomycin A, and maintained
for 24 h period in cell culture. Afterwards, the percentage of cell viability was measured
as MTT reduction [49]. The best antioxidant hybrid 16 (ORAC = 3.2 trolox equiv), the
most potent hChE inhibitor 5 (IC = 76 nM) and ferulic-DBMA hybrid 12 with a well-
5
0
balanced target profile in the micromolar range (hAChE: 6.0 ꢀM; hBuChE: 5.9 ꢀM;
hMAO-A: 4.5 ꢀM; hMAO-B: 7.7 ꢀM) were chosen for this neuroprotective assay.
Melatonin was used as a positive reference [32], and results are shown in Figure 2.
Tested compounds protected around 30-35% of cells at a concentration of 0.3 ꢀM,
although a dose-dependent relationship was not observed in these experiments.
Nevertheless, in any case they did not potentiate the toxicity exerted by
rotenone/oligomycin A, which could be assumed as a lack of toxicity of our newly
obtained hybrids.
1
4

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Figure 2. Effect of compounds 5 (A), 12 (B), and 16 (C) at 0.1, 0.3, 1.0, and 3.0 µM on
cell death induced by the combination of rotenone (30 µM) and oligomycin A (10 µM).
Melatonin (10 nM) is used as positive control in all experiments. Cell viability was
measured as MTT reduction and data were normalized as % of control. Data are
expressed as the means ± SEM of triplicate of at least three different cultures. All
compounds were assayed at increasing concentrations (0.1-3 µM). ***P< 0.001, **P<
0
.01, *P<0.05, with respect to control group. Comparisons between drugs and control
group were performed by one-way ANOVA followed by the Newman–Keuls post-hoc
test.
1
5

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Finally, caffeic – NBP hybrid 9 with a balanced multifunctional profile in hChEs,
hMAOs and ORAC (hAChE: 1.75 ꢀM; hBuChE: 0.69 ꢀM; hMAO-A: 3.5 ꢀM; hMAO-
B: 6.0 ꢀM; ORAC: 1.0 trolox equiv), was chosen to evaluate the ability of new
cinnamic-based hybrids in promoting the differentiation of neural stem-cells towards a
neuronal phenotype. To that purpose, neural stem cells (NSC) isolated from one of the
main neurogenic niches in the adult, the subgranular zone (SGZ) of the dentate gyrus,
were used as described in the experimental section. NSC were grown as neurospheres in
the presence of hybrid 9 (10 µM) during 7 days and later on allowed for 3 days to
differentiate when adhered on a substrate in the presence of serum and compound. At
this point, immunocytochemical analysis was performed to evaluate the ability of tested
compound to promote neuronal differentiation. Our results summarized in figure 3
showed that the new caffeic-based hybrid 9 clearly induced the differentiation of adult
SGZ-derived NSC into a neuronal phenotype. After treatment with compound 9 a
significant increase in the number of β-III-tubulin (early neurogenesis marker) and
MAP-2 (microtubule-associated protein expressed in mature neurons) expressing cells
in the neurospheres in shown. These results suggest that the new caffeic – NBP hybrid 9
has the ability to induce the differentiation of adult neural stem cells into neurons,
promoting its maturation in vitro and showing a great neurogenic effect.
1
6

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Figure 3. Caffeic-based hybrid 9 promotes neuronal differentiation in vitro. Adult
murine neural stem cells isolated from the neurogenic niche of the SGZ of the
hippocampus were grown as neurospheres (NS) during 7 days in the presence of
compound 9 (10 µM). Then, NS were allowed to differentiate on a substrate for another
3
days in the presence of tested compound. Immunocytochemical analysis shows the
expression of two well-known neuronal markers: β-III-tubulin (TuJ clone; green) and
MAP-2 (red) inside the NS (inner part) and in the distal area (outer part). DAPI was
used for nuclear staining. Scale bar, 200µm.
1
7

ACCEPTED MANUSCRIPT
3
. Conclusions
The combination of antioxidant cinnamic-related structures with NBP and DBMA
fragments gave new hybrids with interesting in vitro biological activities. At micro- and
sub-micromolar range they inhibited human AChE, BuChE, MAO-A, and MAO-B, and
showed antioxidant, neuroprotective, and neurogenic properties. In relation to hChEs
inhibition, NBP-based hybrids were more potent than DBMA derivatives and methoxy
derivatives showed better inhibition than their hydroxy counterparts. Regarding hMAOs
the presence of a p-hydroxy group in the cinnamic fragment was essential for getting
inhibition, and introduction of a second hydroxy functionality in position ortho- or
meta- increased the inhibitory potency towards both hMAO-A and hMAO-B shifting
IC s to one-digit-micromolar values. For the radical capture activity, both NBP and
5
0
DBMA hybrids bearing one m-hydroxy group in the cinnamic fragment displayed better
ORAC values than methoxy derivatives and even, better than diphenolic derivatives. In
addition, a selection of hybrids protected the human neuroblastoma cell line SH-SY5Y
against damage provoked by mitochondrial free radicals.
Among the different molecules investigated, we found that hybrids derived from
umbellic – NBP (8), caffeic – NBP (9), and ferulic – DBMA (12) displayed balanced
biological profiles, with IC s in the low-micromolar and submicromolar range for
5
0
hChEs and hMAOs, and an antioxidant potency comparable to vitamin E. Last but not
least, the caffeic – NBP hybrid 9 stimulated the differentiation of adult SGZ-derived
neural stem cells into a neuronal phenotype, showing a great neurogenic effect.
Thus, it is expected that these new cinnamic-based hybrids could increase patient
memory, decrease free radical levels, protect neurons from mitochondrial oxidative
stress, and promote brain auto-repair processes. This biological profile highlights these
new cinnamic-based hybrids as useful multi-target prototypes in the search for new
1
8

ACCEPTED MANUSCRIPT
protective and regenerative drugs for the potential treatment of AD and other
neurodegenerative diseases.
1
9

ACCEPTED MANUSCRIPT
4
. Experimental
4
.1. Chemistry. General Methods
Reagents and solvents were purchased from common commercial suppliers, mostly
Sigma-Aldrich, and were used without further purification. Analytical thin-layer
chromatography (TLC) was carried out using Merck silica gel 60 F254 plates, and the
compounds were visualized under UV-light (λ = 254 or 365 nm) and/or stained with
phosphomolybdic acid 10% wt. in ethanol. Automatized chromatographic separation
was carried out in an IsoleraOne (Biotage) equipment, using different silica Biotage ZIP
KP-Sil 50ꢀ cartridges. High-performance liquid chromatography was performed on a
Waters analytical HPLC-MS (Alliance Watters 2690) equipped with a SunFire C 4.6 x
1
8
5
0 mm column, a UV photodiode array detector (λ = 214–274 nm) and quadrupole
mass spectrometer (Micromass ZQ). HPLC analyses were used to confirm the purity of
all compounds (≥ 95%) and were performed on Waters 6000 equipment, at a flow rate
of 1.0 mL/min, with a UV photodiode array detector (λ = 214–274 nm), and using a
Delta Pak C 5 µm, 300 Å column. The elution was performed in a gradient mixture of
1
8
MeCN/water.
Melting points (uncorrected) were determined in a MP70 apparatus (Mettler
1
13
Toledo). H NMR and C NMR spectra were recorded in CDCl or CD OD solutions
3
3
using the following NMR spectrometers: Varian INOVA-300, Varian INOVA-400,
Varian Mercury-400 or Varian Unity-500. Chemical shifts are reported in δ scale (ppm)
relative to internal Me Si. J values are given in hertz, and spin multiplicities are
4
expressed as s (singlet), broad signal (bs), d (doublet), t (triplet), q (quartet), or m
(
multiplet). High Resolution Mass Spectra (HRMS) were obtained by using an Agilent
200 Series LC system (equipped with a binary pump, an autosampler, and a column
oven) coupled to a 6520 quadrupole-time of flight (QTOF) mass spectrometer.
1
2
0

ACCEPTED MANUSCRIPT
Acetonitrile:water (75:25, v:v) was used as mobile phase at 0.2 mL min-1. The
ionization source was an ESI interface working in the positive-ion mode. The
electrospray voltage was set at 4.5 kV, the fragmentor voltage at 150 V and the drying
gas temperature at 300 °C. Nitrogen (99.5% purity) was used as nebulizer (207 kPa) and
−
1
drying gas (6 L min ).
4
.2. General procedure for the synthesis of cinnamic-based hybrids 1-5 and 10-13.
The corresponding acid (1 mmol) and CDI (1.15 mmol) were mixed into a
microwave tube under nitrogen atmosphere. The tube was sealed up and 3 mL of
anhydrous THF were added using a syringe to dissolve the mixture (CO ↑). This
2
solution was heated into a microwave reactor at 120 ⁰C during 7 min to complete the
acid activation. Afterward, a solution of 1.2 mmol of the corresponding amine in 2 mL
of THF was added with a syringe into the tube; this mixture was heated at 120 ⁰C
during 10 minutes for NBP derivatives and 40 min for DBMA hybrids. After solvent
evaporation under reduced pressure, the crude was re-dissolved in 25 mL of EtOAc and
washed with water (3x5 mL) and brine (3x5 mL), dried over magnesium sulfate and
concentrated under reduced pressure. The crude was purified by column
chromatography using EtOAc:MeOH (9:1) or hexane/EtOAc (0→70%) as eluent.
4
.2.1. (E)-N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(3,4-dimethoxyphenyl)acrylamide (1)
From 3,4-dimethoxy cinnamic acid (0.100 g, 0.48 mmol) and 2-(1-benzylpiperidin-
4
-yl)ethan-1-amine (0.126 g, 0.58 mmol), hybrid 1 was obtained (0.170 g, 87%) as a
1
light yellow solid of mp: 108.1-110.5 ºC. H NMR (500 MHz, MeOD) δ 7.46 (d, J =
1
5.7 Hz, 1H, H ), 7.32 (s, 2H, H ), 7.31 (s, 2H, H ), 7.29 – 7.24 (m, 1H, H ), 7.14 (d, J
3 m o p
2
1

ACCEPTED MANUSCRIPT
=
2.0 Hz, 1H, H ), 7.12 (ddd, J = 8.2, 2.0, 0.5 Hz, 1H, H ), 6.96 (d, J = 8.2 Hz, 1H, H ),
5
9
8
6
.47 (d, J = 15.7 Hz, 1H, H ), 3.86 (s, 3H, H ), 3.85 (s, 3H, H ), 3.51 (s, 2H, H ), 3.35
2 71 61 γ
–
1
6
3.32 (m, 2H, H ), 2.90 (dt, J = 12.1, 3.5 Hz, 2H, H2’eq), 2.05 – 1.99 (m, 2H, H2’ax),
α
.76 – 1.71 (m, 2H, H3’eq), 1.50 (dt, J = 7.9, 6.6 Hz, 2H, H ), 1.37 (dddd, J = 16.4, 9.7,
β
1
3
.4, 3.5 Hz, 1H, H ), 1.28 (qd, J = 12.1, 3.5 Hz, 2H, H ). C NMR (126 MHz,
4
’
3’ax
MeOD) δ 168.86 (C ), 152.20 (C ), 150.70 (C ), 141.56 (C ), 138.19 (C ), 130.92 (C ),
1
6
7
3
i
o
1
29.36 (C ), 129.26 (C ), 128.43 (C ), 123.16 (C ), 119.67 (C ), 112.71 (C ), 111.31
4 m p 9 2 8
(
C ), 64.34 (C ), 56.42 (C ), 56.40 (C ), 54.64 (C ), 38.10 (C ), 37.10 (C ), 34.44
5 γ 61 71 2’ α β
+
+
(
C ), 32.72 (C ). HRMS [ESI+] m/z =408.2407 [M] , Calcd for [C H N O ]
4’ 3’ 25 32 2 3
4
08.2413. HPLC purity 99%.
4
.2.2. (E)-N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(3-methoxyphenyl)acrylamide (2)
From 3-methoxy cinnamic acid (0.250 g, 1.40 mmol) and 2-(1-benzylpiperidin-4-
yl)ethan-1-amine (0.360 g, 1.61 mmol) derivative 2 was obtained (0.430 g, 81%) as a
1
white amorphous solid of mp: 68-70 ºC. H NMR (300 MHz, MeOD) δ 7.48 (d, J =
1
8
5.8 Hz, 1H, H ), 7.39 – 7.21 (m, 6H, Ph, H ), 7.18 – 7.06 (m, 2H, H ), 6.94 (ddd, J =
3 7 5,9
.2, 2.6, 1.0 Hz, 1H, H ), 6.58 (d, J = 15.8 Hz, 1H, H ), 3.82 (s, 3H, H ), 3.51 (s, 2H,
8
2
61
H ), 3.38 – 3.32 (m, 2H, H ), 2.90 (d, J = 11.4 Hz, 2H2’eq), 2.02 (t, J = 11.3 Hz, 2H,
γ
α
H2’ax), 1.74 (d, J = 12.2 Hz, 2H, H3’eq), 1.51 (q, J = 6.8 Hz, 2H, H ), 1.42 – 1.19 (m, 3H,
β
13
H3’ax, 4’). C NMR (75 MHz, MeOD) δ 168.47 (C ), 161.53 (C ), 141.51 (C ), 138.28
1
6
3
(
C ), 137.68 (C ), 130.92 (C ), 130.90 (C ), 129.25 (C ), 128.40 (C ), 122.16 (C ),
i 4 7 o m p 2
1
3
21.32 (C ), 116.48 (C ), 113.83 (C ), 64.37 (C ), 55.73 (C ), 54.66 (C ), 38.13 (C ),
9 8 5 γ 61 2’ α
+
7.06 (C ), 34.46 (C ), 32.75 (C ). HRMS [ESI+] m/z =378.2304 [M] , Calcd for
β
4’
3’
+
[
C H N O ] 378.2307. HPLC purity 100%.
24 30 2 2
2
2

ACCEPTED MANUSCRIPT
4
.2.3. (E)-N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(4-methoxyphenyl)acrylamide (3)
From 4-methoxy cinnamic acid (0.250 g, 1.40 mmol) and 2-(1-benzylpiperidin-4-
yl)ethan-1-amine (0.360 g, 1.70 mmol) derivative 3 was obtained (0.424 g, 77%) as a
1
white amorphous solid of mp: 103-105 ºC. H NMR (500 MHz, MeOD) δ 7.49 (d, J =
8
.5 Hz, 2H, H ), 7.45 (d, J = 15.7 Hz, 1H, H ), 7.34 – 7.28 (m, 4H, Ho, m), 7.28 – 7.23
5 3
(
m, 1H, H ), 6.93 (d, J = 8.5 Hz, 2H, H ), 6.44 (d, J = 15.7 Hz, 1H, H ), 3.81 (s, 3H,
p 6 2
H ), 3.50 (s, 2H, H ), 3.32 – 3.30 (m, 2H, H, H ), 2.89 (dt, J = 12.2, 3.5 Hz, 2H, H2’eq),
7
1
γ
α
2
.01 (td, J = 11.8, 2.4 Hz, 2H, H2’ax), 1.73 (d, J = 12.3 Hz, 2H, H3’eq), 1.50 (q, J = 6.9
Hz, 2H, H ), 1.37 (dtq, J = 13.9, 6.9, 3.5 Hz, 1H, H ), 1.27 (qd, J = 12.2, 3.5 Hz, 2H,
β
4’
1
3
H3’ax). C NMR (126 MHz, MeOD) δ 168.95 (C ), 162.56 (C ), 141.34 (C ), 138.25
1
7
5
(
C ), 130.92 (C ), 130.37 (C ), 129.25 (C ), 128.87 (C ), 128.41 (C ), 119.30 (C ),
i o 5 m 4 p 2
1
3
15.33 (C ), 64.37 (C ), 55.82 (C ), 54.65 (C ), 38.10 (C ), 37.10 (C ), 34.45 (C ),
6 γ 71 2’ α β 4’
+
+
2.74 (C ). HRMS [ESI+] m/z =378.2304 [M] , Calcd for [C H N O ] 378.2307.
3
’
24 30
2
2
HPLC purity 100%.
4
.2.4. (E)-N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(2,4-dimethoxyphenyl)acrylamide (4)
From 2,4-dimethoxy cinnamic acid (0.300 g, 1.44 mmol) and 2-(1-benzylpiperidin-
4
-yl)ethan-1-amine (0.370 g, 1.73 mmol) hybrid 4 was obtained (0.517 g, 88%) as a
1
beige solid of mp: 90-93 ºC. H NMR (300 MHz, MeOD) δ 7.74 (d, J = 15.8 Hz, 1H,
H ), 7.44 (d, J = 8.2 Hz, 1H, H ), 7.36 – 7.28 (m, 4H, Ho, m), 7.29 – 7.18 (m, 1H, Hp),
3
9
6
3
–
.62 – 6.48 (m, 3H, H , H , H ), 3.87 (s, 3H, H ), 3.82 (s, 3H, H ) 3.49 (s, 2H, H ),
2 6 8 51 71 γ
.37 – 3.26 (m, 2H, H ), 2.97 – 2.78 (m, 2H, H2’eq), 2.00 (t, J = 11.2 Hz, 2H, H2’ax), 1.80
α
1.62 (m, 2.5 Hz, 2H, H3’eq), 1.49 (q, J = 6.9 Hz, 2H, H ), 1.39 - 1.19 (m, 3H, H ,
β
4’
2
3

ACCEPTED MANUSCRIPT
13
H3’ax). C NMR (75 MHz, MeOD) δ 169.64 (C ), 163.96 (C ), 161.03 (C ), 138.27(C ),
1
7
5
i
1
36.98 (C ), 130.91 (C ), 130.83 (C ), 129.24 (C ), 128.39 (C ), 119.53 (C ), 117.83
3 o 9 m p 2
(
(
[
C ), 106.69 (C ), 99.23 (C ), 64.37 (C ), 56.01 (C ), 55.91(C ), 54.66 (C ), 38.08
4 6 8 γ 51 71 2’
+
C ), 37.13 (C ), 34.44 (C ), 32.74 (C ). HRMS [ESI+] m/z =408.2416 [M] , Calcd for
α
β
4’
3’
+
C H N O ] 408.2413. HPLC purity 99%.
2
5
32
2
3
4
.2.5.
(E)-N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (5)
From ferulic acid (0.100 g, 0.50 mmol) and 2-(1-benzylpiperidin-4-yl)ethan-1-
amine (0.135 g, 0.6 mmol) ferulic – NBP hybrid 5 was obtained (0.193 g, 95%), as a
1
bright yellow solid of mp: 83.1-84.5 ºC. H NMR (500 MHz, MeOD) δ 7.43 (d, J = 15.7
Hz, 1H, H ), 7.35 – 7.31 (m, 4H, Ho, m), 7.30 – 7.24 (m, 1H, H ), 7.12 (d, J = 2.0 Hz,
3
p
1
1
=
H, H ), 7.03 (dd, J = 8.2, 2.0 Hz, 1H, H ), 6.80 (d, J = 8.2 Hz, 1H, H ), 6.42 (d, J =
5 9 8
5.7 Hz, 1H, H ), 3.89 (s, 3H, H ), 3.54 (s, 2H, H ), 3.35 – 3.33 (m, 2H, H ), 2.92 (dt, J
2
61
γ
α
11.9, 3.5 Hz, 2H, H2’eq), 2.05 (td, J = 11.9, 2.5 Hz, 2H, H2’ax), 1.75 (dt, J = 13.0, 2.5
Hz, 2H, H3’eq), 1.51 (q, J = 7.0 Hz, 2H, H ), 1.39 (dtt, J = 13.5, 6.6, 3.5 Hz, 1H, H ),
β
4’
1
3
1
1
.30 (td, J = 12.5, 3.5 Hz, 2H, H3’ax). C NMR (126 MHz, MeOD) δ 169.11 (C ),
1
49.90 (C ), 149.30 (C ), 141.97 (C ), 138.01 (C ), 130.97 (C ), 129.29 (C ), 128.51
7
6
3
i
o
m
(
C ), 128.23 (C ), 123.16 (C ), 118.70 (C ), 116.49 (C ), 111.52 (C ), 64.29 (C ), 56.36
p 4 9 2 8 5 γ
(
C ), 54.63 (C ), 38.06 (C ), 37.10 (C ), 34.39 (C ), 32.67 (C ). HRMS [ESI+] m/z
6
1
2’
α
β
4’
3’
+
+
=
394.2260 [M] , Calcd for [C H N O ] 394.2256. HPLC purity 99%.
24
30
2
3
4
.2.6.
(E)-N-(4-((Benzyl(methyl)amino)methyl)phenyl)-3-(2,4-
dimethoxyphenyl)acrylamide (10)
2
4

ACCEPTED MANUSCRIPT
Reaction of 2,4-dimethoxy cinnamic acid (0.150 g, 0.72 mmol) and 4-
((benzyl(methyl)amino)methyl)aniline (0.195 g, 0.86 mmol) yielded hybrid 10 (0.180 g,
1
6
7
7
3
0%) as a white amorphous solid of mp: 105-107 ⁰C. H NMR (300 MHz, MeOD) δ
.90 (d, J = 15.8 Hz, 1H, H ), 7.65 (d, J = 8.2 Hz, 2H, H ), 7.51 (d, J = 8.7 Hz, 1H, H ),
3
2’
9
.35 – 7.23 (m, 7H, Ph, H ), 6.77 (d, J = 15.7 Hz, 1H, H ), 6.62 – 6.49 (m, 2H, H ),
3
’
2
8,6
.91 (s, 3H, H ), 3.85 (s, 3H, H ), 3.52 (s, 2H, H ), 3.50 (s, 2H, H ), 2.18 (s, 3H, H ).
5
1
71
β
α
γ
1
3
C NMR (75 MHz, MeOD) δ 167.83 (C ), 164.21 (C ), 161.26 (C ), 139.69 (C ),
1
7
5
i
1
39.33 (C ), 138.31 (C ), 135.31 (C ), 131.19 (C ), 130.84 (C ), 130.40 (C ), 129.30
1’ 3 4’ 9 3’ o
(
(
[
C ), 128.25 (C ), 121.05 (C ), 119.82 (C ), 117.83 (C ), 106.81 (C ), 99.27 (C ), 62.61
m p 2’ 2 4 6 8
C ), 62.21 (C ), 56.05 (C ), 55.93 (C ), 42.31 (C ). HRMS [ESI+] m/z = 416.2099
β
α
51
71
γ
+
+
M] , Calcd for [C H N O ] 416.2100. HPLC purity 100%.
26
28
2
3
4
.2.7.
(E)-N-(4-((Benzyl(methyl)amino)methyl)phenyl)-3-(3,4-
dimethoxyphenyl)acrylamide (11)
,4-Dimethoxy cinnamic
(benzyl(methyl)amino)methyl)aniline (0.065 g, 0.28 mmol) were reacted as described
3
acid
(0.050
g,
0.24
mmol)
and
4-
(
in general procedure to obtain 11 (0.060 g, 60%) as a white amorphous solid of mp:
1
1
07-110 ºC. H NMR (300 MHz, MeOD) δ 7.68 – 7.57 (m, 3H, H ), 7.38 – 7.24 (m,
3
,2’
7
H, Ph, H ), 7.22 – 7.13 (m, 2H, H ), 6.98 (d, J = 8.0 Hz, 1H, H ), 6.67 (d, J = 15.5
3
’
5, 9
8
Hz, 1H, H ), 3.89 (s, 3H, H ), 3.87 (s, 3H, H ), 3.52 (s, 2H, H ), 3.50 (s, 2H, H ), 2.17
2
61
71
α
β
1
3
(
s, 3H, H ). C NMR (75 MHz, MeOD) δ 166.97 (C ), 152.44 (C ), 150.73 (C ), 142.78
γ 1 7 6
(
C ), 139.66 (C ), 139.18 (C ), 135.47 (C ), 130.88 (C ), 130.40 (C ), 129.36 (C ),
3 i 1’ 4’ 3’ o 4
1
29.31 (C ), 128.26 (C ), 123.43 (C ), 121.03 (C ), 119.98 (C ), 112.74 (C ), 111.43
m p 9 2’ 2 8
2
5

ACCEPTED MANUSCRIPT
(
C ), 62.62 (C ), 62.20 (C ), 56.44 (C ), 56.42 (C ), 42.31 (C ). HRMS [ESI+] m/z
5
α
β
61
71
γ
+
+
=
416.2112 [M] , Calcd for [C H N O ] 416.2100. HPLC purity 100%.
26 28 2 3
4
.2.8.
methoxyphenyl)acrylamide (12)
From ferulic acid
(E)-N-(3-((Benzyl(methyl)amino)methyl)phenyl)-3-(4-hydroxy-3-
(0.100
g,
0.50
mmol)
and
3-
((benzyl(methyl)amino)methyl)aniline (0.14 g, 0.60 mmol) ferulic – DBMA hybrid 12
1
was obtained (0.184 g, 89%), as a bright yellow solid of mp: 72 – 74 ºC. H NMR (400
MHz, MeOD) δ 7.64 (s, 1H, H ), 7.56 (s, 1H, H ), 7.56 (d, J = 15.7 Hz, 1H, H ), 7.36 –
2
’
6’
3
7
.21 (m, 6H, Ph, H ), 7.15 (d, J = 2.0 Hz, 1H, H ), 7.10 – 7.05 (m, 2H, H9, 4’), 6.80 (d, J
5’ 5
=
8.1 Hz, 1H, H ), 6.60 (d, J = 15.6 Hz, 1H, H ), 3.88 (s, 3H, H ), 3.51 (s, 2H, H ),
8 2 61 β
1
3
3
.49 (s, 2H, H ), 2.16 (s, 3H, H ). C NMR (101 MHz, MeOD) δ 167.27 (C ), 150.16
α γ 1
(
C ), 149.33 (C ), 143.25 (C ), 140.66 (C ), 140.18 (C ), 139.70 (C ), 130.40 (C ),
7 4 3 3’ i 1’ o
1
29.78 (C ), 129.32 (C ), 128.27 (C ), 128.18 (C ), 126.08 (C ), 123.42 (C ), 121.97
p m 5’ 4 4’ 9
(
C ), 120.06 (C ), 119.04 (C ), 116.54 (C ), 111.65 (C ), 62.74 (C ), 62.62 (C ), 56.36
2’ 6’ 2 8 5 β α
+
+
(
C ), 42.41 (C ). HRMS [ESI+] m/z =402.1934 [M] , Calcd for [C H N O ]
61 γ 25 26 2 3
4
02.1943. HPLC purity 98%.
4
.2.9.
(E)-N-(4-((Benzyl(methyl)amino)methyl)phenyl)-3-(4-hydroxy-3-
methoxyphenyl)acrylamide (13)
From ferulic acid
(0.100
g,
0.50
mmol)
and
4-
((benzyl(methyl)amino)methyl)aniline (0.140 g, 0.60 mmol), ferulic – DBMA hybrid 13
1
was obtained (0.177 g, 86%), as a bright yellow solid of mp: 80 – 83 ºC. H NMR (500
MHz, MeOD) δ 7.63 (d, J = 8.5 Hz, 2H, H ), 7.57 (d, J = 15.6 Hz, 1H, H ), 7.36 – 7.31
2
’
3’
2
6

ACCEPTED MANUSCRIPT
(
m, 6H, Ph, H ), 7.29 – 7.23 (m, 1H, H ), 7.17 (d, J = 2.1 Hz, 1H, H ), 7.08 (dd, J = 8.2,
3
’
p
5
2
.2 Hz, 1H, H ), 6.82 (d, J = 8.2 Hz, 1H, H ), 6.61 (d, J = 15.6 Hz, 1H, H ), 3.90 (s, 3H,
9 8 2
13
H ), 3.56 (s, 2H, H ), 3.55 (s, 2H, H ), 2.20 (s, 3H, H ). C NMR (126 MHz, MeOD) δ
6
1
β
α
γ
1
67.23 (C ), 150.16 (C ), 149.33 (C ), 143.26 (C ), 139.44 (C ), 136.29 (C ), 134.77
1 6 7 3 i 1’
(
C ), 131.03 (C ), 130.54 (C ), 129.40 (C ), 128.48 (C ), 128.19 (C ), 123.43 (C ),
4’ 3’ o m 4 p 9
1
4
21.05 (C ), 119.00 (C ), 116.55 (C ), 111.67 (C ), 62.50 (C ), 62.09 (C ), 56.38 (C ),
2’ 2 8 5 α β 61
+
+
2.12 (C ). HRMS [ESI+] m/z =402.1902 [M] , Calcd for [C H N O ] 402.1943.
γ
25 26
2
3
HPLC purity 97%.
4
.2.10. (E)-N-(4-((Benzyl(methyl)amino)methyl)phenyl)-3-(3-methoxyphenyl)acrylamide
(14)
(E)-3-(3-Methoxyphenyl)acrylic acid (0.150 g, 1.00 mmol) and 4-
((benzyl(methyl)amino)methyl)aniline (0.220 g, 1.00 mmol), were reacted as described
in the general procedure b to obtain 14 (0.255 g, 79%) as a white amorphous solid of
1
mp: 90-93 ºC. H NMR (300 MHz, MeOD) δ 7.68 – 7.60 (m, 3H, H3, 2’), 7.38 – 7.25 (m,
8
H, Ph, H8, 3’), 7.21 – 7.12 (m, 2H, H ), 6.97 (dd, J = 8.1, 2.6 Hz, 1H, H ), 6.79 (d, J =
5, 9 7
1
5.7 Hz, 1H, H ), 3.84 (s, 3H, H ), 3.52 (s, 2H, H ), 3.50 (s, 2H, H ), 2.17 (s, 3H, H ).
2
61
β
α
γ
1
3
C NMR (75 MHz, MeOD) δ 166.55 (C ), 161.56 (C ), 142.70 (C ), 139.70 (C ),
1
6
3
i
1
39.07 (C ), 137.60 (C ), 135.67 (C ), 130.99 (C ), 130.87 (C ), 130.39 (C ), 129.30
1’ 4 4’ 8 3’ o
(
C ), 128.25 (C ), 122.55 (C ), 121.48 (C ), 121.08 (C ), 116.72 (C ), 113.98 (C ),
m p 2 9 2’ 7 5
+
6
2.64 (C ), 62.20 (C ), 55.75 (C ), 42.32 (C ). HRMS [ESI+] m/z =386.1998 [M] ,
β α 61 γ
+
Calcd for [C H N O ] 386.1994. HPLC purity 99%.
2
5
26
2
2
2
7

ACCEPTED MANUSCRIPT
4
.2.11. (E)-N-(4-((Benzyl(methyl)amino)methyl)phenyl)-3-(4-methoxyphenyl)acrylamide
(15)
4
-Methoxy
cinnamic
acid
(0.150
g,
0.84
mmol)
and
4-
((benzyl(methyl)amino)methyl)aniline (0.220 g, 1.00 mmol) were reacted as described
in the general procedure b to obtain hybrid 15 (0.230 g, 70%) as a white amorphous
1
solid of mp: 131-134 ºC. H NMR (300 MHz, MeOD) δ 7.70 – 7.60 (m, 3H, H ), 7.57
3
,2’
(
d, J = 8.6 Hz, 2H, H ), 7.39 – 7.27 (m, 7H, Ph, H ), 6.99 (d, J = 8.6 Hz, 2H, H ), 6.67
5 3’ 6
(
d, J = 15.6 Hz, 1H, H ), 3.86 (s, 3H, H ), 3.54 (s, 2H, H ), 3.52 (s, 2H, H ), 2.19 (s,
2 71 β α
1
3
3
1
1
4
H, H ). C NMR (75 MHz, MeOD) δ 167.09 (C ), 162.80(C ), 142.58(C ), 139.70(C ),
γ 1 7 3 i
39.20(C ), 135.51(C ), 130.87(C ), 130.59(C ), 130.39(C ), 129.31(C ), 128.85(C ),
1
’
4’
3’
o
5
m
4
28.26(C ), 121.08(C ), 119.65(C ), 115.42(C ), 62.64(C ), 62.21(C ), 55.85(C ),
p
2’
2
6
β
α
71
+
+
2.32(C ). HRMS [ESI+] m/z =386.2000 [M] , Calcd for [C H N O ] 386.1994.
γ
25 26
2
2
HPLC purity 99%.
4
.3. General procedure for deprotection of methoxy substituted hybrids.
To the corresponding methoxy derivative (0.1 mmol) dissolved in 3 mL of anhydrous
THF, was added slowly under magnetic stirring, 1 equivalent of BBr per each
3
heteroatom present in the molecule; air was displaced by N and the mixture was
2
allowed to react overnight at room temperature. Reaction was quenched with methanol
(MeOH) (dropwise until end of effervescence) and the solvent evaporated under
reduced pressure to eliminate the remaining BBr , this process was repeated several
3
times depending on the quantity of BBr used, until no fumes were observed when
3
adding MeOH. When necessary, purification was carried out by column
chromatography using a gradient of EtOAc/MeOH 0→10% as eluent
2
8