Design of novel monoamine oxidase-B inhibitors based on piperine scaffold: Structure-activity-toxicity, drug-likeness and efflux transport studies

Article abstract of DOI:10.1016/j.ejmech.2019.111770

Piperine has been associated with neuroprotective effects and monoamine oxidase (MAO) inhibition, thus being an attractive scaffold to develop new antiparkinsonian agents. Accordingly, we prepared a small library of piperine derivatives and screened the inhibitory activities towards human MAO isoforms (hMAO-A and hMAO-B). Structure-activity relationship (SAR) studies pointed out that the combination of α-cyano and benzyl ester groups increased both potency and selectivity towards hMAO-B. Kinetic experiments with compounds 7, 10 and 15 indicated a competitive hMAO-B inhibition mechanism. Compounds 15 and 16, at 10 μM, caused a small but significant decrease in P-gp efflux activity in Caco-2 cells. Compound 15 stands out as the most potent piperine-based hMAO-B inhibitor (IC₅₀ = 47.4 nM), displaying favourable drug-like properties and a broad safety window. Compound 15 is thus a suitable candidate for lead optimization and the development of multitarget-directed ligands.

Full text of DOI:10.1016/j.ejmech.2019.111770

Journal Pre-proof
Design of novel monoamine oxidase-B inhibitors based on piperine scaffold:
Structure-activity-toxicity, drug-likeness and efflux transport studies
Daniel Chavarria, Carlos Fernandes, Vera Silva, Cátia Silva, Eva Gil-Martins, Pedro
Soares, Tiago Silva, Renata Silva, Fernando Remião, Paulo J. Oliveira, Fernanda
Borges
PII:
S0223-5234(19)30922-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111770
EJMECH 111770
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 August 2019
Revised Date: 6 October 2019
Accepted Date: 6 October 2019
Please cite this article as: D. Chavarria, C. Fernandes, V. Silva, Cá. Silva, E. Gil-Martins, P. Soares, T.
Silva, R. Silva, F. Remião, P.J. Oliveira, F. Borges, Design of novel monoamine oxidase-B inhibitors
based on piperine scaffold: Structure-activity-toxicity, drug-likeness and efflux transport studies,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111770.
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Design of novel monoamine oxidase-B inhibitors based on piperine scaffold:
structure-activity-toxicity, drug-likeness and efflux transport studies
Daniel Chavarriaǂ1,2, Carlos Fernandesǂ1, Vera Silva1,3, Cátia Silva1, Eva Gil-Martins1,3,
Pedro Soares1, Tiago Silva1,2, Renata Silva3, Fernando Remião3, Paulo J. Oliveira2,
Fernanda Borges1*
1
CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University
of Porto, Porto, Portugal
2 CNC - Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech,
Biocant Park, Cantanhede, Portugal.
3 UCIBIO-REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences,
Faculty of Pharmacy, University of Porto, Porto, Portugal
ǂ These authors contributed equally.
* Corresponding Authors:
Fernanda Borges, CIQUP/Department of Chemistry and Biochemistry, Faculty of
Sciences, University of Porto, Porto 4169-007, Portugal. E-mail: fborges@fc.up.pt

Graphical abstract
Compound 15
Competitive hMAO-B inhibition (IC50 = 47.4 nM)
Safe cytotoxic profile in SH-SY5Y and Caco-2 cells
Highlights
•
•
•
•
•
A series of piperine derivatives was synthesized, eight of which acted as hMAO-B inhibitors.
α-Cyano and benzyl ester substituents increased both potency and selectivity towards hMAO-B.
Compounds 15 and 16 displayed slight P-gp inhibitory activity in Caco-2 cells.
Compound 15 is highlighted as a potent, selective and competitive hMAO-B inhibitor.
Compound 15 displayed favorable drug-like properties and a safe cytotoxic profile.

Abstract
Piperine has been associated with neuroprotective effects and monoamine oxidase (MAO)
inhibition, thus being an attractive scaffold to develop new antiparkinsonian agents. Accordingly, we
prepared a small library of piperine derivatives and screened the inhibitory activities towards human
MAO isoforms (hMAO-A and hMAO-B). Structure-activity relationship (SAR) studies pointed out that
the combination of α-cyano and benzyl ester groups increased both potency and selectivity towards
hMAO-B. Kinetic experiments with compounds 7, 10 and 15 indicated a competitive hMAO-B inhibition
mechanism. Compounds 15 and 16, at 10 µM, caused a small but significant decrease in P-gp efflux
activity in Caco-2 cells. Compound 15 stands out as the most potent piperine-based hMAO-B inhibitor
(IC50 = 47.4 nM), displaying favorable drug-like properties and a broad safety window. Compound 15 is
thus a suitable candidate for lead optimization and the development of multitarget-directed ligands.
Keywords: Parkinson’s disease, Piperine, Monoamine oxidase, Cholinesterase, Structure-activity
relationship.
Abbreviations: BBB: Blood-brain barrier; CHI: Chromatographic Hydrophobic Index; CNS: Central
nervous system; DMAP: 4-(dimethylamino)pyridine; DCF: 2’,7’-dichlorofluorescein; DCFH-DA: 2’,7’-
dichlorofluorescein diacetate; EDC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; HBA: hydrogen
bond atom acceptor; HBD: hydrogen bond atom donor; Km: Michaelis constant; Ki: inhibition constant;
MAO: monoamine oxidase; MW: molecular weight; NR: eutral Red; PD: Parkinson’s disease; P-gp: P-
glycoprotein; PyBOP: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; RB:
rotatable bonds; RHO 123: Rhodamine 123; RS: reactive species; SAR: Structure-Activity Relationship;
Vmax: maximum velocity.

1.
Introduction
Parkinson’s disease (PD) is the second most common neurodegenerative disorder after
Alzheimer’s disease [1]. PD is characterized by the progressive and selective loss of dopaminergic
neurons in the substantia nigra and by the presence of Lewy bodies [2], which decreases interstitial
dopamine levels and leads to motor dysfunction [3, 4]. PD is also associated with non-motor symptoms
such as cognitive decline, depression and sleep disorders, with some of them preceding the development
of the classical motor symptoms [5, 6]. Current drug treatment of PD almost exclusively focuses on
restoring the dopaminergic function and reducing the severity of motor handicap [7]. The main
pharmacological approaches include dopamine replacement therapy with L-DOPA, inhibition of
dopamine metabolism and reuptake, or the use of direct agonists of post-synaptic dopamine receptors [4,
7].
Monoamine oxidases (MAOs, EC 1.4.3.4) are flavin-dependent enzymes bound to the outer
mitochondrial membrane [8]. Two MAO isoforms (MAO-A and MAO-B) may participate in the
regulation of dopamine levels in neuronal tissue [9]. However, MAO-B is the main MAO isozyme
involved in dopamine metabolism in parkinsonian brains, because MAO-B activity and expression are
increased in most brain areas while MAO-A activity remains unchanged [10]. Selective irreversible
MAO-B inhibitors, like selegiline and rasagiline, are used in PD monotherapy or in combination with
dopamine precursor L-DOPA to reduce dopamine catabolism and thereby increase its half-life [11, 12].
More recently, safinamide, a reversible MAO-B inhibitor, was approved in the European Union to treat
patients with mid- to late-stage PD [13].
Considering that the prevalence of PD is drastically increasing and no disease-modifying
treatment emerged over the last few decades [14], great interest remains in the development of new
antiparkinsonian drugs. Piperine (1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine),
compound 1, Figure 1), the pungent component of several pepper species [15], presents several
biological properties with potential therapeutic interest for PD. Piperine displayed antioxidant, anti-
inflammatory and antiapoptotic activities in cultured neuronal cells [16] and in PD animal models [17-
19]. Moreover, piperine was reported as a competitive, reversible and non-selective rMAO inhibitor [20].
Within this framework, we explored the piperine scaffold in a preliminary study, wherein the unequivocal
structural elucidation of five piperine bioisosteres along with their hMAO inhibitory activities were
assessed [21]. The data acquired put forward the significance of piperidinyl amide group to modulate the
interaction of piperine derivatives with hMAOs [21].

OH
X
Piperine (1)
Figure 1. Rational design of the library based on piperine (compound 1) scaffold.
•
The promising results encouraged a second project aiming to identify the most favourable
chemical modifications on the piperine scaffold required for the development of novel ligands
with potential application in PD. Herein, we report the synthesis of a new library of piperine
derivatives (Figure 1) and the establishment of new structure-activity-relationships (SAR).
Following hMAO-B enzymatic and mechanistic studies, we investigated the cytotoxicity of the
best inhibitors in two different human-derived cell lines (neuroblastoma cells (SH-SY5Y) and
colon adenocarcinoma cells (Caco-2)). The compounds’ ability to induce intracellular oxidative
stress in SH-SY5Y cells and to modulate the P-glycoprotein (P-gp) activity by piperine
derivatives in Caco-2 cells was also evaluated. Finally, we studied the drug-like properties of the
best compounds. The compound´s lipophiliciy was evaluated by chromatographic
hydrophobicity index (CHI).
2.
Results and discussion
2.1.
Chemistry
Piperine-based compounds 2-16 were synthesized following the strategies represented in
Schemes 1 and 2. Several modifications were performed at the diene tether and at the piperidinyl amide
group to attain structural diversity (Figure 1). Piperidinyl moiety was removed to obtain free carboxylic
acids or replaced by lipophilic groups (phenyl, benzyl or phenethyl) linked through ester or amide bonds.
In addition, an α-cyano group was incorporated in the diene tether.
The hydrolysis of piperine in alkaline medium (Scheme 1, step a) yielded piperic acid
(compound 2, Scheme 1), which in turn reacted with the appropriate amines or alcohols to afford the
corresponding amides 3-5 and esters 6-8, respectively. Accordingly, amides 3-5 were synthesized via
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP)-mediated amidation
reaction, under alkaline conditions, between piperic acid and aniline, benzylamine or (2-
bromoethyl)benzene (Scheme 1, step b). Piperic acid ester 6 was obtained by a Steglich esterification
reaction,
assisted
by
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
(EDC)
and
4-

(dimethylamino)pyridine (DMAP), between piperic acid 2 and phenol (Scheme 1, step c). Esters 7 and 8
were synthesized from piperic acid 2 by bimolecular nucleophilic substitution with benzyl bromide and
(2-bromoethyl)benzene, respectively (Scheme 1, step d).
Scheme 1. Synthetic strategy pursued to obtain piperine derivatives 2-8. (a) Methanolic solution of NaOH 2 M,
reflux, 24 h; (b) 1. DMF, DIPEA, PyBOP, CH2Cl2, 0 ºC, 30 min; 2. Amine (aniline, benzylamine or (2-
bromoethyl)benzene), rt, overnight; (c) 1. EDC.HCl, DMAP, CH2Cl2, rt, 30 min; 2. phenol, rt, overnight; (d) 1.
K2CO3, DMF, rt, 30 min; 2. benzylbromide or (2-bromoethyl)benzene, 80 ºC, 8 h.
The synthetic route used to obtain α-cyanopiperic acid (compound 9) and derivatives 10-16 is
shown in Scheme 2. The cinnamic derivative (compound 18) used as starting material was prepared by a
Knoevenagel-Doebner condensation between piperonal (compound 17) and mono-ethyl malonate in
pyridine and catalytic amounts of piperidine (Scheme 2, step a). The reduction of compound 18 with
lithium aluminum hydride (LiAlH4) afford the 3,4-methylenedioxycinnamyl alcohol (compound 19)
(Scheme 2, step b) that was subsequently oxidized to the 3,4-methylenedioxycinnamic aldehyde
(compound 20) with manganese dioxide (MnO2) (Scheme 2, step c). α-Cyanopiperic acid (compound 9)
was obtained by a Knoevenagel condensation between compound 20 and cyanoacetic acid (Scheme 2,
step d). Subsequent amidation (Scheme 2, step e) or Steglich esterification (Scheme 2, step f) reactions
afforded compounds 10-16.
Scheme 2. Synthetic strategy pursued to obtain α-CN derivatives 9-16. (a) mono-ethyl malonate, pyridine, piperidine,
70 ºC, 9 h; (b) 1. Argon atmosphere, dry THF, -78 ºC, 30 min; 2. LiAlH4, -78 ºC to rt, 5 h; (c) MnO2, CH2Cl2, rt,
overnight; (d) cyanoacetic acid, pyridine, piperidine, 60 ºC, 30 min; (e) 1. DMF, DIPEA, PyBOP, CH2Cl2, 0 ºC, 30
min; 2. Amine (aniline, benzylamine or (2-bromoethyl)benzene), rt, overnight; (f) 1. EDC.HCl, DMAP, CH2Cl2, rt,
30 min; 2. Phenol, benzyl alcohol or 2-phenylethanol, rt, overnight.

2.2.
Monoamine oxidase inhibition studies
The potential effects of piperine and its analogues (compounds 1-16) on hMAOs activity were
studied by spectrophotometry, using kynuramine as substrate and recombinant hMAO-A and -B isoforms
[21, 22] . The hMAO-A and hMAO-B inhibitory potency (IC50) and selectivity (SI) data obtained with
the compounds under study and standard inhibitors (clorgyline for hMAO-A and (R)-(−)-deprenyl,
rasagiline, safinamide for hMAO-B) are reported in Table 1.
Table 1. hMAO inhibitory activities of compounds 1-16 and standard inhibitors.
IC50 (µM)
R
Compound
SIb
hMAO-A
hMAO-B
___a
___a
__
2e
1e
___a
___a
___a
___a
1.05±0.08
___a
10d
3e
4e
5
__
__
__
___a
___a
___c
0.156±0.008
0.167±0.011
> 64d
25
6e
7e
4.24±0.56
___a
___a
___a
0.448±0.043
___a
> 22d
__
8
9
0.143±0.080
> 70d
10
___a
___a
___a
___a
__
> 21d
__
11
12
13
0.471±0.008
___a
___a
___a
___a
0.215±0.038
0.0474±0.0042
0.0992±0.0082
>47d
14
15
16
> 211d
> 101d
(R)-(−)-Deprenyle
Rasagiline
__
__
__
__
20.1±1.9
3.65±0.31
0.0386±0.0043
147.3±249
522
24
Safinamide
___a
0.0231±0.0026
2.21±0.26
> 433d
Clorgylinee
0.00274±0.00047
0.00124
a Inactive at 10 ꢀM;
b SI: hMAO-B selectivity index = IC50(hMAO-A)/IC50(hMAO-B).
c Not soluble in phosphate buffer at concentrations higher than 10 ꢀM.

d Values obtained under the assumption that the corresponding IC50 value against hMAO-A or hMAO-B is the highest
concentration tested (10 ꢀM).
e Data from Chavarria et al. [21].
2.2.1. Structure-activity relationship studies
The results obtained show that the compounds that presented hMAOs inhibitory activity were
more selective towards the isoform B. As previously reported, piperine displayed moderate and selective
inhibition towards hMAO-B (compound 1, IC50 = 1.05 µM, SI = 10) in our experimental conditions [21].
Interestingly, the introduction of an α-CN group at the diene tether improved the hMAO-B inhibitory
activity (compound 10: IC50 = 143 nM).
Compounds containing a piperidinyl amide moiety were active towards hMAO-B (compounds 1
and 10). The replacement of the piperidinyl group by a carboxylic acid (compounds 2 and 9) completely
abolished hMAO inhibition. Primary amides were also unable to inhibit both hMAO isoforms
(compounds 3-5, 11 and 13) or have modest inhibitory activity towards hMAO-B (compounds 12). On
the other hand, except for compound 14, esters 6-8, 15 and 16 were more potent and selective hMAO-B
inhibitors than piperidinyl derivatives 1 and 14.
Concerning ester and primary amide substituents, benzyl derivatives displayed the most
promising results. Piperic acid benzyl ester presented an IC50 value similar to that of the phenyl derivative
and lower than the phenethyl analogue (compound 7: IC50 = 167 nM; compound 6: IC50 = 156 nM;
compound 8: IC50 = 448 nM). All α-cyanobenzyl derivatives acted as hMAO-B inhibitors. Compound 12
was the only primary amide of α-cyanopiperic acid acting as hMAO-B inhibitor (compound 12: IC50
=
471 nM). Benzyl ester 15 was more active than phenethyl ester 16, which in turn was more potent than
phenyl ester 14 (compound 15: IC50 = 47.4 nM; compound 16: IC50 = 99.2 nM; compound 14: IC50 = 215
nM). Therefore, we concluded that the combination of the benzyl group with a cyano group enhanced the
hMAO-B inhibitory properties.
In fact, with the exception of compound 14, compounds containing an α-CN presented lower
IC50 values towards hMAO-B than their unsubstituted counterparts, without showing significant hMAO-
A inhibitory activity at 10 µM. This chemical modification also influenced the selectivity of the
compounds. The SI of compounds 10, 15 and 16 were 5 to 8.4-fold higher than that of piperine and
compounds 7 and 8 (compound 1 [SI = 10] vs compound 10 [SI = 70]; compound 7 [SI = 25] vs
compound 15 [SI = 211]; compound 8 [SI = 22] vs compound 16 [SI = 101]. Compound 15 showed the
lowest IC50 value towards hMAO-B and the highest hMAO-B SI (compound 15: IC50 = 47.4 nM; SI =
211).
2.2.2.
Evaluation of human monoamine oxidase-B inhibition mechanism
To further investigate the hMAO-B inhibition mechanism of the best hMAO-B inhibitor
(compound 15), kinetic experiments were performed. The study was extended to compounds 7 and 10 to
evaluate the influence of the α-CN or piperidinyl amide groups in the enzyme inhibition mechanisms. We
measured the initial rates of the hMAO-catalyzed deamination of six different concentrations of
kynuramine in the absence or presence of three different concentrations of the inhibitors. The results
obtained are depicted in Figure 2. Kinetic parameters of Michaelis-Menten reaction (Michaelis constant,

Km, and maximum velocity, Vmax) were determined by the graphical analysis of the double reciprocal
Lineweaver-Burk plots. The presence of increasing concentrations of compounds 7, 10 and 15 resulted in
an increase of Km values and a maintenance of Vmax. The Lineweaver-Burk plots (Figures 2A-C) of these
compounds remained linear and intersected at the y-axis. These observations indicate that the piperine
derivatives 7, 10 and 15 act as competitive inhibitors.
Figure 2. Kinetic studies on the mechanism of hMAO-B inhibition by compounds 7 (A and D), 10 (B and E) and 15
(C and F). (A-C) Double reciprocal plots of the initial velocity of hMAO-B at increasing substrate concentrations
(10-100 µM) in the absence or the presence of the inhibitor. (D-F) Secondary plots of the slope of each Lineweaver-
Burk (Km/Vmax) versus the inhibitor concentration.
The data obtained from the Lineweaver-Burk were then replotted as the slope against the
inhibitor concentrations to obtain the inhibition constants (Ki) of the mentioned compounds. The x-axis
intercept is - Ki (Figures 2D-F). Compounds 7, 10 and 15 displayed Ki values of 75.0 nM, 20.6 nM and
44.1 nM, respectively. The estimated Ki values were well correlated with the inhibition mechanism
established from the kinetic experiments.

2.3.
Cellular studies
2.3.1. Evaluation of cytotoxicity profile
To study the cytotoxicity profile of the most promising piperine-based compounds (hMAO-B
IC50 < 200 nM), we performed cellular cytotoxicity assays in differentiated neuroblastoma (SH-SY5Y)
cells and in colon adenocarcinoma (Caco-2) cells. SH-SY5Y and Caco-2 cells are commonly used to
evaluate the safety of drug candidates [23, 24]. In this study, SH-SY5Y cells were treated with a
differentiation-inducing agent to obtain cells morphologically similar to mature dopaminergic neurons
[25]. Considering that PD mainly affects dopaminergic neurons [26], differentiated SH-SY5Y cells are a
suitable in vitro model to study the neurotoxicity of drug candidates with potential application in PD [27].
Differentiated SH-SY5Y cells and Caco-2 cells were incubated with two different concentrations
(10 and 50 µM) of compounds 6, 7, 10, 15 and 16 for 24 h. Piperine (compound 1) was also tested to
study the effect of the structural modification of piperine scaffold on the cytotoxicity profile of
compounds. Cellular cytotoxicity was evaluated using the resazurin reduction assay, which estimates the
metabolic activity of viable cells [28], and the neutral red (NR) uptake assay, which relies on the
lysosomal accumulation of the dye NR in living cells [29]. The results are presented in Figure 3 and
expressed as mean (% of control) ± SEM of three independent experiments (n = 3).
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
**
**
**
****
****
****
(
0 10 50 0 10 50 0 10 50
M)
(
0 10 50 0 10 50 0 10 50
M)
0 10 50 0 10 50 0 10 50
0 10 50 0 10 50 0 10 50
1
6
7
10
15
16
1
6
7
10
15
16
120
100
80
60
40
20
0
120
100
80
60
40
20
0
***
***
(
M)
( M)
0 10 50 0 10 50 0 10 50
0 10 50 0 10 50 0 10 50
0 10 50 0 10 50 0 10 50
0 10 50 0 10 50 0 10 50
1
6
7
10
15
16
1
6
7
10
15
16
Figure 3. Cellular viability of differentiated SH-SY5Y cells (A-B) and Caco-2 cells (C-D) after incubation with
piperine (1) and compounds 6, 7, 10, 15 and 16 at two different concentrations (10 µM and 50 µM) for 24 h. Cellular
viability was evaluated by measuring metabolic (A and C) and lysosomal activities (B and D) using the resazurin
reduction assay and the neutral red uptake assay, respectively. Statistical comparisons were performed using the
parametric method of two-way ANOVA, followed by the Dunnett’s multiple comparisons test. In all cases, p values
lower than 0.05 were considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs untreated
cells).
The results obtained show that the treatment of differentiated SH-SY5Y cells with compounds 1,
7, 10, 15 and 16 did not markedly decrease the resazurin reduction or the NR uptake when compared to

control cells (% resazurin reduction and NR uptake were always > 85 %) (Figure 3A and 3B). However,
treatment with compound 6 at the highest tested concentration (50 µM) led to significant reductions in
both resorufin fluorescence (79.7 ± 2.1 %, p < 0.0001, Figure 3A) and in NR uptake (59.4 ± 6.5 %, p <
0.0001, Figure 3B). In addition, both cellular metabolic and lysosomal activities in Caco-2 cells
(Figures 3C and 3D, respectively) were not markedly affected by the treatment with any of the tested
piperine derivatives.
Overall, the acquired data suggest that piperine derivatives 7, 10, 15 and 16 present a safe
cytotoxicity profile in both human-derived neuroblastoma and adenocarcinoma cells (% resazurin
reduction and NR uptake were always > 85 %).
2.3.2. Evaluation of intracellular oxidative stress
The toxicity of numerous xenobiotics may be linked to the increased levels of oxidative stress,
which are largely related to the formation of reactive species (RS) [30]. Therefore, we evaluated the
intracellular oxidative stress levels in differentiated SH-SY5Y cells. After exposing the cells to piperine
(compound 1) and compounds 6, 7, 10, 15 and 16 at two different concentrations (10 and 50 µM) for 24
h, the intracellular RS levels were measured using the 2’,7’-dichlorofluorescein diacetate (DCFH-DA)
method. The results are expressed as 2’,7’-dichlorofluorescein (DCF) fluorescence (% of control) ± SEM
(n = 3) and are depicted in Figure 4.
*
Figure 4. Evaluation of intracellular oxidative stress levels in differentiated SH-SY5Y cells evaluated by the DCF
fluorescence assay, after treatment with piperine (compound 1) and compounds 6, 7, 10 and 15 and 16 at two
different concentrations (10 µM and 50 µM) for 24 h. Results are expressed as DCF fluorescence (% of control) ±
SEM (n = 3). Statistical comparisons were performed using the parametric method of two-way ANOVA, followed by
the Dunnett’s multiple comparisons test.
Among the tested piperine derivatives, only compound 10 led to a slight but significant increase
in DCF fluorescence (111.1 ± 3.5 %, p < 0.5) when compared to non-treated cells (Figure 4). Given that
compound 10 did not exhibit cytotoxicity at the tested concentrations (Figures 3A and 3B), the results
obtained suggest that the slightly increased RS levels did not inflict oxidative damage in SH-SY5Y cells.
2.3.3. Evaluation of P-glycoprotein (P-gp) transport activity
The delivery of therapeutic agents into the brain is strongly hampered by the blood-brain barrier
(BBB) [31]. In fact, more than 98 % of all small drugs are unable to cross the BBB [32]. An important

gatekeeper of the BBB is P-gp, an ATP-driven efflux pump localized in the luminal membrane of brain
capillary endothelial cells [33]. P-glycoprotein restricts the access of a large number of prescribed drugs
to the central nervous system (CNS) and contributes to the poor success rates of CNS drug candidates
[33].
Considering that P-gp is highly expressed in Caco-2 cells, this cellular model is widely used not
only to screen the P-gp-mediated transport of drugs [34], but also to evaluate the influence of new drug
candidates in P-gp expression and/or activity [35, 36]. In addition, Caco-2 cells are commonly used as a
surrogate BBB model to study drug permeation [37-39].
We evaluated the P-gp modulatory activity of piperine (compound 1) and compounds 7, 10, 15
and 16 in Caco-2 cells, using Rhodamine 123 (RHO 123) as a P-gp substrate and zosuquidar as a specific
third-generation P-gp inhibitor [40]. Increased P-gp activity enhances RHO 123 efflux, leading to a
decrease in the intracellular fluorescence intensity under normal conditions (FINA). Therefore, the RHO
123 accumulation ratio (FIIA/FINA) will be higher than the control experiments (see supplementary
information, Equation S3). On the other hand, lower P-gp activity is associated with higher FINA, as a
result of the decreased RHO 123 efflux and consequent increase of RHO 123 intracellular accumulation.
Thus, the FIIA/FINA ratio will be lower than the control experiments (see supplementary information,
Equation S3).
The results presented in Figure 5 show that compounds 10, 15 and 16, at 10 µM, significantly
affected the intracellular accumulation of RHO 123, resulting in significant differences in P-gp transport
activity. On the other hand, piperine (compound 1) and compound 7 did not significantly change P-gp-
mediated RHO 123 efflux.
Compound 10 significantly increased P-gp activity in a short-incubation period, resulting in a
decreased RHO123 accumulation and, consequently, in a higher FIIA/FINA ratio. Therefore, compound 10
demonstrated potential for P-gp activation. Given the short incubation period with both the P-gp substrate
and the test compounds, the effect observed with compound 10 did not reflect the potential contribution
of an increased protein expression. (Figure 5).
****
Figure 5. Evaluation of P-gp activity by fluorescence spectroscopy in Caco-2 cells exposed to compounds 1, 7, 10,
15 and 16 (10 ꢀM) only during the incubation period with the fluorescent substrate (RHO 123, 5 ꢀM). Results are
presented as mean ± SEM from at least three independent experiments, performed in triplicate. Statistical

comparisons were made using the parametric method of one-way ANOVA, followed by the Dunnett’s multiple
comparisons test (****p < 0.0001 vs. control cells).
Piperine derivatives 15 and 16, at 10 µM, significantly reduced RHO 123 efflux, resulting in a
decreased FIIA/FINA ratio that indicates mild P-gp inhibition. Together with the data obtained in hMAO
inhibition studies, these findings show that compounds 15 and 16 are potent hMAO-B inhibitors with
mild P-gp inhibitory activity.
Mild P-gp inhibition can be advantageous in combination therapy of PD. Since several
antiparkinsonian agents such as L-DOPA are P-gp substrates [41], mild P-gp inhibition may decrease the
drug efflux at the BBB, improve the drug delivery into the CNS and provide a longer symptomatic relief
[42].
2.4.
Evaluation of drug-like properties
We studied drug-like properties of piperine and the most promising hMAO-B inhibitors
(compounds 7, 15 and 16) to predict their ability to attain the CNS. Compound 10 was excluded because
it presented a positive modulatory effect on P-gp (Figure 5).
First, we determined the chromatographic hydrophobic index logD at pH 7.4 (CHI logD7.4), a
parameter used to assess the compounds’ hydrophobicity [43, 44]. Overall, the tested compounds
presented CHI logD7.4 values between 2.4 and 3.8 (Table 2). Compounds 7, 15 and 16 displayed higher
CHI values than piperine. These results indicate an increase in lipophilicity with the replacement of the
piperidinyl ring by benzyl or phenethyl ester groups.
Table 2. Retention times (tR) obtained by LC/UV at pH 7.4 and calculated CHI and CHI logD7.4 parameters of
compounds 1, 7, 15 and 16
CHIa
CHI LogD7.4
b
Compound
tR (min)
10.61
12.46
12.11
12.37
73.45
102.14
96.73
2.39
3.90
3.61
3.82
1
7
15
16
100.75
a CHI values were calculated using the equation obtained in the linear correlation (see supplementary information,
Figure S1).
b CHI logD7.4 were back-calculated using the equation CHI logD7.4 = (0.0525×CHI)-1.467.
Other physicochemical properties were also estimated. These included molecular weight (MW),
topological polar surface area (TPSA in Å), number of hydrogen acceptors (HBA), number of hydrogen
donors (HBD) and number of rotatable bonds (RB). The values obtained were in line with the reported
limits of CNS-active drugs (Table 3). Another parameter used to assess the ability of a molecule to cross
the BBB by passive diffusion is the logarithm of the ratio of the concentration of the compound in the
brain and in the blood (logBB). Compounds with logBB < −1 have low distribution into the brain and
thus are unlikely to act as CNS drugs [45]. Piperine and compounds 7, 15 and 16 showed estimated
logBB values above −1 suggesting that they may be able to cross the BBB and reach the CNS (Table 3).

Table 3. Predicted drug-like properties of piperine and compounds 7, 10, 15 and 16.
Compound
MWa
285.3
TPSAa
38.77
44.76
68.55
68.55
< 90[46]
HBAa
HBDa
RBa
logBBa
-0.185
0.059
3
0
4
1
308.3
4
0
6
7
333.3
5
5
0
0
6
7
-0.194
-0.175
≥ -1[45]
15
16
347.4
< 500[46]
CNS+ drugs
< 7[47]
< 3[47]
< 8[47]
a
Properties predicted using the Stardrop software. MW: molecular weight; TPSA: topological polar surface area;
HBA: number of H-bond acceptor atoms; HBD: number of H-bond donor atoms; RB: number of rotatable bonds;
logBB: logarithm of the ratio of the concentration of a drug in the brain and in the blood.
3.
Conclusion
We successfully obtained a small library of piperine derivatives and explored their inhibitory
activities towards hMAO-A and hMAO-B. Piperine and derivatives thereof displayed selective hMAO-B
inhibition. SAR studies indicated that the presence of an α-cyano group increase hMAO-B inhibitory
properties and that ester derivatives were considerably more active towards hMAO-B than amides and
carboxylic acids. Concerning the lipophilic group bound to the ester/amide function, benzyl derivatives
were the most potent hMAO-B inhibitors, followed by phenethyl and phenyl derivatives. Remarkably, the
combination of an α-cyano group with ester groups enhanced hMAO-B inhibition potency and selectivity,
leading to an up to 22-fold decrease of hMAO-B IC50 values in comparison to piperine (1). Compounds 6,
7, 10, 15 and 16 were the most potent hMAO-B inhibitors, with IC50 values within the nanomolar range.
Kinetic studies indicated that the piperine derivatives operated via a competitive inhibition mechanism. In
general, piperine and compounds 7, 10, 15 and 16 presented safe cytotoxicity profiles in human-derived
neuroblastoma and colon adenocarcinoma cells. Small decreases of FIIA/FINA ratios associated with
increased intracellular RHO 123 accumulation in Caco-2 cells were also observed for compounds 15 and
16 at 10 µM, indicating a slight P-gp inhibition.
Compound 15 stands out as a non-cytotoxic, potent and selective hMAO-B inhibitor (IC50 = 47.4
nM, SI = 211) with small P-gp inhibitory activity and suitable physicochemical properties for BBB
permeation. Therefore, compound 15 is a valid candidate for lead optimization. In this context, the
modification of the benzyl ester group or the benzodioxole ring can be looked as a valid strategy to obtain
new molecules with improved efficacy and potency. Considering that PD and other neurodegenerative
diseases result from the interplay between multiple pathological mechanisms, compound 15 can also be
fused or merged with other neuroprotective agents to obtain drug candidates with a polypharmacological
profile.
4.
Experimental section
4.1.
Synthesis of piperine derivatives
4.1.1. Synthesis of (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoic acid (2). Synthesis and
structural analysis were previously reported [21].

4.1.2. General procedure for the synthesis of amides. Piperic acid 2 or cyanopiperic acid 9 (1 mmol)
were dissolved in DMF and DIPEA (1.01 mmol). The mixture was cooled at 0 ºC on an ice bath, and a
solution of PyBOP (1.01 mmol) in dichloromethane was slowly added. The mixture was stirred for 30
min, and the appropriate amine (phenyl amine, benzyl amine or phenethyl amine) (1.01 mmol) was
added. The mixture was stirred overnight at room temperature. Purification conditions are described in
literature [21, 48]. In the cases that different conditions were used they were reported.
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-phenylpenta-2,4-dienamide (3). Synthesis and structural
analysis were previously reported [21].
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-benzylpenta-2,4-dienamide (4). Synthesis and structural
analysis were previously reported [21].
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-phenethylpenta-2,4-dienamide (5). ηꢁ=ꢁ82 %. 1H NMR
(CDCl3-d1): δ (ppm) = 2.87 (t, J = 6.9 Hz, 2H, NHCH2CH2Ph), 3.62 (m, 2H, NHCH2CH2Ph), 5.54 (t, J =
5.4 Hz, 1H, NH), 5.84 (d, J = 14.9 Hz, 1H, Hα), 5.97 (s, 2H, OCH2O), 6.65 (dd, J = 11.0 Hz, 15.2 Hz,
1H, Hγ), 6.76 (m, 2H, H5, Hδ), 6.88 (dd, J = 1.6 Hz, 8.1 Hz, 1H, H6), 6.90 (d, J = 1.6 Hz, 1H, H2), 7.23
(m, 3H, H3’, H4’, H5’), 7.33 (m, 3H, H2’, H6’, Hβ). 13C NMR (CDCl3-d1): δ (ppm) = 37.7
(NHCH2CH2Ph), 40.7 (NHCH2CH2Ph), 101.3 (OCH2O), 105.7 (C2), 108.5 (C5), 122.6 (Cα), 123.0 (C6),
124.6 (Cγ), 126.5 (C4’), 128.7 (C2’, C6’), 128.8 (C3’, C5’), 130.9 (C1), 139.0 (Cδ, C1’), 141.1 (Cβ),
•+
148.2 (C3), 148.3 (C4), 166.0 (CONH). EI/MS m/z (%): 321.2 (M , 48), 201.1 (100), 143.1 (19), 115.1
(73), 96.1 (26).
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-2-(piperidine-1-carbonyl)penta-2,4-dienenitrile (10). ηꢁ=ꢁ70 %.
1H NMR (DMSO-d6): δ (ppm) = 1.54 (m, 4H, NCH2CH2CH2), 1.65 (m, 2H, NCH2CH2CH2), 4.00 (m, 4H,
NCH2CH2CH2), 6.10 (s, 2H, OCH2O), 6.98 (d, J = 8.2 Hz, 1H, H5), 7.03 (dd, J = 11.4 Hz, 15.2 Hz, 1H,
Hγ), 7.13 (dd, J = 1.5 Hz, 8.1 Hz, 1H, H6), 7.34 (m, 2H, H2, Hδ), 7.55 (d, J = 11.4 Hz, 1H, Hβ). 13C
NMR (DMSO-d6): δ (ppm) = 24.3 (NCH2CH2CH2), 25.9 (NCH2CH2CH2, NCH2CH2CH2), 102.2
(OCH2O), 105.8 (Cα), 106.9 (C2), 109.2 (C5), 116.0 (CN), 121.7 (C6), 125.2 (Cγ), 130.0 (C1), 146.2
•+
(Cδ), 148.7 (C3), 149.9 (C4), 152.0 (Cβ), 162.3 (CON). EI/MS m/z (%): 310.1 (M , 88), 225.0 (81),
197.0 (65), 159.0 (40), 140.0 (100), 113.0 (30), 84 (32).

(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-2-cyano-N-phenylpenta-2,4-dienamide (11). The crude product
was purified by flash column chromatography (silica gel, dichloromethane). ηꢁ=ꢁ44 %. 1H NMR (DMSO-
d6): δ (ppm) = 6.12 (s, 2H, OCH2O), 7.01 (d, J = 8.1 Hz, 1H, H5), 7.12 (m, 2H, Hγ, Hδ), 7.22 (dd, J = 1.6
Hz, 8.2 Hz, 1H, H6), 7.35 (m, 2H, H2, H4’), 7.43 (m, 2H, H3’, H5’), 7.66 (m, 2H, H2’, H6’), 10.18 (s,
1H, NH). 13C NMR (DMSO-d6): δ (ppm) = 102.3 (OCH2O), 107.1 (Cα), 107.2 (C2), 109.3 (C5), 115.8
(CN), 121.0 (C2’, C6’), 121.7 (C6), 124.6 (C4’), 125.8 (Cγ), 129.2 (C3’, C5’), 129.9 (C1), 138.8 (C1’),
•+
148.1 (Cδ), 148.8 (C3), 150.3 (C4), 152.6 (Cβ), 160.8 (CONH). EI/MS m/z (%): 318.1 (M , 55), 226.1
(100), 196.0 (30), 159.0 (42), 140.0 (54).
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-benzyl-2-cyanopenta-2,4-dienamide (12). The crude product
was purified by flash column chromatography (silica gel, dichloromethane). ηꢁ=ꢁ64 %. 1H NMR (DMSO-
d6): δ (ppm) = 4.39 (d, J = 5.9 Hz, 2H, NHCH2Ph), 6.11 (s, 2H, OCH2O), 6.99 (d, J = 8.1 Hz, 1H, H5),
7.06 (dd, J = 11.5 Hz, 15.2 Hz, 1H, Hγ), 7.18 (dd, J = 1.6 Hz, 8.2 Hz, 1H, H6), 7.31 (m, 7H, Hδ, H2, H2’,
H3’, H4’, H5’, H6’), 7.96 (d, J = 11.1 Hz, 14.8 Hz, 1H, Hβ). 5.86 (t, J = 5.9 Hz, 1H, NH). 13C NMR
(DMSO-d6): δ (ppm) = 43.4 (NHCH2Ph), 102.2 (OCH2O), 106.1 (Cα), 107.1 (C2), 109.2 (C5), 116.0
(CN), 121.6 (C6), 125.6 (Cγ), 127.4 (C4’), 127.8 (C2’, C6’), 128.8 (C3’, C5’), 129.9 (C1), 139.5 (C1’),
•+
147.7 (Cδ), 148.7 (C3), 150.1 (C4), 152.3 (Cβ), 161.5 (CONH). EI/MS m/z (%): 332.1 (M , 100), 241.1
(65), 227.1 (54), 198.1 (32), 140.0 (61), 113.1 (19), 91.0 (98), 65.1 (20).
(2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-2-cyano-N-phenethylpenta-2,4-dienamide (13). The crude
1
product was purified by flash column chromatography (silica gel, dichloromethane). ηꢁ=ꢁ87 %. H NMR
(DMSO-d6): δ (ppm) = 2.81 (m, 2H, NHCH2CH2Ph), 3.42 (dt, J = 6.1 Hz, 7.6 Hz, 2H, NHCH2CH2Ph),
6.11 (s, 2H, OCH2O), 7.00 (d, J = 8.1 Hz, 1H, H5), 7.05 (dd, J = 11.5 Hz, 15.2 Hz, 1H, Hγ), 7.19 (dd, J =
1.6 Hz, 8.3 Hz, 1H, H6), 7.22 (m, 3H, H2, H4’, Hδ), 7.32 (m, 2H, H2’, H6’), 7.38 (m, 2H, H3’, H5’), 7.90
(d, J = 11.5 Hz, 1H, Hβ), 8.36 (t, J = 5.6 Hz, 1H, NH). 13C NMR (DMSO-d6): δ (ppm) = 36.2
(NHCH2CH2Ph), 42.5 (NHCH2CH2Ph), 103.1 (OCH2O), 107.1 (Cα), 108.0 (C2), 110.1 (C5), 116.8 (CN),
122.5 (C6), 126.5 (Cγ), 127.5 (C4’), 129.7 (C2’, C6’), 130.0 (C3’, C5’), 130.8 (C1), 140.6 (C1’), 148.4
•+
(Cβ), 149.6 (C3), 151.0 (C4), 152.4 (Cδ), 162.1 (CONH). EI/MS m/z (%): 346.1 (M , 100), 241.1 (24),
226.1 (100), 196.0 (21), 159.0 (27), 140.0 (35).
4.1.3. General procedures for the synthesis of esters
4.1.3.1. Method A (Steglich esterification). To a stirred solution of piperic acid 2 (1 mmol) in
dichloromethane, EDC.HCl (1.2 mmol) and DMAP (0.1 mmol) were added. The mixture was stirred at
room temperature for 30 min. Then, the phenol (1.2 mmol) was added and the resulting mixture was
stirred, protected from the light, overnight. Purification conditions are described in literature [21, 49].
Phenyl (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoate (6). Synthesis and structural analysis
were previously reported [21].

Phenyl (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-2-cyanopenta-2,4-dienoate (14). ηꢁ=ꢁ66 %. 1H NMR
(CDCl3-d1): δ (ppm) = 6.06 (s, 2H, OCH2O), 6.86 (d, J = 8.0 Hz, 1H, H5), 7.10 (dd, J = 1.6 Hz, 8.2 Hz,
1H, H6), 7.15 (d, J = 1.6 Hz, 1H, H2), 7.21 (m, 4H, Hγ, Hδ, H2’, H6’), 7.27 (m, 1H, H4’) 7.42 (m, 2H,
H3’,H5’), 8.11 (d, J = 11.0 Hz, 1H, Hβ). 13C NMR (CDCl3-d1): δ (ppm) = 102.0 (OCH2O), 102.2 (Cα),
106.8 (C2), 108.9 (C5), 114.6 (CN), 121.3 (C6), 121.3 (C2’, C6’), 126.0 (Cγ), 126.3 (C4’), 129.2 (C1),
129.6 (C3’, C5’), 148.8 (C1’), 149.8 (Cδ), 150.4 (C3), 150.9 (C4), 157.1 (Cβ), 161.3 (COO). EI/MS m/z
•+
(%): 319.0 (M , 16), 226.0 (100), 196.0 (25), 159.0 (38), 140.0 (39).
Benzyl (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-2-cyanopenta-2,4-dienoate (15). η =ꢁ50 %. 1H NMR
(CDCl3-d1): δ (ppm) = 5.31 (s, 2H, OCH2Ph), 6.03 (s, 2H, OCH2O), 6.84 (d, J = 8.1 Hz, 1H, H5), 7.06
(dd, J = 1.6 Hz, 8.2 Hz, 1H, H6), 7.14 (m, 3H, Hγ, Hδ, H2), 7.38 (m, 5H, H2’, H3’, H4’, H5’, H6’), 7.99
(dd, J = 10.9 Hz, 1H, Hβ). 13C NMR (CDCl3-d1): δ (ppm) = 67.6 (OCH2Ph), 101.9 (OCH2O), 102.9 (Cα),
106.7 (C2), 108.8 (C5), 114.7 (CN), 121.3 (C6), 125.6 (Cγ), 128.2 (C2’, C6’), 128.5 (C4’), 128.7 (C3’,
C5’), 129.3 (C1), 135.1 (C1’), 148.7 (C3), 149.0 (Cδ), 150.7 (C4), 156.0 (Cβ), 162.4 (COO). EI/MS m/z
•+
(%): 333.1 (M , 19), 242.0 (29), 175.0 (25), 91.0 (100).
1
Phenethyl (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-2-cyanopenta-2,4-dienoate (16). ηꢁ=ꢁ51 %. H NMR
(CDCl3-d1): δ (ppm) = 3.04 (t, J = 7.0 Hz, 2H, OCH2CH2Ph), 4.46 (t, J = 7.1 Hz, 2H, OCH2CH2Ph), 6.04
(s, 2H, OCH2O), 6.84 (d, J = 8.0 Hz, 1H, H5), 7.11 (m, 4H, H6, H2, Hδ, Hγ), 7.15 (m, 3H, H2’, H4’,
H6’), 7.33 (m, 2H, H3’, H5’), 7.92 (d, J = 10.9 Hz, 1H, Hβ). 13C NMR (CDCl3-d1): δ (ppm) = 35.1
(OCH2CH2Ph), 66.6 (OCH2CH2Ph), 101.9 (OCH2O), 102.9 (Cα), 106.7 (C2), 108.8 (C5), 114.7 (CN),
121.3 (C6), 125.6 (Cγ), 126.8 (C4’), 128.6 (C2’, C6’), 129.1 (C3’, C5’), 129.3 (C1), 137.3 (C1’), 148.7
•+
(C3), 148.8 (Cδ), 150.6 (C4), 155.7 (Cβ), 162.5 (COO). EI/MS m/z (%): 347.1 (M , 34), 243.1 (21),
198.1 (31), 140.0 (37), 105.1 (100).
4.1.3.2. Method B (bimolecular nucleophilic substitution). Piperic acid 2 (1 mmol) was dissolved in
DMF and K2CO3 (1.2 mmol) was added. The mixture was stirred at room temperature for 30 min. Then,
the appropriate alkyl bromide (benzyl bromide or (2-bromoethyl)benzene) (1.2 mmol) was added and the
mixture was stirred, protected from the light, at 80 ºC for 8 h. Purification conditions are described in
literature [21, 50].
Benzyl (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoate (7). Synthesis and structural analysis
were previously reported [21].

Phenethyl (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)penta-2,4-dienoate (8). ηꢁ=ꢁ84 %. 1H NMR (CDCl3-d1):
δ (ppm) = 2.99 (t, J = 7.1 Hz, 2H, OCH2CH2Ph), 4.38 (t, J = 7.1 Hz, 2H, OCH2CH2Ph), 5.93 (d, J = 15.3
Hz, 1H, Hα), 5.98 (s, 2H, OCH2O), 6.79 (dd, J = 10.8 Hz, 15.5 Hz, 1H, Hγ), 6.79 (m, 2H, H5, Hδ), 6.91
(dd, J = 1.6 Hz, 8.2 Hz, 1H, H6), 6.99 (d, J = 1.6 Hz, 1H, H2), 7.23 (m, 3H, H2’, H4’, H6’), 7.32 (m, 2H,
H3’, H5’), 7.39 (dd, J = 10.7 Hz, 15.2 Hz, 1H, Hβ). 13C NMR (CDCl3-d1): δ (ppm) = 35.2
(OCH2CH2Ph), 64.8 (OCH2CH2Ph), 101.4 (OCH2O), 105.9 (C2), 108.6 (C5), 120.2 (Cα), 123.0 (C6),
124.5 (Cγ), 126.5 (C4’), 128.5 (C2’, C6’), 128.9 (C3’, C5’), 130.6 (C1), 138.0 (C1’), 140.2 (Cδ), 144.9
•+
(Cβ), 148.3 (C3), 148.6 (C4), 167.0 (COO). EI/MS m/z (%): 322.1 (M , 65), 218.2 (17), 173.1 (100),
143.1 (38), 115.1 (94), 91.2 (20), 77.2 (19), 65.2 (18).
4.1.4. Synthesis of (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylate (18). Aldehyde 17 (1 mmol) dissolved in
anhydrous pyridine. Then, mono-ethyl malonate (1.5 mmol) and piperidine (0.35 mmol) were added. The
mixture was heated until the maximum aldehyde consumption was observed by TLC. Once the reaction
was complete, the product was purified by column chromatography (silica gel, dichloromethane). The
procedure was adapted from Teixeira et al. [51] with some modifications. ηꢁ= 91 %. 1H NMR (CDCl3-d1):
δ (ppm) = 1.33 (t, J = 7.1 Hz, 3H, OCH2CH3), 4.25 (q, J = 7.1 Hz, 2H, OCH2CH3), 6.00 (s, 2H, OCH2O),
6.26 (d, J = 15.9 Hz, 1H, Hα), 6.81 (d, J = 8.0 Hz, 1H, H5), 7.00 (dd, J = 1.5 Hz, 8.1 Hz, 1H, H6), 7.03
(d, J = 1.7 Hz, 1H, H2), 7.59 (d, J = 15.9 Hz, 1H, Hβ). 13C NMR (CDCl3-d1): δ (ppm) = 14.4
(OCH2CH3), 60.4 (OCH2CH3), 101.5 (OCH2O), 106.5 (C2), 108.5 (C5), 116.3 (Cα), 124.4 (C6), 128.9
•+
(C1), 144.3 (Cβ), 148.3 (C3), 149.6 (C4), 167.2 (COO). EI/MS m/z (%): 220.2 (M , 100), 175.1 (58),
117.0 (31), 89.0 (64), 63.1 (41).
4.1.5. Synthesis of (E)-3-(benzo[d][1,3]dioxol-5-yl)prop-2-en-1-ol (19). Cinnamic ester 18 (1 mmol)
was dissolved in dry THF under argon atmosphere and cooled to −78 °C. Then, LiAlH4 (1.5 mmol) was
added, and the mixture was stirred for 5 h. Once the reaction was complete, the unreacted LiAlH4 was
quenched with methanol until gas evolution stopped, and water (60 mL) was added. The mixture was
neutralized with HCl 6 M, then extracted with dichloromethane (3×30 mL). The combined organic layer
was washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated.
Purification by flash column chromatography (silica gel, dichloromethane) yielded the cinnamyl alcohols
1
19. The procedure was adapted from Jirásek et al. [52] with some modifications. ηꢁ=ꢁ43 %. H NMR
(CDCl3-d1): δ (ppm) = 4.29 (dd, J = 0.9 Hz, 5.8 Hz, 2H, CH2OH), 5.95 (s, 2H, OCH2O), 6.19 (d, J = 5.9
Hz, 15.8 Hz, 1H, Hα), 6.52 (d, J = 15.8 Hz, 1H, Hβ), 6.75 (d, J = 8.0 Hz, 1H, H5), 6.81 (dd, J = 1.6 Hz,
8.1 Hz, 1H, H6), 6.93 (d, J = 1.7 Hz, 1H, H2). 13C NMR (CDCl3-d1): δ (ppm) = 63.8 (CH2OH), 101.1
(OCH2O), 105.8 (C2), 108.3 (C5), 121.2 (C6), 126.7 (Cα), 131.0 (Cβ), 131.2 (C1), 147.3 (C3), 148.0
•+
(C4). EI/MS m/z (%): 178.2 (M , 80). 135.1 (100), 122.1 (42), 91.1 (24).

4.1.6. Synthesis of (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylaldehyde (20). Cinnamyl alcohol 19 (1
mmol) was dissolved in dichloromethane, and MnO2 (9 mmol) was added. The mixture was stirred
overnight at room temperature. Then, the mixture was filtered through celite to remove the MnO2 and
washed with dichloromethane. The filtrate was concentrated and purified by flash column
chromatography (silica gel, dichloromethane). The procedure was adapted from Jirásek et al. [52] with
1
some modifications. ηꢁ=ꢁ62 %. H NMR (CDCl3-d1): δ (ppm) = 6.04 (s, 2H, OCH2O), 6.56 (dd, J = 7.7
Hz, 15.8 Hz, 1H, Hα), 6.86 (d, J = 8.5 Hz, 1H, H5), 7.07 (m, 2H, H2, H6), 7.38 (d, J = 15.8 Hz, 1H, Hβ),
9.05 (d, J = 7.7 Hz, 1H, CHO). 13C NMR (CDCl3-d1): δ (ppm) =101.8 (OCH2O), 106.8 (C2), 108.8 (C5),
125.3 (Cα), 126.9 (C6), 128.5 (C1), 148.6 (C3), 150.5 (C4), 152.5 (Cβ), 193.5 (CHO). EI/MS m/z (%):
•+
176.1 (M , 100), 147.1 (67), 118.1 (48), 89.2 (88), 63.1 (74), 51.1 (20).
4.1.7. Synthesis of (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-2-cyanopenta-2,4-dienoic acid (9).
Aldehyde 20 (1 mmol) was dissolved in anhydrous pyridine. Then, cyanoacetic acid (1.5 mmol) and
piperidine (0.35 mmol) were added. The mixture was heated until the maximum aldehyde consumption
was observed by TLC. Upon completion, the reaction mixture was acidified with HCl 6 M and the solid
formed was isolated by filtration under reduced pressure. The procedure was adapted from Teixeira et al.
[51] with some modifications. ηꢁ=ꢁ87 %. 1H NMR (DMSO-d6): δ (ppm) = 6.12 (s, 2H, OCH2O), 7.01 (d, J
= 8.0 Hz, 1H, H5), 7.08 (dd, J = 11.7 Hz, 15.0 Hz, 1H, Hγ), 7.19 (dd, J = 1.4 Hz, 8.1 Hz, 1H, H6), 7.41
(m, 1H, H2), 7.56 (d, J = 15.1 Hz, 1H, Hδ), 8.03 (d, J = 11.6 Hz, 1H, Hβ), 13.6 (bs, 1H, COOH). 13C
NMR (DMSO-d6): δ (ppm) = 103.2 (OCH2O), 104.3 (Cα), 108.1 (C2), 110.2 (C5), 116.6 (CN), 122.3
(C6), 127.2 (Cγ), 130.7 (C1), 149.7 (C3), 150.8 (Cβ), 151.4 (C4), 157.0 (Cδ), 164.8 (COOH). EI/MS m/z
•+
(%): 243.2 (M , 27), 198.2 (34), 140.1 (100), 113.1 (24).
4.2.
Enzymatic assays
4.2.1. Evaluation of human monoamine oxidase (hMAO) inhibitory activity
The inhibitory activity of piperine and the derivatives thereof on hMAO-A and hMAO-B was
studied using an experimental protocol described elsewhere [21, 22] (see supplementary information).
4.2.2. Evaluation of human monoamine oxidases kinetics and human monoamine oxidase-B-
inhibitor kinetics
To determine the steady-state kinetic parameters (Km, Michaelis constant, and Vmax, maximum
velocity) of hMAO-A and hMAO-B, the enzymatic activity of both isoforms was studied in the presence
six different concentrations of kynuramine (see SI). To evaluate the mechanism of hMAO-B inhibition of
compounds 7, 10 and 15, substrate-dependent kinetic experiments were also performed (see
supplementary information).
4.3.
In vitro toxicology
4.3.1. Cell lines and culture conditions
Human SH-SY5Y neuroblastoma cells and human epithelial colorectal adenocarcinoma (Caco-2)
cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cell

cultures and SH-SY5Y cell differentiation were performed as previously described by Fernandes et al.
[53] and in supplementary information.
4.3.2. Evaluation of cytotoxicity profile
Stock solutions of the test compounds (100 mM) were freshly prepared in DMSO. Final
concentrations of the test compounds were obtained by diluting into culture medium immediately before
use, giving a final maximum concentration of 0.1 % DMSO.
In cytotoxicity studies, differentiated SH-SY5Y cells were exposed to the test compounds (10
µM and 50 µM) for 24 h. Controls were treated with culture media containing 0.1 % DMSO. Cell
viability was estimated using two different methods: resazurin reduction assay and NR uptake assay. The
cytotoxicity end-points were performed as described in literature [40, 53] and in supplementary
information.
4.3.3. Evaluation of intracellular oxidative stress levels
The formation of intracellular RS was evaluated using the 2’,7’-dichlorofluorescein (DCF)
fluorescence assay as previously described by da Fernandes et al. [53] and in supplementary information.
4.3.4. Evaluation of P-glycoprotein (P-gp) transport activity
The effects of the tested compounds on P-gp transport activity was evaluated using rhodamine
123 (RHO 123) as a P-gp fluorescent substrate in Caco-2 cells as previously described by Fernandes et al.
[40] and in supplementary information.
4.4.
Statistical analysis
Data analysis for all the studies are specified in supplementary information.
4.5.
Evaluation of the chromatographic hydrophobicity index
Chromatographic hydrophobicity index logD at pH 7.4 (CHI logD7.4) values were determined using an
experimental protocol described elsewhere [43, 54]. Chromatographic hydrophobicity index logD at pH
7.4 (CHI logD7.4) values were determined using the retention times of samples and a mixture of standard
compounds. The data was acquired on a Shimadzu high-performance liquid chromatograph SPD-M20A
system (Shimadzu, Kyoto, Japan) with a Luna C18 (2) column (Phenomenex, CA, USA) 150×4.6 mm, 5
µm. The mobile phase A was 10 mM ammonium acetate solution (pH 7.4), and mobile phase B was
acetonitrile. The following gradient program was applied: 1 mL/min flow, rt, injection volume 20 ꢀL,
gradient 0−6 min 0-100 % B, 6−14 min 100% B, 14−16 min 100-0 % B.
A calibration plot was obtained using a mixture of the following compounds: paracetamol,
theophylline, caffeine, benzimidazole, colchicine, carbamazepine, indole, propiophenone, butyrophenone,
valerophenone, and heptanophenone (see supplementary information, Figure S1 and Table S1). Stock
solutions of the test compounds (10 µM) were prepared in DMSO and diluted in acetonitrile/water (1:1)
to obtain a final concentration of 250 µM. CHI logD7.4 values were calculated as previously described
[43, 54].

4.6.
Estimation of drug-like properties
The calculation of molecular weight (MW), topological polar surface area (TPSA), number of
hydrogen bond donors (HBD) and acceptors (HBA), number of rotatable bonds (RB) and logarithm of the
ratio of the concentration of a drug in the brain and in the blood (logBB) was performed using the
StarDrop software.
5.
Acknowledgements
This project was supported by Foundation for Science and Technology (FCT) and FEDER/COMPETE
research grants (UID/QUI/00081, NORTE-01-0145-FEDER-000028, POCI-01-0145-FEDER-029164).
D. Chavarria, C. Fernandes, E. Martins, V. Silva, C. Silva, P. Soares and T. Silva grants are supported by
FCT, POPH and FEDER/COMPETE and NORTE-01-0145-FEDER-000028.This article is based upon
work from COST Action CA15135.
6.
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