Synthesis and inhibitory effect of piperine derivates on monoamine oxidase

Article abstract of DOI:10.1016/j.bmcl.2012.02.090

A series of piperine derivates (1-19) have been designed, synthesized and evaluated in vitro for their monoamine oxidase (MAO) A and B inhibitory activity and selectivity. It is worth noting that most of the small amine moieties substituted on the piperidine ring proved to be potent and selective inhibitors of MAO-B rather than of MAO-A. 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid n-propyl amide (3) showed the greatest MAO-B inhibitory activity (IC ₅₀(MAO-B) = 0.045 μM) and good selectivity (IC₅₀(MAO-A) = 3.66 μM). The conjugated double bond and carbonyl group of piperine are proved to be an essential feature for piperine and related alkylamides to exhibit MAO-inhibitory activity. Binding mode of the titled compounds was predicted using FlexX algorithm. The design and optimization of novel small molecule monoamine oxidase inhibitors will be guided by the results of this report.

Full text of DOI:10.1016/j.bmcl.2012.02.090

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3343–3348
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and inhibitory effect of piperine derivates on monoamine oxidase
Li-Hua Mu a, Bo Wang a, , Hao-Yang Ren a, , Ping Liu a, , Dai-Hong Guo a, Fu-Meng Wang a, Lin Bai a,
⇑
Yan-Shen Guo b
a Department of Clinical Pharmacology, General Hospital of PLA, Beijing 100853, China
b Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Xian Nong Tan Street, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of piperine derivates (1–19) have been designed, synthesized and evaluated in vitro for their
monoamine oxidase (MAO) A and B inhibitory activity and selectivity. It is worth noting that most of
the small amine moieties substituted on the piperidine ring proved to be potent and selective inhibitors
of MAO-B rather than of MAO-A. 5-(3,4-methylenedioxyphenyl)-2E,4E-pentadienoic acid n-propyl amide
Received 28 September 2011
Revised 15 February 2012
Accepted 27 February 2012
Available online 8 March 2012
(3) showed the greatest MAO-B inhibitory activity (IC50(MAO-B) = 0.045
lM) and good selectivity
(IC50(MAO-A) = 3.66 M). The conjugated double bond and carbonyl group of piperine are proved to be
l
Keywords:
Monoamine oxidase
Inhibitors
Piperine derivates
Structure–activity relationship
Molecular docking
an essential feature for piperine and related alkylamides to exhibit MAO-inhibitory activity. Binding
mode of the titled compounds was predicted using FlexX algorithm. The design and optimization of novel
small molecule monoamine oxidase inhibitors will be guided by the results of this report.
Ó 2012 Elsevier Ltd. All rights reserved.
Monoamine oxidase (MAO) catalyzes the oxidative deamination
of monoamine neurotransmitters such as serotonin, dopamine, and
norepinephrine, and appears to play important roles in several psy-
chiatric and neurological disorders.1,2 MAO has been divided into
two subtypes, MAO-A and MAO-B, on the basis of their aminoacid
sequence, substrate and inhibitor selectivity, and tissue distribu-
tion.3,4 Selective MAO-A inhibitors are useful in the therapy of
mental disorders, mainly as antidepressants and anxiety, whereas
MAO-B inhibitors are expected to be useful in the therapy of
Parkinson’s and Alzheimer’s disease.5–7 To date, a number of
MAO inhibitors such as coumarins, xanthones, and isoquinoline
alkaloids have been isolated from natural products or synthe-
sized.8–11 The high-resolution structures of MAO-A and MAO-B
has been available and these can be exploited in the design of
selective MAO inhibitors with minimum side effects.12–14
Piperine was the first amide isolated from Piper species and was
reported to display central nervous system depression, antipyretic,
and anti-inflammatory activity.15,16 Moreover, previous studies
have demonstrated that piperine and its derivatives present
sedative-hypnotic, tranquilizing, and muscle-relaxing actions and
can intensify the depressive action of other depressants.17 Amidst
the vast spectrum of piperine’s activities, it was particularly
intriguing to notice the consistent outcome from several indepen-
dent studies, suggesting significant antidepressant-like activity of
this compound. For example, piperine and its analog, antiepilepsi-
rine exhibited antidepressant-like activity in classical behavioral
Figure 1. The piperine structure divided into three key regions.
models such as forced swimming test and tail suspension tests in
rodents.18–20 Piperine treatment also led to increased level of bio-
genic amines such as noradrenaline and serotonin in some regions
of mouse brain, which indirectly suggests its ability to interfere
with the metabolism of these amines.19,20 Lastly, piperine and its
analog—methylpiperate have been directly shown to inhibit the
relevant enzymes that is, monoamine oxidases (MAOs; EC
1.4.3.4) in vitro.21–23
Previously, piperine was found to be more MAO-A selective for
rat21 while it was more MAO-B selective for mouse.22 So, with a
view to having some preliminary idea, we design a series of
derivatives which were from different parts of piperine. MAO
inhibitory activity and structure–activity relationship of these syn-
thesized derivatives were studied. To give structural insights
regarding the binding mode of these inhibitors, we carried out
docking simulations of the most potent and selective MAO-B inhib-
itor 3.
In our present work, several piperine derivatives were prepared,
using a simple coupling method foramide synthesis, and tested for
monoamine oxidase inhibitory activity. The piperine structure
(Fig. 1) can be split into three fragments namely methylenedioxy-
phenyl (MDP) ring (compartment A), side chain with conjugated
⇑
Corresponding author. Tel.: +86 10 66936676; fax: +86 10 66936678.
E-mail address: liuping301@126.com (P. Liu).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.02.090

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L.-H. Mu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3343–3348
Scheme 1. Reagents and conditions: (a) KOH (b) H2SO4/MeOH, EtOH reflux 6 h (c) SOCl2 in CH2Cl2 reflux 1 h (d) R1R2NH, rt 30 min (e) 2-octanol/180 °C 72 h.
Scheme 2. Reagents and conditions: (a) NaBH4/I2, THF, 0 °C nitrogen atmosphere, 48 h (b) BBr3 in CH2Cl2 rt 48 h (c) Ac2O/pyridine.
double bonds (compartment B) and a basic piperidine moiety at-
tached through a carbonylamide linkage to side chain (compart-
ment C). Piperinic acid 2 was obtained by alkaline hydrolysis of
piperine 1 (Scheme 1). The amides (3–6) were readily obtained
from the carboxylic acids through acyl chloride formation with
appropriate amines (Scheme 1).24 The amide functional groups of
piperine were also replaced by a range of ester groups, yielding
the corresponding analogues 10–11 (Scheme 1).25 In. order to
Scheme 3. Reagents NR1R2 = piperidine (13), n-propyl amide (14), n-butyl amide(15), N,N-diethyl amide (16), N-methyl piperazine amide (17).

L.-H. Mu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3343–3348
3345
Table 1
The inhibition of rat main MAO isoforms by the newly synthesized piperine derivativesa.
No.
Compound
IC50 for MAO-Ab
(l
M)
IC50 for MAO-Bb
(
lM)
SIc
MAO-inhibitory selectivity
O
O
O
O
O
N
OH
N
1
0.404
0.26
1.55
Selective for MAO-B
Selective for MAO-B
O
2
18.34
13.59
1.35
O
O
3
4
5
6
7
3.66
>100
3.82
2.04
0.80
0.045
0.33
81.5
—
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-B
Selective for MAO-A
O
O
O
O
O
O
N
O
O
N
0.078
0.96
49.0
2.12
0.51
O
O
N
N
O
HO
N
O
1.57
HO
O
O
O
N
8
1.28
0.75
1.71
Selective for MAO-B
O
O
N
O
O
9
>100
2.74
>100
0.23
—
—
O
O
O
O
O
O
O
10
11.9
Selective for MAO-B
11
12
13
7.55
>100
>100
0.33
>100
1.97
22.8
Selective for MAO-B
O
N
O
O
N
—
—
O
O
O
O
O
O
N
—
Selective for MAO-B
(continued on next page)

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L.-H. Mu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3343–3348
Table 1 (continued)
No.
Compound
IC50 for MAO-Ab
(lM)
IC50 for MAO-Bb
(l
M)
SIc
—
MAO-inhibitory selectivity
—
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
O
O
14
>100
>100
15
16
17
18
19
16.08
5.21
>100
3.17
5.57
3.50
1.75
>100
1.80
1.23
4.59
2.98
—
Selective for MAO-B
Selective for MAO-B
—
N
1.76
4.53
Selective for MAO-B
Selective for MAO-B
O
20
21
Selegiline
Clorgyline
>100
13.91 Â 10À3
—
Selective for MAO-B
Selective for MAO-A
0.42 Â 10À3
0.26
1.62 Â 10À3
a
Selegiline or clorgyline were added at 1
37 °C.
lM to determine the isoenzymes A and B. Newly synthesized compounds were preincubated with the homogenates for 10 min at
b
IC50 values were determined from plots of residual activity percentage, calculated in relation to a sample of the enzyme treated under the same conditions without
inhibitor.
c
SI = IC50 of MAO-A/IC50 of MAO-B.
Figure 2. Lineweaver–Burk plots of the inhibition of MAO-A and MAO-B by compound 3. The reciprocals of MAO-A and MAO-B activities were plotted against the reciprocals
of substrate concentration. Compound 3 concentrations: MAO-A 0 lM (rhombus); 50 lM (rectangle);100 lM (triangle), MAO-B 0 lM (rhombus); 3.5 lM (rectangle);7 lM
(triangle).
achieve the diacetylphenyl containing analogue 8, Piperine 1 was
sequentially treated with BBr3 and Ac2O (Scheme 2). 5-(3,4-meth-
ylenedioxyphenyl)-penta-2E,4E-dienyl piperidine 9 (Scheme 2)
was isolated during an attempt to reduce a single double bond of
piperine according to the method of Das et al.26 Compounds 1,
3–6, 10 and 11 were subjected to hydrogenation(Scheme 3) and
saturated molecules 13–19 thus obtained were bioevaluated.25–30
MAO-A and MAO-B inhibitory activities of the synthesized pip-
erine derivates were determined by a fluorometric assay, using
kynuramine as a substrate, in the presence of their specific inhibi-
according to the method of Clark and Nicklas.32 Protein concentra-
tion was determined according to the method of Bradford33 in
which bovine serum albumin was used as standard. The MAO
inhibitory activities, expressed as IC50 values are summarized in
Table 1.
Some of the synthesized compounds showed potent MAO-B
inhibitory activity with the IC50 value at low micromolar. Among
all the compounds, only compound 7 is selective MAO-A inhibitors
(IC50 (MAO-A) = 0.80 and IC50(MAO-B) = 1.57). Compounds 3 and 5
showed the good MAO-B inhibitory activity (IC50(MAO-B) = 0.045
tors (selegiline 1
lM for MAO-A and clorgyline 1
l
M for MAO-B).31
and 0.078
lM, respectively) and good selectivity (IC50(MAO-
Rat brain mitochondria were isolated from Sprague–Dawley rats
A) = 3.66 and 3.82
l
M, respectively). Compounds 2, 9, 12, 14 and

L.-H. Mu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3343–3348
3347
Figure 3. FlexX-modeled binding mode of compound 3 (carbon atoms colored green) with MAO-A(a) and MAO-B(b). H-bonding interactions are presented with red line.
17 showed no inhibitory activity to both MAO-A and MAO-B
isoforms.
pound 7 is the only compound showing MAO-A inhibition, this
result indicated that the phenolic hydroxyl is likely to increase
the MAO-A inhibitory selectivity.
The inhibitory of mouse and rat brain-derived MAO-B by piper-
ine was competitive in previous studies, the mode of its inhibitory
action of rodent brain-derived MAO-A was found to be either com-
petitive or mixed type for mouse and rat MAO-A.21,22 The Linewe-
aver–Burk plots of the inhibition of MAO-A and MAO-B by
compound 3 showed the inhibitory actions are both competitive
(Fig. 2).34
In order to get some insight for further structure-based modifi-
cation of MAO inhibitors, the binding mode was investigated using
FlexX algorithm implemented in SYBYL 7.2.36 The coordinates of
X-ray co-crystal structure of MAO-A&B (pdb code: 2Z5X & 3PO7)
was employed for docking the synthesized compounds.37,38 The
docking calculations add to the understanding of the structural
requirements for binding to the MAO enzyme. In FlexX calcula-
tions, hydrogen bonding interactions, van der Waals interactions
and steric as well as electrostatic interactions were evaluated.
The topology of the active site for both the proteins MAO-A and
MAO-B are comparable in Figure 3 which shows the binding modes
of compound 3 (a) into the MAO-A binding cavity and (b) into the
MAO-B binding cavity.
Visual inspection of the pose of compound 3 into the MAO-A
binding site revealed that the pyridine ring of 3 is placed in the
‘aromatic cage’ framed by Tyr197, Tyr407, Tyr444, and the aro-
matic ring is oriented to establish p–p stacking interactions with
Tyr407. Conjugated double bonds are embedded in a large hydro-
phobic pocket formed by Ile180, Phe208, Asn181, Gln215, and
Ile335. Moreover, one hydrogen bond is observable for 3 between
the NH of 3 and the carbonyl oxygen of Phe208. Visual inspection
of the pose of compound 3 into the MAO-B binding site revealed
that compound 3 is inserted into the ‘aromatic cage’ framed by
Tyr60, Tyr326, Tyr398, Tyr435, and the aromatic ring. Moreover
the carbonyl oxygen of compound 3 formed hydrogen bonds with
NH on the side chain of SER59 and TYR60. Compound 3 forms more
hydrogen bond interactions with the MAO-B active site compared
to the MAO-A active site which may indicate that 3 interacts more
tightly with MAO-B.
Structure–activity relationships (SARs) were inferred from the
data of enzymatic experiments reported in Table 1.Compounds
3–6, 10 and 11 has a propyl, butyl, diethyl, N-methyl phneyl, meth-
oxy and ethoxyl group in place of the piperidine moiety of piperine
respectively and they have been shown to inhibit both MAO-A and
MAO-B with more selectivity for the latter. Two compounds (3, 5)
with propyl and diethyl substituent showed the better MAO-B
inhibitory activity and good selectivity comparing with piperine.
So, the small molecule amines substitute the piperidine moiety
may improve the MAO-B inhibitory activity. But all the compounds
losing piperidine moiety showed poor MAO-A inhibitory activity
comparing to piperine. Thus, the piperidine moiety (compartment
C, Fig. 1) of piperine seems to aid in achieving better MAO-A selec-
tivity while its omission barely affects the MAO-B selectivity. But
the previous docking of piperine and methyl piperate onto MAO
enzymes by Rahman and Rahmatullah given the contrast conclu-
sion.35 The reasons for such discrepancy are not clear but species
difference is likely to be among plausible explanations. In order
to determine the effect of the compartment B(Fig. 1) of piperine
that can act as an effective inhibitor of MAOs, compounds 12,
13–19 and compound 9 were synthesised. Compounds 13–19 were
obtained from compounds 3–6, 10 and 11 by catalytic hydrogena-
tion but they showed no inhibition or very poor inhibition to both
MAO-A and MAO-B. Compound 9 was synthesised by removing the
carbonyl of piperine and it lost the inhibition to both MAO-A and
MAO-B. Compound 12 (dimer of piperine) also has no MAO-A
and MAO-B inhibition. This result indicated that the conjugated
double bond and carbonyl group (compartment B, Fig. 1) are likely
to be an essential feature for piperine and related alkylamides to
exhibit MAO-inhibitory activity. Compounds 7, 8 screened in our
study had the changed MDP ring (compartment A, Fig. 1) and com-
pounds changing such moiety seem to offer poor inhibitory to
monoamine oxidase. So, the MDP ring of piperine is likely to
impart MAO-B inhibitory activity. This result is analogy with the
previous study of Rahman and Rahmatullah.35 Interestingly com-
Overall, a series of piperine derivatives (1–19) were designed
and evaluated in vitro for their MAO-A and MAO-B inhibitory activ-
ity. Most of the synthesized compounds proved to be potent, and
selective inhibitors of MAO-B rather than of MAO-A. It is worth
noting that the small molecule amines moieties substituted on
the piperidine ring may improve the MAO-B inhibitory activity.
Among those derivatives, compound 3 showed the greatest MAO-
B inhibitory activity (IC50(MAO-A) = 3.66
lM) and good selectivity
(IC50(MAO-B) = 0.045 M). The conjugated double bond and car-
l
bonyl group of piperine are likely to be an essential feature for
piperine and related alkylamides to exhibit MAO-inhibitory
activity.

3348
L.-H. Mu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3343–3348
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.090.
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