Stereoselective Activity of 1-Propargyl-4-styrylpiperidine-like Analogues That Can Discriminate between Monoamine Oxidase Isoforms A and B

Article abstract of DOI:10.1021/acs.jmedchem.9b01886

The resurgence of interest in monoamine oxidases (MAOs) has been fueled by recent correlations of this enzymatic activity with cardiovascular, neurological, and oncological disorders. This has promoted increased research into selective MAO-A and MAO-B inhibitors. Here, we shed light on how selective inhibition of MAO-A and MAO-B can be achieved by geometric isomers of cis-and trans-1-propargyl-4-styrylpiperidines. While the cis isomers are potent human MAO-A inhibitors, the trans analogues selectively target only the MAO-B isoform. The inhibition was studied by kinetic analysis, UV-vis spectrum measurements, and X-ray crystallography. The selective inhibition of the MAO-A and MAO-B isoforms was confirmed ex vivo in mouse brain homogenates, and additional in vivo studies in mice show the therapeutic potential of 1-propargyl-4-styrylpiperidines for central nervous system disorders. This study represents a unique case of stereoselective activity of cis/trans isomers that can discriminate between structurally related enzyme isoforms.

Full text of DOI:10.1021/acs.jmedchem.9b01886

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Article
Stereoselective Activity of 1-Propargyl-4-Styrylpiperidine-Like Analogues
that can Discriminate between Monoamine Oxidase Isoforms A and B
Damijan Knez, Natalia Colettis, Luca Giacinto Iacovino, Matej Sova, Anja Pišlar, Janez
Konc, Samo Lešnik, Josefina Higgs, Fabiola Kamecki, Irene C. Mangialavori, Ana Dolšak,
Simon Žakelj, Jurij Trontelj, Janko Kos, Claudia Binda, Mariel Marder, and Stanislav Gobec
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01886 • Publication Date (Web): 09 Jan 2020
Downloaded from pubs.acs.org on January 9, 2020
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Stereoselective Activity of 1-Propargyl-4-Styrylpiperidine-Like Analogues that can
Discriminate between Monoamine Oxidase Isoforms A and B
Damijan Knez,1,# Natalia Colettis,2,# Luca G. Iacovino,3,# Matej Sova, Anja Pišlar, Janez
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Konc, Samo Lešnik, Josefina Higgs, Fabiola Kamecki, Irene Mangialavori, Ana Dolšak,
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Simon Žakelj, Jurij Trontelj, Janko Kos, Claudia Binda, * Mariel Marder, * and Stanislav
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,
Gobec *
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University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
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Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas.
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Junín 956 (C1113AAD), Buenos Aires, Argentina
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University of Pavia, Department of Biology and Biotechnology, via Ferrata 1, 27100 Pavia,
Italy
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National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia
#
These authors contributed equally to this work.
*
Joint senior authors.
ABSTRACT
The resurgence of interest in monoamine oxidases (MAOs) has been fuelled by recent
correlations of this enzymatic activity with cardiovascular, neurological and oncological
disorders. This has promoted increased research into selective MAO-A and MAO-B
inhibitors. Here, we shed light on how selective inhibition of MAO-A and MAO-B can be
achieved by geometric isomers of cis- and trans-1-propargyl-4-styrylpiperidines. While the
cis isomers are potent human MAO-A inhibitors, the trans analogues selectively target only
the MAO-B isoform. The inhibition was studied by kinetic analysis, UV-Vis spectrum
measurements and X-ray crystallography. The selective inhibition of the MAO-A and MAO-
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B isoforms was confirmed ex vivo in mouse brain homogenates, and additional in vivo studies
in mice show the therapeutic potential of 1-propargyl-4-styrylpiperidines for central nervous
system disorders. This study represents a unique case of stereoselective activity of cis/ trans
isomers that can discriminate between structurally related enzyme isoforms.
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INTRODUCTION
Specific interaction patterns and molecular geometry are critical factors that influence
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ligand binding affinity and ligand selectivity towards a target. Numerous examples of ‘chiral
switching’ in drug development have highlighted the effects of spatial geometry on not only
pharmacodynamics, but also pharmacokinetics and toxicology of drugs.2-3 In addition to
optical isomers, cis and trans isomers can have different pharmacological activities, as
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demonstrated by the well-known cases of cisplatin, tamoxifen, and combretastatin A4 (CA-
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4). This last includes a stilbene motif in the cis conformation (Figure 1A), which efficiently
binds to the colchicine binding site of tubulin and acts as a microtubule-destabilising agent.6
The binding of trans-combretastatin A4 (trans-CA-4, Figure 1A) to this site is
thermodynamically less stable than that of cis-combretastatin A4 (cis-CA-4), and
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consequently the trans conformation does not prevent microtubule assembly. However,
examples of stereoselective activities of cis/ trans isomers are rare in comparison with optical
isomers.
Human monoamine oxidase A (hMAO-A) and human monoamine oxidase B (hMAO-
B) are flavoenzymes that are encoded by separate genes on the X chromosome, and they share
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0% sequence identity. They are differentiated by their substrate specificities and inhibitor
sensitivities. This arises from the differences in the active site size and shape, and the altered
amino-acid residues between these isoforms. The hMAO-A and hMAO-B active sites are
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hydrophobic cavities with volumes of about 550 Å and 700 Å , respectively. The active site
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of hMAO-B is flat and elongated, with a bipartite configuration, where Ile199 and Tyr326 are
the gating residues that determine the shape of the cavity and the specificity of substrate/
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inhibitor binding. hMAO-A has a monopartite and more spherical active site, with Ile335 and
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Phe208 as the gating residues. MAOs catalyse the oxidative deamination of primary and some
secondary amines, and they are responsible for neurotransmitter metabolism in peripheral
tissues and in the central nervous system.10 This reaction produces an imino intermediate,
which is spontaneously hydrolysed to the corresponding aldehyde, with ammonia and
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hydrogen peroxide as side-products. Reduced levels of neurotransmitters, increased activity
and levels of MAOs, and the production of toxic metabolites are linked with various
neurological,12-13 psychological and cardiovascular disorders, and cancers.
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MAO-A inhibitors have been widely used for several decades in the therapy of
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depression and affective disorders. On the other hand, selective MAO-B inhibitors are used
as monotherapies in the early stages of Parkinson’s disease, and as add-on therapy to
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levodopa in advanced forms of Parkinson’s disease. MAO-A and MAO-B are recognized as
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validated targets. In terms of their contribution to increased cellular oxidative stress, novel
inhibitors are emerging as important disease-modifying therapeutic agents in such devastating
neurodegenerative diseases as Alzheimer’s disease, and a number of others.20
To the best of our knowledge, cis/ trans isomerisms of double bonds have never been
used to achieve stereoselective inhibition of MAO isoforms. Here, we demonstrate a unique
case study whereby configuration of the double bond in stilbene-like derivatives can define
the selectivity for either MAO-A or MAO-B. We identify 1-propargyl-4-((E)-styryl)-
piperidines as potent, selective and irreversible hMAO-B inhibitors. Cis analogues with small
substituents on the phenyl ring selectively inhibit hMAO-A. These inhibitors were evaluated
for their MAO inhibitory activities and pharmacological properties.
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RESULTS
Identification, design, synthesis and selectivity profile of potent and selective hMAO-A
and hMAO-B inhibitors
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Compound 1 (Figure 1A) contains a stilbene-like motif and it was identified as a hit
inhibitor in an in-house library screening campaign. Indeed, compound 1 is a potent selective
hMAO-B inhibitor, with no significant inhibition of hMAO-A at 100 µM (Figure 1B).
This hit inhibitor was resynthesised using an efficient and straightforward synthesis
(Scheme 1) that started from readily available isonipecotic acid, which was transformed into
Weinreb’s amide 2, and subsequently reduced to obtain aldehyde 3. The aldehyde then
underwent the Wittig reaction with phosphonium ylide, to yield a mixture of cis (4) and trans
(5) Boc-protected piperidines, which were separated using column chromatography.
Following acidolysis, the corresponding secondary amine was reacted with propargyl bromide
to obtain hit compound 1 and the cis isomer 6. Compound 6 showed preferential inhibition of
hMAO-A over hMAO-B (Figure 1C).
MeO
A
B
MeO
N
OMe
OH
OMe
cis-CA-4
N
F
OH
OMe
MeO
MeO
F
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6
OMe
trans-CA-4
trans (E)
cis (Z)
C
a
a
Selectivity Ratiob
Compound IC50 (hMAO-A) [M] IC50 (hMAO-B) [M]
1 (trans)
(cis)
> 100
0.3422 ±0.0224
6.2215 ±0.7268
>292
0.12
6
0.7261 ±0.0269
Figure 1. Stilbene derivatives and MAO inhibitory potencies. Chemical structures of cis/ trans-
combretastatin A4 (CA-4) (A), and hit inhibitor 1 (B) and cis isomer 6 (B). The stilbene core is blue,
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a
and the stilbene-like moiety of inhibitor 1 is red. (C) IC values are means ±SEM (n = 2, 3).
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Selectivity ratio is defined as IC (hMAO-A)/ IC (hMAO-B).
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Boc
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ii
iii
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COOH
O
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CHO
O
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F
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4: cis (Z)
6: cis (Z)
5: trans (E)
1: trans (E)
Scheme 1. Resynthesis of hit inhibitor 1. (i) 1) Boc O, 1.0 M NaOH, 1,4-dioxane, 0 °C, RT, 1 h
2
(
91%); 2) N,O-dimethyhydroxylamine hydrochloride, Et N, TBTU, CH Cl , 0 °C to RT, overnight
3 2 2
(76%); (ii) LiAlH4, anh. THF,
0
°C,
1
h
(crude product, 83%); (iii) (4-
fluorobenzyl)triphenylphosphonium bromide, sodium bis(trimethylsilyl)amide (NaHMDS, 2 N in
THF), anh. THF, Ar(g), RT, overnight (52%); (iv) 1) 4.0 M HCl in 1,4-dioxane, 80 °C, 2 h (100%); 2)
propargyl bromide (80% solution in toluene), Cs CO , DMF, Ar(g), 0 °C, RT, overnight (yield: 1,
2
3
2
6%; 6, 72%).
The selectivity profile was explained through analysis of the binding cavity using the
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Caver tool. Human (h)MAO-A (Figure 2A) has a wide cavity at the active site near the FAD
cofactor (r = 2.1 Å), which is lined with Tyr407, Phe352 and Gly215. In the direction towards
the entrance, the cavity widens (r = ~2.5 Å), and remains wide to Val210, Gln215 and Ile335,
and then it narrows to Val210, Cys323 and Ile335 (r = 1.9 Å), and remains narrow to the
entrance, which is also the narrowest part of the active site (r = 1.9 Å), and is composed of
Leu97, Gly110 and Val210. In hMAO-B (Figure 2B), the entrance is even wider in the active
site near the FAD cofactor (r = 2.3 Å), with Gln206, Tyr398 and Tyr435, but then narrows in
the direction of the entrance (r = 1.9 Å) at Leu171 and Ile199, and then widens again (r = 2.5
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Å) to Ile199, Ile316 and Tyr326. The narrowest part is at the entrance (r = 1.6 Å), and it is
composed of Glu84, Pro102 and Ser200. The hMAO-A active site entrance is thus
significantly wider (r = 1.9 Å) than the hMAO-B active site entrance (r = 1.6 Å). Both
entrances are also the bottlenecks, which indicate that the active site of hMAO-B might be
more difficult to reach by wider ligands.
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Figure 2. Active sites of hMAO-A (PDB ID: 2Z5X) and hMAO-B (PDB ID: 6FVZ). (A) MAO-A;
FAD cofactor (green sticks); active site (red surface) spans from FAD to the entrance; bottleneck
residues Leu97, Gly110 and Val210 at the entrance (salmon sticks). (B) MAO-B; FAD cofactor (green
sticks); active site (violet surface) spans from FAD to the entrance; bottleneck residues Glu84, Pro102
and Ser200 at the entrance (purple sticks).
The active site of hMAO-B is also significantly more curved than the active site of
hMAO-A, with a bend of 90°, while in hMAO-A, the cavity bends at an angle of ~60°
(compare Figure 2A, B). The narrow entrance of hMAO-B and the higher curvature might
prevent the relatively wider cis isomer 6 from entering the hMAO-B binding site. Indeed, 6
has a maximum width of 4.1 Å when measured as the height of the triangle composed of the
fluorine atom, one of the sp2 carbons, and the carbon bonded to the nitrogen, while the
maximum width of the trans isomer (1) is only 2.5 Å when measured as the distance between
two opposing carbon atoms in the piperidine ring. The cis isomer thus appears to inhibit
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hMAO-A but not hMAO-B because it is too wide to enter the narrower hMAO-B entrance. Its
maximum width is only slightly greater than the 3.8 Å diameter of the hMAO-A entrance,
while it is significantly greater than the 3.2 Å diameter of the hMAO-B entrance, which
suggests that the hMAO-B entrance would need to undergo more substantial conformational
changes to accommodate the cis isomer 6. The active site loop in hMAO-A contains Gly (i.e.,
Gly110), which is replaced by Pro102 in hMAO-B. This produces much higher rigidity in
comparison to hMAO-A, where the active site loop is more flexible. This lack of flexibility of
hMAO-B adds to the restricted access of the cis isomer to the hMAO-B active site. The cis
isomer 6 inactivity against hMAO-B thus appear to be caused by steric hindrance in the active
site, compound 6 being unable to fit into the elongated and rigid cavity (Figure 2).
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However, the selectivity of the trans isomer 1 is not explained only by the analysis of
its access to the active site, as trans isomer 1 can bind to both hMAO-A and hMAO-B. To
simulate the transport process of the inhibitor from the active site to the surface of the protein,
these two isomers were undocked from hMAO-A and hMAO-B using the CaverDock
software.22 Starting from the docked conformations of both ligands at the active sites, the
energetically most favourable trajectories of the ligands to the protein surfaces were
investigated (Figure 3A-H). In hMAO-A, cis isomer 6 is oriented with the propargyl group
facing the FAD cofactor, with a binding energy of –8.3 kcal/mol (Figure 3A, Movie_1_MAO-
A). As cis isomer 6 moves towards the entrance, its energy peaks at 0.4 kcal/mol and then
falls to –3.8 kcal/mol at the entrance (Figure 3B). Trans isomer 1 in the correct orientation has
a binding energy of –6.2 kcal/mol (Figure 3C), which is higher than for 6. As the trans isomer
is undocked, its binding energy peaks at –3.4 kcal/mol and then falls to –4.1 kcal/mol at the
entrance (Figure 3D). Further, the trans ligand in the ‘wrong’ orientation in the active site
(i.e., with the fluorophenyl group facing the FAD cofactor) has a much lower energy of –10.1
kcal/mol (Figure 3D; lower-bounds energy curve). However, in this orientation, the trans
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ligand cannot form the covalent bond with FAD. Thus, in agreement with the experimental
findings, the computational undocking simulation of hMAO-A showed that cis isomer 6 has
lower binding energy at the active site than trans isomer 1, which indicates that the cis isomer
is more likely to bind to hMAO-A than the trans isomer. In addition, the trans isomer might
preferentially access the active site in an orientation that does not allow formation of the
covalent bond, as can be seen by the lower binding energy of the ‘wrong’ conformation
compared to the energy of the correctly oriented trans isomer. This non-covalent only binding
of the trans isomer might lead to the worse performance observed experimentally of this
inhibitor with hMAO-A.
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In hMAO-B, the docked cis ligand 6 in the correct orientation in the active site has
energy of –6.5 kcal/mol (Figure 3E; Movie_2_MAO-B). No energy peaks were observed
along the active site for this isomer, whereby the energy reaches a plateau of 4.6 kcal/mol
near the active site entrance (Figure 3F). Trans isomer 1 in the correct orientation has the
energy of –7.4 kcal/mol (Figure 3G; Movie_2_MAO-B), which is lower than the energy of
the cis isomer, and then this falls further to –8.2 kcal/mol at the lowest point on the way out of
the entrance (Figure 3H). The energy barrier to reach the active site is lower for the trans
isomer than it is for the cis isomer, with the highest peak of 1.5 kcal/mol near the entrance,
which is lower than the 4.6 kcal/mol energy barrier for the cis isomer. This lower energy
barrier of the trans isomer at the entrance might be explained by the smaller maximum width
of the trans isomer compared to the cis isomer, as discussed above. We also note that the
docked trans inhibitor position in the active site has a root mean square deviation of 2.1 Å,
compared to the co-crystal structure of inhibitor 1 in hMAO-B reported in the present study
(PDB-ID: 6RKB). This indicates that the docking successfully predicted the positioning of
this inhibitor in the active site. In agreement with the experimental findings, the lower binding
energies at the active site for the trans isomer, and the lower energy barrier to reach the active
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site for the trans isomer, indicate that the binding of trans isomer 1 to hMAO-B is favored
compared to the binding of cis isomer 6.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Undocking of trans (1) and cis (6) isomers in hMAO-A and hMAO-B. (A) Docked 6 in
hMAO-A: cis isomer (yellow sticks) with propargyl group facing the FAD cofactor (green sticks),
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
with binding energy of –8.3 kcal/mol; bottleneck residues at the active site entrance at the back
(salmon sticks). (B) Energy profile for undocking of 6 from active site of hMAO-A in the correct
conformation (propargyl facing FAD). (C) Docked 1 in hMAO-A: trans isomer (violet sticks) with
propargyl group facing the FAD cofactor (green sticks), with binding energy of –6.2 kcal/mol;
bottleneck residues at active site entrance at the back (salmon sticks). (D) Energy profile for
undocking of 1 from active site of hMAO-A. (E) Docked 6 in hMAO-B: cis isomer (grey sticks) with
propargyl group facing FAD cofactor (green sticks), with binding energy of –6.5 kcal/mol; bottleneck
residues at entrance at the back (violet stick). (F) Energy profile for undocking of 6 from active site of
hMAO-B. (G) Docked 1 in hMAO-B: trans isomer (purple sticks) with propargyl group facing FAD
cofactor (green sticks), with binding energy of –7.4 kcal/mol; bottleneck residues at entrance at the
back (violet sticks). (H) Energy profile for undocking of 1 from active site of hMAO-B.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The distinct activity of this geometric isomer pair prompted us to study the structure–
activity relationships (SARs) through the construction of a focused library of substituted
piperidines (19–26; Scheme 2, Table 1) to probe the effect of piperidine disubstitution pattern
on the inhibitory potency. 1,4-Disubstituted piperidines were the most tolerated and showed
the highest selectivity for both hMAO-A and hMAO-B, and thus several analogues with
various substituents on the phenyl ring were synthesised (Scheme 3, compounds 67–107).
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Boc
N
Boc
N
Boc
N
H
N
i
ii
iii
N
O
COOH
CHO
O
R
nipecotic/
isonipecotic acid
2: 1,4-piperidine
7: 1,3-piperidine
3: 1,4-piperidine
8: 1,3-piperidine
9: 1,3-piperidine, R = 4-F-Ph, cis (Z)
10: 1,3-piperidine, R = 4-F-Ph, trans (E)
11: 1,3-piperidine, R = 2,4,5-F-Ph, cis (Z)
2: 1,3-piperidine, R = 2,4,5-F-Ph, trans (E)
13: 1,4-piperidine, R = 2,4,5-F-Ph, cis (Z)
14: 1,4-piperidine, R = 2,4,5-F-Ph, trans (E)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Boc
N
iv
R
15: 1,3-piperidine, R = 4-F-Ph
6: 1,3-piperidine, R = 2,4,5-F-Ph
7: 1,4-piperidine, R = 4-F-Ph (from 4/5, Scheme 1)
8: 1,4-piperidine, R = 2,4,5-F-Ph
v
1
1
1
v
N
R
N
19: 1,3-piperidine, R = 4-F-Ph, trans (E)
2
2
2
0: 1,3-piperidine, R = 2,4,5-F-Ph, trans (E)
1: 1,4-piperidine, R = 2,4,5-F-Ph, cis (Z)
2: 1,4-piperidine, R = 2,4,5-F-Ph, trans (E)
R
2
2
2
2
3: 1,3-piperidine, R = 4-F-Ph
4: 1,3-piperidine, R = 2,4,5-F-Ph
5: 1,4-piperidine, R = 4-F-Ph
6: 1,4-piperidine, R = 2,4,5-F-Ph
Scheme 2. Synthesis of inhibitors 19–26. (i) 1) Boc O, 1.0 M NaOH, 1,4-dioxane, 0 °C, RT, 1–3 h
2
(86–91%); 2) N,O-dimethyhydroxylamine hydrochloride, Et N, TBTU, CH Cl , 0 °C to RT, overnight
3
2
2
(
76–82%); (ii) LiAlH , anh. THF, 0 °C, 1–2 h (crude products, 79–83%); (iii) Corresponding Wittig
4
salts ((4-fluorobenzyl)triphenylphosphonium bromide or (2,4,5-trifluorobenzyl)triphenylphosphonium
bromide), NaHMDS (2 N in THF), anh. THF, Ar(g), RT, overnight (overall yield for both isomers
combined, 47–56%); (iv) Corresponding mixture of cis and trans isomer (9–14), H (g), Pd/C, EtOH,
2
RT, overnight (97–100%); (v) 1) 4.0 M HCl in 1,4-dioxane, 80 °C, 2 h (100%); 2) propargyl bromide
(
80% solution in toluene), Cs CO , DMF, Ar(g), 0 °C, RT, overnight (26–85%).
2 3
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
X
i
PPh3
Y
MAO inhibitors 67-103
67: R = Ph, cis (Z)
Y
X
68: R = Ph, trans (E)
69: R = 3-F-Ph, cis (Z)
70: R = 3-F-Ph, trans (E)
71: R = 2-F-Ph, cis (Z)
Witting salts
27a-l
Boc
72
: R = 2-F-Ph, trans (E)
73: R = 2-Cl-4-F-Ph, cis (Z)
74: R = 2-Cl-4-F-Ph, trans (E)
Boc
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
N
ii
iv
75
: R = 4-Me-Ph, cis (Z)
76: R = 4-Me-Ph, trans (E)
77: R = 4-iPr-Ph, cis (Z)
78: R = 4-iPr-Ph, trans (E)
CHO
R
R
79: R = 4-Cl-Ph, cis (Z)
8
8
8
8
8
0: R = 4-Cl-Ph, trans (E)
1: R = 4-Br-Ph, trans (E)
2: R = 4-OMe-Ph, trans (E)
3
28-55
67-92
3: R = 4-CF -Ph, cis (Z)
iii
3
4: R = 4-CF -Ph, trans (E)
3
85: R = 3-CF -Ph, cis (Z)
3
Boc
N
86: R = 3-CF -Ph, trans (E)
3
87: R = 4-CN-Ph, cis (Z)
N
88: R = 4-CN-Ph, trans (E)
iv
89: R = 4-SO Me-Ph, cis (Z)
2
9
0: R = 4-SO Me-Ph, trans (E)
2
91: R = 4-cyclopropyl-Ph, cis (Z)
92: R = 4-cyclopropyl-Ph, trans (E)
3: R = Ph
94: R = 3-F-Ph
R
9
R
93-103
5
6-66
95: R = 2-F-Ph
96: R = 4-Me-Ph
97: R = 4-iPr-Ph
4
2 and 43 (mixture):
R = 4-Br-Ph, cis (Z)/trans (E)
v
54
:
R = 4-cyclopropyl-Ph, cis (Z)
55: R = 4-cyclopropyl-Ph, trans (E)
98: R = 4-Cl-Ph
99: R = 4-MeO-Ph
1
00: R = 4-CF -Ph
3
8
8
1
7: R = 4-CN-Ph, cis (Z)
8: R = 4-CN-Ph, trans (E)
02: R = 4-CN-Ph
104: R = 4-CONH -Ph, cis (Z)
101: R = 3-CF -Ph
vi
2
3
105: R = 4-CONH -Ph, trans (E)
102: R = 4-CN-Ph
103: R = 4-n-propyl-Ph
2
106: R = 4-CONH -Ph
2
vii
9
9: R = 4-MeO-Ph
107: R = 4-OH-Ph
Scheme 3. Synthesis of inhibitors 67–107. (i) Benzyl halide, PPh , MeCN, 85 °C, overnight (80–
3
9
7%); (ii) Corresponding Wittig salts (27a–27l), NaHMDS (2 N in THF)/KHMDS (0.5 M in toluene),
anh. THF, Ar(g), RT, overnight (overall yield, 30–66%); (iii) Mixture of cis and trans isomer (28–55),
H (g), Pd/C, EtOH, RT, overnight (83–100%); (iv) 1) 4.0 M HCl in 1,4-dioxane (or HCl in EtOH), 80
2
°C, 2 h (100%); 2) propargyl bromide (80% in toluene), K CO , MeCN, Ar(g), 0 °C, RT, overnight
2
3
(6–89%). (v) Cyclopropylboronic acid, K PO , tricyclohexylphosphine (20% in toluene), Pd(OAc) ,
3
4
2
toluene, water, Ar(g), 100 °C, 3 h (78%); (vi) KOH, tBuOH, 90 °C, 12–24 h, (42–63%); (vii) BBr (1
3
M in CH Cl ), toluene, Ar(g), –20 °C, 1 h, RT, 1 h (57%).
2
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Structure activity relationships and stability of compounds
Hit inhibitor 1 and the synthesised analogues 6, 19–26 and 67–107 were evaluated for
their inhibition of both hMAO-A and hMAO-B using a horseradish peroxidase (HRP)-
Amplex Red coupled assay (Table 1). The most important findings regarding the SARs are
summarised in Figure 4. Scanning the 1,3- and 1,4-disubstitution patterns of piperidine
derivatives with the trans configuration of the double bond revealed at least 5-fold superior
inhibition of hMAO-B for the 1,4-disubstitution (e.g., 19, IC , 1470 nM vs. 1, IC , 342 nM).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
50
Interestingly, reduction of the double bond retained this selective hMAO-B inhibition (IC50,
43 nM, 370 nM, for hit compound 1, analogue 25, respectively. Broadening the series of 1,4-
2
disubsituted piperidines showcased selective inhibition of hMAO-A by cis analogues with
small substituents on the phenyl ring (e.g., fluorine). The most potent was 69, a 3-
fluorophenyl derivative, with an IC50 of 68.4 nM, and almost a 3-log unit selectivity over
hMAO-B. On the other hand, the trans derivatives preferentially inhibited hMAO-B, with a
general trend of improved inhibitory potencies with increasing hydrophobicity/ size of the
substituents on phenyl ring position 4 (-H < -F < -Cl < -Br and -H < -Me < -iPr ≈ -
cyclopropyl). Double-digit nanomolar inhibition of hMAO-B was also obtained for 82, 84,
88, and 90 with -OMe, -CF , -CN, -SO Me substituents, respectively, on position 4 of the
3
2
phenyl ring. Compounds with an ethyl moiety that connected the rings showed comparable
selective inhibition of hMAO-B as their trans congeners. All the N-propargylpiperidine
derivatives inhibited corresponding isoenzyme in an irreversible manner as demonstrated by
the 100-fold dilution assay.
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1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Linker:
-
-
cis linker: selective hMAO-A inhibitiors
trans linker: selective hMAO-B inhibitiors
N
- ethyl linker: selective hMAO-B inhibitors
3
4
Substitution pattern:
1,4-disubstituted superior to 1,3-disubstituted
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
R:
-
-
H, F or 3-CF3 tolerated for selective hMAO-A inhibition in cis derivatives
position 4: Cl, Br, Me, OMe, CN, CF3, iPr tolerated in selective hMAO-B inhibitors
Figure 4. Crucial observations regarding SARs of the stilbene-like MAO inhibitors.
Table 1. Inhibitory potencies and structures of hit compound 1 and all of the synthesised
inhibitors and positive controls.
N
3
linker
4
R
IC ±SEM
IC ±SEM
5
0
50
[
nM]
or
RA ±SD
[nM]
or
%RA ±SD
at 100 µM
Cpd
Structure
Selectivity
%
Ratio1
at 100 µM
Pdp2
Linker
R
hMAO-A
hMAO-B
Trans-
vinyl
1
6
1,4
4-F
60.2 ±1.6%
342.2 ±22.4
>292
1,4
1,4
1,4
Cis-vinyl
Ethyl
4-F
4-F
726.1 ±26.9
76.9 ±2.2%
6221.5 ±726.8
370.1 ±37.3
66.5 ±3.9%
0.12
>270
<0.05
2
2
5
1
Cis-vinyl
Trans-
vinyl
2,4,5-F
5243.7 ±323.4
2
2
1
2
6
9
1,4
1,4
1,3
1,3
1,3
2,4,5-F
2,4,5-F
4-F
81.2 ±3.1%
65.6 ±4.4%
91.0 ±3.1%
93.1 ±3.2%
102.5 ±5.2%
436.2 ±11.7
216.4 ±14.2
>229
>462
>68
Ethyl
Trans-
vinyl
1469.5 ±233.2
7411.6 ±1190.0
2195.6 ±228.3
23
20
Ethyl
4-F
>13
Trans-
vinyl
2,4,5-F
>46
1
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1,3
1,4
Ethyl
Cis-vinyl
Trans-
vinyl
2,4,5-F
H
86.0 ±5.2%
11229.4 ±974.7
>9
168.8 ±8.5
79.1 ±4.0%
<0.002
6
8
1,4
H
32960.7 ±3476.5
3540.2 ±611.8
9
93
1,4
1,4
Ethyl
H
75779.6 ±7566.03
68.4 ±4.3
4603.4 ±371.1
16
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
7
9
9
0
4
Cis-vinyl
Trans-
vinyl
3-F
48912.0 ±2525.4
0.001
1,4
3-F
6906.8 ±860.2
5048.7 ±1224.6
1.4
1,4
1,4
Ethyl
3-F
2-F
19927.3 ±1608.5
3013.7 ±207.3
3363.5 ±310.5
6
71
Cis-vinyl
Trans-
vinyl
65.5 ±0.3%
<0.03
7
9
7
2
5
3
1,4
1,4
1,4
2-F
2-F
27293.2 ±1758.4
11830.4 ±766.5
14159.5 ±708.9
53000.5 ±2662.5
2
Ethyl
5927.3 ±223.8
0.4
0.9
4
6121.5
Cis-vinyl
2-Cl-4-F
±
10141.6
Trans-
vinyl
7
7
7
4
5
6
1,4
1,4
1,4
2-Cl-4-F
4-Me
66028.4 ±8419.0
62.2 ±4.4%
417.6 ±46.9
19162.8 ±5139.7
147.0 ±13.8
158
>5
Cis-vinyl
Trans-
vinyl
4-Me
57.3 ±2.9%
>680
96
1,4
1,4
Ethyl
4-Me
68.1 ±1.6%
83.2 ±0.5%
159.0 ±13.3
>630
>53
7
7
Cis-vinyl
Trans-
vinyl
4-iPr
1883.9 ±278.6
73638.2
7
8
1,4
1,4
1,4
4-iPr
4-iPr
4-Cl
44.4 ±8.2
7.0 ±2.2
1658
23200
10
±11010.13
1
62399.3
±9715.93
18844.3
9
7
7
9
Ethyl
1
Cis-vinyl
11745.1 ±555.9
±26412.73
71.7 ±0.2%
67.7 ±0.0%
54.5 ±1.2%
Trans-
vinyl
8
9
8
0
8
1
1,4
1,4
1,4
4-Cl
4-Cl
4-Br
50.4 ±4.7
38.1 ±6.8
21.2 ±4.2
>1984
>2625
>4717
Ethyl
Trans-
vinyl
Trans-
vinyl
8
9
2
9
1,4
4-OMe
4-OMe
56.1 ±1.6%
68.1 ±11.1
>1468
1,4
1,4
Ethyl
62.2 ±1.9%
80.1 ±4.5%
67.0 ±11.4
>1493
>66
83
Cis-vinyl
Trans-
vinyl
4-CF
4-CF
4-CF
3
3
3
1506.6 ±216.3
84
1,4
1,4
64.9 ±0.5%
62.3 ±0.7%
18.6 ±3.3
10.9 ±3.0
>5376
>9174
1
00
Ethyl
1
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1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
8
8
5
6
1,4
1,4
Cis-vinyl
Trans-
vinyl
3-CF
3-CF
3-CF
3
3
3
858.0 ±29.2
9352.5 ±1512.9
549.7 ±127.5
0.09
64.8 ±4.0%
>182
1
01
1,4
1,4
Ethyl
71.6 ±2.2%
68.1 ±2.7%
129064.0
501.8 ±107.1
1745.3 ±315.4
>199
>57
87
Cis-vinyl
Trans-
vinyl
4-CN
4-CN
4-CN
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
88
02
1,4
57.0 ±7.1
2264
±8079.73
1
1,4
1,4
Ethyl
21478.3 ±6280.3
70.6 ±5.8%
56.6 ±8.7
379
>12
8
9
Cis-vinyl
Trans-
vinyl
4-SO
2
Me
8444.7 ±627.1
9
0
1
2
1,4
1,4
1,4
4-SO
2
Me
68.7 ±0.2%
7398.7 ±661.8
20564.5 ±2315.8
32.7 ±2.5
2640.1 ±342.8
43.7 ±6.4
>3058
3
4-
9
9
Cis-vinyl
Cyclopropyl
4-
Trans-
471
vinyl
Cyclopropyl
4-propyl
1
1
03
04
1,4
1,4
Ethyl
80305.3 ±9033.0
36.1 ±4.1
2225
2
Cis-vinyl
4-CONH
2
2
2
89.5 ±0.6%
51019.0 ±6046.5
Trans-
1
1
05
06
1,4
4-CONH
64.8 ±1.1%
1737.0 ±691.0
58
vinyl
1,4
1,4
Ethyl
4-CONH
4-OH
75.1 ±2.2%
67.7 ±0.5%
5554.5 ±1778.9
7440.7 ±1291.3
195.5 ±19.1
12.1 ±4.2
>18
>13
107
Ethyl
Pargyline
L-Deprenyl
Rasagiline
Clorgiline
/
/
/
/
3967.9 ±275.1
62663.8 ±4113.6
29520 ±8597
3.4 ±0.3
20
5179
820
36.0 ±4.1
13568.4 ±1157.3
0.0003
1
Data are means ±SEM; IC , n = 2, 3; Residual activity (RA), n = 2. Selectivity ratio is defined as
5
0
2
3
IC (hMAO-A)/IC (hMAO-B). Pdn: piperidine disubstitution pattern. Estimated IC values from
5
0
50
50
the RAs bellow 100 µM. Compounds showed precipitation in the assay buffer at concentrations above
00 µM.
1
For additional experiments, compounds 1, 6, 67, 69, 84, 97 and 100 were used in the
form of hydrochloride salts to avoid possible solubility issues. The thermodynamic solubility
of the compounds in PBS (pH 7.4) was at least 17.6 g/250 mL; 10.5 g/250 mL; 8.6 g/250 mL;
12.5 g/250 mL; 14.5 g/250 mL and 16.3 g/250 mL for 6, 67, 69, 84, 97 and 100, respectively,
1
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which is good solubility according to biopharmaceutical classification for any realistically
expected dose of investigated compounds.
Possible chemical instability and photoisomerization of E/Z isomers were also
addressed as these are known liabilities of stilbene derivatives.23 Proton NMRs of 69 (cis
derivative), 84 (trans derivative), and 100 (ethyl analogue) in the form of hydrochloride salts
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
were recorded in D O (Figures S1–S3). Inspection of the spectra confirmed that these
2
compounds are relatively stable after more than one year storage at room temperature
(protected from sun light) as only smaller amounts of impurities were detected for 84 (in the
aromatic area), whereas compounds 69 and 100 showed no signs of chemical instability. The
samples dissolved in the NMR tubes were then left on the benchtop exposed to natural day
light for 7 days, which could induce photoisomerization. No spectral changes were observed
in comparison to spectra of freshly prepared samples. Altogether, these studies imply
reasonable stability of the compounds.
Kinetic evaluation
The exploration of SARs allowed the identification of a few compounds for more in-
depth kinetic analysis with the purified enzyme samples, which allowed their inhibition
mechanism to be better defined. On the basis of their inhibitory potencies and molecular
features, compounds 1, 84 and 97 were selected for hMAO-B, whereas for hMAO-A the
analysis was restricted to 69. The inhibitors were tested using UV-Vis spectrum
measurements (Figure 5) and steady-state enzymatic assays (Table 2). The propargyl unit is
known to react covalently with the N5 atom of the flavin cofactor by rapidly forming a stable
covalent adduct that produces a typical modification of the spectrum; this is seen as bleaching
2
4
of the peak at 450 nm and appearance of a stronger peak at 415 nm. All of these selected
compounds were tested by adding them in 10-fold excess to the protein solution in the
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
spectrophotometer cuvette and measuring the spectra over time until no further modification
was detected. All of the irreversible inhibitors produced a clear change in the enzyme spectra
(Figure 5A-D), similar to other classic acetylenic inhibitors, like L-deprenyl and clorgyline.24
However, there were differences in the times required to complete the reactions for the adduct
formation; i.e., to reach the final spectrum with no further modification. While 69 fully
inactivated hMAO-A almost instantaneously (Figure 5A), similar to clorgyline, the hMAO-B
selective compounds were slower. In particular, 84 initiated the reaction with the flavin within
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
min, showing an intermediate spectrum profile that evolved into the final 415 nm peak in 30
min (Figure 5B). Instead, 1 and 97 did not show any spectral modifications in the first few
minutes, and required half an hour to obtain an intermediate profile, and more than 2 h to
complete the reaction (Figure 5C, D, respectively).
Figure 5. UV-Vis absorption spectra of the hMAO enzymes after FAD cofactor modification by
irreversible covalent inhibitors. The oxidised enzyme (10 µM) before inhibitor addition (100 µM) is
shown as the continuous black line, whereas the grey profile indicates the final spectrum (i.e., no
further changes were observed), which was reached at different times, depending on the inhibitor. The
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intermediate spectra are shown as either dotted or dashed lines. (A) hMAO-A and 69; (B) hMAO-B
and 84; (C) hMAO-B and 1; (D) hMAO-B and 97.
Full kinetic analysis was then undertaken for hMAO-A and hMAO-B inhibition by the
selected compounds, using purified enzyme samples and the spectrophotometric version of
the assay coupled to HRP that was used for IC50 determination, as described above. As
enzyme inhibition by propargyl-based inhibitors involves a first step in which the ligand is
accommodated into the protein active site to be correctly oriented for flavin adduct formation,
competitive inhibition can be measured under steady-state conditions using kynuramine and
benzylamine as substrates for hMAO-A and hMAO-B, respectively. Thus, both the reversible
and irreversible inhibition parameters were determined (Table 2). Compound 69 is a potent
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hMAO-A inhibitor with a reversible K of 0.23 µM, which is 5-fold higher that reported for
i
clorgyline. This difference is in line with the irreversible constant K and it is balanced by the
I,
irreversible inactivation velocity that is 10-fold higher that for clorgyline. Overall, 69 is a very
potent hMAO-A inhibitor with comparable activity to clorgyline. Similar to L-deprenyl, in the
case of hMAO-B selective ligands, the reversible K values are all in the micromolar range.
i
The irreversible constant K for covalent hMAO-B inhibitors is in general 5-fold the
I
reversible K ; however, the k /K indicated comparable inhibitory potencies of the hMAO-B
i
inact
I
inhibitors to L-deprenyl.
Table 2. Kinetic parameters for the reversible and irreversible inhibition of the hMAOs by the selected
-styrylpiperidine inhibitors.
MAO Inhibitor
4
Reversible
inhibition
Irreversible inhibition
kinact/KI
–
1
–1
–1
K (µM)
i
K (µM)
I
kinact (min )
(min µM )
hMAO-A
69
0.23 ±0.02
1.01 ±0.76
1.25 ±0.25
1.24
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Clorgylinea
0.040 ±0.004b
0.22 ±0.08b
0.12 ±0.05b
0.55b
hMAO-B
1
1.92 ±0.32
1.25 ±0.23
1.36 ±0.31
0.97 ±0.11
1.38 ±0.29
17.25 ±4.38
6.55 ±1.62
5.68 ±0.78
0.66 ±0.04
2.03 ±0.38
1.04 ±0.12
2.56 ± 0.24
0.48
0.12
0.16
0.45
8
9
4
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
L-deprenyla
a
Kinetic parameters were compared to clorgyline and L-deprenyl in the case of hMAO-A and hMAO-
b
25
B selective inhibitors, respectively. Data from Esteban et al. (2014).
Structure of hMAO-B in complex with inhibitors
Structural studies were performed by co-crystallisation of hMAO-B with the inhibitors
selected for the kinetic analyses, using previously published procedures.26 Structures for
inhibitors 1, 84 and 97 were solved at resolution ranging from 2.3 Å to 1.7 Å (Figure 6A-C).
The crystallographic statistics are reported in Table S1. Crystallographic studies on hMAO-B
in complex with 1, 84 and 97 showed that these inhibitor molecules are certain to be bound to
the enzyme active site (Figure 6A-C). Ligand binding does not cause any major
conformational changes to the overall protein dimeric structure (Figure S4). As for all of the
inhibitors, the structure of the active site is identical in the two monomers present in the
asymmetric unit that forms the protein dimer, and here we will refer to chain A of each
structure in the following discussion. The electron density map clearly showed that all of
these inhibitors form a covalent adduct with the flavin (Figure 6A-C).
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Figure 6. Crystal structures of the active site of hMAO-B-inhibitor complexes. The hMAO-B
active site cavity is shown as a grey semi-transparent surface, which is lined by the protein residues
(represented in cyan). Water molecules are red spheres, and hydrogen bonds are dashed lines. The
FAD cofactor is in yellow stick representation. The inhibitor molecule bound in the hMAO-B active
site cavity is shown as sticks, with carbon, nitrogen and fluorine in magenta, blue and black,
respectively. The refined 2Fo − Fc electron density map contoured at 1.2σ is shown in blue for the
inhibitor and the FAD molecules. (A) Active site of hMAO-B in complex with 1 (2.3 Å resolution;
PDB code, 6RKB). (B) Active site of hMAO-B in complex with 84 (1.7 Å resolution; PDB code,
6RKP). (C) Active site of hMAO-B in complex with 97 (2.3 Å resolution; PDB code, 6RLE).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All of these inhibitors bind to the hMAO-B active site in a similar fashion, from the
covalent adduct with the flavin, to the 4-substituted phenyl ring at the other end of the
molecule (Figure 6A-C). They adopt an extended conformation, as they occupy both the
substrate and the cavity spaces (Ile199 in the open conformation). The 4-substituted phenyl
ring is constrained by the flat hydrophobic shape of the hMAO-B cavity in a position
perpendicular to the flavin plane, as for other well-known inhibitors, such as L-deprenyl and
safinamide (Figure 7A). The site occupied by the halogen substituent on the aromatic ring of
1
is also conserved, which is a common feature of many hMAO-B inhibitors.27 The more
polar substituents that have lower inhibitory activities (e.g., 82, 105) would be much less
favoured in their binding to this highly hydrophobic site. The enzyme active site architecture
does not undergo any conformational change with respect to that of other hMAO-B structures,
except for minor modifications to the side chains of Cys172 and Tyr188 (Figure 7A). This is
due to the position adopted by the piperidine ring that represents the hallmark of this series of
inhibitors. Despite the flat shape of the hMAO-B cavity, the non-planar conformation of the
piperidine and its pyramidal nitrogen that is involved in the covalent adduct constrain the
piperidine ring to bind perpendicular to the plane of the cavity (Figure 6A-C). Instead, with
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1
1
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
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5
5
5
5
6
other inhibitors such as L-deprenyl and safinamide, there is an aromatic ring that binds co-
planar with the cavity plane, and with any substituted aromatic at the other end of the
molecule, if present (Figure 7A). Therefore, the position of the piperidine in the upper part of
the cavity allows Cys172 and Tyr188 to move slightly upwards. In the structure of the
complex with 97, the conformation of the Cys172 and Tyr188 is conserved to some extent
with respect to the previous structures. This is due to the linker that is devoid of the double
bond, and the consequent tetrahedral conformation of its carbon atoms results in a more
contracted conformation of the inhibitor molecule with respect to 84 (Figure 7B) and 1. As a
result, the piperidine collapses towards the bottom of the cavity, and the covalent linkage is
forced to adopt a slightly different conformation with respect to the other propargyl inhibitors
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 7A, B).
Figure 7. Structural superposition to highlight differences and common features in the binding
modes of the hMAO-B inhibitors. The protein, inhibitor and cofactor atoms are represented as sticks
(colour code as in Figure 6A, except for the carbon atoms of protein and inhibitor molecules, which
are depicted accordingly to distinguish the different structures). For clarity, only Cys172 and Tyr188
of the protein active site residues are shown, which undergo some conformational changes. (A)
Superposition of structures in complex with 84 (green), safinamide (grey) and L-deprenyl (pink). (B)
Superposition of structures in complex with 84 (green) and 97 (light grey).
Cell-based assays
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Following the chemical-space exploration and kinetic and structural evaluations,
several of these structurally diverse hMAO-A and hMAO-B inhibitors were also tested in
vitro in cell models. First, their cytotoxicity profiles were defined using the SH-SY5Y human
neuroblastoma cell line. Using the MTS assay, no significant cytotoxic effects were seen for
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
4-h treatments of these cells with the compounds 1, 6, 67, 69, 84, 97, 100 and 102 at
concentrations of 5 µM and 10 µM (Figure S5). Indeed, at 50 µM, the cell viabilities
remained >80% for almost all of the compounds, which is roughly three log units higher than
the concentrations needed to achieve 50% in vitro inhibition of either of the two MAO
isoforms.
The experimental in vitro cell model with the neurotoxin 6-hydroxydopamine (6-
OHDA) is commonly used to study neurodegenerative processes, as it induces toxicity that
mimics the biochemistry and neuropathology of Parkinson’s disease.28 To determine the
possibility that MAO inhibitors can abrogate 6-OHDA–induced toxicity, we examined lactate
dehydrogenase (LDH) release and propidium iodide (PI) staining in the SH-SY5Y cells after
3
0 min pretreatment with the compounds at the non-cytotoxic concentration of 5 µM,
followed by 6-OHDA treatment for 24 h (Figure 8A, B). The active treatment with 6-OHDA
significantly increased LDH release (135% vs. control), while pretreatment with compounds
1
, 84, 97 and 102 protected against this increased LDH release (76%, 87%, 82%, 82% vs. 6-
OHDA, respectively), thus indicating improved cell viability. In the confirmation using flow
cytometry, the proportion of PI-positive cells increased after the active 24-h treatment with 6-
OHDA, and was significantly reduced following the pretreatments with the hMAO-B
inhibitors 84 and 97 (0.65 and 0.68, respectively), compared to the 6-OHDA–treated cells.
Altogether, this indicates that the selective hMAO-B inhibitors 84 and 97 reduced the 6-
OHDA–induced cytotoxicity and increased the viability of these SH-SY5Y neuronal-like
cells.
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1
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2
2
2
2
2
2
3
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6
A
B
0
1
2
3
4
5
6
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9
0
1
2
3
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9
0
1
2
3
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5
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Effects of inhibitors on 6-OHDA–induced cell death in SH-SY5Y cells. (A, B) SH-SY5Y
cells were pretreated with the compounds (5 µM) for 30 min (as indicated), and then treated with 6-
OHDA (100 µM). After 24 h, LDH assays (A) and PI staining (B) were performed. Data are means
±
standard deviation (n ≥2; each in duplicate). *, p <0.05 (one-way ANOVA, with Bonferroni
correction, followed by t-tests).
In vivo MAO inhibition in mouse brain homogenates, and in vitro permeability
The inhibition of the MAOs in mouse brain homogenates was also investigated both in
vitro and ex vivo (Table 3). Here, the more potent of the inhibitors against hMAO-A (i.e., 69)
and hMAO-B (i.e., 100) were tested based on their IC values and structural features. Brain
5
0
homogenates from male Swiss mice were first characterised in terms of their MAOs activity
and substrate specificity (Table S2, Figure S6).
Table 3. In vitro and ex vivo mouse brain homogenate residual activities of 69 and 100 at 60 mg/kg
i.p. on MAO-A and MAO-B according to substrate.
Compound
Residual activity (%)
MAO-A/B
MAO-A
substrate
MAO-B
substrate
substrate
5
-HT (0.25 mM)
p-Tyramine (0.5 mM)
Benzylamine (0.5 mM)
In vitroa
–1.3 ±0.1
n.d.
Ex vivob
1.6 ±0.02
n.d.
In vitroa
31.3 ±0.2
59.4 ±0.2
Ex vivob
14.2 ±0.2
77.2 ±0.2
In vitroa
n.d.
Ex vivob
n.d.
6
9
1
00
–7.7 ±0.2
24.3 ±0.5
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a
Data are means ±SD (n = 1, in triplicate). n.d., not determined. In vitro: 50 µM compounds (final
concentrations) versus 0.9% saline with mouse brain homogenate from non-treated mice. 2 Ex vivo:
brain homogenates from mice treated (n = 3, per group) with 60 mg/kg i.p. compounds or 0.9% saline
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Compound 69 completely abolished MAO-A activity in vitro at 50 µM when 5-HT
was used as the selective MAO-A substrate. Compound 100 at 50 µM inhibited mice MAO-B
when benzylamine was used as the selective MAO-B substrate (Table 3). When the dual
hMAO-A/B substrate of p-tyramine was used, the inhibition was lower (i.e., higher residual
activities; RAs). The same profile of inhibition was established ex vivo for brain homogenates
from mice treated with these compounds, which were injected intraperitoneally (i.p.) 30 min
before preparation of the brain homogenates. Again, more potent inhibition was seen in these
brain homogenates when the isoenzyme-selective substrates were used. Selective MAO-A
inhibitor 69 was the most active compound when administered at 60 mg/kg i.p. (RA: 1.6%
±
0.02%), while 60 mg/kg i.p. 100 resulted in similar MAO-B inhibition (RA: 24.3% ±0.5%).
Compounds 69 and 100 showed dose-dependent inhibition of these corresponding MAO
isoforms (ED : 0.43 ±0.26 mg/kg and 1.38 ±0.87 mg/kg, respectively).
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MAO inhibitors are in clinical use for treatment of psychiatric and neurological
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disorders, where they are used in chronic, low-dose regimes. These MAO-A and MAO-B
activities in ex vivo mouse brain homogenates were therefore studied after chronic 10-day
administration to the mice of 69 and 100 at the low dose of 0.3 mg/kg i.p.. This chronic
administration of selective MAO-A inhibitor 69 significantly reduced MAO-A activity
compared to 0.9% saline i.p. in the control mice. After the single administration of 69 at 0.3
mg/kg i.p., the ex vivo MAO-A RA was reduced to 67.8% ±31.2% (i.e., compared to 0.9%
saline), whereas the 10-day treatment resulted in MAO-A RA of 9.5% ±23.4%. Similar to 69,
one single i.p. injection of 100 at 0.3 mg/kg showed MAO-B RA of 83.4% ±19.2%, while
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chronic 10-day treatment resulted in RA of 28.3% ±13.8%, which demonstrated accumulation
of inhibition over time.
The ex vivo inhibitory activity of inhibitors 69 and 100 was also demonstrated in the
mouse brain homogenates prepared 90 min after acute oral administration to the mice, via oral
gavage at 30 mg/kg. Inhibitors 69 and 100 showed RAs of 45.3% ±14.6% (vs. 0.9% saline-
treated mice), and 30.5% ±10.4% (vs. 0.9% saline treated mice), respectively. This prompted
us to also study gut permeability of these representative inhibitors (i.e., 69, 84 and 100) in
vitro on rat intestine. The apparent bidirectional permeability coefficient for the rat intestine
in vitro was determined for 69. This was 108 ±13 nm/s (as mean ±SEM) in the absorptive
direction, and 354 ±99 nm/s and in the eliminatory direction. For comparison, the apparent
permeability coefficients of the internal low- and high-permeability standards furosemide and
metoprolol in the absorptive direction were 64 ±11 nm/s and 140 ±18 nm/s, respectively.
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Behavioural studies with the selective MAO-A and MAO-B inhibitors
MAO-A inhibitors are currently used in the therapy of treatment-resistant major
depressive disorder and some specific types of depression.30 Symptoms of depression
31
correlate highly with those of anxiety. These have been shown to be associated with 5-HT
1
A
receptors,32 which are presynaptic and postsynaptic G-protein–coupled receptors that are
involved in neuromodulation. 5-HT1A receptor agonists, such as buspirone, show efficacy for
the relief of anxiety and depression.32 The binding affinities (K values) for this 5-HT
i
1A
receptor subtype were thus determined for these MAO inhibitors, to potentially further
explain their behavioural effects. These ranged from low micromolar to submicromolar Ki
values (Table S3). Following treatments with the selective irreversible MAO-A inhibitors 6,
6
7 and 69, the Swiss mice were subjected to the different behavioural tasks to determine
whether these compounds have any antidepressant-like activity, any activity in an anxiety
model, and whether they affect locomotor activity.33
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The antidepressant-like activity was screened with the widely used tail suspension test.
At 30 min before the behavioural assays, the mice were treated with 6, 67 and 69 at 3 mg/kg
and 10 mg/kg i.p., with 69 also at 1 mg/kg and 30 mg/kg i.p., or were injected with 0.9%
saline as control. Imipramine (30 mg/kg i.p.) was used as the reference drug. Compounds 67
and 69 did not decrease the immobility time, whereas selective MAO-A inhibitor 6 and
imipramine significantly reduced it, which demonstrated the antidepressant-like effects of
these compounds under acute treatment (Figure 9A).
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The performance of mice in the ‘elevated plus maze test’ was used here as a rodent
model of anxiety, which was carried out 30 min after the i.p. treatments with 6, 67 and 69
(Table S4). Compound 6 at the dose of 10 mg/kg i.p. induced a significant decrease compared
to mice injected with 0.9% saline, for both the number of total arm entries (9.7 ±2.9 vs. 22.0
±
1.5; p <0.001) and the proportion of time spent in the centre of the apparatus (13.4 ±2.3 vs.
43.5 ±2.8; p <0.001), and also an increase in the proportion of open-arm entries (2.0-fold; p
0.001). Meanwhile, 69 at 3 mg/kg i.p. produced a significant increase in the proportion of
open-arm entries (2.0-fold; p <0.001) and in the time spent in those entries (2.1-fold; p
0.001), similar to diazepam (reference drug) at 1 mg/kg i.p. (2.1-fold, 2.4-fold, respectively;
p <0.001 for both).
The locomotor activity of the mice was also determined, as it can affect the data
<
<
obtained in these behavioural assays. It was shown that 6 at 10 mg/kg i.p. significantly
decreased spontaneous locomotor activity (Figure 9B), which is correlated with the decrease
in the total arm entries in the elevated plus maze test. This indicates that the effects of 6 after
acute 10 mg/kg i.p. treatment are not associated with antidepressant-like or anxiolytic effects,
but rather with a decrease in the locomotion of the mice. On the other hand, the increased
open arm parameters in the elevated plus maze for 69 (3 mg/kg i.p.) appear to be correlated
with an anxiolytic-like effect, which would be mediated through the 5-HT1A receptor, as
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locomotion was not altered at this dose of 69. Importantly, chronic 10-day treatment with 69
at a low dose (i.e., 0.3 mg/kg i.p., daily) significantly decreased the immobility time in the
treated group (Figure 9C). No significant effects were seen for this chronic low dose of 69 in
the elevated plus maze and locomotor activity tests (Table S5). This confirmed the
antidepressant-like effects of 69 when administered chronically, which were not seen for the
acute treatment regime.
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A
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Figure 9. In-vivo evaluation in mice of antidepressant-like activity of the selective MAO-A
inhibitors 6, 67 and 69. (A) Effects of acute i.p. treatment with 6 (3, 10 mg/kg, n = 13, 9), 67 (3, 10
mg/kg, n = 6, 10) and 69 (1, 3, 10, 30 mg/kg, n = 8, 7, 8, 6) and imipramine (30 mg/kg, n = 8) in the
tail suspension test. Data are means ±SEM of immobility time in comparison to control mice (SAL, n
= 23). *, p <0.05; ***, p <0.001 (one-way ANOVA, followed by Dunnett’s tests). (B) Effects of acute
i.p. treatment with 6 (3, 10 mg/kg, n = 8, 8), 67 (1, 3, 6 mg/kg; n = 5, 5, 5) and 69 (1, 3, 6 mg/kg, n =
1
0, 5, 5) on locomotor activity of the mice. Data are means ±SEM of spontaneous locomotor activity
counts recorded in 5-min sessions. ***, p <0.001 (one-way ANOVA, followed by Dunnett’s tests).
C) Effects of 10-day chronic i.p. treatment with 69 (0.3 mg/kg, daily; n = 10) in the tail suspension
test. Data are means ±SEM of the immobility time, compared to control mice (n = 8). **, p <0.01
unpaired t-test).
(
(
Behavioural studies of the selective hMAO-B inhibitors were performed with
compound 100 (irreversible MAO-B inhibitor). Although trans derivative 84 also showed
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potent irreversible MAO-B inhibition (IC = 18.6 ±3.3 nM; equipotent to 100), it was not
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studied here in vivo due to its poor permeability in the rat gut (see above) and its low binding
potential for the 5-HT1A receptor (Table S3).
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The hole–board test is commonly used to study stress-related changes in exploratory
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behaviour of animals. This was combined here with the locomotor activity test. At acute i.p.
doses ≥10 mg/kg of 100 in the mice, the hole–board test parameters and locomotor activities
were reduced (Table S6, Figure S7A-C). Due to the locomotor impairment at high doses,
compound 100 was further studied in the haloperidol-induced catalepsy test at lower doses
(see Supplementary Results and Discussion). Compound 100 was also tested in the same
battery of behavioural tests after 10 days of low-dose i.p. treatment (0.3 mg/kg, daily), where
it did not affect the locomotor activity of the mice (Figure S7D), nor the parameters of the
hole–board test (Figure S8). During these 10-day treatments (i.e., 69, 100 at 0.3 mg/kg i.p.,
daily), the body mass of the mice was also monitored (Figure S9). No significant differences
were seen for the proportions of body mass variations versus the control mice (0.9% saline
treated). Also, all of these mice survived these 10-day treatments without any apparent signs
of changes in behaviour, which supports the general safety of these compounds.
DISCUSSION AND CONCLUSIONS
Biological receptors differ greatly in shape, polarity and conformational dynamics.1
However, it is a challenging task to obtain selectivity between structurally related targets such
as isoenzymes, which requires different ligand-design approaches. MAO-A and MAO-B are
two closely related isoenzymes where numerous selective inhibitors have been described in
the literature, with some now used in the clinical setting for treatment of neurological
disorders.35
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