Design of enamides as new selective monoamine oxidase-B inhibitors

Article abstract of DOI:10.1111/jphp.13264

Objectives: To develop of new class of selective and reversible MAO-B inhibitors from enamides. Methods: Syntheses of the titled derivatives (AD1–AD11) were achieved by reacting cinnamoyl chloride and various primary and secondary amines in basic medium. All eleven compounds were investigated for in vitro inhibitory activities against recombinant human MAO-A and MAO-B. The reversibilities of lead compound inhibitions were analysed by dialysis. MTT assays of lead compounds were performed using normal VERO cell lines. Key findings: Compounds AD3 and AD9 exhibited the greatest inhibitory activity against MAO-B with IC₅₀ values of 0.11 and 0.10?μm, respectively, and were followed by AD2 and AD1 (0.51 and 0.71?μm, respectively). Most of the compounds weakly inhibited MAO-A, with the exceptions AD9 and AD7, which had IC₅₀ values of 4.21 and 5.95?μm, respectively. AD3 had the highest selectivity index (SI) value for MAO-B (>363.6) and was followed by AD9 (SI 42.1). AD3 and AD9 were found to be competitive inhibitors of MAO-B with K_i values of 0.044?±?0.0036 and 0.039?±?0.0047?μm, respectively. Reversibility experiments showed AD3 and AD9 were reversible inhibitors of MAO-B; dialysis restored the activity of MAO-B to the reference level. MTT assays revealed AD3 and AD9 were non-toxic to normal VERO cell lines with IC₅₀ values of 153.96 and 194.04?μg/ml, respectively. Computational studies provided hypothetical binding modes for AD3 and AD9 in the binding cavities of MAO-A and MAO-B. Conclusions: These results encourage further studies on the enamide scaffold as potential drug candidates for the treatment of Alzheimer's and Parkinson's diseases.

Full text of DOI:10.1111/jphp.13264

Research Paper
Design of enamides as new selective monoamine oxidase-B
inhibitors
a
b
a
a
a
Fathima Sahla Kavully , Jong Min Oh , Sanal Dev , Swafvan Kaipakasseri , Ashique Palakkathondi ,
a
a
c
d
Ajeesh Vengamthodi , Rinshana Fathima Abdul Azeez , Anna Rita Tondo , Orazio Nicolotti , Hoon
Kim and Bijo Mathew
b
e
a
b
Department of Pharmaceutical Chemistry, Al-Shifa College of Pharmacy, Perinthalmanna, India, Department of Pharmacy, and Research Institute
c
of Life Pharmaceutical Sciences, Sunchon National University, Suncheon, Korea, Instituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano,
d
e
Italy, Dipartimento di Farmacia—Scienze del Farmaco, Universit ꢀa degli Studi di Bari “Aldo Moro”, Bari, Italy and Division of Drug Design and
Medicinal Chemistry Research Lab, Department of Pharmaceutical Chemistry, Ahalia School of Pharmacy, Palakkad, India
Keywords
Abstract
docking simulations; enamides; kinetics;
MAO-B inhibitors; reversibilities
Objectives To develop of new class of selective and reversible MAO-B inhibitors
from enamides.
Correspondence
Methods Syntheses of the titled derivatives (AD1–AD11) were achieved by react-
ing cinnamoyl chloride and various primary and secondary amines in basic med-
ium. All eleven compounds were investigated for in vitro inhibitory activities
against recombinant human MAO-A and MAO-B. The reversibilities of lead
compound inhibitions were analysed by dialysis. MTT assays of lead compounds
were performed using normal VERO cell lines.
Bijo Mathew, Division of Drug Design and
Medicinal Chemistry Research Lab,
Department of Pharmaceutical Chemistry,
Ahalia School of Pharmacy, Palakkad
6
78557, Kerala, India.
E-mails: bijovilaventgu@gmail.com;
bijo.mathew@ahalia.ac.in
Key findings Compounds AD3 and AD9 exhibited the greatest inhibitory activ-
ity against MAO-B with IC50 values of 0.11 and 0.10 µM, respectively, and were
followed by AD2 and AD1 (0.51 and 0.71 µM, respectively). Most of the com-
pounds weakly inhibited MAO-A, with the exceptions AD9 and AD7, which had
IC50 values of 4.21 and 5.95 µM, respectively. AD3 had the highest selectivity
index (SI) value for MAO-B (>363.6) and was followed by AD9 (SI 42.1). AD3
Hoon Kim, Department of Pharmacy, and
Research Institute of Life Pharmaceutical
Sciences, Sunchon National University,
Suncheon 57922, Korea.
E-mail: hoon@sunchon.ac.kr
Sanal Dev, Department of Pharmaceutical
Chemistry, Al-Shifa College of Pharmacy,
Perinthalmanna 679325, Kerala, India.
E-mail: sanaldev@gmail.com
and AD9 were found to be competitive inhibitors of MAO-B with K values of
i
0.044 ꢀ 0.0036 and 0.039 ꢀ 0.0047 µM, respectively. Reversibility experiments
showed AD3 and AD9 were reversible inhibitors of MAO-B; dialysis restored the
activity of MAO-B to the reference level. MTT assays revealed AD3 and AD9
were non-toxic to normal VERO cell lines with IC50 values of 153.96 and
Received September 30, 2019
Accepted March 8, 2020
194.04 µg/ml, respectively. Computational studies provided hypothetical binding
modes for AD3 and AD9 in the binding cavities of MAO-A and MAO-B.
Conclusions These results encourage further studies on the enamide scaffold as
potential drug candidates for the treatment of Alzheimer’s and Parkinson’s dis-
eases.
doi: 10.1111/jphp.13264
Fathima Sahla Kavully and Jong Min Oh
contributed equally.
deamination of adrenaline, noradrenaline and serotonin,
whereas MAO-B catalyses the deamination of b-phenethy-
Introduction
[
3]
Monoamine oxidases (MAOs) are flavin adenine dinu-
cleotide (FAD) containing enzymes and play prominent
roles in the oxidative deamination of various dietary and
lamine, dopamine and benzylamine, and the degradation
of these biogenic amines by MAOs can lead to emotional
behavioural changes and various neurodegenerative disor-
[
1]
[4]
biogenic amines. MAOs exist in two isoforms, that is,
MAO-A and MAO-B and are predominantly located in the
outer membrane of mitochondria in platelets, hepatocytes
ders. Furthermore, considerations of the importance of
monoaminergic signalling in the biogenic amine deficien-
cies indicate that MAOs could be involved in the regulation
[2]
[5]
of mood and cognitive function. Based on knowledge of
and brain glial cells.
MAO-A catalyses the oxidative
©
2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**
1

Enamides as new selective MAO-B inhibitors
Fathima Sahla Kavully et al.
the homology and catalytic mechanisms of MAO-A and B,
several research groups have focused on the design and
development of selective MAO inhibitors, and available evi-
dence indicates MAO-A and MAO-B are important drug
targets for the treatment of neuropsychiatric and neurode-
generative disorders like anxiety, depression, Alzheimer’s
reversible type MAO-B inhibitors safinamide and lazabe-
[
19,20]
mide are currently undergoing clinical trials.
Many
privileged structural scaffolds like chalcones, chromones,
coumarins and pyrazolines have been extensively explored
[
21-33]
as the basis of novel MAO-B inhibitors,
and thus,
new classes of selective, reversible MAO-B inhibitors are
viewed with considerable interest in the contexts of AD and
PD.
[6]
disease (AD) and Parkinson’s disease (PD). Oxidative
deamination by MAO-B usually generates ammonia,
hydrogen peroxide and various aldehydes as by-products.
In particular, hydrogen peroxide generation can increase
reactive oxygen species (ROS) levels, which can damage
mitochondrial functions and cause neuronal death and
contribute to neurodegenerative disorders like AD and
Enamides provide a versatile structural linker unit (-NH-
CO-CH = CH-), in which delocalization of the nitrogen
lone pair electrons across the olefinic unit enhances nucle-
[
34-36]
ophilic activity.
Recent pharmacophore-based studies
have shown MAO-A or B inhibitors possessed one or more
hydrophobic rings, preferably aromatic or heteroaromatic
nucleus, hydrogen bond acceptor (HBA) and hydrogen
bond donor (HBD). The HBA or HBD groups may be pre-
sent in the linker, which can connect with hydrophobic
[
7]
PD.
MAO-B inhibition blocks the catabolism of dopamine
[
8]
(
DA) and, thus, increases endogenous dopamine levels,
whereas increased MAO-B activity has been shown to be
associated with the generation of reactive astrocytes leading
to synaptic dysfunction and impaired cognitive function in
[
5,37]
nucleus or hydrophobic zone of the inhibitors.
We
considered that the presence of the two hydrophobic phar-
macophoric features linked with an amide-connected olefi-
nic spacer in enamides would result in recognition by the
inhibitor binding cavity of MAO-B. Recently, some amides
have been reported to exhibit potent selective MAO-B inhi-
[
9]
the AD brain. Furthermore, recent studies have shown
MAO-B mediated c-aminobutyric acid (GABA) produc-
tion is linked to pathological pathways in AD and that sup-
pressing GABA synthesis by inhibiting MAO-B improves
synaptic plasticity and reduces reactive astrocyte numbers
[
38,39]
bitory activity.
Design strategies for novel selective
[
10]
in the AD brain.
selegiline (a selective irreversible MAO-B inhibitor) amelio-
It was also recently documented that
MAO-B inhibitors have focused on the pharmacophoric
groups of known MAO-B inhibitors such as lazabemide
and safinamide (carboxamide moiety), chalcones (a, b-un-
saturated ketone unit) and 1,4-diphenyl-2-butene (olefinic
linkage; Figure 1). In the present study, we utilized the
molecular hybridization principle to design enamide inhi-
bitors of MAO-B.
[10]
rated learning and memory deficits. In addition, a neu-
roimaging study showed that MAO-B overexpression
increased c-secretase-mediated-b-amyloid (Ab) production
[
12]
in the hypothalamus and frontal cortex in AD.
reasons, it has been suggested selective MAO-B inhibitors
For these
[
13,14]
might offer a potential means of treating AD.
Parkinson’s disease is considered the second most preva-
lent neurodegenerative disorder that influences the motor
Materials and Methods
[
15]
system.
The deterioration of dopaminergic neurons and
Synthesis
subsequent DA depletion in basal ganglia influence the gen-
eration and execution of voluntary movements that can
Mixtures of cinnamoyl chloride and various substituted
amines (0.01 M each) in 10% NaOH were stirred using
magnetic stirrer at room temperature for 8 h. Reaction
completions were monitored by TLC. Mixtures were fil-
tered, solids were washed with water until neutral to litmus
paper, and then recrystallized from ethanol.
[
16]
culminate in the motor disorders associated with PD.
DA oxidation can be catalysed by abnormally high MAO-B
activity and results in free radical production, lysosome
dysfunction, mitochondrial impairment, neuroinflamma-
tion, protein aggregation and DA-associated neuron sus-
ceptibility, and the accumulation of these events has been
[
17]
implicated in the pathogenesis of PD.
Interestingly, the
neurotoxic effects of the active metabolite 1-methyl-4-
Enzyme assays
+
phenylpyridinium ion (MPP ) produced from 1-methyl-4-
Monoamine oxidases activities were assayed as described
previously, using recombinant human MAO-A and MAO-
B and kynuramine (0.06 mM) and benzylamine (0.3 mM)
phenyl-1,2,3,6-tetrahydropyridine (MPTP) by MAO-B
have been associated with PD, and the selective inhibition
of MAO-B has been reported to block this conversion and
[
40]
as substrates, respectively.
were 1.79 and 2.09 K , respectively, and their K values
The substrate concentrations
[
18]
suggested to have neuroprotective effects in PD.
The
m
m
FDA approved MAO-B inhibitors selegiline and rasagiline
are currently coadministered with levodopa for the relief of
motor fluctuations in early-stage PD, and selective,
of these substrates were 0.036 and 0.15 mM, respectively.
AChE activity was measured as described previously using
[40]
the enzyme from Electrophorus electricus (Type VI-S).
2
© 2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**

Fathima Sahla Kavully et al.
Enamides as new selective MAO-B inhibitors
Figure 1 Design of enamide-based MAO-B inhibitors.
Analysis of enzyme inhibitions and kinetics
(100 µl) were added to the wells of 96-well microplates.
After incubation for 24 h, cells were centrifuged and the
pellets were diluted in 100 µl of maintenance media. Plates
The inhibitory activities of compounds AD1–11 against
MAO-A, MAO-B and AChE were first determined at a con-
centration of 10 µM, and inhibitory potencies were then
determined using IC50 values. Time-dependencies,
reversibilities and kinetic studies were performed on the
most potent MAO-B inhibitors, as previously
were then incubated in a 5% CO atmosphere for 48 h at
2
37°C, cells were examined every 24 h under a microscope.
Cells were then treated with 20 µl of 3-(4,5-dimethylthia-
zolyl-2)-2,5-diphenyltetrazolium bromide (MTT; 2 mg/ml)
after 48 h. The formazan crystals formed were dissolved by
gently adding 100 µl of DMSO/isopropanol per well and
the absorbances were immediately measured using a micro-
plate reader at 540 nm. Dose–response curves were used to
determine the concentrations required to inhibit cell
[
41,42]
described.
Kinetic experiments were carried out at five
concentrations of the substrates and three inhibitor con-
centrations. We analysed the data using Excel program and
2
obtained their R scores for the lines. For statistical meth-
ods, Kruskal–Wallis test and a post hoc Dunn’s test were
used in reversibility experiments.
[
45]
growth by 50%.
Molecular docking studies
Analysis of inhibitor reversibilities
Initially, the 3D structures of MAO-A (PDB ID: 2Z5X) and
MAO-B (PDB ID: 2V5Z) were retrieved from the Protein
Reversibilities of lead compounds (AD3 and AD9) were
evaluated by dialysis after preincubating them with MAO-B
[46,47]
Data Bank,
ducted using the protein preparation wizard available in
and enzyme pretreatment was then con-
[43]
for 30 min, as previously described.
The concentrations
used were; AD3 0.20 µM, AD9 0.20 µM, lazabemide (a rever-
sible MAO-B reference inhibitor) 0.080 µM and pargyline
[48]
the Schr o€ dinger suite
to optimize and eliminate impre-
cisions in their X-ray crystal structures. During this process,
nine water molecules within MAO-A and eight water mole-
cules within MAO-B were preserved. Ligand docking struc-
tures were prepared and optimized using the LigPrep tool
that enables the generation of ionization states at physio-
(
relative activities of undialysed (A ) and dialysed (A ) sam-
an irreversible MAO-B reference inhibitor) 0.040 µM. The
U
D
[
44]
ples were compared to determine reversibility patterns.
[
49]
logical pH of possible tautomers.
ligand docking protocol in Schr o€ dinger Suite was employed
The QM-polarized
Cytotoxicity studies
[
50]
Tubes containing VERO (African, Green Monkey Kidney)
cells were centrifuged and cell densities were adjusted to
for docking simulations.
While retaining the rigidities of
protein structures, QM-polarized ligand docking allows
ligands a degree of conformational flexibility. The ligand
centre of mass of the X-ray cognate ligand of both PDB
structures was taken as the cubic grid centre for a reference
5
.0 9 10 cells/ml. Cultured cells were diluted using Dul-
1
becco’s modified eagle medium (DMEM) medium contain-
ing 10% fetal bovine serum (FBS). Diluted suspensions
©
2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**
3

Enamides as new selective MAO-B inhibitors
Fathima Sahla Kavully et al.
1
3
point. The QM-polarized ligand docking protocol was used
with default options. For the latter, three computational
steps were followed, namely,: (1) a standard precision (SP)
initial docking using Glide; (2) the calculation of QM par-
tial charges of the docked ligand based on the field gener-
ated by the receptor; (3) a SP redocking phase upon each
CH), 7.6-7.1 (m, 10H, Ar H), 6.6 (d, 1H, CH); C NMR
(500 MHz)CDCl d: 164.34 ,142.33, 138.1 ,134.5, 129.9,
129.01, 128.8, 127.9, 124.48, 121.01, 120.01.
3
N-(P-tolyl) phenyl prop 2-enamide (AD2)
[
51]
ꢁ1
ligand pose considering computed QM based charges.
White powder; M.P. 130–135°C; IR (ZnSe): 3246 cm
ꢁ
1
To estimate ligand-binding affinities, the Molecular
Mechanics/Generalized Born Surface Area (MM-GBSA)
method was added to the calculation of binding free ener-
(NH stretching), 3031 cm
1660 cm
(Aromatic CH stretching),
1
ꢁ1
(Amide carbonyl stretching);
H
NMR
(500 MHz) CDCl dppm: 10.5 (s, 1H, NH), 7.8 (d, IH,
CH), 7.7-7.1 (m, 9H, ArH), 6.6 (d, 1H, CH), 2.3 (s, 3H,
13
CH3); C NMR (500 MHz) CDCl d: 164.04, 142.09, 135.5,
3
[
52]
gies (ΔG) between protein and ligands,
using Prime in
[53,54]
Schrodinger software 2018-2.
Provided that DEMM
,
3
DGsol and DGSA are the terms referred to the minimized
energy of the ligand–protein complex, the solvation energy
and the surface area energy, respectively, the binding free
energies (ΔG) of compounds with MAO-A or MAO-B were
calculated for docking poses minimized by Prime using the
following equation.
134.7, 134.09, 129.89, 129.54, 128.84, 127.95, 121.07,
120.14, 20.9.
N-(4-chlorophenyl) prop2-enamide (AD3)
ꢁ
1
White powder; M.P. 158–165°C; IR (ZnSe): 3252 cm
ꢁ1
(
NH stretching), 3096 cm
(Aromatic CH str),
1
DGbind ¼ DEMM þ DGsolv þ DGSA
ꢁ1
1
659 cm
(Amide carbonyl stretching),
H
NMR
(
500 MHz), DMSO d dppm: 10.3 (s, 1H, NH), 7.7 (d, 1H,
6
Results
13
CH), 7.6-7.3 (m, 9H, ArH), 6.8 (d, 1H, CH); C NMR
500MHz DMSO d ): d 164.3, 140.9, 138.69, 135.1, 130.3,
(
6
Chemistry
1
29.4, 129.1, 128.4, 127 .3, 112.4, 121.2. ESI-MS: 257.7
The selected enamides were synthesized by reacting the
appropriately substituted primary and secondary amines
with cinnamoyl chloride in basic medium (Scheme 1). The
trans geometry of the olefinic linkage of enamides was con-
firmed by large proton–proton coupling constants (d).
High deshielded (ppm) values confirmed the presence of
1
-morpholino-3-phenylprop-2-en-1-one (AD4)
ꢁ
1
White powder; M.P. 130–138°C; IR (ZnSe): 3246 cm
(NH stretching) 2853 cm
ꢁ
1
( aliphatic CH stretching),
ꢁ1
ꢁ1
1644 cm (Amide carbonyl stretching), 1112 cm (C–
O–C bend); H NMR (500 MHz) CDCl dppm: 7.7 (d, 1H,
CH), 7.5-7.2 (m, 5H, ArH), 6.8 ( d, 1H, CH), 3.7 (d, 8H,
2
1
sp carbonyl functional group by C13 NMR. Mass spectra
were consistent with expected molecular weights.
3
13
tetrahydroxazine); C NMR (500 MHz) CDCl : 165.4,
3
143.23, 135.14, 129.18, 128.8, 127.8, 116.8, 66.6, 46.2.
Diphenyl prop-2-enamide (AD1)
ꢁ
1
Off white solid; M.P. 122–130°C; IR (ZnSe): 3236 cm
ꢁ1
N-(4-fluorophenyl) prop-2 enamide (AD5)
(
NH stretching), 3032 cm , (Aromatic CH stretching),
1
ꢁ1
ꢁ1
ꢁ1
1
658 cm
(amide carbonyl stretching);
H
NMR
Off white powder; M.P. 122-126 °C; IR (ZnSe): 3253 cm
ꢁ
1
(
500 MHz) CDCl dppm: 10.2 (s, 1H, NH), 7.7 (d, 1H,
(NH stretching), 3028 cm
(Aromatic), 1658 cm
3
Scheme 1 Synthetic routes used to produce AD1 to AD11.
4
© 2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**

Fathima Sahla Kavully et al.
Enamides as new selective MAO-B inhibitors
1
13
10H, ArH), 6.8 (d, 1H, CH), 2.5 (s, 3H, CH3), C NMR
(
Amide carbonyl stretching);
H
NMR (400 MHz)
CDCl dppm: 10.4 (s, 1H, NH), 7.7 (d, 1H, CH), 7.5-7.2
(500 MHz DMSO d ) d 164.3, 140, 138, 135, 131.1, 130.8,
130.1, 129.4 ,128.1 ,126.4 ,125.4 ,124.8, 122.2, 18.63.
3
6
1
m, 10H, ArH), 6.5 (d, 1H, CH); C NMR (500MHz
3
(
DMSO d ): 162.6 ,147.5, 137.09 ,132.53, 131.30, 130.02
6
,
129.85 ,129.7, 129.07 ,116.49 ,116.32; ESI-MS: 241.2
3
1
-phenyl-1-(piperazin-1-yl)prop-2-en-1-one (AD
1)
N-(4-bromophenyl) prop-2-enamide (AD6)
ꢁ
1
White powder; M.P. 252–260°C; IR (ZnSe): 3254 cm
(NH stretching), 3034 cm
ꢁ1
ꢁ
1
ꢁ1
Off white powder; M.P. 172–180°C; IR (ZnSe): 3249 cm
(Aromatic CH stretching),
ꢁ1
ꢁ
1
(
NH stretching), 3030 cm
(Aromatic CH stretching),
1
1644 cm (Amide carbonyl stretching), 1037 cm (CN
1
ꢁ1
1
660 cm
(Amide carbonyl stretching);
H
NMR
bend); H NMR (500 MHz) CDCl dppm:9.9 (s, 1H, NH),
3
(
500 MHz) CDCl dppm: 10.1 (s, 1H, NH), 7.7 (d, 1H,
7.7 (d ,1H,CH), 7.7-7.3 (m ,5H,Ar H) ,6.8 (d, 1H, CH),
13
3
1
3
CH), 7.5-7.2 (m, 10H, ArH), 6.5 (d, 1H, CH); C NMR
500 MHz DMSO d ) 163.1, 143.3, 137.0, 134.4, 132.0,
3.8-3.6 (t, 4H, CH2); C NMR (500 MHz) CDCl d:165.5,
3
(
148.7, 134.9, 129.9, 128.8, 127.8, 116.3, 45.6 ,42.1.
6
132.9, 129.7, 129.0, 128.9, 128.0; 121.46. ESI-MS: 302.7.
Monoamine oxidase inhibition
N-(pyridinyl) cinnamamide (AD7)
Of the 11 derivatives, nine showed potent inhibitory activ-
ity against MAO-B at 10 µM with residual activities of
<40%, but AD4 and AD11 showed weak inhibitory activi-
ties (Table 1). Compounds AD3 and AD9 showed the
greatest inhibitory activities against MAO-B with IC50 val-
ues of 0.11 and 0.10 µM, respectively, and were followed by
AD2 and AD1 (0.51 and 0.71 µM, respectively). Comparing
substituents at the para-position in ring A, MAO-B inhibi-
tory potencies were increased in order by the presence of -
ꢁ
1
Pale Yellow powder; M.P. 60–65°C; IR (ZnSe): 3468 cm
ꢁ
1
(
NH stretching), 1659 cm
(CN stretching);
(Amide stretching),
NMR (500 MHz)
ꢁ1
1
1
174 cm
H
CDCl dppm: 10.2 (s, 1H, NH), 7.6 (d, 1H, CH), 7.5-7.1
3
1
m, 9H, ArH), 6.6 (d, 1H, CH); C NMR (500 MHz
3
(
DMSO d ): 163.2, 143.2, 137.1, 134.2, 132.8, 132.3, 129.7,
6
129.2, 128.9, 128.2, 121.6.
Cl> -CH > -H> -Br> -F, that is, AD3>AD2>AD1>A-
3
N-(pyridin-2-yl) prop-2 enamide (AD 8)
White powder, M.P. 76–82°C; IR (ZnSe): 3462 cm (NH
D6>AD5, respectively. The presence of an NO group at
2
ꢁ
1
the meta-position in ring A of AD9 increased the potency
to the degree by the chlorine at the para-position in ring A
of AD3. The other five compounds had IC50 values ranging
from 2.30 to 7.20 µM. Nine of the eleven compounds
weakly inhibited MAO-A with IC50 values of >40 µM,
whereas AD9 and AD7 had IC50 values of 4.21 and
5.95 µM, respectively. AD3 had the highest selectivity index
ꢁ1
ꢁ1
stretching), 1651 cm
(Amide stretching), 1170 cm
1
(
(
(
CN stretching); H NMR (500 MHz) CDCl dppm: 10.5
3
s, 1H, NH), 7.7 (d, 1H, CH), 7.5-7.0 (m, 9H, ArH), 6.65
1
d, 1H, CH); C NMR (500 MHz DMSO d ): 163.1, 142.2,
3
6
137.6, 134.5, 132.2, 132.5, 129.8, 129.1, 128.1, 128.0, 121.5;
ESI-MS: 225.22.
(
SI) value for MAO-B at >363.6, followed by AD9 at 42.1.
N-(3-nitrophenyl)prop-2 enamide (AD9)
Kinetics
ꢁ
1
White powder, M.P. 141–144°C; IR (ZnSe): 3250 cm
ꢁ1
(
NH stretching), 3029 cm
(Aromatic CH stretching)
1
Lineweaver–Burk plots and secondary plots showed that
AD3 and AD9 were competitive inhibitors of MAO-B (Fig-
ꢁ1
1
653 cm
(Amide carbonyl stretching);
H
NMR
(
500 MHz), DMSO d dppm; 10.7 (s,1H, NH), 8.7 (d, 1H,
ure 2a and 2c), with K values of 0.044 ꢀ 0.0036 and
6
i
1
3
CH), 8.0-7.4 (m, 9H, ArH), 6.8 (d, 1H, CH); C NMR
500 MHz DMSO d ) d 164.6, 148.1, 141.7, 140.3, 134.9,
0.039 ꢀ 0.0047 µM, respectively (Figure 2b and 2d). These
results suggest that AD3 and AD9 bind to the active site of
the free enzyme and are potent, selective and competitive
inhibitors of MAO-B.
(
6
130.7, 130.39, 129.5, 128.3, 125.4, 121.9, 118.1, 117.7; ESI-
MS: 268.2
N-(o-tolyl) pro-2-enamide (AD 10)
Reversibility studies
ꢁ
ꢁ1
1
White powder; M.P. 140–148°C; IR (ZnSe): 3265 cm
Reversibility studies for MAO-B inhibition were conducted
on AD3 and AD9. In these experiments, inhibitions of
MAO-B by AD3 and AD9 were recovered from 30.8 (value
of A ) to 79.4% (value of A ) and from 34.3 (value of A )
ꢁ
1
(
(
NH stretching), 3028 cm
Amide carbonyl stretching).; H NMR (500 MHz), DMSO
(Aromatic), 1651 cm
1
d6 dppm: 9.47 (s,1H, NH), 7.7 (d, 1H, CH), 7.6-7.1 (m,
U
D
U
©
2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**
5

Enamides as new selective MAO-B inhibitors
Fathima Sahla Kavully et al.
a
Table 1 Inhibitions of recombinant human MAO enzymes and AChE by enamides
Residual activity at 10 µM (%)
IC50 (µM)
b
Compounds
MAO-A
MAO-B
AChE
MAO-A
MAO-B
SI
AD1
94.5 ꢀ 1.47
99.6 ꢀ 4.67
72.9 ꢀ 0.61
85.0 ꢀ 4.43
98.5 ꢀ 1.01
80.6 ꢀ 5.40
34.1 ꢀ 1.09
86.4 ꢀ 1.22
25.7 ꢀ 1.74
92.0 ꢀ 1.12
84.8 ꢀ 2.08
6.53 ꢀ 2.55
8.63 ꢀ 1.49
5.09 ꢀ 1.85
76.1 ꢀ 1.50
22.9 ꢀ 2.38
12.5 ꢀ 2.69
39.7 ꢀ 2.66
18.6 ꢀ 0.80
ꢁ3.16 ꢀ 2.21
38.2 ꢀ 2.45
73.3 ꢀ 3.03
83.1 ꢀ 0.12
78.8 ꢀ 0.20
71.9 ꢀ 1.20
82.7 ꢀ 1.09
72.7 ꢀ 1.01
81.9 ꢀ 0.12
83.1 ꢀ 0.21
83.5 ꢀ 1.31
70.0 ꢀ 0.11
77.6 ꢀ 0.13
75.5 ꢀ 1.69
>40
0.71 ꢀ 0.39
0.51 ꢀ 0.059
0.11 ꢀ 0.024
>40
>56.3
AD2
>40
>78.4
AD3
>40
>363.6
AD4
>40
AD5
>40
4.91 ꢀ 0.021
2.30 ꢀ 0.010
6.52 ꢀ 0.002
2.81 ꢀ 0.81
0.10 ꢀ 0.013
7.20 ꢀ 0.18
>40
>8.2
AD6
>40
>17.4
AD7
5.95 ꢀ 0.83
0.91
AD8
>40
>14.2
42.1
>5.6
AD9
4.21 ꢀ 0.98
AD10
AD11
Toloxatone
Lazabemide
Clorgyline
Pargyline
>40
>40
1.03 ꢀ 0.060
–
–
0.040 ꢀ 0.0019
2.45 ꢀ 0.035
0.020 ꢀ 0.0033
0.0050 ꢀ 0.000052
2.51 ꢀ 0.15
a
Results for reference compounds were determined after preincubation with enzymes for 30 min. Results are expressed as the means ꢀ SE of
b
duplicate experiments by continuous assay methods under standard conditions: data were obtained using Excel program. SI values are expressed
for MAO-B vs MAO-A.
to 84.1% (value of A ), respectively (Figure 3). These
D
Y407 in the proximity of FAD, and more importantly, with
the selective F208 residue, which is mutated to I199 in
MAO-B. For MAO-B, AD3 interacted with Y326 and I199;
the former formed an H-bond with the amide carbonyl
oxygen atom and the latter with the unsubstituted aromatic
ring. In the case of AD9, the docking score difference
between MAO-A and MAO-B was greater than that of
AD3. As shown on the left-hand-side of Figure 5, AD9
formed bidentate H-bonds involving the nitro group and
Y444 and exhibited the p–p interactions described for AD3.
For MAO-B, AD9 interacted with the selective residues
I199 and Y326. In particular, p–p hydrophobic interactions
occurred with I199 and a H-bond was formed with Y236.
recovery values were similar to those of the reversible refer-
ence lazabemide (from 24.4 to 85.0%). Inhibition of MAO-
B by the irreversible inhibitor pargyline was recovered
partly (from 28.8 to 46.7%). These experiments showed
that inhibitions of MAO-B by AD3 and AD9 were recov-
ered to the reversible reference level, indicating both rever-
sibly inhibit MAO-B.
Cytotoxicity
The biocompatibilities of AD3 and AD9 were evaluated
using an MTT assay and normal VERO cell lines. Both
compounds achieved >89% cell viability at 100 µg/ml. The
MTT assay revealed that AD3 and AD9 compounds were
relatively non-toxic to the normal VERO cell lines with
IC50 values of 153.96 and 194.04 µg/ml, respectively.
Discussion
We approached the design of MAO-B inhibitors by incor-
porating different key functional groups in different types
of MAO-B inhibitors; for example, we included the pres-
ence of an a, b-unsaturated ketone and carboxamide and
olefinic linkages in a single scaffold. Of the eleven candidate
compounds produced, enamides with a tertiary nitrogen
(AD4 and AD11) were weaker MAO-A and B inhibitors
than secondary nitrogen containing compounds. In addi-
tion, it was evident from the inhibitory profiles of com-
pounds containing an electron attracting group (e.g. nitro
or chloro (AD9 and AD3, respectively)) on the phenyl sys-
tem most inhibited MAO-B (IC50 = 0.10 and 0.11 µM,
respectively). Replacement with other halogens such as bro-
mine and fluorine rather than chlorine resulted in less
MAO-B inhibitory activities and lower selectivity indices.
Computational studies
With the aim of investigating the binding modes of AD3
and AD9 with MAO-A or MAO-B, computational studies
were performed by QM-polarized docking analyses using
MM-GBSA calculations. Table 2 summarizes calculated
docking scores and the ΔG binding values of the two com-
pounds for MAO-A or MAO-B. In agreement with in vitro
IC50 values, AD3 had a higher docking score for MAO-B
than MAO-A. As shown in Figure 4, AD3 displayed similar
docking poses for MAO-A and MAO-B by approaching the
FAD with its para-chloro phenyl ring. For MAO-A, proba-
ble p–p hydrophobic interactions were established with
6
© 2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**

Fathima Sahla Kavully et al.
Enamides as new selective MAO-B inhibitors
Figure 2 Lineweaver–Burk plots for MAO-B inhibition by AD3 (a) and AD9 (c), and their respective secondary plots (b) and (d) of slopes vs. inhi-
bitor concentrations. Substrate concentrations used were ranging from 0.03 to 0.6 mM, and inhibition tests were carried out at three inhibitor
2
concentrations, that is, ~0.5, 1.0 and 2.0 times IC50 values. Initial velocity was expressed as an increase in absorbance per min. R scores were
more than 0.93 for the lines.
The introduction of the electron donating methyl group
AD2) at the para-position of the phenyl system resulted in
selective MAO-B inhibition in low micromolar range
0.51 µM) with a selectivity index of 78.4. Shifting the
Considering the period for MAO-B biosynthesis in the
human brain (~40 days), irreversible MAO-B inhibitors
have their limitations such as long-lasting enzyme inhi-
(
[
55-58]
(
bition and immunogenic effects.
On the other
methyl group from para (AD2) to ortho (AD10) dramati-
cally reduces MAO-B inhibition by 14-fold and suggested
that substitution positions have great impact on the inhibi-
tory effectiveness of enamides. The potencies of AD3 and
AD9 for MAO-B inhibition were 2.75–5 times lower than
those of lazabemide (reversible) and pargyline (irreversible)
hand, reversible inhibitors enable the recovery of enzyme
activity, and the inhibition may be relieved by competi-
[
59,60]
tive inhibitors.
In the present study, the reversibili-
ties of MAO inhibitions by AD3 and AD9 were
investigated by dialysis by comparing the enzyme activi-
ties of undialysed and dialysed mixtures. Percentage
activities were calculated vs negative controls with no
inhibitor present or vs the irreversible and reversible
positive controls pargyline and lazabemide, respectively.
(
IC50 = 0.04 and 0.02 lM, respectively). However, fine tun-
ing by inserting different electron withdrawing and donat-
ing groups on the two phenyl systems offers the possibility
of tailoring molecules for the treatment of neurodegenera-
tive diseases.
The relative activities for undialysed (A ) and dialysed
U
(A ) of AD3 and AD9 were documented that after
D
©
2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**
7

Enamides as new selective MAO-B inhibitors
Fathima Sahla Kavully et al.
Figure 3 Recoveries from MAO-B inhibitions by AD3 and AD9 as determined by dialysis. AD3 or AD9 was preincubated with MAO-B for
0 min and residual activity was evaluated after dialysis. The concentrations used were; AD3 0.20 µM, AD9 0.20 µM, lazabemide (a reversible
3
MAO-B reference inhibitor) 0.080 µM and pargyline (an irreversible MAO-B reference inhibitor) 0.040 µM. *P < 0.1 to the control using Kruskal–
Wallis test with a post hoc Dunn’s test.
Table 2 Docking scores and ΔG binding values for interactions
between AD3 or AD9 and MAO-A or MAO-B
presenting FAD with a substituted aromatic ring, which
suggests this is the preferred presentation irrespective of
MAO isoform. However, we point out that the weakness of
AD3 in MAO-A inhibition at high concentrations >40 lM,
raises some concerns about the quality of our bioactivity
data, and might explain the mismatch observed between
calculated binding free energies and the experimental selec-
tivity of AD3 and AD9 for MAO-A and MAO-B, respec-
tively. Nevertheless, docking studies carried out on AD3
and AD9 explained at a molecular level their different
experimental affinities for the two MAO isoforms. In par-
ticular, the data in binding for MAO-B were mostly
explained by the chance of forming an H-bond interaction
with Y325, which is a key residue that is changed to I335 in
MAO-A, capable to stabilize the enamide linker for AD3
and AD9 and this is behind the observed high molecular
selectivity for MAO-B.
Docking score (kcal/
mol)
ΔGbinding (kcal/mol)
Compounds
MAO-A
MAO-B
MAO-A
MAO-B
AD3
AD9
ꢁ8.507
ꢁ7.681
ꢁ9.445
ꢁ9.414
ꢁ51.63
ꢁ40.03
ꢁ53.51
ꢁ55.82
dialysis inhibition was greatly recovered, which was
almost similar to the inhibition by lazabemide. The
results obtained during the present study indicate AD3
and AD9 represent a new class of enamide-based reversi-
ble MAO-B inhibitors.
Docking studies were carried out on AD3 and AD9,
which had the greatest MAO-B affinities with selectivity.
Both inhibitors entered the MAO-B binding site by
(
a)
(b)
Figure 4 Top-scored poses for AD3 in binding sites of MAO-A (a) and MAO-B (b). Proteins are rendered as cartoons, whereas residues involved
in the ligand–protein interactions are rendered in stick format. The p–p hydrophobic interactions (black lines) and H-bonds (red lines) are shown
displayed.
8
© 2020 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2020), pp. **–**

Fathima Sahla Kavully et al.
Enamides as new selective MAO-B inhibitors
(
a)
(b)
Figure 5 Top-scored poses for AD9 in the binding sites of MAO-A (a) and MAO-B (b). Proteins are rendered as cartoons, whereas residues
involved in ligand–protein interactions are rendered in stick format. The p–p hydrophobic interactions (black lines) and H-bonds (red lines) are
shown.
Conclusions
Declarations
The present study documents the syntheses of 11 enamide
analogues and presents their human MAO-A and MAO-B
inhibitory activities. A new class of selective MAO-B inhibi-
tors was identified. The most potent derivatives AD3 and
AD9 had IC50 values in the low micromolar range (0.11
and 0.10 µM, respectively) for MAO-B inhibition and satis-
factory selectivity. Kinetics and reversibility studies showed
that AD3 and AD9 are competitive and reversible inhibi-
tors of MAO-B. Furthermore, these compounds were
found to be non-toxic to normal VERO cell lines at
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
Author contributions
BM conceived the original idea, designed the outlines of
the study and prepared the draft of the manuscript. FSK,
SD, SK, AP, AV and RFAA participated the synthesis and
purification of the molecules. JMO and HK participated
the enzyme inhibition study. ART and ON performed the
computational studies.
100 µg/ml. The non-toxic natures and selective, reversible,
competitive modes of MAO-B inhibition of AD3 and AD9
encourage further development of enamides for the treat-
ment of neurodegenerative disorders like AD and PD.
and update. Med Res Rev 2019; 39: 10. Scholl M et al. Early astrocytes in
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