Discovery of N-(1-(3-fluorobenzoyl)-1H-indol-5-yl)pyrazine-2-carboxamide: a novel, selective, and competitive indole-based lead inhibitor for human monoamine oxidase B

Article abstract of DOI:10.1080/14756366.2020.1800666

Herein, two new series of N-substituted indole-based analogues were rationally designed, synthesized via microwave heating technology, and evaluated as noteworthy MAO-B potential inhibitors. Compared to the reported indazole-based hits VI and VII, compounds 4b and 4e exhibited higher inhibitory activities over MAO-B with IC₅₀ values of 1.65 and 0.78?μM, respectively. When compared to the modest selectivity index of rasagiline (II, a well-known MAO-B inhibitor, SI > 50), both 4b and 4e also showed better selectivity indices (SI > 60 and 120, respectively). A further kinetic evaluation of the most potent derivative (4e) displayed a competitive mode of inhibition (inhibition constant (K _i)/MAO-B = 94.52 nM). Reasonable explanations of the elicited biological activities were presented via SAR study and molecular docking simulation. Accordingly, the remarkable MAO-B inhibitory activity of 4e (N-(1-(3-fluorobenzoyl)-1H-indol-5-yl)pyrazine-2-carboxamide), with its selectivity and competitive inhibition, advocates its potential role as a promising lead worthy of further optimization.

Full text of DOI:10.1080/14756366.2020.1800666

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Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Discovery of N-(1-(3-fluorobenzoyl)-1H-indol-5-
yl)pyrazine-2-carboxamide: a novel, selective, and
competitive indole-based lead inhibitor for human
monoamine oxidase B
Ahmed Elkamhawy , Sora Paik , Hyeon Jeong Kim , Jong-Hyun Park , Ashwini
M. Londhe , Kyeong Lee , Ae Nim Pae , Ki Duk Park & Eun Joo Roh
To cite this article: Ahmed Elkamhawy , Sora Paik , Hyeon Jeong Kim , Jong-Hyun Park , Ashwini
M. Londhe , Kyeong Lee , Ae Nim Pae , Ki Duk Park & Eun Joo Roh (2020) Discovery of N-(1-(3-
fluorobenzoyl)-1H-indol-5-yl)pyrazine-2-carboxamide: a novel, selective, and competitive indole-
based lead inhibitor for human monoamine oxidase B, Journal of Enzyme Inhibition and Medicinal
Chemistry, 35:1, 1568-1580, DOI: 10.1080/14756366.2020.1800666
To link to this article: https://doi.org/10.1080/14756366.2020.1800666
© 2020 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1568–1580
https://doi.org/10.1080/14756366.2020.1800666
RESEARCH PAPER
Discovery of N-(1-(3-fluorobenzoyl)-1H-indol-5-yl)pyrazine-2-carboxamide: a novel,
selective, and competitive indole-based lead inhibitor for human monoamine
oxidase B
c
d,e
d
d,f
Ahmed Elkamhawya,b
, Sora Paik , Hyeon Jeong Kim , Jong-Hyun Park , Ashwini M. Londhe , Kyeong
ꢀ
ꢀ
ꢀ
Leea, Ae Nim Paed,f, Ki Duk Parkd,f,g and Eun Joo Rohc,f
b
aCollege of Pharmacy, Dongguk University-Seoul, Goyang, Republic of Korea; Department of Pharmaceutical Organic Chemistry, Faculty of
c
Pharmacy, Mansoura University, Mansoura, Egypt; Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul,
d
Republic of Korea; Convergence Research Center for Diagnosis, Treatment and Care System of Dementia, Korea Institute of Science and
e
f
Technology (KIST), Seoul, Republic of Korea; Department of Biotechnology, Yonsei University, Seoul, Republic of Korea; Division of Bio-Medical
g
Science & Technology, KIST School, Korea University of Science and Technology, Seoul, Republic of Korea; KHU-KIST Department of
Converging Science and Technology, Kyung Hee University, Seoul, Republic of Korea
ABSTRACT
ARTICLE HISTORY
Received 22 April 2020
Revised 7 July 2020
Accepted 8 July 2020
Herein, two new series of N-substituted indole-based analogues were rationally designed, synthesized via
microwave heating technology, and evaluated as noteworthy MAO-B potential inhibitors. Compared to
the reported indazole-based hits VI and VII, compounds 4b and 4e exhibited higher inhibitory activities
over MAO-B with IC50 values of 1.65 and 0.78 mM, respectively. When compared to the modest selectivity
index of rasagiline (II, a well-known MAO-B inhibitor, SI > 50), both 4b and 4e also showed better select-
ivity indices (SI > 60 and 120, respectively). A further kinetic evaluation of the most potent derivative (4e)
displayed a competitive mode of inhibition (inhibition constant (Ki)/MAO-B ¼ 94.52 nM). Reasonable
explanations of the elicited biological activities were presented via SAR study and molecular docking
simulation. Accordingly, the remarkable MAO-B inhibitory activity of 4e (N-(1-(3-fluorobenzoyl)-1H-indol-5-
yl)pyrazine-2-carboxamide), with its selectivity and competitive inhibition, advocates its potential role as a
promising lead worthy of further optimization.
KEYWORDS
Monoamine oxidase B;
carboxamide; MAO-B
inhibitor; microwave
synthesis; molecu-
lar modelling
1. Introduction
(IV), the most commonly used therapies for PD and Alzheimer’s
disease (AD) via blocking dopamine degradation in the nigrostria-
tal pathway. However, numerous undesirable side effects of these
inhibitors have been confirmed in long-term treatment of PD such
as hallucinations, headaches, production of neurotoxic metabo-
lites, sodium channel blockade, calcium channel modulation, and
inhibition of stimulated release of glutamate12–24. Not only for
Parkinson’s disease (PD), a neurodegenerative disorder with no
currently available cure, is characterised by the impairment of
motor function caused by the death of dopaminergic neurons in
the substantia nigra pars compacta. Thus, the available therapies
for ameliorating Parkinson’s symptoms mostly target dopamine
depletion in the brain1–6. Monoamine oxidase B (MAO-B), which is
localized on the outer membrane of mitochondria (mainly in the that reason, but also since there is no disease-modifying treat-
liver and brain), catalyzes the oxidative deamination of mono-
amine neurotransmitters such as dopamine which results in hydro-
gen peroxide (H2O2) production leading to oxidative stress and
neuronal cell death7–9. Recently, MAO-B has been reported to be
highly expressed in the substantia nigra of PD patients. Thus,
inhibition of MAO-B is effective in alleviating the symptoms of PD
patients. Indeed, several substances that can effectively ameliorate
emerging motor symptoms of PD are being used nowadays by
physicians, amongst them levodopa, dopamine agonists, and
MAO-B inhibitors. However, mainly due to their favourable safety
profile in addition to their putative neuroprotective capabilities,
MAO-B inhibitors may constitute a preferable therapeutic option
ment has been discovered yet for PD, there is still a vital need to
develop novel selective MAO-B inhibitors as promising therapeut-
ically active candidates for PD patients. Hence, this encouraged
our institute to launch a discovery project aiming at identification
of novel promising MAO-B inhibitors. As a result, various active
small molecules have been recently reported including unsatur-
ated ketones25, oxazolopyridines and thiazolopyridines26, biaryl
derivatives27, in addition to indole-substituted benzothiazoles and
benzoxazoles28. Moreover, other research groups reported diverse
MAO-B inhibitors belonging to different chemical scaffolds such as
chalcones, pyrazoles, chromones, coumarins, xanthines, isatin
derivatives, thiazolidindiones, (thiazol-2-yl)hydrazones, 1,5-diphe-
nylpenta-2,4-dien-1-ones, in addition to indazole- and indole-5-
for early PD10,11
MAO-B is inhibited by different potent agents such as selegi-
line (I, Figure 1), rasagiline (II), safinamide (III), and sembragiline carboxamides29–34
.
.
CONTACT Ahmed Elkamhawy
a.elkamhawy@gmail.com, a_elkamhawy@mans.edu.eg
College of Pharmacy, Dongguk University-Seoul, Goyang 10326, Republic
of Korea; Eun Joo Roh
r8636@kist.re.kr Chemical Kinomics Research Center, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea
ꢀ
Authors contributed equally to this work.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1569
Figure 1. Chemical structures of well-known MAO-B inhibitors.
In the past few years, the indole moiety attracted the attention or pyrazine), not only anticipated to interact with the binding site
of medicinal chemists as a biologically accepted pharmacophore
possessing wide spectrum of biological activities35,36. However,
only few indole-based MAO-B inhibitors with high efficacy and
promising safety profile were found in literature such as indole-
based chalcones, indole-5-carboxamides, in addition to indole-sub-
stituted benzothiazoles and benzoxazoles28,33,37,38. One of these
studies was carried out by Tzvetkov et al who preliminary pre-
pared some indazole-based compounds (V, VI, and VII, Figure 2)
to be evaluated over human MAO-B. While compound V was
totally inactive, the biological results of VI and VII (MAO-B
IC50¼2590 and 3300 nM, respectively), coupled with their moder-
ate selectivity values, were promising enough to pursue further
structural modifications based on incorporation of a reverse amide
linker to dramatically increase the activity and afford new highly
potent indazole- and indole-5-carboxamides33. However, in their
report, they mainly focussed on development of free NH indazole-
based analogues with only two potent derivatives belonging to
the indole scaffold. Accordingly, the current study took the opti-
mization task for compounds VI and VII as a starting point
towards novel N-substituted indole-based derivatives with poten-
tial inhibition against MAO-B.
The docking model of the most potent indazole derivative dis-
covered by Tzvetkov et al proposed the binding site of MAO-B
having some space remains unoccupied and, in addition, the inda-
zole free NH was predicted not to be incorporated in any type of
H-bond interaction with the binding site. By searching literature,
the scaffold modification of compounds VI and VII into indole
core retaining the same original amide linker at position 5 was
found to be a completely new approach and not to be yet
studied. Inspired by these observations, diverse chemical structural
alterations on compounds VI and VII were planned as depicted in
Figure 2; two novel series were designed via replacing the inda-
zole core with indole scaffold while keeping the original amide
spacer of VI and VII into 5-position of the indole, which have, to
the best of our knowledge, not been reported so far. In addition,
aiming to design our derivatives armed with a potential capability
to bind into the predicted unoccupied space (in the binding site
of MAO-B reported by Tzvetkov et al), the free NH of the indole
moiety was substituted by one carbon spacer (Z ¼ CH2 or CO) con-
necting the indole scaffold to various aromatic substitution pat-
terns in series A, while in series B, the indole nitrogen was linked
to one carbon methyl group. Moreover, we incorporated three dif-
of MAO-B in a similar fashion to the substituted aryl moieties in V
and VI, but also, targeting possible extra-interaction(s) owing to
the heteroatom(s) in these six-membered systems. Accordingly, in
this study, we present the complete synthetic routes of the newly
synthesized compounds through microwave heating technology,
the enzymatic inhibitory activities and selectivity values over MAO
enzymes, structure–activity relationship (SAR), a detailed kinetic
study, in addition to computational docking models of the most
active ligands with MAO enzymes.
2. Materials and methods
2.1. Chemistry
General: All solvents and reagents were obtained from commercial
suppliers and used without further purification. Microwave-
assisted synthetic procedure was applied in
a
Biotage
Initiator þ apparatus operating in single mode; the microwave cav-
ity producing controlled irradiation at 2.45 GHz (Biotage AB,
Uppsala, Sweden). The reactions were carried out in sealed vessels
by employing magnetic stirring with maintaining the desired tem-
perature for the programmed time period. TLC was performed
using glass sheets pre-coated with silica gel 60 F254 purchased by
Merck (Kenilworth, NJ). The NMR spectra were obtained on Bruker
Avance 400 (Billerica, MA). Column chromatography was carried
out on Merck Silica Gel 60 (230–400 mesh) (Kenilworth, NJ). High-
resolution spectra were performed on Waters ACQUITY UPLC BEH
C18 1.7 l–Q-TOF SYNAPT G2-Si High Definition Mass
Spectrometry.
2.1.1. General procedure for synthesis of compounds 2a–c
For compounds 2a–2c; NaH (60% in oil, 48.0 mg, 1.2 mmol) was
added to a solution of 5-nitroindole (1, 162.2 mg, 1.0 mmol) dis-
solved in DMF (11 mL) at 0 ꢁC. The reaction mixture was stirred at
0 ꢁC for 10 min. The appropriate reagent (1.0 mmol; 4-fluorobenzyl
bromide for 2a, 3-fluorobenzoyl chloride for 2b, and 2-(chlorome-
thyl)pyridine hydrochloride for 2c) was added to the mixture. The
mixture was then stirred at 100 ꢁC for 24 h, cooled to room tem-
perature, and partitioned between EA and water using aqueous
NaHCO3. The organic layer was dried over anhydrous Na2SO4 and
ferent six-membered aromatic heterocycles (pyridine, pyridazine, concentrated under reduced pressure. Finally, the residue was

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1570 A. ELKAMHAWY ET AL.
Figure 2. Rational design of the newly synthesized analogues (series A and B).
purified by column chromatography (SiO2, EA/n-Hex) to get the 7.21 (d, J ¼ 2.9 Hz, 1H), 7.33 (d, J ¼ 9.0 Hz, 1H), 8.11 (d, J ¼ 9.0 Hz,
target intermediate.
1H), 8.57 (s, 1H). Reported42.
2.1.1.1. 1-(4-Fluorobenzyl)-5-nitro-1H-indole (2a). Yellow solid,
1
yield: 91%, mp: 126.4–127.0 ꢁC, H NMR (400 MHz, CDCl3) d 5.34 (s,
2.1.3. General procedure of compound 3a–d
Each of compounds 2aꢂd (1.0 mmol) was dissolved in EtOH/H2O
(5/2), followed by adding NH4Cl (267.5 mg, 5.0 mmol) and iron
powder (558.5 mg, 10.0 mmol). The reaction mixture was refluxed
for 1 h and filtered on celite then washed with EtOH. The residue
was extracted using EA, water and aqueous NaHCO3. The organic
layer was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy (SiO2, EA/n-Hex) to get the target intermediate.
2H), 6.74 (s, 1H), 7.03 (d, J ¼ 8.1 Hz, 2H), 7.08 (s, 2H), 7.28 (d,
J ¼ 10.6 Hz, 2H), 8.08 (d, J ¼ 8.6 Hz, 1H), 8.60 (s, 1H). Reported39.
2.1.1.2. (3-Fluorophenyl)(5-nitro-1H-indol-1-yl)methanone (2b).
Yellowish white solid, yield: 67%, mp: 151.0–152.0 ꢁC, 1H NMR
(400 MHz, CDCl3) d 6.79 (d, J ¼ 3.7 Hz, 1H), 7.35–7.40 (m, 1H),
7.47–7.50 (m, 2H), 7.54–7.60 (m, 2H), 8.25 (dd, J ¼ 2.2 Hz, 9.1 Hz,
1H), 8.47–8.51 (m, 2H). 13C NMR (100 MHz, CDCl3) d 109.24, 116.52,
116.53 (JC–F ¼ 24.1 Hz), 117.20, 119.86 (JC–F ¼ 21.1 Hz), 120.27,
125.06 (JC–F ¼ 3.0 Hz), 130.06, 130.69 (JC–F ¼ 5.0 Hz), 130.79, 135.29
(JC–F ¼ 8.1 Hz), 138.97, 144.55, 162.51 (JC–F ¼ 249.5 Hz), 167.08.
2.1.3.1. 1-(4-Fluorobenzyl)-1H-indol-5-amine (3a). Brown solid,
yield: 80%, mp: 76.2–77.4 ꢁC, 1H NMR (400 MHz, DMSO-d6) d 4.49
(s, 2H), 5.27 (s, 2H), 6.18 (d, J ¼ 2.8 Hz, 1H), 6.49 (dd, J ¼ 1.8 Hz,
8.6 Hz, 1H), 6.69 (d, J ¼ 1.6 Hz, 1H), 7.12 (t, J ¼ 9.0 Hz, 3H), 7.20 (t,
J ¼ 8.5 Hz, 2H), 7.28 (d, J ¼ 3.0 Hz, 1H). 13C NMR (100 MHz, DMSO-
d6) d 48.80, 99.98, 104.01, 110.64, 112.32, 115.65 (JC–F ¼ 21.1 Hz),
128.94, 129.41 (JC–F ¼ 8.0 Hz), 129.80, 130.07, 135.35 (JC–F ¼ 3.0 Hz),
2.1.1.3. 5-Nitro-1-(pyridin-2-ylmethyl)-1H-indole (2c). Yellow solid,
yield: 24%, 1H NMR (400 MHz, CDCl3) d 5.49 (s, 2H), 6.76–6.80 (m,
2H), 7.20–7.23 (m, 1H), 7.33 (d, J ¼ 9.0 Hz, 1H), 7.37 (d, J ¼ 3.2 Hz,
1H), 7.59 (td, J ¼ 1.6 Hz, 7.7 Hz, 1H), 8.08 (dd, J ¼ 2.1 Hz, 9.1 Hz, 1H),
142.00, 161.79 (JC–F ¼ 243.5 Hz). Reported43,44
.
8.61 (d, J ¼ 1.9 Hz, 2H). Reported40,41
.
2.1.3.2. (5-Amino-1H-indol-1-yl)(3-fluorophenyl)methanone (3b).
Orange solid, yield: 91%, mp: 91.7–92.9 ꢁC, 1H NMR (400 MHz,
2.1.2. Synthesis of 1-methyl-5-nitro-1H-indole (2d)
K2CO3 (55.3 mg, 0.24 mmol) was added to a solution of 5-nitroin- DMSO-d6) d 5.06 (s, 2H), 6.54 (d, J ¼ 3.7 Hz, 1H), 6.68 (dd,
dole (1, 162.2 mg, 1.0 mmol) in DMF (5 mL). Dimethyl carbonate J ¼ 2.1 Hz, 8.7 Hz, 1H), 6.77 (d, J ¼ 2.0 Hz, 1H), 7.19 (d, J ¼ 3.7 Hz,
(DMC) (0.17 mL, 2.0 mmol) was added and the reaction mixture 1H), 7.49–7.66 (m, 4H), 8.01 (d, J ¼ 8.7 Hz, 1H). 13C NMR (100 MHz,
was refluxed for 3 h, then cooled to 4–5 ꢁC, followed by adding DMSO-d6) d 104.60, 109.35, 113.38, 116.07 (JC–F ¼ 23.1 Hz), 116.81,
ice-cold water to the mixture. Precipitated solid was filtered and 118.88 (JC–F ¼ 21.1 Hz), 125.23 (JC–F ¼ 3.0 Hz), 128.06, 128.23,
washed with water. Yellow solid, yield: 83%, mp: 164.7–165.9 ꢁC, 131.36 (JC–F ¼ 8.1 Hz), 132.34, 137.14 (JC–F ¼ 7.0 Hz), 146.25,
1H NMR (400 MHz, CDCl3) d 3.85 (s, 3H), 6.66 (d, J ¼ 2.8 Hz, 1H), 162.17 (JC–F ¼ 245.5 Hz).