7-Amidocoumarins as Multitarget Agents against Neurodegenerative Diseases: Substitution Pattern Modulation

Article abstract of DOI:10.1002/cmdc.202000454

This study explores the potential of 7-amidocoumarins as multitarget agents against Parkinson's and Alzheimer's diseases, by modulating the substitution patterns within the scaffold. Sixteen compounds were synthesized via 7-amino-4-methylcoumarin acylatio

Full text of DOI:10.1002/cmdc.202000454

Accepted Article
Title: 7-Amidocoumarins as multitarget agents against
neurodegenerative diseases: substitution pattern modulation
Authors: Fernanda Rodríguez-Enríquez, Dolores Viña, Eugenio
Uriarte, Reyes Laguna, and Maria Joao Matos
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000454
Link to VoR: https://doi.org/10.1002/cmdc.202000454
01/2020

10.1002/cmdc.202000454
ChemMedChem
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7-Amidocoumarins as multitarget agents against
neurodegenerative diseases: substitution pattern modulation
Fernanda Rodríguez-Enríquez,[a,b] Dolores Viña,[a,b]* Eugenio Uriarte,[c,d] Reyes Laguna[b] and Maria J.
Matos[c,e]*
[a]
Ms Fernanda Rodríguez-Enríquez; Dr Dolores Viña
Chronic Diseases Pharmacology Group,
Center for Research in Molecular Medicine and Chronic Diseases (CIMUS),
University of Santiago de Compostela,
15782 Santiago de Compostela, Spain
E-mail: mdolores.vina@usc.es
[b]
[c]
Ms Fernanda Rodríguez-Enríquez; Dr Dolores Viña; Dr Reyes Laguna
Departamento de Farmacología, Farmacia y Tecnología Farmacéutica,
Universidade de Santiago de Compostela,
15782 Santiago de Compostela, España
Dr Maria J. Matos; Dr Eugenio Uriarte
Departamento de Química Orgánica, Facultade de Farmacia,
Universidade de Santiago de Compostela,
15782 Santiago de Compostela, España
E-mail: maria.matos@fc.up.pt or mariajoao.correiapinto@usc.es
Dr Eugenio Uriarte
Instituto de Ciencias Químicas Aplicadas,
Universidad Autónoma de Chile,
7500912 Santiago, Chile
[d]
[e]
Dr Maria J. Matos
CIQUP/Department of Chemistry and Biochemistry,
Faculty of Sciences, University of Porto,
4169-007 Porto, Portugal
E-mail: maria.matos@fc.up.pt or mariajoao.correiapinto@usc.es
Abstract: This study explores the potential of 7-amidocoumarins as
multitarget agents against Parkinson’s and Alzheimer’s diseases, by
modulating the substitution patterns within the scaffold. Sixteen
compounds were synthesized via 7-amino-4-methylcoumarin acylation,
and in vitro evaluation of the molecules against hMAO-A, hMAO-B,
hAChE, hBuChE and hBACE1 was performed. Five compounds turned
out to be potent and selective hMAO-B inhibitors in the nanomolar range,
six displayed inhibitory activity of hMAO-A in the low micromolar range,
one showed hAChE inhibitory activity and another one hBACE1
inhibitory activity. MAO-B reversibility profile of 7-(4’-chlorobenzamido)-
4-methylcoumarin (10) was investigated, being this compound a
reversible inhibitor. Neurotoxicity on motor cortex neurons and
neuroprotection against H2O2 were also studied, corroborating the
safety profile of these molecules. Finally, theoretical ADME properties
were also calculated, showing these molecules as good candidates for
the optimization of a lead compound. Results suggest that by
modulating the substitution pattern at position 7 of the scaffold, selective
or multitarget molecules can be achieved.
becoming increasingly prevalent with the aging of the worldwide
population.[4] Within the context of increased longevity, rising
numbers of people with dementia and surviving strokes is adding
further pressure on already stretched health and social care
services.[5]
Alzheimer's disease is the most common cause of dementia (60-
80% of the cases), involving memory loss and deterioration of other
cognitive abilities, interfering with patients’ and caregivers’ daily
life.[6] Despite the advances in the medical sciences, the incidence
of this disease is increasing rapidly and has been estimated to
currently affect 66 million people worldwide and to increase to 115
million by 2050.[7]
The second most prevalent neurodegenerative disease is
Parkinson’s disease. Dorsey et al. reported that the total population
affected by Parkinson’s disease at age 50 in the 5 most populous
nations in Western Europe and in the 10 most populous nations in
the world were between 4.1 and 4.6 million in 2005[8] and estimated
that these numbers will be doubled by 2030.[9]
Understanding some of the molecular changes associated to these
ubiquitous and widespread diseases, has stimulated efforts to
identify targets and develop drugs that especially interact with key
enzymes involved in their development. Knowledge of biochemical
processes brings the opportunity to provide treatments that are
potentially less toxic and more effective than previous therapeutic
approaches. As multifactorial diseases, multitarget approaches
may be interesting solutions.[10]
Introduction
Neurological disorders have been reported as the major cause of
disability-adjusted life-years, and the second leading cause of
deaths worldwide (9.0 million), in 2016.[1] The main responsible for
these number was stroke (~42%), followed by Alzheimer's and
other dementias (~10.4%).[2,3] Neurodegenerative diseases are
1
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Coumarins are a versatile family of compounds and a privileged
scaffold in Medicinal Chemistry.[11] Their multitarget profile,
especially regarding neurodegenerative disease, has already been
described by several groups working in the field.[12-15] For more than
ten years, our research group has been reporting differently
substituted coumarins as monoamine oxidase (MAO),[16-20]
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
inhibitors, as well as neuroprotective agents (Figure 1A-D).[21-26]
Furthermore, 7-substituted coumarins have been raised as potent
multitarget molecules.[27-31] Based on this expertise, a new series of
7-amidocoumarins (Figure 1E) has been synthetized and studied
against different targets. Besides the above-mentioned enzymes,
beta-secretase 1 (BACE1) was also included in this study.
Substitution patterns’ modulation allowed to obtain selective
compounds against one single target, as well as multitarget drugs
with different profiles, with potential against either Alzheimer’s or
Parkinson’s diseases.
O
R
R
R
R
O
O
O
O
MAO-A/MAO-B
inhibitors
MAO-B selective
inhibitors
A
B
O
?
R
N
O
O
H
O
H
N
R
R
E
N
R
R
H
O
O
O
O
O
MAO-B/AChE/BuChE
inhibitors
MAO-B selective
inhibitors
D
C
Figure 1. Rational design of the selection of the studied scaffold. A. 3-Arylcoumarin scaffold; B. 3-Benzoylcoumarin scaffold; C. 3-Carboxamidocoumarin scaffold; D. 3-
Amidocoumarin scaffold and E. 7-Amidocoumarin scaffold.
commercially available 7-amino-4-methylcoumarin, an acylation
reaction in the presence of the conveniently substituted acid
Results and Discussion
chloride, using pyridine in dichloromethane, from 0 C to room
temperature, overnight, afforded the desired 7-amido-4-
Chemistry
methylcoumarins (1-16) in yields between 80–90%. The reaction
mixtures were purified by flash chromatography, using a mixture of
The described derivatives 1-16 were efficiently synthesized
n-hexane and ethyl acetate (9:1), as detailed in the experimental
according to the protocol outlined in Scheme 1. Starting from the
section.
Scheme 1. Synthetic methodology to afford the studied 7-amido-4-methylcoumarins (1-16).
2
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The substituents at position 7 were selected taking into account
their nature, size and volume, directing related to their steric
hindrance. In the case of the 7-benzamido-4-methylcoumarins (7-
14), the substituents present in the aromatic ring at position 7 of the
coumarin scaffold were selected taking into account the different
quadrants of the Craig diagram (methyl vs nitro and chlorine vs
methoxy). Finally, two heterocycles were also selected to complete
the series (thiophene and furan rings, corresponding to compounds
15 and 16).
The in vitro effects of compounds 1-16 on hMAO activity were
studied using an Amplex® Red MAO assay kit and the recombinant
hMAO-A or hMAO-B isoforms.[16] Selegiline and iproniazid were
used as reference controls, and IC50 values and SI are organized in
Table 1. The in vitro effects of compounds 1-16 on hAChE and
hBuChE activities were determined by the Ellman method, using
human recombinant AChE expressed in HEK 293 cells or BuChE
isolated from human serum.[22,32] Tacrine and eserine were used as
reference controls, and all the results are organized in Table 1. To
study the possible effects of our compounds and the reference
inhibitor OM-99 on the enzymatic activity of hBACE1, a method
based on the TR-FRET (Time-Resolved Fluorescence Resonance
Energy Transfer) technique was used. The IC50 values are also
presented in Table 1.[33]
Enzymatic activity inhibition (hMAO-A, hMAO-B, hAChE,
hBuChE and hBACE1)
Table 1. IC50 values of compounds 1-16 and reference inhibitors activity on recombinant hMAO isoforms expressed in baculovirus infected BTI insect cells (BTI-TN-5B1-
4), recombinant hAChE expressed in HEK 293 cells or hBuChE isolated from human serum, and purified baculovirus-expressed hBACE1.
Comp.
1
IC50 hMAO-A (μM)
IC50 hMAO-B (μM)
SI [a]
IC50 hAChE (μM)
IC50 hBuChE (μM)
IC50 hBACE1 (μM)
25.69 ± 1.72
>100
<0.26 [b]
>100
>100
n.d.
2
3
4
5
6
7
8
9
96.65 ± 6.53
24.50 ± 1.64
45.86 ± 10.25
>100
**
0.97 [b]
2.12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
n.d.
n.d.
n.d.
n.d.
>100
n.d.
n.d.
11.55 ± 0.78
0.44 ± 0.03
0.25 ± 0.02
>100
104.23
>400.0 [b]
<0.78 [b]
>278 [b]
>111.36 [b]
-
>100
>100
78.16 ± 5.28
3.78 ± 0.25
>100
0.36 ± 0.02
0.90 ± 0.06
>100
>100
>100
>100
>100
>100
3
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10.1002/cmdc.202000454
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10
>100
>100
0.31 ± 0.02
>322.58 [b]
**
>100
>100
***
**
n.d.
n.d.
11
12
>100
-
-
>100
>100
***
>100
**
n.d.
13
***
***
-
***
14
***
***
-
***
***
***
15
**
1.59 ± 0.11
5.01 ± 0.34
0.017 ± 0.002
7.54 ± 0.36
n.d.
62.89 [b]
6.02
4,043
0.87
n.d.
n.d.
n.d.
>100
>100
n.d.
>100
>100
n.d.
n.d.
34.49 ± 2.31
>100
n.d.
16
30.14 ± 2.03
68.73 ± 4.21
6.56 ± 0.76
n.d.
Selegiline
Iproniazid
Tacrine
Eserine
OM-99
n.d.
n.d.
0.45 ± 0.03
0.12 ± 0.01
n.d.
0.15 ± 0.01
0.15 ± 0.01
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.08 ± 0.002
Each IC50 value is the mean ± S.E.M. from three experiments (n = 3). ** At 100 µM inhibits enzymatic activity by approximately 45-50%. *** The compound proved to be
insoluble at the tested conditions. [a] SI: hMAO-B selectivity index = IC50 (hMAO-A)/IC50 (hMAO-B). [b] Values obtained under the assumption that the corresponding IC50
against hMAO-A or hMAO-B is the highest concentration tested (100 µM). Highest concentration tested: 100 µM; at higher concentration, the compounds precipitate.
n.d.: non determined.
to an increase in the MAO-B inhibitory activity: methyl < ethyl <
Five of the studied compounds (4, 5, 7, 8 and 10) display MAO-B
isopropyl
<
tert-butyl. 7-Benzamido-4-methylcoumarin (7), 4-
(8) and 7-(4’-
activity in the nanomolar range. The first two derivatives, 7-
isobutylacetamido-4-methylcoumarin (4) and 7-tert-
methyl-7-(4’-methylbenzamido)coumarin
chlorobenzamido)-4-methylcoumarin (10) present an aromatic
group attached to the amide at position 7. The first one, without
substituents, and the other two, with a para-methyl (electron donor
group) and a para-chlorine (electron acceptor atom), respectively.
butylacetamido-4-methylcoumarin (5), present small side chains
attached to the amide at position 7. Although, both substitutions on
those side chains are quite bulky. In fact, an increase in the number
of carbon atoms of the alkyl substituent attached to the amide, leads
4
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All these substituents seem to be acceptable for this activity.
However, a para-methoxy or para-nitro substituent leads to inactive
derivatives 9 and 11. Finally, the increase in the number of
substituents attached to the aromatic ring leads to the insoluble
compounds 13 and 14.
[a] Values represent the mean ± S.E.M. of n = 2 experiments, relative to the control;
data show recovery of hMAO-B activity after dilution.
Compound 10 proved to be a reversible hMAO-B inhibitor,
according to the results presented in Table 2. Comparing to isatin,
a reversible MAO-B inhibitor used as reference, its degree of
reversibility is slightly lower. However, compound 10 presents a
better reversibility profile than selegiline, a non-reversible inhibitor
used in therapeutics. Some 3-amidocoumarins already reported by
the group were described as reversible hMAO-B inhibitors.[23]
Recently, a coumarin derivative bearing a pent-2-yn-1-amine group
at position 7, the same position under study in this project, was
reported as a partially reversible MAO-B inhibitor.[26]
The exchange of the benzene ring with thiophene (15) or furan (16)
rings, decreased MAO-B inhibitory activity to the low micromolar
range (IC50 = 1.59 and 5.01 μM, respectively). Furthermore, the
furan ring also decreased the selectivity. Compound 16 has activity
on both isoforms, although it is more potent on MAO-B.
Six of the studied compounds present MAO-A inhibitory activity in
the micromolar range (1-4, 6 and 16). As observed, most of them
present small lateral chains attached to the amide at position 7 of
the coumarin scaffold. From these molecules, 7-acetamido-4-
methylcoumarin (1), 4-methyl-7-propionamidocoumarin (2) and 7-
(2’-bromoacetamido)-4-methylcoumarin (6) proved to be selective
Cellular studies
Neurotoxicity profile
against
this
isoform
whereas
7-isopropylacetamido-4-
methylcoumarin (3) inhibits both MAO isoforms in the same
concentration range.
Neurotoxic activity of compounds 1-16 was measured on rat motor
cortex neurons, obtained from embryos of 19 days pregnant albino
rats (Rattus novergicus) of the Wistar-Kyoto strain.[35] After
treatment and an incubation period of 24 hours, monitorization of
cell viability was performed by using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide method (MTT). The percentage
(%) of cell viability after incubation with the 7-amidocoumarins 1-16,
at 10 μM concentration, is represented in Figure 2.
From the studied series, none of the compounds displays significant
inhibitory activity against BuChE. Regarding AChE inhibition,
compound 6 proved to inhibit this enzyme in the low micromolar
range. Therefore, this compound can be a promising dual target
molecule, presenting activity against both MAO-A and AChE.
Finally, and to push further the multitarget potential of these
molecules, BACE1 inhibitory activity was evaluated for a selection
of
compounds.
From
those,
4-methyl-7-(2’-
150
100
50
thiophenamido)coumarin (15) displays activity in the micromolar
range IC50 = 34.49 μM). This is a very interesting achievement,
since no other coumarin studied by our group ever presented
activity against this target. Compound 15 displays also MAO-B
inhibitory activity in the low micromolar range, being, therefore, a
candidate to explore this dual target activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1
O
1
1
1
1
1
1
S
M
D
Figure 2. Evaluation of the neurotoxic effect of the 7-amidocoumarins 1-16 on rat
motor cortex neurons. Cells were incubated with the compounds at 10 μM,
dissolved in DMSO (1%). The results are expressed as % of viability against the
control group (treated with DMSO 1%) in at least 5 experiments ± S.E.M.
Reversibility profile of the best hMAO-B inhibitor
The type of inhibition exerted by the 7-(4’-chlorobenzamido)-4-
methylcoumarin (10) on hMAO-B isoform was evaluated by using a
dilution method.[34] The selected compound is the most potent and
selective 7-benzamido-4-methylcoumarin within the studied series.
Selegiline and isatin were used as controls, and the results are
shown in Table 2.
As observed in Figure 2, none of the studied derivatives showed
toxicity against the cells used for this study.
Neuroprotective activity
Table 2. Reversibility results for the hMAO-B inhibition of compound 10 and the
reference inhibitors (selegiline and isatin).
Together with the enzymatic inhibition, neuroprotective effect on
neuronal populations affected during the progression of
neurodegenerative diseases is desirable. The neuroprotective
effect of the 7-amidocoumarins 1-16 against H2O2, in the cellular
model previously described, was then evaluated (Figure 3). Cells
were treated with H2O2 (100 μM) previously to the treatment with 7-
amidocoumarins 1-16. Monitorization of cell viability was performed
after 24 hours incubation by using the MTT method.
Compound
10
Slope (ΔUF/t) [%][a]
59.30 ± 3.98
Selegiline
Isatin
12.73 ± 1.85
88.63 ± 5.90
5
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10.1002/cmdc.202000454
ChemMedChem
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by the lack of hydroxyl groups on their structures, as we observed
in some previous projects from our group.
150
100
ADME properties
#
50
0
In order to assess the possible in vivo activity of these compounds,
theoretical physicochemical properties of the 7-amidocoumarins 1-
16 were calculated. These parameters are excellent indicators of
the capacity to cross cellular membranes and therefore of their
ADME (absorption, distribution, metabolism and excretion)
properties. Molinspiration cheminformatics software was used to
calculate the octanol/water partition coefficient (LogP), the polar
surface area (TPSA), the number of atoms and molecular weight
(MW), the number of H-bond acceptors (ON) and H-bond donors
(OHNH), as well as the volume (V) and the number of rotatable links
(rotb). The "CBligand-BBB predictor" program was used to
theoretically predicted the probability to cross through the blood
brain barrier (BBB) of the studied molecules. All the results,
together with the prediction of the violations of Lipinski rules (n viol),
are included in Table 3.
1
2
3
4
5
6
7
8
9
1
2
0
3
4
5
6
1
O
O
S
1
1
1
1
1
1
S
M
M
D
D
_________________________________________________________________________
+H2O2
Figure 3. Neuroprotective effect of the 7-amidocoumarins 1-16 on rat motor cortex
neurons. The cells were incubated with the molecules at 10 μM, dissolved in
DMSO (1%), in the presence of H2O2 (100 μM). The results are expressed as %
of viability against the control group (treated with DMSO 1%) in at least 5
experiments ± S.E.M. #P<0.0001, against the group treated with only DMSO.
As can be seen in Figure 3, all the derivatives, at the studied
concentration, lack neuroprotective activity on the neurons of a
primary culture of the motor cortex neurons. This can be explained
Table 3. Molecular properties of 7-amidocoumarins 1-16 calculated using the Molinspiration software and theoretical prediction of their passage through BBB using the
CBLigand-BBB software.
Compd
LogP
1.59
2.42
2.67
3.20
3.52
2.28
3.36
3.71
3.31
3.94
3.22
2.90
TPSA
59.31
59.31
59.31
59.31
59.31
59.31
59.31
59.31
68.54
59.31
105.13
77.78
n atoms
16
MW
n ON
n OHNH
n rotb
V
n viol
BBB ()
1
2
217.22
231.25
245.28
259.31
273.33
296.12
279.30
293.32
309.32
313.74
324.29
339.35
4
4
4
4
4
4
4
4
5
4
7
6
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
3
3
2
2
2
3
2
3
4
193.10
209.90
226.48
243.29
259.52
211.22
247.94
264.50
273.49
261.48
271.28
299.03
0
0
0
0
0
0
0
0
0
0
0
0
-
+
-
17
3
18
4
19
-
5
20
-
6
17
+
+
+
-
7
21
8
22
9
23
10
11
12
22
+
-
24
25
-
6
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13
4.54
59.31
87.01
59.31
72.45
3.24
23
27
20
20
13
15
20
348.19
369.37
285.32
269.26
173.26
198.27
275.35
4
7
4
5
1
2
5
1
1
1
1
0
2
1
2
5
2
2
3
0
2
275.01
324.58
238.65
229.51
185.84
191.53
261.48
0
0
0
0
0
0
0
+
-
14
2.89
3.16
2.51
2.26
3.05
1.94
15
+
-
16
Selegiline
Tacrine
Eserine
+
+
+
38.92
44.81
Theoretically, all the 7-amidocoumarins 1-16 possess the
desirable physicochemical properties for a good bioavailability,
and none of the compounds violate the Lipinski rule of five (Table
3). 4-Methyl-7-(4’-methylbenzamido)coumarin (8) has the second
highest value of LogP within the series, combined with a low
TPSA. 7-Benzamido-4-methylcoumarin (7), 4-methyl-7-(4’-
presenting all the theoretical properties to have good
bioavailability and cross the BBB. 4-Methyl-7-(2’-
thiophenamido)coumarin (15) is a dual MAO-B/BACE1 inhibitor,
with activities in the low micromolar range. Finally, 7-(2’-
bromoacetamido)-4-methylcoumarin (6) is a dual MAO-A/AChE
inhibitor, presenting IC50 in the low micromolar range. All these
results together help to understand that, by modulating the
substitution pattern at position 7 of the 7-amidocoumarins, it is
possible to design molecules with different profiles against
Parkinson’s and Alzheimer’s diseases.
methylbenzamido)coumarin
(8)
and
7-(4’-
chlorophenylbenzamido)-4-methylcoumarin (10), three of the
MAO-B selective inhibitors (IC50 in the nanomolar range), present
good ADME properties, as well as the theoretical ability to cross
the BBB. Also, 4-methyl-7-(2’-thiophenamido)coumarin (15), the
dual MAO-B/BACE1 inhibitor (IC50 in the low micromolar range),
present both good ADME properties and theoretical ability to
cross the BBB. Finally, the dual MAO-A/AChE inhibitor 7-(2’-
bromoacetamido)-4-methylcoumarin (6, IC50 in the low
micromolar range), also present good ADME properties and
theoretical ability to cross the BBB.
Experimental Section
Chemistry
General information. All reagents were purchased from Sigma-
Aldrich and used without further purification. All solvents were
commercially available grade. All reactions were carried out under
argon atmosphere, unless otherwise mentioned. Reaction
mixtures were purified by flash column chromatography using
Silica Gel high purity grade (Merck grade 9385 pore size 60Å,
230-400 mesh particle size). Reaction mixtures were analysed by
analytical thin-layer chromatography (TLC) using plates
precoated with silica gel (Merck 60 F254, 0.25 mm). Visualization
was accomplished with UV light (254 nm), ninhydrin or potassium
permanganate (KMnO4). 1H NMR and 13C NMR spectra were
recorded on a Bruker AMX spectrometer at 250 and 75.47 MHz
in the stated solvents (CDCl3 or DMSO-d6) using
tetramethylsilane (TMS) as an internal standard. Chemical shifts
were reported in parts per million (ppm) on the δ scale from an
internal standard (NMR descriptions: s, singlet; d, doublet; t,
triplet; q, quadruplet; m, multiplet). Mass spectroscopy was
performed using a Hewlett-Packard 5988A spectrometer. This
system is an automated service utilizing electron impact (EI)
ionization. Elemental analyses were performed using a Perkin-
Elmer 240B microanalyzer and were within (0.4% of calculated
Analyzing the data, it can be observed that, in general, the 7-
benzamidocoumarins (7-16) present better profiles than the 7-
alkylamidocoumarin (1-6). From these molecules (1-6), four
molecules seem to have problems in crossing the BBB. The
molecules with an aromatic ring attached to the 7-amide group (7-
16) seem to be more likely to cross the BBB, as half of the studied
compounds proved to have the right structures to cross this
barrier.
Conclusion
This report describes the MAO-A, MAO-B, AChE, BuChE and
BACE1 inhibitory activities of 7-amidocoumarins, observing that
the nature of the substituents is decisive for their activity and
selectivity. 7-Isobutylacetamido-4-methylcoumarin (4), 7-tert-
butylacetamido-4-methylcoumarin
(5),
7-benzamido-4-
methylcoumarin (7), 4-methyl-7-(4’-methylbenzamido)coumarin
(8) and 7-(4’-chlorobenzamido)-4-methylcoumarin (10) are MAO-
B selective inhibitors, presenting IC50 in the nanomolar range.
Compound 10 proved to be a reversible MAO-B inhibitor,
7
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10.1002/cmdc.202000454
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FULL PAPER
values in all cases). The purity of compounds was assessed by
HPLC and was found to be higher than 99%.
7.65 (dd, 1H, H-6, J=8.5, 1.9), 7.72-7.75 (m, 2H, H-5’, H-6’), 7.93
(s, 1H, H-2’), 10.46 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 18.2, 55.8,
55.9, 111.0, 111.3, 112.5, 115.3, 116.3, 121.5, 125.8, 126.5,
140.0, 148.5, 153.3, 153.7, 154.6, 160.3, 165.6. MS m/z (%): 339
(M+, 73). Ana. Elem. Calc. for C19H17NO5: C, 67.25; H, 5.05.
Found: C, 67.23; H, 5.08.
Synthetic methodology. The 7-amino-4-methylcoumarin
(commercially available, 1.0 mmol) was dissolved in CH2Cl2 (9
mL). Pyridine (1.1 mmol) was then added, and the mixture was
cooled to 0 C. The corresponding acyl chloride (1.1 mmol) was
added dropwise at this temperature, and the mixture was stirred
overnight at room temperature. The batch was evaporated and
purified by column chromatography (hexane/EtOAc, 9:1) to give
the desired compound 1-16.
7-(3’,4’-Dichlorophenylbenzamido)-4-methylcoumarin (13).
1H NMR (DMSO-d6) δ: 2.48 (s, 3H, CH3), 6.27 (s, 1H, H-3), 7.75-
7.83 (m, 2H, H-5, H-6), 7.91-7.98 (m, 2H, H-5’, H-6’), 8.25 (d, 1H,
H-8, J=1.8), 8.88 (s, 1H, H-2’), 10.77 (s, 1H, NH). 13C NMR
(DMSO-d6) δ: 19.5, 111.9, 112.5, 115.2, 118.3, 124.3, 128.2,
128.9, 130.3, 133.5, 133.7, 136.8, 137.3, 152.7, 154.3, 160.8,
164.7. MS m/z (%): 349 (M+, 17), 347 (M+, 30). Ana. Elem. Calc.
for C17H11Cl2NO3: C, 58.64; H, 3.18. Found: C, 58.62; H, 3.22.
7-Acetamido-4-methylcoumarin (1).[36]
4-Methyl-7-propionamidocoumarin (2).[37]
7-Isopropylacetamido-4-methylcoumarin (3). 1H NMR (CDCl3)
δ: 1.27 (s, 3H, CH3), 1.30 (s, 3H, CH3), 2.44 (s, 3H, CH3), 2.65 (q,
1H, CH, J=6.8), 6.22 (s, 1H, H-3), 7.55 (d, 1H, H-5, J=8.7), 7.67
(d, 1H, H-8, J=2.2), 7.86 (dd, 1H, H-6, J=8.7, 2.2), 8.06 (s, 1H,
NH). 13C NMR (CDCl3) δ: 18.5, 19.4, 20.6, 36.5, 106.7, 113.0,
115.7, 121.4, 125.2, 139.0, 145.2, 152.1, 162.1, 175.8. MS m/z
(%): 245 (M+, 76). Ana. Elem. Calc. for C14H15NO3: C, 68.56; H,
6.16. Found: C, 68.52; H, 6.15.
7-(3’,4’,5’-Trimethoxybenzamido)-4-methylcoumarin (14). 1H
NMR (DMSO-d6) δ: 2.41 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 3.87 (s,
36H, 2xOCH3), 6.27 (s, 1H, H-3), 7.30 (s, 2H, H-2’, H-6’), 7.73-
7.75 (m, 2H, H-5, H-6), 7.93 (d, 1H, H-8, J=1.1), 10.54 (s, 1H, NH).
13C NMR (DMSO-d6) δ: 18.2, 56.3, 60.3, 105.7, 106.9, 112.6,
115.5, 116.5, 125.9, 127.0, 129.6, 142.7, 152.8, 153.3, 153.7,
160.0, 165.6. MS m/z (%): 369 (M+, 54). Ana. Elem. Calc. for
C20H19NO6: C, 65.03; H, 5.19. Found: C, 65.06; H, 5.13.
7-Isobutylacetamido-4-methylcoumarin (4). 1H NMR (DMSO-
d6) δ: 0.88 (s, 3H, CH3), 0.91 (s, 3H, CH3), 2.06 (q, 1H, CH, J=7.3),
2.20 (d, 2H, CH2, J=7.3), 2.35 (s, 3H, CH3), 6.20 (s, 1H, H-3), 7.43
(dd, 1H, H-6, J=8.7, 2.0), 7.64 (d, 1H, H-5, J=8.7), 7.73 (d, 1H, H-
8, J=2.0), 10.25 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 18.1, 22.4,
25.6, 45.8, 105.5, 112.2, 114.9, 115.2, 126.0, 142.7, 153.2, 153.8,
160.2, 171.5. MS m/z (%): 259 (M+, 87). Ana. Elem. Calc. for
C15H17NO3: C, 69.48; H, 6.61. Found: C, 69.51; H, 6.58.
4-Methyl-7-(2’-thiophenamido)coumarin
(15).
1H
NMR
(DMSO-d6) δ: 2.39 (s, 3H, CH3), 6.26 (s, 1H, H-3), 7.24 (t, 1H, H-
4’, J=3.8), 7.71-7.74 (m, 2H, H-5, H-6), 7.89-7.91 (m, 2H, H-8, H-
3’), 8.12 (d, 1H. H-5’, J=3.8), 10.65 (s, 1H, NH). 13C NMR (DMSO-
d6) δ: 18.2, 106.7, 112.6, 115.5, 116.2, 125.9, 128.4, 130.1, 132.9,
139.5, 142.4, 153.2, 153.7, 160.2, 160.5. MS m/z (%): 285 (M+,
61). Ana. Elem. Calc. for C15H11NO3S: C, 65.15; H, 3.89. Found:
C, 63.15; H, 3.92.
7-tert-Butylacetamido-4-methylcoumarin (5). 1H NMR (CDCl3)
δ: 1.12 (s, 9H, 3xCH3), 2.30 (s, 2H, CH2), 2.43 (s, 3H, CH3), 7.55
(d, 1H, H-5, J=8.7), 7.61 (d, 1H, H-8, J=2.2), 7.77 (dd, 1H, H-6,
J=8.7, 2.2), 8.09 (s, 1H, NH). 13C NMR (CDCl3) δ: 24.8. 29.6, 31.0,
43.0, 109.9, 112.5, 119.4, 119.7, 125.1, 143.3, 157.2, 161.0,
170.5, 174.0. MS m/z (%): 273 (M+, 91). Ana. Elem. Calc. for
C16H19NO3: C, 70.31; H, 7.01. Found: C, 70.28; H, 7.04.
7-(2’-Furanamido)-4-methylcoumarin (16). 1H NMR (DMSO-d6)
δ: 2.38 (s, 3H, CH3), 6.25 (s, 1H, H-3), 7.22 (t, 1H, H-4’, J=3.5),
7.45 (d, 1H, H-5’, J=3.5), 7.72-7.78 (m, 2H, H-5, H-6), 7.89-7.97
(m, 2H, H-8, H-3’), 10.62 (s, 1H, NH). 13C NMR (DMSO-d6) δ: 18.1,
106.7, 112.6, 115.5, 115.9, 116.2, 125.9, 142.2, 146.4, 147.1,
153.2, 153.6, 156.6, 160.2. MS m/z (%): 269 (M+, 46). Ana. Elem.
Calc. for C15H11NO4: C, 66.91; H, 4.12. Found: C, 66.93; H, 4.16.
7-(2’-Bromoacetamido)-4-methylcoumarin (6).[38]
7-Benzamido-4-methylcoumarin (7).[39]
Pharmacology
Determination of MAO isoforms in vitro activity. The effects of
the 7-amidocoumarins (1-16) on hMAO enzymatic activity were
evaluated by a fluorimetric method following the experimental
protocol previously described by us.[16] Briefly, 50 µL of sodium
phosphate buffer (0.05 M, pH 7.4) containing the test molecules
(new compounds or reference inhibitors) in different
concentrations and adequate amounts of recombinant hMAO-A
or hMAO-B [adjusted to obtain in our experimental conditions the
same reaction velocity (hMAO-A: 1.1 μg protein; specific activity:
4-Methyl-7-(4’-methylbenzamido)coumarin (8).[39]
7-(4’-Methoxybenzamido)-4-methylcoumarin (9).[38]
7-(4’-Chlorobenzamido)-4-methylcoumarin (10).[39]
4-Methyl-7-(4’-nitrobenzamido)coumarin (11).[39]
150
hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 μg
protein; specific activity: 22 nmol of p-tyramine
transformed/min/mg protein)] were incubated for 10 min at 37 C
nmol
of
p-tyramine
oxidized
to
p-
7-(3’,4’-Dimethoxybenzamido)-4-methylcoumarin (12). 1H
NMR (DMSO-d6) δ: 2.40 (s, 3H, CH3), 3.84 (s, 6H, 2xOCH3), 6.25
(s, 1H, H-3), 7.09 (d, 1H, H-5, J=8.5), 7.54 (d, 1H, H-8, J=1.9),
8
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10.1002/cmdc.202000454
ChemMedChem
FULL PAPER
in a flat-black bottom 96-well microtest plate, placed in the dark
fluorimeter chamber. After this incubation period, the reaction was
started by adding 50 µL of the mixture containing (final
concentrations) 200 μM of the Amplex® Red reagent, 1 U/mL of
horseradish peroxidase and 1 mM of p-tyramine. The production
of H2O2 and, consequently, of resorufin, was quantified at 37 C
in a multidetection microplate fluorescence reader (Fluo-star
OptimaTM, BMG LABTECH, Offenburg, Germany) based on the
fluorescence generated (λ excitation, 545 nm, λ emission, 590
nm) over a 10 min period, in which the fluorescence increased
linearly. Control experiments were carried out simultaneously by
replacing the tested molecules with appropriate dilutions of the
vehicles (DMSO ≤ 0.5 %). In addition, the possible capacity of
these molecules to modify the fluorescence generated in the
reaction mixture, due to non-enzymatic inhibition (i.e. for directly
reacting with Amplex® Red reagent), was determined by adding
these molecules to solutions containing only the Amplex® Red
reagent in sodium phosphate buffer. The specific fluorescence
emission (used to obtain the final results) was calculated after
subtraction of the background activity, which was determined
from wells containing all components except the hMAO isoforms,
which were replaced by sodium phosphate buffer solution.
Determination of BACE1 in vitro activity. The assay kit used in
the experiments was LanthaScreen TR-FRET BACE1
(Invitrogen). 30 μL of sodium acetate buffer (50 mM, pH 4.5)
containing different concentrations of the compounds to be
studied (or the reference inhibitor OM-99), the enzyme BACE1
(2.1 U/mL) and the substrate FL- BACE1-biotin (200 nM
concentration) were incubated in 96-well black flat bottom plates
(96-well Half Area Black Flat Bottom Polystyrene NBS Microplate,
Corning®) at room temperature and protected from light for 60
min. Control experiments were simultaneously carried out,
replacing the reference drugs or inhibitors with the appropriate
dilutions of the vehicles (DMSO ≤ 0.5 %). After the incubation
period, the reaction was stopped by adding 10 µL of the stop
solution (Tb-anti-biotin antibody, 5 nM concentration). After
mixing, the solution was kept 60 min at room temperature and
protected from light. Once the incubation was finished,
fluorescence intensity was measured using the TR-FRET
technique (λ excitation 545, λ emission 585) in a multifunction
plate reader (CLARIOstar® Plus, BMG LABTECH, Offenburg,
Germany). The fluorescence emitted by the FL-BACE1-Biotin
substrate was also determined at the same concentration used in
the experiments in the absence of the enzyme and subtracted
from the total fluorescence.
Reversibility. To evaluate whether the 7- (4’-chlorobenzamido)-
4-methylcoumarin (10) is a reversible or irreversible hMAO-B
inhibitor, a dilution method was used.[34] A 100X concentration of
the enzyme used in the above described experiment was
incubated with a concentration of inhibitor equivalent to 10-fold
the IC50 value. After 30 min, the mixture was diluted 100-fold into
a reaction buffer. Enzymatic activity was then evaluated as
previously described. Reversible inhibitors show linear progress
with a slope equal to ~91% of the slope of the control sample,
whereas irreversible inhibition reaches only ~9% of this slope. A
control test was carried out by pre-incubating and diluting the
enzyme in the absence of inhibitor.
Cell viability. Embryos were extracted by caesarean section from
pregnant Wistar Kyoto rats, which were euthanized by CO2
inhalation. Brains were carefully dissected out and after removing
meninges, a portion of motor cortex was isolated. Fragments
obtained from several embryos were mechanically digested and
cells were resuspended in neurobasal medium supplemented
with 2% B-27. Cells were seeded in 96-well plates at a density of
200,000 cells/mL to obtain cortex neuronal cultures. Cultures
were allowed to grow for 7-8 days in an incubator (Form Direct
Heat CO2, Thermo Electron Corporation, Madrid, Spain) under
saturated humidity at a partial pressure of 5% CO2 in air, at 37 C.
Once a dense neuronal network could be observed, motor cortex
cultures were treated with the 7-amidocoumarins (1-16) at 10 µM
to evaluate the cytotoxicity. In order to evaluate their
neuroprotective effect cultures were previously treated with H2O2
(100 µM). After incubation for 24 h under the conditions above-
described, cell viability was evaluated using MTT as follows: 10
µL of a solution containing 0.5 mg/mL MTT in Hank’s was added
to each well containing 100 µL of culture medium and incubation
was performed at 37 C, for 2 h. After incubation, medium was
removed, and formazan salt formed was dissolved in DMSO. The
colorimetric determination was performed at λ 540 nm.
Determination of AChE and BuChE in vitro activity. Ellman´s
method [32] was used to determine in vitro ChE activity. 0.01 U/mL
human recombinant AChE expressed in HEK 293 cells or 0.0005
U/mL BuChE isolated from human serum were added to a 50 mM
phosphate buffer solution (pH 7.2) containing different
concentrations of compounds 1-16 or reference inhibitors. Mixture
was preincubated at 37 ºC for 5 min followed by the addition of 5
mM acetylthiocholine or butyrylthiocholine and 0.25 mM 5,5’-
dithio-bis(2-nitrobenzoic acid) (DNTB). The activity was
measured by the absorbance increasing at λ 412 nm at 1 min
intervals for 10 min at 37 ºC (Fluo-Star OptimaTM, BMG LABTECH,
Offenburg, Germany). Control experiments were performed Acknowledgements
simultaneously by replacing the test drugs (new compounds and
reference inhibitors) with appropriate dilutions of the vehicles
This project was partially supported by the University of Porto and
University of Santiago de Compostela and Consellería de Cultura,
(DMSO ≤ 0.5 %). The specific absorbance (used to obtain the final
results) was calculated after subtraction of the background activity, Educación e Ordenación Universitaria, Centro Singular de
which was determined in wells containing all components except
the AChE or BuChE, which was replaced by a sodium phosphate
buffer solution.
Investigación de Galicia and the European Regional
Development Fund (ERDF) (accreditation 2016-2019,
ED431G/05). MJM would like to thank Xunta de Galicia (Galician
Plan of Research, Innovation and Growth 2011–2015, Plan I2C,
ED481B 2014/086–0 and ED481B 2018/007) and Fundação para
9
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ChemMedChem
FULL PAPER
[25] Y. Duarte, A. Fonseca, M. Gutiérrez, F. Adasme-Carreño, C. Muñoz-
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Matos, Chem. Select. 2019, 4, 551–558.
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[26] M. J. Matos, D. M. Herrera Ibatá, E. Uriarte, D. Viña, ChemMedChem
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Table of Contents
Alzheimer’s and Parkinson’s diseases are the most prevalent neurodegenerative disorders, and there is no cure for them. Therefore,
it is urgent to develop new therapeutic solutions. 7-Amidocoumarins were synthetized and studied as MAO-A, MAO-B, AChE, BuChE
and BACE1 inhibitors, and neuroprotective agents. All the studied compounds proved to be non-neurotoxic on rat motor cortex neurons,
five turned out to be potent and selective hMAO-B inhibitors in the nanomolar range, six displayed hMAO-A inhibition in the low
micromolar range, one showed AChE inhibitory activity and another one BACE-1 inhibitory activity.
Institute and/or researcher Twitter usernames: @mariacmatos @UPorto @cimususc @UniversidadeUSC
11
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