3-Amidocoumarins as Potential Multifunctional Agents against Neurodegenerative Diseases

Article abstract of DOI:10.1002/cmdc.201500408

Monoamine oxidase (MAO) generates reactive oxygen species (ROS), which cause neuronal cell death, causing neurodegeneration. Agents that are able to concurrently inhibit MAO and scavenge free radicals represent promising multifunctional neuroprotective ag

Full text of DOI:10.1002/cmdc.201500408

DOI: 10.1002/cmdc.201500408
Full Papers
3-Amidocoumarins as Potential Multifunctional Agents
against Neurodegenerative Diseases
Maria Jo¼o Matos,*[a] Fernanda Rodríguez-Enríquez,[b] Fernanda Borges,*[a]
Lourdes Santana,[c] Eugenio Uriarte,[c] Martín Estrada,[d] María Isabel Rodríguez-Franco,[d]
Reyes Laguna,[b] and Dolores ViÇa*[b]
Monoamine oxidase (MAO) generates reactive oxygen species
(ROS), which cause neuronal cell death, causing neurodegener-
ation. Agents that are able to concurrently inhibit MAO and
scavenge free radicals represent promising multifunctional
neuroprotective agents that could be used to delay or slow
the progression of neurodegenerative diseases. In this work,
variously substituted 3-amidocoumarins are described that
exert neuroprotection in vitro against hydrogen peroxide in rat
cortical neurons, as well as antioxidant activity in a 1,1-diphen-
yl-2-picrylhydrazyl (DPPH·) radical scavenging assay. Selective
and reversible inhibitors of the MAO-B isoform were identified.
Interestingly, in the case of the 3-benzamidocoumarins, substi-
tution at position 4 with a hydroxy group abolishes MAO-B ac-
tivity, but the compounds remain active in the neuroprotection
model. Further evaluation of 3-heteroarylamide derivatives in-
dicates that it is the nature of the heterocycle that determines
the neuroprotective effects. Evaluation in a parallel artificial
membrane permeability assay (PAMPA) highlighted the need
to further improve the blood–brain barrier permeability of this
compound class. However, the compounds described herein
adhere to Lipinski’s rule of five, suggesting that this novel scaf-
fold has desirable properties for the development of potential
drug candidates.
Introduction
Neurodegenerative diseases (ND) are characterized by a de-
crease in the number of cells of certain neuronal populations,
and are clinically reflected by the appearance of specific symp-
toms, such as modification in the control and coordination of
movement in Parkinson’s disease (PD) or alterations in the lan-
guage and memory processes in Alzheimer’s disease (AD).[1]
Their chronic course produces a gradual but steady deteriora-
tion, the final step of which is death.[2] Because of this, as the
disease progresses, it erodes the quality of life for patients.[3]
Therefore, the development of effective neuroprotective thera-
pies that slow down or stop the disease’s progression at the
earliest stages is one of the main goals of researchers in this
area.[4]
At the cellular level, PD is related to excess production of re-
active oxygen species (ROS), to alterations in catecholamine
metabolism, to modifications in mitochondrial electron trans-
porter chain (METC) function, and to enhancement of iron dep-
osition in the substantia nigra pars compacta (SNpc).[5] The fail-
ure of normal cellular processes that occur in relation to aging
are also believed to contribute to the increased vulnerability of
dopaminergic neurons.[6] Although the precise mechanism cor-
responding to ROS generation related to PD is still unknown,
the major sources of oxidative stress generated by the dopa-
minergic neurons are dopamine metabolism, mitochondrial
dysfunction, and neuroinflammation.[7]
[a] Dr. M. J. Matos, Prof. F. Borges
CIQUP/Departamento de Química e Bioquímica
Faculdade de CiÞncias, Universidade do Porto, 4169-007 Porto (Portugal)
E-mail: mariacmatos@gmail.com
Metabolism of dopamine by monoamine oxidase (MAO)
yields hydrogen peroxide, an oxygen radical that leads to cyto-
toxicity through the peroxidation of lipid membranes. Selegi-
line and rasagiline, two selective MAO-B inhibitors, are current-
ly used to retard the symptoms in PD because they increase
dopamine levels and may exert neuroprotective effects. Inhibi-
tion of MAO-B decreases oxygen radical generation, although
new neuroprotective functions independent of MAO-inhibitory
activity have been reported for these drugs.[8] The occurrence
of oxidative stress in PD patients is supported by postmortem
studies and by preclinical studies showing the ability of oxidiz-
ing toxins to induce cell death in the SN.[9] Accordingly, antioxi-
dants such as tocopherol and ascorbate that scavenge free
radicals and other reactive species may have beneficial thera-
peutic effects in PD by preventing the onset of apoptosis and
fborges@fc.up.pt
[b] F. Rodríguez-Enríquez, Prof. R. Laguna, Prof. D. ViÇa
Departamento de Farmacología, CIMUS
Universidad de Santiago de Compostela
15782 Santiago de Compostela (Spain)
E-mail: mdolores.vina@usc.es
[c] Prof. L. Santana, Prof. E. Uriarte
Departamento de Química Orgµnica, Facultad de Farmacia
Universidad de Santiago de Compostela,
15782 Santiago de Compostela (Spain)
[d] Dr. M. Estrada, Prof. M. I. Rodríguez-Franco
Instituto de Química MØdica
Consejo Superior de Investigaciones Científicas (IQM-CSIC)
C/Juan de la Cierva 3, 28006 Madrid (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500408.
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neuronal degeneration of the dopaminergic nigrostriatal path-
way.[10] Nonetheless, all approved PD pharmacotherapies have
limited efficacy, do not prevent the progression of the disease,
and are associated with adverse motor and non-motor side ef-
fects.[11] Accordingly, there is an urgent need to develop novel
therapies that are superior to current therapies.
Throughout the history of medicine, nature has played a key
role as source of inspiration in the development of drugs with
important biological activities. Coumarins (2H-1-benzopyran-2-
one), common metabolites in plants that have also been de-
tected in microorganisms and animal sources,[12] have been the
focus of much interest owing to several pharmacological activi-
ties they display.[13] The specific pharmacological and biochemi-
cal properties and therapeutic applications of simple coumar-
ins depend on the substituents present on the core scaffold.[13]
Among the thousands of various coumarins present, some nat-
ural and synthetic versions have been evaluated against
a wide range of pharmacological targets of particular interest
in medicinal chemistry.[14] Coumarins have been found to act
as antioxidants and anti-inflammatory agents,[15] neuroprotec-
tive agents,[16,17] antidepressants,[18] anticonvulsants,[19] antibac-
terials,[20] antivirals,[21] anticancer agents,[22,23] anticoagulants,[24]
anti-hypertensives,[25] and enzyme inhibitors,[26–33] among other
activities. More recently it was found that simple coumarins,
usually substituted at positions 3, 4, 6, 7, or 8, have MAO inhib-
itory activities, making them an interesting option in the
search for new drugs for the treatment of ND.[27–33] In recent
years, simple coumarins substituted at positions 3 or 4 have
been described to exhibit activity as inhibitors of cholinesteras-
es (ChE), both acetylcholinesterase (AChE) and butyrylcholines-
terase (BuChE). The planarity and aromaticity of these deriva-
tives proved to be essential for activity. Substitution at posi-
tions 3 and 4 of the coumarin ring also afforded derivatives
found to be b-secretase 1 (BACE-1) inhibitors.[26,29] All these
properties encouraged us to study variously substituted cou-
marins as lead compounds with potential capacities as thera-
peutics to treat ND. In recent years our research group has
been studying 3-substituted coumarins with activity at differ-
ent targets involved in ND. The introduction of an amide
group as a linker between the coumarin skeleton and a phenyl
group at position 3 yielded coumarins with dual activity as
MAO and ChE inhibitors.[29] The introduction of hydroxy groups
enhanced the antioxidant properties of these compounds.[34]
Taking into account the background of our research group
(Figure 1)[28–33] and the knowledge that MAO activity generates
ROS that cause neuronal cell death, the present work provides
an overview on the potential of variously substituted 3-amido-
coumarins as multifunctional agents. They are able to selec-
tively inhibit MAO-B, scavenge free radicals and protect neuro-
nal cells from H2O2-mediated damage, representing a promising
multifunctional scaffold for the development of neuroprotec-
tive agents that could delay or slow the progression of ND.
Herein we describe our studies of the activities of 3-amidocou-
marins with amides introduced at position 3 and a hydroxy
group at position 4.
Figure 1. Rationale behind the design of the compounds studied in this
work.
Results and Discussion
Chemistry
The studied derivatives were efficiently synthesized according
to the protocol outlined in Scheme 1. Coumarins 1–17 were
prepared by starting from commercially available 3-aminocou-
marin, or from 3-amino-4-hydroxycoumarin, which was pre-
pared by reduction of the commercially available 3-nitro-4-hy-
droxycoumarin in ethanol, using palladium on charcoal as a cat-
alyst, under an atmosphere of H2 for 5 h, with a yield of
90%.[35,36] Acylation of the 3-aminocoumarins with the conven-
iently substituted acid chloride, using pyridine in dichlorome-
thane, from 08C to room temperature, afforded the variously
3-substituted coumarins 1–17 in yields between 80 and
90%.[32,37–42] The reaction conditions and characterization of
the new compounds are detailed in the Experimental Section
below.
Pharmacology
MAO in vitro inhibition
Biological evaluation of the test drugs on human MAO (hMAO)
activity was investigated by measuring their effects on the pro-
duction of H2O2 from p-tyramine (a common substrate for
hMAO-A and hMAO-B), using the Amplex Red MAO assay kit
and microsomal MAO isoforms prepared from insect cells (BTI-
TN-5B1-4) infected with recombinant baculovirus containing
cDNA inserts for hMAO-A or hMAO-B.[43] The production of
H2O2 catalyzed by the two MAO isoforms can be detected
using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red re-
agent), a non-fluorescent and highly sensitive probe that
reacts with H2O2 in the presence of horseradish peroxidase to
produce a fluorescent product: resorufin. New compounds and
reference inhibitors were unable to react directly with the
Amplex Red reagent, which indicates that these drugs do not
interfere with the measurements. On the other hand, under
the experimental conditions, hMAO-A displayed a Michaelis–
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(compounds 16 and 17), the in-
troduction of a hydroxy group at
position 4 was found to improve
activity against hMAO-B (IC50:
49.96 and 22.47 mm, respective-
ly). This proved to be the only
case in which the introduction
of a hydroxy group at position 4
led to a slight improvement in
activity.
Reversibility
Reversibility experiments were
performed to evaluate the type
of inhibition effected by deriva-
tives 1, 12, 14, and 17 (Table 2).
These compounds were selected
based on their structure and ac-
tivity against hMAO-B. An effec-
tive dilution method was used,
and selegiline (irreversible inhibi-
tor) and isatin (reversible inhibi-
Scheme 1. Reagents and conditions: a) H2, EtOH, Pd/C, RT, 5 h; b) pyridine, CH2Cl2, 08C!RT, overnight.
Menten constant (KM) equal to 457.17Æ38.62 mm and a maxi-
Table 1. In vitro hMAO-A and hMAO-B inhibitory activity of the synthe-
sized derivatives 1–17 and reference compounds.
mum reaction velocity (Vmax) in the control group of 185.67Æ
12.06 (nmol p-tyramine minÀ1)(mg protein)À1, whereas hMAO-B
showed a KM value of 220.33Æ32.80 mm and a Vmax of 24.32Æ
1.97 (nmol p-tyramine minÀ1)(mg protein)À1 (n=5). Most com-
pounds tested were found to inhibit this enzymatic control ac-
tivity in a concentration-dependent manner. The experimental
IC50 results are listed in Table 1.
Compd
IC50 [mm][a]
SI[b]
hMAO-A
inact.[e]
hMAO-B
1
6
8
10
11
12
13
14
16
17
selegiline
rasagiline
isatin
0.76Æ0.05
36.91Æ2.48
19.00Æ1.27
>131.6[d]
>2.7[d]
>5.3[d]
–
inact.[e]
inact.[e]
inact.[e]
inact.[e]
inact.[e]
inact.[e]
inact.[e]
inact.[e]
inact.[e]
~100[f]
As shown in Table 1, many of the studied compounds dis-
played selective inhibitory activity against hMAO-B in the mi-
cromolar range, with compound 1 being the most active deriv-
ative of the series (IC50 =0.76 mm). Regarding the 3-benzamido-
coumarins and comparing with previously published results,[29]
it was observed that the introduction of a hydroxy group at
position 4 of the coumarin scaffold generally resulted in a loss
of activity against hMAO-B, with derivatives 2–5, 7, and 9 lack-
ing this activity. In fact, from this series, only compound 6 pre-
sented hMAO-B inhibitory activity (IC50 =36.91 mm). An identi-
cal response was observed with the 3-substituent as a heteroar-
ylamide group, with a hydroxy group at position 4. In general,
a decrease in (in the case of the thiophenyl derivative) or loss
of hMAO activity (in the case of the furanyl and pyridyl deriva-
tives) was observed. Moreover, the nature of the heterocycle
determined the activity, and compounds with a thiophene ring
in their structure (12 and 13) proved to be active against
hMAO-B (IC50: 2.27 and 15.50 mm, respectively), whereas those
with a furan ring (10 and 11) lack such activity. Compounds
bearing a pyridine ring (14 and 15) follow the general trend
observed for the hydroxylated compounds. Compound 14,
without a hydroxy group at position 4, displayed activity
against hMAO-B (IC50 =21.11 mm), whereas compound 15, with
a hydroxy group at position 4, did not. For derivatives with an
amide function at position 3 linked to a cyclohexane group
~100[f]
2.27Æ0.15
–
>44.1[d]
>6.5[d]
>4.7[d]
>2.0[d]
>4.5[d]
3539
15.50Æ1.04
21.11Æ1.42
49.96Æ3.35
22.47Æ1.51
0.019Æ0.001[3]
0.069Æ0.004
33.07Æ1.47
67.25Æ1.02[c]
16.44Æ0.85
238
>3.0
inact.[e]
[a] Values are the meanÆSEM of n=5 experiments; compounds 2–5, 7,
9, and 15 proved to be inactive toward both MAO-A and MAO-B at the
highest concentration tested (100 mm). [b] Selectivity index: MAO-B selec-
tivity ratios [IC50 (MAO-A)]/[IC50 (MAO-B)] for inhibitory effects of both new
compounds and reference inhibitors. [c] p<0.01 regarding the corre-
sponding IC50 value obtained against MAO-B, as determined by ANOVA/
Dunnett’s. [d] Values obtained under the assumption that the corre-
sponding IC50 value against MAO-A is >100 mm. [e] Inactive at 100 mm
(highest concentration tested). [f] 100 mm inhibits enzymatic activity by
~50–55%; at higher concentrations the compounds precipitate.
tor) were taken as standards.[44,45] hMAO-B inhibition was ob-
served to be reversible in the presence of all four of these
compounds with their degree of reversibility being lower than
that described for isatin (reversible reference compound).
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Table 2. Reversibility results of hMAO-B inhibition studied for derivatives
1, 12, 14 and 17, and reference inhibitors.
Compd
Slope (AUF/t) [%][a]
1
12
14
17
selegiline
isatin
41.51Æ2.79
63.04Æ4.23
38.43Æ2.58
67.76Æ4.55
3.21Æ0.21
88.63Æ5.94
[a] Values represent the meanÆSEM of n=3 experiments relative to con-
trol; data show recovery of hMAO-B activity after dilution.
Neuronal survival
Compounds 1–17 were studied in vitro to evaluate their neu-
roprotective potential along with their MAO activity and to
assess their effect on oxidative stress. First, to discard any pos-
sible cytotoxic effects of compounds 1–17 against rat cortical
neurons, cell viability was assessed after 24 h treatment with
the new compounds, each at 100 mm. The 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, which
detects cellular dehydrogenase activity, was used. Cells that
are metabolically impaired are less able to reduce MTT than
healthy cells. In comparison with control (1% DMSO), only
compounds 10 and 11 were found to induce a significant de-
crease in cell viability at 100 mm post-treatment, whereas the
rest of the test compounds were devoid of any cytotoxic activ-
ity (Figure 2A).
The neuroprotective effects of compounds 1–17 were then
assessed in cultured rat cortical neurons exposed to H2O2. Neu-
rons incubated with or without compounds 1–17 (100 mm)
were exposed to H2O2 (30 mm) at the same time point and in-
cubated for 24 h. The toxin-treatment group in the cell viability
assays showed a significant difference in the production of tox-
icity relative to those treated with DMSO alone. The results ob-
tained by studying the possible neuroprotective effects of our
new compounds against the effects of H2O2 in the cells are
shown in Figure 2B.
Figure 2. A) Cytotoxicity of compounds 1–17 (100 mm) against cortical neu-
rons, and B) neuroprotective effects of compounds 1–17 (100 mm) and rasa-
giline (ras; 5 mm) on cortical neurons treated with H2O2 and percentage of
living cells in cultures exposed to DMSO/H2O2 relative to cells exposed to
DMSO alone. Results are expressed as the meanÆSEM of at least five inde-
pendent experiments; *p<0.05, **p<0.001 versus the corresponding viabili-
ty obtained in the control group treated with DMSO (panel A) or H2O2 in ad-
#
dition to DMSO (panel B); p<0.001 versus the group treated only with
DMSO.
From the 3-benzamidocoumarins 1–9, the most promising
results against the effects of H2O2 (Figure 2B) corresponded, in
general, to derivatives with a hydroxy group at position 4 of
the coumarin scaffold and a single substituent at the para po-
sition of the benzamide at position 3 (compounds 3, 7, and 9).
In the case of 3-heteroarylamido- and 3-cyclohexanecarboxa-
midocoumarins 10–17, we observed that derivatives with a nic-
otinamide group at position 3 (compounds 14 and 15) exerted
a statistically significant level of neuroprotection, whereas
compounds were found to be inactive against H2O2 if the pyri-
dine ring was substituted for cyclohexane, thiophene, or furan.
Under these conditions, rasagiline (5 mm) did not display signif-
icant neuroprotection. In view of these results it is possible
that other mechanisms beyond MAO inhibition may be in-
volved in the neuroprotective activity of these derivatives.
Neuroprotection exerted by these compounds is concentra-
tion dependent. Therefore, a decrease in neuroprotection is
observed when cultured rat cortical neurons were exposed to
H2O2 but treated with compounds 1–17 at 10 mm. No signifi-
cant differences were found for neurons treated with any com-
pounds and exposed to H2O2 (Table 3).
DPPH· scavenging
Under normal conditions, free radicals are rapidly neutralized
in the mitochondria; over time, however, this neutralization be-
comes less effective, and dysfunction and even cell death
occurs. We studied the 1,1-diphenyl-2-picrylhydrazyl radical
(DPPH·) scavenging activity of those compounds showing the
best neuroprotective effects in cells treated with H2O2 (com-
pounds 3, 7, 9, 14, and 15). As can be seen in Figure 3, most
of the studied compounds were able to scavenge free radicals,
with compounds 3 and 7 (100 mm) being the most active,
showing scavenging activity slightly higher than 50%. These
are also two of the best compounds in the series of 3-benza-
midocoumarins as neuroprotective agents against H2O2. In
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tively easy and straightforward method to predict the passive
central nervous system (CNS) permeation, which had been pre-
viously optimized for application to investigated compounds
with limited aqueous solubility.[47–49] Experimental PAMPA re-
sults are listed in Table 4.
Table 3. Cell viability after treatment with compounds 1–17 at 10 and
100 mm.
Compd
Viability [%]
10 mm
100 mm
DMSO+H2O2
1
2
3
4
5
6
7
72.2Æ1.7
78.3Æ1.7
79.1Æ1.0[c]
73.6Æ3.3[c]
104.4Æ6.3[b]
85.9Æ6.7[c]
83.2Æ5.9[c]
76.3Æ2.3[c]
102.4Æ3.8[b]
99.1Æ5.2[a]
101.9Æ0.8[b]
83.6Æ2.8[c]
83.1Æ3.9[c]
91.3Æ5.3[c]
86.3Æ3.3[c]
100.7Æ10.6[b]
106.1Æ6.3[b]
84.9Æ6.3[c]
80.7Æ3.0[c]
73.3Æ3.1[c]
68.0Æ2.1[c]
63.8Æ2.6[c]
69.7Æ3.8[c]
71.7Æ4.3[c]
78.5Æ6.0[c]
79.4Æ6.5[c]
71.4Æ7.3[c]
72.2Æ4.0[c]
72.4Æ6.4[c]
70.6Æ7.3[c]
68.2Æ3.9[c]
71.3Æ4.7[c]
67.9Æ6.4[c]
73.6Æ6.4[c]
72.9Æ0.7[c]
74.2Æ3.6[c]
Table 4. In vitro evaluation of CNS penetration (experimental permeabili-
ty, Pe) using PAMPA methodology.
[a]
Compd
Pe [10À6 cmsÀ1
]
Prediction
3
12
14
15
7.4Æ0.01
44.2Æ2.1
20.2Æ0.4
9.3Æ0.1
CNSÀ
8
9
CNS+
CNS+
CNS+/À
CNS+
10
11
12
13
14
15
16
17
verapamil
14.8Æ0.1
[a] Results are the mean of the experimental prediction ÆSD of at least
three independent experiments.
The capacity of the compounds to pass through a lipid ex-
tract of porcine brain were determined using a 70:30 mixture
of phosphate-buffered saline solution and ethanol (PBS/EtOH).
In each experiment 10 commercial drugs were also evaluated
for assay validation. A graphical representation of experimental
permeability versus reported values of such well-known drugs
gave a linear correlation: Pe (expt.)=0.72Pe (lit.)+6.70 (R2 =
0.80). From this equation and taking into account the de-
scribed limits for BBB permeation, we established that com-
pounds with permeability values >9.6”10À6 cmsÀ1 could pen-
etrate into the CNS by passive diffusion (CNS+), whereas prod-
ucts with Pe <8.1”10À6 cmsÀ1 could not enter (CNSÀ). Be-
tween these values, the CNS permeation was considered to be
uncertain (CNS+/À). Therefore, from the selected compounds,
[a] p<0.05, [b] p<0.001 versus the corresponding viability obtained in
the control group treated with H2O2 +DMSO. [c] No significant differences
observed with the control group (DMSO+H2O2).
compounds 12 and 14 (Pe: 44.2”10À6 and 20.2”10À6 cmsÀ1
,
respectively) would be able to cross the BBB and reach their
therapeutic targets. In addition, both compounds showed
higher Pe values than verapamil (Pe =14.8”10À6 cmsÀ1), which
is generally used as a standard of high permeability. In the
case of compound 15, passage through the BBB is unlikely
(Pe =9.3”10À6 cmsÀ1).
Figure 3. DPPH· scavenging activity of coumarin derivatives 3, 7, 9, 14, 15,
and vitamin C (positive control).
contrast, for the series of 3-nicotinamidocoumarins, com-
pounds 14 and 15, only if a hydroxy group is present at posi-
tion 4 (compound 15) is free radical scavenging activity ob-
served (~20%). Therefore, the presence of a hydroxy group, in
this specific case at position 4, seems to be crucial for neutrali-
zation of free radicals by these derivatives.
Theoretical evaluation of ADME-related physicochemical/
structural parameters
To better understand the overall properties and the drug-like
characteristics of compounds 1–17, the calculated lipophilicity
(expressed as the octanol/water partition coefficient, or clogP)
and theoretical predictions of other ADME properties (molecu-
lar weight, topological polar surface area (TPSA), number of hy-
drogen bond donors and acceptors, and molecular volume)
were carried out with Molinspiration software, and are present-
ed in the Supporting Information.[50,51] It is significant that all
the described derivatives possess clogP values compatible
with those required to cross membranes. Although the TPSA
(described to be a predictive indicator of membrane penetra-
tion) of selegiline differs greatly from the TPSA values deter-
mined for the studied compounds, all other predicted ADME
In vitro blood–brain barrier permeation
A fundamental requirement for any compound to act on neu-
rodegenerative processes is access to brain tissue, that is, to
be able to cross the blood–brain barrier (BBB). To examine the
capacity of our compounds to pass this barrier, we selected
compounds 3, 12, 14, and 15, some of the most active MAO
inhibitors and/or neuroprotective agents, for use in a parallel
artificial membrane permeation assay (PAMPA).[46] This is a rela-
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using TMS as an internal standard. Coupling constants (J) are ex-
pressed in Hz. Spin multiplicities are given as s (singlet), d (dou-
blet), dd (doublet of doublets), td (triplet of doublets), and m (mul-
tiplet). Mass spectrometry was carried out with a Hewlett–Packard
5972 MSD spectrometer. Elemental analyses were performed with
a PerkinElmer 240B microanalyzer and are within 0.4% of calculat-
ed values in all cases. Flash chromatography (FC) was performed
on silica gel (Merck 60, 230–400 mesh); analytical TLC was per-
formed on pre-coated silica gel plates (Merck 60 F254). Organic solu-
tions were dried over anhydrous Na2SO4. Concentration and evapo-
ration of the solvent after reaction or extraction was carried out on
a rotary evaporator (Büchi Rotavapor) operating at reduced pres-
sure. The analytical results showed >95% purity for all com-
pounds.
values were found to fall in the desirable range. In addition,
we observed no violations of Lipinski’s rules (re. molecular
weight, logP, number of hydrogen bond donors and accept-
ors). The studied compounds, as MAO inhibitors and neuropro-
tective agents, must pass various membranes to reach the
CNS. The obtained information supports the potential of these
derivatives as viable drug candidates. The theoretical informa-
tion obtained is partially in accordance with results of the ex-
perimental in vitro BBB permeation assay. The combination of
both experimental results and calculated values can aid in im-
proving our understanding of the drug likeness of this com-
pound series.
Preparation of the precursor 3-amino-4-hydroxycoumarin. The
commercially available 4-hydroxy-3-nitrocoumarin (2.5 mmol) was
dissolved in EtOH, and a catalytic amount of Pd/C was added to
the mixture. The solution was stirred at room temperature under
H2 atmosphere for 5 h. After completion of the reaction, the mix-
ture was filtered to remove the catalyst. The obtained crude prod-
uct was then purified by FC (hexane/EtOAc, 9:1) to give the de-
sired coumarin in 90% yield.
Conclusions
In this study, a general and efficient synthesis of a new series
of 3-amidocoumarins was developed, using an amidation reac-
tion as a key step. Determination of hMAO isoform activity was
carried out, and many of the compounds exhibited selectivity
for the hMAO-B isoform with activity in the nanomolar (com-
pound 1) or micromolar ranges (compounds 6, 8, 12–14, 16,
and 17). Neuroprotective effects against H2O2 were also stud-
ied. For the series of 3-benzamidocoumarins, the most promis-
ing results in cells treated with H2O2 generally corresponded to
derivatives with a substituent at the para position of the ben-
zamide ring, in addition to a hydroxy group at position 4 of
the coumarin scaffold (compounds 3, 7, and 9). For the series
of 3-heteroarylamidocoumarins, derivatives with a nicotinamide
group at position 3 of the coumarin scaffold (compounds 14
and 15) exerted the most notable neuroprotection. Most of
the selected derivatives exerting neuroprotection also showed
DPPH· scavenging activity (with the exception of compound
14, with no hydroxy groups on its structure). Additionally, the
prediction of BBB accessibility by PAMPA showed the potential
of this compound class to cross the BBB and to thus exert ac-
tivity in the CNS. From the four compounds studied in the
PAMPA, those without a hydroxy group at position 4 proved to
have a greater capacity to cross the BBB (compounds 12 and
14). Compound 15 was found partially able to cross biological
barriers. In addition, prediction of ADME-related physicochemi-
cal/structural parameters provided a preliminary indication of
the potential of this family of compounds as suitable mole-
cules for further development. These results encourage us to
further explore the potential of members of this chemical
family as potential drug candidates for the treatment of Parkin-
son’s disease.
General procedure for the preparation of 3-amidocoumarins 1–
17. The 3-aminocoumarin (commercially available) or the 3-amino-
4-hydroxycoumarin (1 mmol) was dissolved in CH2Cl2 (9 mL). Pyri-
dine (1.1 mmol) was then added, and the mixture was cooled to
08C. Variously substituted acid chloride (1.1 mmol) was added
dropwise at this temperature, and the mixture was stirred over-
night at room temperature. The batch was evaporated and purified
by column chromatography (hexane/EtOAc, 9:1) to give the de-
sired compounds 1–17.
N-(4-Hydroxycoumarin-3-yl)-4’-methylbenzamide (3): white solid
1
(83% yield); Rf =0.26 (9:1, hexane/EtOAc); mp: 210–2118C; H NMR
([D6]DMSO): d=2.49 (s, 3H, CH3), 7.31 (s, 1H, H-4), 7.37–7.44 (m,
4H, H-6, H-8, H-3’, H-5’) 7.64–7.70 (m, 1H, H-7), 7.89–7.92 (m, 3H,
H5, H-2’, H-6’), 9.47 (s, 1H, NH), 12.13 ppm (s, 1H, OH); 13C NMR
([D6]DMSO): d=21.0, 113.1, 116.2, 116.3, 123.7, 124.3, 128.1, 128.8,
130.9, 132.4, 141.7, 151.6, 159.3, 160.4, 166.4 ppm; MS m/z (%): 296
(6), 295 ([M+], 29), 119 (100), 91 (30), 65 (10); Anal. calcd for
C17H13NO4: C 69.15, H 4.44, N 4.74, O 21.67, found: C 69.12, H 4.42,
N 4.77.
N-(4-Hydroxycoumarin-3-yl)-3’,4’-dimethoxybenzamide (5): white
solid (88% yield); Rf =0.25 (9:1, hexane/EtOAc); mp: 247–2488C;
1H NMR ([D6]DMSO): d=3.82 (s, 6H, (CH3)2), 7.07 (d, J=8.3 Hz, 1H,
H-5’), 7.40–7.45 (m, 2H, H-6, H-8) 7.59–7.69 (m, 3H, H-2’, H-6’, H-7),
7.90 (d, J=7.8 Hz, 1H, H-5), 9.44 (s, 1H, NH), 12.20 ppm (s, 1H,
OH); 13C NMR ([D6]DMSO): d=56.3, 103.9, 111.5, 112.0, 116.9, 122.3,
124.3, 125.0, 126.6, 133.0, 148.8, 152.2, 152.4, 159.9, 161.1, 166.8;
DEPT 13C NMR ([D6]DMSO): d=56.3, 111.5, 112.0, 116.9, 122.3, 124.3,
125.0, 133.0 ppm; MS m/z (%): 342 (6), 341 ([M+], 15), 323 (6), 165
(100), 121 (6), 92 (7), 77 (9); Anal. calcd for C18H15NO6: C 63.34, H
4.43, N 4.10, O 28.12, found: C 63.31, H 4.41, N 4.12.
Experimental Section
N-(4-Hydroxycoumarin-3-yl)-3’,4’-dichlorobenzamide (8): white
solid (86% yield); Rf =0.22 (9:1, hexane/EtOAc); mp: 284–2858C;
1H NMR ([D6]DMSO): d=7.37–7.44 (m, 2H, H-6, H-8), 7.64–7.70 (m,
1H, H-5’) 7.79–7.96 (m, 3H, H-6’, H-5, H-7), 8.24 (s, 1H, H-2’), 9.78
(s, 1H, NH), 12.24 ppm (s, 1H, OH); 13C NMR ([D6]DMSO): d=87.0,
116.5, 123.8, 123.9, 124.0, 124.5, 128.4, 130.1, 130.8, 131.3, 132.7,
134.5, 150.5, 151.5, 160.1, 163.2 ppm; MS m/z (%): 351 (54), 350
([M+], 15), 349 (84), 333 (22), 331 (34), 175 (100), 174 (94), 147 (24),
145 (36), 121 (19), 111 (14), 109 (14), 85 (17), 71 (19), 69 (14), 65
Chemistry
Starting materials and reagents were obtained from commercial
suppliers and were used without further purification (Sigma–Al-
drich). Melting points (mp) are uncorrected and were determined
with a Reichert Kofler thermopan or in capillary tubes in a Büchi
510 apparatus. H NMR (300 MHz) and 13C NMR (75.4 MHz) spectra
were recorded with a Bruker AMX spectrometer using CDCl3 or
1
[D6]DMSO as solvent. Chemical shifts (d) are expressed in ppm
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(14), 57 (19); Anal. calcd for C16H9Cl2NO4: C 54.88, H 2.59, Cl 20.25,
N 4.00, O 18.28, found: C 54.85, H 2.55, Cl 20.28, N 3.99.
only the Amplex Red reagent in a sodium phosphate buffer. To de-
termine the kinetic parameters of hMAO-A and hMAO-B (KM and
Vmax), the corresponding activity of both isoforms was evaluated
(under the experimental conditions described above) in the pres-
ence of a wide range of p-tyramine concentrations. The specific
fluorescence emission (used to obtain the final results) was calcu-
lated after subtraction of the background activity, which was deter-
mined from wells containing all components except the hMAO iso-
forms, which were replaced by a sodium phosphate buffer solu-
tion. Under our experimental conditions, this background activity
was practically negligible. MAO activity of the test compounds and
reference inhibitors is expressed as IC50, i.e., the concentration of
each drug required to produce a 50% decrease in control value ac-
tivity for MAO isoforms.
N-(Coumarin-3-yl)furan-2-carboxamide (10): white solid (90%
yield); Rf =0.42 (9:1, hexane/EtOAc); mp: 183–1848C; 1H NMR
(CDCl3): d=6.74 (dd, J=3.6, 1.8 Hz, 1H, H-4’), 7.34–7.58 (m, 4H, H-
5, H-6, H-8, H-5’), 7.77 (td, J=8.0, 1.4 Hz, 1H, H-7), 8.00 (dd, J=1.8,
0.8 hz, 1H, H-3’), 8.58 (s, 1H, H-4), 9.26 ppm (s, 1H, NH); 13C NMR
(CDCl3): d=112.1, 113.2, 115.7, 116.8, 122.9, 124.3, 126.2, 126.8,
127.7, 145.8, 146.5, 149.5, 155.3, 159.8 ppm; MS m/z (%): 256 (16),
255 ([M+], 79), 227 (7), 132 (6), 95 (100), 77 (10); Anal. calcd for
C14H9NO4: C 65.88, H 3.55, N 5.49, O 25.07, found: C 65.87, H 3.56,
N 5.52.
N-(4-Hydroxycoumarin-3-yl)cyclohexanecarboxamide (17): white
solid (91% yield); Rf =0.27 (9:1, hexane/EtOAc); mp: 199–2008C;
1H NMR (CDCl3): d=1.17–1.69 (m, 6H, (CH2)3), 1.75–2.06 (m, 4H,
(CH2)2), 2.30–2.55 (m, 1H, CH), 7.30–7.40 (m, 2H, H-6, H-8), 7.56 (td,
J=7.8, 1.7 Hz, 1H, H-7), 8.01 (dd, J=7.9, 1.7 Hz, 1H, H-5), 8.28 (s,
1H, NH), 13.87 ppm (s, 1H, OH); 13C NMR (CDCl3): d=25.4, 25.7,
29.7, 45.5, 104.7, 116.2, 117.2, 124.4, 124.7, 131.6, 150.5, 152.8,
161.2, 177.6 ppm; MS m/z (%): 288 (5), 287 ([M+], 25), 177 (54), 121
(18), 111 (22), 83 (100), 55 (43); Anal. calcd for C16H17NO4: C 66.89,
H 5.96, N 4.88, O 22.27, found: C 66.91, H 5.99, N 4.85.
Determination of inhibition mode: To evaluate whether compounds
1, 12, 14, and 17 are reversible or irreversible hMAO-B inhibitors,
a dilution method was used.[44] A 100” concentration of the
enzyme used in the above-described experiments was incubated
with a concentration of inhibitor equivalent to 10-fold its IC50
value. After 30 min, the mixture was diluted 100-fold into reaction
buffer containing Amplex Red reagent, horseradish peroxidase,
and p-tyramine, and the reaction was monitored for 15 min. Rever-
sible 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. Control tests were carried out by
pre-incubating and diluting the enzyme in the absence of inhibi-
tor.
Pharmacological assays
Activity against MAO isoforms: The tested compounds were dis-
solved in DMSO (Sigma–Aldrich, Alcobendas, Madrid, Spain) to pre-
pare 10 mm stock solutions, which were kept in storage at À208C.
Percentage of DMSO used in the experiments was never >1%. Se-
legiline and rasagiline, used as reference inhibitors, were acquired
from Sigma–Aldrich (Alcobendas). Human recombinant MAO iso-
forms, used in the experiments, were also purchased from Sigma–
Aldrich (Alcobendas). Resorufin sodium salt, p-tyramine hydrochlo-
ride, sodium phosphate buffer, horseradish peroxidase and Amplex
Red reagent were supplied in the Amplex Red MAO assay kit (Mo-
lecular Probes Inc., Eugene, OR, USA). Briefly, 0.1 mL of sodium
phosphate buffer (0.05m, pH 7.4) containing various concentra-
tions of the test drugs (new compounds or reference inhibitors)
and adequate amounts of recombinant hMAO-A or hMAO-B re-
quired and adjusted to obtain the same reaction velocity under
our experimental conditions, i.e., to oxidize (in the control group)
the same concentration of substrate: 165 pmol of p-tyramine per
min (hMAO-A: 1.1 mg protein; specific activity: 150 nmol of p-tyra-
mine oxidized to p-hydroxyphenylacetaldehyde per min per mg
protein; hMAO-B: 7.5 mg protein; specific activity: 22 nmol of p-tyr-
amine transformed per min per mg protein) were incubated for
15 min at 378C 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 (final concentrations)
200 mm Amplex Red reagent, 1 UmLÀ1 horseradish peroxidase, and
1 mm p-tyramine. The production of H2O2 and, consequently, of re-
sorufin was quantified at 378C in a multi-detection microplate fluo-
rescence reader (FLX800, Bio-Tek Instruments Inc., Winooski, VT,
USA) based on the fluorescence generated (lex 545 nm,
lem 590 nm) over a 15 min period, in which the fluorescence in-
creased linearly.[43] Control experiments were carried out simultane-
ously by replacing the test drugs (new compounds and reference
inhibitors) with appropriate dilutions of the vehicles. In addition,
the possible capacity of the above test drugs to modify the fluo-
rescence generated in the reaction mixture due to non-enzymatic
inhibition (e.g., by direct reaction with the Amplex Red reagent)
was determined by adding these drugs to solutions containing
Neuroprotective study: DMSO, phosphate-buffered saline (PBS,
pH 7.4), Hanks buffer, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT), and H2O2 were acquired from Sigma–Al-
drich (Alcobendas). Poly-d-lysine, neurobasal medium, l-glutamine,
B-27, and fetal bovine serum (FBS) were acquired from Gibco/Invi-
trogen S.A., Barcelona, Spain.
Primary culture of neurons and glia: Pregnant rats (19–20 days)
were euthanized by CO2 inhalation, and embryos were immediately
extracted from the womb by caesarean section, and their brains
were carefully dissected out. Meninges were removed, and a por-
tion of motor cortex was isolated after dissection of the brain.[26]
Fragments obtained from several embryos were subjected to me-
chanic disintegration. Neurobasal medium supplemented with 2%
B-27 (for cortical neurons) was used to seed the cells in 96-well
plates at a density of 100000 cells per mL. Neuronal cultures were
allowed to grow for 8–10 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 378C. Experi-
ments were conducted on female Wistar Kyoto (WKY) rats, ob-
tained from the rat colony maintained at the animal facilities of
our department. Rats were housed, cared for, and acclimatized
(before the experiments). All experiments were carried out in ac-
cordance with European regulations on the protection of animals
(Directive 2010/63/UE), the Spanish Real Decreto 53/2013 (1.Febru-
ary) and/or the Guide for the Care and Use of Laboratory Animals
as adopted and promulgated by the USA.
Determination of neuronal survival: Neuronal cultures were treated
with the compounds in the study at 100 mm (final DMSO concen-
tration ꢀ1%) or with studied compounds and H2O2 (30 mm) over
an incubation period of 24 h. H2O2 was used as reference neurotox-
ic agent for neurons. Cell viability was determined to gauge the
possible cytotoxicity of new compounds or their neuroprotective
effects against a pro-oxidant (H2O2) agent, by reducing MTT to for-
mazan via the mitochondria of viable cells. MTT (5 mgmLÀ1 in
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Full Papers
Hanks buffer) was added to each well to a final concentration of
10%.[52] After incubating for 2 h at 378C, the medium was re-
moved, and formazan crystals formed were suspended in DMSO
(100 mLwellÀ1). The production of formazan by viable cells was
quantified at 378C in an absorbance reader (Fluo-star Optima, BMG
LABTECH, Offenburg, Germany) by measuring the absorbance at
l 570 nm).[53,54] In addition, the possible capacity of the above test
drugs to modify the absorbance generated by reaction with MTT
was determined by adding these drugs to solutions containing
only MTT reagent in neurobasal medium.
for characterizing drug absorption, including intestinal absorption,
bioavailability, Caco-2 permeability, and blood–brain barrier pene-
tration. The method for calculation of molecular volume developed
at Molinspiration is based on group contributions. These were ob-
tained by fitting the sum of fragment contributions to ‘real’ three-
dimensional (3D) volume for a training set of ~12000 mostly drug-
like molecules; 3D molecular geometries for a training set were
fully optimized by the semi-empirical AM1 method.
Statistics: Results are expressed as the mean of at least three ex-
periments ÆSEM or ÆSD for blood–brain barrier permeation assay.
Statistically significant differences between two measurements (P<
0.05, P<0.01, or P<0.001) were determined by analysis of variance
(ANOVA) followed by the multiple comparison Dunnett’s test.
Graphical representation, statistical analysis, and calculation of IC50
values were performed with GraphPad Prism software (ver. 4.03,
San Diego, CA, USA).
Neutralization of free radicals: DMSO, DPPH·, l-ascorbic acid (vita-
min C), and EtOH were acquired from Sigma–Aldrich (Alcobendas).
The DPPH· radical scavenging activity of each compound was de-
termined as previously described, with minor modifications. The
DPPH· radical was dissolved in EtOH (100 mm) and 99 mL of the sol-
utions were transferred to each well of a 96-well microplate. Com-
pounds 3, 7, 9, 14, and 15 (1 mL, 100 mm, final concentration) in
EtOH were added to each well of a 96-well microplate, and the
mixtures were incubated at room temperature for 30 min. Vita-
min C (100 mm) was used as a positive control in the experiments.
The absorbance at 540 nm was measured using a microplate
reader. The radical-scavenging activity of each compound was esti-
mated by comparing the DPPH· absorbance value in the antioxi-
dant–radical reaction mixture after subtraction of the background
activity.[55–57]
Acknowledgements
This work was supported in part by the University of Santiago de
Compostela, the Xunta de Galicia (EM2014/016), the Spanish
Ministry of Economy and Competitiveness (SAF2012-31035), the
Portuguese Foundation for Science and Technology (FCT), and
QREN (FCUP-CIQ-UP-NORTE-07-0124-FEDER-000065), Galician
Plan of Research, Innovation and Growth 2011–2015 (Plan I2C-
ED481B 2014/086-0) and FCT, POPH, and QREN (SFRH/BPD/
95345/2013).
In vitro blood–brain barrier permeation assay: Hydrocortisone, de-
sipramine, promazine, aldosterone, caffeine, ofloxacin, corticoster-
one, imipramine, testosterone, verapamil, piroxicam, lipid pig
brain, PBS (pH 7.4), and dodecane were purchased from Sigma–Al-
drich (Alcobendas) and Acros (Madrid, Spain). Prediction of the
brain penetration was evaluated using a PAMPA BBB assay, in
a manner similar to that described previously.[46–49] Pipetting was
performed with a semiautomatic pipetter (CyBi-SELMA), and UV
reading with a microplate spectrophotometer (Multiskan Spectrum,
Thermo Electron Co.). Millex filter units (PVDF membrane, diameter
25 mm, pore size 0.45 mm) were acquired from Millipore. The por-
cine brain lipid (PBL) was obtained from Avanti Polar Lipids. The
donor microplate was a 96-well filter plate (PVDF membrane, pore
size 0.45 mm), and the acceptor microplate was an indented 96-
well plate, both from Millipore. The acceptor 96-well microplate
was filled with 200 mL PBS/EtOH (70:30), and the filter surface of
the donor microplate was impregnated with 4 mL PBL in dodecane
(20 mgmLÀ1). Compounds were dissolved in PBS/EtOH (70:30) at
10 mgmLÀ1, filtered through a Millex filter, and then added to the
donor wells (200 mL). The donor filter plate was carefully placed on
the acceptor plate to form a sandwich, which was left undisturbed
for 240 min at 258C. After incubation, the donor plate was carefully
removed, and the concentration of compounds in the acceptor
wells was determined by UV/Vis spectroscopy. Every sample was
analyzed at five wavelengths, in four wells and in at least three in-
dependent runs, and the results are given as the mean Æstandard
deviation. In each experiment, 10 quality control standards of
known BBB permeability were included to validate the analysis set.
Keywords: coumarin
· inhibitors · monoamine oxidase ·
neuroprotection · Parkinson’s disease
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Published online on October 23, 2015
ChemMedChem 2015, 10, 2071 – 2079
www.chemmedchem.org
2079
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