Synthesis and adenosine receptors binding affinities of a series of 3-arylcoumarins

Article abstract of DOI:10.1111/jphp.12135

Objectives In the present communication, we report the synthesis, pharmacological evaluation, theoretical evaluation of absorption, distribution, metabolism and excretion properties and structure-activity relationship study of a selected series of 3-arylcoumarins (compounds 1-9). Adenosine receptors (ARs) binding activity and selectivity of the synthesized compounds 1-9 were evaluated in this study. Different substituents were introduced in both benzene rings of the evaluated scaffold, at positions 6 and 3′ or 4′ of the moiety. The lack of data on the 3-arylcoumarin scaffold encouraged us to explore the ARs' binding activity of a selected series of derivatives. Methods A new series of coumarins (compounds 1-9) were synthesized and evaluated by radioligand binding studies towards ARs. Key findings Analysing the experimental data, it can be observed that neither the simple 3-arylcoumarin nor the 4′-nitro derivatives presented detectable binding affinity for the evaluated receptors, although most of the other substituted derivatives have good binding affinity profiles, especially against the hA₁/hA ₃ or only hA₃ AR. Conclusions The most remarkable derivative is compound 2, presenting the best affinity for hA₃ AR (K_i = 2680 nM) and significant selectivity for this subtype. Graphical Abstract In the present communication, we report the synthesis, pharmacological evaluation, theoretical evaluation of ADME properties and SAR study of a selected series of 3-arylcoumarins (compounds 1-9). Adenosine receptors binding activity and selectivity of the synthesized compounds 1-9 were evaluated in this study, and most of the substituted derivatives have good binding affinity profiles, especially against the hA₁/hA₃ or only hA₃ AR. The most remarkable derivative is compound 2, presenting the best affinity for hA₃ AR (K_i = 2680 nM) and significant selectivity for this subtype.

Full text of DOI:10.1111/jphp.12135

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Research Paper
Journal of Pharmacy
And Pharmacology
Synthesis and adenosine receptors binding affinities of a
series of 3-arylcoumarins
Maria João Matosa,b, Veronika Hoggerb, Alexandra Gaspara, Sonja Kachlerc, Fernanda Borgesa,
Eugenio Uriarteb, Lourdes Santanab and Karl-Norbert Klotzc
aCIQUP/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal, bDepartamento de Química
c
Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, Santiago de Compostela, Spain and Institut für Pharmakologie und
Toxikologie, Universität Würzburg, Würzburg, Germany
Keywords
Abstract
3-arylcoumarins; adenosine receptors binding
Objectives In the present communication, we report the synthesis, pharmaco-
logical evaluation, theoretical evaluation of absorption, distribution, metabolism
and excretion properties and structure–activity relationship study of a selected
series of 3-arylcoumarins (compounds 1–9). Adenosine receptors (ARs) binding
activity and selectivity of the synthesized compounds 1–9 were evaluated in this
study. Different substituents were introduced in both benzene rings of the evalu-
ated scaffold, at positions 6 and 3′ or 4′ of the moiety. The lack of data on the
3-arylcoumarin scaffold encouraged us to explore the ARs’ binding activity of a
selected series of derivatives.
activity; absorption, distribution, metabolism
and excretion properties; structure–activity
relationship study
Correspondence
Maria João Matos, Organic Chemistry,
Facultad de Farmacia, University of Santiago
de Compostela, Campus Vida, Santiago de
Compostela, 15782, Spain.
E-mail: mariajoao.correiapinto@rai.usc.es
Methods A new series of coumarins (compounds 1–9) were synthesized and
evaluated by radioligand binding studies towards ARs.
Received January 17, 2013
Accepted July 23, 2013
Key findings Analysing the experimental data, it can be observed that neither the
simple 3-arylcoumarin nor the 4′-nitro derivatives presented detectable binding
affinity for the evaluated receptors, although most of the other substituted deriva-
tives have good binding affinity profiles, especially against the hA1/hA3 or only
hA3 AR.
doi: 10.1111/jphp.12135
Conclusions The most remarkable derivative is compound 2, presenting the best
affinity for hA3 AR (Ki = 2680 nM) and significant selectivity for this subtype.
Introduction
Adenosine receptors (ARs) are G-protein-coupled receptors
that exert a huge number of physiological activity through
activation of four different receptor subtypes: A1, A2A, A2B
and A3.[1] ARs are distributed throughout a wide variety of
tissues in mammalian systems and regulate diverse physio-
logical functions by modulating cell signalling, being acti-
vated by the endogenous agonist adenosine and blocked by
natural antagonists.[2,3] During the last years, the search for
agonist and antagonist ligands binding to individual AR
subtypes has been intensified, as the role of these receptors
in drug discovery is continuously expanding.[4–6] Most of
the AR antagonists are promising new drug candidates for a
number of pathological processes, such as inflammation
(A3),[5] heart and renal failure (A1),[7] or neurological disor-
ders like Parkinson’s[8,9] and Alzheimer’s diseases (A2A or
A1).[10] In particular, A3 AR, the most recently identified
subtype, has been the subject of intensive research during
the last decade as a potential therapeutic target in prevent-
ing ischemic damage in the brain and heart.[11] In addition,
as the A3 AR is expressed in relatively high densities in the
lung, liver, neutrophils, macrophages and glial cells, it can
encompass an important role in inflammation, immuno-
suppression and cell growth.[12] Therefore, A3 AR ligands
can have therapeutic potential against cancer, hepatitis,
myocardial ischemia, rheumatoid arthritis or ophthalmic
diseases.[13] More recently, an involvement of A3 AR in
neuroprotection was also proposed.[14] However, the mixed
A3-mediated protective/damaging effect remains enigmatic
and has to be clarified. Therefore, additional studies must
be performed to verify the modulating effect of the A3 AR
agonists and antagonists in neurodegenerative diseases. As a
consequence, numerous A3 AR agonists and antagonists
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
1

AR binding affinities of 3-arylcoumarins
Maria João Matos et al.
O
O
O
cancer, anti-inflammatory, antimicrobial, vasorelaxant or
enzymatic inhibitory agents, have been ascribed to this type
of structure.[19–33] The fused heterocyclic framework of
coumarins has been widely used as an archetype for the
synthesis of a wide variety of analogues suitable to perform
structure–activity relationship (SAR) studies intended to
improve their biological properties.[19,20] In the last few
years, our research group has been engaged in the synthesis
of new biologically active coumarins. In particular, the
3-arylcoumarin scaffold has been a focal point of our recent
studies demonstrating their importance as monoamine
oxidase (MAO) inhibitors.[27–36] Different synthetic strat-
egies can be used to obtain the arylcoumarin scaffold. The
principal one is the construction of the coumarin nucleus
by condensation: cyclization-type reactions. The classical
Perkin condensation is perhaps the most direct and simple
method known for the preparation of these molecules.[23]
Some variations of this methodology are described in the
chemistry section below.
R
S
O
N
R
(a)
O
N
N
H
O
H
A2B
A1 /A2A
R
R
O
A3
(b)
R
O
O
In the present communication, we report the synthesis,
pharmacological evaluation, theoretical evaluation of
absorption, distribution, metabolism and excretion
(ADME) properties and SAR study of a selected series of
3-arylcoumarins (compounds 1–9). ARs’ binding activity
and selectivity of the synthesized compounds 1–9 were
evaluated in this study.
(c)
Figure 1 Examples of non-classical adenosine receptor ligands.
Quinolinone derivatives (a), chromone derivatives (b) and
3-arylcoumarin derivatives (c).
have been developed and investigated for their potential
therapeutic applications in diseases where the A3 AR seems
to have a positive role.
Materials and Methods
Of the large number of compounds exerting high
potency and selectivity towards ARs, the xanthine and
adenine scaffolds have been used to develop the so-called
classical AR antagonists. In the search for non-classical AR
ligands, novel structures have recently been discovered.
Examples are the quinolinone derivatives (Figure 1a), rec-
ognized as putative A1, A2A and A2B AR ligands.[15] Based on
these findings, and taken into consideration that the cou-
marin nucleus can be a non-nitrogen aromatic bioisoster
scaffold, it was found to be relevant to explore it in the
design of novel AR antagonists. Our interest in this new
scaffold was also motivated by the structure similarity
between the coumarin skeleton and chromone scaffolds
(Figure 1b), previously described as AR antagonists that
present a particular selectivity for the A3 and A2A AR
subtypes.[16–18] Among them, our attention was focused on
the 3-arylcoumarin ring system (Figure 1c), which has
never been proposed as a scaffold for AR ligands. The recent
findings and the lack of data on the 3-arylcoumarin scaffold
encouraged us to explore the ARs’ binding activity of a
selected series of derivatives.
Chemistry
Compounds 1–9 were efficiently synthesized according to
the synthetic strategy outlined in Scheme 1. Perkin con-
densation of different commercially available ortho-
hydroxybenzaldehydes with a conveniently substituted
arylacetic acid, using N,N′-dicyclohexylcarbodiimide
(DCC) as dehydrating agent,[28,29,32] afforded the 3-
arylcoumarins 1–3.[37–39] In fact, these reaction conditions
have been also found to be appropriate when the reagents
encompass in their structure methyl, methoxyl, ethoxyl or
halogen groups. However, in the case of the hydroxyl
substituents, these groups must be previously protected
by acetylation. Therefore, compound 4 was obtained
starting from the 2,5-dihydroxybenzaldehyde and the 4-
methylphenylacetic acid, using potassium acetate in acetic
anhydride, under reflux, for 16 h. Acetylation of the
hydroxyl groups and pyrone ring closure occurred
simultaneously.[40] Nitro-substituted compounds 5 and
6[41,42] were obtained via other modified Perkin condi-
tions, using
a substituted ortho-hydroxybenzaldehyde
Coumarins are an important class of benzopyrone het-
erocyclic compounds, of natural or synthetic origin, associ-
ated with remarkable pharmacological activity.[19,20] Diverse
biological properties, such as antioxidant, anti-HIV, anti-
and 4-nitrophenylacetic acid, in the presence of NaH in
acetic anhydride, for 20 h.[43] Compounds 7 and 8[32] were
obtained by acidic hydrolysis of the respective methoxy/
acetoxy derivatives 3 and 4, using hydriodic acid 57% in the
2
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Maria João Matos et al.
AR binding affinities of 3-arylcoumarins
NO2
R1
O
R1
R
R
R
H
a
b
O
O
OH
O
O
HOOC
5: R = CH3
6: R = OCH3
1: R = R1 = H
2: R = CH3 ; R1 = 4'-CH3
c
3: R = OCH3 ; R1 = 3'-CH3
CH3
d
H3C
O
O
O
4
O
O
O
HO
CH3
O
O
d
7
CH3
HO
O
8
e
CH3
O
O
H3C
O
9
Scheme 1 Reagents and conditions: (a) DCC, DMSO, 110°C, 24 h; (b) CH3CO2K, Ac2O, reflux, 16 h; (c) NaH, Ac2O, r.t., 20 h; (d) HI, AcOH, Ac2O,
110°C, 5 h; (e) chloroacetone, K2CO3, acetone, reflux, 16 h.
presence of acetic acid and acetic anhydride.[29] Finally, the
Williamson reaction of the hydroxycoumarin 8 with
chloroacetone, under reflux of acetone for 16 h, gave the
corresponding ether 9.[32]
Thermo Quest Spectrasystem (Thermo Fisher Scientific,
Waltham, MA, USA) equipped with a P4000 pump, an
UV6000 UV-Vis DAD, and an SN4000 interface to be oper-
ated via
a personal computer. Instrument software
Melting points were determined using a Reichert Kofler
thermopan or in capillary tubes on a Büchi 510 apparatus
(Flawil, Switzerland), and are uncorrected. 1H and 13C NMR
spectra were recorded on a Bruker AMX spectrometer
(Billerica, MA, USA) at 300 MHz and 75.47 MHz, respec-
tively, using TMS as internal standard (chemical shifts in δ
values, J in Hz). Mass spectra were obtained using a
Hewlett-Packard 5988A spectrometer (Palo Alto, CA, USA).
Elemental analyses were performed using a Perkin-Elmer
240B microanalyser (Waltham, MA, USA) and were within
0.4% of calculated values in all cases. Silica gel (Merck 60,
230–00 mesh; Whitehouse Station, NJ, USA) was used for
flash chromatography (FC). Analytical thin layer chroma-
tography was performed on plates precoated with silica gel
(Merck 60 F254, 0.25 mm). The purity of compounds was
assessed by high-performance liquid chromatography
(HPLC) coupled at diode array detector (DAD) on a
ChromQuest 5.0 (Thermo Fisher Scientific) was used for
data acquisition. Different analytical columns and mobile
phases (all solvents were HPLC grade) were tested. The
mobile phase was H2O:CH3CN = 70 : 30, and an Eclipse xdb
C18 column (5 μm particle size, 0.46 mm i.d., 25 cm length;
Agilent Technologies, Santa Clara, CA, USA) was used. The
purity of the compounds was found to be higher than 95%.
Synthetic methodologies
General procedure for the preparation of
3-phenylcoumarins 1–3
A
conveniently substituted ortho-hydroxybenzaldehyde
(7.34 mmol) and the corresponding phenylacetic acid
(9.18 mmol) were dissolved in dimethyl sulfoxide (15 ml).
DCC (11.46 mmol) was added, and the mixture was heated
in an oil bath at 110°C for 24 h. After reaction, ice (100 ml)
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
3

AR binding affinities of 3-arylcoumarins
Maria João Matos et al.
and acetic acid (10 ml) were added to the mixture. The
reaction was kept at room temperature for 2 h, and then
extracted with ether (3 × 25 ml). The organic layer was
washed with sodium bicarbonate solution (50 ml, 5%) and
then water (20 ml). The solvent was evaporated under
vacuum, and the dry residue was purified by FC (hexane/
ethyl acetate 9 : 1) to give the products 1 (mp 131–
132°C),[37] 2 (mp 139–140°C)[38] or 3 (mp 98–99°C)[32,39] as
beige powders, in yields of 70%, 65% and 75%, respectively.
acid 57% (10 ml) was then added dropwise. The mixture
was stirred at 110°C for 5 h. The solvent was evaporated
under vacuum, and the dry residue was purified by CH3CN
crystallization to give 7 or 8 (mp 214–215°C)[32] as beige
powders, in yields of 55% and 61%, respectively.
Synthesis of 3-(4′-methylphenyl)-6-
(2-oxopropoxy)coumarin (9)
To a suspension of anhydrous K2CO3 (0.25 mmol) and the
6-hydroxy-3-(4′-methylphenyl)coumarin (8, 0.13 mmol), in
anhydrous acetone (3 ml), chloroacetone (0.25 mmol) was
added. The suspension was stirred, at reflux temperature,
for 16 h. The mixture was cooled, and the precipitate was
recovered by filtration and washed with anhydrous acetone
(3 × 40 ml). The solvent was evaporated under vacuum, and
the dry residue was purified by FC (hexane/ethyl acetate
85 : 15) to give compound 9 (mp 128–129°C)[32] as a beige
powder, in a yield of 74%.
Synthesis of
6-acetoxy-3-(4′-methylphenyl)coumarin (4)
Compound 4 was synthesized under anhydrous conditions,
using material previously dried at 60°C for at least 12 h and
at 300°C during few minutes immediately before use. A
solution containing anhydrous CH3CO2K (2.94 mmol), the
4-methylphenylacetic acid derivative (1.67 mmol) and the
2,5-dihydroxybenzaldehyde (1.67 mmol) in Ac2O (1.2 ml)
was prepared and refluxed (138°C) for 16 h. The reaction
mixture was cooled, neutralized with 10% aqueous
NaHCO3, and extracted (3 × 30 ml) with EtOAc. The
organic layers were combined, washed with distilled water,
dried (anhydrous Na2SO4), and evaporated under reduced
pressure. The product was purified by recrystallization in
EtOH and dried, to afford 4 as a beige powder.
Biological assays
The adenosine binding affinity of compounds 1–9 for the
human AR subtypes hA1, hA2A, hA2B and hA3, expressed in
Chinese Hamster Ovary (CHO) cells, was determined in
radioligand competition experiments.[44–47] In the binding
affinity assay, it is measured the competition of ligands for
specific binding of [3H]CCPA binding at hA1 ARs, specific
binding of [3H]NECA binding at hA2A ARs, and specific
binding of [3H]HEMADO at hA3 ARs. The results were
expressed as Ki (dissociation constants), which were calcu-
lated with the programme SCTFIT (CA, USA), and given as
geometric means of at least three experiments, including
95% confidence intervals.[46] Due to the lack of a suitable
radioligand for hA2B receptor in binding assay, the potency
of antagonists at hA2B receptor was determined instead by
inhibition of NECA-stimulated adenylyl cyclase activity
with increasing concentrations of antagonist.[44,45] As a
result, cyclic adenosine monophosphate production was
inhibited in a concentration-dependent fashion, and IC50
and Ki values, respectively, were determined.
Synthesis of 3-(4′-nitrophenyl)coumarins 5 and 6
To a dry 100-ml round-bottomed flask, the conveniently
substituted
ortho-hydroxybenzaldehyde
(42.5 mmol),
4′-nitrophenylacetic acid (42.5 mmol) and acetic anhydride
(40.1 ml, 0.43 mol) were added. Then, sodium hydride
(60% dispersion in mineral oil) (42.5 mmol) was added in
small aliquots. After the dissolution of the reagents, an
instantaneous (after 2–5 min) precipitation process was
observed. The reaction mixture was stirred for 20 h, and
then 7 ml of water were added. After the addition of 43 ml
of acetic acid, the mixture was cooled to 4°C for 4 h. The
resulting precipitate was filtered and washed with cold
glacial acetic acid. The acetic acid was then removed as an
azeotrope upon addition of 250 ml toluene and rotary
evaporation to dryness. The process was repeated three
times. The final residue was dried under vacuum and puri-
fied by FC (hexane/ethyl acetate 9:1) to give the products
5 (mp 100–101°C)[41] or 6 (mp 103–104°C)[42] as beige
powders, in yields of 61% and 64%, respectively.
ADME properties
The preliminary data for an ADME profile analysis was
calculated using the Molinspiration property calculation
programme.[48]
Statistical analysis
Synthesis of 6-hydroxy-3-phenylcoumarins
7 and 8
Ki values (dissociation constants) were determined in
radioligand competition experiments with seven to eight
different concentrations of test compound, and each con-
centration was tested in duplicate. Ki values (Table 1) are
reported as geometric means of four independent experi-
A
solution of the 6-methoxy/acetoxy substituted 3-
phenylcoumarin (3 or 4, 0.50 mmol) in acetic acid (5 ml)
and acetic anhydride (5 ml) at 0°C was obtained. Hydriodic
4
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Maria João Matos et al.
AR binding affinities of 3-arylcoumarins
Table 1 Affinity (Ki) to bind human A1 and A3 adenosine receptors expressed in Chinese hamster ovary cells of coumarins 1–9, in radioligand
binding assays
Compounds
R
hA1
hA3
Selectivity
R1
Ki (nM)
Ki (nM)
>30 000
hA1/hA3
1
2
3
4
5
6
7
8
H
H
>30 000
>30 000
>30 000
>30 000
>30 000
>100 000
9 250 (8 270–10 300)
7 900 (4 080–15 300)
>30 000
6 770 (4 070–11 300)
–
CH3
4′-CH3
3′-CH3
4′-CH3
4′-NO2
4′-NO2
3′-CH3
4′-CH3
4′-CH3
–
2 680 (1 440–5 010)
>11.2
>5.9
>4.1
–
OCH3
OCOCH3
CH3
5 050 (3 670–7 000)
7 280 (4 130–12 800)
>30 000
>100 000
14 600 (13 400–15 900)
OCH3
OH
–
0.6
1.6
>6.4
0.08
OH
4 910 (3 970–6 070
4 680 (3 870–5 660)
86 400 (73 600–101 300)
9
OCH2COCH3
Theophylline[44]
–
These results are average results of four experiments.
ments with 95% confidence intervals. For analysis of the
competition curves, the programme SCTFIT was used.[47] Ki
values of the different compounds at the A3 receptor were
analysed with the Kruskal–Wallis test. No statistically sig-
nificant difference between the compounds was detected
associated with this characteristic, a molecule must possess
the appropriate ADME properties. To better understand
the overall properties of the synthesized coumarins, pre-
liminary data for an ADME profile analysis were carried
out. The lipophilicity, expressed as the octanol/water parti-
tion coefficient (represented as logP), was calculated using
the Molinspiration property calculation programme
(Table 2).[48] Therefore, a theoretical prediction of ADME
properties of all compounds was carried out and is
described by different parameters (Table 2).[49,50]
2
(P > 0.05, χ test).
Results
Structural identification
6-Acetoxy-3-(4′-methylphenyl)coumarin (4): Yield 61%. Mp
146–147°C; 1H NMR δ ppm (CDCl3-300 MHz): 2.38 (3H, s,
CH3), 2.44 (3H, s, CH3), 7.24-7.32 (4H, m, H-2′, H-3′, H-5′,
H-6′), 7.40 (1H, dd, J = 8.7, 2.7 Hz, H-7), 7.63 (2H, d,
J = 8.6 Hz, H-5, H-8), 7.77 (1H, s, H-4). 13C NMR δ ppm
(CDCl3-75 MHz): 21.1, 21.3, 117.4, 119.9, 120.2, 124.6, 128.4,
129.1, 129.2, 131.5, 138.4, 138.4, 139.2, 146.7, 150.9, 169.3. MS
(ESI): 295 (M + H+, 20), 294 (M+, 100). Anal. Calcd for
C18H14O4: C, 73.46; H, 4.79. Found: C, 73.41; H, 4.74.
6-Hydroxy-3-(3′-methylphenyl)coumarin (7): Yield 55%.
Mp 132-3°C; 1H NMR δ ppm (CDCl3-300 MHz): 2.24 (3H,
s, CH3), 6.97-7.29 (4H, m, H-5, H-7, H-4′, H-6′), 7.31-7.33
(2H, m, H-2′, H-5′), 7.48 (1H, d, J = 8.8 Hz, H-8), 8.11 (1H,
s, H-4), 9.82 (1H, s, OH). 13C NMR δ ppm (CDCl3-
75 MHz): 25.2, 113.2, 117.4, 120.4, 126.6, 127.5, 128.7,
129.8, 130.2, 135.4, 138.0, 141.1, 147.0, 154.5, 160.6, 169.8.
MS (ESI): 253 (M + H+, 15), 252 (M+, 100). Anal. Calcd for
C16H12O3: C, 76.18; H, 4.79. Found: C, 76.15; H, 4.76.
Discussion
Compounds 1–9 were synthesized and tested for their
in-vitro binding affinity against human AR subtypes hA1,
hA2A, hA2B and hA3, expressed in CHO cells. In the present
communication, studies of the effect on the binding affinity
by the presence of different substituents in the 3-
arylcoumarin scaffold, at positions 3′, 4′ and 6, were per-
formed. As the 3-arylcoumarin scaffold has never been
described as an AR ligand, we were encouraged to explore
the ARs’ binding activity of a selected series of derivatives.
The experimental data show that all the 3-arylcoumarin
derivatives have no detectable binding affinity to hA2A and
hA2B ARs (Ki > 30 000–100 000 nM). Compound 1, the
non-substituted 3-arylcoumarin, presents no binding affin-
ity for any of the evaluated receptors (Ki > 30 000 nM). The
introduction of electron-donating groups in both phenyl
rings (methyl groups at positions 6 and 4′) afforded com-
pound 2, the most interesting compound of this series with
a Ki at the hA3 AR of 2680 nM. The structural modification
of coumarin 1 results in significant affinity and selectivity
for hA3 ARs. The interesting result found for compound 2
was inspiring for the development of new hA3 AR selective
ligands. The changing of the methyl group of the phenyl
substituent for a strong electron-withdrawing nitro group
led to compound 5, which displays no binding affinity for
Biological results
The data from radioligand binding experiments for com-
pounds 1–9 are summarized in Table 1. Details for pharma-
cological experiments were previously described in
materials and methods, and in references 44–47.
One of the important requirements for a molecule to be a
good drug candidate is the ability to cross membranes. Also,
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••
5

AR binding affinities of 3-arylcoumarins
Maria João Matos et al.
Table 2 Structural properties of the coumarin derivatives 1–9
Compounds
R
R1
logP
Molecular weight (g/mol)
TPSA (Å2)
n-OH acceptors
n-OHNH donors
Volume (Å3)
1
2
3
4
5
6
7
8
9
H
H
3.74
4.61
4.19
3.71
4.12
3.73
3.66
3.68
3.76
222.24
250.30
266.30
294.31
281.27
297.27
252.27
252.27
308.33
30.211
30.21
39.44
56.52
76.03
85.27
50.44
50.44
56.52
2
2
3
4
5
6
3
3
4
0
0
0
0
0
0
1
1
0
200.00
233.12
242.10
261.08
239.89
248.88
224.57
224.57
277.89
CH3
4′-CH3
3′-CH3
4′-CH3
4′-NO2
4′-NO2
3′-CH3
4′-CH3
4′-CH3
OCH3
OCOCH3
CH3
OCH3
OH
OH
OCH2COCH3
TPSA, topological polar surface area; n-OH, number of hydrogen bond acceptors; n-OHNH, number of hydrogen bond donors. The data were deter-
mined with Molinspiration calculation software.[48]
any of AR subtypes. The presence of a nitro group at that
position does not seem to be compatible with receptor
binding, as confirmed by the data obtained with compound
6. As the presence of a methyl group at position 4′ seems to
be important for the binding affinity, compounds 4, 8 and 9
were synthesized and studied.
For compound 8, with a hydroxyl group at position 6, a
decrease in the selectivity was observed, although the affin-
ity data for both hA1 and hA3 ARs were interesting
(Ki = 7900 and Ki = 4910 nM, respectively). Compound 4,
with an acetoxy group in the same position of the coumarin
skeleton, is a selective compound, with good binding affin-
ity for the hA3 AR (Ki = 7280 nM). Finally, the presence of a
2-oxopropoxy group (compound 9) in the previously men-
tioned position of the coumarin moiety led also to a note-
worthy hA3 AR ligand (Ki = 4680 nM).
To complete this study, the methyl group at 4′ position
(present in the best compound of the series-compound 2)
was transposed to position 3′ (compounds 3 and 7). In the
case of compound 7, as previously described for compound
8, the presence of a hydroxyl group at position 6 has a
strong influence on the selectivity and affinity towards hA1
and hA3 ARs (Ki = 9250 nM and Ki = 14 600 nM, respec-
tively). Compound 3 with a methyl group at position 3′ of
the 3-aryl ring is a selective hA3 AR ligand (Ki = 5050 nM),
similar to the other non-hydroxylated 4′-methyl derivatives.
In fact, the protection of the hydroxyl groups forming ether
(compound 3) or ester (compounds 4 and 9) derivatives
results in an increase in hA3 selectivity. Compounds 2–4 and
7–9 present better hA3 affinity than theophylline, used as
reference compound. In addition, compounds 2–4 and 9
are hA3 selective ligands.
Moreover, due to the demonstrated importance of the
3-arylcoumarins as MAO inhibitors, these AR ligands can
be further developed to give improvements in both ARs’
activity and ARs vs MAO selectivity. Also, as A3 AR ligands
are involved in neurodegenerative diseases, this study can be
an encouragement for the development of multitarget drugs
as AR ligands and MAO inhibitors. Compound 2, present-
ing the better hA3 affinity/selectivity of this series, was pre-
viously described as MAO-B selective inhibitor, with an IC50
of 308 pM. This compound is one of the best MAO-B
inhibitors described by our research group. This additional
data allow us to consider this compound an excellent candi-
date for future studies in the field of neurodegenerative
diseases.
Finally, analysing the obtained theoretical results, it can
be observed that no violations of Lipinski’s rule (molecular
weight, logP, number of hydrogen donors and acceptors)
were found for the described coumarins. Also, topological
polar surface area, described to be a predictive indicator of
membrane penetration, is found to be positive for these
drug candidates.
Conclusions
In the current study, a series of 3-arylcoumarin derivatives
were synthesized and evaluated as putative AR ligands. A
detailed analysis of the experimental results allows us to
conclude that the affinity or selectivity of the synthesized
coumarins is influenced by the presence and the nature
of the substituents at positions 3′, 4′ and 6 of the
3-arylcoumarin scaffold. The experimental data show that
neither the simple 3-arylcoumarin nor the 4′-nitro deriva-
tives presented detectable binding affinity for the evaluated
receptors, although most of the other substituted deriva-
tives have good binding affinity profiles, especially against
the hA1/hA3 or only hA3 AR. In general, one can conclude
that the presence of a methyl group at positions 3′ or 4′
of the 3-aryl ring is important for the binding affinity.
From these preliminary results, one can conclude that the
presence of the methyl group in the aryl substituent will
provide an important strategy to improve the AR binding
affinity of coumarin-based ligands. In addition, chemical
changes at position 6 are important for modulating the
selectivity of compounds against hA1/hA3 or hA3 ARs.
6
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ••, pp. ••–••

Maria João Matos et al.
AR binding affinities of 3-arylcoumarins
Fulfilling this condition, the presence of a methyl, methoxy,
acetoxy or 2-oxopropoxy groups at position 6 of the scaffold
results in a noticeable affinity and selectivity for hA3 AR.
The presence of a hydroxyl group at position 6 gives rise to
non-selective ligands binding to both hA1 and hA3 ARs. This
study demonstrates that 3-arylcoumarin is an interesting
scaffold for the design of novel ARs ligands. Accordingly,
compound 2 (Ki hA3 AR = 2680 nM), which is also a very
interesting MAO-B selective inhibitor, can be considered the
lead for the design and synthesis of new hA3 AR coumarin-
based selective ligands. This compound, as all compounds
in this series, is in accordance with the Lipinski’s rule.
Therefore, the versatility of the synthetic routes and the
substitution variability of these coumarins allow proposing
them as privileged multitarget scaffolds for drug discovery
in the field of neurodegenerative diseases.
Declarations
Acknowledgements
Partial financial support from Fundação para a Ciência e
Tecnologia (projects PTDC/QUI/70359/2006 and PTDC/
QUI-QUI/113687/2009) is gratefully acknowledged. Partial
financial support from Spanish researchers personal funds
is gratefully acknowledged. M.J. Matos (SFRH/BD/61262/
2009), A. Gaspar (SFRH/BD/43531/2008) and F. Borges
(SFRH/BSAB/1090/2010) thank FCT grants. We thank Leif
Hommers for help with the statistical analysis of the data.
9. Armentero T et al. Past, present and
future of A(2A) adenosine receptor
on chromone scaffold. Biochem
Pharmacol 2012; 84: 21–29.
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