A new series of 3-phenylcoumarins as potent and selective MAO-B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.04.085

6-Methyl-3-phenylcoumarins 3-6 were designed, synthesized and evaluated as monoamine oxidase A and B (MAO-A and MAO-B) inhibitors. The synthesis of these new compounds (resveratrol-coumarin hybrids) was carried out with good yield by a Perkin reaction, fr

Full text of DOI:10.1016/j.bmcl.2009.04.085

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3268–3270
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new series of 3-phenylcoumarins as potent and selective MAO-B inhibitors
Maria Joao Matos a,b, , Dolores Viña a,c, Elias Quezada a,b, Carmen Picciau b, Giovanna Delogu b,
*
Francisco Orallo c, Lourdes Santana a, Eugenio Uriarte a
a Departamento de Química Orgánica, Facultad de Farmacia, 15782 Santiago de Compostela, Spain
b Dipartimento Farmaco Chimico Tecnologico, Facoltà di Farmacia, 09124 Cagliari, Italy
c Departamento de Farmacología, Facultad de Farmacia, 15782 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
6-Methyl-3-phenylcoumarins 3–6 were designed, synthesized and evaluated as monoamine oxidase A
and B (MAO-A and MAO-B) inhibitors. The synthesis of these new compounds (resveratrol–coumarin
hybrids) was carried out with good yield by a Perkin reaction, from the 5-methylsalicylaldehyde and
the corresponding phenylacetic acid. They show high selectivity to the MAO-B isoenzyme, with IC50 val-
ues in the nanomolar range. Compound 5 is the most active compound and is several times more potent
and selective than the reference compound, R-(ꢀ)-deprenyl.
Received 24 March 2009
Revised 17 April 2009
Accepted 20 April 2009
Available online 24 April 2009
Keywords:
Coumarin
Ó 2009 Elsevier Ltd. All rights reserved.
Resveratrol
Monoamino oxidase inhibitors
Perkin reaction
3-Phenylcoumarins
Coumarins (or benzopyrones) are a large family of compounds,
of natural and synthetic origin, that show numerous biological
activities.1 Recent studies pay special attention to their antioxida-
tive, anticarcinogenic and enzymatic inhibition properties.2–6 In re-
gard to the monoamine oxidase (MAO) inhibition, the recent
findings revealed that MAO-A and MAO-B affinity and selectivity
can be efficiently modulated by appropriate substitutions in the
coumarin ring, in particular in the 3:4 and 6:7 positions.7–11
The resveratrol (3,40,5-trihydroxystilbene) is a phytoalexin of
natural origin present in spermatophytes species such as vines,
in response to damage. Resveratrol was already studied as antiox-
idant, anti-inflammatory, cardioprotective (vasodilatory and plate-
let antiaggregatory activities), anticancer, enzymatic inhibitor,
proving to be very efficient in a large group of in vitro, ex vivo
and/or in vivo experiments.12–15 The resveratrol’s cis and trans iso-
mers are inhibitors of the MAO activity. cis-Resveratrol is less effec-
tive than trans-resveratrol as inhibitor of MAO-A and MAO-B
activities.16
inhibitors (iMAO). These modifications were studied to find out
how these changes can contribute to the biological activity of these
molecules, helping to understand a structure–activity relationship
(SAR).
MAO is a FAD-containing enzyme with two known isoforms
(MAO-A and MAO-B) and is present in the mitochondrial outer
membrane of glial, neuronal and other cells.19 MAO enzymes inter-
vene in the monoamines degradation and carry out an important
physiologic function in the adrenaline, noradrenaline and seroto-
nin deamination (preferentially MAO-A) and in the b-phenylethyl-
amine and benzylamine deamination (preferentially MAO-B).20
This enzymatic function increases the synaptic concentration of
these neurotransmitters and conditions to a great extent the neu-
rone’s excitement of those possessing receptors for these
mediators.21
The iMAO are a class of compounds that act by blocking the
MAO enzymatic action, being used by several years in the treat-
ment of the depression and anxiety diseases (iMAO-A) or in Parkin-
son’s disease (iMAO-B).22 Nowadays is being studied also in the
Alzheimer’s disease.23
The active sites of these two isoenzymes (MAO-A and MAO-B)
are not completely known and for this reason there is a lack of
information in respect of how the inhibitors act selectively in
one of the two of them.10 Recent X-ray crystal structures of
MAO-B24,25 and MAO-A,21,26 completed with irreversible and
reversible inhibitors, can be used in the future as a tool to the
structural basis to help understanding the selective enzyme–ligand
recognition and drug development of iMAOs.21
Due to these coincident properties, it seems to be interesting to
design and synthesize hybrids that incorporate the skeleton of
those two kinds of molecules.17,18 In the present compounds the
resveratrol nucleus is blocked by the coumarin ring and can only
assume the trans isomeric form. A series of these molecules, with
different number and position of methoxy groups in the 3-phenyl
ring (compounds 3–6), was synthesized and evaluated as MAO
* Corresponding author. Tel.: +34 981 563100.
E-mail address: mariacmatos@gmail.com (M.J. Matos).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.085

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3268–3270
3269
With the aim of finding out new structural features for the MAO
inhibitory activity and selectivity, we decided in this work to ex-
plore the importance of the number and position of different meth-
oxy groups under the benzenic ring in 3-position (compounds
is an important factor to discriminate the potential therapeutic
application of this kind of molecules.
Comparing the iMAO-B activities of 3 and 4, the introduction of
a p-methoxy group increases the inhibitory activity. Substitution
with two methoxy groups in the 30- and 50-positions on the phenyl
ring, compound 5, improves the iMAO-B activity. Increasing the
number of methoxy substituent to three, compound 6, decreases
the enzymatic inhibitory activity. However, compound 6 is even
better than none substituted coumarin 3. The presence of methoxy
substituent in the 3-phenyl ring seems to be important to modu-
late and improve the inhibitory enzymatic activity of the 6-
methyl-3-phenylcoumarins.
These hybrid compounds with resveratrol–coumarin skeleton
show high selectivity against MAO-B isoenzyme, being active in
the nanomolar range. Introduction of methoxy groups in the phe-
nyl ring improves the activity, giving more active and selective
compounds than the reference ones. These modifications, which
we studying more deeply, can improve the pharmacologic profile
of the synthesized coumarins in the Parkinson’s disease.
4
27,28–6), to establish a relation between them and with the non
substituted analogue (compound 327–29).
The preparation of these 6-methyl-3-phenylcoumarins was per-
formed via the classical Perkin reaction.29 This reaction was carried
out by condensation of the 5-methylsalicylaldehyde 1 and the con-
veniently substituted phenylacetic acids 2, with N,N0-dicyclohexyl-
carbodiimide (DCC) as dehydrating agent, under DMSO reflux,
during 24 h (Scheme 1). The reaction to obtain 3–6 is very clean
and the yields are between 60% and 70%.30–33 The obtained prod-
ucts are easy to purify by flash chromatography, using a mixture
of hexane/ethyl acetate in a proportion 9:1 as eluent.
The inhibitory MAO activity of compounds 3–6 was evaluated
in vitro by the measurement of the enzymatic activity of human re-
combinant MAO isoforms in BTI insect cells infected with baculo-
virus.8,34 Then, the IC50 values and MAO-B selectivity ratios [IC50
(MAO-A)]/[IC50 (MAO-B)] for inhibitory effects of both, new com-
pounds and reference inhibitors, were calculated (Table 1).35
The prepared series of compounds proved to be selective as
inhibitor of the MAO-B isoenzyme. Compound 3, none substituted
in the phenyl ring, is by itself very active and selective against
MAO-B isoenzyme. Compound 4 (with a p-methoxy group) has a
MAO-B IC50 similar to the R-(ꢀ)-deprenyl (reference MAO-B inhib-
itor) and is more selective than this one. The most potent molecule
of this family is compound 5, bearing two methoxy groups in 30-
and 50-positions (IC50 = 8.98 1.42 nM). This one is two times more
active and several times more iMAO-B selective than the R-(ꢀ)-
deprenyl. Compound 6, with 3 methoxy groups, is more active than
3 (none substituted) but it loses activity in respect to the mono and
dimethoxy derivatives (compounds 4 and 5, respectively). None of
the described compounds showed a MAO-A inhibitory activity for
Acknowledgements
Thanks the Spanish Ministerio de Sanidad
y Consumo
(PI061457 and PI061537) and to Xunta da Galicia (PXIB20304PR,
INCITE08PXIB203022PR and 08CSA019203PR) and Fondazione
Banco Sardegna (Italy) for financial support. M.J.M. also thanks to
MIUR the Ph.D. Grant.
References and notes
1. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem.
2005, 12, 887.
2. Hoult, J. R. S.; Payá, M. Gen. Pharmacol. 1996, 27, 713.
3. Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Enzyme Inhib. Med. Chem. 2003, 18, 63.
4. Kabeya, L.; Marchi, A.; Kanashiro, A.; Lopes, N.; Silva, C.; Pupo, M.; Lucisano-
Valim, Y. Bioorg. Med. Chem. 2007, 15, 1516.
the highest concentration tested (100
lM). This iMAO-B selectivity
5. Carotti, A.; Altomare, C.; Catto, M.; Gnerre, C.; Summo, L.; De Marco, A.; Rose, S.;
Jenner, P.; Testa, B. Chem. Biod. 2006, 3, 134.
6. Chilin, A.; Battistutta, R.; Bortolato, A.; Cozza, G.; Zanatta, S.; Poletto, G.;
Mazzorana, M.; Zagotto, G.; Uriarte, E.; Guiotto, A.; Pinna, L.; Meggio, F.; Moro,
S. J. Med. Chem. 2008, 51, 752.
R1
R2
HO
O
R1
R2
Me
CHO
OH
Me
a
7. Santana, L.; Uriarte, E.; González-Díaz, H.; Zagotto, G.; Soto-Otero, R.; Méndez-
Álvarez, E. J. Med. Chem. 2006, 49, 1118.
8. Santana, L.; González-díaz, H.; Quezada, E.; Uriarte, E.; Yáñez, M.; Viña, D.;
Orallo, F. J. Med. Chem. 2008, 51, 6740.
9. Catto, M.; Nicolotti, O.; Leonetti, F.; Carotti, A.; Favia, A.; Soto-Otero, R.;
Méndez-Álvarez, E.; Carotti, A. J. Med. Chem. 2006, 49, 4912.
10. Gnerre, C.; Catto, M.; Leonetti, F.; Weber, P.; Carrupt, P.; Altomare, C.; Carotti,
A.; Testa, B. J. Med. Chem. 2000, 43, 4747.
11. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Befani, O.; Turini,
P.; Alacaro, S.; Ortuso, F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.
12. Frémont, L. Life Sci. 2000, 66, 663.
R3
O
O
R3
2
1
3-6
3: R1 = R2 = R3 = H
4: R1 = R3 = H , R2 = OMe
5: R1 = R3 = OMe , R2 = H
6: R1 = R2 = R3 = OMe
13. Orallo, F. In Resveratrol in Health and Disease; Aggarwal, B. B., Shishodia, S., Eds.;
CRC Press: USA, 2005; p 577.
Scheme 1. Reagents and conditions: (a) DCC, DMSO, 110 °C, 24 h.
14. Leiro, J.; Álvarez, E.; Arranz, J.; Laguna, R.; Uriarte, E.; Orallo, F. J. Leukocyte Biol.
2004, 75, 1156.
15. Orallo, F. Curr. Med. Chem. 2008, 15, 1887.
16. Yánez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Commun. 2006,
344, 688.
17. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yánez, M.; Fraiz, N.; Alcaide, C.;
Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.
18. Vilar, S.; Quezada, E.; Alcaide, C.; Orallo, F.; Santana, L.; Uriarte, E. Qsar Comb.
Sci. 2007, 26, 317.
19. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.
20. Grimsby, J.; Lan, N. C.; Neve, R.; Chen, K.; Shih, J. C. J. Neurochem. 1990, 55,
1166.
21. Edmondson, D. E.; Mattevi, A.; Binda, C.; Li, M.; Hubalek, F. Curr. Med. Chem.
2004, 11, 1983.
Table 1
MAO-A and MAO-B inhibitory activity results for compounds 3–6 and reference
compounds
Compd
MAO-A IC50
MAO-B IC50
Ratio
*
*
*
*
3
4
5
6
283.75 0.98 nM
13.05 0.90 nM
8.98 1.42 nM
160.64 1.01 nM
19.60 0.86 nM
>352b
>7663b
>11,136b
>623b
3431
R-(ꢀ)-Deprenyl
67.25 1.02 l
Ma
Iproniazide
6.56 0.76
l
M
7.54 0.36
l
M
0.87
*
22. Harfenist, H.; Heuseur, D. J.; Joyner, C. T.; Batchelor, J. F.; White, H. L. J. Med.
Chem. 1996, 39, 1857.
23. Wouters, J. Curr. Med. Chem. 1998, 5, 137.
24. Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondson, D. E.; Mattevi, A. Nat.
Struct. Biol. 2002, 9, 22.
25. Binda, C.; Li, M.; Hubalek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 9750.
Inactive at 100 lM (highest concentration tested). At higher concentrations
compounds precipitate.
a
P <0.01 versus the corresponding IC50 values obtained against MAO-B, as
determined by ANOVA/Dunnett’s.
b
Values obtained under the assumption that the corresponding IC50 against
MAO-A is the highest concentration tested (100 lM).

3270
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3268–3270
26. Ma, J.; Yoshimura, M.; Yamashita, E.; Nakagawa, A.; Ito, A.; Tsukihara, T. J. Mol.
Biol. 2004, 338, 103.
27. Hans, N.; Singhi, M.; Sharma, V.; Grover, S. K. Indian J. Chem., Sect. B 1996, 35,
1159.
130.44, 132.38, 134.12, 139.48, 151.45, 153.02, 160.66. DEPT (CDCl3) d (ppm):
20.76, 56.23, 60.77, 105.97, 116.08, 127.63, 132.43, 139.54. MS m/z (%): 326
(M+, 100), 311 (89), 283 (43), 253 (12), 225 (30), 197 (10), 169 (10), 148 (5),
115 (8). Anal. Calcd for C19H18O5: C, 69.93; H, 5.56; O, 24.51. Found: C, 70.14;
H, 5.58.
28. Mohanty, S.; Makrandi, J. K.; Grover, S. K. Indian J. Chem., Sect. B 1989, 28, 766.
´
29. Kamat, S. P.; DSouza, A. M.; Paknikar, S. K.; Beaucahmp, P. S. J. Chem. Res. (S)
34. Determination of human monoamine oxidase (hMAO) isoform activity: The effects
of the test compounds on hMAO isoform enzymatic activity were evaluated by
2002, 242.
30. 6-Methyl-3-phenylcumarin (3). It was obtained with a yield of 68%. Mp 148–
149 °C (biblio. 145–147 °C, 149–150 °C). 1H NMR (CDCl3) d (ppm), J (Hz): 2.42
(s, 3H, –CH3), 7.27 (m, 1H, H-7), 7.34 (m, 2H, H-40 and H-8), 7.43 (m, 3H, H-
20,H-40 and H-5), 7.70 (dd, 2H, H-10 and H-50, J = 7.7 and 1.8), 7.77 (s, 1H, H-4).
13C NMR (CDCl3) d (ppm): 21.29, 116.67, 119.57, 128.17, 128.70, 128.95,
129.02, 129.26, 132.95, 134.65, 135.35, 140.39, 152.16, 161.31. DEPT (CDCl3) d
(ppm): 21.29, 100.60, 116.67, 128.17, 128.95, 129.02, 129.26, 132.95, 140.39.
MS m/z (%): 236 (M+, 100), 208 (50), 178 (8), 165 (8) 76 (6), 51 (69). Anal. Calcd
for C16H12O2: C, 81.34; H, 5.12; O, 13.54. Found: C, 81.44; H, 4.84.
a fluorimetric method following the experimental protocol previously
described by us. Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)
containing the test drugs in various concentrations and adequate amounts of
recombinant hMAO-A or hMAO-B required and adjusted to obtain in our
experimental conditions the same reaction velocity [165 pmol of p-tyramine/
min (hMAO-A: 1.1
lg protein; specific activity: 150 nmol of p-tyramine
oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5
lg
protein; specific activity: 22 nmol of p-tyramine transformed/min/mg
protein)] were placed in the dark fluorimeter chamber and incubated for
15 min at 37 °C. The reaction was started by adding (final concentrations)
31. 3-(40-Methoxy)phenyl-6-methylcumarin (4). It was obtained with a yield of
61%. Mp 144–145oC (biblio. 143 °C). 1H NMR (CDCl3) d (ppm), J (Hz): 2.40 (s,
3H, –CH3), 3.84 (s, 3H, –OCH3), 6.96 (dd, 2H, H-30 and H-50, J = 6.8 and 2.1), 7.26
(m, 3H, H-4, H-7 and H-8), 7.66 (m, 3H, H-3, H-20 and H-60). 13C NMR (CDCl3) d
(ppm): 20.75, 55.32, 113.85, 116.04, 119.54, 127.19, 127.46, 127.66, 129.77,
132.02, 134.03, 138.47, 151.41, 160.05, 160.96. DEPT (CDCl3) d (ppm): 20.76,
55.32, 113.85, 116.04, 127.46, 129.78, 132.01, 138.48. MS m/z (%): 266 (M+,
100), 223 (60), 195 (24), 165 (16), 152 (13), 115 (5), 89 (5), 63 (7), 50 (6). Anal.
Calcd for C17H14O3: C, 76.68; H, 5.30; O, 18.02. Found: C, 76.88; H, 5.32.
32. 3-(30,50-Dimethoxy)phenyl-6-methylcumarin (5). It was obtained with a yield
of 60%. Mp 110–111oC. 1H NMR (CDCl3) d (ppm), J (Hz): 2.40 (s, 3H, –CH3), 3.82
(s, 6H, –(OCH3)2), 6.50 (t, 1H, H-40, J = 2.1), 6.83 (d, 2H, H-20 and H-60, J = 2.1),
7.22–7,33 (m, 3H, H-5, H-7 and H-8), 7.74 (s, 1H, H-4). 13C NMR (CDCl3) d
(ppm): 20.72, 55.41, 100.89, 106.72, 116.06, 119.22, 127.69, 127.93, 132.49,
134.10, 136.68, 140.02, 151.60, 160.48, 160.61. DEPT (CDCl3) d (ppm): 20.72,
55.40, 100.89, 106,71, 116.06, 127.69, 132.49, 140.02. MS m/z (%): 296 (M+,
100), 295 (17), 267 (16), 210 (8), 181 (22), 152 (13), 139 (6), 105 (8), 76 (9).
Anal. Calcd for C18H16O4: C, 72.96; H, 5.44; O, 21.60. Found: C, 73.23; H, 4.90.
33. 6-Methyl-3-(30,40,50-trimethoxy)phenylcumarin (6). It was obtained with a
yield of 70%. Mp 165–166 oC. 1H NMR (CDCl3) d (ppm), J (Hz): 2.43 (s, 3H, –
CH3), 3.90 (s, 3H, –OCH3), 3,92 (s, 6H, –(OCH3)2), 6.93 (d, 2H, H-20and H-60,
J = 2.1), 7.24–7.35 (m, 3H, H-5, H-7 and H-8), 7.76 (s, 1H, H-4). 13C NMR (CDCl3)
d (ppm): 20.70, 56.19, 60.72, 105.94, 116.03, 119.24, 127.48, 127.58, 130.04,
200
tyramine. The production of H2O2 and, consequently, of resorufin was
quantified at 37 °C in multidetection microplate fluorescence reader
(FLX800TM Bio-TekÒ Instruments, Inc., Winooski, VT, USA) based on the
l
M AmplexÒ Red reagent, 1 U/mL horseradish peroxidase and 1 mM p-
a
,
fluorescence generated (excitation, 545 nm, emission, 590 nm) over a 15 min
period, in which the fluorescence increased linearly.Control experiments were
carried out simultaneously by replacing the test drugs with appropriate
dilutions of the vehicles. In addition, the possible capacity of the above test
drugs to modify the fluorescence generated in the reaction mixture due to non-
enzymatic inhibition (e.g., for directly reacting with AmplexÒ Red reagent) was
determined by adding these drugs to solutions containing only the AmplexÒ
Red reagent in a 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 vials containing all
components except the hMAO isoforms, which were replaced by a sodium
phosphate buffer solution.
On the other hand, in our experiments and under our experimental conditions,
the control activity of hMAO-A and hMAO-B (using p-tyramine as a common
substrate for both isoforms) was 165 2 pmol of p-tyramine oxidized to p-
hydroxyphenylacetaldehyde/min (n = 20).
35. All IC50 values shown in the table are expressed as means SEM from five
experiments.