New halogenated 3-phenylcoumarins as potent and selective MAO-B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.07.013

With the aim to find out the structural features for the MAO inhibitory activity and selectivity, in the present communication we report the synthesis and pharmacological evaluation of a new series of bromo-6-methyl-3- phenylcoumarin derivatives (with bro

Full text of DOI:10.1016/j.bmcl.2010.07.013

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5157–5160
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New halogenated 3-phenylcoumarins as potent and selective MAO-B inhibitors
c
Maria João Matos a,b, , Dolores Viña , Patricia Janeiro a,d, Fernanda Borges b, Lourdes Santana a,
*
Eugenio Uriarte a
a Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
b CIQUP/Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, 4169-007 Porto, Portugal
c Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
d Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-535 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim to find out the structural features for the MAO inhibitory activity and selectivity, in the
present communication we report the synthesis and pharmacological evaluation of a new series of
bromo-6-methyl-3-phenylcoumarin derivatives (with bromo atom in both different benzene rings of
the skeleton) with and without different number of methoxy substituent at the 3-phenyl ring. The meth-
oxy substituents were introduced, in this new scaffold, in the meta and/or para positions of the 3-phenyl
ring. The synthesized compounds 3–7 were evaluated as MAO-A and B inhibitors using R-(ꢀ)-deprenyl
(selegiline) and iproniazide as reference inhibitors, showing, most of them, MAO-B inhibitory activities
in the low nanomolar range. Compounds 4 (IC50 = 11.05 nM), 5 (IC50 = 3.23 nM) and 6 (IC50 = 7.12 nM)
show higher activity than selegiline (IC50 = 19.60 nM) and higher MAO-B selectivity, with more than
9050-fold, 30,960-fold and 14,045-fold inhibition levels, with respect to the MAO-A isoform.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 June 2010
Revised 2 July 2010
Accepted 3 July 2010
Available online 8 July 2010
Keywords:
Phenylcoumarins
MAOIs
Halogenation
Perkin reaction
Bromo coumarin derivatives
Coumarins are a large family of compounds, of natural and syn-
thetic origin, that presents different pharmacological activities.1
Due to their structural variability, they occupy an important place
in the realm of natural products and synthetic organic chemistry.
Representatives of these groups of compounds are found to occur
in the vegetable kingdom, either in the free or in the combined
state.2 Recent studies pay special attention to their antioxidative,
anticancer, and enzymatic inhibition properties.3–6 Some couma-
rins proved to be monoamine oxidase (MAO) inhibitors (MAOI).7
Recent findings revealed that MAO-A and MAO-B affinity and
selectivity can be efficiently modulated by appropriate substitu-
tions in the coumarin moiety. The 3/4 and 6/7 positions are partic-
ularly suitable to these modifications.8–14
MAOs are flavoenzymes (FAD-containing enzyme) bound to the
outer mitochondrial membrane of neuronal, glial, and other
cells.23,24 These enzymes are responsible for the oxidative deami-
nation of neurotransmitters and dietary amines.25,26 Two isoforms,
namely MAO-A and MAO-B, have been identified basis on their
amino acid sequences, three-dimensional structure, substrate pref-
erence and inhibitor selectivity.20,27 MAO-A has a higher affinity
for serotonin and noradrenaline, while MAO-B preferentially dea-
minates phenylethylamine and benzylamine.28 These properties
determine the clinical interest of MAOIs. Selective MAO-A inhibi-
tors, such as clorgyline (irreversible) and moclobemide (revers-
ible), are used for the treatment of neurological disorders, like
depression and anxiety,29,30 whilst selective and irreversible
MAO-B inhibitors, such as selegiline and rasagiline, are useful for
the treatment of Parkinson’s31,32 and Alzheimer’s diseases.33,34 As
the ideal drug candidate has not been attained, an intensive search
for new and innovative MAOIs is still needed. This effort has con-
siderably increased in recent years.11
In this context, and in an attempt to develop novel MAO-B
selective inhibitors, we had previously synthesized 3-arylcoumarin
derivatives in which both the coumarin and the resveratrol tem-
plates were present. These compounds proved to be potent and
selective MAO-B inhibitors.8,9 In this Letter, a subsequent project
was developed based on a 6-methyl-3-phenylcoumarin scaffold,
with a bromo pattern in both benzenic rings of the skeleton, and
with several methoxyl substituents located in the 3-phenyl ring
(Scheme 1).
Resveratrol, 3,40,5-trihydroxystilbene, is a natural polyphenolic
compound present in grapes and red wine.15 In in vitro, ex vivo,
and in vivo experiments resveratrol has shown important biologi-
cal activities including antiinflammatory, antioxidant, anticancer,
and cardioprotective properties, besides inhibitory activity to-
wards several enzymes.15–20
Therefore this compound has been attracting a huge interest
since the last decade. Recently, it has been demonstrated that res-
veratrol also has MAO inhibitory activity.16,21 To date, most phar-
macological studies have considered the trans isomer to be more
effective than the cis.22
* Corresponding author. Tel.: +34 981 528070.
E-mail address: mariacmatos@gmail.com (M.J. Matos).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.013

5158
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5157–5160
R
Me
Me
CHO
OH
Me
CHO
OH
(a)
(b)
O
O
Br
Br
1
2
4: R = H
5: R = 4'-OMe
6: R = 3',5'-OMe
(b)
7: R = 3',4',5'-OMe
Me
Br
O
O
3
Scheme 1. Reagents and conditions: (a) NBS, AIBN, CCl4, reflux, 18 h; (b) phenylacetic acids, DCC, DMSO, 110 °C, 24 h.
Table 1
MAO-A and MAO-B inhibitory activity results for compounds 3–7 and reference compounds
Compounds
MAO-A IC50 (nM)
MAO-B IC50 (nM)
Ratio
*
*
*
*
3
4
5
6
4.3 ꢁ 103 0.29 ꢁ 103
>23b
11.05 0.81
>9050b
>30,960b
>14,045b
6.4
3.23 0.49
7.12 0.01
7
31.20 ꢁ 103 2.09 ꢁ 103
67.25 ꢁ 103 1.02 ꢁ 103a
6.56 ꢁ 103 0.76 ꢁ 103
4.89 ꢁ 103 0.22 ꢁ 103
19.60 0.86
R-(ꢀ)-Deprenyl
3431
0.87
Iproniazide
7.54 ꢁ 103 0.36 ꢁ 103
*
Inactive at 100 lM (highest concentration tested). At higher concentrations the 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).
The coumarin derivatives 3–7 were efficiently synthesized
according to the synthetic protocol outlined in Scheme 1. The
experimental details are given in Ref. 35. The treatment of the pre-
cursor 1 with N-bromosuccinimide (NBS) in carbon tetrachloride
(CCl4) under reflux, using 2,20-azo-bis-iso-butyronitrile (AIBN) as
catalyst, afforded the bromo derivative 236 in a yield of 44%. The
preparation of the 8-bromo-6-methyl-3-phenylcoumarins (4–7)
was performed via the classical Perkin reaction.8,9,37–39 This reac-
tion occurs by condensation of the 3-bromo-5-methylsalicylalde-
hyde (2) and the conveniently substituted phenylacetic acids,
with N,N0-dicyclohexylcarbodiimide (DCC) as dehydrating agent,
in dimethyl sulfoxide (DMSO), at 110 °C and during 24 h (Scheme
1).8,9 The reaction is efficient and the compounds 4–7 were ob-
tained with yields between 45% and 50%. Compound 3 was pre-
pared in the same conditions described for compounds 4–7, but
starting from the 5-methylsalicylaldehyde 1 and the o-bromo-
phenylacetic acid.
The inhibitory MAO activity of compounds 3–7 was evaluated
in vitro by the measurement of the enzymatic activity of human
recombinant MAO isoforms in BTI insect cells infected with bacu-
lovirus.8,9,40 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).40–42
In the present communication, the effect of the introduction of a
halogen substituent into the 3-phenylcoumarin was studied. In
fact a superior MAOI activity, regarding the non-halogenated
compounds, was observed.7,8 It was shown that the introduction
of a bromo substituent enhance the MAO-B inhibitory properties
(potency and selectivity) of the recently described 6-methyl-3-
phenylcoumarin (IC50 = 284 nM).7
(IC50 = 4.3 lM). When compared with the 6-methyl-3-phenyl-
coumarin, compound 3 lost at least 15 times the MAO-B inhibitory
activity. The structural change obtained with compound 4, with
the halogen atom in the coumarin nucleus, leads to a significant in-
crease of the MAO-B activity (IC50 = 11.0 nM). In fact it was greater
than the IC50 found for 6-methyl-3-phenylcoumarin and com-
pound 3. So, as consequence, the MAO-B selectivity had increased
too (see Table 1). A change of the bromo atom position in the 3-
phenylcoumarin nucleus, from 20 to 8, was the other strategy per-
formed to improve the activity (compounds 5–7). Compound 5,
with a p-methoxy substituent in the 3-phenyl ring, was the most
potent and selective molecule, against MAO-B isoenzyme, of this
series, with an IC50 in the low nanomolar range (IC50 = 3.2 nM).
This compound is six times more active and to a great extent more
selective than the R-(ꢀ)-deprenyl (IC50 = 19.6 nM, reference MAO-
B inhibitor). Compound 6, with two methoxyl groups in the 30 and
50 positions, reveal also to be a potent MAOI-B. Its activity
(IC50 = 7.1 nM) is still better than the non-phenylsubstituted com-
pound 4. Compound 7, with three methoxyl groups, present a loss
of activity (activity in the micromolar range) and selectivity in re-
gard to the mono and dimethoxyl derivatives (compounds 5 and 6,
respectively). Compounds 3–6 do not exhibit MAO-A inhibitory
activity for the highest tested concentration (100 lM). The MAO
selectivity is an important factor to discriminate the potential
therapeutic application of this kind of molecules. In summary
one can say that the presence of a bromo atom in 8-position and
a restricted number of methoxyl substituents (one or two) in the
3-phenyl ring seems to be important chemical features to
modulate and improve the inhibitory enzymatic activity of the
6-methyl-3-phenylcoumarins.
As it is shown in the Table 1, compound 3, with the halogen
atom in the 3-phenyl ring, has an IC50 in the micromolar range
In conclusion, in the present study it was shown that the
synthesized resveratrol–coumarin hybrid compounds have high

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5157–5160
32. Riederer, P.; Danielczyk, W.; Grunblatt, E. Neurotoxicology 2004, 25, 271.
5159
selectivity for the MAO-B isoenzyme. Most of them present MAO-B
inhibitory activity is in the low nanomolar range. The presence of a
bromo atom in position 8 of the coumarin improves the activity re-
spect to the presence of a bromo atom linked to the 3-phenyl ring.
The introduction of one para-methoxy group in the 3-phenyl ring
of the 8-bromo-6-methyl-3-phenylcoumarin improve to a great
extent the MAO-B inhibitory activity respect to the other prepared
derivatives. In fact, the introduction of a bromo atom improves the
pharmacologic potential of the 6-methyl-3-phenylcoumarins con-
firming that this lead could be effectively optimized in a candidate
for the treatment of neurodegenerative diseases. These finds have
encouraged us to continue the efforts towards the optimization of
the pharmacologic profile of 6-methyl-3-phenylcoumarin.
33. Youdim, M. B. H.; Fridkin, M.; Zheng, H. J. Neural Transm. 2004, 111, 1455.
34. Cesura, A. M.; Pletscher, A. Prog. Drug Res. 1992, 38, 171.
35. 3-(2-Bromophenyl)-6-methylcoumarin (3):
A
solution of 2-hydroxy-6-
methylbenzaldehyde (1, 1.0 g, 7.34 mmol) and 2-bromophenylacetic acid
(1.98 g, 9.18 mmol) in DMSO and DCC (2.36 g, 11.46 mmol) was heated in an
oil-bath at 110 °C for 24 h. Triturate ice (100 mL) and acetic acid (10.0 mL)
were added to the reaction mixture. After keeping it at room temperature for
2 h, the mixture was extracted with ether (3 ꢁ 25 mL). The organic layer was
extracted 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 3 (1.36 g, 59%) as a white
solid. Mp 141–142 °C. 1H NMR (CDCl3) d (ppm): 2.45 (s, 3H, –CH3), 7.28 (m, 1H,
H-40); 7.35 (m, 2H, H-7, H-8); 7.40 (m, 3H, H-5, H-50, H-60); 7.69 (d, 1H, H-30);
7.71 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm): 20.8, 116.4, 118.7, 123.6, 127.4,
127.9, 128.6, 130.1, 131.3, 132.9, 133.0, 134.3, 135.9, 142.6, 152.0, 160.0. MS m/
z (%): 316 (5), 315 (38), 314 (M+, 100), 236 (32), 235 (80), 178 (22), 117 (7), 89
(7), 76 (9). Anal. Calcd for C16H11BrO2: C, 60.98; H, 3.52. Found: C, 60.92; H,
3.47.
Acknowledgments
General procedure for the preparation of 8-bromo-6-methyl-3-phenylcoumarins
(4–7):
A
solution of 3-bromo-2-hydroxy-6-methylbenzaldehyde (2,
0.56 mmol) and the correspondent phenylacetic acid (0.70 mmol) in DMSO
and DCC (0.87 mmol) was heated in an oil-bath at 110 °C for 24 h. Triturate ice
(20 mL) and acetic acid (3.0 mL) were added to the reaction mixture. After
keeping it at room temperature for 2 h, the mixture was extracted with ether
(3 ꢁ 25 mL). The organic layer was extracted 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 a white solid.
Thanks to the Spanish Ministry(PS0900501), to Xunta da Galicia
(PGIDIT09CSA030203PR and INCITE09E2R203035ES) and to FCT
(PTDC/QUI/70359/2006) for financial support. M.J.M. also thanks
FCT for a Ph.D. Grant (SFRH/BD/61262/2009).
References and notes
8-Bromo-6-methyl-3-phenylcoumarin (4): It was obtained with a yield of 45%.
Mp 158–159 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 2.41 (s, 3H, –CH3), 7.28 (d, 1H,
H-7, J = 2.3), 7.45 (m, 3H, H-30, H-40, H-50), 7.58 (m, 1H, H-5), 7.70 (m, 3H, H-4,
H-20, H-60). 13C NMR (CDCl3) d (ppm): 20.5, 109.4, 120.5, 127.1, 128.5, 129.0,
129.1, 134.3, 135.2, 135.6, 139.3, 148.3, 159.7. MS m/z (%): 316 (17), 315 (42),
314 (M+, 100), 313 (99), 288 (36), 286 (15), 285 (36), 207 (30), 178 (26), 89
(10), 76 (10). Anal. Calcd for C16H11BrO2: C, 60.98; H, 3.52. Found: C, 61.01; H,
3.56.
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.
5. Carotti, A.; Altomare, C.; Catto, M.; Gnerre, C.; Summo, L.; De Marco, A.; Rose, S.;
Jenner, P.; Testa, B. Chem. Biodivers. 2006, 3, 134.
8-Bromo-6-methyl-3-(40-methoxyphenyl)coumarin (5): It was obtained with a
yield of 47%. Mp 144–145 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 2.40 (s, 3H, –
CH3), 3.87 (s, 3H, –OCH3), 6.98 (d, 2H, H-30, H-50, J = 7.1), 7.26 (s, 1H, H-7), 7.56
(s, 1H, H-5), 7.67 (dd, 3H, H-4, H-20, H-60, J = 6.9 and J = 1.1). 13C NMR (CDCl3) d
(ppm): 20.5, 55.4, 109.3, 114.0, 120.7, 126.7, 126.9, 128.5, 129.9, 135.2, 137.8,
148.1, 159.9, 160.3. MS m/z (%): 346 (99), 345 (15), 344 (M+, 100), 303 (53), 301
(54), 275 (15), 207 (15), 166 (17), 165 (72), 138 (17), 89 (13), 76 (14), 58 (41).
Anal. Calcd for C17H13BrO3: C, 59.15; H, 3.80. Found: C, 59.17; H, 3.86.
8-Bromo-6-methyl-3-(30,50-dimethoxyphenyl) coumarin (6): It was obtained
with a yield of 46%. Mp 165–166 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 2.40 (s,
3H, –CH3), 3.83 (s, 6H, (–OCH3)2), 6.51 (t, 1H, H-40 J = 2.3), 6.83 (d, 2H, H-20, H-
60, J = 2.3), 7.26 (s, 1H, H-7), 7.57 (s, 1H, H-5), 7.70 (s, 1H, H-4). 13C NMR (CDCl3)
d (ppm): 21.0, 56.0, 101.6, 107.2, 109.9, 120.8, 127.6, 129.2, 135.7, 136.1, 136.6,
140.0, 148.7, 160.0, 161.2. MS m/z (%): 376 (99), 375 (26), 374 (M+, 100), 238
(37), 209 (14), 181 (36), 165 (30), 153 (18), 152 (41), 151 (17), 126 (15), 76
(22). Anal. Calcd for C18H15BrO4: C, 57.62; H, 4.03. Found: C, 57.63; H, 3.98.
8-Bromo-6-methyl-3-(30,40,50-trimethoxyphenyl) coumarin (7): It was obtained
with a yield of 50%. Mp 167–168 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 2.41 (s,
3H, –CH3), 3.91 (d, 9H, (–OCH3)3, J = 5.8), 6.92 (s, 2H, H-20, H-60), 7.28 (d, 1H, H-
7, J = 6.5), 7.59 (d, 1H, H-5, J = 1.3), 7.70 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm):
20.5, 56.1, 56.3, 60.9, 106.0, 106.2, 109.4, 120.4, 127.0, 128.7, 129.7, 135.3,
135.6, 138.9, 148.1, 153.1, 159.7. MS m/z (%): 407 (21), 406 (99), 405 (21), 404
(M+, 100), 391 (56), 389 (56), 363 (19), 361 (19), 303 (21), 247 (13), 187 (15),
186 (15), 139 (19). Anal. Calcd for C19H17BrO5: C, 56.31; H, 4.23. Found: C,
56.28; H, 4.19.
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.
7. Rendenbach-Müller, B.; Traut, M.; Weifenbach, H. Bioorg. Med. Chem. Lett. 1994,
4, 1195.
8. Matos, M. J.; Viña, D.; Quezada, E.; Picciau, C.; Delogu, G.; Orallo, F.; Santana, L.;
Uriarte, E. Bioorg. Med. Chem. Lett. 2009, 19, 3268.
9. Matos, M. J.; Viña, D.; Picciau, C.; Orallo, F.; Santana, L.; Uriarte, E. Bioorg. Med.
Chem. Lett. 2009, 19, 5053.
10. Santana, L.; Uriarte, E.; González-Díaz, H.; Zagotto, G.; Soto-Otero, R.; Méndez-
Álvarez, E. J. Med. Chem. 2006, 49, 1118.
11. Santana, L.; González-Díaz, H.; Quezada, E.; Uriarte, E.; Yáñez, M.; Viña, D.;
Orallo, F. J. Med. Chem. 2008, 51, 6740.
12. Catto, M.; Nicolotti, O.; Leonetti, F.; Carotti, A.; Favia, A.; Soto-Otero, R.;
Méndez-Álvarez, E.; Carotti, A. J. Med. Chem. 2006, 49, 4912.
13. Gnerre, C.; Catto, M.; Leonetti, F.; Weber, P.; Carrupt, P.; Altomare, C.; Carotti,
A.; Testa, B. J. Med. Chem. 2000, 43, 4747.
14. 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.
15. Frémont, L. Life Sci. 2000, 66, 663.
16. 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.
17. Orallo, F. In Resveratrol in Health and Disease; Aggarwal, B. B., Shishodia, S., Eds.;
CRC Press: USA, 2005; p 577.
18. Leiro, J.; Álvarez, E.; Arranz, J.; Laguna, R.; Uriarte, E.; Orallo, F. J. Leukocyte Biol.
2004, 75, 1156.
36. Li, W.; Huanwang, J.; Xiuli, B.; Tao, C.; Lili, J.; Yongmin, L. Catal. Commun. 2007,
8, 80.
19. Orallo, F. Curr. Med. Chem. 2008, 15, 1887.
20. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.
21. Binda, C.; Li, M.; Hubalek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 9750.
37. Hans, N.; Singhi, M.; Sharma, V.; Grover, S. K. Indian J. Chem., Sect B 1996, 35B,
1159.
38. Mohanty, S.; Makrandi, J. K.; Grover, S. K. Indian J. Chem., Sect B 1989, 28B, 766.
´
39. Kamat, S. P.; DSouza, A. M.; Paknikar, S. K.; Beaucahmp, P. S. J. Chem. Res., Synop.
2002, 242.
22. Yánez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Commun. 2006,
344, 688.
23. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.
E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.
24. Novaroli, L.; Daina, A.; Favre, E.; Bravo, J.; Carotti, A.; Leonetti, F.; Catto, M.;
Carrupt, P.; Reist, M. J. Med. Chem. 2006, 49, 6264.
40. Determination of human monoamine oxidase (hMAO) isoform activity: The effects
of the test compounds on hMAO isoform enzymatic activity were evaluated by
a fluorimetric method. 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/
25. Dostert, P.; Strolin Benedetti, M.; Jafre, M. In Monoamine Oxidase: Basic and
Clinical Frontiers; Kamijo, K., Usdin, E., Nagausu, T., Eds.; Excerpta Medica:
Amsterdam, 1982; p 197.
26. Singer, T. P., Muller, F., Eds.; CRC Press: London, 1991; p 437.
27. Binda, C.; Li, M.; Hubálek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 9750.
28. Yamada, M.; Yasuhara, H. Neurotoxicology 2004, 25, 11.
29. Rudorfer, M. V.; Potter, V. Z. Drugs 1989, 37, 713.
30. Palhagen, S.; Heinonen, E.; Hagglund, J.; Kaugesaar, T.; Maki-Ikola, O.; Palm, R.
Neurology 2006, 66, 1200.
min (hMAO-A: 1.1
oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5
lg protein; specific activity: 150 nmol of p-tyramine
l
g
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)
200
tyramine. The production of H2O2 and, consequently, of resorufin was
l
M AmplexÒ Red reagent, 1 U/mL horseradish peroxidase and 1 mM p-
quantified at 37 °C in
a multidetection microplate fluorescence reader
(FLX800™, Bio-TekÒ Instruments, Inc., Winooski, VT, USA) based on the
fluorescence generated (excitation, 545 nm, emission, 590 nm) over a 15 min
period, in which the fluorescence increased linearly. Control experiments were
31. Guay, D. R. Am. J. Geriatr. Pharmacother. 2006, 4, 330.

5160
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5157–5160
carried out simultaneously by replacing the test drugs with appropriate
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).
41. All IC50 values shown in the table are expressed as means SEM from five
experiments.
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
42. Yáñez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Commun. 2006,
344, 688.