MAO inhibitory activity modulation: 3-Phenylcoumarins versus 3-benzoylcoumarins

Article abstract of DOI:10.1016/j.bmcl.2011.05.074

With the aim of finding the structural features for the human MAO inhibitory activity and selectivity, in the present communication we report the synthesis, pharmacological evaluation and a comparative study of a new series of 3-phenylcoumarins (compounds

Full text of DOI:10.1016/j.bmcl.2011.05.074

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4224–4227
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
MAO inhibitory activity modulation: 3-Phenylcoumarins versus
3-benzoylcoumarins
Maria João Matos a, Saleta Vazquez-Rodriguez a, Eugenio Uriarte a, Lourdes Santana a, Dolores Viña b,
⇑
a Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
b Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, 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:
With the aim of finding the structural features for the human MAO inhibitory activity and selectivity, in
the present communication we report the synthesis, pharmacological evaluation and a comparative
study of a new series of 3-phenylcoumarins (compounds 1–4) and 3-benzoylcoumarins (compounds
5–8). A bromo atom and a methoxy/hydroxy substituent were introduced in these scaffolds, at six and
eight positions of the coumarin moiety, respectively. The synthesized compounds 1–8 were evaluated
as MAO-A and B inhibitors using R-(À)-deprenyl and iproniazide as reference compounds. The presence
or absence of a carbonyl group between the coumarin and the phenyl substituent in 3 position remarks,
respectively, the MAO-A or MAO-B inhibitory activity. Some of the new compounds showed MAO-B
inhibitory activities in the low nanomolar range. Compound 2 (IC50 = 1.35 nM) showed higher inhibitory
activity than the R-(À)-deprenyl (IC50 = 19.60 nM) and higher MAO-B selectivity, with more than 74,074-
fold inhibition level, respecting to the MAO-A isoform.
Received 5 April 2011
Revised 18 May 2011
Accepted 20 May 2011
Available online 30 May 2011
Keywords:
Perkin reaction
Knoevenagel reaction
3-Phenylcoumarins
3-Benzoylcoumarins
MAOI activity
Comparative study
Ó 2011 Elsevier Ltd. All rights reserved.
Coumarins, chalcone and their natural and/or synthetic deriva-
tives are biologically interesting compounds because of their struc-
tural diversity. Due to this variability, these heterocyclic
compounds occupy an important role not only in the Organic
Chemistry but also in the Medicinal Chemistry realm.1–6 They are
described as anticancer, anti-inflammatory, antimicrobial, and
antioxidant agents.7–16 A number of studies pay special attention
to coumarin derivatives as monoamine oxidase (MAO)17–23 inhibi-
tors. Recently, chalcone structure has also been identified as a valid
scaffold for monoamine oxidase inhibitors (MAOI).24 Therefore, re-
cent findings reveal that MAO-A and MAO-B affinity and selectivity
can be efficiently modulated by appropriate substitutions in differ-
ent positions of the coumarin and chalcone moiety (Fig. 1).19,25–27
MAO is a FAD-containing enzyme (flavoenzyme) bound to the
outer mitochondrial membrane in neuronal, glial and many other
cells.28,29 Two isoforms namely as MAO-A and MAO-B have been
identified based on their amino acid sequences, three-dimensional
structure, substrate preference and inhibitor selectivity.30,31 These
isoenzymes are responsible for the oxidative deamination of neu-
rotransmitters and dietary amines. Therefore, they are responsible
for the regulation of intracellular levels of biogenic amines in the
brain and the peripheral tissues.32,33 MAO-B preferentially deami-
nates phenylethylamine and benzylamine, while MAO-A has a
higher affinity for noradrenaline and serotonin.34,35 Despite of
these differences, dopamine and tyramine are common substrates
for both isoforms. These properties determine the pharmacological
interest of MAOIs. Selective and irreversible MAO-B inhibitors,
such as selegiline (R-(À)-deprenyl) and rasagiline are useful com-
pounds for the treatment of Parkinson36,37 and Alzheimer’s
diseases.38,39 Selective MAO-A inhibitors, such as clorgyline
(irreversible) and moclobemide (reversible), are useful for the
treatment of neurological disorders, such as depression and anxi-
ety.40,41 All these findings have led us to an intensive search for
novel, selective and efficient MAO inhibitors.
With the aim of finding novel and selective MAO-B inhibitors,
we have previously synthesized 3-arylcoumarin derivatives in
which both the coumarin nucleus and a 3-phenyl ring were differ-
ently substituted. The experimental data show that those com-
pounds are potent and selective MAO-B inhibitors.20–23 In
particular, the 6,8-disubstituted coumarins proved to be very inter-
esting derivatives.22 Based on the previous 3-phenylcoumarins
experimental results, in this paper we describe a new project with
a comparative study between 3-phenylcoumarins (compounds
1–4) and 3-benzoylcoumarins (compounds 5–8), which are
O
O
O
⇑
Corresponding author.
E-mail address: mdolores.vina@usc.es (D. Viña).
Figure 1. Chemical structures of coumarin and trans-chalcone.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.074

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4224–4227
4225
R3
Starting from the same salicylaldehyde, we can afford two series,
differing just in the presence (compounds 5–8) or absence (com-
pounds 1–4) of a carbonyl group between the phenyl substituent
at the 3 position and the coumarin scaffold.
The inhibitory MAO activity of compounds 1–8 was evaluated
in vitro by the measurement of the enzymatic activity of human re-
combinant MAO isoforms expressed in BTI insect cells infected
with baculovirus.51 Subsequently, the IC50 values and MAO-B
selectivity indexes [IC50 (MAO-A)]/[IC50 (MAO-B)] for inhibitory ef-
fects of both new types of compounds and reference inhibitors
were calculated (Table 1).51–53
R3
R1
COOH
(a)
O
O
R2
1: R1 = Br, R2 = OMe, R3 = H
2: R1 = Br, R2 = R3 = OMe
R1
CHO
(b)
OH
3: R1 = Br, R2 = OH, R3 = H
4: R1 = Br, R2 = R3 = OH
R2
In the present communication, the effect of the presence or the
absence of a carbonyl group between the coumarin and the 3-phe-
nyl ring is studied. Substituents and their positions in the 3-phe-
nylcoumarin nucleus have been selected based on previous
results which showed very high MAOI activity for some deriva-
tives. It was shown that introduction of both bromo and methoxy
substituents at 6 and 8 positions respectively (compounds 1 and 2)
enhances the MAO-B inhibitory properties (potency and selectiv-
ity) of the described 3-phenylcoumarins, comparing to some of
the other previously described compounds.20–22 In addition, the
introduction of a substituent in the para position of the 3-phenyl
ring might help to improve the activity. Consequently, when this
substituent is a methoxy group (compound 2), the MAO-B inhibi-
tory activity and selectivity improve in a great extent respecting
to the other derivatives. However, replacement of the methoxy
groups for hydroxyl groups at the indicated positions decreases
the activity (compounds 3 and 4) validating the previous informa-
tion we already had21 and it can even decrease selectivity. On the
other hand, when we analyze the second series where a 3-benzoyl
group has replaced the 3-phenyl substituent, the introduced car-
bonyl group decreases the MAOI activity against B isoform. The
6-bromo-8-hydroxy derivatives 7 and 8 are more active than the
corresponding 6-bromo-8-methoxy ones (compounds 5 and 6),
being the structure activity relationship just the opposite of the
3-phenylcoumarins. Compounds 5 and 6 do not present any MAO
O
O
O
OEt
R1
R3
(c)
O
O
R3
R2
5: R1 = Br, R2 = OMe, R3 = H
6: R1 = Br, R2 = R3 = OMe
(d)
7: R1 = Br, R2 = OH, R3 = H
8: R1 = Br, R2 = R3 = OH
Scheme 1. Reagents and conditions: (a) DCC, DMSO, 110 °C, 24 h; (b) HI, AcOH,
Ac2O, 0 °C to reflux, 4 h; (c) ethanol, piperidine, reflux, 2–5 h; (d) BBr3, DCM, 80 °C,
48 h.
interesting semi-rigid chalcones with the
bond included in the coumarin skeleton (Scheme 1).
a,b-unsaturated double
The 6-bromo-8-methoxycoumarins42 were efficiently synthe-
sized by Perkin43–45 (1 and 2) and Knoevenagel46,47 (5 and 6) reac-
tions. The hydroxy derivatives (3, 4, 7 and 8)42 were obtained by
two different hydrolysis reactions,48–50 according to the synthetic
protocol outlined in Scheme 1. Treatment of the corresponding sal-
icylaldehyde and the conveniently substituted phenylacetic acid
with N,N’-dicyclohexylcarbodiimide (DCC) as dehydrating agent,
in dimethyl sulfoxide (DMSO) at 110 °C during 24 h, afforded the
3-phenylcoumarins 1 and 2. The consequent hydrolysis of 1 and
2 in acetic acid and acetic anhydride, with hydriodic acid 57%, for
4 h, yielded the hydroxy derivatives 3 and 4.42 The synthesis of
the 3-benzoylcoumarins 5 and 6 was performed via condensation
of the conveniently substituted salicylaldehyde with the corre-
sponding b-ketoester, in ethanol at reflux temperature for 5 or
2 h respectively, using piperidine as basic catalyst. The resulting
methoxy derivatives were treated with boron tribromide at 80 °C
for 48 h, to give the corresponding hydroxy derivatives 7 and 8.
inhibitory activity at the higher tested concentration (100 lM).
However, compounds 7 and 8 increase the affinity for the MAO-A
receptor, losing the MAO-B selectivity of the first series. A small
change in the structure causes a big change in the affinity of the
molecules for the receptor. These preliminary results allow us to
understand slightly better interactions between molecule and
receptor and the molecular fragments that are essential to main-
tain and improve the MAO activity and selectivity.
As conclusion, it was verified an important lost of the MAO-B
inhibitory activity and selectivity when the 3-phenyl skeleton is
substituted for the 3-benzoyl one. However, in some of the 3-ben-
zoyl derivatives it was shown not only inactivity against MAO-B
Table 1
MAO-A and MAO-B inhibitory activity results for the synthesized compounds 1–8 and reference inhibitors
Compounds
MAO-A IC50
(lM)
MAO-B IC50
(l
M)
Selectivity Index
*
*
*
1
2
3
83.48x10À3 5.60x10À3
1.35x10À3 0.09x10À3
30.91 2.09
>1,197b
>74,074b
>3.2b
4
5
6
7
20.74 1.40
16.87 1.14
1.2
*
*
—
*
*
—
46.81 3.18a
19.17 1.29
67.25 1.02a
6.56 0.76
73.92 4.99
0.63
**
8
0.19c
3,431
0.87
R-(À)-Deprenyl
19.60x10À3 0.86x10À3
7.54 0.36
Iproniazide
*
Inactive at 100
l
M (highest concentration tested). At higher concentrations compound precipitate.
**
100
lM inhibits enzymatic activity around (by approximately) 45–50%. At higher concentrations compound precipitate.
a
b
c
P < 0.05 versus the corresponding IC50 values obtained against MAO-B, as determined by ANOVA/Dunnett’s.
Values obtained under the assumption that the corresponding IC50 against MAO-A is the highest concentration tested (100
l
M).
Value obtained under the assumption that the corresponding IC50 against MAO-B is the highest tested concentration (100
l
M).

4226
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4224–4227
29. Novaroli, L.; Daina, A.; Favre, E.; Bravo, J.; Carotti, A.; Leonetti, F.; Catto, M.;
Carrupt, P.; Reist, M. J. Med. Chem. 2006, 49, 6264.
30. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.
31. Binda, C.; Li, M.; Hubálek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U. S. A. 2003, 100, 9750.
32. 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.
33. Singer, T. P. F. Muller (Ed.); CRC Press: London, 1991. p. 437.
34. Ma, J.; Yoshimura, M.; Yamashita, E.; Nakagawa, A.; Ito, A.; Tsukihara, T. J. Mol.
Biol. 2004, 338, 103.
35. Weyler, W.; Hsu, Y. P.; Breakefield, X. O. Pharmacol. Ther. 1990, 47, 391.
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isoenzyme, but showed MAO-A inhibitory activity and selectivity.
Therefore, selectivity seems to depend on the nature of the couma-
rins’ substituent. In the present study it was shown that 6-bromo-
8-methoxy-3-phenylcoumarins are an interesting scaffold for
MAO-B inhibitory studies, whereas the 6-bromo-8-hydroxy-3-
benzoylcoumarins are an interesting moiety for MAO-A inhibitory
ones. Compound 2, with a p-methoxy substituent in the 3-phenyl
ring was the most potent and selective molecule of the first series
against MAO-B isoenzyme, with an IC50 in the low nanomolar
range (IC50 = 1.35 nM). This compound is fifteen times more active
and several times more selective than the R-(À)-deprenyl
(IC50 = 19.6 nM, reference MAO-B inhibitor). The MAO selectivity
is an important factor to discriminate the different potential ther-
apeutic applications of these molecules. These findings encourage
us to continue the efforts towards the optimization of the pharma-
cological profile of these structural types as important scaffolds in
the neurodegenerative diseases realm.
41. Palhagen, S.; Heinonen, E.; Hagglund, J.; Kaugesaar, T.; Maki-Ikola, O.; Palm, R.
Neurology 2006, 66, 1200.
42. General procedure for the preparation of 3-phenylcoumarins (1 and 2):
a
solution of the conveniently substituted salicylaldehyde (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 x 25 mL). The
organic layer was extracted with sodium bicarbonate solution (50 mL, 5%)
and then with 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 desired compound.
6-Bromo-8-methoxy-3-phenylcoumarin (1): it was obtained with a yield of
50%. Mp 153–154 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 3.97 (s, 3H, –OCH3),
7.16 (d, 1H, H-7, J = 2.0), 7.26 (d, 1H, H-5, J = 2.0), 7.43–7.48 (m, 3H, H-3’, H-
4’, H-5’), 7.68–7.73 (m, 3H, H-2’, H-6’, H-4). 13C NMR (CDCl3) d (ppm): 57.2,
117.0, 117.3, 121.9, 122.1, 129.2, 129.8, 130.3, 134.8, 139.1, 142.8, 148.3,
160.0. MS m/z (%): 332 (99), 331 (30), 330 (M+, 100), 304 (40), 302 (40), 261
(25), 259 (26), 194 (16), 165 (12), 153 (14), 152 (88), 151 (23), 102 (19), 76
(34). Anal. Calcd for C16H11BrO3: C, 58.03; H, 3.35. Found: C, 58.01; H, 3.30.
6-Bromo-8-methoxy-3-(4’-methoxyphenyl)coumarin (2): it was obtained with
a yield of 53%. Mp 184–185 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 3.85 (s, 3H,
–OCH3), 3.96 (s, 3H, –OCH3), 6.93–6.96 (m, 2H, H-3’, H-5’), 7.12 (d, 1H, H-7,
J = 1.8), 7.23 (d, 1H, H-5, J = 2.0), 7.63–7.67 (m, 2H, H-2’, H-6’), 7.69 (s, 1H,
H-4). 13C NMR (CDCl3) d (ppm): 55.7, 56.8, 114.2, 116.2, 116.8, 121.5, 126.8,
129.4, 130.2, 130.8, 137.3, 142.2, 147.8, 159.8, 160.6. MS m/z (%): 363 (19),
362 (M+, 100), 361 (19), 360 (59), 334 (24), 332 (23), 319 (33), 317 (34), 291
(11), 289 (11), 182 (18), 167 (17), 139 (21), 91 (11). Anal. Calcd for
Acknowledgments
Thanks to the Spanish Ministry (PS09/00501) and to Xunta de
Galicia (PGIDIT09CSA030203PR and 10PXIB203303PR). M.J.M.
thanks FCT for a PhD grant (SFRH/BD/61262/2009). S.V.R. thanks
to Ministerio de Educación y Ciencia for a PhD grant (AP2008-
04263).
References and notes
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C
17H13BrO4: C, 56.53; H 3.63. Found: C, 56.55; H, 3.68.
General procedure for the preparation of hydroxylated 3-phenylcoumarins (3
and 4). solution of the corresponding methoxy-3-phenylcoumarin
a
(0.50 mmol) in acetic acid (5.0 mL) and acetic anhydride (5.0 mL), at 0 °C,
was prepared. Hydriodic acid 57% (10.0 mL) was added dropwise. The
mixture was stirred under reflux temperature for 3 h. The solvent was
evaporated under vacuum and the dry residue was purified by
recrystallization with CH3CN.
6-Bromo-8-hydroxy-3-phenylcoumarin (3): it was obtained with
a yield of
61%. Mp 160–161 °C. 1H NMR (DMSO-d6) d (ppm), J (Hz): 7.19 (d, 1H, H-7,
J = 2.0), 7.43–7.49 (m, 4H, H-5, H-3’, H-4’, H-5’), 7.70 (dd, 2H, H-2’, H-6’,
J = 7.6, J = 1.6), 8.13 (s, 1H, H-4), 10.79 (s, 1H, -OH). 13C NMR (DMSO-d6) d
(ppm): 115.5, 119.9, 120.4, 121.8, 127.9, 128.2, 128.5, 128.8, 134.4, 139.7,
141.1, 145.6, 159.1. MS m/z (%): 319 (16), 318 (99), 317 (17), 316 (M+, 100),
291 (12), 290 (78), 289 (13), 288 (81), 153 (22), 152 (51), 151 (12), 76 (25).
Anal. Calcd for C15H9BrO3: C, 56.81; H, 2.86. Found: C, 56.78; H, 2.82.
6-Bromo-8-hydroxy-3-(4’-hydroxyphenyl)coumarin (4): It was obtained with a
yield of 53%. Mp 249–250 °C. 1H NMR (DMSO-d6) d (ppm), J (Hz): 6.83 (d,
2H, H-3’, H-5’, J = 8.8), 7.14 (t, 1H, H-7, J = 1.5), 7.36 (d, 1H, H-5, J = 2.0), 7.56
(d, 2H, H-2’, H-6’, J = 8.5), 8.00 (s, 1H, H-4). 13C NMR (DMSO-d6) d (ppm):
115.6, 116.0, 119.9, 120.6, 122.5, 125.4, 128.2, 130.4, 138.0, 141.2, 146.0,
158.6, 159.8. MS m/z (%): 335 (35), 334 (99), 333 (45), 332 (M+, 100), 307
(31), 306 (99), 305 (35), 304 (99), 225 (26), 197 (29), 169 (35), 168 (46), 153
(24), 141 (21), 140 (16), 139 (51), 118 (17), 115 (28), 98 (13), 89 (14), 84
(18), 84 (46), 75 (11), 63 (13). Anal. Calcd for C15H9BrO4: C, 54.08; H, 2.72.
Found: C, 54.00; H 2.69.
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General procedure for the preparation of 3-benzoylcoumarins (5 and 6). To a
solution of the appropriate b-ketoester and the corresponding
salicylaldehyde in ethanol was added piperidine in catalytic amount. The
reaction mixture was refluxed for 2–5 h and after completion, the reaction
was cooled and the precipitated was filtered and washed with cold ethanol
and ether to afford the desired compound. Compounds were further
recrystallized in methanol/CH2Cl2.
6-Bromo-8-methoxy-3-benzoylcoumarin (5): it was obtained with a yield of 49%.
Mp 207–208 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 3.82 (s, 3H, –OCH3), 7.09 (s, 1H,
H-7), 7.14 (s, 1H, H-5), 7.24–7.51 (m, 3H, H-3’, H-4’, H-5’), 7.69 (d, 2H, H-2’, H-6’,
J = 7.3), 7.78 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm): 57.2, 116.6, 118.6, 120.5,
123.0, 128.0, 129.2, 130.0, 134.5, 136.3, 143.3, 144.6, 147.8, 157.8, 191.9. MS m/z
(%): 360 (18), 359 (M+, 58), 358 (18), 357 (58), 105 (100), 77 (70). Anal. Calcd for
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Méndez-Álvarez, E.; Carotti, A. J. Med. Chem. 2006, 49, 4912.
28. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.
E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.
C
17H11BrO4: C, 56.85; H, 3.09; Found: C, 56.79; H, 3.06.

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4224–4227
4227
´
6-Bromo-8-methoxy-3-(4-methoxybenzoyl)coumarin (6): It was obtained with
48. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yanez, M.; Fraiz, N.; Alcaide, C.;
Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.
a yield of 90%. Mp 224–225 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 3.71 (s, 3H, –
OCH3), 3.82 (s, 3H, –OCH3), 6.77 (d, 2H, H-3’, H-5’, J = 8.9), 7.14–7.16 (m, 2H, H-5,
H-7), 7.69 (d, 2H, H-2’, H-6’, J = 8.9), 7.72 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm):
55.5, 56.1, 92.8, 95.2, 103.8, 113.7, 121.2, 129.7, 132.1, 141.5, 157.9, 158.3, 159.2,
163.8, 165.8, 190.6. MS m/z (%): 389 (87), 388 (17), 387 (M+, 90), 135 (100), 77
(56). Anal. Calcd for C18H13BrO5: C, 55.55; H, 3.37. Found: C, 55.53; H, 3.34.
General procedure for the preparation of hydroxylated 3-benzoylcoumarins (7 and
8). To the corresponding methoxy-3-benzoylcoumarin in DCM (1 mmol), BBr3 in
DCM (20 mmol) was added in a Schlenk tube. Tube was sealed, and the reaction
mixture was heated at 80 °C for 48 h. The resulting crude was treated with MeOH
and rotated to dryness. The obtained precipitated was recrystallized in MeOH to
afford the desired hydroxy derivative.
49. Martín-Santamaría, S.; Rodríguez, J. J.; de Pascual-Teresa, S.; Gordon, S.;
Bengtsson, M.; Garrido-Laguna, I.; Rubio-Viqueira, B.; López-Casas, P. P.;
Hidalgo, M.; de Pascual-Teresa, B.; Ramos, A. Org. Bio. Chem. 2008, 6, 3486.
50. Albrecht, M.; Mirtschin, S.; de Groot, M.; Janser, I.; Runsink, J.; Raabe, G.; Kogej,
M.; Schalley, C. A.; Fröhlich, R. J. Am. Chem. Soc. 2005, 127, 10371.
51. Determination of human monoamine oxidase (hMAO) isoform activity. The
effects of the tested 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 tested drugs in several 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 mg protein; specific activity: 150 nmol of p-
tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-
B: 7.5 mg 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)
6-Bromo-8-hydroxy-3-benzoylcoumarin (7): it was obtained with a yield of 98%.
Mp 240–242 °C. 1H NMR (DMSO-d6) d (ppm), J (Hz): 7.29 (d, J = 2.2 Hz, 1H, H-7),
7.61–7.39 (m, 3H, H-3’, H-4’, H-5’), 7.76–7.67 (m, 1H, H-5), 7.93 (d, 2H, H-2’, H6’,
J = 8.0), 8.30 (s, 1H, H-4), 10.92 (s, 1H, -OH). 13C NMR (DMSO-d6) d (ppm): 39.3,
39.5, 39.7, 39.9, 40.1, 40.4, 40.6, 116.2, 120.9, 121.8, 122.0, 127.8, 129.2, 130.0,
134.5, 136.3, 142.7, 144.8, 146.3, 157.9, 191.9. MS m/z (%): 345 (46), 343 (M+, 45),
105 (100), 77 (58). Anal. Calcd for C16H9BrO4: C, 55.68; H, 2.63. Found: C, 55.63;
H, 2.59.
200
tyramine. The production of H2O2 and, consequently, of resorufin was
quantified at 37 °C in multidetection microplate fluorescence reader
lM AmplexÒ Red reagent, 1 U/mL horseradish peroxidase and 1 mM p-
a
´
6-Bromo-8-hydroxy-3-(4-hydroxybenzoyl)coumarin (8): it was obtained with a
(FLX800TM, 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
carried out simultaneously by replacing the tested drugs with appropriate
dilutions of the vehicles. In addition, the possible capacity of the above tested
drugs for modifying 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
yield of 40%. Mp 293–295 °C. 1H NMR (DMSO-d6) d (ppm), J (Hz): 6.86 (d, 2H, H-
3’, H-5’, J = 8.7), 7.27 (d, 1H, H-7, J = 2.2), 7.46 (d, 1H, H-5 J = 2.2), 7.82 (d, 2H, H-2’,
H-6’ J = 8.7), 8.18 (s, 1H, H-4), 10.62 (s, 1H, -OH), 10.89 (s, 1H, -OH). 13C NMR
(DMSO-d6) d (ppm): 115.9, 116.2, 121.0, 121.5, 121.6, 127.6, 128.5, 133.0, 142.7,
143.5, 146.3, 157.9, 163.6, 190.0. MS m/z (%): 362 (29), 360 (M+, 30), 121 (100), 93
(16), 65 (15). Anal. Calcd for C16H9BrO5: C, 53.21; H, 2.51. Found: C, 52.97; H,
4.41.
43. Hans, N.; Singhi, M.; Sharma, V.; Grover, S. K. Indian J. Chem., Sect B 1996, 35B,
1159.
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.
44. Mohanty, S.; Makrandi, J. K.; Grover, S. K. Indian J. Chem., Sect B 1989, 28B, 766.
´
45. Kamat, S. P.; DSouza, A. M.; Paknikar, S. K.; Beaucahmp, P. S. J. Chem. Research
(S) 2002, 242.
46. Brunet, E.; Alonso, M. T.; Juanes, O.; Velasco, O.; Rodríguez-Ubis, J. C.
Tetrahedron 2001, 57, 3105.
52. All IC50 values shown in the table are expressed as means SEM from five
experiments.
47. Frederick, R.; Robert, S.; Charlier, C.; de Ruyck, J.; Wouters, J.; Pirotte, B.;
Masereel, B.; Pochet, L. J. Med. Chem. 2005, 48, 7592.
53. Yáñez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Comm. 2006, 344,
688.