Hydroxycoumarins as selective MAO-B inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.11.020

A series of 3-aryl-4-hydroxycoumarin derivatives was synthesized with the aim to find out the structural features for the MAO inhibitory activity and selectivity. Methoxy and/or chloro substituents were introduced in the 3-phenyl ring, whereas the positio

Full text of DOI:10.1016/j.bmcl.2011.11.020

Bioorganic & Medicinal Chemistry Letters 22 (2012) 258–261
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hydroxycoumarins as selective MAO-B inhibitors
Silvia Serra a, , Giulio Ferino b, Maria João Matos c, Saleta Vázquez-Rodríguez c, Giovanna Delogu a,
⇑
Dolores Viña d, Enzo Cadoni b, Lourdes Santana c, Eugenio Uriarte c
a Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
b Dipartimento di Scienze Chimiche, Università degli studi di Cagliari, Complesso Universitario di Monserrato, S.S. 554, I-09042 Monserrato (Cagliari), Italy
c Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
d 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:
A series of 3-aryl-4-hydroxycoumarin derivatives was synthesized with the aim to find out the structural
features for the MAO inhibitory activity and selectivity. Methoxy and/or chloro substituents were intro-
duced in the 3-phenyl ring, whereas the position 6 in the coumarin moiety was not substituted or substi-
tuted with a methyl group or a chloro atom due to their different electronic, steric and/or lipophilic
properties. Most of the synthesized compounds presented MAO-B inhibitory activity. The presence of
methoxy and chloro groups, respectively in the para and meta positions of the 3-phenyl ring, have an
important influence on the inhibitory activity. Moreover, the presence of a chloro atom in the six position
of the moiety (compound 7) improved the inhibitor activity as well as its selectivity against MAO-B com-
pared with iproniazide, used as reference compound. Docking experiments were carried out to under-
stand which are the most energetically preferred orientations adopted by compounds 5, 6 and 7 inside
the MAO-B binding pocket.
Received 12 October 2011
Revised 4 November 2011
Accepted 6 November 2011
Available online 11 November 2011
Keywords:
4-Hydroxycoumarin
MAO inhibitory activity
Suzuki reaction
Docking experiments
Ó 2011 Elsevier Ltd. All rights reserved.
Neurodegenerative diseases (ND) constitute the third most
important health problem in developed countries. Alzheimer’s dis-
ease (AD) is the most prevalent of the ND followed by Parkinson’s
disease (PD). AD is a neurodegenerative and progressive disorder
associated, in the most of the cases, with senile dementia. It seems
to have a multiple etiology although the main cause it is thought
to be the accumulation of b-amyloid plaques in brain that can
provoke a degeneration or atrophy of the cholinergic neurons. PD
is also a chronic and progressive neurodegenerative disorder, char-
acterized by a predominant motor symptomatology, usually accom-
panied by non motor symptoms such as depression and anxiety. It
seems to be caused by a decrease in the dopamine levels in neuronal
synapses. In the treatment of this disease, dopaminergic agonists are
generally employed. However, other therapeutic alternatives can be
employed, such as the use of monoamine oxidase B inhibitors
(MAOI-B), or the use of antioxidant compounds in order to prevent
the oxidative cells damage.
MAO is an important FAD-containing enzyme (flavoenzyme)
present in the outer mitochondrial membrane of neuronal, glial
and many other cells.1,2 It exists in two isoforms namely as
MAO-A and MAO-B that have been identified based on their amino
acid sequences, three-dimensional structure, substrate preference
and inhibitor selectivity.3,4 MAO-A preferentially deaminates sero-
tonin and noradrenaline, whereas MAO-B has a higher affinity to
phenylethylamine and benzylamine.5,6 Therefore, these isoen-
zymes play a vital role in the monoamines degradation and, as con-
sequence, in the inactivation of neurotransmitters. Their regulation
determines the interest of the MAOI compounds as drugs used in
the treatment of neurodegenerative and neurological disorders.
In particularly, MAO-A inhibitors are effective in the treatment of
depression,7,8 while MAO-B inhibitors are useful in the treatment
of PD.9,10 In the last years, it has been proved that in patients
who suffer ND the activity of brain MAO-B is increased. This fact
provokes an increase of free radicals that are responsible for the
oxidative stress, neuronal cell death and also for the development
of the b-amyloid plaques.11 In this sense, it is an accepted theory
that the beneficial effects of the MAO-B inhibitors in the preven-
tion of the neuronal damage is mostly derived to the decrease of
hydrogen peroxide generated by the inhibition of this enzyme.
Coumarins are an important group of organic compounds from
natural and/or synthetic origin, that show, due to their structural
diversity, numerous biological activity.12–14 There are coumarins
described as anticancer, anti-inflammatory, antimicrobial, cardio-
protective, vasorelaxant, and antioxidant agents.15–20 Recently
studies pay special attention to the MAO inhibitory properties of
the 3-arylcoumarin derivatives.21–26
Flavonoids are naturally occurring polyphenolic compounds
that are found in diets and medicinal herbs. The physiological ben-
efits of dietary flavonoids (flavones and isoflavones) have been
attributed to their antioxidant and free radical-scavenging
abilities.27 Additionally, flavonoids have been revealed to exhibit
⇑
Corresponding author.
E-mail address: silvserra@tiscali.it (S. Serra).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.020

S. Serra et al. / Bioorg. Med. Chem. Lett. 22 (2012) 258–261
259
anti-amiloidogenic properties such as inhibitory effects on b-
OH
O
O
secretase enzimes28,29 and also MAO-B inhibitory activity.30
In previous works, we reported the synthesis and biological
evaluation of a series of 3-arylcoumarins as potent and selective
MAO-B inhibitors.23–25 These interesting results prompted us to
synthesize and evaluate new 3-arylcoumarin derivatives, in which
we introduce a hydroxyl group in the 4 position of coumarin skel-
eton (Fig. 1).
R1
R1
I
(a)
O
O
O
I: R1 = H,
II: R1 = Me
1
III
: R = Cl
This modification allows us considering the similarity of the
core structure of the 4-hydroxycoumarin (B) comparing with the
2-hydroxyisoflavone (C). Taking into account these data we could
establish a potential therapeutic application of these analogues
flavonoids/coumarins as a new interesting scaffold with interest
against age-related neurodegeneration such as PD or AD.
The synthesis of 3-aryl-4-hydroxycoumarin derivatives (1–7)31–33
is outlined in Scheme 1.
R3
R2
(b)
B(OH)2
R3
The key step for the synthesis of the 3-arylcoumarin skeleton
was achieved by a palladium-catalyzed coupling Suzuki reaction
between phenyliodonium zwitterions and aryl conveniently
substituted phenyl boronic acids. Initially we have synthesized
the different phenyliodonium coumarinate species (I–III).31,32 They
are electrophilic molecules with a positive charge at iodine, com-
pensated by a internal negative charge that can be localized for-
R2
OH
R1
O
O
1
2
3
mally at the
a-carbon or delocalized to a neighboring oxygen.
1
2
: R = H, R = H, R = H
1
2
3
Then we have carried out the palladium-catalyzed coupling reac-
tion using Pd(OAc)2 as catalyst and P(t-Bu)3 as ligand to afford
the compounds (1–7) with good yields.
: R = H, R = OMe, R = H
3: R1 = Me, R2 = OMe, R3 = H
4: R1 = Cl, R2 = OMe, R3 = H
By 1H and 13C NMR spectroscopy studies we have found out that
the synthesized compounds (1–7) were presented in the 4-hydroxy-
coumarin tautomeric form. Therefore we have centered on their
analysis at the physiologic pH conditions. In the biological inhibitory
assays it was also used the appropriate solvent that guaranties the
presence of this structure.
1
2
3
5
6
: R = H, R = OMe, R = Cl
1
2
3
: R = Me, R = OMe, R = Cl
7 :R1 = Cl, R2 = OMe, R3 = Cl
Scheme 1. Reagents and conditions: (a) PhI(OAc)2, Na2CO3, H2O, rt, 14 h; (b)
Pd(OAc)2, P(t-Bu)3, LiOH, DME/H20, rt, 24–48 h.
The MAO inhibitory activity of compounds 1–7 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.34 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 iproniazide (used as ref-
erence inhibitor) were calculated (Table 1).34,35
In the present Letter, we have studied the effect in the MAOI
activity of the introduction of alkyl, alkoxy or halogen substituents
in different positions of the 3-aryl-4-hydroxycoumarin nucleus.
We have found that the simple 4-hydroxy-3-phenylcoumarin did
not present MAOI activity (compound 1). The introduction of a
methoxy substituent in the para position of the 3-phenyl ring led
to a compound 2 with inhibitory activity against MAO-B. So we
take this compound as a point of reference for the subsequent
modifications. Due to the fact that the 6 position of the coumarin
Table 1
MAO-A and MAO-B inhibitory activity results for the synthesized compounds 1–7 and
reference inhibitor
Compounds
MAO-A IC50
(l
M) MAO-B IC50
(
l
M)
Selectivity index
a
a
a
a
a
a
a
a
1
2
3
4
5
6
7
—
69.59 4.70
>1.4b
>3.1b
—
32.04 2.16
a
9.26 0.63
42.68 2.88
2.79 0.19
7.54 0.36
>10b
>2.3b
>36b
0.87
Iproniazide
R-(ꢀ)-Deprenyl 68.73 4.21
6.56 0.76
17x10ꢀ3 1.9 ꢁ 10ꢀ3 4.043
All IC50 values shown in the table are expressed as means SEM from five experi-
ments. Level of statistical significance:
a
Inactive at 100
l
M (highest concentration tested).
Values obtained under the assumption that the corresponding IC50 against
MAO-A is the highest concentration tested (100 M).
b
l
skeleton is particularly relevant to the MAO-B inhibitor activity
and selectivity,36,37 we have studied the effect of the introduction
of a methyl group or a chloro atom in this position. The introduc-
tion of a methyl group (compound 3) slightly improved the activ-
ity. However, the replacement for a chloro atom at the same
position decreases the activity (compound 4). In addition, when
we analyze the second series (compounds 5–7) where a chloro
atom has been introduced at the meta position of the p-methoxy-
3-phenyl ring, the MAO-B inhibitory activity, in most of the cases,
improves. In this case, if we introduce a methyl group in the 6
position (compound 6) the MAOI-B activity did not change,
whereas if the substituent is a chloro atom (compound 7) the
MAO-B inhibitory activity considerably improves. Therefore
compound 7 is more active against MAO-B isoenzyme than the
O
A
O
OH
O
O
O
O
OH
B
C
Figure 1. Chemical structures of the 3-phenylcoumarin (A), 4-hydroxy-3-phenyl-
coumarin (B) and 2-hydroxyisoflavone (C).

260
S. Serra et al. / Bioorg. Med. Chem. Lett. 22 (2012) 258–261
iproniazide but not than the R-(ꢀ)Deprenyl, used as reference com-
pounds. None of the described compounds showed MAO-A inhibi-
tory activity for the highest concentration tested (100 M).
l
A computational approach, through docking calculations, was
used to better understand which are the most energetically pre-
ferred orientations adopted by compounds 5, 6 and 7 inside the
MAO-B binding pocket.
High resolution crystal structure of hMAO-B (PDB code 2V60)1
was taken as target for docking experiments. Protein Preparation
Wizard of Maestro 9.138 was used to set-up protein before run calcu-
lations. With this helpful tool co-crystallized ligand was removed,
water molecules beyond 5 Å from the ligand were deleted, hydrogen
atoms were added and then minimized using OPLS_2005 force field.
Compounds 5, 6 and 7 were prepared with LigPrep39 tool using
MMFFs force field. The QM-Polarized Ligand Docking (QPLD)40 pro-
tocol was used for docking calculations. In a first step, ligands were
docked into the known26 MAO-B binding site using Glide41 standard
precision (SP); the initial charges were calculated by semiempirical
methods and 8 best poses for each ligand were retained. In the sec-
ond step the Quantum Mechanical (QM) treatment of charges, calcu-
lated by Jaguar42, allowed to take into consideration the polarization
of the charges in the ligand by the protein. The final step consisted to
redock the ligand, with improved charges, with Glide extra precision
(XP) mode, retaining the 4 best poses for each ligand. With the aim of
minimizing the protein–ligand complexes and return the estimate
binding free energy for each ligand, the 4 output complexes derived
from QPLD were subjected to MacroModel-eMBrAcE43 minimiza-
tion. With this flexible-receptor tool all residues with a distance
not exceeding 5Å from the ligand were allowed to move freely, while
residues outside this shell were frozen. The complexes were mini-
mized using OPLS_2005 force field. Results were retrieved using En-
ergy difference mode which calculates separately the energy
associated first on the receptor, than on the ligand and finally on
the complex using the follow equation:
D
E ¼ Ecomplex ꢀ Eligand ꢀ Eprotein
The most stable binding solution for compounds 5, 6 and 7
shows the same pattern (Fig. 2).
The entrance cavity of binding site is occupied by the hydroxy-
coumarin moiety leaving the 3-arylcoumarin rings directed toward
FAD cofactor. Cys172 plays a significant role for the complex stabil-
ization, forming an H-bond with carbonyl oxygen of all the three
coumarins. The higher activity showed by 5 and 7, compared to 6,
could be partially due to involvement of another H-bond in the bind-
ing mode; in this case only 5 and 7 form an H-bond between the 4-
hydroxy group and Ile199. Furthermore, 3-arylcoumarin rings of 5, 6
and 7 share a p–p stacking interaction with Tyr326. Interestingly,
the p-methoxy group occupies the same position in the three
coumarins, while the position adopted by the chloro atom in the
3-phenyl ring appears to be unimportant for the modulation
of MAO-B inhibitor activity, because it assumes an opposite
orientation in the two most actives compounds 5 and 7. Good van
der Waals and electrostatic interactions with Pro102, Ile316 and
Phe343 were also observed for all the docked molecules.
As conclusion, these preliminary results allow us a better under-
standing of the molecular fragments that are essential to maintain
and/or improve the MAO activity and selectivity. These findings
encourage us to continue the efforts towards the optimization of
the pharmacological profile of this structural moiety as an important
scaffold for the potential treatment of ND.
Figure 2. Best docking poses retrieved for compounds 5 (a), 6 (b) and 7 (c) into the
MAO-B (PDB code: 2V60). Coumarins are represented in tube with carbon atoms
colored in plum for 5, turquoise for 6 and purple for 7. Hidden ribbons for a better
visualization from residues Glu159 to Glu179 (excluding Cys172). MAO-B binding
site displayed as gray mesh style. Interacting residues and FAD cofactor labeled in
ball and stick, with carbon atom colored in yellow and gray respectively. Water
molecules depicted in wire rendering. H-bonds displayed in yellow dot line.
Nonpolar hydrogens are omitted.
Acknowledgements
This work was partially supported by the Ministerio de
Sanidad y Consumo (FIS PS09/00501) and Xunta da Galicia

S. Serra et al. / Bioorg. Med. Chem. Lett. 22 (2012) 258–261
261
33. General procedure for the preparation of 3-aryl-4-hydroxycoumarins: a degassed
solution of appropriated phenyl boronic acid (2.2 equiv) and P(t-But)3 (27 mL)
in DME and H2O (4:1, 12.5 mL) was added to a mixture of iodonium ylide
(0.55 mmol), LiOH/H2O (3 equiv) and Pd(OAc)2 (6.2 mg) under argon at room
temperature. After being stirred at the same temperature for 24–48 h. The
resulting mixture was purified by FC (hexane/ethyl acetate, 7:3) to give the
desired compound.
(PGIDIT09CSA030203PR and 10PXIB203303PR). This work was also
partially supported by MIUR and Cagliari University (National
Project ‘‘Stereoselezione in Sintesi Organica metodologie ed
Applicazioni’’) and by CINMPIS and FIRB. S.S. thanks Regione Sarde-
gna for the PhD grant (PR-MAB-A2009-613). M.J.M. thanks to Fun-
dação para a Ciência e Tecnologia for the PhD grant (SFRH/BD/
61262/2009). S.V.R. thanks to Ministerio de Educación y Ciencia
for a PhD grant (AP2008-04263).
3-(30-Chloro-40-methoxyphenyl)-4-hydroxycoumarin (5). It was obtained with
yield 80%. Mp: 298–300 °C. 1H NMR (DMSO-d6) d (ppm): 3.94 (s, 3H, OCH3),
7.21 (s, 1H, H20), 7.28–7.49 (m, 3H, H6, H8, H50), 7.56–7.83 (m, 2H, H7, H60),
7.98 (d, 1H, H5, J = 7.6), 15.7 (s, 1H, OH). 13C NMR (DMSO-d6) d (ppm): 56.2,
105.0, 112.8, 116.6, 116.9, 120.7, 124.2, 124.4, 125.6, 131.5, 132.7, 132.8, 152.7,
154.3, 161.0, 162.3. MS m/z (%): 302 (M+, 100), 182 (64), 121 (95). Anal. Calcd
for C16H11ClO4: C, 63.48; H, 3.66. Found: C, 63.45; H, 3.69.
References and notes
3-(30-Chloro-40-methoxyphenyl)-4-hydroxy-6-methylcoumarin (6). It was
obtained with yield 80%. Mp: 301–304 °C. 1H NMR (DMSO-d6) d (ppm): 2.45
(s, 3H, CH3), 3.90 (s, 3H, OCH3), 7.14–7.24 (m, 1H, H8), 7.25–7.37 (m, 3H, H7,
H20, H50), 7.40–7.52 (m, 1H, H60), 7.80 (s, 1H, H5), 15.4 (s, 1H, OH). 13C NMR
(DMSO-d6) d (ppm): 20.9, 56.6, 105.0, 112.8, 116.4, 120.8, 123.8, 125.6, 129.0,
131.5, 132.7, 133.6, 133.6, 150.8, 154.3, 160.9, 162.4. MS m/z (%): 316 (M+, 59),
182 (80), 135 (100), 97 (36), 83 (37), 71 (51), 57 (91). Anal. Calcd for
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6-Chloro-3-(30-chloro-40-methoxyphenyl)-4-hydroxycoumarin (7). It was
obtained with yield 62%. Mp: 321–323 °C. 1H NMR (DMSO-d6) d (ppm): 3.90
(s, 3H, OCH3), 7.0–7.57 (m, 3H, H8, H20, H50), 7.60–7.84 (m, 2H, H7, H60), 8.0 (s,
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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
tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-
B: 7.5 g protein; specific activity: 22 nmol of p-tyramine transformed/min/mg
lg protein; specific activity: 150 nmol of p-
l
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
quantified at 37 °C in multidetection microplate fluorescence reader
l
M AmplexÒ Red reagent, 1 U/mL horseradish peroxidase and 1 mM p-
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(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
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 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.
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38. Schrödinger Suite 2011 Protein Preparation Wizard; Epik version 2.2,
Schrödinger, LLC, New York, NY, 2011; Impact version 5.7, Schrödinger, LLC,
New York, NY, 2011; Prime version 3.0, Schrödinger, LLC, New York, NY, 2011.
39. LigPrep, version 2.5, Schrödinger, LLC, New York, NY, 2011.
40. Schrödinger Suite 2011 QM-Polarized Ligand Docking protocol; Glide version
5.7, Schrödinger, LLC, New York, NY, 2011; Jaguar version 7.8, Schrödinger, LLC,
New York, NY, 2011; QSite version 5.7, Schrödinger, LLC, New York, NY, 2011.
41. Glide, version 5.7, Schrödinger, LLC, New York, NY, 2011.
30. Desideri, N.; Bolasco, A.; Fioravanti, R.; Proietti Monaco, L.; Orallo, F.; Yañez,
M.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2011, 54, 2155.
31. Zhu, Q.; Wu, J.; Fathi, R.; Yang, Z. Org. Lett. 2002, 4, 3333.
32. General procedure for the preparation of 3-phenyliodonium coumarinates:
iodobenzene diacetate (10 mmol) was suspended in a solution of Na2CO3
(10 mmol) in water (100 mL) and was stirred for 30 min at room temperature.
To this solution was added a mixture of the corresponding 4-hydroxycoumarin
(10 mmol) and Na2CO3 (10 mmol) in water (100 mL). After the mixture was
stirred at room temperature for 14 h, the precipitate was collected by filtration,
washed with water (5 ꢁ 20 mL) and dried under vacuum. The resulting white
solid was used without further purification.
42. Jaguar, version 7.8, Schrödinger, LLC, New York, NY, 2011.
43. MacroModel, version 9.9, Schrödinger, LLC, New York, NY, 2011.