Synthesis, biological evaluation, and molecular simulation of chalcones and aurones as selective MAO-B inhibitors

Article abstract of DOI:10.1111/cbdd.12458

A series of chalcones and aurones were synthesized and evaluated in vitro as monoamine oxidase inhibitors (MAOi). Our results show that aurones, which had not been previously reported as MAOi, are MAO-B inhibitors. Thus, both families inhibited selectively the B isoform of MAO in the micromolar range, offering novel scaffolds for the design of new and potent MAO inhibitors. The main structural requirements for their activity were characterized with the aid of 3D-QSAR and docking studies.

Full text of DOI:10.1111/cbdd.12458

Chem Biol Drug Des 2014
Research Article
Synthesis, Biological Evaluation, and Molecular
Simulation of Chalcones and Aurones as Selective
MAO-B Inhibitors
Nicole Morales-Camilo1, Cristian O. Salas1,*,
known to suppress inflammation and oxidative stress
(16–18) and have anticancer properties (19,20). Aureusidin
has shown antioxidant activity (21), and 4-chloroaurone,
isolated from a marine source, Spatoglossum variabile,
has been modified to obtain compounds with potent anti-
bacterial activity (22).
Claudia Sanhueza2, Christian Espinosa-Bustos1,
2
2,3
ꢀ
Silvia Sepulveda-Boza , Miguel Reyes-Parada
,
Fernando Gonzalez-Nilo4, Marcos Caroli-
5
1,
ꢀ
Rezende and Angelica Fierro *
1
ꢀ
Departamento de Quımica Organica, Facultad de
Quımica, Pontificia Universidad Catolica de Chile, Santiago
ꢀ
ꢀ
ꢀ
Among the many pharmacologically interesting applications
of chalcones, their potential as monoamine oxidase inhibi-
tors (MAOi) has been recently described by different groups
(23–26). MAO is a flavoprotein involved in the degradation of
a wide range of endogenous and exogenous monoamines,
including the neurotransmitters serotonin, dopamine, and
noradrenaline (27). The enzyme exists in two isoforms,
MAO-A and MAO-B (28), which are bound to the mitochon-
drial outer membrane as dimers (29,30). Both proteins have
similar structures and molecular weights (70% amino acid
sequence identity), but differ in their selectivity for substrates
and inhibitors (27). As MAO-A and MAO-B modulate the lev-
els of key signaling molecules, inhibitors of these enzymes
have been developed as drugs for the treatment of neuro-
psychiatric and neurodegenerative disorders (26). Thus,
reversible MAO-A inhibitors such as moclobemide (31) are
currently used as effective antidepressants, whereas MAO-
B inhibitors are useful in the treatment of Parkinson0s dis-
ease (26,32). The latter indication is especially relevant in
elderly patients with Parkinson0s disease, as it has been
shown that brain MAO-B levels increase with age, resulting
in an acceleration of the neurodegenerative processes
(33,34). Therefore, as the detailed chemical structures of
MAO-A and MAO-B are available, this information provides
new possibilities for the development of promising novel
inhibitors (35–37).
de Chile 702843, Chile
Escuela de Medicina, Facultad de Ciencias Medicas,
2
ꢀ
Universidad de Santiago, Casilla 442, Correo 2, Santiago,
Chile
Facultad de Ciencias de la Salud, Universidad Autonoma
de Chile, Santiago 8910124, Chile
3
ꢀ
4
ꢀ
Universidad Andres Bello, Facultad de Ciencias
Biologicas, Centro de Bioinformatica y Biologıa Integrativa,
ꢀ
ꢀ
ꢀ
Santiago 8370146, Chile
ꢀ
5
Departamento de Quımica de los Materiales, Facultad de
ꢀ
ꢀ
Quımica y Biologıa, Universidad de Santiago de Chile,
Casilla 40, Correo 33, Santiago 9170022, Chile
ꢀ
*Corresponding authors: Angelica Fierro, afierroh@uc.cl;
Cristian O. Salas, cosalas@uc.cl
A series of chalcones and aurones were synthesized
and evaluated in vitro as monoamine oxidase inhibitors
(MAOi). Our results show that aurones, which had not
been previously reported as MAOi, are MAO-B inhibi-
tors. Thus, both families inhibited selectively the B iso-
form of MAO in the micromolar range, offering novel
scaffolds for the design of new and potent MAO inhibi-
tors. The main structural requirements for their activity
were characterized with the aid of 3D-QSAR and dock-
ing studies.
Key words: aurones, chalcones, molecular modeling,
monoamine oxidase inhibitors, QSAR
Following previous reports on the activity of chalcones as
MAOi, we decided to investigate a series of synthetic
substituted chalcones. In addition, our study included for
the first time a series of aurones, in order to explore their
pharmacological activity and to compare their putative
MAOi activity with that of the related chalcones. In spite of
some reports describing their therapeutic potential (6,28),
aurones have not yet been reported as MAOi.
Received 7 July 2014, revised 11 September 2014 and
accepted for publication 12 October 2014
Chalcones and aurones are natural and synthetic com-
pounds whose recorded actions on biological targets
make them interesting scaffolds for the design of potential
therapeutic agents (1–7). Examples of the former are xan-
thohumol and cardamonin (Figure 1), of interest as anti-
cancer (8–13) and antibacterial agents (14), and preventing
platelet aggregation (15). Aurones such as sulfuretin are
The evaluation of a total of 16 compounds, 8 chalcones,
and 8 aurones was followed by a selection of the most
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12458
1

Morales-Camilo et al.
Figure 1: Chemical structures of bioactive
chalcone and aurone derivatives.
active compounds of both sets, and by 3D-QSAR and
docking studies, to identify the principal structural features
that might be responsible for their activity.
(E)-3-(2,5-Dimethoxyphenyl)-1-(2-hydroxy-6-
methoxyphenyl)prop-2-en-1-one (3a)
Orange solid, yield 35%, mp: 134.3–135.9 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.81 (s, 3H), 3.87 (s, 3H), 3.94
(s, 1H), 6.42 (d, J = 8.2 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H),
6.87 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 2.9, 9.0 Hz, 1H),
7.15 (d, J = 2.8 Hz, 1H), 7.35 (t, J = 8.3 Hz, 1H), 7.90 (d,
J = 15.7 Hz, 1H), 8.12 (d, J = 15.7 Hz, 1H), 13.19 (s, 1H).
13C NMR (101 MHz; CDCl3) d ppm: 55.8, 55.9, 56.1,
101.5, 110.9, 112.1, 112.4, 113.8, 116.8, 125.0, 128.3,
135.7, 138.2, 153.4, 153.5, 161.0, 164.8, 194.8. IR (KBr,
cmꢀ1): 2932, 2821, 1605, 1497, 1439, 1220. MS (ESI):
314.0 (C18H18O5 [M + H]+). HRMS-ES (m/z): [M + H]+
calcd for C18H18O5, 314.1154; found, 315.1222.
Experimental
Melting points were determined on a Reichert–Thermo-
€
gerate–Kofler apparatus and are uncorrected. Infrared
spectra were recorded on a Bruker Model Vector 22 spec-
trophotometer (Bruker Optik GmbH, Bremen, Germany)
using KBr disks. Nuclear magnetic resonance spectra
were recorded on a Bruker AM-400 apparatus using
CDCl3 solutions containing TMS as internal standard.
HPLC–MS experiments were performed on a Thermo Sci-
entific Exactive Orbitrap MS, Bremen, Germany. Mass
spectra were obtained on a Hewlett-Packard (HP) 5988A
spectrometer. Thin-layer chromatography (TLC) was per-
formed using Merck GF-254 type 60 silica gel. Column
chromatography was carried out using Merck silica gel 60
(70–230 mesh). 2,5- and 2,6-dihydroxyacetophenones and
the substituted aldehydes were purchased from Sigma-
Aldrich, St. Louis, MO, USA. The 2-hydroxy-6-methoxy-
acetophenone and 2-hydroxy-5-methoxyacetophenone
were prepared by methylation of commercial 2,6-dihydr-
oxyacetophenone and 2,5-dihydroxyacetophenone with
methyl iodide, following a reported procedure (38).
(E)-1-(2-Hydroxy-6-methoxyphenyl)-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (3b)
Orange solid, yield 74%, mp: 133.6–133.8 °C [lit. 132–
133 °C (39)] 1H NMR (400 MHz, CDCl3) d ppm: 3.89 (s,
3H), 3.91 (s, 3H), 3.94 (s, 3H), 3.95 (s, 3H), 6.42 (d,
J = 8.2 Hz, 1H), 6.52 (s, 1H), 6.61 (d, J = 8.3 Hz 1H),
7.12 (s, 1H), 7.34 (t, J = 8.3 Hz, 1H), 7.83 (d,
J = 15.6 Hz, 1H) 8.16 (d, J = 15.7 Hz, 1H), 13.33 (s, 1H).
13C NMR (101 MHz; CDCl3) d ppm: 55.8, 56.1, 56.4,
56.5, 96.9, 101.5, 110.9, 111.5, 112.2, 116.0, 125.4,
135.4, 138.6, 143.2, 152.5, 154.8, 160.8, 164.8, 194.5.
IR (KBr, cmꢀ1): 2937, 2785, 1659, 1288, 1218, 1027. MS
(ESI): 344.0 (C19H20O6 [M + H]+). HRMS-ES (m/z):
[M + H]+ calcd for C19H20O6, 344.1260; found, 345.1328.
General synthetic procedure to obtain chalcones
(3a–3h)
To a stirred solution of 2-hydroxy-5-methoxy- or of the 2-
hydroxy-6-methoxyacetophenone (2a or 2b) (0.01 mol)
and the appropriate aldehyde (0.02 mol) in EtOH (30 mL)
was added 50% aqueous KOH (30 mL). The mixture was
stirred for 12 h at room temperature. The reaction mix-
ture was then made acid with dilute HCl, and the precipi-
tated solid was filtered and washed with cold water. The
obtained yellow-orange solid was purified by flash column
chromatography using CH2Cl2 as eluent. In this way, the
following chalcones were prepared.
(E)-3-(2,3-Dimethoxyphenyl)-1-(2-hydroxy-6-
methoxyphenyl)prop-2-en-1-one (3c)
Orange solid, yield 99%, mp: 112.0–114.1 °C [lit. 115–
116 °C (39)] 1H NMR (400 MHz, CDCl3) d ppm: 3.89 (s,
3H), 3.90 (s, 3H), 3.93 (s, 3H), 6.42 (d, J = 8.3 Hz, 1H),
6.61 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 8.0 Hz, 2H), 7.09 (t,
J = 8.0 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.35 (t,
J = 8.3 Hz, 1H), 7.92 (d, J = 15.8 Hz, 1H), 8.11 (d,
J = 15.8 Hz, 1H) 13.17 (s, 1H). 13C NMR (101 MHz;
2
Chem Biol Drug Des 2014

Chalcones and Aurones as Selective MAO-B Inhibitors
CDCl3) d ppm: 55.9, 56.0, 61.3, 101.5, 110.9, 112.1,
113.9, 119.8, 124.2, 128.9, 129.5, 135.8, 137.8, 149.0,
153.2, 161.0, 164.9, 194.7. IR (KBr, cmꢀ1): 2938, 2835,
1632, 1561, 1240, 1093. MS (ESI): 314.0 (C18H18O5
[M + H]+). HRMS-ES (m/z): [M + H]+ calcd for C18H18O5,
314.1154; found, 315.1216.
7.09–7.18 (m, 2H), 7.27 (d, J = 9.8 Hz, 1H), 7.36 (d,
J = 2.9 Hz, 1H), 7.71 (d, J = 15.6 Hz, 1H), 8.19 (d,
J = 15.6 Hz, 1H), 12.42 (s, 1H). 13C NMR (101 MHz;
CDCl3): d ppm: 56.3, 56.5, 61.7, 113.2, 115.0, 119.8,
120.1, 120.5 (2C), 122.2, 124.4 (2C), 124.7, 129.2, 141.1,
152.1, 158.4, 194.2. IR (KBr, cmꢀ1): 2943, 2835, 1573,
1486, 1278, 1175. MS (ESI): 314.0 (C18H18O5 [M + H]+).
HRMS-ES (m/z): [M + H]+ calcd for C18H18O5, 314.1154;
found, 315.1222.
(E)-3-(4-Bromo-2,5-dimethoxyphenyl)-1-(2-hydroxy-
6-methoxyphenyl)prop-2-en-1-one (3d)
Orange solid, yield 93%, mp: 188.3–188.9 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.88 (s, 3H), 3.90 (s, 3H), 3.93
(s, 3H) 6.43 (d, J = 8.2 Hz, 1H), 6.62 (dd, J = 0.6, 8.3 Hz,
1H), 7.11 (s, 1H), 7.15 (s, 1H), 7.36 (t, J = 8.3 Hz, 1H),
7.93 (d, J = 15.7 Hz, 1H) 8.02 (d, J = 15.7 Hz, 1H), 13.14
(s, 1H). 13C NMR (101 MHz; CDCl3) d ppm: 55.8, 56.3,
56.9, 101.6, 111.0, 112.1, 112.2, 114.7, 117.0, 124.1,
128.7, 135.9, 137.6, 150.2, 153.4, 160.9, 164.9, 194.6.
IR (KBr, cmꢀ1): 2961, 2834, 1631, 1561, 1237, 1217. MS
(ESI): 393.0 (C18H17O5Br [M + H]+).
(E)-3-(4-Bromo-2,5-dimethoxyphenyl)-1-(2-hydroxy-
5-methoxyphenyl)prop-2-en-1-one (3h)
Orange solid, yield 42%, mp: 159.9–160.3 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.83 (s, 3H), 3.89 (s, 3H), 3.91
(s, 3H), 6.96 (d, J = 9.0 Hz, 1H), 7.04–7.18 (m, 3H), 7.35
(d, J = 2.8 Hz, 1H), 7.67 (d, J = 15.6 Hz, 1H), 8.06 (d,
J = 15.6 Hz, 1H), 12.38 (s, 1H). 13C NMR (101 MHz;
CDCl3) d ppm: 56.1, 56.3, 57.0, 112.6, 113.4, 115.6,
117.0, 119.2, 119.8, 121.4, 123.3, 123.4, 140.4, 150.2,
151.6, 153.5, 157.9, 193.7. IR (KBr, cmꢀ1): 2917, 2834,
1640, 1573, 1487, 1462, 1050. MS (ESI): 393.0
(C18H17O5Br [M + H]+). HRMS-ES (m/z): [M + 2H]+ calcd
for C18H17O5Br, 392.0259; found, 395.0303.
(E)-3-(2,5-Dimethoxyphenyl)-1-(2-hydroxy-5-
methoxyphenyl)prop-2-en-1-one (3e)
Orange solid, yield 35%, mp: 156.8–158.1 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.82 (s, 3H), 3.83 (s, 3H), 3.88
(s, 3H), 3.94 6.88 (d, J = 8.9 Hz, 1H), 6.96 (d, J = 8.9 Hz,
2H), 7.09–7.17 (m, 2H), 7.35 (d, J = 3.0 Hz, 1H), 7.66 (d,
J = 15.6 Hz, 1H), 8.17 (d, J = 15.7 Hz, 1H), 12.46 (s, 1H).
13C NMR (101 MHz; CDCl3) d ppm: 55.9, 56.0, 56.1,
112.5, 112.9, 114.3, 117.6, 119.2, 119.8, 121.0, 123.7,
124.2, 141.0, 151.7, 153.5, 153.6, 157.9, 193.9. IR (KBr,
cmꢀ1): 2942, 2835, 1617, 1550, 1285, 1238. MS (ESI):
314.0 (C18H18O5 [M + H]+). HRMS-ES (m/z): [M + H]+
calcd for C18H18O5, 314.1154; found, 315.1210.
General synthetic procedure to obtain aurones
(4a–4h)
To a solution of chalcone 3a–h (1 mmol) in pyridine
(10 mL) was added Hg(OAc)2 (1 mmol) at room tempera-
ture, and the mixture was refluxed for 2 h. The cooled
reaction mixture was poured into ice-cold water (50 mL)
and acidified with dil. HCl. The precipitated solid was fil-
tered, washed with cold water, and dried to give the prod-
ucts, which were purified by flash column chromatography
using CHCl3 as eluent. In this way, the following aurones
were prepared.
(E)-1-(2-Hydroxy-5-methoxyphenyl)-3-(2,4,5-
trimethoxyphenyl)prop-2-en-1-one (3f)
Orange solid, yield 61%, mp: 159.3–161.0 °C [lit. 162–
163 °C (39)]. 1H NMR (400 MHz, CDCl3) d ppm: 3.84 (s,
3H), 3.92 (s, 3H), 3.93 (s, 3H), 3.96 (s, 3H), 6.53 (s, 1H),
6.97 (d, J = 9.0 Hz, 1H), 7.13 (m, 2H), 7.40 (d,
J = 2.8 Hz, 1H), 7.56 (d, J = 15.5 Hz, 1H), 8.21 (d,
J = 15.5 Hz, 1H), 12.59 (s, 1H). 13C NMR (101 MHz;
CDCl3) d ppm: 56.5, 56.6, 56.7, 57.1, 97.1, 112.4, 113.9,
115.6, 118.4, 119.5, 120.5, 123.3, 141.5, 143.8, 151.9,
153.5, 155.6, 158.2, 194.2. IR (KBr, cmꢀ1): 2945, 2836,
1557, 1238, 1267, 1172, 1022. MS (ESI): 344.0
(C19H20O6 [M + H]+). HRMS-ES (m/z): [M + H]+ calcd for
(Z)-2-(2,5-Dimethoxybenzylidene)-4-
methoxybenzofuran-3(2H)-one (4a)
Yellow solid, yield 80%, mp: 187.4–188.2 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.84 (d, J = 2.6 Hz, 6H), 3.99
(s, 3H), 6.59 (d, J = 8.3 Hz, 1H), 6.82–6.85 (m, 2H), 6.90
(dd, J = 2.8, 9.0 Hz, 1H), 7.34 (s, 1H), 7.53 (t, J = 8.2 Hz,
1H), 7.84 (d, J = 2.8 Hz, 1H). 13C NMR (50 MHz; CDCl3)
d ppm: 55.8, 56.1, 56.2, 104.8, 105.1, 105.9, 111.0,
111.7, 116.4, 116.9, 122.1, 138.1, 147.0, 153.3, 153.4,
158.5, 166.8, 182.2. IR (KBr, cmꢀ1): 1697, 1600, 1252,
1232, 1073. MS (ESI): 313.0 (C18H16O5 [M + H]+). HRMS-
ES (m/z): [M + H]+ calcd for C18H16O5, 312.0998; found,
313.1067.
C
19H20O6, 344.1260; found, 345.1331.
(E)-3-(2,3-Dimethoxyphenyl)-1-(2-hydroxy-5-
methoxyphenyl)prop-2-en-1-one (3g)
(Z)-2-(2,4,5-Trimethoxybenzylidene)-4-
Orange solid, yield 34%, mp: 159.5–160.1 °C [lit. 102–
103 °C (39)]. 1H NMR (400 MHz, CDCl3) d ppm: 3.84 (s,
3H), 3.91 (s, 3H), 3.92 (s, 3H), 6.99 (t, J = 8.5 Hz, 2H),
methoxybenzofuran-3(2H)-one (4b)
Orange solid, yield 70%, mp: 173.0–174.3 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.76 (s, 3H), 3.83 (s, 3H), 3.88
Chem Biol Drug Des 2014
3

Morales-Camilo et al.
(s, 6H), 6.43 (s, 1H), 7.10–7.17 (m, 3H), 7.36 (s, 1H), 7.82
(s, 1H). 13C NMR (101 MHz; CDCl3) d ppm: 55.9, 56.0,
56.4, 56.6, 96.3, 105.1, 108.0, 113.2, 113.6, 114.6,
122.3, 125.5, 143.2, 146.5, 152.3, 155.2, 155.9, 160.5,
184.4. IR (KBr, cmꢀ1):1696, 1595, 1254, 1229, 1215,
1080. MS (ESI): 343.0 (C19H20O6 [M + H]+).
56.4, 56.6, 96.3, 105.1, 108.0, 113.2, 113.6, 114.6,
122.3, 125.5, 143.2, 146.5, 152.3, 155.2, 155.9, 160.5,
184.4 IR (KBr, cmꢀ1): 1694, 1591, 1488, 1272, 1213,
1194, 1028. HRMS-ES (m/z): [M + H]+ calcd for
C19H18O6, 342.1103; found, 343.3533.
(Z)-2-(2,3-Dimethoxybenzylidene)-5-
(Z)-2-(2,3-Dimethoxybenzylidene)-4-
methoxybenzofuran-3(2H)-one (4c)
methoxybenzofuran-3(2H)-one (4g)
Yellow solid, yield 99%, mp: 170.8–172.3 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.83 (s, 3H), 3.89 (s, 3H), 3.92
(s, 3H), 6.97 (d, J = 8.0 Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H),
7.19–7.26 (m, 3H), 7.35 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H).
13C NMR (101 MHz; CDCl3) d ppm: 55.9, 56.0, 61.7,
105.3, 107.1, 113.7, 114.1, 121.8, 123.5, 124.2, 126.1,
126.6, 148.3, 149.3, 152.8, 156.1, 161.2, 184.9. IR (KBr,
cmꢀ1): 1695, 1645, 1490, 1273, 1115. MS (ESI): 312.0
(C18H16O5 [M + H]+). HRMS-ES (m/z): [M + H]+ calcd for
C18H16O5, 312.0998; found, 313.1058.
Yellow solid, yield 60%, mp: 142.6–148.0 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.89 (s, 3H), 3.91 (s, 3H), 3.99
(s, 3H), 6.60 (d, J = 8.3 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H),
6.95 (d, J = 8.1 Hz, 1H), 7.14 (t, J = 8.1 Hz, 1H), 7.29 (s,
1H), 7.55 (t, J = 8.3 Hz 1H), 7.88 (d, J = 8.0 Hz 1H). 13C
NMR (101 MHz; CDCl3) d ppm: 55.9, 56.3, 61.7, 104.7,
105.2, 105.9, 110.9, 113.8, 123.3, 124.1, 126.7, 138.3,
147.5, 149.2, 152.8, 158.6, 167.0, 182.3. IR (KBr, cmꢀ1):
1704, 1599, 1252, 1077. MS (ESI): 312.0 (C18H16O5
[M + H]+). HRMS-ES (m/z): [M + H]+ calcd for C18H16O5,
312.0998; found, 313.1061.
(Z)-2-(4-Bromo-2,5-dimethoxybenzylidene)-5-
methoxybenzofuran-3(2H)-one (4h)
(Z)-2-(4-Bromo-2,5-dimethoxybenzylidene)-4-
methoxybenzofuran-3(2H)-one (4d)
Yellow solid, yield 70%, mp: 169.4–174.6 °C. 1H NMR
(400 MHz, CDCl3)
d ppm: 3.84 (s, 3H), 3.86 (d,
Yellow solid, yield 99%, mp: 129.8–130.7 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.86 (s, 3H), 3.95 (s, 3H), 4.00
(s, 3H), 6.61 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H),
7.10 (s, 1H), 7.27 (s, 1H), 7.55 (t, J = 8.2 Hz,1H), 7.84 (s,
1H). 13C NMR (101 MHz; CDCl3) d ppm: 56.3, 56.4, 56.9,
104.7, 105.2, 105.3, 111.0, 114.3, 114.7, 116.2, 121.2,
138.2, 147.0, 150.1, 153.4, 158.6, 166.6, 182.0. IR (KBr,
cmꢀ1): 1699, 1600, 1494, 1251, 1215, 1076. HRMS-ES
(m/z): [M + H]+ calcd for C18H15O5Br, 390.0103; found,
391.0168.
J = 2.0 Hz, 6H), 6.85 (d, J = 9.0 Hz, 1H), 6.93 (dd,
J = 3.0, 9.0 Hz, 1H), 7.23 (b.s, 2H), 7.42 (s, 1H), 7.87 (d,
J = 3.0 Hz, 1H). 13C NMR (101 MHz; CDCl3) d ppm: 56.2,
56.4, 56.6, 105.7, 107.6, 112.1, 114.2, 117.2, 117.5,
122.3, 122.4, 126.4, 148.2, 153.9, 154.0, 156.4, 161.4,
185.2. IR (KBr, cmꢀ1): 1691, 1641, 1593, 1490, 1281,
1233, 1098. MS (ESI): 392.0 (C18H15O5 [M + H]+).
Monoamine oxidase inhibition
All compounds were evaluated as previously described
(40). Briefly, a crude rat brain mitochondrial suspension
(male Sprague–Dawley rats weighing 180–220 g, killed by
decapitation) was used as protein source. Serotonin
(5-HT, 100 lM) and 4-dimethylaminophenethylamine
(DMAPEA, 5 lM) were used as selective substrates for
MAO-A and MAO-B, respectively. The reaction mixtures
were analyzed with a Merck-Hitachi HPLC (Tokyo, Japan),
employing a C18 reverse phase column (Lichrospher
250 9 4.6 mm, 5 lm) and an amperometric detector
(Merck–Recipe L3500A). Tetrahydrofuran and acetonitrile
(Merck) were used as eluents. IC50 values were deter-
mined using Prism Graph Pad software, from plots of inhi-
bition percentages (calculated in relation to a sample of
the enzyme treated under the same conditions without
inhibitors) versus ꢀlog inhibitor concentration (from 1 to
100 lM).
(Z)-2-(2,5-Dimethoxybenzylidene)-5-
methoxybenzofuran-3(2H)-one (4e)
Yellow solid, yield 87%, mp: 154.5–157.8 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.86 (s, 6H), 4.00 (s, 3H), 6.85
(d, J = 9.0 Hz, 1H), 6.93 (dd, J = 3.1, 9.0 Hz, 1H), 7.08–
7.17 (m, 2H), 7.38 (dd, J = 1.8, 6.9 Hz, 1H), 7.46 (s, 1H),
7.95 (d, J = 3.0 Hz, 1H). 13C NMR (101 MHz; CDCl3) d
ppm: 55.7, 56.2, 56.5, 107.7, 111.9, 115.9, 116.4, 117.7,
118.7, 121.8, 123.3, 123.8, 146.0, 146.9, 153.5, 153.7,
155.6, 184.7. IR (KBr, cmꢀ1): 1692, 1643, 1489, 1219,
1032. MS (ESI): 312.0 (C18H16O5 [M + H]+). HRMS-ES (m/
z): [M + H]+ calcd for C18H16O5, 312.0998; found,
313.1056.
(Z)-2-(2,4,5-Trimethoxybenzylidene)-5-
methoxybenzofuran-3(2H)-one (4f)
Orange solid, yield 82%, mp: 188.8–192.2 °C. 1H NMR
(400 MHz, CDCl3) d ppm: 3.83 (s, 3H), 3.90 (s, 3H), 3.95
(s, 6H), 6.50 (s, 1H), 7.18–7.24 (m, 3H), 7.43 (s, 1H), 7.89
(s, 1H). 13C NMR (101 MHz; CDCl3) d ppm: 55.9, 56.0,
In silico evaluation
During decades, computational methodologies such as
quantitative structure–activity relationships (QSAR) or
molecular modeling (MM) have been useful tools to advance
4
Chem Biol Drug Des 2014

Chalcones and Aurones as Selective MAO-B Inhibitors
in the understanding of drug–receptor interactions (41–44).
In the present work, 3D-QSAR and docking evaluations
were used to rationalize both the experimental results and
the structural requirements necessary to design new
ligands.
creating an overall neutral system in approximately 0.1 M
NaCl. The ions were equally distributed in a water box.
The final system was submitted to a molecular dynamics
(MD) simulation for 2 ns using the CHARMM31 force field
and the NAMD 2.6 program (49).
The structures of all evaluated compounds were optimized
with Gaussian 03 (45), with the HF–6-31G* method.
The NPT ensemble was used to perform MD calculations.
Periodic boundary conditions were applied to the system
in the three co-ordinate directions. For non-bonded calcu-
ꢁ
lations, a cutoff of 12 A was used. The long-range electro-
Molecular modeling
statics was calculated using the particle mesh Ewald
(PME) method. A pressure of 1 atm was used, and the
temperature was kept at 310 K. The evaluation of the
structure involved analysis of geometry, stereochemistry,
and energy distribution with the VMD program (50), PRO-
SAII server (51), ANOLEA server (52) and Procheck (53).
As the crystal structure of rat MAO-B (rMAO-B) has not
been resolved, a homology model was built from the crys-
tallographic data of human MAO-B (PDB: 2V5Z) obtained
from the Brookhaven Protein Data Bank for all calcula-
tions. The target protein and template were aligned by
amino acid identity. MODELLER9v6 (46) was used to con-
struct the three-dimensional structure using standard
parameters, and the outputs were ranked on the basis of
the internal scoring function of the program. The best
model was submitted to the H++ server (47,48) to com-
pute pK values of ionizable groups in the protein and to
add missing hydrogen atoms according to the specified
pH of the environment. The protein was then inserted into
a POPC membrane, TIP3 solvated, and ions were added
Molecular docking
Docking studies employed AutoDock Vina software (54)
considering the protein to be rigid with flexible ligands. The
ꢁ3
volume chosen for the grid box was 20 A , centered on
N5 of the FAD cofactor located in the ligand-binding site.
The docked compound complexes were built using the
lowest free-energy binding positions. The binding energies
Table 1: Inhibition percentage (IP) of isoforms of rMAO by the synthesized chalcones and aurones, in 10 lM concentrations
Compound
R1
R2
R3
R4
R5
IP(%) of rMAO-A
IP(%) of rMAO-B
3a
3b
3c
3d
3e
3f
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
OCH3
H
H
H
OCH3
H
H
H
OCH3
H
H
H
OCH3
H
H
OCH3
H
Br
H
OCH3
H
Br
H
OCH3
H
Br
H
OCH3
H
Br
OCH3
OCH3
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
H
0
0
14
32
2
11
23
34
23
8
64
62
58
66
49
58
71
60
57
48
54
60
61
58
68
72
3g
3h
4a
4b
4c
4d
4e
4f
0
16
11
16
0
4g
4 h
OCH3
0
Chem Biol Drug Des 2014
5

Morales-Camilo et al.
Scheme 1: Reagents and conditions: (i) MeI, acetone, 12 h; rt (ii) ArCHO, EtOH, KOH 50%, 12 h, rt, 34–99%; (iii) Hg(OAc)2, Py, reflux,
2 h, 60–99%. For compounds 3a–h and 4a–h, the substituents R1–R5 are indicated in Table 1.
in AutoDock Vina are determined using the AMBER force
field. The main interactions were identified using the VMD
program (50).
preparation of aurones from chalcones by treatment with
mercury (II) acetate in acetic acid, but flavanones were a
by-product in these reactions (57). We employed mercury
(II) acetate in pyridine, obtaining only aurones 4a–h in
good yields (58). In all cases, a single geometric isomer
(Z) was obtained as confirmed by analysis of the 1H and
13C NMR spectra, which were in agreement with previ-
ously reported data.
3D-QSAR studies
The pharmacophore models were developed for rat MAO-B
(rMAO-B) inhibitors using the phase 3.4 (55) module imple-
€
mented in the Maestro modeling package (Schrodinger,
Inc., LLC, New York, NY, USA). The ligands were prepared
in LigPrep module at pH 7. Conformer generation was car-
ried out using MacroModel with the force field OPLS_2005.
The solvent model was used with distance-dependent
dielectric. The pharmacophores were defined with a root
Evaluation of chalcones and aurones as MAO
inhibitors
Prior to a complete evaluation of the potential MAOi activ-
ity of all chalcones and aurones studied in this work, a
preliminary screening of all compounds was carried out for
rat MAO-A and MAO-B. The inhibition percentages of iso-
form B by 10 lM solutions of chalcones 3a–h and aurones
4a–h are listed in Table 1.
ꢁ
mean square deviation (RMSD) lower than 1 A. The phar-
macophore features for the molecules applying a set of fea-
tures defined in phase that include hydrogen bond acceptor
(A), hydrogen bond donor (D), hydrophobic moiety (H), neg-
ative group (N), positive group (P) and aromatic ring (R).
Based on the experimental data, the compounds with
IC50 ≤ 12 lM (pIC50 ≥ ꢀ1.08) were considered as active,
while those with IC50 ≥ 13 lM (pIC50 ≤ ꢀ1.11) as inactive.
Hypotheses were obtained for variation of number of sites
and the number of matching active compounds (from 5 to
3). The common pharmacophore was obtained using four
sites that matched in the active ligands. Active and inactive
molecules were then scored for a given pharmacophore
using default parameters: 1 for site, volume, vector, selectiv-
ity, and 1.1 for number of matches. Top ranking hypotheses
were subjected to 3D-QSAR analysis for which grid spacing
All compounds exhibited some degree of selectivity as
MAO-B inhibitors, showing little or no activity vis-a-vis
MAO-A. This selectivity of chalcones has been reported by
different groups before and, aurones seem to follow the
same trend.
ꢂ
These preliminary observations reveal some interesting
structural features regarding the activity of these com-
Table 2: IC50 values for chalcones and aurones with higher activ-
ity as rMAO-B inhibitors
IC50 (lM)a
ꢁ
was 1 A and maximum PLS factors 1.
Compound
MAO-A
MAO-B
Selectivity index (SI)b
Results and Discussion
3a
3d
3g
3h
4a
4d
4g
4h
>100
>100
>100
>100
>100
>100
>100
>100
10.9 ꢁ 0.0
6.1 ꢁ 0.1
6.8 ꢁ 2.1
2.8 ꢁ 0.2
17.8 ꢁ 0.3
26.3 ꢁ 0.3
11.6 ꢁ 0.2
13.5 ꢁ 0.0
>9.2
>16.4
>14.7
>35.7
>5.6
>3.8
>8.6
>7.4
The preparation of the chalcones and aurones listed in
Table 1 is described in Scheme 1 (56). 2-Hydroxy-5- or -
6-methoxyacetophenone (2a–b) was obtained by regiose-
lective alkylation of the respective dihydroxyacetophenone
1a–b (38) and treated with various substituted benzalde-
hydes in ethanolic KOH to give chalcones 3a–h. Prepara-
tion of the corresponding aurones by oxidative cyclization
of the obtained chalcones was achieved by a modifica-
aIC50 values are determined in triplicate in the range of 1–100 lM.
bSelectivity Index: expressed as the ratio IC50 (MAO-A) / IC50
(MAO-B).
tion of
a reported procedure. Sekizaki described a
6
Chem Biol Drug Des 2014

Chalcones and Aurones as Selective MAO-B Inhibitors
A
B
Figure 2: (A) Pharmacophoric landscapes;
(B) Superimposition of these features on the
structure of 3h.
pounds as MAO-B inhibitors. Thus, irrespective of the
substitution pattern of ring B, the inhibitory activity of all
the compounds was stronger when R2 = OCH3 on ring A,
than when R1 = OCH3. The only exception to this was the
pair 3c/3g. Bromination of ring B at R3 also seemed to
improve the inhibitory activity of the chalcone/aurone, as
can be seen by a comparison of pair 3e/h and 4e/h.
Then, we evaluated in more detail the best inhibitors of the
two sets considering the substitution pattern. The obtained
IC50 values of compounds 3a, 3d, 3g, 3h, 4a, 4d, 4g,
and 4h as MAO-B inhibitors and their selectivity indexes
are listed in Table 2.
residues in the active site of MAO-B and a hydroxyl substi-
tuent on ring A. Of all the compounds, chalcone 3h was
the best MAO-B inhibitor, with an IC50 of 2.8 lM. Interest-
ingly, compound 3h exhibited both structural features
mentioned above, an adequate substitution pattern on ring
A and a brominated ring B.
Analysis of the data of Table 2 shows that chalcones (3)
are in general more active than the corresponding aurones
(4). As discussed below, this may be related with the fact
that cyclization in the latter compounds eliminates the pos-
sibility of establishing hydrogen bonds between amino acid
Table 3: Experimental and predicted IC50 values of training set
compounds based on 3D-QSAR hypothesis
Compound
Experimental pIC50 (lM)
Calculated pIC50 (lM)
3a
3d
3g
3h
4a
4d
4g
4h
ꢀ1.03
ꢀ0.78
ꢀ0.83
ꢀ0.45
ꢀ1.28
ꢀ1.41
ꢀ1.06
ꢀ1.13
ꢀ0.86
ꢀ0.82
ꢀ0.72
ꢀ0.65
ꢀ1.34
ꢀ1.34
ꢀ1.17
ꢀ1.27
Figure 3: Plot of predicted versus experimental pIC50 values for
the QSAR model applied to the chalcones and aurones of
Table 3. The dashed line shows the linear fit for the theoretical
versus the experimental values, with
a correlation coefficient
r2 = 0.822 and a standard deviation of 0.16.
A
B
C
Figure 4: Combined effect from 3D-QSAR hypotheses. Visualization of substituents effect: blue cubes showing positive potential while
red cubes showing negative potential of (A) hydrogen bond donor, (B) electron-withdrawing feature, and (C) hydrophobic features.
Chem Biol Drug Des 2014
7

Morales-Camilo et al.
Figure 5: Binding mode of 3h (White)
inside of the active site of hMAO-B. The
amino acid residues lining the active site of
MAO-B are displayed in green.
Our results are in agreement with previous reports where
chalcone derivatives prepared by Chimenti et al. (23), and
Desideri et al. (59), showed a similar pattern, in which an
OH group as R1 and an OCH3 as R3, and a halogen atom
two aromatic groups (R), and hydrophobic substituents
(H). Figure 2A describes the pharmacophoric features
ꢁ
present in the set, with the distances in A between each
pair. Figure 2B depicts these features, as applied to the
structure of 3h, the best inhibitor of the set.
0
as R4 led to the highest inhibitory activity.
The best regression taking into account these features led
to predicted pIC50 values that correlated with the corre-
sponding experimental values listed in Table 3, with a corre-
lation coefficient R2 = 0.822. Figure 3 is a plot of these data.
Computational
3D-QSAR studies
As an attempt to identify a common pharmacophore con-
ferring the MAO-B inhibitory properties, a 3D-QSAR study
was carried out.
A pictorial description of the best regression obtained is
shown in Figure 4, for compound 3h. Blue pharmaco-
phoric features correspond to positive contributions to the
activity, whereas negative ones are represented in red.
Thus, for compound 3h, these features include a hydroxyl
substituent on ring A, capable of forming a hydrogen bond
with the carbonyl group; a second hydrogen-bond-accept-
ing methoxy group on ring A, para to the OH substituent,
and a hydrophobic Br substituent on ring B.
The set of 8 compounds selected as the most active
MAO-B inhibitors was then subjected to a three-dimen-
sional structure–activity relationship (3D-QSAR) study,
aimed to find the structural features responsible for their
activity. This approach allowed us to relate quantitatively,
by means of a partial least squares regression, three-
dimensional descriptors with the measured IC50 values of
the studied compounds. Inspection of the structures
revealed pharmacophoric features that might be responsi-
ble for their activity, and which were adopted as a starting
hypothesis in the search of relevant 3D descriptors. These
features included hydrogen-bond-accepting (A) groups,
Docking studies
We next employed molecular mechanics studies to search
for interactions within the active site of MAO-B that might
support the 3D-QSAR picture developed in the previous
Figure 6: Superimposition of the structure
of chalcone 3h (green), docked into the
active site of hMAO-B (amino acids are
displayed in cyan), with the main
pharmacophoric features of this inhibitor,
obtained from the 3D-QSAR studies.
8
Chem Biol Drug Des 2014

Chalcones and Aurones as Selective MAO-B Inhibitors
section. The active site of MAO-B is formed by an
entrance and a binding cavity, with aromatic amino acid
residues aligned in such a way those inhibitors with long,
planar ring systems, capable of developing p–p interac-
tions with these residues are well accommodated within
the site. This may be the reason why chalcones and auro-
nes are selective MAO-B inhibitors, and are less active vis-
and structure–activity relationships led to the identification
of possible structural modifications to generate more and/
or better interactions of these compounds with the MAO-
B isoform, thereby increasing the activity and selectivity of
these compounds as MAO-B inhibitors. Our suggested
modifications await the future synthesis and evaluation of
novel aurones and chalcones as MAO-B inhibitors, a goal
which is beyond the scope of the present work.
ꢂ
a-vis MAO-A, which does not possess an active site with
these characteristics (60). The MAO-B binding site
includes a pair of tyrosines (Y435 and Y398) close to the
FAD cofactor, which should also be capable of forming p–
p interactions with an electron-poor aromatic ring. This
may also be the reason why the aromatic ring A of chal-
cones is preferentially situated close to the isoalloxazine
molecule. In the case of the less active aurones, our dock-
ing studies revealed the opposite situation, with ring B
positioned close to the FAD fragment. This is probably a
consequence of the lack of coplanarity of rings A and B in
aurones, forcing the molecule to locate the aromatics cen-
ters perpendicularly between them, within the active site of
the enzyme. In fact, optimization of all the aurone struc-
tures investigated in these docking studies led to an aver-
age dihedral angle of 125° between the two rings. When
this planarity is maintained, as in all investigated chalcones
(with an average dihedral angle of 180°), ring A locates
close to the FAD cofactor. Docking of the most active au-
rones and chalcones of the series led in all cases to final
stable conformations which illustrate the points mentioned
above. Thus, for the most active member of the series,
chalcone 3h, ring A is positioned close to the FAD frag-
ment and flanked by Y435 and Y398 (Figure 5).
Acknowledgments
This work was supported by FONDECYT (Research Grant)
No. 1090037 (AF), 1130185 (MR-P), and 1120128 (COS).
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