UNIVERSIDAD DE SANTIAGO DE COMPOSTELA FACULTAD DE FARMACIA DEPARTAMENTO DE QUIMICA ORGANICA Análogos rígidos del resveratrol con interés neuro y cardioprotector. Diseño, síntesis y evaluación farmacológica Memoria para optar al grado de Doctor en Farmacia presenta Maria Carmen Picciau Santiago de Compostela, 2010
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UNIVERSIDAD DE SANTIAGO DE COMPOSTELA
FACULTAD DE FARMACIA
DEPARTAMENTO DE QUIMICA ORGANICA
Análogos rígidos del resveratrol con interés
neuro y cardioprotector. Diseño, síntesis y
evaluación farmacológica
Memoria para optar al grado de
Doctor en Farmacia presenta
Maria Carmen Picciau
Santiago de Compostela, 2010
D. Eugenio Uriarte Villares, Catedrático de Química Orgánica, Dña. Lourdes
Santana Penín, Profesora Titular de Química Orgánica y D. Elías Quezada González,
Investigador contratado dentro del Programa Ángeles Alvariño en el Departamento de
Química Orgánica de la Universidad de Santiago de Compostela
CERTIFICAN:
Que la memoria titulada ANÁLOGOS RÍGIDOS DEL RESVERATROL CON
INTERÉS NEURO Y CARDIOPROTECTOR. DISEÑO, SÍNTESIS Y EVALUACIÓN
FARMACOLÓGICA, que para optar al grado de Doctora en Farmacia presenta
MARIA CARMEN PICCIAU, ha sido realizada bajo nuestra dirección, en el
Departamento de Química Orgánica de la Facultad de Farmacia de la Universidad de
Santiago de Compostela y en el Departamento Fármaco Químico Tecnológico de la
Universidad de Cagliari.
Y considerando que el trabajo constituye tema y labor de Tesis Doctoral,
autorizamos su presentación en la Universidad de Santiago de Compostela. Y para que conste, expedimos el presente certificado en Santiago de Compostela
Å angstrom AcOEt acetato de etilo AcOH ácido acético ADN ácido desoxirribonucleico Ar argón ºC grados Celsius δ desplazamiento químico en ppm d doblete DCC diciclohexilcarbodiimida dd doble doblete DEA dietilanilina DIBAL-H diisobutilaluminio hidruro DMAP dimetilaminopiridina DMF N,N-dimetilformamida DMSO dimetilsulfóxido EtOH etanol g gramo μg microgramo h hora Hz hertzio J constante de acoplamiento m multiplete M molaridad μM micromolar MAO monoaminooxidasa MeOH metanol mg milígramo min minuto mL mililitro mmol milimol m/z relación masa/carga μL microlitros N normalidad NA noradrenalina PE fenilefrina P.f. punto de fusión ppm partes por millón RMN resonancia magnética nuclear s singlete sa singlete ancho t triplete THF tetrahidrofurano TMS tetrametilsilano U unidades de trombina
III.-Relación de compuestos finales obtenidos
III.-Relación de Compuestos Finales Obtenidos
XI
O O
OMe
2
6O O
OMeO O
MeO
5
O O
OMe
OMe
OMe
4O O
OMe
OMe
3O O1
7
O O
Cl
MeO
8
O O
Cl
OMe
9O O
Cl
10O O
Br
MeO
11O O
Br
OMe
12O O
Br
OMe
7a
O O
Cl
HO
8aO O
Cl
OH
9aO O
Cl
OH
10a
O O
Br
HO
11aO O
Br
OH
12aO O
Br
OH
OMe
13aO
S
O
13bO
S
O
13cO
NH
O14a
O
S
OMeO14b
O
S
OMeO14cO
NH
OMeO
15aO
S
O
MeO
15bO
S
O
MeO
15cO
NH
O
MeO
16aO
S
O
OMe
MeO
16bO
S
O
OMe
MeO16cO
NH
O
OMe
MeO
III.-Relación de Compuestos Finales Obtenidos
XII
OMeO
18 19
OMeO
OMe
OMe
OMeO
OMe23
OMe
OMeO
OMe24
25
OOMe
OMeO
OMe
OMe
OMe
OMe26
O
OMe
OMe
OMe28
O
OMe
OMe29
OOMe
30 33
OOMe
O
OMe
OMe34 35
O
OMe
OMe
OMe
O
BrOMe
OMe
OMe
38 39O
BrOMe
OMeO
BrOMe
40
O
OMe
OMe
OMe
O43
O
OMe
OMeO44
OOMe
O45
1.- Introducción
1.-Introducción
3
Las cumarinas son una clase de compuestos de origen natural ó sintético con
actividades farmacológicas y terapéuticas muy diferentes. En los últimos años las
cumarinas han sido aisladas en más de 700 especies de diferentes familias de
Angiosperamae, Umbelliferae, Apiaceae, Rutaceae, donde se pueden encontrar de
forma libre o glicosilada en las hojas, raíces, semillas y frutos. El esqueleto básico de
esta familia de compuestos es la cumarina (2H-1-benzopiran-2-ona) (Figura 1), aislada
por primera vez en el año 1820 de las semillas de Coumarona Odorata Aube (Dpterix
Odorata).1 Desde el punto de vista estructural, las cumarinas se pueden clasificar en
cumarinas simples, o asociadas a otros anillos como por ejemplo: furanocumarinas,
piranocumarinas, biscumarinas, triscumarinas o cumarinolignanos.2
O O
O OO O OO
OR
OR
O O
O OHO
RO
O O
O OHO
RO
OO
O
O OO
O
RO
Ar
R
Cumarina (2H-1-benzopiran-2-ona)
FurocumarinasPiranocumarinas
Biscumarinas Triscumarinas
Cumarinolignanos
Figura 1
1 Soine, T. O.; J. Pharm. Sci. 1964, 53, 231. 2 (a) Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E., Current Medicinal Chemistry 2005, 12, 887; (b) Santana, L.; Uriarte, E; Roleira, F.; Milhazes, N.; Borges, F., Current Medicinal Chemistry 2004, 11, 3239; (c) Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Frontiers in Medicinal Chemistry, 2009, 4, 23. (d) Surya K. De; Richard A. Gibas, Synthesis 2005, 8, 1231.
1.-Introducción
4
1.1.1. Actividad biológica de las cumarinas
Muchas son las actividades biológicas asociadas a las cumarinas: antimicrobianas,
antivirales, inhibidores enzimáticos (como es el caso de la MAO), anticoagulantes,
vasodilatadores y antioxidantes, entre otras.2,3,4
La Warfarina, el Carbocromeno y el Sintrón (Figura 2) son tres conocidos ejemplos
de la familia de las cumarinas con actividad cardioprotectora, debido a su acción
vasodilatadora e inhibidora de la agregación plaquetaria.4
O OO
N CH3
H3C
O
O
H3C O O
Warfarina
OH
O
CH3
Carbocromeno
O O
OH
O
CH3
NO2
Sintrón
Figura 2
Una serie de derivados sintéticos cumarinicos han sido identificados como
inhibidores de la MAO (iMAO). La MAO es una enzima que presenta dos conocidas
isoformas –MAO-A y MAO-B– y que interviene en la degradación de aminas, teniendo
un papel fisiológico muy importante en la desaminación de la adrenalina, noradrenalina
y serotonina (preferentemente la MAO-A) y en la desaminación de la ß-feniletilamina y
bencilamina (preferentemente la MAO-B).5
Los iMAO son una clase de compuestos que actúan bloqueando la acción enzimática
de la MAO de manera que los iMAO-A pueden ser efectivos en el tratamiento de la
depresión y los iMAO-B útiles en el tratamiento del Parkinson.6
Además de estas actividades, algunas cumarinas han mostrado actividad inhibitoria
frente a la tirosinasa. La tirosinasa es un enzima clave en la vía de biosíntesis de la
melanina y cataliza la conversión limitante inicial de tirosina a DOPA.7 Esta enzima
está presente en plantas, animales, hongos y bacterias.8,9 Es una enzima dependiente del
cobre, con dos sitios de unión al mismo.7 Además de su función principal como 3 Leiro, J.; Álvarez, E.; Arranz, J.A.; Laguna, R.; Uriarte, E. y Orallo, F. J. Leukoc. Biol. 2004, 75, 1156. 4 Orallo, F.; Álvarez, E.; Camiña, M; Leiro, J.M.; Gómez, E. y Fernández, P., Mol. Pharmacol. 2002, 61, 294. 5Grimsby, J.; Lan, N.C.; Neve, R.; Chen, K.; Shih, J.C. J. Neurochem. 1990, 55, 1166. 6 Harfenist, H.; Heuseur, D.J.; Joyner, C.T.; Batchelor, J.F.; White, H.L. Journal of Medicinal Chemistry 1996, 39, 1857. 7 Lerch, K. Life Chem. Rep. 1987, 5, 221-234. 8 Whitaker, J. R., In Food Enzymes: Structure and Function, ed. D. Wong. Chapman and Hall 1995, p. 284. 9 Kowalski, S. P.; Eannetta, N. T.; Hirzel, A. T.; Steffens, J. C. Solanum berthaultii. Plant Physiol. 1992, 100, 677.
1.-Introducción
5
tirosinahidroxilasa, la tirosinasa humana tiene actividad DOPA oxidasa, 5,6-
dihidroxiindol oxidasa, y quizá 5,6-dihidroxiindol-2-ácido carboxílico oxidasa, y regula
varias de las etapas de esta vía. El estudio de los inhibidores de la tirosinasa tiene hoy
en día un fuerte impacto en la industria y la economía. Recientemente Masamoto et al.
han estudiado la relación estructura-actividad de 18 cumarinas por su actividad
inhibitoria frente a la tirosinasa de hongo, encontrando que la esculetina tiene la más
fuerte actividad inhibidora de todos los compuestos estudiados.10 En contraste con este
estudio, Sollai et al. han demostrado que la esculetina es un substrato de la tirosinasa
más que un inhibidor, y la umbelliferona es un inhibidor de dicha enzima.11
O O
HO
HO O OHO
Esculetina Umbeliferona
1.1.2. Síntesis de cumarinas
La síntesis de las cumarinas y de sus derivados es una área de gran interés por el gran
número de compuestos tanto de origen natural como sintéticos, que contienen este
núcleo heterocíclico y porque son muchas veces utilizadas en la síntesis de otros
productos como piranocumarinas, furocumarinas, 2-acil-resorcinoles, etc.12
Los primeros métodos de síntesis para la obtención de estos compuestos presentaban
demasiados pasos y en general bajos rendimientos,13 pero hoy en día las rutas sintéticas
más utilizadas recurren a reacciones de condensación de Pechman, Perkin,
Knoevenagel, Reformatsky o Wittig. La primera es, sin duda, la más usada por la
simplicidad de los materiales de partida y por los elevados rendimientos obtenidos.14,15
Los procedimientos sinteticos referidos anteriormente, han sido ampliamente
descritos en la bibliografía.2
10 Masamoto, Y.; Murata, Y.; Baba, K.; Shimoishi, Y.; Tada, M.; Takahata, K. Biol. Pharm. Bull. 2004, 27, 422. 11 Sollai, F.; Zucca, P.; Sanjust, E.; Steri, D.; Rescigno, A. Biol. Pharm. Bull. 2008, 31, 2187. 12 Singh, Pankajkumar R.; Singh, De Vendrapratap U.; Samant, Shriniwas D., Synlett 2004, 11, 1909. 13 Roman, Joc; Kozhkov, V.; Larock, Andrichard C., J. Org. Chem. 2003, 68, 6314. 14 Surya K. De; Richard A. Gibbs, Synthesis 2005, 8, 1231. 15 Pankajkumar, R. Singh; Devendrapratap, U. Singh; Shriniwas, D. Samant., Synlett 2004, 11, 1909.
1.-Introducción
6
1.2. El resveratrol
El resveratrol es un compuesto polifenólico de origen natural producido por algunas
especies de espermatofitas,16 como las vides, en respuesta a un daño sufrido por ejemplo
una infección por hongos o exposición a la radiación UV. Se encuentra en la piel de la
uva y no en la pulpa, por lo cual existe en mayor concentración en el vino tinto que en el
vino blanco (donde el tiempo de contacto con la piel de la uva en la fermentación es
mucho más grande – mayor tiempo de maceración). El interés por este compuesto y sus
derivados empezó cuando los estudios epidemiológicos establecieron una relación
inversa entre el consumo de vino tinto y la incidencia de enfermedades
cardiovasculares,13 además de las propiedades hemostáticas y del aumento del HDL
circulante descrita para el etanol.17 A partir de este momento las propiedades del
resveratrol así como de análogos fenólicos, la mayoría de estructura flavonoide, han
sido extensamente estudiadas.
Estructuralmente el resveratrol es el 3,4’,5-trihidroxiestilbeno que, debido a la
presencia del doble enlace, puede asumir las dos formas isoméricas cis y trans (Figura
3).
HO
OH
OH
HO
OHtrans-resveratrol cis-resveratrol
OH
Figura 3
La forma trans del resveratrol, parece ser la responsable de las principales
propiedades farmacológicas de esta molécula. Es, hoy en día, un producto comercial
común, a partir del que se puede obtener por irradiación UV la forma cis.13
Además del resveratrol, han sido aislados y identificados en muchas especies de
plantas algunos de sus productos de oxidación, oligómeros llamados viniferinas; entre
estas en particular la ε- y la δ-viniferina (Figura 4) y la α-, β-, y γ-viniferina,
respectivamente, trímero, tetrámero y un oligómero polimerizados.
Su biosíntesis está siempre correlacionada a situaciones de estrés o a infecciones y
han demostrado poseer actividad biológica contra un amplio rango de agentes
patógenos18. También están presentes en el vino y en algunos alimentos aunque su papel
ha sido menos estudiado.
O
OH
HO
HO
HO
OH
O
HO
HO
OH
HO
OH
trans-δ-viniferina trans-ε-viniferina
Figura 4
1.2.1. Actividad biológica del resveratrol
Muchas son las actividades biológicas asociadas a este compuesto19, entre ellas:
antioxidante, antiinflamatoria, vasorelaxante, antiagregante plaquetaria, antitumoral e
inhibidora enzimática. Como antiinflamatorio es capaz de discriminar entre las dos
diferentes isoformas de ciclooxigenasa (COX) y es un potente inhibidor de la COX1.20
Las dos isoformas del resveratrol resultan capaces de inhibir la actividad de la MAO. El
cis-resveratrol es menos eficaz que el trans como inhibidor de la MAO-A y de la MAO-
B.21 La actividad inhibitoria de hidroxiestilbenos frente a la tirosinasa de hongo ha sido
también evaluada.22 El resveratrol ha mostrado actividad inhibitoria DOPA oxidasa más
fuerte que el ácido kojico.23
18 Jeandet, P. et al. J. Agric. Food Chem. 2002, 50, 2731. 19 Aggarwal, B.B.; Bhardwaj, A.; Aggarwal, R.S.; Seeram, N.P.; Shishodia, S.; Takada, Y. Anticancer Res. 2004, 24, 2783.; Orallo, F. Biological effects of cis-versus trans-resveratrol. En: Resveratrol in Health and Disease. Editores Aggarwal, B.B., Shishodia, S. University of Texas M.D. Anderson Cancer Center, Houston, USA. CRC Press, Boca Raton, USA, 2005, pág. 577-600.; Orallo, F. Curr. Med. Chem. 2008, 15, 1887. 20 Szewczuk, L.M.; Forti, L.; Stivala, L.A.; Penning, T. M. J. Biol. Chem. 2004, 279, 22727. 21 Yánez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Commun. 2006, 344, 688. 22 Likhitwitayawuid, K. Stilbene with tyrosinase inhibitory activity. Curr. Sci. 2008, 94, 44. 23 Likhitwitayawuid, K.; Sritularak, B.; De-Eknamkul, W. Tyrosinase inhibitors from Artocarpus gomezianus. Planta Med. 2000, 66, 275.
2.- Objetivos
2.-Objetivos
11
Desde hace tiempo nuestro grupo de Química Farmacéutica de la Facultad de
Farmacia se ocupa de la síntesis, caracterización y estudio de compuestos con
estructura cumarínica. En el presente trabajo nos hemos planteado los siguientes
objetivos fundamentales:
1. El diseño y la síntesis de nuevas series de híbridos cumarina-resveratrol en los
cuales el anillo cumarínico comparte el anillo bencénico y el doble enlace con el
resveratrol de forma que este queda bloqueado como isómero trans. Algunos
derivados obtenidos previamente ya han sido sometidos a los correspondientes
ensayos farmacológicos demostrando no solo una muy buena actividad
vasodilatadora e inhibidora de la agregación plaquetaria,1a sino también una
interesante actividad como inhibidores de la MAO1b,1c y de la tirosinasa.2
(Figura 5).
O O
R
R
Figura 5
2. El diseño y la síntesis de 3-heteroarilcumarinas. Son análogos de los compuestos
anteriores en los que uno de los anillos bencénicos del resveratrol se sustituye
por un heterociclo aromático.
3. El diseño y la síntesis de 2-aril-benzofuranos. Estas moléculas también
incorporan el fragmento estilbénico del trans-resveratrol, si bien en este caso es
distinta la posición relativa de ambos anillos aromáticos. También se pretende la
reducción del doble enlace del anillo del furano para obtener una serie de 2-aril-
dihidrobenzofuranos, moléculas análogas a las viniferinas, como ya
mencionamos dímeros del resveratrol (Figura 6).
1 a) Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yanez, M.; Fraiz, N.; Alcaide, C.; Cano, E. and Orallo, F. Bioorganic & Medicinal Chemistry Letters 2006, 16, 257; b) Joao Matos, M.; Viña, D.; Quezada, E.; Picciau, C.; Delogu, G.; Orallo, F.; Santana, L.; Uriarte, E. Bioorganic & Medicinal Chemistry Letters 2009, 19, 3268; c) Joao Matos, M.; Viña, D.; Picciau, C.; Delogu, G.; Orallo, F.; Santana, L.; Uriarte, E. Bioorganic & Medicinal Chemistry Letters 2009, 19, 5053. 2 Fais, A.; Corda, M.; Era, B.; Fadda, M.B.; Matos, M.J.; Quezada, E.; Santana, L.; Picciau, C.; Podda, G. And Delogu, G. Molecules 2009, 14, 2514.
2.-Objetivos
12
RO
OR
OOR
RO
OR
OOR
O
OH
OH
OH
HO
OH
trans-ε-viniferina
Figura 6
4. Las series de moléculas de ambos tipos estructurales que se sintetizarán serán
seleccionados con el objetivo de su estudio en diferentes campos
farmacológicos. Inicialmente dirigidos al estudio de su actividad i-MAO y
efecto cardioprotector.
3.-Discusión de resultados
3.-Discusión de Resultados
15
3.1 Síntesis y evaluación farmacológica de híbridos cumarina-resveratrol
La síntesis de los híbridos cumarina-resveratrol se llevó a cabo utilizando como
reacción clave la reacción de Perkin,1a,1b a partir de un salicilaldehido y un ácido
fenilacético oportunamente sustituidos, utilizando DCC como agente deshidratante en
DMSO a reflujo, durante 24 h (Esquema 1). Los rendimientos obtenidos son entre 50-
70 % y los productos sólidos se purifican fácilmente por cromatografía en columna,
utilizando como disolvente Hexano/AcOEt.
CHO
OH
COOH
O O
XR1
R2R3
X
R1
R2R3
d X=Cle X=Br
a R1 = MeO; R2 = R3 = Hb R2 = MeO; R1 = R3 = Hc R3 = MeO; R1 = R2 = H
7: X = Cl; R1 = MeO; R2 = R3 = H8: X = Cl; R3 = MeO; R1 = R2 = H9: X = Cl; R2 = MeO; R1 = R3 = H10: X = Br; R1= MeO; R2 = R3 = H11: X = Br; R3= MeO; R1 = R2 = H12: X = Br; R2= MeO; R1 = R3 = H
O O
X
R1
R2R3
7a: X = Cl; R1 = OH; R2 = R3 = H8a: X = Cl; R3 = OH; R1 = R2 = H9a: X = Cl; R2 =OH; R1 = R3 = H10a: X = Br; R1= OH; R2 = R3 = H11a: X = Br; R3= OH; R1 = R2 = H12a: X = Br; R2= OH; R1 = R3 = H
DCC, DMSO
110ºC, 24h
HI/AcOH/Ac2Oreflujo, 24h
Esquema 1
Los derivados metoxilados fueron hidrolizados por tratamiento con HI 57 % en
presencia de ácido acético y anhídrido acético. La reacción fue llevada a cabo a reflujo,
durante 24 h, con rendimientos satisfactorios.
1 a)Hans, N.; Singhi, M.; Sharma, V.; Grover, S.K. Ind. J. Chem. 1996, 35B, 1159; b) Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yanez, M.; Fraiz, N.; Alcaide, C.; Cano, E. and Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257; c) Vilar, S.; Quezada, E.; Alcaide, C.; Orallo, F.; Santana, L.; Uriarte, E. QSAR Comb. Sci. 2007, 26, 317.
3.-Discusión de Resultados
16
Algunas 3-arilcumarinas con grupos metoxilos o hidroxilos que fueron sintetizadas,
han sido evaluadas como vasodilatadores y antiagregantes plaquetarios, obteniéndose
muy buenos resultados en los ensayos biológicos realizados, mostrando algunas una
actividad superior al trans-resveratrol.1b,1c
Los efectos vasorelajantes de los compuestos 7a-12a fueron estudiados en anillos
precontraídos de aorta de rata, en presencia de endotelio.2 La adición acumulativa del t-
RESV o de los nuevos compuestos (1-100 μM) causa la relajación dependiente de la
concentración de las contracciones inducidas por fenilefrina (PE, 1 μM) en los anillos.
Los valores de IC50 son mostrados en la Tabla 1.
Tabla 1. Actividad vasorelajante de los compuestos evaluados
Compuesto PE (1μM)
7a 36.63 ± 2.46*
8a **
9a 57.63 ± 3.87*
10a 48.79 ± 3.27*
11a **
12a 46.67 ± 3.13*
t-resv 3.12 ± 0.26 * P<0.01 versus el correspondiente valor de IC50 del t-resv
** Inactivo a 100 μM (más alta concentración evaluada). A concentraciones más altas el compuesto precipita.
Todos los compuestos sintetizados resultan menos activos que el trans-resveratrol y
la introducción de un grupo hidroxilo en la posición 4 del anillo bencénico de la
posición 3 de la cumarina causa la inactividad del compuesto.
Los ensayos de la actividad antiagregante plaquetaria fueron llevados a cabo
utilizando trombina (0.25 μM) como agente estimulante. Los compuestos 7a, 9a y 11a
resultan más activos que el trans-resveratrol. (Tabla 2).
2 a) Orallo, F.; Alvarez, E.; Camina, M.; Leiro, J.M.; Gomez, E.; Fernandez, P. Mol. Pharmacol 2002, 61, 294; b) Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Befani, O.; Turini, P.; Alcaro, S.; Ortuso, F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.
3.-Discusión de Resultados
17
Tabla 2. Actividad antiagregante plaquetaria de los compuestos evaluados
Compuesto Trombina (0.25
U/mL)
7a 91.36 ± 6.13*
8a **
9a 6.41 ± 2.15*
10a **
11a 20.1 ± 1.35*
12a **
t-resv 195.50 ± 13.82 * P<0.01 versus el correspondiente valor de IC50 del t-resv
** Inactivo a 100 μM (más alta concentración evaluada) A concentraciones más altas el compuesto precipita
Experimentos posteriores en estos momentos están en curso, con objeto de clarificar
el mecanismo preciso por el cual los derivados híbridos cumarina-resveratrol producen
efectos de vasodilatación y antiagregación plaquetaria.
Sobre la base de resultados previos obtenidos por nuestro grupo, una serie de nuevos
compuestos han sido sintetizados con el objetivo de estudiar la influencia de distintos
sustituyentes en varias posiciones en la actividad inhibitoria de la MAO. Se trata de una
serie de híbridos cumarina-resveratrol que presentan un grupo metilo en la posicion 6 de
la cumarina y grupos metoxilos en el anillo bencénico de la cumarina en la posición 3,
para compararla con el análogo no substituido (Esquema 2).
Los ensayos enzimáticos demuestran que estos compuestos son en general
inhibidores selectivos de MAO-B ( Tabla 4).
De los resultados obtenidos se deduce que el número y la posición de los grupos
metoxilo sobre la estructura de benzopirano es más importante para la actividad que la
naturaleza del sustituyente heterocíclico en la posición 3.
La sustitución en posición 6 ó 7 del anillo de benzopirano conduce en general a un
incremento de la actividad i-MAO, mientras que la sustitución en posición 5 conduce a
una pérdida de la misma.
3.-Discusión de Resultados
20
Table 4. Valores de IC50 e índice de selectividad MAO-B [IC50 (MAO-A)]/[IC50 (MAO-B)] de los nuevos compuestos y los inhibidores de referencia.
Cada valor de IC50 es la media ± S.E.M. de cinco experimentos (n = 5). Nivel de significación estadística: aP <0,01 en comparación con el IC50 correspondientes a valores obtenidos frente a la MAO-B, fueron determinados por ANOVA / Dunnett s. bLos valores obtenidos bajo el supuesto de que el CI50 correspondiente en contra de la MAO-A es la concentración más alta ensayada (100 µM ó 1 mM) ** Inactivos a 100 mM (la concentración más alta ensayada). A mayores concentraciones los compuestos precipitan. SI: El índice de selectividad hMAO-B = IC50 (hMAO-A) / IC50 (hMAO-B).
3.2 Síntesis y evaluación farmacológica de híbridos benzofurano-resveratrol
La preparación de los 2-arilbenzofuranos intentó llevarse a cabo inicialmente a partir
de 3-fenilcumarinas, utilizando DIBAL-H a -78 °C y a continuación añadiendo H2SO4
2N y calentando la reacción a reflujo durante 3 h (Esquema 5).
A new series of 3-phenylcoumarins as potent and selective MAO-B inhibitors
Maria Joao Matos a,b,*, Dolores Viña a,c, Elias Quezada a,b, Carmen Picciau b, Giovanna Delogu b,Francisco Orallo c, Lourdes Santana a, Eugenio Uriarte a
a Departamento de Química Orgánica, Facultad de Farmacia, 15782 Santiago de Compostela, Spainb Dipartimento Farmaco Chimico Tecnologico, Facoltà di Farmacia, 09124 Cagliari, Italyc Departamento de Farmacología, Facultad de Farmacia, 15782 Santiago de Compostela, Spain
a r t i c l e i n f o
Article history:Received 24 March 2009
a b s t r a c t
6-Methyl-3-phenylcoumarins 3–6 were designed, synthesized and evaluated as monoamine oxidase A
es) are a large family of compounds, inhibitors (iMAO). These modifications were studied to find out
Revised 17 April 2009Accepted 20 April 2009Available online 24 April 2009
and B (MAO-A and MAO-B) inhibitors. The synthesis of these new compounds (resveratrol–coumarinhybrids) was carried out with good yield by a Perkin reaction, from the 5-methylsalicylaldehyde andthe corresponding phenylacetic acid. They show high selectivity to the MAO-B isoenzyme, with IC50 val-ues in the nanomolar range. Compound 5 is the most active compound and is several times more potentand selective than the reference compound, R-(�)-deprenyl.
� 2009 Elsevier Ltd. All rights reserved.
of natural and synthetic origin, that show numerous biologicalactivities.1 Recent studies pay special attention to their antioxida-
2–6
how these changes can contribute to the biological activity of thesemolecules, helping to understand a structure–activity relationship
tive, anticarcinogenic and enzymatic inhibition properties. In re-gard to the monoamine oxidase (MAO) inhibition, the recentfindings revealed that MAO-A and MAO-B affinity and selectivitycan be efficiently modulated by appropriate substitutions in thecoumarin ring, in particular in the 3:4 and 6:7 positions.7–11
The resveratrol (3,40,5-trihydroxystilbene) is a phytoalexin ofnatural origin present in spermatophytes species such as vines,in response to damage. Resveratrol was already studied as antiox-idant, anti-inflammatory, cardioprotective (vasodilatory and plate-let antiaggregatory activities), anticancer, enzymatic inhibitor,proving to be very efficient in a large group of in vitro, ex vivoand/or in vivo experiments.12–15 The resveratrol’s cis and trans iso-mers are inhibitors of the MAO activity. cis-Resveratrol is less effec-tive than trans-resveratrol as inhibitor of MAO-A and MAO-Bactivities.16
Due to these coincident properties, it seems to be interesting todesign and synthesize hybrids that incorporate the skeleton ofthose two kinds of molecules.17,18 In the present compounds theresveratrol nucleus is blocked by the coumarin ring and can onlyassume the trans isomeric form. A series of these molecules, withdifferent number and position of methoxy groups in the 3-phenylring (compounds 3–6), was synthesized and evaluated as MAO
0960-894X/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmcl.2009.04.085
(SAR).MAO is a FAD-containing enzyme with two known isoforms
(MAO-A and MAO-B) and is present in the mitochondrial outermembrane of glial, neuronal and other cells.19 MAO enzymes inter-vene in the monoamines degradation and carry out an importantphysiologic function in the adrenaline, noradrenaline and seroto-nin deamination (preferentially MAO-A) and in the b-phenylethyl-amine and benzylamine deamination (preferentially MAO-B).20
This enzymatic function increases the synaptic concentration ofthese neurotransmitters and conditions to a great extent the neu-rone’s excitement of those possessing receptors for thesemediators.21
The iMAO are a class of compounds that act by blocking theMAO enzymatic action, being used by several years in the treat-ment of the depression and anxiety diseases (iMAO-A) or in Parkin-son’s disease (iMAO-B).22 Nowadays is being studied also in theAlzheimer’s disease.23
The active sites of these two isoenzymes (MAO-A and MAO-B)are not completely known and for this reason there is a lack ofinformation in respect of how the inhibitors act selectively inone of the two of them.10 Recent X-ray crystal structures ofMAO-B24,25 and MAO-A,21,26 completed with irreversible andreversible inhibitors, can be used in the future as a tool to thestructural basis to help understanding the selective enzyme–ligandrecognition and drug development of iMAOs.21
With the aim of finding out new structural features for the MAOinhibitory activity and selectivity, we decided in this work to ex-plore the importance of the number and position of different meth-oxy groups under the benzenic ring in 3-position (compounds427,28–6), to establish a relation between them and with the nonsubstituted analogue (compound 327–29).
The preparation of these 6-methyl-3-phenylcoumarins was per-formed via the classical Perkin reaction.29 This reaction was carriedout by condensation of the 5-methylsalicylaldehyde 1 and the con-veniently substituted phenylacetic acids 2, with N,N0-dicyclohexyl-carbodiimide (DCC) as dehydrating agent, under DMSO reflux,during 24 h (Scheme 1). The reaction to obtain 3–6 is very cleanand the yields are between 60% and 70%.30–33 The obtained prod-ucts are easy to purify by flash chromatography, using a mixtureof hexane/ethyl acetate in a proportion 9:1 as eluent.
The inhibitory MAO activity of compounds 3–6 was evaluatedin vitro by the measurement of the enzymatic activity of human re-combinant MAO isoforms in BTI insect cells infected with baculo-virus.8,34 Then, the IC50 values and MAO-B selectivity ratios [IC50
(MAO-A)]/[IC50 (MAO-B)] for inhibitory effects of both, new com-pounds and reference inhibitors, were calculated (Table 1).35
The prepared series of compounds proved to be selective asinhibitor of the MAO-B isoenzyme. Compound 3, none substitutedin the phenyl ring, is by itself very active and selective againstMAO-B isoenzyme. Compound 4 (with a p-methoxy group) has aMAO-B IC50 similar to the R-(�)-deprenyl (reference MAO-B inhib-itor) and is more selective than this one. The most potent moleculeof this family is compound 5, bearing two methoxy groups in 30-and 50-positions (IC50 = 8.98 ± 1.42 nM). This one is two times moreactive and several times more iMAO-B selective than the R-(�)-deprenyl. Compound 6, with 3 methoxy groups, is more active than3 (none substituted) but it loses activity in respect to the mono anddimethoxy derivatives (compounds 4 and 5, respectively). None ofthe described compounds showed a MAO-A inhibitory activity forthe highest concentration tested (100 lM). This iMAO-B selectivity
* Inactive at 100 lM (highest concentration tested). At higher concentrationscompounds precipitate.
a P <0.01 versus the corresponding IC50 values obtained against MAO-B, asdetermined by ANOVA/Dunnett’s.
b Values obtained under the assumption that the corresponding IC50 againstMAO-A is the highest concentration tested (100 lM).
is an important factor to discriminate the potential therapeuticapplication of this kind of molecules.
Comparing the iMAO-B activities of 3 and 4, the introduction ofa p-methoxy group increases the inhibitory activity. Substitutionwith two methoxy groups in the 30- and 50-positions on the phenylring, compound 5, improves the iMAO-B activity. Increasing thenumber of methoxy substituent to three, compound 6, decreasesthe enzymatic inhibitory activity. However, compound 6 is evenbetter than none substituted coumarin 3. The presence of methoxysubstituent in the 3-phenyl ring seems to be important to modu-late and improve the inhibitory enzymatic activity of the 6-methyl-3-phenylcoumarins.
These hybrid compounds with resveratrol–coumarin skeletonshow high selectivity against MAO-B isoenzyme, being active inthe nanomolar range. Introduction of methoxy groups in the phe-nyl ring improves the activity, giving more active and selectivecompounds than the reference ones. These modifications, whichwe studying more deeply, can improve the pharmacologic profileof the synthesized coumarins in the Parkinson’s disease.
. Lett. 19 (2009) 3268–3270 3269
Acknowledgements
Thanks the Spanish Ministerio de Sanidad y Consumo(PI061457 and PI061537) and to Xunta da Galicia (PXIB20304PR,INCITE08PXIB203022PR and 08CSA019203PR) and FondazioneBanco Sardegna (Italy) for financial support. M.J.M. also thanks toMIUR the Ph.D. Grant.
Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.18. Vilar, S.; Quezada, E.; Alcaide, C.; Orallo, F.; Santana, L.; Uriarte, E. Qsar Comb.
Sci. 2007, 26, 317.19. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.20. Grimsby, J.; Lan, N. C.; Neve, R.; Chen, K.; Shih, J. C. J. Neurochem. 1990, 55,
1166.21. Edmondson, D. E.; Mattevi, A.; Binda, C.; Li, M.; Hubalek, F. Curr. Med. Chem.
2004, 11, 1983.22. Harfenist, H.; Heuseur, D. J.; Joyner, C. T.; Batchelor, J. F.; White, H. L. J. Med.
Chem. 1996, 39, 1857.23. Wouters, J. Curr. Med. Chem. 1998, 5, 137.24. Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondson, D. E.; Mattevi, A. Nat.
Struct. Biol. 2002, 9, 22.25. Binda, C.; Li, M.; Hubalek, F.; Restelli, N.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 9750.
. Che
26. Ma, J.; Yoshimura, M.; Yamashita, E.; Nakagawa, A.; Ito, A.; Tsukihara, T. J. Mol.Biol. 2004, 338, 103.
27. Hans, N.; Singhi, M.; Sharma, V.; Grover, S. K. Indian J. Chem., Sect. B 1996, 35,1159.
28. Mohanty, S.; Makrandi, J. K.; Grover, S. K. Indian J. Chem., Sect. B 1989, 28, 766.29. Kamat, S. P.; D́Souza, A. M.; Paknikar, S. K.; Beaucahmp, P. S. J. Chem. Res. (S)
2002, 242.30. 6-Methyl-3-phenylcumarin (3). It was obtained with a yield of 68%. Mp 148–
34. Determination of human monoamine oxidase (hMAO) isoform activity: The effectsof the test compounds on hMAO isoform enzymatic activity were evaluated bya fluorimetric method following the experimental protocol previouslydescribed by us. Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4)containing the test drugs in various concentrations and adequate amounts ofrecombinant hMAO-A or hMAO-B required and adjusted to obtain in ourexperimental conditions the same reaction velocity [165 pmol of p-tyramine/min (hMAO-A: 1.1 lg protein; specific activity: 150 nmol of p-tyramineoxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5 lgprotein; specific activity: 22 nmol of p-tyramine transformed/min/mgprotein)] were placed in the dark fluorimeter chamber and incubated for15 min at 37 �C. The reaction was started by adding (final concentrations)200 lM Amplex� Red reagent, 1 U/mL horseradish peroxidase and 1 mM p-tyramine. The production of H2O2 and, consequently, of resorufin wasquantified at 37 �C in a multidetection microplate fluorescence reader(FLX800TM, Bio-Tek� Instruments, Inc., Winooski, VT, USA) based on thefluorescence generated (excitation, 545 nm, emission, 590 nm) over a 15 minperiod, in which the fluorescence increased linearly.Control experiments werecarried out simultaneously by replacing the test drugs with appropriatedilutions of the vehicles. In addition, the possible capacity of the above testdrugs to modify the fluorescence generated in the reaction mixture due to non-enzymatic inhibition (e.g., for directly reacting with Amplex� Red reagent) wasdetermined by adding these drugs to solutions containing only the Amplex�
Red reagent in a sodium phosphate buffer.The specific fluorescence emission(used to obtain the final results) was calculated after subtraction of thebackground activity, which was determined from vials containing allcomponents except the hMAO isoforms, which were replaced by a sodiumphosphate 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 commonsubstrate for both isoforms) was 165 ± 2 pmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min (n = 20).
35. All IC50 values shown in the table are expressed as means ± SEM from fiveexperiments.
Synthesis and evaluation of 6-methyl-3-phenylcoumarins as potentand selective MAO-B inhibitors
Maria Joao Matos a,b,*, Dolores Viña a,c, Carmen Picciau a,b, Francisco Orallo c,Lourdes Santana a, Eugenio Uriarte a
a Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spainb Dipartimento Farmaco Chimico Tecnologico, Facoltà di Farmacia, Università di Cagliari, 09124 Cagliari, Italyc 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
Article history:Received 12 June 2009
a b s t r a c t
A series of 6-methyl-3-phenylcoumarins 3-6 were synthesized and evaluated as monoamine oxidase Aand B (MAO-A and MAO-B) inhibitors. A comparative study between the three possible mono methoxy
emarkable amounts in the nature. species, produced in response to an exterior or interior damage.18
Revised 4 July 2009Accepted 7 July 2009Available online 10 July 2009
3-phenyl derivatives and the p-hydroxy analogue is reported. The synthesis of these new resveratrol-cou-marin hybrids was carried out by a Perkin reaction between the 5-methylsalicylaldehyde and the corre-sponding phenylacetic acids. The p-methoxy substituted compound 3 was hydrolyzed to 6 by atraditional reaction with hydriodic acid. The prepared compounds show high selectivity to the MAO-Bisoenzyme, some of them with IC50 values in the low nanomolar range. Compound 4, with the methoxygroup in meta position, is the most active of this series, with an IC50 against MAO-B of 0.80 nM, and isseveral times more potent and MAO-B selective than the R-(�)-deprenyl (reference compound).
� 2009 Elsevier Ltd. All rights reserved.
They have attracted considerable interest due to their numerousbiological activities depending on their substitution pattern.1
Resveratrol shows a large number of pharmacological activities,including antiinflammatory, antioxidant, anticancer, and cardio-
19–23
These compounds have been shown to possess antioxidative andanticarcinogenic properties and to inhibit several enzymes.2–6
Some coumarin derivatives of natural and synthetic origin havebeen characterized as monoamine oxidase inhibitors (MAOIs).7–12
Monoamine oxidase (MAO) is an FAD-containing enzymebound to the mitochondrial outer membrane of neuronal, glial,and other cells.10,13 This enzyme regulates levels of biogenicamines (including neurotransmitters) in the brain and the periph-eral tissues by catalyzing their deamination.11 MAO exists as twodistinct enzymatic isoforms, MAO-A and MAO-B, based on theirsubstrate and inhibitor specificities.14,15
MAO-A preferentially deaminates serotonin, adrenaline andnoradrenaline. That isoenzyme is irreversibly inhibited by low con-centrations of clorgyline. MAO-B preferentially deaminates b-phenylethylamine and benzylamine and is irreversibly inhibitedby R-(�)-deprenyl.16 The MAOIs have been used for several yearsin the treatment of depression and anxiety diseases (MAO-A inhib-itors) and in Parkinson’s disease (MAO-B inhibitors).17
Resveratrol, structurally 3,40,5-trihydroxystilbene, is a naturalphenolic component of Vitis vinifera L. and other spermatophyte
0960-894X/$ - see front matter � 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmcl.2009.07.039
protective properties and enzyme inhibition. cis and trans-res-veratrol proved to be MAO activity inhibitors, the trans isomerbeing more effective than the cis.24
Because of their similar characteristics, it was interesting to de-sign and synthesize hybrids that incorporate the nucleus of thecoumarins and resveratrol molecules.19,25 In previous work, our re-search group had reported a comparative study of the importanceto the MAOI activity of the different number of methoxy groups onthe phenyl ring in the 3 position of coumarin. This study contrib-uted to establish a relationship between them and with the non-substituted analogue.8 Based on this, and with the aim of helpingto better understand a structure/activity relationship for theMAO inhibitory activity and selectivity, in this paper we reportthe synthesis and evaluation of a new series. Maintaining the 6-methyl-3-phenylcoumarin structure, the three possible differentpositions of one methoxy group in 3-phenyl ring were explored.We also explored the importance of the hydrolysis of this methoxygroup.
The synthesis of the 6-methyl-3-phenylcoumarins was carriedout via the classical Perkin reaction.26 This reaction is performedby condensation of the 5-methylsalicylaldehyde 1 and the appro-priately substituted phenylacetic acids 2, with N,N0-dicyclohexyl-carbodiimide (DCC) as dehydrating agent, in DMSO, at 110 �C, for
24 h (Scheme 1). Compounds 3,8 427 and 528 were obtained inyields of 61%, 53%, and 59%, respectively. The reaction mixturewas purified by flash chromatography, using hexane/ethyl acetate,in a proportion of 9:1, as eluent.
The p-methoxy derivative 3 was hydrolyzed with hydriodicacid, in the presence of acetic acid and acetic anhydride, at110 �C, for 5 h (Scheme 1). The residue was purified by crystalliza-tion of acetonitrile, and the phenol derivative 629 was obtainedwith a yield of 63%.
MAO inhibiting activity of compounds 3-6 was evaluatedin vitro by the measurement of the enzymatic activity of human re-combinant MAO isoforms in BTI insect cells infected with baculo-virus.8 Then, the IC50 values and MAO-B selectivity ratio [IC50
(MAO-A)]/[IC50 (MAO-B)] for inhibitory effects of both new com-pounds and reference inhibitors were calculated (Table 1).30
The resveratrol–coumarin hybrid compounds 3, 4, and 6showed high selectivity for the MAO-B isoenzyme and inhibitoryactivity in the nano to picomolar range. Compound 4 was the mostactive compound of this series, making the meta methoxy positionthe most interesting position at which to improve the MAO-B-inhibiting activity. Compound 5 has no MAOI activity up to thehighest tested concentration, proving that the methoxy group inthe ortho position is not favorable to the measured enzymatic inhi-bition. Changes on the methoxy substituent position on the phenylring in coumarin’s 3 position can modulate the pharmacologic po-tential of the synthesized coumarins.
Comparing compound 3 with its hydroxyl derivative 6, it wasshown that the hydrolysis of methoxy groups is not, in this case,a strategy to improve the MAOI activity. The IC50 of compound 6for inhibition of MAO-B activity is approximately 10 times biggerthan compound 3. However, this value is also in the nanomolarrange, and the molecule is also a potent MAOI, selective for theMAO-B isoenzyme.
In conclusion, the synthesized resveratrol–coumarin hybridcompounds show high selectivity for the MAO-B isoenzyme. Most
R
5054 M. J. Matos et al. / Bioorg. Med
OOOH
CHOMe MeOHO
1 2 3: R = p-OMe4: R = m-OMe5: R = o-OMe
6: R = p-OH
a
OMe
b
Scheme 1. Reagents and conditions: (a) DCC, DMSO, 110 �C, 24 h; (b) HI, AcOH,Ac2O, 110 �C, 5 h.
Table 1MAO-A and MAO-B inhibition by the prepared compounds 3–6 and for the referencecompounds
* Inactive at 100 lM (highest concentration tested). At higher concentrations thecompounds precipitate.
a P <0.01 versus the corresponding IC50 values obtained against MAO-B, asdetermined by ANOVA/Dunnett’s.
b Values obtained under the assumption that the corresponding IC50 againstMAO-A is the highest concentration tested (100 lM).
of them present activity in the low nanomolar range. The introduc-tion of one meta methoxy group in the 3-phenyl ring improves sev-eral times the MAO-B inhibitory activity in respect to ortho andpara positions. Compound 4 is about 24 times more active thatR-(�)-deprenyl, and several times more selective than this drug.The hydrolysis of methoxy groups is not a strategy to get betterMAOI activity. These studied modifications can interestingly im-prove the pharmacologic potential of the 3-phenylcoumarins inthe treatment of Parkinson’s disease.
Acknowledgments
Thanks to the Spanish Ministerio de Sanidad y Consumo(PI061457 and PI061537) and to Xunta da Galicia (BTF20303PR,PXIB203022PR, and CSA019203PR) and Fondazione Banco Sarde-gna (Italy) for financial support. M.J.M. also thanks MIUR for aPhD grant.
14. Geha, R. M.; Rebrin, I.; Chen, K.; Shih, J. C. J. Biol. Chem. 2001, 276, 9877.15. Johnston, J. P. Biochem. Pharmacol. 1968, 17, 1285.16. Grimsby, J.; Lan, N. C.; Neve, R.; Chen, K.; Shih, J. C. J. Neurochem. 1990, 55,
1166.17. Harfenist, H.; Heuseur, D. J.; Joyner, C. T.; Batchelor, J. F.; White, H. L. J. Med.
Chem. 1996, 39, 1857.18. Frémont, L. Life Sci. 2000, 66, 663.19. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yánez, M.; Fraiz, N.; Alcaide, C.;
Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.20. Orallo, F. In Resveratrol in Health and Disease; Aggarwal, B. B., Shishodia, S., Eds.;
CRC Press: USA, 2005; p 577.21. Leiro, J.; Álvarez, E.; Arranz, J.; Laguna, R.; Uriarte, E.; Orallo, F. J. Leukocyte Biol.
2004, 75, 1156.22. Orallo, F. Curr. Med. Chem. 2008, 15, 1887.23. De Colibus, L.; Li, M.; Binda, C.; Lustig, A.; Edmondson, D. E.; Mattevi, A. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 12684.24. Yánez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Commun. 2006,
Synthesis and Vasorelaxant and Platelet Antiaggregatory Activities of a New Series of 6-Halo-3-phenylcoumarins †
Elías Quezada 1, Giovanna Delogu 2,*, Carmen Picciau 2, Lourdes Santana 3, Gianni Podda 2, Fernanda Borges 1, Verónica García-Morales 4, Dolores Viña 4 and Francisco Orallo 4
1 Chemistry Department, Faculty of Sciences, University Porto, 4169-007 Porto, Portugal; E-Mails: [email protected] (E.Q.); [email protected] (F.B.)
2 Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy; E-Mails: [email protected] (C.P); [email protected] (G.P.)
3 Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; E-Mail: [email protected] (L.S.)
4 Department of Pharmacology, Faculty of Pharmacy, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; E-Mails: [email protected] (D.V.); [email protected] (F.O.); [email protected] (V.G.M.)
† In memory of Prof. Francisco Orallo.
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +39-0706758566; Fax: +39-0706758553.
Received: 4 December 2009; in revised form: 21 December 2009 / Accepted: 23 December 2009 / Published: 12 January 2010
Abstract: A series of 6-halo-3-hydroxyphenylcoumarins (resveratrol-coumarins hybrid derivatives) was synthesized in good yields by a Perkin reaction followed by hydrolysis. The new compounds were evaluated for their vasorelaxant activity in intact rat aorta rings pre-contracted with phenylephrine (PE), as well as for their inhibitory effects on platelet aggregation induced by thrombin in washed human platelets. These compounds concentration-dependently relaxed vascular smooth muscle and some of them showed a platelet antiaggregatory activity that was up to thirty times higher than that shown by trans-resveratrol and some other previously synthesized derivatives.
Coumarins (or benzopyrones) are a large family of compounds of natural and synthetic origin that show numerous biological activities, including cardiovascular properties [1]. For instance, Carbochromen (3-diethylaminoethyl-7-ethoxycarbonylmethoxy-4-methylcoumarin (Figure 1) is a potent specific coronary vasodilator that has been used for many years in the treatment of angina pectoris [2,3]. Futhermore, warfarin [3-(2-acetyl-1-phenylethyl)-4-hydroxycoumarin (Figure 1) is a coumarin with potent anticoagulant activity and a good pharmacokinetic profile [4].
Figure 1. Chemical structures of trans-resveratrol, carbochromen, and warfarin.
O O
NEt
Et
OO
OEt
Carbochromen
O O
OH O
Warfarin
HO
OH
OH
trans-Resveratrol
trans-Resveratrol (t-RESV; 3,4',5-trihydroxy-trans-stilbene; Figure 1) is a natural phenolic component of Vitis vinifera L. (Vitaceae). It is abundant in the skin of the grapes and it is present in higher concentrations in red than in white wines. trans-Resveratrol has shown a number of biological activities, including protection against coronary heart disease, as a result of different effects: significant antioxidant activity, modulation of lipoprotein metabolism, and vasodilatatory and platelet antiaggregatory properties [5–8].
Because of their similar characteristics, it was of interest to design and synthesize hybrids that incorporate the nucleus of the coumarins and resveratrol molecules. In previous work, our research group had reported the vasorelaxant and platelet antiaggregatory activities of a series of coumarin-resveratrol hybrids (3-arylcoumarins), bearing hydroxy or methoxy groups on the coumarin and/or on the 3-phenyl ring. 6-Hydroxy-3-(3’,5’-dihydroxyphenyl)coumarin showed vasorelaxant and platelet antiaggregatory activity higher than that of trans-resveratrol [9].
Based on this, and with the aim of improving the vasorelaxant and platelet antiaggregatory activities of resveratrol-coumarin hybrids and establishing a relationship between the structure and activity for this type of compounds we have studied the effects of substitution on the 3-arylcoumarin moiety with groups showing different steric and electronics effects. In this paper we report the synthesis and evaluation of a new series of 3-arylcoumarins in which the hydroxy group in the 6 position has been changed for a halogen group, and different positions of hydroxyl group in 3-phenyl ring were explored.
Molecules 2010, 15
272
2. Results and Discussion
The 3-phenylcoumarins 6-11 [10] were prepared from the conveniently substituted phenylacetic acids 1-3, the appropriate salicylaldehyde 4, 5 and dicyclohexylcarbodiimide (DCC) by a Perkin reaction [11–13] in dimethylsulfoxide (DMSO). These reactions gave 46%, 40%, 27%, 29%, 31% and 33% yield, respectively. Hydrolysis of the methoxy groups [14] by treatment with HI in acetic acid/acetic anhydride gave the hydroxy derivatives 12, 13, 14, 15, 16 and 17 [15–16], in 28%, 70%, 60%, 73%, 94% and 57% yield, respectively (Scheme 1).
Scheme 1. General synthetic route to obtain compounds 6-17.
6 X = Cl; R1 = MeO; R2 = R3 = H7 X = Cl; R2 = MeO; R1 = R3 = H8 X = Cl; R3 = MeO; R1 = R2 = H9 X = Br; R1 = MeO; R2 = R3 = H10 X = Br; R2 = MeO; R1 = R3 = H11 X = Br; R3 = MeO; R1 = R2 = H
a
b
12 X = Cl; R1 = OH; R2 = R3 = H13 X = Cl; R2 = OH; R1 = R3 = H14 X = Cl; R3 = OH; R1 = R2 = H15 X = Br; R1 = OH; R2 = R3 = H16 X = Br; R2 = OH; R1 = R3 = H17 X = Br; R3 = OH; R1 = R2 = H
O O
X
R2
R1 R3
Reagents and conditions: (a) DCC, DMSO, 110 °C, 12h; (b) HI/AcOH/Ac2O, 0 °C to rt, 3 h.
The vasorelaxant effects of compounds 12-17 were studied on pre-contracted rat aortic rings with
endothelium [6]. The cumulative addition of t-RESV or the new compounds (1-100 μM) caused a concentration-dependent relaxation of the contractions induced by phenylephrine (PE, 1 μM) in intact rat aortic rings. The corresponding IC50 values are shown in Table 1.
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Table 1. Vasorelaxant activity (IC50 in μM) of tested compounds.
t-RESV 3.12 ± 0.26 * P < 0.01 versus the corresponding IC50 values of t-RESV; ** Inactive at 100 μM (highest concentration tested). At higher concentrations compounds precipitate.
All evaluated compounds resulted less efficient than t-RESV in relaxing the contractions induced
by PE. Furthermore, substitution with a p-hydroxy group in the 3-phenyl ring (compounds 14 and 17) leads to inactive compounds. Platelet aggregation studies were also performed. Compounds 12, 13 and 17 inhibited platelet aggregation more effectively than t-RESV when thrombin (0.25 U/mL) was used as the stimulating agent (Table 2).
Table 2. Antiplatelet activity (IC50 in μM) for tested compounds.
t-RESV 195.50 ± 13.82 * P < 0.01 versus the corresponding IC50 values of t-RESV; ** Inactive at 100 μM (highest concentration tested). At higher concentrations compounds precipitate.
These results indicate that the variation of the position of the hydroxyl group on the 3-phenyl and the substitution with different halogen groups in the 6 position of the coumarin ring, in this type of molecules (resveratrol-coumarins hybrid), can give derivatives with a significantly higher pharmacological potency than t-RESV. This is the case for compound 13, which has a platelet antiaggregatory activity that is more than 32 times higher than that of t-RESV.
3. Experimental
3.1. General
Melting points were determined in a Reichert Kofler thermopan or in capillary tubes in a Buchi 510 apparatus, and are uncorrected. Infrared (IR) spectra were recorded in a Perkin-Elmer 1640FT spectrometer (KBr disks, ν in cm-1).13C and 1H spectra of samples approximately 10% solutions in chloroform-d, were recorder at room temperature in 5 mm o.d. tubes. TMS was used as internal
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standard, chemical shifts are expressed in ppm (δ), J in Hz. NMR spectra were recorded with a Bruker AMX 500 (1H-, 500 MHz; 13C-, 125 MHz) instrument. ). Mass spectra were obtained using a Hewlett-Packard 5988A spectrometer (70 eV). Silica gel (35-60 mesh) was used for flash chromatography (FC). Analytical TLC was performed on plates precoated with silica gel (Merck 60 F254, 0.25 mm). Elemental analyses were performed with a Perkin-Elmer 240B microanalyser. Phenylacetic acids 1-3, salicylaldehydes 4, 5, hydriodic acid (HI), acetic acid (AcOH), acetic anhydride (Ac2O), dicyclohexylcarbodiimide (DCC) and dimethylsulfoxide (DMSO) were commercially available (Aldrich). 3.2. Chemistry
The studied compounds are all known and were prepared by a traditional Perkin reaction carried out
in refluxing dimethylsulfoxide (DMSO) between conveniently substituted phenylacetic acids 1-3 and the corresponding salicylaldehyde 4, 5, using dicyclohexylcarbodiimide (DCC) as dehydrating agent (Scheme 1) [11–13]. Hydrolysis of methoxy groups [14], by treatment with HI in acetic acid/acetic anhydride gave the desired hydroxyl derivatives 12-17 [15–16]. 6-Chloro-3-(2’-methoxy)phenylcoumarin (6). Purified by chromatography using 9:1 hexane/ethyl acetate as eluent; Mp: 177–179 ºC; IR (KBr): 2920, 1705, 1610, 1302, 1125, 780 cm-1; 1H-NMR (CDCl3) δ (ppm): 3.83 (s, 3H, CH3O), 7.00 (d, J = 8.2 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.41 (m, 3H), 7.60 (m, 1H), 7.66 (s, 1H); 13C-NMR (CDCl3) δ (ppm): 55.7, 111.3, 116.7, 118.2, 120.6, 121.0, 126.9, 130.0, 130.4, 130.7, 131.0, 133.8, 140.2, 152.2, 156.9, 157.1; MS m/z (%): 288 ([M+2]+, 33), 286 (M+, 100), 269(16), 251 (8), 240 (5), 193 (21), 152 (16), 118 (14); Anal. Calcd for C16H11ClO3: C, 67.03; H, 3.87. Experimental: C, 67.25; H, 3.99.
Vascular rings were prepared from aortae of male or female Wistar rats weighing 230–270 g. After
an equilibration period of at least 1 h, isometric contractions induced by PE (1 μM) were obtained. When contraction of the tissue in response to the corresponding vasoconstrictor agent had stabilized (after about 20 min), cumulatively increasing concentrations of the compounds were added to the bath at 15–20 min intervals (the time needed to obtain steady-state relaxation). Control tissues were subjected to the same procedure simultaneously, but in this case omitting the compounds and adding the vehicle [appropriate dilutions of dimethylsulfoxide (DMSO)]. Results shown in the tables are expressed as means ± SEM from five experiments. Means were compared by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The inhibitory effects of the tested compounds in rat aorta and human platelet are expressed as IC50 (concentrations that produce a 50%
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inhibition) estimated by least-squares linear regression using the program Origin 5.0, with X = log molar concentration of the tested compounds and Y = % of pharmacological response.
3.4. Antiplatelet activity
Preparation of washed platelets. Washed human platelets were prepared from blood anticoagulated with citrate-phosphate-dextrose, which was obtained from Centro de Transfusion de Galicia (Santiago de Compostela, Spain). Bags containing buffy coat from individual donors were diluted with the same volume of washing buffer (NaCl, 120 mM; KCl, 5 mM; trisodium citrate, 12 mM; glucose, 10 mM; sucrose, 12.5 mM; pH 6) and centrifuged at 400 g for 9 min. The upper layer containing platelet (platelet-rich plasma) was removed and centrifuged at 400 g for 9 min. The resulting platelet pellet was recovered, resuspended with washing buffer, and centrifuged again at 1,000 g for 15 min. Finally, the platelet pellet from this step was resuspended in a modified Tyrode-HEPES buffer (HEPES, 10 mM; NaCl, 140 mM; KCl, 3 mM; MgCl2, 0.5 mM; NaHCO3, 5 mM; glucose, 10 mM; pH 7.4) to afford a cell density of 2.5–3.5 × 108 platelet/mL. The calcium concentration in the extracellular medium was 2 mM.
Platelet aggregation studies. Platelet aggregation was measured using a dual channel aggregometer (Chrono-log, Havertown, PA, USA). Each tested compound, dissolved in DMSO, was incubated with washed platelet at 37 °C for 5 min. Stimulus (thrombin) was then added to induce platelet aggregation and the light transmission was monitored over 5 min period. Platelet aggregation is measured as the maximum change in light transmission during this period. The 100% aggregation value was obtained when vehicle (DMSO) was added instead of the compounds. The final DMSO concentration was below 1% (v/v) in all cases.
4. Conclusions
In conclusion, some of the new synthesized molecules have been characterized as agents with remarkable human platelet antiaggregatory activity and significant vasorelaxant effects in intact rat aorta. Further experiments are in progress aimed at providing new data to clarify the precise mechanism by which coumarin-resveratrol hybrid derivatives produce their characteristic vasorelaxant and platelet antiaggregatory effects.
Acknowledgements
We thank Progetto di Ricerca Scientifica 2007-Università di Cagliari and Fondazione del Banco di Sardegna, Spanish Ministerio de Sanidad y Consumo (FISS PI061537, PI061457), Xunta de Galicia (Spain; INCITE08PXIB203022PR) and Spanish Ministerio de Ciencia e Innovación (FISS PS09/00618). D. Vina acknowledges sponsorships for a tenure-track research position at the University of Santiago de Compostela from the Isidro Parga Pondal Programme of the “Dirección Xeral de Investigación e Desenvolvemento, Xunta de Galicia”. E. Quezada thanks for a postdoctoral grant from FCT (Portugal). Verónica García-Morales thanks for a predoctoral grant (FPU, AP2008-
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02609, Spanish Ministerio de Ciencia e Innovación). C. Picciau thanks for a predoctoral grant Master & Back-Regione Sardegna.
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7. Wu, J.M.; Wang, Z.R.; Hsieh, T.C.; Bruder, J.L.; Zou, J.G.; Huang, Y.Z. Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine. Int. J. Mol. Med. 2001, 8, 3–17.
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10. Oda, N.; Yoshida, Y.; Nagai, S.; Ueda, T.; Sakakibara, J. Synthesis of coumarins by Nucleophilic Denitrocyclization Reaction. Chem. Pharm. Bull. 1987, 35, 1796–1802.
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15. Walter, R.; Zimmer, H.; Purcell, T.C. Synthesis and cyclization reactions of 3-(2-hydroxybenzylidene)-2(3H)-coumaranones. J. Org. Chem. 1966, 31, 3854–3857.
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Synthesis, molecular docking and inhibitory activity against human monoamine
oxidases of 3-heteroarylcoumarins.
Giovanna Delogu1, Carmen Picciau1, Giulio Ferino2, Elías Quezada3, Gianni Podda1,
Eugenio Uriarte2, Dolores Viña*4
1 Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Cagliari, Via Ospedale 72,
09124 Cagliari, Italy 2 Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela,
15782- Santiago de Compostela, Spain 3 Departamento de Química, Facudade de Ciências, 4169-007, Universidade de Porto, Porto, Portugal 4 Departamento de Farmacología, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782-
Santiago de Compostela, Spain
Abstract. Monoamine oxidase (MAO) is an important drug target for the treatment of
neurological disorders. Series of 3-indolyl and 3-thiophenylcoumarins were synthesized
and evaluated as inhibitors of the two MAO isoforms, MAO-A and MAO-B. In general,
the derivatives were found to be selective MAO-B inhibitors with IC50 values in the
nanoMolar (nM) to microMolar (µM) range. Docking experiments were carried out in
order to compare the theoretical and experimental affinity of these compounds to the
human MAO-B protein. According with our results, docking experiments could be an
interesting methodology to try to predict the activity for this class of coumarins against
MAO-B receptor.
1. Introduction.
MAO is a FAD-containing enzyme with two known isoforms (MAO-A and MAO-B)
and it is present in the mitochondrial outer membrane of glial, neuronal and other cells
[1]. MAO enzymes intervene in the monoamines degradation carrying out an important
physiologic function in the adrenaline, noradrenaline and serotonin deamination
(preferentially MAO-A) and in the β-phenylethylamine and benzylamine deamination
(preferentially MAO-B) [2]. The MAO metabolic reaction involves the oxidation of the
amine function via oxidative cleavage of the α-CH bond of the substrate with the
ensuing generation of an imine intermediate. This pathway is accomplished by the
reduction of the flavin cofactor that is reoxidized by molecular oxygen, with
simultaneous hydrogen peroxide release. Subsequently, the imine intermediate is
hydrolyzed by a non-enzymatic pathway yielding ammonia and the corresponding
aldehyde [3]. This enzymatic function increases the synaptic concentration of the
neurotransmitters above mentioned and conditions to a great extent the neurone´s
excitement of those possessing receptors for these mediators [4]. These properties
determine the clinical importance of MAO inhibitors. In fact, interest in selective MAO-
B inhibitors has increased in the last years due to their therapeutic potential in aging
related neurodegenerative diseases such as Alzheimer´s disease (AD) and Parkinson´s
disease (PD) [5]. Selective MAO-A inhibitors have been used because of their
therapeutic potential in the treatment of neurological disorders such as depression [6].
The recent description of the crystal structure of the two isoforms of human MAO
provides to elucidate the mechanism underlying and allows investigation of the
selective interactions between these proteins and their ligands, to probe the catalytic
mechanism, and to gain a complete understanding of the pharmacophoric requirements
necessary for the rational design of new inhibitors [7,8].
Among the different existing inhibitors, those with a (1H)-benzopyran structure have
been deeply studied [9]. Substitution of the coumarin nucleus at position 3 has been
carried with phenyl, methyl, carboxylic acid, ethyl esther or acyl chloride groups [10].
In this work we synthesized and tested for MAO inhibitory activity some coumarin
derivatives substituted at position 3 with different heterocyclic rings. Additionally, we
explore the importance of the number and position of methoxy groups on (1H)-
benzopyran structure.
2. Results and discussion
2.1. Chemistry
The 3-heteroarylcoumarins derivatives were synthesized in moderate yield (25-47%) via
the classical Perkin reaction [11-14] by condensation of the ortho-
hydroxybenzaldehydes bearing the methoxy groups in the appropriate positions and the
conveniently substituted acetic acids, using N,N’-dicyclohexylcarbodiimide (DCC) as
dehydrating agent (Scheme 1). The structures of these compounds were confirmed by 1H NMR, 13C NMR, mass spectrometry and elemental analyses.
2.2. Enzyme inhibition studies
The potential effects of the new synthesized compounds on hMAO activity were
investigated by measuring their effects on the production of hydrogen peroxide (H2O2)
from p-tyramine, using the Amplex® Red MAO assay kit (Molecular Probes, Inc.,
Eugene, Oregon, USA) and MAO isoformas in microsomes prepared from insect cells
(BTI-TN-5B1-4) infected with recombinant baculovirus containing cDNA inserts for
hMAO-A or hMAO-B (Sigma-Aldrich Química S.A., Alcobendas, Spain). The
inhibition of hMAO activity was evaluated using the above method following the
general procedure described previously by us [15]. The tested drugs (new compounds
and reference inhibitors) themselves were unable to directly react with Amplex® Red
reagent. In addition, these test drugs had no effects on the resorufin standard
fluorescence curve which clearly indicates that these compounds do not react with
resorufin and do not quench the fluorescence generated by this product.
The control activity of hMAO-A and hMAO-B (using p-tyramine as common substrate
for both isoforms) was 165 ± 2 pmol of p-tyramine oxidized to p-
hydroxyphenylacetaldehyde/min (n = 20).
The results of the hMAO-A and hMAO-B inhibition studies with compounds 1a-c, 2a-
c, 3a-c and 4a-c are reported in Table 1 together with the selectivity index (SI =
Scheme 1. Reagents and conditions: a) DCC/DMSO 110oC, 24-48 h.
Figure 1. ROC curve relative to synthesized coumarins and 120 ZINC decoys
Table 1. IC50 values and MAO-B selectivity ratios [IC50 (MAO-A)]/[IC50 (MAO-B)] for the inhibitory effects of test drugs (new compounds and reference inhibitors) on the enzymatic activity of human recombinant MAO isoforms expressed in baculovirus infected BTI insect cells.
Each IC50 value is the mean ± S.E.M. from five experiments (n = 5). Level of statistical significance: aP < 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 µM or 1mM). ** Inactive at 100 µM (highest concentration tested). At higher concentrations the compounds precipitate. SI: hMAO-B selectivity index = IC50 (hMAO-A) /IC50 (hMAO-B).
a Docking rank taking into account the study coumarins compounds; b expressed in µM; c expressed in kJ/mol. ** Inactive at 100 µM (highest concentration tested). At higher concentrations the compounds precipitate.