Tesis Definitiva Carmen Picciau

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

a cinco de Octubre de dos mil diez.

Fdo. Eugenio Uriarte Villares Fdo. Lourdes Santana Penín

Fdo. Elías Quezada González

AGRADECIMIENTOS

A Eugenio, Lourdes, Elias per avermi dato la possibilità di realizzare questo dottorato,

per il supporto, l’ affetto e l’ incoraggiamento che mi hanno sempre trasmesso e per la

squisita ospitalità e amicizia durante i periodi trascorsi a Santiago

Al Prof. Gianni Podda per avermi sempre appoggiato in questi anni, per i suoi consigli e

la sua disponibilità.

Alla carissima Giovanna per il suo aiuto nella realizzazione di questo progetto, ma

soprattutto per la sua amicizia sincera, la sua presenza, il suo affetto.

Alla cara Maria Joao per la sua amicizia, l’indispensabile aiuto nella stesura della tesi,

l’affetto e la sua grande disponibilità in ogni circostanza.

A Dolo per la preziosa collaborazione per i saggi biologici della parte farmacologica

A tutti gli amici e colleghi di laboratorio che mi hanno accompagnato in questi anni a

Cagliari e Santiago : Graziella, Michela, Silvana, Valeria, Silvia, Tati, Gabriele, Giulio,

Riccardo, Chachy, Humberto, Fran, per i bei momenti passati insieme.

Ai tanti amici e persone care che porto nel cuore.

Ai miei cari genitori per essermi sempre vicini, per tutto l’appoggio e l’amore che mi

hanno sempre dato.

Alla mia adorata Cristina, il mio angelo.

Ai miei amatissimi Luigi e Stefano, mia famiglia e luce dei miei occhi.

A Luigi e Stefano

Ai miei genitori

A Cristina

I.-Indice

I.-Ìndice

III

I. ÍNDICE………………………………………………………………………….. III II. RELACIÓN DE ABREVIATURAS…………………………………………… VII III. RELACIÓN DE COMPUESTOS FINALES OBTENIDOS………………….. XI 1. INTRODUCCIÓN………………………………………………………………. 1 1.1 Las cumarinas………………………………………………………………... 3 1.1.1. Actividad biológica de cumarinas…………………………………………. 4 1.1.2. Síntesis de cumarinas……………………………………………………… 5 1.2. El resveratrol………………………………………………………………… 6 1.2.1. Actividad biológica del resveratrol………………………………………... 7 2. OBJETIVOS……………………………………………………………………. 9 3. DISCUSIÓN DE RESULTADOS……………………………………………… 13 3.1 Síntesis y evaluación farmacológica de híbridos cumarina-resveratrol……... 15 3.2 Síntesis y evaluación farmacológica de híbridos benzofurano-resveratrol….. 22 4. CONCLUSIONES………………………………………………………………. 25 5. PARTE EXPERIMENTAL……………………………………………………... 29 3-Fenil-6-metilcumarina (1)…………………………………………………….. 33 6-Metil-3-(4’-metoxifenil)cumarina (2)………………………………………... 35 3-(3’,5’-Dimetoxifenil)-6-metilcumarina (3)…………………………………… 37 6-Metil-3-(3’,4’,5’-trimetoxifenil)cumarina (4)……………………………….. 39 6-Metil-3-(2’-metoxifenil)cumarina (5)………………………………………... 41 6-Metil-3-(3’-metoxifenil)cumarina (6)............................................................... 43 6-Cloro-3-(2’-metoxifenil)cumarina (7).............................................................. 45 6-Cloro-3-(4’-metoxifenil)cumarina (8).............................................................. 47 6-Cloro-3-(3’-metoxifenil)cumarina (9).............................................................. 49 6-Bromo-3-(2’-metoxifenil)cumarina (10).......................................................... 51 6-Bromo-3-(4’-metoxifenil)cumarina (11).......................................................... 53 6-Bromo-3-(3’-metoxifenil)cumarina (12).......................................................... 55 6-Cloro-3-(2’-hidroxifenil)cumarina (7a)........................................................... 57 6-Cloro-3-(4’-hidroxifenil)cumarina (8a)........................................................... 59 6-Cloro-3-(3’-hidroxifenil)cumarina (9a)........................................................... 61 6-Bromo-3-(2’-hidroxifenil)cumarina (10a)........................................................ 63 6-Bromo-3-(4’-hidroxifenil)cumarina (11a)....................................................... 65 6-Bromo-3-(3’-hidroxifenil)cumarina (12a)....................................................... 67 3-(2-Tienil)cumarina (13a)……………………………………………………… 69 3-(3-Tienil)cumarina (13b).................................................................................. 71 3-(3-Indolil)cumarina (13c)................................................................................. 73 7- Metoxi-3-(2-tienil)cumarina (14a).................................................................. 75 7- Metoxi-3-(3-tienil)cumarina (14b).................................................................. 77 3-(3-Indolil)-7-metoxicumarina (14c)………………………………………….. 79 6- Metoxi-3-(2-tienil)cumarina (15a).................................................................. 81 6- Metoxi-3-(3-tienil)cumarina (15b).................................................................. 83 3-(3-Indolil)-6-metoxicumarina (15c)................................................................. 85 5,7-Dimetoxi-3-(2-tienil)cumarina (16a)............................................................. 87 5,7-Dimetoxi-3-(3-tienil)cumarina (16b)............................................................. 89 5,7-Dimetoxi-3-(3-indolil)cumarina (16c)........................................................... 91 2-Fenil-6-metoxibenzofurano (18) y 3-Fenil-7-metoxi-2H-cromeno (19)......... 93 2-Acetoniloxi-3-metoxibenzaldehido (20)……………………………………… 95 Acetato de 2-hidroxi-3-metoxibencilo (21)…………………………………….. 97

I.-Ìndice

IV

Bromuro de 2-hidroxi-3-metoxibenciltrifenilfosfonio (22)…………………… 99 7-Metoxi-2-(3’,4’,5’-trimetoxifenil)benzofurano (23)....................................... 101 2-(3’,5’-Dimetoxifenil)-7-metoxibenzofurano (24)…………………………….. 103 7-Metoxi-2-(4’-metoxifenil)benzofurano (25)…………………………………. 105 7-Metoxi-2-(3’,4’,5’-trimetoxifenil)dihidrobenzofurano (26)......................... 107 Bromuro de 2-hidroxibenciltrifenilfosfonio (27)……………………………… 109 2-(3’,4’,5’-Trimetoxifenil)benzofurano (28)…………………………………… 111 2-(3’,5’-Dimetoxifenil)benzofurano (29)……………………………………….. 113 2-(4’-Metoxifenil)benzofurano (30)…………………………………………….. 115 Alcohol 2-hidroxi-5-metilbencílico (31)………………………………………... 117 Bromuro de 2-hidroxi-5-metilbenciltrifenilfosfonio (32)……………………... 119 5-Metil-2-(4’-metoxifenil)benzofurano (33)…………………………………… 121 2-(3’,5’-Dimetoxifenil)-5-metilbenzofurano (34)............................................... 123 5-Metil-2-(3’,4’,5’-trimetoxifenil)benzofurano (35).......................................... 125 Alcohol 5-bromo-2-hidroxibencílico (36)……………………………………… 127 Bromuro de 5-bromo-2-hidroxibenciltrifenilfosfonio (37)…………………… 129 5-Bromo-2-(3’,4’,5’-trimetoxifenil)benzofurano (38)....................................... 131 5-Bromo-2-(3’,5’-dimetoxifenil)benzofurano (39)…………………………….. 133 5-Bromo-2-(4’-metoxifenil)benzofurano (40)………………………………….. 135 Alcohol 3-etoxi-2-hidroxibencílico (41)………………………………………… 137 Bromuro de 3-etoxi-2-hidroxibenciltrifenilfosfonio (42)……………………... 139 7-Etoxi-2-(3’,4’,5’-trimetoxifenil)benzofurano (43).......................................... 141 2-(3’,5’-Dimetoxifenil)-7-etoxibenzofurano (44)............................................... 143 7-Etoxi-2-(4’-metoxifenil)benzofurano (45)…………………………………… 145 6. Anexo: Publicaciones............................................................................................ 147

II.-Relación de abreviaturas

II.-Relación de Abreviaturas

VII

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.

16 Orsini, Fulvia; Pelizzoni, Francesca; Verotta, Luisella; Aburjai, Talal, J. Nat. Prod. 1997, 60, 1082. 17 Leger, A.S.; Cochrane, A.L.; Moore, F., Lancet 1979, 1, 1017.

1.-Introducción

7

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.


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.


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).

O O

H3C

R1R2

R3

1 : R1 = R2 = R3 = R4 = H2 : R1 = R3 = R4 = H R2 = OCH33 : R1 = R3 = OCH3 R2 = R4 = H4 : R1 = R2 = R3 = OCH3 R4 =H5 : R1 = R2 = R3 =H R4 = OCH36 : R1 = R2 = R4 =H R3 = OCH3

R4

Esquema 2


18

La actividad i-MAO de nuestros compuestos fue evaluada in vitro utilizando como

compuestos de referencia el R-(-)-Deprenilo y la Iproniazida. (Tabla 3).

Tabla 3. Actividad i-MAO

Compuestos MAO-A (CI50) MAO-B (CI50) Indice

Selectividad

1 ** 283.75 ± 0.98 nMa >352b

2 ** 13.05 ± 0.90 nMa >7663b

3 ** 8.98 ± 1.42 nMa >11,136b

4 ** 160.64 ± 1.01 nMa >623b

5 ** ***

6 ** 802.60 ± 53.75 pM >124,688b

R-(-)-Deprenilo 67.25 ± 1.02 μMa 19.60 ± 0.86 nMa 3431

Iproniazida 6.56 ± 0.76 μM 7.54 ± 0.36 μMa 0.87

**inactivo a 100 μM (mas alta concentración testada) A concentración más altas el compuesto precipita

a P<0.01 los correspondientes valores IC50 obtenidos contra la Mao-B

b Valores obtenidos asumiendo que los correspondientes IC50 contra Mao-A es la más alta concentración testada

Todos los compuestos sintetizados son potentes inhibidores selectivos de la MAO-B.

Los valores de IC50 de los compuestos son mostrados en la Tabla 3, encontrándose

muchos de ellos en el rango de bajo nanomolar y picomolar. El compuesto 2 tiene una

IC50 MAO-B similar al R-(-)deprenilo y es más selectivo de este. El compuesto más

activo es el 6 que lleva un metoxilo en la posición 3’ y es activo en el rango picomolar.

Ninguno de los compuestos muestra actividad inhibitoria MAO-A. La presencia de

sustituyentes metoxilados parece importante para la actividad inhibitoria MAO-B.

Observando que la sustitución en la posición 3 del anillo cumarínico, parece tener

importancia para su actividad como i-MAO (en la bibliografía aparece además descrita

la sustitución en dicha posición con grupos fenilo, metilo, ácido carboxílico, ester o

cloruro de ácido 2b), pensamos en estudiar el efecto de la introducción de diferentes

anillos heterocíclicos sobre la actividad MAO. Adicionalmente se exploró la

importancia del número y posición de los sustituyentes sobre el anillo de benzopirano


19

sobre dicha actividad, por ello se sintetizaron una nueva serie de 3-heteroarilcumarinas

(Esquema 4).

+O

H

OHR4

OHO

O O

R4R2

R1

R3

R2

R1

R3

13 R1 = R2 = R3 = H14 R1 =OCH3; R2 = R3 = H15 R2 = OCH3; R1 = R3 = H16 R1 = R3 = OCH3; R2 = H

a R4 =

b R4 =

c R4 =

S

S

NH

13a R1 = R2 = R3 = H; R4 =

13b R1 = R2 = R3 = H; R4 =

13c R1 = R2 = R3 = H; R4 =

S

S

NH

14a R1 =OCH3; R2 = R3 = H; R4 =

14b R1 =OCH3; R2 = R3 = H; R4 =

14c R1 =OCH3; R2 = R3 = H; R4 =

S

NH

15a R2 = OCH3; R1 = R3 = H; R4 =

15b R2 = OCH3; R1 = R3 = H; R4 =

15c R2 = OCH3; R1 = R3 = H; R4 =

S

S

NH

16a R1 = R3 = OCH3; R2 = H; R4 =

16b R1 = R3 = OCH3; R2 = H; R4 =

16c R1 = R3 = OCH3; R2 = H; R4 =

S

S

NH

S

DCC/DMSO

110ºC/24-48h

Esquema 4

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.


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).

Compuestos MAO-A (CI50) MAO-B (CI50) Indice Selectividad

13a ** 6.37 ± 0.43 µM > 15b

13b ** 13.3 ± 0.89 µM > 7.5b

13c ** 1.92 ± 0.13 µM > 52b

14a ** 262.95 ± 17.61 nM >380b

14b 4.16 ± 0.28 µMa 55.63 ± 3.73 nM > 75

14c 9.67 ± 0.65 µMa 45.95 ± 3.08 nM > 210

15a ** 633.55 ± 42.43 nM > 158b

15b ** 233.73 ± 15.65 nM > 428b

15c ** ** ---

16a ** ** ---

16b ** ** ---

16c ** ** ---

R-(-)-deprenilo 67.25 ± 1.02 µMa 19.60 ± 0.86 nM 3431

Iproniazida 6.56 ± 0.76 µM 7.54 ± 0.36 µM 0.87


21

OO O

a) DIBAL -78°b) H2SO4 2N, refl.

MeO MeO

18

Esquema 5

Los rendimientos de la reacción son bajos (aprox. 10%), debido a las drásticas

condiciones utilizadas, pues el producto principal de la cumarina comercial 3-fenil-7-

metoxicumarina, resulta ser el 3-fenil-7-metoxi-2H-cromeno 19 (aprox. 90%

rendimiento), que se forma por la reducción del grupo carbonílico de la cumarina a

grupo CH2 (Figura 1).

OMeO O OMeO

19 Figura 1

Los datos 1H-RMN han confirmado la formación del cromeno presentando la señal

de un singlete a 5 ppm correspondiente a los 2 protones metilénicos del C-2.

Los métodos más comunes para la obtención de los 2-arilbenzofuranos recurren a la

reacción de Sonogashira Pd/catalizada entre o-yodo-fenoles y fenilacetilenos variamente

substituidos3, a la ciclodehidrogenación de 2-hidroxiestilbenos4, y a la reacción entre

una sal de fosfonio o-hidroxi substituida y ácidos benzoicos comerciales o los

correspondientes cloruros de los ácidos5. Este último método nos ha parecido el más

sencillo y por lo tanto fue el que elegimos.

El método implica primero la preparación de la sal de fosfonio a partir de un o-

hidroxisalicilaldehido oportunamente sustituido. Se reduce el grupo aldehídico a grupo

bencilico con NaBH4 y luego se trata el producto de reducción con PPH3 HBr

(Esquema 6).

3 i) Kundu, N.; Pal, M.; Mahanty, J.S.; De, M. J. Chem. Soc. Perkin Trans 1, , 1997, 2815 ii) Arcadi, A.; Cacchi, C.; Fabrizi, G.; Marinelli, F.; Moro, L. Synlett, 1999, 9, 1432. 4 Imafuku, K.; Fujita, R. Chemistry Express, 1991, 6, 323 5 a) McAllister, G. D.; Hartley, R.C.; Dawson, M.J.; Knaggs, A.R. J. Chem. Soc. Perkin Trans 1, , 1998, 3453. b) Ono, M.; Kawashima, H.; Nonaka, A.; Kaway, T.; Haratake, M.; Mori, H.; Kung, M.P.; Kung, H.F.; Saji, H.; Nakayama, M. J. Med. Chem. 2006, 49, 2725-2730


22

H

O

OHNaBH4

OHPPh3 HBr PPh3

+ Br-

OH

OH

R RR

Esquema 6

La reacción entre la sal de fosfonio y el cloruro del ácido benzoico en tolueno

utilizando Et3N como catalizador lleva a la formación de los 2-arilbenzofuranos. La

reacción va a reflujo en 2 h y los rendimientos son muy satisfactorios (Esquema 7).

.

PPh3+ Br-

OH

O Cl

O+

Et3NtoluenoR

RR

R

Esquema 7

La reducción del doble enlace en posición 2-3 del benzofurano 23, realizada por

hidrogenación catalítica con Pd/C en EtOH en 24 h llevó a la formación del 2-fenil-

dihidrobenzofurano 26 con un 80 % de rendimiento (Esquema 8).

O

H2/Pd

EtOHOMe OMe

OMe

OMe

OOMe OMe

OMe

OMe

23 26

Esquema 8

Los datos 1H-RMN confirman la formación del producto de reducción con la

aparición de dos doble dobletes a 3.28 y 3.59 ppm relativos a los dos protones del C-3 y

el triplete de C-2 a 5.71 ppm.

En la Tabla 5 se observan lo benzofuranos y también el dihidrobenzofurano 26, que

han sido preparados y que serán sometidos a ensayos biológicos como potenciales

inhibidores de MAO.


23

Tabla 5

OMeO18

OMe

OMe

OMeO

OMe23

OMe

OMeO

OMe 24 25

OOMe

OMe

OOMe

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

OMe

O

BrOMe

40

O

OMe

OMe

OMe

O43

O

OMe

OMeO44

OOMe

O45

4.-Conclusiones

4.-Conclusiones

27

Se obtuvo una nueva serie de derivados híbridos que incorporan en una sola

estructura el núcleo de la cumarina y del resveratrol, con diversos sustituyentes y

con rendimientos aceptables, utilizando como reacción central la reacción de

Perkin (compuestos 1-12).

Se obtuvo una nueva serie de 3-heteroarilcumarinas, isósteros de los híbridos

anteriores, con diversos sustituyentes y buenos rendimientos, utilizando como

reacción clave la reacción de Perkin (compuestos 13-16).

Se obtuvo una nueva serie de derivados híbridos que incorporan en una sola

estructura el núcleo del benzofurano y del resveratrol, con diversos sustituyentes

y con rendimientos aceptables, utilizando como paso clave la reacción de Wittig

(17 compuestos entre el 18 y el 45, véase “III Relación de Compuestos Finales

Obtenidos”).

Se obtuvo un 2-fenildihidrobenzofurano análogo simplificado de la viniferina

(compuesto 26), por reducción de su precursor benzofuránico con un

rendimiento del 80%. La puesta a punto de esta reacción permitirá la síntesis de

una serie seleccionada.

Se caracterizaron todos los derivados obtenidos con punto de fusión, espectros

de masas, 1H-RMN y 13C-RMN.

Ensayos farmacológicos, todavía preliminares, fueron ya realizados para algunos

de los compuestos sintetizados, ofreciendo interesantes perfiles como

vasodilatadores, antigregantes plaquetarios e inhibidores MAO-B. Se continuará

este proceso para todas las series preparadas.

5.-Parte experimental

5.-Parte Experimental

31

Aspectos generales

Las reacciones se llevaron a cabo en atmósfera de argón desoxigenado y seco. Los

disolventes utilizados en las reacciones se secaron por destilación sobre un agente

desecante adecuado, en atmósfera de argón, inmediatamente antes de su uso. Los

agentes desecantes utilizados fueron: Na/benzofenona para THF, CaH2 para CH2Cl2,

CHCl3 y Et3N. El DMSO se destiló al vacío y se guardó sobre tamices moleculares 4Å

bajo argón 1.

El material de vidrio utilizado en las reacciones que exigieron condiciones anhidras

se secó por calentamiento a 140 oC durante 12 horas y posterior enfriamiento en

corriente de argón seco.

Las adiciones de disoluciones y disolvente se llevaron a cabo vía jeringa o cánula.

El secado de las disoluciones obtenidas tras la elaboración de cada reacción se llevó

a cabo mediante Na2SO4 ó MgSO4 anhidros.

Los espectros de RMN se registraron en espectrómetros Bruker WM-250 (250.13

MHz para 1H y 62.89 MHz para 13C). Para los espectros de RMN se emplearon CDCl3

y DMSO-d6 como disolventes. Los desplazamientos químicos están expresados en

unidades δ (ppm), las constantes de acoplamiento J en Hz y tomando como referencia la

señal del TMS (0 ppm) en todos los casos.

Los puntos de fusión se determinaron en un aparato Reichter Kofler y no están

corregidos.

Los espectros de masas de baja redisolución se realizaron en un sistema Hewlett-

Packard 59970-GC/MS operando a 70 eV.

Los análisis elementales se realizaron en analizadores elementales Perkin-Elmer

240B y Carlo-Erba EA1108.

Para cromatografía en capa fina se empleó gel de sílice GF-254 (Merck) y las

manchas se visualizaron bajo luz UV (254 y 366 nm) para los compuestos que absorben

a dicha longitud de onda, y también por el revelado al calor de los cromatofolios de

capa fina previamente tratados con el revelador adecuado2 (ácido fosfomolíbdico, yodo,

según corresponda).

Para cromatografía en columna se empleó gel de sílice 35-70 μm. Para las reacciones

a baja temperatura se utilizaron baños de hielo seco con acetona.

1Perrin, D. y Armarego, W.L.F. Purification of Laboratory Chemicals. 1988,, Pergamon Press. 2 Kirchner, J.G.; Thin-Layer Chromatography. 1978, John Wiley & Sons.


32


33

3-Fenil-6-metilcumarina (1)

DCC/DMSO

H

O

OHCOOH

O O

1

Una disolución de ácido fenilacético (2.50 g, 18.53 mmol), 2-hidroxi-5-

metilbenzaldehido (2.00 g, 14.60 mmol) y DCC (4.72 g, 23.00 mmol) en DMSO (30

mL) fue calentada en un baño de aceite a 110° C por 24 h. Finalizada la reacción se

adicionó agua fria (100 mL) y ácido acético (10 mL). A continuación se dejó con

agitación y a temperatura ambiente durante 4 h, tras lo cual se extrajo con etér etílico (4

x 100 mL). La diciclohexilurea que precipita fue separada de la interfase por filtración y

la fase etérea se extrajo con una disolución al 5 % de NaHCO3 (100 mL). La fase

orgánica fue separada, lavada con agua, secada con Na2SO4 anhidro y el disolvente

evaporado al vacío, obteniéndose un residuo que fue purificado por cromatografía en

columna utilizando como eluyente Hexano/AcOEt 9:1. Se obtuvieron 2.37 g (Rto: 68

%) de 1 como un sólido blanco puro.

P. f.: 148–149 °C 1H-RMN (CDCl3) δ (ppm): 2.42 (s, 3H, CH3), 7.27 (m, 1H, H-7), 7.34 (m, 2H, H-4’ H-8), 7.43 (m, 3H, H-2’ + H-4’ + H-5), 7.70 (dd, 2H, H-1’ + H-5’, J = 7.7 y 1.8), 7.77 (s, 1H, H-4). 13C-RMN (CDCl3) δ (ppm): 21.29, 116.67, 119.57, 128.17, 128.70, 128.95, 129.02, 129.26, 132.95, 134.65, 135.35, 140.39, 152.16, 161.31. MS m/z (%): 236 (M+, 100), 208 (50), 178 (8), 165 (8). Anal. Elem.: Calculado para C16H12O2 C, 81.34; H, 5.12. Experimental C, 81.44; H, 4.84.


34


35

6-Metil-3-(4’-metoxifenil)cumarina (2)

DCC/DMSO

H

O

OHCOOH

O O

OMe

MeO

2

Analogamente a la preparación de 1, la reacción del ácido 4-metoxifenilacético

(2.29 g, 13.80 mmol) con el 2-hidroxi-5-metilbenzaldehido (1.50 g, 11.00 mmol) y

DCC (3.55 g, 17.10 mmol) en DMSO (30 mL), condujo a un residuo que fue purificado

por cromatografía en columna eluyendo con Hexano/AcOEt 9:1. Se obtuvieron 1.78 g

(Rto: 61 %) de 2 como un sólido blanco puro.

P. f.: 144–145 °C 1H-RMN (CDCl3) δ (ppm): 2.40 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 6.96 (dd, 2H, H-3’ + H-5’, J = 6.8 y 2.1), 7.26 (m, 3H, H-4 + H-7 + H-8), 7.66 (m, 3H, H-3 + H-2’ + H-6’). 13C-RMN (CDCl3) δ (ppm): 20.75, 55.32, 113.85, 116.04, 119.54, 127.19, 127.46, 127.66, 129.77, 132.02, 134.03, 138.47, 151.41, 160.05, 160.96. MS m/z (%): M+ 266 (100), 223 (60), 195 (24), 165 (16). Anal. Elem.: Calculado para C17H14O3: C, 76.68; H, 5.30. Experimental: C, 76.88; H, 5.32.


36


37

3-(3’,5’-Dimetoxifenil)-6-metilcumarina (3)

DCC/DMSO

H

O

OHCOOH

O O

MeO

OMe

OMe

OMe

3

Analogamente a la preparación de 1, la reacción del ácido 3,5-

dimetoxifenilacético (1.81 g, 9.20 mmol) con el 2-hidroxi-5-metilbenzaldehido (1.00 g,

7.30 mmol) y DCC (2.35 g, 11.30 mmol) en DMSO (20 mL), condujo a un residuo que

fue purificado por cromatografía en columna eluyendo con Hexano/AcOEt 9:1. Se

obtuvieron 1.29 g (Rto: 60 %) de 3 como un sólido blanco puro.

P. f.: 110–111 °C. 1H-RMN (CDCl3) δ (ppm): 2.40 (s, 3H, CH3), 3.82(s, 6H, OCH3), 6.50 (t, 1H, H-4’, J = 2.1), 6.83 (d, 2H, H-2’ + H-6’, J = 2.1), 7.22–7.33 (m, 3H, H-5 + H-7 + H-8), 7.74 (s, 1H, H-4). 13C-RMN (CDCl3) δ (ppm): 20.72, 55.41, 100.89, 106.72, 116.06, 119.22, 127.69, 127.93, 132.49, 134.10, 136.68, 140.02, 151.60, 160.48, 160.61. MS m/z (%): M+ 296 (100), 295 (17), 267 (16), 210 (8). Anal. Elem.: Calculado para C18H16O4: C, 72.96; H, 5.44. Experimental: C, 73.23; H, 4.90.


38


39

6-Metil-3-(3’,4’,5’-trimetoxifenil)cumarina (4)

DCC/DMSO

H

O

OHCOOH

O O

MeO

OMe

OMe

OMe

MeO

OMe

4

Analogamente a la preparación de 1, la reacción del ácido 3,4,5-

trimetoxifenilacético (2.08 g, 9.20 mmol) con el 2-hidroxi-5-metilbenzaldehido (1.00 g,

7.30 mmol) y DCC (2.35 g, 11.30 mmol) en DMSO (30 mL), condujo a un residuo que

fue purificado por cromatografía en columna con Hexano/AcOEt 9:1. Se obtuvieron

1.66 g (Rto: 70 %) de 4 como un sólido blanco puro.

P. f.: 165–166 °C. 1H-RMN (CDCl3) δ (ppm): 2.43 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 3,92 (s, 6H, (OCH3)2), 6.93 (d, 2H, H-2’ + H-6’, J = 2.1), 7.24–7.35 (m, 3H, H-5 + H-7 + H-8), 7.76 (s, 1H, H-4). 13C-RMN (CDCl3) δ (ppm): 20.70, 56.19, 60.72, 105.94, 116.03, 119.24, 127.48, 127.58, 130.04, 130.44, 132.38, 134.12, 139.48, 151.45, 153.02, 160.66. MS m/z (%): M+ 326 (100), 311 (89), 283 (43), 253 (12). Anal. Elem.: Calculado para C19H18O5: C, 69.93; H, 5.56. Experimental: C, 70.14; H, 5.58.


40


41


DCC/DMSO

H

O

OHCOOH

O O

OMe MeO

5


(1.52 g, 9.15 mmol) con el 2-hidroxi-5-metilbenzaldehido (1.00 g, 7.34 mmol) y DCC

(2.36 g, 11.44 mmol) en DMSO (20 mL), condujo a un residuo que fue purificado por

cromatografía eluyendo con Hexano/AcOEt 9:1. Se obtuvieron 1.15 g (Rto: 59 %) de 5

como un sólido amarillo puro.

P. f.: 177–178 °C. 1H-RMN (CDCl3) δ (ppm): 2.42 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 7.02 (m, 2H, H-7 + H-8), 7.27-7.38 (m, 5H), 7.70 (s, 1H, H-4). 13C-RMN (CDCl3) δ (ppm): 20.80, 55.81, 111.31, 116.21, 119.24, 120.57, 124.20, 126.36, 127.58, 130.14, 130.80, 132.21, 133.89, 141.84, 151.82, 157.22, 160.55. MS m/z (%): M+ 266 (100), 249 (20), 237 (13), 173 (17). Anal. Elem.: Calculado para C17H14O3: C, 76.68; H, 5.30.

Experimental: C, 76.80; H, 5.45.


42


43


6

DCC/DMSO

H

O

OHCOOH

O O

MeOOMe


(1.52 g, 9.15 mmol) con el 2-hidroxi-5-metilbenzaldehido (1.00 g, 7.34 mmol) y DCC


cromatografía en columna con Hexano/AcOEt 9:1. Se obtuvieron 1.04 g (Rto: 53 %) de

6 como un sólido amarillo puro.

P. f.: 84–85 °C. 1H-RMN (CDCl3) δ (ppm): 2.36 (s, 3H, CH3), 3.79 (s, 1H, OCH3), 6.98 (dd, 2H, H-7 + H-8), 7.2-7-4 (m, 5H), 7.7 (s, 1H, H-4). 13C-RMN (CDCl3) δ (ppm): 20.77, 55.37, 114.15, 114.38, 116.10, 119.28, 120.86, 127.70, 129.43, 132.48, 134.12, 136.12, 139.91, 140.10, 151.60, 159.45, 160.66. MS m/z (%): M+ 266 (100), 251 (1), 238 (38), 223 (2). Anal. Elem.: Calculado para C17H14O3: C, 76.68; H, 5.30.



44


45

6-Cloro-3-(2’-metoxifenil)cumarina (7)

7

DCC/DMSO

H

O

OHCOOH

O O

Cl

ClOMe MeO


(0.53 g, 3.20 mmol) con el 5-cloro-2-hidroxibenzaldehido (0.40 g, 2.55 mmol) y DCC


cromatografía eluyendo con Hexano/AcOEt (9:1). Se obtuvieron 0.34 g (Rto: 46 %) de

7 como un sólido amarillo puro

P. f.: 177-179 °C 1H-RMN (CDCl3) δ (ppm): (CDCl3) δ (ppm): 3.83 (s, 3H, OCH3), 7.00 (d, J = 8.2, 1H), 7.05 (d, J = 7.5, 1H), 7.22 (d, J = 8.2, 1H), 7.41 (m, 3H), 7.60 (m, 1H), 7.66 (s, 1H). 13C-RMN (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 (%): [M+2]+ 288 (33), M+ 286 (100), 269 (16), 251 (8). Anal. Elem.: Calculado para C16H11O3Cl: C, 67.03; H, 3.87.



46


47


8

DCC/DMSO

H

O

OHCOOH

O O

Cl

Cl

MeO

OMe


(2.65 g, 15.96 mmol) con el 5-cloro-2-hidroxibenzaldehido (2.00 g, 12.77 mmol) y

DCC (4.11 g, 19.92 mmol) en DMSO (30 mL), condujo a un residuo que fue purificado

por cromatografía eluyendo con Hexano/AcOEt (9:1). Se obtuvieron 1.0 g (Rto: 27 %)

de 8 como un sólido amarillo puro.

P. f.: 194-196 °C 1H-RMN (CDCl3) δ (ppm): 3.85 (s, 3H, OCH3), 6.99 (d, J = 8.9, 2H), 7.29 (d, J = 8.0, 1H), 7.48 (m, 2H), 7.66 (d, J = 8.9, 2H), 7.71 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.4, 113.9 (2C), 117.7, 118.3, 120.3, 121.1, 126.8, 129.8 (2C), 130.8, 136.9, 141.1, 151.9, 156.3, 158.2. MS m/z (%): [M+2]+ 288 (33), M+ 286 (100), 258 (13). Anal. Elem.: Calculado para C16H11O3Cl: C, 67.03; H, 3.87.



48


49


9

DCC/DMSO

H

O

OHCOOH

O O

Cl

ClMeO

OMe


(0.47 g, 2.83 mmol) con el 5-cloro-2-hidroxibenzaldehido (0.35 g, 2.23 mmol) y DCC


cromatografía con Hexano/AcOEt (9:1). Se obtuvieron 0.25 g (Rto: 40 %) de 9 como un

sólido blanco puro.

P. f.: 144-146 °C 1H-RMN (CDCl3) δ (ppm): 3.85 (s, 3H, OCH3), 6.96 (dd, J = 2.5 y 8.2, 1H), 7.25 (m, 2H), 7.35 (m, 2H), 7.48 (m, 2H), 7.72 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.32, 114.2, 114.8, 117.8, 120.6, 120.8, 127.0, 129.2, 129.5, 129.7, 131.3, 135.5, 138.5, 151.8, 159.5, 159.8. MS m/z (%): [M+2]+ 288 (29), M+ 286 (100), 258 (30). Anal. Elem.: Calculado para C16H11O3Cl: C, 67.03; H, 3.87.



50


51

6-Bromo-3-(2’-metoxifenil)cumarina (10)

10

DCC/DMSO

H

O

OHCOOH

O O

Br

BrOMe MeO


(1.03 g, 6.20 mmol) con el 2-hidroxi-5-bromobenzaldehido (1.00 g, 4.90 mmol) y DCC


cromatografía en columna con Hexano/AcOEt (9:1). Se obtuvieron 0.46 g (Rto: 29 %)

de 10 como un sólido amarillo puro.

P. f.: 183-185 °C 1H- RMN (CDCl3) δ (ppm): 3.82 (s, 3H, OCH3), 6.99 (d, J = 7.5, 1H), 7.03 (d, J = 7.4, 1H), 7.24 (d, J = 8.4, 1H), 7.36 (m, 2H), 7.60 (m, 2H), 7.66 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.7, 111.3, 116.7, 118.2, 120.6, 121.0, 123.5, 127.6, 130.0, 130.5, 130.7, 133.8, 140.2, 152.5, 157.1, 159.6. MS m/z (%): [M+2]+ 332 (100), M+ 330 (97), 315 (23), 237 (32). Anal. Elem.: Calculado para C16H11O3Br: C, 58.03; H, 3.35.



52


53


11

DCC/DMSO

H

O

OHCOOH

O O

Br

Br

MeO

OMe


(1.03 g, 6.20 mmol) con el 5-bromo-2-hidroxibenzaldehido (1.00 g, 4.90 mmol) y DCC


cromatografía con Hexano/AcOEt (9:1). Se obtuvieron 0.56 g (Rto: 33 %) de 11 como

un sólido blanco puro.

P. f.: 205-206 °C 1H-RMN (CDCl3) δ (ppm): 3.85 (s, 3H, OCH3), 6.97 (d, J = 8.8, 2H), 7.23 (m, 1H), 7.57 (dd, J = 2.2 y 8.7, 1H), 7.63 (m, 3H), 7.67 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.3, 113.9 (2C), 114.1, 116.9, 118.1, 121.4, 126.5, 128.9, 129.8 (2C), 129.9, 133.6, 136.8, 152.0, 160.4. MS m/z (%): [M+2]+ 332 (100), M+ 330 (13), 302 (12), 288 (40). Anal. Elem.: Calculado para C16H11O3Br: C, 58.03; H, 3.35.



54


55


12

DCC/DMSO

H

O

OHCOOH

O O

Br

BrMeO

OMe

Analogamente a la preparación de 1, la reacción del ácido 4-metoxifenilacetico

(1.03 g, 6.20 mmol) con el 2-hidroxi-5-bromobenzaldehido (1.00 g, 4.90 mmol) y DCC


cromatografía eluyendo con Hexano/AcOEt 9:1. Se obtuvieron 0.53 g (Rto: 31 %) de 12

como un sólido blanco puro.

P. f.: 148-150 °C 1H-RMN (CDCl3) δ (ppm): 3.85 (s, 3H, OCH3), 6.96 (dd, J = 2.3 y 8.2, 1H), 7.20 (m, 2H), 7.36 (m, 2H), 7.60 (d, J = 8.3, 1H), 7.75 (s, 1H), 7.85 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.2, 114.1, 114.6, 116.9, 118.0, 120.7, 121.0, 129.0, 129.4, 130.0, 133.9, 135.3, 138.3, 152.1, 159.3, 159.6. MS m/z (%): [M+2]+ 332 (100), M+ 330 (28), 302 (34). Anal. Elem.: Calculado para C16H11O3Cl: C, 58.03; H, 3.35.



56


57

6-Cloro-3-(2’-hidroxifenil)cumarina (7a)

7a

O O

Cl

O O

Cl HI

Ac2O/AcOH

MeO HO

7

Sobre una didisolución de 7 (0.15 g, 0.52 mmol) en AcOH/Ac2O (1:1, 3 mL), se

adicionó gota a gota HI 57 % (6 mL) a O °C. Finalizada la adición se llevó a

temperatura ambiente y a continuación se mantuvo a reflujo durante 3 h. Finalizada la

reacción, el disolvente se elimina al vacio, obteniendose un residuo sólido que fue

purificado por cromatografía en columna con Hexano/AcOEt 8:2. Se obtuvieron 0.04 g

(Rto: 28 %) del compuesto 7a como un sólido amarillo puro.

P. f.: 225-227 ºC 1H-RMN (DMSO-d6) δ (ppm): 6.86 (m, 2H), 7.23 (m, 2H), 7.39 (d, J = 8.8, 1H), 7.78 (dd, J = 2.3 y 8.8, 1H), 7.97 (s, 1H), 8.00 (s, 1H), 9.64 (sa, 1H, OH). 13C-RMN (DMSO-d6) δ (ppm): 115.7, 116.0, 118.2, 118.7, 121.2, 121.8, 127.1, 129.9, 130.3, 130.7, 133.8, 140.6, 140.7, 152.1, 155.1. MS m/z (%): [M+2]+ 274 (23), M+ 272 (100), 244 (35). Anal. Elem.: Calculado para C15H9O3Cl: C, 66.07; H, 3.33.



58


59


8a

O O

Cl

O O

Cl HI

Ac2O/AcOH

8

OMe OH

Analogamente a la preparación de 7a a partir de 7, la reacción de 8 (0.15 g, 0.52

mmol) y HI (6 mL) en AcOH/Ac2O (1:1, 3 mL), condujo a un residuo que fue

purificado por cromatografía en columna (Hexano/AcOEt 8:2). Se obtuvieron 0.09 g

(Rto: 60 %) de 8a como un sólido amarillo intenso puro.

P. f.: 241-243 ºC 1H-RMN (DMSO-d6) δ (ppm): 6.84 (d, J = 8.6, 2H), 7.44 (d, J = 8.8, 1H), 7.58 (m, 3H), 7.84 (d, J = 2.5, 1H), 8.09 (s, 1H), 9.81 (sa, 1H, OH). 13C-RMN (DMSO-d6) δ (ppm): 115.1 (2C), 117.7, 121.1, 124.8, 127.2, 127.8, 128.1, 129.9 (2C), 130.6, 137.1, 151.2, 158.2, 159.5. MS m/z (%): [M+2]+, 274 (20), M+ 272 (100), 244 (74). Anal. Elem.: Calculado para C15H9O3Cl: C, 66.07; H, 3.33.



60


61


9a

O O

Cl

O O

Cl HI

Ac2O/AcOH

9

OMe OH



purificado por cromatografia (Hexano/AcOEt 8:2). Se obtuvieron 0.10 g (Rto: 70 %) de

9a como un sólido amarillo intenso puro.

P. f.: 221-223 ºC 1H-RMN (DMSO-d6) δ (ppm): 6.82 (dd, J = 2.2 y 8.0, 1H), 7.10 (m, 2H), 7.25 (m, 1H), 7.44 (d, J = 8.8, 1H), 7.61 (dd, J = 2.5 y 8.8, 1H), 7.86 (d, J = 2.5, 1H), 8.14 (s, 1H), 9.60 (sa, 1H, OH); 13C-RMN (DMSO-d6) δ (ppm): 115.5, 115.9, 117.8, 119.2, 120.9, 127.6, 128.0, 128.2, 129.3, 131.1, 135.5, 139.1, 151.5, 157.1, 159.2. MS m/z (%): [M+2]+ 274 (21), M+ 272 (100), 244 (48). Anal. Elem.: Calculado para C15H9O3Cl: C, 66.07; H, 3.33.



62


63

6-Bromo-3-(2’-hidroxifenil)cumarina (10a)

10a

O O

Br

O O

Br HI

Ac2O/AcOH

MeO HO

10

Analogamente a la preparación de 7a a partir de 7, la reacción de 10 (0.10 g,

0.30 mmol) y HI (4 mL) en AcOH/Ac2O (1:1, 2 mL), condujo a un residuo que fue

purificado por cromatografía en columna (Hexano/AcOEt 8:2). Se obtuvieron 0.07g


P. f.: 230-232 ºC. 1H-RMN (DMSO-d6) δ (ppm): 6.87 (m, 2H), 7.23 (m, 2H), 7.40 (d, J = 8.8, 1H), 7.74 (dd, J = 2.3 y 8.8, 1H), 7.99 (s, 1H), 8.02 (s, 1H), 9.64 (sa, 1H, OH). 13C-RMN (DMSO-d6) δ (ppm): 115.7, 116.0, 118.2, 118.7, 121.2, 121.8, 127.1, 129.9, 130.3, 130.7, 133.8, 140.6, 152.1, 155.1, 158.8. MS m/z (%): [M+2]+ 318 (47), M+ 316 (71), 300 (8), 288 (40). Anal. Elem.: Calculado para C15H9O3Br: C, 56.81; H, 2.86.



64


65


11a

O O

Br

O O

Br HI

Ac2O/AcOH

11

OMe OH

Analogamente a la preparación de 7a a partir de 7, la reacción de 10 (0.10 g,

0.30 mmol) y HI (4 mL) en AcOH/Ac2O (1/1, 2 mL), condujo a un residuo que fue


(Rto: 57%) de 11a como un sólido amarillo intenso puro.

P. f.: 226-228 ºC. 1H-RMN (DMSO-d6) δ (ppm): 6.84 (d, J = 8.8, 2H), 7.34 (d, J = 9.1, 1H), 7.54 (d, J = 8.8, 2H), 7.66 (m, 1H), 7.99 (s, 1H), 8.09 (s, 1H), 9.81 (sa, 1H, OH). 13C-RMN (DMSO-d6) δ (ppm): 115.1 (2C), 116.0, 118.0, 121.6, 124.8, 127.7, 129.9 (2C), 130.2, 133.3, 137.0, 151.6, 158.2, 159.4. MS m/z (%): [M+2]+ 318 (31), [M+1]+ 317 (100), M+ 316 (40). Anal. Elem.: Calculado para C15H9O3Cl: C, 56.81; H, 2.86.



66


67


12a

O O

Br

O O

Br HI

Ac2O/AcOH

12

OMe OH





P. f.: 219-221 ºC 1H-RMN (DMSO-d6) δ (ppm): 6.82 (dd, J = 2.0 y 7.7, 1H), 7.10 (m, 2H), 7.25 (m, 1H), 7.38 (d, J = 8.8, 1H), 7.73 (dd, J = 2.3 y 8.8, 1H), 8.00 (d, J = 2.3, 1H), 8.14 (s, 1H), 9.59 (sa, 1H, OH). 13C-RMN (DMSO-d6) δ (ppm): 115.5, 115.9, 116.1, 118.1, 119.2, 121.4, 128.0, 129.3, 130.6, 133.9, 135.5, 139.0, 151.9, 157.1, 159.1. MS m/z (%): [M+2]+ 318 (46), M+ 316 (79), 288 (46). Anal. Elem.: Calculado para C15H9O3Cl: C, 56.81; H, 2.86.



68


69

3-(2-Tienil)cumarina (13a)

O

H

OH

DCC/DMSO

13a

S OHO

O

S

O

Analogamente a la preparación de 1, la reacción del ácido 2-tiofenilacético (1.40

g, 9.82 mmol) con el 2-hidroxibenzaldehido (1.00 g, 8.18 mmol) y DCC (2.53g, 12.27

mmol) en DMSO (10 mL), condujo a un residuo que fue purificado por cromatografía

en columna eluyendo con Hexano/AcOEt 9:1. Se obtuvieron 0.64 g (Rto: 34 %) de 13a

como un sólido blanco puro.

P. f.: 166-167 oC. 1H-RMN (CDCl3) δ (ppm): 7.12 (m, 1H), 7.28 (d, J = 9.4, 1H), 7.35 (d, J = 9.4, 1H), 7.42 (d, J = 5.1, 1H), 7.52 (m, 2H), 7.80 (d, J = 3.8, 1H), 7.99 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 116.4, 119.3, 121.7, 124.6, 127.0, 127.5, 127.7, 129.6, 131.1, 135.5, 135.9, 152.6, 160.1. MS m/z (%): [M + 1]+, 229 (22), M+,228 (30). Anal. Elem.: Calculado para C13H8O2S: C, 68.40; H, 3.53.



70


71

3-(3-Tienil)cumarina (13b)

O

H

OH

DCC/DMSO

13b

S OHO

O

S

O


g, 9.82 mmol) con el 2-hidroxibenzaldehido (1.00 g, 8.18 mmol) y DCC (2.50 g, 12.27


en columna con Hexano/AcOEt 9:1. Se obtuvieron 0.69 g (Rto: 37 %) de 13b como un

sólido blanco puro

P. f.: 175-176 oC. 1H-RMN (CDCl3) δ (ppm): 7.30 (d, J = 7.5, 1H), 7.35 (m, 2H), 7.53 (m, 3H), 7.93 (s, 1H), 8.18 (d, J = 3.0, 1H). 13C-RMN (CDCl3) δ (ppm): 116.3, 119.4, 122.6, 124.5, 125.6, 126.0, 126.2, 127.7, 131.1, 134.3, 137.2, 152.8, 160.0. MS m/z (%): [M + 1]+, 229 (18), M+, 228 (33). Anal. Elem.: Calculado para C13H8O2S: C, 68.40; H, 3.53.

Experimental: C, 68.50; H, 3.60


72


73

3-(3-Indolil)cumarina (13c)

O

H

OH

DCC/DMSO

13c

O

NH

ONH

OH

O

Analogamente a la preparación de 1, la reacción del ácido 3-indolilacético (1.70

g, 9.82 mmol) con el 2-hidroxibenzaldehido (1.00 g, 8.18 mmol) y DCC (2.53g, 12.27


en columna (Hexano/AcOEt 9:1). Se obtuvieron 0.72 g (Rto: 33 %) de 13c como un

sólido blanco puro.

P. f.: 190-191 oC. 1H-RMN (CDCl3) δ (ppm): 7.32 (m, 3H), 7.48 (m, 2H), 7.58 (dd, J = 7.6; 1.3, 1H), 7.98 (m, 1H); 8.16 (s, 1H), 8.20 (d, J = 2.6, 1H), 8.65 (sa, 1H, NH). 13C-RMN (CDCl3) δ (ppm): 111.9, 116.3, 119.5, 120.2, 120.9, 122.7, 122.9, 124.4, 125.5, 127.2, 127.6, 130.1, 134.9, 136.2, 143.5, 152.2, 160.8. MS m/z (%): [M + 1]+, 262 (15), M+, 261 (40). Anal. Elem.: Calculado para C17H11NO2: C, 78.15; H, 4.24.



74


75

7-Metoxi-3-(2-tienil)cumarina (14a)

O

H

OH

DCC/DMSO

14aO

S

O

S OHO MeO

MeO


g, 7.88 mmol) con el 2-hidroxi-4-metoxibenzaldehido (1.00 g, 6.57 mmol) y DCC (2.11

g, 10.25 mmol) en DMSO (10 mL), condujo a un residuo que fue purificado por

cromatografía en columna eluyendo con Hexano/AcOEt 9:1. Se obtuvieron 0.46 g (Rto:

27 %) de 14a como un sólido blanco puro.

P. f.: 155-156 oC. 1H-RMN (CDCl3) δ (ppm): 3.89 (s, 3H), 6.85 (s, 1H), 6.88 (d, J = 8.4, 1H), 7.11 (m, 1H), 7.38 (dd, J = 4.0 y 1.0, 1H), 7.45 (d, J = 8.4, 1H), 7.74 (dd, J = 4.0 y 1.0, 1H), 7.96 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.8, 100.4, 112.9, 113.1, 126.1, 126.5, 126.8, 127.4, 128.7, 136.0, 136.4, 154.5, 161.0, 162.5. MS m/z (%): [M + 1]+, 259 (33), M+, 258 (44). Anal. Elem.: Calculado para C14H10O3S: C, 65.10; H, 3.90.



76


77

7-Metoxi-3-(3-tienil)cumarina (14b)

O

H

OH

DCC/DMSO

14b

S OHO

O

S

O

MeO

MeO




cromatografía en columna (Hexano/AcOEt 9:1). Se obtuvieron 0.42 g (Rto: 25 %) de

14b como un sólido blanco puro.

P. f.: 168-169 oC. 1H-RMN (CDCl3) δ (ppm): 3.87 (s, 3H), 6.85 (m, 2H), 7.36 (dd, J = 5.0 y 3.0, 1H), 7.42 (d, J = 8.4, 1H), 7.50 (dd, J = 5.0 y 1.2, 1H), 7.87 (s, 1H), 8.11(dd, J = 3.0 y 1.2, 1H). 13C-RMN (CDCl3) δ (ppm): 55.7, 100.3, 112.8, 113.0, 119.3, 125.0, 125.5, 126.0, 128.6, 134.6, 137.5, 154.6, 160.8, 162.4. MS m/z (%): [M + 1]+, 259 (25), M+, 258 (47). Anal. Elem.: Calculado para C14H10O3S: C, 65.10; H, 3.90.



78


79

3-(3-Indolil)-7-metoxicumarina (14c)

O

H

OH

DCC/DMSO

14c

S OHO

O

NH

O

MeO

MeO



g, 10.25 mmol) en DMSO (10 mL); condujo a un residuo que fue purificado por


14c como un sólido blanco puro.

P. f.: 185-186 oC. 1H-RMN (CDCl3) δ (ppm): 3.95 (s, 3H), 6.89 (m, 2H), 7.28 (m, 2H), 7.47 (m, 2H), 7.95 (m, 1H), 8.10 (s, 1H), 8.13 (d, J = 3.0, 1H), 8.57 (sa, 1H, NH). 13C-RMN (CDCl3) δ (ppm): 55.7, 100.3, 100.0, 111.7, 112.6, 113.6, 119.4, 119.5, 120.6, 122.5, 126.0, 126.8, 128.1, 135.5, 136.2, 153.8, 161.1, 161.6. MS m/z (%): [M + 1]+, 292 (17), M+, 291 (49). Anal. Elem.: Calculado para C18H13NO3: C, 74.22; H, 4.50.



80


81

6-Metoxi-3-(2-tienil)cumarina (15a)

O

H

OH

DCC/DMSO

15a

S OHO

O

S

O

MeO

MeO





15a como un sólido blanco puro.

P. f.: 150-151 oC. 1H-RMN (CDCl3) δ (ppm): 3.85 (s, 3H), 6.96 (d, J = 2.8, 1H), 7.09 (m, 2H), 7.27 (d, J = 9.0, 1H), 7.42 (dd, J = 5.1 y 1.0, 1H), 7.79 (dd, J = 3.7 y 1.0, 1H), 7.94 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.7, 109.4, 117.3, 119.0, 119.6, 121.9, 127.0, 127.5, 127.7, 135.2, 135.9, 147.1, 156.2, 161.1. MS m/z (%): [M + 1]+, 259 (22), M+, 258 (50). Anal. Elem.: Calculado para C14H10O3S: C, 65.10; H, 3.90.



82


83

6-Metoxi-3-(3-tienil)cumarina (15b)

O

H

OH

DCC/DMSO

15b

S OHO

O

S

O

MeO

MeO






P. f.: 164-165 oC. 1H-RMN (CDCl3) δ (ppm): 3.84 (s, 3H), 6.95 (d, J = 3.0, 1H), 7.06 (dd, J = 9.0 y 3.0, 1H), 7.25 (d, J = 9.0, 1H), 7.37 (dd, J = 5.1 y 3.0, 1H), 7.50 (dd, J = 5.1 y 1.3, 1H), 7.86 (s, 1H), 8.18 (dd, J = 3.0 y 1.3, 1H). 13C-RMN (CDCl3) δ (ppm): 55.7, 109.6, 117.2, 118.9, 119.7, 122.8, 125.6, 126.0, 126.1, 134.3, 136.9, 147.2, 156.0, 160.1. MS m/z (%): [M + 1]+, 259 (27), M+, 258 (52). Anal. Elem.: Calculado para C14H10O3S: C, 65.10; H, 3.90.



84


85

3-(3-Indolil)-6-metoxicumarina (15c)

O

H

OH

DCC/DMSO

15cO

NH

ONH

OH

O

MeO

MeO




cromatografía en columna (Hexano/AcOEt 9:1). Se obtuvieron 0.73 g (Rto: 38 %) de

15c como un sólido blanco puro.

P. f.: 188-189 oC. 1H-RMN (CDCl3) δ (ppm): 3.84 (s, 3H), 5.87 (d, J = 7.7, 1H), 6.93 (d, J = 2.8, 1H), 7.04 (dd, J = 9.0 y 2.8, 1H), 7.30 (m, 3H), 7.79 (d, J = 7.7, 1H), 8.01 (s, 1H), 8.27 (s, 1H), 8.30 (sa, 1H, NH). 13C-RMN (CDCl3) δ (ppm): 55.7, 109.3, 112.6, 115.4, 117.2, 118.7, 119.3, 119.8, 122.7, 124.6, 125.7, 127.3, 135.9, 137.0, 146.7, 150.8, 156.1, 160.5. MS m/z (%): [M + 1]+, 292 (35), M+, 291 (57). Anal. Elem.: Calculado para C18H13NO3: C, 74.22; H, 4.50.



86


87

5,7-Dimetoxi-3-(2-tienil)cumarina (16a)

O

H

OH

DCC/DMSO

16a

S OHO

O

S

O

OMe

MeO

OMe

MeO


g, 6.58 mmol) con el 4,6-dimetoxi-2-hidroxibenzaldehido (1.00 g, 5.49 mmol) y DCC



16a como un sólido blanco puro.

P. f.: 177-178 oC. 1H-RMN (CDCl3) δ (ppm): 3.86 (s, 3H), 3.93 (s, 3H), 6.31 (d, J = 2.1, 1H), 6.46 (d, J = 2.1, 1H), 7.10 (dd, J = 5.0 y 3.6, 1H), 7.36 (d, J = 5.0, 1H), 7.72 (d, J = 3.6, 1H), 8.29 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.6, 56.0, 92.4, 95.1, 126.5, 127.1, 127.3, 127.5, 131.5, 136.9, 153.2, 155.3, 156.9, 160.0, 163.4. MS m/z (%): [M + 1]+, 289 (24), M+, 288 (51). Anal. Elem.: Calculado para C15H12O4S: C, 62.49; H, 4.20.



88


89

5,7-Dimetoxi-3-(3-tienil)cumarina (16b)

O

H

OH

DCC/DMSO

16b

S OHO

O

S

O

OMe

MeOOMe

MeO






P. f.: 158-159 oC. 1H-RMN (CDCl3) δ (ppm): 3.85 (s, 3H), 3.90 (s, 3H), 6.28 (d, J = 1.8, 1H), 6.42 (d, J = 1.8, 1H), 7.35 (dd, J = 4.8 y 2.5, 1H), 7.52 (d, J = 4.8, 1H), 8.10 (d, J = 2.5, 1H), 8.20 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.6, 55.8, 92.2, 94.8, 104.3, 117.3, 124.4, 125.2, 126.1, 132.7, 135.0, 155.3, 156.8, 160.4, 163.2. MS m/z (%): [M + 1]+, 289 (17), M+, 288 (60). Anal. Elem.: Calculado para C15H12O4S: C, 62.49; H, 4.20.



90


91

5,7-Dimetoxi-3-(3-indolil)cumarina (16c)

O

H

OH

DCC/DMSO

16c

O

NH

ONH

OH

O

OMe

MeO

OMe

MeO



(2.11 g, 10.25 mmol) en DMSO (10 mL); condujo a un residuo que fue purificado por

cromatografía en columna eluyendo con Hexano/AcOEt 9:1. Se obtuvieron 0.76 g (Rto:

43%) de 16c como un sólido blanco puro.

P. f.: 179-180 oC. 1H-RMN (CDCl3) δ (ppm): 3.88 (s, 3H), 3.95 (s, 3H), 5.68 (d, J = 7.2, 1H), 6.33 (d, J = 2.0, 1H), 6.48 (d, J = 2.0, 1H), 7.33 (m, 2H), 7.89 (dd, J = 7.2 y 1.2, 1H), 8.17 (s, 1H), 8.33 (dd, J = 7.0 y 1.2, 1H), 8.48 (sa, 1H, NH). 13C-RMN (CDCl3) δ (ppm): 55.8, 56.0, 92.3, 95.0, 104.6, 113.6, 115.3, 115.8, 119.6, 122.6, 124.5, 127.6, 133.3, 136.0, 151.0, 155.0, 156.8, 161.1, 163.2. MS m/z (%): [M + 1]+, 322 (28), M+, 321 (57). Anal. Elem.: Calculado para C19H15NO4: C, 71.02; H, 4.71.



92


93

2-Fenil-6-metoxibenzofurano (18) y 3-Fenil-7-metoxi-2H-cromeno (19)

OMeOO OMeO¡) DiBAL, -780C

¡¡) H2SO4 2N,reflujo

17 18

OMeO+

19

Sobre una didisolución de 17 (0.10 g, 0.40 mmol) en CH2Cl2 anhidro en

atmósfera de argón se añadió gota a gota a -78 ˚C DiBAL-H (0.59 mL, 0.59 mmol) y se

agitó durante 1 h. A continuación se añadieron gota a gota 1.5 equivalentes de DiBAL-

H (0.59 mL, 0.59 mmol) y se mantuvo en agitación 2 h mas a -78 ˚C. Se llevó a -50 ˚C

y se adicionó H2SO4 2N (10 mL). Se dejó subir lentamente la temperatura y a

continuación se calentó a reflujo 3 h. Finalizada la reacción se extrajo con CH2Cl2 (3 x

15 mL). Le fase orgánica fue secada con Na2SO4, filtrada y llevada a sequedad. Se

obtuvo un residuo sólido marrón que fue purificado mediante cromatografía en columna

eluyendo con Hexano/Tolueno 6:4 (2v). Se obtuvieron 35 mg de 18 (Rto: 10%) y 0.34 g

de 19 (Rto: 90%) como sólidos blancos.

Compuesto 18: P. f.: 156-157 ºC 1H-RMN (CDCl3) δ (ppm): 3.80 (s, 3H, OCH3); 6.80 (dd, J = 8.6 y 2.3, 1H); 6.88 (d, J = 1.0, 1H), 7.00 (d, J = 2.0, 1H), 7.24 (m, 1H), 7.37 (m, 3H), 7.74 (m, 2H). 13C-RMN (CDCl3) δ (ppm): 56.4 (OCH3), 96.5, 101.8, 112.65, 121.7, 123.2, 125.1, 128.7, 129.4, 131.4, 140.0, 156.6, 158.7. MS m/z (%): [M+1]+ 224 (14), M+ 223 (80), 208 (100), 151 (32). Anal. Elem.: Calculado para C15H12O2: C, 80.34; H, 5.39.

Experimental C, 80.36; H, 5.44.

Compuesto 19:

P. f.: 189-190 ºC 1H-RMN (CDCl3) δ (ppm): 3.64(s, 3H, OCH3), 5.0 (s, 2H, H-2), 6.32 (s, 1H), 6.36 (d, J = 2.5, 1H), 6.64 (s, 1H), 6.86 (d, J = 7.8, 1H), 7.22 (m, 5H).


94

13C-RMN (CDCl3) δ (ppm): 55.99 (OCH3), 67.88 (OCH2), 102.05, 108.04, 116.83, 120.48, 125.13, 128.23, 128.37, 129.21, 129.32, 137.56, 135.18, 161.31. MS m/z (%): [M+1]+ 239 (15), M+ 238 (91), 237 (100), 194 (13). Anal. Elem.: Calculado para C16H14O2: C, 80.65; H, 5.92.


Compuesto 18

Compuesto 19


95

2-Acetoniloxi-3-metoxibenzaldehido (20)

H

O

OHOMe

Ac2OH

O

OCOCH3OMe

20

Sobre una didisolución de 2-hidroxi-3-metoxibenzaldehido (5.01 g, 32.95 mmol)

y K2CO3 (5.92 g, 42.84 mmol) en etér etílico anhidro (100 mL) se adicionó anhídrido

acético. La reacción se mantuvo en agitación a temperatura ambiente durante 2 h. Luego

se filtró lavando con etér varias veces. La disolución fue llevada a sequedad para dar un

aceite. Se adicionó H2O y se extrajo con CH2Cl2. La fase orgánica fue secada con

Na2SO4, filtrada y llevada a sequedad. El residuo sólido obtenido fue purificado por

cromatografía en columna con Hexano/AcOEt (85:15). Se obtuvieron 5.45 g (Rto: 84

%) del compuesto 20 como sólido blanco.

P. f.: 115-116 ºC 1H-RMN (CDCl3) δ (ppm): 2.19 (s, 3H, CH3CO), 3.66 (s, 3H, OCH3), 7.01 (d, 1H, J = 8.1, H-4), 7.11 (t, J = 8.1, H-3), 7.24 (d, J = 8.1, H-2), 9.92 (s, 1H, CHO). 13C-RMN (CDCl3) δ (ppm): 20.7 (CH3CO), 56.6 (OCH3), 118.2, 121.5, 127.39, 129.5, 141.8, 152.1. 169.0 (CO), 189.1 (CHO). Anal. Elem.: Calculado para C15H12O2: C, 61.85; H, 5.19.



96


97

Acetato de 2-hidroxi-3-metoxibencilo (21)

H

O

OCOCH3OMe

NaBH3CN OCOCH3

OHOMe2120

Sobre una didisolución de 20 (5.45 g, 28.09 mmol) en THF/H2O (100 mL) se

adicionó el cianoborohidruro de sodio, y a continuación se acidificó a pH 3 con

AcOH/THF/HCl (10:8:1). Se mantuvo en agitación a temperatura ambiente durante 1 h.

Se adicionó H2O y se extrajo con CH2Cl2 (3 x 25 mL). La fase orgánica fue lavada con

NaHCO3 (3 x 25 mL) y NaCl saturado (25 mL); secada con Na2SO4, filtrada y llevada a

sequedad. El sólido obtenido fue purificado por cromatografía en columna eluyendo con

Hexano/AcOEt 9:1. Se obtuvieron 2.78 g (Rto: 50 %) del compuesto 21 como un aceite

amarillo.

1H-RMN (CDCl3) δ (ppm): 2.08 (s, 3H, CH3CO), 3.84 (s, 3H, OCH3), 5.19 (s, 2H, CH2), 6.28 (sa, 1H, OH), 6.87 (3H, m). 13C-RMN (CDCl3) δ (ppm): 20.8 (CH3CO), 55.9 (OCH3), 61.4 (CH2), 110.9, 119.5, 121.6, 122.2, 144.2, 146.6, 171.3. Anal. Elem.: Calculado para C10H12O4: C, 61.22; H, 6.16.



98


99

Bromuro de 2-hidroxi-3-metoxibenciltrifenilfosfonio (22)

21

OCOCH3

OHOMe

PPh3HBr PPh3+ Br-

OHOMe

22

Sobre una suspensión de hidrobromuro de trifenilfosfina (4.40 g, 12.82 mmol)

en acetonitrilo anhidro se adicionó una disolución de 21 (2.51 g, 12.82 mmol) en el

mismo disolvente. Se calentó a reflujo durante 2 h. A continuación la reacción se llevó a

sequedad, el residuo obtenido fue solubilizado en CH2Cl2 y precipitado en etér etílico, el

sólido precipitado fue filtrado y secado. Se obtuvieron 5.71 g (Rto: 92 %) del

compuesto 22 como sólido blanco.

P. f.: 203-204 ºC. 1H-RMN (CDCl3) δ (ppm): 3.7 (s, 3H, OCH3), 4.8 (d, J = 14.1, 2H, CH2), 6.52 (m, 1H), 6.65 (m, 1H), 6.89 (d, J = 1.5, 1H), 7.74 (m, 15, H). 13C-RMN (CDCl3) δ (ppm): 24.0 (CH2), 55.0 (OCH3), 112.0, 120.0, 123.4, 123.5, 125.1, 128.0, 129.4, 134.7, 145.0, 155.2.


100


101

7-Metoxi-2-(3’,4’,5’-trimetoxifenil)benzofurano (23)

22

PPh3+ Br-

OHOMe

OMe

OMe

OMeO

OMe

DMAP/DCC

+OHMeO

MeOOMe

O

23

Sobre una didisolución de 22 (1.50 g, 3.13 mmol), ácido 3,4,5-trimetoxibenzoico

(0.67 g, 3.16 mmol) y DMAP (0.06 g, 0.50 mmol), en CH2Cl2 anhidro, se adicionó la

DCC (0.81 g, 3.94 mmol) en CH2Cl2 anhidro. Se mantuvo en agitación a temperatura

ambiente en atmosfera de argón durante una noche. El día siguiente se llevó a sequedad

la reacción y el residuo obtenido fue solubilizado con dioxano anhidro, se adicionó Et3N

(2.45 mL, 17.71 mmol) y se calentó a reflujo 12 h. Finalizada la reacción se llevó a

temperatura ambiente, el precipitado se filtró y la disolución se llevó a sequedad. El

residuo obtenido fue purificado por cromatografía en columna con Hexano/AcOEt

95:15. Se obtuvieron 0.62 g (Rto: 62 %) del compuesto 23 como sólido blanco.

P. f.: 217-218ºC 1H-RMN (CDCl3) δ (ppm): 3.81 (s, 3H, OCH3-C4’), 3.86 (s, 6H, OCH3-C3’ + CH3O-C5’), 3.95 (s, 3H, OCH3-C7), 6.71 (dd, J = 7.2 y 1.7, 1H, H-6), 6.86 (s, 1H, H-3), 7.00 (s, 2H, H-2’ + H-6’), 7.07 (m, 2H, H-4 + H-5). 13C-RMN (CDCl3) δ (ppm): 56.0 (OCH3-C7), 56.3 (OCH3-C3’ + OCH3-C5’), 61.0 (OCH3-C4’), 101.5 (C3), 102.4 (C2’ + C6’), 106.5 (C6), 113.2 (C4), 123.7 (C5), 125.9 (C1’), 131.0 (C3a), 138.7 (C4’), 144.0 (C7a), 145.2 (C7), 153.5 (C3 + C5’), 155.9 (C2). MS m/z (%): [M+1]+ 315 (13), M+ 314 (100), 240 (19), 210 (14). Anal. Elem.: Calculado para C18H18O5: C, 68.78; H, 5.77.



102


103

2-(3’,5’-Dimetoxifenil)-7-metoxibenzofurano (24)

PPh3+ Br-

OHOMe

OMe

OMeO

OMe

DMAP/DCC+ OHMeO

OMe

O

22 24

Analogamente a la preparación de 23, la reacción de 22 (1.50 g, 3.13 mmol),

ácido 3,4,5-trimetoxibenzoico (0.58 g, 3.16 mmol), DMAP (0.06 g, 0.50 mmol), DCC

(0.81 g, 3.94 mmol) en CH2Cl2 anhidro y Et3N (2.45 mL, 17.71 mmol) en dioxano

anhidro condujo a un residuo que fue purificado por cromatografía en columna con

Hexano/AcOEt (95:15). Se obtuvieron 0.47 g (Rto: 53 %) del compuesto 24 como

sólido blanco.

P. f.: 209-211 ºC 1H-RMN (CDCl3) δ (ppm): 3.75 (s, 6H, OCH3-C3’ + OCH3-C5’), 3.93 (s, 3H, OCH3-C7), 6.37 (t, J = 1.9, 1H, H-4’), 6.70 (d, J = 7.5, 1H, H-6), 6.89 (s, 1H, H-3), 6.94 (d, J = 1.9, 2H, H-2’ + H-6’), 7.05 (m, 2H, H-4 + H-5). 13C-RMN (CDCl3) δ (ppm): 55.5 (OCH3-C3’ + OCH3-C5’), 56.1 (OCH3-C7), 101.1 (C4’), 102.2 (C3), 103.1 (C2’ + C6’), 106.8 (C6), 113.4 (C4),123.6 (C5), 130.8 (C3a), 132.1 (C1’), 144.1 (C7a), 145.3(C7), 155.9 (C2), 161.1 (C3 + C5’). MS m/z (%): [M+1]+ 285 (26), M+ 284 (100), 240 (33), 211 (14). An. Elem. Calculado para C17H16O4: C, 71.82; H, 5.67.



104


105

7-Metoxi-2-(4’-metoxifenil)benzofurano (25)

22 25

PPh3+ Br-

OHOMe

OOMe

OMe DMAP/DCC

+OH

O

MeO

Analogamente a la preparación de 23, la reacción de 22 (1.50 g, 3.13 mmol),

ácido 4-metoxibenzoico (0.48 g, 3.16 mmol), DMAP (0.06 g, 0.50 mmol), DCC (0.81 g,

3.94 mmol) en CH2Cl2 anhidro y Et3N (2.45 mL, 17.71 mmol) en dioxano anhidro

condujo a un residuo que fue purificado por cromatografía en columna con

Hexano/AcOEt (95:15). Se obtuvieron 0.40 g (Rto: 49 %) del compuesto 25 como

sólido blanco.

P. f.: 225-226 ºC 1H-RMN (CDCl3) δ (ppm): 3.57 (s, 3H, OCH3-C4’), 3.81 (s, 3H, OCH3-C7), 6.57 (dd, J = 6.6 y 2.4, 1H, H-6), 6.63 (s, 1H, H-3), 6.73 (d, J = 8.8, 2H, H-3’ + H-5’), 6.94 (m, 2H, H-4 + H-5), 7.60 (d, J = 8.9, 2H, H-2’ + H-6’). 13C-RMN (CDCl3) δ (ppm): 55.2 (OCH3-C4’), 56.1 (OCH3-C7), 100.1 (C3), 106.4 (C6), 113.2 (C4), 114.2 (C3’ + C5’), 123.2 (C1’), 123.6 (C5), 26.5 (C2’ + C6’), 131.3 (C3a), 143.9 (C7a), 145.3 (C7), 156.2 (C2), 160.1 (C4’). MS m/z (%): [M+1]+, 255 (22), M+, 254 (100), 239 (53), 211 (19). Anal. Elem.: Calculado para C16H14O3: C, 75.57; H, 5.55.



106


107

7-Metoxi-2-(3’,4’,5’-trimetoxifenil)dihidrobenzofurano (26)

OOMe

OMe

OMe

OMeO

OMe

OMe

OMe

OMe

H2/Pd

23 26

Una disolución de 23 (0.10 g, 0.32 mmol) en etanol absoluto fue hidrogenada

sobre Pd/C (0.30 g) a 4 atm de presión durante 24 h. Una vez finalizada la reacción se

filtró el catalizador, lavando varias veces con etanol absoluto. La fase orgánica fue

llevada a sequedad para dar un sólido que fue purificado por cromatografía en columna

con Hexano/AcOEt 9:1 .Se obtuvieron 0.08 g (Rto: 80 %) del compuesto 26 como un

sólido blanco.

P. f.: 135-136 ºC 1H-RMN (CDCl3) δ (ppm): 3.28 (dd, J = 9.2, 1H, H-3), 3.59 (dd, J = 9.2, 1H, H-3), 3.83 (s, 3H, OCH3-C4’), 3.84 (s, 6H, OCH3-C3’ + OCH3-C5’), 3.89 (s, 3H, OCH3-C7), 5.71 (t, J = 9.2, 1H, H-2), 6.65 (s, 2H, H-2’ + H-6’), 6.79 (d, J = 7.9, 1H, H-6), 6.82 (d, J = 7.6, 1H, H-4), 6.85 (dd, J = 7.9 y 7.6, H-5). 13C-RMN (CDCl3) δ (ppm): 39.5 (C3), 56.6 (OCH3-C7), 56.8 (OCH3-C3’ + OCH3-C5’),61.5 (OCH3-C4’), 85.9 (C2), 103.9 (C2’ + C6’), 112.1 (C6), 117.7 (C4), 122.0 (C5), 128.4 (C3a), 137.6 (C1’), 138.4 (C4’), 145.2 (C7), 148.5 (C7a), 154.0 (C3 + C5’). Anal. Elem.: Calculado para C16H14O3: C, 75.57; H, 5.55.



108


109

Bromuro de 2-hidroxibenciltrifenilfosfonio (27)

OH

OH

PPh3HBr PPh3+ Br-

OH27

Sobre una suspensión de hidrobromuro de trifenilfosfina (13.60 g, 40.00 mmol) en

acetonitrilo anhidro se adicionó una didisolución de alcohol 2-hidroxibencílico (5.00 g, 40.00

mmol) en el mismo disolvente. Se calentó a reflujo 2 h, a continuación se llevo a sequedad la

reacción, el residuo obtenido fue solubilizado en CH2Cl2 y precipitado en etér etílico, el sólido

precipitado fue filtrado y secado. Se obtuvieron 17.00 g (Rto: 94 %) del compuesto 27 como sólido

blanco puro que se utilizó pora la reacción siguiente.

P. f.: 210-211 ºC 1H-RMN (CDCl3) δ (ppm): 4.8 (d, J = 14.1, 2H, CH2), 6.35 (s, 1H), 6.52 (d, J = 2.3, 1H), 6.89 (d, J = 1.5, 1H), 7.74 (m, 2H). 13C-RMN (CDCl3) δ (ppm): 24.9 (CH2), 114.7, 118.0, 122.0, 123.6, 125.1, 127.9, 128.0, 130.5, 134.7, 158.0.


110


111

2-(3’,4’,5’-Trimetoxifenil)benzofurano (28)

PPh3+ Br-

OH

Cl

OMeO

MeOOMe

O

OMe

OMe

OMe

Et3N+

27 28

Una didisolución de 27 (0.50 g, 1.10 mmol) y cloruro de 3,4,5-trimetoxibenzoilo (0.25 g,

1.11 mmol) en tolueno (20 mL) y trietilamina (0.5 mL) fue calentada a reflujo durante 2 h.

Finalizada la reacción se filtró el sólido y la didisolución fue llevada a sequedad. El residuo

obtenido fue purificado por cromatografía en columna con Hexano/AcOEt 95:5, para obtener 28

como sólido blanco puro (0.19 g, Rto: 60 %).

P. f.: 101-102 °C 1H-RMN (CDCl3) δ (ppm): 3.90 (s, 3H, OCH3-C4’), 3.96 (s, 6H, OCH3-C3’ + OCH3-C5’), 6.85 (s, 1H, H-3), 7.09 (s, 2H, H-2’ + H-6’), 7.2-7.6 (m, 6H, Ar-H). 13C-RMN (CDCl3) δ (ppm): 56.2, 56.5, 102.8, 104.6, 111.6, 121.0, 123.3, 123.9, 124.5, 124.8, 139.1, 151.4, 155.5, 156.7. MS m/z (%): M+ 284 (100), 269 (90), 241 (15), 211 (19), 168 (22). Anal. Elem.: Calculado para C17H16O4: C, 71.82; H, 5.67.



112


113

2-(3’,5’-Dimetoxifenil)benzofurano (29)

27 29

PPh3+ Br-

OH

Cl

OMeO

OMeO

OMe

OMe

Et3N+

Una didisolución de 27 (0.50 g, 1.10 mmol) y cloruro de 3,5-dimetoxibenzoilo (0.22 g, 1.11

mmol) en tolueno (20 mL) y trietilamina (0.5 mL) fue calentada a reflujo durante 2 h. Finalizada la

reacción se filtró el sólido y la disolucion fue llevada a sequedad. El residuo obtenido fue purificado

por cromatografía en columna con Hexano/AcOEt 95:5, para obtener 29 como sólido blanco (0.16

g, Rto: 58 %).

P. f.: 110-111 °C 1H-RMN (CDCl3) δ (ppm): 3.87 (s, 6H, OCH3-C3’ + OCH3-C5’), 6.5 (s, 1H, H-3), 7.01 (s, 1H, H-4’), 7.03 (d, 2H, H-2’ + H-6’), 7.2-7.6 (m, 4H, Ar-H). 13C-RMN (CDCl3) δ (ppm): 56.0, 100.6, 102.6, 103.8, 111.5, 121.2, 123.5, 123.9, 124.8, 132.5, 155.4, 156.6, 162.4. MS m/z (%): M+ 254 (100), 225 (12), 197 (20), 168 (15), 139 (10). Anal. Elem.: Calculado para C16H14O3: C, 75.57; H, 5.55.



114


115

2-(4’-Metoxifenil)benzofurano (30)

27 30

PPh3+ Br-

OHCl

O

MeOO

OMeEt3N

Una didisolución de 27 (0.50 g, 1.10 mmol) y cloruro de 4-metoxi-benzoilo (0.19 g, 1.11


reacción se filtró el sólido y la disolución fue llevada a sequedad. El residuo obtenido fue purificado

por cromatografía en columna con Hexano/AcOEt 95:5, obteniendose 30 como sólido blanco (0.17

g, Rto: 69 %).

P. f.: 102-103 °C 1H-RMN (CDCl3) δ (ppm): 3.87 (s, 3H, OCH3-C4’), 6.9 (s, 1H, H3), 6.99 (d, 2H, H-3’ + H-5’), 7.2-7.6 (m, 4H, Ar-H), 7.81 (d, 2H, H-2’ + H-6’). 13C-RMN (CDCl3) δ (ppm): 55.9, 102.6, 111.7, 114.6, 121.2, 122.5, 123.5, 123.6, 124.5, 128.7, 155.6, 156.7, 160.6. MS m/z (%): M+ 254 (100), 225 (12), 197 (20), 168 (15). Anal. Elem.: Calculado para C16H14O3: C, 75.57; H, 5.55.



116


117

Alcohol 2-hidroxi-5-metilbencilico (31)

H

OH

O

OH

OH

NaBH4

31

Sobre una didisolución de 2-hidroxi-5-metilbenzaldehido (0.50 g, 3.60 mmol) en etanol (10

mL) a 0 °C se adicionó borohidruro de sodio (0.13 g, 3.60 mmol) y la reacción se mantuvo en

agitación a temperatura ambiente durante 2 h. Finalizada la reacción la didisolución se llevó a

sequedad, al residuo sólido obtenido se le adicionó HCl 1N (10 mL) y se extrajo con etér etílico (10

mL x 3). La fase orgánica fue secada con Na2SO4, filtrada y evaporada al vacío para dar el

compuesto 31 como un aceite (0.40 g, Rto: 81 %).

1H-RMN (CDCl3) δ (ppm): 2.35 (s, 3H, CH3), 4.85 (s, 2H, CH2), 5.0 (1H, CH2-OH), 6.7 (dd, 2H, H-5 + H-6), 7.0 (s, 1H, H-3), 7.05 (sa, 1H, Ar-OH). 13C-RMN (CDCl3) δ (ppm): 24.8, 58.3, 116.4, 129.6, 128.9, 130.7, 131.5, 151.6.


118


119

Bromuro de 2-hidroxi-5-metilbenciltrifenilfosfonio (32)

PPh3+ Br-

OH

OH

OH31 32

PPh3 HBr

Sobre una suspensión de hidrobromuro de trifenilfosfina (0.90 g, 2.60 mmol) en acetonitrilo

anhidro (25 mL) se adicionó una disolución de 31 (0.40 g, 2.60 mmol) en el mismo disolvente. Se

calentó a reflujo 2 h. A continuación la reacción se llevó a sequedad y el residuo obtenido fue

disuelto en CH2Cl2 (50 mL) y precipitado en etér etílico, el precipitado fue filtrado y secado.

Obteniendose el compuesto 32 como sólido blanco (0.95 g, Rto: 79 %) que se utilizó en la siguiente

reacción.

P. f.: 217-218 ºC 1H-RMN (CDCl3) δ (ppm): 3.77 (s, 3H, OCH3), 4.8 (d, J = 14.1, 2H, CH2), 6.35 (s, 1H), 6.52 (d, J = 1.5, 1H), 6.89 (d, J = 1.5, 1H), 7.74 (sa, 1H, OH). 13C-RMN (CDCl3) δ (ppm): 23.0, 24.9 (CH2), 114.7, 118.4, 123.0, 126.6, 127.1, 128.0, 129.9, 134.0, 135.5, 157.2.


120


121

5-Metil-2-(4’-metoxifenil)benzofurano (33)

32 33

PPh3+ Br-

OHCl

O

MeOO

OMeEt3N

Una didisolución de 32 (0.25 g, 0.50 mmol) y cloruro de 4-metoxibenzoilo (0.09 g, 0.50



por cromatografía en columna eluyendo con Hexano/AcOEt 95:5, obteniendose 33 como sólido

blanco (0.10 g, Rto: 78 %).

P. f.: 157-158 °C 1H-RMN (CDCl3) δ (ppm): 2.16 (s, 3H, CH3-C5), 3.85 (s, 3H, OCH3-C4’), 6.8 (s, 1H, H-3), 6.97 (d, 2H, H-3’ + H-5’), 7.26 (s, 1H, H-5), 7.35 (m, 2H, H-6 + H-7), 7.77 (d, 2H, H-2’ + H-6’). 13C-RMN (CDCl3) δ (ppm): 24.6, 55.8, 102.6, 111.7, 114.6, 120.5, 122.5, 123.8, 125.3, 128.4, 131.7, 152.6, 156.7, 160.5. MS m/z (%): M+ 238 (100), 223 (75), 195 (25), 165 (10). Anal. Elem.: Calculado para C16H14O2: C, 80.65; H, 5.92.



122


123

2-(3’,5’-Dimetoxifenil)-5-metilbenzofurano (34)

PPh3+ Br-

OH

Cl

O

O

Et3NOMe

OMe

+MeO

OMe32 34


mmol) en tolueno (40 mL) y trietilamina (1 mL) fue calentada a reflujo durante 2 h. Finalizada la


por cromatografía en columna con Hexano/AcOEt 95:5, obteniendose 34 como sólido blanco puro

(0.40 g, Rto: 75 %).

P. f.: 142-143 °C 1H-RMN (CDCl3) δ (ppm): 2.45 (s, 3H, CH3-C5), 3.87 (s, 6H, OCH3-C3’+ OCH3-C5’), 6.45 (s, 1H, H-3), 6.92 (s, 1H, H-4’), 7.0 (t, 2H, H-2’ + H-6’), 7.10-7.35 (m, 3H, Ar-H). 13C-RMN (CDCl3) δ (ppm): 24.5, 55.7, 100.6, 102.7, 103.5, 111.8, 120.5, 123.6, 125.3, 131.8, 132.5, 152.2, 156.7, 162.4. MS m/z (%): M+ 268 (100), 237 (20), 225 (15), 181 (25), 134 (10). Anal. Elem.: Calculado para C17H16O3: C, 76.10; H, 6.01.



124


125

5-Metil-2-(3’,4’,5’-trimetoxifenil)benzofurano (35)

32 35

PPh3+ Br-

OHCl

O

MeOO

Et3NOMe

OMe

OMe+ MeO

OMe


2.00 mmol) en tolueno (40 mL) y trietilamina (1 mL) fue calentada a reflujo durante 2 h. Finalizada

la reacción se filtró el sólido y la disolución fue llevada a sequedad. El residuo obtenido fue

purificado por cromatografía en columna eluyendo con Hexano/AcOEt 95:5, obteniendose 35 como

sólido blanco puro (0.33 g, Rto: 56 %).

P. f.: 119-120 °C 1H-RMN (CDCl3) δ (ppm): 2.44 (s, 3H, CH3-C5), 3.89 (s, 3H, OCH3-C4’), 3.96 (s, 6H, OCH3-C3’ + OCH3-C5’), 6.88 (s, 1H, H-3), 7.07 (s, 2H, H-2’ + H-6’), 7.25 (s, 1H, H-4), 7.35-7.4 (m, 2H; H-6 + H-7). 13C-RMN (CDCl3) δ (ppm): 24.6, 56.3, 56.7, 102.6, 104.7, 111.6, 120.5, 123.9, 124.8, 125.6, 131.4, 139.0, 151.4, 152.6, 156.9. MS m/z (%): M+ 298 (100), 283 (80), 255 (15), 169 (20). Anal. Elem.: Calculado para C18H18O4: C, 72.47; H, 6.08.



126


127

Alcohol 5-bromo-2-hidroxibencilico (36)

H

OH

BrO

OH

OH

BrNaBH4

36

Sobre una didisolución de 5-bromo-2-hidroxibenzaldehido (1.00 g, 5.00 mmol) en etanol

(20 mL) a 0 °C se adicionó borohidruro de sodio (0.19 g, 5.00 mmol) y la reacción se mantuvo en

agitación a temperatura ambiente durante 2 h. Finalizada la reacción la disolución se llevó a

sequedad, al residuo sólido obtenido se le adicionó HCl 1N (20 mL) y se extrajo con eter etílico (10

mL x 3). La fase orgánica fue secada con Na2SO4, filtrada y llevada a sequedad para dar el

compuesto 36 como un sólido blanco (0.79 g, Rto: 78 %).

P. f.: 88-89 °C 1H-RMN (CDCl3) δ (ppm): 2.65 (1H, CH2-OH), 4.90 (s, 2H, CH2), 6.74 (d, 1H), 7.15 (s, 1H), 7.26 (m, 1H), 7.4 (1H, OH). 13C-RMN (CDCl3) δ (ppm): 57.3, 115.8, 118.5, 131.1, 132.2, 133.1, 153.4.


128


129

Bromuro de 5-bromo-2-hidroxibenciltrifenilfosfonio (37)

36

PPh3+ Br-

OH

BrOH

OH

Br PPh3 HBr

37

Sobre una suspensión de hidrobromuro de trifenilfosfina (1.17 g, 3.00 mmol) en acetonitrilo

anhidro se adicionó una disolución de 36 (0.79 g, 3.00 mmol) en el mismo disolvente. Se calentó a

reflujo durante 2 h. A continuación la reacción se llevó a sequedad, el residuo obtenido fue

solubilizado en CH2Cl2 y precipitado en etér etílico, el sólido precipitado fue filtrado y secado. Se

obtuvo el compuesto 37 como sólido blanco (1.70 g, Rto: 95 %) que se utilizó en la siguiente

reacción.

P. f.: 219-221 ºC 1H-RMN (CDCl3) δ (ppm): 4.8 (d, J = 14.1, 2H, CH2), 6.35 (s, 1H), 6.52 (d, J = 1.5, 1H), 6.89 (d, J = 1.5, 1H), 7.74 (sa, 1H, OH). 13C-RMN (CDCl3) δ (ppm): 23.4 (CH2), 115.7, 118.4, 118.9, 126.9, 127.9, 128.6, 129.9, 134.0, 135.0, 155.2.


130


131

5-Bromo-2-(3’,4’,5’-trimetoxifenil)benzofurano (38)

Cl

O

MeO

MeO

OMe

PPh3+ Br-

OH O

Et3NBr BrOMe

OMe

OMe+

37 38

Una didisolución de 37 (0.50 g, 0.94 mmol) y cloruro de 3,4,5-trimetoxi-benzoilo (0.21 g,

0.94 mmol) en tolueno (20 mL) y trietilamina (0.5 mL) fue calentada a reflujo durante 2 h.

Finalizada la reacción se filtró el sólido y la disolución fue llevada a sequedad. El residuo obtenido

fue purificado por cromatografía en columna con Hexano/AcOEt 95:5, obteniendose 38 como

sólido blanco (0.26 g, Rto: 75 %).

P. f.: 169-170 °C 1H-RMN (CDCl3) δ (ppm): 3.90 (s, 3H, OCH3-C4’), 3.96 (s, 6H, OCH3-C3’ + OCH3-C5’), 6.89 (s, 1H, H3), 7.06 (s, 2H, H-2’ + H-6’), 7.25 (m, 1H), 7.37 (m, 1H), 7.68 (m, 1H). 13C-RMN (CDCl3) δ (ppm): 56.3, 56.6, 102.9, 104.7, 113.8, 116.6, 124.3, 124.8, 126.2, 129.0, 139.3, 151.4. 154.3, 156.8. MS m/z (%): [M+2]+ 364 (15), M+ 362 (83), 347 (100), 289 (50). Anal. Elem.: Calculado para C17H15BrO4: C, 56.22; H, 4.16.



132


133

5-Bromo-2-(3’,5’-dimetoxifenil)benzofurano (39)

37 39

Cl

OMeO

OMe

PPh3+ Br-

OH O

Et3NBr BrOMe

OMe

+




por cromatografía en columna con Hexano/AcOEt 95:5, obteniéndose 39 como sólido blanco puro

(0.11 g, Rto: 35 %).

P. f. 95-96 °C 1H-RMN (CDCl3) δ (ppm): 3.86 (s, 6H, 2CH3), 6.49 (t, 1H, H-4’), 6.93 (s, 1H, H-3), 6.99 (d, 2H, H-2’ + H-6’), 7.25 (s, 1H), 7.37 (s, 1H), 7.69 (s, 1H). 13C-RMN (CDCl3) δ (ppm): 55.8, 100.3, 102.7, 103.4, 113.9, 116.4, 124.2, 126.2, 129.1, 132.5, 154.4, 156.8, 162.3. MS m/z (%): [M+2]+ 336 (18), 334 M+, 334 (100), 224 (32), 139 (15). Anal. Elem.: Calculado para C16H13BrO3: C, 57.68; H, 3.93.



134


135

5-Bromo-2-(4’-metoxifenil)benzofurano (40)

37 40

PPh3+ Br-

OHCl

O

MeOO

Et3NBr Br+ OMe

Una didisolución de 37 (0.50 g, 0.94 mmol) y cloruro de 4-metoxibenzoilo (0.16 g, 0.95



por cromatografía en columna con Hexano/AcOEt 95:5, obteniendose 40 como sólido blanco puro

(0.13 g, Rto: 50 %).

P. f.: 166-167 °C 1H-RMN (CDCl3) δ (ppm): 3.86 (s, 3H, OCH3-C4’), 6.81 (s, 1H, H-3), 6.99 (d, 2H, H-3’ + H-5’), 7.25 (s, 1H, H-4), 7.35 (m, 2H, H-6 + H-7), 7.79 (d, 2H, H-2’ + H-6’). 13C-RMN (CDCl3) δ (ppm): 55.9, 102.9, 113.6, 114.7, 116.4, 122.8, 124.4, 126.0, 128.8, 129.1, 154.5, 156.8, 160.6. MS m/z (%): [M+2]+ 304 (18), M+ 302 (100), 287 (75), 261 (50). Anal. Elem.: Calculado para C15H11BrO2: C, 59.43; H, 3.66.



136


137

Alcohol 3-etoxi-2-hidroxibencilico (41)

H

OH

O

OH

OH

NaBH4

O O41

Sobre una didisolución de 3-etoxi-2-hidroxibenzilalcohol (2.00 g, 12.00 mmol) en etanol (20

mL) a 0 °C se adicionó borohidruro de sodio (0.45 g, 12.00 mmol) y la reacción se mantuvo en

agitación a temperatura ambiente durante 2 h. Finalizada la reacción se llevó a sequedad la

disolución, se adicionó HCl 1N (40 mL) al residuo sólido obtenido y se extrajo con etér etílico (20

mL x 3). La fase organica fue secada con Na2SO4, filtrada y llevada a sequedad para dar el

compuesto 41 como sólido blanco (1.81 g, Rto: 90 %).

P. f.: 166-167 °C 1H-RMN (CDCl3) δ (ppm): 1.40 (t, 3H, CH3), 3.2 (1H, CH2OH), 4.07 (q, 2H, CH2-CH3), 4.8 (s, 2H, CH2-OH), 6.5 (1H, OH), 6.78 (m, 3H). 13C-RMN (CDCl3) δ (ppm): 14.7, 58.1, 65.1, 114.8, 120.4, 122.3, 129.6, 143.5, 148.6.


138


139

Bromuro de 3-etoxi-2-hidroxibenciltrifenilfosfonio (42)

41

PPh3+ Br-

OH

PPh3 HBrOH

OHO O

42

Sobre una suspensión de hidrobromuro de trifenilfosfina (3.41 g, 10.00 mmol) en

acetonitrilo anidro (40 mL) se adicionó una disolución de alcohol 3-etoxi-2-hidroxibencílico (1.69

g, 10.00 mmol) en el mismo disolvente. Se calentó a reflujo 2 h. A continuación se llevó a sequedad

la reacción, el residuo obtenido fue disuelto en CH2Cl2 y precipitado con eter etílico, el sólido

precipitado fue filtrado y secado. Se obtuvo el compusto 42 como un sólido blanco puro (3.90 g,

Rto: 79 %) que se utilizó en la reacción siguiente.

P. f.: 156-157 °C 1H-RMN (CDCl3) δ (ppm): 1.40 (t, 3H, CH3), 3.2 (1H, CH2OH), 4.07 (q, 2H, CH2-CH3), 4.8 (s, 2H, CH2-OH), 6.5 (1H, OH), 6.78 (m, 3H). 13C-RMN (CDCl3) δ (ppm): 14.7, 58.1, 65.1, 114.8, 120.4, 122.3, 129.6, 143.5, 148.6


140


141

7-Etoxi-2-(3’,4’,5’-trimetoxifenil)benzofurano (43)

42

PPh3+ Br-

OHO

Cl

O

MeO O

Et3NOMe

OMe

OMe+

O

MeO

OMe43


2.00 mmol) en tolueno (40 mL) y trietilamina (1 mL) fue calentada a reflujo durante 2 h. Finalizada

la reacción se filtró el sólido y la disolución fue llevada a sequedad. El residuo obtenido fue

purificado por cromatografía en columna con Hexano/AcOEt 95:5, obteniendose 43 como sólido

blanco puro (0.36 g, Rto: 55 %).

P. f.: 143-144°C 1H-RMN (CDCl3) δ (ppm): 1.55 (t, 3H, CH3), 3.87 (s, 3H, OCH3-C4’), 3.97 (s, 6H, 2CH3), 4.35 (q, 2H, CH2), 6.77 (d, 1H, H-6), 6.95 (s, 1H, H-3), 7.10 (s, 2H, H-2’ + H-6’), 7.12-7.16 (m, 2H, H-4 + H-5). 13C-RMN (CDCl3) δ (ppm): 14.7, 56.2, 56.4, 65.0, 102.8, 104.6, 112.6, 112.8, 123.9, 124.4, 132.4, 139.2, 141.8, 145.2, 151.3, 156.8. MS m/z (%): M+ 328 (100), 313 (75), 285 (15), 150 (10). Anal. Elem.: Calculado para C19H20O5: C, 69.50; H, 6.14.



142


143

2-(3’,5’-Dimetoxifenil)-7-etoxibenzofurano (44)

42 44

PPh3+ Br-

OHO

Cl

O

O

Et3NOMe

OMe

+

O

MeO

OMe



reacción se filtró y la disolución fue llevada a sequedad. El residuo obtenido fue purificado por

cromatografía en columna con Hexano/AcOEt 95:5, obteniendose 44 como sólido blanco puro (0.40

g, Rto: 78 %).

P. f.: 63-64 °C

1H-RMN (CDCl3) δ (ppm): 1.52 (t, 3H, CH3), 3.87 (s, 6H, 2CH3), 4.32 (q, 2H, CH2), 6.47 (t, 1H, H-4’), 6.80 (dd, 1H, H-6), 6.98, (s, 1H, H-3), 7.02 (d, 1H, H-2’ + H-6’), 7.10-7.20 (m, 2H, H-4 + H-5). 13C-RMN (CDCl3) δ (ppm): 14.7, 55.8, 65.2, 100.5, 102.9, 103.5, 110.8, 112.6, 123.8, 126.2, ,132.3, 141.5, 145.3, 156.6, 162.4. Anal. Elem.: Calculado para C18H18O4: C, 72,47; H, 6.08.



144


145

7-Etoxi-2-(4’-metoxifenil)benzofurano (45)

42 45

PPh3+ Br-

OHO

Cl

O

OEt3N

+OMeO

OMe

Una didisolución de 42 (1.00 g, 2.00 mmol) y cloruro de 4-metoxi-benzoilo (0.34 g, 2.00



por cromatografía en columna con Hexano/AcOEt 95:5, obteniendose 45 como un sólido blanco

(0.29 g, Rto: 54 %).

P. f.: 55-56 °C 1H-RMN (CDCl3) δ (ppm): 1.53 (t, 3H, CH3), 3.87 (s, 3H, OCH3-C4’), 4.33 (q, 2H, CH2), 6.60 (dd, 1H, H-6), 6.75 (s, 1H, H-3), 6.98 (d, 2H, H-3’ + H-5’), 7.20 (m, 2H, H-4 + H-5), 7.75 (d, 2H, H-2’+ H-6’). 13C-RMN (CDCl3) δ (ppm): 14.9, 55.8, 65.1, 102.7, 112.8, 112.6, 114.9, 122.7, 123.8, 124.3, 128.4, 141.7, 145.3, 156.6, 160.8. MS m/z (%): M+ 268 (30), 240 (20), 225 (25). Anal. Elem.: Calculado para C17H16O3: C, 76.10; H 6.01.

Experimental C, 76.25; H 6.10.


146

6.-Anexo

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3268–3270

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

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

Keywords:CoumarinResveratrolMonoamino oxidase inhibitorsPerkin reaction3-Phenylcoumarins

Coumarins (or benzopyron

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

* Corresponding author. Tel.: +34 981 563100.E-mail address: [email protected] (M.J. Matos).

(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

mailto:[email protected]

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Chem

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

OHOR1

R2

M. J. Matos et al. / Bioorg. Med.

OOOH

CHOMe Me

1 2 3-6

a R3R1

R2

R3

3: R1 = R2 = R3 = H4: R1 = R3 = H , R2 = OMe5: R1 = R3 = OMe , R2 = H6: R1 = R2 = R3 = OMe

Scheme 1. Reagents and conditions: (a) DCC, DMSO, 110 �C, 24 h.

Table 1MAO-A and MAO-B inhibitory activity results for compounds 3–6 and referencecompounds

Compd MAO-A IC50 MAO-B IC50 Ratio

3 * 283.75 ± 0.98 nM >352b

4 * 13.05 ± 0.90 nM >7663b

5 * 8.98 ± 1.42 nM >11,136b

6 * 160.64 ± 1.01 nM >623b

R-(�)-Deprenyl 67.25 ± 1.02 lMa 19.60 ± 0.86 nM 3431Iproniazide 6.56 ± 0.76 lM 7.54 ± 0.36 lM 0.87

* 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.

References and notes

1. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem.2005, 12, 887.

2. Hoult, J. R. S.; Payá, M. Gen. Pharmacol. 1996, 27, 713.3. Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Enzyme Inhib. Med. Chem. 2003, 18, 63.4. Kabeya, L.; Marchi, A.; Kanashiro, A.; Lopes, N.; Silva, C.; Pupo, M.; Lucisano-

Valim, Y. Bioorg. Med. Chem. 2007, 15, 1516.5. Carotti, A.; Altomare, C.; Catto, M.; Gnerre, C.; Summo, L.; De Marco, A.; Rose, S.;

Jenner, P.; Testa, B. Chem. Biod. 2006, 3, 134.6. Chilin, A.; Battistutta, R.; Bortolato, A.; Cozza, G.; Zanatta, S.; Poletto, G.;

Mazzorana, M.; Zagotto, G.; Uriarte, E.; Guiotto, A.; Pinna, L.; Meggio, F.; Moro,S. J. Med. Chem. 2008, 51, 752.

7. Santana, L.; Uriarte, E.; González-Díaz, H.; Zagotto, G.; Soto-Otero, R.; Méndez-Álvarez, E. J. Med. Chem. 2006, 49, 1118.

8. Santana, L.; González-díaz, H.; Quezada, E.; Uriarte, E.; Yáñez, M.; Viña, D.;Orallo, F. J. Med. Chem. 2008, 51, 6740.

9. Catto, M.; Nicolotti, O.; Leonetti, F.; Carotti, A.; Favia, A.; Soto-Otero, R.;Méndez-Álvarez, E.; Carotti, A. J. Med. Chem. 2006, 49, 4912.

10. Gnerre, C.; Catto, M.; Leonetti, F.; Weber, P.; Carrupt, P.; Altomare, C.; Carotti,A.; Testa, B. J. Med. Chem. 2000, 43, 4747.

11. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Befani, O.; Turini,P.; Alacaro, S.; Ortuso, F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.

12. Frémont, L. Life Sci. 2000, 66, 663.13. Orallo, F. In Resveratrol in Health and Disease; Aggarwal, B. B., Shishodia, S., Eds.;

CRC Press: USA, 2005; p 577.14. Leiro, J.; Álvarez, E.; Arranz, J.; Laguna, R.; Uriarte, E.; Orallo, F. J. Leukocyte Biol.

2004, 75, 1156.15. Orallo, F. Curr. Med. Chem. 2008, 15, 1887.16. Yánez, M.; Fraiz, N.; Cano, E.; Orallo, F. Biochem. Biophys. Res. Commun. 2006,

344, 688.17. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yánez, M.; Fraiz, N.; Alcaide, C.;

Cano, E.; Orallo, F. Bioorg. Med. Chem. Lett. 2006, 16, 257.18. 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–

149 �C (biblio. 145–147 �C, 149–150 �C). 1H NMR (CDCl3) d (ppm), J (Hz): 2.42(s, 3H, –CH3), 7.27 (m, 1H, H-7), 7.34 (m, 2H, H-40 and H-8), 7.43 (m, 3H, H-20 ,H-40 and H-5), 7.70 (dd, 2H, H-10 and H-50 , J = 7.7 and 1.8), 7.77 (s, 1H, H-4).13C NMR (CDCl3) d (ppm): 21.29, 116.67, 119.57, 128.17, 128.70, 128.95,129.02, 129.26, 132.95, 134.65, 135.35, 140.39, 152.16, 161.31. DEPT (CDCl3) d(ppm): 21.29, 100.60, 116.67, 128.17, 128.95, 129.02, 129.26, 132.95, 140.39.MS m/z (%): 236 (M+, 100), 208 (50), 178 (8), 165 (8) 76 (6), 51 (69). Anal. Calcdfor C16H12O2: C, 81.34; H, 5.12; O, 13.54. Found: C, 81.44; H, 4.84.

31. 3-(40-Methoxy)phenyl-6-methylcumarin (4). It was obtained with a yield of61%. Mp 144–145oC (biblio. 143 �C). 1H NMR (CDCl3) d (ppm), J (Hz): 2.40 (s,3H, –CH3), 3.84 (s, 3H, –OCH3), 6.96 (dd, 2H, H-30 and H-50 , J = 6.8 and 2.1), 7.26(m, 3H, H-4, H-7 and H-8), 7.66 (m, 3H, H-3, H-20 and H-60). 13C NMR (CDCl3) d(ppm): 20.75, 55.32, 113.85, 116.04, 119.54, 127.19, 127.46, 127.66, 129.77,132.02, 134.03, 138.47, 151.41, 160.05, 160.96. DEPT (CDCl3) d (ppm): 20.76,55.32, 113.85, 116.04, 127.46, 129.78, 132.01, 138.48. MS m/z (%): 266 (M+,100), 223 (60), 195 (24), 165 (16), 152 (13), 115 (5), 89 (5), 63 (7), 50 (6). Anal.Calcd for C17H14O3: C, 76.68; H, 5.30; O, 18.02. Found: C, 76.88; H, 5.32.

32. 3-(30 ,50-Dimethoxy)phenyl-6-methylcumarin (5). It was obtained with a yieldof 60%. Mp 110–111oC. 1H NMR (CDCl3) d (ppm), J (Hz): 2.40 (s, 3H, –CH3), 3.82(s, 6H, –(OCH3)2), 6.50 (t, 1H, H-40 , J = 2.1), 6.83 (d, 2H, H-20 and H-60 , J = 2.1),7.22–7,33 (m, 3H, H-5, H-7 and H-8), 7.74 (s, 1H, H-4). 13C NMR (CDCl3) d(ppm): 20.72, 55.41, 100.89, 106.72, 116.06, 119.22, 127.69, 127.93, 132.49,134.10, 136.68, 140.02, 151.60, 160.48, 160.61. DEPT (CDCl3) d (ppm): 20.72,55.40, 100.89, 106,71, 116.06, 127.69, 132.49, 140.02. MS m/z (%): 296 (M+,100), 295 (17), 267 (16), 210 (8), 181 (22), 152 (13), 139 (6), 105 (8), 76 (9).Anal. Calcd for C18H16O4: C, 72.96; H, 5.44; O, 21.60. Found: C, 73.23; H, 4.90.

33. 6-Methyl-3-(30 ,40 ,50-trimethoxy)phenylcumarin (6). It was obtained with ayield of 70%. Mp 165–166 oC. 1H NMR (CDCl3) d (ppm), J (Hz): 2.43 (s, 3H, –CH3), 3.90 (s, 3H, –OCH3), 3,92 (s, 6H, –(OCH3)2), 6.93 (d, 2H, H-20and H-60 ,J = 2.1), 7.24–7.35 (m, 3H, H-5, H-7 and H-8), 7.76 (s, 1H, H-4). 13C NMR (CDCl3)d (ppm): 20.70, 56.19, 60.72, 105.94, 116.03, 119.24, 127.48, 127.58, 130.04,

3270 M. J. Matos et al. / Bioorg. Med

130.44, 132.38, 134.12, 139.48, 151.45, 153.02, 160.66. DEPT (CDCl3) d (ppm):20.76, 56.23, 60.77, 105.97, 116.08, 127.63, 132.43, 139.54. MS m/z (%): 326(M+, 100), 311 (89), 283 (43), 253 (12), 225 (30), 197 (10), 169 (10), 148 (5),115 (8). Anal. Calcd for C19H18O5: C, 69.93; H, 5.56; O, 24.51. Found: C, 70.14;H, 5.58.

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.

m. Lett. 19 (2009) 3268–3270

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5053–5055

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

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

Keywords:PhenylcoumarinsMAOIsMonoamino oxidasePerkin reactionCoumarin–resveratrol hybrids

Coumarins are present in r

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

* Corresponding author. Tel.: +34 1918523676.E-mail address: [email protected] (M.J. Matos).

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

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. Che

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

Compounds MAO-A IC50 MAO-B IC50 Ratio

3 * 13.05 ± 0.90 nM >7663b

4 * 802.60 ± 53.75 pM >124,595b

5 * *

6 * 155.59 ± 17.09 nM >643b

R-(�)-Deprenyl 67.25 ± 1.02 lMa 19.60 ± 0.86 nM 3431Iproniazid 6.56 ± 0.76 lM 7.54 ± 0.36 lM 0.87

* 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.

References and notes

1. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem.2005, 12, 887.

2. Hoult, J. R. S.; Payá, M. Gen. Pharmacol. 1996, 27, 713.3. Kontogiorgis, C.; Hadjipavlou-Litina, D. J. Enzyme Inhib. Med. Chem. 2003, 18, 63.4. Kabeya, L.; Marchi, A.; Kanashiro, A.; Lopes, N.; Silva, C.; Pupo, M.; Lucisano-

Valim, Y. Bioorg. Med. Chem. 2007, 15, 1516.5. Carotti, A.; Altomare, C.; Catto, M.; Gnerre, C.; Summo, L.; De Marco, A.; Rose, S.;

Jenner, P.; Testa, B. Chem. Biod. 2006, 3, 134.6. Chilin, A.; Battistutta, R.; Bortolato, A.; Cozza, G.; Zanatta, S.; Poletto, G.;

Mazzorana, M.; Zagotto, G.; Uriarte, E.; Guiotto, A.; Pinna, L.; Meggio, F.; Moro,S. J. Med. Chem. 2008, 51, 752.

7. Santana, L.; Uriarte, E.; González-Díaz, H.; Zagotto, G.; Soto-Otero, R.; Méndez-Álvarez, E. J. Med. Chem. 2006, 49, 1118.

8. Matos, M. J.; Viña, D.; Quezada, E.; Picciau, C.; Delogu, G.; Orallo, F.; Santana, L.;Uriarte, E. Bioorg. Med. Chem. Lett. 2009, 19, 3268.

9. Santana, L.; González-Díaz, H.; Quezada, E.; Uriarte, E.; Yáñez, M.; Viña, D.;Orallo, F. J. Med. Chem. 2008, 51, 6740.

10. Gnerre, C.; Catto, M.; Leonetti, F.; Weber, P.; Carrupt, P.; Altomare, C.; Carotti,A.; Testa, B. J. Med. Chem. 2000, 43, 4747.

11. Catto, M.; Nicolotti, O.; Leonetti, F.; Carotti, A.; Favia, A.; Soto-Otero, R.;Méndez-Álvarez, E.; Carotti, A. J. Med. Chem. 2006, 49, 4912.

12. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Bizzarri, B.; Granese, A.;Carradori, S.; Yanez, M.; Orallo, F.; Ortuso, F.; Alcaro, S. J. Med. Chem. 2009, 52,1935.

13. Chimenti, F.; Secci, D.; Bolasco, A.; Chimenti, P.; Granese, A.; Befani, O.; Turini,P.; Alacaro, S.; Ortuso, F. Bioorg. Med. Chem. Lett. 2004, 14, 3697.

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,

344, 688.25. Vilar, S.; Quezada, E.; Alcaide, C.; Orallo, F.; Santana, L.; Uriarte, E. Qsar Comb.

Sci. 2007, 26, 317.26. Kamat, S. P.; D́Souza, A. M.; Paknikar, S. K.; Beaucahmp, P. S. J. Chem. Res. (S)

2002, 242.27. 3-(30-Methoxy)phenyl-6-methylcumarin (4): It was obtained with a yield of 53%.

Mp 84–85 �C. 1H NMR (CDCl3) d (ppm), J (Hz): 2.44 (s, 3H, –CH3), 3.88 (s, 1H, –OCH3), 6.97 (m, 1H, H-40), 7.26–7.42 (m, 6H, H-5, H-7, H-8, H-20 , H-50 and H-60)7.78 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm): 20.77, 55.37, 114.15, 114.38,116.10, 119.28, 120.86, 127.70, 129.43, 132.48, 134.12, 136.12, 139.91, 140.10,151.60, 159.45 160.66. MS m/z (%): 267 (48), 266 (M+, 100), 239 (16), 238 (70),237 (20), 195 (48), 194 (16), 166 (10), 165 (29), 152 (23). Anal. Calcd forC17H14O3: C, 76.68; H, 5.30. Found: C, 76.76; H, 5.21.

28. 3-(20-Methoxy)phenyl-6-methylcumarin (5): It was obtained with a yield of 59%.Mp 177–178 �C. 1H NMR (CDCl3) d (ppm), J (Hz): 2.41 (s, 3H, –CH3), 3.82 (s, 1H,–OCH3), 7.02 (m, 2H, H-30 , H-40), 7.24–7.41 (m, 5H, H-5, H-7, H-8, H-50 and H-60) 7.69 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm): 20.80, 55.81, 111.31, 116.21,

m. Lett. 19 (2009) 5053–5055

Chem

119.24, 120.57, 124.20, 126.36, 127.58, 130.14, 130.80, 132.21, 133.89, 141.84,151.82, 157.22 160.55. MS m/z (%): 267 (22), 266 (M+, 100), 265 (10), 249 (29),237 (22), 235 (14), 223 (22), 220 (12), 195 (29), 173 (26), 165 (25), 152 (17),145 (19), 118 (19). Anal. Calcd for C17H14O3: C, 76.68; H, 5.30. Found: C, 76.76;H, 5.22.

29. 3-(40-Hydroxy)phenyl-6-methylcumarin (6): It was obtained with a yield of 63%.

M. J. Matos et al. / Bioorg. Med.

Mp 217–218 �C. 1H NMR (CDCl3) d (ppm), J (Hz): 2.37 (s, 3H, –CH3), 6.84 (d, 2H,H-30 and H-50 , J = 8.8), 7.31 (d, 1H, H-8, J = 8.4), 7.40 (dd, 1H, H-7, J = 1.9 and

8.4), 7.56 (m, 3H, H-20 , H-60 and H-5), 8.06 (s, 1H, H-4). 13C NMR (CDCl3) d(ppm): 20.31, 115.04, 115.52, 119.45, 125.29, 126.66, 127.92, 129.83, 132.00,133.67, 138.46, 150.79, 157.95, 160.04. MS m/z (%): 253 (13), 252 (M+, 75), 224(58), 223 (26), 165 (12) 152 (15), 143 (23), 99 (37), 98 (25), 83 (12), 70 (20), 56(100), 55 (27). Anal. Calcd for C16H12O3: C, 76.18; H, 4.79. Found: C, 75.99; H,4.69.

30. All IC50 values shown in the table are expressed as means ± SEM from five

. Lett. 19 (2009) 5053–5055 5055

experiments.

Molecules 2010, 15, 270-279; doi:10.3390/molecules15010270

molecules ISSN 1420-3049

www.mdpi.com/journal/molecules Article

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.

Keywords: resveratrol; coumarin; vasorelaxant; platelet antiaggregatory activity

OPEN ACCESS

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1. Introduction

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.

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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.

OH

O

O OOH

HX

O

+

1 R1 = MeO; R2 = R3 = H2 R2 = MeO; R1 = R3 = H3 R3 = MeO; R1 = R2 = H

R2

R1

R3

4 X = Cl5 X = Br

X

R2

R1 R3

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.

Compound PE (1 μM) 12 36.63 ± 2.46* 13 57.63 ± 3.87* 14 ** 15 48.79 ± 3.27* 16 46.67 ± 3.13* 17 **

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.

Compound Thrombin (0.25 U/mL) 12 91.36 ± 6.13* 13 6.41 ± 2.15* 14 ** 15 ** 16 ** 17 20.1 ± 1.35*

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.

6-Chloro-3-(3’-methoxy)phenylcoumarin (7). Purified by chromatography using 9:1 hexane/ethyl acetate as eluent; Mp: 144–146 ºC; IR (KBr): 2925, 1700, 1615, 1300, 1115, 775 cm-1; 1H-NMR (CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.96 (dd, J = 2.5; 8.2 Hz, 1H), 7.25 (m, 2H), 7.35 (m, 2H), 7.48 (m, 2H), 7.72 (s, 1H); 13C-NMR (CDCl3) δ (ppm): 55.32, 114.2, 114.8, 117.8, 120.6, 120.8, 127.0, 129.2, 129.5, 129.7, 131.3, 135.5, 138.5, 151.8, 159.5, 159.8; MS m/z (%): 288 ([M+2]+, 29), 286 (M+, 100), 258(30), 215 (20), 201 (5), 152 (7); Anal. Calcd for C16H11ClO3: C, 67.03; H, 3.87. Experimental: C, 67.20; H, 3.95.

6-Chloro-3-(4’-methoxy)phenylcoumarin (8). Purified by chromatography using 9:1 hexane/ethyl acetate as eluent; Mp: 194–196 ºC; IR (KBr): 2918, 1710, 1614, 1300, 1117, 779 cm-1; 1H-NMR (CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.99 (d, J = 8.9 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.48 (m, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.71 (s, 1H); 13C-NMR (CDCl3) δ (ppm): 55.4, 113.9 (2C), 117.7, 118.3, 120.3, 121.1, 126.8, 129.8 (2C), 130.8, 136.9, 141.1, 151.9, 156.3, 158.2; MS m/z (%): 288 ([M+2]+, 37), 286 (M+, 100), 258(13), 243 (16), 180 (3), 152 (5); Anal. Calcd for C16H11ClO3: C, 67.03; H, 3.87. Experimental: C, 67.10; H, 3.99.

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6-Bromo-3-(2’-methoxy)phenylcoumarin (9). Purified by chromatography using 9:1 hexane/ethyl acetate as eluent; Mp: 183–185 ºC; IR (KBr): 2909, 1709, 1615, 1305, 1120, 783 cm-1; 1H-NMR (CDCl3) δ (ppm): 3.82 (s, 3H, CH3O), 6.99 (d, J = 7.5 Hz, 1H), 7.03 (d, J = 7.4 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.36 (m, 2H), 7.60 (m, 2H), 7.66 (s, 1H); 13C-NMR (CDCl3) δ (ppm): 55.7, 111.3, 116.7, 118.2, 120.6, 121.0, 123.5, 127.6, 130.0, 130.5, 130.7, 133.8, 140.2, 152.5, 157.1, 159.6; MS m/z (%): 332 ([M+2]+, 100), 330 (M+, 97), 315(23), 237 (32), 152 (15), 118 (16); Anal. Calcd for C16H11BrO3: C, 58.03; H, 3.35. Experimental: C, 58.25; H, 3.49.

6-Bromo-3-(3’-methoxy)phenylcoumarin (10). Purified by chromatography using 9:1 hexane/ethyl acetate as eluent; Mp: 148-150 ºC; IR (KBr): 2898, 1700, 1620, 1300, 1110, 789 cm-1; 1H-NMR (CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.96 (dd, J = 2.3; 8.2 Hz, 1H), 7.20 (m, 2H), 7.36 (m, 2H), 7.60 (d, J = 8.3 Hz, 1H), 7.75 (s, 1H), 7.85 (s, 1H); 13C-NMR (CDCl3) δ (ppm): 55.2, 114.1, 114.6, 116.9, 118.0, 120.7, 121.0, 129.0, 129.4, 130.0, 133.9, 135.3, 138.3, 152.1, 159.3, 159.6; MS m/z (%): 332 ([M+2]+, 100), 330 (M+, 28), 302(34), 195 (10), 180 (10), 152 (17); Anal. Calcd for C16H11BrO3: C, 58.03; H, 3.35. Experimental: C, 58.15; H, 3.50.

6-Bromo-3-(4’-methoxy)phenylcoumarin (11). Purified by chromatography using 9:1 hexane/ethyl acetate as eluent; Mp: 205–206 ºC; IR (KBr): 2900, 1712, 1612, 1298, 1110, 781 cm-1; 1H-NMR (CDCl3) δ (ppm): 3.85 (s, 3H, CH3O), 6.97 (d, J = 8.8 Hz, 2H), 7.23 (m, 1H), 7.57 (dd, J = 2.2, 8.7 Hz, 1H), 7.63 (m, 3H), 7.67 (s, 1H); 13C-NMR (CDCl3) δ (ppm): 55.3, 113.9 (2C), 114.1, 116.9, 118.1, 121.4, 126.5, 128.9, 129.8 (2C), 129.9, 133.6, 136.8, 152.0, 160.4; MS m/z (%): 332 ([M+2]+, 100), 330 (M+, 13), 302(12), 288 (40), 259 (18), 195 (10), 180 (17), 152 (14); Anal. Calcd for C16H11BrO3: C, 58.03; H, 3.35. Experimental: C, 58.15; H, 3.42.

6-Chloro-3-(2’-hydroxy)phenylcoumarin (12). Purified by chromatography using 8:2 hexane/ethyl acetate as eluent. Mp: 225–227 ºC. IR (KBr): 3500, 2920, 1705, 1613, 1300, 1120, 780 cm-1. 1H NMR (DMSO-d6) δ (ppm): 6.86 (m, 2H), 7.23 (m, 2H), 7.39 (d, J = 8.8 Hz, 1H), 7.78 (dd, J = 2.3; 8.8 Hz, 1H), 7.97 (s, 1H), 8.00 (s, 1H), 9.64 (bs, 1H, 1OH). 13C NMR (DMSO-d6) δ (ppm): 115.7, 116.0, 118.2, 118.7, 121.2, 121.8, 127.1, 129.9, 130.3, 130.7, 133.8, 140.6, 140.7, 152.1, 155.1. MS m/z (%): 274 ([M+2]+, 23), 272 (M+, 100), 244(35), 180 (19), 152 (20), 118 (10). Anal. Calcd for C15H9ClO3: C, 66.07; H, 3.33. Experimental: C, 66.25; H, 3.49.

6-Chloro-3-(3’-hydroxy)phenylcoumarin (13). Purified by chromatography using 8:2 hexane/ethyl acetate as eluent; Mp: 221–223 ºC; IR (KBr): 3502, 2925, 1701, 1614, 1310, 1110, 785 cm-1; 1H-NMR (DMSO-d6) δ (ppm): 6.82 (dd, J = 2.2; 8.0 Hz, 1H), 7.10 (m, 2H), 7.25 (m, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.61 (dd, J = 2.5; 8.8 Hz, 1H), 7.86 (d, J = 2.5 Hz, 1H), 8.14 (s, 1H), 9.60 (bs, 1H, 1OH); 13C- NMR (DMSO-d6) δ (ppm): 115.5, 115.9, 117.8, 119.2, 120.9, 127.6, 128.0, 128.2, 129.3, 131.1, 135.5, 139.1, 151.5, 157.1, 159.2; MS m/z (%): 274 ([M+2]+, 21), 272 (M+, 100), 244(48), 180 (14), 152 (23), 118 (10); Anal. Calcd for C15H9ClO3: C, 66.07; H, 3.33. Experimental: C, 66.30; H, 3.45.

6-Chloro-3-(4’-hydroxy)phenylcoumarin (14). Purified by chromatography using 8:2 hexane/ethyl acetate as eluent; Mp: 241–243 ºC; IR (KBr): 3500, 2918, 1708, 1610, 1296, 1119, 783 cm-1; 1H-NMR

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(DMSO-d6) δ (ppm): 6.84 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.8 Hz, 1H), 7.58 (m, 3H), 7.84 (d, J = 2.5 Hz, 1H), 8.09 (s, 1H), 9.81 (bs, 1H, 1OH); 13C-NMR (DMSO-d6) δ (ppm): 115.1 (2C), 117.7, 121.1, 124.8, 127.2, 127.8, 128.1, 129.9 (2C), 130.6, 137.1, 151.2, 158.2, 159.5; MS m/z (%): 274 ([M+2]+, 20), 272 (M+, 100), 244(74), 180 (15), 152 (40), 118 (5); Anal. Calcd for C15H9ClO3: C, 66.07; H, 3.33. Experimental: C, 66.22; H, 3.47.

6-Bromo-3-(2’-hydroxy)phenylcoumarin (15). Purified by chromatography using 8:2 hexane/ethyl acetate as eluent; Mp: 230–232 ºC; IR (KBr): 3510, 2918, 1709, 1620, 1310, 1120, 780 cm-1; 1H-NMR (DMSO-d6) δ (ppm): 6.87 (m, 2H), 7.23 (m, 2H), 7.40 (d, J = 8.8 Hz, 1H), 7.74 (dd, J = 2.3; 8.8 Hz, 1H), 7.99 (s, 1H), 8.02 (s, 1H), 9.64 (bs, 1H, 1OH); 13C-NMR (DMSO-d6) δ (ppm): 115.7, 116.0, 118.2, 118.7, 121.2, 121.8, 127.1, 129.9, 130.3, 130.7, 133.8, 140.6, 152.1, 155.1, 158.8; MS m/z (%): 318 ([M+2]+, 47), 316 (M+, 71), 300(8), 288 (40), 180 (54), 152 (100); Anal. Calcd for C15H9BrO3: C, 56.81; H, 2.86. Experimental: C, 56.95; H, 3.00.

6-Bromo-3-(3’-hydroxy)phenylcoumarin (16). Purified by chromatography using 8:2 hexane/ethyl acetate as eluent; Mp: 219–221 ºC; IR (KBr): 3505, 2919, 1709, 1622, 1312, 1123, 780 cm-1; 1H-NMR (DMSO-d6) δ (ppm): 6.82 (dd, J = 2.0; 7.7 Hz, 1H), 7.10 (m, 2H), 7.25 (m, 1H), 7.38 (d, J = 8.8 Hz, 1H), 7.73 (dd, J = 2.3; 8.8 Hz, 1H), 8.00 (d, J = 2.3 Hz, 1H), 8.14 (s, 1H), 9.59 (bs, 1H, 1OH); 13C- NMR (DMSO-d6) δ (ppm): 115.5, 115.9, 116.1, 118.1, 119.2, 121.4, 128.0, 129.3, 130.6, 133.9, 135.5, 139.0, 151.9, 157.1, 159.1; MS m/z (%): 318 ([M+2]+, 46), 316 (M+, 79), 288 (46), 180 (19), 152 (100); Anal. Calcd for C15H9BrO3: C, 56.81; H, 2.86. Experimental: C, 56.98; H, 3.01.

6-Bromo-3-(4’-hydroxy)phenylcoumarin (17). Purified by chromatography using 8:2 hexane/ethyl acetate as eluent; Mp: 226–228 ºC; IR (KBr): 3520, 2939, 1710, 1622, 1302, 1133, 784 cm-1; 1H-NMR (DMSO-d6) δ (ppm): 6.84 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 9.1 Hz, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.66 (m, 1H), 7.99 (s, 1H), 8.09 (s, 1H), 9.81 (bs, 1H, 1OH); 13C-NMR (DMSO-d6) δ (ppm): 115.1 (2C), 116.0, 118.0, 121.6, 124.8, 127.7, 129.9 (2C), 130.2, 133.3, 137.0, 151.6, 158.2, 159.4; MS m/z (%): 318 ([M+2]+, 31), 317 ([M+1]+, 100), 316 (M+, 40), 288 (39), 180 (14), 152 (46); Anal. Calcd for C15H9BrO3: C, 56.81; H, 2.86. Experimental: C, 56.87; H, 3.0.

3.3. Vasorelaxant activity

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.

References and Notes

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3. Opherk, D.; Schuler, G.; Waas, W.; Dietz, R.; Kubler, W. Intravenous carbochromen: A potent and effective drug for estimation of coronary dilatory capacity. Eur. Heart J. 1990, 11, 342–347.

4. Cao, Y.G.; Liu, X.Q.; Chen, Y.C.; Hao, K.; Wang, G.J. Warfarin maintenance dose adjustment with indirect pharmacodynamic model in rats. Eur. J. Pharm. Scien. 2007, 30, 175–180.

5. Bradamante, S.; Barenghi, L.; Villa, A. Cardiovascular protective effects of resveratrol. Cardiovasc. Drug Rev. 2004, 22, 169–188.

6. Orallo, F.; Alvarez, E.; Camina, M.; Leiro, J.M.; Gomez, E.; Fernandez, P. The possible implication of trans-Resveratrol in the cardioprotective effects of long-term moderate wine consumption. Mol. Pharmacol. 2002, 61, 294–302.

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.

8. Hao, H.D.; He, L.R. J. Mechanisms of cardiovascular protection by resveratrol. Med. Food 2004, 7, 290–298.

9. Vilar, S.; Quezada, E.; Santana, L.; Uriarte, E.; Yanez, M.; Fraiz, N.; Alcaide, C.; Cano, E.; Orallo, F. Design, synthesis, and vasorelaxant and platelet antiaggregatory activities of coumarin-resveratrol hybrids. Bioorg. Med. Chem. Lett. 2006, 16, 257–261.

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.

11. Hans, N.; Singhi, M.; Sharma, V.; Grover, S.K. A novel one-step synthesis of 3-phenyl-, 4-methyl-3-phenyl- and 3-phenyl-4-styrylcoumarins using DCC-DMSO. Indian J. Chem. 1996, 35B, 1159–1162.

12. Perkin, W.H. On the artificial production of coumarin and formation of its homologues. J. Chem. Soc. 1868, 21, 53–63.

13. Perkin, W.H. On the formation of coumarin and of cinnamic and of other analogous acids from the aromatic aldehydes. 1877, 31, 388–427.

14. Begala, M.; Delogu, G.; Maccioni, E.; Podda, G.; Tocco, G.; Quezada, E.; Uriarte, E.; Fedrigo, M.A.; Favretto, D.; Traldi, P. Electrospray ionisation tandem mass spectrometry in the characterisation of isomeric benzofurocoumarin. Rapid Commun. Mass Spectrom. 2001, 15, 1000–1010.

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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16. Mimai, M.; Suzuki, Y.; Hamma, N.; Murayama, E.; Aono, S. Coumarin derivatives. JP Pat. 49054371, 1974.

Sample Availability: Contact the authors.

© 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

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 =

hMAO-B selectivity ratios [IC50(hMAO-A)]/[IC50(hMAO-B)]). Enzymatic assays

revealed that most of tested compounds were moderate to potent hMAO inhibitors at

either low micromolar or nanomolar concentrations, showing a selective inhibitory

activity toward hMAO-B.

We can observe that the number and position of methoxy groups on (1H)-benzopyran

structure resulted more important for the activity than the nature of the heterocycle ring

in position 3. In fact, compounds 4a-c showing methoxy groups at 5 and 7 positions on

(1H)-benzopyran structure are inactive as MAO inhibitors. Introduction of a methoxy

group at either 6 or 7 position on (1H)-benzopyran structure, increases the MAO-B

activity between 10 and 100 fold (compare 1a vs 2a and 3a; 1b vs 2b; 1c vs 2c and 3c)

with the only exception of compounds 3b. Methoxy group at position 7 (compounds 2a-

c) let to improve the MAO-B inhibitory activity compared to the corresponding

compounds with the methoxy group at position 6, however compounds 2b and 2c

showed a weak MAO-A inhibitory activity leading to a small decrease in the B-

selectivity.

2.3. Molecular docking study

Docking experiments were carried out in order to compare the theoretical and

experimental affinity. The Protein Data Bank [16] (PDB) crystallographic structure of

human MAO-B (PDB code 2V60) [8] was considered as receptor for docking

simulations.

The scoring function estimates the affinity between ligands and receptor [17, 18]. With

the aim to verify the usefulness of the scoring function to recognize new molecules

potentially active as MAO-B inhibitors, different ligands were docked to the MAO-B

protein.

The experimental IC50 values were compared with those for the predicted activity

according to the scoring function (see Table 2). The square correlation coefficient (r2)

between the scoring function and the experimental MAO-B inhibitory activity pKi

resulted 0.44. This result is reasonable considering that scoring functions are not able to

provide a good correlation between the predicted and the experimental affinity [19-21].

In our case, a certain tendency of the model to rank the compounds basing on their

respective activity can be appreciated. In fact, when a cut-off value of IC50=1.0µM is

used in order to differentiate the compounds in two groups (high and low affinity), the

model is able to recognize clearly both groups. The most active compounds occupy the

first positions according to the scoring function. However, the compounds with IC50 >

1.0µM, are ranked in the last positions. The only exception is the inactive coumarin 4c

that is ranked in the first positions (see Table 2).

A second study was made in order to asses that the model discriminates between ligands

and decoys. The synthesized coumarins were dispersed in a pool of 120 decoys

extracted from ZINC database [22]. After docking calculations, data were plotted with

the receiver operative characteristics (ROC) curve. This representation is particularly

suited to illustrating the tendency of a binary model to either correctly classify the

compounds or predict false positives [23-25] (see Figure 1). The area under the curve is

0.93, indicating high predictive power for model. Through docking calculations is

possible to differentiate between true ligands and decoys (see Figure 1).

3. Conclusions

In the current study three series of 3-heteroaryl coumarin derivatives were synthesized

and evaluated as inhibitors of MAO-A and –B. In general, the derivatives were found to

be selective for the MAO-B isoform with 7-methoxy-3-heteroarylcoumarins exhibiting

inhibition potencies in the low nanoMolar range. Docking experiments showed that the

affinity of the studied compounds by MAO-B is similar to that obtained experimentally.

According with our results, docking experiments could be an interesting methodology

to try to predict the activity for this class of coumarins in MAO-B receptor.

4. Experimental

4.1. General methods

Melting points (mp) are uncorrected and were determined with a Reichert Kofler

thermopan or in capillary tubes in a Buchi 510 apparatus. 1H NMR (300 MHz) and 13C

NMR (75.4 MHz) spectra were recorder with a Bruker AMX spectrometer. Chemical

shifts (δ) are expressed in parts per million (ppm) using TMS as internal standard. Spin

multiplicities are given as s (singlet), d (doublet), dd (doublet of doublets) and m

(multiplet). Mass spectrometry was carried out with a Kratos MS-50 or a Varian MAT-

711 spectrometer. Elemental analyses were performed by a Perkin-Elmer 240B

microanalyzer and were within ±0.4% of calculated values in all cases. Flash

Chromatography (FC) was performed on silica gel (Merck 60, 230-400 mesh);

analytical TLC was performed on precoated silica gel plates (Merck 60 F254).

4.2. Synthesis

4.2.1. General synthetic method for the Coumarin Skeleton

A solution of ortho-hydroxybenzaldehyde (1-4, 8.18 mmol), substituted acetic acid (a-c,

9.82 mmol) and DCC (12.27 mmol) in dimethylsulfoxide (DMSO, 10 mL), was heated

(oil bath) at 110 oC for 24-48 h. On completion of the reaction, cold water (100 mL) and

acetic acid (15 mL) were added. The reaction mixture was stirred at room temperature

for 4 h and extracted with diethyl ether (4 x 100 mL). The precipitated dicyclohexylurea

was filtered off. The filtrate was extracted with 5% aqueous NaHCO3 (200 mL). The

organic phase was stirred for 1 h with 5% aqueous sodium metabisulfite in order to

remove the unreacted hydroxybenzaldehyde. The organic phase was washed with water,

dried (Na2SO4) and the solvent removed under reduced pressure. The residue was

purified by column chromatography (Hexane/EtOAc, 9:1)

4.2.2. 3-(Thiophen-2-yl)coumarin (1a) [26]: Yield 34%; mp 166-167 oC. 1H

NMR(300MHz)(CDCl3): δ = 7.12 (m, 1H), 7.28 (d, J = 9.4 Hz, 1H), 7.35 (d, J = 9.4 Hz,

1H), 7.42 (d, J = 5.1 Hz, 1H), 7.52 (m, 2H), 7.80 (d, J = 3.8 Hz, 1H), 7.99 (s, 1H). 13C

NMR(300MHz)(CDCl3): δ = 116.4, 119.3, 121.7, 124.6, 127.0, 127.5, 127.7, 129.6,

131.1, 135.5, 135.9, 152.6, 160.1. EI-MS (m/z): 229 [M + 1]+, 228[M+]. Anal. Calcd. for

C13H8O2S: C 68.40, H 3.53; Found: C68.52, H 3.59.

4.2.3. 3-(Thiophen-3-yl)coumarin (1b) [26]: Yield 37%; mp 175-176 oC. 1H

NMR(300MHz)(CDCl3): δ = 7.30 (d, J = 7.5 Hz, 1H), 7.35 (m, 2H), 7.53 (m, 3H), 7.93

(s, 1H), 8.18 (d, J = 3.0 Hz, 1H). 13C NMR(300MHz)(CDCl3): δ = 116.3, 119.4, 122.6,

124.5, 125.6, 126.0, 126.2, 127.7, 131.1, 134.3, 137.2, 152.8, 160.0. EI-MS (m/z): 229

[M + 1]+, 228[M+]. Anal. Calcd. for C13H8O2S: C 68.40, H 3.53; Found: C68.50, H 3.6.

4.2.4. 3-(Indol-3-yl)coumarin (1c): Yield 33%; mp 190-191 oC. 1H

NMR(300MHz)(CDCl3): δ = 7.32 (m, 3H), 7.48 (m, 2H), 7.58 (dd, J = 7.6; 1.3 Hz, 1H),

7.98 (m, 1H); 8.16 (s, 1H), 8.20 (d, J = 2.6 Hz, 1H), 8.65 (br; 1H). 13C

NMR(300MHz)(CDCl3): δ = 111.9, 116.3, 119.5, 120.2, 120.9, 122.7, 122.9, 124.4,

125.5, 127.2, 127.6, 130.1, 134.9, 136.2, 143.5, 152.2, 160.8. EI-MS (m/z): 262 [M +

1]+, 261[M+]. Anal. Calcd. for C17H11NO2: C 78.15, H 4.24; Found: C78.20, H 4.30.

4.2.5. 7-Methoxy-3-(thiophen-2-yl)coumarin (2a) [27]: Yield 27%; mp 155-156 oC. 1H

NMR(300MHz)(CDCl3): δ = 3.89 (s, 3H), 6.85 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H), 7.11

(m, 1H), 7.38 (dd, J = 4.0; 1.0 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 4.0; 1.0

Hz, 1H), 7.96 (s, 1H). 13C NMR(300MHz)(CDCl3): δ = 55.8, 100.4, 112.9, 113.1,

126.1, 126.5, 126.8, 127.4, 128.7, 136.0, 136.4, 154.5, 161.0, 162.5. EI-MS (m/z): 259

[M + 1]+, 258[M+]. Anal. Calcd. for C14H10O3S: C 65.10, H 3.90; Found: C65.17, H

3.96.

4.2.6. 7-Methoxy-3-(thiophen-3-yl)coumarin (2b): Yield 25%; mp 168-169 oC. 1H

NMR(300MHz)(CDCl3): δ = 3.87 (s, 3H), 6.85 (m, 2H), 7.36 (dd, J = 5.0; 3.0 Hz, 1H),

7.42 (d, J = 8.4 Hz, 1H), 7.50 (dd, J = 5.0; 1.2 Hz, 1H), 7.87 (s, 1H), 8.11(dd, J = 3.0;

1.2 Hz, 1H). 13C NMR(300MHz)(CDCl3): δ = 55.7, 100.3, 112.8, 113.0, 119.3, 125.0,

125.5, 126.0, 128.6, 134.6, 137.5, 154.6, 160.8, 162.4. EI-MS (m/z): 259 [M + 1]+,

258[M+]. Anal. Calcd. for C14H10O3S: C 65.10, H 3.90; Found: C65.15, H 3.97.

4.2.7. 3-(Indol-3-yl)-7-methoxycoumarin (2c): Yield 29%; mp 185-186 oC. 1H

NMR(300MHz)(CDCl3): δ = 3.95 (s, 3H), 6.89 (m, 2H), 7.28 (m, 2H), 7.47 (m, 2H),

7.95 (m, 1H), 8.10 (s, 1H), 8.13 (d, J = 3.0 Hz, 1H), 8.57 (br, 1H). 13C

NMR(300MHz)(CDCl3): δ = 55.7, 100.3, 100.0, 111.7, 112.6, 113.6, 119.4, 119.5,

120.6, 122.5, 126.0, 126.8, 128.1, 135.5, 136.2, 153.8, 161.1, 161.6. EI-MS (m/z): 292

[M + 1]+, 291[M+]. Anal. Calcd. for C18H13NO3: C 74.22, H 4.50; Found: C74.29, H

4.56.

4.2.8. 6-Methoxy-3-(thiophen-2-yl)coumarin (3a): Yield 47%; mp 150-151 oC. 1H

NMR(300MHz)(CDCl3): δ = 3.85 (s, 3H), 6.96 (d, J = 2.8 Hz, 1H), 7.09 (m, 2H), 7.27

(d, J = 9.0 Hz, 1H), 7.42 (dd, J = 5.1; 1.0 Hz, 1H), 7.79 (dd, J = 3.7; 1.0 Hz, 1H), 7.94

(s, 1H). 13C NMR(300MHz)(CDCl3): δ = 55.7, 109.4, 117.3, 119.0, 119.6, 121.9, 127.0,

127.5, 127.7, 135.2, 135.9, 147.1, 156.2, 161.1. EI-MS (m/z): 259 [M + 1]+, 258[M+].

Anal. Calcd. for C14H10O3S: C 65.10, H 3.90; Found: C65.15, H 3.94.

4.2.9. 6-Methoxy-3-(thiophen-3-yl)coumarin (3b): Yield 43%. mp 164-165 oC. 1H

NMR(300MHz)(CDCl3): δ = 3.84 (s, 3H), 6.95 (d, J = 3.0 Hz, 1H), 7.06 (dd, J = 9.0;

3.0 Hz, 1H), 7.25 (d, J = 9.0 Hz, 1H), 7.37 (dd, J = 5.1; 3.0 Hz, 1H), 7.50 (dd, J = 5.1;

1.3 Hz, 1H), 7.86 (s, 1H), 8.18 (dd, J = 3.0; 1.3 Hz, 1H). 13C NMR(300MHz)(CDCl3): δ

= 55.7, 109.6, 117.2, 118.9, 119.7, 122.8, 125.6, 126.0, 126.1, 134.3, 136.9, 147.2,

156.0, 160.1. EI-MS (m/z): 259 [M + 1]+, 258[M+]. Anal. Calcd. for C14H10O3S: C

65.10, H 3.90; Found: C65.20, H 3.99.

4.2.10. 3-(Indol-3-yl)-6-methoxycoumarin (3c): Yield 38%; mp 188-189 oC. 1H

NMR(300MHz)(CDCl3): δ = 3.84 (s, 3H), 5.87 (d, J = 7.7 Hz, 1H), 6.93 (d, J = 2.8 Hz,

1H), 7.04 (dd, J = 9.0; 2.8 Hz, 1H), 7.30 (m, 3H), 7.79 (d, J = 7.7 Hz, 1H), 8.01 (s, 1H),

8.27 (s, 1H), 8.30 (br, 1H). 13C NMR(300MHz)(CDCl3): δ = 55.7, 109.3, 112.6, 115.4,

117.2, 118.7, 119.3, 119.8, 122.7, 124.6, 125.7, 127.3, 135.9, 137.0, 146.7, 150.8,

156.1, 160.5. EI-MS (m/z): 292 [M + 1]+, 291[M+]. Anal. Calcd. for C18H13NO3: C

74.22, H 4.50; Found: C74.30, H 4.52.

4.2.11. 5,7-Dimethoxy-3-(thiophen-2-yl)coumarin (4a): Yield 45%; mp 177-178 oC. 1H


(d, J = 2.1 Hz, 1H), 7.10 (dd, J = 5.0; 3.6 Hz, 1H), 7.36 (d, J = 5.0 Hz, 1H), 7.72 (d, J =

3.6 Hz, 1H), 8.29 (s, 1H). 13C NMR(300MHz)(CDCl3): δ = 55.6, 56.0, 92.4, 95.1,

126.5, 127.1, 127.3, 127.5, 131.5, 136.9, 153.2, 155.3, 156.9, 160.0, 163.4. EI-MS

(m/z): 289 [M + 1]+, 288[M+]. Anal. Calcd. for C15H12O4S: C 62.49, H 4.20; Found:

C62.55, H 4.27.

4.2.12. 5,7-Dimethoxy-3-(thiophen-3-yl)coumarin (4b): Yield 35%; mp 158-159 oC. 1H


(d, J = 1.8 Hz, 1H), 7.35 (dd, J = 4.8; 2.5 Hz, 1H), 7.52 (d, J = 4.8 Hz, 1H), 8.10 (d, J =

2.5 Hz, 1H), 8.20 (s, 1H). 13C NMR(300MHz)(CDCl3): δ = 55.6, 55.8, 92.2, 94.8,

104.3, 117.3, 124.4, 125.2, 126.1, 132.7, 135.0, 155.3, 156.8, 160.4, 163.2. EI-MS

(m/z): 289 [M + 1]+, 288[M+]. Anal. Calcd. for C15H12O4S: C 62.49, H 4.20; Found:

C62.53, H 4.24.

4.2.13. 5,7-Dimethoxy-3-(indol-3-yl)coumarin (4c): Yield 43%; mp 179-180 oC. 1H


(d, J = 2.0 Hz, 1H), 6.48 (d, J = 2.0 Hz, 1H), 7.33 (m, 2H), 7.89 (dd, J = 7.2; 1.2 Hz,

1H), 8.17 (s, 1H), 8.33 (dd, J = 7.0; 1.2 Hz, 1H), 8.48 (br, 1H). 13C

NMR(300MHz)(CDCl3): δ = 55.8, 56.0, 92.3, 95.0, 104.6, 113.6, 115.3, 115.8, 119.6,

122.6, 124.5, 127.6, 133.3, 136.0, 151.0, 155.0, 156.8, 161.1, 163.2. EI-MS (m/z): 322

[M + 1]+, 321[M+]. Anal. Calcd. for C19H15NO4: C 71.02, H 4.71; Found: C 71.10, H

4.77.

4.3. Determination of MAO isoforms activity

The effects of the new synthesized compounds on hMAO isoform enzymatic activity

were evaluated by a fluorimetric method following the experimental protocol previously

described by us [15].

Briefly, 0.1 mL of sodium phosphate buffer (0.05 M, pH 7.4) containing the test drugs

(new compounds or reference inhibitors) in various concentrations and adequate

amounts of recombinant hMAO-A or hMAO-B required and adjusted to obtain in our

experimental conditions the same reaction velocity, (hMAO-A: 1.1 μg protein; specific

activity: 150 nmol of p-tyramine oxidized to p-hydroxyphenylacetaldehyde/min/mg

protein; hMAO-B: 7.5 μg protein; specific activity: 22 nmol of p-tyramine

transformed/min/mg protein) were incubated for 15 min at 37 ºC in a flat-black-bottom

96-well microtest™ plate, placed in the dark fluorimeter chamber. After this incubation

period, the reaction was started by adding (final concentrations) 200 μM Amplex® Red

reagent, 1 U/mL horseradish peroxidase and 1 mM p-tyramine. The production of H2O2

and, consequently, of resorufin was quantified at 37 °C in a multidetection microplate

fluorescence reader (FLX800TM, Bio-Tek® Instruments, Inc., Winooski, VT, USA)

based on the fluorescence generated (excitation, 545 nm, emission, 590 nm) over a 15

min period, in which the fluorescence increased linearly.

Control experiments were carried out simultaneously by replacing the test drugs (new

compounds and reference inhibitors) with appropriate dilutions of the vehicles. In

addition, the possible capacity of the above test drugs to modify the fluorescence

generated in the reaction mixture due to non-enzymatic inhibition (e.g., for directly

reacting with Amplex® Red reagent) was determined 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 the background activity, which was determined from vials containing all

components except the hMAO isoforms, which were replaced by a sodium phosphate

buffer solution.

4.4. Ligand docking

All docking calculations were performed with Maestro 9.0 package [28]. The crystal

structure of hMAO-B was prepared for docking with the Protein Preparation Wizard

workflow of Maestro that allows adding hydrogens which were subsequently minimized

with OPLS_2005 force field, and also optimize the protonation state of His residues and

the orientation of hydroxyl groups, Asn residues, and Gln residues. The docking was

performed by means of the Schrödinger program Glide [29], treating the ligands with a

fully flexible all-atom representation, and the receptor with a rigid grid depiction. The

grid was generated applying a van der Waals radii scaling factor of 1.00 with a partial

charge cut-off of less than 0.25e. The co-crystal ligand was used to center docking box

with a size capable of accommodating ligands with a length of ≤ 20Å. Before docking

calculations, all the compounds were subjected to ligand preparation with the LigPrep

tool of Maestro [28]. Different protonation states using a pH value of 7.0 ± 2.0 and

tautomers were generated. The docking calculations were performed with Glide extra

precision (XP) mode. A post-docking minimization was made on the output complexes

in order to reduce the initial 25 poses per ligand to 5.

Acknowledgments.

This work was supported by grants from Xunta de Galicia (Spain;

INCITE08PXIB203022PR), University of Cagliari (ex 60% 2009), Fondazione Banco

Sardegna 2007 and MIUR 2008.

E. Quezada thanks for a postdoctoral grant from FCT (Portugal). G. Ferino is grateful to

Regione Autonoma della Sardegna for a Master & Back scholarship and to S. Vilar for

helpul suggestions on the docking experiments. Technical support from Red Gallega de

Boinformatica and Centro de Supercomputacion de Galicia (CESGA) is gratefully

acknowledged.

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+O

H

OHR4

OHO

O O

R4R2

R1

R3

R2

R1

R3

1 R1 = R2 = R3 = H2 R1 =OCH3; R2 = R3 = H3 R2 = OCH3; R1 = R3 = H4 R1 = R3 = OCH3; R2 = H

a R4 =

b R4 =

c R4 =

S

S

NH

1a R1 = R2 = R3 = H; R4 =

1b R1 = R2 = R3 = H; R4 =

1c R1 = R2 = R3 = H; R4 =

S

S

NH

2a R1 =OCH3; R2 = R3 = H; R4 =

2b R1 =OCH3; R2 = R3 = H; R4 =

2c R1 =OCH3; R2 = R3 = H; R4 =

S

S

NH

3a R2 = OCH3; R1 = R3 = H; R4 =

3b R2 = OCH3; R1 = R3 = H; R4 =

3c R2 = OCH3; R1 = R3 = H; R4 =

S

S

NH

4a R1 = R3 = OCH3; R2 = H; R4 =

4b R1 = R3 = OCH3; R2 = H; R4 =

4c R1 = R3 = OCH3; R2 = H; R4 =

S

S

NH

a)

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).

DRUG MAO-A (IC50) MAO-B (IC50) SI

1a ** 6.37 ± 0.43 µM > 15b

1b ** 13.3 ± 0.89 µM > 7.5b

1c ** 1.92 ± 0.13 µM > 52b

2a ** 262.95 ± 17.61 nM >380b

2b 4.16 ± 0.28 µMa 55.63 ± 3.73 nM > 75

2c 9.67 ± 0.65 µMa 45.95 ± 3.08 nM > 210

3a ** 633.55 ± 42.43 nM > 158b

3b ** 233.73 ± 15.65 nM > 428b

3c ** ** ---

4a ** ** ---

4b ** ** ---

4c ** ** ---

R-(-)-deprenyl 67.25 ± 1.02 µMa 14.80 ± 0.99 nM 4,544

Iproniazide 6.56 ± 0.76 µM 7.54 ± 0.36 µM 0.87

Table 2. Comparison between experimental activity and predicted scoring function

against MAO-B

Docking Ranka Drug IC50 b Doking scorec

1 4c ** -8.4274 2 2a 0.2630 -8.1976 3 2c 0.0459 -7.9367 4 3a 0.6335 -7.9025 5 2b 0.0556 -7.8267 6 3b 0.2337 -7.5740 7 1c 1.9200 -7.5170 8 1b 13.3000 -7.4479 9 3c ** -7.2776 10 1a 6.3700 -6.4593 11 4b ** -5.3446 12 4a ** -5.1865

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.

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