ES2284341B1

ES2284341B1 - PROCEDURE FOR OBTAINING TRANS-COMMUNIC ACID AND DERIVATIVES FROM CUPRESSUS SEMPERVIRENS L.

Info

Publication number: ES2284341B1
Application number: ES200502101A
Authority: ES
Inventors: Enrique Alvarez-Manzaneda Roldan; Rachid Chahboun
Original assignee: Universidad de Granada
Current assignee: Universidad de Granada
Priority date: 2005-08-19
Filing date: 2005-08-19
Publication date: 2008-10-16
Anticipated expiration: 2025-08-19
Also published as: ES2284341A1

Abstract

Procedimiento para la obtención de ácido trans-comúnico y derivados a partir de Cupressus sempervirens L., concretamente un procedimiento de aislamiento y purificación de ácido labda-8(17), 12E,14-trien-19-oico, también conocido como ácido trans-comúnico, en su forma de sal sódica, a partir de las partes aéreas de Cupressus sempervirens L.Procedure for obtaining trans-common acid and derivatives from Cupressus sempervirens L., specifically an isolation and purification procedure for labda-8 (17), 12E, 14-trien-19-oic acid, also known as trans acid -commonic, in its sodium salt form, from the aerial parts of Cupressus sempervirens L.

Description

Procedimiento para la obtención de ácido trans-comúnico y derivados a partir de Cupressus sempervirens L.Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L.

Object of the invention

La presente invención se refiere a un procedimiento de aislamiento y purificación de ácido labda-8(17),12E,14-trien-19-oico, también conocido como ácido trans-comúnico (1), en su forma de sal sódica (4), a partir de las partes aéreas de Cupressus sempervirens L. A partir de (4) se obtienen con elevada pureza el ácido 1 y el éster metílico 5.The present invention relates to a method of isolation and purification of labda-8 (17), 12E, 14-trien-19-oic acid, also known as trans- common acid (1), in its sodium salt form (4 ), from the aerial parts of Cupressus sempervirens L. From (4), acid 1 and methyl ester 5 are obtained with high purity.

1one

State of the art

Los ácidos comúnicos (1-3) son diterpenos labdánicos naturales que se encuentran ampliamente distribuidos en diversas especies vegetales y particularmente en los géneros Juniperus y Cupressaceae. Fue descrito por vez primera su aislamiento en 1961 de la corteza de Juniperus communis L. [V.P. Arya, C. Enzell, H. Erdtman y T. Kubota, 1961, Acta Chem. Scand, 15, 225]. Años después se describió su presencia en los frutos de esta misma especie y en los de Juniperus oxycedrus L. [J. de Pascual Teresa, A. San Feliciano y J.M. Miguel del Corral, 1974, An. Quim., 70, 1015]. Más recientemente se ha descrito su aislamiento de la madera de esta última especie y de la de Juniperus sabina L. [A.F. Barrero, J.F. Sánchez y J. Altarejos, 1987, Ars Pharm., 28, 449].Common acids (1-3) are natural labdanic diterpenes that are widely distributed in various plant species and particularly in the genera Juniperus and Cupressaceae . Its isolation was first described in 1961 from the bark of Juniperus communis L. [VP Arya, C. Enzell, H. Erdtman and T. Kubota, 1961 , Acta Chem. Scand , 15 , 225]. Years later its presence was described in the fruits of this same species and in those of Juniperus oxycedrus L. [J. by Pascual Teresa, A. San Feliciano and JM Miguel del Corral, 1974 , An. Quim ., 70 , 1015]. More recently, its isolation from the wood of this last species and that of Juniperus sabina L. has been described [AF Barrero, JF Sánchez and J. Altarejos, 1987 , Ars Pharm ., 28, 449].

También se ha descrito el aislamiento de ácidos comúnicos (1-3) de la oleorresina [L. Mangoni y M. Belardini, 1964, Tetrahedron Lett., 2643] y de los frutos [A. Ahoud, P. Camero y B. Gastambide, 1964, Bull. Soc. Chim. Fr., 348] de Cupressus sempervirens L.The isolation of common acids (1-3) from oleoresin [L.] has also been described. Mangoni and M. Belardini, 1964 , Tetrahedron Lett ., 2643] and the fruits [A. Ahoud, P. Camero and B. Gastambide, 1964 , Bull. Soc. Chim. Fr. , 348] of Cupressus sempervirens L.

Estudios realizados durante los últimos años han revelado que los ácidos comúnicos (1-3) y algunos compuestos relacionados poseen significativa actividad biológica. Así, se ha comprobado cierto efecto inhibitorio en la activación del antígeno del virus Epstein-Barr (EBV-EA) [R. Tanaka, H. Ohtsu, M. Iwamoto, T. Minami, H. Tokuda, H. Nishino, S. Matsunaga y A. Yoshitaka, 2000, Cancer Lett., 161, 165]. También se ha descrito cierta actividad antibacteriana [B. Trusheva, M. Popova, V. Baukova, 1. Tsvetkova, C. Naydenski y A.G. Sabatini, 2003, Rivista Ital., 36, 3]. Recientemente se ha patentado el uso del aceite esencial de Cupressus sempervirens L, en el que, entre otros muchos terpenoides, se encuentran estos ácidos, como insecticida y repelente de insectos [G. Campresse y S. Campresse, 2004, Patente FR 2003-3149, 20030314].Studies conducted in recent years have revealed that common acids (1-3) and some related compounds have significant biological activity. Thus, some inhibitory effect on the activation of the Epstein-Barr virus (EBV-EA) antigen has been proven [R. Tanaka, H. Ohtsu, M. Iwamoto, T. Minami, H. Tokuda, H. Nishino, S. Matsunaga and A. Yoshitaka, 2000 , Cancer Lett ., 161 , 165]. Some antibacterial activity has also been described [B. Trusheva, M. Popova, V. Baukova, 1. Tsvetkova, C. Naydenski and AG Sabatini, 2003 , Rivista Ital ., 36, 3]. Recently, the use of Cupressus sempervirens L essential oil has been patented, in which, among many other terpenoids, these acids are found as insecticide and insect repellent [G. Campresse and S. Campresse, 2004 , Patent FR 2003-3149, 20030314].

Además de la actividad biológica que presentan los ácidos comúnicos (1-3) y compuestos relacionados, estos diterpenos presentan interés debido a sus características estructurales, que los convierten en productos de partida adecuados para la síntesis de productos de interés. Así, se han empleado en la preparación de productos olorosos tipo ambar-gris [A.F. Barrero, J. Altarejos, E.J. Alvarez-Manzaneda, J.M. Ramos y S. Salido, 1993, Tetrahedron, 49, 6251, 9525; A.F. Barrero, J. Altarejos, S. salido, 1995, Patente ES 93-9300946, 19930504], en la síntesis de lactonas antifúngicas [A.F. Barrero, J.F. Sánchez y J. Elmerabet, 1995, Tetrahedron Lett., 36, 5251; A.F. Barrero, S. Arseniyadis, J.F. Quílez del Moral, M. Mar Herrador, M. Valdivia y D. Jiménez, 2002, J Org. Chem., 67, 2501] y en la preparación de compuestos con actividad antitumoral [T. Katoh, R. Tanaka, M. Takeo, K. Nishida y M. Noda, 2002, Chem. Pharm. Bull., 50, 1625; E.J. Alvarez-Manzaneda, J.L. Romera, A.F. Barrero, R. Alvarez-Manzaneda, R. Chahboun, R. Meneses y M. Aparicio, 2005, Tetrahedron, 61, 837; A.F. Barrero, E.J. Alvarez-Manzaneda, R. Chahboun, R. Meneses, J.M. Cuerva, M. Aparicio y J.L. Romera, 2001, Org. Lett., 3, 647].In addition to the biological activity of common acids (1-3) and related compounds, these diterpenes have an interest due to their structural characteristics, which make them suitable starting products for the synthesis of products of interest. Thus, they have been used in the preparation of aromatic products of the amber-gray type [AF Barrero, J. Altarejos, EJ Alvarez-Manzaneda, JM Ramos and S. Salido, 1993 , Tetrahedron, 49 , 6251, 9525; AF Barrero, J. Altarejos, S. Salido, 1995 , ES Patent 93-9300946, 19930504], in the synthesis of antifungal lactones [AF Barrero, JF Sánchez and J. Elmerabet, 1995 , Tetrahedron Lett ., 36, 5251; AF Barrero, S. Arseniyadis, JF Quílez del Moral, M. Mar Herrador, M. Valdivia and D. Jiménez, 2002 , J Org. Chem., 67 , 2501] and in the preparation of compounds with antitumor activity [T. Katoh, R. Tanaka, M. Takeo, K. Nishida and M. Noda, 2002 , Chem. Pharm. Bull., 50 , 1625; EJ Alvarez-Manzaneda, JL Romera, AF Barrero, R. Alvarez-Manzaneda, R. Chahboun, R. Meneses and M. Aparicio, 2005 , Tetrahedron, 61 , 837; AF Barrero, EJ Alvarez-Manzaneda, R. Chahboun, R. Meneses, JM Cuerva, M. Aparicio and JL Romera, 2001 , Org. Lett., 3 , 647].

       \newpage\ newpage

A pesar del gran interés de este tipo de ácidos diterpénicos y de su abundancia en las fuentes vegetales que los contienen, hasta el momento no se ha descrito un procedimiento eficiente para el aislamiento y purificación del ácido trans-comúnico (1) contenido en Cupressus sempervirens L. Además, hasta la fecha sólo se ha descrito su presencia en los frutos, en la corteza y en la resina de esta especie. Nuestras investigaciones han revelado su presencia en gran cantidad en las hojas de C. sempervirens L, lo que permite un mayor aprovechamiento de la fuente vegetal.In spite of the great interest of this type of diterpenic acids and their abundance in the vegetable sources that contain them, so far an efficient procedure for the isolation and purification of trans- common acid (1) contained in Cupressus sempervirens has not been described L. In addition, to date only its presence in fruits, bark and resin of this species has been described. Our investigations have revealed their presence in large quantities in the leaves of C. sempervirens L, which allows a better use of the plant source.

Description of the invention

La presente invención expone diversos procedimientos para el aislamiento y purificación del ácido trans-comúnico (1), en forma de sal sódica (4), a partir de los frutos de Cupressus sempervirens L., de las hojas de esta especie vegetal, y de la combinación de frutos y hojas, partes aéreas, de esta planta. Se describe asimismo un procedimiento eficaz para la transformación de la sal (4) en el ácido (1) y en el éster metílico (5).The present invention sets forth various procedures for the isolation and purification of trans- common acid (1), in the form of sodium salt (4), from the fruits of Cupressus sempervirens L., from the leaves of this plant species, and from the combination of fruits and leaves, aerial parts, of this plant. An effective process for the transformation of the salt (4) into the acid (1) and the methyl ester (5) is also described.

El estudio de la composición de los frutos de distintas especies de Juniperus y Cupressus ha revelado que J. oxycedrus y C. sempervirens contienen un único isómero. Así, del extracto hexánico de las arcéstidas de J. oxycedrus se obtuvo únicamente ácido mirceo-comúnico (3) (4.2% del peso de planta); mientras que en el correspondiente a las arcéstidas de C. sempervirens sólo se encontró ácido trans-comúnico (1), aislado en forma de sal sódica (2.3% del peso de planta). El procedimiento consistió en la extracción en sistema Soxhlet del material vegetal seco y triturado durante 12 horas, separación de las ceras mediante precipitación de las soluciones concentradas enfriadas a 0°C, y posterior precipitación de la sal del ácido mediante adición de solución acuosa alcalina sobre la solución hexánica concentrada.The study of the composition of the fruits of different species of Juniperus and Cupressus has revealed that J. oxycedrus and C. sempervirens contain a single isomer. Thus, from the hexane extract of the arcestids of J. oxycedrus only mirceo-common acid was obtained (3) (4.2% of the plant weight); while in the corresponding to the arcestids of C. sempervirens only trans- common acid (1) was found, isolated in the form of sodium salt (2.3% of the plant weight). The procedure consisted of the extraction in Soxhlet system of the dried and crushed vegetable material for 12 hours, separation of the waxes by precipitation of the concentrated solutions cooled to 0 ° C, and subsequent precipitation of the acid salt by adding alkaline aqueous solution on the concentrated hexane solution.

Tanto la elevada proporción de ácido trans-comúnico (1) encontrado en los frutos de C. sempervirens L., como la gran abundancia de esta especie vegetal, nos animaron a investigar la composición de los correspondientes extractos de las hojas. Inicialmente se estudió el extracto hexánico, que mediante un procedimiento similar al utilizado en las arcéstidas permitió también el aislamiento de una importante cantidad de sal sódica (4) del ácido trans-comúnico (1.9% del peso de planta).Both the high proportion of trans- common acid (1) found in the fruits of C. sempervirens L., and the great abundance of this plant species, encouraged us to investigate the composition of the corresponding leaf extracts. Initially, the hexane extract was studied, which by means of a procedure similar to that used in the arcestids also allowed the isolation of a significant amount of sodium salt (4) from the trans- common acid (1.9% of the plant weight).

Con objeto de desarrollar procesos menos laboriosos y por tanto, más rentables, se investigó también el aislamiento de ácido trans-comúnico (1) del extracto hexánico de toda la parte aérea de C. sempervirens L., constituída por frutos, hojas y pequeñas ramas. Tras extraer en sistema Soxhlet todo el material vegetal, seco y triturado, se obtuvo, siguiendo el procedimiento ya comentado para frutos y hojas, la correspondiente sal sódica (4) del ácido trans-comúnico (2.0% del peso en planta).In order to develop less laborious and therefore more profitable processes, the isolation of trans- common acid (1) from the hexane extract of the entire aerial part of C. sempervirens L., consisting of fruits, leaves and small branches, was also investigated. . After extracting in the Soxhlet system all the plant material, dried and crushed, the corresponding sodium salt (4) of the trans- common acid (2.0% of the plant weight) was obtained, following the procedure already mentioned for fruits and leaves.

Se han investigado también otros disolventes y condiciones que permitan obtener el ácido trans-comúnico (1) y sus derivados mediante procesos más sencillos y rentables. Los mejores resultados se han alcanzado utilizando cloruro de metileno. Inicialmente se ensayó la extracción en sistema Soxhlet del material vegetal, triturado y seco, durante 6 horas. Tras evaporar el disolvente, se procedió al descerado mediante precipitaciones sucesivas de las soluciones hexánicas concentradas a 0°C. La sal sódica (4) se obtuvo siguiendo el mismo procedimiento indicado en el caso de la extracción con hexano. Así se obtuvo en el caso de los frutos una cantidad de sal sódica de ácido trans-comúnico, correspondiente a un 1.9% del peso de planta, mientras que las hojas proporcionaron el equivalente a un 1.5% del peso de planta y el conjunto de partes aéreas un 1.6% del peso de planta.Other solvents and conditions that allow obtaining trans- common acid (1) and its derivatives by more simple and cost-effective processes have also been investigated. The best results have been achieved using methylene chloride. Initially the Soxhlet system extraction of the plant material, crushed and dried, was tested for 6 hours. After evaporating the solvent, the precipitation was carried out by successive precipitation of the hexane solutions concentrated at 0 ° C. The sodium salt (4) was obtained following the same procedure indicated in the case of hexane extraction. Thus, in the case of the fruits, an amount of sodium salt of trans- common acid, corresponding to 1.9% of the plant weight, was obtained, while the leaves provided the equivalent of 1.5% of the plant weight and the set of parts aerial 1.6% of the plant weight.

Con objeto de simplificar el proceso de extracción, se ensayó la maceración con cloruro de metileno. Tras someter el material vegetal triturado y seco a maceración en este disolvente durante 4 horas, y operando con la solución resultante de modo similar al ya indicado para la extracción con cloruro de metileno en Soxhlet, se obtuvo la correspondiente sal (4). El estado de pureza de la sal obtenida mediante maceración resultó superior al de la obtenida mediante maceración en sistema Soxhlet. Los frutos rindieron un 2.1% en peso de planta, las hojas un 1.8%, y el conjunto de partes aéreas aproximadamente un 1.9% en peso de planta.In order to simplify the process of extraction, maceration was tested with methylene chloride. After subject the crushed and dried plant material to maceration in this solvent for 4 hours, and operating with the resulting solution similar to that already indicated for the extraction with chloride of methylene in Soxhlet, the corresponding salt (4) was obtained. He state of purity of the salt obtained by maceration resulted higher than that obtained by maceration in Soxhlet system. The fruits yielded 2.1% by weight of the plant, the leaves 1.8%, and the set of aerial parts approximately 1.9% by weight of plant.

Embodiment Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the harnesses of C. sempervirens L. by extraction with hexane in Soxhlet system

500 g de material vegetal, seco y finamente triturado, se extrae en sistema Soxhlet con hexano (3 L) durante 12 horas. Transcurrido este tiempo se evapora el disolvente hasta aproximadamente un 20% del volumen inicial. La solución resultante se mantiene durante 12 horas a 0°C, filtrando el precipitado resultante, constituido por ceras. El filtrado obtenido se somete dos veces más a esta operación. A continuación, sobre la solución hexánica descerada se añade, bajo agitación, NaOH 2N (500 mL), separándose un sólido incoloro, constituido por la sal (4). Se filtra a vacío, lavando con pequeñas porciones de hexano frío, primero, y agua fría después, obteniéndose 11.5 g de producto (2.3% del peso de planta). Mediante dos recristalizaciones en MeOH-H_{2}O se obtiene un sólido cristalino de punto de fusión constante.500 g of plant material, dry and finely crushed, extracted in Soxhlet system with hexane (3 L) for 12 hours. After this time the solvent evaporates until approximately 20% of the initial volume. The resulting solution it is maintained for 12 hours at 0 ° C, filtering the precipitate resulting, consisting of waxes. The filtrate obtained is subjected Twice more to this operation. Next, about the solution Hexane drop is added, under stirring, 2N NaOH (500 mL), separating a colorless solid, constituted by salt (4). Be vacuum filter, washing with small portions of cold hexane, first, and cold water later, obtaining 11.5 g of product (2.3% of plant weight). Through two recrystallizations in MeOH-H2O a crystalline solid of constant melting point.

Sólido cristalino incoloro. P.F. 227-228°. [\alpha]_{D} 24° (MeOH).Colorless crystalline solid. P.F. 227-228 °. [α] D 24 ° (MeOH).

IR (KBr): 3085, 2936, 2843, 1642, 1547, 1467, 1404, 1350, 1298, 1256, 1188, 989, 888 cm^{-1}.IR (KBr): 3085, 2936, 2843, 1642, 1547, 1467, 1404, 1350, 1298, 1256, 1188, 989, 888 cm -1.

RMN-^{1}H (CD_{3}COCD_{3}, 300 MHz): \delta 0.71 (3H, s, Me-10), 1.09 (3H, s, Me-4), 1.73 (3H, s, Me-13), 4.41 (1H, sa, H-17), 4.75 (1H, sa, H-17'), 4.82 (1H, da, J = 10.9 Hz, H-15), 5.01 (1H, da, J = 17.5 Hz, H-15'), 5.42 (1H, ta, J = 6.5 Hz), 6.30 (1H, dd, J = 17.5 y 10.9 Hz).1 H NMR (CD 3 COCD 3, 300 MHz): δ 0.71 (3H, s, Me-10), 1.09 (3H, s, Me-4), 1.73 (3H, s, Me-13), 4.41 (1H, sa, H-17), 4.75 (1H, sa, H-17 '), 4.82 (1H, da, J = 10.9 Hz, H-15), 5.01 (1H, da, J = 17.5 Hz, H-15 '), 5.42 (1H, ta, J = 6.5 Hz), 6.30 (1H, dd, J = 17.5 and 10.9 Hz).

RMN-^{13}C (CD_{3}COCD_{3}, 75 MHz): \delta 41.0 (C-1), 21.6 (C-2), 39.8 (C-3), 45.7 (C-4), 57.7 (C-5), 27.4 (C-6), 40.5 (C-7), 150.1 (C-8), 57.7 (C-9), 41.2 (C-10), 24.0 (C-11), 135.4 (C-12), 133.7 (C-13), 142.6 (C-14), 109.9 (C-15), 11.9 (C-16), 107.0 (C-17), 31.0 (C-18), 184.5 (C-19), 13.9 (C-20).NMR - 13 C (CD 3 COCD 3, 75 MHz): δ 41.0 (C-1), 21.6 (C-2), 39.8 (C-3), 45.7 (C-4), 57.7 (C-5), 27.4 (C-6), 40.5 (C-7), 150.1 (C-8), 57.7 (C-9), 41.2 (C-10), 24.0 (C-11), 135.4 (C-12), 133.7 (C-13), 142.6 (C-14), 109.9 (C-15), 11.9 (C-16), 107.0 (C-17), 31.0 (C-18), 184.5 (C-19), 13.9 (C-20).

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the leaves of C. sempervirens L. by extraction with hexane in Soxhlet system

Se sigue un procedimiento similar al descrito para las arcéstidas, pero realizando ahora cinco veces el proceso de descerado. A partir de 500 g de hojas secas se obtienen 9.5 g de (4) (1.9% del peso de planta), que se purifica adecuadamente después de dos recristalizaciones.A procedure similar to that described is followed. for the arcéstidas, but now performing the process five times of descended. From 500 g of dried leaves 9.5 g of (4) (1.9% of plant weight), which is properly purified after two recrystallizations.

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the aerial parts, fruits and leaves, of C. sempervirens L. by extraction with hexane in Soxhlet system

Se utiliza como material vegetal el conjunto de las hojas, frutos y pequeñas ramas que resultan de la poda de C. sempervirens L. Utilizando un procedimiento análogo al descrito en los dos casos anteriores y, tras descerar cinco veces, se obtienen 10.0 g de (4) (2.0% del peso de planta). Tras efectuar tres recristalizaciones en MeOH-H_{2}O se obtuvo el producto en un elevado grado de pureza.The set of leaves, fruits and small branches resulting from pruning of C. sempervirens L. is used as plant material. Using a procedure analogous to that described in the two previous cases and, after descending five times, 10.0 g of ( 4) (2.0% of the plant weight). After performing three recrystallizations in MeOH-H2O the product was obtained in a high degree of purity.

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the harnesses of C. sempervirens L. by extraction with methylene chloride in Soxhlet system

500 g de material vegetal, seco y finamente triturado, se extrae en sistema Soxhlet con cloruro de metileno (3 L) durante 6 horas. Se elimina el disolvente mediante evaporación a vacío, y el residuo resultante se disuelve en 600 mL de hexano y se mantiene a 0°C durante 12 horas, filtrando el precipitado resultante, constituido por ceras. El filtrado obtenido se somete a esta operación tres veces más. A continuación, sobre la solución hexánica descerada se añade, bajo agitación, NaOH 2N (500 mL), separándose un sólido incoloro, constituido por la sal (4). Se filtra a vacío, lavando con pequeñas porciones de hexano frío, primero, y agua fría a continuación, obteniéndose 9.5 g de producto (1.9% del peso de planta). Mediante tres recristalizaciones en MeOH-H_{2}O se obtuvo la sal en un elevado grado de pureza.500 g of plant material, dry and finely crushed, extracted in Soxhlet system with methylene chloride (3 L) for 6 hours. The solvent is removed by evaporation at vacuum, and the resulting residue is dissolved in 600 mL of hexane and kept at 0 ° C for 12 hours, filtering the precipitate resulting, consisting of waxes. The filtrate obtained is subjected to This operation three more times. Next, about the solution Hexane drop is added, under stirring, 2N NaOH (500 mL), separating a colorless solid, constituted by salt (4). Be vacuum filter, washing with small portions of cold hexane, first, and cold water then obtaining 9.5 g of product (1.9% of the plant weight). Through three recrystallizations in MeOH-H2O the salt was obtained to a high degree of purity

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the leaves of C. sempervirens L. by extraction with methylene chloride in Soxhlet system

Se sigue idéntico procedimiento al descrito para las arcéstidas, pero realizando cinco descerados sucesivos. A partir de 500 g de hojas secas se obtiene 7.5 g de (4) (1.5% del peso de planta). Tres recristalizaciones en MeOH-H_{2}O proporcionan el producto con elevada pureza.The same procedure is followed as described for the arcéstidas, but making five successive descents. TO from 500 g of dried leaves you get 7.5 g of (4) (1.5% of plant weight). Three recrystallizations in MeOH-H2O provide the product with high purity.

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the aerial parts, fruits and leaves, of C. sempervirens L. by extraction with methylene chloride in Soxhlet system

Se sigue un procedimiento similar al empleado con las hojas, efectuando cinco descerados. A partir de 500 g de material vegetal se obtienen 8.0 g de (4) ((1.6% del peso de planta). Tres recristalizaciones en MeOH-H_{2}O son suficientes para lograr elevada pureza.A similar procedure to the employee is followed with the leaves, making five drops. From 500 g of Vegetable material is obtained 8.0 g of (4) ((1.6% of the weight of plant). Three recrystallizations in MeOH-H2O They are sufficient to achieve high purity.

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the harnesses of C. sempervirens L. by maceration with methylene chloride

500 g de planta seca y triturada se somete a maceración en cloruro de metileno (2.5 L) a temperatura ambiente durante 4 horas. Se separa el material vegetal mediante filtración y se elimina el disolvente evaporando a vacío. El residuo resultante se disuelve en hexano (500 mL) y se mantiene a 0°C durante 12 horas. El precipitado, constituido por ceras, se filtra y la solución hexánica se somete al mismo proceso dos veces más. A la solución final se adiciona, bajo agitación, NaOH 2N (450 mL), separándose un sólido incoloro, constituido por la sal (4). Se filtra a vacío, lavando con pequeñas porciones de hexano frío, primero, y agua fría luego, obteniéndose 10.5 g de producto (2.1% del peso de planta). Mediante dos recristalizaciones en MeOH-H_{2}O se obtuvo la sal en un elevado grado de pureza.500 g of dried and crushed plant is subjected to maceration in methylene chloride (2.5 L) at room temperature During 4 hours. The plant material is separated by filtration and the solvent is removed by evaporating in vacuo. The residue resulting is dissolved in hexane (500 mL) and kept at 0 ° C for 12 hours The precipitate, consisting of waxes, is filtered and the hexane solution is subjected to the same process two more times. TO the final solution is added, under stirring, 2N NaOH (450 mL), separating a colorless solid, constituted by salt (4). Be vacuum filter, washing with small portions of cold hexane, first, and then cold water, obtaining 10.5 g of product (2.1% of plant weight). Through two recrystallizations in MeOH-H2O the salt was obtained to a high degree of purity

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the leaves of C. sempervirens L. by maceration with methylene chloride

Operando del mismo modo al descrito para las arcéstidas, pero realizando cuatro desceraciones, se obtienen 9,0 g de (4) (1.8% del peso de planta) a partir de 500 g de planta. Dos recristalizaciones en MeOH-H_{2}O permitieron obtener la sal con elevada pureza.Operating in the same way as described for arcéstidas, but making four descents, 9.0 g are obtained of (4) (1.8% of the plant weight) from 500 g of plant. Two recrystallizations in MeOH-H2O allowed get salt with high purity.

Obtaining labda-8 (17), 12 E , 14-trien-19-oato sodium (4) from the aerial parts, harnesses and leaves, of C. sempervirens L. by maceration with methylene chloride

Se sigue un procedimiento análogo al descrito en los dos apartados anteriores, efectuando tres desceraciones. 500 g de material vegetal rindieron 9.5 g de (4) (1.9% del peso de planta). Tras recristalizar dos veces en MeOH-H_{2}O se obtuvo la sal pura.A procedure similar to that described in the two previous sections, making three descents. 500 g of plant material yielded 9.5 g of (4) (1.9% of the weight of plant). After recrystallizing twice in MeOH-H2O was obtained pure salt.

Labda-8 (17), 12 E , 14-trien-19-oic acid (1)

7 g (21.58 mmoles) de labda-8(17),12E,14-trien-19-oato sódico (4) se suspenden en H_{2}O (250 mL) y se añade, con agitación, HCl 2N hasta pH 3. La mezcla resultante se extrae con t-butil metil éter (3 x 60 mL). Las fases orgánicas reunidas se secan sobre sulfato sódico anhidro, se filtran y evaporan a vacío, rindiendo el ácido (1), como un aceite incoloro (6.2 g, 95%).7 g (21.58 mmol) of labda-8 (17), 12 E , 14-trien-19-oato sodium (4) are suspended in H2O (250 mL) and 2N HCl is added with stirring to pH 3. The resulting mixture is extracted with t -butyl methyl ether (3 x 60 mL). The combined organic phases are dried over anhydrous sodium sulfate, filtered and evaporated in vacuo, yielding the acid (1), as a colorless oil (6.2 g, 95%).

[\alpha]_{D} 37° (CHCl_{3}).[α] D 37 ° (CHCl 3).

IR (film): 3600-2800, 3087, 2938, 2845, 1702, 1642, 1543, 1469, 1400, 1354, 1300, 1250, 1185, 991, 884 cm^{-1}.IR (film): 3600-2800, 3087, 2938, 2845, 1702, 1642, 1543, 1469, 1400, 1354, 1300, 1250, 1185, 991, 884 cm -1.

RMN-^{1}H (CDCl_{3}, 300 MHz): \delta 0.65 (311, s, Me-10), 1.22 (311, s, Me-4), 1.74 (3H, s, Me-13), 4.46 (111, sa, H-17), 4.83 (111, sa, H-17'), 4.86 (111, da, J = 10.7 Hz, H-15), 5.04 (111, da, J = 17.3 Hz, 11-15'), 5.40 (1H, ta, J = 6.4 Hz), 6.30 (1H, dd, J = 17.3 y 10.7 Hz).1 H NMR (CDCl 3, 300 MHz): δ 0.65 (311, s, Me-10), 1.22 (311, s, Me-4), 1.74 (3H, s, Me-13), 4.46 (111, sa, H-17), 4.83 (111, sa, H-17 '), 4.86 (111, da, J = 10.7 Hz, H-15), 5.04 (111, da, J = 17.3 Hz, 11-15 '), 5.40 (1H, ta, J = 6.4 Hz), 6.30 (1H, dd, J = 17.3 and 10.7 Hz).

RMN-^{13}C (CDCl_{3}, 75 MHz): \delta 41.2 (C-1), 21.4 (C-2), 40.1 (C-3), 45.9 (C-4), 59.1 (C-5), 27.0 (C-6), 40.6 (C-7), 149.8 (C-8), 57.9 (C-9), 41.0 (C-10), 24.3 (C-11), 135.4 (C-12), 133.9 (C-13), 142.7 (C-14), 110.2 (C-15), 12.1 (C-16), 107.2 (C-17), 29.9 (C-18), 181.3 (C-19), 14.1 (C-20).NMR - 13 C (CDCl 3, 75 MHz): δ 41.2 (C-1), 21.4 (C-2), 40.1 (C-3), 45.9 (C-4), 59.1 (C-5), 27.0 (C-6), 40.6 (C-7), 149.8 (C-8), 57.9 (C-9), 41.0 (C-10), 24.3 (C-11), 135.4 (C-12), 133.9 (C-13), 142.7 (C-14), 110.2 (C-15), 12.1 (C-16), 107.2 (C-17), 29.9 (C-18), 181.3 (C-19), 14.1 (C-20).

Labda-8 (17), 12 E , 14-trien-19-methyl oato (5)

5 g (15.41 mmoles) de labda-8(17),12E,14-trien-19-oato sódico (4) se suspenden en acetona (200 mL) y se añade yodometano (3.28 g, 23.11 mmoles). La mezcla resultante se calienta a reflujo durante 24 horas. Se elimina el disolvente a vacío y el residuo se fracciona en t-butil metil eter (100 mL) y H_{2}O (100 mL), y la fase acuosa se extrae con más t-butil metil éter (2 x 50 mL). Las fases orgánicas reunidas se secan sobre sulfato sódico anhidro, se filtran y evaporan, proporcionando (5) (4.8 g, 98%), como un sólido cristalino.5 g (15.41 mmol) of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) are suspended in acetone (200 mL) and iodomethane (3.28 g, 23.11 mmol) is added. The resulting mixture is heated at reflux for 24 hours. The solvent is removed in vacuo and the residue is divided into t-butyl methyl ether (100 mL) and H2O (100 mL), and the aqueous phase is extracted with more t -butyl methyl ether (2 x 50 mL ). The combined organic phases are dried over anhydrous sodium sulfate, filtered and evaporated, providing (5) (4.8 g, 98%), as a crystalline solid.

Sólido cristalino incoloro. P.F. 105-106°C (MeOH. [\alpha]_{D}) 45° (CHCl_{3}).Colorless crystalline solid. P.F. 105-106 ° C (MeOH. [Α] D) 45 ° (CHCl3).

IR (KBr): 3092, 3038, 2934, 2840, 1720, 1642, 1605, 1543, 1468, 1402, 1348, 1300, 1259, 1195, 985, 885 cm^{-1}.IR (KBr): 3092, 3038, 2934, 2840, 1720, 1642, 1605, 1543, 1468, 1402, 1348, 1300, 1259, 1195, 985, 885 cm -1.

RMN-^{1}H (CDCl_{3}, 300 MHz): \delta 0.56 (3H, s, Me-10), 1.19 (311, s, Me-4), 1.77 (3H, s, Me-13), 3.62 (3H, s, MeO-19), 4.44 (1H, sa, H-17), 4.80 (1H, sa, H-17'), 4.87 (1H, da, J = 10.5 Hz, H-15), 5.01 (1H, da, J = 17.8 Hz, H-15'), 5.40 (1H, ta, J = 6.8 Hz), 6.33 (1H, dd, J = 17.8 y 10.5 Hz).1 H NMR (CDCl 3, 300 MHz): δ 0.56 (3H, s, Me-10), 1.19 (311, s, Me-4), 1.77 (3H, s, Me-13), 3.62 (3H, s, MeO-19), 4.44 (1H, sa, H-17), 4.80 (1H, sa, H-17 '), 4.87 (1H, da, J = 10.5 Hz, H-15), 5.01 (1H, da, J = 17.8 Hz, H-15 '), 5.40 (1H, ta, J = 6.8 Hz), 6.33 (1H, dd, J = 17.8 and 10.5 Hz).

RMN-^{13}C (CDCl_{3}, 75 MHz): \delta 39.2 (C-1), 19.9 (C-2), 38.3 (C-3), 44.2 (C-4), 56.5 (C-5), 26.1 (C-6), 38.4 (C-7), 147.8 (C-8), 56.4 (C-9), 40.1 (C-10), 23.1 (C-11), 133.5 (C-12), 133.2 (C-13), 141.5 (C-14), 109.7 (C-15), 11.8 (C-16), 107.6 (C-17), 28.7 (C-18), 177.4 (C-19), 12.5 (C-20), 50.8 (C-21).NMR - 13 C (CDCl 3, 75 MHz): δ 39.2 (C-1), 19.9 (C-2), 38.3 (C-3), 44.2 (C-4), 56.5 (C-5), 26.1 (C-6), 38.4 (C-7), 147.8 (C-8), 56.4 (C-9), 40.1 (C-10), 23.1 (C-11), 133.5 (C-12), 133.2 (C-13), 141.5 (C-14), 109.7 (C-15), 11.8 (C-16), 107.6 (C-17), 28.7 (C-18), 177.4 (C-19), 12.5 (C-20), 50.8 (C-21).

Claims

1. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from the harnesses of this species through a process that includes the following stages:

a.to.: Extracción de la planta seca y triturada con hexano en sistema Soxhlet.Extraction of the dried plant and crushed with hexane in Soxhlet system.

b.b.: Concentración de la solución resultante.Solution concentration resulting.

c.C.: Sucesivos enfriamientos a 0°C de la solución concentrada y posterior filtración.Successive cooling at 0 ° C of the concentrated solution and subsequent filtration.

d.d.: Adición de solución acuosa de NaOH a la solución hexánica última.Addition of aqueous NaOH solution to the last hexane solution.

e.and.: Filtración del precipitado resultante.Precipitation Filtration resulting.

f.F.: Recristalización en MeOH-H_{2}O del sólido obtenidoRecrystallization in MeOH-H2O of the solid obtained

2. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from the leaves of this species through a process that includes the following stages:

f.F.: Recristalización en MeOH-H_{2}O del sólido obtenido.Recrystallization in MeOH-H2O of the solid obtained.

3. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from the set of the arcéstidas and leaves of this species by means of a process that includes the following stages:

4. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from harnesses of this species through a process that includes the following stages:

a.to.: Extracción de la planta seca y triturada con cloruro de metileno en sistema Soxhlet.Extraction of the dried plant and crushed with methylene chloride in Soxhlet system.

b.b.: Evaporación del disolvente a vacío.Evaporation of the solvent a empty.

c.C.: Disolución en hexano del residuo resultante.Hexane dissolution of the residue resulting.

d.d.: Sucesivos enfriamientos a 0°C de la solución hexánica y posterior filtración.Successive cooling at 0 ° C of the Hexane solution and subsequent filtration.

e.and.: Adición de solución acuosa de NaOH a la solución hexánica última.Addition of aqueous NaOH solution to the last hexane solution.

f.F.: Filtración del precipitado resultante.Precipitation Filtration resulting.

g.g.: Recristalización en MeOH-H_{2}O del sólido obtenido.Recrystallization in MeOH-H2O of the solid obtained.

5. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oate (4) from the leaves of this species through a process that includes the following stages:

6. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from the set of the arcéstidas and leaves of this species by means of a process that includes the following stages:

7. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from the harnesses of this species through a process that includes the following stages:

a.to.: Extracción de la planta seca y triturada mediante maceración con cloruro de metileno a temperatura ambiente.Extraction of the dried plant and crushed by maceration with methylene chloride at temperature ambient.

b.b.: Filtración del material vegetal y evaporación del disolvente a vacío.Filtration of plant material and evaporation of the solvent in vacuo.

8. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from the leaves of this species through a process that includes the following stages:

9. Procedure for obtaining trans- common acid and derivatives from Cupressus sempervirens L., characterized by the isolation of labda-8 (17), 12 E , 14-trien-19-sodium oato (4) from the set of the arcéstidas and the leaves of this species by means of a process that includes the following stages:

10. Process for obtaining trans- common acid and derivatives from Cupressus sempervirens L., according to claims 1 to 9, characterized in that the transformation of labda-8 (17), 12 E , 14-trien-19-oato sodium (4) in labda-8 (17), 12 E , 14-trien-19-oic acid (1) is carried out by a process comprising the following steps:

a.to.: Suspensión de la sal (4) en H_{2}O.Salt suspension (4) in H2O.

b.b.: Adición de HCl 2N sobre la anterior suspensión hasta pH 3.Addition of 2N HCl over the previous one suspension up to pH 3.

c.C.: Extracción de la mezcla anterior con t-butil metil éter.Extraction of the previous mixture with t-butyl methyl ether.

d.d.: Secado de la fase orgánica y evaporación del disolvente.Drying of the organic phase and evaporation of the solvent.

11. Method for obtaining trans- common acid and derivatives from Cupressus sempervirens L., according to claims 1 to 9, characterized in that the transformation of labda-8 (17), 12 E , 14-trien-19-oato sodium (4) in labda-8 (17), 12 E , 14-trien-19-methyl oato (5) is carried out by a process comprising the following steps:

a.to.: Suspensión de la sal (4) en acetona.Salt suspension (4) in acetone.

b.b.: Adición de yodometano a la mezcla anterior y calentamiento a reflujo.Adding iodomethane to the mixture anterior and heating at reflux.

c.C.: Evaporación del disolvente a vacío.Evaporation of the solvent a empty.

d.d.: Fraccionamiento del residuo resultante en H_{2}O-t-butil metil éter.Fractionation of the resulting residue in H2O- t -butyl methyl ether.

e.and.: Extracciones sucesivas de la fase acuosa.Successive phase extractions watery

f.F.: Secado de la fase orgánica y evaporación del disolvente.Drying of the organic phase and evaporation of the solvent.

g.g.: Recristalización en MeOH del sólido resultante.Solid crystal MeOH recrystallization resulting.