5α,7β,9α,10β,13α-Pentaacetoxy-4(20),11-Taxadiene (7β-Acetoxy-Taxusin) and Other Constituents from the Bark of the Mexican Yew, Taxus globosa (Taxaceae)

Bernice Guerrero,¹ Juan Castillo,¹ María Isabel Aguilar² and Guillermo Delgado*¹

¹Instituto de Química de la Universidad Nacional Autónoma de México. Circuito Exterior, Ciudad Universitaria. Coyoacán 04510. México, D. F. and

²Departamento de Farmacia de la Facultad de Química de la Universidad Nacional Autónoma de México. Conjunto E, Circuito Exterior, Ciudad Universitaria, Coyoacán 04510. México, D. F. E-mail: delgado@servidor.unam.mx

Recibido el 24 de abril del 2000. ]]> Aceptado el 5 de junio del 2000.

Abstract

Chemical investigation of the bark of the Mexican yew (Taxus globosa) led to the isolation of sterols, vanillic acid and a per-acetyl taxane identified as 5α, 7β, 9α, 10β, 13α-pentaacetoxy-4(20), 11-taxadiene (7β-acetoxy-taxusin, 2).

Key Words. 5α, 7β, 9α, 10β, 13α-pentaacetoxy-4(20), 11-taxadiene, 7β-acetoxy-taxusin, taxane, Mexican yew, Taxus globosa, Taxaceae.

Resumen

La investigación química de la corteza del tejo mexicano (Taxus globosa) condujo al aislamiento de esteroles, ácido vainíllico y un peracetil taxano identificado como 5α, 7β, 9α, 10β, 13α-pentaacetoxi-4(20), 11-taxadieno (7β-acetoxi-taxusina, 2).

Palabras clave. 5α, 7β, 9α, 10β, 13α-pentaacetoxi-4(20), 11-taxadieno, 7β-acetoxi-taxusina, taxano, tejo mexicano, Taxus globosa, Taxaceae.

Introduction

The taxane diterpenoids are biosynthesized mainly by the members of the Taxus genus (Taxaceae) and represent one of the most interesting groups of bioactive natural products due to the discovery and development of taxol® (1, paclitaxel) [1,2], which has become an efficient therapeutic agent and the best selling anticancer drug in history [3]. Although the chemical study of Taxus species began in the middle of the last century [4], only few taxanes were known around 1971, at the time of the discovery of taxol, and the interest for this substance increased during the late seventies derived from its novel model of antitumoral action [5] and successful clinical trials [6]. Recent reviews have included over 350 taxoids [7], and the investigation on this group of natural products has emerged as an important field in chemistry and biology [8]. Taxoids are relatively scarce in Nature and different strategies to increase the supply of these substances, and in particular of taxol, include total synthesis [9], relay synthesis [10], cultivation of Taxus plants [11], biosynthesis and cell culture production [12,13]. Most of the Taxus species has been chemically investigated, however, the Mexican yew, T. globosa, is perhaps the less studied [14]. From the bark of this plant we have isolated β-sitosterol, β-sitosteryl-β-D-glucoside, vanillic acid and 5α, 7β, 9α, 10β, 13α-pentaacetoxy-4(20),11-taxadiene (7β-acetoxy-taxusin, 2).

Results and Discussion

The geographical distribution of T. globosa is quite sporadic and ranges from the Mexican states of Nuevo León, Tamaulipas, San Luis Potosí, Veracruz, Querétaro, Hidalgo, Oaxaca, and Chiapas to Guatemala, Honduras y El Salvador. It is a small tree up to 15-20 m and its populations are relatively scarce and located in difficult terrains. It is commonly known as granadillo, palmira, romerillo and tlatscal, and it is used locally for fence posts, as ornamental, and for tanning. The continuous harvesting of the trunks represents a threat for the populations [15,16].

The methanolic extract of the bark of the Mexican yew (collected in the State of Chiapas) was partitioned between dichloromethane and water. The dichloromethane fraction was subjected to a silica gel column followed by subsequent chromatographies of selected fractions. This procedure allowed to isolate a white solid, mp 205-207 °C, whose molecular formula C₃₀H₄₂O₁₀ was determined by HRMS. IR absorptions at 1735 and 1247 cm⁻¹ suggested the presence of ester groups. The fragment peaks at m/z 502 [M⁺ - 60], 442 [M⁺ - 120], 382 [M⁺ - 180], 322 [M⁺ - 240] and 262 [M⁺ - 300] in the EIMS, as well as the five methyl singlets at δ_H 2.19, 2.09, 2.08. 2.03 and 1.98 in the ¹H NMR spectrum, established that the structure included five acetates. Additional methyl groups were observed at δ_H 2.20, 2.02, 1.17 and 0.85 (δ_C 14.94, 20.80, 31.16 and 13.29) and the hydrogens of an exomethylene were observed at δ_H 5.28 and 4.95 (δ_C 146.78 and 115.66). ¹H - ¹H COSY spectrum of 2 established connectivities of C(9) to C(10), C(13) to C(14), C(14) to C(1), C(1) to C(2) and C(2) to C(3). HMBC correlations [17] of H(10) with C(11), C(12) and C(15) located the tetrasubstituted double bond at C(11)-C(12) and the C(11)-C(15)-C(1) connectivity, characteristic for the taxane skeleton. This last connectivity was confirmed by the HMBC correlations of H(13) with C(11), C(12) and C(14). The cyclohexane ring was evident from the observed cross-peaks of H(7) with C(6), C(8) and C(19), and from the cross-peaks of the H-20 hydrogens with C(3) and C(5) in the HMBC spectrum. Therefore, the structure corresponded to a taxane with the acetoxy groups attached at C(5), C(7), C(9), C(10) and C(13). The stereochemistry for the tetrasubstituted olefin and the configurations at C(1), C(3), C(8) were defined by the taxane skeleton [7], and the orientation of the acetoxy groups were established by the observed NOESY interactions and comparison with the data informed in the literature. Therefore, the substance isolated from the bark of T. globosa was 5α,7β,9α,10β,13α-pentaacetoxy-4(20), 11-taxadiene (7β-acetoxy-taxusin, 2), previously isolated from T. baccata [18] and T. mairei [19]. β-Sitosterol, β-sitosteryl-β-D-glucopyra-noside and vanillic acid were also isolated from the bark. The chemical and comparative analyses of additional populations of the Mexican yew are currently underway.

Collection and Extraction. The bark of one tree of T. globosa Schlecht. (T. baccata ssp globosa (Schlecht.) Pilger) was collected at Jocosic, State of Chiapas, México, in July 1995, by. J. C., and plant samples were deposited at the National Herbarium, Instituto de Biología de la UNAM (voucher MEXU 669748; JC 726). The identity of the plant material was verified by comparison with authentic material. The air-dried bark (2.2 Kg) was powdered and extracted three times with ethanol. The combined extracts were concentrated at reduced pressure to obtain a dark brown gum. This residue was partitioned three times between dichloromethane and water, and the aqueous fractions were further extracted with dichloromethane. The combined organic fractions were dried (Na₂SO₄) and concentrated, to yield 22 g residue.

Isolation and Characterization. Part of the dichloromethane extract (20 g) was separated over Si-gel by vacuum liquid chromatography (vlc) [20] packed with 60 g of silica (mesh 230-400) using a gradient of n-hexane-EtOAc, and collecting a total of 102 fractions (400 ml each one). Eluates were pooled based on TLC profiles, resulting in a total of 24 sub-fractions, designated A-W. Subfraction D (2 g, eluted with n-hexane-EtOAc 49:1) was separated over Si-gel column using an open column chromatography to afford 390 mg of β-sitosterol, identified by direct comparison with an authentic sample. Fraction K (1 g, eluted with n-hexane-EtOAc 4:1) was purified over Si gel using vacuum liquid chromatography (n-hexane-EtOAc gradient) to obtain a fraction (622 mg) which was rechromatographed by column chromatography using n-hexane-acetone as gradient elution system. This procedure yielded a residue which was further purified by Si gel TLC plates (2 mm, using chloroform-acetone 20:1 as elution system, one development) to afford a total of 40 mg of 2 after several runs (1.8 × 10-3 % yield). Subfraction L (267 mg) was rechromatographed using vacuum liquid chromatography (n-hexane-EtOAc gradient) to obtain a residue which was further purified by prep Si gel TLC (2 mm, using dichloromethane-acetone-acetic acid 9:1:0.1 as eluent, two developments). Usual procedure allowed to isolate 12 mg of vanillic acid (p-hydroxy-m-methoxy-benzoic acid). Mp: 210-212 °C. From subfraction U (eluted with EtOAc from the original column), was isolated β-sitosteryl-β-D-glucopyranoside (30 mg) by sequential crystallizations, and its identity was confirmed by direct comparison with authentic samples.

5α,7β,9α,10β,13α-pentaacetoxy-4(20),11-taxadiene (7β-acetoxy-taxusin, 2). Mp: 205-207 °C (lit: 205-207 °C [18, 19]; IR (CHCl₃) ν_max 3691, 3604, 2956, 1735, 1247 cm⁻¹; 1H NMR (CDCl₃, 500 MHz, assignments by 1H - 1H COSY and HMQC experiments) δ_H 1.88 (1H, m, H-1), 1.72 and 1.86 (1H each, m, H-2a, H-2b), 2.91 (1H, br d, J = 5.1 Hz), 5.39 (1H, t, J = 2.5 Hz), 1.91 and 1.78 (1H each, m, H-6a, H-6b), 5.55 (1H, dd, J = 5, 10.7, H-7), 5.91 (1H, d, J = 10.5, H-9), 6.25 (1H, d, J = 10.5, H-10), 5.94 (1H, m, H-13), 2.66 and 1.03 (1H each, m, H-14a, H-14b), 1.63 (3H, s, H-16), 1.17 (3H, s, H-17), 2.20 (3H, br s, w_1/2 1.0 Hz, H-18), 0.84 (3H, s, H-19), 5.95, 5.28 (1H each, d, J = 1.2, H-20a, H-20b). 2.18, 2.07, 2.06, 2.02, 1.98 (3H each, 5 acetates); 13C NMR (CDCl₃, 125 MHz, assignments by APT and HMQC) δ_C 4.40 (C-1), 27.55 (C-2), 37.60 (C-3), 146.78 (C-4), 74.77 (C-5), 34.13 (C-6), 70.11 (C-7), 46.18 (C-8), 76.84 (C-9), 71.67 (C-10), 134.54 (C-11), 137.40 (C-12), 71.76 (C-13), 31.41 (C-14), 39.45 (C-15), 27.36 (C-16), 31.16 (C-17), 14.94 (C-18), 13.29 (C-19), 115.66 (C-20), 169.82 (OAc at C-5), 169.80 (OAc at C-7), 170.41 (OAc at C-9), 169.2 (OAc at C-10), 170.25 (OAc at C-13), 21.73, 21.58, 21.37, 20.95 and 20.80 (methyls of the acetate groups); HRMS: M+ 562.6416, Calcd for C₃₀H₄₂O₁₀: 562.6558; EIMS m/z (rel. int.): 562 [M]+ (3), 502 (30), 460 (14), 442 (10), 400 (24), 382 (10), 340 (32), 322 (15), 280 (49), 262 (14), 149 (28), 119 (31), 43 (100).

Acknowledgements

The authors thank M. Sc. María Isabel Chávez, Beatriz Quiróz, Rocío Patiño, Luis Velasco, and Francisco Javier Pérez-Flores for technical assistance, and Dr. Héctor G. Barrios for an authentic sample of taxol and field observations of the Mexican yew. Partial financial support was provided by DGAPA-UNAM (IN-212195).

References and Notes

1. (a) Appendino, G. Fitoterapia 1993, 64, 1-25;         [ Links ] (b) Appendino, G. Nat. Prod. Rep. 1995, 12, 349-360.         [ Links ]

2. The name Taxol® is the name registered by Bristol-Myers Squibb Pharmaceuticals for the drug, and it is also the original name used by the discoverers of this substance. Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. Soc. 1971, 93, 2325-2327.         [ Links ] Because it is a tradename, the generic name paclitaxel is also assigned to structure 1.

3. Kingston, D. G. I. J. Nat. Prod. 2000, 63, 726-734.         [ Links ]

4. Lucas, H. Arch. Pharm. (Weinheim, Ger.) 1856, 85, 145-149.         [ Links ]

5. Taxol stabilizes peptidic cell structures named microtubules, altering their dynamic properties, and causing that the cells can no longer divide. Horwitz, S. B.; Fant, J.; Schiff, P. B. Nature 1979, 227, 665-667.         [ Links ]

6. Rowinsky, E. K.; Cazenave, L. A.; Donehower, R. C. J. Natl. Cancer Inst. 1990, 82, 1247-1259.         [ Links ]

7. (a) Baloglu, E.; Kingston, D. G. I. J. Nat. Prod. 1999, 62, 1448-1472;         [ Links ] (b) Kingston, D. G. I.; Molinero, A. A.; Rimoldi, J. M. In Progress in the Chemistry of Organic Natural Products; Herz, W.; Kirby, G. W.; Moore, R. E.; Steglich, W.; Tamm, C. Eds.; Springer-Verlag:New York, 1993, 61, 1-206.         [ Links ]

8. Nicolaou, K. C.; Dai, W.-M.; Guy, R. K. Angew. Chem. Int. Ed. Eng. 1994, 33, 15-44.         [ Links ]

9. Many research groups participated in the synthesis of taxol analogues, but two groups announced practically at the same time the first total synthesis of taxol (1): Holton, R. A.; Somoza, C.; Kim, H.-B.; Liang, F.; Biediger, R. J.; Boatman, P. D.; Shindo, M.; Smith, C. C.; Kim, S.; Nadizadeh, H.; Suzuki, Y.; Tao, C.; Vu, P.; Tang, S.; Zhang, P.; Murthi, K. K.; Gentile, N. N.; Liu, J. H. J. Am. Chem. Soc. 1994, 116, 1597-1599; 1599-1602.         [ Links ] Nicolaou, K. C.; Yang, Z.; Liu, J. J.; Ueno, H.; Nantermet, P. G.; Guy, R. K.; Claiborne, C. F.; Renaud, J.; Couladouros, E. A.; Paulvannan, K.; Sorensen, E. J. Nature 1994, 367, 630-634.         [ Links ]

10. Denis, J. N.; Greene, A. E.; Guénard, D.; Guéritte-Voegelein, F.; Mangatal, L.; Potier, P. J. Am. Chem. Soc. 1988, 110, 5917-5919.         [ Links ]

11. Bormann, S. Chem. Eng. News 1992, 70, 30-32.         [ Links ]

12. Eisenreich, W.; Menhard, B.; Hyland, P. J.; Zenk, M. H. Proc. Natl. Acad. Sci. USA 1996, 93, 6431-6436.         [ Links ]

13. Fett-Neto, A. G.; Melanson, S. J.; Sakata, K.; DiCosmo, F. Bio/Technology 1993, 11, 731-734.         [ Links ]

14. Preliminary studies on this species have been carried out by Robert Nicholson, Smith College, Northampton, Massachussets, USA (personal communication).

15. The information on the geographical distribution of the Mexican yew was obtained from the samples registered at the National Herbarium, Instituto de Biología de la UNAM (MEXU), from field observations, and from personal communication with Dr. Héctor G. Barrios (Instituto de Química de la UNAM).

16. Zamudio, S. Flora del Bajío y de Regiones Adyacentes. Familia Taxaceae. Fasc. 9. Rzedowsky, J.; Calderón de Rzedowski, G., Eds. Instituto de Ecología, A. C. México, 1992.         [ Links ]

17. (a) Bax, A.; Subramanian, S. J. Am. Chem. Soc. 1986, 108, 2093-2094;         [ Links ] (b) Bax, A.; Subramanian, S. J. Mag. Res. 1986, 67, 565-569.         [ Links ]

18. Della Casa de Marcano, D. P.; Halsall, T. G. Chem. Commun. 1969, 1282-1283.         [ Links ]

19. Yang, S. J.; Fang, J. M.; Cheng, Y. S. Phytochemistry 1996, 43, 839-842.         [ Links ]

20. (a) Pelletier, S.W.; Chokshi, H.P.; Desai, H. K. J. Nat. Prod. 1986, 49, 892-900;         [ Links ] (b) Coll, J. C.; Bowden, B. F. J. Nat. Prod. 1986, 49, 934-936.         [ Links ]