Chemotaxonomy of Gonospermum and related genera

Phytochemistry 71 (2010) 627–634

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Chemotaxonomy of Gonospermum and related genera Jorge Triana a, José Luis Eiroa a, Juan José Ortega a, Francisco León b,d, Ignacio Brouard b, Juan Carlos Hernández b, Francisco Estévez c,d, Jaime Bermejo b,* a

Departamento de Química, Universidad de Las Palmas de Gran Canaria, Campus de Tafira, 35017 Las Palmas de Gran Canaria, Canary Islands, Spain Instituto de Productos Naturales y Agrobiología-C.S.I.C.-Instituto Universitario de Bio-Orgánica ‘‘Antonio González”, Universidad de La Laguna, Av. Astrofísico F. Sánchez 3, 38206 La Laguna, Tenerife, Spain c Departamento de Bioquímica, Facultad de Ciencias de la Salud, Universidad de las Palmas de Gran Canaria, 35016, Las Palmas de Gran Canaria, Spain d Instituto Canario de Investigación del Cáncer (ICIC), plaza Sixto Machado 3, 38009, Tenerife, Spain b

a r t i c l e

i n f o

Article history: Received 27 October 2009 Received in revised form 17 December 2009 Available online 22 January 2010 Keywords: Gonospermum fruticosum Gonosperminae Asteraceae Chemotaxonomy Sesquiterpene lactones Sesquiterpene alcohols

a b s t r a c t Aerial parts of Gonospermum fruticosum collected at several locations in the Canary Islands afforded, in addition to known compounds, four sesquiterpene alcohols related to costol and a sesquiterpene lactone, whose structures were established on the basis of their spectroscopic data and chemical transformations. Except for Gonospermum species collected on the island of Tenerife, those collected on the island of El Hierro and, in a previous study those from La Gomera, contain sesquiterpene lactones that can be used as chemotaxonomic markers confirming the inclusion of Gonospermum, Lugoa, and species of Tanacetum endemic to the Canary Islands in a genus that does not support the monophyly of Gonosperminae. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Gonosperminae is one of the 12 presently recognized subtribes of Anthemideae (Asteraceae) and provides one of the putative examples of discontinuous distribution between the Canary Islands and South Africa. As occurred with other members of the Anthemideae tribe, the taxonomic classification of these species has been controversial since the established morphological differences are of little taxonomic value. The Gonosperminae (3 genera, 15 species), comprising the three genera Gonospermum Less (Canary Islands), Lugoa (Canary Islands), and Inulanthera (South Africa), were considered a monophyletic group (Bremer and Humphries, 1993). Inulanthera was initially included in the South African genus Athanasia and subsequently assigned genus level based on chemical and morphological evidence, allowing a connection to be established between the Canarian and South African genera (Källersjö, 1985). However, the phylogenetic analysis of ITS (Internal Transcribed Spacer) sequences reveals that the Canarian genera are not connected to Inulanthera and does not support Gonosperminae monophyly. Rather, the Canarian Gonosperminae seem to be more closely related to the Tanacetum species endemic to the islands than to Inulanthera (Francisco-Ortega et al., 2001), and it has also been recommended * Corresponding author. Tel.: +34 922 318583; fax: +34 922 318571. E-mail address: [email protected] (J. Bermejo). 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.12.013

that Inulanthera be assigned to subtribe Ursiniinae (Oberprieler et al., 2007). The geographical distribution in the Canary Islands of the several Gonospermum and Lugoa species is quite different. Whereas Gonospermum fruticosum (Link) Less is widely distributed and is found on three of the islands (Tenerife, La Gomera and El Hierro), Gonospermum gomerae Bolle is restricted to La Gomera, Gonospermum canariense (DC.) Less to La Palma, Gonospermum elegans (Cass.) Less to El Hierro, and Lugoa revoluta (Link) DC. to Tenerife, while the three endemic Tanacetum species of Gran Canaria are located in very specific areas of Gran Canaria (Bramwell and Bramwell, 2001). Based on cytogenetic studies of the nine endemic taxa of Gonospermum Less, Lugoa DC and Tanacetum L. in the Canary Islands it has recently been proposed that all three taxa be included in the endemic genus Gonospermum (Febles, 2008). Previous chemical study of the Canarian endemics Tanacetum ferulaceum (González et al., 1990), Tanacetum ptarmiciflorum (González et al., 1992a), as well as G. canariense (Triana et al., 2000), G. elegans (Triana et al., 2003), G. gomerae and G. fruticosum collected in La Gomera (Triana et al., 2008) and L. revoluta (Triana et al., 2001) showed a high content of similar sesquiterpene lactones (Table 1). However, a chemical study of G. fruticosum collected on Tenerife (González et al., 1992b) afforded sesquiterpene alcohols rather than lactones and thus, in view of the wide distribution of this species, a new study was undertaken using samples from

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Table 1 Distribution of sesquiterpenoids isolated from Gonospermum, Lugoa and Tanacetum from de Canary Islands. No

Name

Sources

1

5a-Hydroxy-b-costol

2 3 4 5

3a-Hydroxyeudesm-4(15),11-dien-12-al 3a,5b-Dihydroxy-b-costol 3b,5b-Dihydroxy-b-costol Tatridin B-6-O-tiglate

6 7 8

1b,6a-Dihydroxy-eudesm-4(15)-ene 5b-Hydroxycostic acid 3a-Hydroxy-b-costol

9

1b-Hydroxy-b-costol

10

4a-Hydroxy-eudesm-11-en-12-al

11 12 13

4a-Hydroxy-4b-methyldihydrocostol 1b-Hydroxy-b-eudesmol Reynosin

14 15 16 17 18 19

Dentatin A Desacetyl-b-cyclotulipinolide Arbusculin A Arbusculin A 8a-hydroxy Eleganolactone A Desacetyl-b-cyclopyrethrosin

20 21 22 23 24 25 26 27 28 29 30 31

Desacetyl-b-cyclopyrethrosin 6-O-angelate Desacetyl-b-cyclopyrethrosin 6-O-tiglate b-Cyclopyrethrosin 6a-Hydroxy-5,7aH,8bH-eudesm-4(15)-en-8,12-olide Tanapsin Desacetyltanapsin 6-O-tiglate 1b,4a,6a-Trihydroxy-eudesm-11-en 8a,12-olide 4b,6a-Dihydroxy-5,7aH,8bH-eudesman-8,12-olide 4b,6a-Dihydroxy-5,7aH,8bH-11,13- dihydroeudesman-8,12-olide 6a-Hydroxy-5,7aH,8bH-eudesm-4(15)-en-8,12-olide 6a-Hydroxy-5,7aH,8bH-11,13- dihydroeudesm-4(15)-en-8,12-olide Sivasinolide

32

Tatridin A

33

Tatridin B

34

1-epi-Tatridin B

35 36 37 38

Tatridin B 6-O-angelate Tatridin B 6-O-methacrylate 1-epi-Tatridin B 6-O-methacrylate Tamirin

39 40

Tamirin 6-O-angelate Tatridin A 4a,5b-epoxy

41

Spiciformin

G. fruticosum (Anaga-Tenerife) G. fruticosum (Güimar-Tenerife) G. fruticosum (Güimar-Tenerife) G. fruticosum (Güimar-Tenerife) G. fruticosum (Güimar-Tenerife) G. fruticosum (Güimar-Tenerife) G. fruticosum (El Hierro) G. canariense G. gomerae G. fruticosum (Anaga-Tenerife) G. fruticosum (Güimar-Tenerife) G. canariense G. elegans G. fruticosum (Anaga-Tenerife) G. canariense G. fruticosum (Anaga-Tenerife) G. canariense G. fruticosum (Güimar-Tenerife) T. ptarmiciflorum G. fruticosum (Anaga-Tenerife) G. canariense G. elegans G. gomerae T. ferulaceum T. feruleceum T. ferulaceum G. elegans G. fruticosum (El Hierro) G. fruticosum (La Gomera) G. canariense G. elegans G. gomerae T. ferulaceum G. gomerae G. fruticosum (El Hierro) G. gomerae T. ferulaceum L. revoluta G. canariense G. gomerae T. ferulaceum T. ferulaceum T. ferulaceum T. ferulaceum G. canariense G. elegans G. fruticosum (El Hierro) G. fruticosum (La Gomera) G. canariense G. elegans G. gomerae L. revoluta T. ferulaceum T. ptarmiciflorum G. canariense G. elegans G. gomerae L. revoluta T. ferulaceum T. ptarmiciflorum G. canariense G. elegans L. revoluta L. revoluta L. revoluta G. fruticosum (La Gomera) G. gomerae L. revoluta G. gomerae G. fruticosum (La Gomera) G. canariense G. elegans G. gomerae L. revoluta G. canariense G .elegans

References

González et al. (1989) Mahmoud (1997) González et al. (1992a)

González et al. (1992a) González et al. (1992b) González et al. (1992a) Adinarayana and Syamansuudar (1982) Sanz and Marco (1991)

Bohlmann et al. (1983b) González et al. (1992b) Irwin and Geissman (1969) González et al. (1992b) Triana et al. (2003) Konstantinopoulou et al. (2003)

Yunusov et al. (1976) Triana et al. (2008) Doskotch and El-Feraly (1969) González et al. (1990) Bohlmann et al. (1983b) Bohlmann et al. (1984) Gören et al. (1993) González et al. (1990) González et al. (1992b) González et al. (1990) González et al. (1992b) Gören et al. (1992) Sashida et al. (1983)

Sashida et al. (1983)

Sanz and Marco (1991) Bohlmann et al. (1983b) Bohlmann et al. (1983b) Bohlmann et al. (1983b) Gören et al. (1996)

Triana et al. (2008) Rustaiyan et al. (1990)

González et al. (1992b)

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Name

Sources

42

4a,5b-Epoxy desacetyl laurenobiolide

43

1a,10b-Epoxy desacetyl laurenobiolide

44 45 46 47 48 49 50 51 52

11,13-Dihydrotatridin A 11,13-Dihydrotatridin B Costunolide Artemorin 1a-Hydroxy-1-deoxotamirin-4a,5b-epoxide Eleganolactone B Elegain Eleganodiol 1a,5b,8a-Trihydroxygermacra-4,9(7),11(8)-trien-6a,12-olide

different locations. In addition the cytotoxic activity of some of the isolated sesquiterpenes was assessed against three human cancer cell lines. 2. Results and discussion 2.1. Chemical results Purification by column chromatography of the ethanol extract of aerial parts of G. fruticosum collected in Anaga (Tenerife) at the same location as previously (González et al., 1992), afforded in addition to the coumarin scoparone the sesquiterpene alcohols 1b-hydroxycostol and 3a-hydroxycostol previously isolated from this plant, the new sesquiterpene alcohol 1 as well as five known compounds 6-methoxy-7,8-methylenedioxycoumarin (Geissman et al., 1971), 5,7,40 -trihydroxy-3,6-dimethoxyflavone (Van Hereden et al., 2000), sesamin (Kuropka and Glombitza, 1987) 4a-hydroxyeudesm-11-en-12-al (Bohlmann et al., 1983a) and the sesquiterpene lactone reynosin (Sanz and Marco, 1991). From the ethanolic extract of G. fruticosum collected in Güímar (Tenerife) the new sesquiterpene alcohols 1–4 (Fig. 1) and the known compounds sesamin (Kuropka and Glombitza, 1987), stigmasterol (Wilkomirski and Kucharska, 1992), ilicol (Guerreiro et al., 1979) and 3a-hydroxycostol (González et al., 1992b) were obtained. Additionally, from G. fruticosum collected in the island of El Hierro, the new sesquiterpene lactone 5, the known vanillin (Youssef and Frahm, 1995), scoparone (Herz et al., 1970), 5,7,40 -trihydroxy-3,6-dimethoxyflavone (Van Hereden et al., 2000), stigmasterol (Wilkomirski and Kucharska, 1992) and the sesquiterpene lactones tatridin A (Sashida et al., 1983), desacetylb-cyclopyrethrosin (Konstantinopoulou et al., 2003) and 6a-tig(Triana loyloxy-1b-hydroxy-4(15),11-eudesmadien-8a,12-olide et al., 2008) were isolated.

G. gomerae T. ferulaceum T. ptarmiciflorum G. canariense G. elegans G. gomerae L. revoluta T. ferulaceum G. elegans T. ferulaceum G. elegans G. elegans G. elegans G. elegans G. elegans G. gomerae

References

Bohlmann et al. (1982) Bohlmann et al. (1982) Sashida et al. (1983) Sashida et al. (1983) Doskotch and El-Feraly (1970) Geissman (1970) Rustaiyan et al. (1990) Triana et al. (2003) Triana et al. (2003) Triana et al. (2003) Triana et al. (2008)

Compound 1, a colorless oil, had the molecular formula of C15H24O2 [M]+ (236.1784) assigned by HREIMS. The IR spectrum showed absorption bands at 3417, 1653 and 901 cm1 assigned to hydroxyl and olefinic functions. The 1H NMR spectrum of 1 (Table 2) showed the presence of one methyl group at dH 0.89 (3H, s, CH3-14), four exomethylene protons at dH 4.68 (1H, s, H-15a), 4.82 (1H, d, J = 1.5 Hz, H-15b), 4.96 (1H, d, J = 2.5 Hz, H-13a) and 5.08 (1H, s, H-13b) and a hydroxymethyl group at dH 4.16 (2H, s, H12). The 13C NMR spectral data of 1 revealed 15 carbon signals that were resolved by DEPT experiments into one methyl at dC 19.9, nine methylenes at dC 22.2, 26.6, 29.7, 33.0, 34.4, 35.9, 65.3 and 107.7, 108.2 (two terminal double-bond CH2 carbons), one methine at dC 34.9 and four quaternary carbons at dC 37.9, 77.0, 151.8 and 154.0. The 1H NMR data were similar to those of 5a-hydroxycostic acid (Ahmed and Jakupovic, 1990), with the carboxyl on C-12, being replaced by a primary alcohol maintaining an a-orientation of the C-5 hydroxyl group. Thus 1 was identified as 5a-hydroxyb-costol. The 1H NMR spectrum of 2 (Table 2) was similar to that of 3ahydroxy-b-costol (González et al., 1992b) with the primary hydroxyl on C-12 being replaced by an aldehyde at dH 9.54 (1H, s, H-12). Hence, new compound 2 was 3a-hydroxyeudesm-4(15),11-dien12-al. Compound 3 was isolated as a colorless oil whose IR absorption band at 3444 cm1 indicated the presence of hydroxyl groups. The molecular formula of 3, C15H24O3, was established by HREIMS (m/z 252.1737, [M]+) and 13C NMR data. The 1H and 13C NMR spectra in CDCl3 showed signals of a eudesmane sesquiterpene skeleton. The 1 H NMR spectrum of 3 was almost identical with that of 3a-hydroxy-b-costol (González et al., 1992b), but exhibited an additional boriented hydroxyl group at C-5 due to the chemical shift observed for the signals at dH 4.91 (1H, d, J = 1.0 Hz, H-15a) and 5.07 (1H, s, H-15b). Therefore, 3 was identified as 3a,5b-dihydroxy-b-costol.

Fig. 1. Sesquiterpenoids isolated from G. fruticosum.

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Compound 3 was acetylated by treatment with Ac2O and pyridine to give a diacetate 3a isolated as a colorless oil whose NMR data (see Section 3) confirmed the structure proposed for 3. That 4 was a C-3 epimer of 3 was shown by comparison of its spectroscopic data with those of the acetylated derivatives 4a and 3a, respectively. Thus, its 1H and 13C NMR spectroscopic data (see Section 3) confirmed the presence of a b-oriented hydroxyl group at C-3 deduced from the chemical shift and coupling constant of H-3, a multiplet at dH 4.50, which underwent a displacement to d 5.62 (1H, dd, J = 12.1, 5.2 Hz) after acetylation (Zdero et al., 1987). The structure of 4 was therefore confirmed as 3b,5b-dihydroxy-b-costol. The structure of 5 could be easily deduced by comparing its 1H NMR data (Table 2) with those of tatridin B (Sashida et al., 1983). It exhibited four exomethylene protons at dH 5.13 (1H, s, H-14a), 5.17 (1H, s, H-14b), 5.84 (1H, d, J = 2.8 Hz, H-13a) and 6.32 (1H, d, J = 2.8 Hz, H-13b), two olefinic protons at dH 4.97 (1H, m, H-5) and 6.90 (1H, m, tiglate) and a singlet at dH 1.81 (3H, s, CH3-15). The IR spectrum exhibited two absorption bands at 1732 and 1651 cm1 associated with a c-lactone group and a double bond, and in the 1H NMR spectrum two olefinic protons at dH 4.97 (1H, m, H-5) and 6.90 (1H, m, tiglate) and a singlet at dH 1.81 (3H, s, CH3-15). Therefore, 5 was identified as tatridin B-6-O-tiglate (Fig. 1). In summary, the germacrane and eudesmane sesquiterpene lactones isolated from species of the genera Gonospermum and Lugoa, as well as from the endemic Canarian species of Tanacetum (Table 1), are very similar, and are highly oxidized eudesmanolide or germacranolide lactones related to cyclopyrethrosin and tatridin-A and B (Figs. 2 and 3). The presence of these types of lactones supports the proposal, based on cytogenetic studies, to include the afore-mentioned taxa in a single genus endemic to the Canaries (Febles, 2008), which is more closely related to representatives of the Eurasian genus Tanacetum L than to Inulanthera (Oberprieler et al., 2007), in view of the presence in Inulanthera of guaianolide-type sesquiterpene lactones (Bohlmann and Knoll, 1979; Gafner et al., 1998). From the chemical point of view, G. fruticosum is an exception because its collections from the island of Tenerife, yields only sesquiterpene alcohols, whereas collections species from other islands of the archipelago yield lactones (Table 1). This is highly significant when one evaluates the relative importance of inter-island coloni-

zation and insular radiation in the evolution of species endemic to the Canary Islands. 2.2. Biological results Three human tumor cell lines, the hematopoietic cell lines HL60 and U937 and the melanoma SK-MEL-1 cell line, were tested for their sensitivity to sesquiterpene 3. Growth inhibition of human tumor cells in culture was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dyereduction assay (Triana et al., 2008). Etoposide, a chemotherapy drug that is given as a treatment for some types of cancer, was used as a positive control in the cytotoxicity studies. Our results show that the three human tumor cell lines are resistant to this sesquiterpene. The IC50 value in HL-60 cells was 88.9 ± 7.9 lM and > 100 lM in U937 cells and SK-MEL-1 cells. The data shown represent the mean ± SEM of 2–3 independent experiments with three determinations in each. 3. Experimental 3.1. General experimental techniques Optical rotations were recorded in a Perkin–Elmer model 343 polarimeter. IR spectra were recorded using a Bruker model IFS55 spectrophotometer. 1H and 13C NMR spectra were obtained on a Bruker model AMX-400 and AMX-500 spectrometer with standard pulse sequences operating at 400 and 500 MHz in 1H and 100 and 125 MHz in 13C NMR. CDCl3 was used as solvent. EIMS were taken on a Micromass model Autospec (70 eV) spectrometer. Column chromatography (CC) was carried out on silica gel 60 (Merck 230–400 mesh), and prep. TLC on silica gel 60 PF254+366 plates (20  20 cm, 1 mm thickness). 3.2. Plant material Aerial parts of Gonospermum were collected by Prof. José L. Eiroa in the locality and on the dates cited, and were identified by Dr. Rosa Febles, Viera y Clavijo Botanical Garden, Gran Canaria, in whose herbarium the respective voucher specimens have been deposited.

Table 2 1 H NMR data of compounds 1, 2, 3, 4 and 5 (300 MHz in CDCl3).

1a 1b 2a 2b 3a 3b 5 6 7 8a 8b 9a 9b 12 13a 13b 14a 14b 15a 15b OTigl

1

2

3

4

1.47–1.85 m

1.30–1.80 m

1.47–1.85 m

1.30–1.80 m

2.60 m 2.20 m – 1.47–1.85 m 2.15 m 1.47–1.85 m

– 4.30 brs 1.80 brs 1.30–1.80 m 2.30 m 1.30–1.80 m

1.08 2.10 2.04 1.90 4.50

1.47–1.85 m

1.30–1.80 m

4.16 4.96 5.08 0.89

9.54 5.99 6.29 0.74

1.02 d (13.2) 2.03 dd (4.4, 13.7) 1.85 m 1.92 dd (4.6, 18.7) – 4.37 t (2.8) – 1.70–1.80 m 2.58 tt (3.6, 11.8) 1.61 m 1.54 ddd (3.5, 12.0, 16.4) 1.20 dt (3.7, 6.7) 1.80 m 4.14 d (6.7) 4.93 s 5.06 d (1.1) 0.86 s 4.91 d (1.0) 5.07 s –

5.23 s 5.44 s –

s d (2.5) s s

4.68 s 4.82 d (1.5) –

s s s s

4.54 s 4.94 s –

5 d (16.4) m m m m

3.87 m – 2.00–2.30 m 2.00–2.30 m

– 1.70 m 2.46 m 1.70 m

4.97 5.30 3.00 4.08

1.15 1.70 4.19 4.98 5.08 1.04

3.00 d (13.4) 2.38 d (13.4) – 5.84 d (2.8) 6.32 d (3.0) 5.13 s 5.17 s 1.81 s – 6.90 m 1.87 s 1.83 s

m m s s s s

m m m m

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OH

R1 COOH

CH2OH

R2

OH OH 6

8, R1 = H; R2 = OH 9, R1 = OH; R2 = H

7

R1

OH

R2

R HO

O

OH

O 10, R = CHO 11, R = CH2OH

13, R1 = OH; R2 = H 14, R1 = OH; R2 = OH 15, R1 = H; R2 = OH

12

R1

R2

OH O

O

O HO

18, 19, 20, 21, 22, 23,

16, R = H 17, R = OH

R1 = OH; R2 = H R1 = OH; R2 = H R1 = OH; R2 = Ang R1 = OH; R2 = Tigl R1 = OH; R2 = Ac R2 = H R1 = H;

O

27

O O

HO

OR

24, R = Ang 25, R = Tigl 26, R = H

O O

OH

HO

OR2

O

HO

O

O

OH 28

O O

OH 29

O OH 30

Fig. 2. Eudesmane sesquiterpenoids isolated from Gonospermum, Lugoa and Tanacetum from Canary Islands.

3.3. Extraction and isolation Aerial parts of Gonospermum were exhaustively extracted with 95% EtOH in a Soxhlet apparatus for 72 h. The solvents were concentrated under reduced pressure, and the extracts were subsequently fractioned by silica gel column chromatography using hexane and EtOAc mixtures of increasing polarity. The extraction of 1500 g of G. fruticosum collected at Taganana (Tenerife) in May 2003 (Voucher 20914) gave 380 g of a viscous mass which was subjected to column chromatography on silica gel affording four fractions. Fraction 1 (hexane–EtOAc (7:3)) was submitted to preparative TLC using benzene–EtOAc (19:1) to give sesamin (10 mg). Fraction 2 (hexane–EtOAc (3:2)) was eluted with benzene–EtOAc (19:1) on silica gel CC and furnished 6-methoxy7,8-methylendioxycoumarin (18 mg). Purification of fraction 3 (hexane–EtOAc (1:1)) on CC using hexane–EtOAc (4:1) followed by preparative TLC (benzene–EtOAc (9:1)) yielded 4a-hydroxyeudesm-11-en-12-al (8 mg) and 5a-hydroxy-b-costol (1) (7 mg). Fraction 4 (hexane–EtOAc (2:3)) gave, by preparative TLC (benzene–EtOAc (4:1)), 1b-hydroxy-b-costol (25 mg), reynosin (35 mg), 3a-hydroxy-b-costol (82 mg) and a non-separable mixture which was submitted again to preparative TLC using benzene/EtOAc

(7:3) to afford 5,7,40 -trihydroxy-3,6-dimethoxyflavone (12 mg) and scoparone (15 mg). 3.3.1. 5a-Hydroxy-b-costol (1) Colorless oil; ½a25 D  + 46.67° (c 0.11, CHCl3); IR (CHCl3) mmax: 3417, 2925, 1653, 1456, 1375, 1032, 901 cm1; HRESIMS m/z 236.1784 [M]+ (calc. for C15H24O2, 236.17776). EIMS m/z (%): 236 [M]+ (17.0), 218 (29.0), 203 (32.1), 200 (37.4), 187 (29.7), 185 (29.9), 163 (16.4), 161 (29.1), 147 (38.7), 145 (44.2), 133 (49.6), 107 (62.4), 105 (72.9), 95 (100), 93 (89.5), 91 (78.5), 81 (90.7), 79 (78.4), 67 (64.3), 55 (64.0); for 1H NMR see Table 2. 13C NMR (CDCl3) d: 29.7 (C-1), 22.2 (C-2), 34.4 (C-3), 151.8 (C-4), 77.0 (C5), 35.9 (C-6), 34.9 (C-7), 26.6 (C-8), 33.0 (C-9), 37.9 (C-10), 154.0 (C-11), 65.3 (C-12), 108.2 (C-13), 19.9 (C-14), 107.7 (C-15). The extraction of 264 g of G. fruticosum collected at the Mirador de San Martín (Güímar, Tenerife) in April 2003 (Voucher 16253) gave 23 g of a viscous mass which was subjected to CC on silica gel to afford five fractions by elution with hexane–EtOAc in increasing polarity. Fraction 1 gave stigmasterol (29 mg) and ilicol (33 mg) by CC on silica gel using hexane–EtOAc (9:1). Fraction 2, by elution with hexane–EtOAc (4:1), yielded 3a-hydroxyeudesm4(15),11-dien-12-al (2) (1.4 mg). Fraction 3 was submitted to CC

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R1O

HO

O O

O

O

OR2

OH 33,R 1 = 34,R 1 = 35,R 1 = 36,R 1 = 37,R 1 =

32

HO

OR

OH;R2 =H OH;R2 =H OH;R2 =Ang OH;R2 =MeAcr OH;R2 =MeAcr

38,R=H 39,R=Ang

O

O

O O OH 40

HO

O

O

O

O O

O

O

O

O

O

OH

O OH

OH

41

42

43 HO

HO O

O O

O O

OH

OH

44

45

HO

O

O 46

O

47

HO O

O

O O

O O

OH 48

O O

OH OH

49

O

50

HO

OH

HO

O

OH OH OH 51

OH O O 52

Fig. 3. Germacrane sesquiterpenoids isolated from Gonospermum, Lugoa and Tanacetum from Canary Islands.

with hexane–EtOAc (7:3) to give 3a-hydroxy-b-costol (32 mg). Fraction 4, subjected to CC using hexane–EtOAc (3:2), yielded 1 (44 mg) and sesamine (27 mg) and with hexane–EtOAc (1:1) gave 3a,5b-dihydroxy-b-costol (3) (50 mg). Finally, from the fraction 5, eluted with hexane–EtOAc (1:1), 3b,5b-dihydroxy-b-costol (4) (25 mg) was obtained. 3.3.2. 3a-Hydroxyeudesm-4(15),11-dien-12-al (2) Colorless oil; ½a25 D  + 12.86° (c 0.07, CHCl3); IR (NaCl) mmax: 3628, 2922, 2851, 1698, 1649, 1396, 1104, 1036, 971 cm1; HRESIMS m/z 234.1607 [M]+ (calc. for C15H22O2, 234.1620). EIMS m/z (%): 234 [M]+ (15.1), 216 (33.7), 201 (11.0), 177 (14.4), 175 (14.1), 161 (21.5), 147 (16.2), 145 (20.0), 135 (21.6), 133 (25.0), 119 (31.9), 107 (37.3), 105 (35.3), 99 (30.7), 95 (38.4), 93 (41.8), 91 (100), 79 (45.3), 69 (40.7), 57 (41.2), 55 (55.0); for 1H NMR, see Table 2. 3.3.3. 3a,5b-Dihydroxy-b-costol (3) Colorless oil; ½a25 D  + 16.90° (c 0.29, CHCl3); IR (KBr) mmax: 3444, 2949, 1645, 1392, 1157, 1056 cm1; HRESIMS m/z 252.1737 [M]+

(calc. for C15H24O3, 252.1725). EIMS m/z (%): 252 [M]+ (0.9), 234 [MH2O]+(2.4), 219 (5.8), 216 (17.8), 203 (4.6), 201 (10.6), 188 (10.0), 173 (14.9), 161 (5.5), 159 (15.9), 145 (15.4), 133 (21.2), 119 (22.5), 107 (31.2), 95 (100), 93 (40.2), 81 (22.3), 79 (30.2), 67 (18.1), 57 (5.6), 55 (21.8); for 1H NMR see Table 2. 13C NMR (CDCl3) d: 30.0 (C-1), 29.5 (C-2), 75.1 (C-3), 149.5 (C-4), 76.7 (C-5), 35.3 (C6), 34.6 (C-7), 26.4 (C-8), 33.8 (C-9), 38.2 (C-10), 153.8 (C-11), 65.4 (C-12), 108.3 (C-13), 19.8 (C-14), 112.3 (C-15). 3.3.4. Acetylation of compound 3 A solution of 3 (21 mg) in a mixture of acetic anhydride (2 ml) and pyridine (1 ml) was allowed to stand at room temp. overnight. The reaction mixture was treated in the usual way, and the product was purified by silica gel CC with hexane–EtOAc (8:2) to afford the diacetate 3a (12 mg). Colorless oil; IR (KBr) mmax: 3485, 3086, 2931, 2862, 1737, 1651, 1373, 1238, 1026, 987 cm1; HRESIMS m/z 294.1832 [M – C2H2O]+ (calc. for C17H26O4, 294.1831). EIMS m/z (%): 276 [M60]+ (10.8), 258 [M60–18]+ (13.2), 216 [M60– 60]+ (50.0), 201 (12.3), 188 (18.6), 173 (19.3), 159 (29.6), 145

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(31.0), 133 (27.0), 119 (34.3), 105 (44.3), 95 (100), 93 (54.8), 91 (39.4), 79 (35.2), 55 (40.0); 1H NMR (300 MHz, CDCl3) d: 0.89 (3H, s, CH3-14), 1.07 (1H, d, J = 13.2 Hz, H-1a), 1.24 (1H, dt, J = 3.7, 6.7 Hz, H-9a), 1.60 (5H, m, 2  H-6, 2  H-8, H-9b), 1.85 (1H, m, H-2a), 1.98 (1H, dd, J = 4.6, 18.7 Hz, H-2b), 2.09 (3H, s, OAc), 2.10 (1H, m, H-1b), 2.11 (3H, s, OAc), 2.68 (1H, tt, J = 3.63, 11.8 Hz, H-7), 4.61 (2H, s, H-12), 5.03 (1H, br s, H-13a), 5.08 (1H, br s, H-13b), 5.11 (1H, br s, H-15a), 5.28 (1H, br s, H-15b), 5.57 (1H, s, H-3). 13C NMR (CDCl3) d: 30.1 (C-1), 29.4 (C-2), 76.4 (C-3), 145.0 (C-4), 77.0 (C-5), 36.4 (C-6), 34.7 (C-7), 26.6 (C-8), 33.6 (C9), 37.8 (C-10), 148.5 (C-11), 65.9 (C-12), 110.8 (C-13), 19.4 (C14), 116.3 (C-15), 21.3 (CH3CO), 170.0 (COCH3). 3.3.5. 3b,5b-Dihydroxy-b-costol (4) Colorless oil. ½a25 D   4.0° (c 0.40, CHCl3); IR (NaCl) mmax: 3485, 2926, 2874, 1699, 1606, 1430, 1280, 1180, 1025, 987 and 914 cm1; HRESIMS m/z 252.1799 [M]+ (calc. for C15H24O3, 252.1725). EIMS m/z (%): 234 [MH2O]+ (18.6), 219 (9.6), 216 [M2H2O]+ (36.9), 203 (13.5), 201 (19.8), 185 (10.4), 175 (13.6), 173 (18.6), 160 (20.9), 159 (30.7), 150 (44.1), 133 (41.4), 125 (99.8), 109 (58.0), 107 (76.8), 95 (82.1), 93 (96.9), 91 (78.8), 79 (100), 67 (73.8); for 1H NMR see Table 2.

633

z 346.1793 [M]+ (calc. for C20H26O5, 346.1780). EIMS m/z (%): 246 [MRCO2H]+ (3.1), 228 [246-H2O]+ (8.7), 213 [228-Me]+ (2.9), 83 (100), 57 (14.6); for 1H NMR see Table 2. 13C NMR (CDCl3) d: 70.6 (C-1), 30.1 (C-2), 33.8 (C-3), 136.3 (C-4), 131.0 (C-5), 71.2 (C-6), 51.9 (C-7), 78.8 (C-8), 41.3 (C-9), 146.6 (C-10), 136.9 (C-11), 170.1 (C-12), 125.1 (C-13), 114.6 (C-14), 17.4 (C-15), 169.7 (OTigl), 135.4 (OTigl), 125.1 (OTigl), 15.0 (OTigl), 13.6 (OTigl).

Acknowledgments This work was supported in part by grants from the Ministerio de Educación y Ciencia of Spain and Fondo Europeo de Desarrollo Regional (SAF2004-07928 and SAF2007-62536). This work was supported in part by grants from the Programa de Iniciativa Comunitaria INTERREG IIIB Azores-Madeira-Canarias (04/MAC/ 3.5/C5), from the Instituto Canario de Investigación del Cáncer (G-05-09 to J.B.). F.L. was supported by JAE-Postdoctoral Program from the Ministerio de Ciencia e Innovación. J.C.H. was supported by a grant from the Government of Canary Islands.

References 3.3.6. Acetylation of compound 4 A solution of 4 (25 mg) in a mixture of acetic anhydride (2 ml) and pyridine (5 ml) was allowed to stand at room temp. overnight. The reaction mixture was treated in the usual way, and the product was purified by silica gel CC with hexane–EtOAc (8:2) to afford the diacetate 4a (10 mg). Colorless oil; IR (KBr) mmax: 3472, 2938, 1741, 1650, 1447, 1377, 1242, 1032, 968, 914, 878, 756 cm1; HRESIMS m/z 336.1928 [M]+ (calc. for C19H28O5, 336.1973). EIMS m/z: (%): 336 [M]+ (6.4), 293 (3.3), 276 (24.5), 258 (14.1), 216 (62.6), 201 (26.0), 188 (19.7), 173 (25.9), 159 (29.2), 145 (33.7), 131 (28.9), 122 (38.3), 107 (35.3), 105 (40.2), 95 (100), 93 (49.6), 91 (41.9), 79 (42.5), 67 (25.6), 55 (27.0). 1H NMR (500 MHz, CDCl3) d: 1.08 (3H, s, CH3-14), 2.09 (3H, s, OAc), 2.14 (3H, s OAc), 2.40 (1H, m, H-7), 4.60 (2H, s, H-12), 5.05 (1H, s, H-13a), 5.10 (1H, s, H-13b), 5.12 (1H, s, H-15a), 5.15 (1H, s, H-15b), 5.62 (1H, dd, J = 5.2, 12.1 Hz, H-3); 13C NMR (CDCl3) d: 29.5 (C-1), 29.4 (C-2), 75.7 (C3), 147.2* (C-4), 76.3 (C-5), 37.8 (C-6), 33.0 (C-7), 27.5 (C-8), 33.2 (C-9), 38.1 (C-10), 147.1* (C-11), 66.2 (C-12), 111.6 (C-13), 20.8 (C-14), 115.9 (C-15), 21.7 (CH3CO), 170.4 (COCH3) (*values may be interchanged). The extraction of 1250 g of the extract of G. fruticosum collected in Camino del Mocanal (El Hierro) in May 2003 (Voucher 16355) gave 287 g of a viscous mass which was chromatographed by CC on silica gel using hexane–EtOAc in increasing polarity and five fractions were obtained. From fraction 1, stigmasterol (47 mg) was isolated by rechromatography on silica gel and subsequent crystallisation in methanol. Fraction 2 was submitted to CC using benzene–EtOAc (19:1) and preparative TLC benzene–EtOAc (17:3) to yield vanillin (35 mg). Fraction 3 was purified by CC benzene–EtOAc (9:1) and preparative TLC (benzene–EtOAc (4:1)) to afford scoparone (11 mg). Fraction 4 was submitted to CC on silica gel using benzene–EtOAc (3:1) to yield 1a-hydroxydeacetyltulirinol 4a,5b-epoxide (3 mg), tatridin B 6-O-tiglate (5) (2 mg) and 5,7,40 -trihydroxy-3,6-dimethoxyflavone (10 mg). Finally, fraction 5 was purified by CC using hexane–EtOAc (3:2) affording tatridin A (12 mg), desacetyl-b-cyclopyrethrosin (20 mg) and a mixture that was submitted to preparative TLC using hexane–EtOAc (3:2) to give 6a-tigloyloxy-1b-hydroxy-4(15),11-eudesmadien-8a,12olide (5.3 mg). 3.3.7. Tatridin B 6-O-tiglate (5) Colorless oil, [a]D + 5.0° (c 0.10, CHCl3) IR(KBr) mmax: 3400, 2924, 1851, 1732, 1651, 1462, 1377, 1246, 1148, 1028 cm1. HRESIMS m/

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