A New Guaianolide and Other Constituents from Achillea Ligustica

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Biochemical Systematics and Ecology 36 (2008) 461e466 www.elsevier.com/locate/biochemsyseco

A new guaianolide and other constituents from Achillea ligustica

Azzedine Boudjerda a, Hanene Zater a, Samir Benayache b, Jean-Claude Chalchat c, Javier Gonzalez-Platas d, Francisco Leo´n e,f, Ignacio Brouard e, Jaime Bermejo e, Fadila Benayache a,*

a Laboratoire de Phytochimie et Analyses Physico-Chimiques et Biologiques, Equipe Associeé a` l’A. N. D. R. S., Universite´ Mentouri, Route de A€ın El Bey, 25 000 Constantine, Algeria b Laboratoire de Valorisation des Ressources Naturelles et Synthe`se de Substances Bioactives, Equipe Associeé a` l’A. N. D. R. S., Universite´ Mentouri, Route de A€ın El Bey, 25 000 Constantine, Algeria c Laboratoire de Chimie des Huiles Essentielles, Universite´ Blaise Pascal de Clermont, Aubie`re, France d Dpto. Fı´sica Fundamental II, Servicio de Difraccioń de Rayos X. e I.U.B.O ‘‘Antonio Gonzalez’’, Universidad de La Laguna, Avda. Astrofı´sico Francisco Sanchez 3, 38206 La Laguna, Tenerife, Spain e Instituto de Productos Naturales y Agrobiologıá, CSIC, Instituto Universitario de Bio-organica ‘‘Antonio Gonzalez’’, Avda. Astrofı´sico Francisco Sanchez 3, 38206 La Laguna, Tenerife, Spain f Instituto Canario de Investigacioń del Cancer (ICIC), Avda. Astrofı´sico Francisco Sanchez 2, 38206 La Laguna, Tenerife, Spain Received 5 August 2007; accepted 16 November 2007

Keywords: Chlorinated guaianolide; Achillea ligustica; Asteraceae; Anthemideae

1. Subject and source

The aerial parts of Achillea species are widely used in folk medicine (Orkiszewska et al., 1985; Simonpoli, 1993). The species of this genus, contain essential oils (Palic´ et al., 2003; Sadyrbekov et al., 2006), ﬂavonoids (Valant- Vetschera, 1985; Krenn et al., 2003) and sesquiterpene lactones (Balboul et al., 1997; Todorova et al., 2007). As part of our ongoing research on the Asteraceae family (Bentame`ne et al., 2005; Dendougui et al., 2006; Zaiter et al., 2007), we report on the chemistry of Achillea ligustica All. collected in June 2004 in Jijel in the eastern Algeria. A voucher specimen (CAL01/06/04) has been deposited in the Herbarium, Biology Department, Mentouri University of Constantine.

2. Previous work

Previous studies on A. ligustica All. collected from different areas of Europe, reported on essential oils (Maffei et al., 1993; Tzakou et al., 1995a; Tuberoso et al., 2005; Filippi et al., 2006), ﬂavonoids (Tzakou et al., 1995b),

* Corresponding author. Tel./fax: þ213 31 81 88 83. E-mail address: [email protected] (F. Benayache).

0305-1978/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.11.006 462 A. Boudjerda et al. / Biochemical Systematics and Ecology 36 (2008) 461e466 sesquiterpene lactones (Bruno and Herz, 1988; Ahmed et al., 2003), piperidine amide (Greger et al., 1984), lignans (Stojanovic´ et al., 2005) and biological activities of methanolic extract of this species (Conforti et al., 2005).

3. Present study

Air-dried flowering aerial parts (224 g) of A. ligustica (Asteraceae, Anthemideae) were extracted in a Soxhlet apparatus with ethanol (48 h). After removal of solvent up to 40 C in vaccuo, the obtained extract (72 g) was frac- tionated by CC on silica gel 230e400 mesh (hexaneeEtOAc step gradients and then with increasing percentages of MeOH) to yield 21 fractions obtained by combining the eluates on the basis of TLC analysis. Fraction 1 (660 mg) eluted with hexaneeEtOAc (90:10) was submitted to silica gel column chromatography (230e400 mesh) using benzene as eluent. Posterior purification on Sephadex LH-20 column eluted by the hexaneeCH2Cl2eMeOH (2:2:1) system led to the isolation of (E)-ethyl-3,7-dimethyl-3,6-octadienoate (9 mg) (Patel et al., 1979). Fraction 5 (350 mg) eluted with hexaneeEtOAc (70:30) was chromatographed on a silica gel column using hexaneeEtOAc with increasing polarity to obtain three subfractions. These subfractions eluted with hexaneeEtOAc (80:20), (70:30) and (2:1), respectively, gave after purification by preparative TLC developed with CH2Cl2-Me2CO (40:1), borneol (6 mg) (Tsankova and Ognyanov, 1985), 2-oxoisodauc-5-en-12-al (8 mg) (Hansson and Wickberg, 1992) and 1-tricontanol (7 mg) (Ferheen et al., 2005). Fraction 6 (400 mg) eluted with hexaneeEtOAc (65:35), was chromatographed on a silica gel column using CH2Cl2eMe2CO (20:1) followed by preparative TLC developed with hexaneeEtOAc (4:1), to obtain b-sitosterol (5.5 mg) (Ness et al., 1992) and filifolide A (4 mg) (Torrance and Steelink, 1974). Fraction 10 (790 mg) eluted with EtOAceMeOH (95:5) was submitted to a silica gel column using CH2Cl2eMeOH (20:1) followed by preparative TLC developed with hexaneeEt2O (2:3), to obtain Santin (7 mg) (Long et al., 2003) and the new sesquiterpene lactone 1 (10.1 mg). All the structures were identified by spectral analysis, mainly HR-EI-MS and 2D NMR (COSY, ROESY, HSQC, HMBC) as well as by comparing their spectroscopic data with those reported in the literature. In our best knowledge, compound 1 is new and named algerianolide. We report on the isolation and structural identification of 1. 2' 14 1' OH O H HO Cl 10 9 O 2 1 8 H 3 5 7 O 11 H H 13 O 15

O 1

Algerianolide (1) was obtained as colourless crystals. The HR-EI-MS showed the molecular formula C17H21O7Cl by exhibiting ions at m/z 372.0988 (calc. 372.0976) and m/z 374.0932 (calc. 374.0946). This spectrum also exhibited þ þ signals at m/z 337.1299 [M Cl] (calc. 337.1287), 319.1173 [M Cl H2O] (calc. 319.1182), 301.1062 þ [M Cl 2H2O] (cal. 301.1076), 296.0616 (cal. 296.0629) and 294.0669 (cal. 294.0659) [M CH3CO2H þ H2O] , which suggested that this compound contained two hydroxyl and an acetate groups and conﬁrmed the presence of the chlorine atom. The 1H NMR spectrum showed two sets of typical signals for exomethylene protons at dH 6.21 (d, J ¼ 3.5 Hz, H-13a) and dH 5.41 (d, J ¼ 3.2 Hz, H-13b), these two allylic coupled protons were typical for an exomethylene group conjugated with the lactonic carbonyl of a sesquiterpene lactone. In the 1He1H COSY spectrum, the correlations of H-13a and H-13b led to the assignment of H-7 at dH 3.54, which led to H-6 at dH 4.21 as a doublet of doublets (J ¼ 11.0, 9.9 Hz). This proton correlated with the carbon at dC 81.3 in the HSQC spectrum, in- 1 1 dicating a C-6 lactonized sesquiterpene lactone. The correlations of H-6 in the He H COSY spectrum, led to H-5 at dH 3.13 as a doublet (J ¼ 11.0 Hz). The multiplity of H-5 indicated that C-4 was tetrasubstitued. Re-examination of the 1 H NMR spectrum conﬁrmed the presence of the acetate group by exhibiting a 3H singlet at dH 2.16 (dC 21.3 from HSQC) and showed the presence of two other methyl groups. One of the methyl groups (dC 18.0, dH 1.74) which correlated with H-5 in the HMBC experiment spectrum (Fig. 1) must be C-15; while the other one (dC 23.5, dH 1.51) must A. Boudjerda et al. / Biochemical Systematics and Ecology 36 (2008) 461e466 463

14 O Me 1' Cl HO O HO H H 2 10 9 H Me 1 8 2' H 3 4 5 7 H 6 H O 11 H H 13 Me O 15 H 12 O

HMBC

Fig. 1. Representative HMBC correlations for compound 1.

be C-14. In the same spectrum (HMBC), the protons of C-15 correlated with C-5, with the quaternary carbon at dC 67.4 which may be attributed to C-4 and with the carbon of the CH at dC 63.7 (dH 3.62) assigned to C-3. Re-examination of 1 1 the He H COSY spectrum, particularly, the correlations of H-7, led to the attribution of the multiplet at dH 2.21 and the broad doublet (J ¼ 15.6 Hz) at dH 1.96 to H-8b and H-8a, respectively (dC 31.9). In the same spectrum, these two protons showed correlations with the proton corresponding to the doublet of doublets (J ¼ 7.3, 1.7 Hz) at dH 5.09 sug- gesting its attribution to H-9. The value of the chemical shift of H-9 indicated that the acetate group was attached to C-9 (dC 74.6). This was further supported from the presence of the correlation observed in the HMBC spectrum between 0 H-9 and the carbon of the carbonyl at dC 169.9 attributed to the acetate group (C-1 ) because of its correlation with the 0 protons of the methyl group at dH 2.16 (CH3-2 ). In addition, this spectrum showed correlations between H-13a, H-13b 0 and the carbon at dC 169.9. This observation indicated that C-12 and C-1 had the same chemical shift and confirmed the presence of 17 carbon atoms in the structure. This spectrum also showed correlations of both H-8a and the protons of CH3-14 with a hydroxylated quaternary carbon at dC 77.5 which may be attributed to C-10. Hydroxylation of C-10 was supported by the observed correlation between it and the proton of the hydroxyl group at dH 2.33. This assumption was confirmed by the correlation between the hydroxyl proton and C-9. The same spectrum (HMBC) showed correlations of H-3 and the protons of CH3-14 with the quaternary carbon atom at dC 84.5, which may be attributed to C-1. This carbon atom must be hydroxylated because of the value of its chemical shift and its correlation with the proton of the hydroxyl group at dH 4.10. The HMBC spectrum also showed correlations between C-5, C-4, C-3, C-1 and the proton of the broad singlet at dH 4.14 (dc 61.6), which may be assigned to H-2. These observations suggested a guaianolide- type skeleton for this sesquiterpene lactone. The molecular formula C17H21O7Cl of 1 indicated the presence of seven unsaturations, which, in addition to the value of the chemical shifts of C-3 and C-4, suggested an epoxide function at these positions. Consequently, the chlorine atom must be at C-2. The stereochemistry at C-5, C-6, C-7 and C-9 followed from the coupling constants which suggested a trans disposition for H-5, H-6 and H-7 and a b-orientation for H-9. The stereochemistry at C-1, C-2, C-3, C-4 and C-10 1 1 was deduced from the 2D He H ROESY spectrum. The ROESY interactions H-5/H-3, H-5/CH3-15, suggested a3b,4b-epoxy configuration, while the ROESY interactions of H-9/OH-1, H-7/OH-10, and CH3-14/H-2 indicated a b-orientation for OH-1, an a-orientation for OH-10, and an a-orientation for the chlorine atom. Therefore compound 464 A. Boudjerda et al. / Biochemical Systematics and Ecology 36 (2008) 461e466

1 was identified as 2a-chloro-9a-acetoxy-1b,10a-dihydroxy-3b,4b-epoxy-5a,7a-H-guaia-11(13)-en-12,6a-olide. This compound is new and named algerianolide. The molecular structure and relative configuration of 1 were also established by X-ray diffraction analysis (Fig. 2). Diffraction data were collected at room temperature using a Bruker-Nonius Kappa CCD diffractometer with graphite monochromated Mo-Ka radiation (l ¼ 0.71073 A˚ ). Frames were collected with the COLLECT program (Bruker AXS BV, 1997e2004), indexed and processed using Denzo SMN and the files scaled together using the HKL2000 program (Otwinowski and Minor, 1997). Relevant crystal data, experimental conditions and final refined parameters are listed in Table 1 for this compound. The structure solution was obtained by direct methods, using the SIR2004 program (Burla et al., 2005) and refined using the SHELXL-97 program (Sheldrick, 1997). Reflections and Friedel opposites were combined and merged before refinement. All non-hydrogen atoms were refined with anisotropic thermal parameters using full-matrix least squares procedures on F2. All H atoms were placed in geometrically calculated positions. The methyl-H atoms were refined as a rigid groups, which were allowed to rotate but not to tip, with Uiso(H) ¼ 1.5Ueq(C). All other H atoms were allowed to ride on their parent atoms with Uiso(H)¼1.2Ueq(C). 20 1 Algerianolide (1): colourless crystals. mp 215 C, [a]D ¼þ42.35 C(c 0.85 CHCl3), H NMR (400 MHz, CDCl3): d 6.21 (1H, d, J ¼ 3.5 Hz, H-13a), 5.41 (1H, d, J ¼ 3.2 Hz, H-13b), 5.09 (1H, dd, J ¼ 7.3, 1.7 Hz, H-9), 4.21 (1H, dd, J ¼ 11.0, 9.9 Hz, H-6), 4.14 (1H, brs, H-2), 3.62 (1H, brs, H-3), 3.54 (1H, m, H-7), 3.13 (1H, d, J ¼ 11.0 Hz, H-5), 2.21 (1H, m, H-8b), 1.96 (1H, brd, J ¼ 15.6 Hz, H-8a), 1.74 (3H, s, CH3-15), 1.51 (3H, s, CH3- 0 13 14), 2.16 (3H, s, CH3-2 ), 4.10 (1H, s, OH-1), 2.33 (1H, s, OH-10); C NMR (100 MHz, CDCl3): d 169.9 (C-12 & C-10), 138.5 (C-11), 119.4 (C-13), 84.5 (C-1), 81.3 (C-6), 77.5 (C-10, obscured by the signal of the solvent, assigned from HMBC experiment spectrum), 74.6 (C-9), 67.4 (C-4), 63.7 (C-3), 61.6 (C-2), 46.4 (C-5), 41.6 (C-7), 31.9 (C-8), 0 37 23.5 (C-14), 18.0 (C-15), 21.3 (C-2 ); HR-EI-MS m/z 374.0932 (calculated for C17H21O7 Cl, 374.0946), 372.0988 35 þ þ (calculated for C17H21O7 Cl, 372.0976). EI-MS m/z 337 [M Cl] (0.5), 319 [337 H2O] (6.9), 296 þ þ þ [M CH3CO2H H2O] (0.4), 294 [M CH3CO2H H2O] (1.2), 277 [337 CH3CO2H] (4.6), 271 þ þ þ [M CH3CO2H CO CH3] (1.8), 269 [M CH3CO2H CO CH3] (5.5), 259 [277 H2O] (7.7), 250 (7.5), 243 (1.9), 241 (3.7), 231 (5.5), 217 (6.3), 216 (4.9), 215 (4.4), 191 (3.4), 189 (4.0), 183 (5.9), 165 (7.9), 161 (7.0.), 151 (6.8), 137 (15.7), 111 (100).

Fig. 2. A view of the molecule showing the atomic numbering scheme. Displacement ellipsoids are plotted at the 50% probability level. A. Boudjerda et al. / Biochemical Systematics and Ecology 36 (2008) 461e466 465

Table 1 Crystal data and structure reﬁnement for compound 1 Compound 1

Empirical formula C17H21ClO7 M 372.79 T (K) 293 (2) l (A˚ ) 0.71073 Crystal system Monoclinic Space group P21 Unit cell dimensions a (A˚ ) 5.9050 (10) b (A˚ ) 15.5380 (10) c (A˚ ) 10.1160 (10) b () 104.774 (11) V (A˚ 3) 897.5 (2) Z 2 Dc (g cm3) 1.380 m (Mo-Ka) (mm1) 0.248 Crystal size (mm) 0.45 0.20 0.08 q range for data collection () 2.1e28.7 Index ranges 7 h 7; 20 k 21; 13 l 13 Reﬂections collected/unique 15,100/4334 Rint 0.055 Absorption correction None Reﬁnement method Full-matrix least squares on F2 Data/restraints/parameters 4334/0/229 Final R indices [I > 2s(I)] R ¼ 0.052; wR2 ¼ 0.1428; S ¼ 1.13 2 2 2 2 2 Weighting scheme, w 1/[s (Fo) þ (0.0804P) ], P ¼ (Fo þ 2 Fc)/3 Max. and Av. shift/error 0.00/0.00 Flack parameter 0.04 (7) Largest diff. peak and hole/e (A˚ 3) 0.35/0.42 These data have been deposited with the Cambridge Crystallographic Data Centre (deposit number CCDC 655386). Data Acquisition e the Cambridge Crystallographic Data Centre. [email protected], http://www.ccdc.cam.ac.uk/deposit. Tel.: þ44 01223 762910; fax: þ44 01223 336033. Postal address: CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.

4. Chemotaxonomic signiﬁcance

A. ligustica contains compounds present in the other species of the genus. The isolation of the flavonoid santin was in accordance with the results in the literature on the species of Achillea sect. millefolium which reported that most species of the genus Achillea are characterized by the predomination of 6-hydroxyflavones and 6-hydroxyflavonols and their OeMe ethers (Valant-Vetschera, 1987; Ivancheva and Tsvetkova, 2003). The isolated sesquiterpene lactone had a guaianolide skeleton which is common in this genus (Kubelka et al., 1999). It should be noted that the presence of a chlorine atom is rare in the sesquiterpene lactones of Achillea species and that related chlorohydrin guaianolides were isolated previously from only a few Achillea species such as Achillea biebersteinii and Achillea santolina (Yusupov et al., 1979), Achillea clusiana (Todorova et al., 1999), A. ligustica (Ahmed et al., 2003), Achillea depressa (Trifunovic´ et al., 2005) and Achillea clavennae (Trifunovic´ et al., 2006). It is, however, interesting to note that in all the described structures from this genus the chlorine atom was attached at C-1, C-3 or C-4. In our best knowledge, sesquiterpene lactones containing chlorine atom at C-2 were not reported previously from this genus. It should also be noted that the sesquiterpene lactones isolated from A. ligustica All. grow- ing in Algeria, in Sicily (Bruno and Herz, 1988) and Greece (Ahmed et al., 2003) were different.

Acknowledgments

This work was supported in part by Algerian National Agency for Development of Health Research (A. N. D. R. S.) project and grants from the Programa de Iniciativa Comunitaria INTERREG IIIB AzoreseMadeiraeCanaries 466 A. Boudjerda et al. / Biochemical Systematics and Ecology 36 (2008) 461e466

(04/MAC/3.5/C5), and from the Instituto Canario de Investigacio´n del Cancer. The authors wish to thank Dr. Djamel Sarri (Biology Department, University of M’Sila, Algeria) for the identiﬁcation of the plant material.

References

Ahmed, A.A., Gati, T., Hussein, T.A., Ali, A.T., Tzakou, O.A., Couladis, M.A., Mabry, T.J., To´th, G., 2003. Tetrahedron 59, 3729. Balboul, B.A.A.A., Ahmed, A.A., Otsuka, H., Bando, M., Kido, M., Takeda, Y., 1997. Phytochemistry 46, 1045. Bentame`ne, A., Creche, J., Petit, G., Bermejo-Barrera, J., Benayache, S., Benayache, F., 2005. Biochem. Syst. Ecol. 33, 1061. Bruno, M., Herz, W., 1988. Phytochemistry 27, 1871. Burla, M.C., Caliandro, R., Camalli, M., Carrozzini, B., Cascarazo, G.L., De Caro, L., Giacovazzo, C., Polidori, G., Spagna, R., 2005. J. Appl. Crystallogr. 38, 381. Conforti, F., Loizzo, M.R., Statti, G.A., Menichini, F., 2005. Biol. Pharm. Bull. 28, 1791. Dendougui, H., Jay, M., Benayache, F., Benayache, S., 2006. Biochem. Syst. Ecol. 34, 718. Ferheen, S., Ahmed, E., Afza, N., Malik, A., 2005. J. Chem. Soc. Pak. 27, 219. Filippi, J.J., Lanfranchi, D.A., Prado, S., Baldovini, N., Meierhenrich, U.J., 2006. J. Agric. Food Chem. 54, 6308. Greger, H., Zdero, C., Bohlmann, F., 1984. Phytochemistry 23, 1503. Hansson, T., Wickberg, B., 1992. J. Org. Chem. 57, 5370. Ivancheva, S., Tsvetkova, R., 2003. In: Imperato, Filippo (Ed.), Advances in Phytochemistry. Research Signpost, Trivandrum, India, p. 85. Krenn, L., Miron, A., Pemp, E., Petr, U., Kopp, B., 2003. Z. Naturforsch. C J. Biosci. 58, 11. Kubelka, W., Kastner, U., Glasl, S., Saukel, J., Jurenitsch, J., 1999. Biochem. Syst. Ecol. 27, 437. Long, C., Sauleau, P., David, B., Lavaud, C., Cassabois, V., Ausseil, F., Massiot, G., 2003. Phytochemistry 64, 567. Maffei, M., Germano, F., Doglia, G., Chialva, F., 1993. J. Essent. Oil Res. 5, 61. Ness, D.W., Norton, R.A., Benson, M., 1992. Phytochemistry 31, 805. Orkiszewska, A., Lobarzewsky, R., Jedrizejewska, M. Polish Patent, B1 119889, 1985. Otwinowski, Z., Minor, W., 1997. In: Carter Jr., C.W., Sweet, R.M. (Eds.), Methods in Enzymology: Macromolecular Crystallography, Part A, vol. 276. Academic Press, University of Texas, South-western Medical Centre, Dallas, p. 307. Palic´, R., Stojanovic´, G., Naskovic´, T., Ranelovic´, N., 2003. J. Essent. Oil Res. 15, 434. Patel, B.A., Kao, L.C., Cortese, N.A., Minkiewicz, J.V., Heck, R.F., 1979. J. Org. Chem. 44, 918. Stojanovic´, G., Hashimoto, T., Asakawa, Y., Palic´, R., 2005. Biochem. Syst. Ecol. 33, 207. Sadyrbekov, D.T., Suleimenov, E.M., Tikhonova, E.V., Atazhanova, G.A., Tkachev, A.V., Adekenov, S.M., 2006. Chem. Nat. Comp. 42, 294. Simonpoli, P., 1993. Arburi, Arbe, Arbigliule, Savoirs Populaires sur les Plantes de Corse. Parc Naturel Regional de la Corse, Ajaccio, Corsica. Sheldrick, G.M., 1997. SHELXL-97, Program for the Refinement of Crystal Structures. University of Go¨ttingen, Germany. Todorova, M.N., Tsankova, E.T., Taskova, R.M., Peev, D.R., 1999. Z. Naturforsch. C J. Biosci. 54, 1011. Todorova, M., Trendafilova, A., Mikhova, B., Vitkova, A., Duddeck, H., 2007. Phytochemistry 68, 1722. Torrance, S.J., Steelink, C., 1974. J. Org. Chem. 39, 1068. Trifunovic´, S., Aljancic´, I., Vajs, V., Macura, S., Milosavljevic´, S., 2005. Biochem. Syst. Ecol. 33, 317. Trifunovic´, S., Vajs, V., Juranic´, Z., Zˇizak, Zˇ., Tesˇevic´, V., Macura, S., Milosavljevic´, S., 2006. Phytochemistry 67, 887. Tsankova, E., Ognyanov, I., 1985. Planta Med. 2, 180. Tuberoso, C.I.G., Kowalczyk, A., Coroneo, V., Russo, M.T., Dessi, S., Cabras, P., 2005. J. Agric. Food Chem. 53, 10148. Tzakou, O., Loukis, A., Verykokidou, E., Roussis, V., 1995a. J. Essent. Oil Res. 7, 549. Tzakou, O., Couladis, M., Verykokidou, E., Loukis, A., 1995b. Biochem. Syst. Ecol. 23, 569. Valant-Vetschera, K., 1985. Biochem. Syst. Ecol. 13, 15. Valant-Vetschera, K.M., 1987. Biochem. Syst. Ecol. 15, 45. Yusupov, M.I., Mallabaev, A., Kasymov, Sh.Z., Sidyakin, G.P., 1979. Khim. Prir. Soedin. 4, 580. Zaiter, L., Bouheroum, M., Benayache, S., Benayache, F., Leoń, F., Brouard, I., Quintana, J., Este´vez, F., Bermejo, J., 2007. Biochem. Syst. Ecol. 35, 533.