Biochemical Systematics and Ecology 33 (2005) 1061e1065

www.elsevier.com/locate/biochemsyseco

A new guaianolide and other sesquiterpenelactones from Centaurea acaulis L. (Asteraceae)

Ali Bentamene a, Samir Benayache a, Joel Creche b,Genevieve Petit b, Jaime Bermejo-Barrera c,

Francisco Leon c, Fadila Benayache a,*

a Laboratoire de Phytochimie et Analyses Physico-Chimiques et Biologiques, Equipe

Associee a l’A. N. D. R. S., Universite Mentouri, Route de Aın El Bey, 25 000 Constantine, Algeriab Universite Francois Rabelais de Tours, Faculte des Sciences Pharmaceutiques EA 2106 Biomolecules et

Biotechnologies Vegetales, 31 Avenue Monge, 37200 Tours, Francec Consejo Superior de Investigaciones Cientificas, Instituto de Productos Naturales y Agrobiologia, Tenerife, Spain

Received 23 May 2004; accepted 11 March 2005

Keywords: Guaianolides; Sesquiterpene lactones; Centaurea acaulis L.; Asteraceae

1. Subject and source

Centaurea acaulis L., an endemic species of Algeria and Tunisia, flowering fromApril to July (Quezel and Santa, 1963)was collected from the area ofMila in theEast ofAlgeria in June 2000 and authenticated by Prof. M. Kaabeche (Biology Department,University of Setif, Algeria). A voucher specimen (CCA09/06/00) has been depositedin the Herbarium of the Biology Department of Mentouri University of Constantine.

2. Previous work

No reports on the isolation of any secondary metabolites from C. acaulis L. areavailable to date.

* Corresponding author. Tel./fax: C213 31 81 88 83.

E-mail address: fadila.benayache@caramail.com (F. Benayache).

0305-1978/$ - see front matter � 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.bse.2005.03.009

1062 A. Bentamene et al. / Biochemical Systematics and Ecology 33 (2005) 1061e1065

3. Present study

In the continuation of our work on Algerian medicinal plants (Benyahia et al.,2004; Akkal et al., 2003; Benayache et al., 2001; Dendougui et al., 2000) weinvestigated the leaves and the flowers of C. acaulis L. After separation andpurification by chromatographic methods, the chloroform soluble part of theaqueous-methanolic extract afforded a new natural compound, 14-chloro-10b-hydroxy-10(14)-dihydrozaluzanin D (3b-acetoxy-14-chloro-10b-hydroxy-1aH,5aH,6bH,7aH-guaia-4(15),11(13)-dien-6,12-olide) 4 besides the known sesquiterpenelactones: b-cyclocostunolide (5aH, 6bH, 7aH, 10bCH3-eudesma-4(15),11(13)-dien-6,12-olide) 1 (Jain and McCloskey, 1975; Govindan and Bhattacharyya, 1977),costunolide (6bH, 7aH-germacra-1(10),4(5)-dien-6,12-olide) 2 (Akyev et al., 1972),zaluzanin D (3b-acetoxy-1aH,5aH,6bH,7aH-guaia-4(15),10(14),11(13)-trien-6,12-olide) 3 (Romo de Vivar et al., 1967; Kisiel, 1975), santamarin (1b-hydroxy-5aH,6bH, 7aH-10bCH3-eudesma-3(4),11(13)-dien-6,12-olide) 5 (Romo de Vivar andJimenez, 1965; Sanz et al., 1989; Zdero et al., 1991) and kandavanolide (3b-acetoxy-8a-hydroxy-1aH,5aH,6bH,7aH-guaia-4(15),10(14),11(13)-trien-6,12-olide) 6 alreadydescribed for Centaurea species (Rustaiyan and Ardebili, 1984; Danielwski et al.,1993; Vajs et al., 1999), while compounds 1, 2 and 3 are new for the genus Centaurea.

OO

H

1

O

O2

O

O

O

H

HO

H3C

3

134 5

6 7

8

910

1112 1315

O

HO

H

O

HO14CH2Cl

2H3C

O

4

O

O

OH

H

5

O

O

OOH

H

HO

H3C

6

1063A. Bentamene et al. / Biochemical Systematics and Ecology 33 (2005) 1061e1065

Air-dried leaves (6 kg) and air-dried flowers (4 kg) of C. acaulis L. were separatelymacerated at room temperature with EtOH:H2O (80:20 v/v) for 24 h, three times.After filtration, the filtrates were concentrated and re-extracted several times withCHCl3 resulting in 7.8 g and 3.0 g of the chloroform extracts, respectively. Analysisby TLC on silica gel plates showed that the chemical composition of these twoextracts was the same. The extracts were then combined and chromatographedon a 230e400 mesh silica gel (400 g) column eluted with a gradient-CH2Cl2:CH3COCH3, 90:10 to 100% CH3COCH3 to yield 15 major fractions (F1eF15). Fractions F1eF4 which had similar composition (TLC) were submitted topreparative TLC on silica gel GF254 (petrol:Et2O, 2:1, 3:2, 2:3 and 2:3, respectively)to give, after remixing the fractions with the same profile, six fractions (FAeFF).These fractions which were purified by preparative TLC (hexane:AcOEt, 98:2, 95:5,80:20, 70:30, 60:40 and 50:50, respectively) gave 1 (8 mg), 2 (12 mg), 3 (37 mg), 4(40 mg), 5 (72 mg) and 6 (90 mg), respectively.

The positive EIMS spectrum of 4 showed a molecular ion at m/zZ 340/342 (3:1)which agreed with the presence of a chlorine atom in the structure, and with theformula C17H21O5Cl for this compound. In the same spectrum, the ions atm/zZ 322/324 [M�H2O]C, 298/300 [M�CH2CO]C, 280/282 [M�CH3COOH]C,262/264 [M�CH3COOHeH2O]C and 213 [M�CH3COOHeH2OeCH2Cl]

C

suggested that this compound contained a hydroxyl and an acetate group, andconfirmed the presence of the chlorine atom on the sesquiterpene skeleton. Themolecular formula was confirmed by HREIMS as C17H21O5 from the ion atm/zZ 305.1397 [M�Cl]C (calc. for C17H21O5, 305.1389). The

1H NMR spectrumshowed two sets of typical signals for exomethylene protons. At d 6.24 (d,JZ 3.4 Hz, H-13a) and d 5.53 (d, JZ 3.1 Hz, H-13b), these two allylic coupledprotons were typical for an exomethylene group conjugated with the lactoniccarbonyl of a sesquiterpene lactone. The other exomethylene group was character-ized by two doublets at d 5.61 (d, JZ 1.4 Hz, H-15a) and d 5.38 (d, JZ 1.6 Hz, H-15b). In the 1He1H COSY spectrum, the correlations of H-13a and H-13b led to theassignment of H-7 at d 2.71, which correlated with H-6 at d 4.55 as a triplet(JZ 9.4 Hz). This proton correlated with the carbon at d 85.0 in the HSQCspectrum, indicating a C-6 lactonized sesquiterpene lactone. The correlations of H-6in the 1He1H COSY spectrum permitted the assignment of H-5 at d 3.01, whichshowed correlations with the last exomethylene group confirming the presence of anexomethylene double bond at C-4. In the same spectrum, H-5 was also correlatedwith a proton which gave a signal overlapped by the signal for H-7 at d 2.71. Thisproton must be H-1 and suggested that this compound has a guaianolide-typeskeleton. The assignment of H-1 allowed the identification of H-2 at d 2.15 and H-2#at d 2.21. These two protons correlated with the proton at d 5.63 which wasattributed to H-3, indicating the presence of the acetoxy group at C-3. The 1H NMRspectrum also exhibited an AB system (d 3.40 and d 3.46, both d, JZ 10.9 Hz) forthe CH2Cl group which must be at C-10. All these results, in addition to thoseobtained by 13C NMR and DEPT experiments led to the presence of the hydroxylgroup at C-10 (d 74.9). All these assignments were in good agreement with the resultsof an HMBC (400 MHz) experiment optimized for an 8 Hz long-range coupling,

1064 A. Bentamene et al. / Biochemical Systematics and Ecology 33 (2005) 1061e1065

which, in particular, showed correlations of the AB system and the hydroxyl groupwith the same carbon atoms (C-1, C-9 and C-10) and correlations of the protons ofthe second exomethylene group with C-3 and C-5. The same spectrum showedcorrelations between H-13a, H-13b and the protons of the acetoxy group with thecarbon at d 169.3. This observation indicated that C-12 and C-16 had the samechemical shift and confirmed the presence of 17 carbon atoms in the structure. Thestereochemistry at C-1, C-5, C-6 and C-7 followed from the coupling constants. Thestereochemistry at C-3 and C-10 was deduced from the 2D 1He1H NOESYspectrum. The strong NOE-correlation between H-3 and H-5 suggested a b-orientation for the acetoxy group at C-3, while the a-orientation of the CH2Cl groupat C-10 was deduced from the NOE-correlations between the protons of this groupand H-1 and H-5. No NOE-correlations between the protons of CH2Cl and H-8bwere observed which confirmed the proposed stereochemistry. Therefore, compound4 was identified as 14-chloro-10b-hydroxy-10(14)-dihydrozaluzanin D (Romo deVivar et al., 1967; Kisiel, 1975).

3.1. 14-Chloro-10b-hydroxy-10(14)-dihydrozaluzanin D 4.

Colourless oil. [a]20 �l : C15.7 at 589 nm, C16.4 at 578 nm, C18.8 at 546 nm,

C32.0 at 436 nm (CHCl3; c 0.88). 1H NMR (400 MHz, CDCl3): d 6.24 (1H, d,JZ 3.4 Hz, H-13a), 5.63 (1H, m, H-3), 5.61 (1H, d, JZ 1.4 Hz, H-15a), 5.53 (1H, d,JZ 3.1 Hz, H-13b), 5.38 (1H, d, JZ 1.5 Hz, H-15b), 4.55 (1H, t, JZ 9.4 Hz, H-6),3.46 (1H, d, JZ 10.9 Hz, H-14a), 3.40 (1H, d, JZ 10.9 Hz, H-14b), 3.01 (1H, br dd,JZ 9.4, 9.0 Hz, H-5), 2.98 (1H, br s, 10-OH), 2.71 (1H, m, H-1), 2.71 (1H, m, H-7),2.21 (1H, m, H-2a), 2.15 (1H, m, H-2b), 2.10 (1H, m, H-8a), 2.09 (3H, s, H-17), 2.08(1H, m, H-9b), 1.92 (1H, td, JZ 12.4, 3.5 Hz, H-9a), 1.73 (1H, qd, JZ 12.4, 4.1 Hz,H-8b). 13C NMR (100 MHz, CDCl3): d 169.3 (C-12 and C-16), 149.9 (C-4), 139.2 (C-11), 120.0 (C-13), 114.5 (C-15), 85.0 (C-6), 75.6 (C-3), 74.9 (C-10), 53.1 (C-14), 50.0(C-5), 46.0 (C-7), 44.8 (C-1), 37.8 (C-9), 33.8 (C-2), 24.0 (C-8), 21.2 (C-17). EIMS70 eV, m/z: 340 & 342 [M]C, 322 & 324 [M�H2O]C, 298 &300 [M�CH2CO]C, 280& 282 [M�CH3COOH]C, 262 & 264 [M�CH3COOHeH2O]C, 213[M�CH3COOHeH2OeCH2Cl]

C.

4. Chemotaxonomic significance

These results on C. acaulis L. (Compositae) indicate the presence of the lowfunctionalized eudesmanolide, santamarin which is rare in the tribe Cynareae(Appendino et al., 1986) and b-cyclocostunolide which was unknown in this genus,and confirm previous findings that the members of the genus Centaurea containsgermacranolides, eudesmanolides and guaianolides often derived from zaluzanin C.This work also showed that C. acaulis L. contains sesquiterpene lactones withremarkable biological activities like costunolide and zaluzanin D (Hibasami et al.,2003; Krishna Kumari et al., 2003a,b).

1065A. Bentamene et al. / Biochemical Systematics and Ecology 33 (2005) 1061e1065

Acknowledgements

The authors wish to thank A. N. D. R. S. for financial support, Prof. M.Kaabeche (Biology Department, University of Setif, Algeria) for the identification ofthe plant material and professor Paul Mosset (High National School of Chemistry,University of Rennes, France) for optical rotation measurement and NOESYexperiment.

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