New Cytotoxic Sesquiterpene

Lactones from Warionia saharae

Fatima Hilmi, Otto Sticher, Jörg Heilmann

Abstract

Cytotoxicity-guided fractionation of the methanol soluble part of

the dichloromethane extract of the leaves of Warionia saharae led
to the isolation of the two new guaianolide-type sesquiterpene
lactones, 5aH-3b,4b-epoxy-14-oxo-guaia-1(10),11(13)-dien-6a,12-
olide (1), 5aH-2b,4b-epoxy-3a-hydroxy-guaia-1(10),11(13)-dien-
Letter

6a,12-olide (6), and the new eudesmane type sesquiterpene

1b,6a-dihydroxycostic acid (4). In addition, the known sesqui-
terpene lactones 5aH-2b-hydroxyguaia-3(4),10(14),11(13)-trien-
6a,12-olide (2), reynosin (3), 5aH-1a,10a:3a,4a-diepoxyguaia-
11(13)-en-6a,12-olide (5), and dehydroleucodin (7) were isolated
together with the known flavone hispidulin. Cytotoxicity testing
of the sesquiterpene lactones against the KB cancer cell line (ATCC

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CCL17) revealed IC50 values of 3.5 (1), 2.6 (2), 2.7 (3), 4.3 (5), 3.6 (6),
and 1.3 (7) mg/mL. Compound 4 was not active up to 20 mg/mL.

Warionia saharae Benth. & Coss. (Asteraceae) from the monoty-

pic genus Warionia is a shrub endemic in Morocco and South
Oran in Algeria. The leaves are used in Morocco for the treatment
of inflammatory and gastrointestinal disorders [1]. Since the de-
scription of W. saharae in 1872 a phytochemical investigation of
the essential oil [2] as well as the isolation of six new cytotoxic
guaianolide-type sesquiterpene lactones has been reported [3].
462 In the present study we further investigated the methanol solu-
ble part of the dichloromethane extract of W. saharae leaves by
cytotoxicity-guided fractionation.
The 13C-NMR spectrum of compound 1 showed 15 signals, which
could be attributed to one CH3, four CH2, five CH and five quatern-
ary carbons by the DEPT spectra. This corresponds to a molecular
formula of C15H16O4, in agreement with a [M]+ at m/z = 260 in the
EI-MS. Both, the 1H- and 13C-NMR data (see Tables 1 and 2) point-
ed to an oxygenated guaianolide-type sesquiterpene lactone [3],
[4]. The chemical shifts and multiplicities of carbons C-2 ± C-8, C-
11 ± C-13 and C-15 were very similar to those of ludartin isolated
from Stevia yaconensis and Artemisia carruthii [5], [6]. The most
obvious difference was the presence of an a,b -unsaturated alde-
hyde group at C-14 (d = 192.5) in 1 instead of a vinyl methyl
(d = 22.7, in CDCl3) in ludartin [6]. In accordance, a signal of an al-
Affiliation: Department of Chemistry and Applied BioSciences, Institute of
Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich,
Zürich, Switzerland
Correspondence: PD Dr. Jörg Heilmann ´ Swiss Federal Institute of Technology
(ETH) Zurich ´ Institute of Pharmaceutical Sciences ´ Winterthurerstr. 190 ´
8057 Zürich ´ Switzerland ´ Phone: +41-1-6356049 ´ Fax: +41-1-6356882 ´
E-mail: joerg.heilmann@pharma.anbi.ethz.ch
Received: August 3, 2002 ´ Accepted: December 15, 2002
Bibliography: Planta Med 2003; 69: 462±464 ´  Georg Thieme Verlag Stuttgart ´
New York ´ ISSN 0032-0943
dehyde proton resonated in the 1H-NMR at d = 9.90 as a singulet
(H-14). The structure was finally established by 1H,1H-COSY,
1 13
H, C-HSQC, and 1H,13C-HMBC experiments as 5aH-3,4-epoxy-
14-oxo-guaia-1(10),11(13)-dien-6,12-olide. The b-orientation of
the epoxide function and the trans-configuration of the lactone
ring were determined by a ROESY experiment in C6D6.
Compound 6 presented an unusual C-2-O-C-4 ether bridge, which
has only been reported in guaianolide-type sesquiterpene lactones
from W. saharae [3]. Its structure was definitively determined to
be 5aH-2b,4b-epoxy-3a-hydroxyguaia-1(10),11(13)-dien-6a,12-
olide by 1D-, and 2D-NMR spectroscopy and EI-MS data, applying
the same strategy as described for compound 1 (see Tables 1 and 2
for 1H-, 13C-NMR data). Compound 4 was identified as 1b,6a-dihy-
droxycostic acid on the basis of extensive 1D-, 2D-NMR and EI-MS
studies (see Tables 1 and 2 for 1H-, 13C-NMR data) as well as by
comparison of the obtained data with literature values of costic
acid derivatives [7], [8]. The spectral (NMR, MS, UV) and physical
(melting point, optical rotation) data of the known compounds 2,
3, 5, 7 and 8 (hispidulin) were in complete accordance with those
reported in the literature [9], [10], [11], [12], [13], [14], [15]. The
13
C-NMR data of compounds 2 and 5 were hitherto not published
and thus included into Table 2. The cytotoxicity of the sesquiter-
pene lactones and 1b,6a-dihydroxycostic acid (4) was determined
against the KB cancer cell line (ATCC CCL17) and revealed remark-
able activity for all Sls. The determined IC50 values were 3.5 (1), 2.6
 F4.3 (n-hexane-EtOAc, 6 : 4, 100 mL) an impure precipitate was
Table 1 1
H-NMR spectral data of compounds 1, 4, and 6* obtained. Purification of the precipitate by CC on 70 g of silica
gel (40 ± 63 mm, CHCl3-MeOH 92 : 8 1.5 L) yielded pure 8
H 1a 4b 6a (120 mg, ca. 400 ± 600 mL). The remaining part of fraction F4.3
(1566 mg) was subjected to CC on 100 g of Sephadex LH-20 with
1 3.05 (dd; 11.6, 4.8)
cyclohexane-CH2Cl2-MeOH (7: 4:1, 1.1 L) as eluent to give 5 sub-
2a 3.51 (m)c 1.53 (m)c 3.32 (m)d
fractions. Subfraction 3 (758 mg, 300 ± 510 mL) was separated by
2b 3.05 (m)c 1.40 (m)c
3a 3.60 (br s) 2.05 (m) 4.59 (s)
CC on silica gel (75 g, 40 ± 63 mm) using toluene-EtOAc (4 : 1) as
3b 1.76 (m)c eluent to obtain 10 subfractions A1-A10. A1 (0 ± 240 mL) gave
5 3.51 (m)c 1.57 (m)c 3.09 (br d; 10.7) pure 7 (2.9 mg). The subfractions A3 (40 mg, ca. 350 ± 460 mL),
6 3.83 (dd; 10.7, 10) 4.00 (t; 10.3) 3.86 (t; 10.5) A5 (115 mg, ca. 500 ± 720 mL) and A7 (88 mg, ca. 800 ± 1130 mL)
7 3.05 (m)c 2.67 (m) 2.88 (m) were purified by HPLC on a Spherisorb, S 10 ODSII, 5 mm column
8a 2.22 (m) 1.59 (m, 2H)c 2.14 (m)c (250 ” 16 mm) using MeOH-H2O (2 : 1.5, flow 5 mL/min) as elu-
8b 1.15 (m) 1.37 (m)
ent. A3 and A7 gave compounds 1 (tR = 21 min, 1.5 mg) and 6

Letter
9a 3.14 (ddd; 14.6, 5.9, 1.7) 1.82 (m) 2.34 (m)
9b 1.88 (m) 1.02 (m) 2.14 (m)c
(tR = 12 min, 3.7 mg), respectively. A5 yielded compound 5
13a 6.08 (d; 3.4) 6.27 (d; 1.1) 6.05 (d; 3.3) (tR = 11 min, 5.4 mg), moroccolide A [3], a mixture of com-
13b 5.52 (d; 3.2) 5.47 (s) 5.49 (d; 3.2) pounds 3/4 (tR = 19 min, 14.2 mg) and pure 2 (tR = 21 min, 5.2
14a 9.90 (s) 0.79 (s, 3H) 1.88 (d; 1.2, 3H) mg). Compounds 3/4 were separated by liquid-liquid partition
14b between EtOAc and aqueous NH3 (7 %) to obtain 4 (8.6 mg) from

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15a 1.68 (s, 3H) 4.83 (d; 1.2) 1.65 (s, 3H)
the aqueous and 3 (4.7 mg) from the organic phase.
15b 4.70 (d; 1.1)

* J (in Hz) and multiplicity are given in parenthesis. 5aH-3b,4b-Epoxy-14-oxoguaia-1(10),11(13)-dien-6a,12-olide (1):

a
In CD3OD at 500 MHz (295 K). colourless gum (1.5 mg); C15H16O4; [a]D20: + 9.48 (c 0.1, EtOH);
b
In C6D6 at 500 MHz (295 K). 1
c
H-NMR data, see Table 1; 13C-NMR data, see Table 2; EI-MS
Signals overlapped.
d Overlapped by the solvent signal. (CH3OH); m/z = 260 [M]+ (< 1), 243 [M ± OH]+ (< 1), 231 [M ±
CHO]+ (1), 44 (100).

(2), 2.7 (3), 4.3 (5), 3.6 (6), and 1.3 (7) mg/ml. Compound 4 was not 1b,6a-Dihydroxycostic acid (4): colourless gum (8.6 mg);
active up to 20 mg/mL. C15H22O4; [a]D20: + 12.08 (c 0.1, MeOH); 1H-NMR spectral data, see
Table 1; 13C-NMR data, see Table 2; EI-MS (CH3OH): m/z = 266
[M]+ (1), 248 [M ± H2O]+ (2), 230 [M ± 2 H2O]+ (28).
Materials and Methods
5aH-2b,4b-Epoxy-3a-hydroxyguaia-1(10),11(13)-dien-6a,12-olide 463
The leaves of Warionia saharae Benth. & Coss. were collected (6): colourless gum (3.7 mg); C15H18O4; [a]D20: + 12.98 (c 0.1, EtOH);
1
north of Agadir, Morocco, in May 1998. The plant was identified H-NMR spectral data, see Table 1; 13C-NMR data, see Table 2; EI-
by Dr. A. Benchâabane, University Smlallia, Marrakech, Morocco, MS (CH3OH): m/z = 262 [M]+ (6), 244 [M ± H2O]+ (9), 229 [244 ±
and Dr. F. Jacquemoud, Conservatoire et Jardin botaniques de CH3]+ (7).
Gen›ve, Geneva, Switzerland. A voucher specimen is deposited
at the Conservatoire et Jardin botaniques de Gen›ve, with the
identification number 3A/98.
13
Table 2 C-NMR spectral data of compounds 1 ± 2 and 4 ± 6
13
C-NMR spectra of compounds 2, 4, 5 and 6 were measured on a C 1a 2b 4b 5a 6a
Bruker AMX-300 spectrometer at 295 K. All other NMR spectra
were recorded on a Bruker DRX-500 spectrometer and on a Bru- 1 164.5, s 57.9, d 78.4, d 71.0, s 139.7, s
ker DRX-600 spectrometer at 295 K. DEI-MS were measured on a 2 33.1, t 79.7, d 32.2, t 36.2, t 72.3, d
micromass TRIBRID double focusing mass spectrometer at 70 eV. 3 64.7, d 130.4, d 35.2, t 62.7, d 67.1, d
The optical rotations were measured using a Perkin-Elmer 241 4 66.6, s 145.4, s 145.6, s 66.9, s 68.0, s
polarimeter. UV spectra were recorded in MeOH on an Uvikon 5 55.8, d 55.0, d 55.8, d 55.2, d 52.9, d
930 spectrophotometer. 6 80.6, d 84.1, d 69.2, d 82.2, d 82.6, d
7 55.3, d 44.8, d 48.2, d 53.6, d 55.6, d
Air-dried and powdered leaves of W. saharae (1 kg) were perco- 8 26.8, t 30.6, t 26.8, t 24.3, t 26.6, t
lated with CH2Cl2 at room temperature. The extract (157.2 g) was 9 23.5, t 34.9, t 36.5, t 38.1, t 35.1, t
portioned between n-hexane and methanol. The alcoholic phase 10 140.2, s 147.3, s 42.1, s 61.7, s 142.6, s

(30.45 g) was subjected in 3 portions to VLC (silica gel, 3 ” 120 g, 11 140.6, s 140.3, s 143.6, s 140.7, s 141.2, s
12 171.2, s 169.0, s 167.9, s 171.3, s 171.5, s
40 ± 63 mm) using a step gradient of hexane-ethyl acetate (9 : 1 to
13 118.8, t 118.9, t 124.4, t 119.2, t 118.2, t
1 : 1, 3 ” 750 ml each) and final washing with methanol (3 ” 750
14 192.5, d 113.6, t 11.6 q 20.7, q 22.8, q
mL) to give 7 fractions (F1-F7). The fraction F4 (3 g) was further
15 18.9, q 17.1, q 108.4 t 18.9, q 19.1, q
separated by VLC on silica gel (90 g, 40 ± 63 mm) eluting with a
gradient of hexane-EtOAc-MeOH (9 : 1 : 0 to 0 : 9 : 1, 300 mL each) a
In CD3OD at 75 MHz (295 K) for 4 and at 125 MHz for 1 (295 K).
to yield 9 subfractions (F4.1-F4.9). During evaporation of fraction b In C6D6 at 75 MHz (295 K).

Letter ¼ Planta Med 2003; 69: 462 ± 464

 The cytotoxicity of all fractions and compounds was determined
in a KB cell assay (HeLa cells, ATCC CCL17). The test was per-
formed as described in Heilmann et al. [16]. Fractions were test-
ed at 25 and 50 mg/mL, pure compounds in a concentration range
between 0.1 and 20 mg/mL.

Acknowledgements

We thank Dr. A. Benchâabane and Dr. F. Jacquemoud for identifi-

cation of the plant material. Special thanks are given to Drs. O.
Zerbe and K. Winkelmann for recording NMR spectra (both Insti-
tute of Pharmaceutical Sciences, ETH Zurich). The authors thank
Dr. W. Amrein, Mr. R. Häfliger and Mr. O. Greter (Institute of Or-
Letter

ganic Chemistry, ETH Zurich) for recording mass spectra. Further

thanks go to Meherun Grenacher for her effort during her work
as undergraduate student.

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Letter ¼ Planta Med 2003; 69: 462 ± 464