Chemical characterisation of the terpenoid constituents of the Algerian plant Launaea arborescens

Article abstract of DOI:10.1016/j.phytochem.2008.09.025

Chemical investigation of endemic Algerian plant Launaea arborescens resulted in the isolation of a series of triterpenes and sesquiterpenes from both the aerial parts and roots. Five terpenoids have been chemically characterised by means of spectral methods mainly NMR techniques. In addition, the absolute stereochemistry at the chiral carbon in the side chain of 8-deoxy-15-(3′-hydroxy-2′-methyl-propanoyl)-lactucin-3′-sulfate (6) has been determined by comparison of the ¹H NMR spectra of Mosher derivatives of 6 with those of the corresponding MTPA esters of model compounds.

Full text of DOI:10.1016/j.phytochem.2008.09.025

Phytochemistry 69 (2008) 2984–2992
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Chemical characterisation of the terpenoid constituents of the Algerian
plant Launaea arborescens
Fatma Bitam a,b, M. Letizia Ciavatta a, Emiliano Manzo a, Ammar Dibi b, Margherita Gavagnin a,
*
a Istituto di Chimica Biomolecolare, C.N.R., Via Campi Flegrei 34, I-80078 Pozzuoli, Naples, Italy
b Faculté des Sciences, Département de Chimie, Université de Batna, Batna 05000, Algeria
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2008
Received in revised form 29 September 2008
Available online 12 November 2008
Chemical investigation of endemic Algerian plant Launaea arborescens resulted in the isolation of a series
of triterpenes and sesquiterpenes from both the aerial parts and roots. Five terpenoids have been chem-
ically characterised by means of spectral methods mainly NMR techniques. In addition, the absolute ste-
reochemistry at the chiral carbon in the side chain of 8-deoxy-15-(30-hydroxy-20-methyl-propanoyl)-
lactucin-30-sulfate (6) has been determined by comparison of the 1H NMR spectra of Mosher derivatives
of 6 with those of the corresponding MTPA esters of model compounds.
Keywords:
Launaea arborescens
Ó 2008 Elsevier Ltd. All rights reserved.
Oleanane triterpenoids
Ursane triterpenoids
Germacrane sesquiterpenoids
Guaiane sesquiterpenoids
Eudesmane sesquiterpenoids
1. Introduction
dented terpenes, 3b-hydroxy-11
hydroxy-11b,13-dihydro-3-epi-zaluzanin
,15-dihydro-zaluzanin C (3), 3b,14-dihydroxycostunolide-3-O-
a
-ethoxy-olean-12-ene (1), 9
a-
C
(2), -hydroxy-
9a
The genus Launaea belongs to the tribe Lactuceae of the Aster-
aceae family and contains about 40 species, most of which are
adapted to dry, saline and sandy habits (Ozenda, 2004). In the
flora of Algeria, five of the nine Launaea species present are ende-
mic of North Africa and include Launaea arborescens (Batt.) Murb.,
which is a perennial medicinal shrub mainly distributed in the
Southwest part of the country (Quezel and Santa, 1963; Ozenda,
2004). Despite to the pharmacological interest in this plant (local
name ‘‘Oum Lbina”) commonly used in the North African popular
medicine against diarrhoea and abdominal spasms, a very few
chemical studies on L. arborescens have been so far reported. To
the best of our knowledge, only flavonoid (Mansour et al., 1983;
Belboukhari and Cheriti, 2006), phenolic (Giner et al., 1992) and
essential oil (Cheriti et al., 2006) constituents have been de-
scribed in the literature. In addition, very interesting antifungal,
antibacterial and insecticidal activities have been reported for
the methanol extract of the plant (Belboukhari and Cheriti,
2006; Jbilou et al., 2008).
4a
b-glycopyranoside (4), and 3b,14-dihydroxycostunolide-3-O-b-
glucopyranosyl-14-O-p-hydroxyphenylacetate (5), were isolated
and fully characterised by spectral methods, mainly NMR tech-
niques. Biological evaluation of pure novel compounds 1–5 against
gram positive and gram negative bacteria was also carried out.
Finally, a stereochemical analysis was conducted on 8-deoxy-
15-(30-hydroxy-20-methyl-propanoyl)-lactucin-30-sulfate (6) (Zid-
orn et al., 2007), which was the main component of sesquiterpene
fraction, with the aim at assigning the absolute configuration of the
chiral centre in the 30-hydroxy-20-methyl-propanoyl fragment of
the molecule, not previously determined.
2. Results and discussion
The whole plants of L. arborescens were collected in Bechar
(Algeria) during flowering in 2006. The aerial parts of the plants
were separated from the roots and both were allowed to dry before
the extraction. The TLC chromatographic analysis of the liposoluble
extracts obtained from both parts of the plant showed very com-
plex and substantially distinct terpenoid secondary metabolite
patterns even though some analogies were also observed. The ex-
tracts were submitted to subsequent chromatographic steps (see
experimental) and the terpene-containing fractions were further
purified by HPLC to obtain 27 pure metabolites, five of which were
novel compounds.
We describe here the results of our chemical investigation on
the liposoluble extracts of both roots and aerial parts of Algerian
specimens of L. arborescens that led to the finding of a plethora of
sesquiterpenes and triterpenes, which were found to be differently
distributed in both parts. Among these compounds, five unprece-
* Corresponding author. Tel.: +39 0818675096; fax: +39 0818041770.
E-mail address: margherita.gavagnin@icb.cnr.it (M. Gavagnin).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.09.025

F. Bitam et al. / Phytochemistry 69 (2008) 2984–2992
2985
In particular, the terpenoid fraction of the aerial parts extract
was found to be dominated by triterpene lupeol (7) (Reynolds
et al., 1986) (2.9 g, 25% of the extract) co-occurring with a series
of oleanane and ursane triterpenes including the novel 3b-hydro-
phenylacetate (5) were isolated along with known magnolialide
(19) (El-Feraly et al., 1979; Kisiel and Zielinska, 2001a, b), 1b,8
dihydroxy-eudesm-4-en-6b,7 ,11bH-6-olide (20) (Marco, 1989),
a-
a
11b,13-dihydrolactucin 21 (Sarg et al., 1982), picriside C (22)
(Nishimura et al., 1986a; Miyase and Fukushima, 1987), sonchu-
side A 23 (Miyase and Fukushima, 1987), picriside B (24) (Nishim-
ura et al., 1986a), ixerisoside D (25) (Warashina et al., 1990),
crepidiaside A (26) (Adegawa et al., 1985) and macrocliniside A
(27) (Miyase et al., 1984; Kisiel and Gromek, 1993). In addition,
few amounts of triterpenes containing lupeol as main component
were also detected in the ethyl acetate extract of the roots.
The known metabolites were identified by comparison of their
spectral data (1H NMR and MS spectra) with the literature. The
structures of triterpene 1 and sesquiterpenes 2–5 were determined
as described below. In particular, triterpene 1 displayed an olean-
ane skeleton, sesquiterpenes 2 and 3 exhibited a dihydroxylated
guaianolide skeleton and were both related to zaluzanin C (Romo
et al., 1967; Spring et al., 1995) whereas sesquiterpenes 4 and 5
were characterised by a glucosylated germacranolide framework
structurally related to that of the co-occurring picriside C (22)
(Nishimura et al., 1986a; Miyase and Fukushima, 1987).
xy-11a-ethoxy-olean-12-ene (1) and the known: 3-b-hydroxy-
11-oxo-urs-12-ene (8) and 3-b-hydroxy-11-oxo-olean-12-ene (9)
(Bandaranayake, 1980), taraxast-20-ene-3b,30-diol (10) (Dai
et al., 2001; Kisiel and Zielinska, 2001a), oleana-9(11):12-dien-
3b-ol (11) (Tanaka and Matsunaga, 1988), 3b-hydroxy-11
oxyurs-12-ene (12) (Fujita et al., 2000), ursa-9(11):12-dien-3b-ol
(13) (Matsunaga et al., 1988), 3b-11 -dihydroxy-olean-12-ene
(14) (Xiao et al., 1994), 3b-hydroxy-11 -methoxyolean-12-ene
(15) (Fujita et al., 2000), 3b-hydroxy-11 -methoxyurs-12-ene
(16) and 3b-11 -dihydroxy-urs-12-ene (17) (Bohlmann et al.,
a-eth-
a
a
a
a
1984). Stigmasterol (18) (Kojima et al., 1990), was also isolated
as major sterol component of the plant extract.
On the other side the terpenoid fraction of the ethyl acetate ex-
tract of the roots was characterised by a series of sesquiterpene
metabolites exhibiting eudesmane, guaiane and germacrane skele-
tons, the main of which was 8-deoxy-15-(30-hydroxy-20-methyl-
propanoyl)-lactucin-30-sulfate (6) (Zidorn et al., 2007). Four
unprecedented sesquiterpenes,
9
a
a
-hydroxy-11b,13-dihydro-3-
The molecular formula of 3b-hydroxy-11a-ethoxy-olean-12-
epi-zaluzanin C (2), 9 -hydroxy-4
a
,15-dihydrozaluzanin C (3),
ene (1) was C32H54O2 as deduced by both mass spectrum and 13C
NMR spectra. Analysis of the 1H NMR spectrum of compound 1 (Ta-
ble 1) immediately revealed the close structural relationship with
3b,14-dihydroxycostunolide-3-O-b-glycopyranoside (4), and 3b,
14-dihydroxycostunolide-3-O-b-glucopyranosyl-14-O-p-hydroxy-

2986
F. Bitam et al. / Phytochemistry 69 (2008) 2984–2992
Table 1
the co-occurring 3b,11a-dihydroxy-olean-12-ene (14) and 3b-hy-
droxy-11a-methoxy-olean-12-ene (15), suggesting the presence
1H and 13C NMR dataa,b of compound 1 in CDCl3.
Position
1
d c (m)
39.4 (t)
d H (m, J Hz)
HMBCc (C to H)
H-25
of the same functionalised triterpenoid oleanane skeleton. In fact,
eight methyl singlets [d 0.80 (H3-24), 0.83 (H3-28), 0.88 (H3-30),
0.89 (H3-29), 0.99 (6H, H3-23 and H3-26), 1.04 (H3-25), and 1.20
(H3-27)] were observed in the proton spectrum of 1 that also con-
tained two methine signals at d 3.23 (m, H-3) and 3.94 (dd, J = 9.4
and 3.3 Hz, H-11) and the olefinic proton at d 5.31 (1H, d, J = 3.3 Hz,
H-12), similarly to compounds 12 and 14 (Fujita et al., 2000; Xiao
et al., 1994), both isolated from the extract of the plant. The pres-
ence in compound 1 of an ethoxy group replacing the hydroxyl and
the methoxyl function in 14 and in 15, respectively, was indicated
by both two double quartet at d 3.31 and 3.55 (each 1 H, dq, J = 8.7
and 7.2 Hz, CH3CH2O–) and the 3H triplet at d 1.14 (J = 7.2 Hz,
CH3CH2O–). According to the proposed structure, in the 13C NMR
spectrum of 1 two additional signals due to the ethoxy moiety at
d 61.4 (CH3CH2O–) and 16.8 (CH3CH2O–) were observed along with
the typical values of the oleanane skeleton (Table 1).
The relative stereochemistry at C-11 was suggested by analysis
of the coupling constants of H-11 resonating as a double doublet
(J = 9.4 and 3.3 Hz) in agreement with a b-orientation, analogously
with the data reported for the corresponding methoxyl (Fujita
et al., 2000) and hydroxyl (Bohlmann et al., 1984; Xiao et al.,
1994) derivatives. A detailed 2D NMR analysis (1H–1H COSY, HSQC
and HMBC) allowed the assignment of all carbon and proton values
as reported in Table 1. Compound 1 underwent rapidly to an elim-
ination reaction of the ethanol residue at C-11 to give the corre-
sponding conjugated diene 11 (Tanaka and Matsunaga, 1988),
also isolated from the extract. This conversion was also observed
for 14 and 15, thus suggesting that 3b-hydroxy-olean-9(11):12-
diene (11) found in the extract is most likely a work-up derivative.
1.75 (m)
2.02 (m)
1.62 (m)
3.23 (m)
2
3
4
5
6
27.5 (t)
78.6 (d)
38.7 (s)
55.2 (d)
18.4 (t)
H-23, H-24
H-23, H-24
H-23, H-24, H-25
0.77 (m)
1.37 (m)
1.42 (m)
1.57 (m)
1.73 (m)
7
33.3 (t)
8
9
42.6 (s)
51.5 (d)
38.3 (s)
74.8 (d)
122.7 (d)
148.9 (s)
41.8 (s)
26.3 (t)
H-9, H-27
H-25, H-26
H-9, H-25
H-9
1.72 (d, 9.0)
10
11
12
13
14
15
3.94 (dd, 9.4,3.3)
5.31 (d, 3.3)
H-11
H-11, H-27
H-9, H-12, H-27
H-27
1.70 (m)
2.02 (m)
1.61 (m)
16
17
18
19
26.8 (t)
32.3 (s)
46.9 (d)
46.5 (t)
H-28
H-28
H-12, H-28
H-29, H-30
1.99 (m)
1.05 (m)
1.64 (m)
20
21
22
23
24
25
26
27
28
29
30
31.1 (s)
34.7 (t)
37.0 (t)
28.2 (q)
15.6 (q)
15.9 (q)
18.2 (q)
25.3 (q)
28.5 (q)
33.6 (q)
23.7 (q)
H-29, H-30
H-29, H-30
1.11 (m)
2.02 (m)
0.99 (s)
0.80 (s)
1.04 (s)
0.99 (s)
1.20 (s)
0.83 (s)
0.89 (s)
0.88 (s)
H-3, H-24
H-3, H-23
H-9
H-7
H-30
H-29
9a-Hydroxy-3-epi-11b,13-dihydrozaluzanin C (2) had the
molecular formula C15H20O4 as deduced by both ESIMS and 13C
NMR spectra. Analysis of the 1H and 13C NMR spectra (Table 2) of
2 indicated the presence of a guaianolide sesquiterpene skeleton
the same as the co-occurring known metabolites 21–23, 26 and
27. Accordingly, two exomethylene groups [d 4.81 (1H, br s, H-
14a), 5.08 (1H, br s, H-14b), 5.38 (1H, br s, H-15a), and 5.46 (1H,
br s, H-15b)], three oxymethine protons [d 4.58 (1H, sharp m, H-
9), 4.71 (1H, m, H-3) and d 3.81 (1H, t, J = 9.7, H-6)], and a second-
Ethoxy group
OCH2
61.4 (t)
16.8 (q)
3.31 (dq, 8.7,7.2)
3.55 (dq, 8.7,7.2)
1.14 (t, 7.2)
CH3, H-11
CH3
a
Bruker DRX 600 spectrometer in CDCl3, chemical shifts (ppm) referred to
CHCl3(d 7.26) and to CDCl3 (d 77.0).
b
Assignments made by 1H–1H COSY and HSQC experiments.
Significant HMBC correlations (J = 10 Hz).
c
Table 2
1H and 13C NMR dataa,b of compounds 2 and 3 in CDCl3.
Position
2
3
d C (m)
d H (m, J Hz)
HMBCc (C to H)
d C (m)
d H (m, J Hz)
HMBCc (C to H)
1
2
35.6 (d)
39.4 (t)
3.58 (ddd, 12.3,8.7,4.1)
2.20 (m)
H-5, H-14a, H-14b
32.5 (d)
3.41 (t)
3.32 (q, 10.1)
2.00 (m)
H-2a, H-14a, H-14b
H-l, H-4, H-14a
1.95 (m)
2.00 (m)
3
4
5
6
7
8
74.7 (d)
154.0 (s)
49.9 (d)
84.9 (d)
44.3 (d)
39.7 (t)
4.71 (m)
H-15a, H-15b, H-2a
H-5, H-6
H-15a, H-15b
H-5
H-11, H-13
H-6, H-11
74.1 (d)
40.2 (d)
46.9 (d)
82.9 (d)
41.0 (d)
38.6 (t)
4.30 (m)
2.35 (m)
2.35 (m)
4.10 (t, 8.1)
3.15 (m)
H-2a, H-15a, H-15b
H-15
H-1, H-2, H-13
H-4, H-5
H-13a, H-13b
H-6
3.10 (t, 9.7)
3.81 (t, 9.7)
2.32 (m)
1.50 (m)
1.60 (m)
2.23 (m)
2.35 (m)
9
74.3 (d)
150.9 (s)
41.6 (d)
178.4 (s)
13.6 (q)
4.58 (m)
H-8a, H-14a, H-14b
H-l, H-2a
H-6, H-13
74.1 (d)
149.8 (s)
139.3 (s)
169.8 (s)
120.1 (t)
4.72 (br s)
H-14a, H-14b
H-l, H-2a, H-2b, H-14
H-13
10
11
12
13
2.18 (m)
H-13
H-13
1.26 (d,7.2)
5.50 (d, 3.5)
6.21 (d, 3.5)
5.12 (br s)
5.16 (br s)
0.98 (d,7.1)
14
15
112.7 (t)
112.7 (t)
4.81 (br s)
5.08 (br s)
5.38 (br s)
5.46 (br s)
112.7 (t)
8.1 (q)
H-l
H-3b
H-4, H-5
a
Bruker DRX 600 spectrometer in CDCl3, chemical shifts (ppm) referred to CHCl3 (d 7.26) and to CDCl3 (d 77.0).
Assignments made by 1H–1H COSY and HSQC experiments.
Significant HMBC correlations (J = 10 Hz).
b
c

F. Bitam et al. / Phytochemistry 69 (2008) 2984–2992
2987
ary methyl [d 1.26 (3H, d, J = 7.2, H3-13)] were easily recognized by
analysis of the 1H NMR spectrum whereas the presence of a lactone
functionality was suggested by the carbonyl signal at d 178.4 in the
13C NMR spectrum. Comparison of the NMR data in pyridine of 2
with those reported in the literature for related guaianolides (Asada
et al., 1984; Kisiel and Michalska, 2002) showed a close structural
a vinyl hydroxymethyl [d H 4.10 (1H, d, J = 12.3 Hz, H-14a), d H
4.47 (1H, d, J = 12.3 Hz, H-14b] and a secondary hydroxyl group
connected to a glucopyranose moiety by a glycosyl linkage [d H
3.90 (1H, m, H-500), d H 4.10 (1H, m, H-200), d H 4.25 (1H, m, H-
300), d H 4.28 (1H, m, H-400), d H 4.42 (1H, dd, J = 12.1, 5.3 Hz, H-
600a), d H 4.61 (1H, dd, J = 12.1, 4.8 Hz, H-600b), d H 4.83 (1H, d,
J = 8.0 Hz, H-100)]. The spectral data were reminiscent with those
of the co-occurring picriside C (22) (Miyase and Fukushima,
1987) strongly suggesting the presence of the same germacrano-
lide skeleton bearing a glucosylated hydroxyl function at C-3 and
containing an additional hydroxyl group at C-14. The geometry
of the two endocyclic double bonds at C-1 (10) and C-4 (5) was
suggested Z and E, respectively, as reported in formula 4, by anal-
ogy with 22 and further supported by diagnostic NOE effects ob-
served between H2-14 and H-2a and between H-6 and H3-15,
respectively. Analogously, the trans-junction at C-6/C-7 and the
b-orientation of the O-glucosyl residue were suggested to be the
same as picriside C. A detailed analysis of 2D NMR spectra sup-
ported the proposed structure and allowed the attribution of all
carbon and proton resonances (Table 3). Our assignment was in
agreement with those reported in the literature for related ger-
macranolides exhibiting two hydroxyl groups at both C-3 and C-
14 (Nishimura et al., 1986b; Kisiel and Barszcz, 1997; 1998).
Analysis of the spectral data of 3b,14-dihydroxycostunolide-3-
O-b-glucopyranosyl-14-O-p-hydroxyphenylacetate (5) immedi-
ately revealed the close relationship with 4. The molecular formula
C29H36O11 of 5, deduced by HRESIMS spectrum on the sodiated
molecular peak at m/z 560.2155 indicated the presence of an addi-
tional C8H6O2 moiety with respect to 4. The 1H and 13C NMR spec-
tra of the two compounds were very similar according to the
presence of the same germacranolide skeleton bearing the glucosy-
lated hydroxyl function at C-3 and the hydroxyl group at C-14. Fur-
ther three signals at d 3.72 (2H, d, J = 3.5 Hz, CH2b), 7.14 (2H, d,
J = 8.2 Hz, H-30 and H-50), and 7.32 (2H, d, J = 8.2 Hz, H-20 and H-
60) were observed in the proton spectrum of 5 suggesting that
the molecule contained a p-hydroxy-phenyl acetic residue esteri-
fied to an OH group. Accordingly, the carbon spectrum contained
similarity of 2 with 9a-hydroxy-11b,13-diyhdrozaluzanin C-3-O-
b-allopyranoside (28) (Kisiel and Michalska, 2002), which is the
aglycone of glucosides previously isolated from different genera
of Asteraceae family. In particular, the spin-system sequence from
C-1 to C-9 in 2 was the same as 28 whereas differences were ob-
served in the carbon and proton chemical shifts of the 5-membered
ring strongly suggesting the opposite stereochemistry at C-3.
According to this suggestion, H-1 and H-5 values were downfield
shifted [d H-1 4.01, d H-5 3.25 in 2; d H-1 3.65, d H-5 2.91 in 28 (val-
ues in pyridine)] due to the a-oriented 3-OH group. The proposed
relative stereochemical arrangement was further supported by a
series of steric effects observed in the NOESY spectrum of 2. Along
with the expected correlations between
diagnostic cross-peaks were observed between H-2
a
oriented H-1 and H-5,
and H-1, and
a
between H-2b and both H-14 and H-3 according to the b-orienta-
tion of H-3. Analogously, H-9 showed correlations only with H-
14b and both protons H2-8 thus supporting a b-orientation. All car-
bon and proton resonances were assigned by 2D-NMR experiments
(1H–1H COSY, HSQC and HMBC) as reported in Table 2.
The molecular formula C15H20O4 of 9a-hydroxy-4a,15-dihydro
zaluzanin C (3) was established by HRESIMS on the sodiated
molecular peak at 264.1259 m/z. The spectral data of 3 (Table 2)
indicated the same guaianolide framework as compound 2 exhib-
iting two exomethylene groups [d C 112.7 (C-14), 120.1 (C-13),
139.3 (C-11), and 149.8 (C-10)]; d H 5.12 (1H, br s, H-14a), 5.16
(1H, br s, H-14b), 5.50 (1H, d, J = 3.5, H-13a), and 6.21 (1H, d,
J = 3.5, H-13b)], a secondary methyl [d C 8.1 (C-15), d H 0.98 (3H,
d, J = 7.1 Hz, H3-15)] and two secondary hydroxyl functions [d C
74.1 (2C, C-3 and C-9), d H 4.30 (1H, m, H-3) and 4.72 (1H, br s,
H-9)]. Careful analysis of the 1H–1H COSY experiment led us to de-
fine all the proton sequence from H-1 to H-9 indicating that the
two hydroxyl groups were located at C-3 and C-9 analogously with
2 whereas the secondary methyl was at C-4, and the exocyclic dou-
ble bonds were at C-10 and C-11. A survey of the literature on guai-
additional signals at d 170.4 (CO, Ca
), 41.0 (t, Cb), 125.2 (s, C-10),
131.2 (2C, d, C-20 and C-60), 116.5 (2C, d, C-30 and C-50), and
158.3 (s, C-40). Careful comparison of proton and carbon NMR spec-
tra of 5 with those of 4 showed a substantial similarity for d H and d
C values of the glucosyl moieties of both compounds whereas sig-
nificant differences were observed for C-14 values strongly sup-
porting the location of the acyl residue at 14-OH. This suggestion
was further confirmed by diagnostic HMBC correlations observed
anolides showed that 3 was structurally related to known 9
a-
hydroxy-4 ,11b,13,15-tetrahydrozaluzanin C (Kisiel and Barszcz,
a
1996). In particular, strong similarities were observed in the proton
spectra of the two molecules with regards to the perhydroazulene
bicyclic portion clearly suggesting for this part the same substitu-
tion pattern including the relative stereochemistry. The only differ-
ence was the presence in 3 of an additional double bond in the
lactone ring at C-11(13). The suggested relative stereochemistry
was further supported by a NOESY experiment that showed diag-
between the carbonyl signal at d 170.4 (Ca) and the methylene
protons at d 4.57 and 4.76 (H2-14). Thus compound 5 was the
14-O-p-hydroxy-phenyl acetic ester derivative of 4. All NMR reso-
nances were assigned as reported in Table 3 by 2D experiments.
The chemical correlation between the two molecules was finally
confirmed by hydrolysis of 5 that afforded p-hydroxy-phenyl acetic
acid and a glucosyl alcohol that resulted to be identical to 3b,14-
dihydroxycostunolide-3-O-b-glycopyranoside (4).
Finally, a stereochemical analysis was conducted on the known
compound 6 that was the main component of the sesquiterpene
pool of L. arborescens. First of all, with the aim at confirming the
absolute stereochemistry at the 6,7-junction of the guaianolide
framework, suggested as reported for the most literature natural
guaianolides, we decided to apply the Mosher method on the cor-
responding alcohol derivative obtained by opening of the lactone
ring of 6. Unfortunately, due to the rapid dehydration reaction that
was observed to occur under different methanolysis conditions,
every attempt to obtain the free secondary alcohol at C-6 was
unsuccessful. Subsequently we decided to determine the abso-
lute configuration of the chiral centre in 30-hydroxy-20-methyl-
propanoyl fragment as follows. Compound 6 was subjected to
nostic correlations between the
a-oriented protons H-1 and H-3,
and between the b-oriented H3-15 and H-6. Expected NOE interac-
tions between H-5 and both H-1 and H-7 according to the guaiano-
lide skeleton with 1,5-cis and 6,7-trans junctions were also
detected. Full assignment of proton and carbon values (Table 2)
was made by means of detailed analysis of 2D–NMR experiments
(1H–1H COSY, HSQC and HMBC).
The HRESIMS spectrum of 3b,14-dihydroxycostunolide-3-O-b-
glycopyranoside (4) showed
a sodiated molecular peak at
426.1788 m/z according to the molecular formula C21H30O9. The
1H and 13C NMR spectra of 4 indicated the presence of the follow-
ing structural features: two trisubstituted double bonds [d C 126.6
(C-1), 127.3 (C-5), 141.5 (C-4), 135.2 (C-10); d H 5.05 (1H,
overlapped, H-1), 5.02 (1H, overlapped, H-5)], an exocyclic double
bond conjugated to a lactone carbonyl [d C 119.6 (t, C-13), 139.0 (s,
C-11), 168.8 (s, C-12); d H 5.50 (1H, d, J = 2.8 Hz, H-13a) and 6.32
(1H, d, J = 2.8 Hz, H-13b)], a vinyl methyl [d H 1.95 (3H, s, H3-15),

2988
F. Bitam et al. / Phytochemistry 69 (2008) 2984–2992
Table 3
a,b
1H and 13C NMR data
of compounds 4 and 5 in C5D5N.
Position
4
5
d C (m)
d H (m,J Hz)
HMBCc (C to H)
d C(m)
d H (m,J Hz)
HMBCc (C to H)
1
2
126.6 (d)
33.0 (t)
5.05 (m)
2.68 (q, 11.8)
2.55 (m)
131.3 (d)
33.3 (t)
4.95 (dd, 10.6, 5.9)
2.50 (m)
2.58 (q)
H-9
H-3
3
4
5
6
7
8
9
10
11
12
13
83.2 (d)
141.5 (s)
127.3 (d)
81.4 (d)
50.2 (d)
28.9 (t)
4.90 (m)
H-2, H-5, H-10, H-15
H-5, H-15
H-7, H-15
82.8 (d)
140.8 (s)
127.4 (d)
81.2 (d)
50.0 (d)
29.0 (t)
4.83 (m)
H-1, H-1”, H-2, H-15
H-3, H-6, H-15
H-3, H-15
5.02 (m)
4.80 (t, 4.8)
2.52 (m)
2.15 (m) 1.90 (m)
1.90 (m) 3.15 (m)
4.98 (d, 10.2)
4.68 (t, 8.8)
2.46 (m)
1.58 (m) 1.93 (m)
1.90 (m) 2.58 (m)
H-7
H-13
H-6, H-9
H-5, H-9, H-13a, H-13b
H-6, H-7
H-1, H-8, H-14
H-9, H-14
H-6, H-7, H-13a, H-13b
H-13a, H-13b
36.9 (t)
37.0 (t)
135.2 (s)
139.0 (s)
168.8 (s)
119.6 (t)
H-2, H-9
H-13
135.9 (s)
142.2 (s)
172.0 (s)
119.8 (t)
5.50 (d, 2.8)
6.32 (d, 2.8)
4.10 (d, 12.3)
4.47 (d, 12.3)
1.95 (s)
5.49 (d, 3.5)
6.35 (d, 3.5)
4.57 (d, 12.3)
4.76 (d, 12.3)
1.85 (s)
14
58.5 (t)
12.0 (q)
61.5 (t)
12.0 (q)
H-l, H-9
H-3, H-5
15
H-5
Ester moiety
C
a
170.4 (s)
41.0 (t)
125.2 (s)
131.2 (d)
116.5 (d)
158.3 (s)
CH2b, H-14
H-20, H-60
CH2b, H-50
CH2b
CH2b
3.72 (d, 3.5)
10
20, 60
7.32 (d, 8.2)
7.14 (d, 8.2)
30, 50
40
H-20, H-30
H-3, H-2”
Sugar moiety
1”
2”
3”
4”
5”
6”a
6”b
102.8 (d)
75.3 (d)
78.6 (d)
71.7 (d)
78.6 (d)
62.9 (t)
4.83 (d, 8.0)
4.10 (m)
4.25 (m)
4.28 (m)
3.90 (m)
H-3
102.8 (d)
75.4 (d)
78.6 (d)
71.9 (d)
78.7 (d)
63.0 (t)
4.82 (d, 7.6)
4.09 (t, 8.2)
4.23 (m)
4.23 (m)
3.90 (m)
H-5”
H-3”
H-2”
H-3”
H4”, H-6”
H-4”
4.42 (dd, 12.1,5.3)
4.61 (dd, 12.1,4.8)
4.40 (dd, 11.7,5.3)
3.71 (dd, 11.7,2.3)
a
Bruker DRX 600 spectrometer in C5D5N, chemical shifts (ppm) refered to pyridine-d5 (d 8.71, 7.56, 7.19) and (d 149.9, 135.5, 123.5).
Assignmens made by1H–1H COSY and HSQC experiments.
Significant HMBC correlations (J = 10 Hz).
b
c
methanolysis in acid conditions to give the corresponding alcohol
6a, which was fully characterised as acetyl derivative 6b. Com-
pound 6a was allowed to react with R-(ꢀ) and S-(+)- -methoxy-
-trifluoromethyl-phenylacetic acid chlorides to obtain the S-
and R-MTPA ester derivatives 6c and 6d, respectively. Analysis of
the 1H NMR spectra of the two esters showed significant differ-
ences in the multiplet patterns of H2-30 (see Fig. 1). The same reac-
tion conducted on both commercial methyl-(S)-(+)-3-hydroxy-2-
methyl-propionate (I) and methyl-(R)-(ꢀ)-3-hydroxy-2-methyl-
propionate (II) afforded two pairs of MTPA derivatives, (S)/(S)-
MTPA ester (Ia), (S)/(R)-MTPA ester (Ib), (R)/(S)-MTPA ester (IIa)
and (R)/(R)-MTPA ester (IIb). Comparison of the 1H NMR spectra
of each pair with those of S- and R-MTPA esters 6c and 6d clearly
showed that H2-30 multiplet patterns of 6c and 6d were the same
as the pair Ia and Ib, thus inferring the S absolute stereochemistry
at C-20 of 6 (Fig. 1).
All terpenoids 1-5 were tested for both antifungal and antibac-
terial activity at a concentration of 5 lg/ml. No growth inhibition
was exhibited on C. albicans as well as on gram – E. coli and gram
+ S. aureus by the studied metabolites.
a
a
The terpenoid pattern of L. arborescens, described here for the
first time, shows striking similarities with those reported in the lit-
Fig. 1. 1H NMR signals (400 MHz: CDCl3) due to the methylene protons at C-3 for compounds Ia, IIa, Ib, and IIb.

F. Bitam et al. / Phytochemistry 69 (2008) 2984–2992
2989
erature for different species of the same genus (Zaheer et al., 2006;
Sokkar et al., 1993; Abdel-Fattah et al., 1990; Gupta et al., 1989;
Abdel-Salem et al., 1986; Abdel-Salem et al., 1982; Hook et al.,
1984; Majumder and Laha, 1982; Prabhu and Venkateswarlu, 1969).
(27.0 g) that was partitioned between water and ethyl acetate.
An aliquot (2.0 g) of the ethyl acetate soluble part (3.7 g) was sub-
jected to column chromatography using LH-20, to give 9 fractions
from A to I, two of which (C and G) containing terpene compo-
nents. Fraction C (260 mg) was further purified by silica gel column
chromatography using CH2Cl2/MeOH from 100% CH2Cl2 to 20%
MeOH to yield 16 fractions (C1–C16) whereas fraction G afforded
pure compound 6. Fraction C3 (21.4 mg) was subsequently sub-
jected to silica gel column chromatography using n-hexane-EtOAc
(95:5) as eluent to afford pure compound 19 (6.0 mg). Fraction C8
(25.9 mg) was purified by silica gel column chromatography (gra-
dient light petroleum ether/EtOAc) and following RP-HPLC (gradi-
ent MeOH/H2O) to give 2 (1.6 mg), 20 (1.0 mg), 21 (0.8 mg), and 3
(0.6 mg) respectively. Fraction C12 (4.6 mg) was chromatographed
on a silica gel column (CHCl3/MeOH, 95:5) and subsequently RP-
HPLC (gradient MeOH/H2O) to afford pure compounds 22
(0.2 mg), 23 (0.3 mg), 24 (0.2 mg) and 25 (0.6 mg) respectively.
Purification of fraction C13 (22.7 mg) on RP-HPLC column (gradi-
ent MeOH/H2O) led to the isolation of compounds 26 (1.2 mg)
and 5 (7.1 mg). Finally, fraction C14 (17.0 mg) was directly submit-
ted to RP-HPLC chromatography (gradient MeOH/H2O) to afford
compounds 4 (2.1 mg) and 27 (2.2 mg).
3. Experimental
3.1. General experimental procedures
Silica-gel chromatography was performed using pre-coated
Merck F254 plates and Merck Kieselgel 60 powder. Optical rotations
were measured on a Jasco DIP 370 digital polarimeter. IR spectra
were recorded on a Biorad FTS 155 FT-IR spectrophotometer. HPLC
separation was performed on a Shimazdu high-performance liquid
chromatography using a Shimadzu liquid chromatograph LC-10AD
equipped with an UV SPD-10A wavelength detector. NMR experi-
ments were recorded at ICB-NMR Service Centre. 1D and 2D
NMR spectra were acquired in CDCl3, CD3OD and pyridine-d5
(shifts are referenced to the solvent signal) on a Bruker Avance-
400 operating at 400 MHz, using an inverse probe fitted with a gra-
dient along the Z-axis and a Bruker DRX-600 operating at 600 MHz,
using an inverse TCI CryoProbe fitted with a gradient along the Z-
axis.13C NMR were recorded on a Bruker DPX-300 operating at
300 MHz using a dual probe. High resolution ESIMS were per-
formed on a Micromass Q-TOF MicroTM coupled with a HPLC
Waters Alliance 2695. The instrument was calibrated by using a
PEG mixture from 200 to 1000 MW (resolution specification 5000
FWHM, deviation <5 ppm RMS in the presence of a known lock
mass).
3.3.1. 3b-Hydroxy-11a-ethoxy-olean-12-ene (1)
Amorphous powder; ½a D25
ꢁ
ꢀ10° (c 0.10, CHCl3); IR
m
cmꢀ1
:
film
max
3852, 2956, 2919, 1457, 1379, 1163, 1080, 974; 1H and 13C NMR
values (CDCl3) see Table 1; ESIMS positive mode: m/z 425
[MꢀEtOH + H]+.
3.3.2. 9
a
-Hydroxy-11b,13-dihydro-3-epizaluzanin C (2)
Colourless oil; ½a D25
ꢁ
ꢀ3° (c 0.06, CHCl3); IR
m
cmꢀ1: 3407,
film
max
1759, 1456, 1327, 1186, 1064, 991, 910; 1H and 13C NMR values
(CDCl3) see Table 2; 1H NMR values (600 MHz, pyridine-d5): d
1.20 (3H, d, J = 6.7 Hz, H3-13), 1.48 (1H, br t, J = 11.8 Hz, H-8a),
2.17 (1H, m, H-2b), 2.30 (1H, m, H-2a), 2.30 (1H, m, H-8b), 2.30
(1H, m, H-11b), 2.61 (1H, q, J = 10.8 Hz, H-7), 3.25 (1H, t,
J = 9.7 Hz, H-5), 3.95 (1H, t, J = 9.7 Hz, H-6), 4.01 (1H, br t,
J = 8.2 Hz, H-1), 4.71 (1H, br s, H-3b), 5.13 (1H, m, H-9b), 4.88
(1H, s, H-14a), 5.05 (1H, s, H-14b), 5.60 (2H, s, H-15); 13C NMR val-
ues (150.92 MHz, pyridine-d5): d 13.3 (q, C-13), 34.7 (d, C-1), 40.3
(t, C-2), 40.7 (t, C-8), 41.7 (d, C-11), 44.5 (d, C-7), 49.9 (d, C-5),
73.8 (d, C-3), 74.8 (d, C-9), 85.7 (d, C-6), 110.6 (t, C-14), 110.8 (t,
C-15), 154.0 (s, C-10), 157.1 (s, C-4), 178.6 (s, C-12); ESIMS positive
mode: m/z 287 [M + Na]+.
3.2. Plant material
The plant L. arborescens was collected in April 2006 in Bechar
(Laabadla, South of Algeria) and identified by Prof. Bachir Oudjehih,
Institute of Agronomy of University of Batna (Algeria). A voucher
specimen is deposited in the herbarium of the department of the
same University under the number code 423/HIAB.
3.3. Extraction and isolation
Dried and powdered aerial parts (900 g) of L. arborescens were
treated with light petroleum ether (3 l, 3 times) to afford, after
evaporation of the solvent under reduced pressure, 12 g of crude
extract. This extract was subjected to silica gel column using light
petroleum ether and increasing amount of ethyl acetate up to 100%
ethyl acetate to obtain 140 fractions of 50 ml combined together
into five total fractions A (2.9 g), B (100 mg), C (535 mg), D
(300 mg) and E (400 mg). The fractions A, B, D and E containing ter-
penoid molecules were considered for further purification steps.
Fraction A was purified by silica gel column chromatography using
petroleum ether ethyl acetate (98:2) to afford 7 (1.35 g) as main
compound. Fraction B was subjected to silica gel column chroma-
tography (CH2Cl2/MeOH, 98:2) to yield 18 (1.8 mg) and a mixture
of triterpenes that was further separated by RP-HPLC using iso-
cratic elution MeOH/H2O (9:1) to give 8 (1.2 mg) and 9 (1.0 mg).
Fraction D was purified on a silica gel column in the same condi-
tions as fraction B to obtain 10 (13.0 mg). Fraction E was chromato-
graphed by a silica gel column in the same conditions as fraction A
to afford two main fractions (E1 and E2), which were passed
through analytical RP-18-HPLC (Phenomenex) using pure MeOH
as eluent to give 11 (0.6 mg), 12 (0.4 mg) and 13 (0.6 mg) from
E1 and 1 (1.5 mg), 14 (1.0 mg), 15 (1.5 mg), 16 (1.2 mg) and 17
(1.2 mg) from E2.
3.3.3. 9a-Hydroxy-4a,15-dihydro zaluzanin C (3)
Colourless oil; ½a D25
ꢁ
ꢀ11° (c 0.10, CHCl3); IR
m
cmꢀ1: 3047,
film
max
2926, 1757, 1267, 1167, 984, 770; 1H and 13C NMR (CDCl3), see Ta-
ble 2; 1H NMR values (600 MHz, pyridine-d5): d 1.28 (3H, d,
J = 7.0 Hz, H-15), 1.57 (1H, m, H-8a), 2.06 (1H, m, H-2a), 2.34 (1H,
m, H-2b), 2.43 (1H, m, H-5), 2.53 (1H, m, H-8b), 2.54 (1H, m, H-
4a), 3.43 (1H, m, H-7), 3.75 (1H, br q, J = 10.2 Hz, H-1), 4.22 (1H,
t, J = 9.7 Hz, H-6), 4.50 (1H, br s, H-3), 4.87 (1H, m, H-9b), 5.16
(2H, s, H-14), 5.43 (1H, d, J = 3.1 Hz, H-13a), 6.25 (1H, d,
J = 3.1 Hz, H-13b); 13C NMR values (150.92 MHz, pyridine-d5): d
8.41 (q, C-15), 32.6 (d, C-1), 34.4 (t, C-2), 39.4 (t, C-8), 40.4 (d, C-
4), 41.2 (d, C-7), 47.3 (d, C-5), 73.3 (d, C-9), 73.5 (d, C-3), 83.3 (d,
C-6), 110.5 (t, C-14), 118.7 (t, C-13), C-10, C-11 and C-12 were
not detected; ESIMS positive mode: m/z 287 [M + Na]+, HRESIMS:
m/z 287.1214 (calcd for C15H20O4Na, 287.1259).
3.3.4. 3b, 14-Dihydroxycostunolide-3-O-b-glycopyranoside (4)
Amorphous powder; ½a D25
ꢁ
+4° (c 0.10, MeOH); IR
m
cmꢀ1
;
film
max
3420, 2926, 2861, 1757, 1556, 1416; 1286, 1234; 1H and 13C
NMR (pyridine-d5) see Table 3; HRESIMS positive m/z 449.1776
(calcd for C21H30O9Na, 449.1788).
Dried and powdered roots (1 kg) of L. arborescens were macer-
ated with methanol (7 l) to give a crude methanolic extract

2990
F. Bitam et al. / Phytochemistry 69 (2008) 2984–2992
3.3.5. 3b,14-Dihydroxycostunolide-3-O-b-glucopyranosyl-14-O-p-
by Pasteur-pipette silica gel chromatography using light petroleum
hydroxyphenylacetate (5)
ether-ethyl acetate (60:40) to give 1.7 mg of pure 6b.
Amorphous powder; ½a D25
ꢁ
ꢀ0.4° (c 0.55, MeOH); IR
m
cmꢀ1
:
Colourless oil; ½a D25
ꢁ
+8° (c 0.17, CHCl3); IR
m
cmꢀ1: 1718, 1688,
film
max
film
max
3393, 2922, 1738, 1616, 1516, 1448, 1259, 1145, 1018; 1H and
13C NMR (pyridine-d5) see Table 3; 1H NMR values (600 MHz,
CDCl3): d 1.32 (1H, m, H-8a), 1.48 (3H, s, H-15), 1.80 (1H, m, H-
8b), 1.88 (1H, m, H-9a), 2.25 (1H, q, J = 12.0 Hz, H-2a), 2.38 (1H,
m, H-2b), 2.38 (1H, m, H-7), 2.40 (1H, m, H-9b), 4.30 (1H, m, H-
14a), 4.35 (1H, t, J = 8.8 Hz, H-6), 4.38 (1 H, m, H-3), 4.40 (1H, m,
H-14b), 4.82 (1H, d, J = 10.0 Hz, H-5), 4.95 (1H, dd, J = 12.0,
3.5 Hz, H-1), 5.50 (1H, d, J = 3.8 Hz, H-13a), 6.31 (1H, d, J = 3.8 Hz,
H-13b), ester moiety: d 3.40 (2H, s, H2-b), 6.60 (2H, d, J = 8.3 Hz,
H-30 and H-50), 6.90 (2H, d, J = 8.3 Hz, H-20and H-60), sugar moiety:
3.15 (1H, m, H-500), 3.18 (1H, m, H-200), 3.25 (1H, m, H-300), 3.27 (1H,
m, H-400), 3.62 (1H, dd, J = 12.0, 4.8 Hz, H-600a), 3.71 (1H, dd, J = 12.0,
2.6 Hz, H-600b); 4.10 (1H, d, J = 8.0 Hz, H-100); 13C NMR values
(75.46 MHz, CDCl3): d 11.0 (q, C-15), 28.8 (t, C-8), 32.2 (t, C-2),
36.7 (t, C-9), 49.4 (d, C-7), 61.5 (t, C-14), 81.2 (d, C-6), 81.3 (d, C-
3), 120.6 (t, C-13), 126.3 (d, C-1), 131.0 (d, C-5), 134.6 (s, C-10),
141.1 (s, C-4), 139.0 (s, C-11), 170.8 (s, C-12), ester moiety: d 40.6
1621, 1373, 1251, 1135, 989; 1H-NMR spectral data (600 MHz,
CDCl3): d 1.45 (1H, q, J = 12.0 Hz, H8-a), 2.22 (1H, m, H-8b), 2.24
(1H, m, H-9a), 2.44 (3H, s, H-14), 2.56 (1H, br t, J = 13.0 Hz, H-9b),
2.85 (1H, m, H-7), 3.58 (1H, t, J = 10.0 Hz, H-6), 3.70 (1H, d,
J = 10.0 Hz, H-5), 5.05 (1H, d, J = 17.1 Hz, H-15a), 5.31 (1H, d,
J = 17.1 Hz, H-15b), 5.48 (1H, d, J = 3.2, H-13a), 6.20 (1H, d,
J = 3.2 Hz, H-13b), 6.32 (1H, br s, H-3), ester moiety: d 1.25 (3H,
d, J = 7.3 Hz, H-40), 2.85 (1H, m, H-20), 4.22 (2H, d, J = 8.0 Hz, H-30),
2.07 (3H, s, CH3CO); 13C-NMR values (75.46 MHz, CDCl3): d 22.2
(q, C-14), 24.1 (t, C-8), 37.3 (t, C-9), 50.0 (d, C-5), 52.0 (d, C-7),
63.8 (t, C-15), 83.7 (d, C-6), 119.0 (t, C-13), 130.9 (s, C-1), 133.5
(d, C-3), 138.6 (s, C-11), 154.9 (s, C-10), 166.8 (s, C-4), 169.2 (s, C-
12), 195.6 (s, C-2), ester moiety: d 13.5 (q, C-40), 21.5 (q, CH3CO),
39.5 (d, C-20), 63.8 (t, C-30), 171.0 (s, CH3COO), 173.2 (s, C-10); ESIMS
positive mode: m/z 411 [M + Na]+.
3.4.3. 8-Deoxy-15-(30-hydroxy-20-methyl-propanoyl)-lactucin-30-(S)-
MTPA-ester (6c)
Compound 6c was prepared by treating 1 mg of 6a in pyridine
(1 ml) with R-(ꢀ)-MTPA chloride (0.07 ml) at room temperature
overnight. After usual work up, the residue was purified by Pas-
teur-pipette silica gel chromatography using light petroleum
(t, CH2-
a
), 115.2 (d, C-30 and C-50), 124.2 (s, C-10), 130.0 (d, C-20
and C-60), 155.9 (s, C-40), 172.0 (s, C-
a
), sugar moiety: d 61.6 (t,
C00-6), 70.0 (d, C00-4), 73.3 (d, C00-2), 76.3 (d, C00-3), 75.7 (d, C00-5)
100.7 (d, C00-1), ESIMS positive mode: m/z 560 [M + Na]+, HRESIMS:
m/z 583.2181 (calcd for C29H36O11Na, 583.2155).
ether-ethyl acetate (60:40) to give 0.4 mg of pure ester 6c.
3.3.6. 8-Deoxy-15-(30-hydroxy-20-methyl-propanoyl)-lactucin-30-
Colourless oil; ½a D25
ꢁ
+21° (c 0.04, CHCl3); IR
m
cmꢀ1: 1784,
film
max
sulfate (6)
1744, 1684, 1630, 1448, 1381, 1273, 1180, 1113, 970; 1H NMR val-
ues (400 MHz, CDCl3): d 1.44 (1H, m, H8-a), 2.03 (1H, m, H-8b), 2.39
(1H, m, H-9a), 2.45 (3H, s, H-14), 2.56 (1H, br t, 13.0 Hz, H-9b), 2.94
(1H, m, H-7), 3.47 (1H, t, J = 7.0 Hz, H-6), 3.59 (1H, d, J = 8.9 Hz, H-
5), 4.97 (1H, d, J = 17.5 Hz, H-15a), 5.17 (1H, d, J = 17.5 Hz, H-15b),
5.48 (1H, d, J = 3.2 Hz, H-13a), 6.20 (1H, d, J = 3.2 Hz, H-13b), 6.25
(1H, br s, H-3), ester moiety: d 1.23 (3H, d, J = 7.3 Hz, H-40), 2.27
(1H, m, H-20), 4.42 (1H, dd, J = 5.4, 10.8 Hz, H-30a), 4.49 (1H, dd,
J = 7.3, 10.8 Hz, H-30b); ESIMS positive mode: m/z 585 [M + Na]+.
Colourless amorphous solid; ½a D25
ꢁ
+7° (c 0.20, CHCl3); 1H NMR
values (600 MHz, CDCl3): d 1.40 (1H, m, H-8a), 2.15 (1H, m, H-
2a), 2.35 (1H, m, H-9a), 2.55 (3H, s, H-14), 2.56 (1H, t, J = 12.8 Hz,
H9-b), 2.90 (1H, m, H-7), 3.58 (1H, t, J = 10.0 Hz, H-6), 3.77 (1H,
d, J = 10.0 Hz, H-5), 5.00 (1H, d, J = 17.6 Hz, H-15 a), 5.35 (1H, d,
J = 17.6 Hz, H-15 b), 5.45 (1H, s, H-13a), 6.12 (1H, s, H-13b), 6.37
(1H, s, H-3), ester moiety: d 1.17 (3H, d, J = 7.0 Hz, H-40), 2.90
(1H, m, H-20), 4.18 (2H, m, H-30); 13C NMR values (75.46 MHz,
CDCl3): d 22.2 (q, C-14), 24.1 (t, C-8), 37.3 (t, C-9), 50.0 (d, C-5),
52.0 (d, C-7), 63.8 (t, C-15), 83.8 (d, C-6), 119.0 (t, C-13), 130.8 (s,
C-1), 133.5 (d, C-3), 138.6 (s, C-11), 154.9 (s, C-10), 166.9 (s, C-4),
169.3 (s, C-12), 195.6 (s, C-2), ester moiety: d 13.5 (q, C-40), 39.5
(d, C-20), 69.3 (t, C-30), 174.2 (s, C-10).
3.4.4. 8-Deoxy-15-(30-hydroxy-20-methyl-propanoyl)-lactucin-30-(R)-
MTPA-ester (6d)
Compound 6d was prepared by treating 1.0 mg of 6a in pyridine
(0.5 ml) of S-(ꢀ)-MTPA chloride (0.07 ml) at room temperature
overnight. The reaction mixture was purified as described for 6c
3.4. Preparation of the ester derivatives of compound 6
to obtain pure 6d (1.2 mg).
Colourless oil; ½a D25
ꢁ
ꢀ40° (c 0.12, CHCl3); IR
m
cmꢀ1: 1751,
film
max
3.4.1. 8-Deoxy-15-(30-hydroxy-20-methyl-propanoyl)-lactucin (6a)
Compound 6 (2.0 mg) was dissolved in H2SO4/MeOH (3 drops in
1 ml) and stirred for 10 min. After usual work up, the residue was
chromatographed by silica gel column (light petroleum ether/
1684, 1616, 1448, 1381, 1267, 1167, 1113, 1032; 1H NMR values
(400 MHz, CDCl3): d 1.43 (1H, m, H8-a), 2.22 (1H, m, H-8b), 2.40
(1H, m, H-9b), 2.45 (3H, s, H-14), 2.52 (1H, m, H-9a), 2.94 (1H, m,
H-7), 3.59 (1H, m, H-6), 3.66 (1H, d, J = 10.2 Hz, H-5), 5.00 (1H, d,
J = 17.4, H-15a), 5.18 (1H, d, J = 17.4 Hz, H-15b), 5.48 (1H, d,
J = 3.2 Hz, H-13a), 6.20 (1H, d, J = 3.2 Hz, H-13b), 6.27 (1H, br s,
H-3), ester moiety: d 1.24 (3H, d, J = 6.7 Hz, H-40), 2.87 (1H, m, H-
20), 4.40 (1H, dd, J = 5.7, 10.8 Hz, H-30a), 4.53 (1H, dd, J = 6.7,
10.8 Hz, H-30b); ESIMS positive mode: m/z 585 [M + Na]+.
EtOAc, 50:50) to afford 1.2 mg of pure compound 6a.
Colourless oil, ½a D25
ꢁ
+26.1° (c 0.12, CHCl3), IR
m
cmꢀ1: 3434,
film
max
2936, 2836, 2861, 1784, 1738, 1684, 1630, 1549, 1259, 1140,
1051, 978; 1H NMR values (400 MHz, CDCl3): d 1.46 (1H, m, H8-
a), 2.23 (1H, m, H-8b), 2.42 (1H, m, H-9a), 2.45 (3H, s, H-14), 2.54
(1H, br t, J = 12.7 Hz, H-9b), 2.89 (1H, m, H-7), 3.62 (1H, t,
J = 10.2 Hz, H-6), 3.71 (1H, d, J = 10.2 Hz, H-5), 5.09 (1H, d,
J = 16.9 Hz, H-15a), 5.31 (1H, d, J = 16.9 Hz, H-15b), 5.48 (1H, d,
J = 3.2, H-13a), 6.20 (1H, d, J = 3.2 Hz, H-13b), 6.35 (1H, br s, H-3),
ester moiety: d 1.23 (3H, d, J = 7.3 Hz, H-40), 2.77 (1H, m, H-20),
3,76 (2H, m, H-30); ESIMS positive mode: m/z 369 [M + Na]+; HRE-
SIMS: m/z 369.1298 (calcd for C19H22O6Na, 369.1314).
3.5. Preparation of model Mosher esters
3.5.1. Methyl-(S)-(+)-3-hydroxy-2-methyl propionate-S-MTPA-ester
(Ia)
Compound Ia was prepared by treating 0.1 ml of methyl (S)-(+)-
3-methyl propionate with 0.1 ml of R-MTPA chloride in dry CH2Cl2
(1 ml) with catalytic amount of DMAP under stirring overnight at
room temperature. After usual work up, the reaction mixture
was purified by silica gel chromatography using light petroleum
3.4.2. 8-Deoxy-15-(30-hydroxy-20-methyl-propanoyl)-lactucin-30-
acetate (6b)
Acetyl derivative 6b was prepared by treating 2.0 mg of com-
pound 6a with acetic anhydride (2 drops) in pyridine (1 ml) at
room temperature. After usual work-up the product was purified
ether-ethyl acetate (90:10) to get pure Ia (9.0 mg).
Oil; ½a 2D5
ꢁ
ꢀ25° (c 0.90, CHCl3); IR
m
cmꢀ1: 1751, 1643, 1570,
film
max
1436, 1272, 1171, 1123, 1082; Selected 1H NMR values

F. Bitam et al. / Phytochemistry 69 (2008) 2984–2992
2991
(400 MHz, CDCl3): d 4.38 (1H, dd, J = 5.7, 10.8 Hz, H-30a), 4.47 (1H,
dd, J = 7.3, 10.8 Hz, H-30b).
by using the same method as the antifungal test, only differing in
the assay medium (Luria Bertani medium: 10 g/l Bactotryptone,
5 g/l Bactoyeast and 10 g/l NaCl, pH 7.5) and in the incubation tem-
perature (37 °C for 24 h).
3.5.2. Methyl-(S)-(+)-3-hydroxy-2-methyl propionate-R-MTPA-ester
(Ib)
Compound Ib was prepared by treating 0.1 ml of methyl (S)-(+)-
3-methyl propionate with 0.1 ml of S-MTPA chloride in dry CH2Cl2
(1 ml) with catalytic amount of DMAP under stirring overnight at
room temperature. After usual work up the reaction mixture was
purified by silica gel chromatography using light petroleum
ether-ethyl acetate (90:10) to get pure compound Ib (30 mg).
Acknowledgements
The NMR spectra were recorded at the ICB NMR Facility, the
staff of which is gratefully acknowledged. Thanks are also due to
Mr C. Iodice for spectrophotometric measurements and to Mr M.
Zampa for mass spectra and Mrs D. Melck of ICB for antimicrobial
tests. F.B. is deeply grateful to the Ministry of Superior Education
and Scientific Research, Algeria for financial support. The authors
kindly thank Mr A. El Aloui of University of Bechar for collecting
the plant material.
Oil; ½a 2D5
ꢁ
+47° (c 3.0, CHCl3), IR
m
cm : 1751, 1643, 1570, 1436,
-1
film
max
1272, 1171, 1123, 1082; Selected 1H NMR values (400 MHz,
CDCl3): d 4.38 (1H, dd, J = 5.7, 10.8 Hz, H-30a), 4.49 (1H, dd, J = 6.7,
10.8 Hz, H-30b).
3.5.3. Methyl-(R)-(ꢀ)-3-hydroxy-2-methyl propionate-S-MTPA-ester
(IIa)
References
Compound IIa was prepared by treating 0.1 ml of methyl (R)-
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CH2Cl2 (1 ml) with catalytic amount of DMAP under stirring over-
night at room temperature. After the usual work up, pure com-
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Oil, ½a 2D5
ꢁ
ꢀ46° (c 0.23, CHCl3), IR
m
cmꢀ1: 1731, 1583, 1494,
film
max
1436, 1274, 1122, 1082; Selected 1H-NMR values (400 MHz,
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Compound IIb was prepared by treating 0.1 ml of methyl (R)-
(+)-3-methyl propionate with 0.1 ml of S-MTPA chloride in dry
CH2Cl2 (1 ml) with catalytic amount of DMAP under stirring over-
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Oil; ½a 2D5
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m
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max
film
1436, 1274, 1122, 1082; Selected 1H-NMR values (400 MHz,
CDCl3): d 4.38 (1H, dd, J = 5.72, 10.8 Hz, H-30a), 4.47 (1H, dd,
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