Secondary metabolites from two species of pulicaria and their cytotoxic activity

Article abstract of DOI:10.1002/cbdv.201000324

Two new compounds, the sesquiterpene (1E,5E)-8β-acetoxy-4α- hydroxy-7βH-germacra-1(10),5-dien-14-oic acid (2), and a nor-sesquiterpene, (5E)-8β-acetoxy-4α-hydroxy-7βH-germacr-5-en-10-one (3), were isolated from Pulicaria canariensis ssp. lanata, along with ten known compounds, including the flavonoid 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (4). From Pulicaria burchardii, we isolated seven known compounds; the physical and spectroscopic data of the triterpenoid 3β-hydroxytaraxaster-20-en-30-al (1) are reported. The structures of compounds 1-3 were determined on the basis of HR-MS, and 1D- and 2D-NMR studies. The structure of 2 was corroborated by X-ray crystal diffraction. Cell viability experiments revealed that the semisynthetic flavonoid 4b was the most cytotoxic compound against human leukemia cells, and the cytotoxicity was caused by induction of apoptosis, as determined by microscopy of nuclear changes.

Full text of DOI:10.1002/cbdv.201000324

2080
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Secondary Metabolites from Two Species of Pulicaria and Their Cytotoxic
Activity
by Jorge Trianaa), Mariana Lo´peza), Francisco Javier Pe´reza), Francisco Leo´nb), Jose´ Quintanac)d),
Francisco Este´vezc)d), Juan C. Herna´ndezb), Javier Gonza´lez-Platase), Ignacio Brouardb), and
Jaime Bermejo*a)
a) Departamento de Quꢀmica, Universidad de Las Palmas de Gran Canaria, Unidad Asociada al CSIC,
Campus de Tafira, SP-35017, Las Palmas de Gran Canaria
) Instituto de Productos Naturales y Agrobiologꢀa, CSIC - Instituto Universitario de Bio-Organica
b
´
´
´
ꢁAntonio Gonzalezꢂ, Avda. Astrofꢀsico F. Sanchez, 3, SP-38206, La Laguna
(phone: þ34-922-256847 ext. 224; fax: þ34-922-260135; e-mail: jbermejo@ull.es)
c) Departamento de Bioquꢀmica, Facultad de Medicina, Universidad de Las Palmas de Gran Canaria,
´
Avda. S. Cristobal, SP-35016, Las Palmas de Gran Canaria
d
´
´
) Instituto Canario de Investigacion del Cancer, ICIC
e
´
) Departamento de Fꢀsica Fundamental II, Servicio Integrado de Difraccion de Rayos X, Universidad
´
de La Laguna, Avda. Astrofꢀsico F. Sanchez, 4, 38206, SP-La Laguna
Two new compounds, the sesquiterpene (1E,5E)-8b-acetoxy-4a-hydroxy-7bH-germacra-1(10),5-
dien-14-oic acid (2), and a nor-sesquiterpene, (5E)-8b-acetoxy-4a-hydroxy-7bH-germacr-5-en-10-one
(3), were isolated from Pulicaria canariensis ssp. lanata, along with ten known compounds, including the
flavonoid 5,3’-dihydroxy-3,7,4’-trimethoxyflavone (4). From Pulicaria burchardii, we isolated seven
known compounds; the physical and spectroscopic data of the triterpenoid 3b-hydroxytaraxaster-20-en-
30-al (1) are reported. The structures of compounds 1–3 were determined on the basis of HR-MS, and
1D- and 2D-NMR studies. The structure of 2 was corroborated by X-ray crystal diffraction. Cell viability
experiments revealed that the semisynthetic flavonoid 4b was the most cytotoxic compound against
human leukemia cells, and the cytotoxicity was caused by induction of apoptosis, as determined by
microscopy of nuclear changes.
Introduction. – The Pulicaria genus (Asteraceae: Inuleae, Inulinae) consists of ca.
100 species distributed throughout Europe, North Africa, Canary Islands, Cape Verde,
and Asia, and many of these plants are used as traditional herbal medicines [1]. In the
Canary Islands, this genus is represented by two species which are located in the eastern
islands (Lanzarote and Fuerteventura), Pulicaria burchardii Hutch and P. canariensis
Bolle, the latter having two endemic subspecies canariensis and lanata [2]. Phyto-
chemical studies of Pulicaria species have led to the isolation of phenolic derivatives,
flavonoids, terpenes, especially sesqui- and triterpenoids, and fatty acids among others,
and some of them show significant bioactivities [1]. We have previously reported on the
isolation and structure elucidation of flavonoids, sesquiterpenes, and sterols from P.
canariensis ssp. canariensis [3].
As a part of our continuing search for novel, plant-derived cancer chemother-
apeutic agents and our systematic investigation of the composition of Canarian
endemic plants [4], we have studied the constituents of two species of the genus
ꢃ 2011 Verlag Helvetica Chimica Acta AG, Zꢄrich

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2081
Pulicaria. Here, we describe the identification of two new germacrane sesquiterpenoids
and 17 known compounds from the EtOH extracts of the aerial part from P. canariensis
ssp. lanata and P. burchardii.
From P. burchardii, we isolated seven known compounds including triterpenoids,
i.e., 3b-hydroxytaraxaster-20-en-30-al (1) [5], the HR-MS, and 1H- and 13C-NMR data
(Table 1) of which have not been previously reported in the literature, and 11-oxo-a-
amyrin [6]; the aromatic compounds vanillin, 4-hydroxybenzoic acid, and scopoletin
[7]; and the sterols stigmasterol [8], and b-sitosterol [9]. Additionally, from P.
canariensis ssp. lanata, two new sesquiterpenes, 2 and 3, along with the known
compounds 5,3’-dihydroxy-3,7,4’-trimethoxyflavone (4) [10], ferulic acid [7], stigmas-
terol [8], b-amyrin [11], 3b,16b-dihydroxylupeol palmitate [12], 3b,16b-dihydroxylu-
peol 3-myristate [12], calenduladiol [13], and the sesquiterpenoids pulicanadienals A
and B, and pulicanadiene B [3]. The structures of the known compounds were
confirmed by comparison of their spectroscopic data (MS, and 1H- and 13C-NMR) with
those in the literature.
To assess whether some of the isolated compounds and derivatives exhibit cytotoxic
activities, we studied their effects on the viability of the human myeloid leukemia cell
line HL-60.
Results and Discussion. – Isolation and Structure Determination. The aerial parts of
P. canariensis ssp. lanata and P. burchardii were extracted with EtOH and the resulting
extracts were separated by column chromatography, medium-pressure column
chromatography (MPLC), and preparative TLC.

2082
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Compound 1 was obtained as an amorphous solid, a OH (3220 cmꢀ1) and an a,b-
unsaturated CO (1657 and 1630 cmꢀ1) absorptions were observed in its IR spectrum.
The UV absorption at lmax 236 was consistent with the presence of a conjugation in the
molecule. The HR-EI-MS of 1 showed a molecular ion at m/z 440.3657 corresponding
to the molecular formula C30H48O2 (calc. 440.3654). The 13C-NMR spectrum of 1
(Table 1) showed signals for 30 C-atoms. The DEPT spectra indicated the presence of
seven Me, nine CH2, eight CH, groups and six quaternary C atoms. An olefinic signal at
d(C) 149.3 C(21) and 148.5 (C(20)) corresponding to the endocyclic C¼CH, and signals
of an O-bearing CH group at d(C) 79.0 (C(3)) and of a CO C-atom at d(C) 194.1
(C(30)), were observed; this latter showed a correlation in the HSQC experiment with
the signal at d(H) 9.39 (s, HꢀC(30)) in 1H-NMR (Table 1), indicating the presence of
an aldehyde group in the molecule. The 1H-NMR spectrum (Table 1) showed signals of
six tertiary Me groups, one secondary Me group, and a of O-bearing CH group at d(H)
3.22 (dd, J¼5.4, 11.1, HꢀC(3)). All these data suggested the presence of a taraxasterol-
type triterpene skeleton with a Me group oxidized to aldehyde. The presence of a
secondary OH group was confirmed by acetylation of 1 with Ac2O and pyridine,
1
yielding the acetyl derivative 1a, for which the H- and 13C-NMR spectra showed
chemical shifts identical to those reported for 3b-acetoxytaraxaster-20-en-30-al [5].
From the above findings, the structure of 3b-hydroxytaraxaster-20-en-30-al was
assigned to 1. Literature search [5][14] led us to conclude that it is the first time of
reporting the physical and spectroscopic data of 1 (see Exper. Part and Table 1).
a
Table 1. NMR Data of 1 in CDCl3
d(C)
)
d(H) (J in Hz)
d(H) (J in Hz)
d(C)
CH2(1)
CH2(2)
HꢀC(3)
C(4)
HꢀC(5)
CH2(6)
CH2(7)
C(8)
0.88–0.92 (m), 1.70–1.74 (m)
1.60–1.68 (m)
3.22 (dd, J¼5.4, 11.1)
38.7
27.4
79.0
38.8
55.3
18.2
34.2
40.9
50.3
37.1
21.4
27.2
39.0
42.3
26.8
CH2(16)
C(17)
2.04 (br. d, J¼6.1)
29.3
34.8
48.2
29.7
148.5
149.3
43.0
28.0
16.3
16.0
15.4
14.7
17.5
23.1
194.1
HꢀC(18)
HꢀC(19)
CH(20)
HꢀC(21)
CH2(22)
Me(23)
Me(24)
Me(25)
Me(26)
Me(27)
Me(28)
Me(29)
HꢀC(30)
1.15 (dd, J¼8.1, 10.6)
2.25–2.31 (m)
0.70–0.75 (m)
1.35–1.39 (m), 1.51–1.61 (m)
1.40–1.42 (m)
6.73 (dd, J¼3.2, 6.0)
1.35 (s)
0.79 (s)
0.87 (s)
0.99 (s)
1.05 (s)
0.99 (s)
0.69 (s)
HꢀC(9)
1.28–1.30 (m)
C(10)
CH2(11)
CH2(12)
HꢀC(13)
C(14)
1.28–1.32 (m)
1.28–1.32 (m), 1.73–1.77 (m)
1.73–1.77 (m)
1.04 (d, J¼7.3)
CH2(15)
1.07–1.11 (m), 1.66–1.69 (m)
9.39 (s)
a) 1H-NMR at 400 and 13C-NMR at 100 MHz.
Compound 2 was isolated as colorless needles, and its molecular formula was
1
determined as C17H26O5 by HR-FAB-MS (m/z 333.1683 [MþNa]þ ). The H- and
13C-NMR spectra recorded at 258 had a poor resolution; however when the experi-
ments were performed at 508, assignment of the signals was accomplished. The
13C-NMR spectrum (Table 2) and DEPT spectra gave a total of 17 separate C-atom

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2083
resonances (for four Me, three CH2, and six CH groups, and four quaternary C-atoms),
including those of an O-bearing CH group at d(C) 72.7 (C(8))1), an O-bearing
quaternary C-atom at d(C) 74.1 (C(4)), an AcO group at d(C) 21.7 and 173.0, an a,b-
unsaturated carboxylic acid at d(C) 145.8, 128.8, and 173.0 (C(1), C(10), and C(14),
resp.), and an iPr group at d(C) 17.4, 21.3, and 29.4 (Me(13), Me(12), and C(11), resp.).
The 1H-NMR data (Table 2) were similar to those of pulicanadienal B [3], except for
the absence of the aldehyde H-atom signal. This led us to conclude that the aldehyde
group could be oxidized to a COO group. The relative configuration of 2 was
determined from the NOESY spectrum. Important correlations were observed
between HꢀC(8), HꢀC(6), Me(12), and Me(13) as well as between Me(15),
HꢀC(5), and HꢀC(7), leading to the conclusion that the configuration of the
C(5)¼C(6) bond is (E), and the orientation of AcO group as b. The IR absorptions at
3177, 1693, and 1635 cmꢀ1 confirmed the presence of an a,b-unsaturated carboxylic
acid. In fact, methylation with CH2N2 yielded 2a, its 1H-NMR spectrum was recorded at
708 (Table 2) showing a new singlet at d(H) 3.75 (s, MeOC¼O, C(14)). From these data,
the new compound 2 was deduced as (1E,5E)-8b-acetoxy-4a-hydroxy-7bH-germacra-
1(10),5-dien-14-oic acid, and the structure could be confirmed by a single-crystal X-ray
diffraction analysis of 2 (Fig. 1).
Fig. 1. ORTEP Drawing of compound 2
Compound 3 was obtained as amorphous solid. The HR-FAB-MS of 3 exhibited the
[MþNa]þ ion peak at m/z 305.1720 (calc. 305.1729), corresponding to the molecular
formula C16H26O4. The IR absorptions of 3 revealed the presence of OH (3541 cmꢀ1
)
and CO (1729 and 1682 cmꢀ1) groups. The 13C-NMR spectrum (Table 2) displayed 16
separate C-atom resonances (for four Me, four CH2, and five CH groups, and three
quaternary C-atoms), in agreement with the molecular formula and including two
1
)
Germacrane numbering. For systematic names, see the Exper. Part.

2084
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 2. 1H- and 13C-NMR Data of Compounds 2, 2a, and 3 (J in Hz)
Position 2a)
2ab)
3b)
d(H)
d(C) d(H)
d(C)
d(H)
d(C)
1
2
6.34 (dd, J¼5.0, 11.0) 145.8 6.46 (dd, J¼6.7, 10.0) 144.2
2.38–2.42 (m)
45.4
16.3
1.82–1.88 (m),
25.3 2.27–2.35 (m),
1.38–1.44 (m)
25.1
1.53–1.57 (m)c),
2.10–2.20 (m)c)
1.45–1.49 (m)c),
1.76–1.84 (m)c)
2.28 (br. q, J¼11.0)
3
1.75 (dd, J¼8.5, 13.5), 41.4 1.63–1.67 (m),
40.5
42.2
1.53 (dd, J¼10.0, 13.5)
1.82–1.90 (m)
4
5
6
7
8
9
74.1
142.6 5.30 (d, J¼15.3)
74.2
140.7
72.2
138.7
5.16–5.23 (m)c)
5.50 (d, J¼15.5)
5.16–5.23 (m)c)
121.5 5.24 (dd, J¼10.3, 15.0) 121.7
5.23 (dd, J¼10.8, 15.5) 126.6
2.02 (td, J¼3.0, 10.0)
54.1 2.16 (dt, J¼3.3, 10.3)
52.0
71.6
2.19 (dt, J¼5.0, 10.5)
5.39 (dt, J¼3.3, 10.6)
55.7
74.1
4.86 (br. d, J¼10.0)
72.7 5.03–5.07 (m)
2.68 (dd, J¼4.0, 15.0), 30.1 2.63 (dd, J¼3.1, 15.0), 31.0
2.57 (dd, J¼11.0, 14.5), 48.5
2.66 (dd, J¼3.5, 14.5)
207.8
2.57 (dd, J¼10.0, 13.5)
2.83 (dd, J¼4.8, 15.0)
10
11
12
13
14
15
AcO
128.8
128.7
28.3
21.7
17.5
169.9
30.0
1.58–1.64 (m)
0.73 (d, J¼7.0)
0.72 (d, J¼7.0)
29.4 1.70–1.76 (m)
21.3 0.84 (d, J¼6.8)
17.4 0.79 (d, J¼6.9)
173.0
29.1 1.32 (s)
21.7, 2.05 (s)
173.0
1.90–1.94 (m)
0.91 (d, J¼7.0)
0.97 (d, J¼7.0)
26.6
21.7
16.3
–
30.1
21.1,
170.0
–
1.17 (s)
1.90 (s)
1.27 (s)
21.5, 170.9 2.08 (s)
MeO
–
–
3.75 (s)
52.0
–
a) Recorded in CD3OD. b) Recorded in CDCl3. c) Signals overlapped.
olefinic signals at d(C) 126.6 C(6) and 138.7 (C(5)), an O-bearing CH group at d(C)
1
74.1 (C(8)), and Ac group signals at d(C) 21.1 and 170.0. Both the 13C- and H-NMR
(Table 2) data of compound 3 were similar to those of 2. The major differences were the
absence of a C¼C bond between C(1) and C(10), the absence of the COO group signal
for C(14), and the appearance of a new signal at d(C) 207.8 C(10), characteristic of an
isolated ketone CO group. This suggests, together with the molecular formula and the
above mentioned functionalities, that the compound 3 is a germacrane-type norses-
quiterpene. Assignments were carried out based on 2D-NMR data. Thus, HMBCs of
the signals at d(H) 2.40, with those of C(10) (d(C) 207.8); d(H) 2.15, with those of
C(10) (d(C) 207.8) and C(4) (d(C) 72.2); d(H) 5.50, with those of C(15) (d(C) 30.1),
C(6) (d(C) 126.6), and C(7) (d(C) 55.7); d(H) 5.23, with those of C(8) (d(C) 74.1),
C(11) (d(C) 26.6); and d(H) 2.66, with those of C(8) (d(C) 74.1), C(10) (d(C) 207.8)
gave the positions of the different groups. The configuration of the C(5)¼C(6) bond
was determined as (E) by considering the coupling constant (J(5,6)¼15.5). The
relative configuration of 3 was determined by the correlation between Me(15), and
HꢀC(5) and HꢀC(7) observed in a ROESY experiment, indicating that HOꢀC(4) in 3
has the same relative configuration that 2, and leading to the conclusion that 3 is a new
norsesquiterpenoid, (5E)-8b-acetoxy-4a-hydroxy-7bH-germacra-5-en-10-one. Norger-
macrane sesquiterpenoids such 3 are rare, only a few compounds of this type have been

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2085
isolated [15]. To the best of our knowledge, compound 3 is the first example of a 14-
norgermacrane isolated from a natural source.
Our previous data indicated that betuletol 3-methyl ether displays strong cytotoxic
properties against several human leukemia cell lines [16]. The structural similarity of
betuletol 3-methyl ether and 5,3’-dihydroxy-3,7,4’-trimethoxyflavone (4) obtained as
the major product prompted us to prepare derivatives. Thus, typical acetylation gave
acetyl derivatives 3’-acetoxy-5-hydroxy-3,7,4’-trimethoxyflavone (4a) [17] and 5,3’-
diacetoxy-3,7,4’- trimethoxyflavone (4b) [18]. The methylation of 4 with CH2N2 yielded
5-hydroxy-3,7,3’,4’-tetramethoxyflavone (4c) [19], and the acetylation of the latter gave
1
5-acetoxy-3,7,3’,4’-tetramethoxyflavone (4d) [19]. The H- and 13C-NMR data of the
compounds 4a and 4b are given in the Exper. Part.
Antiproliferative and Cytotoxic Activity. We investigated the effect of these
compounds on the growth of human myeloid HL-60 cells. 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) dye-reduction assay. Antiproliferative studies on
compounds 1–4 and 4a–4d indicated that the flavonoids 4 and 4a–4c were cytotoxic
(Table 3), while the cells were resistant to compounds 1–3 and 4d. In previous studies
with naturally occurring and semi-synthetic flavonoids, we had shown that the
cytotoxicity of quercetin, one of the best-studied bio-flavonoid, was greatly improved
by acetylation and methylation of the free OH groups [20]. In the present study, we
demonstrated that the semi-synthetic flavonoid 4b was as potent as quercetin against
HL-60 cells [20]. Etoposide, a chemotherapy drug for the treatment of some types of
cancer, was used as a positive control in the cytotoxicity assays. Since 4b was the most
cytotoxic compound against HL-60 cells, with an IC50 value of ca. 18 mm, we decided to
determine whether this semi-synthetic flavonoid displays cytotoxic properties against
further tumor cell lines. The human lymphoid leukemia cell line Molt-3 (IC50 10ꢁ6 mm)
and the Burkittꢂs lymphoma Raji (IC50 23ꢁ14 mm) cells were as sensitive as HL-60 cells,
while human melanoma SK-MEL-1 (IC50 66ꢁ4 mm) and human breast cancer MCF-7
(IC50 >100 mm) cell lines were highly resistant. For comparison, a representative IC50
plot against HL-60 and SK-MEL-1 cells is shown (Fig. 2,a). To evaluate whether 4b
decreases cell viability through apoptosis activation, morphological changes character-
istic of apoptotic cells (condensed and fragmented chromatin) were analyzed by
fluorescent microscopy using HL-60 cells. As shown in Fig. 2,b, the control cells
appeared normal, with the round and homogeneous nuclei, while cells exposed to
Table 3. Effects of Compounds on the Growth of Human Leukemia HL-60 Cell Linea)
Compound
IC50 [mm]
Compound
IC50 [mm]
1
2
3
4
>100
>100
>100
55ꢁ5
4a
4b
4c
4d
38ꢁ6
18ꢁ7
49ꢁ20
>100
Etoposide
0.24ꢁ0.01
a) Cells were cultured for 72 h, and the IC50 values were calculated as described in Exper. Part. The data
shown represent the meansꢁSE of 3–5 independent experiments with three determinations in each.

2086
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 2. a) Effect of 4b on human HL-60 and SK-MEL-1 cell viability. Cells were cultured in the presence
of the indicated concentrations of 4b for 72 h, and thereafter cell viability was determined by the 3-(4,5-
dimethyl-2-thiazolyl-)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The results of a representa-
tive experiment are shown. Each point represents the mean of triplicate determinations. b) Photo-
micrographs of representative fields of HL-60 cells stained with Hoechst 33258 after treatment with 30 mm
compound 4b for 24 h.
compound 4b displayed condensation of chromatin and the appearance of apoptotic
bodies. This result indicates that the mechanism of cytotoxicity of compound 4b
involves the activation of apoptotic cell death.
This study was supported by a Grant from the Ministry of Education and Science of Spain and from
´
the European Regional Development Fund (SAF2007–62536) to F. E. , Instituto Canario de Investigacion
´
´
´
´
del Cancer to F. L. , and Agencia Canaria de Investigacion, Innovacion y Sociedad de la Informacion
(FEDER) (C200801000174) to J. T. F. L. was supported by JAE-doctoral Program from the Ministerio de
´
Ciencia e Innovacion.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2; 70–230 mesh, Merck), column fractions
were monitored by TLC (silica gel 60 F254). Medium pressure column chromatography (MPLC): silica
gel Merck (40–63 mm). Prep. TLC: silica-gel 60 PF254þ366 plates (20ꢂ20 cm, 1-mm thickness). M.p.:
Bꢀchi Gallenkamp 4A 0865 apparatus; uncorrected. Optical rotations: Perkin-Elmer model 343
polarimeter. UV Spectra: JASCO model V-560 spectrophotometer. IR Spectra: Bruker model IFS-55
1
spectrophotometer. H- and 13C-NMR Spectra: Bruker model AMX-500 and AMX-400 spectrometers
with standard pulse sequences, operating at 500 and 400 MHz for 1H-, and 125 and 100 MHz for 13C, resp.,
CD3OD and CDCl3 were used as solvents, and TMS as internal standard. EI-MS: Micromass model
Autospec (70 eV) spectrometer.
Plant Material. The plant material was identified by Dr. Rosa Febles. P. burchardii was collected at La
Pared (Fuerteventura, Canary Islands) in May 1994. A voucher specimen has been deposited with the
Herbarium of the Viera y Clavijo Botanical Garden in Las Palmas de Gran Canaria (LPA 20915). P.
canariensis ssp. lanata was collected at Famara (Lanzarote, Canary Islands) in May 2005. A voucher
specimen has been deposited with the Herbarium of the Viera y Clavijo Botanical Garden in Las Palmas
de Gran Canaria (LPA 22943).
Extraction and Isolation. The aerial parts of Pulicaria 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 CC (SiO2; hexane/AcOEt mixtures of increasing polarity).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2087
The extraction of 3.125 kg of P. burchardii gave 495 g of a viscous residue, which was subjected to CC
(SiO2) affording two main fractions. Fr. 1 (hexane/AcOEt 7:3) was submitted to MPLC (hexane/AcOEt
7:3) to give stigmasterol (11 mg) and a mixture of compounds that were purified by crystallization, and
further prep. TLC (benzene/AcOEt 9 :1) gave: 11-oxo-a-amyrin (18 mg), 4-hydroxybenzoic acid
(10 mg), and vainillin (10 mg). Fr. 2 (hexane/AcOEt 3 :2) was recrystallized from hexane/AcOEt to
yield scopoletin (20 mg), and the liquid supernatant was subjected to MPLC (hexane/AcOEt 7:3) and
prep. TLC (benzene/AcOEt 4 :1) to give b-sitosterol (7 mg) and 1 (6 mg).
3b-Hydroxytaraxaster-20-en-30-al (¼(3b,18a,19a)-3-Hydroxyurs-20-en-30-al; 1). Amorphous solid.
[a]2D5 ¼ þ66.0 (c¼0.056, CHCl3). UV (MeOH): 236.5 (5.16). IR (KBr): 3220, 2917, 2849, 1657, 1630,
1459, 1374, 1163, 1110, 1041, 971, 761, 717. 1H- and 13C-NMR: see Table 1. EI-MS: 440 (18, Mþ ), 422 (95),
407 (37), 379 (34.5), 340 (24), 271 (10), 207 (40), 189 (100), 135 (64), 121 (55), 107 (53), 95 (61), 81 (49),
69 (50), 55 (39). HR-EI-MS: 440.3657 (C30H48Oþ2 ; calc. 440.3654)
The extraction of 1.504 kg of P. canariensis ssp. lanata gave 115.4 g of a viscous mass which was
subjected to CC (SiO2, hexane/AcOEt gradient). Fractions eluted with hexane/AcOEt 4 :1 were
concentrated and submitted to MPLC (hexane/AcOEt 9 :1) to give three subfractions. The Subfr. 1 was
purified by crystallization from hexane/AcOEt to yield the known compound b-amyrin (14 mg). The
Subfr. 2 gave a solid by precipitation which was separated by filtration. A mixture of fatty acid esters,
3b,16b-dihydroxylupeol 3-palmitate and 3b,16b-dihydroxylupeol 3-myristate (39 mg), was obtained. Last
Subfr. 3 afforded 3b,16b-dihydroxylupeol (20 mg) after crystallization (MeOH). The fraction eluted with
hexane/AcOEt 7:3 was submitted to MPLC (hexane/AcOEt 8 :2) and prep. TLC (benzene/MeCN 9 :1;
two times) to yield 3b,16b-dihydroxylupeol (¼calenduladiol; 12 mg). The fraction eluted with hexane/
AcOEt 3 :2 was subjected to MPLC (hexane/AcOEt 8 :2), and four subfractions were obtained. Subfr. 1
was crystallized from hexane/AcOEt to yield 5,3’-dihydroxy-3,7,4’-trimethoxyflavone (4) (261 mg).
Subfr. 2 was subjected to MPLC (hexane/AcOEt 8 :2) to give pulicanadienal B (33 mg) and the new
norsesquiterpene 3 (11 mg). The next subfraction was separated by MPLC (hexane/AcOEt 7:3) to
afford pulicanadiene B (5 mg), pulicanadienal A (6 mg), and a mixture of compounds that was submitted
to prep. TLC (benzene/MeCN 4 :1; four times) to give ferulic acid (15 mg). Finally, the last subfraction
was chromatographed by MPLC (hexane/AcOEt 3 :2), and the new sesquiterpene 2 (56 mg) was
obtained after further purification by crystallization (hexane/AcOEt).
(1E,5E)-8b-Acetoxy-4a-hydroxy-7bH-germacra-1(10),5-dien-14-oic Acid (¼(5R,6E,8R,9R)-9-
(Acetyloxy)-5-hydroxy-5-methyl-8-(propan-2-yl)cyclodeca-1,6-diene-1-carboxylic Acid; 2). Colorless
crystals (MeOH). M.p. 198–2008 (hexane/AcOEt). [a]2D5 ¼ þ71.4 (c¼0.042, CHCl3). UV (MeOH):
224.0 (2.625). IR (KBr): 3177, 2694, 2935, 2889, 1709, 1693, 1635, 1441, 1398, 1383, 1364, 1279, 1261, 1198,
1163, 1134, 1051, 1022, 983, 912, 881, 827, 735. 1H- and 13C-NMR: see Table 2. EI-MS: 250 (4), 232 (30),
189 (60), 161 (34), 123 (100), 95 (39), 81 (42), 55 (29). HR-FAB-MS: 333.1683 (C17H26NaOþ5 ; calc.
333.1678).
X-Ray Crystal Analysis of Compound 2. Diffraction data were collected at r.t. using a Bruker-Nonius
Kappa CCD diffractometer with graphite monochromated MoKa radiation (l¼0.71073 ꢅ). Crystal
data: C17H26O5, Mr 310.38, orthorhombic, space group P212121, a ¼ 10.0307(7) ꢅ, b ¼ 10.6126(7) ꢅ, c ¼
16.5058(9) ꢅ, V ¼ 1757.1(2) ꢅ3, Z ¼ 4, Dc ¼1.173 g cmꢀ3, m(MoKa)¼0.085 mmꢀ1, F(000)¼672, T¼
293 K; crystal dimensions 0.12ꢂ0.08ꢂ0.06 mm. Frames were collected with the COLLECT program
[21], indexed and processed using Denzo SMN, and the files were scaled together using the HKL2000
program [22]. No absorption correction was applied. Of the 17177 reflections that were collected at
qmax ¼25.928, 3272 were independent, and 2851 were with I>2s(I). The structure was solved by direct
methods (SIR2004) [23] and refined by full-matrix least-squares methods using SHELX97 [24].
Reflections and Friedel opposites were combined and merged before refinement. All H-atoms were
placed in geometrically calculated positions. The Me H-atoms were refined as a rigid groups, which were
allowed to rotate but not to tip, with Uiso(H)¼1.5 Ueq(C). All other H-atoms were allowed to ride on
their parent atoms with Uiso(H)¼1.2 Ueq(C). The rest of atoms were refined with anisotropic thermal
parameters using full-matrix least-squares procedures on F2. Convergence ((D/s)max ¼0.000, av. 0.000) by
full-matrix least squares refinement on F2 reaches R1 ¼0.0653 and wR(F2)¼0.1784 with a goodness-of-fit
of 1.114, and 203 refined parameters. 0.277 and ꢀ0.212 were the values for maximum and minimum
residual density, resp., in the final Fourier synthesis. Crystallographic data (excluding structure factors)

2088
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as
supplementary publication No. CCDC-790568.Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44-(0)1223–336033 or
e-mail:deposit@ccdc.cam.ac.uk).
Methylation of Compound 2. Excess of freshly prepared CH2N2 in Et2O was added to 2 (11 mg) in
Et2O (5 ml). The mixture was kept at r.t. for 12 h. Concentration of the mixture under vacuum furnished
2a (11 mg). Colorless oil. IR (KBr): 3478, 2954, 1739, 1712, 1641, 1436, 1376, 1290, 1255, 1234, 1163, 1050,
1025. 1H- and 13C-NMR: see Table 2. HR-FAB-MS: 347.1827 (C18H28NaOþ5 ; calc. 347.1830).
(1R,2R,3E,5R)-5-Hydroxy-5-methyl-9-oxo-2-(propan-2-yl)cyclodec-3-en-1-yl Acetate (3). Amor-
phous solid. [a]2D5 ¼ ꢀ28.0 (c¼0.15, CHCl3). IR (KBr): 3541, 2963, 2929, 1729, 1682, 1451, 1374, 1248,
1081, 1037, 944, 897. 1H- and 13C-NMR: see Table 2. HR-FAB-MS: 305.1720 (C16H26NaOþ4 ; calc.
305.1729).
Monoacetylation of Compound 4. Compound 4 (57 mg) was dissolved in pyridine (2 ml) and Ac2O
(4 ml). The soln. was stirred for 16 h at r.t. The mixture was concentrated and purified by MPLC (SiO2;
hexane/AcOEt 7.5 :2.5) to furnish 31 mg of 3’-acetoxy-5-hydroxy-3,7,4’-trimethoxyflavone (4a). Yellow
needles. M.p. 154–1568. UV (MeCN): 269 (3.73), 345 (3.82). IR (KBr): 3510, 2930, 2849, 1770, 1734,
1652, 1600, 1506, 1456, 1350, 1308, 1277, 1205, 1161, 1119, 1093, 1016, 923, 904, 811. 1H-NMR (500 MHz,
CDCl3): 12.51 (s, OH); 7.99 (dd, J¼2.0, 9.0, HꢀC(6’)); 7.75 (d, J¼2.0, HꢀC(2’)); 7.01 (d, J¼9.0,
HꢀC(5’)); 6.37 (d, J¼2.0, HꢀC(8)); 6.28 (d, J¼2.0, HꢀC(6)); 3.83 (s, MeOꢀC(4’)); 3.76 (s, MeOꢀC(3),
MeOꢀC(7)); 2.29 (s, AcO). 13C-NMR (125 MHz, CDCl3): 178.8 (C(4)); 168.8 (MeC¼O); 165.6 (C(7));
162.1 (C(5)); 156.7 (C(8a)); 154.8 (C(2)); 153.3 (C(4’)); 139.7 (C(3’)); 139.2 (C(3)); 127.8 (C(6’)); 123.2
(C(1’)); 123.1 (C(2’)); 112.2 (C(5’), C(4a)); 98.0 (C(6)); 92.2 (C(8)); 60.2 (MeOꢀC(3)); 56.0
(MeOꢀC(4’)); 55.8 (MeOꢀC(7)); 20.7 (MeCO). EI-MS: 387 (32, [MþH]þ ), 386 (92, Mþ ), 344 (74),
343 (100), 329 (18), 301 (40), 167 (10). HR-EI-MS: 386.1006 (C20H18Oþ8 ; calc. 386.1002).
Diacetylation of Compound 4. Compound 4 (71 mg) was dissolved in pyridine (3 ml) and Ac2O
(6 ml). The soln. was stirred for 24 h at 408. The mixture was concentrated and purified by MPLC (SO2;
hexane/AcOEt 7.5 :2.5) to furnish 41 mg of 5,3’-diacetoxy-3,7,4’-trimethoxyflavone (4b). Colorless
needles. M.p. 172–1748. UV (MeCN): 326 (3.83), 258 (3.70), 231 (3.84). IR (KBr): 3011, 2916, 2846,
1770, 1634, 1608, 1514, 1436, 1362, 1277, 1227, 1196, 1159, 1145, 1130, 1082, 1043, 924, 820, 665, 623.
1H-NMR (500 MHz, CDCl3): 7.96 (dd, J ¼ 2.0, 9.0, HꢀC(6’)); 7.73 (d, J ¼ 2.0, HꢀC(2’)); 7.00 (d, J ¼ 9.0,
HꢀC(5’)); 6.74 (d, J ¼ 2.5, HꢀC(8)); 6.52 (d, J ¼ 2.5, HꢀC(6)); 3.84 (s, MeOꢀC(4’)); 3.83 (s, MeOꢀC(7));
3.73 (s, MeOꢀC(3)); 2.38 (s, AcOꢀC(3’)); 2.29 (s, AcOꢀC(5)). 13C-NMR (125 MHz, CDCl3): 173.1
(C(4)); 169.7 (MeCOꢀC(3’)); 168.4 (MeCOꢀC(5)); 163.4 (C(7)); 157.8 (C(8a)); 153.09 (C(2)); 153.06
(C(4’)); 150.6 (C(5)); 141.1 (C(3)); 139.7 (C(3’)); 127.6 (C(6’)); 123.3 (C(1’)); 122.8 (C(2’)); 112.1 (C(5’));
111.4 (C(4a)); 108.2 (C(6)); 98.6 (C(8)); 60.0 (MeOꢀC(3)); 56.05 (MeOꢀC(4’)); 56.00 (MeOꢀC(7));
21.2 (MeCOꢀC(3’)); 20.7 (MeCOꢀC(5)). EI-MS: 428 (37, Mþ ), 386 (54), 344 (65), 343 (100), 327 (13),
326 (17), 301 (23). HR-EI-MS: 428.1099 (C22H20Oþ9 ; calc. 428.1107).
Methylation of Compound 4. To a soln. of compound 4 (20 mg) in Et2O (15 ml), excess of freshly
prepared CH2N2 in Et2O was added, and the mixture was kept at r. t. for 12 h. Concentration of the
mixture under vacuum furnished 4c (18 mg) [19].
Acetylation of Compound 4c. Compound 4c (11 mg) was dissolved in pyridine (1 ml) and Ac2O
(3 ml), and the soln. was stirred for 24 h at 408. The product was dried under vacuum to furnish 4d
(10 mg) [19].
Cell Culture and Cytotoxicity Assays. The human leukemia HL-60 and Molt-3 cells, the Burkittꢂs
lymphoma Raji cells, and human SK-MEL-1 melanoma cells were cultured in suspension in RPMI-1640
medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 units/ml), and
streptomycin (100 mg/ml). The human breast cancer cell line MCF-7 was cultured in Dulbeccoꢂs modified
Eagleꢂs medium containing 10% (v/v) heat-inactivated fetal bovine serum, 2 mm l-glutamine, 100 U/ml
penicillin, and 100 mg/ml streptomycin. Cells were maintained at a density of <1ꢂ106 cells/ml.
Cytotoxicity studies were performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide (MTT) assay as described in [25].
Fluorescent Microscopy Analysis. Cells were harvested and fixed in 3% paraformaldehyde and
incubated at r.t. for 10 min. The fixative was removed, and the cells were washed with phosphate-

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
2089
buffered saline (PBS), resuspended in 10 ml of PBS containing 20 mg/ml bis-benzimide trihydrochloride
(Hoechst 33258), and incubated at r.t. for 15 min. Stained nuclei were visualized using a Zeiss fluorescent
microscope.
Supplementary Material. HR-MS, 1H- and 13C-NMR spectra of the compounds 1–3, 4a, and 4b are
available upon request from the corresponding author.
REFERENCES
[1] L.-L. Liu, J.-L. Yang, Y.-P. Shi, Chem. Biodiversity 2010, 7, 327.
[2] D. Bramwell, Z. I. Bramwell, ꢁFlores silvestres de las Islas Canariasꢂ, Ed. S. L. Rueda, Madrid, 2001.
´
´
´
´
´
[3] J. Triana, M. Lopez, F. J. Perez, J. Gonzalez-Platas, J. Quintana, F. Estevez, F. Leon, J. Bermejo, J.
Nat. Prod. 2005, 68, 523.
´
´
´
[4] J. Triana, J. L. Eiroa, J. J. Ortega, F. Leon, I. Brouard, J. C. Hernandez, F. Estevez, J. Bermejo,
Phytochemistry 2010, 71, 627.
[5] K. Shiojima, H. Suzuki, N. Kodera, H. Ageta, H.-C. Chang, Y.-P. Chen, Chem. Pharm. Bull. 1996, 44,
509.
[6] S. Matsunaga, R. Tanaka, M. Akagi, Phytochemistry 1988, 27, 535.
´
[7] R. D. Murray, J. Mendez, S. A. Brown, ꢁThe Natural Coumarins. Occurence, Chemistry and
Biochemistryꢂ, John Wiley & Sons, Chichester, USA, 1982.
[8] H. Kojima, N. Sato, A. Hatano, H. Ogura, Phytochemistry 1990, 29, 2351.
[9] S. Hafez, T. M. Sarg, M. M. El-Domiaty, A. A. Ahmed, F. R. Melek, F. Bohlmann, Phytochemistry
1987, 26, 3356.
[10] G. Lewin, A. Maciuk, S. Thoret, G. Aubert, J. Dubois, T. Cresteil, J. Nat. Prod. 2010, 73, 702.
[11] P. Boiteau, M. B. Pasich, A. R. Ratsimamanga, ꢁLes triterpenoides en physiologie vegetale et
animaleꢂ, Gautier-Villars Ed, Paris, France, 1964.
[12] S.ꢆksꢄz, G. Topcu, Phytochemistry 1987, 26, 3082.
[13] A. P. Datanarayana, N. S. Kumar, M. U. S. Sultanbawa, S. Balasubramaniam, J. Chem. Soc., Perkin
Trans. 1 1981, 2717.
´
´
[14] N. M. Sokkar, E. A. Aboutabl, W. Kubelka, Z. H. Mohamad, S. H. Hilal, Plantes med. phytother.
1993, 26, 368.
[15] T. Ishii, H. Matsuura, Z. Zhaoqi, C. S. Vairappan, Molecules 2009, 14, 4591.
´
[16] S. Rubio, J. Quintana, J. L. Eiroa, J. Triana, F. Estevez, Mol. Carcinog. 2010, 49, 32.
[17] Y. Wang, M. Hamburger, J. Gueho, K. Hostettmann, Phytochemistry 1989, 28, 2323.
[18] E. Malan, D. G. Roux, J. Chem. Soc., Perkin Trans 1 1979, 2696.
[19] T. M. S. Da Silva, M. G. Carvalho, R. Braz-Filho, Quim. Nova 2009, 32, 1119.
´
´
[20] S. Rubio, J. Quintana, M. Lopez, J. L. Eiroa, J. Triana, F. Estevez, Eur. J. Pharmacol. 2006, 548, 9.
[21] Bruker-Nonius (1997–2000): Collect Program Suite, Bruker-Nonius, The Netherlands.
[22] Z. Otwinowski, W. Minor, ꢁProcessing of X-ray Diffraction Data Collected in Oscillation Modeꢂ in
Methods in Enzymology, Volume 276, Macromolecular Crystallography, part A, Eds. C. W. Carter
Jr., R. M. Sweet, Academic Press, New York, 1997, pp. 307–326.
[23] M. C. Burla, R. Caliandro, M. Camalli, B. Carrozzini, G. L. Cascarazo, L. De Caro, C. Giacovazzo,
G. Polidori, R. Spagna, J. Appl. Crystallogr. 2005, 38, 381.
[24] G. M. Sheldrick, SHELXL97, Program for the refinement of Crystal Structures, University of
Gçttingen, Germany, 1997.
´
´
[25] J. Triana, J. L. Eiroa, J. J. Ortega, F. Leon, I. Brouard, F. Torres, J. Quintana, F. Estevez, J. Bermejo, J.
Nat. Prod. 2008, 71, 2015.
Received November 15, 2010