Pyrrolizidine alkaloids of the endemic Mexican genus Pittocaulon and assignment of stereoisomeric 1,2-saturated necine bases

Article abstract of DOI:10.1016/j.phytochem.2007.07.004

The endemic Mexican genus Pittocaulon (subtribe Tussilagininae, tribe Senecioneae, Asteraceae) belongs to a monophyletic group of genera distributed in Mexico and North America. The five Pittocaulon species represent shrubs with broom-like succulent branches. All species were found to contain pyrrolizidine alkaloids (PAs). With one exception (i.e., stems of Pittocaulon velatum are devoid of PAs) PAs were found in all plant organs with the highest levels (up to 0.3% of dry weight) in the flower heads. Three structural types of PAs were found: (1) macrocyclic otonecine esters, e.g. senkirkine and acetylpetasitenine; (2) macrocyclic retronecine esters, e.g. senecionine, only found in roots, and (3) monoesters of 1,2-saturated necines with angelic acid. For an unambiguous assignment of the different stereoisomeric 1,2-saturated necine bases a GC-MS method was established that allows the separation and identification of the four stereoisomers as their diacetyl or trimethylsilyl derivatives. All otonecine esters that generally do not form N-oxides and the 1,2-saturated PAs were exclusively found as free bases, while the 1,2-unsaturated 7-angeloylheliotridine occurring in P. velatum was found only as its N-oxide. In a comparative study the ¹H and ¹³C NMR spectra of the four stereoisomeric necine bases were completely assigned by the use of DEPT-135, H,H-COSY, H,C-HSQC and H,H-NOESY experiments and by iterative analysis of the ¹H NMR spectra. Based on these methods the PA monoesters occurring in Pittocaulon praecox and P. velatum were assigned as 7-O-angeloyl ester respectively 9-O-angeloyl ester of dihydroxyheliotridane which could be identified for the first time as naturally occurring necine base. Unexpectedly, in the monoesters isolated from the three other Pittocaulon species dihydroxyheliotridane is replaced by the necine base turneforcidine with opposite configuration at C-1 and C-7. The species-specific and organ-typical PA profiles of the five Pittocaulon species are discussed in a biogenetic context.

Full text of DOI:10.1016/j.phytochem.2007.07.004

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 154–167
www.elsevier.com/locate/phytochem
Pyrrolizidine alkaloids of the endemic Mexican genus Pittocaulon
q
and assignment of stereoisomeric 1,2-saturated necine bases
a
b
c
c
´
Juan Camilo Marın-Loaiza , Ludger Ernst , Till Beuerle , Claudine Theuring ,
a,d,
c,
*
*, Thomas Hartmann
´
Carlos L. Cespedes
a
Laboratorio de Fitoquı´mica, UBIPRO, FES Iztacala, Universidad Nacional Auto´noma de Me´xico, Av. De Los Barrios 1,
Tlalnepantla 54090, Estado de Me´xico, Mexico
b
NMR-Laboratorium der Chemischen, Institute der Technischen, Universita¨t Braunschweig, Hagenring 30, D-38106 Braunschweig, Germany
Institut fu¨r Pharmazeutische Biologie der Technischen, Universita¨t Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany
c
d
Facultad de Ciencias, Depto. Ciencias Ba´sicas, Universidad del Bio-Bio, Av. Andres Bello s/n, Casilla 447, Chilla´n, Chile
Received 21 May 2007; received in revised form 29 June 2007
Available online 23 August 2007
Abstract
The endemic Mexican genus Pittocaulon (subtribe Tussilagininae, tribe Senecioneae, Asteraceae) belongs to a monophyletic group of
genera distributed in Mexico and North America. The five Pittocaulon species represent shrubs with broom-like succulent branches. All
species were found to contain pyrrolizidine alkaloids (PAs). With one exception (i.e., stems of Pittocaulon velatum are devoid of PAs)
PAs were found in all plant organs with the highest levels (up to 0.3% of dry weight) in the flower heads. Three structural types of
PAs were found: (1) macrocyclic otonecine esters, e.g. senkirkine and acetylpetasitenine; (2) macrocyclic retronecine esters, e.g. seneci-
onine, only found in roots, and (3) monoesters of 1,2-saturated necines with angelic acid. For an unambiguous assignment of the different
stereoisomeric 1,2-saturated necine bases a GC–MS method was established that allows the separation and identification of the four ster-
eoisomers as their diacetyl or trimethylsilyl derivatives. All otonecine esters that generally do not form N-oxides and the 1,2-saturated
PAs were exclusively found as free bases, while the 1,2-unsaturated 7-angeloylheliotridine occurring in P. velatum was found only as its
1
N-oxide. In a comparative study the H and 13C NMR spectra of the four stereoisomeric necine bases were completely assigned by the
1
use of DEPT-135, H,H-COSY, H,C-HSQC and H,H-NOESY experiments and by iterative analysis of the H NMR spectra. Based on
these methods the PA monoesters occurring in Pittocaulon praecox and P. velatum were assigned as 7-O-angeloyl ester respectively 9-O-
angeloyl ester of dihydroxyheliotridane which could be identified for the first time as naturally occurring necine base. Unexpectedly, in
the monoesters isolated from the three other Pittocaulon species dihydroxyheliotridane is replaced by the necine base turneforcidine with
opposite configuration at C-1 and C-7. The species-specific and organ-typical PA profiles of the five Pittocaulon species are discussed in a
biogenetic context.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Pittocaulon; Pyrrolizidine alkaloids; Dihydroxyheliotridane; Turneforcidine; GC–MS analysis; Derivatization; NMR analysis
1. Introduction
q
Taken in part from the PhD thesis of JCML under guidance of Dr.
Pittocaulon (ex Senecio) forms together with several
´
C.L. Cespedes.
*
related genera a monophyletic group within the subtribe
Tussilagininae of the Senecioneae (Asteraceae). The genus
Pittocaulon has been segregated from the huge cosmopoli-
tan genus Senecio. This segregation is supported by means
of classical methods (Robinson and Brettell, 1973) and by
molecular phylogenetic techniques (Bain and Golden,
Corresponding authors. Address: Institut fur Pharmazeutische Biol-
¨
ogie der Technischen, Universita¨t Braunschweig, Mendelssohnstrasse 1,
D-38106 Braunschweig, Germany. Tel.: +49 531 391 5694; fax: +49 531
391 8104 (T. Hartmann); Tel.: +56 42 253256; fax: +56 42 253046
´
(C.L. Cespedes).
´
E-mail addresses: cespedes_leonardo@yahoo.com (C.L. Cespedes),
t.hartmann@tu-bs.de (T. Hartmann).
0031-9422/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2007.07.004

J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
155
2000). All five Pittocaulon species are endemic to Mexico
with one also occurring in Guatemala. They are found in
tropical dry habitats often on rocky outcrops or steep cliffs.
They are shrubs or small trees with broom-like succulent
branches with water-storing pith and bark (Olson, 2005).
The branches bear small clusters of leaves during the rainy
season and are leafless during the drought the rest of the
year. The stems of Pittocaulon praecox, the most abundant
species, are frequently infested by a scale insect, Ceroplastes
albolineatus (Coccidae, Homoptera). We became interested
in the chemistry of Pittocaulon when we observed that the
phloem-feeding scale insect sequesters pyrrolizidine alka-
together with their 1,2-saturated analogs platyphylline
(12) and neoplatyphylline (13) (Fig. 1c). With one excep-
tion (stems of P. velatum) PAs could be detected in all ana-
lyzed plant organs but substantial qualitative and
quantitative differences were observed between different
organs. Flower heads displayed the highest PA concentra-
tions reaching levels up to 0.3% (on a dry weight basis) in
Pittocaulon bombycophole. This corroborates results from
other Senecio species which also showed the highest PA
levels in the inflorescences (Hartmann and Zimmer, 1986).
Whereas senecionine (10) and senkirkine (14) are fre-
quently found in distant taxa within the tribe Senecioneae,
the ligularidines (17, 18) and petasitenines (19, 20) so far
are only found in taxa of the subtribe Tussilagininae, such
as Asian and European Petasites and Homogyne species
and the Asian Farfugium and Ligularia species (Hartmann
and Witte, 1995). From the chemosystematic point of view
this supports the classification of Pittocaulon as member of
the Tussilagininae.
The co-occurrence of macrocyclic otonecine diesters
and retronecine diesters is not surprising since the otone-
cine derivatives originate biogenetically from retronecine
derivatives as it has been demonstrated for the conversion
of senecionine into senkirkine (Toppel et al., 1987; Kelly
et al., 1989). The occurrence of senecionine only in roots
(Table 1) appears reasonable since in all Senecio species
so far studied the roots are the exclusive site of senecio-
nine biosynthesis (Hartmann et al., 1989; Hartmann and
Ober, 2000). The various species-specific macrocyclic ret-
ronecine and otonecine derivatives are formed by periph-
eral modification of the basic backbone structure
senecionine (Hartmann and Dierich, 1998; Pelser et al.,
2005).
´
loids (PAs) from its host plant (Marın-Loaiza et al., 2007).
The occurrence of PAs in most species of the genus
Senecio s.l. is well established (Hartmann and Witte,
1995). Preliminary studies with Pittocaulon revealed the
presence of two structural types of PAs: (i) 1,2-saturated
and unsaturated PAs of the senecionine type (Hartmann
and Witte, 1995) and (ii) simple angeloylmonoesters of
1,2-saturated and unsaturated necine bases. The co-occur-
rence of macrocyclic otonecine derivatives and simple 1,2-
saturated monoesters is rarely observed among Senecio
species. Preliminary observations indicated the presence
of different stereoisomers of the 1,2-saturated necine base
1-hydroxymethyl-7-hydroxypyrrolizidine depending on
the Pittocaulon species analyzed. Since there is a great
inconsistency in the literature about the correct assignment
of these stereoisomeric 1,2-saturated necines occurring in
plants we included in our investigation the elaboration of
a GC–MS method for a complete separation and unequiv-
ocal assignment of the four possible stereoisomers of 1-
hydroxymethyl-7-hydroxypyrrolizidine as well as their
structural identification and assignment by 1D and 2D
NMR techniques.
A particular feature of Pittocaulon is the quantita-
tively more or less balanced co-occurrence of macrocyclic
PA diesters and PA monoesters. In the Senecioneae PA
monoesters or open-chain diesters are rarely found
together with macrocyclic PAs (Hartmann and Witte,
1995). Moreover, it is remarkable that the angeloylesters
(5,6) of dihydroxyheliotridane (4) (Fig. 1a and b) that
are found in two species (i.e., P. praecox and P. velatum)
in the other three species are replaced by the respective
esters (7,8) of turneforcidine (2) (Fig. 1a and b). In the
course of the identification of the saturated necine bases
an unequivocal assignment of the four possible stereoiso-
mers (1–4) (Fig. 1a) was impossible with the tools so far
available in literature. Therefore we established a GC–
MS method allowing the separation and assignment of
the four stereoisomers (see Section 2.2) and confirmed
the structural assignment by comparative NMR (see Sec-
tion 2.3). With these tools in hands an unequivocal
assignment and identification of the stereoisomeric
necines and their esters presented here was accomplished.
The occurrence of dihydroxyheliotridane (4) as a plant
constituent was new, until now it was only known as
synthetic product (Robertson et al., 1996). Turneforci-
dine (2) has been frequently described as necine base of
2. Results
2.1. The PA profiles of the Pittocaulon species
The five Mexican Pittocaulon species were analyzed for
the presence of PAs by GC–MS. The results are summa-
rized in Table 1. The genus was found to contain a rich
diversity of 17 different PAs. Two structural types were
dominating in all species: (1) O7- respectively O9-monoes-
ters of 1,2-saturated necine bases (Figs. 1b), and (2) macro-
cyclic diesters of the 1,2-unsaturated necine base otonecine
(Fig. 1d). In two species, i.e. Pittocaulon filare and P. vela-
tum, the 1,2-saturated monoesters are accompanied by
mono esters of the 1,2-unsaturated necine base retronecine
or heliotridine, respectively (Fig. 1b-1). The macrocyclic
retronecine diester senecionine (10) and its Z/E isomer
integerrimine (11) (Fig. 1c) which are structural analogs
of the otonecine diesters senkirkine (14) and neosenkirkine
(15) could be detected in the roots of three species. In P.
praecox, senecionine and integerrimine were found

Table 1
Organ-specific PA profiles and total PA concentrations in different organs of five Pittocaulon species established by GC–MS
m/z [M]+
RI(ZB1)
Pyrrolizidine alkaloids (relative abundance, %)
P. praecox P. bombycophole
Stea Floa
P. filare
Stea
P. hintonii
P. velatum
Stea Floa
Leaa
Rooa
Stea
Floa
Rooa
Rooa
16
Stea
Floa
Rooa
Rooa
(25)c
7-Angeloylretronecine (9)
7-Angeloylheliotridine (9a)
7-Angeloylturneforcidine (7)
9-Angeloylturneforcidine (8)
7-Angeloyldihydroxyheliotridane (5)
9-Angeloyldihydroxyheliotridane (6)
Senecionine (10)
Integerrimine (11)
Platyphylline (12)
Neoplatyphylline (13)
Senkirkine (14)
Neosenkirkine (15)
Petasitenine (19)
PA-X (X)d
Acetylsenkirkine (16)
Ligularidine (17)e
Neoligularidine (18)e
Acetylpetasitenine (20)
mg/g dry wt:
237
237
239
239
239
239
335
335
337
337
365
365
381
381
407
407
407
423
1809
1810
1780
1806
1810
1837
2284
2342
2335
2363
2459
2534
2447
2542
2632
2708
2709
2632
43
31b
65b
76
8
16
6
38
6
57
23
71
3
39
2
19
1
33
66
11
78
2
tr
tr
(25)c
40
16
4
4
36
tr
tr
59
16
66
14
tr
tr
6
8
10
tr
1
1
1
3
12
3
3
tr
12
4
tr
2
tr
12
1
1
70
3.04
tr
48
0.52
tr
52
0.25
16
2.41
tr
0.16
20
1.12
50
2.89
49
0.99
tr
0.16
0.13
0.97
1.42
0.46
0.32
a
Ste: Stems; Flo: Flowers; Lea: Leaves; Roo: Roots.
b
c
Angeloylesters 9 and 5 are not separated; they were determined after hydrolysis and quantification of the respective necine bases, see Table 2.
Angeloylesters 9 and 5 are not separated; the sum of both compounds account for 25% rel. abundance.
Unknown pyrrolizidine alkaloid.
d
e
Elution sequence of the two E/Z - isomers not assured.

J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
157
Fig. 1. Structures of the stereoisomeric 1,2-saturated necine bases (a) and PA esters identified from genus Pittocaulon.b: 1,2-saturated monoesters; b-1:
1,2-unsaturated monoesters; c: macrocyclic 1,2-unsaturated retronecine, respectively, 1,2-saturated platynecine diesters; d: 1,2-unsaturated macrocyclic
otonecine diesters.
various PA monoesters and open-chain diesters as well
as macrocyclic PAs of the monocrotaline type (Mattocks,
1986; Rizk, 1991; Hartmann and Witte, 1995). The third
stereoisomeric necine base, platynecine (3), is the com-
mon necine base of almost all known 1,2-saturated mac-
rocyclic PAs of the senecionine type like platyphylline
(12) and neoplatyphylline (13) found in roots of P. prae-
cox (Table 1). A biosynthetic relation between the
macrocyclic PAs (Fig. 1c and d) and turneforcidine
(Fig. 1a) appears reasonable since both have
R-configuration at C-7. This applies for three of the Pitt-
ocaulon species but not for P. praecox and P. velatum. In
both species 7R-configured macrocyclic PAs co-occur
with the 7S-configurated dihydroxyheliotridane monoes-
ter. In P. velatum the saturated 7S-configurated monoes-
ter is accompanied by its 1,2-unsaturated analog
7-angeloylheliotridine (Tables 1 and 2). It appears unli-
kely that the 7S-configurated monoesters represent inter-
mediates or off-branching side products in senecionine
biosynthesis, unless one assumes an additional epimeriza-
tion at C-7.
(1,2-unsaturated angeloylesters, e.g. in P. velatum)
(Fig. 1b-1; Tables 1 and 2). It is well documented that in
Senecio species 1,2-unsaturated PAs are synthesized, main-
tained and stored exclusively as N-oxides (Toppel et al.,
1987; Hartmann et al., 1989; Hartmann and Dierich,
1998). However, in this respect 1,2-saturated PAs occurring
in Senecio have never been studied before. All Pittocaulon
samples analyzed contain 1,2-saturated PAs exclusively as
free base (tertiary PA); not even traces of N-oxides were
detected. This is the first evidence from a Senecio species
that 1,2-saturated PAs are solely present as free
bases. The detailed analysis of the 7-angeloylesters from
P. velatum revealed that they represent a mixture of 65% 7-
angeloyldihydroxyheliotridane (5) and 31% 7-angeloylhelio-
tridine N-oxide (9a) (Tables 1 and 2; Fig. 1b-1). Unfortu-
nately the samples from P. filare (stems and roots), P.
praecox (roots) and P. velatum (roots) containing 1,2-unsat-
urated PAs (Table 1) were only analyzed after reduction.
Therefore it was not possible to decide whether the 1,2-
unsaturated PAs were present as N-oxides or as free base.
However the data from P. velatum flowers suggest that 1,2-
unsaturated PAs found in the genus Pittocaulon may be pres-
ent as N-oxides like in other Senecio species. 1,2-Saturated
A particularity of Pittocaulon is the co-occurrence of PAs
as free bases (1,2-saturated angeloylesters) and N-oxides

158
J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
Table 2
Pittocaulon velatum (flowers) contain two 7-angeloylesters that could not be separated by GC (DB1); 32% of the mixture is present as N-oxide
Parameter
M+
RI
PAs (free base)
Total PAs (free base + N-oxide)a
7-Angeloyl esters
237/239
1810
Relative abundance (%)
mg/g dry wt
100
0.65
0
>95
0.95
32
Present as N-oxide (%)
Hydrolysate of 7-angeloyl esters
Dihydroxyheliotridane
Relative abundance (%)
Present as N-oxide (%)
Heliotridine
157
155
1458
1446
100
0
65
0
Relative abundance (%)
Present as N-oxide (%)
nd
31
97
Hydrolysis of the ester mixture yielded the necine bases dihydroxyheliotridane and heliotridine. Dihydroxyheliotridane is present exclusively as free base
(tertiary PA), heliotridine exclusively as N-oxide.
a
The ester fraction contained >5% of a PA tentatively identified as macrophylline (M+ 239; RI 1861); the hydrolysate contained ca. 4% of the respective
necine base most likely macronecine (M+ 157; RI 1261). nd = not detected.
Fig. 2. GC-separation of the di-O-acetyl derivatives of the four stereoisomeric 1,2-saturated necine bases. a and b: Separations of standard mixtures; c:
necine base obtained by hydrolysis of 7-angeloyldihydroxyheliotridane (5) from P. praecox; d: necine base obtained by hydrolysis of 7-
angeloylturneforcidine (7) from P. bombycophole. a and c: separation of a ZB2 column; b and d: separation of a ZB1 column.
PAs occurring in other plant families are present either exclu-
sively as tertiary PAs, i.e. Convolvulaceae (Jenett-Siems
et al., 1998) or as mixtures of free bases and their N-oxides,
i.e. Orchidaceae (Fro¨lich et al., 2006). Otonecine derivatives
like senkirkine due to structural reasons (N-methylation) do
not form N-oxides (Toppel et al., 1987).
2.2. Separation of the 1,2-saturated necine bases and their
esters by GC–MS
The necine bases 3 and 4 were prepared by hydrogena-
tion of retronecine and heliotridine, respectively, using
slightly modified procedures described by Donohoe et al.

J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
159
(2002). Necine base 2 was obtained by hydrolysis of 7, iso-
lated from P. bombycophole and Pittocaulon hintonii.
Necine 1 was a gift from S.E. Denmark. The identity of
the four stereoismeric saturated necine bases were con-
firmed by GC–MS, optical rotation and NMR (see Section
2.3).
Simultaneous gas chromatographic separation of the
four stereoisomeric saturated necine bases was achieved
after O-trimethylsilylation (TMS-derivatives) or acetyla-
tions (acetyl derivatives). Disadvantage of the TMS-deriv-
(Dd = 0.01 and 0.02 ppm, respectively) but large differences
in 1 and 4 (Dd = 0.33 and 0.32 ppm, respectively). While 1
is characterized by its strong shielding of H-1 (dH-1
=
1.94 ppm), isomers 2–4 have dH-1 values between 2.44
and 2.53 ppm. The large sum of the coupling constants
P
involving H-7 ( J7 = 21.6 Hz), typical for 4, needs also
mentioning although this value is decreased to 15.4 Hz in
its ester 5 (see below). Finally, both 1 (dH-2a = 1.53 ppm)
and 4 (dH-2b = 1.53 ppm) feature a substantially higher
shielding of one of their protons at C-2 than the other dia-
stereomers: dH-2a = 1.68 and 1.74 ppm in 2 and 3,
respectively.
atives is
a poor separation of hastanecine (ZB-1,
RI = 1613) and platynecine (ZB-1, RI = 1615). Baseline
separation was obtained with the acetyl derivatives using
either a ZB-1 or a more polar ZB-5 column (Fig. 2).
After hydrolysis of the PA-monoesters this separation
method was used for the assignment of the involved necine
bases of the isolated 1,2-saturated PA monoesters from P.
praecox (Fig. 2c) or the other Pittocaulon species (Fig. 2d).
The procedure can easily be adopted to identify and cor-
rectly assign the saturated necine bases 1–4 from any PA
source. All reference compounds can be easily obtained
by the catalytic hydrogenation starting from retronecine
or heliotridine (see Section 4.4.). The identification of the
necine bases is as well possible for sub-mg quantities from
hydrolyzed PA mixtures since the most common unsatu-
rated necine bases, i.e. retronecine and heliotridine are sep-
arated from the 1,2-saturated necine bases (see RI values in
Section 4.5) and show different fragmentation patterns in
EI-MS.
For the angeloyl esters H,C-HMBC spectra were
recorded to secure the attachment of the acyl moieties to
O-7 or O-9 from the crosspeaks between the carboxyl 13C
1
chemical shift and the H chemical shift of H-7 and H-9,
respectively. For all the necines studied, we found that both
1H and 13C chemical shifts, in particular those of the
a-positions with respect to nitrogen (positions 3, 5 and 8),
were not well reproducible when NMR measurements were
repeated after storing compounds in CDCl3 or in CD3OD
even for a few days. Chemical shift alterations of up to
2 ppm (13C) and up to 0.7 ppm (1H) were observed, which
made many comparisons of spectra meaningless. Such
behavior was also emphasized by S.E. Denmark (personal
communication) and is most likely attributed to the easy
protonation of the necine nitrogen atom. We found that
reproducible NMR spectra of the necines were obtained
when a few milligrams of these are dissolved in CD3OD
and two or three drops of ND3 (25% in D2O, Aldrich)
are added to guarantee that the necines are present in solu-
tion as the free bases. Hence all spectra reported here were
recorded under such conditions. Thus we can present a
compatible set of 1H and 13C NMR data, including stereo-
specific proton assignments, for the stereoisomers dihydr-
oxyheliotridane (4), hastanecine (1), platynecine (3), and
turneforcidine (2) and the three monoesters 5–7. Our 13C
NMR data of the necine bases show excellent agreement
with the following literature values: 4 (Mulzer and Scharp,
1993), 1 (Fleet et al., 1991; Mulzer and Scharp, 1993; Den-
mark and Thorarensen, 1994), 3 (Fleet et al., 1991), 2 (Wee,
2001).
2.3. Identification of the 1,2-saturated necine bases and their
monoesters by NMR spectroscopy
The 1H (Tables 3 and 5) and 13C NMR (Tables 4 and 6)
spectra of the necine bases and their esters were completely
assigned by the use of DEPT-135, H,H-COSY, H,C-HSQC
1
and H,H-NOESY experiments. Moreover, the H NMR
spectra were analyzed by iteration on the full line shape
1
(Weber and Thiele, 1998) to obtain a complete set of H
chemical shifts and H,H coupling constants. This was suc-
cessful for all compounds with the exception of the protons
H-2b, H-6a and H-6b of 7 which had strongly overlapping
proton signals in the region d = 2.12–2.18 ppm. As a con-
sequence, J(6a,6b) is not defined and the values of
J(6a,7) and J(6b,7) are interchangeable for this compound.
The two PA monoesters (5, 6) isolated from P. praecox
1
are both angelates as evidenced by their H and 13C NMR
chemical shifts and EI-MS. The HMBC correlation
between the angeloyl carboxyl carbon atom and the 9-pro-
tons showed that 6 is a 9-O-angeloyl derivative. This was
deduced from the chemical shifts of the 9-protons
(dH = 4.45 and 4.22 ppm) whose signals are easily identi-
fied by their mutual geminal coupling constant of
(ꢀ)11.2 Hz and their vicinal couplings with H-1 of 6.3
and 8.1 Hz, respectively. The H,H-NOEs (Table 5) and
the 13C chemical shifts of the necine part (Table 6) prove
1
The theoretical 400 MHz H NMR spectra of the necine
alcohols are reproduced in Fig. 3. They were obtained by
iterating chemical shifts, coupling constants and line-
widths. The good agreement between calculated and exper-
imental spectra is indicated by R values between 0.8% and
2.0%.
1
Characteristic features in the H NMR spectra are the
very similar (Dd = 0.01 ppm) and the distinctly different
(Dd = 0.18 ppm) chemical shifts of protons H-9a and H-
9b in 3 and 4, respectively, while the differences in 1 and
2 are small and identical (Dd = 0.04 ppm). Protons H-6a
and H-6b have only small shift differences in 2 and 3
that
6 is 9-O-angeloyl-dihydroxyheliotridane: C-9 is
deshielded by 1.9 ppm and C-1 shielded by 3.0 ppm relative
to the values in 4, all other 13C chemical shift differences,
DdC, between 6 and 4 being less than or equal to

160
J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
Table 3
1H NMR data (CD3OD + ND3) of the stereoisomeric 1,2-saturated necine bases
Hastanecine (1)a
1H NMR chemical shifts, dH
Turneforcidine (2)
Platynecine (3)
Dihydroxyheliotridane (4)
1a
1b
2a
2b
3a
3b
5a
5b
6a
6b
7a
7b
8a
9a
9b
–
–
2.45
–
2.44
–
1.94
1.53
1.97
3.05
2.53
3.09
2.70
1.72
2.05
–
2.53
1.68
2.10
3.08
2.58
3.03
2.70
2.00
1.99
4.18
–
1.74
1.98
3.10
2.79
3.23
2.86
1.88
1.90
4.25
–
1.81
1.53
2.89
2.65
3.19
2.59
1.74
2.06
–
4.12
3.25
3.85
3.67
4.09
2.94
3.62
3.58
3.16
3.57
3.53
3.29
3.94
3.93
Coupling constants, J(H,H)
1,2a
9.9
9.5
7.4
6.6
1,2b
1,8
6.6
7.5
7.2
7.1
11.9
8.0
10.5
7.9
1,9a
6.3
6.5
5.9
7.1
1,9b
6.9
7.1
5.1
7.7
2a,2b
2a,3a
2a,3b
2b,3a
2b,3b
3a,3b
5a,5b
5a,6a
5a,6b
5b,6a
5b,6b
6a,6b
6a,7
ꢀ12.6
ꢀ12.3
ꢀ11.8
ꢀ12.4
6.8
6.8
7.0
6.0
10.3
2.9
5.9
ꢀ10.0
ꢀ11.3
5.9
9.1
4.4
7.0
10.2
2.9
6.0
ꢀ9.6
ꢀ9.8
7.3
2.9
10.8
6.3
2.6
9.8
9.0
ꢀ10.8
ꢀ9.9
8.3
1.2
11.9
6.3
3.1
9.9
7.5
ꢀ11.3
ꢀ10.5
7.2
3.7
9.8
6.1
ꢀ13.1
ꢀ12.1
ꢀ13.3
ꢀ12.2
3.6
4.4
3.6
8.1
6b,7
5.3
2.6
1.3
6.4
7,8
2.6
4.6
3.2
7.1
9a,9b
J7
ꢀ10.9
ꢀ10.5
ꢀ11.1
ꢀ11.1
P
11.5
11.6
8.1
21.6
Stereochemically relevant NOEs
1b + 2b,7b
2a,3a
1b,2b
1b,3b
2a,3a
2a,8a
2a,9a+b
2b,3b
3b,5b
7a,8a
8a,9a+b
1a,2a
1a,8a
2a,3a
2b,3b
3b,5b
5a,6a
6a,7a
7a,8a
1a,2a
1a,8a
2a,3a
2b,3b
2b,7b
2b,9a+b
5a,6a
5b,6b
5b,7b
6b,7b
7b,9a+b
2a,9a+b
2b,3b
3b,5b
5a,6a
5b,6b
6b,7b
8a,9a+b
a
To facilitate the comparison of the data of 1–4, the configuration of C-8 is assumed to be always (R) and H-8 is pointing downwards (a). Hence the
stereochemical descriptors in 1 are those for (+)-hastanecine.
1
0.4 ppm. All H chemical shift differences between 6 and 4
are less than or equal to 0.15 ppm apart from those for
H-9a and H-9b, which amount to 0.60 and 0.55 ppm,
respectively. This ester has not been described so far. Sim-
ilar reasons apply for the structural assignment of 5 to be
7-O-angeloyl-dihydroxyheliotridane. The HMBC spectrum
exhibits a cross-peak between C@O and H-7 (dH =
5.26 ppm), H-7 is deshielded by 1.14 ppm relative to 4
(Table 5), and all DdC values between 5 and 4 (Table 6)
are less than or equal to 1.0 ppm with the exception of

J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
161
Table 4
the sum of all coupling constants involving H-7, and which
was close to the value reported for 2 (14.1 Hz) (Aasen et al.,
13C NMR chemical shifts (CD3OD + ND3) of the stereoisomeric necine
bases
1969).
P
C- Hastanecine Turneforcidine Platynecine Dihydroxy-
The J7 rule had been used previously to infer the rel-
ative configurations of C-1, C-7, and C-8 of substituted
PAs (Culvenor and Woods, 1965). However, it can only
hold if the coupling constants do not change substantially
upon esterification or other substitution of the necine alco-
hol, i.e. if the conformational equilibrium is not affected.
The imponderabilities of this method have been empha-
(1)
(2)
(3)
heliotridane (4)
1
2
3
5
6
7
8
9
47.7
30.9
55.3
53.0
33.5
77.9
76.6
65.3
40.8
32.6
56.2
52.8
37.5
72.0
73.7
65.6
45.0
28.9
56.5
54.8
37.3
73.2
72.6
61.6
44.9
28.6
55.1
53.9
35.8
72.0
73.2
63.2
sized (Culvenor et al., 1965; Logie et al., 1995). Our present
P
J7 values for 5 (15.4 Hz, Table 5) and 4 (21.6 Hz, Table
P
3) again confirm the unreliability of the J7 rule. Nuclear
Overhauser effects are a much safer method to derive the
configurations of the pyrrolizidine alkaloids.
C-7 (+3.7 ppm) and C-6 (ꢀ2.3 ppm). Apart from H-7 all
1H chemical shift differences between 5 and 4 are less than
or equal to 0.25 ppm. The H,H-NOEs (Table 5) confirm
the aminoalcohol moiety of this ester to be dihydroxyhelio-
The PA monoester 7 isolated from P. bombycophole is 7-
O-angeloylturneforcidine. Its chemical and NMR spectro-
scopic properties are different from the compound
described by Roeder and Liu (1991). Again, the esterifica-
tion of the 7-OH group follows from the HMBC correla-
tion of the carboxyl carbon chemical shift with the one of
H-7 (dH = 5.28 ppm, Table 5). Proton H-7 is deshielded
by 1.10 ppm relative to turneforcidine (dH = 4.18 ppm,
Table 3), all other proton chemical shifts being affected
by 0.25 ppm or less. Among the 13C chemical shifts of 7,
1
tridane. Compound 5 shows excellent agreement of its H
and 13C NMR data with those of an alkaloid which (Roe-
der and Liu, 1991) had isolated from Senecio integrifolius
var. fauiri and to which they had attributed the structure
of 7-O-angeloyl-turneforcidine. The criterion on which
they based their structural decision was the half-height line
width of the H-7 NMR signal (15 Hz), which equals J7,
P
Fig. 3. Calculated 400 MHz 1H NMR spectra of the 1,2-saturated necine alcohols hastanecine (1), turneforcidine (2), platynecine (3), and
dihydroxyheliotridane (4) in CD3OD/ND3 solutions obtained by iterative fitting of the total line shape.

162
J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
Table 5
1H NMR data (CD3OD + ND3) of the isomeric necine angelates
a
H-
7-O-Angeloyl-dihydroxyheliotridane (5) DdH
9-O-Angeloyl-dihydroxyheliotridane (6) DdH
7-O-Angeloyl-turneforcidine (7) DdH
1a
1b
2a
2b
3a
3b
5a
5b
6a
6b
7a
7b
8a
9a
9b
30
2.49
–
0.05
–
0.07
0.11
0.09
0.07
2.59
–
1.86
1.62
2.90
2.68
0.15
–
–
–
2.32
1.75
ca 2.12
3.12
2.61
3.15
2.70
ca 2.18
ca 2.12
5.28
–
3.41
3.54
3.52
6.18
1.99
1.91
ꢀ0.21
0.07
0.02
0.04
0.03
0.12
0.00
0.18
0.13
1.10
–
1.88
1.64
2.98
2.72
3.17
2.83
1.84
2.20
–
5.26
3.50
3.78
3.63
6.15
1.96
1.87
0.05
0.09
0.01
0.03
0.00
0.02
0.01
0.01
–
ꢀ0.01
0.00
0.60
0.55
ꢀ0.02 3.19
0.24
0.10
0.14
–
2.61
1.75
2.07
–
4.11
3.25
1.14
0.25
ꢀ0.07 4.45
ꢀ0.04 4.22
6.14
0.25
ꢀ0.03
ꢀ0.01
40
1.97
1.89
50
Coupling constants, J (H,H)
1,2a
1,2b
1,8
1,9a
1,9b
6.8
9.4
8.2
6.3
7.3
6.5
10.7
7.8
6.3
8.1
9.1
6.6
6.9
6.8
6.8
2a,2b ꢀ12.5
2a,3a 6.4
2a,3b 4.4
2b,3a 8.6
2b,3b 7.5
3a,3b ꢀ11.1
5a,5b ꢀ11.3
5a,6a 7.3
5a,6b 6.6
5b,6a 5.9
5b,6b 7.0
6a,6b ꢀ13.8
ꢀ12.4
ꢀ12.5
5.9
6.7
2.9
10.0
7.5
ꢀ11.4
ꢀ10.5
7.1
3.9
9.6
6.2
10.2
3.1
6.0
ꢀ9.7
ꢀ10.1
7.6
2.8
10.8
6.3
b
ꢀ12.3
?
6a,7
6b,7
7,8
4.5
6.5
4.4
7.8
4.5c
1.9
6.4
6.9
4.6
9a,9b ꢀ10.9
ꢀ11.2
ꢀ10.7
30,40
30,50
7.3
1.5
1.6
15.4
7.3
7.3
1.5
1.6
21.1
1.5
1.6
11.0
40,50
P
J7
Stereochemically relevant NOEs
1a,2a
1a,3a
1a,8a
2a,3a
2b,3b
5a,6a
5b,6b
6b,7b
7b,9a+b
1a,2a
1a,8a
2a,3a
2b,3b
2b,7b
3b,5b
5a,6a
5b,6b
5b,7b
6b,7b
1b,2b
1b,3b
2a,3a
2a,9a+b
2b,3b
3b,5b
5a,6a
6a,7a
7a,8a
8a,9a+b
a
dH(necine ester)–dH(necine alcohol).
Undetermined.
The values of J(6a,7) and J(6b,7) may be interchanged.
b
c
d
C-7 is increased by 3.5 ppm and dC-6 and dC-8 are decreased
by an esterification of 7-OH. All other 13C shifts are
affected by 0.9 ppm or less. Nuclear Overhauser effects
by 1.7 and 1.8 ppm, respectively, relative to 2 as expected

J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
163
Table 6
13C NMR chemical shifts (CD3OD + ND3) of the isomeric necine angelates
C-
7-O-Angeloyl-dihydroxyheliotridane (5)
DdC
0.0
1.0
ꢀ0.3
0.1
9-O-Angeloyl-dihydroxyheliotridane (6)
DdC
7-O-Angeloyl-turneforcidine (7)
DdC
1
2
3
5
6
7
8
9
10
20
30
40
50
44.9
29.6
54.8
54.0
33.5
75.7
72.7
62.8
169.3
128.9
139.5
16.1
20.7
41.9
28.9
54.9
54.0
36.1
72.3
72.8
65.1
169.6
129.0
139.3
16.1
20.8
ꢀ3.0
0.3
41.7
32.4
56.1
53.0
35.8
75.5
71.9
65.2
168.5
128.8
140.2
16.1
20.9
0.9
ꢀ0.2
ꢀ0.1
0.2
ꢀ0.2
0.1
ꢀ2.3
0.3
0.3
ꢀ1.7
3.7
3.5
ꢀ0.5
ꢀ0.4
ꢀ0.4
ꢀ1.8
ꢀ0.4
1.9
dC(necine ester)–dC(necine alcohol).
(Table 5) confirmed the turneforcidine configuration of the
necine base.
4. Experimental
4.1. Plant materials
3. Conclusion
P. praecox (Cav.) Rob. and Brett. was collected within
´
the Campus of the Universidad Nacional Autonoma de
´
´
´
A specific feature of the PA profiles found in the genus
Pittocaulon is the dominating side-by-side occurrence of
simple angeloylmonoesters of 1,2-saturated necine bases
and 1,2-unsaturated macrocyclic otonecine esters. The
two types of PAs differ in their biological activities. The
1,2-unsaturated PAs are generally pro-toxins that are bio-
activated by cytochrome P450 enzymes of the xenobiotic
detoxification systems of vertebrates (Fu et al., 2004)
and insects (Frei et al., 1992) causing fatal cytotoxic and
mutagenic effects. On the other hand, 1,2-saturated PAs
are not bioactivated and their effects on herbivores is still
not well understood. They represent the only PA type
present in alkaloid-producing orchids (Fro¨lich et al.,
2006) and most PA containing Ipomoea species (Convol-
vulaceae) (Jenett-Siems et al., 1998, 2005). They have been
reported to be insect deterrents (Reina et al., 1997) and
they are sequestered by insects that utilize plant PAs for
their own protection (Hartmann et al., 2005a,b;
Mexico (UNAM) (2277 m), Mexico D.F, Mexico. Flowers,
roots and stems were collected during spring (March 2004),
leaves at fall (October, 2004). Flowers, roots and stems of
P. bombycophole (Bullock) H. Rob. & Brett., P. velatum
(Greenm.) H. Rob. & Brett., and P. hintonii H. Rob. &
Brett. were collected in March 2005. Roots and stems of
P. filare (McVaugh) H. Rob. & Brett. were collected in
March 2005.
P. velatum and P. hintonii, were collected in the state of
Michoacan, Mexico along the road from Ciudad Hidalgo
to Zitacuaro (2235 m) and Municipio of Coalcoman (Ran-
cho la Parota) (1230 m), respectively. P. filare was collected
about 5 km south of the intersection to Los Asmoles on the
non-toll highway Colima-Manzaillo (464 m) in the state of
Colima, Mexico. P. bombycophole was collected along the
road between Buenavista and Coaxcaclan, in the state of
Guerrero, Mexico at 1728 m. The botanical identification
of the plants was done by Dr. Mark Olson (Institute of
Biology, UNAM). Voucher Specimens are deposited at
the Herbarium of the Institute of Biology, UNAM
(MEXU). Plant materials were air-dried, grounded and
stored dry until alkaloid extraction.
´
´
´
´
´
´
´
´
Marın-Loaiza et al., 2007). The latter reference concerns
sequestration of Pittocaulon PAs by a scale insect. The
chemoecological function of the 1,2-saturated PAs needs
further clarification.
From the chemical point of view the presence of the
angeloyl esters of the necine base dihydroxyheliotridane
in two Pittocaulon species and their complete replacement
by the stereoisomeric necine base turneforcidine in the
three remaining species is noteworthy. The unambiguous
assignment of the correct configuration of the respective
1,2-saturated necine bases was achieved by a detailed
GC–MS analysis and confirmation of the structures by
NMR. This method can easily be applied to confirm the
correct configuration of a 1,2-saturated necine base in an
unknown alkaloids sample.
4.2. GC–EI-MS, routine GC and polarimetry
Configuration I. A Hewlett–Packard 5890A gas chro-
matograph equipped with a 30 m · 0.32 mm (ft 0.25 lm)
analytical column (ZB-1, Phenomenex, Aschaffenburg,
Germany) was used. The capillary column was directly
coupled to a TSQ 700 mass spectrometer (Finnigan, Bre-
men, Germany). The conditions applied were: Injector
and transfer line were set at 250 ꢁC; the temperature pro-
gram used was: 100 ꢁC (3 min)–6 ꢁC/min–310 ꢁC (3 min).

164
J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
The injection volume was 1 ll. The split ratio was 1:20, the
carrier gas flow was 1.6 ml minꢀ1 He, and the mass spectra
were recorded at 70 eV.
the dark. The catalyst was removed by centrifugation
(1 min) and the solvent was evaporated under a stream of
N2. The residue was dissolved in 2 ml MeOH then filtrated
and the solvent removed again under a stream of N2. The
oily residue (yield 20 mg) was crystallized from acetone at
ꢀ18 ꢁC. The purity was checked with GC and GC–MS
analysis after derivatization with MSTFA. The resulting
GC–MS chromatograms revealed two peaks with almost
identical mass spectra. The main compound (92%, based
on peak area) was as expected the (ꢀ)-platynecine derivative,
the other compound (8%) the corresponding diasteromere
at C-1 (ꢀ)-turneforcidine. The proportion of (ꢀ)-turne-
forcidine could be reduced to <3% when monocrotaline
was directly hydrogenated with H2 followed by a basic
hydrolysis of the resulting 1,2-dihydromonocrotaline.
(ꢀ)-Dihydroxyheliotridane was prepared from heliotri-
dine using the same method as described above for platyne-
cine. The purity was checked with GC and GC–MS
analysis after derivatization with MSTFA. The resulting
GC–MS chromatograms revealed again two peaks with
almost identical mass spectrua. The main compound
(93%, based on peak area) was the expected (ꢀ)-dihydroxy-
heliotridane derivative, the other compound (7%) the cor-
responding diasteromere at C-1 (+)-hastanecine. The
appearance of the corresponding minor diasteromere did
not interfere with NMR measurements. In fact it showed
to be helpful in establishing the GC-method to separate
all four naturally occurring saturated necine bases.
Configuration II. A Hewlett–Packard 6890N gas chro-
matograph equipped with a 30 m · 0.32 mm (ft 0.25 lm)
analytical column (DB-5, Agilent J&W, Waldbronn, Ger-
many) was used. The capillary column was directly coupled
to a GC Mate II mass spectrometer (Jeol, Tokyo, Japan).
The conditions applied were: Injector and transfer line were
set at 250 and 250 ꢁC, respectively; the temperature pro-
gram used was: 100 ꢁC (3 min)–6 ꢁC/min–310 ꢁC (3 min).
The injection volume was 1 ll. The split ratio was 1:5 to
1:20, the carrier gas flow was 1.5 ml minꢀ1 He, and the
mass spectra were recorded at 70 eV.
Routine gas chromatography (GC) was performed as
described applying a 15 m · 0.25 mm (ft 0.25 lm) fused-sil-
ica column (DB1, J&W Scientific) (Witte et al., 1993; Hart-
mann et al., 2004). Quantitative analyzes were performed
via the FID signals with heliotrine or monocrotaline as
internal standards.
Where possible, the optical activity of the isolated or
synthesized compounds was determined with a Propol
Polarimeter (Dr. Kernchen, Seelze-Letter, Germany) at
25–27 ꢁC. The measurement was not applied to compounds
that showed <95% purity (based on GC and NMR
measurements).
4.3. NMR spectroscopy
589
(ꢀ)-Dihydroxyheliotridane ð½aꢁD ꢀ15.7ꢁ (MeOH)) was
1H and 13C NMR spectra were obtained on a Bruker
DRX-400 spectrometer at 400.1 and 100.6 MHz, respec-
tively, or on a Bruker Avance II-600 spectrometer at
600.1 and 150.9 MHz, respectively. The solvent was
CD3OD (ca. 0.65 ml) to which 2–3 drops of ND3 (25%
also obtained in pure form after hydrolysis of naturally
occurring 7-angeloyldihydroxyheliotridane.
589
(ꢀ)-Turneforcidine ½aꢁD +4.3ꢁ (MeOH) was obtained
by hydrolysis of 7-angeloylturneforcidine isolated from P.
bombycophole and P. hintonii.
589
1
solution in D2O, Aldrich) had been added. H chemical
(+)-Hastanecine ½aꢁD ꢀ10ꢁ (MeOH) was kindly sup-
shifts were referenced to internal tetramethylsilane (TMS,
dH = 0.00 ppm), 13C chemical shifts to the solvent signal
(dC = 49.0 ppm). The quantity of the compounds dissolved
ranged from 1.5 to 20 mg depending on their availablility.
plied as a gift by Dr. S. E. Denmark.
4.5. Separation and characterization of the 1,2-saturated
necine bases by GC/MS
1
One-dimensional H and 13C spectra, DEPT-135 13C spec-
tra and two-dimensional H,H-COSY, H,H-NOESY, H,C-
HSQC and H,C-HMBC spectra were with standard Bruker
pulse programs. The mixing delay in the NOESY experi-
ments was 2.0 s. The one-dimensional H NMR spectra
were analyzed by iterative fitting of the full lineshape using
Bruker’s WIN-DAISY program version 4.05 (1998).
Trimethylsilyl derivatives. The compounds (0.1–0.2 mg)
were dissolved in 100 ll N-methyl-N-(trimethylsilyl)trifluo-
roacetamide (MSTFA) at 75 ꢁC for 30 min and then
directly analyzed by GC–MS.
7,9-Di-O-trimethylsilyl-turneforcidine. RI(ZB1) 1572,
RI(DB5) 1581; GC–EIMS, m/z (rel. int.): 301 (3, [M]+),
286 (6), 211 (15), 198 (2), 185 (65), 170 (2), 147 (3), 143
(6), 122 (3), 82 (100), 73 (16), 55 (2), 45 (2).
1
4.4. Preparation or origin of saturated necine bases
7,9-Di-O-trimethylsilyl-hastanecine. RI(ZB1) 1613,
RI(DB5) 1626; GC–EIMS, m/z (rel. int.): 301 (2, [M]+),
286 (4), 211 (17), 198 (2), 185 (62), 170 (1), 147 (3), 122
(2), 82 (100), 73 (13), 55 (1), 45 (2).
7,9-Di-O-trimethylsilyl-platynecine. RI(ZB1) 1615,
RI(DB5) 1627; GC–EIMS, m/z (rel. int.): 301 (2, [M]+),
286 (7), 211 (15), 198 (2), 185 (73), 170 (2), 147 (3), 143
(1), 122 (4), 82 (100), 73 (16), 55 (2), 45 (2).
The starting compound retronecine was obtained by
hydrolysis of monocrotaline in saturated aqueous BaOH
at 100 ꢁC for 2 h. (ꢀ)-Platynecine was prepared by catalytic
hydrogenation of retronecine using a slightly modified pro-
cedure recently described by Donohoe et al. (2002). A solu-
tion of 23 mg retronecine in 4 ml anhydrous THF
containing 1 mg rhodium on activated charcoal (Fluka)
was stirred under atmospheric pressure of H2 for 16 h in

J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
165
7,9-Di-O-trimethylsilyl-dihydroxyheliotridane. RI(ZB1)
1628, RI(DB5) 1641; GC–EIMS, m/z (rel. int.): 301 (2,
[M]+), 286 (4), 211 (14), 198 (2), 185 (60), 170 (2), 147
(3), 143 (1), 122 (3), 82 (100), 73 (16), 55 (2), 45 (2).
1,2-Unsaturated necine bases (for comparison)
to an Extrelut (Merck) column. The alkaloid fraction was
eluted with CH2Cl2 (l ml/g Extrelut). The solvent was evap-
orated and the residue directly applied to GC–MS analysis
or HPLC.
Semi-preparative HPLC. Samples of 0.5 ml were applied
to a RP-18 Column (Nucleosil 120-7, 250 mm long, 25 mm
i.d.; Macherey & Nagel). Separation was achieved isocrat-
ically by using CH3CN/TFA (25:75 v/v.), pH 2, at a flow
rate of 9 ml minꢀ1; detection: absorbance at 210 nm. Com-
pound 5, RT = 13.7 min; compound 6, RT = 15.7 min.
Monoester 7 was isolated from P. bombyphocole and P.
hintonii using the same procedure as applied for the purifi-
cation and separation of compounds 5 and 6 (HPLC,
RT = 15.1 min).
7,9-Di-O-trimethylsilyl-retronecine. RI(ZB1) 1602,
RI(DB5) 1617; GC–EIMS, m/z (rel. int.): 299 (5, [M]+),
284 (1), 210 (2), 208 (3), 183 (40), 168 (5), 140 (4), 120
(9), 116 (16), 106 (8), 103 (26), 94 (40), 93 (84), 80 (17),
75 (23), 73 (100), 67 (9), 59 (12), 53 (7), 45 (18).
7,9-Di-O-trimethylsilyl-heliotridine. RI(ZB1) 1637,
RI(DB5) 1651; GC–EIMS, m/z (rel. int.): 299 (4, [M]+),
284 (2), 210 (2), 208 (2), 183 (36), 168 (5), 140 (4), 120
(8), 116 (15), 106 (11), 103 (25), 94 (25), 93 (83), 80 (17),
75 (24), 73 (100), 67 (9), 59 (11), 53 (7), 45 (17).
Acetylated derivatives. The compounds (0.5–1 mg) were
dissolved in pyridine/Ac2O (200 ll, 1:1 v/v) at 75 ꢁC for
2 h and then directly analyzed by GC–MS.
7,9-Di-O-acetyl-turneforcidine. RI(ZB1) 1609, RI(DB5)
1659; GC–EIMS, m/z (rel. int.): 241 (3, [M]+), 198 (4), 182
(18), 181 (36), 168 (4), 155 (7), 138 (48), 121 (18), 108 (7), 95
(46), 82 (100), 68 (4), 55 (9).
7,9-Di-O-acetyl-platynecine. RI(ZB1) 1636, RI(DB5)
1687; GC–EIMS, m/z (rel. int.): 241 (2, [M]+), 198 (3),
182 (20), 181 (44), 168 (3), 155 (6), 138 (43), 121 (17), 108
(6), 95 (42), 82 (100), 68 (4), 55 (9).
4.7. Identification of 1,2-saturated PA monoesters
589
7-Angeloyl-dihydroxyheliotridane (5). ½aꢁD
7.5ꢁ
(MeOH). RI(ZB1) 1811, RI(DB5) 1898; GC–EIMS, m/z
(rel. int.): 239 (1, [M]+), 221 (2), 156 (41), 139 (80), 138
(27), 108 (14), 106 (11), 96 (8), 95 (14), 82 (100), 68 (5),
55 (28), 53 (7).
589
9-Angeloyl-dihydroxyheliotridane (6). ½aꢁD
ꢀ8.2ꢁ
(MeOH). RI(ZB1) 1834, RI(DB5) 1911; GC–EIMS, m/z
(rel. int.): 239 (10, [M]+), 221 (17), 195 (3), 140 (11), 122
(13), 119 (6), 106 (4), 96 (41), 95 (100), 82 (90), 67 (3), 55 (23).
7,9-Di-O-acetyl-hastanecine. RI(ZB1) 1652, RI(DB5)
1704; GC–EIMS, m/z (rel. int.): 241 (2, [M]+), 198 (3),
182 (18), 181 (32), 168 (3), 155 (6), 138 (42), 121 (26), 108
(8), 95 (40), 82 (100), 68 (4), 55 (10).
7,9-Di-O-acetyl-dihydroxyheliotridane. RI(ZB1) 1659,
RI(DB5) 1712; GC–EIMS, m/z (rel. int.): 241 (1, [M]+),
198 (2), 182 (21), 181 (41), 168 (2), 155 (4), 138 (38), 121
(32), 108 (9), 95 (35), 82 (100), 68 (5), 55 (11).
589
7-Angeloyl-turneforcidine (7). ½aꢁD +26.6ꢁ (MeOH).
RI(ZB1) 1781, RI(DB5) 1845; GC–EIMS, m/z (rel. int.):
239 (4, [M]+), 156 (60), 139 (100), 138 (17), 114 (3), 113
(20), 108 (7), 82 (60), 68 (2), 55 (9), 53 (2).
9-Angeloyl-turneforcidine (8). RI(ZB1) 1806, RI(DB5)
1869; GC–EIMS, m/z (rel. int.): 239 ([M]+, 14), 221(23),
195 (3), 156 (15), 140 (15), 139 (24), 122 (21), 82 (100), 96
(49), 95 (99), 55 (20).
1,2-Unsaturated necine bases (for comparison).
7,9-Di-O-acetyl-retronecine. RI(ZB1) 1625, RI(DB5)
1712; GC–EIMS, m/z (rel. int.): 239 (18, [M]+), 197 (36),
180 (32), 179 (34), 168 (3), 153 (10), 136 (36), 120 (24),
119 (30), 118 (7), 106 (8), 101 (12), 94 (37), 93 (100), 80
(18), 67 (4), 53 (7).
7,9-Di-O-acetyl-heliotridine. RI(ZB1) 1652, RI(DB5)
1705; GC–EIMS, m/z (rel. int.): 239 (9, [M]+), 197 (24),
180 (25), 179 (23), 168 (3), 153 (11), 136 (35), 120 (21),
119 (49), 118 (6), 106 (13), 101 (13), 94 (38), 93 (100), 80
(21), 67 (6), 53 (10).
4.8. Identification of other PAs
Widely distributed and well characterized PAs like 7-
angeloylretronecine (9), senecionine (10), integerrimine
(11), platyphylline (12), neoplatyphylline (13), senkirkine
(14) and neosenkirkine (15) were identified by their RIs,
molecular ions and mass fragmentation patterns in com-
parison to reference compound and our comprehensive
MS data base of PAs (Witte et al., 1993). Their GC–MS
data are not shown. Some less abundant PAs were also
identified by their GC–MS characteristics in comparison
to reference compounds and the data base. Their GC–
MS data are given below:
Petasitenine (19) RI(ZB1) 2442. GC–EIMS, m/z (rel.
int.): 381 ([M+], 4), 353 (5), 294 (6), 266 (33), 250 (17),
168 (88), 152 (37), 151 (100), 124 (36), 123 (68), 122 (52),
110 (61), 108 (35), 96 (41), 70 (40).
Acetylsenkirkine (16) RI(ZB1) 2641. GC–EIMS, m/z
(rel. int.): 407 ([M+], 1), 320 (2), 303 (8), 266 (6), 249
(11), 221 (5), 166 (25), 153 (48), 122 (30), 110 (48), 107
(26), 83 (24), 82 (29), 81 (24), 53 (29), 43 (100).
4.6. Isolation of the 1,2-saturated PA monoesters
Isolation and identification of monoesters 5 and 6 from
P. praecox: Powdered plant materials (leaves, flowers) were
defatted by Soxhlet extraction with petroleum ether
(40 ꢁC–60 ꢁC) and then the alkaloids Soxhlet-extracted
with MeOH. The solvent was evaporated under reduced
pressure and the residue dissolved in 1 M HCl, filtered
and extracted with CH2Cl2. Zinc dust was added to the
acidic aqueous solution for 3 h to reduce all PA N-oxides.
The mixture was filtered, made basic to pH 11 and applied

166
J.C. Marı´n-Loaiza et al. / Phytochemistry 69 (2008) 154–167
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are separated by GC but the assignment is still uncertain;
the MS fragmentation patterns were identical. RI(DB1)
2720 and 2709. GC–EIMS, m/z (rel. int.): 407 ([M+], 2),
320 (7), 303 (30), 249 (98), 221 (16), 199 (29), 166 (34),
154 (33), 153 (100), 122 (47), 110 (70), 108 (17), 107 (52),
100 (28), 96 (29), 94 (27), 83 (34), 82 (39), 81 (38).
Acetylpetasitenine (20) RI(ZB1) 2632. GC–EIMS, m/z
(rel. int.): 423 ([M+], 5), 336 (79), 319 (43), 304 (44), 296
(13), 281 (11), 264 (20), 250 (61), 238 (22), 168 (61), 154
(34), 150 (86), 141 (42), 94 (37), 83 (35), 81 (34), 123 (46),
122 ( 92), 110 (79), 69 (86), 55 (36), 44 (70), 43 (100).
PA-X (otonecine type PA) RI(ZB1) 2542. GC–EIMS,
m/z (rel. int.): 381 ([M]+, 11), 182 (28), 168 (24), 153
(100), 144 (31), 109 (24), 100 (34), 83 (42), 82 (31), 58
(36), 57 (27).
Acknowledgements
This work was supported by a Grant of the Deutsche
Forschungsgemeinschaft to T.H. We thank Dr. S.E. Den-
mark (University of Illinois, Urbana, USA) for providing
a sample of (ꢀ)-hastanecine and valuable suggestions and
Dr. Mark Olson (Biology Institute, UNAM) for the taxo-
nomic identification of the Pittocaulon species. J.C.M.L
thanks the German Academic Exchange Service (DAAD)
for a Research Fellowship in Braunschweig (01.03.2006–
31.05.2006), as well as DGAPA-PAPIIT IN211105-3 pro-
ject, for the support and DGEP for a doctoral fellowship
(01.02.2005-01.01-2007).
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