The recurvatianes: A suite of oxygenated guaiane sesquiterpenes from Perezia recurvata

Article abstract of DOI:10.1016/j.phytochem.2010.11.021

Six guaiane sesquiterpenes recurvatiane A-F, including the previously known recurvatiane E and xanthomicrol, were isolated from the Andean Perezia recurvata and their structures determined by spectroscopic evidence. The absolute stereochemistry of recurvatiane A was established by derivatization with (R)- and (S)-α-methoxy-α-phenylacetic acids (MPA). The suite of guaiane-based metabolites here reported, which we have named recurvatianes A-F, provide an example of a pathway by which molecular diversity is generated by the occurrence of specific oxidation reactions in the late biosynthetic steps of the frame structure.

Full text of DOI:10.1016/j.phytochem.2010.11.021

Phytochemistry 72 (2011) 284–289
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
The recurvatianes: A suite of oxygenated guaiane sesquiterpenes
from Perezia recurvata
b
b,
Amalia B. Gallardo a,b, Mercedes Cueto a, Ana R. Díaz-Marrero a, , Pedro Cuadra , Victor Fajardo
⇑
⇑
,
José Darias a
a Instituto de Productos Naturales y Agrobiología del CSIC, Avda. Astrofísico F. Sánchez, 3, Apdo. 195, 38206 La Laguna, Tenerife, Spain
b Universidad de Magallanes, Avda. Bulnes 01855, Punta Arenas, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 July 2010
Received in revised form 22 November 2010
Available online 30 December 2010
Six guaiane sesquiterpenes recurvatiane A–F, including the previously known recurvatiane E and xant-
homicrol, were isolated from the Andean Perezia recurvata and their structures determined by spectro-
scopic evidence. The absolute stereochemistry of recurvatiane A was established by derivatization with
(R)- and (S)-a-methoxy-a-phenylacetic acids (MPA). The suite of guaiane-based metabolites here
reported, which we have named recurvatianes A–F, provide an example of a pathway by which molecular
diversity is generated by the occurrence of specific oxidation reactions in the late biosynthetic steps of
the frame structure.
Keywords:
Natural products
Guaiane metabolites
Structure elucidation
Absolute configuration
Perezia recurvata
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
The genus Perezia has been separated (Reveal and King, 1973;
Crisci, 1980; Kim et al., 2002) from genus Acourtia (previously a
The Nassauvieae tribe (Asteraceae) contains approximately 24
genera and 370 species and is an important component in the flora
of the Central and Southern Andean region (Panero and Funk,
2008). Indigenous communities still maintain traditional uses
linked to biodiversity, and medicinal plants are sometimes the only
available treatment for certain illnesses. Some examples are: Pere-
zia multiflora (local name: escorzonera, chanqoroma, used as diure-
tic, antipyretic, expectorant and particularly valued as a means of
controlling bleeding in childbirth and uterine hemorrhages); Pere-
zia coerulescens, Perezia pinnatifida (valeriana: sedative, diuretic,
and diaphoretic) and Perezia purpurata (marancel: antiinflamma-
tory, antiviral, carminative, soft sedative) (De-la-Cruz et al., 2007).
The results from molecular phylogenetic studies of the tribe
Nassauvieae based on chloroplast DNA coding regions show that
the genus Perezia is not monophyletic since Perezia nutans and
Perezia prenanthoides are not related to other species of Perezia.
The removal of P. nutans and P. prenanthoides from Perezia to form
the new genus Calorezia (Panero, 2007) was proposed, so the genus
Perezia includes 30 species instead of the 32 so far recognized, 18 of
which are found in Chile (www.florachilena.cl).
section of Perezia). In earlier literature, Acourtia genus from North
and Central America has usually been treated as Perezia, section
Acourtia, which can be misleading. Chemically, these two genera
differ by the occurrence of isocedrenes in the genus Perezia
(Bohlmann and Zdero, 1979; Zdero et al., 1986, 1988; Bittner
et al., 1989), and perezone (Archer and Thompson, 1965; Joseph-
Nathan, 1974) and related compounds in the species classified in
the genus Acourtia (Zdero et al., 1991).
Thirty years ago the literature available on the chemistry of the
Nassauvieae tribe was almost nonexistent (Heywood et al., 1977).
Since then, contributions to the knowledge of Perezia genus have
been made as a result of the number of chemistry-related studies
of the species Perezia carthamoides, Perezia lactucoides, Perezia line-
aris, Perezia lyrata, Perezia megalantha, Perezia pedicularifolia, Pere-
zia pilifera, Perezia recurvata (Bittner et al., 1989), Perezia
multiflora (Bittner et al., 1989; Joseph-Nathan et al., 1978), P. coe-
rulescens (Angeles et al., 1984), and Perezia ocorzonera (De-Israilev
and González, 1994). These studies indicate that isocedrene deriv-
atives are widely distributed within this genus.
In this paper we describe the structures of six guaiane-derived
sesquiterpenes 1–6 (Fig. 1) which, along with xanthomicrol 7
(Stout and Stout, 1961) were isolated from P. recurvata (Vahl) Less
collected in Sierra Baguales (Magellan Region, Chile). A previous
study (Bittner et al., 1989) of P. recurvata led to the isolation of
compound 5 as the unique guaiane derivative. However, in this
work we have isolated a suite of guaiane-based metabolites that
⇑
Corresponding authors. Tel.: +34 922 252 144; fax: +34 922 260 135 (A.R. Díaz-
Marrero), tel.: +56 61 207 168; fax: +56 61 219 276 (V. Fajardo).
E-mail addresses: ardiaz@ipna.csic.es (A.R. Díaz-Marrero), victor.fajardo@
umag.cl (V. Fajardo).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.11.021

A.B. Gallardo et al. / Phytochemistry 72 (2011) 284–289
285
14
Table 1
13C NMR data of compounds 1–6 [125 MHz, dC ppm, CDCl3].
b
H
H
1
10
9
2
#
1
2
3
4
5
6
O
O
3
8
HO
7
a
12
c
5
6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
47.7
34.3
75.8
135.0
148.5
71.7
47.9
30.3
29.9
34.1
145.1
66.9
114.6
20.1
44.4
37.2
206.7
133.4
181.6
71.6
50.1
29.2
30.2
33.4
145.8
66.2
114.8
20.9
54.7
–
44.5
37.3
205.9
135.0
177.5
72.7
45.9
30.2h
30.2h
33.8
149.1
66.1
112.8
20.9
54.5
170.0
20.8b
–
44.3
37.4
207.8
138.5
174.5
73.0
47.1
30.2c
30.3c
33.8
144.6
66.8
115.6
20.9
55.0
170.1
20.8d
170.8
20.9d
–
44.4
44.3
4
11
H
OAc
37.2
37.2
15
OAc
OAc
AcO
205.5
135.0
177.0
73.0
205.7
135.0
176.5
72.6
AcO
13
1: recurvatiane A
6: recurvatiane F
47.4
41.1
OH
H
MeO
O
30.4
30.1
30.2
29.6
O
MeO
MeO
33.7
33.8
R2
144.4
66.5
115.4
20.6a
54.7
169.8
20.7e
170.5
20.7e
170.4
20.8e
150.7
193.4
134.9
20.9
O
R1
R3
2: recurvatiane B: R1= OH. R2= R3= OAc
3: recurvatiane C: R2= OH, R1= R3= OAc
4: recurvatiane D: R3= OH, R1= R2= OAc
5: recurvatiane E: R1= R2= R3= OAc
59.1
170.0
20.8
170.6
20.8
54.6
OH
6–C@O
6–COCH3
12–C@O
12–COCH3
15–C@O
15–COCH3
170.8g
20.7f
–
7
–
170.7
20.8a
170.7
20.9a
–
–
Fig. 1. Structure of metabolites isolated from Perezia recurvata.
171.2
20.8
171.2
170.9g
20.8f
20.9b
–
a–h
Overlapped signals. Chemical shifts confirmed by 2D HSQC and HMBC
experiments.
provide an interesting example of the pathway by which molecular
diversity is generated by specific oxidation reactions on a guaiane
intermediate in the late biosynthetic steps. Although the known
compound 5 has been referred to by its IUPAC name (Bittner
et al., 1989), it might be convenient to group it together with
1–4, and 6 as recurvatianes A–F.
respectively. Oxygenated functionalities are consistent with the
observed IR absorptions.
All C–H correlations for 1 were inferred from the HSQC spec-
trum. An 1H–1H COSY experiment showed coupling between a
methylene (H2-2) with two well-resolved methine protons (H-1
and H-3). This spin system is indeed extended from H-1 through
H-6. The mutual HMBC correlations of H2-13/C-12, H2-12/C-13
and their long-range correlations of both H2-13 and H2-12 with
C-7 and with an olefinic quaternary carbon C-11 secured an acet-
oxy isopropenyl group at C-7. The long-range correlations of H2-8
and H-6 with C-11 defined the fragment a. Fragment b was estab-
lished by the long range correlation of the secondary methyl group
H3-14 with C-1, C-9 and C-10. Fragments a and b were connected
through C-8 and C-9 by the correlations H-10/C-8, and H-9/C-7.
The remaining carbons of the molecule must form part of a cyclo-
pentene ring with a methyl acetate appendage. As the spin system
indicated three contiguous carbon (C-1 to C-3), the correlation H-
15/C-4, C-3, C-5; H-3/C-4, C-5, C-15 established a cyclopentane
ring fused to fragments a/b by the correlation H-6/C-5, C-4, C-1.
Therefore the connectivity information obtained from COSY, HSQC
and HMBC experiments unambiguously determined the planar
structure of compound 1 as a highly oxidized guaiane network
with a hydroxyl at C-3 and acetate groups at C-6, C-12 and C-15.
The relative stereochemistry of compound 1 was deduced by
NOESY experiments, analysis of coupling constants and molecular
mechanic calculations (PCModel Software). NOE correlation of H-6
with H-7 indicated that the secondary acetate and acetoxy isopro-
penyl groups are located on the same face of the molecule. The
NOE effects observed between H-1 with H-10 and the methylenic
2. Results and discussion
From the methanolic extract of P. recurvata, collected in the
Sierra Baguales (Magellan Region, Chile), recurvatianes A–F (1–6),
were isolated after gel filtration on Sephadex LH-20 chromatogra-
phy followed by normal phase HPLC.
Recurvatiane A (1) was obtained as a colourless oil ½a D20
ꢁ 182
ꢀ
(c, 0.11, CH2Cl2). Its EIMS spectrum showed a peak at 394, which
corresponds to the empirical formula C21H30O7 [M]+ (HREIMS m/z
394.2001 (calcd. for
C21H30O7 394.1992)). The IR spectrum
showed absorption for hydroxyl and carbonyl groups at 3460
and 1740 cmꢁ1, respectively. The 13C NMR and DEPT spectra of
1 (Table 1) showed the presence of 21 carbon signals assigned
to 4 ꢂ CH3 (three from acetate methyl groups), 6 ꢂ CH2 (one ole-
finic), 5 ꢂ CH (two bearing oxygen) and six quaternary carbons
(three olefinic). The lack of 2D NMR spectroscopic information
of most of the guaiane-derived metabolites reported from the
Nassauvieae tribe renders a detailed 2D NMR spectral analysis
of 1 expedient.
The 1H NMR spectrum of 1 displayed singlets at d 5.03 and d
5.09 due to olefinic protons. Signals at d 4.55 (d, J = 6.6) and at
5.82 (s) were assigned to protons geminal to oxygen, as well as
the pair of methylene protons at d 4.50 (d, J = 13.2), 4.60 (d,
J = 13.2) and 4.72 (d, J = 12.9), 4.75 (dd, J = 12.9, 2.2), respectively.
Nine well-resolved signals, each attributed to one of the remaining
aliphatic protons, appeared between 1.14 and 3.11 ppm. Three ace-
tate methyl groups (d 2.01, 2.04 and 2.05) and a secondary methyl
group at d 0.86 (d, J = 7.2) accounted for all the protons of the mol-
ecule (Table 2). According to the spectral data and the molecular
formula, compound 1 has to be bicyclic. Since no IR absorption
for a free acid was observed and only one isoprenic methyl group
(Me-14) was evident, the remaining three methyl groups expected
for a sesquiterpene skeleton must be modified, two of them as
methyl acetate (C-12 and C-15) and the other as a terminal meth-
ylene (C-13). Thus, the remaining three oxygen atoms must be in-
volved in a secondary acetate at C-6, and a hydroxyl group at C-3,
proton H-2b (1.77, m) together with the NOE correlation of H-2
a
(1.61, m) and H-3 established a syn relationship for H-1, H-10
and the hydroxyl group at C-3. On the other hand, the correlation
of the methylenic proton H-9b (1.25, m) with H-10 and the NOE ef-
fect observed between H-9a (1.14, m) with H-7 placed H-7 and H-6
on the opposite side of the molecule relative to H-10, establishing
the whole relative stereochemistry of 1 as depicted in Fig. 2.
Molecular mechanic calculations indicated theoretical dihedral an-
gles of 80° for H-6/H-7 and 86° for H-3/H-2b, respectively. These
theoretical values are in good agreement with the absence of
coupling for H-6 (d 5.82, s, Table 2) and the presence of a doublet

286
A.B. Gallardo et al. / Phytochemistry 72 (2011) 284–289
Table 2
1H NMR data of compounds 1–6 500 MHz, dH ppm, (J) Hz, CDCl3.
#
1
2
3
4
5c
6
1
2
3.11 m
3.32 m
3.03 m
3.00 m
3.00 m
3.04 m
a: 2.17 dd (18.3, 4.1)
a
: 1.61 m
a: 2.15 m
a: 2.15 dd (18.3, 4.1)
a: 2.14 dd (18.3, 3.8)
a
: 2.14 dd (18.3, 4.2)
b: 1.77 m
b: 2.46 dd (18.3, 6.3)
b: 2.44 dd (18.3, 6.6)
b: 2.41 dd (18.3, 6.3)
b: 2.42 ddd (18.3, 6.6, 1.0)
b: 2.45 dd (18.3, 6.6)
3
4
5
6
7
8
4.55 d (6.6)
–
–
5.82 s
2.10 dd (12.9, 4.7)
1.63 m
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5.19 s
2.17 m
6.12 s
2.37 m
6.07 s
2.33 m
6.05 s
2.27 m
5.97 s
2.85 dd (12.9, 4.1)
a
: 1.66 m
b: 1.91 m
: 1.08 m
a
: 1.77 m
b: 1.90 m
: 1.13 m
a
: 1.77 m
b: 1.90 m
: 1.15 m
a
: 1.78 m
b: 1.89 m
: 1.12 m
a
: 1.70 m
b: 1.88 m
: 1.21 m
1.75 m
9
a
: 1.14 m
a
a
a
a
a
b: 1.25 m
b: 1.37 dd (14.8, 6.6)
b: 1.41 dd (14.5,5.7)
b: 1.40 dd (14.2, 5.7)
b: 1.40 dd (5.0, 13.8)
b: 1.43 dd (14.8, 5.33)
10
11
12
2.18 m
–
4.50 d (13.2)
2.53 m
–
4.65 d (13.6)
2.35 m
–
4.12 d (13.2)
2.33 m
–
4.54 d (13.2)
2.32 m
–
4.54 d (13.3)
2.36 m
–
9.50 s
4.60 d (13.2)
4.57 d (13.6)
4.20 d (13.2)
4.68 d (13.2)
4.60 d (13.3)
–
13
5.03 s
5.19 s
5.01 s
5.14 s
5.18 m
6.11 s
5.09 s
5.24 s
5.13 d (1.0)
5.19 s
5.11 s
6.37 s
14
15
0.86 d (7.3)
4.75 dd (12.9, 2.2)
4.72 d (12.9)
0.98 d (7.3)
4.63 d (12.3)
4.81 d (12.3)
0.99 d (7.3)
4.88 dd (12.6,1.6)
4.79 d (12.6)
0.99 d (7.3)
4.37 dd (13.2, 1.3)
4.47 dd (13.2, 1.6)
0.97 d (7.2)
4.73 dd (12.7, 1.1)
4.83 dd (12.7, 1.3)
1.01 d (7.3)
4.94 dd (12.6, 1.3)
4.82 d (12.6)
6-C@O
–
–
–
–
–
6-COCH3
12-C@O
12-COCH3
15-C@O
15-COCH3
2.01 s
–
2.05 s
–
–
–
2.09 s
–
–
–
2.11 s
–
2.09 s
–
–
2.09 s
–
2.10 s
–
–
–
2.10 sa
–
2.05 sb
–
2.04 s
2.04 sa
2.06 s
2.04 sb
2.05 s
a,b
Interchangeable signals.
Recorded at 400 MHz.
c
Table 3
1H NMR
MPA esters 1a and 1b.
D
d (dRꢁdS) values (CDCl3, ppm, recorded at 500 MHz) of the diastereomeric
H
Me14
H
H
O
H
10
1
OAc
H
2
3
6
H
AcO
9
5
H
Me
4
O
H
12
8
H
7
15
O
11
H
13
H
H
Fig. 2. Selected NOEs of recurvatiane A (1).
for H-3 (d 4.55, d, J = 6.6), and therefore with the proposed relative
stereochemistry.
J = 13.2; dH 4.20, d, J = 13.2, dC 66.1 ppm) and C-15 (dH 4.37, dd,
J = 13.2, 1.3; dH 4.47, dd, J = 13.2, 1.6; dC 55.0), for 2, 3, 4, respec-
tively, instead of the corresponding acetate groups found in 5.
We have named compound 5 recurvatiane E.
The absolute stereochemistry of 1 was established by derivati-
zation with (R)- and (S)-
a-methoxy-a-phenylacetic (MPA) acids
(Seco et al., 2001). Subsequent NMR analysis of the
D
d values for
The relative stereochemistry of compounds 2–5 were deduced
by NOESY experiments and analysis of coupling constants. They
all showed the same relative stereochemistry as compound 1, also
confirming the previously proposed (Bittner et al., 1989) for 5. Since
compounds 1–5 were isolated from a unique extract of Perezia, it
should be expected that they all belong to the same enantiomeric
series as recurvatiane A, 1. Thus, the absolute stereochemistry of
these compounds has been assigned as follows: recurvatiane B, 2,
1R, 6R, 7S, 10R; recurvatiane C, 3, 1R, 6R, 7S, 10R; recurvatiane D,
4, 1R, 6R, 7S, 10R; and recurvatiane E, 5, 1R, 6R, 7S, 10R.
the two MPA esters 1a and 1b provided clear evidence to assign
the absolute stereochemistry at C-3 as S, Table 3. This information
together with NOESY data established the absolute stereochemis-
try for recurvatiane A (1) as 1R, 3S, 6R, 7S, 10R.
The fragmentation pattern of the EIMS spectra of recurvatianes
B-D, 2–4, showed a molecular ion at m/z 290 [M–AcOH]+ for each
compound, as well as an ion at m/z 230 [M–2AcOH]+. Furthermore,
comparison of their NMR spectroscopic data with those of the
known compound 5 (Bittner et al., 1989), also isolated in this work,
suggested they are positional isomers, the main differences being a
hydroxyl substituent at C-6 (dH-6 5.19, s, dC 71.6), C-12 (dH 4.12, d,
Recurvatiane F, 6 was isolated as a colourless oil ½a D20
ꢁ 50 (c,
ꢀ
0.03, CH2Cl2). Its EIMS spectrum showed a peak at m/z 305

A.B. Gallardo et al. / Phytochemistry 72 (2011) 284–289
287
germacrene
synthase
[O]
6
R
COOH
12
OH
PPO
8: R= Me
8a: R= CH2OH
8b: R= CHO
9
farnesyl diphosphate
-H2O
germacranolides
eudesmanolides
elemanolides
+ H+, - H+
5
[O]
6
4
O
HO
O
O
O
12
O
O
O
guaianolide
parthenolide
(+)-costunolide
Fig. 3. Biosynthetic pathway for guaianolides in Asteraceae (De Kraker et al., 2002, 2003).
H+
OPP
H
guaiadiene
synthase
H
Enzyme-bound
(+)-germacrene A
farnesyl diphosphate
recurvatianes A-F
-H+
H
H
acylation,
oxidation
specific site
allylic oxidation
HO
HO
*
*
*
OH
OH
10
*
Fig. 4. Proposed biogenetic pathway for recurvatianes A–F.
[M–C2H3O]+, which corresponds to the empirical formula C19H24O6
(HREIMS m/z 305.1401 (calcd. for C17H21O5 305.1389)). Absorption
for carbonyl groups at 1740 and 1720 cmꢁ1 were observed in the IR
spectrum. 13C NMR and DEPT spectra of 6 (Table 1) showed the
presence of 19 carbon signals assigned to 3 ꢂ CH3 (two from acetyl
groups), 5 ꢂ CH2 (one olefinic, one bearing oxygen), 5 ꢂ CH (one
bearing oxygen, one aldehyde) and six quaternary carbons (three
carbonylic, three olefinic). The 1H and 13C NMR data were very sim-
ilar to those of recurvatiane C, 3 particularly the chemical shifts of
the carbon skeleton. Connectivity information obtained from COSY,
HSQC and HMBC experiments unambiguously determined the pla-
nar structure of 6 as an aldehyde derived by oxidation of the
hydroxymethyl side chain of 3. NOESY experiments and coupling
constants indicate the compound possesses the same relative ste-
reochemistry as compounds 1–5, thus we also propose its absolute
configuration as 1R, 6R, 7S, 10R.
lowed by 6,12-lactonization to (+)-costunolide (De Kraker et al.,
2002, 2003), precedes cyclization to the guaiane nucleus and to
other sesquiterpene scaffolds, (Fig. 3) and also that guaianolides
from Asteraceae are generated from parthenolide with a unique
ring closure forming 5 and 7 carbon rings via a 4,5 epoxidation
(De Kraker et al., 2002, 2003). Thus, (+)-costunolide, a 6a,7b-ger-
macranolide, appears to be the presumed branching point in the
biosynthesis of guaianolides from Asteraceae, as well as to the
majority of sesquiterpene lactones found in plants, Fig. 3. The en-
zymes that catalyze the production of (+)-costunolide, have re-
cently been identified (De Kraker et al., 1998, 2002; De-la-Cruz
et al., 2007).
In the Asteraceae family, with the exception of the species
Warionia saharae (Hilmi et al., 2002, 2003a,b; Katinas et al.,
2008), the lactone ring of guaianolides is always 6a,7b, while guai-
anolides with a 6b,7b have only been found in Apiaceae (De Kraker
et al., 2002). Therefore, as the recurvatianes A–F, for example 6, en
route to a guaianolide, possess a 6b,7b stereochemistry, which is
unexpected for metabolites belonging to the Asteraceae family,
an alternative biosynthetic precursor to (+)-costunolide could be
considered. Accordingly, the set of guaiane-derived metabolites
from P. recurvata suggests a possible strategy for the assembling
of a guaiane backbone 10 with the same oxidation state as the ori-
ginal five-carbon building block precursor, Fig. 4. A guaiadiene syn-
thase might be able to facilitate the transformation of FPP to 10
The complete biosynthesis of guaine sesquiterpenes has not yet
been established (Drew et al., 2009). It is assumed that the back-
bone of guaiane sesquiterpene originates from a common germac-
rene precursor that is formed via the acetate-mevalonate-FPP
pathway by
a germacrene synthase, which cyclizes farnesyl
diphosphate (FPP) to (+)-germacrene A, 8 (De Kraker et al., 1998),
Fig. 3.
It has been hypothesized that a number of oxidative steps of the
methyl of the isopropenyl moiety (8 ? 8a ? 8b ?, 9, Fig. 3) fol-

288
A.B. Gallardo et al. / Phytochemistry 72 (2011) 284–289
through an enzyme-bound (+)-germacrane A (FPP? (+)-germac-
rene A? (10) (Rising et al., 2000). The guaiane skeleton 10 can
be further modified by enzymes that catalyze precise and con-
trolled allylic hydroxylation, oxidation, and acylation at the periph-
ery of the bicyclic scaffold leading to the structural diversity of
recurvatianes. Although 10 has not been isolated as a natural prod-
uct it is worth noting that its ent-7-epimer, aciphyllene, has been
isolated from the liverworth Dumortiera hirsuta (Saritas et al.,
1998; Blay et al., 2007).
35 min). Whereas from fraction C, compounds 1 (10.9 mg) (tR
29 min), (0.7 mg) (tR 41 min) (tR 41 min), (4.3 mg) (tR
35 min), 4 (3.5 mg) (tR 37 min), and the aldehyde 6 (6.1 mg) (tR
32 min), were separated after normal phase HPLC using hexane:
EtOAc (6:4) as eluent.
2
3
4.3.1. Recurvatiane A [(–)-6b,12,15-triacetoxy -1b,7
11(13)-dien-3-ol] (1)
a,10b-H-guaia-4,
Colourless oil,
½
a 2D0
ꢀ
¼ ꢁ182 (c 0.11, CH2Cl2); 1H (CDCl3,
500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Tables 2 and
1; EIMS m/z 394 (1) [M]+, 334 (2) [M–AcOH]+, 274 (31) [M–
2AcOH]+, 214 (100) [M–3AcOH]+; HREIMS m/z 394.2001 (calcd.
for C21H30O7 394.1992); IR (film) mmax 3460, 1740, 1373, 1234,
3. Conclusions
In conclusion, five novel highly oxygenated guaiane metabolites
have been isolated from the Andean P. recurvata; their structures
were determined by spectroscopic analysis and their absolute ste-
1030 cmꢁ1
.
reochemistry by derivatization with (R)- and (S)-a-methoxy-a-
4.3.2. Recurvatiane B [(ꢁ)-6b,12-diacetoxy-15-hidroxy-1b,7
a,
phenylacetic acids. This set of guaiane-based metabolites, five of
which are new compounds, which we have named recurvatianes
A–F, 1–6, led us to propose an alternative biosynthetic pathway
that suggests that Nature uses the strategy of assembling the guai-
ane core at the same oxidation state as the original building blocks.
This suite of metabolites provides an example of a pathway by
which molecular diversity is generated by the occurrence of spe-
cific oxidation reactions in the late biosynthetic steps of the frame
structure.
10b-H-guaia-4,11(13)-dien-3-one] (2)
Colourless oil,
½
a 2D0
ꢀ
¼ ꢁ225 (c 0.08, CH2Cl2); 1H (CDCl3,
500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Tables 2 and
1; EIMS m/z 290 (29) [M–AcOH]+, 230 (53) [M–2AcOH]+, 201
(49), 91 (74); HREIMS m/z 290.1510 (calcd. for
C17H22O4
290.1518); IR (film) mmax 3431, 1736, 1375, 1234, 1032 cmꢁ1
.
4.3.3. Recurvatiane C [(ꢁ)-12,15-diacetoxy-6b-hydroxy-1b,7
10b-H-guaia-4,11(13)-dien-3-one] (3)
a,
Colourless oil,
½
a 2D0
ꢀ
¼ ꢁ194 (c 0.16, CH2Cl2); 1H (CDCl3,
500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Tables 2 and
1; EIMS m/z 290 (8) [M–AcOH]+, 230 (100) [M–2AcOH]+, 91 (90);
HREIMS m/z 290.1528 (calcd. for C17H22O4 290.1518); IR (film) mmax
4. Experimental
4.1. General procedures
3420, 1740, 1371, 1232, 1028 cmꢁ1
.
Optical rotations were measured on a Perkin–Elmer model 343
Plus polarimeter using a Na lamp at 25 °C. IR spectra were recorded
in a Perkin–Elmer 1650/FTIR spectrometer. 1H NMR and 13C NMR,
HSQC, HMBC and COSY spectra were measured employing a Bruker
AMX 500 instrument operating at 500 MHz for 1H NMR and at
125 MHz for 13C NMR. Two-dimensional NMR spectra were ob-
tained using the standard Bruker software. EIMS and HRMS data
were taken on a Micromass Autospec spectrometer. HPLC separa-
tions were performed on an Agilent 1200 Series Quaternary LC sys-
4.3.4. Recurvatiane D [(ꢁ)-6b,15-diacetoxy-12-hydroxy-1b,7
10b-H-guaia-4,11(13)-dien-3-one] (4)
a,
Colourless oil,
½
a 2D0
ꢀ
¼ ꢁ247 (c 0.07, CH2Cl2); 1H (CDCl3,
500 MHz) and 13C NMR (CDCl3, 125 MHz) data, see Tables 2 and
1; EIMS m/z 290 (50) [M–AcOH]+, 230 (100) [M–2AcOH]+, 215
(45), 91 (90); HREIMS m/z 290.1531 (calcd. for
C17H22O4
290.1518); IR (film) mmax 3423, 1736, 1375, 1234, 1030 cmꢁ1
.
tem (Jaigel-Sil semipreparative column, 10
l
m, 20 ꢂ 250 mm)
4.3.5. Recurvatiane E [(ꢁ)-6b,12,15-triacetoxy-1b,7
4,11(13)-dien-3-one] (5)
a,10b-H-guaia-
with hexane–EtOAc mixtures. The gel filtration column (Sephadex
LH-20) used hexane-MeOH-CH2Cl2 (3:1:1) as eluent. The spray re-
agent for TLC was H2SO4–H2O–AcOH (1:4:20). PCModel ((v. 7.0)
Serena Software, Bloomington, IN) was used to calculate the en-
ergy-minimized model of recurvatianes A–F (1–6).
Colourless oil, ½a D20
¼ ꢁ67 (c 0.9, CHCl3); lit (Bittner et al., 1989)
ꢀ
½
a 2D0
ꢀ
¼ ꢁ22 (c 1.43, CHCl3); 1H (CDCl3, 400 MHz) and 13C NMR
(CDCl3, 100 MHz) data, see Tables 2 and 1; EIMS m/z 392 (26)
[M]+, 349 (18) [M–C2H3O]+, 332 (25) [M–AcOH]+, 230 (100); HRE-
IMS m/z 392.1844 (calcd. for C21H28O7 392.1835); IR (film) mmax
1740, 1373, 1232, 1030 cmꢁ1. The physical and spectral data agree
with the literature values (Bittner et al., 1989).
4.2. Biological material
Perezia recurvata was collected in the Sierra Baguales (Magellan
Region, Chile) in November 2008. A voucher specimen has been
deposited in the Chemistry Department of the Universidad de
Magallanes (Punta Arenas, Chile) with code PZ-rec-2008/11.
4.3.6. Recurvatiane F [(ꢁ)-6b,15-diacetoxy-3-oxo-1b,7
a,10b-H-guaia-
4,11(13)-dien-12-al] (6)
Colourless oil, ½a D20
ꢀ
¼ ꢁ50 (c 0.03, CH2Cl2); 1H (CDCl3, 500 MHz)
and 13C NMR (CDCl3, 125 MHz) data, see Tables 2 and 1; EIMS m/z
305 (4) [M–C2H3O]+, 277 (29), 246 (100); HREIMS m/z 305.1401
(calcd. for C17H21O5 305.1389); IR (film) mmax 1740, 1720, 1230,
4.3. Extraction and isolation
Dry samples (925 g) were extracted with MeOH at room
temperature, and concentrated to give a dark residue (18.4 g).
The extract was partitioned between EtOAc (3 ꢂ 100 ml) and
water (75 ml). The EtOAc extracts were combined and concen-
trated to obtain a brown oil (8.9 g) that was separated on a
Sephadex LH-20 column, followed by silica-gel chromatography.
Fractions containing guaiane-derivatives, as indicated by their 1H
NMR spectra, were further separated by HPLC. Normal phase
HPLC chromatography of fraction A using hexane:EtOAc (7:3)
1029 cmꢁ1
.
Acknowledgments
This work was supported by the Ministerio de Educación y
Ciencia
(BIO2007-61745, SAF2009-08039, SAF2010-16858).
A.R.D.-M. would like to thank Programa JAE-Doc (CSIC) for financial
support. The authors thank also to Mrs. Stephanie Venables for her
kind revision of this manuscript.
as eluent afforded the known compound
5 (16.1 mg) (tR

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Appendix A. Supplementary data
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