Antiproliferative activity of withanolide derivatives from Jaborosa cabrerae and Jaborosa reflexa. Chemotaxonomic considerations

Article abstract of DOI:10.1016/j.phytochem.2011.12.018

Three withanolides were isolated from the aerial parts of Jaborosa reflexa Phil. Jaborosa cabrerae Barboza yielded five sativolide withanolides (including jaborosalactones R, S, 38, and 39) and two trechonolide withanolides epimeric at C-23 (trechonolide

Full text of DOI:10.1016/j.phytochem.2011.12.018

Phytochemistry 76 (2012) 150–157
Contents lists available at SciVerse ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Antiproliferative activity of withanolide derivatives from Jaborosa cabrerae
and Jaborosa reflexa. Chemotaxonomic considerations
Manuela E. García a, Gloria E. Barboza a, Juan C. Oberti a, Carla Ríos-Luci b, José M. Padrón b,
Viviana E. Nicotra a,b, , Ana Estévez-Braun b,c, , Angel G. Ravelo b,c,
⇑
⇑
⇑
a Facultad de Ciencias Químicas, Instituto Multidisciplinario de Biología Vegetal (IMBIV-CONICET), Universidad Nacional de Córdoba, Casilla de Correo 495, 5000 Córdoba, Argentina
b Instituto Universitario de Bio-Orgánica ‘‘Antonio González’’ IUBO-AG, Universidad de La Laguna, Avda. Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
c Instituto Canario de Investigación del Cáncer (ICIC), Avda. Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2011
Received in revised form 20 December 2011
Accepted 28 December 2011
Available online 8 February 2012
Three withanolides were isolated from the aerial parts of Jaborosa reflexa Phil. Jaborosa cabrerae Barboza
yielded five sativolide withanolides (including jaborosalactones R, S, 38, and 39) and two trechonolide
withanolides epimeric at C-23 (trechonolide A and jaborosalactone 32). In addition, five derivatives were
obtained by chemical derivatization of jaborosalactone 38, and all compounds were fully characterized
by 1D and 2D NMR spectroscopic studies. The in vitro antiproliferative activities of the major natural wit-
hanolides and the semisynthetic derivatives were examined against HBL-100, HeLa, SW1573, T-47D, and
WiDr human solid tumor cancer cell lines. Some chemotaxonomic considerations are discussed.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Jaborosa reflexa
Jaborosa cabrerae
Solanaceae
Phytochemistry
Chemotaxonomy
Antiproliferative activity
Withanolides
1. Introduction
twenty-three species of perennial herbs from semiarid or arid
habits, and five from humid regions (e.g. Jaborosa. oxipetala Speg.,
Withanolides constitute a group of natural C-28 steroids iso-
lated mainly from several genera of Solanaceae. They have an
ergostane-type skeleton with oxidation at positions C-1, C-22,
and C-26. Biogenetic transformations of withanolides can produce
highly modified compounds in both the steroid nucleus and side-
chain, including formation of additional rings. Their chemistry
and occurrence have already been reviewed (Chen et al., 2011;
Misico et al., 2011; Ray and Gupta, 1994; Veleiro et al., 2005).
Many of these compounds exhibit interesting biological activities
such as antifeedant (Bado et al., 2004), antiproliferative (Machín
et al., 2010), cancer chemopreventive (Kennelly et al., 1997; Misico
et al., 2002; Su et al., 2002), and phytotoxic properties (Nicotra
et al., 2007).
Jaborosa sativa (Miers) Hunz. & Barboza, Jaborosa integrifolia Lam.).
So far, more than 50% of the species have been phytochemically
analyzed, withanolides being the compounds most extensively
studied. Jaborosa reflexa is a Patagonian species (Chile and Argen-
tina) with greenish corolla, whereasJaborosa cabrerae, a rosette-like
species with white corolla, is found in a small area of the province of
Catamarca (ca. 3000 m); both grow in very sandy soils.
As part of our program aimed at the discovery of novel withan-
olides from Jaborosa species, reported herein the isolation of a
set of both new and known withanolides from J. reflexa Phil.
and J. cabrerae Barboza. Additionally, some chemotaxonomic con-
siderations are discussed. To get insight into structure–activity
relationships, five semi-synthetic derivatives were prepared from
jaborosactone 38. The antiproliferative activities of semisynthetic
and naturally occurring withanolides were evaluated in human
solid tumor cell lines.
Jaborosa Juss. is one of the five largest genera of South American
Solanaceae (Hunziker, 2001), growing from southern Peru to Argen-
tina with the main center of speciation in the Andean region of
Argentina (Barboza and Hunziker, 1987). The genus comprises of
2. Results and discussion
⇑
Corresponding authors. Address: Facultad de Ciencias Químicas, Instituto
The aerial parts of J. reflexa were air-dried and extracted with
ethanol. After elimination of the solvent, the resulting residue
was defatted and repeatedly chromatographed to yield three new
withanolides (1–3, Fig. 1).
Multidisciplinario de Biología Vegetal (IMBIV-CONICET), Universidad Nacional de
Córdoba, Casilla de Correo 495, 5000 Córdoba, Argentina (V.E. Nicotra). Tel.: +54
351 4334170x147; fax: +54 351 4333030.
E-mail address: vnicotra@fcq.unc.edu.ar (V.E. Nicotra).
0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.12.018

M.E. García et al. / Phytochemistry 76 (2012) 150–157
151
Jaborosalactol 28 (1) had molecular formula C30H40O7 as deter-
mined by HRESIMS. Its 1H NMR spectrum showed characteristic
signals of the H-2, H-3, and H-6 hydrogens for a 1-oxo-2,5-diene-
withanolide at d 5.90 (dd, J = 10.0, 2.4 Hz), 6.81 (ddd, J = 10.0, 4.9,
2.4 Hz), and 5.60 (d, J = 5.5 Hz), respectively (Ramacciotti and Nic-
otra, 2007). Olefinic resonances at d 127.8, 145.5, 135.9, and 124.2,
and a carbonyl carbon at d 202.8 (C-1) were observed in the 13C
NMR spectrum. The correlations found between the H-2/H-4b, H-
Jaborosa rotacea (Nicotra et al., 2006), and jaborosalactones 38
and 39 isolated from Jaborosa caulescens (Nicotra et al., 2007).
Sativolides have an oxygen bridge between C-21 and C-12 in the
steroid nucleus. The additional six-membered hemiketal (or ketal)
ring is presumably formed by intramolecular reaction between a
carbonyl group at C-12 and a hydroxyl group at C-21. This unique
arrangement is characterized by the absence of the methyl-21 1H
NMR doublet at high field and by appearance of resonances at dH
3.95 and 3.63 (H2-21), dC 100.7 (C-12), and dC 59.9 (C-21). The het-
erocyclic ring was confirmed by cross-correlation peaks observed
for H2-21 with C-12 in the HMBC spectrum.
3/H-4
assignment of H-4
Regarding C and D rings, the presence of an
a
, and H-3/H-4b pairs in the COSY spectrum led to the
and H-4 b at d 2.90 and 3.30, respectively.
-acetoxy group at
a
a
C-16 was evidenced by the oxymethine hydrogen at d 5.04 (brt,
J = 7.3 Hz) in the 1H NMR spectrum and the respective signals for
an acetoxy group (d 2.07 (3H); d 21.2 (CH3); d 170.9 (C)). COSY cor-
relations of H3-21 (d 0.87) with H-20 (d 1.75), of H-20 with H-17 (d
2.30), and of H-17 with H-16, clearly placed this acetoxy group at
Jaborosalactone 47 (3) (C28H36O6) could not be obtained pure
through either normal phase TLC or reversed phase TLC. This com-
pound was characterized from 4 mg of a mixture of compounds 2
and 3 in a 3:2 ratio. The 1H and 13C NMR spectra were closely re-
lated to those of compound 2, differing only in the substitution
pattern of rings A and B. A 1-oxo-3,5-diene substitution was in-
ferred from the following observations: (i) two mutually coupled
olefinic signals resonating at d 5.65 (m) and 6.06 (brd, J = 9.6 Hz) as-
signed to H-3 and H-4, respectively; (ii) the downfield shift of the
carbonyl carbon in the 13C NMR spectrum from d 203.9 for com-
pound 2 to d 210.2 for compound 3, assigned to C-1; (iii) the down-
field shift of H3-19 in the 1H NMR spectrum from d 1.23 for 2 to d
1.37 for 3. The 13C NMR signals corresponding to A and B rings
were in agreement with the proposed structure (Pan et al., 2007).
Since compound 3 could not be obtained in pure form and it was
characterized as a mixture with compound 2, its structure was ten-
C-16. The
a orientation of the acetoxy group at C-16 was confirmed
from the NOE effect observed between H-16 and H3-18 in the
NOESY spectrum. In addition, the carbonyl resonance at d 212.6
in the 13C NMR spectrum was assigned to the keto group at C-12
(Ramacciotti and Nicotra, 2007). The correlations observed in the
HMBC spectrum for H-11a (d 3.08), H-11b (d 2.69), H-17 (d 2.30),
and H3-18 (d 1.11), with C-12, confirm this assumption. The reso-
nances shown in the 13C NMR spectrum at d 57.4, d 54.9, d 34.2,
d 78.3, and d 49.3 were attributed to C-13, C-14, C-15, C-16, and
C-17, respectively. Regarding the side-chain, evidence for the pres-
ence of an epoxy lactol was provided by the signal corresponding
to the oxymethine proton at d 4.97 (d, J = 8.1 Hz) assigned to the
hydrogen H-26, and the resonances of two methyl groups at C-24
and C-25 at d 1.41 (H3-27) and 1.42 (H3-28), respectively. H-22
was observed as a double triplet at d 3.58, and H3-21 as a doublet
indicating the absence of a hydroxyl group at C-20. Signals at d 65.0
(C-22), d 65.3 (C-24), d 63.9 (C-25), d 91.8 (C-26), d 16.5 (C-27), and
d 18.9 (C-28) in the 13C NMR spectrum are in agreement with the
epoxy lactol side chain (Nicotra et al., 2003). The side-chain b-
orientation was confirmed by a NOESY experiment; a key cross-
correlation peak was observed between H-17 (d 2.30) and the
tatively established as (20R,22R)-12a,21-epoxy-12ib,17b-dihy-
droxy-1-oxowitha-3,5,24-trien-26,22-olide.
The full and unambiguous proton and carbon NMR assignments
for compounds 1–3 were made using a combination of DEPT, COSY,
HSQC, and HMBC experiments.
Following a similar procedure, the EtOH extract of J. cabrerae
was analyzed and the sativolide withanolides 2, jaborosalactones
R (4), S (5) (Bonetto et al., 1995), 38 (6) and 39 (7) (Nicotra et al.,
2007), together with two trechonolide withanolides epimeric at
C-23 [trechonolide A (8) (Fajardo et al., 1987; Lavie et al., 1987)
and jaborosalactone 32 (9) (Nicotra et al., 2006)] were isolated.
The sativolide withanolides isolated from J. reflexa and J. cabre-
rae are closely interrelated. Compound 2, the main withanolide iso-
lated from J. reflexa, was also isolated from J. cabrerae and could be
considered a biosynthetic precursor of compound 3 (isolated from
J. reflexa) and jaborosalactones R, S, 38, and 39 isolated from J.
cabrerae. With respect to rings A and B, it has been well established
that the 1-oxo-2,5-diene system is a biosynthetic precursors of 1-
cresonance at d 1.68 corresponding to H-14 and H-15
and Lavie, 1992). Thus the structure of 1 was established as
(17R,20S,22R,24S,25S,26R)-16 -acetoxy-22,26:24,25-diepoxy-26-
hydroxyergost-2,5-dien-1-one.
a (Bessalle
a
The molecular formula of jaborosalactone 45 (2) was deter-
mined by HRESIMS as C28H36O6. Comparison of its 1H and 13C
NMR spectra with those of compound 1 showed signals almost
identical for all the carbons and protons of rings A and B, indicating
a 1-oxo-2,5-diene system. On the other hand, the resonances ob-
served for rings C and D and the side-chain closely resemble those
of sativolides, jaborosalactones R, S, and T previously isolated from
J. sativa (Bonetto et al., 1995), jaborosalactone 37 isolated from
oxo-3,5-diene; 1-oxo-5b,6b-epoxy; 1-oxo-5a,6b-dihydroxy; and
1-oxo-2,4-diene-6b-hydroxy systems (Glotter, 1991).
Some genera of the Solanaceae family contain withanolides with
exclusive interesting arrangements which can be considered at a
generic level as chemotaxonomic markers (Misico et al., 2011).
Sativolide and trechonolide withanolides belong particularly to
Jaborosa sect. Lonchestigma (Dunal) Wettstein in which J. reflexa
and cabrerae form part of (Barboza and Hunziker, 1987). The results
obtained in our study agree with this statement since both species
have sativolide withanolides (jaborosalactones R, S, 38, 39, 45, and
46), whereas J. cabrerae also contains trechonolide withanolides
(trechonolide A and jaborosalactone 32). J. cabrerae is closely re-
lated to another Andean species, J. caulescens Gillies & Hook. Phyto-
chemically, both species are similar in composition; they not only
have withanolides with the same skeleton (sativolide- and trechon-
olide-type withanolides), but also share several metabolites
(jaborosalactones R, S, 38, 39 and trechonolide A). In addition, both
are unique among their congeners with trechonolide-type withan-
olide epimeric at C-23 (trechonolide A and jaborosalactone 32 in J.
cabrerae, and jaborosalactones 40 and 41 in J. caulescens). These
similarities are shared with the morphological and ecological
28
O
27
23
24
21
25
21
22
20
O
O
O
O
18
O
OH
26
HO
OH
17
11
16
O
O
12
19
12
13
OAc
9
2
3
1
14
2
3
15
8
10
7
5
4
6
1 jaborosalactol 28
2 2-ene-jaborosalactone 45
3 3-ene-jaborosalactone 46
Fig. 1. New withanolides isolated from Jaborosa reflexa.

152
M.E. García et al. / Phytochemistry 76 (2012) 150–157
features and with the phylogenetical evidence found. Both species
have a similar habitat (sandy mountainous areas above 2500 m);
the same habit with a strong root and underground stems
(rhizomes); the long petiole and pinnatisect leaves; and the
funnel-shaped corolla (Fig. 2 D–G) (Barboza, 1986; Barboza and
Hunziker, 1987). A molecular analysis, based on nuclear and chloro-
plast DNA, indicates J. cabrerae as sister to J. caulescens with very
high support (bootstrap 99%) (Moré et al., 2009). On the other hand,
J. reflexa neither forms a sister group with J. cabrerae nor has a close
chemical relationship with this species.
thetic derivatives 10–14 were evaluated using the well-established
protocol of the National Cancer Institute (NCI) of the United States
(Skehan et al., 1990). As a model, the representative panel of
human solid tumor cell lines HBL-100 (breast), HeLa (cervix),
SW1573 (non-small cell lung), T-47D (breast), and WiDr (colon),
was used. Table 1 summarizes the results expressed as 50% growth
inhibition (GI50). The remaining compounds showed GI50 values
larger than 50 lM. For comparison, the antiproliferative data of
the standard anticancer drugs cisplatin, 5-fluorouracil and campth-
otecin against the same panel of solid tumor cell lines are included.
Viewed as a whole; these results have allowed us to classify the
compounds under three groups. A first group is comprised of
jaborosalactone 38 (6) and derivatives 13 and 14, which caused
significant growth inhibition in all the lines tested with GI50 values
Some withanolides have exhibited antiproliferative activity
against human breast cancer cell lines (Machín et al., 2010). Thus,
jaborosalactone 38, previously isolated from J. caulescens var.
caulescens, was able to induce growth inhibition in all cell lines
tested (GI50 = 2.6–4.1
lM). Considering the activity of jaborosalac-
in the range 1.4–8.3 lM, 4.4–17 lM, and 5.3–19 lM, respectively.
tone 38 and the amount isolated from J. cabrerae (438 mg), a small
set of derivatives (10–14, Fig. 3) was prepared. Chemical modifica-
tions have been introduced on A and B rings (jaborosalactone 38)
since, in previous research, where the in vitro antiproliferative
activity of a series of twenty-two naturally occurring withanolides
was examined, the results underlined the relevance of the substi-
tution pattern on A and B rings on the antiproliferative activity;
the type of side-chain demonstrated no affect on activity (Machín
et al., 2010).
Compound 10 was obtained in low yield by epoxide opening
using NH4Ac/AcH. The Michael type-addition of several nucleo-
philes such as MeOH, BzNH2 and PhSH yielded the corresponding
derivatives 11, 12, and 13. Compounds 11 and 12 were obtained
together with jaborosalactone R (4), while compound 13 was ob-
tained together with methoxy derivative 14.
The second set of compounds, involving jaborosalactones R (4)
and 45 (2), jaborosalactol 28 (1), and derivative 12, exhibited mod-
erate activity with GI50 values in the range 18–33
lM, 17–24 lM,
24–43 M, and 33–50 M, respectively. The third group includes
l
l
jaborosalactones 39 (7) and S (5), and derivatives 10 and 11, which
failed to show any growth inhibition.
The analysis of the antiproliferative activity has allowed us to
establish the following structure–activity relationships: (i) 1-oxo-
2-ene-5b,6b-epoxy system in rings A/B is relevant for the biological
activity jaborosalactone 38 being the most active compound; (ii)
a decrease in or loss of activity was observed for 5-oxygenated
(jaborosalactone S and 10), 5,6-unsaturated (2), or 1-oxo-2,4-diene-
6b-hydroxylated (jaborosalactone R) compounds; (iii) Regarding
compounds having a 1-oxo-2,3 dihidro-5b,6b-epoxy system in
rings A/B, such as jaborosalactone 39 and 11–14 derivatives, in all
cases the antiproliferative activity was lower than that shown by
jaborosalactone 38 with a 1-oxo-2-ene-5b,6b-epoxy system in
The in vitro antiproliferative activities of natural compounds 1,
2, jaborosalactones 38 (6), 39 (7), R (4), and S (5), and semisyn-
Fig. 2. Jaborosa species. A–C: J. reflexa. A: plant; B: branch with flowers; C: root; D–F: J. cabrerae. D: flower; E: plant; F: leaves (note the long purple petiole) and fruit; G:
Flower of J. caulescens.

M.E. García et al. / Phytochemistry 76 (2012) 150–157
153
O
NH4Ac/AcH
OH
5
6
OAc
10 (25%)
O
O
MeOH
+
+
3
H3CO
BzN
O
O
O
O
HO
OH
OH
11 (40%)
jaborosalactone R (4)
O
12
(55%)
O
5
jaborosalactone R (4)
3
(22%)
6
3
O
BzNH2/tol
O
Jaborosalactone 38 (6)
12
(15%)
O
O
O
RO
OH
PhSH
O
12
CH2Cl2/MeOH
13 R=H (25%)
3
14
R=OCH3 (40%)
PhS
O
Fig. 3. Withanolide derivatives obtained from jaborosalactone 38 (6). Yields given in parentheses.
Table 1
In vitro Antiproliferative Activity of Withanolides and Derivatives against Human
by these compounds is of particular interest not only at specific or
generic level but also at higher range (tribes, subtribes, subfamily)
and may contribute to supporting phylogenetic relationships
among the taxa.
With respect to relationships of structure-antiproliferative
activity indicated, most of them agree with those already estab-
lished. However, the activity of withanolides with a 1-oxo-3b-XR
system in ring A has not yet been extensively studied so far. 3b-S
Solid Tumor Cell Lines.a.
Compound
HBL-100
HeLa
SW1573 T-47D
WiDr
Jaborosalactol 28 (1) 34 (6.4) 24 (7.4) 33 (8.7) 43 (8.1) 40 (3.5)
Jaborosalactone R (4) 35 (1.5) 18 (1.7) 33 (1.3) 15 (2.9) 30 (2.8)
Jaborosalactone 38 (6) 4.0 (0.4) 8.3( 1.4) 4.4( 0.1) 8.1 (0.7) 1.4( 0.1)
Jaborosalactone 45 (2) 24 (5.4) 17 (3.8) 23 (8.1) 29 (5.0) 20 (2.4)
11
12
13
90 (11) >100
33 (12) 48 (2.0) 50 (1.3) 25 (5.6) 40 (1.0)
4.7 (1.3) 4.4 (1.6) 4.6 (0.8) 17 (3.2) 14 (4.3)
53 (12) >100
>100
withanolide derivatives showed marked activity (4.4–19 lM) sim-
14
6.0 (2.2) 5.6 (1.0) 5.3 (0.1) 19 (0.1) 16 (0.7)
1.9 (0.16) 2.0 (0.3) 3.4 (0.7) 15 (2.3) 26 (5.6)
5.5 (2.3) 15 (4.7) 4.3 (1.6) 47 (18) 49 (6.7)
0.2 (0.05) 0.5 (0.2) 0.3 (0.1) 2.0 (0.5) 1.8 (0.7)
ilar to that reported by 3b-S withaferin A derivatives (Yousuf et al.,
2011). Accordingly, this type of functionalization should be studied
in depth to assess the biological activity and mechanism of action
of such derivatives.
Cisplatin
5-fluorouracil
Campthotecin
a
Expressed as GI50 values given in M and determined as means of three to five
experiments. Standard deviations are given in parentheses.
4. Experimental
rings A/B. However, the activity of 11–14 derivatives was subject to
the electronegativity of 3b-substituents, with an activity increasing
with decreasing electronegativity of the heteroatom at C-3; (iv) all
compounds tested are sativolides except jaborosalactol 28 (1), an
unmodified skeleton withanolide bearing a six-membered epoxy
lactol ring. However, jaborosalactol 28 (1) showed a similar activity
to jaborosalactone 45 (2). Both withanolides have different skele-
tons but the same substitution pattern in rings A/B. Our findings
demonstrate the relevance of the substitution pattern in A/B rings
to the resulting antiproliferative activity. These results are in line
with those previously reported on the structure–activity relation-
ship of withanolides (Kennelly et al., 1997; Machín et al., 2010; Mis-
ico et al., 2002; Su et al., 2002).
4.1. General
Melting points were measured on a mercury thermometer
apparatus and were uncorr, whereas optical rotations were re-
corded using a JASCO P-1010 polarimeter. UV spectra were ob-
tained with
a Shimadzu-260 spectrophotometer, whereas IR
spectra employed a Nicolet 5-SXC spectrophotometer. NMR exper-
iments were performed on Bruker AVANCE II 400 MHz and AMX
500 MHz instruments. Multiplicity determinations (DEPT) and 2D
spectra (COSY, HSQC, HMBC, and NOESY) were obtained using
standard Bruker software. Chemical shifts are given in ppm (d)
downfield from TMS internal standard. ESIMS and HRESIMS were
measured on an LCT premier XE Micromass spectrometer, and
EIMS was determined at 70 eV on a Finnigan 3300 F-100 mass
spectrometer. UPLC-ESI-QTOF/MS was performed using a system:
UPLC equipment Agilent 1200 l Series equipped with binary pump.
Tandem ESI source (Bruker Daltonics, MA, USA), operated in posi-
tive mode 2 l/min nebulizer gas (Nitrogen) and drying gas
6 l/min at 200 °C (nitrogen), needle voltage (4500), shield voltage
(600 V). Mass spectrometer Micro TOFQ II Bruker Daltonics (MA,
3. Conclusions
With this phytochemical information, the number of Jaborosa
species studied increased to 15 out of 23 species of the genus.
The metabolites isolated here agree with the exclusive withanolide
arrangements of Jaborosa which can be considered chemotaxo-
nomic markers at generic level. The chemical information provided

154
M.E. García et al. / Phytochemistry 76 (2012) 150–157
USA), operating in positive scan mode (from m/z 100 to m/z 800)
calibrated using NaCOOH (10 mM). Software for data analysis:
Hystar 3.2 (Bruker Daltonics, MA, USA). Chromatographic separa-
tions were performed by vacuum–liquid chromatography (VLC),
column chromatography (CC) on silica gel 60 (0.063–0.200 mm),
radial chromatography with a radial Chromatotron Model 7924 T
on silica gel 60 PF254 Merck (1 mm thick), and prep. TLC on silica
gel 60 F254 (0.2 mm thick) plates.
CH2Cl2–MeOH mixtures of increasing polarity, yielding jaborosa-
lactone R (4) (6.6 mg), and jaborosalactone S (5) (6.4 mg).
The mass spectrum of 3, was measured using UPLC-ESI-QTOF/
MS from the compounds 2 and 3 mixture. Detection was carried
out using a PDA detector scanning between 200–800 nm, monitor-
ing at 244 nm. A combination of three columns were used: Eclipse
XDB-C18 (i.d. 3.0 mm ꢀ 100 mm; 1.8
(i.d 3.0 mm ꢀ 50 mm; 1.8 m), and Zorbax Eclipse Plus Phenyl–
Hexyl (i.d. 2.0 mm ꢀ 150 mm; 2 m). The columns oven operated
lm), Zorbax Eclipse XDB-C18
l
l
4.2. Plant material
at 40 °C. Mobile phase and elution conditions were as followed:
(A) 0.5% HCOOH in ultrapure H2O; (B) 0.5% HCOOH in CH3CN (HPLC
grade, Merck). Flow 0.3 ml/min; program: from 90% A (t = 0 min) to
90% B (t = 20 min); 90% A (t = 21 min) and stabilization during
Voucher specimens of J. cabrerae and J. reflexa are deposited at
the herbarium of Museo Botánico Córdoba (CORD), Universidad
Nacional de Córdoba. J. reflexa was collected in Departament Lon-
copué, Neuquén, Argentina, in February 2007 (Barboza et al.
1805). J. cabrerae was collected in Departament Belén, Catamarca,
Argentina, in February 2008 (Barboza et al. 1990).
10 min before next injection. Injection volume was 40
ll (MeOH).
4.3.1. Jaborosalactol 28 (17R,20S,22R,24S,25S,26R)-16 -acetoxy-
a
22,26:24,25-diepoxy-26-hydroxyergost-2,5-dien-1-one (1)
Â
Ã
White amorphous powder; a2D1 + 2.7 (c 0.37, MeOH); UV
4.3. Extraction and isolation of compounds from J. reflexa and J.
cabrerae
(MeOH) kmax (log
e
) 244 (3.18) nm; IR (dry film) vmax 3432 (s),
2961 (s), 2852 (s), 1704 (s), 1665 (s), 1680 (s), 1459 (s), 1381 (s),
1237 (s), 1069 (s), 848 (s) cmꢁ1;1H NMR (CDCl3ꢁD2O (95:05),
400.13 MHz) d 6.81 (1H, ddd, J = 10.0,4.9,2.4 Hz, H-3), 5.90 (1H,
dd, J = 10.0,2.4 Hz, H-2), 5.60 (1H, d, J = 5.5 Hz, H-6), 5.04 (1H, brt,
J = 7.3 Hz, H-16), 4.97 (1H, s, H-26), 3.58 (1H, dt, J = 11.5,2.9 Hz,
H-22), 3.30 (1H, brd, J = 21.3 Hz, H-4b), 3.08 (1H, dd,
The air-dried powdered aerial parts of J. reflexa (85 g) were
exhaustively extracted with EtOH, and the solvent evaporated un-
der reduced pressure. The resulting residue was defatted by parti-
tion in n-hexane-EtOH–H2O (10:3:1) with the EtOH–H2O phase
washed with n-hexane (3 ꢀ 100 ml), and the EtOH evaporated un-
der reduced pressure. The residue was diluted with H2O and ex-
tracted with CH2Cl2 (3 ꢀ 200 ml). The combined CH2Cl2 extracts
were dried (Na2SO4), filtered, and evaporated to dryness under re-
duced pressure. The residue (2.6 g) was fractionated initially by
VLC. Elution with n-hexane–EtOAc mixtures of increasing polarity
(100:0–0:100) and EtOAc–MeOH (100:0–95:05) afforded six frac-
tions containing withanolides. Fractions I-II (380 mg) were applied
to silica gel 60 G using n-hexane–EtOAc mixtures of increasing
polarity to afford a mixture that was further fractionated by prep.
TLC (CH2Cl2–MeOH, 96:4), yielding a mixture (4 mg) which could
not be separated by either normal phase TLC or reversed phase
TLC. The 1H NMR spectrum of this mixture indicated that this mix-
ture consisted of compounds 2 and 3 in a 3:2 ratio. Fractions III–V
(560 mg) were pooled and applied to a silica gel 60 G column. Elu-
tion with n-hexane–EtOAc mixtures of increasing polarity (90:10–
0:100) afforded a mixture (70 mg) that was further processed by
prep. TLC (CH2Cl2-MeOH, 98:02), yielding compound 1 (8.4 mg).
From fraction VI (EtOAc 100%), compound 2 (88 mg) precipitated.
The air-dried powdered aerial parts of J. cabrerae (288 g) were
exhaustively extracted with EtOH; the solvent was evaporated at
reduced pressure. The residue obtained (36 g) was defatted by par-
tition in n-hexane-EtOH–H2O (10:3:1), the resulting EtOH–H2O
phase was washed with n-hexane (3 ꢀ 600 ml), and the EtOH
evaporated at reduced pressure. The residue was diluted with
H2O and extracted with CH2Cl2 (4 ꢀ 200 ml). The CH2Cl2 extract
was dried (Na2SO4), filtered, and evaporated to dryness under re-
duced pressure. The residue (4.9 g) was initially fractionated by
VLC. Elution with n-hexane–EtOAc mixtures of increasing polarity
(100:0–0:100) and EtOAc–MeOH (100:0–80:20) afforded five frac-
tions containing withanolides. These fractions (1.2 g) were pooled
and subjected to silica gel 60 G CC. Elution with n-hexane–EtOAc
mixtures of increasing polarity (100:0–0:100) and EtOAc–MeOH
(100:0–80:20) afforded eleven fractions containing withanolides.
Fraction I (20 mg) was fractionated by prep. TLC with CH2Cl2-
MeOH, 94:6 yielding (in order of elution) compound 2 (2.8 mg),
trechonolide A (8) (2 mg), and jaborosalactone 32 (9) (1.5 mg).
From fraction II jaborosalactone 38 (6) (438 mg) precipitated. Frac-
tion III (22 mg) was fractionated by prep. TLC with CH2Cl2–MeOH,
94:6, yielding jaborosalactone 39 (7) (3.5 mg). Fractions IV–XI
were pooled and processed by radial chromatography with
J = 12.9,5.3 Hz, H-11a), 2.90 (1H, dd, J = 21.3,4.9 Hz, H-4a), 2.69
(1H, t, J = 12.9 Hz, H-11b), 2.30 (1H, dd, J = 11.7,6.8 Hz, H-17),
2.16 (1H, td, J = 12.0,5.3 Hz, H-9), 2.07 (3H, s, H-CH3CO-16), 2.01
(1H, m, H-7b), 1.97 (1H, m, H-15b), 1.96 (1H, m, H-23
(1H, m, H-8), 1.78 (1H, m, H-23b), 1.75 (1H, m, H-20), 1.68 (2H,
m, H-14, H-15 ), 1.67 (1H, m, H-7 ), 1.42 (3H, s, H-28), 1.41 (3H,
a), 1.84
a
a
s, H-27), 1.30 (3H, s, H-19), 1.11 (3H, s, H-18), 0.87 (3H, d,
J = 6.9 Hz, H-21); 13C NMR (CDCl3, 100.63 MHz) d 212.6 (C, C-12),
202.8 (C, C-1), 170.9 (C, CH3CO-16), 145.5 (CH, C-3), 135.9 (C, C-
5), 127.8 (CH, C-2), 124.2 (CH, C-6), 91.8 (CH, C-26), 78.3 (CH, C-
16), 65.3 (C, C-24), 65.0 (CH, C-22), 63.9 (C, C-25), 57.4 (C, C-13),
54.9 (CH, C-14), 50.9 (C, C-10), 49.3 (CH, C-17), 46.5 (CH, C-9),
39.3 (CH2, C-11), 37.7 (CH, C-20), 34.2 (CH2, C-15), 33.3 (CH2, C-
4), 32.5 (CH, C-8), 30.4 (CH2, C-7), 29.7 (CH2, C-23), 21.2 (CH3,
CH3CO-16), 18.9 (CH3, C-28), 18.6 (CH3, C-19), 16.5 (CH3, C-27),
12.8 (CH3, C-21), 12.6 (CH3, C-18); ESIMS m/z 535 [M + Na]+
(100), 454 (28), 413 (13), 342 (20), 315 (9), 277 (5), 105 (64); HRE-
SIMS m/z [M + Na]+ 535.2675 (calcd. for C30H40O7Na, 535.2672).
4.3.2. Jaborosalactone 45 (20R,22R)-12a,21-epoxy-12b,17b-
dihydroxy-1-oxo-witha-2,5,24-trien-26,22-olide (2)
Colorless crystals (n-hexane–EtOAc), mp 243–244 °C;
Â
Ã
a2D1 + 31.9 (c 0.67, MeOH); UV (MeOH) kmax (log
e
) 244 (3.94)
nm; IR (dry film) vmax 3432 (s), 3058 (w), 2965 (s), 1692 (s),
1661 (s), 1385 (s), 1299 (s), 731 (s) cmꢁ1
;
1H NMR (CDCl3,
400.13 MHz) d 6.79 (1H, ddd, J = 10.0,5.0,2.5 Hz, H-3), 5.87 (1H,
dd, J = 10.0,2.5 Hz, H-2), 5.58 (1H, d, J = 5.9 Hz, H-6), 4.36 (1H,
ddd, J = 12.4,8.3,3.4 Hz, H-22), 3.95 (1H, dd, J = 11.2,5.2 Hz, H-
21b), 3.63 (1H, t, J = 11.2 Hz, H-21
4b), 2.94 (1H, ddd, J = 11.2,8.3,5.2 Hz, H-20), 2.85 (1H, dd,
J = 21.3,5.0 Hz, H-4 ), 2.58 (1H, m, H-23b), 2.52 (1H, dd,
J = 12.1,3.2 Hz, H-11 ), 2.21 (1H, dd, J = 17.5,2.0 Hz, H-23 ), 2.12
a), 3.28 (1H, brd, J = 21.3 Hz, H-
a
a
a
(1H, m, H-16b), 2.05 (1H, m, H-7b), 1.94 (3H, s, H-28), 1.91 (1H,
m, H-9), 1.88 (1H, m, H-11b), 1.87 (3H, s, H-27), 1.83 (1H, m, H-
16a), 1.63 (1H, m, H-15a), 1.62 (1H, m, H-8), 1.60 (1H, m, H-7a),
1.52 (1H, m, H-15b), 1.48 (1H, m, H-14), 1.23 (3H, s, H-19), 1.07
(3H, s, H-18); 13C NMR (CDCl3, 100.63 MHz) d 203.9 (C, C-1),
165.2 (C, C-26), 149.3 (C, C-24), 145.4 (CH, C-3), 135.9 (C, C-5),
127.9 (CH, C-2), 124.5 (CH, C-6), 121.9 (C, C-25), 100.7 (C, C-12),
79.0 (C, C-17), 76.5 (CH, C-22), 59.9 (CH2, C-21), 50.8 (C, C-10),
50.6 (CH, C-14), 50.2 (C, C-13), 47.5 (CH, C-20), 41.1 (CH, C-9),

M.E. García et al. / Phytochemistry 76 (2012) 150–157
155
37.9 (CH2, C-11), 35.4 (CH2, C-16), 35.1 (CH2, C-23), 33.3 (CH2, C-4),
4.4.2. Preparation of (20R,22R)-5,6:12
a
,21-diepoxy-12,17-dihydroxy-
31.6 (CH, C-8), 30.6 (CH2, C-7), 24.3 (CH2, C-15), 20.4 (CH3, C-28),
18.9 (CH3, C-19), 12.4 (CH3, C-27), 12.2 (CH3, C-18); EIMS m/z
451 (26), 281 (43), 207 (34), 125 (100), 67 (56), 43 (92); HRESIMS
m/z [M + Na]+ 491.2408 (calcd. for C28H36O6Na, 491.2410).
3-methoxy-1-oxowitha-24-en-26,22-olide (11)
A soln. of jaborosalactone 38 (6) (20 mg, 0.04 mmol) and MeOH
(5 ml) was heated until reflux began, this being maintained for
24 h. After completion of the reaction, the reaction mixture was
conc. and the residue subjected to prep. TLC using EtOAc to afford
11 (8.5 mg, 40%) and jaborosalactone R (4) (11.0 mg, 55%).
4.3.3. Jaborosalactone 46 (20R,22R)-12a,21-epoxy-12,17-dihydroxy-
Â
Ã
White amorphous solid; a2D1 -3.4 (c 1.46, CHCl3); UV (CHCl3)
1-oxowitha-3,5,24-trien-26,22-olide (3)a
1H NMR (CDCl3, 400.13 MHz) d 6.06 (1H, brd, J = 9.6 Hz, H-4),
5.65 (1H, m, H-3), 5.62 (1H, m, H-6), 4.35 (1H, m, H-22), 3.85
kmax (log
e
) 240 (3.62)nm; IR (dry film) vmax 3423, 2931, 1702,
1458, 1303 cmꢁ1
;
1H NMR (CDCl3, 400.13 MHz) d 4.33 (1H, ddd,
J = 12.1,8.4,3.4 Hz, H-22), 3.85 (1H, dd, J = 11.1,5.3 Hz, H-21b),
3.74 (1H, qd, J = 5.9,2.1 Hz, H-3 ), 3.57 (1H, t, J = 11.4, H-21 ),
(1H, dd, J = 11.4,5.4 Hz, H-21b), 3.65 (1H, t, J = 11.4 Hz, H-21a),
3.28 (1H, m, H-2a), 2.95 (1H, m, H-20), 2.76 (1H, dd,
a
a
3.28 (3H, s, OCH3), 3.23 (1H, brs, H-6), 2.93 (1H, ddd,
J = 11.7,8.4,5.3 Hz, H-20), 2.75 (2H, d, J = 6.0 Hz, H-2), 2.59 (1H, t,
J = 14.0 Hz, H-23b), 1.94 (3H, s, H-28), 1.87 (3H, s, H-27), 1.16
(3H, s, H-19), 0.99 (3H, s, H-18); 13C NMR (CDCl3, 100.63 MHz) d
210.4 (C, C-1), 165.0 (C, C-26), 149.2 (C, C-24), 122.0 (C, C-25),
100.4 (C, C-12), 78.7 (C, C-17), 76.3 (CH, C-22), 72.6 (CH, C-3),
62.1 (C, C-5), 61.3 (CH, C-6), 60.2 (CH2, C-21), 56.1 (CH3, OCH3),
51.6 (C, C-13), 51.2 (C, C-10), 50.5 (CH, C-14), 47.2 (CH, C-20),
42.7 (CH2, C-2), 40.8 (CH, C-9), 36.7 (CH2, C-11), 36.2 (CH2, C-4),
35.5 (CH2, C-16), 35.2 (CH2, C-23), 31.3 (CH2, C-7), 27.6 (CH, C-8),
24.2 (CH2, C-15), 20.4 (CH3, C-28), 13.7 (CH3, C-19), 12.4 (CH3, C-
27), 12.1 (CH3, C-18); ESIMS m/z 539 [M + Na]+ (100), 499 (5),
301 (6); HRESIMS m/z [M + Na]+ 539.2629 (calcd. for C29H40O8Na,
539.2621).
J = 20.5,5.4 Hz, H-2b), 2.59 (1H, m, H-23b), 2.11 (1H, m, H-7b),
2.20 (1H, dd, J = 18.0,3.4 Hz, H-23
(1H, m, H-9), 1.95 (3H, s, H-28), 1.88 (3H, s, H-27), 1.80 (1H, m,
H-7 ), 1.71 (1H, m, H-8), 1.69 (1H, m, H-11b), 1.65 (1H, m, H-
15 ), 1.54 (1H, m, H-15b), 1.51 (1H, m, H-14), 1.37 (3H, s, H-19),
a), 2.09 (1H, m, H-11a), 2.08
a
a
1.07 (3H, s, H-18); 13C NMR (CDCl3, 100.63 MHz) d 210.2 (C, C-1),
165.1 (C, C-26), 149.3 (C, C-24), 140.8 (C, C-5), 129.1 (CH, C-4),
126.7 (CH, C-3), 121.8 (C, C-25), 121.6 (CH, C-6), 100.6 (C, C-12),
78.8 (C, C-17), 76.4 (CH, C-22), 59.9 (CH2, C-21), 51.7 (C, C-10),
51,0 (CH, C-14), 50.7 (C, C-13), 47.4 (CH, C-20), 39.5 (CH2, C-2),
39.2 (CH, C-9), 36.9 (CH2, C-11), 35.4 (CH2, C-7), 35.4 (CH2, C-16),
35.3 (CH2, C-23), 30.1 (CH, C-8), 24.1 (CH2, C-15), 20.4 (CH3, C-
28), 20.3 (CH3, C-19), 12.4 (CH3, C-27), 12.2 (CH3, C-18). HRESI-
QTOFMS m/z [M + Na]+ 491.2409 (calcd. for C28H36O6Na,
491.2410).a These data were obtained from a mixture of 2 and 3
compounds.
4.4. Preparation of jaborosalactone 38 derivatives
4.4.3. Preparation of (20R,22R)-5,6:12a,21-diepoxy-12,17-dihydroxy-
3-N-benzyl-1-oxowitha-24-en-26,22-olide (12)
4.4.1. Preparation of (20R,22R)-5
a-Acetoxy-12a,21-epoxy-6,12,17-
To a soln. of jaborosalactone 38 (6) (20 mg, 0.04 mmol) in dry
trihydroxy-1-oxowitha-2,5,24-trien-26,22-olide (10)
toluene (3 ml), benzylamine (10 lL, 0.048 mmol) was added. The
To a soln. of jaborosalactone 38 (6) (20 mg, 0.04 mmol) in AcOH
(3 ml), (NH4)OAc (67 mg, 0.80 mmol) was added. The reaction mix-
ture was heated until reflux began, this being maintained for 48 h
until disappearance of the starting material had occurred. Once the
solvent was removed, the residue was purified by prep. TLC using
EtOAc to obtain compound 10 (5.6 mg, 25%) as an amorphous solid.
reaction mixture was heated until reflux began, this being main-
tained for 72 h until all starting material was consumed. The sol-
vent was then removed and the residue purified by prep.TLC
using EtOAc to render jaborosalactone R (4) (3.0 mg, 15%) and com-
pound 12 (5.4 mg, 22%).
Â
Ã
White amorphous solid; a2D1 + 15.0 (c 0.52, CHCl3); UV (CHCl3)
Â
Ã
White amorphous powder; a2D1 + 33.6 (c 0.56, CHCl3); UV
kmax (log
e
) 239 (3.71) nm; IR (dry film) vmax 3437, 2929, 1706,
(CHCl3) kmax (log
e
) 239 (3.71), 220 (3.73) nm; IR (dry film) vmax
1190, 1014 cmꢁ1
;
1H NMR (CDCl3, 400.13 MHz) d 7.32 (2H, m, H-
3440, 2932, 1688, 1382, 1255, 1023 cmꢁ1
;
1H NMR (CDCl3,
30/H-50), 7.27 (3H, m, H-20/H-60 and H-40), 4.34 (1H, ddd,
J = 12.0,8.4,3.4 Hz, H-22), 3.81 (1H, m, H-21b), 3.75 (1H, d,
J = 13.2 Hz, PhCH2N a), 3.71 ((1H, d, J = 13.2 Hz, PhCH2N b), 3.53
500.13 MHz) d 6.56 (1H, ddd, J = 10.1,5.4,1.7 Hz, H-3), 5.85 (1H,
dd, J = 10.1,2.7 Hz, H-2), 4.82 (1H, brs, H-6), 4.36 (1H, ddd,
J = 11.8,8.2,3.2 Hz, H-22), 3.97 (1H, dd, J = 11.3,5.3 Hz, H-21b),
(1H, dd, J = 11.3,8.5 Hz, H-21
H-6), 2.84 (1H, m, H-20), 2.72 (1H, dd, J = 13.3,6.0 Hz, H-2
(1H, dd, J = 13.3,6.2 Hz, H-2b), 2.52 (1H, m, H-23b), 2.29 (1H, dd,
J = 14.6,5.0 Hz, H-4 ), 2.26 (1H, m, H-11 ), 2.20 (1H, m, H-23 ),
2.17 (1H, m, H-7b), 2.12 (1H, m, H-16b), 1.95 (3H, s, H-28), 1.88
(3H, s, H-27), 1.87 (1H, m, H-11b), 1.75 (1H, m, H-16 ), 1.67 (1H,
m, H-8), 1.58 (2H, m, H-15), 1.55 (1H, m, H-9), 1.41 (1H, m, H-
4b), 1.34 (1H, m, H-14), 1.33 (1H, m, H-7 ), 1.17 (3H, s, H-19),
a), 3.24 (1H, m, H-3
a), 3.23 (1H, brs,
3.70 (1H, t, J = 11.3 Hz, H-21
), 3.10 (1H, dt, J = 20.4,2.5 Hz, H-4b), 2.91 (1H, ddd,
J = 11.3,8.2,5.3 Hz, H-20), 2.66 (1H, m, H-23b), 2.61 (1H, dd,
J = 13.3,4.1 Hz, H-11 ), 2.22 (1H, brd, J = 17.7 H, H-23 ), 2.16 (1H,
m, H-16b), 2.09 (1H, m, H-9), 2.00 (1H, m, H-8), 1.94 (3H, s, H3-
28), 1.92 (3H, s, COCH3), 1.88 (3H, s, H3-27), 1.82 (1H, m, H-16 ),
1.76 (1H, d, J = 13.3 Hz, H-11b), 1.68 (1H, dt, J = 14.0,3.2 Hz, H-
7b), 1.63 (2H, m, H2-15), 1.52 (1H, m, H-14), 1.38 (1H, m, H-7 ),
a), 3.51 (1H, dd, J = 20.4,5.4 Hz, H-
a), 2.55
4a
a
a
a
a
a
a
a
a
a
1.01 (3H, s, H-18); 13C NMR (CDCl3, 100.63 MHz) d 211.1 (C, C-1),
165.0 (C, C-26), 149.2 (C, C-24), 139.8 (C, C-10), 128.5 (x2, CH, C-
30/C-50), 128.0 (x2, CH, C-20/C-60), 127.2 (CH, C-40), 122.0 (C, C-
25), 100.5 (C, C-12), 78.8 (C, C-17), 76.5 (CH, C-22), 62.8 (C, C-5),
62.0 (CH, C-6), 60.1 (CH2, C-21), 51.2 (C, C-10), 51.1 (CH2, PhCH2N),
51.0 (C, C-13), 50.4 (CH, C-14), 49.6 (CH, C-3), 47.3 (CH, C-20), 43.4
(CH2, C-2), 41.0 (CH, C-9), 37.7 (CH2, C-11), 36.9 (CH2, C-4), 35.5
(CH2, C-16), 35.2 (CH2, C-23), 31.2 (CH2, C-7), 27.6 (CH, C-8), 24.2
(CH2, C-15), 20.4 (CH3, C-28), 14.4 (CH3, C-19), 12.4 (CH3, C-27),
12.1 (CH3, C-18);); ESIMS m/z 614 [M + Na]+ (38), 592 (100), 593
(40), 507 (21), 413 (5), 301 (8); HRESIMS m/z [M + Na]+ 614.3093
(calcd. for C35H45NO7Na, 614.3094).
1.33 (3H, s, H3-19), 1.09 (3H, s, H3-18); 13C NMR (CDCl3,
100.63 MHz) d 202.6 (C, C-1), 170.4 (C, COCH3), 165.1 (C, C-26),
149.2 (C, C-24), 140.7 (CH, C-3), 128.3 (CH, C-2), 121.9 (C, C-25),
100.7 (C, C-12), 89.5 (C, C-5), 78.9 (C, C-17), 76.4 (CH, C-22), 67.4
(CH, C-6), 60.0 (CH2, C-21), 51.9 (C, C-10), 51.2 (C, C-13), 50.0
(CH, C-14), 47.3 (CH, C-20), 39.4 (CH, C-9), 37.5 (CH2, C-11), 35.3
(CH2, C-16), 35.1 (CH2, C-23), 33.3 (CH2, C-7), 28.2 (CH2, C-4),
27.8 (CH, C-8), 24.2 (CH2, C-15), 22.3 (CH3, COCH3), 20.3 (CH3, C-
28), 15.4 (CH3, C-19), 12.5 (CH3, C-18), 12.3 (CH3, C-27); ESIMS
m/z 567 [M + Na]+ (100), 507(8), 413 (9), 301(13); HRESIMS m/z
[M + Na]+ 567.2571 (calcd. for C30H40O9Na, 567.2570).

156
M.E. García et al. / Phytochemistry 76 (2012) 150–157
4.4.4. Preparation of (20R,22R)-5,6:12
1-oxo-3-thiophenyl-witha-24-en-26,22-olide (13) and (20R, 22R)-5,
a
,21-diepoxy-12,17-dihydroxy-
1640 medium was purchased from Flow Laboratories (Irvine,
UK), fetal calf serum (FCS) was from Gibco (Grand Island, NY), tri-
chloroacetic acid (TCA) and glutamine were from Merck (Darms-
tadt, Germany), and penicillin G, streptomycin, dimethyl
sulfoxide (DMSO) and sulforhodamine B (SRB) were from Sigma
(St Louis, MO).
6:12 ,21-diepoxy-17-hydroxy-12-methoxy-1-oxo-3-thiophenyl-
a
witha-24-en-26,22-olide (14)
To jaborosalactone 38 (6) (20 mg, 0.04 mmol) dissolved in a
mixture of CH2Cl2/ MeOH (4:1) (6 ml),
8 lL of thiophenol
(0.08 mmol) was added. The reaction mixture was stirred for 5 h
at room temperature. Once the reaction was completed, the reac-
tion mixture was conc. and the residue subjected to prep.TLC puri-
fication using EtOAc to yield compound 13 (6.1 mg, 25%) and
compound 14 (10.0 mg, 40%).
4.5.2. Cells, culture and plating
The human solid tumor cell lines HBL-100 (breast), HeLa (cer-
vix), SW1573 (non-small cell lung), T-47D (breast) and WiDr (co-
lon) were used in this study. These cell lines were a kind gift
from Prof. Godefridus J. Peters (VU Medical Center, Amsterdam,
The Netherlands) and Dr. Rubén P. Machín (HUGC Dr. Negrín, Las
Palmas de Gran Canaria, Spain). Cells were maintained in 25 cm2
culture flasks in RPMI 1640 supplemented with 5% FBS and
2 mM L-glutamine at 37 °C, 5% CO2, 95% humidified air incubator.
Exponentially growing cells were trypsinized and resuspended in
antibiotic containing medium (100 units penicillin G and 0.1 mg
of streptomycin per ml). Single cell suspensions displaying > 97%
viability by trypan blue dye exclusion were subsequently counted.
After counting, dilutions were made to establish the appropriate
cell densities for inoculation onto 96-well microtiter plates. Cells
Â
Ã
Compound 13: White amorphous solid; a2D1 + 33.1 (c 0.61,
CHCl3); UV (CHCl3) kmax (log
e
) 240 (3.64) nm; IR (dry film) vmax
3440, 2937, 1708, 1302, 1099 cmꢁ1
;
1H NMR (CDCl3, 400.13 MHz)
d 7.37 (2H, dt, J = 7.7,1.7 Hz, H-20), 7.31 (2H, brt, J = 7.7 Hz, H-30),
7.27 (1H, brd, J = 7.7 Hz, H-40), 4.34 (1H, ddd, J = 12.0,8.2,3.2 Hz,
H-22), 3.86 (1H, dd, J = 11.3,5.2 Hz, H-21b), 3.73 (1H, qd,
J = 7.0,2.2 Hz, H-3
H-6), 2.94 (1H, m, H-20), 2.77 (1H, dd, J = 14.2,7.0 Hz, H-2
(1H, dd, J = 14.2,7.2 Hz, H-2b), 2.60 (1H, brt, J = 14.3 Hz, H-23b),
2.47 (1H, dd, J = 14.8, 6.3 Hz, H-4 ), 2.25 (1H, d, J = 14.2 Hz, H-7b),
a
), 3.60 (1H, t, J = 11.3 Hz, H-21
a
), 3.28 (1H, brs,
a
), 2.68
a
1.94 (3H, s, H-28), 1.87 (3H, s, H-27), 1.16 (3H, s, H-19), 1.00 (3H, s,
H-18); 13C NMR (CDCl3, 100.63 MHz) d 210.0 (C, C-1), 165.0 (C, C-
26), 149.2 (C, C-24), 133.5 (C, C-10), 132.2 (CH, C-20), 129.2 (CH, C-
30), 127.7 (CH, C-40), 122.0 (C, C-25), 100.3 (C, C-12), 78.7 (C, C-17),
76.3 (CH, C-22), 62.2 (C, C-5), 61.5 (CH, C-6), 60.2 (CH2, C-21), 51.5
(C, C-13), 51.2 (C, C-10), 50.5 (CH, C-14), 47.2 (CH, C-20), 42.0
(CH2, C-2), 41.0 (CH, C-9), 39.3 (CH, C-3), 36.7 (CH2, C-11), 35.9
(CH2, C-4), 35.5 (CH2, C-16), 35.2 (CH2, C-23), 31.1 (CH2, C-7), 27.6
(CH, C-8), 24.2 (CH2, C-15), 20.4 (CH3, C-28), 13.6 (CH3, C-19), 12.4
(CH3, C-27), 12.1 (CH3, C-18);); ESIMS m/z 617 [M + Na]+ (100), 521
(43), 507 (20), 413 (5), 301 (4); HRESIMS m/z [M + Na]+ 617.2546
(calcd. for C34H42O7SNa, 617.2549).
were inoculated in a volume of 100
000 (HBL-100 and SW1573), 15 000 (HeLa and T-47D), and 20
000 (WiDr) cells per well, based on their doubling times.
lL per well at densities of 10
4.5.3. Chemosensitivity testing
Chemosensitivity tests were performed using the SRB assay of
the NCI with slight modifications. Briefly, pure compounds were
initially dissolved in DMSO at 400 times the desired final maxi-
mum test concentration. Control cells were exposed to an equiva-
lent concentration of DMSO (0.25% v/v, negative control). Each
agent was tested in triplicate at different dilutions in the range
Â
Ã
Compound 14: White amorphous solid; a2D1 + 3.6 (c 0.98,
CHCl3); UV (CHCl3) kmax (log
e
) 240 (3.80) nm; IR (dry film) vmax
1–100
Drug incubation times were 48 h, after which cells were precipi-
tated with 25 L ice-cold Cl3CO2H-H2O (1:1, v/v) and fixed for
lM. The drug treatment was started on day 1 after plating.
3465, 2939, 1714, 1306, 1102 cmꢁ1
;
1H NMR (CDCl3,
500.13 MHz) d 7.39 (2H, dt, J = 7.7, 1.6 Hz, H-20), 7.32 (2H, brd,
J = 7.7 Hz, H-30), 7.29 (1H, brd, J = 7.7 Hz, H-40), 4.34 (1H, ddd,
J = 11.4,7.9,3.2 Hz, H-22), 3.77 (1H, dd, J = 11.3,5.5 Hz, H-21b),
l
60 min at 4 °C. The SRB assay was then performed. The optical den-
sity (OD) of each well was measured at 492 nm, using BioTek’s
PowerWave XS Absorbance Microplate Reader. Values were cor-
rected for background OD from wells only containing medium.
The percentage growth (PG) was calculated with respect to un-
treated control cells (C) at each drug concentration level based
on the difference in OD at the beginning (T0) and end of drug expo-
sure (T), according to NCI formulas (Monks et al., 1991). Therefore,
if T is greater than or equal to T0, the calculation is 100 ꢀ [(T ꢁ T0)/
(C ꢁ T0)]. If T is lower than T0 denoting cell killing, the calculation
is 100 ꢀ [(T ꢁ T0)/(T0)]. The effect is defined as percentage of
growth, where 50% growth inhibition (GI50) represents the concen-
tration at which PG is +50. With these calculations a PG value of 0
corresponds to the amount of cells present at the beginning of drug
exposure, while negative PG values denote net cell kill.
3.73 (1H, qd, J = 7.2,1.9 Hz, H-3
3.30 (1H, brs, H-6), 3.22 (3H, s, OCH3), 2.81 (1H, m, H-20), 2.77
(1H, dd, J = 14.0,7.0 Hz, H-2 ), 2.71 (1H, dd, J = 14.0,7.4 Hz, H-2b),
2.60 (1H, brt, J = 14.5 Hz, H-23b), 2.46 (1H, dd, J = 14.7,6.4 Hz, H-
), 2.26 (1H, brd, J = 14.4 Hz, H-7b), 2.19 (1H, m, H-23 ), 2.15
(1H, m, H-16b), 1.95 (3H, s, H-28), 1.88 (3H, s, H-27), 1.82 (1H, m,
H-11 ), 1.80 (1H, m, H-16 ), 1.67 (1H, m, H-8), 1.65 (1H, m, H-
15b), 1.54 (1H, m, H-15 ), 1.52 (1H, m, H-4b), 1.46 (1H, m, H-7 ),
a), 3.62 (1H, t, J = 11.3 Hz, H-21a),
a
4a
a
a
a
a
a
1.45 (1H, m, H-9), 1.40 (1H, m, H-11b), 1.38 (1H, m, H-14), 1.16
(3H, s, H-19), 0.95 (3H, s, H-18); 13C NMR (CDCl3, 100.63 MHz) d
210.1 (C, C-1), 165.1 (C, C-26), 149.1 (C, C-24), 133.6 (C, C-10),
132.3 (CH, C-20), 129.2 (CH, C-30), 127.8 (CH, C-40), 122.0 (C, C-
25), 102.7 (C, C-12), 78.9 (C, C-17), 76.4 (CH, C-22), 62.3 (C, C-5),
61.4 (CH, C-6), 60.2 (t, C-21), 51.8 (C, C-13), 51.4 (C, C-10), 50.5
(CH, C-14), 47.9 (CH3, OCH3), 47.1 (CH, C-20), 42.0 (CH2, C-2),
40.8 (CH, C-9), 39.3 (CH, C-3), 35,9 (CH2, C-4), 35.4 (CH2, C-16),
35.3 (CH2, C-23), 31.2 (CH2, C-7), 30.4 (CH2, C-11), 27.8 (CH, C-8),
24.1 (CH2, C-15), 20.4 (CH3, C-28), 13.4 (CH3, C-19), 12.4 (CH3, C-
27), 12.1 (CH3, C-18); ESIMS m/z 631 [M + Na]+ (100), 617 (5),
413 (4), 301 (5); HRESIMS m/z [M + Na]+ 631.2709 (calcd. for
C35H44O7SNa, 631.2705).
Acknowledgement
This work was supported by Grants from CONICET (Argentina),
Ministerio de Ciencia y Tecnología de Córdoba (Argentina), Myndel
Botanica Foundation, SeCyT-UNC, EU-FEDER: the Spanish MICINN
(SAF2009-13296-C02-01 and CTQ2008-06806-C02-01/BQU), MSC
(RTICC RD06/0020/1046), Canary Islands’ ACIISI (PI 2007/021),
FUNCIS (PI 43/09), and ICIC. Thanks are also given to Dr. A. A. Coc-
ucci (IMBIV-UNC) for providing the photographs of J. caulescens
and L. Ribulgo for preparing the color figures. M.E.G. thanks CON-
ICET (Argentina) for a fellowship. NMR assistance by G. Bonetto
is gratefully acknowledged.
4.5. Biological Assays
4.5.1. Materials
All starting materials were commercially available research-
grade chemicals and used without further purification. RPMI

M.E. García et al. / Phytochemistry 76 (2012) 150–157
157
Misico, R.I., Nicotra, V., Oberti, J.C., Barboza, G., Gil, R.R., Burton, G., 2011.
Withanolides and related steroids. Progress in the Chemistry of Organic
Natural Products 94, 127–229.
Monks, A., Scudiero, D., Skehan, P., Shoemaker, R., Paul, K., Vistica, D., Hose, C.,
Langley, J., Cronise, P., Vaigro-Wolff, A., Gray-Goodrich, M., Campbell, H., Mayo,
J., Boyd, M., 1991. Feasibility of a high-flux anticancer drug screen using a
diverse panel of cultured human tumor cell lines. J. Natl. Cancer Inst. 83, 757–
766.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2011.12.018.
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