Antiproliferative and quinone reductase-inducing activities of withanolides derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.05.045

Two new and five known withanolides (jaborosalactones 2, 3, 4, 5, and 24) were isolated from the leaves of Jaborosa runcinata Lam. We also obtained some derivatives from jaborosalactone 5, which resulted to be the major isolated metabolite. The natural co

Full text of DOI:10.1016/j.ejmech.2014.05.045

European Journal of Medicinal Chemistry 82 (2014) 68e81
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antiproliferative and quinone reductase-inducing activities of
withanolides derivatives
,
b
,
,
,
Manuela E. García a *, Viviana E. Nicotra a b, Juan C. Oberti a b, Carla Ríos-Luci c,
c
d
e
d
ꢀ
Leticia G. Leon , Laura Marler , Guannan Li , John M. Pezzuto ,
Richard B. van Breemen , Jose M. Padron , Idaira Hueso-Falcon , Ana Estevez-Braun
e
c
c
c
ꢀ
ꢀ
ꢀ
ꢀ
a Facultad de Ciencias Qu
ímicas, Universidad Nacional de Cordoba, X5000HUA Cordoba, Argentina
ꢀ ꢀ
b Instituto Multidisciplinario de Biolog
í
ꢀ
a Vegetal (IMBIV-CONICET), Casilla de Correo 495, X5000HUA Cordoba, Argentina
c
ꢀ
ꢀ
ꢀ
Instituto Universitario de Bio-Organica “Antonio Gonzalez” (IUBO-AG), Centro de Investigaciones Biomedicas de Canarias (CIBICAN), Universidad de La
ꢀ
Laguna, C/Astrof
í
sico Francisco Sanchez 2, 38206 La Laguna, Spain
d The Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, 34 Rainbow Drive, Hilo, 96720 HI, USA
e Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, 60612 IL,
USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new and five known withanolides (jaborosalactones 2, 3, 4, 5, and 24) were isolated from the leaves
of Jaborosa runcinata Lam. We also obtained some derivatives from jaborosalactone 5, which resulted to
be the major isolated metabolite. The natural compounds as well as derivatives were evaluated for their
antiproliferative activity and the induction of quinone reductase 1 (QR1; NQ01) activity. Structureeac-
tivity relationships revealed valuable information on the pharmacophore of withanolide-type com-
pounds. Three compounds of this series showed significantly higher antiproliferative activity than
jaborosalactone 5. The effect of these compounds on the cell cycle was determined. Furthermore, the
ability of major compounds to induce QR1 was evaluated. It was found that all the active test compounds
are monofunctional inducers that interact with Keap1. The most promising derivatives prepared from
Received 5 September 2013
Received in revised form
15 May 2014
Accepted 20 May 2014
Available online 20 May 2014
Keywords:
Withanolide derivatives
Jaborosa runcinata
jaborosalactone 5 include (23R)-4b,12b,21-trihydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-
Antiproliferative activity
Quinone-reductase induction
Keap1 protein
26,23-olide (18) and (23R)-21-acetoxy-12
b
-hydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-
26,23-lactame (20).
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
A family of withanolides spiranic at C-23 has been isolated from
Jaborosa runcinata Lam., Jaborosa odonelliana, and Jaborosa arau-
cana [6], and many of these compounds exhibit interesting bio-
logical activities such as antiproliferative [7] and cancer
chemopreventive properties [8]. In addition, some studies have
reported structural modifications of withanolides and structur-
eeactivity relationships. Fundamentally, the ring A of withanolides
has been modified with various nucleophiles and the derivatives
have been evaluated for antiproliferative activity [9,10].
Continuing our search for bioactive compounds, we evaluated
the chemical content of J. runcinata based on its high content of
withanolides. One objective was to obtain enough jaborosalactone
5 to synthesize derivatives and introduce changes at various posi-
tions of the molecule. Two new and five previously reported
withanolides were isolated. From jaborosalactone 5, a set of de-
rivatives was synthesized, and their antiproliferative and chemo-
preventive activities were assessed. Preliminary mechanistic
studies were performed, and the effects on the cell cycle were
Withanolides comprise a group of naturally occurring C28 ste-
roids based on an ergostane skeleton in which C-26 and C-22, or C-
26 and C-23, are oxidized in order to form a d- or g-lactone. They
are known for the diversity of structures involving the steroid nu-
cleus and the side chain, including the formation of additional
rings. Their chemistry and occurrence has been the subject of
several reviews [1e4]. Among the nearly 100 genera included in
Solanaceae, ca. 50% have been investigated [4]. Jaborosa is an
interesting South American genus growing from southern Peru to
Argentina in very diverse habitats [5]. Jaborosa Juss. represents one
of the four major contributors of withanolide structures.
* Corresponding author. Instituto Multidisciplinario de Biología Vegetal (IMBIV-
CONICET), Casilla de Correo 495, X5000HUA Cordoba, Argentina.
ꢀ
E-mail address: manuelagarcia@fcq.unc.edu.ar (M.E. García).
http://dx.doi.org/10.1016/j.ejmech.2014.05.045
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

M.E. Garc
í
a et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
69
Fig. 1. Structures of isolated withanolides.
examined. With respect to quinone reductase (QR1; NQ01)-
inducing compounds, their potential was determined for regu-
lating gene expression through interaction with Keap1 protein.
The antioxidant response element (ARE) controls the expression
of cytoprotective enzymes including QR1, UDP-glucuronyl trans-
ferases and glutathione S-transferases and is regulated, at least in
part, by levels of the transcription factor NrF2 [11]. The cytosolic
protein Keap1 binds Nrf2 and the enzyme, Cul3, which attaches
ubiquitin to Nrf2 thereby marking it for hydrolysis by the proteo-
some. Ubiquitination of Nrf2 can be reduced or prevented by co-
valent modification of one or more of the 37 cysteine residues on
Keap1 [12]. By blocking ubiquitination of Nrf2, cytosolic levels of
Nrf2 increase leading to activation of the ARE. Most ARE inducers
acting through modification of Keap1 cysteine sulfhydryl residues
are electrophilic in nature and include Michael acceptors and iso-
thiocyanates [13,14]. We have developed a mass spectrometry-
based screening assay suitable for the identification of these
Keap1 modifying agents [15].
confirmed from the resonances of carbons 23e28; a similar
arrangement has been described previously [6]. The absence of a
singlet at the high field region of the 1H NMR spectrum and the
appearance of two doublets at 4.15 and 4.24 ppm suggested the
presence of an isolated C-21 hydroxymethylene group. The 13C
NMR spectrum showed only four methyl groups at 14.6, 17.2, 8.6,
and 15.9 ppm corresponding to C-18, C-19, C-27, and C-28,
respectively. The methylene signal at 58.4 ppm (C-21) confirmed
the presence of a hydroxyl group at C-21. The presence of a tet-
rasubstituted double bond at C-17-C-20 was ratified by the
downfield shift for the signals at
d 166.1 and d 127.5, respectively.
The CH2-21 signal was correlated with a signal of conjugated
carbonyl carbon at 193.0 ppm in the HMBC experiment, con-
firming a 17(20)-en-22-keto functionality. The configuration at
the spiranoid center (C-23R) was established by comparison with
spectral data of jaborosalactones 1e6 and 25 [6].
Regarding the A/B rings, the 13C NMR spectrum showed two
carbonyl groups at
carbons, two signals of methine carbons at
(assigned to C-3 and C-6, respectively) and one signal of quaternary
carbon at 77.6 (C-5). These data provided evidence for an oxygen
d
209.2 (C-1) and
d
207.4 (C-4), three carbynolic
d
74.7 and 72.6
d
2. Results and discussion
d
2.1. Chemistry
ether bridge between the A/B rings through the C-3 and C-6 posi-
tions. This assumption was supported by the following cross-
2.1.1. Natural products
correlation peaks in the HMBC experiment: between H3-19 (
0.77) and C-5 ( 77.6) and C-10 ( 50.6), confirming an oxygenated
function at C-5 position; between H2-2 ( 3.04 and 2.42) and C-1
209.2), C-3 ( 74.7), and C-4 ( 207.4), indicating the presence of
an oxygenated methine located at C-3 and two non-conjugated
ketone groups located at C-1 and C-4; and finally the weak but
d
The aerial parts of J. runcinata were air-dried and extracted with
ethanol. After concentration and defatting, the residue was frac-
tionated by a combination of several chromatographic techniques,
ultimately giving two new withanolides, compounds 1 and 2, and
the known spiranic-type withanolides, jaborosalactones 2, 3, 4, 5
and 24 (Fig. 1).
d
d
d
d
(d
d
d
diagnostic HMBC correlation observed between H-3 (
d 4.20) with
The molecular formula of compound 1 was determined by high
resolution electrospray mass spectrometry (HRESIMS) as
C
C-6 ( 72.2) validating C-3/C-6 epoxy functionality. The epoxy
d
group orientation was established by analysis of the coupling
constants of H2-2, H-3, and H-6 and confirmed by comparison with
spectral data of subtrifloralactone K, withanolide reported from
Deprea subtriflora having the same A/B ring substitution pattern
[16]. Compound 1 (jaborosalactone 47) was finally established
28H32O9Na (m/z 535.1946 [MþNa]þ). In its 1H NMR spectrum, the
presence of two methyl signals at d 1.82 and d 2.16 and the absence
of a lactonic hydrogen in the 4e5 ppm region corresponding to the
carbynolic hydrogen H-22 indicated spiranoid dimethyl-
substituted -insaturated -lactone ring comprising C-23 to C-
28. The typical pattern of the spiranoid arrangement was
a
a
,
b
g
as
(23R)-3a,6a-epoxy-5b,12b,21-trihydroxy-1,4,22-trioxo-12,23-
cycloergostan-17,24-dien-26,23-olide.

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M.E. García et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
Scheme 1. Withanolide derivatives obtained from jaborosalactone 5 (3e21). aGeneral reagents and conditions: (a) Ac2O, pyridine, DMAP; (b) RCOCl, (C2H5)3N, DMAP, CH2Cl2; (c)
SOCl2, CH2Cl2, N2, ; (d) PCC, CH2Cl2; (e) H2, 10% Pd/C, THF; (f) SeO2, CH2Cl2, Ar, ; (g) NH4OAc/AcOH, ; (h) LiAlH4, THF-Et2O,
D
D
D
D.
The HRESIMS of compound 2 showed a sodium adduct [MþNa]þ
at m/z 525.2469 corresponding to a formula of C28H38O8Na. The 1H
and 13C NMR of 2 showed a typical chemical shift for C23eC28 of a
and H3-18 (d 1.03). The signals of two oxidized quaternary carbons
at 79.1 and 83.7 ppm were assigned to C-12 and C-17, respectively.
The assignment of C-23R configuration was carried out by analysis
of NOESY spectra, which showed strong correlation between H-22
C-23-spiranoid
similarities, marked differences can be found for A/B rings; the 1H
NMR spectrum of compound 2 exhibited signals at 5.76 and 6.49
g-lactone, such as compound 1. Despite these
and H3-28 (
48) was finally established as (20R,23R)-5
d
2.08). The structure of compound 2 (jaborosalactone
,6 ,12 ,17 ,22 -penta-
d
d
a
b
b
b
a
typical of a 2-en-1-one system without substituent at C-4. These
chemical shifts, plus the presence of a carbonyl carbon signal at
hydroxy-1-oxo-12,23-cycloergostan-2,24-dien-26,23-olide.
d
203.4 and two methine carbon signals at
13C NMR spectrum are in agreement with a conjugated double
bond in ring A. The downfield shift of H-6, which appeared at 3.60
as a broad singlet, revealed a small coupling constant with H2-7,
evidencing an equatorial orientation ( ) of the hydrogen H-6. The
presence of two carbons at 77.2 (C-5) and
74.2 (C-6) in the 13C
NMR spectrum were consistent with a 5 ,6 -diol substitution
typical of many withanolides [17]. The presence of five methyl
groups in the 1H NMR spectrum and the doublet signal at
1.11
assigned to H3-21 confirmed the absence of a hydroxyl group at C-
20. The correlation observed in the COSYexperiment between 1.11
(H3-21), 2.03 (H-20) and 4.01 (H-22), and the absence of
carbonyl carbon corresponding to C-22, suggested a hydroxyl
functionality at C-22, which was assigned in the 13C NMR at
69.2.
This assumption was confirmed for HMBC correlations between H-
22 ( 4.01) and C-20 ( 40.1) and C-21 ( 10.7). The -orientation of
OH-22 was determined by the nOe observed between H-22 ( 4.01)
d
128.5 and 141.1 in the
2.1.2. Derivatization of jaborosalactone 5
An considerable amount (482 mg) of jaborosalactone 5 was
isolated from J. runcinata. The structural arrangement of jabor-
osalactone 5 is closely related to those of jaborosalactone P isolated
from J. odonelliana, one of the most promising inducers of QR1 in
terms of potency and low toxicity in vitro and in vivo [8]. On the
other hand, compounds with the same skeleton have been evalu-
ated as antiproliferative agents against tumor cell lines. In view of
the activity reported by the spiranoid at C-23 withanolides and the
amount of jaborosalactone 5 isolated, a set of derivatives (3e21,
Scheme 1) was prepared. Chemical modifications were introduced
at the C-21 hydroxyl group, the double bonds in the A/B rings, the
lactone of the side chain, and carbonyl groups.
d
a
d
d
a
b
d
d
d
d
d
Compounds 3 and 4 were obtained by acetylation of jabor-
osalactone 5 with Ac2O/Py, while compounds 5e12 were prepared
by acylation with several acyl chlorides of different size, lip-
ophilicity and stereoelectronic properties. The reaction of
d
d
d
a
d

M.E. Garc
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a et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
71
jaborosolactone 5 with SOCl2 afforded the chlorinated compounds
13 (35%) and 14 (5%) while the oxidation of the hydroxyl group at C-
21 with PCC yielded the aldehyde 15 (17%). Compounds 16 (22%)
and 17 (21%) were obtained by hydrogenation of jaborosalactone 5
in the presence of 10% Pd/C. The treatment with SeO2 gave the
derivatives with a hydroxyl group at C-4 18 (33%) and at C-6 19
withanolides spiranic at C-23 showed moderate antiproliferative
activity. A few trends were apparent in the structureeactivity re-
lationships (SAR) at the side chain. i) If one compares the activity of
jaborosalactone 5 to that of the ester derivatives at C-21 position,
for most of the esters obtained, the activity increased (3, 5e7,
9e11), except for adipoyl ester (12), which showed less activity
than its precursor, jaborosalactone 5. Concerning the activity
showed that compounds with aromatic substituents (5e7) are
significantly more active than compounds with aliphatic sub-
stituents (3,9,10). ii) No change of activity was observed when the
lactone was replaced by the lactame functionality at the side chain
(35%). The
b orientations of the corresponding hydroxyl groups
were determined on the basis of the signal splitting and the
coupling constant value of the proton on C-4 or C-6.
Jaborosalactone 4 was obtained in low yield (18%) by epoxida-
tion with MCPBA. Transformation of the lactone into lactam was
made by using ammonium acetate in acetic acid. Thus, compound
20 which also presented an acetate group at C-21 was obtained in
56% yield. When jaborosalactone 5 was treated with LiAlH4 the
carbonyl at C-22 was chemoselectively reduced to compound 21
(3%).
(3 vs 20). iii) Compound 13 (GI50 2.1e16 mM) was the most active
product, in which the hydroxyl group at C-21 position was replaced
by chlorine. iv) The presence of a 17,22-dihydroxy-2-methyl system
proved deleterious for activity, regardless of the A/B ring substit-
uent types [jaborosalactones 24 and 48 (2)].
The A/B rings also appeared to play a role in determining ac-
tivity. i) For compounds with 17 (20)-ene-22-keto-21-hydroxy
system, similar and moderate activity values for 2,5-diene (jabor-
2.2. Biological activity
osalactone 5), 2,5-diene-4
(jaborosalactone 4) compounds were observed. Lower activities
were observed for the 2,4-diene-6 -hydroxy derivative (19) and
the 1,4-dioxo-5 ,hydroxyl-3 ,6 -epoxy compound (1). ii) For
compounds with a methyl group at the C-21 position, moderate
activity for 1-oxo-5 -chloro,6 -hydroxy compound (jabor-
osalactone 3) was observed, while 1-oxo-2-en-5 ,6 -dihydroxy
(jaborosalactone 2), 1-oxo-5-ene (16), and a compound with totally
saturated A/B rings (17) showed weak or no activity. These con-
siderations, based upon substitution patterns, were partly consis-
tent with previous SAR findings [7], and otherwise provide valuable
information on the pharmacophore of withanolide-type com-
pounds and will be helpful for the rational design of more potent
and selective anticancer drugs.
b-hydroxy (18) and 2-ene-5b,6b-epoxy
2.2.1. Antiproliferative activity
The in vitro antiproliferative activities of natural jabor-
osalactones 2, 3, 4, 5, 24, 47 (1), and 48 (2), and semi-synthetic
derivatives (3e7, 9e13, 15, 16e21) were evaluated using the well-
established protocol of the National Cancer Institute (NCI) of the
United States [18]. As models, the representative panel of human
solid tumor cell lines HBL-100 (breast), HeLa (cervix), SW1573
(non-small-cell lung), and WiDr (colon) was used. Table 1 sum-
marizes the results as 50% growth inhibition (GI50).
b
b
a a
a
b
a
b
Viewed as a whole, these results allowed classifying the com-
pounds into three groups. A first group, comprising jaborosalactone
3 and derivatives 5, 7, and 13, caused significant growth inhibition
with GI50 values below 10 mM in most cell lines. The second set of
compounds, consisting of jaborosalactones 4 and 5 and derivatives
3, 4, 6, 9e11, 15e18, 20, and 21, exhibited moderate activity. The
third group, consisting of jaborosalactones 2, 24, 47 (1), and 48 (2)
and derivative 19, failed to show growth inhibition.
The influence of the substitution pattern on the activity of the
withanolides tested in this work was examined. Overall,
2.2.2. Cell cycle disturbances
When cells are exposed to cytotoxic agents, damaged cells may
suffer a block in their cell cycle. If the cell cannot recover from the
damage, cell death results from apoptosis. This blockage in cell
division is known as cell cycle arrest and can take place at any of the
cell cycle phases, namely G0/G1, S, or G2/M phase. The cell cycle
phase distribution by flow cytometry was studied to determine
whether cell growth inhibition involved cell cycle changes. Three
compounds from this series (5, 7, and 13) exhibited potent anti-
proliferative effects on tumor cells and were selected accordingly
for cell cycle studies.
Cells were exposed to each agent at two different drug con-
centrations (high and low), which were chosen based on two
premises [19]: The GI50 values of the compounds (Table 1), and
the sensitivity of the cell line to drug treatment, since at higher
drug doses cell death prevents examination of the cell cycle
phase distribution. The most sensitive cells (HBL-100, HeLa and
Table 1
In vitro antiproliferative activity of withanolides and analogues against human solid
tumor cell lines.a
Compound
HBL-100
HeLa
SW1573
WiDr
Jaborosalactone 2
>100
76 34
11 2.2
22 2.8
49 15
>100
90 14
9.8 1.0
31 3.1
42 6.3
>100
>100
Jaborosalactone 3
Jaborosalactone 4
Jaborosalactone 5
Jaborosalactone 24
2.9 0.7
33 2.5
38 4.9
>100
16 6.2
40 0.2
50 16
>100
>100
>100
Jabrosalactone 47 (1)
Jabrosalactone 48 (2)
82 26
>100
41 5.7
>100
72 12
>100
3
4
5
6
7
9
10
11
12
13
15
16
17
18
19
20
21
31 4.3
27 8.3
3.5 0.3
4.4 1.8
3.7 0.2
18 0.9
19 2.8
26 3.2
60 5.5
2.9 0.7
13 4.9
53 10
43 20
39 5.8
>100
19 0.8
20 1.0
2.40 0.01
13 3.4
4.5 2.3
17 1.7
12 2.8
16 0.6
26 0.5
2.1 0.1
8.6 2.4
>100
>100
77 32
>100
18 0.7
52 6.0
32 3.3
34 3.9
3.5 0.6
14 0.3
4.0 0.4
16 0.8
23 7.5
30 6.0
58 12
3.1 0.4
16 2.7
50 17
39 13
37 7.9
>100
51 8.3
90 14
13 7.4
41 10
34 3.6
29 1.6
31 2.1
41 10
>100
16 6.2
26 0.1
96 5.3
92 11
77 32
>100
SW1573 cell lines) were exposed to 5 (low) and 10
whilst the drug-resistant WiDr cells were treated with 15 (low)
and 30 M (high). Control cells were incubated in the absence of
test drug.
The results varied according to the cell line tested (Fig. 2). In
mM (high),
m
general, test compounds altered the cell cycle with a decrease in the
G1 phase, which was concomitant with an increase in the S or G2/M
compartment. Significant cellular death was observed for the HBL-
100 (7e23%) and HeLa (15e35%) cell lines after exposure to the
compounds. In contrast, reduced cell death was detected in
SW1573 (5e15%) and WiDr (7e12%) treated cells. In HBL-100 and
WiDr cells, there was an accumulation in the G2/M phase whilst for
HeLa and SW1573 cell lines, accumulation was observed in the S
phase of the cell cycle. These differences cannot be explained in
28 6.5
69 4.3
31 3.8
56 12
50 7.4
29 20
a
Expressed as GI50 values given in mM and determined as means of two to five
experiments, standard deviations are given in parentheses.

72
M.E. García et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
Fig. 2. Cell cycle phase distribution in untreated cells (C) and cells treated for 24 h with compounds 5, 7 and 13 at low and high dose concentrations. a) HBL-100; b) HeLa; c)
SW1573; d) WiDr; Areas: black ¼ G0/G1, white ¼ S, gray ¼ G2/M.
terms of GI50 values. Although preliminary, these results suggest
that derivatives 5, 7, and 13 might induce cell death in these cell
lines by different pathways.
and three additional compounds, 6, 9, and 19, exhibited weak ac-
tivity, with CD values of 16.1, 21.0, and 31.7 M, respectively. The
current results indicate that the substitution pattern in A/B rings is
relevant for the activity according to the following observations. i)
The most active compounds have a 1-oxo-2,5-diene system (7 and
m
2.2.3. Induction of quinone reductase 1 (QR1)
20). ii) When 5-ene is replaced by a 5
decreases (jaborosalactone 5 vs jaborosalactone). iii) Similarly,
when 5 -chloro,6 -hydroxy is replaced by a 5 ,6 -dihydroxy sys-
tem, the activity disappears (jaborosalactone 3 vs jaborosalactone).
The low activity observed in compounds with -oxygenated sub-
stituents at C-5 has been reported previously for several with-
anolides [8]. Regarding the side chain, the presence of oxygenated
functions at C-21 positively contributed to inducing activity.
However, highly hydrophobic and/or halogen substituents show a
decrease in activity. Jaborosalactone 24 is a C-23 spiranoid with-
anolide with a 17,22-dihydroxy system. This compound has the
same skeleton and substitution patterns as those of jaborosalactone
P; they only differ in their configuration at C-23. Despite their close
structural similarity, these compounds have shown different
b,6b-epoxy system, activity
QR1 is a flavoprotein which catalyzes two- or four-electron
reduction of a wide variety of quinones, quinone imines, and
other nitrogenous compounds. This enzyme has been demon-
strated to reduce oxidative stress, particularly in the context of
chemoprevention of carcinogenesis [20]. An MTT assay with Hepa
1c1c7 murine hepatoma cells was employed to determine the
expression of QR1 following treatment with 13 compounds
included in this study. An identical assay in mutant Hepa cells was
used to determine whether the observed induction was mono-
functional (acting through activation of the ARE only) or bifunc-
tional (acting through activation of both phase I and phase II
enzymes through alternate pathways). TAOc1 and BPrc1 mutant
cells are defective in a functional Ah receptor or unable to trans-
locate the receptoreligand complex to the nucleus, respectively.
Induction of QR1 seen in these two cell lines indicates the sample
does not work through the XRE pathway.
a
b
a b
a
inducing activity for QR1, with CD values of 6.29 mM and 0.75 mM
for jaborosalactone 24 and jaborosalactone P [8], respectively.
These results suggest that the configuration of the carbon spiranic
is relevant for inducing activity.
Overall, the compounds demonstrating the most promising
profiles of activity are 18 and 20. The compounds are not signifi-
cantly cytotoxic (Table 1) and CD values are relatively low (2.04 and
Ten compounds out of the 13 investigated were found to induce
QR1 with an induction ratio (IR) greater than 2.0. Some compounds
did not demonstrate induction at the highest concentration tested
(20 mg/ml) due to cytotoxicity, yet they were seen to significantly
induce QR1 without killing cells at lower concentrations (e.g.,
0.82 mM, respectively) (Table 2), yielding relatively high CI values
jaborosalactone 3). The most active compound was 7, with a CD
(>20) (Table 2). The compounds are monofunctional inducers
(Table 2), presumably working through interaction with Keap1 (see
below).
(concentration required to double QR1 induction) of 0.59 mM. This
substance was also found to be toxic to cultured cells (Table 1). All
10 active compounds also modulated QR1 activity in both mutant
cell lines. These data indicate that the compounds in this study
induce QR1 monofunctionally, and therefore up-regulate the ARE,
with moderate potency. Therefore, they are promising as cancer
chemopreventive agents.
Jaborosalactones 3, 5, and 24, and derivatives 7, 15, 18, and 20
were promising inducers of QR1 activity with CI (IC50/CD) values of
9.21, 16.85, >6.78, 13.64, 8.11, >20.4, and 22.93 respectively. Jabor-
osalactones 2 and 4 and derivative 13 failed to show QR1 induction,
2.2.4. Keap1 modification by withanolides
The induction of enzymes such as QR, glucuronyl transferases,
glutathione S-transferases, and sulfotransferases can protect cells
against the toxic and neoplastic effects of carcinogens. An increase
in the concentration of Nrf2 in the nucleus of a cell up-regulates the
ARE and induces the expression of these chemopreventive en-
zymes. Based on the hypothesis that ubiquitination and

M.E. Garc
í
a et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
73
Table 2
Concentration required to double induction of QR1 (CD, mM) and chemopreventive index (CI; IC50/CD) for withanolides isolated from Jaborosa runcinata and derivatives in wild-
type Hepa 1c1c7 cells and two mutant cell lines, TAOc1 and BPrc1. Sulforaphane was used as a monofunctional control, and 40-bromoflavone was used as a control in wild-type
cells.
Compound
Hepa 1c1c7
TAOc1
BPrc1
CD (m
M)
IC50
(
mM)
CI (IC50/CD)
CD (m
M)
CD (
mM)
Jaborosalactone 2
N/A
e
N/A
N/A
N/A
Jaborosalactone 3
Jaborosalactone 4
Jaborosalactone 5
Jaborosalactone 24
6
7
9
13
1.26 0.14
N/A
11.57
40.3
>42.7
>30.9
8.1
9.21
N/A
0.96 0.16
N/A
1.44 0.16
N/A
e
2.39 0.90
6.29 0.79
16.1 1.82
0.59 0.19
21.00 1.92
N/A
16.85
>6.78
>1.92
13.64
1.17
2.52 0.75
9.62 1.98
26.50 2.69
1.66 0.69
9.03 1.34
N/A
2.75 0.30
12.46 0.64
25.99 2.56
2.34 0.95
24.40 1.87
N/A
24.6
e
N/A
15
18
19
20
3.55 1.32
2.04 0.29
31.7 3.04
0.82 0.18
0.012 0.005
7.87 0.43
28.8
>41.6
>41.6
18.6
>66.4
>170
8.11
4.15 0.39
0.69 0.13
29.11 2.16
0.91 0.22
>66.4
5.32 0.56
3.22 0.92
31.05 1.91
1.13 0.32
>66.4
>20.41
>1.31
22.93
>5197
>21.65
40-Bromoflavone
Sulforaphane
8.95 0.26
15.09 0.71
proteosome-mediated degradation of Nrf2 in the cytoplasm
decrease upon the covalent modification of 1 or more of the 27
cysteine sulfhydryl groups on Keap1 (a protein that sequesters Nrf2
in the cytoplasm), resulting in higher Nrf2 levels both in the cyto-
plasm and in the nucleus, a high-throughput mass spectrometry-
based screening assay was used to detect covalent modification of
sulfhydryl groups of human Keap1.
Unmodified Keap1 (negative control) was observed at m/z
70 998, and this mass spectrum was compared with that of each
withanolide after incubation with Keap1 (Fig. 3 and Table 3). After
incubation with the positive control isoliquiritigenin (a chalcone
a reference, activity is largely increased by replacing the hydroxyl
group at the C-21 position by a chlorine atom, or by esterification
with furanoic or benzoic acid.
Cell cycle studies of the most potent compounds indicated that
cell death is occurring and its mechanism may vary depending on
the cell line. Although the mechanism of growth alteration at the
cellular level is still unclear, the biological activity and structur-
eeactivity relationships reported provide the basis for further
studies, especially in relation to the search for new derivatives with
enhanced antiproliferative activities.
All of the withanolides reacted with Keap1, probably due to their
containing an
a
,
b-unsaturated ketone), the average mass of Keap1
a,b-unsaturated ketone functional groups. To differentiate their
increased to m/z 71 184. Isoliquiritigenin is known to react with
Keap1 cysteines, especially C151 [21]. Table 3 summarizes the
MALDI mass spectra of Keap1 after incubation with each with-
anolide derivative. It should be noted that mass spectrometry-
based assays do not suffer from interferences such as cytotoxicity
that are common to cell-based assays.
After incubation with each withanolide, the mass of Keap1
increased due to covalent attachment of one or more of these
electrophilic molecules. Since Keap1 contains 27 reduced cysteine
residues, covalent attachment of multiple withanolide molecules to
Keap1 is possible. For example, the MALDI mass spectra in Fig. 3
indicate that an average of 4 molecules of 18 or 20 bound to
Keap1. Studies are in progress to identify the sites of covalent
attachment of each compound. In conclusion, all of the assayed
compounds contain electrophilic groups that reacted with Keap1
(Table 3).
activities in terms of up-regulating the ARE, cell-based function
assays were carried out using QR1 as a marker enzyme. We
observed, once more, that changes in the side chain result in drastic
changes in the activity; the most notable result being the difference
in the inducing activity between jaborosalactone P and jabor-
osalactone 24, compounds which only differ in their configuration
at the C-23 position. We demonstrated that all those derivatives
which acted as QR1 inducers contained electrophilic groups that
react with Keap1. Overall, the most favorable profiles of activity
were observed with compounds 18 and 20.
Based on all these results, the preparation of new withanolide
derivatives as valuable chemopreventive agents is in progress.
4. Experimental
4.1. General experimental procedures
3. Conclusions
Melting points were measured on a mercury thermometer
apparatus and are uncorrected. Optical rotations were measured on
JASCO P-1010 and Perkin-Elmer-343 polarimeters. UV spectra were
obtained using Shimadzu-260 and Jasco V-560 spectrophotome-
ters. IR spectra were obtained in a Nicolet 5-SXC and Bruker IFS 55-
FTIR spectrophotometers. NMR experiments were performed on
Bruker AVANCE II 400 MHz and AMX 500 MHz instruments. Mul-
tiplicity determinations (DEPT) and 2D spectra (COSY, HSQC, HMBC,
and NOESY) were obtained using standard Bruker software.
In the present study, seven natural withanolides were isolated
from J. runcinata and 20 derivatives, including jaborosalactone 4,
were synthesized from jaborosalactone 5. Previous studies funda-
mentally changed withanolide A/B rings; this research aimed at
introducing variability to the side chain, functionalizing the C-21
position with different residues (aliphatic, aromatic and haloge-
nated), and replacing the side-chain lactone with a lactam function.
Regarding antiproliferative activity, previous research has deter-
mined that the most active compounds are those with a 1-oxo-2-
Chemical shifts are expressed in ppm (d) units using tetrame-
thylsilane as the standard. ESIMS and HRESIMS were measured on
an LCT Premier XE Micromass (Manchester, UK) mass spectrometer.
Positive ion MALDI-TOF mass spectra were acquired using an
Applied Biosystems (Foster, CA) Voyager DE-PRO time-of-flight
mass spectrometer. Chromatographic separations were performed
ene-5b,6b-epoxy system or a 1-oxo-3b-XR system on A/B rings.
Here, the most active derivatives (5, 7, and 13) show a 1-oxo-2,5-
diene system in the A/B rings. In addition, if we take the anti-
proliferative activity of the natural precursor (jaborosalactone 5) as

74
M.E. García et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
ꢀ
ꢀ
Cordoba. J. runcinata was collected in Departamento Parana, Entre
Ríos, Argentina, in December 2008 (Barboza et al. 2129).
4.3. Extraction and isolation
The air-dried powdered aerial parts of J. runcinata (233 g) were
exhaustively extracted with EtOH (3 ꢀ 500 mL) and the solvent was
evaporated at reduced pressure. The residue was defatted by
partition in hexane-EtOH-H2O (10:3:1), the resultant EtOHeH2O
phase was washed with hexane (3 ꢀ 100 mL), and EtOH evaporated
at reduced pressure. The residue was diluted with H2O and
extracted with CH2Cl2 (5 ꢀ 300 mL). The CH2Cl2 extract was dried
over anhydrous Na2SO4, filtered, and evaporated to dryness at
reduced pressure. Finally, 3.49 g of crude extract was obtained.
The residue was fractionated initially by molecular exclusion
chromatography. Elution with MeOH afforded 15 fractions. Frac-
tions 7e8 (850 mg) were column chromatographed over silica gel
60 G using hexane:EtOAc and EtOAc:MeOH mixtures of increasing
polarity. Jaborosalactone 5 (482 mg) was precipitated from frac-
tions corresponding to Hexane:EtOAc (4:6 to 2:8) elution.
Remaining fractions were regrouped and chromatographed on
silica gel 60 G, using CH2Cl2:MeOH (10:0 to 8:2) as elution solvent
mixture. The collected eluates were pooled in eleven fractions
containing withanolides which were then purified. Fractions III-IV
were purified by CC with hexane:EtOAc and EtOAc:MeOH mix-
tures of increasing polarity, yielding jaborosalactone 4 (28.2 mg).
The remaining fractions were purified by preparative TLC (EtOA-
c:hexane, 8:2). 39.5 mg of crystallized jaborosalactone 24 was ob-
tained from fraction V; fraction VI yielded 2.5 mg of compound 1
and fraction VII, 6.0 mg of compound 2. Jaborosalactone 2 (30 mg)
and jaborosalactone 3 (16 mg) were isolated from fractions VIII-IX.
The purification of fractions X-XI allowed isolation of 35 mg of
jaborosalactone 24.
Fig. 3. Positive ion MALDI time-of-flight mass spectra of cysteine-rich Keap1 after
incubation with methanol (negative control; top) or withanolides 18 and 20. Note that
the average mass of Keap1 increased due to covalent attachment of multiple with-
anolide molecules.
4.3.1. Jaborosalactone 47 (23R)-3
1,4,22-trioxo-12,23-cycloergostan-17,24-dien-26,23-olide (1)
White amorphous solid; [
(log ε): 221.3 (4.12) nm. IR (film) nmax: 3454, 2957, 2920, 1756, 1677,
1119, 1009, 757 cmꢁ1 1H NMR (CDCl3, 400.13 MHz): 4.24 d (1H,
a,6a-epoxy-5b,12b,21-trihydroxy-
a]21D: e 82.7 (c 0.15, MeOH). UV (MeOH)
lmax
by molecular exclusion chromatography (Sephadex LH-20), column
chromatography on silica gel 60 (0.063e0.200 mm), and prepara-
tive TLC on silica gel 60 F254 (0.2 mm thick) plates.
.
J ¼ 12.7 Hz, H-21a), 4.20 brd (1H, J ¼ 4.4 Hz, H-3), 4.15 d (1H,
J ¼ 12.7 Hz, H-21b), 3.97 d (1H, J ¼ 3.9 Hz, H-6), 3.04 dd (1H,
J ¼ 20.0,4.4 Hz, H-2a), 2.55 m (2H, H2-16), 2.42 dd (1H, J ¼ 20.0,
1.3 Hz, H-2b), 2.31 m (1H, H-14), 2.16 s (3H, H3-28), 1.817 s (3H, H3-
4.2. Plant material
27), 1.815 m (1H, H-11
1.73 m (1H, H-9), 1.728 m (1H, H-11
(1H, H-8), 1.58 m (1H, H-15 ), 1.03 s (3H, H3-18), 0.77 s (3H, H3-19).
a
), 1.79 m (1H, H-7
b
), 1.75 m (1H, H-15
a),
A voucher specimen of J. runcinata is deposited at the herbarium
b), 1.61 m (1H, H-7
a
), 1.60 m
ꢀ
ꢀ
of Museo Botanico Cordoba (CORD), Universidad Nacional de
b
13C NMR (CDCl3 100.03 MHz): 209.2 (C, C-1), 207.4 (C, C-4), 193.0 (C,
C-22), 172.3 (C, C-26), 166.1 (C, C-17), 161.1 (C, C-24), 127.5 (C, C-20),
127.2 (C, C-25), 91.2 (C, C-23), 77.6 (C, C-5), 74.7 (CH, C-3), 74.5 (C, C-
12), 72.2 (CH, C-6), 58.4 (CH2, C-21), 50.6 (C, C-10), 48.9 (C, C-13),
45.8 (CH, C-14), 42.5 (CH, C-9), 39.5 (CH2, C-2), 32.1 (CH2, C-11), 31.2
(CH, C-8), 29.7 (CH2, C-7), 25.1 (CH2, C-16), 22.7 (CH2, C-15), 17.2
(CH3, C-19), 15.9 (CH3, C-28), 14.6 (CH3, C-18), 8.6 (CH3, C-27).
HRESIMS m/z 535.1946 [MþNa]þ (calcd for C28H32O9Na, 535.1944).
Table 3
Covalent modification of Keap1 by electrophile withanolides measured using posi-
tive ion MALDI time-of-flight mass spectrometry.
Compound
m/za
D
Mb
Jaborosalactone 2
Jaborosalactone 3
Jaborosalactone 4
Jaborosalactone 5
Jaborosalactone 24
6
7
9
13
15
18
19
71 289
72 097
72 350
71 807
72 212
71 455
73 069
73 095
75 087
73 131
72 945
71 824
73 220
71 184
70 998
291
1099
1352
809
1214
457
2071
2097
4089
2133
1947
826
4.3.2. Jaborosalactone 48 (20R,22R,23R)-5
pentahydroxy-1-oxo-12,23-cicloergostan-2,24-dien-26,23-olide (2)
White amorphous solid; [
(log ε): 307.3 (3.69), 220 (4.27) nm. IR (film) nmax: 3418, 2966, 2930,
1738, 1668, 1387, 994, 656 cmꢁ1 1H NMR (CDCl3, 400.13 MHz):
a,6b,12b,17b,22-
a]21D: ꢁ70.8 (c 0.24; MeOH), UV (MeOH)
max
l
.
6.49 m (1H, H-3), 5.76 dd (1H, J ¼ 10.0, 2.4 Hz, H-2), 4.01 d (1H,
20
2222
186
J ¼ 11.2 Hz, H-22), 3.60 brs (1H, H-6), 3.15 brd (1H, J ¼ 19.0 Hz, H-
Isoliquiritigenin, positive control
Keap1, negative control
e
4
b
), 2.47 m (1H, H-14), 2.34 dd (1H, J ¼ 13.7,4.0 Hz, H-11
a
), 2.19 m
), 2.03 m (1H, H-20),
), 1.96 s (3H, H3-27), 1.80 m (1H, H-8), 1.73 m (1H,
a
(1H, H-9), 2.08 s (3H, H3-28), 2.03 m (1H, H-4
1.97 m (1H, H-16
a
Mass was measured after 2 h incubation with Keap1 at room temperature.
Change in mass relative to Keap1 control.
b
a

M.E. Garc
í
a et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
75
H-7
b
), 1.52 m (2H, H2-15), 1.502 m (1H, H-7
a
), 1.504 m (1H, H-16
b
),
(CH, C-14), 39.2 (CH, C-2), 39.1 (CH, C-9), 34.0 (CH2, C-11), 30.7 (CH,
C-8), 29.3 (CH2, C-7), 25.6 (CH2, C-16), 23.1 (CH2, C-15), 20.9 (CH3,
CH3COO-21), 19.7 (CH3, C-19), 15.9 (CH3, C-28), 14.4 (CH3, C-18), 9.0
(CH3, C-27). HRESIMS m/z [MþNa]þ 529.2219 (calcd for
1.23 m (1H, H-11
b), 1.19 s (3H, H3-19), 1.11 d (3H, J ¼ 6.6 Hz, H-21),
1.03 s (3H, H3-18). 13C NMR (CDCl3 100.03 MHz): 203.4 (C, C-1),
174.5 (C, C-26), 162.7 (C, C-24), 141.1 (CH, C-3), 128.5 (CH, C-2), 127.3
(C, C-25), 94.1 (C, C-23), 83.7 (C, C-17), 79.1 (C, C-12), 77.2 (C, C-5),
74.2 (CH, C-6), 69.2 (CH, C-22), 52.0 (C, C-10), 50.5 (C, C-13), 43.3
(CH, C-14), 40.1 (CH, C-20), 38.2 (CH, C-9), 35.2 (CH2, C-4), 34.8
(CH2, C-16), 32.8 (CH2, C-7), 32.2 (CH2, C-11), 29.5 (CH, C-8), 23.4
(CH2, C-15), 15.3 (CH3, C-28), 14.5 (CH3, C-19), 12.1 (CH3, C-18), 10.7
(CH3, C-21), 8.8 (CH3, C-27). HRESIMS m/z [MþNa]þ 525.2469 (calcd
for C28H38O8Na, 525.2464).
C30H34O7Na, 529.2226).
4.4.2. Preparation of (23R)-21-benzoyloxy-12
dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (5)
Triethylamine (45 L, 0.34 mmol) and benzoyl chloride (30 mL,
b-hydroxy-1,22-
m
0.25 mmol) were added to a solution of jaborosalactone 5
(19.5 mg, 0.042 mmol) in dry CH2Cl2 (3 mL). The reaction mixture
was stirred until disappearance of the starting withanolide (24 h).
After removal the solvent, the residue was purified by
preparative-TLC using EtOAc-Hexane (7:3) to obtain 6.9 mg of
4.4. Preparation of jaborosalactone 5 derivatives
4.4.1. Preparation of (23R)-21-acetyloxy-12
12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (3) and (23R)-
21-acetoxy-12 -hydroxy-1,22-dioxo-12,23-cicloergostan-3,5,17,24-
tetraen-26,23-olide (4)
DMAP in catalytic amount and excess of acetic anhydride (1 mL,
0.0106 mol) were added to a solution of jaborosalactone 5 (14.3 mg,
b-hydroxy-1,22-dioxo-
compound 5 (29%) as a white amorphous solid; [
a]21D: ꢁ165.6 (c 0.15,
(log ε): 226 (4.51) nm., IR (film) nmax: 3439,
MeOH), UV (MeOH) lmax
b
2969, 2921, 1763, 1726, 1689, 1601, 1272, 1019, 717 cmꢁ1. 1H NMR
(CDCl3, 400.13 MHz): 7.91 brd (2H, J ¼ 8.4 Hz, H-20/H-60), 7.46 m
(1H, H-40), 7.33 m (2H, H-30/H-50), 6.67 dd (1H, J ¼ 10.0,5.0,2.5 Hz,
H-3), 5.75 dd (1H, J ¼ 10.0,2.5 Hz, H-2), 5.47 brd (1H, J ¼ 6.0 Hz, H-
6), 4.98 d (1H, J ¼ 12.2 Hz, H-21a), 4.92 d (1H, J ¼ 12.2 Hz, H-21b),
0.031 mmol) in pyridine (100 mL). The reaction mixture was stirred
for 24 h at room temperature. The reaction mixture was concen-
trated and the residue was subjected to preparative TLC purification
using EtOAc to yield 3.5 mg of compound 3 (22.4%) and 1.4 mg of
compound 4 (9%).
3.19 brd (1H, J ¼ 21.0 Hz, H-4
2.64 m (2H, H2-16), 2.44 dd (1H, J ¼ 14.0, 4.0 Hz, H-11
(1H, H-14), 2.20 s (3H, H3-28), 1.961 m (1H, H-7 ), 1.960 s (3H, H3-
27), 1.81 m (1H, H-9), 1.80 m (1H, H-15 ), 1.67 m (1H, H-7 ), 1.61 m
(1H, H-15 ), 1.47 m (1H, H-11 ), 1.44 m (1H, H-8), 1.09 s (3H, H3-
b
), 2.77 dd (1H, J ¼ 21.0,5.0 Hz, H-4
a),
), 2.37 m
a
b
a
a
b
b
4.4.1.1. (23R)-21-acetyloxy-12
cycloergostan-2,5,17,24-tetraen-26,23-olide (3). White amorphous
solid; [ 21D: ꢁ86.2 (c 0.23, MeOH), UV (MeOH) max (log ε): 209 (3.77) nm.,
b-hydroxy-1,22-dioxo-12,23-
19), 1.087 s (3H, H3-18). 13C NMR (CDCl3 100,03 MHz): 202.9 (C, C-
1), 191.7 (C, C-22), 172.8 (C, C-26), 168.7 (C, C-17) 166.5 (C, COO-
21), 159.5 (C, C-24), 144.9 (CH, C-3), 135.2 (C, C-5), 133.6 (CH, C-
40), 130.0 (C, C-10), 129.5 (CH, C-20/C-60), 128.2 (CH, C-30/C-50),
128.2 (C, C-25), 127.6 (CH, C-2), 124.0 (CH, C-6), 124.1 (C, C-20),
91.1 (C, C-23), 75.6 (C, C-12), 58.8 (CH2, C-21), 49.7 (C, C-10), 48.8
(C, C-13), 46.7 (CH, C-14), 40.6 (CH, C-9), 34.9 (CH2, C-11), 33.1
(CH2, C-4), 31.9 (CH, C-8), 28.9 (CH2, C-7), 25.7 (CH2, C-16), 23.1
(CH2, C-15), 18.3 (CH3, C-19), 15.8 (CH3, C-28), 14.4 (CH3, C-18), 8.8
(CH3, C-27). HRESIMS m/z [MþNa]þ 591.2361 (calcd for
a
]
l
IR (film) nmax: 3364, 2960, 2922, 1737, 1670, 1237, 1010, 542,
515 cmꢁ1. 1H NMR (CDCl3. 400.13 MHz): 6.67 ddd (1H, J ¼ 10.0, 5.0,
2.5 Hz, H-3), 5.75 dd (1H, J ¼ 10.0,2.5 Hz, H-2), 5.48 brd (1H,
J ¼ 5.9 Hz, H-6), 4.72 d (1H, J ¼ 12.2 Hz, H-21a), 4.64 d (1H,
J ¼ 12.2 Hz, H-21b), 3.19 brd (1H, J ¼ 21.0 Hz, H-4
b
), 2.78 dd (1H,
), 2.59 m (2H, H2-16), 2.43 m (1H, J ¼ 14.0,
), 2.35 m (1H, H-14), 2.20 s (3H, H3-28), 1.96 s (3H, H3-
J ¼ 21.0,5.0 Hz, H-4
3.6 Hz, H-11
27), 1.96 m (1H, H-7
1.80 m (1H, H-9), 1.67 m (1H, H-7
H-8), 1.45 m (1H, H-11
), 1.09 s (3H, H3-19), 1.07 s (3H, H3-18). 13
a
a
b), 1.95 s, (3H, CH3COO-21), 1.80 m (1H, H-15
a
),
C
35H36O7Na, 591.2359).
a), 1.63 m (1H, H-15
b
), 1.45 m (1H,
b
C
NMR (CDCl3 100.03 MHz): 203.0 (C, C-1), 191.6 (C, C-22), 172.9 (C, C-
26), 171.0 (C, CH3COO-21), 168.7 (C, C-17), 159.5 (C, C-24), 145.0 (CH,
C-3), 135.3 (C, C-5), 128.4 (C, C-25), 127.7 (CH, C-2), 124.1 (CH, C-6),
123.5 (C, C-20), 91.1 (C, C-23), 75.6 (C, C-12), 58.4 (CH2, C-21), 49.8
(C, C-10), 48.9 (C, C-13), 46.7 (CH, C-14), 40.7 (CH, C-9), 35.1 (CH2, C-
11), 33.3 (CH2, C-4), 32.0 (CH, C-8), 29.1 (CH2, C-7), 25.7 (CH2, C-16),
23.3 (CH2, C-15), 20.9, (CH3, CH3CO-21), 18.4 (CH3, C-19), 15.9 (CH3,
C-28), 14.4 (CH3, C-18), 9.0 (CH3, C-27). HRESIMS m/z [MþNa]þ
529.2219 (calcd for C30H34O7Na, 529.2226).
4.4.3. Preparation of (23R)-21-p-bromobenzoyloxy-12
1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (6)
Triethylamine (153 L, 1.1 mmol), p-bromobenzoyl chloride
(15 mg, 0.07 mmol) and DMAP in catalytic amount were added to a
solution of jaborosalactone 5 (20.5 mg, 0.044 mmol) in dry CH2Cl2
(3 mL). The reaction mixture was stirred at room temperature
under nitrogen until disappearance of the starting material (1 h).
After removing the solvent, the residue was purified by
preparative-TLC using EtOAc to obtain 18.7 mg of compound 6
b-hydroxy-
m
(65.5%) as a white amorphous solid; [a]21D: ꢁ104.3 (c 0,30, MeOH); UV
4.4.1.2. (23R)-21-acetyloxy-12
cicloergostan-3,5,17,24-tetraen-26,23-olido (4). White amorphous
b
-hydroxy-1,22-dioxo-12,23-
max (log ε): 242 (4.35), 228 (4.31) nm.; IR (film) nmax: 3484,
(MeOH) l
2957, 2926, 2857, 1732, 1668, 1268, 1012, 757 cmꢁ1. 1H NMR (CDCl3,
400.13 MHz): 7.83 d (2H, J ¼ 8.5 Hz, H-20/H-60), 7.54 d (2H,
J ¼ 8.5 Hz, H-30/H-50), 6.74 ddd (1H, J ¼ 9.6,4.5,2.2 Hz, H-3), 5.82 brd
(1H, J ¼ 9.6 Hz, H-2), 5.54 brs (1H, H-6), 5.04 d (1H, J ¼ 12.2 Hz, H-
solid; [a]21D: ꢁ51.4 (c 0.09, MeOH), UV (MeOH) max (log ε): 231 (4,51) nm.,
l
IR (film) nmax: 3424, 2916, 1737, 1709, 1681, 1239, 1010, 848 cmꢁ1. 1H
NMR (CDCl3, 400.13 MHz): 5.96 brd (1H, J ¼ 9.8 Hz, H-4), 5.53 m
(1H, H-6), 5.50 m (1H, H-3), 4.72 d (1H, J ¼ 12.2 Hz, H-21a), 4.63 d
(1H, J ¼ 12.2 Hz, H-21b), 3.11 brd (1H, J ¼ 21.0 Hz, H-2a), 2.65 m (1H,
H-2b), 2.60 m (2H, H2-16), 2.37 m (1H, H-14), 2.15 s (3H, H3-28),
21a), 4.96 d (1H J ¼ 12.2 Hz, H-21b), 3.25 brd (1H, J ¼ 21.0 Hz, H-4
b),
2.84 dd (1H, J ¼ 21.0,5.0 Hz, H-4a), 2.27 s (3H, H3-28), 2.03 s (3H,
H3-27), 1.17 s (3H, H3-19), 1.15 s (3H, H3-18). 13C NMR (CDCl3
100.03 MHz): 202.8 (C, C-1),191.6 (C, C-22),172.7 (C, C-26),168.8 (C,
C-17), 165.7 (C, COO-21), 159.4 (C, C-24), 145.0 (CH, C-3), 135.3 (C, C-
5), 131.7 (CH, C-20/C-60), 131.2 (CH, C-30/C-50), 128.9 (C, C-10), 128.3
(C, C-25), 128.0 (C, C-40), 127.7 (CH, C-2), 124.0 (CH, C-6), 123.8 (C, C-
20), 91.1 (C, C-23), 75.5 (C, C-12), 59.1 (CH2, C-21), 49.8 (C, C-10),
48.9 (C, C-13), 46.8 (CH, C-14), 40.6 (CH, C-9), 35.1 (CH2, C-11), 33.2
(CH2, C-4), 32.0 (CH, C-8), 29.0 (CH2, C-7), 25.8 (CH2, C-16), 23.3
(CH2, C-15), 18.4 (CH3, C-19), 15.9 (CH3, C-28), 14.4 (CH3, C-18), 9.0
2.12 m (1H, H-7
CH3COO-21), 1.93 m (1H, H-9), 1.79 m (1H, H-15
), 1.63 m (1H, H-15 ), 1.53 m (1H, H-8), 1.30 m (1H, H-11b
b), 1.99 m (1H, H-11
a
), 1.97 s (3H, H3-27), 1.94 s, (3H,
), 1.74 m (1H, H-
), 1.21 s
a
7
a
b
(3H, H3-19), 1.07 s (3H, H3-18). 13C NMR (CDCl3 100.03 MHz): 209.3
(C, C-1), 191.4 (C, C-22), 172.9 (C, C-26) 170.8 (C, CH3COO-21), 168.6
(C, C-17), 159.3 (C, C-24), 140.0 (C, C-5), 129.1 (CH, C-4), 128.4 (C, C-
25), 126.2 (CH, C-6), 123.8 (C, C-20), 121.5 (CH, C-3), 90.8 (C, C-23),
75.4 (C, C-12), 58.2 (CH2, C-21), 51.1 (C, C-10), 49.1 (C, C-13), 46.9

76
M.E. García et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
(CH3, C-27). HRESIMS m/z [MþNa]þ 669.1468 (calcd for
C-15), 19.8 (CH3, C-19), 15.9 (CH3, C-28), 14.4 (CH3, C-18), 9.0 (CH3,
C-27).
C
35H35O7NaBr, 669.1464).
4.4.4. Preparation of (23R)-21-furoyloxy-12
12,23-cicloergostan-2,5,17,24-tetraen-26,23-olide (7)
Triethylamine (240 L, 1.7 mmol), furoyl chloride (134
1.3 mmol) and catalytic amount of DMAP were added to a so-
lution of jaborosalactone 5 (19.8 mg, 0.043 mmol) in dry CH2Cl2
(3 mL). The reaction mixture was stirred at room temperature
under nitrogen until disappearance of the starting material (1 h).
After removal of the solvent, the residue was purified by
preparative-TLC using EtOAc to obtain 10.2 mg of compound 7
b-hydroxy-1,22-dioxo-
4.4.6. Preparation of (23R)-21-lauroyloxy-12
12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (9)
Triethylamine (188 L, 1.4 mmol), lauroyl chloride (159
0.7 mmol) and catalytic amount of DMAP in were added to a
solution of jaborosalactone 5 (20.7 mg, 0.045 mmol) in dry
CH2Cl2 (3 mL). The reaction mixture was stirred at room tem-
perature under nitrogen until disappearance of the starting ma-
terial (1 h). After removing the solvent, the residue was purified
by preparative-TLC using CH2Cl2:MeOH (9.7:0.3) to obtain
11.5 mg of compound 9 (40%) as a white amorphous solid;
b-hydroxy-1,22-dioxo-
m
mL,
m
mL,
(43%) as a white amorphous solid; [
a]21D:
ꢁ165 (c 0.20, MeOH), UV
max (log ε): 250 (4.38), 226 (4.34) nm., IR (film) nmax: 3473,
(MeOH)
l
[a]21D: ꢁ336.7 (c 0.15, CHCl3), UV (CHCl3) lmax (log ε): 229 (5.51), IR
2963, 1736, 1684, 1296, 1118, 1013, 758 cmꢁ1 1H NMR (CDCl3,
.
(film) nmax: 3497, 2923, 2854, 1742, 1712, 1463, 1111, 723 cmꢁ1. 1H
NMR (CDCl3, 400.13 MHz): 6.67 ddd (1H, J ¼ 10.0, 5.0, 2.6 Hz, H-
3), 5.74 dd (1H, J ¼ 10.0,3.4 Hz, H-2), 5.47 m (1H, H-6), 4.72 d (1H,
J ¼ 12.2 Hz, H-21a), 4.64 d (1H, J ¼ 12.2 Hz, H-21b), 3.18 brd (1H,
400.13 MHz): 7.58 dd (1H, J ¼ 1.8, 0.9 Hz, H-50), 7.14 dd (1H,
J ¼ 3.7, 0.9 Hz, H-30), 6.76 ddd (1H, J ¼ 10.0, 5.0, 2.6 Hz, H-3), 6.49
dd (1H, J ¼ 3.7, 1.8 Hz, H-40), 5.81 dd (1H, J ¼ 10.0, 3.3 Hz, H-2),
5.54 dt (1H, J ¼ 6.1, 2.1 Hz, H-6), 5.01 dd (1H, J ¼ 12.4, 1.3 Hz, H-
21a), 4.95 d (1H, J ¼ 12.4 Hz, H-21b), 3.25 bd (1H, J ¼ 21.0 Hz, H-
H-4
b
), 2.76 m (1H, H-4
a
), 2.57 m (2H, H-16), 2.41 m (1H, H-11
), 1.94 brs
), 1.66 m (1H, H-
), 1.43 m (1H, H-8),
a),
2.32 m (1H, H-14), 2.19 s (3H, H3-28), 1.95 m (1H, H-7
(3H, H3-27), 1.78 m (1H, H-9), 1.78 m (1H, H-15
), 1.61 m (1H, H-15 ), 1.44 m (1H, H-11
b
4
b
), 2.84 dd (1H, J ¼ 21.0, 5.0 Hz, H-4
dd (1H, J ¼ 11.2, 2.4 Hz, H-16
11 ), 2.45 m (1H, H-14), 2.29 bs (3H, H3-28), 2.050 bs (3H, H3-27),
2.047 m (1H, H-7 ), 1.93 m (1H, H-15 ), 1.90 m (1H, H-9), 1.756 m
(1H, H-7 ), 1.75 m (1H, H-15 ), 1.56 m (1H, H-11 ), 1.54 m (1H, H-
a
), 2.78 m (1H, H-16
a), 2.71
a
b
), 2.53 dd (1H, J ¼ 13.8, 3.9 Hz, H-
7
a
b
b
a
1.22e2.18 m (20H, CH2 ꢀ 10, H2-20-110), 1.09 s (3H, H3-19), 1.05 s
(3H, H3-18), 0.81 t (3H, H3-120). 13C NMR (CDCl3 100.03 MHz):
202.8 (C, C-1), 191.5 (C, C-22), 173.8 (C, COO-21), 172.7 (C, C-26),
168.6 (C, C-17), 159.4 (C, C-24), 145.0 (CH, C-3), 135.2 (C, C-5),
128.3 (C, C-25), 127.7 (CH, C-2), 124.1 (CH, C-6), 124.1 (C, C-20),
91.1 (C, C-23), 75.4 (C, C-12), 58.2 (CH2, C-21), 49.8 (C, C-10), 48.9
(C, C-13), 46.9 (CH, C-14), 40.7 (CH, C-9), 35.0 (CH2, C-11),
34.1e22.6 (CH2x10, C-20-110), 33.3 (CH2, C-4), 31.9 (CH, C-8), 29.1
(CH2, C-7), 25.7 (CH2, C-16), 23.3 (CH2, C-15), 18.4 (CH3, C-19),
15.9 (CH3, C-28), 14.4 (CH3, C-18), 14.0 (CH3, C-120), 8.8 (CH3, C-
27). HRESIMS m/z [MþNa]þ 669.3758 (calcd for C40H54O7Na,
669.3767).
b
a
a
b
b
8), 1.19 s (3H, H3-19), 1.17 s (3H, H3-18). 13C NMR (CDCl3
100.03 MHz): 202.9 (C, C-1), 191.4 (C, C-22), 172.8 (C, C-26), 169.3
(C, C-17), 159.4 (C, C-24), 158.5 (C, COO-21), 146.2 (CH, C-50),
144.8 (CH, C-3), 144.2 (C, C-20), 135.2 (C, C-5), 128.1 (C, C-25),
127.5 (CH, C-2), 124.0 (CH, C-6), 123.5 (C, C-20), 118.0 (CH, C-30),
111.4 (CH, C-40), 90.9 (C, C-23), 75.4 (C, C-12), 58.6 (CH2, C-21),
49.7 (C, C-10), 48.9 (C, C-13), 46.5 (CH, C-14), 40.5 (CH, C-9), 34.8
(CH2, C-11), 33.0 (CH2, C-4), 31.9 (CH, C-8), 29.1 (CH2, C-7), 25.6
(CH2, C-16), 23.0 (CH2, C-15), 18.2 (CH3, C-19), 15.7 (CH3, C-28),
14.3 (CH3, C-18), 8.7 (CH3, C-27). HRESIMS m/z [MþNa]þ 581.2158
(calcd for C33H34O8Na, 581.2151).
4.4.7. Preparation of (23R)-21-pivaloyloxy-12
dioxo-12,23-cicloergostan-2,5,17,24-tetraen-26,23-olide (10)
Triethylamine (30 L, 0.2 mmol), pivaloyl chloride (17
0.13 mmol) and catalytic amount of DMAP were added to a solu-
tion of jaborosalactone 5 (20.9 mg, 0.045 mmol) in dry CH2Cl2
(3 mL). The reaction mixture was stirred at room temperature for
b-hydroxy-1,22-
4.4.5. Preparation of (23R)-12
cycloergostan-3,5,17,24-tetraen-26,23-olide (8)
Dimethyl carbamoyl chloride (6.2 L, 0.07 mmol), triethylamine
(18.8 L, 0.14 mmol), and catalytic amount of DMAP were added to
a solution of jaborosalactone 5 (21.1 mg, 0.045 mmol) in dry
CH2Cl2 (3 mL). The reaction mixture was stirred at room temper-
ature under nitrogen for 24 h. Then, an additional amount of
b,21-dihydroxy-1,22-dioxo-12,23-
m
mL,
m
m
48 h, and then additional aliquots of triethylamine (300
mL,
2 mmoL) and pivaloyl chloride (136 L, 1 mmol) were added. After
m
triethylamine (67
m
L, 0.5 mmol) and acid chloride (27
m
L,
completion of reaction, the solvent was removed and the residue
was purified by preparative-TLC using EtOAc:Hexane (7:3) to
obtain 5.4 mg of compound 10 (22%) as a white amorphous solid;
0.3 mmol) were added, and the reaction mixture was left for 24 h.
The solvent was removed and the residue was purified by
preparative-TLC using CH2Cl2:MeOH (9.7:0.3) to obtain 5 mg of a
mixture which could not be separated neither by normal TLC
phase nor by reversed TLC phase. The 1H NMR spectrum of this
mixture indicated that it consisted of jaborosalactone 5 and 8 in a
1:2 ratio. 1H NMR (CDCl3, 400.13 MHz): 5.96 brd (1H, J ¼ 9.7 Hz, H-
4), 5.54 m (1H, H-3), 5.51 m (1H, H-6), 4.27 d (1H, J ¼ 12.5 Hz, H-
21a), 4.14 d (1H, J ¼ 12.5 Hz, H-21b), 3.12 brd (1H, J ¼ 20.5 Hz, H-
2a), 2.65 dd (1H, J ¼ 20.5,4.6 Hz, H-2b), 2.55 m (2H, H2-16), 2.43
[a]21D: ꢁ116.0 (c 0.12, MeOH), UV (MeOH) max (log ε): 225.4 (3.82) nm., IR
l
(film) nmax: 3442, 2961, 2930, 1732, 1457, 1156, 1009 cmꢁ1. 1H NMR
(CDCl3, 400.13 MHz): 6.67 ddd (1H, J ¼ 10.0, 5.0, 2.5 Hz, H-3), 5.75
dd (1H, J ¼ 10.0, 2.5 Hz, H-2), 5.48 m (1H, H-6), 4.69 m (2H, H2-21),
3.19 brd (1H, J ¼ 21.0 Hz, H-4
b
), 2.77 (1H, J ¼ 21.0,5.0 Hz, H-4
a
),
2.61 m (2H, H2-16), 2.43 (1H, J ¼ 14.0, 4.0 Hz, H-11
H-14), 2.20 s (3H, H3-28), 1.95 s (3H, H3-27), 1.95 m (1H, H-7
1.791 m (1H, H-9), 1.790 m (1H, H-15 ), 1.68 m (1H, H-7
(1H, H-15 ), 1.53 m (1H, H-8), 1.44 m (1H, H-11 ), 1.09 s (3H, H3-
a
), 2.32 m (1H,
b
),
a
a), 1.61 m
dd (1H, J ¼ 13.9, 3.9 Hz, H-11
28), 1.97 s (3H, H3-27), 1.96 m (1H, H-7
(1H, H-15 ), 1.65 m (1H, H-7 ), 1.62 m (1H, H-15
11
), 1.43 m (1H, H-8), 1.22 s (3H, H3-19), 1.07 s (3H, H3-18). 13C
a
), 2.32 m (1H, H-14), 2.18 s (3H, H3-
), 1.94 m (1H, H-9), 1.77 m
), 1.45 m (1H, H-
b
b
b
19), 1.07 s (9H, H3-20/H3-30/H3-40), 1.06 s (3H, H3-18). 13C NMR
(CDCl3 100.03 MHz): 202.9 (C, C-1), 191.5 (C, C-22), 178.3 (C, COO-
21), 172.7 (C, C-26), 167.7 (C, C-17), 159.4 (C, C-24), 144.9 (CH, C-3),
135.2 (C, C-5), 128.2 (C, C-25), 127.6 (CH, C-2), 124.4 (C, C-20), 124.0
(CH, C-6), 91.0 (C, C-23), 75.4 (C, C-12), 58.0 (CH2, C-21), 49.7 (C, C-
10), 48.5 (C, C-13), 46.4 (CH, C-14), 40.5 (CH, C-9), 38.7 (C, C-10),
35.0 (CH2, C-11), 33.1 (CH2, C-4), 30.4 (CH, C-8), 29.9 (CH2, C-7),
26.9 (CH3 ꢀ 3, C-20/C-30/C-4), 25.6 (CH2, C-16), 23.2 (CH2, C-15),
18.3 (CH3, C-19), 15.8 (CH3, C-28), 14.2 (CH3, C-18), 8.9 (CH3, C-27).
a
a
b
b
NMR (CDCl3 100.03 MHz): 209.1 (C, C-1), 193.4 (C, C-22), 172.8 (C,
C-26), 166.3 (C, C-17), 159.4 (C, C-24), 140.2 (C, C-5), 129.1 (CH, C-
4), 128.4 (C, C-25), 127.4 (C, C-20), 126.2 (CH, C-3), 121.4 (CH, C-6),
91.1 (C, C-23), 75.5 (C, C-12), 58.7 (CH2, C-21), 51.5 (C, C-10), 48.4
(C, C-13), 46.9 (CH, C-14), 39.2 (CH2, C-2), 39.1 (CH, C-9), 35.0 (CH2,
C-11), 32.0 (CH, C-8), 29.1 (CH2, C-7), 25.2 (CH2, C-16), 23.3 (CH2,

M.E. Garc
í
a et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
77
HRESIMS m/z [MþNa]þ 571.2666 (calcd for C33H40O7Na,
14.0 (CH3, C-18), 8.6 (CH3, C-27). aAssignments may be inter-
changed. HRESIMS m/z [MþNa]þ 615.2561 (calcd for C34H40O9Na,
615.2570).
571.2672).
4.4.8. Preparation of (23R)-21-(2-oxo-cyclopentane-carboxyl)-12
hydroxy-1,22-dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-
olide (11) and (23R)-21-adipoyloxy-12 -hydroxy-1,22-dioxo-12,23-
cycloergostan-2,5,17,24-tetraen-26,23-olide (12)
Triethylamine (20 L, 0.14 mmol), adipoyl chloride (4.3
b-
4.4.9. Preparation of (23R)-21-chloro-12
12,23-cycloergostan-2,5,17,24-tetraen-26,23- olide (13) and (23R)-
21-chloro-12 -hydroxy-1,22-dioxo-12,23-cycloergostan-3,5,17,24-
b-hydroxy-1,22-dioxo-
b
b
m
mL,
tetraen-26,23-olide (14)
0.03 mmol), and DMAP in catalytic amount were added to a solu-
tion of jaborosalactone 5 (26.7 mg, 0.057 mmol) in dry CH2Cl2
(5 mL). The reaction was carried out under argon atmosphere. The
mixture was stirred for 22 h at room temperature. Then, the reac-
tion mixture was refluxed for 4 h and an aliquot of acid chloride
Thionyl chloride (84 mL, 1.14 mmol) was added to a solution of
jaborosalactone 5 (26.4 mg, 0.057 mmol) in dry CH2Cl2 (3 mL). The
reaction was carried out under argon atmosphere. The reaction
mixture was refluxed during 20 h and an aliquot of acid chloride
(3 mL, 0.03 mmol) were added. After 1 h of additional reaction, the
(4.3
m
L, 0.03 mmol) were added. After 1 h of additional reaction, the
solvent was removed and the residue was purified by preparative
TLC using CH2Cl2:MeOH (9.7:0.3) to obtain 9.7 mg (35.3%) of
compound 13 and 1.5 mg (5.4%) of compound 14, which could not
be obtained in pure form and was decomposed after NMR
characterization.
solvent was removed and the residue was purified by preparative
TLC using CH2Cl2:MeOH (9.7:0.3) to obtain 4.6 mg of 11 (14%) and
3 mg of 12 (9%).
4.4.8.1. (23R)-21-(2-oxo-cyclopentane-carboxyl)-12
dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide
White amorphous solid, [a]21D: ꢁ156.5 (c 0.34, MeOH), UV (MeOH) max (log
ε): 226 (3.64) nm; IR (film) nmax: 3360, 2922, 2851, 1733, 1659, 1632,
1382, 1251, 1011, 771 cmꢁ1. 1H NMR (CDCl3, 400.13 MHz: 6.67 ddd
(1H, J ¼ 10.0, 5.0, 2.5 Hz, H-3), 5.74 dd (1H, J ¼ 10.0,2.5 Hz, H-2),
5.47 d (1H, J ¼ 5.7 Hz, H-6), 4.82 dd (1H, J ¼ 12.0, 9.7 Hz, H-21a),
b
-hydroxy-1,22-
(11).
4.4.9.1. (23R)-21-chloro-12
cycloergostan-2,5,17,24-tetraen-26,23-olide (13). White amorphous
solid, [ 21D: ꢁ258.7 (c 0.2, MeOH); UV (MeOH) max (log ε): 224 (4.58) nm;
b-hydroxy-1,22-dioxo-12,23-
l
a]
l
IR (film) nmax: 3469, 2965, 2925, 1742, 1666, 1382, 1250, 1009,
756 cm-1. 1H NMR (CDCl3, 400.13 MHz): 6.74 ddd (1H, J ¼ 10.0, 5.0,
2.5 Hz, H-3), 5.80 dd (1H, J ¼ 10.0,2.4 Hz, H-2), 5.54 brd (1H,
J ¼ 5.4 Hz, H-6), 4.28 d (1H, J ¼ 11.2 Hz, H-21a), 4.12 d (1H,
4.69 d (1H, J ¼ 12.0 Hz, H-21b), 3.17 brd (1H, J ¼ 21.0 Hz, H-4
b), 3.06
t (1H, J ¼ 8.9 Hz, H-10), 2.77 dd (1H, J ¼ 21.0, 5.0 Hz, H-4), 2.61 m
J ¼ 11.2 Hz, H-21b), 3.25 brd (1H, J ¼ 21.0 Hz, H-4
b
), 2.84 dd (1H,
(2H, H2-16), 2.40 dd (1H, J ¼ 14.0, 3.7 Hz, H-11
a
), 2.31 m (1H, H-14),
J ¼ 21.0; 5.0 Hz, H-4
a), 2.64 m (2H, H2-16), 2.49 m (2H, H2-11),
2.21 m (2H, H2-30), 2.17 m (2H, H2-50), 2.19 s (3H, H3-28), 2.02 m
(2H, H2-40), 1.95 s (3H, H3-27), 1.94 m (1H, H-7
), 1.80 m (1H, H-
15 ), 1.78 m (1H, H-9),1.65 m (1H, H-7 ), 1.60 m (1H, H-15 ), 1.45 m
(1H, H-11 ), 1.43 m (1H, H-8), 1.09 s (3H, H3-19), 1.07 s (3H, H3-18).
2.41 m (1H, H-14), 2.26 s (3H, H3-28), 2.02 s (3H, H3-27), 1.16 s (3H,
H3-19), 1.14 s (3H, H3-18). 13C NMR (CDCl3 100.03 MHz): 202.7 (C, C-
1), 190.9 (C, C-22), 172.6 (C, C-26), 168.5 (C, C-17), 159.2 (C, C-24),
144.9 (CH, C-3), 135.2 (C, C-5), 128.3 (C, C-20), 127.6 (CH, C-2), 125.6
(C, C-25), 124.0 (CH, C-6), 90.9 (C, C-23), 75.4 (C, C-12), 49.8 (C, C-
10), 48.9 (C, C-13), 46.6 (CH, C-14), 40.6 (CH, C-9), 37.1 (CH2, C-21),
35.0 (CH2, C-11), 33.2 (CH2, C-4), 31.9 (CH, C-8), 29.1 (CH2, C-7), 25.4
(CH2, C-16), 23.3 (CH2, C-15), 18.4 (CH3, C-19), 15.9 (CH3, C-28), 14.3
(CH3, C-18), 9.0 (CH3, C-27). HRESIMS m/z [MþNa]þ 505.1749 (calcd
for C28H31O5NaCl, 505.1758).
b
a
a
b
b
13C NMR (CDCl3 100.03 MHz): 212.8 (C, C-20), 202.8 (C, C-1), 191.2
(C, C-22), 172.8 (C, C-26), 169.7 (C, C-17), 169.6 (C, COO-21), 159.6 (C,
C-24), 144.9 (CH, C-3), 135.3 (C, C-5), 128.4 (C, C-25), 127.7 (CH, C-2),
124.1 (CH, C-6), 123.6 (C, C-20), 91.1 (C, C-23), 75.5 (C, C-12), 59.1
(CH2, C-21), 54.9 (CH, C-10), 49.8 (C, C-10), 48.9 (C, C-13), 46.7 (CH,
C-14), 40.6 (CH, C-9), 38.0 (CH2, C-30), 34.9 (CH2, C-11), 33.2 (CH2, C-
4), 32.0 (CH, C-8), 29.1 (CH2, C-7), 27.4 (CH2, C-50), 25.9 (CH2, C-16),
23.3 (CH2, C-15), 20.9 (CH2, C-40), 18.4 (CH3, C-19), 15.9 (CH3, C-28),
14.5 (CH3, C-18), 9.0 (CH3, C-27). HRESIMS m/z [MþNa]þ 597.2469
(calcd for C34H38O8Na, 597.2464).
4.4.9.2. (23R)-21-chloro-12b-hydroxy-1,22-dioxo-12,23-
cycloergostan-3,5,17,24-tetraen-26,23-olide (14). 1H NMR (CDCl3,
400.13 MHz): 5.95 brdd (1H, J ¼ 10.1, 2.0 Hz, H-4), 5.53 m (1H,H-6),
5.51 m (1H, H-3), 4.20 d (1H, J ¼ 11.4 Hz, H-21a), 4.05 d (1H,
J ¼ 11.4 Hz, H-21b), 3.01 brd (1H, J ¼ 20.8 Hz, H-2a), 2.65 m (1H, H-
2b), 2.60 m (2H, H2-16), 2.38 m (1H, H-14), 2.15 s (3H, H3-28),
4.4.8.2. (23R)-21-adipoyloxy-12
cycloergostan-2,5,17,24-tetraen-26,23-olide (12). White amorphous
solid, [ 21D: ꢁ167.6 (c 0.25, MeOH); UV (MeOH) max (log ε): 225 (4.36) nm;
b-hydroxy-1,22-dioxo-12,23-
a
]
2.15 m (1H, H-7
(1H, H-9),1.79 m (1H, H-15
1.29 m (1H, H-11
), 1.21 s (3H, H3-19), 1.07 s (3H, H3-18). 13C NMR
b
), 2.00 m (1H, H-11
a
), 1.97 s (3H, H3-27), 1.94 m
l
IR (film) nmax: 3363, 2923, 2854, 1730, 1660, 1411, 1261, 1013,
758 cm-1. 1H NMR (CDCl3, 400.13 MHz: 6.67 ddd (1H, J ¼ 10.0, 5.0,
2.5 Hz, H-3), 5.74 dd (1H, J ¼ 10.0, 2.5 Hz, H-2), 5.48 brd (1H,
J ¼ 5.9 Hz, H-6), 4.72 d (1H, J ¼ 12.0 Hz, H-21a), 4.67 d (1H,
a
),1.76 m (1H, H-7
a
),1.67 m (1H, H-15 ),
b
b
(CDCl3 100.03 MHz): 209.2 (C, C-1), 190.7 (C, C-22), 172.6 (C, C-26),
168.2 (C, C-17), 158.9 (C, C-24), 140.3 (C, C-5), 128.9 (CH, C-4), 128.5
(C, C-25), 126.1 (CH, C-6), 125.8 (C, C-20), 121.4 (CH, C-3), 90.7 (C, C-
23), 75.2 (C, C-12), 51.3 (C, C-10), 49.1 (C, C-13), 46.7 (CH, C-14), 39.1
(CH2, C-2), 38.9 (CH, C-9), 36.9 (CH2, C-21), 34.0 (CH2, C-11), 30.4
(CH, C-8), 29.2 (CH2, C-7), 25.3 (CH2, C-16), 23.3 (CH2, C-15), 19.7
(CH3, C-19), 15.6 (CH3, C-28), 14.3 (CH3, C-18), 8.9 (CH3, C-27).
J ¼ 12.0 Hz, H-21b), 3.18 brd (1H, J ¼ 21.0 Hz, H-4
b
), 2.78 dd (1H,
), 2.55 m (1H, H-16 ), 2.41
), 2.31 m (1H, H-14), 2.26 m (2H, H-
10), 2.23 m (2H, H2-40), 2.19 s (3H, H3-28), 1.96 s (3H, H3-27), 1.96 m
J ¼ 21.0, 5.0 Hz, H-4
a
), 2.64 m (1H, H-16
a
b
dd (1H, J ¼ 14.0, 4.0 Hz, H-11
a
(1H, H-7
b
), 1.84 m (1H, H-15
a
), 1.77 m (1H, H-9), 1.68 m (1H, H-7
a
b
),
),
1.64 m (1H, H-15
), 1.56 m (4H, H2-20/H2-30), 1.44 m (1H, H-11
b
1.42 m (1H, H-8), 1.09 s (3H, H3-19), 1.07 s (3H, H3-18). 13C NMR
(CDCl3 100.03 MHz: 202.8 (C, C-1), 191.5 (C, C-22), 175.0 (C, C-50),
173.3 (C, C-26), 173.2 (C, COO-21), 168.8 (C, C-17), 159.8 (C, C-24),
145.1 (CH, C-3), 134.7 (C, C-5), 128.2 (C, C-25), 127.4 (CH, C-2), 123.9
(CH, C-6), 123.7 (C, C-20), 90.8 (C, C-23), 75.4 (C, C-12), 57.7 (CH2, C-
21), 49.6 (C, C-10), 48.6 (C, C-13), 46.3 (CH, C-14), 40.3 (CH, C-9),
34.7 (CH2, C-11), 33.7 (CH2, C-40), 33.3 (CH2, C-10), 32.9 (CH2, C-4),
31.6 (CH, C-8), 28.6 (CH2, C-7), 25.3 (CH2, C-16), 24.3a (CH2, C-20),
24.1a (CH2, C-30), 22.8 (CH2, C-15), 18.1 (CH3, C-19), 15.6 (CH3, C-28),
4.4.10. Preparation of (23R)-21-carbaldehyde-12b-hydroxy-1,22-
dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (15)
A solution of pyridinium chlorochromate (19.5 mg, 0.09 mmol in
CH2Cl2) was added to jaborosalactone 5 (27.6 mg, 0.059 mmol) and
dissolved in dry CH2Cl2 (3 mL). The reaction mixture was stirred
during 0.5 h at room temperature. After completion of the reaction,
the reaction mixture was filtered through florisil and washed with
ether (20 mL), CH2Cl2 (20 mL), and EtOAc (20 mL). After the solvent
elimination the residue was subjected to preparative-TLC

78
M.E. García et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
purification using CH2Cl2:MeOH (9:1) to yield 4.6 mg (16.7%) of
compound 15 as a white amorphous solid; [a]21D: ꢁ270 (c 0.15, MeOH);
(CH2, C-3), 26.0 (CH2, C-6), 25.6 (CH2, C-16), 24.7 (CH2, C-4), 22.8
(CH2, C-15), 15.8 (CH3, C-28), 14.7 (CH3, C-19), 14.5 (CH3, C-18), 12.0
(CH3, C-21), 8.5 (CH3, C-27). HRESIMS m/z [MþNa]þ 475.2468 (calcd
for C28H36O5Na, 475.2460).
max (log ε): 239 (3.71), 220 (3.73) nm; IR (film) nmax: 3467,
UV (MeOH)
l
2964, 2926, 1743, 1683, 1600, 1247, 1011, 756 cmꢁ1. 1H NMR (CDCl3,
400.13 MHz): 9.86 s (1H, H-21), 6.75 ddd (1H, J ¼ 10.0, 4.9, 2.4 Hz,
H-3), 5.81 dd (1H, J ¼ 10.0,2.4 Hz, H-2), 5.55 brd (1H, J ¼ 5.6 Hz, H-
4.4.12. Preparation of jaborosalactone 4
6), 3.26 brd (1H, J ¼ 21.4 Hz, H-4
b
), 2.87 m (2H, H2-16), 2.85 brd (1H,
Meta-chloroperoxybenzoic acid (18 mg, 0.05 mmol) and
NaHCO3 (16 mg, 0.19 mmol) were added to a solution of jabor-
osalactone 5 (20 mg, 0.043 mmol) in dry CH2Cl2 (5 mL). The reac-
tion mixture was stirred at room temperature for 46 h. Then CH2Cl2
was added and organic phase was washed with saturated aqueous
sodium thiosulfate. Organic phases were pooled, dried with MgSO4,
and concentrated to dryness. The residue was purified by prepar-
ative TLC using EtOAc to obtain 3.7 mg (18%) of jaborosalactone 4.
J ¼ 21.4 Hz, H-4
a
), 2.40 ddd (1H, J ¼ 19.0, 11.0, 7.0 Hz, H-14), 2.29 s
(3H, H3-28), 2.05 s (3H, H3-27), 1.19 s (3H, H3-19), 1.17 s (3H, H3-18).
13C NMR (CDCl3 100.03 MHz): 203.0 (C, C-1), 189.9 (C, C-22), 181.6
(C, C-21), 172.5 (C, C-26), 166.7 (C, C-17), 158.9 (C, C-24), 145.0 (CH,
C-3), 135.2 (C, C-5), 128.7 (C, C-20), 127.6 (CH, C-2), 126.1 (C, C-25),
124.0 (CH, C-6), 91.0 (C, C-23), 75.4 (C, C-12), 50.6 (C, C-13), 49.6 (C,
C-10), 46.3 (CH, C-14), 40.6 (CH, C-9), 35.4 (CH2, C-11), 33.2 (CH2, C-
4), 31.9 (CH, C-8), 29.0 (CH2, C-7), 28.2 (CH2, C-16), 23.5 (CH2, C-15),
18.5 (CH3, C-19), 15.9 (CH3, C-28), 14.4 (CH3, C-18), 9.0 (CH3, C-27).
HRESIMS m/z [M]þ. 462.2037 (calcd for C28H30O6, 462.2042).
4.4.13. Preparation of (23R)-4
12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (18) and (23R)-
,12 ,21-trihydroxy-1,22-dioxo-12,23-cycloergostan-2,4,17,24-
b, 12b, 21-trihydroxy-1,22-dioxo-
6b
b
4.4.11. Preparation of (23R)-12
b
-hydroxy-1,22-dioxo-12,23-
tetraen-26,23-olide (19)
cycloergostan-5,17,24-trien-26,23- olide (16) and (23R)-12
hydroxy-1,22-dioxo-12,23-cycloergostan-17,24-dien-26,23-olide
(17)
Jaborosalactone 5 (25.4 mg, 0.047 mmol) was dissolved in dry
THF (4 mL) and Pd/C (10%) in catalytic amount was added. An H2-
filled ball was connected to the reaction flask and the system was
closed under this atmosphere for 26 h. Then the solution was
filtered and the solvent was removed. The residue was purified by
preparative TLC using EtOAc:hexane (7:3) as eluent to yield 5.4 mg
(22%) of compound 16 and 5.1 mg (21%) of compound 17.
b
-
SeO2 (29 mg, 0.26 mmol) were added to a solution of jabor-
osalactone 5 (35.3 mg, 0.075 mmol) in dry CH2Cl2 (5 mL). The re-
action was carried out with stirring at room temperature under
argon atmosphere. After 24 h of reaction, 0.1 mmol of SeO2 were
added and the reaction mixture was refluxed for 45 h. Then the
reaction mixture was concentrated and the residue was subjected
to preparative TLC purification using EtOAc to yield 12 mg of
compound 18 (32.9%) and 12.8 mg (35,1%) of compound 19.
4.4.13.1. (23R)-4 ,21-trihydroxy-1,22-dioxo-12,23-
cycloergostan-2,5,17,24-tetraen-26,23-olide (18). White amor-
phous solid, [a]21D: ꢁ222.1 (c 0,28, MeOH); UV (MeOH) (log ε): 250
b
, 12
b
4.4.11.1. (23R)-12
b-hydroxy-1,22-dioxo-12,23-cycloergostan-5,17,24-
l
max
trien-26,23-olide (16). White amorphous solid, [
a
]
21D: ꢁ134.6 (c 0.13,
(4,31), 221 (4,50) nm; IR (film) nmax: 3438, 3017, 2964, 2927, 1741,
1668, 1216, 1012, 759 cmꢁ1. 1H NMR (CDCl3, 400.13 MHz): 6.66 dd
(1H, J ¼ 10.5, 4.3 Hz, H-3), 5.83 dd (1H, J ¼ 5.5,1.9 Hz, H-6), 5.81 dd
(1H, J ¼ 10.5,0.5 Hz, H-2), 4.55 brd (1H, J ¼ 4.3 Hz, H-4), 4.26 d (1H,
J ¼ 12.7 Hz, H-21a), 4.14 d (1H, J ¼ 12.7 Hz, H-21b), 2.56 m (2H, H2-
(log ε): 220 (3.99) nm; IR (film) nmax: 3411,
MeOH); UV (MeOH)
lmax
2928, 2869, 1739, 1706, 1677, 1249, 1011, 756 cmꢁ1. 1H NMR (CDCl3,
400.13 MHz): 5.39 brd (1H, J ¼ 6.0 Hz, H-6), 2.42 m (2H, H2-16),
2.42 m (1H, H-4b), 2.42 m (1H, H-2a), 2.27 m (1H, H-14), 2.17 m (1H,
H-2b), 2.14 s (3H, H3-28), 2.08 m (1H, H-4
1.92 s (3H, H3-27), 1.87 m (1H, H-11 ), 1.82 m (1H, H-9), 1.81 m (1H,
H-3a), 1.80 m (1H, H-15 ), 1.67 s (3H, H3-21), 1.61 m (1H, H-15 ),
1.56 m (1H, H-3b), 1.53 m (1H, H-7 ), 1.38 m (1H, H-8), 1.33 m (1H,
H-11
), 1.14 s (3H, H3-19), 1.02 s (3H, H3-18). 13C NMR (CDCl3
a
), 1.93 m (1H, H-7
b
),
16), 2.34 m (1H, H-11
2.09 m (1H, H-7
(1H, H-9), 1.69 m (1H, H-7
1.46 m (1H, H-11
), 1.31 s (3H, H3-19), 1.07 s (3H, H3-18). 13C NMR
a
), 2.32 m (1H, H-14), 2.18 brs (3H, H3-28),
), 1.95 brs (3H, H3-27), 1.83 m (1H, H-15 ), 1.75 m
), 1.65 m (1H, H-15 ), 1.51 m (1H, H-8),
a
b
a
a
b
a
b
a
b
b
(CDCl3 100.03 MHz: 201.9 (C, C-1), 193.9 (C, C-22), 172.7 (C, C-26),
166.0 (C, C-17), 159.8 (C, C-24), 142.7 (CH, C-3), 138.3 (C, C-5), 130.4
(CH, C-6), 128.4 (CH, C-2), 128.3 (C, C-25), 127.6 (C, C-20), 91.1 (C,
C-23), 75.3 (C, C-12), 68.9 (CH, C-4), 58.4 (CH2, C-21), 48.7 (C, C-
10), 48.5 (C, C-13), 46.7 (CH, C-14), 40.5 (CH, C-9), 34.4 (CH2, C-11),
31.3 (CH, C-8), 29.6 (CH2, C-7), 25.2 (CH2, C-16), 22.8 (CH2, C-15),
22.1 (CH3, C-19),15.6 (CH3, C-28), 14.3 (CH3, C-18), 8.9 (CH3, C-27).
HRESIMS m/z [MþNa]þ 503.2048 (calcd for C28H32O7Na,
503.2046).
100.03 MHz: 213.0 (C, C-1), 193.2 (C, C-22), 173.6 (C, C-26), 162.8 (C,
C-17), 160.3 (C, C-24), 140.6 (C, C-5), 127.9 (C, C-25), 124.5 (C, C-20),
122.1 (CH, C-6), 91.1 (C, C-23), 75.5 (C, C-12), 53.0 (C, C-10), 48.5 (C,
C-13), 46.7 (CH, C-14), 37.5 (CH, C-2), 40.2 (CH, C-9), 33.5 (CH2, C-
11), 30.6 (CH2, C-4), 30.3 (CH, C-8), 29.3 (CH2, C-7), 22.9 (CH2, C-15),
25.4 (CH2, C-16), 25.1 (CH, C-3), 18.3 (CH3, C-19), 11.9 (CH3, C-21),
15.5 (CH3, C-28), 14.1 (CH3, C-18), 8.6 (CH3, C-27). HRESIMS m/z
[MþNa]þ 473.2300 (calcd for C28H35O5Na, 473.2304).
4.4.11.2. (23R)-12
dien-26,23-olide (17). White amorphous solid, [
b
-hydroxy-1,22-dioxo-12,23-cycloergostan-17,24-
4.4.13.2. (23R)-6
cycloergostan-2,4,17,24-tetraen-26,23-olide (19). White amorphous
solid, [ 21D: ꢁ310.0 (c 0.18, MeOH); UV (MeOH) max (log ε): 232 (4.22) nm;
IR (film) nmax: 3437, 2925, 1739, 1657, 1012, 769 cmꢁ1
b,12b,21-trihydroxy-1,22-dioxo-12,23-
a]21D: ꢁ134.3 (c 0.23,
(log ε): 221 (4.12) nm; IR (film) nmax: 3469,
a]
MeOH); UV (MeOH)
lmax
l
2928, 2865, 1755, 1696, 1449, 1380, 1247, 1012, 757 cmꢁ1. 1H NMR
(CDCl3, 400.13 MHz: 2.43 t (2H, J ¼ 6.7 Hz, H2-16), 2.27 m (1H, H-
14), 2.14 m (1H, H-2a), 2.08 s (3H, H3-28), 2.07 m (1H, H-2b), 2.00 m
.
1H NMR
(CDCl3, 400.13 MHz): 6.82 dd (1H, J ¼ 9.7, 5.9 Hz, H-3), 6.05 d (1H,
J ¼ 5.9 Hz, H-4), 5.85 d (1H, J ¼ 9.7 Hz, H-2), 4.48 t (1H, J ¼ 2.6 Hz, H-
6), 4.22 d (1H, J ¼ 12.2 Hz, H-21a), 4.13 d (1H, J ¼ 12.2 Hz, H-21b),
(2H, H2-3), 1.89 m (1H, H-9), 1.76 m (1H, H-15
1.66 s (3H, H3-21), 1.62 m (1H, H-5), 1.57 m (1H, H-15
H-7 ), 1.50 m (1H, H-6a), 1.46 m (2H, H2-4), 1.40 m (1H, H-6b),
1.38 m (1H, H-8), 1.29 m (1H, H-11 ), 1.02 s (3H, H3-19), 0.98 s (3H,
H3-18), 0.92 m (1H, H-11
). 13C NMR (CDCl3 100.03 MHz: 215.7 (C,
a), 1.74 s (3H, H3-27),
b), 1.53 m (1H,
2.55 t (2H, J ¼ 6.0 Hz, H2-16), 2.32 dd (1H, J ¼ 14.4, 3.7 Hz, H-11
a),
a
2.17 m (1H, H-14), 2.16 s (3H, H3-28), 2.02 m (1H, H-8), 1.96 m (1H,
H-7 ), 1.79 s (3H, H3-27), 1.77 m (1H, H-15 ), 1.69 m (1H, H-15 ),
1.53 t (1H, J ¼ 14.0 Hz, H-11 ), 1.34 s (3H, H3-19), 1.30 m (1H, H-9),
1.23 m (1H, H-7
), 1.11 s (3H, H3-18). 13C NMR (CDCl3 100.03 MHz):
a
b
a
b
b
b
C-1), 192.2 (C, C-22), 173.2 (C, C-26), 163.0 (C, C-17), 162.6 (C, C-24),
126.4 (C, C-25), 124.2 (C, C-20), 91.4 (C, C-23), 74.6 (C, C-12), 52.3 (C,
C-10), 48.7 (C, C-13), 46.8 (CH, C-14), 45.1 (CH, C-5), 40.0 (CH, C-9),
37.5 (CH2, C-2), 34.4 (CH, C-8), 33.4 (CH2, C-11), 30.2 (CH2, C-7), 26.3
a
205.2 (C, C-1), 193.4 (C, C-22), 172.4 (C, C-26), 166.3 (C, C-17), 160.7
(C, C-24), 156.4 (C, C-5), 139.9 (CH, C-3), 127.4 (C, C-25), 127.2 (C, C-
20), 126.1 (CH, C-2), 119.1 (CH, C-4), 91.2 (C, C-23), 75.1 (C, C-12),

M.E. Garc
í
a et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
79
73.0 (CH, C-6), 58.3 (CH2, C-21), 52.7 (C, C-10), 48.5 (C, C-13), 46.1
(CH, C-14), 45.5 (CH, C-9), 37.5 (CH2, C-7), 32.9 (CH2, C-11), 29.9 (CH,
C-8), 25.0 (CH2, C-16), 23.1 (CH2, C-15), 15.7 (CH3, C-28), 19.0 (CH3,
C-19), 14.4 (CH3, C-18), 8.3 (CH3, C-27). HRESIMS m/z
[MþNaeH2O]þ 485.1933 (calcd for C28H30O6Na, 485.1940).
4.5. Biological assays
4.5.1. Materials
All starting materials were commercially available research-
grade chemicals and used without further purification. RPMI
1640 medium was purchased from Flow Laboratories (Irvine, UK),
fetal calf serum (FCS) from Gibco (Grand Island, NY), trichloroacetic
acid (TCA) and glutamine from Merck (Darmstadt, Germany), and
penicillin G, streptomycin, dimethyl sulfoxide (DMSO) and sulfo-
rhodamine B (SRB) from Sigma (St Louis, MO).
4.4.14. Preparation of (23R)-21-acetoxy-12b-hydroxy-1,22-dioxo-
12,23-cycloergostan-2,5,17,24-tetraen-26,23-lactame (20)
Jaborosalactone 5 (26 mg, 0.056 mmol) and NH4AcO (86.3 mg,
1.1 mmol) were dissolved in acetic acid (5 mL). The reaction
mixture was refluxed for 18 h. Then the reaction mixture was
concentrated and the residue was subjected to preparative TLC
purification using EtOAc to yield 15.9 mg (56.2%) of compound 20
4.5.2. Cells, culture and plating
The human solid tumor cell lines HBL-100 (breast), HeLa (cer-
vix), SW1573 (non-small cell lung), and WiDr (colon) 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.
as a white amorphous solid; [
a]21D: ꢁ211.5 (c 0.26, MeOH); UV (MeOH)
max (log ε): 224 (4.34) nm; IR (film) nmax: 3459, 3367, 2924, 2854,
l
1740, 1666, 1246, 1015, 758 cmꢁ1 1H NMR (CDCl3, 400.13 MHz):
.
ꢀ
Ruben P. Machín (HUGC Dr. Negrín, Las Palmas de Gran Canaria,
6.67 ddd (1H, J ¼ 10.0, 5.0, 2.4 Hz, H-3), 5.74 dd (1H, J ¼ 10.0,
2.4 Hz, H-2), 5.47 brd (1H, J ¼ 5.6 Hz, H-6), 4.71 d (1H, J ¼ 12.0 Hz,
H-21a), 4.63 d (1H, J ¼ 12.0 Hz, H-21b), 3.18 brd (1H, J ¼ 21.0 Hz, H-
Spain). Cells were maintained in 25 cm2 culture flasks in RPMI 1640
supplemented with 5% FBS and 2 mM L
-glutamine in a 37 ꢂC, 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 give the appropriate cell densities for inoculation
onto 96-well microtiter plates. Cells were inoculated in a volume of
4
b
), 2.77 dd (1H, J ¼ 21.0,5.0 Hz, H-4 ), 2.48 dd (1H, J ¼ 13.7,
a
3.3 Hz, H-11), 2.39 ddd (1H, J ¼ 19.0, 11.0, 7.0 Hz, H-14), 2.19 s (3H,
H3-28), 1.97 s (3H, CH3COO-21), 1.94 s (3H, H3-27), 1.09 s (3H, H3-
19), 1.06 s (3H, H3-18). 13C NMR (CDCl3 100.03 MHz): 202.8 (C, C-
1), 191.6 (C, C-22), 172.7 (C, C-26), 170.8 (C, CH3COO-21), 168.5 (C,
C-17), 159.3 (C, C-24), 144.9 (CH, C-3), 135.2 (C, C-5), 127.7 (CH, C-
2), 128.4 (C, C-20), 123.9 (C, C-25), 124.1 (CH, C-6), 90.6 (C, C-23),
75.6 (C, C-12), 58.3 (CH2, C-21), 49.7 (C, C-10), 48.8 (C, C-13), 46.7
(CH, C-14), 40.6 (CH, C-9), 34.9 (CH2, C-11), 31.9 (CH, C-8), 33.2
(CH2, C-4), 29.1 (CH2, C-7), 25.6 (CH2, C-16), 23.2 (CH2, C-15), 20.9
(CH3, CH3COO-21), 15.8 (CH3, C-28), 18.3 (CH3, C-19), 14.3 (CH3, C-
18), 9.0 (CH3, C-27). HRESIMS m/z [MþNa]þ 529.2219 (calcd for
100
mL per well at densities of 10 000 (HBL-100 and SW1573),
15 000 (HeLa), and 20 000 (WiDr) cells per well, based on their
doubling times.
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 maximum
test concentration. Control cells were exposed to an equivalent
concentration of DMSO (0.25% v/v, negative control). Each agent
was tested in triplicate at different dilutions in the range of
C
30H35NO6 Na, 529.2202).
4.4.15. Preparation of (22R,23S)-21,12
b
, 22a-trihydroxy-1,22-
dioxo-12,23-cycloergostan-2,5,17,24-tetraen-26,23-olide (21)
LiAlH4 (9.2 mg, 0.02 mmol) dissolved in 0.3 mL of ether was
added to a solution of jaborosalactone 5 (35 mg, 0.075 mmol) in
anhydrous ether (2 mL) and THF (1.5 mL). The reaction mixture was
stirred at room temperature. After 40 h of reaction, the reaction
mixture was refluxed for an additional 16 h. Then, ice and 1 mL of
distilled H2O were added. The aqueous phase was partitioned with
ether. The resulting organic phase was dried with anhydrous
Na2SO4. The filtrate was evaporated to dryness and purified by
preparative TLC using EtOAc to obtain 1.1 mg (3%) of compound 21
1e100
Drug incubation times comprised 48 h, after which time cells were
precipitated with 25 L ice-cold 50% (w/v) trichloroacetic acid and
mM. The drug treatment was started on day 1 after plating.
m
fixed for 60 min at 4 ꢂC. Then the SRB assay was performed. The
optical density (OD) of each well was measured at 492 nm, using
BioTek's PowerWave XS Absorbance Microplate Reader. Values
were corrected for background OD from wells containing only
medium. The percentage growth (PG) was calculated in relation to
untreated control cells (C) at each of the drug concentration levels
based on the difference in OD at the start (T0) and end of drug
exposure (T), according to NCI formulas1. Therefore, if T is greater
than or equal to T0, the calculation is 100 ꢀ [(T e T0)/(C e T0)]. If T is
less than T0, denoting cell killing, the calculation is 100 ꢀ [(T e T0)/
(T0)]. The effect is defined as percentage of growth, where 50%
growth inhibition (GI50) represents the concentration at which PG
is þ50. With these calculations, a PG value of 0 no difference from
the start of drug exposure, while negative PG values denote net cell
death.
as a white amorphous solid; [a]21D: ꢁ271 (c 0.04, MeOH); UV (MeOH)
lmax
(log ε): 209 (4.37) nm; IR (film) nmax: 3415, 2964, 2921, 1747, 1668,
1439, 1382, 1007, 757 cmꢁ1. 1H NMR (CDCl3, 400.13 MHz): 6.72 ddd
(1H, J ¼ 10.0, 5.0, 2.4 Hz, H-3), 5.81 dd(1H, J ¼ 10.0, 2.4 Hz, H-2),
5.53 brd (1H, J ¼ 5.6 Hz, H-6), 5.14 brs (1H, H-22), 4.39 d (1H,
J ¼ 12.1 Hz, H-21a), 4.165 d (1H, J ¼ 12.1 Hz, H-21b), 3.25 brd (1H,
J ¼ 21.0 Hz, H-4
J ¼ 14.0, 4.0 Hz, H-11
2.13 m (1H, H-14), 2.02 s (3H, H3-27), 1.98 m (1H, H-7
H-9), 1.74 m (1H, H-15 ), 1.66 m (1H, H-7 ), 1.53 m (1H, H-15
1.36 m (1H, H-11 ), 1.35 m (1H, H-8), 1.16 s (3H, H3-19), 0.97 s (3H,
b
), 2,82 dd (1H, J ¼ 21.0,5.0 Hz, H-4
), 2.36 m (2H, H2-16), 2.21 s (3H, H3-28),
), 1.78 m (1H,
),
a), 2.51 dd (1H,
a
b
a
a
b
b
H3-18). 13C NMR (CDCl3 100.03 MHz): 203.0 (C, C-1), 174.2 (C, C-26),
160.8 (C, C-24), 144.7 (CH, C-3), 144.6 (C, C-17), 135.2 (C, C-5), 127.7
(CH, C-2), 127.5 (C, C-25), 124.3 (CH, C-6), 123.9 (C, C-20), 93.5 (C, C-
23), 76.3 (C, C-12), 69.2 (CH, C-22), 60.3 (CH2, C-21), 49.8 (C, C-10),
47.9 (CH, C-14), 47.8 (C, C-13), 35.8 (CH2, C-11), 41.0 (CH, C-9), 33.2
(CH2, C-4), 31.6 (CH, C-8), 29.1 (CH2, C-7), 23.7 (CH2, C-16), 23.0
(CH2, C-15), 18.3 (CH3, C-19), 14.7 (CH3, C-28), 12.6 (CH3, C-18), 8.8
(CH3, C-27). HRESIMS m/z [MþNa]þ 489.2257 (calcd for
4.5.4. Cell cycle analysis
Cells were seeded in six well plates at
a density of
2.5e5 ꢀ 105 cells/well. After 24 h, test compounds were added to
the respective well and incubated for an additional period of 24 h.
Cells were trypsinized, harvested, transferred to test tubes
(12 ꢀ 75 mm) and centrifuged at 1500 rpm for 10 min at 5 ꢂC. The
supernatant was discarded and the cell pellets were resuspended in
200 mL of cold PBS and fixed by the addition of 1 mL ice-cold 70%
C
28H34O6Na, 489.2253).
ethanol. Fixed cells were incubated overnight at ꢁ20 ꢂC followed by

80
M.E. García et al. / European Journal of Medicinal Chemistry 82 (2014) 68e81
centrifugation at 1500 rpm for 10 min. The cell pellets were
resuspended in 500 L PBS. Then, 5 L of DNAse-free RNAse (10 mg/
mL) was added, and the cell suspension was incubated in the dark
at 37 ꢂC for 30 min. After incubation, 5
L of propidium iodide
used to acquire mass spectra over the mass range m/z 65 000 to m/z
80 000. The signals from 300 laser shots were averaged per mass
spectrum.
m
m
m
(0.5%) was added. Flow cytometric determination of DNA content
(25 000 cells/sample) was analyzed using a FACSCalibur Flow Cy-
Acknowledgments
ꢀ
tometer (Becton Dickinson, San Jose, CA, USA). The fractions of the
This work has been financed by CONICET (Argentina), Ministerio
cells in G0/G1, S, and G2/M phase were analyzed using cell cycle
analysis software, ModFit LT 3.0 (Verity Software House, Topsham,
ME, USA).
ꢀ
de Ciencia y Tecnología de Cordoba (Argentina), SeCyT-UNC,
Spanish MINECO (SAF 2012-37344-C03-01), the EU Research Po-
tential (FP7-REGPOT-2012-CT2012-31637-IMBRAIN), the European
Regional Development Fund (FEDER), and the Spanish Instituto de
Salud Carlos III (PI11/00840). M.E.G. thanks CONICET (Argentina)
for the fellowship granted, and Prof. G. Barboza for plant recollec-
tion and identification. NMR assistance by G. Bonetto is gratefully
acknowledged.
4.6. Quinone reductase 1 assay
4.6.1. Cell cultures
Cell culture media and supplements were purchased from Gibco
(Carlsbad, CA, USA). Hepa 1c1c7 cells were obtained from ATCC
(Manassas, VA, USA). Mutant cell lines TAOc1 and BPrc1 were
supplied by Dr. J. P. Whitlock, Jr. (Department of Molecular Phar-
macology, Stanford University School of Medicine, Stanford, Cali-
fornia 94305, USA). All cells were maintained and passaged
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.045.
according to ATCC instructions in MEM-a containing 5% antibiotic-
antimycotic and 10% fetal bovine serum at 37 ꢂC in a 5% CO2
atmosphere.
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