Synthesis and cytotoxic activity evaluation of dihydrocucurbitacin B and cucurbitacin B derivatives

Article abstract of DOI:10.1016/j.bmc.2012.03.001

Two cucurbitacins, dihydrocucurbitacin B (1) and cucurbitacin B (2), which can be obtained in large amounts from the roots of Wilbrandia ebracteata and from the fruits of Luffa operculata, respectively, were used as starting materials for the preparation of a library of 29 semi-synthetic derivatives. The structural changes that were performed include the removal, modification or permutation of functional groups in rings A and B as well as in the side chain. All new semisynthetic compounds, as well as 1 and 2, were tested in vitro for their cytotoxic effects on non-small-cell lung cancer cells (A549 cells). Some of these compound displayed potent to moderate activity against A549 tumor cells, especially those cucurbitacin B derivatives which were modified at ring A.

Full text of DOI:10.1016/j.bmc.2012.03.001

Bioorganic & Medicinal Chemistry 20 (2012) 3016–3030
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and cytotoxic activity evaluation of dihydrocucurbitacin
B and cucurbitacin B derivatives
Karen Luise Lang a, , Izabella Thaís Silva b, Lara Almida Zimmermann b, Vanessa Rocha Machado b,
⇑
Marina Rodrigues Teixeira b, María Ivana Lapuh c, Mariana Alejandra Galetti c, Jorge Alejandro Palermo c,
Gabriela Myriam Cabrera c, Lílian Sibelle Campos Bernardes b, Cláudia Maria Oliveira Simões b,
Eloir Paulo Schenkel b, Miguel Soriano Balparda Caro a, Fernando Javier Durán c
a Programa de Pós-Graduação em Química, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil
b Programa de Pós-Graduação em Farmácia, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil
c UMYMFOR-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Two cucurbitacins, dihydrocucurbitacin B (1) and cucurbitacin B (2), which can be obtained in large
amounts from the roots of Wilbrandia ebracteata and from the fruits of Luffa operculata, respectively, were
used as starting materials for the preparation of a library of 29 semi-synthetic derivatives. The structural
changes that were performed include the removal, modification or permutation of functional groups in
rings A and B as well as in the side chain. All new semisynthetic compounds, as well as 1 and 2, were
tested in vitro for their cytotoxic effects on non-small-cell lung cancer cells (A549 cells). Some of these
compound displayed potent to moderate activity against A549 tumor cells, especially those cucurbitacin
B derivatives which were modified at ring A.
Received 27 December 2011
Revised 23 February 2012
Accepted 1 March 2012
Available online 15 March 2012
Keywords:
Cucurbitacins
Triterpenoids
Semisynthesis
Cytotoxic activity
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
mcl-1 and bcl-xL, and the cell cycle regulators cyclin D1 and c-
Myc.7 Thus, since the most of chemotherapeutic strategies aim to
In recent years, new chemical entities derived from natural
products have gained importance in the search for therapeutic
drugs. In this context, we have investigated the cucurbitacins as
natural product scaffolds for the preparation of new bioactive sub-
stances.1,2 The cucurbitacins are highly oxygenated triterpene
derivatives, based on the cucurbitane skeleton, 19-(10?9b)-
abeo-10-lanost-5-ene, which are known for their bitter taste.3
These compounds are predominantly found in different species
of the Cucurbitaceae family.4 The main components of this family
are cucurbitacins A–T, and there are hundreds of derivatives which
have been classified according to structural features in ring A, side
chain modifications and stereochemistry considerations. These
compounds can be found in free or glycosylated forms.3–5 Recent
works have reported that cucurbitacins B, D, E, I, Q, and related
compounds are active against different tumor cell lines, via sup-
pression of STAT3 (Signal Transducer and Activator of Transcrip-
tion-3) phosphorylation.6 STAT3 is constitutively activated in
multiple human cancers including ovarian, breast, prostate and
lung, playing a pivotal transcriptional role in cancer cell progres-
sion, differentiation and survival by up-regulating several genes,
including those that encode for anti-apoptotic proteins such as
initiate apoptosis, it is now generally accepted that STAT3 repre-
sents a valid target for novel anticancer drug design.8 Some authors
also reported that cucurbitacins can directly modulate the actin
cytoskeleton. Duncan9e demonstrated that cucurbitacin E acts as
a potent disruptor of cytoskeletal integrity by increasing the fila-
mentous or polymerized actin fraction in prostate carcinoma cells.
Other studies carried out with cucurbitacin B also showed the
aggregation of F-actin in various human cancer cell lines.9
In the literature there are three important publications on the
synthesis and modifications of cucurbitacins: the preparation of
hexanorcucurbitacin analogues by Ahn,10 the semisynthetic study,
biological evaluation and QSAR analysis by Halaweish11 and the first
approximation to the total synthesis of the cucurbitane core by
Jung.12 To the best of our knowledge, there are no studies ofthe prep-
aration of cucurbitacin derivatives in which structural changes were
made by removal, modification or permutation of functional groups
in order to explore the influence of modifications in the carbon skel-
eton, conformation, polarity or solubility on the biological activity.
Our research group is interested in the preparation of semisyn-
thetic analogues of bioactive compounds from natural sources with
the aim of improving their biological activity.13 We began our work
with two species of Cucurbitaceae, Wilbrandia ebracteata Cogn. and
Luffa operculata (L.) Cogn. which are known for the production of a
⇑
Corresponding author. Tel.: +55 48 37215076; fax: +55 48 37219350.
E-mail address: karenluise@gmail.com (K.L. Lang).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.001

K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
3017
wide variety of cucurbitacin derivatives, with predominance of
dihydrocucurbitacin B (1) and cucurbitacin B (2), Figure 1.
Both compounds have several oxygenated functional groups,
making them particularly suitable substrates for semisynthetic
modifications, as well as a challenge in terms of chemo- and regi-
oselectivity. Herein, we describe our results in the semisynthesis of
several cucurbitacin derivatives and the evaluation of their in vitro
cytotoxic activity against lung cancer cells (A549). We designed
analogues bearing modifications at positions 2, 3, 7, 16, 20, 22,
and 25 of cucurbitacin B and dihydrocucurbitacin B, as well as by
molecular simplification of the natural products. A preliminary
structure–activity relationship (SARs) is also discussed.
12 and 14 by reaction with 4-toluenesulfonyl chloride in the pres-
ence of DABCO at low temperatures.16 The secondary hydroxyl at
C-16 in compound 12 was acetylated to yield 13 under standard
conditions. These intermediates were treated under SN2 conditions
with a variety of nucleophilic reagents: sulfur nucleofiles like thio-
phenol to give compounds 15 and 16, thioacetate to yield com-
pound 17 and thiourea to obtain 18 and 19 aminothiazoles under
microwave conditions. All the above mentioned products were
synthesized with reasonable yields, between 57 and 84%. Analysis
of the 1H NMR spectra of the products revealed that the coupling
constants of H-2 were identical as in the starting substances
(J = 5.0, 14.0 Hz). Confirmatory NOESY experiments were per-
formed in the case of the thiophenol 15. A NOE correlation was ob-
served between H-2 (4.07 ppm) and the C-28 methyl (1.29 ppm),
2. Results and discussion
2.1. Chemistry
which are both located at the
a face of the molecule. These facts
indicate that the substituent at C-2 is at the b face and that inver-
sion of configuration at C-2 did not take place. It is believed that
the presence of a vicinal carbonyl group at C-3 was responsible
for this result, leading to the more stable intermediate with a pseu-
do-equatorial substituent at C-2 due to enolization under the reac-
tion conditions.
Reaction of sulfonates 12–14 with (Bu)4NBr yielded the bromi-
nated analogues 20 and 21, while treatment with sodium azide in
DMF, afforded the enamino ketones 22, 23 and 24 (Scheme 4). In
the last case the reaction proceeds by base-promoted loss of the
acidic H-2 with a subsequent irreversible nitrogen loss.17
In a previous report, fractionation of the dichloromethane ex-
tract of the roots of W. ebracteata led to the isolation of several cuc-
urbitane-type triterpenoids with cytotoxic properties.14 Two of the
active compounds, dihydrocucurbitacin B (1) and cucurbitacin B
(2) could be obtained with good yields from the roots of W. ebracte-
ata (2.0 g from 3.7 kg) and from the fruits of L. operculata (2.5 g
from 2 kg), respectively, and were used to prepare semisynthetic
derivatives.
2.1.1. Modifications at C-2 and C-16
The first attempted modifications at C-2 and C-16 were oxida-
tions (Scheme 1). The reaction of 1 with PCC yielded the unex-
pected compound 3, instead of the diketone as major compound.
2.1.3. Deoxygenation reactions
As the natural bioactive cucurbitacins have hydroxyls at posi-
tions 2 and 16, the next goal was to study the influence of dehydr-
oxylation at these positions on the biological activity. To
synthesize these compounds, a modified Barton-McCombie deoxy-
genation reaction was used.18 For this purpose, the mono and
dithiocarbonyl intermediates 25 and 26 were prepared from 1.
Treatment of these intermediates with diphenylsilane and lauroyl
peroxide in refluxing toluene yielded the 2-deoxy (27) and 2,16-
dideoxy (28) dihydrocucurbitacin B analogues (Scheme 5).
Treatment of the
a-hydroxy ketones 1 and 3 with KOH in
MeOH–DMF, following the procedure used with triterpenoids hav-
ing a similar A-ring structure, such as maslinic and corosolic acids,
afforded the diosphenols 4 and 5, respectively.15 This sequence is a
simple and practical route to synthesize diosphenol derivatives
from natural abundant cucurbitacins.
In order to protect the hydroxyl groups at C-2 and C-16, com-
pounds 1 and 2 were treated with acetic anhydride to yield dies-
ters 6 and 8, and with succinic anhydride to yield diester 7. In
the same way, acylation of 1 with benzoyl chloride yielded two
new ester derivatives: the benzoyl monoester 9 and diester 10.
In order to explore the use of carbamates, the disubstituted deriv-
ative 11 was prepared by treatment with 1,10-carbonyldiimidazol
(Scheme 2).
2.1.4. Cleavage of the side chain
The presence of an unstauration in the side chain of cucurbita-
cin B results in this compound having a much higher cytotoxic
activity than dihydrocucurbitacin B.12 In order to study the influ-
ence of the side chain on the biological activity of cucurbitacins,
a molecular simplification strategy was applied to obtain new ana-
logues. Starting from 1, the dibenzoyl ester was obtained and re-
duced with lithium tri-tert-butoxyaluminium hydride to yield
2.1.2. Modifications of the
a-hydroxy ketone group in ring A
As it is well known, the
a-hydroxy ketone group is present in
the vicinal diol derivative 29.19 An oxidative cleavage of the
a-diol
on the side chain was performed using periodic acid to give inter-
mediate 30,20 which was then oxidized with PCC to give the 3-keto
compound 31 (Scheme 6).
To avoid the isomerization of C-17, the C-20 ketone was
protected as the ethylene glycol acetal. The benzoates at C-2 and
C-16 were then removed under basic conditions and finally the
carbonyls were deprotected with dilute HCl, yielding hexanorcu-
curbitacin I 33 and hexanorcucurbitacin F 35.21
the most active compounds of the cucurbitacin B family. In order
to study the biological importance of this functionality in ring A,
we performed structural modifications by substitution at C-2.
Ten derivatives were obtained by nucleophilic substitution using
the tosylate as leaving group in a regioselective way, as shown in
Scheme 3. Compounds 1 and 2 were converted to the sulphonates
2.1.5. Modifications in the side chain
In order to observe the importance of some key functionalities
of the side chain, the a,b-unsaturated ketone, the tertiary hydroxyl
at C-20 and the ester at C-25 were modified (Scheme 7). Luche
reduction of 2 yielded the allylic alcohol 36.22 The tertiary hydroxyl
at C-20 in compound 6 was then protected as a triflouroacetate es-
ter to obtain 37.23 The acetate at C-25 was subjected to elimination
conditions using the Alvarez-Manzaneda protocol yielding the al-
kene 38.24
Figure 1. Modifications on Cucurbitacin B and DHB.

3018
K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
Scheme 1. (a) PCC, BaCO3, CH2Cl2; (b) KOH, MeOH, DMF.
development of new drugs. They allow the evaluation of various
types of cancer cells providing leads for the discovery of drugs with
high specificity.28,29
In this work, the cytotoxic activity of the novel synthesized
compounds was evaluated against non-small-cell lung cancer
(A549 cells) using the MTT colorimetric assay.30 The activity is ex-
pressed as 50% growth inhibitory concentration (IC50) values at
48 h and 72 h, and the results are presented in Table 1.
Derivatives of dihydrocucurbitacin B in which both 2-OH and
16-OH were esterified (compounds 6, 7, 8 and 10) together with
the carbamate (compound 11), showed lack of cytotoxic effects.
Compound 3 (16-oxo dihydrocucurbitacin B) was less cytotoxic
than the precursor (compound 1) (26.49 vs 12.09 lM) and the bio-
Scheme 2. (a) Ac2O, Py, DMAP; (b) Succinic anhydride, Py, DMAP; (c) BzCl, Py,
CH2Cl2; (d) CDI, THF, 70 °C.
logical activity was lost when the 2-OH of this compound was
esterified or converted to an enol (compound 4). The influence of
these two hydroxyl groups could also be observed through the
deoxygenation of C-2 and C-16 to give compounds 27 and 28.
The 2-deoxy-dihydrocucurbitacin B (compound 27) showed a
reduction in its cytotoxic activity, while 2,16-dideoxy-dihydrocu-
curbitacin B (compound 28) showed an IC50 value comparable to
the parent compound 1. Further studies should be made combining
the deoxygenation of position 16 and the presence of other molec-
ular modifications to explore the influence of this functionality.
The derivatives of cucurbitacin B with substitution at C-2 (com-
pounds 19, 21 and 24) showed significant cytotoxic effects,
although somewhat lower than the precursor (compound 2). De-
spite the loss of the hydroxyl group at C-2 and the decrease in cyto-
toxicity, the presence of the nitrogenated groups in the case of 19
and 24 opened the possibility to synthesize new derivatives with
higher solubility in biological media. Compound 20, 2-bromo-
dihydrocucurbitacin B derivative, showed moderate activity, about
2.1.6. Introduction of
a,b-unsaturated ketones
The ,b-unsaturated ketone in the side chain appears to have a
a
direct relationship with the strong cytotoxicity of some natural
cucurbitacins.9b,25 In order to explore this observation, this func-
tionality was introduced in other parts of the molecule, such as
rings A and B (Scheme 8). Selective oxidation of 15 with potassium
peroxymonosulfate (OxoneÒ)26 produced the sulfoxide, which was
then converted to the 1-ene-3-one compound 39 by elimination.
The 5-ene-7-one derivative 40 was synthesized from 6 by an allylic
oxidation with CrO3 in pyridine.27
3. Cytotoxic activity
Current in vitro screening methods of cytotoxic effects with the
use of human tumor cells are important tools for research and
two-fold lower than compound 1 (29.09 vs 12.09 lM).
Scheme 3. Substitution at C-2 with sulphur nucleophiles. Reagents and conditions: (a) 4-toluenesulfonyl chloride, DABCO, CH2Cl2, 0 °C; (b) (CH3CO)2O, Py, DMAP; (c) C6H5SH,
THF, NaH; (d) CH3COSK, acetone; (e) SC(NH2)2, EtOH, MW, 100 °C.

K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
3019
2 was acetylated, product 8 showed less cytotoxicity (2.64 vs
0.04 M), although the cytotoxic activity remained high. Modifica-
l
tions on the side chain of compound 1 were conducted to give
compounds 37 and 38, but only 38 showed moderate activity.
Taking into account the side chain relevance for the cytotoxic
activity of cucurbitacins, compounds 33 and 35 were obtained.
These compounds can also be found in natural sources,40 usually
at such low concentrations that hinder biological experimentation.
When the cytotoxicity of both compounds (33 and 35) was evalu-
ated, the activity was lost suggesting once more that the side chain
structure has a huge influence on the cytotoxic activity of this class
of compounds. Compound 5 can also be found in natural sources
and was already reported as active against U937 tumor cell lines,40
NUCG-3 and HONE-1,41 but was inactive in our experiments with
A549 cells.
In summary, we describe here the synthesis of new analogues of
natural products dihydrocucurbitacin B (1) and cucurbitacin B (2)
including structural variations at different positions. From these
results, it is evident that small changes can significantly alter the
cytotoxic effects of cucurbitacins. Among the 29 semisynthetic
compounds tested, some displayed potent to moderate activity
against A549 tumor cells, in particular cucurbitacin B derivatives
8, 19, 21 and 24.
These results allowed a preliminary structure–activity relation-
ship (SAR) evaluation. Further analysis concerning the mechanistic
basis of the cytotoxic activity and a quantitative structure–activity
relationship (QSAR) are being conducted.
Scheme 4. Substitution at C-2. Reagents and conditions: (a) (Bu)4NꢀBr+, DMF; (b)
NaN3, DMF, 70 °C.
For cucurbitacin B, there are several reports on its cyto-
toxic9a,31,32 and antitumoral6f,33 properties, including some exam-
ples where these compounds work in association with other
chemotherapeutic agents.5,31,34–37 Preliminary analyses of
structure–activity relationship pointed out the relevance of the
a
,b-unsaturated ketone in the side chain of cucurbitacin B for its
cytotoxic activity.11,38,39 In an attempt to explore this tendency,
derivatives with an ,b-unsaturated ketone were obtained in other
a
positions of the molecule, specifically in rings A (compound 39)
and B (compound 40). Compound 39 presented a weak activity,
while compound 40 was inactive, confirming the importance of
4. Experimental
4.1. General methods
the a,b-unsaturated ketone location in the side chain. In the same
way, the elimination of this functionality in the side chain (com-
pound 36) also resulted in an inactive product. When compound
All reactions were followed by analytical thin-layer chromatog-
raphy (TLC, Merck Silica Gel 60G F254) with visualization under UV
Scheme 5. Reagents and conditions: (a) 1,10-thiocarbonyldiimidazol, Et2Cl2, 60 °C; (b) [CH3(CH2)10CO]2O2, Ph2SiH2, toluene, 115 °C.
Scheme 6. Reagents and conditions: (a) C6H5COCl, Py, CH2Cl2; (b) C12H28AlO3Li, THF, 0 °C; (c) H5IO6, MeOH; (d) PCC, BaCO3, CH2Cl2; (e) ethylene glycol, HC(OEt)3, TsOH; f) (i)
KOH, MeOH, (ii) HCl, Et2O.

3020
K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
Scheme 7. Reagents and conditions: (a) NaBH4, CeCl3.7H2O, MeOH, ꢀ30 °C; (b) I2, Ph3P, CH2Cl2; (c) TFAA, DBU, CH2Cl2, 0 °C.
Scheme 8. Reagents and conditions: (a) C6H5SH, THF, NaH; (b) Oxone, MeOH, H2O; (c) TEA, toluene, MW, 120 °C, 5 min.; (d) Cr3O, Py, CH2Cl2.
Table 1
Inhibitory effect of dihydrocucurbitacin B and cucurbitacin B analogues on proliferation of A549 cells
a
Compound
IC50
Increase toxicity (CC48/CC72)
48 h
95% Confidence interval
72 h
95% Confidence interval
1
2
3
4
5
6
7
8
9
10
11
16
17
18
19
20
21
22
23
24
26
27
28
30
33
35
36
37
38
39
40
19.91
0.13
16.32–24.87
0.09–0.19
35.65–83.09
—
—
—
—
10.90–19.70
—
—
—
—
—
-
12.09
0.04
10.26–14.25
0.03–0.07
19.80–35.45
—
1.6
3.3
2.1
—
—
—
—
5.5
—
—
—
—
—
—
2.5
1.2
11.1
—
54.42
>100
>100
>100
>100
14.65
>100
>100
>100
>100
>100
>100
28.80
34.93
1.33
26.49
>100
>100
72.52
>100
2.64
>100
>100
>100
>100
>100
>100
11.52
29.09
0.12
—
53.46–98.38
—
1.89–3.70
—
—
—
—
—
—
18.98–43.71
31.40–38.85
0.452–3.94
—
6.80–19.52
25.21–33.58
0.07–0.20
—
33.09–92.02
0.06–0.23
4.49–10.60
51.56–86.39
8.99–14.62
—
>100
>100
0.42
>100
55.19
0.12
—
—
0.16–1.13
10.90–19.69
—
—
—
—
—
—
3.5
2.1
—
—
—
—
—
—
—
—
1.1
—
3.1
6.1
14.65
>100
>100
>100
>100
>100
>100
>100
>100
87.77
>100
3.69
6.90
66.74
11.47
>100
>100
>100
>100
>100
42.6
—
—
—
—
—
—
26.33–68.93
62.83–96.18
—
0.89–1.55
0.11–0.32
75.08–102.62
—
2.34–5.82
0.73–1.84
77.74
>100
1.18
Doxorubicin
Paclitaxel
1.16
0.19
a
IC50: 50% inhibitory concentration (lM); For determination of IC50, data sets from cytotoxicity experiments were analyzed by non-linear regression and calculated using
log(compound) compared with normalized response (variable slope), by GraphPad Prism software.

K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
3021
light and by spraying phosphoric vanillin, followed by heating.
Uncorrected melting points were determined by using a MQAPF-
301 apparatus. IR spectra (KBr) were obtained on a Shimadzu Pres-
tige 2 instrument. NMR spectra were recorded on a Bruker Avance
2 500 MHz spectrometer and on a Varian NMR AS 400. The 2D
NMR spectra were obtained using standard pulse sequences.
High-resolution ESI (ESI-HR-MS) mass spectra were recorded on
a Bruker-Daltonics MicroTOF—Q II mass spectrometer. Column
chromatography was performed using silica gel (MerckÒ). The sol-
vents were purchased from TediaÒ, purified and dried before use.
All other reagents were purchased from Sigma–AldrichÒ.
reduced pressure and the residue was purified by chromatography
on silica gel (40% ethyl acetate/hexane) affording 4 (90 mg, 90%
yield) as a white solid. Mp: 97–98 °C; IR (KBr): 3444, 3974, 1738,
1731, 1714, 1693, 1665, 1397, 1368, 1267, 1252, 1228, 1206,
1127, 1044 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.96 (1H, dd,
;
J = 0.4, 2.7 Hz, H-1), 5.79 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 3.60 (1H,
s, H-17), 3.54 (1H, m, H-10), 3.34 (1H, dd, J = 0.9, 14.8 Hz, H-12a),
2.78 (1H, d, J = 14.8 Hz, H-12b), 2.73 (2H, dt, J = 6.0, 8.5 Hz, H-23),
2.49 (1H, ddt, J = 2.5, 8.5, 19.5 Hz, H-7b), 2.30 (1H, d, J = 8.5, H-8),
2.15 (1H, d, J = 18 Hz, H-15b), 2.09 (1H, d, J = 18 Hz, H-15a), 2.05
(2H, dt, J = 6.0, 8.5 Hz, H-24), 1.98 (1H, m, H-7a), 1.97 (3H, s,
CH3CO2), 1.47 (3H, s, Me-26), 1.46 (3H, s, Me-27), 1.37 (3H, s,
Me-29), 1.37 (3H, s, Me-30), 1.29 (3H, s, Me-20), 1.26 (3H, s, Me-
28), 1.15 (3H, s, Me-19), 1.11 (3H, s, Me-18); 13C NMR
(125.8 MHz, CDCl3): d = 216.1 (C-16), 213.8 (C-22), 210.9 (C-11),
198.4 (C-3), 170.4 (C-31), 144.7 (C-2), 137.1 (C-5), 120.2 (C-6),
114.1 (C-1), 81.4 (C-25), 79.9 (C-20), 61.8 (C-17), 49.9 (C-9), 49.1
(C-15), 47.8 (C-13), 47.6 (C-4), 47.2 (C-12), 44.6 (C-14), 41.3 (C-
8), 35.0 (C-24), 34.7 (C-10), 30.2 (C-23), 27.9 (C-28), 25.9 (C-26),
25.8 (C-27), 23.8 (C-7), 23.4 (C-20), 22.4 (C-32), 20.2 (C-29), 20.1
(C-18), 19.7 (C-19), 18.8 (C-30); ESI-MS (negative ion mode) m/z
555.2984 [MꢀH]ꢀ (calcd for C32H43O8 555.2963).
4.2. Material
Dried and powdered roots of W. ebracteata (3.7 kg) were
exhaustively extracted with 10 L CH2Cl2 of (3ꢁ) at room tempera-
ture, as previously described.14 The isolation procedures resulted
in the purification of 2.0 g of dihydrocucurbitacin B (1).
Additionally, fruits of L. operculata (2 kg) were powdered and
exhaustively extracted with CH2Cl2 (3 ꢁ 4 L) at room temperature
for 5 days. The extract was filtered and taken to dryness under re-
duced pressure and temperature below 45 °C generating the
dichloromethane extract (9 g). This extract was subjected to vac-
uum liquid column chromatography (12 cm id ꢁ 15 cm) on silica
gel: activated charcoal (1:1) using MeOH (500 mL), MeOH:EtOAc
(1:1, 500 mL) and EtOAc (500 mL) as eluent. Fraction EtOAc (4 g)
was subjected to CC using silica gel as adsorbent (particle size
4.3.3. Preparation of 5
Following the procedure described for 4, compound 5 was ob-
tained in 86% yield from 1. Mp: 222–223 °C; IR (KBr): 3547,
2978, 1729, 1712, 1693, 1666, 1660, 1371, 1261, 1231,
63–200
l
m) and hexane/EtOAc 50% as mobile phase (2 L) provid-
1124 cmꢀ1 1H NMR (500 MHz, CDCl3) 5.95 (1H, d, J = 2.5 Hz, H-
;
ing 2.5 g of cucurbitacin B (2).
1), 5.77 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 4.33 (1H, m, H-16), 3.51
(1H, m, H-10), 3.23 (1H, d, J = 14.5 Hz, H-12a), 2.82 (1H, ddd,
J = 6.0, 10.0, 17.5 Hz), 2.73 (1H, d, J = 14.5 Hz, H-12b), 2.53, 2.51
(1H, d, J = 6.9 Hz, H-17), 2.52 (1H, m, H-23b), 2.39 (1H, m, H-7b),
2.05 (2H, m, H-24), 2.02 (1H, d, J = 8.0 Hz, H-8), 1.97 (1H, m, H-
7a), 1.96 (3H, Me-32), 1.86 (1H, dd, J = 9.0, 13.0 Hz, H-15b), 1.46
(3H, Me-26), 1.44 (3H, Me-27), 1.42 (3H, Me-21), 1.40 (3H, Me-
30), 1.36 (3H, Me-28), 1.25 (3H, Me-29), 1.03 (3H, Me-19), 0.99
(3H, Me-18).42
4.3. Preparation of cucurbitacin analogues
4.3.1. Preparation of 3
A mixture of PCC (582 mg, 3.57 mmol) and BaCO3 (706 mg,
3.57 mmol) in dry CH2Cl2 (2.0 mL) was stirred for 5 min. Com-
pound 1 (500 mg, 0.89 mmol) dissolved in 2 mL of CH2Cl2 was then
added and the reaction was stirred for 2 h. The reaction mixture
was diluted with diethyl ether (30.0 mL) and poured through a
short column of FlorisilÒ. The solvent was removed under vacuum
and the residue was purified by column chromatography (50%
ethyl acetate/hexane) to give pure product 3 (240 mg, 48% yield)
as white solid. Mp: 108–109 °C; IR (KBr): 3445, 2975, 1367,
4.3.4. Preparation of 6
A mixture of 1 (300 mg, 0.54 mmol), dry pyridine (2.0 mL), ace-
tic anhydride (2.0 mL) and catalytic amount of DMAP was stirred at
20 °C during 2 h. The mixture was then diluted with 50.0 mL of
EtOAc, washed with HCl 1 M solution (3 ꢁ 30.0 mL), dried over
anhydrous Na2SO4 and filtered. The crude product was purified
by column chromatography (40% ethyl acetate/hexane) to afford
6 (290 mg, 84% yield) as a white solid. Mp: 131–132 °C. IR (KBr)
1266, 1254, 1206, 1125 cmꢀ1
;
1H NMR (500 MHz, CDCl3):
d = 5.81 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 4.45 (1H, dd, J = 5.9,
13.0 Hz, H-2), 3.61 (1H, s, H-17), 3.36 (1H, d, J = 15.0 Hz, H-12a),
2.80 (1H, m, H-10), 2.75 (1H, d, J = 15.0 Hz, H-12b), 2.74 (2H, m,
H-23), 2.52 (1H, m, H-7b), 2.34 (1H, ddd, J = 3.6, 6.1, 13.0, Hz, H-
1), 2.25 (1H, d, J = 8.0 Hz, H-8), 2.14 (1H, d, J = 18 Hz, H-15b),
2.06 (1H, d, J = 18 Hz, H-15a), 2.04 (2H, m, H-24), 1.98 (3H, s,
Me-32), 1.97 (1H, m, H-7a), 1.47 (3H, s, Me-26), 1.46 (3H, s, Me-
27), 1.36 (3H, s, Me-28), 1.34 (3H, s, Me-30), 1.29 (3H, s, Me-21),
1.28 (3H, s, Me-29), 1.28 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b),
1.16 (3H, s, Me-19), 1.13 (3H, s, Me-18); 13C NMR (125.8 MHz,
CDCl3): d = 216.1 (C-16), 213.9 (C-22), 212.6 (C-3), 210.3 (C-11),
170.4 (C-31), 140.7 (C-5), 119.9 (C-6), 81.4 (C-25), 80.0 (C-20),
71.5 (C-2), 61.7 (C-17), 50.3 (C-4), 49.5 (C-9), 48.8 (C-15), 47.7
(C-13), 47.0 (C-12), 44.5 (C-14), 42.0 (C-8), 35.9 (C-1), 34.9 (C-
24), 33.7 (C-10), 30.2 (C-23), 29.3 (C-28), 25.9 (C-26), 25.8 (C-27),
24.0 (C-7), 23.4 (C-21), 22.4 (C-32), 21.2 (C-29), 20.0 (C-19), 19.6
(C-18), 19.2 (C-30); ESI-MS (negative ion mode) m/z 557.3143
[MꢀH]+ (calcd for C32H45O8 557.3119).
3445, 2984, 1731, 1698, 1374, 1245, 1029 cmꢀ1
;
1H NMR
(400 MHz,): d = 5.78 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.48 (1H, dd,
J = 5.5, 13.0 Hz, H-2), 5.14 (1H, m, H-16), 3.25 (1H, d, J = 14.5 Hz,
H-12a), 2.80 (1H, m, H-10) 2.74 (1H, d, J = 14.5 Hz, H-12b), 2.71
(1H, d, J = 7.5, H-17), 2.65 (2H, m, H-23), 2.44 (1H, m, H-7b), 2.15
(3H, s, Me-20), 2.12 (1H, overlapped, H-1a), 2.04 (1H, overlapped,
H-15b), 2.04 (2H, m, H-24), 2.02 (1H, d, J = 7.5 Hz, H-8), 1.99 (3H,
s, Me-32), 1.95 (3H, s, Me-200), 1.93 (1H, m, H-7a), 1.52 (1H, ddd,
J = 13.0, 13.0, 13.0 Hz, H-1b), 1.49 (3H, s, Me-27), 1.46 (3H, s, Me-
26), 1.45 (3H, s, Me-21), 1.41 (1H, m, H-15a), 1.31 (3H, s, Me-28),
1.31 (3H, s, Me-30), 1.29 (3H, s, Me-29), 1.10 (3H, s, Me-19), 1.02
(3H, s, Me-18); 13C NMR (101 MHz, CDCl3): d = 212.3 (C-22),
211.7 (C-11), 205.6 (C-3), 170.1 (C-31), 170.1 (C-10), 170.0 (C-100),
139.7 (C-5), 120.4 (C-6), 81.0 (C-25), 78.6 (C-20), 74.0 (C-16),
73.2 (C-2), 54.0 (C-17), 51.2 (C-4), 49.9 (C-13), 48.6 (C-12), 48.4
(C-9), 47.9 (C-14), 43.2 (C-15), 42.0 (C-8), 35.1 (C-24), 34.3 (C-
10), 32.0 (C-1), 30.4 (C-23), 28.7 (C-28), 26.0 (C-26), 25.8 (C-27),
24.2 (C-21), 23.7 (C-7), 22.4 (C-32), 21.3 (C-29), 20.0 (C-19), 19.6
(C-18), 18.8 (C-30); ESI-MS (negative ion mode) m/z 643.3511
[MꢀH]ꢀ (calcd for C36H51O10 643.3488).
4.3.2. Preparation of 4
To a solution of 3 (100 mg, 0.179 mmol) in MeOH (0.5 mL) and
DMF (0.5 mL) was added KOH (65 mg, 1.79 mmol) with stirring at
20 °C. After 1 h, the reaction mixture was evaporated under

3022
K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
4.3.5. Preparation of 7
reduced pressure. The resulting crude product was purified by
chromatography on silica gel (40% ethyl acetate/hexane) to give
9 as a minor product (230 mg, 19% yield), together with 10 as a ma-
jor product (900 mg, 65% yield) as white solids. (9) Mp: 135–
136 °C; IR (KBr): 3526, 3440, 2975, 1736, 1698, 1370, 1316,
To a solution of 1 (100 mg, 0.18 mmol) in CH2Cl2 (3.0 mL) and
pyridine (0.15 mL, 1.8 mmol) was added succinic anhydride
(180 mg, 1.8 mmol) and a catalytic amount of DMAP with stirring
at 20 °C. After 24 h, the mixture was diluted with CH2Cl2 (20.0 mL)
and washed with HCl 1 M (2 ꢁ 20.0 mL). The organic phase was
dried with anhydrous Na2SO4 and evaporated under reduced pres-
sure. The resulting crude product was purified by column chroma-
tography on silica gel (20% ethyl acetate/hexane), to give the
hemisuccinate 7 (95 mg, 70% yield) as a white solid. Mp: 101–
102 °C; IR (KBr): 3450, 3200, 2978, 2669, 1746, 1729, 1713, 1370,
1270, 1208, 1177, 1123, 1027, 979, 713 cmꢀ1 1H NMR (500 MHz,
;
CDCl3): d = 8.07 (2H, dd, J = 8.4, 1.3 Hz, H-30/H-70), 7.56 (1H, m,
H-50), 7.44 (2H, t, J = 8.4, Hz, H-40/H-60), 5.82 (1H, ddd, J = 2.0, 2.0,
6.0, H-6), 5.72 (1H, dd, J = 5.5, 13.6 Hz, H-2), 4.31 (1H, m, H-16),
3.28 (1H, d, J = 14.6 Hz, H-12a), 2.91 (1H, m, H-10), 2.83 (1H, m,
H-23a), 2.71 (1H, d, J = 14.6 Hz, H-12b), 2.55 (1H, d, J = 6.9 Hz, H-
17), 2.52 (1H, m, H-23b), 2.43 (1H, ddt, J = 2.5, 8.0, 19.5 Hz, H-
7b), 2.27 (1H, ddd, J = 3.6, 5.5, 13.0, Hz, H-1a), 2.06 (2H, m, H-
24), 2.0 (1H, d, J = 8.0 Hz, H-8), 1.98 (1H, m, H-7a), 1.96 (3H, s,
Me-32), 1.85 (1H, dd, J = 9.0, 13.0 Hz, H-15b), 1.69 (1H, ddd,
J = 13.0, 13.0, 13.0 Hz, H-1b), 1.46 (3H, s, Me-27), 1.44 (3H, s, Me-
30), 1.42 (3H, s, Me-26), 1.40 (1H, m, H-15a), 1.40 (3H, s, Me-21),
1.39 (3H, s, Me-28), 1.32 (3H, s, Me-29), 1.13 (3H, s, Me-19), 0.98
(3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3): d = 213.9 (C-22),
212.3 (C-11), 205.5 (C-3), 170.3 (C-31), 165.7 (C-10), 139.8 (C-5),
133.2 (C-50), 129.9 (C-30/C-70), 129.5 (C-20), 128.3 (C-40/60), 120.6
(C-6), 81.3 (C-25), 78.9 (C-20), 73.9 (C-2), 71.0 (C-16), 57.8 (C-
17), 51.4 (C-4), 50.7 (C-13), 48.8 (C-12), 48.5 (C-9), 48.4 (C-14),
45.5 (C-15), 42.4 (C-8), 34.8 (C-24), 34.4 (C-10), 32.1 (C-1), 30.7
(C-23), 28.8 (C-28), 26.2 (C-26), 25.9 (C-27), 24.5 (C-21), 23.9 (C-
7), 22.4 (C-32), 21.3 (C-29), 20.0 (C-19), 19.9 (C-18), 18.7 (C-30);
ESI-MS (negative ion mode) m/z 663.3557 [MꢀH]ꢀ (calcd for
1250, 1164, 1017, 980 cmꢀ1 1H NMR (400 MHz, CDCl3): d = 5.80
;
(1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.51 (1H, dd, J = 13.5, 5.3 Hz, H-2),
5.23 (1H, m, H-16), 3.29 (1H, d, J = 14.8 Hz, H-12a), 2.84 (1H, m,
H-10), 2.75 (1H, d, J = 14.8 Hz, H-12b), 2.74 (1H, d, J = 7.5, H-17),
2.72 (2H, m, H-23), 2.72 (2H, m, H-30), 2.68 (2H, m, H-300), 2.60
(2H, m, H-20) 2.45 (2H, m, H-200), 2.43 (1H, m, H-7b), 2.12 (2H, m,
H-1a), 2.03 (2H, m, H-24), 2.01 (1H, overlapped, H-15b), 2.02
(1H, d, J = 8.0 Hz, H-8), 2.0 (1H, overlapped, H-15b), 1.99 (3H, s,
Me-32), 1.95 (1H, m, H-7a), 1.53 (1H, ddd, J = 13.5, 13.5, 13.5 Hz,
H-1b), 1.48 (3H, s, Me-27), 1.47 (3H, s, Me-26), 1.45 (3H, s, Me-
21), 1.41 (1H, overlapped, H-15a), 1.32 (3H, s, Me-29), 1.31 (3H,
s, Me-30), 1.28 (3H, s, Me-28), 1.09 (3H, s, Me-19), 1.0 (3H, s,
Me-18); 13C NMR (100 MHz, CDCl3): d = 213.0 (C-22), 212.6 (C-
11), 205.9 (C-3), 174.6 (C-40), 174.4 (C-400), 171.8 (C-10), 171.7 (C-
100), 170.7 (C-31), 139.3 (C-5), 120.4 (C-6), 81.2 (C-25), 78.6 (C-
20), 74.3 (C-16), 73.5 (C-2), 54.1 (C-17), 51.2 (C-4), 50.0 (C-13),
48.4 (C-12), 48.3 (C-9), 47.6 (C-14), 42.9 (C-15), 41.9 (C-8), 35.1
(C-24), 34.0 (C-10), 31.9 (C-1), 30.7 (C-23), 29.0 (C-30), 28.8 (C-
300), 28.4 (C-28), 25.7 (C-20), 25.6 (C-200), 24.2 (C-21), 23.6 (C-7),
22.2 (C-32), 21.1 (C-29), 19.8 (C-19), 19.6 (C-18), 18.6 (C-30);
ESI-MS (negative ion mode) m/z 759.3612 [MꢀH]ꢀ (calcd for
C
39H51O9 663.3539). (10) Mp: 122–123 °C; IR (KBr): 2978, 1737,
1723, 1714, 1696, 1369, 1268, 1176, 1115, 1068, 1024, 978 cmꢀ1
;
1H NMR (500 MHz, CDCl3): d = 8.07 (2H, dd, J = 8.5, 1.3 Hz, H-30/
H-70), 7.92 (2H, dd, J = 8.5, 1.3 Hz, H-300/H-700), 7.57 (2H, m, H-50/
H-500), 7.44 (4H, m, H-40/H-400/H-60/H-600), 5.78 (1H, ddd, J = 2.0,
2.0, 6.0, H-6), 5.74 (dd, J = 13.0, 5.5 Hz, H-2), 5.53 (1H, m, H-16),
3.34 (1H, d, J = 14.7 Hz, H-12a), 2.93 (1H, m, H-10), 2.92 (2H, d,
J = 7.2 Hz, H-17), 2.80 (1H, d, J = 14.6 Hz, H-12b), 2.63 (1H, ddd,
J = 17.5, 10.0, 6.0 Hz, H-23a), 2.52 (1H, m, H-23b), 2.45 (1H, ddt,
J = 2.5, 8.0, 19.0 Hz, H-7b), 2.30 (1H, ddd, J = 3.6, 5.5, 13.0 Hz, H-
1a), 2.13 (1H, dd, J = 13.8, 9.0 Hz, H-15b), 2.08 (1H, d, J = 8.0 Hz,
H-8), 1.96 (1H, dd, J = 5.0, 19.0 Hz, H-7a), 1.95 (3H, s, Me-32),
1.77 (1H, m, H-24a), 1.72 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b),
1.54 (1H, m, H-24b), 1.52 (1H, d, J = 13.8 Hz, H-15a), 1.47 (3H, s,
H-30), 1.44 (3H, s, H-27), 1.39 (3H, s, H-26), 1.30 (3H, s, H-21),
1.27 (3H, s, H-28), 1.27 (3H, s, H-29), 1.16 (3H, s, H-19), 1.10 (3H,
s, H-18); 13C NMR (125.8 MHz, CDCl3): d = 212.5 (C-22), 211.8 (C-
11), 205.4 (C-3), 170.2 (C-31), 165.7 (C-100), 165.5 (C-10), 139.8 (C-
5), 133.2 (C-50/C-500), 129.8 (C-30/C-70), 129.4 (C-300/C-700), 128.5
(C-40/C-60), 128.4 (C-40/C-600), 120.5 (C-6), 80.9 (C-25), 78.9 (C-20),
74.4 (C-16), 73.8 (C-2), 54.7 (C-17), 51.4 (C-4), 50.4 (C-13), 48.7
(C-12), 48.4 (C-9), 48.0 (C-14), 43.5 (C-15), 42.0 (C-8), 35.3 (C-
24), 34.4 (C-10), 32.1 (C-1), 30.5 (C-23), 28.8 (C-28), 25.8 (C-26),
25.5 (C-27), 24.3 (C-21), 23.8 (C-7), 22.4 (C-32), 21.3 (C-29), 20.1
(C-19), 19.8 (C-18), 18.7 (C-30); ESI-MS (negative ion mode) m/z
767.3803 [MꢀH]ꢀ (calcd for C46H55O10 767.3801).
C40H55O14 759.3597).
4.3.6. Preparation of 8
Following the procedure described for 6, compound 8 was ob-
tained in 80% yield from 2. Mp: 121–122 °C; IR (KBr): 3439,
2979, 1738, 1728, 1690, 1633, 1367, 1241, 1131, 1024 cmꢀ1 1H-
;
NMR (400 MHz, CDCl3): d = 7.15 (1H, d, J = 15.6 Hz, H-24), 6.40
(1H, d, J = 15.6, H-23), 5.77 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.47
(1H, dd, J = 5.2, 13.5 Hz, H-2), 5.17 (1H, m, H-16), 3.24 (1H, d,
J = 14.6 Hz, H-12a), 2.80 (1H, m, H-10), 2.74 (1H, d, J = 14.6 Hz, H-
12b), 2.69 (1H, d, J = 7.5 Hz, H-17), 2.41 (1H, ddt, J = 2.5, 8.0,
19.0 Hz, H-7b), 2.14 (3H, s, Me-20), 2.11 (1H, overlapped, H-1a),
2.03 (3H, s, Me-32), 1.97 (1H, d, J = 8.0 Hz, H-8), 1.97 (1H, m, H-
7a), 1.91 (1H, overlapped, H-15b),1.86 (3H, s, Me-200), 1.58 (3H, s,
H-27), 1.57 (3H, s, H-26), 1.52 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-
1b), 1.42 (3H, s, Me-21), 1.38 (1H, d, J = 13.0 Hz, H-15a), 1.31 (3H,
s, H-30), 1.30 (3H, s, H-28), 1.28 (3H, s, H-29), 1.09 (3H, s, H-19),
1.02 (3H, s, H-18); 13C NMR (101 MHz, CDCl3): d = 211.8 (C-11),
205.6 (C-3), 200.8 (C-22), 170.3 (C-31), 170.1 (C-10), 169.6 (C-100),
152.7 (C-24), 139.7 (C-5), 120.4 (C-6), 119.2 (C-23), 79.1 (C-25),
77.6 (C-20), 73.5 (C-16), 73.3 (C-2), 54.1 (C-17), 51.2 (C-4), 49.9
(C-13), 48.6 (C-12), 48.4 (C-9), 48.0 (C-14), 43.1 (C-15), 42.1 (C-
8), 34.3 (C-10), 31.9 (C-1), 28.8 (C-28), 26.6 (C-26), 26.3 (C-27),
23.7 (C-21), 23.6 (C-7), 21.9 (C-32), 21.3 (C-29), 20.7 (C-20), 20.6
(C-200), 19.9 (C-19), 19.7 (C-18), 18.7 (C-30); ESI-MS (positive ion
mode) m/z 660.3742 [M+NH4]+ (calcd for C36H54NO10 660.3742).
4.3.8. Preparation of 11
To a solution of 1 (100 mg, 0.18 mmol) in THF (3.0 mL), 1,10-car-
bonyldiimidazole (120 mg, 0.72 mmol) was added and the mixture
was heated at 70 °C. After 4 h, the reaction was diluted by adding
30 mL of diethyl ether and washed with brine (2 ꢁ 30.0 mL). The
organic phase was dried with anhydrous Na2SO4 and evaporated
under reduced pressure. The resulting crude product was purified
by chromatography on silica gel (70% ethyl acetate/hexane) to give
11 (67 mg, 50% yield) as a white solid. Mp: 148–149 °C; IR (KBr):
2935, 1758, 1727, 1395, 1370, 1316, 1289, 1240, 1223, 1172,
4.3.7. Preparation of 9 and 10
To a solution of compound 1 (1.0 g, 1.79 mmol) in CH2Cl2
(10.0 mL) and pyridine (5.0 mL), benzoyl chloride (0.41 mL,
3.57 mmol) was added dropwise under nitrogen atmosphere, at
0 °C. The reaction was kept at 20 °C for 4 h, diluted by adding
50 mL of CH2Cl2 and washed with HCl 1 M (3 ꢁ 30 mL). The organic
phase was dried with anhydrous Na2SO4 and evaporated under
1041, 1002, 938 cmꢀ1
;
1H NMR1H NMR (400 MHz, CDCl3):
d = 8.17 (1H, m, H-40), 8.07 (1H, m, H-400), 7.45 (1H, s, H-20), 7.37

K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
3023
(1H, s, H-200), 7.09 (2H, m, H-30/H-300), 5.84 (1H, ddd, J = 2.0, 2.0, 6.0,
H-6), 5.65 (1H, dd, J = 5.0, 13.0 Hz, H-2), 5.63 (1H, m, H-16), 3.31
(1H, d, J = 14.5 Hz, H-12a), 2.91 (1H, d, J = 7.2 Hz, H-17), 2.87 (1H,
m, H-10), 2.80 (1H, d, J = 14.5 Hz, H-12b), 2.65 (2H, m, H-23),
2.48 (1H, ddt, J = 2.5, 8.0, 19.0 Hz, H-7b), 2.37 (1H, ddd, J = 3.6,
5.5, 13.0 Hz, H-1a), 2.15 (1H, dd, J = 13.8, 9.0 Hz, H-15b), 2.10
(1H, d, J = 8.0 Hz, H-8), 1.99 (1H, dd, J = 5.0, 19.0 Hz, H-7a), 1.98
(3H, s, Me-32), 1.75 (1H, overlapped, H-15a), 1.73 (2H, m, H-24),
1.68 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b), 1.50 (3H, s, Me-30),
1.39 (3H, s, Me-27), 1.37 (3H, s, Me-26), 1.37 (3H, s, Me-21), 1.34
(3H, s, Me-29), 1.32 (3H, s, Me-28), 1.17 (3H, s, Me-19), 1.08 (3H,
s, Me-18); 13C NMR (101 MHz, CDCl3): d = 213.4 (C-22), 211.3 (C-
11), 204.0 (C-3), 170.2 (C-31), 147.8 (C-10), 147.7 (C-100), 139.0 (C-
5), 137.2 (C-40), 136.8 (C-400), 130.8 (C-30), 130.6 (C-300), 120.9 (C-
6), 117.2 (C-20), 117.0 (C-200), 80.9 (C-25), 78.9 (C-20), 78.5 (C-2),
76.6 (C-16), 54.7 (C-17), 51.3 (C-4), 50.2 (C-13), 48.4 (C-12), 48.2
(C-9), 47.9 (C-14), 43.0 (C-15), 41.9 (C-8), 35.0 (C-24), 34.1 (C-
10), 31.8 (C-1), 31.0 (C-23), 28.5 (C-28), 25.7 (C-26), 25.6 (C-27),
24.3 (C-21), 23.7 (C-7), 22.3 (C-32), 21.1 (C-29), 20.2 (C-19), 19.7
(C-18), 18.7 (C-30); ESI-MS (positive ion mode) m/z 749.3754
[M+H]+ (calcd for C40H53N4O10 749.3756).
s, Me-70), 2.40 (1H, ddt, J = 19.5, 8.5, 2.5 Hz, H-7b), 2..24 (1H, m,
H-1a), 2.04 (2H, m, H-24), 2.0 (1H, m, H-15b), 2.0 (1H, d,
J = 8.5 Hz, H-8), 1.99 (3H, s, Me-32), 1.94 (3H, s, Me-200), 1.92 (1H,
m, H-7a), 1.50 (3H, s, Me-26), 1.47 (3H, s, Me-27), 1.45 (3H, s,
Me-21), 1.40 (1H, dd, J = 14.0, 1.0 Hz, H-15a), 1.29 (3H, s, Me-30),
1.25 (3H, s, Me-28), 1.25 (3H, s, Me-29), 1.04 (3H, s, Me-19), 1.01
(3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3): d = 212.5 (C-22),
211.4 (C-11), 203.9 (C-3), 170.3 (C-31), 170.1 (C-100), 145.0 (C-40),
139.1 (C-5), 134.1 (C-10), 129.8 (C-30/C-50), 128.0 (C-20/C-60),
121.1 (C-6), 81.2 (C-25), 78.8 (C-2), 78.7 (C-20), 74.2 (C-16), 54.2
(C-17), 51.7 (C-4), 50.0 (C-13), 48.8 (C-12), 48.4 (C-9), 48.0 (C-
14), 43.3 (C-15), 42.1 (C-8), 35.3 (C-24), 34.4 (C-10), 33.6 (C-1),
30.6 (C-23), 28.6 (C-28), 26.2 (C-26), 26.0 (C-27), 24.4 (C-21),
23.8 (C-7), 22.5 (C-32), 21.9 (C-70), 21.5 (C-29), 21.1 (C-200), 20.1
(C-19), 19.8 (C-18), 18.9 (C-30). ESI-MS (positive ion mode) m/z
779.3436 [M+Na]ꢀ (calcd for C41H56Na11S 779.3435).
4.3.11. Preparation of 14
Following the procedure described for 12, compound 14 was
obtained in 77% yield from 2. Mp: 133–134 °C; IR (KBr): 3450,
3279, 1738, 1730, 1693, 1632, 1368, 1250, 1175, 1126, 1092,
968, 891, 679, 560 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 7.85 (2H,
;
4.3.9. Preparation of 12
d, J = 8.4 Hz, H-30/H-50), 7.34 (2H, d, J = 8.4 Hz, H-20/H-60), 7.07
(1H, d, J = 15.6 Hz, H-24), 6.48 (1H, d, J = 15.6 Hz, H-23), 5.78 (1H,
ddd, J = 2.0, 2.0, 6.0, H-6), 5.40 (1H, dd, J = 13.0, 6.0 Hz, H-2), 4.35
(1H, m, H-16), 3.24 (1H, d, J = 14.7 Hz, H-12a), 2.81 (1H, m, H-
10), 2.70 (1H, d, J = 14.7 Hz, H-12b), 2.50 (1H, d, J = 7.1 Hz, H-17),
2.45 (3H, s, H-70), 2.38 (1H, ddt, J = 19.5, 8.2, 2.4 Hz, H-7b), 2.24
(1H, ddd, 12.5, 5.5, 3.7 Hz, H-1a), 2.03 (3H, s, Me-32), 1.97 (1H, d,
J = 8.5 Hz, H-8), 1.96 (1H, m, H-7a), 1.86 (1H, dd, J = 13.3, 9.3, H-
15b), 1.58 (3H, s, Me-26), 1.56 (1H, overlapped, H-1a), 1.55 (3H,
s, Me-26), 1.45 (3H, s, Me-26), 1.42 (1H, overlapped, H-15b), 1.42
(3H, s, Me-21), 1.26 (3H, s, Me-28), 1.26 (3H, s, Me-29), 1.03 (3H,
s, Me-19), 0.97 (3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3):
d = 211.8 (C-11), 203.9 (C-3), 202.4 (C-22), 170.2 (C-31), 152.0 (C-
24), 144.8 (C-40), 138.9 (C-5), 134.0 (C-10), 129.6 (C-30/C-50), 127.9
(C-20/C-60), 121.1 (C-6), 120.2 (C-23), 79.3 (C-25), 78.8 (C-2), 78.1
(C-20), 71.3 (C-16), 58.2 (C-17), 51.5 (C-4), 50.6 (C-13), 48.7 (C-
12), 48.4 (C-9), 48.0 (C-14), 45.3 (C-15), 42.3 (C-8), 34.2 (C-10),
33.5 (C-1), 28.5 (C-28), 26.4 (C-27), 25.9 (C-26), 23.9 (C-21), 23.8
(C-7), 22.0 (C-32), 21.7 (C-70), 21.4 (C-29), 19.9 (C-18), 19.9 (C-
19), 18.6 (C-30); ESI-MS (positive ion mode) m/z 730.3613
[M+NH4]+ (calcd for C39H56NO10S 730.3619).
To a solution of compound 1 (1.0 g, 1.79 mmol) in CH2Cl2
(8.0 mL), DABCO (1.0 g, 8.95 mmol) was added and the mixture
was cooled at 0 °C. p-Toluenesulfonyl chloride (1.36 g, 7.16 mmol)
was then added (3 ꢁ 1.3 mmol each 15 min). After 15 min of the
last addition the mixture was diluted by adding 60.0 mL of cold
CH2Cl2, washed with cold HCl 1 M (3 ꢁ 50.0 mL) and cold brine
(3 ꢁ 50.0 mL). The organic phase was dried with anhydrous Na2SO4
and evaporated under reduced pressure. The resulting crude prod-
uct was purified by chromatography on silica gel (40% ethyl ace-
tate/hexane) to afford 12 (1.02 g, 80% yield) as a white solid. Mp:
122–123 °C; IR (KBr): 3549, 3449, 1279, 1732, 1697, 1598, 1370,
1357, 1179, 967, 889 cmꢀ1 1H NMR (400 MHz, CDCl3): d = 7.65
;
(2H, d, J = 8.4 Hz, H-30/H-50), 7.35 (2H, J = 8.4 Hz, H-20/H-60), 5.79
(1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.39 (1H, dd, J = 13.0, 6.0 H-2),
4.31 (1H, m, H-16), 3.25 (1H, d, J = 14.5, H-12a), 2.82 (1H, m,
23a), 2.81 (1H, m, H-10), 2.70 (1H, d, J = 14.5, H-12b), 2.54 (1H, d,
J = 8.2 Hz, H-17), 2.53 (1H, overlapped, H-23b), 2.45 (3H, s, Me-
70), 2.40 (1H, m, H-7b), 2.24 (1H, ddd, J = 13.0, 6.1, 3.6 Hz, H-1a),
2.06 (2H, m, H-24), 1.99 (1H, m, H-7a), 1.98 (1H, d, J = 8.5 Hz, H-
8), 1.96 (3H, s, Me-32), 1.55 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-
1b), 1.85 (1H, dd, J = 13.0, 9.0 Hz, H-15b), 1.47 (3H, s, Me-27),
1.45 (3H, s, Me-26), 1.43 (3H, s, Me-21), 1.40 (1H, m, H-15a),
1.35 (3H, s, Me-30), 1.27 (3H, s, Me-28), 1.26 (3H, s, Me-29), 1.24
4.3.12. Preparation of 15
To a solution of 12 (80 mg, 0.11 mmol) in anhydrous THF
(1.0 mL), thiophenol (0.01 mL, 10.3 mmol) was added with stirring
at 20 °C under nitrogen atmosphere. After 2 h, the reaction was di-
luted by adding 20 mL of CH2Cl2 and washed with NaOH 20%
(2 ꢁ 25 mL). The organic phase was dried with anhydrous Na2SO4
and evaporated under reduced pressure. The resulting crude prod-
uct was purified by chromatography on silica gel (30% ethyl ace-
tate/hexane) to afford 15 (62 mg, 84% yield) as a white solid. Mp:
105–106 °C; IR (KBr): 3454, 2976, 1730, 1713, 1704, 1694, 1470,
(1H, m, H-1a
), 1.03 (3H, s, Me-19), 0.97 (3H, s, Me-18); 13C NMR
(125.8 MHz, CDCl3): d = 213.9 (C-22), 211.8 (C-11), 203.9 (C-3),
170.4 (C-31), 144.9 (C-40), 138.9 (C-5), 133.9 (C-10), 129.7 (C-30/C-
50), 127.9 (C-20/C-60), 121.0 (C-6), 81.3 (C-25), 78.9 (C-2), 78.8 (C-
20), 71.0 (C-16), 57.7 (C-17), 51.5 (C-4), 50.6 (C-13), 48.7 (C-12),
48.3 (C-9), 48.3 (C-14), 45.4 (C-15), 42.3 (C-8), 34.8 (C-24), 34.2
(C-10), 33.5 (C-1), 30.7 (C-23), 28.5 (C-28), 26.1 (C-26), 25.8 (C-
27), 24.4 (C-21), 23.8 (C-7), 22.4 (C-32), 21.7 (C-29), 21.3 (C-200),
19.9 (C-19), 19.8 (C-18), 18.6 (C-30); ESI-MS (positive ion mode)
m/z 732.3780 [M+NH4]+ (calcd for C39H58NO10S 749.3776).
1462, 1366, 1252, 1210, 1174, 1128, 1022, 749, 692 cmꢀ1 1H
;
NMR (CDCl3): d = 7.40 (2H, m, H-20/H-60), 7.30 (2H, m, H-30/H-50),
7.26 (1H, m, H-40), 5.78 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 4.30 (1H,
m, H-16), 4.07 (1H, dd, J = 13.5, 5.3 Hz, H-2), 3.18 (1H, d,
J = 14.6 Hz, H-12a), 2.80 (1H, m, H-23a), 2.63 (1H, d, J = 14.6 Hz,
H-12b), 2.62 (1H, m, H-10), 2.51 (1H, d, J = 7.1 Hz, H-17), 2.50
(1H, m, H-23b), 2.40 (ddt, J = 19.6, 8.5, 2.9 Hz, H-7b), 2.26 (1H,
ddd, J = 13.5, 5.3, 3.6 Hz, H-1a), 2.05 (2H, m, H-24), 1.97 (1H, over-
lapped, H-7a), 1.96 (3H, s, Me-32), 1.95 (1H, d, J = 8.0 Hz, H-8), 1.83
(1H, dd, J = 12.7, 9.5 Hz, H-15b), 1.54 (1H, ddd, J = 13.5, 13.5,
13.5 Hz, H-1b), 1.45 (3H, s, Me-26), 1.43 (3H, s, Me-27), 1.39 (3H,
s, Me-21), 1.37 (1H, d, J = 12.7 Hz, H-15a), 1.33 (3H, s, Me-29),
4.3.10. Preparation of 13
Following the procedure described for 6, compound 13 was ob-
tained in 95% yield from 12. Mp: 121–122 °C; IR (KBr) 3315, 2979,
1735, 1697, 1369, 1254, 1175, 1091, 1014, 970 cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d = 7.85 (2H, d, J = 8.4 Hz, H-30/H-50), 7.35 (2H,
d, J = 8.4 Hz, H-20/H-60), 5.77 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.70
(1H, dd, J = 13.0, 6.0 Hz, H-2), 5.14 (1H, m, H-16), 3.25 (1H, d,
J = 14.7 Hz, H-12a), 2.79 (1H, m, H-10), 2.75 (1H, d, J = 14.7 Hz, H-
12b), 2.71 (1H, d, J = 7.5 Hz, H-17), 2.69 (2H, m, H-23), 2.45 (3H,

3024
K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
1.29 (3H, s, Me-28), 1.05 (3H, s, Me-19), 0.94 (3H, s, Me-18). 13C
NMR (125.8 MHz, CDCl3): d = 213.9 (C-22), 212.2 (C-11), 208.2
(C-3), 170.3 (C-31), 140.3 (C-5), 133.8 (C-10), 132.5 (C-30/C-50),
129.0 (C-20/C-60), 127.5 (C-40), 120.1 (C-6), 81.2 (C-25), 78.9 (C-
20), 71.0 (C-16), 57.7 (C-2), 54.6 (C-17), 51.7 (C-4), 50.6 (C-13),
48.7 (C-9), 48.6 (C-12), 48.4 (C-14), 45.5 (C-15), 42.3 (C-8), 36.4
(C-10), 34.8 (C-24), 33.7 (C-1), 30.6 (C-23), 28.7 (C-28), 26.1 (C-
26), 25.8 (C-27), 24.4 (C-21), 23.8 (C-7), 23.1 (C-29), 22.4 (C-32),
19.8 (C-19), 19.8 (C-18), 18.7 (C-30); ESI-MS (positive ion mode)
m/z 675.3349 [M+Na]+ (calcd for C38H52NaO7S 675.3326).
(C-18), 18.8 (C-30); ESI-MS (negative ion mode) m/z 659.3264
[MꢀH]ꢀ (calcd for C36H51O9S 559.3259).
4.3.15. Preparation of 18
A
sealed 10 mL glass tube containing thiourea (213 mg,
2.8 mmol) and compound 12 (100 g, 0.140 mmol) in EtOH was
introduced in the cavity of a microwave reactor (CEM Co., Dis-
cover) and irradiated with stirring under a maximum potency
(300 W) for 10 min. The temperature was raised up to 100 °C. After
cooling of the mixture to room temperature, the reaction vessel
was opened and the product was evaporated under reduced pres-
sure. The resulting crude product was purified by chromatography
on FlorisilÒ (50% ethyl acetate/hexane) to afford 18 (64 mg, 76%
yield) as a white solid. Mp: 142–143 °C; IR (KBr): 3445, 3354,
2978, 1730, 1713, 1693, 1681, 1652, 1534, 1531, 1372, 1262,
4.3.13. Preparation of 16
Following the procedure described for 15, compound 16 was
obtained in 78% yield from 13. Mp: 101–102 °C; IR (KBr): 2978,
1738, 1730, 1714, 1684, 1370, 1250, 1024 cmꢀ1 1H NMR (CDCl3):
;
d = 7.40 (2H, dd, J = 8.0, 1.5 Hz, H-20/H-60), 7.30 (2H, m, H-30/H-50),
7.26 (1H, m, H-40), 5.76 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.12 (1H, m,
H-16), 4.05 (1H, dd, J = 13.2, 5.3 Hz, H-2), 3.17 (1H, d, J = 14.6 Hz, H-
12a), 2.68 (1H, d, J = 7.2 Hz, H-17), 2.67 (1H, d, J = 14.6 Hz, H-12b),
2.58 (2H, m, H-23), 2.57 (1H, m, H-10), 2.40 (ddt, J = 19.6, 8.5,
2.9 Hz, H-7b), 2.26 (1H, ddd, J = 13.2, 5.3, 3.6 Hz, H-1a), 2.02 (2H,
m, H-24), 2.0 (1H, overlapped, H-15b), 1.99 (3H, s, Me-32), 1.97
(1H, d, J = 8.0 Hz, H-8), 1.96 (3H, s, Me-200), 1.90 (1H, ddd, J = 13.2,
6.0, 1.5 Hz, H-7a), 1.54 (1H, ddd, J = 13.2, 13.2, 13.2 Hz, H-1b),
1.48 (3H, s, Me-26), 1.45 (3H, s, Me-27), 1.41 (3H, s, Me-21), 1.37
(1H, dd, J = 14.0, 1.0 Hz, H-15a), 1.32 (3H, s, Me-29), 1.28 (3H, s,
Me-28), 1.26 (3H, s, Me-30), 1.06 (3H, s, Me-19), 0.98 (3H, s, Me-
18); 13C NMR (125.8 MHz, CDCl3): d = 212.3 (C-22), 211.6 (C-11),
208.1 (C-3), 170.0 (C-31), 140.3 (C-5), 133.7 (C-10), 132.6 (C-30/C-
50), 129.0 (C-20/C-60), 127.6 (C-40), 120.1 (C-6), 81.0 (C-25), 78.6
(C-20), 74.0 (C-16), 54.5 (C-2), 54.0 (C-17), 51.6 (C-4), 49.9 (C-
13), 48.6 (C-9), 48.5 (C-12), 47.9 (C-14), 43.2 (C-15), 42.1 (C-8),
36.4 (C-10), 35.1 (C-24), 33.6 (C-1), 30.4 (C-23), 28.6 (C-28), 26.0
(C-26), 25.8 (C-27), 24.2 (C-21), 23.7 (C-7), 23.2 (C-29), 22.3 (C-
32), 20.9 (C-200), 19.9 (C-19), 19.6 (C-18), 18.8 (C-30); ESI-MS (neg-
ative ion mode) m/z 693.3459 [MꢀH]ꢀ (calcd for C40H53O8S
663.3467).
1208, 1128, 1024 cmꢀ1 1H NMR (CDCl3): d = 5.76 (1H, ddd,
;
J = 2.0, 2.0, 6.0, H-6), 4.31 (1H, t, J = 7.5 Hz, H-16), 3.24 (1H, d,
J = 14.5 Hz, H-12a), 2.82 (1H, ddd, J = 6.8, 9.0, 17.5 Hz, H-23a),
2.67 (1H, J = 14.5 Hz, H-12b), 2.59 (1H, m, H-10), 2.54 (d,
J = 7.5 Hz, H-17), 2.51 (1H, m, H-23), 2.45 (1H, overlapped, H-2),
2.41 (1H, m, H-7b), 2.39 (1H, overlapped, H-2), 1.98 (1H, m, H-
7a), 1.97 (3H, s, Me-32), 1.96 (1H, d, J = 8.5 Hz, H-8), 1.89 (1H,
ddd, J = 14.0, 9.0, 4.5 Hz, H-1a), 1.84 (1H, m, H-15b), 1.82 (1H, over-
lapped, H-15a), 1.51(1H, m, H-1b), 1.46 (3H, s, Me-27), 1.44 (3H, s,
Me-26), 1.42 (3H, s, Me-21), 1.37 (3H, s, Me-30), 1.27 (3H, s, Me-
29), 1.24 (3H, s, Me-28), 1.09 (3H, s, Me-19), 0.97 (3H, s, Me-18);
13C NMR (125.8 MHz, CDCl3): d = 213.9 (C-3), 213.6 (C-11), 213.0
(C-22), 170.3 (C-31), 165.6 (C-10), 140.7 (C-5), 119.7 (C-6), 115.6
(C-2), 81.2 (C-25), 78.9 (C-20), 71.1 (C-16), 57.7 (C-17), 50.9 (C-
4), 50.7 (C-14), 49.0 (C-9), 48.8 (C-12), 48.4 (C-13), 45.6 (C-15),
42.3 (C-8), 38.0 (C-2), 36.0 (C-10), 34.8 (C-24), 30.6 (C-23), 28.5
(C-28), 26.1 (C-26), 25.8 (C-27), 24.5 (C-1), 24.4 (C-21), 23.9 (C-
7), 22.9 (C-29), 22.4 (C-32), 19.8 (C-18), 19.6 (C-19), 18.7 (C-30);
ESI-MS (negative ion mode) m/z 599.3188 [MꢀH]ꢀ (calcd for
C33H47N2O6S 599.3160).
4.3.16. Preparation of 19
Following the procedure described for 18, compound 19 was
obtained in 62% yield from 14. Mp: 198–199 °C; IR (KBr): 3642,
3467, 3363, 2972, 1727, 1691, 1613, 1537, 1370, 1289, 1257,
4.3.14. Preparation of 17
To a solution of 13 (80 mg, 0.106 mmol) in anhydrous acetone
(1.0 mL), potassium thioacetate (121 mg, 1.06 mmol) was added
with stirring at 20 °C under nitrogen atmosphere. After 8 h, the
mixture was filtered and evaporated under reduced pressure. The
resulting crude product was purified by column chromatography
on silica gel (30% ethyl acetate/hexane), to give 17 (51 mg, 73%
yield) as a white solid. Mp: 113–114 °C; IR (KBr): 3479, 2978,
1127, 990 cmꢀ1 1H-NMR (CDCl3): d = 7.02 (1H, d, J = 15.6 Hz, H-
;
24), 6.50 (1H, d, J = 15.6, H-23), 4.36 (1H, t, J = 7.1 Hz, H-16), 3.40
(2H, s, NH2), 3.16 (1H, d, J = 14.8 Hz, H-12a), 2.62 (1H, m, H-10),
2.60 (1H, d, J = 14.5 Hz, H-12b), 2.50 (1H, d, J = 7.1 Hz, H-17), 2.47
(1H, overlapped, H-1a), 2.46 (1H, overlapped, H-7b), 2.35 (1H, dd,
J = 15.2, 11.5 Hz, H-1b) 2.02 (1H, m, H-7a), 2.01 (3H, s, Me-32),
1.98 (1H, d, J = 8.5 Hz, H-8), 1.87 (1H, dd, J = 13.3, 9.3, H-15b),
1.55 (3H, s, Me-26), 1.54 (3H, s, Me-27), 1.43 (3H, s, Me-21), 1.41
(3H, s, Me-28), 1.37 (3H, s, Me-29), 1.33 (3H, s, Me-30), 1.33 (1H,
overlapped, H-15a) 1.18 (3H, s, Me-19), 0.95 (3H, s, Me-18); 13C
NMR (125.8 MHz, CDCl3): d = 214.1 (C-11), 202.8 (C-22), 170.5
(C-32), 166.0 (C-10), 151.3 (C-24), 150.6 (C-2), 141.0 (C-5), 120.3
(C-23), 120.2 (C-6), 115.3 (C-3), 79.5 (C-25), 78.3 (C-20), 70.7 (C-
16), 57.8 (C-17), 50.4 (C-14), 48.5 (C-9), 48.0 (C-12), 47.9 (C-13),
45.0 (C-15), 43.0 (C-8), 39.8 (C-4), 36.1 (C-10), 31.8 (C-28), 27.6
(C-29), 26.7 (C-26), 26.1 (C-227), 25.8 (C-21), 23.7 (C-7), 23.5 (C-
1), 22.9 (C-32), 21.7 (C-32), 20.4 (C-19), 19.6 (C-18), 18.9 (C-30);
ESI-MS (positive ion mode) m/z 599.3135 [M+H]+ (calcd for
1737, 1730, 1714, 1693, 1682, 1369, 1249 cmꢀ1
;
1H NMR
(500 MHz, CDCl3): d = 5.78 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.14
(1H, ddd, J = 1.2, 7.6, 9.3 Hz, H-16), 4.58 (1H, dd, J = 5.0, 14.0 Hz,
H-2), 3.30 (1H, dd, J = 0.6, 14.6 Hz, H-12a), 2.85 (1H, m, H-10),
2.75 (1H, d, J = 14.6 Hz, H-12b), 2.72 (1H, d, J = 7.6 Hz, H-17), 2.66
(2H, m, H-23), 2.41 (1H, ddt, J = 2.5, 8.0, 19.5 Hz, H-7b), 2.36 (3H,
s, Me-20), 2.13 (1H, ddd, J = 5.0, 8.5, 13.0 Hz, H-1a), 2.05 (2H, m,
H-24), 2.01 (1H, overlapped, H-15b), 2.0 (3H, s, Me-32), 1.99 (1H,
d, J = 8.0 Hz, H-8), 1.95 (3H, s, Me-200), 1.93 (1H, m, H-7a), 1.51
(1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b), 1.50 (3H, s, Me-26), 1.47
(3H, s, Me-27), 1.45 (3H, s, Me-21), 1.40 (1H, dd, J = 1.2, 14.2, H-
15a), 1.36 (3H, s, Me-28), 1.33 (3H, s, Me-30), 1.31 (3H, s, Me-
29), 1.06 (3H, s, Me-19), 1.01 (3H, br s, Me-18); 13C NMR
(125.8 MHz, CDCl3): d = 212.4 (C-22), 211.7 (C-11), 206.7 (C-3),
194.6 (C-10), 170.1 (C-31), 170.0 (C-100), 140.2 (C-5), 120.3 (C-6),
81.0 (C-25), 78.6 (C-20), 74.1 (C-16), 54.0 (C-17), 51.9 (C-4), 50.6
(C-2), 50.0 (C-13), 48.7 (C-12), 48.6 (C-9), 47.9 (C-14), 43.3 (C-
15), 42.1 (C-8), 36.5 (C-10), 35.2 (C-24), 33.8 (C-1), 30.6 (C-20),
30.4 (C-23), 29.3 (C-28), 26.0 (C-26), 25.8 (C-27), 24.2 (C-21),
23.8 (C-7), 22.4 (C-29), 22.3 (C-32), 20.9 (C-200), 19.8 (C-19), 19.6
C33H47N2O6S 599.3149).
4.3.17. Preparation of 20
To a solution of 12 (100 mg, 0.140 mmol) in DMF (1.0 mL),
(Bu)4NꢀBr+ (91 mg, 2.80 mmol) was added with stirring at 20 °C.
After 35 h, the reaction was diluted by adding 20 mL of diethyl
ether and washed with brine (2 ꢁ 25 mL). The organic phase was
dried with anhydrous sodium sulfate and evaporated under

K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
3025
reduced pressure. The resulting crude product was purified by
chromatography on silica gel (40% ethyl acetate/hexane) to afford
20 (70 mg, 80% yield) as a white solid. Mp: 113–114 °C; IR (KBr):
1.32 (3H, s, Me-29), 1.24 (3H, s, Me-28), 0.99 (3H, s, Me-19), 0.98
(3H, s, Me-18); 13C NMR (CDCl3): d = 213.9 (C-22), 213.6 (C-11),
198.6 (C-3), 170.3 (C-31), 137.6 (C-5), 136.9 (C-2), 119.6 (C-6),
112.9 (C-1), 81.2 (C-25), 78.9 (C-20), 71.1 (C-16), 57.8 (C-17),
51.9 (C-4), 50.6 (C-13), 49.2 (C-9), 48.9 (C-12), 48.4 (C-14), 48.0
(C-4) 45.7 (C-15), 41.6 (C-8), 34.8 (C-24), 34.0 (C-10), 30.6 (C-23),
27.9 (C-28), 26.1 (C-27), 25.8 (C-26), 24.5 (C-21), 23.6 (C-7), 22.4
(C-32), 20.5 (C-29), 19.9 (C-19), 19.8 (C-18), 18.3 (C-30); ESI-MS
(negative ion mode) m/z 556.3280 [MꢀH]ꢀ (calcd for C32H46NO7
556.3280).
3289, 2978, 1730, 1697, 1368, 1260, 1218, 1128, 1021 cmꢀ1 1H
;
NMR (CDCl3): d = 5.81 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 4.85 (1H,
dd, J = 5.2, 11.5 Hz, H-2), 4.31 (1H, t, J = 7.1 Hz, H-16), 3.22 (1H, d,
J = 14.7 Hz, H-12a), 2.82 (1H, m, H-23a), 2.70 (1H, m, H-10), 2.70
(1H, d, J = 14.5 Hz, H-12b), 2.52 (1H, m, H-23b), 2.53 (1H, d,
J = 7.1 Hz, H-17), 2.52 (1H, m, H-1a), 2.41 (1H, m, H-7b), 2.06
(2H, m, H-24), 2.01 (3H, s, Me-32), 1.98 (1H, d, J = 7.8 Hz, H-8),
1.99 (1H, m, H-7a), 1.86 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b),
1.85 (1H, dd, J = 9.0, 13.0 Hz, H-15b), 1.46 (3H, s, Me-27), 1.44
(3H, s, Me-26), 1.42 (3H, s, Me-21), 1.40 (1H, m, H-15a), 1.34
(3H, s, Me-30), 1.38 (3H, s, Me-29), 1.29 (3H, s, Me-28), 1.10 (3H,
s, Me-19), 0.97 (3H, s, Me-18); 13C NMR (125 MHz, CDCl3):
d = 213.9 (C-22), 212.2 (C-11), 203.6 (C-3), 170.3 (C-31), 139.4 (C-
5), 120.8 (C-6), 81.3 (C-25), 78.9 (C-20), 71.0 (C-16), 57.8 (C-17),
52.8 (C-2), 51.9 (C-4), 50.6 (C-13), 48.8 (C-12), 48.5 (C-14), 48.3
(C-9), 45.5 (C-15), 42.4 (C-8), 37.9 (C-1), 37.1 (C-10), 34.8 (C-24),
30.7 (C-23), 28.9 (C-28), 26.2 (C-27), 25.9 (C-26), 24.5 (C-21),
23.8 (C-7), 23.2 (C-29), 22.4 (C-32), 20.0 (C-19), 19.9 (C-18), 18.7
(C-30); ESI-MS (negative ion mode) m/z 621.2409 [MꢀH]ꢀ (calcd
for C32H46BrO7 621.2432).
4.3.20. Preparation of 23
Following the procedure described for 22, compound 23 was
obtained in 51% yield from 16 as a white solid. Mp: 129–130 °C;
IR (KBr): 3458, 3363, 2978, 1740–1640, 1372, 1260, 1022 cmꢀ1
;
1H NMR (CDCl3): d = 5.71 (1H, ddd, J = 2.5, 2.5, 5.0, H-6), 5.64
(1H, d, J = 2.5 Hz, H-1), 5.16 (1H, m, H-16), 3.45 (1H, m, H-10),
3.23 (1H, d, J = 14.3 Hz, H-12a), 2.75 (1H, d, J = 14.5 Hz, H-12b),
2.71 (1H, d, J = 7.7 Hz, H-17), 2.64 (1H, ddd, J = 17.4, 10.0, 6.0, H-
23a), 2.37 (ddt, J = 19.6, 8.5, 2.9 Hz, H-7b), 2.05 (2H, m, H-24),
2.04 (1H, d, J = 8.5 Hz, H-8), 2.03 (1H, overlapped, H-15b), 1.98
(3H, s, Me-32), 1.95 (3H, s, H-20), 1.96 (1H, m, H-7a), 1.49 (3H, s,
Me-26), 1.46 (3H, s, Me-27), 1.45 (3H, s, Me-21), 1.44 (1H, over-
lapped, H-15a), 1.35 (3H, s, Me-30), 1.31 (3H, s, Me-29), 1.23
(3H, s, Me-28), 1.03 (3H, s, Me-18), 1.00 (3H, s, Me-19); 13C NMR
(125.8 MHz, CDCl3): d = 213.0 (C-11), 212.3 (C-22), 198.4 (C-3),
170.1 (C-31), 170.0 (C-10), 137.6 (C-5), 136.9 (C-2), 119.6 (C-6),
112.6 (C-1), 81.0 (C-25), 78.6 (C-20), 74.1 (C-16), 54.1 (C-17),
50.0 (C-4), 49.1 (C-9), 48.8 (C-12), 47.9 (C-13), 47.9 (C-14), 43.5
(C-15), 41.3 (C-8), 35.1 (C-24), 34.9 (C-10), 30.4 (C-23), 27.8 (C-
28), 26.0 (C-27), 25.7 (C-26), 24.3 (C-21), 23.5 (C-7), 22.3 (C-32),
20.9 (C-20), 20.5 (C-29), 19.9 (C-19), 19.6 (C-18), 18.4 (C-30); ESI-
MS (negative ion mode) m/z 598.3391 [MꢀH]ꢀ (calcd for
4.3.18. Preparation of 21
Following the procedure described for 20, compound 21 was
obtained in 86% yield from 14. Mp: 131–132 °C; IR (KBr): 3510,
2978, 1729, 1693, 1633, 1370, 1250, 1125 cmꢀ1 1H-NMR (CDCl3):
;
d = 7.06 (1H, d, J = 15.6 Hz, H-24), 6.47 (1H, d, J = 15.6, H-23), 5.81
(1H, ddd, J = 2.0, 2.0, 6.0, H-6), 4.86 (1H, dd, J = 5.5, 13.0 Hz, H-2),
4.36 (1H, t, J = 7.1 Hz, H-16), 3.21 (1H, d, J = 14.5 Hz, H-12a), 2.72
(1H, m, H-10), 2.68 (1H, d, J = 14.5 Hz, H-12b), 2.51 (1H, ddd,
J = 3.6, 6.1, 13.0, Hz, H-1a), 2.49 (1H, d, J = 7.1 Hz, H-17), 2.41
(1H, m, H-7b), 2.01 (3H, s, Me-32), 1.99 (1H, m, H-7a), 1.97 (1H,
d, J = 8.0 Hz, H-8), 1.88 (1H, dd, J = 7.1, 13.0 Hz, H-15b), 1.86 (1H,
ddd, J = 13.0, 13.0, 13.0 Hz, H-1b), 1.57 (3H, s, Me-27), 1.55 (3H,
s, Me-26), 1.44 (3H, s, Me-21), 1.43 (1H, d, J = 13.0 Hz, H-15a),
1.38 (3H, s, Me-29), 1.33 (3H, s, Me-30), 1.29 (3H, s, Me-28), 1.09
(3H, s, Me-19), 0.98 (3H, s, Me-18); 13C NMR (CDCl3): d = 212.1
(C-11), 203.7 (C-3), 202.4 (C-22), 170.2 (C-31), 152.0 (C-24),
139.4 (C-5), 120.9 (C-6), 120.3 (C-23), 79.3 (C-25), 78.2 (C-20),
71.3 (C-16), 58.2 (C-17), 52.8, (C-2), 51.9 (C-4), 50.6 (C-14), 50.2
(C-4), 48.7 (C-12), 48.5 (C-9), 48.1 (C-13), 45.3 (C-15), 42.5 (C-8),
37.9 (C-1), 37.1 (C-10), 28.9 (C-28), 26.4, (C-27), 26.0 (C-26), 24.0
(C-21), 23.8 (C-7), 23.3 (C-29), 22.0 (C-32), 20.0 (C-19), 19.9 (C-
18), 18.8 (C-30); ESI-MS (negative ion mode) m/z 619.2292
[MꢀH]ꢀ (calcd for C32H44BrO7 619.2276).
C34H48NO8 598.3385).
4.3.21. Preparation of 24
Following the procedure described for 22, compound 24 was
obtained in 54% yield from 14. Mp: 121–122 °C; IR (KBr): 3468,
2979, 1733, 1695, 1651, 1371, 1254, 1127 cmꢀ1
;
1H NMR
(500 MHz, CDCl3): d = 7.06 (1H, d, J = 15.6 Hz, H-24), 6.47 (1H, d,
J = 15.6, H-23), 5.80 (1H, ddd, J = 2.5, 2.5, 5.0, H-6), 5.73 (1H, m,
H-1), 4.37 (1H, m, H-16), 3.49 (1H, s, H-10), 3.25 (1H, d,
J = 14.7 Hz, H-12a), 2.71 (1H, d, J = 14.5 Hz, H-12b), 2.49 (1H, d,
J = 7.0 Hz, H-17), 2.36 (1H, m, H-7b), 2.02 (1H, d, J = 7.8 Hz, H-8),
2.01 (1H, m, H-7a), 1.99 (3H, s, Me-32), 1.88 (1H, dd, J = 9.0,
13.0 Hz, H-15b), 1.57 (3H, s, Me-27), 1.54 (3H, s, Me-26), 1.56
(1H, overlapped, H-15a), 1.44 (3H, s, Me-21), 1.39 (3H, s, Me-30),
1.32 (3H, s, Me-29), 1.25 (3H, s, Me-28), 1.0 (3H, s, Me-19), 0.99
(3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3): d = 213.9 (C-11),
202.5 (C-22), 198.8 (C-3), 170.3 (C-31), 151.9 (C-24), 137.4 (C-5),
137.3 (C-2), 120.4 (C-23), 120.4 (C-6), 119.9 (C-1), 79.3 (C-25),
78.2 (C-20), 71.4 (C-16), 58.2 (C-17), 50.8 (C-14), 49.2 (C-9), 48.9
(C-12), 48.1 (C-13), 48.0 (C-4), 45.6 (C-15), 41.6 (C-8), 35.0 (C-
10), 27.9 (C-28), 26.4 (C-27), 25.9 (C-26), 24.0 (C-21), 23.6 (C-7),
22.0 (C-32), 20.4 (C-29), 20.0 (C-19), 19.9 (C-18), 18.4 (C-30);
ESI-MS (negative ion mode) m/z 554.3121 [MꢀH]ꢀ (calcd for
4.3.19. Preparation of 22
To a solution of 15 (80 mg, 0.11 mmol) in DMF (1.0 mL), sodium
azide (72 mg, 1.11 mmol) was added. The mixture was heated at
70 °C and after 1 h was diluted by adding 20.0 mL of diethyl ether
and washed with brine (2 ꢁ 25.0 mL). The organic phase was dried
with anhydrous Na2SO4 and evaporated under reduced pressure.
The resulting crude product was purified by chromatography on
silica gel (50% ethyl acetate/hexane) to afford 22 (37 mg, 60% yield)
as a white solid. Mp: 133–134 °C; IR (KBr): 3452, 3347, 2925, 1727,
1694, 1582, 1368, 1260, 1022 cmꢀ1; 1H NMR (CDCl3): d = 5.73 (1H,
ddd, J = 2.5, 2.5, 5.0, H-6), 5.64 (1H, d, J = 2.5 Hz, H-1), 4.33 (1H, t,
J = 7.1 Hz, H-16), 3.47 (1H, br s, H-10), 3.25 (1H, d, J = 14.7 Hz, H-
12a), 2.83 (1H, ddd, J = 17.4, 10.0, 6.0, H-23a), 2.71 (1H, d,
J = 14.5 Hz, H-12b), 2.53 (1H, d, J = 7.1 Hz, H-17), 2.53 (1H, m, H-
23b), 2.36 (1H, m, H-7b), 2.07 (2H, m, H-24), 2.02 (1H, d,
J = 7.8 Hz, H-8), 1.96 (3H, s, Me-32), 1.99 (1H, m, H-7a), 1.85 (1H,
dd, J = 9.0, 13.0 Hz, H-15b), 1.46 (3H, s, Me-27), 1.44 (3H, s, Me-
26), 1.43 (1H, m, H-15a), 1.42 (3H, s, Me-21), 1.40 (3H, s, Me-30),
C32H44NO7 554.3123).
4.3.22. Preparation of 25 and 26
To a solution of 1 (400 mg, 0.71 mmol) in 1,2-dichloroethane
(6.0 mL), 1,10-thiocarbonyldiimidazole (1.0 g, 5.68 mmol) was
added and the mixture was heated at 70 °C. After 6 h, the reaction
was cooled down, evaporated under reduced pressure and sub-
jected to column chromatography on silica gel (30% ethyl ace-
tate/hexane) to give 25 (200 mg, 42% yield) and 26 (250 mg, 45%
yield) as white solids. (25): Mp: 122–123 °C; IR: 3140, 2974,

3026
K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
1733, 1712, 1700, 1464, 1388, 1334, 1286, 1234, 986 cmꢀ1
;
1H
Me-19), 0.97 (3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3):
d = 213.9 (C-3), 213.6 (C-11), 213.0 (C-22), 170.3 (C-31), 140.7 (C-
5), 119.7 (C-6), 81.2 (C-25), 78.9 (C-20), 71.1 (C-16), 57.7 (C-17),
50.9 (C-4), 50.7 (C-13), 49.0 (C-9), 48.8 (C-12), 48.4 (C-14), 45.6
(C-15), 42.3 (C-8), 38.0 (C-2), 36.0 (C-10), 34.8 (C-24), 30.6 (C-
23), 28.5 (C-28), 26.1 (C-26), 25.8 (C-27), 24.5 (C-1), 24.5 (C-21),
23.9 (C-7), 22.9 (C-29), 22.4 (C-32), 19.8 (C-18), 19.6 (C-19), 18.7
(C-30); ESI-MS (negative ion mode) m/z 543.3353 [MꢀH]ꢀ (calcd
for C32H47O7 543.3327).
NMR (200 MHz, CDCl3): d = 8.41 (1H, s, H-40), 7.66 (1H, s, H-20),
7.06 (1H, s, H-30), 6.22 (1H, dd, J = 5.5, 13.6 Hz, H-2), 5.85 (1H,
ddd, J = 2.0, 2.0, 6.0, H-6), 4.32 (1H, m, H-16), 3.30 (1H, d,
J = 14.5 Hz, H-12a), 2.92 (1H, m, H-10), 2.83 (1H, m, H-23a), 2.71
(1H, d, J = 14.5 Hz, H-12b), 2.52 (1H, d, J = 6.9 Hz, H-17), 2.52 (1H,
m, H-23b), 2.43 (1H, m, H-7b), 2.37 (1H, m, H-1a), 2.05 (2H, m,
H-24), 2.0 (1H, d, J = 8.0 Hz, H-8), 1.97 (3H, s, Me-32), 1.91 (1H,
m, H-7a), 1.83 (1H, m, H-15b), 1.77 (1H, ddd, J = 13.0, 13.0,
13.0 Hz, H-1b), 1.46 (3H, s, Me-27), 1.44 (3H, s, Me-30), 1.44 (3H,
s, Me-26), 1.39 (3H, s, Me-21), 1.39 (3H, s, Me-28), 1.33 (3H, s,
Me-29), 1.28 (1H, overlapped, H-15a), 1.14 (3H, s, Me-19), 0.89
(3H, s, Me-18); 13C NMR (50 MHz, CDCl3): d = 213.7 (C-22), 212.3
(C-11), 203.3 (C-3), 183.2 (C-10), 170.3 (C-31), 138.9 (C-5), 136.9
(C-40), 130.3 (C-30), 121.2 (C-6), 118.0 (C-20), 81.2 (C-25), 81.2 (C-
16), 78.9 (C-20), 70.6 (C-2), 57.7 (C-17), 51.5 (C-4), 50.5 (C-13),
48.7 (C-12), 48.3 (C-9), 48.2 (C-14), 45.4 (C-15), 42.2 (C-8), 34.7
(C-24), 34.2 (C-10), 31.9 (C-1), 30.7 (C-23), 28.1 (C-28), 26.1 (C-
26), 25.8 (C-27), 24.5 (C-21), 23.8 (C-7), 22.4 (C-32), 21.1 (C-29),
20.0 (C-19), 19.9 (C-18), 18.6 (C-30); ESI-MS (positive ion mode)
m/z 671.3353 [M+H]+ (calcd for C36H51N2O8S 671.3361). (26) Mp:
134–135 °C; IR: 3130, 2974, 1728, 1712, 1697, 1464, 1393, 1329,
4.3.24. Preparation of 28
Following the procedure described for 27, compound 28 was
obtained in 19% yield from 26 as a colourless oil. Mp: 98–99 °C;
IR (KBr): 3439, 2974, 1730, 1714, 1693, 1370, 1254, 1213, 1123,
1020, 950 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.76 (1H, ddd,
;
J = 2.0, 2.0, 6.0, H-6), 3.12 (1H, dd, J = 0.8, 14.3 Hz, H-12a), 2.67
(1H, J = 14.3 Hz, H-12b), 2.56 (1H, overlapped, H-10), 2.54 (2H,
overlapped, H-23), 2.40 (2H, overlapped, H-2), 2.40 (1H, over-
lapped, H-7b), 2.35 (1H, t, J = 7.5 Hz, H-17), 2.04 (2H, overlapped,
H-24), 2.02 (1H, m, H-8), 1.99 (1H, m, H-7a), 1.97 (3H, s, Me-32),
1.86 (1H, ddd, J = 14.0, 9.0, 4.5 Hz, H-1a), 1.64 (1H, overlapped,
H-16), 1.52 (1H, m, H-1b), 1.47 (3H, s, Me-27), 1.44 (3H, s, Me-
26), 1.42 (3H, s, Me-21), 1.39 (2H, m, H-15), 1.36 (1H, m, H-16),
1.27 (3H, s, Me-29), 1.23 (3H, s, Me-28), 1.13 (3H, s, Me-30), 1.09
(3H, s, Me-19), 0.99 (3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3):
d = 213.9 (C-3), 213.7 (C-11), 213.1 (C-22), 170.4 (C-31), 140.8 (C-
5), 120.1 (C-6), 81.3 (C-25), 80.1 (C-20), 51.0 (C-4), 50.5 (C-14),
49.1 (C-13), 49.0 (C-9), 49.0 (C-12), 48.8 (C-17), 42.5 (C-8), 38.2
(C-2), 36.2 (C-10), 35.3 (C-24), 34.1 (C-15), 30.3 (C-23), 28.5 (C-
28), 26.1 (C-26), 26.0 (C-27), 24.7 (C-21), 24.5 (C-1), 24.0 (C-7),
23.2 (C-29), 22.5 (C-32), 21.5 (C-16), 19.6 (C-19), 19.0 (C-18),
18.4 (C-30). ESI-MS (positive ion mode) m/z 551.3343 [M+Na]+
(calcd for C32H48NaO6 551.3342).
1286, 1246, 1231, 981, 734 cmꢀ1 1H NMR (200 MHz, CDCl3):
;
d = 8.49 (1H, s, H-400), 8.40 (1H, s, H-40), 7.69 (1H, s, H-200), 7.59
(1H, s, H-20), 7.10 (2H, s, H-30/H-300), 6.22 (1H, dd, J = 5.0, 13.0 Hz,
H-2), 6.04 (1H, m, H-16), 5.83 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 3.37
(1H, d, J = 14.5 Hz, H-12a), 3.08 (1H, d, J = 5.5 Hz, H-17), 2.92 (1H,
m, H-10), 2.81 (1H, d, J = 14.5 Hz, H-12b), 2.63 (2H, m, H-23a),
2.5 (1H, overlapped, H-7b), 2.45 (1H, overlapped, H-1a), 2.1 (1H,
d, J = 8.0 Hz, H-8), 2.05 (2H, m, H-24), 1.97 (3H, s, Me-32), 1.91
(1H, m, H-7a), 1.83 (1H, m, H-15b), 1.73 (1H, ddd, J = 13.0, 13.0,
13.0 Hz, H-1b), 1.97 (3H, s, Me-32), 1.50 (3H, s, Me-30), 1.39 (3H,
s, Me-21), 1.39 (3H, s, Me-26), 1.35 (3H, s, Me-21), 1.33 (1H, over-
lapped, H-15a), 1.31 (3H, s, Me-28), 1.31 (3H, s, Me-29), 1.17 (3H, s,
Me-19), 1.08 (3H, s, Me-18); 13C NMR (50 MHz, CDCl3): d = 214.2
(C-22), 211.4 (C-11), 203.1 (C-3), 183.2 (C-10), 182.4 (C-100), 170.1
(C-31), 139.0 (C-5), 136.9 (C-40), 136.9 (C-400), 136.6 (C-), (C-),
130.6 (C-30), 130.5 (C-300), 120.8 (C-6), 118.0 (C-20), 117.4 (C-200),
83.6 (C-2), 80.9 (C-16), 80.8 (C-25), 79.2 (C-20), 54.9 (C-17), 51.4
(C-4), 50.0 (C-13), 48.4 (C-12), 48.1 (C-9), 47.7 (C-14), 42.7 (C-
15), 41.9 (C-8), 34.8 (C-24), 34.1 (C-10), 31.8 (C-1), 31.5 (C-23),
27.9 (C-28), 25.5 (C-26), 25.4 (C-27), 24.5 (C-21), 23.7 (C-7), 22.3
(C-32), 21.0 (C-29), 20.0 (C-19), 19.7 (C-18), 18.7 (C-30); ESI-MS
(positive ion mode) m/z 781.3302 [M+H]+ (calcd for C40H53N4O8S2
781.3299).
4.3.25. Preparation of 29
A solution of compound 10 (800 mg, 1.04 mmol) in anhydrous
THF (15.0 mL) was cooled at 0 °C and stirred for 5 min. Lithium tri-
tert-butoxyaluminium hydride (1.59 g, 6.24 mmol) was then added
and the mixture was stirred on an ice bath under nitrogen atmo-
sphere. After 2 h, the mixture was diluted by adding 100.0 mL of
EtOAc, washed with of saturated solution of sodium potassium tar-
trate (3 ꢁ 50.0 mL) and water (2 ꢁ 40.0 mL). The organic phase
was thendried withNa2SO4 and evaporatedunder reducedpressure.
The resulting crude product was purified by chromatography on sil-
ica gel (70% ethyl acetate/hexane) to afford 29 (750 mg, 93% yield) as
a white solid. Mp: 137–138 °C; IR (KBr): 3515, 2975, 1722, 1713,
1694, 1688, 1601, 1583, 1371, 1315, 1275, 1214, 1175, 1113, 1070,
4.3.23. Preparation of 27
To a solution of 25 (190 mg, 0.28 mmol) in toluene (7 mL),
diphenylsilane (0.4 mL, 2.24 mmol) and lauroyl peroxide
(6 ꢁ 0.2 mL of a solution 0.160 g/mL in toluene, each 15 min) were
added. The reaction mixture was stirred under argon atmosphere
at 115 °C for 1.5 h. After cooling, the solution was evaporated un-
der reduced pressure and subjected to column chromatography
on silica gel to obtain 27 (45 mg, 67% yield) as a white solid. Mp:
108–109 °C; IR (KBr): 3439, 2974, 1730, 1714, 1693, 1370, 1254,
1025, 966, 714 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 8.02 (4H, m,
;
H-30/H-70/H-300/H-700), 7.58 (2H, m, H-50/H-500), 7.45 (4H, m, H-40/H-
400/H-60/H-600), 5.75 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.71 (1H, dd,
J = 7.5, 7.5 Hz, H-16), 5.12 (1H, ddd, J = 11.5, 10.0, 4.3 Hz, H-2), 3.33
(1H, d, J = 10.0 Hz, H-3), 3.23 (1H, dd, J = 10.0, 1.3 Hz, H-22), 3.19
(1H, d, J = 12.7 Hz, H-12a), 2.90 (1H, d, J = 7.7 Hz, H-17), 2.67 (1H,
d, J = 12.7 Hz, H-12b), 2.50 (1H, m, H-10), 2.43 (1H, ddt, J = 19.6,
8.5, 2.9 Hz, H-7b), 2.12 (1H, dd, J = 14.0, 7.5 Hz, H-15b), 2.01 (1H,
overlapped, H-1a), 2.0 (1H, d, J = 8.5 Hz, H-8), 1.94 (3H, s, Me-32),
1.93 (1H, overlapped, H-7a), 1.74 (1H, m, H-23a), 1.61 (1H, over-
lapped, H-23b), 1.60 (1H, overlapped, H-24a), 1.59 (1H, d,
J = 14 Hz, H-15a), 1.44 (1H, m, H-24b), 1.36 (3H, Me-26), 1.36 (3H,
Me-27), 1.35 (3H, Me-30), 1.32 (1H, overlapped, H-1b), 1.31 (3H,
Me-21), 1.22 (3H, Me-28), 1.12 (3H, Me-19), 1.05 (3H, Me-29), 1.04
(3H, Me-18); 13C NMR (125.8 MHz, CDCl3): d = 212.6 (C-11), 170.4
(C-31), 166.9 (C-100), 166.0 (C-10), 140.0 (C-5), 133.2 (C-500), 133.2
(C-50), 130.3 (C-200), 130.0 (C-20), 129.7 (C- C-300/C-700), 129.3 (C- C-
30/C-70), 128.6 (C- C-400/C-600), 128.3 (C-40/C-60), 119.6 (C-6), 82.1
(C-25), 79.3 (C-22), 78.4 (C-3), 76.0 (C-20), 75.8 (C-16), 74.9 (C-2),
1213, 1123, 1020, 950 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.77
;
(1H, ddd, J = 2.0, 2.0, 6.0, H-6), 4.31 (1H, m, H-16), 3.24 (1H, d,
J = 14.5 Hz, H-12a), 2.82 (1H, m, H-23a), 2.67 (1H, d, J = 14.3 Hz,
H-12b), 2.59 (1H, m, H-10), 2.53 (1H, d, J = 7.5 Hz, H-17), 2.51
(1H, m, H-23b), 2.46 (1H, overlapped, H-2a), 2.40 (1H, overlapped,
H-7b), 2.39 (1H, overlapped, H-2b), 2.06 (2H, m, H-24), 1.98 (1H,
ddd, J = 19.6, 5.0, 1.5 Hz, H-7a), 1.96 (3H, s, Me-32), 1.96 (1H, d,
J = 8.5 Hz, H-8), 1.89 (1H, m, H-1a), 1.85 (1H, dd, J = 13.3, 9.0 Hz,
H-15b), 1.53 (1H, m, H-1b), 1.46 (3H, s, Me-26), 1.44 (3H, s, Me-
27), 1.42 (3H, s, Me-21), 1.41 (1H, d, J = 13.3 Hz, H-15a), 1.37 (3H,
s, Me-30), 1.27 (3H, s, Me-29), 1.24 (3H, s, Me-28), 1.09 (3H, s,

K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
3027
52.5 (C-17), 50.6 (C-13), 48.9 (C-12), 48.1 (C-9), 47.6 (C-14), 44.0 (C-
15), 42.6 (C-8), 42.4 (C-4), 38.7 (C-23), 33.8 (C-10), 30.6 (C-1), 26.1
(C-26), 25.7 (C-27), 24.5 (C-28), 23.7 (C-7), 22.6 (C-32), 22.4 (C-21),
21.5 (C-29), 20.2 (C-19), 19.8 (C-18), 18.5 (C-30); ESI-MS
(positive ion mode) m/z 795.4088 [M+Na]+ (calcd for C46H60NaO10
795.4079).
128.5 (C-400/C-600), 128.4 (C-40/C-60), 120.3 (C-6), 75.4 (C-16), 73.7
(C-2), 64.0 (C-17), 51.4 (C-4), 49.7 (C-14), 48.8 (C-9), 48.5 (C-13),
46.9 (C-12), 43.3 (C-15), 42.7 (C-8), 34.4 (C-10), 32.1 (C-1), 31.5
(C-21), 28.7 (C-28), 23.9 (C-7), 21.3 (C-29), 20.1 (C-19), 19.9 (C-
18), 18.7 (C-30); ESI-MS (positive ion mode) m/z 633.2838
[M+Na]+ (calcd for C38H42NaO7 633.2823).
4.3.26. Preparation of 30
4.3.28. Preparation of 32
To a solution of 29 (700 mg, 0.91 mmol) in MeOH (14.0 mL),
H5IO6 (622 mg, 2.73 mmol) was added with stirring at 20 °C. After
1 h, the reaction was diluted with 50.0 mL of EtOAc and poured
over aq 5% NaHSO3. The organic phase was dried with anhydrous
Na2SO4 and evaporated under reduced pressure. The resulting
crude product was purified by chromatography on silica gel (60%
ethyl acetate/hexane) to afford 30 (480 g, 86% yield) as a white so-
lid. Mp: 129ꢀ130 °C; IR (KBr): 3449, 2979, 1737, 1730, 1714, 1693,
Compound 31 (180 mg, 0.29 mmol) was stirred at 20 °C with
triethyl orthoformate (0.96 mL, 5.8 mmol), ethylene glycol
(0.65 mL, 11.6 mmol) and a catalytic amount of p-toluenesulfonic
acid. After 3 h the reaction mixture was diluted with EtOAc and
washed with brine (2 ꢁ 25.0 mL) and water (2 ꢁ 25.0 mL). The or-
ganic phase was dried with anhydrous Na2SO4 and evaporated un-
der reduced pressure. The resulting crude product was purified by
chromatography on silica gel (40% ethyl acetate/hexane) to afford
32 (160 mg, 79% yield) as white solid. Mp: 152–153 °C; IR (KBr):
1368, 1247, 1118, 1022, 950 cmꢀ1 1H NMR (500 MHz, CDCl3):
;
d = 8.04 (2H, dd, J = 8.5, 1.3 Hz, H-30/H-70), 7.99 (2H, dd, J = 8.5,
1.3 Hz, H-300/H-700), 7.59 (2H, m, H-50/H-500), 7.44 (4H, m, H-40/H-
400/H-60/H-600), 5.90 (1H, ddd, J = 9.0, 6.5, 1.2 Hz, H-16), 5.78 (1H,
ddd, J = 2.0, 2.0, 6.0, H-6), 5.13 (1H, ddd, J = 11.5, 10.0, 4.3 Hz, H-
2), 3.48 (1H, d, J = 6.5 Hz, H-17), 3.34 (1H, dd, J = 10.0, 3.5 Hz, H-
3), 3.30 (1H, dd, J = 14.5, 0.8, Hz, H-12a), 2.52 (1H, m, H-10), 2.52
(1H, d, J = 14.5 Hz, H-12b), 2.47 (ddt, J = 19.5, 7.8, 3.0 Hz, H-7b),
2.20 (3H, Me-21), 2.16 (1H, dd, J = 14.0, 9.0 Hz, H-15b), 2.0 (1H,
J = 7.8 Hz, H-8), 2.0 (1H, overlapped, H-1a), 1.97 (1H, overlapped,
H-7a), 1.70 (1H, br d, J = 14.0 Hz, H-15a), 1.36 (3H, Me-30), 1.33
(1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b), 1.25 (3H, Me-28), 1.13
(3H, Me-19), 1.07 (3H, Me-29), 0.76 (3H, Me-18); 13C NMR
(125.8 MHz, CDCl3): d = 210.9 (C-11), 206.1 (C-20), 167.1 (C-100),
166.0 (C-10), 140.3 (C-5), 133.3 (C-500), 133.3 (C-50), 130.1 (C-20),
130.1 (C-200), 129.8 (C-30/C-70), 129.7 (C-300/C-700), 128.6 (C-40/C-
60), 128.5 (C-400/C-600), 119.5 (C-6), 78.5 (C-3), 75.5 (C-16), 75.0
(C-2), 64.2 (C-17), 49.9 (C-13), 48.8 (C-9), 48.6 (C-14), 47.1 (C-
12), 43.5 (C-15), 43.0 (C-8), 42.8 (C-4), 33.9 (C-10), 31.6 (C-21),
30.7 (C-1), 24.7 (C-28), 24.0 (C-7), 21.6 (C-29), 20.3 (C-19), 20.0
(C-18), 18.9 (C-30); ESI-MS (negative ion mode) m/z 611.3014
[MꢀH]ꢀ (calcd for C38H43O7 611.3014).
2983, 1721, 1714, 1696, 1271, 1173, 1115, 714 cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d = 8.07 (2H, m, H-30/H-70), 8.03 (2H, m, H-300/
H-700), 7.56 (2H, m, H-50/H-500), 7.44 (4H, m, H-40/H-400/H-60/H-600),
5.79 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.76 (1H, m, H-16), 5.75 (1H,
dd, J = 6.0, 13.0 Hz, H-2), 4.04 (2H, m, H-1000), 3.80 (2H, m, H-2000),
3.18 (1H, d, J = 15.0, H-12a), 2.93 (1H, d, J = 6.7 Hz, H-17), 2.91
(1H, m, H-10), 2.70 (1H, d, J = 14.8 Hz, H-12b), 2.46 (ddt, J = 19.5,
8.0, 2.9 Hz, H-7b), 2.29 (1H, ddd, J = 13.0, 6.0, 3.5 Hz, H-1b), 2.14
(1H, dd, J = 9.0, 14.0, Hz, H-15b), 2.08 (1H, d, J = 8.0 Hz, H-8), 1.99
(1H, ddd, J = 20.0, 6.0, 1.5 Hz, H-7a), 1.71 (1H, ddd, J = 13.0, 13.0,
13.0 Hz, H-1b), 1.60 (1H, d, J = 14.0 Hz, H-15a), 1.43 (3H, s, Me-
30), 1.39 (3H, s, Me-28), 1.32 (3H, s, Me-21), 1.30 (3H, s, Me-29),
1.15 (3H, s, Me-19), 0.94 (3H, s, Me-18); 13C NMR (125.8 MHz,
CDCl3): d = 212.5 (C-11), 205.5 (C-3), 166.1 (C-1’’), 165.7 (C-10),
139.8 (C-5), 133.2 (C-50), 132.9 (C-500), 130.5 (C-20), 129.9 (C-300/C-
700), 129.5 (C-30/C-70), 129.5 (C-), 128.4 (C-), 128.3 (C-), 120.5 (C-),
110.6 (C-20), 75.2 (C-16), 73.9 (C-2), 65.2 (C-1000), 62.8 (C-2000),
57.2 (C-17), 51.4 (C-4), 48.8 (C-12), 48.5 (C-13), 47.8 (C-14), 47.7
(C-9), 43.7 (C-15), 42.4 (C-8), 34.4 (C-10), 32.2 (C-1), 28.7 (C-28),
24.5 (C-21), 23.9 (C-7), 21.3 (C-29), 20.1 (C-19), 19.3 (C-18), 18.4
(C-30); ESI-MS (positive ion mode) m/z 655,3282 [M+H]+ (calcd
for C30H47O8 655.3265).
4.3.27. Preparation of 31
A mixture of PCC (200 mg, 1.23 mmol) and BaCO3 (250 mg,
1.23 mmol) in dry CH2Cl2 (5.0 mL) was stirred for 5 min. Com-
pound 30 (250 mg, 0.41 mmol) dissolved in 2 mL of CH2Cl2 was
then added and the reaction was stirred at 20 °C. After 20 h the
reaction mixture was diluted with diethyl ether (30.0 mL) and
poured through a short column of FlorisilÒ. The solvent was re-
moved under vacuum and the residue was purified by column
chromatography on silica (50% ethyl acetate/hexane) to give pure
product 31 (195 mg, 78% yield) as a white solid. Mp: 156–157 °C;
IR (KBr): 2974, 1973, 1917, 1737, 1720, 1699, 1692, 1275, 1205,
4.3.29. Preparation of 33
Compound 32 (100 mg, 0.143 mmol) was dissolved in 1 M KOH
methanolic solution (3.0 mL) with stirring at 20 °C. After 1 h, the
reaction was diluted with EtOAc and washed with brine
(2 ꢁ 25.0 mL), water (2 ꢁ 25.0 mL) and 1 M HCl solution
(3 ꢁ 30.0 mL). The organic phase was dried with anhydrous Na2SO4
and evaporated under reduced pressure. The resulting crude prod-
uct was purified by chromatography on silica gel (50% ethyl ace-
tate/hexane) to afford 33 (30 mg, 52% yield) as a white solid. Mp:
95–96 °C; IR (KBr): 3563, 3458, 2978, 1704, 1692, 1650, 1399,
1175, 1114, 1026, 978, 713 cmꢀ1
;
1H NMR (500 MHz, CDCl3):
1362, 1219, 1191, 1100, 1089, 1039, 1025 cmꢀ1 1H NMR (CDCl3):
;
d = 8.1 (2H, dd, J = 8.5, 1.3 Hz, H-30/H-70), 8.03 (2H, dd, J = 8.5,
1.3 Hz, H-300/H-700), 7.60 (2H, m, H-50/H-500), 7.47 (4H, m, H-40/H-
400/H-60/H-600), 5.91 (1H, ddd, J = 8.6, 6.5, 1.2 Hz, H-16), 5.82 (1H,
ddd, J = 2.0, 2.0, 6.0, H-6), 5.75 (dd, J = 13.0, 5.5 Hz, H-2), 3.50
(1H, d, J = 6.5 Hz, H-17), 3.36 (1H, dd, J = 14.5, 0.9 Hz, H-12a),
2.93 (1H, m, H-10), 2.61 (1H, d, J = 14.5 Hz, H-12b), 2.49 (ddt,
J = 19.6, 8.5, 2.9 Hz, H-7b), 2.28 (1H, ddd, J = 13.0, 5.5, 3.5 Hz, H-
1a), 2.22 (3H, s, Me-21), 2.19 (1H, dd, J = 13.8, 9.0 Hz, H-15b),
2.07 (1H, d, J = 8.0 Hz, H-8), 2.02 (1H, ddd, J = 19.5, 6.0, 1.1 Hz, H-
7a), 1.74 (1H, overlapped, H-15a), 1.72 (1H, ddd, J = 13.0, 13.0,
13.0 Hz, H-1b), 1.45 (3H, s, Me-30), 1.39 (3H, s, Me-28), 1.32 (3H,
s, Me-29), 1.16 (3H, s, Me-19), 0.8 (3H, s, Me-18); 13C NMR
(125.8 MHz, CDCl3): d = 210.7 (C-11), 205.8 (C-20), 205.3 (C-3),
165.9 (C-1’’), 165.7 (C-10), 139.8 (C-5), 133.3 (C-50), 133.2 (C-5’’),
129.9 (C-200), 129.9 (C-300/C-700), 129.5 (C-20), 129.5 (C-30/C-70),
d = 5.90 (1H, d, J = 2.4 Hz, H-1), 5.78 (1H, ddd, J = 2.5, 2.5, 5.0 Hz, H-
6), 4.98 (1H, ddd, J = 1.2, 6.5, 9.0 Hz, H-16), 3.53 (1H, m, H-10), 3.29
(1H, dd, J = 1.1, 14.3 Hz, H-12a), 3.16 (1H, d, J = 6.5 Hz, H-17), 2.56
(1H, J = 14.3 Hz, H-12b), 2.40 (ddt, J = 2.9, 8.5, 19.6 Hz, H-7b), 2.18
(3H, s, Me-21), 2.02 (ddd, J = 1.5, 4.9, 19.6 Hz, H-7a), 2.01 (d,
J = 8.5 Hz, H-8), 1.95 (ddd, J = 0.7, 9.2, 13.5 Hz, C-15b), 1.56 (dd,
J = 1.2, 13.5 Hz, C-15a), 1.42 (3H, s, Me-30), 1.36 (3H, s, Me-29),
1.26 (3H, s, Me-28), 1.03 (3H, s, Me-19), 0.7 (3H, s, Me-18); 13C
NMR (125.8 MHz, CDCl3): d = 211.8 (C-11), 207.9 (C-20), 198.7
(C-3), 144.7 (C-2), 137.0 (C-5), 120.7 (C-1), 114.6 (C-6), 71.8 (C-
16), 67.6 (C-17), 50.0 (C-9), 49.4 (C-13), 48.9 (C-14), 47.7 (C-4),
47.4 (C-12), 45.3 (C-15), 42.1 (C-8), 34.8 (C-10), 31.6 (C-21), 28.0
(C-28), 23.8 (C-7), 20.3 (C-29), 20.2 (C-19), 19.9 (C-18), 18.5 (C-
30); ESI-MS (positive ion mode) m/z 401.2324 [M+H]+ (calcd for
C24H33O5 401.2322).

3028
K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
4.3.30. Preparation of 34
(1H, dd, J = 16.0, 6.0 Hz, H-23), 5.76 (1H, ddd, J = 2.0, 2.0, 6.0, H-
6), 4.63 (1H, m, H-16), 3.97 (1H, dd, J = 6.0, 1.2 Hz, H-22), 3.55
(1H, ddd, J = 11.5, 9.3, 4.2 Hz, H-2), 3.24 (1H, d, J = 14.6 Hz, H-
12a), 2.86 (1H, d, J = 9.3, H-3), 2.49 (1H, m, H-10), 2.48 (1H, d,
J = 14.6 Hz, H-12b), 2.43 (1H, m, H-7b), 2.37 (1H, d, J = 6.5 Hz, H-
17), 2.0 (1H, overlapped, H-7a), 1.96 (3H, s, Me-32), 1.95 (1H, d,
J = 8.2 Hz, H-8), 1.89 (1H, dd, J = 12.2, 9.0 Hz, H-15b), 1.78 (1H, dt,
J = 12.4, 4.2 Hz, H-1a), 1.53 (3H, s, Me-27), 1.52 (3H, s, Me-26),
1.50 (1H, overlapped, H-15a), 1.30 (3H, s, Me-30), 1.23 (3H, s,
Me-21), 1.18 (3H, s, Me-28), 1.07 (3H, s, Me-19), 0.96 (3H, s, Me-
29), 0.94 (3H, s, Me-18); 13C NMR (125.8 MHz, CD3OD): d = 216.6
(C-11), 171.9 (C-31), 142.7 (C-5), 137.4 (C-24), 128.8 (C-23),
120.0 (C-6), 81.9 (C-3), 81.5 (C-22), 77.2 (C-20), 72.3 (C-16), 71.6
(C-2), 56.4 (C-17), 52.5 (C-13), 50.0 (C-9), 49.6 (C-12), 49.1 (C-
14), 45.6 (C-15), 44.2 (C-8), 43.4 (C-4), 34.9 (C-10), 34.7 (C-1),
27.5 (C-26), 27.3 (C-27), 25.3 (C-28), 24.8 (C-7), 24.2 (C-21), 22.3
(C-32), 22.3 (C-29), 20.4 (C-19), 20.3 (C-18), 19.6 (C-30); ESI-MS
(negative ion mode) m/z 561.3438 [MꢀH]ꢀ (calcd for C32H49O8
561.3433).
Following the procedure described for 32, compound 34 was
obtained in 88% yield from 30. Mp: 145–146 °C; IR (KBr): 3563,
2978, 1713, 1694, 1601, 1581, 1378, 1314, 1278, 1177, 1114,
1068, 886, 711 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 8.04 (2H, dd,
;
J = 8.5, 1.3 Hz, H-30/H-70), 8.03 (2H, dd, J = 8.5, 1.3 Hz, H-300/H-700),
7.56 (2H, m, H-50/H-500), 7.44 (4H, m, H-40/H-400/H-60/H-600), 5.76
(1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.73 (1H, m, H-16), 5.13 (1H, ddd,
J = 4.5, 10.0, 12.0 Hz, H-2), 4.04 (2H, m, H-1000), 3.79 (2H, m, H-
2000), 3.32 (1H, d, J = 10.0 Hz, H-3), 3.11 (1H, d, J = 14.8, H-12a),
2.91(1H, d, J = 6.5 Hz, H-17), 2.61 (1H, d, J = 14.8 Hz, H-12b), 2.50
(1H, m, H-10), 2.44 (ddt, J = 19.5, 8.0, 2.9 Hz, H-7b), 2.10 (1H, dd,
J = 9.0, 14.0, Hz, H-15b), 2.0 (1H, d, J = 8.0 Hz, H-8), 1.99 (1H, over-
lapped, H-1a), 1.94 (1H, dd, J = 19.5, 5.5 Hz, H-7a), 1.57 (1H, d,
J = 14.0 Hz, H-15a), 1.35 (3H, s, Me-30), 1.32 (1H, ddd, J = 13.0,
13.0, 13.0 Hz, H-1b), 1.30 (3H, s, Me-21), 1.23 (3H, s, Me-28),
1.12 (3H, s, Me-19), 1.06 (3H, s, Me-29), 0.90 (3H, s, Me-18); 13C
NMR (125.8 MHz, CDCl3): d = 212.6 (C-11), 167.0 (C-1’’), 166.2 (C-
10), 140.1 (C-5), 133.1 (C-50), 132.9 (C-500), 130.6 (C-20), 130.0 (C-
200), 129.7 (C-300/C-700), 129.5 (C-30/C-70), 128.4 (C-400/C-600), 128.3
(C-40/C-60), 119.5 (C-6), 110.7 (C-20), 78.5 (C-3), 75.2 (C-16), 75.0
(C-2), 65.2 (C-1000), 62.8 (C-2000), 57.2 (C-17), 48.8 (C-13), 48.3 (C-
14), 47.8 (C-12), 47.6 (C-9), 43.7 (C-15), 42.6 (C-8), 33.8 (C-10),
30.6 (C-1), 24.5 (C-21), 24.5 (C-28), 23.8 (C-7), 21.5 (C-29), 20.2
(C-19), 19.2 (C-18), 18.5 (C-30); ESI-MS (positive ion mode) m/z
657.3441 [M+H]+ (calcd for C40H49O8 657.3422).
4.3.33. Preparation of 37
To a solution of 6 (100 mg, 0.15 mmol) in anhydrous CH2Cl2,
DBU (1.16 mL,7.75 mmol) and trifluoroacetic anhydride (1.08 mL,
7.75 mmol) were added with stirring at 0 °C under nitrogen atmo-
sphere. After 10 h, the reaction was diluted by adding icewater
(20.0 mL). The aqueous phase was extracted with CH2Cl2
(3 ꢁ 20 mL) and the combined organic layers were washed with
HCl 1 M (2 ꢁ 25.0 mL) and brine (2 ꢁ 25.0 mL). The organic phase
was dried with anhydrous Na2SO4 and evaporated under reduced
pressure. The resulting crude product was purified by chromatog-
raphy on silica gel (40% ethyl acetate/hexane) to afford 37 (48 mg,
42% yield) as a white solid. Mp: 86–87 °C; IR (KBr): 2976, 1788,
1741, 1731, 1695, 1369, 1246, 1215, 1170, 1149, 1113, 1082,
4.3.31. Preparation of 35
Compound 34 (150 mg, 0.23 mmol) was dissolved in 1 M KOH
methanolic solution (3.0 mL) with stirring at 20 °C. After 1 h, the
reaction was diluted with EtOAc and washed with brine
(2 ꢁ 25.0 mL), water (2 ꢁ 25.0 mL) and 1 M HCl solution
(3 ꢁ 30.0 mL). The organic phase was dried with anhydrous Na2SO4
and evaporated under reduced pressure. The resulting crude prod-
uct was purified by chromatography on silica gel (50% ethyl ace-
tate/hexane) to afford 35 (60 mg, 64% yield) as a white solid. Mp:
145–146 °C; IR (KBr): 3491, 1695, 1681, 1363, 1267, 1192, 1092,
1048, 1028, 977 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.79 (1H,
;
ddd, J = 2.0, 2.0, 6.0, H-6), 5.48 (1H, dd, J = 5.5, 13.0 Hz, H-2), 5.28
(1H, m, H-16), 3.27 (1H, d, J = 14.5 Hz, H-12a), 2.79 (1H, m, H-10)
2.75 (1H, d, J = 7.5, H-17), 2.66 (H, d, J = 14.5 Hz, H-12b), 2.60
(1H, m, H-23a), 2.53 (1H, m, H-23b), 2.45 (1H, m, H-7b), 2.15
(3H, s, Me-20), 2.12 (1H, overlapped, H-1a), 2.06 (1H, overlapped,
H-15b), 2.04 (2H, m, H-24), 2.02 (1H, d, J = 7.5 Hz, H-8), 1.99 (3H,
s, Me-32), 1.96 (1H, m, H-7a), 1.95 (3H, s, Me-200), 1.94 (3H, s,
Me-21), 1.54 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b), 1.48 (3H, s,
Me-27), 1.46 (1H, m, H-15a), 1.45 (3H, s, Me-26), 1.32 (3H, s,
Me-30), 1.31 (3H, s, Me-28), 1.29 (3H, s, Me-29), 1.10 (3H, s, Me-
19), 1.02 (3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3) d 210.8 (C-
11), 205.4 (C-3), 202.1 (C-22), 170.2 (C-31), 170.1 (C-10), 169.7
(C-100), 155.5 (C-1000), 139.7 (C-5), 120.3 (C-6), 114.3 (C-2000), 90.6
(C-20), 80.9 (C-25), 73.1 (C-2), 73.1 (C-16), 55.0 (C-17), 51.2 (C-
4), 50.2 (C-13), 48.6 (C-12), 48.3 (C-9), 47.6 (C-14), 43.1 (C-15),
42.1 (C-8), 34.6 (C-24), 34.3 (C-10), 32.1 (C-23), 31.9 (C-1), 28.7
(C-28), 26.0 (C-26), 25.8 (C-27), 23.6 (C-7), 22.3 (C-32), 21.3 (C-
29), 21.0 (C-20), 20.9 (C-200), 20.6 (C-20), 20.0 (C-19), 19.8 (C-18),
18.4 (C-30); ESI-MS (negative ion mode) m/z 739. 3280 [MꢀH]ꢀ
(calcd for C38H50F3O11 739.3311).
1058, 1028 cmꢀ1 1H NMR (500 MHz, CDCl3): d = 5.74 (1H, ddd,
;
J = 2.0, 2.0, 6.0, H-6), 4.95 (1H, ddd, J = 9.1, 6.5, 1.6 Hz, H-16), 3.60
(1H, ddd, J = 4.3, 9.2, 9.2 Hz, H-2), 3.25 (1H, dd, J = 1.15, 14.3 Hz,
H-12a), 3.15 (1H, d, J = 6.5 Hz, H-17), 2.98 (1H, d, J = 9.2 Hz, H-3),
2.45 (1H, d, J = 14.3 Hz, H-12b), 2.42 (1H, m, H-7b), 2.36 (1H, m,
H-10), 2.16 (3H, s, Me-21), 1.95 (1H, m, H-7a), 1.94 (1H, dd,
J = 9.0, 13.0 Hz, H-15b), 1.90 (1H, J = 7.5 Hz, H-8), 1.90 (1H, m, H-
1a), 1.50 (1H, dd, J = 7.1, 13.0 Hz, H-15a), 1.30 (3H, s, Me-30),
1.21 (3H, s, Me-28), 1.10 (1H, m, H-1b), 1.10 (3H, s, Me-19), 0.97
(3H, s, Me-29), 0.66 (3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3):
d = 211.6 (C-11), 208.1 (C-20), 140.7 (C-5), 119.1 (C-6), 80.9 (C-3),
71.7 (C-16), 70.9 (C-2), 67.6 (C-17), 49.9 (C-13), 48.8 (C-9), 48.6
(C-14), 47.1 (C-12), 45.0 (C-15), 43.0 (C-8), 41.9 (C-4), 33.9 (C-
10), 33.3 (C-1), 31.5 (C-21), 24.6 (C-28), 23.8 (C-7), 21.5 (C-29),
20.4 (C-19), 19.7 (C-18), 19.1 (C-30); ESI-MS (negative ion mode)
m/z 405.2616 [MꢀH]ꢀ (calcd for C24H37O5 405.2635).
4.3.32. Preparation of 36
A mixture of 2 (100 mg, 0.18 mmol) and CeCl3.7H2O (200 mg,
0.537 mmol) in MeOH (2.5 mL) was cooled at -30 °C and NaBH4
(81 mg, 2.15 mmol) was then added. After stirring for 2 h, the reac-
tion was diluted by adding 20.0 mL of EtOAc and washed with
brine (2 ꢁ 20.0 mL). The organic phase was dried with anhydrous
Na2SO4 and evaporated under reduced pressure. The resulting
crude product was purified by chromatography on silica gel using
ethyl acetate as mobile phase to afford 36 (72 mg, 72%) as a white
solid. Mp: 199–200 °C; IR (KBr): 3448, 3279, 1732, 1717, 1698,
4.3.34. Preparation of 38
Iodine (79 mg, 0.31 mmol) was added to a solution of Ph3P
(81 mg, 0.31 mmol) in CH2Cl2 (0.5 mL) and the mixture was stirred
at room temperature for 10 min.
A solution of 6 (100 mg,
0.155 mmol) in CH2Cl2 (1.0 mL) was then added and the mixture
was stirred at room temperature. After 8 h, Aq 5% NaHSO3 was
added and the reaction was stirred for 10 min. The mixture was
then diluted with CH2Cl2 (20.0 mL) and washed with H2O
(2 ꢁ 15.0 mL) and brine (2 ꢁ 15.0 mL). The organic phase was dried
with anhydrous Na2SO4 and evaporated under reduced pressure.
The resulting crude product was purified by chromatography on
1567, 1556, 1403, 1281, 1128, 1081, 750 cmꢀ1
;
1H NMR
(500 MHz, CD3OD): d = 5.92 (1H, dd, J = 16.0, 1.2 Hz, H-24), 5.77

K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
3029
silica gel (40% ethyl acetate/hexane) to afford 38 (32 mg, 35%) as a
white solid. Mp: 119–120 °C; IR (KBr): 3445, 2979, 1738, 1731,
NMR (500 MHz, CDCl3): d = 6.70 (1H, dd, J=2.1, 10.4 Hz, H-2),
5.98 (1H, dd, J = 3.0, 10.4 Hz, H-2), 5.79 (1H, ddd, J = 2.5, 2.5, 5.0,
H-6), 4.34 (1H, t, J = 7.0 Hz, H-16), 3.36 (1H, m, H-10), 3.26 (1H,
d, J = 15.0 Hz, H-12a), 2.83 (1H, ddd, J = 6.5, 9.0, 17.5 Hz, H-23a),
2.75 (1H, d, J = 15.0 Hz, H-12b), 2.54 (1H, d, J = 7.0 Hz, H-17), 2.52
(1H, m, H-23b), 2.36 (1H, ddt, J = 3.0, 8.5, 19.5 Hz, H-7b), 2.06
(2H, overlapped, H-24), 2.03 (1H, overlapped, H-7a), 2.02 (1H, d,
J = 8.5 Hz, H-8), 1.96 (3H, s, Me-32), 1.87 (1H, J = 9.0, 13.0 Hz, H-
15b), 1.47 (1H, overlapped, H-15a), 1.46 (3H, s, Me-26), 1.44 (3H,
s, Me-27), 1.42 (3H, s, Me-21), 1.41 (3H, s, Me-30), 1.29 (3H, s,
Me-29), 1.20 (3H, s, Me-28), 1.01 (3H, s, Me-19), 1.00 (3H, s, Me-
18); 13C NMR (125.8 MHz, CDCl3): d = 213.8 (C-22), 213.1 (C-11),
202.4 (C-3), 170.3 (C-31), 145.7 (C-2), 137.2 (C-5), 127.3 (C-1),
120.5 (C-6), 81.2 (C-25), 78.9 (C-20), 71.0 (C-16), 57.8 (C-17),
50.6 (C-), 49.0 (C-12), 48.8 (C-4), 48.4 (C-14), 48.3 (C-9), 45.7 (C-
15), 41.5 (C-8), 36.5 (C-10), 34.8 (C-24), 30.7 (C-23), 27.4 (C-28),
26.1 (C-27), 25.8 (C-26), 24.5 (C-21), 23.6 (C-7), 22.4 (C-32), 20.2
(C-29), 20.0 (C-19), 19.9 (C-18), 18.1 (C-30); ESI-MS (negative ion
mode) m/z 541.3146 [MꢀH]ꢀ (calcd for C32H45O7 541.3171).
1696, 1373, 1238, 1110, 1028 cmꢀ1 1H NMR (500 MHz, CDCl3):
;
d = 5.78 (1H, ddd, J = 2.0, 2.0, 6.0, H-6), 5.48 (1H, dd, J = 5.5,
13.5 Hz, H-2), 5.34 (1H, m, H-24), 5.13 (1H, ddd, J = 1.5, 8.2,
9.5 Hz, H-16), 3.37 (1H, m, H-23a), 3.27 (1H, m, H-23b), 3.24 (1H,
d, J = 15.0 Hz, H-12a), 2.80 (1H, m, H-10), 2.75 (1H, d, J = 8.2 Hz,
H-17), 2.74 (1H, d, J = 15.0 Hz, H-12b), 2.43 (1H, ddt, J = 2.5, 8.0,
19.5 Hz, H-7b), 2.15 (3H, s, Me-20), 2.13 (1H, overlapped, H-1a),
2.02 (1H, d, J = 8.0 Hz, H-8), 2.0 (1H, m, H-15b), 1.95 (1H, m, H-
7a), 1.90 (3H, s, Me-200), 1.76 (3H, d, J = 1.5 Hz, Me-26), 1.67 (3H,
br s, Me-27), 1.53 (1H, ddd, J = 13.0, 13.0, 13.0 Hz, H-1b), 1.46
(3H, s, Me-21), 1.40 (1H, dd, J = 1.5, 14.0, H-15a), 1.32 (3H, s, Me-
28), 1.31 (3H, s, Me-30), 1.29 (3H, s, Me-29), 1.10 (3H, s, Me-19),
1.02 (3H, s, Me-18); 13C NMR (125.8 MHz, CDCl3): d = 211.8 (C-
11), 211.3 (C-22), 205.6 (C-3), 170.2 (C-10), 170.1 (C-100), 139.7 (C-
5), 135.5 (C-25), 120.4 (C-6), 115.5 (C-24), 78.5 (C-20), 73.9 (C-
16), 73.3 (C-2), 54.0 (C-17), 51.3 (C-4), 49.9 (C-13), 48.6 (C-12),
48.4 (C-9), 47.9 (C-14), 43.2 (C-15), 42.1 (C-8), 35.2 (C-23), 34.3
(C-10), 32.0 (C-1), 28.7 (C-28), 25.7 (C-26), 24.0 (C-21), 23.7 (C-
7), 21.3 (C-29), 20.8 (C-200), 20.7 (C-20), 20.0 (C-18), 19.6 (C-19),
18.9 (C-30), 18.3 (C-27); ESI-MS (negative ion mode) m/z
585.3421 [MꢀH]ꢀ (calcd for C34H49O8 585.3422).
4.3.36. Preparation of 40
CrO3 (46 mg, 0.47 mmol) was dissolved in a mixture of pyridine
(0.4 mL, 0.31 mmol) and CH2Cl2 (1.0 mL). After stirring for 10 min,
a solution of 9 (100 mg, 0.16 mmol) in 0.5 mL of CH2Cl2 was added
slowly. The mixture was stirred at room temperature for 72 h, fil-
tered and the filtrate was neutralized with 5% HCl, washed (NaCl,
NaHCO3, and water), dried with anhydrous Na2SO4, and evaporated
under reduced pressure. The residue was subjected to column
chromatography (40% ethyl acetate/hexane) to give 44 (20 mg,
20% yield) as a white solid. Mp: 131–132 °C; IR (KBr) 3445, 2984,
4.3.35. Preparation of 39
To a solution of compound 15 (150 mg, 0.224 mmol) in metha-
nol (2.0 mL), a suspension of OxoneÒ (179 mg, 0.29 mmol) in water
(2.0 mL) was added at 0 °C. After 5 min, a saturated solution of so-
dium bisulfite (5.0 mL) was added. After evaporation under re-
duced pressure, the crude mixture was dissolved in water and
extracted with diethyl ether (2 ꢁ 20 mL). The organic phase was
then dried with anhydrous Na2SO4 and evaporated under reduced
pressure. The resulting crude product was purified by chromatog-
raphy on silica gel (40% ethyl acetate/hexane) to afford the mix-
tures of sulfoxides (86 mg, 57% yield) Major isomer 1H NMR
(CDCl3) d = 7.65 (2H, dd, J = 8.0, 1.5 Hz, H-20/H-60), 7.56 (2H, m,
H-30/H-70), 7.53 (1H, m, H-40), 5.80 (1H, ddd, J = 2.0, 2.0, 6.0, H-6),
4.28 (1H, m, H-16), 6.43 (1H, dd, J = 13.2, 6.0 Hz, H-2), 3.06 (1H,
d, J = 14.6 Hz, H-12a), 2.78 (1H, m, H-23a), 2.56 (1H, d,
J = 14.6 Hz, H-12b), 2.51 (1H, m, H-10), 2.49 (1H, overlapped, H-
23b), 2.46 (1H, d, J = 7.2 Hz, H-17), 2.41 (1H, overlapped, H-7b),
2.03 (2H, m, H-24), 1.96 (1H, d, J = 8.0 Hz, H-8), 1.96 (1H, m, H-
7a), 1.95 (3H, s, Me-32), 1.91 (2H, m, H-1), 1.80 (1H, dd, J = 9.0,
13.5 Hz, H-15b), 1.44 (3H, s, Me-26), 1.42 (3H, s, Me-27), 1.37
(1H, m, H-15a), 1.35 (3H, s, Me-20), 1.32 (3H, s, Me-29), 1.27
(3H, s, Me-30), 1.22 (3H, s, Me-28), 1.11 (3H, s, Me-19), 0.92 (3H,
s, Me-18); 13C NMR (125.8 MHz, CDCl3): d = 213.8 (C-22), 211.8
(C-11), 207.6 (C-3), 142.3 (C-10), 139.3 (C-5), 131.2 (C-40), 129.1
(C-30/C-50), 124.6 (C-20/C-60), 121.0 (C-6), 81.2 (C-25), 78.9 (C-20),
71.0 (C-16), 71.0 (C-2), 57.7 (C-17), 51.6 (C-4), 50.4 (C-13), 48.8
(C-12), 48.7 (C-9), 48.3 (C-14), 45.5 (C-15), 42.3 (C-8), 35.3 (C-
10), 34.8 (C-24), 30.6 (C-23), 27.9 (C-28), 26.1 (C-26), 25.8 (C-27),
24.4 (C-20), 23.8 (C-7), 23.2 (C-29), 21.7 (C-1), 19.9 (C-20), 19.8
(C-18), 18.6 (C-30), CH3CO2 (170.3, 22.4); ESI-MS (positive ion
mode) m/z 669.3470 [M+H]+ (calcd for C38H53O8S 669.3456).
A sealed 10 mL glass tube containing the mixture of isomers
(80 mg, 0.12 mmol) and triethylamine (0.03 mL, 0.24 mmol) in tol-
uene was introduced in the cavity of a microwave reactor (CEM Co.,
Discover) and irradiated with stirring under a maximum potency
(300 W) for 5 min. The temperature was raised up to 120 °C. After
cooling the mixture to 20 °C, the reaction vessel was opened and
the product was evaporated under reduced pressure. The resulting
crude product was purified by chromatography on silica (40% ethyl
acetate/hexane) to afford 39 (46 mg, 71% yield) as a white solid.
Mp:133–134 °C; IR (KBr): 3420, 1972, 1931, 1723, 1702, 1698,
1731, 1698, 1374, 1245, 1029 cmꢀ1 1H NMR (CDCl3): d = 6.23
;
(1H, d, J = 2.2 Hz, H-6), 5.52 (1H, dd, J = 13.0, 5.3 Hz, H-2), 5.19
(1H, m, H-16), 3.24 (1H, d, J = 15.5 Hz, H-12a), 3.19 (1H, ddd,
J = 13.0, 3.8, 2.2 Hz, H-10), 2.86 (1H, d, J = 15.5 Hz, H-12b), 2.66
(1H, d, J = 7.5 Hz, H-17), 2.63 (2H, m, H-23), 2.59 (1H, s, H-8),
2.45 (1H, dd, J = 15.2, 9.0 Hz, H-15b), 2.31 (1H, ddd, = 13.0, 5.3,
3.8 Hz, H-1a), 2.18 (CH3COO), 2.03 (2H, m, H-24), 1.98 (3H, s,
Me-20), 1.92 (3H, s, Me-200), 1.72 (1H, ddd, J = 13.0, 13.0, 13.0 Hz,
H-1b), 1.48 (3H, s, Me-26), 1.46 (3H, s, Me-27), 1.45 (3H, s, Me-
21), 1.44 (3H, s, Me-28), 1.41 (3H, s, Me-29), 1.34 (3H, s, Me-30),
1.33 (1H, overlapped, H-15a), 1.15 (3H, s, Me-19), 1.05 (3H, s,
Me-18); 13C NMR (125.8 MHz, CDCl3): d = 212.0 (C-22), 209.5 (C-
11), 202.9 (C-3), 198.3 (C-7), 170.4 (C-10), 170.0 (C-31), 169.5 (C-
100), 163.9 (C-5), 125.1 (C-6), 81.0 (C-25), 78.5 (C-20), 73.6 (C-16),
72.4 (C-2), 57.2 (C-8), 53.9 (C-17), 52.0 (C-4), 49.5 (C-13), 48.8
(C-9), 48.6 (C-12), 47.5 (C-14), 43.1 (C-15), 36.5 (C-10), 35.2 (C-
24), 30.5 (C-23), 30.2 (C-1), 29.7 (C-28), 26.0 (C-26), 25.8 (C-27),
24.3 (C-21), 22.4 (C-20), 21.1 (C-29), 21.0 (C-19), 20.8 (C-200), 20.6
(C-32), 19.7 (C-18), 19.2 (C-30); ESI-MS (positive ion mode) m/z
681.3280 [M+Na]+ (calcd for C36H50NaO11 681.3245).
4.4. Biological activity
4.4.1. Cell line
Human non-small-cell lung cancer A549 cells were kindly pro-
vided by Dr. Rosina Gironès from Microbiology Department of Uni-
versity of Barcelona, Spain. A549 cells (American Type Culture
Collection [ATCC] CCL185) were grown in Minimal Essential Med-
ium supplemented with 10% fetal bovine serum, 100 U/mL penicil-
lin G, 100 lg/mL streptomycin and 25 lg/mL amphotericin B in a
humidified 5% CO2 atmosphere at 37 °C.
4.4.2. Cell viability assay
The effect of compounds treatment on the viability of A549 cells
was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide (MTT) (Sigma, MO, USA) assay based on the ability
1694, 1674, 1661, 1366, 1275, 1217, 1210, 1181, 1025 cmꢀ1 1H
;

3030
K. L. Lang et al. / Bioorg. Med. Chem. 20 (2012) 3016–3030
Oncogene 2005, 24, 3236; (e) Blaskovich, M. A.; Sun, J. Z.; Cantor, A.; Turkson, J.;
Jove, R.; Sebti, S. M. Cancer Res. 2003, 63, 1270; (f) Jayaprakasan, B.; Seeram, N.
P.; Nair, M. G. Cancer Lett. 2003, 189, 11; (g) Duncan, M. D.; Duncan, K. L. K. J.
Surg. Res. 1997, 69, 55.
of live cells to cleave the tetrazolium ring to a molecule that absorb
at 540 nm.30 Briefly, cells were plated in 96-well culture plates
(1x104 cells/well). After 24 h incubation, the cells were treated
with different concentrations of the compounds. After 48 h and
7. (a) Yu, H.; Jove, R. Nature Rev. Cancer. 2004, 4, 97; (b) Fletcher, S.; Drewry, J. A.;
Shahani, V. M.; Page, B. D. G.; Gunning, P. T. Biochem. Cell Biol. 2009, 87, 825.
8. Siddiquee, K. Z.; Turkson, J. Cell. Res. 2008, 18, 254.
72 h at 37 °C, the medium was removed and 50
(1 mg/mL) was added to each well, and cells were further incu-
bated at 37 °C for 4 h. The MTT solution was removed, 100 L of di-
lL of MTT reagent
9. (a) Haritunians, T.; Gueller, S.; Zhang, L.; Badr, R.; Yin, D.; Xing, H.; Fung, M. C.;
Koeffler, H. P. Leukemia Res. 2008, 32, 1366; (b) Wakimoto, N.; Yin, D.; O0Kelly,
J.; Haritunians, T.; Karlan, B.; Said, J.; Xing, H.; Koeffler, H. P. Cancer Sci. 2008,
99, 1793; (c) Yin, D.; Wakimoto, N.; Xing, H.; Lu, D.; Huynh, T.; Wang, X.; Black,
K. L.; Koeffler, H. P. Int. J. Cancer. 2008, 123, 1364; (d) Knecht, D. A.; LaFleur, R.
A.; Kahsai, A. W.; Argueta, C. E.; Beshir, A. B.; Fenteany, G. PLoS ONE. 2010, 5,
e14039; (e) Duncan, K. L. K.; Duncan, M. D.; Alley, M. C.; Sausville, E. A. Biochem.
Pharmacol. 1996, 52, 1553; Dinan, L.; Whiting, P.; Girault, J.-P.; Lafont, R.;
Dhadialla, T. S.; Cress, D. E.; Mugat, B.; Antoniewski, C.; Lepesant, J.-A. Biochem.
J. 1997, 327, 643; (g) Maloney, K. N.; Fujita, M.; Eggert, U. S.; Schroeder, F. C.;
Field, C. M.; Mitchison, T. M.; Clardy, J. J. Nat. Prod. 2008, 71, 1927.
10. Ryu, S. Y.; Choi, S. U.; Lee, S. H.; Lee, C. O.; No, Z.; Ahn, J. W. Arch. Pharm. Res.
1995, 18(1), 60.
11. Bartalis, J.; Halaweish, F. T. Bioorg. Med. Chem. 2011, 19, 2757. and publications
and patents cited therein..
12. Jung, M. E.; Lui, R. M. J. Org. Chem. 2010, 75, 7146.
13. (a) Schenkel, E. P.; Farias, M. R.; Mayer, R.; Breitmaier, E.; Rucker, G.
Phytochemistry 1992, 31, 1329; (b) Farias, M. R.; Schenkel, E. P.; Mayer, R.;
Rücker, G. Planta Med. 1993, 59, 272; (c) Santos, R. I.; Santos, M. A.; dos
Schenkel, E. P. Int. J. Pharmacog. 1996, 34, 300.
l
methyl sulfoxide (Merck, Germany) was added to each well to
dissolve formazan crystals, and the plates were gently shaken,
whereby crystals were completely dissolved, followed by reading
on a scanning multiwell spectrophotometer (Infinite 1200 TECAN,
Grödje, Austria). The 50% inhibition concentration (IC50) was de-
fined as the concentration that inhibited cell proliferation by 50%
when compared to untreated controls. Untreated cells were used
as controls. Paclitaxel (Glenmark, Brazil) (at 0–10
lM) and doxoru-
bicin (Zodiac, Brazil) (at 0–10 M) were used as positive control
l
(purity > 98%). The final concentration of DMSO showed no inter-
ference with the growth of cells.
4.5. Statistical analysis
14. Lang, K. L.; Guimarães, T. R.; Rocha Machado, V.; Zimmermann, L. A.; Silva, I. T.;
Teixeira, M. R.; Durán, F. J.; Palermo, J. A.; Simões, C. M. O.; Caro, M. S. B.;
Schenkel, E. P. Planta Med. 2011, 77, 1648.
15. (a) Wen, X.; Zhang, P.; Liu, J.; Zhang, L.; Wu, X.; Ni, P.; Sun, H. Bioorg. Med. Chem.
Lett. 2006, 16, 722; (b) Wen, X.; Xia, J.; Cheng, K.; Zhang, L.; Zhang, P.; Liu, J.;
Zhang, L.; Ni, P.; Sun, H. Bioorg. Med. Chem. Lett. 2007, 17, 5777.
16. Hartung, J.; Hünig, S.; Kneuer, R.; Schwarz, M.; Wenner, H. Synthesis 1997, 12,
1433.
The IC50 values and 95% confidence intervals of the tested com-
pounds on A549 cells were calculated with a log (compound) ver-
sus normalized response (variable slope) curve fit (GraphPad
Prism, Software Inc., San Diego, CA).
Acknowledgments
17. Drögemüller, M.; Flessner, T.; Jautelat, R.; Scholz, U.; Winterfeldt, E. Eur. J. Org.
Chem. 1998, 12, 2811.
The authors acknowledge Conselho Nacional de Desenvolvi-
mento Científico e Tecnológico (CNPq, MCT, Brazil), Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, MEC,
Brazil), Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET, Argentina), Agencia Nacional de Promoción Científica y
Tecnológica (ANPCyT, Argentina) and UBA (Univerdad de Buenos
Aires, Argentina) for financial support and fellowships. The authors
declare no conflict of interest with respect to this publication.
18. Barton, D. H. R.; Jang, D. O.; Jaszberenyi, J. C. Tetrahedron 1993, 49, 7193.
19. Seyden-Penne, J. Reductions by the Alumino- and Borohydrides in Organic
Synthesis; Wiley-VCH: France, 1997.
20. Králíková, S.; Budesinsky, M.; Tomecková, I.; Rosenberg, I. Tetrahedron 2006, 62,
9742.
21. Veleiro, A. S.; Rosenstein, R. E.; Jaliffa, C. O.; Grilli, M. L.; Speroni, F.; Burton, G.
Bioorg. Med. Chem. Lett. 2003, 13, 343.
22. Gemal, A. L.; Luche, J. L. J. Am. Chem. Soc. 1981, 18, 5454.
23. Weyermann, P.; Keese, R. Tetrahedron 2011, 67, 3874.
24. Alvarez-Manzaneda, E. J.; Chahboun, R.; Cabrera Torres, E.; Alvarez, E.; Alvarez-
Manzaneda Roldán, R.; Haidour, A.; Ramos, J. Tetrahedron Lett. 2005, 46, 1075.
25. Jayaprakasam, B.; Seeram, N. P.; Nair, M. G. Cancer Lett. 2003, 189, 11.
26. Durán, F. J.; Ghini, A. A.; Coirini, H.; Burton, G. Tetrahedron 2006, 62, 4762.
27. Cuia, J.; Fana, L.; Huanga, L.; Liub, H.; Zhoub, A. Steroids 2009, 74, 62.
28. Itharat, A.; Houghton, P. J.; Houghton, E.; Burke, P. J.; Sampson, J. H.; Raman, A.
J. Ethnopharmacol. 2004, 90, 33.
29. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose,
C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich, M.; Campbell, H.;
Mayo, J.; Boyd, M. J. Nat. Cancer Inst. 1991, 83, 757.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.03.001.
References and notes
30. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
31. Liu, T.; Zhang, M.; Zhang, H.; Sun, C.; Deng, Y. Eur. Arch. Otorhinol. 2008, 265,
1225.
32. Duangmano, S.; Dakeng, S.; Jiratchariyakul, S.; Suksamrarn, A.; Smith, D.;
Patmasiriwat, P. Int. J. Mol. Sci. 2010, 11, 5323.
33. Chan, K. T.; Meng, F. Y.; Li, Q.; Ho, C. Y.; Lam, T. S.; To, Y.; Lee, W. H.; Li, M.; Chu,
K. H.; Toh, M. Cancer Lett. 2010, 294, 118.
34. Iwanski, G. B.; Lee, D. H.; En-Gal, S.; Doan, N. B.; Castor, B.; Vogt, M.; Toh, M.;
Bokemeyer, C.; Said, J. W.; Thoennissen, N. H.; Koeffler, P. Brit. J. Pharm. 2010,
160, 998.
35. Sadzuka, Y.; Hatakeyama, H.; Sonobe, T. Int. J. Pharm. 2010, 383, 186.
36. Liu, T.; Peng, H.; Zhang, M.; Deng, Y.; Wu, Z. Eur. J. Pharm. Sci. 2010, 641, 15.
37. Dang, G. V.; Rode, B. M.; Stuppner, H. Eur. J. Pharm. Sci. 1994, 2, 331.
38. Chen, C.; Qiang, S.; Lou, L.; Zhao, W. J. Nat. Prod. 2009, 72, 824.
39. (a) Fang, X.; Phoebe, C. H.; Pezzuto, J. M.; Fong, H. H. S.; Farnsworth, N. R.;
Yellin, B.; Hecht, S. M. J. Nat. Prod. 1984, 47, 988; (b) Kanchanapoom, T.; Kasaia,
R.; Yamasakia, K. Phytochemistry 2002, 59, 215.
40. Matsuda, H.; Nakashima, S.; Abdel-Halim, O. B.; Morikawa, T.; Yoshikawa, M.
Chem. Pharm. Bull. 2010, 58, 747.
41. Wu, P. L.; Lin, F. W.; Wu, T. S.; Kuoh, C. S.; Lee, K. H.; Lee, S. J. Chem. Pharm. Bull.
2004, 52, 345.
1. (a) Butler, M. S. Nat. Prod. Rep. 2008, 25, 475; Ganesan, A. Curr. Opin. Chem. Biol.
2008, 12, 306; (b) Harvey, A. L. Drug Disc. Today 2008, 13, 894; (c) Lam, K. S.
Trends Microbiol. 2007, 15, 279; (d) Newman, D. J.; Cragg, G. M. J. Nat. Prod.
2007, 70, 461; (e) Chin, Y.-W.; Balunas, M. J.; Chai, H. B.; Kinghorn, A. D. AAPS J.
2006, 8, E239.
2. Wermuth, C. G. The Practice of Medicinal Chemistry; Elsevier Academic Press,
2008.
3. (a) Miró, M. Phytother. Res. 1995, 9, 159; Valente, L. M. M. Quím. Nova 2004, 27,
944; (b) Chen, J. C.; Chiu, M. H.; Nie, R. L.; Cordell, G. A.; Qiu, S. X. Nat. Prod. Rep.
2005, 22, 386; Lee, K.-H. J. Nat. Prod. 2010, 73, 500; (c) Lee, D. H.; Iwanski, G. B.;
Thoennissen, N. H. Sci. World J. 2010, 10, 413.
4. (a) Dinan, L.; Harmatha, J.; Lafont, R. J. Chromatogr. A 2001, 935, 105; (b) Ríos, J.
L.; Escandell, J. M.; Recio, M. C. Stud. Natural Prod. Chem. 2005, 32, 429.
5. (a) Lee, D. H.; Thoennissen, N. H.; Goff, C.; Iwanski, G. B.; Forscher, C.; Doan, N.
B.; Said, J. W.; Koeffler, P. Cancer Lett. 2011, 306, 161; (b) Lavie, D.; Glotter, E.
Fortschr. Chem. Org. Naturst. 1971, 29, 307.
6. (a) Thoennissen, N. H.; Iwanski, G. B.; Doan, N. B.; Okamoto, R.; Lin, P.; Abbassi,
S.; Song, J. H.; Yin, D.; Toh, M.; Xie, W. D.; Said, J. W.; Koeffler, P. Cancer Res.
2009, 69, 5876; (b) Siqueira, J. M.; Peters, R. R.; Gazola, A. C.; Krepsky, P.; Farias,
M. R.; Rae, G. A.; Brum-Fernandes, A. J.; Ribeiro-do-Valle, R. M. Life Sci. 2007, 80,
1382; (c) Yang, L.; Wu, S.; Zhang, Q.; Liu, F.; Wu, P. Cancer Lett. 2007, 256, 267;
(d) Sun, J.; Blaskovich, M. A.; Jove, R.; Livingston, S. K.; Coppola, D.; Sebti, S. M.
42. Velde, V.; Lavie, D. Tetrahedron 1983, 39, 317.