Synthesis and cytotoxic evaluation of C-9 oxidized podophyllotoxin derivatives

Article abstract of DOI:10.1016/j.bmc.2006.12.008

A series of podophyllotoxin and podophyllic aldehyde derivatives, lacking the lactone ring and oxidized at C-9 position, has been prepared. The functionalities considered at C-9 were carboxylic acids and several derivatives such as esters, amides, nitrile

Full text of DOI:10.1016/j.bmc.2006.12.008

Bioorganic & Medicinal Chemistry 15 (2007) 1670–1678
Synthesis and cytotoxic evaluation of C-9 oxidized
podophyllotoxin derivatives
a
*
´
´
M Angeles Castro, Jose M. Miguel del Corral, Marina Gordaliza, Pablo A. Garcıa,
a
´
M Antonia Gomez-Zurita and Arturo San Feliciano
Departamento de Quı´mica Farmace´utica, Facultad de Farmacia, Campus Miguel de Unamuno,
Universidad de Salamanca, 37007 Salamanca, Spain
Received 10 July 2006; revised 4 December 2006; accepted 8 December 2006
Available online 12 December 2006
Abstract—A series of podophyllotoxin and podophyllic aldehyde derivatives, lacking the lactone ring and oxidized at C-9 position,
has been prepared. The functionalities considered at C-9 were carboxylic acids and several derivatives such as esters, amides, nitriles
or anhydrides. The synthesized compounds were cytotoxic at the micromolar level, though less potent and selective than the parent
compounds, revealing the influence of the C-9 electrophilic character on the potency and selectivity of these cyclolignans.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
tant for the anti-HIV activity described for those
derivatives.7 Also, there are natural cyclolignans that
present a free carboxylic group at C-94 for which, as
far as the authors knows, no biological activities are
described.
Podophyllotoxin 1 and its semisynthetic derivatives,
etoposide, teniposide and etopophos, are antiviral
and cytotoxic compounds in clinical use that belong
to the cyclolignan family of natural products
(Fig. 1).1 Podophyllotoxin derivatives are widely used
as anticancer agents, but they still have several sec-
ondary effects. Because of that, the aryltetralin lignans
are the subject of extensive research and nearly every
ring of the cyclolignan skeleton (A–E) has been mod-
ified2,3 and in those cases in which the lactone ring re-
mained, it used to be like in podophyllotoxin, that is a
90,9-lactone.
Over the years, our group has been involved in the
chemical transformation of podophyllotoxin and has
prepared a large number of cyclolignans by modifica-
tions of nearly all the rings of the skeleton looking for
more potent, less toxic and more selective analogues.8,9
In this sense, we prepared a potent cytotoxic and selec-
tive cyclolignan,10,11 the podophyllic aldehyde 2, that
lacked the c-lactone ring, generally considered an
important feature for the bioactivity of podophyllotoxin
analogues.1–3 Thus, the podophyllic aldehyde became
our lead compound for further modifications from
which the imine derivatives are worth to point out be-
cause they not only maintained the level of cytotoxicity
of the parent aldehyde, but have also considerably im-
proved the selectivity against the HT-29 colon
carcinoma.12
Furthermore, there have been described several natu-
ral cyclolignans,4 such as justicidin E, retrojusticidin
B or formosolactone5 among others (Fig. 1), that pos-
sess a 9,90-lactone instead of a 90,9-lactone present in
podophyllotoxin-like lignans. In general, the retrolac-
tones have been less studied, with only a few examples
as that of the synthesis and antiproliferative effects of
retroetoposide.6 In those cases, the compounds were
less cytotoxic although the retrolactone seemed impor-
Those facts prompted us to prepare new analogues of
podophyllic aldehyde with higher oxidation degree at
C-9, in order to analyse how the electrophilic character
at that position could influence the antineoplastic selec-
tivity previously observed for the aldehyde derivatives.
Thus, we now report the synthesis of new cyclolignans
with several carboxyl-related derivatives (esters, amides,
nitriles, etc.) at C-9 position and their cytotoxicity.
Keywords: Podophyllotoxin; Podophyllic aldehyde; C-9 oxidized
cyclolignans; Cytotoxicity.
*
Corresponding author. Tel.: +34 923 294528; fax: +34 923
294515; e-mail: macg@usal.es
´
Present address: Centro de Investigacion Biomolecular Aplicada
(CIBASA), Campus Miguel de Unamuno, Salamanca, Spain.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.12.008

M. A. Castro et al. / Bioorg. Med. Chem. 15 (2007) 1670–1678
1671
OH
7
9
CHO
1
2
5
O
O
O
A
B
3
C
R1
D O
O
O
9'
O
COOCH3
8'
1'
O
HO
O
O
6'
2'
OH
E
O
H3CO
OCH3
OCH3
H3CO
OCH3
O
4'
O
OCH3
O
Podophyllic aldehyde 2
Podophyllotoxin 1
H3CO
OCH3
OR2
O
O
O
O
O
O
O
O
R1
R2
H
CH3
Etoposide
Teniposide
Etopophos
H
O
S
H3CO
OCH3
OCH3
O
O
OH
OH
CH3
P
Justicidin E
Formosolactone
Figure 1. Structures of podophyllotoxin and related compounds.
2. Results and discussion
2.1. Chemistry
experiments and by preparation of their acetyl deriva-
tives 9.
On the other hand, the reduction of the methyl ester 2,
after transient protection of the aldehyde group as its
dithiolane, yielded the hydroxy-aldehyde 1011 which
was treated with TsOH to give a very complex reaction
mixture, from which only the 9,90-lactone 11 was isolat-
ed after column chromatography, probably due to a fur-
ther oxidation, under atmospheric oxygen, of the desired
lactol (Scheme 1).
Podophyllic aldehyde 2 was the starting material for the
preparation of C-9 carboxylic acid derivatives. It was
obtained from podophyllotoxin
described.10–12
1
as previously
The aldehyde group was oxidized13 to the carboxylic
acid 3 with NaClO2 using 2-methyl-2-butene as scaven-
ger, then transformed into its methyl ester 4 by treat-
ment with an ethereal solution of diazomethane. The
1H NMR data of diester 4 were in agreement with those
published by Jones14 and now, its 13C NMR data are
reported. The acid 3 was also transformed into the
mixed anhydride 5 by reaction with acetic anhydride
(Scheme 1).
Other functions considered for being introduced at C-9
were nitriles and amides. The nitrile 13 was obtained
from the aldehyde 2, in good yield through its interme-
diate oxime 12 (Scheme 2), either by isolation of 1211a
followed by its dehydratation with acetic anhydride/so-
dium acetate,16 or directly in a one-pot reaction under
microwave irradiation17 which afforded the nitrile 13
with a considerable reduction on the reaction time.
Another procedure described in the literature18,19 to ob-
tain nitriles from aldehydes with good yields is the treat-
ment with nitroethane and sodium acetate, however,
when it was applied to 2, the nitrile 13 was obtained in
lower yields and accompanied by the corresponding aro-
matised nitrile 13a in a 55:45 ratio.
In previous works10,15 we proposed that the podophyllic
aldehyde 2 could be cytotoxic by transformation into its
lactol-lactone analogue, giving rise to a more rigid mol-
ecule, similar to the trans-lactones, with the four rings
almost coplanar and thus more reactive and more sus-
ceptible to nucleophilic attack by several biomolecules.
Under these premises, we tried to prepare several deriv-
atives with a lactol function at C-9.
Amides 14–20 were obtained from the acid 3 using CDI20
or DCC/HOBT21 as activating agents (Scheme 3) and
their structures confirmed by two-dimensional NMR
experiments. Thus, reaction of 3 with ethyl-, phenyl-,
4-methylphenyl- and 3,4,5-trimethoxyphenyl-amines in
THF and CDI yielded the amides 14, 16–18 with low
yields (9–30%). When ethanolamine was used, the only
isolated product was the phthalimide 19, in which again
the aromatisation of the C ring took place, together with
the aminolysis of the C-90 ester group.
Attempts to transform directly the aldehyde 2 into the
lactol by treatment with TsOH under different reac-
tion conditions were unsuccessful, recovering either
the starting material or products derived from the
C-ring aromatisation. Consequently we decided to
modify the functionality at C-90 and the acid 3 was
saponified to the diacid 6 and further treatment with
acetyl chloride afforded the anhydride 7 which was re-
duced with NaBH4 to yield the lactol-lactones 8, in
which
a
migration of the double bond from
C-7 to C-8(80) took place. The presence of the lactol
at C-9 was confirmed by two-dimensional NMR
Treatment of 3 with hexylamine under reflux, without
any activating agent, afforded the diamide 20; however

1672
M. A. Castro et al. / Bioorg. Med. Chem. 15 (2007) 1670–1678
OH
1) KOH / MeOH
NaClO2
CHO
COOR1
O
O
O
O
O
O
+
O
Me2C=CHMe
2) H3O , pH 4
COOCH3
COOCH3
3) CH2N2 / Et2O
O
NaH2PO4 / H2O
t-BuOH
4) Swern oxidation
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
81%
R1
H
3
1
2 (96%)
3
CH2N2 / Et2O (99%)
CH3
4
KOH / MeOH, Δ
Ac2O (64%)
COCH3
5
OR2
O
O
COOH
COOH
O
O
O
O
O
O
NaBH4
AcCl
O
THF, - 10 ˚C
Me2CO
O
O
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
8 (36%)
9 (90%)
R2: COCH3
7 (71%)
6 (98%)
R2: H
Ac2O / Py
O
1) (CH2SH)2, Me3SiCl
CH2Cl2
CHO
CHO
O
O
O
O
O
O
O
TsOH
COOCH3
CH2OH
C6H6, Δ
[O]
2) LiAlH4 / Et2O
3) HgO, BF3-Et2O
THF / H2O
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
2
10 (88%)
11 (25%)
Scheme 1. Synthesis of cyclolignans 3–11.
.
NH2OH HCl, N-methyl-2-pyrrolidinone / MW (89%)
CN
O
O
O
CHO
O
O
N-OH
.
O
NH2OH HCl
COOCH3
NaOAc
COOCH3
COOCH3
7'
Py / EtOH, Δ
Ac2O, Δ
(91%)
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
12 (95%)
13
13a: Δ7'
2
EtNO2, NaOAc / AcOH, Δ (40%)
Scheme 2. Preparation of nitriles 13 and 13a.
when the reaction was performed in the presence of
DCC and HOBT, the corresponding amide 15 was ob-
tained in good yield (70%). When the latter conditions
were applied to prepare the ethylamide 14, an important
increase in the yield (from 9% with CDI to 87% with
DCC) was also observed.

M. A. Castro et al. / Bioorg. Med. Chem. 15 (2007) 1670–1678
1673
COOH
CONHR
R
method
O
O
O
O
R-NH2 / THF
Et
n-C6H13
Ph
A (9%) / B (85%) 14
COOCH3
COOCH3
method A (CDI)
or
method B (DCC, HOBT)
B (70%)
15
16
A (30%)
H3CO
OCH3
OCH3
H3CO
OCH3
OCH3
A (26%)
A (22%)
17
3
14 - 18
OCH3
OCH3
OCH3
18
HO-(CH2)2-NH2
CDI, THF
n-C6H13-NH2
Δ
O
OH
CONHC6H13
CONHC6H13
O
O
O
O
N
O
H3CO
OCH3
H3CO
OCH3
OCH3
OCH3
20 (20%)
19 (23%)
Scheme 3. Preparation of the amides 14–20.
2.2. Cytotoxicity
with respect to the parent compounds podophyllotoxin
1 and podophyllic aldehyde 2.
Most of the compounds prepared were evaluated in vitro
to establish their antineoplastic cytotoxicity22,23 against
cell cultures of P-388 murine leukaemia, A-549 human
lung carcinoma, HT-29 human colon carcinoma and
MEL-28 human melanoma. The results obtained are
shown in Table 1, expressed in lM.
Among the carboxylic acid derivatives, there were no
significant differences in the cytotoxicity measured for
amides, nitriles or anhydrides. The less potent com-
pounds of the series are those having a free carboxylic
group at C-9 position (3 and 6) and the reverse lactone
11 resembling justicidin E. Those results indicate that
the electrophilic character at that position clearly influ-
ences the cytotoxic potency and the selectivity of these
cyclolignans.
The first general observation that can be made is that no
differences are observed among the IC50 values for the
four cell lines, which means that the selectivity observed
for 2 has been lost. On the other hand, although all the
derivatives tested were cytotoxic, a decrease in the
potency of one or two orders of magnitude was observed
The lactol-lactone 8 displayed the best cytotoxicity of
the series but was less potent than we expected based
on the postulation made before, although we have to
take in account that 8 is not exactly the lactol expected
from 2, because the double bond in C ring is located at a
different position, making the molecule less rigid. This
may be compared to the cis-lactones, all of them being
less potent than the trans-lactone analogues.1–3,8,9
Table 1. Cytotoxic activity of cyclolignan derivatives (IC50, lM)
Compound
P-388
A-549
HT-29
MEL-28
1
2
0.012
0.23
>20
5.1
0.012
0.12
>20
5.1
0.012
0.012
>20
5.1
n.d.
0.23
>20
5.1
3
4
In summary, we have prepared a series of podophyllo-
toxin and podophyllic aldehyde derivatives, lacking the
lactone ring and oxidized at C-9 position to carboxylic
acid derivatives such as esters, amides, nitriles or anhy-
drides. The compounds synthesized were cytotoxic at
the micromolar range, although less potent and selective
than the parent compounds.
5
10
10
10
10
6
>20
12
>20
12
>20
12
>20
12
7
8
1.2
1.2
1.2
n.d.
0.25
>20
2.3
10
11
12
13
15
16
17
18
19
20
0.25
>20
2.3
0.25
>20
2.3
0.25
>20
2.3
n.d.
1.9
2.4
1.9
2.4
1.9
2.4
n.d.
n.d.
1.9
3. Experimental
3.1. Chemistry
1.9
1.9
1.9
1.9
1.9
1.9
1.7
2.2
1.7
2.2
1.7
2.2
n.d.
n.d.
n.d.
2.0
2.0
2.0
NMR spectra were recorded on a Bruker AC 200 at
200 MHz for 1H and 50.3 MHz for 13C in deuterio-
n.d., not determined.

1674
M. A. Castro et al. / Bioorg. Med. Chem. 15 (2007) 1670–1678
chloroform with TMS as an internal standard. Opti-
cal rotations were recorded on a Perkin-Elmer 241
polarimeter in chloroform solution and UV spectra
on a Hitachi 100-60 spectrophotometer in ethanol
solution. IR spectra were obtained on a Nicolet
Impact 410 spectrophotometer. HRMS were run in
a VG-TS-250 spectrometer working at EI (70 eV) or
FAB and in a hybrid quadrupole time of flight spec-
trometer QSTAR XL (Applied Biosystems) by ESI in
positive mode (the ionization voltage was 5.0 kV).
Column chromatography (CC) was performed on sil-
ica gel (Merck No. 9385). TLC was carried out on
silica gel 60F254 (SDS 0.20 mm thick). Solvents and
reagents were purified by standard procedures as
necessary.
anhydrous Na2SO4 and the solvent evaporated off to
20
D
give 5 (35 mg, 64%). ½aꢀ ꢁ110 (c 1.14). UV kmax (lge):
211 (4.5), 242 (4.0), 336 (4.0) nm. IR (film) mmax: 1732
(COOCH3, C@C–COOCO), 1677 (C@C–COOCO)
cmꢁ1
.
ESI-HRMS: calcd for C25H24O10 + Na,
507.1261; found, 507.1250. 1H NMR: Table 2. 13C
NMR: Table 3.
3.1.6. (70R,80S)-30,40,50-Trimethoxy-4,5-methylenedioxy-
2,70-cyclolign-7-ene-9,90-dioic acid (6). Acid 3 (105 mg,
0.238 mmol) was dissolved in a methanolic solution of
5% KOH (5.0 mL), stirred and heated under reflux for
6 h. After cooling to room temperature, concentration
under reduced pressure and dilution with EtOAc, it
was extracted with aq satd Na2CO3. The aqueous phase
was acidified to pH 2 with 2 N HCl and extracted with
EtOAc. The organic layer was washed with brine, dried
over anhydrous Na2SO4 and the solvent was removed
3.1.1. Podophyllotoxin (1). It was isolated from
Podophyllum emodi in the same way as we previously
published.24
under reduced pressure to yield the diacid 6 (100 mg,
20
D
98%). ½aꢀ ꢁ145 (c 0.11). UV kmax (lge): 212 (4.5), 241
3.1.2. 90-Methyl (70R,80S)-30,40,50-trimethoxy-4,5-methyl-
enedioxy-9-oxo-2,70-cyclolign-7-en-90-oate (2). Podo-
phyllotoxin 1 was transformed into podophyllic alde-
hyde 2 after saponification, esterification and Swern
oxidation by a previously described method (96% over-
all yield).11
(4.4), 336 (4.1) nm. IR (KBr) mmax: 3600–2500 (COOH),
1703 (COOH) cmꢁ1
C22H20O9 + Na, 451.0999; found, 451.1004. H NMR:
Table 2. 13C NMR: Table 3.
.
ESI-HRMS: calcd for
1
3.1.7. (70R,80S)-30,40,50-Trimethoxy-4,5-methylenedioxy-
2,70-cyclolign-7-ene-9,90-dioic anhydride (7). Compound
5 (124 mg, 0.290 mmol) was treated with acetyl chloride
(0.50 mL, 7.0 mmol) in dry acetone (2.0 mL) and stirred
at room temperature for 4 d. After concentration of the
reaction mixture under reduced pressure, methanol was
added and a yellow precipitate was formed and separat-
3.1.3. (70R,80S)-30,40,50,90-Tetramethoxy-4,5-methylene-
dioxy-90-oxo-2,70-cyclolign-7-en-9-oic acid (3). To a solu-
tion of 2 (500 mg, 1.17 mmol) and 2-methyl-2-butene
(1.5 mL, 12 mmol) in t-butanol (25 mL), a solution of
sodium chlorite (0.44 mL, 1.5 mmol) in aq 5% NaH2PO4
(10 mL) was slowly added. After the mixture was stirred
at room temperature for 4 d, it was concentrated under
reduced pressure, dissolved in EtOAc and extracted with
aq satd Na2CO3. The aqueous phase was acidified with
2 N HCl and extracted with EtOAc. The organic layer
was washed with brine, dried over anhydrous Na2SO4
ed by filtration to yield the anhydride 7 (85 mg, 71%).
20
D
½aꢀ ꢁ230 (c 0.65). UV kmax (lge): 214 (4.4), 246 (4.3),
283 (4.2), 328 (3.9) nm. IR (KBr) mmax: 1848 (COOCO),
1770 (COOCO), 1668 (C@C–COOCO) cmꢁ1. EI-
HRMS: calcd for C22H18O8, 410.0992; found,
1
410.0992. H NMR: Table 2. 13C NMR: Table 3.
and the solvent removed under reduced pressure to give
20
D
3 (420 mg, 81%). ½aꢀ ꢁ129 (c 0.96). UV kmax(lge): 213
3.1.8. (70R)-9-Hydroxy-30,40,50-trimethoxy-4,5-methylene-
dioxy-2,70-cyclolign-8(80)-eno-90,9-lactone (8). To a solu-
tion of 7 (70 mg, 0.17 mmol) in dry THF (12 mL),
sodium borohydride (1.7 mg, 0.043 mmol) was added
and stirred at ꢁ10 ꢁC for 6 h. The reaction was
quenched with aq satd NH4Cl and extracted with
CH2Cl2. The organic phase was washed with brine,
dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The reaction product was purified
by CC on silica gel (CH2Cl2–EtOAc, 8:2) to yield
(4.4), 241 (4.3), 336 (4.0) nm. IR (KBr) mmax: 3600–
2500 (COOH), 1732 (COOCH3), 1683 (C@C–COOH)
cmꢁ1. ESI-HRMS: calcd for C23H22O9 + Na, 465.1156;
found, 465.1145. 1H NMR: Table 2. 13C NMR: Table 3.
3.1.4. Dimethyl (70R,80S)-30,40,50-trimethoxy-4,5-methyl-
enedioxy-2,70-cyclolign-7-ene-9,90-dioate (4). The com-
pound 3 (30 mg, 0.068 mmol) was treated with an
ethereal solution of diazomethane for 1 h. After remov-
ing the solvent, the dimethyl diester 4 (31 mg, 100%) was
the hydroxylactones 8 (25 mg, 36%). IR (film) mmax:
20
D
obtained. ½aꢀ ꢁ115 (c 0.88). UV kmax (lge): 210 (3.4),
3392 (OH), 1762 and 1714 (c-hydroxy-a,b-unsaturat-
243 (4.3), 312 (3.7), 342 (3.9) nm. IR (KBr) mmax: 1734
(COOCH3), 1708 (C@C–COOCH3) cmꢁ1. ESI-HRMS:
calcd for C24H24O9 + Na, 479.1312; found, 479.1301.
1H NMR: Table 2. 13C NMR: Table 3.
ed-c-lactone) cmꢁ1 1H NMR: Table 2. 13C NMR:
.
Table 3.
3.1.9. (70R)-9-Acetoxy-30,40,50-trimethoxy-4,5-methylenedi-
oxy-2,70-cyclolign-8(80)-eno-90,9-lactone (9). The hydroxy-
lactones 8 (15 mg, 0.037 mmol) were treated with
acetic anhydride (1.0 mL) in pyridine (1.0 mL) and stir-
red at room temperature for 1 h. Then, ice was added
and after it melted, the solution was diluted with EtOAc.
The organic layer was washed with 2 N HCl, aq satd
NaHCO3 and brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure to give the
3.1.5. Acetic (70R,80S)-30,40,50,90-tetramethoxy-4,5-methyl-
enedioxy-90-oxo-2,70-cyclolign-7-en-9-oic anhydride (5).
The acid 3 (50 mg, 0.11 mmol) was dissolved in acetic
anhydride (5.0 mL) and stirred at room temperature
for 1 d. Then, ice was added and after it melted, the mix-
ture was diluted with EtOAc. The organic layer was
washed with aq satd NaHCO3 and brine, dried over

M. A. Castro et al. / Bioorg. Med. Chem. 15 (2007) 1670–1678
Table 2. 1H NMR data of compounds 3–9 and 11–20 (d ppm (J Hz))
1675
H
3
4
5
6
7
8
9
11
12
3
6
7
6.65 s
6.84 s
7.72 s
6.65 s
6.83 s
7.62 s
6.69 s
6.86 s
7.69 s
6.66 s
6.84 s
7.72 s
6.31 s
6.87 s
6.61/6.59 s
6.73 s
6.62/6.61 s 6.30 s
6.72/6.70 s 6.86 s
6.63 s
6.73 s
3.65–3.85 m 7.40 d (3.2) 6.71 s
7.53 d (2.6) 3.93 dd (23, 4.6)
3.62 dd (23, 4.6)/ 3.65–3.90 m
6.09 br s
6.40–6.60 m 6.34 s
6.40–6.60 m 6.34 s
9
6.94/6.93 s
6.37/6.35 s 6.48 s
6.37/6.35 s 6.49 s
7.86 s
6.25 s
6.25 s
20
60
70
80
90
6.24 s
6.24 s
6.24 s
6.24 s
6.22 s
6.22 s
6.20 s
6.20 s
4.63 d (2.6) 4.60 d (2.7) 4.66 d (2.6) 4.62 s
3.98 d (2.6) 4.01 d (2.7) 4.01 d (2.6) 3.96 s
4.18 s
4.13 d (2.6)
4.78/4.71 t (4.6)
4.70–4.90 m 3.78 d (2.2) 4.47 d (2.2)
3.39–3.59 m 3.98 d (2.2)
4.44 t (8.9)
3.98 t (8.9)
5.98 d (1.3) 5.94 s
5.96 d (1.3) 5.93 s
–OCH2O– 6.00 d (1.7) 5.98 d (1.1) 6.02 d (1.3) 5.99 s
5.98 d (1.7) 5.97 d (1.1) 6.01 d (1.3) 5.98 s
6.01 d (1.1) 5.95 s
5.99 d (1.1) 5.94 s
3.70–3.90 m 3.78 s
5.96 s
5.94 s
3.80 s
3.80 s
3.80 s
CH3O-30 3.74 s
CH3O-40 3.78 s
CH3O-50 3.74 s
CH3O-90 3.65 s
3.74 s
3.79 s
3.74 s
3.65 s
3.75 s
3.80 s
3.75 s
3.67 s
3.72 s
3.77 s
3.72 s
3.89 s
3.90 s
3.82 s
3.71 s
3.76 s
3.91 s 3.77 s
3.70–3.90 m 3.73 s
3.71 s
3.62 s
8.24 br s
OH
Others
9.07 br s
9.71 br s
4.45 br s
3.77 s
2.31 s
2.19/2.20 s
H
13
14
15
16
17
18
19
20
3
6.59 s
6.74 s
7.23 s
6.24 s
6.24 s
6.64 s
6.64 s
6.65 s
6.80 s
6.67 s
6.80 s
7.29 s
6.32 s
6.32 s
6.58 s
6.78 s
7.30 s
6.30 s
6.30 s
7.11 s
6.90 s
7.10 s
8.00 s
6.60 s
6.60 s
6
6.75 s
7.11 s
6.30 s
6.30 s
6.75 s
7.11 s
6.31 s
6.31 s
7.34 s
8.17 s
6.55 s
6.55 s
7
7.30 s
6.31 s
6.31 s
20
60
70
80
4.60 d (4.2) 4.51 d (3.5) 4.51 d (3.4)
3.61 d (4.2) 4.04 d (3.5) 4.03 d (3.4)
4.55 d (3.3)
4.13 d (3.3)
5.98 s
4.56 d (3.3) 4.49 d (4.4)
4.11 d (3.3) 4.12 d (4.4)
–OCH2O– 5.99 d (1.3) 5.97 s
5.98 d (1.3)
5.97 s
5.96 s
3.75 s
3.79 s
3.75 s
3.63 s
6.09 br s
5.99 s
5.99 s
5.98 s
3.77 s
3.80 s
3.77 s
3.66 s
8.21 br s
6.13 s
6.05 s
CH3O-30
CH3O-40
CH3O-50
CH3O-90
NH
3.76 s
3.81 s
3.76 s
3.73 s
3.75 s
3.79 s
3.76 s
3.79 s
3.76 s
3.79 s
3.85 s
3.97 s
3.85 s
3.84 s
3.92 s
3.84 s
3.75 s
3.63 s
6.05 br s
3.76 s
3.66 s
8.15 br s
3.76 s
3.66 s
8.02 br s
6.09 br s
Others
3.35 q (6.5) 3.32 dd (13.1, 5.7) 7.55 d (8.0)
1.21 t (6.5) 1.05–1.98 m (8 H) 7.31 dd (8.0, 7.1) 7.13 d (8.4) 3.83 s (6 H) (4 H)
0.87 t (6.6) 7.09 t (7.1) 2.31 s 3.79 s (3 H)
7.45 d (8.4) 6.86 s (2H) 3.60–4.05 m 3.27–3.37 m (4 H)
1.00–1.80 m (16 H)
0.75–0.90 m (6 H)
1
acetoxylactones 9 (15 mg, 90%). H NMR: Table 2. 13C
NMR: Table 3.
3.1.12. Methyl (70R,80S)-9-hydroxyimino-30,40,50-trimeth-
oxy-4,5-methylenedioxy-2,70-cyclolign-7-en-90-oate (12).
Over a solution of 2 (156 mg, 0.368 mmol) in dry EtOH
(3.0 mL), dry pyridine (28 lL, 0.35 mmol) and hydroxyl-
amine hydrochloride (28 mg, 0.40 mmol) were added.
The mixture was stirred under reflux for 2 h and then al-
lowed to cool to room temperature and the solvent re-
moved under reduced pressure. Water was added and
the product was extracted with EtOAc, washed with
2 N HCl and brine, dried over anhydrous Na2SO4 and
filtered off. The evaporation of the solvent afforded the
oxime 12 (153 mg, 95%).11a
3.1.10. (70R,80S)-90-Hydroxy-30,40,50-trimethoxy-4,5-methyl-
enedioxy-2,70-cyclolign-7-en-9-al (10). It was prepared as
previously described from 2 by reduction with LiAlH4 of
its dithiolane protected derivative and final
deprotection.11
3.1.11. (70R,80S)-30,40,50-Trimethoxy-4,5-methylenedioxy-
2,70-cyclolign-7-eno-9,90-lactone (11). A mixture of 10
(170 mg, 0.428 mmol) and TsOH (10 mg, 0.058 mmol)
was stirred in benzene (15 mL) under reflux for 5 h.
Then, solvent was removed under reduced pressure
and the product was redissolved in EtOAc. The organic
layer was washed with aq satd NaHCO3 and brine, dried
over anhydrous Na2SO4, filtered and evaporated until
dryness. CC on silica gel (CH2Cl2–EtOAc, 96:4) of the
residue afforded 11 (43 mg, 25%). IR (film) mmax: 1752
3.1.13. Methyl (70R,80S)-8-cyano-30,40,50-trimethoxy-4,5-
methylenedioxy-9-nor-2,70-cyclolign-7-en-90-oate (13). (a)
From 12. The oxime 12 (153 mg, 0.349 mmol) was re-
fluxed in acetic anhydride (2.0 mL) with sodium acetate
(2 mg, 0.02 mmol) for 21 h. Then, it was extracted with
EtOAc and the organic phase was washed with brine
and dried over anhydrous Na2SO4 to obtain, after evap-
oration of the solvent, the nitrile 13 (134 mg, 91%). IR
(C@C–COO)
cmꢁ1
.
ESI-HRMS:
calcd
for
1
C22H20O7 + Na, 419.1101; found, 419.1131. H NMR:
Table 2. 13C NMR: Table 3.
(film) mmax: 2212 (C@C–CN), 1735 (COOCH3) cmꢁ1
.

1676
M. A. Castro et al. / Bioorg. Med. Chem. 15 (2007) 1670–1678
Table 3. 13C NMR data of compounds 3–9 and 11–20 (d ppm)
C
3
4
5
6
7
8
9
11
12
13
14
15
16
17
18
19
20
1
125.1 125.1 124.6 125.1 125.7 124.0
132.1 131.8 132.8 132.1 134.6a 129.2
109.8 109.9 109.9 110.0 103.2b 109.3
149.8 149.5 153.1 150.0 150.8 147.0
147.1 147.2 150.4 147.3 147.3 147.0
108.9 108.8 109.0 109.1 109.2b 108.0
123.2
129.0
109.3
147.3
147.2
107.9
125.7 131.1 124.0 125.2 125.0 125.0 125.1 125.0 126.7 129.7
134.3a 126.8 131.2 131.1 131.3 131.5 131.6 131.5 130.3a 130.4
108.7 109.6 109.9 109.5 109.6 109.6 109.7 109.6 106.2 104.3
149.5 148.3 150.1 148.7 148.7 149.1 149.2 149.2 150.5 149.4
146.8 147.1 147.3 146.9 146.9 147.0 147.1 147.1 150.1 148.5
109.2 107.8 108.2 108.1 108.2 108.4 108.4 108.3 105.1 103.1
2
3
4
5
6
7
139.3 137.3 140.6 139.8 135.7 28.3/27.9 28.2/27.8 132.3 133.2 142.2 130.9 131.0 132.3 132.2 132.3 123.0 128.1
122.0 122.9 121.6 121.4 120.5 156.8 154.5 127.0 126.4 103.6 127.7 127.7 127.7 127.9 127.8 123.6 130.1
171.8 167.0 162.0 172.0 162.5 97.6/97.4 92.9/92.5 169.5 151.4 119.0 167.1 167.1 165.2 165.2 165.4 168.0b 168.0
8
9
10
20
30
40
50
60
70
80
90
137.6 137.8 137.2 137.8 135.0a 138.3
104.5 104.7 104.5 104.5 109.8 105.4a
153.0 153.1 153.1 153.2 153.5 153.2
136.8 136.9 137.2 136.9 137.6 137.3
153.0 153.1 153.1 153.2 153.5 153.2
104.5 104.7 104.5 104.5 109.8 105.6a
137.6
105.6
153.3
137.2
153.2
105.6
135.0a 138.2 136.6 137.7 137.7 137.5 137.6 137.4 139.0 136.4
108.0 105.3 104.8 104.9 104.8 104.8 104.8 104.9 107.0 107.6
154.2 153.3 153.4 153.1 153.1 153.2 153.3 153.2 153.2 153.1
137.6 137.6 137.3 137.0 137.0 138.0 137.1 137.1 138.0 137.6
153.3 153.3 153.4 153.1 153.1 153.2 153.3 153.2 153.2 153.1
103.1 105.3 104.8 104.9 104.8 104.8 104.8 104.9 107.0 107.6
51.3 46.5 45.5 46.3 46.2 46.2 46.3 46.5 133.4a 133.1
40.8 46.5 48.8 47.1 47.1 47.2 47.3 47.7 133.4a 130.9
71.9 172.5 170.8 172.9 172.8 173.0 173.0 173.5 168.8b 170.1
101.6 101.3 101.8 101.3 101.4 101.5 101.6 101.6 102.3 101.6
56.2 56.3 56.1 56.1 56.1 56.1 56.2 56.1 56.3 56.2
60.9 60.7 60.8 60.7 60.7 60.8 60.9 61.0 61.1 61.0
56.2 56.3 56.1 56.1 56.1 56.1 56.2 56.1 56.3 56.2
52.3 52.9 52.4 52.4 52.7 52.8 52.8
46.5a 47.0a 46.2a 46.4a 46.9c 42.8/42.6 42.8
46.3a 46.5a 46.1a 46.1a 45.2c 130.6
172.5 172.7 171.9 177.7 168.8 169.9
131.6
169.3a
101.4
56.2
–OCH2O– 101.5 101.6 101.7 101.7 102.1 101.3
CH3O-30
CH3O-40
CH3O-50
CH3O-90
Others
55.9 56.1 56.0 56.1 56.2 56.2
60.6 60.8 60.6 60.8 60.9 60.8
55.9 56.1 56.0 56.1 56.2 56.2
52.5 52.6 52.6
60.8
56.2
52.0 166.2
22.2
168.6a
20.8
34.7 39.9 129.0 135.5 134.2 41.1 40.3, 40.0
14.7 31.4 119.9 120.0 97.6 61.5 31.5, 31.4
29.3 129.0 129.5 153.2
26.5 124.2 134.0 134.6
29.4, 29.0
26.7, 26.5
22.6
22.5
14.0
21.0 56.1
61.0
14.0
a–cExchangeable assignments.
FAB-HRMS: calcd for C23H21NO7 + H, 424.1396;
found, 424.1379. 1H NMR: Table 2. 13C NMR: Table 3.
was redissolved in CH2Cl2. The organic layer was
washed with brine, dried over anhydrous Na2SO4,
filtered and evaporated to obtain a mixture that was
purified by silica gel CC to obtain the amides in
9–30% yield.
(b) From 2 and NH2OH. A mixture of 2 (42 mg,
0.10 mmol), hydroxylamine hydrochloride (10 mg,
0.14 mmol) and N-methyl-2-pyrrolidinone (100 lL,
1.04 mmol) was irradiated at 750 W in a domestic
microwave oven for 10 min (5 · 2 min). Then, it was
diluted with water and extracted with EtOAc. The
organic layer was washed with brine, dried, filtered
and concentrated in vacuum to yield the nitrile 13
(37 mg, 89%).
Method B. To a solution of 3 (0.10–0.25 mmol) in dry
THF (3.0 mL), N,N0-dicyclohexylcarbodiimide (DCC,
1.0 equiv), 1-hydroxybenzotriazole (HOBT, 1.0 equiv)
and the corresponding amine (2.0 equiv) were added
and the mixture stirred at room temperature for 24 h.
The mixture was then filtered and the solvent evaporat-
ed. The residue was redissolved in CH2Cl2 and this
organic phase was washed with aq satd NaHCO3, brine,
dried over anhydrous Na2SO4, filtered and evaporated.
Purification by silica gel CC afforded the amides in 70-
85% yield.
(c) From 2 and EtNO2. Aldehyde 2 (94 mg, 0.22 mmol),
nitroethane (82 lL, 1.1 mmol) and sodium acetate
(71 mg, 0.87 mmol) were refluxed in glacial acetic acid
(2.0 mL) for 22 h. The mixture was allowed to cool to
room temperature and then diluted with water, washed
with aq satd NaHCO3 and brine, dried over anhydrous
Na2SO4, filtered and the solvent evaporated off. Purifi-
cation by silica gel CC (CH2Cl2–EtOAc, 95:5) afforded
the mixture 13 + 13a in a 55:45 ratio (37 mg, 40%).
3.1.15. Methyl (70R,80S)-9-ethylamino-30,40,50-trimeth-
oxy-4,5-methylenedioxy-9-oxo-2,70-cyclolign-7-en-90-oate
(14). Following the method A, the reaction between 3
(51 mg, 0.12 mmol) and ethylamine 2.0 M in THF
(87 lL, 0.17 mmol) yielded, after CC purification
(n-hexane–EtOAc, 6:4), the ethylamide 14 (5 mg, 9%).
3.1.14. General procedures for the synthesis of amides 14–
18. Method A. To a solution of 3 (0.07–0.15 mmol) in
dry THF (4.0 mL), N,N0-carbonyldiimidazol (CDI, 1.5
equiv) was added in small portions and the mixture stir-
red at room temperature for 1 h. The corresponding
amine (1.5 equiv) was then added and the mixture stir-
red at room temperature for 24 h more. The solvent
was evaporated under reduced pressure and the residue
Application of the method B to 3 (95 mg, 0.21 mmol)
afforded 14 (87 mg, 85%). IR (film) mmax: 3370 (NH),
1731 (COOCH3), 1651 (CONH), 1532 (CONH)
cmꢁ1
.
ESI-HRMS: calcd for C25H27NO8 + Na,
492.1634; found, 492.1641. 1H NMR: Table 2. 13C
NMR: Table 3.

M. A. Castro et al. / Bioorg. Med. Chem. 15 (2007) 1670–1678
1677
3.1.16. Methyl (70R,80S)-9-hexylamino-30,40,50-trimeth-
oxy-4,5-methylenedioxy-9-oxo-2,70-cyclolign-7-en-90-oate
(15). Following method B, 3 (48 mg, 0.11 mmol) and
n-hexylamine (29 lL, 0.22 mol) yielded 15 (40 mg,
(CONH), 1552 (CONH) cmꢁ1.ESI-HRMS: calcd for
C34H44N2O7 + Na, 615.3046; found, 615.3047. 1H
NMR: Table 2. 13C NMR: Table 3.
70%) after CC (CH2Cl2–EtOAc, 85:15). IR (film) mmax
3319 (NH), 1731 (COOCH3), 1622 (CONH), 1537
(CONH) FAB-HRMS: calcd for
cmꢁ1
C29H35NO8 + H, 526.2441; found, 526.2487. H NMR:
Table 2. 13C NMR: Table 3.
:
3.2. Cytotoxic assays
.
A screening method previously described was used to as-
sess the antitumoral activity against the following cell
lines: P-388 (lymphoid neoplasma from DBA/2 mouse),
A-549 (human lung carcinoma), HT-29 (human colon
carcinoma) and MEL-28 (human melanoma).
1
3.1.17. Methyl (70R,80S)-30,40,50-trimethoxy-4,5-methylene-
dioxy-9-oxo-9-phenylamino-2,70-cyclolign-7-en-90-oate (16).
From 3 (40 mg, 0.090 mmol) and aniline (13 lL,
0.14 mmol) using method A, phenylamide 16 was ob-
Cells were seeded into 16-mm wells (multidishes NUNC
42001) at concentrations of 1 · 104 (P-388), 2 · 104
(A-549, HT-29 and MEL-28) cells/well, respectively, in
1 mL aliquots of MEM-10% FCS medium containing
the compound to be evaluated at different concentra-
tions. In each case, a set of control wells was incubated
in the absence of drug and counted daily to ensure expo-
nential cell growth. After 4 days at 37 ꢁC, under a 10%
CO2, 98% humid atmosphere, P-388 cells were observed
by inverted microscopy and the degree of inhibition was
determined by comparison with the controls, while
A-549, HT-29 and MEL-28 were stained with crystal
violet before examination. All calculations represent
the average of duplicate wells.
tained (14 mg, 30%) after CC (n-hexane–EtOAc, 7:3).
20
D
½aꢀ ꢁ90 (c 0.20). IR (film) mmax: 3353 (NH), 1731
(COOCH3), 1662 (CONH), 1537 (CONH) cmꢁ1. FAB-
HRMS: calcd for C29H27NO8 + H, 518.1815; found,
1
518.1863. H NMR: Table 2. 13C NMR: Table 3.
3.1.18. Methyl (70R,80S)-30,40,50-trimethoxy-4,5-methyl-
enedioxy-9-oxo-9-(p-tolylamino)-2,70-cyclolign-7-en-90-
oate (17). Chromatographic purification of the reaction
product (CH2Cl2–EtOAc, 92:8) between 3 (44 mg,
0.10 mmol) and p-toluidine (16 mg, 0.15 mmol) follow-
20
D
ing method A gave 17 (14 mg, 26%). ½aꢀ ꢁ126 (c
0.26). IR (film) mmax: 3343 (NH), 1731 (COOCH3),
1659 (CONH), 1525 (CONH) cmꢁ1. FAB-HRMS: calcd
for C30H29NO8 + H, 532.1971; found, 532.1953. 1H
NMR: Table 2. 13C NMR: Table 3.
Acknowledgments
´
´
´
Financial support came from Consejerıa de Educacion y
Cultura de la Junta de Castilla y Leon (SA-49/01 and
3.1.19. Methyl (70R,80S)-30,40,50-trimethoxy-4,5-methyl-
enedioxy-9-oxo-9-(3,4,5-trimethoxyphenylamino)-2,70-
cyclolign-7-en-90-oate (18). Using method A, 3 (65 mg,
0.15 mmol) and 3,4,5-trimethoxyaniline (41 mg,
0.22 mmol) afforded 18 (20 mg, 22%) after CC
(CH2Cl2–EtOAc, 8:2). IR (film) mmax: 3341 (NH), 1732
(COOCH3), 1659 (CONH), 1539 (CONH) cmꢁ1. FAB-
HRMS: calcd for C32H33NO11 + H, 608.2131; found,
´
´
SA-068/04), Fundacion Ramon Areces (predoctoral
grant to P.A.G.) and Universidad de Salamanca
(USAL2005-03).
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608.2192. H NMR: Table 2. 13C NMR: Table 3.
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´
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´
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